key: cord-322573-1fw1ehzd authors: vicente, diego; cilla, gustavo; montes, milagrosa; pérez-yarza, eduardo g.; pérez-trallero, emilio title: human bocavirus, a respiratory and enteric virus date: 2007-04-17 journal: emerg infect dis doi: 10.3201/eid1304.061501 sha: doc_id: 322573 cord_uid: 1fw1ehzd in spain, human bocavirus (hbov) was detected in 48 (9.1%) of 527 children with gastroenteritis at similar frequency as for children with respiratory illness (40/520, 7.7%). fecal excretion adds new concern about the transmission of hbov. to our knowledge, this report is the first to document hbov in human feces. h uman bocavirus (hbov), a recently described new virus species belonging to the parvoviridae family, was identified as a human pathogen in september 2005 (1) . since then, this parvovirus has been found in children with respiratory tract illness in practically all areas of the world in which it has been investigated (2) (3) (4) (5) , an indication of its wide dissemination. to determine the prevalence and clinical characteristics of hbov, we investigated the presence of this virus in children with respiratory tract infection in our region (gipúzkoa, basque country, spain). among the first patients in whom hbov was detected in nasopharyngeal aspirates, we found two 12-month-old children with diarrhea in addition to respiratory symptoms. because animal parvoviruses are frequently associated with enteritis in young animals (6), we investigated the presence of hbov in the diarrheal feces of both children. hbov was detected in both samples, and no other intestinal pathogens were identified. to rule out the possibility that this result could have been due to fecal contamination resulting from swallowing respiratory secretions, and to determine whether the gastrointestinal tract is affected by this new respiratory virus, we studied its presence in patient feces in 527 episodes of acute gastroenteritis, unrelated to respiratory infection, in children <3 years of age, mainly from nonhospital centers (ambulatory clinics). our analyses were conducted from december 2005 through march 2006. viral dna and rna were obtained from nasopharyngeal aspirates and stool specimens with an automatic extractor biorobot m48 (qiagen, hilden, germany) by using the magattract virus mini m48 kit (qiagen). cdna was obtained by using m-mulv reverse transcriptase (promega, madison, wi, usa) and random primers. aliquots of the dna and cdna were frozen at −40°c until pcr for hbov detection was performed. respiratory samples were investigated for respiratory syncytial virus, influenza viruses a and b, parainfluenza virus types 1-4, and adenovirus by cell culture and pcr. rhinovirus, coronavirus (nl63 coronavirus included), and metapneumovirus were studied by pcr alone. fecal specimens were examined for shigella spp., salmonella spp., yersinia enterocolitica, campylobacter spp., and enteroinvasive escherichia coli o157 by standard culture methods. rotavirus was investigated by enzyme immunoassay and norovirus by reverse transcriptase pcr. hbov detection was performed by pcr with primers derived from the np1 gene (1). positive samples were retested and confirmed as positive by using a second pcr assay with primers derived from another location in the hbov genome (vp1 gene) (7) . amplified np1 and vp1 gene fragments (354 bp and 403 bp, respectively) were sequenced and analyzed by using the blast software (www.ncbi.nlm.nih.gov/ blast). each pcr run included a negative control (water) that was treated as the clinical sample throughout, and pcr was performed with the usual precautions to avoid contamination. strain spain001 (genbank accession no. ef186830) was included as positive control in each pcr run. of the 527 stool samples analyzed from december 2005 through march 2006, hbov was detected in 48 (9.1%). from a second group of 520 children <3 years of age who came to the pediatric emergency unit of our hospital with an episode of acute respiratory infection during the same period, a similar frequency of hbov detection was obtained (40/520, 7.7%) when nasopharyngeal aspirates were tested. analysis of np1 and vp1 partial gene sequences obtained from all fecal and respiratory hbovpositive samples showed a similarity of >95% with previously published hbov sequences. of 40 hbov-positive respiratory samples, 25 (62.5%) showed coinfection with other viruses (respiratory syncytial virus in 13, rhinovirus in 3, influenza a in 3, coronavirus oc43 in 2, adenovirus in 1, influenza b in 1, respiratory syncytial virus and coronavirus oc43 in 1, and influenza a and rhinovirus in 1). of the 48 hbov-positive fecal samples, 28 (58.3%) showed coinfection with another intestinal pathogen (salmonella enteritidis in 1, campylobacter jejuni in 5, rotavirus in 14, norovirus in 7, and c. jejuni and norovirus in 1). in this study, simultaneous detection of hbov and other agents was frequent for respiratory or enteric specimens. the incidence of coinfection in respiratory illness was similar to that observed in studies that were not limited to specimens that had already tested negative for other microorganisms and in which a wide number of agents were investigated (4) . adenoviruses have been associated with infection of the colon and the gut and are a cause of severe gastroenteritis in nonindustrialized countries. in this study, coinfection of adenovirus and hbov was detected in 1 respiratory specimen but these viruses together were not detected in any fecal sample. hbov and parvovirus b19 are the only 2 species of the parvoviridae family that have been associated with disease in humans. to date, hbov has only been detected in samples from the respiratory tract and has been associated with both upper and lower respiratory tract disease in infants and young children. the results of our study show that hbov is also present in the gastrointestinal tract in children with gastroenteritis with or without symptoms of respiratory infection. the fecal excretion adds new concern about the transmission of hbov. to our knowledge, this report is the first to document hbov in human feces. the high frequency of hbov detection in the feces of children with gastroenteritis and the absence of any other intestinal pathogen suggest that this new virus species is an enteric, as well as a respiratory, pathogen. further investigations to confirm this preliminary hypothesis and gain greater knowledge of the association between hbov and enteric disease are required. genus in the family parvoviridae. previously identified members of this genus are pathogens of bovines and canines. a parvovirus of human origin was recently discovered and called human bocavirus because it is closely related to bovine parvovirus and canine minute virus. human bocavirus is associated with respiratory tract infections, particularly in infants and young children. articles should be no more than 1,200 words and need not be divided into sections. if subheadings are used, they should be general, e.g., "the study" and "conclusions." provide a brief abstract (50 words); references (not to exceed 15); figures or illustrations (not to exceed 2); tables (not to exceed 2); and a brief biographical sketch of first author-both authors if only 2. dispatches are updates on infectious disease trends and research. the articles include descriptions of new methods for detecting, characterizing, or subtyping new or reemerging pathogens. developments in antimicrobial drugs, vaccines, or infectious disease prevention or elimination programs are appropriate. case reports are also welcome. cloning of a human parvovirus by molecular screening of respiratory tract samples detection of human bocavirus in japanese children with lower respiratory tract infections human bocavirus: prevalence and clinical spectrum at a children's hospital frequent detection of human rhinoviruses, paramyxoviruses, coronaviruses, and bocavirus during acute respiratory tract infections human bocavirus in hospitalized children pathological and virological studies of experimental parvoviral enteritis in calves human bocavirus infection address for correspondence: emilio pérez-trallero, hospital donostia -microbiology, paseo dr beguiristain, s/n san sebastian gipúzkoa this work was partly financed by the "convenio diputación gipúzkoa-hospital donostia" and by a grant from the spanish ministerio de sanidad y consumo ciber cb06/06. dr vicente is a medical microbiologist at the hospital donostia, gipúzkoa, spain. his research focuses on viral respiratory infections and meningococcal infection epidemiology. key: cord-324898-bjxpuqhr authors: lazzeri, marta title: how italian respiratory physiotherapists have faced and are facing the coronavirus disease 2019 pandemic date: 2020-08-03 journal: arch physiother doi: 10.1186/s40945-020-00086-8 sha: doc_id: 324898 cord_uid: bjxpuqhr nan how italian respiratory physiotherapists have faced and are facing the coronavirus disease 2019 pandemic marta lazzeri 1, 2 dear editor: the whole world is facing an unknown disease that often manifests itself in a serious and deadly form: the coronavirus disease 2019, or covid-19, which is caused by viral infection (sars-cov-2) and presents with severe acute respiratory syndrome. symptoms of covid-19 can vary. the most frequent are dry cough, asthenia, fever, and in more severe cases (approximately 25%) pneumonia characterized by bilateral interstitial infiltrates with hypoxic respiratory failure (i.e., acute respiratory distress syndrome; ards) after serious alteration of the ventilation/perfusion ratio (va/q). it is associated with a systemic inflammatory response that can induce microcirculation alterations, arrhythmia, myocarditis, renal and liver failure. non-invasive intervention such as high-flow nasal oxygen therapy, continuous positive airway pressure, and non-invasive mechanical ventilation, aimed to correct hypoxemia and to reduce the work of breathing, can help to avoid endotracheal intubation with its potential complications and wasting effects on outcomes or survival. however, these interventions must be managed in appropriate settings with a careful monitoring, since rapid and unexpected worsening often occurs, necessitating immediate endotracheal intubation. in the acute phase, having specialized health professionals with specific respiratory skills has been critical. where present, respiratory physiotherapists (rph) made a great contribution by working together with other health professionals in assisting patients, providing "quick basic training sessions" about respiratory care for non-specialist healthcare professionals, locating and assessing all kinds of equipment (ventilators, interfaces, oxygen delivery systems, among others) to assist patients with respiratory failure and implementing early intervention with those patients in healthier conditions. the expertise previously gained in the treatment of acute and chronic respiratory failure conditions due to different etiopathogeneses (such as fibrosis, exacerbation of chronic obstructive pulmonary disease, interstitial lung diseases, difficult to wean post-acute patients) was extremely valuable, because the clinical functional manifestations of many of the aforementioned conditions are not different to those deficits observed in covid-19. knowledge about the oxygen delivery systems and interfaces, non-invasive mechanical ventilation, weaning from assisted ventilation and tracheostomy, airway clearance strategies and techniques, early mobilization and posture variation is applied every day to optimize ventilation, to improve gas exchanges, to prevent secondary impairments and possible post-acute sequelae, and even to reduce the intensive care unit and hospital stay. moreover, when clinical conditions allow it, all actions to promote the patient autonomy in respiration, exercise tolerance and mobility in the daily life activity and other basic functions including coughing, phonation, and swallowing are ensured. many hospital departments have been converted to admit covid-19 patients: intensive and respiratory subintensive care beds expaned exponentially; pre-triage to access emergency rooms and separate routes for covid+ and covid-patients were established; scheduled activities, both for outpatients and for medical/surgical hospitalization were suspended, except for emergencies. some hospitals planned physiotherapists' work schedules to cover a 12-day service (morning and afternoon), and re-organized physiotherapist teams (also with newly hired physiotherapists) to adequately cover weekends and holidays. in addition, all therapeutic interventions, distinguishing between essential and non-essential services, have been reprogrammed. the telephone counseling or telemonitoring and tele-rehabilitation interventions have been established for children, elderly people and patients with chronic diseases. unfortunately, and very quickly, a number of posts appeared on social networks and on other websites offering "new" respiratory therapies actually devoid of a scientific basis, or suggesting obsolete "respiratory exercises" protocols widely rejected in literature many years ago; some of these procedures are useless, if not even risky, for patients with postacute respiratory failure. physiotherapists working in covid-19 units urgently sought out information about protective measures to be adopted and correct physiotherapeutic interventions to be used in different situations. despite the absolute novelty of the pathology and the lack of scientific evidence, based on the available knowledge of physiopathology and respiratory care strategies, in collaboration with other scientific societies (associazione italiana fisioterapisti -aifi-, associazione italiana pneumologi ospedalieri -aipo-, società italiana pneumologia -sip-, european respiratory society-ers-), documents have been published in open-access journals and platform aimed to promote safe use of personal protective equipment (ppe) and to support the decision process in the management of covid-19 patients in the acute and postacute phases [1] [2] [3] [4] . now hospitalizations are decreasing and many patients are discharged: assessment tools are being prepared to measure respiratory, cardiological, neuromotor, cognitive, and emotional impairment, and to stratify patients in order to direct more impaired individuals to rehabilitation facilities, and to discharge those patients who recovered at least partial independence to centralized quarantine facilities or to home. as subjects affected by covid-19 can be contagious for a long time, even after two consecutive negative swabs, remote management and follow-up for at least after 3 months after discharge are ongoing. after 3 months of experience with covid-19, data show typical neuromuscular sequelae and critical illnesses seen mostly in ards survivors after being bedridden in intensive care units, as well as severe complications affecting the cardio-respiratory system, even in patients with few or non-symptoms. already, control ct scans performed before discharge often show signs of pulmonary fibrosis, lung damage that can seriously limit functional capacity and quality of life. given the novelty of covid-19 pneumonia, the impact of permanent lung injury after recovery is unknown. after middle east respiratory syndrome coronavirus infection (which is similar to covid-19 in some ways), about 1/3 of the patients showed signs of pulmonary fibrosis at radiological follow-up [5, 6] . scientific societies have recommended low-intensity exercise training (< 3.0 mets), with a gradual increase in workload based on symptoms (dyspnea and/or fatigue borg score ≤ 3-4) and objective measures such as oxygen saturation level and heart rate. a careful monitoring by an expert healthcare professional is mandatory, as many patients have unexpected reactions to physical activity and exercise. severe desaturation or marked tachycardia occurs also in nonsymptomatic patients. in addition, non-comorbid young patients recovered from covid-19, without having been intubated or sedated during hospitalization, show severe fatigue for weeks after discharge. many of these people might require a long-term monitoring and management related to functional outcomes, such as fatigue, weakness and pre-existing disabilities (e.g. a program of aerobic exercises, strength and balance training, strategies for energy conservation, functional rehabilitation). during this pandemic (and for a long time going forward) healthcare workers need to wear protection that limit their movement, that scar their faces, that increase their fatigue and sweat for many hours every day, and "hide" them to patients. the emotional load is great assisting lonely people, far from their most loved ones, undergoing life-saving procedures. people die without being able to receive a last hug, a caress from their family, or one last greeting "we love you. we will not forget you," they can only receive a "smile" through our eyes as the last comfort. in addition, there is the grief for relatives and friends who have fallen ill and died, and there is the fear of being infected and infecting our families, but also the awareness and the great pride to be key actors, together with all the other health professionals, with our valuable contribution in this awful war. now, more than ever before, physiotherapists are called to action. nevertheless, we need to widen our competences to answer to these new health needs, we need to rewrite organizational models in the acute settings, and to empower our role in the community. once again, the history of this pandemic reminded us of the vital role of the scientific community, with its communication channels, such as peer-reviewed scientific journals. all health professionals are full-fledged members of this scientific community, each with a privileged viewpoint, and each one with the potential to acquire and interpret unique and indispensable information to make the overall picture more complete. now that the emergency gradually makes room for a new normality, it will be essential to continue collecting data, documenting successes and failures of our work, during these and the next months, especially through the publication of empirically derived observations and results. there is much to be learned from this experience, and it will make us more ready to face future challenges, including those pertaining to physiotherapy and pulmonary rehabilitation. respiratory physiotherapy in patients with covid-19 infection in acute setting: a position paper of the italian association of respiratory physiotherapists (arir) joint statement on the role of respiratory rehabilitation in the covid-19 crisis: the italian position paper report of an ad hoc international task force to develop an expert-based opinion on early and short-term rehabilitative interventions (after the acute hospital setting) in covid 19 survivors on behalf of aipo, arir, sip, aifi and sifir. italian suggestions for pulmonary rehabilitation in covid-19 patients recovering from acute respiratory failure: results of a delphi process middle east respiratory syndrome-coronavirus infection: a case report of serial computed tomographic findings in a young male patient follow-up chest radiographic findings in patients with mers-cov after recovery. the indian journal of radiology & imaging publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations none.author's contributions no other authors have been involved in this letter. the author(s) read and approved the final manuscript. no funding.availability of data and materials not applicable.ethics approval and consent to participate not applicable. not applicable. author has no competing interests to declare.received: 11 june 2020 accepted: 21 july 2020 key: cord-347509-2ysw9a0a authors: aronen, matti; viikari, laura; vuorinen, tytti; langen, henriikka; hämeenaho, mira; sadeghi, mohammadreza; söderlund‐venermo, maria; viitanen, matti; jartti, tuomas title: virus etiology of airway illness in elderly adults date: 2016-06-20 journal: j am geriatr soc doi: 10.1111/jgs.14175 sha: doc_id: 347509 cord_uid: 2ysw9a0a nan to the editor: several new respiratory viruses have been discovered in humans since 2001: metapneumovirus (mpv), new coronavirus (cov) strains nl63 and hku1, bocavirus (bov) 1, and rhinovirus (rv) species c. 1, 2 susceptibility to respiratory viral infections may be important especially in older age, but the viral etiology and clinical significance of respiratory illnesses in elderly adults is poorly documented. [2] [3] [4] the aims of this study were to investigate the presence of viruses in elderly adults and to assess the association between viral infection and respiratory illness and between viral infection and chronic illness in individuals with an illness that requires hospitalization. the study was conducted at turku city hospital between july 2007 and april 2009. inclusion criteria included aged 65 and older and any disease necessitating hospitalization. hospital episodes were divided in two groups depending on whether the individual had respiratory symptoms. the ethics committee of turku university hospital approved the study protocol. nasopharyngeal swab and serum samples were collected on hospital admission and after 2 weeks or at discharge from the hospital. swab samples were analyzed for adenovirus, cov nl63 and oc43, bov, enteroviruses, mpv, rv, influenza a and b, piv1-3, and respiratory syncytial virus (rsv) a and b using polymerase chain reaction in the department of virology, turku university hospital. bov infections were serologically confirmed in the department of virology, helsinki university hospital. seven hundred twenty-nine swab samples were collected from 663 individuals. the mean age of individuals with and without respiratory symptoms was 83 ae 7. asthma, chronic obstructive pulmonary disease (copd), and rheumatic disease were more common in episodes with respiratory symptoms than in those without, and hypertension, stroke, dementia, and depression were less common (all p < .05). overall, 160 of 438 (37%) episodes with respiratory symptoms had more virus detections than 67 of 291 (23%) episodes without respiratory symptoms (p < .001, table 1 ). influenza (p = .006), cov (p = .005), and mpv (p = .02) were detected more often when respiratory symptoms were present than when not. no acute hbov1-4 infections occurred in the 396 episodes with respiratory symptoms studied using serology. virus epidemics in elderly adults followed documented epidemics in the region. in episodes with respiratory symptoms two or more viruses were present in 22 (5%) samples, the most common viruses in coinfections being rv (59%) and influenza a virus (32%). the most common virus combination was rv with cov, accounting for 25% of the coinfections. during episodes without respiratory symptoms, two or more viruses were detected in 14 (5%). the most common viruses were rsv and rv being present in 9 (64%) and 8 (57%) coinfections, respectively. in the 729 episodes, age (range 65-100) was not associated with detection of respiratory viruses (p = .49), but the presence of a virus, especially influenza (odds ratio (or) = 1.02, 95% confidence interval (ci) = 0.01-1.04) and parainfluenza viruses in the upper airways (or = 1.02, 95% ci = 1.00-1.03), was positively associated with the individual's weight. a virus was detected in 23% of episodes without respiratory symptoms, which is consistent with findings in individuals of all ages. 5 a striking difference between these elderly adults and children was the low number of bov and the high number of rsv coinfections in subjects without respiratory symptoms. the detection rate for the newly discovered bov 1 was low, possibly because of short-term local replication or mere mucosal contamination of virus from grandchildren; there were no genuine acute bov 1 infections. this is consistent with previous studies of adults; 6,7 bov 1 respiratory infection seems to be more a pediatric problem. 8 no studies have investigated bov infections in individuals with a mean age of 80 and older. an association has been reported between body mass index and severe influenza-like illness, 9 although only in people younger than 60, and the viruses were not identified. obesity may impair vaccine-induced immunity and make obese individuals more susceptible to influenza. 10 the current study shows that there is an association between respiratory virus detection and weight in elderly adults. in conclusion, influenza virus and rv were the most detected viruses in episodes with respiratory symptoms, but overall, symptomatic and asymptomatic respiratory virus infections are relatively uncommon in elderly adults. weight was associated with virus detection. the detection rates for the newly discovered bov 1-4 were low in elderly adults. to the editor: disclosure of cancer diagnosis and treatment options is critical for people with cancer and physicians. an information strategy customized to patient preferences may improve treatment adherence, coping strategies, and quality of life at the terminal stage. 1 older adults seem to wish to know their diagnosis 2 and perceive this information to be important, whereas physicians and families view full disclosure with some reluctance. 3, 4 the objective of the current study was to assess what older adults with cancer know about their diagnosis and treatment and to identify factors associated with the completeness of this information. this was a cross-sectional analysis of the prospective elderly cancer patient (elcapa) cohort 5 survey of individuals aged 70 and older with as-yet untreated malignancy between 2007 and 2012. individuals were referred to geriatric oncology clinics in teaching hospitals near paris, france. each participant had received information during a dedicated visit with the oncologist. the endpoint was the completeness of self-reported information about cancer diagnosis and treatment, assessed by a geriatrician. complete information was defined as correct information about diagnosis and planned treatment, partial information as correct information about either of these points, and no information as no or incorrect information about both points. demographic, tumorrelated, and geriatric characteristics were recorded prospectively at baseline. planned cancer treatments were categorized as curative, palliative, or exclusive supportive care. there were 615 patients with a median age of 80; 52% were men, 38% were inpatients, 52% had metastases, and 42% had a performance status (ps) of 2 or greater. the four most common cancer types were colorectal (24%), breast (17%), urinary tract (14%), and prostate (11%). median activity daily living (adl) score was 4 (interquartile range (iqr) 5-6) and median cumulative illness rating scale for geriatrics (cirs-g) score was 11 (iqr 8-15). twenty-eight percent of patients had two or more major comorbidities, and 19% had a mini-mental state examination (mmse) score of less than or equal to 23. treatment intent was curative in 51%, palliative in 28%, and exclusive supportive care in 21%. overall, 548 (89.1%, 95% confidence interval (ci) = 86.4-91.5%) reported complete information, 31 (5.0%, 95% ci = 3.5-7.1%) partial information, and 36 (5.9%, 95% ci = 4.1-8.0%) no information. proportions of patients reporting complete information were similar for all tumor sites (86-95%, p = .50) except for carcinoma of unknown primary (cup) (50%, p < .001) and lung cancer (63.5%, p < .001). table 1 shows the factors associated with partial and no or incorrect information in univariate ordinal logistic regression. because strong correlations linked ps, metastatic status, and treatment intent (cramer v >0.40), only treatment intent was introduced into the multivariate model. the partial and no information categories had small numbers of patients, with no marked differences according to univariate analysis, and were collapsed into a single group. there was a significant interaction between age and treatment intent (p = .03). according to multivariate analysis, factors independently associated with not reporting complete information were cup or lung cancer (adjusted odds ratio (aor) = 7.70, 95% ci = 2.73-21.72), two or more major comorbidities (cirs-g grade 3 or 4) (aor = 2.72, 95% ci = 1.26-5.87), mmse score less arterial stiffness is associated with low thigh muscle mass in middle-aged to elderly men is frailty associated with cardiovascular drug use? frailty in older adults: evidence for a phenotype antihypertensive medications and differences in muscle mass in older persons: the health, aging and body composition study relation between use of angiotensin-converting enzyme inhibitors and muscle strength and physical function in older women: an observational study effect of perindopril on physical function in elderly people with functional impairment: a randomized controlled trial association between frailty and carotid central structure changes: the three-city study associations of central and brachial blood pressure with cognitive function: a population-based study cognitive function in the prefrailty and frailty syndrome antihypertensive drugs, prevention of cognitive decline and dementia: a systematic review of observational studies, randomized controlled trials and meta-analyses, with discussion of potential mechanisms a diverse group of previously unrecognized human rhinoviruses are common causes of respiratory illnesses in infants new respiratory viruses and the elderly the diagnosis of viral respiratory disease in older adults respiratory viral infections in the elderly identification of respiratory viruses in asymptomatic subjects: asymptomatic respiratory viral infections human bocavirus: a novel parvovirus epidemiologically associated with pneumonia requiring hospitalization in thailand respiratory viruses in bronchoalveolar lavage: a hospital-based cohort study in adults human bocavirus-the first 5 years obesity as a risk factor for severe influenza-like illness the weight of obesity on the human immune response to vaccination the authors wish to thank maarit wuorela, md, evangelos margaritis, md, heli yl€ a-outinen, md, laboratory assistant heidi jokinen, biostatistician tero vahlberg, and colleagues from the geriatrics department, turku city hospital.financial support came from uulo arhio foundation, turku; special government transfers for turku city hospital, turku; jalmari and rauha ahokas foundation, helsinki; finnish anti-tuberculosis association, helsinki; academy of finland, helsinki; research funds of university of helsinki, helsinki; sigrid jus elius foundation, helsinki; all in finland.conflict of interest: the authors have no conflict of interest in connection with this paper.author contributions: aronen, jartti, and viikari take responsibility for the content. aronen, viikari, jartti, vuorinen: study concept and design. aronen, viikari, langen, sadeghi, h€ ameenaho: acquisition of data. aronen, jartti, vuorinen, s€ oderlund-venermo: analysis and interpretation of data. aronen, jartti: drafting of manuscript. aronen, jartti, viikari, vuorinen, viitanen, s€ oderlund-venermo: critical revision of manuscript for important intellectual content. jartti, viikari: administrative, technical, or material support; study supervision.sponsor's role: the sponsors and employers had no role in the design, methods, subject recruitment, data collections, analysis, or preparation of paper. key: cord-328086-ji2emajn authors: zhou, jie‐ying; peng, ying; peng, xiao‐you; gao, han‐chun; sun, ya‐ping; xie, le‐yun; zhong, li‐li; duan, zhao‐jun; xie, zhi‐ping; cao, you‐de title: human bocavirus and human metapneumovirus in hospitalized children with lower respiratory tract illness in changsha, china date: 2018-01-11 journal: influenza other respir viruses doi: 10.1111/irv.12535 sha: doc_id: 328086 cord_uid: ji2emajn background: lower respiratory tract illness is a major cause of morbidity and mortality in children worldwide, however, information about the epidemiological and clinical characteristics of lrtis caused by hmpv and hbov in china is limited. objectives: human bocavirus (hbov) and human metapneumovirus (hmpv) are two important viruses for children with lower respiratory tract infections (lrti). we aimed to assay the correlation between viral load and clinical characteristics of hbov and hmpv with lrti in changsha, china. methods: nasopharyngeal aspirates (npas) from children with lrti were collected. real‐time pcr was used to screen hbov and hmpv. analyses were performed using spss 16.0 software. results: pneumonia was the most frequent diagnosis. there was no significant difference between hbov‐ and hmpv‐positive patients in age (p = .506) or hospitalization duration (p = .280); 24.1% and 18.2% were positive for hbov and hmpv. hbov infections peaked in summer (32.2%), and hmpv infections peaked in winter (28.9%). the hbov‐positive patients had a shorter hospitalization duration than the hbov‐negative patients (p = .021), and the hmpv‐positive patients had a higher prevalence of fever than the hmpv‐negative patients (p = .002). the hbov viral load was significantly higher among patients aged <1 year (p = .006). the mean hbov and hmpv viral loads were not significantly different between patients with single infections and coinfections. patients infected with hbov only were older than those coinfected with hbov and other respiratory viruses (p = .005). no significant difference was found in the clinical characteristics of patients infected with hmpv only and those coinfected with hmpv and other respiratory viruses. conclusion: pneumonia was the most frequent diagnosis caused by hbov and hmpv. neither hbov nor hmpv viral load was correlated with disease severity. hmpv, which was originally isolated in the netherlands 1 and is closely related to avian metapneumovirus, was the first human disease-causing pathogen identified in the genus metapneumovirus. 2 hmpv causes various clinical symptoms, such as cough, wheezing, and fever. 2 young children and the elderly are more susceptible to hmpv infection. 2, 3 indeed, hmpv mainly infects children under 5 years of age and causes upper respiratory tract and severe lower respiratory tract infections. [4] [5] [6] [7] [8] hbov was discovered in sweden and classified in the family parvoviridae, 9 and causes various clinical symptoms, including cough, wheezing, and fever. 2 hbov infections are largely confined to infants and young children (<24 months). globally, the average prevalence of hbov in respiratory tract samples ranges from 1.0 (ci: 0.0-2.0) to 56.8% (ci: 46.9-66.8). 10 however, information about the epidemiological and clinical char(table 1) . to standardize quantification of hbov and hmpv, known numbers of dna or rna transcripts containing the primer targets were used in 10-fold serial dilutions (10 0 to 10 7 copies/μl). any amplification detected before 40 cycles was considered positive. quantification of >10 0 copies/μl (10 3 copies/ml) was considered a positive result. an internal positive control gene (gapdh), positive control, and negative (water) control were included in all reactions. viral loads were expressed as initial copy numbers per real-time pcr reaction, and quantitative results were transformed as the log number of viral copies/μl. continuous variables are expressed as means ± standard deviation (sd) and were compared by independent samples t test. categorical variables are expressed as frequencies or percentages. comparisons were performed by chi-squared or fisher's exact test. analyses were performed using spss 16.0 software. two-sided p-values <.05 were considered to indicate statistical significance. in total, we analyzed 1092 samples collected from lrti patients patients were divided into two groups: those with and without hbov or hmpv infections (table 2) (table 5 ). according to the british thoracic society guidelines for the management of community-acquired pneumonia in children 11 and the clinical diagnosis, lrtis were classified as mild to moderate or severe. the mean hbov and hmpv viral loads did not differ significantly between the two groups (p = .053 and p = .231) (figure 3 ). in this study, we used real-time pcr to explore the etiology and exwith lrtis. this is higher than previous study (62.34%) in changsha that used traditional pcr, 18 and lower than that a study in lanzhou that used real-time pcr. 6 on the one hand, the high detection rate in our study could be caused by the greater sensitivity of real-time pcr compared with traditional pcr. on the other hand, it is possible that the detection rate differs geographically. rsv was the most frequently virus detected in patients with respiratory infections, followed by piv3, hbov, adv, and hmpv. our data support previous reports of rsv, piv3, hbov, adv, and hmpv as the major agents associated with lrtis among children in a hospital setting. 12, [19] [20] [21] [22] the hbov infection rate (24.1%) in the present study is consistent with earlier reports (1.9%-24.6% 8, 9, 16, [23] [24] [25] [26] , and the incidence of hmpv (18.2%) in patients with respiratory tract infections is similar to that in other regions (1.5%-18% 2, 9, 17, 23, [26] [27] [28] . therefore, hmpv and hbov are major causes of lrtis worldwide. the seasonal peaks of hbov and hmpv infections vary among countries because of differences in climatic and geographic factors. in this study, hbov activity peaked in summer, in agreement with the report by jiang. 29 in contrast, detection of hbov in lanzhou peaked in december and april, 30 possibly due to the dry, cold weather in lanzhou and the warm, humid weather in changsha. hmpv detection peaked in winter, which is in line with previous studies. 2, 7, 18, 31 in contrast, in hong kong, hmpv detection peaks in spring/summer. 32 the seasonality of hbov differed geographically, possibly due to climatic factors. the most frequent symptoms of hbov-and hmpv-positive patients were cough and fever, in accordance with previous reports. 6, 18, 33 however, deng reported that in chongqing, wheezing was the most frequent symptom exhibited by hbov-positive patients with severe lrtis. 16 there was no difference between the hbov-and hmpv-positive patients in the incidence of fever, tachypnea, cyanosis, o 2 therapy, or wbc count. the hbov-positive patients had a shorter hospitalization duration than hbov-negative patients (p = .021). in contrast, deng reported that hbov-positive patients had a longer hospitalization duration. the longer hospitalization duration of hbov-negative patients in our study may have been caused by the presence of other viruses (such as rsv and piv3) in many of them. also, hospitalization duration was significantly associated with age (≤6 months), maternal smoking during pregnancy, and a family history of asthma. 19 a high hmpv viral load contributes to development of fever. 4, 5 indeed, hmpv-positive patients had a higher incidence of fever than hmpv-negative patients (p = .002) in our study. we assessed the relationship between viral load and clinical features (age, gender, respiratory rate, temperature, cyanosis, hospitalization duration, and wbc count). the only significant association of hbov-or hpmv-positive patients was a higher viral load in <1-year-old hbov-positive patients. this is in agreement with jiang's report that patients with a high viral load were significantly younger. 29 in contrast, the duration of wheezing and hospitalization was longer in children with a high than a low hbov viral load in the study by deng, 16 possibly due to inclusion of only patients with severe lrtis. hmpv and hbov coinfections with other viruses are common in this study has the following strengths: a considerable duration, large number of patients, and comparison of the most common viruses. its limitation includes the lack of a control group without clinical evidence of illness. further studies should compare a symptomatic group with a control group and a symptomatic period with an asymptomatic period. in summary, we present a prospective study of lrtis caused by hbov and hmpv. a further comprehensive and in-depth study of the role of hbov and hmpv in lrtis in china is warranted. a newly discovered human pneumovirus isolated from young children with respiratory tract disease human metapneumovirus: insights from a ten-year molecular and epidemiological analysis in germany outbreak of human metapneumovirus infection in a severe motor-and-intellectual disabilities ward in japan viral load and acute otitis media development after human metapneumovirus upper respiratory tract infection clinical disease and viral load in children infected with respiratory syncytial virus or human metapneumovirus clinical characteristics and viral load of respiratory syncytial virus and human metapneumovirus in children hospitaled for acute lower respiratory tract infection detection of human metapneumovirus and respiratory syncytial virus by real-time polymerase chain reaction among hospitalized young children in iran incidence, morbidity, and costs of human metapneumovirus infection in hospitalized children high human bocavirus viral load is associated with disease severity in children under five years of age human bocavirus: current knowledge and future challenges british thoracic society guidelines for the management of community acquired pneumonia in children: update 2011 etiology, seasonality and clinical characterization of viral respiratory infections among hospitalized children in beirut, lebanon infections and coinfections by respiratory human bocavirus during eight seasons in hospitalized children abdel baset rw. respiratory syncytial virus and human metapneumovirus in severe lower respiratory tract infections in children under two human metapneumovirus infection in jordanian children: epidemiology and risk factors for severe disease high viral load of human bocavirus correlates with duration of wheezing in children with severe lower respiratory tract infection burden of human metapneumovirus infection in young children clinical features of human metapneumovirus genotypes in children with acute lower respiratory tract infection in changsha severe lower respiratory tract infection in infants and toddlers from a non-affluent population: viral etiology and co-detection as risk factors viruses in community-acquired pneumonia in children aged less than 3 years old: high rate of viral coinfection associations between co-detected respiratory viruses in children with acute respiratory infections multiple viral respiratory pathogens in children with bronchiolitis viral etiologies of hospitalized acute lower respiratory infection patients in china bocavirus in children with respiratory tract infection high prevalence of human bocavirus detected in young children with severe acute lower respiratory tract disease by use of a standard pcr protocol and a novel real-time pcr protocol viral coinfection in acute respiratory infection in mexican children treated by the emergency service: a cross-sectional study comparison of severe pneumonia caused by human metapneumovirus and respiratory syncytial virus in hospitalized children detection of human bocavirus and human metapneumovirus by real-time clinical significance of different virus load of human bocavirus in patients with lower respiratory tract infection human bocavirus infection in young children with acute respiratory tract infection in lanzhou the prevalence of human bocavirus, human coronavirus-nl63, human metapneumovirus, human polyomavirus ki and wu in respiratory tract infections in kuwait children with respiratory disease associated with metapneumovirus in hong kong human metapneumovirus (hmpv) infection in immunocompromised children human metapneumovirus and human bocavirus associated with respiratory infection in apulian population real-time pcr for diagnosis of human bocavirus infections and phylogenetic analysis human metapneumovirus viral load is an important risk factor for disease severity in young children association between high nasopharyngeal viral load and disease severity in children with human metapneumovirus infection development and assay of rna transcripts of enterovirus species a to d, rhinovirus species a to c, and human parechovirus: assessment of assay sensitivity and specificity of real-time screening and typing methods application of taqman lowdensity arrays for simultaneous detection of multiple respiratory pathogens human coronavirus infections in rural thailand: a comprehensive study using real-time reverse-transcription polymerase chain reaction assays impact of human coronavirus infections in otherwise healthy children who attended an emergency department real-time quantitative pcr detection of four human bocaviruses human bocavirus and human metapneumovirus in hospitalized children with lower respiratory tract illness in changsha, china we thank li-li li, na-liu, jie-mei yu, li-li pang, and other staff members in department of viral diarrhea (national institute for viral disease control and prevention) for detection of virus pathogens. the authors have no competing interests to report. the process obtained families' informed consent, and the study protocol was approved by the first affiliated hospital of hunan normal university, changsha, china. http://orcid.org/0000-0002-5152-3093 key: cord-344271-5aynmdsk authors: de souza luna, luciano kleber; panning, marcus; grywna, klaus; pfefferle, susanne; drosten, christian title: spectrum of viruses and atypical bacteria in intercontinental air travelers with symptoms of acute respiratory infection date: 2007-03-01 journal: j infect dis doi: 10.1086/511432 sha: doc_id: 344271 cord_uid: 5aynmdsk respiratory infections after air travel are frequent, but epidemiological data are incomplete. using sensitive polymerase chain reactions, we studied the spectrum of atypical bacteria and respiratory viruses in travelers fulfilling the case definition of severe acute respiratory syndrome. a pathogen was identified in 67 travelers (43.2%). influenza and parainfluenza viruses were most prevalent, at 14.2% and 15.5%, respectively. prevalences of adenoviruses, human metapneumovirus, coronaviruses, and rhinoviruses ranged between 2.6% and 4.8%. human bocavirus, respiratory syncytial virus, and legionella, mycoplasma, and chlamydophila species were absent or appeared at frequencies of <1%. to our knowledge, these are the first specific baseline data for the mentioned agents in the context of air travel. istered, laboratory data on the spectrum of causative agents are actually not available [1, 4, 5] . the epidemic of severe acute respiratory syndrome (sars) in 2003 involved a period of heightened awareness of respiratory infections after air travel. samples for laboratory testing were routinely obtained from patients, which provided a unique opportunity for studying their disease etiologies. after initial characterization of the causative agent of sars, we acted as a diagnostic reference laboratory for the world health organization (who) [6] . during the epidemic, we accepted samples for initial and confirmatory testing exclusively from those patients who fulfilled the who case definition of suspected or probable sars. the definition was designed to be sensitive and thus to prevent any possible transmission, but its specificity was low. it covered most respiratory illnesses compatible with viral or atypical bacterial infection. respiratory agents are best diagnosed by direct assays, the most sensitive of which is polymerase chain reaction (pcr). because of the rapid pace at which the technique evolves, we searched the literature for the most up-to-date pcr assays that cover the broadest possible range of genetic diversity for each respective agent. high sensitivities had to be clearly proved in studies. where assays fulfilling these criteria were not available, we established sensitive real-time pcrs de novo. these assays were used to determine a point prevalence of the full spectrum of respiratory viruses and atypical bacteria in sars-compatible patients. patients and methods. respiratory samples ( ) from n p 214 172 patients were available. all patients fulfilled the who case definition of suspected or probable sars, which required a combination of fever and lower-respiratory-tract symptoms (e.g., cough or difficulty breathing) plus either a stay in an affected area during the preceding 10 days or close contact with suspected patients. probable cases additionally had radiological evidence of respiratory distress syndrome without other reason. samples were prepared as described elsewhere [6] . assays included adenovirus (adv) [7] ; rhinovirus (rv) [8] ; human bocavirus (hbov) [9] ; human coronaviruses (hcov) 229e, nl63, and oc43 (table 1) ; influenza virus (inf) a (table 1) ; and a combined assay for inf a and b, parainfluenzaviruses (pivs) 1-3, respiratory syncytial virus (rsv), and human metapneumovirus (hmpv) (hexaplex plus; prodesse). the commercial system was chosen because several published respiratory multiplex assays gave instable results in our laboratory. we also tested for the presence of atypical bacteria, including mycoplasma, chlamydophila, and legionella species [10] . fortable 1 . formulations of reverse-transcription (rt)-polymerase chain reaction (pcr) assays designed for the study. sense primer probe antisense primer formulation hcov 229e and oc43 tactatgactggcagaatgtttca and tactatgactactagacagtttca, mulations of new assays established for the present study are provided in table 1. their limits of detection (lods) were determined using in vitro-transcribed rna [6] . lods for hcovs nl63 and inf a were !5 copies of synthetic rna genomes per reaction, corresponding to ∼50 rna copies/swab sample. lods for hcov 229e and oc43 were !20 copies/ reaction, corresponding to ∼200 copies/swab sample. results. travel histories were reconstructed by telephone interviews with hospitals, family physicians, or patients themselves. for 164 patients (hereafter called "flight patients"), additional information was retrieved. this included 124 for whom the exact dates and destinations of departure to germany were known and 22 for whom only destinations could be reconstructed. seventeen patients had not traveled as initially reported but had been in contact with patients who had suspected sars. for 8 patients, no additional information could be re-trieved. they were included in the cohort because a travel history had been confirmed before samples were received. only those patients who had reportedly not traveled were evaluated separately. they are hereafter called "contact patients." the average age of flight patients was 42.2 years (range, 1-79 years). the average age of contact patients was 41.8 years (range, 4-79 years). neither the average or median ages nor the sds in both groups were significantly different. figure 1 shows the age distributions in both groups. some 71% of flight patients were between 19 and 60 years old, as were 76% of contact patients ( ). only 12 patients were !19 years old, 11 of p p .57 whom were flight patients. they contributed 13.4% of all positive findings in flight patients, which was significantly more than the mean positivity rate in all age groups ( , stu-p ! .012 dent's t test). at least 1 pathogen was detected in 67 flight patients (43.2%) and in 8 contact patients (47%) ( , student's t test). table p p .7 2 shows the global pathogen detection rates by age group, as well as the relative and absolute prevalences of pathogens in flight patients. in contact patients ( ), adv, human piv, n p 17 inf b, and hmpv were detected in 2 patients each and inf a in 1 patient. double infections occurred in only 2 patients: rv/ piv in a flight patient and adv/piv in a contact patient. inf and piv were clearly the most prevalent agents in flight patients, at 14.2% and 15.5%, respectively, without significant differences between age groups (1-way analysis of variance [anova], 95% significance level). equal distribution of these viruses was also seen in contact patients. covs were more frequent than one would expect in a mostly adult cohort. detection rates did not differ between age groups at the 95% significance level. rsv and hmpv appeared to be significantly more frequent in flight patients !18 years old than in the other age groups ( and .01, f test). for hmpv, this was also p p .006 seen in contact patients ( , f test). the novel hbov was p p .02 not detected in any patient, which indicates that this agent may be restricted to children. indeed, all data available so far about bov have been derived from cohorts of young infants [9, 11, 12] . a complete absence was also observed of chlamydophila species, but it cannot be derived from our data what fraction of patients had already received preemptive antibiotic treatment at the time of sampling. there was no association between the airport of departure and the detection of pathogens in general ( , 1-way anova) p ! .3 or of any pathogen in particular. to identify any possible influence of sampling on detection rates, we analyzed common categories of clinical samples separately for both flight and contact patients. samples were categorized as follows: upper-respiratory-tract samples (category 1), throat-wash fluids (category 2), and lower-respiratory-tract samples (sputum and bronchoalveolar lavage [bal] fluids; category 3). a comparison of age versus sample type yielded a significant lack of lower-respiratory-tract samples in patients !19 years old (1-way anova, 95% significance level). because a significantly higher detection rate of viruses had been identified in these patients, they were eliminated from the analysis, to avoid a bias. after elimination, no significant age differences in the 3 categories of samples remained ( , f test). the p p .3 average ages for categories 1, 2, and 3 were 42.1, 43.9, and 47.0 years, respectively. upper-respiratory-tract samples analyzed included 39 pharyngeal, nasal, and nasopharyngeal swabs and 99 throat-wash fluids. lower-respiratory-tract samples included 50 sputum specimens and 4 bal fluids. the detection frequencies of every pathogen in the 3 categories were compared by separate anovas. global detection frequencies did not differ significantly between categories (any pathogen: 51.3%, 34.3%, and 46.3% in category 1, 2, and 3, respectively). only for inf was the detection frequency in swabs was significantly higher than that for other samples (category 1, 30.1%; category 2, 10.1%; category 3, 14.8%; , f test). the same was observed for p p .01 inf a only (21%, 5%, and 9%, respectively; ) but not p p .01 for inf b alone. discussion. baseline data on the prevalence of respiratory viruses and atypical bacteria after air travel are not currently available. these patients constitute a specific subcohort of patients with community-acquired respiratory disease. unlike general cohorts, children and elderly persons are underrepresented, and adults of working age constitute the majority of patients (71% in our study). normally, respiratory infections in adults are mild and infrequent, but this may change in the context of travel. there is evidence that air travel increases the incidence of respiratory disease in general and that significant transmission in modern aircraft has occurred of sars, inf, and other agents of respiratory infection [2, 4] . because morbidity during working life is economically relevant and new thera-peutic options are at hand, investigation into the etiology of travel-associated respiratory disease seems to be well justified. the criteria of the who sars case definition, including fever, possibly caused an underrepresentation of milder infections in our cohort (rsv and hmpv). however, this made our patients representative of those who are likely to be absent from work. we used up-to-date diagnostic assays to determine the spectrum of viral or atypical agents in these patients. the rate of resolved etiologies was markedly higher than that in studies of community-acquired respiratory infections, including recent collective analyses and some of the latest original studies [5, [13] [14] [15] . this may be due to the extended spectrum of highly sensitive assays applied. our main finding is that the spectrum of agents in returning travelers is broad. almost one-half of all patients were infected with respiratory viruses. agents with treatment options, such as influenza or atypical bacteria, were present in only 15.4% of flight patients and 15.7% of all patients, making even upto-date diagnostics unrewarding. in this context, it is interesting that lower-respiratory-tract samples did not yield increased detection rates. the general assumption that nasopharyngeal washes are more sensitive than swabs in patients with community-acquired respiratory disease could not be confirmed in our study [5] . good detection rates and a low risk of aerosolization suggest that swabs should be the preferred in this type of patients. it cannot be told whether the high prevalence and diversity of respiratory viruses seen in our study is specific to patients with recent intercontinental air travel. because otherwise healthy adults of working age are usually not tested for respiratory viruses, comparison data from similar cohorts are not available. our contact patients provided a small control group, and the observed prevalence of viruses confirms the findings seen in flight patients. however, it should be noted that all contact patients fulfilled the case definition of sars, requiring recent contact with a suspected patient [6] . in germany, this means that all of them most likely had contact with a recent intercontinental traveler. it is unclear why and how patients acquire viral respiratory disease in the context of air travel [2, 4] . in our study, it was interesting that significant clusters of patients with the same diagnosis, who could have been on the same flight, did not exist. similarly, no association was detected between any pathogen and a particular airport. it would thus be likely that viruses are picked up during prior travel rather than being acquired in flight. in any case, data from this unique cohort suggest that the prevalence of known, emerging, and potentially novel respiratory viruses in adults must be carefully studied. understanding the spectrum and the etiological contribution of viruses in adult respiratory disease will be of growing importance in the future. with new antiviral drugs at hand, more consultations will occur for respiratory diseases. targeted therapy will require broad-spectrum pathogen detection with rapid results, most likely by pcr. given the large range of assays required for this study and considering current reagent costs, it is obvious that developing more efficient diagnostic assays is as essential as developing drugs themselves. today, diagnostic technology is far from attaining this aim. respiratory infections during air travel aircraft cabin air recirculation and symptoms of the common cold prevalence of respiratory symptoms among female flight attendants and teachers transmission of infectious diseases during commercial air travel community-acquired pneumonia identification of a novel coronavirus in patients with severe acute respiratory syndrome internally controlled real-time pcr monitoring of adenovirus dna load in serum or plasma of transplant recipients amplicon sequencing and improved detection of human rhinovirus in respiratory samples cloning of a human parvovirus by molecular screening of respiratory tract samples single-run, parallel detection of dna from three pneumonia-producing bacteria by real-time polymerase chain reaction evidence of human coronavirus hku1 and human bocavirus in australian children detection of human bocavirus in japanese children with lower respiratory tract infections enhanced identification of viral and atypical bacterial pathogens in lower respiratory tract samples with nucleic acid amplification tests surveillance of respiratory virus infections in adult hospital admissions using rapid methods characterization of viral agents causing acute respiratory infection in a san francisco university medical center clinic during the influenza season we are grateful to laurent kaiser, eric claas, and harald kessler for providing detailed bench protocols for the published polymerase chain reaction assays used in the study. key: cord-323112-e78zpa9c authors: waterer, grant; wunderink, richard title: respiratory infections: a current and future threat date: 2009-07-16 journal: respirology doi: 10.1111/j.1440-1843.2009.01554.x sha: doc_id: 323112 cord_uid: e78zpa9c despite all the medical progress in the last 50 years pulmonary infections continue to exact and extremely high human and economic cost. this review will focus on the human, pathogen and environmental factors that contribute to the continued global burden or respiratory diseases with a particular focus on areas where we might hope to see some progress in the coming decades. despite all the advances in medical science in the past four decades, lower respiratory tract infections remain the fourth most common cause of death in middle to high income countries and the leading cause of death in low income countries. 1 mortality aside, respiratory tract infections have an enormous economic cost. the annual global cost is unknown, but in the usa the direct cost of community and nosocomial lower respiratory tract infections was estimated at us$15 billion in 1985. 2 by 2001 the economic burden of non-influenza viral respiratory tract infection alone had increased to us$40 billion in the usa. 3 including direct and indirect costs (such as loss of work time), the cost of influenza alone in the usa is currently estimated at us$87 billion per year, 4 although total indirect costs have been estimated by others at over us$100 billion annually. 5 clearly, the global cost of respiratory infection must be in the trillions (us$) per year. in this final article in the pulmonary infectious diseases series we will discuss why pulmonary infections remain a major health problem and are likely to continue to be so well into the foreseeable future. the first and most obvious reason why pulmonary infections remain a problem is that potential pathogens abound in our everyday environment. while some pathogens are dependant on human-to-human spread, there are numerous examples of pathogens that normally invade their host directly from an environment source. typical examples include legionella spp., non-tuberculous mycobacteria and a plethora of fungi and molds, including cryptococcus, histoplasma, aspergillus and coccidiomycosis, just to name a few. while our immune systems have adapted in response to environmental threats, these pathogens will always remain a threat, particularly in subjects whose immune response becomes compromised by age or disease. human disease may also result from pulmonary pathogens crossing directly from animals to humans. among the well-recognized respiratory zoonoses are chlamydia psittaci (psittacosis), coxiella burnetti (q-fever), fracisella tularensis (tularaemia) and of course viral infections such as influenza and hanta. while many of these infections can be avoided by strict hygiene protocols, wherever there is close proximity between humans and animals, such as in large parts of the developing world, some inter species transmission inevitable. the largest risk in the environment, however, comes from other humans, as the vast majority of pulmonary infections are acquired from other infected individuals. this is particularly true of viral infections. the more crowded the human environment, the faster the spread of respiratory pathogens through the population. during the severe acute respiratory syndrome (sars) outbreak, extensive public health campaigns aimed at reducing spread of respiratory droplets by good cough hygiene, the avoidance of work, school or day care during acute infections, and a massive uptake in mask wearing in the general populace had a dramatic effect on the rates of all viral respiratory infections in hong kong. 6 as relaxing these efforts led to a return to normal rates of viral transmission, much more can clearly be done to reduce community spread of pulmonary pathogens, especially during pan/epidemics. nosocomial transmission of pulmonary pathogens is also a major environmental problem. hospital outpatient clinics are a well-documented source of transmission of multi-resistant bacteria between patients with cystic fibrosis 7 and bronchiectasis. 8 intensive care units, particularly those with long-term ventilated patients, are also a frequent source of recurrent cross infection with multi-resistant bacteria. 9 environmental pools of organisms within hospitals themselves have also been commonly associated with outbreaks of nosocomial pneumonia with pathogens, such as legionella, 10,11 aspergillus 12,13 and mucormycosis. 14 while it is clear that strict infection control can reduce nosocomial infection rates, 15 the practical necessity of pooling vulnerable hosts together combined with the inevitable ageing of health-care facilities will ensure that nosocomial outbreaks continue to be a problem. not only is the average age increasing, but the number of very elderly people in whom senescencerelated immune compromise is common has dramatically increased over the past few decades in most western countries. a very large number of age-related immune deficits have been described. key changes increasing the risk of pulmonary infections include the production of lower affinity antibodies, reduced phagocytic ability and reduced responsiveness of naïve cd4-positive t cells. 16 what is also important is that the pro-inflammatory response becomes progressively less regulated in the elderly, 17 often termed 'inflamm-ageing'. 18 the excess pro-inflammatory cytokine response is almost certainly a significant factor in the increased the risk of septic shock, ards and multi-organ failure in elderly patients with pulmonary sepsis. 19 further complicating the fact that we have more vulnerable aged people in our communities is that we cluster them together. the close contact between elderly individuals in nursing homes and other agedcare residences frequently leads to rapid spread of new pathogens throughout the facility, as many case series have documented. [20] [21] [22] [23] [24] [25] while this close clustering is to some extent an economic necessity, much more study is required into how to limit the spread of respiratory pathogens in aged-care facilities, particularly in epidemic circumstances. age aside, advances in medical care have also meant a vastly expanded pool of people with chronic organ failure living in the community. cardiac failure, chronic renal failure and diabetes in particular are all associated with a significantly greater risk of death from pneumonia. 26 unfortunately, it also appears that immunization, at least against influenza, has substantially reduced efficacy in these vulnerable groups. [27] [28] [29] adding to increasing age and increasing numbers of people with chronic organ failure in our communities is the additional factor of deliberate immunosuppression. in recent years the marked increase in tumour necrosis factor antagonists and monoclonal antibodies targeting specific lymphoid populations in patients with inflammatory arthritis (and especially rheumatoid disease) has significantly over taken patients on immunosuppressant therapy after solid organ transplantation as the major cause of iatrogenic immunosuppression. an increased risk of tuberculosis in particular has been a major problem with tnf antagonist therapy. 30 at the milder end of iatrogenic immunosuppression is recent evidence that inhaled corticosteroids, commonly used in asthma and copd, probably increase the incidence of pneumonia. 31 however, whether this statistical increase in pneumonia is clinically important remains unclear as total mortality is not increased and total hospitalizations are lower, suggesting that the small increase in risk of pneumonia is more than compensated for by other beneficial effects. austrian and gold demonstrated that it takes time, possibly days, for antibiotics to alter the natural course of pneumonia. 32 more recent papers demonstrating potential benefits of combination antibiotic therapy in pneumococcal pneumonia show a similar delay between the onset of antibiotic therapy and an identifiable benefit, 33, 34 and a review of all deaths from community-acquired pneumonia in young adults in the uk also found that many presented too late to benefit from any available therapy. 35 patients delay presentation to medical care when they have pneumonia for many different reasons. in some cases it is possible that the onset of disease is so swift that they are unable to seek help, especially if they live alone. however, many complex psychological and practical factors, including financial ability to access health-care, factor into the decision to delay medical treatment. 36 the lack of potential for new antibiotics to alter early mortality requires new approaches to be developed. drotecogen alpha does appear to reduce some of the organ damage in patients with pneumonia and sepsis but the survival benefit is modest. 37 as discussed in the review on streptococcus pneumoniae, 38 reducing the virulence of invading pathogens is one promising line of research. in nosocomial pneumonia, and especially ventilator-associated pneumonia, delayed recognition and hence delayed initiation of therapy is also associated with increased mortality. 39, 40 due to the frequent lack of significant inflammatory response and often the very non-specific nature of patient symptoms (if any), diagnosis of nosocomial pneumonia remains a clinical challenge. all recent guidelines have called for significant research in new diagnostic methods; [41] [42] [43] however, this remains a significant unmet need. human pulmonary pathogens are so well adapted to their human hosts that many cause little or no disease in animals. some pathogens have also developed substantial adaptations to evade our immune responses to them. structural change to the cell wall of mycobacterium tuberculosis enabling it to resist digestion after phagocytosis is one well-characterized adaptation. an example of the extent to which human pathogens can adapt is the production by some viruses of an il-10 like protein that can directly downregulate immune response. 44 as well as adapting to our innate immune response, pathogens also modify their genome in direct response to our attempts to reduce their virulence. the multitude of mechanisms by which bacteria have become antibiotic resistant is well documented, and due to these adaptations we now have problems with a wide range of pulmonary pathogens such as panresistant pseudomonas aeruginosa 45 and extensively drug-resistant m. tuberculosis. 46 even in the community setting drug-resistant pathogens, such as methicillin-resistant staphylococcus aureus, are beginning to become a significant concern as a cause of pneumonia in some regions. 47 viruses can also adapt to anti-viral agents, for example neuraminidase inhibitor-resistant influenza. 48 while not responding to any external pressure, the constant modification of viral genomes also ensures a steady supply of pathogens. antigenic drift and antigenic shift continue to keep influenza near the top of the list of pulmonary pathogens. new pathogens, like the coronavirus responsible for sars, 49 are also certain to continue to emerge, much as hiv did in the 1980s. finally, if new threats do not arise naturally, there is always the unfortunate possibility that humans will deliberately introduce them. 50 given the capacity for pathogens to adapt, it seems likely that regardless of what antimicrobials we develop, the development of resistance is inevitable. however, even if antibiotic resistance does not develop, clearing out one pathogen simply creates space for another to move in. bronchiectasis and cystic fibrosis are classic examples of a procession of bacteria occupying vacated niches. non-tuberculous mycobacteria, which have increased significantly in the past few decades as problematic pulmonary pathogens, 51 are another example of bacteria finding new niches. the emerging data on serogroup replacement in pneumococci in response to pneumococcal vaccination 52 are further evidence that the efficacy of any strategy we develop to reduce bacterial infections is likely to reduce over time as bacteria adapt to the niche available. human pathogens have evolved with us and are well adapted to overcome our innate immune responses. when pressure has been applied, either through antibiotics, antivirals or vaccination, pathogens have either shown the capacity to adapt to them or new pathogens have occupied the vacated niche. as we continue to increase the population of vulnerable hosts, pulmonary infections will remain a major health problem for the foreseeable future. new antibiotics and antivirals may help with specific threats, but will not address most of the fundamental problems. new therapeutic and diagnostic approaches coupled with clinical vigilance, strict infection control and solid public health measures are the hopes for reducing the burden of pulmonary infectious disease over the coming decades. world health organization. top ten causes of death economic costs of respiratory tract infections in the united states the economic burden of non-influenza-related viral respiratory tract infection in the united states the annual impact of seasonal influenza in the us: measuring disease burden and costs economic burden of respiratory infections in an employed population respiratory infections during sars outbreak environmental contamination with an epidemic strain of pseudomonas aeruginosa in a liverpool cystic fibrosis centre, and study of its survival on dry surfaces pseudomonas cross-infection from cystic fibrosis patients to non-cystic fibrosis patients: implications for inpatient care of respiratory patients acquisition and cross-transmission of staphylococcus aureus in european intensive care units prospective 3-year surveillance for nosocomial and environmental legionella pneumophila: implications for infection control nosocomial legionnaires' disease: lessons from a four-year prospective study construction-related nosocomial infections in patients in health care facilities. decreasing the risk of aspergillus, legionella and other infections impact of air filtration on nosocomial aspergillus infections. unique risk of bone marrow transplant recipients nosocomial pulmonary mucormycosis with fatal massive hemoptysis the international nosocomial infection control consortium (inicc): goals and objectives, description of surveillance methods, and operational activities immunosenescence: emerging challenges for an ageing population innate immunity and inflammation in ageing: a key for understanding age-related diseases inflammageing and lifelong antigenic load as major determinants of ageing rate and longevity early mortality in patients with community-acquired pneumonia: causes and risk factors respiratory syncytial virus outbreak in a longterm care facility detected using reverse transcriptase polymerase chain reaction: an argument for real-time detection methods outbreak of chlamydia pneumoniae infection in a japanese nursing home two outbreaks of severe respiratory disease in nursing homes associated with rhinovirus a preventable outbreak of pneumococcal pneumonia among unvaccinated nursing home residents in new jersey during an outbreak of multidrug-resistant pneumococcal pneumonia and bacteremia among unvaccinated nursing home residents sequential outbreak of influenza a and b in a nursing home: efficacy of vaccine and amantadine prognosis and outcomes of patients with community-acquired pneumonia. a meta-analysis t cell responses are better correlates of vaccine protection in the elderly response to influenza vaccination in community and in nursing home residing elderly: relation to clinical factors the comparison of antibody response to influenza vaccination in continuous ambulatory peritoneal dialysis, hemodialysis and renal transplantation patients. scand role of cytokines in rheumatoid arthritis: an education in pathophysiology and therapeutics long-term use of inhaled corticosteroids and the risk of pneumonia in chronic obstructive pulmonary disease: a meta-analysis pneumococcal bacteremia with special reference to bacteremic pneumococcal pneumonia monotherapy may be suboptimal for severe bacteremic pneumococcal pneumonia combination antibiotic therapy lowers mortality among severely ill patients with pneumococcal bacteremia a national confidential enquiry into community acquired pneumonia deaths in young adults in england and wales. british thoracic society research committee and public health laboratory service i really should've gone to the doctor': older adults and family caregivers describe their experiences with community-acquired pneumonia severe community-acquired pneumonia as a cause of severe sepsis: data from the prowess study new insights into pneumococcal disease appropriateness and delay to initiate therapy in ventilator-associated pneumonia clinical importance of delays in the initiation of appropriate antibiotic treatment for ventilator-associated pneumonia guidelines for the management of hospital-acquired pneumonia in the uk: report of the working party on hospital-acquired pneumonia of the british society for antimicrobial chemotherapy clinical practice guidelines for hospitalacquired pneumonia and ventilator-associated pneumonia in adults. can guidelines for the management of adults with hospital-acquired, ventilator-associated, and healthcare-associated pneumonia viral interleukin 10 (il-10), the human herpes virus 4 cellular il-10 homologue, induces local anergy to allogeneic and syngeneic tumors pan-drug-resistant pseudomonas aeruginosa causing nosocomial infection at a university hospital in taiwan management of multidrug-resistant tuberculosis: update staphylococcus aureus community-acquired pneumonia during the 2006 to 2007 influenza season emergence of resistance to oseltamivir among influenza a(h1n1) viruses in europe identification of a novel coronavirus in patients with severe acute respiratory syndrome bioterrorism for the respiratory physician when and how to treat pulmonary nontuberculous mycobacterial diseases serotype replacement and multiple resistance in streptococcus pneumoniae after the introduction of the conjugate pneumococcal vaccine key: cord-343042-9mue4eiv authors: bertozzi, giuseppe; maglietta, francesca; baldari, benedetta; besi, livia; torsello, alessandra; di gioia, cira rosaria tiziana; sessa, francesco; aromatario, mariarosaria; cipolloni, luigi title: mistrial or misdiagnosis: the importance of autopsy and histopathological examination in cases of sudden infant bronchiolitis-related death date: 2020-05-27 journal: front pediatr doi: 10.3389/fped.2020.00229 sha: doc_id: 343042 cord_uid: 9mue4eiv pediatrics, among all the branches of medicine, is a sector not particularly affected by a high number of claims. nevertheless, the economic value of the compensation is significantly high, for example, in cases of children who suffered multiple disabilities following perinatal lesions with a long life expectancy. in italy, most of the claims for compensation concern surgical pathologies and infections. among these latter, the dominant role is taken by respiratory tract infections. in this context, the purpose of this manuscript is to present a case series of infant deaths in different emergency-related facilities (ambulances, emergency rooms) denounced by relatives. following these complaints, the autopsy was performed, and subsequent histological examinations revealed the presence of typical and pathognomonic histological findings of acute viral bronchiolitis, whose morphological appearance is poorly reported in the literature. the analysis of these cases made it possible to highlight the following conclusions: the main problems in diagnosing sudden death causes, especially in childhood, are the rapidity of death and the scarce correlation between the preexistent diseases and of the cause of death itself. for all these reasons, the autopsy, either clinical or medicolegal, is mandatory in cases of sudden unexpected infant death to manage claim requests because only the histological examinations performed on samples collected during the autopsy can reveal the real cause of death. pediatrics, among the branches of medicine, is not particularly affected by a high number of claims. nevertheless, the economic value of compensation is significantly high, for example, in cases of children who suffered multiple disabilities following perinatal lesions with a long life expectancy. according to carroll and buddenbaum (1) and moriani et al. (2) , examining the data collected by an association of several american insurance companies (physician insurers association of america), it was found that only 28% of the cases resulted in compensation; among these, in cases where no damages had been paid, the average cost per the only defense was $28,779, while it results in $67,502 for paid claims (3) . the medical diagnoses, most commonly involved in civilian pediatric trials in the united states, were brain damage (average damages $440,379) and meningitis ($437,423) . respiratory problems in newborns account for $270,607. in italy, data from the insurance company carige spa, in the period 2005-2012, excluding neonatology, highlighted how the main pathologies for which legal action was referred to surgery (gastrointestinal and testicular) and infections (more respiratory ones). moreover, the data on litigation have also shown a different stratification of the number of requests for compensation, which were greater in the north and minors in central italy, mostly involving the public health system (4) . according to the italian study performed during the 2005-2010 period, neonatology has also shown an overlapping geographical stratification, with the greatest interest for the public sector. the claims for damages following death, concerning the neonatal intensive care unit (nicu), mainly concern respiratory diseases (30.7% of cases) (5, 6) . both in the pediatric population in general, but especially in the neonatological one, the dominant role is taken by the respiratory tract infections. in this context, the purpose of these case series is to demonstrate how the identification of the correct cause of death in the sudden unexpected infant deaths (suids) allowed evaluating the absence of medical liability. particularly, the definition of gold standard methods in similar cases could be considered very important to avoid the compensation in unjustified claim requests. all procedures performed in the study were in accordance with the ethical standards of the institution and with the 1964 helsinki declaration and its later amendments or comparable ethical standards. written informed consent was obtained from the first-degree relatives. a 10-month-old male infant died during emergency medical services (ems) transport to the hospital. when parents had been asked for any modification in their child habits, a mild "rhinitis" for a few days was told. for this reason, they went to their trusted pediatrician 2 days earlier, who suggested saline nasal rinses and a short turn check. the medical examiner documented no relevant external sign to explain death. therefore, the parents sued the pediatrician for both penal and civil liability. during the forensic autopsy, the macroscopic examination was unremarkable except for mild edema affecting both lungs. on the contrary, histological examination showed in both lungs a diffuse transmural inflammation in the bronchiolar wall. other tissue sections showed chronic inflammation, and bronchiolar wall fibrosis primarily restricted to bronchioles (figure 1 ). an ambulance was called for a 9-month-old female infant, who lived in a nomad camp; her parents referred that suddenly she did not respond to external stimuli. relatives did not refer to any symptoms neither clinical signs in the previous days. during the resuscitation maneuvers in place, the infant died. thus, the prosecutor ordered the autopsy for alleged medical liability; her parents demanded the civil compensation to the local health insurance, thinking that during the ambulance transportation, there was medical responsibility. the macroscopic examination, both at the external corpse and internal organs, only showed severe pulmonary edema. the histology was characterized by lymphocytic infiltration of the bronchioles (figure 2 ). an 18-month-old female infant was admitted to the emergency room of a pediatric hospital for severe cough and pharyngitis; she died after a few hours. symptoms onset occurred the day before hospitalization. she was a preterm infant (29.3 weeks, birth weight 1,400 g) who suffered from severe respiratory distress at birth (apgar score 1 ′ = 4) and needed a long period of hospitalization. after discharge, she showed neurodevelopmental impairment; moreover, a month before death, she suffered from many viral infective pathologies such as influenza and mononucleosis: all pathologies were successfully treated with standard pharmacological therapies. in this case, the judicial authority disposed of the forensic examination, suspecting medical liability to clarify penal and civil aspects: indeed, at the time of death, a claim for damages has been made to the hospital by the family of the patient. the autopsy showed congestion of tracheal and bronchial mucosa. at histological examination, focal edema and diffuse congestion of both lungs, acute emphysema, and peribronchial and intrabronchial wall leukocyte infiltrates were found; the same results involved nearby septal vessels (figure 3 ). the method used in these cases, as in all cases of sudden death, consists of a rigorous and multidisciplinary methodological approach (7, 8) : -anamnestic collection and clinical findings: the clinical symptomatology presented by the subject before his death or in close chronological concurrence, clinical history of the case, and previous medical records; -anatomical evidence: a macroscopic examination of all organs, their weight, consistency, and color at the autopsy, such as the appearance of fluids (blood, urine, vitreous humor). -histomorphological examination h&e of the organ samples to study any alteration due to pathological condition; -immunohistochemistry: to evaluate, in particular, the presence and the location of the main white blood cells via antibody anti-leukocyte common antigen (cd45). the adherence to this diagnostic procedure not only allows you to check and evaluate a larger quantity of data but allows a complete evaluation on all fronts, from the study of which can confirm the suspicion and/or an unexpected result but crucial for investigations. in the discussed cases, following both the autoptic and especially the microscopic examination, the cause of death was identified in all investigated cases: a rapidly progressive acute bronchiolitis was ascertained. these findings allowed exonerating doctors from any penal liability. the bronchiolitis was defined in 2006, from a collaboration between the american academy of pediatrics (aap) and the european respiratory society (ers), as "a constellation of clinical symptoms and signs including a viral upper respiratory prodrome followed by increased respiratory effort and wheezing in children <2 years of age" (9) . acute viral bronchiolitis (avb) is a lower respiratory tract infective disorder, typically affecting infants <2 years old (90% of cases). respiratory syncytial virus (rsv) is involved in up to 70% of cases, followed by rhinovirus (up to 25%); the remaining cases are related to coronavirus, adenovirus, influenza, and parainfluenza virus, and human metapneumovirus. coinfections are common (10) . however, the seasonality of bronchiolitis, generally more frequently found during the winter months, coincides with the seasonal pattern of rsv diffusion (11) . the infection starts in the upper respiratory tract, spreading to the lower airways in a few days. the bronchiolar damage is determined by the direct action of the virus on the epithelium of the same tract; alternatively, it was indirectly immunemediated, and it was characterized by a peribronchial infiltration of white blood cell types, mainly mononuclear cells, with edema of the submucosa and adventitia (9) . the pathophysiological continuation is caused by a mixture of edema, increased production of mucus, and progressive damage of the epithelium even to necrosis, which determines obstruction of the airflow, entrapment of distal air, atelectasis, and alteration of the ventilation/perfusion. the results are hypoxemia and increased respiratory work, which in turn worsens hypoxemia (9) . the most important extrapulmonary symptoms involve the brain (apnea, epileptic status) and heart (ventricular tachycardia, ventricular fibrillation, cardiogenic shock, complete heart block, and pericardial tamponade) and are common in children with severe infections (12) . the most dreadful complications of bronchiolitis are central apnea, a respiratory pause with bradycardia, cyanosis, pallor, and hypotonia that often requires hospitalization (13) . bronchiolitis represents a disease with high morbidity but low mortality. death from respiratory failure in bronchiolitis is rare and varies from deaths from 2.9:100,000 in the uk to 5.3:100,000 in the us, for children under 12 months, with a relationship that goes hand in hand, reducing itself to the improvement of good intensive practices (9, (14) (15) (16) . in all these cases, in the absence of the clinical-anamnestic data that can guide the clinical diagnosis, the external examination data and the autopsy macroscopic data could point toward a diagnosis of suid. in the case of özdemir et al. (17) , in fact, on the totality of the cases of malpractice claims, 57.5% of the children had died and 59.3% were subjected to autopsy. in these cases, the causes of death reported before and after the autopsies were different in 68%, and the medical staff was found to be responsible for 46.1% of the claims. therefore, the determination of the exact cause of death assumes fundamental importance to ascertain the causal link of any conduct of both health facilities and individual professionals in determining death (18) (19) (20) . this allows not only a measurement of the quality of care provided by promoting public trust for the health system but also as a measure of clinical governance; moreover, it is possible to better manage the medicolegal disputes as a guarantee of ascertaining the truth. indeed, in italy, the data on the frequency of adverse events (aes), preventable adverse events (paes), and negligent adverse events (naes) are available; nevertheless, data about malpractice claims are not available both under the penal and civil points of view. furthermore, the epidemiological purposes cannot be forgotten, considering that it is the only reliable method of data collection. indeed, a complete methodological approach, integrating clinical data, autopsy, and histological findings could be considered the best way to solve similar cases. in fact, in the reported case studies, histopathologic diagnostics identified pathognomonic signs of acute bronchiolitis characterized by edema, congestion, leukocytic infiltration in the bronchiolar wall, leukocytes in the peribronchial interstitial pulmonary space, allowing the identification of the exact cause of death. therefore, these pieces of evidence have allowed excluding the medical responsibility in the reported cases, demonstrating that there are events not related to the supplied health care. the analysis of the presented cases shows that the autopsy is mandatory in suid occurrence, in which the absence of anamnestic data and/or acute clinical signs does not allow to identify the cause of death. hypothesizing medical negligence in each case, the autopsy was performed following the judicial appointment after the relative's complaint. the subsequent histological examinations revealed the presence of typical and pathognomonic histological findings of avb, whose morphological appearance is poorly described in the literature. only the postmortem examinations have allowed excluding medical liability and therefore the compensation for damage. in light of these findings, it could be considered essential an accurate evaluation of similar cases, collecting all data to avoid compensation in unjustified claims made against the hospital. in this way, it is possible to contain the hospital costs related to this kind of accident. for all these reasons, the autopsy combined with the subsequent examination represents a gold standard method to identify the absence of the hospital's responsibility in suid cases. all datasets generated for this study are included in the article/supplementary material. written informed consent was obtained from the first-degree relatives for the publication of this case report. gb, fm, ma, and lc contributed to the conception of the study and wrote the manuscript. fs, bb, lb, at, and cd contributed significantly to literature review and manuscript preparation. gb, fm, fs, bb, lb, at, cd, ma, and lc helped perform the analysis with constructive discussions and approved the final version. malpractice claims involving pediatricians: epidemiology and etiology suicide by sharp instruments: a case of harakiri medical diagnoses commonly associated with pediatric malpractice lawsuits in the united states pediatric claims in italy during a 8-years survey neonatal malpractice claims in italy: how big is the problem and which are the causes? post-mortem magnetic resonance foetal imaging: a study of morphological correlation with conventional autopsy and histopathological findings a multidisciplinary approach is mandatory to solve complex crimes: a case report italian mafia: a focus on apulia mafia with a literature review acute bronchiolitis in infants, a review prospective multicenter study of viral etiology and hospital length of stay in children with severe bronchiolitis effect of prematurity on respiratory syncytial virus hospital resource use and outcomes extrapulmonary manifestations of severe respiratory syncytial virus infection -a systematic review frequency of apnea and respiratory viruses in infants with bronchiolitis clinical predictors of radiographic abnormalities among infants with bronchiolitis in a paediatric emergency department variation in the management of infants hospitalized for bronchiolitis persists after the american academy of pediatrics bronchiolitis guidelines risk factors for bronchiolitis-associated deaths among infants in the united states medical malpractice claims involving children medical records quality as prevention tool for healthcare-associated infections (hais) related litigation: a case series personalised healthcare: the dima clinical model healthcare-associated infections: not only a clinical burden, but a forensic point of view key: cord-327493-v2iatbol authors: kwon, hyo jin; rhie, young jun; seo, won hee; jang, gi‐young; choi, byung min; lee, jung hwa; lee, chang‐kyu; kim, yun kyung title: clinical manifestations of respiratory adenoviral infection among hospitalized children in korea date: 2013-08-05 journal: pediatr int doi: 10.1111/ped.12108 sha: doc_id: 327493 cord_uid: v2iatbol background: the objective of our study was to understand the epidemiological and clinical features of respiratory adenoviral infections among children at a single institution over the course of several years. methods: from january 2005 to april 2009, 1836 children (≤15 years old) who had been admitted to korea university ansan hospital were tested for acute respiratory infection. the patients who were positive for an adenovirus infection were enrolled in this study, and their medical records were retrospectively reviewed. results: adenoviruses were isolated from 310 patients. the male to female ratio was 1.6:1 and mean age was 32 ± 24 months. children under 5 years of age had the highest prevalence. in 2007, adenovirus infections occurred endemically throughout the year. the clinical diagnoses were primarily upper respiratory tract infections (45.4%), lower respiratory tract infections (48.1%), and neurologic disease (5.2%). associated symptoms, signs and laboratory findings included fever (91.9%), cough (83.9%), pharyngeal injection (62.3%), rale (32.6%) and elevated c‐reactive protein (93.9%). the most common radiologic findings were perihilar and peribronchial infiltrates (42.6%). co‐infections were observed in 29 cases. the mean durations of hospitalization and fever were 6.2 ± 6.5 and 4.8 ± 3.1 days, respectively. the lengths of hospitalization were similar for patients admitted for upper respiratory tract infections with severe morbidity and those admitted for lower respiratory tract infections. no children in the study died. conclusion: our study demonstrates that respiratory adenovirus infections are an important cause of hospitalization in young children, and contribute to a significant morbidity. adenovirus is a major cause of acute respiratory illness in children worldwide. 1, 2 adenovirus-related deaths in children are rare, but adenovirus-attributable hospitalizations in young children and high-risk populations are quite high. 3, 4 a previous study found a high burden of hospital admission among children with adenoviral infection. 5 the spectrum of adenoviral infection in children ranges from subclinical illness to complicated disease involving multiple organs, with pneumonia being one of the most common presentations. although most infections are selflimited, adenovirus can be associated with severe conditions in both immunocompromised and immunocompetent individuals. 6, 7 sporadic serotype analysis and some case reports have been performed in korea since 1995. 8, 9 however, little information on the epidemiology and clinical characteristics of respiratory adenoviral infection, especially in hospitalized children, has been published. the study was performed to more fully characterize the epidemiological pattern, clinical features and complications associated with hospitalization for adenoviral infection in korean children. nasal aspirate specimens were collected from patients presenting with acute respiratory symptoms at ansan hospital, which is affiliated with korea university and serves the communities of ansan city, a city neighboring seoul. nasal aspirates were routinely tested to identify adenovirus, parainfluenza, respiratory syncytial virus (rsv) and influenza a/b by viral culture using three standard cell lines (hep-2, mdck and llc-mk2). we retrospectively analyzed data from hospitalized children 15 years of age or younger who had a laboratory-confirmed adenovirus infection between january 2005 and april 2009. study approvals were obtained from the institutional review boards of korea university ansan hospital. clinical, laboratory and radiological data were extracted from electronic medical records. clinical information regarding diagnosis, management during hospitalization, intensive care unit (icu) stay, the need for mechanical ventilator assistance and any underlying conditions were obtained from the hospital records. the statistical analysis was performed using spss version 12.0 (spss, chicago, il, usa). data were presented as numbers (percentage), mean ϯ sd or median (range) as appropriate. comparisons between groups were carried out using the mann-whitney u-test. a p-value of < 0.05 was considered to be significant in the analysis. from january 2005 to april 2009, a total of 1836 specimens were collected from patients admitted with respiratory viral infection. of these samples, 310 episodes were confirmed as adenoviral infections, an overall proportion of 16.9% (310/1836). ages ranged from 1 month to 11.3 years, and the mean age was 32 ϯ 24 months. the patients in this study were grouped by age as 0-<1, 1-<2, 2-<3, 3-<4, 4-<5, and ն5 years. about 90% of all the adenovirus-related cases occurred primarily in children aged less than 5 years, and the peak incidence was in the 1-<2year group (23.5%) (fig. 1 ). when the <1-year-old group was divided into two subgroups, the subgroup younger than 6 months represented only 8.4% of the total cases, while the 6-<12 month subgroup was 14.2%. the ratio of boys to girls was 1.6:1. twenty-six patients (8.4%) had one or more underlying conditions, including asthma, chronic neurologic disease and/or prematurity. the annual detection (detection rate, numbers of positive/ numbers of specimen) of adenovirus was as follows: 68 cases diagnoses at admission were based on clinical, laboratory, and radiographic information. adenovirus was associated with a wide variety of diagnoses, ranging from upper respiratory tract infections (urti) to severe pneumonia and encephalitis ( table 1 ). the most frequent presentation was lower respiratory tract infections (lrti) (149/310, 48.1%), followed by urti (141/310, 45.4%). among the lrti, the admitting diagnosis was pneumonia for 107 patients (107/310, 34.5%), acute bronchiolitis in 20 (20/310, 6.5%), and acute bronchitis in 22 (22/310, 6.1%). urti included pharyngitis (92/310, 29.6%), otitis media (26/310, 8.4%), sinusitis (13/310, 4.2%) and croup (10/310, 3.2%). twenty patients (6.5%) presented with other diagnoses, including febrile convulsions, neonatal sepsis, gastroenteritis and encephalitis. over 90% of adenoviral infections were accompanied by fever, which was the most frequent clinical finding. the mean duration of fever was 4.8 ϯ 3.1 days (range 0-15 days). a high fever (ն39°c) was described in one-third of all patients, and prolonged fever (ն10 days) was reported in 6.8% of cases. other common features were cough (83.9%), rhinorrhea (63.2%) and sputum (61.3%). respiratory distress was seen in 19 cases (6.1%). gastrointestinal symptoms were also commonly found. sixty-five (21%) patients presented with diarrhea, anorexia was seen in 63 (20.3%), and vomiting was found in 10 cases (3.2%). other clinical symptoms included seizure (8.4%), headache (4.5%) and skin rash (2%). crackles on auscultation were heard in 32.6% (101/310) of patients. wheezing was noted in 37 (12%) patients, and retraction of the chest wall was seen in 14 (4.5%). other abnormal findings on physical examination were pharyngeal injection (62.3%), redness of the tympanic membrane (12.6%) and conjunctival injection (8.7%). in addition, cervical lymphadenopathy was detected in eight (2.6%) patients, and hepatosplenomegaly was found in two (0.6%). leukocytosis was detected in 69 (22.3%) patients, and leukopenia was seen in four (1.3%) ( table 2) . 10 elevated c-reactive protein (crp) and erythrocyte sedimentation rate (esr) were the most common laboratory findings (93.9%). liver enzyme, aspartate aminotransferase (ast) and alanine aminotransferase (alt) levels were elevated in about 10% of the patients. co-infections with other pathogens were identified in 29 cases. the most common cause was mycoplasma pneumoniae (m. pneumoniae) (12/29). influenza a (2/29) and b (4/29), parainfluenza virus (2/29), rsv (2/29) and rotavirus (7/29) were also concomitantly identified with adenovirus infection. five cases of rotavirus infection were identified as hospital-acquired infection, while other bacterial isolates were not detected at the time of admission. chest radiography was performed in all patients at admission, and abnormal findings were found in 42.6% (132/310); 104 (35.5%) had perihilar or peribronchial infiltrates, 23 (7.4%) had lobar infiltration or consolidation, three (1%) had hyperinflation and two (0.6%) had pleural effusion. the mean duration of hospital stay was 6.2 ϯ 6.5 days (range 3-98 days). the majority of patients (93.9%, 291/310) received antibiotics. two or more antimicrobial agents were used in 34.2% of patients. the most commonly used agents were ampicillin/ sulbactam (80.8%), third-generation cephalosporin (24.7%), aminoglycoside (16.2%) and macrolide (8.6%). oxygen supplementation was provided to 11 patients (3.6%). five patients (1.6%) were admitted or transferred to the icu, and three of them received mechanical ventilation. the characteristics of icu patients are detailed in table 3 . two of the icu patients were admitted in 2005, while the remaining three patients were admitted in 2007. all were male, and had no underlying conditions, except one who was born prematurely. the first case was diagnosed with severe pneumonia, which progressed to ischemic-hypoxic encephalopathy. the second case was pneumonia complicated with bronchiolitis obliterans. the third case was a 23-month-old premature infant with bronchopulmonary dysplasia who required ventilator care for 24 days. the fourth case was pneumonia with a long-lasting fever (15 days) that was improved without complication. the fifth case was admitted for fever and seizure, and adenovirus from csf was identified. no deaths were reported. duration of hospital stay and fever were used as parameters for determining disease severity. duration of hospital stay was significantly longer in children with lrti (p < 0.001) and co-infection (p = 0.001). however, the length of hospitalization was not related to age, sex or pre-existing condition. on the other hand, prolonged fever was associated with underlying disease (p = 0.026) and asthma (p = 0.016). age, sex, diagnosis and co-infection, including m. pneumoniae, were not related to the duration of fever. this study revealed that adenoviral infection accounted for a relatively high proportion (16.9%) of all respiratory virus infections in hospitalized children compared to the results of previous reports. 11, 12 other pathogens included rsv (48%), influenza (19%), and parainfluenza (16.1%). in our study, 81.3% of the adenoviral infections occurred in young children between 6 months and 5 years of age. similar demographic features have been reported in other studies. 13, 14 according to pereira's report, children aged 5 years had higher antibody positivity against adenovirus, and most infections happened in children younger than 5 years. 15 infants less than 6 months of age are known to have a neutralizing antibody by maternal transmission, which appears to be protective during the first 6 months of life. the frequency of infection was low in infants under 6 months of age; however, a severe case, such as encephalitis, as reported in this study, may occur. the male to female ratio was 1.6:1. the tendency for male predominance has been described in the literature concerning adenovirus infection, as well as in studies of other respiratory viruses. [16] [17] [18] we observed that more cases of adenoviral infectious disease occurred in 2007. adenovirus was the most common in the spring and summer of 2007, but occurred throughout the year in other years examined in this study. it seems that adenovirus shows a seasonal variation, with sporadic epidemics. in korea, small outbreaks of adenovirus infection were reported in the summer of 1995 and in the spring of 1996. 19 in a temperate climate, such as korea, adenovirus infections are known to occur in the spring, early summer and winter. 20 our data showed similar findings, illustrating a slight tendency toward variations in the seasons in which adenovirus was detected more frequently. in one study, 21 adenoviral diseases in children were characteristically accompanied by a high and persistent (mean 5.4 days) fever. over one-third of the children in our study showed a high fever despite the supportive treatment, and a prolonged fever (ն10 days) was found in 6.8% of cases. larrañaga et al. 22 reported that 70% of hospitalized children with adenoviral infection had pneumonia, while our results revealed a significant proportion of patients (45.4%) with urti. the prolonged fever associated with adenovirus infection highlights the primary cause for hospital treatment in patients with urti. also, many patients appeared to have gastrointestinal symptoms with hepatic involvement, as determined by a mild to high elevation of liver enzymes. adenovirus was detected in the csf of one infant who was diagnosed with encephalitis. according to our study, the clinical manifestation of adenovirus infection varied, with multiple organ involvement. several studies have demonstrated that adenovirus is unique among the common respiratory viruses in that it can involve other organs, resulting in conjunctivitis, gastroenteritis, acute hemorrhagic cystitis and meningoencephalitis. 23, 24 although the white blood cell count was within the normal range in most patients (76.4%), elevated crp and esr levels were shown in 93.9% of all the patients. elevated crp and esr levels are also generally thought to be related to bacterial infection, as they are inflammatory markers. others have reported that adenoviral infection typically results in elevated esr and crp levels, unlike what is seen in other viral diseases. 25, 26 therefore, it is not surprising that many patients were initially treated with antibiotics. the clinical use of antigen detection tests is expected to make earlier diagnosis of adenoviral infection, and to reduce unnecessary treatment of antibiotics. the relation to co-infections has been reported by some authors. korppi et al. reported that mixed infection was common (55%) in children with adenovirus infection, and bacterial co-infection was demonstrated in 45% of patients. 27 another study noted that co-infection with measles was a risk factor for mortality in the acute stage of adenovirus respiratory infections. 28 in our study, the most common cause of co-infection was m. pneumoniae. we found no significant difference in disease severity between adenovirus-infected patients with co-infection and those without. when more than one pathogen is identified with adenovirus, it is difficult to determine whether it is true co-infection because adenovirus can persist in the respiratory or gastrointestinal tract after active infection. further study will be necessary to determine whether co-infection can affect the disease course and prognosis. there were several limitations to our study. first, the serotypes of adenovirus were not determined. therefore, we were unable to make more refined observations regarding differences in age distributions, clinical characteristics and determinants of severity based on the serotypes. second, nearly all the children received antibiotic treatment, and the judicious use of antibiotics and resistant development is becoming increasingly important in korea. finally, we did not test for human bocavirus, coronavirus, metapneumovirus, or aerobic bacteria; therefore, some co-infections may have been overlooked. in conclusion, our study contributes to critical epidemiological baseline on respiratory adenoviral infection in korean children, and highlights the importance of adenovirus as a major cause of hospitalization. adenovirus was more frequently detected in young children and was associated with significant morbidity. prolonged fever was associated with urti and necessity of hospitalization, as well as lrti. large-scale investigation through a multicenter approach is required to determine the optimal monitoring and treatment strategies and to achieve a better understanding of the clinical course of adenoviral infection in children. multicentered study of viral acute lower respiratory infections in children from four cities of argentina, 1993-1994 genetic heterogeneity of the hexon gene of adenovirus type 3 over a 9-year period in korea adenovirus bronchiolitis in manitoba: epidemiologic, clinical, and radiologic features severe diffuse adenovirus 7a pneumonia in a child with combined immunodeficiency: possible therapeutic effect of human immune serum globulin containing specific neutralizing antibody burden of viral respiratory disease hospitalizations among children in a community of seoul severe adenovirus bronchiolitis in children adenovirus: an increasingly important pathogen in paediatric bone marrow transplant patients ten cases of severe adenoviral pneumonia in the spring 1995 comprehensive serotyping and epidemiology of human adenovirus isolated from the respiratory tract of korean children over 17 consecutive years (1991-2007) nathan and oski's hematology of infancy and childhood, 7th edn the association of newly identified respiratory viruses with lower respiratory tract infections in korean children clinical picture and epidemiology of adenovirus infections (a review) adenovirus infection in hospitalized immunocompetent children adenovirus infections in hospitalized patients in israel: epidemiology and molecular characterization adenovirus infections genotype prevalence and risk factors for severe clinical adenovirus infection, united states risk factors associated with severe influenza infections in childhood: implication for vaccine strategy interleukin-9 polymorphism in infants with respiratory syncytial virus infection: an opposite effect in boys and girls clinical characteristics of acute viral lower respiratory tract infections in hospitalized children in seoul, 1996-1998 adenoviral diseases in children: a study of 105 hospital cases adenovirus surveillance on children hospitalized for acute lower respiratory infections in chile (1988-1996) lower respiratory tract infections due to adenovirus in hospitalized korean children: epidemiology, clinical features, and prognosis disseminated adenovirus disease in immunocompromised and immunocompetent children the differentiation of classic kawasaki disease, atypical kawasaki disease, and acute adenoviral infection: use of clinical features and a rapid direct fluorescent antigen test serum c-reactive protein in children with adenovirus infection mixed infection is common in children with respiratory adenovirus infection lower respiratory infections by adenovirus in children. clinical features and risk factors for bronchiolitis obliterans and mortality we thank the doctors and laboratory staff of korea university ansan hospital for their participation in the retrospective adenoviral infection study. we also thank our collaborators at myung moon pediatrics for their support. key: cord-340280-m1j6v33y authors: jeon, jae‐hyun; han, minje; chang, ho‐eun; park, sung‐soo; lee, jae‐woong; ahn, young‐joon; hong, duck‐jin title: incidence and seasonality of respiratory viruses causing acute respiratory infections in the northern united arab emirates date: 2019-04-07 journal: j med virol doi: 10.1002/jmv.25464 sha: doc_id: 340280 cord_uid: m1j6v33y background: the data on the seasonality of respiratory viruses helps to ensure the optimal vaccination period and to monitor the possible outbreaks of variant type. objectives: this study was designed to describe the molecular epidemiology and seasonality of acute respiratory infection (ari)‐related respiratory viruses in the united arab emirates (uae). methods: both upper and lower respiratory specimens were collected for the analysis from all the patients who visited the sheikh khalifa specialty hospital (sksh) with ari for over 2 years. the multiplex real‐time reverse transcription polymerase chain reaction (rrt‐pcr) test was used to detect respiratory viruses, which include human adenovirus, influenza virus (flu) a and b, respiratory syncytial virus, parainfluenza viruses, human rhinovirus (hrv), human metapneumovirus, human enterovirus, human coronavirus, and human bocavirus. results: a total of 1,362 respiratory samples were collected from 733 (53.8%) male and 629 (46.2%) female patients with ari who visited the sksh between november 2015 and february 2018. the rrt‐pcr test revealed an overall positivity rate of 37.2% (507/1362). the positive rate increased during winter; it was highest in december and lowest in september. flu was the most frequently detected virus (273/1362 [20.0%]), followed by human rhinovirus (146/1362 [10.7%]). the flu positivity rate showed two peaks, which occurred in august and december. the peak‐to‐low ratio for flu was 2.26 (95% confidence interval: 1.52‐3.35). conclusions: the pattern of flu in the uae parallels to that of temperate countries. the trend of the small peak of flu in the summer suggests a possibility of semi‐seasonal pattern in the uae. accordingly, understanding the epidemiology of respiratory viruses is important for promoting preparedness to tackle this public health threat. [3] [4] [5] [6] [7] [8] the epidemiology of ari-related respiratory viruses in developed countries with temperate climates has been well studied. 1, 5, 9 contrary to the accumulating knowledge of aris in temperate regions, epidemiological research on acute respiratory viral illness in tropical and subtropical areas is limited, although the epidemiological diversity, according to local climate and latitude, has been well studied. 8, [10] [11] [12] the knowledge of the regional distribution of respiratory viruses is essential not only for local prevention and control of aris but also for global health decision-making. 13 to our knowledge, limited information is available on the epidemiology and clinical characteristics of respiratory viral infections in the united arab emirates (uae). although a few studies in nearby countries with similar meteorology have described the epidemiology of aris, the reports cover a limited population group. 14 our study was designed to describe the molecular epidemiology of ari-related respiratory viruses, including the seasonality of the viruses in the northern uae for over 2 years. we collected both upper and lower respiratory specimens for the analysis from all the patients who visited the sheikh khalifa specialty hospital (sksh) with acute respiratory illness between november 2015 and february 2018. physicians were frequently encouraged throughout the year via sms and e-mails to perform viral real-time reverse transcription polymerase chain reaction (rrt-pcr) tests to diagnose suspected acute ari cases. an ari was defined as the simultaneous occurrence of at least one respiratory symptom or sign (cough, purulent sputum, sore throat, nasal congestion, rhinorrhea, dyspnea, wheezing, or injected tonsils) and at least one of the following systemic symptoms: fever, chills, myalgia, or malaise. we used the edwards harmonic technique method to measure the peak-to-low ratio. 15, 16 the edwards technique is a geometrical model, which is an approach that fits a sine curve to a time series of frequencies by the use of ordinary regression methods. the peakto-low ratio was interpreted as a measure of relative risk that compares the month with the highest incidence (peak) with the month with the lowest incidence (low or trough). 15 the positivity rates for respiratory viruses during the discrete peak and low periods were compared using a direct method (χ 2 -test) to analyze statistical significance. 17 we applied the chi-squared (χ 2 ) test and fisher's exact test to paired nominal data. student t test was applied to analyze the means of continuous data. a two-sided alpha level of 0.05 defined statistical significance. all comparative statistical analyses were conducted using spss version 18.0 software (spss inc, chicago, il). among the virus-positive cases, the admission rate was significantly higher in the elderly group than in the other two age groups ( table 3 ). the number of flu and hrv cases was large enough to evaluate the associated seasonality. the peak-to-low ratio of flu was 2.26 (95% ci: 1.52-3.35) and it had significant seasonality (p < 0.01). hrv was detected year-round and showed no significant seasonality. the peak-to-low ratio of hrv was 1.44 (95% ci: 1.00-2.34), which was insignificant (p = 0.22). ari is highly prevalent and is responsible for a high burden of disease in many countries. respiratory viral infection is the most common cause of ari, and predicting the timing of peak respiratory virus activity is important for improving disease control. in this study, the overall positivity rate of respiratory viruses and the incidence of each virus by age group were similar to those reported by previous studies describing the epidemiology of respiratory viruses. 6, 7, 9, 18 several studies have reported that men are more susceptible to viral infection, and have more vigorous immune and behavioral responses. [19] [20] [21] however, there was no difference in the positivity rate or the incidence of ari by sex in our study. the higher positivity rate of respiratory viruses in the pediatric group was compatible with that reported in other studies. 9, 22 of the 1362 patients included in our study, 388 (28.5%) with ari were admitted for further management. our data showed a lower admission rate for virus-positive than virus-negative patients (13.6% vs 37.3%), implying a better prognosis for virus-related aris than that for aris of other etiologies, as noted in several previous studies. 23, 24 the higher admission rate in the elderly group is also in agreement with that reported by earlier studies. 2, 24, 25 flu was the most common respiratory virus in all age groups, and the positivity rate was 20.0%, which is similar to previous data reported from studies in oman. 14,26 a/h3n2 was the most common subtype detected throughout the year. the high incidence of flu and a/h3n2 may be related to the severe symptoms characteristic of these infections, which increase the likelihood of patients visiting the emergency department. many studies have reported that ari symptoms are more likely to be experienced by individuals infected with flu than by those infected with other respiratory viruses. in addition, a/h3n2 infections are more severe than a/h1n1 or b infections. 27,28 flu vaccination could have influenced the incidence of flu and its subtypes in the present study. however, regional information on flu vaccination is not available in the uae. in this study, hrv was the second-most commonly detected respiratory in all age groups. this finding is in agreement with other studies. 29, 30 good accessibility of patients with common cold to emergency department might explain the higher incidence of hrv in the uae. however, the incidence of hrv could have been overestimated. many patients tested positive for hrv at the emergency department, but actually had bacterial coinfections, and their more severe symptoms could have been attributable to the bacterial infections or combined comorbidities like asthma. rsv is known as the leading cause of ari. 1, 9, 31 however, the incidence of rsv among positive cases in our study was lower than that reported in other publications. 31, 32 it is possible that the incidence of rsv was underestimated in the current study because comparatively small numbers of infants and young children were included. the hot and dry climate of the uae could also be a possible explanation for the low incidence of rsv in this region. rsv is more common during the rainy season in tropical and subtropical areas, and is reported to have a low survival rate at high temperatures. 32, 33 small numbers of hcov, hadv, hmpv, hbov, hev, and hpiv cases were sporadically detected in our study. reports suggest that because of the mildness of symptoms and the self-limiting nature of these infections, patients may not seek medical care, and this may lead to an underestimation of the incidence of infection. 7,34 however, our data suggest that in the uae, like in other temperate countries, a diverse set of respiratory viruses contribute to the ari cases that compel patients to visit medical facilities, because of their severity. because we have reported the incidence of such severe infections, we believe that the data in this study will be of value to medical institutions in the uae. among the virus-positive cases in our study, the coinfection rate was 7.9% overall and 14.7% in the pediatric group. these are compatible with the values mentioned in previous reports. 9 however, several similarly designed studies showed higher rates of coinfection (from 13.2%-42.5%), particularly in young children. 7, 22, 35 hrv was the most common virus to be found in cases of coinfection with other viruses in our study; this finding was in agreement with that reported in the literature. 9, 30 coinfection with flu and hrv was common in our study although other studies have reported a negative association between the two viruses. 5,36 flu and hrv may have been detected in coinfected patients more frequently because the incidence of both viruses was higher than that of the other respiratory viruses in this study. a characteristic semi-seasonality pattern on respiratory viral infection rates was observed in our study. flu was the main contributing organism to the seasonal pattern; the subtypes a/h1n1/pdm09 and flu b contributed predominantly to the winter peak. low temperatures and low specific humidity during the winter season in the uae may be the cause of the peak in the number of flu cases in the winter. 12 in addition to the major peak in the winter, a small peak in august was also observed for flu (semi-seasonal pattern), which parallels the patterns reported from temperate and tropical countries in the northern hemisphere. 5, 8, 12 most of the respiratory viral infections, including flu, are more common in the winter in temperate countries. 8 however, flu and rsv infections tend to show annual peaks in association with the rainy season in the tropical area. 8, 33, 37 in subtropical areas, previous studies have observed that respiratory viruses, including flu, peaked in the coldest months in temperate areas. 38, 39 the uae is located in a subtropical area and does not have a rainy season. thus, we expected a peak of flu in the winter season, as is common in other subtropical areas. 12 however, flu in the uae showed a two-peaks semi-seasonal pattern, which has also been observed in some other regions located at similar latitudes (taiwan and nepal). 8 the majority of the population of the uae is exposed to a dry, air-conditioned environment for most of the day, especially during the summer season, because outside temperatures range from 39 to 45°c. despite this relatively controlled environment, the summer peak of flu in the uae could be explained by prolonged effective contact rates, because of the increased indoor activity, and lowered relative humidity, which have been reported to be related to the incidence of viral illnesses. 40, 41 this finding implies the potential necessity for sustained flu vaccination campaigns and repeated vaccinations to reduce the severity of flu infections, especially for high-risk individuals. 42, 43 nationwide data are needed to confirm the semi-seasonal pattern of flu in the uae. our study was subject to some limitations. first, this was a single center study with relatively small sample size; moreover, the decision of whether to collect a sample from the patient for viral testing was at the physician's discretion. physicians in the emergency department may have preferred to perform a rapid influenza antigen test rather than to conduct a multiplex rrt-pcr test, which requires a longer time to produce results. hence, the incidence of respiratory viruses may have been underestimated because of the low sensitivity of the rapid antigen test, and because viruses, other than the flu, may not have been identified even if present in the samples. 25, 44 second, our results may not be generalizable to the entire population of the uae because most of the study specimens were taken from patients in the northern emirates. in addition, our study could have underestimated the degree of seasonality in the uae because the seasonality of viral infections tends to be more apparent when analyses include mild cases that require minimal conservative care (for example, outpatient care in the primary clinic). 45 to our knowledge, this is the first study to describe the rrt-pcrbased seasonal pattern of respiratory viruses related to ari in the uae. moreover, the high sensitivity of the detection method enabled the collection of more reliable epidemiologic data. to date, very few studies have provided such data for the middle-eastern region. the seasonality of flu was similar to that observed in temperate countries and was also consistent with a semi-seasonal pattern of incidence. this knowledge can serve as baseline data for more expansive future surveillance studies of respiratory viruses in the uae and in the middle-eastern region. we would like to acknowledge the physicians and employees of the department of pulmonology, intensive care units, and the emergency department of the sheikh khalifa specialty hospital who participated in the collection of study specimens. this project did not receive any financial or other support from any organization. the economic burden of non-influenza-related viral respiratory tract infection in the united states burden of respiratory viruses in patients with acute respiratory failure evaluation of the bioactivity of influenza vaccine strains in vitro suggests that the introduction of new strains in the 2010 southern hemisphere trivalent influenza vaccine is associated with adverse events seasonal variations of respiratory viruses and etiology of human rhinovirus infection in children respiratory viral infections during the 2009-2010 winter season in central england, uk: incidence and patterns of multiple virus co-infections nation-wide surveillance of human acute respiratory virus infections between 2013 and 2015 in korea writing committee of the who consultation on clinical aspects of pandemic(h1n1)2009 influenza latitudinal variations in seasonal activity of influenza and respiratory syncytial virus (rsv): a global comparative review epidemiology of viral respiratory infections in a tertiary care centre in the era of molecular diagnosis humidity and respiratory virus transmission in tropical and temperate settings correlations between climate factors and incidence--a contributor to rsv seasonality environmental predictors of seasonal influenza epidemics across temperate and tropical climates incidence and risk factors for acute respiratory illnesses and influenza virus infections in australian travellers to asia seasonal variations of respiratory viruses detected from children with respiratory tract infections in riyadh, saudi arabia simple estimators of the intensity of seasonal occurrence the recognition and estimation of cyclic trends methods to assess seasonal effects in epidemiological studies of infectious diseases--exemplified by application to the occurrence of meningococcal disease etiology of community-acquired pneumonia: increased microbiological yield with new diagnostic methods epidemiologic and clinical features among patients hospitalized in wisconsin with 2009 h1n1 influenza a virus infections hospitalization incidence, mortality, and seasonality of common respiratory viruses over a period of 15 years in a developed subtropical city sex differences in the response to influenza virus infection: modulation by stress multiple simultaneous viral infections in infants with acute respiratory tract infections in spain epidemiology and clinical outcome of virus-positive respiratory samples in ventilated patients: a prospective cohort study the burden of influenzaassociated hospitalizations in oman viral pneumonia estimating the burden of influenza-associated hospitalization and deaths in oman (2012-2015). influenza other respir viruses thrombotic risk and immobility in residents of long-term care facilities clinical signs and symptoms predicting influenza infection seasonal variations of 15 respiratory agents illustrated by the application of a multiplex polymerase chain reaction assay influenza a viruses dual and multiple infections with other respiratory viruses and risk of hospitalisation and mortality. influenza other respir viruses viral etiology of respiratory infections in children in southwestern saudi arabia using multiplex reverse-transcriptase polymerase chain reaction epidemiology of respiratory virus infections among infants and young children admitted to hospital in oman epidemiology and seasonality of respiratory tract virus infections in the tropics the common cold prevalence and seasonality of six respiratory viruses during five consecutive epidemic seasons in belgium human rhinoviruses seasonal trends of viral respiratory tract infections in the tropics viral etiology of acute respiratory infections among children in porto alegre, rs, brazil respiratory viral infections among pediatric inpatients and outpatients in taiwan from 1997 to 1999 seasonality of viral infections: mechanisms and unknowns airborne spread of infectious agents in the indoor environment prevention and control of seasonal influenza with vaccines: recommendations of the advisory committee on immunization practices -united states, 2017-18 influenza season repeated influenza vaccination for preventing severe and fatal influenza infection in older adults: a multicentre case-control study rapid and sensitive method using multiplex real-time pcr for diagnosis of infections by influenza a and influenza b viruses, respiratory syncytial virus, and parainfluenza viruses 1, 2, 3, and 4 deviations in influenza seasonality: odd coincidence or obscure consequence? incidence and seasonality of respiratory viruses causing acute respiratory infections in the northern united arab emirates the authors declare that there are no conflict of interests. http://orcid.org/0000-0002-1725-8468 key: cord-341765-ml6eo8r3 authors: widhidewi, ni wayan; wiyatno, ageng; dewantari, aghnianditya kresno; paramasatiari, lila; aryastuti, sri agung; artika, i nengah; setiawan, wayan doddy; soebandrio, amin; aye myint, khin saw; safari, dodi title: identification of viral etiology of acute respiratory tract infections in children and adults in tabanan, bali, indonesia date: 2020-03-25 journal: access microbiol doi: 10.1099/acmi.0.000120 sha: doc_id: 341765 cord_uid: ml6eo8r3 acute respiratory tract infection (arti) is the most common infectious disease in humans worldwide. the morbidity and mortality rates are high, especially in developing countries from southeast asia and africa. while arti is commonly associated with viruses, there is limited data on the spectrum of viruses causing arti in developing countries, including indonesia. this study was based on utilizing molecular techniques targeting a panel of 11 endemic and emerging respiratory viral pathogens including zoonotic viruses in a cohort of children and adults presenting at tabanan general hospital, bali, with acute respiratory illness, from january to november 2017. in total, 98 out of 200 samples (49.0 %) tested positive for viruses. our study confirmed 64.3 % viral etiology in children and 12.2 % in adults. viruses that were detected were herpesviridae (15.0 %) followed by enteroviruses (12.0 %), influenza a virus (11.5 %), respiratory syncytial virus (8.0 %), adenoviridae (6.5 %), human metapneumovirus (3.5 %), paramyxoviridae (2.0 %), bocavirus (1.0 %) and coronaviridae (0.5 %). the study sheds light on the viral spectrum of arti in children and adults in tabanan, bali, indonesia acute respiratory tract infection (arti), the most common infectious disease in humans, is a growing global health problem [1, 2] . it is easily transmitted between humans, and known to cause high morbidity and mortality in all age groups [3] . arti is predicted to be the cause of 4.5 million deaths every year worldwide [4] . in 2015, pneumonia was responsible for 15 % of all deaths of children under 5 years old [4, 5] with most of the cases occurring in developing countries especially in africa and southeast asia [2, 5, 6 ]. in indonesia, arti is reported as one of the top five common diseases associated with high mortality in children and adults. according to the republic of indonesia health system review in 2013 [7] , arti is ranked second after prematurity in causing death in children under 5 years. whereas in adults, lower respiratory tract infection is the fourth cause of death after stroke, ischemic heart disease and diabetes mellitus. altogether respiratory infections accounted for the death of 81 100 indonesians in 2012 [4] . respiratory viruses including influenza a and b, respiratory syncytial virus (rsv), adenovirus, and parainfluenza virus are the most frequently detected viruses in patients with arti in southeast asia [8] . on the other hand, new emerging viruses are on the rise, such as human metapneumovirus (hmpv) and human bocavirus [3, 9] . in this study, throat swab specimens were collected from patients with respiratory symptoms to identify viral etiological agents of arti. the data will provide insights on the epidemiology of respiratory viruses in subjects with arti in tabanan region, bali, indonesia. throat swab specimens were collected from 200 out-patients admitted to tabanan general hospital, a regency hospital in tabanan regency, bali, between january to november 2017. patients with symptoms of arti with an onset of illness ≤7 days were included in the study. the enrollment criteria included fever (≥37.5 °c) or history of fever with one or more of the following respiratory symptoms: cough, rhinorrhea, nasal obstruction and sore throat. data on demography, risk factors, exposure to wild animals and clinical symptoms were also collected on day of admission by trained hospital staff. throat swabs from patients were preserved in 2 ml of viral transport medium (vtm) prepared in-house, containing bovine brain heart infusion and antibiotics. specimens were immediately stored at −20 °c at the hospital. every two days, the specimens were transported to the biology molecular laboratory, faculty of medicine, university of warmadewa, bali on ice and stored at −80 °c. samples in batches of 50 were transported using dry ice to the eijkman institute for molecular biology laboratory in jakarta and stored at −80 °c prior to testing. viral nucleic acid was extracted using qiaamp viral rna minikit (qiagen, hilden, germany) according to the manufacturer's instructions. viral specific targets were identified by using reverse transcription-pcr (rt-pcr). in summary, 60 µl of viral dna-rna was obtained and 4 µl used as a template for complementary dna (cdna) synthesis using goscript reverse transcription system (promega, madison, usa) and random hexamers. singleplex pcr assays were used for detection of a panel of respiratory viruses using family-level primers for paramyxoviridae, herpesviridae, coronaviridae, hantaviridae, adenoviridae, arenaviridae; genus-level primers for enterovirus, henipavirus, influenza a virus, bocavirus; and pneumovirinae sub-family primer including respiratory syncytial virus (rsv) and human metapneumovirus (hmpv). all of the primers and positive controls that were used in the amplification reaction were based on previous reports [10] [11] [12] [13] [14] [15] [16] [17] . singleplex pcr reaction was performed in thermal cycler proflex pcr system with appropriate run controls. a recombinant plasmid representing sequence fragments of all family viruses was constructed and used as the positive control. for amplification, 2 µl of cdna template was added to 23 µl of promega go taq green polymerase master mix (promega, madison, usa). all pcr products were analysed using electrophoresis in 1.5 % agarose gel. visualization of positive band was performed using gel imaging biorad gel doc xr system and quantity one 1-d analysis software (bio-rad, california, usa). all samples with a positive band were followed up for further characterization by fragment sequencing based on sanger method using bigdye terminator v3.1 and applied biosystem (abi) sequencing machine. sequencing results were analysed using geneious software r8 version 8.1 (biomatters ltd, auckland, new zealand) and compared with genbank database by blast for sequence homology. in this study, the age of patients ranged from 8 months to 80 years, with a median 9 years. among the patients enrolled, 112 (56.0 %) were males. children (age group 0-17 years) accounting for 159 (79.5 %) and adults (age group ≥18 years) for 41 (20.5 %) ( table 1) . most patients had fever less than five days (89.5 %) with 191 (95.5 %) of patients developing fever before admission and 79 (39.5 %) at admission. the respiratory symptoms at admission were tabulated in table 1 . among 200 throat swabs tested, 98 samples (49.0 %) tested positive for viruses (table 2) . we found that viral detection rate in children (64.3 %) was significantly higher than adults (35. table 2) . almost all of the herpesviridae were further characterized to be cytomegalovirus (cmv) (n=28, 14.0 %). in this study, cmv was the most prevalent among children and enteroviridae was the most prevalent among adults ( table 2 ). the subtypes of the influenza a virus were identified as h3n2 (n=14, 7.0 %), h1n1 (n=8, 4.0 %), and h1n2 (n=1, 0.5 %) ( table 2 ). majority of enteroviruses were characterized as rhinovirus a (n=10, 5.0 %) and rhinovirus c (n=10, 5.0 %) followed by enterovirus 84, coxsackievirus a6, coxsackievirus a24, and coxsackievirus b3 with each of them accounting for 0.5 % (n=1). both strains of rsv were detected in this study, rsv a (n=12, 6.0 %) and rsv b (n=4, 2.0 %). the adenoviridae positives were found to be human adenovirus b2 (n=4, 2.0 %), b3 (n=8, 4.0 %) and c (n=1, 0.5 %). single virus detection was observed in 40.5 % samples (n=81), and co-detection in 7.0 % (n=14) for two viruses, 1.0 % (n=2) for three viruses and 0.5 % (n=1) for four viruses. co-detection of influenza-cmv (n=3) and hmpv-cmv (n=3) were the most common co-detection identified in this study. among 17 cases of co-detection in this study, 85.7 % were from children with the majority of under 6 years age group. the study identified the epidemiology of respiratory virus infections in patients presenting to a regency hospital in bali, indonesia. bali is one of the most popular domestic and international tourist destinations in indonesia which has a significant role for the country's economics and healthcare. the virus detection rate of 49.0 % from the total number of samples was similar to those previously reported [18] [19] [20] [21] . although the specimens were collected during the acute phase of the disease following strict eligibility criteria, more than 50.0 % of patients remain undiagnosed which was likely to be associated with bacteria or other viruses not included in our panels [22] . as reported in an earlier study, the virus detection rate was significantly higher in children than adults as they are more prone to arti because of their immature immune system and lack of hygiene [22, 23] . in our study, 16 (8.0 %) patients were rsv-positive and more frequently in children under 5 years old (n=15, 7.5 %). although rsv outbreaks are known to vary from year to year according to geographical pattern, majority of rsv infection in this study was observed in march during the height of the rainy season which is consistent with earlier studies in tropical countries [24, 25] . using rsv pcr panel, we also detected seven hmpv all in children with three of them developing bronchopneumonia (data not shown). hmpv is known to pose significant threat causing upper and lower respiratory illness in children and elderly, especially those with underlying respiratory and immunocompromised conditions [26, 27] . two genotypes of hmpv have been identified to date, which can be classified into five lineages, a1, a2a, a2b, b1 and b2. however, their association with disease severity remains unclear [28, 29] . there was only a single report on hmpv from indonesia documenting the association of hmpv lineage a1 and a2 with asthma exacerbations and pneumonia [30] indicating that hmpv should be screened in those with chronic and severe respiratory infection. such data on clinically important pathogens could become critical to implement vaccines once they become available. further characterization of positive enterovirus specimens revealed one coxsackievirus a24 subtype (0.5 %) that is known to be associated with acute hemorrhagic conjunctivitis outbreaks [31, 32] and acute flaccid paralysis [33] . coxsackievirus a6 was commonly linked with respiratory infection, herpangina and hand, foot and mouth disease (hfmd) outbreaks [34, 35] . in one study, the virus was observed in a patient with lower respiratory tract infection [36] . cmv, although not a respiratory pathogen, was detected in a number of cases (14 %) in our study, and is most likely associated with a suppressed immune system. in addition, the majority of the coinfection were associated with cmv, which might be a reinactivation rather than an etiological agent. studies have reported co-detection rates of respiratory viruses ranging from 7 to 40 % [18, 35, 37] . co-detection was observed in the form of influenza-cmv and hmpv-cmv in 8.5 % of specimens especially in children under the age of 6 years, none of which were associated with severe clinical manifestations. majority of the infections were associated with cmv, which may raise susceptibility to other infections, cmv might not be an etiological agent, and its occurrence can be reactivation, prolonged shedding of carrier state or be due to coincidental infection. this observation on co-infection of respiratory viruses in children was supported by zimmerman et al. and esposito et al. [21] and is most likely due to their increased susceptibility to viral infection. in this study, we identified and characterized viral pathogens to determine the etiology of acute respiratory infections including the zoonotic respiratory viruses which are poorly characterized. the positive detection rate of respiratory viruses was 49.0 %, with 8.5 % co-detection rate. the most common viruses detected in all ages were herpesviridae, enterovirus, influenza a virus and rsv. in addition to the influenza virus routinely screened in arti studies, other viral agents associated with severity like herpesviridae, enterovirus and rsv should be screened in respiratory illnesses. the study sheds lights on the viral spectrum of arti in children and adults in tabanan regency, bali. additional studies are required to determine nationwide epidemiology of respiratory viruses and association between viruses and clinical severity to guide prevention strategies in indonesia. the research was supported by eijkman institute of molecular biology and faculty of medicine universitas warmadewa. we also would like to thank ministry of research, technology, and higher education, republic of indonesia, and predict, u.s. agency for international development (usaid) emerging pandemic threats program for their generous support on molecular testing used in this study. estimates of the global, regional, and national morbidity, mortality, and aetiologies of lower respiratory infections in 195 countries, 1990-2016: a systematic analysis for the global burden of disease study global, regional, and national causes of under-5 mortality in 2000-15: an updated systematic analysis with implications for the sustainable development goals aetiology of acute respiratory tract infections in hospitalised children in cyprus world health organization. indonesia: who statistical profile unicef manson`s tropical diseases identification of new respiratory viruses in the new millennium detection and identification of coxsackievirus b3 from sera of an indonesian patient with undifferentiated febrile illness sensitive and broadly reactive reverse transcription-pcr assays to detect novel paramyxoviruses a novel immunochromatographic system for easy-to-use detection of group 1 avian influenza viruses with acquired human-type receptor binding specificity identification and characterization of a new bocavirus species in gorillas multiple organ infection and the pathogenesis of sars characterization of arenaviruses using a family-specific primer set for rt-pcr amplification and rflp analysis. its potential use for detection of uncharacterized arenaviruses emergence of fatal avian influenza in new england harbor seals hantavirus in african wood mouse, guinea epidemiology characteristics of respiratory viruses found in children and adults with respiratory tract infections in southern china detection of respiratory viruses using a multiplex real-time pcr assay in germany new epidemiological and clinical signatures of 18 pathogens from respiratory tract infections based on a 5-year study viral infections in outpatients with medically attended acute respiratory illness during the 2012-2013 influenza season clinical significance of multiple respiratory virus detection risk factors for acute respiratory infection in the australian community seasonality of influenza and respiratory syncytial viruses and the effect of climate factors in subtropical-tropical asia using influenza-like illness surveillance data epidemiology and seasonality of respiratory viral infections in hospitalized children in kuala lumpur, malaysia: a retrospective study of 27 years [internet]. bmc pediatrics the role of human metapneumovirus genetic diversity and nasopharyngeal viral load on symptom severity in adults epidemiological, clinical and genotypic features of human metapneumovirus in patients with influenza-like illness in senegal comparison of clinical characteristics of human metapneumovirus and respiratory syncytial virus infections in hospitalized young children prophylactic and therapeutic approaches for human metapneumovirus respiratory viruses and torque teno virus in adults with acute respiratory infections a recent epidemic of coxsackie virus type a24 acute haemorrhagic conjunctivitis in singapore genetic characteristics of the coxsackievirus a24 variant causing outbreaks of acute hemorrhagic conjunctivitis in jiangsu, china rhinoviruses and respiratory enteroviruses: not as simple as abc an outbreak of coxsackievirus a6 hand, foot, and mouth disease associated with onychomadesis in taiwan clinical and epidemiologic features of coxsackievirus a6 infection in children in northern taiwan between prevalence and characterization of enterovirus infections among pediatric patients with hand foot mouth disease, herpangina and influenza like illness in thailand etiology and clinical characteristics of single and multiple respiratory virus infections diagnosed in croatian children in two respiratory seasons the authors thank ni putu aniek mahayani, md, pediatrician; i wayan karya, md, ent; nyoman kertanadi,md, ent; i gede agus sastrawan, md, m.biomed, internist; i n. gede sumardika, md, clinical pathology from tabanan general hospital for their contribution in enrollment and specimen collection. the authors are also grateful to dr. agus haryono and prof. dr. myrtha karina for their suggestions and reviewing this manuscript. the authors declare that they have no conflicting interests. this study was approved by the ethic committee of faculty of medicine university of udayana, bali, ethical number: 1847/un.14.2/ litbang/2016. written informed consent was obtained from all of the patients and for children under 18 years old, written consent was provided by their parents or legal guardians. five reasons to publish your next article with a microbiology society journal 1 . the microbiology society is a not-for-profit organization. 2. we offer fast and rigorous peer review -average time to first decision is 4-6 weeks. 3. our journals have a global readership with subscriptions held in research institutions around the world. 4. 80% of our authors rate our submission process as 'excellent' or 'very good'. 5. your article will be published on an interactive journal platform with advanced metrics.find out more and submit your article at microbiologyresearch.org. key: cord-323551-22v2hn3v authors: galanti, m.; birger, r.; ud-dean, m.; filip, i.; morita, h.; comito, d.; anthony, s.; freyer, g. a.; ibrahim, s.; lane, b.; matienzo, n.; ligon, c.; rabadan, r.; shittu, a.; tagne, e.; shaman, j. title: rates of asymptomatic respiratory virus infection across age groups date: 2019-04-15 journal: epidemiol infect doi: 10.1017/s0950268819000505 sha: doc_id: 323551 cord_uid: 22v2hn3v respiratory viral infections are a leading cause of disease worldwide. a variety of respiratory viruses produce infections in humans with effects ranging from asymptomatic to life-treathening. standard surveillance systems typically only target severe infections (ed outpatients, hospitalisations, deaths) and fail to track asymptomatic or mild infections. here we performed a large-scale community study across multiple age groups to assess the pathogenicity of 18 respiratory viruses. we enrolled 214 individuals at multiple new york city locations and tested weekly for respiratory viral pathogens, irrespective of symptom status, from fall 2016 to spring 2018. we combined these test results with participant-provided daily records of cold and flu symptoms and used this information to characterise symptom severity by virus and age category. asymptomatic infection rates exceeded 70% for most viruses, excepting influenza and human metapneumovirus, which produced significantly more severe outcomes. symptoms were negatively associated with infection frequency, with children displaying the lowest score among age groups. upper respiratory manifestations were most common for all viruses, whereas systemic effects were less typical. these findings indicate a high burden of asymptomatic respiratory virus infection exists in the general population. respiratory viral infections are a leading cause of disease worldwide, affecting all age groups and representing a serious threat to human health, particularly for infants, older adults and the immunocompromised. the effects of infection on individuals can vary considerably and include completely asymptomatic manifestations, mild upper respiratory effects and severe symptoms requiring hospitalisation. the epidemiology of respiratory viral infections is generally analysed through passive symptom-based surveillance. when studying the burden of infection, many observational studies focus on severe outcomes, such as cardiac and pulmonary complications in hospitalised patients [1] [2] [3] [4] , or the role of respiratory viruses in the exacerbation of pre-existing respiratory conditions [5] [6] [7] . additionally, community-based longitudinal studies have generally been restricted to young children or households and involve the sampling of specimens to identify viruses after an index symptomatic episode occurs [8] [9] [10] . investigation of the prevalence and effects of respiratory viral infections in the broader population, not just among individuals seeking medical attention, is needed to more fully understand the burden of these infections within the community and to develop adequate preventive measures against these pathogens. in particular, the proportion of the population that is infected and yet does not develop symptoms must be determined to better quantify transmission risk, forecast future disease incidence and pathogen spread, and support public health response efforts. here, we document rates of asymptomatic respiratory virus infection through a large-scale community study across multiple age groups. we use data from a cohort of individuals who were tested weekly for respiratory viruses irrespective of symptom status. we combine these test results with participant-provided daily records of cold and flu symptoms and use this information to characterise symptom outcomes by virus and age category. we enrolled 214 individuals from multiple locations in manhattan borough, new york city. we refer to the participants as healthy as they were enrolled from the general population, as opposed to individuals seeking clinical care. the cohort included children attending two daycares, along with their siblings and their parents, teenagers and teachers from a high school, adults working at two emergency departments (a paediatric er and an adult er), and adults working at a university medical centre. the study period spanned 2 years from october 2016 to april 2018 with some individuals enrolled for a single cold and flu season (october-april) and others for the entire period. all individuals from the selected facilities who were willing to participate were enrolled in the study. enrolled individuals were asked to complete a baseline survey and provide two nasopharyngeal swab samples (one from each nostril). following this preliminary step, two nasopharyngeal samples were again collected weekly from each participant irrespective of symptoms. the baseline questionnaire completed at the time of enrolment included information on ethnicity, general health, daily habits, travel history and household structure. for the entire duration of the study, participants provided a daily report rating nine respiratory illness-related symptoms (fever, chills, muscle pain, watery eyes, runny nose, sneezing, sore throat, cough, chest pain), which were recorded on a likert scale (0 = none, 1 = mild, 2 = moderate, 3 = severe). participants were also asked to note if they had sought medical attention, stayed home or taken influenza-like illness (ili)-related medications as a consequence of their listed symptoms. parents provided consent, baseline questionnaire answers and the daily survey information for their enrolled children. a total daily score was generated for each participant by summing the scores of the individual symptoms (total daily score ranges from 0 to 27). details on the participants are summarised in table 1 . two nasopharyngeal samples per participant were collected on a weekly basis using minitip flocked swabs. both samples were stored jointly in 2 ml dna/rna shield (zymo research, irvine, ca, usa) at 4-25°c for up to 30 days and then stored at −80°c in two aliquots. nucleic acids were extracted from 200 µl of sample and 10 µl of internal control using the easymag nuclisens system (biomerieux, durham, nc, usa). samples were then screened for viruses using the esensor xt-8 respiratory viral panel (rvp; genmark dx, carlsbad, ca, usa) [4] , a multiplex pcr assay. the rvp system separately detects influenza a (any subtype, a/h1n1, a/h3n2, a/h1n1pdm2009) and b; rsv a and b; parainfluenza (piv) 1, 2, 3 and 4; human metapneumovirus (hmpv); human rhinovirus (hrv); adenovirus b/e and c; and coronaviruses (cov) 229e, nl63, oc43 and hku1. samples positive for a particular virus were identified by an electrical signal intensity of ⩾2 na/mm 2 (with the exception of cov oc43 for which positive results were identified by an intensity of ⩾25 na/mm 2 , per manufacturer specifications). we classified all specimens, irrespective of result, as symptomatic or asymptomatic according to the individual symptom score in the days surrounding the date of swab collection. we used multiple definitions as a standard for symptomatic infection does not exist. definitions are summarised in table 2 : definitions 1-4 consider a time window of 7 days around the day of the collection, whereas definitions 5-8 use a window of 11 days. while some definitions use raw metrics, definitions 4 and 8 normalise scores by the average symptom score for each participant (average weekly total symptom scores for each participant ranged from 0 to 39). we refer to infections that do not satisfy one or more specified definitions as asymptomatic infections. the association between reporting respiratory symptoms and testing positive was calculated with the χ 2 test. a 'symptomatic week' was defined as a calendar week where the total symptom score was ⩾10. analyses were conducted using the total number of positive samples, as well as the number of illness-events. we defined an illness-event as a group of consecutive weekly swab specimens for a given individual that were positive for the same virus (allowing for a 1-week gap to account for false negatives and temporary low shedding). the effects of different viruses and the severity of symptoms among different age groups were assessed using analysis of variance (anova). the χ 2 statistic was also used to assess pairwise differences. participants were divided into four groups: children (under 10 years of age), teenagers (10-17 years of age), adults with daily contact with children (parents and pediatric er doctors) and adults without daily contact with children. to assign adult participants to the correct category, we used information on household composition derived from the initial questionnaire. in the analysis of symptoms by virus, specimens positive for more than one virus were excluded. to analyse the specific effects of different viruses, we grouped symptoms into upper respiratory symptoms (runny nose, sneezing, sore throat, watery eye), lower respiratory symptoms (cough, chest pain) and systemic symptoms (fever, chills, muscle pain). of the 4215 nasopharyngeal samples collected, 737 (17.5%) were positive for one or multiple respiratory viruses. among the positive results, between 69% and 74% of the samples were classified as asymptomatic depending on the chosen definition (table 2) . overall, 55% of positive specimens were asymptomatic by all definitions. testing positive for one or more respiratory viruses was associated with an increased likelihood of being symptomatic (p < 0.0001); however, for the majority of symptomatic weeks (67%), rvp did not identify the presence of any respiratory virus. there was a weak association between pre-existing respiratory conditions (asthma, allergies) and the likelihood of experiencing symptomatic infections. however, the association was significant only for some definitions, and the effect on symptom score was marginally significant (p = 0.08 anova). coinfections accounted for 10% of positive samples and were found most frequently among children; they occurred throughout the year and were predominantly a combination of hrv and adenovirus. pairwise comparisons between single infections and coinfections across all eight definitions showed that testing positive for multiple viruses was not associated with more severe symptoms. among the viruses considered, influenza and hmpv were associated with significantly higher symptom scores than rsv, hrv, cov, adenovirus and piv. as shown in figure 1 , more than 50% of influenza and hmpv infections were classified as symptomatic by a majority of definitions, whereas the other viruses were mostly asymptomatic according to all definitions (p < 0.01 and p < 0.001 when comparing, respectively, the ratio of symptomatic influenza and hmpv infections to the other viral infections). the analysis of specific symptoms confirmed the higher severity of influenza and hmpv for lower, upper and systemic 2 m. galanti et al. symptoms (fig. 2a) . upper respiratory symptoms were most commonly associated with positive results for all viruses and for all age groups, followed by lower respiratory symptoms and then systemic symptoms ( fig. 2a and b) . infected children showed a similar percentage of upper and lower respiratory symptoms, and teenagers showed a similar percentage of lower respiratory and systemic symptoms. the majority of hmpv infections presented with both upper and lower respiratory symptoms. higher severity of symptoms was not associated with higher frequency of infection. figure 3 shows that while children were most frequently infected with a respiratory virus (they presented with the highest number of viral shedding events per season), they recorded (as reported by their parents) the lowest symptom scores on average. adults without daily contact with children reported the highest symptom score (the difference was significant only between children and adults without contacts with children, p = 0.003). this finding holds when controlling for length of infection, as longer-lasting infections were more frequent in children. host response to respiratory viruses is heterogeneous: some presumed infections, measured by detectable shedding of virus, exhibit no symptoms or signs of disease, whereas others result in more serious symptoms. when assessing the burden of an [11] [12] [13] . in [11] we showed that most individuals, particularly children and their close contacts, contract multiple respiratory infections per year. here, we analysed the symptoms reported by the same infected individuals and characterised them by virus and by age group. the presence of respiratory symptoms was associated with testing positive for one or more respiratory viruses. however, the majority of symptomatic manifestations were not paired with a positive rvp result. the origin of these symptomatic, negative rvp results could be due to allergies, bacterial infections or potential viral infections with pathogens that have not yet been identified or for which the viral panel does not test. one of the main goals of this analysis was to estimate the asymptomatic ratio, that is, the fraction of infections occurring without symptoms. the asymptomatic ratio is an important indicator for constraining both true respiratory virus prevalence within a community and the potential for further disease transmission [14] . asymptomatic carriers can, in fact, contribute to disease spread by generating ( possibly symptomatic) secondary infections. estimates of the asymptomatic ratio vary widely not only across diseases (from 95% of polio virus infections to <10% of measles), but also within the same disease due to different diagnostic testing procedures (pcr vs. serological tests [15] ) and sampling approaches (household vs. longitudinal studies). among respiratory viruses, the role of asymptomatic infection is poorly understood. for influenza alone, the prevalence of asymptomatic infection has been estimated to be as low as 9.4% and as high as 90% depending on the virus type, study, season and definition of asymptomatic infection [15, 16] . there is also some evidence that viral shedding correlates with symptom severity and that the contagiousness of asymptomatic individuals is less than for symptomatic persons [17, 18] . the rationale is that respiratory symptoms (coughing, sneezing, runny nose) help spread pathogens through droplet transmission, either inhaled or settled [19] . however, the absence of symptoms might bring asymptomatic infected individuals into greater contact with susceptible persons outside the home. this is particularly true for infants and toddlers whose behaviour, hygiene habits and close physical contact typically favour the spread of germs, especially in childcare settings. whether greater contact occurs and makes asymptomatic individuals effectively as contagious as symptomatic persons is not known. an additional difficulty is that a standard, accepted definition of symptomatic infection does not exist. further, perception of symptoms is highly subjective, and it may be difficult to assess whether a symptom is caused by the pathogen. for example, chronic symptoms can occurrunny nose is a common sign in children that does not necessary imply viral infectionand allergies can cause sneezing. fever, muscle pain and chills may also be caused by infections other than respiratory viruses. for these reasons, we adopted multiple definitions of 'symptomatic episodes', including personalised metrics. more than half of the rsv, adenovirus, piv, cov and hrv infections documented here were asymptomatic according to all definitions. influenza and hmpv infections caused significantly more symptoms than these other viruses; however, given its high prevalence, hrv was responsible for more than half of symptomatic episodes. in general, all viral infections presented with similar symptoms, with upper respiratory manifestations being more frequent, whereas systemic symptoms such as fever, muscle pain and chills were less typical. for surveillance in which ili (defined as fever plus cough and/or sore throat) is used to record respiratory infections among people seeking care, this constraint may mean that many viral infections go undetected. consistent with previous findings [20, 21] we did not find an association between viral co-infection and the likelihood of being symptomatic or presenting with more severe symptoms. regardless of the definition, our findings underscore the extremely high proportion of respiratory viral infections that are asymptomatic. further analysis is required to capture the role played by asymptomatic individuals in outbreak transmission dynamics, specifically the relative infectiousness of asymptomatic vs. symptomatic infections. there was considerable heterogeneity in individual immune response: some individuals seemed to be consistently less predisposed to developing respiratory symptoms upon viral infection, whereas others were always symptomatic when testing positive. infections in children were less symptomatic than in adults, even though children proved to be more frequently infected with respiratory viruses [11] . frequent infections may enhance the ability of the immune system to identify and respond to pathogens; hence, the group subjected to the fewest respiratory viral infections (adults without children) reported the highest average symptom scores. however, the apparent lower pathogenicity among children may simply be an artefact introduced due to parents reporting symptoms for their children. some symptoms, such as sore throat, muscle pain and chest pain, are difficult to identify in young children, and in fact they were less reported in this age category than among adults and teenagers. this possible bias in the reporting of symptoms is one limitation of our study. the daycare and workplace connections among some individuals in our cohort are another possible bias: some infections were likely caused by the same strain; thus the symptom profiles could be due to specific features of the pathogen rather than individual immune response. further, all the children in our cohort were attending daycare or were siblings of children attending daycare. interactions within the daycare setting could be a confounder in this analysis. future studies should investigate the genetic basis of heterogeneity in host response to respiratory virus infection in order to identify the regulatory pathways controlling reactions to these infections. moreover, longitudinal studies of this type, involving large networks of connected individuals, are needed to assess the role of asymptomatic infections in the transmission of respiratory viruses. the burden of respiratory syncytial virus infection in young children estimates of the global, regional, and national morbidity, mortality, and aetiologies of lower respiratory tract infections in 195 countries: a systematic analysis for the global burden of disease study mortality associated with influenza and respiratory syncytial virus in the united states comparison of the biofire filmarray rp, genmark esensor rvp, luminex xtag rvpv1, and luminex xtag rvp fast multiplex assays for detection of respiratory viruses role of viral respiratory infections in asthma and asthma exacerbations respiratory viruses and exacerbations of asthma in adults role of viruses in exacerbations of chronic obstructive pulmonary disease molecular epidemiology of respiratory syncytial virus transmission in childcare viral shedding and clinical illness in naturally acquired influenza virus infections epidemiology of viral respiratory tract infections in a prospective cohort of infants and toddlers attending daycare longitudinal active sampling for respiratory viral infections across age groups asymptomatic summertime shedding of respiratory viruses asymptomatic shedding of respiratory virus among an ambulatory population across seasons estimating pathogen-specific asymptomatic ratios the fraction of influenza virus infections that are asymptomatic: a systematic review and meta-analysis household transmission of the 2009 pandemic a/h1n1 influenza virus: elevated laboratory-confirmed secondary attack rates and evidence of asymptomatic infections does influenza transmission occur from asymptomatic infection or prior to symptom onset? world health organization writing group (2006) nonpharmaceutical interventions for pandemic influenza, international measures dynamics of infectious disease transmission by inhalable respiratory droplets epidemiology of multiple respiratory viruses in childcare attendees infection with multiple viruses is not associated with increased disease severity in children with bronchiolitis author orcids.m. galanti, 0000-0002-9060-1250.financial support. this work was supported by the defense advanced research projects agency contract w911nf-16-2-0035. the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.conflict of interest. js and columbia university disclose partial ownership of sk analytics. all other authors declare no competing interests. key: cord-322657-q4aeood2 authors: jartti, tuomas; lehtinen, pasi; vuorinen, tytti; österback, riikka; van den hoogen, bernadette; osterhaus, albert d.m.e.; ruuskanen, olli title: respiratory picornaviruses and respiratory syncytial virus as causative agents of acute expiratory wheezing in children date: 2004-06-17 journal: emerg infect dis doi: 10.3201/eid1006.030629 sha: doc_id: 322657 cord_uid: q4aeood2 we studied the viral etiology of acute expiratory wheezing (bronchiolitis, acute asthma) in 293 hospitalized children in a 2-year prospective study in finland. a potential causative viral agent was detected in 88% of the cases. eleven different viruses were represented. respiratory syncytial virus (rsv) (27%), enteroviruses (25%), rhinovirus (24%), and nontypable rhino/enterovirus (16%) were found most frequently. in infants, rsv was found in 54% and respiratory picornaviruses (rhinovirus and enteroviruses) in 42% of the cases. in older children, respiratory picornaviruses dominated (65% of children ages 1-2 years and 82% of children ages >3 years). human metapneumovirus was detected in 4% of all children and in 11% of infants. to prevent and treat acute expiratory wheezing illnesses in children, efforts should be focused on rsv, enterovirus, and rhinovirus infections. a cute expiratory wheezing illnesses (bronchiolitis, acute asthma) are the primary causes of hospitalization in children. an estimated 3% of children without other medical conditions are hospitalized for bronchiolitis (1) . the annual hospitalization rate for exacerbation of asthma is 0.15% in children (2) . in the united states alone, ≈200,000 children are hospitalized for bronchiolitis and acute asthma each year, which causes a substantial impact on families and the community. respiratory viruses are the most important precipitants of acute expiratory wheezing in children (3, 4) . bronchiolitis is reportedly induced in infants mainly by respiratory syncytial virus (rsv), and asthma in older children is induced mainly by rhinovirus. the role of rhi-novirus in infants is not clear. furthermore, the roles of other respiratory viruses, e.g., enteroviruses, and the recently discovered human metapneumovirus (hmpv) in the etiology of acute wheezing are not well established (5, 6) . investigating the viral origin of acute expiratory wheezing is useful because some antiviral treatments and vaccination are available, and the efficacy of anti-inflammatory treatments may be related to viral origin. the purpose of the study was to investigate the role of 11 respiratory viruses in children hospitalized for acute expiratory wheezing. the viral etiology was studied for 2 years prospectively to cover outbreaks of all major respiratory viruses. virus culture, virus antigen detection, polymerase chain reaction (pcr) techniques, and serologic testing were used to optimize the diagnosis of viral infection. as part of a randomized clinical trial evaluating the efficacy of systemic corticosteroid in the treatment of acute expiratory wheezing in children, we investigated the viral etiology of the infections. from september 1, 2000, through may 31, 2002, a total of 293 children participated in the study in the department of pediatrics, turku university hospital. study breaks occurred from june to july 2001 and during christmas week 2001. inclusion criteria were the following: age from 3 months to 16 years, hospitalization for acute expiratory wheezing, and written informed consent from the parents. exclusion criteria were the following: chronic diseases other than asthma or allergy, systemic glucocorticoid treatment within 4 weeks before the study, severe wheezing necessitating intensive-care unit treatment, and previous participation in this study. the study protocol was approved by the ethics committee of the turku university hospital. acute expiratory wheezing was called bronchiolitis when it occurred in children <3 years of age. when it recurred >2 times in persons of any age or occurred in persons >3 years of age, the diagnosis of asthma was used (7) . to some extent, bronchiolitis and asthma are expressions of the same pathologic process, and no rigid criteria separate these illnessess. all patients were examined by one of the two study physicians (t.j. and p.l.). on patient's admission, a nasopharyngeal aspirate sample was taken through a nostril by inserting a disposable catheter connected to a mucus extractor to a depth of 5 to 7 cm and retracting it slowly while applying gentle suction with an electric suction device. all specimens were obtained without inserting any solution into the nostrils. disposable plastic gloves were used, and all surfaces were wiped with disinfectant to prevent contamination. immediately after the secretion was suctioned, two sterile cotton swabs were dipped in the aspirate. the swabs were then placed in vials containing 2 ml of viral transport medium (5% tryptose phosphate broth, 0.5% bovine serum albumin, and antimicrobial agents in phosphate-buffered saline) for virus culture and pcr assays. the rest of the mucus was used for virus antigen detection. the specimens were transported to the laboratory on the same day at room temperature. the tubes for rsv and hmpv pcr assays were frozen at -70°c before processing. blood samples were collected on patient's admission and 2-3 weeks after discharge from the hospital. viral antigens for adenovirus; influenza a and b viruses; parainfluenza virus types 1, 2, and 3; and rsv were detected by time-resolved fluoroimmunoassay (8) . immunoglobulin (ig) g antibodies to the same viruses were measured from paired serum samples by enzyme immunoassays as described earlier (9) (10) (11) . purified heattreated coxsackievirus a9, coxsackievirus b3, echovirus 11, and poliovirus 1 were used as an antigen mixture in enterovirus igg assays and purified heat-treated coxsackievirus a16, coxsackievirus b3, and echovirus 11 in igm assays (12) . virus culture was performed according to routine protocols in a549, hela, and llc-mk2 cell lines and human foreskin fibroblasts (13) . the supernatants of cell cultures exhibiting a cytopathogenic effect were further studied by antigen detection for adenovirus; influenza a and b viruses; parainfluenza virus types 1, 2, and 3; and rsv or by reverse transcription (rt)-pcr for enterovirus-es and rhinovirus. nucleic acids for rt-pcr were isolated from the nasopharyngeal samples with a commercial kit (high pure viral nucleic acid kit, roche diagnostics, mannheim, germany) according to the manufacturer's instructions. rt-pcr was used to detect enteroviruses and rhinovirus, coronavirus, rsv, and hmpv, as described previously (6, 14, 15) . a case was defined as virus positive if at least one of the tests used was positive for virus. the rates of hmpv, respiratory picornaviruses, and rsv detected during the first study season 2000-2001 have been published (16) . the chi-square test was used for intergroup comparisons of differrent age groups in specific virus groups. the results were analyzed by using sas software (version 8.2, sas institute, cary, nc). from september 2000 through may 2002, a total of 661 children were hospitalized for acute expiratory wheezing ( figure 1 ). of the 661 patients, 341 did not meet the study criteria: 87 had already participated in the study, 79 were <3 months of age, 55 were not enrolled during study breaks, 48 had had systemic glucocorticoid treatment within 4 weeks, 24 did not need hospitalization, 17 had a chronic disease, 12 had guardians with language difficulties, 11 needed treatment in our intensive care unit, 3 had guardians who were not present, 2 were exposed to varicella, 2 patients' cases were not reported to the study physician, and 1 child was not eligible because of social reasons. the remaining 320 were eligible, but the parents of 27 (8%) children did not give their consent for participation in the study. eventually, 293 children participated in the study. the median age of the 293 study patients was 1.6 years (range 3 months-15.2 years). seventy-six (26%) children were <12 months of age, 152 (52%) children were 12-35 months old, and 65 (22%) children were >3 years. in 179 children, the clinical diagnosis was bronchiolitis and in 114, acute asthma. of the children with asthma, 49 were <3 years of age, 53% were boys, 38% experienced atopy, and 41% had parents who smoked. a potential causative viral agent was detected in 88% of the cases (table 1) . rsv (27%), enteroviruses (25%), and rhinovirus (24%) were the most common causative agents, resulting in 31%, 28%, and 28% of 258 virus-positive cases, respectively. the viruses in samples identified by the primary picornavirus pcr test but not identifiable in the liquid-hybridization assay were named rhino/ enteroviruses (16%). according to our sequence data, these amplicons have shown >90% homology to human rhinoviruses. the remaining eight viruses studied accounted for 18% of the cases, and none of these viruses was detected in >5% of all cases. mixed viral infections were found in 57 (19%) cases and were usually associated with respiratory picornaviruses. coinfection with enteroviruses and rsv was the most common mixed infection (19%), followed by rhinovirus and rsv (14%), rhino/enterovirus and rsv (11%), and enteroviruses and rhinovirus (9%). of 12 hmpv infections, 5 were associated with other respiratory viruses. most of the viruses (84%), respiratory picornaviruses especially, were detected by using pcr (table 1) . rhinovirus was cultivated in 25 (38%) of 65 specimens with pcr-positive results, enteroviruses in 14 (24%) of 59 specimens with pcr-positive results, and rhino/ enterovirus in 1 (2%) of 46 specimens with pcr-positive results. to compare different methods of detecting rsv infection, we selected the patients whose samples were studied with four methods (n = 257). the recovery rate of rsv by igg serologic testing was 22%; by virus antigen detection, 21%; by virus culture, 20%; and by pcr, 18%. typical of the situation in finland, a minor rsv epidemic occurred during the spring of 2000, followed by a major epidemic during the winter of 2001 to 2002 ( figure 2 rsv (54%), respiratory picornaviruses (42%), and hmpv (11%) were the most common viruses in infants ( figure 3 ). respiratory picornaviruses were detected in 65% and rsv in 22% of the cases in children ages months. in children aged >3 years, respiratory picornaviruses (82%) were found most frequently. comparisons between age groups showed that rsv (p < 0.001) and hmpv (p = 0.0030) infected infants significantly more often than children in other age groups, adenovirus infected children ages 1-2 years significantly more often (p = 0.022), and enteroviruses infected children ages >3 years more often (p = 0.0018; figure 3 ). no other significant differences were found. our prospective study produced four notable findings. first, respiratory virus infection was detected in up to 90% of hospitalized children with acute expiratory wheezing. second, respiratory picornaviruses were commonly associated with wheezing in infants. third, one third of the wheezing children ages >3 years were infected with enteroviruses. fourth, hmpv infections occurred in infants, but mainly during the first study year, and they were associated with only 4% of all cases with expiratory wheezing. all major studies of the viral origins of expiratory wheezing are presented in table 2 . in studies from the 1960s to the 1990s, viral diagnosis was based on conventional virus culture, antigen detection, and serologic testing, and a viral agent could be established in 20% to 50% of children with expiratory wheezing. bronchiolitis was mainly considered an rsv infection, with recovery in up to 73% of patients with confirmed cases (30) . lower rsv recovery rates were seen in older children (17, 18, 21, 22, 24, 27) . in the 1990s, viral detection rates increased to 75% to 85%, mainly as result of the increased detection of rhinoviruses by pcr (3, 5) . our data confirm that rsv plays a key role in the etiology of bronchiolitis during rsv epidemics. our findings regarding bronchiolitis give a prominent role also to rhinovirus, which has earlier been considered a common causative agent of wheezing in older children only (3, 4, 28, 31) . we found no differences in the distribution of rhinovirus infections by patient age. this is the first long-term study to report a high association of enterovirus infections with acute expiratory wheezing in children. enteroviruses, which replicate most prolifically in the gastrointestinal tract, have recently been shown to be associated with upper respiratory infections in 25% to 35% of the cases (32, 33) . our findings are in agreement with those of rawlinson et al. (4) , who found enteroviruses by pcr in 29% of the young children with well-documented asthma during the summer. we found enteroviruses mostly in older children. hmpv was detected in 4% of our patients. a recent study of children hospitalized for acute respiratory tract disease found hmpv in 6% of the cases (34) . bronchiolitis and pneumonitis were the main diagnoses. hmpv predominantly infected infants as seen in our study and in previous studies (4, 6) . hmpv outbreaks have been reported mainly in mid-winter, which was supported by our study. notably, the hmpv outbreak with 10 cases was seen during the first study year, and two cases were found during the second year, which suggests that epidemics do not occur every year. the use of pcr has markedly increased the recovery rates of viruses in acute respiratory infections (3, 5) . the clinical value of positive respiratory picornavirus pcr tests is, however, questionable as picornavirus rna has also been detected in 5% to 30% of asymptomatic children (3, 35) . we recently found that the number of positive pcr results for picornavirus markedly decreased over 2 to 3 weeks and disappeared over 5 to 6 weeks after an acute respiratory infection, which suggests that a positive pcr result for picornaviruses is related to acute infection (36) . none of the 79 healthy controls were infected with enteroviruses, but 16% were positive for rhinovirus or nontypeable rhino/enterovirus (36) . in detecting rsv infections, pcr was no more sensitive than virus culture, antigen detection, or serologic testing. this finding is in contrast to the results of previous studies, especially in adults (37) . in children too, pcr has been almost 1.5 times more sensitive than culture and antigen detection (38) . these differences may be explained by the greater sensitivity of the nested rt-pcr used in those studies. compared to children, adults may also have lower titers of viruses in the nasopharynx, which favors pcr diagnosis over virus culture or antigen detection. we likely did not miss many cases of adenovirus, parainfluenza virus, or influenza virus infections because pcr has only modestly increased sensitivity to those viruses compared to virus culture and antigen detection (39, 40) . our study has some limitations. first, and most important, we studied fewer than half of the children admitted to our hospital for acute expiratory wheezing. however, throughout the study, we enrolled approximately half of the patients hospitalized for expiratory wheezing each month. since the respiratory virus season is the main factor determining the viral cause of acute illness, any season-ality bias is largely excluded. several infants with rsv infection were missed, because infants <3 months of age were not included. however, during the summer study break, when rhinovirus and enteroviruses are normally circulating in the community, children were not enrolled. this balances the ratio of missed rsv cases to picornavirus cases. we therefore believe that our sample reliably represents the whole patient population hospitalized during the study years. furthermore, we only analyzed viral infections. chlamydia pneumoniae and mycoplasma pneumoniae have been detected in 5% to 25% of children with acute wheezing, but the clinical importance of these findings remains to be determined (7, 28) . in conclusion, this study showed that acute expiratory wheezing necessitating hospitalization was most often associated with rsv, enterovirus, and rhinovirus infections. acute expiratory wheezing in infants may be a risk factor for childhood asthma (31) . therefore, efforts should focus on developing antiviral agents and vaccines against rsv and respiratory picornaviruses. the study was supported by the academy of finland, the pediatric research foundation, and the foundation of jalmari and rauha ahokas. dr. jartti is a fellow in pediatric allergology at turku university hospital, turku, finland. he is interested in the pathogenesis and treatment of acute expiratory wheezing illnesses with a special interest in respiratory viral infections. rates of hospitalization for respiratory syncytial virus infection among children in medicaid trends in childhood asthma: prevalence, health care utilization, and mortality rhinovirus and respiratory syncytial virus in wheezing children requiring emergency care. ige and eosinophil analyses asthma exacerbations in children associated with rhinovirus but not human metapneumovirus infection community study of role of viral infections in exacerbations of asthma in 9-11 year old children a newly discovered human pneumovirus isolated from young children with respiratory tract disease recurrent wheezy bronchitis and viral respiratory infections laboratory diagnosis of infectious diseases. principle and practice enzyme immunoassays for detection of igg and igm antibodies to parainfluenza types 1, 2 and 3 purification of adenovirus hexon protein by high-performance liquid chromatography immunoglobulin class-specific antibody response in respiratory syncytial virus infection measured by enzyme immunoassay enterovirus infection as a risk factor for beta-cell autoimmunity in the prospective birth-cohort trial dipp picornaviridae: rhinoviruses-common cold viruses detection of enteroviruses and rhinoviruses in clinical specimens by pcr and liquid-phase hybridization detection of rhinovirus, respiratory syncytial virus, and coronavirus infections in acute otitis media by reverse transcriptase polymerase chain reaction human metapneumovirus and acute wheezing in children a collaborative study of the aetiology of acute respiratory infections in britain 1961-4. a report of the medical research council working party on acute respiratory virus infections epidemiologic patterns of acute lower respiratory disease of children in a pediatric group practice respiratory viral infection in children: a survey in general practice, roehampton viral infections in wheezy bronchitis and asthma in children the etiologic and epidemiologic spectrum of bronchitis in pediatric practice respiratory viral infection and wheezy bronchitis in childhood role of viruses and bacteria in acute wheezy bronchitis in childhood: a study of sputum respiratory virus infections and aeroallergens and acute bronchial asthma the association of viruses with acute asthma risk factors for acute wheezing in infants and children: viruses, passive smoke, and ige antibodies to inhalant allergens wheezing bronchitis in children. incidence, viral infections, and other risk factors in a defined population detection of viral, chlamydia pneumoniae and mycoplasma pneumoniae infections in exacerbations of asthma in children differential detection of rhinoviruses and enteroviruses rna sequences associated with classical immunofluorescence assay detection of respiratory virus antigens in nasopharyngeal swabs from infants with bronchiolitis association of rhinovirus infection with increased disease severity in acute bronchiolitis rhinovirus-induced wheezing in infancy-the first sign of childhood asthma? intranasal fluticasone propionate does not prevent acute otitis media during viral upper respiratory infection in children diagnosis of group a coxsackieviral infection using polymerase chain reaction human metapneumovirus infections in hospitalized children use of polymerase chain reaction for diagnosis of picornavirus infection in subjects with and without respiratory symptoms persistence of rhinovirus and enterovirus rna after acute respiratory illness in children diagnosis of respiratory syncytial virus infection: comparison of reverse transcription-pcr to viral culture and serology in adults with respiratory illness improved detection of respiratory syncytial virus in nasal aspirates by seminested rt-pcr direct detection of respiratory syncytial virus, parainfluenza virus, and adenovirus in clinical respiratory specimens by a multiplex reverse transcription-pcr assay effectiveness of reverse transcription-pcr, virus isolation, and enzyme-linked immunosorbent assay for diagnosis of influenza a virus infection in different age groups key: cord-351046-yq7287k9 authors: schubert, gena; haverland, josh; wyst, craig vander; mcgreevy, jon title: how much drool is too much?() date: 2019-12-13 journal: clin pediatr emerg med doi: 10.1016/j.cpem.2019.100742 sha: doc_id: 351046 cord_uid: yq7287k9 a 3-year-old boy presented to the emergency department with a chief complaint of “lethargy” and was found to have ptosis with eventual respiratory failure and need for emergent intubation. there is a broad differential for a patient with respiratory failure, and careful physical examination and history are imperative to reduce morbidity and prevent mortality. after further evaluation and workup, the diagnosis is ultimately revealed. 3-year-old boy with a history of allergic rhinitis and persistent asthma presented to a pediatric emergency department (ed) in arizona with parental concern for congestion and drooping red eyes at 10:00 pm. his father noted that earlier in the day, his speech was more difficult to understand but returned to normal after a nap. vital signs on arrival to the ed were temperature 36.9°c, heart rate 108 beats per minute, respiratory rate 24 breaths per minute, and pulse oximetry of 99% on room air. on physical examination, there were bilateral conjunctival erythema and swollen nasal turbinates, and the patient was noted to be rubbing his eyes. he was breathing comfortably on room air with clear breath sounds, and his heart had a regular rate and rhythm. cranial nerves 2-12 were intact, and he ambulated appropriately for age and followed commands. there were no focal neurologic deficits on examination. the patient was continued on home cetirizine, started on tetrahydrozoline eye drops, and discharged home with the diagnosis of allergic rhinitis and viral conjunctivitis. the patient returned to the same ed with his father the next day at around 10:00 am for a right forehead laceration after hitting his head on the edge of the bed. the injury occurred while in the care of his grandparents. there was no report of vomiting or loss of consciousness. both the father and the provider felt the patient's behavior was at neurologic baseline for a 3-year-old at the time of arrival to the ed. similar to his previous visit, he was afebrile to 36.9°c with a heart rate of 107 beats per minute, respiratory rate of 26 breaths per minute, and pulse oximetry of 98% on room air. the physical examination noted a 3-cm linear forehead laceration over frontal bone with surrounding hematoma. there was no mention of any other abnormal physical examination findings including eye findings. he met low-risk criteria according to the pediatric emergency care applied research network (pecarn) head trauma algorithm and a 3-year-old boy presented to the emergency department with a chief complaint of "lethargy" and was found to have ptosis with eventual respiratory failure and need for emergent intubation. there is a broad differential for a patient with respiratory failure, and careful physical examination and history are imperative to reduce morbidity and prevent mortality. after further evaluation and workup, the diagnosis is ultimately revealed. myasthenia gravis; respiratory distress; ptosis; myasthenic crisis thus did not receive imaging or observation. his laceration was repaired with skin adhesive, and he was discharged home with his father. the patient returned 9 hours later to the same ed for his third visit within 24 hours. this time, he presented with his mother and with a chief complaint of lethargy. the patient lives in a split household and had been in the care of the mother since 4:00 pm. over the last 3 hours, she had noted new-onset drooling, garbled speech which was difficult to understand, difficulty swallowing water, and increased fatigue. she also felt his eyes appeared more erythematous and watery with droopy eyelids. she denied a history of any of the above symptoms prior to this visit. since the patient had been in her care, he had 4 episodes of urinary incontinence despite previously being fully potty-trained. the mother denied any recent illness including cough, fever, ear pain, diarrhea, or vomiting. the father arrived and recalled the child having increased secretions under his care as well. the patient pointed to back of his throat when asked if he was in pain, which the father thought may be related to molar teeth eruption. the initial vital signs on his third ed presentation were as follows: temperature 36.7°c, heart rate 130 beats per minute, respiratory rate 30 breaths per minute, pulse oximetry 99%, and blood pressure of 113/81 mm hg. on examination, the patient was awake and calm in his mother's arms with no acute distress. he appeared appropriate for a 3-year-old and was interactive and did not appear altered. the provider was able to understand some of his speech, although some speech was garbled, making it difficult to understand fully. his head was atraumatic other than the previously repaired laceration. his pupils were of normal size, equal, round, and reactive to light bilaterally without significant conjunctival injection. he had ptosis bilaterally. the tympanic membranes were pearly. there was no rhinorrhea, but he did have drooling and clear oral secretions. no oropharyngeal erythema, exudates, palatal petechiae, uvular deviation, or tonsillar hypertrophy or asymmetry was appreciated. there was normal work of breathing and clear lungs sounds bilaterally. when agitated, he had intermittent stridor, but at rest, he did not exhibit stridor, tracheal tugging, accessory muscle use, wheezes, or crackles. he had tachycardia but no murmurs, rubs, or gallops. there was no lymphadenopathy. his abdomen was soft, nontender, nondistended, and without hepatosplenomegaly. neurologically, the patient had bilateral ptosis but no other gross cranial nerve deficits. he had 5/5 strength bilaterally in his upper and lower extremities. gross sensation was intact. his patellar reflexes were 2+ bilaterally, and providers were unable to reliably elicit other reflexes. anal sphincter tone was normal. he was able to ambulate without difficulty and had no ataxia. because of the history of head injury earlier in the day with new-onset neurologic complaints, a computerized tomography (ct) study of the head without contrast was ordered in addition to soft tissue neck radiographs due to the increased drooling and stridor. a respiratory pathogen polymerase chain reaction panel (rpp) nasopharyngeal swab; complete blood count; complete metabolic panel; urine drug screen; and ingestion laboratory tests including aspirin, acetaminophen, and alcohol levels were sent. an electrocardiogram (ecg), as part of the toxicological workup, was also performed. the result of the ct head was normal. the neck radiographs showed "mild narrowing of the subglottic airway with loss of normal shouldering, suggesting croup" (figure 1 ). complete blood count and complete metabolic panel were unremarkable, and aspirin, acetaminophen, and alcohol levels were negative. rpp was positive for coronavirus. ecg result was likewise normal. approximately 2 hours after initial evaluation, the providers were called back into the room by his nurse who had noticed labored breathing, tachycardia to 146 beats per minute, and a decreased respiratory rate to 20, and he was now on 1.5 l oxygen flow via nasal cannula to maintain oxygen saturations above 90%. the patient was less active and alert than his previous examination and had more copious clear oral secretions, labored pattern of breathing, diffuse crackles throughout bilateral lung fields, and inspiratory stridor. he was tachycardic but without any murmurs, rubs, or gallops and no appreciable hepatomegaly. other than being less active, there were no other acute changes in his neurologic examination. the decision was made to intubate the patient for airway protection, and he underwent rapid sequence intubation with fentanyl, midazolam, and rocuronium. he was easily intubated on the first attempt with a 4.0fr microcuffed endotracheal tube (ett), which was downsized intentionally because of concern for increasing stridor and potentially narrowed airway. no caustic burns or other airway abnormalities were appreciated during direct laryngoscopic visualization. the patient oxygenated and ventilated without difficulty following intubation, although when the ett cuff was deflated for repositioning of the ett following radiograph, copious amounts of clear secretions from the patient's mouth were noted. the rapid urine drug screen was negative, and the patient was admitted to the pediatric intensive care unit for further care. the differential for a patient presenting to the ed with acute respiratory distress is broad and includes infection, ingestion, trauma, envenomation, muscular disorders, and autoimmune etiologies. scorpion envenomations along with other insect bites are common in arizona and can be a cause of drooling and increased secretions. this was at the top of the differential upon evaluation and the need for emergent intubation. scorpion envenomation often presents with respiratory distress along with increased drooling, abnormal eye movements, muscle twitching, and agitation. the patient did have respiratory distress, drooling, and ptosis of his eyes, but no abnormal eye movements were appreciated. additionally, the patient initially presented without respiratory distress and progressed, which is not characteristic of envenomation. grading and treatment of scorpion envenomation (table 1 ) are based on location of pain, location of paresthesia, cranial nerve involvement (oral secretions, blurry vision, tongue fasciculations, nystagmus), and skeletal neuromuscular dysfunction (abnormal movements or flailing of the extremities). 1 additionally, with stridor and drooling, infectious etiologies were on the top of the differential. most common bacterial etiologies include streptococcus species and staphylococcus aureus. 2 there are many viral etiologies as well, including rhinovirus, adenovirus, enterovirus, coronavirus, and influenza. the patient was coronavirus positive on rpp. additionally, the patient had stridor along with an radiograph concerning for subglottic narrowing, most consistent with croup. croup, also referred to as laryngotracheitis, is an upper respiratory illness caused by parainfluenza 1 or 2. 2 the other etiologies for croup include influenza a and b, measles, adenovirus, and respiratory syncytial virus. croup is diagnosed based on signs and symptoms including stridor, barking cough, or hoarseness. a neck radiograph will show the traditional narrowing of the trachea known as a steeple sign. 3 however, a radiograph is not routinely performed for diagnosis because croup is usually a clinical diagnosis. although the patient had coronavirus and concern for croup, this did not explain his ptosis and other neurologic findings. ingestion along with trauma is often also at the top of the differential as it typically presents acutely, and prompt diagnosis and treatment are critical. in 4 additionally, poisoning with selenium and mercury can present similarly. treatment includes administration of atropine along with pralidoxime. 4 drugs that can cause hypersalivation include morphine, pilocarpine, methacholine, haloperidol, and clozapine. cocaine and phencyclidine can also cause increased secretions. there was no history to suggest ingestion in the patient. muscular disorders can also cause respiratory distress and failure. muscular dystrophy, polymyositis, and myasthenia gravis should be on the differential. the most common muscular dystrophies in children are duchene and becker. both are x-linked recessive and caused by a mutation in the dystrophin gene which clinically presents with progressive weakness. 5 weakness often affects proximal limb muscles before distal, and lower extremities before upper extremities. the classic finding is gowers sign, where a child uses their hands and arms to walk up their body to get to an upright position from sitting because of the lack of hip and thigh muscle strength. 5 physical examination findings include pseudohypertrophy of the calf, lumbar lordosis, waddling gait, shortening achilles tendon, hypotonia, and hyporeflexia. 5 the patient did not have any secondary symptoms of the above, and the patient had a normal gait. polymyositis, on the other hand, is a rare autoimmune myopathy with direct t-cell invasion of the muscle fibers. 6 the hallmark of this disease is weakness, primarily proximal muscle weakness. lastly, myasthenia gravis is an antibody-mediated autoimmune disease that affects postsynaptic neuromuscular junction. 7 usually, symptoms are gradual, but patients can present in myasthenic crisis leading to respiratory failure. most common symptoms include ptosis and diplopia which are worse with activity and improved with rest. myasthenic crisis is a life-threatening condition defined by worsening weakness and requires noninvasive ventilation or intubation. 8 the patient arrived to the pediatric intensive care unit intubated on a ventilator with a rate of 28 breaths per minute, tidal volume of 100 ml (7 ml/kg), peep of 7, pressure support of 10, and fio 2 of 60%. he was sedated but had clear and equal breath sounds bilaterally. his heart had a regular rate and rhythm, and he had good perfusion throughout. no abnormal physical examination findings were found. his orogastric tube was set to low intermittent suction, and he remained on a dexmedetomidine drip at 0.4 μg/kg/h and ampicillin/sulbactam 50 mg/kg intravenously every 6 hours pending culture results. a ct of the neck was obtained to rule out other etiologies for respiratory distress, which was negative for peritonsillar or retropharyngeal abscess. an extubation trial was done on day 2 of admission. because of inspiratory stridor and increased work of breathing, he was given inhaled dexamethasone and racemic epinephrine with minimal improvement. he continued with poor pharyngeal tone, drooling, dysarthria, and hypoxia with oxygen saturations 80%, and he was reintubated for airway protection. during extubation, the patient was moving all his extremities, but there was concern for hypotonia. the patient was on sedating medications, which were a confounding factor. neurology was consulted at that time because of concern for a neuromuscular process. their recommendations included to obtain negative inspiratory force studies, magnetic resonance imaging of the thoracic and lumber spine with gadolinium contrast, acetylcholine receptor antibody, ganglioside (gm1, gd1b, gq1b) antibodies, lumbar puncture with standard indices plus cerebrospinal fluid (csf) igg, oligoclonal bands, igg/albumin ratio, igg index, and electromyography. csf studies were normal with wbc 0, rbc 0, protein 19, and glucose 97. csf west nile ab igg and igm were negative. csf oligoclonal bands were negative, and the patient had a normal igg to albumin ratio. a comprehensive drug screen was negative. negative inspiratory force was reported at −40. electromyography with 2-hz repetitive stimulation was abnormal, which showed a greater than 10% decrement for the left deltoid/axillary, left tibialis anterior/peroneal, and left adduction digiti minimi/ulnar. ganglioside (gm1, gd1b, gq1b) igg and igm antibodies were negative. initial acetylcholine binding and block antibodies were negative, but positive for acetylcholine receptor muscle binding (0.15 nmol/l, normal b0.02), negative striated muscle ab titer, and achr muscle modeling ab 17%, therefore 17% loss of ach receptor. given these results, the diagnosis of myasthenia gravis was made. after diagnosis, the patient was treated with ivig and prednisone for 3 days with improvement of symptoms. he was successfully extubated on day 9 of hospitalization after treatment and remained stable throughout his 18-day hospitalization. he did not require plasma exchange. he was placed on pyridostigmine along with tapering doses of steroids prior to discharge. after discharge, the patient was followed by neurology, physical therapy, occupational therapy, general pediatrics, and immunology. he regained his baseline strength, his ptosis resolved, and he returned to his normal behavior and activities. he was tapered off steroids in the year after discharge from the hospital and remained on pyridostigmine. myasthenia gravis is the most common disorder of neuromuscular transmission. the hallmark of the disorder is fluctuating degrees and variable combination of weakness in ocular, bulbar, limb, and respiratory muscles. the weakness is from the antibody-mediated t-cell response to acetylcholine receptors or receptor-associated proteins at the postsynaptic neuromuscular junction. 7 the incidence of myasthenia gravis is about 20 per 100 000 in the united states, which includes children and adults, and a prevalence of roughly 60 000 patients affected. it is estimated that the risk of myasthenic crisis in myasthenia gravis patients may be as high as 10 to 20%, and 13 to 20% of myasthenia gravis presents with myasthenic crisis as the first manifestation. 9, 10 precipitating factors include infection, thymectomy, pregnancy, childbirth, and tapering of immunosuppressive medications. 11 medications most often associated are antibiotics (aminoglycosides, fluoroquinolones, erythromycin, azithromycin), cardiac medications (β-blockers, procainamide, quinidine), and magnesium. 12 in the case of our patient, no specific cause or precipitating factor was found except for potentially an upper respiratory illness with coronavirus and signs and symptoms of croup. the diagnosis of myasthenia gravis is complex. bedside tests including the ice pack test and edrophonium test are sensitive but have major limitations with false-positive results. thus, a confirmatory test with antibodies and electrophysiological studies (repetitive nerve stimulation studies and single-fiber electromyography) are needed. an immunologic assay to detect the presence of circulating acetylcholine receptor antibodies is the first step in the laboratory confirmation of myasthenia. on the other hand, muscle specific kinase antibodies and receptor tyrosine kinases are found in less than 50% of generalized myasthenia gravis without acetylcholinesterase receptor antibodies but are rare in ocular myasthenia gravis. 13, 14 the treatment for myasthenia gravis begins with respiratory support including intubation or noninvasive ventilation. once disabling and often fatal, myasthenia gravis can be managed effectively with therapies that include anticholinesterase agents, rapid immunomodulatory therapies, chronic immunosuppressive agents, and thymectomy. treatment varies based on the age of the patient, severity of disease, respiratory involvement, bulbar involvement, and progression of the disease. goals of treatment are to minimize adverse effects of the drug while having improvement in symptoms and return to baseline function. symptomatic treatment with acetylcholinesterase inhibition increases the amount of acetylcholine at the neuromuscular junction, thereby improving symptoms. pyridostigmine is often the treatment of choice and sometimes is used as monotherapy. with generalized myasthenia gravis, steroids and nonsteroidal immunosuppressive agents are used to target immune dysregulation. likewise, plasma exchange or ivig is an immunomodulating treatment used for bridge therapy or during acute exacerbations of myasthenia gravis. approximately 15% of patients with myasthenia gravis are found to have a thymoma. surgery with complete resection is indicated for these patients when possible. myasthenic crisis is an uncommon yet important diagnosis to keep on the differential of a patient with respiratory distress, especially in a patient with ptosis and sialorrhea. it is also imperative to take a closer look at patients who present for multiple visits to the ed in a short time period even if they appear to be unrelated or benign. our patient presented 3 times to the ed in a 24-hour period. he was diagnosed with allergic rhinitis and a facial laceration in his first 2 visits. individually, these were not concerning, but retrospectively, they were red herrings. when assessing a child with respiratory distress requiring intubation, the differential should remain broad, but the physical examination and history are paramount for diagnosis and differentiating infectious vs other etiologies. early recognition and consultation with specialists can help with diagnosis, treatment, and management. goldfrank's toxicologic emergencies clinical courses of croup caused by influenza and parainfluenza viruses the usefulness of lateral neck roentgenograms in laryngotracheobronchitis annual report of the american association of poison control centers' national poison data system (npds): 26th annual report diagnosis and management of duchenne muscular dystrophy, part 1: diagnosis, and neuromuscular, rehabilitation, endocrine, and gastrointestinal and nutritional management monoclonal antibody analysis of mononuclear cells in myopathies. i: quantitation of subsets according to diagnosis and sites of accumulation and demonstration and counts of muscle fibers invaded by t cells myasthenia gravis on the concept of myasthenic crisis myasthenic crisis the management and outcome of patients with myasthenia gravis treated acutely in a neurological intensive care unit medications and myasthenia gravis (a reference for health care professionals) detection and characterization of musk antibodies in seronegative myasthenia gravis anti-musk myasthenia gravis presenting with purely ocular findings key: cord-347465-yu6oj30v authors: kurskaya, olga; ryabichenko, tatyana; leonova, natalya; shi, weifeng; bi, hongtao; sharshov, kirill; kazachkova, eugenia; sobolev, ivan; prokopyeva, elena; kartseva, tatiana; alekseev, alexander; shestopalov, alexander title: viral etiology of acute respiratory infections in hospitalized children in novosibirsk city, russia (2013 – 2017) date: 2018-09-18 journal: plos one doi: 10.1371/journal.pone.0200117 sha: doc_id: 347465 cord_uid: yu6oj30v background: acute respiratory infections (aris) cause a considerable morbidity and mortality worldwide especially in children. however, there are few studies of the etiological structure of aris in russia. in this work, we analyzed the etiology of aris in children (0–15 years old) admitted to novosibirsk children’s municipal clinical hospital in 2013–2017. methods: we tested nasal and throat swabs of 1560 children with upper or lower respiratory infection for main respiratory viruses (influenza viruses a and b, parainfluenza virus types 1–4, respiratory syncytial virus, metapneumovirus, four human coronaviruses, rhinovirus, adenovirus and bocavirus) using a rt-pcr kit. results: we detected 1128 (72.3%) samples were positive for at least one virus. the most frequently detected pathogens were respiratory syncytial virus (358/1560, 23.0%), influenza virus (344/1560, 22.1%), and rhinovirus (235/1560, 15.1%). viral co-infections were found in 163 out of the 1128 (14.5%) positive samples. we detected significant decrease of the respiratory syncytial virus-infection incidence in children with increasing age, while the reverse relationship was observed for influenza viruses. conclusions: we evaluated the distribution of respiratory viruses in children with aris and showed the prevalence of respiratory syncytial virus and influenza virus in the etiological structure of infections. this study is important for the improvement and optimization of diagnostic tactics, control and prevention of the respiratory viral infections. we detected 1128 (72.3%) samples were positive for at least one virus. the most frequently detected pathogens were respiratory syncytial virus (358/1560, 23.0%), influenza virus (344/1560, 22.1%), and rhinovirus (235/1560, 15.1%). viral co-infections were found in 163 out of the 1128 (14.5%) positive samples. we detected significant decrease of the respiratory syncytial virus-infection incidence in children with increasing age, while the reverse relationship was observed for influenza viruses. we evaluated the distribution of respiratory viruses in children with aris and showed the prevalence of respiratory syncytial virus and influenza virus in the etiological structure of plos acute respiratory infections (aris) pose a significant public health problem worldwide, causing considerable morbidity and mortality among people of all age groups [1] . children are on average infected two to three times more frequently than adults. [2] . there are more than 200 respiratory viruses that can cause aris. human respiratory syncytial virus (hrsv), human rhinovirus (hrv), human metapneumovirus (hmpv), human parainfluenza virus (hpiv), human enterovirus (ev), influenza virus (ifv), human coronavirus (hcov), adenovirus (hadv), and human bocavirus (hbov) are the most common viral agents associated with aris, accounting for around 70% of aris [3, 4] . the frequency of mixed respiratory viral detection varies from 10% to 30% in hospitalized children [5] [6] [7] . in addition, several new human respiratory viruses have been described in recent years, including human metapneumovirus [8, 9] , human bocavirus [10] , and novel human coronaviruses, including severe acute respiratory syndrome coronavirus (sars-cov) [11] , human coronaviruses nl63 (hcov-nl63), hku1 (hcov-hku1) [12] , and middle east respiratory syndrome coronavirus (mers-cov) [13] . although the majority of aris are associated with respiratory viruses, antibiotics are often used in the clinical treatment of aris. as children with aris often have similar clinical symptoms, studying the clinical hallmarks of children with virus-related aris and the spectrum of respiratory viruses will help in developing more accurate treatments for aris [14] . rapid diagnosis is important not only for timely treatment starting but also for the detection of a beginning influenza epidemic and the avoidance of unnecessary antibiotic treatment [15, 16] . western siberia plays a key role in ecology, epizootiology and epidemiology of emerging diseases. this territory was involved in the circulation of a/h5n1 and a/h5n8 avian influenza viruses in 2005-2017 [17, 18] . these viruses were spread by wild birds' migration. in western siberia migratory flyways of birds' wintering in different regions of the world: south east asia, central asia, middle east, hindustan, europe, and africa-cross. for this reason, there is high probability of the emergence of humans and animal influenza viruses reassortants, as well as emergence of local outbreaks of human morbidity caused by uncommon variants of influenza viruses. furthermore, novosibirsk is the largest transport hub in this part of russia with numerous international connections, that is important for the spread of aris [19, 20] . the prevalence of respiratory viruses among children with aris differs in different regions and varies over time [21] [22] [23] [24] [25] . thus, to better understand the epidemiology of acute respiratory infections in novosibirsk region, we investigated etiology of aris in children admitted to novosibirsk children's municipal clinical hospital in 2013-2017. all aspects of the study were approved by the ethics committee of the federal state budgetary institution "research center of clinical and experimental medicine" (2013-23). accordingly, written informed consent was obtained from parents prior to sample taking. children 0-15years of age were enrolled in the study within 3 days of illness onset and had at least two of the following symptoms: fever, sore throat, cough, rhinorrhea, nasal congestion, sputum, shortness of breath, lung auscultation abnormalities, tachypnea, and chest pain. paired nasal and throat swabs were collected from each patient admitted to novosibirsk children's municipal clinical hospital by hospital nurses. a total of 1560 samples collected during four epidemic seasons of 2013-2017 (october-april) were enrolled to the study. the epidemiological and clinical information including case history, symptoms, physical signs, and examination were included in a standardized questionnaire. the samples were placed immediately in viral transport medium (eagle mem, bsa and antibiotics) and stored at 4-8˚c prior transportation to the laboratory (not more than 24 hours). detection of respiratory viruses was performed immediately after delivery to the laboratory. all specimens were tested for 15 common respiratory viruses, including influenza virus types a, b (ifva and ifvb), human parainfluenza virus (hpiv) types 1-4, human respiratory syncytial virus (hrsv), human metapneumovirus (hmpv), four human coronaviruses (hco), human rhinovirus (hrv), human adenovirus (hadv) and human bocavirus (hbov), using a real-time pcr assay-kit. viral nucleic acids were extracted from nasal and throat swabs using rna/dna extraction kit «ribo-sorb» (interlabservice, russia) according to the manufacturer's instructions. the extracted viral nucleic acid was immediately used to perform the reaction of reverse transcription using commercial kit "reverta-l" (interlabservice, russia). detection of respiratory viruses, including hpiv 1-4, hrsv, hmpv, hcov-oc43, hcov-229e, hcovnl63, hcov-hku1, hrv, hadv, and hbov was performed using a rt-pcr kit «amplisens arvi-screen-fl» (interlabservice, russia), and ifva and ifvb virus detection was performed using a rt-pcr kit « amplisens influenza virus a/b-fl» (interlabservice, russia) according to the manufacturer's instructions. positive and negative controls were included in each run. two-tailed chi-square test (two by two table) was performed to compare the infection rates for respiratory viruses among different age groups. p-value <0.05 was considered to be statistically significant. totally, 1560 samples collected from patients with aris during the period from december 2013 to april 2017 were enrolled in the investigation. there were 824 males (52.8%) and 736 females (47.2%), and the patient's ages ranged from 3 months to 15 years. the most numerous age group (43.2%) was between 1 and 3 years old. the age distribution is shown in table 1 . the incidence of respiratory viral infection between boys (601/824; 72.9%) and girls (527/736; 71.6%) (χ 2 = 0.345, p>0.05). the respiratory virus positive rate appeared to decrease with age. the lowest positive rate was observed in the age group of more than 6 years old (167/302; 55.3%), while the positive rates in age groups less than 6 years old were more than 70% (table 1 ). statistically significant difference was observed between the age group of more than 6 years old and other age groups (χ 2 = 54.113, p<0.01). no statistically significant difference was observed among the age groups less than 1 year old, 1-3 years, and 4-6 years. among all the samples, single infections accounted for 61.9% (965/1560), while co-infections accounted for 10.4% (163/1560) with the lowest rate of incidence in children more than 6 years old compared to children younger than 6 years (χ 2 = 20.389, p<0.01) (fig 1) . hrsv and ifv were the most frequently detected viruses with high incidence of 23.0% (358/ 1560) and 22.1% (344/1560), respectively, among all patients with aris. hrv was detected in 15.1% (235/1560), followed by hmpv, hpiv and hbov with the detection rates higher than 5.0%. the positivity rates of hcov and hadv were lower than 5.0% (fig 2) . the data were analyzed with regard to the age and gender distribution of virus infection. no difference in the etiological distribution of viral pathogens was observed between males and females. among detected respiratory viruses, hrv, hpiv, hcov, and hadv did not have statistically significant difference in the distribution among the different age groups. hmpv was detected in age group less than 1 year old much more frequently than in children 1-3 years old (χ 2 = 4.986, p<0.05) and 7-15 years old (χ 2 = 8.174, p<0.05). hbov was significantly more frequently observed in children younger than 3 years old compare with children of 4-15 years old (χ 2 = 28.523, p<0.005). the incidence of hrsv decreased significantly with increasing age (p < 0.05) dropping from 35.1% in children younger than 1 year old to 5.3% in the school-age children (7-15 years old group), while the reverse relationship was observed for ifv (fig 3) . the data were analyzed with regard to the seasonality. overall, virus detection positive rate did not differ significantly between seasons, ranging from 71.3% to 73.3%. co-infections with two or more viruses were detected in 163 out of the 1128 (14.5%) positive samples (table 1) . dual infections accounted for 11.4% (129/1128) of all positive samples and acute respiratory infections are a serious health and economic problem, causing high morbidity and significant economic losses due to temporary disability payments and medical costs. children are the most vulnerable group to the development of the disease. as several authors mentioned previously [26] , aris can lead to serious diseases such as bronchiolitis and pneumonia and sometimes even cause death in infants and children worldwide. nevertheless, most of the data on the epidemiological features and etiological structure of aris were from moredeveloped countries, and less is known about the etiology of aris in russia. in the present study, we examined the viral etiology of aris in hospitalized children in novosibirsk by real-time rt-pcr assay. we detected at least one of the tested viruses in 72.3% (1128/1560) of the samples. in similar studies conducted in different regions of the world, the virus detection rate ranged from less than 50% to 75% [27] [28] [29] . for example, in studies performed in china, the proportion of positive samples in children with aris ranged from 37.6% to 78.7% [15, 17, 30, 31] . the previous study of respiratory infections among children in european part of russia revealed the 71.5% detection rate of respiratory viruses [32] . the percentage of the respiratory viruses' detection varies in different years in different regions, which may be associated with climatic and environmental factors, population distribution, economic status and diagnostic methods used [1] . in addition, seasonality of sampling can also lead to differences in the level of viruses' detection in various studies. thus, ju x. et al. carried out a study continuously from july 2011 through july 2013 and found 48.66% of samples to be positive for at least one respiratory virus [33] . in contrast, we collected samples only during epidemic seasons of aris, so the positivity rate in our study was considerably higher. furthermore, acute respiratory infections can be caused by viruses that are not yet known, as well as bacteria that have not been included in these studies [14] . we found that the prevalence of respiratory viruses did not differ between boys and girls (72.9% and 71.6% respectively), which confirms the absence of a gender-based susceptibility to respiratory viral infections [14] . however, we observed a decrease in the respiratory viruses' detection rate with age, with the lowest detection rate in school-age children compared to children under 7 years of age (55.3% versus 76.4%, respectively). these data are consistent with findings obtained in other regions of russia [32] and it may be due to decreased sensitivity to respiratory virus infections in older children. etiology of aris and the respiratory virus prevalence varies in different studies. in the united states, ifv, hrsv and hpiv were the most frequently detected [34] . studies conducted in france have shown that metapneumovirus and respiratory syncytial virus are the most common [35] . ifv, hrsv and hrv were the most commonly detected respiratory viruses among children with aris in most regions of china [14] . the study of aris etiologic structure showed that hrsv, hrv, hpiv and ifv were registered significantly often among children in western part of russia [32] . in our study the most common viruses detected were hrsv and ifv, followed by hrv. the age distribution of aris indicated that children under 3 years old were more likely to be infected by hrsv confirmed the importance of rsv in children with aris, especially in children < 4 years of age [36] [37] [38] [39] . we observed a high rate of hrsv-detections in 2013-2014 (44.4%), while in 2016-2017 it was less than 10% which could be due to the annual variation in the circulation pattern of hrsv. such year-to-year variation in the epidemiological patterns of viral infections confirms importance of the long-term study of the aris epidemiology [40] . influenza virus is one of the major causative agents of respiratory disease in humans and may lead to serious illness [41] . in temperate countries influenza outbreaks usually occur during the winter season. finally, in our study, ifv (344/1560, 22.1%) was the second most frequent detected pathogen with markable seasonality in winter months. during the 2013-2014 epidemic season, influenza virus detection rate was low-6.9% of all respiratory viruses, which was in accordance with the official influenza surveillance results of ministry of health, russia [42] . in our study in 2014-2015, influenza viruses were detected in 17.2% among all respiratory viruses. among those, the percentage of influenza a virus accounted for 73.3% and influenza b virus made up 26.7% of all detected influenza viruses. at the same time, in russia influenza b was the main etiological agent, accounting for 50.6% of all detected influenza viruses. in 2015-2016, influenza a(h1n1)pdm09 virus was predominant in russia, accounting for 79% of all influenza-positive samples consistent with our results (82%). in 2016-2017 influenza a(h3n2) virus was dominant in russia detected in 61.3% of all influenza virus cases [42] . in our study influenza a and b viruses were detected with approximately the same frequency (48% and 52% respectively). with the introduction of molecular techniques, the detection of multiple co-infecting viruses has become common, though the prevalence of each virus varies between studies [43]. in our study detection rate of viral co-infection was 14.5% among the positive samples. according to the previous reports, the incidence of viral co-infection in children can reach 30% [44] . co-infection is most often found in children under the age of 5, due to the immaturity of the immune system and, thus, greater susceptibility to infection [45] . in our study, we significantly more frequently detected cases of simultaneous infection with two or more viruses in children under 7 years of age compared with children of school age (12.2% versus 3.4%), while there was no significant difference in the incidence of viral co-infection between the age groups of 0-1 year, 1-3 years and 4-6 years. in conclusion, in our study we investigated the etiological structure of acute respiratory viral infections in hospitalized children in novosibirsk, russia, and evaluated age and seasonal distribution of the various respiratory viruses. systematic monitoring of respiratory viruses is necessary to better understand the structure of respiratory infections. such studies are important for the improvement and optimization of diagnostic tactics, as well as measures for the control and prevention of the respiratory viral infections. prevalence of respiratory viruses among children hospitalized from respiratory infections in shenzhen, china estimates of world-wide distribution of child deaths from acute respiratory infections role of respiratory viruses in acute upper and lower respiratory tract illness in the first year of life: a birth cohort study multiplex real-time pcr for detection of respiratory tract infections epidemiology of human respiratory viruses in children with acute respiratory tract infections in jinan, china respiratory virus infections in hospitalized children and adults in lao pdr. influenza other respir viruses single and multipathogen viral infections in hospitalized children with acute respiratory infections a newly discovered human pneumovirus isolated from young children with respiratory tract disease ten years of human metapneumovirus research cloning of a human parvovirus by molecular screening of respiratory tract samples identification of a novel coronavirus in patients with severe acute respiratory syndrome the novel human coronaviruses nl63 and hku1 coronaviruses: important emerging human pathogens epidemiological and clinical characteristics of respiratory viral infections in children in shanghai seasonal influenza in adults and children-diagnosis, treatment, chemoprophylaxis, and institutional outbreak management: clinical practice guidelines of the infectious diseases society of america reducing antibiotic use in influenza: challenges and rewards genetic and biological characterization of avian influenza h5n1 viruses isolated from wild birds and poultry in western siberia novel reassortant clade 2.3.4.4 avian influenza a(h5n8) virus in wild aquatic birds monitoring of influenza viruses in western siberia virological evaluation of avian influenza virus persistence in natural and anthropic ecosystems of western siberia high incidence of multiple viral infections identified in upper respiratory tract infected children under three years of age in viral etiology of acute respiratory infection in gansu province, china respiratory virus infections among children in south china aetiology of acute respiratory tract infections in hospitalised children in cyprus plos one epidemiology and seasonality of respiratory viral infections in hospitalized children in kuala lumpur, malaysia: a retrospective study of 27 years molecular epidemiology of respiratory viruses in virus-induced asthma. front microbiol detection and typing by molecular techniques of respiratory viruses in children hospitalized for acute respiratory infection in rome, italy viral aetiology of influenza-like illness in belgium during the influenza a(h1n1)2009 pandemic etiology and seasonality of viral respiratory infections in rural honduran children the epidemiology and etiology of influenza-like illness in chinese children from 2008 to 2010 epidemiological analysis of respiratory viral etiology for influenza-like illness during 2010 in zhuhai the features of arvd etiological structure in different age and professional population groups in saint-petersburg during 2013-2014 epidemic season viral etiology of influenza-like illnesses in huizhou aetiology of influenza-like illness in adults includes parainfluenzavirus type 4 nationwide surveillance of 18 respiratory viruses in patients with influenzalike illnesses. a pilot feasibility study in the french sentinel network viral etiologies of acute respiratory infections among hospitalized vietnamese children in a 3-year prospective study of the epidemiology of acute respiratory viral infections in hospitalized children in shenzhen, china influenza other respir viruses relative frequency, possible risk factors, viral codetection rates, and seasonality of respiratory syncytial virus among children with lower respiratory tract infection in northeastern brazil. medicine (baltimore) respiratory viruses associated hospitalization among children aged< 5 years in bangladesh viral aetiology influenza like illnesses in santa cruz understanding the symptoms of the common cold and influenza on the outcome of the epidemic season of influenza and aris viral co-infections in pediatric patients hospitalized with lower tract acute respiratory infections clinical disease severity of respiratory viral co-infection versus single viral infection: a systematic review and meta-analysis surveillance of 16 respiratory viruses in patients with influenza-like illness in nanjing we thank the clinicians of novosibirsk children's municipal clinical hospital for their assistance with sample collection. we thank dr. galina skosyreva and elena timofeeva (department of propaedeutic of childhood diseases, novosibirsk state medical university, novosibirsk, russia) for their assistance with sample collection. key: cord-332361-pdoln3nr authors: khor, chee-sieng; sam, i-ching; hooi, poh-sim; quek, kia-fatt; chan, yoke-fun title: epidemiology and seasonality of respiratory viral infections in hospitalized children in kuala lumpur, malaysia: a retrospective study of 27 years date: 2012-03-20 journal: bmc pediatr doi: 10.1186/1471-2431-12-32 sha: doc_id: 332361 cord_uid: pdoln3nr background: viral respiratory tract infections (rti) are relatively understudied in southeast asian tropical countries. in temperate countries, seasonal activity of respiratory viruses has been reported, particularly in association with temperature, while inconsistent correlation of respiratory viral activity with humidity and rain is found in tropical countries. a retrospective study was performed from 1982-2008 to investigate the viral etiology of children (≤ 5 years old) admitted with rti in a tertiary hospital in kuala lumpur, malaysia. methods: a total of 10269 respiratory samples from all children ≤ 5 years old received at the hospital's diagnostic virology laboratory between 1982-2008 were included in the study. immunofluorescence staining (for respiratory syncytial virus (rsv), influenza a and b, parainfluenza types 1-3, and adenovirus) and virus isolation were performed. the yearly hospitalization rates and annual patterns of laboratory-confirmed viral rtis were determined. univariate anova was used to analyse the demographic parameters of cases. multiple regression and spearman's rank correlation were used to analyse the correlation between rsv cases and meteorological parameters. results: a total of 2708 cases were laboratory-confirmed using immunofluorescence assays and viral cultures, with the most commonly detected being rsv (1913, 70.6%), parainfluenza viruses (357, 13.2%), influenza viruses (297, 11.0%), and adenovirus (141, 5.2%). children infected with rsv were significantly younger, and children infected with influenza viruses were significantly older. the four main viruses caused disease throughout the year, with a seasonal peak observed for rsv in september-december. monthly rsv cases were directly correlated with rain days, and inversely correlated with relative humidity and temperature. conclusion: viral rtis, particularly due to rsv, are commonly detected in respiratory samples from hospitalized children in kuala lumpur, malaysia. as in temperate countries, rsv infection in tropical malaysia also caused seasonal yearly epidemics, and this has implications for prophylaxis and vaccination programmes. acute respiratory tract infection (rti) is a major cause of morbidity and mortality worldwide, particularly in children [1, 2] . an estimated 1.9 million children die from acute rti every year, with 70% of the mortality occurring in africa and southeast asia [2] . most respiratory tract infections are caused by viruses [3] . respiratory viruses are generally transmitted through inhalation or direct contact with respiratory aerosols or secretions. transmission is often associated with geographic and climatic factors. lower temperatures, lower ultraviolet b radiance, and higher humidity prolong the survival rate of respiratory viruses in the environment [4, 5] . in temperate countries, seasonal activity of respiratory viruses has been reported, particularly in association with temperature [4] . in tropical countries, correlation of respiratory viral activity with climatic factors are not so well defined, which may suggest that more complex interactions are involved [6] . relatively few studies on viral rtis have been conducted in southeast asian countries like malaysia [7] [8] [9] , despite reports from the ministry of health that rti is one of the main causes of hospitalization in malaysia [10] . the epidemiology of respiratory viruses needs to be established to increase the effectiveness of any planned vaccination and prophylaxis programmes. the lack of up-to-date basic epidemiological data on viral rtis in malaysian children needs to be addressed. in this study, we describe etiological agents, demographic details of patients, and seasonality (including association with meteorological factors) due to viral rtis in a teaching hospital in kuala lumpur, over the last 27 years. over a 27-year period (1982-2008) , 10269 samples from children ≤ 5 years were sent for respiratory virus detection, of which 2708 (26.4%) were positive for the common respiratory viruses. the numbers of samples received at the laboratory have been increasing in recent years, from an annual mean of 319 between 1982-1999 to an annual mean of 503 between 2000-2008, as the number of admissions has increased. the mean annual positive rate has been reasonably stable at 25.4% (standard deviation, 8.1%; range, 8.9-38.1%). the viruses detected were rsv (1913, 70.6%), parainfluenza viruses 1-3 (357, 13.2%), influenza a and b viruses (297, 11.0%), and adenovirus (141, 5.2%). the 310 typeable parainfluenza virus cases consisted of 93 (30.0%) parainfluenza-1 virus (piv-1), 23 (7.4%) parainfluenza-2 (piv-2) virus and 194 (62.6%) parainfluenza-3 (piv-3) virus, while a further 47 cases could not be typed by immunofluorescence (if). influenza cases consisted of 233 (78.1%) influenza a and 64 (21.9%) influenza b. coinfections were only detected in 26 cases, with co-infection of rsv and influenza a (n = 6), rsv and piv-1 (n = 2), rsv and piv-3 (n = 7), rsv and adenovirus (n = 3), influenza a and piv-3 (n = 2), influenza a and adenovirus (n = 1), influenza b and piv-1 (n = 1), influenza b and adenovirus (n = 1), piv-3 and adenovirus (n = 1), rsv and influenza a and piv-1 (n = 1). among 792 samples with positive viral isolation, 442 samples were also positive by if, giving an overall if sensitivity of 55.8% (with viral isolation as a gold standard). the demographic data of these children are summarized in table 1 . the mean age of the study population was 1.14 ± 1.06 years old, and 76.2% of the positive cases were seen in children ≤ 1 year old. rsv was by far the commonest identified respiratory virus in children ≤ 6 months, accounting for 81.3% of the positive samples in this age group, but this declined to 56.7% in those aged 1-5 years, respectively. correspondingly, the relative importance of influenza viruses and adenovirus increased with age in the ≤ 6 months to the 1-5 years age groups, from 5.5% to 20.2%, and 2.6% to 8.8%, respectively. the mean ages of children infected by each of the common viruses ranged from 0.96 to 1.51 years, and were significantly different by one-way anova testing (f = 24.632, df = 4, p < 0.05). post-hoc testing showed that children infected with rsv were significantly younger (mean difference = 0.178, se = 0.024, p < 0.05), while children infected with influenza viruses were significantly older than the study population (mean difference = 0.372, se = 0.065, p < 0.05). amongst the positive cases, 59.8% were male and 41.2% were female. overall, the most commonly infected ethnic group were malays (61.5%), followed by chinese (20.6%), and indians (16.0%), reflecting the ethnicities of the patients sampled. there were no significant differences in terms of gender and ethnicity between patients infected with different viruses. due to the relatively small numbers of laboratory-confirmed cases for all other respiratory viruses other than rsv over the study period, particularly in the early years, it was hard to discern multi-year cycles of individual viruses ( figure 1, figure 2 ). in the last decade, most viruses were detected every year, except for piv2. to obtain a clearer picture of seasonality within a year, the cases due to each virus were also aggregated into months ( figure 3 ). disease activity due to the main respiratory viruses was present throughout the year, with yearly peaks of activity. rsv showed the most pronounced seasonality, with peak activity at the year-end (september-december), and lowest activity in mid-year (april-june). piv-1 and piv-3 virus activity was mainly seen in march-may. the number of piv-2 cases was too small to detect any seasonality. influenza a was seen throughout the year, with peak activity in may, while there was more obvious increased influenza b virus activity between november-march. adenovirus activity was present all year-round, with a peak in february-march. since rsv activity showed the clearest seasonal trend, and had the highest number of cases, the association of rsv with climatic factors was further analyzed ( table 2 ). spearman's rank correlation and multiple regression showed direct correlation of monthly rsv cases with rain days and inverse correlation with temperature. additionally, regression analysis identified a further significant inverse correlation of rsv cases with relative humidity. multiple regression showed that a monthly increase of one rain day was associated with a 0.469 increase in monthly rsv cases. however, increases of 1% in relative humidity and 1°c in temperature were associated with 1.070 and 2.426 decreases in rsv cases, respectively. a total of 14.3% of explained variance (r 2 = 0.143) in the number of monthly rsv cases could be attributed to these three factors. other climatic factors were not significantly associated with rsv cases. the malaysian ministry of health reports that respiratory infections are one of the principal causes of hospitalization (9.4% in 2009), and pneumonia is one of the ten principal causes of death (10.4% in 2009) in public hospitals [10] . however, as diagnostic capacity for respiratory viruses is extremely limited, little is known about the epidemiology of viral rtis in malaysia, which are well known to have high financial and clinical impact [11] [12] [13] . this retrospective study of respiratory viruses at a teaching hospital over the past 27 years is the most comprehensive study carried out in malaysia. our findings support a previous local study carried out over a year, which showed that rsv was the most commonly detected respiratory virus, followed by parainfluenza viruses, influenza viruses and adenovirus [7] . we sd, standard deviation * p < 0.05 ** of the 10269 total number of samples received, 85 and 56 were missing gender and ethnicity data, respectively. found that this trend has remained for the past three decades. in our laboratory, the average virus detection rate (annually) by if and/or virus isolation was 25.4% in all respiratory samples tested. this considerably underestimates the true burden of respiratory viruses, as lack of resources precluded the routine use of more sensitive diagnostic methods such as shell vial culture [14] and molecular detection methods, and testing of a wider range of viruses. respiratory virus detection rates of more than 50% can be achieved with molecular detection [15, 16] . other studies in tropical countries have reported that emerging respiratory viruses such as metapneumovirus (5.3-5.4%) [17, 18] , coronaviruses (0.6%) [19] , bocavirus (8.0%) [19] and human rhinovirus c (12.8-30%) [20, 21] also contribute substantially to morbidity. most studies, including those in asia, show that the most common causes of respiratory viral infections are rsv and rhinoviruses [3, [22] [23] [24] . our laboratory does not routinely detect rhinoviruses, but rsv was the most frequently detected respiratory virus, particularly in infants less than one year old. this suggests that maternal antibodies were ineffective in preventing rsv infections. older children may be less prone to rsv infection due to the maturity of their immune system or natural immunity obtained through repeated infections of rsv. the burden of rsv in young infants in tropical countries, including malaysia, emphasizes the likely global benefits of developing a safe and effective vaccine for rsv. our data also showed a slight male preponderance (59.8%) in children with respiratory viral infections, consistent with other studies [25] . in temperate countries, respiratory viral infections have clear seasonal variations with most cases occurring during winter. possible explanations for this include seasonal variations in host immune response to infection [26] , climatic factors such as ambient temperature and low relative humidity which increase viral survival in the environment [27] , and changes in host behaviour which increase contact with others. seasonal trends are more variable in the tropics, with some studies showing that respiratory virus infections occur all year round, while some show clearer seasonality. located in southeast asia, kuala lumpur (latitude 3°n ) has a mean annual temperature of 27.4°c, constant high relative humidity (> 71.6%), and heavy rainfall throughout the year. in our study, the common respiratory viruses were detected throughout the year. the rsv annual epidemics are strongly seasonal, while seasonality is less clear for influenza, parainfluenza, and adenovirus, which may be due to the smaller number of cases. for adenovirus, most studies show sporadic occurrence without any seasonal trend [28, 29] . in the tropics, influenza occurs all year-round [29] , similar to our data. in singapore, parainfluenza virus type 3 was the most commonly detected parainfluenza virus type, with seasonal peaks in february-march similar to our results [29] . in kuala lumpur, rsv peaks at the end of the year. in contrast, in neighbouring singapore (latitude 1°n) and lombok, indonesia (latitude 8°s), rsv peaks around march-august [29, 30] . in most rsv studies carried out in temperate countries, where temperature varies widely between seasons, temperature is highly inversely correlated to rsv cases [4, 31] . we also found a negative correlation between rsv and temperature in tropical kuala lumpur, where the much reduced temperature variation may explain the weaker correlation (correlation coefficient of -0.116). as previously observed in indonesia [32] , we also found that the number of rain days was significantly associated with rsv cases, but not rainfall. malaysia experiences brief, intense showers of rainfall, as well as prolonged episodes of light rainfall. therefore, the number of rain days may have a greater influence than the absolute amount of rainfall on behaviours such as children staying indoors, thus increasing close contact and indoor transmission of respiratory viruses [33] . while humidity was inversely correlated with rsv in our study, as with respiratory infections in singapore [5] , a positive correlation was reported in lombok, indonesia [32] . there are well recognized inconsistencies in reported associations of respiratory infections with meteorological factors in different settings [34] . clearly, seasonality of respiratory viruses in the tropics cannot be explained by climatic factors alone, as associations vary widely between geographic locations [35] . there are likely to be multiple, poorly understood interactions between climatic, environmental and behavioural factors, and complex interplay between different circulating viruses and population immunity. the local epidemiology of respiratory viruses needs to be determined for each site, for effective planning of interventions such as potential vaccines. this study has several limitations. as the data was collected retrospectively, there was some missing data. we were unable to obtain dates of admission for the earlier patients, and thus could not differentiate between nosocomial and community-acquired viral infections. however, we have previously found that nosocomial cases made up 25/157 (15.9%) influenza cases from 2002-2007 [13] and 17/146 (11.6%) rsv cases from 1989-2010 (khor cs, sam ic and chan yf, unpublished observations). these nosocomial rates of respiratory viral infections are similar to previously published rates of 12.1-13.8% [36, 37] , thus supporting the likelihood that the majority of infections seen in our study were community-acquired. over 27 years, there may have been changes in physicians' practices, such as criteria for taking specimens and admitting patients, and types of samples collected. furthermore, with a relatively low if sensitivity of 55.8% using virus isolation as the gold standard, there is likely to be considerable underdiagnosis of viral infections compared to molecular methods [38] . nevertheless, despite these limitations, these unusually extensive records kept over 27 years provide valuable insight into current and historical respiratory virus epidemiology in a tropical southeast asian country, particularly with the limited available facilities for virus diagnosis. to extend these findings, we are currently carrying out prospective studies using molecular methods to detect a wider range of respiratory viruses. respiratory viral infections due to rsv, parainfluenza viruses, influenza viruses and adenovirus are significant causes of morbidity in hospitalized children in kuala lumpur, and are likely to be underdiagnosed. the most common cause of viral rti was rsv, which causes annual seasonal epidemics and predominantly infects young infants ≤ 6 months. further studies, both hospital-based and population-based, are required in malaysia to fully understand the clinical and community impact of respiratory viruses in children. university malaya medical centre (ummc) is a 900bed tertiary hospital in kuala lumpur, the capital of malaysia. in this retrospective study, we analysed records of all respiratory samples from hospitalized children aged ≤ 5 years sent to the hospital's diagnostic analysis was carried out with spss v16.0 (spss inc., chicago, usa). demographic factors such as gender, age, and ethnicity were analyzed by univariate anova. levene's test, brown-forsythe test and welch test were done to confirm the homogeneity of variance across samples. since the data variance is unequal, the games-howell test was selected as the post hoc test to compare cases with each virus type to the population separately. the seasonal trends for each respiratory virus were evaluated. as the number of rsv cases was highest, rsv activity was further analyzed to determine its associations with meteorological factors such as rain days, relative humidity, temperature, pressure, rainfall, and ultraviolet radiance. meteorological data were provided by the malaysian meteorological department, from two weather stations located in petaling jaya and subang jaya, about 5-15 km away from the hospital. the monthly data from both stations were averaged before correlation with monthly rsv cases. spearman's rank correlation and stepwise multiple regression analysis and were performed with rsv cases as the dependant variable, and the climatic factors as independent variables. a p value of < 0.05 was considered significant. the smaller numbers of cases precluded similar analyses for the other respiratory viruses. hannah moore 1. the methods section needs to follow the background section and not be placed at the end of the manuscript. emerging respiratory agents: new viruses for old diseases? estimates of worldwide distribution of child deaths from acute respiratory infections respiratory viral infections in infants: causes, clinical symptoms, virology, and immunology the relationship of meteorological conditions to the epidemic activity of respiratory syncytial virus correlations between clinical illness, respiratory virus infections and climate factors in a tropical paediatric population influenza seasonality: underlying causes and modeling theories respiratory viruses detected in hospitalised paediatric patients with respiratory infections viral agents of acute respiratory infections in young children in kuala lumpur seasonal variation in respiratory syncytial virus chest infection in the tropics health facts cost of hospitalization for respiratory syncytial virus chest infection and implications for passive immunization strategies in a developing nation high direct healthcare costs of patients hospitalised with pandemic (h1n1) 2009 influenza in malaysia clinical features of malaysian children hospitalized with community-acquired seasonal influenza prospective comparison of r-mix shell vial system with direct antigen tests and conventional cell culture for respiratory virus detection surveillance of eight respiratory viruses in clinical samples of pediatric patients in southeast brazil population-based surveillance for hospitalizations associated with respiratory syncytial virus, influenza virus, and parainfluenza viruses among young children human metapneumovirus in children human metapneumovirus in infants and young children in thailand with lower respiratory tract infections; molecular characteristics and clinical presentations the incidence of human bocavirus infection among children admitted to hospital in singapore high prevalence of human rhinovirus c infection in thai children with acute lower respiratory tract disease human rhinovirus group c in hospitalized children viral pathogens associated with acute respiratory infections in central vietnamese children the association of newly identified respiratory viruses with lower respiratory tract infections in korean children respiratory viruses in acute respiratory tract infections in western india respiratory syncytial virus infection in tropical and developing countries epidemic influenza and vitamin d absolute humidity modulates influenza survival, transmission, and seasonality viral etiology and epidemiology of acute lower respiratory tract infections in korean children seasonal trends of viral respiratory tract infections in the tropics ingerani: incidence and clinical features of hospitalization because of respiratory syncytial virus lower respiratory illness among children less than two years of age in a rural asian setting are meteorological parameters associated with acute respiratory tract infections? climatic, temporal, and geographic characteristics of respiratory syncytial virus disease in a tropical island population bridges cb: rainfall, household crowding, and acute respiratory infections in the tropics seasonality -still confusing the relationship of meteorological conditions to the epidemic activity of respiratory syncytial virus virus cross-infection in paediatric wards nosocomial respiratory syncytial virus infection: impact of prospective surveillance and targeted infection control comparative study of nasopharyngeal aspirate and nasal swab specimens for diagnosis of acute viral respiratory infection the authors would like to thank the diagnostic virology laboratory of university malaya medical centre for providing laboratory records; and the malaysian meteorological department for providing the meteorological data. we have submitted the manuscript in accordance with the bmc pediatrics instructions for authors, which asks for the methods section to be placed after the conclusions section (http://www.biomedcentral.com/bmcpediatr/authors/instructions/researcharticle).2. table 1 column headings can still be improved. column headers should include "n(%)" included in the table. 3. i believe it would be useful to display data < 6 mths and 6-12 mths, however i will leave that decision to the associate editor we agree that the age group data would make the paper more useful. the data for < 6 mths and 6-12 mths have been included in table 1. 4. figure 3 is useful but is difficult to see in grayscale. it needs to be produced in colour or different shading patterns are required. it appears that adenovirus testing did not occur until 1991. if that is so, this needs to be described in results. we have provided figure 3 in colour. adenovirus testing has been performed since 1982, and in fact figure 3 does show that adenovirus was detected in very low numbers in 1985, 1987 and 1990. we have modified the colour of the adenovirus bars to make it clearer. 5 . results text describing the increase of number of samples over the years and the positive detection rate remaining stable at 25.4 +/-8.1%, needs to be described more clearly. presumably this is overall rate and standard deviation? a range of the virus positivity rate between the years would be more useful. we have clarified that 8.1% is the standard deviation, and added the range to the sentence, as suggested. we hope that it is now clearer."quality of written english: needs some language corrections before being published" the manuscript has been reviewed and corrected by the two main authors who conceived the study (ics and yfc), who are both native english speakers. furthermore, ics received his secondary, university and postgraduate education in english in the united kingdom. reviewers this article was reviewed by hong yan zhang and hannah moore (both nominated by associate editor dat tran). open peer review reviewed by hong yan zhang and hannah moore (both nominated by associate editor dat tran). for the full reviews, please go to the reviewers' comments section. the study was conceived by yfc and ics, who supervised csk in study design and data collation. psh collected the original diagnostic laboratory data. kfq planned the statistical analysis. all authors were involved in analysis of data and writing of the manuscript. all authors have read and approved the final manuscript. the authors declare that they have no competing interests. key: cord-332926-8hdkpkgz authors: meissner, h. cody title: bronchiolitis date: 2017-07-18 journal: principles and practice of pediatric infectious diseases doi: 10.1016/b978-0-323-40181-4.00033-5 sha: doc_id: 332926 cord_uid: 8hdkpkgz nan bronchiolitis is a syndrome of inflammation and obstruction of the lower respiratory tract that usually is caused by a viral infection occurring in the first 12 months of life. 1 between 2% and 3% of infants younger than 1 year of age are hospitalized with bronchiolitis annually, accounting for 57,000 to 175,000 hospitalizations in the united states. [2] [3] [4] yearly hospital admissions attributable to bronchiolitis increased more than twofold between 1980 and 1996, likely reflecting the increased use of childcare centers and changes in criteria for hospital admission for children with bronchiolitis. 5 however, during the first 16 years of the 21st century, bronchiolitis hospitalization rates have fallen by 20% to approximately 20 to 30 admissions per 1000 children younger than 12 months of age. 6 one explanation for lower rates of bronchiolitis hospitalization is the improved health of neonates at the time of discharge compared with several years earlier. this can be attributed to the use of antenatal glucocorticoids, surfactant replacement, improvements in methods for ventilatory support, corrective cardiac surgery performed earlier in life, and a better understanding of neonatal nutrition. during seasonal outbreaks of bronchiolitis from november through march in north america, respiratory syncytial virus (rsv) is identified as the etiologic agent in up to 80% of hospitalized children 7 (table 33 .1). other respiratory viruses that cause a largely indistinguishable syndrome include human rhinoviruses, parainfluenza viruses, human metapneumoviruses, coronaviruses, adenoviruses, influenza viruses, enteroviruses, and human bocaviruses. [8] [9] [10] [11] the yearly cycles of respiratory virus circulation are depicted in fig. 33.1 . no evidence has been found for a primary role for bacteria as a cause of bronchiolitis. however, bordetella pertussis, chlamydia trachomatis, and mycoplasma pneumoniae should be included in the differential diagnosis of a young child with lower respiratory tract infection. the peak incidence of severe disease occurs before 6 months of age, and the highest rates of hospitalization occur among infants between 60 and 90 days of age. 1 most of those hospitalized for bronchiolitis are term infants with no known risk factors. chronologic age is the single most important determinant for severe bronchiolitis; about two thirds of hospitalizations occur before 6 months of age. 1,2 several reasons may account for this age distribution. birth shortly before or soon after the onset of the rsv season results in a longer period of exposure to rsv earlier in life. maternal antibody concentrations to rsv also show seasonal variation, and infants born early in the rsv season are more likely to be born to mothers with low neutralizing antibody concentrations. [12] [13] [14] certain comorbidities, including prematurity (<29 weeks' gestation), chronic lung disease of prematurity, and hemodynamically significant congenital heart disease, may result in more severe disease compared with children without these comorbities. 15, 16 worldwide, 65,000 to 200,000 deaths due to bronchiolitis were estimated to occur in 2005 20 20 week no. number of infections among children younger than 5 years of age. 17, 18 fewer than 100 us pediatric deaths occur annually as a complication of rsv-associated bronchiolitis. 19 occurrence of the respiratory virus season is predictable even though the severity of the season, the date of onset, the peak of activity, and the end of the season cannot be predicted with precision. 20 there can be variation in timing of community outbreaks of disease due to rsv from year to year in the same community and among neighboring communities, even in the same season. southern us communities tend to experience the earliest onset of rsv activity, and those in the midwest tend to experience the latest onset. the duration of the season in western and northeastern areas is typically between that in the south and the midwest. nevertheless, these variations occur within the overall pattern of rsv outbreaks, usually beginning in november or december, peaking in january or february, and ending by the end of march or april. [20] [21] [22] [23] limited numbers of cases of bronchiolitis may occur during late spring, summer, and early fall, and they often are caused by viruses other than rsv, such as rhinoviruses or parainfluenza viruses. these cases often are milder than rsv-related cases. in tropical countries, the annual epidemic of rsv coincides with the rainy season, although cases can occur throughout the year. household crowding is a risk factor for severe viral lower respiratory tract illness due to rsv and other respiratory viruses. [24] [25] [26] [27] as the number of household members increases, the likelihood of close exposure to infectious respiratory secretions also increases. childcare attendance has been correlated with an increased risk of bronchiolitis in some studies. unlike other respiratory viral infections, exposure to passive household tobacco smoke has not been associated with an increased risk of rsv hospitalization on a consistent basis. exposure to secondhand smoke has been associated with an increased risk of respiratory infections part ii clinical syndromes and cardinal features of infectious diseases: approach to diagnosis and initial management hemodynamically significant congenital heart disease are at increased risk of more severe bronchiolitis. 15, 16, 33, 34 infants with hemodynamically insignificant heart disease, including secundum atrial septal defect, small ventricular septal defect, pulmonic stenosis, uncomplicated aortic stenosis, mild coarctation of the aorta, and patent ductus arteriosus, and infants with lesions adequately corrected by surgery (unless they continue to require medication for management of congestive heart failure) are not considered to be at increased risk for hospitalization. 35 severe respiratory distress with bronchiolitis can be the presenting manifestation of previously unrecognized congenital heart disease. rsv-infected infants have various courses after hospitalization. 22 among otherwise healthy infants, intensive care unit admission because of respiratory deterioration is uncommon (approximately 2% of patients). the decision to admit to the intensive care unit is based on the possible need for intubation because of progressive hypercapnia, increasing hypoxemia despite supplemental oxygen, or episodes of apnea. the typical course for a previously healthy infant older than 6 months of age is one of improvement over 2 to 3 days, as evidenced by a lower respiratory rate and fewer retractions. pulmonary function abnormalities and evidence of mild desaturation may persist for several weeks. the differential diagnosis of bronchiolitis includes airway hypersensitivity to environmental irritants, anatomic abnormality of the airway, cardiac disease with pulmonary edema, cystic fibrosis, foreign-body aspiration, and gastroesophageal reflux. a diagnosis of bronchiolitis should be based on the history and physical examination. serial examinations may reveal fluctuations in disease acuity over a short period, reflecting rapid changes in blockage of the lumens of the small airways. 1 specific signs and symptoms at the time of presentation have a limited ability to predict disease severity. assessment of the likelihood of progressive disease should consider the risk factors, including: age younger than 12 weeks, a history of prematurity, underlying cardiopulmonary disease, or immunodeficiency. 1 routine radiographic studies are not recommended. infants with bronchiolitis may have abnormalities (e.g., atelectasis) on chest radiography, but the changes have little correlation with disease severity. 36, 37 radiography should be reserved for infants who require intensive care management and for those who do not improve as expected. routine virologic testing is not recommended because the result is unlikely to influence management. 1 respiratory isolation recommendations are similar for most viral infections and are based largely on the symptoms, not the specific cause. the positive predictive value of an antigen detection assay decreases as disease incidence goes down. specificity of antigen detection assays is lowest during the off-season and at the onset and the end of the respiratory virus season. polymerase chain reaction (pcr) assays have improved sensitivity and specificity compared with antigen detection, but they are expensive, and results are unlikely to influence management. using pcr assays, coinfections with more than one virus have been identified in as many as one third of hospitalized patients with bronchiolitis. the significance of coinfections is incompletely understood. [38] [39] [40] [41] [42] pcr assays should be interpreted with caution because positive results can be found in more than 30% of asymptomatic young children. 43 whether this reflects prolonged shedding from a previous infection, the incubation period for a pending infection, infection by a strain with limited ability to cause disease, or a low-grade infection producing small amounts of virus is unknown. most infants with bronchiolitis can be managed at home with supportive care, but concerns regarding increasing respiratory effort, apnea, or inability to feed adequately may precipitate hospitalization of a young child. 1 disease severity may be estimated by respiratory rate, use of accessory muscles, and degree of hypoxemia. however, the course of bronchiolitis varies, ranging from mild dyspnea to progressive respiratory distress. certain underlying conditions such as prematurity, immunodeficiency, chronic lung disease of prematurity, and congenital due to other viruses. in contrast to the well-documented benefit of breastfeeding against some viral illnesses, existing data are conflicting regarding the specific protective effect of breastfeeding against rsv infection. other reported risk factors for bronchiolitis include poverty, malnutrition, maternal smoking during pregnancy, congenital malformation of the airways, neuromuscular impairment, household crowding, childcare attendance, size of the childcare facility, lower level of maternal education, and living at an increased altitude. however, most of these risk factors are inconsistent from study to study, and in most instances, the impact on increased hospitalization is small. 15 acute bronchiolitis is caused by an infectious agent, usually a respiratory virus with specific tropism for bronchiolar epithelium. because most infants experience mild illness and recover from bronchiolitis, information regarding the pathologic changes caused by infection is not available. largely based on results from animal studies, rsv infection of the epithelial cells of the human respiratory tract mucosa results in a lymphocytic infiltration of the bronchiolar walls and edema of the surrounding tissue. disease progression is associated with proliferation and necrosis of the bronchiolar epithelium. the sloughed necrotic epithelium and the increased mucus production lead to obstruction of the lumen of the infant's small airways. air movement is restricted during inspiration and expiration but is more restricted during expiration, when the lumen is further compromised by positive expiratory pressure, resulting in expiratory wheezing. obstruction results in air trapping and the characteristic appearance of hyperinflation on chest radiographs. as this air is absorbed, the radiographic pattern evolves to show atelectasis. 23, [27] [28] [29] chapter 225 discusses the pathophysiology of rsv bronchiolitis. upper respiratory tract symptoms consisting of nasal congestion and discharge and a low-grade fever begin about 3 to 5 days after the onset of infection. bronchiolitis represents the later stage of a respiratory viral infection that develops after 2 to 4 days of illness. approximately 30% to 40% of rsv-infected infants experience progression of disease to the lower respiratory tract. spread to the lower airway occurs by aspiration of sloughed rsv-infected epithelial cells from the upper airway or by cell-to-cell spread of the virus. lower airway involvement is marked by an increase in the work of breathing, cough, tachypnea, wheezing, crackles, use of accessory muscles, and nasal flaring. 1 the respiratory rate often exceeds 60 to 70 breaths/ min in young infants. intercostal, supracostal, and subcostal retractions are evident. initially, wheezing occurs during the expiratory phase only and is audible only through a stethoscope. as wheezing progresses, it can be heard without a stethoscope. the chest becomes hyperexpanded and hyperresonant, respirations become more labored, and retractions become more severe. mild hypoxemia occurs in most infants with bronchiolitis. respiratory failure can occur due to progressive hypercapnia and respiratory muscle fatigue. 1 disease severity may be recognized by the absence of audible air exchange on auscultation; flaring of the alae nasi; expiratory grunting; severe subcostal, supraclavicular, and intercostal retractions; and hypoxemia. a child with these findings may require intubation and ventilatory support. apnea can be an early manifestation of rsv infection, sometimes resulting in respiratory failure. [30] [31] [32] because the severity of bronchiolitis often waxes and wanes before consistent improvement, serial assessment of respiratory status should be performed. the ability of a young infant to breastfeed or bottle-feed without distress over time often provides a practical guide to disease severity and management. an infant who has substantial difficulty feeding as a result of respiratory distress has moderate or severe illness and usually requires hospitalization. otherwise healthy infants younger than 2 months of age, infants born prematurely (<29 weeks′ gestation), and infants with chronic lung disease of prematurity or infants born with congenital heart disease are most likely to experience severe rsv disease. 1 infants born with infection. these studies were challenged on the basis that control groups received water aerosols, which can produce bronchospasm in individuals with hyperreactive airways. clinical trials with ribavirin have not demonstrated a consistent decrease in the need for mechanical ventilation, decrease in length of stay in the intensive care unit, or reduction in days of hospitalization. conflicting results from efficacy trials, concern about the potential toxic effects among exposed healthcare professionals, aerosol route of administration, and high cost have resulted in the limited use of ribavirin. 63 chapter 225 discusses ribavirin. options for the treatment of bronchiolitis if caused by influenza a or b viruses are discussed in chapter 229. most infants recover completely from acute bronchiolitis. severe bronchiolitis early in life is associated with an increased risk of asthma, especially after rsv or rhinovirus bronchiolitis, and the risk can persist into early adulthood. it is not understood whether bronchiolitis injures the lung such that normal lung development does not occur and predisposes to subsequent episodes of wheezing or whether certain infants have a pre-existing aberration of the immune response or airway function that predisposes to severe bronchiolitis and recurrent wheezing. [64] [65] [66] [67] [68] [69] strategies that reduce exposure of vulnerable infants to contagious individuals with respiratory tract infections offer opportunities to reduce bronchiolitis morbidity. encouraging breastfeeding and avoiding passive exposure to cigarette smoke are critical aspects of proper healthcare for all children. the role of monoclonal antibodies in reducing the risk of rsv infection in certain high-risk infants and young children is discussed in chapter 225. no safe and effective vaccine is available to prevent infection with rsv or most other viral causes of bronchiolitis. the influenza vaccine is the only exception, and this vaccine should be administered annually to all infants starting at 6 months of age. because influenza vaccine is not approved for use in infants younger than 6 months of age, annual influenza vaccination is important for the family members and caregivers of young patients. all references are available online at www.expertconsult.com. heart disease are associated with an increased risk of progression to severe disease. despite the high burden of disease from bronchiolitis, no available treatment shortens the course or hastens the resolution of symptoms. therapy is supportive, and most children with bronchiolitis do well regardless of management. 1 therapies bronchodilator therapy. most randomized, controlled trials have failed to demonstrate a consistent benefit from αor β-adrenergic agents. although transient improvement in respiratory status has been reported, most infants treated with bronchodilators do not benefit from their use. [44] [45] [46] [47] the potential adverse effects (i.e., tachycardia and tremors) and cost of these agents outweigh the potential benefits. recommendations from the american academy of pediatrics state, "clinicians should not administer albuterol to infants and children with a diagnosis of bronchiolitis." 1 epinephrine. epinephrine is an adrenergic agent with αand β-receptor agonist activity. it has been evaluated in children with bronchiolitis after systemic administration and when delivered directly into the respiratory tract. large, multicenter trials comparing nebulized epinephrine with albuterol or placebo in hospitalized children with bronchiolitis found no improvement in outcomes. [48] [49] [50] available evidence does not support a role for epinephrine use among hospitalized patients or outpatients with bronchiolitis. corticosteroid therapy. although corticosteroids may benefit selected patients with asthma or croup, no evidence supports the use of corticosteroid therapy in the general population of children with bronchiolitis. numerous studies have documented a lack of benefit. [51] [52] [53] oxygen therapy. various degrees of hypoxia are commonly seen in infants and young children with bronchiolitis. intermittent periods of hypoxemia (≥90% oxyhemoglobin saturation) as measured by pulse oximetry should not be interpreted as a need for supplemental oxygen. transient desaturation is common in healthy infants. oxygen saturation has much less impact on respiratory drive than carbon dioxide concentration in the blood. oxygen saturation and respiratory distress have poor correlation among infants with lower respiratory tract infections. continuous monitoring with pulse oximetry of children with mild degrees of oxygen desaturation has been associated with prolonged length of stay compared with children who do undergo continuous monitoring. 54, 55 chest physiotherapy. suctioning of the nasopharynx to remove secretions and temporarily improve airflow is a common practice for children with bronchiolitis. chest physiotherapy using vibration or percussion or deep suctioning is not recommended. 56, 57 hydration. as many as one third of patients admitted to the hospital with a diagnosis of bronchiolitis require fluid replacement therapy. the ability to swallow may be compromised when the respiratory rate exceeds 60 breaths/min, especially if the child has nasal congestion. for infants who are unable to maintain hydration orally, fluid may be administered intravenously or through a nasogastric tube. 58, 59 antibiotic therapy. antibacterial therapy should not be administered to an infant or young child with bronchiolitis unless there is a strong suspicion of a concomitant bacterial infection. 1 nebulized hypertonic saline. data suggest that nebulized hypertonic saline may have a modest benefit in infants with rsv bronchiolitis. [60] [61] [62] hypertonic saline may facilitate mucociliary clearance of the bronchioles that results from the inflammatory response to infection. some evidence suggests that 3% inhaled saline may be safe and may reduce length of hospital stay when a hospitalization lasts longer than 3 days. antiviral therapy. ribavirin is a nucleoside analogue with in vitro activity against rsv, adenovirus, influenza a and b viruses, and parainfluenza viruses. early trials indicated that ribavirin therapy was associated with a modest improvement in clinical scores, oxygenation, and duration of mechanical ventilation for infants with severe bronchiolitis due to rsv clinical practice guideline: the diagnosis, management and prevention of bronchiolitis rsv-associated hospitalization among children less than 24 months prospective multicenter study of viral etiology and hospital length of stay in children with severe bronchiolitis technical report: updated guidance for palivizumab prophylaxis among infants and young children at increased risk of hospitalization for respiratory syncytial virus infection polymicrobial acute respiratory infections in a hospital based pediatric population bronchodilators for bronchiolitis nebulized hypertonic saline for acute bronchiolitis in infants nebulized hypertonic saline for acute bronchiolitis: a systematic review rhinovirus wheezing illness and genetic risk of childhood-onset asthma the microbiology of asthma clinical practice guideline: the diagnosis, management and prevention of bronchiolitis the burden of rsv infection in young children rsv-associated hospitalization among children less than 24 months rsv-associated hospitalizations among infants and young children in the united states bronchiolitis associated hospitalizations among u.s. children, 1980-1996 trends in bronchiolitis hospitalizations in the united states community-acquired pneumonia requiring hospitalization among united states children prospective multicenter study of viral etiology and hospital length of stay in children with severe bronchiolitis multicenter study of viral etiology and relapse in hospitalized children with bronchiolitis epidemiology of multiple respiratory viruses in childcare attendees polymicrobial acute respiratory infections in a hospital based pediatric population rsv transplacental antibody transfer and kinetics in mother-infant pairs in bangladesh the role of neutralizing antibody in protection of american indians against rsv disease maternal rsv antibody titers: season and children matter technical report: updated guidance for palivizumab prophylaxis among infants and young children at increased risk of hospitalization for respiratory syncytial virus infection palivizumab prophylaxis reduces hospitalization due to rsv in young children with hemodynamically significant congenital heart disease global burden of acute lower respiratory infections due to respiratory syncytial virus in young children: a systematic review and meta-analysis rsv circulation in seven countries with global disease detection regional centers rsv-associated mortality in hospitalized infants and young children the relationship of meteorological conditions to the epidemic activity of rsv bronchiolitis: recent evidence on diagnosis and management respiratory syncytial virus and parainfluenza virus environmental and demographic risk factors for rsv lower respiratory tract disease the unresolved issue of risk factors for hospitalization of infants with rsv infection born after 33-35 weeks gestation review of epidemiology and clinical risk factors for severe rsv infection progress in understanding and controlling rsv: still crazy after all these years severe human lower respiratory tract illness caused by rsv and influenza virus is characterized by the absence of pulmonary cytotoxic lymphocyte responses feigin and cherry's textbook of pediatric infectious diseases apnea in children hospitalized with bronchiolitis incidence of apnea in infants hospitalized with respiratory syncytial virus bronchiolitis: a systematic review identifying hospitalized infants who have bronchiolitis and are at high risk for apnea low incidence of rsv hospitalizations in hemodynamically significant congenital heart disease palivizumab in congenital heart disease: should international guidelines be revised? report of the committee on infectious diseases randomised controlled trial of clinical outcome after chest radiograph in ambulatory acute lower-respiratory infection in children evaluation of the utility of radiography in acute bronchiolitis human metapneumovirus infection in young children hospitalized with acute respiratory tract disease: virologic and clinical features polymicrobial acute respiratory infections in a hospital based pediatric population multi-pathogen infections in hospitalized children with acute respiratory infections frequency of detection of picornaviruses and seven other respiratory pathogens in infants viruses in community acquired pneumonia in children aged less than 3 years old: high rates of viral co-infections frequent detection of respiratory viruses without symptoms: toward defining clinically relevant cutoff values pharmacologic treatment of bronchiolitis in infants and children: a systematic review acute viral bronchiolitis in children: a very common condition with few therapeutic options comparison of nebulized epinephrine to albuterol in bronchiolitis bronchodilators for bronchiolitis epinephrine and dexamethasone in children with bronchiolitis a multicenter, randomized, doubleblind, controlled trial of nebulized epinephrine in infants with acute bronchiolitis racemic adrenaline and inhalation strategies in acute bronchiolitis bronchiolitis study group of the pediatric emergency care applied research network (pecarn). a multicenter, randomized, controlled trial of dexamethasone for bronchiolitis oral dexamethasone for bronchiolitis: a randomized trial glucocorticoids for acute viral bronchiolitis in infants and young children effect of oxygen supplementation on length of stay for infants hospitalized with acute viral bronchiolitis observational study of two oxygen targets for discharge in bronchiolitis chest physiotherapy using passive expiratory techniques does not reduce bronchiolitis severity: a randomised controlled trial evaluation of an alternative chest physiotherapy method in infants with respiratory syncytial virus bronchiolitis intravenous fluids versus gastric-tube feeding in hospitalized infants with viral bronchiolitis: a randomized, prospective pilot study nasogastric hydration versus intravenous hydration for infants with bronchiolitis: a randomised trial hypertonic saline or high volume normal saline for viral bronchiolitis: mechanisms and rationale nebulized hypertonic saline for acute bronchiolitis in infants nebulized hypertonic saline for acute bronchiolitis: a systematic review ribavirin for respiratory syncytial virus infection of the lower respiratory tract in infants and young children rhinovirus wheezing illness and genetic risk of childhood-onset asthma the microbiology of asthma lung function prior to viral lower respiratory tract infections in prematurely born infants diminished lung function as a predisposing factor for wheezing respiratory illness in infants diminished lung function, rsv infection and respiratory morbidity in prematurely born infants lung function in prematurely born infants after viral lower respiratory tract infections key: cord-349560-8n65rgfz authors: kleines, michael; häusler, martin; krüttgen, alexander; scheithauer, simone title: wu polyomavirus (wupyv): a recently detected virus causing respiratory disease? date: 2009-11-04 journal: viruses doi: 10.3390/v1030678 sha: doc_id: 349560 cord_uid: 8n65rgfz the wu polyomavirus (wupyv) is a novel member of the family polyomaviridae recently detected in respiratory tract specimens by shotgun sequencing. intriguingly, viral genome has been detected in 0.4% to 11.5% of respiratory tract specimens from children with respiratory disease. the levels of co-infection with established respiratory viruses were in the range between 30.8% and 91.7%. moreover, some studies report detection of wupyv in stool or serum. so far, wupyv infections can not be distinguished from other viral infections by means of clinical symptoms. respiratory tract disease like pneumonia or bronchitis is frequently observed in patients harbouring wupyv. detection of viremia suggests systemic infections. however, the available data do not prove wupyv to be a human pathogen. further investigations are necessary. respiratory tract infections are a major cause of hospital admission in infants and young children. they are caused by a broad spectrum of established respiratory tract pathogens, particularly by viruses including the respiratory syncytial virus (rsv), the most important cause of severe respiratory tract infections in young children, influenza viruses, parainfluenza viruses, rhinoviruses, coronaviruses (229e, oc43), and adenoviruses. studies investigating the entire spectrum of established respiratory tract pathogens are rare and mostly based on the detection of pathogen specific antibodies. only in recent years, a few studies became available presenting data based on the direct detection of relevant infectious agents. creer and co-workers [1] identified at least one potential pathogen in 69% of specimens from adults suffering from lower respiratory tract infections (lrti, 63% contained viruses, 26% contained bacteria) still leaving a significant diagnostic gap to be filled with so far unidentified pathogenic microorganisms. additional agents associated with respiratory tract infections have been discovered recently, e.g. the human metapneumovirus (hmpv) [2] , coronaviruses (nl63, hku1) [3, 4] , and the human bocavirus (hbov) [5] . furthermore, herpes viruses may contribute to fill this gap as they are assumed to trigger respiratory tract infections [6, 7] . unfortunately, relevant studies did not often include them. by now, the pathogenic impact of the human metapneumovirus and its relevance on respiratory tract infections has been well established. it is a common cause of upper (urti) and lower respiratory tract infections (lrti) in children, elderly, and immunocompromised persons; asymptomatic infections seem to be uncommon [8] . hmpv has been identified to be an important cause of bronchiolitis in young children. the novel human coronavirus hcov-nl63 is also widely accepted to be a significant respiratory tract pathogen. a strong association with croup has been shown [9] . human coronavirus hcov-hku1 infections are observed rather infrequent, often in children with underlying diseases [4] . several reports link the virus with pneumonia and bronchiolitis. for the human bocavirus (hbov) an increasing number of reports present evidence for an association with respiratory tract infections; however, co-infections with established respiratory tract pathogens are frequent. recently, the wu polyomavirus was detected in respiratory tract specimens [10] . the first polyomaviruses were isolated in 1971 and named jc virus and bk virus. in 2007 and 2008 3 further viruses of this particular family were described for the first time: wu polyomavirus, ki polyomavirus, and merkel cell polyomavirus [11] . wupyv was discovered by shot gun sequencing of nasopharyngeal aspirate of a three-years-old child from australia suffering from pneumonia [10] . the investigators isolated total nucleic acids from the nasopharyngeal aspirate, randomly amplified fragments, and cloned them. sequences of the obtained clones gained by shot gun sequencing were subjected to automated editing and database searches. the combination of these technologies led to the identification of a dna-sequence distant to all sequences included in the sequence database, but related to viruses of the family polyomaviridae. thus, the identification of a novel virus belonging to this viral family has been proposed, exclusively based on molecular data. so far, no system for in vitro or in vivo propagation has been discovered, neither a cell line nor an animal model. thus, until now koch's postulates could not be fulfilled. however, the molecular data form reasonable evidence to propose the existence of the novel virus wupyv. large-scale screening based technologies as the one used for the discovery of wupyv may be a powerful tool in discovering new infectious agents causing respiratory tract diseases particularly with regard to the etiological gap of up to 40%. the novel virus has a 5229 bp dsdna genome with a gc-content of 39%. the genome contains an early coding region encoding the t-antigens, a late coding region encoding structural proteins, and a non-coding control region [10] . all features are typical for members of the polyomavirus family. the sequence identity of wupyv at the amino acid level to all of the well-known polyomaviruses ranged from 15% -49%. the closest relative within the virus family is the ki polyomavirus (kipyv) with a sequence identity of 68%, which was described briefly before wupyv. thus, both viruses were grouped into a new subclass of the polyomavirus family. however, the international committee on taxonomy of viruses (ictv) has not recognised wupyv or kipyv yet. typical members of the family polyomaviridae form small, non-enveloped, icosahedral particles with 40-45 nm in diameter containing a supercoiled dsdna genome [11] . the virion is made up by 72 pentamers of the structural protein vp1, each pentamer binds to a single copy of vp2 or vp3. the genome is attached to cellular histones. there is no evidence hinting at a virus structure for wupyv differing from other polyomaviruses. however, wupyv virions have not been displayed by electron microscopy, thus there is no evidence for the precise virion structure at this stage. based on the genome organisation, two subclasses of the polyomavirus family have been postulated: the primate-like group and the mouse polyoma-like group [10] . the genome of members of both subclasses is divided into three regions. the early coding region encodes at least the small tantigen and the large t-antigen. both are binding partners of the rb-protein and p53. the late coding region encodes at least the structural proteins vp1-vp3, and the non coding control region contains viral promoters and the origin of replication. the early coding region of members of the mouse polyoma-like group additionally encodes the medium t-antigen, whereas the late coding region of members of the primate-like group additionally encodes the structural protein agnoprotein. apart from kipyv, the jc virus and the bk virus are the closest relatives of wupyv based on genome organisation and amino acid sequence. the early coding region contains a splice site for the large tantigen and binding domains for the rb-protein and p53. however, wupyv neither encodes open reading frames for the middle t-antigen nor the agnoprotein. thus, wupyv and kipyv were provisionally grouped into a third subclass of the polyomavirus family. so far, no expression studies have been conducted on wupyv to clarify whether the predicted open reading frames are in fact expressed during the viral infection of the target. interestingly, as shown for hbov the predicted transcripts of a virus are not necessarily identical to the transcripts detectable in an infected cell [12] . there is the need for an appropriate experimental system to carry out expression studies on wupyv for further characterization. polyomaviruses have a restricted host range. the bk virus and the jc virus, the closest relatives of wupyv within the well established members of the virus family, are human-specific. this suggests a host range restricted to humans for wupyv, too. infection with bkv takes place in early childhood; 90% of 5-9 years old children are seropositive. wupyv infections have been reported predominantly in children <3 years of age [10] suggesting a strong increase of seropositivity during early childhood for this virus, too. the incubation period of wupyv infections is unknown. respiratory transmission has been suggested for jc virus and bk virus although the diseases related to these viruses are not involving the respiratory tract. both viruses can be extracted from tonsil tissue [13, 14] . the viral entry route for wupyv is not completely defined. it could be defined by uptake through dividing cells of the upper respiratory tract and the oropharynx. in this case it would be likely that mostly children are at risk of infection as, due to their growing up, their anabolism is enhanced involving increased cell division. alternatively transmission may occur occasionally by blood transfusion and organ transplantation. jc virus and bk virus primary infections are typically subclinical or linked to mild respiratory illness [13, 15] . viral dissemination to the target cells occurs via blood [16] . sites of lifelong persistence are epithelial cells of the kidney for bk virus [17] and lymphocytes, oligodendrocytes as well as epithelial cells of kidney for jc virus [18] . 5% of immunocompetent bk virus carriers show viruria [19] , 20 -30% show shedding of jc virus [20] . this indicates that viral persistence can be terminated and viral reactivation can take place even in immunocompetent persons. reactivation can be assumed to an obviously higher extent in immunocompromised individuals. concordantly the related diseases, hemorrhagic cystitis and polyomavirus nephropathy for bk virus and progressive multifocal leukencephalopathy (pml) for jc virus, predominantly occur in persons with reduced immunocompetence. as wupyv was detected in a respiratory tract specimen the respiratory tract can be assumed to be the entry site for this virus, too. the virus was detected in serum indicating that the dissemination occurs via blood. so far, sites of persistence have not been identified. the virus has not been detected in urine [10] . thus, epithelial cells of kidney may not be the site of persistence for wupyv. wupyv is made responsible for upper and lower respiratory tract diseases, e.g. bronchiolitis, croup, and pneumonia [10] . besides nasopharyngeal samples, wupyv could also be detected in stool samples [21] , even in patients without any apparent clinical respiratory symptoms [22] . however, it has to be determined, whether wupyv is able to replicate in the intestine or whether it accumulates there by oral ingestion. for detection of wupyv-infections several conventional pcr formats as well as real-time pcr protocols were developed reporting sensitivities up to 100% and specificities up to 97.7% [10, [23] [24] [25] . antigen or antibody detection formats have not been reported, so far. no culture system is known at this time. the diagnostics of established respiratory viruses is based on a broad spectrum of available assays, aiming at the detection of virus-specific antibodies, viral protein, or viral nucleic acids. acute respiratory tract infections commonly have a short incubation period. thus, antibodies are usually not present at the beginning of the acute phase of the disease. for this reason, detection of virus-specific antibodies by complement fixation assays, enzyme linked immunosorbent assays (elisa), or immunofluorescence assays (ifa), all of which are available for established respiratory viruses, is inappropriate for the identification of the etiologic agent causing an acute respiratory tract infection, but useful for retrospective or epidemiological studies. direct detection of viral protein or viral nucleic acids is the mode of choice for respiratory virus diagnostics. viral cell culture represents the gold standard for the well established respiratory viruses, but is too slow for in time diagnostics. less time consuming methods have been developed. these comprise polymerase chain reaction (pcr), antigenspecific elisa, antigen-specific ifa, and, available only for a few respiratory viruses, very fast immunochromatographic assays. sensitivity and specificity of these assays vary considerably. the pcr displays the highest sensitivity. thus, being restricted to pcr formats for detection of wupyv is no bias to sensitivity. particularly real time pcr formats have an established track record in both, very sensitive detection and differentiation between colonisation and acute infection based on the quantitative data [26, 27] . availability of automated extraction of nucleic acids permits short processing times and, by this, handling of large specimen cohorts [28] . following the discovery of wupyv in australia, the virus was detected in specimens from patients with respiratory tract disease on all continents suggesting a worldwide distribution [10, [29] [30] [31] . so far, wupyv-dna was reported to be found in respiratory tract specimens (e.g. nasopharyngeal washes, tracheal secretion, bal), serum, and faeces. the virus could not be detected in urine or from uv lightassociated primary malignant lymphomas [10, 32] . specimens from other malignant diseases have not been investigated. the use of tracheal secretion for diagnostics has been shown to lead to an underestimation of the rate of positive specimens compared to other respiratory materials for hbov [33] . this may be true for wupyv, too. the data available are mainly based on retrospective studies exclusively including symptomatic patients. the detection rate in respiratory samples from children with respiratory disease varies from 0.4% to 11.5% [34, 35] . the age of wupyv infected patients ranged from a few weeks to 53 years, children <3 years of age were dominating. infections were predominantly detected in late winter, spring, and early summer [36] . high infection rates were reported for study populations preselected for lack of immunocompetence. hiv positive patients had detection rates of up to 35.7% in respiratory tract specimens and 8.3% in blood [37, 38] . the rates of co-infection with established respiratory viruses lay between 30.8% and 91.7% [39, 40] , commonly exceeding 50%. wupyv was detectable in blood, possibly indicating its potential for systemic infections. le and co-workers [41] presented evidence for viral persistence, wattier et al. [36] for nosocomial infections with wupyv. the real time protocols available allow the quantification of wupyv. quantification of viral loads in respiratory tract specimens revealed viral titers up to 10 10 copies/ml, but low and medium viral loads were dominating [21, 24] . no correlation between viral load and the rate of co-infection or clinical diagnoses was observed. few studies included asymptomatic control groups [34, 36, [42] [43] [44] . the results were not concordant and reached from higher detection rates in the control group to higher detection rates among the group of patients with respiratory tract diseases. prospective studies have been published only recently. van der zalm and co-workers [34] reported the detection of wupyv in a cohort of 18 children. their parents were contacted twice a week over a 6-months period (november to april) and asked for symptoms of respiratory tract disease in their children. every two weeks respiratory tract specimens were collected, regardless of respiratory symptoms. 11.5% of the specimens of children with symptoms were wupyv positive, but only 3.1% of specimens of healthy children, indicating at least an association of wupyv with disease. to this time there are no studies available concerning therapeutic interventions. this is mainly due to the retrospective nature of most investigations. further, no causal association between wupyv and respiratory disease has been shown yet [45] . as the need for therapeutics is driven by associating infections with disease, studies on therapeutic interventions are not to be expected in the near future. for bk virus associated nephropathy in renal transplant patients, reduction of immunosuppressive therapy is recommended if possible. in the case of progressive renal dysfunction fluoroquinolones (antibacterials), intravenous immunoglobulines, leflunomide, and -in otherwise refractory casescidofovir could be administered. for jc virus caused progressive multifocal leukoencephalopathy (pml) no specific therapy exists. in hiv-positive patients haart induced improvement of cellular function may lead to an at least temporary improvement. failure of treatment approaches with interferon alfa-2b, cytarabine, cidofovir, and topotecan has been documented [46] . the discovery of novel respiratory viruses has the potential to diminish the diagnostic gap for respiratory tract infections. creer and co-workers [1] , who omitted the recently identified respiratory viruses in their study on adults, reported a diagnostic gap of 31%. the share of specimens from children negative for any respiratory pathogen investigated was 22% when hmpv, hbov, and hcov-nl63 were included [47] . no study included the novel polyomaviruses or herpes viruses until now. thus, a further reduction of the respiratory tract infections of unknown origin seems reasonable. however, a substantial gap is remaining leaving sufficient room for additional respiratory viruses to be discovered in the future. so far it has not been finally proven, if wupyv is a real causative agent for respiratory diseases. association of virus detection with previously unexplained respiratory disease led to the tempting idea of wupyv representing a new etiologic agent, particularly in cases where no other respiratory tract pathogen could be identified. furthermore, the association of mouse pneumotropic polyomavirus with intestinal pneumonia and significant mortality [48] indicates that polyomaviruses have the capacity to be respiratory tract pathogens. many studies correlating the detection of wupyv with the incidence of respiratory symptoms argue for this hypothesis, but it remains difficult to prove as asymptomatic control groups have not been investigated sufficiently. additionally, the studies including control groups report dissenting findings. however, in two studies asymptomatic individuals display a lower frequency of virus detection compared to symptomatic patients. one of these is the only prospective study available. the difficulty to prove wupyv to be a respiratory pathogen may be due to the fact that most studies available so far try to correlate the virus with respiratory tract disease in general. in case of wupyv being the causative agent of a particular entity of respiratory tract disease, inclusion of patients displaying any kind of respiratory disease may bias the investigation. focussing on a single entity of respiratory disease proved successful for hcov-nl63, which was shown to cause croup. no association of wupyv viral loads and clinical symptoms could be observed so far, and coinfections with other viruses were described frequently. this weakens the hypothesis of wupyv being a respiratory tract pathogen. however, collection of samples was not performed by means of a standardized protocol among the various groups or at a defined time point, after onset of symptoms. as viral loads decrease during the acute phase of infection, the combination of longitudinal data from different time points in one analysis may bias the correlation of viral loads and symptoms. furthermore, detection of co-infection does not exclude pathogenic potential for wupyv, as early childhood is characterized by subsequent episodes of respiratory infections. co-detection of the declining pathogen responsible for the last episode of respiratory disease and the pathogen responsible for the present, acute disease has to be expected. only determination of the viral load kinetics would allow defining the clinical impact of a detectable microorganism. the large t-antigen of wupyv contains binding sites for the rb protein and p53. thus, tumourigenic potential cannot be excluded. however, the association of wupyv with other diseases, particularly tumour diseases has not been sufficiently investigated yet. taken together, the currently available data neither prove nor deny wupyv to be a respiratory tract pathogen. several scenarios may describe the role of the virus best. wupyv may be (1) part of the endogenous viral flora without pathogenic potential; (2) an opportunistic pathogen with pathogenic potential in the respiratory tract under conditions still to be defined; (3) a pacemaker for secondary infections; (4) a viral pathogen using the respiratory tract only as an entry route to reach the final target cell; infection of this cell type is the basis of a disease not related to the respiratory tract. to reach a final conclusion more powerful, controlled studies need to be performed prospectively. however, retrospective analysis of age-and time-matched control populations also permits conclusions if the data collected at the time are related to respiratory illness. ideally, prospective studies would be carried out in a multicenter-design including control groups concentrating on the correlation of wupyv with single entities of respiratory tract diseases. criteria for inclusion of specimens have to be comparable, as well as the time point and the protocol for extraction of specimens. all known or suspected respiratory tract pathogens should be included and follow-up investigations should be performed. the detection method of choice would be quantitative pcr to allow discrimination between colonisation and productive infection. additionally, serological investigations should be included to show that the patients seroconvert in response to an infection. the study participants should be evaluated before enrolment. beside investigation of respiratory tract disease, the cell type hosting the persisting virus has to be identified and a possible association with tumour diseases has to be investigated. aetiological role of viral and bacterial infections in acute adult lower respiratory tract infection (lrti) in primary care a newly discovered human pneumovirus isolated from young children with respiratory tract disease identification of a new human coronavirus characterization and complete genome sequence of a novel coronavirus, coronavirus hku1, from patients with pneumonia cloning of a human parvovirus by molecular screening of respiratory tract samples herpes simplex virus (hsv) pneumonia in a heart transplant: diagnosis and therapy. heart lung epstein-barr-virus-induced interstitial lung disease human metapneumovirus and lower respiratory tract disease in otherwise healthy infants and children croup is associated with the novel coronavirus nl63 identification of a novel polyomavirus from patients with acute respiratory tract infections the role of polyomaviruses in human disease human bocavirus can be cultured in differentiated human airway epithelial cells the role of bk virus in acute respiratory tract disease and the presence of bkv dna in tonsils detection of jc virus dna in human tonsil tissue: evidence for site of initial viral infection human papovavirus isolated from urine of a child with acute tonsillitis human endothelial cells allow passage of an archetypal bk virus (bkv) strain-a tool for cultivation and functional studies of natural bkv strains bk virus infection of the human urinary tract cultivation of papovalike virus from human brain with progressive multifocal leucoencephalopathy human polyomavirus jc and bk persistent infection discovery and epidemiology of the human polyomaviruses bk virus (bkv) and jc virus (jcv) wu polyomavirus in fecal specimens of children with acute gastroenteritis detection of wu polyomavirus dna by real-time pcr in nasopharyngeal aspirates, serum, and stool samples a single-tube real-time pcr assay for detection of the two newly characterized human ki and wu polyomaviruses low to medium wu-virus titers in young children with lower respiratory tract infections development and evaluation of real-time pcr assays for the detection of the newly identified ki and wu polyomaviruses detection of cytomegalovirus dna in human specimens by lightcycler pcr detection of cytomegalovirus dna in human specimens by lightcycler pcr: melting point analysis is mandatory to detect virus strains with point mutation in the target sequence of the hybrdization probes efficient extraction of viral dna and viral rna by the chemagic viral dna/rna kit allows sensitive detection of cytomegalovirus, hepatitis b virus, and hepatitis g virus by pcr wu polyomavirus infection in children human polyomaviruses, wu and ki in hiv exposed children with acute lower respiratory tract infections in hospitals in south africa wu polyomavirus in children with acute lower respiratory tract infections dna from bk virus and jc virus and from ki, wu, and mc polyomaviruses as well as from simian virus 40 is not detected in non-uv-light-associated primary malignant melanomas of mucous membranes high prevalence of human bocavirus detected in young children with severe acute lower respiratory tract disease by use of a standard pcr protocol and a novel real-time pcr protocol prevalence and pathogenicity of wu and ki polyomaviruses in children; the netherlands discovery and identification of wu polyomavirus in children from zhejing region role of human polyomaviruses in respiratory tract disease in young children wu polyomavirus in patients infected with hiv or hepatitis c virus reactivation and mutation of newly discovered wu, ki, and merkel cell carcinoma polyomaviruses in immunosuppressed individuals wu and ki polyomavirus present in the respiratory tract of children, but not in immunocompetent adults clinical and epidemiologic characterization of wu polyomavirus infection human bocavirus and ki/wu polyomaviruses in pediatric intensive care patients wu polyomavirus in children with acute lower respiratory tract infections wu polyomavirus in children no evidence for an association between infection with wu and ki polyomaviruses and respiratory disease treatment options for aids patients with progressive multifocal leukencephalopathy molecular epidemiology and disease severity of respiratory syncytial virus in relation to other potential pathogens in children hospitalized with acute respiratory infection in jordan effect of host age on experimental k virus infection in mice this study was supported by an eu-grant to m.k. (contract no. 037276). key: cord-325635-don9qjpz authors: turner, paul; turner, claudia; watthanaworawit, wanitda; carrara, verena; cicelia, naw; deglise, carole; phares, christina; ortega, luis; nosten, francois title: respiratory virus surveillance in hospitalised pneumonia patients on the thailand-myanmar border date: 2013-09-16 journal: bmc infect dis doi: 10.1186/1471-2334-13-434 sha: doc_id: 325635 cord_uid: don9qjpz background: pneumonia is a significant cause of morbidity and mortality in the developing world. viruses contribute significantly to pneumonia burden, although data for low-income and tropical countries are scarce. the aim of this laboratory-enhanced, hospital-based surveillance was to characterise the epidemiology of respiratory virus infections among refugees living on the thailand-myanmar border. methods: maela camp provides shelter for ~45,000 refugees. inside the camp, a humanitarian organisation provides free hospital care in a 158-bed inpatient department (ipd). between 1st april 2009 and 30th september 2011, all patients admitted to the ipd with a clinical diagnosis of pneumonia were invited to participate. clinical symptoms and signs were recorded and a nasopharyngeal aspirate (npa) collected. npas were tested for adenoviruses, human metapneumovirus (hmpv), influenza a & b, and rsv by pcr. results: seven hundred eight patient episodes (698 patients) diagnosed as pneumonia during the enhanced surveillance period were included in this analysis. the median patient age was 1 year (range: < 1-70), and 90.4% were aged < 5 years. at least one virus was detected in 53.7% (380/708) of episodes. virus detection was more common in children aged < 5 years old (<1 year: or 2.0, 95% ci 1.2-3.4, p = 0.01; 1-4 years: or 1.4, 95% ci 0.8-2.3, p = 0.2). rsv was detected in 176/708 (24.9%); an adenovirus in 133/708 (18.8%); an influenza virus in 68/708 (9.6%); and hmpv in 33/708 (4.7%). twenty-eight episodes of multiple viral infections were identified, most commonly adenovirus plus another virus. rsv was more likely to be detected in children <5 years (or 12.3, 95% ci 3.0-50.8, p = 0.001) and influenza viruses in patients ≥5 years (or 2.8, 95% ci 1.5-5.4, p = 0.002). ipd treatment was documented in 702/708 cases; all but one patient received antimicrobials, most commonly a beta-lactam (amoxicillin/ampicillin +/−gentamicin in 664/701, 94.7%). conclusions: viral nucleic acid was identified in the nasopharynx in half the patients admitted with clinically diagnosed pneumonia. development of immunisations targeting common respiratory viruses is likely to reduce the incidence of pneumonia in children living refugee camps and similar settings. pneumonia remains a leading cause of mortality globally: 4.2 million pneumonia deaths were recorded in 2004 [1] . the highest incidence of disease occurs in young children [2, 3] . an estimated 156 million pneumonia episodes occur annually in children less than five years old. over 95% of these occur in the developing world, where the incidence of clinical pneumonia is estimated to be 0.29 episodes per child-year. almost three-quarters of childhood pneumonia deaths occur in sub-saharan africa and southeast asia [4] . bacterial pathogens, most notably streptococcus pneumoniae and haemophilus influenzae type b, are important vaccinepreventable causes of pneumonia [5] . viruses, in particular influenza and respiratory syncytial virus (rsv), are also responsible for a large number of pneumonia cases each year [6] . using global population data for 2005, for children under the age of five years, it was estimated that rsv was responsible for over 30 million episodes of lower respiratory tract infections (lrti), with~3 million of these requiring hospital admission, and 66,000-199,000 deaths [7] . by similar analyses of data from 2008, influenza viruses were estimated to cause 20 million lrti and 1 million severe lrti, with 28,000-111,500 deaths, in children aged less than five years [8] . in both of these reviews, 99% of deaths from either influenza-or rsvassociated lrti occurred in the developing world. these viruses may be responsible for up to 35% of lrti (rsv 22% [7] and influenza 13% [8] ) in children under the age of five years. other viral pathogens associated with childhood pneumonia include adenoviruses, human metapneumovirus (hmpv), and parainfluenza viruses [5, 9] . approximately one-third of the worldwide refugee population of 15 million live in camps [10] . these camps are often crowded with poor sanitation, providing ideal conditions for transmission of respiratory pathogens [11, 12] . there have been refugees from myanmar (burma) living in camps in thailand since 1984. in 2006, lower respiratory tract infections were estimated to be the cause of 9% of deaths, and were responsible for 25% of all reported morbidity, in the under-5 age group of the border refugee population. the overall under-5 year mortality rate was 6 per 1,000, giving an estimated lrtispecific mortality rate of 0.5 per 1,000 [13] . in 2007, the us centers for disease control and prevention (cdc) and the shoklo malaria research unit (smru) established a respiratory virus surveillance programme in the burmese refugee population living in maela camp, northwest thailand. the programme included patients admitted to hospital with pneumonia during april 2009-september 2011. the aim of in-patient surveillance was to determine the relative burden of virusassociated pneumonia. the results of 30 months of in-patient surveillance are presented here. maela camp is located in rural tak province approximately 500 km from bangkok. it is the largest of the nine camps on the thailand-myanmar border, housing approximately 45,000 people in a 4 km 2 area, and has been in continuous operation since 1984. karen is the predominant ethnicity in the camp population. general healthcare is provided by the non-governmental organisation première urgence-aide médicale internationale (pu-ami). camp residents receive world health organisation (who) expanded programme on immunisation (epi) immunisations, but immunisations against respiratory pathogens (haemophilus influenzae type b, influenza viruses, and streptococcus pneumoniae) are not available. from april 2009 to september 2011, laboratory-enhanced respiratory surveillance was undertaken at the in-patient department (ipd) of the maela pu-ami hospital. throughout this period, trained local health workers reviewed ipd admission logs on six days each week to identify patients with an admission diagnosis of pneumonia, including those who were admitted on the seventh day. health workers invited all pneumonia patients to participate in enhanced surveillance and enrolled all who agreed. for enrolled patients, health workers completed a brief symptoms questionnaire by patient interview and record review, and collected nasopharyngeal aspirates (npa) as previously described [14] . patient episodes were subsequently excluded from analyses if they (a) failed to meet the surveillance case definition for pneumonia (table 1) , (b) occurred within 14 days of previous episode in the same patient, or (c) lacked adequate laboratory specimens. surveillance case definitions were based on those devised by who for children under five years of age [15, 16] . for older individuals, in whom a satisfactory clinical case definition is lacking, the surveillance case definition was based on that described by the british thoracic society [17] . npa specimens, in 1 ml viral transport medium (vtm, prepared in-house), were transported daily to the smru microbiology laboratory, which is located in the town of mae sot, approximately 50 km from maela. specimens were placed into an insulated cool box immediately after collection and were transported back to the mae sot laboratory within eight hours of collection, where they were stored at−80°c until analysis. viral nucleic acid was extracted from thawed npa-vtm specimens using commercial kits, following the manufacturer's instructions (qiaamp viral rna minikit [qiagen, hilden, germany], april 2009 until september 2010; viral nucleic acid extraction protocol of the magcore hf16 automated extractor [rbc bioscience, taiwan], october 2010 until september 2011). extracts were analysed by real-time reverse-transcription pcr (rrt-pcr) for adenoviruses, hmpv, influenza viruses (a and b, with typing of influenza a viruses to detect seasonal h1/h3 and pandemic h1 strains), and rsv as described elsewhere [14, 18, 19 ]. an internal control human rnasep pcr was included to confirm the specimen adequacy and to identify pcr inhibition [20] . specimens were considered positive if a virus pcr ct value was < 40 with appropriate run control results. to ensure the reproducibility of results approaching the assay limits of detection (approximately 100 copies per reaction for each target; smru internal qc data), specimens with low positive pcr results (ct values of [35] [36] [37] [38] [39] were repeated and only if the ct was < 40 in both runs was the virus pcr considered positive. clinical and laboratory data were recorded on paperbased case record forms and subsequently entered into an access 2003 database (microsoft, redmond, wa, usa) and systematically checked for errors by comparison with the original case record forms. data were analysed by stata/ic version 12.1 (statacorp, college station, tx, usa). proportions were analysed by chi-squared or fisher's exact tests as appropriate. logistic regression was used to calculate odds ratios (or) and their 95% confidence intervals (ci). multivariate models were constructed to determine relationships between age, viral detection, pneumonia severity (<5 years old only), and antimicrobial use prior to admission. two-tailed p-values of < 0.05 were considered significant. this surveillance program underwent ethical and regulatory review at cdc, and was determined not to meet the definition of research. local ethical review in maela was not possible at the commencement of surveillance. however, the surveillance activity was discussed with pu-ami staff, and all concerns were addressed, prior to the beginning of the project. verbal consent was obtained from each potential participant, or their parent/legal guardian in the case of children aged <15 years, prior to enrolment in the surveillance programme. among all ipd pneumonia admissions, 835 patient episodes were enrolled in enhanced surveillance. after review of the symptom questionnaire, 117 patient episodes were excluded because of failure to meet the case definition and one episode was excluded for a patient who presented twice within 14 days. in three episodes, npa specimens were not collected and in another six, the specimens were technically inadequate (internal control pcr negative). the remaining 708 patient episodes were included in the following analyses. a total of 698 individuals were sampled (689 patients with single episodes, eight patients with two episodes, and one with three episodes). the median age at presentation was one year (iqr < 1-2; range < 1 to 70). six hundred and forty patients (90.4%) were aged < 5 years (table 2 ). there were significantly more males in the < 1 year age group compared with the older patients (64.7% vs. 52.2%, p = 0.001). patients presented at a median of 4 days after symptom onset (iqr 2-6). eighty five percent of pneumonia episodes in the patients aged < 5 years were classified as severe or very severe; this did not vary by gender or the duration of illness prior to admission (data not shown). children aged 1-4 years were less likely to have severe or very severe pneumonia compared with those aged < 1 year (or 0.6, 95% ci 0.4-0.9, p = 0.03). treatment was documented for 702/708 (99.2%) episodes: all but one patient received an antimicrobial drug, most commonly amoxicillin or ampicillin +/−gentamicin (664/702, 94.6%). viral nucleic acid was detected in 53.7% (380/708) npa specimens. the rank order of detection was rsv (176, 24.9% of npa), adenovirus (133, 18.8%), influenza a (58, 8.2%), hmpv (33, 4 .7%), and influenza b (10, 1.4%). detection of viruses varied considerably by age (table 2 ). patients aged < 5 years were more likely to have viral adenoviruses were also more likely to be detected in those aged one year or more (1-4 years old: or 1.9, 95% ci 1.2-2.9, p = 0.003; ≥ 5 years old: or 1.7, 95% ci 0.8-3.3, p = 0.1) (figure 1 ). diagnosis of severe or very severe pneumonia in the < 5 year age group was associated with detection of rsv (or 1.9, 95% ci 1.1-3.2, p = 0.03). the trend remained the same, although the association became statistically non-significant, when controlling for age and detection of other viruses in a multivariate logistic regression model (aor 1.5, 95% ci 0.8-2.7, p = 0.2). in the same multivariate model, detection of adenovirus (aor 0.5, 95% ci 0.3-0.9, p = 0.01) or an influenza virus (aor 0.5, 95% ci 0.3-1.0, p = 0.05) were associated with a lower likelihood of severe or very severe pneumonia diagnosis. multiple viruses were detected in 4.0% (28/708) specimens. two viruses were detected in 26 specimens (13 adenovirus + rsv; 8 influenza + rsv; 3 adenovirus plus influenza; 2 adenovirus + hmpv) and three viruses were detected in two specimens (1 adenovirus + influenza + hmpv; 1 adenovirus + influenza + rsv). there were no associations between multiple virus detection with age or severity of pneumonia (data not shown). virus detection varied by season. rsv, influenza viruses, and hmpv were all detected in the wet (june-october) and cool (november-february) seasons, whereas adenovirus detection occurred year round and peaked in the late cold and hot (march-may) seasons ( figure 2 ). interestingly, in an age-adjusted analysis, patients who had received an antimicrobial in the two weeks preceding admission were more likely to be rsv pcr positive (aor 1.7, 95% ci 1.2-2.5, p = 0.003). no such association was seen for the other viruses. (figure 3 ). twenty five patients with influenza a associated pneumonia (admitted may-october 2009) have been described in detail in a previous manuscript [14] . laboratory-enhanced surveillance has documented the contribution of respiratory viruses to 708 hospitalised clinical pneumonia episodes occurring in a crowded refugee camp on the thailand-myanmar border during april 2009 to september 2011. as expected, the vast majority of patients were aged less than five years [2] . viruses were detected in the nasopharynx of half of the cases. virus detection was significantly more likely in infants compared to older children and adults, although the number of adult cases was small. during the same period, routine hospital surveillance detected 2,081 in-patient lrti episodes, including 32 (1.5%) deaths and 1,340 (64.4%) episodes in children under five years [21] . therefore, ipd pneumonia episodes under enhanced surveillance reported here accounted for 34.0% of all ipd lrti episodes (47.8% of ipd lrti episodes in children aged less than five years and 9.2% of episodes in persons aged five years or older). the maela data add to the scant data on the aetiology of pneumonia in refugee populations. the results are broadly consistent with a similar surveillance programme conducted in two kenyan refugee camps [22] , where 51.3% patients with severe acute respiratory infection (sari) had at least one of adenovirus, hmpv, influenza a/b, parainfluenza virus 1-3, or rsv detected. in the kenya surveillance, adenovirus was the commonest virus detected (22.7% of specimens), followed by rsv (14.1%). an influenza virus was detected in 11.6% of specimens. the majority (82%) of hospitalised sari cases were children less than five years of age. results from pneumonia aetiology studies from various locations in the developing world have confirmed the high prevalence of virus detection in hospitalised pneumonia episodes in young children [23, 24] . rsv has been frequently identified as the commonest virus and is associated with severe infections [25] [26] [27] [28] . a significant proportion of rsv infections occur in the first year of life, although both primary infection and re-infections are common in older children [26, 29, 30] . rsv has previously been documented to be a significant pathogen in maela, with an incidence of rsv-associated pneumonia of 0.24 episodes per child-year at risk in a cohort of infants followed from birth until two years of age [31, 32] . our results confirm that rsv is the leading virus associated with pneumonia in the general maela camp population. the majority of infections were in young children and only two (2/176, 1.1%) rsv infections occurred in patients aged five years or older. influenza viruses were detected in almost 10% of patient episodes of pneumonia. this figure is consistent with previously published data on influenza hospitalisations. simmerman and uyeki determined that influenza viruses were detected in 6-14% of hospitalised pneumonia cases in a recent review of east and southeast asian data [33] . the role of adenoviruses in the aetiology of pneumonia remains unclear. frequent re-infection and persistence in young children makes their detection in npa specimens at the time of pneumonia diagnosis difficult to interpret [34, 35] . a recent case-control study from kenya, undertaken as part of a multi-centre paediatric pneumonia aetiology study (perch), did not find a higher odds of adenovirus detection in pneumonia cases compared with controls [36] . in that study, only the detection of rsv in the upper respiratory tract had a significant association with hospitalisation for pneumonia. who definitions for clinical pneumonia in childhood were used in our enhanced surveillance in maela. these definitions were designed to have optimal sensitivity for the diagnosis of potentially life-threatening bacterial infection in resource-poor settings [15] . it was accepted that some over-diagnosis and unnecessary treatment would occur [37] . as was demonstrated in a south african study, where virus-associated pneumonias were reduced by a third in children given a nine-valent pneumococcal conjugate vaccine, a proportion of virus-positive individuals would have had a secondary bacterial infection [38] , but many will have received an unnecessary course of antibiotics. recent work from pakistan has confirmed that placebo and amoxicillin had equivalent efficacy for who non-severe pneumonia and that oral amoxicillin at home is an acceptable alternative to hospital admission and parenteral antibiotics for severe pneumonia [39, 40] . in maela, almost all patients were treated with at least one antimicrobial drug, yet over a third (excluding adenovirus) had a proven viral infection. collectively, these findings point to the likely over treatment of viral infections in children with who defined pneumonia, which may contribute to the rising prevalence of antimicrobial resistance in the developing world [41] . the enhanced surveillance system had several limitations. not all hospitalised pneumonia episodes were captured and therefore it was not possible to calculate virus-specific incidence rates. estimating the representativeness of the patients enrolled by comparison of enhanced surveillance results with routine surveillance figures is problematic. however, as we note, if a direct comparison is made, enhanced surveillance included 47.8% (640/1,340) of all lrti episodes identified through routine surveillance in children under five years of age but only 9.2% (68/741) of episodes in patients aged five years or older. differences in case definitions used in the routine surveillance compared with enhanced surveillance is the most likely explanation for this discrepancy [21] . for the under-5 age group the definitions were almost identical (i.e. who-based) but for the older age group lrti was defined in routine surveillance by the presence of a fever plus cough or sore throat and shortness of breath/difficulty breathing. absence of a requirement for abnormal chest signs likely resulted in inclusion of many non-pneumonia cases within the lrti category. the low number of deaths recorded in the his system suggests that severity was not a significant reason for non-inclusion. also, since pu-ami hospital was the only general medical admissions unit within the camp, there were not significant numbers of patients missed as a result of admission to other hospitals. however, it remains possible that the older participants were not representative of the entire hospitalised pneumonia patient group. additional severity data (e.g. need for supplemental oxygen and length of stay) and outcome data were not collected, which further limit the conclusions that can be drawn from the surveillance and the comparisons that can be made with other studies. the panel of viruses tested for included the key pathogens for which evidence of an association with pneumonia is proven, but inclusion of pcr assays to detect additional respiratory viruses would have added value and may have identified a higher prevalence of multiple infections. studies using multiplexed virus pcr assays have detected both an increased proportion of children with single and multiple viral lower respiratory infections [42] . both human bocavirus and rhinoviruses may be detected in a large proportion of pneumonia cases, although data regarding causality from case-control studies are still limited [36, 43] . despite this, it was demonstrated that influenza virus and/or rsv were associated with a third of hospitalised pneumonia episodes in maela. the cost of in-patient pneumonia treatment is high. in two recent studies, the estimated average cost per district hospital pneumonia admission was us$99.26 in kenya and us$490.80 in thailand [44, 45] . influenza infections are vaccine preventable. a vaccine to prevent rsv infection remains elusive, although progress continues to be made [46, 47] . inclusion of influenza immunisation, and rsv immunisation should it become available, in the immunisation programme for refugees on the thailand-myanmar border would likely significantly reduce the burden of pneumonia requiring hospitalisation. viruses were commonly identified in burmese refugees admitted to hospital with clinically-diagnosed pneumonia. use of influenza immunisation and the development of vaccines targeting other common respiratory viruses would be likely to reduce the incidence of pneumonia in children living in refugee camps and similar settings. who: the global burden of disease: 2004 update. geneva: world health organisation unicef: pneumonia: the forgotten killer of children. geneva: unicef/who childhood pneumonia mortality-a permanent global emergency epidemiology and etiology of childhood pneumonia respiratory tract infections in children in developing countries viral pneumonia global burden of acute lower respiratory infections due to respiratory syncytial virus in young children: a systematic review and meta-analysis global burden of respiratory infections due to seasonal influenza in young children: a systematic review and meta-analysis human metapneumovirus-associated lower respiratory tract infections among hospitalized human immunodeficiency virus type 1 (hiv-1)-infected and hiv-1-uninfected african infants unhcr: global trends: refugees, asylum-seekers, returnees, internally displaced and stateless persons. geneva: united nations high commissioner for refugees a community-based study of acute respiratory tract infection in thai children influenza pandemic plans: what about displaced populations? committee for coordination of services to displaced persons in thailand (ccsdpt) influenza in refugees on the thailand-myanmar border who: integrated management of childhood illness handbook in pocket book of hospital care for children: guidelines for the management of common illnesses with limited resources anonymous: bts guidelines for the management of community acquired pneumonia in adults real-time reverse transcriptase pcr assay for detection of human metapneumoviruses from all known genetic lineages comparison of nasopharyngeal and oropharyngeal swabs for the diagnosis of eight respiratory viruses by real-time reverse transcription-pcr assays real-time reverse transcription-polymerase chain reaction assay for sars-associated coronavirus unhcr: health information system epidemiology of respiratory viral infections in two long-term refugee camps in kenya rna viruses in community-acquired childhood pneumonia in semi-urban nepal; a cross-sectional study the etiology of pneumonia in malnourished and well-nourished gambian children respiratory syncytial virus infection in tropical and developing countries incidence and severity of respiratory syncytial virus pneumonia in rural kenyan children identified through hospital surveillance viral etiology of severe pneumonia among kenyan infants and children clinical presentation and severity of viral community-acquired pneumonia in young nepalese children respiratory syncytial virus infection and disease in infants and young children observed from birth in kilifi district respiratory syncytial virus epidemiology in a birth cohort from kilifi district, kenya: infection during the first year of life high rates of pneumonia in children under two years of age in a south east asian refugee population a high burden of respiratory syncytial virus associated pneumonia in children less than two years of age in a south east asian refugee population the burden of influenza in east and south-east asia: a review of the english language literature. influenza other respi viruses persistence of adenovirus nucleic acids in nasopharyngeal secretions: a diagnostic conundrum respiratory viral infection in lower airways of asymptomatic children a preliminary study of pneumonia etiology among hospitalized children in kenya acute lower respiratory tract infections in children: possible criteria for selection of patients for antibiotic therapy and hospital admission a role for streptococcus pneumoniae in virus-associated pneumonia comparison of oral amoxicillin with placebo for the treatment of world health organization-defined nonsevere pneumonia in children aged 2-59 months: a multicenter, double-blind, randomized, placebo-controlled trial in pakistan effectiveness of community case management of severe pneumonia with oral amoxicillin in children aged 2-59 months in matiari district, rural pakistan: a cluster-randomised controlled trial antimicrobial resistance in developing countries part ii: strategies for containment spectrum of respiratory viruses in children with community-acquired pneumonia human bocavirus infections the economic burden of inpatient paediatric care in kenya: household and provider costs for treatment of pneumonia, malaria and meningitis the incidence of pneumonia in rural thailand new strategies for control of respiratory syncytial virus infection an evaluation of the emerging interventions against respiratory syncytial virus (rsv)-associated acute lower respiratory infections in children respiratory virus surveillance in hospitalised pneumonia patients on the thailand-myanmar border we are grateful for the hard work of say paw and mallika (smru clinic, maela) and for the support of the clinical staff at the pu-ami hospital in maela. this work was supported by a us-cdc cooperative agreement (5u50ci000473). smru is part of the mahidol oxford university tropical medicine research unit, supported by the wellcome trust of great britain. the findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the centers for disease control and prevention. the authors declare that they have no competing interests.authors' contributions pt, vc, ct, cd, cp, lo, and fn conceived the surveillance project. nc and ct were responsible for specimen and data collection. ww performed the laboratory work. pt and vc did the data analysis. pt prepared the first draft of the manuscript. all authors reviewed and contributed to revisions of the manuscript. all authors read and approved the final manuscript. key: cord-336335-spap39b7 authors: silva, denise r; viana, vinícius p; müller, alice m; livi, fernando p; dalcin, paulo de tarso r title: respiratory viral infections and effects of meteorological parameters and air pollution in adults with respiratory symptoms admitted to the emergency room date: 2013-08-26 journal: influenza other respir viruses doi: 10.1111/irv.12158 sha: doc_id: 336335 cord_uid: spap39b7 background: respiratory viral infections (rvis) are the most common causes of respiratory infections. the prevalence of respiratory viruses in adults is underestimated. meteorological variations and air pollution are likely to play a role in these infections. objectives: the objectives of this study were to determine the number of emergency visits for influenza-like illness (ili) and severe acute respiratory infection (sari) and to evaluate the association between ili/sari, rvi prevalence, and meteorological factors/air pollution, in the city of porto alegre, brazil, from november 2008 to october 2010. methods: eleven thousand nine hundred and fifty-three hospitalizations (adults and children) for respiratory symptoms were correlated with meteorological parameters and air pollutants. in a subset of adults, nasopharyngeal aspirates were collected and analyzed through ifi test. the data were analyzed using time-series analysis. results: influenza-like illness and sari were diagnosed in 3698 (30·9%) and 2063 (17·7%) patients, respectively. thirty-seven (9·0%) samples were positive by ifi and 93 of 410 (22·7%) were ifi and/or pcr positive. in a multivariate logistic regression model, ifi positivity was statistically associated with absolute humidity, use of air conditioning, and presence of mold in home. sunshine duration was significantly associated with the frequency of ili cases. for sari cases, the variables mean temperature, sunshine duration, relative humidity, and mean concentration of pollutants were singnificant. conclusions: at least 22% of infections in adult patients admitted to er with respiratory complaints were caused by rvi. the correlations among meteorological variables, air pollution, ili/sari cases, and respiratory viruses demonstrated the relevance of climate factors as significant underlying contributors to the prevalence of rvi. respiratory tract infections are the most common causes of infection, and viruses account for the majority of these infections, leading to significant levels of morbidity and mortality. 1 emergency rooms (ers) serve as the frontline for patients at highest risk for respiratory infection diseases, especially because of the acute nature of these illnesses. 2 the prevalence of respiratory viral infection (rvi) in adults admitted to the er is largely unexplored, as most relevant data concern infants and children. 3, 4 rvi can be severe in elderly patients, especially in those with underlying respiratory or cardiac disease. during winter months, rvi can account for many of the admissions to hospitals. 5, 6 the etiology of respiratory infections in adults remains undetermined in more than 50% of cases. 7, 8 in a study with 510 adults hospitalized with pulmonary diseases, an overall prevalence of respiratory viruses (rvs) in the lower respiratory tract was of 42á2%, with rhinoviruses and influenza a virus being the most common. 9 in adults with acute asthma admitted to the er, a prevalence of 12á2% of rvi was found. 10 in another study, with adults admitted to hospital with respiratory symptoms, viruses accounted for 15% of hospital admissions for respiratory infections. 11 seasonality of certain acute respiratory tract infection pathogens can be explained by meteorological variations. in a study, temperature was highly inversely correlated with respiratory syncytial virus (rsv), influenza a, and adenovirus frequency; rhinovirus was also associated with relative humidity (rh). climatic factors may influence the interaction among the host, pathogen, and environment, increasing the probability of exposure, susceptibility, and infection. 12 in addition, experimental data have shown that air pollutants affect lung immune responses and inflammatory reactions and that these effects may underlie the increased risk for respiratory infections. 13, 14 because of the large impact respiratory virus infections have on morbidity and even mortality, it is important to understand whether and how meteorological factors and exposure to air pollutants could influence respiratory virus infections. the aims of this study were to determine the number of emergency room visits for influenza-like illness (ili) and severe acute respiratory infection (sari) and to evaluate the association between ili/sari frequency, respiratory virus prevalence, and meteorological factors/air pollution, especially in adult population, in a humid subtropical climate. the present study was divided into two parts: in the first one, we characterized the symptomatic respiratory subjects attending the er at hospital de cl ınicas de porto alegre (hcpa), during 1 year (november 2008 to october 2009). in the second part, we included patients with respiratory symptoms ≤5 days to determine the prevalence of rvi; this part was conducted during 2 years (november 2008 to october 2010). in the first year of the study, we also collected climate and air pollution data. the study was conducted at hcpa, in the city of porto alegre, southern brazil. the hcpa is a general, tertiary care, university-affiliated hospital with 750 beds and approximately 30 000 hospitalizations/year. with a population of 1 360 590 inhabitants, porto alegre is surrounded by a metropolitan area that encompasses 31 municipalities (3 717 430 inhabitants). porto alegre has a humid subtropical climate, with the hallmark of the great variability (classification cfa in k€ oppen-geiger). 15 the ethics committee at hcpa has approved access to patient records. all subjects selected for the study gave written informed consent to participate. in the first part of the study, the clinical records of all daily visits (adults ≥18 years and children) to the er were reviewed by the research team. patients with respiratory symptoms (cough, coryza, nasal obstruction, odynophagia, dyspnea, chest pain, dysphonia, wheezing, and fever), regardless of the onset time, were included in the study. patients presenting only with fever were not included. demographic, clinical, and laboratorial characteristics were registered in a standardized questionnaire: sex, age, race, years of schooling, smoking status, symptoms at admission, duration of symptoms, presence of comorbidities, admission vital signs (temperature, heart rate, respiratory rate, and peripheral oxygen saturation measured by a digital oximeter), breath sounds, radiological findings, length of hospital stay, intensive care unit (icu) admission, hospitalization outcome (death or discharge). in the second part, adults ≥18 years with respiratory symptoms ≤5 days were included, and nasopharyngeal aspirates were collected. every day, a daily shift (morning, afternoon, or evening) was randomized for inclusion of patients. the patients were interviewed by a member of the research team, and the following data were registered in a standardized questionnaire (in addition to the data already registered in the first part): family income, influenza vaccine, previous antibiotic use, and occupational exposure, flu symptoms in the family, presence of smokers at home, family history of respiratory disease, air conditioning at home or at work, use of wood stoves, mold in home. the total cases of ili (fever >38°c and cough or sore throat) and sari (fever >38°c and cough or sore throat and shortness of breath or difficulty breathing) were registered. nasopharyngeal aspirates were obtained according to a standard protocol for the second part of study. an indirect immunofluorescence assay (ifi) was carried out using the respiratory daily meteorological parameters, like temperature (maximum, average, and minimum, in°c), relative (%) and absolute (g/kg) humidity, rainfall (mm), and sunshine duration (number of sunshine hours per day), were obtained from the climate laboratory at the federal university of rio grande do sul. also, the mean concentration of pollutants (lm/m 3 ) was recorded. the methodology used by this laboratory informs automatically the pollutant that reached the highest concentration in the last 24 hours. in the city of porto alegre, it is probable that the pollutant that is responsible for more than 90% of the days is ozone, and in the rest of days, particulate matter of <10 lm dynamic diameter (pm 10 ). data were presented as number of cases, mean ae standard deviation (sd), or median with interquartile range (iqr). categorical comparisons were carried out by chi-square test using yates's correction if indicated or by fisher's exact test. continuous variables were compared using the t-test or wilcoxon test. odds ratios (ors) and nominal 95% confidence intervals (ci) were presented. a two-sided p value <0á05 was considered significant for all analyses. data analysis was performed using spss 18á0 (statistical package for the social sciences, chicago, il, usa) and the free statistical software r (http://www.r-project.org). the data were analyzed using time-series analysis, through a generalized linear model (glm) to examine the association between ili or sari and air pollution/meteorological variables, using logistic regression. an autoregressive integrated moving average (arima) model was developed for ili or sari. pollution and meteorological parameters were inserted as explanatory variables into these arima models. for the second part of the study, further models were explored to include ifi and/or pcr results as outcome variables. volatility of the mean model variance error(s) was addressed using autoregressive conditional heteroscedasticity (arch) models within an arima modeling framework. an arima was developed for ili and sari. arima (p, d, q) are useful tools for analyzing time-series data containing non-stationary common trends, and these models were proposed by box and jenkins in 1976. the arima (p, d, q) allow to make predictions from their parties ar (autoregressive) and ma (moving average). for both sg and sars, we made the identification of the order of the parts and autoregressive moving average following the box-jenkins approach. first we identified the need for differentiation, for ili and sari, the graphical analysis of time-series and autocorrelation functions, as well as for testing the unit root (dickey-fuller) who did not reject the hypothesis of non-stationarity for ili and sari. in a second moment, it was noted that there was no seasonality for ili and sari, by analyzing the periodograms and autocorrelation functions and partial autocorrelation. subsequently, variable selection models arima (p, d, q) for ili and sari followed the approach backward. the akaike information criterion (aic) which acts to penalize the number of parameters in the modeland the schwarz information criterion (sic) (or bayesian information criterion, bic) were also used to select models. sample size requirements were estimated from the literature review. 10, 11 using a prevalence of rvi in adults of 15%, with a significance level of 99%, and total confidence interval amplitude of 0á10, we calculated that 339 patients would be needed in the second part of the study. during the 12-month study period, there were 37 059 admissions to the er (24 189 adults and 12 870 children), of which 11 953 (32á3%) presented with respiratory symptoms. the most common symptoms were cough (73á4%), fever (56á1%), dyspnea (40á9%), chest pain (24á5%), and coryza (20á9%). the median duration of symptoms before admission was 3 days (iqr: 1-6 days). a total of 2205 (18á5%) patients admitted to er needed to be hospitalized; of these patients, 242 (2á0%) required icu admission. ili and sari were diagnosed in 3698 (30á9%) and 2063 (17á7%) patients, respectively. the overall mortality rate among all study participants was 280 of 11 953 (2á3%). demographic and clinical characteristics of the study population are shown in table 1 . according to the selection criteria of the study, 425 patients met the inclusion criteria and were invited to participate, and 15 patients declined to participate. then, 410 adults were enrolled for virological investigation, 255 in the first year and 155 in the second one. there were no differences in age, sex, race, years of schooling, and symptoms between selected sample and all adults admitted to the er in the same period. however, as expected, the median duration of symptoms was lower in the selected seven samples considered to be unsatisfactory for ifi were positive by pcr. another 50 samples that were negative in ifi were positive by pcr. of the 37 samples positive in ifi, 18 were sent for pcr too, and in nine, the result was positive. in seven of these cases, the results of ifi and pcr were concordant. in one patient, adenovirus was identified on ifi and influenza a was found in pcr. in another case, ifi detected piv type 2, and pcr identified piv types 1 and 3. the characteristics of patients with pcr positive and negative were shown in table 3 . figure 1 shows timesseries graph for virus percent positive by ifi and by pcr by month. table 4 shows the descriptive statistics corresponding to the environmental variables considered in this study. figure 2 shows the modeled and observed values for ili and sari cases. figures 3 and 4 show the daily number of patients with ili and sari and meteorological parameters. we checked the autocorrelation between the covariates of climate data and observations from previous days, and we considered the following lags (in days) for each variable: average temperature (5), rainfall (2), sunshine duration (6), relative humidity (5), mean concentration of pollutants (3), and absolute humidity (2) . the number of ili and sari cases tends to be higher between july 5, 2009, and august 22, 2009 . in this period, the mean temperatures (tmin: 9á52 ae 3á89°c and tmax: 18á9 ae 4á92°c) and the sunshine duration (4á13 ae 3á31 hours of sun per day) were lower, as expected in winter. the rainfall tends to be higher than the median calculated for the entire year (0 mm, iqr: 0-5á4 mm). in addition, the absolute humidity (ah; 7á8 ae 2á1 g/kg) and mean concentration of pollutants (20á0 ae 7á0 lm/m 3 ) were lower in this period compared with the annual values. table 5 shows multivariate logistic regression model for ifi and pcr. we included 255 patients in logistic regression for ifi and 180 patients in logistic regression for pcr, because we have climate data only for the first year of study. ifi positivity was statistically associated with ah (or: 0á72; 95% ci 0á59-0á86), use of air conditioning (or: 4á16; 95% ci 1á45-11á83), and presence of mold in home (or: 2á95; 95% ci 1á10-8á29). on the other hand, pcr positivity was statistically associated with use of air conditioning (or: 2á27; 95% ci 1á04-4á97), average temperature (or: 0á92; 95% ci 0á86-0á98), and mean concentration of pollutants (or: 1á04; 95% ci 1á00-1á08). the multivariate time-series models for ili and sari cases are summarized in table 6 . sunshine duration was the only independent covariate that was significantly associated with the frequency of ili cases. the b-coefficient for this parameter was negative, indicating increasing ili frequency with decreasing sunshine duration. in the model for sari cases, the following variables proved to be significant: mean temperature (b = 0á399; p = 0á025), sunshine duration (b = à0á392; p = 0á007), rh (b = à0á098; p = 0á05), and mean concentration of pollutants (b = à0á079; p = 0á018). acute rvis are responsible for causing significant levels of morbidity and mortality. the most common respiratory syndrome caused by these pathogens is ili. a more severe presentation, named sari, was also related to some rvs. 16, 17 in this study, we have examined the relationship between ili and sari cases, meteorological variables, and air pollution using multivariate time-series analyses. we found that ili cases were inversely correlated with sunshine duration. in addition, sari cases were significantly associated with mean temperature, sunshine duration, rh, and concentration of pollutants. seasonal cycles of infectious diseases have been attributed to changes in atmospheric variables, the prevalence or virulence of the pathogen, or the behavior of the host. 18 earlier investigations have demonstrated that lower temperatures and sunshine duration, conditions usually encountered in winter, were associated with admissions for rvi. 12, 19 temperature was found to be highly inversely correlated with rsv, influenza a, and adenovirus frequency. 12 interestingly, we found a positive correlation between temperature and sari cases. one possible explanation is that it was demonstrated that for every one degree celsius rise in temperature, the risk of premature death and acute morbidity especially among respiratory patients is up to six times higher than in the rest of the population. second, evidence is emerging that increasing temperature is associated with increases in air pollution, especially ground-level ozone, and can amplify the adverse effects of poor air quality. 20 taking this evidence into account, we could expect that higher temperatures may have increased concentration of pollutants, leading to more sari cases. however, our data showed a decrease in air pollution during the months with a higher prevalence of sari. the third hypothesis to explain the relationship between higher temperatures and sari cases was related to el niño southern oscillation (enso) phenomenon. enso undergoes cycles between warm phases (el niño episodes) and reverse cold phases (la niña episodes). in the southern region of brazil, this phenomenon is associated with elevated temperatures and rainfall, especially in spring and in the period between may and july. previous reports have determined that el niño events were associated with increased hospitalizations and more severe influenza epidemics. 21, 22 severe acute respiratory infection cases were found to be negatively related to rh in our study. previous studies have demonstrated that higher rh decreases the survival of lipidenveloped virus, like influenza a, influenza b, rsv, and piv. [23] [24] [25] the use of indoor heating in winter lowers the rh; breathing dry air could cause desiccation of the nasal mucosa, epithelial damage, and reduced mucociliary clearance, increasing the host susceptibility to rvis. 19 however, even in tropical regions with humid climate (rh >70%), a higher activity of influenza can be found. this observation could be explained by the variation of viral stability in different rh levels. the stability of aerosolized influenza virions is maximal at lower rh (20-40%), moderate at higher rh (60-80%), and minimum at a mid-range rh (50%). 23 in a multivariate logistic regression model for ifi-positive patients, we found that ah was a protect factor for rvi. a recent study suggested that ah may better correlate with influenza virus survival and transmission. unlike rh, ah measures the actual water vapor content of air irrespective of temperature and has a prominent wintertime low, both indoor and outdoor. such findings suggest that humidification measures could be helpful decreasing survival and transmissibility of influenza. 26 air pollution has been associated with adverse health outcomes. studies have suggested acute effects causing respiratory symptoms, cardiovascular events, hospital admissions, and mortality. although the available evidences indicate associations between exposure to pollutants and increased risk of rvi, potential mechanisms mediating these effects are largely unexplored. 27, 28 surprisingly, our results showed that sari cases were associated with a decrease in mean concentration of pollutants. in fact, this could be a reflection of higher rainfall in the same period, as rain acts washing out or scattering pollutants from atmosphere. 29 on the other hand, we cannot exclude an effect of indoor pollution. in the last years, indoor pollution has been recognized as an emerging health problem, as about 90% of our time is spent indoors where we are exposed to chemical and biological contaminants. 30 we estimated indoor pollution indirectly in our study, questioning patients about the use of wood stoves and air conditioning, and the presence of mold in home. our findings suggested that ifi-positive patients were more prone to live in a residence with mold growth. dampness and mold are two important sources of indoor pollution, consistently associated with respiratory symptoms. home dampness may be a marker for mold growth, dust mites, endotoxins, and reduced ventilation, which could increase concentrations of indoor pollutants. 31 cough, wheezing, and upper respiratory symptoms were associated with dampness and mold in a metaanalysis. 32 according to these results, the prevalence of cough and wheezing was higher in patients with mold in home and ifi positive. air conditioning was also positively related to ifi test in this study. air conditioning use was associated with fewer hospital admissions for cardiovascular diseases, chronic obstructive pulmonary disease, and pneumonia on days with high concentrations of pm 10 , 33 as individuals are less exposed to outdoor pollutants. nevertheless, the majority of virus transmission occurs within indoor, air-conditioned (i.e., cooler, lower humidity) environments that favor airborne virus survival and transmission. 24, 25 in hot and humid conditions, indoor transmission in air conditioning environments may account for most of the transmission. 34 we found a prevalence of 22% of rv, which is higher than that previous studies have demonstrated (between 12% and 15% in adults). 12, 13 moreover, the length of stay was lower in our ifi-and/or pcr-positive patients. this finding is consistent with existing knowledge that virus identification allows the prompt initiation of therapy when indicated and avoids the unnecessary use of antibiotics, decreasing the length of hospital stay. the present study has some limitations. first, it was based on data collected from a single center, which may have potential biases because of the characteristics of the catchment population, like vaccination coverage. second, it is also important to note that this investigation was performed in a group of hospitalized patients, which is a bias toward the most severe disease cases. additionally, we do not have the concentrations of individual air pollutants, but it is implausible to reliably separate the effects of air pollutants because they frequently react with each other, sometimes potentiating individual effects. 10, 35 the short study period should also be considered a limitation. finally, the use of molecular techniques (pcr) in all study patients could be useful, increasing the number of viruses detected, as limited sensitivity of ifi method is well known. 10, 36 despite these limitations, this is the first study, to our knowledge, to analyze the relationship between rv, meteorological parameters, and air pollution in an adult population. in conclusion, we found that in adult patients admitted to er with respiratory complaints, at least 22% of infections were caused by rv. the correlations found among meteorological variables, air pollution, ili/sari cases, and rv demonstrated the relevance of climate factors as significant underlying contributors to the prevalence of rvi in a temperate region. there is still a need of additional investigations to clarify and confirm these data, perhaps using longer time-series observations. fundo de incentivo a pesquisa -sbpt; fipe-hcpa; fapergs. communicable respiratory threats in the ed: tuberculosis, influenza, sars, and other aerosolized infections role of respiratory viruses in acute upper and lower respiratory tract illness in the first year of life: a birth cohort study detection and typing by molecular techniques of respiratory viruses in children hospitalized for acute respiratory infection in rome, italy the contribution of influenza to combined acute respiratory infections, hospital admissions, and deaths in winter excess hospital admissions for pneumonia and influenza in persons > or = 65 years associated with influenza epidemics in three english health districts: 1987-95 community-acquired pneumonia prospective epidemiologic survey of patients with community-acquired pneumonia requiring hospitalization in switzerland frequency of detection of respiratory viruses in the lower respiratory tract of hospitalized adults incidência de infecc ßão viral do trato respirat orio em asma aguda atendida em sala de emergência surveillance of respiratory virus infections in adult hospital admissions using rapid methods are meteorological parameters associated with acute respiratory tract infections? increased susceptibility to rsv infection by exposure to inhaled diesel engine emissions ultrafine carbon black particles enhance respiratory syncitial virus-induced airway reactivity, pulmonary inflammation, and chemokine expression updated world map of the k€ oppen-geiger climate classification influenza activity-united states and worldwide, 2007-08 season influenza pandêmica (h1n1) 2009 -an alise da situac ßão epidemiol ogica e da resposta no ano de seasonal variation in host susceptibility and cycles of certain infectious diseases influenza virus transmission is dependent on relative humidity and temperature climate change and respiratory disease: european respiratory society position statement association of normal weather periods and el nino events with hospitalization for viral pneumonia in females: california, 1983-1998 respiratory virus and the effects of climate ª 2013 the authors. influenza and other respiratory viruses published by survival of airborne influenza virus: effects of propagating host, relative humidity, and composition of spray fluids the effect of environmental parameters on the survival of airborne infectious agents aerosol transmission of influenza a virus: a review of new studies absolute humidity modulates influenza survival, transmission, and seasonality air pollution and health ambient air pollution and health climate change and human health the index project: executive summary of a european union project on indoor air pollutants variations of formaldehyde and voc levels during 3 years in new and older homes meta-analyses of the associations of respiratory health effects with dampness and mold in homes air conditioning and source-specific particles as modifiers of the effect of pm(10) on hospital admissions for heart and lung disease incidence of common respiratory viral infections related to climate factors in hospitalized children in hong kong air pollution and cardiovascular disease: a statement for healthcare professionals from the expert panel on population and prevention science of the american heart association advances in the laboratory diagnosis of viral respiratory disease the authors have no competing interests to disclose. key: cord-353308-e4s8el0s authors: parashar, umesh d; anderson, larry j title: severe acute respiratory syndrome: review and lessons of the 2003 outbreak date: 2004-05-20 journal: int j epidemiol doi: 10.1093/ije/dyh198 sha: doc_id: 353308 cord_uid: e4s8el0s nan , the chinese ministry of health notified the world health organization (who) of an outbreak of atypical pneumonia that likely emerged in guandong province, china, in november 2002. 1 during late february to early march 2003, clusters of atypical pneumonia were recognized in vietnam, hong kong, canada, and singapore. [2] [3] [4] [5] [6] epidemiological investigations revealed that the index patients for each of these clusters had stayed on the ninth floor of a hotel in hong kong on 21-22 february (figure 1 ). further investigation indicated that the likely source of the outbreak was a physician from guandong province (case a) who stayed on the same hotel floor on 21 february. this physician had cared for patients affected by the respiratory disease outbreak and he had been symptomatic with a febrile, respiratory illness since 15 february. this dramatic chain of transmission brought to the world's attention this new respiratory disease, called severe acute respiratory syndrome (sars), and clearly illustrated the potential for sars to spread extensively from a single infected person and to rapidly disseminate globally through air travel. the who issued an historic global alert 7 and, together with its international partners, initiated a rapid and intense response to this global public health emergency. the response led within 2 weeks to the identification of the aetiological agent, sars-associated coronavirus (sars-cov), [8] [9] [10] [11] and to a series of decisive and effective containment efforts that interrupted the last chain of human transmission in less than 4 months. 12 in this article, we review what has been learned about the aetiological agent and its pathogenesis and pathology, clinical manifestations, epidemiology, and diagnosis plus strategies for control of sars. sars-cov is an enveloped, positive-stranded, rna virus in the coronaviridae family ( figure 2 ). coronaviruses are associated with a variety of enteric and respiratory disease syndromes in several animal species. the two recognized human coronaviruses other than sars-cov, oc 43, and 229 e, have definitively been associated only with upper respiratory illness. sars-cov was first cultured in vero e6 cells from autopsy tissue, bronchoalveolar lavage, and nasal swab samples obtained from patients hospitalized in hong kong, canada, germany, and singapore, with identification by electron microscopy and complete genomic sequencing. the aetiological role of sars-cov was confirmed by the replication of an illness similar to that seen among humans in experimentally infected cynomolgus macaques, followed by re-isolation of the virus and detection of a specific immune response in the infected macaques. 13, 14 the genomic sequence of sars-cov is quite distinct from that of other human and animal coronaviruses, 15, 16 suggesting that the virus has likely circulated in its natural reservoir for a considerable period of time. the search for the origins of sars-cov and its potential reservoir(s) is ongoing. as many as 42% of the early sars cases in guandong occurred among people who were involved in animal trade or in food preparation, 17 and people involved in animal trade in guandong were more likely to have antibodies to sars-cov than those who did not trade animals or general population controls. 18 in addition, a coronavirus with 99% homology with human sars-cov isolates was recovered from civet cats and a racoon dog sold live for food in markets in guangdong. 19 collectively, these observations support the hypothesis that sars-cov was first transmitted from wild animals used for food to humans, with subsequent personto-person transmission. experimental studies have shown that ferrets and domestic cats are also susceptible to infection by sars-cov and that they can efficiently transmit the virus to previously uninfected animals that are housed with them. 20 further clues to the animal reservoir for sars-cov might be provided by studies of wild and domesticated animals from animal markets in guandong and the genomic sequence analysis of any viral isolates obtained from these species as well as from human patients. studies have also demonstrated the presence of antibody to sars-cov or a sars-cov-like virus in a small proportion of healthy adults in hong kong who had sera banked in may 2001, at least 2 years prior to the sars outbreak. 21 this finding suggests that sars may have been transmitted periodically in the past from animals to humans and the virus may have evolved and adapted to human infection and transmission 22 or that chance events resulting in efficient transmission led to the outbreak in 2003. angiotensin-converting enzyme 2 (ace2) was recently identified as a functional sars-cov receptor, 23 and ace2 is efficiently expressed in lung, heart, kidney, and gastrointestinal tissue in humans. sars-cov has been isolated in cell culture in autopsy specimens from lung, intestinal, and kidney tissue of patients who died of sars. 24,25 sars-cov rna can also be detected by reverse transcription-polymerase chain reaction (rt-pcr) assays in the plasma and serum of more than 50% of patients during the first week of illness. 26 pathological findings in the lungs of sars patients during the first 10 days of illness include pneumocyte proliferation and desquamation, hyaline membrane formation, mixed inflammatory infiltrate, and intra-alveolar oedema. 27, 28 increased numbers of interstitial and alveolar macrophages, with focal haemophagocytosis in interstitial macrophages, have also been described. in cases of longer duration, diffuse alveolar damage, squamous metaplasia, and multinucleated giant cells, of macrophage origin, are observed. biopsy and autopsy specimens from the colon and terminal ileum of sars patients show relatively normal architecture with no evidence of villous atrophy or inflammatory infiltrates. 29 no distinct histopathological changes have been described in other tissues. the role of cytokines and immunopathogenic mechanisms in sars are being investigated. lymphopaenia is common among patients with sars. while cd4 t lymphocytes counts are reduced in nearly all sars patients, reductions in circulating levels of cd8 t lymphocytes, b lymphocytes, and natural killer cells are also common. 30, 31 cd4 and cd8 lymphocyte counts fall early in the course of illness, and low cd4 and cd8 levels have been associated with more severe illness. the presence of lymphopaenia and pathological manifestations of diffuse alveolar damage, destruction of the white pulp of the spleen, and haemophagocytosis suggest that proinflammatory cytokines released by stimulated alveolar macrophages may have a major role in the pathogenesis of sars. 27 death from sars usually occurs late in the course of illness (ͼ1 week after onset) and has been attributed to adult respiratory distress syndrome, multiorgan failure, thromboembolic complications, secondary infections, and septic shock. coinfection with human metapneumovirus has been reported among patients with laboratory-confirmed sars; 32 however, the role of co-infection in amplifying the pathogenicity of sars-cov is unknown. sars-cov disease has a median incubation period of approximately 4-6 days; most patients become ill within 2-10 days after exposure. the most common initial symptom is fever, often accompanied by headache, myalgias, malaise, chills, and rigor. respiratory symptoms typically do not begin until 2-7 days after onset of fever, but may be present at onset in up to one-third of patients. lower respiratory tract symptoms are common and typically include a non-productive cough with later onset of dyspnoea. upper respiratory symptoms such as rhinorrhoea and sore throat are seen in less than 25% of patients. diarrhoea has been reported at presentation in approximately 25% of patients, although this symptom was observed in as many as 73% of all patients affected by an outbreak at an apartment complex in hong kong that is believed to have resulted from fecal-oral/respiratory transmission of sars-cov. 33 respiratory signs such as rales and rhonchi are present in less than one-third of cases, and their severity often does not correlate with the findings seen on chest radiographs. elderly patients and those with underlying chronic illnesses such as renal failure sometimes present with atypical symptoms, including the absence of fever. 34 in general, sars appears to be milder in children. 35, 36 haematological abnormalities include lymphopaenia (70-90% of patients), thrombocytopaenia (30-50%), prolongation of the activated partial thromboplastin time (40-60%), and elevated levels of lactate dehydrogenase (70-90%), alanine or aspartate aminotransferases (20-30%), and creatinine phosphokinase (30-40%). none of these laboratory findings can reliably discriminate between sars-cov disease and other atypical pneumonias. chest radiographs may be normal in up to 30% of patients who are evaluated early in illness, and high-resolution chest computed tomography scans can detect abnormalities in up to two-thirds of patients with normal chest x-rays. [37] [38] [39] [40] [41] [42] [43] [44] nearly everyone with sars-cov disease develops radiographic evidence of pneumonia by illness day 7-10. the radiographic abnormalities typically begin as isolated, focal, ground glass opacities predominately in a peripheral location, often in the lower lobes, and progress over several days to focal, multifocal, or diffuse consolidation involving additional lobes or both lungs; mediastinal lymphadenopathy, cavitation, atelectasis, and pleural effusions are uncommon. some 15-25% of patients with sars-cov disease require mechanical ventilation, and about half of these people die despite ventilation therapy. severity of illness and risk of death from sars increases with advancing age and in the presence of certain underlying medical conditions, particularly diabetes mellitus. 45 the overall case-fatality rate of approximately 10% can increase to ͼ50% in people older than age 60. during the 2003 global epidemic, 8098 cases of probable sars with 774 (9.6%) deaths were reported in 29 countries. 46 the countries with the greatest number of reported cases included mainland china (n = 2674), hong kong special administrative region (n = 977), taiwan (n = 218), singapore (n = 161), and canada (n = 151). health care facilities were severely affected and transmission in hospitals was a major factor in the amplification of outbreaks. in addition to health care workers, who accounted for 21% of all cases globally, other patients on the same ward as sars patients and visitors to the hospital were affected. for example, of 74 cases reported from 15 april to 9 june in toronto, canada, 39% occurred among health care workers, 38% resulted from exposure during hospitalization, and 23% occurred among hospital visitors. 47 after health care facilities, households of sars patients were the second most common setting of sars-cov transmission. surveys in hong kong and singapore indicated that 6-8% of people in the households of sars patients may have been secondarily infected. 48, 49 the risk of transmission was greatest among those involved in direct patient care or other close contact with the patient. transmission to casual and social contacts appears to be uncommon, but secondary cases have been documented after exposures in the workplace and on airplanes and other conveyances. 50, 51 the epidemiology of sars outbreaks suggests that sars-cov is transmitted primarily through droplets and close personal contact, a conclusion that is supported by the finding that surgical masks confer protection against infection. 51, 52 studies documenting stability of the virus for days in the environment raise the possibility of fomite transmission, and in a few instances, possible transmission by small-particle aerosols cannot be excluded. in particular, aerosol-generating medical procedures (e.g. endotracheal intubation, bronchoscopy) may be associated with an increased risk of transmission in health care settings. [53] [54] [55] [56] given that profuse watery diarrhoea is seen in a significant proportion of patients and sars-cov can be shed in large quantities in stool, faeces remain a possible source of virus and fecal-oral or fecal-respiratory spread are the leading hypotheses for a large outbreak affecting more than 300 people at an apartment complex in hong kong. 9 mathematical modelling studies indicate that each sars case infects an average of two to four people. [57] [58] [59] however, some sars patients are very efficient in transmitting sars-cov to susceptible people under certain circumstances, leading to socalled 'super-spreading events'. 51, [53] [54] [55] [56] 60 super-spreading events have occurred in a number of settings, including the hotel in hong kong, from which the sars outbreak disseminated globally, and hospitals in many locations. the factors contributing to super-spreading events are not completely understood but may be related to the inherently greater infectiousness of some patients, alternative modes of transmission, or the exposure of large numbers of contacts in an environment conducive to transmission. asymptomatic sars-cov infections have been documented, 61 but they appear to be uncommon and their epidemiological significance in disease transmission has not been established. the risk of transmission from patients appears to be greatest during the second week of illness, 51 which correlates with the timing of peak viral load in respiratory secretions. 9 the timing of peak infectiousness might also explain, in part, the predilection for sars-cov to spread in health care settings, as most patients seek medical care by the end of the first week of illness. although sars-cov rna can be detected in fecal specimens by rt-pcr for more than a month after the onset of illness, no transmission has been documented more than 10 days after the resolution of fever. genome sequencing analyses indicate that sars-cov isolates from the outbreaks in hong kong, vietnam, singapore, toronto, and taiwan are closely related and match the viral isolate obtained from the ill physician from guandong province, supporting the epidemiological conclusion that each of these outbreaks was directly or indirectly linked to the ill physician. 62 on the other hand, sars-cov isolates from guangdong province exhibit greater diversity and other viruses belonging to genetic lineages different from the global outbreak strain were introduced into hong kong prior to march 2003, but these introductions did not lead to large outbreaks. these data raise the question of whether some viral strains may have an inherently greater epidemic potential; this hypothesis needs further evaluation. alternatively, differences in transmission unrelated to virus strain (i.e. super-spreading events) could explain the presence or absence of outbreaks associated with independent human infections. the clinical manifestations of sars-cov disease are not sufficiently distinct from those of other respiratory pathogens to permit a reliable differential diagnosis. however, a combination of clinical and epidemiological features can provide clues to the diagnosis of sars. 63 with no documented person-to-person transmission of sars-cov worldwide, the predilection for sars-cov to cause unusual clusters of pneumonia in health care settings provides a means to focus on surveillance efforts. if the re-emergence of sars-cov is confirmed, then a history of exposure to known sars casepatients or sars-affected areas may be helpful in early recognition of patients. the laboratory diagnosis of sars-cov is based on either detection of viral rna in clinical specimens or the finding of antibodies directed against sars-cov in serum. viral isolation is not recommended for routine diagnosis because of its low sensitivity and the biosafety hazards it poses. available real-time rt-pcr assays for sars-cov are highly specific and can detect as few as 1-10 copies of viral rna in clinical specimens, but a low viral load in respiratory and fecal specimens during the first week of illness limits the clinical sensitivity and utility of these assays. limited data suggest that sars-cov rna can be detected in the serum of more than 50% of sars patients during the first week of illness. 26 because of the potential for contamination, positive rt-pcr results should be confirmed by testing a second specimen in a reference laboratory. 64 the identification of serological antibody to sars-cov remains the reference standard for confirmation of sars-cov infection. however, antibody is usually detectable only after the first week of illness and some patients may not mount an antibody response for up to 21-28 days after illness onset. it is important to note that neither a negative rt-pcr test nor a negative test of serum obtained ͻ28 days after illness onset can reliably exclude infection with sars-cov. body fluids that should be submitted for testing from patients with suspected sars include respiratory tract secretions, serum, whole blood, and stool. lower respiratory tract specimens (e.g. sputum, bronchial alveolar lavage fluid) appear to have higher yield than upper respiratory tract specimens (e.g. nasal or pharyngeal aspirates or swabs). collection of multiple specimens of different types is likely to increase the overall diagnostic yield, and different specimens will have a greater diagnostic yield at different times in illness (table 1) . sars-cov test results should always be considered in the context of clinical and epidemiological findings. in the setting of no sars human-to-human transmission worldwide, the positive predictive value of a sars-cov test is extremely low, and thus any positive laboratory result must be interpreted with extreme caution because of its implications for global public health. diagnostic assays for other respiratory pathogens may be helpful in differentiating sars-cov disease from other illnesses, but sars patients may be simultaneously infected with sars-cov and another respiratory pathogen. factors that should be considered in the evaluation of patients with dual infections include the strength of the epidemiological exposure criteria for sars-cov disease, the specificity of the alternate diagnostic test, and the compatibility of the clinical presentation and course of illness with the alternative diagnosis. in the absence of a vaccine, effective drugs, or natural immunity to sars-cov, the key to controlling sars is the classic public health control strategy of case identification and containment. these measures proved effective and were associated with cessation of transmission throughout the world by july 2003. the key to containment efforts is a surveillance system that provides ready access to timely information on the number of new cases, the likely source of exposure for cases, the number of cases not previously identified as contacts, and the number of contacts with high-risk exposures to known cases (potential prospective cases). since sars anywhere has implications locally, nationally, and globally, it is essential that the health care and public health communities exchange information on individual sars cases and the status of sars transmission in the community and globally. once a potential case of sars is detected, appropriate infection control measures must be implemented immediately to prevent transmission. sars patients often require hospitalization because of the severity of their illness; those with less severe illness are sometimes hospitalized to ensure that strict isolation procedures are followed. in other settings, patients have been managed in residential settings after evaluation of their suitability for this purpose. because the possibility of airborne transmission cannot be excluded, patients who require hospitalization should preferably be admitted to an airborne infection isolation room or specially adapted sars unit or ward where they can be treated safely and appropriately. in some settings, a lack of isolation rooms and/or a need to concentrate infection control efforts and resources may lead to a strategy of grouping patients in individual rooms on the same floor, which proved effective in many settings in the 2003 outbreak. health care workers should strictly adhere to use of appropriate personal protective equipment, and the potential for transmission to other non-sars patients and hospital visitors should be minimized through administrative controls. contact tracing, the identification of people potentially exposed to a case of sars, is essential to prevent spread. this provides a means to focus control efforts on people who are at high risk of sars, to identify people early in the course of their illness, and to implement control measures before they can spread the virus to others. contact tracing, evaluation for illness, and monitoring should be an immediate high priority to maximize the chance to rapidly control an outbreak. during the 2003 outbreak, quarantine of exposed people was one of the contact management strategies used to prevent inadvertent sars-cov exposures by separating those exposed from the unexposed. this potentially decreased the interval between the onset of symptoms and the institution of control measures. other key components of an effective sars control strategy include the following: (1) systems for rapid and frequent communication of crucial information about sars; (2) education and training of public health and health care workers about the clinical and epidemiological aspects of sars, appropriate use and interpretation of laboratory tests, and best practices for effective use of infection control strategies; and (3) efficient information technology systems that provide a means to link clinical, epidemiological, and laboratory data on sars cases and to disseminate this information locally, nationally, and globally, and systems that allow rapid identification, tracking, evaluation, and monitoring of contacts of sars cases. the emergence of sars-cov dramatically illustrates the potential for a new disease to suddenly appear and spread, leading to widespread health, social, and economic consequences. fortunately, the experience also demonstrates the power of traditional public health measures-including surveillance, infection control, isolation, and quarantine-to contain and control a sars outbreak. it is not possible to predict when and where sars-cov will reappear and whether it will cause similar outbreaks in the future. possible sources of virus for a re-emergence of sars-cov include its original animal reservoir, persistent infection in humans, or the laboratory. the recently detected sars-cov cases in china are hypothesized to represent animal-to-human transmission, 65, 66 and each of the laboratory-acquired cases in singapore and taiwan highlight the need for stringent biosafety precautions. 67, 68 to achieve the type of swift and decisive response that is required to control a sars outbreak, we must be prepared and have learned from the many lessons of the 2003 sars outbreak. preparation for a response to an outbreak of sars requires co-ordination and co-operation among public health, health care, and other emergency response entities at all levels of government. investments in sars preparedness are likely to yield additional dividends in preparedness to battle other emerging infectious diseases and other threats to public health. epidemiological and aetiological studies of patients with severe acute respiratory syndrome (sars) from guangdong in a major outbreak of severe acute respiratory syndrome in hong kong a cluster of cases of severe acute respiratory syndrome in hong kong identification of severe acute respiratory syndrome in canada clinical features and short-term outcomes of 144 patients with sars in the greater toronto area severe acute respiratory syndrome-singapore world health organization. who issues a global alert about cases of atypical pneumonia [press release world health organization multicentre collaborative network for sars diagnosis. a multicentre collaboration to investigate the cause of severe acute respiratory syndrome coronavirus as a possible cause of severe acute respiratory syndrome identification of a novel coronavirus in patients with severe acute respiratory syndrome a novel coronavirus associated with severe acute respiratory syndrome update 96-taiwan, china: sars transmission interrupted in last outbreak area koch's postulates fulfilled for sars virus newly discovered coronavirus as the primary cause of severe acute respiratory syndrome the genome sequence of the sars-associated coronavirus characterization of a novel coronavirus associated with severe acute respiratory syndrome role of china in the quest to define and control severe acute respiratory syndrome prevalence of igg antibody to sars-associated coronavirus in animal traders-guangdong province isolation and characterization of viruses related to the sars coronavirus from animals in southern china sars virus infection of cats and ferrets sars-related virus predating sars outbreak, hong kong. emerg infect dis the chinese sars molecular epidemiology consortium. molecular evolution of the sars coronavirus during the course of the sars epidemic in china angiotensin-converting enzyme 2 is a functional receptor for the sars coronavirus fatal severe acute respiratory syndrome is associated with multiorgan involvement by coronavirus (sars-cov) sars coronavirus-infected cells in lung detected by new in situ hybridization technique detection of sars coronavirus in plasma by real-time rt-pcr lung pathology of fatal severe acute respiratory syndrome lung pathology of severe acute respiratory syndrome (sars): a study of 8 autopsy cases from singapore enteric involvement of severe acute respiratory syndrome-associated coronavirus infection expression of lymphocytes and lymphocyte subsets in patients with severe acute respiratory syndrome haematological manifestations in patients with severe acute respiratory syndrome: retrospective analysis human metapneumovirus detection in patients with severe acute respiratory syndrome clinical progression and viral load in a community outbreak of coronavirus-associated sars pneumonia: a prospective study atypical sars in geriatric patient. emerg infect dis [serial online children hospitalized with severe acute respiratory syndrome-related illness in toronto severe acute respiratory syndrome (sars) in children: epidemiology, presentation and management thin-section ct of severe acute respiratory syndrome: evaluation of 73 patients exposed to or with the disease severe acute respiratory syndrome: radiographic appearances and pattern of progression in 138 patients thin-section ct in patients with severe acute respiratory syndrome following hospital discharge: preliminary experience radiologic manifestations of severe acute respiratory syndrome sars: imaging of severe acute respiratory syndrome severe acute respiratory syndrome: radiographic and ct findings severe acute respiratory syndrome: radiographic review of 40 probable cases in toronto imaging of severe acute respiratory syndrome in hong kong outcomes and prognostic factors in 267 patients with severe acute respiratory syndrome in hong kong world health organization. cumulative number of reported probable cases of severe acute respiratory syndrome (sars) update: severe acute respiratory syndrome-toronto, canada secondary household transmission of sars probable secondary infections in households of sars patients in hong kong. emerg infect dis [serial online transmission of the severe acute respiratory syndrome on aircraft consensus document on the epidemiology of severe acute respiratory syndrome (sars) who/cds/csr/gar/2003.11. geneva: world health organization effectiveness of precautions against droplets and contact in prevention of nosocomial transmission of severe acute respiratory syndrome (sars) investigation of a nosocomial outbreak of severe acute respiratory syndrome (sars) in toronto, canada illness in intensive care staff after brief exposure to severe acute respiratory syndrome cluster of sars among medical students exposed to single patient, hong kong. emerg infect dis [serial online possible sars coronavirus transmission during cardiopulmonary resuscitation. emerg infect dis [serial online transmission dynamics and control of severe acute respiratory syndrome transmission dynamics of the etiological agent of sars in hong kong: impact of public health interventions modeling the sars epidemic emerg infect dis [serial online asymptomatic severe acute respiratory syndrome-associated coronavirus infection molecular epidemiology of the novel coronavirus that causes severe acute respiratory syndrome combining clinical and epidemiologic features for early recognition of sars. emerg infect dis [serial online sars international reference and verification laboratory network: policy and procedures in the inter-epidemic period update 4: review of probable and laboratory-confirmed sars cases in southern china new case of laboratory-confirmed sars in guangdong, china-update 5 world health organization. severe acute respiratory syndrome (sars) in singapore world health organization. severe acute respiratory syndrome (sars) in taiwan, china key: cord-318591-ssnlfjap authors: pecego, ac; amâncio, rt; costa, dm; bozza, fa; siqueira, mm; oliveira, ml; cerbino-neto, j; japiassu, a title: etiology, clinical, and epidemiological characteristics of severe respiratory infection in people living with hiv date: 2020-01-22 journal: int j std aids doi: 10.1177/0956462419882587 sha: doc_id: 318591 cord_uid: ssnlfjap people living with hiv (plwh) are more prone to severe respiratory infections. we used the severe acute respiratory infection (sari) definition to describe the etiology, clinical, and epidemiological characteristics in this population. this was a prospective observational study including plwh hospitalized with fever and cough. those with symptom onset up to 10 days were classified as severe acute respiratory infection and 11–30 days as non-severe acute respiratory infection. blood, urine samples and nasopharyngeal swabs were collected. data were extracted from patient charts during their hospital stay. forty-nine patients were included, median cd4 cell count: 80 cells/mm(3), median time since hiv diagnosis and hospital admission: 84 months and 80% were antiretroviral therapy exposed. twenty-seven patients were classified as sari. etiology was identified in 69%, 47% were polymicrobial. respiratory virus (9 sari vs. 13 non-sari), bacteria (5 sari vs. 4 non-sari), mycobacterium tuberculosis (6 sari group vs. 7 non-sari group), pneumocystis jirovecii (4 sari vs. 1 non-sari), cryptococcus neoformans (1 sari vs. 3 non-sari), and influenza a (1 sari vs. 2 non-sari). dyspnea was statistically more prevalent in sari (78% vs. 36%, p = 0.011) but the risk of death was higher in the non-sari (4% vs. 36%, p = 0.0067). in the severely immunocompromised plwh, severe acute respiratory infection can be caused by multiple pathogens and codetection is a common feature. people living with hiv (plwh) are highly susceptible to respiratory infections. 1 even in the antiretroviral therapy (art) era, tuberculosis (tb) has fallen disproportionately among seropositive patients 2 and pneumonia remains five times more common among this population, despite achieving cd4 cell counts above 500 cells/mm 3 . 3 plwh are also at increased risk for poor influenza outcomes, which may be particularly risky for those living in countries with limited resources. [4] [5] [6] after the recent influenza a (h1n1, h5n1, and h7n9) and the middle east respiratory virus (mers-cov) outbreaks, the who is encouraging and supporting countries to strengthen surveillance on severe acute respiratory infections (sari) 7 but with limited information on plwh regarding etiology and prognosis, despite their increased risk for respiratory infections and adverse outcomes. [8] [9] [10] so, in this study, we described how sari is represented, according to clinical presentation, epidemiology and etiology in a population of plwh with respiratory infection residing in a high-prevalence tb area. a prospective observational study was conducted at the instituto nacional de infectologia evandro chagas (ini-fiocruz), rio de janeiro, brazil, from may 2012 to december 2013. ini-fiocruz is a national reference center for infectious diseases and has been a reference center for care, research, and training related to hiv/aids since 1986. the aids program at ini is one of the largest providers of primary, specialty, and tertiary care for plwh patients in rio de janeiro state with a cohort of about 4000 patients in active followup. we conducted surveillance of every hiv-positive patient hospitalized during the study period above. every monday, wednesday, and friday we carried out reviews of inpatient medical records and all plwh hospitalized with respiratory symptoms were screened by the principal investigator, a physician specialized in infectious diseases. in the presence of fever and/or cough and onset of symptoms for less than 30 days, the patient was invited to participate in the study, which involved the collection of clinical material as detailed below in the sample collection and processing section. patients were excluded if symptoms occurred more than 30 days ago or if no fever and/or no cough were reported by the patient. patients were classified as sari if the onset of symptoms was 10 days, according to who global standards for sari definition. 7 those with symptom onset greater than 10 days but up to 30 days prior were considered as non-sari patients ( figure 1 ). participants were screened by a physician regarding respiratory viral symptoms such as myalgia, diarrhea, arthralgia, coryza, and odynophagia. data on dyspnea, respiratory frequency, and peripheral oxygen saturation (spo 2 ) using pulse oximetry were collected by a respiratory therapist. patients were considered to have dyspnea if rated 5 on an ascending scale from 0 to 10. clinical and epidemiological data were collected during hospitalization using standardized forms, until hospital discharge or death ( table 1 ). variables of interest were age, gender, time since hiv diagnosis based on the first hiv-positive result, cd4 nadir cell count, and cd4 cell count at hospital presentation or up to 2 months before, having had a previous tb diagnosis (pulmonary or disseminated) or any other previous opportunistic infection. viral load was considered to be undetectable if less than 400 copies/ml, and we considered being art exposed if patient ever used art regardless of adherence. prophylaxis of respiratory infection was recorded, such as pneumocystis jirovecii pneumonia (pjp) prophylaxis and pneumococcal and influenza vaccines. data about the respiratory infection included leucocyte count, c-reactive protein, pulse oximetry, and crb-65 and the combined crb-65 þ o 2 score at admission. outcome variables of interest were: intensive care unit (icu) admission, noninvasive ventilation use beginning within 72 h of hospital admission and death. microbiologically-confirmed pneumonia was defined as fever (axillary temperature) 38 c, (new) pulmonary infiltrate on chest x-ray or computed tomography scan plus an etiological agent identified through the tests mentioned below. for every patient included we collected blood cultures for bacteria, fungi, and mycobacteria processed with the automated bactalert v r system (healthcare-bio merieux), urine samples for urinary streptococcus pneumoniae and legionella pneumophila serogroup 1 testing (binaxnow kit v r ) and triple respiratory swab (nasopharyngeal, one for each nostril and one for posterior pharynges). respiratory swabs were tested for respiratory syncytial virus (rsv), adenovirus (adeno), influenza a (flua), parainfluenza 1-3 (piv 1-3), human-metapneumovirus (hmpv), and rhinovirus (rv) using rt-pcr. probes were labeled at the 5 0 terminus with 6 carboxyfluorescein (fam) and with quencher blackhole-1 dt (biosearch technologies, inc., novato, ca). the tests were done following centers for disease control and prevention (cdc) guidance. sputum or induced sputum and bronchoalveolar fluid were tested for fungus and mycobacterium spp. under direct visualization followed by inoculation into sabouraud culture medium for fungi and bactec v r mgit tm 960 and lowenstein-jensen for mycobacterial culture. mycobacterium spp. identification was performed under conventional techniques (kent and kubica, 1985, cdc atlanta). all clinical samples were collected up to 72 h after hospital admission except for p. jirovecii detection which was done through staining and indirect immunofluorescence assay (ifl) on bronchoalveolar samples, which were performed only in mechanically-ventilated patients. table 1 . clinical and epidemiological characteristics of patients with less than 10 days since symptom onset (sari) vs patients with more than 10 days since symptom onset (non-sari). total we conducted a descriptive analysis of the convenience sample from the studied population. categorical variables were presented as proportion and continuous variables summarized as median and interquartile ranges for sari and non-sari patients. we compared the distribution of continuous variables by using u or mann-whitney test, and categorical variables by using the chi-squared or fisher's exact test when appropriate at univariate analysis. the analyses were processed using statistical package for social science (spss) and graphpad prism 3.0 for windows (graphpad software, san diego, ca, usa). statistical significance was considered whenever p value was less than 0.05. a total of 149 patients were screened and 74 patients met our inclusion criteria. among those, 8 patients refused to participate because of discomfort and pain during the nasal swab collection and 15 patients could not be included due to temporary lack of supplies. a total of 49 patients with fever and cough and symptoms lasting up to 30 days were included (figure 1 ), of which 27 (55%) met the sari case definition. a profound and prolonged immunosuppression is reflected by the median nadir lymphocyte cd4 cell count (58 cells/mm 3 ) and at hospital admission (80 cells/mm 3 ) and time since hiv diagnosis with a median of 84 months, except for five patients with recent hiv diagnosis (in the previous three months). this finding is supported by the fact that 38 (80%) patients were art exposed, but only 12 (25%) had an undetectable viral load. prevention of respiratory infection was observed in only 7 (14%) patients taking prophylactic sulfamethozaxole-trimethoprim for pjp, 7 patients (14%) who previously received s. pneumoniae vaccine and 9 (18%) received influenza seasonal vaccine. about half of the patients had a previous history of tb and 18 (37%) had a history of other opportunistic infections. pneumonia was confirmed in 46 (94%) patients and 31 (63%) had x-rays with multilobar infiltrates. in the remaining three patients, the chest x-ray was normal and ct scan could not be done (data not shown). pulse oximetry showed that 15 patients (30%) had spo 2 less than 90% and non-invasive ventilation was initiated in 67% of the patients in the first 72 h of admission. twenty (41%) patients were classified as crb-65 ¼ 0 points, 19 patients classified as crb-65 ¼ 1 (39%) point, 9 (18%) as crb-65 ¼ 2 points and 1 as crb-65 ¼ 3 points. although 39 patients had crb-65 0-1, the majority required non-invasive ventilation (niv) in the first 72 h of hospitalization and 25 had chest x-rays with multilobar infiltrates. the mortality risk for those 10 patients (20.4%) with crb-65 2-3 points was 4.5 higher (95% confidence interval [ci]: 0.94-21.92) compared to crb-65 0-1 points, p value ¼ 0.069, with sensitivity (s) and specificity (sp) of 85% and 44% and positive predictive value (ppv) and negative predictive value (npv) of 87% and 40%, respectively. when combined with pulse oximetry, 15 patients (30.6%) had crb65þo 2 of 2-4 points and a 10.5 higher risk of dying (95% ci: 1.87-59.1), p value ¼ 0.0051 with a s:75% and sp:78% and ppv: 94% and npv: 42% (data not shown). in total, nine patients (18.6%) died during hospitalization. the median time between symptom onset and hospital admission was 10 days (6) (7) (8) (9) (10) (11) (12) (13) (14) (15) (16) (17) (18) (19) (20) . according to sari classification, we saw that clinical and epidemiological characteristics were evenly distributed with no statistical difference between sari and non-sari patients, except for mortality rate, which was significantly more prevalent among non-sari patients (table 1) . symptoms related to respiratory viral infection were analyzed among sari and non-sari patients (figure 2 ). fever and cough were present in 100% of cases because they were used as inclusion criteria. dyspnea was the only symptom that was statistically more prevalent in the sari subgroup, p ¼ 0.011 ( figure 2) . arthralgia, conjunctivitis, odynophagia, and coryza were all infrequent in both subgroups. microbiological confirmation was possible in 34 (69%) patients and 16 (47%) patients had polymicrobial infections ( table 2 ). viral pathogens were the most common agents, occurring in 17 (35%) patients with 22 isolates. rv was the most common virus among sari (4; 15%) and non-sari (7; 32%) patients, followed by rsv in (5; 10%); the majority (4/5) in the non-sari group, influenza a in 3 (6%), hmpv in 2 (4%), and parainfluenza-2 in 1 (2%) patient. all three cases of influenza a happened in non-influenzavaccinated patients, but since the vaccination was infrequent, we could not explore its impact. influenza a (pdm09)h1n1 was confirmed in one patient classified as sari, and the other two influenza viruses were identified among patients whose symptom onset was greater than 10 days ago, both seasonal ah3n2 strains. the two isolates of hmpv and the only piv-2 were identified in patients with sari. mycobacterium tuberculosis (mtb) was the most common bacterial pathogen identified in 13 (27%) patients (table 2) . among the 24 patients (49%) who had been previously diagnosed with tb, 8/24 (33%) were readmitted with mtb infection the majority in the non-sari group (7/8 patients, 88%). s. pneumoniae was evenly distributed among sari (four isolates) and non-sari (three isolates), but pjp was more frequent in the sari group, while cryptococcus neoformans was more common in the non-sari group ( table 2) . among polymicrobial infections, 13 patients had 2 pathogens and 3 patients had 3 or more pathogens identified. polymicrobial infection was more frequent in the non-sari group, 9 (41%) vs. 7 (26%), but with no statistical difference. no single agent was significantly more prevalent according to this classification. the main agents associated with severity and icu admission were pjp in 80% (4/5); cryptococcus neoformans in 75% (3/4), s. pneumoniae in 43% (3/7), and pseudomonas aeruginosa in 100% (1/1) of the cases. infections were polymicrobial in 41% vs. 30% of the patients being admitted to the icu (p ¼ 0.52) and in 44% of the patients who died vs. 25% among survivors (p ¼ 0.25). we found that plwh hospitalized with fever and cough, with less than 30 days of symptoms were mostly young, with severe and prolonged immunosuppressed caused by hiv infection. there were no clinical or epidemiological aspects that could distinguish the classification of sari and non-sari. in terms of etiology, sari was represented by a broad range of infectious agents in this population and much of the lung infections were polymicrobial. in one-third of the cases, we found viruses in nasopharyngeal samples and two out of three influenza viruses were detected in non-sari patients. our findings highlight the importance of viral pathogens in community-acquired respiratory infections: viruses were implicated in 35% of respiratory symptoms in plwh and rv was the most common one. this may be due to the fact that the rv is present throughout the year regardless of specific seasonality. 11, 12 although being historically related to the common cold and to mild respiratory illness, [13] [14] [15] recent data suggest that rv can be related to more severe cases. 16 according to cohen et al. it was the principal agent of sari during a hospital surveillance period 2009-2012 in south africa, where 74% were plwh 9 and also an important agent in elderly hospitalized with sari in chile 17 as well as the causal agent of more severe cases requiring icu treatment, in codetection with s. pneumoniae. 18 most of our cases were found in co-detection with another agent suggesting that rv may contribute to more severe cases but the possibility of only being a bystander cannot be rule out in this context. in accordance to our findings, hmpv remains an infrequent cause of sari even in plwh. 19 apart from viral detection, s. pneumoniae is reemphasized as the main bacterial pathogen found in patients with cap who presented with sari, regardless of the cd4 lymphocyte count as shown by l opez-aldeguer et al. 20 another important aspect of our study was the fact this pyogenic bacterium was found in patients with late onset of symptoms (non-sari) and three out of seven patients presented with bacteremia and all three of them died, a feature often correlated with poor prognosis. 21 l. pneumophila remains an uncommon cause of community-acquired lung infection but we acknowledge that some cases might have been missed since the urinary antigen test can only detect type 1 infections and up to 14 days of onset. 20, 22 we found a high frequency of mtb in patients with few days of symptoms and in a population with a previous history of pulmonary or disseminated tb, suggesting that prolonged immunosuppression may reactivate the bacillus. [22] [23] [24] our findings are in agreement with coelho et al. who showed that tb was the most common opportunistic infection in the total cohort followed at our center. in high tb prevalence settings, such as the one observed in our context, this diagnosis should be pursued in plwh regardless of symptom duration. 25 another important aspect of our findings was to emphasize the polymicrobial aspect of respiratory infections in this population. co-detection of multiple pathogens can occur in the context of hiv infection 20, 25, 26 but also in the general adult population as recently demonstrated by karhu et al. (39% of severe acquired pneumonia patients) and in children with sari. [26] [27] [28] in our sample, coinfection could be associated with death (44% vs. 25%, p ¼ 0.25) but did not reach statistical significance, probably due to the small sample size. whether the presence of multiple pathogens leads to a more severe presentation with increased risk of poor outcomes is still unclear. 29 when looking at coinfection involving bacteria plus a respiratory virus, mortality seems to be higher in the bacterial coinfection subgroup, 30 32, 33 but these findings may not be applied to viral-viral coinfection. 34 since sari and non-sari were similar according to clinical and epidemiological aspects, the delay between symptom onset and the demand for care (non-sari group) could have contributed to the higher mortality observed in our findings, as shown previously by several authors. 6, 35, 36 although 39 patients had crb-65 of 0-1 points and could have been sent home with oral antibiotic therapy, the majority required niv in the first 72 h of hospital admission and 63% had x-rays with multilobar infiltrates, another predictor of unfavorable prognosis. similar findings were found by almeida et al. in 49 plwh admitted with pneumonia in an emergency department with a median crb65 score lower then hiv-negative patients but which were also associated with a higher risk of mortality. 37 in the paper of yone et al., 54 out 62 plwh admitted with community acquired pneumonia (cap) had crb65 of 0-1. 38 in our study, adding the pulse oximetry to crb-65 score, as suggested by the consensur ii, the latin america pneumonia working group, increased the ability to confidently predict the 30-day in-hospital mortality. 39 in the context of sari surveillance target at influenza, a possible explanation for our late detection relies on the fact that immunosuppressed and critically ill patients there seems to be prolonged viral shedding. 40 the median cd4 cell count in this study population was 80 (24-274) cells/mm 3 and the proportion of patients with undetectable viral load was 25% despite 80% being exposed to art. compared to the whole cohort followed at our center for the period 2012-2013, where 69.5% were male and the median age was 42.5 years, the median cd4 cell count was higher (542 cells/mm 3 ), and so was the proportion who had undetectable hiv viral load (69.5%) and ever being exposed to art (90.8%). 41 this shows that the population in this study is severely immunocompromised and found it harder to adhere to art at the time. we observed two cases of influenza in the non-sari group, respectively 20 and 30 days after symptom onset, and both patients developed respiratory failure that led to death (data not shown). this highlights the fact that immunosuppressed plwh with respiratory symptoms may not seek hospital attention in time to be captured by sari surveillance. finally, regarding symptom frequency, we found a low percentage of sore throat, runny nose and conjunctivitis and a high percentage of dyspnea, diarrhea, and myalgia. in fact, dyspnea was the only symptom more prevalent in the sari group (p < 0.05). although gastrointestinal involvement may have been influenced by the advanced immunodeficiency syndrome, other authors studying the symptoms of influenza in lessimmunocompromised plwh reached similar results. 42, 43 it is important to note that our study tried to address viral symptoms in a context of multiple pathogens. on one hand, it is difficult to assign a symptom to a specific virus or bacteria but on the other hand, it may reflect what actually happens in clinical practice, because as already highlighted above, a significant percentage of respiratory infections, especially in plwh are of mixed type. our study has limitations. the small sample size conducted at a single center does not allow generalization of the results and the detection of respiratory viruses from the lower respiratory tract would have been more representative of the etiology of respiratory infection. patients with advanced hiv infection may have multiple infections at the same time and an overlap of signs and symptoms is frequent, so the need for hospitalization may not be attributed exclusively to the respiratory infection per se. all cases in this study occurred in a 19-month period at a single institution, and viral epidemiology at this site and during this timeframe may not be representative of all seasons and locations. finally, due to the constraints of the respiratory viral kit used, we may not have identified all possible and important viral infections such as adenovirus, coronavirus and bocavirus and pcr technique may detect a virus that is representative of several conditions such as the true agent itself, a bystander or facilitating bacterial superinfection. 44 in severely immunocompromised plwh, sari can be represented by a wide variety of respiratory pathogens and the codetection of multiple pathogens is common. viruses must be considered as a common cause of respiratory infection either alone or in combination with other pathogens and in scenarios where tb is endemic, this agent should be pursued regardless of cough duration. hiv-1 and bacterial pneumonia in the era of antiretroviral therapy aidsrelated tuberculosis in rio de janeiro hospitalization for pneumonia among individuals with and without hiv infection, 1995-2007: a danish population-based, nationwide cohort study mortality associated with seasonal and pandemic influenza and respiratory syncytial virus among children <5 years of age in a high hiv prevalence setting-south africa deaths associated with respiratory syncytial and influenza viruses among persons 5 years of age in hiv-prevalent area mortality amongst patients with influenza-associated severe acute respiratory illness who|who global epidemiological surveillance standards for influenza surveillance for severe acute respiratory infections in southern arizona epidemiology of severe acute respiratory illness (sari) among adults and children aged 5 years in a high hiv-prevalence setting the role of influenza, rsv and other common respiratory viruses in severe acute respiratory infections and influenza-like illness in a population with a high hiv sero-prevalence viral etiology of influenza-like illnesses in cameroon the seasonality of rhinovirus infections and its implications for clinical recognition respiratory viruses in adults with community-acquired pneumonia etiology of community-acquired pneumonia: increased microbiological yield with new diagnostic methods viral etiology of community-acquired pneumonia among adolescents and adults with mild or moderate severity and its relation to age and severity communityacquired pneumonia requiring hospitalization among u.s. adults clinical relevance of rhinovirus infections among adult hospitalized patients viral infection in patients with severe pneumonia requiring intensive care unit admission human metapneumovirus-associated severe acute respiratory illness hospitalisation in hiv-infected and hiv-uninfected south african children and adults outcomes in hiv-infected patients admitted due to pandemic influenza changing global epidemiology of pulmonary manifestations of hiv/ aids pyogenic bacterial lower respiratory tract infection in human immunodeficiency virus-infected patients hiv and tuberculosis: a deadly human syndemic tuberculosis and hiv co-infection prevalent tuberculosis at hiv diagnosis in rio de janeiro, brazil: the tb/hiv in rio (thrio) cohort respiratory viruses in hiv-infected patients with suspected respiratory opportunistic infection lower respiratory tract virus findings in mechanically ventilated patients with severe community-acquired pneumonia the role of respiratory viral infections among children hospitalized for community-acquired pneumonia in a developing country does virus-bacteria coinfection increase the clinical severity of acute respiratory infection?: virus-bacteria coinfection and respiratory infection different pattern of viral infections and clinical outcomes in patient with acute exacerbation of chronic obstructive pulmonary disease and chronic obstructive pulmonary disease with pneumonia: respiratory viral infections in copd patients does viral co-infection influence the severity of acute respiratory infection in children? influenza virus infection is associated with increased risk of death amongst patients hospitalized with confirmed pulmonary tuberculosis in south africa epidemiology, co-infections, and outcomes of viral pneumonia in adults: an observational cohort study clinical disease severity of respiratory viral co-infection versus single viral infection: a systematic review and meta-analysis severe 2009 pandemic influenza a (h1n1) infection and increased mortality in patients with late and advanced hiv disease outcomes of influenza a(h1n1)pdm09 virus infection: results from two international cohort studies curb-65 and other markers of illness severity in community-acquired pneumonia among hiv-positive patients influence of hiv infection on the clinical presentation and outcome of adults with acute community-acquired pneumonia in yaounde, cameroon: a retrospective hospitalbased study neumon ıa aguda adquirida en la comunidad en adultos: actualizaci on de los lineamientos para el tratamiento antimicrobiano inicial basado en la evidencia local del grupo de trabajo de sudame´rica influenza susceptibility, severity, and shedding in hiv-infected adults: a review of the literature hospitalization rates, length of stay and in-hospital mortality in a cohort of hiv infected patients from rio de janeiro, brazil influenza a h1n1 in hiv-infected adults*: influenza a h1n1 in hiv-positive adults hiv-infected hospitalized patients with 2009 pandemic influenza a (ph1n1)-united states, spring and summer editorial commentary: what is the real role of respiratory viruses in severe community-acquired pneumonia? we thank rogerio valls and jorge salluh for their valuable suggestions during the study period. authors' contribution acp collected data, performed the analysis, interpreted results, and wrote the manuscript. rta contributed to data management, performed the analysis and drafting the figures and tables. fab and aj participated in the conception of this study and revised the manuscript. aj and jcn participated in the interpretation of the results and writing. ms and mlo processed the respiratory samples. dcm participated in the data collection. all authors read and approved the final manuscript. de-identified data and materials are available on a case-bycase basis. please contact the corresponding author with requests. the authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. the study was conducted in accordance with the declaration of helsinki. written informed consent was obtained from all patients prior to any study-related procedure. the national institute of infectious diseases review board approved study protocol (ini, caae 0014.0.009.000-08). participants who refused to participate or were excluded by exclusion criteria were not harmed by not participating in the study. the authors received no financial support for the research, authorship, and/or publication of this article. https://orcid.org/0000-0002-4131-1425 key: cord-347262-q88g1561 authors: schutzer‐weissmann, j.; magee, d.j.; farquhar‐smith, p. title: severe acute respiratory syndrome coronavirus 2 (sars‐cov‐2) infection risk during elective peri‐operative care: a narrative review date: 2020-07-11 journal: anaesthesia doi: 10.1111/anae.15221 sha: doc_id: 347262 cord_uid: q88g1561 the protection of healthcare workers from the risk of nosocomial severe acute respiratory syndrome coronavirus 2 (sars‐cov‐2) infection is a paramount concern. sars‐cov‐2 is likely to remain endemic and measures to protect healthcare workers against nosocomial infection will need to be maintained. this review aims to inform the assessment and management of the risk of sars‐cov‐2 transmission to healthcare workers involved in elective peri‐operative care. in the absence of data specifically related to the risk of sars‐cov‐2 transmission in the peri‐operative setting, we explore the evidence‐base that exists regarding modes of viral transmission, historical evidence for the risk associated with aerosol‐generating procedures and contemporaneous data from the covid‐19 pandemic. we identify a significant lack of data regarding the risk of transmission in the management of elective surgical patients, highlighting the urgent need for further research. widely reported and is an issue of great concern to clinicians and policy-makers. community surveillance in the uk suggests an increased incidence of sars-cov-2 infection among patient-facing healthcare workers [1] . of confirmed covid-19 cases in china, 3.8% were healthcare workers and higher proportions have been reported in italy and spain [2] . up to 11 may 2020, 203 healthcare worker deaths had been reported in the uk [3] . whilst none of these were anaesthetists or intensivists, 53/1718 (3.1%) healthcare workers performing or involved in tracheal intubation of patients with confirmed or suspected covid-19 subsequently reported laboratory-confirmed sars-cov-2 infection [4] . more than 20% of all severe acute respiratory syndrome (sars) cases were healthcare workers [5] and nosocomial sars infection was prominent among healthcare workers looking after patients who required respiratory support [6] . following the sars epidemic, the world health organization (who) published a list of aerosol-generating procedures [7] , a concept originally developed to protect against transmission of tuberculosis, an obligate airborne pathogen [8] . the who subsequently commissioned a systematic review of the evidence for the association between aerosol-generating procedures and nosocomial sars coronavirus-1 (sars-cov-1) transmission [9] . this incorporated 10 retrospective observational studies, five case-control and five cohort studies. given its genesis, the evidence is inevitably associative and imprecise. nonetheless, there was a consistent and strong signal (pooled odds ratio 6.6) that tracheal intubation was associated with an increased risk of transmission of sars-cov-1 to healthcare workers. understandably, this has been given considerable weight by policy-makers during the current pandemic [10] [11] [12] . the possibility that this increased risk may, in part, be due to airborne transmission has informed not only the use of personal protective equipment (ppe) but also procedural modifications of the peri-operative pathway. guidance continues to evolve [13] and, although not all are explicitly advised in national or international guidelines, these have included: tracheal intubation and extubation in the operating theatre to avoid contamination of anaesthetic rooms; an 'aerosol clearance time' defined in terms of room ventilation during which no one should enter, and some suggest even leave, the room following an aerosol-generating procedure; avoiding manual (bag-mask) ventilation before tracheal intubation; and, by inference, avoiding intra-operative positive pressure ventilation via supraglottic airway devices. these may be associated with adverse consequences or resource cost, some of which are outlined in table 1 . this article is protected by copyright. all rights reserved unlike sars-cov-1, sars-cov-2 is likely to become an endemic threat. healthcare systems face the challenge of increasing activity to accommodate the backlog that has built up due to service disruption [14] whilst protecting patients and staff from nosocomial infection. however, there is limited evidence concerning transmission risk in the elective peri-operative setting. here, we review the evidence from sars and contemporaneous data from covid-19 to inform assessment and management of the risk of sars-cov-2 transmission to healthcare workers involved in elective peri-operative care. according to the who, sars-cov-2 is predominantly transmitted by contact with infected respiratory fluids or exposure to infected respiratory droplets [15] . environmental contamination is widespread [16, 17] and this can be mitigated by hand hygiene, gloves, aprons and environmental decontamination. exposure to infective droplets emitted by coughing may be mitigated by wearing a fluid-resistant surgical mask [18] . airborne transmission is via aerosols, particles "that remain infectious when suspended in air over long distance and time" [19] and may, therefore, transmit infection further than the two-metre range of larger droplets [20, 21] . aerosol deposition is also an important source of surface contamination [22] . airborne viral transmission is complex, uncertain and controversial [21] . respiratory bioaerosols are generated by wind shear forces arising from the passage of air over infected mucosa in the respiratory tract. the number and size distribution of aerosols and their viral content vary according to the site and force of generation, environmental conditions and the degree of viral shedding at the site of aerosol generation. the infectivity of aerosols depends upon the aerosol viral load, where they deposit in the respiratory tract and tissue tropic factors (such as, in the case of sars-cov-2, cellular angiotensinconverting enzyme-2 (ace-2) receptor expression) [23] . airborne viral spread has been demonstrated in animal models [24] and healthy human volunteers [25] and epidemiological studies suggest that this is a transmission route in other viruses [20, 26] . sars-cov-2 infects and replicates in both lower respiratory tract and nasopharynx [27] . under experimental conditions, the persistence of viable sars-cov-2 in aerosols for up to 3 h has been demonstrated [28] . sars-cov-2 ribonucleic acid (rna) has been detected in air samples from clinical environments [22] . other studies were unable to detect sars-cov-2 in air samples but swabs from air outlets were positive [16] . it is important to note that presence of detectable sars-cov-2 rna does not necessarily imply the presence of viable virus and there are no reports to date of viable sars-cov-2 isolated from air samples this article is protected by copyright. all rights reserved collected in the clinical environment. there is no direct evidence of sars-cov-2 airborne transmission but there is epidemiological evidence that airborne sars-cov-1 transmission may have occurred, both in the community and in the healthcare environment. for example, modelling of airflow dynamics correlated with the sars-cov-1 transmission dynamics in an apartment block, where spread by contact or respiratory droplet was unlikely [29] . similarly, the transmission of sars-cov-1 to medical students who were not in direct contact with an infected patient correlated with airflow modelling [30] . in both cases, defective engineering created environmental conditions that may have contributed to these events (in the first, faulty drain seals allowing faecal aerosols to be drawn into the air conditioning; in the second, imbalance between ventilation inflow and outflow) and they are not necessarily representative of normal transmission dynamics [31, 32] . whilst the predominant route of sars-cov-2 transmission may be contact/droplet-mediated, environmental conditions -including those associated with aerosol-generating procedures -may promote 'opportunistic' airborne transmission [33] . aerosol-mediated airborne transmission has been a source of great anxiety among healthcare workers. national guidelines recommend 'airborne precautions' [34] for those involved in aerosol-generating procedures but contact/droplet precautions for most other clinical activity [11, 12, 35] . the who list of aerosol-generating procedures is based on epidemiological evidence of transmission to healthcare workers caring for sars patients [30, [36] [37] [38] [39] [40] [41] [42] [43] [44] . this evidence is related to the risk of transmission whilst caring for patients with respiratory failure and critical appraisal of how this sars data applies to the risk of sars-cov-2 transmission in the elective peri-operative environment is necessary. table 2 summarises the raw data from the who-commissioned systematic review by tran et al. [9] related to transmission risk associated with tracheal intubation. tracheal intubation has been highlighted here because it is the most relevant aerosol-generating procedure in the context of elective peri-operative care. other procedures, such as extubation, rely on this evidence by extension. moreover, it is notable as an example of the methodology that tracheal intubation is a discrete and identifiable event and was common among sars patients. it is therefore liable to proxy assumptions: for example, if the majority of sars patients had developed appendicitis, such methodology might identify appendicectomy as an aerosol-generating procedure. this article is protected by copyright. all rights reserved the studies were limited by heterogeneous populations, poorly-defined and variable exposure, and recall bias. across eight studies, there were 76 infections associated with tracheal intubation among a population of 2250 healthcare workers. the number of healthcare workers exposed to tracheal intubation is relatively small compared with those who were not, such as non-clinical staff. across all studies, 22 patients transmitted sars-cov-1 to 99 healthcare workers. in the second-largest study, the 26 healthcare workers who developed sars all looked after seven -and 23 looked after only four -of the 45 sars patients whose tracheas were intubated [41] . in the largest case-control study caring for a "superspreading patient" (no definition offered in the paper) was associated with healthcare worker infection, and in multivariate analysis, this association was stronger than tracheal intubation [36] . this evidence, therefore, rests on a small number of infections associated with a yet smaller number of highly infectious patients, among a heterogeneous population of healthcare workers, matched in some cases according to profession, in others by presence during rather than performance of the procedures under investigation. all identify other measures of proximity or contact with patients which are associated with increased transmission risk of a comparable order of magnitude to that of being involved in tracheal intubation. the authors discuss the "difficulty in identifying the specific part of a given procedure, which may be complex and involve several manoeuvres, that imparts the greatest risk of transmission." they "acknowledge that the findings presented may have been influenced by direct and indirect contact transmission" and conclude that their "findings serve to highlight the lack of precision in the definition for aerosol generating procedures" [9] . a recent systematic review led by health protection scotland appraised the evidence base for the who list of aerosol-generating procedures [45] . they only identified four additional reports relating to transmission risk during tracheal intubation. three are case reports and in each of these they found that "the multiple factors that could have led to infection transmission in this case make it very difficult, if not impossible to identify the most high risk elements." the other study that they singled out [42] was included in the who systematic review. health protection scotland could only identify "weak evidence for an increased risk of respiratory infection transmission" from performing tracheal intubation. this article is protected by copyright. all rights reserved there is a consistent and strong signal throughout these studies that involvement in tracheal intubation of sars patients with respiratory failure was associated with an increased risk of viral transmission. in a recent international study, 10.7% healthcare workers involved in tracheal intubation during the covid-19 pandemic reported lab-confirmed sars-cov-2 infection or hospitalisation or self-isolation due to covid-19 symptoms [4] . how the risk of infection associated with tracheal intubation is mediated, however, is not clear. the elective peri-operative environment is different from the acute settings from which this evidence is drawn. the preconditions and purpose of tracheal intubation for elective surgery are different from emergent/urgent tracheal intubation for respiratory failure. pre-admission measures such as selfisolation, symptomatic screening and viral rna testing prior to admission aim to reduce the risk that patients undergoing elective surgery are infected with sars-cov-2 and the risk of healthcare worker exposure to the virus in the elective peri-operative environment. however, it is important to note that these measures do not eliminate these risks. in order to provide effective and efficient protection to healthcare workers in this environment, it is imperative to consider how and why involvement in tracheal intubation and related airway procedures is associated with increased risk of viral transmission. coughing is the common denominator of a number of defined aerosol-generating procedures, including tracheal intubation, extubation and bronchoscopy. indeed, the tuberculosis guidelines which introduced the term refers to 'cough-inducing and aerosol-generating procedures' [8] . the recent reiteration [46] that tracheal intubation is aerosol-generating was based on simulated tracheal intubation of a 'coughing' manikin [47] . modelling studies confirm that the risk of airborne transmission depends upon cough frequency [48] . air sampling demonstrates increased bio-aerosol concentrations during coughing on bronchoscope insertion [49] . coughing is not a procedure but its exclusion from discussions of aerosol-generating procedures may also reflect the misconception that it does not produce aerosols, which originates from early studies that were technically insensitive to smaller particles [50] . any respiratory activity, including breathing, produces aerosols and more recent studies have demonstrated that coughing produces large numbers of both droplets and aerosols [51, 52] . the dichotomy between droplets that deposit within a short distance and aerosols that travel further may be an over-simplification [53, 54] . this article is protected by copyright. all rights reserved h1n1 avian influenza viral titres in air samples taken during tracheal intubation were not significantly higher than background levels in intensive care units [55] although this study may have been underpowered to detect this difference [45] . another study found no link between influenza aerosols in sampled air and aerosol-generating procedures [56] . in canada, use of muscle relaxants for tracheal intubation was increased during the second sars outbreak which may have contributed to the decrease in healthcare worker infections [57] and support the notion that coughing is the prime aerosol (and/or droplet) generator. the studies upon which the who list of aerosol-generating procedures is based do not provide any direct evidence that tracheal intubation itself increases the risk of sars transmission. rather, this data implies that proximity and time in proximity to desaturation after induction of anaesthesia can be minimised by effective pre-oxygenation [74, 75] and measures which facilitate apnoeic oxygenation (for example, maintaining airway patency, minimising air entrainment via mask leak and head-up position [76] ). recent guidance supports the judicious use of supraglottic airway devices [13] . some have advocated against the use of supraglottic airway devices in favour of cuffed tracheal tubes on the basis that there may be a lower risk of an aerosol leak during positive pressure ventilation [77] . there is evidence that, when used appropriately, this is not the case. the mean oropharyngeal leak pressure of the i-gel® (intersurgical, wokingham, uk) is 25 cmh 2 o in non-paralysed patients and 28 cmh 2 o in paralysed patients [78] . the leak fraction with i-gel® was no higher when ventilating with peak pressures below 25 cmh 2 o compared with cuffed tracheal tube [79] . several tests of leak have been shown to be sensitive and reliable in clinical settings [80] . the risk of aerosol generation may be greater on insertion or removal where poor seal or coughing may facilitate generation and dispersal of aerosols. therefore, perhaps more important than leak fraction is the primary failure rate where supraglottic airway devices do not achieve an adequate seal on insertion. for i-gel® this has been estimated to be 4-7% [81] but in this regard, supraglottic airway devices with an inflatable cuff may be more reliable [82] . an 'aerosol clearance time' -waiting for a period of time for room ventilation defined in terms of air changes -has been recommended [83, 84] . uk national guidance from public health england advocates (as 'pragmatic') 20 minutes in a room with 10 to 12 air changes per hour following an aerosol-generating procedure [85] . this corresponds to approximately four air changes and a clearance of 96-98%. this is similar to the three to five air changes recommended by the australian and new zealand college of this article is protected by copyright. all rights reserved anaesthetists [86] . there is evidence to support this where there has been extensive aerosol generation, e.g. intensive care rooms [55] or bronchoscopy suites [49] . if aerosols are generated by associated respiratory activities rather than the act of tracheal intubation or extubation itself, requiring an aerosol clearance time following these procedures but not in other situations, for example in a recovery ward where patients are breathing and coughing, may seem logically inconsistent: in both environments, patients generate potentially infectious aerosols by breathing and coughing. there are, however, arguments for maintaining aerosol clearance times in the elective perioperative environment. foremost is the precautionary principle: there is epidemiological evidence that tracheal intubation and other airway manoeuvres are consistently associated with an increased viral transmission risk and there may be elements of these procedures which increase risk that we do not appreciate. current uk guidelines do not recommend airborne precautions for healthcare workers where aerosolgenerating procedures are not taking place, for example in recovery wards, outpatient suites or general practice consultation rooms [11] . other international authorities, however, advise airborne precautions for all healthcare workers coming into close proximity to an 'open' airway and not just after aerosolgenerating procedures [87] and this would include healthcare workers in recovery wards and many outpatient and community healthcare environments. this guidance is supported by the evidence presented here which emphasises the importance of proximity to patients' airways over the procedure itself. a further example of this might be a recent study of 44 anaesthetists who performed awake spinal anaesthesia (not an aerosol-generating procedure) on 49 sars-cov-2 positive patients which found that only 1/37 (2.7%) who used aerosol precaution ppe subsequently tested positive for sars-cov-2 compared to 4/7 (57.1%) who used droplet precaution ppe [88] . this may have implications for other regional anaesthetic techniques, a subject which has been recently reviewed by uppal et al. [89] . the focus on aerosol-generating procedures may also risk neglecting other practices to reduce transmission that are equally important. these control measures include frequent handwashing [90] , double-gloving during tracheal intubation [91] , surface cleaning of anaesthetic machines, monitors and other equipment in the immediate vicinity after tracheal intubation [92] and patient use of fluid-resistant surgical mask following extubation [93] . basic infection control practices are often poorly observed [94] . sampling studies consistently identify extensive surface contamination warranting greater emphasis on this element of infection control [16, 17] . evidence-based guidance [92] includes simple, inexpensive this article is protected by copyright. all rights reserved measures such as placing alcohol-gel dispensers near anaesthetists which have been shown to dramatically increase hand decontamination [95] . strict adherence to standard infection control precautions and frequent, thorough surface cleaning may reduce contact transmission [96] . one product of the sars experience was the concept of the aerosol-generating procedure. this epidemiological evidence, graded as very low quality, provides useful guidance in the management of symptomatic acutely unwell patients. in the elective peri-operative and other healthcare settings, however, restricting airborne precautions to healthcare workers undertaking aerosol-generating procedures may under-estimate risks to those who are in close proximity to patients but not involved in these procedures. the emphasis on aerosol-generating procedures also potentially risks neglecting the primary barriers to covid-19 transmission of contact precautions and hand washing. the limitations of this review reflect the limitations of the data. there is very limited evidence related to the risk of sars-cov-2 transmission in the elective peri-operative environment. the mechanism of infection transmission and the factors that influence it is inferred from physical studies of aerosol generation and behaviour and clinical studies of other viruses in other settings. as anaesthetists, our understanding of the complexities of aerodynamics and virology is necessarily limited and highlights the need for multidisciplinary research in this area. coronavirus (covid-19) infection survey pilot: 28 rapid risk assessment -coronavirus disease 2019 covid-19 in the eu/eea and the uk -eighth update at least 23 nationalities among nhs staff killed by covid risks to healthcare workers following tracheal intubation of patients with covid-19: a prospective international multicentre cohort study summary of probable sars cases with onset of illness from 1 critically ill patients with severe acute respiratory syndrome infection prevention and control of epidemic-and pandemic-prone acute respiratory diseases in health care guidelines for preventing the transmission of mycobacterium tuberculosis in health-care facilities aerosol generating procedures and risk of transmission of acute respiratory infections to healthcare workers: a systematic review consensus guidelines for managing the airway in patients with covid-19 covid-19: infection prevention and control (ipc) european centre for disease control prevention and contol. infection prevention and control and accepted article this article is protected by copyright. all rights reserved preparedness for covid-19 in healthcare settings -third update use of supraglottic airways during the covid-19 pandemic -icm anaesthesia covid-19 elective surgery cancellations due to the covid-19 pandemic: global predictive modelling to inform surgical recovery plans modes of transmission of virus causing covid-19: implications for ipc precaution recommendations surface environmental, and personal protective equipment contamination by severe acute respiratory syndrome coronavirus 2 (sars-cov-2) from a symptomatic patient aerosol and surface distribution of severe acute respiratory syndrome coronavirus 2 in hospital wards the role of community-wide wearing of face mask for control of coronavirus disease 2019 (covid-19) epidemic due to sars-cov-2 infection prevention and control of epidemic-and pandemic-prone acute respiratory infections in health care recognition of aerosol transmission of infectious agents: a commentary airborne transmission and precautions: facts and myths aerodynamic analysis of sars-cov-2 in two wuhan hospitals multiorgan and renal tropism of sars-cov-2 mode of parainfluenza virus transmission determines the dynamics of primary infection and protection from reinfection airborne transmission of respiratory accepted article this article is protected by copyright. all rights reserved infection with coxsackievirus a type 21 viral infections in workers in hospital and research laboratory settings: a comparative review of infection modes and respective biosafety aspects virological assessment of hospitalized patients with covid-2019 aerosol and surface stability of sars-cov-2 as compared with sars-cov-1 evidence of airborne transmission of the severe acute respiratory syndrome virus cluster of sars among medical students exposed to single patient, hong kong epidemiology: dimensions of superspreading a pandemic in times of global tourism: superspreading and exportation of covid-19 cases from a ski area in austria airborne transmission of communicable infection -the elusive pathway personal protective equipment during the coronavirus disease (covid) 2019 pandemic -a narrative review infection control: severe acute respiratory syndrome coronavirus 2 (sars-cov-2) which preventive measures might protect health care workers from sars? investigation of the influencing factors on severe acute respiratory syndrome among health care workers factors associated with transmission of severe acute respiratory syndrome among health-care workers in singapore. epidemiology and infection accepted article this article is protected by copyright risk factors for sars infection among hospital healthcare workers in beijing: a case control study a case-control study on the risk factors of severe acute respiratory syndromes among health care workers. chung-hua liu hsing ping hsueh risk factors for sars transmission from patients requiring intubation: a multicentre investigation in toronto transmission of severe acute respiratory syndrome during intubation and mechanical ventilation sars among critical care nurses illness in intensive care staff after brief exposure to severe acute respiratory syndrome assessing the evidence base for medical procedures which create a higher risk of respiratory infection transmission from patient to healthcare worker covid-19 and risks posed to personnel during endotracheal intubation exposure to a surrogate measure of contamination from simulated patients by emergency department personnel wearing personal protective equipment toward understanding the risk of secondary airborne infection: emission of respirable pathogens evaluation of bioaerosol exposures during hospital bronchoscopy examinations droplet fate in indoor environments, or can we prevent the spread of infection? exhaled droplets due to talking and coughing size distribution and sites of origin of droplets expelled from the human respiratory tract during expiratory activities airborne transmission of severe acute respiratory syndrome coronavirus-2 to healthcare workers: a narrative review turbulent gas clouds and respiratory pathogen emissions: potential implications for reducing transmission of covid-19 influenza aerosols in uk hospitals during the h1n1 (2009) pandemic -the risk of aerosol generation during medical procedures exposure to influenza virus aerosols in the hospital setting: is routine patient care an aerosol generating procedure intubation of sars patients: infection and perspectives of healthcare workers exposure to influenza virus aerosols during routine patient care aerosol transmission of influenza a virus: a review of new studies dispersion and exposure to a cough-generated aerosol in a simulated medical examination room health and social care committee. oral evidence: management of the coronavirus outbreak epidemiological research priorities for public health control of the ongoing global novel coronavirus (2019-ncov) outbreak epidemiology, transmission dynamics and control of sars: the 2002-2003 epidemic accepted article this article is protected by copyright. all rights reserved estimating the generation interval for coronavirus disease (covid-19) based on symptom onset data covid-19: four fifths of cases are asymptomatic, china figures indicate the relative transmissibility of asymptomatic covid-19 infections among close contacts sars and mers: recent insights into emerging coronaviruses symptom screening at illness onset of health care personnel with sars-cov-2 infection in king county screening of healthcare workers for sars-cov-2 highlights the role of asymptomatic carriage in covid-19 transmission. elife 2020. epub 11 may detection of sars-cov-2 in different types of clinical specimens covid-19-guidance/personal-protective-equipment-public-health-england-and-covid-19-montgomery-in-reverse medications to reduce emergence coughing after general anaesthesia with tracheal intubation: a systematic review and network meta-analysis exhaled air dispersion during bag-mask ventilation and sputum suctioning-implications for infection control preoxygenation techniques: comparison of three minutes and four breaths difficult airway society 2015 guidelines for management of unanticipated difficult intubation in adults peri-operative management of the obese surgical patient anaesthesia and caring for patients during the covid-19 outbreak comparison of oropharyngeal leak pressure and clinical performance of lma proseal tm and i-gel® in adults: meta-analysis and systematic review comparison of the i-gel with the cuffed tracheal tube during pressure-controlled ventilation comparison of four methods for assessing airway sealing pressure with the laryngeal mask airway in adult patients greif r. i-gel tm supraglottic airway in clinical practice: a prospective observational multicentre study success rate of airway devices insertion: laryngeal mask airway versus supraglottic gel device consensus statement: safe airway society principles of airway management and tracheal intubation specific to the covid-19 adult patient group personal protective equipment (ppe) for both anesthesiologists and other airway managers: principles and practice during the covid-19 pandemic infection-preventionand-control/reducing-the-risk-of-transmission-of-covid-19-in-the-hospital-setting australian and new zealand college of anaesthetists and the faculty of pain medicine. anzca statement on personal protection equipment during the sars-cov-2 pandemic evaluating the national ppe guidance for nhs healthcare workers during the covid-19 pandemic accepted article this article is protected by copyright. all rights reserved spinal anaesthesia for patients with coronavirus disease 2019 and possible transmission rates in anaesthetists: retrospective, single-centre, observational cohort study neuraxial anaesthesia and peripheral nerve blocks during the covid-19 pandemic: a literature review and practice recommendations physical interventions to interrupt or reduce the spread of respiratory viruses: systematic review anaesthesia and covid-19: infection control perioperative covid-19 defense: an evidence-based approach for optimization of infection control and operating room management minimising droplet and virus spread during and after tracheal extubation frequency of hand decontamination of intraoperative providers and reduction of postoperative healthcare-associated infections: a randomized clinical trial of a novel hand hygiene system the effect of improving basic preventive measures in the peri-operative arena on staphylococcus aureus transmission and surgical site infections: a randomized clinical trial this article is protected by copyright. all rights reserved table 2 summary of the raw data incorporated by tran et al. [9] of evidence relating aerosol-generating procedures to sars-cov-1 infection among healthcare workers.*p < 0.05; or, odds ration; rr, relative risk; na, not available; ecg, electrocardiograph ma et al. [40] this study is based on the same dataset used by liu et al. [39] . the exposure definition used is different: it reports a risk estimate for exposure to a composite of four procedures (intubation, tracheotomy, airway care, and cardiac resuscitation). to avoid duplication and false comparison, its data is not presented here. healthcare workers exposed to confirmed sars-covwong et al. [30] not applicable to tracheal intubation but reports a cohort of medical students exposed to a single inpatient; 16 key: cord-342314-nx0a3nvd authors: gebru, mu’uz; tefera, genene; dawo, fufa; tessema, tesfaye sisay title: aerobic bacteriological studies on the respiratory tracts of apparently healthy and pneumonic camels (camelus dromedaries) in selected districts of afar region, ethiopia date: 2017-11-16 journal: trop anim health prod doi: 10.1007/s11250-017-1476-4 sha: doc_id: 342314 cord_uid: nx0a3nvd a cross-sectional study was conducted to isolate and identify bacterial species from the respiratory tract of apparently healthy and pneumonic camels in asayita and dubti woredas in the afar region, ethiopia. from a total of 74 lung tissue and 74 tracheal swab samples staphylococcus aureus, 16.3%, streptococcus equi subsp. equi, 13.0%, and pasteurella multocida, 10.9%, were dominant isolates from pneumonic lungs; escherichia coli, 12.7%, proteus species, 10.9%, and klebsiella pneumoniae, 9.1%, were the majority in the normal lungs. the majority of the isolates colonized both anatomical sites investigated. there was a statistically significant association between the health status of the camels as well as the anatomical site studied with the isolation rates of the major respiratory pathogens (p < 0.05). furthermore, the isolates were susceptible to norfloxacin, streptomycin, and gentamicin but resistant to ampicillin and tetracycline on in vitro test. further studies on the pathogenicity of the major isolates are recommended. generally, camels are assumed to be well adapted to their environment and resist infectious diseases that inflict other livestock species in the same region; however, a number of economically important diseases affect camels (alharbi 2016) . recently, respiratory tract diseases have emerged among camels worldwide causing considerable loss of life and production (funk et al. 2016; ali et al. 2017) . camel respiratory problem is an emerging disease in ethiopia causing extensive loss of production and deaths with limited efforts to study the causative agents (bekele 2008) . outbreaks have been reported in 1995 in the afar region (roger et al. 2000; roger et al. 2001 roger et al. ), in 2005 roger et al. /2006 in the afar and oromia regions (wernery et al. 2006) , in the somali and oromia regions, as well as in 2011 in afar region in the country. a definitive etiology of the problem has not yet been conclusively determined though previous reports indicate the involvement of peste des petits ruminant (ppr)-like virus, streptococcus equi subsp. equi, pasteurella, mannheimia, and mycoplasma species (yigezu et al. 1997; roger et al. 2001) . despite some efforts made far, there was no substantial result obtained from the investigation of the disease. more investigative efforts are still in need for the identification of the true cause of the problem so as to design sustainable control strategies. streptococcus species have been isolated from clinically healthy camels although they were not definitely identified and characterized. besides, streptococcus species have been isolated from active respiratory disease of camels (wareth et al. 2014) . but there is no data on the isolation of s. equi subsp. equi from camels. therefore, the present study was conducted to determine comparatively the bacterial species associated with camels with and without pneumonic lungs, identify the streptococci spp. prevalent in camel respiratory tract, as well as determine the antibiotic sensitivity pattern of the leading bacterial agents recovered from camels with pneumonic lungs. the study was conducted in selected pastoral and agropastoral residences of asayita and dubti districts of afar national regional state (anrs), located in northeast part of ethiopia (fig. 1) . animal husbandry in the region is characterized by extensive pastoral production system and seasonal mobility due to the shortage of rainfall. the camel population of asayita and dubti districts are about 3277 and 5966, respectively (csa 2004) . dubti woreda is about 610 km northeast of addis ababa, the capital city of ethiopia, at 11°49′ 59.99″ n and 41°00′ 0.00″ e, with an average elevation of 503 m above sea level (masl) and high temperature which ranges from 25 to 42°c. may-june is the driest season of the year; the main rainy season is from july to september. asayita woreda is about 667 km northeast of addis ababa, at 11°3 4′ n and 41°26′ e. the study population included camels slaughtered in the study areas during the study period. in the region, all camels were kept under extensive pastoral and agro-pastoral production conditions. nearly all of the slaughtered camels were adult males and clinically healthy during antemortem examination. the community in the area keeps a higher proportion of breeding females and few males for mating and transportation. the owners release adult camels (> 2 years) for browsing to the field during the day time; fig. 1 administrative location of afar region in the country showing the study zone and woredas however, young camels (< 2 years) are kept in a confined and fenced area around the homesteads. a cross-sectional study was conducted in dubti and asayita woredas. these areas were chosen conveniently based on access to transport to conduct the laboratory analysis of collected samples at samara veterinary regional laboratory and due to previous reports of camel respiratory infection outbreaks in the areas. a total of ten kebelles (pastoral associations (pas)) were selected from the two woredas (five pas from each woreda). the pas were selected purposively based on accessibility to the villages by vehicle or proximity to road, previous and existing report of camel respiratory problems, and awareness of the society and size of the camel population. however, as the number of slaughtered camels was so small, only 74 camels were used in the study. consequently, 46 and 28 lungs together with their analogous trachea were sampled from dubti and asayita, respectively. the procedures followed by tesfaye et al. (2013) were used in collecting and processing samples for isolation and identification of bacterial spp. ante-mortem and post-mortem examinations were carried out to identify animals and/or lungs with respiratory problems using clinical examination before slaughter and visualization, palpation and incision methods after slaughter. afterwards, tracheal swabs and lung tissue samples were collected. after 24 h of incubation in broth, the cultures were mixed; a loopful of the culture was streaked over a 7% sheep blood agar (oxide, hampshire, england) and incubated aerobically at 37°c for 24 to 72 h. single colony from each type of the observed colonies was sub-cultured on blood agar plates. the pure cultures were processed by gram's staining; gram-negative bacteria were sub-cultured on macconkey agar plates (oxide, hampshire, england) for further characterization. primary identification of the bacteria was performed using colony morphology, gram reaction, cellular morphology, catalase and oxidase tests, and growth on macconkey agar. final identification was carried out by subjecting pure cultures of single colony type into a series of secondary biochemical tests, namely, methyl-red, indole, citrate utilization, coagulase, motility, tsi, esculin hydrolysis, as well as sugar fermentation tests, as required for each bacterial spp. considered. in addition, growth characteristics on mannitol salt agar and edward's medium were used for identification of staphylococci and streptococci spp., respectively. all the tests were conducted following standard procedures. pure isolates of streptococcus species were prepared for biolog-microstation identification system (biolog, california, usa), at the institute of biodiversity conservation, addis ababa, ethiopia, which is a bacterial identification technique based on surface fatty acid substrates of each microbial species. the colonies of streptococcus species were inoculated on the biolog universal growth (bug) agar with 5.0% sheep blood and incubated at 37°c for 24 h. sub-culturing was done using the same culture media to have pure culture colonies before identification was done by omnilog. bacterial suspension was prepared from the grown pure culture colonies with an appropriate level of bacterial density (20.0% turbidity in 16-18 inoculating fluid), as recommended in the protocol of the instrument. the suspension was inoculated in to biolog micro plates aseptically. then, the micro plates were loaded in to the omnilog and incubated at 37°c for 24 h after the necessary information regarding the bacteria was registered on the software of the instrument. finally, the micro plates were read by the omnilog identification system and the bacterial species were identified using the inbuilt biolog database. antibiotic susceptibility tests were conducted on the representative isolates of the dominant bacterial spp. isolated following disk diffusion method using oxoid antibiotic disks, namely, ampicillin, erythromycin, kanamycin, norfloxacin, tetracyclin, chloramphenicol, streptomycin, gentamicin, vancomycin, and doxycyclin. the bacterial isolates were cultivated on a blood agar to obtain fresh culture. three to five identical colonies were harvested and transferred to a tube containing 4-5 ml sterile distilled water. the suspension was mixed gently until turbidity corresponding to 0.5 mcfarland standard was reached to obtain a homogeneous suspension and then poured onto mueller-hinton agar plate (oxoid, hampshire, england). seven percent sheep blood was used for streptococcus species (janosi and kaszanyitzki 2003) . after the inoculum on the plate was dried, antibiotic disks were distributed over the surface of the inoculated plates at equal distance from each other using sterile forceps. the disks were gently pressed onto the agar to ensure firm contact and then incubated for 24 h at 37°c. after 24 h, the diameters of the zones of complete inhibition around the disks were measured from the back of the plate to the nearest whole millimeter using a millimeter-gauged ruler. zone margins were read as areas showing no obvious growth detected by the unaided eye. it was read again after 48 h of incubation if the isolates were not sufficiently grown. every test was conducted twofold, and then an average of the two results was taken as a final result. growth inhibition zones were interpreted according to the manufacturer's recommendation. the data were analyzed using spss® version 17.0 software. the results generated from the study were summarized using descriptive statistics. percentages were used to express the relative abundance of each genera/species to the total number of isolates. furthermore, chi-square test was computed so as to observe the relationship between the variants. a p value of < 0.05 was considered indicative of a statistically significant difference. bacterial species isolated from the trachea and lungs of camels twenty six (35.1%) of the lungs possessed one or more types of lesion. the remaining 48 (64.9%) of the lungs had no evidence of gross pathological lesion. the collected tracheal swabs and lung tissue samples were processed for aerobic bacteriological isolation and characterization. accordingly, only 39 (52.7%) of the lung tissue samples and 33 (44.6%) of the tracheal swabs yielded bacterial colonies. the remaining 4 (5.4%) of the lung lesions, 31 (41.9%) of the apparently healthy lungs, and 41 (55.4%) of the tracheal samples examined did not reveal any bacterial growth. more than one bacterial species were isolated from 20 (27.0%) pneumonic lungs, 6 (8.1%) apparently healthy lungs, and 18 (24.3%) tracheal samples. on the other hand, only one bacterial isolate was identified from 2 (2.7%) pneumonic lungs, 11 (14.9%) apparently healthy lungs, and 15 (20.3%) tracheal samples investigated. a total of 262 bacterial isolates were identified from 74 lung and 74 corresponding tracheal samples; out of which 115 (43.9%) and 147 (56.1%) were recovered from the trachea and lung, respectively. totally, 92 bacteria were isolated from the pneumonic camel lungs from which s. aureus, 15 (16.3%), s. equi subsp. equi, 12 (13.0%), and p. multocida, 10 (10.9%), were the dominant isolates. s. equi subsp. zooepidemicus and s. pneumoniae were recovered at the lowest rate; each comprised 1% (table 1) . e. coli and proteus species accounted for 7 (12.7%) and 6 (10.9%), respectively, among 55 bacterial isolates demonstrated from the healthy lungs. on the other side, s. equi subsp. equi, b. bronchiseptica, and p. aeruginosa, each 1 (1.8%), were isolated at the lowest proportion. m. haemolytica was not recovered from the normal lungs. there was a statistically significant difference between the isolation rates of s. equi subsp. equi, p. multocida, m. haemolytica, b. bronchiseptica, s. aureus, and k. pneumonia and the health status of the lung (p < 0.05), but no difference was observed for the above isolates of the trachea samples in relation to the health status of the corresponding lungs (p > 0.05). among 262 bacterial isolates recovered from the lungs and trachea, the majority colonized both the anatomical sites investigated although differently (fig. 2) . however, a general decrease in the isolation rate of the normal flora and increase in isolation of potential respiratory pathogens, namely, s. equisubsp. equi, p. multocida, m. hemolytica, and s. pneumonia, was observed in the lungs than the trachea. there was a statistically significant difference between the anatomical sites examined with the isolation rate of the major respiratory pathogens (except b. bronchiseptica) (p < 0.05). generally, the present study showed that the isolation frequency of the major respiratory pathogenic bacteria was higher in lungs with different lesions than clinically normal lungs. on the other hand, the normal bacterial flora was more prevalent in the respiratory passage ways of apparently healthy camels than the pneumonic ones. besides, there was a statistically significant difference between pneumonic and apparently healthy camels with the isolation rate of the major respiratory pathogens (except b. bronchiseptica and k. pneumoniae) (p < 0.05) (fig. 3) . the antibiogram of the isolates studied indicated that the s. equi subsp. equi isolates were found to be sensitive to vancomycin (100.0%), norfloxacin (94.1%), erythromycin (58.8%), gentamicin (52.9%), and ampicillin (52.0%). most of the isolates of p. multocida have shown a high level of sensitivity to gentamicin, streptomycin, norfloxacin, and chloramphenicol. most of the isolates of m. haemolytica were sensitive to streptomycin, kanamycin, gentamicin, and norfloxacin (table 2 ). the respiratory tract of apparently healthy animals acted as a reservoir for many species of microorganisms that reached the nasal cavity through various ways. this study has shown that a wide variety of bacterial species colonize the respiratory passageways of camels in the study area. this is supported by several researchers who previously demonstrated diverse bacterial species from various regions of the camel respiratory tract: nasal tracts, tonsil, trachea and lungs (ismail et al. 2014; wareth et al. 2014) , and normal and pneumonic lungs (wareth et al. 2014; ahmed and musa 2015) . the consistent isolation of these organisms from the pneumonic lungs of various species of animals might indicate their role in causing different respiratory infections especially when the immune system of the animal is compromised by some other external factors. the normal bacterial flora of a healthy individual can be altered by several factors such as changes in the hygienic condition, environmental and climatic conditions, and nutritional and immunological status of the animal. such factors could lower the resistance of the lung tissue and the existing organism most probable would get the upper hand, leading to the presentation of a variety of pathologies (bosch et al. 2013 ). the current study revealed that the majority of the isolated bacteria colonize both anatomical sites and a general increase in the isolation rate of the pathogenic bacteria as one goes down the respiratory tract (fig. 2) . the normal bacterial flora was more prevalent in the respiratory passage ways of apparently healthy than the pneumonic camels. this is quite suggestive of the accepted idea that these organisms live as a commensal in the upper respiratory tract, invading the lung under conditions of stress. stress factors have been reported to suppress the mucocilliary clearance mechanism and the overall respiratory defensive system which allows the proliferation of bacterial commensals in the respiratory tract and an abrupt shift from commensal to pathogen (caswell 2014) . similar trend of bacterial distribution was also observed by azizollah et al. (2009) and al-tarazi (2001) during their bacteriological studies on the respiratory tracts of camels. considering the stress of weather, disease, and poor management conditions to which the camels are constantly subjected to in the study area, the pathogenic role of several bacterial species that inhabit the upper respiratory tract of apparently normal camels could be enormous. therefore, s. equi subsp. equi, m. haemolytica, p. multocida, k. pneumoniae, s. aureus, and b. bronchiseptica are described as normal inhabitants of upper respiratory tract as well as causative agents of pneumonia during stressful conditions. in line with this, these bacteria were also isolated at a higher incidence rate from the pneumonic lungs in the present investigation. the pathogenic bacteria isolated during the present study period are comparable to those previously reported from the respiratory tract of apparently healthy camels (ahmed and musa 2015) . most species of the genus streptococcus are considered potential pathogens, occur in nature, and some are commensal in the respiratory, genital, and alimentary tracts and skin of animals and man (parks et al. 2015) . streptococcus species have been isolated from clinically healthy camels although they were not definitely identified and characterized (azizollah et al. 2009 ). on the other hand, streptococcus species such as β-haemolytic streptococci, s. viridans, s. pneumoniae, and s. pyogenes were described in active respiratory disease of camels elsewhere (wareth et al. 2014; ahmed and musa 2015) . yigezu et al. (1997) have reported the bacteria from camel lungs and suggested its role in the epizootic occurring in camels in ethiopia. in the present work, s. equi subsp. equi was recovered from the lungs of camels, at a much higher rate in pneumonic lungs compared to healthy ones, but was not from the trachea. this could suggest low level occurrence of the organism as normal flora and involvement in camel pneumonia under optimum conditions. therefore, the bacteria could be considered as one of the important players in camel respiratory infections. streptococcus equi subsp. equi has been identified as the causative agent in equine strangles. the agent is directly transmissible via oral or nasal routes and by indirect means through contamination of the environment by excretions from sick horses. regrouping of horses is usually a risk factor in a strangles outbreak (yigezu et al. 1997) . although the camel rearing areas of asayita and dubti are uninhabited by horses, they are inhabited by donkeys and used as pack animals; this could favor the transmission of the agent by contact. pasteurella species reside as commensals on the mucosa of the upper respiratory of diverse animal species and play a role as a primary or secondary agent in pneumonia (poliquin et al. 2015) . the isolation rate of pasteurella multocida from the pneumonic camel lung (10.9%) was higher than that of healthy lungs (7.3%). abubakar et al. (2008) have also demonstrated p. multocida at a rate of 4.4% from pneumonic lungs of camels in nigeria but failed to isolate it from normal lungs. the recovery rate of the bacteria from lung lesions in the present work is higher compared to that of previous reports from camels (abubakar et al. 2008; wareth et al. 2014) , sheep (merga et al. 2006) , and goats (demissie et al. 2014 ). this discrepancy might be attributed to the variation in sample size, difference in agro-ecological zones of study areas, and host species diversity as well as health status of the study animals. mannheimia haemolytica are commensals, residing in the nasopharyngeal micro flora and are all capable of causing infection when the body defense mechanisms are impaired ( c a s w e l l 2 0 1 4 ) a l t h o u g h m a i n l y i n r u m i n a n t s . m. haemolytica was isolated at a high rate from the lungs of clinically diseased camels in this study; the isolation rate from pneumonic lungs (8.7%) is to a great extent higher than the report of desissa et al. (2009) , 0.5% from donkeys, and abubakar et al. (2008) , 0.3% from camels. however, the finding is consistent with that of al-tarazi (2001), who found 6.6% from pneumonic lungs of camels in jordan. much higher rates have been reported from ovine pneumonic lungs (demissie et al. 2014 ) and lungs of healthy goats (merga et al. 2006 ). these bacteria were not isolated from the trachea and apparently normal lungs. the occurrence of m. haemolytica in higher proportion in the clinically ill camels can make the bacteria to be considered as one of the major factors for occurrence of camel respiratory disease complex. k. pneumoniae was recovered at an enormously comparable frequency from the pneumonic and healthy lungs (table 1) ; this is comparable with that of al-doughaym et al. (1999) from the lungs of pneumonic camels, 10.9%. higher isolation rates were reported by wareth et al. (2014), 26.7%, and azizollah et al. (2009) , 25.0%. k. pneumoniae has been reported from the respiratory tracts of diverse animal spp. such as donkeys (desissa et al. 2009 ), at higher rate from donkeys having respiratory problems. lower figures have also been reported by abubakar et al. (2008) , 6.3%, from nigerian camel lungs and ismail et al. (2014) from egypt. b. bronchiseptica has been reported by nesibu et al. (2010) at a rate of 3.5% from the pulmonary lesions of camels compared to the current isolation rate, 4.3%. in addition to this it has been also isolated from the trachea (3.0%) and lung (1.8%) of apparently healthy camels. ahmed and musa (2015) reported a lower rate (0.1%). staphylococcus aureus is known to occur as a commensal on the skin, the nose, and mucous membranes of healthy humans and animals and also an opportunistic pathogen in multiple infectious diseases (lozano et al. 2016) . in this study, s. aureus was the commonest bacteria in the pneumonic lungs, 16.3%, which is higher when compared the report of al-tarazi (2001), 10.6%, from lung lesions of camels. higher isolation rates were reported by wareth et al. (2014), 37.1%, and al-doughaym et al. (1999) , 24.8%, from pneumonic camel lungs. ismail et al. (2014) and azizollah et al. (2009) also recovered higher rates, 14.5 and 14.0%, respectively, of s. aureus from lungs of apparently healthy camels. the present and previous data suggest that the bacteria reside as a normal inhabitant of upper respiratory tract and possibly as a causative agent of secondary pneumonia. the failure to isolate bacteria from four lung tissues with lesions might be due to the involvement of other pathogens such as mycobacterium, anaerobic bacteria, virus, mycoplasma, and fungi, and may be parasites. however, most normal lung tissues yielded one or more bacterial types even though lopéz (2001) reported that normal lungs were ideally supposed to be sterile as the pulmonary macrophages continue to scavenge for bacterial agents in the lungs thereby making the lung free from pathogens as a defensive mechanism. in addition to the above mentioned major bacterial pathogens, other bacterial species have been isolated from the lungs and trachea of both apparently healthy and pneumonic camels. this signifies that a number of factors may be involved in camel respiratory infections. despite the fact that micrococcus, enterococcus, coagulase-negative staphylococcus species, bacillus, proteus, corynebacterium, e. coli, p. aeruginosa, and neisseria species are considered non-pathogenic, their isolation from pneumonic camel lungs by various workers (al-doughaym et al. 1999; al-tarazi 2001; abubakar et al. 2008; azizollah et al. 2009; wareth et al. 2014 ) indicated that they could have a role in the development of respiratory infections. hence, the possible role of these bacteria in camel respiratory disease complex under various conditions should not be overlooked though they have not been considered as major causative agents. very recently, an emerging middle-east respiratory syndrome coronavirus (mers-cov) has been reported in camels of the middle east and beyond (funk et al. 2016; ali et al. 2017) . we have also isolated a variety of viruses from respiratory tracts of ethiopian camels (ayelet et al. 2013) . therefore, viruses could play a significant role as a primary or complicating causative agents in camel respiratory infections. furthermore, the study assessed the antibiotic susceptibility profiles of the bacterial isolates in order to choose the most effective antimicrobial agents that could be used to treat camels with respiratory problems in the area. accordingly, increased level of resistance among the respiratory pathogens against the commonly used antimicrobials in respiratory tract infections was observed ( table 2 ). the majority of camel respiratory pathogens were found susceptible to norfloxacin, streptomycin, and gentamicin but resistant to the action of ampicillin and tetracycline. this is in agreement with the results of el-mahmood et al. (2009) and serin et al. (2010) . in addition, a similar resistance of s. equi subsp. equi and k. pneumoniae to tetracycline was recorded (nkang et al. 2009 ). fazlani et al. (2006) reported a high level of resistance of m. haemolytica and p. multocida to streptomycin. in this study, however, both organisms were found to be susceptible to streptomycin, while they were resistant to tetracycline and ampicillin. this represents a shift of resistance from streptomycin to tetracycline and ampicillin. this may be associated with the present lesser usage or misuse of streptomycin for animal diseases unlike tetracycline which is commonly used for most animal diseases today. the high rate of resistance observed in many of the isolates to tetracycline and ampicillin could be either because they are frequently and unnecessarily prescribed or sold over the counter in the open market and private veterinary drug shops without prescription. the people in the study areas have easy access to purchase tetracycline and ampicillin from the private pharmacies and any open market without prescription. they have also the opportunity of getting many drugs from ngo's working in livestock production, but they have a strong desire to tetracycline. as a result, they provide health care by themselves. this probably means that the selective pressure of these commonly used antibiotics on the bacteria circulating in the community could have resulted in high frequency of resistance among the isolates. apart from ease of accessibility, these drugs have been found to be adulterated and therefore used at a very low dosage. therefore, there is a need for practitioners and researchers to be aware of the bacterial flora of the camels and of their antibiotic sensitivities so as to be informed of the appropriate antibiotics to be used in the course of respiratory infections and control programs. in conclusion, the present and previous studies in this area pointed out that respiratory infection is considered as the major cause of morbidity and mortality in camels. it is a multifactorial process among which a variety of bacterial species have been associated with respiratory problems. in the present investigation, s. equi subsp. equi, p. multocida, m. haemolytica, k. pneumoniae, s. aureus, and b. bronchiseptica have been isolated at much higher proportions from the lungs of pneumonic than the apparently healthy camels. therefore, these bacterial spp. could be considered as candidate pathogens causing camel respiratory infections in the study area. furthermore, the isolates have shown considerable resistance to commonly prescribed antimicrobials in the country calling for the need to conduct susceptibility testing for control of camel respiratory infections in the area. therefore, we believe the data in this work contributes to diagnosis and control of respiratory problems in camels, which are important livestock spp. in arid and semi-arid regions in ethiopia, in particular, and the tropics, in general. funding information this research project was financially supported by the subtr project entitled binvestigation on major camel diseases in ethiopia and development of intervention strategies^under the tr project of banimal health improvement,^granted within the 1st thematic research (tr) of addis ababa university, ethiopia. hence, the authors are indebted to the project for its financial support as well as the college of veterinary medicine and agriculture, addis ababa university, and semera regional veterinary laboratory for hosting and providing all the necessary facilities for smooth and effective execution of this study. conflict of interest the authors declare that they have no conflict of interest. camel pneumonia in nigeria: epidemiology and bacterial flora in normal and diseased lung characterization of bacteria isolated from dromedary camels affected with pneumonia for the first time in sudan aetiological study on pneumonia in camel (camelus dromedarius) and in vitro antibacterial sensitivity pattern of the isolates vaccines against middle east respiratory syndrome coronavirus for humans and camels systematic, active surveillance for middle east respiratory syndrome coronavirus in camels in egypt bacteriological and pathological study on pneumonia in the one-humped camel (camelus dromedarius revue d' elevage et de medicine veterinaire des pays tropicaux investigation of respiratory viruses in camel slaughtered at addis ababa akaki abattoir the aerobic bacterial population of the respiratory passageways of healthy dromedaries in najaf-abbad abattoir, central iran gross and microscopic pulmonary lesions of camels from eastern ethiopia. tropical animal health and production viral and bacterial interactions in the upper respiratory tract failure of respiratory defenses in the pathogenesis of bacterial pneumonia of cattle central statistical agency (csa) 2004. agricultural sample survey report on livestock and livestock characteristics biochemical and antigenic characterization of mannheimia, pasteurella and mycoplasma species from naturally infected pneumonic sheep and goats isolation and identification of aerobic bacterial flora from the upper respiratory tract of donkeys in central ethiopia antimicrobial susceptibility of some respiratory tract pathogens to commonly used antibiotics antimicrobial susceptibility of bacterial species identified from mastitic milk samples of camel mers-cov at the animal-human interface: inputs on exposure pathways from an expert-opinion elicitation bacteriological examination of respiratory tract of apparently healthy camels in egypt comparison of e-test and agardisc diffusion methods for antibiotic susceptibility testing of thermophilic campylobacter of animal origin respiratory system, thoracic cavity, and pleura staphylococcus aureus in animals and food: methicillin resistance, prevalence and population structure the aerobic bacterial flora of the respiratory passage ways of healthy goats in dire dawa abattoir bacteriological studies on pulmonary lesions of camel (camelus dromedarius) slaughtered at addis ababa abattoir assessment of antibiotics susceptibility profiles of some selected clinical isolates from laboratories in nigeria invasive streptococcal disease: a review for clinicians pasteurella species peritoneal dialysis-associated peritonitis: household pets as a risk factor investigation of a new pathological condition of camels in ethiopia detection of antibodies of rinderpest and peste des petits ruminants viruses (paramyxoviridae, morbillivirus) during a new epizootic disease in ethiopian camels (camelusdromedarius) preputial bacterial flora and antibiotic susceptibility in wrestling dromedary bulls in aydin region of turkey diversity of bacterial species in the nasal cavity of sheep in the highlands of ethiopia and first report of histophilus somni in the country subclinical pulmonary pathogenic infection in camels slaughtered in cairo isolation of streptococcus equi subsp. equi (strangles agent) from an ethiopian camel key: cord-348490-dqabq6d8 authors: maeder, muriel n.; rabezanahary, henintsoa m.; zafindraibe, norosoa j.; randriatiana, martin raoelina; rasamoelina, tahinamandranto; rakotoarivo, andry t.; vanhems, philippe; hoffmann, jonathan; bénet, thomas; rakoto andrianarivelo, mala; rakoto-alson, olivat a. title: sickle-cell disease in febrile children living in a rural village of madagascar and association with malaria and respiratory infections date: 2016-12-01 journal: bmc hematol doi: 10.1186/s12878-016-0069-1 sha: doc_id: 348490 cord_uid: dqabq6d8 background: in madagascar, the last study on sickle cell disease (scd) was done in the early 1980s. the country is known as endemic for malaria and respiratory infections. the main objective of this study was to estimate the prevalence of scd; the secondary objective was to evaluate its association with malaria and respiratory infections. methods: this is a cross-sectional study which was carried out in a rural village in the south east coast of madagascar between may 2011 and november 2013. participants were children aged between 2–59 months presenting with fever measured by axillary temperature ≥37.5 °c at inclusion. genotyping of haemoglobin s was done by pcr and malaria was diagnosed by rapid diagnostic test. research for viral and atypical bacterial respiratory pathogens was performed on nasopharyngeal swabs. uni-and multivariate polytomous logistic regression was done to assess associations between microbiological results and scd status, with hbaa phenotype as reference. results: a total of 807 children were analysed. prevalence of scd among febrile children was 2.4% (95% ci, 1.5–3.7%) and that of sct was 23.8% (95% ci, 20.9–26.9%). there was no difference in the prevalence of malaria infection according to haemoglobin status (p = 0.3). rhinovirus (22.5%), adenovirus (14.1%), and bocavirus (11.6%) were the most common respiratory pathogens detected. after univariate analysis, patients with scd were more frequently infected by parechovirus (p = 0.01), while patients with sct were more prone to rsv a or b infection (p = 0.01). after multivariate analysis, hbas phenotype was associated with higher risk of rsv a and b infection compared to hbaa (adjusted or = 1.9; 95% ci: 1.2–3.1, p = 0.009), while hbss phenotype was associated with higher risk of parechovirus infection (adjusted or = 6.0; 95% ci: 1.1–31.3, p = 0.03) compared to hbaa, independently of age, gender, period per quarter, and the other viruses. conclusion: the prevalence of scd among under-five children presenting with fever was high in the study population. no association was found between sct and malaria but few viruses, especially parechovirus, seem to play an important role in the occurrence of pneumoniae among scd patients. during its 59th world health assembly held in 2006, who recognized sickle-cell disease (scd), an inherited disorder of haemoglobin as a priority of public health. a resolution was adopted to develop and strengthen efforts for its prevention and management. sickle-cell anaemia, one of the most common forms of scd, is due to a point mutation within the sixth codon of the β-globin chain. the produced abnormal varianthaemoglobin s (hbs)is responsible for chronic haemolytic anaemia and vaso-occlusion which are the underlying causes of the clinical presentation of scd. individuals who express the homozygous form (hbss) manifest the disease, while those with the heterozygous form (hbas), also known as sickle-cell trait (sct), are usually asymptomatic carriers. initially limited to the sub-saharan africa, the middle-east and some parts of india [1] , scd has currently spread to all continents with the migration of populations. the βs-globin gene is found on five common haplotypes in africa (bantu, benin, cameroon and senegal) and asia (saudi arab-indian) [2] that can be used as a marker of genetic diversity and population origin. approximately 300,000 children with severe haemoglobin disorders are born every year worldwide [3] and over 75% of scd occur in africa where carrier frequency ranged from 1% (central african republic, senegal) to 38% (united republic of tanzania) [1] . in madagascar, several studies conducted in 1950s had estimated the general prevalence of scd from 4% in the central highlands [4] to 11-13% in the south-eastern coast [5] . significant ethnical variation and high prevalence in asymptomatic carrier up to 28% were also reported [6] . to our knowledge, the last published study was done in the early 1980s [7] . these studies, however, were based on the detection of sickle red blood cells in hypoxic conditions, also known as "sickling test", and the sickle solubility test [3] now considered as obsolete techniques. in area where malaria is endemic, its relation with sct was often studied, in particular the high degree of resistance to severe and complicated malaria in sickle cell trait [8, 9] . malaria is endemic in madagascar with strong and persistent transmission in the east coast [10] . from may 2011 to november 2013, a cross-sectional study was conducted in the rural village of ampasimanjeva in the south east cost of madagascar aiming to identify blood-borne protein biomarkers that can differentiate the causes of unexplained acute febrile illness in children. the study site is known for its high endemicity to malaria, acute respiratory infections and scd. the main objective of this study was to estimate the prevalence of scd; the secondary objective was to evaluate its association with malaria and respiratory infections. this is an observational cross-sectional study with prospective data collection of children aged between 2 to 59 months presenting with fever (axillary temperature ≥37.5°c according to who criteria). the study was conducted from may 2011 to november 2013 in the rural village of ampasimanjeva located in the south east cost of madagascar. ampasimanjeva is 320 km away from the capital city antananarivo with a total inhabitant estimated to be 22,000 according to the last census. the east coast has hot and humid subequatorial climate and an annual high cumulative rainfall of 4,000 mm per year. transmission of malaria in this region is reported to be strong and intense all over time. a standardized questionnaire on socio-demographic and clinical data was fulfilled for each individual. the ampasimanjeva community hospital is a hospital district centre receiving each year around 950 children between 2 and 5 years old. acute respiratory infections were the main cause of morbidity accounting for 35% of consultation all ages combined. ethical approval for the study was obtained from the national ethical committee of the ministry of health of madagascar (authorization 019-ce/minsan as of 09/ 04/2010). individual written informed consent was provided by the parents of all study participants. for each child, venous blood was collected with safety blood containers on dry and spray-dried edta. the samples were transported in a refrigerated container at +4°c to the centre d'infectiologie charles mérieux for analysis. transport of samples was organized twice a week. after dna extraction (qiaamp dna mini kit, qiagen, germany) of the edta blood samples, the mutation responsible for the generation of hbs was identified by restriction fragment length polymorphism (rflp) assay of pcr-amplified dna (bio-rad, usa) [11] . the rflp pattern of the normal profile (hbaa) of healthy subject is characterized by the presence of the restriction endonuclease ddei site and cleaved in 2 fragments (189 and 93 bp). the homozygous profile (hbss) responsible of the disease has no restriction site and only one fragment of 282 bp is observed. the heterozygous profile (hbas) also known as sickle-cell trait combines restriction and no restriction site and reveals 3 fragments (93, 189, and 282 bp). rapid diagnostic test (rdt) of malaria was performed on site using carestart™ malaria (access bio, inc., new jersey, usa) and parasite density was estimated microscopically from the positive samples as previously described [12] . bacterial and viral dnas as well as viral rna were extracted from the nasopharyngeal swabs using rtp® pathogen kit (stratec molecular, germany). a multiplex realtime pcr assay allowing the identification of 22 respiratory pathogens (fast-track diagnostics respiratory pathogens kit, ftd luxembourg) was used as previously described [13] . following nucleic extraction of edta-whole blood samples, another multiplex real-time pcr assay was performed to identify staphylococcus aureus, streptococcus pneumoniae and haemophilus influenzae type b as previously described [14] . all rna/dna amplifications and detections were done using bio-rad cfx96 machine (bio-rad, usa). categorical variables were described as number and percentage with their 95% confidence intervals (ci); continuous covariates were described as median and interquartile range (iqr). the chi2 test or fisher's exact test was used to compare categorical variables. continuous covariates were compared by mann-whitney u-test or kruskal-wallis one-way analysis of variance. univariate and multivariate polytomous logistic regression analysis was done to assess microbiological results associated with haemoglobin status, where the normal phenotype was the reference (hbaa). statistical analyses were done using epi info™ 7.1.3 (centers for disease control and prevention, atlanta, georgia) and stat 13.0 (statacorp). all tests were 2-tailed and a p-value of less than 0.05 was considered significant. there were 862 children enrolled in the survey. among them, 55 were excluded because not meeting the age and temperature criteria and because of inadequate blood sample. subsequently, a total of 807 children were selected for the final analysis. out of the 807 children participating in the survey, 401 (49.7%) were male and the median age was 20 months (iqr: 10-36 months). the median temperature at inclusion was 38.8°c (iqr: 38.5-39.4°c). to assess the weight gain and growth, anthropometric parameters of children were measured [15] . the median upper arm circumference was 140 mm (iqr: 132-146 mm). the median weight-for-height z-score was−1.5 (iqr:−2,2; 0.7); and 245 (30.5%) had weight-for-height z-score ≤−2sd. the main reasons for medical consultation were malaria (36.6%), pneumonia (33.2%), acute respiratory infection (28.5%), and asthma (1.6%). pneumococcal conjugate vaccine was introduced in 2012 and the national coverage was 76% in 2013 according to who and unicef estimates. however no information on the vaccination status against s. pneumoniae is available among the study population. to identify the genotype of haemoglobin, the pcrproducts from all 807 blood samples were digested and analyzed by the rflp method. the results of the first 100 genotyping assay were compared to those of the electrophoresis of haemoglobin on alkaline (ph 8.5) agarose gels (hydragel 7 hemoglobine, sebia, evry, france) and 100% concordant identification was observed (data not shown). overall, 73.9% (95% ci, 70.7-76.8%) children expressed the normal phenotype hbaa, whereas the prevalence of sct (heterozygous hbas) was 23.8% (95% ci, 20.9-26.9%) and that of scd (homozygous hbss) was 2.4% (95% ci, 1.5-3.6%). to classify the nutritional status of the study population, we evaluated the mean weight-for-height z-score. (table 1) . these findings were supported by a significant difference in the values of mid-upper arm circumference between the 3 groups (p = 0.001). we also found a higher prevalence of males among individuals with hbss (68.4%) compared to the hbas (42.7%) and hbaa (51.3%) groups (p = 0.03). out of the 807 children, rdt showed 295 (36.6%; 95% ci, 33.3-39.9%) positive results of which 273 giemsastained thick smear tests were done. the microscopic results showed that 197/273 (72.2%) infections were caused by plasmodium falciparum, 2/273 (0.7%) were due to both p. falciparum and p. vivax, and the others were negative (74/273, 27.1%). there was no association between haemoglobin status and malaria infection (table 1) . strikingly, no children with hbs below 2 years of age were infected; however, a trend in an increased detection rate of p. falciparum in older children with hbas and hbss was observed (fig. 1) . overall out of 764 nasopharyngeal samples tested, rhinovirus (22.5%; 95% ci, 19.7-25.6%; n = 172), adenovirus (14.1%; 95% ci, 11.8-16.8%; n = 108), bocavirus (11.6%; 95% ci, 9.6-14.1%; n = 89), respiratory syncytial virus a or b (11.3%; 95% ci, 9.2-13.7%; n = 86), and human metapneumovirus a and b (9.0%; 95% ci, 7.1-11.3%; n = 69) were the most common respiratory pathogens detected; chlamydia pneumoniae and mycoplasma pneumoniae accounted for < 2.0% of cases (fig. 2) . parechovirus was most frequently detected from respiratory sample in hbss patients (n = 2, 11.1%) compared to the hbaa (n = 9, 1.6%) and hbas (n = 1, 0.6%) patients (p = 0.02). similar results were observed with human coronavirus 229e where patients in the hbss group yielded high positive result (n = 1, 5.6%) compared to the hbaa (n = 6, 1.1%) and hbas (n = 0) groups (p = 0.07) ( table 1) . conversely, respiratory syncytial virus a and b infection were more frequent in hbas individuals (n = 30, 17.0%) compared to hbaa (n = 56, 9.8%) and hbss (n = 0) patients (p = 0.01). viral coinfection were frequent in the hbss group (n = 8, 44.4%) compared to the hbaa (n = 144, 25.3%) and hbas (n = 34, 19.2%) groups (p = 0.03). univariate polytomous regression disclosed the risk of respiratory syncytial virus a and b which was 2-fold more higher in hbas compared to hbaa groups (crude odds ratio [or] = 1.9; 95% ci: 1.2-3.0, p = 0.01), and the risk of parechovirus infection which was higher in hbss compared to hbaa groups (crude or = 7.8; 95% ci: 1.6-38.9; p = 0.01) [ table 2 ]. after multivariate analysis, hbas compared with hbaa phenotype remained associated with higher risk of rsv a and b infection (adjusted or = 1.9; 95% ci: 1.2-3.1, p = 0.01), independently of age, gender, period per quarter, and parechovirus infection. hbss compared with hbaa phenotype remained associated with higher risk of parechovirus infection (adjusted or = 6.0; 95% ci: 1.1 our study shows that prevalence of scd was 2.4% and that of sct 23.8% among children aged 2-59 months presenting with fever and living in high endemic area for malaria. to our knowledge, this study is the first to document the prevalence of hbss in febrile young children. although no similar age group is available for comparison, previous results report high carrier rate of 29.6% to 31.9% [16, 17] from the same ethnic group in which the βs-globin gene frequency was reported to be 0.159 [17] . we cannot certify however the ethnicity of our studied population because it has not been queried during the investigation. in an extensive study of 1,214 our results demonstrate a significantly lower mean weight-for-height z-score for patients with scd than those of normal children and individuals with sct. previous studies from other developing countries have reported a state of undernutrition among patients with scd [18, 19] that may lead to slow growth and maturational abnormality, [20] . these results confirm previous observations that nutritional supplementation is a major component in the management of scd. studies reporting gender differences in scd incidence are rare and mainly focus on the occurrence of crises or comorbidities [21, 22] . however, in a cross-sectional study carried out over 735 students in india, the prevalence of sct using the sickling test was higher among male (7.3%) compared to that among female (5.9%) [23] . our study found higher scd prevalence among male population, but no significant difference between male and female for sct prevalence. this should be taken into account in the gender-selective use of medical facilities and management of patients. previous studies have shown that sct protects against severe forms [8] and mild malaria infections [24, 25] , although the precise mechanism remains poorly understood. in our study, a high proportion of p. falciparum positive result (36.6%) has been found among the studied population. overall, we did not find any statistical difference between malaria-infected children in the hbas group compared to the normal phenotype. however, as the age increases, the frequencies of children in the hbas and hbss groups who experienced malaria increases. the absence of malaria infection in the patients with scd up to 2 years of age may have several causes including an early death due to malaria or other infections. individuals with scd are strongly susceptible to encapsulated bacterial organisms such as s. pneumoniae, a common colonizer of the nasopharynx in healthy children. here we report a very high pneumococcal carriage rate among the febrile children studied. strikingly, all children (100%) with homozygous sickle cell disease in our study were carriers. in previous reports, a much lower rate was found in the us (12%) [26] and uganda (33%) [27] during study conducted on patients with homozygous sickle cell disease. in our study, pneumonia and acute respiratory infections were the main reasons for medical consultation after malaria. however, the role of s. pneumoniae in the development of the disease in the majority of patients is hypothetical due to the absence of healthy matched control group. it should be mentioned that 1.9% of the patients had bacteraemia with s. pneumoniae during the course of infection but its causative role has not been proven up as up to 5% of febrile children may also develop asymptomatic or occult bacteraemia [28] . the overall prevalence of viral infection/detection among the study population was high independent of haemoglobin status. however, among the 23 viral and atypical bacteria pathogens studied, there was no statistical difference in prevalence between the three groups for 20 of them and include hrv the most common respiratory virus detected or influenza virus a (h1n1)/ pdm09 known to increase disease severity in children with scd [29] . parechovirus, a member of the family picornaviridae, was significantly associated with a high risk of infection in scd patients in our study. parechovirus infections are usually asymptomatic or lead to a mild gastro-intestinal or respiratory diseases. only two patients had parechovirus detected, both presenting with symptoms of pneumoniae, suggesting that larger prospective study is required to better describe the real impact of parechovirus infection among scd population. conversely, rsv a or b belonging to the family paramyxoviridae, was significantly associated with a high risk of infection in sct patients compared to the normal population whereas no virus was detected in scd patients. rsv a and b are a major cause of lower respiratory tract infection during infancy and childhood and were associated with acute chest syndrome similar in severity to influenza infection in febrile children with scd [30] . the reason why rsv a and b were not detected in our study using a sensitive molecular tool is unknown but it may be linked either to the small number of identified scd patients, or the possible death of those children. however no follow-up of the study population was carried out to ascertain the latter hypothesis. detection of respiratory viruses has been facilitated by the recent widespread availability of nucleic acid amplification techniques. in a study carried out previously in ampasimanjeva to describe the aetiology of a clinically defined cohort of children with fever and acute respiratory infections, the most representative pathogens were human metapneumovirus (hmpv) for pneumonia, human parainfluenzavirus (hpiv) for other acute lower respiratory infections, respiratory syncytial virus (rsv) for flu-like illnesses and human adenovirus (hadv) for upper respiratory tract infections [13] . however, the clinical significance of viral detection in biological samples from patients with respiratory infections remains often unclear. for example, when detected in a patient with severe pneumonia, respiratory viruses may represent subclinical infection, persistent shedding after a prior infection, infection restricted to the upper respiratory tract, or infection involving the lower respiratory tract [31] [32] [33] . while some pathogens, such as rsv, hmpv and flu seem to be strict pathogens, other viruses such as hrv are widely identified in both cases and controls. it was beyond the scope of this study to evaluate the viral distribution in asymptomatic patients to study proportion of viral shedding in a control group, or to distinguish between upper and lower respiratory tract infections. nevertheless, the same study conducted a year ago in the same population has confirmed our findings that a wide range of respiratory viruses might have played a role in escalating respiratory tract viral infections among patients with scd and sct. our study was limited by the fact that the genotyping method used may not be totally specific to detect haemoglobin s as the presence or the absence of the restriction site ddei may be found in other hemoglobinopathies. however, according to one recent publication [34] , hbs seemed to be the most prevalent in africa where over 700 haemoglobin variants were identified. additionally, the association between sickle cell trait and malaria is not conclusive for the following reasons: the initial design was probably not optimal to detect this association in the current study, and more accurate methods for detecting hbs should have been used (i.e. hemoglobin electrophoresis). further independent and well-structured research should be considered in that matter. a second limitation of this study is the absence of long-term follow-up to estimate the outcomes of children and the proportion and causes of death mainly among those living with scd. our findings show that the prevalence of scd among under-five children presenting with fever was high and a better knowledge of the role and distribution of viral and atypical bacteria pathogens in the study population has been found. this study has helped moving towards a better understanding of the epidemiology of scd, and we hope it will trigger further research in that direction, for the following reasons. firstly, as scd is a major public health concern in madagascar, a large prospective study on the general population to estimate the true magnitude of the disease should be done. secondly, it is important to document data about the clinical course and mortality associated to scd in regions where it is endemic. thirdly, study involving increasing laboratory capacity should be conducted to help a better understanding of scd epidemiology, as well as other hemoglobinopathies. finally, conclusions from these studies will be used for awareness-raising among national authorities in order to strengthen the national control program. this work was supported by grants from the fondation mérieux, lyon, france. the authors thank the children's families for their consent to participate in this study, and the entire medical staff at medical foundation of ampasimanjeva for assistance and cooperation. this paper is dedicated to doctor bénédicte contamin who died in august 2015 for her complete devotion to patients. the datasets supporting the conclusions of this article are available at the supplemental materials. authors' contributions mnm, jh and ora contributed to the design of the study, participated in data collection and analysis; mra, tb, pv, and jh contributed to data analysis and writing of the final manuscript; hmr, njz, tr and atr carried out the experimental work and analyzed the data; mrr acquired data and contributed to the clinical management of patients. all authors read and approved the final version. inherited haemoglobin disorders: an increasing global health problem the co-inheritance of alpha-thalassemia and sickle cell anemia is associated with better hematological indices and lower consultations rate in cameroonian patients and could improve their survival sickle cell disease: new opportunities and challenges in africa initial research on sicklemia in madagascar blood groups and drepanocytosis on saint mary's island (madagascar) new research on drepanocytosis in madagascar epidemiological survey and sanitary problems in a village in east central madagascar protective effects of the sickle cell gene against malaria morbidity and mortality sickle cell anaemia and malaria epidemiological stratification of malaria in madagascar rapid and nonradioactive prenatal diagnosis of beta thalassaemia and sickle cell disease: application of the polymerase chain reaction (pcr) evaluation of carestart tm malaria pf/pv combo test for plasmodium falciparum and plasmodium vivax malaria diagnosis in butajira area, south-central ethiopia viral and atypical bacterial etiology of acute respiratory infections in children under 5 years old living in a rural tropical area of madagascar multicenter case-control study protocol of pneumonia etiology in children: global approach to biological research, infectious diseases and epidemics in lowincome countries (gabriel network) length/height-for-age, weight-for-age, weight-for-length, weight-for-height and body mass index-for-age: methods and development new hypothesis on its introduction in africa beta-globin haplotype analysis suggests that a major source of malagasy ancestry is derived from bantu-speaking negroids an observational study of children with sickle cell disease in kilifi, kenya nutritional status, hospitalization and mortality among patients with sickle cell anemia in tanzania malnutrition in sickle cell anemia: implications for infection, growth, and maturation gender differences in pain and healthcare utilization for adult sickle cell patients: the pisces project an analysis of fetal hemoglobin variation in sickle cell disease: the relative contributions of the x-linked factor, beta-globin haplotypes, alpha-globin gene number, gender, and age do gender differences influence the prevalence of sickle cell disorder and related morbidities among school children in rural central india? red blood cell polymorphisms in relation to plasmodium falciparum asymptomatic parasite densities and morbidity in senegal differential impact of sickle cell trait on symptomatic and asymptomatic malaria colonization with antibiotic-resistant streptococcus pneumoniae in children with sickle cell disease nasopharyngeal carriage rate of streptococcus pneumoniae in ugandan children with sickle cell disease occult pneumococcal bacteremia: a review severe pandemic h1n1 and seasonal influenza in children and young adults with sickle cell disease respiratory syncytial virus and seasonal influenza cause similar illnesses in children with sickle cell disease frequent detection of respiratory viruses without symptoms: toward defining clinically relevant cutoff values identification of respiratory viruses in asymptomatic subjects: asymptomatic respiratory viral infections respiratory viruses in adults with community-acquired pneumonia global epidemiology of haemoglobin disorders and derived service indicators the authors declare that they have no competing interests. • we accept pre-submission inquiries • our selector tool helps you to find the most relevant journal submit your next manuscript to biomed central and we will help you at every step: key: cord-342133-khrljehj authors: principi, nicola; piralla, antonio; zampiero, alberto; bianchini, sonia; umbrello, giulia; scala, alessia; bosis, samantha; fossali, emilio; baldanti, fausto; esposito, susanna title: bocavirus infection in otherwise healthy children with respiratory disease date: 2015-08-12 journal: plos one doi: 10.1371/journal.pone.0135640 sha: doc_id: 342133 cord_uid: khrljehj to evaluate the role of human bocavirus (hbov) as a causative agent of respiratory disease, the importance of the viral load in respiratory disease type and severity and the pathogenicity of the different hbov species, we studied all hbov-positive nasopharyngeal samples collected from children who attended an emergency room for a respiratory tract infection during three winters (2009–2010, 2011–2012, and 2013–2014). human bocavirus was detected using the respiratory virus panel fast assay and real-time pcr. of the 1,823 nasopharyngeal samples, 104 (5.7%) were positive for hbov; a similar prevalence was observed in all three periods studied. among hbov-infected children, 53.8% were between 1–2 years old, and hbov was detected alone in 57/104 (54.8%) cases. all of the detected hbov strains belonged to genotype 1. the median hbov load was significantly higher in samples containing strains with both the n546h and t590s mutations compared to other samples (p<0.05). children with a single hbov-1 infection more frequently had upper respiratory tract infections (urtis) than those who were co-infected (37.0% vs 17.8%, respectively, p = 0.04). the duration of hospitalization was longer among children with high viral loads than that observed among children with low viral loads (8.0 ±2.2 days vs 5.0 ±1.5 days, respectively, p = 0.03), and the use of aerosol therapy was more frequent among children with high viral loads than among those with low viral loads (77.1% vs 55.7%, respectively, p = 0.04). this study shows that hbov is a relatively uncommon but stable infectious agent in children and that hbov1 seems to be the only strain detected in italy in respiratory samples. from a clinical point of view, hbov1 seems to have in the majority of healthy children relatively low clinical relevance. moreover, the viral load influences only the duration of hospitalization and the use of aerosol therapy without any association with the site of the respiratory disease. human bocavirus (hbov) is a recently identified viral agent that belongs to the family parvoviridae and contains a single linear positive-sense or negative-sense single-stranded deoxyribonucleic acid genome [1] . this virus has been detected mainly in younger children, in nasopharyngeal secretions, in sera and blood samples of patients with upper (urti) and lower (lrti) respiratory tract infections and in faecal specimens of subjects with gastroenteritis [2] . currently, hbovs are classified into species 1 through 4; hbov1 is predominantly found in the respiratory tract, and hbov2, hbov3, and hbov4 are found mainly in stool [3] . despite there are studies suggesting that hbov is able to infect the lower airways causing severe infections in both children and adults, the role of hbov as a causative agent of respiratory disease is frequently questioned due to its common detection with other potential pathogens [4] and the evidence that in some studies co-infections can have a significantly greater clinical and socioeconomic impact on infected children and their households than hbov infection alone [5] . moreover, the importance of the viral load in determining the type and severity of respiratory disease as well as the pathogenicity of the different hbov species [6] are not precisely defined. the main aim of this study was to contribute to resolving these problems. the circulation of hbov during several winter seasons in italy was investigated, and a phylogenetic analysis of detected strains was performed. in addition, correlations between different hbov strains and the severity of disease in cases with infections due to hbov alone or due to co-infections were studied. finally, the role of the viral load was analysed. to evaluate the circulation of the different hbov types and the possible relationships between viral load, virus genetic characteristics, and the severity of infection, nasopharyngeal swabs were collected from otherwise healthy children attending the emergency room of the fondazione irccs ca' granda ospedale maggiore policlinico, university of milan, italy, due to a respiratory tract infection arising between november 1 and march 31 during 3 winters (2009-2010, 2011-2012, and 2013-2014) . the study was approved by the ethics committee of the fondazione irccs ca' granda ospedale maggiore policlinico, milan, italy. written informed consent of a parent or legal guardian was required, and children 8 years of age were asked to give their written assent. patients' demographic characteristics and medical histories were retrieved from hospital charts and were systematically recorded before and after the first visit to the emergency room using standardized written questionnaires [7] . the study patients were classified into disease groups (i.e., acute otitis media, rhinosinusitis, pharyngitis, croup, infectious wheezing, acute bronchitis, pneumonia) on the basis of signs and/or symptoms using well-established criteria and were finally subdivided into two subgroups: upper (urtis) and lower respiratory tract infections (lrtis) [8] . nasopharyngeal secretions were collected from all of the children immediately after admission to the emergency room using a paranasal flocked swab (1 swab per child), which was stored in a tube containing 1 ml of universal transport medium (kit cat. no. 360c, copan italia, brescia, italy). viral nucleic acids were extracted from each swab by means of a nuclisens easymag automated extraction system (biomeriéux, craponne, france), and the extract was tested for respiratory viruses using the respiratory virus panel xtag rvp fast v2 (luminex molecular diagnostics, inc., toronto, canada), which simultaneously detects influenza a virus (subtypes h1 or h3); influenza b virus; respiratory syncytial virus (rsv) types a and b; human parainfluenza virus types 1-4 (hpiv1-4); adenovirus (adv); human metapneumovirus (hmpv); coronaviruses (hcov) 229e, nl63, oc43 and hku1, enterovirus/rhinovirus (ev/hrv); and hbov, in accordance with the manufacturer's instructions [9, 10] . samples that were positive for hbov were stored at -80°c. hbov real-time polymerase chain reaction (pcr) viral nucleic acid extracts previously testing positive for hbov were re-tested for confirmation by two different singleplex real-time pcrs using taqman universal master mix ii (applied biosystems, california, usa). amplification and detection of viral dna were performed with a 7900ht real-time pcr system machine (applied biosystems, california, usa). conserved regions for rt-pcr primers and probes were identified in the hbov ns-1 and np-1 genes from the nucleotide sequence alignments available from genbank (for ns1, dq206700-08, dq000495-96, and dq200648, and for np-1, dq000495-96, ab243566-72, dq296618-35, dq353695-99, dq299885, dq267760-75, dq284856, dq295844, and am109958-66; http:// www.ncbi.nlm.nih.gov/genbank/). each 25 μl singleplex reaction mixture consisted of 0.5 μl of forward primer 5'-tgcagacaacgcytagttgttt-3' and reverse primer 5'-ctgtcccg cccaagataca-3' for the 88 base pair ns-1 target or forward primer 5'-agcatcgctcctacaaaagaaaag-3' and reverse primer 5'-tcttcatcacttggtctga ggtct-3' for the np-1 target, 0.125 μl of probe 5'-ccaggattgggtggaacctgcaaa-3' or 5'-aggctcgggctcatatcatcaggaaca-3', and 2.5 μl of sample viral dna. pcrs were conducted at 50°c for 2 min and then at 95°c for 10 min, followed by 45 cycles at 95°c for 15 sec and at 60°c for 1 min. taqman probes were labelled at the 5' ends with the reporter molecule 6-carboxyfluorescein and at the 3' ends with black hole quencher 1 (biosearch technologies, inc., novato, ca). each run included one synthetic template control and one no-template control for each target. specimen extracts were also tested by real-time pcr for the human rnase-p gene to monitor specimen quality and the presence of pcr inhibitors. a positive test for both the ns1 and np-1 targets or for a single target confirmed from a second extraction from a new sample aliquot was considered definitive evidence of hbov infection. the viral load was obtained using real-time pcr with the ns1 primers and probe previously described and a dna plasmid used as the standard calibrator. the amplified target fragment of the plasmid was verified by sequencing. plasmid dna concentrations were detected with an nd-1000 spectrophotometer (nanodrop products, wilmington, de, usa). each run included plasmid and negative controls. standard precautions were taken throughout the pcr process to avoid cross-contamination. negative controls and serial dilutions of the positive controls were included in every pcr assay. finally, quantitative results were reported as dna copies/ ml of respiratory samples. the viral load was defined as low for values 10 6 log (copies/ml) and as high for values >10 6 log (copies/ml). for genotyping, the viral vp-1/2 gene was amplified using a conventional pcr assay. briefly, 4 sets of forward and reverse primers (5'-cacagacagaagcagacgagat-3' and 5'-ggtg agaagtgacagctgtattg-3'; 5'-ttcagaatggtcacctctaca-3' and 5'-ctgtgc ttccgttttgtctta-3'; 5'-aactttgactgtgaatgggtta-3' and 5'-aaatagtgcc tggaggatgat-3'; 5'-ctatcaccagagaaaatccaatc-3' and 5'-gagacggtaaca ccacta-3') were used in pcr amplification and the quantitect probe master mix (qia-gen, venlo, netherlands) was used as the basis for the reaction mix. viral products were analysed by electrophoresis on a 1.5% agarose gel and purified with the qiaquick gel extraction kit (qiagen, venlo, netherlands). sequencing reactions were set up with purified dna, one of the specific primers used in the pcr and bigdye terminator v3.1 cycle sequencing kit (applied biosystems, california, usa) according to the protocol recommended by the manufacturer. sequencing and sequence analysis were performed on a 3130 genetic analyser (applied biosystems, california, usa). all alignments were performed using clustalx 2.1 and bioedit (version 7.1.3.0) software (ibis biosciences, carlsbad, ca). phylogenetic trees of the vp-1/2 protein gene were generated using the neighbour-joining method and p-distance model of the molecular evolutionary genetics analyses (mega) software, version 5.05 [11] . bootstrap probabilities for 1,000 iterations were calculated to evaluate confidence estimates. the graphs were made using graphpad prism version 5.01 for windows (graphpad software, san diego, ca). all genotyped sequences of the hbov vp-1/2 gene were submitted to genbank (accession numbers kr014412-kr014516). tests for positive selection were conducted on the datamonkey server [12] using the singlelikelihood ancestor (slac), and the fixed-effects likelihood (fel) [13] , the internal branch fixed-effects likelihood (ifel) [14] , the mixed effects model of evolution (meme) [15] , and fast unconstrained bayesian approximation methods (fubar). the dn/ds ratios were calculated using the slac and fel codon-based maximum likelihood approaches. the slac approach counts the number of non-synonymous changes per non-synonymous site (dn) and tests whether it is significantly different from the number of synonymous changes per synonymous site (ds). the fel approach estimates the ratios of non-synonymous to synonymous changes for each site in an alignment. the ifel method is similar to the fel, but tests site-bysite selection along only the internal branches of the phylogeny. in order to avoid an excessive false-positive rate, sites with slac, fel, ifel and meme p-values of <0.1 and a fubar posterior probability of >0.90 were accepted as candidates for selection. descriptive statistics of the responses were generated. continuous variables were presented as mean values and standard deviations (sds) and categorical variables as numbers and percentages. for categorical data, comparisons between groups were performed using a contingency table analysis with the χ 2 or fisher's exact test when appropriate. for ordered categorical data, a cochran-armitage test for trend was used to compare the groups. continuous data were analysed using a two-sided student's t-test after ensuring the data were normally distributed (based on the shapiro-wilk statistic) or using a two-sided wilcoxon's rank-sum test if the data were non-normal. all analyses were two tailed, and p-values of 0.05 or less were considered to be statistically significant. all analyses were conducted using sas version 9.2 (cary, nc, usa). during the three study periods, 1,823 nasopharyngeal samples were collected in the emergency room. of these, 104 (5.7%) tested positive for hbov (table 1) . among hbov infected children, 53.8% were between 1-2 years old, whereas 28.8% and 17.3% were aged <1 and 3 years, respectively. the prevalence of hbov detection was quite similar in the three studied periods; hbov was the only virus detected in 57/ 104 (54.8%) cases and was detected in association with one (89.5%) or more (10.5%) viruses in 47 (45.2%) cases. ev/hrv and rsv were the most common co-infecting viral agents and were found, respectively, in 20 and 18 samples. subjects with co-infection were younger than those without (p = 0.03). considering 10 6 dna copies/ml as a cut-off, the viral load was classified as low in 66 (63.5%) cases and as high in 38 (36.5%) cases. the phylogenetic tree constructed using the vp1/vp2 sequences showed that all of the italian hbov strains detected during the three study periods belonged to hbov genotype 1 (fig 1) . no unusual clustering was observed among the identified strains; hbovs circulating in 2009 were closely related to strains circulating in 2014. the sequence identity matrix of the vp1/vp2 gene showed minimum to maximum identity ranges of 97.8-100% between the italian strains and 98.4-99.7% with respect to hbov st1 reference strains (dq000495). in comparison to the reference strain, 8/105 (7.6%) strains had only one amino acid difference, 32/105 (30.4%) strains had two amino acid differences, and the remaining strains (65/105; 61.9%) had at least three amino acid changes. a total of 61/672 (9.1%) amino acid positions were observed to have at least one change in the vp1/vp2 sequence alignments (fig 2) . of these, 7/61 (11.5%) changes occurred within the vp1 unique (vp1u) region corresponding to the first 129 amino acids at the n-terminus of the vp1 gene. specifically, the following changes were observed: r17k, g28d, e29k, l40s, h43q, d72n, and g126e (fig 2) . the vp1u region includes the conserved phospholipase a 2 (pla 2 ) motif (nt 21-63). the vp1u sequences of all hbov isolates identified in this study revealed conserved yxgxg (nt [16] [17] [18] [19] [20] and hdxxy (nt 41-45) motifs in the catalytic site of secreted pla 2 . in addition, the amino acid residues at positions 21, 41, 42 and 63 have been hypothesized to form the catalytic network for enzymatic activity. in our hbov strains, all the sequences had amino acids associated with efficient enzymatic activity (p21, h41, d42, and d63). of note, two hbov strains (mi-267-jan2014 and mi-272-jan2014) had a peculiar amino acid sequence in the 19-amino acid segment starting at amino acid 411 (kvptrrvqpyirqtnwkhr), which has not been previously reported in hbov strains included in the genbank database (in red, fig 1) . overall, these two strains had 13 and 14 amino acid changes compared to the hbov st1 strain, and almost all these changes were included in the region described below. the origin of this highly divergent region, which occurred in spite of the conservation displayed in the rest of the hbov dna genome, remains to be defined. a global analysis of selective pressure made using the slac model indicated an estimated overall dn/ds ratio of 0.18. overall, the site-specific analyses identified three sites (411, 536, and 546) as under positive selection by at least two methods used (slac, fel, fubar, and meme). the ifel model was used to determine the selection pressure acting on the vp1/vp2 codons along the internal branches of the tree. two positively selected codons (411 and 546) were identified. the selected sites, highlighted with arrows in fig 2, were presumably located in of these strains, 6/13 (46.1%) were also characterized by the n534k, q535p, and q535d mutations. several negatively selected sites were identified by different methods (table 2 ). regarding the viral load, a wide range of hbov dna levels from 3.5x10 2 to 7.5x10 9 copies/ml were found in the clinical samples. in fig 2 (right side) , the viral load of each italian hbov strain is reported near the aligned mutations. in the group of strains (n = 70; 66.6%) harbouring at least 2 mutations in addition to a149t, the values of the hbov load greater than 1x10 8 dna copies/ml are reported in grey. a total of 16 strains had a very high viral load, and 13/16 (81.3%) harboured the t590s mutation. this percentage is nearly significantly different than the overall frequency (42/70; 53.8%) of t590s in the group of strains described below (p = 0.08). seven of the 13 strains with the t590s mutation had an additional mutation in one of the sites under positive selection (reported in fig 2 with an asterisk) . in detail, 4/13 (30.8%) had the n546h change, 2/13 (15.4%) had the a411d change, and 1/13 (7.7%) had the a411t change. based on the observed data, we hypothesize that the double mutation of n546h with t590s may positively affect viral replication and specific immune response. as shown in table 3 , the median hbov load was significantly higher in samples of strains with the n546h and t590s changes than that in samples of wild type t590 strains, strains with only the t590s mutation, and strains with only the n546h mutation (p-values of 0.0078, 0.016 and 0.018, respectively). finally, the two divergent strains (mi-267-jan2014 and mi272-jan2014) with unusual amino acid changes were observed with viral loads lower than 10 6 dna copies/ml. this could suggest that these mutations do not confer a replicative advantage in these virus strains. in table 4 , data regarding demographic, clinical and laboratory characteristics of children infected by hbov-1 alone or co-infected with hbov-1 and one or more other respiratory viruses are reported. because a preliminary evaluation did not find any differences among subjects co-infected with ev/hrv, rsv or other viruses, all co-infections were considered together. as shown, children infected with only hbov-1 had urtis more frequently than those with a co-infection (37.0% vs 17.8%, respectively, p = 0.04). moreover, a similar illness within the family in the 7 days since patient enrolment was significantly more common among co-infected children than among those with a single infection (48.9% vs 26.5%, respectively, p = 0.02). no other significant differences between the groups were observed. table 5 shows the demographic, clinical, and laboratory characteristics of the enrolled subjects according to the hbov load. subjects with low and high viral loads were quite similar. the only significant differences were found in the duration of hospitalization, which was longer among children with a high viral load than among those with a low viral load (8.0 ±2.2 days vs 5.0 ±1.5 days, respectively, p = 0.03), and in the use of aerosol therapy, which was more frequent among children with a high viral load than among those with a low viral load (77.1% vs 55.7%, respectively, p = 0.04). moreover, mutations leading to a high or low viral load were not associated with atypical clinical characteristics. this study shows that in italy during the winter periods 2009-2010, 2011-2012, and 2013-2014, the incidence of hbov infection among children with respiratory disease was relatively low, limited to approximately 5% of cases, and did not significantly vary from year to year. the phylogenetic analysis showed that all of the strains detected in this study belonged to hbov genotype 1 and were closely related to the prototype strain identified by allander et al. [1] . this was expected because this genotype is the most common among hbovs associated with respiratory infections [1] . most of the patients in whom hbov1 was identified were younger than 3 years of age, further highlighting that younger children are the individuals most frequently infected by this viral agent [1] . serological studies have shown evidence that the number of subjects positive for anti-hbov1 antibodies continuously increases with increasing age group from the ages of 6 months to 6 years, and by the age of 2 years approximately 80% of children have been infected with hbov1 [16, 17] . more than 50% of the children infected by hbov1 in this study were coinfected with at least one other respiratory virus. moreover, co-infected patients had lrti more frequently than those infected by hbov alone. these findings are not surprising because simultaneous detection of hbov1 and other viruses in children with respiratory disease and greater severity in co-infected cases have been already reported in studies in which it was also demonstrated that hbov1 can frequently be identified in the respiratory secretions of asymptomatic subjects [18] [19] [20] [21] [22] [23] . recently, it has been reported that hbov1 can be shed for several days or months after a previous infection [24] , which could explain the simultaneous identification of hbov1 and other respiratory viruses, the frequency of asymptomatic infections and the generally greater severity of infections in co-infected individuals compared to those with hbov1 alone. in most of the co-infected cases, detection of hbov in the respiratory secretions with the new sensitive molecular methods able to identify very low viral loads might be a consequence of a previous clinically resolved disease, and a virus other than hbov was therefore the real cause of the disease. however, reports of severe clinical manifestations in patients infected with only hbov have been published [25] , highlighting that the assessment of the real importance of hbov infection in a single patient remains very difficult. studies on children with severe pneumonia, acute wheezing, asthma and/or bronchiolitis suggested that hbov1 is able to infect the lower airways down to the bronchioles [26] [27] [28] [29] [30] [31] [32] . moreover, hbov1 has been found as the only infectious agent in adult lung transplant recipients with severe lrti, whereas it was not detected in respiratory secretions of asymptomatic transplanted subjects [33] . this would indicate that hbov1 is not always a bystander or the cause of mild respiratory problems but rather a real, although relatively rare, causative agent of severe disease in both children and adults, particularly when they are immunocompromised. on the other hand, hbov can cause serious neurological infections [34] and contribute to chronic disease in adult patients mainly because it can persist after childhood infection and reactivate [35] . evaluation of the viral load has been considered a possible method to define when this virus is the real cause of a respiratory disease and when it is only a secondary infection. unfortunately, this approach has had no success because although some studies have shown evidence for a strict correlation between high viral load and severe lrti in children with a single hbov infection [36] [37] [38] , others, including the present study, did not show a clear relationship between these two variables [39] . however, the evolution of virulence appears to involve a variety of mechanisms in different viral systems, including mutations in regulatory regions and viral adaptation for utilization of alternative or expanded repertoires of cellular receptors. an alternative hypothesis to evaluate the importance of hbov1 concerns the correlation between viral load levels and the presence of specific mutations. however, mutations associated with increased or reduced replication are rarely reported for hbov. recently, hao et al. have reported that few nucleotide changes were correlated with a lower viral load [40] . in the present study, a double mutant (n546h and t590s) was observed in samples with a significantly higher viral load. however, further phenotypic validation studies are required to draw major conclusions regarding the impact of these mutations on viral replication. furthermore, as reported by qu et al. [41] , it seems that nucleotide changes in the vp1u region could affect the replication efficiency of hbov. likewise, in our strains all the amino acids of the catalytic site were conserved, and no mutations that affect spla 2 activity were identified. in agreement with others [42] , the phylogenetic analysis of this study confirmed the very low degree of variability in the hbov genomic region encoding proteins that are exposed to the virus surface and are therefore under immunologic pressure. only 9% (62 codons) of amino acids were found to have at least one change in the vp1/vp2 gene, a finding not substantially different from that reported by hao et al. in a different geographic area [40] . in our study, several amino acid changes were observed in strains circulating in almost all the respiratory seasons. this finding provides evidence that the selection of those variants best adapted to each particular environment might select for variants with an evolutionary advantage. seven of these mutations were located in a genomic region (i.e., vp1u) previously reported to be involved in the mechanisms of virus replication. for instance, the vp1u amino acid variations r17k and l40s have been previously reported [43] , whereas the remaining variations have not yet been described. interestingly, two hbov strains identified in respiratory samples collected in january 2014 had unusual amino acid sequences in a somewhat conserved genomic region. the reason for this genetic diversity is still undefined. however, as described for hbov, other parvoviruses [44] , and enteroviruses, a series of α-helices and β-barrels in the vp2 protein were intercalated by an external loop, in which the majority of the genetic variability accumulated. nevertheless, these two divergent strains were found in samples with low viral loads, and we might hypothesize a loss of replication advantage for these strains. in the present study, the dn/ds ratios for all pairwise comparisons were <1, which is in line with previous results showing that positive selection was extremely limited in parvoviruses [45] . in fact, selective pressure analyses have identified 3 codons under positive selection. in a previous paper, a different codon (l40) was identified as being under positive selection [46] . nevertheless, the great majority of codons were under negative or neutral selection, which has also been confirmed by others [47] . this finding suggests that only a few amino acids of the vp1/vp2 proteins present on the surface of the virion are potentially subjected to a strong selective pressure by the host immune response. in conclusion, this study confirms that hbov is less common than other respiratory viruses but that the frequency of its detection in children with respiratory disease is in time stable. it was detected with a prevalence of about 5% in several consecutive seasons and no unusual clustering was observed among identified strains, with strains circulating in 2009 being closely related to those circulating in 2014. moreover, only a minority of virus sites were found to be under positive selective pressure, and all the strains detected in respiratory tract infections of this italian study belonged to genotype 1. from a clinical point of view, this study highlights that in otherwise healthy children, hbov1 seems to have relatively low clinical relevance, because patients infected with hbov alone mainly suffered from an urti. the viral load was not associated with clinical characteristics of the infection, and viral mutations, despite affecting viral replication, did not affect the conditions or severity of the clinical presentation. further studies are needed to clarify the clinical relevance of hbov in children, particularly in those at risk for severe chronic underlying disease, and to evaluate the role of viral modification in conditioning the degree of viral virulence and the specific immune response. conceived and designed the experiments: np se. performed the experiments: ap az as. analyzed the data: fb. contributed reagents/materials/analysis tools: sbi gu as sbo ef. wrote the paper: np ap se. cloning of a human parvovirus by molecular screening of respiratory tract samples human bocavirus infections human bocaviruses are highly diverse, dispersed, recombination prone, and prevalent in enteric infections impact of viral infections in children with community-acquired pneumonia: results of a study of 17 respiratory viruses impact of human bocavirus on children and their families the human bocavirus role in acute respiratory tract infections of pediatric patients as defined by viral load quantification clinical and socioeconomic impact of different types and subtypes of seasonal influenza viruses in children during influenza seasons textbook of pediatric infectious diseases comparison of the luminex respiratory virus panel fast assay with in-house realtime pcr for respiratory viral infection diagnosis comparison of the luminex xtag respiratory viral panel with xtag respiratory viral panel fast for diagnosis of respiratory virus infections mega5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods datamonkey: a suite of phylogenetic analysis tools for evolutionary biology not so different after all: a comparison of methods for detecting amino acid sites under selection adaptation to different human populations by hiv-1 revealed by codon-based analyses detecting individual sites subject to episodic diversifying selection seroepidemiology of human bocavirus in hokkaido prefecture seroepidemiology of human bocavirus infection in jamaica human bocavirus: a novel parvovirus epidemiologically associated with pneumonia requiring hospitalization in thailand multiple versus single virus respiratory infections: viral load and clinical disease severity in hospitalized children human bocavirus in children: mono-detection, high viral load and viraemia are associated with respiratory tract infection human bocavirus detection in nasopharyngeal aspirates of children without clinical symptoms of respiratory infection human bocavirus infections in hospitalized children and adults detection of bocavirus in saliva of children with and without respiratory illness human bocavirus 1 primary infection and shedding in infants single detection of human bocavirus 1 with a high viral load in severe respiratory tract infections in previously healthy children human bocavirus detection in an atopic child affected by pneumonia associated with wheezing detection of human bocavirus in hospitalised children human bocavirus in children suffering from acute lower respiratory tract infection in beijing children's hospital clinical characteristics of human bocavirus infections compared with other respiratory viruses in spanish children role of human metapneumovirus, human coronavirus nl63 and human bocavirus in infants and young children with acute wheezing role of emerging respiratory viruses in children with severe acute wheezing etiology of bronchiolitis in a hospitalized pediatric population: prospective multicenter study absence of humanbocavirus in bronchoalveolar lavage fluid of lung transplant patients neurological manifestations in acute onset of viral gastroenteritis frequency and clinical relevance of human bocavirus infection in acute exacerbations of chronic obstructive pulmonary disease human bocavirus in children: mono-detection, high viral load and viraemia are associated with respiratory tract infection human bocavirus in patients with respiratory tract infection comorbidity and high viral load linked to clinical presentation of respiratory human bocavirus infection frequent and prolonged shedding of bocavirus in young children attending daycare correlation between nucleotide mutation and viral loads of human bocavirus 1 in hospitalized children with respiratory tract infection phospholipase a2-like activity of human bocavirus vp1 unique region human bocavirus amongst an allages population hospitalised with acute lower respiratory infections in cambodia complete coding sequences and phylogenetic analysis of human bocavirus (hbov) human bocavirus capsid structure: insights into the structural repertoire of the parvoviridae evolutionary relationships among parvoviruses: virus-host coevolution among autonomous primate parvoviruses and links between adeno-associated and avian parvoviruses epidemic and molecular evolution of human bocavirus in hospitalized children with acute respiratory tract infection rapid molecular evolution of human bocavirus revealed by bayesian coalescent inference key: cord-331973-avjw4kx1 authors: das, shubhagata; dunbar, sherry; tang, yi-wei title: laboratory diagnosis of respiratory tract infections in children – the state of the art date: 2018-10-18 journal: front microbiol doi: 10.3389/fmicb.2018.02478 sha: doc_id: 331973 cord_uid: avjw4kx1 in the pediatric population, respiratory infections are the most common cause of physician visits. although many respiratory illnesses are self-limiting viral infections that resolve with time and supportive care, it can be critical to identify the causative pathogen at an early stage of the disease in order to implement effective antimicrobial therapy and infection control. over the last few years, diagnostics for respiratory infections have evolved substantially, with the development of novel assays and the availability of updated tests for newer strains of pathogens. newer laboratory methods are rapid, highly sensitive and specific, and are gradually replacing the conventional gold standards, although the clinical utility of these assays is still under evaluation. this article reviews the current laboratory methods available for testing for respiratory pathogens and discusses the advantages and disadvantages of each approach. acute respiratory tract infections are one of the leading causes of childhood morbidity and mortality worldwide, and it has been estimated that globally, respiratory infections are responsible for about 2 million deaths in children between 0 and 5 years of age (bryce et al., 2005; kallander et al., 2008) . approximately 80% of these respiratory infection cases are caused by viral pathogens such as influenza a and b, respiratory syncytial virus (rsv) a and b, parainfluenza virus types 1-3, adenovirus, rhinovirus, human metapneumovirus (hmpv), and others (mahony, 2008) . the non-specific clinical presentation of respiratory infections poses a considerable challenge to the differential diagnosis of these pathogens. early and accurate diagnosis of the causative pathogens in respiratory infections is essential to administer appropriate antiviral or antibacterial therapy, initiate effective infection control measures, and reduce the length of hospital stay (barenfanger et al., 2000; byington et al., 2002; akers et al., 2017) . in the last decade, there has been a remarkable improvement in the diagnosis of respiratory pathogens with the availability of molecular and pointof-care (poc) testing. although an increasing number of laboratories are adopting rapid molecular assays, conventional testing methods, such as culture and immunodiagnostics are still used. this review focuses on current laboratory methods for testing for respiratory pathogens, and discusses the advantages and disadvantages of each approach. electron microscopy (em) is one of the oldest direct examination methods that has been implemented for both clinical viral diagnosis and study of viral ultrastructure and pathogenesis in developed countries (roingeard, 2008) . historically, em has played an instrumental role in identifying novel viral strains in several outbreak situations, such as the coronavirus associated with the severe acute respiratory syndrome (sars) outbreak (falsey and walsh, 2003; ksiazek et al., 2003) . however, despite several advantages, the use of em has been limited in respiratory viral diagnosis as it is expensive, laborious, time-consuming, and has a greater turnaround time (approximately 3-16 h including specimen preparation), and is often insensitive when compared to other diagnostic methods (goldsmith and miller, 2009; zhang et al., 2013) . additionally, em requires strict control of experimental conditions, a high concentration of viral particles (> 10 5 l −1 ), and considerable technical skill and expertise for accurate analysis. detection of viruses by observing the cytopathic effect and hemadsorption in cell culture has been considered the "gold standard" for diagnosis of respiratory viral pathogens for decades. viruses such as adenovirus, influenza a/b, rsv, and human parainfluenza viruses are the most common respiratory viruses that are isolated and detected by cell culture (olsen et al., 1993; winn and koneman, 2006) . the traditional tube culture method is advantageous for growing a wide variety of viruses, including novel or unknown viruses, but takes days and often weeks to provide results. over the years, modified cell culture methods such as the centrifugation enhanced shell-vial method has reduced the turnaround time from 5 to 10 days to 24 h (dilnessa and zeleke, 2017) . shell-vial culture using combination cell lines allows simultaneous detection of multiple respiratory viruses and, as compared to conventional culture, has similar sensitivity for parainfluenza 1-3 (87% vs. 83%) and influenza a/b (78% vs. 75%), and significantly higher sensitivity for rsv (73% vs. 42%) (lasala et al., 2007) . despite these advantages, many clinically relevant viruses are difficult to grow in culture (such as rhinovirus and coronavirus) and may produce variable results (ieven and goossens, 1997; hodinka, 2013) . additionally, multiple freezing and thawing of the samples prior to testing can reduce the viral titer, thus effecting the growth in culture. therefore, as compared to molecular tests, both traditional tube and shell-vial culture methods are laborious, exhibit higher false negative rates, and have longer turnaround times, making viral culture less clinically relevant (leland and ginocchio, 2007; ginocchio, 2007; hematian et al., 2016) . culture is also the gold standard for detection of atypical (bacterial) respiratory pathogens, which is followed by identification and antimicrobial susceptibility testing by various manual or automated methods (tenover, 2011) . bacterial culture can also often be insensitive, especially when specimen adequacy is not determined by gram stain or if specimens were collected after antibiotic exposure (ats and idsa, 2005; woodhead et al., 2005; harris et al., 2017) . bacterial culture is also labor intensive, requires substantial technical expertise, and has a typical turnaround time of 48 -96 h if antibiotic susceptibility testing is performed, and can therefore be considered inadequate for optimal patient care and guiding effective antimicrobial therapy (tenover, 2011) . rapid immunoassays (rias) can deliver test results in less than 30 min and are usually performed in the poc setting, thus allowing the test results to be incorporated into the clinical decision-making algorithm (weinberg and walker, 2005) . among the four primary ria formats (latex agglutination, horizontal flow devices, lateral flow devices, and optical immunoassays), the lateral-flow immunoassay (lfia) is the most versatile and popular immunochromatographic method. rias are relatively inexpensive, easy to perform, and most of them have waived status in the united states according to the clinical laboratory improvement act (clia) guidelines, thereby making them invaluable in outpatient clinics, primary care, emergency, and low resource settings (ginocchio, 2007) . currently, commercially available rias are mostly limited to the detection of influenza a virus, influenza b virus, and rsv. numerous studies have revealed that rias demonstrate overall poor sensitivity for influenza and rsv (44-95%); however, they have a higher median specificity (90 to 95%) as compared to cell culture (who, 2005; leland and ginocchio, 2007; ginocchio, 2007) . in the pediatric population, commercially available immunoassays have demonstrated high sensitivity (93%) for detection of rsv, and a systematic review of published studies has further revealed that the sensitivity of rsv rias is relatively higher for children (81%) than adults (29%) (slinger et al., 2004; chartrand et al., 2015) . the higher sensitivity can be attributed to the fact that pediatric patients often shed higher titers of respiratory viruses and for a longer time as compared to adults (englund et al., 1996; kawai et al., 2007) . despite the overall lower sensitivity, rias have been deemed a valuable diagnostic tool in the emergency department as they can significantly decrease the length of stay, additional ancillary testing, and antibiotic prescriptions for those children who do test positive for influenza (abanses et al., 2006; blaschke et al., 2014) . direct fluorescent antibody (dfa) testing of nasopharyngeal wash specimens is considered a rapid and reliable method for detecting respiratory viral infections. commercial dfa kits have demonstrated high sensitivity and specificity for multiple respiratory viruses such as hmpv (95 and 100%), adenovirus (62 and 100%), rsv (94 and 96%), and parainfluenza viruses (88 and 99.7%), although the results can be subjective and require technical expertise for accurate interpretation (landry and ferguson, 2000; ohm-smith et al., 2004; rocholl et al., 2004; aslanzadeh et al., 2008; vinh et al., 2008) . the high specificity (99-100%) of dfa indicates that the test can be used as a reliable detection method, especially during the initial days of the illness, as was shown by shafik et al. (2011) for rsv in children. pathogen-specific antibodies typically appear about 2 weeks after the initial infection and can be detected by serological tests. serological tests can successfully identify antibodies to most respiratory pathogens such as rsv, adenovirus, influenza a and b, parainfluenza 1-3 virus, etc., and can detect mixed infections from hospitalized children suffering from acute respiratory infections, with the exception of infants for whom an antibody response is usually undetected (hall et al., 1991; chkhaidze et al., 2006) . however, it has been reported that serological assays are significantly less sensitive for the detection of parainfluenza virus and adenovirus when compared to molecular methods, such as rt-pcr (kuypers et al., 2006) . kuypers et al. (2006) found that rt-pcr detected 40% more specimens from pediatric patients that were positive for at least one respiratory virus than were detected by fluorescent antibody assay (fa). fa testing, in addition to rt-pcr, is useful for epidemiological studies as it increases the probability of identifying acute viral infections and has been used for accurate assessment of respiratory viruses other than influenza in children (sawatwong et al., 2012; feikin et al., 2013; zhang et al., 2017) . for bacteria, serological testing is particularly challenging, especially for identifying atypical bacterial agents such as mycoplasma pneumoniae. with varied sensitivity (14% -77%) and specificity (49% -97%) as compared to pcr, serological testing has limited usefulness clinically (beersma et al., 2005; wellinghausen et al., 2006) . the clinical utility of serologic tests is further limited because they require both acute and convalescent sera to monitor seroconversion or to identify a four−fold increase in antibody titer (loeffelholz and chonmaitree, 2010) . additionally, serological tests are not relevant for identifying frequently recurring viral infections as the serum igm levels are lower due to repeated exposure to vaccines or circulating viruses. for optimal virus specific igm testing, an acute-phase serum specimen should be obtained early in the course of illness (dunn, 2015) . a wide variety of newer, nucleic acid amplification tests (naats) for the detection of respiratory pathogens are commercially available. these tests are listed in table 1 and described below according to complexity and pathogen coverage. the accuracy of respiratory virus detection by molecular tests is not only dependent on their specific assay chemistry, but is also critically affected by the type, quantity, and quality of specimens collected (dunn, 2015) . several types of specimens can be used for detection of respiratory viruses, including: bronchoalveolar lavage (bal), throat swab, nasopharyngeal (np) washes, np aspirates, lung aspirates, and np swabs, although the appropriate specimen type depends on the specific patient population. for example, in pediatric patients, obtaining nasal washes and nasal aspirates is more technically challenging when compared to np swabs, especially in severely ill children and in poor resource settings (hammitt et al., 2012) . it has been observed that for optimal test results, specimens should be collected within 3-5 days after onset of symptoms to ensure that the sample has a high concentration of virus particles, viral antigen or viral nucleic acids, should be transported to the testing laboratory in refrigerated condition (2-8 • c) in appropriate transport media, and testing should be performed within 48 h (grys and smith, 2009 ). detection of respiratory pathogens by naats such as pcr, nucleic acid sequence-based amplification (nasba), transcription mediated amplification (tma), strand displacement amplification (sda), loop-mediated isothermal amplification (lamp), rolling circle amplification (rca), etc., have gained immense popularity over the past decade. large syndromic panels that can detect multiple pathogens simultaneously are beneficial for infection control, timely treatment decisions, and are substantially less expensive than detecting individual pathogens by monoplex real-time rt-pcr (reijans et al., 2008; schreckenberger and mcadam, 2015) . these multiplex respiratory panels vary in terms of their complexity (high, moderate, or waived), throughput, pathogens detected, ability to subtype, instrumentation (batched or random access), workflow (stepwise or integrated sample-to-answer), ease of use, and time to result (several hours to minutes) (caliendo, 2011; krunic et al., 2011; mahony et al., 2011) . studies have reported increased diagnostic yield (60% vs. 35%) and considerably higher sensitivity (80-100%) and specificity (82-100%) for these assays when compared to conventional diagnostic methods such as dfa, viral isolation, and immunoassays (reijans et al., 2008; gharabaghi et al., 2011) . in comparison to other naat-based molecular methods, high-plex syndromic panels demonstrate comparable performance, although sensitivity and specificity might vary for individual targets (sails et al., 2017) . for example, greater than 99% specificity was observed for all targets except enterovirus/rhinovirus (94%), and the overall sensitivity ranged between (83-100%) when the nxtag rpp and the anyplex ii rv16 assays were evaluated for detection of respiratory pathogens in hospitalized children (brotons et al., 2016) . syndromic panels can also detect bacterial infections and viral-bacterial coinfections, which is vital for developing effective treatment plans and prevention strategies (brealey et al., 2015; brotons et al., 2016) . however, for novel pathogen strains, such as flu a(h1n1pdm2009), panel tests often fail to identify the subtype and require confirmation by other tests (bryce et al., 2012) . syndromic respiratory panels with high sample throughput can usually run 1-96 specimens and are mostly classified as high complexity assays according to clia guidelines, which indicates that running these panel requires considerable knowledge, training, and experience (fda, 2018b) . it has been noted that these high throughput panels are ideal for batch testing, especially during outbreak situations and respiratory viral seasons when sample volumes are unusually high (ginocchio et al., 2009; crawford et al., 2010; tang et al., 2016) . despite the advantages, implementation of high throughput panels can be challenging because of demanding sample preparation, processing, and result interpretation procedures, and the turnaround time varies from approximately 5-16 h (beckmann and hirsch, 2016) . moderate complexity sample-to-answer molecular test systems have gained immense popularity over the last few years because of their ease of use, faster turnaround time, and efficient workflow. currently, the united states. fda-cleared sampleto-answer test systems include both low-plex, targeted assays and high-plex panels that can detect multiple pathogens and pathogen classes. these tests usually have low to moderate sample throughput (e.g., 1-12 samples/run), but have faster turnaround times when compared to batched, high throughput, multiplex panels (popowitch et al., 2013) . the high-plex, sampleto-answer panels can detect up to 12-20 pathogens based on the panel size. most of these qualitative assays allow random access that ensures that tests can be performed on demand, and the reactions occur in closed cassettes or cartridges, thereby minimizing the risk of contamination (poritz et al., 2011; popowitch and miller, 2015) . comparative studies evaluating the performance of these assays has reported more than 95% agreement between different commercial platforms, and greater than 85% sensitivity and 99% specificity in most cases (babady et al., 2018) . however, some high-plex sample-to-answer tests have demonstrated lower sensitivity for certain pathogens such as adenovirus (57%), influenza a(h1n1pdm09) (73%), and influenza b virus (77%) (popowitch et al., 2013) . cross-reactivity between adenovirus subtypes has also been reported for some assays, but the differentiation of these groups is not deemed clinically important for routine diagnosis (lin et al., 2004; gray et al., 2007) . despite a short turnaround time of 1-2 h, the clinical utility of these assays has been questioned since no statistically significant reduction in antibiotic prescription rates or mortality has been observed between patients who tested positive for a non-influenza virus and those who tested negative (48% vs. 49% respectively) (green et al., 2016) . in some cases, a reduction in length of hospital stay (7 days vs. 9 days) and duration of isolation (75 h vs. 82 h) was observed (rogers et al., 2015; akers et al., 2017) . the primary concerns for the highplex sample-to-answer assays are high cost per test, and the inability to order customized, targeted panels (ramanan et al., 2018) . multiplex assays such as the verigene rp flex panel (luminex) offer flexible testing and payment options by allowing users to order any combination of targets for an individual sample. low-plex integrated respiratory test systems typically target 1-4 pathogens per assay. comparative studies evaluating the performance of these assays has reported more than 90% concordance between different commercial platforms, and greater than 95% sensitivity and specificity in most cases (dugas et al., 2014; selvaraju et al., 2014; mcmullen et al., 2017) . the total turnaround time per sample for these assays varies between ∼1-2 h, and the number of samples that can be tested in a standard 8-h work shift depends on the number of instruments available in the testing laboratory (ling et al., 2018) . smaller panels targeting a few pathogens that can be run independently (random access) or simultaneously (random batch) could be a viable option for controlling laboratory costs. therefore, when considering among various testing options, it is recommended that in addition to clinical performance, other assay characteristics such as ease of use, panel composition, total turnaround time, hands-on time, and cost be considered before implementing an assay in various clinical settings (juretschko et al., 2017) . nucleic acid amplification based poc testing is relatively new in the realm of respiratory viral diagnosis and has generated contradicting opinions regarding their implementation and clinical utility. molecular poc assays have extremely short turnaround times (<30 min), minimal hands-on time (1-2 min), and can be easily operated by non-laboratory staff members, thereby making them suitable for near patient implementation and testing (brendish et al., 2015; peters et al., 2017; ling et al., 2018) . currently most of the united states. fdacleared commercial poc assays are limited to the detection of influenza a/b and rsv viruses, with the exception of the filmarray r rp ez (biofire) which detects 14 targets (fda, 2018c) . most of the studies evaluating the clinical performance of these assays have reported high sensitivity (87-100%) and specificity (>98%) for detecting influenza a/b and rsv in pediatric and adult patients (bell et al., 2014; nie et al., 2014; popowitch and miller, 2015; gibson et al., 2017; ling et al., 2018) . however, these studies have also shown that the clinical performance varies and sensitivity is significantly low for influenza b (45.2-54.5%); therefore, the results should be interpreted with caution and additional confirmatory testing should be considered, depending on the clinical circumstances (jokela et al., 2015; busson et al., 2017; davis et al., 2017) . molecular poc tests can be costly and more prone to incorrect results and contamination, as the assays are usually performed by non-laboratory personnel and poor training, failure to follow manufacturer's instructions, and failure to perform adequate quality control might affect the accuracy of results (azar and landry, 2018) . currently, more evidence-based studies are required to understand the overall clinical utility of molecular poc testing, since no correlation has been observed between implementation of these tests and reduction of antibiotic usage or hospital length of stay (andrews et al., 2017; brendish et al., 2017) . molecular testing has considerably improved the diagnosis of respiratory pathogens and is being considered as the new "gold standard". although these tests have gained immense popularity, factors such as the patient population (adult, pediatric, and immunocompromised), the size of the laboratory, the purpose of testing (routine or urgent care), and cost/benefit ratio should be considered before implementing a particular assay. for example, during the 2012-2013 influenza outbreak, two nosocomial cases were identified at memorial sloan kettering cancer center on january 1, 2013. despite implementing appropriate infection control measures, five more cases were identified few days later on january 7, 2013. a delay in reporting the positive influenza result was observed as the laboratory was following the routine diagnostic algorithm, and this might have resulted in the subsequent cases of nosocomial infection. following the 2012-2013 outbreak, the laboratory implemented the filmarray r rp for routine diagnosis and the xpert r xpress flu/rsv, a rapid molecular poc system, for seasonal use during epidemics and pandemics because the needs for routine testing and urgent care are completely different in these situations. molecular tests are highly sensitive and specific and result in higher pathogen detection, and therefore physicians and attending clinicians should evaluate test results before initiating a particular course of treatment. rapid molecular tests are best suited for routine diagnosis and outbreak situations; whereas, conventional methods, such as cell culture or em could be considered for identifying novel strains of the pathogens. with the introduction of new technologies, newer assays will be developed for respiratory and other pathogens. for example, using np swab specimens from children, graf et al. (2016) demonstrated that untargeted next-generation sequencing-based metagenomics (mngs) testing had excellent agreement (93%) with multiplex molecular panels and detected more viruses that were either not targeted by the panel or missed due to highly divergent genome sequences. they concluded that mngs can be used for accurate and unbiased detection of expected or unexpected pathogens. implementing mngs downstream of rapid molecular panel testing has been proven useful for characterizing the nature of hospital-associated transmission events and directing the control strategies in pediatric populations during outbreak situations (greninger et al., 2017) . however, diagnostic implementation of ngs is currently limited by incomplete understanding of analytical performance, high cost of the system and complexity of sequence data analysis. future research will be necessary to focus on demonstrating the clinical utility of these new and upcoming assays in various patient populations and in resource limited settings. impact of rapid influenza testing at triage on management of febrile infants and young children influence of time to diagnosis of severe influenza on antibiotic use, length of stay, isolation precautions, and mortality: a retrospective study multiplex pcr point of care testing versus routine, laboratory-based testing in the treatment of adults with respiratory tract infections: a quasi-randomised study assessing impact on length of stay and antimicrobial use prospective evaluation of rapid antigen tests for diagnosis of respiratory syncytial virus and human metapneumovirus infections guidelines for the management of adults with hospitalacquired, ventilator-associated, and healthcare-associated pneumonia detection of influenza a and b viruses and respiratory syncytial virus using clia-waived point-of-care assays: a paradigm shift to molecular tests multicenter evaluation of the eplex respiratory pathogen panel for the detection of viral and bacterial respiratory tract pathogens in nasopharyngeal swabs clinical and financial benefits of rapid detection of respiratory viruses: an outcomes study comparing luminex nxtagrespiratory pathogen panel and respifinder-22 for multiplex detection of respiratory pathogens evaluation of 12 commercial tests and the complement fixation test for mycoplasma pneumoniae-specific immunoglobulin g (igg) and igm antibodies, with pcr used as the "gold standard multicenter clinical evaluation of the novel alere tm i influenza a&b isothermal nucleic acid amplification test a national study of the impact of rapid influenza testing on clinical care in the emergency department viral bacterial coinfection of the respiratory tract during early childhood routine molecular point-of-care testing for respiratory viruses in adults presenting to hospital with acute respiratory illness (respoc): a pragmatic, open-label, randomised controlled trial point-of-care testing for respiratory viruses in adults: the current landscape and future potential comparison of nxtag respiratory pathogen panel and anyplex ii rv16 tests for multiplex detection of respiratory pathogens in hospitalized children who estimates of the causes of death in children a large-scale study of respiratory virus infection over 2 years using the luminex xtagrvp assay contribution of a rapid influenza diagnostic test to manage hospitalized patients with suspected influenza the effect of rapid respiratory viral diagnostic testing on antibiotic use in a children's hospital multiplex pcr and emerging technologies for the detection of respiratory pathogens diagnostic accuracy of rapid antigen detection tests for respiratory syncytial virus infection: systematic review and meta-analysis serodiagnosis of acute respiratory infections in children in georgia laboratory surge response to pandemic (h1n1) 2009 outbreak diagnostic accuracy and cost analysis of the alere tm i influenza a&b near-patient test using throat swabs cell culture, cytopathic effect and immunofluorescence diagnosis of viral infection evaluation of the xpert flu rapid pcr assay in highrisk emergency department patients specimen collection, transport, and processing: virology, " in manual of clinical microbiology, 11th edn rapid diagnosis of respiratory syncytial virus infections in immunocompromised adults novel coronavirus and severe acute respiratory syndrome clia categorizations [online nucleic acid based tests additional diagnostic yield of adding serology to pcr in diagnosing viral acute respiratory infections in kenyan patients 5 years of age and older evaluation of multiple commercial molecular and conventional diagnostic assays for the detection of respiratory viruses in children multi-center evaluation of the cobas r liat r influenza a/b & rsv assay for rapid point of care diagnosis detection of respiratory viruses using non-molecular based methods evaluation of multiple test methods for the detection of the novel 2009 influenza a (h1n1) during the new york city outbreak modern uses of electron microscopy for detection of viruses unbiased detection of respiratory viruses using rna-seq-based metagenomics: a systematic comparison to a commercial pcr panel genotype prevalence and risk factors for severe clinical adenovirus infection, united states clinical utility of on-demand multiplex respiratory pathogen testing among adult outpatients rule-out outbreak: 24-hour metagenomic next-generation sequencing for characterizing respiratory virus source for infection prevention specimen requirements: selection, collection, transport, and processing immunity to and frequency of reinfection with respiratory syncytial virus specimen collection for the diagnosis of pediatric pneumonia influence of antibiotics on the detection of bacteria by culturebased and culture-independent diagnostic tests in patients hospitalized with community-acquired pneumonia traditional and modern cell culture in virus diagnosis point: is the era of viral culture over in the clinical microbiology laboratory? relevance of nucleic acid amplification techniques for diagnosis of respiratory tract infections in the clinical laboratory performance of the alere i influenza a&b assay and maripoc test for the rapid detection of influenza a and b viruses multicenter clinical evaluation of the luminex aries flu a/b & rsv assay for pediatric and adult respiratory tract specimens delayed care seeking for fatal pneumonia in children aged under five years in uganda: a case-series study longer virus shedding in influenza b than in influenza a among outpatients treated with oseltamivir advances in the diagnosis of respiratory tract infections: role of the luminex xtag respiratory viral panel a novel coronavirus associated with severe acute respiratory syndrome comparison of real-time pcr assays with fluorescent-antibody assays for diagnosis of respiratory virus infections in children simulfluor respiratory screen for rapid detection of multiple respiratory viruses in clinical specimens by immunofluorescence staining prospective comparison of r-mix tm shell vial system with direct antigen tests and conventional cell culture for respiratory virus detection role of cell culture for virus detection in the age of technology use of oligonucleotide microarrays for rapid detection and serotyping of acute respiratory disease-associated adenoviruses parallel validation of three molecular devices for simultaneous detection and identification of influenza a and b and respiratory syncytial viruses advances in diagnosis of respiratory virus infections detection of respiratory viruses by molecular methods molecular diagnosis of respiratory virus infections the performance of luminex aries r flu a/b & rsv and cepheid xpert r flu/rsv xc for the detection of influenza a, influenza b, and respiratory syncytial virus in prospective patient samples evaluation of alere i influenza a&b for rapid detection of influenza viruses a and b evaluation of the binax now, bd directigen, and bd directigen ez assays for detection of respiratory syncytial virus isolation of seven respiratory viruses in shell vials: a practical and highly sensitive method evaluation of alere i rsv for rapid detection of respiratory syncytial virus in children hospitalized with acute respiratory tract infection performance characteristics of xpert flu/rsv xc assay comparison of the biofire filmarray rp, genmark esensor rvp, luminex xtag rvpv1, and luminex xtag rvp fast multiplex assays for detection of respiratory viruses filmarray, an automated nested multiplex pcr system for multipathogen detection: development and application to respiratory tract infection syndromic panel-based testing in clinical microbiology respifinder: a new multiparameter test to differentially identify fifteen respiratory viruses adenoviral infections in children: the impact of rapid diagnosis impact of a rapid respiratory panel test on patient outcomes viral detection by electron microscopy: past, present and future comparison of the luminex nxtag respiratory pathogen panel and a multiplex in-house real-time pcr panel for the detection of respiratory viruses in symptomatic patients serology as an adjunct to polymerase chain reaction assays for surveillance of acute respiratory virus infections point-counterpoint: large multiplex pcr panels should be first-line tests for detection of respiratory and intestinal pathogens comparison of the simplexa tm flu a/b & rsv kit (nucleic acid extraction-dependent assay) and the prodesse proflu + tm assay for detecting influenza and respiratory syncytial viruses comparison of direct fluorescence assay and real-time rt-pcr as diagnostics for respiratory syncytial virus in young children evaluation of the quicklab rsv test, a new rapid lateral-flow immunoassay for detection of respiratory syncytial virus antigen clinical evaluation of the luminex nxtag respiratory pathogen panel developing molecular amplification methods for rapid diagnosis of respiratory tract infections caused by bacterial pathogens evaluation of a commercial direct fluorescent-antibody assay for human metapneumovirus in respiratory specimens evaluation of three immunoassay kits for rapid detection of influenza virus a and b low prevalence of chlamydia pneumoniae in adults with communityacquired pneumonia who recommendations on the use of rapid testing for influenza diagnosis koneman's color atlas and textbook of diagnostic microbiology guidelines for the management of adult lower respiratory tract infections electron microscopy: essentials for viral structure, morphogenesis and rapid diagnosis serology enhances molecular diagnosis of respiratory virus infections other than influenza in children and adults hospitalized with community-acquired pneumonia all authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication. the study was supported in part by a research agreement between the memorial sloan kettering cancer center and the luminex corporation (sk2013-0929) and by an nih/nci cancer center support grant p30 (ca008748). conflict of interest statement: sda and sdu are employees of luminex corporation.the remaining author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © 2018 das, dunbar and tang. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord-330343-p7a8chn4 authors: kelly-cirino, cassandra; mazzola, laura t; chua, arlene; oxenford, christopher j; van kerkhove, maria d title: an updated roadmap for mers-cov research and product development: focus on diagnostics date: 2019-02-01 journal: bmj glob health doi: 10.1136/bmjgh-2018-001105 sha: doc_id: 330343 cord_uid: p7a8chn4 diagnostics play a central role in the early detection and control of outbreaks and can enable a more nuanced understanding of the disease kinetics and risk factors for the middle east respiratory syndrome-coronavirus (mers-cov), one of the high-priority pathogens identified by the who. in this review we identified sources for molecular and serological diagnostic tests used in mers-cov detection, case management and outbreak investigations, as well as surveillance for humans and animals (camels), and summarised the performance of currently available tests, diagnostic needs, and associated challenges for diagnostic test development and implementation. a more detailed understanding of the kinetics of infection of mers-cov is needed in order to optimise the use of existing assays. notably, mers-cov point-of-care tests are needed in order to optimise supportive care and to minimise transmission risk. however, for new test development, sourcing clinical material continues to be a major challenge to achieving assay validation. harmonisation and standardisation of laboratory methods are essential for surveillance and for a rapid and effective international response to emerging diseases. routine external quality assessment, along with well-characterised and up-to-date proficiency panels, would provide insight into mers-cov diagnostic performance worldwide. a defined set of target product profiles for diagnostic technologies will be developed by who to address these gaps in mers-cov outbreak management. ► the middle east respiratory syndrome-coronavirus is a high-priority pathogen identified by the who r&d blueprint because of its high fatality rate, large geographical range of the dromedary camel reservoir and lack of medical interventions. ► accurate and accessible diagnostic tests are essential to outbreak containment and case management, as well as surveillance in both humans and animals, but available diagnostic tests are limited by inconsistent quality assessment, specimen acquisition issues and infrastructure requirements. ► diagnostic research and development (r&d) needs to include point-of-care testing options, syndromic panels for differential diagnosis, a greater understanding of viral and antibody kinetics, improved access to clinical specimens, and establishment of international reference standards. diagnostics play a central role in the early detection and control of outbreaks and can enable a more nuanced understanding of the disease kinetics and risk factors for the middle east respiratory syndrome-coronavirus (mers-cov), one of the high-priority pathogens identified by the who. in this review we identified sources for molecular and serological diagnostic tests used in mers-cov detection, case management and outbreak investigations, as well as surveillance for humans and animals (camels), and summarised the performance of currently available tests, diagnostic needs, and associated challenges for diagnostic test development and implementation. a more detailed understanding of the kinetics of infection of mers-cov is needed in order to optimise the use of existing assays. notably, mers-cov point-of-care tests are needed in order to optimise supportive care and to minimise transmission risk. however, for new test development, sourcing clinical material continues to be a major challenge to achieving assay validation. harmonisation and standardisation of laboratory methods are essential for surveillance and for a rapid and effective international response to emerging diseases. routine external quality assessment, along with well-characterised and up-to-date proficiency panels, would provide insight into mers-cov diagnostic performance worldwide. a defined set of target product profiles for diagnostic technologies will be developed by who to address these gaps in mers-cov outbreak management. the middle east respiratory syndrome-coronavirus (mers-cov) is an emerging virus associated with severe respiratory illness, first detected in 2012 in saudi arabia. 1 5 provides an overview to the current status of mers-cov diagnostics, including feedback from subject matter expert and developer interviews on the common challenges with test development and implementation, and identifies gaps for further research and development (r&d). mers-cov is a zoonotic virus, and dromedary camels (camelus dromedarius) are the reservoir host and the source of zoonotic transmission to humans. [6] [7] [8] dromedaries appear to be only mildly symptomatic following infection and present a significant reservoir risk for spillover events. 2 6 9 mers-cov rna has been detected in dromedary camels in a number of countries, including egypt, oman, qatar and saudi arabia, with evidence suggesting that mers-cov is also widespread in the middle east, africa and south asia. 5 8 10-35 infection in camels is notifiable to the oie. 36 individuals with close and frequent contact with dromedaries are at a higher risk for mers-cov infection than the general population. 37 38 clinical indications and management coronaviruses are a family of viruses that can cause diseases in humans, ranging from the common cold to severe acute respiratory syndrome (sars). the clinical spectrum of mers ranges from no symptoms (or asymptomatic infection), mild symptoms including fever, cough, gastrointestinal illness and shortness of breath, to severe disease including pneumonia, acute respiratory distress syndrome and death. 2 39 severe cases of mers can result in respiratory failure, requiring mechanical ventilation and support in intensive care. risk factors for severe disease include a weakened immune system, older age (>60 years), and comorbidities such as diabetes, cancer, renal disease and chronic lung disease. 40 41 human-tohuman transmission spreads through close and unprotected human contact, and more than half of reported mers cases have occurred through nosocomial transmission. [42] [43] [44] [45] to prevent nosocomial infections, who and others recommend using standard infection and prevention control measures when caring for patients. [46] [47] [48] who also recommends that contact tracing of all symptomatic and asymptomatic close contacts of the primary patient should be conducted routinely. 49 the molecular epidemiology for mers-cov has not changed significantly since the initial human cases were detected in 2012. the current virus remains 99% identical to the sequences seen in the first human cases from 2012 as well as archived camel sera from 1983, with no increase in pathogenicity observed in the animal host. [50] [51] [52] as genetic mutations could impact detection, bmj global health immunotherapy and vaccine development efforts, 53 sequencing of mers-cov strains from camels and humans (after a zoonotic spillover) is important and is regularly being conducted in affected member states (who, personal communication, 2018). there are currently no prophylactic or therapeutic interventions of proven efficacy for any coronavirus infections. without a specific therapy for mers, treatment is supportive. 5 54 55 effective mers therapeutics are still in the early stages of research and evaluation. several broad-spectrum antiviral agents including nitazoxanide, 56 viral methyltransferase inhibition 57 and nucleotide prodrugs 58 have shown in vitro activity against mers-cov. early results for novel mers-specific therapeutics that inhibit viral replication or have specific neutralising activity are promising. 47 59 60 the who r&d blueprint for mers has called for three types of vaccines: (1) dromedary camel vaccine to prevent zoonotic transmission, (2) human vaccine for long-term protection of persons at high exposure risk and (3) human vaccine for reactive use in outbreak settings. 55 61 mers-cov vaccines are in the early stages of development, 55 62 63 with one candidate vaccine in phase i clinical trials (nct02670187). 64 neutralising monoclonal antibodies have been designed to target the mers-cov spike protein, 53 65 with chadox1 and modified vaccinia ankara vectors also strong vaccine candidates, 60 66 but none have yet advanced to clinical trials. to accelerate the process, the coalition for epidemic preparedness innovation has recently launched a call for proposals for the development of a human mers-cov vaccine in order to engage with developers interested in supporting these efforts. 67 the who laboratory guidelines recommend nucleic acid amplification tests (naat) for diagnosis, using serology for diagnosis only when naat is not available. 68 in suspected patients, a single negative test result does not exclude diagnosis. repeat sequential sampling and testing is strongly recommended. the kinetics of mers-cov infection has been shown to vary widely across cases, 40 69-72 prompting a more detailed investigation of viral and antibody dynamics across the broad range of sample types, disease states and host factors. 73 74 the best naat test sensitivity is achieved using specimens from the lower respiratory tract (sputum, tracheal aspirates or bronchoalveolar lavage), where mers-cov replication occurs at higher and more prolonged levels of mers-cov rna, typically between 10 6 and 10 10 copies/ml. 72 75 mers-cov viral load is generally higher for severe cases, with more prolonged viral shedding than mild cases. viral load concentrations, which may be undetectable at early-stage infection, generally peak in the second week after symptom onset, and then drop to undetectable in survivors by the fourth week from onset. upper respiratory tract specimens (nasopharyngeal or oropharyngeal swabs) may also be used, but demonstrate 100×-1000× lower viral load and can test negative for mild cases. 76 77 if possible, both upper and lower respiratory tract sampling are advised. specimens outside the respiratory tract are not recommended for diagnosis, as they can test negative in both severe and mild presentation. viral rna has been detected in stool samples (10 4 copies/ml), serum samples (10 3 copies/ml) and urine (10 2 copies/ml), more likely an indicator of severity as it typically precedes a poor clinical outcome. 71 76 78 serological diagnosis can be made using paired samples, more often used for research rather than diagnostic purposes, preferably with the initial sample collected in the first week of illness and the second collected 3-4 weeks later. if only a single serum sample can be collected, this should occur at least 3-4 weeks after onset of symptoms for determination of a probable case. table 1 presents an overview of the implementation requirements for mers-cov diagnostics (detailed commercial product information is presented in online supplementary tables s1 and s2). molecular diagnostics such as naat (eg, pcr) typically require sophisticated laboratory infrastructure including biosafety cabinets, 79 while most serological tests (elisa, indirect immunofluorescence test (iift)) can be run on the benchtop in a more modest laboratory environment, depending on sample preparation precautions. 80 81 point-of-care (poc) tests are designed to be used outside of a traditional laboratory; near-poc tests are defined for rapid use in a laboratory near the patient, but are more automated and easy to use than the traditional laboratory test. 72 75 poc tests such as low-complexity rapid diagnostic tests (rdts) can be used at the bedside, typically with non-invasive samples after minimal training. inhouse tests are described in sections below; commercial sources are listed in online supplementary tables s1 and s2. naats are currently the standard for mers-cov diagnosis, as these tests (typically reverse transcriptase pcr (rt-pcr)) have the highest sensitivity at the earliest time point during the acute phase of infection. following the who guidelines, two different targets on the mers-cov need to be detected by rt-pcr to confirm a case. mers-cov assays to detect the upstream envelope gene (upe) followed by confirmation of open reading frame 1a (orf1a), 1b (orf1b) genes or nucleocapsid (n) genes for confirmation have been developed. 55 82 most commercial pcr tests perform parallel screening for the upe gene with confirmation by the orf1a, orf1b or n genes (most commonly upe + orf1a). initial naat tests for mers-cov were developed as inhouse tests, following the first detection of mers-cov in the middle east. [83] [84] [85] [86] inhouse tests are not necessarily subject to quality control or regulation, and may not be rigorously validated; in some cases, inhouse tests are eventually developed into commercial products through collaboration and licensing efforts. 50 83 84 87-89 commercial assays may undergo an international and/or incountry regulatory process; once on the market they can be independently evaluated for sensitivity, specificity and limit of detection. 78 90 as of 2018, there are several commercial naat tests available for mers-cov, including duplex and multiplex panels (see online supplementary table s1). serology is not widely performed for diagnosing acute mers-cov infection; however, it has been a useful tool bmj global health to determine the extent of infection around clusters and in seroepidemiological studies in animals and humans. seroconversion typically occurs during the second and third week after symptom onset; data suggest that low antibody titre in the second week or delayed seroconversion is more closely associated with mortality than high viral load. 71 74 mers-cov seroconversion may not be observed for some patients, notably with mild or asymptomatic infection, and can show cross-reactivity with antibodies to other coronaviruses. 42 69 serological methods for the detection of antibodies against mers-cov include elisa, iift and neutralisation tests. mers-cov serological assays can employ commercial reagents or proprietary monoclonal antibodies as capture agents. 87 91 92 many mers-cov elisa tests are labelled for research use only, with little or no clinical validation data available. similar to the elisa, iift is used when it is difficult to evaluate specific antigens individually by enzyme immunoassays or there is a preference for broader analysis of an immobilised specimen. iift microscopy assay can probe the entire antigen spectrum of the specimen, and is often designed for simultaneous detection of antibodies against biochemically distinct antigens. neutralisation is a method for detecting anti-mers-cov antibody activity via inhibition of infection or replication, 69 93 performed as plaque reduction neutralisation, microneutralisation (mn) and pseudoparticle neutralisation (ppnt). mn is labour-intensive and slow, requiring at least 3-5 days for results; neutralisation techniques other than ppnt require biosafety level 3 containment as they involve live virus cultures. 94 rdts can leverage the same antibody/antigen capture agents as elisa but in a lateral flow strip cartridge. 95 this enables a fast 10-30 min time to result, but with a 100-fold lower detection sensitivity than elisa. 91 92 follow-up confirmatory testing is therefore required. rdts are typically paired with minimally invasive specimen collection (blood, oral fluid, nasal swabs) so that they can be used with minimal training outside of laboratory settings. early prototypes for mers-cov rdts have been developed, 87 92 96 with commercial rdts for detection of mers-cov in camels and humans available (online supplementary table s2). the human mers-cov rdt does not appear to be widely used, perhaps due to the more invasive processing required for lower respiratory specimens, as well as sensitivity issues for upper respiratory specimens. the camel mers-cov rdt is used with upper respiratory specimens; however, test sensitivity varies depending on specimen sampling and infection kinetics. 97 multiplex panels at the early stages, the symptoms of mers-cov infection can mimic diseases such as influenza, pneumonia, sars and other respiratory infections. a syndromic approach involves testing for pathogens based on a syndrome such as fever or acute respiratory distress; a shift from individual tests to multiplex panels can quickly identify or eliminate likely pathogens from a single specimen. for analysis of circulating reservoirs, multiplex microbead-based immunoassays have been used to detect igg antibodies for multiple pathogens. 98 99 multiplex, syndromic panels that include mers-cov have been demonstrated using pcr-based panels including mers-cov, showing similar limits of detection to single assays. 89 100 101 commercial respiratory panel tests including mers-cov have also recently been developed (see online supplementary table s1). there is a need for international consensus and adoption of minimum standards for tests used in diagnosis, surveillance and research, following who's recommended algorithm for human cases 82 and oie recommendation for animal health. 36 harmonisation of the testing process can be achieved by building consensus and capacity across international and incountry laboratories. in order to enable and sustain the capacity for a rapid outbreak response, laboratories must have access to high-quality reagents and instrumentation, along with technical support and cold-chain transport when necessary. in addition, international reference panels would achieve a more standardised training for external quality assessment (eqa) and quality control. building on mandatory case reporting, 102 an international mers-cov data sharing platform that includes case exposure history and sequence data would greatly facilitate the knowledge base across the mers-cov community. [103] [104] [105] [106] clinical validation understanding mers-cov viral dynamics across a broad range of specimen types is critical to establishing the limits of detection and timing of diagnostics in order to make the greatest impact for diagnosis, case management and surveillance. ensuring a test has appropriate sensitivity and specificity is a major challenge in the development of diagnostics for novel and rare pathogens, as there is often a very limited supply of well-characterised clinical material. especially during the early stage of an outbreak, clinical evaluation must often be performed in the affected countries by laboratories working closely with the ministries of health. typically only a small number of patient specimens are shared outside of the affected countries due to strict import and export regulations, particularly for 'dual-use' pathogens. 107 108 specifically, the provisions of the nagoya protocol have significant impact on the access to genetic materials for both commercial and non-commercial applications. 109 110 in particular, the development and validation process for new diagnostics could be accelerated if well-characterised specimens and reference standards could be more easily obtained. eqa can be useful for evaluation of test performance, as shown with evaluations of both inhouse and commercial assays for mers-cov, [111] [112] [113] and bmj global health more recently a global proficiency testing programme used to assess laboratory detection of mers-cov. 114 even after validation, a substantial amount of reference material is required for quality control; often manufacturers must develop their own calibration standards to maintain supply and to control lot-to-lot variability. international reference standards and qualified specimen panels can accelerate the development and validation of diagnostic tests. in particular, the who international biological reference preparations (as provided by member states) serve as reference sources for ensuring the reliability of in vitro biological diagnostic procedures used for diagnosis of diseases and treatment monitoring, including mers-cov. several international institutes also provide specimens for validation; these groups typically have a defined pathogen/disease focus with a corresponding archive of biological reference materials; however, the supplies may be limited (see online supplementary material 1). currently, mers-cov diagnosis by pcr requires a laboratory with sophisticated facilities and biosafety cabinets. the turnaround time to receive a test result can take days to weeks, depending on laboratory proximity, sample transport options and laboratory processing capacity, 72 75 and infrastructure requirements place most pcr systems in reference laboratories, which may not be ideal for diseases like mers-cov that recommend immediate isolation for infections detected across a variety of settings. 81 115 116 a more nimble approach is needed for mers-cov case detection and triage, 92 117 and at border crossings for animal surveillance, quarantine and targeted vaccination. 11 21 87 118 the fao-oie-who mers technical working group has given a clear call for the development of an rdt to improve identification and isolation of primary human cases in healthcare facilities. 5 serological rdts are ideal for low infrastructure settings such as a primary health clinic, home or field testing. however, specimen collection remains a key challenge for mers-cov, as the recommended lower respiratory specimens are difficult to obtain outside of a hospital setting. upper respiratory specimens such as nasal swabs are easy to obtain and work well in conjunction with rdts for camels, but these specimens generally have low virus titre in humans, thus limiting current use of rdts to animal testing. 87 92 96 improvement of the current rdt detection chemistry, if feasible, may support the future use of these tests in humans, at least for rapid triage in highly infectious cases. poc and near-poc microfluidic platforms enable a more flexible, but still highly sensitive approach for near-patient naat testing in decentralised settings. near-poc naat platforms are compact and self-contained, with automated sample preparation for processing in minimal laboratory settings, which most healthcare workers can be trained to operate within a day. [119] [120] [121] recent publications describe mers-cov assays designed for poc pcr, 89 loop-mediated isothermal amplification assay 122 and paper-based sensor detection 123 ; however, no mers-cov assays are currently available for the existing near-poc platforms. given that pcr is now the standard for mers-cov diagnosis, it would be highly desirable to have an automated, self-contained naat assay that can be readily deployed in a field or clinic setting. syndromic testing can be valuable during the early stages of an outbreak, in order to distinguish mers-cov from other respiratory infections or identify cases of coinfection. 100 124 a syndromic panel could be effective in expediting pathogen and outbreak identification, especially with technologies that can screen for multiple pathogens simultaneously. 125 using the panel approach, a definitive diagnosis could enable timely decisions about triage, treatment, infection control and contact tracing. 126 while the per-test cost rises with test complexity, including additional reagents and more sophisticated instrumentation, a rapid and efficient diagnosis scheme can impact intervention and infection control and can be cost-saving overall. 127 128 as respiratory diseases are both regional and seasonal, 129-131 region-specific panels may be more cost-effective. 132 multiplex panels offer the alternative for a 'bundled' testing paradigm; however, if not routinely used (if the market is small), then developers may be reluctant to support the test for diagnostic use, which requires additional investment for validation and regulation. surveillance can be an effective method to identify the initial stages of outbreak, but it requires routine and effective sampling. the impact of surveillance testing depends on the test sensitivity and specificity, sampling rates, kinetics of the disease, and whether the target is animal or human populations. most surveillance sampling is performed in the field, either through population-based or 'hot spot' sampling. for mers-cov, it may be difficult and expensive to implement routine surveillance in dromedary camel stock, as they represent a significantly large reservoir but may suffer only mild effects from mers-cov infection, if any. the ideal surveillance tool would be a highly sensitive and field-appropriate screening test. per-test cost is also an important factor along with ease of implementation. this review has identified diagnostics currently available for mers-cov and highlighted ongoing challenges caused by critical gaps in diagnostics to support outbreak management. rdts offer the potential for rapid poc screening for mers-cov; however, there are practical limits to implementing lower respiratory sample acquisition outside of a hospital setting, limiting feasibility. poc or near-poc naat platforms provide an opportunity for implementation of automated, self-contained bmj global health testing in hospitals and clinics with limited training in endemic-prone areas. expansion of test menu options for existing poc or near-poc naat platforms will strengthen incountry response capacity to endemic diseases and simultaneously ensure countries are prepared for future pandemics. syndromic multiplex panels may expedite differential diagnosis of mers-cov from other endemic respiratory diseases, but further analysis is needed to inform implementation and cost-effectiveness in the context of regional and seasonal detection. there is also a need for more sensitive serological assays with lower cost and minimum cross-reactivity that can be used as surveillance tools. a more detailed understanding of mers-cov viral and antibody kinetics is needed across the broad range of sample types in order to optimise the use of existing assays and to address ongoing technical challenges in the detection of mild and asymptomatic infections. surveillance continues to be important for the detection of mers-cov spillover events; however, questions remain on the cost-effectiveness of routine screening of the large reservoir camel population. in addition, support towards sample biobanks with well-characterised specimens and reference standards will facilitate diagnostic development and quality assurance for mers-cov diagnostics worldwide. in order to achieve the goals of the r&d blueprint efforts, who is identifying key target product profiles for diagnostics in order to mobilise funding and resources to support the development and implementation of the most critically needed tests. isolation of a novel coronavirus from a man with pneumonia in saudi arabia who | middle east respiratory syndrome coronavirus (mers-cov). who mers-cov r&d blueprint plan of action r&d blueprint for action to prevent epidemics progress on the global response, remaining challenges and the way forward evidence for camel-tohuman transmission of mers coronavirus human-dromedary camel interactions and the risk of acquiring zoonotic middle east respiratory syndrome coronavirus infection middle east respiratory syndrome coronavirus neutralising serum antibodies in dromedary camels: a comparative serological study middle east respiratory syndrome coronavirus (mers-cov) origin and animal reservoir risk factors for mers coronavirus infection in dromedary camels in burkina faso cross-sectional surveillance of middle east respiratory syndrome coronavirus (mers-cov) in dromedary camels and other mammals in egypt mers coronaviruses in dromedary camels geographic distribution of mers coronavirus among dromedary camels middle east respiratory syndrome coronavirus (mers-cov) serology in major livestock species in an affected region in jordan absence of middle east respiratory syndrome coronavirus in camelids antibodies against mers coronavirus in dromedary camels serological evidence of mers-cov antibodies in dromedary camels (camelus dromedaries) in laikipia county middle east respiratory syndrome coronavirus infection in dromedary camels in saudi arabia middle east respiratory syndrome coronavirus quasispecies that include homologues of human isolates revealed through whole-genome analysis and virus cultured from dromedary camels in saudi arabia longitudinal study of middle east respiratory syndrome coronavirus infection in dromedary camel herds in saudi arabia middle east respiratory syndrome (mers) coronavirus seroprevalence in domestic livestock in saudi arabia lack of middle east respiratory syndrome coronavirus transmission from infected camels mers coronavirus in dromedary camel herd, saudi arabia cross-sectional study of mers-cov-specific rna and antibodies in animals that have had contact with mers patients in saudi arabia human infection with mers coronavirus after exposure to infected camels, saudi arabia dromedary camels in northern mali have high seropositivity to mers-cov middle east respiratory syndrome coronavirus (mers-cov) in dromedary camels in nigeria middle east respiratory syndrome coronavirus (mers-cov) in dromedary camels serologic evidence for mers-cov infection in dromedary camels middle east respiratory syndrome coronavirus in dromedary camels: an outbreak investigation mers-cov situation update, map 1. mers-cov livestock field surveys by country epidemiological investigation of middle east respiratory syndrome coronavirus in dromedary camel farms linked with human infection in abu dhabi emirate middle east respiratory syndrome coronavirus antibody reactors among camels in dubai identification of diverse viruses in upper respiratory samples in dromedary camels from united arab emirates mers cov: oie -world organisation for animal health occupational exposure to dromedaries and risk for mers-cov infection risk factors for primary middle east respiratory syndrome coronavirus infection in camel workers in qatar during 2013-2014: a case-control study middle east respiratory syndrome (mers) | symptoms & complications | cdc middle east respiratory syndrome coronavirus disease in children middle east respiratory syndrome coronavirus disease is rare in children: an update from saudi arabia transmission of merscoronavirus in household contacts hospital-associated outbreak of middle east respiratory syndrome coronavirus: a serologic, epidemiologic, and clinical description the role of super-spreaders in infectious disease super-spreading events of mers-cov infection mers-cov outbreak following a single patient exposure in an emergency room in south korea: an epidemiological outbreak study development of medical countermeasures to middle east respiratory syndrome coronavirus who | infection prevention and control (ipc) guidance summary. who who recommended surveillance standards an observational, laboratorybased study of outbreaks of middle east respiratory syndrome coronavirus in jeddah and riyadh, kingdom of saudi arabia microevolution of outbreakassociated middle east respiratory syndrome coronavirus, south korea middle east respiratory syndrome coronavirus: virology, pathogenesis, and epidemiology middle east respiratory syndrome coronavirus vaccines: current status and novel approaches challenges presented by mers corona virus, and sars corona virus to global health a roadmap for mers-cov research and product development: report from a world health organization consultation nitazoxanide, a new drug candidate for the treatment of middle east respiratory syndrome coronavirus toward the identification of viral cap-methyltransferase inhibitors by fluorescence screening assay broad-spectrum antiviral gs-5734 inhibits both epidemic and zoonotic coronaviruses coronaviruses -drug discovery and therapeutic options a novel neutralizing monoclonal antibody targeting the n-terminal domain of the mers-cov spike protein vaccine development for emerging virulent infectious diseases rapid development of vaccines against emerging pathogens: the replication-deficient simian adenovirus platform technology report from the world health organization's third product development for vaccines advisory committee (pdvac) meeting middle east respiratory syndrome vaccines chadox1 and mva based vaccine candidates against mers-cov elicit neutralising antibodies and cellular immune responses in mice emerging infectious diseases: a proactive approach laboratory testing for middle east respiratory syndrome coronavirus kinetics of serologic responses to mers coronavirus infection in humans viral load kinetics of mers coronavirus infection viral shedding and antibody response in 37 patients with middle east respiratory syndrome coronavirus infection the role of laboratory diagnostics in emerging viral infections: the example of the middle east respiratory syndrome epidemic mers coronavirus: data gaps for laboratory preparedness predictors of mortality in middle east respiratory syndrome (mers) mers-cov diagnosis: an update clinical features and virological analysis of a case of middle east respiratory syndrome coronavirus infection kinetics and pattern of viral excretion in biological specimens of two mers-cov cases spread of mutant middle east respiratory syndrome coronavirus with reduced affinity to human cd26 during the south korean outbreak who | laboratory biosafety manual -third edition challenges and opportunities for the implementation of virological testing in resource-limited settings advances in addressing technical challenges of point-of-care diagnostics in resource-limited settings who | laboratory testing for middle east respiratory syndrome coronavirus. who mers-cov lab detection of a novel human coronavirus by real-time reverse-transcription polymerase chain reaction performance and clinical validation of the realstar mers-cov kit for detection of middle east respiratory syndrome coronavirus rna real-time reverse transcription-pcr assay panel for middle east respiratory syndrome coronavirus development of dual taqman based one-step rrt-pcr assay panel for rapid and accurate diagnostic test of mers-cov: a novel human coronavirus, ahead of hajj pilgrimage development and validation of a rapid immunochromatographic assay for detection of middle east respiratory syndrome coronavirus antigen in dromedary camels an isothermal, label-free, and rapid one-step rna amplification/detection assay for diagnosis of respiratory viral infections comparison of eplex respiratory pathogen panel with laboratory-developed real-time pcr assays for detection of respiratory pathogens clinical validation of 3 commercial real-time reverse transcriptase polymerase chain reaction assays for the detection of middle east respiratory syndrome coronavirus from upper respiratory tract specimens a sensitive and specific antigen detection assay for middle east respiratory syndrome coronavirus a highly specific rapid antigen detection assay for on-site diagnosis of mers seroepidemiology for mers coronavirus using microneutralisation and pseudoparticle virus neutralisation assays reveal a high prevalence of antibody in dromedary camels in egypt seroepidemiology of middle east respiratory syndrome (mers) coronavirus in saudi arabia (1993) and australia (2014) and characterisation of assay specificity lateral flow assays development of monoclonal antibody and diagnostic test for middle east respiratory syndrome coronavirus using cell-free synthesized nucleocapsid antigen middle east respiratory syndrome (mers) coronavirus and dromedaries identification of mycoplasma suis antigens and development of a multiplex microbead immunoassay serosurveillance of viral pathogens circulating in west africa two-tube multiplex real-time reverse transcription pcr to detect six human coronaviruses a multiplex liquid-chip assay based on luminex xmap technology for simultaneous detection of six common respiratory viruses surveillance and testing for middle east respiratory syndrome coronavirus using healthmap to analyse middle east respiratory syndrome (mers) data progress in promoting data sharing in public health emergencies who | influenza surveillance outputs data sharing: make outbreak research open access the weapon potential of a microbe biological agents: weapons of warfare and bioterrorism explanation of the nagoya protocol on access and benefit sharing and its implication for microbiology global scientific research commons under the nagoya protocol: towards a collaborative economy model for the sharing of basic research assets first international external quality assessment of molecular diagnostics for mers-cov external quality assessment of mers-cov molecular diagnostics during the 2015 korean outbreak external quality assessment for the molecular detection of mers-cov in china proficiency testing for the detection of middle east respiratory syndrome coronavirus demonstrates global capacity to detect middle east respiratory syndrome coronavirus point-of-care testing for infectious diseases: diversity, complexity, and barriers in low-and middle-income countries diagnostic point-of-care tests in resource-limited settings response to emergence of middle east respiratory syndrome coronavirus an orthopoxvirusbased vaccine reduces virus excretion after mers-cov infection in dromedary camels evaluation of the whole-blood alere q nat point-of-care rna assay for hiv-1 viral load monitoring in a primary health care setting in mozambique point-of-care cepheid xpert hiv-1 viral load test in rural african communities is feasible and reliable performance of the samba i and ii hiv-1 semi-q tests for viral load monitoring at the point-of-care one-pot reverse transcriptional loop-mediated isothermal amplification (rt-lamp) for detecting mers-cov multiplex paperbased colorimetric dna sensor using pyrrolidinyl peptide nucleic acid-induced agnps aggregation for detecting mers-cov, mtb, and hpv oligonucleotides the impact of co-infection of influenza a virus on the severity of middle east respiratory syndrome coronavirus wpro | second meeting on laboratory strengthening for emerging infectious diseases in the asia pacific region point-counterpoint: large multiplex pcr panels should be first-line tests for detection of respiratory and intestinal pathogens cost analysis of multiplex pcr testing for diagnosing respiratory virus infections impact of a rapid respiratory panel test on patient outcomes pcr for detection of respiratory viruses: seasonal variations of virus infections global mortality estimates for the 2009 influenza pandemic from the glamor project: a modeling study prevalence and seasonal distribution of respiratory viruses during the 2014 -2015 season in istanbul development of a respiratory virus panel test for detection of twenty human respiratory viruses by use of multiplex pcr and a fluid microbead-based assay acknowledgements we gratefully acknowledge input to the roadmap from all those who attended the fao-oie-who global technical meeting on mers-cov in september 2017. the opinions expressed in this article are those of the authors and do not necessarily reflect those of the institutions or organisations with which they are affiliated. editorial assistance for later drafts was provided by rachel key: cord-339009-wcoch07b authors: file, thomas m.; tsang, kenneth w. t. title: severe acute respiratory syndrome: pertinent clinical characteristics and therapy date: 2012-08-23 journal: treat respir med doi: 10.2165/00151829-200504020-00003 sha: doc_id: 339009 cord_uid: wcoch07b severe acute respiratory syndrome (sars) is a newly emerged infection that is caused by a previously unrecognized virus–a novel coronavirus designated as sars-associated coronavirus (sars-cov). from november 2002 to july 2003 the cumulative number of worldwide cases was >8000, with a mortality rate of close to 10%. the mortality has been higher in older patients and those with co-morbidities. sars has been defined using clinical and epidemiological criteria and cases are considered laboratory-confirmed if sars coronavirus is isolated, if antibody to sars coronavirus is detected, or a polymerase chain reaction test by appropriate criteria is positive. at the time of writing (24 may 2004), no specific therapy has been recommended. a variety of treatments have been attempted, but there are no controlled data. most patients have been treated throughout the illness with broad-spectrum antimicrobials, supplemental oxygen, intravenous fluids, and other supportive measures. transmission of sars is facilitated by close contact with patients with symptomatic infection. the majority of cases have been reported among healthcare providers and family members of sars patients. since sars-cov is contagious, measures for prevention center on avoidance of exposure, and infection control strategies for suspected cases and contacts. this includes standard precautions (hand hygiene), contact precautions (gowns, goggles, gloves) and airborne precautions (negative pressure rooms and high efficiency masks). in light of reports of new cases identified during the winter of 2003–4 in china, it seems possible that sars will be an important cause of pneumonia in the future, and the screening of outpatients at risk for sars may become part of the pneumonia evaluation. severe acute respiratory syndrome (sars) was first recog-1. definition nized in the guangdong province in southeast china in late 2002, for surveillance purposes and before the availability of laboraand it subsequently spread globally rapidly during the early tory tests to detect the causative agent, sars was originally months of 2003. [1, 2] sars captured worldwide attention as a defined using clinical and epidemiologic criteria from suspect or highly infectious disease with high mortality and as an occupationprobable cases. [3, 4] a suspect case included a respiratory illness of al hazard among healthcare providers. the impact of sars was unknown etiology and with the following criteria: extensive from both a sociological and economical consequence -• measured temperature >100.4ºf (>38.0ºc) particularly in asia. because the causative agent of sars is • one or more clinical findings of respiratory illness (e.g. cough, contagious, preventative measures focus on avoidance of exposhortness of breath, difficulty in breathing, or hypoxia) sure, and infection control strategies for suspected patients and • travel within 10 days of onset of symptoms to an area with contacts. the emergence of sars illustrates the need for global suspected or documented community transmission of sars cooperation of healthcare systems to ensure the public health of (excluding areas with secondary cases limited to healthcare local regions, and the need to be prepared to rapidly institute workers or direct household contacts and close contact within policies to respond to newly emerging infectious threats. 10 days of onset of symptoms with either a person with a respiratory illness or a person under investigation or suspected recombination event between mammalian-like and avian-like parof having sars). ent viruses which may have been responsible for the switch of host of the sars-cov from animals to humans. a probable case was defined as a suspect case with either radiographic evidence of pneumonia or respiratory distress syndrome, or autopsy findings consistent with respiratory distress 3. epidemiology syndrome without an identifiable cause. [4] once the virus was identified and laboratory tests became as of july 2003 the cumulative number of worldwide sars available for detection, the surveillance case definition for sars cases was 8437, with a mortality of 9.6%. [12] of the reported cases was updated to include laboratory criteria for evidence of infection 64% were from china, 19% from hong kong, 8% from taiwan, with the sars-associated coronavirus (sars-cov). initially, 3% from canada, and 2% from singapore. the us has been since it was unclear whether sars infection could be present in relatively spared from the clinical impact of sars. at july 2003, people who were asymptomatic, the definition included the possi-27 probable cases had been reported, of which only eight had bility of asymptomatic ('subclinical') infection. however, subselaboratory confirmation of acute coronavirus infection. [13] quent investigations suggest that asymptomatic infection is very the initial cases reported in hong kong were linked to an index uncommon. [1] as a consequence, the latest surveillance case defipatient, a medical doctor from the guangdong province of china nition for sars by the us center for disease control and who traveled to hong kong to attend a wedding in late february prevention (cdc) has been updated to include clinical criteria for 2003. [14] he had previously treated patients with 'atypical' pneuearly illness, mild-to-moderate illness, and severe respiratory illmonia in guangdong. subsequently several guests who had stayed ness, which are then characterized by epidemiological and laboraat the same hotel became ill with sars. these patients subsetory criteria (table i) . [5] quently infected numerous healthcare workers and family members or became index cases in other countries (canada, vietnam, singapore, etc.) [ figure 1 ]. the mortality has been higher in older patients and those with co-morbidities. the syndrome has been observed primarily in soon after the recognition of the clinical syndrome of sars, adults aged 25-70 years, and children have been relatively spared. several different laboratories identified a novel coronavirus, desig-there appears to be no significant underlying predisposing condinated sars-cov, in vero e6 cell cultures inoculated with respirtion for the development of sars, however the elderly and atory secretions and lung tissue of infected patients. [6, 7] other patients with underlying conditions are at greater risk for mortalitechniques, including electron microscopy, reverse transcriptionty. in one study from hong kong the mortality for those >60 years polymerase chain reaction (rt-pcr), and serovonversion, have of age was 43%. [15] in another study, multivariate analysis showed also pointed to this as the causative agent. sero-epidemiological that age >60 years, presence of diabetes mellitus or heart disease, data indicate the sars-cov was not previously found in and the presence of other co-morbid conditions were independenthumans. [8] preliminary reports of detection of the sars ly associated with mortality. [16] early in the evaluation of this coronavirus in himalayan palm civet cats and a number of other syndrome, most of the descriptions were of patients who required species are suggestive of interspecies transmission of this new hospitalization. however, as more cases were identified, particuvirus. investigators from hong kong reported a coronavirus relarly in the western countries, the majority of patients have not sembling sars virus isolated from several himalayan palm civet required hospitalization. cats and a raccoon dog obtained in a market in the guangdong province (such animals are considered as food delicacies in that sars appears to be transmitted by close contact with patients region. [9] ) they also reported that several of the handlers at the who have illness due to sars virus. [1, 2] the greatest risk of market had antibody to the sars virus. studies of the genetic transmission is most probably via direct contact with respiratory sequence of the two viruses show a similar pattern, suggesting a secretions. there is no evidence of spread from patients before species jump from wild animals to humans. furthermore, experi-they develop symptoms. the majority of cases have been reported mental infection of macaques with sars-cov produced a pneu-among healthcare workers and family members of affected permonia that was pathologically similar to sars in humans. [10] sons. however, evidence of community spread of the disease is stavrinides and guttman compared the sars-cov genome with emerging, suggesting that other modes of transmission, such as related coronaviruses and found about half the dna resembled airborne or direct contact, may also have a role. [1] clusters of cases coronavirus sequences from mammals, while the other half looked in community settings such as hotels and apartment buildings like virus found in birds. [11] these data suggest a possible past demonstrate that transmission can be efficient. many household [5] clinical criteria presence of two or more of the following features: fever (might be subjective), chills, rigors, myalgia, headache, diarrhea, sore throat, or rhinorrhea temperature >100.4°f (>38°c), and one or more clinical findings of lower respiratory illness (e.g. cough, shortness of breath, or difficulty breathing) meets clinical criteria of mild-to-moderate respiratory illness, and one of more of the following findings: radiographic evidence of pneumonia, or acute respiratory distress syndrome, or autopsy findings consistent with pneumonia or acute respiratory distress syndrome without an identifiable cause one or more of the following exposures in the 10 days before onset of symptoms: travel to a foreign or domestic location with documented or suspected recent transmission of sars-cov, or close contact with a person with mild-to-moderate or severe respiratory illness and history of travel in the 10 days before onset of symptoms to a foreign or domestic location with documented or suspected recent transmission of sars-cov one or more of the following exposures in the 10 days before onset of symptoms: close contact with a person with confirmed sars-cov disease, or close contact with a person with mild-to-moderate or severe respiratory illness for whom a chain of transmission can be linked to a confirmed case of sars-cov disease in the 10 days before onset of symptoms detection of serum antibody to sars-cov by a test validated by cdc (e.g. enzyme immunoassay), or isolation in cell culture of sars-cov from a clinical specimen, or detection of sars-cov rna by a rt-pcr test validated by cdc and with subsequent confirmation in a reference laboratory (e.g. cdc) case classification b probable case of sars-cov disease: meets the clinical criteria for severe respiratory illness and the epidemiologic criteria for likely exposure to sars-cov confirmed case of sars-cov disease: clinically compatible illness (i.e. early, mild-to-moderate, or severe) that is laboratory confirmed a tests to detect sars-cov are being refined and, therefore, criteria for laboratory diagnosis of sars-cov are changing. b asymptomatic infection or clinical manifestations other than respiratory illness may be identified in future as more is learned about sars-cov. cdc = center for disease control; rt-pcr = reverse transcription polymerase chain reaction; sars-cov = sars-associated coronavirus. contacts have become ill. epidemiologic evidence indicates that quired sars via contact with a fomite. of still unexplained significance, a few patients have been involved in the transmission the transmission of sars is facilitated by face-to-face contact, of an unusually large number of secondary cases -so-called superand this still appears to be the most common mode of spread in the spreading events. form of droplet transmission. [1] however, airborne or fecal transmission may have a role in some settings, and it could account for peiris et al. [17] studied the viral load of sars-cov over time in the extensive spread within buildings and other confined areas that respiratory secretions from 14 sars patients and found the load in has been observed in some places in asia. transmission via casual nasopharyngeal aspirates increased to a peak on the tenth day after contact is uncommon, but has been documented on an airplane or onset of symptoms, then decreased gradually. notwithstanding the in a taxi. [1] some healthcare workers also appeared to have ac-small sample size and the effects of concomitant administration of ribavirin and corticosteroid, this suggests that sars patients of virology in beijing. the virus was apparently transmitted to the student's mother and a nurse caring for the student (all three had might be most contagious in the second week of illness. laboratory-confirmed cases). at the time of writing, a total of nine after the termination of the initial outbreak of sars in july possible cases of sars have been identified and the health author-2003, there were two further cases of sars-cov infection which ities were involved in active surveillance to identify other possible were likely acquired in a laboratory setting, one case in late august cases. [20] and the other in december. [18] this reinforces the necessity for careful laboratory practices when working with this virus. it 4. clinical manifestations appears there was no evidence of secondary transmission associated with either case, despite the active social activities undertaken the initial descriptions of the clinical manifestations of sars by these two scientists after their exposure to sars-cov. an have come from reports of patients who have required hospitalizaadditional four community-acquired cases from the guangdong tion. [8, [21] [22] [23] [24] in such patients the disease is often reported as a bi-province of china were subsequently diagnosed from december phasic or tri-phasic illness with an initial acute febrile phase 2003 through january 2004. of interest, no close contacts of these followed by a lower respiratory illness phase then progression in cases was found to have fever or respiratory symptoms after home approximately 20-30% of patients to a phase characterized by quarantine to date. [19] more recently, in april 2004, the chinese acute respiratory distress syndrome (ards) necessitating ventilator support. ministry of health reported additional cases of sars which seemed to be initially associated with an index case of a 26-year-it is imperative to appreciate that individual patients do not old female graduate student who worked at the national institute necessarily display these 'phases', which could be highly individpeiris et al. [17] (n = 50) lee et al. [23] (n = 138) poutanen et al. [22] (n = 10) tsang et al. [21] (n = 10) ualized, from hyperacute to indolent presentation in time course. festations as the patients described above (figure 2). although the at the time of writing, the mortality of probable sars cases was majority of patients developed symptoms of respiratory tract inapproximately 10%, but was much higher in older individuals and fection during admission, only a minority of patients had sympthose with significant co-morbidities. toms at the time of admission to the hospital. diarrhea was present the mean incubation period of sars is estimated to be 6 days, in only 10% at the time of admission, but 50% developed this with a usual range up to approximately 10 days after exposymptom while in the hospital. these investigators also observed sure. [1, 2, 15, 25] the illness generally begins with a prodrome of fever, the duration of time from the onset of illness until the evolution of often associated with chills, rigors and myalgia. headache and various endpoints of their disease: fever, 0.3 days; admission to the severe malaise may accompany this phase; rash has been absent in most cases. in one outbreak within an apartment complex in hong kong, diarrhea was found in 66% of cases. [1] after a typical period of 3-7 days, a lower respiratory phase may begin with the onset of non-productive cough and progressive pneumonia. in the initial reports of cases, 20-30% of patients required intensive care unit management and mechanical ventilation. the presenting symptoms of patients admitted to the hospital from four published series are listed in table ii. [8, [21] [22] [23] most patients were admitted to the hospital several days after the onset of symptoms. the most common complaints were fever and chills or rigors. upper respiratory tract symptoms such as rhinorrhea and sore throat were less common. at the time of examination, abnormal auscultatory findings were present in about one-third of patients. although fever and progressive respiratory manifestations are a hallmark of most cases of sars, patients with more indolent characteristics (including absence of fever) have been describedespecially in elderly or immunocompromised patients. [1, 26] the first report of a complete outbreak of sars (from beginning of the outbreak until declaration of containment) was recently published by vu et al. [27] they report a cohort of patients, all of whom required hospitalization, who presented with similar manihospital, 4.3 days; onset of radiographic change, 4.4 days; onset of multivariate analysis, the only factors that were predictive of an respiratory symptoms, 4.5 days; onset to maximal radiographic adverse outcome were advanced age, a high peak lactate change, 10 days; onset to intubation, 10.5 days; onset to end of dehydrogenase level, and a higher absolute neutrophil count. fever, 12.7 days; and onset to death, 18.8 days. [27] 5. diagnosis chest x-ray abnormalities are usually absent during the initial phase of illness but become progressively abnormal during the the initial manifestations of sars are not specific and cannot phase of lower respiratory illness. initially this is characterized by easily be distinguished from those of other respiratory infections. early focal interstitial infiltrates, usually seen as ground glass the first clinical definition developed by the who (see section 1) opacity, and progressing to bilateral disease (figure 3). a typical was found to be only 29% sensitive and approximately 70% ards picture has emerged in many very seriously ill patients. in specific for identifying laboratory documented cases. [30] clinicians these patients, high-resolution computed tomography (hrct) is should conduct thorough diagnostic testing to rule out other etiolomore sensitive in early disease when the chest x-ray could be gies in patients suspected of having sars. initial recommended normal or only showing inconclusive consolidation. characteristidiagnostic testing procedures include chest radiograph and pulse cally, hrct shows peripheral and, most commonly, lower lobe oximetry. since sars often progresses rapidly, repeated chest xconsolidation. [28, 29] although non-diagnostic and closely mimickrays within the first or second day, sometimes twice daily, may be ing the appearance of bronchiolitis obliterans with organizing helpful in documenting the course of disease. indiscriminate use of pneumonia, hrct is also helpful for showing no evidence of hrct to detect 'radiographically occult disease' is to be discourpleural effusion or intrathoracic lymphadenopathy, which are very aged in view of infection control issues, and the rapidly progresrarely seen in sars. [2, 21, 28, 29] spontaneous pneumomediastinum sive nature of sars, thus making it likely that radiographic also occurs as a rare complication with sars. [8] abnormalities would be more apparent within a few days after laboratory abnormalities most often associated with sars hospitalization. [2] tests for evaluation of specific organisms assoinclude absolute lymphopenia, mild neutropenia, and thrombociated with pneumonia should be performed, and include: blood cytopenia. mild to moderately elevated plasma levels of creatine cultures, sputum gram stain and culture, and testing for viral phosphokinase, lactate dehydrogenase, and transaminases were respiratory pathogens -especially influenza and respiratory synseen in 30-80% of cases (table ii) . cytial virus. urinary antigen for both pneumococcus spp. and lee et al. [23] found that advanced age, male sex, and high levels legionella spp. should also be considered. acute and convalescent of serum creatine phosphokinase, serum lactate dehydrogenase, a serum (preferably 28 days after onset of symptoms) should be relatively high initial neutrophil count (i.e. mean 4.6 vs 3.7 × collected from each patient who meets the sars clinical case 10 -9 l), and a low levels of serum sodium were significant predic-definition. however, if acute and convalescent phase sera are tive factors for intensive care unit admission, and death. on collected at least 8-10 days apart, a 4-fold or greater rise in different rt-pcr assays performed on different specimen aliquots identify the coronavirus rna. [33] because of the possibility of false-negative cultures and rt-pcr assays, only the absence of antibody in a serum specimen obtained >21 days after symptom onset is considered by the cdc to be a negative laboratory test for sars coronavirus. the likelihood of detecting sars-cov is increased if multiple specimens (e.g. stool, serum, respiratory tract specimens) are collected during the course of illness. clinicians should consult with their local laboratory personnel and health department about obtaining such tests. the priority of specimens for sars-cov testing and optimal timing for collection are presented in table iii. of the 50 patients with clinical sars described by peiris et al., [8] 45 had serological or pcr evidence of sars-associated coronavirus infection; and of the 5 who were unconfirmed, 4 had serological testing prior to 14 days of onset of illness (possibly prior to the time of seroconversion). another series of 72 cases in hong kong showed that despite significantly high dosages of corticosteroid therapy, a seroconversion of 95.8% occurred on day 21 after onset of illness. [35] this differs from the us experience where of the 74 probable cases of sars reported by 15 july 2003, only 8 had been confirmed by laboratory diagnosis (all by serology); 38 were negative and 28 had no result. b antibody testing should also be carried out on a serum sample collected >28 days after symptom onset. antibody titer when tested in parallel should be considered indicative of a confirmed case. a variety of methods for detection of coronavirus infection are now available. these include culture methods, pcr-based methods, and serological tests. [31] culture of the sars coronavirus is considered solid evidence of infection, but there have been problems with the various generations of rt-pcr assays, both with false-positive results and with inconsistent detection of viral genome in the first days of illness as well as later in the convalescent phase. it is therefore recommended that detection of sars-cov rna by rt-pcr be validated by a second reference laboratory. [32] because antibodies to sars coronavirus have not been found in the general population, background sars coronavirus antibodies do not appear to be a substantial concern. however, the current serologic assays (both elisa and ifa [indirect fluorescent antibody] formats) do not reliably detect antibodies until the titers rise substantially after the second week of illness. according to the uscdc, suspect or probable cases are considered laboratory-confirmed if sars coronavirus is isolated, if antibody to sars coronavirus is detected and confirmed by a second reference laboratory, or if two table iv . a summary of infection control precautions for patients hospitalized with suspected/probable severe acute respiratory syndrome (sars) [reproduced from sampathkumar et al., [39] with permission] place patient in a negative pressure, specially vented, room a number of other agents have been suggested for therapy of sars-cov, including interferon-α, glycyrrhizin, and protease inhibitors. [1] in one preliminary, uncontrolled study by loutfy et al. [38] from toronto, use of interferon 9 μg/day for a minimum of 2 days and increased to 15 μg/day for a total of 10 days, plus corticosteroids (oral prednisone 50mg twice a day, or intravenous methylprednisolone 40mg every 12 hours) was associated with reduced disease-associated impaired oxygen saturation and more rapid resolution of radiographic lung abnormalities. the authors acknowledge, however, that these findings need to be interpreted cautiously in view of lack of randomization, the retrospective dosing, and the limited sample size (a total of 21 patients). table v . a summary of protective measures taken by severe acute respiratory syndrome (sars)-infected and non-infected staff in hong kong hospitals (reproduced from seto et al., [40] c comparing proportion of infected (n = 11) over non-infected staff (n = 72), with those without mask. since the causative agent of sars is contagious, in the absence of effective drugs or vaccines the only currently effective strategy 6. treatment for limiting the impact of sars is implementation of preventive at the time of writing (24 may 2004), no specific therapy is recommended. a variety of treatments have been attempted, but there are no controlled data. most patients have been treated throughout the illness with broad-spectrum antimicrobials, supplemental oxygen, intravenous fluids, and other supportive measures. some clinicians have advocated a combination of ribavirin and corticosteroids, but the efficacy of these drugs has not been established. the use of systemic corticosteroids in sars is controversial, and the efficacy based on controlled studies is unavailable. [36] one study found initial use of pulse-dosed methyl prednisolone (≥500 mg/day) to be more efficacious and equally well tolerated as a lower dose of methyl prednisolone, but this was based on retrospective observational evaluation. [35] the use of corticosteroids in patients with viral infections can be hazardous when not accompanied by an effective anti-viral agent. [36] early testing of ribavirin and other antiviral compounds against the novel coronavirus have not produced evidence of in vitro activity. [31] an evaluation of the use of ribavirin was published by knowles et al., [37] who reported adverse events in 110 patients with suspected or probable sars treated with ribavirin. of those 110 patients 61% had evidence of hemolytic anemia; hypocalcemia and hypomagnesemia were detected in 58% and 46% of patients, respectively. [33] the authors felt the benefits of ribavirin may not outweigh the risk of adverse events. there was a potential for ribavirin to have negative clinical and economic consequences because of the adverse events. it is now considered among the table vi . recommendations for the evaluation of patients with communityacquired respiratory illness in the presence or absence of severe acute respiratory syndrome-associated coronavirus (sars-cov) transmission in the world [34] in the absence of sars-cov transmission anywhere in the world, the diagnosis of sars-cov disease should be considered only in patients who require hospitalization for radiographically confirmed pneumonia and who have an epidemiologic history that raises the suspicion of sars-cov disease. in the absence of sars-cov transmission anywhere in the world, suspicion of sars infection is raised if, within 10 days of symptom onset: the patient had a history of recent travel to mainland china, hong kong, or taiwan; was in close contact with ill people with a history of recent travel to such areas; is employed in an occupation at particular risk for sars-cov exposure, including a healthcare worker with direct contact or a worker in a laboratory that contains live sars-cov; or is part of a cluster of cases of atypical pneumonia without an alternative diagnosis once sars-cov transmission has been documented in the world, a diagnosis of sars should still be considered in patients who require hospitalization for pneumonia and who have an epidemiologic history described above. in addition, all patients with fever or respiratory symptoms should be questioned about whether within 10 days of symptom onset they have had: close contact with someone suspected of having sars-cov disease; a history of foreign travel (or close contact with an ill person with a history of travel) to a location with documented or suspected sars-cov infection; exposure to a domestic location with documented suspected sars-cov (including a laboratory that contains live sars-cov); or close contact with an ill person with such an exposure history fig. 4 . algorithm for evaluating and managing patients requiring hospitalization for radiographically confirmed pneumonia, in the absence of person-toperson transmission of severe acute respiratory syndrome-associated coronavirus (sars-cov) anywhere in the world. [34] measures centered on avoidance of exposure and infection. such hospitalized patients with suspected sars should be isolated measures include global and regional surveillance, early detection in negative pressure rooms; healthcare workers should wear masks of new cases, identification of patient contacts, and strict adher-(a high efficiency mask such as the n-95 respirator used for ence to infection control policies. guidelines for infection control tuberculosis) to prevent air-droplet and airborne acquisition. behave been published and should be consulted for updated recom-cause coronaviruses can survive on environmental surfaces, good mendations. [34, 39] healthcare workers encountering a possible case hand-washing with soap and water or use of an alcohol-based hand of sars (suspected or probable) should take meticulous safety rub is highly recommended as well. environmental surfaces that precautions and seek advice from an expert in sars infection are frequently touched by the patient or are soiled with body fluids control. should be cleaned and disinfected with a household disinfectant. the early symptoms of sars-cov disease usually include fever, chills, rigors, myalgia, and headache. respiratory symptoms often do not appear until 2-7 days after the onset of illness. 2 in settings with more extensive transmission, all patients with fever or respiratory symptoms should be evaluated for possible sars-cov disease. 3 depending on symptoms and exposure history, initial diagnostic testing for patients may include complete blood count, chest x-ray (cxr), pulse oximetry, blood cultures, sputum gram stain and culture, testing for viral respiratory pathogens (notably influenza and respiratory syncytial virus), legionella sp. and pneumococcal urinary antigen. 4 an alternative diagnosis should be based on laboratory tests with high-predictive value (e.g. blood culture, urinary antigen). 5 chest computed tomography (ct) may show evidence of an inflitrate before a cxr. therefore, a chest ct should be considered in patients with a strong epidemiologic link to a known case of sars and a negative cxr 6 days after onset of symptoms. 6 sars isolation precautions should be discontinued after consultation with local public health authorities and the evaluating physician. an important characteristic of the recent sars outbreaks has been infection transmission. table iv lists precautions for patients hospitalized with sars. the predilection for transmission to healthcare providers after patient care. for the most part this has occurred after close, patients with sars-cov disease who do not otherwise need to unprotected contact with symptomatic individuals. healthcare be hospitalized can be managed appropriately as outpatients. workers who have had unprotected exposure and who develop these patients should limit interactions outside the home. they fever or respiratory symptoms should not come to work, and should be instructed to wear surgical masks in the presence of should report their symptoms to the infection control/employee household contacts, contain respiratory secretions in facial tissues, health service and their physician immediately. healthcare workand wash hands frequently. they should stay away from work, ers who have had unprotected exposure during procedures with school, or other public places for 10 days after resolution of fever. high risk of aerosolization (e.g. intubations, bronchoscopy) should household members or other close contacts of these patients should wear gloves and practice good hand hygiene. in the abbe quarantined for a 10-day period, since there is a high risk of sence of fever or respiratory symptoms, they need not limit their sars. [20] this policy has been running since the middle of march 2003 at queen mary hospital of the university of hong kong, activities. despite the disappearance of sars in hong kong since june the importance of sars precautions was demonstrated in a 2003. only authorized and minimum number of staff working in case-control study in five hong kong hospitals, with 241 nonthese wards may enter the premises. all staff entering these infected and 13 infected healthcare providers who had documentrestricted areas follow strict and stepwise 'gowning' and 'degowned contacts with sars patients (table v) . [40] all of the healthcare ing' procedures, and use standard personal protection equipment providers were surveyed concerning the use of masks, gloves, (disposable surgical paper cap, n-95 mask, and reusable eye gowns, and hand-washing, as recommended under droplet precaugoggles and cotton surgical gown). patients are treated with potent tions. no staff member who reported use of all four measures was antibiotics, usually in the form of a combination of cephalosporin infected. in contrast, all 13 infected staff members had omitted at and macrolide, or in the event of allergy to these antibiotics with least one of the measures (p = 0.0224). the authors observed that levofloxacin. patients who improve clinically and radiologically both surgical and high efficiency masks (n-95 masks) were proare unlikely to have sars, and are moved to wards that don't tective against infection, whereas paper masks did not significantrequire the level of intensive care and/or isolation as would be ly reduce the transmission (such masks are easily wet with saliva required for those patients who have sars, for observation for and are not recommended for precautions against droplets). 5-7 days before discharge. in the event of confirmed or suspected in order to be prepared for the recurrence of sars and the need sars, a patient will be diverted to the appropriate wards to for early implementation of control measures, the us cdc reminimize exposure of fellow patients, if no single-room accomleased clinical guidelines for the identification and evaluation of modation could be provided. possible sars-cov disease among patients presenting with community-acquired illness. [34] the key principles upon which control 8. conclusion measures are based have taken into consideration the fact that in the year 2003 a vast majority of patients with sars-cov disease because a new virus causes sars, it is very difficult to predict had a clear history of exposure either to a sars patient or to a the eventual significance of this infection. however, the resetting in which srs-cov transmission occurred (i.e. hospital); emergence of sporadic cases in china, has caused great concern as and developed pneumonia. recommendations for the evaluation to its future impact. it has had enormous economic and political of patients with community-acquired respiratory illness were deimpact on the affected areas of the world. although important veloped for two primary circumstances: firstly in the absence of progress has been made concerning the etiology, epidemiology, sars-cov transmission anywhere in the world, and secondly and prevention of this virus, many important questions remain. once transmission had been documented (these were released prior without answers to some of these questions, the eventual province in china (or close contact with an ill person with a outcome of sars remains unclear. history of recent travel to the area) in the 10 days before onset of symptoms. [19] when such patients are identified, appropriate isolaa major outbreak of severe acute respiratory syndrome references in hong kong outcomes of 144 patients with sars in the greater toronto area management of severe acute respiratory syndrome: the center for disease control and prevention. outbreak of severe acute respiratory 417-24 syndrome: worldwide case definitions for surveillance of severe acute respiratory syndrome sars clinical description of a completed outbreak sever acute respiratory syndrome (sars) [online]. available from url: of sars in vietnam severe acute respiratory syndrome: radiographic high-resolution ct findings of severe acute cases: united states and worldwide a novel coronavirus associated with severe acute respiratory syndrome evaluation of who criteria for identifying patients with severe respiratory syndrome out of hospital: prospective observa identification of a novel coronavirus in patients with severe acute respiratory syndrome coronavirus as a possible cause of severe (sars) and coronavirus testing: united states center for disease control and prevention. interpreting sars-cov test results the sars coronavirus from animals in southern china koch's postulates fulfilled for sars 20] virus detection of sars coronavirus in plasma by mosaic evolution of the severe acute respiratory real-time rt-pcr available level preparedness and response to severe acute respiratory syndrome (sars) from url high-dose pulse versus nonpulse corticosteroid regimens in severe acute respiratory syndrome the use of corticosteroids in sars severe acute respiratory syndrome common adverse events associated with the use of ribavirin for severe acute respiratory syndrome in canada short term outcome and risk factors for adverse clinical outcomes in adults with severe acute respiratory syndrome clinical progression and viral load in a roids in severe acute respiratory syndrome: a preliminary study sars: epidemiology, clinical 18. center for disease control and prevention / presentation, management, and infection control measures center for disease control and prevention. recent sars cases in china accessed 2004 and contact in prevention of nosocomial transmission of severe acute respirato-may 20] ry syndrome (sars) center for disease control and prevention. china reports ninth recent possible sars case correspondence and offprints: professor thomas m. file, jr, infectious a cluster of cases of severe acute respiratory disease service identification of severe acute respiratory syndrome in canada key: cord-327264-j0efi5vc authors: waterer, grant title: respiratory infections in the asia‐pacific region date: 2017-05-24 journal: respirology doi: 10.1111/resp.13077 sha: doc_id: 327264 cord_uid: j0efi5vc nan i frequently tell young prospective research students that respiratory infections are a great area to develop expertise in because unlike cancer or cardiovascular disease they can be guaranteed the problem will not be solved and go away in their lifetime. the burden of respiratory infectious disease is large, not only acutely, but also due to their chronic complications. lower respiratory tract infections remain the number one cause of death in the developing world. 1 in the developed world, lower respiratory tract infections are still the fifth leading cause of death. 1 the increasing age of the general population in most countries, our ability to keep people from succumbing to chronic organ failure and the ever increasing array of immunosuppressant drugs being used in a variety of neoplastic and immune diseases ensure that there is an ever increasing pool of vulnerable hosts. as well as host factors, it is the speed of adaptation of the pathogens that makes it most likely that we will not conquer death from respiratory infection any time soon. while some progress has been made in reducing pneumococcal disease with the newer conjugate vaccines, 2 there is evidence that the speed of adaptation (as measured by the proportion of strains not covered by the vaccines) is increasing. [3] [4] [5] adaptation in other pathogens is posing challenges in multiple areas including drug-resistant tuberculosis, pan-resistant gram-negative pathogens and ever increasing rates of community-acquired methicillin-resistant staphylococcus aureus in many western countries. not only is there adaptation of existing pathogens, the progressive encroachment of humans on other habitats has seen the rise of new pathogens such as coronaviruses causing severe acute respiratory syndrome and middle east respiratory syndrome, as well as novel influenza strains such as h1n1 09 and the avian h5, h7 and h9 strains. with the notable exception of h1n1 09, all of these viral infections are associated with severe disease and high mortality rates. in this series on respiratory infections in respirology, many of the above-mentioned challenges will be covered including the burden of disease due to respiratory pathogens (both acute and long-term), drug-resistant pathogens and newly emerging pathogens. other reviews will focus on key areas such as the evidence around what is best practice in caring for patients with respiratory infections and key differences in respiratory pathogens in different areas of particular relevance to the asia-pacific region. i hope readers of respirology will find these reviews a useful resource clinically and an inspiration to get involved in research as since the bugs will not stand still, neither can we! disclosure statement g.w. has been paid to attend advisory boards for menarini pharmaceuticals. school of medicine, the university of western australia, perth, western australia, australia world health organisation. the top 10 causes of death hospitalizations for pneumonia after a decade of pneumococcal vaccination effect of high-valency pneumococcal conjugate vaccines on invasive pneumococcal disease in children in spidnet countries: an observational multicentre study the herd effects of infant pcv7/pcv13 sequential implementation on adult invasive pneumococcal disease, six years post implementation; a nationwide study in israel the rise and fall of pneumococcal serotypes carried in the pcv era key: cord-327685-fymfqvp3 authors: channappanavar, rudragouda; perlman, stanley title: pathogenic human coronavirus infections: causes and consequences of cytokine storm and immunopathology date: 2017-05-02 journal: semin immunopathol doi: 10.1007/s00281-017-0629-x sha: doc_id: 327685 cord_uid: fymfqvp3 human coronaviruses (hcovs) can be divided into low pathogenic and highly pathogenic coronaviruses. the low pathogenic covs infect the upper respiratory tract and cause mild, cold-like respiratory illness. in contrast, highly pathogenic hcovs such as severe acute respiratory syndrome cov (sars-cov) and middle east respiratory syndrome cov (mers-cov) predominantly infect lower airways and cause fatal pneumonia. severe pneumonia caused by pathogenic hcovs is often associated with rapid virus replication, massive inflammatory cell infiltration and elevated pro-inflammatory cytokine/chemokine responses resulting in acute lung injury (ali), and acute respiratory distress syndrome (ards). recent studies in experimentally infected animal strongly suggest a crucial role for virus-induced immunopathological events in causing fatal pneumonia after hcov infections. here we review the current understanding of how a dysregulated immune response may cause lung immunopathology leading to deleterious clinical manifestations after pathogenic hcov infections. coronaviruses belong to the virus family coronaviridae and are enveloped, positive-sense rna viruses. the coronavirus genome is approximately 31 kb, making these viruses the largest known rna viruses [1, 2] . coronaviruses infect a variety of host species, including humans and several other vertebrates. these viruses predominantly cause respiratory and intestinal tract infections and induce a wide range of clinical manifestations [3, 4] . coronaviruses infecting the respiratory tract have long been recognized as significant pathogens in domestic and companion animals and as the cause of mild and severe respiratory illness in humans [4, 5] . in general, coronaviruses infecting humans can be classified into low pathogenic hcovs, which include hcov-229e, hcov-oc43, hcov-nl63, and hcov-hku and highly pathogenic covs such as severe acute respiratory syndrome cov (sars-cov) and middle east respiratory syndrome cov (mers-cov) [6, 7] . low pathogenic hcov infect upper airways and cause seasonal mild to moderate cold-like respiratory illnesses in healthy individuals. in contrast, the highly pathogenic hcovs (pathogenic hcov or hcov hereafter) infect the lower respiratory tract and cause severe pneumonia, which sometimes leads to fatal acute lung injury (ali) and acute respiratory distress syndrome (ards), resulting in high morbidity and mortality [8] [9] [10] [11] [12] . highly pathogenic hcovs pose a substantial threat to public health. during the 2002-2003 epidemic, sars-cov infected approximately 8400 individuals with a 9.6% overall mortality rate [13, 14] . more recently, mers-cov crossed species to infect 1936 individuals resulting in 690 deaths (∼36% mortality rate) as of april 5, 2017 [15, 16] . recent identification of sars-like coronaviruses in bats and mers-cov in domesticated camels makes it likely that these viruses will continue to cross species barriers and cause additional outbreaks in human populations [17] [18] [19] [20] . these highly pathogenic hcovs cause a wide spectrum of clinical manifestations in humans, with a large fraction of patients developing short period of moderate clinical illness and a small but a substantial number of patients experiencing severe disease characterized by ali and ards [21] [22] [23] 10] . thus, there are basically two groups of patients, those developing milder disease, which resolved and those with severe disease, which was commonly fatal. the disease severity in pathogenic hcov infections was also influenced by several factors such as initial viral titers in the airways and age and comorbid conditions of the infected individual. while younger individuals below 18 years experience mild-moderate clinical illness, elderly individuals exhibit worse outcomes after infection with sars-cov or mers-cov [22, 10, 24] . additionally, individuals with comorbid conditions such as diabetes, obesity, heart failure, and renal failure among others experience severe disease, particularly after mers-cov infection [25, 26] . despite several years of research, specific factors causing the unusually high morbidity and mortality following pathogenic hcovs are incompletely understood. virus-induced direct cytopathic effects and viral evasion of host immune responses are believed to play major roles in disease severity. however, studies from humans who died of sars and more recent studies in animal models suggested that a dysregulated immune response occurred, resulting in an exuberant inflammation and lethal disease. in this review, we discuss recent advances in our understanding of hcov pathogenesis, with a special emphasis on cytokine storm and immunopathology as causes for deleterious consequences during hcov infections. sars-cov infection in humans resulted in an acute respiratory illness that varied from mild febrile illness to ali and in some cases ards and death [27, 10] . the clinical course of sars presents in three distinct phases. the initial phase was characterized by robust virus replication accompanied by fever, cough, and other symptoms, all of which subsided in a few days. the second clinical phase was associated with high fever, hypoxemia, and progression to pneumonia-like symptoms, despite a progressive decline in virus titers towards the end of this phase [28] . during the third phase, ∼20% of patients progressed to ards, which often resulted in death [29, 30] . because of a progressive decline in virus titers, the third phase is thought to have resulted from exuberant host inflammatory responses. the most common clinical manifestations of mers include flu-like symptoms such as fever, sore throat, nonproductive cough, myalgia, shortness of breath, and dyspnea, which rapidly progress to pneumonia [25, 21] . other atypical presentations include mild respiratory illness without fever, chills, wheezing, and palpitations. mers-cov in humans also causes gastrointestinal symptoms such as abdominal pain, vomiting, and diarrhea. the majority of mers patients with dyspnea progress to develop severe pneumonia and require admission to an intensive care unit (icu). although most healthy individuals present with mild-moderate respiratory illness, immunocompromised and individuals with comorbid conditions experience severe respiratory illness, which often progressed to ards [21] . overall, mers-cov caused severe disease in primary index cases, immunocompromised individuals and in patients with comorbid conditions, but secondary cases of household contacts or healthcare workers were mostly asymptomatic or showed mild respiratory illness. gross and microscopic pathology of sars typically, analyses of lungs from patients who succumbed to sars showed lung consolidation and edema with pleural effusions, focal hemorrhages, and mucopurulent material in the tracheobronchial tree. diffuse alveolar damage (dad) was a prominent histological feature in sars lungs [31, 32] . other changes included hyaline membrane formation, alveolar hemorrhage, and fibrin exudation in alveolar spaces with septal and alveolar fibrosis observed during later stages [32, 33] . staining for viral antigen revealed infection of airway and alveolar epithelial cells, vascular endothelial cells, and macrophages [31, 32] . furthermore, sars-cov viral particles and viral genome were also detected in monocytes and lymphocytes [31] . in addition to these changes, histological examination of lungs from patients who died of sars revealed extensive cellular infiltrates in the interstitium and alveoli. these cellular infiltrates included neutrophils and macrophages with macrophages being the predominant cell type [31, 32] . these results correlated with increased numbers of neutrophils and monocytes and lower cd4 and cd8 t cell counts in the peripheral blood samples of patients with fatal sars [34-36]. despite numerous laboratory-confirmed cases and deaths due to mers-cov infection in several countries, only one autopsy report of mers in humans is available. analysis of lung tissue from this patient showed pleural, pericardial, and abdominal effusions associated with generalized congestion, edema, and consolidation of lungs [37] . similar to sars-cov infection, dad was a prominent feature in the lungs. additionally, epithelial cell necrosis, sloughing of bronchiolar epithelium, alveolar edema, and thickening of alveolar septa were also noted. immunohistochemical examination showed that mers-cov predominantly infected airways and alveolar epithelial cells, and endothelial cells and macrophages. the severity of lung lesions correlated with extensive infiltration of neutrophils and macrophages in the lungs and higher numbers of these cells in the peripheral blood of mers patients [37] . cytokines and chemokines have long been thought to play an important role in immunity and immunopathology during virus infections. a rapid and well-coordinated innate immune response is the first line of defense against viral infections, but dysregulated and excessive immune responses may cause immunopathology [38] [39] [40] . although there is no direct evidence for the involvement of pro-inflammatory cytokines and chemokines in lung pathology during sars and mers, correlative evidence from patients with severe disease suggests a role for hyper-inflammatory responses in hcov pathogenesis. additionally, sars-cov-infected airway epithelial cells (aecs) also produce large amounts of ccl3, ccl5, ccl2, and cxcl10 [43]. the delayed but excessive production of these cytokines and chemokines is thought to induce a dysregulated innate immune response to sars-cov infection. high serum levels of pro-inflammatory cytokines (ifn-γ, il-1, il-6, il-12, and tgfβ) and chemokines (ccl2, cxcl10, cxcl9, and il-8) were found in sars patients with severe disease compared to individuals with uncomplicated sars [44] [45] [46] [47] . conversely, sars patients with severe disease had very low levels of the anti-inflammatory cytokine, il10 [44] . in addition to pro-inflammatory cytokines and chemokines, individuals with lethal sars showed elevated levels of ifn (ifn-α and ifn-γ) and ifn-stimulated genes (isgs) (cxcl10 and ccl-2) compared to healthy controls or individuals with mild-moderate disease [48-51]. these results were the first to suggest a possible role for ifns and isgs in the immunopathogenesis of sars in humans. thus, it appears from these studies that dysregulated and/or exaggerated cytokine and chemokine responses by sars-cov-infected aecs, dcs, and macrophages could play an important role in sars pathogenesis. similar to sars, mers-cov infection of human airway epithelial cells induces significant but delayed ifn and proinflammatory cytokine (il-1β, il-6, and il-8) responses [52] . while mers-cov replicates both in naïve and activated human monocyte-macrophages and dcs, only activated t cells support mers-cov replication [53] [54] [55] . this is in contrast to sars-cov, which abortively infected monocyte-macrophages, dcs, and t cells. mers-cov infection of thp-1 cells, a monocyte cell line, and human peripheral blood monocyte-derived macrophages and dendritic cells induced delayed but elevated levels of pro-inflammatory cytokines and chemokines such as ccl-2, ccl-3, ccl-5, il-2, and il-8 [54, 55]. however, induction of ifn-α/β by monocytemacrophages and dcs was not substantial except for plasmacytoid dendritic cells, which produced copious amounts of ifns upon mers-cov infection [56] . recent studies showed elevated levels of serum pro-inflammatory cytokines (il-6 and ifn-α) and chemokines (il-8, cxcl-10, and ccl5) in individuals with severe mers compared to those with mild to moderate disease [57, 58] . high serum cytokine and chemokine levels in mers patients correlated with increased neutrophil and monocyte numbers in lungs and in the peripheral blood, suggesting a possible role for these cells in lung pathology [57, 58, 37 ]. several inbred mouse strains have been evaluated to study sars-cov pathogenesis. mice infected with the human strain of sars-cov (sars-cov-urbani) were permissive to virus replication but developed only mild lung pathology and clinical illness [59] . subsequently, isolation of mouseadapted strains of sars-cov (e.g., sars-cov-ma15) allowed studies of lethal sars [60] [61] [62] . ma15 infects airway and alveolar epithelial cells and epithelial cells of other organs [62] . young mice of many strains (e.g., c57bl/6, 129) support ma15 replication in the lungs but are resistant to developing significant clinical disease [63, 64] . in contrast, young balb/c mice infected with ma15 develop lethal disease characterized by diffuse alveolar damage, enhanced monocyte/macrophage and neutrophil accumulation, pulmonary edema, and hyaline membrane formation [62] . furthermore, aged mice of all strains develop lethal clinical disease and succumb to infection [65, 66, 64] . in addition to mouse models, sars-cov infection of aged rhesus macaques resulted in significantly more pathology than young adult animals [67] . these animal models replicated several key features of sars-cov infection in humans and were thus useful for investigating sars pathogenesis. studies in animal models have been particularly useful in elucidating the role of cytokines and chemokines in mediating lung immunopathology following hcov infections. infection of non-human primates (nhps) with sars-cov induced a dysregulated immune response resulting in increased disease severity in aged but not young nhps, despite similar viral titers in the airways [67] . since enhanced expression of genes regulating inflammation but not virus titers correlated with disease severity, an exaggerated immune response is thought to induce lethal disease in aged nhps [67] . similarly, in sars-cov-infected balb/c mice, disease severity in aged mice correlated with early and disproportionately strong upregulation of ards-associated inflammatory gene signatures [66] . in a recent study, we identified a pathogenic role for ifn-i in mice infected with ma15. our results showed that rapid sars-cov replication in balb/c mice induced a delayed ifn-α/β response accompanied by an excessive influx of pathogenic inflammatory monocyte-macrophages (imms) [38] . the accumulating imms themselves produced additional levels of monocyte chemo-attractants such as ccl2, ccl7, and ccl12 (through ifn-α/β receptor stimulation), resulting in further accumulation of pathogenic imms, which in turn enhanced disease severity. these infiltrating imms produced elevated levels of pro-inflammatory cytokines such as tnf, il-6, il1-β, and inos. blocking ifn signaling, depleting imms, or neutralizing a single inflammatory cytokine, tnf, protected mice from lethal sars-cov infection. additionally, ifn-α/β or imm-derived pro-inflammatory cytokines sensitized t cells to undergo apoptosis, further impeding virus clearance [38] . in another study of sars-cov infection, loss of tir-domain-containing adapter-inducing interferon-β (trif), an adapter molecule for tlr3 and tlr4 signaling, resulted in a distinct inflammatory signature characterized by neutrophil and other inflammatory cell infiltration [68] . a dysregulated immune response to sars-cov in trif-deficient mice was associated with aberrant antiviral ifn (ifn-α and ifnβ), pro-inflammatory cytokine and chemokine (il-6, tnf, ifn-γ, and ccl5), and interferonstimulated gene (rsad2, ifit1, and cxcl10) responses. notably, virus titers were significantly higher in tlr3 −/− and trif −/− mice compared to their wt controls [68] . although the viral factors regulating the pro-inflammatory response of neutrophils and monocyte-macrophages remain to be identified, the e protein of sars-cov has been shown to enhance pro-inflammatory cytokine and chemokine and inflammasome activity via its ion channel activity [69] [70] [71] . these results support the notion that higher virus titers and dysregulated cytokine/chemokine responses cause a bcytokine storm^with lung immunopathological changes following sars-cov infection. animal models employed to study mers include rhesus macaques, rabbits, marmosets, and mice among others. mers-cov challenged rhesus macaques developed mild to moderate disease [72] . similarly, mers-cov-infected rabbits displayed mild clinical disease with mild-moderate perivascular, peribronchiolar infiltration, and to a lesser extent lung interstitial inflammation [73, 74] . in contrast, marmosets displayed moderate-severe respiratory disease characterized by broncho-interstitial pneumonia, alveolar edema, and fibrin deposition [75] . marmosets with severe disease showed increased neutrophil and macrophage infiltration in alveoli and interstitial septa, although whether marmosets develop severe disease remains controversial [75, 76] . although gross and histological lesions and inflammatory cell infiltration in mers-cov infected marmosets resemble human disease, there are no data available describing cytokine and chemokine responses in these animals. small laboratory animals, particularly rodents, do not support mers-cov replication due to inability of mers-cov-spike protein to bind to human dpp4 (hdpp4) orthologs in these animals [77] . the first mouse model to study mers was generated by intranasal transduction of adenovirus encoding hdpp4. these mice developed mild to moderate pneumonia, especially in immunodeficient mice [78] . several hdpp4 transgenic mouse models developed thereafter exhibited variable organ tropism and disease severity, depending on the promoter driving the hdpp4 expression [79, 80] . more recently, hdpp4 knock-in mice in which hdpp4 is expressed under the mouse hdpp4 promoter have also been described. these mice also developed moderate clinical disease after infection with human isolates of mers-cov [81] . we and others recently developed a similar mouse model and showed that serial passage of human isolate of mers-cov resulted in mouse adaptation. mice infected with this adapted virus caused lethal respiratory illness and will be useful for studies of pathogenesis [82, 83] . overall, delayed and aberrant antiviral and proinflammatory cytokine production in mers-cov-infected human macrophages and dendritic cells and high serum proinflammatory cytokine levels in patients with severe disease compared to mild-moderate clinical disease suggesting that possible dysregulated and enhanced cytokine responses promote lung pathology following mers-cov infection. to counter innate antiviral cytokine responses, sars-cov and mers-cov encode several structural and non-structural proteins (nsps) that antagonize antiviral immune response. sars-cov encoded nsp1, nsp3-macrodomain, nsp3deubiquitinase (dub), and orf3b, orf6, and orf9b subvert antiviral response by antagonizing ifn and isg responses [84] [85] [86] [87] [88] [89] . while nsp3 impairs ifn responses by unknown mechanism, nsp1 inhibits ifn responses by blocking stat1 phosphorylation [90, 91] . additionally, structural proteins such as the membrane (m) and nucleocapsid (n) proteins dampen ifn signaling by inhibiting tbk1/ikke and by unknown mechanisms, respectively [92] [93] [94] [95] . similarly, mers-cov structural proteins m and n and accessory proteins orf3, orf4a, and orf4b antagonize ifn responses [85, 96, 97] . it should be noted that most if not all of these putative antiviral mechanisms were demonstrated in transient expression assays and whether they are actually important in the context of infectious virus remains to be determined. structural and nonstructural protein antagonism of ifn responses further amplifies inflammatory responses by promoting unrestrained virus replication resulting in increased viral pamps that further dampen ifn signaling and stimulate prrs to induce an aberrant inflammatory response. lack of ifn signaling also leads to an excessive accumulation of ly6c low monocytes and neutrophils. despite several years of research studying sars and mers pathogenesis, specific host factors that drive lung pathology following hcov infections are relatively unknown. however, a careful review of the literature related to sars-cov and mers-cov pathogenesis in humans and animal models highlights several key factors that may play a crucial role in the initiation and progression of an exacerbated inflammatory responses. studies from animal models, especially mouse models, provide correlative evidence for differential disease outcome if the viruses predominantly infect airway epithelial cells versus both airway and alveolar epithelial (type i and type ii pneumocytes) cells. in b6 and 129 strains, both of which are permissive to virus replication but resistant to developing clinical disease, viral antigen is predominantly located in airway epithelial cells early after infection. in contrast, in highly susceptible balb/c mice, virus antigen is detected in the lung airways and in alveolar type i and ii pneumocytes (fig. 1) . these results suggest a critical role for hcov-infected type i and ii pneumocytes in mediating lung pathology and host susceptibility. cov-specific t cells are crucial for virus clearance and limit further damage to host [64, 104] . additionally, t cell responses also dampen overactive innate immune responses [105, 106] . exuberant inflammatory responses caused by pathogenic hcov diminish the t cell response, in the case of sars-cov infection via tnf-mediated t cell apoptosis, thus resulting in uncontrolled inflammatory response. 3. accumulation of alternatively activated macrophages and altered tissue homeostasis: in some sars patients with extended duration of disease, dad was accompanied by fibrosis of interstitial and alveolar spaces and hyperplasia of pneumocytes. similar histological features were noticed in lungs of sars-cov-challenged stat −/− mice on b6 and 129 backgrounds. lungs from these mice revealed an enhanced perivascular infiltration of alternatively activated macrophages, neutrophils, and fibroblasts accompanied by extensive fibrin deposition and alveolar collapse, features observed during end stage ali and ards in humans [63, 107] . further studies revealed that abrogation of stat1 signaling, specifically in myeloid cells, resulted in alternative activation of macrophages [108] . in addition, a delicate balance between host coagulation and fibrinolysis processes regulates tissue remodeling and ali [109] . 4. ards: inflammatory mediators play a key role in the pathogenesis of ards, a primary cause of death in patients infected with sars-cov or mers-cov [110, 111] . several pro-inflammatory cytokines, including il-6, il-8, il-1β, and gm-csf, reactive oxygen species, and chemokines such as ccl2, ccl-5, ip-10, and ccl3 contribute to ards [48, 112, 113] . additionally, uncontrolled epithelial cell proliferation and impaired tissue remodeling during later stages induce ards leading to pulmonary fibrosis and death. a summary of causes and consequences of cytokine storm and immunopathology to hcov pathogenesis is demonstrated in fig. 2 . high virus titers and subsequent exuberant inflammatory cytokine and chemokine responses correlate with high morbidity and mortality observed during pathogenic hcov infections. a systematic review of therapeutic effects of several commonly used antiviral and immunomodulatory agents used during sars outbreak showed inconclusive results [114] . similarly, therapeutic interventions aimed towards reducing viral load were somewhat beneficial when administered early but not during later stages of mers-cov infection [115] [116] [117] . these results suggest that besides controlling viral load, novel strategies directed at attenuating inflammatory responses will likely improve clinical outcomes. here, we describe agents that have the potential to mitigate hcov-induced inflammation. corticosteroid therapy corticosteroids are a class of steroidal hormones that exert anti-inflammatory functions and are generally used to suppress inflammatory conditions. during the 2003 sars epidemic, corticosteroids were the mainstay of immunomodulatory therapy. the timely administration of corticosteroids often leads to early improvement in terms of reduced fever, resolution of radiographic lung infiltrates, and better oxygenation [118] [119] [120] . however, while some studies showed no beneficial effect, other demonstrated adverse outcomes following corticosteroid therapy during sars-cov infection in humans. early treatment of corticosteroids in sars patients enhanced plasma viral load in non-icu patients, thus leading to exacerbated disease [118] . overall, these results show that the timing, dosage, and duration of corticosteroid therapy are critical if this intervention is to be beneficial in hcov infections. in general, corticosteroid therapy is not recommended for treatment of hcov respiratory infections. interferons pegylated and non-pegylated interferons have been under investigation for therapeutic purposes in hcovinfected individuals. however, therapeutic use of these agents produced mixed results both in humans and animal models of hcov infections. early administration of ifn was marginally beneficial in reducing viral load and resulted in moderate improvement in clinical manifestations. in contrast, delayed administration of ifn did not have any advantage compared to placebo controls. similarly, early administration of combination of ifn and ribavirin modestly ameliorated disease severity but did not affect mortality [115, 121, 117, 122] . other possible therapeutics ifn-αβ inhibitors and ifn-λ ifn-αβ restrict virus replication through induction of isgs. however, ifn-αβ can also exacerbate disease by enhancing recruitment and function of imms and other innate immune cells. while an early interferon response was protective in sars-cov-infected mice, delayed ifn-αβ signaling dysregulated the anti-sars-cov immune response suggesting that timing of ifn therapy is critical in determining the disease outcome. based on these results, the administration of ifn-αβ receptor blockers or antagonists should be considered as an option to prevent exuberant inflammatory responses during later stages of severe disease, particularly during sars [38] . in contrast to ifn-αβ, ifn-λ mainly activates epithelial cells and lacks monocytemacrophage-mediated pro-inflammatory activity of ifn-αβ [123] . additionally, ifn-λ suppresses neutrophil recruitment to the site of inflammation [124] . since sars-cov and mers-cov predominantly infect aecs and ifn-λ stimulates antiviral gene in epithelial cells without over-stimulating the immune system, use of ifn-λ may be an ideal therapeutic option. suppression of oxidized phospholipids oxidized phospholipids (oxpl) have been shown to promote ali by increasing lung macrophage cytokine/chemokine production via tlr4-trif signaling in influenza a virus (iav)-infected mice [125] . in a recent study, therapeutic administration of the tlr4 antagonist, eritoran, protected mice from lethal iav infection by reducing the levels of oxpl and inflammatory cytokines and chemokines [126] . despite potent immunomodulatory functions, eritoran has no direct antiviral activity, suggesting its use in the amelioration of inflammatory responses. since pathogenic human coronaviruses cause acute lung injury and promote oxpl production in the lungs [125] , strategies to suppress oxpl either by using eritoran or other similar compounds could be of value in dampening hcovinduced inflammation. sphingosine-1-phosphate receptor 1 agonist therapy in mice infected with iav, sphingosine-1-phosphate receptor 1 fig. 2 schematic representation of protective versus pathogenic inflammatory responses to pathogenic hcov infections (s1p1) signaling in endothelial cells was shown to orchestrate pathogenic inflammatory responses [127] . targeted s1p1 agonism restrained excessive inflammatory cell recruitment, suppressed pro-inflammatory cytokines and chemokines, and reduced iav induced morbidity and mortality [127, 128] . , so that sars-cov infection of endothelial cells may drive s1p1-mediated inflammatory cytokine/chemokine responses and neutrophil and macrophage accumulation. therefore, s1p1 agonism could be a potential therapeutic agent in hcov patients to dampen pathogenic cytokine and chemokine responses, if a role for an excessive immune response by these cells is demonstrated. inhibitors of monocyte recruitment and function studies in animal models demonstrate pathogenic roles for imms during lethal hcov infections. in a mouse model of cardiac inflammation, systemic delivery of optimized lipid nanoparticles containing a ccr2-silencing short interfering rna (sirna) efficiently degraded ccr2 mrna and impaired imm recruitment to sites of inflammation thus resulting in improved disease outcome [129, 130] . since hcovs are single-stranded rna (ssrna) viruses and stimulation of imms with the tlr7 agonist, r837 (a synthetic ssrna mimic), induces strong inflammatory responses, it is possible that immspecific tlr-7 signaling promotes excessive inflammation in response to hcov infection. thus, a tlr7 antagonisttargeted approach to mitigate inflammation could prove beneficial. other immunomodulatory agents several other immunomodulatory agents that could ameliorate inflammatory responses following pathogenic hcov infections include cytokine/chemokine inhibitors and danger-associated molecular pattern (damp) antagonists [131] . studies from animal models show a significant contribution of tnf to acute lung injury and impaired t cell responses in sars-covchallenged mice. in vivo neutralization of tnf activity or infection of mice lacking tnfr provides protection against sars-cov-induced morbidity and mortality [38, 132] . however, it is to be noted that tnf was not detected in the serum of sars patients at least during later stages of infection. inflammation is an indispensable part of an effective immune response, without which successful elimination of an infectious agent is difficult. the inflammatory response begins with the initial recognition of a pathogen, which then mediates immune cell recruitment, eliminates pathogens, and ultimately results in tissue repair and return to homeostasis. however, certain viruses such as highly pathogenic covs, iav, and ebola viruses induce excessive and prolonged cytokine/ chemokine response known as bcytokine storms,^which results in high morbidity and mortality due to immunopathology. although studies reviewed in this manuscript provide evidence that bcytokine storms^and immunopathology can occur during pathogenic hcov infections, we do not yet have a sufficient understanding of the specific factor/s responsible for exuberant inflammatory responses. studies from human autopsies and animal models strongly suggest a pathogenic role for inflammatory cytokines/chemokines derived from imm and neutrophils. therefore, therapeutic interventions targeting these pro-inflammatory cytokines and chemokines could prove beneficial in ameliorating undesirable inflammatory responses. additionally, since high virus titers at early and later stages of infection strongly correlate with disease severity in humans, strategies directed at controlling viral load as well as attenuating the inflammatory response might prove beneficial. therefore, future studies should focus on identification of specific signaling pathways that mediate inflammatory responses in hcov-infected patients and animals. (9) coronaviridae coronaviruses, toroviruses, and arteriviruses coronavirus host range expansion and middle east respiratory syndrome coronavirus emergence: biochemical mechanisms and evolutionary perspectives epidemiology, genetic recombination, and pathogenesis of coronaviruses coronavirus pathogenesis and the emerging pathogen severe acute respiratory syndrome coronavirus coronavirus-associated pneumonia in previously healthy children clinical disease in children associated with newly described coronavirus subtypes identification of a novel coronavirus in patients with severe acute respiratory syndrome newly discovered coronavirus as the primary cause of severe acute respiratory syndrome coronavirus as a possible cause of severe acute respiratory syndrome genomic characterization of a newly discovered coronavirus associated with acute respiratory distress syndrome in humans cytokines and chemokines in human macrophages: implications for pathogenesis high secretion of interferons by human plasmacytoid dendritic cells upon recognition of middle east respiratory syndrome coronavirus clinical progression and cytokine profiles of middle east respiratory syndrome coronavirus infection comparative and kinetic analysis of viral shedding and immunological responses in mers patients representing a broad spectrum of disease severity aged balb/c mice as a model for increased severity of severe acute respiratory syndrome in elderly humans a new mouse-adapted strain of sars-cov as a lethal model for evaluating antiviral agents in vitro and in vivo mouse-passaged severe acute respiratory syndrome-associated coronavirus leads to lethal pulmonary edema and diffuse alveolar damage in adult but not young mice a mouse-adapted sars-coronavirus causes disease and mortality in balb/c mice sars-cov pathogenesis is regulated by a stat1 dependent but a type i, ii and iii interferon receptor independent mechanism age-related increases in pgd(2) expression impair respiratory dc migration, resulting in diminished t cell responses upon respiratory virus infection in mice a live, impaired-fidelity coronavirus vaccine protects in an aged, immunocompromised mouse model of lethal disease early upregulation of acute respiratory distress syndrome-associated cytokines promotes lethal disease in an aged-mouse model of severe acute respiratory syndrome coronavirus infection exacerbated innate host response to sars-cov in aged non-human primates toll-like receptor 3 signaling via trif contributes to a protective innate immune response to severe acute respiratory syndrome coronavirus infection the pdz-binding motif of severe acute respiratory syndrome coronavirus envelope protein is a determinant of viral pathogenesis severe acute respiratory syndrome coronavirus envelope protein ion channel activity promotes virus fitness and pathogenesis severe acute respiratory syndrome coronavirus e protein transports calcium ions and activates the nlrp3 inflammasome middle east respiratory syndrome coronavirus (mers-cov) causes transient lower respiratory tract infection in rhesus macaques asymptomatic middle east respiratory syndrome coronavirus infection in rabbits prophylaxis with a middle east respiratory syndrome coronavirus (mers-cov)-specific human monoclonal antibody protects rabbits from mers-cov infection infection with mers-cov causes lethal pneumonia in the common marmoset intratracheal exposure of common marmosets to mers-cov jordan-n3/2012 or mers-cov emc/ 2012 isolates does not result in lethal disease receptor variation and susceptibility to middle east respiratory syndrome coronavirus infection rapid generation of a mouse model for middle east respiratory syndrome animal models for sars and mers coronaviruses animal models of middle east respiratory syndrome coronavirus infection pre-and postexposure efficacy of fully human antibodies against spike protein in a novel humanized mouse model of mers-cov infection a mouse model for mers coronavirusinduced acute respiratory distress syndrome mouse-adapted mers coronavirus causes lethal lung disease in human dpp4 knockin mice severe acute respiratory syndrome coronavirus orf6 antagonizes stat1 function by sequestering nuclear import factors on the rough endoplasmic reticulum/golgi membrane interaction of sars and mers coronaviruses with the antiviral interferon response severe acute respiratory syndrome coronavirus nsp1 suppresses host gene expression, including that of type i interferon, in infected cells coronavirus papain-like proteases negatively regulate antiviral innate immune response through disruption of sting-mediated signaling interferon and cytokine responses to sars-coronavirus infection sars coronavirus pathogenesis: host innate immune responses and viral antagonism of interferon severe acute respiratory syndrome coronavirus evades antiviral signaling: role of nsp1 and rational design of an attenuated strain the conserved coronavirus macrodomain promotes virulence and suppresses the innate immune response during severe acute respiratory syndrome coronavirus infection severe acute respiratory syndrome coronavirus papain-like protease ubiquitin-like domain and catalytic domain regulate antagonism of irf3 and nf-kappab signaling severe acute respiratory syndrome coronavirus open reading frame (orf) 3b, orf 6, and nucleocapsid proteins function as interferon antagonists sars-cov nucleocapsid protein antagonizes ifn-beta response by targeting initial step of ifn-beta induction pathway, and its c-terminal region is critical for the antagonism suppression of innate antiviral response by severe acute respiratory syndrome coronavirus m protein is mediated through the first transmembrane domain middle east respiratory syndrome coronavirus m protein suppresses type i interferon expression through the inhibition of tbk1-dependent phosphorylation of irf3 the structural and accessory proteins m, orf 4a, orf 4b, and orf 5 of middle east respiratory syndrome coronavirus (mers-cov) are potent interferon antagonists initial viral load and the outcomes of sars proliferative growth of sars coronavirus in vero e6 cells viral load kinetics of mers coronavirus infection lung epithelial apoptosis in influenza virus pneumonia: the role of macrophage-expressed tnf-related apoptosis-inducing ligand macrophage-expressed ifn-beta contributes to apoptotic alveolar epithelial cell injury in severe influenza virus pneumonia cellular inhibitor of apoptosis protein ciap2 protects against pulmonary tissue necrosis during influenza virus infection to promote host survival t cell responses are required for protection from clinical disease and for virus clearance in severe acute respiratory syndrome coronavirus-infected mice adaptive immune cells temper initial innate responses not so fast: adaptive suppression of innate immunity transcriptomic analysis reveals a mechanism for a prefibrotic phenotype in stat1 knockout mice during severe acute respiratory syndrome coronavirus infection induction of alternatively activated macrophages enhances pathogenesis during severe acute respiratory syndrome coronavirus infection genome wide identification of sars-cov susceptibility loci using the collaborative cross clinical features and virological analysis of a case of middle east respiratory syndrome coronavirus infection acute respiratory distress syndrome in critically ill patients with severe acute respiratory syndrome characterization of cytokine/chemokine profiles of severe acute respiratory syndrome expression profile of immune response genes in patients with severe acute respiratory syndrome sars: systematic review of treatment effects ribavirin and interferon therapy in patients infected with the middle east respiratory syndrome coronavirus: an observational study treatment with interferon-alpha2b and ribavirin improves outcome in mers-cov-infected rhesus macaques ribavirin and interferon alfa-2a for severe middle east respiratory syndrome coronavirus infection: a retrospective cohort study the use of corticosteroid as treatment in sars was associated with adverse outcomes: a retrospective cohort study high-dose pulse versus nonpulse corticosteroid regimens in severe acute respiratory syndrome corticosteroid treatment of severe acute respiratory syndrome in hong kong pegylated interferon-alpha protects type 1 pneumocytes against sars coronavirus infection in macaques coronaviruses-drug discovery and therapeutic options ifnlambda is a potent anti-influenza therapeutic without the inflammatory side effects of ifnalpha treatment ifn-lambda resolves inflammation via suppression of neutrophil infiltration and il-1beta production identification of oxidative stress and toll-like receptor 4 signaling as a key pathway of acute lung injury the tlr4 antagonist eritoran protects mice from lethal influenza infection endothelial cells are central orchestrators of cytokine amplification during influenza virus infection suppression of cytokine storm with a sphingosine analog provides protection against pathogenic influenza virus silencing of ccr2 in myocarditis therapeutic sirna silencing in inflammatory monocytes in mice immunomodulatory therapy for severe influenza the effect of inhibition of pp1 and tnfalpha signaling on pathogenesis of sars coronavirus acknowledgements we thank dr. anthony fehr for careful review of this manuscript. this work was supported in part by grants from the n.i.h. (po1 ai060699, ro1 ai091322). key: cord-349279-wbb7h2zu authors: walker, gregory j.; stelzer‐braid, sacha; shorter, caroline; honeywill, claire; wynn, matthew; willenborg, christiana; barnes, phillipa; kang, janice; pierse, nevil; crane, julian; howden‐chapman, philippa; rawlinson, william d. title: viruses associated with acute respiratory infection in a community‐based cohort of healthy new zealand children date: 2019-05-07 journal: j med virol doi: 10.1002/jmv.25493 sha: doc_id: 349279 cord_uid: wbb7h2zu acute respiratory infections (aris) are a major cause of morbidity among children. respiratory viruses are commonly detected in both symptomatic and asymptomatic periods. the rates of infection and community epidemiology of respiratory viruses in healthy children needs further definition to assist interpretation of molecular diagnostic assays in this population. children otherwise healthy aged 1 to 8 years were prospectively enrolled in the study during two consecutive winters, when aris peak in new zealand. parents completed a daily symptom diary for 8 weeks, during which time they collected a nasal swab from the child for each clinical ari episode. a further nasal swab was collected by research staff during a clinic visit at the conclusion of the study. all samples were tested for 15 respiratory viruses commonly causing ari using molecular multiplex polymerase chain reaction assays. there were 575 aris identified from 301 children completing the study, at a rate of 1.04 per child‐month. swabs collected during an ari were positive for a respiratory virus in 76.8% (307 of 400), compared with 37.3% (79 of 212) of swabs collected during asymptomatic periods. the most common viruses detected were human rhinovirus, coronavirus, parainfluenza viruses, influenzavirus, respiratory syncytial virus, and human metapneumovirus. all of these were significantly more likely to be detected during aris than asymptomatic periods. parent‐administered surveillance is a useful mechanism for understanding infectious disease in healthy children in the community. interpretation of molecular diagnostic assays for viruses must be informed by understanding of local rates of asymptomatic infection by such viruses. metapneumovirus (hmpv), human enterovirus (ev), and adenovirus (adv), are the most common viral aetiological agents associated with aris. 8 although most respiratory viral infections are mild, they can potentially cause life-threatening illness, particularly in high-risk groups such neonates, the elderly, and those with chronic respiratory disease. advances in molecular diagnosis have made it possible to easily and quickly identify viruses in people with aris, with quantitative polymerase chain reaction (pcr) now considered by most to be the reference standard for detection of respiratory viruses over traditional methods such as viral culture. 9 however, the clinical significance of virus detection is not always clear, as respiratory viruses are also detected in asymptomatic subjects. 10, 11 further understanding of the role of viral detection in aris may improve clinical decision-making around infection control and therapy. direct comparisons between studies of respiratory virus epidemiology can be difficult for a number of reasons. climatic and social factors affect the survival and transmission of respiratory viruses, as well as host mobility and susceptibility. 12, 13 this results in many viruses having characteristic seasonal patterns that vary by geographical location. reported epidemiology is also affected by study design. only one-fifth of aris result in a health care visit, 14 were recruited for the study. this was made up of 138 children from a previous study, 17 while the remaining 167 participants replied to advertising via staff email for the local district health boards, via posters and through referral from family and friends. there were 50 of 372 participants deemed ineligible for various reasons including that they slept in more than one bedroom, the bedroom was already heated, or they were going away for more than 2 weeks during the study period. before commencement of the study 17 of 372 participants withdrew, while 4 of 305 participants commenced the program but were later excluded after failing to comply with study conditions. the remaining cohort for analysis was 301 children, pcr, respectively, as previously described. 18 sequences were compared to the ncbi nonredundant nucleotide (nr/nt) database using the basic local alignment search tool (blast) on the ncbi website (https://blast.ncbi.nlm.nih.gov/blast.cgi) and genotype assigned according to the closest identifying sequence. details of primers used in detection of respiratory viruses are included in the supplementary material (table s1 ). any period in which participants had a multi-day cumulative symptom score ≥4, with a single day symptom score ≥2 was defined as an ari episode. the end of an ari episode was marked by three consecutive symptom-free days. a symptom score correction (daily baseline score of −1) was applied to participants who were considered to have background rhinitis. this included children who had (i) rhinitis symptoms (runny nose or sneezing) for ≥49 of the 56 study days, (ii) no more than 7 days with no symptoms, or (iii) 7 or more days of only rhinitis symptoms. in cases where multiple swabs were taken during a single ari episode, only the first swab taken was included in analysis. the exception to this was if a new virus was detected in subsequent swabs, or if swabs were taken >30 days apart. risk ratio (rr) was calculated as the proportion of samples positive for a virus with ari symptoms divided by the proportion of samples negative for a respiratory virus with ari symptoms. there were 16 856 child-days of symptom observed and a total of 612 nasal swabs collected from the 301 participants. there were 575 aris identified (table 1) . nasal swabs were received from 400 of 575 of these episodes (69.6%). in addition, 212 swabs were taken when children were asymptomatic, most of which were performed during the clinical visit by researchers at the conclusion of the study. due to inclusion of children from a previous study of wheezing, those with asthma were highly represented within our cohort (33.6%). overall, children with asthma displayed a small increase in the rate of aris per child-month (1.13 vs 1.00) compared with nonasthmatics (table s2 ), but this increase was not significant (p = 0.057). of the 400 swabs taken during an ari, 307 (76.8%) were positive for a virus. during an ari, detection of respiratory viruses in nasal swabs was highest amongst the youngest age group (1 year old), and decreased to age 4 ( table 1 ). during aris, multiple viruses were codetected in 37 (9.3%) swabs. the most commonly detected viruses in samples collected during ari were hrv (52.8%), hcov (11.0%), parainfluenza virus (pif) (6.0%), ifv (4.5%), rsv (3.8%), and hmpv (3.5%). these were all significantly more likely to be associated with ari episodes than asymptomatic detection (table 2 ). adv was detected with similar frequency in both ari (3.3%) and asymptomatic samples (3.8%), while detection of hbov was relatively low in both groups. detection of any virus and codetection of viruses were both significantly associated with swabs collected during ari episodes. sequencing was performed on 193 of a possible 284 picornavirus positive samples (68.0%). rhinovirus strains hrv-a and hrv-c were significantly associated with ari episodes (table 2) . overall, detection of individual viruses was similar between asthmatic and nonasthmatic children in swabs taken during an ari (table s3) . hrv-b (p = 0.042) and rsv (p = 0.034) were significantly over represented in asthmatics during aris. aris were most frequent in may (1.39), which was also the month in which the least data was collected. this was followed by june (1.22) august (1.21), and july (1.07). the rate of aris steadily decreased following the winter season ( figure 1 ). hrv was the predominant virus detected in each month, and was most prevalent in late-winter and spring (figure 1 ). hcov and rsv had peaks during early winter, while adv peaked in spring. ifv and hmpv were most prevalent in july, but were also detected in other months. pif was detected consistently throughout study months, although not in may. there is a need for improved population data regarding respiratory viruses in children, particularly in the southern hemisphere. this is the first study assessing the epidemiology of an extended range of respiratory viruses in a community cohort of healthy children in new zealand. infection with respiratory viruses was common in both symptomatic and asymptomatic children, and this study demonstrated these relatively high frequencies were true of children with asthma (1.12) and those without asthma (1.00). the proportion of aris associated with a virus-positive swab in this population declined with age. children 1 year of age were positive for a respiratory virus in 88.9% of samples taken during an ari, compared to 69.8% in participants aged greater than or equal to 5 years age. a similarly designed household surveillance study showed that weekly detections of viruses in young (aged <5) and older (aged 5-17) children were significantly more frequent than in adults. 20 in the current study swabs were taken at the onset of illness, when viral load reportedly peaks for some respiratory viruses. 21 it is possible that this method is more likely to detect viruses associated with ari than other studies obtaining weekly swabs. sample sizes of each virus detected here were too small to compare individual viruses between age groups. however, a multi-country community surveillance study has previously shown that with the exception of influenza, prevalence of individual respiratory viruses was highest in younger children. 3 the rates of ari per child-month were similar between age groups, suggesting that other factors such as development of allergy may play a more significant role than age in acute respiratory health. the study was conducted during the winter/spring seasons of 2 consecutive years. consistent with previous reports in the southern hemisphere rsv peaked in this pediatric population in early winter. 4, 22 interestingly, we found a similar pattern for detection of hcov. it has recently been reported in china that while overall hcov infection is most prevalent in winter, hcov subtypes display different epidemic patterns. 23 as only a small number of hcovs were detected (n = 50), we did not distinguish between subtypes when reporting our results. influenza was detected from june to october and peaked in july. a limitation was that no data were collected for the summer and autumn periods, capturing only the times of highest respiratory virus activity. this may account for the low detection of adv, hbov, and ev in comparison to other year-round epidemiological studies. 24 a yearround analyses may also allow for investigation of phenomena such as the february back to school hrv peaks. 25 our study differs from a previous nz-based analysis of viruses in hospitalized children with lrti, 22 where relatively high rates of rsv (39%) and hmpv (7%) were detected. in our community-based study, incidence of these viruses was considerably lower. previous groups have reported that rsv and hmpv rarely occur in asymptomatic subjects, 11, 26, 27 suggesting that detection of these viruses is almost always of clinical relevance. ifv may be present in people with few or no symptoms. however, the duration and copy number of viral shedding is considerably lower in these cases, 28 and further research is needed to understand the contribution of asymptomatic detection to community transmission. in the present study, we found these viruses in very few samples of asymptomatic subjects, and demonstrated these three viruses (and others) to be significantly associated with aris. respiratory viruses were detected in a large proportion of swabs taken during asymptomatic periods (37.3%), with the vast majority of these detections caused by hrv. this is unsurprising as hrvs were the most frequently detected virus overall, and have been commonly detected in asymptomatic children before. 11 it has previously been unclear whether subtypes of hrv have varying clinical impact on their hosts, with studies of healthy, 5 and asthmatic 18 children reporting no differences in symptom risk between hrv subtypes. our data, along with another recent study 19 suggest that hrv-a and hrv-c subtypes are significantly more likely to cause symptoms related to ari. in our study hrv-b, adv, and hbov were detected similarly in samples from both ari episodes and asymptomatic periods. while their corresponding risk ratios are not considered significant, the number of detections of these viruses is relatively small, and a larger analysis would be required to rule out the clinical significance of detecting hrv-b, adv and hbov in aris. the effect of viral coinfection on respiratory disease severity in children has not been well established. a number of studies have found increased hospitalizations, longer length of stay in hospital, and higher mortality when two or more respiratory viruses were detected. [29] [30] [31] however, others report no association between viral coinfection and clinical outcomes 32,33 including a systematic review of 43 such publications. 34 in this study we found a codetection was observed in 9.3% of total virus detections during aris, which is similar to rates reported previously. 4 we also codetected viruses in a small number of swabs collected during asymptomatic periods, and show that codetections are significantly more likely to be associated with aris. our sample size for codetection of viruses is relatively small, and larger studies have demonstrated a more significant association. 19 respiratory viruses cause significant morbidity, particularly for disability-adjusted life years (dalys) for 291 diseases and injuries in 21 regions, 1990-2010: a systematic analysis for the global burden of disease study global burden of respiratory infections due to seasonal influenza in young children: a systematic review and meta-analysis respiratory viruses and influenza-like illness: epidemiology and outcomes in children aged 6 months to 10 years in a multi-country population sample community epidemiology of human metapneumovirus, human coronavirus nl63, and other respiratory viruses in healthy preschool-aged children using parentcollected specimens community-wide, contemporaneous circulation of a broad spectrum of human rhinoviruses in healthy australian preschool-aged children during a 12-month period epidemiology of human respiratory viruses in children with acute respiratory tract infection in a 3-year hospital-based survey in northern italy viral bacterial coinfection of the respiratory tract during early childhood the role of infections and coinfections with newly identified and emerging respiratory viruses in children molecular diagnosis of respiratory viruses new molecular virus detection methods and their clinical value in lower respiratory tract infections in children respiratory viral detections during symptomatic and asymptomatic periods in young andean children environmental factors affecting the transmission of respiratory viruses impact of pollution, climate, and sociodemographic factors on spatiotemporal dynamics of seasonal respiratory viruses incidence and viral aetiologies of acute respiratory illnesses (aris) in the united states: a population-based study implementing hospital-based surveillance for severe acute respiratory infections caused by influenza and other respiratory pathogens in new zealand southern hemisphere influenza and vaccine effectiveness research and surveillance. influenza other respir viruses indoor visible mold and mold odor are associated with new-onset childhood wheeze in a dose-dependent manner rhinoviruses significantly affect day-to-day respiratory symptoms of children with asthma viruses causing lower respiratory symptoms in young children: findings from the orchid birth cohort community surveillance of respiratory viruses among families in the utah better identification of germs-longitudinal viral epidemiology (big-love) study viral shedding and clinical illness in naturally acquired influenza virus infections respiratory virus detection during hospitalisation for lower respiratory tract infection in children under 2 years in south auckland epidemiology characteristics of human coronaviruses in patients with respiratory infection symptoms and phylogenetic analysis of hcov-oc43 during 2010-2015 in guangzhou viral etiology of common cold in children absence of back to school peaks in human rhinovirus detections and respiratory symptoms in a cohort of children with asthma frequent detection of respiratory viruses without symptoms: toward defining clinically relevant cutoff values respiratory pathogens in children with and without respiratory symptoms viral shedding and transmission potential of asymptomatic and paucisymptomatic influenza virus infections in the community acute viral lower respiratory tract infections in cambodian children: clinical and epidemiologic characteristics the impact of dual viral infection in infants admitted to a pediatric intensive care unit associated with severe bronchiolitis high incidence of multiple viral infections identified in upper respiratory tract infected children under three years of age in concurrent detection of other respiratory viruses in children shedding viable human respiratory syncytial virus human rhinovirus and disease severity in children respiratory viral coinfection and disease severity in children: a systematic review and metaanalysis real-time reverse transcription-pcr assay for comprehensive detection of human rhinoviruses screening respiratory samples for detection of human rhinoviruses (hrvs) and enteroviruses: comprehensive vp4-vp2 typing reveals high incidence and genetic diversity of hrv species c detection of viral and bacterial respiratory pathogens in patients with cystic fibrosis viruses associated with acute respiratory infection in a community-based cohort of healthy new zealand children http://orcid.org/0000-0003-0044-1394 key: cord-333286-lr32e0w4 authors: lehtoranta, liisa; latvala, sinikka; lehtinen, markus j. title: role of probiotics in stimulating the immune system in viral respiratory tract infections: a narrative review date: 2020-10-16 journal: nutrients doi: 10.3390/nu12103163 sha: doc_id: 333286 cord_uid: lr32e0w4 viral respiratory tract infection (rti) is the most frequent cause of infectious illnesses including the common cold. pharmacological solutions for treating or preventing viral rtis are so far limited and thus several self-care products are available in the market. some dietary supplements such as probiotics have been shown to modulate immune system function and their role in reducing the risk and the course of rtis has been investigated extensively within the past decade. however, the mechanism of action and the efficacy of probiotics against viral rtis remains unclear. we searched pubmed, google scholar, and web of knowledge for pre-clinical and clinical studies investigating the effect of probiotics on respiratory virus infections, immune response, and the course of upper and lower respiratory tract illness. the literature summarized in this narrative review points out that specific probiotic strains seem effective in pre-clinical models, through stimulating the immune system and inhibiting viral replication. clinical studies indicate variable efficacy on upper respiratory illnesses and lack proof of diagnosed viral infections. however, meta-analyses of clinical studies indicate that probiotics could be beneficial in upper respiratory illnesses without specific etiology. further studies aiming at discovering the mechanisms of action of probiotics and clinical efficacy are warranted. respiratory viruses cause the most common infectious illnesses in humans-acute rtis that can be divided into upper rtis (urti), e.g., the common cold, and lower rtis (lrti), e.g., bronchitis and pneumonia. these illnesses affect all age groups annually and cause a high burden on health care systems and global economics due to absenteeism from daycares, school, and work. over 200 virus types have been identified as causative agents for respiratory illnesses [1, 2] . in most cases, especially illnesses of the upper respiratory tract are mild to moderate and often self-limiting. on the other hand, lrtis leading to pneumonia can be especially fatal among children and the elderly, or immunocompromised subjects [2] . in the past decade, studies linking the microbiota and immune system function have laid the foundation for the opportunities in microbiota modulation and bacterial therapeutics in health and disease. further, studies have associated the gut and airway microbiota with upper and lower respiratory tract health and immunity [3, 4] . thus, modulation of the gut microbiota and immunity by dietary supplements or pharmaceuticals is of increasing interest in finding novel solutions to manage rtis. among the promising candidates are probiotics that have been studied for immune function modulation and viral infections. meta-analyses suggest that probiotics could be beneficial in the context of acute urti typically caused by viruses [5, 6] . viruses that cause rtis are found in various virus families, which differ in virulence and utilize variable strategies to infect the host cells and to evade the host immune system [14, 15] . respiratory viruses spread via nasal secretions that can be transmitted through the air or by hand-to hand and surface-to-hand contact [16] . infection requires penetration of the virus through the host mucus layer, including the microbiota, and antiviral molecules in the mucus, such as antibodies and collectins. once on the mucosal epithelial cells, respiratory viruses attach to specific receptors, such as intercellular adhesion molecule (icam)-1 (rhinoviruses), peptidases (coronaviruses), or sialic acids (influenza viruses), that mediate the internalization of the virus by endocytosis [17, 18] . the viral receptors are differentially expressed on host cells resulting in virus-specific host cell tropism that is one key factor in viral pathogenesis. for example, influenza viruses typically infect bronchial cells, whereas rhinoviruses infect the epithelial cells of the upper airways, resulting in differences in illness presentation. viral genomic structure can be, for example, positive-or negative-sense single-stranded (ss) or double-stranded (ds) rna or dna [19] . most rti causing viruses, picornaviruses, influenza viruses, and coronaviruses are ssrna viruses, whereas adenoviruses are dsdna viruses. the genomic structures are recognized by different receptors in the host and activate different types of immune responses. some respiratory viruses, e.g., coronaviruses, are surrounded by a viral envelope which confers additional protection from the host immune system. respiratory viruses have further developed molecules that help in evading the immune response, for example, by disrupting the interferon (ifn) response and hijacking the host's cellular machinery for the production of virus copies [15] . once the viruses have penetrated into the host cells, the epithelial and immune cells detect the viral structures by pattern recognition receptors (prr) of which toll-like receptors (tlrs) and retinoic acid-inducible gene i (rig-i)-like receptors (rlrs) play a central role. tlr3, tlr7, tlr8, and tlr9 are located in the endosomes and can identify viral ss (tlr7 and 8) and ds (tlr3) rna structures, and dna (tlr9) [18] . the recognition by tlrs leads to activation of transcription factor nuclear factor kappa-light-chain-enhancer of activated b cells (nf-κb) and ifn regulatory factors (irf) 3, 5, and 7 [12, 20, 21] , resulting in expression of pro-inflammatory cytokines and type i ifns, ifn-α and ifn-β. type i ifns are broadly secreted by cells, but epithelial cells further secrete type iii lambda ifns in response to viral infections. cytoplasmic rnas, on the other hand, are recognized by rlrs, of which rig-i recognizes ssrna and melanoma differentiation associated 5 (mda-5) dsrna. the activation of rlrs leads to type i (and type iii in epithelial cells) ifn production via mitochondrial antiviral signaling protein (mavs). type i and iii ifns induce an antiviral state in the surrounding cells which is not, however, necessarily sufficient to resist the infection, but delays the spreading of the infection [12, 22] . the activation of rlrs, and tlrs by viral infection and cellular stress, leads to formation of nucleotide-binding oligomerization domain (nod)-, leucine-rich repeat (lrr)-, and pyrin domain-containing protein 3 (nlrp3) inflammasome [23] . although the role of nlrp3 is still somewhat unclear in viral rtis, it seems to play a role at least in rhinovirus, influenza, adenovirus, and rsv infections. nlrp3 inflammasome activation drives caspase 1-dependent il-1β and il-18 cytokine response and inflammatory programmed cell death (pyroptosis) [24] . epithelium-derived pro-inflammatory cytokines tumor necrosis factor (tnf)-α, interleukin (il)-1β, il-6, chemokine (c-c motif) ligand (ccl) 2, ccl5, chemokine (c-x-c motif) ligand (cxcl)8, and cxcl10 induce innate cellular responses by attracting and activating natural killer (nk) cells, macrophages, and neutrophils that further amplify the innate cytokine and chemokine response [17] . the role of other innate cells like, for example, intraepithelial lymphocytes, γδt cells, mucosa associated invariant t cells (mait), and innate lymphoid cells (ilc) is less well described in viral infections, however, they are likely to contribute to innate and adaptive responses against viral infections, as exemplified by the nk cells [25, 26] and by the role of ilc2 cells in overcoming an influenza infection [27] . if the innate immune or memory responses cannot clear the pathogen effectively and the adaptive immune system is unexperienced with the virus, an adaptive immune response is initiated and required. key are dendritic cells (dcs) that present the viral antigens and induce b and t cell responses against the pathogen in the secondary lymph nodes. b and t cell responses initiate within four-six days post-infection and peak later at days 7-14 depending on the respiratory virus [28] [29] [30] . typically, common respiratory viruses, such as rhinovirus and influenza virus, are cleared before adaptive immune responses are activated [22, 30] indicating that memory responses and innate immunity are essential in viral eradication. however, the induction of cytotoxic cd8 t cells, cd4 t cells, and antibody responses is key for virus eradication by adaptive immunity and for establishing protective immunity for secondary infections. the activation of the epithelium, innate immune cells, and adaptive responses is important for defense against respiratory viruses, but on the other hand, the host inflammatory response is the major cause of symptoms and more severe pathologies [12, 18, 31] . chronic activation of cd8 t cell responses and adaptive immunity may lead to pulmonary damage and acute respiratory distress syndrome, like in severe cases of coronavirus infections (e.g., sars-cov or sars cov-2) and pandemic influenza virus infections [18, 29, 32] . in milder colds, rhinoviruses are not cytolytic and do not actually cause considerable damage to host cells and may pass asymptomatically. presentation of cold symptom severity seems to correlate with host inflammatory response. specifically, the early expression of pro-inflammatory il-8 [33] and high levels of neutrophils in nasal aspirates [34] have been shown to correlate with symptom severity of rhinovirus and influenza infection [35, 36] . production of anti-inflammatory il-10, resolvins, and regulatory t cell responses acts as a natural mechanism to control lung inflammation during acute influenza virus (and others) infection [37] [38] [39] . virus-host immune interactions are key to viral pathogenesis and to ultimately determine the outcome of the infection. probiotics, by definition, are live microorganisms that, when administered in adequate amounts, confer a health benefit on the host [40] . most probiotics are lactic acid bacteria, belonging to lactobacillus spp., (now with new taxonomy including lacticaseibacillus spp., lactiplantibacillus spp., levilactobacillus spp., ligilactobacillus spp., limosilactibacillus spp. [41] ) or bifidobacterium spp. furthermore, some strains of other microbial genera, such as propionibacterium spp., and bacillus spp., have been reported to have probiotic properties. traditional lactic acid bacteria have long been considered safe and suitable for human consumption as very few instances of infection have been associated with these bacteria, and several published studies have specifically addressed their safety (reviewed, e.g., by [42] [43] [44] ). regarding probiotics safety in rtis, meta-analyses conclude that the reported side effects related to the consumption of probiotics were minor [5, 6, 45] . probiotics are mostly consumed orally in the form of dietary supplements and food (e.g., yoghurt). therefore, their primary site of action is in the gastrointestinal (gi) tract [46] . however, probiotics have been detected with pcr-based methods from nasopharyngeal mucosa, adenoids, and tonsils after oral consumption [13, 47, 48] , but it is unclear what the contribution to upper respiratory tract immune stimulation against viral rti by probiotics is, as oral ingestion results in the stimulation of the intestinal immune system as well. the small intestine, that is naturally exposed to microbes and nutrients due to the thin mucosal layer, seems to play an important role in immune stimulation by probiotics [49, 50] . however, dissecting the relative contributions of the small and large intestine and upper gi tract on immune stimulation against rti is challenging with available research data. independent of the actual mucosal inductor site of the probiotic, it has been shown that lymphocytes circulate between mucosal tissues [51] . thus, local mucosal stimulatory effects may influence immune responses at other mucosal tissues and contribute to antiviral immunity. even very closely related bacteria have differences in their antigenic structures and thus influence the immune system uniquely. probiotics are thought to influence immune function primarily in a strain-specific manner [40] . although these effects in general are strain-specific, probiotics also share common mechanisms of immune stimulation, such as the secretion of metabolites. for instance, short chain fatty acids are known to have immunomodulatory effects [52] . direct effects of the probiotics on immune function are driven by interactions of bacterial structures or metabolites with receptors, like tlrs, on the host epithelial and immune cells. on the other hand, probiotics may influence immune function indirectly by changing the composition and/or activity of the host microbiota [53] . for example, in the small intestine where the number of endogenous bacteria is lower than in the large intestine, ingestion of probiotics temporally changes the microbiota composition and influences the host immune response [49, 50] . in rtis, orally consumed probiotics may elicit systemic effects from the gi tract via the "gut-lung axis" by modulating mucosal immune function [54, 55] . probiotics are taken up by m cells or by cx3c chemokine receptor 1 (cx3cr1)+ macrophages located in the gut epithelium and then transferred to dcs in the subepithelial tissue. probiotics are able to modulate dc polarization and function [56] that influence the subsequent t and b cell responses [57] in the inductive sites (peyer's patch, and mesenteric lymph nodes). t and b cells can also enter the circulation and migrate to extraintestinal sites, such as the respiratory tract [51, 58] . however, the exact mechanisms of probiotics (and their metabolites) in respiratory infections has not been clearly established and may be influenced by the investigated probiotic strain, microbiota composition, and immunological status of an individual. in the following chapters, we review the current pre-clinical evidence of immunomodulatory and antiviral mechanisms of probiotics against respiratory virus infections with a focus on the direct stimulatory effects of probiotics on virus-infected immune cells and animal models. probiotic bacteria can engage and activate tlrs leading to the activation of nf-κb and irfs in immune cells that are essential in antiviral defense. for example, it has been demonstrated in murine bone marrow-derived dcs that lactobacillus acidophilus ncfm and l. acidophilus x37 induce the upregulation of tlr3, il-12, and ifn-β in a tlr2-dependent manner [59] . in macrophage-derived raw264.1 cells, lactobacillus gasseri sbt2055 upregulated ifn-β and myxovirus resistance (mx)1 mrna expression [60] and in human monocyte-derived macrophages, two lacticaseibacillus rhamnosus strains, gg and lc705, induced type i ifn-dependent gene activation [61] . pro-inflammatory and ifn-regulated genes including il-6, il-12, il-1β, il-8, ccl20, cxcl10, mx1, and mx2 were induced by both lacticaseibacillus strains. in addition, the gene expression of tlr3 and tlr7, receptors recognizing viral dsrna and ssrna, respectively, was upregulated by these strains. tlr3 gene expression was upregulated only by l. rhamnosus lc705, the strain with higher antiviral potential, while tlr7 was moderately upregulated by both strains. in vitro studies with immune cells have also shown the ability of probiotics and their components to restrict viral replication. in human monocyte-derived macrophages, lacticaseibacillus strains showed the ability to prevent influenza a virus replication which correlated with the ability to activate type i ifn-dependent antiviral genes [61] . similarly, mouse adapted influenza a virus (pr8) titer was reduced in raw264.1 cells by l. gasseri sbt2055 [60] . in mouse bone marrow dcs, the inhibition of viral replication by l. acidophilus atcc4356 s-layer protein was demonstrated [62, 63] . priming of cells with s-layer protein prior to h9n2 avian influenza virus infection inhibited the invasion and replication of the virus, stimulated the type i ifn signaling pathway, increased il-10 mrna, and decreased tnf-α mrna expression [62] . polyinosinic/polycytidylic acid (poly i/c), a synthetic mimic of dsrna, is widely used in in vitro studies to stimulate tlr3. it induces characteristic inflammatory responses associated with virus infections such as increased production of inflammatory cytokines. stimulation of airway epithelial cells with poly i/c has been found to closely mimic inflammatory responses associated with respiratory virus infections [64] . poly i/c challenge in peripheral blood mononuclear cells (pbmcs) induced changes in the gene expression of tlr3, ifn, and nf-kb-dependent pathways similar to acute viral infections [63] . moreover, poly i/c was able to induce a pulmonary dysfunction similar to rsv in a mouse model [65] . probiotic bacteria have shown the ability to modulate poly i/c-induced responses. studies with heat-killed lacticaseibacillus casei crl431 showed that the strain reduced tnf-α, ifn-γ, and il-17 levels when it was introduced before or simultaneously with poly i/c challenge in pbmcs alone and in a co-culture system with alveolar epithelial cell line a549 [66] . il-10 and il-29 (also known as ifn-λ1) were induced in response to poly i/c together with heat-killed l. casei crl431, indicating a boost in pro-inflammatory responses and the activation of anti-inflammatory and antiviral mechanisms. in the intestinal human colon cell line (hct116), regulation of poly i/c response by lactiplantibacillus plantarum subsp. plantarum du1, latilactobacillus sakei du2, and weissella cibaria du1 was examined [67] . these strains modified poly i/c-induced expression of cytokines and antiviral genes by upregulating ifn-β, tlr3, and rig-i while dampening the inflammatory response. moreover, the probiotic strains induced ifn-α, ifn-β, and il-10 and reduced the expression of inflammatory cytokines il-1β and tnf-α in human monocytic thp-1 macrophages [67] . in addition to the pro-inflammatory and antiviral gene activation described above, also direct interactions of viruses and probiotic bacteria have been demonstrated between porcine influenza a virus and enterococcus faecium in vitro [68] . similar upregulation of ifn response by probiotics has been shown in several studies in intestinal epithelial cells [69] [70] [71] and macrophages [68, 72] . overall, the in vitro studies indicate that probiotics may stimulate similar innate immune pathways to respiratory viruses and potentially modulate virus-induced immune responses. several mouse studies have shown that the administration of probiotics can help to fight against viral rtis. the beneficial effects of oral probiotic supplementation on mouse survival and health status is well demonstrated [60, [73] [74] [75] [76] [77] . for example, oral administration of lacticaseibacillus paracasei subsp. paracasei cncm-i-1518 [74] , l. gasseri lg2055 [60] , and bifidobacterium longum mm-2 [75] reduced mortality and improved immune control in influenza-infected mice. probiotic administration resulted in better health status of the mice and lower virus loads in the lungs after influenza infection. in addition, the immune response to viruses was modulated by probiotic administration. for instance, l. paracasei cncm-i-1518 modified the pro-and anti-inflammatory cytokine release in the lungs before and after influenza infection and affected total cell counts in the lungs after probiotic treatment [74] . similarly, b. longum mm-2 suppressed inflammation in the lower respiratory tract through the decrease in influenza virus proliferation and pulmonary il-6 and tnf-α cytokine production [75] . activation of host defense systems by increased ifn-γ, il-2, il-12, and il-18 gene expression and nk cell activation in lungs was also demonstrated. in non-infected mice, b. longum mm-2 significantly enhanced ifn-γ production by peyer's patch cells and splenic nk cell activity. in the infected mice, nk cell activity was significantly enhanced both in the spleen and lungs by the probiotic strain. another bifidobacterium (b. bifidum) improved anti-influenza immune responses by inducing both humoral and cellular immunities [76] . decreased il-6 levels were detected in the lung and higher igg1 and igg2 levels in the sera of probiotic-treated mice compared with control mice. furthermore, l. gasseri sbt2055 has been found effective in preventing both influenza a virus [60] and rsv [78] infections in mice. pre-treatment with l. gasseri sbt2055 induced the expression of antiviral genes mx1 and 2 -5 oligoadenylate synthase (oas)1a in lung tissues before viral infection and reduced lung inflammatory responses after viral infection [60] . the same l. gasseri strain was found effective in preventing rsv infection in mice by reducing lung viral loads and pro-inflammatory cytokines and by stimulating ifns and ifn responsive gene expression such as ifn-β1, ifn-γ, interferon-inducible transmembrane protein (ifitm)3, oas1a, and interferon-stimulated gene (isg)15 [78] . in addition to live probiotics, also orally administered heat-killed bacteria seem to confer protection against viral rtis in mice [73] . heat-killed l. paracasei mcc1849 reduced symptom scores and lung virus titers in influenza-infected mice and induced antigen-specific iga production in the small intestine, serum, and lungs. the proportion of iga+ b cells and follicular helper t cells (tfh) in peyer's patches was increased as was the gene expression of il-12p40, il-10, il-21, signal transducer and activator of transcription (stat)4, and b cell lymphoma protein (bcl)-6, which are associated with tfh cell differentiation. to conclude, different probiotic strains have been shown effective in inhibiting the replication of various respiratory viruses including influenza viruses and rsv in vitro. similar effects have been demonstrated in several mouse studies with the ability to reduce virus titers in lung tissues and modulation of antiviral and pro-inflammatory gene expression before and after viral infection. accumulating clinical evidence suggests that probiotics in general may have favorable effects against rtis. for instance, several systematic reviews and/or meta-analyses have evaluated the effects of prophylactic ingestion of probiotics on the rti-associated outcomes, e.g., either only in children [79, 80] , or both in children and adults [5, 6, 45] (table 1) . of note, the majority of the outcomes in these analyses are related to urti, and data on lrti outcomes are either not available or are very limited. therefore, in the below chapter, we primarily focus on clinical trials on probiotics' effects on urti symptoms/episodes/duration. probiotic consumption had no effect on the duration of rtis (9 rcts, n = 3529, md -0. 81 in children (below 18 years), the meta-analysis by wang et al., 2016 , reported that probiotic use compared with placebo significantly decreased the number of subjects having at least one rti episode, had fewer numbers of days of rtis per person, and had fewer numbers of days absent from daycare or school [80] . however, the meta-analysis did not find a statistically significant difference on the illness episode duration between the probiotic and the placebo. laursen and hojsak [79] limited the analysis to children up to 7 years old and reported that probiotic use was associated with reduced risk of at least one urti and reduced the risk of antibiotic use, but the use was not associated with a reduction in rti duration or missed days of daycare due to rti [79] . this meta-analysis also discussed the effects of the individual probiotic strains on rti outcomes. the results of the analysis showed that the most effective probiotic strains on rti-related outcomes were l. rhamnosus gg (rti duration) and l. acidophilus ncfm as a single supplement and in combination with b. lactis bi-07 (rti duration and antibiotic use). interestingly, these strains have shown in vitro the ability to induce antiviral ifn signaling pathways (see section 4.2) which may potentially explain their beneficial effects observed in rtis. however, as multiple studies with probiotic strains other than l. rhamnosus gg are limited or lacking, comparison and interpretation of the strain specific results should be made carefully. meta-analyses that pool data from clinical trials conducted with children, adults, and the elderly show that probiotic use is more beneficial over placebo in reducing the number of participants experiencing episodes of acute urti [5, 6] , reducing antibiotic prescription rates for acute urtis [5, 6] , and reducing the mean duration of an episode of an acute urti as well as cold-related school absences [5, 45] . when the literature search was conducted, meta-analyses were not found in the databases searched on probiotic effects on respiratory infections restricted to the elderly population, potentially due to the fact that data are fairly limited regarding this age group. while there is consensus that probiotics could have potential in reducing the risk for rtis, it should be noted that clinical trials in the meta-analyses have been conducted in populations of different ages and genetic backgrounds, with various strains and/or their combinations, supplementation matrices, and doses. moreover, the measured outcomes and data collection procedures between the trials (i.e., infection episode definition) are not harmonized and therefore may vary considerably. consequently, pooling all the data creates a bias, as the probiotic effect is generally dependent on the dose, population, and strain. moreover, as discussed above, the probiotics effects on the immune system are strain-specific which affects the interpretation of the results. with regard to probiotics effects to specific respiratory viruses in clinical settings, several trials have characterized the respiratory infection etiology in infants [81] , in children [82] [83] [84] , in adults [85] , and in the elderly [86] . in addition, two clinical trials have investigated the efficacy of probiotics in an experimental rhinovirus challenge model [87] [88] [89] (table 2) . children had less days with respiratory symptoms per month (6.5 vs. 7.2, p < 0.001). no effect on the occurrence of respiratory viruses during the study or respiratory symptoms associated with viral findings. l. rhamnosus gg 10 9 cfu of live or heat-inactivated (by spray-drying) in 100 ml of fruit juice or control juice daily for 6 weeks. in the clinical trials conducted in free-living subjects in the community, no consistent data exist that show that specific probiotics would reduce the incidence of laboratory-confirmed respiratory virus infections as such. in preterm infants, the use of l. rhamnosus gg for 60 days was associated with lower incidence of rhinovirus-induced episodes (comprising 80% of all rti episodes) compared with the placebo. however, l. rhamnosus gg had no effect on rhinovirus rna load during infections, duration of rhinovirus rna shedding, duration or severity of rhinovirus infection, or the occurrence of rhinovirus rna in asymptomatic infants. in children attending daycare, l. rhamnosus gg [83] consumption for 28 weeks did not reduce the occurrence of any of the common respiratory viruses either. in otitis-prone children, supplementation of a combination of l. rhamnosus gg, l. rhamnosus lc705, b. breve 99, and propionibacterium jensenii js, for six months, reduced the number of human bocavirus-positive nasopharyngeal samples when compared with placebo, but not the number of rhino/enterovirus-positive samples [84] . furthermore, in schoolchildren, the consumption of levilactobacillus brevis kb290 during influenza season was associated with lower incidence of physician-diagnosed influenza virus cases [82] . in adults attending military service, the use of a combination of l. rhamnosus gg and b. lactis bb-12 for either 90 or 150 days was not overall associated with lower occurrence of common respiratory viruses upon presentation of cold symptoms [85] . however, in a subgroup, there was a lower occurrence of rhino/enteroviruses after three months in the probiotic group when compared with the placebo. in nursing home residents, wang et al., 2018 , reported that the use of l. rhamnosus gg for six months was not associated with the reduction in occurrence of confirmed viral respiratory infections [86] . the differences between the findings in these trials may be explained by the fact that these studies were conducted in various age groups with different immune system statuses (infants vs. children vs. healthy adults vs. the elderly), different seasons, as well as variable probiotic strains, strain combinations, doses, and variable lengths of intervention. furthermore, most of the studies were not designed for analyzing the viral infection etiology as the primary outcome and the diagnosis for the identification of the viral agent was not applied. since over 200 respiratory virus types can cause respiratory infections and, in many cases, the infections and symptoms overlap, or the etiology is undiagnosed, the potential antiviral effects of probiotics against specific viruses can be difficult to determine in clinical trials targeting free-living subjects within the community. to overcome this caveat, two probiotics have been investigated in an experimental rhinovirus challenge model that allows investigation of the effect of a probiotic strain to a specific viral pathogen. in a rhinovirus (type 39) challenge model, b. lactis bl-04 was administered for 28 days prior and during five days of experimental rhinovirus infection to healthy volunteers [89] . b. lactis bl-04 supplementation resulted in significantly lower rhinovirus titers in nasal washes during the infection as well as in a lower number of infected participants shedding the virus compared with the placebo. moreover, b. lactis bl-04 induced a significantly higher concentration of il-8 in nasal washes after 28 days of supplementation and prior to infection. given the reduced viral titer, an increase in il-8 could indicate priming of the mucosal immune system prior to infection. this hypothesis is in line with a clinical study conducted in healthy active adults, where supplementation of b. lactis bl-04 reduced the risk of urti episodes compared with placebo [90] . in another similar experimental rhinovirus type 39 challenge pilot trial, no significant antiviral effect was seen with live or inactivated l. rhamnosus gg supplementation compared with placebo [87, 88] , suggesting potential strain-specific differences on the efficacy of probiotics in respiratory virus infections. nevertheless, further adequately powered trials with harmonized study designs are necessary to draw conclusions on the efficacy of probiotics against specific respiratory viruses. viral rtis are the most common infections of mankind and the health and financial impact of seasonal epidemics and global pandemics on society is high. due to the large number of various respiratory viruses, the development of efficient therapies, such as vaccines, is challenging. when preventative measures are scarce or lacking, the role of a well-functioning immune system becomes crucial for providing resistance to an infection. within the past decade, research highlighting the importance of the microbiota on immune system function has raised interest in understanding the role of microbiota modulation and bacterial therapeutics by dietary and pharmaceutical solutions in health and disease. of the available solutions, probiotic bacteria have been studied for immune function modulation in the context of respiratory viral infections. in this review, we have summarized the current evidence on the effects of probiotics on antiviral immune function in vitro and in vivo, and clinical evidence on the effect of probiotics on viral rtis and on the course of rti (figure 1 ). importance of the microbiota on immune system function has raised interest in understanding the role of microbiota modulation and bacterial therapeutics by dietary and pharmaceutical solutions in health and disease. of the available solutions, probiotic bacteria have been studied for immune function modulation in the context of respiratory viral infections. in this review, we have summarized the current evidence on the effects of probiotics on antiviral immune function in vitro and in vivo, and clinical evidence on the effect of probiotics on viral rtis and on the course of rti (figure 1 ). in vitro data indicate that probiotics have strain-specific immunomodulatory effects on the host and immune cells by engaging tlrs that stimulate ifn pathways. the upregulation of ifn response seems to prime cells for better resistance against virus infection as probiotics were shown effective in inhibiting the replication of various respiratory viruses, including influenza viruses and rsv. similar effects have been demonstrated in mice with the ability of the probiotics to reduce virus titers in lung tissues and to modulate antiviral and pro-inflammatory gene expression before and after viral infection. interestingly, some studies in mice show an increase in il-10 response, suggesting control of the pro-inflammatory response that typically drives lung pathology in severe infections. most likely probiotics' effects in the gut are transferred into the respiratory tract via the gut-respiratory tract axis, however, this mechanism of action remains to be studied in more detail. the pre-clinical studies further show improvement in the symptom scores of mice, suggesting potential clinical benefits. indeed, some evidence exists for specific probiotic strains, e.g., from the species of l. rhamnosus, l. acidophilus, and b. lactis for their ability to induce antiviral immune responses in preclinical models, which is in agreement with their effects observed in clinical trials in reducing the risk of rti-associated outcomes. however, translation of probiotic effects from cell culture and animal studies to humans can be challenging and variable confounding factors, e.g., age, diet, microbiome, genetic and epigenetic immune status of an individual, study season, and variable viral epidemiology, all have an impact on the study outcome and are difficult to standardize. the clinical studies that have diagnosed and characterized viral etiology are limited, nevertheless, the metaanalyses investigating probiotic clinical interventions on rtis show that probiotic use is associated with lower incidence and duration of mild rtis, both in children and in adults. further studies aiming at discovering the mechanism of action of probiotics and establishing the association of immune system function stimulation and clinical efficacy are warranted. in vitro data indicate that probiotics have strain-specific immunomodulatory effects on the host and immune cells by engaging tlrs that stimulate ifn pathways. the upregulation of ifn response seems to prime cells for better resistance against virus infection as probiotics were shown effective in inhibiting the replication of various respiratory viruses, including influenza viruses and rsv. similar effects have been demonstrated in mice with the ability of the probiotics to reduce virus titers in lung tissues and to modulate antiviral and pro-inflammatory gene expression before and after viral infection. interestingly, some studies in mice show an increase in il-10 response, suggesting control of the pro-inflammatory response that typically drives lung pathology in severe infections. most likely probiotics' effects in the gut are transferred into the respiratory tract via the gut-respiratory tract axis, however, this mechanism of action remains to be studied in more detail. the pre-clinical studies further show improvement in the symptom scores of mice, suggesting potential clinical benefits. indeed, some evidence exists for specific probiotic strains, e.g., from the species of l. rhamnosus, l. acidophilus, and b. lactis for their ability to induce antiviral immune responses in pre-clinical models, which is in agreement with their effects observed in clinical trials in reducing the risk of rti-associated outcomes. however, translation of probiotic effects from cell culture and animal studies to humans can be challenging and variable confounding factors, e.g., age, diet, microbiome, genetic and epigenetic immune status of an individual, study season, and variable viral epidemiology, all have an impact on the study outcome and are difficult to standardize. the clinical studies that have diagnosed and characterized viral etiology are limited, nevertheless, the meta-analyses investigating probiotic clinical interventions on rtis show that probiotic use is associated with lower incidence and duration of mild rtis, both in children and in adults. further studies aiming at discovering the mechanism of action of probiotics and establishing the association of immune system function stimulation and clinical efficacy are warranted. the common cold epidemiology of viral pneumonia. clin microbiome and disease in the upper airway the influence of the microbiome on respiratory health probiotics for preventing acute upper respiratory tract infections probiotics for preventing acute upper respiratory tract infections respiratory virus infections emerging respiratory viruses other than influenza sars-cov-2 viral load in upper respiratory specimens of infected patients epidemiology of viral respiratory infections epidemiologic, clinical, and virologic characteristics of human rhinovirus infection among otherwise healthy children and adults: rhinovirus among adults and children pathogenesis of rhinovirus infection nasal microbiota clusters associate with inflammatory response, viral load, and symptom severity in experimental rhinovirus challenge mechanisms of viral mutation viral manipulation of the host immune response transmission routes of respiratory viruses among humans epithelial cells and airway diseases the host immune response in respiratory virus infection: balancing virus clearance and immunopathology the morphology of virus particles. classification of viruses immune responses to influenza virus infection sars coronavirus pathogenesis: host innate immune responses and viral antagonism of interferon pathogenesis of influenza virus infections: the good, the bad and the ugly inflammasome control of viral infection the nlrp3 inflammasome: molecular activation and regulation to therapeutics cytokine networks between innate lymphoid cells and myeloid cells early local immune defences in the respiratory tract group 2 innate lymphoid cell production of il-5 is regulated by nkt cells during influenza virus infection protective and harmful immunity to rsv infection the cd8 t cell response to respiratory virus infections host defense function of the airway epithelium in health and disease: clinical background immunopathology in influenza virus infection: uncoupling the friend from foe the trinity of covid-19: immunity, inflammation and intervention relationship of upper and lower airway cytokines to outcome of experimental rhinovirus infection role of nasal interleukin-8 in neutrophil recruitment and activation in children with virus-induced asthma elevated levels of interleukins il-1 beta, il-6 and il-8 in naturally acquired viral rhinitis symptom pathogenesis during acute influenza: interleukin-6 and other cytokine responses a distinct function of regulatory t cells in tissue protection effector t cells control lung inflammation during acute influenza virus infection by producing il-10 the role of pro-resolution lipid mediators in infectious disease expert consensus document. the international scientific association for probiotics and prebiotics consensus statement on the scope and appropriate use of the term probiotic a taxonomic note on the genus lactobacillus: description of 23 novel genera, emended description of the genus lactobacillus beijerinck 1901, and union of lactobacillaceae and leuconostocaceae absence of adverse events in healthy individuals using probiotics-analysis of six randomised studies by one study group a systematic review of the safety of probiotics safety assessment of probiotics for human use effectiveness of probiotics on the duration of illness in healthy children and adults who develop common acute respiratory infectious conditions: a systematic review and meta-analysis understanding immunomodulatory effects of probiotics recovery of probiotic lactobacillus rhamnosus gg in tonsil tissue after oral administration: randomised, placebo-controlled, double-blind clinical trial lactobacillus rhamnosus gg in adenoid tissue: double-blind, placebo-controlled, randomized clinical trial identification of the transcriptional response of human intestinal mucosa to lactobacillus plantarum wcfs1 in vivo regulation of intestinal homeostasis and immunity with probiotic lactobacilli mucosal immunity: induction, dissemination, and effector functions short chain fatty acids (scfas)-mediated gut epithelial and immune regulation and its relevance for probiotic mechanisms of action the gut-lung axis in health and respiratory diseases: a place for inter-organ and inter-kingdom crosstalks microbiota and gut-lung connection lactobacilli differentially modulate expression of cytokines and maturation surface markers in murine dendritic cells human antigen-presenting cells respond differently to gut-derived probiotic bacteria but mediate similar strain-dependent nk and t cell activation location, location, location: the impact of migratory heterogeneity on t cell function lactobacillus acidophilus induces virus immune defence genes in murine dendritic cells by a toll-like receptor-2-dependent mechanism oral administration of lactobacillus gasseri sbt2055 is effective for preventing influenza in mice nonpathogenic lactobacillus rhamnosus activates the inflammasome and antiviral responses in human macrophages inhibition of h9n2 virus invasion into dendritic cells by the s-layer protein from l. acidophilus atcc 4356 a pathway analysis of poly(i:c)-induced global gene expression change in human peripheral blood mononuclear cells the airway epithelium: soldier in the fight against respiratory viruses double-stranded rna induces similar pulmonary dysfunction to respiratory syncytial virus in balb/c mice non-viable lactobacillus casei beneficially modulates poly i: c immune response in co-cultures of human cells immunobiotic strains modulate toll-like receptor 3 agonist induced innate antiviral immune response in human intestinal epithelial cells by modulating ifn regulatory factor 3 and nf-κb signaling lactobacillus casei zhang modulate cytokine and toll-like receptor expression and beneficially regulate poly i: c-induced immune responses in raw264. 7 macrophages immunobiotic lactic acid bacteria beneficially regulate immune response triggered by poly (i: c) in porcine intestinal epithelial cells immunobiotic bifidobacteria strains modulate rotavirus immune response in porcine intestinal epitheliocytes via pattern recognition receptor signaling exopolysaccharides from lactobacillus delbrueckii oll1073r-1 modulate innate antiviral immune response in porcine intestinal epithelial cells immunobiotic lactobacillus rhamnosus strains differentially modulate antiviral immune response in porcine intestinal epithelial and antigen presenting cells orally administered heat-killed lactobacillus paracasei mcc1849 enhances antigen-specific iga secretion and induces follicular helper t cells in mice lactobacillus paracasei feeding improves immune control of influenza infection in mice consecutive oral administration of bifidobacterium longum mm-2 improves the defense system against influenza virus infection by enhancing natural killer cell activity in a murine model immunomodulatory and prophylactic effects of bifidobacterium bifidum probiotic strain on influenza infection in mice oral intake of lactobacillus rhamnosus m21 enhances the survival rate of mice lethally infected with influenza virus prevention of respiratory syncytial virus infection with probiotic lactic acid bacterium lactobacillus gasseri sbt2055 probiotics for respiratory tract infections in children attending day care centers-a systematic review probiotics for prevention and treatment of respiratory tract infections in children: a systematic review and meta-analysis of randomized controlled trials prebiotic and probiotic supplementation prevents rhinovirus infections in preterm infants: a randomized, placebo-controlled trial effects of probiotic lactobacillus brevis kb290 on incidence of influenza infection among schoolchildren: an open-label pilot study the use of the probiotic lactobacillus rhamnosus gg and viral findings in the nasopharynx of children attending day care human bocavirus in the nasopharynx of otitis-prone children specific probiotics and virological findings in symptomatic conscripts attending military service in finland probiotics to prevent respiratory infections in nursing homes: a pilot randomized controlled trial effect of live and inactivated lactobacillus rhamnosus gg on experimentally induced rhinovirus colds: randomised, double blind, placebo-controlled pilot trial human rhinovirus in experimental infection after peroral lactobacillus rhamnosus gg consumption, a pilot study effect of probiotic on innate inflammatory response and viral shedding in experimental rhinovirus infection-a randomized controlled trial probiotic supplementation for respiratory and gastrointestinal illness symptoms in healthy physically active individuals key: cord-324432-k0g3r1lw authors: maykowski, philip; smithgall, marie; zachariah, philip; oberhardt, matthew; vargas, celibell; reed, carrie; demmer, ryan t.; stockwell, melissa s.; saiman, lisa title: seasonality and clinical impact of human parainfluenza viruses date: 2018-08-29 journal: influenza other respir viruses doi: 10.1111/irv.12597 sha: doc_id: 324432 cord_uid: k0g3r1lw background: widespread availability of rapid diagnostic testing for respiratory viruses allows more in‐depth studies of human parainfluenza viruses (hpiv). objectives: this study aimed to assess seasonality of hpiv types 1‐4, clinical outcomes by hpiv type, and risk factors for illness severity. patients/methods: this retrospective study was performed from january 2013 to december 2015 in children and adults with hpiv, detected by multiplex reverse transcription polymerase chain reaction, participating in a community surveillance study of acute respiratory infections (aris) in new york city and patients admitted to a tertiary care center in the same neighborhood. seasonality trends by hpiv type were compared between the community and hospital groups. the associations between hpiv type, demographics, clinical characteristics, and illness severity were assessed. results: hpiv was detected in 69 (4%) of 1753 community surveillance participants (median age 9.2 years) and 680 hospitalized patients (median age 6.8 years). seasonality for hpiv types 1‐3 agreed with previously described patterns; hpiv‐4 occurred annually in late summer and fall. in the community cohort, 22 (32%) participants sought medical care, 9 (13%) reported antibiotic use, and 20 (29%) reported ≥1 day of missed work or school. among hospitalized patients, 24% had ≥4 chronic conditions. multivariable ordinal logistic regression demonstrated that increased severity of illness was significantly associated with hpiv‐4 and chronic cardiovascular and respiratory conditions in children and with age ≥65 years and chronic respiratory conditions in adults. conclusions: hpiv‐4 presented late summer and early fall annually and was associated with increased severity of illness in hospitalized children. human parainfluenza viruses (hpiv) types 1-4 are common respiratory pathogens that cause both upper and lower respiratory tract illnesses, especially in young children. 1 hpiv-1 and hpiv-2 are more common in children and more often associated with croup. 1 hpiv-3 is more frequently associated with bronchiolitis, bronchitis, and pneumonia. hpiv-4, while least well-characterized, has been described as associated with both mild illness in children and lower respiratory tract disease. 2 hpiv types have varying seasonality, prevalence, and clinical manifestations. in the united states, hpiv-1 usually occurs in the fall of odd numbered years, hpiv-2 occurs every fall, and hpiv-3 occurs in spring and summer. 1, 3 the seasonality of hpiv-4 has not been as well defined, often due to its low prevalence. 4 a recent study conducted in colorado found a year-round prevalence of hpiv-4 with peaks in the fall of odd numbered years. 5 however, estimates of hpiv-4 prevalence are increasing, potentially due to improved and increased diagnostic testing, but few recent studies have assessed this. 6, 7 widespread availability of multiplex reverse transcription polymerase chain reaction (rt-pcr) assays allows for more in-depth studies of the epidemiology and impact of hpiv types. the population of this study included both a community-based cohort and hospitalized children and adults with laboratory-detected hpiv. the inclusion of the community cohort allowed for a broader evaluation of the impact of hpiv than has been previously assessed. the study's objectives were to (a) assess the seasonality of hpiv types 1-4, (b) determine clinical outcomes by hpiv type, and (c) identify risk factors for increased severity of illness. a retrospective study of participants in a community surveillance cohort and hospitalized patients with hpiv types 1-4 detected from january 1, 2013 to december 31, 2015 was performed. the community cohort was derived from the mobile surveillance for acute respiratory infections (aris) and influenza-like illness (ili) in the community (mosaic) study, a 5-year community-based surveillance ordinal logistic regression demonstrated that increased severity of illness was significantly associated with hpiv-4 and chronic cardiovascular and respiratory conditions in children and with age ≥65 years and chronic respiratory conditions in adults. conclusions: hpiv-4 presented late summer and early fall annually and was associated with increased severity of illness in hospitalized children. epidemiology, parainfluenza, respiratory, seasonality, viruses f i g u r e 1 flowcharts depicting the overall number of respiratory viral panel (rvp) tests ordered which yielded the final number of human parainfluenza virus (hpiv) types in the community cohort (1a) and in hospitalized patients (1b) study in new york city (nyc) that includes 250 households annually. 8, 9 households were identified by contacting a random sample of participants who had taken part in a large population-based survey of an urban, primarily immigrant latino community. for this study, eligible households had 3 or more members with at least 1 member under 18 years of age, were spanish-or english-speaking, and had a cellular telephone with text messaging. 8 text messages were sent twice weekly inquiring about possible respiratory illness among household members. nasal swabs were collected from ill participants by research staff if at least two of the following were reported to be present: fever/feverishness, runny nose/congestion, sore throat, cough, and/or myalgia. for infants, either runny nose or congestion prompted collection of a swab. hospitalized adults and children with laboratory-detected hpiv admitted to a university-affiliated medical center in nyc located in the same neighborhood as the community cohort were included. testing was requested by treating clinicians, and as per the standard of care, is generally recommended for all hospitalizations to evaluate acute respiratory symptoms and guide transmission precautions. patients hospitalized within 2 calendar-days of hpiv detection were included. those with hpiv detected >2 days before admission, >2 days after admission and those with a second positive test for the same hpiv type within 4 weeks of the first positive test were excluded. nasopharyngeal swabs for both the community cohort and the hospitalized patients were tested for respiratory pathogens using multiplex rt-pcr (biofire diagnostics, inc. salt lake city, utah) which has been reported to have 91%-100% sensitivity and 98%-100% 10, 11 swabs from the community were tested in a research laboratory at columbia university medical center (cumc). swabs from hospitalized patients were tested in the clinical microbiology laboratory at newyork-presbyterian hospital at cumc. blood, urine, and respiratory tract cultures for bacteria were sent by treating clinicians as per the standard of care for suspected infections and also processed by the clinical microbiology laboratory. for the community cohort, days of missed school or work; medical care in primary care clinics, urgent care, or emergency departments; hospitalizations; and use of antibiotics were collected by follow-up calls at least 15 days after the resolution of respiratory illness. for hospitalized patients, demographic and clinical characteristics were obtained from electronic medical records (emr). primary icd-9 diagnoses were categorized as respiratory or nonrespiratory. patients' primary and secondary diagnoses were categorized into groups of chronic conditions previously described as risk factors for severe respiratory disease in adults and children with respiratory viruses. [12] [13] [14] conditions were excluded if they could not be classified within a chronic condition category, had <15 occurrences across all patients, or have not been associated with severe respiratory disease, for example bipolar disorder. manual chart review of readmitted patients was performed to determine whether readmissions within 4 weeks were due to respiratory illness. hospital course and healthcare utilization data were obtained from the emr including radiographs within 2 days of hpiv detection; all antibiotics within 2 days prior to or 7 days after hpiv detection; icu admission and 30-day mortality. respiratory support including use of continuous positive airway pressure (cpap), mechanical ventilation, or extracorporeal membrane oxygenation (ecmo) was determined by billed procedure codes. use of bilevel positive airway pressure (bipap) or oxygen supplementation was not assessed, as accurate usage data were unavailable in structured electronic sources. ta b l e 1 comparison of demographic characteristics, symptoms, and outcomes of participants in a community cohort, by human parainfluenza viruses (hpiv) type to assess seasonality, epidemiologic curves that modeled the detection of each hpiv type in the community cohort versus hospitalized patients were created. demographic and clinical characteristics of the two groups were analyzed as proportions, means, and/or medians; associations between hpiv type and clinical course were examined using chi-square, fisher's exact, and anova tests, as appropriate. ta b l e 2 comparison of demographic and clinical characteristics of hospitalized patients with different human parainfluenza viruses (hpiv) in the community cohort, 1805 swabs were obtained, of which 69 (4%) of 1753 participants were positive for at least one hpiv type ( figure 1a ). the 69 hpiv-positive swabs were from 59 households; hospital community at least one hpiv type ( figure 1b) the mean length of hospitalization was 5.3 (sd ± 8.4) days and varied by hpiv type ( were associated with higher odds of increased severity of illness. among the 290 adults, 12 (4%) had severe, 41 (14%) had moderate, and 237 (82%) had mild illness (table 5 ). in the final adjusted model for adults, age ≥65 years and respiratory conditions were associated with higher odds of increased severity of illness. we had a unique opportunity to compare the epidemiology and clinical impact of hpiv types among individuals in a communitybased surveillance study and among hospitalized patients during the same time and in the same geographic area. the seasonality of hpiv types 1-3 was consistent with published trends, 1,3 but we found that hpiv-4 occurred annually in late summer and fall, suggestive of a local epidemiologic trend that has not been previously described. we found that hpiv-3 was most common among hospitalized patients (52%) while hpiv-4 was most common in the community cohort (36% we modified a previously validated severity of illness score to further explore the predictors of illness severity. in children, increased severity of illness was associated with respiratory conditions, cardiovascular conditions and hpiv-4 while in adults, increased severity of illness was associated with age ≥65 years and cardiovascular conditions. the association of comorbid conditions and severe illness in those with hpiv is consistent with other respiratory viruses, most notably rsv and influenza, which both can exacerbate underlying cardiac and pulmonary conditions. [12] [13] [14] 21, 22 this may also explain why 21% of hospitalized patients had a nonrespiratory primary diagnosis. the rate of co-detection was similar across hpiv strains and as others have shown, viral co-detections did not impact severity of disease. 23 however, due to limited sample size, we could not assess the impact of specific co-infections. while only 17% of hpiv-positive patients had a positive bacterial culture, 73% received at least one dose of an antibiotic between 2 days before and 7 days after hpiv detection. thus, most antibiotic usage represented empiric therapy, suggesting an opportunity for antibiotic stewardship. this study had limitations. the community cohort was 100% hispanic/latino, reducing the study's generalizability, and small, limiting our ability to find differences in severity of illness or symptomatology associated with hpiv types. our hospital is a referral center and cares for many patients with underlying conditions. furthermore, its referral status could impact accurate interpretation of "local" epidemiology as only 32% of the hospitalized patients lived in the same zip codes as the mosaic study participants (m. stockwell, personal communication). testing of hospitalized patients relied on clinicians' judgement. we did not review radiographic findings or capture bipap use which may have biased assessment of severity of illness. the severity of illness score was developed for children and not previously tested in adults. use of administrative data restricted our ability to assess patients' symptomatology. although most patients had a respiratory diagnosis, we may have misclassified hpiv infections that actually represented prolonged hpiv shedding from previous illnesses. we did not classify positive bacterial cultures as consistent with infection vs. colonization vs. contamination. in this study, each hpiv type demonstrated seasonality, but each was similar in the community and among hospitalized patients. we found that hpiv-4 had distinct epidemiology, not previously described, and was associated with increased severity of illness in hospitalized children. as demonstrated for other respiratory viruses, older age and underlying conditions, particularly respiratory and cardiac conditions, were associated with increased severity of illness. additional studies exploring hpiv incidence and severity in both children and adults could help reinforce the need for vaccine development. the authors thank alexandra hill-ricciuti for analytic assistance. seasonal trends of human parainfluenza viral infections: united states estimates of parainfluenza virus-associated hospitalizations and cost among children aged less than 5 years in the united states epidemiology and clinical presentation of parainfluenza type 4 in children: a 3-year comparative study to parainfluenza types 1-3 epidemiology of parainfluenza infection in england and wales, 1998-2013: any evidence of change? human parainfluenza type 4 infections mosaic: mobile surveillance for acute respiratory infections and influenza-like illness in the community community -and hospital laboratory-based surveillance for respiratory viruses. influenza other respir viruses filmarray respiratory panel information sheet multicenter evaluation of the eplex respiratory pathogen panel for the detection of viral and bacterial respiratory tract pathogens in nasopharyngeal swabs respiratory syncytial virus infection in elderly and high-risk adults children with complex chronic conditions in inpatient hospital settings in the united states pediatric deaths attributable to complex chronic conditions: a population-based study of washington state predicting severe pneumonia outcomes in children human parainfluenza virus types 1-4 in hospitalized children with acute lower respiratory infections in china a prospective study of parainfluenza virus type 4 infections in children attending daycare severe pneumonia after cardiac surgery as a result of infection with parainfluenza virus type 4 human parainfluenza virus 4 outbreak and the role of diagnostic tests defining the risk and associated morbidity and mortality of severe respiratory syncytial virus infection among infants with chronic lung disease influenza virus infections among patients attending emergency department according to main reason to presenting to ed: a 3-year prospective observational study during seasonal epidemic periods single-and multiple viral respiratory infections in children: disease and management cannot be related to a specific pathogen seasonality and clinical impact of human parainfluenza viruses key: cord-331228-wbd0s4fo authors: shehata, mahmoud m.; gomaa, mokhtar r.; ali, mohamed a.; kayali, ghazi title: middle east respiratory syndrome coronavirus: a comprehensive review date: 2016-01-20 journal: front med doi: 10.1007/s11684-016-0430-6 sha: doc_id: 331228 cord_uid: wbd0s4fo the middle east respiratory syndrome coronavirus was first identified in 2012 and has since then remained uncontrolled. cases have been mostly reported in the middle east, however travel-associated cases and outbreaks have also occurred. nosocomial and zoonotic transmission of the virus appear to be the most important routes. the infection is severe and highly fatal thus necessitating rapid and efficacious interventions. here, we performed a comprehensive review of published literature and summarized the epidemiology of the virus. in addition, we summarized the virological aspects of the infection and reviewed the animal models used as well as vaccination and antiviral tested against it. coronaviruses (cov) became known to cause human disease in the twentieth century. hcov-229e and hcov-oc43 were discovered in the 1960s and shown to cause respiratory infections in humans [1, 2] . with the emergence of sars-cov in 2003 [3] , two other human coronaviruses were discovered, hcov nl63, hcov hku1 [4] . in 2012, a new type of coronavirus was detected as the cause of severe respiratory illness in humans. the first case was a 60-year-old male from saudi arabia admitted to hospital with acute respiratory illness leading to pneumonia and acute renal failure. the virus initially named as human corona virus-emc [5] , is currently known as the middle east respiratory syndrome coronavirus (mers-cov) [6] . classification and nomenclature of mers-cov phylogenetically, mers-cov is a lineage c β coronavirus (β-cov) and is closely related to bat coronaviruses hku4 and hku5. the rooted phylogenetic analysis showed that mers-cov had an amino acid sequence identity less than 90% to all other known covs [7] . the virus initially named by many different working groups as novel coronavirus, human coronavirus emc, human b coronavirus 2c emc, human b coronavirus 2c england-qatar, human b coronavirus 2c jordan-n3, and b coronavirus england 1, which represented the places where the first complete viral genome was sequenced (erasmus medical center, rotterdam, the netherlands) or where the first laboratory-confirmed cases were identified or managed (jordan, qatar, and england) was later named as mers-cov by the coronaviruses study groups of ictv [5, 6, 8] . mers-cov is an enveloped virus with a positive sense rna genome. coronavirus genomes range between 25 to 32 kb in size. the complete sequence of hcov-emc-2012 resulted in 30 119 nucleotides sequence [7] . coronavirus genomes are polycistronic with large replicase open reading frames orf1a and orf1b which are subsequently cleaved into 15 or 16 nonstructural proteins (nsps). the region downstream of orf1b encode smaller genes including the spike (s), envelope (e), membrane (m), and nucleocapsid (n) structural protein [9] [10] [11] . the functional receptor for mers-cov is the dipeptidyl peptidase 4 (dpp4) which is present on human nonciliated bronchial epithelial cells surfaces [12] . the dpp4 protein displays high amino acid sequence conservation across different species, including the sequence that was obtained from bat cells. cell lines susceptibility studies showed that mers-cov infected several human cell lines, including histiocytes as well as respiratory, kidney, intestinal, and liver cells [13] . the range of tissue tropism in vitro was broader than that for any other known human coronavirus [14] . mers-cov can also infect nonhuman primate, porcine, bat, civet, rabbit, and horse cell lines all possessing the dpp4 receptor [15] . the replication cycle of mers-cov consists of numerous steps as illustrated by lu et al. [30] . the mers-cov s protein is a class i fusion protein composed of two subunits: the amino n-terminal receptor binding s1 and carboxyl c-terminal membrane fusion s2 subunits. the s1/s2 junction is a protease cleavage site which is responsible for membrane fusion activation, virus entry, and syncytium formation. the s1 c domain contains the receptor binding domain (rbd), and an n domain [13] . neutralizing monoclonal antibodies against the rbd may inhibit virus entry into cells and receptor-dependent syncytium formation in cell culture, hence vaccines containing the rbd induced high levels of neutralizing antibodies in mice and rabbits [16] [17] [18] . dpp4 is the cell key functional receptor for the mers-cov s protein [19] . mers-cov is the first cov that has been identified to use dpp4 as a receptor [19, 20] . dpp4 has important roles in glucose metabolism, t cell activation, chemotaxis modulation, cell adhesion, and apoptosis [19, 21] . the s2 subunit contains five domains: a fusion peptide, the heptad repeat 1 (hr1) and hr2 domains, a transmembrane domain, and a cytoplasmic domain, which form the stalk region of s protein that facilitates fusion of the viral and cell membranes [22, 23] . the binding of the s1 subunit to the cellular receptor triggers conformational changes in the s2 subunit, which inserts its fusion peptide into the target cell membrane to form a six-helix bundle fusion core between the hr1 and hr2 domains that approximates the viral and cell membranes for fusion. mers-cov utilizes many pathways for membrane fusion depending on available host proteases, such as transmembrane protease serine protease 2 (tmprss2), trypsin, chymotrypsin, elastase, thermolysin, endoproteinase lys-c, and human airway trypsin-like protease. proteases cleave the s protein into the s1 and s2 subunits to activate the mers-cov s protein for endosome-independent host cell entry at the plasma membrane [24] [25] [26] . in addition to the pervious fusion proteases furin has been identified recently to play an essential role in the mers-cov s protein cleavage activation into their biologically active forms [27, 28] . after cell entry, the virion particle disassembles to release the nucleocapsid and viral rna into the cytoplasm for expression of viral polyproteins pp1a and pp1ab. doublemembrane vesicles and convoluted membranes are formed by the attachment of the hydrophobic domains of the mers-cov replication machinery to the limiting membrane of auto-phagosomes [29] . the viral polyproteins pp1a and pp1ab are cleaved by papain-like protease and 3c-like protease into nsp1 to nsp16 [7, 30, 31] . these nonstructural proteins form the replication-transcription complex, which regulates transcription and viral protein expression [29] . after the production of abundant viral rna and structural and accessory proteins, the n protein binds to the genomic rna in the cytoplasm to form the helical nucleocapsid (viral core). the viral core is enveloped by budding through intracellular membranes between the endoplasmic reticulum and golgi apparatus [32] . the s, e, and m proteins are transported to the budding virion, where the nucleocapsid probably interacts with m protein to generate the basic structure and complexes with the s and e proteins to induce viral budding and release from the golgi apparatus [33] . mers-cov replication cycle is completed by releasing the progeny virions through the cell membrane via exocytosis pathway. mice mers-cov strain hcov-emc/2012 was inoculated to three different mouse strains (immunocompetent balb/c mice, 129s6/svev and innate immune-deficient 129/ stat1 -/mice) intranasally. no significant weight loss was observed and infectious virus could not be detected in the lungs. only moderate pathological lesions were observed in the lungs. hence no viral replication was observed in these strains of mice [34] . zhao et al. developed a mouse model transduced with a recombinant adenovirus vector expressing hdpp4 (ad5-hdpp4) in lung tissue. inoculation of mers-cov in these mice resulted in mers-cov replication but without mortality. young mice cleared from mers-cov in 6-8 days and old mice in 10-14 days. perivascular and peribronchial lymphoid infiltration was observed, with progression to an interstitial pneumonia postinfection [35] . in another study, transgenic mice expressing hdpp4 were susceptible to mers-cov infection. infectious virus was isolated from lung and brain tissue and weight loss was observed [36] . pascal et al. developed humanized transgenic mouse. no mortality or clinical signs was observed but interstitial pneumonia and significant lung disease were observed histopathologically, suggesting that humanized dpp4 mouse is a model for mers-cov infection in which pathological changes resembles mers-cov infection in humans [37, 38] . the rhesus macaque was the first animal model used for mers-cov infection as it possessed dpp4 receptor [38, 39] . in infected animals, an increase in respiratory rates, body temperature, cough and reduced appetite was observed with mild to moderate severity. infectious virus isolated only from the lower respiratory tract. viral rna was detected in the conjunctiva, nasal mucosa, tonsils, pharynx, trachea, bronchus and lungs. mild to marked interstitial pneumonia with dark red lesions appeared in lungs. seroconversion of neutralizing antibodies began at 7 dpi and increased in titer with time. the development of a transient pneumonia, rapid replication, and tropism of mers-cov for the lower respiratory tract resembled the severity of the disease observed in humans [38, 40, 41] . similarly, the common marmoset was shown to possess the dpp4 receptor [42] . radiographic imaging showed mild to severe bilateral interstitial infiltration and extensive bronchointertitial pneumonia in infected animals. infectious virus was detected in lower and upper respiratory tract tissue and viral rna was detected in nasal mucosa, oropharyngeal swabs, blood, conjunctiva, lymph nodes, gastrointestinal tract, kidney, heart, adrenal gland, liver, spleen, brain and lungs [42] . inoculation of syrian hamsters and ferrets with mers-cov did not result in infection [12, 43] . rabbits may be used as a model to study pathogenesis, transmission, and disease control strategies of mers-cov in vivo as they seroconvert and shed virus after inoculation [44] . in september 2012, a novel coronavirus infection was noted in promed mail [45] . the virus was isolated from the sputum of a 60-year-old saudi male, who was admitted to a hospital with pneumonia and acute kidney injury in june 2012. a few days later, another report appeared describing an almost identical virus detected in a patient in qatar with acute respiratory syndrome and acute kidney injury. the patient had a recent travel history to saudi arabia and then traveled to uk for further medical care [5, 46, 47] . two cases from jordan (april 2012) were retrospectively diagnosed as mers patients. since that time, more than 1542 cases of mers-cov infection have been reported including 544 deaths [48] . the actual number of cases could be higher than those reported [49] . an outbreak of more than 180 confirmed cases including 36 deaths occurred in south korea in may and june 2015. the median age of korean cases were 55 years (range: 16 to 87 years), 60% were men, and 14% were health care professionals. the index case was a 68-year-old male who had recently traveled to several countries in the arabian peninsula [50] . disease control and prevention (cdc), and the ministry of health of saudi arabia (mohsa) as asymptomatic, mild, severely symptomatic, or mortal. cases may be classified into suspected, probable, and confirmed [52, 53] . any person with laboratory confirmation of infection with mers-cov irrespective of clinical signs and symptoms is considered as a confirmed case. who criteria for laboratory confirmation require detection of viral rna or acute and convalescent serology. the presence of nucleic acid can be confirmed by positive results from at least two sequence-specific rrt-pcrs or a single sequence-specific rrt-pcr test and direct sequencing from a separate genomic target [54] . a case confirmation by serological methods requires demonstration of seroconversion in two samples collected at least 14 days apart using at least one screening assay (enzyme-linked immunoassay, immunofluorescence assay) and a neutralization assay. a probable case is defined by the following criteria, a febrile acute respiratory illness as pneumonia or acute respiratory distress syndrome, direct contact with a confirmed mers-cov case and unavailability of mers-cov testing or results being inconclusive for a single inadequate specimen. any person who developed a fever and pneumonia or acute respiratory distress syndrome with a history of travel to countries in or near the arabian peninsula within 14 days before symptom onset or was in contact with a traveler from this region who developed a febrile respiratory illness is considered as a mers-cov suspected case. the who, cdc, and mohsa recommended laboratory diagnostics for mers-cov infection [6, 9, 51, 55, 56] . mers-cov cases must be confirmed by at least two positive qrt-pcr tests on two different specific genomic regions or single positive qrt-pcr with a sequence of another positive genome fragment [57] . the who algorithm for testing mers-cov relies on qrt-pcr and sequencing [58] . available real-time tests include an assay targeting the rna upstream of the e gene (upe) as a highly sensitive screening assay and three confirmatory assays targeting open reading frames (orf 1a and 1b) and/ or n gene. the orf 1a assay is of equal sensitivity to the upe assay. the orf 1b assay is less sensitive but is useful for confirmation. these assays are specific for mers-cov and have not shown cross-reactivity with other respiratory human coronaviruses. for sequencing, two target genes, the rna-dependent rna polymerase (rdrp, present in orf 1b) and n genes are enough to confirm the existence of mers-cov rna in the samples of a patient [57] . several serologic assays including immunofluorescence assays, protein microarray assay, enzyme-linked immunosorbent assay (elisa) have been developed for the detection of mers-cov antibodies [57, [59] [60] [61] . any positive test by one of these assays should be confirmed with a neutralization assay. single serological result may be valuable for definition of probable case and should be followed by further testing for confirmation of mers-cov infection [62] [63] [64] . incubation period of mers-cov infections was studied by assiri et al. in 2013. the median incubation period was 5.2 days (95% ci 1.9-14.7 days) [65] . in another report from france of a secondary case, a patient who shared a room with an infected patient, the incubation period was estimated at 9 to 12 days [66] . in the recent outbreak in south korea during may/june 2015, the median incubation period was 6.3 days [67] . who and cdc recommended that individuals that returned from the arabian peninsula and other affected countries must be evaluated for mers-cov infections up to at least 14 days [68] . clinical features of mers-cov infections range from asymptomatic cases to mildly ill, severe pneumonia, acute respiratory distress syndrome, septic shock and mortal with multi-organ failure (table 1) [64, 65] . many other clinical features such as gastrointestinal symptoms (anorexia, nausea, vomiting, abdominal pain, diarrhea), pericarditis, and disseminated intravascular coagulation were reported [65, 69, 70] . specific clinical conditions (comorbidities) were apparently proportionate with high severity of mers-cov infections. a study by assiri et al. in saudi arabia showed that of a total of 47 patients with mers-cov infection in 2013, 45 (96%) had underlying clinical conditions, including diabetes mellitus (68%), hypertension (34%), chronic cardiac disease (28%), and chronic kidney disease (49%) [65] . this high rate of comorbidities must be interpreted with some caution, since diabetes mellitus is common in saudi arabia, and because approximately half of those 47 were part of an outbreak in a hemodialysis unit, where rates of comorbidities might be high due to chronic kidney disease [65, 71] . in another study, being on dialysis, diabetes mellitus, and age > 50 years was associated with mortality [72] . in this study, testing positive for mers-cov in a plasma sample was a predictor of severe outcome [72] . younger adults and children appeared to be less susceptible to mers-cov infection. only one study described mers-cov infection in children [73] . all of those children were discovered during contact investigations of older patients. only 2 of 11 children developed symptoms of mers-cov infection. these two children had underlying conditions (cystic fibrosis and down syndrome). the other 9 children were asymptomatic. there are few reports of mers-cov infections in pregnant women. a five-month pregnant female developed vaginal bleeding and abdominal pain after one week, then delivered a stillborn infant [74] . another case in the united arab emirates was near term phase, she gave birth to an apparently healthy baby, and died after delivery [52] . mild and asymptomatic mers-cov infections have been reported, a majority of whom were identified among the contacts of patients [62, 75, 76] . in a report from mohsa, more than 3000 contacts of patients were screened using qrt-pcr and seven healthcare workers with mers-cov infection were identified, two of whom were asymptomatic and five of whom had mild upper respiratory tract symptoms [75] . epidemiological and virological studies were conducted in attempts to determine person to person transmission of mers-cov. they studied case clustering in household and hospital outbreaks in the uk, tunisia, italy, and in healthcare facilities in saudi arabia, france, iran, and lately in south korea. those studies provided strong evidence that human-to-human transmission occurs [70, [77] [78] [79] [80] . the number of contacts infected by individuals with confirmed infections, however, appears to be limited [62] , except the outbreak of south korea in may/ june 2015, where most cases were secondary and some cases were tertiary infections [67, 81] . secondary cases often were milder or symptomless [62] . possible modes of transmission include droplet and close contact transmission, air borne transmission, and fomite transmission [82] . the majority of all laboratory-confirmed secondary cases have been associated with healthcare settings [82] . the majority of cases of jeddah, saudi arabia hospital outbreak during the spring of 2014 were acquired through human-to-human transmission due to systematic weaknesses in infection control [76] . secondary transmission rates were assessed within households and the transmission rate was around 4%, suggesting that the actual number of infection is greater than reported [62] . during the outbreak in south korea during may/june 2015, 25 secondary infections were associated with the index case, who was hospitalized from may 15 to may 17 and 11 were tertiary [83] . the median incubation period was six days for secondary cases and six days for tertiary cases. this outbreak also clearly demonstrated roles of "superspreaders," who may be responsible for a high proportion of cases [83] . for instance, a single patient infected more than 70 other people while being treated in the emergency room of a hospital in south korea for three days, 27-29 may 2015. transmissibility and epidemic potential studies of mers-cov revealed that the reproduction number (r 0 ) of patients infected with mers-cov ranged between 0.6 to 0.69 [84, 85] . the finding of an r 0 < 1 suggests that mers-cov does not yet have pandemic potential. other study suggested that r 0 values might reach to 0.8 to 1.3 in the absence of infection control [49] . shedding periods of mers-cov in humans was reported to be long as viruses were detected in lower respiratory samples of symptomatic patients for more than two weeks [86] . at instances, prolonged shedding for 6 weeks was detected in an asymptomatic healthcare worker. these findings raise concerns that asymptomatic persons could transmit infection to others in a silent manner [87] . the majority of cases have occurred in saudi arabia and united arab emirates [88] [89] [90] . many cases have also been reported outside the arabian peninsula in north africa, europe, asia, and north america as shown in table 2 . almost all cases reported outside the arabian peninsula had a travel history to it. the first cluster was in october/november 2012 in four men of the same family in riyadh, saudi arabia, two of whom died [75] . the second cluster was reported in jordan in april 2012 involving 10 healthcare workers exposed to fatal patients. in addition, seven surviving hospital contacts seroconverted suggesting that they had mers-cov infection [91] . the third cluster was reported in uk during january/ february 2013. an english resident had a travel history to saudi arabia and pakistan in january, developed a severe respiratory illness, and tested positive for both mers-cov and h1n1 influenza a, and died in march 2013 after infecting several contacts [92] . a cluster of 43 cases of mers-cov was reported in al-hasa in saudi arabia during april 2013. all those cases were directly linked to human to human contact in the same hospital. there were only two confirmed cases of healthcare workers, and three family members were detected by a survey of over 200 household contacts that visited this hospital [77] . in france, may 2013, an infection of mers-cov was reported in a patient who recently traveled to the united arab emirates. a second case who shared the hospital room with the first case tested positive. the first patient died and the second patient was critically ill. a survey of 100 healthcare workers found no other infections with mers-cov, despite the lack of use of personal protective equipment [70] . a surge in mers-cov cases was reported in saudi arabia and the united arab emirates during march and april 2014 [55, 76] . the majority of cases were associated with hospital-based outbreaks jeddah, riyadh, tabuk, and madinah in saudi arabia as well as in al ain, and abu dhabi in united arab emirates. cases included several healthcare workers, visitors, patients, and ambulance staff. person to person transmission was confirmed in > 75% of cases. the majority of infected health care workers developed mild symptomatic or asymptomatic infection, but about 15% had severe illness or died [93] . the recent outbreak of south korea occurred in may 2015. the index case was a man who had recently traveled to bahrain, the united arab emirates, saudi arabia, and qatar [55] . as of late july 2015, > 180 secondary cases were reported including 36 death and many cases had been reported among household and hospital contacts [55, 67] . in china, one case occurred in a man who traveled to china from korea following exposure to two relatives with mers-cov infection [55] . in spite of reporting of mers-cov infections throughout the year, some evidence on disease seasonality occurred. the first identified cases of mers-cov infection were reported in april and june 2012 [5, 46, 47] . a high increase in cases was reported in april and may 2013 followed by a surge in case reporting in april and may 2014. increase in case reporting in march to may 2013 were attributed to infection from infected young camels [94, 95] sources and modes of transmission of mers-cov are still unclear. initially, a bat origin of mers-cov was suggested based on the relation of genome sequences between mers-cov and bat coronaviruses [96] . cell tropism studies showed that both bat coronavirus hku4 and mers-cov shared the same cell type receptors, dpp4 [4, 7, 75] . mers-cov grows readily in several bat-derived cell lines [14] . there is no evidence for direct or indirect transmission of mers-cov from bats to humans. virological studies performed in europe, africa, and asia, including the middle east, have shown that coronavirus rna sequences are found frequently in bat feces. some of the sequences were closely related to mers-cov sequences [97] [98] [99] . in a survey from saudi arabia, 823 fecal and rectal samples were tested by pcr for mers-cov, many coronaviruses sequences were detected [97] . most of the detected sequences were unrelated to mers-cov, but one sequence of 190 nucleotide in the rna-dependent rna polymerase (rdrp) gene had a 100% identity with a mers-cov. this sequence was detected from feces of a taphozous perforatus bat captured near the home of the index saudi patient. uncommon contact of humans with bats indicates that bats are not the intermediate host of mers-cov transmission but may be the reservoir of the virus [100] . dromedary camels (camelus dromedarus) appear to be the source of mers-cov. other animals like sheep, goats, and cows tested negative to anti-mers-cov antibodies. camel sera from oman, canary islands, and egypt were positive for anti-mers-cov antibodies in about 100%, 14%, and > 90% of the samples respectively [63, 101, 102] . retrospective studies on archived human sera showed no evidence of infection with mers-cov before 2012 [103] , but anti-mers-cov antibodies were detected in archived camel sera in saudi arabia in 1993 [95] , and united arab emirates in 2003 [104] , indicating circulation of mers-cov in camels for many years. bactrian camels in mongolia tested negative for mers-cov antibodies [105] . serologic studies from around the middle east suggested that camels are one of the sources of mers-cov as > 90% of adult camels tested positive and had high titers of antibodies. seropositivity was different in juvenile camels and was usually lower than in adults. these results suggested that mers-cov infections in camels occurred in young ages followed by frequent boosting [94, 95, 102, 104] . camels in other parts of the world, far from the middle east like in europe, australia, and the americas do not have mers-cov antibodies and have no evidence of infection [106] . table 3 summarizes camel serologic studies. in a study aimed to evaluate virus infectivity and shedding in camels, three adult dromedary camels were inoculated with mers-cov intratracheally, intranasally, and conjunctivally. those camels shed large quantities of virus from the upper respiratory tract and infectious virus was detected in nasal secretions for 7 days post-inoculation and viral rna for up to 35 days post-inoculation [113] . human infections with mers-cov were linked to camels. the first evidence was a study in saudi arabia in which the mers-cov full genome sequences of isolates from a man with fatal infection and from one of his camels were identical. this patient had a direct contact with his deceased camels some days before the onset of symptoms. these results suggested that mers-cov can infect dromedary camels and can be transmitted from them to humans by direct close contact [86] . in other studies, phylogenetic analyses of camel and human isolates of the mers-cov genome demonstrated that the viruses were highly identical or in some cases were similar to each other [107, 108, 114] . seroepidemiological studies shown low prevalence of mers-cov antibodies in humans in saudi arabia [103, 115] . a survey of 10 009 individuals representative of the general population of saudi arabia resulted in 15 seropositive subjects (0.15%), however, seropositivity increased 15-23 folds in camel-exposed individuals [78] . in a separate report, 7 of 87 camel shepherds and 140 slaughterhouse workers (3.1%) tested positive for mers-cov antibodies [103] . an overview of mers-cov transmission routes is illustrated in fig.2 . the development of an effective vaccine is critical for prevention of a mers-cov pandemic. some investigators have indicated that the rbd protein of mers-cov s protein is a good candidate antigen as a subunit vaccine. various rbd fragments showed the highest dpp4 binding affinity and induced the highest-titer of igg ab and neutralizing ab in mice and rabbits [17, 18, [116] [117] [118] [119] [120] . a robust neutralizing antibody response was elicited in balb/c mice against mers-cov after immunization with purified full s protein nanoparticles produced in sf9 cells infected with specific recombinant baculovirus containing the s gene [121] or a recombinant human adenoviral vectors (rad5 or rad41) containing the s or s1 genes [122, 123] . vaccinia ankara was encoded with full s protein and inoculated to balb/c mice that developed high levels of neutralizing antibodies and had induction of humoral and cell-mediated immunity [124, 125] . another study using ad5-hdpp4-transduced balb/c mice immunized with venezuelan equine encephalitis virus replicon particles containing s protein elucidated a reduction of viral titers to nearly undetectable levels and increased neutralizing antibodies [35] . recently, wang et al. developed two candidate vaccines, a subunit (full s and s1 protein fraction) and a dna vaccine (full s and s1 gene in a mammalian vrc8400 vector). the vaccine containing the full s dna and s1 protein was the most efficacious in mice and rhesus macaques [126] . using antibodies to deter mers-cov infection appears to have some promise. transfer of sera containing anti-mers-cov-s protein to or seropositive camel sera to ad5-hdpp4-transduced mice accelerated virus clearance, inhibited virus attachment, and reduced weight loss [35, 37, 127] . recently, corti et al. successfully isolated monoclonal antibodies from serum obtained from a mers-cov survivor after 200 days of infection [128] . transduced ad5-hdpp4 balb/c mice were immunized with 15 mg/kg of the mab and showed decreased lung [105] viral titers, no weight loss, and decreased peribronchial lymphoid infiltration [128] . no approved antivirals for use against mers-cov infection are yet available. the first approach performed when a new unknown virus like mers-cov emerges is testing drugs used as antiviral for similar viruses [29, 129, 130] . type i interferons and ribavirine combination exhibited acceptable results in cell culture and rhesus macaques by decreasing the host inflammatory response, replication of virus, and improved clinical outcome [129, 131, 132] . a human cohort study in saudi arabia showed that treatment with combination of ribavirin and interferon-α2b to 5 did not improve clinical outcomes but this may have been due to late treatment or due to the immunocompromised state of the patients [133] . in a retrospective study of 20 mers-cov infected patients treated with ribavirin and interferon α-2a, results showed 14-day and 28-day survival was improved by 70% and 28% in the treated group as compared to an untreated group [134] . the second approach is screening of approved drugs with known safety profiles and transcriptional signatures in different cell lines. several drugs, including antiparasitics, neurotransmitters, antibacterials, inhibitors of clathrinmediated endocytosis estrogen receptor, lipid or sterol metabolism, protein processing, and dna synthesis or repair were tested on culture cells [119, [135] [136] [137] [138] [139] . lopinavir-ritonavir combined with pegylated interferon and ribavirin therapy showed improved outcomes in infected marmosets [140] . the third approach involves in vitro inhibition of s protein to block virus entry into host cells using designed antiviral peptides targeting the hr2 domain of the s2 subunit of the mers-cov and preventing the interaction between the hr1 and hr2 domains required for the formation of the heterologous six-helix bundle in viral fusion core formation [22, 23] . other drugs that act as inhibitors for viral proteases and helicase to suppress mers-cov infection were tested [141] [142] [143] [144] [145] . other investigators studied inhibition of mers-cov infection by competitive inhibition of dpp4 cell receptor using compounds such as sitagliptin, vildagliptin, and saxagliptin [19, 146] . more than three years have passed since the first detection of mers-cov human infection and the virus, uncontrolled, continues to cause major outbreaks in the middle east. the recent outbreak in korea demonstrated that a single index case can lead to 185 more infections in a short period of time, hence raising questions about the accuracy of the number of cases being reported in the middle east. furthermore, the korean outbreak confirmed the high fatality rate of mers-cov infection as being true rather than overestimated in case only the more severe cases are detected. in all, public health, veterinary health, and research efforts need to be consolidated in order to answer the following high priority questions: -what is the true extent of human infection with mers-cov? -what antivirals and vaccines are to be used in humans? -what infection control measures are needed in healthcare settings to prevent nosocomial outbreaks? -what measures need to be in place in order to prevent zoonotic infections from camels? -is it possible to control the virus in the camel population and if so, how? -are there other animal species involved in the mers-cov transmission cycle? a new virus isolated from the human respiratory tract recovery in tracheal organ cultures of novel viruses from patients with respiratory disease coronavirus as a possible cause of severe acute respiratory syndrome discovery of seven novel mammalian and avian coronaviruses in the genus deltacoronavirus supports bat coronaviruses as the gene source of alphacoronavirus and betacoronavirus and avian coronaviruses as the gene source of gammacoronavirus and deltacoronavirus isolation of a novel coronavirus from a man with pneumonia in saudi arabia middle east respiratory syndrome coronavirus (mers-cov): announcement of the coronavirus study group genomic characterization of a newly discovered coronavirus associated with acute respiratory distress syndrome in humans transmission and evolution of the middle east respiratory syndrome coronavirus in saudi arabia: a descriptive genomic study first confirmed cases of middle east respiratory syndrome coronavirus (mers-cov) infection in the united states, updated information on the epidemiology of mers-cov infection, and guidance for the public nidovirales: evolving the largest rna virus genome virus-encoded proteinases and proteolytic processing in the nidovirales adenosine deaminase acts as a natural antagonist for dipeptidyl peptidase 4-mediated entry of the middle east respiratory syndrome coronavirus differential cell line susceptibility to the emerging novel human betacoronavirus 2c emc/2012: implications for disease pathogenesis and clinical manifestation human coronavirus emc does not require the sars-coronavirus receptor and maintains broad replicative capability in mammalian cell lines serologic assessment of possibility for mers-cov infection in equids identification of a receptor-binding domain in the s protein of the novel human coronavirus middle east respiratory syndrome coronavirus as an essential target for vaccine development the receptor binding domain of the new middle east respiratory syndrome coronavirus maps to a 231-residue region in the spike protein that efficiently elicits neutralizing antibodies searching for an ideal vaccine candidate among different mers coronavirus receptor-binding fragments-the importance of immunofocusing in subunit vaccine design dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-emc interspecies transmission and emergence of novel viruses: lessons from bats and birds dipeptidylpeptidase iv from bench to bedside: an update on structural properties, functions, and clinical aspects of the enzyme dpp iv structure-based discovery of middle east respiratory syndrome coronavirus fusion inhibitor structure of the fusion core and inhibition of fusion by a heptad repeat peptide derived from the s protein of middle east respiratory syndrome coronavirus the spike protein of the emerging betacoronavirus emc uses a novel coronavirus receptor for entry, can be activated by tmprss2, and is targeted by neutralizing antibodies inhibition of proprotein convertases abrogates processing of the middle eastern respiratory syndrome coronavirus spike protein in infected cells but does not reduce viral infectivity role of the spike glycoprotein of human middle east respiratory syndrome coronavirus (mers-cov) in virus entry and syncytia formation host cell entry of middle east respiratory syndrome coronavirus after two-step, furin-mediated activation of the spike protein furin at the cutting edge: from protein traffic to embryogenesis and disease mers-coronavirus replication induces severe in vitro cytopathology and is strongly inhibited by cyclosporin a or interferon-α treatment middle east respiratory syndrome coronavirus (mers-cov): challenges in identifying its source and controlling its spread crystal structure of the papain-like protease of mers coronavirus reveals unusual, potentially druggable active-site features characterization of the coronavirus m protein and nucleocapsid interaction in infected cells sars-beginning to understand a new virus wildtype and innate immune-deficient mice are not susceptible to the middle east respiratory syndrome coronavirus rapid generation of a mouse model for middle east respiratory syndrome generation of a transgenic mouse model of middle east respiratory syndrome coronavirus infection and disease pre-and postexposure efficacy of fully human antibodies against spike protein in a novel humanized mouse model of mers-cov infection pneumonia from human coronavirus in a macaque model host species restriction of middle east respiratory syndrome coronavirus through its receptor, dipeptidyl peptidase 4 middle east respiratory syndrome coronavirus (mers-cov) causes transient lower respiratory tract infection in rhesus macaques an animal model of mers produced by infection of rhesus macaques with mers coronavirus infection with mers-cov causes lethal pneumonia in the common marmoset the middle east respiratory syndrome coronavirus (mers-cov) does not replicate in syrian hamsters asymptomatic middle east respiratory syndrome coronavirus infection in rabbits novel coronavirus-saudi arabia: human isolate. archive number: 20120920.1302733. published date patient with new strain of coronavirus is treated in intensive care at london hospital middle east respiratory syndrome coronavirus (mers-cov)-saudi arabia middle east respiratory syndrome coronavirus: quantification of the extent of the epidemic, surveillance biases, and transmissibility mers-cov in the republic of korea at a glance. 2015 51. who. laboratory testing for middle east respiratory syndrome coronavirus -interim recommendations (revised) revised case definition for reporting to who-middle east respiratory syndrome coronavirus-interim case definition as of 14 middle east respiratory syndrome (mers), case definition middle east respiratory syndrome coronavirus, case definition for reporting to who, interim case definition as of 14 middle east respiratory syndrome coronavirus (mers-cov): summary of current situation, literature update and risk assessment interim guidelines for collecting, handling, and testing clinical specimens from patients under investigation (puis) for middle east respiratory syndrome coronavirus (mers-cov) assays for laboratory confirmation of novel human coronavirus (hcov-emc) infections laboratory testing for middle east respiratory syndrome coronavirus cross-reactive antibodies in convalescent sars patients' sera against the emerging novel human coronavirus emc (2012) by both immunofluorescent and neutralizing antibody tests contact investigation of a case of human novel coronavirus infection treated in a german hospital specific serology for emerging human coronaviruses by protein microarray transmission of mers-coronavirus in household contacts seroepidemiology for mers coronavirus using microneutralisation and pseudoparticle virus neutralisation assays reveal a high prevalence of antibody in dromedary camels in egypt laboratory testing for middle east respiratory syndrome coronavirus epidemiological, demographic, and clinical characteristics of 47 cases of middle east respiratory syndrome coronavirus disease from saudi arabia: a descriptive study che d; investigation team. first cases of middle east respiratory syndrome coronavirus (mers-cov) infections in france, investigations and implications for the prevention of human-to-human transmission preliminary epidemiological assessment of mers-cov outbreak in south korea update: severe respiratory illness associated with middle east respiratory syndrome coronavirus (mers-cov)-worldwide family cluster of middle east respiratory syndrome coronavirus infections clinical features and viral diagnosis of two cases of infection with middle east respiratory syndrome coronavirus: a report of nosocomial transmission is mers another sars? ifn-α2a or ifn-β1a in combination with ribavirin to treat middle east respiratory syndrome coronavirus pneumonia: a retrospective study middle east respiratory syndrome coronavirus disease in children cov investigation team stillbirth during infection with middle east respiratory syndrome coronavirus middle east respiratory syndrome coronavirus infections in health care workers mers-cov outbreak in jeddah-a link to health care facilities hospital outbreak of middle east respiratory syndrome coronavirus presence of middle east respiratory syndrome coronavirus antibodies in saudi arabia: a nationwide, cross-sectional, serological study world health organization global outbreak alert and response network middle east respiratory syndrome coronavirus international investigation team. family cluster of middle east respiratory syndrome coronavirus infections spread of mers to south korea and china summary and risk assessment of current situation in republic of korea and china notice to health care providers: updated guidelines for evaluation of severe respiratory illness associated with middle east respiratory syndrome coronavirus (mers-cov) epidemiological investigation of mers-cov spread in a single hospital in south korea interhuman transmissibility of middle east respiratory syndrome coronavirus: estimation of pandemic risk assessing the pandemic potential of mers-cov evidence for camel-to-human transmission of mers coronavirus a case of long-term excretion and subclinical infection with middle east respiratory syndrome coronavirus in a healthcare worker update on the epidemiology of middle east respiratory syndrome coronavirus (mers-cov) infection, and guidance for the public, clinicians, and public health authorities middle east respiratory syndrome middle east respiratory syndrome coronavirus (mers-cov): summary of current situation, literature update and risk assessment-as of 5 hospital-associated outbreak of middle east respiratory syndrome coronavirus: a serologic, epidemiologic, and clinical description severe respiratory illness caused by a novel coronavirus middle east respiratory syndrome coronavirus (mers-cov) mers coronavirus in dromedary camel herd, saudi arabia seroepidemiology of middle east respiratory syndrome (mers) coronavirus in saudi arabia ) and characterisation of assay specificity rooting the phylogenetic tree of middle east respiratory syndrome coronavirus by characterization of a conspecific virus from an african bat middle east respiratory syndrome coronavirus in bats, saudi arabia human betacoronavirus 2c emc/2012-related viruses in bats, ghana and europe close relative of human middle east respiratory syndrome coronavirus in bat the lancet infectious diseases. need for global cooperation in control of mers-cov middle east respiratory syndrome coronavirus (mers-cov) serology in major livestock species in an affected region in jordan koopmans mp. middle east respiratory syndrome coronavirus neutralising serum antibodies in dromedary camels: a comparative serological study investigation of anti-middle east respiratory syndrome antibodies in blood donors and slaughterhouse workers in jeddah and makkah, saudi arabia, fall 2012 antibodies against mers coronavirus in dromedary camels absence of mers-coronavirus in bactrian camels, southern mongolia mers coronavirus neutralizing antibodies in camels mers coronaviruses in dromedary camels middle east respiratory syndrome coronavirus in dromedary camels: an outbreak investigation middle east respiratory syndrome (mers) coronavirus seroprevalence in domestic livestock in saudi arabia middle east respiratory syndrome coronavirus antibody reactors among camels in dubai geographic distribution of mers coronavirus among dromedary camels antibodies against mers coronavirus in dromedary camels replication and shedding of mers-cov in upper respiratory tract of inoculated dromedary camels middle east respiratory syndrome coronavirus infection in dromedary camels in saudi arabia lack of mers coronavirus neutralizing antibodies in humans, eastern province, saudi arabia intranasal vaccination with recombinant receptor-binding domain of mers-cov spike protein induces much stronger local mucosal immune responses than subcutaneous immunization: implication for designing novel mucosal mers vaccines potent neutralization of mers-cov by human neutralizing monoclonal antibodies to the viral spike glycoprotein a truncated receptor-binding domain of mers-cov spike protein potently inhibits mers-cov infection and induces strong neutralizing antibody responses: implication for developing therapeutics and vaccines a safe and convenient pseudovirus-based inhibition assay to detect neutralizing antibodies and screen for viral entry inhibitors against the novel human coronavirus mers-cov tailoring subunit vaccine immunity with adjuvant combinations and delivery routes using the middle east respiratory coronavirus (mers-cov) receptor-binding domain as an antigen purified coronavirus spike protein nanoparticles induce coronavirus neutralizing antibodies in mice immunogenicity of an adenoviral-based middle east respiratory syndrome coronavirus vaccine in balb/c mice systemic and mucosal immunity in mice elicited by a single immunization with human adenovirus type 5 or 41 vectorbased vaccines carrying the spike protein of middle east respiratory syndrome coronavirus middle east respiratory syndrome coronavirus spike protein delivered by modified vaccinia virus ankara efficiently induces virus-neutralizing antibodies protective efficacy of recombinant modified vaccinia virus ankara (mva) delivering middle east respiratory syndrome coronavirus spike glycoprotein evaluation of candidate vaccine approaches for mers-cov passive immunotherapy with dromedary immune serum in an experimental animal model for middle east respiratory syndrome coronavirus infection prophylactic and postexposure efficacy of a potent human monoclonal antibody against mers coronavirus inhibition of novel β coronavirus replication by a combination of interferon-α2b and ribavirin cyclophilins as modulators of viral replication broad-spectrum antivirals for the emerging middle east respiratory syndrome coronavirus treatment with interferon-α2b and ribavirin improves outcome in mers-cov-infected rhesus macaques ribavirin and interferon therapy in patients infected with the middle east respiratory syndrome coronavirus: an observational study ribavirin and interferon α-2a for severe middle east respiratory syndrome coronavirus infection: a retrospective cohort study cell host response to infection with novel human coronavirus emc predicts potential antivirals and important differences with sars coronavirus antiviral potential of erk/mapk and pi3k/akt/mtor signaling modulation for middle east respiratory syndrome coronavirus infection as identified by temporal kinome analysis testing of middle east respiratory syndrome coronavirus replication inhibitors for the ability to block viral entry screening of an fda-approved compound library identifies four small-molecule inhibitors of middle east respiratory syndrome coronavirus replication in cell culture interferon-β and mycophenolic acid are potent inhibitors of middle east respiratory syndrome coronavirus in cell-based assays treatment with lopinavir/ritonavir or interferon-β1b improves outcome of mers-cov infection in a nonhuman primate model of common marmoset prediction and biochemical analysis of putative cleavage sites of the 3c-like protease of middle east respiratory syndrome coronavirus assessing activity and inhibition of middle east respiratory syndrome coronavirus papain-like and 3c-like proteases using luciferase-based biosensors evaluation of ssya10-001 as a replication inhibitor of severe acute respiratory syndrome, mouse hepatitis, and middle east respiratory syndrome coronaviruses thiopurine analogs and mycophenolic acid synergistically inhibit the papain-like protease of middle east respiratory syndrome coronavirus the newly emerged sars-like coronavirus hcov-emc also has an "achilles' heel": current effective inhibitor targeting a 3c-like protease inhibition of middle east respiratory syndrome coronavirus infection by anti-cd26 monoclonal antibody mahmoud m. shehata, mokhtar r. gomaa, mohamed a. ali, and ghazi kayali declare that they have no conflict of interest. this manuscript is a review article and does not involve a research protocol requiring approval by the relevant institutional review board or ethics committee. key: cord-315934-h70j2jmt authors: yu, jianxing; liu, chunyan; xiao, yan; xiang, zichun; zhou, hongli; chen, lan; shen, kunling; xie, zhengde; ren, lili; wang, jianwei title: respiratory syncytial virus seasonality, beijing, china, 2007–2015 date: 2019-06-17 journal: emerg infect dis doi: 10.3201/eid2506.180532 sha: doc_id: 315934 cord_uid: h70j2jmt during july 2007–june 2015, we enrolled 4,225 hospitalized children with pneumonia in a study to determine the seasonality of respiratory syncytial virus (rsv) infection in beijing, china. we defined season as the period during which >10% of total pcrs performed each week were rsv positive. we identified 8 distinctive rsv seasons. on average, the season onset occurred at week 41 (mid-october) and lasted 33 weeks, through week 20 of the next year (mid-may); 97% of all rsv-positive cases occurred during the season. rsv seasons occurred 3–5 weeks earlier and lasted ≈6 weeks longer in rsv subgroup a–dominant years than in rsv subgroup b–dominant years. our analysis indicates that monitoring such rsv subgroup shifts might provide better estimates for the onset of rsv transmission. pcr-based tests could be a flexible or complementary way of determining rsv seasonality in locations where rsv surveillance is less well-established, such as local hospitals throughout china. r espiratory syncytial virus (rsv) is a major cause of lower respiratory tract infection in young children worldwide (1-3); 2.7-3.8 million hospitalizations and 94,600-149,400 deaths occur each year among children <5 years of age as a result of rsv infection (4) . studies have also demonstrated the contribution of rsv to respiratory tract infections in adults (5, 6) . however, no licensed rsv vaccine is available (7) , and the only approved specific therapy, palivizumab (anti-rsv antibody), has limited uses among infants at high risk for severe respiratory illness in high-resource settings (8) . rsv causes epidemics in the winter in regions with temperate climates (8, 9) . however, spatiotemporal variations have been observed in the timing of rsv activity (10, 11) , and knowledge of the exact timing is helpful for guiding healthcare providers and health officials on the timing of diagnostic testing and immunoprophylaxis for infants at high risk for infection (12) . rsv circulation is monitored in the united states year-round through the national respiratory and enteric virus surveillance system (10) and in 15 countries of europe through the european influenza surveillance network (13) . since 2017, the world health organization has also conducted rsv surveillance to guide its global rsv prevention strategy (9) using the global influenza surveillance and response system (14) . however, most of the realtime data on rsv seasonality comes from rsv surveillance, and data are lacking in many places of the world. because disease surveillance is labor-and resource-intensive, information on seasonality from other sources is needed. china has a high burden of rsv infection (4) , but rsv surveillance in this country is less established, and implementation of diagnostic tests is limited. several previous studies reported an rsv prevalence of 17%-33% among children with severe acute respiratory illness (15, 16) , but few have assessed the seasonality or trends of rsv infections in china. not having data available on rsv seasonality in china could encumber implementation of therapy and prophylactic interventions for rsv. since july 2007, we have been monitoring for rsv infection among hospitalized children with pneumonia in beijing, china. in this study, we evaluated the pcr results collected in 8 consecutive years (2007) (2008) (2009) (2010) (2011) (2012) (2013) (2014) (2015) to characterize the seasonality of rsv by year. also, because disease and death attributable to rsv varies from year to year (4, 11, 17) , we explored the factors that might affect rsv activity. during july 1, 2007-june 30, 2015, we enrolled children with pneumonia who were admitted into wards of the departments of respiratory medicine, infectious diseases, and emergency medicine and the pediatric intensive care unit (picu) of beijing children's hospital (beijing, china). we recruited children 28 days-13 years of age who had symptoms of acute infection, defined as fever (body temperature ≥38.0°c) or hypothermia (body temperature <35.5°c), leukocytosis (leukocyte count >15,000 cells/ ml for children <5 years of age or >11,000 cells/ml for children ≥5 years of age), or leukopenia (leukocyte count <5,000 cells/ml for children <5 years of age or <4,000 cells/ml for children ≥5 years of age); had >1 respiratory sign or symptom (i.e., cough, sputum production, shortness of breath, tachypnea [>60 breaths/min for patients <2 months of age, >50 breaths/min for patients 2-11 months of age, >40 breaths/min for patients 12-59 months of age, and >30 breaths/min for patients >5 years of age], wheezing or crackles, dyspnea, or chest pain); and had radiographic evidence suggestive of pneumonia (e.g., chest radiograph showing consolidation, infiltrates, or pleural effusion). we excluded children known to be immunosuppressed (defined as having received a solid organ or hematopoietic stem cell transplant, undergoing chemotherapy, having a history of hiv or aids, or using steroids for >30 days). we obtained informed consent from each child's parents or guardians before enrollment. the study protocol was approved by the ethics review committee at the institute of pathogen biology, chinese academy of medical sciences, beijing, china. we transferred the nasopharyngeal aspirates of each enrolled patient into universal transport medium (copan group, https://www.copangroup.com), distributed them into aliquots, and stored them at -80°c. we screened for rsv subgroups a (rsv-a) and b (rsv-b) and other common respiratory viruses, including influenza virus (a and b), human rhinovirus, human parainfluenza viruses 1-4, human adenovirus, human enterovirus, human bocavirus, human metapneumovirus, and human coronavirus (229e, oc43, nl63, and hku1), using multiplex reverse transcription pcr and pcr assays as described (18) . at enrollment, using a standardized reporting form, we collected demographic data (sex and age), epidemiologic data (date of illness onset and history of prematurity [defined as birth at gestational age <37 weeks]), and clinical data (signs, symptoms, and concurrent medical conditions). concurrent medical conditions included congenital heart disease (chd; i.e., children with an international classification of diseases, ninth revision [icd-9], diagnostic code 745.xx, 746.xx, or 747.xx), chronic lung disease (e.g., bronchopulmonary dysplasia), chromosomal anomalies (e.g., down syndrome), moderate to severe anemia (hemoglobin <90 g/l), and malnutrition. we also collected data on clinical outcomes, including picu admission, noninvasive ventilation (e.g., continuous positive airway pressure), invasive ventilation (i.e., mechanical ventilation involving tracheostomy or endotracheal tube), acute respiratory failure (icd-9 code 518.81), shock (icd-9 code 785.5x), sepsis (icd-9 code 038.xx), and death, by abstracting data from medical charts. we fitted a logistic regression model (with sine and cosine functions of the illness onset week) to individual patient data and retained a seasonal curve as described previously (19) . to profile the seasonality of rsv with a smooth seasonal curve, we adopted an approach described previously by the us centers for disease control and prevention: we defined the rsv season as consecutive weeks during which the percentage of tests positive for rsv per week exceeded a threshold of 10% (20, 21) . the peak was expected to occur between the dates of onset and offset, unless an outbreak took place outside of the season. we used a multivariable logistic regression model to explore the factors that could affect rsv transmission over a period of years. we introduced a pair of sine and cosine functions of illness onset week into the model as described in the previous paragraph. we modeled age and year using a restricted cubic spline with 5 knots (22) . we also introduced into the model other factors that differed significantly (p<0.10 in bivariate analysis), such as sex, dominant rsv subgroup in each season, concurrent medical conditions, and picu admission during hospitalization. we conducted all analyses in r version 2.15.3 (https://cran.r-project.org) using mgcv package version 1.8-15 (23) . during the study period, 9,950 children with a primary diagnosis of pneumonia (icd-9 codes 480-488) were admitted to beijing children's hospital (figure 1, overall, rsv was identified in 1,270 (30%) children among the enrolled cohort. we observed a holiday effect for the chinese spring festival (occurring in late january or early february) each year. a sharp decrease in sample number and samples positive for rsv were observed each year during this event, resulting in a bimodal distribution curve ( figure 1, during the study, 335 (8%) hospitalized children with pneumonia were admitted into the picu, and 8 died (median age 1.4 years, range 4 months-13 years). rsv-positive children were more likely than rsv-negative children to be admitted into the picu (positive 10% vs. negative 7%; p = 0.001), receive noninvasive ventilation (positive 20% vs. negative 10%; p<0.001), and have respiratory failure (positive 16% vs. negative 9%; p<0.001) ( table 2 ). of the 8 deceased children, 2 were positive for adenovirus, 1 for enterovirus, and 1 for both human parainfluenza virus 1 and human bocavirus; none of the deceased children tested positive for rsv. all 8 deceased children were born at term, and only 2 had concurrent medical conditions at hospital admission (1 chd, 1 iga nephropathy). (18) 154 (22) 89 (17) 84 (13) 68 (14) 60 (14) 28 (7) 28 (10) 12-23 54 (8) 94 (14) 71 (14) 104 (16) 69 (14) 48 (11) 39 (9) 19 (7) 24-59 78 (11) 82 (12) 61 (12) 118 (18) 60 (12) 63 (14) 110 (26) 55 (20) >60 155 (22) 135 (19) 91 (17) 204 (31) 151 (31) 159 (36) 212 (50) 120 (43) underlying medical condition 68 (9) 103 (15) 65 (12) 88 (14) 71 (14) 44 (10) 32 (7) 22 (8) <0.001 history of prematurity ‡ 8 (1) 23 (3) 24 (5) 36 (6) 29 (6) 27 (6) 22 (5) 13 (5) <0.001 congenital heart disease § 49 (7) 76 (11) 38 (7) 47 (7) 38 (8) 13 (3) 10 (2) 7 (2) <0.001 chronic lung diseases ¶ 6 (1) we identified 8 distinctive rsv seasons during the study period (july 1, 2007-june 30, 2015) using our model (p<0.001 for all), even though the number of rsv-positive children varied (from 41 to 280) by study year. the percentage of pcr tests positive for rsv each week throughout the summer months typically exceeded 3% but was <10%; once the 10% positivity threshold was exceeded, the percentage of tests positive for rsv increased rapidly. overall, 97% of rsv-positive pcrs occurred within the period defined by the 10% threshold (table 3 ; figure 3 ). using the 10% cutoff point and a fitted seasonal curve, we determined the following rsv season parameters for each of the 8 years of our study: season onset (first of 2 consecutive weeks during which rsv positivity in seasonal curve exceeded 10%), duration, peak (week with highest rsv positivity in seasonal curve), offset (last week that rsv positivity in seasonal curve exceeded 10%), and percentage of rsv-positive samples captured within the season. data show that the average season onset occurred at calendar week 41 (mid-october) and lasted 33 weeks, through week 20 (mid-may) of the next year ( table 3 ). the peak of rsv activity occurred at calendar week 3 (mid-january). rsv circulated at low levels during off-seasons. overall, 24 (3.3%) of 724 children tested were positive for rsv in summer, a finding consistent with broberg et al. (table 3) . we explored factors that significantly affected rsv activity by using multivariable modeling. after adjusting for week of illness onset, year, picu admission, and chd, age had a strong decreasing monotonic effect on rsv infection (p<0.001; figure 5 , panel a). we also observed a yearly cyclic pattern with a distinct periodicity of 4 years for rsv year in the response plot (p<0.001; figure 5 , panel b). however, when we introduced rsv-a-dominant year into the model as a factor to be adjusted for, the cyclic trends and the year's association with rsv activity diminished (p = 0.11). we performed a pcr-based rsv screening in a cohort of children with pneumonia in beijing to assess rsv seasonality. our findings show that on average the rsv season starts at calendar week 41 (mid-october) and lasts 33 weeks through week 20 of the next year (mid-may); 97% of total rsv-positive pcrs occur during this period. this seasonal pattern is highly consistent with that reported in the united states (10) , a country at the same latitude as china in the northern hemisphere. the world health organization is expecting an rsv vaccine on the market within 5-10 years (24); ≈62 rsv vaccine candidates are under development, and 19 of them are undergoing clinical trials. our study of rsv seasonality and trends in beijing could inform vaccine development and the optimization of future vaccination strategies, such as the timing of administration (year-round or seasonal), target population (mothers or infants), and ingredients, for china, the country with the largest population in the world. we used rsv percent positivity to determine rsv seasonality because this method is highly sensitive and can be used to define rsv season not only retrospectively at the end of the season but also during the year in real-world practice. the main disadvantage of this strategy is that results can be driven in large part by the denominator; the presence of other cocirculating pathogens that cause pneumonia, especially influenza viruses, can influence the parameters of the rsv season. although the number of pcrs performed each year varied considerably and a shift toward older age was evident among affected children in the last 2-3 years of the study, the seasonal pattern each year remained consistent and reproducible when classifying the study year by the prevailing rsv subgroup. during the 8 consecutive years of the study, the number of children admitted with pneumonia dropped from 718 in the first year to 282 in the last; rsv positivity also decreased remarkably, from 280 in the first year to 41 in the last. the reasons for this decrease might be attributable to a decrease in the occurrence of rsv-associated pneumonia in the pediatric population or, more likely, a fall in pediatrician interest for enrolling patients into the study over time, given we did not observe a simultaneously drastic drop of pneumonia cases in the hospital (figure 1, panel a) . moreover, the demographic characteristics of patients also differed throughout the study years. in the first study year, more children 28 days-5 months of age (42%) were enrolled, and in the last, more children >5 years of age (43%) were enrolled. despite this change, the observed seasonal characteristics in each year changed little if any, indicating that viral factors rather than demographic factors had more of an influence over rsv seasonality. when study years were classified by the prevailing rsv subgroup, the observed season onset, peak, and offset showed good reproducibility. we found that >90% of rsv-positive pcrs could be captured during the rsv season for all 8 study years, and the average capture was 97% for 2007-2015 combined. the method we used in our study (pcr-based testing) can be used by local hospitals in china where surveillance data are lacking to compile the extensive amount of data needed to assess rsv seasonality. the shifting in dominance from rsv-a to rsv-b every 2 years was repeatedly observed in our study and others (25) . these shifts have been shown to be strongly associated with changes in the dominant rsv strain circulating (11, 25) ; however, this phenomenon has not been investigated in detail. similar to previous studies (8, 26, 27) , the positivity for rsv-a (19%, 785/4,225) was significantly higher than that for rsv-b (11%, 485/4,225; p<0.001) in our study. the season onset and peak in rsv-a-prevailing years occurred ≈3-5 weeks earlier and duration was ≈6 weeks longer than those observed in rsv-b-prevailing years. the response plot showing the effect of year on rsv activity also indicated patterns of regular peaks in the years rsv-a prevailed (i.e., 2007-08, 2010-11, 2011-12, and 2014-15) , but the effect of the year diminished when rsv-a-prevailing year was adjusted for as a factor in the model, suggesting that repeated shifting between rsv-a and rsv-b might play a substantial role in driving rsv transmission dynamics in populations. this observation is also supported by a model proposed by white et al., who found that the transmission rate of rsv-a (8%) was slightly higher than that of rsv-b (25) . just like the influencing factors explored in other studies (e.g., geographic latitude and longitude, social and demographic factors, population density, and climate) (11, 13, 22) , rsv subgroup replacement might play a key role in the activity of rsv. the alternating nature of rsv subgroups could explain the alternating early-big or late-small pattern and year-to-year variation in epidemic size and timing of rsv transmission observed previously (11, 25) . our finding that rsv subgroup shifting was associated with rsv activity suggests that when using rsv seasonal data or conducting rsv surveillance, one should pay attention to the prevailing subgroups in the season to optimize the timing of immunoprophylaxis. this finding indicates the significance of genotyping in rsv surveillance. although rsv-a cases outnumbered rsv-b in our study, we did not determine the reason for this finding, whether rsv-a caused more symptomatic illness or transmitted more quickly than rsv-b among children. the relationship between rsv subgroup infection and disease severity is still controversial (8, 26, 28, 29) , and this issue warrants further study. in our study, more than half of the rsv hospitalizations occurred in infants 28 days-5 months of age. a monotonic decreasing effect on the activity of rsv with age was observed in the response plot ( figure 5 , panel a), indicating that younger age is a risk factor for rsv infection. children who were in their first months of life had the highest risk for rsv infection (3, 30) . administration of 1 dose of palivizumab (15 mg per kg body weight) each month for 5 months has been recommended to protect children at high risk for severe respiratory infection (e.g., preterm infants and infants with chd in their first year of life) (12) . as of march 2019, palivizumab is not licensed for use in china, and no immunoprophylaxis is available to prevent severe rsv infection. one third (30%) of children in our study with pneumonia requiring hospitalization had rsv infection; this finding is similar to previous estimates of 28%-34% in other countries (1, 2) . considering the high positivity rate of rsv in children with pneumonia, rsv-associated illness should be considered a high priority for public health authorities in china. our study of the relationship between age and rsv infection gives urgency to developing rsv diagnostics and indicates the need to study long-lasting and high-affinity new therapeutics and vaccines in the future (9, 31) . our study has some limitations. first, our study was conducted at 1 local hospital. because no national rsv seasonality data were available for comparison, whether our results could represent other regions in china with an rsv burden is unknown. however, overall, our data are comparable with those observed in the united states. in addition, a previous study conducted in 15 countries of europe showed that rsv seasons peaked later and lasted longer with increasing latitude (13) . because china is a large country that spans several geographic zones and climates, further studies are needed at other locations to fully characterize rsv seasonality in china. second, our study was conducted in children with pneumonia, a population that usually has a high rsv positivity rate. seasonality should also be evaluated in children with mild symptoms and in adults. third, we did not evaluate the size of the local hospital or the minimum number of tests needed each year to conduct a more reliable analysis of rsv seasonality. fourth, we excluded children without fever from our study. because illness caused by rsv can manifest without fever, particularly among infants (32) , the case definition used in our study was suboptimal, and we might have missed some children infected with rsv. considering no international rsv case definition exists (13), we encourage researchers in future studies to determine a working case definition that can balance the many attributes (e.g., accuracy, feasibility, flexibility, and usefulness) of the various definitions that have been used to conduct rsv surveillance or study disease burden. in conclusion, rsv infection showed distinctive seasonal patterns in beijing, china. the prevailing rsv subgroup in a given season appears to affect the timing of rsv activity. monitoring alterations of rsv subgroups might provide a better and more comprehensive description of rsv transmission and trends. a pcr-based diagnostic test at local hospitals could be a useful tool to determine rsv seasonality in circumstances where rsv season is unknown or surveillance is less established. viral etiology of severe pneumonia among kenyan infants and children centers for disease control and prevention epic study team. community-acquired pneumonia requiring hospitalization among u.s. children respiratory syncytial virus-associated hospitalizations among infants and young children in the united states respiratory syncytial virus global epidemiology network. global, regional, and national disease burden estimates of acute lower respiratory infections due to respiratory syncytial virus in young children in 2015: a systematic review and modelling study respiratory syncytial virus infection in elderly and high-risk adults mortality associated with influenza and respiratory syncytial virus in the united states respiratory syncytial virus network. lower respiratory tract infection caused by respiratory syncytial virus: current management and new therapeutics respiratory syncytial virus: infection, detection, and new options for prevention and treatment advances in rsv vaccine research and development-a global agenda respiratory syncytial virus seasonality-united states environmental drivers of the spatiotemporal dynamics of respiratory syncytial virus in the united states updated guidance for palivizumab prophylaxis among infants and young children at increased risk of hospitalization for respiratory syncytial virus infection european influenza surveillance network. seasonality and geographical spread of respiratory syncytial virus epidemics in 15 european countries world health organization. who global respiratory syncytial virus surveillance viral etiologies of hospitalized acute lower respiratory infection patients in china clinical and epidemiologic characteristics of respiratory syncytial virus infection among children aged <5 years centers for disease control and prevention. respiratory syncytial virus activity-united states prevalence of human respiratory viruses in adults with acute respiratory tract infections in beijing studying seasonality by using sine and cosine functions in regression analysis centers for disease control and prevention. respiratory syncytial virus-united states determining the seasonality of respiratory syncytial virus in the united states: the impact of increased molecular testing temporal trends of respiratory syncytial virus-associated hospital and icu admissions across the united states generalized additive models: an introduction with r meeting report: who consultation on respiratory syncytial virus (rsv) vaccine development the transmission dynamics of groups a and b human respiratory syncytial virus (hrsv) in england & wales and finland: seasonality and cross-protection distribution of respiratory syncytial virus subtypes a and b among infants presenting to the emergency department with lower respiratory tract infection or apnea characteristics and their clinical relevance of respiratory syncytial virus types and genotypes circulating in northern italy in five consecutive winter seasons distribution and clinical impact of human respiratory syncytial virus genotypes in hospitalized children over 2 winter seasons respiratory syncytial virus and other viral infections among children under two years old in southern vietnam 2009-2010: clinical characteristics and disease severity the burden of respiratory syncytial virus infection in young children group b streptococcus and respiratory syncytial virus immunisation during pregnancy: a landscape analysis evaluation of case definitions for estimation of respiratory syncytial virus associated hospitalizations among children in a rural community of northern india we are thankful to all the patients participating in the study. we are also thankful to the clinicians and other medical staff who helped collect the samples and clinical data. key: cord-319675-mwy3t1ny authors: gu, li; qu, jiuxin; sun, bing; yu, xiaomin; li, hui; cao, bin title: sustained viremia and high viral load in respiratory tract secretions are predictors for death in immunocompetent adults with adenovirus pneumonia date: 2016-08-17 journal: plos one doi: 10.1371/journal.pone.0160777 sha: doc_id: 319675 cord_uid: mwy3t1ny the predictors for fatal adenovirus (adv) pneumonia among immunocompetent adults are unclear. laboratory-confirmed, hospitalized adv pneumonia adults were prospectively enrolled in beijing chao-yang hospital from march to june 2013. clinical data and serial whole blood and respiratory tract secretions from such patients were collected. quantitative real-time polymerase chain reaction was performed to quantify the viral load. a total of 14 adv pneumonia cases were consecutively enrolled, and four of them were fatal. ten cases were caused by adv-55, three by adv-7 and one by adv-3. there were no differences in age, gender or underlying diseases between the patients in the fatal cases and surviving cases. at admission (on day 5–7 after illness onset), the patients in fatal cases presented higher initial viral loads in respiratory tract secretions (8.578 ± 2.115 vs 6.263 ± 1.225 log(10) copies/ml, p = 0.023). all patients in fatal cases presented with viremia on day 12–14 (100% vs 66.7%, p = 0.017). a higher initial viral load in the respiratory tract and sustained viremia (more than 2 weeks) may be predictors for fatal clinical outcomes. severe adenovirus (adv) infections causing significant acute respiratory distress syndrome have raised concerns for immunocompetent adults [1] . most severe cases were previously reported to be associated with adv-3, 4, 7 and 21 [2, 3] . a new strain, adv-55 (formerly known as adv-11a), has been a major adv pneumonia pathogen in immunocompetent adolescents and adults in china since 2008 [4] . our previous study identified a fatal adv-55 infection case with a patient who presented with systemic infection and high-level viremia [5] . however, even though adv-55 as well as adv-3, 4, 7 and 21 can cause severe cases, those strains are not necessarily associated with bad outcomes, and the pathogenesis of fatality is still unknown. we have already ascertained that adv can be detected in whole blood specimens of severe cases, but we still lack the knowledge about the viral shedding history and the relationship between the viral clearance in the respiratory tract and viremia duration and clinical outcome. in this study, we focus on the dynamic virological changes in whole blood and respiratory tract secretions to see if lethal cases experienced a high viral load and longer duration of viral shedding compared to the non-lethal cases. we hypothesize that sustained virus shedding in the blood and/or respiratory tract can appear in severe immunocompetent adult cases, and it may be a risk factor for fatal outcome. the study was reviewed and approved by the institutional review board of beijing chao-yang hospital (the project approval number is 10-ke-49). written informed consent was provided by all adults and the parents of patients aged less than 18 years. adults with communityacquired pneumonia (cap) (age14yrs) admitted to beijing chao-yang hospital from march to june 2013 were prospectively included. patients with hiv infection or neutropenia; receiving immunosuppressive chemotherapy or steroids equivalent to prednisone >15 mg/d for 30 days; who were pregnant or breast feeding women; or who were known or suspected to have active tuberculosis were excluded. methods of etiological evaluation followed the standard for adults suspected with cap [4] [5] [6] . in short, sputum or respiratory tract aspiration, blood and urine were collected at admission and submitted to the infectious disease and clinical microbiology laboratory. microbiological methods were based on the following tests: (1)sputum specimens for gram stain and cultures considered valid only if microscopy showed > 25 neutrophils and <10 epithelial cells per low field microscopy; (2)urine specimens for the rapid detection of s. pneumonia and legionella pneumophila antigen; (3) blood culture; (4) sputum and tracheal aspiration for virus real-time polymerase chain reaction (pcr) detection, including rhinovirus, influenza a and b, respiratory syncytial virus a and b, adenovirus, parainfluenza 1-4, coronavirus oc43 and 229e, and metapneumovirus; and (5) sputum for mycoplasma pneumoniae pcr detection. only subjects with positive adenovirus results who were negative for other etiologies during the study period were enrolled. the results of pcr testing for respiratory viruses were reported to clinicians within 6 hours after sputum collection. for those with positive adv pcr testing, serial whole blood and respiratory tract samples were collected until death or discharge. in this study, we tried to collect the same type of respiratory tract samples from each patient to carry out serial viral load testing, to lower bias from different types of samples. for patients with mechanical ventilation, we collected serial tracheal aspirations, and for patients without mechanical ventilation, we collected sputum samples. we also collected data regarding age, gender, co-morbidities, clinical symptoms, vital signs, antimicrobial treatment, chest radiographic findings, and laboratory results. recorded complications included the following: use of mechanical ventilation and extracorporeal membrane oxygenation (ecmo), and second bacterial or fungal infection. patients were also followed up to discharge or death. respiratory tract samples, including sputum and tracheal aspirations, were processed by equal volume 1% trypsin digestion. the viral dna was extracted from 1 milliliter (ml) digested respiratory sample and 1 ml whole blood sample using a qiaamp dna mini kit (qiagen, valencia, ca, usa). first, we amplified the entire hexon genes of the samples by pcr. the primer sequences are listed in table 1 [7] . then, the adv type was determined using blast (http:// blast.ncbi.nlm.nih.gov/blast.cgi). regarding the quantification of adv in the samples, we used a commercial fq-pcr kit (daan gene, cat. #da-b067, guangzhou, china) targeting the dna polymerase gene [genebank: kf279555.1] two-tailed independent samples t-test or mann-whitney u-test (on condition of non-normal distributions) was used to compare continuous variables between the two groups. for the categorical data, univariate analysis was performed using the chi-square test or fisher's exact test. significance was fixed at p value < 0.05. data analysis was performed using spss 15.0 (spss inc; chicago, il). from march 2013 to june 2013, a total of 14 admitted cases were confirmed with adenovirus as the only pathogen of pneumonia; four of the patients died in the icu. the mean age was 30 years old. males predominated in number over females, with a sex ratio of approximately 6:1. only two patients had co-morbidities, one with tuberculous pleuritis for 2 months and one with congenital heart disease. there were no differences in age, gender or underlying diseases between fatal cases and surviving cases. there are three types of adv found in this study, adv-55 (n = 10), adv-7 (n = 3) and adv-3 (n = 1). the adv-55 ratio in the fatal group was similar to that in the surviving group (100% vs 60%, p = 0.251) ( table 2) . clinical features: comparison between surviving and fatal cases most clinical symptoms and signs noted in the surviving cases and fatal cases had no differences, except for the pneumonia severity index (psi) score and dyspnea. the fatal cases had a higher psi score (101.3 ± 8.0) and dyspnea incidence (4/4) compared to the psi score (46.5±26.7) and dyspnea incidence (2/10) in surviving cases (p = 0.002 and 0.015, respectively) ( table 2 ). all of the fatal cases described bilateral involvement on chest radiography, and 75% noted pleural effusion (table 2) . for the surviving cases, 40% (4/10) noted bilateral involvement, and 20% (2/10) described pleural effusion. there were no significant differences between the two groups in bilateral involvement or pleural effusion. the adv load tracking in the respiratory tract samples and whole blood the mean time from disease onset to initial pcr test for respiratory tract samples and whole blood was 6.23 days, and there were no significant difference between the fatal group (7.5±1.8days) and surviving group (5.5±1.3days). we measured the adenoviral load in respiratory tract samples and whole blood, expressed as log 10 dna copies per ml samples. for initial viral load in respiratory tract samples on day 5-7 after disease onset, the results showed that, compared to the surviving cases, the fatal cases had a higher initial viral load (8.578 ± 2.115 vs 6.263 ± 1.225, p = 0.023) (fig 1a) . however, for the initial viral load in whole blood, there was no significant difference between the two groups (3.71±2.67 vs 5.978±1.610, p = 0.162, fig 1b) . we also investigated viral shedding duration evaluated by positive ratio on day 5-7 and on day 12-14 after illness onset. for respiratory tract samples, there was no significant difference of viral positive ratio between the two groups either on day 5-7 (100% vs 100%) or on day 12-14 after onset of disease (60% vs 100%, p = 0.126) (fig 1c) . viremia was common at admission (on 5-7 day after onset of illness), as noted in 100% (4/4) of the fatal cases and 71.4% (5/7) of the surviving cases (p = 0.491). on day 12-14 after onset of illness, however, it persisted in 100% (4/4) of the fatal cases with 33.3% (2/6) of the surviving cases (p = 0.017) (fig 1d) . for the surviving severe patients, we found that the clinical manifestation recovered gradually with a downward trend in viral load in respiratory tract and whole blood samples. as shown in fig 2, a 25 -year-old male with acute respiratory distress syndrome (ards) had a high initial viral load (10 8.32 copies/ml) in tracheal aspiration. his condition improved, with the sputum viral load going down on day 14, and ecmo was withdrawn on day 14. with the viral load in blood going down to negative on day 22, the clinical condition was significantly improved, and the patient did not need oxygen therapy; the chest x-ray was also remarkably resolved. as shown in fig 3, the respiratory tract viral load of a 34-year-old male with ards also had a high initial viral load (10 9.25 copies/ml) in tracheal aspiration. the viral load in tracheal aspiration gradually decreased during the first 20 days, and the lung infiltrate absorbed partially. however, the viral load trended up again and the patient died. he also maintained a viral load in whole blood before death. antibiotics were given to all of the patients empirically before and after confirmed diagnosis. there is currently no formally approved antiviral therapy for the treatment of severe lifethreatening adenovirus infection in china. acyclovir, ganciclovir or ribavirin is commonly chosen by the physician to treat adenoviral infection. in this study, all of the fatal patients were administered antiviral drugs-one was treated with ganciclovir, two with acyclovir and one with ribavirin. in surviving patients, 50% were treated with antiviral drugs-three with ganciclovir, one with acyclovir and one with ribavirin. all of the fatal patients (4/4) were complicated with ards and admitted to the icu. they needed mechanical ventilation, and three of them received ecmo to maintain oxygenation. for surviving patients, one of them (1/10) was admitted to the icu due to ards, and had mechanical ventilation and ecmo; one patient with respiratory failure was treated with non-invasive ventilation, and one patient was treated for myocarditis. the myocarditis patient presented with peak levels of creatine kinase isoenzyme (ck-mb) and cardiac troponin i (ctni) on day 7-8 after disease onset, and eight days later, with viral load going down to negative, ck-mb and ctni went down to normal levels in parallel (fig 4) . for the four fatal patients, the times of death were on days 14, 16, 22 and 28 after disease onset, respectively. there was no significant difference in length of stay in-hospital between the two groups (13.5±6.5 days vs 13.3±9.2 days, p = 0.969) ( table 2 ). there were three patients complicated with bacterial or fungal infections among the fatal cases. the first patient presented with consecutive aspergillus fumigatus in tracheal aspirate cultures, the second patient had a cavity present on chest ct, and the third patient had acinetobacter baumannii in tracheal aspirate and pleural effusion cultures. there were only two patients complicated with superinfections in surviving patients. one patient (a 16-year-old male) presented with a typical crescent sign and halo sign with chest ct follow-up; voriconazole was administered to resolve this condition (fig 5) . another patient with ecmo presented with acinetobacter baumannii in tracheal aspirates. we investigated the relationship between the virological factors and clinical outcomes in a cohort of 14 hospitalized adults with adv pneumonia. our results suggest that a higher initial viral load (10 8 copy/ml) in the respiratory tract samples on day 5-7 after disease onset is a predictor for fatal clinical outcome. we also reported that viremia is common and sustained viremia for 14 days or more may be associated with mortality the pathogenesis of mortality in adv pneumonia is still unknown. virological factors, e.g., a new strain with new genetics, viral load, slow virus clearance and systemic infection with viremia likely play key roles for severe adv [2] [3] [4] [5] [6] [7] [8] . however, no study has evaluated the viral shedding history among immunocompetent adults with adv pneumonia. this study first monitored the consecutive viral load in respiratory tract samples and whole blood samples. our previous clinical study demonstrated that on day 5-7 after disease onset, the peak stage of illness presented for patients with shortness of breath or severe dyspnea [5] . again, in this study, we showed that the viral load on day 5-7 could also provide an insight into the severity of illness. evidence even proved that a higher level of viral load in respiratory tract samples on day 5-7 after disease onset was significantly associated with fatal outcome. we have noted that viremia is quite common on day 5-7 after disease onset, when 9 out of 11 (81.8%) patients had viremia. adenovirus viremia has been found in hematopoietic stem cell transplantation recipients and associated with adv disease [8] . compared with previous reports of viremia and clinical outcomes, another novel finding is that we demonstrated that fatal outcomes could be predicted by sustained viremia, but not by viremia itself. in this study, we showed that 100% (4/4) of patients in fatal cases presented with viremia on day 12-14 after disease onset, compared with 60% (p = 0.126) of the patients in surviving cases. in one case, as shown in fig 2, even though the patient presented with a higher viral load (10 8.32 copies / ml) in tracheal aspiration, which may be associated fatal outcome, his clinical manifestation recovered gradually with a downward trend in the viral load in respiratory tract and whole blood samples. compared to this case in fig 3, the patient described in fig 3 not only had a higher viral load (10 9.25 copies/ml) in tracheal aspiration but also presented with sustained elevated viral copies, especially in whole blood. shike et al. also reported a 6-monthold infant with systemic infection by adenovirus who had high-level viremia and showed reduction in viral load paralleling her clinical recovery [9] . therefore, in severe cases, dynamic monitoring of viral shedding, especially in whole blood, could help predict the clinical outcome. patients might have bad outcomes if the viral load in whole blood does not present a significant downward trend around two weeks after disease onset. there is currently no formally approved antiviral therapy for the treatment of severe lifethreatening adenovirus infection in china. cidofovir is considered the medicine of choice for severe infection in immunocompromised patients. cidofovir is not available in most hospitals in china, including our hospital. acyclovir, ganciclovir or ribavirin is usually prescribed in china. in this study, antiviral drugs were administered in all of the fatal cases-one patient was treated with ganciclovir, two with acyclovir and one with ribavirin. in surviving patients, 50% were treated by antiviral drugs-three with ganciclovir, one with acyclovir and one with ribavirin. the choice of the antiviral medicine was decided by the patient's physician. as none of these three medicines have been confirmed to be effective for adv infection, the relationship between viral shedding and clinical outcomes in this study was not associated with anti-adenoviral treatment effect. our study has two limitations. as adv 55 was the most common infection type (10/14, 71.4%) in this study, results might be more significant in adv 55-associated pneumonia and might not be generalizable to other types of adv pneumonia. in our previous study, adults infected with adv 55 were 10 years older and presented with higher psi scores compared with adults infected with other serotypes [4] . another limitation of this descriptive work may be the small number of analyzed patients, especially in the group of fatal cases (n = 4). more cases are needed to confirm our findings. in conclusion, our data provide new insight into the virology of adv pneumonia. a higher initial viral load (10 8 copy/ml) in the respiratory tract on day 5-7 after disease onset and sustained viremia for 2 weeks or more may be associated with fatal clinical outcomes. two fatal cases of adenovirus-related illness in previously healthy young adults-illinois severe adenovirus pneumonia in immunocompetent adults: a case report and review of the literature severe pneumonia due to adenovirus serotype 14: a new respiratory threat? emergence of community acquired adenovirus type 55 as a cause of community-onset pneumonia severe community-acquired pneumonia caused by adenovirus type 11 in immunocompetent adults in beijing viral and mycoplasma pneumoniae community-acquired pneumonia and novel clinical outcome evaluation in ambulatory adult patients in china outbreak of acute respiratory disease in china caused by b2 species of adenovius type 11 adenovirus viremia and disease: comparison of t celle depleted and conventional hematopoietic stem cell transplantation recipients from a single institution quantitation of adenovirus genome during acute infection in normal children we thank drs. ran li, chen ma, yudong yin, lin wu, and yiqun guo for their contributions to specimen collection.notation of publication: this work has been accepted as an oral presentation at the third isirv-avg conference on influenza and other respiratory virus infections: advances in clinical management on june 4 th -6 th , 2014 at the keio plaza hotel, tokyo, japan. conceived and designed the experiments: bc. lg jxq bs xmy.analyzed the data: lg hl.contributed reagents/materials/analysis tools: jxq bc.wrote the paper: lg jxq bc. competing interests: the authors have declared that no competing interests exist. key: cord-352684-4o623x3n authors: tan, m. y.; tan, l. n.; aw, m. m.; quak, s. h.; karthik, s. v. title: bocavirus infection following paediatric liver transplantation date: 2016-10-23 journal: pediatr transplant doi: 10.1111/petr.12840 sha: doc_id: 352684 cord_uid: 4o623x3n hbov is an emergent virus, which is frequently detected as a co‐infective agent. however, it can cause disease on its own. it is associated with respiratory and diarrhoeal illness in children and adults, whether immunocompetent or immunocompromised. we report hbov infection in a child post‐liver transplantation, who presented with persistent fever and mild tachypnea, 3 weeks after a successful transplant. she recovered spontaneously with no graft dysfunction. hbov is a recently discovered virus of the parvoviridae family that has been recognized as a co-infective virus along with other established viruses causing respiratory tract infection. 1 it has been associated with respiratory tract and/or diarrhoeal illnesses in children and adults, although having a predilection for children, in both immunocompetent and immunocompromised patients. 1 after solid organ transplant, children are immunocompromised. they are predominantly on calcineurin inhibitor-based immunosuppressive regimens, with or without corticosteroids, to avoid graft rejection. this puts them at increased risk of infections. although with improved molecular diagnostic techniques, newer viruses have been detected with increasing incidence, more so in the transplant population. 2 we report our experience with hbov following paediatric liver transplantation. a 15-month-old girl underwent liver transplant for decompensated chronic liver disease, secondary to underlying biliary atresia. her pretransplant clinical course had been complicated by sepsis with renal impairment. her initial sepsis was attributed to intra-abdominal collections. she had episodes of prolonged fever even after her collections were drained, and a respiratory multiplex (pcr-based test) to look for viruses, from nasopharyngeal aspirates, was negative (3 weeks prior to transplant). she had eventual full recovery. in terms of immunosuppression, intravenous basiliximab was used as a renal-sparing agent in the immediate post-transplant period with tacrolimus being introduced later. intravenous mycophenolate mofetil was used as adjuvant immunosuppression, with corticosteroids. her course was complicated by biopsy-proven acute cellular rejection on the 7th postoperative day, requiring pulsed intravenous methylprednisolone (10 mg/kg/day). steroids were subsequently weaned off. she made steady progress post-transplant, with normal graft function. she developed persistent low-grade pyrexia up to 38.5°c (101.3 fahrenheit), 3 weeks after her transplant. clinically, she had mild tachypnea even when not febrile, with normal breath sounds on examination. she had mild ascites, which was improving from her preliver transplant state, thus unlikely contributing to her tachypnea. her abdomen was otherwise soft and non-tender. there was no diarrhoea or other clinical focus of infection. her inflammatory markers were unremarkable-full blood count showed a white cell count of 9.72×10 9 /l (normal range 5.00-15.00), with mild lymphopenia at 2.00×10 9 /l (normal range 6.00-9.00), without neutropenia. crp was minimally raised, at 14 mg/l. investigations were done to look for a source of the fever. this included a blood and urine culture which returned negative. an ultrasound of the abdomen did not show significant intra-abdominal collections or abscesses. chest x-ray showed patchy bibasal atelectasis ( figure 1 ), with mild peribronchial haziness, without any consolidation. a nasal swab sample sent for immunofluorescence for common respiratory viruses was negative as well. she remained clinically well, but was started empirically on broadspectrum antibiotics, while cultures were pending. as the fever persisted, a ct scan of the abdomen was obtained to look for collections that could have been missed earlier. this showed a possible collection; however, when drained, it did not show any evidence of bacterial or fungal infection; and her fever persisted despite this drainage. her ct scan also showed bilateral atelectasis that was seen in her chest x-ray. a respiratory multiplex pcr to look for other viruses was also sent off from nasopharyngeal fluid. the pcr (the xtag rvp fast v2 nucleic acid multiple test) for hbov was positive, with no other viruses detected (negative for influenza, parainfluenza, and coronavirus groups). her fever settled after a week, and she was well at discharge. we did not have to modify her immunosuppression, and her graft function remains good on follow-up with no other clinical concerns. clinical presentation is still not clearly defined, but has been associated with respiratory tract infections (both upper and lower tracts) as well as gastroenteritis in children. 3 it is also commonly seen as a co-infection 3 and found shed in the stool and respiratory secretions of asymptomatic children. 4 however, in recent years, it has been emerged that hbov alone is pathogenic and can cause disease in immunocompetent children, most commonly presenting with respiratory symptoms, such as wheeze and bronchiolitis. 4 in our patient, there were many potential sources of fever, one being the abdominal collection which was drained. we acknowledge that she was on broad-spectrum antibiotics and hence cultures might be negative. however, her fever persisted for a week after, despite drainage and antibiotics, leading us to conclude hbov as the most likely pathogen causing her fever. while we recognize hbov still has unclear clinical significance, in our patient, we think hbov is most likely pathogenic given the consistent respiratory symptoms, single pathogen isolated, with no other source to explain symptoms. interesting to note is that she did have respiratory samples sent for if as well as for multiplex testing 3 weeks prior to transplant, which were negative. thus, it is likely that hbov was acquired and not a reflection of asymptomatic carriage. hence, the new detection of hbov coinciding with her respiratory symptoms and fever further supports our hypothesis that hbov is pathogenic. we do acknowledge the limitation that we did not perform subsequent advanced testing such as serological analysis, quantitative single real-time pcr, immunofluorescence assays or sequencing to confirm hbov-positive results. unfortunately, these advanced tests are not routinely offered in our laboratory and available only at highly specialized diagnostic laboratories overseas. however, that would incur a high cost, and our patient was an overseas patient from a neighbouring country with limited financial resources. the value and the role of quantitative assay in assessing the severity of the hbov disease are also debated. 5, 6 more importantly, the patient's fever settled after a week, and she was discharged well. thus, we did not pursue further advanced analysis of subsequent respiratory samples by pcr or other methods to confirm the hbov or gain information on duration of viral shedding in the respiratory tract or of blood samples to monitor for hbov dissemination. we also did not send blood samples to assess for viraemia. the message that we want to give across through the case is, even in the absence of availability or requirement of specific antiviral therapy in the given setting, being vigilant about newer emerging viruses that can potentially cause severe disease is needed to avoid unnecessary and often expensive investigations. the hbov is associated with higher hospitalization days, 6 can cause neurological infections, 7 and may contribute to chronic disease in adulthood as it can persist and reactive during later period of life. 8 there are a few reports of hbov infections in immunocompromised hosts wherein they have caused respiratory or diarrhoeal illness, and rarely even life-threatening conditions in the stem cell transplant population. severe diarrhoeal illness was reported in a 9-year-old boy with underlying erythropoietic protoporphyria, who had undergone orthotopic liver transplant and previous stem cell transplant, 9 months and 4 months, respectively, prior to presentation. 9 hbov has also been shown to cause disseminated illness in a child, post-stem cell transplant. 10 hbov infection has also been postulated to be related to hepatitis in a child with t-cell immunodeficiency. 11 there are no reports of hbov-associated respiratory infections in children after liver transplantation. our patient had an atypical presentation with pyrexia and only mild tachypnea-she had no cough nor symptoms or signs of a lower respiratory tract infection. the commonly available if-based testing for respiratory viruses was negative as well. the diagnosis was achieved only with the extended pcr-based test. her chest x-ray did not show any significant changes, although there were subtle findings of bibasal atelectasis ( figure 1 ). these are frequently seen in children with significant ascites preliver transplant, especially in the immediate post-transplant period, as they are weaned off mechanical ventilation. moreover, our patient had significant ascites pretransplant which was actually improving when her persistent fevers began, thus unlikely to explain her tachypnea. our experience highlights the importance of awareness to have a lower threshold for screening for atypical newer pathogens, which could potentially cause infectious complications post-liver transplantation. our patient will add to the international experience of hbov in transplant recipients. human bocavirus-the first 5 years rare and emerging viral infections in transplant recipients human bocavirus: passenger or pathogen in acute respiratory tract infections? chapter 15 -human bocavirus comorbidity and high viral load linked to clinical presentation of respiratory human bocavirus infection bocavirus infection in otherwise healthy children with respiratory disease neurological manifestaions in acute onset of viral gastroentritis frequency and clinical relevance of human bocavirus infection in acute exacerbations of chronic obstructive pulmonary disease human bocavirus in an immunocompromised child presenting with severe diarrhea disseminated bocavirus infection after stem cell transplant hepatitis and human bocavirus primary infection in a child with t-cell deficiency we thank dr dimple rajgor for her assistance in editing, formatting, and submission of the manuscript for publication. my tan and sv karthik: initiated and drafted the report; ln tan, m aw, and sh quak: involved in patient care and critical input on draft case report. all authors contributed to the writing, reviewing, editing, and approval of the case report submitted. key: cord-354918-129inbwq authors: kotsimbos, t.; mccormack, j. title: respiratory infectious disease: complacency with empiricism in the age of molecular science. we can do better! date: 2007-06-04 journal: intern med j doi: 10.1111/j.1445-5994.2007.01424.x sha: doc_id: 354918 cord_uid: 129inbwq nan the recently launched document, 'burden of respiratory infectious disease in australia' is a timely reminder of the full impact of respiratory infection on the health of australians, both as multiple specific disease entities that are significant in their own right and as an important complicating factor that cuts across all areas of respiratory health. 1 the document outlines the dramatic mismatch between the enormity of total disease burden and the relative paucity of effective management strategies. in the first instance this mismatch may seem surprising, but on further reflection, this should not be the case. a few home truths (i) viral infections are an important cause of respiratory infection and yet a specific diagnosis is rarely made, (ii) few antiviral therapies exist and when they do -such as in the case of influenza -they are either underused or overused because of diagnostic inefficiencies, (iii) our approach to using antibacterial agents is guided by 'what should reasonably be covered' or 'what organisms are being missed' rather than a treatment regimen targeted for a particular organism, (iv) in association with diagnostic uncertainty, we do not make sufficient allowances for immunocompromised states or overexuberant immune responses to respiratory infectious disease (rid) and (v) current antibiotic guidelines encourage an approach of increasingly covering all potential organisms depending on the severity of illness. these issues lead to increased direct costs both to the individual (unnecessary exposure to side-effects) and the community (exposure to antibiotic selection pressure and future antibiotic resistance, financial cost) and indirect costs in that other potentially useful treatment approaches are not considered (e.g. anti-inflammatory and immunemodifying drugs). development of antibiotic guidelines for rid has progressively taken these home truths into consideration. such guidelines recommend minimizing antibiotic use when the primary pathogen is likely to be a virus and maximizing antibiotic use when there is a high probability of significant bacterial infection. 2-5 individual risk stratification, according to patient comorbidities and illness severity is used to varying degrees in all guidelines. physi-cians managing patients with rid are encouraged to do the best for both the individual and society where antibiotic usage is concerned. it is suggested that continuing these approaches, while incorporating new value adding information, will ensure the development of future significant improvements in both our knowledge and management of all forms of rid. in addition, such a strategy may have the added benefit of prioritizing ongoing antimicrobial drug development and even streamlining regulatory drug approvals. despite the many different syndromes and aetiological agents covered, the 'burden of respiratory infectious disease in australia' document presents an overwhelming theme of diagnostic inefficiency in spite of advances in molecular technology. 1 as an example, numerous studies have shown that a specific microbial diagnosis is routinely made in very few cases of patients hospitalized with community-acquired pneumonia. although this number approaches 50% with detailed microbiological and molecular testing in some studies, a microbial diagnosis eludes us in approximately half of all patients. 6 whether this is because 'uncommon' organisms have not been tested for, or 'common' organisms have been missed because of an overexuberant host response is not known. diagnostic inefficiency not only leads to a heavy reliance on empirical treatment strategies, but also contributes to lack of knowledge. diagnostic inefficiency handicaps innovative thinking regarding the management of acute rid, particularly in those who are severely ill and not responding to empirical antimicrobial therapy. our current reliance on empirical antibiotic strategies to cover 'likely bacterial pathogens' as set out in numerous guidelines is unavoidable in the short term given the current diagnostic limitations for respiratory infection syndromes. in the long term, however, there is a need to move away from an almost absolute requirement for empirical antibiotics in rid and move towards targeted antibiotic strategies as is the case for most other organbased infections. 5 to achieve this, there needs to be considerable improvement in the development of diagnostic tools for rid. these tools need to be rapid, reliable and available at the point of care. this is not impossible as has >been shown by the development of rapid hiv/cd4 diagnostic tests to help stratify limited antiretroviral use in developing countries. 7, 8 improvements in the sensitivity and specificity of diagnostic assays for specific respiratory pathogens are also required. improved sensitivity would enhance our ability to avoid using antibacterials in viral infections. improved specificity would enhance our ability to use specific antibiotics and/or antivirals in the first instance and to use them to their maximum potential according to pharmacodynamic principles. although the recent developments of multiplex polymerase chain reaction assays for viral and atypical bacterial organisms and enzyme-linked immunosorbent assays for some bacterial antigens (e.g. legionella, streptococcus pneumoniae) are promising, this area requires considerably more basic and applied research. even if the logistic issues associated with obtaining a test sample and immediate test results mean that empirical antibiotics continue to be used at least initially in rid, improved diagnostic tests can possibly allow for treatment to be subsequently tailored. there would also be a longerterm benefit as epidemiological data on local respiratory infections could enhance decision-making regarding empirical antibiotic use. another potential spin-off from enhanced data collection would be a better understanding of the pathogenetic mechanisms associated with specific virus or bacterial infections. this information may also positively influence the development of the antimicrobial pipeline of pharmaceutical companies. all the issues related to acute respiratory infections in otherwise-healthy individuals apply to an even greater degree to 'at-risk' groups. these persons have either an underlying lung disease (e.g. asthma, chronic obstructive pulmonary disease (copd) or immune impairment (e.g. being elderly and having immunodeficiency syndromes). compared with the rest of the population, the 'at-risk' population is susceptible to a wider range of infections and more severe disease from any given infecting organism. there is often a greater imperative to make a specific diagnosis in these patients as the risk-benefit ratio of an ongoing reliance on empirical antibiotic strategies is usually not acceptable. improvements in the sensitivity and specificity of diagnostic assays for rid have been shown to offer dramatic benefits to the clinical management of 'at risk' patients. as an example, the management of hiv/aids and its associated opportunistic infections has been revolutionized over the last 15 years by an approach that combines quantitative viral load and cd4 testing and effective antiretroviral therapy. 9 similarly, lung and other organ transplantation has now become commonplace in western countries and one of the commonest infections posttransplantation used to be cytomegalovirus (cmv) pneumonitis due to reactivation of this ubiquitous dna virus. twenty years ago, transplant physicians relied on poorly sensitive histopathological and non-specific viral culture diagnostics for cmv disease and relatively blunt treatment approaches using antiviral treatments. the whole field was transformed when sensitive molecular testing was routinely applied and combined with more focused antiviral strategies yielding both better results and a richness of information regarding pathobiological events. 10 first, subclinical cmv reactivation was identified as being very common in the lung allograft and hence, a period of universal prophylaxis was appropriate for all cmv 'at-risk recipients' (>90% lung transplant recipients). second, an understanding of the dynamics of cmv reactivation following cessation of antivirals had immediate direct benefits to the patient (i.e. quantitative cmv detection to determine the need for specific antiviral use) and indirect benefits (subtle reactivation syndromes could now be related to specific immunological profiles and long-term lung allograft outcomes). third, the efficacy of specific intervention strategies could now be easily monitored. this conceptual framework is now being extended to other reactivating dna viruses post-transplantation and in the case of lung transplants is being increasingly used to delineate the overall influence of community respiratory rna viruses on the lung allograft with a view to developing novel antiviral intervention strategies. in the setting of lung transplantation, the potentially direct and indirect (i.e. allograft rejection) life-threatening consequences of respiratory infection are an important drive for innovation in management. 11 these concepts relating to the specific example of lung transplantation can be extended to more general respiratory conditions. in addition to diagnostic improvements, the management of at-risk patients could be enhanced by obtaining disease-susceptibility information in welldefined 'at-risk' populations (e.g. cystic fibrosis, allergic bronchopulmonary aspergillosis, bronchiectasis, tuberculosis and even immunosuppression treatment profiles). this information could aid decision-making in terms of whether to use prophylactic or pre-emptive antibiotics to avoid specific acute infection syndromes in certain 'at-risk' patient groups, a strategy already used in hiv/aids. this information could also provide insights into hostpathogen interactions in acute conditions, such as pneumonia as well as asthma and copd, where acute exacerbations and chronic progression are an important burden of respiratory disease. this research could also provide a greater interpandemic influenza is a yearly public health concern and continues to be associated with significant morbidity and mortality despite the benefits that have come from an active influenza surveillance network, national influenza vaccination strategies and the availability of antivirals against influenza. one can only imagine the potential nightmare scenarios that may result from a new influenza pandemic (e.g. h5n1 'avian' influenza), particularly if the emergent virus achieves human-to-human transmission while retaining a high degree of pathogenicity. so there is a great deal of government investment in 'pandemic flu preparation plans' around the world. these protocols are invariably built around an appropriately staged plan of action concentrating on surveillance, containment and antiviral strategies in the early phases of any epidemic outbreak to buy as much time as possible for appropriate vaccines to be developed and distributed. more subtly, there is also a great deal of research and development aimed at developing novel antiviral interventions and optimizing vaccine efficacy and production. clearly, when there is an ever-present threat to the nation's future health, every effort is made to eliminate as much diagnostic and management uncertainty as possible. the risks posed by the outbreak of severe acute respiratory syndrome (sars) in 2003 were responsible for marshalling all the newly available technologies at the time to successfully isolate, identify and sequence the new strain of coronavirus that was responsible in record time. 12 all this was achieved with a view to fast track the development of effective antiviral and other treatments for sars. finally, the potential threat of bioterrorism has also led to government-sponsored dramatic alignment of molecular science and new technologies to provide rapid diagnostic testing for 'suspicious' samples. 13 in conclusion, in our day-to-day management of rid problems, we can learn much from our approach to infectious disease complications in 'at-risk' individuals and to emerging viral threats. in the former case, we seem to have become complacent with an empirical treatment approach that is compounded by lack of knowledge and a relatively blunt approach to matching specific conditions to specific treatments. the latter cases, however, indicate that we currently can obtain and use value-adding information so that we can begin to do better; all that is required is a sense of vision and a prioritization to do so. identification of rid as an important health priority will simultaneously raise the awareness of the unmet clinical need posed by respiratory infection across many areas of medicine and offer a clear path forward to focus efficiently and coordinate future clinical science research and health policy initiatives directed at reducing the current and future burden of rid. investing today in reducing this burden will pay future dividends many times over for us all -irrespective of whether we become ill or not. guidelines for the management of adults with community acquired pneumonia:diagnosis, assessment of severity, antimicrobial therapy, and prevention practice guidelines for the management of community acquired pneumonia in adults. infectious disease society of america ats/idsa cap guidelines for adults melbourne: therapeutic guidelines ltd etiology of community-acquired pneumonia in hospitalized patients in chile: the increasing prevalence of respiratory viruses among classic pathogens hiv-1 viral load assays for resource limited settings a reliable and inexpensive easycd4 assay for monitoring hiv-infected individuals in resource-limited settings aids treatment enters its 25th year hcmv dna load in plasma and bronchoalveolar lavage fluid: a longitidinal study of lung transplant recipients cytomegaloviral infection & other infections related to lung allograft survival severe acute respiratory syndrome current and developing technologies for monitoring agents of bioterrorism and biowarfare key: cord-346290-my8ow5ee authors: nelson, philipp p.; papadopoulos, nikolaos g.; skevaki, chrysanthi title: respiratory viral pathogens date: 2020-05-28 journal: reference module in biomedical sciences doi: 10.1016/b978-0-12-801238-3.11635-6 sha: doc_id: 346290 cord_uid: my8ow5ee respiratory viruses are responsible for a variety of clinical syndromes including the common cold, acute otitis media, laryngitis, sinusitis, pneumonia, bronchiolitis, influenza-like illness, and exacerbations of asthma and chronic obstructive pulmonary disease. diagnosis of respiratory viral infections is primarily clinical and is further supported by laboratory techniques such as antigen detection, serology, and nucleic acid detection. preventive strategies are based on avoidance of risk factors and, in case of influenza, vaccination. treatment modalities include over-the-counter and non-specific remedies along with a small number of specific antiviral medications such as the influenza neuraminidase inhibitors or palivizumab against respiratory syncytial virus. orthomyxoviridae's major representatives are the influenza viruses, which are grouped into four genera with one member per genus: influenza a virus (iav, alphainfluenzavirus), influenza b virus (ibv, betainflunenzavirus), influenza c virus (icv, gammainfluenzavirus) , and the recently discovered influenza d virus (idv, deltainflunezavirus) . while iav has a wide host range with water fowl as main reservoir, ibv mainly infects humans. icv and idv have also zoonotic potential but cause mostly mild respiratory diseases (asha and . orthomyxoviridae form enveloped, spherical or pleomorphic virions with 80-120 nm in diameter. their linear, negative-sense rna genome has a total length of 10-15 kb and is divided into eight (iav, ibv) and seven (icv, idv) segments, respectively. it encodes for up to 12 proteins, among others, in iav and ibv, hemagglutinin (ha) and neuraminidase (na) for attachment, cell entry, and release of new particles. the na and ha proteins are regularly subjected to small changes, which are capable of producing viral strains causing annual epidemics. this phenomenon is called "antigenic drift," while "antigenic shift" is the process by which a sudden major change in the ha or na proteins of iav occurs due to genetic reassortment (king, 2011) . instead of ha and na, which bind and cleave sialic acid (n-acetylneuraminic acid), icv and idv express one combined protein, the hemagglutinin-esterase-fution glycoprotein (hef). this protein requires an acetylated derivative of n-acetylneuraminic acid for attachment to the host cells (su et al., 2017) (fig. 2) . 1 rhinovirus (rv). the rhinovirus capsid is arranged in an icosahedron composed of 60 copies of each of the three subunits vp1-3 (shown in red, blue, and yellow). reproduced with permission from papadopoulos ng and skevaki cl (2006) viruses of the lung. in: encyclopedia of respiratory medicine, 483-488, https:// doi.org/10.1016/b0-12-370879-6/00494-4. (iav) . hemagglutinin spikes (green) radiate all over the surface and are interspersed by neuraminidase (yellow) and matrix protein m2 (light blue). the latter are embedded in the envelope's lipid bilayer(light yellow), which in turn surrounds a layer of matrix protein m1 (dark blue). the segmented rna (orange) of the virus is located in the interior. human parainfluenza viruses (hpivs) are respiratory viruses in the family of paramyxoviridae. they have enveloped, pleomorphic but mostly spherical virions with a diameter of 300-500 nm. the single, linear, negative-sense rna genome consists of 14.5-20 kb in a helical nucleocapsid. for efficient replication, the genome length is confined to multiples of 6 nucleotides. among others, it encodes for the glycosylated fusion (f) protein and the glycosylated receptor-binding protein (rbp), also called haemagglutininneuraminidase (hn) protein, which binds to neuraminic acid on the cell surface. four types of hpivs are known: hpiv-1 and 3 belong to the genus respirovirus in the subfamily of orthoparamyxovirinae, hpiv-2 and 4, like mumps virus, to the genus orthorubulavirus in the subfamily of rubulavirinae (rima et al., 2019) . viruses of the family of pneumoviridae form enveloped, spherical or filamentous virions with 100-200 nm in diameter, which contain a single, linear, negative-sense rna genome. this genome is bound in a complex with the nucleocapsid (n) protein, the polymerase (l), and a necessary co-factor (p). the glycosylated fusion (f) and attachment (g) proteins in the envelope mediate cell entry. in contrast to paramyxoviruses, almost all pneumoviruses lack a hemagglutinin and neuraminidase (rima et al., 2017) . human respiratory syncytial virus (hrsv or rsv) belongs to the genus orthopneumovirus. the genome of $15 kb contains 10 genes for 11 proteins, two of them being non-strucutural proteins (g protein, two groups, rsv-a and rsv-b, are distinguished (pangesti et al., 2018) . another pneumovirus is human metapneumovirus (hmpv) in the genus metapneumovirus. it forms paramyxovirus-like pleomorphic to spherical particles of 150-600 nm diameter. the genome of hmpv comprises roughly 13 kb and encodes for 9 proteins (shafagati and williams, 2018) . until the beginning of the 21st century, only two human coronaviruses (hcov-229e and oc43) were known. since then, many other members of the family of coronaviridae have been indentified, most of them infecting animals and only four others infecting humans: hcov-nl63 and hku1 cause respiratory diseases worldwide, severe acute respiratory syndrome (sars) coronavirus was discovered in an outbreak in 2003-2004, and middle east respiratory sondrome (mers) coronavirus, so far constricted to the arabian peninsula. coronaviruses form enveloped, spherical virions with a diameter of 120-160 nm. the size of the single, linear positive-sense rna genome ranges between 26 and 32 kb, which represents the largest genome of known rna viruses. the trimeric glycosylated spike (s) protein forms characteristic 15-20 nm long protrusions, which mediate receptor binding and membrane fusion. common to all coronaviruses are also the membrane (m) and envelope (e) glycoproteins and the nucleocapsid (n) protein. depending on the species, other proteins are included, e.g., a hemagglutinin-esterase (he) for reversible attachment to o-acetylated sialic acid in hcov-oc43 and hku1. during replication, two polyproteins are produced from the replicase gene and then cleaved into several small products; among them nsp1, which is used for genus demarcation (king, 2011) . high estimated mutation rates and possible genetic recombinations during replication increase the probability of the emergence of new (zoonotic) coronaviruses (su et al., 2016) (fig. 3) . hcov-229e (subgenus duvinacovirus) and hcov-nl63 (subgenus setracovirus) belong to the genus alphacoronavirus. the four other coronaviruses belong to the genus betacoronavirus. hcov-oc43 and hcov-hku1 are subspecies of the species betacoronavirus 1 in the subgenus embecovirus. mers-cov is a member of the subgenus merbecovirus and sars-cov of the subgenus sarbecovirus (walker et al., 2019) . the cell entry receptor differs between those viruses as well. hcov-229e binds human aminopeptidase n (apn, cd13) while hcov-nl63 and sars-cov use angiotensin-converting enzyme (ace) 2 as their receptor. according to their he protein, hcov-oc43 and hku1 use 9-o-acetylated sialic acids attached to cell surface proteins. mers-cov requires dipeptidyl peptidase 4 (dpp4, cd26) for cell entry (hulswit et al., 2019; raj et al., 2013) . human adenoviruses (hadv) belong to the genus mastadenovirus and are further classified into 6 species (a-g) with more than 100 types identified so far (lion, 2019). the symptoms of an infection vary between different types and range from respiratory disease to diarrhea or conjunctivitis. adenoviruses are non-enveloped, double-stranded dna viruses with a genome of 26-48 kb in icosahedral virions ranging from 70 to 90 nm in diameter (king, 2011). fiber-like protrusions extend from each of the 12 penton base capsomers at the corners of the icosahedron. the knobs at the ends of these fibers confer attachment to the host cells, among others to coxsackie and adenovirus receptor (car) or membrane cofactor protein (mcp, cd46). while most hadvs only use one of the two receptors, group d viruses use both receptors simultaneously (khanal et al., 2018) . during replication $40 polypeptides are produced through alternative splicing (fig. 4) . although most polyomaviruses (pyvs) have been associated with different types of tumors, two recently discovered polyomaviruses of the genus betapolyomavirus, wupyv and kipyv, have been found in respiratory samples worldwide, most frequently in samples from immunocompromised patients. while both alpha-and betapolyomaviruses may infect humans, gamma-and deltapolyomaviruses do not (babakir-mina et al., 2013) . pyvs are non-enveloped, icosahedral viruses of 40-45 nm diameter and contain a single, circular double-stranded dna genome of $5 kb. inside the virion, the genome is packed with histone proteins h2a, h2b, h3, and h4. the capsid consists of 72 pentameric capsomers of the major capsid protein vp1 and the two minor capsid proteins vp2 and vp3. typically, the two regulatory proteins large and small tumor antigen (ltag and stag, respectively), which are expressed early during infection, are used for phylogenetic analyses (moens et al., 2017) . even though detected frequently, a clear association between wupyv and kipyv on one hand and respiratory disease on the other hand is still missing (babakir-mina et al., 2013) . parvoviridae are non-enveloped, icosahedral viruses of around 25 nm diameter. the capsid encloses a single, linear, mostly negativesense dna-genome with approximately 5.5 kb. the genome encodes for three capsid proteins (vp1-3) and five non-structural proteins (ns1-4, np1). human bocavirus 1 (hbov1), a member of the species primate bocaparvovirus 1, in the genus bocaparvovirus and the subfamily of parvovirinae, is strongly associated with upper and lower respiratory tract infections in young children. the related viruses, hbov2-4, are only found in stool samples. by exploiting the cellular dna repair machinery, hbov1 is independent from the cell cycle for genome replication (qiu et al., 2017) . generally, respiratory viruses are transmitted between humans by the following routes: direct or indirect contact to infected persons, via droplets, or by aerosols. the main route for each virus differs and is still in discussion (kutter et al., 2018) . in case of zoonotic mers-cov infection, the consumption of uncooked meat or milk from or direct contact to infected animals are other suspected routes (song et al., 2019) . control of the transmission of respiratory infections may be partly achieved through adequate hygiene practices and avoidance of congregation and stress. upon entering a susceptible host through eyes, nose, or mouth, respiratory viruses replicate in nasopharyngeal epithelial cells or in the epithelium of the lower respiratory tract. in more severe cases, a subsequent systemic spreading of the virus is possible. the site of the deposition of inhaled particles also depends on their size. while inhalation of aerosols with influenza virus may lead to infection of the lower respiratory tract, larger droplets are retained in the upper respiratory tract (paules and subbarao, 2017) . between respiratory viruses, patterns of infection differ, too. for example, epithelial infection by rvs has a "patchy" distribution and is not overtly cytotoxic, in contrast to many other respiratory viruses, including influenza viruses, paramyxoviruses, and adenoviruses, which cause extensive inflammation and epithelial shedding (royston and tapparel, 2016; pawełczyk and kowalski, 2017; shim et al., 2017) . several pathogen recognition receptors (prrs) contribute to the detection of viral structures within the infected cell, among them specific toll-like receptors (tlrs), which bind viral nucleic acids and stimulate the production of type i interferons (pichlmair and sousa, 2007) . one way of iav to suppress interferon expression and finally cell death is the production of non-structural protein 1 (ns1) within a few hours after infection. ns1 binds viral rna and prevents recognition by prrs and inhibits expression of interferon-induced genes (shim et al., 2017) . respiratory virus infection causes an increase in both vascular permeability mediated by vasoactive amines and glandular secretion under the influence of cholinergic reflexes and neuropeptides. several cytokines and chemokines have also been implicated in virus-induced inflammation. inflammatory cells further aggravate events by release of additional mediators. humoral immunity is also activated in response to viral infections with production of serotype-specific antibodies. the nature of the response is associated with age, previous infection, and vaccination status of the host. recovery from respiratory viral illness is usually achieved prior to the detection of specific antibodies indicating that cellular and/or non-specific immune responses are primarily responsible for viral eradication. nevertheless, antibodies are able to provide protection from secondary infections. however, different aspects limit the protective effect of neutralizing antibodies against specific viruses: (a) the effect may not be long-lasting (e.g., for hmpv), (b) high mutation rates lead to the occurrence of escape-mutants (e.g., in influenza viruses), (c) many different serotypes of one virus are circulating and antibodies are lacking cross-reactivity (e.g., against other rvs). respiratory viruses not only induce a local inflammatory reaction at the level of infected epithelial cells but may also act at a distance through neuronal pathways. all parasympathetic, sympathetic, and nonadrenergic noncholinergic nervous supply of the respiratory tree may be affected by viral infections, suggesting an important neuroimmune interaction, which may contribute to the virus-mediated reactive airway disease (fig. 5) . pathogenesis of respiratory viral infections. respiratory viruses enter the human body mainly through the nose. thereafter, they replicate in nasopharyngeal epithelial cells, but also in the lower respiratory tract. immune mediators such as cytokines, chemokines, and bradykinins will both elicit an "immune" response leading to effective virus eradication and contribute to the development of associated symptomatology, through vascular leakage and increased glandular secretions. neurogenic pathways are also involved in the development of clinical manifestations. reproduced with permission from papadopoulos ng and skevaki cl (2006) viruses of the lung. in: encyclopedia of respiratory medicine, 483-488, https://doi.org/10.1016/b0-12-370879-6/00494-4. respiratory viruses are related to various distinct as well as non-specific clinical presentations. while acute infections are usually selflimited, severe forms may occur. there is considerable overlap between viruses and clinical presentations. although differences exist, all agents may cause any of several clinical syndromes (table 1) . among these, the common cold is by far the commonest with an enormous socioeconomic impact. clinical presentation ranges from asymptomatic to upper respiratory tract (urt) symptoms such as nasal congestion, rhinorrhea, and nasopharyngeal irritation, lower respiratory tract symptoms such as cough, to systemic symptoms including general malaise, fever, headache, and sleep impairment depending on the viral culprit and host. the incubation period is 24-48 h and symptoms usually last for 5-7 days with a peak of viral replication before or together with the first clinical symptoms. the common cold is a rather benign clinical entity, which may however be complicated by secondary bacterial infections, otitis media, sinusitis, pneumonia, and asthma exacerbations; severe courses of disease and death may occur in young children and immunocompromised patients. viral urt infections often result in impairment of the function of the eustachian tube, which predisposes to the development of acute otitis media. the latter is one of the commonest infections among children and is very often attributed to viral etiology. respiratory viruses may also predispose to nasopharyngeal bacterial colonization and alteration of the host's immune response. finally, viruses may be responsible for antibiotic treatment failure in cases of concurrent bacterial and viral infections. likewise, respiratory viruses have been implicated in the development of sinusitis, either directly as an extension of urt infection or as a result of secondary bacterial infection. laryngitis and laryngotracheobronchitis-croup have more frequently been associated with the parainfluenza virus group. pneumonia in infants and young children is most commonly attributed to viruses. disease burden is also significant among the elderly and the immunocompromised. rvs and rsvs are the agents most frequently isolated in viral pneumonias, although age, season, year, and other variations can be considerable. the presence of adenovirus is a risk factor for severe disease in infants. influenza occurs in yearly winter epidemics and pandemics every 10-40 years resulting in enormous morbidity and mortality, especially among the very young, the elderly, and the immunocompromised. infections with ibv or icv are mostly milder than those with iav (paules and subbarao, 2017) . the zoonotic potential of idv is yet unknown. so far, this virus could not be found in patients with respiratory symptoms although a high seroprevalence in people working with cattle has been observed (su et al., 2017) . rsv is the major pathogen implicated in acute bronchiolitis in infants up to 1 year and is responsible for a great number of hospitalizations and deaths in this age group. in the second year of life, rv and hbov become more frequent pathogens (jartti et al., 2019) . host immune response skewed towards type 2 cytokine production seems to play a central role in the pathogenesis of the disease. bronchiolitis is further associated with recurrent wheezing. the great majority of acute asthma exacerbations in children and a considerable proportion in adults is preceded by a viral urt infection. such exacerbations may often result in hospitalization. the most frequently isolated offending agent is rv, a fact that reflects the preponderance of the virus among common cold cases. finally, exacerbations of chronic obstructive pulmonary disease may also have a viral etiology, most commonly due to rvs. wupyv and kipyv have so far only been associated with increased sputum production and wheezing; a clear role as causative agent has not been shown (cook, 2016) . the diagnosis of respiratory viral infections relies upon clinical criteria and is further supported by laboratory techniques such as direct viral, nucleic acid or antigen detection, culture, and serology. direct detection is based on the identification of whole virus or inclusion bodies with electron or light microscopy, respectively. viral antigens may also be detected by the use of immunofluorescence and enzyme immunoassays but an adequate viral load is required. viral cultures usually demonstrate the biological effects of an agent, such as the cytopathic effect or hemagglutination, in cultured cells. culture methods, however, have drawbacks due to table 1 disease syndromes of common respiratory viruses in upper and lower respiratory tract. their low sensitivity, extensive time length (days to weeks), and sometimes complex culture systems. determination of specific viral antibodies in a host's blood sample and comparison between acute and convalescent phases of respiratory illness is another diagnostic modality, which is mainly used for epidemiological purposes. serological tests include immunoassays, complement fixation, and passive agglutination assays as well as hemagglutination inhibition. nucleic acid amplification tests (naats), like realtime polymerase chain reactions (real-time pcrs), are gaining more and more importance due to their high sensitivity and specificity, combined with low turnaround times. together with antigen-based diagnostic tests, naats are used in point-of-care tests (pocts), which are able to provide results much faster than lab-based tests and directly at the site of sample collection. in the field of genetic epidemiology and virus discovery, next-generation sequencing (ngs) became the method of choice, as no susceptible cell lines are required. usually, samples from the respiratory tract are taken for diagnostic purposes. these include swabs from nose or throat, nasopharyngeal aspirates, nasal washes, sputum, bronchial aspirates, and bronchoalveolar lavages. upon infection with hpevs or hadvs systemic infections or other complications, e.g., gastrointestinal or neurological symptoms, may occur. in this case, additional specimens, ranging from stool, blood, urine, and cerebrospinal fluid, are taken (olijve et al., 2018; khanal et al., 2018) . the development of effective vaccines is hampered by the large number of viral serotypes. currently, licensed vaccines are available only against influenza a and b viruses, as well as an oral live vaccine against hadvs for u.s. military personell (paules and subbarao, 2017; crenshaw et al., 2019) . following the regular recommendations of the world health organization (who), the composition of the seasonal influenza vaccines is updated every year to include the most important strains and to confer the best protection. however, as the actual frequency of the circulating influenza strains in each season may differ from this recommendation, the efficacy of these vaccines vary from year to year. mostly, tri-and quadrivalent influenza vaccines, inactivated, liveattenuated, or recombinant vaccines are used. to overcome the need for a yearly vaccination, the search for alternative approaches and universal influenza vaccines is ongoing. vaccine candidates against other respiratory viruses, like hrsvs, hpivs, hmpv, and mers-cov are in development or under clinical investigation. over-the-counter remedies for the alleviation and reduction of duration of common cold symptoms include vitamin c, zinc, and echinacea, although these agents are only moderately effective. nasal decongestants, first generation antihistamines, anticholinergic nasal sprays, oral and intranasal a-adrenergic agonists, inhalation of humidified hot air at 42-44 c, nonsteroidal antiinflammatory drugs, and cromones have been proposed for symptomatic relief with moderate results. a combination of specific antivirals with one or more anti-inflammatory drugs may offer the ideal therapeutic approach since it would both inhibit viral replication and block inflammatory events, although cost and compliance are considerable obstacles. unfortunately, only a limited number of antiviral agents against respiratory viruses is available, due to the frequent mutations resulting in the constant emergence of new and often resistant strains. nevertheless, many new drugs and experimental approaches are under investigation (papadopoulos et al., 2017) . the licensed antiviral chemotherapy against influenza viruses can be grouped into four classes, which prevent uncoating (adamantanes like amantadine and rimantadine), inhibit na (e.g., oral oseltamivir and inhaled zanamivir), prevent membrane fusion (arbidol), and interfere with the viral rna-dependent rna polymerase (the broad-spectrum antiviral favipiravir). adamantanes are only active against iva, however, today all circulating seasonal strains of iav are resistant against this antiviral, so the use is not recommended any more. na inhibitors are available for treatment and prophylactic purposes, but prophylactic applications are more effective as randomized controlled trials showed only marginal effects on the duration of symptoms (paules and subbarao, 2017) . new experimental antiviral agents target both the virus and the host, e.g., by inducing apoptosis only in infected cells (shim et al., 2017) . another experimental host-targeted strategy, against infections with hbov, is repurposing of kinase inhibitors developed for tumor treatments (qiu et al., 2017) . palivizumab is a monoclonal antibody against rsv f glycoprotein, which may be administered prophylactically to reduce the risk of severe bronchiolitis and hospitalization in specially vulnerable children, i.e. preterm infants and children with chronic lung or heart diseases (jartti et al., 2019) . the antivirals cidofovir and ribavirin have been used against rsv especially in immunocompromised patients, but little to no beneficial and many adverse effects have been observed. consequently, their widespread use is not recommended (khanal et al., 2018) . the clinical usefulness of the capsid binding antivirals pleconaril, vapendavir, and pocapavir against rvs still has to be shown. a high likelihood for drug resistances limits their clinical applicability (papadopoulos et al., 2017) . a long-known effective compound against a variety of respiratory viruses and the resulting common cold is interferon alpha-2b, administered intranasally, before or shortly after exposure. however, due to its high cost, dosage frequency (six times daily), and side effects (mainly nasal irritation and bleeding) clinical application has been abandoned (mossad, 1998) . the human polyomaviruses ki and wu: virological background and clinical implications perspective on adenoviruses: epidemiology, pathogenicity, and gene therapy enteroviruses and parechoviruses human coronaviruses oc43 and hku1 bind to 9-o-acetylated sialic acids via a conserved receptor-binding site in spike protein domain a bronchiolitis needs a revisit: distinguishing between virus entities and their treatments the repertoire of adenovirus in human disease: the innocuous to the deadly virus taxonomy. ninth report of the international committee on taxonomy of viruses. international union of microbiological societies transmission routes of respiratory viruses among humans adenovirus persistence, reactivation, and clinical management viruses and bacteria in the etiology of the common cold proposals for the classification of human rhinovirus species a, b and c into genotypically assigned types ictv virus taxonomy profile: polyomaviridae treatment of the common cold human parechovirus: an increasingly recognized cause of sepsis-like illness in young infants molecular epidemiology of respiratory syncytial virus promising approaches for the treatment and prevention of viral respiratory illnesses the role of human parainfluenza virus infections in the immunopathology of the respiratory tract innate recognition of viruses human parvoviruses dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-emc ictv virus taxonomy profile: pneumoviridae royston l and tapparel c (2016) rhinoviruses and respiratory enteroviruses: not as simple as abc human metapneumovirus-what we know now influenza virus infection, interferon response, viral counter-response, and apoptosis from sars to mers, thrusting coronaviruses into the spotlight epidemiology, genetic recombination, and pathogenesis of coronaviruses novel influenza d virus: epidemiology, pathology, evolution and biological characteristics changes to virus taxonomy and the international code of virus classification and nomenclature ratified by the international committee on taxonomy of viruses ictv virus taxonomy profile: picornaviridae microbiologic diagnosis of respiratory illness: practical applications influenza burden, prevention, and treatment in asthma-a scoping review by the eaaci influenza in asthma task force european scientific working group on influenza (eswi) /research_projects/study_groups/respiratory_viruses-european society of clinical microbiology and infectious diseases (escmid) study group on respiratory viruses (esgrev) hadvwg.gmu.edu-human adenovirus working group. talk.ictvonline.org-international committee on taxonomy of viruses (ictv). www.picornaviridae.com-picornavirus study group. www.resvinet.org-respiratory syncytial virus network the authors would like to thank the european society of clinical microbiology and infectious diseases (escmid) study group on respiratory viruses (esgrev) for providing a platform for scientific discussion on the topic. key: cord-335055-gzuug3p5 authors: kwiyolecha, elizabeth; groendahl, britta; okamo, bernard; kayange, neema; manyama, festo; kidenya, benson r.; mahamba, dina c.; msanga, delfina r.; gehring, stephan; majigo, mtebe; mshana, stephen e.; mirambo, mariam m. title: patterns of viral pathogens causing upper respiratory tract infections among symptomatic children in mwanza, tanzania date: 2020-10-28 journal: sci rep doi: 10.1038/s41598-020-74555-2 sha: doc_id: 335055 cord_uid: gzuug3p5 upper-respiratory tract infections (urti) are the leading causes of childhood morbidities. this study investigated etiologies and patterns of urti among children in mwanza, tanzania. a cross-sectional study involving 339 children was conducted between october-2017 and february-2018. children with features suggestive of urti such as nasal congestion, dry cough, painful swallowing and nasal discharge with/without fever were enrolled. pathogens were detected from nasopharyngeal and ear-swabs by multiplex-pcr and culture respectively. full blood count and c-reactive protein analysis were also done. the median age was 16 (iqr: 8–34) months. majority (82.3%) had fever and nasal-congestion (65.5%). rhinitis (55.9%) was the commonest diagnosis followed by pharyngitis (19.5%). viruses were isolated in 46% of children, the commonest being rhinoviruses (23.9%). nineteen percent of children had more than 2 viruses; rhinovirus and enterovirus being the commonest combination. the commonest bacteria isolated from ears were staphylococcus aureus and pseudomonas aeruginosa. children with viral pathogens had significantly right shift of lymphocytes (73%—sensitivity). majority (257/339) of children were symptoms free on eighth day. viruses are the commonest cause of urti with rhinitis being the common diagnosis. rapid diagnostic assays for urti pathogens are urgently needed in low-income countries to reduce unnecessary antibiotic prescriptions which is associated with antibiotic resistance. | (2020) 10:18490 | https://doi.org/10.1038/s41598-020-74555-2 www.nature.com/scientificreports/ in many low-and middle-income countries. in tanzania, there is only one study that has documented the viral etiologies of rti 3 . therefore, there is a paramount need to establish information on the common etiologies of rtis in tanzania, the information that can stimulate further studies and possible control interventions including introduction of cheap and reliable methods to detect these pathogens in clinical settings. in addition due to increased use of antibiotic without a support of a diagnostic test in the treatment of urti as observed in number of previous studies [11] [12] [13] , make the availability of epidemiological data on the patterns of etiology of urti of paramount important. overuse of antibiotics without prescriptions for urti is widespread in developing countries 14 , this is partially contributed by lacking of data on the etiologies of urtis. therefore, these data are relevant to clinicians in developing countries and policy makers in order to invest on the improved diagnostic facilities and reduce antibiotic prescriptions for urtis. study area, design and study population. a cross sectional hospital based study involving 339 children aged 1-59 months presenting with rti symptoms was conducted from october 2017 to february 2018 in the city of mwanza, tanzania. the study was conducted in two health facilities namely: buzuruga health center (bhc), and nyamagana district hospital (ndh). these are public health facilities providing free services to children below 5 years of age. the study included all children presented with nasal congestion or runny nose, hoarseness of voice with dry cough, painful swallowing with tender cervical lymph nodes and enlarged tonsils on examination, ear pain or ear discharge, nasal discharge with or without fever (axilla body temp of 37.5 °c and above). in this study we defined: pharyngitis as painful swallowing dry cough, plus or minus hoarseness of the voice (sore throat), tonsillitis as painful swallowing with tender cervical lymph nodes and enlarged tonsils(primarily tonsillar inflammation) and rhinitis as presence of one or more symptoms including sneezing, itching, nasal congestion, and rhinorrhea. sample size estimation and sampling procedures. sample size was calculated using yamane taro (1967) with precision level of 5%. the minimum sample size estimated was 270 children. however, a total of 339 children were enrolled. all children who met the inclusion criteria were serially enrolled until the desired sample size was attained. data collection and sample collection. sociodemographic and clinical information were collected using pretested structured data collection tool. nasopharyngeal swabs (copan diagnostics inc. usa, canada) were obtained as previously described 15 by inserting the swab into one nostril straight back along the floor of the nasal passage until reaching the nasopharynx. the swabs were rotated gently for 5-10 s to loosen the epithelial cells and collect the sample. the swabs were then inserted into viral transport medium and stored at − 80 °c until processing. in children presenting with ear discharge; ear swabs were collected using flexible shaft swab via an auditory speculum in case of inner ear while a sample from outer ear was obtained by firmly rotating swab in outer canal. swabs were immediately taken to the laboratory for bacterial culture and sensitivity. for each consenting participant, about 4 mls of blood was also collected for blood cell counts and quantitative c reactive protein analysis. a thorough general and physical examination was performed to all enrolled children to establish clinical features. all children were managed as per local hospitals protocol. patients were followed two times; the first follow up was on the 4th day, where these patients were fully examined and their full blood count, crp and ear swab culture results were revealed. in case of positive ear swab culture treatment was changed based on susceptibility patterns. the second follow up was done on the 8th day as clinical review to evaluate for disease progression. laboratory procedures. ear swab specimens were inoculated onto chocolate agar, blood agar (ba) and macconkey agar (mca) plates and incubated aerobically at 37 °c for 24-48 h. in-house biochemical identification tests were used to identify isolated bacteria to their species level 16 . the identified isolates were tested for antimicrobial susceptibility following clsi guidelines, the tested antibiotic discs included: amikacin (30 µg), gentamicin (10 µg), erythromycin (15 µg), vancomycin (30 µg), clindamycin (2 µg), ciprofloxacin (5 µg) which were used for gram positive bacteria and ampicillin (10 µg), ceftazidime (30 µg), meropenem (10 µg), amikacin (30 µg), piperacillin-tazobactam (100/10 µg) and ciprofloxacin (5 µg) for gram negative bacteria 17 . bacterial isolates obtained were inoculated into brain heart infusion broth with 20% glycerine and stored at -80 °c freezer. e. coli atcc 25992 and staphylococcus aureus atcc 25923 were used as control strains. nasopharyngeal swabs were transported to mainz university germany and were tested to detect enterovirus (ev), influenza virus type a (iva), influenza virus type b (ivb), respiratory syncytial virus (rsv), parainfluenza virus type 1 (piv1), parainfluenza virus type2 (piv2), parainfluenza virus type 3 (piv3), parainfluenza virus type 4 (piv4), adenovirus (av), rhinovirus (rv), human metapneumovirus (mpv), coronavirus (cv), bocavirus, mycoplasma pneumoniae (mpn), chlamydophila pneumoniae (cpn), bordetella pertussis (bp), bordetella parapertussis (bpp) and legionella pneumophila (lpn) using multiplex pcr as previously described 18 . blood samples were quantitatively tested for c-reactive protein following manufacturer instructions (medical instruments co., ltd, shanghai, china). blood in edta container (bd vacutainer, nairobi, kenya) was used to estimate complete blood count (fbc) using hematological analyzer (beckman coulter (uk) ltd) 19 . www.nature.com/scientificreports/ statistical data analysis. data entry was done using microsoft excel then exported to stata version 13 for analysis. continuous variables (age and temperature) were summarized using median with interquartile ranges. categorical variables (sex and level of education) were summarized using frequency and proportions. to determine the utility of fbc, and crp in the determination of causative agents among children below 5 years of age, a 2 by 2 table and receiver operating curve (roc) characteristic analysis were used to determine the sensitivity, specificity, positive and negative predictive values. children presenting with symptoms for more than 7 days were classified as having chronic illness. a child who had no any symptom on day eight of follow was declared cure. ethical approval and consent to participate. the approval for conducting the research was sought from the joint cuhas/bmc research ethics and review with ethical clearance number: crec/255/2017. permission to conduct the study was also sought from the pediatrics departments at bhc and ndh. the aim and importance of the study was explained to parents/caretakers before enrollment of children to the study, followed by a signed informed consent by the parent/caretaker. all information regarding the patient remained confidential. patient's records were kept such that the identity of the patient was not disclosed. for those who refused to participate, were provided with services similar to the participants and had equal chance to treatment regardless of their inclusion status. all methods were carried out in accordance with relevant guidelines and regulations. socio-demographic characteristics of study participants. the www.nature.com/scientificreports/ ing from 1 to 7 days and were classified as acute illness in this study ( table 2 ). the slightly majority 184 (55.9%) of children presented with rhinitis ( fig. 1) . lymphocytes, neutrophils and crp were used to predict the possible causative agents. children with viral pathogens had significantly elevated lymphocytes, with normal or elevated crp. the sensitivity of elevated lymphocytes in detecting viral pathogens was 73.4% (fig. 3a,b ). etiologies of urti among under five children in mwanza city. this is the first study to establish etiologies of rtis in the lake victoria zone tanzania. findings from this study shows that, number of viruses are responsible for rtis among children below 5 years of age attending outpatient clinics in the city of mwanza. the prevalence of 46.9% reported in the current study is low compared to a previous study conducted in ifakara and dar es salaam, which reported prevalence of 70.5% 3 . the possible explanation for these differences could be criteria used in the enrollment of the study participants. in the previous study fever was the main inclusion criterion which was not the case in the current study. in addition, this study was conducted in different season of the year (october 2017 through february 2018) compared to the previous which was conducted from april to august and from june to december at two different sites, respectively. viral infections have been found to be influenced by season variations [20] [21] [22] [23] [24] . further studies to establish seasonality of these viruses are warranted in developing countries. the findings in this study are comparable to the previous study in kenya among children below 5 years of age, whereby viruses were isolated in 45% of children with rtis 25 . in comparison to a previous study in refugee www.nature.com/scientificreports/ camp in kenya the prevalence of viral infection reported in this study is low (46% vs. 66.6%) 26 . the possible explanation could be overcrowding conditions in refugee camp which has been found to facilitate transmission of rtis viruses 27, 28 . regarding distribution of viruses; rhinovirus, adenovirus and parainfluenza 3 were the commonest viral pathogens in the current study which is contrary to a previous study in kenya 26 whereby influenza a virus, respiratory syncytial virus and influenza b were the commonest. the predominance of rhinovirus, influenza viruses was also reported in a previous study in tanzania 3 . the distribution of respiratory viruses is associated with climatic changes; the peak of infection usually occurs in winter period in temperate regions which is equivalent to wet season in hot climates like tanzania 29, 30 . as documented earlier, a significant proportion of children who had viral infection presented with acute illness 27 . however, it should be noted that some studies have shown that some viral diseases may last for several weeks 27, 28 which may account for the few children in the present study who presented with chronicity. regarding ear bacterial infections, staphylococcus aureus, pseudomonas aeruginosa, serratia marcescens, klebsiella pneumoniae, providencia spp. and streptococcus spp. were isolated. this is contrary to the previous study whereby streptococcus pneumoniae was the commonest isolate 3 . the possible explanation could be wide coverage of pneumococcal vaccines. decrease in streptococcus pneumoniae infections has been observed after introduction of the vaccine. a previous study by mushi et al. 31 in similar settings established that staphylococcus aureus and pseudomonas aeruginosa were the commonest pathogens causing csom among adult patients. in the current study, only one nasopharyngeal specimens yielded bacterial isolate (bordetella parapertussis) which is contrary to a previous observation by ndossa et al. 32 that detected streptococcus pneumoniae to be colonizing the nasopharynx of children in the city of mwanza. moreover, the bacterial isolates in the current study were highly resistant to the readily available and the over counter antibiotics like amoxicillin, trimethoprim/ sulphamethoxazole and ampicillin with majority being susceptible to ciprofloxacin ear drops. this could be explained by the fact that, ciprofloxacin is not readily used in children below 12 years of age. in the current study, rhinitis was the commonest presenting disease, followed by pharyngitis and pneumonia. this is further supported by findings in the current study which reported rhinovirus to be the commonest pathogen. a previous study 33 , documented rhinovirus to cause up to 25-85% of the upper respiratory tract infections. systemic responses of th1 for rhinovirus results into stimulation of specific clones of cd4 t cells and secretions of large amount of granulocytes macrophage colony stimulating factor (gm-csf) which are also responsible for the urti symptoms 34 . moreover, rhinovirus infection has also been associated with lower respiratory tract disease, asthma exacerbations and fatal pneumonia [33] [34] [35] . on the other hand, the spectrum of diseases in this study could be also explained by the commonly isolated viruses; influenza viruses, rsv, parainfluenza viruses and adenoviruses which are the common viruses responsible for most of the upper respiratory diseases as previously reported 36, 37 . in the current study, children with viral pathogens had a right shift of lymphocytes with an increased sensitivity of 78.0%, with left deviation of the neutrophils or slightly raised crp (not more than 10 mg/dl). these findings are important and could be used to predict these infections in resource limited setting and assist in decision making on the management of the patients which eventually might reduce unnecessary prescriptions of antibiotics. the observation is further supported by the fact that the majority of children were free from the initial symptoms on day eighth, underscoring that viral caused urti have mild symptoms which tend to disappear with time 38,39 . study limitations. diagnosis of patterns of urti was done clinically with no imaging to support the diagnosis, this might have caused misclassification. however, efforts were made to minimize this by consulting senior pediatricians whenever overlap of symptoms occurred. another potential limitation was failure to perform multiplex pcr for all ear swabs therefore viral pathogens might have been missed in these samples. urti are common in children below 5 years of age and are predominantly caused by viruses. elevated lymphocytes, normal neutrophils with elevation or normal crp levels can predict viral causes of urti while the raise in neutrophils and crp are more likely to predict bacterial infections. clinicians should suspect urti caused by viral infections whenever the children present with runny nose with congestion, fever and dry cough. the use of antibiotics should be minimized in children with urti symptoms since most of the symptoms disappears within a week. further studies to determine etiologies of lower respiratory tract infections are warranted in this setting. all data are included in the manuscript. raw data is available upon request and the request should be made to the director of research and innovation, catholic university of health and allied sciences. scientific reports | (2020) 10:18490 | https://doi.org/10.1038/s41598-020-74555-2 www.nature.com/scientificreports/ human bocavirus symptomatic treatment of upper respiratory tract symptoms in children beyond malaria-causes of fever in outpatient tanzanian children estimates of world-wide distribution of child deaths from acute respiratory infections antibiotic use in acute upper respiratory tract infections pneumonia case management trials group: effect of pneumonia case management on mortality in neonates, infants, and preschool children: a meta-analysis of community-based trials global estimate of the incidence of clinical pneumonia among children under five years of age disease control priorities in developing countries the clinical impact of human respiratory virus infections disease control priorities in developing countries physicians' antibiotic prescribing habits for upper respiratory tract infections in turkey trends in antimicrobial prescribing for bronchitis and upper respiratory infections among adults and children assessing the overuse of antibiotics in children with urtis in saudi arabia: development of the parental perception on antibiotics scale (papa scale) prescription and non-prescription antibiotic dispensing practices in part i and part ii pharmacies in moshi municipality, kilimanjaro region in tanzania: a simulated clients approach comparison of nasopharyngeal and oropharyngeal swabs for the diagnosis of eight respiratory viruses by real-time reverse transcription-pcr assays evaluation of the bruker maldi biotyper for identification of gram-positive rods-development of a diagnostic algorithm for the clinical laboratory performance standards for antimicrobial susceptibility testing validation of a multiplex reverse transcriptase pcr elisa for the detection of 19 respiratory tract pathogens evaluation of a multiplex reverse transcriptase pcr elisa for the detection of nine respiratory tract pathogens viral infections in relation to age, atopy, and season of admission among children hospitalized for wheezing seasonality of viral infections: mechanisms and unknowns an explanation for the seasonality of acute upper respiratory tract viral infections the seasonality of rhinovirus infections and its implications for clinical recognition epidemiology and seasonality of respiratory viral infections in hospitalized children in kuala lumpur, malaysia: a retrospective study of 27 years aetiology of acute respiratory infections in children under five years in nakuru, kenya improved detection of respiratory viruses in pediatric outpatients with acute respiratory illness by real-time pcr using nasopharyngeal flocked swabs risk factors for acute respiratory tract infections in under-five children in enugu southeast nigeria respiratory tract infections in the tropics seasonal variations of respiratory viruses detected from children with respiratory tract infections in riyadh, saudi arabia epidemiology and seasonality of respiratory tract virus infections in the tropics predictors of disease complications and treatment outcome among patients with chronic suppurative otitis media attending a tertiary hospital factors associated with colonization of streptococcus pneumoniae among under-fives attending clinic in mwanza city rhinovirus: more than just a common cold virus rhinovirus infections in the upper airway an update on the pathophysiology of rhinovirus upper respiratory tract infections adenoviral infections in children: the impact of rapid diagnosis epidemiological analysis of respiratory viral etiology for influenza-like illness during 2010 in zhuhai incubation periods of acute respiratory viral infections: a systematic review estimating incubation period distributions with coarse data the authors acknowledge the assistance provided by department of pediatrics mainz university, the department of microbiology and immunology-cuhas-bugando, and the department of pediatrics and child health-cuhas-bugando. this study was supported by the departments of pediatrics-university medical center of the johannes gutenberg university mainz, mainz, germany, department of microbiology and immunology cuhas-bugando and the department of pediatrics and child health of the cuas-bugando. the authors declare no competing interests. correspondence and requests for materials should be addressed to m.m.m.reprints and permissions information is available at www.nature.com/reprints.publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution 4.0 international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons licence, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons licence, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/4.0/. key: cord-324775-3x5os79m authors: crowe, j.e. title: human respiratory viruses date: 2008-07-30 journal: encyclopedia of virology doi: 10.1016/b978-012374410-4.00488-x sha: doc_id: 324775 cord_uid: 3x5os79m viruses are the leading causes of acute lower respiratory-tract infection in infancy. respiratory syncytial virus (rsv) is the most common pathogen, with hmpv, piv-3, influenza viruses, and rhinoviruses accounting for the majority of the remainder of acute viral respiratory infections. humans generally do not develop lifelong immunity to reinfection with these viruses; rather, specific immunity protects against severe and lower respiratory-tract disease. respiratory virus infections of humans are the most common and frequent infections of man. hundreds of different viruses can be considered respiratory viruses. viruses that enter through the respiratory tract include viruses that replicate and cause disease that is restricted to the respiratory epithelium, and other viruses that enter through the mucosa but also spread by viremia causing systemic disease. an example of the latter is measles virus. sars coronavirus is another. in general, viruses that do not cause viremia are capable of reinfecting the same host multiple times throughout life. in contrast, infections with systemic viruses induce lifelong immunity. probably, the high rate of reinfection of mucosally restricted viruses reflects the difficulty and metabolic cost of maintaining a high level of immunity at the vast surface area of the mucosa. virus-specific iga levels are maintained at high levels generally only for several weeks or months after infection. the anatomy and the cell types of the respiratory tract dictate to a large degree the type of disease observed during respiratory virus infection. the demarcation between the upper and lower respiratory tracts is the vocal cords. the structures of the upper respiratory tract, which are all interconnected, include the nasopharynx, the larynx, the eustachian tube and middle ear space, and the sinuses. significant collections of lymphoid tissue reside in the upper respiratory tract, the tonsils and the adenoids. the lower respiratory tract structures include the trachea, bronchi, bronchioles, alveoli, and lung tissue. the cell types that line the respiratory tract are complex, and exhibit different susceptibilities to virus infection. the predominant cell types are ciliated and nonciliated epithelial cells, goblet cells, and clara cells. smooth muscle cells are prominent features of the airways down to the level of the bronchioles, and the lung possesses type i and ii pneumocytes. the disease syndromes that are associated with respiratory viruses generally follow the anatomy of the respiratory tract. different viruses appear to have tropisms for different cells or regions of the respiratory tract; therefore, there are particular associations of viruses with clinical syndromes. the clinical diagnoses for infections with disease manifestations in the respiratory tract are rhinitis and the common cold, sinusitis, otitis media, conjunctivitis, pharyngitis, laryngitis, tracheitis, acute bronchitis, bronchiolitis, pneumonia, and exacerbations of reactive airway disease or asthma. clinical syndromes with more systemic illness due to respiratory viruses include the influenza syndrome, measles, severe acute respiratory syndrome (sars), and hantavirus pulmonary syndrome (hps). the principal causes of acute viral respiratory infections in children became apparent through large epidemiologic studies conducted soon after cell culture techniques became available. the landmark studies of association of viruses with clinical syndromes were performed in the 1960s and 1970s. recent studies have increased our understanding of the causes of viral respiratory infection in infants, especially because of the advent of molecular tests such as the polymerase chain reaction (pcr), which is more sensitive than cell culture. respiratory syncytial virus (rsv), parainfluenza viruses (pivs), adenoviruses, and influenza viruses were identified initially as the most common causes of serious lower respiratory tract disease in infants and children. more recently, human metapneumovirus (hmpv) was identified as a major cause of serious illness. in the last 10 years, a number of additional viruses have been associated with respiratory illness, as discussed below. however, still, infectious agents are not identified in 30-50% of clinical illnesses in large surveillance studies, even using sensitive diagnostic techniques such as viral culture on multiple cell lines, antigen detection assays, and rt-pcr based methods. it is not known if these illnesses are due to identified pathogens that are simply not detected due to low titers of virus in patient samples or if there are novel agents that are yet to be identified. reactivation of latent viruses, such as the herpesviruses hsv and cmv, and adenoviruses occurs in immunocompromised humans, particularly subjects with late-stage hiv infection, those with organ transplantation, and patients with leukopenia and neutropenia caused by chemotherapy. cmv is the most frequently recovered virus from diagnostic procedures such as bronchoalveolar lavage in immunosuppressed patients with pneumonia. these patients also suffer more frequent and more severe disease including mortality with common respiratory viruses, including rsv, hmpv, piv, influenza viruses, rhinoviruses, and adenoviruses. nosocomial transmission including large unit outbreaks is not uncommon, and can result in high frequency of transmission. picornaviridae a wide variety of picornaviruses cause respiratory disease, including rhinoviruses, the enteroviruses a to d including coxsackieviruses a/b, echoviruses, non-polio enteroviruses, and parechoviruses 1-3. enterovirus infections occur most commonly in the summer months in temperate areas, which differs from the season of many of the other most common respiratory viruses such as paramyxoviruses and influenza virus. rhinovirus infections occur year-round. rhinovirus is a genus of the family picornaviridae of viruses. rhinoviruses are the most common viral infective agents in humans, and a causative agent of the common cold. there are over 105 serologic virus types that cause cold symptoms, and rhinoviruses are responsible for approximately half of all cases of the common cold. rhinoviruses have single-stranded positive-sense rna genomes. the viral particles are icosahedral in structure, and they are nonenveloped. rhinovirus-induced common colds may be complicated in children by otitis media and in adults by sinusitis. most adults, in fact, have radiographic evidence of sinusitis during the common cold, which resolves without therapy. therefore the primary disease is probably best termed rhinosinusitis. rhinovirus infection is associated with exacerbations of reactive airway disease in children and asthma in adults. it is not clear whether rhinovirus is restricted to the upper respiratory tract and induces inflammatory responses that affect the lower respiratory tract indirectly, or whether the viruses spread to the lower respiratory tract. in the past, it was thought that these viruses did not often replicate or cause disease in the lower respiratory tract. however, recent studies discern strong epidemiological associations of rvs with wheezing and asthma exacerbations, including episodes severe enough to require hospitalization. likely, rhinoviruses can infect the lower airways to some degree, inducing a local inflammatory response. another possibility is that significant local infection of the upper respiratory tract might induce regional elaboration of mediators that causes lower airways disease. association of rhinovirus infection with lower respiratory tract illness is difficult to study because cell diagnosis by cell culture is not sensitive. rt-pcr diagnostic tests are difficult to interpret because they are often positive for prolonged periods of time and even asymptomatic individuals may have a positive test. comprehensive serologies to confirm infection are difficult because of the large number of serotypes. nevertheless, most experts believe rhinoviruses are a common cause of lower respiratory tract illness. these viruses cause oral lesions and often are associated in children with a disease syndrome termed 'hand-footand-mouth disease'. the pharyngitis associated with this infection often is marked by the very characteristic findings of herpangina, a clinical syndrome of ulcers or small vesicles on the palate and often involving the tonsillar fossa associated with the symptoms of fever, difficulty swallowing, and throat pain. outbreaks commonly occur in young children, in the summer. non-polio enteroviruses are common and distributed worldwide. although infection often is asymptomatic, these viruses cause outbreaks of clinical respiratory disease, sometimes with fatal consequences. studies have associated particular types with clinical syndromes, as enterovirus 68 with wheezing and enterovirus 71 with pneumonia. the term 'echo' in the name of the virus is an acronym for enteric cytopathic human orphan, although this may be an archaic notion since most echoviruses are associated with human diseases, most commonly in children. there are at least 33 echovirus serotypes. echoviruses can be isolated from many children with upper respiratory tract infections during the summer months. echovirus 11 has been associated with laryngotracheitis or croup. epidemiology studies also have associated echoviruses with epidemic pleurodynia, an acute illness characterized by sharp chest pain and fever. these viruses have been assigned a new genus of the family picornaviridae because of distinctive laboratorybased molecular properties. the most common member of the genus parechovirus, human parechovirus 1 (formerly echovirus 22) is a frequent human pathogen. the genus also includes the closely related virus, human parechovirus 2 (formerly echovirus 23). human parechoviruses usually cause mild respiratory or gastrointestinal illness. most infections occur in young children. there is a high seroprevalence for parechoviruses 1 and 2 in adults, and a few clear descriptions of neonatal cases of severe disease. respiratory syncytial virus rsv is a single-stranded negative-sense nonsegmented rna genome virus of the family paramyxoviridae, genus pneumovirus. it is one of the most infectious viruses of humans and infects infants at a very young age, often in the first weeks or months of life. it is the most common viral cause of severe lower respiratory tract illness in children and one of the most important causes of hospitalization in infants and children throughout the world. there is one serotype, but circulating viruses exhibit an antigenic dimorphism such that there are two antigenic subgroups designated a and b. reciprocal cross-neutralization studies using human sera showed that the antigenic groups are about 25% related. reinfection is common and can be caused by viruses of the same subgroup. yearly, epidemics of disease often peak between january and march in temperate regions. rsv infection causes mild upper respiratory tract infection in most infants and young children, but results in hospitalization in 0.5-1% of infants. most children have been infected by the age of 2 years. there is an association of rsv infection early in life and subsequent asthma, although a causal relationship is controversial. most hospitalized infants are otherwise healthy, but some groups are considered high risk for severe disease such as premature infants especially those with chronic lung disease and infants born with congenital heart disease. immunocompromised patients of any age are at risk of severe disease. these viruses constitute a group of four distinct serotypes (types 1-4) of single-stranded rnaviruses belonging to the family paramyxoviridae. when considered as a group, they are the second most common cause of lower respiratory tract infection in young children. piv3 is the most common cause of severe disease. repeated infection throughout life is common. first infections are more commonly associated with lower respiratory tract disease, especially croup, while subsequent infections typically are limited to the upper respiratory tract. pivs are detected using cell culture with hemadsorption or immunofluorescent microscopy, and rt-pcr. in 2001, investigators in the netherlands described a new human respiratory virus, hmpv. evidence of near universal seroconversion was found in the general population by 5 years of age, suggesting ubiquitous infection in early childhood. this virus, a member of the genus pneumovirus with rsv, differs from rsv in that it lacks the ns1 and ns2 nonstructural genes that counteract host interferons and it possesses a slightly different gene order. studies of the role of hmpv in pediatric lower respiratory tracts infection (lri) in otherwise healthy children in the united states, using a prospectively collected 25-year database and sample archive representing about 2000 children, revealed that nearly 12% of lri in children was associated with a positive hmpv test. this and similar studies suggested that the virus is one of the major respiratory pathogens of early childhood. the clinical features of hmpv lri were similar to those of other paramyxoviruses, most often resulting in cough, coryza, and a syndrome of bronchiolitis or croup. interestingly, hmpv seemed to be clinically intermediate between rsv and piv in that it tended to cause bronchiolitis with similar frequency to rsv but more frequently than piv, while causing croup less often than the latter. studies in subjects with conditions predisposing to increased risk of respiratory illness suggest that hmpv plays a significant role in exacerbations of asthma in children and adults, lri in immunocompromised subjects, and in the frail and elderly. measles virus, a paramyxovirus of the genus morbillivirus causes infection with systemic disease, also known as rubeola. the virus is spread both by direct contact/fomite transmission and by aerosol transmission, and therefore is one of the most highly contagious infections of man. the classical symptoms of measles include 3 or more days of fever that is often quite high and a clinical constellation of symptoms termed 'the three cs': cough, coryza, and conjunctivitis. a characteristic disseminated maculopapular rash appears soon after onset of fever. transient mucosal lesions in the mouth of a characteristic appearance (koplik's spots) are considered diagnostic when identified by an experienced clinician. the virus causes a number of systemic effects and can be complicated by severe pneumonia, especially when primary infection occurs in an unvaccinated adult or immunocompromised person of any age. mortality in developing countries is high, especially when infection occurs in the setting of malnutrition. these emerging pathogens that are grouped in their own new genus henipaviruses may not be respiratory pathogens in a conventional sense, but they are paramyxoviruses that probably infect humans by the respiratory route. nipah virus is a newly recognized zoonotic virus, named after the location in malaysia where it was first identified in 1999. it has caused disease in humans with contact with infectious animals. hendra virus (formerly called equine morbillivirus) is another closely related zoonotic paramyxovirus that was first isolated in australia in 1994. the viruses have caused only a few localized outbreaks, but their wide host range and ability to cause high mortality raise concerns for the future. the natural host of these viruses is thought to be a certain species of fruit bats present in australia and the pacific. pigs may be an intermediate host for transmission to humans in nipah infection, and horses in the case of hendra. although the mode of transmission from animals to humans is not defined, it is likely that inoculation of infected materials onto the respiratory tract plays a role. the clinical presentation usually appears to be an influenza-like syndrome, progressing to encephalitis, may include respiratory illness, and causes death in about half of identified cases. influenza is a single-stranded segmented negative-sense rna genome virus of the family orthomyxoviridae. there are three types of influenza viruses: influenza virus a, influenza virus b, and influenza virus c. influenza a and c infect multiple species, while influenza b infects humans almost exclusively. the type a viruses are the most virulent human pathogens among the three influenza types, and cause the most severe disease. the influenza a virus can be subdivided into different subtypes based on the antibody response to these viruses. the subtypes that have been confirmed in humans in seasonal influenza, ordered by the number of known human pandemic deaths, are: h1n1 which caused the 1918 pandemic, and h2n2 which caused the 1957 pandemic of avian influenza that originated in china, h3n2 which caused the pandemic of 1968. currently, h3n2, h1n1, and b viruses cause annual seasonal epidemics. in addition, h5n1 virus infection of humans occurred during an epizootic of h5n1 influenza in hong kong's poultry population in 1997. the disease affected animals of many species and exhibited a high rate of mortality in humans. the virus is spreading throughout asia, carried by wild birds. human-to-human transmission does not occur efficiently at this time; however, there is widespread current concern about the potential for an h5n1 pandemic if the virus acquired transmissibility among humans. the h7n7 avian virus also has unusual zoonotic potential. in 2003 this virus caused an outbreak in humans in the netherlands associated with an outbreak in commercial poultry on several farms. one death occurred and 89 people were confirmed to have h7n7 influenza virus infection. h1n2 virus appears to endemic in pigs and humans. h9n2, h7n2, h7n3, and h10n7 human infections have been reported. influenza b virus is almost exclusively a human pathogen, and is less common than influenza a. it mutates less rapidly than influenza a, and there is only one influenza b subtype. in humans, common symptoms of influenza infection and syndrome are fever, sore throat, myalgias, headache, cough, and fatigue. in more serious cases, influenza causes pneumonia, which can be fatal, particularly in young children and the elderly. influenza pneumonia has an unusually high rate of complication by bacterial superinfection with staphylococcal and streptococcal bacterial pneumonia occurring in as many as 10% of cases in some clinical series. viruses of the family adenoviridae infect both humans and animals. adenoviruses were first isolated in human lymphoid tissues from surgically removed adenoids, hence the name of the virus. in fact, some serotypes establish persistent asymptomatic infections in tonsil and adenoid tissues, and virus shedding can occur for months or years. these double-stranded dna viruses are less than 100 nm in size, and have nonenveloped icosahedral morphology. the large dsdna genome is linear and nonsegmented. there are six major human adenovirus species (designated a through f) that can be placed into 51 immunologically distinct serotypes. human respiratory tract infections are mainly caused by the b and c species. adenovirus infections can occur throughout the year. sporadic outbreaks occur with many of the serotypes, while others appear to be endemic in particular locations. respiratory illnesses include mild disease such as the common cold and lower respiratory tract illness, including croup, bronchiolitis, and pneumonia. conjunctivitis is associated with infection by species b and d. there is a particular constellation of symptoms called 'pharyngoconjunctival fever' which is very frequently associated with acute adenovirus infection. in contrast, gastroenteritis has been associated most frequently with the serotype 40 and 41 virus of species f. immunocompromised subjects are highly susceptible to severe disease during infection with respiratory adenoviruses. the syndrome of acute respiratory disease (ard), especially common during stressful or crowded living conditions, was first recognized among military recruits during world war ii and continues to be a problem for the military following suspension of vaccination. ard is most often associated with adenovirus types 4 and 7. members of the genus coronavirus also contribute to respiratory illness including severe disease. there are dozens of coronaviruses that affect animals. until recently, only two representative strains of human coronaviruses were known to cause disease, human coronavirus 229e (hcov-229e) and hcov-oc43. a recent outbreak of sars-associated coronavirus (sars-cov) showed that animal coronaviruses have the potential to cross species to humans with devastating effects. there has been one major epidemic to date, between november 2002 and july 2003, with over 8000 cases of the disease, and mortality rates approaching 10%. sars-cov causes a systemic illness with a respiratory route of entry. sars is a unique form of viral pneumonia. in contrast to most other viral pneumonias, upper respiratory symptoms are usually absent in sars, although cough and dyspnea occur in most patients. typically, patients present with a nonspecific illness manifesting fever, myalgia, malaise, and chills or rigors; watery diarrhea may occur as well. recently, investigators reported the identification of a fourth human coronavirus, hcov-nl63, a new group 1 coronavirus. evidence is emerging that hcov-nl63 is a common respiratory pathogen of humans, causing both upper and lower respiratory tract illness. human coronavirus (hcov) hku1 was first described in january 2005 following detection in a patient with pneumonia. several cases of respiratory illness have been associated with the virus, but the infrequent identification suggests to date that this putative group 2 coronavirus causes a low incidence of illness. several herpes viruses cause upper respiratory infections, especially infection of the oral cavity. herpes simplex pharyngitis is associated with characteristic clinical findings, such as acute ulcerative stomatitis and ulcerative pharyngitis. herpes simplex virus 1 (hsv-1) and herpes simplex virus 2 (hsv-2), also called human herpesvirus 1 (hhv-1) and human herpesvirus 2 (hhv-2), respectively, cause oral lesions, although over 90% of oral infections are caused by hsv-1. primary oral disease can be severe, especially in young children, who sometimes are admitted for rehydration therapy due to poor oral intake. a significant proportion of individuals suffer recurrences of symptomatic disease consisting of vesicles on the lips. epstein-barr virus (ebv) mononucleosis syndrome is often marked by acute or subacute exudative pharyngitis; in some cases, the swelling of the tonsils in ebv pharyngitis is so severe that airway occlusion appears imminent. most of the viruses of the family herpesviridae can cause severe disease in immunocompromised patients (especially hematopoietic stem cell transplant patients), including cytomegalovirus (cmv), ebv, varicella-zoster virus, herpesvirus 6, herpesvirus 7, and herpesvirus 8. a new virus was identified recently in respiratory samples from children with lower respiratory tract disease in sweden. sequence analysis of the viral genome revealed that the virus is highly related to canine minute virus and bovine parvovirus and is a member of the genus bocavirus, subfamily parvovirinae, family parvoviridae. this virus was tentatively named human bocavirus (hbov). hbov has been identified as the sole agent in a limited number of respiratory samples from children hospitalized with respiratory tract disease. it remains to be seen whether the virus is causative of or merely associated with disease in these preliminary studies. over 400 cases of hps have been reported in the united states. the disease was first recognized during an outbreak in 1993. about a third of recognized cases end in death. the four corners area outbreak is well known; however, cases now have been reported in 30 states. patients with hps usually present with a febrile illness beginning with symptoms of a flu-like illness. physical examination is not specific, often only with findings of fever, and increased heart and respiratory rates. in addition to the respiratory symptoms, abdominal pain and fever are common. diagnosis is often delayed until a severe illness occurs requiring mechanical ventilation. rotaviruses are dsrna enteric viruses that are the most common cause of severe viral infectious gastroenteritis in children. clinical series suggest that some children with gastroenteritis suffer upper respiratory symptoms during the prodrome of disease manifestation, and virus can be recovered from respiratory secretions. some reports suggest that rotavirus infection is associated with lower respiratory tract illness, although this association is unclear. these dsrna viruses (named using an acronym for respiratory enteric orphan virus) are not clearly associated with respiratory disease, but seroconversion rate is high in the first few years of life, and they probably cause minor or subclinical illness. pharyngitis occurs with primary hiv infection and may be associated with mucosal erosions and lymphadenopathy. polyomaviruses are small dsdna genome nonenveloped icosahedral viruses that may be oncogenic. there are two polyomaviruses known to infect humans, jc and bk viruses. eighty percent or more of adult us subjects are seropositive for these viruses. jc virus can infect the respiratory system, kidneys, or brain. bk virus infection causes a mild respiratory infection or pneumonia and can involve the kidneys of immunosuppressed transplant patients. given the overlap in the winter season of these viruses in temperate areas, it is not surprising that co-infections with two or more viruses occur. in general, when careful studies using cell culture techniques were used for virus isolation, more than one virus was isolated from respiratory secretions of otherwise healthy subjects with acute respiratory illness in about 5-10% of cases in adults and 10-15% in children. there is little evidence that more severe disease occurs during co-infections, although there is insufficient evidence on this point to be definitive. the incidence of two molecular diagnostic tests being positive (generally rt-pcr, for these rna viruses) is expected to be higher than that of culture, because molecular tests can remain positive for an extended period of time after virus shedding has ended. respiratory viruses generally have two main modes of transmission, large particle aerosols of respiratory droplets transmitted directly from person-to-person by coughing or sneezing, or by fomites. fomite transmission occurs indirectly when infected respiratory droplets are deposited on hands or on inanimate objects and surfaces with subsequent transfer of secretions to a susceptible subject's nose or conjunctiva. most respiratory viruses, unlike measles virus or varicella zoster virus, do not spread by small particle aerosols across rooms or down halls. therefore, contact and droplet precautions are sufficient to prevent transmission in most settings; handwashing is critical in healthcare settings during the winter season. ribavirin is a nucleoside antimetabolite pro-drug that is activated by kinases in the cell, resulting in a 5 0 triphosphate nucleotide form that inhibits rna replication. the drug was licensed in an aerosol form in the us in 1986 for treatment of children with severe rsv lower respiratory tract infection. the efficacy of aerosolized ribavirin therapy remains uncertain despite a number of clinical trials. most centers use it infrequently, if ever, in otherwise healthy infants with severe rsv disease. intravenous ribavirin has been used for adenovirus, hantavirus, measles virus, piv, and influenza virus infections, although a good risk/benefit profile has not been established clearly for any of these uses. a humanized mouse monoclonal antibody directed to the f protein of rsv, 'palivizumab', is licensed for prevention of rsv hospitalization in high-risk infants. it is efficacious in half or more of high-risk subjects. a more potent second-generation antibody is being studied in clinical trials. experimental treatment of both immunocompetent and immunocompromised rsv-infected subjects has been reported but the efficacy of this approach is not established. there are four licensed drugs in the us for treatment or prophylaxis of influenza. 'amantadine' and 'rimantadine' are two of the drugs that interfere with the ion channel activity caused by the viral m2 protein of influenza a viruses, which is needed for viral particle uncoating following endocytosis. the other two drugs, 'oseltamivir' and 'zanamivir', are neuraminidase inhibitors that act on both influenza a and b viruses by serving as transition state analogs of the viral neuraminidase that is needed to release newly budded virion progeny from the surface of infected cells. the cell surface normally is coated heavily with the viral receptor sialic acid. resistance to the ion channel inhibitors arises rapidly during prophylaxis or treatment, and in 2006 resistance levels became so common in circulating viruses that the cdc no longer recommends use of these drugs. 'interferon-a' has been shown to protect against rhinovirus infections when used intranasally. this biological drug causes some side effects, such as nasal bleeding, and resistance to the drug developed during experimental use, so the molecule is no longer being developed for this purpose. 'pleconaril' has been tested for treatment of rhinovirus infection, as it is an oral drug with good bioavailability for treating infections caused by picornaviruses. this drug acts by binding to a hydrophobic pocket in the vp1 protein and stabilizing the protein capsid, preventing release of viral rna into the cell. the drug reduced mucus secretions and other symptoms and is being further examined. 'acyclovir' and related compounds are guanine analog antiviral drugs used in treatment of herpes virus infections. hsv stomatitis in immunocompromised patients is treated with 'famciclovir', or 'valacyclovir', and immunocompetent subjects with severe oral disease compromising oral intake are sometimes treated. these compounds have also been used prophylactically to prevent recurrences of outbreaks, with mixed results. intravenous acyclovir is effective in hsv or varicella zoster virus pneumonia in immunocompromised subjects. 'ganciclovir' with human immunoglobulin may reduce the mortality associated with cmv pneumonia in hematopoietic stem cell transplant recipients and has been used as monotherapy in other patient groups. 'cidofivir' is a nucleotide analog with activity against a large number of viruses, including adenoviruses. intravenous cidofovir has been effective in the management of severe adenoviral infection in immunocompromised patients but may cause serious nephrotoxicity. there are licensed vaccines for influenza viruses. in the us, both a trivalent (h3n2, h1n1, and b) inactivated intramuscular vaccine and a live attenuated trivalent vaccine for intranasal administration is available. the efficacy of these vaccines is good when the vaccine strains chosen are highly related antigenically to the epidemic strain. antigenic drift caused by point mutations in the ha and na molecules leads to antigenic divergence, requiring new vaccines to be made each year. the influenza genome is segmented, which allows reassortment of two viruses to occur during co-infection, which sometimes leads to a major antigenic shift resulting in a pandemic. pandemics occur every 20-30 years on average. there is current concern about the potential for an h5n1 pandemic, and experimental vaccines are being tested for this virus. to date, h5n1 vaccines have been poorly immunogenic compared to comparable seasonal influenza vaccines. vaccines were developed for adenovirus serotypes 4 and 7, and these were approved for preventing epidemic respiratory illness among military recruits. essentially, these were unmodified viruses given by the enteric route in capsules, instead of the respiratory route, which is the natural route of infection leading to disease. inoculation by the altered route resulted in an immunizing asymptomatic infection. all us military recruits were vaccinated against adenovirus from 1971 to 1999 with near complete prevention of the disease in this population, but the sole manufacturer of the vaccine halted production in 1996 and supplies ran out 3 years later. since 1999, adenovirus infection has reemerged as a significant problem in the military with approximately 10% of all recruits suffering illness due to adenovirus infection during basic training; some deaths have occurred. live attenuated vaccine candidates are under development and being tested in phase i and ii clinical trials for rsv and the pivs. mutant strains with reduced pathogenicity were isolated in the laboratory, tested, and sequenced. now, vaccine candidates are being optimized by combining mutations from separate biologically derived viruses into single strains using recombinant techniques for generating rnaviruses from cdna copies, a process called reverse genetics. subunit vaccines have been developed for rsv, but there are safety concerns about their use in young infants because formalin inactivated vaccine induced a more severe disease response to infection in the 1960s. there are no vaccines against rhinoviruses as there is little or no cross-protection between serotypes, and it is not feasible to develop a vaccine for over 100 serotypes. efforts to develop coronavirus vaccines are in the preclinical stage. viruses are the leading causes of acute lower respiratory tract infection in infancy. rsv is the most common pathogen, with hmpv, piv-3, influenza viruses, and rhinoviruses accounting for the majority of the remainder of acute viral respiratory infections. humans generally do not develop lifelong immunity to reinfection with these viruses; rather, specific immunity protects against severe and lower respiratory tract disease. risk of respiratory syncytial virus infection for infants from low-income families in relationship to age, sex, ethnic group, and maternal antibody level viral infections in relation to age, atrophy, and season of admission among children hospitalized for wheezing parainfluenza viruses what have we learned from the tucson children's respiratory study? epidemiology of respiratory syncytial virus infection in washington, dc. part ii. infection and disease with respect to age, immunologic status, race, and sex characterization of an avian influenza a (h5n1) virus isolated from a child with a fatal respiratory illness human metapneumovirus and lower respiratory tract disease in otherwise healthy infants and children picornavirus infections in children diagnosed by rt-pcr during longitudinal surveillance with weekly sampling: association with symptomatic illness and effect of season fields virology human t-cell leukemia viruses: general features m yoshida, university of tokyo, chiba, japan 2008 elsevier ltd. all rights reserved.glossary px region htlv sequence between the env and 3 0 ltr, encoding tax, rex and other small regulatory proteins. rex trans-modulator of viral rna splicing and transport. tax pleiotropic regulator activating viral and cellular replication interacting with cellular transcription factors, tumor suppressor proteins, and cell cycle checkpoints. human t-cell leukemia virus 1 (htlv-1) is the first established tumorigenic retrovirus of humans; exogenous to humans this virus is classified as the species human t-cell leukemia virus, in deltaretroviridae, within the family retroviridae. htlv-1 infection is associated with leukemia and neural disease, adult t-cell leukemia (atl) and htlv-1-associated myelopathy/tropical spastic parapasis (ham/tsp), respectively. the genomic structure of the virus with genes for nonstructural proteins established a distinct viral genus that includes bovine leukemia virus. htlv-1 has no oncogene, but nevertheless transforms t cells rather efficiently and is identified as the etiologic agent of atl. htlv-1 has unique regulatory proteins, tax and rex, and tax has been identified as a critical molecule not only in regulation of viral replication but also in induction of atl. after long and enormous efforts to identify a retrovirus in human tumors, htlv was described in t-cell lines as a convincing human retrovirus. the first report of the virus (htlv) was from a patient with mycosis (mf) in the us, and another (adult t-cell leukemia virus (atlv)) was from a patient with atl in japan. subsequently, the mf case was characterized as atl and the two isolates were established to be the same following a comparison of their genomes.a prototypical retroviral genome contains the gag, pol, and env genes encoding the virion proteins including core proteins, reverse transcriptase, and surface glycoprotein, respectively. acute leukemia viruses generally have an oncogene acquired from cellular genes that substitutes a part of the gag, pol, and env sequences. in contrast to these genomes, htlv has additional genes in an extra px region between env and the 3 0 ltr (ltr -long terminal repeat). this unique genomic structure classified htlv as a member of a distinct genus of the retroviridae, which includes htlv-1, and-2, bovine leukemia virus (blv), and simian t-cell leukemia viruses (stlv-1, -2, and -3). htlv-2 was isolated from a patient with hairy t-cell leukemia and its genome similarity to the type 1 is about 60%.stlvs have been isolated from various species of old world nonhuman primates, including the japanese macaque, african green monkey, pig-tailed macaque, gorilla, and chimpanzee. their genomes share 90-95% homology.blv infects and replicates in b cells of cows and sheep and induces b-cell lymphoma. key: cord-346887-dl0wc4u2 authors: cho, hye jung; shim, so‐yeon; son, dong woo; sun, yong han; tchah, hann; jeon, in‐sang title: respiratory viruses in neonates hospitalized with acute lower respiratory tract infections date: 2012-12-11 journal: pediatr int doi: 10.1111/j.1442-200x.2012.03727.x sha: doc_id: 346887 cord_uid: dl0wc4u2 background: the burden of respiratory syncytial virus (rsv) in neonates has not been clearly studied. the aims of this study were to determine the overall distribution of respiratory viruses in neonates hospitalized with acute lower respiratory tract infectiosns (alri) and to describe the clinical characteristics of rsv infections in these neonates. methods: from january 2009 through may 2010, neonates aged <1 month who were hospitalized with alri and did not have underlying disease were included in the study. viruses were identified on multiplex reverse transcription polymerase chain reaction using nasal swab samples. clinical variables were evaluated between the rsv and non‐rsv infection groups. results: of the 108 infants included in the study, 46 (42.6%) had rsv; human rhinovirus (18.5%), human parainfluenza virus 3 (7.5%), and human metapneumovirus (3.7%) were the next most common infections. codetections accounted for 8.3% of the cases. crowding increased the risk of rsv infection compared to the non‐rsv group (or, 16.5; p = 0.001). the rsv group had a greater incidence of dyspnea (p = 0.027), pneumonia (p < 0.001), requirement for oxygen (p < 0.001), and prolonged hospitalization (p = 0.011) than the non‐rsv group. conclusions: rsv was the most common viral etiology in neonates without underlying diseases who were hospitalized with alri. the disease severity of rsv infection was worse than that of other detected viral infections. strict prevention strategies should be considered in overcrowded situations. in recent years we have developed an understanding of respiratory infections through the detection of viruses using reverse transcription-polymerase chain reaction (rt-pcr). in addition to respiratory syncytial virus (rsv), human rhinovirus (hrv), human parainfluenza virus (hpiv), and human metapneumovirus (hmpv) have been found to be major causes of acute lower respiratory tract infections (alri) in infants and young children. [1] [2] [3] alri are leading causes of hospitalization and mortality among children worldwide, particularly young children. 4, 5 only a few studies, however, have reported on respiratory viruses in neonates with alri. understanding the overall spectrum of respiratory viruses causing alri in hospitalized neonates is essential to establish preventive and therapeutic strategies in this population. rsv is the most common etiological agent of alri in infants and young children. 6, 7 to date, many reports have described the burden of rsv infections and its risk factors. prematurity, low birthweight, and chronic lung disease (cld) have been suggested as risk factors for rsv infections. additionally, epidemiologic factors, such as crowding, having older siblings, day-care center attendance and exposure to smoke are important risk factors. [8] [9] [10] [11] very few studies have focused on neonatal rsvassociated alri among healthy neonates aged <1 month. 5, 12 the primary objective of this study was to determine the distribution of respiratory viruses in otherwise healthy neonates hospitalized with alri. a secondary aim was to identify the clinical characteristics of rsv infections in this population. recorded the following variables: history of premature birth, the presence of underlying medical conditions, breastfeeding, number of siblings, siblings attending day-care centers, number of residents at home, exposure to smoke, and residence in a neonatal day-care center in the previous 48 h. in south korea, neonatal day-care centers are common places that provide caregivers for mothers and neonates after discharge from delivery hospitals. in these facilities, assistants take care of many neonates in a room, similar to a nursery. respiratory infections were considered to be acquired at a neonatal day-care center if the neonates became symptomatic within 48 h of discharge from such a center. the clinical features and clinical courses during the hospitalization were summarized by one of the attending physicians who were blinded to this study. clinical variables, such as rhinorrhea and/or cough, fever (defined as axillary temperature ն38°c), and requirement for oxygen therapy, were recorded. apnea was defined as a lack of breathing activity for at least 20 s. dyspnea referred to tachypnea (respiration rate ն60 breaths/min) combined with the presence of pronounced chest wall retractions. pneumonia was documented only if the clinical diagnosis was confirmed on chest radiography. on admission, a blood sample was taken to measure total white blood cell count and c-reactive protein, and for blood cultures. neutropenia was defined as an absolute neutrophil count <1000/mm 3 . all included neonates were divided into the rsv group and non-rsv group based on the results of multiplex rt-pcr. neonates with codetection or negative multiplex rt-pcr were excluded from the grouping. demographics, clinical variables, and laboratory data were compared between groups. ethics approval was obtained from the ethics committee of gachon university gil hospital. for the multiplex rt-pcr, rna was extracted from nasal swab samples using the viral gene-spin tm viral dna/rna extraction kit (intron, seoul, south korea), and cdnas were synthesized. rt was performed using the revert aid tm first strand cdna synthesis kit (fermentas, burlington, on, canada). pcr amplification was performed using 10 ml of seeplex rv master mix 12 (seegen, seoul, south korea), 4 ml of 5x multiplex primer sets, 3 ml of 8-methoxypsoralen solution, and 3 ml of newly synthesized first-strand cdna. seeplex rv contained a and b sets of primers designed from highly conserved regions of genetic sequences for 12 respiratory viruses, including human coronavirus (hcov)-229e/nl63, hcov-oc43, hmpv, hrv, rsv a/b, influenza a/b, hpiv-1, 2, 3, and adenovirus. an initial pre-pcr step of 94°c for 15 min was performed followed by a total of 40 pcr cycles under the following conditions: 95°c for 30 s, 60°c for 1.5 min, and 72°c for 1.5 min. the final cycle was followed by an extension step at 72°c for 10 min to complete any partial polymerization. the amplified pcr products were separated on 2% agarose gel and stained with ethidium bromide. the type of respiratory virus was identified by comparison with the reference band size provided by the manufacturer. statistical analysis was performed using spss version 14.0 (spss, chicago, il, usa). significance was examined using the chi-squared test for categorical variables, fisher's exact test for binary variables, and the independent sample t-test for continuous variables. normally distributed data are expressed as mean ϯ sd. non-parametric data are expressed as median (25-75th percentiles). differences were considered significant for p < 0.05. in the study period from january 2009 to may 2010, a total of 140 neonates with alri were admitted to the nicu (fig. 1 ). table 1 lists the distribution of 108 viral agents isolated from the enrolled infants. among the viruses, rsv was the predominant agent (42.6%), followed by hrv (18.5%), hpiv-3 (7.5%) and hmpv (3.7%). in nine neonates (8.3%), two viruses were simultaneously present, while no virus was identified in 21 neonates (19.4%). most rsv cases appeared in november and persisted until may, and then gradually diminished. most hrv cases occurred in the spring (march-april). four hmpv cases newly appeared during the late spring of 2010 (april-may; fig. 2 ). table 2 lists the neonates' demographic data. the median age at diagnosis in the rsv and non-rsv groups was 22 days and 24 days, respectively, and the proportion of males was 69.6% and 65.6%, respectively. no significant difference was observed between the groups according to the presence of environmental risk factors, such as number of siblings and siblings attending day-care center, number of residents at home, or exposure to smoke. a significantly higher proportion of infants in the rsv group had contracted infections from neonatal day-care centers (34.8% vs 3.1%, p = 0.001, odds ratio, 16.5; 95% confidence interval: 2.06-132.6). clinical data were compared between the rsv group (n = 46) and non-rsv group (n = 32; table 3 ). dyspnea was documented significantly more often in the rsv group (34.8% vs 12.5%, p = 0.027). in contrast, fever ն38°c was less frequent in the rsv group (15.2% vs 43.8%, p = 0.005). pneumonia and particularly consolidation in the right upper lobe were more often found in the rsv group (54.3% vs 15.6%, p < 0.001; 45.7% vs 6.3%, p < 0.001, respectively). the rsv group more frequently required oxygen therapy (45.7% vs 6.3%, p < 0.001) and had longer hospital stay (9.41 ϯ 3.43 days vs 7.34 ϯ 3.51 days, p = 0.011). all nine neonates with viral codetections were diagnosed with pneumonia, and three of them (33.3%) needed supplemental oxygen therapy. no neonates died during the study period. two neonates from each group required mechanical ventilation. both neonates pre sented apnea on admission and definite respiratory symptoms, such as cough and rhinorrhea, were subsequently followed. to the best of our knowledge, this is the first study to focus exclusively on previously healthy neonates with alri. to demonstrate the clinical impact of respiratory viral infection on neonates, we included only full-term neonates without underlying disease. in the present study, 80.5% of the neonates who were tested on multiplex rt-pcr had positive viral results. this detection rate is slightly higher than other similar reports for infants and small children. 1, 13 one study based on hospitalized children with alri in korea reported that rsv (33.2%) was the most common virus among 13 respiratory viruses and was followed by hrv (19.1%), influenza virus (16.9%), hmpv (15.4%), and hpiv (8.3%). 14 this distribution was similar to the present results, although the proportion of rsv was greater in the present study. we found that most respiratory viral infections in neonates are acquired from the community. in the present study, hrv was the second most commonly detected virus, accounting for 18.5% of detected viruses. previous studies reported that hrv was the second most commonly detected virus, 2,15 and could result in serious lower respiratory tract disease in early life. 3, 16 recently, several studies reported that most young infants with hrv detection had symptomatic illness, whereas half of the children in which hrv was detected were asymptomatic. 17, 18 in young infants, including neonates, the chances of colonization may be lower than in older children. regarding these findings, hrv should be considered the main virus in the present study. interestingly, in this study, influenza was not detected, although the study period coincided with the pandemic of influenza a (h1n1) virus. according to the ko et al. korean, nationwide data for 2009, infants <6 months old accounted for only 4.7% of hospitalized children with h1n1 influenza infection. 19 it appears that younger infants have relatively lower risk for influenza because of lower exposure, but additional studies, including outpatient studies, are needed to support this conclusion. previous studies reported that viral codetections were found in 11-40% of subjects; in the present study only 8.3% presented codetection with two viruses. the relatively lower percentage of codetection in the present study may be due to lower colonization in neonates and lower chances of exposure to various viruses. an association between dual viral detection and more severe disease has not been properly established. some authors consider that any dual viral infection increases severity of disease. 20, 21 other researchers, however, have found no difference when compared to single viral infections. 13, 22 in the present study all neonates with codetection were diagnosed with lobar pneumonia. based on these findings, we support an association of viral codetection with a more severe clinical course. notably, rsv is the most common cause of alri-associated mortality, especially in preterm infants. in the present study there were no cases of mortality. a high level of maternal rsv igg antibodies was considered one reason for this finding because it may offer protection from severe illness in term infants during the first few months of life. 23 in south korea, the rsv season ranges from october to march, 24 but it is reported that the season of rsv outbreaks varies considerably from year to year. 9 the present findings were similar to that expected in temperate climate zones; the incidence of rsv infections peaked from january to february 2009. this pattern, however, changed in 2010; there was no significant variation in the monthly distribution of rsv throughout the period from january to may. according to previous reports, male gender, presence of older siblings, tobacco smoke exposure, and siblings attending daycare center were important risk factors for rsv infection. 8, [25] [26] [27] [28] [29] [30] in the present study there were no significant differences in these factors between the rsv and non-rsv groups. this implies that such risk factors could influence not only rsv infections but also other viral respiratory infections. in the present study a significantly higher proportion of the rsv group obtained infections from neonatal day-care centers. neonates from neonatal day-care centers tended to be admitted as a group. crowding situations in neonatal day-care centers may affect the transmission of rsv infections. in agreement with previous studies, we found that rsv infections had a more severe clinical course than other viral respiratory infections. 3, 6 fattouh et al. reported that compared with non-rsv neonates, rsv neonates frequently had dyspnea and required supplemental oxygen with a higher rate of hospitalization. 31 in the present study, 54.3% of neonates in the rsv group were diagnosed with pneumonia, whereas only 15.6% were diagnosed with pneumonia in the non-rsv group. we performed chest radiography in all enrolled neonates and concluded that rsv infections were capable of causing pneumonia, especially in the right upper lobe. to date, the majority of studies on rsv in neonates have been focused on neonates with underlying, high-risk medical conditions, such as premature infants, especially those with cld or congenital heart disease. immunoprophylaxis, which is effective at reducing rsv hospitalization, is recommended only for the highest risk infants because of cost-effectiveness. 32 most infants who are hospitalized with rsv-related disease, however, have no underlying medical conditions. 9 therefore, until a safe and effective vaccine is developed, prevention is essential to reduce rsv infections. in the present study, overcrowded environments, such as neonatal day-care centers, was a strong risk factor for rsv infection. education regarding transmission-based precautions, such as hand hygiene and standard contact precautions, should be considered. there were several limitations in this study. because we focused only on neonates with infections severe enough to require hospitalization, we could not estimate the burden of viral respiratory infections not requiring hospitalization. although our regional center is the largest one in the study area, it is unknown whether these data represent regionwide demographics. in the present study, only eight cases of codetection were confirmed. we could not determine the severity of codetection due to the small sample size. further studies with larger sample sizes, including asymptomatic control groups and detailed subgroup analyses, are necessary. viruses in community-acquired pneumonia in children aged less than 3 years old: high rate of viral coinfection role of rhinovirus in hospitalized infants with respiratory tract infections in spain viral etiology of acute respiratory infections with cough in infancy: a community-based birth cohort study estimates of world-wide distribution of child deaths from acute respiratory infections global burden of acute lower respiratory infections due to respiratory syncytial virus in young children: a systematic review and meta-analysis risk factors in children hospitalized with rsv bronchiolitis versus non-rsv bronchiolitis respiratory syncytial virus-associated hospitalizations among infants and young children in the united states risk factors for severe rsv-induced lower respiratory tract infection over four consecutive epidemics epidemiology and prevention of respiratory syncytial virus infections among infants and young children hospitalization rates for respiratory syncytial virus infection in premature infants born during two consecutive seasons flip-2 study: risk factors linked to respiratory syncytial virus infection requiring hospitalization in premature infants born in spain at a gestational age of 32 to 35 weeks clinical prediction rule for rsv bronchiolitis in healthy newborns: prognostic birth cohort study frequent detection of viral coinfection in children hospitalized with acute respiratory tract infection using a real-time polymerase chain reaction clinical characteristics of acute lower respiratory tract infection due to 13 respiratory viruses detected by multiplex pcr in children respiratory syncytial virus and human rhinoviruses are the major causes of severe lower respiratory tract infections in kuwait rhinovirus associated with severe lower respiratory tract infections in children respiratory pathogens in children with and without respiratory symptoms rhinovirus transmission within families with children: incidence of symptomatic and asymptomatic infections characteristics of hospitalized children with 2009 pandemic influenza a (h1n1): a multicenter study in korea the impact of dual viral infection in infants admitted to a pediatric intensive care unit associated with severe bronchiolitis mixed respiratory virus infections the association of newly identified respiratory viruses with lower respiratory tract infections in korean children trends in chronologic age and infant respiratory syncytial virus hospitalization: an 8-year cohort study clinical and epidemiological comparison of human metapneumovirus and respiratory syncytial virus in seoul nosocomial infection: a risk factor for a complicated course in children with respiratory syncytial virus infection: results from a prospective multicenter german surveillance study respiratory syncytial virus infection in 406 hospitalized premature infants: results from a prospective german multicentre database clinical relevance of prevention of respiratory syncytial virus lower respiratory tract infection in preterm infants born between 33 and 35 weeks gestational age respiratory syncytial virus infections in hospitalized infants: association between viral load, virus subgroup, and disease severity the pediatric investigators collaborative network on infections in canada study of predictors of hospitalization for respiratory syncytial virus infection for infants born at 33 through 35 completed weeks of gestation effect of prematurity on respiratory syncytial virus hospital resource use and outcomes acute lower respiratory tract infection due to respiratory syncytial virus in a group of egyptian children under 5 years of age prevention of serious respiratory syncytial virus-related illness. ii: immunoprophylaxis the overall distribution of respiratory viruses in neonates hospitalized with alri has been described in the present study. rsv was the most common viral alri etiology and hrv was the second most common. rsv had a more severe course than other detected viruses. crowding situations increased the risk of rsv detection. strict prevention strategies should be considered in overcrowded situations. key: cord-344889-1y4ieamp authors: cameron, robert j.; de wit, deo; welsh, toni n.; ferguson, john; grissell, terry v.; rye, peter j. title: virus infection in exacerbations of chronic obstructive pulmonary disease requiring ventilation date: 2006-05-24 journal: intensive care med doi: 10.1007/s00134-006-0202-x sha: doc_id: 344889 cord_uid: 1y4ieamp objectives: we aimed to characterise and quantify the incidence of common infectious agents in acute exacerbations of chronic obstructive pulmonary disease (copd) requiring ventilation, with a focus on respiratory viruses. design: an epidemiological study conducted over 3 years. setting: a 12-bed intensive care unit (icu). participants: icu patients over 45 years of age with a primary diagnosis of copd exacerbation requiring non-invasive ventilation (niv) or ventilation via endotracheal tube (ett). materials and methods: nasopharyngeal aspirates (npa) and posterior pharyngeal swabs (ps) were tested for viruses with immunofluorescence assay (ifa), virus culture (vc) and polymerase chain reaction (pcr). paired virus and atypical pneumonia serology assays were taken. blood, sputum and endotracheal aspirates were cultured for bacteria. results: 107 episodes in 105 patients were recorded. twenty-three (21%) died within 28 days. a probable infectious aetiology was found in 69 patient episodes (64%). a virus was identified in 46 cases (43%), being the sole organism in 35 cases (33%) and part of a mixed infection in 11 cases (10%). a probable bacterial aetiology was found in 25 cases (23%). there was no statistically significant difference in clinical characteristics or outcomes between the group with virus infections and that without. conclusion: forty-six (43%) of the patients with copd exacerbation requiring mechanical ventilation had a probable viral pathogen. prodromal, clinical and outcome parameters did not distinguish virus from non-virus illness. pcr was the most sensitive whilst virus culture was the least of virus assays. electronic supplementary material: the electronic reference of this article is http://dx.doi.org/10.1007/s00134-006-0202-x. the online full-text version of this article includes electronic supplementary material. this material is available to authorised users and can be accessed by means of the esm button beneath the abstract or in the structured full-text article. to cite or link to this article you can use the above reference. abstract objectives: we aimed to characterise and quantify the incidence of common infectious agents in acute exacerbations of chronic obstructive pulmonary disease (copd) requiring ventilation, with a focus on respiratory viruses. design: an epidemiological study conducted over 3 years. setting: a 12-bed intensive care unit (icu). participants: icu patients over 45 years of age with a primary diagnosis of copd exacerbation requiring non-invasive ventilation (niv) or ventilation via endotracheal tube (ett). materials and methods: nasopharyngeal aspirates (npa) and posterior pharyngeal swabs (ps) were tested for viruses with immunofluorescence assay (ifa), virus culture (vc) and polymerase chain reaction (pcr). paired virus and atypical pneumonia serology assays were taken. blood, sputum and endotracheal aspirates were cultured for bacteria. results: 107 episodes in 105 patients were recorded. twenty-three (21%) died within 28 days. a probable infectious aetiology was found in 69 patient episodes (64%). a virus was identified in 46 cases (43%), being the sole organism in 35 cases (33%) and part of a mixed infection in 11 cases (10%). a probable bacterial aetiology was found in 25 cases (23%). there was no statistically significant difference in clinical characteristics or outcomes between the group with virus infections and that without. conclusion: forty-six (43%) of the patients with copd exacerbation requiring mechanical ventilation had a probable viral pathogen. prodromal, clinical and outcome parameters did not distinguish virus from non-virus illness. pcr was the most sensitive introduction chronic obstructive pulmonary disease (copd) is defined as the presence of progressive, incompletely reversible obstructive lung disease from diffuse causes, including chronic bronchitis, emphysema and small airways disease. some 3-5% of approximately 1000 patients presenting annually with exacerbations of copd are admitted to our intensive care unit (icu) for ventilatory assistance with either bilevel non-invasive positive pressure ventilation (niv) or ventilation via an endotracheal tube (ett). despite several studies in the unventilated population [1, 2, 3, 4, 5, 6] , and two studies in ventilated copd patients [7, 8] , the viral aetiology of many copd exacerbations remains unclear. over 200 respiratory viruses [9] have been shown to cause upper and lower respiratory tract infections via inflammatory and neural mediators [10] . of these, influenza types a and b (inf a, b), parainfluenza types 1, 2 and 3 (para 1, 2, 3), rhinovirus (rv), adenovirus (av), respiratory syncytial virus (rsv), coronavirus (cov) [11, 12] and, less commonly, human metapneumovirus (hmpv) [13] , and enterovirus (ev) [14, 15] have been shown to play significant roles in airway infections. we hypothesised that viral infections may be associated with a significant disease burden in patients with exacerbations of copd admitted to icu for ventilatory support. we aimed to investigate the incidence of these viruses. we also sought other known biological and atypical pathogens responsible for copd exacerbations to build up as complete a microbiological profile as possible. any patient over the age of 45 years requiring mechanical ventilation for an exacerbation of copd within 48 h of admission to hospital was considered for the study. these patients were admitted to the icu for ventilatory support. we defined copd as baseline fev 1 < 70% predicted with incompletely reversible obstructive spirometry as per american thoracic society/ european respiratory (ats/ers) copd consensus guidelines [16] . in the absence of prior spirometry, we assessed a smoking history of at least 20 pack years, physical examination, review of previous hospital records, chest radiograph changes suggestive of copd, exclusion of other major co-morbidities as a primary cause of chronic breathlessness and chronic reduction in exercise tolerance to make the diagnosis of copd. icu admission for ventilatory support was considered when, despite optimal medical therapy and oxygen administration, there was acidosis (ph < 7.35), hypercapnia (paco 2 > 45 mmhg), increasing respiratory distress and/or worsening fatigue or reduced consciousness [16] . the decision to mechanically ventilate was made by the attending icu specialist based upon past history and current clinical and radiological information. each patient was assessed for either niv or endotracheal intubation using the guidelines of the british thoracic society [17] . patients were excluded from the study who themselves elected not to have mechanical ventilation, or who were not mechanically ventilated because they were not expected to survive due to the severity of their underlying copd or concurrent illnesses, after consultation with either themselves, if capable and conscious, or with their relatives/care givers. patients with asthma completely reversible with bronchodilators, those less than 45 years old, those with restrictive lung disease or surgery or trauma within the previous 4 weeks and those admitted to general wards before icu admission for more than 48 h (and therefore at risk of a hospital-acquired infection) were excluded. the presence of clinical pneumonia or the finding of chest radiograph changes did not exclude patients, as virus infections are well recognised to cause pneumonic changes [17] . the study was conducted between july 2000 and november 2003. approval for the study was obtained from our hospital ethics committee and consent obtained from patients or their next-of-kin. the duration of prodromal symptoms of increased cough, worsening dyspnoea, increased quantity or discolouration of sputum or increased wheeze was estimated from the start of their symptomatic deterioration. prior smoking history, exercise tolerance, baseline functional capability and the presence of other co-morbidities were recorded. treatment prior to hospital admission was listed, including home oxygen, corticosteroid therapy and antibiotics in the previous 3 days. baseline clinical parameters, blood results, apache ii score and arterial blood gas prior to commencement of mechanical ventilation were noted. every effort was made to exclude other causes of respiratory failure, such as respiratory depressants, uncontrolled oxygen therapy, pulmonary embolism, increased atmospheric pollution, acute left ventricular failure and pneumothorax by using careful history taking, clinical acumen, radiology, angiography and echocardiography where appropriate. full blood count, chemistry, blood cultures and chest radiography were performed on admission to hospital. sputum specimens were collected on admission, if possible, or within 48 h of hospital admission, and viral nasal and pharyngeal specimens were taken after patient stabilisation and enrolment in the study. sputum for bacterial analysis was collected via endotracheal tube suction in intubated patients and by spontaneous or voluntary cough from patients who received niv. all other specimen collection was identical. nasopharyngeal aspirate (npa) and posterior pharyngeal swab (ps) (copan venturi transystem, italy) were collected from all patients. immunofluorescence assay (ifa), either direct (merifluor bioscience, cincinnati, ohio) or indirect (chemicon international, temecula, ca), and virus culture (vc) were performed on the npa. virus culture only was performed on the ps and polymerase chain reaction (pcr) on both specimens. all patients were routinely treated with bronchodilators and empirical antibiotics for at least 5 days, changed or ceased according to the subsequent microbiological profile. all patients received physiotherapy when clinically stable. new chest radiograph changes on admission implied pneumonic change or collapse which was not present on previous chest films (if available) or which was clearly abnormal and had the hallmarks of a primary pulmonary pathological process. whether changes were new or chronic was agreed by consensus between a thoracic physician and an intensivist. we have defined a probable pathogen as "the most likely biological agent(s) to have caused the exacerbation of copd". acute serological titres for inf a and b, para 1, 2 and 3, rsv, av, chlamydia, legionella and mycoplasma antibodies were followed 3 weeks later with a paired convalescent titre. a fourfold rise in titre signified a probable causative agent. virus nucleic acid extraction with or without reverse transcriptase and amplification was performed on npa and ps samples. pcr detection was performed for inf a and b, para 1, 2 and 3 and rsv types a and b (hexaplex assay) and human ev, hmpv, cov, rv and av (real-time pcr). appendix 1 in the electronic supplementary material (esm) contains further details. a positive pcr viral assay was considered evidence for a probable virus pathogen. a screening genus-specific chlamydia assay (both igg and iga) was initially performed. for the purpose of this study, seroconversion of either or both igg and iga between acute and convalescent sera was considered evidence of acute chlamydia genus infection. a single acute or convalescent specimen with either iga, or iga and igg present was indicative of probable recent infection with chlamydia. a positive igg without iga was not considered evidence of recent chlamydia infection. sputum processing was performed using standard laboratory media. microscopy of gram stain smears as well as identification of all significant isolates was reported. sputum samples were considered non-representative of a lower respiratory tract specimen if there were fewer than 20 neutrophils per high-power field or more than 25 squamous epithelial cells per low-power field. the significance of a culture result was based on the quality of the sputum and whether the culture matched the smear result [18] . identified bacteria were defined as probable pathogens if the predominant growth of a potential respiratory pathogen was obtained in the presence of gram stain microscopy showing profuse organisms with a consistent morphotype. other bacterial isolates were considered as commensals or improbable pathogens. statistical analysis was performed using r (version 2.0.1) (www.r-project.org). categorical variables were analysed using the chi-squared test or fisher's exact test as appropriate, depending on whether numbers in the contingency tables satisfied the chi-squared test assumptions. continuous variables were analysed using the mann-whitney u-test for non-normally distributed variables or the unpaired ttest for normally distributed variables. a p value of < 0.05 was considered significant. of 118 potential participants (120 potential episodes), a total of 13 were not entered in the study for the following reasons: participation was declined (4); death before inclusion (6); enrolment more than 48 h after hospital admission (2); or failure to meet the criteria for the diagnosis of copd (1). two patients were admitted twice with separate episodes. three patients were included in this study without hypercarbia > 45 mmhg. the indications for niv in these cases were a combination of hypoxia (po 2 < 55) in spite of adequate oxygen therapy, ph < 7.35 and symptomatic relief. of the 105 patients (107 episodes), 23 (21%) died within 28 days of admission. the mean apache ii score was 20 (range 9-40) ( table 1) . less than 40% of the patients had recent or any pre-morbid spirometry values, and these data were therefore omitted in reporting. convalescent serological follow-up of initial viral and atypical titres was 52%. we were unable to obtain a sputum specimen suitable for bacterial analysis in 27/107 patient episodes (25%). all these patients received niv. a probable infectious aetiology was found in 69 episodes (64%). a virus was the probable pathogen in 46 cases (43%), being the sole organism in 35 cases (33%) ( table 2 ) and part of a mixed infection in 11 cases (10%) ( table 3 ). virus cultures for herpes simplex virus type 1 (hsv1) were positive in 8 cases (7%). six hsv1 cases also had another probable pathogen identified and the remaining two had no distinguishable clinical or radiological features that identified hsv as a probable pathogen. these eight hsv1 results were therefore not included as probable pathogens. 23 (21) only two other viral cultures were positive, one for inf a and one for rv, both of which correlated with a positive pcr. inf a was the most common organism in 14 cases (13%) with 7 (8%) rv, 3 (3%) cov, 3 (3%) hmpv and 2 (2%) ev. twenty-four (22%) of 107 episodes occurred in the australian summer and autumn months from december to may, whilst the bulk of exacerbations, 83/107 (78%), occurred in the winter and spring months between june and november (p = 0.0000007). figure 1 shows the seasonal breakdown. there were two discrete clusters of influenza a that occurred each within a 3-week period, both in early spring and separated by 3 years, and comprising 9/14 (65%) of the a probable bacterial aetiology was found in 25 cases (23%) ( table 2) , being a sole agent in 16 cases (15%) and in a mixed infection in a further 9 (8%) ( table 3) . haemophilus influenzae was the most common bacterium in10/107 cases (11%). chlamydia species were the only atypical organisms found, being the sole aetiology in 3 cases (3%) and in 3 mixed infections (3%). mixed infections constituted 13 (12%) of all cases (table 3) . there was no statistically significant difference between the group with probable viral aetiology and those without a viral aetiology in length of prodrome, initial clinical parameters, apache ii score, changes on chest radiograph, length of stay or mortality (table 4 ). there are only two other published series of infective agents in ventilated copd patients [7, 8] . a subsequent study also examined the outcomes of treatment in one of these initial series [19] . all 50 patients in the soler study [7] and all 54 in the fagon study [8] were intubated, facilitating protected brush and bronchoalveolar lavage specimens. no bronchoscopic specimen collection was performed on the smaller subset of intubated patients in our study, where 61 patients (57%) received niv alone, with 17 (16%) receiving both niv and ett and only 29 (27%) being intubated without niv. the collection of appropriate respiratory samples has been complicated by the introduction of niv as an accepted modality for the treatment of severe copd exacerbations, as niv patients may not produce suitable sputum. fibre-optic bronchoscopy may not be easily tolerated by these patients, in spite of the use of modified face masks [20] . inducement of sputum specimens with hypertonic saline, although an effective method of sample collection in unintubated patients [21, 22] , may induce or worsen bronchospasm and was not considered a practicable option in our cohort with initial respiratory instability. bacteria grown from sputum or endotracheal aspirate may not be representative of the pathogenic organism that is causing the exacerbation [23, 24, 25] , with up to half of stable copd patients colonised by a potential bacterial pathogen [26, 27] . to reduce this possibility, we examined our sputum specimens for the presence of neutrophils and correlated the gram stain with culture in an effort to determine whether the organism was a commensal or a probable pathogen [18] . twenty-five (24%) of the 105 patients received antibiotics within the 3 days prior to icu admission. of these 25, 3 (12%) had a positive bacterial culture (all h. influenzae). the possibility that recent antibiotic therapy may have reduced the bacterial yield in the other 22 episodes must be considered. both soler [7] and fagon [8] focused primarily on the bacterial yield. fagon did not investigate for viruses. soler obtained serology for chlamydia, mycoplasma, legionella, inf, para, rsv and hsv. av, hmpv, cov, ev and rv were not sought. soler found potential pathogens in 72% of cases, compared with 64% in our study, and community-acquired bacteria in 56% of cases in his cohort, against 23% in our study. fagon found bacteria in 27/54 (50%) of protected brush specimens, predominantly haemophilus and streptococcus species. soler did not find significant clinical or outcome differences between the bacteria-positive and -negative groups, whilst fagon noted significantly greater pyrexia on presentation in the bacteria-positive group, but in no other parameters. protected brush specimens may well increase the overall yield of bacteria, whilst ensuring the false-positive rate is minimised. the limited range of viruses sought and the lack of pcr assay may well have underrepresented virus incidence (16%) in soler's study, compared with 43% in ours. he found atypical organisms in 18% of cases, compared with our 5%. with adequate follow-up serology in only 67% of the survivors in our study, this may account for the paucity of proven atypical organisms. some 33% of cases were polymicrobial in soler's study, compared with 13% in ours. the mechanisms for viral upper respiratory tract infections (urtis) causing lower respiratory tract infection (lrti) symptoms include cytokine and paracrine release, altered cell-mediated immunity and lymphocyte recruitment [28] , neurohormonal reflexes and direct epithelial damage of the lower respiratory tract [10, 29, 30, 31] . we may therefore assume that the recovery of common viruses from the upper respiratory tract samples (npa and ps) reflects a probable viral aetiology for lower respiratory tract symptoms. a lower sensitivity for viral diagnosis than for direct bronchoscopic sampling of lower respiratory tract specimens is possible [22, 32, 33] . a virus aetiology has been implicated in non-ventilated copd exacerbations in between 29 and 64% of cases [6, 32, 34, 35] , with the most common virus detected in each series varying due to seasonal variation, method of virus detection and influenza vaccination status. infa [6, 36] , rsv [32, 33, 36] , cov [37] and rv [5, 22, 32, 33] were the most frequently detected viral pathogens in more recent studies of copd exacerbations. in our study, inf a, para 3, rsv and rv were most often detected ( table 2 ). one study suggests that patients with evidence of virus infection during copd exacerbations have greater symptom severity at onset than those with non-virus exacerbations, and a significantly longer median time to symptom recovery [32] . we did not find this in our study, with no significant differences in prodromal symptoms, length of icu and hospital stay or mortality observed between virus and non-virus exacerbations. our study is the first to use pcr to detect viruses in ventilated copd patients. pcr was the most sensitive method for virus detection, with virus culture the least sensitive. garbino reports a 10% virus culture positivity from bronchoalveolar lavage (bal) specimens in a group of hospitalised patients with lrtis, whilst reverse transcriptase pcr (rt-pcr) identified a virus in 29% of the same specimens [33] . this may be because the presence of only small quantities of virus is not detectable by culture. most viral pathogens are more difficult to culture after more than 72 h of illness and pcr is able to detect infection for a longer period due to its high sensitivity. pcr is consistently associated with high detection rates in other studies of the copd exacerbation population, with a 40-64% virus yield [5, 6, 32] . a rising antibody titre over 3 weeks is considered strong evidence for a probable virus or atypical pathogen, but is obviously not possible in deceased patients or those lost to follow-up. whilst a useful epidemiological tool, it provides a retrospective diagnosis and therefore cannot affect clinical decisions in the critically unwell. we used new genetic techniques to enhance the diagnostic yield and provide a more accurate picture of the role of viral infection (see appendix 1, esm). however, most cases were diagnosed by rt-pcr alone. this heavy re-liance on rt-pcr to establish a diagnosis of viral infection may be too sensitive, detecting small amounts of residual viral nucleic acid without other clinical evidence of viral infection. extensive precautions were taken against contamination in our study, and negative pcr controls showed no evidence of false positivity. respiratory viruses may be commensal in stable copd patients. seemungal found respiratory viruses more often in the sputum and nasal lavage of patients with exacerbations of copd (56%) than of patients with stable copd (19%) [6, 32] . latent sequences of common respiratory viruses such as adenovirus [5, 37] have been detected by pcr. it has been well demonstrated that copd patients may excrete rsv for extended periods of time [19] , so one cannot be absolutely certain that the rsv demonstrated in our study was necessarily pathogenic. for other viruses, persistent colonisation is not well described and it is reasonable to assume that they played a pathogenic role. quantitative real-time pcr may allow differentiation between latent or former infection and current infection. advances in the availability and rapidity of molecular sequencing may also allow changes in infectious agent serotype or antibiotic resistance to be discerned [33, 38] . there is no evidence to date to suggest that treatment with antiviral agents in this subset of copd patients with virus exacerbations requiring ventilatory support will significantly alter the course of their acute illness. it is nevertheless important to have a better understanding of the pattern of disease in order to target more efficiently any potential treatments. the early detection of virus infection by pcr is in its infancy, but the development of more sophisticated anti-viral agents may allow early targeting of virus pathogens. a probable virus pathogen was found in 46 cases (43%) and a probable bacterial aetiology was found in 25 cases (23%) in this study of ventilated copd exacerbation patients. no prodromal or initial clinical parameters distinguished viral from non-viral illness in our study. there were no significant differences between virus and non-virus exacerbations in ventilation time/length of stay or 28-day mortality. niv made protected specimen collection more difficult than in intubated patients, which may have decreased the bacterial and viral yield. pcr offered a more detailed view of the spectrum of virus illness in this group of patients, including previously unsought viruses such as ev and hmpv. respiratory syncytial and other virus infections in persons with chronic cardiopulmonary disease time series analysis of the relation between influenza virus and hospital admissions of the elderly in ontario, canada, for pneumonia, chronic lung disease, and congestive heart failure effect of viral infections in patients with chronic obstructive pulmonary diseases bronchial hyperreactivity and bronchial obstruction in respiratory viral infection. an attempt to evaluate the relationship infectious exacerbations of chronic obstructive pulmonary disease associated with respiratory viruses and non-typeable haemophilus influenzae respiratory viruses in exacerbations of chronic obstructive pulmonary disease requiring hospitalisation: a case-control study bronchial microbial patterns in severe exacerbations of chronic obstructive pulmonary disease (copd) requiring mechanical ventilation characterization of distal bronchial microflora during acute exacerbation of chronic bronchitis. use of the protected specimen brush technique in 54 mechanically ventilated patients the classification of viruses infecting the respiratory tract virusinduced airway hyperresponsiveness. role of inflammatory cells and mediators a model of viral wheeze in nonasthmatic adults: symptoms and physiology virus-induced asthma attacks human metapneumovirus and lower respiratory tract disease in otherwise healthy infants and children respiratory picornaviruses and respiratory syncytial virus as causative agents of acute expiratory wheezing in children field's virology, 4th edn standards for the diagnosis and treatment of patients with copd: a summary of the ats/ers position paper noninvasive ventilation in acute respiratory failure evaluation of antimicrobial treatment in mechanically ventilated patients with severe chronic obstructive pulmonary disease exacerbations noninvasive positivepressure ventilation via face mask during bronchoscopy with bal in high-risk hypoxemic patients evaluation of a quantitative real-time pcr for the detection of respiratory syncytial virus in pulmonary diseases detection of rhinovirus in induced sputum at exacerbation of chronic obstructive pulmonary disease recent advances in diagnosis and management of chronic bronchitis and emphysema evidencebased approach to acute exacerbations of copd exacerbations of chronic obstructive pulmonary disease: when are bacteria important? impact of sputum bacteria on airway inflammation and health status in clinical stable copd relationship between bacterial colonisation and the frequency, character, and severity of copd exacerbations natural and experimental rhinovirus infections of the lower respiratory tract virus-induced airway hyperresponsiveness in man low grade rhinovirus infection induces a prolonged release of il-8 in pulmonary epithelium detection of rhinovirus rna in lower airway cells during experimentally induced infection respiratory viruses, symptoms, and inflammatory markers in acute exacerbations and stable chronic obstructive pulmonary disease lower respiratory viral illnesses: improved diagnosis by molecular methods and clinical impact impact of respiratory virus infections on persons with chronic underlying conditions epidemiology and treatment of chronic bronchitis and its exacerbations respiratory viral infections in adults respiratory viral infections in adults with and without chronic obstructive pulmonary disease rapid and sensitive routine detection of all members of the genus enterovirus in different clinical specimens by real-time pcr key: cord-351990-aham72b9 authors: radin, jennifer m.; hawksworth, anthony w.; kammerer, peter e.; balansay, melinda; raman, rema; lindsay, suzanne p.; brice, gary t. title: epidemiology of pathogen-specific respiratory infections among three us populations date: 2014-12-30 journal: plos one doi: 10.1371/journal.pone.0114871 sha: doc_id: 351990 cord_uid: aham72b9 background: diagnostic tests for respiratory infections can be costly and time-consuming. improved characterization of specific respiratory pathogens by identifying frequent signs, symptoms and demographic characteristics, along with improving our understanding of coinfection rates and seasonality, may improve treatment and prevention measures. methods: febrile respiratory illness (fri) and severe acute respiratory infection (sari) surveillance was conducted from october 2011 through march 2013 among three us populations: civilians near the us–mexico border, department of defense (dod) beneficiaries, and military recruits. clinical and demographic questionnaire data and respiratory swabs were collected from participants, tested by pcr for nine different respiratory pathogens and summarized. age stratified characteristics of civilians positive for influenza and recruits positive for rhinovirus were compared to other and no/unknown pathogen. seasonality and coinfection rates were also described. results: a total of 1444 patients met the fri or sari case definition and were enrolled in this study. influenza signs and symptoms varied across age groups of civilians. recruits with rhinovirus had higher percentages of pneumonia, cough, shortness of breath, congestion, cough, less fever and longer time to seeking care and were more likely to be male compared to those in the no/unknown pathogen group. coinfections were found in 6% of all fri/sari cases tested and were most frequently seen among children and with rhinovirus infections. clear seasonal trends were identified for influenza, rhinovirus, and respiratory syncytial virus. conclusions: the age-stratified clinical characteristics associated with influenza suggest that age-specific case definitions may improve influenza surveillance and identification. improving identification of rhinoviruses, the most frequent respiratory infection among recruits, may be useful for separating out contagious individuals, especially when larger outbreaks occur. overall, describing the epidemiology of pathogen specific respiratory diseases can help improve clinical diagnoses, establish baselines of infection, identify outbreaks, and help prioritize the development of new vaccines and treatments. acute respiratory infections make up a huge proportion of disease burden in the united states and globally, with an estimated 94 037 000 disability adjusted life years and 3.9 million deaths worldwide each year [1] . respiratory infections are often difficult to diagnose clinically due to nonspecific and overlapping symptoms. additionally, diagnostic tests can be time-consuming and costly and often require trained and well-equipped laboratories, making laboratory confirmation of each case impractical. however, laboratory results from various surveillance populations can be paired with clinical, demographic, and seasonality variables to create models that can give timely predictions of disease outcomes. preventive measures and treatments to reduce respiratory disease burden can also be improved through routine surveillance by gaining a better understanding of the percent positivity of pathogens among acute respiratory cases, seasonality, and coinfection occurrence. currently, limited respiratory disease etiology studies have been done in the united states [2, 3, 4] , despite many being done in other countries [5, 6, 7, 8, 9, 10, 11, 12, 13, 14] . additionally, few viral etiology studies have collected clinical signs and symptoms and assessed their association with a broad range of respiratory pathogens [9, 13, 14] . most descriptive studies and predictive models for respiratory diseases have focused on identifying influenza using clinical signs and symptoms [15, 16, 17, 18, 19, 20] , however few have been age-stratified and therefore may have missed some important differences in clinical presentation by age. understanding us-specific disease burden and seasonality is important, since disease incidence, distribution, and seasonality may vary between populations, regions, and climates. this study aimed to describe characteristics associated with specific respiratory pathogens, as well as the etiology, seasonality, and coinfection rates among three us populations: military recruits, department of defense (dod) beneficiaries, and civilians living near the us-mexico border. the results of this study can help improve timely and more accurate diagnosis, inform treatment plans, establish baselines of infection, identify outbreaks, and help prioritize the development of new vaccines and future treatments. participants fri and sari surveillance was conducted between october 2011 and march 2013 among three surveillance groups in the united states: civilians near the us-mexico border, dod dependents, and military recruits. separate seasonality data from the same populations was available for january 2012 through december 2013. recruits are typically young and healthy adults who enter into an 8-12 week ''boot camp'' training program, which involves strenuous, and physically demanding activities and living in high-density barracks. during the first week of training, recruits receive a series of vaccinations, including influenza (seasonally) and adenovirus. most training centers also administer at least one dose of bicillin to incoming trainees as prophylaxis against streptococcus bacteria. the dod dependent population is made up of the families of active duty and retired military personnel. this population consists of all ages and has good access to health care through the tricare health care program. this research was conducted in compliance with all applicable federal and international regulations governing the protection of human subjects in research. the research conducted in the recruit and dod beneficiary populations underwent nhrc irb approval (nhrc protocols 31230 and nhrc.1999.0002) and written consent or parental guardian consent for minors was obtained for all participants. the data collected from the border population was part of a surveillance program run by the us centers for disease control and prevention (cdc) and was considered non-research by the nhrc irb. nhrc staff members only provided diagnostic support and only received non-personally identifiable data. case definitions were slightly different for each of the three populations. in the recruit population, an fri case was defined as a person who sought medical care and had an oral temperature $38.0uc (100.5uf) and either cough or sore throat. for the beneficiary population and border populations, the same fri case definition was used; however, a fever $37.8uc (100.0uf) or subjective fever was used. additionally, inpatients who met sari case definition at select border sites were also sampled. the sari case definition included people who presented with either fever $37.8uc (100.0uf) or feeling feverish/chills, in addition to cough, and hospital admission, with onset in the last 10 days. additionally, children under age five were included if they were admitted to the hospital with clinical suspicion of pneumonia. nasal or combination nasal/pharyngeal swabs and questionnaire data were collected from a convenience sample of up to 20 patients per week per site who sought medical attention, met the fri or sari case definition, and provided written informed consent. specimens were placed in universal transport medium [21] , preserved at 280uc, and later transported on dry ice to the reference laboratory at the nhrc every one to two weeks for testing. additionally, the following demographic and clinical signs and symptoms were collected from each fri and sari case: sex, age, study population, month of illness, pneumonia, sore throat, cough, nausea, shortness of breath, congestion, pink eye, body ache, headache, temperature, number of days of symptoms before seeking care, and date of seeking care. samples were extracted using the roche magna pure lc extraction system following manufacturer's instructions. samples were then tested for a broadspectrum panel consisting of standard pcr gel tests: hmpv (single plex), covnl63 (single plex), and cov229e and covoc43 (multiplex). real-time polymerase chain reaction (pcr) assays were run on the applied biosystems 7500 fast real-time pcr system testing for influenza a, influenza b, adenovirus, rsv, rhinovirus, and a bacterial multiplex consisting of m. pneumoniae, c. pneumoniae, and b. pertussis. the rhinovirus primers used in our study have been found to correctly identify 87 of the 100 distinct rhinovirus subtypes and to accurately distinguish rhinoviruses from enteroviruses [22] . all pcr tests were done using in-house primers, except for the influenza a and influenza b which were done using cdc primers. additionally, viral culture testing for parainfluenza was done for a systematic sample of specimens not positive for influenza or adenovirus. the frequencies and means of clinical and demographic characteristics across participants positive for each of the six viruses, bacterial infections, co-infections and no/unknown pathogen were compared. (table 1) . additionally, characteristics of influenza, other pathogen, or no/unknown pathogen were compared among age stratified groups of dod beneficiaries and us-mexico border populations ( table 2) and characteristics of rhinovirus, other pathogen, and no/ (24) 14 (15) 4 (5) 6 (10) 4 (3) 13 (27) 19 (43) unknown pathogen were compared among military recruits (table 3) . chi-square tests for categorical variables and analysis of variance (anova) tests for continuous variables were used to identify signs, symptoms, associated with each pathogen group from all populations. variables that were univariately associated with the pathogens (p,0.15) were investigated further in a multinomial logistic regression model. variables with a p,0.05 were considered in the final adjusted model (tables s1-s2 in s1 file). coinfections were coded as ''other pathogen'' even if one pathogen was influenza or rhinovirus. statistical analysis was conducted using sas software (version 9.3, sas institute, inc., cary, north carolina). proc logistic with link5glogit was used for the multinomial modeling and proc anova and proc freq were used for anova and chi-squared analyses, respectively. during the 18 months of the study, october 2011 through march 2013, 1444 patients met the fri or sari case definitions and were enrolled in this study, consisting 406 (28%) from the fri/sari border, 423 (29%) from the dod beneficiary, and 615 (43%) from the fri recruit populations. the percent positivity for each pathogen out of all specimens tested was rhinoviruses (16%), influenza (14%), rsv (6%), adenovirus (2%), coronaviruses (5%), bacterial (2%), hmpv (1%), and coinfections (6%). influenza a (h3n2) was the most common influenza subtype, making up 54% of influenza specimens, followed by influenza b (31%), and ph1n1 (13%). among coronaviruses, covoc43 was the most commonly identified strain, making up 67% of coronavirus specimens, seasonal patterns were apparent for influenza, rhinovirus, and rsv, whereas more consistent low levels of infection were seen for adenovirus and bacterial infections. the recruit population had a more constant number of fri cases sampled than the other two populations throughout the study period. overall, they also had a greater percent positivity for rhinovirus compared with the other two populations, but lower percent positivity for influenza and rsv. rhinovirus appeared to have two peaks: one in spring and one in summer/fall among the border and beneficiary populations and consistently higher levels among the recruit population. in all populations influenza and rsv peaked in the winter (fig. 1) . influenza a typically peaked before influenza b. rhinovirus and bacterial infections were more frequently isolated from recruits and men who make up the majority of the recruit population; whereas influenza and rsv were less frequent among these groups compared with the dod beneficiary and border populations. fewer rhinovirus and more rsv pathogens were identified from sari cases compared to fri. pneumonia and shortness of breath were more frequent among rhinovirus cases and less common among influenza cases. however, time to seeking care was shorter for influenza and hmpv and longest for bacterial infections. influenza, rsv, adenovirus, and hmpv cases all had higher temperatures compared with no/unknown pathogens, and rhinovirus has significantly lower temperature (table 1) . descriptive statistics for influenza, other, and no/unknown pathogen among dod beneficiaries and us-mexico border populations showed different clinical presentation across three age groups. among 0-4 year olds, influenza was more frequent among us-mexico border populations compared to dod beneficiaries. in the youngest age group, sore throat and fever were also more frequent among those positive for influenza compared to other or no/unknown pathogen and among 5-24 year olds, cough, fever, and short time to seeking care were more frequent. finally, among the 25 and older age group, sore throat, nausea, congestion, pink eye, body ache, and head ache were more frequently seen in the influenza group compared to the other groups. interestingly, fever was the least frequent among influenza positive participants in the oldest age group (table 2) . clinical presentation among recruits also differed by those with laboratory confirmed rhinovirus, other pathogen, or no/unknown pathogen. a higher percentage of males were diagnosed with rhinovirus or other pathogen compared to the other and no/unknown pathogen. additionally, participants with rhinovirus had higher percentages of pneumonia, shortness of breath, congestion, and less fever and longer time to seeking care than the other groups and participants with either rhinovirus or other pathogen had higher cough than the no/unknown pathogen group (table 3) . building on the descriptive statistics, we found that fever was predicative of influenza compared to no/unknown pathogen among 0-4 year olds. among 5-24 year olds, fever, cough, and short time to seeking care were predicative of influenza compared to no/unknown pathogen. finally, among those 25 years and older, sore throat and nausea were predictive of influenza compared to no/ unknown pathogen. (table s1 in s1 file). among the recruit population, cough epidemiology of respiratory infection and less fever were predictive of rhinovirus, and cough, fever, and less shortness of breath were predictive of other pathogen compared to no/unknown pathogen. (table s2 in s1 file). at least one pathogen was identified in 51% of all fri and sari cases. coinfections were found in 81 (6%) of all fri/sari cases tested, among which were 76 double, four triple, and one quadruple coinfections. the most frequent pathogen associated with a coinfection was rhinovirus, followed by rsv and covoc43. the top three most common coinfections were rhinovirus and rsv (15% of all coinfections, 11 cases), rhinovirus and adenovirus (13% of all coinfections, 10 cases), and rhinovirus and covoc43 (12% of all coinfections, 9 cases). interestingly, our study found an overall coinfection percent positivity of 6% which declined with age: 11% of 0-4 year olds, 5% of 5-24 year olds, 3% of 25-49 year olds and 2% of 50+ year olds. (table 4 ). in order to reduce respiratory disease burden, it is necessary to gain a better understanding of the percent positivity, coinfection rates, and seasonality of specific respiratory pathogens. additionally, a predictive clinical model that uses symptoms, seasonality, and patient demographics can also help improve prevention efforts and patient treatment. timeliness is especially important for influenza antivirals, which work best if given within the first 48 hours of symptoms. however, rapid diagnostic tests have poor sensitivity [23] and multiplex pcr tests are impractical for most clinical settings. additionally, treatment with antibiotics can often be incorrectly prescribed for viral infections, leading to increased antibiotic resistance. therefore, creating models to better predict the type of pathogen using symptoms and characteristics easily collected by a clinician at the time of visit could improve treatment accuracy and help protect the effectiveness of existing antibiotics and antivirals. many respiratory etiology studies have been done outside the united states among patients with both severe, lower respiratory illness [24, 25, 26] , as well as more mild, upper respiratory disease [7, 8, 9, 10, 11, 12] . these studies are important because viral etiologies vary across populations and regions, depending on factors such as population susceptibility, age, circulating strains, climate, comorbidities, and vaccination coverage. our study adds to the very limited number of etiology studies done in the united states [2, 3, 4] by examining viral etiology among three different us populations: military recruits, dod dependents, and a us-mexico border civilians and including all ages. our study is also unique in that it collected clinical signs, symptoms, and demographics for each case tested with the broad-spectrum respiratory panel. similar studies have been limited and have used smaller sample sizes, focused on one age group, and did not test for as many respiratory pathogens. identifying population-specific baselines of infection enables us to identify elevated rates, which may indicate an outbreak or the start of a pandemic. recognizing associated symptoms can help determine the most likely pathogen, as was seen in 2009 with the pandemic influenza (h1n1) strain first identified in brawley and san diego, california, in two of the populations in this study [27] . there were several key differences of infection among the three populations. the recruit population had consistently higher levels of rhinovirus and bacterial infections than the other groups, which may be reflective of close living conditions and a younger age group, mostly 18-24 years old. this population is also highly vaccinated for influenza and showed the smallest amount of influenza infection compared with the other two populations. the higher frequency of influenza among 0-4 year olds in the us-mexico border population (61%) compared to beneficiaries (39%) may be reflective of different exposures or differences in vaccination uptake. however, the overall frequency of influenza found between the two groups was similar. adenovirus, which historically had a large impact on recruits, was also low as a result of resumption of the adenovirus vaccines in october 2011 [28] . rsv, which usually infects young children, was more commonly found in the dod dependent (48%) and border populations (48%) compared with the recruits (4%). overall, rhinoviruses were the most common respiratory pathogen identified (236 cases, 16%), followed by influenza (197 cases, 14%), rsv (85 cases, 6%), and coronaviruses (66 cases, 5%), which coincides with other etiology studies [13] . the results of the age-stratified influenza, other pathogen, and no/unknown results showed some interesting differences across age groups. most interesting was the relatively low percentage of influenza positive participants with fever (42%) in the 25 and older year age group, compared to the 0-4 (74%), and 5-25 year olds (76%). fever may sometimes be masked by the consumption of antipyretics, although this group also complained of high headache (81%) and body ache (90%). the presentation differences of influenza by age could have some important implications for influenza surveillance, as the standard influenzalike illness and sari case definitions require [29] . consequently, an age-stratified case definition may be more appropriate as the standard case definitions may be underestimating the burden of influenza in older age groups. circulating strains may also influence clinical presentation across age groups. (table 2, table s1 in s1 file). in our 5-24 year olds we found fever, cough, and short time to seeking care to be predictive of influenza infection. unfortunately, previous studies assessing the diagnostic accuracy of fever, cough, and acute onset to predict flu have found them to have low sensitivity (20%), and high specificity (96%) [18] . another study, found that sore throat and fever in participants less than 5 years old had a sensitivity of 51% and specificity of 54% and cough and fever among those greater than 5 years had a sensitivity of 80% and specificity of 42% [30] . although identifying predictive symptoms can be useful, it is important to recognize that diagnostic accuracy may still be low due to overlapping symptoms of many respiratory infections and may change due to fluctuations in circulating strains, age of the population and comorbidities. ( table 2, table s1 in s1 file). rhinovirus was the most frequently identified pathogen among recruits, although it typically does not present with severe illness or acute onset, which is reflected in the long time to seeking care among rhinovirus positive participants. interestingly, we also found the lowest percentage of fever among participants positive for rhinovirus compared to any other respiratory pathogen (table 1) . these findings are similar to other studies [13, 14, 31] and could have important implications for designing respiratory disease surveillance systems to capture outbreaks of viruses in the same family. for example, the recent outbreak of enterovirus 68 has similar symptoms of low fever [32] . (table 3, table s2 in s1 file). understanding coinfections can also be useful for preventing respiratory disease. our study found around 30% to 40% of coronavirus and adenovirus infections occurred as coinfections and they most frequently occurred with rhinovirus. similarly, other studies have also found the highest ratio of coinfections among adenovirus and coronaviruses [33, 34] , and have found rhinovirus to be part of the most frequently occurring coinfections [26, 33] . these results suggest that infection with some viruses, such as rhinoviruses, could create opportunistic environments for colonization with other viruses and bacteria. interestingly, coinfections were most common among the youngest age group, newborn to four years, which did not have the highest rhinovirus rate. targeting rhinovirus infection through creation of new vaccines or treatment could have more far-reaching benefit in protecting a person from other infections. respiratory coinfection rates have varied across studies and are likely influenced by age, type of case definition used to enroll participants, and pathogens tested. previous studies with a median age of 2-3 years old have found coinfections among sari and acute respiratory tract infections of 17% and 19%, respectively [26, 33] . a third study among childcare attendees in the us found even higher coinfection rates of 47% [4] . finally, a study from scotland that tested respiratory specimens from all age groups found a coinfection rate of 5% [34] . these studies coincide with ours and show high coinfection rates among children. declining coinfection rates with age may be a result of overall increased immunity to a broad range of respiratory pathogens as people get older. one limitation of this study is that it only captured people with fri/sari who sought medical care. military recruits may be less likely to seek care than other groups due to concern over losing training time or having to restart the program. therefore, the etiology of more mild infections may be underrepresented for these two groups. additionally, the case definitions were slightly different for the three populations, which may have influenced which pathogens were identified in each group. although this study involved three different us populations, the results of this study may be less generalizable to the general us public who are not associated with the military or living on the us-mexico border. despite this, signs and symptoms from these pathogens should be similar across other populations in similar age groups and with similar vaccination coverage. additionally, we found that seasonality of infection for recruits was similar to that of the border and beneficiary populations for several pathogens, but with different intensity. consequently, illness surveillance in recruits, which made up the largest proportion of our study population, can be beneficial in informing disease trends in the general public. although we tested for many pathogens, there are likely still circulating viruses and bacteria for which we did not test, such as bocavirus, covhku1, or potentially unrecognized viruses; therefore, these cases were likely incorrectly classified as part of the ''no pathogen'' group. additionally, the timing of sample collection in the course of illness could impact whether or not viruses were identified by pcr. symptom collection may also be biased in the youngest age group of 0-4 year olds, who may not be able to articulate how they are feeling. despite this, our study was part of a well-established existing surveillance program that consistently collected and tested a substantial number of specimens at many different sites across the united states. in the future, additional years of surveillance data will continue to improve our understanding of seasonality. although nothing will replace the accuracy of laboratory diagnostics, a broader understanding of seasonality, clinical presentation, demographics, and coinfection rates of pathogen specific respiratory diseases can improve diagnosis and treatment, by informing clinicians on appropriate antiviral and antibiotic treatment during the patient's visit. this can ultimately reduce the number of lost work days and transmission. additionally, describing baseline of disease and seasonality for specific pathogens can improve our ability to detect outbreaks. identifying the percent positivity of each pathogen, coinfection rates, and risk factors for disease will help inform vaccination programs, and possible investment in the development of future vaccines or treatments. supporting information s1 file. tables s1 and s2. of defense, or the us government. approved for public release; distribution is unlimited. u.s. government work (17 usc 105) . not copyrighted in the u.s. this research was conducted in compliance with all applicable federal and international regulations governing the protection of human subjects in research (protocols nhrc 31230 and nhrc.1999.0002). world health organization (who) the frequency and seasonality of influenza and other respiratory viruses in tennessee: two influenza seasons of surveillance data viruses associated with acute respiratory infections and influenza-like illness among outpatients from the influenza incidence surveillance project epidemiology of multiple respiratory viruses in childcare attendees viral etiology of severe pneumonia among kenyan infants and children acute respiratory infection and influenza-like illness viral etiologies in brazilian adults epidemiological analysis of respiratory viral etiology for influenzalike illness during 2010 in zuhai, china influenza-like illness sentinel surveillance in peru acute viral infections of upper respiratory tract in elderly people living in the community: comparative, prospective population based study of disease burden viral etiology of influenza-like illnesses in cameroon viral etiology of influenza-like illnesses in antananarivo rapid detection of respiratory tract viral infections and coinfections in patients with influenza-like illnesses by use of reverse transcription-pcr dna microarray systems clinical characteristics and outcomes of influenza and other influenza-like illness in mexico city a household-based study of acute viral respiratory illnesses in andean children a clinical diagnostic model for predicting influenza among young adult military personnel with febrile respiratory illness in singapore predicting influenza infections during epidemics with use of a clinical case definition clinical signs and symptoms predicting influenza infection determination of clinical and demographic predictors of laboratory-confirmed influenza with subtype analysis etiology and clinical characteristics of influenza-like illness (ili) in outpatients in beijing does this patient have influenza? diagnostic hybrids. collection and transport: universal transport medium real-time reverse transcription-pcr assay for comprehensive detection of human rhinovirus low sensitivity of rapid diagnostic test for influenza community-acquired pneumonia distinguished from influenza infection based on clinical signs and symptoms during a novel (swine) influenza a/h1n1 pandemic multicentered study of viral acute lower respiratory infections in children from four cities of argentina, 1993-1994 respiratory viral coinfections identified by a 10-plex real-time reverse-transcription polymerase chain reaction assay in patients hospitalized with severe acute respiratory illness-south africa initial identification and characterization of an emerging zoonotic influenza virus prior to pandemic spread history of the restoration of adenovirus type 4 and type 7 vaccine, live oral (adenovirus vaccine) in the context of the department of defense acquisition system world health organization. who surveillance case definitions for ili performance of case definitions used for influenza surveillance among hospitalized patients in a rural area of india rhino/enteroviruses in hospitalized children: a comparison to influenza viruses severe respiratory illness associated with enterovirus d68 -missouri and illinois epidemiology of respiratory viral infection using multiplex rt-pcr in epidemiology and clinical presentations of four human coronaviruses 229e, hku1, and oc43 detected over 3 years using a novel multiplex real-time pcr method we thank maria rosario araneta, patrick blair, daisy cabrera, johnnie conolly, jennifer cortinas, larivhie delacruz, daniel edgeworth, maria fierro, james key: cord-321284-0y69n1ea authors: el kholy, a. a.; mostafa, n. a.; ali, a. a.; soliman, m. m. s.; el-sherbini, s. a.; ismail, r. i.; el basha, n.; magdy, r. i.; el rifai, n.; hamed, d. h. title: the use of multiplex pcr for the diagnosis of viral severe acute respiratory infection in children: a high rate of co-detection during the winter season date: 2016-06-10 journal: eur j clin microbiol infect dis doi: 10.1007/s10096-016-2698-5 sha: doc_id: 321284 cord_uid: 0y69n1ea respiratory tract infection is a major cause of hospitalization in children. although most such infections are viral in origin, it is difficult to differentiate bacterial and viral infections, as the clinical symptoms are similar. multiplex polymerase chain reaction (pcr) methods allow testing for multiple pathogens simultaneously and are, therefore, gaining interest. this prospective case-control study was conducted from october 2013 to february 2014. nasopharyngeal (np) and oropharyngeal (throat) swabs were obtained from children admitted with severe acute respiratory infection (sari) at a tertiary hospital. a control group of 40 asymptomatic children was included. testing for 16 viruses was done by real-time multiplex pcr. multiplex pcr detected a viral pathogen in 159/177 (89.9 %) patients admitted with sari. there was a high rate of co-infection (46.9 %). dual detections were observed in 64 (36.2 %), triple detections in 17 (9.6 %), and quadruple detections in 2 (1.1 %) of 177 samples. seventy-eight patients required intensive care unit (icu) admission, of whom 28 (35.8 %) had co-infection with multiple viruses. adv, hbov, hrv, hev, and hcov-oc43 were also detected among asymptomatic children. this study confirms the high rate of detection of viral nucleic acids by multiplex pcr among hospitalized children admitted with sari, as well as the high rate of co-detection of multiple viruses. adv, hbov, hrv, hev, and hcov-oc43 were also detected in asymptomatic children, resulting in challenges in clinical interpretation. studies are required to provide quantitative conclusions that will facilitate clinical interpretation and application of the results in the clinical setting. respiratory tract infection is a major cause of hospitalization in children. most such infections are of viral origin, but viral infection is often hard to distinguish from bacterial infection [1] . pathogen-specific clinical symptoms are often lacking [1] . specific diagnosis, therefore, relies almost entirely on laboratory investigation [2] . treatment with antibiotics induces the development of antibiotic resistance in bacteria [3] and has a negligible effect on most acute respiratory infections (aris), which are generally of viral origin [4] . nevertheless, antibiotics are frequently prescribed due to a lack of clinically valid diagnostic tests verifying a viral etiology [5] . sensitive methods, such as quantitative real-time polymerase chain reaction (qpcr) analyses on nasopharyngeal samples, for a number of viruses have been introduced in hospitals as a sensitive diagnostic tool among children with respiratory tract infection [6] . investigations using specific pcr for individual viruses (bmonoplex pcr^) are too time consuming and elaborate for the laboratory, and are usually used to detect influenza and respiratory syncytial virus (rsv). when the viruses targeted by monoplex pcr are negative, no specific etiology is identified to help clinical management of the patient and epidemiological monitoring of infections. multiplex pcr methods, on the other hand, enable testing for many pathogens in parallel in a single analysis and are, therefore, increasingly gaining importance [1] . this prospective case-control study was conducted during the period from october 2013 to february 2014 (winter season) at cairo university pediatric hospitals (800 beds) and included all children who were hospitalized in intensive care units (icus) and hospital wards with severe acute respiratory infections (sari) according to the world health organization (who) case definition for sari [7] . a standardized study protocol was developed to record the demographic characteristics and medical history of the patients, clinical features including symptoms and signs, outcome of the illness, hospital course, and laboratory and radiological findings. forty healthy age-matched asymptomatic children with no history of a recent respiratory tract infection during the previous 2 weeks, who were not admitted to the hospital, and who do not have any chronic underlying illness were included as a control group. informed verbal consent was obtained from the parents of all the patients and the controls. the study design conformed to the revised helsinki declaration of bioethics and was approved by the scientific ethics committee of the department of pediatrics, faculty of medicine of cairo university. sampling and sample processing: nasopharyngeal (np) and oropharyngeal (throat) swabs were obtained, transported, and preserved on viral transport media. the swabs inside the 15-ml tube were agitated vigorously for 10 s using a vortex mixer to free cells from the swab tip. viral testing was done by real-time multiplex pcr using anyplex™ ii rv16 detection (v1.1) (cat. no. rv7g01y) supplied by seegene, operated on a cfx96™ real-time pcr detection system (bio-rad), which simultaneously detects 16 respiratory viruses [adenovirus (adv), influenza a virus (flu a), influenza b virus (flu b), parainfluenza viruses 1-4 (piv-1, -2, -3, -4), rhinovirus (hrv), respiratory syncytial virus (rsv), bocavirus (hbov), metapneumovirus (mpv), coronavirus 229e (cov 229e), coronavirus nl63 (cov nl63), coronavirus oc43 (cov oc43), and enterovirus (hev)]. nucleic acid extraction was done automatically using seeprep12™ viral (#spn1004) supplied by nordiag, using the extraction seeprep machine (seegene), as indicated as a nucleic acid extraction option by the manufacturer. protocol viral na was operated using 530 μl from the sample to result in an eluted volume of 60 μl. reverse transcription was done using the cdna synthesis kit for manual set up cdna synthesis premix (sgrt801) from seegene. interpretation of the results was done according to the manufacturer's instructions, in addition to automatic analysis using the seegene viewer software after exporting the run data to it [8] . data were coded and entered using the statistical package spss version 15 (ibm corp., armonk, ny, usa). data were summarized using mean [standard deviation (sd)] or median (range) for quantitative variables, and number and percentage for qualitative variables. comparison between groups was done using the chi-square test for qualitative variables, and the mann-whitney u-test was used for quantitative variables that were not normally distributed. a p-value < 0.05 was considered significant. the turnaround time for multiplex pcr was 6 h, but the test was done in batches three times per week. nucleic acids of respiratory viruses causing sari were detected in 159/177 (89.9 %) patients enrolled in the study. the demographic and clinical characteristics of the patients are shown in table 1 , and the frequency and monthly distribution of pathogens among the patients are shown in table 2 and fig. 1 . the median interval between the onset of symptoms and testing in our study was 3 days. the nucleic acids of more than one virus were detected in 83 patients (46.9 %). dual detections were observed in 64 (36.2 %), triple detections in 17 (9.6 %), and quadruple detections in 2 (1.1 %) of 177 samples. the frequency of co-detection with each of the studied viruses among the patients is shown in table 2 . important combinations included rsv with other viruses in 45 patients. rhinovirus was mostly detected in association with other viruses (64 out of 78 patients positive for hrv). bocavirus was detected in 16 patients. it was the only virus detected in three of them, while it was detected in association with another virus in 13 patients. a large proportion of the patients had received antibiotic therapy prior to hospital admission (121 patients, 68.4 %), and almost all the patients received antibiotic therapy during hospital admission (169 patients, 95.5 %). seventy-eight patients required icu admission, of whom 28 (35.8 %) had co-infection with multiple viruses as follows; 24 had co-infection with two viruses, three had co-infection with three viruses, and one patient had co-infection with four viruses. twenty-one of the patients who were admitted to the icu had rsv in association with other viruses, 14 of whom had co-infection of rsv and rhinovirus. among the 40 healthy asymptomatic children who were included as a control group, no virus was detected in 12 of them, while a viral pathogen was detected in 28 controls. the only viruses that were detected among the control group were adv, hbov, hev, hcov-oc43, and hrv, either as a single detection or as a co-detection (table 3) . a dual detection of these viruses was present in six controls, while triple detections of these viruses were present in four controls. the frequency of detection of these viruses among the patients and the controls is shown in fig. 2 . the following viruses were not detected at all among the control group, either alone or as a co-detection with other viruses: rsv, hmpv, flu a, flu b, hpiv-1-2-3-4, hcov-nl63, hcov-229e. we used multiplex pcr for the detection of respiratory viruses among patients admitted with sari. we also assessed the presence of viral nucleic acids by pcr in healthy asymptomatic children in order to better understand the etiologic role of the tested viruses in respiratory disease. viral nucleic acids were detected in 89.9 % of patients admitted with severe lower respiratory tract infections. this is in keeping with a previous study that reported the detection of a viral pathogen by pcr in 72.3 % of children aged ≤5 years [9] , and with bierbaum et al., who reported viral pathogen detection in children in excess of 80 %, compared to only 20 % in adults presenting with acute respiratory illness [10] . similar to previous reports [10, 11] , the most frequently detected virus in our study was rsv (48 %). flu a was detected in 6.2 % of the samples and flu b was detected in 2.3 % of the samples. these results are similar to those of lam et al., who reported a positive rate of 6.3 % for flu a by pcr and a positive rate of 3.3 % for flu b [2] . some 6-12 % of all children use healthcare services every year because of influenza [12] . additionally, many cases of infection with influenza virus are not diagnosed as such [1] . early confirmation of influenza viruses is helpful because treatment with neuraminidase inhibitors then becomes an option [1] . hpivs are important causes of upper respiratory tract illness and lower respiratory tract illness, especially in children [13, 14] . collectively, the four types of hpivs were detected in 8 % of the patients with sari in the current study. similar to previous studies, multiplex pcr allowed the detection of hrv, hcov-oc43, and hmpv, which were not detectable by conventional cell culture isolation or direct detection using immunofluorescence assay. the improvement in the diagnostic yield by adding hrv was confirmed in our study, and also reported previously by gruteke et al. [15] . [16] . these findings are comparable to previous reports [10, 11] . in view of the fact that respiratory tract infections in children occur in close succession during the winter season, it can be assumed that infections actually overlap. this may provide an explanation for the fact that viral nucleic acids of different viruses in one specimen are found virtually only in childrenfor example, in 8 % of all specimens analyzed by bierbaum et al. [17] and 46.9 % of the samples in our study. co-detections were reported more commonly than single infections in children than older adults (≥65 years; p = 0.01) [16] . co-infection of rsv with other respiratory viruses is common and can increase the severity of the disease [18] . in the current study, co-infection with rsv was present in 52.9 % of the patients who tested positive for rsv. this may have increased the severity of the disease, resulting in hospital admission. moreover, 26.9 % of the patients who were admitted to the icu had rsv in association with other viruses. for a long time, it was assumed that symptomatic infection with respiratory tract viruses was regularly shown by the confirmation of viral nucleic acids in respiratory secretions. however, in recent years, there have been increasing indications that this is not necessarily the case [19] . data are available from only a few case-control studies that examined to what extent specific respiratory viruses cause disease [9, [20] [21] [22] [23] . in the current study, adv, hbov, hrv, hev, and hcov-oc43 were not only detected in patients admitted with sari, but were also detected in asymptomatic children. the clinical importance of bocaviruses remains the subject of controversy. bocavirus dna can be confirmed in the respiratory tract of asymptomatic children; a high concentration of these viruses in respiratory specimens seems to be associated with symptoms [19] . only quantitative monoplex pcr or multiplex pcr can provide information about virus concentration. bocavirus was detected by pcr in nasopharyngeal swabs samples of children suffering from acute respiratory tract infection in saudi arabia [24] . in our study, hbov was detected among symptomatic children admitted with sari, as well as in asymptomatic children. six different human pathogenic coronavirus species (hcov) are known to date [1] . as a rule, they cause benign disorders of the upper respiratory tract [25] . however, after the sars (severe acute respiratory syndrome) epidemic in 2003 with the sars cov, hcov-nl63 was described in 2004 and hcovhku1 in 2005 [21] . the middle eastern respiratory syndrome (mers) is caused by mers-cov [26] . human corona viruses (hcov-nl63, hcov-oc43, hcov-229e) were detected in seven patients admitted with sari. however, hcov-oc43 was also detected in six asymptomatic children. adenovirus was the third most frequently detected virus in our study population. adenovirus dna is also often found in the respiratory secretions of asymptomatic children [1] , and was detected among patients as well as asymptomatic children in our study. hrvs are frequently found in asymptomatic children [27] , but are also detected in patients with symptoms ranging from mild common colds [28] to serious lower respiratory tract disease [29] . since the development of molecular assays for the detection of hrvs, the detection rate of hrv in patients with respiratory infections has increased to up to 50 % [30] . hrv was only second to rsv in frequency of detection by multiplex pcr in swab samples of children admitted with sari in our study. however, hrv was also detected in 4/40 controls. hrvs are frequently detected in asymptomatic children, making it difficult to interpret the clinical significance of a positive pcr finding [9] . moreover, hrvs could be detected until 2-5 weeks after the onset of symptoms [31] . in spite of these facts, rhedin et al. [9] reported that their data indicate that 39 % [95 % confidence interval (ci): 1 to 62] of acute respiratory infections in their population could be attributed to hrv. the potentially important rate of excreters of viral nucleic acids of specific pathogens that are not directly associated with the acute illness presents a problem. to date, quantitative conclusions are not reliably established, neither for monoplex pcr nor for multiplex pcr [1] . rhedin et al. showed that the nucleic acids of rsv, hmpv, and parainfluenza virus can be confirmed almost exclusively in symptomatic children and very rarely in asymptomatic children. their results indicate that a qpcr finding of rsv, hmpv, or piv is likely to be causative of disease in children with acute respiratory infection, and that detection of several other viruses, such as hbov, hrv, adv, hcov, and hev, must be interpreted with caution, due to the high detection rates among healthy children [9] . the detection of adv, hbov, hrv, hcov-oc43, and hev among asymptomatic children in the current study and the absence of rsv, hmpv, flu a, flu b, hpiv-1-2-3-4, hcov-nl63, and hcov-229e in these children support these findings. whereas the detection of some viruses, such as influenza virus and respiratory syncytial virus (rsv), is clearly predictive for respiratory disease [32] [33] [34] , the clinical significance upon the detection of several other viruses needs further investigation. the interpretation of a viral detection is complicated by the fact that infections with multiple viruses are common in children with acute respiratory infection and that many viruses have lately been reported to be found also in asymptomatic children [20] . most respiratory tract infections in children are of viral origin, especially during the winter season, which, in egypt, is from november to february. almost all the patients in our study received antibiotic therapy during hospital admission. the identification of specific viral pathogens will limit the unnecessary use of antibiotics and will facilitate giving specific antiviral therapy when indicated (e.g., neuraminidase inhibitors in confirmed influenza virus infection). better understanding of how to interpret viral findings by the use of new technologies is important to improve management decisions, which, in turn, will ameliorate patient outcomes and reduce unnecessary use of antibiotics. this study confirms the high rate of detection of viral nucleic acids by multiplex pcr) among hospitalized children admitted with severe acute respiratory infection, as well as the high rate of detection of multiple viruses. adenovirus, hbov, hrv, hev, and hcov-oc43 were also detected in asymptomatic controls, resulting in challenges in clinical interpretation. further studies are required to provide quantitative conclusions that will facilitate clinical interpretation and application of the results in the clinical setting. funding source none. the authors declare that they have no conflict of interest. ethical approval informed verbal consent was obtained from the parents of all the patients and the controls. the study design conformed to the revised helsinki declaration of bioethics and was approved by the scientific ethics committee of the department of pediatrics, faculty of medicine of cairo university. the role of multiplex pcr in respiratory tract infections in children rapid multiplex nested pcr for detection of respiratory viruses effect of azithromycin and clarithromycin therapy on pharyngeal carriage of macrolide-resistant streptococci in healthy volunteers: a randomised, double-blind, placebo-controlled study antibiotics for bronchiolitis in children the effect of rapid respiratory viral diagnostic testing on antibiotic use in a children's hospital new molecular virus detection methods and their clinical value in lower respiratory tract infections in children who regional office for europe guidance for sentinel influenza surveillance in humans comparison of anyplex ii rv16 with the xtag respiratory viral panel and seeplex rv15 for detection of respiratory viruses clinical utility of pcr for common viruses in acute respiratory illness detection of respiratory viruses using a multiplex real-time pcr assay in germany frequent detection of viral coinfection in children hospitalized with acute respiratory tract infection using a real-time polymerase chain reaction new vaccine surveillance network (2006) the underrecognized burden of influenza in young children respiratory syncytial virus and parainfluenza virus progress in the development of respiratory syncytial virus and parainfluenza virus vaccines practical implementation of a multiplex pcr for acute respiratory tract infections in children influenza and other respiratory virus infections in outpatients with medically attended acute respiratory infection during the 2011-12 influenza season performance of a novel microarray multiplex pcr for the detection of 23 respiratory pathogens (symp-ari study) does respiratory virus coinfection increases the clinical severity of acute respiratory infection among children infected with respiratory syncytial virus? human bocavirus and acute wheezing in children frequent detection of respiratory viruses without symptoms: toward defining clinically relevant cutoff values a case-control study of acute respiratory tract infection in general practice patients in the netherlands respiratory pathogens in children with and without respiratory symptoms human bocavirus: a novel parvovirus epidemiologically associated with pneumonia requiring hospitalization in thailand detection of bocavirus in children suffering from acute respiratory tract infections in saudi arabia antiviral strategies against human coronaviruses isolation of a novel coronavirus from a man with pneumonia in saudi arabia predominance of rhinovirus in the nose of symptomatic and asymptomatic infants viruses and bacteria in the etiology of the common cold rhinovirus illnesses during infancy predict subsequent childhood wheezing role of respiratory viruses in acute upper and lower respiratory tract illness in the first year of life: a birth cohort study highly frequent infections with human rhinovirus in healthy young children: a longitudinal cohort study the burden of respiratory syncytial virus infection in young children influenza a and b virus infections in children viral etiology of severe pneumonia among kenyan infants and children key: cord-328829-yywxmioq authors: boixeda, ramon; rabella, nuria; sauca, goretti; delgado, maria; martínez-costa, xavier; mauri, montserrat; vicente, vanessa; palomera, elisabet; serra-prat, mateu; capdevila, josep antón title: microbiological study of patients hospitalized for acute exacerbation of chronic obstructive pulmonary disease (ae-copd) and the usefulness of analytical and clinical parameters in its identification (virae study) date: 2012-05-25 journal: int j chron obstruct pulmon dis doi: 10.2147/copd.s30568 sha: doc_id: 328829 cord_uid: yywxmioq purpose: respiratory infection is the most common cause for acute exacerbation of chronic obstructive pulmonary disease (ae-copd). the aim of this work was to study the etiology of the respiratory infection in order to assess the usefulness of the clinical and analytical parameters used for copd identification. patients and methods: we included 132 patients over a period of 2 years. the etiology of the respiratory infection was studied by conventional sputum, paired serology tests for atypical bacteria, and viral diagnostic techniques (immunochromatography, immunofluorescence, cell culture, and molecular biology techniques). we grouped the patients into four groups based on the pathogens isolated (bacterial versus. viral, known etiology versus unknown etiology) and compared the groups. results: a pathogen was identified in 48 patients. the pathogen was identified through sputum culture in 34 patients, seroconversion in three patients, and a positive result from viral techniques in 14 patients. no significant differences in identifying etiology were observed in the clinical and analytical parameters within the different groups. the most cost-effective tests were the sputum test and the polymerase chain reaction. conclusion: based on our experience, clinical and analytical parameters are not useful for the etiological identification of copd exacerbations. diagnosing copd exacerbation is difficult, with the conventional sputum test for bacterial etiology and molecular biology techniques for viral etiology providing the most profitability. further studies are necessary to identify respiratory syndromes or analytical parameters that can be used to identify the etiology of new ae-copd cases without the laborious diagnostic techniques. chronic obstructive pulmonary disease (copd) is associated with significant morbidity and mortality, with the world health organization estimating its rise from being the fifth to the third leading cause of death by 2030. 1 the overall prevalence of copd in spain is estimated to be 10.2%. 2 copd is a slow, progressive disease, with patients experiencing episodes of acute deterioration known as exacerbations, 3 which increase in frequency and severity with disease progression. the causes of acute exacerbations of copd (ae-copd) are multifactorial. half of the ae-copd cases are attributed to respiratory infections (50%), but exacerbations are also associated with pollution, temperature changes, allergens (30%), and other comorbidities (26%) such as heart failure and pulmonary thromboembolism. 4 in several studies, the presence of bacteria in ae-copd has been associated with purulence of the sputum and the presence of inflammatory markers. 5, 6 in recent years, emerging new diagnostic techniques have revealed a relationship with respiratory viruses. [7] [8] [9] [10] in addition, the etiology of respiratory infections in ae-copd patients differs according to the geographic area. 11 currently, ae-copd patients are treated with antibiotics as the first line of defense, depending on the severity of the copd and the severity of the exacerbation itself. 12 with the emergence of multidrug resistance and increased economic spending on antibiotic therapy, numerous studies have assessed the benefit of antibiotic treatment, 13, 14 recommending a short course of antibiotic treatment for slight exacerbations. 15 similarly, biological markers such as procalcitonin have been proposed as markers for antibiotic administration in the ae-copd, 16 allowing a reduction in antibiotic prescriptions. the identification of respiratory viruses as a cause for ae-copd may help reduce the use of antibiotics. therefore, it is important to find clinical and analytical parameters that could guide us in identifying the etiology of new ae-copd cases, especially considering the laborious diagnostic techniques currently used for diagnosis. the aims of our study were to identify the etiology of respiratory infections in patients hospitalized for ae-copd using different diagnostic tests and to evaluate the usefulness of the clinical and analytical parameters of the diagnostic process. we included patients who were consecutively admitted to the hospital of mataró with ae-copd between april 1, 2005 and march 31, 2007. copd was defined according to the global initiative for chronic obstructive pulmonary disease (gold) criteria, 17 with patients exhibiting compatible spirometry measurements and a smoking history of at least ten packs/year. a diagnosis of acute exacerbation (ae) was assumed when a minimum of two anthonisen criteria 18 were present. any patient with copd decompensation that was caused by a noninfectious disease was excluded from the study, assuming that the selected patients presented with an upper or lower respiratory tract infection. identification was made based on the respiratory infection and dyspnea admission diagnoses from the international statistical classification of diseases, ninth revision, clinical modification (icd-9-cm) 19,20 (491, 492, 493, 496, 518.81, 464, 465, 466, 519.11, 786 .0), excluding the patients who had a known cause of respiratory failure that was different from infectious exacerbation (heart failure, pulmonary thromboembolism, pneumonia). finally, the patients who met the inclusion criteria and none of the exclusion criteria (severe immunosuppression, the need for mechanical ventilation or admittance to the intensive care unit, arrival from a nursing home, or a terminal stage of the disease) were asked for their informed consent. the study was approved by the consorci sanitari del maresme ethics committee of the hospital of mataró. upon admission, a complete clinical history and physical exam were performed. each patient's demographic and l ifestyle characteristics, baseline dyspnea (based on the dyspnea scale from the medical research council 21 ), exacerbation history, history of pneumonia, and hospital admissions during the previous year were evaluated. the contact with family members at home suffering from an upper respiratory tract infection was collected. we obtained information on each patient's upper respiratory tract (nasal congestion, rhinorrhea, and sneezing), lower respiratory tract (cough and expectoration), and constitutional symptoms (dysthermia, fever, chills, asthenia, anorexia, headache, arthromyalgia, and impaired consciousness). upon admission, the severity of the exacerbation was classified (depending on the presence of respiratory failure, severe cyanosis, baseline dyspnea deterioration, the utilization of accessory muscles, the worsening of blood gas levels, and a blood ph , 7.35), as were the severity of copd (according to the gold criteria) 17 and copd prognosis (according to the body mass, airflow obstruction, dyspnea, and exercise capacity [bode] multidimensional index). 22 vital signs and anthropometric data were collected from each patient, and a chest radiograph was taken to rule out pneumonia. baseline spirometry measurements were collected, and each patient's treatment before and during hospitalization for ae-copd was recorded. submit your manuscript | www.dovepress.com a baseline blood gas reading, a complete cell blood count (cbc), and basic biochemistry readings (ast, alt, creatinine, urea, glucose, and electrolytes) were collected from each patient upon arrival to the emergency room. a routine blood analysis that included total protein and a protein profile was performed the following day. upon hospital admission, the sputum was collected upon spontaneous expectoration in a conventional manner with or without using mucolytic agents. sputa were collected before starting antibiotic treatment at the hospital. the sputa were cultured only if the quality criteria were met in a sputum gram stain (,10 epithelial cells and .25 polymorphonuclear leukocytes). 23 sera were collected from patients at admission and 4 weeks after the initial collection for a second paired serology. passive agglutination techniques were used to detect mycoplasma pneumoniae, and microimmunofluorescence was used to detect chlamydia species. the npl and the nasal exudate that were collected 24 hours after admission were used for viral detection. the different techniques that were performed are detailed below. immunochromatography was used to rapidly detect antigens from influenza viruses a and b or respiratory syncytial virus (rsv). the binaxnow influenza a and b ® and binaxnow rsv ® tests (binax inc, scarborough, me) were used according to the manufacturer's instructions. immunofluorescence techniques were used to detect influenza viruses a and b, adenovirus, parainfluenza virus, and rsv. the replication of influenza viruses a and b, adenovirus, parainfluenza, rhinovirus, and rsv was detected in cell culture. we determined the presence of nucleic acids from influenza viruses a and b, rsv a and b, parainfluenza 1, 2, 3, and 4, coronaviruses 229e and oc43, rhinovirus, and metapneumovirus. the realaccurate™ respiratory rt-pcr kit (pathofinder, maastricht, netherlands) was used for the nucleic acid and respiratory virus amplification test, and the qiaamp viral rna mini kit (qiagen iberia sl, madrid, spain) was used to extract rna from clinical samples. the realaccurate™ respiratory rt-pcr kit (pathofinder) consists of ready-to-use solutions that contain primers and taqman probes that were used in accordance with the conditions set by the manufacturer. in a control visit performed one week after admission or coinciding with hospital discharge, we recorded the evolution of the episode with regards to clinical symptoms, physical examination, and treatment. in conjunction with the earlier episode evaluation, we identified cases of treatment failure (identified as the persistence of hemodynamic alterations, respiratory failure, severe adverse effects, or a lack of treatment response). the length of hospital stay and possible complications were also included. the data were collected in a microsoft access database and analyzed using spss for windows, version 14.0 (ibm corporation, armonk, ny). the qualitative variables are expressed as counts and percentages, while the quantitative variables are expressed as means and standard deviations or interquartile range. comparisons between means were performed using the student's t-test for independent samples or the mann-whitney u test for variables that did not meet the criteria of normality. for comparisons of proportions, the chi-square or fisher's exact test was used. in all cases, we considered values of p , 0.05 to indicate significant differences. to study the relationship between the analytical and clinical parameters and the etiology of ae-copd, three groups were categorized. group 1 contained patients for whom a virus was detected with diagnostic tests, group 2 included the patients who exhibited the detection of bacteria only, and group 3 contained patients with unknown etiologies. during the study period, 718 consecutive patients were admitted to the hospital with a diagnosis of respiratory infection and dyspnea according to icd-9-cm guidelines. 18 we included 155 patients based on the inclusion and exclusion criteria. in six patients with the clinical criteria for chronic bronchitis, spirometry was not performed in the follow-up. finally, 17 of the 148 remaining patients were submit your manuscript | www.dovepress.com dovepress dovepress excluded because the follow-up spirometry readings were not compatible with an infection. thus, we studied 132 patients with ae-copd of a probable infectious origin who met the inclusion criteria and had no other causes of acute decompensation (figure 1 ). the demographics of the patients and the baseline characteristics of ae-copd are presented in tables 1 and 3 . the patients were hospitalized in the following departments: internal medicine (48.5%), pneumology (33.3%), and the short stay unit (18.2%). over the course of two years, the admissions peaks coincided with seasonal variations, exhibiting two annual peaks (spring and winter) (figure 2) . a total of 51 pathogens were isolated. of these, 37 were bacterial and 14 were viral. of the 73 sputum samples surveyed, 19 were grampositive, 17 were gram-negative, and 37 contained polymorphonuclear cells without bacteria. in addition, ziehl-neelsen staining was performed in 26 samples, all of which had a negative result. the culture results were positive in 34 patients (table 2) , with higher numbers of patients exhibiting h. influenzae and p. aeruginosa. we obtained two positive serologies for mycoplasma pneumoniae (1.5%) and one for chlamydia pneumoniae (0.8%). an npl was collected from all patients to detect a possible viral etiology. we identified a positive result in 14 patients and found herpes virus type-1 in two patients. the detection of herpes virus type-1 was attributed to contamination. so then, we identified a positive result from viral techniques in 12 patients. an etiological agent was identified in 48 of the patients (36.3%), and an unknown ae-copd etiology was assigned to 84 patients (63.7%). a bacterial agent was identified as the etiological agent in 33 patients ( (1.5%), e. coli plus influenza a virus and p. aeruginosa plus coronavirus. the efficiencies of the diagnostic tests are shown in table 2 . we compared the characteristics of the patients using bacterial and viral isolation. when we assessed the clinical and analytical parameters of ae-copd according to etiological diagnoses, no significant differences were observed, with the exception of the lymphocyte count for the patients whose ae-copd was attributed to a virus (table 3) . we obtained statistically significant differences between the analytical datasets for the patients with known and unknown etiologies. in patients with an unknown etiology, we observed a greater decrease in the ph and po2 in the baseline arterial blood gas upon arrival at the emergency room, as well as a greater leukocytosis and increased heart rate ( table 3) . the evaluation of ae-copd after a week of hospital admission revealed clinical improvement in the majority of patients (92.3%). treatment failure was observed in seven patients (5.4%), and no positive changes were observed in three patients (2.3%). treatment failure was evidenced by the worsening of respiratory failure in three patients, severe adverse effects in two patients, and a lack of treatment response in four patients. only one patient died during hospitalization. this prospective observational study of patients admitted for ae-copd (vir-ae) included a 2-year follow-up period and was intended to identify the infectious etiology of copd exacerbations (whether viral or bacterial), as well as to describe the clinical features and analytical variables used to differentiate the cause of exacerbation. an infectious cause was identified in 48 of the 132 patients included in this study (36.3%). a bacterial etiology was identified in 33 patients, a viral infection was observed in 12 patients, and two patients had mixed etiology. a higher sensitivity was observed with the conventional sputum analysis and the polymerase chain reaction technique (pcr) for the npl analysis. the clinical and laboratory variables that were evaluated for the diagnosis were practically the same for the bacterial and viral etiology cases, with the exception of a relative lymphocyte count that was lower in the group with viral etiology and a longer hospitalization period in patients with bacterial infections. we attributed the longer hospital stay to parenteral treatment after finding multiresistant bacteria in some patients with bacterial etiology. other studies have identified clinical symptoms such colds or a sore throat upon the isolation of rhinovirus, 8 or even when rhinorrhea was associated with a bacterial etiology. 24 however, we have not identified any clinical symptom as indicative of a viral etiology in this study, probably because of the sample size. we have also identified a greater involvement of baseline arterial blood gas (a decrease in ph and po 2 ) in the group with an unknown etiology, as well as increased leukocytosis and heart rate, which could mean that a bronchospasm component contributes to the ae-copd within this group. in addition to bronchospasm, other causes of ae-copd such as heart failure or pulmonary thromboembolism were likely to be excluded from our study due to the fact that we only selected a population with a highly suspicious respiratory infection. similarly, we excluded patients coming from residential centers, patients admitted to the hospital in the 30 days prior to their current admission, and patients transferred from the intensive care unit in order to eliminate any hospital-acquired infections. this comprehensive selection of patients may limit the external validity of our results. we observed a mean age of 73 years (range 51-88 years) and a smoking history in all of our patients (23.5% were active smokers), which is probably due to the high percentage of men in our study. the diagnosis of copd exacerbation is a matter of debate. the most frequent cause of copd exacerbation is considered to be viral or bacterial bronchial infection. 25 this fact is based on the regular presence of purulent coughing and bacterial isolation from the cough of more than half of the patients with exacerbations. 26 in addition, up to 30% of patients with stable copd had evidence of bronchial bacterial colonization in the absence of ae-epoc. 6 however, authors such as sethi et al have demonstrated that pathogen colonization is not responsible for the exacerbation, as for haemophilus, where infection with an additional bacterial strain is necessary to elicit an exacerbation. 27 the identification of a bacterial etiology for ae-copd is primarily obtained through the study of sputum. we obtained sputum in 65% of patients, which shows the difficulty of obtaining it in clinical practice. a mixed respiratory flora was obtained in 37% of these patients and was diagnostic in 24.2%, with the samples exhibiting a predominance of h. influenzae and p. aeruginosa. it is important to note that our patients had severe copd exacerbations that caused respiratory failure and required hospital admission. h. influenzae and p. aeruginosa were detected mainly in the patients with severe copd, which could explain the inability to isolate bacteria such as pneumococcus and viruses, as severe copd benefits the enterobacteria and p. aeruginosa. the routine use of serology is not useful for the diagnosis of acute mycoplasma pneumoniae or chlamydia infection. from the point of view of viral etiology, pcr techniques have been performed on the bronchial exudate of copd streptococcus pneumoniae 2 -----2 haemophilus influenza 11 -----11 moraxella catarrhalis 3 -----3 pseudomonas aeruginosa 13 respiratory syncytial virus -28 in our study, the result was lower, as we detected a virus in 10% of all the ae-copd cases, with little evidence of influenza virus in our sample. this could be explained due to the fact that an influenza epidemic was not identified during the study period. 29 the discrepancies between our data and the data from other studies could also be explained by the different samples and techniques used. for example, higher percentage were obtained of sputum samples than npl samples (47% and 31%, respectively). 9 in reference to the literature, we probably could have obtained better results by analyzing the sputum rather than obtaining the npl for virological diagnostic techniques. even so, based on the results obtained, we can state that viral infections could be the cause of ae-copd. we should have this in mind at the time of prescribing antibiotics, especially in a slightly sick patient who presents no purulent sputum. viral disease has a seasonal distribution, and therefore, efforts to confirm the diagnosis in routine clinical practice notes: data are expressed as number (%) unless otherwise indicated. *we compared the patients with known diagnostic (33 as bacterial, 12 as viral, 2 as mixed viral and bacterial, and 1 as mycobacterium spp.) and unknown diagnostic. abbreviations: ae-copd, acute chronic obstructive pulmonary disease exacerbation; crp, c-reactive protein; nat, nucleic acid amplification test; paco 2 , partial pressure of carbon dioxide; po 2 , partial pressure of oxygen; post-bd fev 1 , post-bronchodilator forced expiratory volume in the first second; sato 2 , saturated oxygen; sd, standard deviation; u/l, units per litre. submit your manuscript | www.dovepress.com dovepress dovepress are to be reserved for situations in which viruses are present in the community; otherwise, this diagnosis could lead to a significant economic cost. rapid viral antigen detection with immunochromatography tests has a low diagnostic sensitivity, is not very useful, and is too expensive to be used in nonepidemic situations. 30 in conclusion, we stress that differentiating the etiology of ae-copd on the basis of clinical and laboratory data is difficult in common clinical practice. in our experience, the most profitable diagnostic tests to identify the possible cause of the acute decompensation of a patient with copd are the conventional sputum test for bacteria and molecular biology techniques for viruses. prevalence of copd in spain: impact of undiagnosed copd on quality of life and daily life activities toward a consensus definition for copd exacerbations outcomes following acute exacerbation of severe chronic obstructive pulmonary disease exacerbations of chronic obstructive pulmonary disease: when are bacteria important? bacterial infection in chronic obstructive pulmonary disease. a study of stable and exacerbated outpatients using the protected specimen brush respiratory viral infections in adults with and without chronic obstructive pulmonary disease respiratory viruses, symptoms, and inflammatory markers in acute exacerbations and stable chronic obstructive pulmonary disease respiratory viruses in exacerbations of chronic obstructive pulmonary disease requiring hospitalisation: a case-control study infections and airway inflammation in chronic obstructive pulmonary disease severe exacerbations prevalence of viral infection detected by pcr and rt-pcr in patients with acute exacerbation of copd: a systematic review diagnosis and management of chronic obstructive pulmonary disease: joint guidelines of the spanish society of pulmonology and thoracic surgery (separ) and the latin antibiotics in chronic obstructive pulmonary disease exacerbations. a meta-analysis antibiotics for exacerbations of chronic obstructive pulmonary disease short-course antibiotic treatment in acute exacerbations of chronic bronchitis and copd: a meta-analysis of double-blind studies antibiotic treatment of exacerbations of copd: a randomized, controlled trial comparing procalcitonin-guidance with standard therapy the global initiative for obstructive lung disease home page antibiotic therapy in exacerbations of chronic obstructive pulmonary disease catàleg de diagnòstics i procediments positive predictive value of icd-9-cm codes to detect acute exacerbation of copd in the emergency department usefulness of the medical research council (mrc) dyspnoea scale as a measure of disability in patients with chronic obstructive pulmonary disease the body-mass index, airflow obstruction, dyspnea, and exercise capacity index in chronic obstructive pulmonary disease microscopic and bacteriologic analysis of expectorated sputum identifying viral infections in vaccinated chronic obstructive pulmonary disease (copd) patients using clinical features and inflammatory markers bacteria and exacerbations of chronic obstructive pulmonary disease chronic obstructive pulmonary disease. 6: the aetiology of exacerbations of chronic obstructive pulmonary disease new strains of bacteria and exacerbations of chronic obstructive pulmonary disease detection of rhinovirus in induced sputum at exacerbation of chronic obstructive pulmonary disease system of influenza surveillance in spain low sensitivity of rapid diagnostic test for influenza the authors report no conflicts of interest in this work. this project was funded by a mataró tv3 foundation grant (042710).our thanks to the pneumology department of the hospital de mataró for its assistance with this research and agustí viladot for the bibliographic revision. submit your manuscript here: http://www.dovepress.com/international-journal-of-copd-journalthe international journal of copd is an international, peer-reviewed journal of therapeutics and pharmacology focusing on concise rapid reporting of clinical studies and reviews in copd. special focus is given to the pathophysiological processes underlying the disease, intervention programs, patient focused education, and self management protocols.this journal is indexed on pubmed central, medline and cas. the manuscript management system is completely online and includes a very quick and fair peer-review system, which is all easy to use. visit http://www.dovepress.com/testimonials.php to read real quotes from published authors. key: cord-325783-pqonn0as authors: nicholls, john m; poon, leo lm; lee, kam c; ng, wai f; lai, sik t; leung, chung y; chu, chung m; hui, pak k; mak, kong l; lim, wilna; yan, kin w; chan, kwok h; tsang, ngai c; guan, yi; yuen, kwok y; malik peiris, js title: lung pathology of fatal severe acute respiratory syndrome date: 2003-05-24 journal: lancet doi: 10.1016/s0140-6736(03)13413-7 sha: doc_id: 325783 cord_uid: pqonn0as background: severe acute respiratory syndrome (sars) is a novel infectious disease with global impact. a virus from the family coronaviridae has been identified as the cause, but the pathogenesis is still unclear. methods: post-mortem tissue samples from six patients who died from sars in february and march, 2003, and an open lung biopsy from one of these patients were studied by histology and virology. only one full autopsy was done. evidence of infection with the sars-associated coronavirus (sars-cov) and human metapneumovirus was sought by reverse-transcriptase pcr and serology. pathological samples were examined by light and electron microscopy and immunohistochemistry. findings: all six patients had serological evidence of recent infection with sars-cov. diffuse alveolar damage was common but not universal. morphological changes identified were bronchial epithelial denudation, loss of cilia, and squamous metaplasia. secondary bacterial pneumonia was present in one case. a giant-cell infiltrate was seen in four patients, with a pronounced increase in macrophages in the alveoli and the interstitium of the lung. haemophagocytosis was present in two patients. the alveolar pneumocytes also showed cytomegaly with granular amphophilic cytoplasm. the patient for whom full autopsy was done had atrophy of the white pulp of the spleen. electron microscopy revealed viral particles in the cytoplasm of epithelial cells corresponding to coronavirus. interpretation: sars is associated with epithelial-cell proliferation and an increase in macrophages in the lung. the presence of haemophagocytosis supports the contention that cytokine dysregulation may account, at least partly, for the severity of the clinical disease. the case definition of sars should acknowledge the range of lung pathology associated with this disease. published online may 16, 2003 http://image.thelancet.com/extras/03art4347web.pdf background severe acute respiratory syndrome (sars) is a novel infectious disease with global impact. a virus from the family coronaviridae has been identified as the cause, but the pathogenesis is still unclear. methods post-mortem tissue samples from six patients who died from sars in february and march, 2003 , and an open lung biopsy from one of these patients were studied by histology and virology. only one full autopsy was done. evidence of infection with the sars-associated coronavirus (sars-cov) and human metapneumovirus was sought by reverse-transcriptase pcr and serology. pathological samples were examined by light and electron microscopy and immunohistochemistry. findings all six patients had serological evidence of recent infection with sars-cov. diffuse alveolar damage was common but not universal. morphological changes identified were bronchial epithelial denudation, loss of cilia, and squamous metaplasia. secondary bacterial pneumonia was present in one case. a giant-cell infiltrate was seen in four patients, with a pronounced increase in macrophages in the alveoli and the interstitium of the lung. haemophagocytosis was present in two patients. the alveolar pneumocytes also showed cytomegaly with granular amphophilic cytoplasm. the patient for whom full autopsy was done had atrophy of the white pulp of the spleen. electron microscopy revealed viral particles in the cytoplasm of epithelial cells corresponding to coronavirus. since nov 1, 2002 , an outbreak of severe acute respiratory syndrome (sars) has affected 33 countries in five continents, with 7053 reported cases and 506 deaths at the time of writing. 1 local transmission has occurred in at least six countries. the first cases of sars in hong kong special administrative region were recognised in february, 2003. as of may 8, 2003 , there had been 1661 cases and 208 deaths in the region. clinically, the disease is characterised by fever, dyspnoea, lymphopenia, and rapidly progressing changes on radiography. 2, 3 upper-respiratory-tract symptoms are not prominent, but diarrhoea has been reported by some patients. there is no response to conventional antibiotics used to treat atypical pneumonia. the sars-associated coronavirus (sars-cov) has been consistently associated with this disease. 3 although this virus seems to be necessary for the development of sars, it has not been localised at the site of lung pathology. 3, 4 poutanen and colleagues 5 found human metapneumovirus (hmpv) as a second pathogen in patients with sars and postulated that hmpv potentiates the progression or severity of coronavirus infection. the use of steroids together with ribavirin has been reported to confer clinical benefit, although randomised clinical trials to support its clinical efficacy are not available. to aid understanding of the pathogenesis of the disease and the relation to current therapy, we report the pathological and virological findings in six patients with sars who died. all patients who met a modified who case definition of sars 6 and who underwent lung biopsy or post-mortem examination during march, 2003, at four major hospitals in the kowloon hospital cluster were eligible for inclusion. the case definition was fever (temperature 38°c or higher), cough or shortness of breath, new pulmonary infiltrates on chest radiograph, and either a history of exposure to a patient with sars or a lack of response to empirical antimicrobial coverage for typical and atypical pneumonia (beta-lactams and macrolides, fluoroquinolones or tetracyclines). 3 owing to the potentially infectious nature of the disorder, full autopsy was done in only one case. in the other cases, the post-mortem examination was limited to the lungs. we included only those patients for whom paired serum samples were submitted for virological studies during the course of the illness. patients with only needle-biopsy samples of organs available were excluded. during the period under study, there were six patients with pathological and virological investigation; one of these also underwent lung biopsy earlier in the course of the illness. in all cases the autopsy material was fixed in 10% neutral buffered formalin and later processed for electron microscopy. separate pieces of fresh tissue were sent for virological study; if sufficient quantity was available, they were stored at -70°c for immunofluorescence studies. patients patient 1, a 37-year-old woman, had good health before the illness. she was admitted on march 13, 2003, with a history of fever, cough, and general muscle pain for 10 days. oxygen saturation on admission was 89% on 50% oxygen. the chest radiograph showed patchy haziness. the patient soon developed respiratory failure necessitating mechanical ventilation; ribavirin and antibiotic treatment was then started. the ratio of partial arterial pressure of oxygen (pao 2 ) to fraction of inspired oxygen (fio 2 ) was 24·2 kpa on the day before death. she died on march 23, after 10 days of assisted ventilation. patient 2, a 39-year-old woman who was previously well, had indirect contact with a person who developed fever after return from guangzhou, china. she subsequently developed fever with shortness of breath and diarrhoea. she had lymphopenia at admission, and the chest radiograph showed infiltrates in the left upper zone. treatment with ribavirin and steroids was started on march 21. she developed respiratory failure the next day and was intubated. she had repeated cardiac arrests and died on march 24. patient 3, a 64-year-old man, developed influenza-like symptoms and left pleuritic chest pain on feb 15. a chest radiograph taken a day later showed haziness in the left lower zone. he subjectively improved after self-medication with levofloxacin and penicillin. he visited hong kong on feb 21 as a tourist, but his condition then deteriorated. he was admitted directly to the intensive-care unit and intubated on feb 22. investigation showed diffuse bilateral ground-glass changes on chest radiograph and severe oxygen desaturation. despite empirical antimicrobial coverage for typical, atypical, and hospital-acquired pneumonia, his condition continued to deteriorate. he was empirically treated with oseltamivir, foscarnet, intravenous immunoglobulin and, at a later stage, ribavirin and steroids. after ventilation for 10 days with a peak airway pressure of 36 cm water, tidal volume of 730 ml, and pao 2 /fio 2 9·8 kpa, he died on march 4 with acute respiratory distress syndrome and multiorgan failure. patient 4 was a 53-year-old man with a history of hypertension. he had developed influenza-like symptoms a few days before admission on feb 28 with chest radiographic changes of diffuse bilateral pneumonia and respiratory failure. he did not respond to doxycycline, amantadine, and cefotaxime. an open lung biopsy was done on march 4. routine microbiological and virological investigations were negative. he was empirically treated with oseltamivir, steroids, and ribavirin. non-oliguric renal failure developed and was later complicated by nosocomial pneumonia due to pseudomonas aeruginosa. he died on march 19, after ventilation for 16 days with a peak airway pressure of 34 cm water, tidal volume of 640 ml, and pao 2 /fio 2 17·0 kpa patient 5 was a 49-year-old businessman who had a history of hepatitis b liver cirrhosis and portal hypertension. he developed influenza-like symptoms while in hong kong but still travelled overseas on feb 26. investigation at his destination showed lymphopenia and thrombocytopenia in the peripheral blood examination, and bilateral pulmonary infiltrates on chest radiograph. he went into respiratory failure on march 2, and on march 5 was transferred back to hong kong, where he was ventilated. the ventilation pressure was 20/10 cm water most of the time. positive end-expiratory pressure was 15-17 cm water. the tidal volume was generally kept at 450-650 ml, and the pao 2 was between 70% and 90% and fio 2 between 80% and 100%. his renal function deteriorated progressively from march 9 to march 12, and at the time of death the urea concentration was 10·6 mmol/l and creatinine 218 µmol/l. total bilirubin varied between 18 µmol/l and 27 µmol/l. despite intensive support and antimicrobial coverage with piperacillin-tazobactam, azithromycin, and oseltamivir, he died on march 13. patient 6 was a 77-year-old man with good health previously. on march 2 he was admitted to hospital for increasing shortness of breath, fever, and cough for routine microbiological investigations included culture of blood and sputum for bacterial pathogens. 3 serology was done with complement-fixation tests for respiratory viruses, chlamydiae, and mycoplasma pneumoniae. total rna and dna were extracted from nasopharyngeal aspirates with the viral rna minikit and qiamp dna minikit (qiagen, hilden, germany). reverse-transcriptase (rt) pcr was done for influenza a, 7 adenovirus, 8 hmpv, and a newly recognised coronavirus subsequently reported to be associated with sars. 3 the rt-pcr protocol has been described previously. 3 the biopsy sample or post-mortem lung tissue was homogenised in a tissue grinder, and the supernatant was inoculated onto cell cultures. viral rna was extracted from the tissues with the rneasy minikit (qiagen) according to the manufacturer's tissue protocol instructions. rt-pcr for the novel coronavirus and for hmpv was carried out as described above. serial dilutions of serum from acute and convalescent patients were tested in parallel for antibodies to the coronavirus and hmpv by an indirect immunofluorescence test on frhk-4 cells infected with the respective virus. 3 role of the funding source the sponsors of the study had no role in the study design, data collection, analysis, or interpretation, or in the writing of the report. ante-mortem investigations for routine bacterial and viral respiratory pathogens proved unremarkable. however, all six patients had four-fold or greater rises in antibody titre the clinical presentations of two of these patients (patients 3 and 4) have been reported previously. 2 only one patient (patient 4) had an open lung biopsy done before death. the material was divided into four pieces; one was sent for viral studies, one frozen at -70°c, one fixed in formalin, and the one fixed in 2·5% glutaraldehyde. the latter was processed for transmission electron microscopy. for immunohistochemistry, paraffin-embedded blocks of the lung tissue were sectioned at 5 µm and dewaxed by standard procedures. monoclonal antibodies (dako, carpintaria, ca, usa) to keratin ae1/3 (1:50), cd3 t-cell (1:5), and cd68 macrophage marker (1:50) were applied on each section, and staining was done with the ventana nexes automated immunohistochemical stainer. frozen post-mortem lung tissue from one patient (patient 5) was available for study. cryostat sections were stained with directly conjugated monoclonal antibodies to influenza viruses a and b, adenovirus, and parainfluenza virus (dako, usa) and examined under a nikon eclipse e800m fluorescent microscope. in the biopsy sample from patient 4, most of the material was pleural tissue with a small amount of lung parenchyma. the architecture was preserved with a mild increase in interstitial lymphocytes. cytomegaly was present in a very few pneumocytes; it was characterised by nuclear enlargement, prominent nucleolus, and amphophilic granular cytoplasm (figure 1). no typical viral inclusions were identified. there was a mild to moderate increase in alveolar macrophages and early hyaline-membrane formation. gross findings in the post-mortem lung tissue were similar among patients (table 2) . where the lungs were removed en bloc, they weighed 1000-2100 g and were oedematous, with a greyish brown consolidated cut surface. the consolidation was irregular and patchy, with foci of pale tissue measuring up to several millimetres in diameter. more diffuse involvement was seen in one case (patient 4); mucopurulent material was seen in the tracheobronchial tree. in cases of disease duration of less than 10 days, the histological involvement of the lung varied, with a mixed inflammatory infiltrate, oedema, and hyaline-membrane formation seen (figure 2; table 2). the intra-alveolar oedema was granular or vacuolated. desquamation of pneumocytes was a prominent and consistent feature (figure 3). all cases showed scattered single enlarged cells that, in most, were associated with large nuclei and prominent nucleoli, similar to the findings in the open lung biopsy sample. clearing of the chromatin was also seen. giantcell formation was seen within the alveolar lumen in four cases, and there were a few cells that contained amphophilic to basophilic cytoplasmic granules within enlarged pneumocytes. one patient (patient 3) showed fibrin thrombi within pulmonary vessels, and another (patient 1) showed intimal swelling of pulmonary vessels. in three of four cases, the giant cells were cytoplasm. we therefore hypothesise that this cytoplasmic amphophilia may be due to viral assembly within the golgi apparatus-a feature seen on electron microscopy of the open lung biopsy sample from patient 4. 3 the histological changes of uncomplicated viral pneumonias are rarely described, and reports tend to be derived from post-mortem examination of patients who succumb to the pneumonia; thus, they may not be representative of the majority of pneumonia patients, who survive. 3, 15 the same is true for sars, because the patients admitted to hospital have not been in a suitable medical condition to warrant biopsy. however, several features of fatal coronaviral pneumonia can be identified. coronavirus infection in the early stages seems to stimulate epithelial cells and results in cellular proliferation and squamous metaplasia in the lung. this type-2 pneumocyte hyperplasia and hypertrophy has also been identified with porcine reproductive and respiratory syndrome virus (an arterivirus tentatively classified under the coronaviridae 16 ) as well as porcine respiratory coronavirus (previously known as transmissible gastroenteritis virus, respiratory variant). in those infections mild type-2 pneumocyte proliferation is associated with squamous metaplasia. 17 in sars, macrophage proliferation is more prominent in the consolidated areas of the lung; this distribution is also seen with porcine respiratory coronavirus. 18 in contrast to typical diffuse alveolar damage in which neutrophils and fibroblasts are the main cellular agents and macrophages have a lesser role, 9 in patients with fatal sars macrophages are the prominent leucocyte in the alveoli, even in the early stages of the disease. the finding of haemophagocytosis in the lung and white-pulp atrophy of the spleen identified in sars is reminiscent of that reported in fatal influenza subtype h5n1 disease in 1997. 19 haemophagocytosis has been attributed to cytokine dysregulation. 20 lymphopenia is another feature common to both sars-cov and h5n1 influenza pneumonia. 21 both viruses have crossed to human beings from animals or birds. 3, 21 experimental studies in which macrophages are infected in vitro suggest that, compared with conventional human influenza viruses, the subtype h5n1 influenza a viruses isolated in 1997 are hyperinducers of proinflammatory cytokines. 22 human coronavirus oc43 can replicate in human macrophages in vitro. 23 taken together, the similarity of clinical and pathological changes in sars-cov pneumonia and h5n1 pneumonia suggest that proinflammatory cytokines released by stimulated macrophages in the alveoli have a prominent role in pathogenesis of sars, resulting in cytokine dysregulation. this idea has implications for the management of coronaviral pneumonia. intervention with steroids might modulate this cytokine response and prevent a fatal outcome, as has been proposed for nonviral acute respiratory distress syndrome. 24 although we did not find pcr evidence of a secondary viral infection, such as hmpv, in pigs infected with some isolates of porcine respiratory coronavirus, the respiratory lesions were severe enough to predispose the pigs to secondary bacterial or mycoplasmal infections or to lead to combined viral infections. 25 as we have already mentioned, the histological features of porcine reproductive and respiratory syndrome virus infection are similar to those of sars-cov infection; it is possible that sars-cov infection in human beings may "open the door" to secondary infections, as previously reported. 5 the patients who received ribavirin therapy still had pcr and electron-microscopic evidence of sars-cov in the lung. this finding supports clinical evidence that a search articles the lancet • vol 361 • may 24, 2003 • www.thelancet.com positive for the macrophage marker cd68 (figure 4); in one case, they were positive for the epithelial marker ae1/3. in the patient who had frozen tissue available, no staining was identified to adenovirus, parainfluenza virus, or influenza viruses a or b on frozen-section immunofluoresence. one patient, in whom the time from onset of symptoms to death was 8 days, showed squamous metaplasia of the bronchi with loss of cilia ( figure 5 ). in addition, within the parenchyma and also within the interstitium there were increased numbers of cd68positive mononuclear cells and focal haemophagocytosis ( figure 6 ). because of the risk of infection, most postmortem examinations were limited to the chest, but in patient 5, for whom a full autopsy was done, pronounced white-pulp atrophy of the spleen was seen. in this case, electron microscopy showed dilated golgi apparatus in the cytoplasm of pneumocytes, and within these organelles, 90 nm viral particles with small external spikes corresponding to coronavirus were identified (figure 7). these particles were of similar morphology to those reported previously. 3 all of the six patients described had serological evidence of recent infection with the sars-cov. five had evidence of viral rna detectable in samples taken before or after death. in previous studies, healthy controls and patients with unrelated disease had no evidence of sars-cov antibody or rna in the serum or the respiratory tract, respectively. 3 in patients with disease of duration less than 10 days there was hyaline-membrane formation, pneumocyte proliferation, and oedema. diffuse alveolar damage was seen in cases of longer duration. this diffuse alveolar damage typically goes through an exudative phase followed by a proliferative phase, 9 although others have described a three-stage process: an inflammatory or exudative phase, a proliferative phase, and a final fibrotic phase. 10 viral infections resulting in diffuse alveolar damage typically fall into two categories; influenza viruses a and b and adenovirus cause most cases of viral pneumonia in immunocompetent adults. immunocompromised hosts are susceptible to pneumonias caused by cytomegalovirus and other herpesviruses, measles virus, and adenovirus. 11 in addition to the diffuse alveolar damage here, morphological features of giant cells and pneumocyte hyperplasia, which appear to be prominent in this viral infection, were seen in these patients. 4, 12 multinucleate giant cells within the alveoli have previously been reported in patients with sars, 4,12 but their derivation has remained unclear. we have documented that in three of the patients these giant cells are of macrophage origin and in one other patient they are epithelial in origin. these giant cells were not present in the ante-mortem lung biopsy sample and were present only in patients with disease progression for longer than 8 days from admission. some animal coronaviruses induce syncytium formation in cell culture, 13 and sars-cov also induces focal syncitium formation 4 in vero cells. bronchial epithelial denudation, loss of cilia, and squamous metaplasia were early features. another notable feature was the presence of large pneumocytes showing an enlarged nucleus and granular amphophilic cytoplasm. these changes were also seen in the open lung biopsy sample from patient 4. coronavirus assembly takes place in the golgi apparatus of the cytoplasm; 14 in the sections of the coronavirus-infected culture this process was characterised by swelling and increased granularity of the for a more promising antiviral agent is of high priority in the treatment of this disease. the recent who case definition of sars when an autopsy is done states that autopsy features showing changes of respiratory distress syndrome without an identifiable cause can be included, whereas those without respiratory distress syndrome should be excluded. 26 previous reports have featured late cases, in which diffuse alveolar damage is pronounced. 4, 12 our series of serologically confirmed cases shows there is a range of morphological changes in sars, and that in disease of less than 10 days' duration the changes of respiratory distress syndrome may be focal and dissimilar to those previously published cases. pathologists undertaking these autopsies should be aware of the varied range of these morphological changes. cumulative number of reported cases of severe acute respiratory syndrome a cluster of cases of severe acute respiratory syndrome in hong kong severe acute respiratory syndrome (sars) is associated with a coronavirus a novel coronavirus associated with severe acute respiratory syndrome identification of severe acute respiratory syndrome in canada severe acute respiratory syndrome (sars) detection of influenza a virus from different species by pcr amplification of conserved sequences in the matrix gene clinical assessment of a generic dna amplification assay for the identification of respiratory adenovirus infections common pathways and patterns of injury the pulmonary physician in critical care: 6, the pathogenesis of ali/ards viral pneumonias in adults: radiologic and pathologic findings a major outbreak of severe acute respiratory syndrome in hong kong world organization for animal health the cytoplasmic tail of infectious bronchitis virus e protein directs golgi targeting influenza pneumonia in lung transplant recipients: clinical features and association with bronchiolitis obliterans syndrome comparison of the pathogenicity of two us porcine reproductive and respiratory syndrome virus isolates with that of the lelystad virus experimental reproduction of pneumonia in gnotobiotic pigs with porcine respiratory coronavirus isolate ar310 transmissible gastroentertitis virus (respiritory variant) pathology of fatal human infection associated with avian influenza a h5n1 virus hemophagocytic syndromes and infection clinical features and rapid viral diagnosis of human disease associated with avian influenza a h5n1 virus induction of proinflammatory cytokines in human macrophages by influenza a (h5n1) viruses: a mechanism for the unusual severity of human disease human macrophages are susceptible to coronavirus oc43 role for macrophage migration inhibitory factor in acute respiratory distress syndrome pathogenicity of three isolates of porcine respiratory coronavirus in the usa case definitions for surveillance of severe acute respiratory syndrome (sars) we thank klaus stöhr and who for initiating and coordinating the information exchange between members of the who sars laboratories network, which has allowed rapid understanding of the aetiology of this disease; s y lam, s w kwan, k f lo, h y ng, c y cheung, k fung, and o k wong for their assistance; and trevor ellis of the department of agriculture and fisheries, government of the hong kong special administrative region for advice.we acknowledge research funding from public health research grant a195357 from the national institute of allergy and infectious diseases, usa, the wellcome trust grant gr067072/d/02/z, the university of hong kong, and the hospital authority of hong kong special administrative region. none declared. key: cord-339039-6gyo9rya authors: bonvehí, pablo e.; temporiti, elena r. title: transmission and control of respiratory viral infections in the healthcare setting date: 2018-04-30 journal: curr treat options infect dis doi: 10.1007/s40506-018-0163-y sha: doc_id: 339039 cord_uid: 6gyo9rya purpose of the review: viral respiratory infections have been recognized as a cause of severe illness in immunocompromised and non-immunocompromised hosts. this acknowledgement is a consequence of improvement in diagnosis and better understanding of transmission. available vaccines and antiviral drugs for prophylaxis and treatment have been developed accordingly. viral respiratory pathogens are increasingly recognized as nosocomial pathogens as well. the purpose of this review is to describe the most frequent and relevant nosocomial viral respiratory infections, their mechanisms of transmission and the infection control measures to prevent their spread in the healthcare setting. recent findings: although most mechanisms of transmission and control measures of nosocomial viral infections are already known, improved diagnostic tools allow better characterization of these infections and also lead to the discovery of new viruses such as the coronavirus, which is the cause of the middle east respiratory syndrome, or the human bocavirus. also, the ability to understand better the impact, dissemination and prevention of these viruses, allows us to improve the measures to prevent these infections. summary: healthcare viral respiratory infections increase patient morbidity. each virus has a different mechanism of transmission; therefore, early detection and prompt implementation of infection control measures are very important in order to avoid their transmission in the hospital setting. healthcare-associated viral infections cause an increase in morbidity and mortality among hospitalized patients and also in healthcare-associated costs. viral respiratory pathogens are increasingly recognized as etiological agents of nosocomial infections. they can be acquired in different settings inside the hospital and they can produce upper (uri) or lower respiratory tract infections (lri) [1•]. transmission can occur by air through aerosolization, by drops of respiratory secretions or by inoculation after touching contaminated surfaces [2] . among immunocompromised hosts, these types of infections can present themselves with subclinical manifestations and can be associated with high morbidity and mortality. for this reason, a high suspicion vigilance must be kept to confirm viral infections in these hosts. it is also known that viral shedding is more prolonged [3, 4•] . there are several etiological agents that cause these infections, with different ways of transmission depending on each virus. for this reason, it is mandatory to suspect and correctly identify the etiological agent and promptly implement isolation precautions. molecular methods have improved sensitivity for the diagnosis of viral infections and have also facilitated the identification of other viral pathogens that cause respiratory tract infections [5] . in the clinical setting, it is sometimes difficult to establish if a respiratory viral infection was acquired in the hospital or in the community. this situation is most commonly observed when the length of hospitalization is less than or equal to the incubation period of the disease. in some cases, virological studies such as next generation sequencing can be used to differentiate between these situations [6] . as a measure to establish the magnitude of respiratory viral infections in the healthcare setting, it must be noted that 20% of all nosocomial pneumonias are of viral origin. in this context, the incidence of each virus is related to its circulation in the community [2, 7] . the objective of this revision is to describe the means of transmission and the measures to control the dissemination of respiratory viral infections in the healthcare setting. there are several viruses associated with upper and lower respiratory infections in humans. we describe the most relevant ones for the clinician. this virus belongs to the mononegavirales order, paramyxoviridae family and pneumovirinae subfamily. main characteristics of rsv include the number and order of genes and lack of hemagglutinin and neuraminidase activity [8] . rsv is the main cause of respiratory tract disease in small children. viral propagation is so efficient that humans can acquire this infection in their first years of life. immunity achieved after rsv infection is incomplete and reinfections are frequent. although life threatening infections occur mainly during the first 2 years of life, rsv infections can cause a high morbidity due to acute upper and lower respiratory tract infections. in children, rsv causes bronchiolitis and pneumonia and it is a common pathogen in older adults and immunocompromised hosts, where it can either cause upper respiratory infections or pneumonia [9] . influenza viruses are single-stranded, negative-sense rna viruses belonging to the orthomyxoviridae family and are classified into three main types, a, b and c, based on their antigenic different characteristics. these three types show significant differences related to their genetic organization, structure, possible hosts, epidemiology and clinical manifestations, with influenza a and b the most common human pathogens. these viruses share certain features such as the presence of an envelope derived from the host cell and surface glycoproteins essential for replication. they have a spheric or filamentous shape and measure 8 to 120 nm with surface projections that are spicules of hemagglutinins (ha) and neuraminidases (na), also important for their antigenic mutations, as they are used as antigens for the influenza vaccine [10] . this virus is an important cause of infections in immunocompetent and in immunocompromised hosts causing mild upper respiratory infections (pharyngitis, coryza, conjunctivitis) and complicated infections (pneumonia, hepatitis, hemorrhagic cystitis or disseminated diseases), respectively. there are seven species of hadv (a to g) and an increasing number of different serotypes were identified through genomic assays. besides this, the improved understanding of viral persistence sites and reactivation requires continuous adaptations of diagnostic tools in order to facilitate timely detection and control of hadv infections. hadv infections are more frequent in closed communities such as the armed forces, nurseries and hospitals with immunocompromised hosts [11••] . this is a single-stranded rna virus from the paramyxoviridae family classified into five types: respirovirus genus (piv 1 and 3) and rubulavirus (piv 2, 4a, and 4b). [12] piv serotypes may cause bronchiolitis, laryngotracheobronchitis (croup) and pneumonia in different age groups in immunocompetent hosts, with piv 3 more frequently associated with lower respiratory tract infections. among immunocompromised hosts, piv has been found in upper and lower respiratory tract infections in 2.2 -7.1% of hsct recipients. in solid organ transplant patients, lung recipients have demonstrated to be at risk for piv disease and this infection was also associated to allograft rejection or dysfunction [13] . these viruses are non-segmented rna viruses, belonging to the mononegavirales order, paramyoviridae family, pneumovirinae subfamily and metapneumovirus genus. initially described as a pathogen in children, where it can cause upper respiratory disease, bronchiolitis and pneumonia, has also been later identified as an adult respiratory viral pathogen [14] . in immunocompromised hosts, hmpn has also been associated to produce upper and lower respiratory tract infections among hsct patients and solid organ recipients, mainly lung transplant patients [13] . picornaviridae family includes nine genuses, four of which (rhinovirus, enterovirus, parechovirus and hepatovirus) can infect humans. human rhinoviruses (hrv) were classified based on immune serotypes, type of receptor, antiviral sensitivity and genotype. rhinovirus infectivity can be neutralized by specific antisera and the new serotypes are defined based on the absence of cross reactivity with known specific antisera. hrv are rna viruses and more than 160 serotypes have been identified. clinical importance of hrv infections is well demonstrated in children where they are responsible for more than 70% of asthma exacerbations [15] . in immunocompetent adults, these are the most common cause of selflimited upper respiratory tract infections and could be responsible for more than 80% of common colds in autumn and spring seasons [16] . immunocompromised hosts are particularly prone to severe infections. in lung transplant patients can be a common infection and lead to graft dysfunction. among hematological malignancies hrv can produce lower respiratory tract infections with potential adverse consequences [11••, 17, 18] . hcov are positive-stranded rna viruses from the coronaviridae family, causing common colds; in children, they have also been associated with bronchiolitis and croup [19] . there are five hcov identified as hcov-oc43, hcov-229e, hcov-hku1, hcov-nl63 and hcov-sars. the latter virus was responsible of an epidemic of severe respiratory disease syndrome in some countries in asia (china, hong kong sar, taiwan, singapore, vietnam) and canada in 2003, with approximately 10% mortality and high attack rate in healthcare workers and family members; it was detected in humans for the last time in 2004 [19, 20] . more recently, one coronavirus was able to cause severe respiratory disease in humans in the middle east. this coronavirus was named as mers-cov and was isolated in saudi arabia in 2012 from respiratory secretions in a man who died from pneumonia. this finding, combined with a retrospective assessment of stored samples from an outbreak observed in healthcare workers in jordan, allowed the recognition of mers-cov as a cause of this new severe respiratory disease [21• ]. the origin of mers-cov has been widely debated. it was originally believed that the reservoir was bats due to the similarity between mers-cov and bat coronavirus. however, the strain found in humans was not related to the one described in bats. another possible source for this type of coronavirus is the camel because they have antibodies against mers-cov similar to those found in humans. most human cases of mers-cov were described in saudi arabia and, to a lesser degree, in the united arabs emirates. later on, other cases outside the arabian peninsula were notified, but all of them were imported from endemic countries in the middle east, and were related to close contact with sick people. most of the patients were healthy males around 50 years [22] . this is a single-stranded dna virus that has recently been found as a human pathogen. it belongs to the parvoviridae family, parvovirinae subfamily, bocaparvovirus genus; four genotypes have been described (hbov 1 -4) [23, 24] . hbov has been associated with upper and lower respiratory tract infections, sometimes as a single agent or as a co-pathogen with other viruses. it is also found in high proportion in fecal samples of children who had the virus already detected in respiratory samples. it has also been reported in stool and blood of immunosuppressed hosts [13] . this virus is transmitted through droplets or by direct contact (table 1) [25] . droplet transmission is possible when the source is within 1 m. the size of droplets, produced by coughing, sneezing or any other procedure that moves respiratory secretions, is larger than 5 μm. recent data has demonstrated the transmission of rsv through aerosolized particles based on the fact that they appear close to hospitalized children with bronchiolitis and rsv infections. these particles can remain suspended in the air for prolonged periods of time and can infect human epithelial cells [26] . the survival of rsv in the environment seems to be partly dependent on drying time and dew point. at room temperature rsv in patients' secretions can be viable on polished surfaces for 3 to 30 h. on porous surfaces such as cloth or tissue papers viability is shorter, usually less than an hour. viral infectivity in the hands varies from person to person, generally less than an hour [27] . the risk of transmission is not the same in all the hospital areas: in pediatric and neonatal units, risk of transmission occurs between 6% and 56%, in transplant and oncohematological units fluctuates between 6% and 12%, and in other adult wards between 30% and 32% [28] . it must also be considered that healthcare workers can be a source of transmission for rsv, considering that shedding occurs between 15% to 20% of them, even without symptoms [29] . in high transmission sites, as in oncohematological adult patient wards, it is recommended to manage these infections in ambulatory care to decrease the risk of transmission within the hospital. nevertheless, it has been demonstrated that this group of patients can acquire rsv and piv infections in spite of their short time of hospitalization [30, 31] . influenza virus can be transmitted through infectious droplets eliminated by patients when coughing or sneezing, or through direct contact with surfaces contaminated by respiratory secretions from symptomatic infected subjects (table 1 ) [32] . airborne transmission was observed with 2009 influenza a h1n1p strain; therefore, airborne precautions should also be implemented in case of a pandemic situation where high risk of aerosolization can occur (e.g. intubation) [13] . in closed areas with high risk patients, such as bone marrow transplant units about 50% of the infections come from healthcare personnel or infected visitors [33•] . another source of nosocomial transmission of influenza are hospitalized patients with prolonged viral shedding, sometimes for weeks or months. high risk patients for prolonged shedding include those under high doses of steroids, oncohematological patients and children [13, 28, 32] . another potential source of transmission at the hospital are those individuals not yet symptomatic, but within 1-3 days prior the infection. these cases are difficult to detect, and hence, preventive control measures are usually not implemented [34] . different studies show that healthcare workers are one of the main vehicles of transmission of influenza within the hospital. there is data showing that many of them continue to work being ill, which certainly amplifies the rate of transmission [35] . it is transmitted through direct contact with a contaminated patient's environment and also by fomites containing the virus (table 1) . adenovirus can also be transmitted through the fecal-oral route and by organ transplantation from infected donors. it can infect an individual, remain latent for a period of time and later cause disease during immunosuppression [36] . the ability of adenovirus to survive in the environment on certain surfaces like non-porous ones can be as prolonged as 49 days, facilitating its transmission [37] . it is transmitted through direct contact with patients' secretions or by direct contact with the contaminated environment (table 1) . this virus can survive more than 4 h on porous surfaces and more than 10 h on non-porous surfaces. however, it is difficult to recover it from the hands and only 5% has been detected after 10 min of contact with contaminated secretions [38] . piv can also be transmitted through droplets, and nosocomial outbreaks have been described [13, 39, 40] . nosocomial transmission has been reported in pediatric and neonatal emergency wards, as well as in nurseries [41••] . immunosuppressed hosts, particularly hsct and oncohematological patients, are more prone to present piv infections with an incidence of 4% and 2%, respectively [42] . viral asymptomatic excretion has been described in hsct and oncohematological patients, further increasing transmission in the hospital setting [43] . hmpn this virus is transmitted by respiratory secretions from coughing, sneezing or through direct contact with contaminated surfaces (table 1) . it is transmitted mainly within the community, but a few reports of nosocomial outbreaks have been described [44] . autoinoculation is the most common way of transmission after contact with contaminated objects. aerosolization also contributes to viral spread (table 1 ) [45] . some epidemiologic data support the possibility of transmission by contact or by air (table 1) . however, means of transmission of mers-cov have not been elucidated yet. the epidemiology of mers-cov infection since isolation of the pathogen in 2012 is consistent with multiple introductions of the virus into humans from an animal reservoir, with no long-term sustained human-to human transmission [46] . hbov is probably transmitted through respiratory droplets; however; nosocomial transmission of hbov has not been reported (table 1 ) [13] . education of healthcare workers, close monitoring of compliance to preventive measures, and infection surveillance are useful tools to reduce the risk of transmission for most respiratory viruses that cause nosocomial infections [2] . in the following section, preventive and control measures for each respiratory virus are described. the precautions recommended by the cdc to avoid rsv transmission include standard and contact precautions (handwashing with water and soap or alcohol hand rub; appropriate use of gloves in order to decrease autoinoculation and use of gown) [2] . use of facemask (droplet precautions) and goggles when direct contact with the patient are also recommended by others (table 2 ) [29, 47] . eye protection should always be worn when performing procedures that might generate sprays from respiratory secretions, considering that nose and eyes are the main sites for virus inoculation [2, 47] . cdc also recommends that infected patients be hospitalized in single rooms or kept in cohort isolation if no individual isolation rooms are available. when cohorting, there should be at least 0.9 m of separation between beds [2] . another measure to be considered is the use of individual equipment for each patient, including toys. other strategies that help prevent rsv transmission in the healthcare setting include: reducing the quantity of persons in contact with hospitalized patients (especially oncohematological), avoiding the presence of children under 12 years of age in the hospital when rsv circulates either in the community or the hospital, and using single-bed rooms for infected patients [2, 29, 48] . some studies have demonstrated that healthcare workers use of eye protection might more efficient than contact precautions in decreasing the risk of rsv transmission within the hospital setting [28] . other recommendations, with very little or no evidence at all, suggest that healthcare personnel caring for rsv infected patients be exclusive for these individuals and that hospital visitor restrictions be taken during rsv season. the use of drugs for prophylaxis is limited to palivizumab in the high risk pediatric population although there are some data that demonstrates its effectiveness in the prevention of rsv in hsct adult patients [13, [49] [50] [51] . annual influenza vaccination of healthcare workers and high-risk patients for influenza complications and their households is the most effective measure to prevent the disease and its complications. it also contributes to decrease viral transmission in the healthcare setting [2] . vaccination of healthcare workers is the second most important strategy, just behind handwashing, to prevent nosocomial dissemination of influenza [41••] . several studies have demonstrated a reduction in morbidity and mortality among patients in different healthcare settings when healthcare workers have been vaccinated against influenza [41••] . different strategies were successfully implemented in order to increase vaccine uptake in healthcare personnel. among these were educational campaigns regarding benefits of the vaccine for patients and personnel, actions addressed to remove administrative barriers for vaccination, implementation of accessible locations for vaccine administration and of signed declination statements, and mandatory vaccination policies for healthcare workers [52] . however, the scientific support for this last strategy has been questioned recently [53] . antivirals against influenza have proved effective to reduce influenza viral transmission. the ones now available are the neuraminidase inhibitors such as oseltamivir, zanamivir and peramivir. oseltamivir which is administered through the oral route has proved to effectively prevent secondary cases of influenza among household contacts if it is given within 48 h of symptoms initiation of the index case. zanamivir, which is administered by inhalation per mouth, has also proved effective in preventing secondary influenza cases in households and healthcare workers [54, 55] , but caution is advised, as its efficacy has only been demonstrated in particular circumstances. during a nosocomial outbreak, in high-risk patients and non-vaccinated healthcare workers, antiviral administration should be implemented. when a mismatch between the circulating influenza strain and the vaccine strain is identified, antiviral prophylaxis should be given to all healthcare workers if an outbreak occurs. duration of antiviral administration during a nosocomial outbreak of influenza should be two weeks or one week after the outbreak control [2] . besides vaccination and antivirals, infection control measures are of utmost importance in order to prevent influenza virus transmission and dissemination in the hospital environment. these measures include handwashing, limitation of visitors and of health personnel in contact with infected patients, cohort or single bed rooms isolation, and droplet precautions. precautions to avoid aerosolization should also be implemented in case of a new pandemic strain or when performing high risk procedures (e.g. intubation) ( table 2 ) [13] . preventive measures to avoid adenovirus nosocomial infections include patient cohorting, reduction of visitors and contact and droplet precautions, along with the exclusion of infected healthcare workers from clinical duties (table 2 ) [13, 36] . handwashing seems to be non-effective in removing adenovirus from contaminated fingers [56] . there are no available vaccines or antiviral prophylaxis to help prevent adenovirus infection [33•, 57, 58] . in order to prevent nosocomial transmission of piv several measures are proposed. they include contact precautions, reinforcement of hand hygiene and standard precautions in all patients to avoid an outbreak in the hospital setting (table 2 ) [2] . there are some vaccines under the initial phases of development that take advantage of mucosal immunity, but there are no efficacy data yet [59] . hmpv infection control measures to prevent nosocomial transmission of this virus include handwashing and contact precautions ( table 2 ) [42] . there is no consensus regarding recommendations to prevent mers-cov transmission. while who recommends contact isolation and droplet precautions for any suspected case and respiratory precautions only for aerosol generating procedures, the cdc promotes air and contact precautions for all the activities related to patient care (table 2 ). other centers recommend intensification of handwashing, contact precautions, ocular protection and the use of facemasks. currently, any suspected or confirmed mers-cov case should be treated in a medical facility. transfer and transportation of an infected patient in the hospital environment should include a minimum crew, all wearing surgical facemasks. healthcare workers with direct patient contact should follow the recommendations mentioned before and keep updated on new recommendations. any transference of mers-cov infected patients from one healthcare setting to another should be coordinated with public health authorities [22, 57, 60] . although nosocomial transmission has not been reported, it seems prudent to implement at least contact precautions in patients when this virus is detected (table 2) . the epidemiology of viral respiratory infections has been better understood and modified in the last years for multiple reasons, including vaccination, improvement in diagnostic assays such as molecular techniques and better knowledge and understanding of mechanisms of transmission. cdc/nhsn surveillance definitions for specific types of infections surveillance definitions janu healthcare infection control practices advisory committee. guidelines for preventing health-careassociated pneumonia, 2003: recommendations of cdc and the healthcare infection control practices advisory committee viral pneumonia in patients with hematologic malignancy or hematopoietic stem cell transplantation respiratory viral infections in solid organ and hematopoietic stem cell transplantation reference is important because nosocomial respiratory tract infections are frequent and severe in this group of patients. this article allows the reader to get updated on respiratory viral infections in immunosuppressed hosts detection of respiratory viruses and legionella spp. by real-time polymerase chain reaction in patients with community acquired pneumonia the role of next generation sequencing in infection prevention in human parainfluenza virus 3 infections in immunocompromised patients nosocomial viral respiratory infections: perennial weeds on pediatric wards adenovirus 1784-1793. in: mandell, douglas, and bennett's principles and practice of infectious diseases a case control study assessing the impact of non-ventilated hospital-acquired pneumonia on patient outcome treanor: influenza (including avian influenza and swine influenza) 2272-2296. in: mandell, douglas, and bennett's principles and practice of infectious diseases this reference reviews all the aspects related to respiratory viral infections including those which are of nosocomial origin. it is a very detailed and exhaustive description of the most common and frequent viral respiratory tract infections. it describes each virus, their mechanisms of transmission ison parainfluenza viruses. 2580-87. in: mandell, douglas, and bennett's principles and practice of infectious diseases respiratory viral infections in hematopoietic stem cell and solid organ transplant recipients. semin respir crit care med rhinovirus and respiratory syncytial virus in wheezing children requiring emergency care. ige and eosinophil analyses frequency and natural history of rhinovirus infections in adults during autumn chronic rhinoviral infection in lung transplant recipients clinical and molecular epidemiology of human rhinovirus infections in patients with hematologic malignancy world health organization. summary of probable sars cases with onset of illness from 1 middle east respiratory syndrome is a severe and emerging respiratory viral infection that can also be transmitted in the health care setting. this article describes in detail the latest advances in the research of mers-cov, its epidemiology, mechanisms of transmission, virus characteristics middle east respiratory syndrome coronavirus: review of the current situation in the world cloning of a human parvovirus by molecular screening of respiratory tract samples risk of acute gastroenteritis associated with human bocavirus infection in children: a systematic review and metaanalysis outbreak of respiratory syncytial virus (rsv) infection in immunocompromised adults on a hematology ward evidence of respiratory syncytial virus spread by aerosol. time to revisit infection control strategies? breese hall respiratory syncytial virus (rsv) risk of nosocomial respiratory syncytial virus infection and effectiveness of control measures to prevent transmission events: a systematic review. influenza other respir viruses nosocomial respiratory syncytial virus infections: the "cold war" has not ended prolonged outbreak of human parainfluenza virus 3 infection in a stem cell transplant outpatient department: insights from molecular epidemiologic analysis molecular characterization of strains of respiratory syncytial virus identified in hematopoyetic stem cell transplant outpatient unit over 2 years: community or nosocomial infection guidelines for preventing infectious complications among hematopoietic cell transplant recipients: a global perspective the authors of this article address the importance of rsv, adenovirus, pai, hrv, influenza and hmpn in both sot and hsct patients. they point out the main characteristics of these viruses in immunosuppressed hosts such as prolonged excertion and higher morbidity and mortality in relation to normal hosts the influenza viruses and influenza incidence and recall of influenza in a cohort of glasgow healthcare workers during the 1993-4 epidemic: results of serum testing and questionnaire adenovirus in solid organ transplant recipients prolonged recovery of desiccated adenoviral serotypes 5, 8, and 19 from plastic and metal surfaces in vitro potential role of hands in the spread of respiratory viral infections: studies with human parainfluenza virus 3 and rhinovirus 14 infection control of nosocomial respiratory viral disease in the immunocompetent host an outbreak of human parainfluenza virus 3 infection in an outpatient hematopoietic stem cell transplantation clinic dare and talbot make a very good description of health care acquired viral respiratory infections, their different control measures(education, hand-washing, isolation, ppe, cohorting of patients and personnel) and other measures such as influenza vaccination parainfluenza virus infections in hematopoietic cell transplant recipients and hematologic malignancy patients: a systematic review respiratory virus infection among hematopoietic cell transplant recipients: evidence for asymptomatic parainfluenza virus infection viral respiratory tract infections in transplant patients: epidemiology, recognition and management how contagious are common respiratory tract infections? clinical features and viral diagnosis of two cases of infection with middle east respiratory syndrome coronavirus: a report of nosocomial transmission an outbreak of respiratory syncytial virus in a bone marrow transplant center detection and control of a nosocomial respiratory syncytial virus outbreak in a stem cell transplantation unit: the role of palivizumab respiratory syncytial virus infection in adults immunization of health-care personnel: recommendations of the advisory committee on immunization practices (acip) influenza vaccination of healthcare workers: critical analysis of the evidence for patient benefit underpinning policies of enforcement safety and effectiveness of neuraminidase inhibitors for influenza treatment, prophylaxis, and outbreak control: a systematic review of systematic reviews and/or meta-analyses oseltamivir and inhaled zanamivir as influenza prophylaxis in thai heaith workers: a randomized, double-blind, placebo-controlled safety trial over 16 weeks adenovirus type 8 epidemic keratoconjunctivitis in an eye clinic: risk factors and control adenovirus: current epidemiology and emerging approaches to prevention and treatment emerging viral respiratory tract infections-environmental risk factors and transmission safety and immunogenicity of an intranasal sendai virus-based human parainfluenza virus type 1 vaccine in 3-to 6-year-old children an opportunistic pathogen afforded ample opportunities: middle east respiratory syndrome coronavirus this is also observed in the healthcare setting where respiratory viruses are recognized as a cause of uri and lri. these infections are not only limited to children and may cause severe infections in immunocompromised patients.knowing the mechanisms of respiratory viral transmission in the healthcare setting allows to implement infection control measures to prevent its dissemination and reduce the risk of a hospital outbreak.early detection and strict compliance to infection control measures limit the spread of nosocomial viral respiratory infections. other measures, such as healthcare workers receiving an annual influenza vaccination, should be encouraged. the authors declare that they have no competing interests. this article does not contain any studies with human or animal subjects performed by any of the authors. key: cord-343074-dsubeaso authors: lee, wan‐ji; jung, hee‐dong; cheong, hyang‐min; kim, kisoon title: molecular epidemiology of a post‐influenza pandemic outbreak of acute respiratory infections in korea caused by human adenovirus type 3 date: 2014-06-01 journal: j med virol doi: 10.1002/jmv.23984 sha: doc_id: 343074 cord_uid: dsubeaso an outbreak of upper respiratory tract infections associated with human adenovirus (hadv) occurred on a national scale in korea from september to december 2010, following a major h1n1 influenza pandemic. data from the korea influenza and respiratory surveillance system (kinress) showed an unusually high positive rate accounting for up to 20% of all diagnosed cases. to determine the principal cause of the outbreak, direct polymerase chain reaction (pcr) amplification followed by sequence analysis targeting parts of the hexon gene of hadv was performed. serotypes of 1,007 pcr‐diagnosed hadv‐positive samples from patients with an acute upper respiratory tract illness were determined and epidemiological characteristics including major aged group and clinical symptoms were analyzed. the principal symptom of hadv infections was fever and the vulnerable aged group was 1–5 years old. based on sequence analysis, hadv‐3 was the predominant serotype in the outbreak, with an incidence of 74.3%. from the beginning of 2010 until may, the major serotypes were hadv‐1, 2, and 5 (70–100%) in any given period. however, an outbreak dominated by hadv‐3 started between july and august and peaked in september. phylogenetic analysis revealed that there was no genetic variation in hadv‐3. the results demonstrated that an outbreak of upper respiratory illness followed by h1n1 influenza pandemic in korea was caused mainly by emerged hadv‐3. j. med. virol. 87: 10–17, 2015. © 2014 wiley periodicals, inc. human adenoviruses (hadvs) in the family adenoviridae, genus mastadenovirus are responsible for various illnesses, including respiratory tract infections, conjunctivitis, cystitis, and gastroenteritis [kunz and ottolini, 2010] . typically, hadv-b species (types 3, 7, 12, 14, and 55) , hadv-c (types 1, 2, 5, and 6), and hadv-e (type 4) are closely associated with respiratory tract infections [metzger et al., 2007; kajon et al., 2010; walsh et al., 2010; tang et al., 2013] . identification of serotypes is important because the pathogenesis of hadv infection is determined mainly by serotypes. based on nucleotide and amino acid sequences of the hypervariable region in the hexon gene, phylogenetic analysis has been used for the clustering and typing of hadvs [crawford-miksza and schnurr, 1996a; biere and schweiger, 2010] . accordingly, phylogenetic analysis can facilitate molecular epidemiological investigations of hadv outbreaks. respiratory hadv infections have been closely associated with acute respiratory infections such as febrile illness, pharyngitis, and coryza (common cold). repeated outbreaks of such diseases have been reported worldwide [ryan et al., 2002; kim et al., 2003; chang et al., 2008] . in the same context, respiratory hadv infections can be identified from patients with an upper respiratory tract infection throughout the entire year, with an average detection rate of 4% in korea [lee et al., 2010] . however, in 2010-after a major h1n1 influenza pandemic in 2009-an outbreak of hadv infections occurred in korea. this study aimed to identify the major type of hadv involved through direct pcr amplification followed by hexon gene sequence analysis. the public health impact based on clinical features of the outbreak is also emphasized. this is believed to be the first report of a nationwide outbreak of respiratory adenovirus infections in korea. in 2010, 13,502 throat swabs from patients with an acute respiratory infections exhibiting a fever (>38˚c), coughing, sore throat, and runny nose were collected and used to detect viral causative agents through the korea influenza and respiratory viruses surveillance system (kinress), which obtains routinely clinical specimens from outpatients with acute respiratory infections who seek primary clinical care from about 100 hospitals nationwide in korea. specimens were placed in virus transport medium (bd, sparks, md) and sent to 17 local institutes of health and environmental research maintained at 4˚c to be tested for major respiratory viruses including hadvs. the remained samples were stored at à70˚c until use. because an abnormally high positive rate of hadv was observed nationwide from july 2010 until the end of the year, all positive cases (n ¼ 1,007) of hadvs were selected and then subjected to differentiate serotypes retrospectively. to compare any difference in hadv prevalence between the pre-and postpandemic phases, 91 samples of nasal aspirates taken in 2008 before the h1n1 influenza pandemic were collected through the acute respiratory infection network. the study was approved by the ethics committee of the korean national institute of health (approval # 2008; 2012 -09con-03-4c, 2010 ; 2010-03exp-1-r). characteristics of the clinical symptoms associated with hadv infections as well as records of all enrolled patients with acute respiratory infections were analyzed from the clinical records compiled via acute respiratory infections network for 2008 and kinress for 2010. logistic regression analysis with odds ratio values were performed to test the relationships between hadv infection and clinical symptoms using sas software (version 9.2; sas institute, cary, nc). total viral dna was extracted from 140 ml aliquots of specimens using quickgene-810 (fujifilm, tokyo, japan) following the manufacturer's protocol. to amplify part of the hexon gene, primer pairs av3r (5 0 -atgtggaaicaggcigtigacag-3 0 ) and av5l (5 0 -cggtggtgittiaaiggittiacittgtccat-3 0 ) [craford-miksza et al., 1999] were used to produce a 458 base pair (bp) fragment (position 19,552-20,009; accession # dq086466). the pcr was performed using sp-taq polymerase (cosmogenetech, seoul, korea) to maximize fidelity of dna amplification. the amplification products were analyzed by electrophoresis in 2% agarose gels (gendepot, barker, tx) and they were visualized using sybr safe dna gel stain (invitrogen, carlsbad, ca) under ultraviolet light. the pcr products were then purified using a qiaquick spin column purification system (qiagen, hilden, germany) to remove any trace of primers and analyzed directly on an abi3730 sequencer (applied biosystems, foster city, ca). partial hexon gene sequences were analyzed using dnastar 8.0 (lasergene, madison, wi) and aligned against other available hadv sequences using clus-talw. to type hadv, the nucleotide sequence homology was inferred from the identity scores obtained using the blastn program (national center for biotechnology information, bethesda, md). mega4 was used to create phylogenetic trees through the neighbor-joining method based on the kimura 2parameter distance matrix listed in the software, and bootstrap values were obtained from a random sampling of 1,000 replicates [tamura et al., 2007] . reference hadv sequences were obtained from genbank (http://www.ncbi.nlm.nih.gov/genbank/) and used for phylogenetic analysis. the nucleotide sequences of the partial hexon genes for strains identified in this study have been deposited in the genbank nucleotide sequence database under serial accession numbers from kc747631 to kc747649. reference sequences with accession number used in this study were given in supplementary table si . from the beginning of 2010 to the end of the year, an outbreak of respiratory infections associated with hadv in korea was examined retrospectively because of the significantly high positive rate of hadv found in samples. the monthly positive rates of hadv from throat swabs at the 17 local institutes of health and environmental research units during the period are shown in figure 1 and compared with those taken in 2008 as a baseline. the median positive rate of hadv in patients with an acute respiratory infection in the baseline period (previous 3-year average) was 2.0% (range 1.0-5.0%); however, during the outbreak period from june to december 2010, the rate increased significantly to 16.7% (range 3.8-31.3%; p < 0.001; fig. 2 ). the majority of these hadv positive cases (up to 77.9%) were identified from children (1-5 years old aged group; fig. 3 ) and the highest positive rate was observed at 2 years old aged group (24.2% in 1-5 years old aged group) in 2010. no gender bias was identified in a given aged group. human respiratory syncytial viruses (hrsvs), human rhinoviruses (hrvs), human metapneumovirus (hmpv), human para-influenzaviruses (hpivs), human coronaviruses (hcovs), human bocaviruses (hbovs), and influenza viruses were also detected through the kinress databank. of these, hrvs was the leading co-infecting agent among the cases of hadv infection with a positive rate of 25.3% in 2008 and 6.6% in 2010. other respiratory viruses were also identified as coor multi-infecting agents with positive rates ranging from 1.1% to 2.2%. the serotype of each adenovirus was determined by sequencing analysis followed by direct pcr of a part of the hexon gene. from the beginning of 2010 until may, the major serotypes were types 1, 2, and 5 (70-100%) in any given period. however, an outbreak dominated by type 3 started between july and august and peaked in september. based on the sequence analysis, hadv-3 was the predominant serotype, in this phase of the outbreak, at 74.3%. besides hadv-3, 12 more serotypes were also identified: hadv-2 (10.1%), hadv-1 (8.0%), hadv-5 (3.2%), hadv-4 (2.3%), hadv-6 (0.8%), hadv-8 (0.6%), hadv-7 (0.2%), hadv-11 (0.1%), hadv-19 (0.1%), hadv-34 (0.1%), hadv-41 (0.1%), and hadv-55 (0.1%). by contrast, only six serotypes (hadv-1, hadv-2, hadv-3, hadv-4, hadv-5, and hadv-6) were identified in samples from 2008. hadv-3 was also the major serotype at that time at 40.7%. however, hadv-2 (25.3%) and hadv-1 (23.1%) comprised larger proportions in 2008 than in 2010 (fig. 4) . interestingly, hadv-41, which is known to be a gastroenteritic cause of hadv infection, was also confirmed from a patient with acute respiratory infections. the clinical features of overall adenovirus infections are summarized in table i . most of the patients had a fever, coughing, runny nose, nasal congestion, and/or sore throat. over 80% of patients had a febrile illness and approximately half of them had a cough and a runny nose. symptomatic comparison of patients who were infected with hadv-3 in the baseline period (2008), there was no significantly elevated proportion of patients with fever and severe infections in the outbreak period (83.8% vs. 89.8%, p ¼ 0.24). instead, coughing, a runny nose, and nasal congestion were significantly lower in 2010 (p < 0.001; fig. 5 ). the partial hexon gene sequences of 1,007 pcrpositive cases of hadv from patients with acute respiratory infections were determined. in terms of species a through g, hadv sequences from the outbreak period did not form a cluster differing from reference sequences. sequence alignment was also performed to determine the sequence similarities for the leading serotype, hadv-3. all the hadv-3 strains isolated in the outbreak period showed high sequence identity (98.15-100%) compared with those in most other years (fig. 6 ). one isolate of the gastroenteritis strain hadv-41 also showed no significant variation from formerly reported sequence information. there were no sequence differences between the hadvs that were isolated in 2008 and 2010 for all types (hadv-1, hadv-2, hadv-3, hadv-4, hadv-5, and hadv-6). hadv infection is one of the leading causes of respiratory illness with typical clinical features, seen in community-based surveillance [cao et al., 2014] . to date, over 57 serotypes of hadv have been identified, and different serotypes have been correlated with different clinical manifestations [chu and pavan-langston, 1979; wood, 1988; gonçalves and de vries, 2006 ]. frequent outbreaks of acute respiratory infections caused by hadv have been described worldwide. the most frequent serotypes associated with respiratory outbreaks in various countries listed below have been mainly classified into subspecies b (hadv-11: china, hadv-7: korea, hadv-3: taiwan), c (hadv-1 and 2: malaysia), and e (hadv-4: usa) chang et al., 2008; yang et al., 2009; abd-jamil et al., 2010; lee et al., 2010] . symptoms caused by hadv infection range from mild symptoms to severe pneumonia. furthermore, respiratory illness associated with hadv can be confused with an influenza-like illness (ili) including fever, coughing, sore throat, and muscle pain [tohma et al., 2012] . to avoid the misdiagnosis of hadv infection from patients with an ili, a molecular biology technique such as direct pcr is used for discriminating possible causative viral agents. in korea, the laboratory surveillance system named kinress has been targeting outpatients with acute respiratory infections since 2005. annually, in 1-5% of patients with acute respiratory illness the cause is infection with hadv and this proportion is consistent with previous reports [lee et al., 2010a,b] . in the first post-influenza pandemic period, an abrupt outbreak of hadv was identified starting in june 2010. the median positive rate of adenovirus infections in the baseline period (previous 3-year average) was 2.0%. however, during the hadv outbreak-from june to december 2010-the mean positive rate increased significantly to 16.7%, ranging from 3.8% to 31.3%. a total of 1,007 hadv-positive cases were diagnosed from 13,502 clinical specimens (7.5%) in 2010. among these, hadv-3 (74.3% of positive cases) was identified as the major causative agent responsible for this outbreak of respiratory illness. interestingly, hadv-41 belonging to subspecies f was isolated from a patient with respiratory illness without diarrhea. because the hadv-41 is known to be a causative agent for viral gastroenteritis [uhnoo et al., 1984] rather than respiratory illness, it remains to be clarified whether this finding solely caused by pathological changes of the virus or not. phylogenetic analysis revealed that the outbreak caused by hadv type 3 during 2010 in korea seemed not to be related to any specific changes in viral genotypes. instead, in 2010 this strain played a unique role as a causative agent of respiratory illness. similar to other reports regarding hadv infection in patients with respiratory illness, the most affected age group was children aged 1-5 years old (77.9%). although 1-5 years old aged group took a higher proportion than other aged groups, the positive rate of hadv-3 in each age group also higher than other aged groups. these result was consistent with previous report that the positive rate of hadv was the most higher among children less than 5 years of age [cooper et al., 2000; chang et al., 2008; cheng et al., 2008] . to confirm hadv-3 as the sole causative agent for this outbreak, multiple co-infections by other respiratory viruses together with hadvs were analyzed. dual or more multiple infections with hrsvs, hrvs, hmpv, hpivs, hcovs, hbovs, and influenza viruses were studied. the results showed that hrvs were the leading cause of co-infection with hadv in 2008 (25.3%) and 2010 (6.6%). the incidence of this single infection rate was significantly higher in 2010 (86.4%) than in 2008 (70.3%) (p < 0.001). thus, it is highly feasible that hadv-3 infection was the sole cause of the outbreak in 2010. epidemiological and molecular data presented in this study confirmed that the outbreak in 2010 was not associated with genetic alterations causing a change in the pathology of the major causative agent, hadv-3, nor with multiple infections with other respiratory viruses. because the present study focused on outpatients with acute respiratory infections, severe manifestations such as bronchitis, pneumonia, and bronchiolitis were not scored in this study. nevertheless, several symptoms associated with hadv-3 infection such as nasal congestion, runny nose, and coughing were fewer in 2010 when compared with 2008 (p < 0.001). because little is known about the association between disease severity and the virulence-determining factors of hadv, it is unclear whether these reduced clinical symptoms arose from a particular change in virulence. in recent years, pcr and sequence analyses targeting the hadv hexon gene encoding a serotype-specific epitope have enabled rapid identification and classification of the genotypes and serotypes of hadvs [craford-miksza et al., 1999] . the strategy of serotype analysis used in this study was also based on a hexon gene sequence and allowed basic molecular biological information on circulating hadvs to be obtained. even though hadvs have dna as their genetic material, recombination is very frequently reported for this virus, which could be important for hadv evolution resulting in shifts in its pathology and virulence [craford-miksza and schnurr, 1996b; walsh et al., 2009; rebelo-de-andrade et al., 2010] . one recent report indicated that recombinant hadvs introduced into the community could produce highly virulent strains with epidemic and lethal potential [halstead et al., 2010] . therefore, there might be a limit to explaining the relationship between clinical manifestations and hadv infections through pcrbased molecular typing targeting just one gene. it would be better to use two or more genomic point comparisons for serotype determination to obtain better evidence for the pathogenicity and disease severity of particular serotypes of hadvs. the unusually high incidence of hadv-3 in 2010 following a pandemic of influenza (h1n1) in 2009 might have had several causes. one possibility is there was a micro-environmental change in hosts following the pandemic era. as presented in this study, the outbreak caused by hadv-3 following the pandemic era seemed not to be associated with specific changes in the viral hexon gene, which is known to encode major antigenic sites of hadv. instead, extensive changes in the overall host population's immune status resulting from the primary introduction of h1n1 influenza into the community could have mediated this unusually high incidence of hadv. there was no significant changes were observed in the hadv genome supports this hypothesis. the other possibility is that the original introduction of hadv could lead to fluctuations in population immunity triennially, regardless of the influence of the influenza pandemic. during the preparation of this manuscript, repetitive hadv outbreaks caused by hadv-3 were also detected in 2013 (data not shown). again, no significant mutations were observed using hexon sequence analysis targeting randomly sampled hadv-3 viruses. this repetitive outbreak caused by the hadv-3 suggests that when hadv-3 was the major circulating virus, population immunity might have been diluted. thus, newborn infants might be a newly susceptible group to hadv-3 and this might have contributed to the triennial outbreak of hadv-3 infection. to validate this hypothesis, a birth cohort-derived seroepidemiological study targeting specific antibodies against hadvs should be carried out to test the present data further. there are published data about the prevalence and distribution of hadv serotypes in korea [kim et al., 2003; lee et al., 2010a] , but those descriptions are limited by being hospital based and not representative of the general korean population. this study is believed to be the first nationwide report regarding an outbreak of hadv infection based on the kinress data. in conclusion, through a nationwide surveillance system during 2010 in korea, this study has documented that in an outbreak of hadv infection following a pandemic era, hadv-3 was the predominant serotype based on the hexon gene. further investigations to verify which factor(s) were associated with this abrupt outbreak following the h1n1 influenza virus pandemic in 2010 are needed to understand the major causes. molecular identification of adenovirus causing respiratory tract infection in pediatric patients at the university of malaya medical center human adenoviruses in respiratory infections: sequencing of the hexon hypervariable region reveals high sequence variability emergence of community-acquired adenovirus type 55 as a cause of community-onset pneumonia a community-derived outbreak of adenovirus type 3 in children in taiwan between molecular and clinical characteristics of adenoviral infections in taiwanese children in 2004-2005 ocular surface manifestations of the major viruses the epidemiology of adenovirus infections in greater manchester, uk 1982-96 adenovirus serotype evolution is driven by illegitimate recombination in the hypervariable regions of the hexon protein strain variation in adenovirus serotypes 4 and 7a causing acute respiratory disease analysis of 15 adenovirus hexon proteins reveals the location and structure of seven hypervariable regions containing serotype-specific residues adenovirus: from foe to friend recombinant adenovirus type 3 and type 14 isolated from a fatal case of pneumonia molecular epidemiology of adenovirus type 4 infections in us military recruits in the postvaccination era molecular epidemiology and brief history of emerging adenovirus 140associated respiratory disease in the united states genome type analysis of adenovirus types and 7 isolated during successive outbreaks of lower respiratory tract infections in children the role of adenovirus in respiratory tract infection molecular classification of human adenovirus type 7 isolated from acute respiratory disease outbreak (ard) in korea comprehensive serotyping and epidemiology of human adenovirus isolated from the respiratory tract of korean children over 17 consecutive years (1991-2007) clinical severity of respiratory adenoviral infection by serotypes in korean children over 17 consecutive years abrupt emergence of diverse species b adenoviruses at us military recruit training centers outbreak of acute respiratory infection among infants in large epidemic of respiratory illness due to adenovirus types 7 and 3 in healthy young adults meg a4: molecular evolutionary genetics analysis (mega) software version 4.0 genome and bioinformatic analysis of a hadv-b14p1 virus isolated from a baby with pneumonia in beijing detection and serotyping of human adenoviruses from patients with influenza-like illness in mongolia importance of enteric adenovirus 40 and 41 in acute gastroenteritis in infants and young children evidence of molecular evolution driven by recombination events influencing tropism in a novel human adenovirus that causes epidemic keratoconjunctivitis computational analysis identifies human adenovirus type 55 as a re-emergent acute respiratory disease pathogen adenovirus gastroenteritis genomic analyses of recombinant adenovirus type 11a in china outbreak of acute respiratory disease in china caused by b2 species of adenovirus type 11 we thank 17 local institutes of health and environmental research for supporting kinress. we thank you-jin kim for help in preparation of this paper. additional supporting information may be found in the online version of this article at the publisher's web-site. key: cord-345211-4ivqlsgt authors: murdoch, david r. title: how recent advances in molecular tests could impact the diagnosis of pneumonia date: 2016-03-07 journal: expert rev mol diagn doi: 10.1586/14737159.2016.1156536 sha: doc_id: 345211 cord_uid: 4ivqlsgt molecular diagnostic tests have been the single major development in pneumonia diagnostics over recent years. nucleic acid detection tests (nats) have greatly improved the ability to detect respiratory viruses and bacterial pathogens that do not normally colonize the respiratory tract. in contrast, nats do not yet have an established role for diagnosing pneumonia caused by bacteria that commonly colonize the nasopharynx due to difficulties discriminating between pathogens and coincidental carriage strains. new approaches are needed to distinguish infection from colonization, such as through use of quantitative methods and identification of discriminating cut-off levels. the recent realization that the lung microbiome exists has provided new insights into the pathogenesis of pneumonia involving the interaction between multiple microorganisms. new developments in molecular diagnostics must account for this new paradigm. pneumonia continues to provide a huge global burden of disease [1] . although the incidence of pneumonia increases with increasing age, it is not widely appreciated that pneumonia is also the world's biggest killer of young children [2] . a wide variety of microorganisms are listed as pneumonia pathogens [3] , and identification of pneumonia etiology is useful for both patient management and surveillance purposes. however, despite increased recognition of the burden of disease and advances in effective vaccines against major pneumonia pathogens, we continue to struggle in our efforts to identify the pathogens that cause pneumonia in individual patients [4] . indeed, historically, we have been unable to define a causative pathogen in a significant proportion of pneumonia episodes, even with the best methods. determining the microbial etiology of pneumonia in children has been a particular challenge [5] . the implications of poor pneumonia diagnostics extend to the population level as well; assessment of interventions, such as vaccines, is hindered by suboptimal measures of disease impact that often rely on accurate surveillance data [6] . why is it so difficult to determine the microbial etiology of pneumonia? the inability to obtain good quality specimens from the lower respiratory tract is one fundamental problem with pneumonia diagnostics. whereas, it is relatively easy to get specimens from the site of infection with diseases such as meningitis, gastroenteritis, and endocarditis, obtaining representative and uncontaminated specimens from the lungs in pneumonia is a challenge. sputum and bronchoscopic specimens may be contaminated by normal respiratory flora, and transthoracic lung aspirates are rarely performed despite a good safety profile [7] . instead, we are currently reliant on testing more distant clinical specimens, particularly from the upper respiratory tract, blood, or urine. in general, testing these specimens has suboptimal sensitivity and/or specificity for determining the microbial etiology of pneumonia with confidence. a second important issue hindering our ability to determine pneumonia etiology is the fact that some major pneumonia pathogens, such as streptococcus pneumoniae, haemophilus influenzae, and staphylococcus aureus, may also asymptomatically colonize the upper respiratory tract as part of normal oropharyngeal flora. consequently, the detection of these microorganisms is insufficient by itself in order to attribute pneumonia causation. this perspective reviews the current use of molecular diagnostics for determining the microbial etiology of pneumonia and future prospects for this purpose. the focus is on recent advances and their clinical applications rather than detailed description of specific technologies. presently, we are still reliant on traditional diagnostic tools that have been used for decades to determine the microbial etiology of pneumonia. current guidelines for the management of community-acquired pneumonia in adults typically recommend that microbiologic testing should be largely restricted to patients with more severe disease, and give guidance about the judicious use of blood cultures, sputum microscopy and culture, urinary antigen tests, and serology [8] [9] [10] . guidelines for the management of community-acquired pneumonia in children are even more restrictive, again recommending that tests should mainly be used on patients with severe disease, with a focus on blood cultures and detection of respiratory viruses [11, 12] . common to these guidelines is a cautious approach to the use of molecular diagnostics, although this may change with ongoing reviews. this caution is driven partly by the perceived and real lack of commercial and standardized assays and partly by lack of good data on diagnostic accuracy. there is also a lingering perception that molecular diagnostics are expensive tests and that use should be restricted on this basis. cost is not the barrier it used to be, and the relative price of molecular diagnostics has actually fallen over recent years. indeed, many molecular assays are now comparable in cost to conventional culture-based methods. molecular methods have been a particularly welcome addition to the pneumonia diagnostic toolbox. collectively, they represent the single biggest recent advance in the field. while not exactly new (polymerase chain reaction (pcr) assays for respiratory pathogens have been around for over 20 years), the widespread adoption of nucleic acid detection tests (nats) by diagnostic laboratories has been relatively slow. this contrasts to nats for other infectious diseases, which have been more quickly embraced by laboratories and clinicians [13] . a major reason for the slow uptake and acceptance of respiratory nats has been the lack of commercial assays. this situation is changing rapidly, undoubtedly spurred on by recent outbreaks of global concern, such as pandemic influenza, middle east respiratory syndrome (mers), and ebola [14] [15] [16] . indeed, the increased availability of commercial assays is the major change in the area since i first started reviewing the role of molecular diagnostics in pneumonia over 10 years ago [17, 18] . to date, molecular tests for pneumonia have mainly focused on detection of specific known pathogens by nats. nats have several advantages over other existing diagnostic tools. they potentially detect low levels of all-known pneumonia pathogens in clinical specimens, do not depend on the viability of the target microbe, and can provide results within a clinically relevant time frame. they are also probably less affected by previous antimicrobial therapy than are culturebased diagnostic methods. there are now a wide variety of user-friendly platforms that have enabled nats to be deployed in laboratories outside of specialist tertiary referral centers. although nats have mainly focused on detecting the presence of a particular microorganism, they may also provide additional information, such as data on antimicrobial resistance and strain typing. the nats that are most widely used in diagnostic laboratories are those that detect potential pneumonia pathogens that are not part of the normal flora, namely respiratory viruses and selected non-colonizing bacteria. for these microbes, simply detecting their presence in a respiratory sample has been regarded as sufficient evidence to assign causation. in contrast, nats for other bacteria, including some of the most important pneumonia pathogens, have struggled for a defined role outside research laboratories. nats for detection of the following respiratory pathogens now have established roles. nats have revolutionized the diagnosis of viral respiratory tract infections [19, 20] . with high sensitivity and specificity, rapid turnaround time, and availability as commercial assays, nats are now the testing method of choice for respiratory viruses. respiratory viruses commonly detected by nats, often in large multiplex panels, include influenza a and b viruses, respiratory syncytial virus, parainfluenza viruses, human metapneumovirus, human rhinoviruses, enteroviruses, adenoviruses, human bocavirus, and several coronaviruses (oc43, 229e, nl63, and hku1). nats are also established diagnostic tools for detection of severe acute respiratory syndromecoronavirus and mers-coronavirus [16, 20] . in the context of pneumonia, the detection of a respiratory virus in an upper respiratory specimen by a nat has been regarded as sufficient to assign causation in both children and adults [21] . however, this assumption is not always reliable. there is still debate about the exact role (if any) of some viruses in the pathogenesis of pneumonia, including human rhinoviruses and human bocavirus [22] . this has led some to question the wisdom of using large multiplex nat panels as first-line tests for respiratory pathogens given potential problems with interpretation of positive results [23] . in addition, when control groups are used in pneumonia etiology studies, respiratory viruses are often detected in a similar proportion of both subjects with and without pneumonia, especially in children [24, 25] . these findings vary by specific virus, with influenza a and b viruses, respiratory syncytial virus, and human metapneumovirus being typically detected in a significantly higher proportion of cases with pneumonia than controls. environmental bacteria of the genus legionella are the cause of legionnaires' disease, a pneumonia that requires specific antimicrobial treatment and is often associated with outbreaks [26] . of the many legionella species that can cause human infection, legionella pneumophila is the most common cause globally, with infection usually acquired from water sources. other species predominate in some geographic locations, particularly legionella longbeachae which is an inhabitant of soil and compost. there is no human-to-human spread of legionnaires' disease, and human infection follows environmental exposure to the causative microorganism. legionella spp. are fastidious organisms and the traditional reliance on culture, serology, and urinary antigen tests has led to underdiagnosis of legionnaires' disease globally [27] . indeed, the almost sole reliance on the urinary antigen test, which can only detect l. pneumophila serogroup 1, in some parts of the world has created a 'blind spot' for legionnaires' disease caused by other species and serogroups [27] . nats have long been used to detect legionella infection [27] and are well-suited for this purpose. legionella are not regarded as human colonizers [28] , and the detection of any amount of legionellae in a clinical specimen is regarded as diagnostic for infection, assuming contamination has not occurred during the testing process. furthermore, all species and serogroups can be detected. legionella dna has been detected in both upper and lower respiratory samples, urine, and blood from patients with legionnaires' disease [29] [30] [31] [32] [33] [34] , although sputum and other lower respiratory samples are regarded as the specimens of choice [31] . recently, the systematic use of pcr [31] and collection of induced sputum from patients unable to expectorate [35] have uncovered a hidden burden of legionnaires' disease and have demonstrated the diagnostic utility of nats for this disease. arguably, nats are now the test of choice for legionnaires' disease. being an environmental organism, there has been concern about legionella contamination during testing, highlighted by the occasional documented contamination of nucleic acid extraction kits [36] . this problem can be overcome by strict adherence to good laboratory practice. mycoplasma pneumoniae is associated with a variety of both upper and lower respiratory tract infections and is an important cause of pneumonia, frequently occurring in outbreaks. nats are now widely regarded as the methods of choice for detection of m. pneumoniae infections [37, 38] , and a m. pneumoniae target has been incorporated alongside viral targets in many large respiratory multiplex panels. in practice, nats have also been useful for m. pneumoniae outbreak identification and management [39] . both upper and lower respiratory tract specimens are suitable for testing by nats, and a positive result in the context of pneumonia is regarded as being diagnostic, as asymptomatic carriage of m. pneumoniae is uncommon [40] . chlamydophila pneumoniae is a relatively common cause of community-acquired pneumonia in some geographic regions, but is not typically associated with severe disease [41, 42] . nats have long been used for diagnostic purposes and, indeed, c. pneumoniae nats were the focus of some of the earliest efforts to standardize nats for respiratory pathogens [43] . perhaps because of the association with milder disease, c. pneumoniae targets have not been a priority for incorporation into multiplex respiratory panels. colonization with pneumocystis jirovecii is common among the general population and is associated with pneumonia in immunocompromised individuals. nats have advantages over traditional microscopy-based diagnostic tools, detecting p. jirovecii in induced sputum, bronchoscopic, or oropharyngeal specimens with high sensitivity [44] . indeed, it was pcrbased methods that provided some of the early evidence of the existence of p. jirovecii colonization [45] . the use of quantitative methods has been necessary in order to discriminate between colonization and disease; thus, at least partially overcoming the problem of false-positive results. although promising, the cut-off values remain to be standardized for quantitative nats and may vary with patient population [46] . microscopy and culture of lower respiratory samples remain the standard diagnostic tools for tuberculosis. despite improvements in culture-based methods, the slow turnaround time for laboratory diagnosis and global concern about disease burden and multidrug resistance has provided the impetus for the development of rapid testing methods [47] . considerable effort has been put into the development of nats for mycobacterium tuberculosis, and several commercial assays are now widely available [48] . interestingly, tuberculosis is unusual among infectious diseases in that nats are less sensitive than culture, a possible consequence of difficulty with dna extraction. sensitivities are typically 92-100% for smear-positive specimens and 40-93% for smear-negative specimens [48] . importantly, small, user-friendly platforms (such as the xpert mtb/rif assay) have been developed to an extent that they have been successfully deployed in many resource-poor locations where rapid diagnostics for tuberculosis are most needed [47, 49] . this is a real success story and highlights what can be achieved with molecular diagnostics outside major laboratories. nats are now established tests for the detection of the causative agents of pertussis [50] [51] [52] . while bordetella pertussis is the major cause of pertussis in humans and can be complicated by pneumonia [53, 54] , bordetella bronchiseptica, bordetella holmesii, and bordetella parapertussis have all been occasionally associated with (often milder) pertussis-like illnesses [50] . nats are the most sensitive methods for the detection of b. pertussis and, with rapid turnaround times, have been invaluable tools in outbreak management [55, 56] . the main limitation of b. pertussis nats is specificity, particularly the ability of the most widely used (is481-based) assays to also detect b. holmesii, which is generally considered a false positive result [50] . the use of dual target assays has been advocated to overcome this potential problem [52] . what are the limitations of molecular diagnostic tests for pneumonia and what needs to be addressed in order to progress with development? common to all pneumonia diagnostic testing, the inability to obtain good quality specimens from the lower respiratory tract is a major problem that will only be overcome through new innovative and safe methods of specimen collection and a greater understanding of the relationships between changes in the lung and in more distant specimens in pneumonia. a second-major limitation of nats is their inability to distinguish pathogens from innocent bystanders. in essence, we need two pieces of information. first, is a particular microorganism present in the clinical specimen? second, if the microorganism is present, is it causing this episode of pneumonia? to date, diagnostic efforts have focused almost exclusively on the first question and have ignored the second, which is usually much more difficult to answer unless the microorganism is never present as a colonizer (e.g. legionella spp.). the efforts to use nats as a diagnostic for pneumococcal pneumonia illustrate some of the key issues. s. pneumoniae is regarded as the most common cause of pneumonia in all age groups and yet, molecular tests do not have an established diagnostic role [57] . s. pneumoniae is also a common nasopharyngeal colonizer, with carriage prevalence exceeding 50% among children in some regions of the world [58] . earlier pneumococcal pcr assays, such as those targeting the pneumolysin gene, had problems of poor specificity due to detection of other, closely related streptococci [59] , although false-positive results are less likely with some of the currently more widely used targets (e.g. autolysin gene) [60] . s. pneumoniae is frequently detected by nats in both upper and lower respiratory specimens from patients with pneumonia [59] , but the clinical implications of a positive result are uncertain given the relative high prevalence of pneumococcal carriage. testing of blood for s. pneumoniae by pcr has been promoted as an alternative approach that can potentially avoid issues of contamination. among italian children, blood pcr showed promise as a diagnostic for invasive pneumococcal disease [61] [62] [63] [64] with high specificity [65] . however, in other populations, positive results have been reported in control participants who do not have suspected pneumococcal disease [66] , raising concerns about the broader utility of this approach. in particular, false-positive results are relatively common in children from developing countries where pneumococcal carriage is common [67] . consequently, a positive pneumococcal nat result alone is usually not enough to diagnose pneumococcal pneumonia. the possible exception is the use of nats to detect s. pneumoniae in pleural fluid [68, 69] . another important limitation with current nats is their focus, by nature, on specific known or suspected pneumonia pathogens. implicit with this focus is the assumption that we know the full range of key potential pathogens that cause pneumonia. this may well be correct, but there is increasing interest in the use of modern techniques, such as next generation sequencing, which provide the opportunity for discovering new or unexpected pathogens [70, 71] . the broader approach of these technologies may provide novel insights into pneumonia etiology, but have yet to show added advantage as a routine diagnostic tool [72] . no major new pneumonia pathogens have been uncovered over recent years. a key reason for the slow adoption of respiratory nats by diagnostic laboratories is the lack of commercial assays that have been approved by regulatory bodies. the impact has probably been felt more in the usa given the strict requirements of the us fda. the level and importance of regulation have been debated [73, 74] , and it is clear that a balance is needed between appropriate regulation of new diagnostics, while avoiding unnecessary barriers to the deployment of useful tests. the use of molecular tests for legionnaires' disease is a good example of how assay regulation has had an impact on diagnostic strategies. despite nats being the diagnostic test of choice for legionnaires' disease, the shortage of fda-approved assays has severely limited the use of these tests in the usa, resulting in the continued use of suboptimal diagnostics and underdiagnosis of this disease [27] . moreover, the absence of a prominent role in diagnostic algorithms has possibly led to an incorrect perception that the performance of nats is inadequate. hopefully, increased experience with these tests will reverse this perception. a major recent revelation in respiratory medicine has been recognition of the lung microbiome [75] . until recently, the lungs in health were regarded as sterile. the use of modern culture-independent techniques has not supported this concept, consistently finding evidence of bacteria in the lower airways [75] . this important realization has challenged our traditional paradigm of pneumonia pathogenesis. the tradition view that pneumonia is caused by a single invasive pathogen in a normally sterile site is likely wrong. increasing recognition that bacteria and viruses frequently interact in the causative pathway to pneumonia [76, 77] adds additional complexity, as does the frequent finding of polymicrobial infections [78] . under the new paradigm, dominant species emerge from the lung ecosystem in pneumonia through uncertain mechanisms, and the bacterial versus viral pneumonia concept is too simplistic. consequently, we probably need to use more sophisticated approaches to pneumonia diagnosis than assays that simply target single specific putative pathogens. we have a lot to learn and are only just beginning to understand changes in the microbiome during acute infections [75, 79, 80] . analysis of the lung microbiome may provide insights into pneumonia etiology and reveal novel markers for pneumonia prognosis and for treatment guidance [81] . molecular diagnostic techniques clearly have a central role in these metagenomic analyses [82, 83] , providing another opportunity for next generation sequencing technology. any future developments in pneumonia diagnostics must be cognizant of new knowledge about the lung microbiome and about changes in the lungs during the pathway to pneumonia. there is likely to be less focus on just the detection of specific known pathogens, with more interest in the search for markers of change in the lung microbial ecology in the diseased state. the potential application of metagenomics in the diagnostic laboratory is still uncertain and will dependent on the emergence of improved sequencing technology and bioinformatics software. whole genome sequencing of bacterial isolates is already being increasingly used for strain characterization and epidemiological analyses [84] . for detection of specific pathogens, existing molecular tests can detect very low microbial loads with high analytical specificity. it is unlikely that further test developments will lead to significantly improved performance than we have now. indeed, the interpretation of low-level positive results from nats already provides a dilemma for diagnostic laboratories. instead, the focus of developers should be on making test platforms that are more user-friendly and that allow shorter turnaround times. we also need to place greater attention on the meaningful interpretation of positive results in order to determine which microorganisms are actually causing individual episodes of pneumonia. one approach to help distinguish infection from contamination or colonization is to quantify microbial load by molecular methods. the situation is analogous to culturebased methods, whereby organisms isolated in greater quantities from certain cultures are regarded as more likely to be clinically significant. this approach depends on the determination of cut-off microbial load levels that will provide sufficient diagnostic accuracy to distinguish infection from colonization/ contamination. however, given the lack of suitable comparator 'gold' standards, these cut-off values are very difficult to identify in a scientific manner. they may also vary for different microorganisms. quantitative multiplex pcr has been used to determine the etiology of community-acquired pneumonia in adults using cutoffs developed for interpretation of culture results from lower respiratory tract specimens [85, 86] . not surprisingly, the addition of pcr targets for common bacterial pneumonia pathogens, such as s. pneumoniae, resulted in an etiological diagnosis being made in a high proportion of cases. however, these cutoffs have been determined by expert opinion and, although generally intuitive, might be quite misleading. despite the challenges in doing so, we need more objective assessment of cut-off values using good scientific and epidemiologic principles. quantitative approaches have also been applied to nasopharyngeal specimens. among hiv-infected adults in south africa, quantitative pcr testing of nasopharyngeal samples distinguished between pneumococcal pneumonia and asymptomatic pneumococcal colonization with reasonable diagnostic accuracy [87, 88] . data from other populations are needed in order to assess whether this method can be used in clinical practice. microbial load in pneumonia can also be a prognostic marker. for example, pneumococcal density in the nasopharynx [89] and blood [90, 91] are associated with disease severity in adults. we can probably also be more innovative with statistical methods to help interpret molecular test results. statistical modeling techniques can be used to help overcome the limitations of diagnostic testing for pneumonia, particularly the lack of good comparator standards. latent class analysis has been used to determine the prevalence of pneumonia caused by specific pathogens in epidemiologic studies [92, 93] and for the assessment of diagnostic tests in tuberculosis [94, 95] . possibly the most sophisticated use of statistical modeling techniques in the context of pneumonia etiology is currently being undertaken as part of the pneumonia etiology research for child health (perch) study [96] . perch is a large 7-country case-control study focused on the causes of severe pneumonia in young children from developing countries, the findings of which will be published in 2016. in this study, partially latent class models have been designed for estimating the population etiology distribution and the individual etiology probabilities for specific pneumonia pathogens, with a major focus on molecular diagnostic test results [97] . the findings of perch are awaited with great anticipation, as this approach has never before been so extensively applied to the etiology of an infectious disease. we have entered a new age in pneumonia diagnostics that needs to look beyond the targeting of a limited number of potential pathogens. new knowledge about the lung microbiome and pneumonia pathogenesis, together with emerging developments in sequencing technology, provide opportunities for novel diagnostic tools to help better guide the management of pneumonia. molecular tests are now mainstream diagnostics for many respiratory infections, although we still have much to learn about using them more effectively to assist with the management of pneumonia. those involved in molecular diagnostic test development for pneumonia need to look beyond the simple detection of specific known pathogens and also provide means to accurately interpret the clinical significance of a positive result. it is not good enough to simply assess a new test by measuring analytical sensitivity and making comparisons to the performance of other similar assays. diagnostic tests must be evaluated properly following good epidemiological principles. new developments in molecular diagnostics must also be cognizant of emerging information about the lung microbiome, which will likely provide opportunities for the discovery of novel markers to help guide pneumonia management. the recent expansion in the number and variety of commercial nats is likely to continue, leading to greater uptake by diagnostic laboratories and greater incorporation into diagnostic test algorithms and guidelines for the management of pneumonia. the provision of even more user-friendly platforms will enable deployment of these tests in laboratories that have not traditionally used molecular diagnostics. further efforts are needed to evaluate the clinical application of microbial load measurement for the purpose of distinguishing pathogens from colonizing microorganisms and as a prognostic marker. it is still unclear about how new knowledge about the lung microbiome will affect the development of pneumonia diagnostics, but the impact may be profound. while it will be some time before next generation sequencing becomes a standard tool in diagnostic laboratories, this technology may provide valuable insights into the lung microbial ecology in health and disease and a greater understanding of the pathogenesis of pneumonia. with this comes a shift away from the targeting of a limited number of putative pneumonia pathogens toward the identification of signal patterns characteristic of different etiologies and stages of pneumonia. the hope is that these signals will have sufficient diagnostic accuracy to help guide pneumonia management. the author has no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. this includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties. â�¢ identifying the microbial etiology of pneumonia is challenging, largely due to difficulty in obtaining uncontaminated specimens from the site of infection and in discriminating between colonizing microorganisms and true pathogens. â�¢ molecular tests, particularly nats, have been the major advance in pneumonia diagnostics over recent years. â�¢ although uptake has been relatively slow, nats now have established roles (often as the diagnostic of choice) for the detection of respiratory viruses and several non-colonizing bacteria (e.g. legionella species). in contrast, nats have yet to have an established role for several important bacterial pneumonia pathogens (e.g. s. pneumoniae) that also are asymptomatic colonizers of the upper respiratory tract. â�¢ further developments in molecular tests need to focus on methods to help interpret the significance of positive results. the use of quantitative nats and microbial load cutoffs has shown promise as one means to discriminate between colonizing and pathogenic microorganisms. â�¢ the recent revelation that the lung microbiome exists and new knowledge about the interaction between bacteria and viruses has changed the traditional view of pneumonia pathogenesis. new diagnostics need to account for this new paradigm and be less focused on just detecting specific known pathogens. global, regional, and national incidence, prevalence, and years lived with disability for 301 acute and chronic diseases and injuries in 188 countries, 1990-2013: a systematic analysis for the global burden of disease study 2013 global burden of childhood pneumonia and diarrhoea community-acquired pneumonia breathing new life into pneumonia diagnostics this commentary summarizes some of the key challenges in determining the microbial etiology of pneumonia laboratory methods for determining pneumonia etiology in children vaccine effects and impact of vaccination programmes in post-licensure studies transthoracic lung aspiration for the aetiological diagnosis of pneumonia: 25 years of experience from the gambia bts guidelines for the management of community acquired pneumonia in adults: update infectious diseases society of america/american thoracic society consensus guidelines on the management of community-acquired pneumonia in adults pneumonia in adults: diagnosis and management. manchester: national institute for health and care excellence the management of community-acquired pneumonia in infants and children older than 3 months of age: clinical practice guidelines by the pediatric infectious diseases society and the infectious diseases society of america british thoracic society guidelines for the management of community acquired pneumonia in children: update rapid identification of pathogens using molecular techniques development and deployment of a rapid recombinase polymerase amplification ebola virus detection assay in guinea in 2015 update on influenza diagnostics: lessons from the novel h1n1 influenza a pandemic mers coronavirus: diagnostics, epidemiology and transmission nucleic acid amplification tests for the diagnosis of pneumonia molecular genetic methods in the diagnosis of lower respiratory tract infections molecular diagnosis of respiratory viruses nucleic acid amplification-based diagnosis of respiratory virus infections viral pneumonia viral infections of the lower respiratory tract: old viruses, new viruses, and the role of diagnosis point-counterpoint: large multiplex pcr panels should be first-line tests for detection of respiratory and intestinal pathogens a preliminary study of pneumonia etiology among hospitalized children in kenya respiratory viruses associated with community-acquired pneumonia in children: matched case-control study this recent case-control study highlights the relatively high prevalence of respiratory virus detection among community controls and some of the challenges with interpretation of positive pcr results in children with pneumonia epidemiology and clinical management of legionnaires' disease current and emerging legionella diagnostics for laboratory and outbreak investigations environmental legionellosis and oropharyngeal colonization by legionella in immunosuppressed patients utility of real-time pcr for diagnosis of legionnaires' disease in routine clinical practice detection of legionella dna in peripheral leukocytes, serum, and urine from a patient with pneumonia caused by legionella dumoffii impact of routine systematic polymerase chain reaction testing on case finding for legionnaires' disease: a pre-post comparison study this study illustrates a pragmatic application of pcr for the diagnosis of legionnaires' disease use of the polymerase chain reaction to detect legionella dna in urine and serum samples from patients with pneumonia diagnosis of legionella pneumophila, mycoplasma pneumoniae, or chlamydia pneumoniae lower respiratory infection using the polymerase chain reaction on a single throat swab specimen sensitivity of legionella pneumophila dna detection in serum samples in relation to disease severity enhanced detection of legionnaires' disease by pcr testing of induced sputum and throat swabs contamination of qiagen dna extraction kits with legionella dna acute respiratory infection due to mycoplasma pneumoniae: current status of diagnostic methods molecular methods for the detection of mycoplasma and ureaplasma infections in humans detection and characterization of mycoplasma pneumoniae during an outbreak of respiratory illness at a university polymerase chain reaction is superior to serology for the diagnosis of acute mycoplasma pneumoniae infection and reveals a high rate of persistent infection standardizing chlamydia pneumoniae assays: recommendations from the centers for diseases control and prevention (usa) and the laboratory centre for disease control (canada) recent advances in the diagnosis of pneumocystis jirovecii pneumonia in hiv-infected adults pneumocystis carinii carriage in immunocompromised patients with and without human immunodeficiency virus infection impact of hiv infection status on interpretation of quantitative pcr for detection of pneumocystis jirovecii tuberculosis diagnostics in 2015: landscape, priorities, needs, and prospects molecular diagnostics for tuberculosis automated real-time nucleic acid amplification technology for rapid and simultaneous detection of tuberculosis and rifampicin resistance: xpert mtb/rif assay for the diagnosis of pulmonary and extrapulmonary tb in adults and children: policy update. geneva: world health organization towards improved accuracy of bordetella pertussis nucleic acid amplification tests nucleic acid amplification tests for diagnosis of bordetella infections laboratory diagnosis of pertussis bordetella pertussis in adult pneumonia patients pertussis: review of epidemiology, diagnosis, management and prevention a boarding school outbreak of pertussis in adolescents: value of laboratory diagnostic methods epidemiology and control of pertussis outbreaks in a tertiary care center and the resource consumption associated with these outbreaks laboratory diagnosis of invasive pneumococcal disease carriage of streptococcus pneumoniae and other respiratory bacterial pathogens in low and lower-middle income countries: a systematic review and meta-analysis evaluation of a pcr assay for detection of streptococcus pneumoniae in respiratory and nonrespiratory samples from adults with community-acquired pneumonia evaluation and improvement of real-time pcr assays targeting lyta, ply, and psaa genes for detection of pneumococcal dna real-time pcr is more sensitive than multiplex pcr for diagnosis and serotyping in children with culture negative pneumococcal invasive disease molecular detection methods and serotyping performed directly on clinical samples improve diagnostic sensitivity and reveal increased incidence of invasive disease by streptococcus pneumoniae in italian children bacteremic pneumococcal community-acquired pneumonia in children less than 5 years of age in italy detection of streptococcus pneumoniae and identification of pneumococcal serotypes by realtime polymerase chain reaction using blood samples from italian children â�¤5 years of age with community-acquired pneumonia pneumococcal dna is not detectable in the blood of healthy carrier children by real-time pcr targeting the lyta gene pcr using blood for diagnosis of invasive pneumococcal disease: systematic review and meta-analysis the perch study group. detection of streptococcus pneumoniae by whole blood lyta pcr and association with pediatric pneumonia detection of bacteria and viruses in the pleural effusion of children and adults with community-acquired pneumonia pleural fluid nucleic acid testing enhances pneumococcal surveillance in children viral pathogen discovery the changing face of pathogen discovery and surveillance the use of next generation sequencing in the diagnosis and typing of respiratory infections genetic testing and fda regulation: overregulation threatens the emergence of genomic medicine fda regulation of laboratory-developed diagnostic tests: protect the public, advance the science the lung microbiome: new principles for respiratory bacteriology in health and disease a recent review of recent current knowledge about the lung microbiome the co-pathogenesis of influenza viruses with bacteria in the lung viral bacterial co-infection of the respiratory tract during early childhood polymicrobial community-acquired pneumonia: an emerging entity the microbiome and critical illness towards an ecology of the lung: new conceptual models of pulmonary microbiology and pneumonia pathogenesis precision respiratory medicine and the microbiome analysis of the upper respiratory tract microbiotas as the source of the lung and gastric microbiotas in healthy individuals the microbiome and the respiratory tract whole genome sequencing in clinical and public health microbiology comprehensive molecular testing for respiratory pathogens in community-acquired pneumonia comparison of sputum and nasopharyngeal aspirate samples and of the pcr gene targets lyta and spn9802 for quantitative pcr for rapid detection of pneumococcal pneumonia genomic load from sputum samples and nasopharyngeal swabs for diagnosis of pneumococcal pneumonia in hiv-infected adults use of a rapid test of pneumococcal colonization density to diagnose pneumococcal pneumonia â�¢ the first study to demonstrate the potential application of nasopharyngeal colonization density, using quantitative pcr, to diagnose pneumococcal pneumonia in adults pneumococcal colonisation density: a new marker for disease severity in hiv-infected adults with pneumonia association between pneumococcal load and disease severity in adults with pneumonia severity of pneumococcal pneumonia associated with genomic bacterial load classical and latent class analysis evaluation of sputum polymerase chain reaction and urine antigen testing for diagnosis of pneumococcal pneumonia in adults estimating the proportion of pneumonia attributable to pneumococcus in kenyan adults: latent class analysis comparison of tb-lamp, genexpert mtb/rif and culture for diagnosis of pulmonary tuberculosis in the gambia a bayesian framework for estimating the incremental value of a diagnostic test in the absence of a gold standard the pneumonia etiology research for child health project: a 21st century childhood pneumonia etiology study the pneumonia etiology research for child health core team. partially latent class models for case-control studies of childhood pneumonia aetiology key: cord-333026-9f6ecg30 authors: kompanikova, j.; zumdick, a.; neuschlova, m.; sadlonova, v.; novakova, e. title: microbiologic methods in the diagnostics of upper respiratory tract pathogens date: 2017-03-03 journal: clinical research and practice doi: 10.1007/5584_2017_10 sha: doc_id: 333026 cord_uid: 9f6ecg30 upper respiratory tract infection (uri) is a nonspecific term used to describe acute infections involving the nose, paranasal sinuses, pharynx, and larynx above the vocal cords. the aim of this study was to provide a summary of the most common pathogens of uri and to compare advantages and disadvantages of traditional and new rapid microbiological tests used to identify them. blood samples were simultaneously examined by the enzyme-linked immunosorbent assay (elisa) and by the filmarray respiratory panel for eight different pathogens in a total of 15 tests performed in nasopharyngeal swabs. the elisa method is unable to identify the pathologic agent until the host’s immune system elicits a response. the method is readily available in many laboratories at a low cost, which puts less strain on economic resources. the filmarray(®) panel, on the other hand, is more expensive, but it is fast and exact in the identification of a broad spectrum etiologic agents. nonetheless, since most repiratory tract infections are viral in origin and there is no treatment available, the diagnosis provided by the filmarray panel does not provide any additional clinical benefit and thus should be used only whenever necessary on the individual basis. upper respiratory tract infections (uris) involve the moist surface of the eyes and eyelids, the nasolacrimal ducts, the middle ear, paranasal sinuses, mastoid air cells, and the main respiratory passage of the nose and throat as far as the epiglottis and vocal cords. acute uris include the common cold, pharyngitis, epiglottitis, and laryngotracheitis (nester et al. 1995) . a variety of viruses, bacteria, fungi, and parasites can infect the respiratory tract. transmission of organisms occurs by aerosol droplet or direct hand-to-hand contact with infected secretions, with subsequent passage to the nares or eyes. most uris are of viral etiology (dasaraju and liu 1996) . epiglottitis and laryngotracheitis are exceptions with severe cases likely caused by haemophilus influenzae type b. bacterial pharyngitis is often caused by streptococcus pyogenes. bacterial and viral upper respiratory infections produce highly variable clinical symptoms that cannot be used to identify the etiologic agent. proper treatment depends on the correct identification of a pathogen involved as antibiotics provide little or no benefit with viral infections (nester et al. 1995) . the uris are in 69% of viral origin (mäkelä et al. 1998) . orthomyxoviruses (influenza a and b), paramyxoviruses (parainfluenza and respiratory syncytial viruses), coronaviruses, adenoviruses, and enteroviruses (coxsackie and echo viruses) cause common cold. however, most colds are caused by more than 89 types of rhinoviruses (musher 2003; cooper et al. 2001 ). more than 40 strains of adenoviruses cause pharyngitis resembling a strep throat. rhinoviruses are unresponsive to antibiotics and other medications that control bacterial infections. antibiotic treatment of adenovirus infections is of no value and sometimes can even be harmful, because it supresses normal bacterial flora and enables the resistant opportunistic pathogens to grow in an uncontrolled way (nester et al. 1995 (murray et al. 2005) . the most common uri of bacterial origin is pharyngitis caused by group a beta-hemolytic streptococci, with streptococcous pyogenes as a main representative, which accounts for 5-10% of pharyngitides (poole and portugal 2005 the filmarray panel is a multiplexed nucleic acid test intended for the simultaneous qualitative detection and identification of multiple respiratory pathogen nucleic acids in nasopharyngeal swabs. this new platform combines automated sample preparation, nucleic acid extraction, and polymerase chain reaction (pcr)-based detection from a single unprocessed sample in 1 h (biofire diagnostics; salt lake city, ut). this method allows for identification of 21 different respiratory pathogens from a nasopharyngeal swab, 18 of viral etiology and three of bacterial origin (idaho technology 2007). in the present study we seek to determine the individual advantages and disadvantages of different diagnostic tools for pathogens underlying the uris, as well as under which circumstances a specific method would have an advantage over another one. table 1 . these results corresponded with a number of respiratory infections, as diagnosed before hand, such as uris, pneumonia, acute inflammation of nasopharynx, acute bronchitis, hypothermia unrelated to external temperature, and undefined fever. for comparison, specimens obtained from two patients were tested simultaneously with traditional elisa and filmarray panel methods. a venous blood sample and a nasopharyngeal swab were taken from both patients for either method, respectively. in one of these patients, all results were negative and no etiologic agent could be identied by the elisa method. in total, 28 days were was required to obtain the full panel of results (table 2 ; patient 1). in addition, culture of the specimen obtained from a nasopharyngeal swab from the same patient also was negative for any pathogens. however, nasal swab specimen, examined by the filmarray panel, yielded a positive result for human rhinovirus or human enterovirus (table 3 ; patient 1), which was negative when with elisa. elisa performed in a second patient showed an elevation of igg against parainfluenza virus 1 (igg positive -1.53, cut off -0.709, index -2.1). this result, however, is inconclusive regarding an acute current infection as it rather confirms a previous encounter with the virus. the result regarding influenza a infection was also negative (igm -0.355, cut off -0.832, index -0.4; and igg -0.083, cut off -0.381, index -0.2). the testing time for all eight pathogens amounted to 22 days (table 2 ; patient 2). the examination with the filmarray panel in this patient identified the etiologic agent as influenza a/h1-2009 (table 3 ; patient 2). the identification had to be confirmed with a serum hemagglutination inhibition test (hit), which was done at the department of virology of the regional institute of public health, in banská bystrica, slovakia. the hit was performed twice 2 weeks apart to assess a possible change in antibody titers. while in the first sample influenza a and b titers were negative, there was a significant rise in the antibody titer against table 4 . health insurance gave 600 points for each of the 15 tests, which makes a total 9000 points, plus 150 points for sample culture from upper respiratory tract and 320 points for a culture form lower respiratory tract. each insurance point has a value of 0.0066 €. the laboratory therefore received 59.40 € for running the elisa and additionally 3.10 € for the cultures, which sums up to a total of 62.50 € or 224% of the real material costs. on the other hand, health insurance gave 2500 points for each of the 21 pathogen targets in the filmarray panel, which makes a total of 52,500 points and comes to 346.50 € or 210% of the real material costs amounting to 165.00 € per sample. the insurance reimbursed 11.70 € for influenza a and b testing each. each type of influenza was tested twice giving the cost of 46.80 € in total. the actual laboratory costs for all hit tests were 8.00 €, which is about 6 times less than the insurance reimbursement. laboratory testing is generally not recommended in the evaluation of upper respiratory infections. tests for specific pathogens are helpful when therapy depends on the results. targeted therapy is not available for most viruses that cause uri. therefore, viral testing is rarely indicated for uncomplicated uris in the outpatient setting. however, confirmation of a viral condition such as influenza may reduce inappropriate use of antibiotics (balentine and siamak 2015) . considering the benfits for patient, the speed to identify the etiologic agent clearly favors the filmarray system. this method readily identified the etiologic agent in both patients in whom it was applied in the present study, whereas the hit identified the virus only in the repeat sample of the second patient. however, identification of the etiologic agent did not have any benefit for the first patient with mild symptomatology of coryza because no targeted treatment for human rhinovirus/enterovirus infection was needed. the symptomatology in the second patient was severe and the speed of pathogen identification is crucial for the commencement of appropriate treatment, especially in suspected cases of influenza where early administration of neuraminidase inhibitors significantly reduces mortality rates. in such cases, accuratelly targeted therapy has an enormous benefit for the patient. the laboratory costs to run one examination with different methods showed that the filmarray multiplex pcr respiratory panel is more expensive than the elisa, hit, and the cultivation. one examination with the filmarray panel brought a 181.50 € per patient profit for the laboratory, whereas the profit from running elisa together with cultivation was 34.62 €, and that from hit was 38.80 € per patient. therefore, filmarray respiratory panel is best in terms of profit margin for the laboratory and least favorable for health insurance. serologic diagnostic methods, such as elisa and hit, cannot identify the pathologic agent until the host's immune system elicits a response. however, advantages of those methods are that they are readily available in many laboratories and are least pricey for health insurance. the disadvantage is that the spectrum of pathogens detected is small. a clinical benefit of the filmarray respiratory panel is that it is quick and exact and may identify a broader spectrum of possible pathogens. since most uris are viral in origin and there is no treatment available, the diagnosis provided by the filmarray panel is not always necessary and the method should be used on an individual basis when clinically justified. from the economic standpoint, filmarray respiratory panel is the most profitable for the laboratory. in critically ill patients, a spectrum of diagnostic methods should be used to obtain diagnosis as fast as possible. in patients with minor respiratory tract infections, a more rational approach should be undertaken since the speed and accuracy of diagnosis are less crucial. the decision to choose a specific diagnostic method rests with the medical caregiving staff. upper respiratory tract infection principles of appropriate antibiotic use for acute pharyngitis in adults: background infections of the respiratory system lippincott's illustrated reviews: microbiology viruses and bacteria in the etiology of the common cold upper respiratory tract infection workup medical microbiology, 5th edn how contagious are common respiratory tract infections? microbiology: a human perspective treatment of rhinosinusitis in the outpatient setting characteristics of streptococcus pneumoniae, haemophilus influenzae, moraxella catarrhalis and staphylococcus aureus isolated from the nasopharynx of healthy children attending day-care centres in the czech republic acknowledgments this study was supported by department of microbiology and immunology. we are thankful to our colleagues who provided expertise that greatly assisted the research. the authors declare no conflicts of interest in relation to this article. key: cord-329877-vish6v8e authors: lapinsky, stephen e.; hawryluck, laura title: icu management of severe acute respiratory syndrome date: 2003-05-09 journal: intensive care med doi: 10.1007/s00134-003-1821-0 sha: doc_id: 329877 cord_uid: vish6v8e background: severe acute respiratory syndrome (sars) is a contagious viral illness first recognized in late 2002. it has now been documented in 26 countries worldwide, with significant outbreaks in china, hong kong, singapore, and toronto. research into identifying the etiological agent, evaluating modes of disease transmission, and treatment options is currently ongoing. discussion: the disease can produce a severe bilateral pneumonia, with progressive hypoxemia. up to 20% of patients require mechanical ventilatory support, with a fatal outcome occurring in about 5% of cases. conclusions: we review the current knowledge about this disease, with particular emphasis on icu management and infection control precautions to prevent disease transmission. electronic supplementary material: supplementary material is available if you access this article at http://dx.doi.org/10.1007/s00134-003-1821-0. on that page (frame on the left side) a link takes you directly to the supplementary material. severe acute respiratory syndrome (sars) is a viral illness characterized by a syndrome of fever and respiratory symptoms that can progress to respiratory failure and death. initial reports of a highly contagious atypical pneumonia originated from guangdong province, people's republic of china, in november 2002. the condition remained isolated to china until february 2003, when an infected physician traveled to hong kong. since then, until 20 april 2003 , the disease has spread to affect over 3,500 individuals in 26 countries (who 19 april 2003 , updates available at www.who.int/csr/sars/en). the largest outbreaks have been described in hong kong, toronto, canada, and singapore, related to a contact with this infected physician in a hong kong hotel. while the western world has been aware of this condition for only several weeks, considerable progress has been made in the identification of the responsible viral organism. there is less known about the mode of transmission and treatment of this disease, but likely mechanisms include droplet spread, surface contact, and possibly airborne transmission. this review describes the current state of knowledge of sars, with particular reference to the management of the critically ill patient and the safety and protection of the icu staff. the recommendations are based on the sparse published data available, collaborations between physicians in many affected centers, recommendations from the world health organization and centers for disease control, and local experience. a novel coronavirus has been isolated from patients meeting the case definition for sars, using electron microscopy and polymerase chain reaction (pcr) of virus isolated in cell culture [1, 2] . preliminary serological studies suggest that this virus has not previously infected the population of the united states [1] . the viral genome has been sequenced, and early pcr-based tests are in an evaluation phase. case definitions of sars are currently based on the presence of epidemiological risk factors (close contact with sars cases or travel to sars "affected" areas) along with a combination of fever and respiratory symptoms, with or without hypoxia and/or chest radiographic changes [3] . however, as sars spreads into the general population, our ability to distinguish it from other community-acquired pneumonias based on such epidemiological linkages will become increasingly tenuous. at this time sars must be considered in the differential diagnosis of any community-acquired or nosocomial pneumonia. a "typical" history (see below), suggestive laboratory values (see below), and failure to respond to conventional antibiotics should raise suspicion. diagnostic tests will be crucial in the future both to ensure that patients are isolated rapidly, and that treatment is initiated. the incubation period has been reported as 2-10 days, and early manifestations include influenzalike symptoms such as fever, myalgias, and headache. it is presently not clear at what stage of the disease viral shedding occurs, or whether someone who is infected but asymptomatic can infect others. as our knowledge of sars and the etiological coronavirus evolves, we will be able to answer these important questions. the viral load may play a role in both the transmission and severity of subsequent disease. the notion of "super spreaders" has been suggested to describe the occasional patient who is associated with spread to large numbers of contacts. our current understanding of the illness is that fever occurs in virtually all patients and is often the presenting symptom. fever may occasionally be absent in the elderly. some patients have mild respiratory symptoms at the onset, and gastrointestinal manifestations are relatively uncommon. diarrhea has been reported with increased frequency in recent outbreaks. the respiratory phase starts after 3 -7 days with dry cough and shortness of breath. in some cases these symptoms are followed by hypoxia and radiological evidence of progressive pulmonary infiltrates. the radiological picture is patchy, of focal infiltrates or consolidation often with a peripheral distribution, which may progress to diffuse infiltration. pulmonary infiltrates may worsen during the first 10 days, and acute respiratory distress requiring mechanical ventilation has occurred in about 10-20% of patients. the case fatality rate is approximately 3-12% depending on whether the denominator includes both suspect and probable cases (3%) or probable cases alone (12%) [4, 5, 6] . while the mortality rate is higher in older patients particularly those with preexisting comorbidity (e.g., diabetes and immunosuppression), we have also seen young, previously healthy persons succumb to the disease. this may be due to higher viral loads or to their host response. the disease runs for 7-14 days, and a biphasic course has been described in some patients, with an initial illness, improvement, and subsequent deterioration. this worsening can present as recurrent fever 4-7 days after initial defervescence, new chest infiltrates on radiography, or recurrent respiratory failure. in some patients an initial mild to moderate respiratory illness may initially improve, later to be followed by a progressive deterioration requiring ventilatory support. laboratory findings in patients with sars include thrombocytopenia and leukopenia (in particular lymphopenia). elevated creatine kinase, lactate dehydrogenase, and transaminases have been noted. there is evidence that the peak lactate dehydrogenase and an initial elevated white cell count may carry a poor prognosis [6] . epidemiological studies are currently underway to help determine prognostic factors. diagnostic testing initial diagnostic testing should include a search for other respiratory pathogens, including blood cultures, sputum gram's stain and culture, and serological tests. bronchoscopy is valuable to exclude other diagnoses but is not recommended in patients with a typical clinical picture and clear epidemiological link, due to the high risk that bronchoscopy poses to icu staff. in patients who are immunosuppressed and when concerns regarding other diagnoses are high, the risk of bronchoscopy may be acceptable. clinicians should save any available clinical specimens (respiratory, blood, and serum) for additional testing until a specific diagnosis is made. specific tests for the virus, including antibody tests (enzyme-linked immunosorbent assay and immunofluorescence), and pcr tests are in development and evaluation stages. if the diagnosis is uncertain, empirical therapy for community-acquired pneumonia should be considered using antibiotics with activity against both typical and atypical respiratory pathogens. in all series of sars described to date therapy has included broad spectrum antibiotics, including a fluoroquinolone or macrolide [4, 5, 6] . the treatment regimen for sars that we follow, as of 11 april 2003, is as follows: -antibiotic therapy -respiratory fluoroquinolone or macrolide -ribavirin -400 mg intravenously every 8 h for 3 days, followed by -1200 mg orally every 12 h for 7days -corticosteroids -methylprednisolone 40 mg intravenously every 12 h for 3 days followed by prednisone 50 mg orally per day for 7 days. the antiviral drug ribavirin has been used in the majority of patients treated in hong kong and in toronto, without evidence of efficacy or even a strong anecdotal suggestion that patients benefit. the adverse effects of ribavirin are significant, particularly hemolytic anemia and electrolyte disturbances such as hypokalemia and hypomagnesemia. the drug is also teratogenic, and this should be considered when evaluating treatment options. anecdotal evidence suggests a benefit of corticosteroids, particularly in the patients with progressive pulmonary infiltrates and hypoxemia. in some but certainly not all patients a dramatic improvement has been noted following steroid therapy. various regimens have been used in different centers, with doses ranging from methyprednisolone 40 mg twice daily (similar to pneumocystis pneumonia therapy) [8] to 2 mg/kg per day (similar to late-phase therapy for acute respiratory distress syndrome) [7] to pulse doses of 500 mg intravenously per day). patients with a late deterioration have usually been restarted on ribavirin and increased doses of steroids. the evidence supporting such practices currently does not exist, and benefit is uncertain. physicians must remain vigilant and search for another cause of fever and secondary sources of infection. management is affected by the increased risk of droplet transmission of virus by certain procedures. oxygen therapy using aerosol humidifiers may increase the risk of droplet spread. other high-risk procedures include obtaining nasopharyngeal swabs, bag-mask ventilation, intubation, suctioning, chest physiotherapy in nonintubated patients, nebulized drug therapy, noninvasive ventilation, and extubation (see table 1 ). if a ventilated patient desaturates and requires manual bag ventilation, it is important to turn the ventilator to "standby" prior to disconnecting, to avoid droplet spray. in fact, in an intubated patient with sars we recommend avoiding disconnecting the ventilator unless there is an obvious mechanical ventilator failure, even in the event of a cardiac arrest. the ventilatory management of patients with sars does not differ from that for patients with acute respiratory distress syndrome. the use of high-frequency oscillation may be associated with increased risk of droplet spread and exposure to respiratory secretions, and our practice is to avoid this intervention. jet ventilation for those failing conventional ventilation may be used safely. little experience exists with the use of interventions such as nitric oxide and prone positioning. anecdotally, the experience in toronto and singapore has been that nitric oxide offers little benefit. infectious disease consultation is essential, for up-todate advice on management and to advise on infection control precautions. this organism appears to be transmitted by droplet spread, although surface contamination and possibly airborne spread may play a role. recent data suggest that the virus may remain viable for considerable periods on a dry surface (up to 24 h). staff education and continued vigilance are essential. infection control measures for healthcare workers exposed to patients should include (see table 2 ). the antechambers should be equipped with: -sinks/disposal units for gowns, gloves, masks, antibacterial soap, and alcohol hand wash -fresh boxes of gloves -an instruction sheet to staff on how to "undress" and "redress" without contamination to avoid repeatedly breaking the negative pressure barrier individual rooms should be stocked with basic supplies. modified cardiac arrest carts containing emergency drugs such as epinephrine, atropine, and bicarbonate should be available in the room in the event of urgent need. staff should remain outside the negative pressure rooms as much as possible. this means timing blood analysis and administration of any therapies to minimize entries and use of video camera equipment or windows to monitor sars patients without direct staff exposure. an antechamber (preferably with a sink) helps to maintain strict infection control precautions and avoids the potential contamination of a single door room. pens, paper, and other personal items should not be allowed into or removed from the room. dress precautions -airborne precautions using a n95 respirator [9] or equivalent (ffp2 respirator): it is important that manufacturer specifications be adhered to, for example, some n95 masks maintain protection for 8 h, some in the icu the risk of droplet spread is increased by various procedures (table 1 ). efforts to avoid viral spread include the avoidance of nebulizers for drug administration and limitation or avoidance of the use of noninvasive ventilation. nebulized humidification for oxygen therapy may carry similar risks, and our practice is to provide nonhumidified oxygen using nasal prongs or a venturi mask. a non-rebreather mask with expiratory port allowing gas filtration is available and may be of value. during bag-mask-valve ventilation a filter should be used on the expiratory port. high-risk procedures include endotracheal intubation and bronchoscopy. intubation should be performed by the most skilled person available using the method with which they are most comfortable. awake intubation may be associated with patient agitation and coughing, which can severely compromise infection control precautions. a powered air purification respirator (e.g., 3m airmate) can be used for these procedures. ventilators should have two filters (e.g., conserve 50 pall filters) placed so as to eliminate the exhalation of viral particles into the environment and protect as much as possible the inside of the ventilator from contamination. one filter should be interposed between the distal end of the expiratory tubing and the ventilator itself and the second placed on the exhalation outlet of the ventilator. ideally the exhalation port should then be connected to a central scavenging system which would eliminate release of viral particles into the icu. many ventilators have an expiratory filter as an integral component of the exhalation circuit. the use of disposable circuits and humidifiers may also help reduce the risk of contamination to patients and staff. transporting a sars patient for testing is an infection control challenge. sars patients should never be transported while being supported by bag-valve-mask ventilation, and should preferably be intubated. the risks and benefit of any procedure should be considered prior to transporting a patient. if bag ventilation is used, a filter should be placed between the bag and the endotracheal tube. infection control should be also consulted for their advice on proper precautions. an important component of infection control is the limitation of personnel and visitors having contact with the patient. it is crucial that staff members do not work if they are ill even if the diagnosis is not clear. staff members with unprotected contact with a sars patient are subject to a compulsory 10-day quarantine period at home. visitors are currently restricted in toronto hospitals. all visitors are screened for symptoms of sars and adhere to the same precautions as hospital staff in gowning, gloving, wearing n95 masks and goggles. visits with sars patients are prohibited even on compassionate grounds. sars has resulted in significant challenges for critical care medicine and likely will change the icu environment for some time to come. in affected areas the poli-cies are changing on a daily basis as more information about the virus and the disease is obtained. the ability of this disease to incapacitate staff has resulted in staff safety becoming a priority to maintain adequate critical care services. the concept of "universal precautions" now includes strict respiratory and contact precautions. the guidelines and recommendations will change as our knowledge grows. information and communication technology have played an important role in allowing collaboration and rapid transfer of information. it is only through such sharing that we can hope to improve the mortality and morbidity of our patients and stay healthy and well ourselves. updated information on sars can be obtained from the following websites: -centers for disease control: www.cdc.gov -world health organization: www.who.int/csr/sars/ -new england journal of medicine: nejm.org/earlyrelease/sars.asp a novel coronavirus associated with severe acute respiratory syndrome preliminary clinical description of severe acute respiratory syndrome a cluster of cases of severe acute respiratory syndrome in hong kong identification of severe acute respiratory syndrome in canada a major outbreak of severe acute respiratory syndrome in hong kong effect of prolonged methylprednisolone therapy in unresolving acute respiratory distress syndrome: a randomized controlled trial la voie l (1990) corticosteroids as adjunctive therapy for severe pneumocystis carinii pneumonia in the acquired immunodeficiency syndrome. a double-blind, placebo-controlled trial laboratory performance evaluation of n95 filtering facepiece respirators acknowledgements we acknowledge input from dr. wilf demajo, dr. tom stewart, and dr. rob fowler as well as information from colleagues in toronto, hong kong, and singapore. key: cord-349226-xzlc1pni authors: khatiwada, saroj; subedi, astha title: lung microbiome and coronavirus disease 2019 (covid-19): possible link and implications date: 2020-08-05 journal: hum microb j doi: 10.1016/j.humic.2020.100073 sha: doc_id: 349226 cord_uid: xzlc1pni coronavirus disease 2019 (covid-19) is a rapidly emerging disease caused by severe acute respiratory syndrome coronavirus 2 (sars-cov-2). the disease begins as an infection of lungs, which is self-limiting in the majority of infections; however, some develop severe respiratory distress and organ failures. lung microbiome, though neglected previously have received interest recently because of its association with several respiratory diseases and immunity. lung microbiome can modify the risk and consequences of covid-19 disease by activating an innate and adaptive immune response. in this review, we examine the current evidence on covid-19 disease and lung microbiome, and how lung microbiome can affect sars-cov-2 infection and the outcomes of this disease. to date there is no direct evidence from human or animal studies on the role of lung microbiome in modifying covid-19 disease; however, related studies support that microbiome can play an essential role in developing immunity against viral infections. future studies need to be undertaken to find the relationship between lung microbiome and covid-19 disease. the rapid outbreak of the coronavirus disease has crippled global health and economy [1] . as of 21 st july 2020, it has affected 188 countries and infected 14,727,753 people causing death of 610,560 patients [2] . the rapid increase in the number of severely ill patients after covid-19 infection across the globe has resulted in heavy investment in health care infrastructures and medical supplies. to stop the spread of the virus, many countries have imposed a strict international travel ban and put the population into home restriction [3] . the covid-19 disease is caused by a virus called severe acute respiratory syndrome coronavirus 2 (sars-cov-2), which emerged in wuhan, china at the end of 2019 [4] . the disease is highly transmissible among persons through aerosols, and evidence shows the virus can remain viable for up to 3 days on plastic and stainless steel, all of which facilitates the easy propagation of this virus [5] . the usual symptoms of covid-19 include fever, dry cough, and tiredness. some patients may have aches and pains, nasal congestion, anosmia, sore throat or diarrhea [6] . a large majority of patients may not develop any symptoms after the viral infection, thereby acting as a hidden carrier for this devastating disease [7] . the common complications of covid-19 that lead to death include acute respiratory failure (arf), pneumonia, acute respiratory distress syndrome (ards), acute liver injury, acute cardiac injury, acute kidney injury, septic shock, disseminated intravascular coagulation, blood clots and rhabdomyolysis [8] [9] [10] [11] . the development of pneumonia or acute respiratory illness is consistently observed after covid19 infection with the majority of the patient showing lesions involving bilateral lungs, which is often the common cause of covid-19 death [10, 12, 13] . microbiome refers to the collective genomes of all the microorganisms. it includes every organism (not only bacteria, but also archaea, fungi and viruses), dna and rna species, and 4 proteins that can affect health and development of diseases [14] . microbiota refers to the community of microorganisms, including bacteria, viruses, fungi, and protozoans, that live in a host body [15] . the covid-19 disease begins with the invasion of lungs by sars-cov-2 virus, and the major complications that develop subsequently are related to lung infection and immune response generation, therefore, lung microbiome might play an important role from initiation to the progression of this disease [16] . this review focuses on the potential relationship between covid-19 disease and lung microbiome. we summarized the most important findings on lung microbiome and its role in immunity and lung disease including covid-19. we searched for the keywords (sars-covfor the articles until 20 july 2020. all the original research articles reporting microbiome in covid-19 were included. we highlight the role of the lung microbiome, and how the microbiome can modulate sars-cov-2 attack along with the consequences of viral attack. finally, we seek the potential role of lung microbiome and immunity in severity of covid-19 disease. we also discuss the relevant studies supporting the role of microbiome against viral infection and disease complications. until very recently, the lung was considered to be a sterile site free from bacteria [17] . being an environment with favourable temperature, moisture and mucus, and a large surface area that is in regular contact with the external environment, the lung is indeed a microbiome rich site [18] . lung microbiome is more dynamic and transient than that of the gastrointestinal tract because of bidirectional movement of air and mucus [19, 20] . in a healthy human, the lung microbiota has a low density but harbours a prominent diversity of interacting microbiota. at the phylum level, firmicutes, bacteroidetes and proteobacteria are the most common phyla. at the genus level, prevotella, veillonella and streptococcus are the predominant microorganisms [17, 21] . the composition of lung microbiome is mainly determined by microbial immigration, elimination, and relative growth rates of its members. in lung diseases, these factors can change, and therefore overgrowth of one species with reduction of microbial diversity occurs (shown in figure 1 ) [22] . during lung pathology, drastic changes in the local environment occur, creating a favourable environment for a specific microbial growth. in one of the common lung disease, asthma, bronchoscopy samples have shown an increased abundance of haemophilus, neisseria, fusobacterium, and porphyromonas [23, 24] . recent evidence shows microbiota administration through the oral or intranasal route as probiotics can lower allergic inflammation seen in asthma, highlighting the intricate relationship between microbiota and asthma [25] . in another common lung disease, chronic obstructive pulmonary disease (copd), lactobacillus, fusobacteria, leptotrichia and fusobacterium were observed in abundant numbers [26, 27] . the most abundant bacteria seen in cystic fibrosis were pseudomonas, staphylococcus, stenotrophomonas, achromobacter, and streptococcus [28, 29] . recent evidence shows that lung microbiome is altered in lung cancer, and the microbiome can influence cancer development and progression [30, 31] . emerging evidence shows the relationship between lung microbiota and susceptibility to pneumonia [32] . pneumonia is a widespread lung disease characterised by infection of lung alveoli leading to an infiltration of inflammatory cells and fluids into the alveoli. this disease can be caused by bacteria, virus, fungi or protozoans [33] . streptococcus pneumoniae and haemophilus influenzae type b are the most common bacterial causative agents, whereas, 6 respiratory syncytial virus (rsv) is the most common viral agent for pneumonia [34, 35] . the endotracheal aspirates of patients with ventilator associated pneumonia had a higher bacterial load but a lower abundance of bacterial class, bacilli, than matched controls [36] . similarly, the microbiota profile of endotracheal aspirates was different between the mechanically ventilated pneumonia group and the non-pneumonia group. in the intubated patients with pneumonia, pseudomonas, corynebacterium, and rothia were more abundant, while streptococcus and prevotella were less abundant as compared to those without pneumonia [37] . the abundance of firmicutes phyla was reduced in the patients with interstitial pneumonia including the overall phyla richness as compared to healthy group. the abundance of streptococcus was significantly reduced in interstitial pneumonia patients, while prevotella and veillonella were enriched in comparison to healthy volunteers [38] . in addition, the microbial diversity and composition tended to differ according to the causative agent of pneumonia, which suggests that lung microbiome can vary according to the type of pneumonia [39] . overall, pneumonia is characterized by a shift in lung microbiome, with domination by pneumonia etiologic agent, high microbial biomass, and lowering of microbial diversity [33] . the composition and diversity of upper respiratory tract and lung microbiome have been found to shift in response to respiratory viral infections in humans and animals [40] [41] [42] . some of the common viruses invading respiratory tract and lungs include rhinovirus, influenza virus, adenovirus, parainfluenza virus and rsv. these respiratory viral infections are quite common during childhood, and they often increase susceptibility to secondary bacterial infection of lungs [41] . the upper respiratory tract microbiome of rhinovirus and rsv infected infants was found to be largely dominated by moraxella, streptococcus, corynebacterium, haemophilus, and dolosigranulum [43] . in another study, rhinovirus infection was found to increase the relative abundance of haemophilus and neisseria, both of which are associated with secondary lung infections [44] . in mice, influenza virus infection of lungs caused a shift in the 7 composition and diversity of microbial community. the viral infection changed the dominant bacterial class from alphaproteobacteria to gammaproteobacteria and actinobacteria, and this was followed by an increase in the relative abundance of streptococcus and staphylococcus [45] . in summary, the lung microbiome is highly dynamic, and infections (bacterial, viral) can lead to rapid alterations in the lung microbiome, and the microbial communities can play a critical role in those infections (shown in figure 1 ). the alveolar surface is the largest surface area in direct contact to the external environment, so it is constantly exposed to invading microorganisms. therefore, it harbours several lines of defence against potential infections. a continuous layer of pulmonary epithelial cells constitutes a physical and biological barrier for inhaled substances and pathogens. the mucus coating of the pulmonary epithelium, proteolytic enzymes, defence proteins (immunoglobulins, 8 lactoferrin, defensins) and lysozymes present on the surface and in the fluids around alveoli protect against infection [18] . pulmonary epithelial cells are in close contact with cells of the immune system, and they also secrete a wide range of cytokines and chemokines. they express cell surface receptors called pattern-recognition receptors (prrs), such as the toll-like receptors (tlrs), which enable them to recognize pathogen-associated molecular patterns (pamps) from viruses, bacteria, fungi, protozoa, and multicellular parasites [46] . apart from the above mentioned immune mechanisms, lung contains immune cells such as dendritic cells, macrophages (alveolar and interstitial), lymphocytes (t-cells, γδ t cells, nk cells, b-cells), innate lymphoid cells and neutrophils that support innate and adaptive immune mechanism [47]. all these immune cells are involved in eliminating antigens and pathogens from the respiratory tract through phagocytosis or activation of effectors molecules that remove antigens. regulatory t cells resident within the lungs are vital for the maintenance of immune tolerance to airborne particles, and they are directly influenced by local microbiota [46] . another type of t cells, called lung resident memory γδ t cells provide a rapid immune response at barrier surfaces to recall antigens previously encountered through lung mucosa, which is apart from its role in cytokine and interferon production to stop viral and bacterial infection [48] . despite the existing immunity mechanisms, lung infection is unavoidable. in the next section, we will discuss how sars-cov-2 initiates lung infection and escape the immune response. in the respiratory tract, sars-cov-2 bind to the specific host receptors called angiotensinconverting enzyme 2 (ace2). this virus has an envelope-anchored spike protein that facilitates virus entry into host cells by first binding to ace2. the virus then enters the host cell through endocytosis [49] . inside the cells, the viral genome replicates and makes necessary proteins, 9 which assemble to form new virions. the newly released virions can invade other lung cells [4, 50] . the stress of viral production will lead to cell death, which in turn will trigger a series of immune responses and initiate inflammation reflected as lymphopenia, elevated c reactive protein (crp) and erythrocyte sedimentation rate [13] . the viral rnas and pamps are detected by the prrs, such as tlrs inside the cells [51] . the recognition will lead to the activation of the transcription factor nuclear factor-κb (nf-κb) and interferon regulatory factor 3 (irf3) to induce proinflammatory cytokines such as interferonα and tumour necrosis factor-beta (tnf-β). the production of type i interferons (ifn) will cause suppression of viral multiplication and prevent the severity of disease [52] . increase in interleukin-6 (il-6), crp, and chemokines such as interferon-γ-inducible protein 10 (ip-10), monocyte chemoattractant protein 1 (mcp-1), macrophage inflammatory protein 1-alpha (mip-1a), and tumour necrosis factor-alpha (tnf-α) were observed in covid-19 patients [10, 53] . if the body immune response cannot stop the sars-cov-2 multiplication, then a massive degree of lung injury occurs. this degree of damage can generate a substantial immune response from immune effector cells which is characterized by a large amount of cytokines (ifn-α, ifn-γ, il-1β, il-6, il-12, il-18, il-33, and tnf-α) and chemokines (ccl2, ccl3, ccl5, cxcl8, cxcl9, and cxcl10) in the body [54] . this condition of hyperinflammation manifested as 'cytokine storm' is called as covid-19 ards. this is one of the most dangerous and potentially life-threatening events related to covid-19 disease [55] . besides, sars-cov-2 infection damages the pulmonary lining altering the composition of normal microbiota, and can promote the growth of specific bacteria to initiate secondary pneumonia [41] . the formation of double-membrane vesicles that lack prrs, where the virus replicates, leading to failure of recognition by immune cells and therefore avoid host detection [54] . another feature of this virus, eight surface proteins, can block the action of interferon i, causing the cells to fail to stop viral multiplication [56] . the virus has also developed a strategy to stop host cell recognition by mimicking host cell capping machinery [52] . the sars-cov-2 viral infection occurs amid the local environment of diverse microbiota; therefore, it is apparent that lung microbiota can have an impact on the initiation, development, and progression of the covid-19 disease. lungs are at the frontline of immunity as they are constantly exposed to a wide variety of external environment. the microbiome has a principal role in shaping pulmonary immunity, and healthy lung has a vast array of microbiota. microbes boost innate and adaptative immunity (site-specific in lungs and systemic), release factors which assist respiratory functions, and prevent an invasion of lungs from pathogens [57] . the normal microbiota restricts the growth of harmful pathogens that may make their way into the lungs. this growth restriction may occur by several mechanisms, such as, limiting nutrients access and secreting growth inhibitors [58] . activation of lung immune cells that initiates innate and adaptive immunity requires microbe exposure [59] . the function of one of the vital effector and regulator cells of the lung, γδ t cells, depends on pathogen invasion [48, 60] . evidence shows that exposure to certain bacterial strains in neonate protects against excessive airway inflammation through the alteration of immune cells [61] . furthermore, microbial exposure is required for developing innate immunity, as children exposed to the microbial rich environment had a lower rate of asthma and allergic sensitization, and stronger immunity [62] . one of the products of lung's normal flora showed an allergo-protective effect in the animal model of airway inflammation [63] . in summary, previous studies highlight that lung immunity development requires exposure to diverse microbes (shown in figure 1 ). [67] . moreover, this study showed the severity of covid-19 disease is directly correlated with the predominance of opportunistic pathogens (clostridium ramosum and clostridium hathewayi) and inversely with the predominance of beneficial commensals (alistipes onderdonkii and bacteroides ovatus) [67] . similarly, covid-19 patients had increased proportions of opportunistic fungal pathogens, candida albicans, candida auris, and aspergillus flavus as compared to controls [66] . in summary, the role of gut microbiome against sars-cov-2 infection and disease severity is rapidly emerging, and therefore gut microbiota may be developed as therapeutics for the management of covid-19 disease in future. till date, only two studies have analysed the lung microbiome of covid-19 patients [64, 65] . in one of the study, bronchoalveolar lavage fluid from covid-19 patients (n=8), communityacquired pneumonia patients (n=25) and healthy controls (n=20) was investigated, and a significant difference in microbiota composition was observed [65] . both covid-19 and community-acquired pneumonia patients had enrichment of pathogenic and commensal bacteria, indicating a degree of microbial dysbiosis in both diseased states [65] . in the other study, the lung post-mortem biopsies from 20 deceased covid-19 patients were used to study lung microbiome. the most common bacterial genera were acinetobacter, chryseobacterium, burkholderia, brevundimonas, sphingobium, and enterobacteriaceae. the most common fungal genera were cutaneotrichosporon, followed by issatchenkia, wallemia, cladosporium, alternaria, dipodascus, mortierella, aspergillus, naganishia, diutina, and candida [64] . therefore, a mix of bacterial and fungal infection was seen in the covid-19 patients of this study. overall, there is very little information about the composition of lung microbiome in covid-19 patient. further studies with bigger sample size are required to know more precisely the composition and the role of lung microbiome in the severity of sars-cov-2 infections. although there is no direct evidence if lung microbiome can affect potential sars-cov-2 infections and the outcomes of this disease, yet several lines of evidence support that bacteria in lungs have important roles in this disease. firstly, microbiota dwelling on the respiratory surface can acts as a barrier, thereby preventing viral attachment to host cells. secondly, microbiota prime the lung immunity, which will fight against viral infection, and exposure to 13 a diverse range of microbiota may build a wider immunity. such phenomenon is particularly common in the gut, where gut microbiota can protect against potential flu viral infections [68] . in mice, the nasal application of a respiratory normal flora, corynebacterium pseudodiphtheriticum, increased the tlr3 antiviral response against rsv and enhanced the production of tnfα, il-6, ifnγ, and ifnβ [69] . another respiratory tract flora, staphylococcus aureus, protected against influenza induced lung injury [70] . recent evidence of the association between covid-19 disease severity with the gut microbiota seems highly promising [67] . thus, it appears that lung microbiome may modify propensity to sars-cov viral infection. till date, it is unknown if lung microbiota can modify the risk of developing severe respiratory complications such as ards after sars-cov-2 infection. the dysbiosis of lung microbiome may contribute to covid-19 ards because microbial dysbiosis is found to provoke a dysregulated immune response leading to inflammation [22, 55, 71] . in the ards unrelated to covid-19 disease, lung microbiome is found to be enriched with gut-associated bacteria, which demonstrates the relationship between lung microbiome and ards [72] . recently, the altered microbiome (increase in bacterial burden and a decrease in alpha diversity) was shown to be associated with inflammation and mortality in ards patients. the change in microbiota population (a reduction in betaproteobacteria and an increase in staphylococcus, streptococcus and enterobacteriaceae) was significantly associated with serum cytokine il-6 [73] . lung microbiome seems to have a critical role in severely ill patients because a recent study found that lung microbiota predicts the clinical outcome and death in those patients [74] . this study showed that increased lung bacterial burden and lung enrichment with gut-associated bacteria were predictive of adverse consequences of ards. thus, in covid-19 ards, the microbiome may have a significant role in determining the severity of the disease, and the outcomes of ards. in the future, studies will find if the presence of a specific bacterial population is associated with low risk for sars-cov-2 infection and ards development. based on the current evidence, it appears that immunity is at the forefront for protection against covid-19 disease. immunity development is a continuous process and requires exposure to a range of microbiota and environments [75] . exposure of human population to diverse air microbes that may occur in unhygienic environment is believed to develop more robust immunity, and this may partly account for the variation in the covid-19 death rate among countries [76] . several other factors such as the method of delivery at birth, air microbiota of the immediate environment, prenatal and childhood exposures to microbes, respiratory viral illness, indoor and outdoor pollution can have a critical influence in the microbiome composition and immunity development in children [46] . exposure to air with high air microbial load is found to reduce asthma risk in children [77] . in many developed countries, over the years, there has been a change in diet, sanitary conditions, antibiotics use, exposure to environmental chemicals and the change in the living environment, all of which may have ultimately reduced the exposure to diverse microbes. this factor is believed to be one of the principal factors for the current rise in the number of autoimmune lung diseases, and weak immunity [32, 75] . till date little is known about the role of lung microbiome in covid-19 disease. future studies are highly warranted to solve the complex relationship between lung immunity, lung microbiome and sars-cov-2 infection along with this disease complications. because of the therapeutic benefits of microbiota as probiotics, future studies should also focus on the potential benefits and applicability of microbiota transplantation for the covid-19 disease. we also emphasize collecting bronchoalveolar samples from the covid-19 infected patients and perform 16s rrna gene sequencing in a large sample size. we conclude that lung microbiome can have a profound impact on the susceptibility to sars who. coronavirus disease (covid-2019) situation reports 2020 wuhan coronavirus (2019-ncov) global cases the socio-economic implications of the coronavirus and covid-19 pandemic: a review covid-19 infection: origin, transmission, and characteristics of human coronaviruses aerosol and surface stability of sars-cov-2 as compared with sars-cov-1 who. q&a on coronaviruses (covid-19) 2020 covid-19: four fifths of cases are asymptomatic, china figures indicate coronavirus disease 2019 coronavirus disease 2019: retrospective study clinical features of patients infected with 2019 novel coronavirus in wuhan epidemiology and clinical features of covid-19: a review of current literature clinical and epidemiological features of 36 children with coronavirus disease 2019 (covid-19) in zhejiang, china: an observational cohort study. the lancet infectious diseases clinical characteristics of 3,062 covid-19 patients: a meta-analysis the lung microbiome in health and disease the role of the lung microbiota and the gut-lung axis in respiratory infectious diseases covid-19 pathophysiology: a review the microbiome and the respiratory tract respiratory microbiome and epithelial interactions shape immunity in the lungs the respiratory tract microbiome and lung inflammation: a two-way street the lung microbiome: new principles for respiratory bacteriology in health and disease spatial variation in the healthy human lung microbiome and the adapted island model of lung biogeography the role of the microbiome in exacerbations of chronic lung diseases features of the bronchial bacterial microbiome associated with atopy, asthma, and responsiveness to inhaled corticosteroid treatment disordered microbial communities in asthmatic airways probiotics against airway allergy: host factors to consider the lung microbiome in moderate and severe chronic obstructive pulmonary disease the lung tissue microbiome in chronic obstructive pulmonary disease sputum dna sequencing in cystic fibrosis: non-invasive access to the lung microbiome and to pathogen details the lower airway microbiota in early cystic fibrosis lung disease: a longitudinal analysis interaction between the microbiome and tp53 in human lung cancer characterization of microbiome in bronchoalveolar lavage fluid of patients with lung cancer comparing with benign mass like lesions the lung microbiome and its role in pneumonia integrative physiology of pneumonia comprehensive molecular testing for respiratory pathogens in community-acquired pneumonia identification of respiratory microbiota markers in ventilator-associated pneumonia the dynamics of respiratory microbiota during mechanical ventilation in patients with pneumonia alveolar microbiota profile in patients with human pulmonary tuberculosis and interstitial pneumonia streptococcus pneumoniae caused different microbial structure and correlation network in lung microbiota differences in the nasopharyngeal microbiome during acute respiratory tract infection with human rhinovirus and respiratory syncytial virus in infancy respiratory viral infection-induced microbiome alterations and secondary bacterial pneumonia patients with coronavirus disease 2019 or h1n1 influenza. clinical infectious diseases differences in the nasopharyngeal microbiome during acute respiratory tract infection with human rhinovirus and respiratory syncytial virus in infancy changes in microbiota during experimental human rhinovirus infection dynamic changes in the microbiome and mucosal immune microenvironment of the lower respiratory tract by influenza virus infection lung homeostasis: influence of age, microbes, and the immune system lung defences: an overview lung-resident gammadelta t cells and their roles in lung diseases receptor recognition by the novel coronavirus from wuhan: an analysis based on decade-long structural studies of sars coronavirus novel 2019 coronavirus structure, mechanism of action, antiviral drug promises and rule out against its treatment coronavirus infections and immune responses severe acute respiratory syndrome coronavirus an overview of viral structure and host response epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a descriptive study molecular immune pathogenesis and diagnosis of covid-19 the cytokine storm in covid-19: an overview of the involvement of the chemokine/chemokine-receptor system sars coronavirus pathogenesis: host innate immune responses and viral antagonism of interferon. current opinion in virology the influence of lung microbiota on lung carcinogenesis, immunity, and immunotherapy innate immunity in the lungs the lung microbiota: role in maintaining pulmonary immune homeostasis and its implications in cancer development and therapy lung microbiota promotes tolerance to allergens in neonates via pd-l1 innate immunity and asthma risk in amish and hutterite farm children farm-derived grampositive bacterium staphylococcus sciuri w620 prevents asthma phenotype in hdm-and ova-exposed mice the lung tissue microbiota features of 20 deceased patients with covid-19 genomic diversity of severe acute respiratory syndrome-coronavirus 2 in patients with coronavirus disease alterations in fecal fungal microbiome of patients with covid-19 during time of hospitalization until discharge alterations in gut microbiota of patients with covid-19 during time of hospitalization tonic interferon signals in lung stromal cells protect from influenza virus infection respiratory commensal bacteria corynebacterium pseudodiphtheriticum improves resistance of infant mice to respiratory syncytial virus and streptococcus pneumoniae superinfection bacterial colonization dampens influenza-mediated acute lung injury via induction of m2 alveolar macrophages the impact of lung microbiota dysbiosis on inflammation enrichment of the lung microbiome with gut bacteria in sepsis and the acute respiratory distress syndrome unique patterns of lower respiratory tract microbiota are associated with inflammation and hospital mortality in acute respiratory distress syndrome lung microbiota predict clinical outcomes in critically ill patients role of the microbiota in immunity and inflammation hay fever, hygiene, and household size exposure to environmental microorganisms and childhood asthma we, the authors declare that we have no known competing financial interests or personal relationships that could have appeared to influence the work not applicable key: cord-335116-c83xyev5 authors: proença-módena, josé luiz; buzatto, guilherme p.; paula, flávia e.; saturno, tamara h.; delcaro, luana s.; prates, mirela c.; tamashiro, edwin; valera, fabiana c.p.; arruda, eurico; anselmo-lima, wilma t. title: respiratory viruses are continuously detected in children with chronic tonsillitis throughout the year date: 2014-07-21 journal: int j pediatr otorhinolaryngol doi: 10.1016/j.ijporl.2014.07.015 sha: doc_id: 335116 cord_uid: c83xyev5 objective: to evaluate the oscillations on the viral detection in adenotonsillar tissues from patients with chronic adenotonsillar diseases as an indicia of the presence of persistent viral infections or acute subclinical infections. study design: cross-sectional prospective study. setting: tertiary hospital. methods: the fluctuations of respiratory virus detection were compared to the major climatic variables during a two-year period using adenoids and palatine tonsils from 172 children with adenotonsillar hypertrophy and clinical evidence of obstructive sleep apnoea syndrome or recurrent adenotonsillitis, without symptoms of acute respiratory infection (ari), by taqman real-time pcr. results: the rate of detection of at least one respiratory virus in adenotonsillar tissue was 87%. the most frequently detected viruses were human adenovirus in 52.8%, human enterovirus in 47.2%, human rhinovirus in 33.8%, human bocavirus in 31.1%, human metapneumovirus in 18.3% and human respiratory syncytial virus in 17.2%. although increased detection of human enterovirus occurred in summer/autumn months, and there were summer nadirs of human respiratory syncytial virus in both years of the study, there was no obvious viral seasonality in contrast to reports with ari patients in many regions of the world. conclusion: respiratory viruses are continuously highly detected during whole year, and without any clinical symptomatology, indicating that viral genome of some virus can persist in lymphoepithelial tissues of the upper respiratory tract. objective: to evaluate the oscillations on the viral detection in adenotonsillar tissues from patients with chronic adenotonsillar diseases as an indicia of the presence of persistent viral infections or acute subclinical infections. study design: cross-sectional prospective study. setting: tertiary hospital. methods: the fluctuations of respiratory virus detection were compared to the major climatic variables during a two-year period using adenoids and palatine tonsils from 172 children with adenotonsillar hypertrophy and clinical evidence of obstructive sleep apnoea syndrome or recurrent adenotonsillitis, without symptoms of acute respiratory infection (ari), by taqman real-time pcr. results: the rate of detection of at least one respiratory virus in adenotonsillar tissue was 87%. the most frequently detected viruses were human adenovirus in 52.8%, human enterovirus in 47.2%, human rhinovirus in 33.8%, human bocavirus in 31.1%, human metapneumovirus in 18.3% and human respiratory syncytial virus in 17.2%. although increased detection of human enterovirus occurred in summer/autumn months, and there were summer nadirs of human respiratory syncytial virus in both years of the study, there was no obvious viral seasonality in contrast to reports with ari patients in many regions of the world. conclusion: respiratory viruses are continuously highly detected during whole year, and without any clinical symptomatology, indicating that viral genome of some virus can persist in lymphoepithelial tissues of the upper respiratory tract. ß 2014 elsevier ireland ltd. all rights reserved. we have previously reported high rates of detection of respiratory virus genomes in tonsils and adenoids from patients with chronic adenotonsillar diseases, suggesting a significant association of viruses, particularly picornaviruses, with severe tonsillar hypertrophy [3] . however, no conclusive evidence of productive -acute or persisting -viral infection, as opposed to virus latency, has been established. in general, peaks of respiratory virus detection in children with ari occur with marked seasonal variations in temperate and subtropical regions [12, 13] . in tropical areas, the seasonal pattern of viral detection is more difficult to be analysed, due to the heterogeneity of data in several parts of the world. however, respiratory viruses have been mainly observed during the rainy seasons [14] . in southeast brazil, a region of transition between tropical and subtropical climates, peaks of viral ari tend to occur during cooler months [15] [16] [17] . the present paper reports analyses of over time variations in rates of detection of respiratory viruses in tissues and secretions removed from children undergoing tonsillectomy while in the absence of ari symptoms. the rationale was that if detection of respiratory viruses in hypertrophic tonsillar tissues oscillated with variations in temperature and rainfall, in a way similar to what occurs among ari patients, this would suggest an association with acute subclinical respiratory viral infections, rather than prolonged asymptomatic harbouring of viral nucleic acids in the tissues. respiratory virus genomes were searched in adenoids (ad), palatine tonsils (pt) and nasopharyngeal secretions (nps) obtained from all 172 children (91 males) aged 1-13 years (mean 5.8 years) who underwent adenotonsillectomy to treat adenotonsillar hypertrophy with clinical evidence of obstructive sleep apnoea syndrome [6] or recurrent adenotonsillitis according to paradise criteria [18] . patients were treated at the division of otorhinolaryngology of the school of medicine of ribeirão preto, university of são paulo, between may 2010 and june 2012. patients with signs/symptoms of acute respiratory infections within the last four weeks prior to surgery and patients with immunodeficiencies were excluded from the study. indeed, the exclusion of these patients was a safety criterion for surgery. all clinical samples obtained in this study were maintained in a preservative solution (rna later -invitrogen, carlsbad, ca, usa) at à70 8c until nucleic acid extraction. the study was conducted according to the principles expressed in the declaration of helsinki and was approved by the university hospital clinical research ethics committee (file number 10466/ 2008). a written informed consent was obtained from all parents and guardians prior to enrolment. the detailed description of each pcr, including the primers sequences, can be obtained in a previously published paper [3] . in the present study were included 51 patients, extending the observation period to two years, allowing that the seasonal pattern of viral circulation was determined in these patients. the analysis of the seasonality of respiratory viruses in adenotonsillar tissues was performed cross matching the virus presence to the temperature and rainfall. ribeirão preto is a city in the state of são paulo, southeast brazil with a population of 619,746, located at 21810 0 40 00 s and 47848 0 36 00 w, 500 m above sea level. the climate is a transition between tropical and subtropical conditions, with annual average temperature of 23 8c, with dry mild winters and hot rainy summers. during this study, the mean monthly minimal and maximal daily temperatures were respectively 18.3 8c (range, 13.5-20.5) and 25.3 8c (range, 23.5-26.8). rainfall is the major climate variable, with yearly rainy seasons between november and march and dry season from june to september. the mean monthly accumulated rainfall throughout the study was 108 mm, ranging from 0 to 533 mm. accumulated rainfall and mean seasonal temperatures were obtained from the site of the integrated center of agrometeorological information of são paulo sate (http:// www.ciiagro.sp.gov.br). rates of detection of respiratory viruses in adenoids, tonsils and respiratory secretions were determined for 172 children. genomes of at least one respiratory virus were detected in over 87% of the patients, without discernible seasonal variations (fig. 1 ). remarkably, high rates of virus detection were obtained from all three kinds of clinical samples throughout the study. the frequencies of virus detection ranged from 71.5% to 94.1% in adenoids, and from 37.5% to 86.6% in secretions and palatine tonsils. of the three sample kinds, adenoid tissue yielded the highest frequencies of virus detection during almost the whole study, except for autumn months (march-june) of 2011, when nasal secretions yielded higher rates of positivity (fig. 1) . the present analysis is based on results from a total of 172 patients, covering a 2-year period, and raises new issues made clear upon inclusion of additional cases to a study that was underway [3] . overall, the most frequent viruses were human adenovirus (hadv) detected in 52.8%, followed by human enterovirus (hev) in 47.2%, human rhinovirus (hrv) in 33.8%, human bocavirus (hbov) in 31.1%, human metapneumovirus (hmpv) in 18.3%, human respiratory syncytial virus (hrsv) in 17.2%, influenza virus (flu) in 4.5%, human parainfluenzavirus (hpiv) in 4.5%, and human coronavirus (hcov) in 2.7%. the frequencies of hadv, hbov and hrsv were higher in adenoids, whereas hrv was more frequently detected in nasal secretions and hev in palatine tonsils. the rates of viral co-infections and the agreement between results from different tissues were high. in this 2-year study period, two or more viruses were detected in 62.2% of the patients, and 54% of them had the same virus detected in adenoids and palatine tonsils. overall, hadv detection rates fluctuated from summer troughs of approximately 37.5% up to peaks of greater than 70% in spring-2011 and autumn-2012, without clearly seasonal periods (fig. 2 ). hbov detection rates were usually above 20% (12.5-46%) without discernible seasonality (fig. 3) . detection frequencies of hadv and hbov were consistently higher in adenoids than in other samples. rates of detection of the picornaviruses hrv and hev were opposite in the summer, while the rate of hev detection was at its [ ( f i g . _ 1 ) t d $ f i g ] peak, hrv was at its lowest ( figs. 4 and 5) . the overall hev detection rates were composed mostly by results obtained from adenoids and palatine tonsils, while detection in respiratory secretions was found in a smaller proportion of the hev-positive patients (fig. 4) . although peaks of hev detection occurred in summer/autumn months, the rates of hev positivity were always above 30%, indicating that a great proportion of tonsil tissues harbour hev, independently of the season of the year. contrary to hev, hrv overall rates showed no clear seasonal variations, and were mostly composed by results obtained from secretions, with correspondingly lower rates of positivity in tonsillar tissues (fig. 5) . rates of detection of the paramyxoviruses hmpv and hrsv varied from 0% to 31% during the study period (figs. 6 and 7 ). hrsv detection reached maximum level in spring/winter months, mostly composed by detection in respiratory secretions, with summer nadirs in both years of the study (fig. 6) . differently, hmpv overall detection rates did not vary significantly during the study period (fig. 7) , and the nadir observed in the summer of 2012 probably reflects the low number of samples analysed in that season. flu, hpiv and hcov were detected in very low frequencies, with sporadic cases distributed during the 2-year study period without seasonal pattern. several studies have shown that some respiratory viruses circulate seasonally, with a typically increase of the viral incidence in colder months, mainly in temperate regions [12] , but also in subtropical regions [15] [16] [17] . in tropical regions the results are more difficult to interpret, with several studies indicating higher viral circulation in rainy seasons [14, [19] [20] [21] , while others show that respiratory viruses are prevalent year-round [22] . in salvador for instance, a tropical city in the northeast of brazil, the presence of viral infections was significantly associated with precipitation during the rainy season in patients with community-acquired pneumonia [19] . it is broadly accepted that respiratory viruses spread by shedding in secretions from acutely symptomatic patients [12] , but respiratory viruses are also frequently detected in asymptomatic individuals [23] [24] [25] , raising the hypothesis that the viral shedding by people without acute symptoms can be important to the viral dissemination. in fact, high frequencies of detection of respiratory viruses have also been observed in secretions and tissues from patients with chronic adenotonsillar diseases [3, 5, 26, 27] , but no analysis of seasonality had been done in that particular setting. although the presently reported analyses confirmed previously published findings that found high rates of viral detection in patients with chronic adenotonsillar diseases, the analysis of the fluctuations in viral detection rate during these 2 years showed that most of respiratory viruses have no obvious seasonal pattern, supporting the notion that such high frequency of virus genome detection can be related to virus persistence in lymphoepithelial tissues of the upper respiratory tract. the discovery of hadv was consequence of its recovery from adenoid explants [28] , and several studies have documented that several adenovirus species, especially adenovirus c, can persist in mucosal lymphoid tissues, possibly by maintenance of the quiescent viral genomes in non-dividing lymphocytes [29, 30] . in fact, hadv causes persistent/latent infection in tonsillar t-cell subpopulations [29] , and can infect continuous b-cell and myeloid cell lineages in vitro [30] , suggesting that several different cell populations in tonsils may carry the virus genome. therefore, it was no surprise that in the present study hadv was the most frequent respiratory virus detected in adenoids, and the second most frequent in palatine tonsils. in addition, the lack of seasonal trends in rates of hadv detection, both in tissues and secretions, is confirmatory that most of the high hadv frequency is attributable to persistence. in tropical regions, adenovirus is frequently associated with the rainy season in patients with acute respiratory infections [19] . hbov is a parvovirus that occurs worldwide in association with respiratory and gastrointestinal disorders [31, 32] . in addition to the general propensity of parvoviruses to persist and even endogenise into host genomes [33] , at least two lines of evidence support persistence of hbov in humans. only around 25% of patients who are pcr-positive for hbov have mrna for a viral structural protein detectable as a marker of active viral replication [34] and hbov episomes have been found in human clinical samples, including tissue biopsies [35] [36] [37] . therefore, it is not surprising that in the present study hbov was frequently detected, without discernible seasonality, suggesting that, at least part of [ ( f i g . _ 6 ) t d $ f i g ] this high frequency could be attributed to virus persistence, mainly in adenoids. while persistence and latency of dna viruses in lymphoepithelial tissues have long been known, the same is not the case for rna viruses. in the present study, hev was the second most frequently detected agent at overall frequencies consistently above 30%. although there are very few studies about seasonality of hev in tropical regions, the observed trend for an increase in hev detection towards the summer is in keeping with what happens in temperate regions of the world [38] . however, the high rates of hev positivity were consistently due to detection in tissue fragments, which was less frequently accompanied by shedding in secretions, supporting the idea that hev can persist in adenoids and tonsils in a high proportion of patients. while confirmation of hev persistence in tonsillar tissues will require detailed molecular investigation, the present findings of such consistently and non-seasonal hev detection over time, coupled with the already reported higher frequencies of hev detection in the most highly hypertrophic tonsils [3] indicates that perhaps hev persistence can be associated with the pathogenesis of chronic adenotonsillar diseases. hrv, the other picornavirus included in the present analyses, is frequently detected in acute respiratory infections of children and adults, usually with marked seasonal variation, especially in subtropical and temperate areas [39, 40] , as well as in asymptomatic patients [24] . in tropical regions, the literature results are controversial. in trinidad, west indies, hrv was prevalent throughout the year, without seasonal association [41] , whilst in salvador hrv was associated with relative humidity (p = 0.05) [19] . in the present analysis hrv was frequently detected in all seasons, mostly in secretions, rather than in tissues. it is interesting that hrv and hev are both picornaviruses, currently classified in the same genus, with very similar replication cycles, and yet showed dissimilar frequencies of detection in tissues. hrv was detected in lower frequencies in lymphoid tissues as compared to hev, suggesting the existence of other sites of infection as sources of hrv shedding into secretions, such as nasal epithelium. this is remarkable, since the patients with hypertrophic adenotonsillar diseases had no acute nasal symptoms at the time of surgery. although the overall rates of detection of hmpv and hrsv in the present analyses were lower than those of other viruses, they were still frequently higher than 20%. moreover, hrsv rates were frequently higher than 10%, with significant contribution of positivity in adenotonsillar tissues, again suggesting that these tissues may be regarded as sites of persistence of paramyxoviruses. pertaining to this issue, it is interesting that phylogenetic studies of respiratory syncytial virus have pointed to the existence of reservoirs to maintain the virus during inter-seasonal periods, thus creating potential for its reintroduction in the susceptible population [42] . it is therefore reasonable to think that adenoids and tonsils of children with chronic adenotonsillar diseases would be natural reservoirs of respiratory viruses and that, by shedding viruses in respiratory secretions, these children would become sources of infection for their siblings and schoolmates. to the best of our knowledge, this has been the most comprehensive study so far conducted on the variations of respiratory virus detection rates over time in children with chronic adenotonsillar diseases in a subtropical/tropical region. although further studies are in order to clarify whether these findings result from long term virus shedding consequent to persistence, or to current asymptomatic viral infections, the lack of obvious seasonal patterns of respiratory viruses in hypertrophic adenotonsillar tissues supports the view that some respiratory viruses may persist in adenoids and tonsils. in the absence of results from molecular markers of active viral replication, the mere detection of viral genomes is not enough to establish that such infections were productive. however, the existence of genomes of numerous viruses in adenotonsillar tissues is an exciting finding, and deserves further investigation. in addition to its obvious epidemiological importance as possible sources of community respiratory virus outbreaks, persistence of these viruses could have pathogenic potential in the development of tonsillar hypertrophy, functioning as chronic stimuli for inflammation. alternatively, for reasons not yet understood, respiratory viruses could more readily establish an asymptomatic carrier states in patients with chronic tonsillar hypertrophy. however, the lack of control group, with samples of tonsils obtained from healthy patients, makes any inference about the development of disease difficult to be explored in the present study. thereby, studies about viral persistence in adenoids and tonsils, mainly those including tissue samples from healthy subjects, can bring new insights to the understanding of poorly understood chronic tonsillar diseases that affect large numbers of children worldwide. respiratory viral infections in infants: causes, clinical symptoms, virology, and immunology tonsillectomy compared to acute tonsillitis in children: a comparison study of societal costs high rates of detection of respiratory viruses in tonsillar tissues from children with chronic adenotonsillar disease rhinovirus/enterovirus rna in tonsillar tissue of children with tonsillar disease microbiological profile of adenoid hypertrophy correlates to clinical diagnosis in children update in obstructive sleep apnea syndrome in children impaired right ventricular function in adenotonsillar hypertrophy age-dependent changes in the size of adenotonsillar tissue in childhood: implications for sleep-disordered breathing pathologic evaluation of routine tonsillectomy and adenoidectomy specimens in the pediatric population: is it really necessary? relevance of biofilms in pediatric tonsillar disease is there any correlation between allergy and adenotonsillar tissue hypertrophy? impact of pollution, climate, and sociodemographic factors on spatiotemporal dynamics of seasonal respiratory viruses correlations between climate factors and incidence -a contributor to rsv seasonality epidemiology and seasonality of respiratory tract virus infections in the tropics acute respiratory infection and influenza-like illness viral etiologies in brazilian adults occurrence and severity of infections caused by subgroup a and b respiratory syncytial virus in children in southeast brazil viral etiology of acute respiratory infections among children in porto alegre efficacy of tonsillectomy for recurrent throat infection in severely affected children. results of parallel randomized and nonrandomized clinical trials seasonal patterns of viral and bacterial infections among children hospitalized with community-acquired pneumonia in a tropical region respiratory syncytial virus circulation in seven countries with global disease detection regional centers etiology and seasonality of viral respiratory infections in rural honduran children latitudinal variations in seasonal activity of influenza and respiratory syncytial virus (rsv): a global comparative review detecting respiratory viruses in asymptomatic children risk factors for wheezing in a subtropical environment: role of respiratory viruses and allergen sensitization picornavirus infections in children diagnosed by rt-pcr during longitudinal surveillance with weekly sampling: association with symptomatic illness and effect of season frequent detection of respiratory viruses by real-time pcr in adenoid samples from asymptomatic children detection of viruses in human adenoid tissues by use of multiplex pcr group name proposed for new respiratory-tract viruses latent species c adenoviruses in human tonsil tissues modeling adenovirus latency in human lymphocyte cell lines andersson, cloning of a human parvovirus by molecular screening of respiratory tract samples human bocavirus, a respiratory and enteric virus widespread endogenization of densoviruses and parvoviruses in animal and human genomes detection of human bocavirus mrna in respiratory secretions correlates with high viral load and concurrent diarrhea bocavirus episome in infected human tissue contains non-identical termini detection of head-to-tail dna sequences of human bocavirus in clinical samples detection of a bocavirus circular genome in fecal specimens from children with acute diarrhea in beijing enterovirus surveillance -united states respiratory picornaviruses and respiratory syncytial virus as causative agents of acute expiratory wheezing in children acute viral respiratory infection in children under 5 years: epidemiological study in two centers in distribution and seasonality of rhinovirus and other respiratory viruses in a cross-section of asthmatic children in trinidad, west indies phylodynamics and dispersal of hrsv entails its permanence in the general population in between yearly outbreaks in children the authors thank maria cecília onofre and helder g. de souza for secretarial assistance; lú cia lopes, jamila mendonça de souza and maria lú cia silva for expert technical support. in addition, the authors thank fapesp (fundação de amparo à pesquisa do estado de são paulo) for financial support (grant number 2009/51818-8). the authors have no conflicts of interest to disclose and have no financial disclosures to make. key: cord-335505-s013j5ex authors: zhang, chen; zhu, na; xie, zhengde; lu, roujian; he, bin; liu, chunyan; ma, xuejun; tan, wenjie title: viral etiology and clinical profiles of children with severe acute respiratory infections in china date: 2013-08-22 journal: plos one doi: 10.1371/journal.pone.0072606 sha: doc_id: 335505 cord_uid: s013j5ex background: no comprehensive analysis is available on the viral etiology and clinical characterization among children with severe acute respiratory infection (sari) in china during 2009 h1n1 pandemic and post-pandemic period. methods: cohort of 370 hospitalized children (1 to 72 months) with sari from may 2008 to march 2010 was enrolled in this study. nasopharyngeal aspirate (npa) specimens were tested by a commercial assay for 18 respiratory viral targets. the viral distribution and its association with clinical character were statistically analyzed. results: viral pathogen was detected in 350 (94.29%) of children with sari. overall, the most popular viruses were: enterovirus/rhinovirus (ev/rv) (54.05%), respiratory syncytial virus (rsv) (51.08%), human bocavirus (boca) (33.78%), human parainfluenzaviruse type 3 (piv3) (15.41%), and adenovirus (adv) (12.97%). pandemic h1n1 was the dominant influenza virus (ifv) but was only detected in 20 (5.41%) of children. moreover, detection rate of rsv and human metapneumovirus (hmpv) among suburb participants were significantly higher than that of urban area (p<0.05). incidence of vsari among suburb participants was also significant higher, especially among those of 24 to 59 months group (p<0.05). conclusion: piconaviruses (ev/rv) and paramyxoviruses are the most popular viral pathogens among children with sari in this study. rsv and hmpv significantly increase the risk of sari, especially in children younger than 24 months. higher incidence of vsari and more susceptibilities to rsv and hmpv infections were found in suburban patients. acute respiratory infection (ari) is the leading causes of children death globally [1] [2] [3] [4] [5] . even in developed country, severe acute respiratory infection (sari), with its high mortality and morbidity among children younger than 5, impose great burden on the society [2, 3] . lacking of etiologic diagnosis is the main reason that more than 50% of ari cases suffered unnecessary or inappropriate prescription of antibiotics, since most acute respiratory tract infections are caused by viruses [3] . this often leads to severe consequence such as high rate of resistance [6] , especially in those with severe acute respiratory infection (sari), which frequently happens in virus-infected children [3] . therefore etiology studies, especially those of viruses, have been performed to help diagnosis and proper antiviral treatment of sari [7, 8] , as well as prevention of nosocomial infections among in-patients [9] . diagnosis of ari is complicated by the wide range of potential pathogens that can present with similar clinical symptoms [10, 11] . in recent years, the introduction of nucleic acid based diagnostic tests has markedly improved our ability in understanding viral etiology among ari patients [12] . xtag® rvp fast, a test based on multiplex pcr and luminex molecular diagnostics universal array that detects 18 most commonly seen viral targets in respiratory infection, is approved by the us food and drug administration (fda) and has shown improved viral detection ability as compared to traditional methods like direct fluorescent antibody (dfa) and culture methodology [13] [14] [15] [16] [17] [18] [19] . shortly after the advent of severe acute respiratory syndrome (sars) and the avian influenza, the emergence of the influenza virus a (h1n1) 2009 pandemic caused significant vibrations to the public health authorities and stressed the health systems worldwide. this highlighted our weaknesses regarding the diagnosis and assessment of sari. china is the biggest developing country with largest population of child. however, we have found no published case-control studies regarding comprehensive viral etiology and clinical characterization of children sari using commonly acknowledged reliable test in china. to better understand the role of respiratory viruses in children with sari during 2009 h1n1 pandemic and postpandemic era, and help diagnosis and antiviral treatment, we conducted a comprehensive evaluation of viral etiology and clinical characterization among hospitalized children with sari admitted to the beijing children hospital from may 2008 to march 2010. this study has increased our knowledge on the management of sari and community-acquired pneumonia. the study protocol was approved by the institutional review board of national institute for viral disease control and prevention, china cdc, and the scientific committee of the beijing pediatric research institute, and the ethical review committee of beijing children's hospital. individual written informed consent was obtained from the parents or guardians of all participants. all participants in this study were inpatients from beijing children's hospital between may 2008 and march 2010, diagnosed as sari based on clinical grounds recommended by the world health organization (who) (20) . eligibility and classification of the clinical syndromes of sari were determined from individual's original record of medical history and examination. the criteria of hospitalized patient inclusion were: sudden onset of fever >38 o c and cough or sore throat and difficulty breathing (dyspnea, oxygen saturation < 90%). additional criteria were a normal or low leukocyte count, or lower chest wall indrawing. among these sari cases, 120 were defined as very severe acute respiratory infection (vsari) cases with any of the following criteria: 1) incidence of complication such as vomit or diarrhea, 2) respiratory failure or anhelation or heart failure, 3) icu admissions. nasopharyngeal aspirate (npa) or blood or induced sputum (is) was collected from the patients at the first day of admission and transferred into virus transport medium and stored at -70 o c until tested. demographic information and medical test result were collected with standardized forms. nucleic acid was extracted from 200 µl of the virus transport medium using qiaamp minelute virus spin kit (qiagen, mississauga, ontario, canada) according to the manufacturer's instructions. 10 µl of the nucleic acids were tested using xtag® rvp fast assay according to the manufacturer's instructions (abbott molecular inc., usa) and analyzed on bio-plex 200 system (bio-rad laboratories, inc., usa). the rvp fast assay simultaneously detects the following viruses: respiratory syncytial virus (rsv); influenza(ifv) a (h1, h3, and h5) and b viruses; parainfluenza viruse (piv) 1, 2, 3, and 4; human metapneumovirus (hmpv); adenovirus(adv); piconavirus(pic) which includes enterovirus (ev) and rhinovirus (rv); human coronaviruse(hcov) nl63, hku1, 229e, and oc43; and human bocavirus(boca). the assay also includes an internal positive control added to each specimen at the extraction stage (escherichia coli phage ms2 rna) and a positive run control that is added to each run (bacteriophage lambda dna). comparisons between urban and suburb patients, as well as severe and very severe infection, were performed using chisquare test. correlation of virus detected and clinical signs or diagnosis was performed using binary logistic regression of spss statistics 17.0. comparison of continuous variables like body temperature was conducted using variance analysis. the clinical symptoms of all 370 participants presented with different severe respiratory infection signs including convulsion, shock, pleural effusion, cough, wet rale, dry rale, expectoration, vomiting, respiratory failure, and heart failure. the three most commonly observed symptoms were cough (97.57%), wet rale (62.43%), and expectoration (54.32%). 341 participants showed parenchymal infiltration in chest radiography (data not shown). viral prevalence is also presented in table 1 .5-16.2%), orthomyxoviruses (7.84%, ifva,7.03%; ifvb, 0.81%) and hcovs (6.76%: hcov-oc43, 4.32%; hcov-229e, 0.81%; hcov-nl63, 1.08%; and hcov-hku1, 1.08%). pandemic h1n1 was the dominant influenza virus(ifv) but only detected in 20 (5.41%) of children enrolled in this study. we noticed that all pandemic h1n1 infection took place during 2009(from january through august). figure 1 shows the seasonal distribution of few dominant respiratory viruses and incidence of sari. ev/rv was the most frequently discovered pathogen in this study; it was active throughout whole year, with a flat line of infection rate around 50% ( figure 1a) . rsvb, in contrast to ev/rv, only prevailed in winter. it infected 60-100% patients in winter, but almost disappeared in summer ( figure 1b) . hmpv showed similar distribution as rsvb, but the infection rate was much lower ( figure 1d ). of all the other 3 dominant pathogens: both piv3 ( figure 1c ) and boca( figure 1e ) showed a peak in the august of 2009, however adv ( figure 1f ) infection was always active with a flat line from september of 2008 to december of 2009. patients from urban area are obviously older than suburb/ rural ones, average age 15.34 vs. 8.72 months ( table 2 ). further analysis of age distribution based on chi-test showed that ratio of young children (24 to 59 months) is obviously higher in rural area (21.6% vs. 3.9%, p < 0.001, figure 2a) . several viruses are more frequently detected among suburb/ rural patients( to find the reason that causes severe infection, we performed complete comparison between vsari patients and the sari, including clinical signs, number of viral target, gender, and age(table 3, figure 2b ). no difference in gender or average age was discovered between vsari and sari patients. also multiple infection does not have significant impact in severity of infection (p=0.11). however, comparison of age group showed some difference (p = 6.93e-05). judging from detailed list of figure 2b , it is obvious that patients older than 24 months are more resistant to very severe infection (3.16% vs. 19.64%). to find the association between virus infection and clinical signs in sari, binary logistic regression was performed between 4 commonly diagnosed respiratory abnormality, including anhelation, respiratory failure, heart failure and pleural effusion, and the viral target detected by xtag® rvp fast. as shown in table 4 association between commonly diagnosed respiratory disease and virus detection was also performed by binary logistic regression. as shown in table 5 , both rsva (p=0.00, or=11.11; 95%ci, 2.08-50) and rsvb (p=0.00, or=10; 95%ci, 2.38-50) is associated with bronchiolitis and hmpv associated with pneumonia/bronchopneumonia (p=0.00, or=14.29, 95%ci, 2.78-100). in this study, we performed systematic study of viral etiology and clinical profiles among children with sari admitted in beijing children's hospital between may 2008 and march 2010. unlike most previous studies in china, which primarily focused on common acute respiratory infections in children [20] [21] [22] [23] , we exclusively studied the in-hospital patients with deeper infection in the respiratory tract and severer symptoms. 32.34% of the cases in this study suffered very severe symptoms such as heart failure and respiratory failure, as well as complications like diarrhea and vomiting. another result that is different from previous study is a co-infection rate of 64.32%. besides the special focus on in-patients, a broader viral spectrum and higher sensitivity of xtag® rvp fast test may also give rise to this [15] [16] [17] [18] 24] . the most supportive evidence is an unprecedented high infection rates of ev/rv (54.05%), which is consistent with previous report that xtag rvp fast is extremely sensitive in ev/rv detection compared to other molecular based methods [19, 25] . virus infection in children with sari plos one | www.plosone.org who and the pan american health organization recommend hospital-based surveillance of severe acute respiratory infections (sari) as a tool to monitor severe disease caused by influenza. although the study was performed during 2009 h1n1 pandemic period, our data showed that the special cohort of sari patients resulted in relatively low incidence of influenza viruses (7.03%). pandemic h1n1 was the dominant influenza virus(ifv) but was only detected in 20 (5.41%) of children. as reported in previous study [5, 26] , children infected with influenza viruses are usually older than those suffered from rsv, while more than 80% patients in this study were under 24 months old. paramyxoviruses, especially rsv and hmpv, are might prevalence and might be the main reason of sari outbreak during 2008 winter to 2009 spring in this study. similar to previous studies [5, [27] [28] [29] , rsv played an important role in children of sari. it showed obvious seasonal distribution and was obviously related to several respiratory symptoms. infection of rsvb increased and peaked with increase of sari cases during september 2008 through march 2009. both rsva and rsvb increased the risk of bronchiolitis with high odds ratio ( table 5 ). the obvious relationship between rsv and severe respiratory symptoms coincides with a similar retrospective study performed on kenya children of severe pneumonia, in which rsv, among all commonly discovered chi-square test respiratory viruses, was found to be the only associated virus with children pneumonia [7, 30] . another paramyxoviruse, hmpv, showed similar seasonality as rsvb, although the infection rate was not high among all cases. what's more important is that infection of hmpv is obviously related to all three vsri-related symptoms: anhelation, heart failure, and respiratory failure ( table 4) . the third commonly detected paramyxovirus was piv3, a viral target infects about 1-13% of children with pulmonary diseases. infection rate varies with age and severity of cohort among different studies [5, 31] . piv3 is the predominant subtype among parainfluenza viruses, and was responsible for 83.8% parainfluenza infection in this cohort. to our knowledge, piv3 was more related with immunocompromised children than common sari patients in previous studies [32] [33] [34] [35] . it has frequently been reported that infection of rsv and hmpv result in same or similar symptoms and were indistinguishable on clinical basis [9] [10] [11] 36, 37] . however, our analysis of association produced somehow different results: hmpv was more directly related with vsari and thus improved risk of pneumonia, while rsv was more related to risk of bronchiolitis. in contrast to paramyxoviruses, infection rate of ev/rv distributed relatively even throughout the whole year, in spite of its high total detection rate (50.4%). this assumption is supported by association study, in which no confident relationship was found between ev/rv and any severe symptoms (table 4 ) or any diagnosis (table 5 ). in reference to previous studies, ev/rv in respiratory tract is frequently detected by pcr among children of upper respiratory infection and asymptomatic controls. however, few evidence has been found supporting association between ev/rv detection and severe lower respiratory symptoms [36, 37] . further studies are needed to address the relevance of the ev/rv single and coinfections on clinical outcomes. great difference was discovered between urban and suburb patients in this study. children from suburb area are apparently younger, with higher incidence of sari, and were more susceptible to rsv, hmpv, and ev/rv. bronchiolitis and asthmatic bronchitis also happened more frequently in suburb area ( table 2) . for each of the five most popular pathogens: boca, adv, rsv, ev/rv (piconaviruses), and piv3, we performed binary logistic regression between existence of co-infection and all clinical signs. unfortunately, no statistical significance was observed in any run of regression data not shown). to our knowledge, this is the first report on the viral etiology, epidemiological and clinical profiles of hospitalized children with sari using a commercial assay for 18 respiratory viral targets in asia. improved laboratory test highlighted the significance of viral etiology and its distribution among children with sari in china. our data is similar to the recent surveillance report on sari in south africa by use of the validated rt-pcr multiplex assay(39), which is also consistent with several studies in other parts of the world (3, 7, 30) . secondary, comparison analysis was firstly performed between urban and suburban/rural patients in china; in addition, this is also the first study on association analysis estimates of mortality in children younger than 5 years for burkina faso a molecular epidemiological study of respiratory viruses detected in japanese children with acute wheezing illness respiratory viral infections in infants: causes, clinical symptoms, virology, and immunology clinical and epidemiological comparison of human metapneumovirus and respiratory syncytial virus in seoul epidemiology and seasonality of respiratory viral infections in hospitalized children in kuala lumpur, malaysia: a retrospective study of 27 years the global problem of antibiotic resistance a preliminary study of pneumonia etiology among hospitalized children in kenya etiology and epidemiology of viral pneumonia among hospitalized children in rural mozambique: a malaria endemic area with high prevalence of human immunodeficiency virus the burden of respiratory syncytial virus infection in young children human metapneumovirus and severity of respiratory syncytial virus disease detection of human metapneumovirus in infants with acute respiratory tract infection detection of respiratory viruses by molecular methods development of a respiratory virus panel test for detection of twenty human respiratory viruses by use of multiplex pcr and a fluid microbead-based assay comparison of the bd directigen flu a+b kit and the abbott testpack rsv with a multiplex rt-pcr elisa for rapid detection of influenza viruses and respiratory syncytial virus comparison of the luminex respiratory virus panel fast assay with in-house real-time pcr for respiratory viral infection diagnosis evaluation of multiple test methods for the detection of the novel 2009 influenza a (h1n1) during the new york city outbreak enhanced viral etiological diagnosis of respiratory system infection outbreaks by use of a multitarget nucleic acid amplification assay performance of a rapid molecular multiplex assay for the detection of influenza and picornaviruses comparison of the luminex xtag rvp fast assay and the idaho technology filmarray rp assay for detection of respiratory viruses in pediatric patients at a cancer hospital the epidemiology and etiology of influenza-like illness in chinese children from 2008 to 2010 three years surveillance of viral etiology of acute lower respiratory tract infection in children from clinical study on interstitial lung disease in children of china performance of the luminex xtag respiratory viral panel fast in a clinical laboratory setting comparison of three multiplex pcr assays for the detection of respiratory viral infections: evaluation of xtag respiratory virus panel fast assay, respifinder 19 assay and respifinder smart 22 assay etiology and incidence of viral and bacterial acute respiratory illness among older children and adults in rural western kenya molecular monitoring of causative viruses in child acute respiratory infection in endemo-epidemic situations in shanghai respiratory viruses in hospitalized children with acute lower respiratory tract infections in harbin, china viral etiology of bronchiolitis among pediatric inpatients in northern taiwan with emphasis on newly identified respiratory viruses viral etiology of severe pneumonia among kenyan infants and children impact of parainfluenza virus infection in pediatric cancer patients parainfluenza 3 virus and other common respiratory pathogens in children with human immunodeficiency virus infection parainfluenza and influenza virus infections in pediatric organ transplant recipients parainfluenza virus infection in adult lung transplant recipients: an emergent clinical syndrome with implications on allograft function comparison of results of detection of rhinovirus by pcr and viral culture in human nasal wash specimens from subjects with and without clinical symptoms of respiratory illness persistence of rhinovirus and enterovirus rna after acute respiratory illness in children we are grateful to the staff and patients of the pediatric wards of beijing children hospital, and to the clinical, information and communication technology, and laboratory staff who contributed to this study. we thank dr lyna zhang, cdc of usa, for their proofreading of this manuscript. no compensation was received for participation in this research. conceived and designed the experiments: cz zx xm wt. performed the experiments: cz nz xz rl bh cl. analyzed the data: cz nz zx wt. contributed reagents/materials/ analysis tools: rl. wrote the manuscript: cz nz wt. key: cord-343365-4y9fedcr authors: chang, christopher title: unmet needs in respiratory diseases: “you can’t know where you are going until you know where you have been”—anonymous date: 2013-11-30 journal: clin rev allergy immunol doi: 10.1007/s12016-013-8399-2 sha: doc_id: 343365 cord_uid: 4y9fedcr the care of patients with respiratory diseases has improved vastly in the past 50 years. in spite of that, there are still massive challenges that have not been resolved. although the incidence of tuberculosis has decreased in the developed world, it is still a significant public health problem in the rest of the world. there are still over 2 million deaths annually from tuberculosis, with most of these occurring in the developing world. even with the development of new pharmaceuticals to treat tuberculosis, there is no indication that the disease will be eradicated. respiratory syncytial virus, severe acute respiratory syndrome, and pertussis are other respiratory infectious diseases with special problems of their own, from vaccine development to vaccine coverage. asthma, one of the most common chronic diseases in children, still accounts for significant mortality and morbidity, as well as high health care costs worldwide. even in developed countries such as the usa, there are over 4,000 deaths per year. severe asthma presents a special problem, but the question is whether there can be one treatment pathway for all patients with severe asthma. severe asthma is a heterogeneous disease with many phenotypes and endotypes. the gene for cystic fibrosis was discovered over 24 years ago. the promise of gene therapy as a cure for the disease has fizzled out, and while new antimicrobials and other pharmaceuticals promise improved longevity and better quality of life, the average life span of a patient with cystic fibrosis is still at about 35 years. what are the prospects for gene therapy in the twenty-first century? autoimmune diseases of the lung pose a different set of challenges, including the development of biomarkers to diagnose and monitor the disease and biological modulators to treat the disease. the study and management of lung diseases is of concern to allergist/immunologists and pulmonologists. the most common chronic lung disease is asthma, but there is an immunological basis for many other lung diseases, which span a vast clinical spectrum of lung disorders ranging from infectious lung disease to cancer. several significant challenge areas include the diagnosis and treatment of certain specific infectious lung diseases, including viral lower respiratory infections caused by respiratory syncytial virus, rhinovirus, metapneumovirus, coronovirus, and enterovirus. severe acute respiratory syndrome (sars) coronavirus (sars-coa) was responsible for the outbreak of severe acute respiratory syndrome in the early 2000s that originated in asia and led to significant mortality in those afflicted. other viruses such as bird flu and swine flu have the ability to cause respiratory tract disease as well. tuberculosis is another primarily respiratory infection that has been resistant to eradication. new strains of multidrug-resistant tuberculosis have emerged. other challenges involve the genetic diseases cystic fibrosis and alpha-1-antitrypsin deficiency, autoimmune lung diseases, lung diseases that are part of a systemic autoimmune disease, and chronic obstructive pulmonary disease (copd). the diagnosis and treatment of hypersensitivity pneumonitis still poses problems to clinicians. biomarkers are continually being developed in lung and other cancers, but more research is needed [1] . the genetics of cystic fibrosis are now well elucidated, but the development of a successful gene therapy has been unexpectedly slow (table 1) . for many of these conditions, it was and still is expected that the ongoing development of new waves of molecular biology techniques, coupled with computerized automated analysis that can provide transcription signatures, will help identify differences between patients with these diseases and spur on the development of relevant and effective treatments. the subsequent discussion takes on some of the more interesting and challenging issues in respiratory disease over the past two decades, with a focus on what to expect in the future as well. asthma is one of the most common chronic diseases worldwide. the who estimates that there are over 300 million asthmatics worldwide and 250,000 deaths per year. in the usa, there are 22 million asthmatics, 6.5 million of whom are children. until recently, the mortality rates from asthma had been steadily increasing. it is only in the past 5-10 years that the mortality rates have begun to stabilize and are perhaps even on a decline. the increasing incidence of asthma has been attributed to various theories, perhaps the most popular of which is the "hygiene hypothesis" [2] . this attributes the rise in incidence to the reduced rate of infectious diseases, cleaner living environments, and, in general, those features that accompany a higher standard of living. but, conversely, the annual death rates per 100,000 asthmatics tend to be higher in the economically poorer countries. this emphasizes the most significant global concern regarding asthma care, which is access to care and medications. in developed countries, this problem is less prevalent, and in countries such as the usa, we are primarily focused on asthma education and compliance issues. in both situations, severe asthma is still a problem in that it impacts quality of life and still contributes to considerable morbidity and mortality. one of the problems of severe asthma is related to defining the disease. asthma is inherently very heterogeneous, and many phenotypes of severe asthma have been described. it is increasingly clear that there is no single medication that works for all patients with severe asthma. while patient compliance is an issue, we frequently fall into the trap of believing the problem is always patient compliance, when in fact, it may be that patients are not taking their medications because they perceive that they do not work. nevertheless, the explosive growth in the availability of new pharmaceutical interventions provides us with an arsenal of controller medications, at least in the developed world. more medications are being developed every day, including the newer class of biological modulators against cytokines and chemokines [3] , such as anti-il5 [4, 5] and anti-il4rα [6, 7] . the next challenge will involve the identification of suitable patients for these new and existing treatments. but how will this be done? researchers over the years have attempted to define asthma phenotypes, trying to categorize asthma patients based on demographic and clinical characteristics in an attempt to define certain patient groups for certain preferential treatments [8] [9] [10] [11] [12] [13] [14] [15] [16] . in general, this has led to more confusion as the different research groups do not necessarily come up with the same classifications. moreover, asthma in children is also significantly different from that in adults, and the response to medications varies based on multiple and not a single factor. the choice and exclusion of confounding variables has been a thorn in the side of those attempting to define a specific asthma phenotype. it has also not been found that specific childhood asthma phenotype will develop into a specific adult asthma phenotype. the concept of asthma endotypes has now been the subject of much research because endotypes address the underlying mechanism of a disease [17] [18] [19] [20] . however, this is also confusing because of the highly redundant inflammatory pathways that can lead to disease. the use of biomarkers to identify those patients who are at high risk for severe, lifethreatening exacerbations has not been entirely fruitful. the recent commercial introduction of fractional exhaled nitric oxide (feno) to identify patients with eosinophilic inflammation is a potentially useful tool, but more is needed to guide treatment. the identification of a gene for asthma has revealed that this is more complex than one might have expected. there are now over 100 genes that have been attributed to asthma. some of the more likely candidates include adam33, ormdl3, dennd1b, filaggrin, chi3l1, and il-33. moreover, the existence of shared genes between asthma and other comorbid conditions such as obesity has also been demonstrated [21] . ultimately, asthma, like many other diseases, is a heterogeneous disease with many phenotypes and endotypes [17, 18] . not one single treatment will fit all, and the art of medicine may be to find the right peg for the corresponding hole [22] . the twenty-first century has ushered the concept of personalized or genomic medicine, which utilizes advancements in molecular biology, such as proteomics, metabolomics, and genomics, to identify the optimal therapy for each individual patient. cystic fibrosis-what happened to gene therapy? it has now been 24 years since the identification of the gene responsible for cystic fibrosis (cf) [23] [24] [25] [26] [27] [28] [29] . the initial study describing the gene named "cystic fibrosis transmembrane conductance receptor (cftr)" was first published in the late 1980s by tsui et al. [26, [29] [30] [31] [32] [33] [34] [35] [36] . this discovery led to a localization of the gene to chromosome 7, leading to widespread belief that a cure for cf utilizing gene therapy was right around the corner. the landmark development of a molecular biology technique which became known as genetic targeting in mice, pioneered by capecchi [37] [38] [39] [40] , evans [41, 42] , and smithies [43] [44] [45] [46] [47] , promised gene replacement in humans in a similar way to that performed in mice. twentyfour years later, there are new advancements in the management of cystic fibrosis with regard to pharmacological and supportive respiratory treatment, but still no cure. these advancements have prolonged the longevity of patients with cf, but their life expectancy still only averages to be about 35 years. so, what happened to gene therapy? what happened to all that promise that buoyed the cystic fibrosis community back in the 1980s? the idea behind gene therapy is to introduce a missing of malfunctioning gene into the cells of a patient using a "harmless" virus that can be manufactured to carry a normal copy of the diseased gene. this technique can either target cells globally or be restricted to a certain group or location of cells. but gene therapy has not progressed as smoothly or as quickly as anticipated. previously unrecognized barriers became apparent [48] . these included the fact that studies were initially focused on molecular or biochemical outcomes and not on clinical efficacy. moreover, the administration of the gene would need to be repeated due to epithelial turnover. repeated administration leads to host recognition, which may inhibit gene expression. questions on which cells to target in order to achieve efficacy has slowed research. the use of viral material, including viral dna and liposomes, could potentially lead to inflammatory responses. the point is that unexpected consequences were discovered as research proceeded, and this has had an impact on the progress of gene research. in 2012, a group of british investigators began large-scale trials of gene therapy delivered by encompassing the gene in fat globules and delivering the gene by nebulization. future methodologies will utilize a viral delivery strategy, but this is still several years into the future. gene therapy for cystic fibrosis is not dead, but certainly moving at a far slower pace than originally expected. current treatments are not curative for cystic fibrosis. gene therapy and stem cell transplants are two techniques that are still under investigation, and it may turn out that only certain mutations may be candidates for gene therapy. mutations in the cf gene have in fact been subclassified into six classes [49] , each with its own pathogenic or physiologic characteristics. for example, the common f508del mutation results in reduced amounts of cftr channel expression, which leads to exacerbation of the disease [50] . respiratory syncytial virus-how to develop an effective vaccine? respiratory syncytial virus is an infection of the lower respiratory tract that causes significant morbidity and mortality in infants and young children [51] . globally, there are over 30 million new lower respiratory tract infections per year and approximately 200,000 deaths per year in children fewer than 5 years of age. the majority of these deaths occur in lowincome countries where access to care may be limited. the search for a vaccine for respiratory syncytial virus (rsv) has been ongoing for many years, but like the previous case of gene therapy in cystic fibrosis, this also has been a challenge to achieve. in the absence of a vaccine, researchers have developed passive immunization to the virus in the form of a monoclonal antibody to rsv, named palivizumab. the current global strategies for the development of an rsv vaccine now target four areas: infants <6 months of age; infants >6 months of age and young children; pregnant women for whom passive immunization can be implemented; and the elderly, in whom rsv can also have significant morbidity [52] [53] [54] . the main challenges that have prevented the development of an effective vaccine so far revolve around the fact that even natural infection to rsv does not provide long-term protection from reinfection. an earlier study actually found that the vaccine may actually accentuate the disease, especially in young infants who may not have a fully mature immune system and may be unable to effectively engage in somatic mutation, leading to a suboptimal b cell repertoire [55, 56] . this may also apply to older infants or young children who may still be rsv-naive. the issues in adults are just the opposite as most adults have been exposed to rsv and thus have rsv antibodies. the effectiveness of passively immunizing pregnant women in order to deliver igg across the placenta to the fetus must be weighed against possible adverse effects of such a "vaccine" on the fetus. in the elderly, the challenge has been the ability to boost immunity through active immunization in an individual who is becoming immunosenescent. there are now several vaccine development programs that pursue a variety of vaccine strategies. a live virus vaccine trial has shown that a particular vaccine candidate, medi-559, is safe, although effectiveness has not been proven [57] . other strategies include subunit rsv vaccines, the use of dnaconjugate vaccines to boost antigen presentation, and further development of passive antibody prophylaxis that is focused on f or g proteins [52, [58] [59] [60] [61] [62] [63] [64] [65] [66] [67] [68] [69] [70] [71] [72] . an additional challenge with all of these strategies is that effective vaccine studies in animals have not been translated into successful human trials. tuberculosis is a global health problem. tuberculosis is an infection caused by the bacterium mycobacterium tuberculosis, which primarily affects the lung, but can also affect other tissues, including bone and the nervous system. tuberculosis is believed to be one of the oldest infections to reportedly affect mankind, with archeological and anthropological studies showing evidence of infection in humans over 4,000 years ago. it continues to top the charts of mortality and morbidity in developing nations. it is estimated that in 2007, there were 9.27 million new cases of tb, as well as a prevalence of 13.7 million. the 1.32 million deaths in 2007 from tb in patients without hiv and 450,000 deaths in patients with hiv is a shocking statistic that shows how far we have come, yet how far we still are from effectively eradicating the disease [73] . two very effective drugs have been developed to treat tuberculosis, isoniazid (inh) and rifampin. however, tuberculosis continues to be a global health problem. tuberculosis is second to hiv/aids as the infection with the highest mortality globally. more recently, an increase in multiple drug-resistant tuberculosis (mdr-tb) has been observed, with most cases from india, china, and russia. there were 450,000 reported cases of mdr-tb in the world in 2012. of these, it is estimated that 9.6 % fall into the category of xdr-tb, which is an even more resistant form. thus, drug resistance is a significant challenge in the treatment of tb in the twenty-first century. another challenge stems from the fact that about one third of patients with hiv/aids are infected with tb, although many may not yet be symptomatic. the who defines six core functions and six strategic approaches to combat tb (table 1) . overall, 80 % of tb cases occur in just 22 countries, and 60 % of cases occur in china. however, as overall healthcare accessibility and technology improves, there have been significant declines in the rate of tb in some of the asian countries, including cambodia and china. there is no dispute that the dots and "stop tb strategy," as outlined in table 1 , have contributed to significant progress in the control of tb, but significant challenges remain. one of the areas that are being focused on now has to do with the identification of biomarkers. biomarkers can play several roles in the improvement of treatment of tb in the world. the main areas of focus for research in biomarkers are to identify those that can perform the following functions: (1) prediction of a curative state, (2) prediction of reactivation of tb, and (3) prediction of immunity to tb. in the past, biomarkers for tb would normally involve culturing for the organism, but the development of molecular biology techniques has afforded us the use of non-culture biomarkers. the types of approaches, as described by wallis et al. in 2013 [74] , categorize biomarker development into functional categories ( table 2) . non-culture biomarkers include cytokine levels, quantifiable genetic measures of the presence of tb, other serological tests, imaging techniques, gene transcription profiles, and micro-rna profiles. the development of a vaccine has been an ongoing challenge in the quest for a cure for tb, and limitations in these studies include the lack of valid biomarkers to assess protection in clinical trials. the cytokines interferon-γ and il-18 have been studied as biomarkers for latent tb infection. a tuberculosis-stimulated interferon-γ release assay can detect sensitization to tb antigens, but, unfortunately, cannot differentiate between resolved and persistent latent infection. interleukin-18 is a marker of innate immunity. elevated levels of plasma interleukin levels seen in tb patients indicate increased activation of innate immunity, which was not observed in controls. a chemokine that was observed to be elevated in tb were ccr7 [75] , while the levels of the apoptosis inhibitor bcl2 expression were reduced [76, 77] . il-18 levels actually correlated with the radiographic extent of the disease [78, 79] . hiv appears also to modify cytokine production in patients with tb [80] . other cytokines and chemokines have been profiled as potential biomarkers for tb disease activity [81] [82] [83] [84] [85] [86] , although to this date, none has been shown to be useful for widespread clinical application. fifty years ago, it was the introduction of pyrazinamide and rifampicin that reduced the duration of therapy from 18 months to 6 months. the value of biomarkers may be reflected in the implications of being able to predict cure and latency of infection, as well as active infection. from a therapeutic standpoint, this may mean the difference between the current regimen of 6 months of treatment versus a possible reduction in the duration of therapy for certain patient groups. but this depends on the biomarker being significantly accurate as to not lead to inadequate treatment of affected individuals. though a half century has passed since the development of effective treatment for drug-susceptible tb, the period of treatment is still at least 6 months of standard therapy, i.e., rifampicin and isoniazid for 6 months and pyrazinamde and ethambutol during the initial 2 months of treatment. efforts to reduce the length of treatment are dependent on the discovery of more effective treatment as well as being able to monitor the treatment. at the same time, more and more patients with tb are demonstrating multiple drug resistance forms including mdr or xdr tb. the addition of second-line drugs for the treatment of these more difficult to treat patients is plagued by longer periods of treatment, higher cost, and reduced efficacy. the drugs often used for second-line treatment include aminoglycosides, terizidone, protionamide, capreomycin, fluroquinolones, cycloserine, and ethionamide. the mortality of mdr and xdr tb in hiv patients is particularly high, and researchers have focused on novel approaches to combat this global health issue, including revamping of efforts to develop a vaccine for tb [87] [88] [89] [90] [91] [92] , treatment of latent infection to reduce reservoir pools of infection, and the development of predictive biomarkers. one should not forget that for years we have had a vaccine for tb, the bcg vaccine named after albert calmette and camille guerin. while this vaccine has been used primarily in underdeveloped countries and has been administered to over 4 billion people worldwide, the limitations include an inability to protect against adult tb and adverse effects when used in hiv-positive tb patients. further research is ongoing to develop an improved replacement or enhancement vaccine for bcg [91, [93] [94] [95] [96] [97] [98] [99] [100] . therefore, as we develop new biomarkers and new treatment strategies, one should remember that the infective landscape of tb is not a static target either. the emergence of multidrug-resistant strains as mentioned above means that the disease may become more difficult to treat and that the development of biomarkers must take new medication use into consideration. moreover, the development of biomarkers requires the availability of large patient samples, such as those that can be found in biobanks of large high-volume treatment centers. the willingness of patients to contribute to research, and the regulatory landscape for the collection of samples for research, could potentially negatively impact the ability to conduct large-scale studies on biomarkers. for a comprehensive review of biomarkers, diagnostics, drug treatment, and vaccine development, the reader is referred to an excellent series of articles published in lancet in 2010 [74, 89, 101] . the incidence of pertussis appears to be increasing over the past 30 years. this trend flies in the face of an availability of, first, the killed whole organism pertussis vaccine dtwp and, subsequently, the acellular vaccine (dpap). the acellular vaccine was less reactogenic because it incorporated a step during manufacturing that removed endotoxin, which was a major reason for the adverse side effect profile. it was clear that vaccination against pertussis afforded much improved prevention of the disease, as illustrated by the 157-fold decreased rate for pertussis between the 1930s and 1940s. dtwp vaccines were used until the 1990s, when technological development in the 1970s and 1980s led to the introduction of commercially available acellular pertussis vaccines [102] . the continuing increase in pertussis cases leads to an obvious question. is this a failure of an immunization program? earlier, we discussed several examples that illustrate the difficulty of bringing an effective vaccine to market (tb and rsv). the problems associated with pertussis are clearly different. there are several reasons that may explain why we have not eradicated the disease. it is well documented that the dtap vaccine is not as immunogenic as the whole organism dtwp vaccines. studies have indicated that the overall [149] efficacy of dtap vaccines is below 70 %. we do not normally test for pertussis titers following vaccination, except in studies. but there are those who suggest that the increase in pertussis cases is mainly due to the increased ability to diagnose the disease and to an increased awareness. yet another challenge may be that pertussis has mutated over the years, rendering the vaccine less effective. because our current vaccine is <70 % effective, the ability of mutant strains to evade destruction would lead to their preferential survival. it has been shown that pertussis vaccine which contains more antigens, such as the five-component vaccine that contains pertussis toxin (pt), filamentous hemagglutinin (fha), pertactin (prn), and fimbriae (fim 2/3), is superior to those containing fewer antigens. it has also become apparent that the balance of antigens in the vaccine may play a role in determining efficacy. for example, a whole-cell pertussis vaccine with antigenic components pt, fha, and fim was compared to an acellular pertussis vaccine in children. the whole-cell vaccine produced high levels of antibodies to prn and fim, but low levels to pt and fha, in contrast to the acellular pertussis vaccine which produced high levels of antibody to pt and fha. more than one study has suggested that the vaccine components may antagonize each other. it has been found that the antibody levels to fha do not correlate with the efficacy of the vaccine; furthermore, it has been suggested that adding fha to a vaccine may actually suppress efficacy. linked epitope suppression is another concept to explain the failure of pertussis vaccines. while one would expect that the presence of antigens that do not deliver a brisk vaccine response should not affect the response to other vaccine antigens, this may not be the case. if there were a linkage suppression between two epitopes on the pertussis vaccine, then this could be a significant consideration in vaccine design as one would then need to exclude certain antigens from the vaccine component mix. suffice to say that vaccine development is a very complex topic, and it involves various techniques, including the use of classical adjuvants such as aluminum salts, emulsions or liposomes, or novel adjuvants such as toll-like-receptor agonists, saponins, or immune stimulating complexes [103] . one cannot discuss challenges in pertussis without commenting on the fear of immunizations held by many families, even in developed countries such as the usa. the refusal of many parents to immunize their children, for whatever reason they may have, plays a significant role in our inability to eradicate several diseases, including pertussis, measles, rubella, and so on. this phenomenon is not limited to the uneducated population as many highly educated individuals appear to choose not to subject their children to the relatively miniscule real or imagined risks of immunizations. certainly, one has an astronomically higher risk of being killed in a car accident than dying from a vaccination. and yet, people who refuse to vaccinate their children do not stop driving or riding in cars. the problem with all this is that achieving "herd" immunity is important in total eradication. this means that if only a very small portion of the population do not immunize their children, there is still a possibility of eradicating a disease, but as more people choose to remain unimmunized, the likelihood of successful eradication becomes less and less. unfortunately, people who choose to remain unimmunized fail to understand that in order that everyone not have to get immunized in the future (as in the case of smallpox), everyone must be immunized in the present [104] . severe acute respiratory syndrome, or sars, was an infection that reached epidemic proportions during the early part of the 2000s. it originated in china and rapidly spread to neighboring countries, and eventually, it was reported in many other distant nations, including europe and the usa. although the number of cases was far fewer, by several orders of magnitude, than the common flu, it caught the attention of healthcare professionals and public health officers because of its high mortality, with 8,000 cases and 774 deaths reported between 2002 and 2003, for a mortality rate of 9.6 %. sars was later identified to be caused by a coronavirus and was named sars-coa. travel histories of infected people were instrumental in tracing the origin of the disease [105] . but where did this virus come from and how did it suddenly cause so much havoc? initially, in may of 2003, the virus was traced to civets, a cat-like mammal that is occasionally consumed as food by the chinese. however, these creatures were eventually dismissed as the original source because of the lack of appearance of further infected civets. in 2005, reservoirs of sars-like viruses were found in chinese horseshoe bats. these were often brought to market, and it was thought that the virus was passed to humans at that time. phylogenetic studies further provided evidence that genetically, it was likely that the sars coronavirus evolved from viruses that infected the horseshoe bats [105] [106] [107] [108] [109] [110] [111] [112] . so can sars happen again? in fact, smaller outbreaks that do not attract as much attention may already be occurring in a manner that mimics sars. a middle east respiratory syndrome or mers has been reported to also be caused by a coronavirus. the mortality in this case is high as well, 38 of 64 cases, or >50 % [112] [113] [114] [115] [116] [117] [118] [119] [120] [121] [122] . mers is also thought to have originated in bats. interestingly, many other viruses are thought to be transmitted by bats, including ebola, hendra, and rabies. the cellular receptor for mers is cd26, or dipeptidyl peptidase 4 [123] . lessons learned from the 2003 sars epidemic included the fact that transmission can often be facilitated by the very medical workers who are trying to save patients [109, 110] . strict infection control measures are critical in limiting the spread of the disease, including the use of negative pressure rooms, n95 masks, and gowns. the optimal dosing of antiviral medications and the timing of supportive measures of respiratory care are still unknown, but previous experience should guide us to be more vigilant if this ever occurs in a large-scale fashion in the future. autoimmune lung diseases-biomarkers and biologics-sparing the steroids? anti-neutrophil cytoplasmic antibody (anca) was discovered in 1982 [124, 125] . the antigenic targets of anca include myeloperoxidase or proteinase-3, and the antibodies against these antigens were for a long time referred to as p-anca and c-anca, respectively, the p and c indicating their cellular staining pattern (perinuclear or cytoplasmic). this latter nomenclature has since been discouraged, and the antibodies should be referred to in the context of their specific antigenic target [126, 127] . an understanding of the pathogenesis of anca-associated vasculitis (aav) would help in the development of more effective drugs to treat this group of diseases [125] , which include granulomatosis with polyangiitis (formerly wegener's granulomatosis), eosinophilic granulomatosis with polyangiitis (egpa or churg-strauss disease), and microscopic polyangiitis [128] . for toxin-mediated aav, a meta-analysis has shown a link between crystalline silica and aav [129] . silica is an inflammasome activator and is believed to trigger the activation of pro-inflammatory cytokines leading to the disease. other triggers include drugs, most commonly hydralazine, propylthiouracil, and penicillamine (all of which are known to stimulate b lymphocyte activation), and infectious trigger such as staphylococcus aureus. avoidance of exposure may prove to be a useful methodology to prevent disease, but as of this time, the triggers of aav are too diverse and too controversial to recommend any particular avoidance strategy. another disease state that affects millions, perhaps billions, of people worldwide is allergies. allergies that affect the respiratory tract can take the form of allergic asthma or simply allergic rhinitis and conjunctivitis. the hygiene hypothesis would indicate that because of our cleaner living environments, we are developing higher rates of sensitivities to common allergens, both indoor and outdoor, as well as higher rates of autoimmune diseases [130] [131] [132] [133] [134] [135] [136] . one of the main avenues of treatment has always been avoidance, but we have not really been able to define what exactly constitutes effective avoidance. many of the studies done on dust mite and animal dander avoidance have not been able to identify a single avoidance measure that can make a significant impact on outcome, although perhaps a comprehensive avoidance plan may provide some relief. studies are needed to (1) determine whether indeed effective avoidance is possible in the face of indoor and outdoor exposures to allergens and (2) the extent to which avoidance of allergens, if possible, provides a significant benefit to patients. the diagnosis and management of respiratory diseases is plagued by numerous unmet needs, affecting both common diseases such as asthma to uncommon occurrences such as sars. some of the problems are common and ultimately involve improving our understanding of the mechanisms of disease. only then can we develop tools to help us manage these patients. the advent of molecular biology has allowed us to develop genetic analyses of patients, and this has led to a realization that these diseases are frequently not one disease, but many [137] . thus, the genomic and personalized medicine acts of 2006 and 2010 are important legislatures that will hopefully divert increased funding into studies that will help us identify which treatment should optimally be used on which patient [138, 139] . understanding the pathogenesis of these diseases is another significant unmet need which requires commitment of funding and resources [140] . the respective contributory roles of genes, epigenetics, and the environment in the pathogenesis of lung diseases must be better elucidated [141] [142] [143] [144] [145] [146] [147] [148] . with better understanding of disease comes better diagnostic markers and better treatment. other unmet needs circle around the availability of biomarkers to both diagnose disease and vaccines and other novel treatments to manage disease. new techniques for vaccination which will hopefully alleviate concerns of vaccines among vaccine-apprehensive patients may help lead to the eradication of infectious respiratory disease such as tb, pertussis, and rsv. there is much work yet to be done, and it is important to remember that diseases are not static either, especially in the context of pathogen-related diseases as pathogens are also very adept at evading our efforts to destroy them. the challenges outlined in this article are far from comprehensive, and specific unmet needs exist for a variety of other respiratory diseases, including alpha-1-antitrypsin deficiency, hypersensitivity pneumonitis, chronic obstructive pulmonary disease, lung cancer, pulmonary hypertension in the newborn, and others. while each disease state may have its own unique set of challenges, in general, one can categorize these into three major areas of need, namely, diagnosis, treatment, and prevention (fig. 1 ). more specifically, these would include the identification of biomarkers to provide less invasive methods for the diagnosis and monitoring of the disease, as well as determining prognosis, the development of new drugs and treatment modalities, and also the development of vaccines or other environmental controls to reduce the incidence of the disease. autoantibodies to tumor-associated antigens as biomarkers in cancer immunodiagnosis early life exposure to antibiotics and the risk of childhood allergic diseases: an update from the perspective of the hygiene hypothesis chemokines and their receptors in the allergic airway inflammatory process profile of reslizumab in eosinophilic disease and its potential in the treatment of poorly controlled eosinophilic asthma mepolizumab in eosinophilic disorders inhibiting interleukin-4 and interleukin-13 in difficult-to-control asthma dupilumab in persistent asthma with elevated eosinophil levels the practical understanding and treatment of asthma asthma in children and adolescents: a comprehensive approach to diagnosis and management asthma: developments in targeted therapy asthma and pregnancy the patient with asthma in the emergency department occupational asthma the critically ill asthmatic-from icu to discharge the challenge of asthma in minority populations the adult asthmatic characterizing wheeze phenotypes to identify endotypes of childhood asthma, and the implications for future management multiple atopy phenotypes and their associations with asthma: similar findings from two birth cohorts methylation of il-2 promoter at birth alters the risk of asthma exacerbations during childhood asthma endotypes: the right direction towards personalized medicine for asthma analyses of shared genetic factors between asthma and obesity in children symptom-adjusted therapy in asthma: it is time to listen to our patients physical localization of two dna markers closely linked to the cystic fibrosis locus by pulsed-field gel electrophoresis identification of the cystic fibrosis gene: genetic analysis the search for the cystic fibrosis gene tracing the mutations in cystic fibrosis by means of closely linked dna markers progress towards cloning the cystic fibrosis gene in situ hybridization of two cloned chromosome 7 sequences tightly linked to the cystic fibrosis locus molecular approaches to the cystic fibrosis gene cystic fibrosis locus defined by a genetically linked polymorphic dna marker a polymorphic dna marker linked to cystic fibrosis is located on chromosome 7 cystic fibrosis: analysis of linkage of the disease locus to red cell and plasma protein markers cystic fibrosis: progress in mapping the disease locus using polymorphic dna markers. i genetic analysis of cystic fibrosis using linked dna markers mapping of the cystic fibrosis locus on chromosome 7 progress towards cloning the cystic fibrosis gene gene targeting in mice: functional analysis of the mammalian genome for the twenty-first century how close are we to implementing gene targeting in animals other than the mouse? targeted gene replacement the new mouse genetics: altering the genome by gene targeting production of a severe cystic fibrosis mutation in mice by gene targeting disruption of the cystic fibrosis transmembrane conductance regulator gene in embryonic stem cells by gene targeting gene targeting approaches to complex genetic diseases: atherosclerosis and essential hypertension targeted gene duplication and disruption for analyzing quantitative genetic traits in mice toward an animal model of cystic fibrosis: targeted interruption of exon 10 of the cystic fibrosis transmembrane regulator gene in embryonic stem cells gene conversions and their relation to homologous chromosome pairing direct alteration of a gene in the human genome gene therapy for the treatment of cystic fibrosis assessing the disease-liability of mutations in cftr progress in cystic fibrosis and the cf therapeutics development network respiratory syncytial virus-a comprehensive review the path to an rsv vaccine strategic priorities for respiratory syncytial virus (rsv) vaccine development influenza and respiratory syncytial virus (rsv) vaccines for infants: safety, immunogenicity, and efficacy understanding respiratory syncytial virus (rsv) vaccine-enhanced disease o l s z e w s k a w ( 2 0 0 1 ) immunopathogenesis of vaccine-enhanced rsv disease nonclinical phenotypic and genotypic analyses of a phase 1 pediatric respiratory syncytial virus vaccine candidate medi-559 (ra2cp248/404/1030deltash) at permissive and non-permissive temperatures rsv vaccine in development: assessing the potential costeffectiveness in the dutch elderly population translational sciences approach to rsv vaccine development a protective and safe intranasal rsv vaccine based on a recombinant prefusion-like form of the f protein bound to bacterium-like particles implication of respiratory syncytial virus (rsv) f transgene sequence heterogeneity observed in phase 1 evaluation of medi-534, a live attenuated parainfluenza type 3 vectored rsv vaccine the recent progress in rsv vaccine technology new insights for development of a safe and protective rsv vaccine challenges in developing a pediatric rsv vaccine protective effect of a rsv subunit vaccine candidate g1f/m2 was enhanced by a hsp70-like protein in mice development of a piv-vectored rsv vaccine: preclinical evaluation of safety, toxicity, and enhanced disease and initial clinical testing in healthy adults immunogenicity and efficacy of recombinant rsv-f vaccine in a mouse model effects of alveolar macrophage depletion on liposomal vaccine protection against respiratory syncytial virus (rsv) evaluation of the live attenuated cpts 248/404 rsv vaccine in combination with a subunit rsv vaccine (pfp-2) in healthy young and older adults replacement of the f and g proteins of respiratory syncytial virus (rsv) subgroup a with those of subgroup b generates chimeric live attenuated rsv subgroup b vaccine candidates immunological properties of plaque purified strains of live attenuated respiratory syncytial virus (rsv) for human vaccine current approaches to the development of vaccines against disease caused by respiratory syncytial virus (rsv) and parainfluenza virus (piv). a meeting report of the who programme for vaccine development tuberculosis: a global health problem biomarkers and diagnostics for tuberculosis: progress, needs, and translation into practice expression of cxc and cc type of chemokines and its receptors in tuberculous and nontuberculous effusions identification of probable early-onset biomarkers for tuberculosis disease progression immunohistochemical analysis of cytokines and apoptosis in tuberculous lymphadenitis serum il-18 levels in tuberculosis: comparison with pneumonia, lung cancer and healthy controls il-18 production in human pulmonary and pleural tuberculosis hiv alters plasma and m. tuberculosisinduced cytokine production in patients with tuberculosis production of tnf-alpha, il-12(p40) and il-17 can discriminate between active tb disease and latent infection in a west african cohort nk cell activity in tuberculosis is associated with impaired cd11a and icam-1 expression: a regulatory role of monocytes in nk activation impaired interleukin-1beta converting enzyme (ice) activity in patients with pulmonary tuberculosis profiles of ifn-gamma and its regulatory cytokines (il-12, il-18 and il-10) in peripheral blood mononuclear cells from patients with multidrug-resistant tuberculosis stimulation of dendritic cells via cd40 enhances immune responses to mycobacterium tuberculosis infection depressed interleukin-12 (il-12), but not il-18, production in response to a 30-or 32-kilodalton mycobacterial antigen in patients with active pulmonary tuberculosis toward novel vaccines against tuberculosis: current hopes and obstacles ten years of the global alliance for vaccines and immunization: challenges and progress new vaccines for tuberculosis novel tuberculosis vaccines on the horizon tuberculosis: current state of knowledge: an epilogue 2012: the year in review. part ii: tuberculosis and lung disease history of bcg vaccine genome sequencing and analysis of bcg vaccine strains tuberculosis vaccine trials tuberculosis vaccine trials the current state of tuberculosis vaccines vaccine development for tuberculosis: current progress tuberculosis vaccines: time to reset the paradigm? bettering bcg: a tough task for a tb vaccine global tuberculosis drug development pipeline: the need and the reality pertussis: challenges today and for the future unmet needs in modern vaccinology: adjuvants to improve the immune response immunization coverage levels among 19-to 35-month-old children in 4 diverse, medically underserved areas of the united states severe respiratory syndromes: travel history matters detection of coronaviruses in bats of various species in italy ecology, evolution and classification of bat coronaviruses in the aftermath of sars isolation and characterization of a bat sars-like coronavirus that uses the ace2 receptor clinical management and infection control of sars: lessons learned severe acute respiratory syndrome (sars): lessons learnt in hong kong tracing the sars-coronavirus from sars coronavirus to novel animal and human coronaviruses middle east respiratory syndrome coronavirus (mers-cov): a perpetual challenge middle east respiratory syndromecoronavirus infection: an overview a novel human coronavirus: middle east respiratory syndrome human coronavirus update: recommendations for middle east respiratory syndrome coronavirus (mers-cov) middle east respiratory syndrome: new disease, old lessons middle east respiratory syndrome coronavirus (mers-cov): challenges in identifying its source and controlling its spread middle east respiratory syndrome coronavirus (mers cov): update 2013 first cases of middle east respiratory syndrome coronavirus (mers-cov) infections in france, investigations and implications for the prevention of human-to-human transmission update: severe respiratory illness associated with middle east respiratory syndrome coronavirus (mers-cov)-worldwide from sars to mers: 10 years of research on highly pathogenic human coronaviruses molecular basis of binding between novel human coronavirus mers-cov and its receptor cd26 anti-neutrophil cytoplasmic antibody (anca)-associated vasculitis: where to go? pathogenesis of anca-associated vasculitis, an update antineutrophil cytoplasmic autoantibodies: how are they detected and what is their use for diagnosis, classification and follow-up? treatment of anca-associated vasculitis, where to go? pathogenesis of ancaassociated vasculitis the association between silica exposure and development of ancaassociated vasculitis: systematic review and meta-analysis hydroxychloroquine: from malaria to autoimmunity the hygiene theory harnessing helminths and their ova to treat autoimmunity hygiene hypothesis and autoimmune diseases the role of the environment in the development of pediatric inflammatory bowel disease do infections facilitate the emergence of systemic sclerosis? air pollution in autoimmune rheumatic diseases: a review the role of environmental estrogens and autoimmunity systemic lupus erythematosus one disease or many? the genomics of autoimmune disease in the era of genome-wide association studies and beyond the genomics and personalized medicine act of genes, epigenetic regulation and environmental factors: which is the most relevant in developing autoimmune diseases? the influence of sex and gender on the immune response genes, tolerance and systemic autoimmunity the x chromosome and immune associated genes twin studies in autoimmune disease: genetics, gender and environment genetics of asthma susceptibility and severity the genetic and environmental causes of pulmonary fibrosis epigenomics of idiopathic pulmonary fibrosis who's new stop tb strategy key: cord-314190-fvdock94 authors: florin, todd a; plint, amy c; zorc, joseph j title: viral bronchiolitis date: 2017-01-01 journal: the lancet doi: 10.1016/s0140-6736(16)30951-5 sha: doc_id: 314190 cord_uid: fvdock94 viral bronchiolitis is a common clinical syndrome affecting infants and young children. concern about its associated morbidity and cost has led to a large body of research that has been summarised in systematic reviews and integrated into clinical practice guidelines in several countries. the evidence and guideline recommendations consistently support a clinical diagnosis with the limited role for diagnostic testing for children presenting with the typical clinical syndrome of viral upper respiratory infection progressing to the lower respiratory tract. management is largely supportive, focusing on maintaining oxygenation and hydration of the patient. evidence suggests no benefit from bronchodilator or corticosteroid use in infants with a first episode of bronchiolitis. evidence for other treatments such as hypertonic saline is evolving but not clearly defined yet. for infants with severe disease, the insufficient available data suggest a role for high-flow nasal cannula and continuous positive airway pressure use in a monitored setting to prevent respiratory failure. acute bronchiolitis, a viral infection of the lower respiratory tract, is one of the most substantial health burdens for infants and young children worldwide. 1 respiratory syncytial virus is the most prevalent viral cause of bronchiolitis in infants. estimates suggest that about 34 million new cases of lower respiratory infection due to respiratory syncytial virus occur globally in children younger than 5 years, with 3·4 million admissions to hospitals and about 199 000 deaths per year, predominantly in the developing world. 2 in developed countries such as the usa, bronchiolitis is the most common reason for admission to hospital in the fi rst 12 months of life, 3 accounting for approximately 100 000 infant admissions annually. although admissions to hospital have declined from 2000 to 2010, emergency department visits have increased, in addition to increased use of mechanical ventilation and hospital charges. 4, 5 the clinical management remains challenging despite the frequency, global reach, economic cost, and morbidity and mortality associated with bronchiolitis. several treatment strategies (including bronchodilators and corticosteroids) showed no eff ect in pooled metaanalyses, making supportive care the hallmark of current therapy. in this seminar, we aim to summarise the current evidence for the epidemiology, pathophysiology, diagnostic approach, and management of acute viral bronchiolitis. bronchiolitis is a seasonal infection, with the season typically beginning in late october in the temperate northern hemisphere, peaking in january or february, and ending in april. 6 globally, independent of region, respiratory syncytial virus infection peaks consistently during annual or biannual epidemics. although the peak and duration of these epidemics vary worldwide, they are consistent year-to-year within a country. 7 some data suggest that climate might also be associated with prevalence of respiratory syncytial virus infection, with global surveillance suggesting that infection peaks during wet months in areas with high precipitation and during cooler months in hot regions. 7 indoor crowding in population-dense areas during rainy seasons or cooler months might be one factor that facilitates viral transmission. 8 additionally, weather-related factors, such as inhalation of cold and dry air that might impair ciliary function, the airway mucosa, and inhibition of temperature-dependent antiviral responses, might infl uence both disease transmission and severity. 9, 10 altitude, climate, and meteorological conditions (such as wind speed and dew point) have been shown to have a modest association with bronchiolitis. 11, 12 furthermore, air pollutants, such as ozone and traffi c pollutants, have been associated with exacerbations of respiratory infections in children younger than 5 years. [13] [14] [15] environmental tobacco smoke has been associated with increased risk for respiratory syncytial virus-attributable admission to hospital and disease severity in those admitted. 16, 17 as with other respiratory viral infections, the risk of severe respiratory syncytial virus bronchiolitis might be greater in boys than in girls. 18, 19 this diff erence might be due to diff erences in lung and airway development, and by genetic factors. 8, 20 pathophysiology bronchiolitis is characterised by extensive infl ammation and oedema of the airways, increased mucus production, and necrosis of airway epithelial cells. 21 respiratory syncytial virus binds to epithelial cells and replicates, resulting in epithelial necrosis and ciliary destruction. 21, 22 the cell destruction triggers an infl ammatory response with proliferation of polymorphonuclear cells and lymphocytes. the submucosa and adventitial tissues become oedematous with increased mucus secretion. 22 plugs composed of cellular debris and mucus form in the bronchiole lumens leading to bronchiolar obstruction, air trapping, and diff erent degrees of lobar collapse. 21 microbiology molecular testing has led to an improved understanding of the viruses associated with bronchiolitis. respiratory syncytial virus remains the most commonly identifi ed virus, detected in 41-83% of patients. [23] [24] [25] [26] [27] [28] other viruses associated with bronchiolitis include rhinovirus, metapneumovirus, coronavirus, human bocavirus, infl uenza virus, adenovirus, and parainfl uenza virus. 24, 25, 27, 29, 30 studies have investigated whether severity of illness, as measured by need for hospital admission, length of hospital stay, intensive care unit admission, repeated emergency department visits, and apnoea, is associated with specifi c viral infections or co-infections, but the evidence is confl icting. data from some studies have shown that in infections involving a single virus, respiratory syncytial virus is associated with a more severe course compared with other viruses. 31, 32 up to 30% of children with bronchiolitis are found to have co-infections with two other viruses, 29 with the combination of respiratory syncytial virus and rhinovirus being the most commonly reported. 29 some evidence suggests that co-infection in bronchiolitis, particularly respiratory syncytial virus in combination with rhinovirus or metapneumovirus, could be associated with a more severe disease course compared with infection by a single virus. 25, 30, 31, 33, 34 however, other studies do not confi rm this association. 24, 26 furthermore, although use of nucleic acid amplifi cation tests has greatly improved our ability to detect viruses present in respiratory infections, studies using these technologies have also found at least one respiratory virus in up to 30% of children younger than 6 years with no respiratory symptoms. [35] [36] [37] these viruses might be detected because of asymptomatic colonisation, incubation before clinical infection, or prolonged viral shedding post-infection. the confl icting evidence and high prevalence of respiratory viruses in asymptomatic children suggest no indication at this time that management should vary based on presumed viral cause and presence, or absence of viral co-infections. the diagnosis of bronchiolitis is clinical and thus requires a clinician to recognise signs and symptoms of viral lower respiratory tract infection in young children. peak incidence occurs between 3 months and 6 months of age. 38 since the early clinical defi nition by court, and as noted in recent practice guidelines, the most specifi c defi nition is in infants. [38] [39] [40] although the same physiology can occur in toddlers older than 12 months, many clinical trials have excluded these children or have included them as a small subgroup of patients. bronchiolitis in toddlers can overlap with other conditions such as viral-induced wheezing and asthma, and application of evidence from trials predominantly assessing infants might not be appropriate. further eff orts to focus the defi nition might assist eff orts to standardise care. the classic clinical presentation of bronchiolitis starts with symptoms of a viral upper respiratory infection, such as nasal discharge, that progress to the lower respiratory tract over several days (fi gure 1). timing of symptom progression can vary, and young infants can present with apnoea. lower respiratory tract symptoms of bronchiolitis include persistent cough, tachypnoea, and increased work of breathing, as shown by intercostal or supraclavicular retractions, use of abdominal muscles, grunting, or nasal fl aring. auscultatory fi ndings include crackles and wheeze. a hallmark characteristic of bronchiolitis is the minute-to-minute variation in clinical fi ndings, as mucus and debris in the airways are cleared by coughing or as the child's state changes from sleep to agitation. this variation can confound assessment, and often requires several examinations over a period of observation. nasal congestion can also confound the clinical assessment. nasal suctioning might help to ascertain which fi ndings are truly from the lower airways. fever can be present in about a third of infants with bronchiolitis, but it is usually present early in the illness with a temperature less than 39°c. 38 the median duration of symptoms is about 2 weeks, with 10-20% of infants still having symptoms at 3 weeks after onset. 38, 41 various clinical scores have been shown in studies and clinical protocols to correlate with disease severity and improvement. 42, 43 although documentation of a score can be useful as an objective measure, individual scores are not highly predictive, and they should be repeated and combined with other measures of severity for a universal assessment to guide decision making. the diff erential diagnosis for bronchiolitis includes considerations of various infectious and non-infectious causes. absence of upper respiratory symptoms should raise suspicion of other causes of respiratory distress in young infants, including cardiac disease, congenital airway abnormalities such as a vascular ring, or foreign body aspiration. other infections can resemble or complicate bronchiolitis. pertussis should be considered in infants with severe or paroxysmal cough, or with known exposure. bacterial infections complicating viral bronchiolitis, including otitis media or pneumonia, might present as a new fever or worsening status later in the course of illness. various risk factors have been associated with progression to severe bronchiolitis. those supported by the strongest evidence include presence of chronic lung disease of prematurity and haemodynamically important congenital heart disease, with immunodefi ciency and neuromuscular disorders also considered as high risk in practice guidelines. 38, 39 young infants (aged <2-3 months) and those with a history of premature birth (especially <32 weeks' gestation) are also at high risk for progression and can present with apnoea without other clinical fi ndings. studies assessing the risk for further apnoea in hospital have found it to be limited to infants less than 1 month for full-term infants, 48 weeks postconceptional age for preterm infants, or those with apnoea observed before admission. 44 bronchiolitis is a clinical diagnosis based on history and physical examination, according to consensus across national guidelines. for infants with a typical presentation of bronchiolitis, routine imaging or laboratory testing is not recommended, as they increase costs without evidence for benefi t (table). appropriate use of pulse oximetry monitoring and initiation of oxygen for bronchiolitis have received increasing attention in studies and practice guidelines. findings suggest that arbitrary thresholds for oxygen administration might drive hospital admissions and prolong hospital length of stay. these outcomes represent only part of the morbidity of bronchiolitis, but developing evidence suggests that intermittent hypoxaemia might occur commonly in otherwise stable infants with bronchiolitis and raises questions as to whether this factor should be used as a sole indication for admission to hospitals. a canadian randomised trial found reduced admissions to hospital from the emergency department without any increase in revisits when pulse oximeters displayed values 3% higher than the actual value, suggesting that arbitrary pulse oximetry thresholds result in unnecessary admissions. 51 a similar trial in the uk in the hospital setting found that reduction of the oxygen threshold from 94% to 90% resulted in earlier discharge from the hospital without any evidence of adverse outcomes. 52 a us trial comparing intermittent versus continuous pulse oximetry in non-hypoxaemic infants in hospitals found similar outcomes between the groups. 53 this us trial and other evidence supports recommendations in us practice guidelines that clinicians use a threshold of 90% for initiation of oxygen, whereas uk guidelines recommend 92%. 38, 39 as the child improves, reduction in intensity of monitoring to intermittent checks is appropriate. a recent study using blinded oximetry at home showed that a substantial proportion of infants with bronchiolitis who are otherwise doing well have oxygen desaturations less than 90%, particularly during sleep, further calling into question arbitrary thresholds for hospital admissions and initiation of oxygen. 54 this evidence will probably lead to eff orts to reduce continuous monitoring in children without other indications for monitoring. the majority of children with bronchiolitis have either normal radiographs or radiographic fi ndings consistent with simple bronchiolitis, 55-58 including peribronchial thickening, hyperinfl ation, and atelectasis. one prospective study 58 of routine radiographs as part of the assessment for bronchiolitis in the emergency department reported airspace disease in 17 (7%) of 246 patients. despite the low prevalence of radiographic pneumonia, this and other studies have reported an increase in antibiotic prescription after radiographs are performed, because of non-specifi c fi ndings that infl uence clinicians' decisions. 58, 59 factors that have been associated with defi nite focal infi ltrates consistent with pneumonia include hypoxia (oxygen saturation <92%), [56] [57] [58] 60 grunting, persistently focal crackles, and fever (especially >39°c). 38 chest radiographs should only be considered in patients when the presentation is not classic for bronchiolitis. these situations include when another diagnosis (such as foreign body aspiration) is high on the diff erential diagnosis, when a child is severely ill and respiratory failure is imminent, and when symptoms are progressing or not resolving according to the typical disease course expected for bronchiolitis. lung ultrasound is increasingly used to assess cardiopulmonary conditions in adults and children. several studies have investigated the use of lung ultrasound in the diagnosis of bronchiolitis. two small studies found that ultrasound fi ndings in infants with bronchiolitis correlate with clinical fi ndings, and might be more specifi c than chest radiography, 61,62 but further studies are needed to establish whether there is a role for ultrasound in diagnosis or assessment of severity. with the development of pcr to detect respiratory viruses in the nasopharynx, interest in the use of viral testing for causative diagnosis in bronchiolitis has increased. virological testing, however, does not generally assist in management and is insuffi cient to predict outcomes. 30, 63 many national guidelines therefore recommend against routine virological testing in bronchiolitis (table). recent studies suggest that higher respiratory syncytial virus genomic load, measured using quantitative pcr, might be associated with increased length of stay, use of respiratory support, and need for intensive care, in addition to recurrent wheezing, compared with lower viral loads. 28, 31, 32, 64 further study is warranted to confi rm this association and clarify whether viral load measurement improves understanding of disease pathophysiology and severity. several guidelines recommend using respiratory syncytial virus testing to guide cohorting of patients; however, the viruses most likely to cause bronchiolitis are all transmitted in a similar fashion (close contact with large-particle aerosols or direct contact with contaminated fomites). [65] [66] [67] thus, infection control might not be dependent on the identifi cation of specifi c viruses, but rather on following strict precautions including hand hygiene, separating infants in shared hospital rooms by more than 1 m, and other infection control procedures. 65 additionally, given the sensitivity of pcr testing, results should be interpreted with caution. certain viruses, such as rhinovirus, might be detected because of viral shedding from an unrelated illness or colonisation; whereas certain other viruses, such as respiratory syncytial virus and metapneumovirus, are almost always associated with an acute infection. blood and urine testing is not routinely recommended as part of standard practice in the diagnostic work-up of bronchiolitis (table) . a blood gas measurement should not be routinely obtained in infants with bronchiolitis, unless there are signs of impending respiratory failure or severe distress. proportions of serious bacterial infections, especially bacteraemia and meningitis, are very low in infants with bronchiolitis. 68 abnormal white blood cell count is rarely useful in predicting serious bacterial infections in children infected with respiratory syncytial virus. 69 guidelines universally do not recommend complete blood counts in infants with bronchiolitis unless blood count is part of assessment for a fever in infants younger than 1-2 months. similarly, given that bacteraemia is exceedingly rare (with cited proportions of <0·1% in the post-pneumococcal vaccine era), blood cultures should not be routinely performed, except in the septic work-up of infants younger than 1-2 months, 70, 71 or in those with severe illness and signs of sepsis. hydration status is an important consideration in infants with bronchiolitis and should be determined by clinical examination. routine measurement of serum electrolytes is of little value in the majority of infants. urinary tract infections in infants with bronchiolitis occur with greater frequency than do bacteraemia and meningitis, with proportions ranging from 1% to 7%. 68, 71, 72 it is reasonable to obtain a urinalysis and urine culture for infants aged less than 60 days with fever and for older febrile infants who have risk factors for urinary tract infections, 73 but urine should not be routinely obtained in all infants with bronchiolitis. current recommendations for management of bronchiolitis focus on agents to treat the patho physiological eff ects of viral lower respiratory infection (eg, bronchodilators and hypertonic saline). specifi c antivirals such as ribavirin to treat respiratory syncytial virus infection are not recommended in practice guidelines for typical cases of bronchiolitis because of challenging delivery methods, high cost, and potential health risks to caregivers. multiple new agents for prevention and treatment are under investigation and might become available in the future, including immunoglobulins, small interfering rna interference, fusion inhibitors, and small molecules. 74 numerous studies have assessed the role of bronchodilators for the treatment of bronchiolitis, and systematic reviews have found no consistent benefi t. a 2014 cochrane collaboration systematic review identifi ed 30 studies assessing bronchodilators, predominantly salbutamol and excluding epinephrine, and 21 studies that looked specifi ally at clinical scores found no evidence of benefi t in any outcomes for infants admitted to hospitals. 75 in outpatients, oxygen saturation, admission to hospital, or time to resolution of symptoms did not improve with bronchodilator usage compared with placebo. for outpatient studies assessing short-term change in pooled clinical scores, the reviewers found a small signifi cant diff erence in mean score (z=2·26; p=0·024) that was of small eff ect with minimal clinical importance (fi gure 2). outpatient studies were heterogeneous (i²=81%; p<0·00001), and those showing benefi t in scores tended to include older children and children with recurrent wheezing. nebulised epinephrine was assessed in another cochrane collaboration systematic review. 76 this review found no benefi t for epinephrine compared with placebo for inpatients in hospital length of stay or other outcomes. a multicentre scandinavian study published after this cochrane review found that inpatients receiving standing doses of epinephrine had longer length of stay compared with inpatients receiving as-needed epinephrine or placebo. 77 for outpatients, the cochrane review found a diff erence in the numbers of admissions associated with epinephrine treatment during the time of an emergency department visit, but not during the overall course of illness when assessed at 1 week. clinical practice guidelines including those from the usa, uk, and canada do not recommend treatment with bronchodilators for bronchiolitis because of this evidence (table) . 38 nebulised hypertonic saline is thought to reduce airway oedema, decrease mucus plugging, improve mucociliary clearance, and rehydrate the airway surface liquid in infants with bronchiolitis. 78 these physiological changes are extrapolated from the cystic fi brosis literature, 79, 80 and the pathophysiological processes in acute bronchiolitis are diff erent. therefore, the theoretical benefi ts of hypertonic saline seen in cystic fi brosis might not be present in infants with acute viral bronchiolitis. although initial trials demonstrated some ability of hypertonic saline to decrease hospital length of stay and transiently improve clinical severity score, 81-83 more recent trials demonstrated confl icting results. [84] [85] [86] [87] [88] [89] [90] the trials that showed the largest benefi t were done in hospitals with lengths of stay more than 72 h; thus, hypertonic saline for infants in countries and institutions in which the length of stay approaches or exceeds 72 h might be benefi cial at reducing length of stay. the confl icting results are test for heterogeneity in the inpatient studies demonstrated low inconsistency between the nine studies (i²=36%; p=0·13) and the summary eff ect was not signifi cant (z=1·06; p=0·29), the outpatient studies demonstrated very high inconsistency between the 12 studies (i²=81%; p<0·00001) and the summary eff ect was signifi cant (z=2·26; p=0·024), and the overall heterogeneity of the meta-analysis demonstrated high inconsistency between the 21 studies (i²=73%; p<0·00001) and the overall summary eff ect was signifi cant (z=2·4; p=0·016). ipr=ipratropium. sal=salbutamol. neb=nebulised. reproduced from gadomski and scribani, 75 refl ected in the diff erences in recommendations across national guidelines (table), with some countries not recommending hypertonic saline, some recommending use for all inpatients, and some recommending use only in moderate to severe illness. the largest systematic review and meta-analysis, published in 2015, examined 24 trials and 3209 patients. 91 infants receiving hypertonic saline had a signifi cant diff erence in hospital length of stay of -0·45 days (95% ci -0·82 to -0·08; p=0·01) compared with those receiving 0·9% saline or standard care. in seven trials, hypertonic saline reduced the risk of admission to hospital by 20% (risk ratio 0·8; 95% ci 0·67-0·96) compared with 0·9% saline. no substantial adverse eff ects of hypertonic saline were noted in this systematic review. although the results of this meta-analysis were signifi cant, attention should be paid to the outer limits of the confi dence intervals, which approach no diff erence, and to the clinical relevance of these diff erences. a second 2015 meta-analysis of 15 trials and 1922 patients found a smaller, but signifi cant, decrease in length of stay by -0·36 days (95% ci -0·5 to -0·22; p<0·001) in those who received hypertonic saline (fi gure 3). 92 this study found a discrepancy between the overall combined eff ect of all studies on length of stay and the negative results from the largest trials, allowing the authors to conclude that neither individual trials nor pooled estimates provide a strong evidence-based foundation for the use of hypertonic saline. both meta-analyses showed substantial heterogeneity across studies (i²=82·1%, p<0·001; 91 and i²=77·8%, p=0·029 92 ). a recent reanalysis of the fi rst 2015 meta-analysis removed two outlying chinese studies and accounted for imbalances in day of illness at presentation. 93 these analyses resolved the heterogeneity and found that hypertonic saline does not reduce length of stay in infants admitted to hospitals with bronchiolitis (mean diff erence in length of stay removing outliers -0·21 days; 95% ci -0·43 to 0·02; mean diff erence in length of stay accounting for day of illness imbalance 0·02 days; 95% ci -0·14 to 0·17). large trials have not demonstrated benefi t for hypertonic saline and this meta-analysis found no eff ect of hypertonic saline on length of stay after adjustment for outliers and imbalances. the decision to undertake future trials is controversial given the positive results of some metaanalyses and negative results of others. multiple studies have examined the role of corticosteroids in the management of children with bronchiolitis. data from two large multicentre trials have shown no benefi t to corticosteroids alone in reducing admissions to hospital, 94, 95 and a 2013 cochrane collaboration review supports the results of these studies. 96 this review included only studies that enrolled children younger than 24 months with a fi rst episode of wheezing and signs of a viral illness. among the included eight outpatient studies (1824 participants) comparing corticosteroids with placebo there was no reduction in admission at day 1 (z=1·05; p=0·3) or day 7 (z=1·38; p=0·17) after enrolment (fi gure 4), clinical scores, length of stay in the emergency department, or length of time to resolution of symptoms. among the nine inpatient studies (772 participants), length of hospital stay was not reduced. 96 on the basis of this evidence, multiple clinical practice guidelines recommend against the use of corticosteroids for infants with bronchiolitis (table) . although clinicians report considering a family or personal history of atopy when deciding whether to treat infants with bronchiolitis with corticosteroids, 41 there is no evidence that such infants receive any benefi t from corticosteroid treatment. [94] [95] [96] evidence for the presence or absence of respiratory syncytial virus infection in these infants being associated with a response to corticosteroids is also unavailable. 94, 95 authors from a large study with a factorial design have suggested, in an unadjusted analysis, that high-dose corticosteroids in combination with nebulised epinephrine might reduce admissions for outpatients with bronchiolitis by day 7, 95 but these results are considered exploratory. 39, 45 high-fl ow oxygen and respiratory support non-invasive technologies to improve oxygenation and ventilation for bronchiolitis include humidifi ed high-fl ow nasal cannula oxygen and continuous positive airway pressure. 97 high-fl ow nasal cannula allows delivery of high fl ows (usually 1-2 l/kg per min) with humidifi cation and a cannula designed to improve patient tolerance. it has been used widely in premature infants, but the mechanisms of action are unclear, in particular whether it might deliver positive end-expiratory pressure in some conditions. evidence for effi cacy of high-fl ow nasal cannula is predominantly observational, with studies documenting corneli (2007) goebel (2000) kuyucu (2004) plint (2009) improved respiratory parameters and reduced intubation rates after implementation. 97 one small randomised trial compared high-fl ow nasal cannula with hypertonic saline and found no diff erence in change in respiratory score. 98 concerns about high-fl ow nasal cannula include the potential for rapid deterioration if the infant is not closely monitored and costs associated with overuse. continuous positive airway pressure has been studied in intensive care settings in observational studies and several small trials, with some evidence of improved respiratory parameters. 97 the uk guidelines recommend considering continuous positive airway pressure in children with impending respiratory failure from bronchiolitis. antibiotic overuse in children with bronchiolitis probably occurs because of concerns about the presence of fever, the young age of aff ected patients, diffi culty diff erentiating atelectasis from infectious consolidation on chest radiograph, and concern for undetected secondary bacterial infection. bronchiolitis, however, has a clear viral cause and the occurrence of secondary bacterial infections is low, with a risk of bacteraemia or meningitis of less than 1%. 99 a detailed review of randomised clinical trials found that routine use of antibiotics did not improve duration of symptoms, length of hospital stay, need for oxygen therapy, or hospital admission. 38 overuse of antibiotics is known to result in unnecessary adverse eff ects on the patient, and the development of antimicrobial resistance. routine use should be avoided unless there is clear evidence of a secondary bacterial infection (table) . acute otitis media has been documented in up to 60% of infants with bronchiolitis. 100, 101 antibiotic use for acute otitis media in bronchiolitis should follow established evidence and guidelines for acute otitis media. 102 macrolide antibiotics have anti-infl ammatory properties that might have potential benefi t in mitigating the infl ammation present in bronchiolitis. two randomised trials found that there was no diff erence between azithromycin and placebo in hospital length of stay, need for oxygen, or hospital re-admission. 103, 104 another randomised trial found that azithromycin lowered nasal interleukin-8 concentrations, prolonged time to subsequent wheezing episodes, and resulted in fewer days with respiratory symptoms in the year following the bronchiolitis episode compared with placebo. 105 finally, a us multicentre study found that in children aged 12-72 months with a history of recurrent severe lower respiratory tract infections, early administration of azithromycin during a lower respiratory tract infection reduced the likelihood of progression to a severe infection, but it is not clear whether the underlying disease in these children was bronchiolitis or some other disease process. 106 given the current evidence, routine use of macrolides is not recommended in bronchiolitis and more research is needed to clarify any potential role it might have in the future. hydration, suctioning, and chest physiotherapy have been suggested as supportive therapies. infants with bronchiolitis might have diffi culty feeding because of nasal congestion and increased work of breathing; thus, hydration remains a cornerstone of therapy. a multicentre study of 759 infants younger than 12 months admitted to hospital with bronchiolitis showed no benefi t of intravenous fl uids compared with administration of fl uids by nasogastric tube in mean length of stay, admission to the intensive care unit, need for ventilatory support, and adverse events. this trial also found that a nasogastric tube might be easier to place than an intravenous line in these infants. 107 most guidelines recommend either nasogastric or intravenous fl uids to maintain hydration, with the uk and scottish guidelines preferring nasogastric or orogastric hydration in those that can tolerate it compared with intravenous hydration (table). if intravenous fl uids are used, isotonic fl uids are preferred to avoid risk of hyponatraemia. 38 because infants are obligate nasal breathers, nasal suctioning has been suggested to help with clearing of the nares, improve the work of breathing, and improve feeding; however, suctioning might irritate the nasal mucosa and result in oedema. no randomised controlled trials have examined the role of nasal suctioning in bronchiolitis. the insuffi cient available evidence includes a retrospective cohort study of 740 infants 108 and a small observational study of 40 infants. 109 these studies suggest that deep suctioning might increase length of stay for inpatients, 108 infrequent suctioning is associated with an increased length of stay, 108 and oxygen saturation might increase after suctioning. 109 to draw conclusions about causality from these observational studies is diffi cult, because the potential for confouding by indication exists (eg, sicker children might be more likely to receive deep suctioning). evidence suggests that oxygen saturation increases after nasal irrigation even without suctioning. 110 current guidelines give diff ering recommendations with regard to suctioning; those that support its use recommend only superfi cial suctioning rather than deep suctioning (table) . 38, 39, 45 chest physiotherapy use in bronchiolitis appears to vary by country. 111, 112 a recent cochrane collaboration review of 12 studies (1249 participants) demonstrated no evidence of benefi t to any type of chest physiotherapy among inpatients in length of stay, oxygen saturation, or respiratory parameters. 113 no published guidelines routinely recommend chest physiotherapy for the management of uncomplicated bronchiolitis in otherwise healthy children without respiratory comorbidities (table) . 38, 39, 45 prognosis much work has been published about the risk of developing recurrent wheezing and asthma following bronchiolitis in infancy. [114] [115] [116] studies have followed birth cohorts to determine the risk of subsequent wheezing after lower respiratory tract infection in young childhood, and cohorts of children admitted to hospital with bronchiolitis. [114] [115] [116] [117] overall, admission to hospital with bronchiolitis at a young age is associated with an increased risk of recurrent wheezing. studies report that 17-60% of children with bronchiolitis might develop recurrent wheezing in the years following their initial admission to hospital. a large study from taiwan that followed up 1981 children admitted with bronchiolitis before age 3 years found that by age 10 years, 351 (17·7%) of 1981 children with bronchiolitis had a diagnosis of asthma compared with 2159 (11·7%) of 18 527 controls (hazard ratio 1·58; 95% ci 1·41-1·71). 118 one small cohort study of 138 patients has suggested that 18 (39%) of 46 children admitted with bronchiolitis before 12 months have asthma by 18 years compared with eight (9%) of 92 controls. 115 however, another study that followed a birth cohort of 1246 children found that although lower respiratory tract infection in childhood was associated with an increased risk of recurrent wheezing, this association decreased with age and was not signifi cant by age 13 years. 116 most children in this cohort had mild illness not requiring hospital admission, and severity of illness might be associated with the increased risk of asthma. 119 the question remains whether respiratory infection at a young age itself predisposes children to asthma through damage or alteration of lung function, or whether children with severe bronchiolitis might have individual risk factors (such as altered immune response or airway function) that predisposes them to both severe bronchiolitis and recurrent wheezing. 8 substantial knowledge gaps and controversies exist in the management of acute bronchiolitis. the role of nebulised hypertonic saline in acute management is not clear, resulting in confl icting recommendations across clinical guidelines (table) . although meta-analyses suggest a small reduction of length of stay, these analyses are limited by heterogeneity, not accounting for duration of illness, and not considering the role of outlying study populations. 91 two large multicentre trials do not support a clinically signifi cant diff erence in length of stay for inpatients and there is no clear evidence of cost benefi t. 4, [89] [90] [91] 120 while the same meta-analysis suggests a possible reduction in admissions for outpatients, the confi dence intervals were wide and the studies were also heterogeneous. 91 no multicentre studies of nebulised hypertonic saline have been completed in outpatients. although the evidence is increasing that hypertonic saline has little role in meaningfully reducing the length of stay, its role in outpatients is less clear. a large outpatient multicentre study could clarify whether there is any benefi t. evidence also suggests that combined therapy with nebulised epinephrine and corticosteroids might reduce admissions to hospital. 95 synergy between corticosteroids and β2 agonists is well documented in clinical trials of asthma management. [121] [122] [123] [124] basic science literature also shows that β2 agonists and corticosteroids enhance each other's eff ectiveness, particularly with regard to antiinfl ammatory gene expression. 125, 126 because of the economic burden of bronchiolitis and the plausible basic and clinical evidence for synergy, a large and multicentre trial is needed to ascertain whether combined therapy with epinephrine and corticosteroids is benefi cial. oxygen saturation and the use of pulse oximetry play an important role in the decision to admit infants with bronchiolitis to hospital and in the length of their hospital stay. [51] [52] [53] [54] [55] [56] [57] [58] [59] clinical practice guidelines also give confl icting guidance on the level of oxygen saturation at which admission should be considered. furthermore, a substantial proportion of discharged infants have episodes of transient desaturation. 54 again, in view of the large health-care costs associated with hospital admission in bronchiolitis, further research is needed to clarify the level of oxygen saturation requiring admission, the role of continuous and spot measurements of oxygen saturation, and the clinical importance of transient desaturations in otherwise stable young infants. taf contributed to the design and coordinated the writing of this manuscript. all authors contributed to the literature search and writing of this manuscript. we declare no competing interests. taf, acp, and jjz have participated in trials of bronchiolitis funded by unrestricted public or academic institutional funding sources that had no infl uence on the conception and writing of this manuscript. viral bronchiolitis in children temperature-dependent innate defense against the common cold virus limits viral replication at warm temperature in mouse airway cells nasal mucociliary transport in healthy subjects is slower when breathing dry air altitude and environmental climate eff ects on bronchiolitis severity among children presenting to the emergency department the relationship of meteorological conditions to the epidemic activity of respiratory syncytial virus air pollution and acute respiratory infections among children 0-4 years of age: a 1-8 year time-series study air pollution interacts with past episodes of bronchiolitis in the development of asthma exposure to traffi c and early life respiratory infection: a cohort study systematic literature review assessing tobacco smoke exposure as a risk factor for serious respiratory syncytial virus disease among infants and young children severity of respiratory syncytial virus bronchiolitis is aff ected by cigarette smoke exposure and atopy respiratory syncytial virus-associated hospitalizations among infants and young children in the united states respiratory syncytial virus-associated hospitalizations among children less than 24 months of age interleukin-9 polymorphism in infants with respiratory syncytial virus infection: an opposite eff ect in boys and girls kendig and chernick's disorders of the respiratory tract in children pathological changes in virus infections of the lower respiratory tract in children bronchiolitis: lingering questions about its defi nition and the potential role of vitamin d detection of new respiratory viruses in hospitalized infants with bronchiolitis: a three-year prospective study respiratory syncytial virus, human bocavirus and rhinovirus bronchiolitis in infants in very young infants severity of acute bronchiolitis depends on carried viruses viral etiologies of infant bronchiolitis, croup and upper respiratory illness during 4 consecutive years virus type and genomic load in acute bronchiolitis: severity and treatment response with inhaled adrenaline prospective multicenter study of the viral etiology of bronchiolitis in the emergency department prospective multicenter study of viral etiology and hospital length of stay in children with severe bronchiolitis respiratory syncytial virus genomic load and disease severity among children hospitalized with bronchiolitis: multicenter cohort studies in the united states and finland evaluation of viral load in infants hospitalized with bronchiolitis caused by respiratory syncytial virus association of rhinovirus infection with increased disease severity in acute bronchiolitis dual infection of infants by human metapneumovirus and human respiratory syncytial virus is strongly associated with severe bronchiolitis frequent detection of respiratory viruses without symptoms: toward defi ning clinically relevant cutoff values detection of respiratory syncytial virus and rhinovirus in healthy infants respiratory viral detection in children and adults: comparing asymptomatic controls and patients with community-acquired pneumonia bronchiolitis in children: diagnosis and management. nice guideline clinical practice guideline: the diagnosis, management, and prevention of bronchiolitis the defi nition of acute respiratory illnesses in children bronchodilator and steroid use for the management of bronchiolitis in canadian pediatric emergency departments validity of bronchiolitis outcome measures severity scoring systems: are they internally valid, reliable and predictive of oxygen use in children with acute bronchiolitis? identifying hospitalized infants who have bronchiolitis and are at high risk for apnea bronchiolitis: recommendations for diagnosis, monitoring and management of children one to 24 months of age bronchiolitis in children: a national clinical guideline inter-society consensus document on treatment and prevention of bronchiolitis in newborns and infants quality plan for the spanish national healthcare system of the spanish ministry for health and social policy; catalan agency for health technology assessment evidence based guidelines for the management of bronchiolitis prise en charge de la bronchiolite aiguë du nourrisson de moins 1 an: actualization et consensus medical au sein des hôpitaux universitaires du grand ouest (hugo) eff ect of oximetry on hospitalization in bronchiolitis: a randomized clinical trial oxygen saturation targets in infants with bronchiolitis (bids): a double-blind, randomised, equivalence trial use of intermittent vs continuous pulse oximetry for non-hypoxemic infants and young children hospitalized for bronchiolitis: a randomized clinical trial eff ect of oxygen desaturations on subsequent medical visits in infants discharged from the emergency department with bronchiolitis chest radiographs in the pediatric emergency department for children < or = 18 months of age with wheezing clinical predictors of pneumonia among children with wheezing clinical predictors of radiographic abnormalities among infants with bronchiolitis in a paediatric emergency department evaluation of the utility of radiography in acute bronchiolitis impact of chest radiography for children with lower respiratory tract infection: a propensity score approach clinical factors associated with focal infi ltrates in wheezing infants and toddlers lung ultrasound in bronchiolitis: comparison with chest x-ray lung ultrasound: a useful tool in diagnosis and management of bronchiolitis virologic testing in bronchiolitis: does it change management decisions and predict outcomes viral load in infants hospitalized for respiratory syncytial virus bronchiolitis correlates with recurrent wheezing at thirty-six-month follow-up the spread of infl uenza and other respiratory viruses: complexities and conjectures boston: exposure, epidemiology risk program. department of environmental health harvard school of public health transmission of infl uenza a in human beings risk of serious bacterial infection in young febrile infants with respiratory syncytial virus infections lack of usefulness of an abnormal white blood cell count for predicting a concurrent serious bacterial infection in infants and young children hospitalized with respiratory syncytial virus lower respiratory tract infection appropriateness of testing for serious bacterial infection in children hospitalized with bronchiolitis occult serious bacterial infection in infants younger than 60 to 90 days with bronchiolitis a systematic review assessing the utility of urine testing in febrile infants aged 2 to 12 months with bronchiolitis urinary tract infection: clinical practice guideline for the diagnosis and management of the initial uti in febrile infants and children 2 to 24 months lower respiratory tract infection caused by respiratory syncytial virus: current management and new therapeutics bronchodilators for bronchiolitis epinephrine for bronchiolitis racemic adrenaline and inhalation strategies in acute bronchiolitis hypertonic saline or high volume normal saline for viral bronchiolitis: mechanisms and rationale sodium chloride increases the ciliary transportability of cystic fi brosis and bronchiectasis sputum on the mucus-depleted bovine trachea hydrator therapies for cystic fi brosis lung disease nebulized 3% hypertonic saline solution treatment in hospitalized infants with viral bronchiolitis nebulized hypertonic saline in the treatment of viral bronchiolitis in infants nebulized hypertonic saline/salbutamol solution treatment in hospitalized children with mild to moderate bronchiolitis nebulized hypertonic saline for bronchiolitis: a randomized clinical trial nebulized hypertonic saline for bronchiolitis in the emergency department: a randomized clinical trial the tale of 2 trials: disentangling contradictory evidence on hypertonic saline for acute bronchiolitis 3% hypertonic saline versus normal saline in inpatient bronchiolitis: a randomized controlled trial a randomized trial of nebulized 3% hypertonic saline with salbutamol in the treatment of acute bronchiolitis in hospitalized infants sabre: a multicentre randomised control trial of nebulised hypertonic saline in infants hospitalised with acute bronchiolitis the eff ect of 3% and 6% hypertonic saline in viral bronchiolitis: a randomised controlled trial nebulized hypertonic saline for acute bronchiolitis: a systematic review hypertonic saline (hs) for acute bronchiolitis: systematic review and meta-analysis association between hypertonic saline and hospital length of stay in acute viral bronchiolitis: a reanalysis of 2 meta-analyses a multicenter, randomized, controlled trial of dexamethasone for bronchiolitis epinephrine and dexamethasone in children with bronchiolitis glucocorticoids for acute viral bronchiolitis in infants and young children cpap and high-fl ow nasal cannula oxygen in bronchiolitis high fl ow therapy versus hypertonic saline in bronchiolitis: randomised controlled trial antibiotics for bronchiolitis in children under two years of age acute otitis media in children with bronchiolitis the clinical course of bronchiolitis associated with acute otitis media the diagnosis and management of acute otitis media a single dose of azithromycin does not improve clinical outcomes of children hospitalised with bronchiolitis: a randomised, placebo-controlled trial azithromycin therapy in hospitalized infants with acute bronchiolitis is not associated with better clinical outcomes: a randomized, double-blinded, and placebo-controlled clinical trial randomized trial to evaluate azithromycin's eff ects on serum and upper airway il-8 levels and recurrent wheezing in infants with respiratory syncytial virus bronchiolitis early administration of azithromycin and prevention of severe lower respiratory tract illnesses in preschool children with a history of such illnesses: a randomized clinical trial emergency departments international collaborative (predict) nasogastric hydration versus intravenous hydration for infants with bronchiolitis: a randomised trial suctioning and length of stay in infants hospitalized with bronchiolitis is nasal suctioning warranted before measuring o 2 saturation in infants with bronchiolitis? nasal irrigation with saline solution signifi cantly improves oxygen saturation in infants with bronchiolitis decreasing unnecessary utilization in acute bronchiolitis care: results from the value in inpatient pediatrics network eff ectiveness of chest physiotherapy in infants hospitalized with acute bronchiolitis: a multicenter, randomized, controlled trial chest physiotherapy for acute bronchiolitis in paediatric patients between 0 and 24 months old acute bronchiolitis: predisposing factors and characterization of infants at risk asthma and allergy patterns over 18 years after severe rsv bronchiolitis in the fi rst year of life respiratory syncytial virus in early life and risk of wheeze and allergy by age 13 years acute bronchiolitis in infancy as risk factor for wheezing and reduced pulmonary function by seven years in akershus county a longitudinal study on early hospitalized airway infections and subsequent childhood asthma the severity-dependent relationship of infant bronchiolitis on the risk and morbidity of early childhood asthma saline in acute bronchiolitis rct and economic evaluation: hypertonic saline in acute bronchiolitis-randomised controlled trial and systematic review added salmeterol versus higher-dose corticosteroid in asthma patients with symptoms on existing inhaled corticosteroid. allen & hanburys limited uk study group eff ect of inhaled formoterol and budesonide on exacerbations of asthma. formoterol and corticosteroids establishing therapy (facet) international study group scientifi c rationale for using a single inhaler for asthma control a holy grail of asthma management: toward understanding how long-acting beta(2)-adrenoceptor agonists enhance the clinical effi cacy of inhaled corticosteroids long-acting beta 2-adrenoceptor agonists synergistically enhance glucocorticoiddependent transcription in human airway epithelial and smooth muscle cells beta 2-adrenoceptor agonist-induced rgs2 expression is a genomic mechanism of bronchoprotection that is enhanced by glucocorticoids taf receives funding from the national institutes of health-national institute of allergy and infectious diseases (grant 1k23ai121325-01). the nih had no role in the writing of this manuscript, and in the decision to submit the paper for publication. we would like to thank kerry aicholtz for her administrative support. key: cord-326163-u8nh8cr3 authors: van benten, inesz j.; van drunen, cornelis m.; koopman, laurens p.; kleinjan, alex; van middelkoop, barbara c.; de waal, leon; osterhaus, albert d.m.e.; neijens, herman j.; fokkens, wytske j. title: rsv‐induced bronchiolitis but not upper respiratory tract infection is accompanied by an increased nasal il‐18 response date: 2003-08-18 journal: j med virol doi: 10.1002/jmv.10482 sha: doc_id: 326163 cord_uid: u8nh8cr3 the aim of this study was to investigate potential differences in the local nasal immune response between bronchiolitis and upper respiratory tract infection induced by respiratory syncytial virus (rsv). nasal brush samples were obtained from 14 infants with rsv bronchiolitis and from 8 infants with rsv upper respiratory tract infection. the samples were taken during infection (acute phase) and 2–4 weeks later (convalescent phase). cytospin preparations were stained immunohistochemically for t cells, macrophages, and eosinophils. staining also took place for intercellular adhesion molecule‐1 (icam‐1), t‐helper 1 (th1)‐like (interleukin‐12 [il‐12], interferon‐γ [ifn‐γ]), th2‐like (il‐4, il‐10), and proinflammatory cytokines (il‐6, il‐8, il‐18). during both rsv‐induced bronchiolitis and upper respiratory tract infection, cellular inflammation was observed. this was characterised by an increase in the numbers of nasal macrophages, which tended to be higher in bronchiolitis than in upper respiratory tract infection. numbers of t lymphocytes and icam‐1 positive cells increased during both bronchiolitis and upper respiratory tract infection. there were no differences between numbers in the groups. interestingly, a distinct nasal proinflammatory cytokine response was observed in rsv‐induced bronchiolitis. this is characterised by an increase in the number of il‐18 positive cells. this increase is specific for bronchiolitis, as a similar increase could not be detected in rsv‐induced upper respiratory tract infection. numbers of il‐6 and il‐12 positive cells were higher in both bronchiolitis and upper respiratory tract infection, and there were no differences between the groups. by contrast, the number of il‐8, ifn‐γ, il‐4, and il‐10‐positive cells remained constant. in conclusion, clear differences were found in nasal immune responses of children with rsv‐induced upper respiratory tract infection or bronchiolitis. the induction of a strong il‐18 response was typical for bronchiolitis, as this could not be observed in rsv‐induced upper respiratory tract infection, and could explain the eosinophilia that is observed frequently during bronchiolitis. j. med. virol. 71:290–297, 2003. © 2003 wiley‐liss, inc. respiratory syncytial virus (rsv) infection in young children can either be restricted to the upper respiratory tract, leading to a simple cold, or include the lower airways and result, for example, in bronchiolitis [fisher et al., 1997] . although rsv-induced bronchiolitis is nearly always preceded by symptoms of upper respiratory tract infection, it is not clear why an rsv infection becomes more severe in some cases. an interesting question is whether there could be differences between the nasal immune responses of rsv-induced bronchiolitis and rsv-induced upper respiratory tract infection. the immunological effects of rsv infections are quite diverse and seem to encompass t-helper 1 and 2 (th1, th2), and proinflammatory responses. increased levels of typical th2 markers, such as eosinophil cationic protein (ecp) and rsv-specific ige, have been found in nasal lavage samples during bronchiolitis in infants [welliver and duffy, 1993; garofalo et al., 1994] . in line with the increased levels of ecp, higher peripheral blood eosinophil counts have also been observed [garofalo et al., 1994; ehlenfield et al., 2000] . in addition, stimulated peripheral blood mononuclear cells (pbmcs) revealed enhanced th2 cytokine responses in infants during rsv bronchiolitis compared with controls [roman et al., 1997] . proinflammatory and th1 responses have also been found in infants with bronchiolitis. for example, the cytokines interferon-g (ifn-g), interleukin-2 (il-2), il-6, and il-8, and chemokines macrophage inflammatory protein-1a (mip-1a) and regulated upon activation, normal t cell expressed and secreted (rantes) were found in nasal lavage and blood samples during rsv bronchiolitis [abu-harb et al., 1999; van schaik et al., 1999; brandenburg et al., 2000; garofalo et al., 2001; sung et al., 2001; tripp et al., 2002] . during upper respiratory tract infection in infants, elevated levels of proinflammatory and th1 cytokines il-1b, il-6, il-8, and tumour necrosis factor-a (tnf-a) have been observed in nasal samples [noah et al., 1995] . limited data are available comparing rsvinduced bronchiolitis and rsv-induced upper respiratory tract infection. this study investigated whether differences could be observed on the cellular level between the nasal immune responses of infants with rsv-induced bronchiolitis and rsv-induced upper respiratory tract infection. for this purpose, the numbers of nasal inflammatory cells, the numbers of th1-and th2-like cytokine-positive cells, and the numbers of proinflammatory cytokine-positive cells were determined in both groups of children. the results show distinct nasal immunological effects during rsv-induced bronchiolitis versus rsv-induced upper respiratory tract infection. during the winters of 1998 and 1999, 14 infants (median age 9 weeks) seen at the sophia's children hospital in rotterdam (the netherlands) with rsvinduced lower respiratory tract infection (bronchiolitis) were selected for inclusion in the study. the diagnosis of bronchiolitis was based on the diagnosis of rsv infection in the presence of clinical symptoms and radiological findings characteristic for lower respiratory tract disease. in nearly all cases, the associated respiratory problems required hospitalisation of the affected children. eight infants (median age, 26 weeks) were selected for comparison. they had participated in a prospective birth cohort study and presented with mild upper respiratory tract infection symptoms caused by rsv without bronchiolitis. none of the children with upper respiratory tract infection went to hospital. physical examination was performed and nasal brush samples were taken from the upper respiratory tract infection group within the first few days after the onset of infection (median, 3 days). this was done within 24 hr after arrival in the hospital for the bronchiolitis group. to obtain baseline measurements, nasal brush samples were taken from all infants with upper respiratory tract infection and from 11 of the 14 infants with bronchiolitis during the convalescent phase of the disease (2 to 4 weeks later). the study was approved by the medical ethical committee of the erasmus university medical centre rotterdam. written informed consent was given by all parents for participation of their child in this study. cells were harvested from the nose with a cytobrush (medscand medical, sweden) and processed as described elsewhere [godthelp et al., 1996] . cells were washed in rpmi 1640 medium (life technologies), and cytospin preparations were made on 10% (w/v) poly-l-lysine (sigma)-coated microscope slides. rsv infection was confirmed by direct immunofluorescent staining of nasal brush cells with antiviral antibodies [rothbarth et al., 1988] and/or by virus isolation from nasal brush supernatant. immunohistochemical staining of cd3, cd68, mbp, icam-1, and il-18 slides were fixed in acetone and placed in a semiautomatic stainer (sequenza, shandon, amsterdam, the netherlands). immunohistochemical staining was carried out as described elsewhere [godthelp et al., 1996] . briefly, slides were pre-incubated with 10% (v/v) normal goat serum (ngs; clb, the netherlands) (10 min) and subsequently for 60 min with mouse anti-human monoclonal antibodies directed against cd3, cd68, major basic protein (mbp), intercellular adhesion molecule-1 (icam-1), or il-18 diluted in phosphate-buffered saline (pbs) supplemented with 1% (w/v) blocking reagent (boehringer-mannheim, germany) (table i) . after 30-min incubation for with biotinylated goat anti-mouse ig serum, slides were incubated for 30 min with either streptavidin alkaline phosphatase for cd3, cd68, and mbp or with polyclonal goat anti-biotin antibody for icam-1 and il-18. after new fuchsin (chroma, germany) staining, sections were counterstained with gill's hematoxylin and were mounted in glycerin-gelatin. isotypic control antibody was used for control staining. tyramide signal amplification (tsa) staining for il-4, il-8, il-10, il-12, and ifn-g a sensitive protocol was used based on the alkaline phosphatase method described above. slides were incubated with mouse anti-human monoclonal antibodies directed against il-4, il-8, il-10, il-12, ifn-g, or an isotypic control antibody for 60 min (table i) . after incubation with biotinylated goat anti-mouse ig serum, endogenous peroxidase was blocked using 0.2% (w/v) azide, 0.02% (v/v) hydrogen peroxide and 50% (v/v) methanol in pbs. slides were then incubated with streptavidin conjugated peroxidase (30 minutes) (nen, usa), biotinyl tyramide in tris-hcl buffer (10 min) for amplification of the signal, with alkaline-phosphatase conjugated goat-anti-biotin and new fuchsin substrate (30 min). sections were stained for il-6 (table i) in this study, 1,000 cells stained with a purple-blue nucleus were counted in every nasal brush sample. all slides were blinded and counted by two independent investigators in order to ensure an objective analysis. the number of positively stained cells was calculated as a percentage of 1,000 nasal brush cells. cd3 and icam-1 positively stained cells had a red cell membrane. red cytoplasmic staining was found for cd68, mbp, il-4, il-8, il-10, il-12, il-18, and ifn-g. il-6 positive cells had dark brown cytoplasmic staining. on the basis of morphology, both inflammatory and ciliated epithelial cells were found to stain positive for cytokines. statistical analysis of cell numbers carried out with spss. percentages of positive cells were log-transformed to obtain a normal distribution among all data. the paired sample t-test was used to analyse differences between the two sampling moments. differences in cytokine-positive cells between patients with bronchiolitis and upper respiratory tract infection and between different patient characteristics were analysed with the independent sample t-test. correlations between percentages of cells and the age of the child were tested using spearman's correlation coefficient. differences between patient groups and sampling moments were considered statistically significant when the p value was 0.05. all but one of the 14 patients with rsv bronchiolitis needed hospital admission. six infants were admitted to the medium-care unit and seven to the intensive care unit (icu). three of the infants admitted to the icu required mechanical ventilation. all the bronchiolitis infants suffered from a runny nose and cough, and three infants had wheezing symptoms. none of the eight infants with mild rsv upper respiratory tract infection symptoms was admitted to the hospital. patient characteristics are summarised in table ii. during the acute phase of bronchiolitis as well as during upper respiratory tract infection, there was a marked increase in the numbers of macrophages (cd68positive cells) in nasal brush samples compared with convalescent samples (fig. 1a) . the median numbers increased from 0.9 to 4.4% in upper respiratory tract infection (p ¼ 0.009) and from 1.4 to 9.6% in bronchiolitis (p ¼ 0.001). although the median number of macrophages in the acute phase was higher during bronchiolitis than during upper respiratory tract infection, this was not statistically significant. the influx of macrophages was paralleled by a similar influx of t lymphocytes (cd3-positive cells). statistically significant increases in numbers of t lymphocytes were observed in upper respiratory tract infection (from median 1.7-2.3%; p ¼ 0.02), and a trend was also found toward increased numbers of t lymphocytes in bronchiolitis (from median 1.3-2.3%; p ¼ 0.06) (fig. 1b) . no differences were found between the two groups. the recruitment of inflammatory cells to the site of infection is often accompanied by an increased local expression of adhesion molecules that facilitates the migration of these cells [wang et al., 2000] . this also seems to be the case in this study. an increase was observed in the number of icam-1 positive cells during both upper respiratory tract infection (from median 11.7-18.4%) and bronchiolitis (from median 12.3-22.7%). however, compared with convalescence, significantly elevated numbers of icam-1 positive cells were only found during the acute phase of bronchiolitis (p ¼ 0.04) (fig. 1c) . small numbers of eosinophils (bmk-13 positive cells; median, 0%; range, 0-0.4%, fig. 1d ) were also found in nasal brush samples. no differences were observed in the number of eosinophils, either between acute and convalescent samples or between bronchiolitis and upper respiratory tract infection in the acute phase (fig. 1d) . however, during the convalescent phase in bronchiolitis, a small but significantly higher number of eosinophils were detected compared with the convalescent phase of patients presenting with upper respiratory tract infection (median 0% vs. 0.01%; p ¼ 0.05). nasal brush samples were also stained for th1-like cytokines il-12 and ifn-g, and for th2-like cytokines il-4 and il-10 (fig. 2) . in bronchiolitis, median numbers of il-12 positive cells increased from 3.2% at baseline (convalescence) to 6.6% during the acute phase of infection (p ¼ 0.04). there were no differences between bronchiolitis and upper respiratory tract infection in terms of numbers of il-12 positive cells. by contrast, no differences were found for il-4, il-10 and ifn-g positive cells between the acute and convalescent phase or between upper respiratory tract infection and bronchiolitis. similarly, no differences were found in the th2/th1 ratios (il-4/ifn-g or il-10/il-12) for patients with rsv bronchiolitis and upper respiratory tract infection at either sampling time point. however, there was a fall in the il-10/il-12 ratio during the acute phase of bronchiolitis compared with the convalescent phase (p ¼ 0.04). proinflammatory cytokines: il-6, il-8, and il-18 in contrast with the th1-and th2-like cytokine positive cells, an increase in numbers of proinflammatory il-18 positive cells was found during bronchiolitis compared with convalescence (from median 27.0-68.5%; p ¼ 0.01; fig. 3a ). interestingly, this increase was only observed during bronchiolitis, and not during upper respiratory tract infection. during the acute phase of infection, numbers of il-18 positive cells were higher during bronchiolitis than upper respiratory tract infection (p ¼ 0.001). although children with bronchiolitis and with upper respiratory tract infection differed in age at the moment of sampling, the increase in numbers of il-18 positive cells during bronchiolitis is not a consequence of the differences in age between the two groups. as shown in figure 4 , numbers of il-18 positive cells did not relate to the age of the child during infection in either children with bronchiolitis or children with upper respiratory tract infection. during baseline (convalescence), no differences in numbers were observed between bronchiolitis and upper respiratory tract infection. moreover, no age-dependent maturation was observed either. among infants with bronchiolitis, numbers of il-18 positive cells were not related to the severity of infection, as determined by the need for mechanical ventilation or admittance to the icu. in contrast to il-18, the numbers of il-6 positive cells increased for both bronchiolitis (from median 9.9-18.5%) and upper respiratory tract infection (from median 19.0-30.9%) (fig. 3b) . however, this increase was only statistically significant for bronchiolitis (p ¼ 0.03) but not for upper respiratory tract infection. no differences were observed in terms of il-6 positive cells between bronchiolitis and upper respiratory tract infection during either the acute or convalescent phase. median numbers of il-8 positive cells ranged between 0.5% and 45.1%, and no differences were found between acute and convalescent sampling or between bronchiolitis and upper respiratory tract infection (data not shown). during both rsv-induced upper respiratory tract infection and bronchiolitis, a general nasal inflammation was observed. this was characterised by increased numbers of macrophages, t lymphocytes, and icam-1 positive cells, a finding that is in line with previous observations [de weerd et al., 1998; grigg et al., 1999] . most importantly, this study showed a striking difference in cytokine responses between both types of infection, i.e., an increase in the number of il-18 positive cells in nasal brush samples during bronchiolitis. this increase was not evident during upper respiratory tract infection. the strong il-18 response was accompanied by an increase in proinflammatory cytokine il-6 and th1-like cytokine il-12 responses during bronchiolitis, but these responses were not different from those in patients with upper respiratory tract infection. il-18 is a proinflammatory cytokine, and its effect is closely related to that of il-1, i.e., the induction of tnfa, il-1, il-6, il-8, granulocyte-macrophage-colony-stimulating factor (gm-csf), icam-1, fas ligand, and several chemokines and the inhibition of il-10 and ige production [nakanishi et al., 2001; wang et al., 2001] . il-18 is also involved in antiviral mechanisms and is, in combination with il-12, a powerful inducer of ifng production from t lymphocytes and natural killer (nk) cells [dinarello, 1999] . il-18 can therefore stimulate th1 responses. in these nasal brush samples, an increase was observed in il-18 and il-12 responses during bronchiolitis, but this was not accompanied by a corresponding increase in the numbers of ifn-g positive cells. garofalo et al. [2001] did find higher levels of ifn-g protein in nasopharyngeal samples of infants with rsv bronchiolitis compared with rsv-induced upper respiratory tract infection. it is possible that the increase in the expression of ifn-g per cell was too small to be detected in this study due to the immaturity of the children's immune systems. this is supported by the observation that when mononuclear cells or t lymphocytes from cord blood are stimulated with mitogen, lower levels of ifn-g protein are produced compared with cells isolated from adult blood [chalmers et al., 1998; pit et al., 2000] . however, the approach in the present study did not allow us to determine whether levels of ifn-g expression rather than cell numbers could have been upregulated. despite this uncertainty, the observation of increasing numbers of il-18 and il-12 positive cells during rsv-induced bronchiolitis could explain the underlying mechanism of increased ifn-g production observed during general bronchiolitis in children [van schaik et al., 1999] . with respect to the other proinflammatory cytokines il-6 and il-8, there was no similar distinction between bronchiolitis and upper respiratory tract infection. although il-6 responses are increased during the acute phase of an rsv bronchiolitis compared with convalescence, this increase has also been observed for upper respiratory tract infection. there were no differences in the number of il-8 positive cells. however, others have found increased levels of il-8 protein in nasal samples during bronchiolitis [abu-harb et al., 1999] . noah et al. [1995] found elevated protein levels for both cytokines in nasal pharyngeal samples of infants with upper respiratory tract infection. furthermore, levels of il-6 and il-8 proteins in plasma were higher in infants with severe, as compared with mild, rsv infection [bont et al., 1999; brandenburg et al., 2000] . this discrepancy with the findings in this study is probably a consequence of the differences in the methods used to detect changes in the immune response. where this study determined changes in cell numbers, the other studies examined protein levels. nasal brushes are easy to carry out in infants and can adequately document cellular immune responses in the nose. in these brushes, it was observed on the basis of morphology that cytokines are not only produced by inflammatory cells but also by ciliated epithelial cells. this is in line with other studies indicating that cytokines are produced by epithelial cells after infection with rsv [arnold et al., 1994; fujishima et al., 1998 ]. this explains the higher percentages of cytokine-positive cells observed in cytospins in the present study, as would be expected purely on the basis of the number of inflammatory cells present in such samples. a possible caveat of the study is the age difference between infants in the two groups, which could have skewed the results. although cytokine responses could depend on the age of the child [buck et al., 2002] , no evidence was found that the il-18 data were affected. as shown in the figure 4 , no age-related maturation of il-18 responses was observed, either in infants with bronchiolitis or in infants with upper respiratory tract infection. moreover, at baseline (convalescence), numbers of il-18 positive cells did not differ between the two groups, and there was no age-related maturation. this shows that the differences in il-18 response between the two groups are not due to differences in age, but rather that this response represents an increase in il-18 positive cells in children with severe rsv-induced infection. similarly, possible confounders, such as gender, birth weight, duration of pregnancy, cigarette smoking by the parents, and allergic disease in the family of the children, did not differ significantly between the groups and did not affect the results (data not shown). it remains unclear whether the distinct nasal immune response for il-18 in rsv-induced bronchiolitis is functionally relevant. it would be interesting to determine whether this nasal increase in il-18 could be a direct reflection of the immune response in the lower airway during rsv bronchiolitis or the severity of infection. a potential link between il-18 expression and severity of disease has been postulated before. during tuberculosis, for instance, higher levels of il-18 were found in patients with high-grade fever compared with patients without fever [yamada et al., 2000 ]. an additional example of the link between il-18 and severity of disease was observed in asthmatic patients. in patients with asthma, not only were il-18 levels significantly higher in serum during an acute exacerbation than on remission days, but il-18 levels were also related inversely to peak expiratory flow . although patients were excluded who were positive for some of the respiratory viruses (rsv, parainfluenza virus, influenza virus), the study did not look for the most prevalent inducers of respiratory infection and asthma exacerbations, namely rhinovirus and coronavirus [nicholson et al., 1993; makela et al., 1998] . it is therefore possible that the increase in levels of il-18 during exacerbations is a direct reflection of a respiratory viral infection. the increased il-18 response during bronchiolitis could also be linked to the eosinophilia in nasal and peripheral blood samples. this has often described as accompanying bronchiolitis [garofalo et al., 1994; ehlenfield et al., 2000] . until recently, the increase in numbers of eosinophils was thought to result from enhanced th2 responses during infection [roman et al., 1997] . the present study could not confirm this hypothesis, as no th2-skewed response during bronchiolitis were observed. a previous study in our group showed that rsv-stimulated peripheral blood t lymphocytes taken during bronchiolitis produced predominantly th1, and some th2, cytokines, whatever the clinical severity of the underlying disease [brandenburg et al., 2000] . similar results were recently published by tripp et al. [2002] , who found an increase in both rsv-specific th1 and th2 cytokine-positive t lymphocytes during bronchiolitis compared with controls. on the other hand, the present data do suggest an alternative explanation for the observed eosinophil influx. il-18 has been shown to induce the expression of the eosinophil chemoattractants rantes and eotaxin [campbell et al., 2000; alaaeddine et al., 2001] . in combination with the increase in levels of icam-1 expression by il-18 [yoshida et al., 2001] , this cytokine could be responsible for the eosinophilia observed during and after bronchiolitis. in conclusion, despite the limitations of this study, clear differences were found in the local nasal immune response of children presenting with either rsvinduced upper respiratory tract infection or bronchiolitis. rsv bronchiolitis was characterised by a strong increase in cells positive for the proinflammatory cytokine il-18. the increase in il-18 positive cells is specific for bronchiolitis, as a similar increase could not be detected in rsv-induced upper respiratory tract infection. although il-6 and il-12 responses also increased during bronchiolitis, these were not different from patients with upper respiratory tract infection. the strong il-18 responses during bronchiolitis could be a direct reflection of the severity of infection and could explain the eosinophilia frequently observed during bronchiolitis. il-8 and neutrophil elastase levels in the respiratory tract of infants with rsv bronchiolitis production of the chemokine rantes by articular chondrocytes and role in cartilage degradation interleukin-8, interleukin-6, and soluble tumour necrosis factor receptor type i release from a human pulmonary epithelial cell line (a549) exposed to respiratory syncytial virus peripheral blood cytokine responses and disease severity in respiratory syncytial virus bronchiolitis type 1-like immune response is found in children with respiratory syncytial virus infection regardless of clinical severity longitudinal study of intracellular t cell cytokine production in infants compared to adults differential roles of il-18 in allergic airway disease: induction of eotaxin by resident cell populations exacerbates eosinophil accumulation intracellular cytokine profile of cord and adult blood lymphocytes t cell subset analysis in peripheral blood of children with rsv bronchiolitis il-18: a th1-inducing, proinflammatory cytokine and new member of the il-1 family eosinophilia at the time of respiratory syncytial virus bronchiolitis predicts childhood reactive airway disease twenty years of outpatient respiratory syncytial virus infection: a framework for vaccine efficacy trials respiratory syncytial virus-induced interleukin-4 production by human conjunctival epithelial cells contributes to allergy: preliminary study peripheral blood eosinophil counts and eosinophil cationic protein content of respiratory secretions in bronchiolitis: relationship to severity of disease macrophage inflammatory protein-1alpha (not t helper type 2 cytokines) is associated with severe forms of respiratory syncytial virus bronchiolitis dynamics of nasal eosinophils in response to a nonnatural allergen challenge in patients with allergic rhinitis and control subjects: a biopsy and brush study bronchoalveolar lavage fluid cellularity and soluble intercellular adhesion molecule-1 in children with colds viruses and bacteria in the etiology of the common cold interleukin-18 is a unique cytokine that stimulates both th1 and th2 responses depending on its cytokine milieu respiratory viruses and exacerbations of asthma in adults nasal cytokine production in viral acute upper respiratory infection of childhood prenatal immune priming with helminth infections: parasite-specific cellular reactivity and th1 and th2 cytokine responses in neonates respiratory syncytial virus infection in infants is associated with predominant th-2-like response rapid diagnosis of infections caused by respiratory syncytial virus a comparison of cytokine responses in respiratory syncytial virus and influenza a infections in infants il-18 might reflect disease activity in mild and moderate asthma exacerbation peripheral blood mononuclear cells from infants hospitalized because of respiratory syncytial virus infection express t helper-1 and t helper-2 cytokines and cc chemokine messenger rna increased production of ifn-gamma and cysteinyl leukotrienes in virus-induced wheezing adhesion molecule expression on epithelial cells infected with respiratory syncytial virus interleukin-18 enhances the production of interleukin-8 by eosinophils the relationship of rsv-specific immunoglobulin e antibody responses in infancy, recurrent wheezing, and pulmonary function at age 7-8 years increased levels of circulating interleukin-18 in patients with advanced tuberculosis il-18-induced expression of intercellular adhesion molecule-1 in human monocytes: involvement in il-12 and ifn-gamma production in pbmc the authors thank all the children and parents who participated in this study, afke brandenburg for collecting nasal brush and blood samples of infants with bronchiolitis, and frank kalthoff (novartis) for kindly providing the il-4 antibody. key: cord-321756-a7eh4dkb authors: kwofie, theophilus b; anane, yaw a; nkrumah, bernard; annan, augustina; nguah, samuel b; owusu, michael title: respiratory viruses in children hospitalized for acute lower respiratory tract infection in ghana date: 2012-04-10 journal: virol j doi: 10.1186/1743-422x-9-78 sha: doc_id: 321756 cord_uid: a7eh4dkb background: acute respiratory tract infections are one of the major causes of morbidity and mortality among young children in developing countries. information on the viral aetiology of acute respiratory infections in developing countries is very limited. the study was done to identify viruses associated with acute lower respiratory tract infection among children less than 5 years. method: nasopharyngeal samples and blood cultures were collected from children less than 5 years who have been hospitalized for acute lower respiratory tract infection. viruses and bacteria were identified using reverse transcriptase real-time polymerase chain reaction and conventional biochemical techniques. results: out of 128 patients recruited, 33(25.88%%, 95%ci: 18.5% to 34.2%) were positive for one or more viruses. respiratory syncytial virus (rsv) was detected in 18(14.1%, 95%ci: 8.5% to 21.3%) patients followed by adenoviruses (adv) in 13(10.2%, 95%ci: 5.5% to 16.7%), parainfluenza (piv type: 1, 2, 3) in 4(3.1%, 95%ci: 0.9% to 7.8%) and influenza b viruses in 1(0.8%, 95%ci: 0.0 to 4.3). concomitant viral and bacterial co-infection occurred in two patients. there were no detectable significant differences in the clinical signs, symptoms and severity for the various pathogens isolated. a total of 61.1% (22/36) of positive viruses were detected during the rainy season and respiratory syncytial virus was the most predominant. conclusion: the study has demonstrated an important burden of respiratory viruses as major causes of childhood acute respiratory infection in a tertiary health institution in ghana. the data addresses a need for more studies on viral associated respiratory tract infection. acute respiratory infections (ari) are one of the major causes of morbidity and mortality in young children throughout the world especially in developing countries [1, 2] . data from who estimated the burden of ari at 94,037,000 disability-adjusted life years (dalys) and 3.9 million deaths in 2001 [3] . similar report from a metaanalysis study demonstrates that throughout the world 1.9 million (95% ci 1.6-2.2 million) children died from ari in 2000, 70% of them in africa and southeast asia [2] . a further systematic analysis also estimated 1.575 million (uncertainty range: 1.046 million -1.874 million) deaths of children worldwide in 2008 as due to ari [4] . majority of acute lower respiratory tract infections (alrti) in developed countries have been reported to be often due to viral pathogens of which most common are rsv, piv, influenza viruses, adv, human coronaviruses and bocaviruses [5] [6] [7] . on the contrary, information on these viruses in developing countries is limited probably due to paucity of modern diagnostic molecular techniques. these infections are therefore treated unsuccessfully with antibiotics based on suspicion of bacterial causes [8] . apart from the public health concern of nosocomial infections that are associated with viral respiratory infections [9, 10] , significant costs derived from long duration of hospitalization and several healthcare visits could also aggravate the poor socio-economic status and increased child mortality in developing countries including ghana. lessons from the outbreak of the severe acute respiratory syndrome (sars) epidemics which resulted in the death of 776 individuals [11] and the recent emergence of a novel swine flu pandemic emphasize the risk posed by respiratory viral infections in humans [12] . this study was done to determine the burden of respiratory viruses among children hospitalized at the komfo anokye teaching hospital for acute lower respiratory illness using the real time polymerase chain reaction (rt-pcr). this was a hospital based cross-sectional study of children less or equal to five years and hospitalized for acute lower respiratory tract illness. the study was performed at the children's ward of the komfo anokye teaching hospital (kath), ghana from january to december 2008. kath is approximately a thousand bed tertiary medical facility located in ashanti region, kumasi, the confluence of the transportation network in the central part of ghana. its position makes it the most accessible tertiary and referral medical facility in ghana attending to a population of over 4.4 million in the ashanti region and beyond [13] . to have a year round incidence of the various viral agents, recruitment was done throughout the year 2008. screening and recruitment were started at the beginning of every week till two or three patients were recruited. after a maximum of three patients have been obtained it was suspended till the beginning of the next week. this cycle was repeated till the maximum of 11 patients were recruited for each month. at the recruitment station all patients arriving at the unit were screened but only those less than five completed years were recruited. patients recruited into the study should have features of severe pneumonia or very severe pneumonia as defined by the who-imci (world health organisation integrated management of childhood illness) protocol [14, 15] . severe pneumonia was said to be present if the child has a history of cough and/ or difficult breathing of less than 3 weeks duration, with lower chest wall recession. severe pneumonia in addition to cyanosis and/or inability to feed or drink was classified as very severe pneumonia. a child was recruited into the study only after the guardian or parents had consented after the objectives of the study had been explained in english or the local dialect. a standardized case record form was then used to record the history of the illness as well as the presenting clinical features of pneumonia. five (5 ml) of blood sample was then taken into 25 ml of brain heart infusion broth (bhib) and quickly sent to the bacteriological laboratory for culture. nasopharyngeal specimens were also taken using the nasopharyngeal flocked swab (copan, italy). the swab was gently inserted up the nostril towards the pharynx until resistance was felt and then rotated 3 times to obtain epithelial cells. it was then withdrawn and put into 2.5 ml phosphate buffered saline (x1). the samples were transported on ice to the laboratory within few hours of collection. they were then vortexed, transferred into 1.5 ml eppendorf tubes (eppendorf, germany) and kept at -80°c for polymerase chain reaction (pcr). both samples were taken by experienced prior trained nurses of the children ward. children were excluded if both samples could not be obtained for any reason. viral nucleic acids were extracted from samples using qiaamp viral mini kit (qiagen, germany) according to the manufacturers' instruction [16] . rt-pcr amplification was performed for the detection of adv, rsv, influenza a (flu a), influenza b (flu b), parainfluenza 1(piv 1), parainfluenza 2 (piv 2) and parainfluenza 3 (piv 3) using the light cycler version 1.5 (roche, germany). primers highly specific to each of the viruses were used ( table 1 ). the reaction mixture for rna viruses was made up of 5 μl of rna extract, 1 μl deoxynucleotide (dntp) triphosphate (10 mm) (qiagen), 1 μl bovine serum albumin (bsa)(1 mg/ml)(qiagen), 12.5 μl qiagen one step rt-pcr buffer x5 (containing 12.5 mm mgcl 2 ) (qiagen), 1 μl qiagen one step enzyme mix, 1 μl each of sense and antisense primer(10 μm each), 0.5 μl probe and 2.0 μl of rnase free water. dna virus reaction mixture was made up of 5 μl pcr buffer (10x), 1 μl of dntp mix (10 mm each), 1 μl of mgcl 2 (50 mm), 0.5 μl of hot star taq dna polymerase, 0.5 μl of probe, 1 μl of bsa (1 mg/ml) and 1 μl each of sense and antisense primers. the pcr conditions for piv, rsv and influenza viruses consisted of an initial reverse transcription at 20 minutes for 50°c, taq dna polymerase activation at 95°c followed by forty five (45) cycles of denaturation at 15 seconds for 95°c and annealing at 15 seconds for 58°c. the initial reverse transcription step was omitted in the case of adv. for each batch of tests that were run, rnase free water (qiagen, germany) was used as negative control and in vitro transcribed gene of the various viruses was used to determine the detection limit of the assay as positive control. the in vitro transcripts were prepared in our collaborative institution (bonn institute of virology, bonn, germany) and transported to ghana via cooling chain. the in vitro transcripts were prepared by amplifying the genes of interest of the viruses with specific set of primers. the products were ligated to the pcr2.1 vector using the topo ta cloning kit (invitogen corp, carlsbad, ca) and transformed into competent e.coli cells. after overnight incubation, desirable colonies were selected and purified using qiamp spin-miniprep kit (qiagen, germany). pcr was performed on the purified product and rna transcripts were then synthesized from the lowest dilution (with clear band) using ambion megascripts t3 kit (invitogen corp, calsbad, ca). the final products were purified using rneasy mini kit (qiagen, germany) and the concentrations of the transcripts were determined using a spectrophotometer (themoscientific, u.s.a). the copy numbers of the rna transcripts were determined following the method of fronhoffs [21] . ten-fold dilutions of the rna transcripts were prepared in carrier rna-rnase free water (310 μg of carrier rna in 310 ml of rnase free water) and tested. the detection limit was determined as the last dilution after which all other replicates gave negative results. the detection limits were respiratory syncytial virus (rsv): 20 copies/μl, piv 1: 1 -78 copies/μl, piv 2: 400 copies/μl, piv 3: 30 copies/μl, influenza a: 120 copies/μl and influenza b: 480 copies/μl. to determine the specificities, our assays were tested on other different viruses and they turn out negative. bacterial isolates were identified using conventional biochemical methods including urease and indole production, citrate utilization, hydrogen sulphide, gas production and fermentation of sugars. the biochemical media used included simon's citrate medium, urea and triple sugar iron agar (tsi). coagulase tests were performed for all staphylococci organisms. data obtained was double entered into a spreadsheet database prepared with microsoft ® excel. it was then compared and cleaned for abnormal wrongful entries. statistical analysis was done using stata se statistical software version 11.2(texas, usa) after the data had been imported. categorical variables such as age groups and their association with respiratory agents were analyzed using the fischer's exact test. continuous variables were expressed as medians with their inter-quartile ranges. a non-parametric k-sample test on the equality of medians was used to evaluate the differences in the medians of the various subgroups of the continuous variables. for all analysis done, a p-value of less than 0.05 was considered statistically significant. the study protocol was approved by the six (20.0%) out of 30 children less than six months of age were found to be positive for viral infection ( table 2 ). the highest number of children infected were between 6 months and 2 years, the proportions infected for the various age groups were however not significantly different. clinical presentation of patients were compared to general pathogens identified (table 3 ) and the individual viral pathogens (table 4) . generally, there were no detectable significant differences in the clinical presentation as well as the gender and duration of illness of patients for the various pathogens isolated. the study also investigated the seasonal variation of viruses. rsv infections were detected throughout the year however the peak infection rate occurred during the minor rainy seasons (october) (figure 1 ). adenovirus infections on the other hand had two main peaks occurring in the month of april and october. viral agents play an important role in acute lower respiratory infections and may herald the onset of pneumonia caused by secondary bacterial infections [22] . although information on the causes of respiratory illness in tropical countries is very scanty, available data indicates about one -third of the cases of respiratory tract infections are due to viruses [23] . the present study identified viruses in 25.8% of patients hospitalized for alrti with rsv being the most predominant. the overall prevalence is comparable to previous studies done in other developing countries [24] and the predominance of rsv is in accordance with the assertion that this virus is the single most frequent lower respiratory tract pathogen in infants and young children worldwide [25] [26] [27] . adenoviruses, the second highest viruses detected in this study have been reported to be responsible for 5-10% of lower respiratory tract infections with the highest rate occurring in younger children [28] [29] [30] [31] . our study similarly recorded a 10.2% detection rate of adenoviruses however there was no statistical difference in the age specific prevalence. this could possibly be due to the few number of younger children (less than one year) enrolled in this study. this study generally recorded higher cases of tachypnoea, chest recessions and very severe pneumonia in rsv compared to adenovirus infected patients but the differences were not statistically significant. although similar clinical presentations have been found to be associated with rsv [32, 33] , our small sample size could account for our inability to detect these differences. among patients who were poorly fed, those without viral or bacterial infections were found to be higher (p = 0.03). poor feeding and malnutrition has generally been associated with less risk for respiratory viral infection in developing countries [34] [35] [36] however the possibility of increased mortality could occur especially when bacterial infections are involved. further case controlled studies are therefore needed in developing countries to investigate this observation there have been fewer studies of piv infections in developing countries and most of them do not differentiate the subtypes of the viruses. our study recorded a 3.1% prevalence of piv infections with the predominant type being piv-3. similar hospital based studies have been reported in other developing countries [37] [38] [39] [40] . piv infections have been strongly associated with croup [41, 42] . the present study recorded no case of croup among piv patients. viral associated bacteremia was detected in two patients (1.7%). low rate of secondary bacterial infections has generally been reported in developing countries with the isolation rate varying between 2 and 10% [43] [44] [45] . studies in some developed countries however reported the converse [46, 47] probably due to the higher sensitivity of the bacteria identification techniques used. the blood culture identification system used in this study may be limited in the detection of bacteria associated with lower respiratory tract infection. perhaps the detection rate could have increased if the present study had identified bacterial pathogens from nasopharyngeal aspirates or washings as reported by hishinki et al. [47] . the isolation of staphylococcus aureus in an rsv positive patient in the present study was similar to studies reported by berman et al., [48] and cherian et al., [43] . in their case, the bacterium was associated with fatality in the children. the present study observed that all subjects enrolled in this study were treated empirically with antibiotics in accordance with the paediatric emergency treatment protocol for managing severe and very severe pneumonia. similar occurrences have been reported where patients were treated unsuccessfully with multiple antibiotics [8] . policy makers may therefore consider reviewing the clinical algorithm for patient management as the economic cost both to the health service and the patient's household derived from the use of antibiotics could be enormous. the period of this study experienced major rainfalls in the months of may to july and minor rainfalls in the month of september to october. the month of october recorded the highest detection of viral infections (57.6%; 19/33) with the commonest being rsv. this phenomenon has been reported in other developing countries [43, 49] . the occurrence of rsv infections in the rainy and cold season could be due to overcrowding as a result of populations staying more to their homes. more studies are however needed to define the seasonality of respiratory viruses in tropical countries. our study had some limitations which include underestimation of the overall prevalence of respiratory viruses since we did not test for coronaviruses, human metapneumovirus, bocaviruses and rhinoviruses which have also been reported in hospitalized patients. also the negative results recorded for influenza a and piv 2 and the low prevalence of influenza b in our samples could be due to the low sensitivity of the assay for these viruses. the limit of detections of influenza a, influenza b and piv 2 are 120 copies/μl, 480 copies/μl and 400 copies/μl respectively as such viral copies below these limits of detections could be missed by our assay. this study has demonstrated that respiratory viruses are associated with a considerable number of hospital admissions in ghana. hospitalized children presenting with symptoms of alrti may not necessarily need treatment with antibiotics but rather antiviral drug options could be explored. the importance of further cross-sectional and longitudinal studies of viral-associated respiratory infections among out-patients and healthy populations cannot be over emphasized. this will contribute immensely to overcoming the paucity of data on the importance of respiratory associated viruses to the burden of disease and the morbidity and mortality of under five children. acute respiratory infections are the leading cause of death in children in developing countries estimates of worldwide distribution of child deaths from acute respiratory infections world health organisation: burden of disease in dalys by sex and mortality stratum in who regions, estimates for 2001. the world health report global, regional, and national causes of child mortality in 2008: a systematic analysis progress in the surveillance of respiratory syncytial virus (rsv) in europe development of three multiplex rt-pcr assays for the detection of 12 respiratory rna viruses population-based surveillance for hospitalizations associated with respiratory syncytial virus, influenza virus, and parainfluenza viruses among young children risk of secondary bacterial infection in infants hospitalized with respiratory syncytial viral infection the clinical spectrum of respiratory syncytial virus disease in the gambia an outbreak of acute respiratory disease in trinidad associated with para-influenza viruses newly discovered coronavirus as the primary cause of severe acute respiratory syndrome emergence of a novel swine-origin influenza a (h1n1) virus in humans komfo anokye teaching hospital, centre of excellence integrated approach to child health in developing countries integrated management of childhood illness by outpatient health workers: technical basis and overview. the who working group on guidelines for integrated management of the sick child qiagen: protocol: purification of viral rna (spin protocol) a method for the rapid construction of crna standard curves in quantitative real-time reverse transcription polymerase chain reaction epidemiology of acute respiratory infections in children of developing countries. review of infectious diseases viral respiratory infections and their role as public health problem in tropical countries (review) respiratory syncytial virus infection in tropical and developing countries update: respiratory syncytial virus activity-united states, 1996-97 season epidemiology and aetiology of acute bronchiolitis in hong kong infants viral etiology of acute lower respiratory tract infections in hospitalized young children in northern taiwan respiratory adenoviral infections in children: a study of hospitalized cases in southern taiwan in 2001-2002 frequent detection of human rhinoviruses, paramyxoviruses, coronaviruses, and bocavirus during acute respiratory tract infections detection of a broad range of human adenoviruses in respiratory tract samples using a sensitive multiplex real time pcr assay adenovirus pneumonia in infants and factors for developing bronchiolitis obliterans: a 5-year follow-up epidemiology and clinical presentation of respiratory syncytial virus infection in a rural area of southern mozambique clinical presentation and severity of viral community-acquired pneumonia in young nepalese children the etiology of pneumonia in malnourished and well-nourished gambian children respiratory syncytial virus infections in malnourished children viruses associated with acute lower respiratory tract infections in children from the eastern highlands of papua new guinea (1983-1985) epidemiology of acute respiratory tract infections among young children in kenya. review of infectious diseases viral etiology and epidemiology of acute respiratory infections in children in etiology of acute lower respiratory tract infections in gambian children: i. acute lower respiratory tract infections in infants presenting at the hospital viral etiology of respiratory infections in children under 5 years old living in tropical rural areas of senegal: the evira project respiratory syncytial virus and parainfluenza virus parainfluenza viruses bronchiolitis in tropical south india etiology of acute respiratory infections in children in tropical southern india. review of infectious diseases diagnosis of childhood pneumonia in the tropics association of invasive pneumococcal disease with season, atmospheric conditions, air pollution, and the isolation of respiratory viruses incidence of bacterial coinfection with respiratory syncytial virus bronchopulmonary infection in pediatric inpatients acute lower respiratory tract illnesses in cali, colombia: a two-year ambulatory study seasonal variation in respiratory syncytial virus infections in children in evaluation of quantitative and typespecific real-time rt-pcr assays for detection of respiratory syncytial virus in respiratory specimens from children pring-akerblom p: rapid and quantitative detection of human adenovirus dna by real-time pcr comparison of real-time pcr assays with fluorescent-antibody assays for diagnosis of respiratory virus infections in children simultaneous detection of influenza viruses a and b using real-time quantitative pcr respiratory viruses in children hospitalized for acute lower respiratory tract infection in ghana the study was partly supported by the komfo anokye teaching hospital (ghana) and the kumasi center for collaborative research in tropical medicine (ghana). we will like to thank all directorate of child health doctors, nurses and patients at the komfo anokye teaching hospital. we also thank the bonn institute of tropical medicine (germany) for supporting us with laboratory materials. authors' contributions yaa, aa and bn co-worked on the data collection and contributed to laboratory analysis of the samples. sbn performed statistical analysis of the data and contributed to writing of the manuscript. tbk planned, initiated the study and contributed to writing of the manuscript. ow carried out the laboratory analysis of the samples and contributed to writing of the manuscript and interpretation of the data. all authors have read and approved the manuscript. the authors declare that they have no competing interests. key: cord-345472-qrddwebe authors: sebina, ismail; phipps, simon title: the contribution of neutrophils to the pathogenesis of rsv bronchiolitis date: 2020-07-27 journal: viruses doi: 10.3390/v12080808 sha: doc_id: 345472 cord_uid: qrddwebe acute viral bronchiolitis causes significant mortality in the developing world, is the number one cause of infant hospitalisation in the developed world, and is associated with the later development of chronic lung diseases such as asthma. a vaccine against respiratory syncytial virus (rsv), the leading cause of viral bronchiolitis in infancy, remains elusive, and hence new therapeutic modalities are needed to limit disease severity. however, much remains unknown about the underlying pathogenic mechanisms. neutrophilic inflammation is the predominant phenotype observed in infants with both mild and severe disease, however, a clear understanding of the beneficial and deleterious effects of neutrophils is lacking. in this review, we describe the multifaceted roles of neutrophils in host defence and antiviral immunity, consider their contribution to bronchiolitis pathogenesis, and discuss whether new approaches that target neutrophil effector functions will be suitable for treating severe rsv bronchiolitis. respiratory syncytial virus (rsv) is the leading viral cause of lower respiratory infections (lri) among young infants [1, 2] . in 2015, an estimated 33 million cases and 120,000 deaths due to rsv infection were reported worldwide [2] . the estimated cost of treating severe cases of rsv-induced bronchiolitis is over $300 million per year [3] , and an fda-approved vaccine against rsv remains elusive [4, 5] . a greater understanding of the molecular mechanisms underlying the immunopathogenesis of rsv-bronchiolitis may reveal novel pathways that can be targeted for improving treatment therapies for severe rsv patients. severe viral bronchiolitis in infancy is a major independent risk factor for the development of chronic asthma in later life [6] . therefore, ameliorating severe bronchiolitis in infancy may also act as a preventative strategy for later asthma in susceptible individuals. the prevailing view is that severe bronchiolitis results from excessive inflammation and consequent immunopathology, rather than virus-induced cytology. neutrophils are the dominant inflammatory cell in the airways of paediatric patients with rsv bronchiolitis [7] [8] [9] , accounting for up to 80% of the cellular infiltrate at peak symptomatology [8] , and yet the precise contribution of neutrophils to antiviral immunity remains ill-defined. in the context of anti-bacterial or anti-fungal immunity, neutrophils mediate protection through the production of antimicrobial peptides, respiratory oxygen species (ros), cytokines and chemokines, and the formation of neutrophil extracellular traps (nets). new information from omics-based technologies and advances in multi-parameter flow cytometry are shedding new light on neutrophil diversity and function, suggesting a greater complexity to a cell that is often unfavourably viewed as a blunt tool on account of its spectacular prowess at microbial killing and short life-span. here, we review the multifaceted roles of the neutrophil in host defence, antiviral immunity, and immunopathogenesis in the context of viral lris. we find that neutrophils make an important contribution to innate antiviral immunity and help to optimise the induction of an effective adaptive immune response. however, as a consequence of their ability to initiate and amplify the magnitude of an inflammatory reaction, it is clear that a failure to adequately regulate this response can lead to excessive collateral damage and a loss of tissue function, leading to significant morbidity from an rsv infection. we consider a number of novel approaches that might be employed to either harness or suppress the actions of neutrophils to promote antiviral immunity and/or ameliorate the immunopathology associated with severe rsv infection. an estimated four billion neutrophils are produced in the bone marrow every hour [10, 11] , and accordingly, neutrophils are by far the most abundant leukocyte in blood, representing approximately 60% of the total white blood cell count. as well as circulating in large numbers, neutrophils enter and surveil tissues in the steady state [11] . neutrophils are one of the host's first defensive measures against a sterile or microbial insult. neutrophils employ three major defensive mechanisms: phagocytosis, degranulation (secreting an array of anti-microbial peptides), and netosis [12, 13] . during phagocytosis, neutrophils utilise a combination of surface-expressed opsonic receptors, intracellular signalling cascades, and cytoskeletal rearrangements to engulf and internalise microbes [14, 15] . neutrophils contain cytoplasmic granules, which store antimicrobial molecules and facilitate cross-talk with other immune cells [16] . these granules (summarised in table 1 ) are subdivided into primary or azurophilic granules (containing, e.g., myeloperoxidase, cd63), secondary or specific granules (containing, e.g., lipocalin-2 and lactoferrin), and tertiary or gelatinase granules (containing, e.g., cathelicidins and neutrophil collagenase) [16] . this classification is based on their production during granulopoiesis and expression of distinct protein markers [16] . as a consequence of phagocytosis, the internalised microbe is trapped in 'bag-like' structures known as phagosomes, which fuse with neutrophil granules for the safe destruction of microbes [14] . upon fusion, antimicrobial molecules (summarised in table 1 ) are released into the phagosome, whereupon they kill the trapped microbe [16, 17] . table 1 . neutrophil granules and their anti-microbial properties. neutrophils can also eliminate microbes by releasing extracellular dna traps composed of decondensed nuclear chromatin, histones, and protein granules, in a process termed netosis [28, 29] . classical activation of netosis depends on nadph oxidase-mediated production of ros [30] , which promotes chromatin decondensation via histone deamination or citrullination. this process is aided by the downstream activation of the protein-arginine deiminase type 4 (pad4), a nuclear enzyme that citrullinates arginine residues, converting amine groups to ketones [31] [32] [33] . ros production can also activate azurophilic granules to release myeloperoxidase (mpo) and neutrophil elastase (ne). if these enzymes translocate to the nucleus, they can disrupt chromatin packaging [15, 17] , and promote the release of chromatin, which then interacts with other granular and cytosolic proteins to form 'nets' [30] . these nets are important for immunity against fungi and some bacterial species [22, 32, 34] . for example, people who are unable to produce nets develop chronic granulomatous disease and are highly susceptible to invasive aspergillosis [23, 35] . similarly, individuals lacking mpo are susceptible to recurrent fungal infections [23] . in pad4-deficient mice, neutrophil killing of shigella flexneri or group a streptococcus is diminished [36] . collectively, these studies demonstrate that netosis is an important weapon in the host's defensive armoury. however, if left unchecked, net-induced responses may also mediate severe pathology. excessive netosis can damage the epithelium in pulmonary aspergillosis [37] , damage the endothelium in transfusion-related acute lung diseases [38] , and exacerbate rhinovirus infection-induced allergic asthma [39] . therapeutic approaches to block netosis are now being assessed for the treatment of infectious diseases (including bacterial sepsis), autoimmune diseases (including systemic lupus erythematosus [40] , rheumatoid arthritis [41] ) and chronic lung disorders (such as cystic fibrosis [42] ). neutrophils are a vital component of the innate immune system, with key roles in pathogen recognition, the killing of invading pathogens, the presentation of antigens to t-cells, the recruitment of other inflammatory cells, and the production of cytokines [18, 43] . for sensing invading pathogens, neutrophils employ a vast array of pattern recognition receptors (prrs), including toll-like receptors (tlrs), c-type lectin receptors (e.g., dectin-1) and cytoplasmic sensors of ribonucleic acids (rig-i and mda5) [18, 44, 45] . neutrophils also express nucleotide-binding oligomerisation domain (nod)-like receptors, important for inflammasome formation [46] . the sensing of pathogens through these prrs activates the effector functions of neutrophils, including ros production, net formation, and degranulation (as highlighted in section 2). activated neutrophils may also influence the quality of innate and adaptive immune responses by affecting the trafficking and function of other innate cells, such as dendritic cells (dcs) [47] [48] [49] [50] [51] [52] [53] . for example, the neutrophil-derived chemokine ccl3 supports the rapid recruitment of dcs to the inoculation site during leishmania major infection [54] . dc function can also be affected: in the context of an infection with toxoplasma gondii, neutrophil-depletion attenuated interleukin (il)-12 and tnf production by splenic dcs [55] . neutrophils also affect the recruitment of other cells too, which they accomplish via the secretion of pro-inflammatory mediators such as danger-associated molecular patterns (damps; e.g., high mobility group box 1 (hmgb1), double stranded dna, and s100 complex proteins), pro-inflammatory cytokines (e.g., il-1α, il-1β, il-6, il-17, and tnf) and chemokines (e.g., cxcl1, cxcl2, cxcl8, ccl2, and ccl3) [12, 56, 57] . neutrophils can also initiate adaptive immune responses by directly presenting antigens themselves, or by acting as accessory cells, to support t-cell responses. for instance, human neutrophils upregulate mhc-ii and co-stimulatory molecule (cd40 and cd80) expression and can present antigens to cd4 + t-cells following phagocytosis [58] . neutrophil acquisition of these antigen-presenting properties (mhc-ii, cd40, and cd80 expression) is associated with increased activation and proliferation of cd4 + t-cells in response to tetanus toxoid. neutrophils can also direct t-cell recruitment to the site of infection. for example, in response to influenza virus infection, lung-infiltrating neutrophils were found to deposit a long-lasting chemoattracting trail (expressing the chemokine cxcl12) in the lung to guide antigen-specific cd8 + t-cells into specific niches [59] . in the absence of neutrophils, influenza-specific cd8 + t-cells were lower in the lung, leading to increased viral load and delayed viral clearance. neutrophils can also influence cd4 + t cell helper (th) responses, in particular, th17 immune responses [50, 51, 60] . in a mouse model of allergic asthma, neutrophil cytoplasts (enucleated cell bodies) augmented dc-mediated th17 responses in the lymph nodes, which subsequently increased asthma-like pathology in the lung [51] . collectively, these studies demonstrate that neutrophils are important participants in innate immunity and contribute to effective adaptive immune responses. the vast majority of human respiratory viruses, including rsv, rhinovirus, influenza, coronavirus, adenovirus, and parainfluenza virus, can cause bronchiolitis [61]. however, rsv-induced bronchiolitis is the leading cause of hospitalisation and death among infants within the first two years of life [1, 62] . rsv is highly contagious and persists outside of the host for almost six hours [63] . this prolonged survival facilitates its spread to susceptible individuals, mainly via inoculation of open mucous membranes lining the eyes and buccal cavity. upon inoculation, rsv infects the nasopharyngeal epithelium of the upper respiratory tract, replicates in epithelial cells, and then spreads to the lrt via the bronchiolar epithelium [9, 64] . this occurs within 1-3 days post infection, with peak infectivity occurring at 5-7 days post-inoculation [3, 65] . during this period, rsv triggers extensive inflammation (characterised by increased neutrophilia and levels of inflammatory cytokines), mucus hypersecretion, and oedema in the airways [1, 9, 66] . in severe cases, increased mucus production and deposition of cellular debris can occlude the bronchiole lumen, contributing to bronchiolar obstruction, air trapping, and lobar collapse [9] . patients with severe disease experience dyspnea, wheezing, and cough, the latter often persisting for three or more weeks. mild cases of bronchiolitis are manageable in outpatient departments. however, severe disease can be life threatening, necessitating urgent mechanical ventilation and admission to intensive care units for some patients. individuals with severe rsv-induced bronchiolitis during infancy are more likely to develop impaired lung function, recurrent wheezing, and asthma in adulthood [67] . whether neutrophils play a beneficial or detrimental role during rsv bronchiolitis remains difficult to ascertain clinically due to difficulties in sampling young infants. however, the predominance of neutrophils in the airway wall and alveoli of lung autopsy samples from fatal cases of rsv-related lri [9, 68] or in the airways of paediatric patients (~80% of the total cell infiltrate) with severe rsv-induced bronchiolitis [8] would suggest that they are not innocent bystanders. we postulate that neutrophils promote lung pathophysiology during rsv bronchiolitis through the production of pro-inflammatory cytokines and the releasing of cytotoxic molecules (illustrated in figure 1 ). these products, released through distinct cellular processes (e.g., netosis, degranulation) contribute to mucus hyperproduction, airway epithelial cell (aec) death and sloughing, and oedema ( figure 1 ). in rsv-infected mice, the depletion of neutrophils decreases ne, mpo, and mmp9 levels in the airways [69] . depletion of neutrophils has also been shown to lower tnfα levels and attenuate airway mucin production in the lungs of rsv infected mice [70] . in addition, in vivo antagonism of cxcr2 (a chemokine receptor important for neutrophil recruitment into the lung) reduces mucus production in the airways of rsv-infected mice [71] . in vitro, co-culture of rsv-infected aecs with neutrophils increases aec damage compared with rsv-infected aecs cultured without neutrophils [72] . collectively, these findings suggest that neutrophils can contribute to pathogenesis; however, a limitation of modelling rsv in mice is that the virus replicates poorly, and hence this is not a particularly tractable system to assess the beneficial properties of neutrophils, such as their antiviral activities. (1), degranulation (2), respiratory oxygen species (ros) production (3), and the release of neutrophil extracellular traps (netosis) (4) are associated with increased lung inflammation, systemic fever, mucus hypersecretion, airway obstruction, and epithelial cell death. together, these factors contribute to increased lung damage during severe rsv bronchiolitis. infantile severe viral bronchiolitis remains a major risk factor for persistent wheezing and chronic asthma in childhood [73, 74] . therefore, excessive neutrophilic inflammation in infants with severe rsv bronchiolitis may contribute to the onset of type 2 inflammation and cause long-term defects in lung development that predispose susceptible infants to the development of asthma in later childhood. thus, regulating excessive neutrophilic responses during severe rsv-bronchiolitis might protect against the loss of lung function, curtail the development of allergic sensitisation, and reduce the prevalence of asthma. in light of this, it is important to understand (1) the molecular events governing neutrophil recruitment to the lung, (2) the type of neutrophils recruited to the lung, (3) the molecular signals that control optimal neutrophil activation and function, and (4) whether specific neutrophil subtypes can be targeted therapeutically to improve the management of severe rsv bronchiolitis. we discuss these questions in the sections below. the earliest stages of neutrophil recruitment are initiated by damps (table 2) , including hmgb1, dna, and histones, which are released by damaged or dying cells [75] . indeed, hmgb1 is elevated in the nasopharynx of rsv-infected children compared with those infected with other viruses [76] . animal models of rsv-bronchiolitis have improved our knowledge on the mechanisms of disease development and progression (reviewed extensively in [77] [78] [79] ); however, as figure 1 . potential roles of neutrophils in the pathophysiology of severe respiratory syncytial virus (rsv) bronchiolitis. excessive neutrophil-derived inflammatory cytokine production (1), degranulation (2), respiratory oxygen species (ros) production (3), and the release of neutrophil extracellular traps (netosis) (4) are associated with increased lung inflammation, systemic fever, mucus hypersecretion, airway obstruction, and epithelial cell death. together, these factors contribute to increased lung damage during severe rsv bronchiolitis. infantile severe viral bronchiolitis remains a major risk factor for persistent wheezing and chronic asthma in childhood [73, 74] . therefore, excessive neutrophilic inflammation in infants with severe rsv bronchiolitis may contribute to the onset of type 2 inflammation and cause long-term defects in lung development that predispose susceptible infants to the development of asthma in later childhood. thus, regulating excessive neutrophilic responses during severe rsv-bronchiolitis might protect against the loss of lung function, curtail the development of allergic sensitisation, and reduce the prevalence of asthma. in light of this, it is important to understand (1) the molecular events governing neutrophil recruitment to the lung, (2) the type of neutrophils recruited to the lung, (3) the molecular signals that control optimal neutrophil activation and function, and (4) whether specific neutrophil subtypes can be targeted therapeutically to improve the management of severe rsv bronchiolitis. we discuss these questions in the sections below. the earliest stages of neutrophil recruitment are initiated by damps (table 2) , including hmgb1, dna, and histones, which are released by damaged or dying cells [75] . indeed, hmgb1 is elevated in the nasopharynx of rsv-infected children compared with those infected with other viruses [76] . animal models of rsv-bronchiolitis have improved our knowledge on the mechanisms of disease development and progression (reviewed extensively in [77] [78] [79] ); however, as pneumoviruses are host-specific, a major limitation with using mice to study human rsv stems from its inability to replicate efficiently in this setting. to better understand the pathogenic processes that underlie viral bronchiolitis, we and others have preferred to inoculate mice with pneumonia virus of mice (pvm), which is orthologous to human rsv [9, 80] . because pvm is a natural pathogen in rodents, a low inoculum dose (e.g., 10 pfu) can be employed, and this allows for greater insights into host-pathogen interactions over time as the virus replicates in the airway epithelium. importantly, inoculation with a low dose of pvm can induce the more severe pathologies of infant bronchiolitis, such as alveolar epithelial cell apoptosis, bronchial epithelial necrosis, multifocal acute alveolitis, intra-alveolar oedema, haemorrhage, and increased neutrophilia [76, 78, 81] . these pathological features are similar to those observed in autopsy samples obtained from fatal cases of rsv bronchiolitis [9] . although there are limitations with the use of pvm (highlighted in [78] ), it is proving a useful model to understand the cellular and molecular processes that underlie pathogenesis. unlike wild-type (wt) control mice, plasmacytoid dendritic cell (pdc)-depleted, toll-like receptor (tlr)7-deficient, or interferon regulatory factor (irf)7-deficient neonatal mice develop severe pathology, characterised by increased neutrophilia and lung inflammation in response to acute pvm infection [80] [81] [82] . in the absence of the tlr7-irf7 signalling pathway, massive amounts of preformed airway epithelial cell hmgb1 is released into the extracellular environment, increasing the level of neutrophilic inflammation and amplifying tissue pathology, most notably tissue oedema, epithelial sloughing, and cell death [76, 80, 82, 83] . the cell death occurs through programmed necroptosis and further increases the levels of hmgb1, perpetuating the inflammatory response [76] . rsv infection of primary human airway epithelial cells also induces necroptosis-dependent hmgb1 release [76] . intriguingly, therapeutic inhibition of necroptosis during severe viral bronchiolitis protected mice against the subsequent development of experimental asthma in later-life [76] , indicating that severe bronchiolitis is causally associated with subsequent asthma. examination of the infant bronchiolitis revealed that pharmacological inhibition of necroptosis or immunoneutralisation of hmgb1 decreases the expansion of il-13-producing type-2 innate lymphoid cells (ilc2s), and prevents alterations to the airway wall such as thickening of the airway smooth muscle (asm) layer [76, 83] . in vitro, asm cells cultured with il-33/il-2-activated ilc2s isolated from the lungs of pvm-infected mice undergo increased cell division, elucidating a cellular and molecular pathway by which excessive inflammation and immunopathology promotes both type 2 inflammation and an aberrant repair response that alters the architecture of the developing lung [83] . therefore, therapeutic targeting of the hmgb1 signalling axis may act as a novel asthma preventative by dampening ilc2-mediated type-2 inflammation and associated asm remodelling. low circulating pdc numbers in infancy are associated with acute lris [84] . interestingly, the temporal depletion of pdc in neonatal mice predisposes to severe pvm bronchiolitis due to a lack of immunoregulation by regulatory t-cells [80] . the adoptive transfer of splenic regulatory t-cells to infected pdc-depleted neonatal mice decreased hmgb1 and il-6 levels, epithelial sloughing, and lung neutrophil infiltration. despite the lack of effect on viral load, the attenuated inflammatory response improved the clinical score. additionally, the adoptive transfer of regulatory t-cells in early life was sufficient to protect against the later development of experimental asthma [80] . these data suggest that therapies that enhance immunoregulation will both ameliorate the severity of bronchiolitis and break its nexus with the development of asthma. damps can indirectly promote neutrophilic inflammation by inducing the production of neutrophil-active chemoattractants (table 2) , such as il-8 [85] and leukotriene b4 (ltb4) [86] . il-8 levels correlate with neutrophil numbers in the airways and are elevated in nasal washes of rsv-infected children [68] . moreover, genetic polymorphisms in the il-8-encoding gene that increase il-8 production are associated with increased severity of rsv bronchiolitis [87] , and pharmacological inhibition of il-8 in humans reduces neutrophilia and improves clinical outcomes [88] . levels of the eicosanoid ltb4 are elevated in endotracheal aspirates of infants with rsv bronchiolitis compared with healthy controls [89] , and are associated with increased neutrophilic responses [90] . treatment of rsv-infected mice with the leukotriene inhibitor zileuton reduces cellular inflammation (including neutrophils) in the lung, prevents rsv-induced weight loss and decreases airway pathology compared with untreated control mice [91] . mice lacking ccl3 or its receptor ccr1 display significantly reduced numbers of airway neutrophils in response to infection with pvm [92, 93] . moreover, the blockade of ccl3/ccr1 in combination with antiviral therapy improves the survival of mice infected with a lethal pvm dose [94] . damps can also promote γδ-t cells [95] and cd4 + th17 cells [96] to produce il-17a [96] [97] [98] , which is elevated in nasal aspirates from rsv-infected infants in comparison with uninfected controls [99] . il-17a augments neutrophil recruitment into the lung by stimulating the production of il-8 and leukotrienes by microvascular endothelial and lung epithelial cells [100, 101] . in addition, il-17 activates p38 mapk-induced expression of endothelial adhesion markers (e-selectin, vcam-1, and icam-1) on lung endothelial cells, promoting trans-endothelial migration of neutrophils. lack of il-17 in rsv-infected mice reduces neutrophil numbers in the lung, limits mucus production, and improves the cytotoxic t-cell (ctl) responses against rsv [99] . however, the mechanism by which il-17 deficiency enhances ctl responses to rsv remains unknown. nonetheless, inhibiting il-17 signalling may prevent neutrophil-induced inflammation and pathology during rsv infection. induce secretion of pro-inflammatory cytokines, drive ilc2 responses, induce necroptosis and aec death [76, 83] neutrophils have traditionally been viewed as a homogenous cell population. however, it is now appreciated that diversity exists at the molecular, phenotypic, and functional level [11, 13, 112, 113] . this heterogeneity is a consequence of differences in proliferative capacity, maturation status, transcriptional and epigenetic properties, and environmental cues in tissues [11] . in one study that employed fucci-474 reporter mice to identify cells at different stages of the cell cycle, subsequent mass cytometry and transcriptomic analysis revealed the existence of three distinct neutrophil subsets in the bone marrow [114] . these subsets consisted of a highly proliferative precursor that differentiated into two non-proliferating subsets, one immature and one mature. the precursor neutrophils were ly6gand cxcr2-negative, but expressed high levels of ckit and cxcr4. the immature subset displayed low levels of surface ly6g and cxcr2 and was positive for cxcr4. the mature subset displayed higher levels of ly6g, cxcr2, and cd101 expression and lacked surface cxcr4. immature and mature neutrophil subsets were subsequently identified in human blood, with immature neutrophils associating with increased tumour burden [114] , suggesting that neutrophil heterogeneity influences disease outcomes. importantly, different neutrophil subsets have been identified in circulation and in the lung during acute rsv-bronchiolitis [115, 116] . flow cytometric analysis revealed four distinct neutrophil subsets according to their differential expression of cd16 and cd62l [115] . these included a mature subset (defined as cd16 hi cd62l hi ), an immature one (cd16 lo cd62l hi ), a suppressive subset (cd16 hi cd62l lo ), and a progenitor subset (cd16 lo cd62l lo ). the relative frequency of these subsets changed over the course of infection, suggesting that they may play different roles, although functional differences between these subsets remain to be shown. in a separate study in rsv-infected patients, geerdink and colleagues identified that neutrophils in balf, compared to those in blood, displayed heightened expression of the inhibitory immune receptor leukocyte-associated immunoglobulin-like receptor-1(lair-1) [117] . flow cytometry analysis also revealed increased sirp-α, siglec-9, cd11b, and reduced cd62l and cd31 expression on activated lair-1-expressing neutrophils. agonistic ligation of lair-1 was shown to limit net formation by balf neutrophils in vitro. of note, rsv-infected lair-1-deficient mice present with greater neutrophilic inflammation upon inoculation with rsv [118] , supporting the notion that agonism of lair-1 is a logical strategy to ameliorate the severity of viral bronchiolitis. in a separate study performed in infants with an rsv infection, the neutrophils in the nasopharyngeal aspirates were shown to exhibit lower cd62l and cd31 (pecam-1) and higher cd54 (icam-i), cd11b, and cd18 expression compared to those in peripheral blood [116] . cd18 and icam-1 expression on neutrophils are also increased in rsv-infected infants compared with uninfected control infants [119] . in a more recent study, the addition of physiological concentrations of neutrophils to human rsv-infected nasal cultures in vitro increased epithelial cell damage, characterised by lower ciliary activity, cilium loss, less tight junction expression, and greater detachment of epithelial cells, compared to cultures with rsv-infected epithelial cells alone [120] . high-throughput analysis of lung-infiltrating neutrophils using the latest transcriptomic and high-dimensional flow cytometry is now needed to better identify different neutrophil subsets and associate these with beneficial and deleterious outcomes. such knowledge, together with an understanding of the molecular signals controlling neutrophil recruitment and function, is likely to reveal novel prognostic biomarkers and targets for therapeutic intervention. neutropenic individuals are highly susceptible to bacterial and fungal infections. the evidence with regard to viral infections is less clear [18] , suggesting that neutrophils are not critical participants. however, neutrophils do appear to be required for optimal host immunity against an rsv infection. to accomplish this, neutrophils detect virus-associated molecular patterns through pattern recognition receptors (such as tlrs), produce an array of antimicrobial products, and assist the adaptive immune responses. for example, tlr4 expression on neutrophils is important for generating optimal immunity against rsv infection [121, 122] . the expression of tlr4 is lower on blood and airway neutrophils of infants with severe rsv infection compared with uninfected controls [122] . in humans, two tlr4 gene mutations are associated with an increased risk of developing severe rsv bronchiolitis [123] . of note, the beneficial or detrimental roles of tlr-signalling in neutrophils during respiratory viral infections may be context-dependent. for instance, tlr4-signalling may be detrimental in rsv infection but protective against influenza. therapeutic tlr4 antagonism lowers tnf-α, il-1α, ptgs2, and cxcl1 gene expression and improves survival of mice infected with a lethal dose of influenza a [124] . some neutrophil anti-microbial products may be exploited for boosting anti-viral immunity during rsv infection. for instance, inflammatory neutrophils produce cathelicidins; cationic proteins important for host defence. intriguingly, in infants hospitalised with viral bronchiolitis, those with the lowest levels of ll-37 were more likely to be infected with rsv, suggesting that ll-37 is beneficial [125] . human neutrophils stimulated in vitro with rsv virions secrete ll-37, inhibiting the formation of new viral particles and reducing the apoptosis of infected epithelial cells [126, 127] . neutrophil-derived ll-37 may support the production of interferons, which are required for viral control. in mice, treatment with an exogenous ll-37 peptide protected against body weight loss, increased interferon-lambda production, and lowered rsv load [128] . neutrophils may also influence the quality of adaptive immune responses that develop during rsv infection. for example, neutrophils may aid in the rapid recruitment of virus-specific cd8 + t-cells. in infants with rsv infection, a systemic influx of bone marrow-derived neutrophil precursors in blood precedes robust cd8 + t-cell activation and a reduction in severe disease symptoms [129] . however, the precise mechanisms through which neutrophils influence cd8 + t-cells remain unclear. collectively, these studies suggest that neutrophils can act beneficially in mediating immune protection against rsv infection. however, perturbation of optimal neutrophil function during infection may amplify their effector responses and thus contribute to the pathology that occurs in severe rsv patients. excessive neutrophilia is associated with increased tissue damage during severe rsv bronchiolitis [130] . in part, this is attributed to molecular dysregulation of neutrophil-effector pathways, including ros production, netosis, degranulation, and over-secretion of proteolytic enzymes [101, 111, [131] [132] [133] [134] , (highlighted in figure 1 ). excessive ros production activates indiscriminate oxidative stress in the lung, contributing to lung damage [108] [109] [110] . indeed, markers of oxidative stress are elevated in the lungs and blood of patients with severe rsv [108] . in vivo treatment of mice with antioxidants significantly reduces rsv-induced inflammation and pathology [108] . ne levels are heightened in nasal aspirates and serum from paediatric patients with acute rsv infection compared with uninfected controls [68, 106] , and nets are readily detected in balf samples obtained from patients with an rsv infection [111] . in another study that sampled patients with rsv bronchiolitis, blood neutrophils showed little net formation, whereas neutrophils from the airways underwent netosis [117] . intriguingly, in an ex vivo model, this process was diminished, with an agonistic antibody directed against lair-1, which was shown to be elevated on airway neutrophils but not on circulating neutrophils [117] . in vitro stimulation of human neutrophils with rsv virions induces ros-dependent netosis via pad-4 citrullination and downstream activation of the pi3k/akt, erk, and p38 mapk pathways [131] . the deposition of net products in the culture was shown to trap rsv virions in dna lattices coated with ne and mpo, implicating a beneficial antiviral effect of nets [131] . however, in calves with a severe bovine rsv infection, the widespread airway obstruction is caused by luminal plugs composed of mucins, cellular debris, and nets [111] . as dna-rich mucus is associated with airway obstruction [111, 121] , excessive net release likely contributes to pathogenesis, although this has yet to be demonstrated clinically or in an experimental animal model of bronchiolitis. findings from other experimental systems such as allergic asthma have demonstrated that nets can exacerbate immunopathology [39] . virus-associated exacerbations of asthma are attenuated following inhibition of netosis, either through blockade of ne or degradation of nets with dnase [39] . mechanistically, netosis inhibition reduced type 2-mediated allergic inflammation and lowered airway hyper-reactivity [39] . clearly, as with most inflammatory processes, netosis is a double-edged sword. a greater understanding of netosis in the context of acute lris is needed; however, on balance it appears that limiting netosis in severe rsv bronchiolitis would be advantageous. neutrophil-derived toxic granules and proteolytic enzymes also contribute to the pathogenesis of rsv bronchiolitis. rsv increases the expression of matrix metalloproteinase-9 (mmp-9), which augments the influx of neutrophils to the site of infection [107] . elevated levels of mmp-9 are detected in the respiratory secretions of rsv-infected paediatric patients and are associated with increased risk of requiring mechanical ventilation [133] [134] [135] . hence, mmp-9 activity is considered a useful predictor of disease severity in children with rsv-induced respiratory failure [133] . genetic mmp-9 deficiency in mice decreases viral burden and lung inflammation [134] , although it is still unclear whether neutrophil-derived mmp-9 plays a dominant role in the mmp-9-induced pathology during rsv infection. specific ablation of mmp-9 in neutrophils using cre-recombinase-loxp systems would help address this question. in summary, severe neutrophilia and excessive production of powerful antimicrobial mediators appears to cause significant collateral damage, resulting in airway obstruction and the clinical symptoms that characterise severe rsv bronchiolitis. pharmacological tools to inhibit the excessive neutrophilic response in the lung are likely to ameliorate the severity of rsv bronchiolitis. to achieve this goal, therapeutic strategies should seek to modify the expression of cytokines and/or chemokines, which regulate neutrophil trafficking/recruitment into the lung, and neutrophil activation and function in response to infection (as proposed in figure 2 ). macrolide antibiotics, in particular azithromycin, which is efficacious against il-8 and neutrophil-induced inflammation, decrease the severity of symptoms during viral bronchiolitis [88, 136, 137] . a randomised trial in infants hospitalised with rsv bronchiolitis demonstrated that, compared with the placebo-control group, a 14-day treatment with azithromycin significantly decreased wheezing episodes, time to recovery, and overall respiratory morbidity over the subsequent year in comparison [88] . notably, this was associated with lower il-8 levels in nasal lavage fluid, suggesting that neutrophil numbers were attenuated, although this was not specifically assessed [88] . preclinical studies in mice have also demonstrated that azithromycin decreases virus-induced neutrophil accumulation in the lung by abrogating the expression of neutrophil inflammatory mediators such as cxcl1 [136] . in a follow-up study to the clinical trial, azithromycin treatment modified the composition of the upper airway microbiome, reducing the abundance of moraxella [138] . this phenotype was associated with lower odds of developing recurrent wheezing episodes over the next 12 months [138] . ongoing phase ii clinical trials are evaluating whether the addition of azithromycin to routine bronchiolitis care in hospitalised infants reduces the frequency of recurrent wheeze episodes during preschool years (clinicaltrials.gov identifier: nct01486758). viruses 2020, 12, x for peer review 10 of 20 would help address this question. in summary, severe neutrophilia and excessive production of powerful antimicrobial mediators appears to cause significant collateral damage, resulting in airway obstruction and the clinical symptoms that characterise severe rsv bronchiolitis. pharmacological tools to inhibit the excessive neutrophilic response in the lung are likely to ameliorate the severity of rsv bronchiolitis. to achieve this goal, therapeutic strategies should seek to modify the expression of cytokines and/or chemokines, which regulate neutrophil trafficking/recruitment into the lung, and neutrophil activation and function in response to infection (as proposed in figure 2 ). macrolide antibiotics, in particular azithromycin, which is efficacious against il-8 and neutrophil-induced inflammation, decrease the severity of symptoms during viral bronchiolitis [88, 136, 137] . a randomised trial in infants hospitalised with rsv bronchiolitis demonstrated that, compared with the placebo-control group, a 14-day treatment with azithromycin significantly decreased wheezing episodes, time to recovery, and overall respiratory morbidity over the subsequent year in comparison [88] . notably, this was associated with lower il-8 levels in nasal lavage fluid, suggesting that neutrophil numbers were attenuated, although this was not specifically assessed [88] . preclinical studies in mice have also demonstrated that azithromycin decreases virusinduced neutrophil accumulation in the lung by abrogating the expression of neutrophil inflammatory mediators such as cxcl1 [136] . in a follow-up study to the clinical trial, azithromycin treatment modified the composition of the upper airway microbiome, reducing the abundance of moraxella [138] . this phenotype was associated with lower odds of developing recurrent wheezing episodes over the next 12 months [138] . ongoing phase ii clinical trials are evaluating whether the addition of azithromycin to routine bronchiolitis care in hospitalised infants reduces the frequency of recurrent wheeze episodes during preschool years (clinicaltrials.gov identifier: nct01486758). therapeutic inhibition strategies may aim to: (1) block excessive neutrophil granulopoiesis in the bone marrow (e.g., neutralising g-csf production and function); (2) antagonise chemokine-mediated neutrophil activation and chemotaxis to the lung microenvironment (e.g., using cxcr2 small molecule inhibitors); (3) (4) (5) (6) regulate neutrophil function in the lung (e.g., blocking the production of antimicrobial products such as mmps (3), ros (4), inflammatory cytokines (5) , and deposition of nets (6)). therapeutic anti-viral products (7) may also limit rsv-induced recruitment of neutrophils in the lung during infection. targeting chemokine receptors, for instance cxcr2, which is important for neutrophil migration, may also be a therapeutic option (figure 2 ). danirixin, a selective and reversible antagonist of cxcr2, decreases the activation and transmigration of neutrophils to the lungs of rats treated with aerosolised lipopolysaccharide [139] . a clinical trial to evaluate the efficacy of danirixin in rsv-infected infants <2 years of age (clinicaltrials.gov identifier: nct02201303) is ongoing. pharmacological inhibition of mmp activity has also been employed as a therapeutic strategy for treating chronic lung diseases [25, 140, 141] . for example, marimastat, a broad-spectrum mmp inhibitor, reduces bronchial hyperresponsiveness to inhaled allergens in atopic asthmatic individuals [140] . similarly, mice treated with another broad-spectrum mmp inhibitor, r-94138, also exhibited reduced airway inflammation, characterised by reduced eosinophils and lymphocytes recruited to the lung airways during experimental allergic asthma [141] . as mmp-9 levels are significantly elevated in rsv-infected children [133] [134] [135] , selective inhibition of mmp-9 activity may be of significant benefit for the treatment of rsv bronchiolitis (figure 2 ). inhibition of netosis may also be a therapeutic option. strategies to achieve this might involve the targeting of neutrophil granules (including elastases or mpo), neutrophil-activating pattern recognition receptors (e.g., tlrs) or enzymes (nadph-oxidase and pad4). the ne inhibitor azd9668 improves lung function in patients with bronchiectasis [142] ; however, it is yet to be tested for efficacy in infants with a severe rsv infection. similarly, selective inhibition of mpo with pf-1355, which attenuates tissue injury in experimental immune complex-mediated alveolitis [143] , may offer a therapeutic benefit against severe rsv bronchiolitis ( figure 2 ). the detection of pathogen-associated molecular patterns by pattern recognition receptors on neutrophils can induce netosis. therefore, pharmacological disruption of these interactions may attenuate netosis. for instance, tak-242, a small molecule tlr4 inhibitor [144] that reduces inflammation, may also be used to regulate netosis. considering that the formation of nets is largely dependent on the activity of nadph oxidase or pad4, the inhibition of either enzyme is likely to decrease the magnitude of neutrophil-associated tissue damage. genetic deletion of pad4 or pad4 inhibition by cl-amidine or bb-cl-amidine improves clinical outcomes in several different mouse models of inflammatory disease [40, 145, 146] , although whether pad4 inhibitors are beneficial against a severe rsv infection remains unknown. the modulation of neutrophil numbers or function is evidently a promising target to ameliorate the severity of rsv bronchiolitis. however, several feasibility issues remain, particularly in relation to safety and specificity. selective targeting of neutrophils is challenging due to their close relationship to other myeloid lineages, such as monocytes and macrophages. this may alter tissue homeostasis and immune defence against other intracellular pathogens such as mycobacterium tuberculosis or listeria monocytogenes. moreover, since neutrophils are important for robust antimicrobial host defence, their therapeutic inhibition may predispose the host to other infectious agents or effect colonisation by commensals, which can become pathobionts if left unchecked. to overcome this, one strategy would be to target specific neutrophil subsets, and thus ablate the pathogenic population without compromising the beneficial effect of neutrophils in mediating antimicrobial host defence. new insights are needed to identify approaches that can support this possibility. most therapeutic approaches in the pipeline have focused on inhibiting excessive neutrophil function in treating disease pathology. however, an alternative approach is to modify neutrophil function to boost host immunity against pathogenic microbes, particularly in the elderly. for instance, the cholesterol-lowering medication simvastatin has been associated with improved neutrophil function and immunity against bacterial infection in humans [147] . in this study, adding simvastatin to the normal treatment regimen significantly reduced the severity of bacterial pneumonia and sepsis in patients older than 62 years [147] . mechanistically, simvastatin acted by augmenting the migratory accuracy of neutrophils into tissues, albeit with reduced netosis and ne release, which promoted optimal non-inflammatory bacterial control. therefore, pharmacological approaches to rejuvenate neutrophils in the elderly might be beneficial in boosting immunity against rsv. there remains a pressing need to develop new treatments to reduce the significant morbidity and mortality associated with severe rsv bronchiolitis. neutrophils contribute to host immunity against rsv; however, they are strongly associated with the development of severe disease and are thus a logical target for therapeutic intervention. new technological approaches, such as single cell rna sequencing, combined with proteomics, metabolomics, and advances in flow cytometry that allow for multi-parameter analyses, must now be applied to clinical samples from infants with mild and severe disease to gain a greater understanding of the full repertoire of neutrophil heterogeneity and function in the pathogenesis of rsv bronchiolitis. such information will reveal important insights into neutrophil diversity and identify novel tractable and druggable targets to ameliorate the severity of rsv-bronchiolitis. viral bronchiolitis global, regional, and national disease burden estimates of acute lower respiratory infections due to respiratory syncytial virus in young children in 2015: a systematic review and modelling study respiratory syncytial virus and parainfluenza virus respiratory syncytial virus vaccine development novel antigens for rsv vaccines role of viral infections in the development and exacerbation of asthma in children respiratory syncytial virus binds and undergoes transcription in neutrophils from the blood and airways of infants with severe bronchiolitis bronchoalveolar lavage cellularity in infants with severe respiratory syncytial virus bronchiolitis the histopathology of fatal untreated human respiratory syncytial virus infection neutrophil kinetics in man heterogeneity of neutrophils neutrophils: between host defence, immune modulation, and tissue injury neutrophil diversity in health and disease phagocytosis by neutrophils cell intrinsic functions of neutrophils and their manipulation by pathogens biological roles of neutrophil-derived granule proteins and cytokines neutrophil degranulation, plasticity, and cancer metastasis neutrophils in the activation and regulation of innate and adaptive immunity neutrophils in cancer: neutral no more neutrophil-derived cathelicidin protects from neointimal hyperplasia the role of neutrophils in inflammation resolution mice lacking neutrophil elastase reveal impaired host defense against gram negative bacterial sepsis myeloperoxidase is required for neutrophil extracellular trap formation: implications for innate immunity neutrophil-induced genomic instability impedes resolution of inflammation and wound healing a therapeutic role for matrix metalloproteinase inhibitors in lung diseases? ll-37, the only human member of the cathelicidin family of antimicrobial peptides human neutrophil collagenase. a distinct gene product with homology to other matrix metalloproteinases an emerging role for neutrophil extracellular traps in noninfectious disease neutrophils: new insights and open questions neutrophil extracellular traps: the biology of chromatin externalization the pseudokinase mlkl activates pad4-dependent net formation in necroptotic neutrophils infection-induced netosis is a dynamic process involving neutrophil multitasking in vivo histone hypercitrullination mediates chromatin decondensation and neutrophil extracellular trap formation neutrophil extracellular traps contain calprotectin, a cytosolic protein complex involved in host defense against candida albicans novel cell death program leads to neutrophil extracellular traps pad4 is essential for antibacterial innate immunity mediated by neutrophil extracellular traps neutrophils sense microbe size and selectively release neutrophil extracellular traps in response to large pathogens extracellular dna traps are associated with the pathogenesis of trali in humans and mice host dna released by netosis promotes rhinovirus-induced type-2 allergic asthma exacerbation peptidylarginine deiminase inhibition is immunomodulatory and vasculoprotective in murine lupus effect of interleukin-6 receptor inhibition with tocilizumab in patients with rheumatoid arthritis (option study): a double-blind, placebo-controlled, randomised trial cxcr2 mediates nadph oxidase-independent neutrophil extracellular trap formation in cystic fibrosis airway inflammation neutrophils and immunity: challenges and opportunities toll-like receptors stimulate human neutrophil function activation of an immunoregulatory and antiviral gene expression program in poly(i:c)-transfected human neutrophils recognition of peptidoglycan from the microbiota by nod1 enhances systemic innate immunity s100a9 and s100a12 activate airway epithelial cells to produce muc5ac via extracellular signal-regulated kinase and nuclear factor-kappab pathways secretion of s100a8, s100a9, and s100a12 by neutrophils involves reactive oxygen species and potassium efflux interleukin (il)-22 and il-17 are coexpressed by th17 cells and cooperatively enhance expression of antimicrobial peptides neutrophils mediate immune modulation of dendritic cells through glycosylation-dependent interactions between mac-1 and dc-sign neutrophil cytoplasts induce th17 differentiation and skew inflammation toward neutrophilia in severe asthma neutrophils acquire the capacity for antigen presentation to memory cd4(+) t cells in vitro and ex vivo an in vitro transepithelial migration assay to evaluate the role of neutrophils in respiratory syncytial virus (rsv) induced epithelial damage respiratory syncytial virus bronchiolitis in infancy is an important risk factor for asthma and allergy at age 7 respiratory syncytial virus in early life and risk of wheeze and allergy by age 13 years il-1alpha, il-33 and s100 proteins: dual-function alarmins respiratory syncytial virus infection promotes necroptosis and hmgb1 release by airway epithelial cells animal models of human respiratory syncytial virus disease animal models of respiratory syncytial virus infection animal models of respiratory syncytial virus infection and disease plasmacytoid dendritic cells protect from viral bronchiolitis and asthma through semaphorin 4a-mediated t reg expansion toll-like receptor 7 gene deficiency and early-life pneumovirus infection interact to predispose toward the development of asthma-like pathology in mice plasmacytoid dendritic cells promote host defense against acute pneumovirus infection via the tlr7-myd88-dependent signaling pathway hmgb1 amplifies ilc2-induced type-2 inflammation and airway smooth muscle remodelling plasmacytoid dendritic cells during infancy are inversely associated with childhood respiratory tract infections and wheezing il-8) mediates neutrophil recruitment and behavior in the zebrafish inflammatory response leukotriene b4-mediated neutrophil recruitment causes pulmonary capillaritis during lethal fungal sepsis il-8/il-17 gene variations and the susceptibility to severe viral bronchiolitis randomized trial to evaluate azithromycin's effects on serum and upper airway il-8 levels and recurrent wheezing in infants with respiratory syncytial virus bronchiolitis airway eicosanoids in acute severe respiratory syncytial virus bronchiolitis neutrophil swarms require ltb4 and integrins at sites of cell death in vivo zileuton reduces respiratory illness and lung inflammation, during respiratory syncytial virus infection, in mice interferon-gamma coordinates ccl3-mediated neutrophil recruitment in vivo the chemokine macrophage-inflammatory protein-1 alpha and its receptor ccr1 control pulmonary inflammation and antiviral host defense in paramyxovirus infection altered pathogenesis of severe pneumovirus infection in response to combined antiviral and specific immunomodulatory agents allergen-induced il-6 trans-signaling activates gammadelta t cells to promote type 2 and type 17 airway inflammation interleukin-1 and il-23 induce innate il-17 production from gammadelta t cells, amplifying th17 responses and autoimmunity hmgb1-il-23-il-17-il-6-stat3 axis promotes tumor growth in murine models of melanoma s100a9 gene silencing inhibits the release of pro-inflammatory cytokines by blocking the il-17 signalling pathway in mice with acute pancreatitis il-17-induced pulmonary pathogenesis during respiratory viral infection and exacerbation of allergic disease local il-17a potentiates early neutrophil recruitment to the respiratory tract during severe rsv infection neutrophils in respiratory syncytial virus infection: a target for asthma prevention interleukin-1 alpha mediates the enhanced expression of intercellular adhesion molecule-1 in pulmonary epithelial cells infected with respiratory syncytial virus primary infection of mice with high titer inoculum respiratory syncytial virus: characterization and response to antiviral therapy. can reduced nasal viral load and ifn responses in infants with respiratory syncytial virus bronchiolitis and respiratory failure rsv vaccine-enhanced disease is orchestrated by the combined actions of distinct cd4 t cell subsets human neutrophil elastase in rsv bronchiolitis matrix metalloprotease 9 mediates neutrophil migration into the airways in response to influenza virus-induced toll-like receptor signaling respiratory syncytial virus induces oxidative stress by modulating antioxidant enzymes respiratory syncytial virus and cellular stress responses: impact on replication and physiopathology baicalein triazole prevents respiratory tract infection by rsv through suppression of oxidative damage neutrophil extracellular traps cause airway obstruction during respiratory syncytial virus disease neutrophil heterogeneity in health and disease: a revitalized avenue in inflammation and immunity human neutrophils in the saga of cellular heterogeneity: insights and open questions developmental analysis of bone marrow neutrophils reveals populations specialized in expansion, trafficking, and effector functions neutrophil subset responses in infants with severe viral respiratory infection shedding of l-selectin and pecam-1 and upregulation of mac-1 and icam-1 on neutrophils in rsv bronchiolitis lair-1 limits neutrophil extracellular trap formation in viral bronchiolitis respiratory syncytial virus infection enhances neutrophil and eosinophil adhesion to cultured respiratory epithelial cells. roles of cd18 and intercellular adhesion molecule-1 immune profiles provide insights into respiratory syncytial virus disease severity in young children respiratory syncytial virus fusion protein promotes tlr-4-dependent neutrophil extracellular trap formation by human neutrophils neutrophil tlr4 expression is reduced in the airways of infants with severe bronchiolitis association between common toll-like receptor 4 mutations and severe respiratory syncytial virus disease the tlr4 antagonist eritoran protects mice from lethal influenza infection serum ll-37 levels associated with severity of bronchiolitis and viral etiology the human cathelicidin ll-37 has antiviral activity against respiratory syncytial virus human cathelicidin, ll-37, inhibits respiratory syncytial virus infection in polarized airway epithelial cells cathelicidins have direct antiviral activity against respiratory syncytial virus in vitro and protective function in vivo in mice and humans t-cell activation during natural respiratory syncytial virus infection in infants neutrophil-mediated inflammation in respiratory syncytial viral bronchiolitis respiratory syncytial virus induces the classical ros-dependent netosis through pad-4 and necroptosis pathways activation neutrophil extracellular traps in respiratory disease: guided anti-microbial traps or toxic webs? pulmonary matrix metalloproteinase-9 activity in mechanically ventilated children with respiratory syncytial virus matrix metalloproteinase-9 mediates rsv infection in vitro and in vivo early elevation of matrix metalloproteinase-8 and -9 in pediatric ards is associated with an increased risk of prolonged mechanical ventilation azithromycin attenuates airway inflammation in a mouse model of viral bronchiolitis clarithromycin targets neutrophilic airway inflammation in refractory asthma azithromycin therapy during respiratory syncytial virus bronchiolitis: upper airway microbiome alterations and subsequent recurrent wheeze danirixin: a reversible and selective antagonist of the cxc chemokine receptor 2 the effect of marimastat, a metalloprotease inhibitor, on allergen-induced asthmatic hyper-reactivity inhibition of matrix metalloproteinases prevents allergen-induced airway inflammation in a murine model of asthma phase ii study of a neutrophil elastase inhibitor (azd9668) in patients with bronchiectasis pf-1355, a mechanism-based myeloperoxidase inhibitor, prevents immune complex vasculitis and anti-glomerular basement membrane glomerulonephritis tak-242, a small-molecule inhibitor of toll-like receptor 4 signalling, unveils similarities and differences in lipopolysaccharide-and lipid-induced inflammation and insulin resistance in muscle cells neutrophil-mediated ifn activation in the bone marrow alters b cell development in human and murine systemic lupus erythematosus locally instructed cxcr4(hi) neutrophils trigger environment-driven allergic asthma through the release of neutrophil extracellular traps simvastatin improves neutrophil function and clinical outcomes in pneumonia. a pilot randomized controlled clinical trial this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license key: cord-351323-cbejbm5v authors: roy mukherjee, tapasi; chanda, shampa; mullick, satarupa; de, papiya; dey‐sarkar, malay; chawla‐sarkar, mamta title: spectrum of respiratory viruses circulating in eastern india: prospective surveillance among patients with influenza‐like illness during 2010–2011 date: 2013-06-13 journal: j med virol doi: 10.1002/jmv.23607 sha: doc_id: 351323 cord_uid: cbejbm5v in developing countries, viruses causing respiratory disease are a major concern of public health. during january 2010–december 2011, 2,737 patients with acute respiratory infection from the outpatient departments as well as patients admitted to hospitals were screened for different respiratory viruses. nasal and or throat swabs were collected and transported to the laboratory where initial screening of influenza a and influenza b viruses was performed. the samples were tested further for influenza c virus, parainfluenza viruses 1–4, human rhinovirus, metapneumovirus and respiratory syncytial virus by conventional rt‐ pcr. the study revealed that the majority of the patients were under 5 years of age; both due to their higher susceptibility to respiratory infections and presentation to hospitals. out of 2,737 patients enrolled in this study, 59% were found positive for one or more respiratory viruses. influenza b infection was detected in 12% of patients followed by influenza a (11.7%), respiratory syncytial virus (7.1%), parainfluenza virus‐2 (6%), metapneumovirus (3%), parainfluenza virus‐3 (1%), parainfluenza virus‐4 (0.6%), parainfluenza virus‐1 (0.3%), influenza c (0.2%) and human rhinovirus (0.2%). distinct seasonal infection was observed only for influenza a and influenza b viruses. j. med. virol. 85:1459–1465, 2013. © 2013 wiley periodicals, inc. acute respiratory infections are a leading cause of morbidity and mortality in infants and children, especially in developing countries. according to the world health organization (who), acute respiratory infections account for approximately 20% of all deaths among children under 5 years [who, 2010] , and 70% of these deaths occur in africa and southeast asia [williams et al., 2002] . viruses are a leading cause of acute respiratory infections with epidemiological variability that depends on the climate [armstrong et al., 1999] . to date, most of the known data are based on studies with children and infants only [choi et al., 2006; costa et al., 2006; kusel et al., 2006; ma et al., 2006; ordás et al., 2006; pierangeli et al., 2007] whereas the etiology remains largely undetermined for infections in the elderly [fine et al., 1999; garbino et al., 2002; file, 2003] . in children, respiratory syncytial virus and influenza viruses induce bronchiolitis, asthma exacerbation and pneumonia, leading to high rates of hospital admission [el-hajje et al., 2008; di carlo et al., 2009] . human rhinovirus, believed previously to cause only mild upper respiratory illnesses, has also been found in association with acute and chronic lower respiratory tract infections, including asthma exacerbations and chronic obstructive pulmonary disease (copd); although the role of human rhinovirus as a cause has only been established for asthma [papadopoulos et al., 2002; friedlander and busse, 2005; xatzipsalti et al., 2005; pierangeli et al., 2007; singh and busse, 2007; matthew et al., 2009] . it was reported that metapneumovirus accounted for approximately 5-10% of all acute respiratory infections in children and adults [van den hoogen et al., 2001] whereas adenoviruses were responsible for 5-10% of acute respiratory infections only in children [chen et al., 2004] . in addition, infections caused by other respiratory viruses such as parainfluenza viruses and coronaviruses occur worldwide. furthermore, coronavirus nl63 [van der hoek et al., 2004] , coronavirus hku [fouchier et al., 2004; woo et al., 2005] and bocavirus [allander et al., 2007] are also responsible for acute respiratory infections in pediatric, elderly and immunosuppressed patients. most studies based on laboratory diagnosis in hospitals are still restricted to influenza and respiratory syncytial virus. on the other hand, emerging respiratory viruses have been a subject of concern because of the risk of rapid spread and high fatality rates due to lack of both diagnosis and effective antiviral therapy. in developing countries where diagnosis of viruses causing respiratory disease is restricted to a few laboratories, the etiology is undefined in a significant proportion (>60%); which undermines the impact of acute respiratory infections on health and economic burden [monto, 1994; henrickson et al., 2004] . in eastern india, frequency and genetic diversity of influenza viruses during [2005] [2006] [2007] [2008] [2009] have been reported , but no information is available from the indian subcontinent regarding other respiratory viruses. the present study aimed to identify common circulating respiratory viruses in addition to influenza during january 2010 through december 2011 in the eastern region of india. nasal and/or throat swabs were collected from patients with influenza like illness attending the outpatients departments of hospitals in kolkata, west bengal, as described previously [agrawal et al., 2009a] . in addition, swabs from patients with severe respiratory illness admitted to different hospitals in west bengal were also referred to the laboratory for diagnosis. [mukherjee et al., 2010] . informed consent forms and detailed case histories were recorded before the collection of a specimen. the study was approved by the institutional ethical committees. viral rna was extracted by using commercially available qiaamp viral rna mini kit (qiagen, hilden, germany) as per manufacturer's instructions. real-time pcr for initial detection of influenza viruses, amplification of the matrix (m) gene of influenza a and influenza b viruses were carried out by real-time rt-pcr as described previously [agrawal et al., 2009a] . respiratory viruses other than influenza were detected using a multiplex rt-pcr technique. firststrand cdna synthesis was achieved using random primers and reverse transcription system (invitrogen, carlsbad, ca). the resulting cdna was then subjected to multiplex pcr to enable simultaneous detection of multiple viruses. primer sequences used in the study are described in table i . the amplified products were then separated by agarose gel electrophoresis and visualized under uv after ethidium bromide staining. all samples were tested independently three times, using negative and positive controls to exclude cross-contamination of samples. distribution frequencies of respiratory viruses were compared using pearson's chi square test and fisher's exact test. continuous variables for population parameters such as age, laboratory investigations and other parameters were compared using one-way analysis of variance. from january 2010 through december 2011, 2,737 swab samples from patients with respiratory infections were obtained; 1,574 (57.5%) of the patients were out of 2,737 samples, 1,616 (59%) were positive for one or more respiratory viruses; of which 11.7% were positive for influenza a; which comprised both influenza a(h1n1)pdm09 (6.7%) and influenza a(h3n2) (5%), influenza b was detected in 12% cases, influenza c (0.2%), parainfluenza virus-1 (0.3%), parainfluenza virus-2 (6%), parainfluenza virus-3 (1.0%), and parainfluenza virus-4 (0.6%), respiratory syncytial virus (7.1%), metapneumovirus (3.0%) and human rhinovirus were found in 0.2% cases (fig. 1) . during the study period, 64 samples were found to be co-infected with respiratory syncytial virus and influenza b viruses. metapneumovirus was found together with respiratory syncytial virus (n ¼ 16), influenza a (n ¼ 1), and influenza b (n ¼ 3) viruses. mixed infection with influenza a and influenza b was detected in 13 cases. the prevalence of viral infection in different age groups has been shown in figure 2 . infections due to influenza a(h3n2), influenza b, metapneumovirus and respiratory syncytial virus was high in children under 5 years of age followed by 5-15 years and 15-35 years; whereas, adults (15-35 years) and the elderly people were infected mainly with influenza a (h1n1)pdm09 virus. influenza c virus infection predominantly occurred in children under the age of 5 years; although the frequency is very low. similarly higher percentage of parainfluenza virus-1 infection was observed among children (under 5 years of age) followed by 15-35 years of age. in other age groups parainfluenza virus-1 was not observed in this study. elderly persons (>60 years of age) were at high risk for infection with parainfluenza virus-2 and -4, followed by the patients of 45-60 and patients under 5 years of age; whereas the infection rate for parainfluenza virus-3 was high among the patients under 5 years of age. for human rhinovirus infection, the risk group was 35-45 years followed by 45-60 years. during this study, all mixed infections were observed only in children aged less than 5 years. the correlation of meteorological variations with the prevalence of respiratory viruses in eastern india is shown in figure 3 . due to the pandemic in 2009, only influenza a(h1n1)pdm09 strain was found in 2010, however influenza a(h3n2) emerged again in 2011. influenza b was found mainly after the monsoon season (october-december). seasonal fluctuations for influenza c virus, parainfluenza virus 1-4, metapneumovirus and respiratory syncytial virus infection have not been observed during this study. in 2004, surveillance for influenza viruses was expanded to india, as part of the global influenza surveillance network. however specific diagnosis which requires laboratory tests, were not widely available in eastern india. hence the information on epidemiology and clinical features of respiratory virus infection in india is based entirely on research studies and the disease burden or seasonal prevalence of respiratory viruses remains largely undefined. this study initiated to complete the information on circulating respiratory viruses among patients attending the outpatients departments of different hospitals with acute respiratory infections in the eastern region of india during 2010 through 2011. during this surveillance, influenza b infection was found to be most prevalent followed by influenza a. in 2010, only influenza a(h1n1)pdm09 virus circulated whereas in 2011, only influenza a/h3n2 was found. this was in contrast with the previous report from eastern india, where higher prevalence of influenza a was observed compared to influenza b . increased frequency of influenza b infection could be due to the post-pandemic effect of influenza a(h1n1)pdm09 following which seasonal influenza a/h1n1 and influenza a/h3n2 viruses disappeared during 2009-2010. this is consistent with studies from vietnam [do et al., 2011] and other asian countries [mathisen et al., 2010] . metapneumovirus has been associated with both upper and lower respiratory tract infections in children (3-13%) [van den hoogen et al., 2001; bosis et al., 2005; gerna et al., 2005; choi et al., 2006; sarasini et al., 2006; boivin et al., 2007] . during this study period, metapneumovirus was found in only 3% samples. this finding corroborates with reports from canada, england and the usa, where the infection rate due to metapneumovirus varies from 2% to 4.5% [stockton et al., 2002; falsey et al., 2003; boivin et al., 2004] , but is significantly less compared to the prevalence in netherlands (10%), australia (9.7%), and chile (5.4%) [van den hoogen et al., 2001; luchsinger et al., 2005; mackay et al., 2006] . respiratory syncytial virus which is the most common etiological agent of viral lower respiratory tract infections in infants and young children in the world caused 33.8 million new episodes in children <5 years of age [nair et al., 2010] and is also associated with hospital admission of 3-9 per 1,000 children below 1 year [pancer et al., 2011] . in thailand alone, 417.1/100,000 incidences of pneumonia per year are attributed to respiratory syncytial virus [olsen et al., 2010] . worldwide, respiratory syncytial virus has been identified as the cause of 3.4 million cases of acute lower respiratory infections requiring hospital admission. among all age groups, 7.1% cases were found positive for respiratory syncytial virus during 2010-2011 in this study. following respiratory syncytial virus, parainfluenza virus-2 was the most predominant virus (6%) compared to parainfluenza virus-1, -3 or -4. consistent with surveillance results during 2006-2009, in eastern india roy et al., 2011] and in bangladesh [zaman et al., 2009] children under 5 years old were found to be most vulnerable to infections due to influenza b and influenza a (h3n2) viruses. whereas, influenza a (h1n1)pdm09 affected adults and the elderly which is consistent with findings from other studies [cauchemez et al., 2009; john and moorthy, 2010; mukherjee et al., 2010] . similar to influenza viruses, metapneumovirus infection was also detected mainly in children (under 5 year of age), suggesting early acquisition of infection [van den hoogen et al., 2001; lu et al., 2011; zappa et al., 2011] . during 2007-2008, only 8.7% of respiratory syncytial virus infection was found among children [agrawal et al., 2009b] ; whereas in the same geographical region, 13.7% samples were found to be positive for respiratory syncytial virus in children (under 5 years) during this study period (2010) (2011) . the higher prevalence of respiratory syncytial virus during this study period could be due to lower activity of influenza a viruses compared to the activity of influenza a viruses in 2007-2008 [agrawal et al., 2009b] . among children with acute respiratory infections, co-infection with one or more respiratory viruses has been observed [bonzel et al., 2008; canducci et al., 2008] which correlates with results of present study, where predominant mixed infections were observed only in children. unlike temperate countries where the prevalence of seasonal influenza may reach epidemic proportions during the winter months [john and moorthy, 2010] , in tropical countries like india, year round circulation of strains has been reported, though the infection peaks during rainy season (june-september) [agrawal et al., 2009a . there could be several factors such as socio-economic, environmental, education, overcrowding and other factors, which could affect the seasonal incidence and distribution of viral infections. in eastern india, influenza b was found to be prevalent after the monsoon season as well as in the winter months [roy et al., 2011] , and influenza a viruses predominated during the monsoon (june-july) [agrawal et al., 2009a; mukherjee et al., 2010] . during 2010-2011 seasonal infection due to influenza a [influenza a(h1n1)pdm09 or influenza a/h3n2] correlated with previous reports. however the seasonal pattern of infection with respiratory syncytial virus during 2010-2011 was found to be different from the previous reports from kolkata, india [agrawal et al., 2009a,b] and bangladesh [huq et al., 1990] , where respiratory syncytial virus infection was found more commonly in the winter months or the dry seasons. such variations in seasonal incidence are difficult to explain, as virus infection could be affected by a large number of different factors [weber et al., 1998 ]. previous studies in temperate climates showed that parainfluenza virus-1 and -2 infection occurs annually [laurichesse et al., 1999; karron and collins, 2007] , parainfluenza virus-4 infection occurs twice-yearly during the late fall and winter [aguilar et al., 2000; vachon et al., 2006; lau et al., 2009] and parainfluenza virus-3 infection occurs mainly during late spring and summer [laurichesse et al., 1999] . these differences may be attributed to different geographical regions and study years. however, as the numbers of positive cases were very low in the present study, the seasonal prevalence of influenza c, parainfluenza viruses and human rhinovirus could not be determined. hence large-scale studies over a broader geographical range and longer time period are required to understand the seasonal infection pattern of respiratory viruses in india. comparative evaluation of real-time pcr and conventional rt-pcr during 2 year surveillance for influenza and respiratory syncytial virus among children with acute respiratory infections in kolkata, india, reveals a distinct seasonality of infection prevalence of respiratory syncytial virus group b genotype ba-iv strains among children with acute respiratory tract infection in kolkata, eastern india genetic characterization of circulating seasonal influenza a viruses (2005-2009) revealed introduction of oseltamivir resistant h1n1 strains during 2009 in eastern india detection and identification of human parainfluenza viruses 1, 2 3, and 4 in clinical samples of pediatric patients by multiplex reverse transcription-pcr human bocavirus and acute wheezing in children trends in infectious disease mortality in the united states during the 20th century global genetic diversity of human metapneumovirus fusion gene an outbreak of severe respiratory tract infection due to human metapneumovirus in a long-term care facility frequent detection of viral coinfection in children hospitalized with acute respiratory tract infection using a real-time polymerase chain reaction impact of human metapneumovirus in childhood: comparison with respiratory syncytial virus and influenza viruses twoyear prospective study of single infections and co-infections by respiratory syncytial virus and viruses identified recently in infants with acute respiratory disease household transmission of 2009 pandemic influenza a (h1n1) virus in the united states respiratory adenoviral infections in children: a study of hospitalized cases in southern taiwan in 2001-2002 the association of newly identified respiratory viruses with lower respiratory tract infections in korean children respiratory viruses in children younger than five years old with acute respiratory disease from epidemiological assessment of respiratory syncytial virus infection in hospitalized infants, during the season viral etiologies of acute respiratory infections among hospitalized vietnamese children in respiratory syncytial virus in hospitalized children. a 3-year study human metapneumovirus infections in young and elderly adults community-acquired pneumonia processes and outcomes of care for patients with community-acquired pneumonia: results from the pneumonia patient outcomes research team (port) cohort study a previously undescribed coronavirus associated with respiratory disease in humans the role of rhinovirus in asthma exacerbations prospective epidemiologic survey of patients with community-acquired pneumonia requiring hospitalization in switzerland changing circulation rate of human metapneumovirus strains and types among hospitalized pediatric patients during three consecutive winterspring seasons national disease burden of respiratory viruses detected in children by polymerase chain reaction acute lower respiratory tract infection due to virus among hospitalized children in dhaka pandemic influenza in india parainfluenza viruses role of respiratory viruses in acute upper and lower respiratory tract illness in the first year of life: a birth cohort study clinical and molecular epidemiology of human parainfluenza virus 4 infections in hong kong: subtype 4b as common as subtype 4a epidemiological features of parainfluenza virus infections: laboratory surveillance in england and wales, 1975-1997 large-scale seroprevalence analysis of human metapneumovirus and human respiratory syncytial virus infections in beijing, china detection of human metapneumovirus in children hospitalized for acute lower respiratory infection in detection of human bocavirus in japanese children with lower respiratory tract infections genetic diversity of human metapneumovirus over 4 consecutive years in australia clinical presentation and severity of viral community-acquired pneumonia in young nepalese children distribution and seasonality of rhinovirus and other respiratory viruses in a cross-section of asthmatic children in trinidad, west indies studies of the community and family: acute respiratory illness and infection prevalence and epidemiology of pandemic h1n1 strains in hospitals of eastern india global burden of acute lower respiratory infections due to respiratory syncytial virus in young children: a systematic review and meta-analysis incidence of respiratory pathogens in persons hospitalized with pneumonia in two provinces in thailand role of metapneumovirus in viral respiratory infections in young children infections caused by rsv among children and adults during two epidemic seasons association of rhinovirus infection with increased disease severity in acute bronchiolitis detection and typing by molecular techniques of respiratory viruses in children hospitalized for acute respiratory infection in surveillance and molecular characterization of human influenza b viruses during 2006-2010 revealed co-circulation of yamagata-like and victoria-like strains in eastern india detection and pathogenicity of human metapneumovirus respiratory infection in pediatric italian patients during winter-spring season human rhinovirus models in asthma human metapneumovirus as a cause of community-acquired respiratory illness a newly discovered human pneumovirus isolated from young children with respiratory tract disease identification of a new human coronavirus respiratory syncytial virus infection in tropical and developing countries acute respiratory infections in children estimates of world-wide distribution of child deaths from acute respiratory infections characterization and complete genome sequence of a novel coronavirus, coronavirus hku1, from patients with pneumonia rhinovirus viremia in children with respiratory infections influenza in outpatient ili case-patients in national hospital-based surveillance co-circulation of genetically distinct human metapneumovirus and human bocavirus strains in young children with respiratory tract infections in italy key: cord-325830-mrtpihc7 authors: nelson, philipp p.; rath, barbara a.; fragkou, paraskevi c.; antalis, emmanouil; tsiodras, sotirios; skevaki, chrysanthi title: current and future point-of-care tests for emerging and new respiratory viruses and future perspectives date: 2020-04-29 journal: front cell infect microbiol doi: 10.3389/fcimb.2020.00181 sha: doc_id: 325830 cord_uid: mrtpihc7 the availability of pathogen-specific treatment options for respiratory tract infections (rtis) increased the need for rapid diagnostic tests. besides, retrospective studies, improved lab-based detection methods and the intensified search for new viruses since the beginning of the twenty-first century led to the discovery of several novel respiratory viruses. among them are human bocavirus (hbov), human coronaviruses (hcov-hku1, -nl63), human metapneumovirus (hmpv), rhinovirus type c (rv-c), and human polyomaviruses (kipyv, wupyv). additionally, new viruses like sars coronavirus (sars-cov), mers coronavirus (mers-cov), novel strains of influenza virus a and b, and (most recently) sars coronavirus 2 (sars-cov-2) have emerged. although clinical presentation may be similar among different viruses, associated symptoms may range from a mild cold to a severe respiratory illness, and thus require a fast and reliable diagnosis. the increasing number of commercially available rapid point-of-care tests (pocts) for respiratory viruses illustrates both the need for this kind of tests but also the problem, i.e., that the majority of such assays has significant limitations. in this review, we summarize recently published characteristics of pocts and discuss their implications for the treatment of rtis. the second key aspect of this work is a description of new and innovative diagnostic techniques, ranging from biosensors to novel portable and current lab-based nucleic acid amplification methods with the potential future use in point-of-care settings. while prototypes for some methods already exist, other ideas are still experimental, but all of them give an outlook of what can be expected as the next generation of pocts. the availability of pathogen-specific treatment options for respiratory tract infections (rtis) increased the need for rapid diagnostic tests. besides, retrospective studies, improved lab-based detection methods and the intensified search for new viruses since the beginning of the twenty-first century led to the discovery of several novel respiratory viruses. among them are human bocavirus (hbov), human coronaviruses (hcov-hku1, -nl63), human metapneumovirus (hmpv), rhinovirus type c (rv-c), and human polyomaviruses (kipyv, wupyv). additionally, new viruses like sars coronavirus (sars-cov), mers coronavirus (mers-cov), novel strains of influenza virus a and b, and (most recently) sars coronavirus 2 (sars-cov-2) have emerged. although clinical presentation may be similar among different viruses, associated symptoms may range from a mild cold to a severe respiratory illness, and thus require a fast and reliable diagnosis. the increasing number of commercially available rapid point-of-care tests (pocts) for respiratory viruses illustrates both the need for this kind of tests but also the problem, i.e., that the majority of such assays has significant limitations. in this review, we summarize recently published characteristics of pocts and discuss their implications for the treatment of rtis. the second key aspect of this work is a description of new and innovative diagnostic techniques, ranging from biosensors to novel portable and current lab-based nucleic acid amplification methods with the potential future use in point-of-care settings. while prototypes for some methods already exist, other ideas are still experimental, but all of them give an outlook of what can be expected as the next generation of pocts. keywords: virus diagnostics, innovative approaches, biosensors, bedside testing, poct, commercial point-ofcare tests introduction respiratory viruses such as influenza a viruses (iav) or human respiratory syncytial virus (rsv) are well-known, circulate worldwide, and are associated with significant morbidity and mortality (iuliano et al., 2018; shi et al., 2019) . on the other hand, there are emerging infectious diseases which were, according to the definition by the who, hitherto unknown or rare but are now rapidly spreading either in number of cases or geographically (who, 2014a) . in the last 20 years, in addition to the emergence of novel influenza and coronaviruses, advances in molecular detection methods have led to the discovery of new respiratory viruses already circulating worldwide (jartti et al., 2012) . in 2001 human metapneumovirus (hmpv) was discovered (van den hoogen et al., 2001) . the outbreaks of severe acute respiratory syndrome coronavirus (sars-cov) in 2003, middle east respiratory syndrome coronavirus (mers-cov) since april 2012, and sars-cov-2 since december 2019 highlight the danger of emerging (zoonotic) and highly pathogenic respiratory viruses (fouchier et al., 2003; zaki et al., 2012; who, 2020c) . two other coronaviruses, human coronaviruses (hcov) nl63 and hku1 were discovered in 2004 and 2005, respectively van der hoek et al., 2004; woo et al., 2005) . in 2005, allander et al. described a new member of the family parvoviridae, human bocavirus (hbov) (allander et al., 2005) . the two new human polyomaviruses ki and wu (kipyv, wupyv) were first discovered in 2007 (allander et al., 2007; gaynor et al., 2007) and although often detected in samples, their role in causality of respiratory illness is still under discussion (reviewed in jartti et al., 2012) . in 2007, the human rhinovirus group c (rv-c) was introduced when newly sequenced strains differed significantly from the existing groups a and b (lee et al., 2007) . finally, avian influenza viruses (aiv) such as iav h5n1, h7n7, or h9n2 crossed the species barrier to infect humans several times in the last years (reviewed in kim et al., 2016) . lab-based techniques still dominate the field of virus diagnostics. classical methods such as virus cultures, electron microscopy, and serology have been complemented by nucleic acid amplification tests (naats), sequencing (including next generation sequencing), and different antigen detection methods. today, in most clinical settings, naats have replaced virus cultures as the gold standard, due to their high specificity, faster turnaround times, and absence of limitations posed by the need for susceptible cell lines (fox, 2007) . in contrast to lab-based tests, point-of-care tests (pocts) are performed at the site of sample collection (e.g., bedside, physician's office, or emergency department) and provide results usually in <2 h (basile et al., 2018; vos et al., 2019) . furthermore, they require only little hands-on time and no specific laboratory training as most critical steps are automated in a single device. the latter may range from handheld to benchtop size and is not designed for high-throughput sample processing. pocts and other fast diagnostic tests performed in laboratories but provide results within 1-2 h may be called near-pocts. prompt identification of the causative pathogen may help the responsible healthcare professional choosing the appropriate treatment or take the right decisions in outbreak situations, regarding hospitalization and quarantine (brendish et al., 2015) . in this review, we provide a brief overview of currently available pocts for the diagnosis of emerging and new respiratory viruses along with their advantages and limitations and discuss recently published approaches and techniques with a potential use in future pocts. for the diagnosis of commonly encountered respiratory viruses such as iav, influenza b virus (ibv), and rsv many commercially available pocts and near-pocts with different sensitivities and specificities for each virus are available (supplementary table 1 ). however, a recent meta-analysis has demonstrated that three newer generation rapid multiplex polymerase chain reaction systems (mpcrs) (biomérieuxbiofire r filmarray r rp, nanosphere verigene r rv+ test, and hologic gen-probe prodesse assays) are highly accurate, though usually more expensive, and may provide important diagnostic information for early identification of iav, ibv, and rsv (huang h. s. et al., 2018; rabold and waggoner, 2019) . diagnosis of emerging and novel viruses, including hbov, rv-c, coronaviruses (e.g., hcov-hku1, hcov-nl63, sars-cov-2) as well as specific subtypes of aiv (h5n1, h7n9, h10n8) and reassortant iav strains remains challenging (schuster and williams, 2018; who, 2020a) . the diagnosis of most of these viruses is based on molecular techniques that can only be performed at specialized referral centers. recently, there has been increased interest in using pocts in other settings, such as emergency departments, although implementation might be hampered by the need for specific training (bouzid et al., 2020) . naats have higher sensitivity than immunochromatographic assays, but generally, they require a higher degree of technical skills and training (drancourt et al., 2016) . polymerase chain reaction (pcr) remains the gold standard technique for the diagnosis of aiv subtypes while who recommends against the utilization of rapid tests in avian flu diagnosis (who, 2005; monne et al., 2008; schuster and williams, 2018) . rv-c, is typically detected from nasopharyngeal specimens using reverse transcriptase pcr (rt-pcr) and specific species and serotypes can be further identified by semi-nested pcrs or sequencing (bochkov et al., 2011; jartti et al., 2012; schuster and williams, 2018) . diagnosis of hbov was based on the detection of the specific igm antibody along with a 4-fold increase of the igg titer or low igg avidity indicative of seroconversion, but adaptive immune response requires several hours or even days to develop, thus the utility of such tests in acute settings is limited (soderlund-venermo et al., 2009) . as hbov dna persists in airway secretions for months after an acute infection, quantitative pcr along with serology are currently the preferred diagnostic methods (christensen et al., 2019) . viral dnaemia, mrna detection via rt-pcr, and antigen immunodetection assays have shown some promising results but further studies to define their sensitivity, specificity, and applicability in clinical practice are needed (soderlund-venermo et al., 2009; christensen et al., 2010 christensen et al., , 2013 christensen et al., , 2019 proenca-modena et al., 2011; xu et al., 2017) . the detection of kipyv and wupyv, found in secretion from both symptomatic and asymptomatic patients, is based on pcr and serology testing (neske et al., 2010; touze et al., 2010; jartti et al., 2012) . sars-cov-2, hcov-nl63, and -hku1 as well as hmpv diagnosis is based primarily on rt-pcr methods (van den hoogen et al., 2004; nichols et al., 2008; jartti et al., 2012; who, 2020a) . no data is available for fluorescent antibody, rapid cultures and enzyme immunoassay (eia) diagnostic tests for coronaviruses nl63 and hku1, whereas fluorescent antibody assays may be of some utility for hmpv (nichols et al., 2008; jartti et al., 2012) . for hmpv specifically, multiplex ligationdependent probe amplification (mlpa) on a nasopharyngeal swab has high sensitivity and specificity rates (100 and 96%, respectively) (reijans et al., 2008; panda et al., 2014; hoppe et al., 2016) . available tests for the now extinct sars-cov include antibody testing using an eia and rt-pcr tests in respiratory, blood, and stool specimens (cdc, 2004) . for the detection of mers-cov, that has its epicenter in the arabian peninsula, the united states centers for disease control and prevention (cdc) and the who recommend sampling from the lower respiratory tract and real-time rt-pcr (rrt-pcr) testing with specific primers since this appears to be more sensitive than testing of upper respiratory tract specimens (who, 2014b,c; cdc, 2015) . naats have been proven to be highly accurate and easily scalable tests during large outbreaks of novel or emerging viruses, like the sars-cov-2 pandemic. rapid genome sequencing analysis accommodates the fast development of reliable in-house and commercially available naats reagents shortly after an outbreak onset (cdc, 2020; who, 2020b). biosensors can be a reliable and cost-effective way to detect specific pathogens in point-of-care settings. different types of sensors for rapid identification of respiratory viruses have been developed recently. by using a gold-coated array of carbon electrodes, the authors were able to detect mers-cov spike protein in the picogram range within 20 min (layqah and eissa, 2019) . this electrochemical assay is based on the competitive binding of a mers-cov antibody either to the virus in the sample or to the immobilized antigen on the electrode, which can be measured by a reduced peak current through the chip. in theory, this technique can be easily expanded to simultaneously detect multiple viruses, however, its diagnostic performance needs to be validated with the use of patient samples. different types of biosensors for aiv detection use nanobio hybrid materials (reviewed in lee et al., 2018) . in one of those approaches, a dna probe coupled to a field-effect transistor enabled detection of target dna down to 1 fm (lin et al., 2009) . another sensitive technique is based on surface plasmon resonance (spr), in which biomolecules bound to a metal surface lead to the reduction of the reflection of an incident light beam (tang et al., 2010; chang et al., 2018) . with a new antibody against a recombinant aiv h7n9, the authors were able to reach a detection limit of a few hundred copies per ml nasal fluid within 10 min of processing time. although still experimental, the characteristics of this approach render it a promising candidate for a future rapid poct. in a capillary convective pcr (ccpcr), the reagents circulate across a temperature gradient in a simple capillary tube, which allows run times shorter than 30 min (chou et al., 2011) . together with a self-made dipstick detection method, this principle was already used to test for non-respiratory viruses like hepatitis c virus (zhang et al., 2013 (zhang et al., , 2014 . zhou et al. integrated this method into a 1.5 kg device for the automation of the detection of different iav strains (zhuo et al., 2018) . although fast and sensitive, manual rna extraction limits the use as poct so far. hardick et al. presented another portable naat device not only for the detection of iav, but also for ibv, rsv, and mers-cov. it is based on a rt-pcr in microfluidic cards but likewise lacks automated nucleic acid extraction (hardick et al., 2018) . alternatively to nucleic acid-based techniques, giant magnetoresistive (gmr) biosensors function comparable to an enzyme-linked immunosorbent assay but use magnetic labels instead of enzymes or fluorophores coupled to detection antibodies (hall et al., 2010) . with such a sensor, wu et al. constructed a handheld device which, in connection with a computer or smartphone, was able to detect iav h3n2 in purified and disrupted virus solutions (wu et al., 2017) . although h3n2 strains are circulating already since 1968 in the human population (smith et al., 2004) , this method is likely adaptable to emerging aivs with the use of appropriate capture antibodies. by designing a prototype for a lateral flow assay for approximately 5 us$, huang et al. proved that the simultaneous detection of iav and ibv in swab samples is possible at very low costs (huang et al., 2017) . however, the sensitivity of this prototype needs significant improvements before it can be used under clinical conditions. instead of constructing a completely new apparatus, cui et al. used commercially available glucose test strips together with specifically designed glucose-bearing substrates to test for the cleavage activity of iav neuraminidase in spiked samples (cui et al., 2017) . although the majority of the presented approaches and prototypes focuses on the detection of influenza viruses, most of them can theoretically be applied to emerging or new viruses with only minor changes. in comparison to pcrs, isothermal naats do not require complex devices when working with extracted nucleic acids. reverse transcription strand invasion-based amplification (rt-siba) and reverse transcription loop-mediated isothermal amplification (rt-lamp) are two examples which have been used to detect e.g., hmpv (song et al., 2014) , iav (eboigbodin et al., 2016) , and mers-cov (huang p. et al., 2018) . wang et al. went on to integrate seven rt-lamp assays into a microfluidic chip for the multiplex detection of different respiratory viruses in a device weighing <3 kg . another chipbased system, named iroad, uses reverse transcription-based recombinase polymerase amplification (rt-rpa) and was able to rapidly identify iav, different hcovs, and other respiratory viruses in extracted nucleic acids (koo et al., 2017) . the development of new laboratory and point-of-care diagnostic tests for influenza, rsv, and emerging respiratory viruses has taken up pace in recent years. healthcare professionals, hospital managers, and laboratory directors will need to update and re-evaluate best practices regularly. the advancement of diagnostic capabilities may change the way we identify, document, and communicate respiratory viral infections in the future. along with these developments, the expectations of patients and healthcare professionals may change as well, i.e., patients will want to know the responsible pathogen and their clinical prognosis. with the development of specific antiviral therapies and vaccines, new diagnostic algorithms will be needed to ensure the highest quality of care while containing costs. from a viewpoint of quality of care and clinical management, timely infection control, and the ability to act upon results, the future will likely belong to portable, clia-waived rapid diagnostic tests that take 10-20 min. a key criterion for the evaluation of diagnostic tests will be their clinical utility, i.e., their ability to identify the current culprit for the patient's symptoms, and to distinguish relevant pathogen(s) from bystander pathogens. more research is needed to correlate clinical outcomes and laboratory data. a second concern will be the correct timing of diagnostic testing with regards to a patient's course of illness. the sensitivity and positive predictive value of a diagnostic test depend on specimen quality and virus load (which is usually higher in children and early in the course of illness), duration of viral shedding, and patient's immune status. future diagnostic algorithms will need to consider these factors in addition to the epidemiology of viruses in the respective season or region. the initial criticism of pocts was directed toward a lack of sensitivity and a high degree of variability in test results. early studies during the 2009/10 flu pandemic reported sensitivities for influenza pocts ranging from 20 to 70% for the same test kit (rath et al., 2012) . in published evaluation studies, it was not entirely clear whether pocts were performed at the bedside or whether samples were sent to the laboratory, which would constitute a near-poct. these methodological inconsistencies also impaired the value of meta-analyses comparing the point-ofcare performance of different commercial tests (chartrand et al., 2015) . the reported performance differences also hint to the fact that the training and experience of the staff performing the test have an impact. procedural concerns were most pronounced with early-stage lateral flow ("strip") tests, where the result was read manually. second-generation antigen pocts and modern molecular pocts achieve significantly higher reproducibility and sensitivity through automation of key steps in the process. in 2018, the fda implemented new performance regulations setting new sensitivity thresholds required for pocts to maintain approval (fda, 2017) . as a result, only several influenza pocts were taken off the market. the role of regulators in setting quality standards for pocts cannot be underestimated (zhang et al., 2016; azar and landry, 2018) . in addition to the reliability of the poct result itself, the hands-on time and the time-to-result are still critical for useracceptance at the bedside. it is expected that future pocts will be more robust and easier to use. as the majority of acute respiratory infections (ari) is of viral origin, these infections are common reasons for inappropriate antibiotic use (harris et al., 2016; tief et al., 2016) . studies have raised the expectation that expanded use of virus diagnostics at the point-of-care may help to limit the use of antibiotics (bonner et al., 2003) . the european health action plan on amr (european parliament, 2018) and the o'neill report on "tackling drug-resistant infections globally" (o'neill, 2016) both point to rapid diagnostics as a key instrument in tackling amr. including pocts in antimicrobial stewardship programs might increase their acceptance by physicians. the ability to direct "the right treatment to the right patient at the right time" facilitates precision medicine. in the future, advanced virus diagnostics may be combined with biomarker pocts for the prediction of individual-level host responses to further target treatment to those who are most likely to benefit from it. a major obstacle to expanding the use of pocts is cost. in fact, pocts would be most impactful in settings where the majority of early treatment decisions are made. the current pricing schemes, however, seem too high for broadscale testing in the community and emergency departments. in addition to pricing, reimbursement strategies need to be reconsidered. even in hospital emergency rooms, percapita standard reimbursements disincentivize the use of virus diagnostics in patients with ari. up-to-date economic models are needed to clarify the cost-effectiveness of different types of pocts. the steepest increase in antimicrobial resistance is being observed in low-middle income countries (lmic) (who-paho, 2018). for lmic, portable pocts with multi-modality for known and emerging pathogens, or simple lab-based instrumentation with no/minimal need for cold chain or refrigeration of reagents, may be the most likely to succeed. none of the currently commercially available pocts covers all viruses discussed here (tables 1, 2, supplementary table 1 ). to date, the same holds true for new technologies as well. however, it is conceivable that these techniques may be adapted to other respiratory viruses after having shown their usefulness in practice. especially biosensors have the potential for a wider spectrum of applications. they also do not encounter the major limitation of naats as pocts: the extraction of nucleic acids (ali et al., 2017) . for these tests, even less obvious complications, like the stability of the plastic materials against required chemicals, have to be overcome for future highly specific nucleic acidbased pocts. any ideal poct should fulfill the assured criteria of the world health organization to be applicable in resource-limited settings (mabey et al., 2004) . currently, this is most likely true for tests based on lateral flow immunochromatography but coming improvements e.g., in miniaturization and battery capacity may facilitate the use of other test principles (basile et al., 2018) . while economic benefits of pocts and better outcomes for the patients are still discussed, it is likely that these tests will gain further importance with decreasing processing costs and improved robustness. cs and pn planned, structured, and edited the manuscript. pn searched the literature and integrated all contributions. pn, br, pf, ea, and st participated in the writing of the first draft of the manuscript and subsequent revisions. br and pf contributed equally to this work. all authors critically read, reviewed, and approved the submitted final version of the manuscript. authors would like to thank the escmid study group on respiratory viruses (esgrev) for providing a platform for interaction between authors. the supplementary material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fcimb. 2020.00181/full#supplementary-material influenza ag a/ b/ a(h1n1) pandemic. product page virus not included in this assay; fia, fluorescent immunoassay; lfic, lateral flow immunochromatography assay; ci, chromatographic immunoassay; pe, photofluorescent excitation; icmi, immunochromatographic membrane immunoassay human bocavirus; (h)cov, (human) coronavirus human metapneumovirus; rv-c, human rhinovirus type c influenza a virus avian influenza virus please refer to supplementary table 1 for detailed reliability parameters. a two different kits for influenza (a, b) and rsv; b performance current nucleic acid extraction methods and their implications to pointof-care diagnostics identification of a third human polyomavirus cloning of a human parvovirus by molecular screening of respiratory tract samples detection of influenza a and b viruses and respiratory syncytial virus by use of clinical laboratory improvement amendments of 1988 (clia)-waived point-of-care assays: a paradigm shift to molecular tests multicenter evaluation of the eplex respiratory pathogen panel for the detection of viral and bacterial respiratory tract pathogens in nasopharyngeal swabs point-of-care diagnostics for respiratory viral infections molecular modeling, organ culture and reverse genetics for a newly identified human rhinovirus c impact of the rapid diagnosis of influenza on physician decisionmaking and patient management in the pediatric emergency department: results of a randomized, prospective, controlled trial rapid diagnostic tests for infectious diseases in the emergency department point-of-care testing for respiratory viruses in adults: the current landscape and future potential severe acute respiratory syndrome interim guidelines for collecting, handling, and testing clinical specimens from patients under investigation (puis) for middle east respiratory syndrome coronavirus (mers-cov) -version 2.1 frequently asked questions on covid-19 testing at laboratories assay manual simple strategy for rapid and sensitive detection of avian influenza a h7n9 virus based on intensity-modulated spr biosensor and new generated antibody diagnostic accuracy of rapid antigen detection tests for respiratory syncytial virus infection: systematic review and meta-analysis rapid dna amplification in a capillary tube by natural convection with a single isothermal heater detection of spliced mrna from human bocavirus 1 in clinical samples from children with respiratory tract infections human bocaviruses and paediatric infections human bocavirus in children: mono-detection, high viral load and viraemia are associated with respiratory tract infection highly specific and rapid glycan based amperometric detection of influenza viruses the pointof-care laboratory in clinical microbiology reverse transcription strand invasion based amplification (rt-siba): a method for rapid detection of influenza a and b microbiology devices; reclassification of influenza virus antigen detection test systems intended for use directly with clinical specimens. docket no. fda-2014-n-0440 a previously undescribed coronavirus associated with respiratory disease in humans aetiology: koch's postulates fulfilled for sars virus nucleic acid amplification tests for detection of respiratory viruses identification of a novel polyomavirus from patients with acute respiratory tract infections assay manual gmr biosensor arrays: a system perspective initial performance evaluation of a spotted array mobile analysis platform (map) for the detection of influenza a/b, rsv, and mers coronavirus appropriate antibiotic use for acute respiratory tract infection in adults: advice for high-value care from the american college of physicians and the centers for disease control and prevention human metapneumovirus in haematopoietic stem cell transplantation recipients: a case series and review of the diagnostic and therapeutic approach multiplex pcr system for the rapid diagnosis of respiratory virus infection: systematic review and meta-analysis a rapid and specific assay for the detection of mers-cov disposable autonomous device for swab-to-result diagnosis of influenza estimates of global seasonal influenzaassociated respiratory mortality: a modelling study new respiratory viral infections avian influenza a viruses: evolution and zoonotic infection an isothermal, label-free, and rapid one-step rna amplification/detection assay for diagnosis of respiratory viral infections an electrochemical immunosensor for the corona virus associated with the middle east respiratory syndrome using an array of gold nanoparticle-modified carbon electrodes recent advances in aiv biosensors composed of nanobio hybrid material a diverse group of previously unrecognized human rhinoviruses are common causes of respiratory illnesses in infants poly-silicon nanowire field-effect transistor for ultrasensitive and labelfree detection of pathogenic avian influenza dna cepheid assays detect a broad range of contemporary human and avian influenza strains diagnostics for the developing world development and validation of a one-step real-time pcr assay for simultaneous detection of subtype h5, h7, and h9 avian influenza viruses high prevalence of antibodies against polyomavirus wu, polyomavirus ki, and human bocavirus in german blood donors respiratory viruses other than influenza virus: impact and therapeutic advances tackling drug-resistant infections globally, final report and recommendations human metapneumovirus: review of an important respiratory pathogen detection of human bocavirus mrna in respiratory secretions correlates with high viral load and concurrent diarrhea qiastat-dx r respiratory panel instructions for use: version 2. manual rapid diagnostic tests for infectious diseases early detection of influenza a and b infection in infants and children using conventional and fluorescence-based rapid testing respifinder: a new multiparameter test to differentially identify fifteen respiratory viruses emerging respiratory viruses in children global disease burden estimates of respiratory syncytial virus-associated acute respiratory infection in older adults in 2015: a systematic review and metaanalysis mapping the antigenic and genetic evolution of influenza virus clinical assessment and improved diagnosis of bocavirus-induced wheezing in children identification of human metapneumovirus genotypes a and b from clinical specimens by reverse transcription loop-mediated isothermal amplification surface plasmon resonance: an introduction to a surface spectroscopy technique an inception cohort study assessing the role of pneumococcal and other bacterial pathogens in children with influenza and ili and a clinical decision model for stringent antibiotic use generation of merkel cell polyomavirus (mcv)-like particles and their application to detection of mcv antibodies rapid molecular tests for influenza, respiratory syncytial virus, and other respiratory viruses: a systematic review of diagnostic accuracy and clinical impact studies rapid detection of multiple respiratory viruses based on microfluidic isothermal amplification and a real-time colorimetric method recommendations on the use of rapid testing for influenza diagnosis a brief guide to emerging infectious diseases and zoonoses. world health organization, regional office for south-east asia interim surveillance recommendations for human infection with middle east respiratory syndrome coronavirus laboratory testing for middle east respiratory syndrome coronavirus: interim recommendations (revised) coronavirus disease (covid-19) technical guidance: laboratory testing for 2019-ncov in humans novel coronavirus (2019-ncov) in suspected human cases: interim guidance report of the who-china joint mission on coronavirus disease 2019 (covid-19) recommendations for implementing antimicrobial stewardship programs in latin america and the caribbean: manual for public health decision-makers characterization and complete genome sequence of a novel coronavirus, coronavirus hku1, from patients with pneumonia portable gmr handheld platform for the detection of influenza a virus comparative diagnosis of human bocavirus 1 respiratory infection with messenger rna reverse-transcription polymerase chain reaction (pcr), dna quantitative pcr, and serology isolation of a novel coronavirus from a man with pneumonia in saudi arabia external quality assessment for the molecular detection of mers-cov in china a convenient nucleic acid test on the basis of the capillary convective pcr for the on-site detection of enterovirus 71 a one-step dipstick assay for the on-site detection of nucleic acid a rapid on-site assay for the detection of influenza a by capillary convective pcr key: cord-342853-n3e6yawi authors: naghipour, mohammadreza; cuevas, luis e.; bakhshinejad, tahereh; dove, winifred; hart, c. anthony title: human bocavirus in iranian children with acute respiratory infections date: 2007-03-26 journal: j med virol doi: 10.1002/jmv.20815 sha: doc_id: 342853 cord_uid: n3e6yawi human bocavirus (hbov), a virus discovered in sweden in 2005, has been associated with acute respiratory infections in young children and subsequent reports suggest that hbov may have a worldwide distribution. this report describes the frequency and clinical presentation of hbov in 261 iranian children<5 years old with acute respiratory infections attending two regional hospitals in rasht, iran in the winter of 2003–2004. polymerase chain reaction (pcr) and reverse transcription pcr (rt‐pcr) were used for the detection of hbov and other respiratory pathogens from nasopharyngeal specimens. hbov was detected in 21 (8%) children. fifteen (12%) of these children were identified among 122 children admitted to hospital and 6 (4%) from 139 outpatients (p < 0.05). most children with hbov were less than 2 years (17/21, 81%) and 7 (33%) were less than 1 year old. although hbov was identified in all ages it affected slightly older children than the respiratory syncytial virus (rsv). the frequency of the virus varied from 1 (3%) in 40 patients in november to 7 (12%) of 61 in february, suggesting a seasonal pattern during the autumn and early winter. seven children had co‐infections with rsv, adenovirus or influenza a. the relatively high frequency of hbov suggests that the virus may contribute substantially to acute respiratory infections in children. j. med. virol. 79:539–543, 2007. © 2007 wiley‐liss, inc. acute respiratory infections are among the most important causes of childhood morbidity and mortality and are responsible for one-fifth of all deaths in children under five, resulting mainly from pneumonia and bronchiolitis [bryce et al., 2005] . viruses play a significant role in these infections and the number of viruses associated with severe acute respiratory infections has increased in recent years with the detection of new pathogens such as human metapneumovirus (hmpv) [van den hoogen et al., 2001] and severe acute respiratory syndrome associated with a coronavirus [chan-yeung and yu, 2003] . despite the advances in understanding the aetiology of acute respiratory infections, a significant proportion of episodes remain unclassified and it is likely that more ''new'' viruses will be discovered [snell, 2004] . human bocavirus (hbov) was first described in 2005 [allander et al., 2005] and it was suggested that the virus might be a cause of acute respiratory infections. a case series of 21 patients infected with hbov who presented to two referral hospitals in iran is described. the study was conducted from november 2003 to march 2004 based on 17-shahrivar and rasoul-e-akram hospitals. 17-shahrivar is a paediatric reference university hospital with 200 beds and rasoul-e-akram is a general regional referral hospital in rasht, guilan in northern iran. the original aim of the study was to describe the etiology of acute viral respiratory infections in children in northern iran. these included respiratory syncytial virus (rsv), hmpv, influenza a and b, parainfluenza, and adenovirus, plus chlamydia spp. and mycoplasma pneumoniae. children less than 5 years of age with acute respiratory infections of less than 7 days duration attending the outpatient department or being admitted to hospital from saturday to thursday were enrolled after informed parental consent. acute respiratory infections were defined following the world health organization (who) protocol and were classified into upper and lower tract infections on the basis of the children's respiratory frequency and the presence of subcostal indrawing [pio, 2003] . oxygen saturations (po 2 ) were measured in all patients admitted using a pulse oximeter (nonin medical, inc. mpl, mn, model 8500) before initiation of oxygen therapy. children were classified as having mild/moderate (po 2 !94%) or severe hypoxia (po 2 <94%) and a questionnaire containing socio-demographic, clinical, therapeutic, and outcome data was completed for each patient. ethical approval for the study was obtained from the research ethics committees of the liverpool school of tropical medicine and guilan university of medical sciences and informed consent was obtained from all parents. nasopharyngeal aspirates or swabs (medical wire & equipment co. ltd., corsham, wilts, uk) were collected from all children using sterile mucus extractors for nasopharyngeal aspirates and stored at à808c until processed. samples were processed in the department of medical microbiology, university of liverpool, uk. dna and rna were extracted using qiaamp1 dna and rneasy mini kits (qiagen ltd., crawly, west sussex, uk). extracted rna was processed using reverse transcriptionpolymerase chain reaction (rt-pcr) for detection of rsv and hmpv [greensill et al., 2003] , influenza a & b, and parainfluenza 1-4 viruses [templeton et al., 2004] , using primers and methods described previously. pcr assays were used to amplify the dna of adenovirus, chlamydia spp, and mycoplasma pneumoniae [couroucli et al., 2000] . the hbov np-1 gene was detected by pcr using the primers 188 f (gagctctgtaagtactattac) and 542 r (ctctgtgttgactgaatacag) and the same reactions and thermal cycler programme as described and corrected subsequently by allander et al. [allander et al., 2005] to detect a 345-bp product. one-third of the hbov amplicons were sequenced to confirm the identity of the virus (lark technologies, essex, uk). epi info 2002 (cdc, atlanta) was used for the descriptive analysis of characteristics of the children included means, standard deviations (sd), median, inter quartile range (iqr), and percentages. cross tabulation of the frequency of the virus in hospitalized and ambulatory children and their clinical presentations were compared using parametric tests. p values <0.05 were considered statistically significant. respiratory specimens were collected from 261 children. of these, 139 were ambulatory and 122 were hospitalized. their median (iqr) age was 14 (7-32) months and 167 (64%) were male. hbov dna was detected in 21 (8%) children; 15 (12%) were identified among the 122 hospitalized children and 6 (4%) among the 139 outpatients (p < 0.05). fourteen (67%) cases were male and seven female. five patients were known to have a history of asthma and six had been hospitalized previously with asthma or pneumonia. the age distribution of the children with hbov is shown in figure 1 . most cases (17, 81%) were less than 2 years of age and 7 (33%) were less than 1 year old. the numbers of children enrolled and those with hbov by month are shown in figure 2 . the proportion of children infected with hbov each month ranged from 1 (3%) out of 40 patients in november to 7 (12%) out of 61 in february, suggesting a seasonal pattern (chi square for trend, p ¼ 0.05). in total, 39 (15%) children with rsv, 37 (14%) with adenovirus, 11 (4%) with influenza a, 4 (2%) with chlamydia spp, 2 (1%) with m. pneumoniae, and none with hmpv or parainfluenza were identified. seven children with hbov were co-infected with another respiratory pathogen (1 with inf a and 3 each for rsv and adenovirus). none of the hbov positive children were co-infected with hmpv, parainfluenza viruses, chlamydia spp, or m. pneumoniae [naghipour et al., in press] . the clinical presentation of the patients is described in table i and are compared to children without hbov in table ii . all children had a history of cough (100%) with a mean duration of 4 days (range 1-7), 17 (81%) had fever with a mean duration of 2 days (range 1-5), and 11 (52%) had tachypnoea. the po 2 concentrations <94% were recorded in 3 of the 15 hospitalized children, and one was co-infected with influenza a. the clinical diagnoses on discharge were pneumonia in ten, upper respiratory infections in six, tracheobronchitis in three, and asthma in two cases. fourteen of the children admitted had chest radiography performed and hyperinfiltration was the most frequent finding reported in 11 children (8 infected with hbov alone), in addition to consolidation in 3. the mean (sd) duration of admission in the patients was 5 (2) days with a range from 3 to 9 days. a total of six of the amplicons were subjected to dna sequencing of both strands. of these, four were identical to those of the swedish reference strain. two showed changes, one with a point mutation at codon 47 (r ! k) and the other at codon 78 (s ! n). these are available at www.ddbj.nig.ac.jp with accession numbers of ab257721 and ab257722, respectively. this case series describes the presence of hbov outside industrialized countries. the frequency of hbov in the study was 8%, which is slightly higher than in earlier reports (canada 1.5%, sweden 3.1%, australia 5.6%, and japan 5.7%) [allander et al., 2005; bastien et al., 2006; ma et al., 2006; sloots et al., 2006] , but lower than reported recently from germany (10.3%) [weissbrich et al., 2006] and korea (11.3%) [choi et al., 2006] indicating that more studies are required to assess its relative importance. hbov was observed more frequently in patients admitted to the hospitals, some of whom had severe hypoxia, than in ambulatory children. this higher frequency however could also reflect the sampling technique used, as samples for admitted patients were collected using nasopharyngeal aspirates while ambulatory patients were tested with throat swabs, and further studies would be required to confirm these findings. similar to previous reports, a higher proportion of the cases were male, although this was a reflection of the higher number of male children enrolled in the study. overall, there was no significant difference in the prevalence by sex. most children infected with hbov were also less than 2 years of age, which is similar to the swedish (12 out of 14 cases), japanese (16 out of 18), and australian children. however, as acute respiratory infections in general mostly affect young children [bryce et al., 2005] , it is not surprising that most of the children with hbov had this age distribution. interestingly, different to rsv, which affects mostly children <6 months of age [constantopoulos et al., 2002] , hbov seems to affect slightly older children, as has been observed with hmpv [al-sonboli et al., 2005] . although, the study only enrolled children for 5 months, there seems to be a seasonal pattern during the late autumn and early winter, as reported from earlier studies and larger studies are warranted to confirm these findings. co-infections with other respiratory viruses have been observed in previous studies [allander et al., 2005; bastien et al., 2006; ma et al., 2006; sloots et al., 2006 ] and a relatively high proportion of co-infections is reported in this study (33%). this noticeable proportion of co-infections needs to be further elucidated as it might suggest that hbov is an incidental finding without a significant role in the causation of acute respiratory infections or, as suggested for hmpv [greensill et al., 2003] , it might play a role modifying the clinical presentation of children who have co-infections with other viruses. given the high frequency of hbov in iran, this virus might play a significant role as a cause of acute respiratory infections in children. however, given that up to now all the information is based on small case series, without a negative control group of healthy children to confirm that hbov is indeed responsible for the clinical manifestation observed, the aetiological role of hbov as a cause of acute respiratory infections still needs to be demonstrated conclusively. from the cover: cloning of a human parvovirus by molecular screening of respiratory tract samples respiratory syncytial virus and human metapneumovirus in children with acute respiratory infections in yemen who estimates of the causes of death in children outbreak of severe acute respiratory syndrome in hong kong special administrative region: case report the association of newly identified respiratory viruses with lower respiratory tract infections in korean children burden of respiratory syncytial viral infections on paediatric hospitals: a twoyear prospective epidemiological study detection of microorganisms in the tracheal aspirates of preterm infants by polymerase chain reaction: association of adenovirus infection with bronchopulmonary dysplasia human metapneumovirus in severe respiratory syncytial virus bronchiolitis detection of human bocavirus in japanese children with lower respiratory tract infections contribution of viruses, chlamydia spp. and mycoplasma pneumoniae to acute respiratory infections in iranian children standard case management of pneumonia in children in developing countries: the cornerstone of the acute respiratory infection programme evidence of human coronavirus hku1 and human bocavirus in australian children novel and re-emerging respiratory infections rapid and sensitive method using multiplex realtime pcr for diagnosis of infections by influenza a and influenza b viruses, respiratory syncytial virus, and parainfluenza viruses 1,2,3,and 4 frequent detection of bocavirus dna in german children with respiratory tract infections this study was supported by the iranian ministry of health and medical education through a study scholarship for dr. naghipour. key: cord-346673-kyc1wks5 authors: nickbakhsh, s.; thorburn, f.; von wissmann, b.; mcmenamin, j.; gunson, r. n.; murcia, p. r. title: extensive multiplex pcr diagnostics reveal new insights into the epidemiology of viral respiratory infections date: 2016-03-02 journal: epidemiol infect doi: 10.1017/s0950268816000339 sha: doc_id: 346673 cord_uid: kyc1wks5 viral respiratory infections continue to pose a major global healthcare burden. at the community level, the co-circulation of respiratory viruses is common and yet studies generally focus on single aetiologies. we conducted the first comprehensive epidemiological analysis to encompass all major respiratory viruses in a single population. using extensive multiplex pcr diagnostic data generated by the largest nhs board in scotland, we analysed 44230 patient episodes of respiratory illness that were simultaneously tested for 11 virus groups between 2005 and 2013, spanning the 2009 influenza a pandemic. we measured viral infection prevalence, described co-infections, and identified factors independently associated with viral infection using multivariable logistic regression. our study provides baseline measures and reveals new insights that will direct future research into the epidemiological consequences of virus co-circulation. in particular, our study shows that (i) human coronavirus infections are more common during influenza seasons and in co-infections than previously recognized, (ii) factors associated with co-infection differ from those associated with viral infection overall, (iii) virus prevalence has increased over time especially in infants aged <1 year, and (iv) viral infection risk is greater in the post-2009 pandemic era, likely reflecting a widespread change in the viral population that warrants further investigation. acute respiratory infections are the commonest cause of illness in all ages, and a leading cause of mortality in children aged <5 years, creating a significant global healthcare burden [1] [2] [3] . various aetiological pathogens (viruses, bacteria and some fungi) are recognized, causing largely indistinguishable symptoms. in most settings, viruses are the most frequently detected agent [4, 5] . although most infections are mild, respiratory viruses have the potential to cause severe illness in high-risk groups. although influenza is a major research focus [6] , the advent of polymerase chain reaction (pcr) technology has led to improved awareness that non-influenza viruses are also important contributors to disease burden, and of the role of viral subtype in clinical severity [7] [8] [9] . the use of pcr testing as part of routine diagnostics provides an important resource for monitoring respiratory viruses as part of national surveillance [10] . multiplex pcr methods in particular provide a valuable resource for epidemiological enquiry [11] . all patients requiring microbiological diagnosis are tested for all pathogens included in the panel, ensuring consistency in testing across patients. the collation of multiplex diagnostic data from a large patient population and over an extended time-frame therefore enables robust comparisons of infection trends temporally and across patient subgroups. furthermore, when testing is implemented over multiple years, sufficient data can be accrued to investigate the clinical relevance of co-infections and their epidemiological patterns [12] . although the utility of diagnostic data in the epidemiology of respiratory infections has been demonstrated [11, [13] [14] [15] [16] , studies that cover all major viruses, patient age and illness severity groups, and that span multiple years, are lacking. the largest nhs health board in scotland, greater glasgow and clyde (nhsggc), has used multiplex pcr testing as part of their routine diagnostic services since 2005. this health board serves ∼1·1 million people, representing ∼1·7% of the total uk population [17] . the resultant accumulation of data provides a novel opportunity to investigate viral respiratory infections in a more comprehensive fashion than previously possible. these data also provide a unique opportunity to compare the periods before and after the introduction of the novel pandemic influenza virus [a(h1n1) pdm09] into scotland (see [18] ). we analysed diagnostic data generated by nhsggc using multiplex pcr from 2005 to 2013 with the following objectives: (i) to describe testing and virus prevalence trends, (ii) to examine temporal and patient subgroup distributions for each individual virus, and (iii) to compare factors associated with overall viral infection and co-infection using statistical modelling, in order to provide robust and timely estimates of who is most at risk of viral-associated respiratory illness, and when, within a major urban uk population. in this study we used virological diagnostic data generated by the west of scotland specialist virology centre (wossvc) for nhsggc during 2005-2013 [19] . during this period, a total of 61 427 clinical samples were received from 40 962 patients attending primary and secondary healthcare services for respiratory diagnostic purposes (i.e. excluding pathology-origin samples). most (98%) clinical samples were taken from the upper or lower respiratory tract: primarily nasal and/or throat swabs (67%), gargles (13%), nasopharyngeal aspirates (7%), sputum (5%), bronchoalveolar lavage (3%) and nasopharyngeal/ endotracheal secretions (2%). in a minority of cases (n = 142 samples), plasma was additionally taken for follow-up investigation; most (89%) of these samples related to the 2009 influenza a pandemic period which was excluded from statistical modelling analyses. each sample was tested by real-time rt-pcr for 11 groups of respiratory viruses: human rhinovirus (rv); influenza a virus [iav; a generic assay detecting seasonal h3n2 and h1n1 subtypes and one specific to a(h1n1)pdm09], influenza b virus (ibv), human respiratory syncytial virus (rsv), human coronavirus (cov; aggregating 229e, nl63, hku1 and oc43 species), adenovirus (adv), human metapneumovirus (mpv) and human parainfluenza types 1-4 (piv1-4). details of nucleic acid extraction methods and the real-time pcr assays are provided elsewhere [20] . complete testing coverage across viruses was largely maintained throughout the study period. however, high frequencies of partial testing did arise due to the burden placed on laboratory resources during the major waves of a(h1n1)pdm09 virus circulation. the laboratory protocols were consistent throughout the study period, with the exception of the rv assay which was modified during 2009 to detect a wider array of rv and enteroviruses (including d68), and the cov-hku1 assay which was discontinued in 2012. for each of the 61 427 clinical samples, positive/negative pcr test results were recorded by the laboratory for each virus group. information was also provided on the sampling date, patient's age at sampling, gender, and the origin of the sample [whether the patient had attended a general practice (gp), hospital outpatient or non-critical-care inpatient services, or was admitted to a critical care ward]. in the case of inconclusive/absent test results or other patient information, the corresponding data were coded as missing. all patient identifiers were anonymized. of the 40 962 patients, 8394 had multiple samples submitted for virological testing during the study period (range 1-37 samples, median 1, s.d. = 1·22). for 70% of these patients, the samples were received within a 30-day window. we aggregated the pcr test results to within this time-frame generating single 'episodes' of respiratory illness, using the collection date of the first sample when assigning temporal information. episodes were classified as positive for a given virus if at least one sample tested positive. following data exclusions, 44 230 patient episodes, representing 36 157 individual patients, were retained for analysis of temporal distributions. we conducted descriptive statistical analyses of viral infection prevalence in the patient population providing time-and agestratified estimates. by the end of april 2009, scotland was afflicted by the influenza pandemic [20] . figure 1a highlights the resultant upsurge in testing frequencies during the summer and autumn waves of 2009, and during a third wave of a(h1n1)pdm09 virus circulation in the winter of 2010/2011. during these periods, testing was primarily directed towards iav and only subsets of iav-negative patients were tested for other viruses. due to this disruption in regular testing procedures, we focused our description of viral infection distributions across patient subgroups on the 26 974 patient episodes tested outside this period, and refer readers to a previous report for details of viruses detected during the 2009 pandemic [20] . for each virus group, we compared the frequency of mono-infection episodes (one virus group detected) and co-infection episodes (more than one virus group detected). to correctly classify episodes into these subgroups, we excluded all partially tested patients. in more detailed analyses, we counted the frequency of each possible virus pair and quantified the statistical correlation between mono-infection and co-infection frequencies across viruses. we investigated statistical associations between time period, season, patient age, gender, and gp/general hospital/critical care origin (a proxy for illness severity), and two outcomes: (i) virus-positive vs. virusnegative episodes, and (ii) co-infection vs. monoinfection episodes. with respect to time, we split sampling dates into two major periods either side of the influenza pandemic and periods of high partial testing: pre-pandemic [prior to may 2009 when the a(h1n1)pdm09 virus was established in scotland] and post-pandemic [following subsidence of the third major wave of the a(h1n1)pdm09 virus in january 2011]. associations with each factor were first assessed by crude unadjusted odds ratios, and then adjusted for confounding using multivariable logistic regression models that included all factors to assess their independence. statistical interactions were examined using mantel-haenszel stratification methods (based on p < 0·05, results not shown). the potential interactions were added to the main effects models and their significance assessed based on an interaction parameter p < 0·05. model fit was assessed by le cessie van houwelingen global goodness-of-fit tests [21] . all statistical analyses were carried out in r v. 3·1·1 [22] . to correctly classify patients into outcome groups, all partially tested patients were excluded. of the 36 157 fully tested patients, 90% sought healthcare facilities once during the study period thereby contributing a single episode. however, 4218 patients had attended healthcare facilities more than once, providing information for multiple episodes (range 2-26 episodes, median 2, s.d. = 2·04). we retained the first observed episode per patient in the statistical analyses to ensure the patient-level interpretation of statistical associations was not influenced by the nonindependence of data relating to the same individual. see supplementary figure s1 for full details of data preparation. we analysed 44 230 episodes of respiratory illness tested by wossvc during 2005 to 2013. full details of patient distributions across subgroups and per study year are provided in supplementary table s1. patients' median age was 27 years (range 0-98 years, s.d. = 25·5 years) and 49% were male. excluding the three major waves of influenza a(h1n1)pdm09 virus circulation, episode frequencies increased year-by-year from 2472 cases tested in 2005 to 6149 cases tested in 2013. however, the age patterns were not consistent over this period; the percentage of adult episodes was greater in 2013 than in 2005 (e.g. 21% vs. 8% in patients aged 565 years), while the percentage of child episodes was fewer in 2013 than 2005 (e.g. 16% vs. 26% in patients aged 1-5 years) ( fig. 1b) . at least one virus was detected in 35% (15 302/44 230) of tested patients; these patients had a median age of 17 years (range 0-96 years, s.d. = 25 years) and 49% were male. the prevalence of confirmed viral infection in the patient population was greater in the 2013 influenza season than in 2005 in all age groups (fig. 1c) ; the absolute difference in prevalences was 22% (infants aged <1 year), 12% (1-5 years), 14% (6-16 years), 18% (17-45 years), 12% (46-64 years) and 17% (565 years). overall virus-specific prevalences in the patient population were ranked as follows: rv (14%, n = 4847); iav (9·7%, n = 4244); rsv (4·9%, n = 1786); cov (4·1%, n = 1339); adv (3·6%, n = 1221); ibv (3%, n = 1019); mpv (2·6%, n = 345); piv-3 (2·2%, n = 757); piv-4 (0·86%, n = 286); piv-1 (0·84%, n = 295) and piv-2 (0·35%, n = 122). age distributions for each viral infection group are presented in supplementary table s2 . the most common infection in each 6-month period (excluding 2009) was rv, constituting a low of 19% of infections during the typical influenza period of 2005/2006, to a high of 59% during the typical non-influenza period of 2010 (fig. 1d) . for most virus groups, detections were most frequent in the 1-5 years group (with the exception of iav, ibv and cov), males, and hospital attendees not admitted to a critical care ward (fig. 2) . seasonally, virus detections were most common in december (45% in gp attendees, 43% in hospital attendees) and least common in august (11% in gp attendees, 22% in hospital attendees) (fig. 3a,c) . the most commonly detected viral infection in each month was rv, peaking in september in both gp and hospital attendees (fig. 3b,d) . influenza a and b were the most common detections in december-march in gp attendees (combined proportion: range 31-45%), and in january-february in hospital attendees (combined proportion of 30%). of the remaining non-influenza viral infections, a large proportion was attributed to rsv, rv and cov during periods of high influenza activity; their combined proportions ranged from 39% to 52% in gp attendees (december-march) and from 51% to 55% in hospital attendees (january-february). of 9094 positive patients (in 26 974 patients outside of the pandemic period), 1952 were gp attendees, 6560 were general hospital attendees (outpatients and non-critical-care inpatients), and 1282 were inpatients admitted to a critical care ward [an intensive care unit (icu), intensive therapy unit (itu), high dependency unit (hdu), or coronary care unit (ccu)]. the latter group provided a proxy for classifying episodes of severe respiratory illness. eighty-eight percent (n = 4443) of gp attendees and 69% (n = 15 027) of hospital attendees were aged >5 years. as shown in figure 4 , the prevalence of severe episodes in all viruspositive patients, regardless of origin, was greater in patients with rv (7·5%), rsv (7·5%), piv1 (11·8%) and piv4 (7·4%) infections than in virus-negative patients or other viral infections including iav (5·5%) and ibv (4·1%). investigating further the rv/ iav and rv/piv1 comparisons, we found the observed difference in prevalence was statistically significant based on pearson's χ 2 tests (p = 0·036 and p = 0·05, respectively). age-specific prevalence of severe episodes was greatest at the extremes of age (<5 and 565 years) for all viruses except hpiv2 (we note the particularly small sample size for this virus group). of 9654 virus-positive patients from 27 284 episodes tested for all 11 viruses, 11% (1086/9654) had a co-infection. the median age in co-infected patients was 3 years (range 0-91 years, s.d. = 22 years) and 58% were male. co-infections were more commonly detected in those aged ≤5 years overall (18% compared to 7% in the >5 years group) and for each viral infection, particularly rv, rsv, adv and cov (detected in 6%, 3%, 3% and 2% of these infections, respectively, in those aged >5 years) (fig. 5a,b) . a total of 1389 virus pairs were detected in 1086 episodes of co-infection; most episodes involved two viruses (87%, 964/1086), the remaining involved three (n = 105), four (n = 15) and five (n = 2) viruses. all viruses were detected with most others at least once (fig. 5c) ; however, a clustering pattern was evident in which rv, adv, rsv and cov were frequently detected with one another. the most common virus detection in a co-infection was rv (56% of coinfections), the majority of which were with adv (n = 195, 25%) and rsv (n = 181, 23%). other viruses relatively frequently detected in co-infections were adv, rsv and cov; constituting 31%, 30% and 28% of co-infections, respectively. we found a significant positive correlation between virus detection frequencies in mono-infections and coinfections [pearson's product-moment correlation = 0·88 (95% ci 0·60-0·97, p < 0·001) and fitted linear regression model slope = 0·85 (p < 0·001)] (fig. 5d) . however, iav and ibv were identified in co-infections at relatively low frequencies (n = 121 and n = 68, respectively) compared to non-influenza viruses (e.g. rv, n = 678) (fig. 5d ). table 1 summarizes the results of univariable and multivariable logistic regression analyses for associations with viral infection. season, age group, and patient origin were significantly associated with the odds of viral infection based on unadjusted odds ratio estimates. in the multivariable analysis, several independently significant factors were identified based on the adjusted odds ratios. viral respiratory infections were more likely to be detected in winter, in children aged 1-5 years, and in gp attendees, irrespective of the other factors. following adjustment for multiple factors, time period was also a significant predictor (because of a negative confounding by age): the odds of viral infection were significantly greater postpandemic than pre-pandemic. significant statistical interactions (based on p < 0·05) revealed that the effect of age was not homogeneous across gender or patient-origin subgroups. this variation in age association across other factors is shown in figure 6a ,b where age-specific infection prevalences are stratified by the third factor. these figures show that the age distribution of infection differed according to gender and patient-origin subgroups. table 2 summarizes the results of univariable and multivariable logistic regression analyses for associations with co-infection. several differences were found in comparison with viral infections overall. based on unadjusted odds ratio estimates, time period, season (autumn only), age group, gender and patient origin were significantly associated with co-infection. however, in the multivariable analysis time period and gender were confounded by age and were therefore not identified as significant independent factors. in contrast to viral infection overall, co-infections were equally likely to be detected in spring and winter, were less likely to be detected in the 1-5 years age group than infants, and were more likely to be detected in general hospital attendees (outpatients and those not admitted to critical care wards) than gp attendees. significant statistical interactions (based on p < 0·05) revealed that the effect of age on co-infection status was not homogeneous across gender and patient-origin groups. in contrast to viral infection overall, co-infections were relatively more common in males than females in those aged 46-64 years and hospital attendees in all age groups (fig. 6c-d) . there was no evidence of a poor model fit based on the global goodness-of-fit tests: (i) p values = 0·147, 0·07, 0·07 for the main effect model and two models with interaction terms, respectively, for associations with viral infection overall, and (ii) p values = 0·940, 0·985, 0·746 for the main effect model and two models with interaction terms, respectively, for associations with co-infection. the advent of multiplex pcr as part of routine diagnostics provides an unprecedented opportunity for studying the epidemiology of multiple respiratory viruses simultaneously within a single population. previous uk-based studies have highlighted the utility of laboratory-based surveillance for monitoring respiratory infection trends, and in comparing the relative burdens between viruses [10, 13, 23] . our study is the first to compare the epidemiologies of different respiratory virus groups utilizing extensive diagnostic data generated by multiplex rt-pcr from patients attending both primary and secondary healthcare services. the collation of test-negative results by diagnostic laboratories provides valuable denominator information for measuring disease occurrence, to estimate the relative contribution of different pathogens to healthcare usage (such as gp consultations) and to provide an early warning for periods of increased healthcare pressures. importantly, the diagnostic test data utilized in this study were generated by a single laboratory, permitting a more consistent comparison of trends across patient and virus groups because testing methods were on the whole standardized throughout the study. our study has revealed changes in the frequency of virological testing of respiratory illnesses in the nhsggc health board during 2005-2013, with adults representing an increasingly greater percentage of episodes. however, age-specific prevalences were greater in the 2013 influenza season than in 2005 for all age groups. it is possible that there is raised awareness in the public and/or clinicians, and consequently greater healthcare seeking and/or sampling behaviour in adults. alternatively these results could reflect a true increase in non-viral causes of respiratory illness in this age group. we note that a shift in the demography of the glasgow population has been reported [24] . our observations might indicate the impact of an ageing population on respiratory-related healthcare services, through an increase in gp/hospital consultations, or a genuine increase in the incidence of adult respiratory infections. rv was the most prevalent virus overall, corroborating previous uk-based studies that include patients attending both primary and secondary healthcare services [10, 12] . the clinical significance of rv is disputed, although severe cases of disease are recognized depending on virus species, patient subgroups, and season [7, [25] [26] [27] . in additional analyses (fig. 4) we found the prevalence of severe respiratory illness (patients located in critical care wards) was significantly greater in rv infections than iav, supporting the proposition that rv is associated with more severe disease than traditionally accepted. of the other non-influenza viruses, rsv and cov were relatively highly prevalent. we note that the extent of research into the commonly circulating covs is small compared to iav and rsv, although severe clinical cases are recognized [28] . our study is the first comparative analysis in the uk to include cov, providing an important opportunity to quantify its temporal and patient subgroup distributions and co-infection patterns in comparison to the other common virus groups. we confirm that cov contributes a large fraction of infections during periods of high influenza activity and that cov is relatively frequently co-detected with other viruses. the contribution of different respiratory viruses to the healthcare burden in scotland has previously been studied [23] . further investigation on a seasonal basis is needed to help elucidate the public health relevance of rv and cov, particularly since cov has a similar age distribution as the influenza viruses. the remaining viruses (adv, mpv, piv1-4) were detected in comparatively smaller numbers on a yearly basis and during months of high influenza activity. the 9-year study period provided a novel opportunity to compare the epidemiology of respiratory viruses before and after the 2009 influenza a pandemic [18] . in our multivariable statistical analysis we found viral infections to be more likely in the post-pandemic era. this result was independent of other factors such as patient's age implying non-patient factors, such as a change in the underlying virus population, have increased the likelihood that a patient seeking healthcare services will have a viral infection (as opposed to non-viral causes). whether this is a direct consequence of the pandemic virus, its impact on the epidemiologies of others viruses, or a consequence of long-term changes in the non-influenza virus population, remains to be elucidated. seasonal and patient-related factors corroborate existing knowledge and were independent of time, indicating the generality of these factors as predictors of viral infection. it is well recognized that the burden of viral respiratory illness lies predominantly in young children [29] . we found that in patients with respiratory illness attending healthcare facilities, those aged 1-5 years were more likely than other age groups to have a viral infection independent of season or time period. the most commonly detected viruses in this age group were rv, rsv, adv and mpv (20%, 9·3%, 9·1% and 4·7% of infections, respectively) corroborating previous reports [23, 30] . together with a recent study that found bacterial-viral co-infections were relatively uncommon in children with pneumonia [31] , these findings support the concern regarding the overprescription of antibiotics in children [32] . that the increasing trend in virus prevalence was most notable in infants (<1 year) also warrants further attention. while it is possible that these findings are influenced by changes in clinical testing decisions, we note that this trend is particularly pertinent in relation to recent european outbreaks of enterovirus d68 in children [33] ; investigation into the contribution of individual viruses will be the focus of future work. we further note that, based on the multivariable statistical analyses, the increasing trend in prevalence in children explained why co-infections were more likely detected in the post-2009 pandemic era. there are very few studies describing co-infection patterns in respiratory viruses. our study provides the largest examination to date, confirming that around 11% of viral infections in patients attending healthcare services in an urban setting involve more than one virus, similar to the 10·4% reported by a previous uk-based study [12] . that nearly all respiratory viruses were co-detected with all other viruses highlights the sufficient opportunities for co-infections. we would expect co-infection frequencies to reflect individual virus prevalences. indeed, in line with the aforementioned study [12] , rv was the most common detection in co-infections, rv/rsv was a frequent pairing, and most co-infections were in children aged <5 years. our study also reveals that covs are relatively frequently involved in co-infections. however, co-infections with influenza viruses were relatively few, perhaps explained by differences in their age and seasonal distributions, or an inter-viral interference [34] . we found that the average age of co-infection was 3 years, compared to 17 years for viral infections overall, and co-infections were more likely in infants than in those aged 1-5 years. that co-infections were more likely in young children is probably explained by (i) a greater opportunity for co-infection due to a shorter exposure lifetime and consequently greater susceptibility to a wider array of viruses, and (ii) a greater chance of co-infections being detected because children tend to shed virus for longer periods. in adults, the age distribution of co-infections differed according to gender and patient origin; the prevalence was greatest in males and in general hospital attendees not admitted to critical care wards for those aged 46-64 years (fig. 6c,d) . this result provides insight into an age-dependent factor in co-infection patterns in adults but must be viewed with some caution; it is potentially influenced by a bias in multiple specimens submitted in relation to single episodes of illness in adults, most likely as a result of comorbidities. interestingly, co-infections were more likely in general hospital attendees not admitted to critical care wards than gp attendees, supporting the potential role of co-infections in illness severity [35] . there are several limitations to our study to be noted. detection of viral nucleic acid may not represent active infection for all viruses in all cases [36] , potentially introducing detection biases temporally and across patient groups. furthermore, the timing of infection events, and variation in shedding duration across virus and patient groups [37, 38] , could potentially bias the observed co-infection patterns. we also note that our study lacked information on the presence/absence of bacterial pathogens which are also significant contributors to respiratory infections. one further important consideration is that laboratory diagnostic data cannot inform on the epidemiology of asymptomatic infections in the community, or in symptomatic people who do not attend healthcare services. furthermore, that viral populations are not static could also impact on the generalisability of the observed trends and associations; the introduction of new strains can alter disease outcomes, and consequently healthcare seeking behaviour, influencing the stability of healthcare consultation rates in patient subgroups. given the dynamic nature of virus populations, the epidemiological information generated through surveillance must be maintained to ensure future vaccine and antiviral developments are directed to where they are most needed [39, 40] . 1·15 (0·75-1·79, p = 0·521) or, odds ratio; ci, confidence interval. * distribution of patient numbers, with corresponding % in parentheses, across factor levels for all patients (summary) and for co-infection and mono-infection groups. † unadjusted or based on univariable logistic regression. ‡ adjusted or based on multivariable logistic regression. § patients' location corresponding with first clinical sample: gp, general practitioner's surgery; hospital (general), outpatients and non-critical-care patients; hospital (critical care), patients admitted to an intensive care, intensive therapy, high dependency, or coronary care unit. our study provides the most comprehensive description of viral respiratory infections in the uk to date, revealing new epidemiological insights with public health relevance. of particular concern is a greater viral prevalence in 2013 compared to 2005, particularly in infants, and a greater risk of viral infection in the post-2009 pandemic era. further investigation into the long-term temporal dynamics of individual viruses and the epidemiological consequences of virus cocirculation is needed. for supplementary material accompanying this paper visit http://dx.doi.org/10.1017/s0950268816000339. global burden of respiratory infections due to seasonal influenza in young children: a systematic review and meta-analysis global burden of acute lower respiratory infections due to respiratory syncytial virus in young children: a systematic review and meta-analysis disability-adjusted life years (dalys) for 291 diseases and injuries in 21 regions, 1990-2010: a systematic analysis for the global burden of disease study viruses and bacteria in the etiology of the common cold adults hospitalised with acute respiratory illness rarely have detectable bacteria in the absence of copd or pneumonia; viral infection predominates in a large prospective uk sample investments in respiratory infectious disease research 1997-2010: a systematic analysis of uk funding association between human rhinovirus c and severity of acute asthma in children human metapneumovirus: review of an important respiratory pathogen newly discovered coronavirus as the primary cause of severe acute respiratory syndrome a new laboratory-based surveillance system (respiratory datamart system) for influenza and other respiratory viruses in england: results and experience from seasonal variations of 15 respiratory agents illustrated by the application of a multiplex polymerase chain reaction assay single, dual and multiple respiratory virus infections and risk of hospitalization and mortality assessing the burden of influenza and other respiratory infections in england and wales epidemiology characteristics of respiratory viruses found in children and adults with respiratory tract infections in southern china viral etiologies of hospitalized acute lower respiratory infection patients in china epidemiology and viral etiology of the influenza-like illness in corsica during the 2012-2013 winter: an analysis of several sentinel surveillance systems impact of the 2009 h1n1 pandemic on age-specific epidemic curves of other respiratory viruses: a comparison of pre-pandemic, pandemic and postpandemic periods in a subtropical city during the summer 2009 outbreak of 'swine flu' in scotland what respiratory pathogens were diagnosed as h1n1 a goodness-of-fit test for binary regression models, based on smoothing methods r: a language and environment for statistical computing. r foundation for statistical computing disease burden of the most commonly detected respiratory viruses in hospitalized patients calculated using the disability adjusted life year (daly) model population trends by age group in glasgow human rhinovirus species and season of infection determine illness severity clinical features and complete genome characterization of a distinct human rhinovirus (hrv) genetic cluster, probably representing a previously undetected hrv species, hrv-c, associated with acute respiratory illness in children the significance of rhinovirus detection in hospitalized children: clinical, epidemiological and virological features epidemiology and clinical presentations of the four human coronaviruses 229e, hku1, nl63, and oc43 detected over 3 years using a novel multiplex real-time pcr method respiratory viral infections in infants: causes, clinical symptoms, virology, and immunology respiratory viral infections during the 2009-2010 winter season in central england, uk: incidence and patterns of multiple virus co-infections community-acquired pneumonia requiring hospitalization among u.s. children respiratory virus infections in febrile children presenting to a general practice out-of-hours service emergence and epidemic occurrence of enterovirus 68 respiratory infections in the netherlands in 2010 do rhinoviruses reduce the probability of viral co-detection during acute respiratory tract infections? single and multiple respiratory virus infections and severity of respiratory disease: a systematic review clinical utility of pcr for common viruses in acute respiratory illness influence of age, severity of infection, and co-infection on the duration of respiratory syncytial virus (rsv) shedding epidemiology of multiple respiratory viruses in childcare attendees emerging respiratory viruses: challenges and vaccine strategies respiratory viruses other than influenza virus: impact and therapeutic advances the authors are grateful to dominic mellor, emma thomson, louise matthews and richard reeve for their helpful critique of the manuscript and discussions. a subset of the clinical samples was provided by health protection scotland as part of the scottish enhanced respiratory viral infection surveillance programme.this work was supported by the medical research council uk (grant g0801822). none. key: cord-355165-xc6ythgp authors: van den wijngaard, cees; van asten, liselotte; van pelt, wilfrid; nagelkerke, nico j.d.; verheij, robert; de neeling, albert j.; dekkers, arnold; van der sande, marianne a.b.; van vliet, hans; koopmans, marion p.g. title: validation of syndromic surveillance for respiratory pathogen activity date: 2008-06-17 journal: emerg infect dis doi: 10.3201/eid1406.071467 sha: doc_id: 355165 cord_uid: xc6ythgp syndromic surveillance is increasingly used to signal unusual illness events. to validate data-source selection, we retrospectively investigated the extent to which 6 respiratory syndromes (based on different medical registries) reflected respiratory pathogen activity. these syndromes showed higher levels in winter, which corresponded with higher laboratory counts of streptococcus pneumoniae, respiratory syncytial virus, and influenza virus. multiple linear regression models indicated that most syndrome variations (up to 86%) can be explained by counts of respiratory pathogens. absenteeism and pharmacy syndromes might reflect nonrespiratory conditions as well. we also observed systematic syndrome elevations in the fall, which were unexplained by pathogen counts but likely reflected rhinovirus activity. earliest syndrome elevations were observed in absenteeism data, followed by hospital data (+1 week), pharmacy/general practitioner consultations (+2 weeks), and deaths/laboratory submissions (test requests) (+3 weeks). we conclude that these syndromes can be used for respiratory syndromic surveillance, since they reflect patterns in respiratory pathogen activity. syndromic surveillance is increasingly used to signal unusual illness events. to validate data-source selection, we retrospectively investigated the extent to which 6 respiratory syndromes (based on different medical registries) refl ected respiratory pathogen activity. these syndromes showed higher levels in winter, which corresponded with higher laboratory counts of streptococcus pneumoniae, respiratory syncytial virus, and infl uenza virus. multiple linear regression models indicated that most syndrome variations (up to 86%) can be explained by counts of respiratory pathogens. absenteeism and pharmacy syndromes might refl ect nonrespiratory conditions as well. we also observed systematic syndrome elevations in the fall, which were unexplained by pathogen counts but likely refl ected rhinovirus activity. earliest syndrome elevations were observed in absenteeism data, followed by hospital data (+1 week), pharmacy/general practitioner consultations (+2 weeks), and deaths/laboratory submissions (test requests) (+3 weeks). we conclude that these syndromes can be used for respiratory syndromic surveillance, since they refl ect patterns in respiratory pathogen activity. e arly warning surveillance for emerging infectious disease has become a priority in public health policy since the anthrax attacks in 2001, the epidemic of severe acute respiratory syndrome in 2003, and the renewed attention on possible infl uenza pandemics. as a result, new surveillance systems for earlier detection of emerging infectious diseases have been implemented. these systems, often labeled "syndromic surveillance," benefi t from the increasing timeliness, scope, and diversity of health-related registries (1) (2) (3) (4) (5) (6) . such alternative surveillance uses symptoms or clinical diagnoses such as "shortness of breath" or "pneumonia" as early indicators for infectious disease. this approach not only allows clinical syndromes to be monitored before laboratory diagnoses, but also allows disease to be detected for which no additional diagnostics were requested or available (including activity of emerging pathogens). our study assessed the suitability of different types of healthcare data for syndromic surveillance of respiratory disease. we assumed that syndrome data-to be suitable for early detection of an emerging respiratory disease-should refl ect patterns in common respiratory infectious diseases (7) (8) (9) (10) . therefore, we investigated the extent to which time-series of respiratory pathogens (counts per week in existing laboratory registries) were refl ected in respiratory syndrome time-series as recorded in 6 medical registries in the netherlands. we also investigated syndrome variations that could not be explained by pathogen counts. as an indication for syndrome timeliness, we investigated the delays between the syndrome and pathogen time-series. we defi ned syndrome data as data in health-related registries that refl ect infectious disease activity without identifying causative pathogen(s) or focusing on pathogenspecifi c symptoms (such as routine surveillance data for infl uenza-like illness [11] or surveillance of acute fl accid paralysis for polio [12] ). registries for syndrome data were included if they met the following criteria: 1) registration on a daily basis; 2) availability of postal code, age, and sex; 3) availability of retrospective data (>2 years); and 4) (potential) real-time data availability. six registries were selected ( table 1 ) that collected data on work absenteeism, general practice (gp) consultations, prescription medications dispensed by pharmacies, diagnostic test requests (laboratory submissions) (13), hospital diagnoses, and deaths. in all registries, data were available for all or a substantial part of 1999-2004. for the gp, hospital, and mortality registry, defi nition of a general respiratory syndrome was guided by the case defi nitions and codes found in the international classifi cation of diseases, 9th revision, clinical modifi cation (icd-9-cm), as selected by the centers for disease control and prevention (atlanta, ga, usa) (www.bt.cdc.gov/surveillance/syndromedef). for the laboratory submissions and the pharmacy syn-drome, we selected all data that experts considered indicative of respiratory infectious disease (for detailed syndrome defi nitions, see online technical appendix, available from www.cdc.gov/eid/content/14/6/917-techapp.pdf). as a reference for the syndrome data, we included specifi c pathogen counts for 1999-2004 from the following sources: 1) weekly sentinel surveillance system of the dutch working group on clinical virology (which covers 38%-73% of the population of the netherlands [14] respiratory disease-related counts of streptococcus pneumoniae (data in 2003-2004 were interpolated for 2 laboratories during short periods of missing data; total coverage 24%); and 3) national mandatory notifi cations of pertussis. the networks for respiratory pathogen counts are other networks than the earlier described laboratory submissions network for syndrome data. data were aggregated by week and analyzed by using sas version 9.1 (sas institute inc., cary, nc, usa). for the gp, pharmacy, and laboratory submissions registries, we expressed the respiratory counts as a percentage of total weekly counts to adjust for the infl uence of holidays and, for laboratory submissions, changes in the number of included laboratories over time. by looking at the graphs, we explored the relationship between the time-series of respiratory pathogens and syndromes and calculated pearson correlation coeffi cients. to investigate whether the respiratory syndromes refl ect patterns in respiratory pathogen counts, we constructed multiple linear regression models. these models estimated respiratory syndrome levels at a certain time with, as explanatory variables, the lagged (range of -5 to +5 weeks) pathogen counts as explanatory variables. we used linear regression of the untransformed syndrome to estimate the additive contributions of individual pathogens to the total estimated syndrome. we assumed a constant syndrome level attributable to factors other than the respiratory pathogens and constant scaling factors for each of the lagged pathogens. a forward stepwise regression approach was used, each step selecting the lagged pathogen that contributed most to akaike's information criterion of model fi t (15) . each pathogen entered the model only once and only if it contributed signifi cantly (p<0.05). negative associations (e.g., between enteroviruses, which peak in summer, vs. respiratory syndromes, which peak in winter) were excluded to avoid noncausal effects. to discriminate between primary and secondary infections by s. pneumoniae (as a complication of respiratory virus infection) (16) (17) (18) (19) , we used the residuals from regressing s. pneumoniae counts on other pathogens as the variable for s. pneumoniae (instead of its counts) for all the earlier described models for respiratory syndromes. we checked for autocorrelation in the residuals of the models with hierarchical time-series models (using splus 6.2) (20,21). we calculated r 2 values to estimate to what extent respiratory pathogen counts explain variations in syndromes. to explore to what extent seasonal variation could be a confounder, we also calculated r 2 values of the models after adding seasonal variables (sine and cosine terms) and r 2 values for seasonal terms alone. we also investigated the pathogen-specifi c effects in the models, by calculating the standardized parameter estimates before and after adding seasonal terms. the models were used to estimate the expected syndrome level with 95% upper confi dence limits (ucls). we considered distinct syndrome elevations that exceeded the ucls, as unexplained by the models (for model details, see online technical appendix). we investigated the timeliness of the registry syndromes in 2 ways: 1) as a measure of differences in timeliness between registries, we evaluated the time delays of the syndromes relative to each other by calculating for each of the syndromes the time lag that maximized pearson correlation coeffi cient with the hospital registry (as a reference); 2) by estimating the time delays between each of the syndromes and the lagged pathogens included in its regression model. respiratory syndrome time series were plotted for all registries (figure 1 ). the christmas and new year holidays coincided with peaks and dips in the pharmacy and absenteeism syndromes (not shown). because these results were probably artifacts, we smoothed these yearly peaks and dips and censored them in the analyses performed on the absenteeism registry, in which they had a strong infl uence on outcomes. for all registries, the respiratory syndromes demonstrated higher levels of activity in winter, which overlapped or coincided roughly with the seasonal peaks of infl uenza a, infl uenza b, rsv, and (albeit less pronounced) s. pneumoniae laboratory counts ( figure 1 ). infections with parainfl uenza virus, m. pneumoniae, adenovirus, and rhinovirus were detected slightly more frequently during winter (data not shown). bordetella pertussis and enterovirus showed seasonal peaks only in summer (data not shown). the seasonal peaks in laboratory counts of infl uenza a, infl uenza b, and rsv corresponded with peaks in the gp, pharmacy, and hospital syndromes. other syndromes did have less obvious correspondence. each year, around october, the respiratory syndrome showed a peak in the gp we calculated pearson correlation coeffi cients between the different unlagged time series of respiratory pathogens and syndromes (table 2) . syndrome time series in all reg-istries correlated strongly with s. pneumoniae (unadjusted total counts). the hospital, gp, pharmacy, and laboratory submissions data strongly correlated with rsv and infl uenza a counts (table 2) . mortality data correlated strongly with infl uenza a (r = 0.65) and infl uenza b (r = 0.50) infections. the highest correlations between pathogen time series were between s. pneumoniae and the other pathogens (up to 0.51 with infl uenza a, table 3 ). table 4 presents, for each registry, the time lag (in weeks) that maximized the model fi t of regressing syndrome on pathogens. for the gp, hospital, mortality, and pharmacy data, the respiratory pathogens explained the syndrome variation very well (78%-86%). variations in the absenteeism syndrome could be explained for 68% by variations in the pathogen counts. although the laboratory submissions syndrome had the lowest explained variance, still 61% of the variations in this syndrome were explained by variations in pathogen counts. hierarchical time-series models did not show signifi cant autocorrelation in the residuals of the models with pathogen counts as explanatory variables (20, 21) . when seasonal terms were added to the model, the variations in the mortality syndrome were just as well ex-920 emerging infectious diseases • www.cdc.gov/eid • vol. 14, no. 6, june 2008 plained as by the model with only pathogen counts (table 5 ; r 2 remains 78%), while by the model with only seasonal terms, the explained variance was much lower (only 52%, table 5 ). for the hospitalizations, laboratory submissions, and gp data, only slightly more syndrome variation was explained by adding seasonal terms. with only seasonal terms, the explained variance for these syndromes was clearly lower than with only pathogens in the models (8%-11% lower, table 5 ). however, for the absenteeism and, to a lesser extent, the pharmacy data, the model with both pathogen and seasonal terms clearly explained more syndrome variations (table 5 , absenteeism 68% vs. 80%; pharmacy 80% vs. 87%). furthermore, for the absenteeism data, the model with only seasonal terms had an even higher r 2 than the model with only pathogens, whereas for the pharmacy data, the r 2 with only seasonal terms was only slightly lower (3%, table 5 ). table 6 shows that for mortality, hospitalizations, laboratory submissions, and gp data, the pathogens with the highest effect clearly were rsv, infl uenza a, and infl uenza b, with no or only modest decline in standardized parameter estimates after adding seasonal terms. for the gp and hospital data, some pathogens became insignifi -cant after seasonal terms were added (gp: rhinovirus and adenovirus; hospital: parainfl uenza virus). for the pharmacy data, half of all pathogen variables became insignifi cant after seasonal terms were added, whereas for the absenteeism data, almost all pathogens became insignificant (table 6) . several syndrome observations exceeded the 95% ucls of the models (0-10/registry/year), which indicates that those syndrome observations deviated strongly from model predictions. the recurrent elevation in october of the absenteeism, gp, and pharmacy syndrome several times exceeded the ucls (october 2001: pharmacy and gp; 2002: absenteeism; 2003: gp, absenteeism; not shown), which indicated that the model could not explain these elevations. in figure 2 , for each registry, the difference in timeliness with the hospital registry is indicated by the lag that maximizes r 2 . the absenteeism syndrome (green line) preceded the hospital syndrome by 1 week, followed by the gp-based and prescription-based syndromes at +1 week and the syndrome based on mortality and laboratory submission data at +2 weeks after the hospital syndrome (projected on x-axis, figure 2 ). the differences in timeliness between the syndromes and the pathogen surveillance data were refl ected by the regression models relating the syndromes to the (positive or negative) lagged pathogens (table 4 ). infl uenza a and infl uenza b had lags of 0-5 weeks, which suggests that the registry-syndromes were 0-5 weeks ahead of laboratory counts for these infections. fluctuations in the time series of respiratory hospitalizations and the laboratory rsv counts seemed to appear in the same week (lag = 0). all other syndromes appeared to be 1-3 weeks later than the rsv counts, except absenteeism, which is 2 weeks earlier. again, absenteeism seemed to be the earliest syndrome (2-5 weeks earlier than rsv, infl uenza a, and infl uenza b), followed by the hospital syndrome (0-2 weeks earlier), the gp-based and prescription-based syndromes (2 weeks earlier until 1 week later), the laboratory submission syndrome (1 week earlier until 2 weeks later), and the mortality syndrome (0-3 weeks later than rsv, infl uenza a, and infl uenza b). we explored the potential of 6 dutch medical registries for respiratory syndromic surveillance. although several other studies also evaluated routine (medical) data for syndromic surveillance purposes (22) (23) (24) (25) (26) (27) , most evaluated only 1 syndrome and correlated this only to infl uenza data. an exception is bourgeois et al. (24) , who validated a respiratory syndrome in relation to diagnoses of several respiratory pathogens in a pediatric population, and cooper et al. (27) , who estimated the contribution of specifi c respiratory pathogens to variations in respiratory syndromes. both studies concluded that rsv and infl uenza explain most of the variations in these syndromes, consistent with our fi ndings. our study shows that all syndrome data described in this study showed higher levels in winter, which corresponded to the seasonal patterns of rsv, s. pneumoniae, and infl uenza a and b viruses. linear regression showed that the syndromes can be explained by lagged laboratory counts for respiratory pathogens (up to 86%, highest effect of infl uenza a, infl uenza b, and rsv), which indicates their potential usefulness for syndromic surveillance. timeliness differed, with up to 5 weeks potential gain in early warning by syndromic data, compared with routine laboratory surveillance data. a limitation of our study is the short duration of our time series, especially for absenteeism and pharmacy data. therefore, whether our observed associations between syndromes and pathogen counts can be generalized remains unclear. we relied on laboratory pathogen counts as a proxy for their prevalence and the illness they cause. changes in test volume over time would result in misclassifi cation bias (as noncausative pathogens will be detected as well). however, such changes are presumably dwarfed by changes during "truly" epidemic elevations of common respiratory pathogens. additionally, laboratory diagnostics are mostly performed on hospitalized patients, and thus results inadequately refl ect activity of pathogens that predominantly cause mild illness. by adding seasonal terms, we observed that for the absenteeism and, to a lesser extent, the pharmacy registry, the associations between the respiratory syndromes and the pathogen counts might be biased to some extent. for the gp, hospital, laboratory submission, and mortality data, 922 emerging infectious diseases • www.cdc.gov/eid • vol. 14, no. 6, june 2008 absenteeism 2 5 4 2 4 5 ---gp -1 1 2 -1 1 2 2 --3 pharmacy -1 0 2 0 2 5 2 -5 3 hospitalization 0 2 1 --2 3 ---laboratory submissions -2 0 1 -3 -2 -5 mortality 3 1 0 -----*s. pneumoniae, streptococcus pneumoniae; rsv, respiratory syncytial virus; rv, rhinovirus; piv, parainfluenza virus; gp, general practice; -, pathogen not included in model. †the lag time (in weeks) is indicated, that showed optimal fit between syndrome time-series and lagged pathogen counts included in the linear regression model; e.g., according to the model, the trend in hospitalizations precedes the influenza a laboratory counts by 2 weeks. season is probably not an important confounder for the association between the syndromes and pathogens, because including seasonal terms in the models resulted in the same or only slightly higher explained syndrome variance (measured by r 2 ). models with seasonal terms alone mostly had lower explained variance than the pathogen models. for the gp and hospital data, some pathogens became insignifi cant after seasonal terms were added (table 6 ) but not those pathogens with the largest effect estimates (rsv, infl uenza a and b). therefore, we are confi dent in concluding that the gp, hospital, laboratory submission, and mortality syndromes do refl ect pathogen activity suffi ciently for use in syndromic surveillance. the higher r 2 value of the absenteeism model with seasonal terms alone suggests seasonality of absenteeism caused by several nonrespiratory conditions (28, 29) . to some extent, this also applies to the pharmacy syndrome, which includes medications that are not specifi c for respiratory infections (e.g., antimicrobial drugs). this could be validated in future studies by linking medications to illness. however, for both the absenteeism and pharmacy syndromes, the variation explained by seasonal terms is probably overestimated to some extent because data for only 2 and 3 years were used. consequently, these time series contained less information on variation between different years than for the other registries, which benefi ts fi tting of a model with several sine and cosine terms. to our knowledge, laboratory submission data (test requests) have not been evaluated before as a data source for syndromic surveillance. the modest explained variance for the laboratory submissions syndrome could possibly refl ect the limited use in our country of laboratory testing algorithms, which leads to substantial differences in diagnostic regimes for patients with similar clinical symptoms. in addition, occasional extra alertness by clinicians can make these data unreliable for surveillance. for instance, an unusual peak was observed in the laboratory submissions syndrome in 1999, after the offi cial announcement of an outbreak of legionnaires' disease (30) . an unexpected increase was also observed in the absenteeism, gp, and pharmacy syndromes, which occurred consistently each year around october (2001) (2002) (2003) (2004) . these peaks preceded the syndrome peaks concurring with peaks in infl uenza a, infl uenza b, and rsv counts and may be caused by rhinovirus activity-and asthma exacerbations caused by rhinovirus-which usually rises in the fall (31) (32) (33) . rhinovirus might go undetected because gp physicians rarely ask for diagnostics if they suspect a nonbacterial cause for relatively mild respiratory disease. although (table 1) . measured by the syndrome lag with the maximized r 2 , the timeliness differed between the registries in the following order: absenteeism, hospital, pharmacy/general practice (gp), mortality/ laboratory submissions (as projected on the x-axis). specifi c asthma diagnoses were excluded from the respiratory syndrome defi nitions, exacerbations of asthma might affect other respiratory categories in the gp or pharmacy syndrome. this observation illustrates that additional diagnostics are needed for identifying the causes of unexplained respiratory disease elevations. several novel respiratory pathogens for which diagnostics are not yet widely available have been discovered in recent years, underlining that it is quite possible that "hidden" epidemics occur (34) (35) (36) . the extra october peak and several other syndrome elevations above the 95% ucls in our study may well refl ect such hidden epidemics. the fact that these occur is supported by studies showing that many individual syndrome cases cannot be linked to known pathogens. for example, cooper et al. (37) , who investigated syndromic signals by using patient self-sampling (at home), could only obtain diagnostic results for 22% of these cases. for early warning surveillance, timeliness is crucial. absenteeism data seem to have the best timeliness, but their lack of medical detail complicates interpretation. unexpectedly, the hospital data refl ect respiratory pathogen activity earlier than the gp data. although in the netherlands patients are encouraged to consult their gp before going to the hospital, elderly persons, for whom respiratory infections are more likely to cause severe illness, may often go to a hospital directly. therefore, hospital data may prove to be an earlier marker for respiratory disease than gp data, but this possibility needs further exploration. an important concern when using syndromic surveillance is that it may generate nonspecifi c alerts, which, if they happen regularly, would lead to lack of confi dence in a syndrome-based surveillance system. here, we see a clear advantage of using data from multiple registries in parallel so that signal detection can be made more specifi c by focusing on signals that occur concurrently in >1 data source. to illustrate this we defi ned every exceeding of the ucls of the regression models as a "signal," i.e., a syndrome elevation unexplained by known pathogen activity and therefore possibly refl ecting activity of underdiagnosed or emerging infectious disease. over 2002-2003 (the period that all 6 registries were in the study), only 5 "concurrent" signals occurred versus 34 "single" signals over all registries. we did not evaluate whether the syndromes indeed detect outbreaks of infectious diseases earlier than clinical or laboratory pathogen surveillance. such an evaluation is often performed by testing the ability to detect historical natural outbreaks or simulated outbreaks (10, 38) . however, historical natural outbreaks are rare and simulated outbreaks may be unrealistic. nevertheless, further research into the outbreak detection performance of these syndromes would be worthwhile. the results of this study suggest that it might be best to combine syndromic data and pathogen counts in a prospective surveillance system. such surveillance can identify distinct syndrome elevations that cannot be explained by respiratory pathogen activity as indicated by routine laboratory pathogen surveillance. overall, the gp, hospital, mortality and, to a lesser extent, laboratory submission syndromes refl ect week-toweek fl uctuations in the time-series of respiratory pathogens as detected in the laboratory. registries monitoring trends of these syndromes will therefore most likely refl ect illness caused by emerging or underdiagnosed respiratory pathogens as well and therefore are suited for syndromic surveillance. further research would be required to assess to what extent absenteeism and pharmacy data refl ect respiratory illness. investigating the actual outbreak detection performance of the syndromes in this study would also be worthwhile. data from the registries in this study are not yet realtime available, although given modern information technology, this availability is clearly feasible. our study can help prioritize which type of healthcare data to include in future syndromic real-time surveillance systems. syndromic surveillance and bioterrorism-related epidemics using automated medical records for rapid identifi cation of illness syndromes (syndromic surveillance): the example of lower respiratory infection surveillance of the bioterrorist threat: a primary care response evaluation of australia's national notifi able disease surveillance system experimental surveillance using data on sales of over-the-counter medications-japan syndromic surveillance in public health practice use of ambulance dispatch data as an early warning system for communitywide infl uenzalike illness public health assessment of potential biological terrorism agents use of automated ambulatory-care encounter records for detection of acute illness clusters, including potential bioterrorism events outbreak detection through automated surveillance: a review of the determinants of detection surveillance of respiratory pathogens and infl uenza-like illnesses in general practices-the netherlands, winter 1997-98 the global eradication of polio by the year automated, laboratory-based system using the internet for disease outbreak detection, the netherlands reporting virus diagnostics in the netherlands: representativeness of the virological weekly reports a new look at statistical model identifi cation respiratory viruses augment the adhesion of bacterial pathogens to respiratory epithelium in a viral species-and cell type-dependent manner enhanced adherence of streptococcus pneumoniae to human epithelial cells infected with respiratory syncytial virus association of invasive pneumococcal disease with season, atmospheric conditions, air pollution, and the isolation of respiratory viruses respiratory viral infection predisposing for bacterial disease: a concise review automated detection of infectious disease outbreaks: hierarchical time series models bayesian statistical instrument for trend detection and time-series modelling syndromic surveillance for infl uenzalike illness in ambulatory care network modeling emergency department visit patterns for infectious disease complaints: results and application to disease surveillance validation of syndromic surveillance for respiratory infections developing a national primary care-based early warning system for health protection-a surveillance tool for the future? analysis of routinely collected data medication sales and syndromic surveillance the contribution of respiratory pathogens to the seasonality of nhs direct calls seasonality of infectious diseases seasonal variation in cause-specifi c mortality: are there high-risk groups? 25-year follow-up of civil servants from the fi rst whitehall study a large outbreak of legionnaires' disease at a fl ower show, the netherlands rhinovirus infections in an industrial population. i. the occurrence of illness respiratory infections and the autumn increase in asthma morbidity a case-control study of acute respiratory tract infection in general practice patients in the netherlands cloning of a human parvovirus by molecular screening of respiratory tract samples identifi cation of a novel coronavirus in patients with severe acute respiratory syndrome identifi cation of a new human coronavirus linking syndromic surveillance with virological self-sampling systematic review: surveillance systems for early detection of bioterrorism-related diseases we thank daan notermans for his expert opinion on providing syndrome defi nitions; mariken van der lubben for reading and commenting on the manuscript; statistics netherlands (cbs, ingeborg deerenberg and john kartopawiro), the foundation for pharmaceutical statistics (sfk, jan-dirk kroon and fabiënne griens), the dutch national medical register (lmr, willem hoogen stoevenbeld), and the national information network of gps (linh, robert verheij) for providing data; and the members of the dutch working group on clinical virology for collecting and providing weekly positive diagnostic results.mr van den wijngaard is an epidemiologist at the center for infectious disease control at the national institute of public health and the environment (rivm), bilthoven. his main research interests include the use of healthcare data for infectious disease surveillance and monitoring. all material published in emerging infectious diseases is in the public domain and may be used and reprinted without special permission; proper citation, however, is required. key: cord-337562-pkejb0a9 authors: bialasiewicz, seweryn; lambert, stephen b.; whiley, david m.; nissen, michael d.; sloots, theo p. title: merkel cell polyomavirus dna in respiratory specimens from children and adults date: 2009-03-17 journal: emerg infect dis doi: 10.3201/eid1503.081067 sha: doc_id: 337562 cord_uid: pkejb0a9 merkel cell polyomavirus (mcpyv) dna was detected in 7 (1.3%) of 526 respiratory tract samples from patients in australia with upper or lower respiratory tract symptoms. partial t antigen and major capsid protein sequences of mcpyv identified in respiratory secretions showed high homology (99%–100%) to those found in merkel cell carcinoma. merkel cell polyomavirus (mcpyv) dna was detected in 7 (1.3%) of 526 respiratory tract samples from patients in australia with upper or lower respiratory tract symptoms. partial t antigen and major capsid protein sequences of mcpyv identifi ed in respiratory secretions showed high homology (99%-100%) to those found in merkel cell carcinoma. i n the past 2 years, several previously unknown human polyomaviruses (pyvs) have been identifi ed: ki virus (kipyv) (1) and wu virus (wupyv) (2) from respiratory samples, and more recently, merkel cell polyomavirus (mcpyv), most commonly from merkel cell carcinoma tissue (3) . mcpyv has been found in integrated and episomal states (3); however, a mode of transmission for mcpyv has not yet been proposed. in this study, we sought to identify mcpyv in respiratory specimens by using real-time pcr. this study was conducted exclusively at the sir albert sakzewski virus research centre, royal children's hospital and health service, brisbane, queensland, australia. the necessary ethical approval for this study was obtained from the ethics committee of the royal children's hospital. specimens tested in this study (n = 526) were collected from january through december 2003 from 418 pediatric patients (birth to 14 years of age) (n = 450; age range 3 days-13.5 years; mean 1.7 years; median 0.8 years) and 71 adult patients (n = 76; age range 14.3 years-80.1 years; mean 47.1 years; median 52.5 years) who were hospital-ized or sought treatment at hospitals in queensland, australia, for upper or lower respiratory tract symptoms. most (95.6%) samples were nasopharyngeal aspirates (npas); the remainder were bronchoalveolar lavage specimens, bronchial washing specimens, and endotracheal aspirates. these samples were collected as part of a previous study that tested for infl uenza viruses a and b, adenoviruses, human metapneumovirus, respiratory syncytial virus, and parainfl uenza viruses 1, 2, and 3, in addition to kipyv and wupyv (4) . samples were screened by using an mcpyv realtime pcr that was specifi c for the vp2/3 region. briefly, 10 pmol of each primer mcpyv-2.0-4367f (5′-ggcagcatcccggctta-3′) and mcpyv-2.0-4399r (5′-ccaaaaagaaaagcatcatcca-3′) and 4 pmol of dual-labeled probe mcpyv-2.0-4371-prb (5′-fam-atacattgccttttgggtgtttt-bhq1-3′) in a 25-μl reaction using qiagen quantitect probe master mix (qiagen, doncaster, victoria, australia) with 2 μl of template were run on a lightcycler 480 (roche diagnostics, castle hill, new south wales, australia) under the following conditions: incubation at 95°c for 15 min, followed by 45 cycles of 95°c for 15 s and 60°c for 1 min. quantitative real-time pcr was not performed because of limited applicability due to inherent variability in respiratory sample collection. the presence of mcpyv in samples positive by real-time pcr was then confi rmed by using partial large t antigen (lt3) and major capsid protein (vp1) conventional mcpyv detection pcr assays of feng et al. (3) . all pcrs were performed in a unidirectional workfl ow through dedicated suites for reagent preparation, pcr setup, and amplifi cation. ten random real-time pcr-negative samples, 10 water control samples, and template-negative control samples were used to exclude amplicon or sample cross-contamination. a clinical sample that was positive for mcpvv by all 3 assays and confi rmed by sequencing was used as a positive control for all pcrs. thirty-one (5.9%) samples produced positive results in the real-time pcr screening. of these, 8 (1.5%) could be confi rmed by only 1 conventional pcr, and 7 (1.3%) yielded positive results in all 3 mcpyv pcrs. all positive detections were in npa samples. this variation in detection rates could have been due to the unforeseen nonspecifi city of the real-time pcr, or to the inherent lower sensitivity of conventional pcrs, because most real-time pcr-positive samples produced late signals at cycle threshold values ≈40. we chose a conservative algorithm, in which a sample must have been positive in all 3 assays to be considered a true positive. this rule may have led to an underestimation of mcpyv prevalence in this sample population. all mcpyv-positive samples were collected during the spring and early summer months (october-december . five (1.1%) of these samples originated from pediatric patients, which suggests early acquisition of virus. in fact, all 4 mcpyv-positive patients who were not immunosuppressed were <2 years of age (table 1) . this age range coincides with ages of those who experience primary infections of many other human viruses (5, 6) . of the 3 immunosupressed mcpyv-positive patients, 2 were adults (2.6%), and 1 was a 6.6-year-old child. further information on each mcpyv-positive patient is listed in table 1 . we could not discern whether the higher frequency of detections in adults was representative of the general adult population or whether the detections were overrepresented due to a small sample size or the immune status of the adult cohort. the integration status of the detected mcpyv dna was not able to be ascertained; however, for 1 patient for whom multiple samples were tested, mcpyv was neither detected 2 months before nor 3 months after mcpyv-b4 was detected, which suggests that, at least in this particular patient, mcpyv may have had transitory presence or activity. multiple samples were not available for any of the other mcpyv-positive patients. three mcpyv (mcv-b1/b2/b3)-positive samples were further investigated through bidirectional sequencing of their lt3 (fj009185, fj009186, and fj009187, respectively) and vp1 (fj009188, fj009189, and fj009190, respectively) assay amplifi cation products. the sequences of the 3 samples showed high homology to those of the 2 previously described mcpyv strain sequences, mcc350 (eu375803) and mcc339 (eu375804) (3); similarities in the lt3 and vp1 target regions ranged from 99.6%-100.0% and 99.0%-99.6%, respectively ( table 2) . clinical notes were available for 5 of the 7 mcpyvpositive patients and indicated that these patients exhibited a variety of upper and lower respiratory tract symptoms. mcpyv was the sole viral agent detected in 6 of the 7 samples (table 1 ). however, due to limited remaining volumes, study samples were not screened for other known respiratory pathogens, including coronaviruses and rhinoviruses, which may have led to a higher codetection rate, similar to that for kipyv (1, 4) and wupyv (2, 4) . to date, mcpyv has been most commonly studied and detected in the context of merkel cell carcinoma (3, 7) , but it has also been identifi ed in kaposi sarcoma tissue (3), primary squamous cell carcinoma tissue (7), keratoacanthoma tissue from an organ recipient (8) , in normal sunexposed skin (7) , and in a small number of control tissues (normal skin, small bowel, hemorrhoids, appendix, and gall bladder) (3) . in this study, mcpyv was detected in 1.3% of respiratory samples collected from symptomatic persons. this description of the presence of mcpyv in the respiratory tract raises questions about the transmission and respiratory pathogenicity of this newly described pyv. analogous to mcpyv, both kipyv (1,4) and wupyv (2,4) have been found globally in respiratory specimens with a high prevalence of codetected viruses, but clear evidence for a causal association with respiratory illness has yet to be identifi ed. these pyvs may be merely transmitted through the respiratory route or detected during periods of reactivation (9) . similarly, jc and bk pyvs are suspected of being transmitted by inhalation and are occasionally detected in respiratory samples, yet patients generally remain asymptomatic or exhibit nonspecifi c upper respiratory tract symptoms (10, 11) . like these other pyvs, mcpyv may potentially be transmitted through the respiratory route and become latent in other sites, such as epidermal tissue, by systemic spread, in a similar fashion to murine pyv (12) . clearly, further studies using larger sample populations, expanded respiratory pathogen detection methods, and control groups are needed to elucidate what role, if any, mcpyv plays in upper and lower respiratory tract illness. on the basis of the ages and immune status of the mcpyv-positive patients, we can hypothesize that mcpyv may have an infectious cycle similar to that of other human pyvs, in which the virus is acquired early in life, undergoes a period of latency, and then becomes reactivated in the event of immunosuppression (10) . sequence data from positive specimens indicate that mcpyv found in respiratory secretions is similar to the viruses identifi ed within merkel cell carcinomas. whether and how the virus is translated from the respiratory tract to skin cells, if it is able to be transported systemically, and its role in the malignant transformation of merkel cells are all questions worthy of further investigation. search past issues of eid at www.cdc.gov/eid identifi cation of a third human polyomavirus identifi cation of a novel polyomavirus from patients with acute respiratory tract infections clonal integration of a polyomavirus in human merkel cell carcinoma presence of the newly discovered human polyomaviruses ki and wu in australian patients with acute respiratory tract infection role of human metapneumovirus, human coronavirus nl63 and human bocavirus in infants and young children with acute wheezing clinical and epidemiologic characterization of wu polyomavirus infection in nghiem p. merkel cell polyomavirus is more frequently present in north american than australian merkel cell carcinoma tumors the presence of polyomavirus in nonmelanoma skin cancer in organ transplant recipients is rare emerging respiratory agents: new viruses for old diseases? fields virology bk virus: a clinical review the primary site of replication alters the eventual site of persistent infection by polyomavirus in mice we thank the staff of the microbiology division of the queensland health pathology service central for access to the samples used in this study.this study was supported by the royal children's hospital foundation grant i 922-034, sponsored by the "woolworths fresh futures" appeal. mr bialasiewicz is a phd candidate and scientist in the research section of the queensland paediatric infectious diseases laboratory, sir albert sakzewski virus research centre, brisbane, australia. his main research is focused on the characterization of emerging pediatric viral respiratory disease and molecular diagnostics. key: cord-339386-sxyeuiw1 authors: mcintosh, kenneth; perlman, stanley title: 157 coronaviruses, including severe acute respiratory syndrome (sars) and middle east respiratory syndrome (mers) date: 2015-12-31 journal: mandell, douglas, and bennett's principles and practice of infectious diseases doi: 10.1016/b978-1-4557-4801-3.00157-0 sha: doc_id: 339386 cord_uid: sxyeuiw1 nan the family coronaviridae, within the order nidovirales, contains two subfamilies, the coronavirinae and the torovirinae. coronaviruses (covs) are a large group of viruses infecting mammals and birds and producing a wide variety of diseases. they have been divided into four genera, two of which contain viruses infecting humans (see later). all human coronaviruses (hcovs) are primarily respiratory pathogens. during the winter of 2002 to 2003, an alarming new disease appeared, severe acute respiratory syndrome (sars), which was quickly attributed to a new cov, the sars-cov. the outbreak originated in southern people's republic of china, with evidence that the virus was first derived from bats and was transmitted to man through an intermediate host, probably the palm civet (paguma larvata) or raccoon dog (nyctereutes procyonoides). [1] [2] [3] the sars epidemic was controlled through a massive effort at case identification and containment, and the last known case occurred in mid-2004. in retrospect, the emergence of sars is consistent with what is known about covs as a group: they are important pathogens in animals causing a wide variety of diseases through a wide variety of pathogenic mechanisms, and they have been noted to mutate frequently and infect new species. 4, 5 more recently, a related but different cov producing severe respiratory disease has emerged, the middle east respiratory syndrome coronavirus (mers-cov). mers-cov was grown in june 2012, from a sputum sample obtained from a man in saudi arabia who died of overwhelming pneumonia. the virus was quickly identified as a new cov most closely related to several bat covs. 6 this report was followed by a number of other reports identifying a total of 537 infected individuals, all of whom had acute respiratory symptoms, severe in most, and fatal in 145 (as of may 11, 2014) . 7 human-to-human transmission has been documented, especially in hospital settings, 7a but appears to be inefficient. in 1965, tyrrell and bynoe 8 cultured a virus obtained from the respiratory tract of a boy with a common cold by passage in human embryonic tracheal organ cultures. the media from these cultures consistently produced colds in volunteers. the agent was ether sensitive but not related to any known human virus. subsequently, electron microscopy of fluids from infected organ cultures revealed particles that resembled infectious bronchitis virus of chickens. 9 at about the same time, hamre and procknow 10 recovered a cytopathic agent in tissue culture from medical students with colds. the prototype virus was named 229e and was found on electron microscopy to have a similar or identical morphology ( fig. 157-1) . 9 using techniques similar to those used by tyrrell and bynoe, mcintosh and colleagues 11 reported the recovery of several infectious bronchitis-like agents from the human respiratory tract, the prototype of which was named oc43 (oc for organ culture). at much the same time, mouse hepatitis virus and transmissible gastroenteritis virus of swine were shown to have the same morphology on electron microscopy. 12, 13 shortly thereafter, the name coronavirus (the prefix corona denoting the crownlike appearance of the surface projections) was chosen to signify this new genus. 14 the number of animal covs quickly grew, including viruses causing diseases in rats, mice, chickens, turkeys, various other bird species, cattle, several wild ruminants, beluga whales, dogs, cats, rabbits, and pigs, with manifestations in the respiratory and gastrointestinal tracts, central nervous system (cns), liver, reproductive tract, and others. through sequencing and antigenicity studies, the animal and human covs (hcovs) initially were divided into three groups: group 1, which • covs are members of the nidovirales order, single-stranded, positive-sense rna viruses with a large genome. they mutate and also recombine frequently. • laboratory diagnosis is best accomplished by finding viral rna through polymerase chain reaction. • there are no accepted effective antiviral drugs for covs. • prevention is through epidemiologic methods. the sars epidemic was halted through careful case finding, quarantine, and use of barrier precautions. 15 a cov was independently and almost simultaneously isolated from sars patients by several laboratories and found by sequencing to be only distantly related to previously characterized covs. [16] [17] [18] [19] [20] the sars outbreak stimulated a rapid and intense public health response coordinated by the world health organization, and by july 2003, transmission had ceased throughout the world. despite this effort, however, 8096 probable cases had occurred in 29 countries, with 774 deaths. 15 with the identification of the sars-cov, the hcov field became much more active. sensitive molecular methods were developed to detect rna from viruses identical or closely related to hcov-229e and hcov-oc43 in the respiratory tract, and two new species were discovered: nl63, an alphacoronavirus, and hku1, a betacoronavirus. [21] [22] [23] hcov-nl63 was found independently by three groups, two in the netherlands and, somewhat later, the third in new haven, figure 157-2 phylogenetic relationships among members of the subfamily coronavirinae. a rooted neighbor-joining tree was generated from amino-acid sequence alignments of coronaviridae-wide conserved domains in replicase polyprotein 1 (adrp, nsp3; mpro, nsp5; rdrp, nsp12; hel, nsp13; exon, nsp14; nendou, nsp15; o-mt, nsp16) for 21 coronaviruses, each a representative of a currently recognized coronavirus species. five of the six known human coronaviruses (hcov-229e, hcov-nl63, hcov-hku1, sars-cov, and mers-cov) are indicated. hcov-oc43 is closely related to bovine coronavirus, which is shown in the figure. equine torovirus berne served as the outgroup. virus names are given with strain specifications; species and genus names are in italics as per convention. the tree shows the four main monophyletic clusters, corresponding to genera alpha-, beta-, gamma to be established numerous reports of cov-like particles (covlps) found by electron microscopy in human fecal matter, but these particles have been difficult to characterize further. more recent efforts to detect cov rna in feces using polymerase chain reaction (pcr) and primers for respiratory hcovs have had limited success and have failed to associate covs with gastrointestinal disease. 28, 29 toroviruses were, like covs, first described in animals. they were first detected in the feces of cattle (breda virus) and horses (berne virus). 30, 31 shortly thereafter, beards and colleagues 32 examined human fecal material and reported finding particles with a similar appearance that aggregated in the presence of antiserum to the bovine and equine viruses. the human toroviruses, like the bovine toroviruses, do not grow in tissue culture, and thus almost all existing information about them is based on electron micrographic data. unlike animal toroviruses, 33,34 pcr-amplified torovirus rna sequences have not been found in human stool samples. a report of genome sequences amplified from particles purified from human stool 35 was subsequently retracted and considered to reflect laboratory contamination from porcine strains. 36 at this time, there are no reports definitively showing the existence of human toroviruses. the cov nucleic acid is rna, approximately 30 kilobases in length, of positive sense, single stranded, polyadenylated, and infectious. the rna, the largest known viral rna ( fig. 157-3) , codes for (in order from the 5′ end) a large polyprotein that is cleaved by virus-encoded connecticut. 24 in all three cases, positive samples were from infants and children with respiratory disease. notably, hcov-nl63 and hcov-229e were estimated to originate from a common precursor and diverge approximately 1000 years ago. 25 hcov-hku1 was found in hong kong in an adult with respiratory disease. these two new hcov strains subsequently have been found worldwide and appear to have pathogenicity similar to that of hcov-229e and hcov-oc43, with the possible exception that nl63 is more frequently found in children with croup. 26, 27 the mers-cov was found when a man was admitted in june 2012 to a hospital in jeddah, saudi arabia with overwhelming acute pneumonia with renal failure, and a sample of sputum grew a cytopathic virus that, on sequencing, proved to be a cov, classified as a betacoronavirus, and most closely related to two bat covs, hku-4 and hku-5. 6 over the next 23 months 536 additional cases (145 fatal) were found, all but a few of them sporadic or hospital-based and in individuals living or traveling in the middle east. in the remainder of this chapter, the group of respiratory hcovs first discovered in the 1960s and containing hcovs 229e, oc43, nl63, and hku-1 are referred to as community-acquired hcovs to distinguish them from the sars-cov and the mers-cov. in view of the prominence of covs in animal enteric diseases, there have been extensive efforts to identify enteric hcovs. there are figure 157-3 genome organization of representative human coronaviruses. all coronavirus genomes have the same basic structure and mechanism of replication. 4 the 5′ end of each genome encodes a leader sequence, which is attached to each virus-specific messenger rna transcript by a novel mechanism of discontinuous replication. the first two thirds of each genome encode replicase-associated genes. gene 1 is translated as two large polyproteins, with the first expressed from orf1a and the second from orf1a/b following a −1 frameshift event. these polyproteins are then cleaved into individual proteins by two virus-encoded proteases. the major structural genes, the hemagglutinin-esterase (he), surface (s), envelope (e), transmembrane (m), and nucleocapsid (n) proteins, are indicated in green. the nonreplicase, accessory genes located at the 3′ end of the genome are indicated with open boxes. the functions of these proteins are largely not known, and there is no sequence homology between accessory proteins of different coronaviruses. some of these proteins are virion associated, but none are required for virus replication. the open reading frames (orfs) encoding these proteins are numbered in order of appearance from the 5′ end of the genome, with the exception that ns12.9 of hcov-oc43. i is an internal protein expressed from an alternative reading frame located within the n gene. it is equivalent to sars-cov-specific protein 9b and the mers-cov-specific protein 8b. (figure prepared by 4 (dpp-4), which, like ace2 and apn, is an ectopeptidase that is abundantly expressed in the respiratory tract. 46 all the community-acquired respiratory hcovs grow only with difficulty in tissue culture. despite this, both 229e and nl63 were discovered because they produced a detectable cytopathic effect, the first in human embryonic kidney 10 and the second in llc-mk2 cells. 21 both the sars-cov and the mers-cov were initially isolated and grew readily in vero cells. 6, 17 hcovs oc43 and hku-1 have been grown in tissue culture after laboratory adaptation or in primary human airway epithelial cells. [47] [48] [49] detection of all these viruses in clinical specimens is most conveniently and sensitively achieved using pcr. likewise, the enteric covs have been difficult to cultivate in vitro. all but a few strains have been detected only by electron microscopy of human fecal material. [50] [51] [52] 53, 54 some strains have been characterized by immune electron microscopy and found to be related to hcov-oc43. 55 two strains obtained from an outbreak of necrotizing enterocolitis in texas and passaged in intestinal organ cultures were reported to contain four or five proteins with apparent molecular weights similar to those of other covs but not related antigenically to known human strains or mouse hepatitis virus a59. 56 the evidence favors the view that these isolates, as well as particles antigenically related to hcov-oc43, are members of the family coronaviridae, although their association with human disease is not yet proven. surveys of children with diarrhea using pcr imply that diarrhea associated with the four well-described hcov strains is unusual. 28, 29 epidemiology evidence of community-acquired respiratory cov infections has been found wherever in the world it has been sought. in temperate climates, respiratory cov infections occur more often in the winter and spring than in the summer and fall. the contribution of cov infections to the total number of upper respiratory illnesses may be as high as 35% during times of peak viral activity. overall, the proportion of adult colds produced by covs may be reasonably estimated at 15%. early studies of hcov-oc43 and 229e in the united states demonstrated periodicity, with large epidemics occurring at 2-to 3-year intervals. 57 strain hcov-229e tended to be epidemic throughout the united states, whereas strain hcov-oc43 appeared in localized outbreaks. similar studies of nl63 and hku1 have not been done, but it seems from the available data that they also vary widely in incidence from year to year and place to place. reinfection is common and may be due to the rapid diminution of antibody levels after infection. 58 infection occurs at all ages but is most common in children. the ratio of symptomatic to total infections varies between 50% and 90%, depending on the age of the population studied, the method of virus detection, and the definition of "infection. " 59, 60 among adult volunteers 72% of those infected with hcov-229e developed colds. 61 the first report of a new cov causing severe pneumonia appeared in promed mail on september 20, 2012. a man from jeddah, saudi arabia had developed pneumonia in june and died of respiratory and renal failure, and a virus was grown from a sputum sample that was subsequently sequenced and found to be a betacoronavirus thought at the time to be most closely related to bat covs hku4 and hku5. 6 between then and may 2014, a total of 537 cases occurred, all infected by this virus, now termed the middle east respiratory syndrome coronavirus (mers-cov). one hundred forty-five of the cases were fatal. 7 more than 400 of these have been acquired and diagnosed in the kingdom of saudi arabia, with most of the remainder in the united arab emirates, qatar, jordan, and kuwait. cases originating in the arabian peninsula have also occurred in travelers to egypt, tunisia, germany, italy, great britain, malaysia, the philippines, and the united states, with a few secondary cases sometimes occurring in those locations through close family or hospital spread. in the united states,these include two unrelated mers cases, and a third case related to one of these. 61a while infections with severe respiratory involvement have proteases to form several nonstructural proteins, including an rnadependent rna polymerase, methyltransferases, and a helicase, followed by either four or five structural proteins intermingled with a variable number of nonstructural and minor structural proteins. 37 the first of the major structural proteins is a surface hemagglutinin-esterase protein, present on hcovs oc43 and hku1 and some animal betacoronaviruses, that may play some role in the attachment or release of the particle, or both, at the cell surface. this gene contains sequences similar to the hemagglutinin of influenza c virus, likely evidence of an interfamily recombinational event that occurred many years ago. the next gene encodes the surface glycoprotein that forms the petal-shaped surface projections and is responsible for attachment and the stimulation of neutralizing antibody. this is followed by a small envelope protein, a membrane glycoprotein, and a nucleocapsid protein that is complexed with the rna. there are several other open reading frames whose coding functions are not clear. the strategy of replication of covs is similar to that of other nidoviruses, in that all messenger rnas form a nested set with common polyadenylated 3′ ends, with only the unique portion of the 5′ end being translated. 4 as in other rna viruses, mutations are common in nature, although the mutation rate is much lower, approximately 2 × 10 −6 per site per replication cycle. 38 unlike other rna viruses, covs encode a 3′-5′ exonuclease that has proofreading activities, playing a critical role in maintaining replication fidelity in cell cultures and in animals. 39 covs are also capable of genetic recombination if two viruses infect the same cell at the same time. all covs develop exclusively in the cytoplasm of infected cells (fig. 157-4) . they bud into cytoplasmic vesicles from membranes of the pre-golgi endoplasmic reticulum. these virus-filled vesicles are then extruded by the exocytic secretory pathway. 40 the resultant virus particles have a diameter of 70 to 80 nm on thin-section electron microscopy and 60 to 220 nm on negative staining. they are pleomorphic, with widely spaced, petal-shaped projections 20 nm long (see fig. 157-1) . the cellular receptor for 229e and most other alphacoronaviruses is aminopeptidase n (apn). 41 interestingly, nl63, the other known human alphacoronavirus, uses as its cellular receptor angiotensinconverting enzyme ii (ace2), 42 the same receptor as is used by the sars-cov. 43 mouse hepatitis virus, a betacoronavirus related to strain oc43, uses as its receptor a member of the carcinoembryonic antigen family. 44 hcov-oc43 and bcov, which is closely related to hcov-oc43, bind to 9-o-acetylated neuraminic acid as part of the entry process. 45 the host cell receptor for mers-cov is dipeptidyl peptidase as 50%. there was no mortality in children or young adults younger than the age of 24 years. in response to the global spread and associated severe disease, the world health organization coordinated a rapid and effective control program that included isolation of cases, careful attention to contact, droplet and airborne infection control procedures, quarantine of exposed persons in some settings, and efforts to control spread between countries through travel advisories and travel alerts. presumably as a result of these efforts, global transmission ceased by july 2003. a few subsequent cases of sars were detected, but all were either a result of laboratory spread or individual cases related to presumed contact with civet cats or other intermediate hosts. the last known case occurred in mid-2004. spread of sars to humans is thought to have occurred primarily through droplet or contact transmission, with a possible role for fomites. in most instances, an individual case transmitted to very few others, although several well-documented instances of small-particle airborne transmission occurred, resulting in super-spreading events. 67 spread in hospital settings appeared to be surprisingly efficient, but it could be effectively suppressed with the enforcement of droplet and contact precautions. 68 containment measures were efficacious, in part, because patients were most contagious only after lower respiratory disease developed. 69 the chain of spread was finally broken in the people's republic of china, the last country to experience endemic spread, in june 2003. it now seems almost certain that the human epidemic began with the spread of a closely related bat virus first to palm civets or other animals sold in live wild game markets and then to humans in guangdong province in the people's republic of china, and that the virus adapted itself through mutation and possibly recombination, until it transmitted readily among humans. [70] [71] [72] the virus that spread worldwide came largely from a single infected individual who traveled from occurred at all ages, the elderly and those with underlying conditions (diabetes, renal disease, immunosuppression) have been most often severely or fatally affected. the who and the cdc have published a case definition, as well as surveillance instructions to aid in epidemiologic control of the mers-cov. 62, 63 the putative bat origin of this virus was strengthened by the finding that the virus grew readily in primary bat tissue culture. 64 nevertheless, and while bats sampled in the middle east, africa, and europe were found to carry viruses closely related to the mers-cov, 65 epidemiologic studies suggested there was likely to be at least one intermediate host. serologic and virologic studies indicated that camels in the middle east and africa were frequently infected by viruses very similar to some of those found in human mers cases. 65a acquisition of mers from camels appears likely, although the proportion of camel-acquired cases (versus those acquired from person-to-person contact or through another animal intermediate) is not clear. case clusters indicate that person-to-person hospital spread is more common than spread within families, and that casual-contact spread is unusual. 7a the sars epidemic began in guangdong province in the people's republic of china in mid-november 2002. 66 it came to worldwide attention in march 2003 when cases of severe, acute pneumonia were reported to the world health organization from hong kong, hanoi, and singapore. disease spread in hospitals to health care workers, visitors, and patients, among family members, and, on occasion, in hotels, apartment complexes, markets, and airplanes. worldwide spread was rapid but focal ( fig. 157-5) . the largest numbers of cases were reported from the people's republic of china, hong kong, taiwan, singapore, and toronto, canada. the overall case-fatality rates in these locations ranged from 7% to 17%, but persons with underlying medical conditions and those older than 65 years of age had mortality rates as high involvement in other organ systems, including diarrhea, leukopenia, thrombocytopenia, and, most notably, pan-lymphopenia. 83 virus has been detected in respiratory secretions, blood, stool, and urine specimens and tissue from the lung and kidney. on the basis of pcr testing, virus titer is highest during the second week of illness 84 and can often be detected into the third week of illness and sometimes for as long as several months. 18, 85 pulmonary symptoms may worsen late in the course of the illness, with the development of adult respiratory distress syndrome. 84 there may also be late evidence of liver and kidney involvement. the pulmonary pathology of infection by the sars-cov has been described extensively, 17,86,87 but little has been published about the pathology in other organ systems. [87] [88] [89] the extrapulmonary pathologic changes found most consistently at autopsy are extensive necrosis of the white pulp of the spleen and a generalized small vessel arteritis. 87, 88 in the lung, there is hyaline membrane formation, interstitial infiltration with lymphocytes and mononuclear cells, and desquamation of pneumocytes in the alveolar spaces. giant cells are a constant finding and usually have macrophage markers. in bronchoalveolar lavage, biopsy, and autopsy specimens viral particles have been noted in type i and ii pneumocytes. 90 at this point, little is known about the pathogenesis of mers-cov because the infection was only recently described and no pathological specimens are yet available. it is anticipated that the pathologic changes in the lungs of patients with severe disease will be similar to those observed in patients with sars or other patients with acute respiratory distress syndrome (ards). almost all the antigenically distinct respiratory cov strains that were isolated in the 1960s have been administered to volunteers, and all these produce illness with similar characteristics. 8,91 a summary of these characteristics is given in table 157 -1, in which a comparison is made with colds produced by rhinoviruses in similarly inoculated volunteers. the incubation period of cov colds was longer and their duration somewhat shorter, but the symptoms were similar. asymptomatic infection was sometimes seen and, indeed, has been a feature guangdong province to hong kong and infected a large number of individuals before himself succumbing to the disease. in contrast, the virus that was epidemic in the people's republic of china was more variable. although an etiologic role is not proven, enteric covs (or covlps) have been most frequently associated with gastrointestinal disease in neonates and infants younger than 12 months. particles have been found in the stools of adults with the acquired immunodeficiency syndrome. 73, 74 asymptomatic shedding is common, particularly in tropical climates 75 and in populations living in poor hygienic conditions. 76 the viruses can be detected for prolonged periods 51,53,77 and without any apparent seasonal pattern. 78 community-acquired respiratory covs (hcov-229e, oc43, nl63, hku-7) generally replicate in ciliated and nonciliated (hcov-229e) epithelial cells of the nasopharynx, 79 probably producing both direct cell degeneration 80 and an outpouring of chemokines and interleukins, with a resultant common-cold symptom complex similar to that produced by rhinovirus infection. 81 the incubation period is, on average, 2 days, and the peak of respiratory symptoms, as well as viral shedding, is reached at approximately 3 or 4 days after inoculation. 61 the pattern of virus replication of covs must be at least in part determined by virus-receptor interaction. the two best-defined receptors for the respiratory covs are aminopeptidase n for strain hcov-229e and angiotensin-converting enzyme ii for nl63. the pathogenicity of sars is more complex and involves systemic spread. the route of infection of the sars-cov is probably through the respiratory tract. after an incubation period that is usually 4 to 7 days, but can be as long as 10 to 14 days, the disease begins, starting usually with fever and other systemic (influenza-like) symptoms, with cough and dyspnea developing a few days to a week later. 82 although the lung is the focus of the disease process, there are often signs of levels decreased, suggesting that disease was partly immune mediated. 84 clinical improvement was associated with the onset of a virusspecific antibody response. 119 pediatric disease was, interestingly, significantly less severe than adult disease, although the features were similar. 121 disease during pregnancy was severe, with high mortality in both the mother and fetus. 122 congenital transmission was not described. the nature of the illness associated with enteric cov infection is much less clear. one study found a significant association of gastroenteritis in infants 2 to 12 months of age with the presence of covlps in the stool. 55 another study, confined to infants in a neonatal intensive care unit, found highly significant associations between the presence of covlps in the stool and the presence of water-loss stools, bloody stools, abdominal distention, and bilious gastric aspirates. 53 a further study of symptomatic infants shedding covlps pointed to possible differences between covlp-associated diarrhea and rotavirus diarrhea: although fever and vomiting were of similar incidence, stools were more often occult blood positive (18% in covlp-associated vs. 0% in rotavirus-associated disease), less often watery (66% vs. 92%), and more often mucoid (32% vs. 8%). 77 finally, covs have been associated with at least three outbreaks of necrotizing enterocolitis in newborns, 52, 53, 56 and the best characterized strains 56 were isolated in infants with this illness. surveys seeking hcov rna by pcr using primers that would detect the known community-acquired respiratory hcovs in stool have been quite disappointing. in one study of 878 fecal samples from children with gastrointestinal complaints tested over 2 years, all four hcov species were found, but in all but 4 of 22 hcov-positive cases, either rotavirus or norovirus was also present. 28 in addition, about half of the children in this survey had respiratory and gastrointestinal symptoms. in the same study, 112 asymptomatic children were sampled and 2 were positive. another study sampled 151 symptomatic children, and 2 were found to have hku1 rna. 29 molecular studies using primers that would broadly detect new covs should be considered to resolve questions about the role of covs in human gastrointestinal disease. 123 like many other viruses, covs have been sought as possible etiologic agents in multiple sclerosis. the search has been stimulated by the capacity of jhm, a well-studied strain of mouse hepatitis virus, to produce in mice and rats an immune-mediated chronic demyelinating encephalitis histologically similar to multiple sclerosis. 124 hcov-oc43 125, 126 and hcov-229e 127 have been detected in brain tissue from multiple sclerosis patients using virus isolation, 125 in situ hybridization, immunohistology, 126 and pcr. 127 moreover, t-cell lines established from patients with multiple sclerosis by stimulation with myelin basic protein or hcov-229e were found to be cross-reactive with the opposite antigen, suggesting that molecular mimicry might be a possible pathogenic mechanism for the disease association. 128 an adolescent boy with acute demyelinating encephalitis was found to have hcov-oc43 rna in both the respiratory tract and the cerebrospinal fluid. 129 despite these intriguing reports, compelling evidence is lacking to establish an etiologic or pathogenetic association of covs with cns disease in humans. although some human respiratory covs grow in tissue culture directly from clinical samples and although antigen detection systems have been developed for both hcov-oc43 and hcov-229e, 130, 131 laboratory diagnosis of cov respiratory infections is best accomplished by molecular methods. reverse-transcriptase pcr (rt-pcr) systems have been developed using many different primers and detectors. from a clinical point of view, a single generic test for respiratory covs would be desirable, and such tests have been developed. however, when tested side by side with specific systems, the generic systems have a somewhat lower sensitivity. 95 systems that combine primers and probes specific for several covs have also had considerable success. 132 of both serologic surveys and pcr-based studies of natural infection of infants, children, and adults. 92, 93 more serious respiratory tract illness is probably also caused by all four strains of community-acquired hcov. the evidence for this is not conclusive, but it seems likely that all strains can produce pneumonia and bronchiolitis in infants, 24, 27, 94, 95 otitis and exacerbations of asthma in children and young adults, [96] [97] [98] pneumonia in healthy adults, 99 exacerbations of asthma and chronic bronchitis in adults, 100, 101 both serious bronchitis and pneumonia in the elderly, 102, 103 and pneumonia in the immunocompromised host. 104, 105 hcovs are found in asymptomatic individuals of all ages, and, when accompanied by illness, are also sometimes accompanied by infections with other potential respiratory pathogens. these characteristics (infection without disease, coinfection during disease) are features of many respiratory pathogens, including particularly rhinoviruses, adenoviruses, human metapneumovirus, human bocavirus, and parainfluenza viruses, but also (although less frequently) respiratory syncytial virus and influenza virus. because infections with respiratory hcovs are so common, however, it is possible that they are responsible for a significant portion of these serious lower respiratory tract diseases, even though the basic pathogenicity of hcovs (judging from volunteer studies) is similar to that of rhinoviruses, and clearly less than that of respiratory syncytial virus, influenza viruses, and certain adenovirus types. there is some evidence that hcov-oc43 is more pathogenic in the elderly than hcov-229e 106 and also some evidence that infection with nl63 in children is different from the other respiratory hcovs in that several series have found an excess of children with croup. 26, 27 information about the clinical presentation of patients infected with the mers-cov is limited. it is clear that there is a spectrum of illness with some infections consisting of mild upper respiratory symptoms only, and others characterized by cough and fever with progression to respiratory failure over about a week. 6, 7, 62, 107 renal failure, as well as pericarditis and adult respiratory distress syndrome has been part of the reported clinical picture. the mers-cov host cell receptor, dpp-4, is expressed at high levels in the kidney, 108 raising the possibility that direct infection of this organ contributes to renal disease. a case definition that will lead to further epidemiologic studies has been published by the world health organization. 109 severe acute respiratory syndrome coronavirus the first symptom in most cases of sars was fever, usually accompanied by headache, malaise, or myalgia. this was followed, usually in a few days, but as long as a week later, by a nonproductive cough and, in more severe cases, dyspnea. approximately 25% of patients had diarrhea. interestingly, upper respiratory symptoms such as rhinorrhea and sore throat usually did not occur. 82, 84, [110] [111] [112] [113] the chest radiograph was frequently abnormal, showing scattered air-space opacification, usually in the periphery and lower zones of the lung. 114 spiral computed tomography demonstrated both ground-glass opacification and consolidation, often in a subpleural distribution. [115] [116] [117] lymphopenia was common, 84, 86, 110 with normal or somewhat depressed neutrophils. paradoxically, neutrophilia was associated with poor outcomes. 83 the decrease in lymphocytes in the blood was most marked for cd4 cells but was seen in all t-cell phenotypes, including cd3 and cd8, as well as natural killer cells. creatine kinase was often abnormal, as were lactic dehydrogenase and aspartate aminotransferase. levels of proinflammatory cytokines were elevated at early times during infection in patients with severe clinical disease 118 and decreased in those patients who resolved the infection. 119 approximately 25% of patients developed severe pulmonary disease that progressed to adult respiratory distress syndrome. adult respiratory distress syndrome with sars-cov infection was most likely to develop in patients older than 50 years or with underlying disease such as diabetes, cardiac disease, and chronic hepatitis. 84, 110, 111, 120 the overall mortality rate was between 9% and 12%, with the highest rates in the elderly and adults with underlying liver disease. in some patients, clinical deterioration occurred during the second week of illness, as virus ribavirin. 134 it is now known that ribavirin has little activity against sars-cov in vitro, and there is no evidence that either intervention improved outcomes. 135, 136 lopinavir/ritonavir and intravenous immune globulin were also used in some patients, again without conclusive evidence that they were helpful or harmful. there is anecdotal and at least partially controlled evidence of the benefit of either interferon-α or interferon-β treatment. further, treatment of sars-cov-infected cynomolgus monkeys with pegylated interferon-α resulted in improved outcomes, 137 lending credence to the use of this therapy if sars recurred. treatment of mers-cov infection depends entirely on supportive measures. no antiviral drugs are recommended, although several studies have indicated that mers-cov is more sensitive to interferonα or interferon-β than sars-cov. 138, 139 standard droplet precautions should be used, with aerosol precautions during certain high-risk procedures. 109 rigorous application of hospital infection control procedures, particularly those directed at contact and droplet spread, was shown to have a major beneficial effect on the spread of the sars-cov. 68 the containment of the global sars outbreak is a testament to the power of the cooperation and collaboration engendered by the world health organization to address a major public health threat. similar precautions are recommended for patients with suspected or confirmed mers-cov infections. 109 vaccines for animal covs have been developed and widely used with variable efficacy. in one instance, a vaccine for feline infectious peritonitis appeared to lead to enhanced disease with subsequent natural infection. if sars does return or mers reaches epidemic proportions, an effective vaccine would be extremely helpful in control efforts, and a variety of vaccination strategies, including inactivated, subunit, and live-attenuated vaccines, are being pursued. 135, 140 in addition, hospitals have been advised on improvement of infection control procedures so that in future epidemics of respiratory viruses, they will not be a major source of spread of infection, as occurred in the 2002 epidemic of sars. the mers-cov was originally isolated in vero and llc-mk2 cells, and there are several published methodologies for detection and identification by pcr. 133 current recommendations from who are that definition of a possible case should be immediately reported to national authorities, and clinical, epidemiologic, and microbiologic investigations should be carried out. 63 although sars-cov was grown from respiratory tract specimens in vero e6 and fetal rhesus monkey kidney cells, the more sensitive and rapid rt-pcr assays were most widely used to detect infection. virus was detected by rt-pcr in upper and lower respiratory tract, blood, stool, and urine specimens. early in the illness, specimens were found positive only in approximately one third of patients. 84 use of samples from multiple sources increased the yield. virus was detected most frequently during the second week of illness. 18, 84, 85 antibody tests have been developed using tissue culture-grown virus and indirect immunofluorescence or enzyme-linked immunosorbent assay. immunoglobulin m antibody can be detected in most patients for a limited period of time, and immunoglobulin g antibody appears first approximately 10 days after onset of fever in patients with good outcomes and becomes essentially universal after 4 weeks. 84 laboratory diagnosis of the gastrointestinal covs depends now entirely on electron microscopy of stool specimens and detection of characteristic particles in negatively stained specimens. such testing is best performed in laboratories with extensive previous experience. given the severity of sars, clinicians throughout the world empirically treated most patients with corticosteroids and intravenous or oral the complete reference list is available online at expert consult. severe acute respiratory syndrome coronavirus-like virus in chinese horseshoe bats isolation and characterization of viruses related to the sars coronavirus from animals in southern china coronaviruses post-sars: update on replication and pathogenesis isolation of a novel coronavirus from a man with pneumonia in saudi arabia hospital outbreak of middle east respiratory syndrome coronavirus cultivation of a novel type of common-cold virus in organ cultures the morphology of three previously uncharacterized human respiratory viruses that grow in organ culture a new virus isolated from the human respiratory tract recovery in tracheal organ cultures of novel viruses from patients with respiratory disease severe acute respiratory syndrome coronavirus as a possible cause of severe acute respiratory syndrome a novel coronavirus associated with severe acute respiratory syndrome identification of a novel coronavirus in patients with severe acute respiratory syndrome characterization of a novel coronavirus associated with severe acute respiratory syndrome identification of a new human coronavirus a previously undescribed coronavirus associated with respiratory disease in humans characterization and complete genome sequence of a novel coronavirus, coronavirus hku1, from patients with pneumonia mosaic structure of human coronavirus nl63, one thousand years of evolution croup is associated with the novel coronavirus nl63 detection of human coronaviruses in children with acute gastroenteritis unique and conserved features of genome and proteome of sarscoronavirus, an early split-off from the coronavirus group 2 lineage infidelity of sars-cov nsp14-exonuclease mutant virus replication is revealed by complete genome sequencing morphogenesis of avian infectious bronchitis virus and a related human virus (strain 229e) human aminopeptidase n is a receptor for human coronavirus 229e human coronavirus nl63 employs the severe acute respiratory syndrome coronavirus receptor for cellular entry angiotensin-converting enzyme 2 is a functional receptor for the sars coronavirus crystal structure of mouse coronavirus receptor-binding domain complexed with its murine receptor dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-emc association of coronavirus infection with neonatal necrotizing enterocolitis medical reviews: coronaviruses a prospective, community-based study on virologic assessment among elderly people with and without symptoms of acute respiratory infection effects of a "new" human respiratory virus in volunteers available at www.cdc .gov.coronavirus/mers/index middle east respiratory syndrome (mers): case definitions interim surveillance recommendations for human infection with middle east respiratory syndrome coronavirus human betacoronavirus 2c emc/2012-related viruses in bats, ghana and europe middle east respiratory syndrome coronavirus quasispecies that include homologues of human isolates revealed through whole-genome analysis and virus cultured from dromedary camels in saudi arabia effectiveness of precautions against droplets and contact in prevention of nosocomial transmission of severe acute respiratory syndrome (sars) a review of studies on animal reservoirs of the sars coronavirus molecular evolution of the sars coronavirus during the course of the sars epidemic in china clinical progression and viral load in a community outbreak of coronavirusassociated sars pneumonia: a prospective study multiple organ infection and the pathogenesis of sars time course and cellular localization of sars-cov nucleoprotein and rna in lungs from fatal cases of sars role of respiratory viruses in acute upper and lower respiratory tract illness in the first year of life: a birth cohort study detection of rhinovirus, respiratory syncytial virus, and coronavirus infections in acute otitis media by reverse transcriptase polymerase chain reaction respiratory viruses and exacerbations of asthma in adults a prospective, community-based study on virologic assessment among elderly people with and without symptoms of acute respiratory infection clinical impact of human coronaviruses 229e and oc43 infection in diverse adult populations interferon-mediated immunopathological events are associated with atypical innate and adaptive immune responses in patients with severe acute respiratory syndrome clinical disease in children associated with newly described coronavirus subtypes sars: systematic review of treatment effects the spike protein of sars-cova target for vaccine and therapeutic development severe acute respiratory syndrome coronavirus-like virus in chinese horseshoe bats evolutionary insights into the ecology of coronaviruses isolation and characterization of viruses related to the sars coronavirus from animals in southern china coronaviruses post-sars: update on replication and pathogenesis isolation of a novel coronavirus from a man with pneumonia in saudi arabia middle east respiratory syndrome coronavirus (mers-cov) summary and literature update-as of 9 hospital outbreak of middle east respiratory syndrome coronavirus cultivation of a novel type of common-cold virus in organ cultures the morphology of three previously uncharacterized human respiratory viruses that grow in organ culture a new virus isolated from the human respiratory tract recovery in tracheal organ cultures of novel viruses from patients with respiratory disease growth in suckling-mouse brain of "ibv-like" viruses from patients with upper respiratory tract disease morphologic characteristics and nucleic acid type of transmissible gastroenteritis virus of pigs severe acute respiratory syndrome coronavirus as a possible cause of severe acute respiratory syndrome a novel coronavirus associated with severe acute respiratory syndrome identification of a novel coronavirus in patients with severe acute respiratory syndrome the genome sequence of the sars-associated coronavirus characterization of a novel coronavirus associated with severe acute respiratory syndrome identification of a new human coronavirus a previously undescribed coronavirus associated with respiratory disease in humans characterization and complete genome sequence of a novel coronavirus, coronavirus hku1, from patients with pneumonia evidence of a novel human coronavirus that is associated with respiratory tract disease in infants and young children mosaic structure of human coronavirus nl63, one thousand years of evolution croup is associated with the novel coronavirus nl63 the association of newly identified respiratory viruses with lower respiratory tract infections in korean children detection of human coronaviruses in children with acute gastroenteritis human coronaviruses are uncommon in patients with gastrointestinal illness studies with an unclassified virus isolated from diarrheic calves purification and partial characterization of a new enveloped rna virus (berne virus) an enveloped virus in stools of children and adults with gastroenteritis that resembles the breda virus of calves identification and characterization of a porcine torovirus phylogenetic and evolutionary relationships among torovirus field variants: evidence for multiple intertypic recombination events characterization of torovirus from human fecal specimens notice of retraction to "the novel hemagglutinin-esterase genes of human torovirus and breda virus unique and conserved features of genome and proteome of sarscoronavirus, an early split-off from the coronavirus group 2 lineage infidelity of sars-cov nsp14-exonuclease mutant virus replication is revealed by complete genome sequencing a live, impaired-fidelity coronavirus vaccine protects in an aged, immunocompromised mouse model of lethal disease morphogenesis of avian infectious bronchitis virus and a related human virus (strain 229e) human aminopeptidase n is a receptor for human coronavirus 229e human coronavirus nl63 employs the severe acute respiratory syndrome coronavirus receptor for cellular entry angiotensin-converting enzyme 2 is a functional receptor for the sars coronavirus receptor for mouse hepatitis virus is a member of the carcinoembryonic antigen family of glycoproteins crystal structure of mouse coronavirus receptor-binding domain complexed with its murine receptor dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-emc the adaptation of two human coronavirus strains (oc38 and oc43) to growth in cell monolayers detection of the new human coronavirus hku1: a report of 6 cases culturing the unculturable: human coronavirus hku1 infects, replicates, and produces progeny virions in human ciliated airway epithelial cell cultures pleomorphic virus-like particles in human faeces chronic enterocyte infection with coronavirus: one possible cause of the syndrome of tropical sprue? association of coronavirus infection with neonatal necrotizing enterocolitis pleomorphic, enveloped, virus-like particles associated with gastrointestinal illness in neonates human enteric coronaviruses: antigenic relatedness to human coronavirus oc43 and possible etiologic role in viral gastroenteritis isolation and propagation of a human enteric coronavirus medical reviews: coronaviruses the time course of the immune response to experimental coronavirus infection of man a prospective, community-based study on virologic assessment among elderly people with and without symptoms of acute respiratory infection human coronavirus in young children hospitalized for acute respiratory illness and asymptomatic controls effects of a "new" human respiratory virus in volunteers available at www.cdc.gov. coronavirus/mers/index middle east respiratory syndrome (mers): case definitions interim surveillance recommendations for human infection with middle east respiratory syndrome coronavirus human coronavirus emc does not require the sars-coronavirus receptor and maintains broad replicative capability in mammalian cell lines human betacoronavirus 2c emc/2012-related viruses in bats, ghana and europe middle east respiratory syndrome coronavirus quasispecies that include homologues of human isolates revealed through whole-genome analysis and virus cultured from dromedary camels in saudi arabia description and clinical treatment of an early outbreak of severe acute respiratory syndrome (sars) in guangzhou, pr china severe acute respiratory syndrome-singapore effectiveness of precautions against droplets and contact in prevention of nosocomial transmission of severe acute respiratory syndrome (sars) viral loads in clinical specimens and sars manifestations a review of studies on animal reservoirs of the sars coronavirus cross-host evolution of severe acute respiratory syndrome coronavirus in palm civet and human molecular evolution of the sars coronavirus during the course of the sars epidemic in china detection of coronavirus-like particles in homosexual men with acquired immunodeficiency and related lymphadenopathy syndrome stool viruses, coinfections, and diarrhea in hiv-infected patients. berlin diarrhea/wasting syndrome study group coronaviruslike particles and other agents in the faeces of children in efate, vanuatu coronavirus-like particles in adults in melbourne, australia coronaviruslike particles in human gastrointestinal disease: epidemiologic, clinical, and laboratory observations an eight-year study of the viral agents of acute gastroenteritis in humans: ultrastructural observations and seasonal distribution with a major emphasis on coronavirus-like particles isolation and characterization of current human coronavirus strains in primary human epithelia cultures reveals differences in target cell tropism ultrastructure of human nasal epithelium during an episode of coronavirus infection signs and symptoms in common colds epidemiological determinants of spread of causal agent of severe acute respiratory syndrome in hong kong haematological manifestations in patients with severe acute respiratory syndrome: retrospective analysis clinical progression and viral load in a community outbreak of coronavirusassociated sars pneumonia: a prospective study detection of sars-cov rna in stool samples of sars patients by nest rt-pcr and its clinical value a major outbreak of severe acute respiratory syndrome in hong kong lung pathology of fatal severe acute respiratory syndrome the clinical pathology of severe acute respiratory syndrome (sars): a report from china multiple organ infection and the pathogenesis of sars time course and cellular localization of sars-cov nucleoprotein and rna in lungs from fatal cases of sars antigenic relationships amongst coronaviruses a case-control study of acute respiratory tract infection in general practice patients in the netherlands role of respiratory viruses in acute upper and lower respiratory tract illness in the first year of life: a birth cohort study coronavirus infection in acute lower respiratory tract disease of infants genetic variability of human coronavirus oc43-, 229e-, and nl63-like strains and their association with lower respiratory tract infections of hospitalized infants and immunocompromised patients the association of viral and bacterial respiratory infections with exacerbations of wheezing in young asthmatic children recurrent wheezy bronchitis and viral respiratory infections detection of rhinovirus, respiratory syncytial virus, and coronavirus infections in acute otitis media by reverse transcriptase polymerase chain reaction coronavirus infections in military recruits: three-year study with coronavirus strains oc43 and 229e respiratory viruses and exacerbations of asthma in adults pan-viral screening of respiratory tract infections in adults with and without asthma reveals unexpected human coronavirus and human rhinovirus diversity rhinovirus and coronavirus infection-associated hospitalizations among older adults a prospective, community-based study on virologic assessment among elderly people with and without symptoms of acute respiratory infection coronavirus 229e-related pneumonia in immunocompromised patients clinical impact of community-acquired respiratory viruses on bronchiolitis obliterans after lung transplant clinical impact of human coronaviruses 229e and oc43 infection in diverse adult populations the united kingdom public health response to an dipeptidyl-peptidase iv from bench to bedside: an update on structural properties, functions, and clinical aspects of the enzyme dpp iv world health organization. global alert and response (gar): infection prevention and control clinical features and short-term outcomes of 144 patients with sars in the greater toronto area acute respiratory distress syndrome in critically ill patients with severe acute respiratory syndrome severe acute respiratory syndrome in children: experience in a regional hospital in hong kong severe acute respiratory syndrome among children severe acute respiratory syndrome: radiographic appearances and pattern of progression in 138 patients imaging of severe acute respiratory syndrome in hong kong severe acute respiratory syndrome: radiographic and ct findings thin-section ct of severe acute respiratory syndrome: evaluation of 73 patients exposed to or with the disease early enhanced expression of interferon-inducible protein-10 (cxcl-10) and other chemokines predicts adverse outcome in severe acute respiratory syndrome interferon-mediated immunopathological events are associated with atypical innate and adaptive immune responses in patients with severe acute respiratory syndrome critically ill patients with severe acute respiratory syndrome clinical presentations and outcome of severe acute respiratory syndrome in children severe acute respiratory syndrome and pregnancy viral discovery and sequence recovery using dna microarrays corona virus induced subacute demyelinating encephalomyelitis in rats: a morphological analysis two coronaviruses isolated from central nervous system tissue of two multiple sclerosis patients detection of coronavirus rna and antigen in multiple sclerosis brain human coronavirus gene expression in the brains of multiple sclerosis patients long-term human coronavirus-myelin cross-reactive t-cell clones derived from multiple sclerosis patients detection of coronavirus in the central nervous system of a child with acute disseminated encephalomyelitis diagnosis of human coronavirus infection by immunofluorescence: method and application to respiratory disease in hospitalized children surveillance of community-acquired viral infections due to respiratory viruses in rhone-alpes (france) during winter 1994 to 1995 clinical disease in children associated with newly described coronavirus subtypes detection of a novel human coronavirus by real-time reversetranscription polymerase chain reaction development of a standard treatment protocol for severe acute respiratory syndrome treatment and vaccines for severe acute respiratory syndrome sars: systematic review of treatment effects pegylated interferon-alpha protects type 1 pneumocytes against sars coronavirus infection in macaques efficient replication of the novel human betacoronavirus emc on primary human epithelium highlights its zoonotic potential human cell tropism and innate immune system interactions of human respiratory coronavirus emc compared to those of severe acute respiratory syndrome coronavirus the spike protein of sars-cova target for vaccine and therapeutic development key: cord-318229-29cgwivt authors: baier, claas; haid, sibylle; beilken, andreas; behnert, astrid; wetzke, martin; brown, richard j. p.; schmitt, corinna; ebadi, ella; hansen, gesine; schulz, thomas f.; pietschmann, thomas; bange, franz-christoph title: molecular characteristics and successful management of a respiratory syncytial virus outbreak among pediatric patients with hemato-oncological disease date: 2018-02-13 journal: antimicrob resist infect control doi: 10.1186/s13756-018-0316-2 sha: doc_id: 318229 cord_uid: 29cgwivt background: respiratory syncytial virus (rsv) is responsible for upper and lower respiratory tract infection in adults and children. especially immunocompromised patients are at high risk for a severe course of infection, and mortality is increased. moreover rsv can spread in healthcare settings and can cause outbreaks. herein we demonstrate the successful control and characteristics of a rsv outbreak that included 8 patients in our department of pediatric hematology and oncology. methods: we performed an epidemiologic investigation and a molecular analysis of the outbreak strains. moreover we present the outbreak control bundle and our concept for rsv screening in the winter season. results: rsv a and b strains caused the outbreak. rsv b strains affected 3 patients, 2 of whom were co-infected with rsv a. exactly this rsv a strain was detected in another 5 patients. our multimodal infection control bundle including prophylactic rsv screening was able to rapidly stop the outbreak. conclusion: an infection control bundle in rsv outbreaks should address all potential transmission pathways. in pediatric settings the restriction of social activities might have a temporal negative impact on quality of life but helps to limit transmission opportunities. molecular analysis allows better understanding of rsv outbreaks and, if done in a timely manner, might be helpful for guidance of infection control measures. respiratory syncytial virus (rsv) of the family pneumoviridae is a single stranded rna-virus with two antigenic different subtypes (a and b). it causes upper and lower respiratory tract infection (urti and lrti) in children and adults in a seasonal pattern [1] [2] [3] [4] . the median incubation period is 4.4 days [5] , ranging from 2 to 8 days. human to human transmission takes place via droplets as well as direct and indirect contact (e.g. contaminated surfaces or hands of medical staff ). patients with hemato-oncological disease are at risk for severe rsv-caused infection -especially in the context of hematopoietic stem cell transplantation (hsct) [6, 7] . in the literature varying rsv-related case fatality rates are reported in children with cancer to range from 5% to 33% [8] [9] [10] . respiratory tract infection (rti) due to rsv is typically community/household-acquired. rsv is a member of the so called community-acquired respiratory viruses such as influenza virus. nevertheless, hospital (nosocomial) acquisition is possible as well and transmission may occur by other infected patients, staff or visitors [11, 12] . rsv outbreaks in inpatient pediatric oncologic care facilities and in adult hematology and oncology units have been described [9, [12] [13] [14] [15] [16] . an understanding of transmission pathways helps to guide adequate outbreak control measures and to implement prophylactic measures. finally, rsv-caused respiratory tract infections are a differential diagnosis worth considering in neutropenic cancer patients with fever [17, 18] . therapeutic options for patients in hematopoietic stem cell transplantation and with intensive cancer therapy, who are severely infected by rsv, include the use of systemic or aerosolized ribavirin and polyclonal intravenous immunoglobulins (ivig) [19, 20] . the rsvspecific monoclonal antibody palivizumab has been used for treatment and for passive immunization (prophylaxis) in high risk pediatric patient groups [19] [20] [21] . here we describe the successful management and characteristics of a rsv outbreak in a single non-hsct pediatric hematology and oncology ward including 8 patients in march and april 2016. moreover, we show the results of the molecular strain analysis. an outbreak case is a patient with a positive rsv laboratory testing in samples from the upper or lower respiratory tract and a definite or possible nosocomial onset. a definite nosocomial case was defined as a positive rsv laboratory testing on day 5 or later of the hospital stay. a possible nosocomial case was defined as a positive rsv laboratory testing on day 2 to 4 of hospital stay. patients who were admitted to the ward with a new positive rsv laboratory testing, and had been on the ward within 8 days prior to admission were also considered a possible nosocomial case. all patients that were accommodated in the same room with cases were considered contacts. a combined nose/throat swab was taken for routine viral diagnostics. material from the lower respiratory tract was suitable as well. samples taken for diagnostic purposes were processed at the institute of virology using real-time rt-pcr or direct fluorescent antibody (dfa) staining. rna was extracted from the specimens using a qiaamp viral rna mini kit in a qiacube according to the manufacturer's instruction (qiagen, hilden, germany). cdna synthesis, amplification and detection of nucleic acid were performed in an applied biosystems® 7500 real-time pcr system (life technologies, carlsbad, california) by a commercially available one-step real-time rt-pcr kit (rsv/hmpv r-gene® pcr kit, biomérieux, nürtingen, germany) according to the manufacturer's instructions. for dfa staining a ready to use fitc (fluorescein isothiocyanate) -labeled monoclonal rsv antibody (light diagnostics, merck, darmstadt, germany) was used according to a protocol described before [22] . pcr and dfa did not differentiate between rsv a and b. one diagnostic specimen was tested using a point-of-care test (poct) system (sofia, quidel, kornwestheim, germany), which is available in the pediatric emergency room. nasopharyngeal aspirates from 6 outbreak patients (case 1, 2, 3, 5, 6, and 7) were taken only for strain typing purposes on one occasion (march18 th ), which were exclusively processed at the institute for experimental virology, twincore -centre for experimental and clinical infection research. in addition, selected archived (frozen) samples taken for diagnostic purposes from outbreak patients (case 2, 3, 4, 5, 7, 8) were provided by the institute of virology and processed at the institute for experimental virology, twincore -centre for experimental and clinical infection research. linearized acrylamide (ambion, thermo fisher scientific; 35 μg/ml final concentration) was added to the sample and total rna was extracted from 140 μl of aspirate according to the manufacturer's description (qiaamp viral rna mini kit, qiagen, hilden, germany). cdna synthesis was performed using the superscript iii kit from invitrogen (invitrogen, darmstadt, germany) and random hexamer primers. next, a nested pcr was performed first amplifying the rsv-g and f protein coding region and in a second round amplifying the g protein gene. pcr products were sent for sanger sequencing (gatc, konstanz, germany) and the sequences were analyzed using mega software and the highlighter analysis tool [23] . we obtained ethical approval for this study from the ethics committee of the hannover medical school. the outbreak occurred in the clinic for pediatric hematology and oncology which is a tertiary referral center for children from 0-18 years with hematologic and solid neoplasia. the affected ward harbors 5 singleand 5 two-bed rooms. each single room has an anteroom and a high-efficiency particulate air filtration with the air flow directed to the hallway. the same floor houses the outpatient clinic and a recreation room for the hemato-oncological pediatric patients. exchange from patients between the ward and the outpatient clinic occurs regularly. moreover two recreation rooms for social activities are part of the ward. most patients receive antineoplastic therapy during their stay. a substantial number of patients have a neutrophil count below 500 cells / μl. the ward is serviced by permanent health care workers (hcws) and house-keeping staff. external personnel (such as medical consultants or physiotherapists) enter the ward when necessary. autologous and allogenic hsct are performed on a separate ward with 6 single rooms. parents are allowed to stay overnight with their children. patients with a positive rsv test are electronically marked in the hospital alert system. besides single room accommodation, contact and droplet precautions (surgical mask, gown, gloves) are used at any time by visitors and hcws when entering the room of a rsv positive patient. affected patients are encouraged to stay in their room and are trained in hygienic hand washing to minimize spread via contact. when leaving the room becomes necessary (e.g. for examination), patients wear a surgical mask. these measures also apply for patients with typical respiratory symptoms (e.g. cough or sneezing) before a pathogen is identified. measures are usually suspended when there are two negative rsv pcr-based test results at a minimum 2-day interval and therefore patients are not anymore considered infectious. hcws with symptoms of urti are suspended from direct patient care and wear a surgical mask while on the ward. visitors with symptoms of acute rti are not permitted to enter the ward. strict hand hygiene following who guidelines is implemented. targeted rsv diagnostics are performed in case of suspected viral rti. positive testing for respiratory viruses is regularly reported for epidemiologic and infection control reasons to the infection control staff. in march a total of 8 patients (cases 1 to 8) were tested rsv-positive in respiratory samples. patient characteristics are shown in table 1 . 6 patients fulfilled criteria for nosocomial acquisition. the remaining 2 patients had a possible nosocomial acquisition, as they had been on the ward within 8 days prior to admission. epidemic curve and an outbreak timeline with the diagnostic results can be seen in figs 1 and 2. case 1 was neutropenic and developed severe lrti with a rsv positive bronchoalveolar lavage and a requirement for oxygen. case 2 also suffered from lrti, which was less severe. cases 3 to 8 had respiratory symptoms of an urti such as cough, sneeze and a positive rsv testing in secretions from the upper respiratory tract. case 6 was co-infected with influenza a virus . 4 patients received oral ribavirin therapy (case 1, 2, 5, 8) and 5 patients (case 1 to 5) temporarily required supportive oxygen administration via nasal cannula. ivig or palivizumab was not administered. no direct rsvassociated mortality was observed. in addition, all patients were empirically treated by antibiotics presuming bacterial co-respectively superinfection according to inhouse standards (table 1) . viral persistence (viral shedding) is defined as the time period from first positive diagnostic test to sustained negativity. duration of viral persistence was minimally 4 days (case 7) and maximally at least 63 days (case 1)see also table 1 . active outbreak management was started after detection of 3 new rsv infected patients in calendar week 10 (see fig. 1 ). an outbreak control team consisting of the infection control unit and the physicians in charge was established. the head of the clinic for pediatric hematology and oncology, the medical director of our institution, and the public health authority were informed. in addition to the existing standard infection control measures described above, interventional measures were introduced. all hcws (including permanent staff members and external personnel), visitors and outpatients were required to wear a surgical mask at any time (patient care and non-patient care activities) when on the ward and in the outpatient clinic (preemptive barrier precaution). moreover, roommates of patients tested positive for rsv in their clinical course were moved to single rooms for 8 days (typical maximum incubation period; so called quarantine). they were repeatedly tested for rsv using pcr. all newly admitted patients were tested for rsv (admission screening). twice weekly pcr rsv prevalence screening for all patients on ward was established (prevalence screening). if possible, elective patient admissions were delayed to reduce patientto-nurse ratio. moreover, only parents were allowed as visitors. all social activities for the patients and relatives were suspended. during outbreak, all two bed-rooms were occupied by one patient only, meaning single room isolation for all patients on the ward. repeated training sessions for staff were provided by the infection control team. they addressed rsv transmission pathways and underlined the importance of droplet precautions (e.g. masks and cough etiquette for hcws and visitors) as well as hand hygiene. intervention measures were fully implemented on 22 nd of march. the last nosocomial case occurred on 29 th of march (case 8). however, the patient had been discharged from the ward on 22 nd of march, and readmitted on 29 th of march (fig. 2) . thus, after intervention measures had been in place no further nosocomial rsv for phylogenetic analysis of the rsv genome we focused on the viral glycoprotein g of rsv as this gene is highly variable and shows the highest sequence variance between the rsv subgroups a and b (53% amino acid sequence identity [24] ). from the total of 8 patients, five of them were infected with a rsv a strain (case 1, 3, 4, 5, 6), one was tested positive for rsv b (case 8) and two patients were infected with rsv a and b (case 2 and 7) dependent on the time point of sampling. despite the coexistence of genetically definite genotypic strains with many nucleotide exchanges especially in the c-terminal variable region of rsv-g [25] [26] [27] , we detected the very same nucleotide sequence for the coding region of the rsv-g protein for all patients infected with rsv a (fig. 3a) . only one single nucleotide exchange was detected in the intergenic region of the virus infecting patient c3 and only at one time point of sampling ( fig. 3a; c3_13_3 ). in the specimen taken 5 days later (c3_18_3), this variation was no longer detectable. among the three patients infected with rsv b, we observed 4 nucleotide differences in the coding region of the g protein (fig. 3b) . two additional variations were observed in the intergenic region of the g gene (fig. 3b) . the rsv b viruses infecting patients c2 and c7 were almost identical with merely three nucleotide differences between them. notably, the chromatograms of the sanger sequencing from these patients revealed sequence ambiguity at exactly these three positions: between g/a at position 907, between t/a at position 974 and g/a at residue 979. while in case c7, residues g, t and g were dominant, in patient c2, residues a, a, a predominated. this result suggested that these two patients were infected with an essentially identical viral quasispecies and that merely the relative number of viruses with g, t, g residues compared to viruses with a, a, a nucleotides at these positions varied between these two patients. in contrast, patient c8 carried a rsv b virus with an unambiguous sequence at these three positions g, t, g (fig. 3d) . moreover, it displayed three additional polymorphisms in the g protein coding region relative to the virus infecting patients c2 and c7, thus supporting the conclusion that this patient was infected with a different rsv b virus. noticeably, case 2 was tested rsv negative in two samples taken and processed for routine diagnostic on march 15 th and 18 th . rsv strain sequences available from other, nonoutbreak pediatric patients (same season as the outbreak) were used for comparison. these strains show a predominantly polyclonal pattern for rsv a (fig. 3c) . interestingly, one patient (rsv_02_1) was infected with the very same rsv b strain as cases 2 and 7 (fig. 3c , lower part), whereas there were clear sequence differences for the other rsv b strains isolated from non-outbreak pediatric patients. for our clinic of pediatric hematology and oncology this was the first actively managed rsv-outbreak. in the previous two winter seasons in total only 3 rsv-positive patients were detected on the affected ward. we studied the epidemiologic and molecular background of this outbreak. considering bed and room occupancy on the ward during the outbreak, direct patient to patient transmission (e.g. via droplets or contaminated surfaces) in cases 1 and 2 as well as 3 and 4 seemed epidemiologically possible as each pair was accommodated in the same room before samples were tested positive for rsv. cases 5 and 7 acquired rsv at day 6 and 5 of their stay on the ward, respectively, suggesting nosocomial rsv acquisition. these two patients did not share rooms with other infected patients but were on ward during the outbreak. cases 6 and 8 were tested positive for rsv on admission. nosocomial acquisition was considered possible, as case 6 and 8 had been discharged 8 and 7 days, respectively, from the affected ward prior to re-admission. during this previous stay patients with symptoms of rti and a positive rsv test were already on the ward. nonetheless, community-onset still was an option for case 6 and 8. based on this epidemiologic background, our main hypothesis was that direct and indirect patient to patient transmission (the latter for example via the hcws' hands) caused the outbreak. however, at this point transmission by an infected visitor or hcws acting as a point source could not be excluded. moreover, taking all epidemiological data into account, a random introduction of several different community-acquired strains seemed unlikely to us. we suspected ongoing transmission of a single rsv variant and sequencing was used retrospectively to test this hypothesis (see below). the standard, pre-outbreak infection control measures regarding rsv were mainly in line with previously made recommendations for hospitalized patients with hematooncologic disease [19, 28] . the additionally implemented fig. 3 highlighter plot depicting nucleotide mismatches comparing the sequence of the strain obtained from patient c1 to all other rsv a strains, and the sequence of the strain obtained from patient c7 to all other rsv b strains. a rsv a and (b) rsv b glycoprotein sequences were aligned using mega and depicted as highlighter plot using the highlighter analysis tool [23] . nucleotide exchanges compared to a reference sequence (c1_18_3 (1) for rsv a and c7_18_3 (1) for rsv b) are depicted in color. absence of sequence information is depicted as grey bar. a schematic of the rsv g protein with the different domains is depicted on top [modified from [39] ]. (1) indicates samples taken exclusively for strain typing at the 18 th of march and (2) indicates samples collected for routine viral diagnostics. c rsv-g sequence alignment from other pediatric, non-outbreak patients compared to the references c1 and c7, respectively. d sequencing chromatograms for rsv b cases. the depicted area is highlighted by * and ** in figure 3b . overlying sequence information from different quasispecies detected in the samples are highlighted in a box measures, in particular single room accommodation for contact patients (quarantine), suspension of all social activities, and surgical masks for all hcws and visitors at any time, addressed the postulated rsv transmission pathways during this outbreak. these postulated pathways were direct patient to patient transmission (e.g. roommate to roommate), but also transmission via hcws and visitors. direct patient to patient transmission as the most probable route of infection has been shown by lehners et al. in a large rsv outbreak in a german hematology and transplant unit [11] . jensen et al. described direct patient to patient transmission, mixed with introduction of strains from outside, in an outbreak affecting immunocompromised adults [29] . we therefore focused on patient to patient transmission early during the outbreak by strict isolation precautions for rsv infected patients and contacts. isolation for infected patients was also a key measure in a multimodal control bundle described by inkster et al. [15] . contact patients were isolated for 8 days and repeatedly tested in order to disrupt infection chains as described in literature [11] . this so called quarantine concerned 2 patients in our outbreak. one of them (case 4) was eventually tested rsv-positive at day 8 of quarantine while being negative at day 2 and 5. this underlines the value of the measure. finally, we reemphasized in training sessions the need for preemptive isolation of patients with respiratory symptoms. as all these measures required more isolation capacity on the ward, we restricted elective admissions and located all patients in single rooms. as another measure we reduced direct patient to patient contacts on the ward by suspending community events, as active social behavior can be a risk factor for nosocomial rsv acquisition [30] . even so this noticeably restricted the social life for the patients and their families during the outbreak, we enforced this measure. we further restricted social contacts by temporally limiting visits of infants to the ward, as (especially young) infants are known to be the main reservoir for rsv and as our outbreak was approximately concurrent (slightly delayed) to the rsv community peak. only parents were allowed to the ward, which is in line with an intervention done by kelley et al. [12] . a restrictive visiting policy is as well described by singh et al. in a pediatric rsv outbreak [14] . the use of surgical masks for everyone on the ward is an important measure to prevent droplet associated nosocomial rsv transmissions. this is even more rational as rsv may be transmitted via symptomless or oligosymptomatic persons (e.g. hcws or visitors) and the infectious period can in fact already begin 1 to 2 days before actual onset of symptoms. a literature review by french et al. concluded that personal protective equipment might be advantageous for reducing nosocomial rsv transmission [31] . kelly et al. showed that five hcws showing only mild symptoms were involved in a rsv outbreak on an adult stem cell transplant unit [12] . this underlines the necessity that hcws with respiratory symptoms should not participate in direct patient care activities, at least in a high risk patient care setting. we re-emphasized this issue in training sessions for the hcws. although staff screening is described in literature [15] , we were able to terminate this outbreak without staff screening. a cohort of hcws to take care of solely rsv-positive patients as reported before [9] had also not been established but would have been another option in case of an ongoing outbreak. temporal survival of respiratory viruses in general [32] and specifically rsv [33] on inanimate surfaces is described, thus contact transmission via the hands of staff was conceivable for nosocomial acquisition. this is especially of importance as cough etiquette and compliance to basic hygienic principles may be reduced for obvious reasons in pediatric patients, so a higher environmental rsv burden is probable. nonetheless we did not implement changes in the well established cleaning and disinfection procedures on the ward. we detected prolonged rsv persistence (virus shedding), which has been reported in patients with hematological disorders [34] . this finding needs to be considered for efficient outbreak control and favors the practice of repeated testing in immunocompromised patients as we did. likewise, this is important as pediatric hemato-oncologic patients are often readmitted several times for cancer treatment cycles or fever in neutropenia. when symptoms are no longer present or mild but viruses are still being shed, rsv may be re-introduced to the ward. thus, for termination of isolation precautions during the outbreak, we required negative results as reported before [35] . in fact, two subsequent negative results at a minimum 2-day interval were necessary. the usefulness of this requirement is supported by the longitudinal course of the samples from patient 5 which were obtained in april and may. this patient produced positive specimens on two occasions, after one specimen had been tested negative (see fig. 2 ). active rsv-surveillance by screening on admission and twice weekly for all patients on the ward insured rapid detection of rsv-positive patients. this is in line with successful infection control measures reported in literature [9, 12] . we presume that a prophylactic admission and prevalence rsv screening for all patients in the winter season might be helpful as a preventive measure in high risk populations. therefore, one consequence of this outbreak was the implementation of an active rsv surveillance (admission and prevalence screening once weekly) in our clinic for pediatric hematology and oncology during the rsv season. the beginning and ending of this seasonal screening period is determined by in-house and regional/national rsv epidemiology [36] . moreover, pre-rsv-season audits involving clinicians, infection control staff and the institute of virology take place to ensure timely beginning of screening procedures and adherence to the existing infection control practices. molecular characterization of rsv strains, for instance by whole genome sequencing [37] or characterization of rsv g-protein [16, 38] , has been used to investigate nosocomial rsv outbreaks. we were able to collect and examine selected outbreak strains by g-protein gene sequencing. we found that cases 1 to 7 were infected with an rsv a virus with identical g protein coding region. in case of patient c3 one nucleotide difference in the intergenic region of the g gene was observed in one of two samples collected five days apart (fig. 3a . it is possible that this change was due to natural drift of the infecting virus over time or that this polymorphism is indicative of the presence of two slightly different viruses replicating in parallel and dominating on the one and the other day of sampling, respectively. moreover we found that case 8 had a rsv-b infection and that cases 2 and 7 were co-infected by rsv a and rsv b viruses. while sequence analysis of the earlier samples of case 2 and 7 revealed infection by the rsv a virus, the sequence analysis of the later specimen showed infection by an rsv b virus. with the available specimen, we were unable to distinguish if these two patients had a prolonged co-infection between these viruses or if they were sequentially infected by rsv a and rsv b. these findings became available only after the outbreak ended, as routine virological testing during the outbreak did not include molecular differentiation of rsv a and b. in retrospect, these results indicated the decision not to cohort rsv-patients during the outbreak, as we probably might have cohorted rsv-patients with different subtypes. detailed sequencing analysis suggests that cases 2 and 7 were infected by an almost identical rsv b virus population. we observed three nucleotide differences between these viruses; however, nucleotides of the viruses at these three positions were ambiguous in both cases (g,t,g versus a,a,a residues). thus, both patients were likely infected by a highly similar rsv b quasispecies which was characterized by two different nucleotide signatures varying in abundance between patients. in contrast, the rsv b virus infecting patient c8 differed in two key criteria. first, it did not show any sequence ambiguity at the three above mentioned residues that was characteristic for the rsv b virus population observed in patients c2 and c7. second, it displayed three additional polymorphisms in the coding region of the g protein. taken together, this suggests that patient c8 was infected by another rsv b virus and that there was no transmission from patients c2 and c7 to patient c8. outbreak strains of the subtype rsv a were highly similar and different from polyclonal strains from other nonoutbreak pediatric patients (fig. 3c) . we therefore conclude that a single rsv a strain was introduced to the ward and then spread within the ward. interestingly, the rsv b isolate c7_18_3 (1) was identical to the community strain rsv_02_1, however further epidemiologic and clinical information are not accessible for the non-outbreak patient. taken together, the nucleotide analysis suggests independent introductions of at least 2 different rsv b strains into the ward affecting patient c2, c7 and c8, and transmission of one rsv a strain on the ward between patients c1 to c7. looking exclusively at the molecular analysis, it is not possible to disclose the exact transmission pathway of rsv a. rsv a might have been introduced to the ward by an infected patient (index patient) on the ward (maybe case 1) and was then successively transmitted from patient to patient. alternatively, a point source, such as a rsv-positive hcw, may have caused the outbreak. however, in correlation with the epidemiologic observations such as overlapping patient stays on the ward, stay of case 1 and 2, and case 3 and 4 in a double room, and social activity on the ward in the initial phase of the outbreak, we consider a direct and indirect patient to patient transmission most likely. rsv poses a significant infectious threat to pediatric patients with an underlying oncologic disease. this outbreak and other outbreaks reported in literature demonstrate the potential of rsv to spread in a hospital. we strictly enforced our existing infection control practices and implemented temporally additional measures to terminate the outbreak. according to our experiences an outbreak control bundle for rsv should include (preemptive) barrier precautions (especially masks), prevalence and admission screenings for all patients, and strict isolation procedures for infected patients and contact patients. quarantine for contacts should at least be for 8 days, the usual maximal incubation period of rsv. in pediatric settings the restriction of visitors (especially siblings) and social activities on the ward can be helpful to prevent transmission and rsv introduction from outside, but definitely limits social life quality. as shown in other outbreaks with viral and bacterial pathogens restriction of admissions still is a very effective measure as it enables single room accommodation for all or the majority of the patients. moreover, a decrease of the patient to nurse ratio makes transmission more unlikely. in our case the molecular analysis was very helpful to verify the true outbreak character of the rsv cluster and revealed ongoing transmission of an unique rsv a strain on the ward, and an probable independent introduction of different rsv b strains into the ward. respiratory syncytial virus -a comprehensive review respiratory syncytial virus: the virus, the disease and the immune response defining the epidemiology and burden of severe respiratory syncytial virus infection among infants and children in western countries respiratory syncytial virus: how, why and what to do incubation periods of acute respiratory viral infections: a systematic review respiratory syncytial virus infection in patients with hematological diseases: single-center study and review of the literature respiratory syncytial virus infection in recipients of allogeneic stem-cell transplantation: a retrospective study of the incidence, clinical features, and outcome respiratory syncytial virus infection in infants with acute leukemia: a retrospective survey of the japanese pediatric leukemia/lymphoma study group detection, control, and management of a respiratory syncytial virus outbreak in a pediatric hematology-oncology department respiratory syncytial virus infections in children with cancer risk factors and containment of respiratory syncytial virus outbreak in a hematology and transplant unit respiratory syncytial virus outbreak on an adult stem cell transplant unit respiratory syncytial virus infection outbreak among pediatric patients with oncologic diseases and/or bmt hospital outbreak of human respiratory syncytial virus (hrsv) illness in immunocompromised hospitalized children during summer consecutive yearly outbreaks of respiratory syncytial virus in a haemato-oncology ward and efficacy of infection control measures the clinical and phylogenetic investigation for a nosocomial outbreak of respiratory syncytial virus infection in an adult hemato-oncology unit respiratory viral infections and co-infections in children with cancer, fever and neutropenia frequent respiratory viral infections in children with febrile neutropenia -a prospective follow-up study fourth european conference on infections in leukaemia (ecil-4): guidelines for diagnosis and treatment of human respiratory syncytial virus, parainfluenza virus, metapneumovirus, rhinovirus, and coronavirus how i treat respiratory viral infections in the setting of intensive chemotherapy or hematopoietic cell transplantation updated guidance for palivizumab prophylaxis among infants and young children at increased risk of hospitalization for respiratory syncytial virus infection comparison of the performance of direct fluorescent antibody staining, a point-of-care rapid antigen test and virus isolation with that of rt-pcr for the detection of novel 2009 influenza a (h1n1) virus in respiratory specimens identification and characterization of transmitted and early founder virus envelopes in primary hiv-1 infection the g glycoprotein of human respiratory syncytial viruses of subgroups a and b: extensive sequence divergence between antigenically related proteins evolution of subgroup a respiratory syncytial virus: evidence for progressive accumulation of amino acid changes in the attachment protein genetic diversity of the attachment protein of subgroup b respiratory syncytial viruses identification of variable domains of the attachment (g) protein of subgroup a respiratory syncytial viruses guidelines for preventing infectious complications among hematopoietic cell transplantation recipients: a global perspective outbreak of respiratory syncytial virus (rsv) infection in immunocompromised adults on a hematology ward contributing and terminating factors of a large rsv outbreak in an adult hematology and transplant unit risk of nosocomial respiratory syncytial virus infection and effectiveness of control measures to prevent transmission events: a systematic review how long do nosocomial pathogens persist on inanimate surfaces? a systematic review possible transmission by fomites of respiratory syncytial virus long-term shedding of influenza virus, parainfluenza virus, respiratory syncytial virus and nosocomial epidemiology in patients with hematological disorders community acquired respiratory virus infections in cancer patients-guideline on diagnosis and management by the infectious diseases working party of the german society for haematology and medical oncology defining the timing of respiratory syncytial virus (rsv) outbreaks: an epidemiological study investigation of respiratory syncytial virus outbreak on an adult stem cell transplant unit using whole genome sequencing molecular characterization of a respiratory syncytial virus outbreak in a hematology unit in heidelberg structure and function of respiratory syncytial virus surface glycoproteins not applicable. this research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.availability of data and materials all data generated or analysed during the current study are included in this published article. all authors contributed to the manuscript according to the icmje (international committee of medial journal editors) recommendations: all authors were involved in data acquisition, analysis and interpretation. sh, rb and tp carried out molecular analysis. cb, sh, tp and f-cb prepared the manuscript. cb organized the drafting process. asb and anb, cb and f-cb were involved in the active outbreak management. ts and cs carried routine rsv diagnostic. mw collected specimens for typing purposes. all authors critically revised the manuscript and account for accuracy and correctness. all authors have read and agreed to the final draft before submission. we obtained ethical approval for this study from the ethics committee of the hannover medical school (nr. 3450-2016). submit your next manuscript to biomed central and we will help you at every step: key: cord-336420-1a2u9p4t authors: söderman, martina; rhedin, samuel; tolfvenstam, thomas; rotzén-östlund, maria; albert, jan; broliden, kristina; lindblom, anna title: frequent respiratory viral infections in children with febrile neutropenia a prospective follow-up study date: 2016-06-16 journal: plos one doi: 10.1371/journal.pone.0157398 sha: doc_id: 336420 cord_uid: 1a2u9p4t objective: febrile neutropenia is common in children undergoing chemotherapy for the treatment of malignancies. in the majority of cases, the cause of the fever is unknown. although respiratory viruses are commonly associated with this condition, the etiologic significance of this finding remains unclear and is therefore the subject of this study. study design: nasopharyngeal aspirates were collected during 87 episodes of febrile neutropenia in children age 0–18 years, being treated at a children’s oncology unit between january 2013 and june 2014. real-time polymerase chain reaction was used to determine the presence of 16 respiratory viruses. follow-up samples were collected from children who tested positive for one or more respiratory viruses. rhinoviruses were genotyped by vp4/vp2 sequencing. fisher’s exact test and mann-whitney u test were used for group comparisons. results: at least one respiratory virus was detected in samples from 39 of 87 episodes of febrile neutropenia (45%), with rhinoviruses the most frequently detected. follow-up samples were collected after a median of 28 days (range, 9–74 days) in 32 of the 39 virus-positive episodes. the respiratory viral infection had resolved in 25 episodes (78%). the same virus was detected at follow-up in one coronavirus and six rhinovirus episodes. genotyping revealed a different rhinovirus species in two of the six rhinovirus infections. conclusion: the frequency of respiratory viral infections in this group of patients suggests an etiologic role in febrile neutropenia. however, these findings must be confirmed in larger patient cohorts. febrile neutropenia is a common complication in children undergoing chemotherapy for the treatment of malignancies. because septicemia (which is potentially lethal) is difficult to rule out at the onset of fever, empiric treatment with broad-spectrum antibiotics is promptly initiated based on wide indications [1] . however, in most cases, no underlying cause of the fever can be identified [2] . febrile neutropenia is associated with long hospitalization [3] [4] [5] which has negative social effects for the child and its family [6] . in addition, hospitalization and the use of broad-spectrum antibiotics increase the patient's risk of subsequent infection with antibiotic-resistant bacteria [7, 8] and fungal infections [9, 10] . a better understanding of the etiology of febrile neutropenia is thus needed in order to decrease unnecessary hospitalization and excessive antibiotic use. as infections with respiratory viruses are a common morbidity in children [11] respiratory viruses likely play a significant role in childhood febrile neutropenia. advances in molecular methods have increased the sensitivity of viral diagnostics tests, with recent studies reporting the detection of respiratory viruses in the nasopharynx in 44-57% of childhood febrile neutropenia episodes using real-time polymerase chain reaction (pcr) [3] [4] [5] 12] . however, the clinical significance of positive pcr findings is unclear, as respiratory viruses have also been detected in asymptomatic immunocompetent children [13] [14] [15] [16] . in addition, some respiratory viruses can be detected weeks after the first infection, which is suggestive of prolonged viral shedding [17, 18] . here, we describe the results of a longitudinal study involving repeated sampling and the assessment of a broad panel of respiratory viruses by pcr to clarify whether respiratory viruses play a causal role in childhood febrile neutropenia. children aged 0-18 years, who were treated for a malignancy at the childhood cancer unit at astrid lindgren children's hospital in stockholm, sweden, between january 2013 and june 2014, were eligible for enrollment in this study. all the patients who met the criteria for febrile neutropenia were asked to participate. patients could be enrolled multiple times if recurrent episodes of febrile neutropenia occurred during the study period. to be enrolled for a new episode, the patient needed to have been afebrile for more than 72 hours and have completed the antibiotic treatment for the previous episode of febrile neutropenia. febrile neutropenia was defined as a body temperature of 38.5°c on one occasion or 38.0°c on two occasions at least 60 minutes apart, combined with an absolute neutrophil count of either 0.5×10 9 /l on one occasion or 1.0×10 9 /l with a decline to less than 0.5×10 9 /l over a subsequent 48-hour period. oral and written information regarding the study were provided to each patient prior to enrollment, and signed consents were obtained from their caretakers. the study was approved by the regional ethical review board in stockholm. university laboratory (iso: 15189:2012) for microbiological analysis. viral nucleic acids were extracted from the npa with a magattract virus mini m48 kit (qiagen, sollentuna, sweden) and analyzed with in-house real-time pcrs for the following 16 viruses: adenovirus (hadv); bocavirus (hbov); coronaviruses nl63/oc43/229e/hku1 (hcov); enterovirus (ev); influenza virus a, including a(h1n1)pdm09 and b (flu); metapneumovirus (hmpv); parainfluenza viruses 1-3 (piv); respiratory syncytial virus (rsv) and rhinovirus (rv) [19] . a semiquantitative assay was used and the actual cycle thresholds-values (ct-values) were provided, however, the npa has not been validated for quantitative data. in all study subjects who initially tested positive for one or more respiratory viruses, a follow-up sample was collected at the time of their next visit to the hospital. this follow-up sample was collected regardless of the patient's absolute neutrophil count or symptoms and was analyzed in the same manner as the first sample. however, if the patient had a new episode of febrile neutropenia at the time of the follow-up, this sample was considered both a follow-up sample and a new episode sample, which required an additional follow-up sample if the new episode tested positive for a respiratory virus. rv/ev genotyping was performed by vp4/vp2 sequencing for samples that were pcr-positive for rv and/or ev using a recently published method [20] based on a method and primers originally published by wisdom et al. [21] . briefly, viral rna was extracted as described above for most samples, but the rneasy lipid tissue mini kit (qiagen, sollentuna, sweden) was used for samples that were negative in the vp4/vp2 pcr after magattract extraction. the rna was used for the nested reverse transcriptase (rt)-pcr with one-step superscript-platinum taq (life technologies, stockholm, sweden) for the first (outer) pcr, and platinum taq for the second (nested) pcr. sequencing was done on an abi 3730 instrument and the rv/ev species and type was determined by maximum likelihood phylogenetic trees constructed using phyml [22] and the sequences have been deposited in genbank under accession numbers kx15472-kx154585 for rv-a and kx290514-kx290520 for rv-c. two of the rv-c samples were not submitted to the genbank because the sequences were of suboptimal quality due to a probable infection with more than one rv genotype. blood cultures were collected from all patients for the detection of bacterial infections and analyzed at the karolinska university laboratory, as per routine clinical procedures. clinical data, including the results of biochemical and microbiological analyses, patient's characteristics, treatment during the febrile episode, fever characteristics, and the duration of hospitalization, were collected from the medical records. data were analyzed using graphpad prism 6.0 software (graphpad prism, san diego, ca). fisher's exact test and the mann-whitney u test were used for group comparisons of categorical and continuous data, respectively. a p-value less than 0.05 was considered statistically significant. a total of 56 patients representing 92 episodes of febrile neutropenia were enrolled in the study. five episodes were excluded due to incomplete sampling. therefore, the analyses included 54 patients with 87 episodes of febrile neutropenia (ranges, 1-5 episodes per patient and 8-214 days between episodes). the characteristics of the study participants are listed in table 1 . the median age across all episodes was 7 years (range, 0.5-17.7 years), and 59% of the episodes occurred in females. the patient was undergoing treatment for a hematologic malignancy in 51% of the episodes and for a solid tumor in 49% of the episodes. respiratory symptoms were noted in 70% of the episodes of febrile neutropenia ( table 1) . at least one respiratory virus was identified in 39 (45%) of the 87 episodes. a single respiratory virus was detected in 34 (39%) of the episodes (table 1) . multiple viruses (range, 2-4 viruses) were detected in two episodes (2%), whereas co-presence with a respiratory virus and septicemia was detected in three episodes (3%) ( table 1) . of the 87 episodes, rv was the most frequently detected respiratory virus (n = 21, 24%), followed by hcov (n = 7, 8%), flu (n = 4, 5%), rsv (n = 3, 3%), piv (n = 3, 3%), hmpv (n = 2, 2%), hbov (n = 2, 2%), and hadv tumor type 0.14 0.04 hematologic d malignancy days with antibiotics (n = 1, 1%) ( table 2 ). in 12 of the virus-positive episodes, the patient was <4 years of age. the most common respiratory virus identified in the group <4 years of age was rv (n = 9), followed by piv (n = 2), hcov (n = 2), hadv (n = 1), hbov (n = 1) and rsv (n = 1). in addition, one episode with co-presence with a respiratory virus and septicemia was identified in the group <4 years of age. of the 36 episodes involving only respiratory viral infection (i.e., infection with either a single respiratory virus or multiple respiratory viruses) respiratory symptoms were detected in 31 (86%) ( table 1) . no symptoms were apparent in four episodes involving rv and one episode involving hcov (table 2) . of the 31 episodes involving respiratory symptoms, 26 reported the appearance of respiratory symptoms at time of fever onset ( table 2 ). in the remaining five episodes, the respiratory symptoms appeared over 6 days before the onset of fever. these five episodes represented one flu b and four rv ( table 2 ). bacterial blood cultures were positive in 13 (15%) of the episodes of febrile neutropenia. five cultures were excluded from further analysis and not defined as septicemia because they were determined to be either contaminants or of no clinical relevance by the treating clinician and/ or the laboratory: micrococcus species (n = 1), coagulase-negative staphylococcus (n = 1), staphylococcus epidermidis (n = 2), and unspecified gram-positive bacteria (n = 1). eight episodes were therefore considered true septicemia. of these, five episodes involved only septicemia (6%) ( table 3 ) (i.e. they tested negative for respiratory viruses by pcr). three episodes involved co-presence with a respiratory virus and septicemia; gram-positive bacteria (staphylococcus epidermidis (n = 2) and alpha streptococcus (n = 1)) were detected in all three episodes and one episode was also positive for gram-negative bacteria (escherichia coli). in all three episodes involving co-presence of respiratory virus and septicemia the patients were being treated for a hematologic malignancy, and all had respiratory symptoms ( table 1 ). the viruses found in this group were rv (n = 2) and hbov (n = 1) ( table 2 ). in 43 episodes (49%), no respiratory virus or septicemia were detected and the febrile episode was therefore defined as a fever of unknown origin (table 1) . when comparing episodes involving only respiratory viral infection (i.e., infection with either a single respiratory virus or multiple respiratory viruses) with episodes involving only septicemia or fever of unknown origin, no statistically significant differences were observed with respect to age, gender, days with fever, or maximum temperature (table 1) . however, the presence of respiratory symptoms was significantly higher in the episodes involving only respiratory viral infection (86%) compared with both the episodes involving only septicemia (40%) and fever of unknown origin (58%) (p = 0.043 and p = 0.007, respectively). the episodes with only respiratory viral infection were also more often treated for a hematological malignancy (58%) compared to the fever of unknown origin episodes (35%) (p = 0.04 (table 4) . a new respiratory virus was detected in six follow-up samples: flu a (n = 2), rv (n = 2), hcov (n = 1), and rsv (n = 1). of these six, the episodes for all except one involving rv exhibited respiratory symptoms, and one (flu) represented a new episode of febrile neutropenia. rv genotyping was performed to determine whether the episodes exhibiting repeated pcr positivity involved a new or persistent infection. of samples from the 21 rv-positive episodes, 20 were successfully sequenced, resulting in the identification of 11 rv-a and 9 rv-c species ( table 2 ). before sequencing, four patients, representing six episodes, remained rv-positive at follow-up (table 4 ). one patient was rv-positive in three follow-up samples; however sequencing revealed that two of these follow-up samples contained a new rv specie (fig 1) . sequencing also revealed that the three remaining patients all had the same genotype (fig 1) . all of the rv-c had cleared by the time of follow-up (table 2) , and the four persistent episodes involving rv were associated with rv-a infections ( table 2 ). to the best of our knowledge, this is the first longitudinal study to assess respiratory viral persistence in children presenting with febrile neutropenia. we found that respiratory viral infections are common in children with febrile neutropenia, in the majority of episodes accompanied by respiratory tract symptoms and that the infection had resolved at follow-up in the majority of the episodes. these results support the theory holding that there is a causal relationship between respiratory viral infections and episodes of febrile neutropenia, but proving this theory will require more longitudinal studies with asymptomatic neutropenic control cohorts. in addition, prolonged viral persistence with regard to these respiratory tract infections seems to be uncommon in children undergoing chemotherapy for the treatment for malignancies. in a previous study we detected respiratory viruses in 46% of episodes of febrile neutropenia [3] . subsequent studies have confirmed this finding, with detection rates ranging between 52-57% [4, 5] . the detection rate of 45% in our current study is consistent with earlier studies. it is crucial to correctly diagnose febrile neutropenia in immunosuppressed children, as infections with viruses [23, 24] , bacteria [25, 26] or fungi can be fatal [26] in this group of patients. the clinical significance of a single time-point pcr finding of certain pathogens has been debated. studies on immunocompetent children have reported that viruses such as hbov, hcov, and rv are detected in asymptomatic children at rates ranging between 27-40% [13] [14] [15] [16] , which could be suggestive of prolonged viral shedding from an earlier infection or the incubation period prior to a symptomatic[17] , or asymptomatic infection [13, 14, 17] . to clarify this issue, more longitudinal studies with appropriate control groups are needed. rhedin et al. conducted a case-control study to investigate the etiologic role of respiratory viruses in a group of children with respiratory tract symptoms seeking medical care at a pediatric emergency center [13] . the control group consisted of children on routine visits to child welfare centers for vaccination within the childhood immunization program. the detection rate of respiratory viruses in the control group was high (35.4%) with rv and hcov the most commonly detected viruses in asymptomatic children and hmpv, piv and rsv detected only rarely. the authors concluded that hmpv, piv and rsv play an etiologic role and suggested that findings of rv and hcov must be carefully interpreted [13] .we chose a longitudinal design for the present study, and found that 78% of the infections had resolved by the median follow-up time of 28 days. in addition, two of the rv infections identified at follow-up represented new species of rv from the previous infection, which increases the proportion of resolved infections to 84%. one major limitation of the current study was the lack of a control cohort of neutropenic patients without fever to address the question as to whether respiratory viruses are frequently found in asymptomatic immunosuppressed children. however, the use of a longitudinal design allowed us to collect follow-up samples from patients both with and without respiratory symptoms. only five patients at the first sampling and two patients at follow-up sampling were positive for a respiratory virus without the presence of respiratory symptoms, which suggests that the frequency of asymptomatic infections is low in this group of patients. interestingly, rv was detected in five of these seven episodes and hcov was detected in the other two, which are in line with other studies [13] . the presence of respiratory symptoms was significantly higher in the episodes involving only respiratory viral infection, compared with both episodes involving only septicemia and fever of unknown origin, with symptoms appearing with the onset of fever in the majority of the episodes. peck et al. investigated the incidence of respiratory virus in patients receiving a hematopoietic stem cell transplant using longitudinally collected respiratory tract samples regardless of symptoms [27] . in line with our results, a majority of the respiratory viruses detected could be correlated with respiratory tract symptoms, with the exception of piv, which was detected in asymptomatic patients. infection with rv or hcov was not investigated in that study. in addition, high viral loads were correlated to more symptoms. our real-time-pcr technique was not validated for use in reporting clinical viral loads; thus the presented ct-values must be interpreted with extreme caution. nevertheless, the ct-values <30 observed in 27 of the 39 episodes of febrile neutropenia are suggestive of high viral loads ( table 2) . considered together, our data suggests that respiratory viruses play an etiologic role in febrile neutropenia, but our results should be interpreted carefully, especially those regarding rv, which is commonly detected in asymptomatic patients [13] . despite the above-mentioned limitations, we believe that our findings of a respiratory virus together with other clinical parameters such as respiratory symptoms, negative blood cultures after 48 hours, and a history of short duration of myelosuppression, may help to reduce the treatment time with broad-spectrum antibiotics and the need for prolonged hospitalizations. we were particularly interested in rv in our study because it was the most frequently detected virus, was still detected at follow-up, and has been reported in asymptomatic immunocompetent children [13] [14] [15] [16] . the clinical impact of rv has been correlated with severe respiratory diseases in children under 5 years of age [28] . in our study, only the a and c species of rv were identified. these species are believed to be more pathogenic and more strongly associated with hospital treatment than rv-b [28] [29] [30] . rv-a and rv-c causing both upper and lower respiratory tract infections were the most frequently detected species in another study that investigated rv infection in immunosuppressed children [31] . in the only case of rv-b reported in that study, the patient had an upper respiratory tract infection. in our study cohort, all episodes with a detected rv infection were hospitalized as a result of their febrile neutropenia, and therefore the severity of the disease in relation to the rv specie was difficult to evaluate. respiratory viruses and their shedding times have not been thoroughly investigated in immunosuppressed children. earlier studies on rsv have shown prolonged viral shedding in immunosuppressed children compared with healthy children [32] . martin et al. conducted a longitudinal study of healthy immunocompetent children attending a daycare center [33] and found prolonged shedding (i.e., persistent virus >7 days) of all viruses examined except flu a and b. jartti et al. reported shedding times of 2-3 and 5-6 weeks for ev and rv, respectively, in immunocompetent children [17] . however, rv was not sequenced in that study; therefore, it is unclear whether the sample at 5 weeks represented the same or a new genotype of rv. another study reported that prolonged persistence (>30 days) of the same rv strain is uncommon (<5%) [34] . shedding times were of special interest in our cohort of immunosuppressed children because viral shedding necessitates isolation from other immunosuppressed children. the design of our present study did not allow us to determine the exact shedding time, which is a major limitation. however, the clearance of all viruses except rv-a and hcov at a median follow-up time of 28 days, together with respiratory symptoms appearing at the time of fever onset in a majority of the patients, suggests that the shedding time for viruses such as flu, hmpv, rsv and piv is limited. the same rv genotypes were detected from follow-up samples in four episodes, with a follow-up time of 12-51 days. furthermore, five patients positive for rv reported symptom appearance 6 days or longer before fever onset. that could indicate longer shedding times for rv, which is in line with results from studies on immunocompetent children [17] . however, this needs to be addressed in additional studies with repeated followup sampling, preferable on a weekly basis. hbov has been associated with acute wheezing in immunocompetent children [35] . in immunosuppressed children, hbov has been detected both together with other viruses and also detected repeatedly, suggesting prolonged shedding or reactivation [36, 37] . in this study, hbov was detected twice, with hadv, rsv and rv in one episode and together with septicemia in another episode; both episodes involved respiratory symptoms. at follow-up sampling, hbov was no longer detectable. in our previous study [3] , we detected hbov in three episodes: together with one other virus (rv) with septicemia and as the only agent, making it difficult to ascertain the clinical relevance. our data strengthen the evidence suggesting that respiratory viruses play an etiologic role in febrile neutropenia in children receiving treatment for a malignancy. if confirmed in future studies, the findings reported here will have implications for the clinical management of these patients. our finding could lead to a decrease in the duration of both hospitalization and treatment with broad-spectrum antibiotics, leading to positive social effects for the child and his or her family as well as decreasing the risk of subsequent infection with antibiotic-resistant bacteria. clinical practice guideline for the use of antimicrobial agents in neutropenic patients with cancer: 2010 update by the infectious diseases society of america a prospective study on the epidemiology of febrile episodes during chemotherapy-induced neutropenia in children with cancer or after hemopoietic stem cell transplantation respiratory viruses, a common microbiological finding in neutropenic children with fever frequency and clinical outcome of respiratory viral infections and mixed viral-bacterial infections in children with cancer, fever and neutropenia nasopharyngeal detection of respiratory viruses in febrile neutropenic children health-related quality of life anticipated with different management strategies for paediatric febrile neutropaenia european guidelines for empirical antibacterial therapy for febrile neutropenic patients in the era of growing resistance: summary of the antibiotic resistance is associated with longer bacteremic episodes and worse outcome in febrile neutropenic children with cancer invasive fungal infections in paediatric acute myeloid leukaemia the clinical feature of invasive fungal infection in pediatric patients with hematologic and malignant diseases community-acquired pneumonia requiring hospitalization among u.s. children respiratory viral infections in children with leukemia clinical utility of pcr for common viruses in acute respiratory illness frequent detection of respiratory viruses without symptoms: toward defining clinically relevant cutoff values upper respiratory virus detection without parent-reported illness in children is virus-specific respiratory pathogens in children with and without respiratory symptoms persistence of rhinovirus and enterovirus rna after acute respiratory illness in children frequent and prolonged shedding of bocavirus in young children attending daycare development and implementation of a molecular diagnostic platform for daily rapid detection of 15 respiratory viruses coexistence of two clades of enterovirus d68 in pediatric swedish patients in the summer and fall of 2014 screening respiratory samples for detection of human rhinoviruses (hrvs) and enteroviruses: comprehensive vp4-vp2 typing reveals high incidence and genetic diversity of hrv species c new algorithms and methods to estimate maximum-likelihood phylogenies: assessing the performance of phyml 3.0 management of respiratory viral infections in hematopoietic cell transplant recipients and patients with hematologic malignancies influenza a/h1n1 in pediatric oncology patients pathogenesis of bloodstream infection in children with blood cancer a prospective study of septicaemia on a paediatric oncology unit: a three-year experience at the royal liverpool children's hospital, alder hey, uk respiratory virus infection among hematopoietic cell transplant recipients: evidence for asymptomatic parainfluenza virus infection human rhinovirus species associated with hospitalizations for acute respiratory illness in young us children human rhinovirus c infections mirror those of human rhinovirus a in children with community-acquired pneumonia high prevalence of human rhinovirus c infection in thai children with acute lower respiratory tract disease human rhinovirus c infections in pediatric hematology and oncology patients respiratory syncytial viral infection in children with compromised immune function epidemiology of multiple respiratory viruses in childcare attendees duration of rhinovirus shedding in the upper respiratory tract in the first year of life clinical assessment and improved diagnosis of bocavirus-induced wheezing in children human bocavirus in children with acute lymphoblastic leukemia persistence of human bocavirus dna in immunocompromised children key: cord-324843-r43u7sld authors: kockuzu, esra; bayrakcı, benan; kesici, selman; cıtak, agop; karapınar, bulent; emeksiz, serhat; anıl, ayşe berna; kendirli, tanıl; yukselmis, ufuk; sevketoglu, esra; paksu, şukru; kutlu, onur; agın, hasan; yıldızdas, dincer; keskin, halil; kalkan, gokhan; hasanoglu, arzu; yazıcı, mutlu uysal; sık, guntulu; kılınc, arda; durak, fatih; perk, oktay; talip, mey; yener, nazik; uzuner, selcuk title: comprehensive analysis of severe viral infections of respiratory tract admitted to picus during the winter season in turkey date: 2019-06-17 journal: indian j crit care med doi: 10.5005/jp-journals-10071-23177 sha: doc_id: 324843 cord_uid: r43u7sld objectives: to analyze the course of seasonal viral infections of respiratory tract in patients hospitalized in pediatric intensive care units (picu) of 16 centers in turkey. materials and methods: it is a retrospective, observational, and multicenter study conducted in 16 tertiary picus in turkey includes a total of 302 children with viral cause in the nasal swab which required picu admission with no interventions. results: median age of patients was 12 months. respiratory syncytial virus (rsv) was more common in patients over one year of age whereas influenza, human bocavirus in patients above a year of age was more common (p <0.05). clinical presentations influencing mortality were neurologic symptoms, tachycardia, hypoxia, hypotension, elevated lactate, and acidosis. the critical ph value related with mortality was ≤7.10, and critical pco(2) ≥60 mm hg. conclusion: our findings demonstrate that patients with neurological symptoms, tachycardia, hypoxia, hypotension, acidosis, impaired liver, and renal function at the time of admission exhibit more severe mortal progressions. presence of acidosis and multiorgan failure was found to be predictor for mortality. knowledge of clinical presentation and age-related variations among seasonal viruses may give a clue about severe course and prognosis. by presenting the analyzed data of 302 picu admissions, current study reveals severity of viral respiratory tract infections and release tips for handling them. how to cite this article: kockuzu e, bayrakcı b, kesici s, cıtak a, karapınar k, emeksiz s, et al. comprehensive analysis of severe viral infections of respiratory tract admitted to picus during the winter season in turkey. indian j crit care med 2019;23(6):263–269. microbial agents that most commonly lead to childhood infections are viruses. in developed countries, infants and preschool children have viral infection 6-10 times in a year, and school-age children and adolescents have 3-5 times in a year. 1 in a study published by american centers for disease control and prevention in 2015, pathogens were detected in 80% of 2,254 pneumonia cases in patients aged between a day and 17 years, 66% of these pathogens were viruses. 2, 3 viruses may cause several clinical conditions that require hospitalization in a picu such as bronchiolitis, pneumonia, chronic lung diseases, and more severe manifestations such as myocarditis, encephalitis, and sepsis. besides the known agents, recently new generation viruses including human metapneumovirus (hmpv), coronavirus, and bocavirus have also been lead to cause these manifestations. 4, 5 in this study, it is aimed to bring out the clinical characteristics of patients who had viral infections requiring picu admission during winter season in turkey. initially descriptive properties of variables (mean, median, number, and percentage) were found. numerical variables were checked for normal distribution. student t test was used for normally distributed numerical variables. mann-whitney u test was used for variables with no normal distribution when two groups were compared. in comparison with the numerical values of multiple groups, anova was used for variables with normal distribution and kruskal vallis test was used for variables with no normal distribution. comparison of categorical variables was carried out by chi-square test and fisher exact test. the most effective presenting finding on mortality was revealed with regression analysis. multivariate regression analysis was performed for determining mortality risk factor. critical ph and pco 2 value were found with the roc curve. roc analysis was performed to evaluate the relationship between blood ph and mortality. the p-value <0.05 was considered as significant. the results were evaluated using the statistical package for social sciences -spss 17 (chicago, usa) program. data of 302 patients with severe viral infections were followed in picu of 16 centers in turkey. out of 302 patients, 113 (37%) were girls and 189 (63%) were boys. mortality rate was 7.6% (23/302). the mean prism score was 37.2±13.2 in patients who died and 11.1±10.3 in patients discharged (p <0.001). median age of patients was 12 months. forty-seven percent of patients were under one year of age and 53% of patients were over one year of age. mortality rate was 9.2% and 6.3% in each age groups, respectively (p = 0.34). it was seen that 45.7% of the patients were admitted to the emergency department, 24.2% were admitted to the inpatient service of the same hospital, and 23.3% of the patients were accepted from the inpatient or emergency services of other hospitals. the mean duration of symptoms before admission to the hospital was 3.9±2.8 days. duration of stay in picu was between one day and 116 days, hospital duration was between two days and 120 days. symptoms were cough in 78.1%, fever in 62.5%, nasal discharge in 45.6%, gastrointestinal symptoms in 20.8%, neurological symptoms (seizures, changes in consciousness) in 17.5%, and rash in 4.6% of all patients. hypoxia was found in 49%, tachypnea in 43%, fever in 40%, tachycardia in 34%, and hypotension in 7% of patients. physical examinations revealed respiratory findings in 86%, prolonged capillary filling in 32%, neurological findings in 14%, and gastrointestinal system findings in 10% of all patients. indication of picu admission was respiratory dysfunction in 71.5%, circulatory dysfunction in 10.2%, neurologic dysfunction in 7.6%, and mods in 10.5% of patients. at the time of admission and in follow-up, respiratory failure was observed in 91.3%, cardiovascular failure in 22.1%, hematological failure in 15.2%, renal failure in 10.2%, and hepatic failure in 9.9% of patients. in regression analysis, mods was found as a predictor for mortality [odds ratio: 20.5(95%), confidence interval (ci): 5.8-72.1; p = 0.00]. anemia was found in 45.4%, leukocytosis in 17.8%, leukopenia in 14.2%, thrombocytosis in 19.8%, thrombocytopenia in 14.9%, prolonged inr in 26.6%, and electrolyte imbalance in 74.8% of patients. acidosis was found in 55.2%, alkalosis in 5.9%, high pco 2 in 37.7%, and elevated lactate in 56.9% of patients. the mean lactate value was 6.3±6.1 in patients who died and 2.7±2.5 in patients who were discharged (p <0.001). in regression analysis, acidosis was found to be a predictor for mortality (odds ratio 2.5(95%) ci, 1.2-4.8; p = 0.007). it was found that ph ≤7.10 negatively affected survival (specificity 97.9% and sensitivity 26.3%). pco 2 ≥60 mm hg was also shown to have negative effect on survival (specificity 78.1% and sensitivity 68.4%). clinical and laboratory findings at the time of admission of patients associated with mortality are shown in table 1 and age-related discriminating factors are shown in table 2 . anterior-posterior chest radiograph revealed positive findings in 86% of patients (infiltration in 74%, atelectasis in 9%, pleural effusion in 5%, and pneumothorax in 4%). no statistically significant difference was found between the patients who died and discharged in terms of chest radiograph findings. out of all, 53.3% patients had underlying disease [neurologic in 17.8%, respiratory in 16.5%, cardiovascular in 12.5%, immunological in 6.2%, hematological-oncological in 5.9%, renal in 2.3%, and others (metabolic disease, chromosomal anomalies) 9.5%]. sixty percent of patients who died had underlying disease. this rate was 52% in discharged patients (p >0.05). the total number of viruses detected in nasal swabs of patients hospitalized in picu, number of viruses when multiple reproductions were included and excluded in the patients who died, mortality rates when the viruses were evaluated in themselves are shown in table 3 . multiple viruses were detected in 13% of all patients. multiple viruses isolated was in 17.3% of the patients who died and in 13.2% of the discharged patients (p >0.05). for patients detected with a single-virus infection, there was statistically no difference between the species of the virus, between patients who survived, and patients who died (p = 0.195). when the viruses were evaluated among themselves and patients with multiple viruses were excluded, 37% of patients with coronavirus were found to have died (table 3) . median age of patients with influenza was 41 (11-75) months and median age of patients with the viruses other than influenza was 10 (3.5-28) months (p <0.001). the median prism score was 22 (8) (9) (10) (11) (12) (13) (14) (15) (16) (17) (18) (19) (20) (21) (22) (23) (24) (25) (26) (27) and median pelod score was 11 (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13) (14) (15) (16) (17) (18) (19) (20) (21) in patients with influenza. the median prism score was 9 (3-17) and median pelod score was 10 (1-15) in patients with the viruses other than influenza (p <0.005). the relationships between the indication of picu admission, organ failures at the time of admission or revealed at follow-up, and species of viruses isolated are shown in table 4 . among all, 60.2% of patients received antiviral therapy, 94.3% antibiotic therapy, 15.8% antifungal therapy. 47.4% of patients who died, and 61.6% of the patients discharged received antiviral therapy (p = 0.22). the median duration of stay on mv was 4 (2-5) days, median duration of stay in picu was 7 (4-15), and median duration of hospital was 15 (10-27) days in patients who received antiviral therapy. the median duration of stay on mv was 3 (2-5) days, median duration of stay in picu was 7 (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13) , and median duration of hospital was 15 days in the patients who did not receive antiviral therapy (p >0.05). it was found that virus species did not affect mortality between patients who received and did not receive antiviral therapy (table 5) . of all, 92.3% of patients required respiratory support treatments. renal replacement therapy was performed in 6.6%, plasma exchange in 5.2%, and ecmo in 1.9% of patients. viral respiratory tract infections are the most common infections worldwide. they are the most important cause of mortality in all age groups, particularly in children. 7 this retrospective observational study was conducted in order to analyze the course of seasonal viral infections of respiratory tract in patients hospitalized in picus of 16 centers representing the whole country. there are a bunch of studies in literature investigating the relationship between viruses and morbidity, comparison of single-multiple viruses or relationship between diseases and viruses. [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] current study is multi-directional and has comprehensively analyzed all these issues in a very large population requiring picu. viral respiratory tract infections constitute significant proportion of patients admitted to picus in winter, and these patients require advanced intensive care support with high mortality and morbidity rates. in line with this, our study group shows a very high mortality rate as 7.6%, which supports the justification of this multicenter survey. although respiratory tract viruses commonly cause lower respiratory tract infections, presentation with extrapulmonary clinical manifestations are also likely in children under one year of age. 8, 19 similarly, the most common cause of hospitalization was respiratory symptoms in our study. fever, neurological symptoms, circulatory disorders, and shock were more common indications for hospitalization in patients under one year compared to older children (table 2 ). in patients over one year of age presenting with respiratory symptoms, hypoxia as well as a need for mv were more common. we attributed this situation to rsv, which was more frequently observed in this age group. in the literature, rsv is more prevalent in infancy for picu admission though influenza and bocavirus that was more common in our patients less than one year of age. 1, 9, 10 we statistically evaluated all initial symptoms (clinical, laboratory, and radiological) and virus species through four parameters (duration of stay on mechanical ventilation (mv), duration of stay in picu, duration of hospitalization, and mortality). accordingly, we assessed relationship among presenting symptoms, virus species, and severity of disease. it was observed that presenting symptoms that have an effect on mortality were neurologic symptoms, hypotension, tachycardia, and hypoxia. it was also found that liver and renal function test abnormalities, higher lactate values were related with death. eighty-seven percent of mortalities are presented with acidosis ( table 1 ). the critical ph value related with mortality and critical pco 2 was ≤7.10 and ≥60 mm hg, respectively. in regression analysis, acidosis alone was found to increase mortality by 2.5 folds. in the light of these findings, it can be predicted that patients with neurological symptoms, tachycardia, hypotension, low levels of pulse so 2 , acidosis, and impaired liver or renal function at the time of admission will have a higher morbidity and mortality. kumar et al. showed that organ failure in patients hospitalized in icu with h1n1 infection increased mortality. 11 also, kendirli et al. demonstrated the correlation between organ failures and mortality in the patients who presented with h1n1 infection. 12 similarly, in our study, mortality rates were higher and in regression analysis, we demonstrated that initial presentation with mods increased mortality by 20-folds. in a study, it was shown that rsv was the most common agent in patients who needed intensive care. 20 this is definitely compatible with our findings. spaeder and fackler reported that virus species did not affect durations of stay in picu and hospital, as well as development of mods and mortality. 21 in our study, we also investigated virus species organ failure relationship and emerging indications for picu admission. we demonstrated that there was no correlation between virus species and diagnosis of admission to picu or type of organ failure developed (table 4 ). however, 63% of the patients who died in 2017 winter season had rsv, parainfluenza or influenza. secondary bacterial agent was observed in about half of the patients who died. it is not easy to blame viral agents to be the leading causes of mortality because of the frequent accompaniment of secondary bacterial agents. similar to the literature, we found that secondary bacterial infections negatively affected mortality. 15, 22, 23 in the present study, multiple viruses were detected in 13% of the patients. although there are also contrary opinions, [24] [25] [26] detection of multiple viruses does not affect mortality and duration of stay in picu. 22, 27, 28 similarly, we showed that multiple viruses did not affect mortality. when multiple virus isolated patients were excluded, coronavirus revealed to be the most lethal virus as three out of eight patients with coronavirus died (table 3 ). coronavirus is known as the primary agent of upper respiratory tract infection and common cold. 29 coronavirus had been defined as the causal agent of sars (severe acute respiratory syndrome), which more severely progresses in adults, while the disease lasts shorter and shows milder progression in children. 30 unlike the literature, coronavirus presented severe symptoms with organ failures in our group of patients (table 4 ). lower respiratory tract infections caused by coronavirus types out of sars are rare and some studies found the incidence of pneumonia and bronchitis as <5%. [31] [32] [33] [34] [35] [36] therefore, in our study, the incidence of coronavirus was low that is consistent with the literature, but unlike that coronavirus caused lower respiratory tract infections and emerged as the most lethal viral agent (table 3) . however, 36% of the patients with coronavirus had secondary bacterial infections; so, coronavirus is not solely responsible for mortality. although secondary infections were found in only 16% of all patients, we found that 94% of the patients who presented with viral infections received antibiotics. sixty-two percent of the patients received antiviral treatment (oseltamivir) because antiviral therapy was started before nasal swab results in patients with severe infection findings. we found that antiviral therapy did not affect mortality (table 5) including the influenza subgroup. though, there are studies in literature showing that oseltamivir initiated in the early period was effective. [37] [38] [39] however, weakness of our study is that the data we gathered is not capable of differentiating the initiation timing for antiviral therapy. further studies are needed for this issue. influenza is already known as the only agent that benefits from oseltamivir. when we separately evaluated the influenza subgroup, we found the median patient age as 41 months and higher prism and pelod scores compared with rest of the viral agents. influenza was more common in older children and progressed more severely. therefore, oseltamivir can be preferred in children especially over one year of age and who presented with more severe symptoms because they are more likely to have influenza. consistent with the literature, comorbidities were common in the present study. 9, [12] [13] [14] 20 although 60% of the patients who died had an underlying disease, a statistical correlation is lacking as described in the literature. 9, 12 in our study, it was found that mechanical respiratory support was administered to the majority of the patients and renal replacement therapy was applied in the group, which had organ failure and progression. as expected, we found that prism and pelod scores were higher in patients who died. we found that tachycardia, hypotension, hypoxia, acidosis, impaired liver or renal tests, and mods at the time of admission are the factors associated with mortality and predictors of a severely progressing disease. it was found that acidosis increased mortality by 2.5-folds and mods by 20-folds. respiratory symptoms were common and besides respiratory symptoms, incidence of circulatory disorders and shock findings were also high in patients less than one year of age. fever, tachycardia, neurologic symptoms, the incidence of anemia, leukocytosis, thrombocytopenia, impaired renal tests, and electrolyte imbalance were high in patients less than one year of age. consequently, acidosis and mods at the time of admission or a patient less than one year of age with symptoms mentioned above should alert clinicians for a possible poor prognosis. although we could not show the relationship of virus type with severity and mortality, rsv, parainfluenza, and influenza were the commonly isolated viruses in the patients who died. coronavirus was found as the most lethal virus. although coronavirus is known as the primary agent of upper respiratory tract infection and common cold, this result of our study can be attributed to the incidence of secondary bacterial infections being high in the patients with coronavirus detected. with this context, further studies are needed to investigate the relationship between coronavirus and mortality. in our study, we could not demonstrate effects of oseltamivir on mortality, duration of hospitalization, and stay in the picu including influenza subgroup but there are studies reporting that oseltamivir initiated within first 48 hours is beneficial. therefore, oseltamivir can be preferred in children who present with more severe symptoms because they are more likely to have influenza. viral infections of the lower respiratory tract: old viruses, new viruses, and the role of diagnosis respiratory viruses detected in mexican children younger than 5 years old with community-acquired pneumonia: a national multicenter community acquired pneumonia requiring hospitalization among u.s. children frequency of respiratory viruses in children with lower respiratory tract infection prevention and control of influenza: recommendations of the immunization practices advisory committee (acip) international pediatric sepsis consensus conference: definitions for sepsis and organ dysfunction in pediatrics who estimates of the causes of death in children extrapulmonary manifestations of severe respiratory syncytial virus infection-a systematic review atypical extrapulmonary presentations of severe respiratory syncytial virus infection requiring intensive care human bocavirus infection as a cause of severe acute respiratory tract infection in children critically ill patients with 2009 influenza a(h1n1) infection in canada critically ill children with pandemic influenza (h1n1) in pediatric intensive care units in turkey the critically ill child with novel h1n1 influenza a: a case series continued high incidence of children with severe influenza a(h1n1) pdm09 admitted to paediatric intensive care units in germany during the first three post-pandemic influenza seasons paediatric mortality related to pandemic influenza a h1n1 infection in england: an observational populationbased study human bocavirus and ki/wu polyomaviruses in pediatric intensive care patients human metapneumovirus infection among children hospitalized with acute respiratory illness human coronavirus in the 2014 winter season as a cause of lower respiratory tract infection respiratory viral infections in infants: causes, clinical symptoms, virology, and immunology premorbid factors and outcomes associated with respiratory virus infections in a pediatric intensive care unit hospital-acquired viral infection increases mortality in children with severe viral respiratory infection an investigation into the prevalence and outcome of patients admitted to a pediatric intensive care unit with viral respiratory tract infections in cape town, south africa the frequency of influenza and bacterial coinfection: a systematic review and meta-analysis. influenza other respir viruses the impact of dual viral infection in infants admitted to a pediatric intensive care unit associated with severe bronchiolitis detection of multiple respiratory viruses associated with mortality and severity of illness in children the impact of multiple viral respiratory infections on outcomes for critically ill children respiratory and gastrointestinal epithelial modulation of the immune response during viral infection diagnostic value of real time polymerase chain reaction to detect viruses in young children admitted to the paediatric intensive care unit with lower respiratory tract infection frequent detection of human coronaviruses in clinical specimens from patients with respiratory tract infection by use of a novel real-time reverse-transcriptase polymerase chain reaction seasonality of infectious diseases and severe acute respiratory syndrome -what we don't know can hurt us clinical picture, diagnosis, treatment and outcome of severe acute respiratory syndrome (sars) in children impact of human coronavirus infections in otherwise healthy children who attended an emergency department clinical disease in children associated with newly described coronavirus-subtypes clinical manifestation of human coronavirus nl63 infection in children in taiwan role of human metapneumovirus, human coronavirus nl63 and human bocavirus in infants and young children with acute wheezing detection of the new human coronavirus hku1: a report of 6 cases outcomes of adults hospitalized with influenza early versus late oseltamivir treatment in severely ill patients with 2009 pandemic influenza a (h1n1): speed is life very low pandemic influenza a (h1n1) 2009 mortality associated with early neuraminidase inhibitor treatment in japan: analysis of 1000 hospitalized children authors would like to thank dr. ersoy civelek for statistical analysis and all participants for their contributions. key: cord-353698-gj8sx3zy authors: bibiano-guillen, c.; arias-arcos, b.; collado-escudero, c.; mir-montero, m.; corella-montoya, f.; torres-macho, j.; buendía-garcia, m.j.; larrainzar-garijo, r. title: adapted diving mask (adm) device as respiratory support with oxygen output during covid-19 pandemic date: 2020-10-28 journal: am j emerg med doi: 10.1016/j.ajem.2020.10.043 sha: doc_id: 353698 cord_uid: gj8sx3zy nan at the end of 2019, several cases of pneumonia were identified in wuhan (hubei, china) [1], caused by a new orthocoronavirinae, commonly known as coronavirus, from the coronaviridae family. in january 2020, there was a public health emergency declaration [2] and, as of march 2020, a pandemic [3, 4] . at present, more than 2 million cases have been confirmed globally (2.160 .2017 april 18th) [5] [6] [7] [8] [9] . in spain, the first spots of epidemiological interest were identified in madrid. some of them were registered in the sanitary region where both hospital universitario infanta leonor and virgen de la torre hospitals belong. the first pcr-positive patient was detected on march 4 th . after two weeks, on 18 th march, the number of positive cases increased to 302, and 41 patients died. by 1 st april, one month after the outbreak, the total number of cases was 1714. these data confirmed the explosive progression of the pandemic and the high mortality of patients who were hospitalized and made it necessary to implement several and different therapeutic measures to try and revert the catastrophic progression of this infection. the most extended therapeutic approach for covid-19 is based on two main strategies [10] [11] [12] [13] : pharmacological treatment directed toward several physiological targets (viremia, immunological reactions, prothrombotic reactions) and hemodynamic and respiratory support with positive end-expiratory pressure (peep) in addition to mechanical ventilation. this is vitally necessary until pharmacological treatment or patient immune responses become effective. china and italy have already described that acute respiratory distress syndrome (ards) is the most common manifestation in the clinical course of covid-19 pneumonia [11] [12] [13] [14] ; however, this syndrome has a different progression than other j o u r n a l p r e -p r o o f respiratory diseases. the first-choice treatment for ards is mechanical ventilation (mv) with the use of orotracheal intubation (oti). the ards mortality rate is over 50%, and the delay in this procedure is related to an even worse prognosis [15, 16] . the main limiting factors that healthcare systems must face when handling these critical patients are the limited access to ventilators and icu resources and the fact that they are already overwhelmed by massive hospitalization due to respiratory distress and oti needs [17, 18] . this is why current lines of work are focused on developing respiratory support alternatives that will gain time or allow the maintenance of an acceptable respiratory status until patients can access the icu [19, 20] . in the absence of approved mechanical devices, such as continuous positive airway pressure (cpap), positive end-expiratory pressure (peep) devices are being used [14, 16, 21] . in this paper, we describe our experience with the adaptation of diving masks (figure 1 ) and predesigned and 3d-printed pieces (annex) that work as respiratory peep valves and high-flow oxygen connectors. during the inspiratory phase, oxygen reaches the fitted mask via a high-flow tube connected to a flowmeter at an outlet on a high-pressure hospital circuit or in a portable oxygen bottle. the residual volume of the mask becomes a reservoir. during the expiratory phase, the peep valve resists airflow. the exhaled air flows decontaminated through an n99 high-efficiency filter, reducing the possibility of contagion by viral aerosolization. the main objective was to assess the efficacy of this alternative prototype for respiratory support in the context of the covid-19 pandemic. the secondary outcomes were the clinical profiles of patients who could benefit from this device, as well as the safety parameters and potential adverse events. a descriptive case series study of twenty-five patients with acute respiratory syndrome secondary to sars-cov2 infection was performed at a spanish center, hospital universitario infanta leonor of madrid, between march 30 and april 18, 2020. we tested the easybreath first-generation snorkel mask (decathlon©) adapted with 3dprinted appliances as a respiratory support device with oxygen output and a peep valve. all patients provided written informed consent, and the study was approved by the hospital committee for medical and health research ethics. there were a total of twenty-five patients: twenty-one men and four women. the demographics, baseline characteristics and comorbidities are shown in table 1 . we established two clinical settings for the use of an adapted diving mask (adm) at the emergency department: 1. patients with an oxygen saturation under 93% and oxygen reserve to 15 l. 2. patients using pulmodyne® cpap who presented with mild to severe intolerance to their interface or with an oxygen saturation under 92% despite maximum oxygen airflow. the mask was used with interrupted discontinuous character only to feed or hydrate the patient, aspirate secretions, if necessary, and administer artificial tears in the case of dry eye. in the absence of this nursing care, the patient was advised to maintain mask therapy for as long as possible. severe clinical status was established when radiographic pulmonary alterations and serologic alterations such as lymphocyte count, transaminases, ldh and d-dimer were present. to assess the initial response of patients, we analyzed two variables: 1. oxygen saturation improvement by comparing previous measurements and those immediately after oxygen saturation to the use of the mask. a. qualitative variable: at least a 3-point improvement in oxygen saturation (yes/no). b. continuous variable: improvement in mean-score mean from pre-mask saturation values. 2. arterial blood gas analysis (abga) at one hour. gas parameters were measured after one hour of mask use. to assess patient improvement, we used the following variables: the medical records of patients were analyzed by the research team of the emergency department, hospital universitario infanta leonor, madrid. epidemiological, clinical, laboratory, radiological and treatment outcome data were obtained via data collection forms from the electronic medical records. the data were reviewed by a trained team of physicians. the recorded information included demographic data, medical history, exposure history, underlying comorbidities, symptoms, signs, laboratory findings, and treatment measures. descriptive statistical analysis was performed using ibm spss statistics version 22 software (ibm corporation usa). in this case series, the most common radiological pattern was the bilateral interstitial pattern (92,85%), followed by the multilobular pulmonary consolidation pattern (7.14%). a total of 3.57% of patients had less than 1500/µl lymphocytes, 85.7% had less than 150000/µl platelets, 78.5% had over 250 ldh u/l, 46.4% had d-dimer levels above 500 ng/ml, and 82.1% had a got over 40 u/l. the mean baseline oxygen saturation on admission was 83.6%, distributed in severity ranges as shown in table 2 . there were two patients who had missing data. the distribution of patients according to the clinical setting is described in table 2 . adm use was higher in patients with an oxygen saturation under 93% with reservoir masks at 15 lpm (18 patients). oxygen saturation was measured before initiating adm therapy. the mean value was 90.24 %, and the patient distribution was made according to severity at saturation levels, as shown in table 2 . after adm initiation, patients had a very positive initial response ( table 3 ). the immediate saturation value after adm placement was 95.8%. in twenty-one patients, the saturation improvement was 3 or higher. the mean po2 after one hour of adm treatment was 93.75 mmhg, as measured by arterial abga (in five patients, we were not able to collect these data), the mean pco2 was 40.91 mmhg, and all patients surpassed the 50 mmhg limit. table 4 shows the different variables used to analyze the improvement in the intervention and the mean value of all oxygen saturation measurements for all days that adm therapy was used in those patients who prolonged its time of use. more than 50% of patients tolerated the mask for 24 hours, switching to 3-hour minimum rotational use in combination with other therapies or devices. the main cause of adm cessation was physical intolerance (nine patients). eight patients needed invasive mechanical ventilation due to disease progression. finally, five patients experienced clinical respiratory improvement. during the time of the study, eight patients died, five of whom were dismissed for icu admission for prevalent comorbidities and advanced disease stage. three of the patients who were admitted to the icu for invasive mechanical ventilation died. there were no adverse events related to oxygen system failure during clinical use of the adm device or any eye or skin adverse events. four patients presented with isolated mild hypercapnia (pco2 > 45 mmhg<5° mmhg) during adm use, two of whom presented in the first hour of therapy and two during the adm rotation period with other therapies and interfaces, without any additional clinical or analytical repercussions. the mask was not indicated in any patients who, in their baseline situation, already had hypercapnia of any degree or respiratory acidosis. one patient presented with respiratory acidosis (ph <7.35) and moderate hypercapnia (pco2> 50 mmhg) during mask use. this patient was not considered for icu care due to late disease progression and poor respiratory dynamics, which ended in death a few hours later (table 5 ). the adapted easybreath™ system is an efficient and safe alternative for respiratory support in the shortage of official devices in the current state of the pandemic. it has already been described that covid-19 disease has two differentiated phases: initial pulmonary alveolar affectation caused by viral access to ac2 receptors [22, 23] and, second, an inflammatory phase mediated by macrophages [24] . both have a common characteristic, hypoxemia, and this is why choosing the best respiratory support as soon as possible is of utmost importance for the clinical course of patients. regrettably, sanitary activity during the covid-19 pandemic is conditioned by what the who has defined as "multiple victim incidence", where more patients are generated than those who can be treated in optimal conditions [18] [20] . for this j o u r n a l p r e -p r o o f reason, many of the recent publications reference overwhelmed healthcare systems and compare the current situation to battlefield medicine practice. the first-choice treatment for ards is mechanical ventilation (mv) with the use of orotracheal intubation (oti) [14] [15] [16] 21] . in the clinical event of resource and device shortage (full icu, respirator shortage and lack of healthcare professionals), noninvasive respiratory support can be considered a valid alternative despite the worsening prognosis already published [11, 21, 25] . both nimv and cpap or high-oxygen airflow therapy can be administered to patients by several devices depending on the availability and indication: oronasal masks, facial masks or helmets [26] [27] [28] . through release valves, less nebulization is produced by helmets and the highest with oronasal masks. it is of high importance to take into account these devices in the covid-19 pandemic context, as potential viral contamination to patients is caused by aerosolized particles, which is a limiting factor, as well as the limited availability of the cpap/bipap device [29] [30] [31] . with the use of the easybreath™ mask (adm), we can address both issues. on the one hand, adms minimize contamination in the patient surroundings, as the facial silicone almost completely seals the faces of patient and because it is a semi-open device with just one output through the exhalatory port where air comes out already filtrated by a highly efficient n99,99 filter. this prevents exposure to other patients and healthcare professionals [29, 32, 33] . on the other hand, the technical requirements for adms are minimal: an oxygen flowmeter with as much oxygen airflow possible. all necessary pieces needed for the adaptation of adms have been designed by a multidisciplinary team of engineers and doctors (see annex). 3d impression technology for medical use needs to work with nontoxic, inert materials that would prevent leaching of one or more substances contained in the adaptor that was originally of powder base. to avoid this and at the same time comply with regulatory rules en 5356 and en 13544, the preferred material is pa12 (sls) [34, 35] . our results show a rapid improvement in oxygen saturation maintained in time without any adverse events. in addition, adaptation of the adm is safe for two main j o u r n a l p r e -p r o o f reasons. first, a 3d anti-suffocation valve adapted to our device allows for patients to inspire atmospheric air in case there is a pressure drop in the system (accidental decoupling of the oxygen source). second, there was no hypercapnia or respiratory acidosis in any of the follow-up tests performed in patients, meaning that rebreathing did not take place. the mild hypercapnia that was detected in some patients may pertain to the highoxygen airflow administered (fio2 administered close to 100%). this may also occur with common reservoir masks. therapy should be suspended only if hypercapnia progressively increases or increases to moderate hypercapnia levels (pco2> 50 mmhg) or if it is associated with acidosis of any degree (ph <7.35). in other words, patients should remove their masks if hypercapnia is present, despite being mild, if it increases in the first hours of use, if it is moderate or if any of the above factors are also associated with respiratory acidosis. patients will be able to maintain masks with stable mild hypercapnia in the first 3 hours and if a lack of acidosis remains. one alveolar recruitment technique that helps patient oxygenation in icus for patients with ards is pronation [13] . few studies have shown that the pronation maneuver in a non-icu setting in patients treated with any type of respiratory support in the emergency room or who are hospitalized may improve the ventilatory/perfusion gradient of lungs and, therefore, hypoxemia [16] . therefore, pronation, when necessary, is best tolerated by patients wearing masks. in our series, patients had low saturation levels even in pronation and used adms with up to >90% oxygen saturation in the supine position and with head elevated to 30 degrees. some starting resources are necessary to be able to adapt to masks, but once the adaptation process is complete, masks can be completely reused by another patient, except for a high-efficiency filter following the matachana sequence designed for our center: 1. enzymatic detergent wash (mediclean forte™) 2. rinse. 3. wash the disinfector at 80° for 50 minutes to guarantee ao=3000. this is the reference value used for thermoresistant germs, including hepatitis b or mycobacteria. 4. 85° drying turbine. this study has some limitations. the main limitation is derived from the design. we did not have a control group, as this was not possible in the critical clinical setting that has developed. therefore, we cannot compare respiratory support with other therapies with the same characteristics, which would have been highly desirable. additionally, 3d printing allows for the creation and testing of many prototypes in a rapid manner, but for midterm provisions and in a global pandemic setting, the final solution should be able to increase the production rate to respond to high demand when 3d printing is not available, perhaps via injection molding. injection molding will make it possible to produce thousands of pieces per day at a lower cost, although initial investment (design and production of the mold) needs to be perfectly validated before starting the production process. another disadvantage of 3d printing is that the production error has to be reduced to ±0,005 mm with a surface finish of 0,4 μm. this is not possible to guarantee with 3d printing methods, but it would be possible to produce with injection molding. we are aware of the limitations of the adm proposal as an alternative to respiratory support, and assuming its use in pandemics and the deep shortage of certified alternatives, we can conclude that it complies with clinical requirements to be produced in other centers with limited resources [17] [18] [19] 36] , especially in developing countries. this is a therapy that provides an adm device; it only requires oxygen connection with a flowmeter with a minimum airflow of 15 lpm. it has also shown a lack of significant adverse events, presenting an optimal initial tolerability in twentythree patients, and we propose it as a more comfortable interface option for those patients highly dependent on noninvasive respiratory support. j o u r n a l p r e -p r o o f j o u r n a l p r e -p r o o f mild hypercapnia (%) 20 skin irritation (%) 0 eye irritation (%) 0 j o u r n a l p r e -p r o o f who declares covid-19 a pandemic covid-19: towards controlling of a pandemic covid-19 and community mitigation strategies in a pandemic first case of 2019 novel coronavirus in the united states estimation of covid-19 outbreak size in italy outbreak investigation for covid-19 in northern vietnam covid-19 and italy: what next? lancet. lancet publishing group covid-19 in europe: the italian lesson novel 2019 coronavirus sars-cov-2 (covid-19): an overview for emergency clinicians clinical course and outcomes of critically ill patients with sars-cov-2 pneumonia in wuhan, china: a single-centered, retrospective, observational study xiaobo yang* hospitalized patients with 2019 novel coronavirus-infected pneumonia in wuhan, china management of respiratory failure due to covid-19 respiratory support for patients with covid-19 infection recommendations for endotracheal intubation of covid-19 patients respiratory physiotherapy in patients with covid-19 infection in acute setting: a position paper of the italian association of respiratory physiotherapists clinical ethics recommendations for the allocation of intensive care treatments in exceptional, resource-limited circumstances: the italian perspective during the covid-19 epidemic the response of ilan's emer ency edical system to the ovid-19 outbreak in italy hospital surge capacity in a tertiary emergency referral centre during the covid-19 outbreak in italy emergency departments and the covid-19 pandemic: making the most of limited resources clinical consensus recommendations regarding non-invasive respiratory support in the adult patient with acute respiratory failure secondary to sars-cov-2 infection structural basis of receptor recognition by sars-cov-2. nature receptor recognition by the novel coronavirus from wuhan: an analysis based on decade-long structural studies of sars coronavirus covid-19: consider cytokine storm syndromes and immunosuppression high-flow nasal cannula may be no safer than noninvasive positive pressure ventilation for covid-19 patients helmet cpap to treat acute hypoxemic respiratory failure in patients with covid-19: a management strategy proposal treatment of acute hypoxemic nonhypercapnic respiratory insufficiency with continuous positive airway pressure delivered by a face mask: a randomized controlled trial high-flow nasal cannulae for respiratory support in adult intensive care patients protecting health-care workers from subclinical coronavirus infection high-flow nasal cannula for covid-19 patients: low risk of bio-aerosol dispersion more awareness is needed for severe acute respiratory syndrome coronavirus 2019 transmission through exhaled air during non-invasive respiratory support: experience from china staff safety during emergency airway management for covid-19 in hong kong activities of daily living after hip fracture: profile and rate of recovery during 2 years of follow-up three-dimensional (3d) printing of polymer-metal hybrid materials by fused deposition modeling. materials (basel) enhancement of thermal, mechanical and physical properties of polyamide 12 composites via hybridization of ceramics for bone replacement how emergency departments prepare for virus disease outbreaks like covid-19 acknowledgement e ac no led e ristina rce for her re ision and translation of the manuscript and anuel ere -espa a uniesa d, for their support in the early phases of covid 19 pandemia. the authors want to acknowledge the effort and creativity in the design of the adapting parts of the mask to juan manuel canalejo-bautista (airbus) and alberto molina parga (ct engineers) key: cord-330079-pdaowkop authors: xu, lin; he, xia; zhang, ding-mei; feng, fa-shen; wang, zhu; guan, lin-lin; wu, jue-heng; zhou, rong; zheng, bo-jian; yuen, kwok-yung; li, meng-feng; cao, kai-yuan title: surveillance and genome analysis of human bocavirus in patients with respiratory infection in guangzhou, china date: 2012-09-11 journal: plos one doi: 10.1371/journal.pone.0044876 sha: doc_id: 330079 cord_uid: pdaowkop human bocavirus (hbov) is a novel parvovirus associated with respiratory tract diseases and gastrointestinal illness in adult and pediatric patients throughout the world. to investigate the epidemiological and genetic variation of hbov in guangzhou, south china, we screened 3460 throat swab samples from 1686 children and 1774 adults with acute respiratory infection symptoms for hbov between march 2010 and february 2011, and analyzed the complete genome sequence of 2 hbov strains. specimens were screened for hbov by real-time pcr and other 6 common respiratory viruses by rt-pcr or pcr. hbov was detected in 58 (1.68%) out of 3460 samples, mostly from pediatric patients (52/58) and inpatient children (47/58). six adult patients were detected as hbov positive and 5 were emergency cases. of these hbov positive cases, 19 (32.76%) had co-pathogens including influenza virus (n = 5), rsv (n = 5), parainfluenza (n = 4), adenovirus (n = 1), coronavirus (n = 7). the complete genome sequences of 2 hbovs strains (genbank no. jn794565 and jn794566) were analyzed. phylogenetic analysis showed that the 2 hbov strains were hbov1, and were most genetically close to st2 (genbank accession number dq0000496). recombination analysis confirmed that hbov strain gz9081 was an intra–genotype recombinant strain among hbov1 variants. human bocavirus (hbov), recently identified as a new member of the parvoviridae family from the respiratory secretions of children suffering from lower respiratory tract infection [1] , has a single-stranded dna genome of ,5.2kb, which contains three open reading frames (orfs), encoding two non-structural proteins (ns1 and np1) and two viral capsid proteins (vps) [2] . the nucleotide sequences are highly conserved among hbovs circulating in different geographic regions [3] , with the vp1/ vp2 gene displaying relatively commonly found nucleotide polymorphisms [2] . since its first identification, hbov has been detected in 1.5%-19% of respiratory tract secretions [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] and 0.8%-9.1% of fecal samples [13, [16] [17] [18] [19] , respectively, from patients with acute respiratory tract illnesses or gastroenteritis worldwide, it is of note that most of these reports were mainly derived from young children and infants, with only a few exceptions testing adult patients. more recently, three more genotypes of hbov (hbov2-4) were found, [2, 20, 21] , and reports have shown that inter-genotype and intra-genotype recombinations are present among bocavirus [22] . all 4 genotypes of hbov have been identified in children with acute gastroenteritis (age), whereas only hbov1 and hbov2 were reported in respiratory tract samples. due to the higher rates of co-infections with other pathogens, it remains to be clarified whether in these diseases hbovs are the key etiologic agent or just a concomitant virus bystander. to better understand the epidemiology of the hbov infection, in conjunction of a viral surveillance program, we investigated the presence of hbov in patients with acute respiratory infection in guangzhou, a city located in south china. geographically, the city is characteristics of a tropical-subtropical climate, with the average annual temperature of 20-22uc and average relative humidity of 77%. the city is also highly populated, with a resident population of 12.70 million, plus a non-residential population of 4.76 million. these socio-natural factors make the region generally vulnerable to air-borne as well as food-borne viral infection. the epidemiological status and genomic characteristics of hbov prevailing in pediatric and adult patients with respiratory infection in the region, however, remains unknown. in our current study, we screened throat swab specimens from patients with acute respiratory tract infection symptoms for hbov and other common respiratory viruses over a 12-month period using polymerase chain reaction (pcr) methods, and in addition, the molecular phylogeny and complete genome sequences of 2 hbov strains were also analyzed. all research involving human participants was approved by the medical ethics review board of zhongshan school of medicine, sun yat-sen university, in accordance with the guidelines for the protection of human subjects. written informed consent was obtained from each participant/guardian. from march 2010 to february 2011, 3460 throat swabs were obtained from 1686 children and 1774 adult patients who had been admitted to five hospitals in guangzhou, china. they were only taken from individuals with # 3 days of fever (temperature $37.5uc), and with cough, sputum, throat sore or other respiratory tract infection symptoms. there were 2009 male and 1451 female patients with age ranging from 1 day to 95 years. demographic, epidemiology and clinical information including case history, symptoms, physical signs and examination results etc. were collected using a standardized questionnaire. all specimens were added to 2 ml vtm (consists of earle's balanced salt solution (biosource international, usa), 4.4% bicarbonate, 5% bovine serum albumin, 100 mg/ml vancomycin, 30 mg/ml amikacin, and 40 u/ml nystatin) according to a standard protocol and transported within 8 hr at 4uc to biosafety laboratory of sun yat-sen university, where they were divided into aliquots, and stored at 280uc until processing further. all specimens were tested for 7 common respiratory viruses, including influenza virus types a, b and c (inf-a, inf-b and inf-c), parainfluenza (piv) types 1-4, respiratory syncytial virus (rsv), human metapneumovirus (hmpv), human coronavirus (hcov), adenovirus (adv) and hbov using pcr, rt-pcr or real-time pcr methods as described below. information of patients whose throat swabs were found positive for hbov was analyzed retrospectively. dna and rna were simultaneously extracted from 200 ml of throat swab specimen using qiaamp minielute virus spin (qiagen, germany). reverse transcription of virus rna was conducted by superscript iii transcriptase and random hexamer primers (invitrogen, life technology, usa), both kits were used according to the manufacturer's instructions. inf-a, -b and -c, piv -1, -2, -3 and -4, rsv-a and -b, hmpv, hcov, and adv were detected by a standard reverse transcription-pcr (rt-pcr) or pcr techniques as previously described using specific primers listed in table s1 [11, [23] [24] [25] , and amplified products were detected by agarose gel electrophoresis. screening of hbov used real-time pcr. the full sequence of hbov was referred from the st2 strain (genbank accession number dq000496, or nc_007455). taqman real-time pcr primers (np1-f and np1-r) and probe (synthesized by invitrogen, life technology, usa) were designed to bind the np1 highly conserved region of different hbov strains and analyzed by primer express software (version 3.0, applied biosystems, usa) (for primer and probe sequences, see table 1 ), with regard to optimal g-c content (55%, 44% and 60% for np1-f, np1-r and probe, respectively), melting temperature (58.4uc, 59.3uc and 70.0uc for np1-f, np1-r and probe, respectively), and amplicon length (88bp). each reaction mixture consisted of 10 ml 26 iq supermix reaction mixture (bio-rad, usa), 2 ml of viral dna, 0.5 mm each of the forward and reverse primers, and 0.3 mm of the probe, and nuclease-free water to a final volume of 20 ml. real-time pcr was conducted for 95uc for 15 min, followed by 45 cycles of 95uc for 15 s, 60uc for 1 min on the abi7500 realtime pcr system. the complete genomes of hbov strains were amplified using primers designed for complete genome by the primer premier 5.0 software to bind relatively conserved regions of hbov as available in the genbank database (primer sequences shown in table 1 ). the pcr was carried out using the platinum pfx taq polymerase (invitrogen, usa) in a prepared reaction mix according to the following condition: 95uc for 5 min, followed by 40 cycles of 94uc for 15 s, 53uc to 58uc (see table 1 for melting temperature of different primers) for 45 s, and 68uc for 2 min, and a final extension at 72uc for 7 min. pcr products for genome analysis was purified by agarose gel dna purification kit (takara, china), and the pcr products of terminal sequences were cloned into pcr-blunt 4-topo vector (zero blunt topo pcr cloning kit for sequencing, invitrogen, usa). all pcr products used for cloning and sequencing were from three independent pcr reactions. sequencing was performed by a commercial service of invitrogen co. according to the method described in ref. [26] (guangzhou, china) and submitted to the genbank database. the genomic sequences and orfs of hbov were comparatively analyzed with complete genome sequences of other hbov strains in the genbank (including hbov reference strains st1, st2, parvovirus b19, bovine parvovirus, canine minute virus, and virus strains obtained from several countries). these sequences were aligned by the clustal x program, and a neighbor-joining tree was constructed using the mega 4.0 software. potential recombinant sequences and parental sequences were analyzed using the recombination detection program (rdp) [27] . rdp scanning was performed by geneconv, bootscan, max-chi, chimaera, and siscan methods. a multiple comparison corrected p-value cutoff of 0.001 was used throughout. simplot checking was used to confirm and evaluate localization of possible recombination break points by bootscan program [28] . recombinant validation was done by checking the bilateral gene sequences of the recombinant site using phylogenetic trees. of the 3460 samples collected from patients with respiratory tract infection symptoms and signs enrolled in the study during the period between march 2010 and february 2011, detection for 7 viruses, namely, influenza, piv, rsv, hmpv, hcov, adv and hbov, showed that 1275 (36.8%) were found positive for one single virus and 112 (3.2%) were infected by more than one virus. as shown in table 2 , 21.1% of patients tested were found positive for inf-a, -b, or -c (median age 25 years), 7.7% for rsv (median age 0.91 years), 4.2% for adv (median age 5 years), 3.5% for piv (median age 1.2 years), 2.7% for hmpv (median age 2 years), and 2.6% for hcov (median age 3 years). hbov dna was detected in 58 samples (1.68%) (median age 1.5 years) by real-time pcr, including 52 pediatric (47 inpatient, 4 outpatient and 1 emergency patient) and 6 adult (1 inpatient and 5 emergency patients) cases. the monthly distribution of 7 respiratory viruses tested in patients with indications for respiratory infection from march, 2010 to february, 2011 showed biannual peaks. the highest peak of total positive rate of hbov and other 6 common respiratory viruses appeared in august (56.2%, chi-square test, p,0.05 compared with other months), and another lower peak appeared in winter to spring (january to march). interestingly, influenza virus was prevalent throughout the year, and peaked in august (fig. s1 ). it is also of note that hbov was detected in nearly all months except january during the study year, and the peak was present in may and june (5.4% and 6.3% respectively, fig. 1 ). patients enrolled in this study aged from 1 day to 95 years, including 1686 children (#15 years old) and 1774 adults (.15 years old) with a median age of 16 years. the total infection rate of common respiratory virus in children is 41.6% (701 positive out of 1686 pediatric subjects), as compared to that of 38.7% (686 positive out of 1774 adult subjects) in adults, for most of the screened respiratory viruses, the infection rate of pediatric patients was higher than adult patients (p,0.05) except influenza virus, which tended to infect adults (see fig. s2 for the age distribution of common respiratory viruses). in contrast to influenza virus, which displayed a higher infection rate (540 out of 731 influenza-infected individuals) in adult patients aged from 15 to 64 years old (fig. s2) , hbov tended to mostly infect infants younger than 2 years of age (41 out of 58 all hbov-positive subjects) with a few adult infection (6 in age .15 years old group amongst 58 all hbov-positive subjects). (fig. 2 ). in this study, the common symptoms of patients detected as hbov positive included cough (91.4%), fever (100%),rhinorrhea (36.2%), sputum (36.2%). it is noteworthy that while 43 (74.1%) out of the 58 hbov-positive patients were clinically diagnosed as lower respiratory tract infection including bronchopneumonia, acute asthmatic bronchopneumonia, and severe pneumonia, only 15 (25.9%) patients met the criteria of severe respiratory infection as featured by dyspnoea. in addition, 9 (15.5%) of the hbovpositive patients were clinically presented as acute upper respiratory tract infection, and 3 were diagnosed, respectively, as bronchial asthma, herpangina and infectious mononucleosis. of note, a 77-year-old patient with acute exacerbation of copd was found infected by hbov without the presence of any of other 6 respiratory viruses tested in this study. this patient displayed a normal hemogram but a chest radiograph of coarse lung marking. to better understand the hbov pathogenicity, clinical characteristics of hbov-positive outpatients/emergency cases were compared with those of hbov-positive inpatients ( table 3 ). the hbov positive rate in outpatients/emergency was 0.56% (10 out of 1800 outpatients/emergency cases), significantly lower than that of 2.89% in inpatients (48 out of 1660 inpatients) (chi-square test, p,0.01). the odds of infection with hbov resulting in severe disease (or admission) were 5.21 (95% ci 2.64-10.25). hbov positive pediatric patients were more diagnosed as lower respiratory tract illness than adults in both outpatients and inpatients (3 out of 5 pediatric vs 0 out of 5 adults in outpatient/emergency hbov-positive patients and 40 out of 47 pediatric vs 0 out of 1 adult in inpatients), especially in infants under 2 years old (35 out of 52 hbov-positive pediatric patients) ( table 3 ). in contrast, hbov positive adults were mainly diagnosed as acute upper respiratory tract infection (auri) ( table 3) . hbov-positive pediatric inpatients were more likely to be co-infected with other viruses than adult inpatients. however, hbov-positive adult outpatients were more frequently co-infected with influenza than pediatric outpatients, and the reason may lie in the high influenza infection rate in adults ( table 2 and table 3 ). the surveillance data of 7 common respiratory viruses showed that among the 3460 samples, 112 were tested as more than one virus positive. although influenza was the most common coinfecting virus, the highest co-infection rate occurred in hcov and hbov (table 4 ). in 19 of 58 hbov positive specimens (32.76%), other virus can be found, and hcov was the most commonly co-detected virus with hbov, accounting for 7 out of 19 (36.84%) hbov co-infection cases. there were totally 8 triple virus co-infection cases (table 4 ). it is noteworthy that despite of the high co-infection rate, in as high as 67.24% hbov-positive patients, no other screened common respiratory virus was found, especially in pediatric patients (36/52, 69.2%), and most of them the complete genomes of two hbov strains gz4785 (genbank no. jn794565) and gz9081 (genbank no. jn794566) obtained in this study were highly conserved with 98.8% identity to each other, and showed more than 99% nucleotide identity to st2 strain of hbov1 (genbank accession no. dq0000496). hbov strains used in the phylogenetic analysis included the strains obtained in this study in guangzhou (gz4785 and gz9081), representative strains of hbov1-4, human parvovirus b19, bovine, and canine minute virus. based on complete genome, the phylogenetic analysis results showed that the two guangzhou strains gz4785 and gz9081 were genetically close to hbov1 (fig. 3) , consistent with the sequence comparison analysis results. from the phylogenetic tree based on complete genome, different strains of hbov1 were clearly divided into three groups (fig. 4a) , and the representative strain of group i and group ii was the prototype strains st1 and st2, respectively. group iii included 4 strains which came from taiwan, thailand, and guangzhou. most chinese strains obtained from respiratory specimens belonged to group i, but it is noteworthy that gz9081 strain obtained in this study belonged to group iii. no apparently genotypic differences existed between the phylogenetic trees based on the 3 hbov orfs (ns1, np1 and vp1/vp2) and the complete genome of 23 hbov1 strains (fig. 4b-d) . similar to previous studies [2, 24] , ns1 appeared to be the most conserved gene, whereas vp1/vp2 had the most nucleotide polymorphisms. the phylogenetic trees were almost identical between vp1/vp2 gene and complete genome, which indicated that vp1/vp2 can be used instead of complete genome to analyze the genetical relationship of hbovs. in phylogenetic analysis, we found that hbov strain gz9081 belonged to a group different from most of chinese strains obtained from respiratory specimens. interestingly, full-length genome analysis showed that gz9081 strain contained an ns1 gene closely homologous to that of the st1 group, and that the rest of its genome (np1 and vp1/vp2 genes) resembled the cu74 group, suggesting that gz9081 might represent a hybrid virus, prompting us to conduct the recombinant analysis on this strain. therefore, rdp was further carried out. the bootscan plot of the recombination event was showed in fig. 5 , which confirmed the daughter linage gz9081 was a recombinant of parental strains st1 and cu74. two trees of the relevant strain were constructed respectively on the recombinant region (position: 1-1272+4385end) and non-recombinant region (position: 1272-4385), which further confirmed that gz9081 was closely related with st1 on the recombinant region (fig. 5) . it was very probable that gz9081 was an hbov1 intra-genotypic recombinant. human bocavirus (hbov) was a newly discovered parvoviridae virus. by now, little is known about its epidemiology and genetic characteristics in guangzhou, china. the pathogenicity of hbov is still in uncertain because of its high co-infection rate with other pathogens, and it remains unclear whether hbovs are sole etiologic agent or just a concomitant virus bystander. therefore, to understand the prevailing status and pathogenicity of hbov, other pathogens needed to be simultaneously examined. so in this study, a surveillance of 12 months period during 2010-2011 in guangzhou was established to understand the prevalence and pathogenicity of hbov in patients with acute respiratory infection symptoms. other 6 common respiratory viruses (influenza virus, parainfluenza virus, adenovirus, coronavirus, rsv, metapneumovirus) were screened at the same time to understand their coinfection status with hbov. the results revealed that the overall monthly distribution of hbov and other 6 common respiratory viruses were typical, in accordance with the epidemics of respiratory viruses in tropical/subtropical areas like guangzhou, which demonstrated that the surveillance data in this study were highly reliable. it is notable that hbov, like rsv, tended to mainly infect #2 year old infants, with only a few adult infections, and the majority of the hbov positive patients (67.24% of all hbov positive patients and 69.23% of hbov positive children) were hbov single positive, indicating it may have potential pathogenicity, especially in infants. therefore, to better elucidate its pathogenic roles, the clinical characteristics of hbov-positive outpatients/emergency cases were analyzed for comparison with hbov-positive inpatients ( table 3 ). the results showed that the hbov positive rate in outpatients/emergency was statistically lower than inpatients, and the odds of infection with hbov resulting in severe disease (or admission) were as high as 5.21. hbov positive pediatric patients were more diagnosed as lower respiratory tract illness than adults in both inpatients and outpatients, and pediatric inpatients were more likely coinfected with other viruses. although there was possibility that other copathogens not be screened in this study may exist, these results showed that hbov may have pathogenic role in causing severe disease/admission or lower respiratory tract illness of children. in addition, our results showed that hbov was related to asthma or its exacerbation, which occurred in 10 pediatric patients. previously, only limited data in adults were available for the study of hbov, especially in large samples. many reports of adult hbov infection enrolled no more than 100 samples [2, 8, 12, 29, 30] . the prevalence and associated illness of hbov in adults have not been well characterized in guangzhou. in our study, we found 6 hbov-positive adult cases in 1774 adult patients with upper respiratory tract infection, and the 3.4% prevalence rate was in accordance with those previously reported for adults [2, 8, 12, 29, 30] . although hbov infection was rare in adults with respiratory infection in our study population, it was still notable that hbov may cause exacerbation in adults with basic or primary pulmonary disease like copd. we found 52 hbov-positive pediatric patients, which represented 3.1% positive rate in pediatric patients with respiratory infection. this is similar to other reports of 1.5%-19%. consistent with other studies [2, 8, 30] , the prevalence rates were higher in children under 2 years of age, but generally decrease with the increase of age (fig. 2) , which implied that antibodies against hbov acquired during early life may provide protection. in our study, up to 32.76% co-infection rate of hbov was observed from throat samples, and the rate may be higher if more viruses were screened. the most frequently detected co-pathogen was hcov, different from previous reports in china [12, 15] , and the reason may lie in different climate/geography conditions and virus distribution. there was no obvious evidence that co-infection of hbov and other common respiratory viruses can increase the disease severity, since no correlation was found between coinfection and clinical symptoms, and the rate of lower respiratory tract illness did not increase in co-infection cases. in order to improve the diagnostic sensitivity, our present study employed real-time pcr to screen hbov with the primers and probes binding the np1 conserved region. to exclude pcr contamination and prevent false positive and false negative results in hbov and other 6 common respiratory viruses screening, the following strategies were used. firstly, all pcr was strictly carried out in 4 separate rooms, namely, reagent preparation, sample preparation, pcr and pcr-product rooms. secondly, each pcr/ real-time pcr assay was performed in duplicates and repeated three times. thirdly, positive samples were further confirmed by pcr using another set of primers, and for some of the 6 common respiratory viruses positive samples, viral isolation and determination was performed to further confirm the pcr positive results. complete genome sequence of 2 hbov strains were obtained in our study. gene analysis showed high identity (98.8%) between each other, and phylogenetic analysis demonstrated that they belonged to hbov1, which was more frequently detected in respiratory tract illness than other genotypes. phylogenetic tree of complete genome showed that different strains of hbov1 can be divided into three groups. it is noteworthy that the two strains identified in this study (gz4785 and gz9081), which were circulating at the same time during the 1 year study period, belonged to different groups. furthermore, our phylogenetic analysis results showed that most chinese hbov1 strains obtained from respiratory samples belonged to group i which was genetically closely to st2, but gz9081 was different, which belonged to group iii. phylogenetic trees based on different hbov orfs showed that its ns1 gene was closely homologous to the st1 group, whereas its np1 and vp1/vp2 genes resembled the cu74 group, suggesting it may be a recombinant. although generally hbov1 sequences were highly conserved, there were still evidences that the recombination existed among hbovs [22] , and co-infection of hbovs [20] might increase the chance of recombination between hbovs. therefore, we suspect that gz9081 may be a hybrid virus. so a recombinant detection program was performed to analyze gz9081. the result confirmed that gz9081 was an intra-genotype of hbov1 recombinant, originated from the parental strains st1 and cu74 (fig. 5) . as far as we know, this is the first time that recombination between hbov1 is reported and this is the first recombinant strain of hbov1 reported in china. since recombination could change virulence or antigenicity of viruses, we believe that the finding of hbov recombination might have significance on the epidemiology, seroprotection and pathogenicity study of hbov. further studies are needed to examine the virulence and antigenic changes of gz9081 strain. continuous surveillance and genome sequence analysis are needed to obtain more information on the genotypic variation and molecular evolution of hbov in china. human bocavirus and acute wheezing in children cloning of a human parvovirus by molecular screening of respiratory tract samples human bocavirus infection in young children in the united states: molecular epidemiological profile and clinical characteristics of a newly emerging respiratory virus realtime pcr for diagnosis of human bocavirus infections and phylogenetic analysis realtime pcr assays for detection of bocavirus in human specimens human bocavirus: prevalence and clinical spectrum at a children's hospital human bocavirus in patients with respiratory tract infection human bocavirus in italian patients with respiratory diseases human bocavirus in french children high prevalence of human bocavirus detected in young children with severe acute lower respiratory tract disease by use of a standard pcr protocol and a novel real-time pcr protocol frequent detection of human rhinoviruses, paramyxoviruses, coronaviruses, and bocavirus during acute respiratory tract infections clinical and molecular epidemiology of human bocavirus in respiratory and fecal samples from children in hong kong clinical and genetic analysis of human bocavirus in children with lower respiratory tract infection in taiwan bocavirus infection in hospitalized children human bocavirus infection in young children with acute respiratory tract infection in lanzhou human bocavirus infection in children with acute gastroenteritis in japan and thailand human bocavirus infection in children with acute gastroenteritis and healthy controls human bocavirus infection in children hospitalized with acute gastroenteritis in china human bocavirus in children hospitalized for acute gastroenteritis: a case-control study human bocaviruses are highly diverse, dispersed, recombination prone, and prevalent in enteric infections human bocavirus species 2 and 3 in brazil recombination analysis based on the complete genome of bocavirus characterization and complete genome sequence of a novel coronavirus, coronavirus hku1, from patients with pneumonia children with respiratory disease associated with metapneumovirus in hong kong simultaneous detection of influenza a, b, and c viruses, respiratory syncytial virus, and adenoviruses in clinical samples by multiplex reverse transcription nested-pcr assay dna sequencing with chainterminating inhibitors rdp2: recombination detection and analysis from sequence alignments a modified bootscan algorithm for automated identification of recombinant sequences and recombination breakpoints human bocavirus infections in hospitalized children and adults human bocavirus: a novel parvovirus epidemiologically associated with pneumonia requiring hospitalization in thailand all enrollees participating in the surveillance project are appreciated. we owe our special thanks to the participated doctors and nurses of the 5 hospitals conceived and designed the experiments: k-yc lx. performed the experiments: lx xh d-mz f-sf zw l-lg. analyzed the data: lx xh k-yy m-fl. contributed reagents/materials/analysis tools: rz b-jz j-hw m-fl. wrote the paper: lx xh m-fl k-yy. key: cord-324950-ux7shvji authors: saade, georges; deblanc, céline; bougon, juliette; marois-créhan, corinne; fablet, christelle; auray, gaël; belloc, catherine; leblanc-maridor, mily; gagnon, carl a.; zhu, jianzhong; gottschalk, marcelo; summerfield, artur; simon, gaëlle; bertho, nicolas; meurens, françois title: coinfections and their molecular consequences in the porcine respiratory tract date: 2020-06-16 journal: vet res doi: 10.1186/s13567-020-00807-8 sha: doc_id: 324950 cord_uid: ux7shvji understudied, coinfections are more frequent in pig farms than single infections. in pigs, the term “porcine respiratory disease complex” (prdc) is often used to describe coinfections involving viruses such as swine influenza a virus (swiav), porcine reproductive and respiratory syndrome virus (prrsv), and porcine circovirus type 2 (pcv2) as well as bacteria like actinobacillus pleuropneumoniae, mycoplasma hyopneumoniae and bordetella bronchiseptica. the clinical outcome of the various coinfection or superinfection situations is usually assessed in the studies while in most of cases there is no clear elucidation of the fine mechanisms shaping the complex interactions occurring between microorganisms. in this comprehensive review, we aimed at identifying the studies dealing with coinfections or superinfections in the pig respiratory tract and at presenting the interactions between pathogens and, when possible, the mechanisms controlling them. coinfections and superinfections involving viruses and bacteria were considered while research articles including protozoan and fungi were excluded. we discuss the main limitations complicating the interpretation of coinfection/superinfection studies, and the high potential perspectives in this fascinating research field, which is expecting to gain more and more interest in the next years for the obvious benefit of animal health. bacterial and viral respiratory diseases are a major health issue in species reared under confined conditions in large groups. most often multiple infectious agents are involved in the development of these clinical conditions making unsuited the common reductionist approach of host-pathogen interactions by the study of single infection [1] . infection by more than one type of pathogen (viruses, bacteria and parasites amongst others) is described as a mixed infection. however, the term coinfection is frequently used to describe concomitant infection of a cell or a host by separate pathogens [2] . since in the literature the definitions of coinfection and mixed infection have been both used to describe the same events, we will use the term "coinfection" in the current review. additionally, in virology, the term superinfection is used if one virus infects the cell or the host before infection by the second superinfecting virus. we will also use the term "superinfection" in the review. finally, an opportunistic pathogen is usually considered as a pathogen that would not have infected animals in absence of the primary infection, or alternatively, "pathogen" that would have been asymptomatic in the absence of the primary infection. in some studies, however, the use of the terms "coinfection" is not suitable and "superinfection" should be used instead, as we will see later. this semantic point is responsible for a lot of confusion and makes comparisons between studies sometimes tricky. the outcome of any coinfection or superinfection can be affected by the interactions taking place between the infectious agents, the nature of the cell/host, adverse environmental and management conditions, intestinal and respiratory microbiomes, and the triggered immune response-innate and adaptive-developed afterwards [2, 3] . when occurring at the same time or with a delay, infections can impact the virulence of causative pathogens with subsequent consequences on the host immune response and its ability to clear the infections [2] . the first contact with a pathogen can change the cell/host response against any other second pathogen, possibly causing a more virulent infection, reducing its severity or suppressing it completely [4] . thus, different scenarios concerning the pathogen interactions can be observed, the first infectious agent can promote the second one, attenuate its effects or simply prevent its establishment. conversely, the second pathogen may also influence the first one directly or indirectly. coinfections have been described in both humans and animals [1, 2] . moreover, bacterial and viral infections might be followed by secondary bacterial or viral infections, which in some cases are responsible for the pathology development and the observed clinical signs. in this review, the current knowledge regarding frequent coinfections that occur in the porcine respiratory tract and particularly in the lungs are reviewed. when possible, we focused on the interactions between the mentioned pathogens and the various mechanisms justifying these interactions and their consequences on the host's response. we especially discussed coinfections involving main bacteria and viruses associated with the so-called porcine respiratory diseases, excluding coinfections involving parasites and fungi (including their metabolites, such as mycotoxins). moreover, we do not discuss the impact of adverse environmental and management conditions which have been shown to be of major importance in the modulation of respiratory infections' severity [3]. respiratory diseases have been formally described in pigs as early as the 1960′s [5] and several studies have been carried out to identify associated agents. the role of the infectious pathogens has been assessed by using two main approaches: direct research of the pathogens (by culture or polymerase chain reaction-pcr for instance) from tissue samples of diseased (acute or chronic stage) and non-diseased pigs or indirect detection by serological tests to look for antibodies produced after exposure to specific pathogens. these studies indicated that frequently under field conditions, several infectious pathogens are simultaneously detected from lung lesions (see [6] [7] [8] causative respiratory infectious agents can be divided into primary and secondary or opportunistic pathogens. primary pathogen being defined here as pathogen that can infect the animal as first unique pathogen and then facilitate secondary or opportunistic coinfection. these primary pathogens include common bacteria such as highly virulent actinobacillus (a.) pleuropneumoniae, m. hyopneumoniae, bordetella (b.) bronchiseptica in young piglets and common viruses such as swiav [1] . prrsv and pcv2 are not strictly respiratory pathogens as swiav, however, since they also frequently affect the respiratory system and since they can act as facilitators of secondary respiratory infections, they must be considered too. other primary pathogens such as aujeszky's disease virus (adv) and prcov are reported but they are far less frequently encountered today or they have less impact on porcine health [1]. then, some viruses like the porcine cytomegalovirus can also inhibit host immune functions-particularly the action of t lymphocytes-and promote respiratory diseases such as the porcine reproductive and respiratory syndrome [9] . among the secondary pathogens common bacteria such as lower virulence strains of a. pleuropneumoniae, a. suis, glaesserella parasuis, pasteurella multocida, and streptococcus (s.) suis are reported. together primary and secondary pathogens are involved in the "porcine respiratory disease complex" (prdc) [10] . several studies have assessed the nature of the infectious agents directly or indirectly associated with respiratory diseases in pigs [7, 8, 11, 12] . in one of these studies involving breeding sows in five french farrow-to-finish herds [12] , results indicated that s. suis, a secondary pathogen, was quite widespread among sows-67.1% of the animals being positive using a pcr assay-and pcv2 and swiav infections were highly prevalent (75% of the sows with antibodies against pcv2 and between 91.7% and 100% of the sows with antibodies against swiav). other infectious agents such as a. pleuropneumoniae, g. parasuis and p. multocida were detected in 31%, 25%, and 23% of the sows, respectively [12] . in another study evaluating infectious agents associated with respiratory diseases in 125 farrow-to-finish pig herds in france, it has been shown that m. hyopneumoniae, prrsv, and swiav subtype h1n1 were the major pathogens involved in pneumonia-like gross lesions [8] . for extensive pleuritis, prrsv was frequently associated with a. pleuropneumoniae [8, 12] . regarding bacteria associated with lung lesions in 3731 french slaughter pigs [8] , a report mentioned lesions of pneumonia and pleuritis as the most frequent lesions. in these lesions, bacteria such as m. hyopneumoniae, p. multocida, a. pleuropneumoniae, s. suis, and g. parasuis were detected in 69.3%, 36.9%, 20.7%, 6.4%, and 0.99% of the lungs, respectively [13] . in a retrospective analysis of the etiologic agents associated with respiratory diseases in pigs in usa, two or more infectious agents were identified in 88.2% of the analyzed cases [7] . prrsv (35.4% of the samples), p. multocida (31.6%), m. hyopneumoniae (27%), swiav (22.2%), g. parasuis (22.0%) and pcv2 (18.6%) were the infectious agents most frequently encountered [7] . in korean pigs, prrsv and pcv2 were frequently identified associated or not to various bacteria such as s. suis (25.2%), m. hyopneumoniae (20.1%), p. multocida (12.9%), and a. pleuropneumoniae (5%) [11] . below we review the main primary pathogens as defined above, common viruses such as prrsv, pcv2, swiav, prcov and adv as well as bacteria like a. pleuropneumoniae, m. hyopneumoniae and b. bronchiseptica. conversely, other pathogens involved in the prdc are not presented in the following sections while considered in additional file 1 presenting the different coinfections' situations. prrsv is an enveloped single stranded positive rna virus belonging to the arteriviridae family. two different species, prrsv-1 (also known as betaarterivirus suid 1), from european origin, and prrsv-2 from american origin, are now distinguished [14] . this enveloped virus replicates mainly or exclusively in macrophages such as alveolar macrophages (ams), but also macrophages from the nasal mucosa and pulmonary intravascular macrophages (pims) [15, 16] . in vitro, prrsv can also replicate in cultured monocytes and monocyte-derived cells including macrophages [17] and in vitro-derived dendritic cells (dcs) generated either from bone marrow hematopoietic cells (bmdcs) or blood monocytes (modcs), depending on the in vitro culture conditions [18, 19] . however, such in vitro generated dcs are not representative of in vivo primary dcs which do not seem to be permissive to viral replication [20] . in fact, modc and bmdc (at least when generated using granulocyte macrophage colony-stimulating factor, gm-csf) although possessing functional overlaps with the dc family, do not represent bona fide dcs, which represent an own lineage of hematopoietic cells distinct from the monocytic lineage [21] . different cell surface molecules are involved in prrsv entry and infection of cells: heparan sulfate, porcine sialoadhesin-also known as sialic acid-binding immunoglobulin-type lectin 1 (siglec-1), siglec-10, cd151 and cd163 [22, 23] . heparan sulfate is a glycosaminoglycan (gag) that seems to play a modest or secondary role in prrsv infection since the blocking of this receptor on ams induced only a mild decrease in prrsv infectivity. moreover, this effect was not observed with all the prrsv isolates tested, suggesting that the involvement of heparan sulfate depends on the antigenic diversity of prrsv [22] . siglec-1/cd169 is a member of the sialic acid-binding lectins (siglecs) family and is expressed on macrophages [22] and siglec-10 has been identified as an alternative receptor to siglec-1 [23] . binding of prrsv to siglecs induces its internalisation by clathrin-mediated endocytosis. expression of recombinant porcine sialoadhesin is sufficient to induce the internalisation of prrsv by non-permissive cells, but not replication [24] . cd163 is a scavenger receptor involved in prrsv infection [22] . its expression on nonpermissive cells makes them susceptible to infection with prrsv and allows productive replication of the virus [22] . moreover, cd169-ko animals are still susceptible to prrsv-2 infection [22] , whereas cd163-ko animals are resistant to prrsv-1 and prrsv-2 [25, 26] . finally, myh9 has been recently identified as an indispensable partner of cd163 for prrsv cell entry for both prrsv-1 and prrsv-2 [27] . prrs clinical signs can be nearly absent to severe depending on the considered prrsv species and strains. when observed, there are, amongst the most frequent, lethargy, dyspnea, tachypnea, as well as a reproductive disease [16] . prrsv can persist in infected pigs for several months after the initial infection particularly in lymphoid tissues and has the ability to alter the host's immune system to escape it (for review see [16] ). prrsv interferes with the porcine innate immune response through downregulation of type i interferons (ifns-ifnα and ifnβ mostly), which are cytokines known for their antiviral properties [28] . prrsv-infected macrophages also had a reduced capacity to produce the pro-inflammatory cytokines tnfα and il1β [28] and the production of the anti-inflammatory cytokine il10 was found enhanced during infection [29] . nevertheless, the role of cytokine modulation during prrs is unclear considering that the effects appeared to depend on the prrsv species, as well as on the prrsv isolates, since opposite results can be found with different prrsv strains [30, 31] . in fact, some prrsv-2 isolates were shown to enhance ifnα production while other prrsv-1 isolates suppressed it. results seemed also very variable for the immunoregulatory il10 along different isolates of prrsv-1 [30, 31] , making general conclusions about how prrsv alters innate immune responses difficult. prrsv impact on adaptive cellular immunity seems also to be highly variable according to the species and the strain [20] . conversely, whereas non-protective antibody response against the viral nucleocapsid is found within a week post-infection, neutralizing antibodies appearance is highly delayed for all prrsv species and strains, appearing only after 3 or 4 weeks of infection and peaking even later [16] . pcv2 is a naked circular single stranded dna virus belonging to the circoviridae family and responsible for porcine circovirus disease (pcvd). the attachment of pcv2 to target cells occurs through chondroitin sulfate b and probably other receptors [32] . internalisation is not fully known but it does not seem to involve a specific receptor and the gags could mediate internalisation and binding to the target cells [33] . most of the time the infection is subclinical but in some circumstances such as coinfections with other respiratory pathogens it can cause the post-weaning multisystemic wasting syndrome (pmws), clinically characterized by wasting respiratory disease, and enteritis [34] . infection with pcv2 can occur in utero, resulting in stillborn piglets and mummified fetuses, or death at different ages after birth [34] . in young and older animals, pcv2 was found in cells expressing monocytes (cd14 + ), and t and b cells (cd4 + , cd8 + , igm + ) markers [35] . further results showed that active replication of the virus was supported by t and b cells, with enhanced replication in proliferative cells [36] . in vitro, pcv2 can also infect many other cell types including endothelial cells, gut epithelial cells, fibrocytes, and dcs [37] . in dcs the virus seems to persist and remain infective for a prolonged period without replication indicating that these cells might serve as a vehicle for virus spread in the host [38] . pmws is characterized by the depletion of lymphoid cells affecting t cells, b cells, and nk cells [39] . this lymphopenia was also associated with impaired responses of peripheral blood mononuclear cells (pbmcs) to mitogen stimulation with lower levels of il2, ifnγ, and il4 production compared to pbmcs from non-infected pigs [40] . another feature of pmws is an elevated level of il10 found in lymphoid organs, especially in the t cells rich areas [41] . il10-mediated immunosuppression could play an important role in the pcv2 infection and the development of pmws. pcv2 has also the ability to alter the innate immune response [42] . even though the virus does not productively infect dcs, evidence shows that it can interfere with the normal plasmacytoid dcs (pdcs) response. upon stimulation with cpg-odn, pdcs' ability to produce ifnα and tnfα was impaired in cells previously infected with pcv2 [43] . pcv2 dna isolated from infected cells induced the suppression of pdc ifnα production [43] . influenza a viruses are enveloped single stranded negative rna viruses belonging to the orthomyxoviridae family. these enveloped viruses can infect a broad range of hosts, with pigs being one of their natural hosts (for a review see [44] ). the three main iav subtypes encountered in pigs are h1n1, h1n2, and h3n2 [44] , but many genetic lineages and antigenic variants within these subtypes are co-circulating in the pig population worldwide. subclinical infections with swiavs are common in pigs, but they can also induce a disease similar to what is observed in humans, with upper respiratory tract distress associated with fever, cough, rhinitis, high morbidity, and low mortality [44] . the main targets of swiavs are epithelial cells of the respiratory tract but iavs can also non-productively infect alveolar macrophages [45] . two major glycoproteins are present at the surface of the virus: hemagglutinin (ha) and neuraminidase (na). binding of ha with the sialic acid molecules at the surface of the host cells will induce the endocytosis of the viral particle [44] . the na molecule plays the main role in the budding of the virus by removing the sialic acid, allowing the release of neoformed virus particles from the infected cell [44] . the innate response against the virus includes production of high levels of pro-inflammatory cytokines such as ifnα, tnfα, and il6. dcs, in particular pdcs play an important role in this response [46] . an important observation was that the production of these cytokines correlated to the viral loads and the severity of the disease. infection with swiav induces cellular and humoral specific immune responses in pigs recovering from the disease and the serum igg and the mucosal iga can protect the animal from re-infection [44] . ns1 and pa-x are the main viral proteins that alter the innate immune response, mainly by blocking the type i ifn response [47] as well as the nlrp3 inflammasome activation [48] in infected-epithelial cells and alveolar macrophages. finally, the main mechanisms through which the swiav escapes the adaptive host immune system are the antigenic drift and the antigenic shift concerning mainly ha and na which are also the two major antigenic proteins expressed on the surface of the virus and against which the neutralizing humoral response is directed [44] . prcov is an enveloped single stranded positive rna virus belonging to the coronaviridae family. in pigs, four alphacoronavirus, one betacoronavirus and one deltacoronavirus have been described [49, 50] . thus, most of the porcine coronaviruses are from the genus alphacoronavirus. the only respiratory porcine coronavirus, prcov, is a variant of transmissible gastroenteritis virus (tgev) where a large 5′ region deletion (nucleotides 621-681) in the spike gene of the virus altered the tropism and the virulence. even if pigs have been shown to be susceptible to the first sars-cov (serological evidence and isolation of the virus in a pig farm in the xiqing county of tianjin, china) [51] they have not been successfully experimentally infected, at this stage, by sars-cov-2 [52] . prcov uses aminopeptidase-n (cd13) domain iv to enter cells [53] and replicates to high titers in the lungs (1 × 10 7 -10 8 tissue culture infectious dose 50-tcid 50 ) specifically in type 1 and 2 pneumocytes. moreover, it can infect epithelial cells of the nares, trachea, bronchi, bronchioles, alveoli, and, occasionally, alveolar macrophages [49] . infections with the prcov are usually subclinical, but there is variation between strains and some can induce a more severe disease. prcov can infect pigs of all ages by direct contact transmission or aerosol [49] . the clinical signs are associated to the respiratory system and are mild to severebronchointerstitial pneumonia-depending the strain and the context (environmental and management factors as well as the presence of other pathogens). suid herpesvirus 1, usually known as prv or adv is the responsible agent of aujeszky's disease in pigs. it is a double stranded enveloped dna virus from the herpesviridae family and alphaherpesvirinae subfamily targeting respiratory and/or genital mucosae for its replication [54] . adv has a very broad host range varying from domestic animals like pigs, cattle, goats, sheep, cats and dogs to wild animals such as ferrets, foxes, hares, raccoons, and wild deer, and where it induces different diseases [54] . infected animals usually show fever, sneezing, coughing and vomiting accompanied occasionally with typical nervous manifestations like convulsions, aggressiveness and lack of coordination. mortality rate can reach 100% in suckling piglets while in mature pigs the infection is inapparent or mild [54] . adv possesses eleven types of envelope glycoproteins playing major roles in the interaction with host cells and the induction of immune response [54] . viral binding and fusion with the plasma membrane of the target cell-epithelial cells, neurons and alveolar macrophages-are controlled by a cascade of events orchestred by glycoproteins c (gc), gb, gd, gh and gl. the binding process starts with an interaction of gc with heparin sulfate proteoglycans [54, 55] . stabilization of this interaction is then assured by the binding of gd to specific cellular receptors known as herpesvirus entry mediators such as hvea (tnfrsf14), hveb (prr2, nectin 2), hvec (prr1, nectin 1), hved (pvr, cd55), and 3-o-sulfated heparin sulfate [54, 56] . at this stage, tyrosine-based or dileucine-based endocytosis in parallel with clathrin-mediated endocytosis occur by the mediation of gb, gh and gl, leading to the penetration of the capsid and the tegument into the cellular cytoplasm. finally, the interaction of the capsid with dynein leads to the release of viral dna into the cellular nucleus after a transport along microtubules from the periphery to the nuclear pores [55] . porcine humoral immune response is induced by adv and neutralizing antibodies are mainly directed against gc [57] . specific cell mediated immune responses are also triggered and mhc class i restricted, gc-specific, cytotoxic cells are induced. adv also alters the ifn signaling pathway by suppressing stat1 tyrosine phosphorylation leading to an inhibition of ifn-stimulated genes (isgs) expression [54, 57] . adv may be involved in the prdc and can be isolated alone or with other pathogens. accordingly, a study conducted in taiwan reported the association of adv with pcv2 in 10.3% of the evaluated pigs using a multiplex pcr [58] . animals affected with this gram negative bacterium develop a pleuropneumonia characterized by fibrinohemorrhagic necrotizing bronchopneumonia and fibrinous pleuritis which can reach a high mortality rate [59, 60] . although the disease is best known in its acute/peracute forms, subacute and/or chronic presentations with low or no mortality are highly prevalent, especially in the presence of antibiotic treatments. many herds are subclinically infected without previous or present episodes of clinical disease and in the absence of suggestive lesions at the slaughter house. animals are, nevertheless, carriers of the pathogen. this happens in several conventional herds which may be simultaneously infected not only with several low/intermediate virulent strains, but also, in some cases, with strains highly likely to cause disease. in the latter case, outbreaks may suddenly appear in the presence of concomitant diseases or as a consequence of changes in management and/or environment [59, 60] . eighteen serotypes of the bacterium have been described, which can all induce disease, although clear differences in virulence have been described [59, 60] . these bacteria can be found mainly in tonsils of carrier animals; virulent strains have a tropism for the lower respiratory tract where they preferentially bind to ciliated cells of the terminal bronchioli and pneumocytes [59, 60] . different virulence factors expressed by a. pleuropneumoniae are involved in the colonization and the development of the disease. adhesion to cells could be mediated by type iv fimbriae that are expressed upon contact with respiratory epithelial cells in vitro and during lung infection [59, 60] . adhesion of a. pleuropneumoniae to respiratory epithelial cells also involves the binding of bacterial lipopolysaccharides to glycosphingolipids on the surface of the cells [59, 60] . the formation of biofilm by the bacteria is likely to play an important role in the colonization of the host [61] . after attachment to the target cells, the bacteria can produce four different pore-forming exotoxins (apx i, ii, iii and iv) inducing the lysis of alveolar epithelial cells, thus allowing the acquisition of nutrients by the bacteria, but also participating in the development of the lesions [60, 62] . some of the virulence factors expressed by a. pleuropneumoniae interfere with the host's immune response. the toxins apx i, ii and iii induce the lysis of not only respiratory epithelial cells, but also of cells involved in the innate immune response such as macrophages and neutrophils [60, 63] . at lower concentrations, these toxins lose their lytic properties but can still impair macrophages chemotactic activity and their phagocytic abilities [64] . the capsular polysaccharides of a. pleuropneumoniae interfere with macrophage phagocytosis and enable resistance to complement-mediated killing [60] . a. pleuropneumoniae may also interfere with the antibody response by producing proteases that can degrade porcine iga and igg [59, 60] . this cell wall-free bacterium is considered to play a primary role in prdc and is the causative agent of porcine enzootic pneumonia (ep), a disease with high morbidity but low mortality rates [65] . the main pathological mechanisms involved in m. hyopneumoniae infections are: (i) adhesion to the ciliated cells of the tracheal epithelium inducing ciliostasis, loss of cilia and exfoliation, dysregulation of cellular homeostasis (with increased intracellular calcium concentration) and secretion of cytotoxic factors, (ii) alteration of the mucociliary tract, (iii) inflammatory reactions sometimes exacerbated and prolonged, and (iv) manipulation of the innate and adaptive immune responses [65, 66] . among the adhesins described in m. hyopneumoniae, p97 is reported to be a major determinant of cell adhesion [65] [66] [67] . several other adhesins were reported: p102 linked to p97, lpps, lppt, mgpa, p65, p76, p110, p146, p159, and p216 [65, 66] . most adhesins are transcribed and translated during m. hyopneumoniae infection and then undergo posttranslational cleavage to result in diverse products on the membrane surface [65, 67, 68] . the diversity of surface proteins can also derive from the variation in the number of repeats in genes encoding adhesins [69] . these mechanisms of antigenic variation enable the bacterium to escape from immune system recognition and to invade the host [66] . adhesins can also recruit extracellular matrix components (plasminogen, fibronectin and actin amongst others), and therefore can promote invasion and inflammatory response [65, 70] . the immune response induced against m. hyopneumoniae, may have a double action: over-activation of the local immune response resulting in a pathologic inflammatory reaction or local immunosuppression explaining the chronic nature of the associated pathologies [65, 66] . acute m. hyopneumoniae infection leads to the recruitment and activation of various innate immune cells, essentially through the involvement of a large range of cytokines: il1, il6 and tnfα in lungs; cxcl8, il1, il2, il4, il6, tnfα and il10 in bronchus-associated lymphoid tissue (balt) or tracheobronchial lavage fluid (tblf) [65, 71, 72] . some of these inflammatory cytokines (tnfα, cxcl8, il1β, il6) are produced chronically in the lungs and play powerful roles in apoptosis (tnfα), differentiation and chemotaxis of neutrophils (respectively, il6 and il8), and macrophage activation (tnfα, il1β). chronic infections are typically associated with intense lymphoid hyperplasia [71] and are characterized by an accumulation of igg-and iga-expressing plasma cells, cd4 + t cells, macrophages and dcs in the balt of inflamed lung tissue [73] . involvement of t cell activation in chronic inflammation is also supported by the presence of t-cell cytokines such as il-2 and il-4 in bronchoalveolar exudates [72] . in vitro studies conducted with macrophages cocultured with m. hyopneumoniae highlighted a strong activation of inflammatory pathways inducing the production of cytokines and chemokines, and expression of receptors or pathways inducing cell apoptosis [65, 66, 74, 75] . moreover, m. hyopneumoniae is described as an inhibitor of macrophages phagocytic activity, which may explain the chronicity of m. hyopneumoniae infections and the greater host susceptibility to other pathogens [65, 66, 74] . mycoplasma hyopneumoniae was found to activate costimulatory molecule expression on bona fide dcs with poor tnfα production, contrasting with monocytes. interestingly, a strong mitogenic activity for b cells was observed [76] . altogether, these data indicate that m. hyopneumoniae is well sensed by the innate immune system, but the presence of immune evasion mechanisms targeting antigen presenting cells remains a possibility that needs further investigations. antibody responses after infection develop slowly and do not appear to correlate with protection [65, 66] . the literature on m. hyopneumoniae infections coupled with information from mouse models indicates that adaptive immune responses represent a fragile balance between pathogenic and protective th-cell responses, probably belonging to the th1 or th17 types [65, 66] . this aerobic gram-negative bacterium can be found in the respiratory tract of several animal species and it presents a worldwide distribution in the porcine rearing [77] . b. bronchiseptica has a strong tropism for ciliated cells from the respiratory tissue and is mostly detected in the apical portion of the ciliated cells of turbinates, trachea and lungs [77, 78] . it can also be found in the cytoplasm of neutrophils and macrophages and rarely in the alveolar lumen associated with small tufts of cilia [77, 78] . hence, infected pigs show cilia loss in the bronchial and bronchiolar epithelium associated with multifocal erosion, fibrosis, and hyperplasia. neutrophil infiltrates are noted in the peri-conchal meatus and the submucosa of the bronchioles and alveoli, while lymphocyte and plasma cell infiltrations occur at the level of the lamina propria [77, 78] . cell adhesion of b. bronchiseptica is a multifactorial process involving two main virulence factors; filamentous hemagglutinin (fha) and pertactin (prn) [77, 79] . the expression of both adhesins is controlled by the bordetella virulence genes (bvg)as signal transduction system. fha is an adhesin with several binding domains including a carbohydrate-recognition domain responsible of the adhesion to macrophages and ciliated epithelial cells, a heparin-binding domain that mediates the binding to sulfated polysaccharides, and an arg-gly-asp domain (rgd) regulating the intercellular adhesion molecule 1 (icam1) by epithelial cells after interaction with the nf-κb signalling pathways [79] . this rgd domain is also present in the structure of prn and contributes to the binding process [77, 79] . on the other hand, non-opsonic adhesion mechanisms play a role in binding to the host cells such as carbohydrate-specific mechanisms and those involving sialic acid-containing compounds [77] . virulence of the bacteria depends on the strains; therefore, clinical signs can be different going from sneezing and transient nasal discharge for moderate and non-toxic strains to bronchopneumonia and atrophy of the nasal turbinate bones for virulent strains, especially if they are associated to other bacteria such as p. multocida [77] . thus, b. bronchiseptica is usually described as primary lung pathogen in young pigs where it causes necrohemorrhagic bronchopneumonia whereas in older pigs this bacterium is mostly known as an opportunistic pathogen contributing to the prdc [77] . the immune response against b. bronchiseptica is mainly triggered by the different toxins expressed such as adenylate cyclase, tracheal cytotoxin and dermonecrotic toxin (dnt). in the following section and additional file 1, we have used the published studies evaluating multiple infections including viral-viral, bacterial-viral, viral-bacterial and bacterial-bacterial respiratory coinfections and superinfections in swine. both in vivo and in vitro studies comparing single to multiple infections were included. studies evaluating vaccinations and the development of diagnostic techniques such as elisa or qpcr were excluded as well as trials testing antiviral or antibacterial molecules when there was no clear comparison between single and multiple infections. an attempt to present a synthetic view of coinfections is depicted in the heat maps (see figures 1, 2, 3) . however, we recommend readers to refer to additional file 1 for each coinfection couple to get a more detailed view. in these heat maps, we were interested in the effect of the first pathogen on the multiplication of the second one (named "assessed pathogen" in the figures) and on the host immune response and/or clinical signs. these effects were evaluated and a grade from − 5 to + 5 was given to every pathogen depending on the intensity of its impact on the multiplication of the second agent and on the immune response or on the clinical signs. negative grades were given to pathogens decreasing the multiplication of other pathogens, while positive grades were given to those inducing an increase. similarly, negative grades were attributed to pathogens with a tendency to decrease clinical signs or immune response related to the other pathogen. positive grades were given in case of an increase. the sum of the grades was calculated if the same pathogen combination was evaluated in several studies except in the case of discordant results. this grading was represented in the following heat maps and the number of the identified studies for the same pathogen combination is shown on the maps. in the heat maps, other pathogens, that are less associated to prdc such as g. parasuis, m. hyorhinis, m. floculare, p. multocida, and s. suis or even not considered as respiratory pathogens like staphylococcus aureus, classical swine fever virus, hepatitis e virus, porcine rubulavirus, ppv, and torque teno sus virus 1 have also been included. indeed, these pathogens can also impact the outcome of respiratory infections and deserve, at least, to be mentioned. viral/viral respiratory coinfections have always had an important role in the porcine respiratory disease complex [1] . several studies assessed the presence of two or more viral pathogens in pigs showing respiratory clinical signs in farms located in endemic regions [7, 8, 13] . the main viruses contributing to the porcine respiratory disease are swiav, prrsv, pcv2, and to a lower extent the prcov and the adv. due to their fast-spreading and their economic consequences, some viruses were more studied than others in the last 20 years, especially pcv2, prrsv, and swiav as shown in additional file 1a and b. we will thus put more emphasis on these three viruses as causes of primary infections. many in vivo studies were carried out to assess the severity of the clinical signs and the development of the microscopic/macroscopic lesions. this approach enabled a comparison between coinfection/superinfection and single-infection conditions. then, viral interference was progressively more frequently measured as a way to better understand the consequences of coinfections. in the last decades, the strong development of molecular biology and various tools enabled the evaluation of the immune response developed following polyviral infections. in additional file 1, the selected studies that were carried out on viral coinfections are presented from the oldest in vivo experiments to the latest in vitro and ex vivo experiments (additional file 1a and b). this data synthesis highlights the major impact of prrsv primary infection, which can both increase the titre of the following virus (pcv2, hepatitis e virus-hev) in vitro [80] and in vivo [81] [82] [83] (figure 1a ), but can also worsen the clinical score associated to the disease ( figure 3a ). interestingly, even when the prrsv does not increase the viral production of the other virus, as observed in coinfections involving swiav [84] or prcov [85] (figure 1a) , it can also worsen the associated clinical signs. swiav and pcv2 as primary infectious agents have been less studied. however, it can be observed that swiav can interfere with other virus productions (prrsv and prcov) [85, 86] whereas pcv2 has some detrimental impact on the clinical outcome of secondary viral infections (prrsv, swiav, and ppv) [87] [88] [89] (see additional file 1 and figure 1a) . then, regarding the inflammation induced in coinfection conditions, various outcomes were observed depending which viruses were considered (figure 2a) . many in vitro and in vivo experiments, with different bacterium-virus and virus-bacterium combinations, have been performed to identify the underlying mechanisms of the prdc (see figures 1b, c, 2b , c, and 3b). the main studies are presented in additional file 1c and d. bacterium-viral coinfections can also involve various primary respiratory pathogens. among them, the most frequently studied bacterium is m. hyopneumoniae, a pathogen that induces a chronic respiratory disease and can influence the outcome of a subsequent viral infection. however, mycoplasma infection needs to be already well established in the respiratory tract at the time of the viral infection to potentiate it. indeed, m. hyopneumoniae inoculated to pigs simultaneously or shortly before the virus did not strongly impact the severity of the viral infections (pcv2, swiav, prrsv) [90] [91] [92] , while its impact was clearly evidenced when inoculated 3 weeks before viral infections [93] . it is well-known that viral infections can induce an ideal environment for a bacterial superinfection through different mechanisms such as the destruction of the epithelial barrier, the over-expression of the receptors involved in the bacterial adhesion to the cells, and the alteration of the host immune response [1, 2, 94, 95] . the swiav infection has been shown, for instance, as a way to facilitate the colonization of epithelial cells by s. suis, but only for the serotypes containing sialic-acid in their capsule [96] . the swiav infection induces a loss of ciliated cells leading to the impairment of the mucociliary clearance function, but induces also the presence of the viral ha on the surface of infected cells that interacts with the sialic acid of the bacterial capsule, leading to increased adherence of s. suis [96, 97] . although these swiav effects on s. suis have been clearly shown in vitro, no clear in vivo impact of swiav infection on s. suis pulmonary load has been described [98] . it was clearly shown that the presence of both pathogens significantly induces more inflammation than single infections [98, 99] . overall, studies carried out in pigs showed that a bacterium-virus or a virus-bacterium coinfection frequently induces an aggravation of pulmonary lesions ( figure 3b ) and a higher inflammation ( figure 2b ) and immune response, with increased production of pro-inflammatory cytokines. in many bacterium-virus and virus-bacterium associations, this worsened outcome seems to be the result of additive effects from both pathogens rather than a real synergy [100, 101] . however, a potentiation of the viral infection by bacteria can also be observed in other cases, such as in the m. hyopneumoniae-prrsv coinfection [102] . in that case, higher amounts of prrsv genomes were detected in lymphoid tissue and blood [102] and a slower viral clearance was observed [75] (figure 1b) , suggesting that the recruitment of immune cells in the lung parenchyma upon established m. hyopneumoniae infection may provide a steady supply of susceptible cells for prrsv [1] . then, in porcine ams and in the "african green monkey" (originally described as porcine origin) st-jude porcine lung (sjpl) cell line, prrsv infection has been shown to be blocked by a pre-infection with a. pleuropneupmoniae, this antiviral activity being due to the a. pleuropneumoniae metabolites [103] ( figure 1b) . given the fact that in vivo studies involving prrsv and a. pleuropneumoniae did not always investigate the impact of an a. pleuropneumoniae pre-infection on the subsequent prrsv infection [104, 105] , as done in experiments performed in vitro [103] , it cannot be easily concluded if this interference would be observed in the target species. however, in vivo, prrsv-a. pleuropneumoniae interactions were reported as absent or mild [104, 105] (figure 3b ). in virus-bacterium coinfections, the dogma usually encountered is that viruses play an immunomodulatory role, which favors bacterial superinfections. nevertheless, a pre-disposing effect is also described for m. hyopneumoniae, which promotes viral but also bacterial superinfections [65] (figure 1d ). few studies of experimental coinfections or superinfections with m. hyopneumoniae and/or other bacteria involved in prdc were performed compared to coinfections involving viruses. these studies are reported in additional file 1e. overall, these coinfections or superinfections induce more clinical signs and lung lesions and poorer technical performances when compared to single infections with the same infectious pathogens ( figure 3c ). the bacterialbacterial coinfections are also responsible for immune response alterations (for reviews see [106, 107] ). for example, macrophages from pigs infected by m. hyopneumoniae decrease their phagocytosis capacity against a. pleuropneumoniae [60, 65] . m. hyorhinis and m. flocculare, two mycoplasmas commonly co-isolated with m. hyopneumoniae in gross pneumonia-like lesions, may also impact the immune response by inducing the cytotoxicity of immune cells and/or the secretion of cytokines affecting its outcome [108] . co-stimulation of porcine bmdcs with m. hyopneumoniae and m. hyorhinis induces a strong il12 production. in this last in vitro model, m. hyopneumoniae associated with m. flocculare reduces tnfα production compared to bmdcs stimulation by m. flocculare alone producing a tnfα concentration greater than that observed after stimulation with m. hyopneumoniae alone and m. hyorhinis alone [108] . therefore, m. flocculare might play an initial role in pulmonary inflammation by inducing the production of tnfα by resident myeloid cells. supplementary investigations will be needed to elucidate the role of this cytokine in the pathogenesis of the disease [108] . other examples of bacterium-bacterium in vivo coinfections are presented in additional file 1e. regarding coinfections and superinfections, most studies assessed the clinical outcome of the process but less is known about the mechanisms of interactions between pathogens and the consequences for the pathogens themselves, the infected cells and more generally for the host. the outcome of dual infection is variable depending on the antagonism, neutrality or synergy between the infectious agents. on the host side, coinfection can make the host response ineffective, and vice versa. if we look now at the possible interactions that can occur between pathogens we have to consider the nature of the infectious agents (summary provided in figure 4) . different situations can be observed and coinfections can involve virus with virus, bacterium with virus and vice versa, and bacterium with bacterium. regarding virus-virus interactions, consequences are diversified and many studies looking at virus replication in coinfection situations have been carried out [2]. the first consequence of coinfection could be the so-called viral interference, a situation whereby one virus interferes with the replication of the other one making the cells resistant to the superinfecting virus [109] . the most common way for viral interference is indirect and based on the production of type 1 and 3 ifns which induce the expression of isgs after interacting with their cognate receptors [110, 111] . these proteins then activate numerous mediators of the cellular antiviral system that may non-specifically block the replication of viruses. they may also interfere, to a certain extent, with bacterial multiplication since ifn can also be induced by intracellular bacterial pathogens (ibps) or some extracellular bacteria [107, 112] . nevertheless, in some situations type 1 ifns can also increase the host susceptibility to subsequent bacterial infection [113] through impaired macrophage recruitment with a reduced cxcl1 and cxcl2 transcription [114] and a reduced il17 [115] production. then, there is also the non-interferon-mediated viral interference (or intrinsic interference) which is a cellular state of resistance induced by the virus to new viral infection by closely related or unrelated viruses [116] . various mechanisms are described to explain this cellular state of direct or indirect resistance (for examples see [2]). in this type of interference, which can occur between viruses but also between viruses and bacteria [107, 117] , there is a competition between pathogens for the metabolites and all the host factors that allow their multiplication. besides the mechanisms involving a competition for common cellular factors, there are also several other mechanisms of interference described. these relies on viral defective interfering (di) particles [118] , rna interference (rnai) [119] , non-specific double stranded rna (dsrna) [120] as well as trans-acting proteins [121] . interference can occur at specific steps or multiple steps of the viral replication cycle (attachment, penetration, genome replication and/or budding) and can be direct or indirect. inhibition of superinfection (superinfection exclusion and superinfection suppression) is one of several consequences that can be observed in the interference between related and unrelated microorganisms. in superinfection exclusion, an established infection interferes with a subsequent, closely related infection [2, 122] . an example of this phenomenon in pigs is the exclusion of highly virulent classical swine fever virus strain margarita in wild boars persistently infected with this virus upon a challenge infection with the same margarita strain [123] . superinfection suppression is a quite close concept where this time persistently infected cells resist to a challenge with a heterologous virus [2] . furthermore, when the host immune response-innate or adaptive-is considered in the study of the complex interactions taking place in viral coinfections, additional mechanisms of indirect interference linked to cellular and humoral cross-protection-resulting from a first viral contact with a wild-type or a vaccine strain-can be described. conversely, in some situations, viral coinfections can directly or indirectly result in enhanced replication and virulence for one or both pathogens as observed in several studies involving porcine viruses [80, 83, [124] [125] [126] . in other cases, coinfection/superinfection has no effects on virus replications and the viruses can coexist in a relation called accommodation [2] . besides consequences in terms of viral replication, there are also consequences for the genetic of the viruses and their evolution through events of recombination between closely related viral genomes. recombination, the parameters influencing it and its consequences were reviewed in rna and dna viruses [127, 128] . then, as a result of all these possible interactions between viruses, the severity of the resulting disease and its epidemiology can be altered as exemplified in additional file 1. in the pig studies, most often, however, the exact mechanisms controlling interactions between viruses were not elucidated. several mechanisms explaining bacterium-virus and virus-bacterium interactions have been identified (for reviews see [1, 94, 117] ). the interactions can have either a positive or a negative impact on both pathogens depending on the bacterial and viral species involved. usually, when the interactions are direct they promote viral infection without affecting the bacterial species [1, 94, 117] . examples of these direct interactions are (i) direct binding of the virus to a bacterium or (ii) the utilization of a bacterial product by the virus. an example of direct interactions in the respiratory tract is the cleavage of the iav ha into ha1 and ha2 by a staphylococcus aureus protease helping the viral particle to become infectious [129] . on the contrary, when interactions are indirect they often provide an advantage to bacterial infections. four mechanisms dealing with indirect interactions have been described: (i) viral alteration of the epithelial barrier, (ii) reduction or suppression of the host immune response, (iii) viral alteration/displacement of the microbiota, and (iv) virus-induced alteration of bacterial cellular receptor expression [94] . all these mechanisms can operate together for the benefit of the superinfecting bacteria. a typical example of these indirect interactions is provided by pcv2 and swiav and porcine pathogenic bacteria such as a. pleuropneumoniae [130] and s. suis [96, 97, 131] where the bacteria benefit from the prior viral infections. however, bacteria can also directly benefit from a previous viral infection as observed in a study demonstrating that staphylococcus aureus was able to bind viral ha [132] . the consequence of that binding was an enhanced bacterial internalisation by two mechanisms: (i) binding to ha exposed at the surface of infected cells, and (ii) binding to free extracellular virions. in some other situations, non-pathogenic bacteria can also directly or indirectly protect the host from viral infection as typically observed with probiotic bacteria which can show antiviral activity through the binding/ capture of the viruses and/or the competition for cell adhesion (for a review see [117] ). this type of interaction has been frequently observed with enteric bacteria [117] and an example in pigs is the reduced infection of ipec-j2 cells by vesicular stomatitis virus after pre-incubation of the cells with multiple probiotic bacteria [133] . an intriguing relationship is occurring between ibp and viruses where metabolic reprogramming in host cells triggered by viruses might support or conversely limit coinfection by an intracellular bacterial partner (for a review see [107] ). different possibilities can be identified in that type of interaction [107] : (i) the first pathogen can reprogram cellular metabolism related to cellular immunity and decrease the defense against the other pathogen, (ii) the metabolic changes triggered by the first pathogen can facilitate the adhesion, the penetration, and the replication of the other, and (iii) the coinfection transform the active replicative state of the first pathogen into a stable persistent state. the first possibility associated to a decrease of the cellular defense is a commonly accepted mechanism [134] while the second and the third possibilities are less experimentally demonstrated [107] . looking at bacterium-bacterium interactions, they are extremely complex to assess because of the large diversity of the bacterial world and because little is known about the mechanisms underpinning these interactions during infections. moreover, it is now also clear that intestinal and respiratory microbiomes affect the interactions between pathogenic bacteria and the porcine host [135] . some examples of the complex interactions occurring in bacteria-bacteria coinfections are presented in additional file 1e, but little is known about the mechanisms controlling these interactions. however, some mechanisms were provided above and interesting reviews dealing with that subject were published recently [106, 107] discussing the possible direct interactions between bacteria-mainly chemical and physical. indirect interactions between bacteria were not reviewed in these articles but were discussed to some extent in other review papers focusing on polymicrobial infections [1, 136]. the first observation coming from this review must be, even if several studies have been carried out on the subject, a lack of data about some specific coinfections and many discrepancies between studies. for instance, there are only a few in vivo studies about pcv2 in virus/virus coinfections and about pcv2 and prrsv in virus/bacterium coinfections (additional file 1 and heat maps in figures 1, 2, 3) . discrepancies are not surprising because of the definition of coinfection is not always the same between studies in addition to huge variations in the coinfection parameters amongst studies. in this review we focused on experimental (in vivo and in vitro) coinfections, it is worth to underline that these studies are inspired by field veterinarians and epidemiologists observations. however, the definition of epidemiologist coinfection can also vary between studies. indeed, in some cases there is concomitant direct identification of two microorganisms in the same animals, sometimes in the same farms, while in other cases it is just an indirect identification of the microorganisms' presence at some points through indirect serological assays. moreover, as stated before, the term coinfection is sometimes used to describe some situations of superinfections where the delay can be significant. regarding the experimental parameters, the multiplicities of infection (moi), the strains, the potential delays between successive infections, the routes of inoculations, the types of cellular hosts considered (more or less susceptible to one of the microorganism), the genetic background (breeds) and the sanitary status (specific pathogen free or conventional breeding) of the pigs, and the readout to assess coinfection outcome varied a lot between studies. to fully compare studies, a standardization of the assays would be needed. interestingly also, whereas in vitro studies' usefulness is not in question, it is important to underline here that in vitro observed interplay between pathogens cannot be automatically applied in vivo. for instance, whereas m. hyopneumoniae decreases the prrsv titre in vitro [75] , it increases prrsv shedding in vivo and indeed worsens the clinical signs upon coinfection [102] . consequently, the use of intermediary settings, such as co-culture of different cell types (see [2] for examples), precision cut lung slices (pcls) [137] or organoids [138] , could help to understand the complexity of coinfections in the respiratory tissue. in vitro approaches usually consider one or a few cell types with some limitations during the evaluation process of the coinfection consequences. some viruses can contribute to the elimination of other viruses just because of their ability to replicate faster on a particular cell type [139] . thus, results obtained in vitro cannot mimic the field situation when both agents coinfect the same pig, providing inaccurate conclusions about the coinfection dynamics. under such circumstances, pathogens may simply have different host cells and no longer be under direct competition for resources [140] . besides the different interactions that infecting agents can have between them through a competition to resources, studies showed clearly that the immune system and the immunological responses can highly affect these interactions by inducing the competitive power of a pathogen or abolishing it and making it less competitive on the resource [141, 142] . the effects of the immune system (especially humoral parameters) are often not taken into consideration in selected in vitro models [140] . on the other hand, in vivo coinfection experiments have to deal with numerous constraints (health status of the animals used, cost, husbandry, and ethics amongst others) and therefore are not always easy to perform. hence, although in vivo experiments are required in this very complex field, they surely need to be combined to in silico/in vitro/ex vivo analyses of potential interactions between pathogens. moreover, multiple parameters of the coinfection protocols appeared difficult to set without any a priori such as the choice of the pathogen that will be inoculated first and the delay between infections. one possibility to deal with multiple parameters is to use intra-host infection mathematical modelling [143] allowing to play, at limited cost, with the different parameters of the coinfections. however, these models need to be fed with data coming from conventional in vitro experiments as well as more complex in vivo studies. the other possibility is to rely on field prevalence studies monitoring the very presence of the pathogens (isolation, pcr) instead of the sero-conversion, in order to have a clear epidemiological picture of when and where coinfections occur. consequently, ex vivo models such as pcls generated from freshly sacrificed pigs [137] or organoids [138] are developing. these models are closer to mimic the in vivo situation than usual in vitro approaches, combining different types of cells and providing the pathogens with a wider range of cell hosts. however, the contribution of the immune response to the interaction between different pathogens is rarely considered [97] . furthermore, the moi cannot be controlled because the number of infected cells in the slice or the organoid cannot be monitored easily either. another limiting factor in coinfection studies is the cell regeneration, which can vary between in vivo and in vitro models. cell regeneration can highly affect the dynamics of a coinfection, giving some pathogens extra target cells guarantying their longer existence while contributing to the clearance of others [140] . finally, other potential technical limitations could always be discussed such as the lack of precision or sensitivity in the different diagnostic techniques especially in the presence of multiple agents. hence, the detection of coinfecting pathogens could be compromised or reduced as compared to their detection level in the context of single infections. as shown in this review many works have been dedicated to the study of coinfections and superinfections in pigs. usually, when the experiments were carried out in vivo, the researchers were more interested in the clinical outcome than in the interactions occurring between pathogens. indeed, in most of the cases the fine interactions between pathogens and especially the mechanisms behind these interactions and its potential consequences, at the molecular level, on the immune response were not studied for several reasons including technical limitations. also, in the studies assessing the occurrence of coinfections/superinfections in the fields, coinfection identification based on molecular tools such as pcr would be more accurate than sero-prevalence approaches which are less prone to identify currently present pathogens and thus coinfective pathogens. then, a better knowledge of each pathogen involved is crucial. we thus would like to make recommendations for future studies dealing with respiratory coinfections in pigs: (i) authors should clearly summarize their coinfection or superinfection experimental setup-doses of pathogens, delays between infections-in their materials and methods section; (ii) in this summary they would need to clearly present the pathogens they use and they should, as often as possible, select well-characterized strains; (iii) environmental and management conditions would need a strict control and monitoring; (iv) animal genetic and sanitary status would need to be carefully described and monitored during the study; and (v) the multiplications of all the pathogens shall be followed during the experiment using highly sensitive and specific assays. a clear description of all these parameters would help the scientific community to compare studies and progress in the understanding of the complex interactions between microorganisms. in the last years, the concept of innate immune memory or trained immunity has gained a lot of interest. this concept is coming from old observation, in 1946 [144] , recognizing that the bacterial vaccine strain "bacille de calmette et guérin" (bcg) was protecting not only against mycobacterium tuberculosis but also against antigenically different microorganism causing childhood mortality, suggesting an "adaptation" of the cellular innate immune system. since then, many interesting studies about innate immune memory or trained immunity have been published (for a review see [145] ) and it is recognized that cells such as myeloid cells, nk cells, and even epithelial cells [146] can have a higher and quicker response upon re-exposure to a pathogen. trained immunity is accompanied by epigenetic changes and most often associated with modifications in cellular metabolism. a close look at potential epigenetic changes and cellular metabolism modifications would be of high interest in respiratory coinfection studies in the porcine species. recently an alternative to the mechanism of trained immunity in resident lung innate immune cells named "epigenetic legacy" has been described [147] . in that study, the authors demonstrated that following iav clearance and clinical recovery (1-month post-infection), mice were better protected from streptococcus pneumoniae infection by adult bone-marrow-derived ams displaying transient transcriptional and epigenetic distinct profiles. this newly described consequence of a first viral infection also needs additional studies about prdc with an identification of the mechanisms shaping the complex interactions between pathogens. supplementary information accompanies this paper at https ://doi. org/10.1186/s1356 7-020-00807 -8. additional file 1. studies about coinfections in the pig respiratory tract and their consequences. microorganisms associated with pneumonia in slaughter weight swine retrospective analysis of etiologic agents associated with respiratory diseases in pigs infectious agents associated with respiratory diseases in 125 farrow-to-finish pig herds: a cross-sectional study transcriptome analysis of porcine thymus following porcine cytomegalovirus infection interaction between mycoplasma hyopneumoniae and swine influenza virus epidemiological characteristics of pulmonary pneumocystosis and concurrent infections in pigs in jeju island longitudinal study of respiratory infection patterns of breeding sows in five farrow-to-finish herds bacterial pathogens associated with lung lesions in slaughter pigs from 125 herds reorganization and expansion of the nidoviral family arteriviridae porcine alveolar macrophage-like cells are pro-inflammatory pulmonary intravascular macrophages that produce large titers of porcine reproductive and respiratory syndrome virus porcine reproductive and respiratory syndrome virus (prrsv): pathogenesis and interaction with the immune system infection of monocytes with european porcine reproductive and respiratory syndrome virus (prrsv-1) strain lena is significantly enhanced by dexamethasone and il-10 phenotypic and functional modulation of bone marrow-derived dendritic cells by porcine reproductive and respiratory syndrome virus porcine reproductive and respiratory syndrome virus productively infects monocyte-derived dendritic cells and compromises their antigen-presenting ability porcine reproductive and respiratory syndrome virus type 1.3 lena triggers conventional dendritic cells 1 activation and t helper 1 immune response without infecting dendritic cells dendritic cells, monocytes and macrophages: a unified nomenclature based on ontogeny prrs virus receptors and their role for pathogenesis molecular cloning of porcine siglec-3, siglec-5 and siglec-10, and identification of siglec-10 as an alternative receptor for porcine reproductive and respiratory syndrome virus (prrsv) involvement of sialoadhesin in entry of porcine reproductive and respiratory syndrome virus into porcine alveolar macrophages gene-edited pigs are protected from porcine reproductive and respiratory syndrome virus replacement of porcine cd163 scavenger receptor cysteine-rich domain 5 with a cd163-like homolog confers resistance of pigs to genotype 1 but not genotype 2 porcine reproductive and respiratory syndrome virus direct interaction between cd163 n-terminal domain and myh9 c-terminal domain contributes to porcine reproductive and respiratory syndrome virus internalization by permissive cells differential production of proinflammatory cytokines in the pig lung during different respiratory virus infections: correlations with pathogenicity increased production of proinflammatory cytokines following infection with porcine reproductive and respiratory syndrome virus and mycoplasma hyopneumoniae porcine reproductive and respiratory syndrome virus field isolates differ in in vitro interferon phenotypes cytokine profiles and phenotype regulation of antigen presenting cells by genotype-i porcine reproductive and respiratory syndrome virus isolates strain-dependent porcine circovirus type 2 (pcv2) entry and replication in t-lymphoblasts cell tropism and entry of porcine circovirus 2 porcine circovirus diseases characteristics of porcine circovirus-2 replication in lymphoid organs of pigs inoculated in late gestation or postnatally and possible relation to clinical and pathological outcome of infection porcine circovirus type 2 (pcv2) distribution and replication in tissues and immune cells in early infected pigs porcine circovirus type 2 displays pluripotency in cell targeting dendritic cells harbor infectious porcine circovirus type 2 in the absence of apparent cell modulation or replication of the virus association of lymphopenia with porcine circovirus type 2 induced postweaning multisystemic wasting syndrome (pmws) cytokine profiles of peripheral blood mononuclear cells from pigs with postweaning multisystemic wasting syndrome in response to mitogen, superantigen or recall viral antigens distribution and characterization of il-10-secreting cells in lymphoid tissues of pcv2-infected pigs porcine circovirus type 2 (pcv2) viral components immunomodulate recall antigen responses subset-dependent modulation of dendritic cell activity by circovirus type 2 review: influenza virus in pigs influenza a virus interactions with macrophages: lessons from epithelial cells dendritic cells in innate and adaptive immune responses against influenza virus specificity and functional interplay between influenza virus pa-x and ns1 shutoff activity protein of 2009 pandemic influenza a virus inhibits porcine nlrp3 inflammasome-mediated interleukin-1 beta production by suppressing asc ubiquitination porcine coronaviruses. emerging transbounding animal viruses fatal swine acute diarrhoea syndrome caused by an hku2-related coronavirus of bat origin sars-associated coronavirus transmitted from human to pig susceptibility of ferrets, cats, dogs, and other domesticated animals to sars-coronavirus 2 further characterization of aminopeptidase-n as a receptor for coronaviruses diseases of swine molecular biology of pseudorabies virus: impact on neurovirology and veterinary medicine three classes of cell surface receptors for alphaherpesvirus entry the porcine humoral immune response against pseudorabies virus specifically targets attachment sites on glycoprotein gc multiplex pcr for rapid detection of pseudorabies virus, porcine parvovirus and porcine circoviruses update on actinobacillus pleuropneumoniae-knowledge, gaps and challenges actinobacillosis biofilm formation is prevalent among field isolates of actinobacillus pleuropneumoniae influence of actinobacillus pleuropneumoniae and its metabolites on porcine alveolar epithelial cells phagocytosis by pig alveolar macrophages of actinobacillus pleuropneumoniae serotype 2 mutant strains defective in haemolysin ii (apxll) and pleurotoxin (apxiii) functional and structural changes of porcine alveolar macrophages induced by sublytic doses of a heat-labile, hemolytic, cytotoxic substance produced by actinobacillus pleuropneumoniae diseases of swine update on mycoplasma hyopneumoniae infections in pigs: knowledge gaps for improved disease control in vivo expression analysis of the p97 and p102 paralog families of mycoplasma hyopneumoniae post-translational processing targets functionally diverse proteins in mycoplasma hyopneumoniae genotype distribution of mycoplasma hyopneumoniae in swine herds from different geographical regions mycoplasma hyopneumoniae resides intracellularly within porcine epithelial cells immunohistochemical labelling of cytokines in lung lesions of pigs naturally infected with mycoplasma hyopneumoniae cytokine expression in porcine lungs experimentally infected with mycoplasma hyopneumoniae a morphologic and immunohistochemical study of the bronchus-associated lymphoid tissue of pigs naturally infected with mycoplasma hyopneumoniae transcription analysis of the porcine alveolar macrophage response to mycoplasma hyopneumoniae differential production of proinfammatory cytokines: in vitro prrsv and mycoplasma hyopneumoniae co-infection model differential innate immune responses induced by mycoplasma hyopneumoniae and mycoplasma hyorhinis in various types of antigen presenting cells diseases of swine a porcine model for the evaluation of virulence of bordetella bronchiseptica contribution of bordetella bronchiseptica filamentous hemagglutinin and pertactin to respiratory disease in swine matrine displayed antiviral activity in porcine alveolar macrophages coinfected by porcine reproductive and respiratory syndrome virus and porcine circovirus type 2 effect of an interferon-stimulated response element (isre) mutant of porcine circovirus type 2 (pcv2) on pcv2-induced pathological lesions in a porcine reproductive and respiratory syndrome virus (prrsv) coinfection model experimental inoculation of conventional pigs with porcine reproductive and respiratory syndrome virus and porcine circovirus 2 hepatitis e virus chronic infection of swine co-infected with porcine reproductive and respiratory syndrome virus vaccine efficacy and immune response to swine influenza virus challenge in pigs infected with porcine reproductive and respiratory syndrome virus at the time of siv vaccination dual infections of feeder pigs with porcine reproductive and respiratory syndrome virus followed by porcine respiratory coronavirus or swine influenza virus: a clinical and virological study comparison of mono-and co-infection by swine influenza a viruses and porcine respiratory coronavirus in porcine precision-cut lung slices expression of toll-like receptor signaling-related genes in pigs co-infected with porcine reproductive and respiratory syndrome virus and porcine circovirus type 2 infection of cesarean-derived colostrum-deprived pigs with porcine circovirus type 2 and swine influenza virus effect of porcine parvovirus vaccination on the development of pmws in segregated early weaned pigs coinfected with type 2 porcine circovirus and porcine parvovirus simultaneous porcine circovirus type 2 and mycoplasma hyopneumoniae co-inoculation does not potentiate disease in conventional pigs experimental dual infection of pigs with an h1n1 swine influenza virus (a/sw/hok/2/81) and mycoplasma hyopneumoniae mycoplasma hyopneumoniae potentiation of porcine reproductive and respiratory syndrome virus-induced pneumonia preinfection of pigs with mycoplasma hyopneumoniae induces oxidative stress that influences outcomes of a subsequent infection with a swine influenza virus of h1n1 subtype virus-bacteria interactions: an emerging topic in human infection the co-pathogenesis of influenza viruses with bacteria in the lung capsular sialic acid of streptococcus suis serotype 2 binds to swine influenza virus and enhances bacterial interactions with virus-infected tracheal epithelial cells dynamic virus-bacterium interactions in a porcine precision-cut lung slice coinfection model: swine influenza virus paves the way for streptococcus suis infection in a two-step process investigation of pathogenesis of h1n1 influenza virus and swine streptococcus suis serotype 2 co-infection in pigs by microarray analysis transcriptional approach to study porcine tracheal epithelial cells individually or dually infected with swine influenza virus and streptococcus suis mycoplasma hyopneumoniae does not affect the interferon-related anti-viral response but predisposes the pig to a higher level of inflammation following swine influenza virus infection cytokine and chemokine mrna expression profiles in tracheobronchial lymph nodes from pigs singularly infected or coinfected with porcine circovirus type 2 (pcv2) and mycoplasma hyopneumoniae (mhyo) the impact of animal age, bacterial coinfection, and isolate pathogenicity on the shedding of porcine reproductive and respiratory syndrome virus in aerosols from experimentally infected pigs actinobacillus pleuropneumoniae possesses an antiviral activity against porcine reproductive and respiratory syndrome virus enriched housing reduces disease susceptibility to co-infection with porcine reproductive and respiratory virus (prrsv) and actinobacillus pleuropneumoniae (a. pleuropneumoniae) in young pigs dual infections of prrsv/influenza or prrsv/actinobacillus pleuropneumoniae in the respiratory tract polybacterial human disease: the ills of social networking how viral and intracellular bacterial pathogens reprogram the metabolism of host cells to allow their intracellular replication intra-species and inter-species differences in cytokine production by porcine antigen-presenting cells stimulated by mycoplasma hyopneumoniae viral interference and interferon type i interferons in infectious disease type iii interferons in viral infection and antiviral immunity the roles of type i interferon in bacterial infection a role for ifn-alpha beta in virus infection-induced sensitization to endotoxin influenza "trains" the host for enhanced susceptibility to secondary bacterial infection type i interferon induction during influenza virus infection increases susceptibility to secondary streptococcus pneumoniae infection by negative regulation of γδ t cells intrinsic interference: non-interferon mediated viral interference virus-bacteria interactions: implications and potential for the applied and agricultural sciences the antiviral and antitumor effects of defective interfering particles/genomes and their mechanisms rnai, a new therapeutic strategy against viral infection non-specific dsrna-mediated antiviral response in the honey bee roles of nonstructural polyproteins and cleavage products in regulating sindbis virus rna replication and transcription superinfection prevents recombination of the alphaherpesvirus bovine herpesvirus 1 classical swine fever virus vs. classical swine fever virus: the superinfection exclusion phenomenon in experimentally infected wild boar porcine reproductive and respiratory syndrome virus (prrsv) influences infection dynamics of porcine circovirus type 2 (pcv2) subtypes pcv2a and pcv2b by prolonging pcv2 viremia and shedding microbiome associations in pigs with the best and worst clinical outcomes following co-infection with porcine reproductive and respiratory syndrome virus (prrsv) and porcine circovirus type 2 (pcv2) synergistic effects of sequential infection with highly pathogenic porcine reproductive and respiratory syndrome virus and porcine circovirus type 2 mechanisms and consequences of positivestrand rna virus recombination recombination in the alphaherpesvirus bovine herpesvirus 1 role of staphylococcus protease in the development of influenza pneumonia porcine circovirus type 2 promotes actinobacillus pleuropneumoniae survival during coinfection of porcine alveolar macrophages by inhibiting ros production sialic acid-dependent interactions between influenza viruses and streptococcus suis affect the infection of porcine tracheal cells viral hemagglutinin is involved in promoting the internalisation of staphylococcus aureus into human pneumocytes during influenza a h1n1 virus infection a novel eukaryotic cell culture model to study antiviral activity of potential probiotic bacteria intramacrophage survival for extracellular bacterial pathogens: mgtc as a key adaptive factor role of the microbiome in swine respiratory disease co-infection subverts mucosal immunity in the upper respiratory tract innate immune response to a h3n2 subtype swine influenza virus in newborn porcine trachea cells, alveolar macrophages, and precision-cut lung slices organoids in immunological research interspecific recombination between two ruminant alphaherpesviruses, bovine herpesviruses 1 and 5 plos one 11:e0155589 research data, including large and complex data types • gold open access which fosters wider collaboration and increased citations maximum visibility for your research: over 100m website views per year ready to submit your research ? choose bmc and benefit from dominance of hepatitis c virus (hcv) is associated with lower quantitative hepatitis b surface antigen and higher serum interferon-γ-induced protein 10 levels in hbv/hcv-coinfected patients dual infections of cd163 expressing nptr epithelial cells with influenza a virus and prrsv why, when and how should exposure be considered at the within-host scale? a modelling contribution to prrsv infection results of bcg immunization in new york city trained immunity: a program of innate immune memory in health and disease trained immunity carried by non-immune cells influenza-induced monocyte-derived alveolar macrophages confer prolonged antibacterial protection publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations the authors would like to thank the colleagues who shared their experience regarding coinfections in pigs. fm is supported by an establishment grant from the région pays de la loire (rfi food for tomorrow-cap aliment). gs phd is funded by foundation philippe jabre (liban). all the authors were involved in the writing of the review. gs, nb, and fm generated the figures. gs, cd, jb, cf, cm-c, gsi, nb and fm prepared the additional file 1. all the authors read and approved the final manuscript. key: cord-329263-o5e2go23 authors: kaplan, nasser m.; dove, winifred; abd‐eldayem, sawsan a.; abu‐zeid, ahmad f.; shamoon, hiyam e.; hart, c. anthony title: molecular epidemiology and disease severity of respiratory syncytial virus in relation to other potential pathogens in children hospitalized with acute respiratory infection in jordan date: 2007-11-26 journal: j med virol doi: 10.1002/jmv.21067 sha: doc_id: 329263 cord_uid: o5e2go23 human respiratory syncytial virus (hrsv) is the major viral cause of acute lower respiratory tract infections in children. few data about the molecular epidemiology of respiratory syncytial virus in developing countries, such as jordan, are available. the frequency and severity of infections caused by hrsv were assessed in hospitalized jordanian children <5 years of age compared with other potential etiological agents. overall a potential pathogen was detected in 78% (254/326) of the children. hrsv was detected in 43% (140/326) of the nasopharyngeal aspirates. hrsv was found more frequently during the winter (january/february), being less frequent or negligible by spring (march/april). analysis of 135 hrsv‐positive strains using restriction fragment length polymorphism showed that 94 (70%) belonged to subgroup a, and 41 (30%) to subgroup b. there were also two cases of mixed genotypic infection. only four of the six previously described n genotypes were detected with np4 predominating. there were no associations between subgroup or n‐genogroup and disease severity. hrsv was significantly associated with more severe acute respiratory infection and the median age of children with hrsv was lower than for those without. next in order of frequency were adenovirus (116/312: 37%), human bocavirus (57/312: 18%), rhinovirus (36/325: 11%), chlamydia spp. (14/312: 4.5%), human metapneumovirus (8/326: 2.5%), human coronavirus nl63 (4/325: 1.2%), and influenza a virus (2/323: 0.6%). influenza b; parainfluenza viruses 1–4, human coronavirus hku1 and mycoplasma pneumoniae were not detected. j. med. virol. 80:168–174, 2008. © 2007 wiley‐liss, inc. acute respiratory infection is the major cause of death in children <5 years of age, and occurs predominantly in developing countries [bryce et al., 2005] . human respiratory syncytial virus (hrsv) is the leading viral cause of acute respiratory infection in infants and young children in terms of prevalence and effect [shay et al., 2001] . hrsv causes substantial annual winter epidemics in temperate climates, representing a major cause of pediatric hospitalizations and a serious economic burden [viegas et al., 2004] . hrsv can cause reinfections throughout the child's life and it can infect infants in the presence of maternal antibodies. this could be due to either an inadequate immune response or to the extensive genetic variability of the virus. although antigenic variation is not essential for reinfection, growing evidence suggests that it may contribute to reinfections by immune evasion [viegas and mistchenko, 2005] . it has also been reported that hrsvspecific t-cell responses do not provide protection against reinfection, and reinfection does not boost hrsv-specific t-cell proliferation [bont et al., 2002] . hrsv is an enveloped rna virus that is classified within the pneumovirus genus as a member of the family paramyxoviridae [collins et al., 2001] . the virus has a non-segmented, single-stranded, negative-sense genome. its genome encodes 10 proteins, including two major surface glycoproteins (g and f), two matrix proteins (m 1 and m 2 ), a small hydrophobic protein (sh), and three nucleocapsid associated proteins (n, p, and l) [collins et al., 1984] . the two antigenic glycoproteins g and f are responsible for host cell attachment and viral entry by membrane fusion, respectively [palomo et al., 2000] . hrsv is divided into two major antigenic subgroups, a and b, on the basis of reactivity with monoclonal antibodies against the major structural glycoproteins g and f [mufson et al., 1985] . however, these two subgroups can be further subdivided into genotypes by restriction analysis and nucleotide sequence variability [sullender et al., 1993; peret et al., 1998] . the n (nucleoprotein) gene is relatively well conserved between virus isolates, but the g gene shows much greater variability. the variation in these genes has been used to define hrsv typing schemes as n gene differences resulting in the np1-np6 genotypes. the purpose of the present study was to examine the molecular epidemiology of hrsv in jordan. we also compared the disease severity of hrsv subgroups a and b and their associated genotypes in hospitalized jordanian children set in the context of other potential respiratory pathogens. this prospective cross-sectional study was conducted over six consecutive months from december 2003 to may 2004. children younger than 5 years of age with acute respiratory infection admitted to the pediatric wards of king hussein medical centre and queen alia hospital, amman, jordan were enrolled in the study irrespective of the severity of their illness. king hussein medical centre, a tertiary referral hospital, and queen alia hospital, a district general hospital, provide hospital pediatric care for amman, the capital city of jordan, and its surroundings. the study was approved by the medical research ethical committee of the king hussein medical centre, amman, jordan and signed informed consent was obtained from each of the children's parents or legal guardians for participation in the study. the clinical diagnosis of acute respiratory infection and assessment of its severity was made by using the world health organization standard protocol for acute respiratory infection based on the presence of cough, tachypnoea, chest indrawing, and wheezing for <7 days duration [pio, 2003] . severe disease was defined as present in children with a respiratory rate >60/min and chest indrawing. oxygen saturation (po 2 ) was measured by using pulse oximetry (nellcor puritan bennett npb-195, uk) and a po 2 85% used as the cut-off for giving supplementary oxygen. a standardized questionnaire containing clinical, socio-demographic, therapeutic, and outcome data was completed for each patient. nasopharyngeal aspirates were collected by instilling 1 ml sterile phosphate-buffered saline through a sterile nasopharyngeal mucous extractor. the aspirates were frozen at à808c until analyzed in the department of medical microbiology, university of liverpool, uk. the genome of hrsv was detected in nasopharyngeal aspirates by reverse transcription-polymerase chain reaction (rt-pcr) [greensill et al., 2003] . hrsvpositive strains were classified into subgroup a and b by restriction fragment length polymorphism analysis [cane and pringle, 1992] . total rna and dna were extracted separately from nasopharyngeal aspirates by using the commercial rneasy and qiaamp dna mini kits (qiagen, crawley, west sussex, uk) according to manufacturer's instructions. chain reaction for hrsv the primers: n1 (5 0 -gga aca agt tgt tga ggt tta tga ata tgc-3 0 ) and n2 (5 0 -ctt ctg ctg tca agt cta gta cac tgt agt-3 0 ) were used to amplify the nucleocapsid (n) gene between nucleotides 858 and 1,135 giving a 278-bp product [cane and pringle, 1991 ]. an aliquot of 10 ml of the extracted viral rna serving as a template for cdna synthesis was added to 40 ml of pcr mixture. the final 50 ml mastermix reaction contained 1â pcr buffer, 3 mm mgcl 2 , 5 mm dithiothreitol (dtt), 0.4 mm deoxyribonucleotide triphosphates (dntps), 0.4 mm of primers mixture containing equal volumes of the two primers at a concentration of 20 mm each, 20 units (u) of rnase inhibitor (rnasin), 25 u of murine leukemia virus reverse-transcriptase, and 2.5 u of amplitaq gold dna polymerase (roche diagnostics ltd., burgess hill, uk). the rt-pcr was performed in a perkin elmer gene-amp 2400 thermal cycler (norwalk, ct) according to the following program: 30 min reverse transcription cycle at 508c, followed by 5 min reverse transcriptase inactivation and dna polymerase activation cycle at 948c, then forty pcr cycles (1-min denaturation at 948c, 1 min of primer annealing at 558c, and 1 min of primer extension at 728c), and a final extension cycle of 728c for 10 min. ten microliter volumes of each amplified dna pcr product were separated by electrophoresis on a 2% (wt/ vol) agarose-tris-borate-ethylene diamine tetracetic acid gel, stained with ethidium bromide, and visualized under ultraviolet light (syngene gel documentation and analysis system, ingenius, cambridge, uk). analysis of hrsv aliquots of 2 ml from hrsv positive amplified dna were separately digested directly and without prior purification by a set of five restriction endonuclease enzymes namely hindiii, psti, bglii, rsai, haeiii (roche). the restriction patterns were analyzed by electrophoresis on a 2% (wt/vol) agarose-tris-borateethylene diamine tetracetic acid gel, stained with ethidium bromide, and visualized under ultraviolet light. hrsv amplicons were typed into six nucleoprotein (np) genotypes on the basis of their restriction endonuclease digestion profiles [cane and pringle, 1992] . hrsv subgroup a included np2, np4, and np5, while subgroup b included np1, np3, and np6 genotypes. detection of other respiratory pathogens was performed according to previously published protocols. rt-pcr was used for the detection of human metapneumovirus (hmpv) [greensill et al., 2003] , influenza a and b, and parainfluenza virus 1-4 [templeton et al., 2004] , human rhinovirus (hrv) and human coronaviruses nl63 and hku1 [choi et al., 2006; sloots et al., 2006] . pcr was used to detect human bocavirus (hbov) [allander et al., 2005] , adenoviruses, chlamydia spp., and mycoplasma pneumoniae [couroucli et al., 2000] . epi info version 3.3.2 (centers for disease control, atlanta, ga) was used for statistical analysis by applying chi-square and student's t-tests. p values <0.05 were considered statistically significant. a total of 326 children (188, 58% male) with a median age of 5 months were recruited, and 326 nasopharyngeal aspirates were collected and analyzed, however the volume of some nasopharyngeal aspirates was inadequate for both dna and rna extractions. of the 326 children studied 15 (5%) had some underlying condition which might have contributed to disease severity. of these 13 had cardiopulmonary disease, predominantly congenital heart disease (but none had bronchopulmonary dysplasia), one was premature and one had immunodeficiency. in all 8 (53%) of those with an underlying condition had severe disease. a total of 72/326 (22%) patients had no pathogens detected by pcr (table i) , but 254 (78%) had at least one potential respiratory pathogen detected which consisted of 140/ 326 (43%) hrsv, 116/312 (37%) adenoviruses, 57/312 (18%) hbov, 36/325 (11%) rhinovirus, 14/312 (4.5%) chlamydia spp., 8/326 (2.5%) hmpv, 4/325 (1.2%) human coronavirus nl63, and 2/323 (0.6%) influenza a virus. overall 106/326 (33%) infants had mixed infections (table ii) and of these 83 were infected with 2 potential pathogens, 22 with 3, and 1 with 4 potential pathogens. a total of 67 hrsv co-infections (48% of all hrsv infections) were detected; 30 with adenoviruses, 10 with hbov, 10 with adenovirus and hbov, 8 with rhinoviruses, 3 with chlamydia spp., 1 with rhinovirus and hbov, 1 with chlamydia spp. and adenovirus, 1 with chlamydia spp. and hbov, 1 with chlamydia spp. and rhinovirus, 1 with adenovirus and rhinovirus, and 1 with chlamydia spp., rhinovirus and hbov. no hrsv/hmpv co-infections were detected. influenza b, parainfluenza viruses 1-4, human coronavirus hku1 and m. pneumoniae were not detected in this study. restriction fragment length polymorphism analysis was performed for 98% (137/140) of hrsv-positive strains; three weakly positive strains were not subjected to further subgrouping analysis. hrsv subgroup a was detected in 70% (94/135) and subgroup b in 30% (41/135) of the samples. there were two cases of mixed genotypic infections, one np2/np4 (a/a) and one np4/np3 (a/b). genotyping analysis of hrsv strains showed that 63 (47%) were np4, 31 (23%) np2, 26 (19%) np1, and 15 (11%) np3. each of the four genotypes co-circulated during the january-march period of the study (table iii) . all the children in the study had lower respiratory tract infections mainly bronchiolitis and bronchopneumonia and routine cultures of their blood and respiratory secretions had detected no potential bacterial or fungal pathogens. although there were no deaths, three children were admitted to the intensive care unit, one infected only with hrsv subgroup b (np1), one infected only with hbov, and one who had congenital heart disease with no pathogens detected. a total of 139 (43%) children had severe, and 187 (57%) had mildmoderate acute respiratory infection. significantly (p < 0.0005) more hrsv-infected children had severe disease (100/140; 71%) compared to those uninfected with hrsv (39/186; 21%). the median age of hrsvinfected patients was 4.8 months (range 1-48 months) and 82 (59%) were male, compared with a median age of 6 months and 104 (56%) male patients in the hrsvnegative patients (p < 0.05). severe hrsv infections occurred in all age groups, however it was significantly more likely to occur in those under 6 months of age (p < 0.01). thus 62 (62%) in the 0-6 months age group, 30 (30%) in the 7-12 months group, and 8 (8%) in those older than 1 year had severe disease. hrsv subgroup a and b were associated with 66 and 33 cases of severe disease, respectively (p ¼ 0.2). there was no significant difference between the individual hrsv genotypes as potential causes of severe disease. in the 100 patients with severe acute respiratory infection in whom hrsv was detected, it was the sole pathogen detected in 53 (53%) patients, however in the remaining 47 cases, it was found as a mixed infection with adenovirus (20 patients), hbov and adenovirus (9 patients), hbov (8 patients), chlamydia spp. (3 patients), rhinovirus (2 patients), hbov and rhinovirus (1 patient), chlamydia spp. and hbov (1 patient), chlamydia spp. and adenovirus (1 patient), adenovirus and rhinovirus (1 patient), and chlamydia spp., hbov and rhinovirus (1 patient). there was no significant difference (p > 0.5) in the prevalence of severe disease between those where hrsv was the sole pathogen (53/ 73:73%) and those with hrsv and another potential pathogen (47/67:70%). the median age of children with severe acute respiratory infection was 4 months for those infected only with hrsv, and 6 months for those co-infected with hrsv and other potential respiratory pathogens (p < 0.05). in the 40 patients with mildmoderate acute respiratory infection in whom hrsv was detected, it was the only pathogen in 20 (50%) patients, however in the remaining 20 cases, it was found as a mixed infection with adenovirus (10 patients), rhinovirus (6 patients), hbov (2 patients), adenovirus and hbov (1 patient), and chlamydia spp. and rhinovirus (1 patient). the median age of children with mild-moderate acute respiratory infection was 4 months for those infected only with hrsv, and 9.5 months for those co-infected with hrsv and other potential respiratory pathogens (p ¼ 0.05). of the 36 children infected with rhinovirus 24 (67%) had mild/moderate and 12 (33%) severe disease. the median age of those infected with rhinovirus was 4.5 months not significantly different from that of those infected with hrsv. although 12 of the children infected with rhinovirus had severe disease, only one of these severe infections occurred in the absence of coinfection with any of the other eleven potential respiratory pathogens. of the 24 children with mild/moderate disease 17 (71%) were co-infected with other pathogens. there was a similar prevalence of rhinovirus infection in january-april. this study has confirmed that hrsv is the most frequent cause of severe acute respiratory infection in young children in jordan. the fact that a large proportion of hrsv infections (71%) were associated with more severe disease is not unexpected because only hospitalized patients were involved in this study. however significantly more hospitalized infants with severe respiratory disease than mild/moderate disease due to hrsv were detected compared to infection with the other potential pathogens. worldwide, two subgroups of hrsv circulate independently within human populations, with group a being the more prevalent [peret et al., 2000] . in a 3-year study conducted in the usa [walsh et al., 1997] , group b viruses predominated for 2 years, while group a predominated in the third year. information on the relative frequencies of subgroups a and b in the middle east and africa is scarce. in the present study, subgroup a was predominant (70% vs. 30%), as previously shown in jordan [bdour, 2001] , and yemen [al-sonboli et al., 2005] . this predominance of subgroup a hrsv is the most common pattern worldwide [cane, 2001] . only four of the six previously described n genotypes [fletcher et al., 1997] were found among the hrsv strains. all four genotypes were identified during the january-march period of the study, with genotype np4 predominating (table ii) . however all four genotypes co-circulated at the same time. some authors have concluded that infection with group a hrsv is associated with more severe disease [peret et al., 2000] but others have found no such association [fletcher et al., 1997; walsh et al., 1997 ]. in the present study hrsv subgroup a infection was not associated with more severe disease than infection with subgroup b (table iii) . the medical staffs were unaware of group and genotype results and thus potential bias leading to a differential misclassification of the disease severity is unlikely. although the study extended through only 6 months of the year, there was a demonstrable peak of acute respiratory infection due to hrsv during january and february, as shown in a previous study in jordan [al-toum et al., 2006] . hrsv was less frequent or almost absent in march-may. hrsv-infected children were significantly younger than hrsv-negative children, and severe hrsv infections most frequently (90%) affected children younger than 12 months of age in agreement with a previous report from jordan [meqdam and nasrallah, 2000] . although it has been shown previously that children infected with group a hrsv were significantly older than were those infected with group b hrsv [hall et al., 1990] , there was no significant difference in the ages of these groups (p ¼ 0.2) in the present study. there were no significant differences between the different hrsv genotypes as potential causes of severe disease, so it was difficult to establish a relationship between genotypes and disease severity. overall 102 (31.2%) of the children had more than one potential pathogen detected in their nasopharyngeal aspirates. this is perhaps not surprising as the respiratory viral seasons often co-incide [choi et al., 2006] , and increasing the range of potential pathogens sought (in this study 10 viral and 2 bacterial pathogens) will reveal more co-infections. in 68 children the copathogens were hrsv with one or more other pathogen. however in no case was a co-infection with hrsv and hmpv encountered. such co-infection has previously been linked with more severe acute respiratory infection in some [greensill et al., 2003; semple et al., 2005; foulongne et al., 2006] but not all studies [maggi et al., 2003; wilkesmann et al., 2006] . hmpv was less frequently detected (2.5% of cases) than in some other hospital based studies [al-sonboli et al., 2005; semple et al., 2005] . however the peak prevalence of hmpv infections does seem to vary year by year and the hrsv and hmpv seasons are not always concurrent [serafino et al., 2004; choi et al., 2006] . after adenovirus the second most common hrsv copathogen was the newly described hbov. the finding of hbov in jordanian children has already been reported [kaplan et al., 2006] . it was infrequently found as sole pathogen which suggested that further studies were needed to assess its role as a respiratory pathogen. however recent report from edinburgh, uk has demonstrated that it is an important respiratory pathogen in children and not found in children without acute respiratory infection [manning et al., 2006] . adenoviruses were detected in 116 of the 312 children (37%), a prevalence that is significantly higher than those reported recently from germany (12.9%), brazil (6%), and india (1.5%) [grondahl et al., 1999; maitreyi et al., 2000; straliotto et al., 2002] . however only the german study used rt-pcr. the latter two employed immunofluoresence and viral culture, respectively. in 75 (65%) of the cases of adenovirus infection it was found together with another potential pathogen. the german study showed that in 5% of the cases when an agent was detected there was more than one potential pathogen and in most cases this was adenovirus with another pathogen [grondahl et al., 1999] . it is unclear why we had such a high detection rate of adenovirus infection and it warrants further study. rhinoviruses were the fourth commonest respiratory pathogen detected (36:11%). this prevalence is double that (5.8%) described for lower respiratory tract infections in korean children [choi et al., 2006] but somewhat lower than that (44%) found recently in australia . rhinovirus was present as a copathogen in 24 (67%) of jordanian children whereas in australia in almost one-third of the rhinovirus infections a co-pathogen was detected . hrvs are well-recognized causes of upper respiratory tract infection. however in the australian study it was detected in almost half the samples from patients with lower respiratory tract infection , again somewhere higher than the 11% prevalence in jordanian children, hayden [2004] has recently reviewed the role of rhinoviruses in lower respiratory tract infection and found that they were responsible for between 12% and 24% of such infections in children. in one study it was present as a co-pathogen in almost 50% of infections [papadopoulos et al., 2002] . better therapies and prevention strategies are needed to decrease the burden of acute respiratory infection particularly that due to hrsv. thus further molecular epidemiological studies over longer periods of time are warranted to better determine the role of the different hrsv genotypes in the epidemiology and the severity of disease and their inter-relationship with other respiratory pathogens. this could inform better therapeutic approaches and vaccine development. cloning of a human parvovirus by molecular screening of respiratory tract samples respiratory syncytial virus and human metapneumovirus in children with acute respiratory infections in yemen epidemiology and clinical characteristics of respiratory syncytial virus infections in jordan frequent detection of human rhinoviruses, paramyxoviruses, coronaviruses and bocavirus during acute respiratory tract infections respiratory syncytial virus subgroup a in hospitalized children in zarqa natural reinfection with respiratory syncytial virus does not boost virus-specific t-cell immunity who estimates of the causes of death in children molecular epidemiology of respiratory syncytial virus respiratory syncytial virus heterogeneity during an epidemic: analysis by limited nucleotide sequencing (sh gene) and restriction mapping (n gene) molecular epidemiology of respiratory syncytial virus: rapid identification of subgroup a lineages the association of newly identified respiratory viruses with lower respiratory tract infections in korean children identification of a tenth mrna of respiratory syncytial virus and assignment of polypeptides to the 10 viral genes respiratory syncytial virus detection of microorganisms in the tracheal aspirates of preterm infants by polymerase chain reaction: association of adenovirus with bronchopulmonary dysplasia respiratory syncytial virus genotypes and disease severity among children in hospital human metapneumovirus infection in young children hospitalized with respiratory tract disease human metapneumovirus in severe respiratory syncytial virus bronchiolitis rapid identification of nine microorganisms causing acute respiratory tract infections by single tube multiplex reverse transcription-pcr: feasibility study occurrence of groups a and b of respiratory syncytial virus over 15 years: associated epidemiologic and clinical characteristics in hospitalized and ambulatory children rhinovirus and the lower respiratory tract human bocavirus infection among children human metapneumovirus associated with respiratory tract infections in a 3-year study of nasal swabs from infants in italy rapid detection of respiratory viruses by centrifugation enhanced cultures from children with acute lower respiratory tract infections epidemiological profile and clinical associations of human bocavirus and other human parvoviruses enhanced detection of respiratory syncytial virus by shell vial in children hospitalized with respiratory illnesses in northern jordan two distinct subtypes of human respiratory syncytial virus evaluation of the antibody specificities of human convalescent-phase sera against the attachment (g) protein of human respiratory syncytial virus: influence of strain variation and carbohydrate side chains association of rhinovirus infection with increased disease severity in acute bronchiolitis circulation patterns of genetically distinct group a and b strains of human respiratory syncytial virus in a community circulation patterns of group a and b human respiratory syncytial virus genotypes in 5 communities in north america standard case management of pneumonia in children in developing countries: the cornerstone of the acute respiratory infection programme dual infection in infants by human metapneumovirus and human respiratory syncytial virus is associated with severe bronchiolitis respiratory syncytial virus and metapneumovirus in children over two seasons with a high incidence of respiratory infections in brazil bronchiolitis-associated mortality and estimates of respiratory syncytial virus-associated deaths among us children, 1979-1997 evidence of human coronavirus hku1 and human bocavirus in australian children viral etiology of acute respiratory infections among children in porto alegre, rs, brazil analysis of respiratory syncytial virus genetic variability with amplified cdnas rapid and sensitive method using multiplex real-time pcr for diagnosis of infections by influenza a and influenza b viruses, respiratory syncytial virus, and parainfluenza viruses 1, 2,3 and 4 molecular epidemiology of human respiratory syncytial virus subgroup a over a six-year period (1999-2004) in argentina respiratory viruses seasonality in children under five years of age in severity of respiratory syncytial virus infection is related to virus strain human metapneumovirus infections cause similar symptoms and clinical severity as respiratory syncytial virus infections key: cord-346539-kxnrf5g5 authors: riggioni, carmen; comberiati, pasquale; giovannini, mattia; agache, ioana; akdis, mübeccel; alves‐correia, magna; antó, josep m.; arcolaci, alessandra; kursat azkur, ahmet; azkur, dilek; beken, burcin; boccabella, cristina; bousquet, jean; breiteneder, heimo; carvalho, daniela; de las vecillas, leticia; diamant, zuzana; eguiluz‐gracia, ibon; eiwegger, thomas; eyerich, stefanie; fokkens, wytske; gao, ya‐dong; hannachi, farah; johnston, sebastian l.; jutel, marek; karavelia, aspasia; klimek, ludger; moya, beatriz; nadeau, kari; o'hehir, robyn; o'mahony, liam; pfaar, oliver; sanak, marek; schwarze, jürgen; sokolowska, milena; torres, maría j.; van de veen, willem; van zelm, menno c.; wang, de yun; zhang, luo; jiménez‐saiz, rodrigo; akdis, cezmi a. title: a compendium answering 150 questions on covid‐19 and sars‐cov‐2 date: 2020-06-14 journal: allergy doi: 10.1111/all.14449 sha: doc_id: 346539 cord_uid: kxnrf5g5 in december 2019, china reported the first cases of the coronavirus disease 2019 (covid‐19). this disease, caused by the severe acute respiratory syndrome‐related coronavirus 2 (sars‐cov‐2), has developed into a pandemic. to date it has resulted in ~6.5 million confirmed cases and caused almost 400,000 related deaths worldwide. unequivocally, the covid‐19 pandemic is the gravest health and socio‐economic crisis of our time. in this context, numerous questions have emerged in demand of basic scientific information and evidence‐based medical advice on sars‐cov‐2 and covid‐19. although the majority of the patients show a very mild, self‐limiting viral respiratory disease, many clinical manifestations in severe patients are unique to covid‐19, such as severe lymphopenia and eosinopenia, extensive pneumonia, a “cytokine storm” leading to acute respiratory distress syndrome, endothelitis, thrombo‐embolic complications and multiorgan failure. the epidemiologic features of covid‐19 are distinctive and have changed throughout the pandemic. vaccine and drug development studies and clinical trials are rapidly growing at an unprecedented speed. however, basic and clinical research on covid‐19‐related topics should be based on more coordinated high‐quality studies. this paper answers pressing questions, formulated by young clinicians and scientists, on sars‐cov‐2, covid‐19 and allergy, focusing on the following topics: virology, immunology, diagnosis, management of patients with allergic disease and asthma, treatment, clinical trials, drug discovery, vaccine development and epidemiology. over 140 questions were answered by experts in the field providing a comprehensive and practical overview of covid‐19 and allergic disease. the first cases of the coronavirus disease 2019 (covid19) , caused by the novel severe acute respiratory syndrome-related coronavirus 2 (sars-cov-2), were reported in china in december 2019 1 and rapidly led to pandemic. currently, ~6.8 million confirmed cases of covid-19 and near 400,00 covid-19-related deaths have been reported globally. 2 these numbers, which are still rising, likely underestimate the cumulative incidence of covid-19 due to several factors; these include limitations of current diagnostic tests, the extent of population testing and reporting, and the type and timing of community mitigation strategies adopted by each country, among others. 3 covid-19 shows a complex clinical profile with many different presentations. like in many other viral infections, subclinical, mild, moderate, or severe cases (10-20% of patients require hospitalization and 2-4% intensive care unit, icu) presenting with or without pneumonia are observed. asymptomatic cases are common but, to date, there is a lack of epidemiological surveys that provide a clear percentage of asymptomatic cases. 4, 5 the covid-19 pandemic is the world's gravest public health crisis of the 21st century, and there is an urgent need for reliable and updated scientific and clinical information. covid-19 is a zoonosis to cd147 (known as basigin or extracellular matrix metalloproteinase inducer), which is expressed in human airway and kidney epithelium, as well as in innate cells and lymphocytes, 14 and to tmprss4, which is highly expressed in intestinal epithelial cells. 15 in addition, antibody-dependent enhancement of sars-cov-2 cell entry may also contribute to infection as reported for sars-cov. 16 sars-cov-2 may use receptors that have been reported for other coronaviruses, such as cd26, aminopeptidase n and glutamyl aminopeptidase for cell invasion. 13, 17, 18 among these, cd26 (encoded by dpp4) has emerged as a putative receptor for sars-cov-2 because structural analyses predict that the spike protein of sars-cov-2 binds to cd26. 19 this receptor has been shown to be expressed in the human epithelium and immune cells. 14 there is limited evidence about covid-19-associated polymorphisms. ace might be one of the candidate genes that influences pneumonia progression in sars. it is conceivable that the d allele influences the renin-angiotensin system via elevation of serum or local ace levels, which may damage the endothelium or epithelium of the lungs. 20 the variance in covid-19 prevalence and mortality cannot be explained by an ace insertion or deletion polymorphism alone, or one polymorphism of any single gene. however, polymorphisms in genes of toll-like receptors, inflammasome, intracellular molecular sensors, interferons (ifns) 21 and interleukins (ils) may contribute. structural proteins of sars-cov-2 virions, such as the spike glycoprotein, envelope, membrane and nucleocapsid, are the main immunogenic molecules (figure 1) . 22 ,23 sars-cov-2 adaptive responses develop mainly to the spike protein, and immunodominant t and b cell epitopes have been reported. 24 intracellularly, the viral rna replicase complex, and non-structural and translated proteins, activate innate immune pathways. this leads to an ifn type i response, nf-kb activation in epithelial cells, as well as activation of nlrp3 and other inflammasomes, in macrophages and dendritic cells. 23 this article is protected by copyright. all rights reserved the spike protein of sars-cov-2 has a receptor-binding domain that binds ace2 with higher affinity than sars-cov. 8 in addition, the sars-cov-2 spike protein harbors a polybasic furin cleavage site (prrar) with an insertion of 4 amino acid residues, which is distinct from that found in sars-cov and other sars-like viruses. this allows effective cleavage by furin and other proteases and determines viral infectivity and host range. 6 the severe lymphopenia observed in covid-19 25 is similar to that reported in hiv infection and acquired immune deficiency syndrome. the latter is characterized by cd4+ t cell lymphopenia, whereas covid-19 causes general lymphopenia. however, severe lymphopenia development in covid-19 happens in weeks, whereas hiv-induced lymphopenia takes years. 26 hiv and sars-cov-2 are both rna viruses and share some similarities in their replication pathways; hence certain rna replication drugs may work in both diseases (figure 1) . 27 there are 2 strains of sars-cov-2 that are clinically relevant. genome analysis of sars-cov-2 from human samples shows high rates of mutation and deletion in several viral genes, including the spike-glycoprotein gene. 28 covid-19 treatments, including future vaccination against sars-cov-2, may drive the genetic evolution of the virus affecting virulence and pathogenicity. for example, a report on a 382-nt deletion in orf8 (figure 1 ) of sars-cov2 isolated from patients in singapore implied that mutations may arise as a result of human adaptation and could be associated with attenuation. 29 nevertheless, the emergence of a sars-cov-3 is possible as long as there is close contact between humans and living animals that harbor coronaviruses. data from 96 covid-19 patients in china show sars-cov-2 detection in respiratory samples for a median of 18 days (13-29 days) . in this study, sputum and saliva were not analyzed separately. viral shedding was significantly longer in patients with severe disease, with a median of 21 days (14) (15) (16) (17) (18) (19) (20) (21) (22) (23) (24) (25) (26) (27) (28) (29) (30) days), compared to mild disease, 14 days (10-21 days). furthermore, glucocorticoids treatment this article is protected by copyright. all rights reserved longer than 10 days significantly extended the duration of sars-cov-2 shedding. 30 viral load differed significantly by sample type, with respiratory samples showing the highest, followed by stool samples, and serum samples showing the lowest (figure 2) . 30 another study of 78 patients with covid-19 (33 asymptomatic vs 42 symptomatic) has estimated that the duration of viral shedding from nasopharynx swabs was 8 days (3-12 days) for asymptomatic vs 19 days (16-24 days) for symptomatic patients. 31 the viral load range from 1.34 × 10 11 copies per ml to 7.52 × 10 5 in sputum of patients who died or survived, respectively. 32 tmprss2 and tmprss4 promote sars-cov-2 infection of ace-expressing human enterocytes 15 causing diarrhea in adults and children. 33,34 sars-cov-2 has been detected in stool samples by reverse transcription polymerase chain reaction (rt-pcr) (figure 2) . the median duration of the virus in stool samples (22 days, interquartile range 17-31 days) was significantly longer than in respiratory samples (18 days, 13-29 days) . 30 however, sars-cov-2 released into the intestinal lumen was inactivated by simulated human colonic fluid, and infectious virus was not recovered from the stool specimens of patients with covid-19. therefore, the intestine is a potential site of sars-cov-2 replication, which may contribute to local and systemic illness and overall disease progression but unlikely to contribute to the spreading of covid19. 15 section 2: immunology of covid-19 from previous sars studies, it is known that the median seroconversion time for detectable igg was 17 days after infection. 35 detectable levels of sars-specific igg and neutralizing antibodies persisted for up to 720 days. this suggests that there is antibody-mediated protection from sars-cov recurrent infection for up to 2 years. 36 there are inconsistent reports on the humoral response to sars-cov-2. one study with 285 covid-19 patients reported that sars-cov-2 virus-specific igg and igm peaked 17-19 days and 20-22 days after symptom onset, respectively. 37 on the other hand, another study of 26 hospitalized covid-19 patients showed that seroconversion could take up accepted article to 50 days. 38 these discrepancies may be related to the time of sars-cov-2 diagnosis or the clinical characteristics of each cohort and warrant additional studies. systemic iga responses may play a relevant role in the pathogenesis of covid-19. 39 mucosal iga likely exerts a protective role by preventing sars-cov-2 adherence to epithelial cells. circulatory iga may also contribute to sars-cov-2 neutralization. in addition, iga has the ability to either promote inflammation, through the formation of immune complexes, or to dampen it via fc-mediated inhibitory itam-signaling. 40, 41 a seroconversion study in covid-19 patients has found and association between disease severity and sars-cov-2-specific iga levels. these were significantly higher than sars-cov-2-specific-igm and -igg levels in critically ill covid-19 patients. 39 whether this association, previously unseen in sars-cov infection, 42 is due to a protective or detrimental role of iga in covid-19 remains to be elucidated. preliminary findings indicate that asymptomatic and mild cases of covid-19 can generate detectable levels of sars-cov-2-specific antibodies in serum. however, seroconversion is observed less frequently in asymptomatic compared to mild or severe cases, and many asymptomatic cases yield undetectable sars-cov-2-specific antibody responses. 37, [43] [44] [45] so far, no robust data are available on the qualitative differences in humoral responses between asymptomatic and symptomatic covid-19 patients. it is not clear which molecular mechanisms underlie the milder symptoms of covid-19 in children as compared to adults. children may mount a sars-cov-2 antibody response characterized by more efficient production of the so-called natural antibodies, which arise from activated igm+ memory b cells. 46 these cells, which are more prevalent in children than in adults, presumably produce broadly neutralizing antibodies early during the infection. this article is protected by copyright. all rights reserved b cell receptor-sequencing has been conducted in the blood of covid-19 patients. naive b cells exhibited little clonal expansion, whereas cd27+cd38+ memory b cells showed the highest expansion levels among diverse b cell subsets. covid-19 patients significantly expanded specific bcell receptor clones compared to those in the healthy controls. these findings suggest that b cells experience unique clonal variable, diversity, and joining gene segment rearrangements upon sars-cov-2 infection. 47 the lifespan and functionality of these b cells remain to be elucidated. the term "herd immunity" refers to the generation of population immunity that protects a region, or country, from infection. 48 the number of confirmed covid-19 cases has reached approximately 6.5 million. 2 the world population is estimated to be 7.8 billion. to ascertain the extent of herd immunity, it is pivotal to define the prevalence of sars-cov-2-exposed humans. it is thought that 67% is the minimum percentage of symptomatic or asymptomatic covid-19 population required for herd immunity. 48 that is to say that worldwide herd immunity may occur when ⁓5 billion humans have a protective immune response against sars-cov-2. to date, there are no reliable data, particularly on the number of asymptomatic individuals that show seroconversion, to determine the degree of herd immunity. 49 il-4 is pleiotropic and could theoretically cause negative effects on immune responses. however, based on phase ii and iii studies with dupilumab (an il-4rα-specific monoclonal antibody that blocks il-4 and il-13 signaling) in the context of atopic dermatitis, chronic rhinosinusitis with nasal polyps and asthma, no increased risk of infections to viral or bacterial pathogens have been documented. 50 furthermore, dupilumab had no impact on responses to non-live vaccines. 51 this article is protected by copyright. all rights reserved allergic airway disease patients appear to be underrepresented among covid-19 patients. 52-54 this could be partly attributed to the low ace2 expression detected in allergic patients, with or without concomitant asthma. 55 furthermore, allergen challenge, which induces t helper (th)-2 inflammation, 56 has been shown to reduce ace2 expression in a murine model of asthma, and ace2 expression was inversely associated with type 2 biomarkers (il-13, ige, exhaled nitric oxide fraction). 57 these results are in line with previous work showing that decreased ace expression in the airway epithelium of asthmatic subjects was associated with eosinophilic inflammation. 58 on the other hand, the analysis of nasal airway transcriptome data from 695 children identified that tmprss2 is highly upregulated by type 2 inflammation through the action of il-13. therefore, the reduced ace2 expression seen in asthmatic patients may be compensated by an increase in tmprss2 production. 59 eosinopenia has been reported in ⁓50-70% of severe covid-19 patients. a minority of covid-19 patients present with eosinophilic inflammation. 25,60 the th1/th2 cytokine balance may play a role, particularly as it pertains to il-5, which promotes eosinophilopoiesis and eosinophil survival and activation. eosinophilic inflammation suggests the dominance of type 2 inflammation, which may play a protective role against sars-cov-2. on the other hand, it may be the result of a hypersensitivity reaction to drugs used to treat covid-19. 61-63 anti-il-5 treatment, which induces eosinophil deficiency, results in a higher viral load in influenza and rhinovirus infection. this might be due to the ability of eosinophils to bind and inactivate the influenza a virus and respiratory syncytial virus (rsv). 64 a similar role seems possible in sars-cov-2 infection, where type-2 asthma patients potentially benefit from antiviral eosinophil responses. on the other hand, covid-19 post-mortems did not show lung eosinophilia 62 , which argues against its local protective role in sars-cov-2 infection, although it is important to control for glucocorticoid-driven eosinophil reduction in these studies. 61 this article is protected by copyright. all rights reserved eosinopenia is commonly reported in severe covid-19. 65, 66 the underlying mechanisms are largely unknown and most likely multifactorial. a number of possible explanations have been proposed: decreased eosinophilopoiesis; defective eosinophil egression from the bone marrow; and eosinophil apoptosis induced by type 1 ifn released during the acute infection. 61 also, increased eosinophil migration and retention within inflamed tissues has been described, 67 but disputed for the aforementioned reasons. 62 there is no evidence for an enhanced susceptibility of patients on anti-il-5/il-5r treatment to develop viral infections. observational studies in covid-19 patients reported elevated eosinophil counts with a favorable outcome, whereas eosinopenia was observed in more severe cases. 25, 68 neither was there proof of causation nor evidence for enhanced tissue presence in lungs of covid-19 patients. 69 there is neither evidence for a protective effect of these biologicals nor a negative effect regarding sars-cov-2 infection. importantly, maintaining proper asthma control is imperative and so is to follow up on severe asthmatics during the covid-19 pandemic, for example via telemedicine. 50 more than 1 billion people worldwide are infected with helminths, with those living in resource-poor tropical areas being disproportionately affected. helminth co-infection has been shown to influence the severity of viral infection in mice. for example, murid herpesvirus 4 respiratory infection, prior infection with schistosoma mansoni, reduced disease severity. 70 however, immune responses to pulmonary coronaviruses and murid herpesvirus 4 are different and therefore the impact of helminth co-infection is yet to be determined. this is particularly important as the pandemic is now spreading through the helminth-endemic regions of the word. 71 this article is protected by copyright. all rights reserved sars-cov-2 infects human t cells via cd147-binding. 72 t cells are severely affected by sars-cov-2, which reduces t cell counts nearly 2 times below the reference limit. this effect is more pronounced in critically ill covid-19 patients. 60, 73, 74 in addition to the reduction in t cell numbers, a recent study found that cd4+ and cd8+ t cells as well as natural killer cells displayed reduced antiviral cytokine production in covid-19 patients. a reduced cytotoxic potential was identified in covid-19 patients, particularly in those that required icu, and was associated with high il-6 serum levels. 75 circulating sars-cov-2−specific cd8+ and cd4+ t cells have been reported in ∼70% and 100% of covid-19 convalescent patients, respectively. 76 cd4+ t cell responses to the spike protein were robust and correlated with sars-cov-2-specific-igg and -iga titers. the m, spike and n proteins each accounted for 11-27% of the total cd4+ response, with additional responses commonly targeting nsp3, nsp4, orf3a and orf8, among others. for cd8+ t cells, spike and m proteins were recognized, with at least 8 sars-cov-2 orfs targeted. interestingly, sars-cov-2−reactive cd4+ t cells were detected in ∼40-60% of unexposed individuals, which indicate cross-reactive t cell recognition between circulating 'common cold' coronaviruses and sars-cov-2. 76 three sars-recovered individuals, 9-and 11-years post-infection, were analyzed for t-cell responses against 550 sars-cov peptides that may share homology with mers-cov. sarsspecific memory t cells persisted at 9 and 11 years post-sars infection in the absence of antigen exposure. 77 based on these data, it is likely that specific sars-cov-2 epitopes elicit a persistent t cell response, which may also confer protection against other 'common cold' coronaviruses. 76 however, long-term studies on the natural history of sars-cov-2 infection are pending. different mechanisms have been proposed for lymphopenia: 1) t cell exhaustion. the expression of programmed cell death-1 marker (also known as pd-1), which is associated with t-cell exhaustion, was higher in t cells from covid-19 patients than in healthy controls; the expression of pd-1 and tim-3 (another exhaustion marker) increased as covid-19 progressed. 78 2) activation of p53 signaling in lymphocytes, which suggests a role for apoptosis for in lymphopenia. 3 this article is protected by copyright. all rights reserved pyroptosis, which induces lymphopenia and may be proinflammatory. 79 cov-2, which may also cause a cytopathic effect on infected t cells. 5) other mechanisms of lymphopenia that remain to be studied are bone marrow suppression during cytokine storm syndrome (css; see below) and sequestration in the lungs during extensive bilateral pneumonia. 60 lymphopenia can be used as an early predictor of severity and clinical outcome. a significant reduction in lymphocyte counts was common in severe and critically ill covid-19 patients. a continuing or gradual decrease of lymphocyte counts was indicative of poor prognosis and usually required icu admission ( table 1) 60 . in agreement with this, a number of studies have identified lymphopenia as an independent risk factor for mortality in covid-19. 25,80 in covid-19 patients, decreases were observed in total lymphocytes, cd4+ and cd8+ t cells, b cells and natural killer cells. t cell and natural killer cell counts were below normal levels, while b cell counts were at the low end of the normal range. a reduction in specific subsets of lymphocytes, such as cd16+cd56+ natural killer cells and regulatory t cells, was reported in severe covid-19 patients. 60 css is associated with a wide variety of diseases, both infectious and noninfectious. it is a complex cascade of multicellular activation events that leads to an excessive or uncontrolled release of proinflammatory cytokines. css-associated inflammation begins at a local site and spreads throughout the body via the systemic circulation and can cause multi-organ failure and hyperferritinemia. 60,81 css encompasses the activation of large numbers of blood cells, including b cells, natural killer cells, macrophages, dendritic cells, neutrophils, monocytes, resident tissue cells and epithelial and endothelial cells. their activation cause a massive release of pro-inflammatory cytokines, which this article is protected by copyright. all rights reserved drives pathology. 82 the cells involved in css during covid-19 have not been fully determined yet. were the most important cell types releasing a large amount of proinflammatory cytokines. 60, 83 which cytokines are most elevated during css? multiple proinflammatory cytokines and inflammasome activation may contribute to css pathogenesis. 60 elevated serum ferritin, il-6, il-1β, ifn-γ , cxcl10 (known as ip-10) and ccl2 immunosuppression is a double-edged sword in viral infections. 86 this article is protected by copyright. all rights reserved primary immunodeficient patients are a high-risk group in the current pandemic, but to date it is unknown if a particular immunodeficiency poses a higher risk of severe disease. international primary immunodeficiency monitoring is being carried out and few cases have been documented. patients at higher risk are those with complications resulting from their primary immunodeficiency and strict follow-up must be done in those cases. a consensus has been established that baseline chronic treatment should be continued in those patients if they are asymptomatic or mildly symptomatic. furthermore, recommendations regarding primary immunodeficient patients adhere to individual national guidelines emphasizing social distancing and strict hygiene measures. systematic testing of primary immunodeficient patients is not advised, however recommendations may change as the pandemic evolves. 89 there are no longitudinal studies analyzing t regulatory cells in covid19 systemic dysregulation of metabolism, such as that seen in obesity and diabetes is a risk factor of sars-cov-1 and sars-cov-2 infection and of covid-19 severity 69 . these diseases lead to chronic systemic inflammation, upregulation of sars-cov-2 receptors in the lungs and the periphery, and they disturb the glucose and lipid metabolism of tissues and immune cells. 14, 91, 92 ards is an acute life-threatening inflammation of the lung due to infection, trauma, or inflammatory conditions. excessive inflammation leads to alveolar damage and increased permeability of endothelial and epithelial cells. this results in protein-rich fluid accumulation in the interstitium and the air space, which causes impaired gas exchange and hypoxemia. reactive oxygen species, leukocyte proteases, chemokines, and cytokines also contribute to lung injury. the barrier impairment of the lung microvascular barrier is central to the pathogenesis of ards. 93 in covid-19 patients, ards is more common in the elderly, those with multiple comorbidities, and those with continuing or gradually progressing neutrophilia and lymphopenia, and a higher level of creactive protein, lactate dehydrogenase, d-dimer and procalcitonin. 60, 88 there are at least 2 clinical phenotypes of ards: 1) near normal pulmonary compliance with isolated viral pneumonia; 2) decreased pulmonary compliance. 95, 96 what specific therapies can be suggested for ards? different treatments were suggested for ards. corticosteroid treatment is generally not recommended, although widely used in critically ill patients. convalescent plasma (cp) was administered to a small number of patients and was associated with virus clearance and clinical improvement ( table 2) . low tidal mechanical ventilation, positive end-expiratory pressure, prone positioning ventilation, and fluid management guidelines were associated with improved outcomes. extracorporeal membrane oxygenation could be used according to the inclusion and exclusion criteria of the eolia trial. other potential therapies such as mesenchymal stem cell therapy and cytokine inhibitors are still in trials and without definite results. 60,97 bcg is a live attenuated vaccine that was developed against tuberculosis at the beginning of the 20th century. bcg vaccination induces metabolic and epigenetic modifications by enhancing trained immunity (innate immunity to subsequent infections). 98 it was hypothesized that general bcg vaccination policies adopted by different countries might have impacted the transmission patterns and/or covid-19-associated morbidity and mortality. 99 the mechanisms underlying kawasaki disease -a generalized vasculitis, in young children, of unknown, potentially post-viral etiology-are poorly understood. the rare covid-19-associated inflammatory syndrome also features vasculitic changes, affects older children too and is often only associated with positive sars-cov-2 serology, but not viral shedding. its mechanisms need to be elucidated and may include post-infectious, antibody and immune-complex mediated pathology. in adults, there are occasional cases of covid-19-associated cutaneous vasculitis, possibly a localized manifestation of the disease that leads to severe generalized vasculitis in some children. [104] [105] [106] interestingly kawasaki-like disease was not reported in chinese cases and the first months of european cases. the season of the disease and environmental factors should be considered. the chinese epidemic was mainly from january to march whereas the usa epidemic started in mid-march and is still ongoing. initial results of acute phase reactants such as c-reactive protein, alanine transaminase, lactate dehydrogenase, d-dimer, procalcitonin, serum ferritin and il-6 on admission were used to evaluate the severity and predict the mortality. however, dynamic changes of these variables will be more precise in predicting the recovery or progression of covid-19. continuing or progressively increasing levels of c-reactive protein, procalcitonin, d-dimer and lactate dehydrogenase were shown to be associated with a high risk of death in severe covid-19 patients. 25,60,107 patients with acute respiratory illness (i.e., fever and at least one sign/symptom of respiratory disease such as cough or shortness of breath) and a history of contact with a confirmed or probable covid-19 case during the 14 days before symptom onset. patients with any acute respiratory illness in the context of a pandemic should have sars-cov-2 infection in their differential diagnosis. special attention should be given to patients with sudden onset of anosmia, loss of taste, gastrointestinal symptoms or skin lesions without respiratory symptoms who also have epidemiological links. 5,25,108 smell loss is now a well-established diagnostic symptom of covid-19 and can be present in otherwise asymptomatic patients, making it a useful tool in initial diagnosis. 109 this has resulted in anosmia to be included in the list of symptoms used in early screening tools for possible covid-19 in many international bodies. 109 rapidly progressive respiratory failure and sepsis, elevated serum proinflammatory cytokine levels, elevated acute phase reactants (e.g. c-reactive protein), cell-free-hemoglobin-leukopenia and markers of disseminated intravascular coagulation. 110 rt-pcr to generate cdna from sars-cov-2 rna extracted from respiratory samples, followed by quantitative pcr (figure 2 ). 111 common gene targets for sars-cov-2 include the envelope, nucleocapsid, spike, rna-dependent rna polymerase, and orf1 genes. it is recommended to include in the analysis, at least, 2 target genes. 112 nasopharyngeal and oropharyngeal (throat) swabs are the primary specimens for sars-cov-2 rt-pcr testing. lower respiratory tract specimens (i.e. sputum, endotracheal aspirate or bronchoalveolar lavage) may have higher viral loads and be more likely to yield positive tests ( figure 2 ). however, these locations carry a high risk of aerosolization and therefore should be reserved for severe patients with a negative test on an upper respiratory tract specimen and high suspicion for lower respiratory tract sars-cov-2 infection. 113, 114 serology is useful to determine prior exposure to sars-cov-2 within a given period of time (the length of time following infection that one remains positive is unknown) (figure 2) . detection of this article is protected by copyright. all rights reserved antibodies specific to the receptor binding domain of the spike protein indicates neutralization capacity, hence informing better about the development of protective immunity. 37, 111, 115 the antibody response occurs later than initiation of symptoms as well as of the detection of viral rna by rt-pcr in respiratory tract specimens, which usually peaks within the first week of symptom onset (figure 2) . although antibodies to sars-cov-2 have been detected as early as the first week after symptom onset, igm, iga and igg seroconversion commonly occurs between the 2 nd and 3 rd week of clinical illness onset. thereafter, igm starts to decline, reaching low levels by week 5 and almost disappears by week 7, while iga and igg persist beyond this period. 37, 39, 111, 116 the main approaches include nucleic acid amplification on respiratory samples using mobile devices (rt-pcr or isothermal nucleic acid amplification) and viral antigens or host antibodies (viral protein fragments) detection using immunoassays. 117 however, individual tests need validation in large populations before use and their sensitivity, specificity, positive and negative predictive values have to be accurately ascertained. otherwise, they may lead to covid-19 under or over diagnosis, thus undermining the public health efforts to control the disease. 118 a high rate of false negatives with antigen point-of-care assays may be due to the fact that the majority of patients produce antibodies against sars-cov-2 only after the second week after of infection ( figure 2 ). 119 furthermore, an effective antibody response is connected with several determinants, comprising severity of the disease, age and nutritional status of the patient, medications administered and concomitant infections. 118 nucleic acid amplification using rt-pcr directly targeting the virus is not affected by the above-mentioned limitations. 120 this article is protected by copyright. all rights reserved positive by the fifth and final test. 121 patients with an initial positive sars-cov-2 result had an increased risk of progressing to severe cases. altogether, these findings underscore how the timing of the immune response influences rt-pcr tests for sars-cov-2, and the importance of combining rt-pcr and seroconversion data for covid-19 diagnosis. the decision to discontinue home isolation/quarantine should be adapted to specific groups of patients based on factors such as symptom severity, healthcare systems´ capacity, laboratory diagnostic resources and local epidemic status. patients with suspected or confirmed symptomatic covid-19 can discontinue self-isolation/quarantine if all the following 4 conditions are met: a) resolution of fever (without the use of fever-reducing medications) for at least 3 days; b) clinical improvement in respiratory symptoms (e.g., cough, shortness of breath) for at least 3 days; c) at least 8 days have passed since the onset of symptoms for mild cases or at least 14 days for severe cases and immunocompromised patients; d) 2 negative rt-pcr tests from respiratory specimens taken 24 hours apart. if there is limited or no testing capacity, the combined symptom/test-based strategy should be reserved to hospitalized covid-19 cases and healthcare workers, whereas for mild or asymptomatic covid-19 cases (suspected or confirmed) the symptom-based strategy (condition a) and b) and c)) without lab testing is considered acceptable to end the self-isolation period. 122 pandemic strategies for risk minimization should be elaborated, harmonized and followed as such in allergy clinics, centers and practices. 123 in the eaaci/aria position paper by pfaar et al. 124 experts in the field have developed practical recommendations for optimizing allergic patients 'care whilst ensuring the safety of all health care professionals (figure 3) . general guidance from national health authorities should be strictly followed (i.e., world health organization, who; european centre for disease prevention). in-person consultations should be minimized to the lowest necessary level and triaged by telemedicine whenever possible (figure 4 ). 125 special attention should be paid to dataprotection in adherence to national data-security and -protection laws. non-delayable diagnostic and therapeutic measures should strictly follow reasonable preventive measures. several specific considerations regarding diagnostic and therapeutic measures are important in different allergic diseases ( figure 5) . moreover, socio-psychological aspects play a fundamental role in the care of allergic patients during the current pandemic and should be especially recognized and followed. stress caused by isolation and stigmatization due to allergic symptoms may amplify the development of allergic symptoms. 126 virtual doctor consultations have been regarded as an alternative to on-site clinical encounters and are increasing during the covid-19 pandemic. 124 initially, pre-visit telephonic communication is helpful to screen for patients with potential sars-cov-2 infection. 127 the epidemiological history should be investigated to determine if patients have fever or respiratory symptoms. in addition, previsit specific triage improves the efficiency of the patient's visit, thus reducing the length of stay in the hospital. to reduce face-to-face meetings, physicians can train some patients to self-treat at home based on the diagnosis obtained through a telephone consultation (figure 4) . a strict screening protocol is needed to identify sars-cov-2 infected patients (figure 4) . ideally, only sars-cov-2 negative patients (diagnosed via rt-pcr and/or rapid test) should come to the clinic. in places where systematic testing is unavailable, at least, normal temperature and negative epidemiological history should be mandatory to proceed to the outpatient departments. patients with a body temperature higher than 37.3ºc should have additional screening examinations, including routine blood tests, chest computed tomography scanning and even throat swabs for sars-cov-2 rt-pcr testing. 128 the indication and urgency of the tests for diagnosis should be considered. contraindications for skin, provocation and lung function tests can be explained beforehand to the patient, which helps to accepted article avoid unnecessary in-person consultations. 124 any test generating aerosol particles should be avoided because it is considered high risk (figure 4) . personal protective equipment (ppe) must be used when collecting biological samples. biological samples collected on-site from suspected or confirmed covid-19 patients (e.g. antibody assays, rna isolation, flow cytometry) should be processed following bsl-2 practices. during and after the covid-19 pandemic, the usage of bsl-2 facilities is mandatory for all newly arriving patient samples to prevent spreading the disease. research procedures involving sars-cov-2 isolation or culture should be conducted in a bsl-3 facility. 124,129 patients with common allergic diseases do not develop distinct symptoms or severe outcomes. allergic children show a mild course similar to non-allergic children 5 . in a recent study of 182 hospitalized children, 43 of them were reported with allergies. allergic rhinitis was the most prevalent allergic disease (83.7%), followed by drug allergy, atopic dermatitis, food allergy and asthma. in this study, allergic children showed a reduced increase in acute phase reactants, procalcitonin, d-dimer and aspartate aminotransferase levels compared to all patients. there were no deaths in allergic children in that study. 130 clinical history is very helpful to identify seasonality-and exposure-related symptoms driving the diagnosis of pollen-induced allergic rhinitis. an atopy test (in vivo or in vitro) reinforces the diagnosis. however, covid-19 can be superimposed on allergic rhinitis symptoms. 124 symptoms such as fever, fatigue and sudden loss of smell, are suggestive of covid-19 and should be closely monitored. pandemic? this article is protected by copyright. all rights reserved n95 facial masks have been proven useful in reducing allergen exposure by blocking pollen access to nose and mouth. on the other hand, surgical masks do not protect against inhalation of small airborne contaminants and are not designed to seal tightly against the user´s face, hence the contaminated air can pass through the gaps. 131 there are no conclusive data on the impact of allergic rhinitis on covid-19 susceptibility 132 . a recent study with 24 allergic rhinitis patients demonstrated a reduction of ace2 expression in nasal brush samples following an allergen challenge. 55 also, this study reported lower ace2-expression in the epithelium of asthmatic patients. on the other hand, tmprss2 is highly upregulated by type 2 inflammation through the action of il-13. 59 therefore, further studies are necessary to determine if allergic rhinitis patients have an altered risk of sars-cov-2 infection as compared to non-allergic individuals. although limited, the available evidence suggests that, compared to non-allergic individuals, allergic there is no scientific evidence that treatments for allergic rhinitis either increase susceptibility to sars-cov-2 infection or the severity of covid-19. therefore, allergen avoidance measures, nasal saline douches, and background controller therapies recommended by current guidelines for allergic rhinitis, such as nasal corticosteroids or second-generation h1-blockers, should be continued as prescribed, both in non-infected and covid-19 diagnosed patients. 124 the loss of smell in chronic rhinosinusitis is caused by type-2 inflammation of the olfactory epithelium. 135 in covid-19, the exact mechanism of potential olfactory neuropathy is still unclear. 136 however, a study found that sustentacular cells of the olfactory epithelium express ace2 and tmprss2, which enable sars-cov-2 entry and may subsequently impair the sense of smell. 137 a considerable percentage of covid-19 patients experience loss of smell as an early sign of the disease. 109 in many patients smell recovers in 1-2 weeks and there is no indication that intranasal corticosteroid treatment has a positive impact on the recovery. 138 on the other hand, there is no evidence suggesting that this treatment has a negative impact on symptomatology and/or accepted article development of covid-19. consequently, it is recommended to continue regular intranasal corticosteroid treatment for chronic rhinosinusitis (figure 5) . 124 an asthma exacerbation is difficult to differentiate from covid-19 ards or pneumonia by the patient, especially if it is triggered by rhinovirus, or other common respiratory viruses, because both conditions have dry cough and dyspnea. the british thoracic society advises patients with asthma experiencing fever, fatigue and loss of taste or smell to alert their physician as these are indicative of covid-19. 144 the distinction can be made by the physician based on the presence of wheeze, which is generally (but not always) absent in covid-19 pneumonia, as well as high-resolution chest tomography and viral diagnostic tests. 124 patients with controlled asthma are not at higher risk of severe infection than the general population. 53,54 ace2 expression was shown to be decreased in patients with allergic asthma 55 and in those receiving inhaled corticosteroids. 145 on the other hand, ace2 expression in asthmatic patients was increased in african-americans, in males and associated with diabetes, 55 and type 2 inflammation in children is associated with increased expression of tmprss2. 59 it is clear though that uncontrolled asthma is a risk factor of severe covid-19, thus all efforts should be focused on treating asthma by regular use of controller medication, including inhaled corticosteroids and biologicals. 53,146,147 there is no evidence available that patients on inhaled corticosteroids are at higher risk of covid-19 infection or of more severe symptoms than the general population. it is strongly advised by international scientific societies that patients continue with their routine control medication including inhaled corticosteroids during the pandemic (figure 5) . 132,148 this article is protected by copyright. all rights reserved recent evidence indicates that inhaled corticosteroid treatment reduces the expression of viral membrane receptors used to infect the human airways in a dose-dependent manner. 145 on the other hand, the immune suppression exerted by corticosteroids may impair anti-viral responses. 141 however, there are no clinical studies investigating the effect of inhaled corticosteroid on sars-cov-2 infection rates. spirometry is essential for the diagnosis of new asthma cases as stated by the global initiative for asthma guidelines. therefore, it should be conducted, but under special conditions (negative pressure chamber, etc.) and only in areas with low sars-cov-2 infection incidence. healthcare providers performing lung function testing need to wear maximum ppe (filtering face-piece particles 2 or 3 face mask, goggles, or disposable face shield covering the front and sides of the face, clean gloves, and clean isolation gowns), and the spirometer devices should be properly disinfected between patients (figure 3) . 149 an alternative, less precise, is monitoring morning and evening peak expiratory flow variability over a week. 150, 151 the global initiative for asthma guidelines state that routine spirometry should be avoided, especially in high-risk areas of covid-19 transmission. if spirometry needs to be performed, maximum ppe should be used (figures 3 and 4) . 148 the treatment of asthmatic patients can be monitored using personal devices measuring forced expiratory volume and peak expiratory flow. many of these devices are equipped with remote transmission functions and thus are amenable for the telemedicine management of patients. 152 pandemic? this article is protected by copyright. all rights reserved there is no evidence suggesting that the current approach to treat asthmatic patients during an exacerbation should change during the covid-19 pandemic. moreover, there is no proof that a short course of systemic corticosteroids impacts the evolution of covid-19. thus, oral corticosteroids should be given as usual for the treatment of an asthma exacerbation ( figure 5) . 144, 148 in the few cases in which patients are treated with long-term oral corticosteroids in addition to their high dose inhaled corticosteroids this should be continued in the lowest dose possible to prevent exacerbations. 148 the cause of the asthma exacerbation should be studied thoroughly to rule out potential exacerbations due to viral infections. 89 the preferred treatment is a pressurized metered-dose inhaler with a spacer. each patient should have an individual spacer, and this should not be shared at home. the use of nebulizers should be avoided when possible because they increase the risk of disseminating viral particles, which could affect other patients and healthcare personnel. 148 anti-ige treatment with omalizumab (or other biologics indicated for asthma) should be continued in non-infected patients. self-administration devices at home, whenever this option is available, are preferred, to minimize face-to-face contact in the clinic. in infected patients, omalizumab administration should be delayed until complete clinical recovery and viral clearance is achieved ( figure 5) . 50,153 endotype is associated with severe asthma in obese patients, are obese asthmatic patients more likely to develop severe covid-19? obesity, as part of the metabolic syndrome, increases the risk of severe covid-19. this is due to the pre-existent systemic low-grade inflammation and increased expression of sars-cov-2 entry receptors (ace2, tmprss2 and cd147). 154, 155 obese patients tend to have worse asthma control, increased hospitalizations and suboptimal response to standard controller therapy. thus, both difficult-to-control asthma and underlying metabolic syndrome are risk factors for severe covid-19. this article is protected by copyright. all rights reserved the il-6/th17 endotype encountered in late-onset obese asthma might be an additional risk factor. 156,157 the dermatological manifestations of covid-19 range from an un-specific macular erythematous rash, urticarial lesions, chickenpox-like vesicles and acro-ischemic lesions. 158, 159 they can result from local inflammation due to circulating immune complexes or from systemic manifestations leading to vasculitis and thrombosis. 160 these patients are also at increased risk of drug hypersensitivity lesions ( figure 6 ). 161 there is no evidence that patients with barrier defects such as atopic eczema have a higher risk for sars-cov-2 infection or skin complications during covid-19. however, patients with atopic dermatitis are often on systemic immunosuppressants and should be monitored closely. optimal topical treatment regime should also be encouraged in all patients. 162 hand hygiene procedures are pivotal to prevent self-infection and virus spreading. however, extensive water contact enhances dry skin, disturbs the commensal microbiota and leads to barrier disruption in healthy individuals. moreover, it exacerbates diseases with an intrinsic barrier defect such as atopic dermatitis. 163, 164 effective skin-care after hand hygiene is therefore essential to prevent barrier disruption and sensitization events. here, emollients containing hyaluronic acid, vitamin e, ceramide or urea are recommended. 165 dupilumab is approved for the treatment of moderate-to-severe atopic dermatitis. first data from italy on dupilumab-treated non-infected in high epidemic areas, and current evidence from dupilumab trials, suggest no negative effect of dupilumab regarding viral infections 166 with reports on a reduced number of herpes simplex superinfections and less bacterial superinfections. [167] [168] [169] accepted article this article is protected by copyright. all rights reserved the current eaaci statement on the usage of biologicals in the context of covid-19 advices no change of therapy in non-infected individuals and to withhold/delay the application of biologicals for a minimum of two weeks or the resolution of the disease in case of sars-cov-2 infection (figure 5) . 50 this is based on expert opinion in the light of missing data and may be adapted if more information becomes available. acro-ischemic lesions on toes and fingers have been identified in a subgroup of covid-19 patients. 25, 170 the data available are scarce and it is unclear if preventive or active anticoagulation should be initiated. however, acro-ischemic lesions could predate other sars-cov-2 symptoms in children and young adults. covid-19-induced skin lesions can be related to thrombovascular events (i.e. petechiae, acroischemia, dry gangrene) or to typical viral infections (i.e. erythematous rash, urticaria, maculopapular exanthema). 161 drug hypersensitivity has to be considered as a differential diagnosis, mainly in the second group, being a distinction difficult during the acute phase. diagnosis relies mostly on clinical observations. in that regard, an accurate chronology of the reaction and the drug exposure timeline is very informative 63 . laboratory and histopathological findings may also help. immunomodulatory drugs (including azithromycin), hydroxychloroquine/chloroquine and ifns, are the ones most frequently involved in hypersensitivity reactions. most reactions are non-immediate and further studies are required to clarify whether this increased frequency is caused by the drug immunogenicity or simply derives from a greater consumption as compared to other treatments. 161 this article is protected by copyright. all rights reserved drug provocation tests are not recommended because reactions can occur during the tests, including the generation and spreading of virus-containing aerosols. however, they may be considered after careful risk-benefit assessment in cases of urgent need, such as chemotherapy in cancer patients, perioperative drugs and radiocontrast media in subjects needing urgent procedures, and antibiotics if no effective alternative drug is available. 124 most ait products authorized for use in europe indicate that ait should be discontinued in case of infection; the same principle will apply to the covid-19 pandemic. patients on subcutaneous or sublingual ait, who are diagnosed with covid-19, those suspected of sars-cov-2 infection or symptomatic patients with a positive contact to sars-cov-2 individuals, ait should be interrupted until the patient has recovered. in patients not infected or who have recovered from the infection, ait could be continued ( table 3) . these recommendations are conditional and could change as clinical data evolve. 124 ,134 ait should continue in non-infected patients or those recovered from covid-19 ( figure 5 ). this is especially important in patients with life-threatening conditions such as venom allergy. it is possible to extend the intervals between vaccines during subcutaneous ait, as done for inhalant allergens, to minimize visits to the allergy clinic. if venom ait was stopped due to sars-cov-2 infection, it is unclear when it should be re-initiated because data from convalescent patients is scarce. 134 in patients diagnosed with covid-19 or cases with suspected sars-cov-2 infection, oral immunotherapy dosing should continue as indicated in the dosing plan and in coordination with the treating physician. oral immunotherapy can be continued in non-infected patients and those who have recovered from covid-19 ( figure 5 ). in areas with high level of sars-cov-2 community transmission, visits to the allergy clinic for oral immunotherapy up-dosing should be postponed. 124, 134 accepted article this article is protected by copyright. all rights reserved these patients are generally on symptomatic treatment. they need to look out for symptoms suggesting hypoxia or pneumonia, such as shortness of breath, deep shallow breathing, chest pains or persistent tachycardia. special attention needs to be given to those with risk factors for disease progression, such as patients older than 65 years, cardiac or pulmonary comorbidities and immunosuppression. 171, 172 prophylactic low molecular weight heparin, or heparin, has been recommended by the who in severe to critically ill covid-19 patients. 141 however, the international society on thrombosis and haemostasis recommended that all hospitalized covid-19 patients, not just those in icu, should receive prophylactic low molecular weight heparin in the absence of contraindications. 173 during the sars outbreak in 2003, corticosteroids did not change the course of the viral infection and delayed viral clearance. 174 on the other hand, a retrospective study on sars patients in hong kong suggested a better survival rate in patients treated with prednisolone for milder pneumonia or methylprednisolone in more severe cases. 175 recently, chinese experts stated that, in covid-19 patients, systemic corticosteroids should be considered on individual indications in a low-to-moderate dose and for no longer than a week. 176 the national institutes of health in their covid-19 treatment guidelines advises against the use of systemic corticosteroids in non-critically ill patients. 177 there are over 170 clinical trials on covid-19 treatment registered now in the international databases and very few have been completed. currently promoted pharmacological treatments are, at the most, based on anecdotic data collected in small numbers of covid-19 patients. these studies did not satisfy evidence-based medicine criteria, but caught general attention through news media, for example hydroxychloroquine (see below). tocilizumab is a humanized monoclonal antibody specific for il-6r, and it is approved for the treatment of rheumatoid arthritis. a positive response to tocilizumab points towards an imbalanced innate immune response in severe covid-19. luo et al. 178 reported that of the 15 patients treated with tocilizumab, 7 of them critically ill, 11 of the patients recovered within a week. prompt resolution of symptoms and encouraging results have also been reported in uncontrolled or retrospective trials. [179] [180] [181] [182] [183] [184] [185] [186] [187] [188] [189] [190] these zoonotic beta-coronaviruses share structural and genomic similarities that are useful to patients? this article is protected by copyright. all rights reserved fang et al. 192 suggested that there is ace2 overexpression upon treatment with ace inhibitors, thiazolidinediones and ibuprofen. there were concerns pertaining to the use of nonsteroidal antiinflammatory drugs in covid-19 patients. the european medicines agency clarified that no scientific evidence established a link between ibuprofen, or other nonsteroidal anti-inflammatory drugs, and a risk to worsen covid19. 193 section 7: clinical trials and drug discovery in covid-19 adaptations for clinical trials during the pandemic must include all concerned parties such as there are drugs that interfere with ace2 and tmprss2, which are molecules used by the virus to enter the cell. 9, 195 for example, camostat mesylate is a clinically proven serine protease inhibitor with affinity for tmprss2. it has shown activity against sars-cov-2 in human lung calu-3 cells. 9 several drugs that target virus internalization are being investigated, including chloroquine phosphate and hydroxychloroquine, which have shown limited efficacy in humans and raised concerns due to side effects (see below). 196 drugs designed to inhibit the viral replication machinery may be effective against sars-cov-2. for example, remdesivir inhibits viral rna polymerases, which prevents sars-cov-2 replication (see below). it is uncertain whether lopinavir-boosted ritonavir and other antiretrovirals improve clinical outcomes or prophylaxis among patients at high risk of sars-cov-2 infection. 200 additional potential candidates include other broad-spectrum antiviral drugs such as arbidol and favipiravir and phytochemicals with anti-viral activity such as resveratrol ( figure. 1) . 197 in a cohort of severe covid-19 patients, compassionate-use of remdesivir showed clinical improvement in 68% of patients (36 out of 53). 201 of note, a double-blind, randomized, placebocontrolled trial of intravenous remdesivir was conducted in 1,063 adults hospitalized with covid-19 with evidence of lower respiratory tract involvement; remdesivir was superior to placebo in shortening the time to recovery in adults hospitalized with covid-19 and evidence of lower respiratory tract infection. 202 furthermore, in a study of 5 hiv-positive hospitalized patients with severe covid-19, three of them were given lopinavir-boosted ritonavir and 2 darunavir-boosted cobicistat for 14 days. four patients recovered and 1 remained hospitalized. 203 meplazumab is a cd147-specific humanized monoclonal antibody that has been shown to prevent sars-cov-2 infection of fibroblasts (veroe6 cells). 72 currently, there is insufficient evidence to draw any conclusions on the benefits of meplazumab for the therapy of covid-19 patients. in an observational chinese study, adults hospitalized with covid-19 pneumonia (n=17) who were treated with an intravenous infusion of meplazumab as an add-on therapy showed a higher recovery rate compared to controls (n=11). 198 however, these results should be interpreted with caution because they were generated in a non-randomized, non-stratified study, with a small sample size. large-scale studies are needed to assess the effectiveness and safety profile of meplazumab as a potential therapy for covid-19. cp therapy for covid-19 treatment has yielded promising results. for example, in a trial of 10 severe covid-19 patients, 205 cp therapy was well tolerated and improved the clinical outcomes. the viral load was undetectable after cp transfusion in 7 patients who had viremia. no severe adverse effects were observed. other clinical trials have shown the beneficial effect of cp therapy in covid-19 patients and ongoing clinical trials will provide additional data on its efficacy, safety and optimal timing for treatment ( table 2) . in this regard, it is unclear whether in patients with a high viral load, such as severely ill patients, cp therapy may drive tissue pathology through immune complexes or complement activation. baricitinib, fedratinib, and ruxolitinib are potent and selective jak-stat signaling inhibitors approved for indications such as rheumatoid arthritis and myelofibrosis. these drugs are powerful antiinflammatory medications that may reduce the systemic levels of cytokines associated with covid-19. 206 indeed, in a pilot study of 12 covid-19 patients, baricitinib limited the css and was beneficial for the patients. 207 the use of jak inhibitors has been associated with a higher risk of opportunistic viral infections, such as herpes zoster, which suggests that the reduced inflammation caused by jak inhibitors may limit, to some extent, anti-viral responses. 208 this article is protected by copyright. all rights reserved ivermectin (avermectin b1a and avermectin b1b) is an anti-parasitic drug that has shown broadspectrum anti-viral activity in vitro. in sars-cov-2-infected fibroblasts (vero-hslam cells), a single addition of ivermectin at 2 h post-infection reduced viral rna ~5000-fold at 48 hours. 209 however, plasma concentrations of total and unbound ivermectin did not reach the ic50 determined in vitro, even at a 10-times higher dose than approved by the food and drug administration (usa). 210 consequently, the likelihood of a successful clinical trial using ivermectin is low. in an observational study of 1,446 covid-19 patients, 811 received hydroxychloroquine treatment, which did not change the risk of intubation or death. 211 furthermore, in a brazilian randomized control study evaluating 2 different doses of chloroquine in covid-19 patients with severe respiratory symptoms, mortality was 2.5 times higher in the high-dose chloroquine arm. 212 moreover, pre-published results from us veterans health administration hospitals did not support any advantages of hydroxychloroquine administered alone or with azithromycin. 213 in addition, the results of a clinical study conducted in 821 individuals showed that hydroxychloroquine did not prevent illness compatible with covid-19 or confirmed infection when used as postexposure prophylaxis within 4 days after exposure. 214 however, because of the retraction of two main papers on hydroxychloroquine treatment for covid-19 patients, this area requires further attention by the european medicines agency and the food and drug administration. mesenchymal stem cells may exert antiviral mechanisms in the context of sars-cov-2 infection. the basal ifn-stimulated gene expression of mesenchymal stem cells is high, which enhances their responsiveness to ifn signaling, potentially inducing broad viral resistance. mesenchymal stem cell therapy may potentiate the low ifn-i and -iii levels and moderate ifn-stimulated gene response reported in sars-cov-2-infected ferrets and covid-19 patients. 215 it is is being used in some centers but its efficacy in covid-19 has not been proven. data available are mainly experimental with few records in humans and no reports on its efficacy in randomized clinical trials. 216 common anti-hypertensive drugs inhibit ace, but not ace2. importantly, ace2 opposes ace actions and lowers blood pressure by converting angiotensin-ii (a vasoconstrictor peptide) into its metabolites-angiotensin (1-7) (vasodilators). 217 other common related antihypertensive drugs are angiotensin-2 receptors blockers, which block at-1, a receptor for angiotensin-ii, through which it exerts its vasoconstrictor effect. however, at-1 is not known to be used by sars-cov-2 to infect cells. it was shown in animal models that ace inhibitors might increase ace2 expression, thus increasing susceptibility to infection. it has not been proven in humans but it raised the concerns during the covid-19 pandemic. 217 based on the data available to date, antihypertensive treatment with these medications should be continued. 218 at the moment, the animal model that resembles more closely human covid-19 is the rhesus macaque, whose ace2 receptor is identical to that in humans. this model recently showed that sars-cov-2 reinfection was hampered due to infection-acquired immunity and demonstrated the therapeutic effect of remdesivir in covid-19 prior use in human clinical trials. 219, 220 the murine ace2 receptor is different from humans, hence humanized murine models with recombinant human ace2 are necessary. 221 previous vaccine research for sars/mers facilitates rapid translation. 222 in the who vaccine single-domain antibodies have been investigated as potential therapeutics for influenza, rsv and hiv in addition to coronaviruses. sars-cov-2 mainly targets the respiratory tract, hence the development of vaccines directed to the respiratory epithelia and lung parenchyma using a nebulizer has been considered to maximize bioavailability and function. 229 although active research against respiratory viruses has focused on aerosolized plasmid dna vaccines, other forms of vaccine administration are currently further advanced in clinical trials. 222 veterinary medicine commonly uses aerosolized coronavirus vaccines for chicken farms. 230 a novel vaccine platform requires careful evaluation and should ideally include toxicological studies in valid animal models. early progress towards sars vaccines has facilitated a "running start" but standards of care and safety must be maintained. acceleration rather than omission of clinical trials is key. preliminary data from oxford university is anticipated by mid-2020. 222 of note, a doseescalation, single-center, open-label, non-randomized, phase 1 was conducted in 108 healthy individuals that received an ad5 vectored covid-19 vaccine. the vaccine was tolerable and this article is protected by copyright. all rights reserved immunogenic at 28 days post-vaccination. sars-cov-2-specific antibodies peaked at day 28 postvaccination and specific t-cell responses were detected from day 14 post-vaccination. 231 an important aspect is that covid-19-associated mortality is very high, almost unavoidable when the pandemic control fails. this is due to rapid community spread, high community virus, especially in the elderly and co-morbid, but also in younger non-comorbid persons, including healthcare workers, young adults and children. the covid-19 pandemic also seems to be characterized by a significant level of asymptomatic spread. [232] [233] [234] the iceberg of covid-19: are there asymptomatic cases below the surface? the the differences are almost entirely due to the timing and effectiveness of public health interventions. countries that failed to control did too little, too late, and allowed sars-cov-2 to rip through their population, with catastrophic outcomes. those that intervened early effectively stopped the disease transmission. 235 this article is protected by copyright. all rights reserved it is difficult to determine as it varies greatly from country to country, depending on how well countries control their epidemics with widespread testing, case isolation and vigorous contact tracing, testing and isolation if positive. in countries that do this well, the r0 can be very low indeed. in countries that fail to control the spread of the virus, the r0 is high but unknown as sars-cov-2 spreads untested and therefore undetected. it has been estimated to be ~2.2. 236 sars-cov-2 transmits more readily than either sars-cov or mers-cov. the r0 of sars-cov-2 is controversial but if left unchecked it is likely to be greater than 3-4. however, the r0 number cannot be precisely defined as no country has left it to spread completely unchecked. in any case, even when preventative measures are taken, the r0 of sars-cov-2 is higher than that of sars-cov (1.7-1.9) and mers-cov (<1). 237 there is a considerable frequency of very mild covid-19 patients as well as asymptomatic sars-cov-2-infected people. this makes transmission control more challenging than either sars-cov or mers-cov, where illness is frequently more severe. children are at low risk of severe covid-19 outcomes. 238, 239 most patients in pediatric age with sars-cov2 infection presented with no or mild clinical manifestations, including fever, fatigue and dry cough. they were typically managed with supportive treatments only and they had generally a favorable prognosis with a recovery within 2 weeks. [240] [241] [242] young children also frequently carry other respiratory viruses, which potentially limit sars-cov-2 infection, as reported for other viral infections. 243 differences between children and adults in the regulation of ace2 expression may also play a role. 46 ace2 mrna expression was high in type i and ii alveolar epithelial cells, in nasal and oral mucosa and nasopharynx, in smooth muscle cells and endothelium of vessels from the stomach, small intestine, colon, and in the kidney of human adults (mean age 52±22). 244 interestingly, a recent study demonstrated age-dependent ace2 gene expression in the nasal epithelium, which was lowest in younger children and increased with age. 245 in addition, cd147, cd26 and their molecular interaction proteins seem to be differently expressed in peripheral blood mononuclear cells and t cells in children in comparison with adults. 14 many children remain asymptomatic, even when they have radiologic pneumonia detected on screening. 238 given that children are effective transmitters of other respiratory viruses, 246 it is expected that they will be just as good at transmitting sars-cov-2. bats are likely the natural reservoir of sarsseverity (see questions below). data on ethnicity and covid-19 are scarce and further research on ethnicity and covid-19 outcomes is needed. 250 however, the data available show a disproportionate number of covid-19 deaths in black, asian and minority ethnic backgrounds. in fact, one third of uk icu admissions are reportedly from them. 251 in the usa, african americans had more covid-19 diagnoses and deaths, after adjusting for age, poverty, comorbidities, and epidemic duration. these disparities are also seen in the hispanic and asian communities. 252 pregnant women may be at a higher risk of poorer covid-19 outcomes because they have deficient ifn-α and ifn-λ responses to viral infections. 253 however, reported pregnancy outcomes in covid-19 are reassuring as they appear similar to non-pregnant adult females. 254 this article is protected by copyright. all rights reserved testing treatments is problematic because pregnant women are excluded from most trials. 255 it is known that azithromycin doubles innate ifn production from virus-infected lung cells. 256 it is safe for all trimesters of pregnancy 257 and has been shown effective in high-quality clinical trials of virusinduced lung disease. 258, 259 given that the human ace2 protein is encoded on the x chromosome, this may be relevant for malefemale differences in outcomes. particularly in males with rare ace2 coding variants as they will express those variants in all ace2-expressing cells compared to a mosaic pattern of expression in females. 260 males may also have differences in certain innate antiviral responses compared to female counterparts. 261 there is reasonably robust data of covid-19 deaths in hospitals because most people who die in hospital are tested. deaths outside hospitals are likely underestimated as people are dying in care homes where mortality approaches ~40%, 267 and may die without being tested and diagnosed. it is difficult to determine prevalence as testing practices vary so much from country to country. seroprevalence studies will help to collect these data. covid-19 was introduced rapidly to many industrialized countries as a result of air travel. 268 most of europe and the usa probably did not react in a timely and efficient manner, resulting in the rapid spread and subsequent high mortality rates. in light of the devastating situation in many european countries and the usa, less industrialized countries had a little more time to better prepare to control the pandemic. 269 an important factor for prevalence studies is the percentage of the population that has undergone a diagnostic test, which seems to be at lower levels in developing countries. this article is protected by copyright. all rights reserved respiratory viruses spread less readily in summer than in winter for reasons that are not well understood. dry air and higher temperatures are slowing down the spread of respiratory viruses. absence of school attendance, more time outdoors, greater household ventilation, warmer temperatures facilitating virus inactivation and higher vitamin d levels are all likely to play a part. although social distancing measures are implemented, the summer weather should play a role in hampering the spread of covid-19. however, based on the analogy of previous influenza pandemic, it is unlikely that summer, on its own, could stop transmission of sars-cov-2. [270] [271] [272] it largely depends on the sars-cov-2 seroprevalence developed in each country, which is still unknown. countries that have had widespread transmission may be hit by a second wave, but presumably with less severe consequences. countries that effectively controlled the pandemic are at a higher risk of second wave of covid-19 if those effective controls are relaxed due to the limited viral transmission and lack of active immunization. sars-cov-2 has spread worldwide in humans, causing mild or no disease in many cases. it will continue circulating similar to other human coronaviruses (229e, hku1, nl63, oc43), and it may well become an endemic, seasonal virus. 273 the main route of sars-cov-2 transmission is via respiratory droplets and aerosols. [274] [275] [276] avoidance of high virus loads, acquired through aerosol and droplet transmission, is paramount to prevent severe outcomes. consequently, social distancing, masks and hand sanitation are undoubtedly effective because they prevent the droplet and surface contact-associated initial high virus load and the increased risk of severe disease. [277] [278] [279] what is the evidence supporting social distancing and face mask to prevent sars-cov-2 infection? this article is protected by copyright. all rights reserved a systematic review and meta-analysis has found that transmission of viruses was lower with physical distancing of 1 m or more, compared with a distance of less than 1 (or 0.18) and protection was increased as distance was lengthened. in addition, face mask use could result in a large reduction in risk of infection (or 0.15), with stronger associations with n95 or similar respirators compared with disposable surgical masks or similar. eye protection also was associated with less infection (or 0.22). 280 therefore, the covid-19 pandemic can be controlled if social distancing is combined with widespread testing, case isolation, vigorous contact tracing and personal protection. indeed, severe and critical illness among chinese healthcare workers before january 10 th was 45%, a time when personal protection equipment and infectious control measures were likely not implemented. after february 1 st , when personal protection measures were in place, the percentage of severe and critically ill chinese healthcare workers dropped to 8.7%. 281 sars-cov-2 remained viable in aerosols for 3 h with a ~10-fold reduction in infectious titre. 282 sars-cov-2 was more stable on plastic and stainless steel than on copper and cardboard; viable virus was detected up to 3 days after application to plastic and 2 days to stainless steel, on each surface the virus titer was reduced nearly ~100-fold. 282 importantly, sunlight exposure inactivated 98% of infectious sars-cov-2 every 6.8 minutes in simulated saliva and every 14.3 minutes in culture media. this study suggests that persistence, and subsequently exposure risk, may vary significantly between indoor and outdoor environments. 272 therefore, it is convenient to minimize contact with surfaces touched by others (even before sars-cov-2 existed), particularly at indoor environments, for example when using public transportation. in 248 covi̇d-19 patients, the estimated median time from symptom onset to viral clearance in the nasal swabs was 11 days, while in asymptomatic cases it was 2 days. 283 in patients that recovered, the median duration of viral shedding was ⁓20 days, while in non-survivors it was detected until death. the longest duration of viral shedding in survivors was 37 days. 110 accepted article and found that a slow viral clearance is associated with an increased risk of high disease severity with a 1% mortality rate. 285 the individual variation in the transmission of an infection is described by a factor called "dispersion factor or k". the lower "k" value, the more transmission comes from a small proportion of individuals acting like superspreaders. superspreading clusters have been observed in past coronavirus outbreaks (sars/mers), where a small number of infected individuals was responsible for a large proportion of secondary transmissions, with an estimated "k" of about 0.16 for sars and 0.25 for mers. 286 it is unclear whether superspreading clusters have contributed to the covid-19 outbreak. a simulation of early outbreak trajectories estimated that "k" for covid-19 is higher than for sars and mers. 286 however, in a recent preprint study, the estimate of "k" for sars-cov-2 was around 0.1, suggesting that around 10% of infected patients may have been responsible for 80% of secondary transmissions. 287 individual variation in infectiousness is difficult to measure, as it is mostly empirical, but the identification of any sars-cov-2 superspreading will be of primary importance for pandemic control. the designation of covid-19-dedicated wards and personnel within hospitals is useful to limit nosocomial sars-cov-2 infections. 124 it also allows other non-covid-19 conditions to be treated using routine healthcare resources more safely and effectively. maintaining such separation requires intensive sars-cov-2 testing in view of the high asymptomatic infection rate. 288 community-based strategies are effective at controlling the transmission of sars-cov-2. australia, hong kong, japan, singapore, south korea, and new zealand have all controlled effectively. their cumulative covid-19 mortality is >100-fold less than that in belgium, france, italy, spain and the uk, countries which have had difficulties to adequately control the pandemic. 147, 289, 290 it is important to implement measures to contain the spread of the virus, such as developing models to predict sars-cov-2-related mortality. 291 closing live animal markets is likely to reduce the risk of future viral outbreaks although this is not a practical way to prevent viral outbreaks for multiple reasons including social and economic. lifestyle factors that may influence sars-cov-2 infection susceptibility and covid-19 severity include smoking, stress, diet and alcohol intake, among others. for example, smoking has been shown to increase the susceptibility to respiratory tract infections and its severity, 294 and it is a risk factor for severe covid-19. 295 moreover, alcohol consumption may impair anti-viral immunity; 296 in vitro studies with human monocytes have shown that both acute and prolonged alcohol exposures inhibit type i ifn induction upon toll-like receptor-8 and -4 stimulation 297 . dietary habits may also play a role as obese patients have been shown to have a higher risk of developing severe covid-19. 298 furthermore, there are bioactive food compounds with antiviral activity, such as resveratrol, 299 although the amount of them obtained through the diet is unlikely to play a relevant role in covidit is known that respiratory virus infection causes perturbations in the gut microbiota and that germfree mice are more susceptible to viral infections, which intimates a role for the microbiota in covid19 . 300 however, the impact of the commensal microbiota on sars-cov-2 infection susceptibility and covid-19 severity is unknown. 301 an essential step is identifying the bacterial species interacting with sars-cov-2. this is rather challenging given the large number of bacterial species in the lung and respiratory tract, 302 and especially in the gut. however, a number of lung metagenomic studies have reported an abundance of prevotella in the lung of sars-cov-2 infected patients 303 . while in accepted article silico analysis have revealed that prevotella proteins may promote viral infection 304 , prospective studies are necessary to ascertain if this is a consequence of the infection or a risk factor for it. it is well-established that epithelial barrier defects and/or damage favor the development of th2 immunity. 305, 306 increased hygiene, in general, as well as overexposure to epithelial barrier opening molecules, such as detergents, can promote the onset of allergic disease. 307 to date, there is no evidence linking the covid-19 protective measures (gloves, hand-sanitizers, etc.) with increased allergy prevalence. in this regard, multifactorial epidemiological studies are needed. these studies should consider the impact on allergic diseases of virus-specific type 1 responses and psychosocial and environmental changes caused by the pandemic and efforts to contain it. although there has been a significant change in pollution parameters, unfortunately this reduction in pollution is transient and consequently unlikely to be significant. the exposome-related allergy and asthma risk is multifactorial. it includes climate change, biodiversity, the microbiome and nutrition among others, which have not changed during the pandemic. 308 in addition, although pollution levels have dropped, climate change still occurs at an accelerated pace. lifestyle changes during the lockdown 309 , weight gain and increased exposure to indoor allergens and pollutants may even increase the incidence of allergic diseases in the long-run. with the rapid spread of covid-19 at a pandemic scale, we are overwhelmed and drowned with a wealth of information. a global fight to contain the pandemic has started in which we need international solidarity and prompt sharing of accurate scientific information. we strongly support the this article is protected by copyright. all rights reserved continue scit or slit: non-infected individuals this article is protected by copyright. all rights reserved hypersensitivity reactions to drugs may occur more often during the pandemic due to the increased use of drugs and drug interactions, which can result in morbilliform rash, erythroderma, early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia effect of changing case definitions for covid-19 on the epidemic curve and transmission parameters in mainland china: a modelling study presumed asymptomatic carrier transmission of covid-19 eleven faces of coronavirus disease 2019 the proximal origin of sars-cov-2 phylogenetic network analysis of sars-cov-2 genomes structural and functional basis of sars-cov-2 entry by using human ace2 sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor distribution of ace2, cd147, cyclophilins, cd26 and other sars-cov-2 associated molecules in various human tissues and immune cells in health and disease sars-cov-2 entry factors are highly expressed in nasal epithelial cells together with innate immune genes sars-cov-2 receptor ace2 is an interferon-stimulated gene in human airway epithelial cells and is detected in specific cell subsets across tissues single cell rna sequencing of 13 human tissues identify cell types and receptors of human coronaviruses distribution of ace2, cd147, cd26 and other sars-cov-2 associated molecules in tissues and immune cells in health and in asthma, copd, obesity, hypertension, and covid-19 risk factors tmprss2 and tmprss4 promote sars-cov-2 infection of human small intestinal enterocytes the potential danger of suboptimal antibody responses in covid-19 resistance to coronavirus infection in amino peptidase n-deficient pigs mammalian glutamyl aminopeptidase genes (enpep) and proteins: comparative studies of a major contributor to arterial hypertension emerging wuhan (covid-19) coronavirus: glycan shield and structure prediction of spike glycoprotein and its interaction with human cd26 ace1 polymorphism and progression of sars longitudinal profile of antibodies against sars-coronavirus in sars patients and their clinical significance antibody responses to sars-cov-2 in patients with covid-19 long-term coexistence of sars-cov-2 with antibody response in covid-19 patients distinct features of sars-cov-2-specific iga response in covid-19 patients the multifaceted b cell response in allergen immunotherapy covid-19 autopsies eaaci position paper on how to classify cutaneous manifestations of drug hypersensitivity lung eosinophils-a novel "virus sink" that is defective in asthma? covid-19, chronic inflammatory respiratory diseases and eosinophils -observationsfrom reported clinical case series the role of peripheral blood eosinophil counts in covid-19 allergy differentiation and activation of eosinophils in the human bone marrow during experimental human endotoxemia clinical features of 85 fatal cases of covid-19 from wuhan. a retrospective observational study clinical features of patients infected with 2019 novel coronavirus in wuhan helminth-induced il-4 expands bystander memory cd8(+) t cells for early control of viral infection will helminth co-infection modulate covid-19 severity in endemic regions? cd147 as a target for covid-19 treatment: suggested effects of azithromycin and stem cell engagement lymphocytosis and lymphopenia induced by imported infectious diseases: a controlled cross-sectional study of 17,229 diseased german travelers returning from the tropics and subtropics platelet-to-lymphocyte ratio is associated with prognosis in patients with coronavirus disease-19 impaired immune cell cytotoxicity in severe covid-19 is il-6 dependent targets of t cell responses to sars-cov-2 coronavirus in humans with covid-19 disease and unexposed individuals. cell. 2020. accepted article this article is protected by copyright. all rights reserved 77 memory t cell responses targeting the sars coronavirus persist up to 11 years post-infection reduction and functional exhaustion of t cells in patients with coronavirus disease 2019 (covid-19) research progress of the relationship between pyroptosis and disease lymphopenia predicts disease severity of covid-19: a descriptive and predictive study into the eye of the cytokine storm review: cytokine storm syndrome: looking toward the precision medicine era the pathogenesis and treatment of the `cytokine storm immune responses in covid-19 and potential vaccines: lessons learned from sars and mers epidemic exuberant elevation of ip-10, mcp-3 and il-1ra during sars-cov-2 infection is associated with disease severity and fatal outcome immunosuppression for hyperinflammation in covid-19: a double-edged sword? management of critically ill adults with covid-19 treatment for severe acute respiratory distress syndrome from covid-19 managing childhood allergies and immunodeficiencies during respiratory virus epidemics -the 2020 covid-19 pandemic: a statement from the eaaci-section on pediatrics dysregulation of immune response in patients with covid-19 in wuhan, china obesity and disease severity magnify disturbed microbiome-immune interactions in asthma patients altered lipid metabolism in recovered sars patients twelve years after pathogenesis of acute respiratory distress syndrome pulmonary vascular endothelialitis, thrombosis, and angiogenesis in covid-19 immunology of covid-19: mechanisms, clinical outcome, diagnostics and perspectives -a report of the 1 covid-19 pneumonia: ards or not? acute respiratory distress syndrome: advances in diagnosis and treatment bcg-induced trained immunity: can it offer protection against covid-19? is bcg vaccination affecting the spread and severity of covid-19? is global bcg vaccination-induced trained immunity relevant to the progression of sars-cov-2 pandemic? sars-cov-2 rates in bcg-vaccinated and unvaccinated young adults considering bcg vaccination to reduce the impact of covid-19 hyperinflammatory shock in children during covid-19 pandemic accepted article this article is protected by copyright. all rights reserved an outbreak of severe kawasaki-like disease at the italian epicentre of the sars-cov-2 epidemic: an observational cohort study kawasaki-like disease: emerging complication during the covid-19 pandemic hematological findings and complications of covid-19 clinical characteristics of coronavirus disease 2019 in china anosmia as a presenting symptom of sars-cov-2 infection in healthcare workers -a systematic review of the literature, case series, and recommendations for clinical assessment and management clinical course and risk factors for mortality of adult inpatients with covid-19 in wuhan, china: a retrospective cohort study interpreting diagnostic tests for sars-cov-2 comparative performance of sars-cov-2 detection assays using seven different primer-probe sets and one assay kit detection of sars-cov-2 in different types of clinical specimens laboratory testing for 2019 novel coronavirus (2019-ncov) in suspected human cases sars-cov-2 antibody testing -questions to be asked iga-ab response to spike glycoprotein of sars-cov-2 in patients with covid-19: a longitudinal study diagnosing covid-19: the disease and tools for detection advice on the use of point-of-care immunodiagnostic tests for covid-19 serological immunochromatographic approach in diagnosis with sars-cov-2 infected covid-19 patients us cdc real-time reverse transcription pcr panel for detection of severe acute respiratory syndrome coronavirus 2 distinct characteristics of covid-19 patients with initial rrt-pcr-positive and rrt-pcr-negative results for sars-cov-2 guidance for discharge and ending isolation in the context of widespread community transmission of covid-19 an academic allergy unit during covid-19 pandemic in italy covid-19 pandemic: practical considerations on the organization of an allergy clinic -an eaaci/aria position paper telemedicine in the era of covid-19 how stress and anxiety can alter immediate and late phase skin test responses in allergic rhinitis infection prevention and control during health care when novel coronavirus (ncov) infection is suspected management practice of allergic rhinitis in china during the covid-19 international consensus (icon) on management of otitis media with effusion in children clinical practice guideline: tympanostomy tubes in children advice for healthcare professionals treating people with asthma (adults) in relation to covid-19 covid-19 related genes in sputum cells in asthma: relationship to demographic features and corticosteroids asthma and covid-19: is asthma a risk factor for severe outcomes? allergy allergy and asthma in children and adolescents during the covid outbreak: what we know and how we could prevent allergy and asthma flares persistence of coronaviruses on inanimate surfaces and their inactivation with biocidal agents the relationship between fev1 and pef in the assessment of the severity of airways obstruction circadian variability in airways characteristics: a spirometric study the role of mobile health technologies in allergy care: an eaaci position paper covid-19, asthma, and biologic therapies: what we need to know covid-19 and the role of chronic inflammation in patients with obesity the role of adipocytes and adipocyte-like cells in the severity of covid-19 infections interleukin-17-producing innate lymphoid cells and the nlrp3 inflammasome facilitate obesity-associated airway hyperreactivity obesity and adult asthma: diagnostic and management challenges cutaneous manifestations in covid-19: a new contribution cutaneous manifestations in covid-19: a first perspective cutaneous manifestations in covid-19: lessons learned from current evidence diagnosis and management of the drug hypersensitivity reactions in coronavirus disease 19 european task force on atopic dermatitis (etfad) statement on severe acute respiratory syndrome coronavirus 2 (sars-cov-2)-infection and atopic dermatitis water exposure on the hands in adolescents: a report from the bamse cohort the skin microbiome: impact of modern environments on skin ecology, barrier integrity, and systemic immune programming consensus of chinese experts on protection of skin and mucous membrane barrier for health-care workers fighting against coronavirus disease no evidence of increased risk for covid-19 infection in patients treated with dupilumab for atopic dermatitis in a high-epidemic area efficacy and safety of dupilumab in adolescents with uncontrolled moderate to severe atopic dermatitis: a phase 3 randomized clinical trial comparative safety of systemic immunomodulatory medications in adults with atopic dermatitis long-term management of moderate-tosevere atopic dermatitis with dupilumab and concomitant topical corticosteroids (liberty ad chronos): a 1-year, randomised, double-blinded, placebo-controlled, phase 3 trial pharmacologic treatments for coronavirus disease 2019 (covid-19): a review role of adjunctive treatment strategies in covid-19 and a review of international and national clinical guidelines isth interim guidance on recognition and management of coagulopathy in covid-19 sars: systematic review of treatment effects corticosteroid treatment of severe acute respiratory syndrome in hong kong on the use of corticosteroids for 2019-ncov pneumonia tocilizumab treatment in covid-19: a single center experience a single center observational study of the clinical characteristics and short-term outcome of 20 kidney transplant patients admitted for sars-cov2 pneumonia impact of low dose tocilizumab on mortality rate in patients with covid-19 related pneumonia tocilizumab for treatment of severe covid-19 patients: preliminary results from smatteo covid19 registry (smacore). microorganisms off-label use of tocilizumab in patients with sars-cov-2 infection extracorporeal membrane oxygenation in the treatment of severe pulmonary and cardiac compromise in covid-19: experience with 32 patients tocilizumab therapy reduced intensive care unit admissions and/or mortality in covid-19 patients use of subcutaneous tocilizumab in patients with covid-19 pneumonia covid-19 in solid organ transplant recipients: initial report from the us epicenter clinical presentation and initial management critically ill patients with severe acute respiratory syndrome coronavirus 2 (sars-cov-2) infection in brescia, italy pilot prospective open, single-arm multicentre study on offlabel use of tocilizumab in patients with severe covid-19 tocilizumab for the treatment of severe covid-19 pneumonia with hyperinflammatory syndrome and acute respiratory failure: a single center study of 100 patients in effective treatment of severe covid-19 patients with tocilizumab insight into 2019 novel coronavirus -an updated interim review and lessons from sars-cov and mers-cov are patients with hypertension and diabetes mellitus at increased risk for covid-19 infection? accepted article this article is protected by copyright. all rights reserved 193. agency em. ema gives advice on the use of non-steroidal anti-inflammatories for covid covid-19clinical trials: quality matters more than quantity coronavirus disease 2019 (covid-19): a clinical update chloroquine or hydroxychloroquine for covid-19: why might they be hazardous? the lancet candidate drugs against sars-cov-2 and covid-19 meplazumab treats covid-19 pneumonia: an openlabelled, concurrent controlled add-on clinical trial inhibition of sars-cov-2 infections in engineered human tissues using clinical-grade soluble human ace2 systematic review of the efficacy and safety of antiretroviral drugs against sars, mers or covid-19: initial assessment compassionate use of remdesivir for patients with severe covid-19 remdesivir for the treatment of covid-19 -preliminary report covid-19 in patients with hiv: clinical case series a trial of lopinavir-ritonavir in adults hospitalized with severe covid-19 effectiveness of convalescent plasma therapy in severe covid-19 patients covid-19: combining antiviral and anti-inflammatory treatments accepted article this article is protected by copyright. all rights reserved baricitinib therapy in covid-19: a pilot study on safety and clinical impact growing evidence of the safety of jak inhibitors in patients with rheumatoid arthritis the fda-approved drug ivermectin inhibits the replication of sars-cov-2 in vitro the approved dose of ivermectin alone is not the ideal dose for the treatment of covid-19 observational study of hydroxychloroquine in hospitalized patients with covid-19 effect of high vs low doses of chloroquine diphosphate as adjunctive therapy for patients hospitalized with severe acute respiratory syndrome coronavirus 2 (sars-cov-2) infection: a randomized clinical trial outcomes of hydroxychloroquine usage in united states veterans hospitalized with covid-19. medrxiv a randomized trial of hydroxychloroquine as postexposure prophylaxis for covid-19 imbalanced host response to sars-cov-2 drives development of covid-19 current status of cell-based therapies for respiratory virus infections: applicability to covid-19 angiotensin-converting enzyme 2 (ace2) as a sars-cov-2 receptor: molecular mechanisms and potential therapeutic target covid-19 and the use of angiotensin-converting enzyme inhibitors and receptor blockers lack of reinfection in rhesus macaques infected with sars-cov-2 clinical benefit of remdesivir in rhesus macaques infected with sars-cov-2 on the way from sars-covsensitive mice to murine covid-19 model the covid-19 vaccine development landscape structures of human antibodies bound to sars-cov-2 spike reveal common epitopes and recurrent features of antibodies human neutralizing antibodies elicited by sars-cov-2 infection a human neutralizing antibody targets the receptor binding site of sars-cov-2 structural basis for potent neutralization of betacoronaviruses by single-domain camelid antibodies a human monoclonal antibody blocking sars-cov-2 infection delivery of alx-0171 by inhalation greatly reduces respiratory syncytial virus disease in newborn lambs vaccine efficacy against ontario isolates of infectious bronchitis virus safety, tolerability, and immunogenicity of a recombinant adenovirus type-5 vectored covid-19 vaccine: a dose-escalation, open-label, nonrandomised, first-in-human trial presymptomatic sars-cov-2 infections and transmission in a skilled nursing facility accepted article this article is protected by copyright. all rights reserved asymptomatic transmission, the achilles' heel of current strategies to control covid-19 asymptomatic seroconversion of immunoglobulins to sars-cov-2 in a pediatric dialysis unit suppressing the epidemic in new south wales covid-19 -navigating the uncharted covid-19, sars and mers: are they closely related? sars-cov-2 infection in children epidemiology of covid-19 among children in china coronavirus infection in pediatric emergency departments research g. children with covid-19 in pediatric emergency departments in italy severe acute respiratory syndrome coronavirus 2 (sars-cov-2) infection in children and adolescents: a systematic review multicentre italian study of sars-cov-2 infection in children and adolescents, preliminary data as at 10 virus-virus interactions impact the population dynamics of influenza and the common cold tissue distribution of ace2 protein, the functional receptor for sars coronavirus. a first step in understanding sars pathogenesis nasal gene expression of angiotensin-converting enzyme 2 in children and adults the september epidemic of asthma hospitalization: school children as disease vectors susceptibility of ferrets, cats, dogs, and other domesticated animals to sars-coronavirus 2 coronavirus disease 2019 (covid-19) in italy patients with diabetes are at higher risk for severe illness from covid-19 ethnicity and covid-19: an urgent public health research priority is ethnicity linked to incidence or outcomes of covid-19? assessing differential impacts of covid-19 on black communities impaired type i and iii interferon response to rhinovirus infection during pregnancy and asthma coronavirus disease 2019 in pregnancy consider pregnancy in covid-19 therapeutic drug and vaccine trials azithromycin induces anti-viral responses in bronchial epithelial cells azithromycin plus chloroquine: combination therapy for protection against malaria and sexually transmitted infections in pregnancy early administration of azithromycin and prevention of severe lower respiratory tract illnesses in preschool children with a history of such illnesses: a randomized clinical trial effect of azithromycin on asthma exacerbations and quality of life in adults with persistent uncontrolled asthma (amazes): a randomised, doubleblind, placebo-controlled trial accepted article this article is protected by copyright. all rights reserved ace 2 coding variants: a potential x-linked risk factor for covid-19 disease tlr7 ligands induce higher ifn-alpha production in females baseline characteristics and outcomes of 1591 patients infected with sars-cov-2 admitted to icus of the lombardy region risk factors for severity and mortality in adult covid-19 inpatients in wuhan spread of sars-cov-2 in the icelandic population updates by select demographic and geographic characteristics clinical characteristics of patients who died of coronavirus disease 2019 in china epidemiology of covid-19 in a long-term care facility in king county, washington coast-to-coast spread of sars-cov-2 during the early epidemic in the united states outbreak dynamics of covid-19 in europe and the effect of travel restrictions absolute humidity and pandemic versus epidemic influenza the signature features of influenza pandemics--implications for policy simulated sunlight rapidly inactivates sars-cov-2 on surfaces potential impact of seasonal forcing on a sars-cov-2 pandemic accepted article this article is protected by copyright. all rights reserved high sars-cov-2 attack rate following exposure at a choir practice airborne transmission of severe acute respiratory syndrome coronavirus-2 to healthcare workers: a narrative review aerodynamic analysis of sars-cov-2 in two wuhan hospitals changes in contact patterns shape the dynamics of the covid-19 outbreak in china evaluating the effectiveness of social distancing interventions to delay or flatten the epidemic curve of coronavirus disease social network-based distancing strategies to flatten the covid-19 curve in a post-lockdown world physical distancing, face masks, and eye protection to prevent person-to-person transmission of sars-cov-2 and covid-19: a systematic review and meta-analysis. the lancet epidemiology working group for ncip epidemic response ccfdc, prevention aerosol and surface stability of sars-cov-2 as compared with sars-cov-1 clinical progression of patients with covid-19 in shanghai distinct regulation of tonsillar immune response in virus infection covid-19 in a designated infectious diseases hospital outside hubei province pattern of early human-to-human transmission of wuhan estimating the overdispersion in covid-19 transmission using outbreak sizes outside china disease control, civil liberties, and mass testing -calibrating restrictions during the covid-19 pandemic evaluation of the effectiveness of surveillance and containment measures for the first 100 patients with covid-19 in singapore a dynamic residential community-based quarantine strategy: china's experience in fighting covid-19 advanced forecasting of sars-cov-2-related deaths in italy peer-to-peer contact tracing: development of a privacy-preserving smartphone app ethics of instantaneous contact tracing using mobile phone apps in the control of the covid-19 pandemic how cigarette smoke skews immune responses to promote infection, lung disease and cancer smoking is associated with covid-19 progression: a meta-analysis. medrxiv alcohol's effect on host defense inhibition of tlr8-and tlr4-induced type i ifn induction by alcohol is different from its effects on inflammatory cytokine production in monocytes high prevalence of obesity in severe acute respiratory syndrome coronavirus-2 (sars-cov-2) requiring invasive mechanical ventilation antiviral activity of resveratrol gut microbiota and covid-19-possible link and implications considering the effects of microbiome and diet on sars-cov-2 infection: nanotechnology roles the microbiome and the respiratory tract a familial cluster of pneumonia associated with the 2019 novel coronavirus indicating person-to-person transmission: a study of a family cluster covid-2019 associated overexpressed prevotella proteins mediated hostpathogen interactions and their role in coronavirus outbreak the initiation of th2 immunity towards food allergens igg1(+) b-cell immunity predates ige responses in epicutaneous sensitization to foods laundry detergents and detergent residue after rinsing directly disrupt tight junction barrier integrity in human bronchial epithelial cells emerging concepts and challenges in implementing the exposome paradigm in allergic diseases and asthma: a practall document will changes in alcohol and tobacco use be seen during the covid-19 lockdown? treatment of 5 critically ill patients with covid-19 with convalescent plasma treatment with convalescent plasma for critically ill patients with severe acute respiratory syndrome coronavirus 2 infection use of convalescent plasma therapy in two covid-19 patients with acute respiratory distress syndrome in korea treatment with convalescent plasma for covid-19 patients in wuhan effect of convalescent plasma therapy on viral shedding and survival in covid-19 patients treatment of covid-19 patients with convalescent plasma the authors thank the european academy of allergy and clinical immunology this article is protected by copyright. all rights reserved this article is protected by copyright. all rights reserved this article is protected by copyright. all rights reserved this article is protected by copyright. all rights reserved this article is protected by copyright. all rights reserved this article is protected by copyright. all rights reserved this article is protected by copyright. all rights reserved this article is protected by copyright. all rights reserved this article is protected by copyright. all rights reserved this article is protected by copyright. all rights reserved key: cord-328742-r4ht266w authors: heo, jung yeon; noh, ji yun; jeong, hye won; choe, kang-won; song, joon young; kim, woo joo; cheong, hee jin title: molecular epidemiology of human adenovirus–associated febrile respiratory illness in soldiers, south korea(1) date: 2018-07-17 journal: emerg infect dis doi: 10.3201/eid2407.171222 sha: doc_id: 328742 cord_uid: r4ht266w during january 2013–april 2014, we subjected nasopharyngeal specimens collected from patients with acute febrile respiratory illness in a military hospital to pcr testing to detect 12 respiratory viruses and sequence a partial hexon gene for human adenovirus (hadv) molecular typing. we analyzed the epidemiologic characteristics of hadv infections and compared clinical characteristics of hadv types. among the 305 patients with acute febrile respiratory illness, we detected respiratory viruses in 139 (45.6%) patients; hadv was the most prevalent virus (69 cases). of the 40 adenoviruses identified based on type, hadv-55 (29 cases) was the most prevalent, followed by hadv-4 (9 cases). hadv-55 was common in patients with pneumonia (odds ratio 2.17; 95% ci 0.48–9.86) and hospitalized patients (odds ratio 5.21; 95% ci 1.06–25.50). in soldiers with hadv infection in korea, hadv-55 was the most prevalent type and might be associated with severe clinical outcomes. during january 2013-april 2014, we subjected nasopharyngeal specimens collected from patients with acute febrile respiratory illness in a military hospital to pcr testing to detect 12 respiratory viruses and sequence a partial hexon gene for human adenovirus (hadv) molecular typing. we analyzed the epidemiologic characteristics of hadv infections and compared clinical characteristics of hadv types. among the 305 patients with acute febrile respiratory illness, we detected respiratory viruses in 139 (45.6%) patients; hadv was the most prevalent virus (69 cases). of the 40 adenoviruses identified based on type, hadv-55 (29 cases) was the most prevalent, followed by hadv-4 (9 cases). hadv-55 was common in patients with pneumonia (odds ratio 2.17; 95% ci 0.48-9.86) and hospitalized patients (odds ratio 5.21; 95% ci 1.06-25.50). in soldiers with hadv infection in korea, hadv-55 was the most prevalent type and might be associated with severe clinical outcomes. h uman adenoviruses (hadvs) are considered the most important causative agent of acute respiratory infection in soldiers, particularly in new recruits (1, 2) . a total of 79 hadv types have been documented (3) . the distribution of hadv types differs substantially by geographic region and environmental factors (4, 5) . hadvs are prevalent in training facilities or military barracks for soldiers, but the prevalent types of hadv have changed over time (6) . historically, hadv-4 and hadv-7 have been the most prevalent causes of acute febrile respiratory illness (afri) among us military personnel since the 1950s (7) . vaccination against hadv-4 and hadv-7 has been effective in reducing afri among us military trainees to date (8, 9) . however, since 2007, the emergence of new adenovirus types such as hadv -14, which is distinct from the prototype in the united states, has been associated with outbreaks of afri and severe pneumonia (including several deaths) in military populations (10, 11) . hadv is most prevalent in patients with acute lower respiratory tract infection and is the most common cause of pneumonia among military personnel in south korea (12) . however, studies evaluating the types of hadv in this population are limited because of the lack of knowledge about hadvs among military physicians. in 2012, hadv-55 was identified in patients with severe pneumonia, and an outbreak of afri among military personnel in south korea was recorded (13, 14) . hadv-55, which is a novel hadv type characterized by genome recombination between hadv-b11 and hadv-b14, caused outbreaks of acute respiratory diseases in military camps in turkey, china, and singapore (15) (16) (17) (18) . although cases of pneumonia caused by hadv-55 among military personnel in south korea have been recorded, information on the epidemiology and characteristics of type-specific hadv respiratory infections among military personnel in south korea is limited. thus, our study aimed to investigate the epidemiology of hadv infections and to compare the clinical characteristics by type of hadvs in soldiers in south korea via hospital-based surveillance on viral respiratory infections. the study was approved by the institutional review board of the south korea armed forces medical command. during january 2013-april 2014, we enrolled in the study all new recruits and active duty soldiers with afri who were required to visit the emergency department or to undergo we collected nasopharyngeal or throat swab specimens from the patients with afri within 24 hours after their hospital visit by using a flocked swab. we stored the specimen at 4°c in viral transport media until further testing. within 3 days of collection, we sent specimens to a commercial laboratory center (gc labs, yongin, south korea), where they were subjected to respiratory virus multiplex reverse transcription pcr. we extracted total viral nucleic acid from the specimens by using the chemagic viral dna/rna extraction kit (chemagen inc., baesweiler, germany) and performed cdna synthesis by using the capfishing full-length cdna premix kit (seegene inc., seoul, south korea). we performed pcr by using the seeplex rv12 ace detection kit (seegene inc., south korea), which is used for identifying influenza viruses a and b, respiratory syncytial viruses a and b, adenovirus, parainfluenza virus types 1-3, rhinovirus group a, human coronavirus 229e/nl63, human coronavirus oc43, and human metapneumovirus. we extracted dna from the adenovirus-positive respiratory specimens by using the qiaamp dna blood mini kit (qiagen, hilden, germany). we amplified the partial nucleotides of the hexon gene by using pcr as described elsewhere with some modifications (19) . we amplified viral sequences by using oligonucleotide primers producing a 475-bp fragment: adhex1f (5′-caacacctay-gastacatgaa-3′) and adhex1r (5′-katgggg-taragcatgtt-3′). pcr conditions were as follows: initial denaturation at 94°c for 1 min, followed by 35 cycles of denaturation at 94°c for 1 min, annealing at 50°c for 1 min, elongation at 68°c for 1 min, and final extension at 68°c for 5 min. for samples that tested negative in the first pcr reaction, we performed heminested pcr by using primers (273 bp) adhex1f (5′-caacacctay-gastacatgaa-3′) and adhex2r (5′-acatcctt-bckgaagttcca-3′) with the same temperature and time profiles. we determined dna sequences in both directions by using the applied biosystems automatic sequencer abi 3730xl and abi prism bigdye terminator v3.1 sequencing system (applied biosystems, foster city, ca, usa). we identified the type of hadv by using blast (http://blast.ncbi.nlm.nih.gov/blast.cgi). we generated phylogeny on the basis of the 232-bp nucleotide sequences of the hexon gene of hadvs. for the phylogenetic analysis, we selected the sequences of each type of hadv from genbank. we used mega 6 software to generate the phylogenetic tree and evaluated topologies by performing a bootstrap analysis of 1,000 iterations (20) . we obtained clinical information of the patients with hadv respiratory infection during the study period from standardized case report forms. the case report forms, which included clinical diagnosis, intensive care unit stay, requirement for mechanical ventilation or vasopressor, and symptoms at presentation, were completed within 7 days of the hospital visit by an attending physician. we conducted pearson's χ 2 and fisher exact test for the demographic and clinical variables by using the spss for windows version 20 (ibm corp., armonk, ny, usa). for all analyses, we defined statistical significance as p<0.05. during january 2013-april 2014, we enrolled 305 patients with afri in the study. we detected a total of 157 respiratory viruses in 139 (45.6%) soldiers with afri. hadv was the most prevalent virus (49.6% [69/139]), followed we commonly observed cases of hadv infection during winter and spring ( figure 1 ). among the tested cases of afri, the monthly positive rate for hadv ranged from 0% to 53.8%; average positivity rate was 22.6%. a peak positive rate for hadv infection occurred in soldiers with afri during march-april 2014. the mean age of the patients with hadv infection was 21.7 years. among the 69 patients with hadv infection, 40.6% were new recruits and 75.4% were hospitalized (table 2) . from the 69 patients with hadv infection, 51 respiratory specimens were available for further molecular analysis. for the 51 samples tested, we identified the hadv type in 40 samples (figure 2 ). hadv-55 (72.5% [29/40] ) was the most prevalent type in soldiers with hadv infection, followed by hadv-4 (22.5% [9/40]). we detected hadv-55 and hadv-4 in 2013, but we did not detect hadv-4 in 2014. we detected hadv-5 and hadv-6 in 1 case each. we observed no statistically significant difference in the demographic characteristics or signs and symptoms of the patients with hadv-55 infection compared with those with other types of hadv infection (table 3) . furthermore, we observed no statistically significant difference between the patients in terms of laboratory and radiographic findings. coinfections with other bacteria or viruses were similar in both groups. the patients with hadv-55 infection were more likely to have onset pneumonia (44.8% vs. 27.3% [odds ratio (or) 2.17; 95% ci 0.48-9.86]) and be hospitalized (86.2% vs. 54.5% [or 5.21; 95% ci 1.06-25.50]) than those infected with other types of hadv. in particular, we identified hadv-55 infection in all patients who required hospitalization in the intensive care unit or mechanical ventilation caused by acute respiratory distress syndrome (ards). in our study, hadv was the most prevalent virus detected among soldiers with afri in south korea, representing 49.6% of the cases. hadv-55 was the most prevalent type among the cases that could be identified using pcr. although hadv-55 has recently received public attention as an emerging pathogen that causes outbreaks of respiratory illness and severe pneumonia in the general population and soldiers, acute respiratory illness associated with hadv-55 has rarely been reported in the civilian population in south korea (6, 16, (21) (22) (23) . in children in south korea, hadv-3 and hadv-7 are prevalent serotypes and have also been associated with the outbreaks of adenoviral respiratory illness since the 1990s (24, 25) . severe pneumonia cases associated with hadv-55 infection were recently reported in soldiers in south korea (13, 14) . our finding that hadv-55 was the most prevalent type in soldiers in south korea differs from the epidemiology of adenovirus in children in south korea. another notable finding of this study was the changing epidemiologic trend from the co-circulation of hadv-4 and hadv-55 in 2013 to the predominant circulation of hadv-55 in 2014. hadv-4 was the second most common type after hadv-55 among soldiers with afri in south korea in 2013. however, hadv-4 has not been identified in soldiers in south korea since november 2013. although the shift in the hadv types in soldiers in south korea could not be fully understood because of the relatively short study period, hadv-55 infection has been prevalent among soldiers in south korea since 2014 (14, 26) . pneumonia and hospitalization associated with hadv-55 infection were more frequent than those associated with the other types of hadv infection in soldiers in south korea. in particular, hadv-55 infection was associated with severe pneumonia or ards. nevertheless, we observed no significant differences between hadv-55 cases and non-hadv-55 cases in terms of the frequency of clinical diagnosis of pneumonia, hospitalization, and ards. these findings could be explained by 2 assumptions. first, specific hadv types such as hadv-3, hadv-7, or hadv-14 might be more virulent than other types (25, 27, 28) . considering that the hadv-55 genome is more similar to the hadv-14 genome than the hadv-11 genome, hadv-55 infection could be associated with severe respiratory infection in certain patients (17) . second, the lower levels of herd immunity against hadv-55 could have an influence on the epidemic of hadv-55-associated respiratory infection in soldiers in south korea (29, 30) . severe respiratory illness and outbreaks associated with hadv-55 in soldiers in south korea might be similar to those observed in military personnel in china (13, 14, 21, 22) . in this study, the proportion of new recruits with hadv infection among soldiers in south korea was not as high as expected. more than half of the south korean soldiers with hadv infection were on active duty. these findings contrast with previous data in which hadv-associated respiratory infection has been common among new recruits 1224 emerging infectious diseases • www.cdc.gov/eid • vol. 24, no. 7, july 2018 . the patients included in this study might have had clinically severe illness rather than mild illness because our institute is the only central referral hospital in the military system in south korea. moreover, hadv easily spreads to advanced training sites or military barracks and it can spread in geographically dispersed military barracks by the movement of soldiers because of prolonged viral shedding (31) . in this study, some of the patients with hadv-55 infection who were active duty soldiers were identified in the clusters of patients in 4 military barracks (data not shown). although the outbreak of hadv-55 infection was not directly confirmed in this study, outbreaks of hadv-55-associated respiratory infection might be occurring among active duty soldiers. our study has some limitations. first, the study was performed for a relatively short period (16 months) . although the data might be insufficient to reflect the epidemiology of respiratory hadv infection among military personnel in south korea, the variation of hadv type over time and the impact of the emergence of hadv-55 on clinical severity can be observed. considering the long-term experience of the us military with respiratory hadv infection, the distribution of hadv types might show a substantial difference in the military and community populations in south korea. thus, a surveillance system must be established to detect the circulation of the hadv type among military personnel. second, the study population included patients who needed hospitalization or an emergency department visit. among soldiers in south korea with hadv infection, patients who had severe clinical signs and symptoms might be those who were primarily enrolled in this study. nevertheless, our results suggest that of the several hadv types, hadv-55 and hadv-4 might be implicated in hadv respiratory infections among soldiers in south korea. furthermore, active duty soldiers and new recruits could have a substantial disease burden caused by respiratory hadv infection. third, we could not determine hadv types in 11 cases. the viral titer in the clinical specimen might have been low and the viral dna degraded because some respiratory specimens were not frozen until the molecular experiment. using the 2-step hemi-nested pcr, we did not observe a dna band in 11 samples. this study is important because it is a prospective study on patients with afri. studies on the hadv type in soldiers in south korea since 2012 are limited. however, the previous studies provided data on the epidemiology of the hadv type in patients with severe clinical manifestations or in those with hadv respiratory infection. these conditions might result in bias in the epidemiology of hadv infections among military personnel in south korea. in conclusion, our study found that hadv was the most prevalent virus among soldiers with afri in south korea. in particular, hadv-55 and hadv-4 were the prevalent types in soldiers with hadv-associated respiratory infection. hadv-55 was associated with severe clinical outcomes. further studies are needed to verify which hadv types are associated with afri in military recruits. in addition, studies on the introduction or development of an effective vaccine against hadv-55 and hadv-4 should be considered. outbreak of febrile respiratory illness caused by adenovirus at a south korean military training facility: clinical and radiological characteristics of adenovirus pneumonia respiratory infections in the u.s. military: recent experience and control first isolation of a new type of human adenovirus (genotype 79), species human mastadenovirus b (b2) from sewage water in japan a two decade survey of respiratory adenovirus in taiwan: the reemergence of adenovirus types 7 and 4 genotype prevalence and risk factors for severe clinical adenovirus infection, united states adenovirus: epidemiology, global spread of novel serotypes, and advances in treatment and prevention respiratory diseases among u.s. military personnel: countering emerging threats adenovirus vaccines in the u.s. military dramatic decline of respiratory illness among us military recruits after the renewed use of adenovirus vaccines acute respiratory disease associated with adenovirus serotype 14-four states abrupt emergence of diverse species b adenoviruses at us military recruit training centers acute lower respiratory tract infections in soldiers outcomes of early administration of cidofovir in non-immunocompromised patients with severe adenovirus pneumonia clinical features and courses of adenovirus pneumonia in healthy young adults during an outbreak among korean military personnel respiratory disease caused by a species b2 adenovirus in a military camp in turkey outbreak of febrile respiratory illness associated with adenovirus 11a infection in a singapore military training camp computational analysis identifies human adenovirus type 55 as a re-emergent acute respiratory disease pathogen an outbreak of acute respiratory disease in china caused by human adenovirus type b55 in a physical training facility molecular identification of adenoviruses in clinical samples by analyzing a partial hexon genomic region mega6: molecular evolutionary genetics analysis version 6.0 outbreak of acute respiratory disease in china caused by b2 species of adenovirus type 11 emergence of community-acquired adenovirus type 55 as a cause of community-onset pneumonia severe pneumonia associated with adenovirus type 55 infection genome type analysis of adenovirus types 3 and 7 isolated during successive outbreaks of lower respiratory tract infections in children clinical severity of respiratory adenoviral infection by serotypes in korean children over 17 consecutive years (1991-2007) febrile respiratory illness associated with human adenovirus type 55 in south korea military fatal type 3 adenoviral pneumonia in immunocompetent adult identical twins a community-based outbreak of severe respiratory illness caused by human adenovirus serotype 14 prevalence of antibodies to adenovirus serotypes 4 and 7 among unimmunized us army trainees: results of a retrospective nationwide seroprevalence survey epidemic of adenovirus-induced respiratory illness among us military recruits: epidemiologic and immunologic risk factors in healthy, young adults spread of adenovirus to geographically dispersed military installations address for correspondence: hee jin cheong similarities of t cell function in cell-mediated immunity and antibody production lymphokines: non-antibody mediators of cellular immunity generated by lymphocyte activation a lymphocyte-stimulating factor produced in vitro virus interference. i. the interferon a factor stimulating dna synthesis derived from the medium of leukocyte cultures interferon-like virus-inhibitor induced in human leukocytes by phytohemagglutinin although the term cytokine had not yet even been defined, interferon-α, the first cytokine known, was identified in 1957 as a protein that interfered with virus replication. activities of interferon-γ and interleukin-2 were identified in 1965. macrophage migratory inhibitory factor was identified in 1966 key: cord-342476-0rupk21u authors: van rijn, anneloes l.; van boheemen, sander; sidorov, igor; carbo, ellen c.; pappas, nikos; mei, hailiang; feltkamp, mariet; aanerud, marianne; bakke, per; claas, eric c. j.; eagan, tomas m.; hiemstra, pieter s.; kroes, aloys c. m.; de vries, jutte j. c. title: the respiratory virome and exacerbations in patients with chronic obstructive pulmonary disease date: 2019-10-24 journal: plos one doi: 10.1371/journal.pone.0223952 sha: doc_id: 342476 cord_uid: 0rupk21u introduction: exacerbations are major contributors to morbidity and mortality in patients with chronic obstructive pulmonary disease (copd), and respiratory bacterial and viral infections are an important trigger. however, using conventional diagnostic techniques, a causative agent is not always found. metagenomic next-generation sequencing (mngs) allows analysis of the complete virome, but has not yet been applied in copd exacerbations. objectives: to study the respiratory virome in nasopharyngeal samples during copd exacerbations using mngs. study design: 88 nasopharyngeal swabs from 63 patients from the bergen copd exacerbation study (2006–2010) were analysed by mngs and in-house qpcr for respiratory viruses. both dna and rna were sequenced simultaneously using an illumina library preparation protocol with in-house adaptations. results: by mngs, 24/88 samples tested positive. sensitivity and specificity, as compared with pcr, were 96% and 98% for diagnostic targets (23/24 and 1093/1120, respectively). additional viral pathogens detected by mngs were herpes simplex virus type 1 and coronavirus oc43. a positive correlation was found between cq value and mngs viral normalized species reads (log value) (p = 0.002). patients with viral pathogens had lower percentages of bacteriophages (p<0.001). no correlation was found between viral reads and clinical markers. conclusions: the mngs protocol used was highly sensitive and specific for semi-quantitative detection of respiratory viruses. excellent negative predictive value implicates the power of mngs to exclude any pathogenic respiratory viral infectious cause in one test, with consequences for clinical decision making. reduced abundance of bacteriophages in copd patients with viral pathogens implicates skewing of the virome during infection, with potential consequences for the bacterial populations, during infection. a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 chronic obstructive pulmonary disease (copd) is characterized by exacerbations with high morbidity and mortality, and over 65 million patients suffer from this disease worldwide [1] . a copd exacerbation is an acute event leading to worsening of the respiratory symptoms and is associated with a deterioration of lung function [2] . exacerbations are mainly associated with infections, of which a large part is caused by viruses (22-64%) [3] [4] [5] [6] . however, in part of the exacerbations an etiologic agent is not detected. current routine virus diagnostics is based on polymerase chain reactions (pcr) and inherently the number of detectable pathogens is restricted to the ones included in the assay. rare, mutated and pathogens with an uncommon clinical presentation will be missed, along with new and currently unknown ones. over the last decades, several previously unidentified viruses have been discovered as respiratory pathogens, including metapneumovirus [7] , middle-east respiratory syndrome coronavirus [8] and human bocavirus [9] . metagenomic next generation sequencing (mngs) is an innovative method, which enables the detection of all genomes in a given sample. proof of principle studies have shown that mngs on respiratory samples can confirm and extend pcr results and deliver typing and resistance data at the same time [10] [11] [12] [13] [14] . the performance of mngs in the clinical diagnostic setting, especially the positive and negative predictive value, has not yet been elucidated and is likely to differ per clinical syndrome and sample. previous data from reports on 16s rrna analysis from the respiratory tract have led to increased insight in the microbiome in patients with copd [15] . changes in bacterial populations have been associated with exacerbation events and clinical phenotypes [15] . however, these studies are intrinsically limited to analysis of the bacterial part of the microbiome. so far only a few studies using shotgun metagenomics have focussed on the respiratory virome in children with acute respiratory infections [16, 17] . in this study, we analyse the composition of the virome in adult patients with exacerbations of copd. the aim of this study was to correlate the respiratory virome in copd patients as found by mngs with qpcr and clinical data. patients with copd were included in the bergen copd exacerbation study (bces) between 2006 and 2010 in bergen, norway [18] . all patients lived in the haukeland university hospital district. baseline data taken during the first visit while in the stable state included amongst others exacerbation history, medications, comorbidities, spirometry and global initiative for chronic obstructive lung disease (gold 2007) categorisation. patients were given a telephone number to a study nurse, whom they would contact in case of an exacerbation. patients with an exacerbation according to a predefined set of symptoms were scheduled for an appointment with a study physician the next working day. during exacerbations, nasopharynx swabs were sampled and two different markers for the severity of the exacerbation were scored. after an exacerbation a control visit was scheduled. during the study period 154 patients had at least one exacerbation and in total 325 exacerbations were included in bces, of which 88 exacerbation samples were tested in the current study. nasopharyngeal samples were frozen and stored at -80˚c. in total 88 nasopharyngeal samples of patients at the time of exacerbation were selected based on the availability of other samples (outside the current focus) and sent to the leiden university medical center (the netherlands) for further testing. the viral respiratory panel covered by the multiplex real-time pcr (qpcr) developed in our laboratory consists of coronavirus 229e, coronavirus hku1, coronavirus nl63, coronavirus oc43, influenza a, influenza b, human metapneumovirus, parainfluenza 1-4 (differentiation with probes), respiratory syncytial virus, and rhinovirus [19] . total nucleic acids (na) were extracted directly from 200 μl clinical sample, using the total nucleic acid extraction kit on the magnapure lc system (roche diagnostics, almere, the netherlands) with 100 μl output eluate. nucleic acid amplification and detection by real-time pcr was performed on a biorad cfx96 thermocycler, using primers, probes and conditions as described previously [19] . cq values were normalized using a fixed baseline fluorescence threshold. the metagenomics protocol used has been described and optimized for simultaneously rna and dna detection previously [14] . in short, internal controls, equine arteritis virus (eav) for rna and phocid herpesvirus-1 (phhv) for dna (kindly provided by prof. dr. h.g.m. niesters, the netherlands), were spiked in 200 μl of the virus transport medium in which the nasopharyngeal swab was stored. nucleic acids were extracted directly from 200 μl clinical sample using the magnapure 96 dna and viral na small volume extraction kit on the mag-napure 96 system (roche diagnostics, almere, the netherlands) with 100 μl output eluate (an updated version of the isolation method used for qpcr, tested previously [20] ). extraction buffer was used as negative control (for extraction, library preparation, and sequencing). for library preparation, 7 μl of nucleic acids were used, using the nebnext 1 ultra™ directional rna library prep kit for illumina 1 , with several in-house adaptations to the manufacturers protocol in order to enable simultaneous detection of both dna and rna. the following steps were omitted: poly a mrna capture isolation, rrna depletion and dnase treatment step. this resulted in a single tube per sample throughout library preparation containing both dna and rna. metagenomic sequencing was performed on an illumina nextseq 500 sequencing system (illumina, san diego, ca, usa), and approximately 10 million 150 bp paired-end reads per sample were obtained. after quality pre-processing, sequencing reads were taxonomically classified with centrifuge [21] using an index constructed from ncbi's refseq and taxonomy databases (accessed february 2019) with reference nucleotide sequences for the viruses, bacteria, archaea, fungi, parasites, and protozoa. reads with multiple best matches were uniquely assigned to the lowest common ancestor (k = 1 centrifuge setting; previously validated [14] ). both negative and positive results were confirmed using genomedetective website [22] version 1.111 (accessed december 2018-january 2019) and horizontal coverage (%) was determined using genomedetective. read counts were normalized, dividing the rawread count by the total number of reads in the sample and by the (average) genome size, and multiplied by 10^11 (to achieve comprehensible read counts in the same order of magnitude as the raw read counts). for samples with dubious or inconclusive classification results a de novo assembly was performed. pre-processed short reads assigned to a higher taxonomic level of a suspected viral target were extracted and de novo assembled with spades version 3.11.1 [23] into longer stretches of contiguous sequences (contigs). the resulting contigs were then run against the blast ncbi's nucleotide (nt) database (accessed 2017) using blastn 2.7.1 [24] . after identification of a putative target sequence, all the reads from the original sample were mapped against the identified best blast hit for further confirmation using bwa 0.7.17 software package [25] . sensitivity, specificity, positive and negative predictive values were calculated based on 24 pcr positive and 1120 pcr negative target results of 88 samples. correlation between qpcr cq value and logarithm of normalized numbers of mngs viral reads was tested with population pearson correlation coefficient. potential correlations of mngs data with clinical variables were tested as follows. cq value/ viral reads and clinical parameters (exacerbation severity, duration of exacerbation or decrease/increase in forced expiratory volume in 1 second (fev 1 , control visit compared to baseline) were tested with one-way anova and kruskal-wallis test when appropriate (depending on distribution). comparison of the percentage of phages of all viral reads (after subtraction of the internal control eav and phhv reads) between mngs virus positive samples and negative samples was tested with mann-whitney u test, comparison with clinical parameters with kruskal-wallis test. diversity of the virome in different patient groups was characterized by shannon diversity index (h) and tested with welch two sample t-test. statistical analyses were performed using ibm spss statistics version 25 software for windows <0.05 were considered statistically significant. prior to inclusion all subjects received written and oral information and signed informed consent. the bces study was approved by the regional ethical committee in western norway (rek-vest, case-number 165.08). the performance of this study, including mngs, was approved by the medical ethics review committee of the leiden university medical center (cme number b16.004); no additional consent was necessary. in total 63 patients with 88 exacerbations were included with a median of one exacerbation per patient (range 1-5). baseline patient characteristics and exacerbation characteristics are shown in tables 1 and 2 respectively. of the 88 samples, 23 (26%) tested positive with in-house pcr: 14 (61%) were rhinovirus positive, three influenza a, two coronavirus nl63, one coronavirus oc43, two parainfluenza 3 and one parainfluenza 4. cq values ranged from 19-38 (table 3 ). a median of 11 million (7,522,643-20,906,019) sequence reads per sample were obtained. of the 11 million reads, approximately 93% were homo sapiens reads, 3% were bacterial and 0.1% viral (table 4) . a median of 3% of the reads could not be assigned to sequences in the centrifuge index database (ncbi refseq). of the 23 qpcr positive samples, 22 tested positive with mngs, resulting in a sensitivity of mngs of 96%. only one sample, that was rhinovirus positive by qpcr (cq 38), could not be detected by mngs ( table 3) . coverage of reference genomes was high (93-100%) with the exception of three samples: 30% coverage of rhinovirus c (1,401,120 mapped reads, accession ay391777.1). a coverage plot of all reads against this reference strain (fig 1) showed good horizontal and vertical coverage (read coverage depth 428). the original oc43 qpcr amplification appeared to have been inhibited, and repeated oc43 qpcr confirmed the positive mngs result (cq 25). sensitivity, specificity and predictive value. the sensitivity, specificity and predictive values of mngs were calculated based on 24 pcr positive and 1120 pcr negative target results of 88 samples and the normalized read counts (table 5 ). calculations were made using different cut-off values of respectively �0, �15 and �50 normalized read counts. with a cutoff of �15, the sensitivity was 92% and specificity 100% and the positive predictive value (ppv) increased to 92%. the negative predictive value (npv) was 100% for all cut-off levels. a roc curve (fig 2) , using youden's index [26] demonstrated that the optimal sensitivity and specificity were achieved using a cut-off of 5 reads (96% (23/24) and 100% (1115/1120) respectively). typing mngs provides additional typing data, as compared to qpcr. of the 13 rhinoviruses detected with mngs, 6 (46.2%) were rhinovirus a, 2 (15.4%) rhinovirus b and 5 (38.5%) rhinovirus c. the three influenza viruses were assigned to be h3n2 strains by mngs. in order to analyse the semi-quantitative quality of the mngs assay, the number of the normalized sequence reads (log) mapping to qpcr target viruses (species level) as obtained with mngs were compared to the cq values of qpcr. a significant negative correlation was found (fig 3; pearson correlation coefficient ρ = -0.6, p = 0.002). the following markers were tested for potential associations with clinical severity of exacerbation (exacerbation severity, self-reported exacerbation severity), length of exacerbation and a decrease/increase in fev 1 (control visit compared to baseline): mngs pathogen positive versus negative exacerbation (qpcr targets), the number of normalized reads (log, cutoff of �5normalized reads) for the different target viruses (species level). no correlation was found between these markers and the different disease severity parameters (results not shown). overall proportions of normalized read counts of viral families (excluding eav and phhv control reads, cut-off of �5normalized reads) detected by mngs per patient are shown in fig 4. patients with viral pathogens (pcr target viruses) had significantly reduced proportions of bacteriophages when compared to patients without viral pathogen:0% and 79% bacteriophages respectively (p<0.001) bacteriophage reads vs. all viral reads, normalized reads excluding eav and phhv control reads. the shannon diversity scores for bacteriophages (normalized reads, cut-off of �5normalized reads) were comparable for copd exacerbations of viral aetiology in pcr positive versus negative patients (fig 5) . shannon diversity (normalized reads, excluding internal controls) was significant lower for all viral reads (p<0.001) and eukaryotic viruses (p = 0.028) in patients with viral pathogens (pcr target viruses positive). no significant association was found between the diversity scores, nor the percentage of bacteriophages, and the following parameters: disease severity, length of exacerbation, number of exacerbations during the study period, difference in fev 1 , gold stage, smoking, crp level, and the virus species (results not shown). the most prevalent phyla were proteobacteria, firmicutes actinobacteria and bacteroidetes, see fig 6. the normalized bacterial read count of the most prevalent phyla was not significantly different between patients with a pcr-target virus positive and pcr-target negative patients. pathogenic bacterial species detected with an abundance of >10% of the bacterial reads were: h. influenza (five samples); m. catarrhalis (20 samples); s. pneumoniae (one sample); and the respiratory virome in copd patients s. aureus (one sample). no apparent association with bacteriophages was found, or was a high abundance of bacteriophages associated with copd exacerbations of viral cause. the raw sequence data of the samples, after removal of human reads have been deposited to sequence read archive database (http://www.ncbi.nlm.nih.gov; accession number srx6713943-srx6714030). in this study, the respiratory virome in patients with copd exacerbations was analysed with both mngs and qpcr, and combined with clinical data. the incidence of viral pathogens was 26% with both mngs and qpcr. mngs failed to detect one rhinovirus with low load (cq 38) and pcr failed to detect one betacoronavirus oc43 (72644 reads), due to one of the limitations of pcr, i.e. inhibition of amplification. one additional viral pathogen, not present in the respiratory pcr panel, was detected: herpes simplex virus 1, found by others to be associated with copd [27] . the incidence of viral pathogens was comparable to that in previous publications (22-64%) [3, 5, 6] . the viral pathogen with the highest incidence was rhinovirus, followed by influenza, coronaviruses and para-influenza viruses. interestingly, subtyping data was readily available by mngs, accentuating the high resolution of mngs, with rhinovirus (rv) species a and c being most frequent, followed by rv-b. rv-c was first identified in 2006 and associated with high symptom burdens in children and asthmatics [28, 29] . recently, an asthma-related cadherin-related family member 3 (cdhr3) gene variant [30] was associated with greater rv-c receptor display on pulmonary cell surfaces of children and adults, and associated with higher susceptibility to severe virus-triggered asthma episodes [31, 32] . in line, romero-espinoza et al detected predominantly rv-c in children with acute asthma exacerbations by mngs [33] . the significance of rv-c infection in the adult population is less well studied. although rv-c has been previously associated with exacerbations of copd [34, 35] , to our knowledge, to date, cdhr3 polymorphisms have not yet found to be associated with copd. the sensitivity, specificity and positive and negative predictive values of mngs were high: 96%, 100%, 82% and 100%, respectively, when encountering a cut-off of �5normalized reads, with a detection limit of approximately cq 38. the high negative predictive value implicates the potential of mngs to exclude the most prevalent viral respiratory infections in one test. the potential to exclude any infectious cause, both viral and bacterial, would have significant consequences for starting and/or continuation of antimicrobial or, at the other end of the spectrum, immune-modulating treatment. the normalized viral species sequence read count might give an indication of the viral burden and the clinical relevancy of the detected virus. although in our dataset we could not find any correlation with disease severity, several paediatric studies demonstrated a correlation between virus load and severity in respiratory infections [36] [37] [38] [39] . further analysis with a larger number of infected patients and/or a different spectrum of exacerbation severity will be needed to demonstrate or exclude such an association in copd patients. furthermore, the complete respiratory virome showed a high bacteriophage abundance that could be linked to the absence of viral pathogens. lower bacteriophage abundance may be the result of viral expansion. hypothetically, a healthy virome size and diversity fits a certain size and diversity of bacteriophages, while during viral infection, pathogens predominate the virome. alternatively, others have hypothesized that viral and microbial diversity may play a role in infection susceptibility and the development of acute and chronic respiratory diseases [33] . our results indicate that virome dysbiosis may be accompanied by bacteriome dysbiosis, though no significant differences were detected in line with other reports [40, 41] . however, these studies don't compare between copd exacerbation with and without viral infections. others have found a higher phage abundance in a patient with severe copd when compared with one patients with moderate copd and healthy controls, dna sequencing, in line with the hypothesis of a state of dysbiosis that increases with disease progression [27] . in copd patients, viral infections have been suggested to trigger bacterial overgrowth and infections [42, 43] , demonstrating the significance of viral-bacterial interactions. moreover, hypothetically, bacteriophages play a role in the horizontal gene transfer of bacterial virulence factors. the most abundant bacterial phyla detected in this study were comparable with other reports. although the percentage of proteobacteria was relatively high when compared to other studies of the nasopharyngeal microbiome, our swabs are sampled during copd exacerbations [44, 45] . study of the lower airways by means of e.g. protected brushes during bronchoscopy is needed for further analysis of bacterial and viral (sub)populations including comparison with pcr and culture results. studies comparing the respiratory virome during stable disease and exacerbations are needed to determine a potential correlation between the virome/bacteriome during stable state and disease progression or exacerbation frequency. in the current study, most respiratory pathogens detected were rna viruses. this is in line with previous literature [3, 5, 6] . however, it must be noted that, despite the fact that a wide range of dna viruses have been detected with the current protocol (dna bacteriophages with high abundance, herpes simplex virus, bocavirus, anelloviruses, cmv, ki polyomavirus [14] ), we cannot exclude suboptimal detection of some dna viruses. furthermore, highly divergent viruses with sequences deviating from their representative ncbi refseq sequences may have been missed, as has been described by others [46] . however, bioinformatic classification using alternative databases (both genomedetective and local databases) did not result in additional findings. though mngs renders the possibility to detect all viruses in direct respiratory material, this revolutionary method is not yet used as routine accredited diagnostic procedure for pathogen detection. before mngs can be implemented as a routine diagnostics, the optimal protocol must be defined and analysis and interpretation of the metagenomic data must be standardized, followed by external quality assessment. this study demonstrates good performance of our mngs protocol, in line with other studies [37, 38, 47, 48] and seems to overcome some of the current thresholds for implementation in clinical diagnostics. the mngs protocol used was highly sensitive and specific for semi-quantitative detection of respiratory pathogenic viruses. excellent negative predictive value implicates the potential of mngs to exclude a known viral infectious cause in one test, with consequences for clinical decision making. reduced abundance of bacteriophages in copd patients with viral pathogens implicates skewing of the virome, and speculatively the bacterial population, during infection. management and prevention of exacerbations of copd copd exacerbations: defining their cause and prevention copd exacerbations. 2: aetiology the role of viral infections in exacerbations of chronic obstructive pulmonary disease and asthma. therapeutic advances in respiratory disease a newly discovered human pneumovirus isolated from young children with respiratory tract disease isolation of a novel coronavirus from a man with pneumonia in saudi arabia cloning of a human parvovirus by molecular screening of respiratory tract samples exploring the potential of next-generation sequencing in detection of respiratory viruses genomic characterization of a newly discovered coronavirus associated with acute respiratory distress syndrome in humans unbiased parallel detection of viral pathogens in clinical samples by use of a metagenomic approach metagenomic sequencing complements routine diagnostics in identifying viral pathogens in lung transplant recipients with unknown etiology of respiratory infection. plos one metagenomic sequencing for combined detection of rna and dna viruses in respiratory samples from paediatric patients. biorxiv lung microbiome dynamics in copd exacerbations metagenomic analysis of viral genetic diversity in respiratory samples from children with severe acute respiratory infection in china characterization of the viral microbiome in patients with severe lower respiratory tract infections, using metagenomic sequencing systemic inflammatory markers in copd: results from the bergen copd cohort study performance of different mono-and multiplex nucleic acid amplification tests on a multipathogen external quality assessment panel metagenomic sequencing for combined detection of rna and dna viruses in respiratory samples from paediatric patients centrifuge: rapid and sensitive classification of metagenomic sequences genome detective: an automated system for virus identification from high-throughput sequencing data spades: a new genome assembly algorithm and its applications to single-cell sequencing basic local alignment search tool fast and accurate short read alignment with burrows-wheeler transform understanding diagnostic tests 3: receiver operating characteristic curves the human virome protein cluster database (hvpc): a human viral metagenomic database for diversity and function annotation frequent detection of human rhinoviruses, paramyxoviruses, coronaviruses, and bocavirus during acute respiratory tract infections masstag polymerase-chain-reaction detection of respiratory pathogens, including a new rhinovirus genotype, that caused influenza-like ill a genome-wide association study identifies cdhr3 as a susceptibility locus for early childhood asthma with severe exacerbations cadherin-related family member 3, a childhood asthma susceptibility gene product, mediates rhinovirus c binding and replication genetic association of the functional cdhr3 genotype with early-onset adult asthma in japanese populations unbiased detection of respiratory viruses by use of rna sequencing-based metagenomics: a systematic comparison to a commercial pcr panel we thank our generade project partners floyd wittink, wouter suring (hogeschool leiden), danny duijsings (baseclear) and christiaan henkel (leiden university). we also like to thank tom vreeswijk, lopje höcker and mario van bussel (kml, lumc) for help with the pre-sequencing experiments and jeroen laros (human genetics, lumc) for help with the bioinformatics. we thank caroline brouwer for technical assistance (kml, lumc). key: cord-342993-deuytbml authors: maffey, alberto f.; barrero, paola r.; venialgo, carolina; fernández, francisco; fuse, valentina a.; saia, mariana; villalba, analía; rodríguez fermepin, marcelo; teper, alejandro m.; mistchenko, alicia s. title: viruses and atypical bacteria associated with asthma exacerbations in hospitalized children date: 2010-05-06 journal: pediatr pulmonol doi: 10.1002/ppul.21236 sha: doc_id: 342993 cord_uid: deuytbml objectives and working hypothesis: to evaluate the prevalence of respiratory viruses mycoplasma pneumoniae and chlamydophila pneumoniae and gain insight into their seasonal circulation pattern in children with acute asthma exacerbations in a temperate southern hemisphere region. study design: patients hospitalized between 3 months and 16 years of age were included in a 1‐year prospective, observational, cross‐sectional study. respiratory secretions were collected and the presence of different viruses and atypical bacteria analyzed by immunofluorescence and polymerase chain reaction. results: two hundred nine patients (118 females) aged (mean ± sd) 4.4 ± 4 years were included. a potential causative agent was detected in 78% of the patients. the most frequently detected viruses were respiratory syncytial virus (hrsv) (n = 85; 40%) and rhinovirus (hrv) (n = 52; 24.5%); m. pneumoniae and c. pneumoniae were detected in 4.5% and 2% of the cases, respectively. patients with hrsv (vs. hrv) were hospitalized for a longer time (6.7 vs. 5.2 days, p = 0.012), required more days of oxygen supply (5.1 vs. 3.4, p = 0.005), had a longer duration of the exacerbation before hospitalization (3.6 vs. 1.9 days, p = 0.001) and were younger (3.7 vs. 5.1 years, p = 0.012). three peaks of admissions were observed. a first peak (early autumn) caused by hrv, a second peak (winter) caused mainly by hrsv and a third one (spring), caused by hrsv, an increase in hmpv together with a second outbreak of hrv. conclusions: children with an acute asthma exacerbation presented a high prevalence of respiratory viruses. most hospitalizations corresponded to seasonal increases in prevalence of hrv and hrsv. pediatr pulmonol. 2010; 45:619–625. © 2010 wiley‐liss, inc. asthma is the most-frequent pediatric chronic disease, and its prevalence is increasing in numerous regions of the world. 1 the international study of asthma and allergies in childhood (isaac) has reported that in argentina the current prevalence of asthma is 16.4% in children aged 6 years old and 10.9% in children aged 13. 2 respiratory exacerbations are the main source of morbidity, mortality, school absence, and health expenses associated with asthma. infections associated with respiratory viruses and atypical bacteria are the main cause of asthma exacerbation. about 85-90% of the wheezing episodes in infants and 65-70% in children and adolescents are triggered by respiratory viruses. 3 various viral agents, including the respiratory syncytial virus (hrsv), influenza a and b (fluav and flubv), parainfluenza (hpiv), adenovirus (hadv), rhinovirus (hrv), enterovirus (hev), coronavirus (hcov), and the recently described metapneumovirus (hmpv) 4 and bocavirus (hbov) 5 have been detected in patients presenting with asthma exacerbation. while hrsv is the main agent associated with wheezing in infants and preschool children hrv is the most-frequent agent 1 detected in school children, adolescents, and adults. 3 infections caused by atypical bacteria (mycoplasma pneumoniae and chlamydophila pneumoniae) have been associated not only with asthmatic exacerbations but also with the development of chronic infections that may contribute to the persistence and severity of asthma. 6, 7 numerous adequately treated asthmatic patients continue to experience wheezing episodes, so studies are necessary to understand the relationship between the inflammatory process triggered by various infectious agents and the events that lead to the loss of asthma control. 8, 9 this knowledge will assist the development of prevention and treatment strategies to help decrease the morbidity caused by bronchial asthma. [10] [11] we performed a cross-sectional study to determine by means of immunofluorescence assay (ifa) and molecular techniques the prevalence of traditional and newly described respiratory viruses and atypical bacteria in children hospitalized due to an acute asthma exacerbation in buenos aires, a temperate southern hemisphere city. the study was performed at the ricardo gutiérrez children's hospital in buenos aires between january 1 and december 31, 2006. the study protocol was approved by the institutional review board of the hospital. legally authorized representatives of the children provided informed consent. patients included in the study were between 3 months and 16 years of age, with a history of two or more previous wheezing episodes diagnosed by a physician and presenting with a new episode severe enough to require hospitalization. forty-two (20%) patients were under 12 months, an age group where it is difficult to establish a definite diagnosis of asthma. because of the difficulty in differentiation of asthma from wheezing episodes of other origin in very young children, 12 to be included in the study patients under 3 years of age needed to have an index for the prediction of asthma according to the criteria of castro-rodríguez et al. 13 children over three needed to comply with the diagnostic criteria of bronchial asthma according to the gina guidelines. 14 the exclusion criteria were premature birth and the presence of a chronic disease of the pulmonary or cardiovascular system, metabolic disorders, immunosupression, genetic or neurological disorders. we recorded number of weeks of gestation and weight at birth, familial history of asthma, allergic rhinitis or atopic eczema in first degree relatives, passive smoking, medications used to achieve asthma control, age at first wheezing episode, and hospitalizations in the last 12 months by interviewing the parents and reviewing medical records. during hospitalization, data on clinical evolution (days of hospitalization and oxygen requirement), treatment required (bronchodilators, systemic corticosteroids, antibiotics, mechanical respiratory assistance), and complications presented (pneumonia and atelectasis) were recorded. the interval of time (days) between the onset of the respiratory exacerbation and admission and time between admission and the collection of the respiratory specimen were also recorded. the results of the characterization of the 12 respiratory viruses studied (hrsv, hadv, fluav and flubv, hpiv-1, 2, and 3, hmpv, hrv, hev, hcov, hbov) as well as of the atypical bacteria m. pneumoniae and c. pneumoniae were registered. due to the wide range of age of the patients included, for statistical analysis of the different variables the patients were arbitrarily divided into three groups: children under 1 year of age, children between 1 and 4 years of age, and children between 4 and 16 years of age. a nasopharyngeal aspirate (bsn 30 1 , laboratorio barcat, buenos aires, argentina) was obtained from each patient on admission. on 12 patients in whom respiratory secretions could not be collected by this method, a nasal swab (transport swab 1 , copan, brescia, italy) was obtained. the sample was kept at 48c until submission to the laboratory. the respiratory sample was processed the day it was collected by the ifa to detect hrsv, hadv, hpiv-1, 2, and 3, fluav, and flubv (light diagnostics, chemicon int., temecula, ca). a total of 500,000 cells per ml were considered adequate for ifa detection. the secretions were kept at à208c for further studies with polymerase chain reaction (pcr) and reverse transcription pcr (gotaq 1 , promega, madison, wi; onestep rt-pcr, qiagen, valencia, ca). the viruses (hrsv, fluav, flubv, hpiv, hadv, hrv, hev, hcov, hmpv, and hbov) and atypical bacteria (m. pneumoniae and c. pneumoniae) were determined in separate reactions according to the protocols previously described. 15 a positive case was defined as that in which at least one infectious agent was detected. descriptive statistics were performed using epi 2000. for comparison of groups data were imported into r statistical programming language environment, version 2.6.2 (available from http://www.r-project.org). variables examined to compare patients with single agents versus co-detections and between hrsv and hrv single detections were age, gender, passive smoking, familial history of asthma or atopy, duration of exacerbation before hospitalization, days on oxygen supply, length of hospitalization and complications (atelectasis and pneumonia). chi-square and fisher's exact tests were used for comparison of categorical variables. for numerical variables we used student's t-test or wilcoxon test when it was not possible to assume a normal distribution. the values are expressed as mean ae standard deviation. the probability level to determine statistical significance was 0.05. a total of 217 patients that fulfilled the inclusion criteria were admitted during the study period. five patients, all less than 2 years of age, were excluded once studies were initiated to rule out differential diagnosis of asthma. two patients declined to participate, and parents of another patient were not present to give the informed consent. a total of 42 (20%) out of the 209 patients studied were under 1 year of age, 77 (37%) between 1 and 4 years of age, and 90 (43%) between 4 and 16 years of age. table 1 shows the demographic and clinical characteristics and complications present in the population studied. additional information on the first wheezing episode was obtained on 205 patients: 181 (88%) had their first wheezing episode in the first 3 years of life and 62% of these wheezed in their first year. the interval of time between the onset of the respiratory exacerbation and admission was found to be (mean ae sd): 2.9 ae 2.2 days, between the onset of the respiratory exacerbation and the collection of the respiratory specimen 4.4 ae 2.4 days, and between admission and the specimen collection 1.5 ae 1.1 days. a total of 69 (33%) patients had been prescribed an asthma controller therapy, but only 37 (18%) were complying with it adequately: 6 (14%) of the 42 children under 1 year of age, 7 (9%) of the 77 children between 1 and 4 years of age, and 24 (27%) of the 90 children between 4 and 16 years of age. sixteen (44%) of those who were complying with therapy received budesonide, 13 (35%) fluticasone, 6 (16%) fluticasone plus salmeterol, and 2 (5%) montelukast. in the physical exam carried out at admission, 199 (95%) of the patients presented cough, 177 (85%) rhinorrhea, 128 (61%) fever !388c, 57 (28%) pharyngitis, and 24 (11%) conjunctivitis. in addition, 22 (10.5%) were diagnosed with acute otitis media. overall, 197 (94%) were found to have an upper respiratory infection. during hospitalization, all the patients received oxygen treatment through a nasal cannula or a facemask, shortacting bronchodilators (nebulized albuterol) and systemic corticosteroids (hydrocortisone, dexametasone, or methyl-prednisolone); but none of them required mechanical ventilation. there were no significant differences when evaluating the presence of complications (atelectasis and pneumonia) when an infectious agent was detected or when codetections occurred (p ¼ 0.37 and p ¼ 0.75, respectively). samples of respiratory secretions were obtained through a nasopharyngeal aspirate from 197 (94%) patients. in 12 (6%) patients respiratory secretions could not be obtained by this method, so a nasal swab was pediatric pulmonology table 2) . patients with co-detections were significantly younger than those without co-detections (3.1 vs. 4.8 years of age; p ¼ 0.008). no hpiv-2 was detected. the four cases of hcov and the only case of flubv were present in co-detections. the importance of the two more-prevalent respiratory viruses (hrsv and hrv) as single detection, as well as that of the viral co-detections, was analyzed related to the main variables studied. we found that the patients with hrsv were hospitalized later in the course of their disease (3.6 vs. 1.9 days, p ¼ 0.001), experienced a longer hospitalization (6.7 vs. 5.2 days, p ¼ 0.012), required more days of oxygen supplementation ( the number of hospitalizations varied seasonally and was associated with the circulation of the more frequently isolated viruses (fig. 2) . in the present study, a potential causative agent of an acute asthma exacerbation was identified in 78% of the patients studied. this rate of detection is in agreement with those reported in the literature, which vary between 65% and 90% of positive cases according to the age of the population studied, the number of infectious agents evaluated, and the diagnosis techniques used. 16, 17 the pcr technique allowed the detection of hrv, hev, hmpv, hbov, hcov, m. pneumoniae, and c. pneumoniae, and also contributed to the identification of cases that had not been detected by ifa. however, it should be pointed out that while the ifa technique detects viral antigens in patients that present an active viral infection, the higher sensitivity of the pcr technique allows the detection of nucleic acids that may correspond either to a current infection or to a past infection. 18 in effect, nokso-koivisto et al. 19 described the presence of positive picornavirus rna in respiratory samples obtained from children without concurrent respiratory symptoms. in our study, 94% of the patients had an active upper respiratory infection at the time of hospital admission, thereby lowering the risk of diagnosing false positive cases. the most frequently detected agent was hrsv, in both the group of children between 1 and 4 years of age and the group of children between 4 and 16 years of age. this finding supports previous observations that hrsv can cause acute exacerbations of chronic diseases at any age. 20 community-based studies in school children 21 have found that hrv is the most prevalent virus, responsible for up to 70% of the asthma exacerbations. in our study, we detected hrv in 25% of the patients. this difference could be that our patients were not only hospitalized, but also younger. some concerns could be regarded about the sample size that could lack sufficient statistical power, specially in smaller subgroups of analysis. however, several differences between groups reached statistical significance. patients with exacerbations caused by hrv had a significantly shorter number of days from the beginning of the respiratory symptoms to hospital admission, and also shorter hospitalization and less oxygen requirement days than those with exacerbations caused by hrsv. although the children affected by hrv were older and thus could be expected to have a wider airway caliber, this difference may be because the exacerbations triggered by hrv show a better response to treatment than those triggered by other respiratory viruses due to different pathogenic mechanisms of airway obstruction. 22 thus, it was not possible to determine if the difference in clinical outcome observed might be related to the age of the patients' group or to the different pathogenic mechanisms of hrv and hrsv. in our cohorting, other common respiratory viruses, such as fluv, hbov, hcov, hmpv, and hpiv, exhibited a much lower prevalence than hrsv and hrv. it is well known that fluv triggers asthma exacerbations less frequently than other respiratory viruses. 23 due to its frequent presence in co-detection and its long period of excretion after an active infection, the role of hbov as a causative agent of asthmatic exacerbations requires further studies. the detection of hmpv, hcov, hpiv, and hadv, while higher in infants and school children, was significantly lower than that of hrsv and hrv. 24 in asthmatic hospitalized children, studies have reported prevalence of 20% for m. pneumoniae and 5-15% for c. pneumoniae, 25 which is higher than that found by our group (4.5% for m. pneumoniae and 2% for c. pneumoniae). this difference may be due to a lower circulation of these agents during the year in which the study was performed or to a lower detection rate, as we did not use serological methods. another possible explanation could be the preponderance of young children sampled, as m. pneumoniae is usually less frequent under 4 years of age than in school age children. therefore, treatment with macrolides is not justified in all patients presenting an asthma exacerbation but should be considered in children presenting persistent symptoms that cannot be controlled with conventional treatment. 26 in almost a quarter of patients (22%) we found evidence of a co-detection between respiratory viruses and/or atypical bacteria. we did not find significant differences when analyzing the occurrence of co-detections between the different agents involved and the hospitalization or pediatric pulmonology oxygen requirement days and the development of complications. 27, 28 however, the presence of such a high index of co-detection points to the need to revise the concept of ''cohortization'' or grouping patients in hospital rooms. co-detections in which any of the viral agents recently described is involved can favor the development of nosocomial infections. 29 although previously described in other regions of the world, to our best knowledge, this is the first report of respiratory viruses and atypical bacteria associated to wheezing in children described in the southern cone of america. in the near future, when antiviral therapies become more readily available, to know the regional patterns of circulation of viral agents might be useful to treat specific viral respiratory infections, helping to prevent asthmatic exacerbations. [30] [31] in conclusion, the present study confirms the high prevalence of respiratory viruses in hospitalized children with an acute asthma exacerbation, highlights the importance both of hrsv and hrv in all age groups, and describes their seasonal pattern in a temperate southern hemisphere location. the asthma epidemic prevalence of asthma symptoms in latin america: the international study of asthma and allergies in childhood (isaac) respiratory picornaviruses and respiratory syncytial virus as causative agents of acute expiratory wheezing in children human metapneumovirus and acute wheezing in children human bocavirus and acute wheezing in children increased frequency of detection of chlamydophila pneumoniae in asthma detection of viral, chlamydia pneumoniae and mycoplasma pneumoniae infections in exacerbations of asthma in children new treatment regimes for virus-induced exacerbations of asthma viruses and asthma, inception, exacerbation, and possible prevention the role of viral infections in the natural history of asthma early identification of atopy in the prediction of persistent asthma in children definition, assessment and treatment of wheezing disorders in preschool children: an evidence-based approach a clinical index to define risk of asthma in young children with recurrent wheezing global strategy for asthma management and prevention: gina executive summary new respiratory viruses in children 2 months to 3 years old with recurrent wheeze prevalence of viral respiratory tract infections in children with asthma role of viruses and atypical bacteria in exacerbations of asthma in hospitalized children: a prospective study in the nord-pas de calais region (france) comparison of real-time pcr assays with fluorescentantibody for diagnosis of respiratory virus infections in children human picornavirus and coronavirus rna in nasopharynx of children without concurrent respiratory symptoms respiratory syncytial virus infection in elderly and high-risk adults community study of role of viral infections in exacerbations of asthma in 9-11 year old children prednisolone reduces recurrent wheezing alter a first wheezing episode associated with rhinovirus infection or eczema influenza virus and acute asthma in children respiratory picornaviruses and respiratory syncytial virus as causative agents of acute expiratory wheezing in children importance of acute mycoplasma pneumoniae and chlamydia pneumoniae infections in children with wheezing the effect of telithromycin in acute exacerbations of asthma impact of human metapneumovirus and respiratory syncytial virus co-infection in severe bronchiolitis absence of human metapneumovirus co-infection in cases of severe respiratory syncytial virus infection a comparison of nested polymerase chain reaction and immunofluorescence for the diagnosis of respiratory infections in children with bronchiolitis and the implications for a cohorting strategy respiratory viruses seasonality in children under five years of age in buenos aires, argentina. a five-years analisis managing childhood asthma: challenge of preventing exacerbations the authors thank dr. a. sonia buist and the staff of the ats methods in epidemiologic, clinical and operations research (mecor) for encouraging our research, and very specially dr. shannon carson for kindly correcting the manuscript. also, we would like to thank mrs. patricia riveiro, bioq. m. angeles marques, and bioq. andrea c. entrocassi for their skillful assistance in diagnostic procedures. we are indebted to dr. van der hoek for kindly providing the positive control to detect coronavirus. key: cord-336743-udokbcki authors: lilitsis, emmanouil; stamatopoulou, vaia; andrianakis, eleftherios; petraki, adamantia; antonogiannaki, elvira-markela; georgopoulos, dimitrios; vaporidi, katerina; kondili, eumorfia title: inspiratory effort and breathing pattern change in response to varying the assist level: a physiological study date: 2020-06-10 journal: respir physiol neurobiol doi: 10.1016/j.resp.2020.103474 sha: doc_id: 336743 cord_uid: udokbcki aim: to describe the response of breathing pattern and inspiratory effort upon changes in assist level and to assesss if changes in respiratory rate may indicate changes in respiratory muscle effort. methods: prospective study of 82 patients ventilated on proportional assist ventilation (pav+). at three levels of assist (20%-50%-80%), patients’ inspiratory effort and breathing pattern were evaluated using a validated prototype monitor. results: independent of the assist level, a wide range of respiratory rates (16-35br/min) was observed when patients’ effort was within the accepted range. changing the assist level resulted in paired changes in inspiratory effort and rate of the same tendency (increase or decrease) in all but four patients. increasing the level in assist resulted in a 31% (8-44%) decrease in inspiratory effort and a 10% (0-18%) decrease in respiratory rate. the change in respiratory rate upon the change in assist correlated modestly with the change in the effort (r = 0.5). conclusion: changing assist level results in changes in both respiratory rate and effort in the same direction, with change in effort being greater than that of respiratory rate. yet, neither the magnitude of respiratory rate change nor the resulting absolute value may reliably predict the level of effort after a change in assist. assisted mechanical ventilation aims to assure adequate ventilation by assisting and partially unloading the respiratory muscles (pierson, 2002) . ideally, the level of assist should not only result in normal or near-normal arterial blood gases but also to normal or near-normal levels of inspiratory effort, to prevent both over-and under-assistance induced injury of the diaphragm . setting the level of ventilator assist in everyday practice relies mostly on the clinical estimation of inspiratory effort, as indicated by the breathing pattern -tidal volume (vt) and respiratory rate (rr)-and clinical signs of respiratory distress (boles et al., 2007; hansen-flaschen, 2000; hess, 2001; ray et al., 2006) . however, the relationship between ventilatory assist and inspiratory effort, tidal volume, and respiratory rate is complicated and multifactorial, affected by the patient's respiratory drive, ability to generate alveolar ventilation, and the mode of support (vaporidi et al., 2020) . therefore, although tidal volume and respiratory rate are commonly used in everyday practice to titrate the level of assist, the scientific evidence guiding assist titration to the patient effort is limited, derived from studies with a relatively small number of patients (berger et al., 1996; j o u r n a l p r e -p r o o f 4] carteaux et al., 2016; giannouli et al., 1999; marantz et al., 1996) and do not consider the random/normal variability in breathing pattern, unrelated to the level of assist. the aim of this study was to 1) characterize the responses of respiratory drive, respiratory effort, and breathing pattern to changing levels of ventilatory assist in critically ill patients and 2) assess if changes in respiratory rate may indicate changes in respiratory drive and effort. to this end, during proportional assist ventilation with adjustable gain factors (pav+), noninvasive measurements of respiratory drive, effort (as indicated by inspiratory muscle pressure) were obtained at different levels of assist, using a validated prototype monitor (pvi) (kondili et al., 2010; younes et al., 2007) . contrary to conventional modes of assisted mechanical ventilation, pav+ permits the patients to select their desired breathing pattern as determined by feedback mechanisms of control of breathing (younes, 1992) . this study was conducted in the medical-surgical icu of the university hospital of heraklion between january 2011 and july 2012 after approval by the hospital ethics committee (11118). since the study protocol does not involve a therapeutic intervention and clinical or diagnostic interventions, it was considered as carrying no more than minimal risks, and informed consent was waived. patients were included in the study after at least 48 hours of mechanical ventilation, when they fulfilled standard criteria for initiation of weaning from controlled mechanical ventilation (hemodynamic stability, reversal of cause of respiratory failure). exclusion criteria were: age under 18 years, pregnancy, severe muscle weakness or cervical spine injury, uncontrolled pain, fever, or delirium, readiness for spontaneous breathing j o u r n a l p r e -p r o o f 5] trial, and when pvi monitor was unavailable. the study was interrupted if the patient could not trigger the ventilator, developed respiratory distress, or became for any reason unstable (increase in vasopressor requirements, deterioration of oxygenation, etc.). at the time of the study, all patients were ventilated on assisted modes (pressure support or pav+). all patients were studied on pav+ at three different levels of assist 20%, 50%, and 80% at the peep set by the treating physician. each level of assist, applied randomly, was maintained for 10 min. moreover, recordings were obtained for 10 minutes at the mode selected by the treating physician (ps or pav+) before and immediately after pav+. arterial blood gases were obtained during the last minute of low (20%) and high (80%) level of assist. the pressure generated by the inspiratory muscles per breath (pmus) was evaluated as an index of effort and calculated on a breath-by-breath basis by a research prototype monitor (pvi monitor, yrt limited, winnipeg, canada) using a method described in detail previously. briefly, the inputs required by the monitor to calculate pmus are the airway pressure (paw) and flow (v′), whereas volume (v) was obtained by v′ integration. at least two points during expiration that satisfied passive conditions (i.e., flow was driven by the elastic recoil pressure) were automatically identified by the monitor. at these points, the equation of motion was applied, and elastance (erspvi) and resistance (rrspvi) of the respiratory system were calculated. using these values and the equation of motion, pmus was calculated in each breath. provide non-invasively measurement of pmus, as well as of tidal volume and respiratory rate in breath by breath basis (kondili et al., 2010; younes et al., 2007) . patient mechanical inspiratory time (ti) was measured as the interval between the beginning of pmus increase and the point at which pmus started to decline rapidly. patient mechanical expiratory time was measured as the remainder of the respiratory cycle. the rate of rise of pmus (dp/dt,) was calculated as the change in pmus during inspiration divided by ti and was used as an index of respiratory drive. the level of peepi during the different levels of assist was measured as the positive deflection of pmus from the onset of mechanical inspiration to the point of zero flow. additionally, the following data were collected: severity score on admission, etiology of respiratory failure, days on mechanical ventilation, icu, and hospital length of stay and outcome. the output of pvi monitor data was processed before analysis to optimize data quality (e.g., artifact rejection). from each 10-min period, the 7th to 10th min of the recording was selected for analysis. in each patient, for each variable and a given level of assist, the distribution of values was evaluated. means and sd were calculated after examining for normal distribution. the calculated means in each variable were used for comparisons among patients. between groups of patients or levels of assist, differences in continuous variables were as determined by the study design, at the end of the study period (30 min), patients were placed again on the same ventilator settings used by the treating physician. thus, in each patient, recordings were available with the same ventilator settings at approximately 30 min apart, while the patient's condition was considered stable (pre-and post-study). an analysis of ventilation variables in the pre-and post-study recordings was used to determine the variability (range of difference) of each variable in the 'stable' critically ill patient. we used ibm spss statistics for windows version 25 (armonk, ny) for analysis. during 18 months, 82 critically ill patients were included in the study. the patients' characteristics are presented in table 1 . patients were studied on the 8±6 day of mechanical ventilation; the baseline measurements from the ventilator and the pvi monitor are presented in table 2 . in the analysis of single patient data (3-min breath by breath analysis), and for all pvi variables, a normal distribution was found in more than 90% of cases, and thus, the mean values were used for comparison among patients. results were qualitatively similar when median values were used (data not shown). ventilatory variables in the pre-and post-study period were not different (table 2) . this was true when the two ventilation modes (ps, pav+) were examined independently (data not shown). arterial blood gases were also not different before and after the study. the variability in tidal volume and respiratory rate, indicated by the coefficient of variation (tobin et al., 1988) , was 9% and 13%, respectively. the 5-95 range of change in each variable was calculated as an indicator of the variability in breathing pattern that can be expected in a relatively stable critically ill patient when ventilation settings have not been modified (table 2) . we subsequently analyzed, independently of the level of assist, the correlation between indices of respiratory drive (dp/dt), effort (pmuspeak), and breathing pattern (tidal volume -vt and respiratory rate -rr). a very strong linear correlation was observed between respiratory drive and pmuspeak at each level of assist (r=0.9). respiratory drive correlated weakly with respiratory rate (r= 0.4), but not with tidal volume. when respiratory drive and pmuspeak were within the accepted 'target' range for mechanically ventilated patients , a broad range of respiratory rate (r5-95: 16-35 br/min) was observed (figure 1 ). finally, we examined the patterns of change of respiratory drive, pmuspeak, rr, and vt in response to changes in the level of assist. with increasing assist, small but statistically significant changes were observed in all variables (table 3 ). the change in effort, in response to change in assist, was greater than the change in respiratory rate ( figure 2 ). respiratory drive decreased as assist increased, and this change correlated strongly with the change in the effort (r=0.8), but moderately with the change in respiratory rate (r=0.5), and not with the change in tidal volume. the patterns of response to assist increase were characterized by a concomitant increase in tidal volume and decrease in respiratory rate, yet, these changes were mostly within the expected range of variation, and not specific for the change in effort (figure 3) 4). a significant change in respiratory rate, opposite to that of effort, was observed only in four patients (5%). the sensitivity and specificity of a decrease in respiratory rate to predict a decrease in effort was 88% and 71%, respectively, with a positive predictive value of 90%. yet, the change in respiratory rate correlated only modestly with the change in effort (r=0.5) and the resulting inspiratory effort at high assist (r=0.4, figure 4 ). in some patients (n=16, 20%), effort did not decrease with increasing assist. the development of intrinsic peep due to increase in tidal volume (12 cases) and the presence of high respiratory drive due to metabolic acidosis (7 cases) were the most common causes of this phenomenon. this study in critically ill patients in the post-acute phase examined the changes in breathing pattern, respiratory drive and effort and their correlations, induced by changes of the level of assist. patients were studied using pav+, a mode that permits patients to choose their breathing pattern and monitored with pvi, a prototype monitor validated to estimate inspiratory muscle pressure (kondili et al., 2010; younes, 1992; younes et al., 2007) . the main findings of the study are: 1) patients respond to changes in ventilatory assist mainly by changing effort per breath; 2) when respiratory drive and/or effort are normal, a wide range of respiratory rate is observed; 3) although respiratory rate changes towards the same direction as effort, neither the magnitude of change, nor the resulting value of respiratory rate are related to the level of effort, suggesting the limited role of respiratory rate in titrating assist to a target level of effort. in this study the correlation of indices of respiratory drive (dp/dt) and effort (pmus) was, as expected, very strong (de vries et al., 2018; mauri et al., 2016; vaporidi et al., 2020) . respiratory rate correlated weakly with respiratory drive, and most importantly, a wide range of breathing frequencies was observed in patients with relatively normal respiratory drive and effort. tachypnea was present in several patients with normal respiratory drive and pmus, and, although no patient exhibited hypoxemia, hyperactive delirium or fever at the time of the study, other causes of tachypnea, such as systemic inflammation, receptor stimulation or cortical stimulation could not be excluded (telias et al., 2018; vaporidi et al., 2020) . in addition, in our study, respiratory drive was estimated using the rate of increase of pmus, which in the presence of neuromuscular weakness underestimates the actual respiratory drive (the rate of increase of electrical activity of the respiratory center output). therefore, at the presence of neuromuscular weakness, respiratory center activity during inspiration may be high causing an increase in respiratory rate despite normal inspiratory pressure during inspiration. this observation is in line with several studies showing a higher than normal respiratory rate in critically ill patients (akoumianaki et al., 2019; giannouli et al., 1999; marantz et al., 1996) . moreover, a low respiratory rate was not uncommon, despite a high respiratory drive and effort, highlighting the inadequacy of respiratory rate as an indicator of respiratory drive and effort. this is not an unexpected finding as the respiratory rate in critically ill patients may be affected by several factors, most commonly sedation and opioid analgesia, which were also present in these patients (grap et al., 2012; mcgrane and pandharipande, 2012; vaporidi et al., 2020) . the response of critically ill patients to changes in assist was characterized overall by a decrease in respiratory drive, effort and respiratory rate, and an increase in tidal volume. patients changed their effort more often and to a greater extent than their respiratory rate upon change of assist and drive. this pattern of response is the same as the one observed in healthy subjects (duffin et al., 2000) . in our study in most patients, these changes of rr and vt were within the range of variability observed without a change in ventilator settings. moreover, this variability, for both rate and tidal volume, was found higher in the critically ill than in healthy individuals (tobin et al., 1988) . in mechanically ventilated patients a decrease in the respiratory rate in response to the increasing level of assist is usually considered as indicative of respiratory muscle unloading (esteban et al., 1997; esteban et al., 1999; sellares et al., 2012; yang and tobin, 1991) . this study identified that, indeed, a decrease in respiratory rate has a positive predictive value of 90% to predict a decrease in the inspiratory effort. however, this study also identified that it is only the direction of change and not the magnitude of the effect that can be predicted. thus, these results indicate that, although a change in effort in response to a change in assist can be predicted by the change in respiratory rate with reasonable accuracy, neither the absolute value of respiratory rate nor its change can indicate if the resulting effort is low, normal or high. some methodological issues and limitations of this study should be considered in the interpretation of results. first, the changes in breathing pattern were studied while the patients were ventilated only in pav+ mode. therefore, different changes in breathing pattern in response to changes in ventilator assist may be present during ventilation on psv. yet, a previous study has shown similar changes in patients' neural respiratory rate in response to varying the assist level between pav and psv (giannouli et al., 1999) . second, the estimation of inspiratory effort and respiratory drive was performed using the measurement of pmuspeak, instead of the gold standard method of trans-diaphragmatic pressure (pdi). however, we have previously shown that pmuspeak derived by pvi monitor provides an accurate estimation of inspiratory effort (kondili et al, 2010) . at different levels of assist during ventilation on pav+, pmuspeak was compared with transdiaphragmatic pressure (pdi), and the pressure developed by all respiratory muscles (pmus) -calculated with the campbell diagram and using chest wall mechanics measured at active and passive ventilation. no significant difference between pmuspeak, and pdi and pmus, in terms of timing, magnitude, shape, and the rate of pressure increase. setting the level of ventilator assist aims in adequate unloading of the respiratory muscles. in everyday clinical practice, the titration of assist usually aims to achieve a tidal volume of 6-8 ml/kg and a respiratory rate of 15-25 breaths/min. moreover, automated systems have been introduced to facilitate titration of assist, relying on measurements of tidal volume and respiratory rate. it is widely believed that high breathing rate may be indicative of excessive work of breathing, and that a decrease in respiratory rate with increasing the level of assist indicates unloading of respiratory muscles and thus adequate assist level. however, it has been shown that frequently tachypnea may be due to factors unrelated to respiratory load. the results of this study emphasize that respiratory rate is not a sensitive indicator of the patient's effort and that neither the magnitude nor the absolute change in the respiratory rate in response to varying the level of assist reflects the changes in inspiratory effort accurately. it is, therefore important, in clinical practice to avoid oversimplifications and assumptions such as that a relatively normal respiratory rate assures a normal level of inspiratory effort. during the j o u r n a l p r e -p r o o f 13] recent covid-19 epidemic, it has been reported that patients with severe disease 'surpisingly' did not develop tachypnea and respiratory distress, an observation which is in line with the findings of this study, and not at all unexpected. in conclusion, the present study, in mechanically ventilated patients on the recovery phase of acute illness, showed that changing the assist level results in changes of inspiratory effort in more patients and to a greater magnitude, than of respiratory rate, though both effort and rate change almost always in the same tendency (either increase or decrease). yet, neither the magnitude of change nor the absolute value of respiratory rate can be used to quantitatively estimate the resulting respiratory effort after a change in the level of assist. the injurious effects of elevated or nonelevated respiratory rate during mechanical ventilation mechanism of relief of tachypnea during pressure support ventilation weaning from mechanical ventilation comparison between neurally adjusted ventilatory assist and pressure support ventilation levels in terms of respiratory effort assessing breathing effort in mechanical ventilation: physiology and clinical implications a model of the chemoreflex control of breathing in humans: model parameters measurement extubation outcome after spontaneous breathing trials with t-tube or pressure support ventilation. the spanish lung failure collaborative group effect of spontaneous breathing trial duration on outcome of attempts to discontinue mechanical ventilation. spanish lung failure collaborative group response of ventilator-dependent patients to different levels of pressure support and proportional assist sedation in adults receiving mechanical ventilation: physiological and comfort outcomes dyspnea in the ventilated patient: a call for patient-centered mechanical ventilation ventilator modes used in weaning estimation of inspiratory muscle pressure in critically ill patients response of ventilator-dependent patients to different levels of proportional assist oxygenation patients recovering from severe acute respiratory distress syndrome sedation in the intensive care unit indications for mechanical ventilation in adults with acute respiratory failure acute respiratory failure in the elderly: etiology, emergency diagnosis and prognosis diaphragm-protective mechanical ventilation lung-and diaphragm-protective ventilation in acute respiratory distress syndrome: rationale and challenges predictors of weaning after acute respiratory failure is my patient's respiratory drive (too) high? variability of resting respiratory drive and timing in healthy subjects respiratory drive in critically ill patients. pathophysiology and clinical implications a prospective study of indexes predicting the outcome of trials of weaning from mechanical ventilation proportional assist ventilation, a new approach to ventilatory support. theory a method for monitoring and improving patient: ventilator interaction the authors declare that there is no conflict of interest. this research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. key: cord-320950-x02zp349 authors: esposito, susanna; mencacci, antonella; cenci, elio; camilloni, barbara; silvestri, ettore; principi, nicola title: multiplex platforms for the identification of respiratory pathogens: are they useful in pediatric clinical practice? date: 2019-06-04 journal: front cell infect microbiol doi: 10.3389/fcimb.2019.00196 sha: doc_id: 320950 cord_uid: x02zp349 respiratory tract infections (rtis) are extremely common especially in the first year of life. knowledge of the etiology of a rti is essential to facilitate the appropriate management and the implementation of the most effective control measures. this perspective explains why laboratory methods that can identify pathogens in respiratory secretions have been developed over the course of many years. high-complexity multiplex panel assays that can simultaneously detect up to 20 viruses and up to four bacteria within a few hours have been marketed. however, are these platforms actually useful in pediatric clinical practice? in this manuscript, we showed that these platforms appear to be particularly important for epidemiological studies and clinical research. on the contrary, their routine use in pediatric clinical practice remains debatable. they can be used only in the hospital as they require specific equipment and laboratory technicians with considerable knowledge, training, and experience. moreover, despite more sensitive and specific than other tests routinely used for respiratory pathogen identification, they do not offer significantly advantage for detection of the true etiology of a respiratory disease. furthermore, knowledge of which virus is the cause of a respiratory disease is not useful from a therapeutic point of view unless influenza virus or respiratory syncytial virus are the infecting agents as effective drugs are available only for these pathogens. on the other hand, multiplex platforms can be justified in the presence of severe clinical manifestations, and in immunocompromised patients for whom specific treatment option can be available, particularly when they can be used simultaneously with platforms that allow identification of antimicrobial resistance to commonly used drugs. it is highly likely that these platforms, particularly those with high sensitivity and specificity and with low turnaround time, will become essential when new drugs effective and safe against most of the respiratory viruses will be available. further studies on how to differentiate carriers from patients with true disease, as well as studies on the implications of coinfections and identification of antimicrobial resistance, are warranted. respiratory tract infections (rtis) are extremely common especially in the first year of life (everard, 2016) . most of these infections are due to one of the many respiratory viruses, mainly respiratory syncytial virus (rsv) (griffiths et al., 2017) , influenza virus (iv) (antonova et al., 2012) , and rhinovirus (rv) . however, parainfluenza virus (piv) (branche and falsey, 2016) , adenovirus (adv) (esposito et al., 2016b) , human metapneumovirus (hmpv) , bocavirus (bv) (principi et al., 2015a) , and enterovirus (ev) (hellferscee et al., 2017) can also play a relevant role, particularly during epidemics. when bacteria are the cause, streptococcus pyogenes is a common cause of pharyngitis (paradise, 1992) , and streptococcus pneumoniae is a typical cause of lower rtis (esposito and principi, 2012) . in some cases, coinfections with two or more viruses (scotta et al., 2016) or with viruses and bacteria (brealey et al., 2015) can occur. for many years, it was thought that knowledge of the etiology of a respiratory infection was essential to facilitate the appropriate management and the implementation of the most effective control measures. it was presupposed that evidence showing that a given viral pathogen was the cause of a respiratory infection could reduce the prescription of further diagnostic tests and the use of antibiotics. theoretically, clinician uncertainty and the anxiety of patients and their family members could also be reduced (gill et al., 2017) . this perspective explains why laboratory methods that can identify pathogens in respiratory secretions have been developed over the course of many years. initially, cell cultures, immunofluorescence assays, and rapid antigen direct tests were used. these tests were mainly used for virus identification due to the higher frequency of viral rtis. however, none of these tests were considered completely satisfactory for clinical use. although cell cultures exhibited a high specificity and good sensitivity, they were very expensive and had a long turnaround time (leland and ginocchio, 2007; gharabaghi et al., 2011) . immunofluorescence assays achieved moderate sensitivity, identified no more than eight viruses, and sometimes required a long turnaround time (ginocchio and mcadam, 2011) . finally, rapid antigen direct tests, although able to provide results in few minutes with high specificity, were available only for rsv, iv, and adenovirus and have low sensitivity (gharabaghi et al., 2011; ginocchio and mcadam, 2011) . these problems have been overcome, at least in part, in recent years, when methods based on nucleic acid amplification became available. such methods exhibit enhanced sensitivity and specificity, and they can detect a broad range of pathogens within an acceptable turnaround time. single polymerase chain reactions (pcrs) for all the known respiratory viruses and several multiplex platforms using pcr and methods for nucleic acid amplification for the simultaneous detection of two or more viruses have been developed (hanson and couturier, 2016) . high-complexity multiplex panel assays that can simultaneously detect up to 20 viruses (mahony et al., 2007) , 18 viruses and two or three atypical bacteria (gonsalves et al., 2019) , 18 viruses and four bacteria (beckmann and hirsch, 2016 ) and a total of 33 pathogens including 12 bacteria (fast track diagnostic, 2018 ) within a few hours have been developed. however, not all the bacteria that play a relevant role in the determination of respiratory infections are systematically included. some of these multiplex platforms have been marketed and largely evaluated in clinical practice in patients admitted to the emergency departments, hospital wards and intensive care units. despite these evaluations, the actual role of these diagnostic measures is not precisely defined. in particular, it has not yet been established whether the availability of a laboratory measure able to identify several potential etiologic agents of a respiratory infection offers real advantages in term of diagnostic accuracy, choice of appropriate therapy and reduction of the social and economic problems strictly associated with pediatric respiratory diseases. moreover, in the majority of the cases they don't allow the identification of antimicrobial resistance to commonly used drugs. in this paper, what can be derived from the presently available studies in this regard is discussed. multiplex platforms based on molecular methods can be used only in the hospital, as they require specific equipment and laboratory technicians with considerable knowledge, training, and experience (beckmann and hirsch, 2016; esposito et al., 2016a; biomerieux, 2018; fast track diagnostic, 2018) . moreover, these platforms have a turnaround time that is significantly shorter than that of culture but generally much longer than that of rapid tests as they take some hours to give reliable results. this can be a limitation in the emergency department or in the intensive care unit, where many patients require immediate diagnostic and therapeutic decisions (beckmann and hirsch, 2016; esposito et al., 2016a; biomerieux, 2018; fast track diagnostic, 2018) . only the most recently developed platforms, such as the biofire r filmarray r respiratory panel 2, have an acceptable turnaround time of about 1 h, not much longer than a rapid test (biofire, 2018) . finally, the number of samples that can be processed per run can significantly vary from assay to assay. in some cases, such in the case of the already cited biofire r assay, only one sample could be processed per run, while most platforms have higher sample throughput (up to 96 samples) (chan et al., 2018) . this can be a problem during epidemics when several patients have to be tested simultaneously. multiplex assays are significantly more sensitive and specific compared with rapid immunochromatographic tests and immunofluorescence assays; however, as multiplex assays detect both viable and non-viable viruses and bacteria, they can lead to debatable results (beckmann and hirsch, 2016; esposito et al., 2016a; biomerieux, 2018; fast track diagnostic, 2018) . generally, the presently developed and marketed multiplex assays exhibit comparable performance with regards to sensitivities and specificities, and detection of coinfections. a previous comparison of the (luminex, 2018) nx tag rpp assay, biofire film array respiratory panel (fa-rp) (biomerieux, 2018) , respifinder22 (rf22) (beckmann and hirsch, 2016) , and the (luminex, 2018) rvp fast assay v2 (esposito et al., 2016a) in terms of the ability to detect common pathogens revealed that the discordance was lower than 10%, although the turnaround time, workflow simplicity and risk of contamination were lower for the (luminex, 2018) nx tag rpp (chen et al., 2016; tang et al., 2016) . a systematic review and meta-analysis (huang et al., 2018) of studies on the accuracy of fa-rp, nanosphere verigene rv+ test (hologic, 2018; luminex, 2018) gen-probe prodesse assays (hologic, 2018) in the detection of iv a, iv b virus, rsv, hmpv, and av showed that all of these assays had high diagnostic accuracy, with an area under the receiver operating characteristic curve (auroc) equal to or >0.98 for all tested viruses. the only exception was adenovirus, for which the auroc was 0.89. finally, a similarly high accuracy was demonstrated for anyplex ii rv16, advansure rv, and real-q rv (yun et al., 2018) . the allplex respiratory panels (allplex; seegene, republic of korea), a recently released one-step real-time reverse transcription-pcr method for the simultaneous detection of multiple pathogens, has been demonstrated to be a rapid and accurate method for detecting respiratory viruses, particularly in case of multiple viral infections (lee and lee, 2019) . a study comparing allplex rp1 assay with prodesseproflu+ and profast+ (hologic, madison, wi, usa) , and genexpert flu/rsv xc (cepheid, usa) for iv and rsv detection, found accuracy of 95, 91, and 96% and sensitivity of 94, 88, and 95%, respectively. the three assays showed a 100% specificity and positive predictive value (ppv), while the negative predictive values (npv) were 84, 73, and 86% for allplex rp1, prodesse and genexpert, respectively (gimferrer et al., 2018) . in general, the accuracy of multiplex platforms in identifying respiratory pathogens is similar to those of single pcr, although in some cases, a slightly lower efficiency has been shown. for example, when the luminex (2018) nx tag rpp was compared with single pcr, it was shown that the multiplex assay had a lower sensitivity against cv hku1 and cv o43 and was oversensitive for several other viruses, leading to several false-positive results. the worst accuracy was found for hmpv, for which the ppv was <50% (chen et al., 2016) . similarly, compared with standard pcr assays, the sensitivity of taqman array card was 54, 56, and 75% for adv, piv-1 and−2, respectively, and 82-95% for the other tested viruses. assay specificity was 99%, and coefficients of variation for virus controls ranged from 1.5 to 4.5% (weinberg et al., 2013) . however, as previously reported, not all the bacterial pathogens that cause respiratory infections are systematically included in the available multiplex platforms. this is a relevant limitation in clinical practice for most of these assays as it is the lack of information regarding antimicrobial resistance to the most commonly used anti-infective drugs. evidence showing that a virus is present in the respiratory secretions of a patient with an rti does not necessarily mean that that particular infectious agent is the cause of the disease. a virus can be the etiologic agent, but viruses can also be asymptomatically carried or shed for several weeks after an infection that has been cured. therefore, viruses can be identified in the asymptomatic incubation period without having an actual role in the disease. contrary to what was thought some years ago, viruses can asymptomatically colonize the airways. both children admitted to the hospital for nonrespiratory diseases and healthy children attending day care were shown to be carriers of at least one respiratory virus in ∼30% of cases, although the viral load was generally lower in carriers than in symptomatic subjects (jansen et al., 2011; moe et al., 2016) . notably, the rate of asymptomatic colonization varies significantly from virus to virus. the detection of iv, rsv, hmpv, and piv is generally indicative of disease, as the frequency of asymptomatic carriage of these agents is relatively uncommon. in contrast, the detection of certain other viruses raises many doubts due to the high frequency with which these viruses can be identified in asymptomatic children (rhedin et al., 2014) . despite the capacity to cause upper and lower rtis and to trigger asthma and chronic obstructive pulmonary disease exacerbations (esposito et al., 2012b; parker et al., 2014; , rv is the virus that is most frequently found in asymptomatic patients. principi et al. (2015b) studied 88 healthy children <2 years old who were followed with weekly nasopharyngeal samples during the months of the winter season of 2013-2014. a total of 1,408 nasopharyngeal samples were obtained, and 326 samples tested positive for rv (23.1%). of these, 209 (64.1%) were not associated with respiratory symptoms, suggesting asymptomatic colonization. carriage is common also for bv (chow and esper, 2009 ), which can be detected in the respiratory secretions of healthy children with the same frequency as that observed in patients with rti (von linstow et al., 2008) . similar findings have been reported for adv, which has been detected in the nasopharynx of healthy children in up to 11% of cases (colvin et al., 2012; rhedin et al., 2014) . pathogens that are asymptomatically carried are also the pathogens that can be most commonly detected with the most recent multiplex platforms. the carriage of mycoplasma pneumoniae (mp) has been identified in many asymptomatic children, although the prevalence varied according to the site of the study [21% in denmark (spuesens et al., 2013) , and 56% in the usa (wood et al., 2013) ] and the period of respiratory sample collection [3% in the spring, and 58% in the summer (colvin et al., 2012) ]. similarly, a certain percentage of healthy children (4-6%) has been shown to test positive for chlamydophila pneumoniae (cp) (emre et al., 1994; block et al., 1997; falck et al., 1997) . long-term viral shedding after a previous infection can further render the results of multiplex platforms poorly effective in identifying the etiology of a disease. however, there are significant differences in long-term shedding among the various infectious agents. generally, those agents that are uncommonly carried are the same agents that are shed only for a few days, and the opposite trend applies for those agents that are frequently asymptomatically carried. although certain influenza patients can shed the virus up to 8 days, shedding typically peaks on day 1 after the onset of symptoms (ip et al., 2017) . although there are exceptions (munywoki et al., 2015) , rsv shedding has been estimated to last between 3.4 and 7.4 days (hall et al., 1976; okiro et al., 2010) . in contrast, rv and bc are generally shed for a longer time. in a study involving 46 children aged 6-36 months with bocavirus infection, it was shown that in 22% of cases, the virus persisted in the respiratory secretions for more than 30 days, despite the rapid disappearance of clinical manifestations (wagner et al., 2016) . the same prolonged shedding has been reported for rv (loeffelholz et al., 2014) , although there are differences according to the type of the infecting strain (daleno et al., 2013) . long-lasting persistence in the respiratory secretions after acute infection has been demonstrated for adv (kalu et al., 2010) and atypical bacteria. the persistence of mp dna in the throat is common, with a median carriage time of 7 weeks after the disease onset (range 2 days−7 months) (nilsson et al., 2008) . cp can be detected for months, as this pathogen temporarily interrupts its replication cycle when exposed to antimicrobials but later resumes replication, leading to the generation of infectious particles (panzetta et al., 2018) . finally, viruses can be detected during the incubation period without playing any role in the determination of the actual disease. the incubation period for viral respiratory infections is generally short. it has been calculated (lessler et al., 2009 ) that this period lasts 5.6 days (95% confidence interval [ci], 4.8-6.3) for adv, 3.2 days (95% ci, 2.8-3.7) for human coronavirus, 1.4 days (95% ci, 1.3-1.5) for iv a, 0.6 days (95% ci, 0.5-0.6) for iv b, 2.6 days (95% ci, 2.1-3.1) for piv, 4.4 days (95% ci, 3.9-4.9) for rsv, and 1.9 days (95% ci, 1.4-2.4) for rv. however, during this period, these viruses are detectable in the respiratory secretions and can lead to diagnostic mistakes. defining the etiology of disease is further complicated by the detection of coinfections, as it is practically impossible to establish which agent is the true causative agent. unfortunately, coinfections are common. in a study involving 592 children with radiographically confirmed cap, viral coinfections were demonstrated in 117 cases (19.7% of the enrolled patients and 26.9% of those with viral infections). similar findings were reported when viral-bacterial coinfections were studied (nolan et al., 2018) . these findings are not surprising, as a previous or concurrent viral rti can favor the development of a secondary bacterial coinfection throughout the airway. augmented bacterial adherence and colonization, dysregulation of the innate and adaptive immune response, immunosuppression, the release of bacteria from biofilms, and alteration of the microbiome are mechanisms through which viruses can favor bacterial superinfection (bakaletz, 2017) . in conclusion, multiplex platforms, despite significantly increasing the possibility to detect which pathogens are present in the respiratory secretions of a child with a respiratory infection, do not offer any advantage in comparison to tradition diagnostic tests regarding the identification of the true etiologic agent of the disease. when multiplex assays are used, the benefits of determining which infectious agent(s) is (are) potentially responsible for an rti are strongly limited by the low number of drugs that are active against the respiratory targets that are currently available on these diagnostic platforms. presently, only drugs against influenza virus, rsv and atypical bacteria are licensed. furthermore, it is debated that these drugs should be used in all the subjects suffering from infections due to sensitive agents. the systematic use of neuraminidase inhibitors (european center for disease prevention and control, 2018) and baloxavir marboxil (hayden et al., 2018) is not recommended for all the cases of influenza because influenza is frequently a mild disease, and the advantage of drug administration is limited to a marginal reduction in the disease duration. consequently, the use of these drugs is reserved only for extremely severe cases, although their true efficacy in these cases has not been definitively demonstrated (lessler et al., 2009; european center for disease prevention and control, 2018) . rsv infection can be treated with ribavirin, which is the only licensed drug for the treatment of this virus. however, ribavirin is difficult to use, costly, and teratogenic, and there is weak evidence for its efficacy. ribavirin is typically used in severe cases that occur in immunocompromised subjects (brendish and clark, 2017) . although several new drugs against rsv are in development, and it is likely that in next few years some of them will be licensed for universal use, the present treatment of rsv infection remains based on supporting measures, such as hydration and o 2 administration (xing and proesmans, 2019) . antibiotics that are effective in vitro against atypical bacteria are generally recommended and largely used when these pathogens are the suspected or demonstrated cause of an rti (esposito et al., 2006 (esposito et al., , 2012a kohlhoff and hammerschlag, 2015) . however, the actual relevance of these drugs in clinical practice is debated, and there is evidence that seems to suggest that they are only slightly effective or not effective (spuesens et al., 2014; gardiner et al., 2015) . in children, only macrolides can be used against mp and cp, as other drugs that are effective against atypical bacteria cannot be prescribed to these patients, particularly the youngest. the administration of tetracyclines and chloramphenicol can lead to severe adverse events; ketolides and streptogramins have limited use in pediatrics; and fluoroquinolones are not licensed for use in subjects <18 years of age (principi and esposito, 2001) . however, many studies that have compared the clinical course of mp and cp infections in children treated with macrolides or other drugs that are ineffective against these pathogens have reported no difference between the two groups, suggesting that macrolides are useless (principi and esposito, 2001) . in addition, macrolides should be used only when their use is presumed to be effective in reducing the abuse of antibiotics and the emergence of resistant strains . finally, despite its high risk of nephrotoxicity, cidofovir, which is a drug licensed for the treatment of cytomegalovirus (cmv) retinitis in hiv-infected patients, has been used to treat adv infections in immunocompromised subjects (ganapathi et al., 2016) . a retrospective evaluation including 16 children showed that in 10/16 cases, viral clearance and clinical response were achieved. however, four patients expired despite the viral clearance, and one of these deaths was directly ascribed to the adv infection. however, cidofovir is not licensed for the treatment of adv; this drug is reserved only for very severe cases occurring in immunocompromised children and cannot be considered as a potential solution to treat mild-to-moderate cases of respiratory disease in otherwise healthy children (ganapathi et al., 2016) . a recent prospective study including 284 children and 232 adults with rti, aimed to determine antibiotic misuse, showed that viral infection was more common in children than in adults, while antibiotic overuse occurred both in children (37%) and, at a significantly higher level, in adults (83%). the study highlights the needing for effective interventions to decrease antibiotic overuse in rti patients of all ages (van houten et al., 2019) . it has been proposed that the use of tests that can identify viruses reduce the use of antibiotics, the prescription of other diagnostic measures, the risk of hospital acquisition among other patients and the length of stay in the pediatric clinical setting. the advantages of these tests from a medical, social, and economic point of view should therefore be enormous. however, the results of studies on multiplex assays are few and conflicting. when these tests were used in pediatric patients admitted to the intensive care unit, no advantage in antibiotic prescriptions was demonstrated (byington et al., 2002) . in contrast, a retrospective evaluation of the use of these tests in pediatric inpatients showed that a positive test result was associated with a decreased length of stay in the hospital and a shorter duration of antibiotics, at least in patients with common respiratory diagnoses (schulert et al., 2013) . however, in wishaupt et al. (2011) the availability of results within 12-36 h did not lead to any advantage in terms of hospital admissions, length of hospital stay or the duration of antibiotic use when antibiotic treatment had been initiated. a more recent retrospective study in which a multiplex assay was tested in children admitted to the emergency department prior to admission or within the first 2 days of hospitalization revealed that patients for whom the results of the test were available were less likely to receive antibiotics for ≥2 days; moreover, these patients were more likely to be in isolation for ≥2 days compared with the controls (subramony et al., 2016) . finally, a recent study conducted in special high-risk settings such as hematology and oncology units, suggests that the diagnoses of asymptomatic virus infections, such as rsv and influenza, can be useful to lower the risk of hospital acquisition. the screening program proved useful for identifying asymptomatically infected patients with viral shedding, thus reducing the risk of transmission and potential nosocomial clusters of rsv and influenza virus on hemato-oncological wards (baier et al., 2018) . on the other hand, less than satisfactory results were also reported in studies that evaluated the impact of rapid tests or immunofluorescence assays, although marketed preparations have been shown to exhibit sensitivity and specific higher than 90% (vos et al., 2019) . a cochrane review (doan et al., 2014) that analyzed four studies regarding the impact of rapid viral diagnosis in children admitted to the emergency department reported that the only advantage of the rapid diagnosis was a lower rate of chest radiography (relative risk [rr], 0.77; 95% ci, 0.65-0.91). no effect on the length of the visit or blood or urine testing was demonstrated. moreover, a trend toward decreased antibiotic use was demonstrated, but the difference between the tested and untested children was not significant. the knowledge that a bronchiolitis case was associated with the detection of rsv in the nasopharyngeal secretions did not lead to any advantage. children with positive results from the rapid test received similar blood tests and radiological examinations. moreover, although the children who tested positive had a more severe disease, virologic testing for rsv did not identify children at risk. globally, the rapid test for rsv is considered useless, and this attitude explains why the test is not recommended by several experts (ralston et al., 2014) . the only rapid test for a single virus that seems to positively impact physician attitudes and have a satisfactory effect on antibiotic consumption the clinical course of disease is the rapid test for influenza. several years ago, esposito et al. (2003) showed that children with a positive rapid influenza test were significantly less likely than those with a negative test or no test to undergo routine blood examinations (2.3% vs. 14.5% and 15.0%; p = 0.045 and p = 0.038) or receive antibiotics (32.6% vs. 64.8% and 61.8%; p < 0.0001 and p = 0.0003). children with positive tests also tended to have a lower incidence (although not significant) of chest radiographs (4.6% vs. 11.5% and 11.7%) and a lower likelihood of admission (0% vs. 4.6% and 5.8%). similar results were reported more recently by cantais et al. (2018) , who showed that the diagnosis of influenza was followed by a 47.9% reduction in blood puncture, a 69.0% reduction in chest x-rays, a 77.8% reduction in lumbar puncture, a 79.2% reduction in urine culture, a 70.1% reduction in antibiotic treatments, and a 25.0% reduction in hospital stay; altogether, these reductions resulted in a reduction of medical costs estimated to be >€69,000 per season. finally, no studies have evaluated the impact of multiplex platforms on the expectations of patient and parents. generally, laboratory tests are requested because they are considered to be effective measures to improve the judgement of clinicians. however, the diagnosis of a viral disease by a laboratory test can be interpreted as a simplification of a more complex clinical problem. bronchiolitis due to rsv can be very severe, and its inclusion among a viral diagnosis may lead to parental dissatisfaction, especially given that no specific therapies are available for rsv, and only supportive measures are prescribed (cabral et al., 2014) . moreover, no definitive information is available on the impact of multiplex assays on clinician anxiety. all these data are needed for a more complete evaluation of the impact of multiplex assays in clinical practice. multiplex platforms for the identification of respiratory viruses and atypical bacteria allow for the identification of most of the infectious agents that cause respiratory infections in infants and children. it is highly likely that these platforms can be particularly important for studies specifically planned to evaluate epidemiology of respiratory pathogens and clinical research. on the contrary, their routine use in pediatric clinical practice remains debatable. they cannot be used in the community where most of the pediatric respiratory diseases are diagnosed. moreover, they cannot allow to overcome the limitation of the traditional diagnostic tests for respiratory pathogens as they do not differentiate carriage from infection, do not seem to influence therapy as effective drugs are available only for iv and rsv, and do not seem to significantly impact of the socioeconomic problems strictly related to pediatric respiratory infections. they seem, however, justified in the presence of severe clinical manifestations, and in immunocompromised patients for whom specific treatment option can be available, particularly when they can be used simultaneously with platforms that allow identification of antimicrobial resistance to commonly used drugs. it is highly likely that these platforms, particularly those with high sensitivity and specificity and with low turnaround time, will become essential when new drugs effective and safe against most of the respiratory viruses will be available. se wrote the first draft of the manuscript. am, ec, and bc gave a substantial scientific contribution. es performed the literature review. np co-wrote the first draft of the manuscript and supervised the project. all authors approved the final submitted version of the manuscript. this work has received funding from the european union's horizon 2020 research and innovation programme under grant agreement no. 701088. burden of paediatric influenza in western europe: a systematic review influenza and respiratory syncytial virus screening for the detection of asymptomatically infected patients in hematology and oncology viral-bacterial co-infections in the respiratory tract comparing luminex nxtag-respiratory pathogen panel and respifinder-22 for multiplex detection of respiratory pathogens the biofire r filmarray r respiratory panels (rp & rp2) chlamydia pneumoniae in acute otitis media viral bacterial co-infection of the respiratory tract during early childhood antiviral treatment of severe noninfluenza respiratory virus infection the effect of rapid respiratory viral diagnostic testing on antibiotic use in a children's hospital they just say everything's a virus"-parent's judgment of the credibility of clinician communication in primary care consultations for respiratory tract infections in children: a qualitative study impact of bedside diagnosis of influenza in the paediatric emergency ward comparison of the biofire filmarray respiratory panel, seegene anyplexii rv16, and argene for the detection of respiratory viruses clinical evaluation of the new high-throughput luminex nxtag respiratory pathogen panel assay for multiplex respiratory pathogen detection the human bocaviruses: a review and discussion of their role in infection detection of viruses in young children with fever without an apparent source phylogenetic analysis of human rhinovirus isolates collected from otherwise healthy children with community-acquired pneumonia during five successive years rapid viral diagnosis for acute febrile respiratory illness in children in the emergency department the association of chlamydia pneumoniae infection and reactive airway disease in children acute tonsillopharyngitis associated with atypical bacterial infection in children: natural history and impact of macrolide therapy antibiotic therapy for pediatric communityacquired pneumonia: do we know when, what and for how long to treat? impact of rhinoviruses on pediatric communityacquired pneumonia effect of a rapid influenza diagnosis unsolved problems in the approach to pediatric community-acquired pneumonia partial comparison of the nxtag respiratory pathogen panel assay with the luminex xtag respiratory panel fast assay v2 and singleplex realtime polymerase chain reaction for detection of respiratory pathogens epidemiology and clinical characteristics of respiratory infections due to adenovirus in children expert opinion on neuraminidase inhibitors for the prevention and treatment of influenza. review of recent systematic reviews and meta-analyses paediatric respiratory infections prevalence of chlamydia pneumoniae in healthy children and in children with respiratory tract infections human line. ftd respiratory pathogens 33 use of cidofovir in pediatric patients with adenovirus infection. version 2. f1000res antibiotics for community-acquired lower respiratory tract infections secondary to mycoplasma pneumoniae in children evaluation of multiple commercial molecular and conventional diagnostic assays for the detection of respiratory viruses in children testing for respiratory viruses in children: to swab or not to swab evaluation of seegene allplex respiratory panel 1 kit for the detection of influenza virus and human respiratory syncytial virus current best practices for respiratory virus testing multiplexed detection and identification of respiratory pathogens using the nxtag r respiratory pathogen panel respiratory syncytial virus: infection, detection, and new options for prevention and treatment respiratory syncytial virus infections within families multiplexed molecular diagnostics for respiratory, gastrointestinal, and central nervous system infections baloxavir marboxil for uncomplicated influenza in adults and adolescents enterovirus genotypes among patients with severe acute respiratory illness, influenza-like illness, and asymptomatic individuals in south africa prodesse proflu+ assay multiplex pcr system for the rapid diagnosis of respiratory virus infection: systematic review and meta-analysis viral shedding and transmission potential of asymptomatic and paucisymptomatic influenza virus infections in the community frequent detection of respiratory viruses without symptoms: toward defining clinically relevant cutoff values persistence of adenovirus nucleic acids in nasopharyngeal secretions: a diagnostic conundrum treatment of chlamydial infections: 2014 update performance evaluation of allplex respiratory panels 1, 2, and 3 for the detection of respiratory viral infection role of cell culture for virus detection in the age of technology incubation periods of acute respiratory viral infections: a systematic review duration of rhinovirus shedding in the upper respiratory tract in the first year of life verigene respiratory pathogens flex test development of a respiratory virus panel test for detection of twenty human respiratory viruses by use of multiplex pcr and a fluid microbead-based assay respiratory virus detection and clinical diagnosis in children attending day care influence of age, severity of infection, and co-infection on the duration of respiratory syncytial virus (rsv) shedding polymerase chain reaction is superior to serology for the diagnosis of acute mycoplasma pneumoniae infection and reveals a high rate of persistent infection etiology and impact of coinfections in children hospitalized with community-acquired pneumonia duration of shedding of respiratory syncytial virus in a community study of kenyan children chlamydia persistence: a survival strategy to evade antimicrobial effects in-vitro and in-vivo etiology and management of pharyngitis and pharyngotonsillitis in children: a current review rhinoviral infection and asthma: the detection and management of rhinoviruses by airway epithelial cells human rhinoviruses and severe respiratory infections: is it possible to identify at-risk patients early? emerging role of mycoplasma pneumoniae and chlamydia pneumoniae in paediatric respiratory-tract infections macrolide-resistant mycoplasma pneumoniae: its role in respiratory infection paediatric human metapneumovirus infection: epidemiology, prevention and therapy bocavirus infection in otherwise healthy children with respiratory disease prospective evaluation of rhinovirus infection in healthy young children clinical practice guideline: the diagnosis, management, and prevention of bronchiolitis clinical utility of pcr for common viruses in acute respiratory illness role of a respiratory viral panel in the clinical management of pediatric inpatients respiratory viral coinfection and disease severity in children: a systematic review and meta-analysis carriage of mycoplasma pneumoniae in the upper respiratory tract of symptomatic and asymptomatic children: an observational study mycoplasma pneumoniae infections -does treatment help? impact of multiplex polymerase chain reaction testing for respiratory pathogens on healthcare resource utilization for pediatric inpatients clinical evaluation of the luminex nxtag respiratory pathogen panel antibiotic misuse in respiratory tract infections in children and adults-a prospective, multicentre study (tailored treatment) clinical and epidemiologic characteristics of human bocavirus in danish infants: results from a prospective birth cohort study rapid molecular tests for influenza, respiratory syncytial virus, and other respiratory viruses: a systematic review of diagnostic accuracy and clinical impact studies determining persistence of bocavirus dna in the respiratory tract of children by pyrosequencing field evaluation of taqman array card (tac) for the simultaneous detection of multiple respiratory viruses in children with acute respiratory infection clinical impact of rt-pcr for pediatric acute respiratory infections: a controlled clinical trial mycoplasma pneumoniae in children with acute and refractory asthma new therapies for acute rsv infections: where are we? comparison of three multiplex pcr assays for detection of respiratory viruses: anyplex ii rv16, advansure rv, and real-q rv key: cord-352837-a29d5dkv authors: hirsch, hans h title: spatiotemporal virus surveillance for severe acute respiratory infections in resource-limited settings: how deep need we go? date: 2019-04-01 journal: clin infect dis doi: 10.1093/cid/ciy663 sha: doc_id: 352837 cord_uid: a29d5dkv nan following the discovery of molecular cloning, nucleic acid sequencing, and polymerase chain reaction, the wonderland of molecular biology has opened its doors again to virtually unlimited possibilities, as next-generation/deep sequencing or, better, massive parallel sequencing allows for the comprehensive analysis of all genomic nucleic acids of any origin in any given sample in real time. initially applied to the bacterial microbiome, deep sequencing has similarly detected a multitude of viral genome sequences, and changed hitherto common views on persisting viral infections [1] , putting forward the concept of the "virome" in general, and of the "human viromes" in particular [2] . while the numeric summary of viral genome sequences is mind-blowing, even after careful annotation and curation through refined bioinformatic tools, the utility of massive parallel sequencing has been prominently demonstrated in epidemiologic cluster analysis, for example, leading to the identification of a novel arenavirus as the cause of donor-derived death of 3 solid organ transplant recipients [3] , or of a novel human polyomavirus causing lethal multiorgan failure in a pancreas transplant recipient [4] . given the technical and bioinformatic advances as well as the declining laboratory costs, the application of deep sequencing to identify etiologic agents in clinical samples has been approached in different pathologies, including those caused by community-acquired respiratory viruses (carvs). despite the general enthusiasm for metagenomics, it remains important to realize that there are caveats. in fact, multiple technical and conceptual aspects influence the results of deep sequencing, may cause significant differences in the read-outs, and hence bias their interpretation. conceptually, it is clear that the human virome is different in different organ sites and body location (eg, the gastrointestinal, urogenital, or respiratory tract) and is subject to dynamic changes of the viral constituents, not only as a result of physiological changes (eg, dietary, hormonal, aging), but also due to complex pathological alterations such as trauma, inflammation, and immune responses [5] , exemplified in inflammatory bowel disease or transplantation [6, 7] . technically, nucleic acid extraction is presumed to be representative across all different agents, whereas in fact different sample preparations, buffers, purification, single fragment partitioning, and sequence analytic procedures may introduce an as-yet little-understood bias, which can lead to underrepresentation of certain viral genomes-for example, human coronavirus compared with paramyxovirus genomes (h. h. hirsch, unpublished observation). thus, every step of the wet and dry laboratory procedure is dependent on the methodology, and may yield potentially different results [8] . independent confirmatory testing has become an essential part of the presumably hypothesis-free deep sequencing [9] . these aspects are important when discussing the results of massive parallel sequencing to either rule in or rule out the involvement of a pathogen in a given pathology. with these caveats in mind, and given the significant global burden of viral respiratory tract disease in the very young and the very old [10] [11] [12] oropharyngeal (np/op) samples of sari cases were identified through a national surveillance study conducted by the uganda virus research institute from 2010 through 2015. the virome present in influenza virus-negative np/op samples was analyzed by deep sequencing enriching for viral sequences. the epidemiologic data were collected by sentinel hospitals in 9 different geographic areas, and hence focused on sari (eg, pediatric and adult patients admitted to hospital for severe influenza-like illness), thereby combining feasibility and clinical significance. the numbers are impressive, as 3921 sari cases were identified, 335 (8.5%) of which were influenza-positive np/op samples. of the remaining 3586 cases of influenza-negative sari, 2901 had spatial and temporal data available to pinpoint 9 clusters involving 301 (10.4%) influenza-negative sari cases. notably, the demographic characteristics of the ugandan sari cases indicated that mostly young children were enrolled, 80% of whom were <1 year of age, both within and outside of clusters. although coughing and shortness of breath were leading features of sari cases of both clustered and nonclustered cases, fever with temperatures >38°c and stridor were significantly more frequent in cluster cases than in noncluster cases. in two-thirds of the sari cluster cases, a virus-capturing technique and massive parallel sequencing using the hiseq illumina system yielded informative genome sequences in 82%, identifying human rhinovirus in 34.5%, human paramyxoviruses consisting of respiratory syncytial virus in 17.9%, and parainfluenza viruses in 11.7%, metapneumovirus in 6.9%, followed by coronaviruses (5.5%), adenoviruses (4.1%), and coxsackie virus (5.5%) and other enteroviruses (4.8%). these numbers compare well with results from resource-rich settings testing symptomatic children and adults using multiplex nucleic acid testing (nat) [13] [14] [15] , whereby the detection rate in children was around 70% and significantly higher than the 30% found in adults [13] , and where human rhinoviruses/enteroviruses together accounted for >45% of the detected carvs [13] . while this suggests that, by and large, a significant part of the pathogens could have been detected by multiplex nat for carvs, the deep sequencing approach of this study revealed a number of remarkable surprises, which may help to close the gap on at least 30%-50% of the carvnegative multiplex nat results [16] . cytomegalovirus (cmv) was identified in 26.9% of the sari clusters, and may either reflect reactivation as a bystander of significant inflammatory states including infections with other including viral pathogens [17] [18] [19] , or may in fact reflect the manifestation of cmv primary infections as sari, a hypothesis viable in view of the high seroprevalence in developing countries [20] and the very young age of 80% of the sari cluster patients described here. similarly, the discovery modus of deep sequencing identified more somewhat expected pathogens in that age group, such as human herpesvirus 6 and parvovirus b19, for which similar arguments could be made. by the same token, it is surprising that other viruses were not reported, some of which had been proposed to be associated with respiratory symptoms or febrile tonsillitis such as bk virus and other polyomaviruses [21] . similarly, the complete absence of the apparently ubiquitous anelloviruses, which have received much attention in studies of respiratory samples from patients with inflammatory conditions or transplantation, is notable [7, [22] [23] [24] . however, no samples from the lower respiratory tract were available for this analysis. the discovery potential of deep sequencing of this us-ugandan study is, however, highlighted impressively by the identification of picobirnavirus in 2 cases, as well as measles, as the cause of sari clusters in 18 cases, of which 13 belonged to genotype b3, which is endemic in eastern africa. remarkably, 5 cases were caused by genotype b4, and could be traced back to an unvaccinated tourist from england and an outbreak in manchester, united kingdom, in 2011. thus, global travel and outbreak have been linked. even though undervaccination may play an important role in the ugandan setting, it is clear that measles outbreaks have been a problem in resource-rich settings such as in switzerland, where 105 cases have occurred in the year 2017 (incidence of 12.5 per million inhabitants), causing outbreaks among mostly unvaccinated individuals, despite the current 2-dose vaccination coverage of 85% and 93% of 2-and 16-year-old children, respectively [25] . of note, measles virus also belongs to the human paramyxovirus family and is transmitted via respiratory fluids, but was detected alone as well as together with other viral pathogens in the present study by cummings et al. taken together, this report from resource-limiting settings is also of relevance for resource-rich countries and raises the question about how to best expand current first-or second-line testing for respiratory viral pathogens including cmv, parvovirus b19, and measles, and how to move to more deep sequencing virome analysis and comprehensive metagenomics in the near future. the spatiotemporal cluster analysis, combined with viral metagenomics presented in this study, is of interest also for other settings, and raises the question of how and when this is can be best delivered in real time. such efforts are under way for influenza virus vaccine prediction [26] , the pacemaker of respiratory viral research, and will be of utility for characterizing vaccine failure and antiviral drug resistance. the average ugandan cluster consisted of 30 cases within a 13-km radius and 38 days, and viruses were detected in only some of the patients. not unexpectedly, certain factors were associated with the ugandan sari clusters, such as human immunodeficiency virus (hiv) infection, urban location, higher population density (eg, 431.5 vs 250 persons/km 2 ), or higher median annual rainfall of 1293 vs 1150 mm/year. these differences, though statistically significant, need to be put in perspective, as the study was anchored in sentinel hospitals, typically located in urban areas where hiv status is more likely to be known, whereas sari cases in rural areas may not have been equally able to seek hospital admission. given the >10-fold higher average population density of boston (5000 persons/km 2 ), or basel (7500 persons/km 2 ), one might suspect that the associations in the ugandan setting may have been a surrogate for other factors such as general hygiene standards, other medical conditions, and socioeconomic factors in certain suburban quarters. similarly, the differences in annual rainfall of <10% need to be balanced against larger seasonal changes in short-term higher rainfall, which may be more important for case clustering and should be addressed in future studies. despite a critical appreciation, this collaborative us-ugandan study combining epidemiology with state-of-the-art modeling and virology not only sets standards for resource-rich industrial settings, but provides next to hiv, ebola, and malaria another proof-of-concept of feasibility and impact of innovative joint projects and partnerships between advanced research groups and dedicated institutions in resource-limited countries. potential conflicts of interest. author certifies no potential conflicts of interest. the author has submitted the icmje form for disclosure of potential conflicts of interest. conflicts that the editors consider relevant to the content of the manuscript have been disclosed. redefining chronic viral infection translational aspects of the human respiratory virome a new arenavirus in a cluster of fatal transplant-associated diseases identification of a novel polyomavirus in a pancreatic transplant recipient with retinal blindness and vasculitic myopathy the human gut virome: inter-individual variation and dynamic response to diet intestinal domination and the risk of bacteremia in patients undergoing allogeneic hematopoietic stem cell transplantation temporal response of the human virome to immunosuppression and antiviral therapy full-length haplotype reconstruction to infer the structure of heterogeneous virus populations evaluation of unbiased next-generation sequencing of rna (rna-seq) as a diagnostic method in influenza virus-positive respiratory samples global burden of acute lower respiratory infections due to respiratory syncytial virus in young children: a systematic review and meta-analysis mortality associated with influenza and respiratory syncytial virus in the united states global mortality estimates for the 2009 influenza pandemic from the glamor project: a modeling study comparing viral respiratory tract infections in symptomatic children and adults: multiplex nat from 2010-2015 comparing luminex nxtag-respiratory pathogen panel and respifinder-22 for multiplex detection of respiratory pathogens community-acquired respiratory paramyxovirus infection after allogeneic hematopoietic cell transplantation: a single-center experience comprehensive diagnostics for respiratory virus infections after transplantation or after potential exposure to swine flu a/h1n1: what else is out there? high incidence of active cytomegalovirus infection among septic patients sepsis and cytomegalovirus: foes or conspirators? viral impact on long-term kidney graft function human cytomegalovirus: clinical aspects, immune regulation, and emerging treatments bk virus: opportunity makes a pathogen reactivation of multiple viruses in patients with sepsis torque teno virus in children who underwent orthotopic liver transplantation: new insights about a common pathogen viral infection in acute exacerbation of idiopathic pulmonary fibrosis the evolution of seasonal influenza viruses key: cord-320286-9tumplp3 authors: bastien, nathalie; brandt, ken; dust, kerry; ward, diane; li, yan title: human bocavirus infection, canada date: 2006-05-17 journal: emerg infect dis doi: 10.3201/eid1205.051424 sha: doc_id: 320286 cord_uid: 9tumplp3 human bocavirus was detected in 18 (1.5%) of 1,209 respiratory specimens collected in 2003 and 2004 in canada. the main symptoms of affected patients were cough (78%), fever (67%), and sore throat (44%). nine patients were hospitalized; of these, 8 (89%) were <5 years of age. human bocavirus was detected in 18 (1.5%) of 1,209 respiratory specimens collected in 2003 and 2004 in canada. the main symptoms of affected patients were cough (78%), fever (67%), and sore throat (44%). nine patients were hospitalized; of these, 8 (89%) were <5 years of age. a new parvovirus, human bocavirus (hbov), was recently identified in sweden (1) . the virus was identified in clinical specimens from infants and children with respiratory tract illness. phylogenetic analyses of the complete genome of hbov showed that that the virus is most closely related to canine minute virus and bovine parvovirus, which are members of the genus bocavirus, family parvoviridae (1). to date, the only parvovirus known to be pathogenic in humans is b19, which is responsible for fifth disease in children (2) . the role of hbov in respiratory tract illnesses is unknown. we retrospectively investigated hbov in canadian patients with acute respiratory infection (ari) in 2003 and 2004 to assess the impact of hbov infections on respiratory tract illnesses and identify the signs and symptoms of this illness. a total of 1,209 specimens from patients with ari from january 2003 to december 2004 were tested for hbov. the specimens originated from the saskatchewan provincial public health laboratories. specimen types analyzed included throat swabs, nasopharyngeal swabs, nasopharyngeal aspirates, and auger suctions. all specimens were negative for influenza viruses a and b; parainfluenza viruses 1, 2, and 3; adenovirus; and respiratory syncytial virus (rsv) by direct or indirect fluorescence assays or virus isolation and for human metapneumovirus (hmpv) by reverse transcription-polymerase chain reaction. specimens were collected from all age groups: 290 (24%) from those <5 years of age, 59 (5%) from those 6-10 years of age, 90 (7.4%) from those 11-15 years of age, 86 (7.1%) from those 16-20 years of age, 358 (29.6%) from those 21-50 years of age, and 324 (27%) from those >50 years of age, the age of the patients was unknown for 2 (0.2%) specimens. hbov was detected by polymerase chain reaction (pcr) using primers specific for 2 different regions of the genome. the screening primers 188f (2281-5′-gac-ctctgtaagtactattac-3′-2301) and 542r (2634-5′-ctctgtgttgactgaatacag-3′-2614), reported by allander et al. (1) , were based on the sequence of the putative noncapsid protein 1 (np-1) gene. the second set of primers, vp1/vp2f (4492-5′-gcaaacccatcactct-caatgc-3′-4513) and vp1/vp2r (4895-5′-gctctct-cctcccagtgacat-3′-4875), was used for confirmation and was based on the published hbov putative vp1/vp2 gene sequences (dq000495) (1). viral dna was extracted from 285 µl of original samples with a biorobot mdx and the qiaamp virus biorobot mdx kit (qiagen, valencia, ca, usa). we used 5 µl of dna in a volume of 50 µl containing 20 pmol of each primer. the thermocycler conditions were 95°c for 15 min for activation of hotstarttaq dna polymerase (qiagen); 35 cycles of 94°c for 1 min, 54°c for 1 min, and 72°c for 2 min; and extension at 72°c for 10 min. nucleotide sequences of np-1 gene amplicons were determined with an abi 377 sequencer and a fluorescent dye terminator kit (applied biosystems, foster city, ca, usa). dna sequences were assembled and analyzed with seqman, editseq, and megalign programs in lasergene (dnastar, madison, wi, usa). to avoid cross-contamination, specimen processing, dna extraction, amplification, and analyses were conducted in different rooms. for dna extraction and pcr procedures, we included 12 negative controls per 96-well plate. a total of 18 (1.5%) of the 1,209 specimens tested were positive for hbov by pcr. hbov activity was found throughout the year with no apparent seasonal prevalence ( table 1 ). the sex distribution of patients was 61% (11) male and 39% (7) female (table 2) . patients with hbov ranged in age from 10 months to 60 years (median 11.5 years), and no significant difference in infection rates was observed between age groups. the main clinical symptoms were cough (78%), fever (67%), and sore throat (44%) ( table 2 ). other clinical symptoms included flulike symptoms (28%), headache (22%), nausea (17%), and myalgia (11%). five patients had rhinitis, 1 had pneumonia, and 1 had bronchiolitis. one patient had rhinitis, bronchiolitis, and pneumonia, and 1 patient had rhinitis and pneumonia. nine (50%) hbov patients were hospitalized; 8 (89%) were <5 years of age, and 1 was between 21 and 50 years of age. the incidence of lower respiratory tract infection was lower in outpatients: 1 with bronchiolitis and no pneumonia ( table 2) . although the infection rates were similar in all age groups, a significant increase in hospitalization rates was seen in those <5 years of age compared with those >6 years of age (8/8 vs. 1/10, p = 0.001) ( pneumonia (3/3) and half of those with bronchiolitis (1/2) were also in this age group. nucleotide sequences were determined for nucleotides 2342-2581 that encode the np-1 gene of hbov (genbank accession nos. dq267760-dq267775). no differences in nucleic acid sequences were found between different canadian isolates. these isolates were also identical to 2 swedish isolates (st1 and st2, genbank accession nos. dq000495-dq000496) (1). although a causal relationship still needs to be demonstrated by including a control group of healthy persons, detection of hbov in respiratory tract specimens from patients with undiagnosed ari suggests that this virus may be associated with respiratory illness. this finding supports those of allander et al. with regard to the association of hbov with respiratory disease (1) . it also demonstrates that hbov was present in canada in 2003 and 2004, which suggests that it may be circulating worldwide. since this study used only samples from ari patients who tested negative for influenza viruses a and b, parainfluenza viruses 1-3, adenovirus, rsv, and hmpv, dual infection cannot be excluded. in addition, whether hbov is present asymptomatically in humans cannot be excluded because samples from healthy persons were not tested. allander et al. reported hbov only in infants and children, which was probably the result of testing fewer specimens from adults patients (1) . most respiratory viruses show a seasonal distribution with peak activity in winter. human parvovirus b19, the only parvovirus that is pathogenic in humans, is also seasonal, with peak occurrences in spring and summer (3) . in contrast, no seasonal prevalence was observed for hbov infection; the virus was found throughout the year. the lack of seasonality observed for hbov may have been caused by the low prevalence in this study. thus, additional year-round studies are needed to better understand the epidemiology of hbov. most (89%) hospitalizations were in persons <5 years of age, which suggests that hbov may cause more severe respiratory illness in infants and children, similar to disease caused by rsv (4, 5) , hmpv (6,7), human coronavirus nl63 (8) (9) (10) (11) (12) (13) (14) , and human coronavirus 229e (15) . more comprehensive studies with data on prevalence, risk factors, and use of health services are needed to determine the role of hbov in ari and its effect on the healthcare system. this study was supported by the public health agency of canada. dr bastien is a scientist at the national microbiology laboratory of the public health agency of canada in winnipeg. her research interests include the diagnosis and pathogenesis of respiratory viruses. cloning of a human parvovirus by molecular screening of respiratory tract samples parvoviruses human parvovirus b19 respiratory syncytial virus impact of respiratory virus infections on persons with chronic underlying conditions human metapneumovirus infection in the canadian population a 1-year experience with human metapneumovirus in children aged <5 years new human coronavirus, hcov-nl63, associated with severe lower respiratory tract disease in australia human coronavirus nl63 infection in canada human coronavirus nl63 infection and other coronavirus infections in children hospitalized with acute respiratory disease in hong kong, china detection of human coronavirus nl63 in young children with bronchiolitis spectrum of clinical illness in hospitalized patients with "common cold" virus infections coronavirus infection in acute lower respiratory tract disease of infants identification of a new human coronavirus coronavirus 229e-related pneumonia in immunocompromised patients key: cord-329306-p5wmqmvj authors: kim, kiwook; song, yeon han; park, joo-hyun; park, hye kyeong; kim, su young; jung, hun; lee, sung-soon; koo, hyeon-kyoung title: rhinovirus associated severe respiratory failure in immunocompetent adult patient date: 2014-09-30 journal: tuberc respir dis (seoul) doi: 10.4046/trd.2014.77.3.132 sha: doc_id: 329306 cord_uid: p5wmqmvj rhinovirus infection is typically associated with the common cold and has rarely been reported as a cause of severe pneumonia in immunocompetent adults. a 55-year-old previous healthy woman, who consumed half a bottle of alcohol daily, presented with respiratory failure after one week of upper respiratory infection symptoms. radiography revealed bilateral, diffuse ground glass opacity with patchy consolidation in the whole lung field; bronchoalveolar lavage fluid analysis indicated that rhinovirus was the causative organism. after five days of conservative support, the symptoms and radiographic findings began to improve. we report this rare case of rhinovirus pneumonia in an otherwise healthy host along with a review of references. rhinovirus infection is typically associated with the common cold 1,2 and exacerbation of asthma symptoms 3 . rhinovirus infection can extend to lower respiratory tract in children [4] [5] [6] or immunocompromised hosts 7, 8 , but is generally not concerned as singular cause of severe pneumonia, especially in immunocompetent adults. although polymerase chain reaction (pcr) methods have enhanced the detection of respiratory viral infection, pcr cannot help distinguish between with patchy consolidations in the whole lung field, but pleural effusion was not present (figure 2 ). laboratory tests revealed a white cell count of 20,930/mm 3 (differential count: 91.5% neutrophils, 6.4% lymphocytes, and 0% eosinophil), hemoglobin concentration 12.7 g/dl, platelet count 366,000/mm 3 , erythrocyte sedimentation rate 84 mm/hr, and c-reactive protein level 43.1 mg/dl. analysis of arterial blood gases indi-cated a pao 2 of 33.5 mm hg, paco 2 26.1 mm hg, hco 3 19.7 mmol/l, and sao 2 71.1% without acidosis. the patient' s liver and renal function tests were within normal range. high-flow o 2 therapy and broad-spectrum antibiotics with oseltamivir (tamiflu, roche, utley, nj, usa) were started empirically, and bronchoscopy was performed immediately. total cell counts of bronchoalveolar lavage (bal) fluid were 240 cells/mm 3 , www.e-trd.org including 75% neutrophils, 4% mononuclear cells, 1% eosinophil, and 20% macrophages. cultures for common bacteria, acid-fast bacilli, and fungi were all negative. pcr for pneumocystis jirovecii, and mycobacterium tuberculosis were also negative. however, pcr of bal fluid indicated the presence of rhinovirus, which was not detected following pcr of the nasopharyngeal specimen. mycoplasma antibody and urinary pneumococcal antigen test were negative. serologic test for venereal disease research laboratory test, hepatitis b surface antigen, hepatitis b, c antibodies, and human immunodeficiency virus, as well as for autoantibodies such as anti-nuclear antibody, rheumatoid factor, and anti-neutrophil cytoplasmic antibody were all negative. clinical symptoms and infiltration on chest x-ray began to improve after five days ( figure 1b) , and the patient was discharged from hospital after three weeks. although technical advances have allowed for increased detection of viral pneumonia, viral infections other than influenza are generally not considered as causes of severe respiratory tract infection in immunocompetent hosts, because viral clearance usually occurs rapidly in healthy individuals. however, the outbreak of severe acute respiratory syndrome, avian influenza a (h5n1) virus, and the 2009 pandemic influenza a (h1n1) virus raised the importance of lower respiratory tract viral infections. additionally, new respiratory viruses, such as human metapneumovirus, coronavirus, and human bocavirus were found to be causes of severe viral pneumonia. the use of molecular diagnostic assays has enhanced the detection of respiratory virus infection; however, the identification of virus does not necessarily mean that it is a lower respiratory tract infection pathogen [9] [10] [11] . therefore, it is still believed that severe viral pneumonia caused by frequently exposed rhinovirus could hardly occurs in immunocompetent adults. according to choi et al. 12 , viruses are frequently detected in the airway of pneumonia patients who require intensive care unit admission, and may cause severe forms of pneumonia. in this study, rhinovirus was identified in 8.6% of respiratory specimens evaluated, but most of patients were the elderly individuals who had underlying comorbidities including structural lung disease, diabetes mellitus, solid cancer, or hematologic malignancies. on the contrary, our relatively young immunocompetent patient suffered severe rhinovirus pneumonia without bacterial co-infection, which was confirmed by bal fluid analysis, and not by the nasopharyngeal specimen. two possible explanations exist for the occurrence of this extremely rare case. first, despite prior indications, severe rhinovirus pneumonia may actually occur in an immunocompetent host. second, chronic alcoholic ingestion could be a major immune modulator for respiratory viral infection, apart from alcoholic liver damage. some previous reports revealed the influence of alcohol on innate immunity in the respiratory system [13] [14] [15] , but the unfavorable impact could play a more critical role in viral defense mechanisms than expected. in conclusion, although the clinical significance of habitual alcoholic ingestion in viral pneumonia requires further evaluation, viral etiology should be considered in cases of severe pneumonia even though in immunocompetent adults, especially if alcoholics. no potential conflict of interest relevant to this article was reported. virological studies in natural common colds in sheffield in 1960 patterns of illness in rhinovirus infections of military personnel respiratory viruses and exacerbations of asthma in adults perez-brena p. role of rhinovirus in hospitalized infants with respiratory tract infections in spain rhinovirus associated with severe lower respiratory tract infections in children rhinovirus and the lower respiratory tract two outbreaks of severe respiratory disease in nursing homes associated with rhinovirus rhinovirus outbreaks in long-term care facilities incidence and characteristics of viral community-acquired pneumonia in adults viral infection in adults hospitalized with community-acquired pneumonia: prevalence, pathogens, and presentation viral pneumonia viral infection in patients with severe pneumonia requiring intensive care unit admission the alcoholic lung: epidemiology, pathophysiology, and potential therapies chronic ethanol ingestion increases superoxide production and nadph oxidase expression in the lung inhibition of tlr8-and tlr4-induced type i ifn induction by alcohol is different from its effects on inflammatory cytokine production in monocytes key: cord-330919-dep3v1pt authors: whyte, claire s; morrow, gael b; mitchell, joanne l; chowdary, pratima; mutch, nicola j title: fibrinolytic abnormalities in acute respiratory distress syndrome (ards) and versatility of thrombolytic drugs to treat covid‐19 date: 2020-04-23 journal: j thromb haemost doi: 10.1111/jth.14872 sha: doc_id: 330919 cord_uid: dep3v1pt the global pandemic of coronavirus disease 2019 (covid‐19) is associated with the development of acute respiratory distress syndrome (ards), which requires ventilation in critically ill patients. the pathophysiology of ards results from acute inflammation within the alveolar space and prevention of normal gas exchange. the increase in proinflammatory cytokines within the lung leads to recruitment of leukocytes, further propagating the local inflammatory response. a consistent finding in ards is the deposition of fibrin in the air spaces and lung parenchyma. covid‐19 patients show elevated d‐dimers and fibrinogen. fibrin deposits are found in the lungs of patients due to the dysregulation of the coagulation and fibrinolytic systems. tissue factor (tf) is exposed on damaged alveolar endothelial cells and on the surface of leukocytes promoting fibrin deposition, while significantly elevated levels of plasminogen activator inhibitor 1 (pai‐1) from lung epithelium and endothelial cells create a hypofibrinolytic state. prophylaxis treatment of covid‐19 patients with low molecular weight heparin (lmwh) is important to limit coagulopathy. however, to degrade pre‐existing fibrin in the lung it is essential to promote local fibrinolysis. in this review, we discuss the repurposing of fibrinolytic drugs, namely tissue‐type plasminogen activator (tpa), to treat covid‐19 associated ards. tpa is an approved intravenous thrombolytic treatment, and the nebulizer form has been shown to be effective in plastic bronchitis and is currently in phase ii clinical trial. nebulizer plasminogen activators may provide a targeted approach in covid‐19 patients to degrade fibrin and improving oxygenation in critically ill patients. in early december 2019 multiple cases of pneumonia of unknown aetiology were reported in wuhan, hubei province, china [1] [2] [3] . in january 2020 the world health organisation declared that this was caused by a new type of coronavirus, (sars-cov-2). the spread of sars-cov-2 has been exponential resulting in a global pandemic with more than two million confirmed cases. while most people with covid-19 develop only mild illness, characterised by a fever and continuous cough [2] , approximately 14% develop severe disease that requires hospitalisation and oxygen support and 5% require admission to intensive care. covid-19 patients with respiratory distress present primarily with severe hypoxemia, yet respiratory system compliance can vary from near normal to exceptionally low [4] . in severe cases, patients with covid-19 develop a type of acute respiratory distress syndrome (ards), sepsis and multiorgan failure. older age and co-morbidities are associated with higher mortality [5] . a hallmark of ards is increased alveolar-capillary permeability triggered by exudation of fluid rich in cells and plasma proteins, including albumin, fibrinogen, proinflammatory cytokines and coagulation factors [6, 7] (figure 1 ). this leads to recruitment of inflammatory leukocytes, including neutrophils [8] alveolar macrophages [9] , monocytes and platelets, which propagate the local inflammatory response [10] . fibrin deposition in the air spaces and lung parenchyma, are consistently observed with ards and contributes to hyaline-membrane formation and subsequent alveolar fibrosis [11] [12] [13] [14] . this promotes the development and progression of respiratory dysfunction and right heart failure [15] . fibrin deposition is the net result of an alteration in the balance of the coagulation and fibrinolytic pathways, and several therapeutic strategies have been explored to target the dysfunction of these systems in ards [16] [17] [18] [19] . recent case studies describe fibrin deposits in biopsies of lung tissue from patients with covid-19 [20, 21] , with ards commonly reported [22, 23] . consistent with this large numbers of infiltrating immune cells have been found in covid-19 positive lung tissues, particularly monocytes and macrophages, [21, [23] [24] [25] alongside the formation of fibrin, [15, 21, 25] proteinaceous hyaline membranes and pulmonary fibrosis [24, 25] . ct scans of covid-19 patient's lungs reveal characteristic ground glass opacities (gco), indicating partial filling of the bronchoalveolar airspace with exudate [26, 27] . the timing of the accidental sampling in the covid-19 patients with lung cancer suggests these early fibrin lung depositions present prior to clinical symptoms of pneumonia [21] . therefore, biomarkers to allow early identification of these changes would be highly beneficial in early diagnosis and timely treatment of covid-19 patients. this review will focus on the molecular mechanisms and role of inflammatory cells in underpinning fibrin deposition and persistence in the lungs of critically ill covid-19 patients and discuss potential therapeutic strategies to help support these patients. this article is protected by copyright. all rights reserved sequestration of leukocytes, particularly neutrophils, within the microvasculature of the lung is central to the development of ards, leading to a massive insult to the alveolar-capillary membrane, unrestricted inflammation and microthrombus formation (reviewed by [28] ). neutrophils, resident alveolar macrophages and monocyte-derived macrophages, as well as recruited monocytes, infiltrate the lungs, enhance lung injury, and play a key role in the pathogenesis of ards [29] [30] [31] [32] . release of proinflammatory cytokines, including macrophage inflammatory protein 2 (mip-2), interleukin 8 (il-8), interleukin-6 (il-6), interleukin-10 (il-10) and tumour necrosis factor  (tnf-), encourage ongoing infiltration of immune cells from the intravascular compartment to the alveolar airspaces [33] [34] [35] . indeed, these proinflammatory cytokines are used as biomarkers of ards and have been suggested to be important in progression of covid-19 associated ards [28] . accumulation of coagulation factors in the lungs can also drive ards through the activation of proteaseactivated receptors (pars) which are expressed on cells in the lungs including alveolar epithelial cells, fibroblasts, monocytes and macrophages [36, 37] . par signalling induced by tissue factor, coagulation factor xa, factor viia or thrombin can augment fibrosis in addition to driving fibrin generation. fibrosis is characterised by fibroblast migration, proliferation and deposition of collagen in the intra-alveolar spaces. par-1 can be acted upon in fibroblasts by both thrombin and factor xa to promote their proliferation, induce production of pro-collagen and amplify expression of various growth factors including connective tissue growth factor (ctgf) [38, 39] . par signalling can enhance inflammation in acute lung injury (ali) by increasing the expression of pro-inflammatory cytokines, including il-6 [40], il-8, [41] [42] [43] and platelet derived growth factor [44] . accumulation of neutrophils in the lungs further contributes to the pathophysiology of ards [28] . neutrophils release their dna alongside their nuclear and cytoplasmic contents into the extracellular environment during the cell death process, netosis. these web like cellular extrusions, termed neutrophil extracellular traps (nets) form a scaffold of chromatin decorated with cytoplasmic and granule proteins and histones. nets play a role in the fight against invading pathogens. however, if not tightly regulated, nets can contribute to the pathogenesis of non-infectious diseases where they can exacerbate coagulation and inflammation [45] and have recently been reported as a contributing player in the pathogenesis of ards and ali where they cause further damage to the lungs [46, 47] . net production has accepted article been identified in the lungs during ards, where levels of nets are greatly increased in the bronchoalveolar lavage (bal) of both ards patients and mouse models of induced ali and ards [46, [48] [49] [50] . increased nets correlate with the severity of ards [46, 49] and disease severity is reduced in mouse models when nets are degraded using dnase1 [46] . a hypercoagulable state exists in the lungs of ards patients, leading to the deposition of fibrin in the intra-alveolar space [51] (figure 1 ). inflammation modulates coagulation by activating c-reactive protein (crp), thereby augmenting tissue factor exposure on monocytes and alveolar macrophages [52, 53] which in turn promote thrombin generation and deposition of fibrin. hepatic synthesis of fibrinogen, an acute phase protein, is increased 2-10-fold in plasma during infection [54] and local synthesis in the lung epithelium is evident during pneumonia [55] thereby further exacerbating fibrin deposition. fibrin deposition augments inflammation and fibrosis as well as damaging lung surfactant [50, 56, 57] . this is coupled with a hypofibrinolytic state in the alveolar space, where fibrinolytic inhibitors have been shown to be elevated. levels of thrombin activatable fibrinolysis inhibitor (tafi) and protein c inhibitor were found to be significantly elevated in the bronchoalveolar fluid of patients with interstitial lung disease when compared to healthy controls [58] . furthermore, it has been reported that α2-macrogloblin levels are increased in obstructive lung disease, which may correlate with the increase in plasminogen observed in the bal of ards patients [59, 60] . however, the principal fibrinolytic inhibitor described in the pathogenesis of ards is plasminogen activator inhibitor 1 (pai-1), which is known to be elevated in severe acute respiratory syndrome coronavirus (sars-cov) and ali [11, 61] . in ards, crp promotes local release of pai-1 from endothelial cells [62, 63] . additionally, infiltration of platelets, the major circulating pool of pai-1, may result in local release. we have recently shown that a significant amount of this active pai-1 remains associated with the stimulated platelet membrane [64, 65] . attenuation of the plasminogen activation system leads to abnormal turnover of fibrin in the alveolar space. plasma pai-1 levels have been reported as an independent risk factor for poor prognosis and mortality in ali [60, 62, 63, [66] [67] [68] [69] . prabhakaran et al [62] reported a significant increase in pai-1 antigen and activity in plasma and the edema fluid in ali, with evidence of significant pulmonary production [62] . a clear role for pai-1 as a prognostic marker in ards was confirmed by a prospective observational study this article is protected by copyright. all rights reserved which demonstrated 5-fold higher levels in patients who progressed to ards than those with uncomplicated aspiration pneumonitis (2687 vs. 587 ng/ml, respectively) [68] . importantly, a hypofibrinolytic state and increased pai-1 was observed in the sars-cov epidemic in 2002 and 2003 [61] . gralinski et al used a non-biased systems biology approach to study the dysfunctional fibrinolytic pathway in an infection model of sars-cov [61] . fibrin persistence was mediated by overexpression of pai-1 which overcomes local upa and tissue-type plasminogen activator (tpa) [61] . sars-cov infected cells contain high levels of tgf-β1, which in turn stimulates expression of extracellular matrix protease inhibitors, including pai-1 [70] which has been specifically linked to ards induced by sars-cov [71] . these studies illustrate a clear role for pai-1 in the aetiology of ards and suggest it is a key protein contributing to abnormal turnover of fibrin in the alveolar space. plasma pai-1 has been reported as a potential biomarker for predicting disease progression in ali to ards, with one study concluding that pai-1 antigen > 640 ng/ml was a 100 % positive predictor for mortality [62] . similar pathology of fibrin depositions in the lungs has been identified in covid-19 [21, 25] , suggesting pai-1 may be a useful prognostic marker for patients at risk of developing ards and thus requiring critical care and ventilation. a common finding with covid-19 patients requiring hospitalisation is increased levels of d-dimers and and fibrin degradation products (fdp) which are associated with a higher risk of mortality [72] . prothrombin time and activated partial thromboplastin time show a mild elongation [72] . coupled with the fact critically ill covid-19 patients will be immobilised, there is an increased risk of hospital-associated venous thromboembolism (vte) [73] . these findings have led to a recent recommendation for prophylactic anticoagulant therapy with low molecular weight heparin (lmwh) for patients hospitalised with covid-19, without contraindications, to limit the extent of the coagulopathy [74, 75] . heparin treatment (both unfractionated and lmwh) reduces inflammatory biomarkers [76] , and therefore may be beneficial in reducing the inflammatory state in covid19 this article is protected by copyright. all rights reserved anticoagulant therapy is essential to limit ongoing fibrin deposition and microthrombi formation in ards and treat the systemic prothrombotic complications in these patients. however, lmwh will be ineffective in clearing fibrin clusters deposited in the alveolar space. there is therefore a requirement to readdress the balance of fibrinolysis in the lung, either by enhancing plasminogen activation or downregulating fibrinolytic inhibitors. the significant increase in pai-1 in ards and ali curtails local upa, but also tpa, activity [11, 17, 80, 81] . in a pig model of trauma, administration of tpa or upa prevented development of ards, with animals displaying normal pao 2 [82] . a phase 1 clinical trial revealed a significant improvement in pao 2 at 24 hours in 19 out of 20 patients with severe ards secondary to trauma or sepsis following administration of upa or streptokinase [83, 84] . these patients had a pao 2 of less than 60 mmhg, usually considered fatal, which increased to 231.5 mmhg following thrombolytic therapy with an overall 30 % survival rate and no incidence of bleeding [84] . the use of tpa to treat ards in covid-19 patients has recently been proposed by moore et al [15] . an initial case report from 3 patients from the current sars-cov-2 pandemic, demonstrates a transient improvement in p/f ratio in 2 cases and sustained 50 % improvement in 1 case following administration of a 25 mg bolus of intravenous tpa followed by a further 25 mg infusion [85] . the authors suggest that there is a precedent for increasing the dose of the bolus of tpa whilst maintaining heparin infusion, as the anticoagulant is effective against submassive pulmonary embolism [85, 86] . in addition to readdressing the fibrinolytic balance, administration of tpa to ards patients may confer anti-inflammatory effects, as it has been shown to suppress neutrophil activation in a rat model of ali induced by il-1α [87] . a major consideration in anticoagulant or thrombolytic therapy is the undesirable complication of bleeding. in respiratory medicine, treatments are often delivered as aerosolised protein therapeutics as diffusion of proteins from the blood to the lungs can be limited [88] . interestingly, nebulised anticoagulant therapy with antithrombin or heparin has been shown to reduce lung injury without an increase in systemic bleeding in animal models [89] [90] [91] and ali patients [92] . however, as discussed, heparin will prevent further fibrin deposition but will be ineffective in the removal of pre-existing fibrin. a recent publication compared the efficacy of the nebulised form of the plasminogen activator, streptokinase and nebulised heparin in the treatment of ards [93] . the primary outcome in this trial was the change in pao 2 /fio 2 ratio, which was significantly higher in the streptokinase group from day 1 to day 8, compared to the heparin and standard-of-care groups. importantly, icu mortality was significantly lower in streptokinase patients compared to other groups [93] . a 1999 case report [94] describes a young woman with ards who was resistant to conventional therapeutics and was treated with nebulised and intravenous tpa, followed by continuous treatment with nebulized unfractionated heparin. the patient this article is protected by copyright. all rights reserved stabilized following fibrinolytic treatment and demonstrated a significant enhancement in pulmonary gas exchange. plastic bronchitis is a condition that can develop from several respiratory disorders, resulting in casts of compacted mucous that have been shown to contain fibrin [95] . plastic bronchitis is primarily observed in children and has been described in cases of influenza a (h1n1) [96, 97] and human bocavirus [98] . nebulised tpa has been shown to be effective in preventing recurrent cast formation in plastic bronchitis [95] . reports thus far are from single case studies, however, there is an ongoing phase ii clinical trial of nebulised tpa (platypus; alteplase, nct02315898) for treatment of plastic bronchitis. these data clearly indicate that use of nebulised fibrinolytics could allow a more targeted approach to correct the haemostatic imbalance that results in fibrin deposition, while limiting the risk of systemic activation of fibrinolysis that may trigger unwanted bleeding ( figure 2 ). inhaled tpa is absorbed into the vasculature thus increasing fibrinolytic capacity in the plasma [99] and the potential to lyse the microthrombi observed in covid-19 patients. however, it is conceivable that intravenous infusions of tpa may be necessary to disperse larger thrombi in the circulation. a potential caveat of a nebulizer formulation is that aerosolised proteins are susceptible to degradation so the formulation and excipient used must be considered [88] . however, in the case of tpa, an extreme advantage is that a formulation of nebulised alteplase has been developed and is currently being tested in a phase ii clinical trial [88] . the covid-19 global pandemic has necessitated a demand for novel therapeutics to limit the complications of ards and/or reduce the burden on ventilatory support in intensive care units. the indication that fibrin deposits occur prior to symptoms [21] of the disease, suggests that targeting the fibrinolytic system to promote fibrin resolution could limit severity and improve pulmonary function. given the urgent time scale of the clinical requirement, repurposing of existing therapies, such as nebulised tpa, to promote fibrin dissolution in the lung and improve oxygenation is a pragmatic approach in addressing the ards complications associated with covid-19. this article is protected by copyright. all rights reserved outbreak of pneumonia of unknown etiology in wuhan, china: the mystery and the miracle bin cao. clinical features of patients infected with 2019 novel coronavirus in wuhan alimuddin zumla, eskild petersen. the continuing 2019-ncov epidemic threat of novel coronaviruses to global health -the latest 2019 novel coronavirus outbreak in wuhan, china covid-19 does not lead to a "typical" acute respiratory distress syndrome clinical course and outcomes of critically ill patients with sars-cov-2 pneumonia in wuhan, china: a single-centered, retrospective, observational study activation and regulation of systemic inflammation in ards: rationale for prolonged glucocorticoid therapy lung parenchyma remodeling in acute respiratory distress syndrome pulmonary fibrosis correlates with outcome in adult respiratory distress syndrome. a study in mechanically ventilated patients nf-kappab regulatory mechanisms in alveolar macrophages from patients with acute respiratory distress syndrome the role of macrophages in the pathogenesis of ali/ards accepted article this article is protected by copyright. all rights reserved 11 depressed bronchoalveolar urokinase activity in patients with adult respiratory distress syndrome intravascular coagulation associated with the adult respiratory distress syndrome pathophysiology of acute lung injury and the acute respiratory distress syndrome the acute respiratory distress syndrome tissue plasminogen activator (tpa) as a novel treatment for refractory covid-19 associated acute respiratory distress syndrome (ards)? j trauma acute care surg anticoagulant therapy in acute lung injury emerging role of anticoagulants and fibrinolytics in the treatment of acute respiratory distress syndrome pulmonary coagulopathy as a new target in therapeutic studies of acute lung injury or pneumonia--a review bench to bedside: targeting coagulation and fibrinolysis in acute lung injury immunostaining in the lung of a patient with covid-19 pulmonary pathology of early-phase 2019 novel coronavirus (covid-19) pneumonia in two patients with lung cancer clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus-infected pneumonia in wuhan pathological findings of covid-19 associated with acute respiratory distress syndrome zhonghua bing li xue za zhi = chinese journal of pathology zhonghua bing li xue za zhi = chinese journal of pathology radiological findings from 81 patients with covid-19 pneumonia in wuhan, china: a descriptive study acute respiratory distress syndrome high levels of interleukin-8 in the blood and alveolar spaces of patients with pneumonia and adult respiratory distress syndrome phenotypic characterization of alveolar monocyte recruitment in acute respiratory distress syndrome pulmonary macrophage subpopulations in the induction and resolution of acute lung injury role of alveolar macrophage and migrating neutrophils in hemorrhage-induced priming for ali subsequent to septic challenge inhibition of alveolar neutrophil immigration in endotoxemia is macrophage inflammatory protein 2 independent interleukin-8 and development of adult respiratory distress syndrome in at-risk patient groups relative production of tumour necrosis factor alpha and interleukin 10 in adult respiratory distress syndrome tissue factor-dependent coagulation protease signaling in acute lung injury proteinase-activated receptors in fibroproliferative lung disease thrombin is a potent inducer of connective tissue growth factor production via proteolytic activation of protease-activated receptor-1 factor xa stimulates fibroblast procollagen production, proliferation, and calcium signaling via par1 activation accepted article this article is protected by copyright. all rights reserved 40 activation of protease-activated receptor (par)-1, par-2, and par-4 stimulates il-6, il-8, and prostaglandin e2 release from human respiratory epithelial cells tissue factorfactor viia-specific up-regulation of il-8 expression in mda-mb-231 cells is mediated by par-2 and results in increased cell migration activation of protease-activated receptor (par)-1, par-2, and par-4 stimulates il-6, il-8, and prostaglandin e2 release from human respiratory epithelial cells thrombin stimulates production of interleukin-8 in human umbilical vein endothelial cells factor xa is a fibroblast mitogen via binding to effector-cell protease receptor-1 and autocrine release of pdgf neutrophil extracellular traps in immunity and disease maladaptive role of neutrophil extracellular traps in pathogen-induced lung injury neutrophil extracellular traps (nets) are increased in the alveolar spaces of patients with ventilator-associated pneumonia extracellular traps are elevated in patients with pneumonia-related acute respiratory distress syndrome neutrophil extracellular traps contribute to the pathogenesis of acid-aspiration-induced ali/ards alteration of surfactant function due to protein leakage: special interaction with fibrin monomer therapeutic modulation of coagulation and fibrinolysis in acute lung injury and the acute respiratory distress syndrome diagnostic and prognostic utility of tissue factor for severe sepsis and sepsis-induced acute lung injury serial abnormalities of fibrin turnover in evolving adult respiratory distress syndrome acute phase reactants in inflammation and infection induction of fibrinogen expression in the lung epithelium during pneumocystis carinii pneumonia surfactant incorporation markedly alters mechanical properties of a fibrin clot fibroblast migration in fibrin gel matrices thrombinactivatable fibrinolysis inhibitor and protein c inhibitor in interstitial lung disease alpha 2-macroglobulin in patients with obstructive lung disease, with and without alpha 1-antitrypsin deficiency local abnormalities in coagulation and fibrinolytic pathways predispose to alveolar fibrin deposition in the adult respiratory distress syndrome mechanisms of severe acute respiratory syndrome coronavirus-induced acute lung injury elevated levels of plasminogen activator inhibitor-1 in pulmonary edema fluid are associated with mortality in acute lung injury pathogenetic and prognostic significance of altered coagulation and fibrinolysis in acute lung injury/acute respiratory distress syndrome functional plasminogen activator inhibitor 1 is retained on the activated platelet membrane following platelet activation plasminogen associates with phosphatidylserine-exposing platelets and contributes to thrombus lysis under flow accepted article this article is protected by copyright. all rights reserved 66 increased plasminogen activator inhibitor-1 concentrations in bronchoalveolar lavage fluids are associated with increased mortality in a cohort of patients with pseudomonas aeruginosa bronchoalveolar levels of plasminogen activator inhibitor-1 and soluble tissue factor are sensitive and specific markers of pulmonary inflammation alveolar plasminogen activator inhibitor-1 predicts ards in aspiration pneumonitis plasminogen activator inhibitor (pai-1) predict a poor prognosis in clinical acute lung injury expression of elevated levels of proinflammatory cytokines in sars-cov-infected ace2+ cells in sars patients: relation to the acute lung injury and pathogenesis of sars severe acute respiratory syndrome-associated coronavirus nucleocapsid protein interacts with smad3 and modulates transforming growth factor-beta signaling abnormal coagulation parameters are associated with poor prognosis in patients with novel coronavirus pneumonia practical guidance for the prevention of thrombosis and management of coagulopathy and disseminated intravascular coagulation of patients infected with covid-19 anticoagulant treatment is associated with decreased mortality in severe coronavirus disease 2019 patients with coagulopathy isth interim guidance on recognition and management of coagulopathy in covid-19 anti-inflammatory effects of heparin and its derivatives: a systematic review haematological manifestations in patients with severe acute respiratory syndrome: retrospective analysis the severe acute respiratory syndrome coronavirus 3a protein up-regulates expression of fibrinogen in lung epithelial cells accepted article this article is protected by copyright. all rights reserved 79 a major outbreak of severe acute respiratory syndrome in hong kong extravascular coagulation and fibrin deposition in acute lung injury coagulation, fibrinolysis, and fibrin deposition in acute lung injury prevention of adult respiratory distress syndrome with plasminogen activator in pigs a brief overview of acute respiratory distress syndrome treatment of severe acute respiratory distress syndrome: a final report on a phase i study tissue plasminogen activator (tpa) treatment for covid-19 associated acute respiratory distress syndrome (ards): a case series heparin plus alteplase compared with heparin alone in patients with submassive pulmonary embolism tissue plasminogen activator (tpa) inhibits interleukin-1 induced acute lung leak heuze-vourc'h n. designing inhaled protein therapeutics for topical lung delivery: what are the next steps? effects of nebulized antithrombin and heparin on inflammatory and coagulation alterations in an acute lung injury model in rats aerosolized anticoagulants ameliorate acute lung injury in sheep after exposure to burn and smoke inhalation heparin nebulization attenuates acute lung injury in sepsis following smoke inhalation in sheep nebulized anticoagulants in lung injury in critically ill patients-an updated systematic review of preclinical and clinical studies streptokinase versus unfractionated heparin nebulization in patients with severe acute respiratory distress syndrome (ards): a randomized controlled trial with observational controls inhalation/intravenous recombinant tissue plasminogen activator and inhaled heparin in a patient with acute respiratory distress syndrome management of plastic bronchitis with nebulized tissue plasminogen activator: another brick in the wall plastic bronchitis in three children associated with 2009 influenza a(h1n1) virus infection bronchial casts and pandemic (h1n1) 2009 virus infection human bocavirus dna detected in a boy with plastic bronchitis meta-analysis of preclinical studies of fibrinolytic therapy for acute lung injury with thanks to wai-lum sum from medical illustration at the university of aberdeen with help in compiling figure 2 of the manuscript. njm and pc conceived the idea. csw, gbm, jlm and njm all wrote and edited the manuscript. this article is protected by copyright. all rights reserved this article is protected by copyright. all rights reserved key: cord-319814-tyqb473m authors: zhang, dingmei; he, zhenjian; xu, lin; zhu, xun; wu, jueheng; wen, weitao; zheng, yun; deng, yu; chen, jieling; hu, yiwen; li, mengfeng; cao, kaiyuan title: epidemiology characteristics of respiratory viruses found in children and adults with respiratory tract infections in southern china date: 2014-06-11 journal: int j infect dis doi: 10.1016/j.ijid.2014.02.019 sha: doc_id: 319814 cord_uid: tyqb473m background: the world health organization (who) ranks respiratory tract infection (rti) as the second leading cause of death worldwide for children under 5 years of age. the aim of this work was to evaluate the epidemiology characteristics of respiratory viruses found in children and adults with rti from july 2009 to june 2012 in southern china. methods: in this work, a total of 14 237 nasopharyngeal swabs (14 237 patients from 25 hospitals) were analyzed, and seven respiratory viruses (influenza virus, respiratory syncytial virus, parainfluenza virus, adenovirus, human metapneumovirus, human coronavirus, human bocavirus) were detected using pcr/rt-pcr from nasopharyngeal swabs. results: the demographic characteristics, viral prevalence, age distribution, seasonal distribution, and pathogen spectrum of the patients with rtis were analyzed. co-infection was observed in 483 specimens, but it was more common in male patients, inpatients, children, and young adults. it varied by season, being more prevalent in the spring and summer and less so in the winter. human coronavirus and human bocavirus were the most common pathogens, tending to occur in co-infection with other respiratory viruses. conclusions: this work adds to our knowledge of the epidemiology characteristics of these seven common respiratory viruses among patients with rti in southern china. the detection of the specific viral causes of infection provides a useful starting point for an understanding of illness attributable to respiratory infection, and might also provide data relevant to the development of prevention strategies. of risk factors is critical to the successful implementation of a prevention and control program. southern china is believed to be the origin of some important respiratory viruses, such as severe acute respiratory syndrome coronavirus (sars-cov) 16 and influenza virus. 17 southern china has large populations of humans and domestic and wild animals, as well as a large transient population that includes laborers and business people from different provinces of china and from other countries. the mixing of these large regional populations may favor the transmission of respiratory viruses. the prevalence and clinical presentation of human viral infections in china have been reported previously. 8, 10, [18] [19] [20] however, there are no previously published reports describing the etiology of the seven common respiratory tract viruses of inpatient and outpatient rti across the seasons among the various age and gender categories. information on rti from southern china is also sparse. to directly address this situation, nasopharyngeal swabs were collected continuously from children and adults seeking medical attention for rti from a total of 25 hospitals in southern china between july 2009 and june 2012. seven respiratory viruses were detected by pcr/rt-pcr and their epidemiological characteristics were analyzed. all research involving human participants was approved by the institutional review board of zhongshan school of medicine, sun yat-sen university, in accordance with the guidelines for the protection of human subjects. participants provided written informed consent after being briefed on the purpose of the study and of their right to keep information confidential. written consent was obtained from all study participants or their guardians. study participants had all been admitted to one of the 25 hospitals covering southern china. selection criteria included having one or more respiratory symptoms, such as headache, cough, expectoration, and pharyngodynia, combined with a body temperature above 37.5 8c. symptoms, history of illness, results of a clinical examination and laboratory tests, and demographic data were collected for each patient using a standardized form. nasopharyngeal swabs were collected according to a standard procedure, kept in viral transport medium, and stored at à80 8c prior to analysis (one swab was collected from each patient). for patients with viral infections, some additional clinical information was abstracted retrospectively from the medical treatment records. dna or rna was extracted from 200 ml of the nasopharyngeal swab specimen using the qiaamp minielute virus spin kit (qiagen). reverse transcription of virus rna was conducted using superscript iii rt (invitrogen, life technology) in order to detect rna viruses (flu, rsv, piv, hmpv, and hcov). dna samples extracted using the kit were used directly to detect dna viruses (adv and hbov). both kits were used in accordance with the manufacturer's instructions. flu (a, b, c), rsv, piv, adv, hmpv, hcov, and hbov were detected by standard pcr or reverse transcription pcr (rt-pcr), as described previously, using specific primers listed in the supplementary material table s1 , [21] [22] [23] [24] [25] and amplified products were detected using agarose gel electrophoresis. statistical analysis was performed using spss 13.0 (spss inc., chicago, il, usa). viral prevalences were compared using the chisquare test for categorical variables, and the cartogram was drawn using excel software. a p-value of <0.05 was considered statistically significant. a total of 14 237 nasopharyngeal swabs were collected from july 2009 to june 2012. in all, 7323 specimens were from outpatients and 6914 specimens were from inpatients. more specimens were collected from males than from females (ratio 1.48). the median patient age was 21 years (range 0-110 years). the number of children (age 14 years) was 6117 and the number of other patients (>14 years) was 8120. the seasonal distribution of patients sampled was 3756 in spring (january to march), 2617 in summer (april to june), 3993 in autumn (july to september), and 3871 in winter (october to december). an estimated 99.9% of eligible patients volunteered to participate. our data showed a significant difference in inpatient proportion by age, and showed the difference in sex distribution by age. they did not show the 20-30 and 50-55 years groups, and the relative numbers of male participants in other age groups were all over 50%. the clinical characteristics of the patients are listed in the supplementary material table s2 . most patients presented with symptoms of respiratory tract illness (rti), including fever (!37.5 8c; 100.0%), cough (84.1%), expectoration (42.1%), runny nose (36.4%), and sore throat (19.8%). the total rate of detection of all seven viruses for all specimens was 39.24% (5582/14 237). flu viruses were detected in 2632 specimens (18.50%), rsv in 1120 (7.86%), piv in 494 (3.47%), adv in 493 (3.47%), hmpv in 319 (2.24%), hcov in 351 (2.47%), and hbov in 180 (1.26%). the total viral detection rate (all seven viruses) for all inpatients was 40.01% (2772/6914), which was higher than that of outpatients (38.47%, 2817/7323) (chi-square = 3.937, p = 0.047). with the exception of flu, the viruses were more common in inpatients than in outpatients ( figure 1 ). the total viral detection rate (all seven viruses) was higher in male patients (40.11%, 3403/8485) than in female patients (37.95%, 2179/5742) (chi-square = 6.687, p = 0.010). the rates of detection of rsv, piv, and hmpv were higher in male patients than in female patients, and the detection rate of flu was lower in male patients than in female patients (chi-square = 19.262, p < 0.0001). there was no difference in the detection rates for adv, hcov, and hbov ( figure 2 ). a decline in the incidence of viral infections with age was observed for respiratory viruses, except for flu. the detection rates of rsv, piv, adv, hmpv, hcov, and hbov among children ( 14 years) were higher than among adults (>14 years old). the detection rates for rsv and hbov were highest among children <5 years old. the detection rate for flu was highest among patients aged 15-35 years ( figure 3 ). a combined graph for all three study years showing the monthly distribution of the seven respiratory viruses was drawn. the total detection rate of the seven respiratory viruses was highest in february (48.03%) and august (47.16%), and lowest in october (23.14%). the highest rate of rsv was detected in february (16.10%); the highest rate of hmpv was detected in march (4.30%); piv, hcov, and hbov showed the highest rates of detection in june: 7.87%, 3.68%, and 3.43%, respectively. flu showed the highest rate of detection in august (32.03%), and adv showed the highest rate of detection in december (4.38%). the total detection rates for the seven respiratory viruses in spring, summer, autumn, and winter were 44.31%, 41.15%, 41.66%, and 30.52%, respectively. the rates of detection were different during the four seasons (chi-square = 177.859, p < 0.0001). however, the detection rates in summer and autumn were similar in all four seasons, the detection rate of flu was the highest of the seven respiratory viruses, followed by rsv. the detection rate of flu was highest in autumn and there was no statistically significant difference in rates between spring/summer and summer/winter. however, the detection rate of rsv was highest in spring. the detection rate for piv was highest in summer. there was no difference in the rate of detection of piv during spring, autumn, and winter. for hmpv, the detection rates during spring and summer were higher than during autumn and winter. there was no difference between spring/summer and autumn/winter. the detection rate for hcov was lowest in autumn; there was no difference among spring, summer, and winter. the detection rate for hbov was lower in spring than in summer, autumn, and winter. the pathogen spectrum differed with patient age. the proportion of patients infected with flu increased with age, peaking in the group including age 15 years (86.31%), after which it fluctuated slightly but remained stable, and declined significantly after age 60 years. however, the rates of detection of rsv, piv, adv, hmpv, and hcov relative to the total rate of detection decreased after the patients reached adulthood, and then fluctuated only slightly until age 60 years, after which they declined significantly. rsv was the most common pathogen detected in children, accounting for 31.72% of all viruses detected in children. flu accounted for 25.91% of all the viruses detected in children. flu was the most common pathogen detected in adults, accounting for 74.97% of all viruses detected. flu was the most common pathogen detected in outpatients (68.25%). rsv was the most common pathogen detected in inpatients (31.78%). however, rsv was the most common pathogen detected in inpatient children (34.77%). flu was the most common pathogen detected in inpatient adults (81.04%), outpatient children (34.46%), and outpatient adults (42.02%). the proportion of flu infections among female cases (53.33%) was greater than among male cases (43.20%). however, there were fewer rsv, piv, hcov, and hmpv among female cases than among male cases. the pathogen spectrum also differed across the seasons. rsv accounted for a larger proportion of the total number of infections during spring (32.27%); piv, hmpv, and hbov accounted for a larger proportion during summer (12.92%, 9.01%, and 5.11%, respectively). flu accounted for a larger proportion during autumn (61.23%). adv and hcov accounted for a larger proportion during winter (11.35% and 9.99%, respectively) ( figure 4 ). co-infections occurred in 483 specimens, with detection rates of 3.39% (483/14 237) of all specimens and 9.55% (483/5057) of positive specimens. among the 483 co-infected specimens, 442 were double infections, 40 were triple infections, and one was a quadruple infection ( table 1) there were 175, 121, 113, and 74 cases of co-infection in spring, summer, autumn, and winter, respectively; the co-infection rates were 4.66%, 4.63%, 2.83%, and 1.91%, respectively (chi-square = 60.209, p < 0.0001). the co-infection rates in spring and summer were higher than in autumn and winter, and the rate of coinfection in winter was the lowest. the who ranks rti as the second leading cause of death worldwide for children younger than 5 years of age. 26 however, the influenza viruses are the only viral respiratory pathogens for which vaccines are currently available. 27 ongoing vaccine research and development are focusing on many other leading viral pathogens. [27] [28] [29] the detection of the specific viral causes of infection provides a useful starting point for an understanding of illness attributable to respiratory infection. it also provides data relevant to the development of prevention strategies. the objective of the study was to estimate the prevalence of respiratory viruses in people who presented with acute respiratory tract infections in southern china over a 3-year study period. human piv is a major cause of respiratory tract illness in infants and young children worldwide. 30 all children experience at least one piv infection by the age of 5 years, and re-infection may occur throughout life because of incomplete immunity. 31 the virus is associated with a wide variety of rtis, but most frequently with croup and pneumonia. annual epidemics of hpiv-3 infection are responsible for considerable economic losses as a result of hospitalization, medication costs, work and school absence, and mortality. however, piv infections have been less well studied compared to rsv and influenza viral infections. in the present study, piv was associated with 10.00% of all the rti hospitalizations and 15.00% of the rti in outpatient children. a study in the usa found piv infections to be associated with 11.50% of all pediatric rti hospitalizations. 32 human bocavirus (hbov) was first reported by allander et al. in 2005. 33 subsequently, hbov was reported in respiratory specimens collected from different countries and regions worldwide, [34] [35] [36] and detected in 1.50% to 8.30% of respiratory specimens from individuals with acute rti, especially young children and infants. in the present study, hbov was detected in 3.85% of all children <2 years old, who provided 78.33% of all the hbov-positive specimens. hbov infection has recently attracted attention worldwide. however, the incidence and clinical presentation of this infection varies widely, often involving co-infection with other potential pathogens. 37 such characteristics have led to debate over the role of hbov as a true pathogen. in the present study, among the 180 hbov-positive specimens, there were 66 (36.67%) co-infections with at least one other respiratory virus. elderly people have also been reported to be susceptible to hbov. a single lineage of hbov was detected among a wide age distribution of patients with acute rti. in this study, a total of 18 adult specimens were positive for hbov. the virus has also been found in stool specimens from patients with gastrointestinal illness. 38, 39 therefore, additional evidence and studies are needed throughout the world to gain a better understanding of this virus. this study adds to the knowledge of seasonal variations of respiratory viral infections in southern china. the epidemiology of respiratory viral infection was found to vary tremendously by geographical region. in temperate climates, the prevalence of these viruses is well documented as a cause of yearly winter epidemics of acute lower rtis. 40 this study clearly showed evidence of seven viruses throughout the 3-year study period. the highest rate of infection was in spring, with a peak in february (48.03%), and the lowest rates of infection were in winter, with a nadir in october (23.14%), which may be attributed to the fact that february is the coldest month in southern china. the detection rates for summer and autumn were similar and this is likely due to high humidity and a lack of significant delimitation between summer and autumn. with recent advances in the detection of respiratory agents, numerous studies have shown that some pediatric patients with acute lower rtis become infected simultaneously with multiple respiratory viruses. 41, 42 multiple viral infections have been linked in some reports to higher fever, a longer hospital stay, more frequent use of antibiotics, and an increased risk of admission to the icu. 43 however, there is no consensus regarding the effect of co-infection on the severity of disease. the effect may depend upon which viruses co-infect. 44 in the present study, 3.39% of specimens had multiple or dual infections, with a predominance of flu compared to the other co-viruses. however, in the positive specimens, the co-infection rates for hcov and hbov were higher than those of other respiratory viruses. the co-infection rates were higher in male patients, inpatients, and patients aged 14 years. the rates of co-infection were higher during spring and summer than during autumn and winter. the rates of co-infection were lowest during winter, which corresponded with the overall low detection rates in winter. ethical approval: all research involving human participants was approved by the institutional review board of zhongshan school of medicine, sun yat-sen university, in accordance with the guidelines for the protection of human subjects. participants received 'written informed consent' about the purpose of the study and their right to keep information confidential. written consent was obtained from all participants or their guardians. conflict of interest: the authors declare that they have no conflicts of interest. global burden of acute respiratory infections in children: implications for interventions estimates of world-wide distribution of child deaths from acute respiratory infections population-based surveillance for hospitalizations associated with respiratory syncytial virus, influenza virus, and parainfluenza viruses among young children medical burden of respiratory syncytial virus and parainfluenza virus type 3 infection among us children. implications for design of vaccine trials global burden of respiratory infections due to seasonal influenza in young children: a systematic review and meta-analysis global burden of acute lower respiratory infections due to respiratory syncytial virus in young children: a systematic review and meta-analysis epidemiology of respiratory viral infections in two long-term refugee camps in kenya etiology and clinical characterization of respiratory virus infections in adult patients attending an emergency department in beijing parainfluenza virus infection of young children: estimates of the populationbased burden of hospitalization characterization of human coronavirus etiology in chinese adults with acute upper respiratory tract infection by real-time rt-pcr assays epidemiology of respiratory virus infections among infants and young children admitted to hospital in oman novel human bocavirus in children with acute respiratory tract infection epidemiology of human metapneumovirus respiratory syncytial virus and parainfluenza virus mortality attributable to 9 common infections: significant effect of influenza a, respiratory syncytial virus, influenza b, norovirus, and parainfluenza in elderly persons characterization of a novel coronavirus associated with severe acute respiratory syndrome the 1918 'spanish' flu: pearls from swine human respiratory syncytial virus in children with acute respiratory tract infections in china genetic variability of respiratory syncytial viruses (rsv) prevalent in southwestern china from 2006 to 2009: emergence of subgroup b and a rsv as dominant strains molecular monitoring of causative viruses in child acute respiratory infection in endemo-epidemic situations in shanghai simultaneous detection of influenza a, b, and c viruses, respiratory syncytial virus, and adenoviruses in clinical samples by multiplex reverse transcription nested-pcr assay simultaneous detection of fourteen respiratory viruses in clinical specimens by two multiplex reverse transcription nested-pcr assays characterization and complete genome sequence of a novel coronavirus, coronavirus hku1, from patients with pneumonia detection of adenoviruses in stools from healthy persons and patients with diarrhea by two-step polymerase chain reaction bocavirus infection in hospitalized children who estimates of the causes of death in children seasonal influenza vaccines the absence of enhanced disease with wild type respiratory syncytial virus infection occurring after receipt of live, attenuated, respiratory syncytial virus vaccines current status of vaccines for parainfluenza virus infections parainfluenza viruses parainfluenza virus type 3: seasonality and risk of infection and reinfection in young children molecular characterization and phylogenetic analysis of human parainfluenza virus type 3 isolated from saudi arabia cloning of a human parvovirus by molecular screening of respiratory tract samples human bocavirus infection in young children in the united states: molecular epidemiological profile and clinical characteristics of a newly emerging respiratory virus human bocavirus: a novel parvovirus epidemiologically associated with pneumonia requiring hospitalization in thailand human bocavirus in italian patients with respiratory diseases frequent detection of human rhinoviruses, paramyxoviruses, coronaviruses, and bocavirus during acute respiratory tract infections human bocavirus infection in children with acute gastroenteritis in japan and thailand human bocavirus 1 and 3 infection in children with acute gastroenteritis in brazil respiratory syncytial virus infection in tropical and developing countries evaluation of viral co-infections in hospitalized and non-hospitalized children with respiratory infections using microarrays multipathogen infections in hospitalized children with acute respiratory infections dual infection of infants by human metapneumovirus and human respiratory syncytial virus is strongly associated with severe bronchiolitis multiple versus single virus respiratory infections: viral load and clinical disease severity in hospitalized children we owe our special thanks to the four collaborating institutes (the centre for disease control and prevention of guangdong province, guangzhou centre for disease control and prevention of the city, the affiliated pearl river hospital of southern medical university, and the supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ijid.2014.02.019. key: cord-319877-izn315hb authors: de wit, emmie; van doremalen, neeltje; falzarano, darryl; munster, vincent j. title: sars and mers: recent insights into emerging coronaviruses date: 2016-06-27 journal: nat rev microbiol doi: 10.1038/nrmicro.2016.81 sha: doc_id: 319877 cord_uid: izn315hb the emergence of middle east respiratory syndrome coronavirus (mers-cov) in 2012 marked the second introduction of a highly pathogenic coronavirus into the human population in the twenty-first century. the continuing introductions of mers-cov from dromedary camels, the subsequent travel-related viral spread, the unprecedented nosocomial outbreaks and the high case-fatality rates highlight the need for prophylactic and therapeutic measures. scientific advancements since the 2002–2003 severe acute respiratory syndrome coronavirus (sars-cov) pandemic allowed for rapid progress in our understanding of the epidemiology and pathogenesis of mers-cov and the development of therapeutics. in this review, we detail our present understanding of the transmission and pathogenesis of sars-cov and mers-cov, and discuss the current state of development of measures to combat emerging coronaviruses. supplementary information: the online version of this article (doi:10.1038/nrmicro.2016.81) contains supplementary material, which is available to authorized users. mers 12 , and a cluster of three cases of mers in the uk was identified in september 2012 (ref. 13 ). mers-cov continued to emerge and spread to countries outside of the arabian peninsula as a result of travel of infected persons; often, these imported mers cases resulted in nosocomial transmission. in may 2015, a single person returning from the middle east started a nosocomial outbreak of mers in south korea that involved 16 hospitals and 186 patients 14 . as of 26 april 2016, there have been 1,728 confirmed cases of mers, including 624 deaths in 27 countries 15 . this review highlights the pandemic and epidemic potential of emerging coronaviruses and discusses our current knowledge of the biology of sars-cov and mers-cov, including their transmission, their pathogenesis and the development of medical countermeasures. key features of these viruses are the dominance of nosocomial transmission, and pathogenesis that is driven by a combination of viral replication in the lower respiratory tract and an aberrant host immune response. several potential treatments for sars and mers have been identified in animal and in vitro models, including small-molecule protease inhibitors, neutralizing antibodies and inhibitors of the host immune response. however, efficacy data from human clinical trials are lacking but are needed to move these potential countermeasures forward. respectively (fig. 1a) . similarly to all viruses in the order nidovirales, sars-cov and mers-cov have a unique coding strategy: two-thirds of the viral rna is translated into two large polyproteins, and the remainder of the viral genome is transcribed into a nested set of subgenomic mrnas 16, 17 (fig. 1b) . the two polyproteins, pp1a and pp1ab, encode 16 non-structural proteins (nsp1-nsp16) 18 that make up the viral replicase-transcriptase complex. the polyproteins are cleaved by two proteases, papainlike protease (plpro; corresponding to nsp3) and a main protease, 3c-like protease (3clpro; corresponding to nsp5). the nsps rearrange membranes that are derived from the rough endoplasmic reticulum (rer) into double-membrane vesicles, in which viral replication and transcription occur 19 . one unique feature of coronaviruses is the exoribonuclease (exon) function of nsp14 (ref. 20) , which provides the proofreading capability required to maintain a large rna genome without the accumulation of detrimental mutations 21, 22 . sars-cov and mers-cov transcribe 12 and 9 subgenomic rnas, er-golgi intermediate compartment (ergic) . a cellular compartment that facilitates transport between the endoplasmic reticulum (er) and the golgi complex. infected individuals who each infect a disproportionately large number of secondary cases. respectively, and these encode the four structural proteins spike (s), envelope (e), membrane (m) and nucleocapsid (n), as well as several accessory proteins that are not involved in viral replication but interfere with the host innate immune response or are of unknown or poorly understood function. the envelope spike glycoprotein binds to its cellular receptor, angiotensin-converting enzyme 2 (ace2) for sars-cov and dipeptidyl peptidase 4 (dpp4) for mers-cov 23 . after membrane fusion, either directly with the host cell membrane or with the endosome membrane, the viral rna genome is released into the cytoplasm, and the rna is uncoated to allow translation of the two polyproteins, transcription of the subgenomic rnas and replication of the viral genome (fig. 1b) . newly formed envelope glycoproteins are inserted in the rer or golgi membranes; genomic rna and nucleocapsid proteins combine to form nucleocapsids, and the viral particles bud into the er-golgi intermediate compartment (ergic). virion-containing vesicles subsequently fuse with the plasma membrane to release the virus 24 . the first indication of the source of sars-cov was the detection of the virus in masked palm civets and a raccoon dog and the detection of antibodies against the virus in chinese ferret badgers in a live-animal market in shenzhen, china 25 . however, these animals were only incidental hosts, as there was no evidence for the circulation of sars-cov-like viruses in palm civets in the wild or in breeding facilities 26 . rather, bats are the reservoir of a wide variety of coronaviruses, including sars-cov-like and mers-cov-like viruses 27 (fig. 2) . thus, the search for the reservoir of mers-cov initially focused on bats, but a serological survey in dromedary camels from oman and the canary islands showed a high prevalence of mers-cov-neutralizing antibodies in these animals 28 . in addition, mers-cov rna was detected in swabs that were collected from dromedary camels at a farm in qatar that was linked to two human cases of mers, and infectious virus was isolated from dromedary camels in saudi arabia and qatar [29] [30] [31] [32] . serological evidence for the circulation of a mers-cov-like virus in dromedary camels has been obtained in the middle east, eastern africa and northern africa, dating back as far as 1983 (ref. 33 ). dromedary camels in saudi arabia harbour several viral genetic lineages 34 , including those that have caused human outbreaks. taken together, these data strongly point to the role of dromedary camels as a reservoir for mers-cov. the ubiquity of infected dromedary camels close to humans and the resulting continuing zoonotic transmission may explain why mers-cov continues to cause infections in humans, whereas sars-cov, without the continuing presence of an infected intermediate host and with relatively infrequent human-bat interactions, has caused no more infections in humans. human-to-human transmission of sars-cov and mers-cov occurs mainly through nosocomial transmission; 43.5-100% of mers cases in individual outbreaks were linked to hospitals, and very similar observations were made for some of the sars clusters 35, 36 . transmission between family members occurred in only 13-21% of mers cases and 22-39% of sars cases. transmission of mers-cov between patients was the most common route of infection (62-79% of cases), whereas for sars-cov, infection of health care workers by infected patients was very frequent (33-42%) 35 . the predominance of nosocomial transmission is probably due to the fact that substantial virus shedding occurs only after the onset of symptoms 37, 38 , when most patients are already seeking medical care 39 . an analysis of hospital surfaces after the treatment of patients with mers showed the ubiquitous presence of viral rna in the environment for several days after patients no longer tested positive 40 . moreover, many patients with sars or mers were infected through super spreaders 14, 35, 37, [41] [42] [43] . the clinical courses of sars and mers are remarkably similar, although there are subtle differences (box 1) . owing to the current sparsity of data on human mers-cov infections 44 , the pathogenesis of this virus is poorly understood; however, similar mechanisms may underlie the pathogenesis of both mers and sars. the binding of spike protein to ace2 and the subsequent downregulation of this receptor contribute to lung injury during sars 45 . although it seems counterintuitive that receptor downregulation would increase pathology, it has been shown that ace2 can protect against acute lung injury. the downregulation of ace2 results in the excessive production of angiotensin ii by the related enzyme ace, and it has been suggested that the stimulation of type 1a angiotensin ii receptor and middle east respiratory syndrome coronavirus (mers-cov) encode two large polyproteins, pp1a and pp1ab, which are proteolytically cleaved into 16 non-structural proteins (nsps), including papain-like protease (plpro), 3c-like protease (3clpro), rna-dependent rna polymerase (rdrp), helicase (hel) and exonuclease (exon). an additional 9-12 orfs are encoded through the transcription of a nested set of subgenomic rnas. sars-cov and mers-cov form spherical particles that consist of four structural proteins. the envelope glycoprotein spike (s) forms a layer of glycoproteins that protrude from the envelope. two additional transmembrane glycoproteins are incorporated in the virion: envelope (e) and membrane (m). inside the viral envelope resides the helical nucleocapsid, which consists of the viral positive-sense rna ((+)rna) genome encapsidated by protein nucleocapsid (n). b | following entry of the virus into the host cell, the viral rna is uncoated in the cytoplasm. orf1a and orf1ab are translated to produce pp1a and pp1ab, which are cleaved by the proteases that are encoded by orf1a to yield 16 nsps that form the rna replicase-transcriptase complex. this complex localizes to modified intracellular membranes that are derived from the rough endoplasmic reticulum (er) in the perinuclear region, and it drives the production of negative-sense rnas ((-)rnas) through both replication and transcription. during replication, full-length (-)rna copies of the genome are produced and used as templates for full-length (+)rna genomes. during transcription, a subset of 7-9 subgenomic rnas, including those encoding all structural proteins, is produced through discontinuous transcription. in this process, subgenomic (-)rnas are synthesized by combining varying lengths of the 3′end of the genome with the 5′ leader sequence necessary for translation. these subgenomic (-)rnas are then transcribed into subgenomic (+)mrnas. although the different subgenomic mrnas may contain several orfs, only the first orf (that closest to the 5′end) is translated. the resulting structural proteins are assembled into the nucleocapsid and viral envelope at the er-golgi intermediate compartment (ergic), followed by release of the nascent virion from the infected cell. one of a panel of recombinant inbred mouse strains derived from a genetically diverse set of founder strains and designed for the analysis of complex traits. the aggregation of leukocytes around blood vessels. (agtr1a) increases pulmonary vascular permeability, thus potentially explaining the increased lung pathology when the expression of ace2 is decreased 46 . immunopathology. the immune response is essential for the resolution of an infection, but it can also result in immunopathogenesis. one indication that immunopathogenesis may contribute to sars was the observation that viral loads were found to be decreasing while disease severity increased 39, 47 . it is unclear whether a similar trend applies to mers 48, 49 . moreover, progression to acute respiratory distress syndrome (ards) is associated with the upregulation of pro-inflammatory cytokines and chemokines, particularly interleukin-1β (il-1β), il-8, il-6, cxc-chemokine ligand 10 (cxcl10) and cc-chemokine ligand 2 (ccl2) 50, 51 ; increased plasma levels of these molecules have been detected in patients with sars [52] [53] [54] [55] . retrospective longitudinal studies in patients who recovered from sars versus those who succumbed to the disease have shown an early expression of interferon-α (ifnα), ifnγ, cxcl10, ccl2 and proteins that are encoded by ifn-stimulated genes (isgs) in all patients, but only patients who survived then had gene expression profiles that are indicative of the development of an adaptive immune response. by contrast, patients who succumbed maintained high levels of cxcl10, ccl2 and isg-encoded proteins, whereas spike-specific antibodies were present at low levels or were absent 56 , which suggests that severe disease is related to the lack of a switch from an innate immune response to an adaptive immune response. experiments using collaborative cross mouse lines and mouse-adapted sars-cov identified one host gene, trim55, as important for sars pathogenesis. although there was no difference in clinical signs or viral replication in trim55 −/− mice compared with wild-type mice, perivascular cuffing and the number of inflammatory cells in the lungs were reduced in the trim55 −/− mice 57 . a biological process in which small rna molecules induce the degradation of specific mrna molecules, thereby inhibiting gene expression. systems in which a dna molecule is produced that contains the viral leader and trailer sequences, with an assayable reporter replacing the viral orfs. when combined with the expression of viral proteins in trans, this system can be used to model the viral life cycle without the necessity of using infectious virus. the involvement of the host immune response in the pathogenesis of sars, and most likely also that of mers, suggests that drugs which inhibit viral replication will need to be combined with treatments that control detrimental immune responses. immune evasion. sars-cov and mers-cov use several strategies to avoid the innate immune response. viral pathogen-associated molecular patterns (pamps), such as double-stranded rna (dsrna) or uncapped mrna, are detected by pattern recognition receptors (prrs), such as retinoic acid-inducible gene i protein (rig-i; also known as ddx58) or melanoma differentiation-associated protein 5 (mda5; also known as ifih1) 58 . this triggers complex signalling cascades involving myd88 that lead to the production of type i ifns and the activation of the transcription factor nuclear factor-κb (nf-κb). in turn, active nf-κb induces the transcription of pro-inflammatory cytokines (fig. 3a) . type i ifns signal through ifnα/β receptor (ifnar) and downstream molecules such as signal transducer and activator of transcription (stat) proteins to stimulate the production of antiviral proteins that are encoded by isgs, such as ifn-induced protein with tetratricopeptide repeats 1 (ifit1 ; fig. 3b ). collectively, this establishes an antiviral immune response that limits viral replication in infected and in neighbouring cells (fig. 3) . infection of knockout mice revealed the importance of innate immunity. infection of myd88 −/− and stat1 −/− mice, but not mice that were deficient in ifn receptors, with a mouse-adapted strain of sars-cov resulted in more severe disease than infection with a non-adapted sars-cov strain 59, 60 . moreover, mers-cov infection of wild-type mice that were transduced with human dpp4 caused mild disease, but symptoms were more severe in myd88 −/− mice and ifnar1 −/− mice 61 . sars-cov and mers-cov avoid host detection of their dsrna by replicating in virus-induced doublemembrane vesicles that lack prrs 19, 62, 63 . moreover, the recognition of sars-cov mrnas, for example, by mda5 and ifit1 is prevented by capping of the viral mrnas by nsp14 and the nsp10-nsp16 complex 64 . recombinant sars-cov that lacks the methylation activity of nsp16 is attenuated and exhibits increased sensitivity to type i ifns. this effect is dependent on ifit1 or mda5, as the same virus is not attenuated in mice that are deficient in either molecule 65 . although mrna capping has not yet been shown for mers-cov, structural similarity between the mers-cov nsp10-nsp16 complex and the sars-cov nsp10-nsp16 complex suggests that a similar mechanism exists to avoid host recognition of mers-cov mrnas by cytosolic prrs 66 . sars-cov encodes at least eight proteins that interact with the signalling cascades downstream of prrs; in mers-cov, several proteins have been identified with similar functions (fig. 3) . the nucleocapsid protein of sars-cov has been associated with the suppression of rnai in mammalian cells 67 . furthermore, this protein antagonizes ifn induction, probably early in the signalling cascade, as downstream signalling molecules relieve the inhibition 68 . mers-cov orf4a has a similar ifn-antagonistic function, involving the binding of dsrna and subsequent inhibition of mda5 activation 69 , potentially through interaction with ifn-inducible dsrna-dependent protein kinase activator a (prkra; also known as pact), which interacts with mda5 and rig-i 70 . moreover, mers-cov orf4a, orf4b, orf5 and membrane protein inhibit the nuclear trafficking of ifn-regulatory factor 3 (irf3) and activation of the ifnb promoter 71 . these viral proteins, except for orf5, also inhibit the expression of genes that are under the control of an ifn-stimulated response element (isre), and orf4a reduces the expression of genes that are stimulated by nf-κb 71 . finally, mers-cov orf4b interacts with tbk1 and inhibitor of nf-κb kinase-ε (ikkε), thereby suppressing the interaction between ikkε and mitochondrial antiviral-signalling protein (mavs) and inhibiting the phosphorylation of irf3 (ref. 72 ). the membrane protein of sars-cov inhibits the formation of a signalling complex that contains ikkε, thus repressing the activation of irf3 and irf7 and their induction of type i ifn expression. the membrane protein of mers-cov inhibits irf3 function and the expression of genes that are regulated by an isre, including ifnβ 71 , but whether this occurs through a mechanism similar to that of sars-cov is unclear. sars-cov plpro disrupts nf-κb signalling 73 and blocks the phosphorylation of irf3 indirectly 73, 74 . furthermore, sars-cov plpro inhibits the induction of type i ifns, potentially through the deubiquitylation of phosphorylated irf3 (refs 73, 75) . similar functions have been described for mers-cov plpro 76 . experiments involving recombinantly expressed proteins, in vitro translation, protein overexpression and minireplicon systems have shown that nsp1 of sars-cov blocks the ifn response through the inhibition of severe acute respiratory syndrome coronavirus (sars-cov) and middle east respiratory syndrome coronavirus (mers-cov) have an incubation period of ~5 days, and 95% of patients develop disease within 13 days of exposure 14, 38, [144] [145] [146] . common early symptoms are fever, chills, coughing, malaise, myalgia and headache, and less common symptoms include diarrhoea, vomiting and nausea 2, 6, 39, 89, 90, 92, 95, 144, [146] [147] [148] . upper respiratory tract symptoms and viral shedding are rare, which explains difficulties in obtaining a laboratory diagnosis from nasal or nasopharyngeal swabs 149 . abnormal chest x-rays are more common in patients with mers (90-100%) 144,148 than in those with sars (60-100%) 39, 89 . accordingly, only 20-30% of patients with sars require intensive care and subsequent mechanical ventilation, whereas 50-89% of patients with mers require intensive care 2, 39, 89, 90, 95, 144, 147, 148 . the higher incidence of acute respiratory distress syndrome (ards) in individuals with mers is reflected in the case fatality rate: this is ~36% for mers compared with ~10% for sars 15, 145 . comorbidities have an important role in both sars and mers. several risk factors are associated with poor disease outcome, especially advanced age and male sex 2, 14, 39, 144, 146, 148, 150, 151 . for mers, additional risk factors for a poor outcome include diabetes mellitus, hypertension, cancer, renal and lung disease, and co-infections 14, 144, 146, 148, 150, 151 . health care settings seem to increase the risk of viral transmission owing to aerosol-generating procedures such as intubation and bronchoscopy. appropriate hospital hygiene practices and awareness are crucial to limit future nosocomial outbreaks. both nsp7 and nsp15 from sars-cov were also suggested to be ifn antagonists, but the underlying mechanism is unknown 73 . nsp15 is an inhibitor of mavsinduced apoptosis; however, this occurs through an ifn-independent mechanism 84 . finally, transcriptomic and proteomic analysis of human airway cell cultures showed that mers-cov but not sars-cov induces repressive histone modifications that downregulate the expression of certain isgs 85 . it should be noted that most of the interactions of sars-cov and mers-cov proteins with innate immune pathways were established in in vitro systems, which rely on the overexpression of viral and, . following prr-mediated detection of a pamp, the resulting interaction of prrs with mitochondrial antiviral-signalling protein (mavs) activates nuclear factor-κb (nf-κb) through a signalling cascade involving several kinases. activated nf-κb translocates to the nucleus, where it induces the transcription of pro-inflammatory cytokines. the kinases also phosphorylate (p) ifn-regulatory factor 3 (irf3) and irf7, which form homodimers and heterodimers and enter the nucleus to initiate the transcription of type i interferons (type i ifns). both severe acute respiratory syndrome coronavirus (sars-cov) and middle east respiratory syndrome coronavirus (mers-cov) have developed mechanisms to interfere with these signalling pathways, as shown; these subversion strategies involve both structural proteins (membrane (m) and nucleocapsid (n)) and non-structural proteins (nsp1, nsp3b, nsp4a, nsp4b, nsp5, nsp6 and papain-like protease (plpro); indicated in the figure by just their nsp numbers and letters). b | binding of type i ifns to their dimeric receptor, ifnα/β receptor (ifnar), activates the janus kinase (jak)-signal transducer and activator of transcription (stat) signalling pathway, in which jak1 and tyk2 kinases phosphorylate stat1 and stat2, which form complexes with irf9. these complexes move into the nucleus to initiate the transcription of ifn-stimulated genes (isgs) under the control of promoters that contain an ifn-stimulated response element (isre). collectively, the expression of cytokines, ifns and isgs establishes an antiviral innate immune response that limits viral replication in infected and in neighbouring cells. again, viral proteins have been shown to inhibit these host signalling pathways to evade this immune response. iκbα, nf-κb inhibitor-α. a broadly active antiviral nucleoside analogue with several direct and indirect mechanisms of action; mainly used for the treatment of hepatitis c, in combination with interferon. having polyethylene glycol (peg) attached, to a drug for example; this moiety improves the solubility, decreases the immunogenicity and increases the stability, of the drug of interest, thereby allowing a reduced dosing frequency to be used. (table 1) and sars-cov, including the use of antibodies, ifns, inhibitors of viral and host proteases, and host-directed therapies. in the absence of a clinically proven effective antiviral therapy against sars-cov and mers-cov, patients mainly receive supportive care, which is often supplemented by different combinations of drugs. ribavirin 86 and various types of ifn have been given to patients with mers in saudi arabia 87 and china 88 , typically in combination with a broad-spectrum antibiotic and oxygen. the efficacy of treatments for sars-cov and mers-cov infection currently remains unclear. in addition, treatment for mers is typically started only in a late disease stage, when immunopathology predominates and antiviral drugs are likely to provide little benefit. ribavirin was used most frequently during the sars outbreak, often in combination with corticosteroids, which have an anti-inflammatory effect 2,89-92 . ifnα was also given, usually in combination with immunoglobulins or thymosins, which stimulate the development of t cells, and in a small number of cases in combination with ribavirin 93, 94 . none of these treatments was tested in a clinical trial, which makes it difficult to assess their efficacy. in fact, retrospective analysis did not yield a treatment combination that was clearly effective. moreover, the data from patients are contradictory about whether ribavirin, when used alone, provided a benefit or was possibly even detrimental 89, 90, 92, 95 . in vitro coronaviruses have a lower sensitivity to ribavirin than other viruses. deletion of the nsp14-encoding sequence increases the sensitivity of coronaviruses to ribavirin; however, the underlying mechanism is unclear and is not related to the proofreading function of nsp14 (ref. 96 ). therefore, ribavirin should be considered only in combination with other antiviral treatments. although ifns are effective against mers-cov in vitro [97] [98] [99] , their effect in humans has yet to be proved. the effectiveness of ifn is increased in vitro if ribavirin is added 98, 100 , and a combined use of the two drugs reduces disease severity in a rhesus macaque model of mers 101 . the potential side effects of these treatments, such as fatigue, depression and anaemia, have inhibited their use as a first-line treatment for mers, and they are generally administered only after a patient's condition starts to deteriorate. for example, one study of five patients who were infected with mers-cov indicated no survival following ribavirin and ifnα2b therapy; however, therapy was not started until 10 days after admission 87 . a separate study found an improvement in survival 14 days after mers diagnosis and the start of treatment, but not 28 days after 102 . in a third study, a combination of ifnα2a and ribavirin or ifnβ1a and ribavirin did not improve survival; however, some of the patients were more than 50 years old and had preexisting renal failure 103 . in a single case in which ribavirin and ifnα2b were started shortly after admission to hospital, the patient started to improve on day 6 after admission and made a complete recovery 104 . ifnβ1b is a more potent inhibitor of mers-cov replication in vitro than other types of ifn 97, 99 , and an improved outcome of disease was observed in common marmosets after challenge with mers-cov 105 . thus, the type of ifn that is used for treatment in humans should be reconsidered (usually, ifnα is used). furthermore, ribavirin and/or ifns should be tested in clinical trials to determine their efficacy in mers treatment and to establish treatment protocols. additional antiviral treatments. the protease inhibitors lopinavir and ritonavir, which are used in combination to treat infection with hiv, improved the outcome of patients with sars when combined with ribavirin, compared with patients who were treated with ribavirin alone 106, 107 . lopinavir showed no clear antiviral activity against mers-cov in vitro 97 , and it is thus rarely used in patients with mers. however, lopinavir and ritonavir improve the outcome in common marmosets when treatment is initiated 6 hours after infection with mers-cov 105 . thus, the testing of lopinavir and ritonavir in clinical trials in patients with mers should be reconsidered. one patient who received pegylated ifnα, ribavirin, lopinavir and ritonavir in combination had undetectable levels of mers-cov in the blood 2 days after the initiation of therapy; however, this patient did not survive 108 . the combination of ifnα, ribavirin, lopinavir and ritonavir was also used for mers treatment in south korea, but efficacy data are not yet available. however, three case reports indicate recovery in five out of seven patients who were treated with this combination [109] [110] [111] . as 3clpro and plpro are essential for cleavage of the viral polyproteins and are distinct from cellular proteases, they are ideal drug targets, in particular plpro, compounds that mimic biologically active peptides or proteins. a complement-activated molecule that is important for the recruitment to and activation of inflammatory cells in the lungs. which is involved in both viral replication and ifn antagonism. indeed, most antiviral drug-like molecules have been developed against 3clpro and plpro, which was aided by the rapid report of crystal structures of these proteases 112 . plpro was initially identified as a drugable target for sars-cov; recently, it has been noted that some of the compounds that target plpro from sars-cov are also active against plpro from mers-cov. for example, both 6-mercaptopurine and 6-thioguanine inhibit mers-cov and sars-cov in vitro 113 ; however, the efficacy of these molecules has not yet been tested in vivo. mycophenolic acid also inhibits the replication of mers-cov in vitro 97, 99 through the inhibition of plpro 113 , but it had no effect in marmosets 105 . as new coronaviruses are likely to emerge from bats, protease inhibitors were designed against bat coronaviruses with the goal of developing a universal antiviral compound against emerging zoonotic coronaviruses. this approach yielded an inhibitor of tylonycteris bat coronavirus hku4 (hku4-cov), which is closely related to mers-cov 11 . this inhibitor, named sg85, indeed inhibits mers-cov replication in vitro 112 . similarly, peptidomimetics that target and inhibit 3clpro of mers-cov, hku4-cov and pipistrellus bat coronavirus hku5 (hku5-cov) have also been identified, but have not yet progressed beyond the in vitro stage 114 . several other drugs that were approved for use in humans were shown to inhibit the replication of mers-cov in vitro, notably chloroquine, chlorpromazine, loperamide and cyclosporine a 62, 99, 113, 115 , although their mechanisms of action are unknown, and the benefit of cyclosporine a in patients is debatable owing to the immunosuppressive effect of the drug. although cyclophilin inhibitors that do not result in immunosuppression are available, their activity against mers-cov has not yet been tested. antibody and plasma therapy. plasma from convalescent patients and/or antibody therapies have been the leading proposed treatment for mers so far 116 . there are several potential advantages to this approach. for example, as case numbers increase, the pool of survivors becomes larger; provided these individuals have sufficiently high antibody titres and are willing and able to donate plasma, this is a low-tech, reasonably safe treatment option. furthermore, generation of monoclonal antibodies for use in humans is well established, with a fairly straightforward path to safety and efficacy testing. however, to date, there are very few reports on the use of convalescent plasma and none on the use of monoclonal antibodies as treatments for acute, severe respiratory disease in humans. a post hoc meta-analysis of 32 studies of either sars or severe influenza found a significant reduction in the pooled odds of mortality when convalescent plasma was used 117 . however, study design was rated as low or very low quality, as there were generally a lack of control groups and a moderate-to-high risk of bias, which suggests that a properly designed clinical trial of convalescent plasma use in severe respiratory infections is needed 117 . potent monoclonal antibodies that neutralize the mers-cov spike protein in vitro have been developed [118] [119] [120] [121] . however, with a few exceptions, in vivo data relating to the use of convalescent plasma or monoclonal antibodies in the treatment of mers are currently lacking. serum from high-titre dromedary camels decreased mers-cov loads in the lungs of mice that had been transduced with human dpp4 (ref. 122 ). human polyclonal antibodies against the spike protein were generated by vaccinating transchromosomic bovines, and treatment with these antibodies reduced viral titres in the lungs of dpp4-transduced mice when treatment was administered 24 or 48 hours after challenge with mers-cov 123 . dpp4-transduced mice were also treated with humanized neutralizing monoclonal antibody 4c2h, which is directed against the receptor-binding domain of the mers-cov spike protein, 1 day after mers-cov challenge, and this treatment also decreased viral titres in the lungs 124 , as did the neutralizing antibody lca60, which was obtained from a convalescent patient and produced recombinantly 125 . human neutralizing monoclonal antibodies regn3048 and regn3051 also provided a benefit in mice that expressed human dpp4 and were challenged with mers-cov 126 . the human neutralizing monoclonal antibody m332 reduced mers-cov replication in the lungs of rabbits following prophylactic, but not therapeutic, treatment 127 . treatment of rhesus macaques with the human monoclonal antibody 311b-n1 day after challenge resulted in reduced lung pathology 128 . in all of these studies, viral replication was not completely inhibited, and there were some pathological alterations to the lungs, despite the therapy. furthermore, none of the studies addressed the potential emergence of escape mutants in vivo. alternatively, antibodies that target the region of dpp4 that binds to the spike protein could be used to prevent entry of mers-cov; this approach was successful in vitro 129 . however, whether such a treatment would be feasible and would not have substantial adverse effects in humans remains to be determined. host-directed strategies can also limit viral replication. for example, the spike protein of sars-cov is cleaved by cathepsin b and cathepsin l, transmembrane protease serine 2 (tmprss2) and possibly other host proteases 130, 131 . inhibition of host serine proteases by camostat reduced the entry of sars-cov and increased survival in a mouse model 132 . however, the targeting of host proteases is more likely to result in undesirable side effects than the targeting of viral proteases. another underappreciated strategy is attenuation of detrimental host responses. the development of such treatments would require a thorough understanding of the host responses that are involved in acute lung injury and ards, processes that are unfortunately poorly understood at the moment. nonetheless, in vitro studies and limited studies in animal models with other respiratory viruses have shown that anaphylatoxin c5a is important for the development of acute lung injury, and blocking anaphylatoxin c5a can reduce lung pathology 133 . vaccines that contain immunogenic parts of a pathogen rather than the entire pathogen. vaccines based on the direct introduction of a plasmid encoding an antigen; following in situ production of this antigen, an immune response is mounted against it. changes in gene expression during in vitro mers-cov infection were used to predict potential effective drugs. one of the drugs with predicted efficacy, the kinase inhibitor sb203580, modestly inhibited sars-cov and mers-cov replication following the treatment of cells prior to infection, but treatment after infection inhibited replication of only sars-cov and not mers-cov 134 . vaccines. vaccination could be used to prevent infection or to reduce disease severity, viral shedding and thereby transmission, thus helping to control mers outbreaks. several vaccination strategies were developed against sars-cov and tested in animals, such as an inactivated virus, a live-attenuated virus, viral vectors, subunit vaccines, recombinant proteins and dna vaccines 135, 136 . similar approaches have been used for the development of experimental mers-cov vaccines 137 . to date, three mers-cov vaccines have been evaluated in non-human primates. in one study, rhesus macaques were primed with dna encoding the spike protein, followed by boosts with spike dna and with recombinant protein consisting of the spike subunit containing the receptor-binding domain, or primed and boosted once with the subunit protein. both approaches reduced pathological changes in lung function in animals that were infected with mers-cov 19 weeks after the last vaccination 138 . moreover, three vaccinations with a recombinantly expressed protein that contains the receptor-binding domain of the spike protein reduced viral loads and lung pathology in rhesus macaques that were infected 2 weeks after the last vaccination 139 . three dna vaccinations with a construct encoding the full-length spike sequence reduced viral loads and pathology in the lungs after challenge with mers-cov 5 weeks after the last vaccination 140 . one concern of vaccination in humans is vaccinemediated enhancement of disease, a process in which the disease following infection is more severe in vaccinated individuals than in unvaccinated individuals. although this was observed in only a small subset of vaccine studies that were carried out for sars-cov 136 and has not yet been observed in any of the published mers-cov vaccine studies, it is an important concern. moreover, it is unclear who to vaccinate against mers-cov, as healthy individuals seem to be at little risk of severe disease. older patients or patients with underlying disease, who have the highest risk of severe mers, would be important target populations. however, vaccination in such patients can be problematic owing to their poor immune responses, as has been established for influenza virus 141 . in addition, vaccination of people with a high risk of exposure to mers-cov, such as health care workers, slaughterhouse workers and camel herders, is advisable 142 . as our understanding of the pathogenesis of emerging coronaviruses increases, so will the opportunities for the rational design of therapeutics that target viral replication or immunopathology. the rational design of new drugs and the repurposing of existing compounds have already resulted in the development of plpro inhibitors and the identification of kinase inhibitors that inhibit the replication of sars-cov and mers-cov in vitro. however, only a few potential treatments have progressed past the identification of an effect in vitro, and in vivo studies to select the most promising treatment options are required. the development of several mouse models of mers is thus an important step forward (box 2) . owing to the acute nature of mers and the important role of immunopathology, combination therapies aimed at simultaneously inhibiting viral replication, limiting viral dissemination and dampening the host response are likely to yield the best results. furthermore, treatment should be started as early as possible, rather than waiting until the patient has already developed extensive lung damage. the development of therapies against sars and mers needs to focus on testing in humans, in properly controlled clinical trials. the current non-standardized, uncontrolled approach to treatment is not informative and may not be beneficial to the patient. the recent ebola outbreak has demonstrated that rapid clinical trial design and approval are possible and that exceptional situations call for deviations from normal procedures . while treatments are being developed and evaluated, the prevention of viral transmission is key to reducing the burden of mers. the large proportion of nosocomial mers-cov infections indicates that preventive measures in hospitals are currently either not fully implemented or insufficient. prevention of zoonotic transmission from dromedary camels is another possibility to decrease the number of mers cases. the most of our understanding of the pathogenesis of severe acute respiratory syndrome (sars) and middle east respiratory syndrome (mers) comes from animal studies. ideally, these models recapitulate all or specific aspects of human disease. several mouse models have been established, for example by using mouse-adapted sars coronavirus (sars-cov) or expressing human receptors in mice 152 . although it has been recognized that mice might poorly mimic specific human responses to infection, the availability of knockout and transgenic mice enables the targeted study of virus-host interactions. several non-human primate models have been developed for sars-cov and mers coronavirus (mers-cov) 152 . the close relationship of non-human primates to humans often allows faithful recapitulation of a disease and the host response. however, these benefits are countered by the need for specialized husbandry, the sometimes limited availability of the animals and reagents, and high costs. the pathogenesis of sars-cov and mers-cov in their respective reservoir hosts is not nearly as well studied as that in humans. currently, only one experimental-infection study has been carried out in bats with mers-cov 153 , and none has been carried out for other coronaviruses. thus, data are mostly limited to the detection of coronaviruses in naturally infected bats. the detection of coronaviruses mainly in faecal samples from bats and not in oral swabs suggests that replication in bats occurs predominantly in the gastrointestinal tract 9,154,155 . by contrast, a combination of intratracheal and intranasal inoculation of masked palm civets with sars-cov resulted in interstitial pneumonia, with oral and rectal viral shedding 156 . the pathogenesis of mers-cov in dromedary camels has been studied experimentally in a limited number of animals. these animals developed transient mild disease; however, large quantities of mers-cov were shed from the upper respiratory tract, in line with the predominant replication of mers-cov in the nasal turbinates and larynx in these animals, which explains the frequent zoonotic transmission 157 . first vaccines against mers-cov have been tested in dromedary camels 140, 143 ; indeed, when camels were vaccinated with a modified vaccinia virus that expresses the mers-cov spike protein, subsequent challenge of these animals with mers-cov resulted in less viral shedding than in unvaccinated animals 143 , thereby potentially limiting the transmission to naive animals or to humans. certainly, there has been progress in the development of vaccines and therapies against emerging coronaviruses, but more research and rigorous testing is required if we are to successfully combat these novel pathogens. when the severe acute respiratory syndrome (sars) outbreak developed into the first pandemic of the twenty-first century, it became clear that the medical and scientific communities were not adequately prepared for the emergence of highly pathogenic viruses. whereas several months elapsed and several thousand cases of sars were observed before the causative agent was identified as sars coronavirus (sars-cov) 4-6 , subsequent advances in molecular diagnostic tools, such as next generation sequencing, meant that middle east respiratory syndrome coronavirus (mers-cov) was identified before it caused a large outbreak of mers 11 . the availability of the full-length genome of mers-cov enabled the rapid development and distribution of diagnostic assays. the first animal models of disease, several treatment efficacy studies and the identification of the reservoir followed soon after. unfortunately, the sars pandemic did not yield solid clinical data on the efficacy of treatment regimens. these data are urgently needed for the treatment of mers, as well as to prepare for novel coronaviruses that may emerge. several studies have used synthetic biology to study the zoonotic transmission potential of sars-cov-like viruses from bats 9,10,158,159 . the ebola virus outbreak in west africa has highlighted the need for fast-tracking of potential treatments, as several clinical trials were started only towards the end of the outbreak. the combined experiences of the outbreaks of sars, mers and ebola provide a blueprint for the response to emerging pathogens: after the identification of the causative agent, diagnostic assays need to be developed and distributed rapidly, and simultaneously, awareness of the new syndrome and reporting of (suspected) cases must be increased. in addition, infection control measures in health care facilities are essential. research needs to focus on understanding the epidemiology, including pathogen transmission and identification of the reservoir and/or intermediate hosts. animal models need to be developed, as well as therapeutic and prophylactic measures. finally, promising treatments need to be fast-tracked into clinical trials. epidemiology and cause of severe acute respiratory syndrome (sars) in guangdong, people's republic of china a major outbreak of severe acute respiratory syndrome in hong kong molecular epidemiology of the novel coronavirus that causes severe acute respiratory syndrome identification of a novel coronavirus in patients with severe acute respiratory syndrome a novel coronavirus associated with severe acute respiratory syndrome coronavirus as a possible cause of severe acute respiratory syndrome summary of probably sars cases with onset of illness from 1 sars-cov infection in a restaurant from palm civet the isolation of a bat sars-cov-like virus that uses the human ace2 as a receptor without prior adaptation a sars-like cluster of circulating bat coronaviruses shows potential for human emergence the first identification of mers-cov as the cause of severe lower respiratory disease in humans patient with new strain of coronavirus is treated in intensive care at london hospital middle east respiratory syndrome coronavirus outbreak in the republic of korea who. coronavirus infections: disease outbreak news. who nidovirus transcription: how to make sense…? coronaviruses post-sars: update on replication and pathogenesis coronaviruses: an overview of their replication and pathogenesis sars-coronavirus replication is supported by a reticulovesicular network of modified endoplasmic reticulum unique and conserved features of genome and proteome of sars-coronavirus, an early split-off from the coronavirus group 2 lineage infidelity of sars-cov nsp14-exonuclease mutant virus replication is revealed by complete genome sequencing insights into rna synthesis, capping, and proofreading mechanisms of sars-coronavirus dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-emc isolation and characterization of viruses related to the sars coronavirus from animals in southern china ecology, evolution and classification of bat coronaviruses in the aftermath of sars middle east respiratory syndrome coronavirus neutralizing serum antibodies in dromedary camels: a comparative serological study middle east respiratory syndrome coronavirus in dromedary camels: an outbreak investigation evidence for camel-to-human transmission of mers coronavirus mers coronavirus in dromedary camel herd, saudi arabia isolation of mers coronavirus from a dromedary camel co-circulation of three camel coronavirus species and recombination of mers-covs in saudi arabia transmission characteristics of mers and sars in the healthcare setting: a comparative study an analysis of the predominant role for nosocomial transmission in the epidemiology of both sars and mers transmission of middle east respiratory syndrome coronavirus infections in healthcare settings epidemiology, transmission dynamics and control of sars: the 2002-2003 epidemic preliminary epidemiological assessment of mers-cov outbreak in south korea clinical progression and viral load in a community outbreak of coronavirus-associated sars pneumonia: a prospective study a description of the clinical representation of sars-cov respiratory disease in patients from hong kong environmental contamination and viral shedding in mers patients during mers-cov outbreak in south korea evidence that infectious mers-cov can be detected on common hospital surfaces during an outbreak, which highlights the potential for nosocomial transmission and stresses the need for infection control the role of superspreading in middle east respiratory syndrome coronavirus (mers-cov) transmission middle east respiratory syndrome coronavirus superspreading event involving 81 persons the role of super-spreaders in infectious disease clinicopathologic, immunohistochemical, and ultrastructural findings of a fatal case of middle east respiratory syndrome coronavirus infection in the united arab emirates a crucial role of angiotensin converting enzyme 2 (ace2) in sars coronavirus-induced lung injury angiotensin-converting enzyme 2 protects from severe acute lung failure temporal relationship of viral load, ribavirin, interleukin (il)-6, il-8, and clinical progression in patients with severe acute respiratory syndrome clinical features and virological analysis of a case of middle east respiratory syndrome coronavirus infection kinetics and pattern of viral excretion in biological specimens of two mers-cov cases biomarkers in acute respiratory distress syndrome the mercurial nature of neutrophils: still an enigma in ards? sars-cov virus-host interactions and comparative etiologies of acute respiratory distress syndrome as determined by transcriptional and cytokine profiling of formalin-fixed paraffin-embedded tissues distinct immune response in two mers-cov-infected patients: can we go from bench to bedside? elucidating the molecular physiopathology of acute respiratory distress syndrome in severe acute respiratory syndrome patients early enhanced expression of interferon-inducible protein-10 (cxcl-10) and other chemokines predicts adverse outcome in severe acute respiratory syndrome interferon-mediated immunopathological events are associated with atypical innate and adaptive immune responses in patients with severe acute respiratory syndrome genome wide identification of sars-cov susceptibility loci using the collaborative cross sensing of rna viruses: a review of innate immune receptors involved in recognizing rna virus invasion sars-cov pathogenesis is regulated by a stat1 dependent but a type i, ii and iii interferon receptor independent mechanism myd88 is required for protection from lethal infection with a mouse-adapted sars-cov rapid generation of a mouse model for middle east respiratory syndrome a study in which the dpp4-based host restriction is overcome in mice by expression of the human variant of dpp4, leading to the development of several transgenic mouse models mers-coronavirus replication induces severe in vitro cytopathology and is strongly inhibited by cyclosporin a or interferon-α treatment ultrastructure and origin of membrane vesicles associated with the severe acute respiratory syndrome coronavirus replication complex in vitro reconstitution of sars-coronavirus mrna cap methylation attenuation and restoration of severe acute respiratory syndrome coronavirus mutant lacking 2′-o-methyltransferase activity coronavirus non-structural protein 16: evasion, attenuation, and possible treatments the nucleocapsid protein of coronaviruses acts as a viral suppressor of rna silencing in mammalian cells sars-cov nucleocapsid protein antagonizes ifn-β response by targeting initial step of ifn-β induction pathway, and its c-terminal region is critical for the antagonism middle east respiratory syndrome coronavirus accessory protein 4a is a type i interferon antagonist middle east respiratory syndrome coronavirus 4a protein is a double-stranded rna-binding protein that suppresses pact-induced activation of rig-i and mda5 in the innate antiviral response the structural and accessory proteins m, orf 4a, orf 4b, and orf 5 of middle east respiratory syndrome coronavirus (mers-cov) are potent interferon antagonists middle east respiratory syndrome coronavirus orf4b protein inhibits type i interferon production through both cytoplasmic and nuclear targets severe acute respiratory syndrome coronavirus papain-like protease ubiquitin-like domain and catalytic domain regulate antagonism of irf3 and nf-κb signaling regulation of irf-3-dependent innate immunity by the papain-like protease domain of the severe acute respiratory syndrome coronavirus the sars coronavirus papain like protease can inhibit irf3 at a post activation step that requires deubiquitination activity crystal structure of the middle east respiratory syndrome coronavirus (mers-cov) papain-like protease bound to ubiquitin facilitates targeted disruption of deubiquitinating activity to demonstrate its role in innate immune suppression sars coronavirus nsp1 protein induces template-dependent endonucleolytic cleavage of mrnas: viral mrnas are resistant to nsp1-induced rna cleavage a two-pronged strategy to suppress host protein synthesis by sars coronavirus nsp1 protein severe acute respiratory syndrome coronavirus nsp1 facilitates efficient propagation in cells through a specific translational shutoff of host mrna severe acute respiratory syndrome coronavirus evades antiviral signaling: role of nsp1 and rational design of an attenuated strain middle east respiratory syndrome coronavirus nsp1 inhibits host gene expression by selectively targeting mrnas transcribed in the nucleus while sparing mrnas of cytoplasmic origin molecular determinants for subcellular localization of the severe acute respiratory syndrome coronavirus open reading frame 3b protein severe acute respiratory syndrome coronavirus open reading frame (orf) 3b, orf 6, and nucleocapsid proteins function as interferon antagonists mavs-mediated apoptosis and its inhibition by viral proteins pathogenic influenza viruses and coronaviruses utilize similar and contrasting approaches to control interferon-stimulated gene responses mechanisms of action of ribavirin against distinct viruses ribavirin and interferon therapy in patients infected with the middle east respiratory syndrome coronavirus: an observational study clinical analysis of the first patient with imported middle east respiratory syndrome in china clinical features and short-term outcomes of 144 patients with sars in the greater toronto area identification of severe acute respiratory syndrome in canada development of a standard treatment protocol for severe acute respiratory syndrome a cluster of cases of severe acute respiratory syndrome in hong kong interferon alfacon-1 plus corticosteroids in severe acute respiratory syndrome: a preliminary study description and clinical treatment of an early outbreak of severe acute respiratory syndrome (sars) in guangzhou, pr severe acute respiratory syndrome (sars) in singapore: clinical features of index patient and initial contacts coronaviruses lacking exoribonuclease activity are susceptible to lethal mutagenesis: evidence for proofreading and potential therapeutics broad-spectrum antivirals for the emerging middle east respiratory syndrome coronavirus inhibition of novel β coronavirus replication by a combination of interferon-α2b and ribavirin interferon-β and mycophenolic acid are potent inhibitors of middle east respiratory syndrome coronavirus in cell-based assays ribavirin and interferon-β synergistically inhibit sars-associated coronavirus replication in animal and human cell lines treatment with interferon-α2b and ribavirin improves outcome in mers-cov-infected rhesus macaques the first application of a potential treatment option for mers through the repurposing of ifnα2b and ribavirin in a non-human primate model ribavirin and interferon alfa-2a for severe middle east respiratory syndrome coronavirus infection: a retrospective cohort study ifn-α2a or ifn-β1a in combination with ribavirin to treat middle east respiratory syndrome coronavirus pneumonia: a retrospective study ribavirin and interferon-α2b as primary and preventive treatment for middle east respiratory syndrome coronavirus: a preliminary report of two cases treatment with lopinavir/ritonavir or interferon-β1b improves outcome of mers-cov infection in a non-human primate model of common marmoset treatment of severe acute respiratory syndrome with lopinavir/ritonavir: a multicentre retrospective matched cohort study role of lopinavir/ritonavir in the treatment of sars: initial virological and clinical findings virological and serological analysis of a recent middle east respiratory syndrome coronavirus infection case on a triple combination antiviral regimen middle east respiratory syndrome-coronavirus infection: a case report of serial computed tomographic findings in a young male patient combination therapy with lopinavir/ ritonavir, ribavirin and interferon-α for middle east respiratory syndrome: a case report clinical implications of five cases of middle east respiratory syndrome coronavirus infection in south korea outbreak from sars to mers: crystallographic studies on coronaviral proteases enable antiviral drug design thiopurine analogs and mycophenolic acid synergistically inhibit the papainlike protease of middle east respiratory syndrome coronavirus ligand-induced dimerization of middle east respiratory syndrome (mers) coronavirus nsp5 protease (3cl pro ): implications for nsp5 regulation and the development of antivirals screening of an fda-approved compound library identifies four small-molecule inhibitors of middle east respiratory syndrome coronavirus replication in cell culture international severe acute respiratory & emerging infection consortium. treatment of mers-cov: decision support tool. international severe acute respiratory & emerging infection consortium the effectiveness of convalescent plasma and hyperimmune immunoglobulin for the treatment of severe acute respiratory infections of viral etiology: a systematic review and exploratory meta-analysis a conformation-dependent neutralizing monoclonal antibody specifically targeting receptorbinding domain in middle east respiratory syndrome coronavirus spike protein potent neutralization of mers-cov by human neutralizing monoclonal antibodies to the viral spike glycoprotein identification of human neutralizing antibodies against mers-cov and their role in virus adaptive evolution exceptionally potent neutralization of middle east respiratory syndrome coronavirus by human monoclonal antibodies passive immunotherapy with dromedary immune serum in an experimental animal model for middle east respiratory syndrome coronavirus infection human polyclonal immunoglobulin g from transchromosomic bovines inhibits mers-cov in vivo a humanized neutralizing antibody against mers-cov targeting the receptor-binding domain of the spike protein prophylactic and postexposure efficacy of a potent human monoclonal antibody against mers coronavirus pre-and postexposure efficacy of fully human antibodies against spike protein in a novel humanized mouse model of mers-cov infection prophylaxis with a mers-covspecific human monoclonal antibody protects rabbits from mers-cov infection 3b11-n, a monoclonal antibody against mers-cov, reduces lung pathology in rhesus monkeys following intratracheal inoculation of mers-cov jordan-n3/2012 inhibition of middle east respiratory syndrome coronavirus infection by anti-cd26 monoclonal antibody identification of a broadspectrum antiviral small molecule against severe acute respiratory syndrome coronavirus and ebola, hendra, and nipah viruses by using a novel high-throughput screening assay evidence that tmprss2 activates the severe acute respiratory syndrome coronavirus spike protein for membrane fusion and reduces viral control by the humoral immune response protease inhibitors targeting coronavirus and filovirus entry the role of c5a in acute lung injury induced by highly pathogenic viral infections cell host response to infection with novel human coronavirus emc predicts potential antivirals and important differences with sars coronavirus a decade after sars: strategies for controlling emerging coronaviruses sars vaccines: where are we? middle east respiratory syndrome: current status and future prospects for vaccine development evaluation of candidate vaccine approaches for mers-cov recombinant receptor binding domain protein induces partial protective immunity in rhesus macaques against middle east respiratory syndrome coronavirus challenge a synthetic consensus anti-spike protein dna vaccine induces protective immunity against middle east respiratory syndrome coronavirus in nonhuman primates immune efficacy of first and repeat trivalent influenza vaccine in healthy subjects and hemodialysis patients presence of middle east respiratory syndrome coronavirus antibodies in saudi arabia: a nationwide, cross-sectional, serological study the finding that vaccination of dromedary camels reduces mers-cov shedding on infection, which provides a proof-of-principle for the vaccination of dromedary camels to block zoonotic transmission epidemiological, demographic, and clinical characteristics of 47 cases of middle east respiratory syndrome coronavirus disease from saudi arabia: a descriptive study a report of the clinical presentation of mers in patients in saudi arabia the epidemiology of severe acute respiratory syndrome in the 2003 hong kong epidemic: an analysis of all 1755 patients middle east respiratory syndrome hospital-associated outbreak of middle east respiratory syndrome coronavirus: a serologic, epidemiologic, and clinical description clinical aspects and outcomes of 70 patients with middle east respiratory syndrome coronavirus infection: a single-center experience in saudi arabia respiratory tract samples, viral load, and genome fraction yield in patients with middle east respiratory syndrome association of higher mers-cov virus load with severe disease and death mortality risk factors for middle east respiratory syndrome outbreak, south korea animal models for sars and mers coronaviruses replication and shedding of mers-cov in jamaican fruit bats (artibeus jamaicensis) severe acute respiratory syndrome coronavirus-like virus in chinese horseshoe bats bats are natural reservoirs of sars-like coronaviruses civets are equally susceptible to experimental infection by two different severe acute respiratory syndrome coronavirus isolates the first description of mers-cov replication and shedding in the respiratory tract of dromedary camels synthetic recombinant bat sars-like coronavirus is infectious in cultured cells and in mice sars-like wiv1-cov poised for human emergence the work was supported by the intramural research program of the national institute of allergy and infectious diseases (niaid), us national institutes of health. the authors declare no competing interests. key: cord-343050-1pfqgvie authors: huang, qiu sue; turner, nikki; baker, michael g; williamson, deborah a; wong, conroy; webby, richard; widdowson, marc-alain title: southern hemisphere influenza and vaccine effectiveness research and surveillance date: 2015-06-09 journal: influenza other respir viruses doi: 10.1111/irv.12315 sha: doc_id: 343050 cord_uid: 1pfqgvie the 2009 influenza a(h1n1)pdm09 pandemic highlighted the need for improved scientific knowledge to support better pandemic preparedness and seasonal influenza control. the southern hemisphere influenza and vaccine effectiveness research and surveillance (shivers) project, a 5-year (2012–2016) multiagency and multidisciplinary collaboration, aimed to measure disease burden, epidemiology, aetiology, risk factors, immunology, effectiveness of vaccination and other prevention strategies for influenza and other respiratory infectious diseases of public health importance. two active, prospective, population-based surveillance systems were established for monitoring influenza and other respiratory pathogens among those hospitalized patients with acute respiratory illness and those enrolled patients seeking consultations at sentinel general practices. in 2015, a sero-epidemiological study will use a sample of patients from the same practices. these data will provide a full picture of the disease burden and risk factors from asymptomatic infections to severe hospitalized disease and deaths and related economic burden. the results during the first 2 years (2012–2013) provided scientific evidence to (a) support a change to nz's vaccination policy for young children due to high influenza hospitalizations in these children; (b) contribute to the revision of the world health organization's case definition for severe acute respiratory illness for global influenza surveillance; and (c) contribute in part to vaccine strain selection using vaccine effectiveness assessment in the prevention of influenza-related consultations and hospitalizations. in summary, shivers provides valuable international platforms for supporting seasonal influenza control and pandemic preparedness, and responding to other emerging/endemic respiratory-related infections. the 2009 influenza a(h1n1)pdm09 pandemic highlighted the need for improved scientific knowledge to support better pandemic preparedness and seasonal influenza control. the southern hemisphere influenza and vaccine effectiveness research and surveillance (shivers) project, a 5-year (2012-2016) multiagency and multidisciplinary collaboration, aimed to measure disease burden, epidemiology, aetiology, risk factors, immunology, effectiveness of vaccination and other prevention strategies for influenza and other respiratory infectious diseases of public health importance. two active, prospective, population-based surveillance systems were established for monitoring influenza and other respiratory pathogens among those hospitalized patients with acute respiratory illness and those enrolled patients seeking consultations at sentinel general practices. in 2015, a sero-epidemiological study will use a sample of patients from the same practices. these data will provide a full picture of the disease burden and risk factors from asymptomatic infections to severe hospitalized disease and deaths and related economic burden. the results during the first 2 years (2012-2013) provided scientific evidence to (a) support a change to nz's vaccination policy for young children due to high influenza hospitalizations in these children; (b) contribute to the revision of the world health organization's case definition for severe acute respiratory illness for global influenza surveillance; and (c) contribute in part to vaccine strain selection using vaccine effectiveness assessment in the prevention of influenza-related consultations and hospitalizations. in summary, shivers provides valuable international platforms for supporting seasonal influenza control and pandemic preparedness, and responding to other emerging/endemic respiratory-related infections. keywords disease burden, epidemiology, immunology, influenza, risk factors, vaccine effectiveness. the 2009 influenza a(h1n1)pdm09 pandemic provided a test of global preparedness to assess the epidemiology of a pandemic and to respond appropriately and rapidly. the world was illprepared to respond to a severe influenza pandemic or to any similarly global, sustained and threatening public health emergency. 1 one fundamental constraint highlighted during the pandemic was the limited understanding of the epidemiology and severity of the pandemic which in turn hampered international efforts to mount an appropriate response. 2 rapid assessment of the epidemiologic, virologic and clinic features of a pandemic is essential to provide critical information to decision-makers on how to minimize morbidity and mortality, and mitigate potential economic and societal disruption. soon after the pandemic virus emerged in april 2009 in mexico and spread globally, public health leaders, anxious to understand the full breadth of influenza epidemiology, turned their attention to countries in southern temperate areas with an upcoming influenza season. this demonstrated the absence of an established real-time system in the southern hemisphere to provide more complete surveillance of an influenza pandemic. also, such a system would help monitor the epidemiology of new strains of seasonal influenza and the effectiveness of vaccination, both for the southern hemisphere and for upcoming northern hemisphere seasons. in december 2010, the us centers for disease control and prevention (us-cdc) made a funding opportunity announcement for a temperate southern hemisphere site to conduct research on the disease burden, epidemiology, and prevention of influenza and other respiratory diseases of public health importance. new zealand (nz) is a temperate southern hemisphere country with a population of 4á4 million people. the influenza season mainly occurs from june to september. [3] [4] [5] [6] nz's predominantly public-funded healthcare system with associated integrated health information systems is a strong asset in conducting population-based research. all new zealanders are assigned a unique health identifier allowing tracking of healthcare utilization over time and confidential record linkage to multiple databases including hospitalization and surveillance data. additionally, patients are registered with primary care providers who maintain highly computerized records with detailed demography, immunization status and clinical information. the nz population is well characterized in terms of demographic structure, particularly by ethnicity and socio-economic status. indigenous maori and pacific peoples (collectively about 20% of the population) appear particularly vulnerable to influenza and other respiratory infections. 3, 7 in in this article, we describe the objectives and study designs of shivers. we also describe lessons learned from the first 2 years and planned future studies as well as international applications. the overarching aim of shivers is to comprehensively investigate the disease burden, epidemiology, aetiology, risk factors, immunology, effectiveness of vaccination and other prevention strategies for influenza and other respiratory viral diseases of public health importance. the project consists of 9 objectives as detailed in table 1 . they can be divided into five main streams: influenza disease burden data are essential to allocate limited health resources, assist influenza vaccination policy development and improve vaccine uptake, particularly for subpopulations at risk. however, the evidence to support valid and precise estimates of influenza disease burden globally remains weak with low quality, partly due to the short duration of studies and the heterogeneity of study settings and methods (statistical modelling, active versus passive case findings, virological versus clinical detection). [8] [9] [10] [11] [12] [13] [14] in addition, there is scarce information on sero-epidemiologic investigation of seasonal influenza at a population level. serology can detect both symptomatic and asymptomatic infections, thus estimating the true incidence of influenza infection. this parameter cannot be determined by either disease surveillance programmes or detection of virologically confirmed cases as they would vastly underestimate influenza incidence and overestimate severity. [15] [16] [17] [18] shivers allows calculation of rates of infection and different clinical presentations in the same population at the same time for an accurate picture of the relative severity of influenza infection in the population and vulnerable subpopulations at four levels: (a) severe hospitalized disease; (b) moderate disease requiring a general practice visit; (c) mild disease not requiring a general practice visit; (d) incidence of infection (symptomatic and asymptomatic). aetiology shivers provides an integrated platform for the study of respiratory diseases caused by influenza and other common and emerging respiratory pathogens. the aetiological component allows us to (1) monitor antigenic drift of seasonal influenza viruses, contributing to who's annual vaccine strain selection; (2) support pandemic preparedness including surveillance for new subtypes of influenza a viruses (e.g. a(h7n9)); (3) monitor common non-influenza respiratory pathogens to understand their impact on the disease and epidemiology; and (4) provide early detection for emerging respiratory viruses (e.g. mers-cov). there is increasing evidence in the literature for the importance of polymicrobial infections. however, there remain gaps in our understanding of respiratory virus codetection and whether this represents co-infection and affects clinical disease manifestations and severity. there are contradictory reports with some suggesting that co-infections increase the severity of respiratory disease, 19-23 while others have found either no association [24] [25] [26] [27] [28] or that co-infections may actually be protective. 29 additionally, bacterial coinfections associated with cases of influenza are a leading cause of severe morbidity and mortality: bacterial coinfections complicated nearly all influenza deaths in the 1918 pandemic and up to 34% of the 2009 a(h1n1)pdm09 infections managed in intensive care units worldwide. 30, 31 shivers will help our understanding of the potential role of pathogen co-detection in patient outcome, severity, aetiology, demography and underlying risk conditions. influenza vaccine strain selection requires annual consultations and frequent updates to match the antigenic drift of the circulating viral strains, and ample evidence indicates that influenza vaccine effectiveness (ve) varies not only by virus type (subtype) but also from year to year. 32, 33 robust and timely vaccine effectiveness estimates are important to measure the public health benefit of seasonal influenza control strategies, pandemic preparedness and vaccine strain selection. 34 many ve estimates derive from observational studies with existing data collecting systems which often have multiple limitations and biases, and there are international calls for more rigorous ve studies. [34] [35] [36] [37] [38] [39] shivers is providing robust and timely estimations of the protective effect of seasonal influenza vaccine in the prevention of hospitalizations and general practice consultations for laboratory-confirmed influenza using case test-negative control methods. 40, 41 immune response an individual's immune response to influenza infection can vary depending on many factors (e.g. age, underlying conditions and ethnicity). clinical observation during the 2009 pandemic indicated that the unexpected low morbidity and mortality rates in the elderly were in part due to their crossreactive immunity. 3, 42, 43 there are knowledge gaps regarding each component of adaptive immune responses in determining an individual's risk of acquiring influenza virus infection and the severity of the resulting disease: antihemagglutinin (ha) antibodies, antineuraminidase (na) antibodies, isotypes of responding antibodies, influenza-specific cd4+, cd8+ t cells, surface markers and key cytokines. 44, 45 additionally, there are scarce data on the correlation of cellular immunologic and neuraminidase targeted antibody responses in individuals with serologically (anti-ha antibodies) defined influenza infection. 46, 47 by interconnecting the epidemiological and immune studies in severe and moderate disease cases and high-risk subgroups (e.g. pacific and maori ethnic groups) and individuals with serologically defined influenza infection, shivers will facilitate our understanding of host immune responses that determine an individual's risk of acquiring influenza infection or developing severe disease. identification and quantification of risk factors for influenza infection and poor outcomes (hospitalization, icu treatment, death) provides evidence to inform decisions on targeted pharmaceutical (vaccinations, antivirals), healthcare (e.g. improved treatment of comorbidities) and non-pharmaceutical (e.g. exposure to infections) interventions to reduce the risk of seasonal and pandemic influenza. elderly people have a significantly higher risk of influenza-associated death compared with non-elderly people. 48 additionally, the 2009 pandemic in nz revealed that the risk of hospitalization and death was markedly higher for maori and pacific people, and those from the most deprived socioeconomic groups. 3, 42, 49 however, it is not clear whether these sociodemographic factors are independent risk factors for influenza. furthermore, some chronic health conditions (high body mass index, asthma and pregnancy) have been shown to increase the risk of having a poor outcome from influenza infection. [50] [51] [52] [53] in nz, household crowding has been identified as a risk factor for transmission of meningococcal disease, 54 rheumatic fever 55 and tuberculosis 56 and may also be contributing to higher rates of influenza for some populations. the household setting (crowding, housing conditions) may influence transmission of influenza, but these effects remain poorly understood. 57-59 shivers will provide a multifaceted understanding of influenza risk that considers organism, host and environmental factors and opportunities for intervention. this comprehensive and quantitative approach will include detailed consideration of the independent contributions of host ethnicity, socioeconomic position, chronic illness status, obesity, household environment exposures and infecting virus. study sites shivers study sites are located within two district health boards of the auckland region of nz: adhb and cmdhb ( figure 1 ). this is a predominantly urban population of 906 000 people, with a wide spectrum of socio-economic, cultural, ethnic and demographic groups broadly similar to the new zealand population. 60 we established two surveillance platforms (hospital and sentinel general practice) in the two dhbs: four publicly funded hospitals serve the secondary healthcare needs for all residents of the two dhbs: auckland city hospital and the associated starship children's hospital (adhb), and middlemore hospital and the associated kidz first children's hospital (cmdhb). in 2012, we began active, prospective, continuous, population-based surveillance for influenza and other respiratory pathogens among persons residing in the two dhbs hospitalized for respiratory disease (figure 2 ). research nurses reviewed daily records of all overnight acutely admitted inpatients to identify any inpatient with a suspected acute respiratory illness (ari). they interviewed these patients by applying the world health organization (who) interim sari case definition: 'an acute respiratory illness with a history of fever or measured fever of ≥38°c, and cough, and onset within the past 7 days, and requiring inpatient hospitalization'. 61 since 2013, the who final sari case definition has been used with onset changed from '7 days' to '10 days'. the patients were differentiated into sari cases (those who met the sari case definition) and non-sari patients (those with ari who did not meet the sari case definition). a case report form captured information on demography, history of presenting illness, comorbidities, influenza vaccination history, disease outcome and risk factors. 60 if a patient met the sari case definition, a respiratory specimen (nasopharyngeal swab or aspirate) was collected and tested simultaneously for influenza and other respiratory viruses by real-time reverse transcription (rt) polymerase chain reaction (pcr) techniques: influenza virus, respiratory syncytial virus (rsv), rhinovirus, parainfluenza virus types 1-3, adenovirus and human metapneumovirus ( figure 3) . 60 a systematic sample of about 50% of non-sari patients were also interviewed and provided a respiratory sample, in addition to those from whom a specimen was collected for clinical purposes. some sari cases and non-sari patients were also tested for clinical purposes for a range of respiratory bacteria (e.g. streptococcus pneumonia, in 2013, we began active, prospective, population-based surveillance for influenza and other respiratory pathogens among persons enrolled in sentinel general practices who seek medical consultations (figure 4 ). eighteen sentinel general practices situated within adhb and cmdhb were recruited. these practices have a combined total of 103 752 enrolled patients, covering approximately 12% of the adhb and cmdhb population. the participating general practitioners (gp) and practice nurses (pn) assessed all consultationseeking patients. if a patient met the influenza-like illness (ili) case definition: 'an acute respiratory illness with a history of fever or measured fever of ≥38°c, and cough, and onset within the past 10 days, and requiring consultation in that general practice', a respiratory specimen (nasopharyngeal swab or throat swab) was collected to test for influenza and other respiratory viruses by real-time rt-pcr ( figure 3 ). gp/pn documented the components of the case definition that were present and recorded patients who met the ili case definition in an advanced electronic form designed for the practice management system (pms). patient information already captured in the pms was automatically retrieved, including demography, comorbidities, vaccination history and regular medication list. further data were captured by interviewing ili patients regarding influenza vaccination obtained elsewhere, pregnancy and a clinician's judgement of obesity. to more fully understand the epidemiology of influenza, these two platforms will be leveraged for other studies: (a) sero-epidemiological study: to obtain rates of mild influenza illness that do not trigger gp visits as well as symptomatic/asymptomatic infections, the enrolled patients in those sentinel general practices will be used to randomly select a cohort of persons (stratified by age and ethnicity) and followed through one influenza season. the serologic surveys will measure preand post-season antibodies to circulating influenza strains using relevant vaccine reference strains as antigens; (b) immunology study: a subset of samples from severe, moderate influenza cases, related risk groups and individuals with serologically defined influenza infection are selected for the study of humoral and cellular immune responses ( figure 3) ; and (c) remaining studies: the combined laboratory testing results and metadata collected from these platforms are used to study vaccine effectiveness, interaction between respiratory pathogens, respiratory mortality, risk factors and economic burden and vaccine cost-effectiveness. our innovative study design interconnecting multiple objectives, in addition to exploiting nz's unique healthcare structure, will maximize efficiency and study power. the two surveillance platforms provided specimens and data to serve the nine objectives of shivers. figure 5 shows how each of the objectives is linked to each other and maps data and specimen flows between them. since its conception in 2006, sari surveillance has become a recognized international standard for monitoring hospitalized severe respiratory disease related to influenza and other pathogens. 61 occurred over this time. [65] [66] [67] it was designed to monitor trends in severe influenza disease and to best capture the majority of influenza respiratory disease to estimate the burden of influenza-associated respiratory hospitalizations, and risk factors for severe disease. 63 the initial sari case definition included the symptom onset of acute respiratory illness within 7 days. 61 the shivers results in 2012 showed that a small proportion (7%) of influenza cases had specimens collected 8-9 days after the symptom onset. these specimens only consisted of a small proportion (9%) of total specimens; thus, the cost of testing was minimal (manuscript in preparation). this result was shared with the who global influenza programme. it contributed to a change in the final who sari case definition, with onset shifting from 'within 7 days' to 'within 10 days'. 63 burden and epidemiology shivers allows the estimation of influenza disease burden and risk factors at various levels of severity. firstly, the disease burden of severe influenza is estimated from the hospital surveillance platform. it measured population-based incidence for sari-associated influenza hospitalizations including icu admissions and in-hospital deaths as it provided reliable numerators and denominators, thus without a need for additional healthcare utilization surveys. [68] [69] [70] [71] our first-year findings (30 april 2012 to 28 april 2013) showed that the sari-associated influenza hospitalization rate was substantial with the overall adjusted annual incidence of 54/ 100 000 persons (manuscript in preparation). this rate was similar to us data on influenza-associated hospitalizations during 1979-2001, with an average annual incidence of 36á8/ 100 000. 12 the very young (0-4 years) and elderly (≥65 years) had the highest sari-associated influenza hospitalization rates, consistent with trends identified in international literature, particularly those from developed countries with temperate climates. 10, [72] [73] [74] [75] a high rate of influenza-related hospitalizations and low vaccine uptake (6%) in young children (6 months to 4 years) from shivers led the nz government to change vaccination policy by extending free influenza vaccination to those in this age group who have been hospitalized or have a history of significant respiratory illness. 76 sari surveillance is likely to underestimate the true burden of severe influenza resulting in hospitalization. some patients will present with non-respiratory symptoms or respiratory disease that does not meet the sari case definition, or stay briefly in emergency department. 63, 66 shivers has begun to address this gap; a pilot study in 2012 testing persons with respiratory disease who did not meet the sari case definition showed that a small proportion (6%) of non-sari patients were positive for influenza viruses, compared with 18% of sari cases (manuscript in preparation). future work to expand the case definition to all acute hospital admissions in a sample of very young children will further expand our knowledge of the burden of influenza in this important group potentially protected by maternal immunization. additionally, sari and associated influenza cases will be linked to the hospital discharge data to determine the accuracy and validity of the discharge data by determining proportions of the principal discharge diagnosis code categories that are sari and influenza cases. this will help inform modelling studies of icd-coded data and help provide some validation of these with laboratory-confirmed data. sari surveillance is also likely to grossly undercount the actual number of influenza-associated deaths because only a minority of influenza-related sari deaths are correctly diagnosed, tested and recorded as such. additional influenza deaths resulting from secondary bacterial infections and exacerbation of pre-existing chronic conditions and atypical clinical presentations are not captured. 77 this limitation presents a challenge in accurately measuring influenzarelated mortality. future work on statistical modelling may allow for indirect estimation of 'excess' mortality attributable to influenza in those broad categories such as pneumonia, respiratory or circulatory deaths during influenza seasons. 78 secondly, the disease burden of moderate influenza is estimated using data from shivers ili surveillance. our findings from the 2013 season (29 april to 29 september) showed that the ili-associated influenza consultation rate was about 14 times higher than sari-associated influenza hospitalization rate (manuscript in preparation). additionally, ili-associated influenza consultations and sari-associated influenza hospitalizations showed contrasting socio-demographic patterns: higher rates of ili-associated influenza consultations were shown in preschoolers (aged 1-4 years), school-age children and adults (<65 years), those of asian ethnicity and those from least deprived socio-economic status (ses) groups. this was a different picture from sari surveillance where sari-associated influenza hospitalizations were more frequent in the very young (under 1 year), the elderly, m aori and pacific peoples and those from most deprived ses groups. 79 these results provided insights into the interplay between healthcare access opportunities and related health-seeking behaviours and the differential effect of the predominant strains on various age groups. thirdly, the disease burden of mild influenza not requiring medical consultation (e.g. school/work-related absenteeism) and influenza infection (symptomatic/asymptomatic) will be estimated from the shivers serosurvey. additionally, we will also conduct severity assessment using true numbers of infections as the denominator to calculate case/fatality and case/hospitalization ratios. furthermore, the data on influenza disease burden will allow us to estimate direct medical costs and indirect societal costs (e.g. loss of productivity, loss of earning and loss of life) for the study population and subpopulations. 80, 81 aetiology preliminary results in 2013 identified an under-recognized burden of non-influenza respiratory viruses, particularly rsv and rhinoviruses, in sari and ili cases in nz as we have never had active population-based study on these viruses previously although substantial burden of rsv and rhinovirus has been described elsewhere. 82, 83 ili-associated consultations and sari-associated hospitalizations for rsv and rhinovirus show different socio-demographic patterns (age, ethnicity and ses) from that of influenza. for example, both ili-and sari-associated rsv incidences were similarhigh rates for very young (<1 year and 1-4 years) followed by elderly (≥65 years). this presented a very different agespecific incidence profile from that of influenza (indicated above). this result, together with subsequent multiyear surveillance data, may provide insights on differential effects of various respiratory viruses on the age distribution of the host and disease severity. shivers surveillance platforms provided a systematic opportunity to estimate ve for the prevention of general practice visits and hospitalizations for rt-pcr-confirmed influenza from the same population in the same influenza season. 40, 41 a case test-negative control design is used to estimate annual propensity-adjusted vaccine effectiveness in both hospital and community settings. the data in 2013 showed moderate effectiveness of influenza vaccine against medically attended and hospitalized influenza in nz with 56% (95% ci 34,70) against influenza presenting to general practice and 52% (95% ci 32,66) protection against laboratory-confirmed influenza hospitalization. 41 immune responses shivers surveillance platforms also provided sera and whole-blood samples during acute and convalescent phases of infection to study humoral and cellular immune responses from a subset of severe (n = 39) and moderate (n = 29) influenza cases in 2013. with these samples, and using a combination of serological and immunological assays, we were able to (a) estimate the relative contribution of early adaptive and cellular immune responses to disease severity; (b) identify differences in the immune profiles between these diseases groups; and (c) identify immunological correlates of disease severity in subpopulations. data acquired so far indicate that sari cases may experience a more robust immunologic response during infection (i.e. greater increases in ha-and na-specific antibody titres as well as magnitude of t-cell response). the ability to parse out immunological differences between severe and moderate influenza cases in this pilot cohort highlights the value of adding the active immunology study to the shivers platforms. as nz has a well-characterized socio-demographic distribution (age, sex, ethnicity, deprivation) from population census data, socio-demographic risk factors can be characterized quite easily. shivers will use the results obtained from hospital and sentinel general practice surveillance to disentangle the effect of ethnic and socio-economic gradients. for other more specific risk factors (e.g. host factors such as comorbidities, and environmental factors such as housing conditions), there are limited data available on their distribution in the population. consequently, it is difficult to assess the importance of the risk factor data collected. there are several comparison/control groups such as hospital-based control populations without respiratory illness, serosurvey participants as a control group and sari/ili testnegative controls. these controls could be compared with sari/ili cases to estimate the importance of specific risk factors for influenza infection and related severe/moderate diseases including socio-economic, underlying medical conditions, health intervention, health service utilization, and environmental and behavioural factors. we have established active, prospective, population-based surveillance systems for a wide range of respiratory disease presentations and designed a portfolio of influenza studies based on these platforms. the shivers results during the first 2 years (2012-2013) provided scientific evidence to support change to nz's vaccination policy for young children due to high sari-associated influenza hospitalizations in these children; contribute to the finalization of the world health organization's sari case definition for global influenza surveillance; and contribute in part to vaccine strain selection with vaccine effectiveness assessment in the prevention of general practice visits and hospitalizations for laboratoryconfirmed influenza. in the next 3 years (2014-2016), this project will continue to help us to understand (1) the burden of influenza infection including symptomatic/asymptomatic infection and mild disease not requiring medical consultation; (2) influenza risk that considers organism, host and environmental factors; (3) the impact of viral-viral and viral-bacteria co-infections on clinical disease and severity; and (4) the nature of responding adaptive immune responses in determining individual's risk of acquiring influenza virus infection. over 5 years, we hope this project will shed more light on the burden of influenza and other respiratory pathogens in our study population and subgroups and estimate key epidemiologic parameters such as relative rates of infection, clinical disease, general practice visits and hospitalizations as well as risk factors for illness, effectiveness of vaccination, mechanisms of immunity and monitoring for new influenza viruses with pandemic potential such as a(h7n9) and other emerging viruses (e.g. mers-cov) and provide a framework for timely assessment of severity which is essential in an event of emergence of these pathogens. the platforms established here are relevant not only for new zealand policy, but also for the region and the world. it will provide robust systematic virologic, epidemiologic and vaccine effectiveness data on circulating pandemic or seasonal influenza viruses at a time when circulation in the northern hemisphere is low. this could provide valuable information on all emergent respiratory pathogens that have some winter seasonality. moreover, the data elements on a range of disease severities collected in the same population at the same time will generate epidemiologic parameters that maybe broadly generalizable and translatable to similar developed, southern and northern temperate countries worldwide. this will help enormously to better understand more basic surveillance data and to extrapolate those data in models to plan and predict influenza behaviour, generate burden estimates, model the impact of seasonal influenza vaccination to support more global use and better prepare for the next pandemic. in summary, shivers is expected to provide extensive data to guide improved methods for disease surveillance; improve clinical case management, early detection and optimization of laboratory diagnosis; inform vaccine strain selection and vaccine development; guide targeted vaccination strategies for population and subgroups; understand host immune responses and identify better immune diagnostic markers. korrapadu, louise optland, cecilia dela cruz. special thanks to it staff and sari surveillance participants. also, a special thanks to dr dean erdman from gastroenteritis and respiratory viruses laboratory branch, the u.s. centers for disease control and prevention who provided the real-time pcr assay for non-influenza respiratory viruses. support in kind is provided by the nz ministry of health. report of the review committee on the functioning of the international health regulations (2005) in relation to pandemic (h1n1) pandemic preparedness and response-lessons from the h1n1 influenza of 2009 surveillance for the 2009 pandemic influenza a (h1n1) virus and seasonal influenza viruses -new zealand influenza surveillance and immunisation in new zealand influenza surveillance in new zealand in 2005 pandemic influenza a(h1n1)v in new zealand: the experience from increasing incidence of serious infectious diseases and inequalities in new zealand: a national epidemiological study incidence of influenza in healthy adults and healthcare workers: a systematic review and metaanalysis pandemic influenza a (h1n1) in pregnancy: a systematic review of the literature global burden of respiratory infections due to seasonal influenza in young children: a systematic review and meta-analysis background paper on influenza vaccines and immunization influenza-associated hospitalizations in the united states mortality associated with influenza and respiratory syncytial virus in the united states assessment of the burden of influenza in children influenza infection rates, measurement errors and the interpretation of paired serology comparative community burden and severity of seasonal and pandemic influenza: results of the flu watch cohort study influenza-related deaths-available methods for estimating numbers and detecting patterns for seasonal and pandemic influenza in europe epidemiological characteristics of 2009 (h1n1) pandemic influenza based on paired sera from a longitudinal community cohort study human metapneumovirus in hospitalized children in amman, jordan viruses in community-acquired pneumonia in children aged less than 3 years old: high rate of viral coinfection dual respiratory virus infections the impact of dual viral infection in infants admitted to a pediatric intensive care unit associated with severe bronchiolitis dual infection of infants by human metapneumovirus and human respiratory syncytial virus is strongly associated with severe bronchiolitis severity of viral coinfection in hospitalized infants with respiratory syncytial virus infection simultaneous multiple viral infections in childhood acute lower respiratory tract infections in southern taiwan broad respiratory virus detection in infants hospitalized for bronchiolitis by use of a multiplex rt-pcr dna microarray system pandemic (h1n1) 2009 and seasonal influenza a (h1n1) co-infection rapid detection of respiratory tract viral infections and coinfections in patients with influenza-like illnesses by use of reverse transcription-pcr dna microarray systems multiple versus single virus respiratory infections: viral load and clinical disease severity in hospitalized children bacterial coinfection in influenza: a grand rounds review bacterial and viral infections associated with influenza influenza vaccine strain selection and recent studies on the global migration of seasonal influenza viruses efficacy and effectiveness of influenza vaccines: a systematic review and metaanalysis new strategies are needed to improve the accuracy of influenza vaccine effectiveness estimates among seniors vaccines for preventing influenza in healthy adults the case test-negative design for studies of the effectiveness of influenza vaccine the test-negative design for estimating influenza vaccine effectiveness vaccine effectiveness in older individuals: what has been learned from the influenza-vaccine experience challenges in evaluating influenza vaccine effectiveness and the mortality benefits controversy the effectiveness of seasonal trivalent inactivated influenza vaccine in preventing laboratory confirmed influenza hospitalisations in auckland the effectiveness of seasonal trivalent inactivated influenza vaccine in preventing laboratory confirmed influenza hospitalisations and primary care visits in auckland risk factors and immunity in a nationally representative population following the 2009 influenza a(h1n1) pandemic severe pandemic 2009 h1n1 influenza disease due to pathogenic immune complexes the lower serum immunoglobulin g2 level in severe cases than in mild cases of pandemic h1n1 2009 influenza is associated with cytokine dysregulation association between severe pandemic 2009 influenza a (h1n1) virus infection and immunoglobulin g(2) subclass deficiency host adaptive immunity deficiency in severe pandemic influenza the role of polymorphisms in host immune genes in determining the severity of respiratory illness caused by pandemic h1n1 influenza populations at risk for severe or complicated influenza illness: systematic review and meta-analysis the second wave of 2009 pandemic influenza a(h1n1) in new zealand a novel risk factor for a novel virus: obesity and 2009 pandemic influenza a (h1n1) critical care services and 2009 h1n1 influenza in australia and new zealand risk factors and outcomes among children admitted to hospital with pandemic h1n1 influenza pandemic influenza a (h1n1) in pregnancy: a systematic review of the literature household crowding a major risk factor for epidemic meningococcal disease in auckland children acute rheumatic fever associated with household crowding in a developed country tuberculosis associated with household crowding in a developed country household transmission of the 2009 pandemic a/h1n1 influenza virus: elevated laboratory-confirmed secondary attack rates and evidence of asymptomatic infections comparative epidemiology of pandemic and seasonal influenza a in households impact of pollution, climate, and sociodemographic factors on spatiotemporal dynamics of seasonal respiratory viruses implementing hospitalbased surveillance for severe acute respiratory infections (sari) caused by influenza and other respiratory pathogens in new zealand interim global epidemiological surveillance standards for influenza generic protocol for influenza surveillance global epidemiological surveillance standards for influenza who regional office for europe guidance for influenza surveillance in humans validity of clinical case definitions for influenza surveillance among hospitalized patients: results from a rural community in north india what are the most sensitive and specific sign and symptom combinations for influenza in patients hospitalized with acute respiratory illness? results from western kenya lessons from a one-year hospital-based surveillance of acute respiratory infections in influenza study in southern hemisphere ª 2015 the authors. influenza and other respiratory viruses published by berlin-comparing case definitions to monitor influenza incidence of influenza-like illness and severe acute respiratory infection during three influenza seasons in bangladesh estimation of the national disease burden of influenza-associated severe acute respiratory illness in kenya and guatemala: a novel methodology the cost of influenza in thailand the substantial hospitalization burden of influenza in central china: surveillance for severe, acute respiratory infection, and influenza viruses the underrecognized burden of influenza in young children burden of seasonal influenza hospitalization in children adult hospitalizations for laboratory-positive influenza during the virologically confirmed populationbased burden of hospitalization caused by influenza a and b among children in hong kong high influenza hospitalisations lead to extension of free influenza vaccination to young children estimated global mortality associated with the first 12 months of 2009 pandemic influenza a h1n1 virus circulation: a modelling study global mortality estimates for the 2009 influenza pandemic from the glamor project: a modeling study influenza-associated primary care consultations and hospitalisations show contrasting socio-demorgraphic patterns medical and economic burden of influenza in the elderly population in central and eastern european countries influenza cost and cost-effectiveness studies globally-a review disease burden of the most commonly detected respiratory viruses in hospitalized patients calculated using the disability adjusted life year (daly) model acute viral infections of upper respiratory tract in elderly people living in the community: comparative, prospective, population based study of disease burden the shivers investigation team (listed in an alphabetic order) the shivers project is funded by us cdc (1u01ip000480-01). the project is a 5-year research cooperative agreement between the authors declare that they have no competing interests. key: cord-333868-qrnsmhws authors: rothman, richard e.; irvin, charlene b.; moran, gregory j.; sauer, lauren; bradshaw, ylisabyth s.; fry, robert b.; josephson, elaine b.; ledyard, holly k.; hirshon, jon mark title: respiratory hygiene in the emergency department date: 2006-08-23 journal: ann emerg med doi: 10.1016/j.annemergmed.2006.05.018 sha: doc_id: 333868 cord_uid: qrnsmhws the emergency department (ed) is an essential component of the public health response plan for control of acute respiratory infectious threats. effective respiratory hygiene in the ed is imperative to limit the spread of dangerous respiratory pathogens, including influenza, severe acute respiratory syndrome, avian influenza, and bioterrorism agents, particularly given that these agents may not be immediately identifiable. sustaining effective respiratory control measures is especially challenging in the ed because of patient crowding, inadequate staffing and resources, and ever-increasing numbers of immunocompromised patients. threat of contagion exists not only for ed patients but also for visitors, health care workers, and inpatient populations. potential physical sites for respiratory disease transmission extend from out-of-hospital care, to triage, waiting room, ed treatment area, and the hospital at large. this article presents a summary of the most current information available in the literature about respiratory hygiene in the ed, including administrative, patient, and legal issues. wherever possible, specific recommendations and references to practical information from the centers for disease control and prevention are provided. the “administrative issues” section describes coordination with public health departments, procedures for effective facility planning, and measures for health care worker protection (education, staffing optimization, and vaccination). the patient care section addresses the potentially infected ed patient, including emergency medical services concerns, triage planning, and patient transport. “legal issues” discusses the interplay between public safety and patient privacy. emergency physicians play a critical role in early identification, treatment, and containment of potentially lethal respiratory pathogens. this brief synopsis should help clinicians and administrators understand, develop, and implement appropriate policies and procedures to address respiratory hygiene in the ed. respiratory tract infections are common presentations among emergency department (ed) patients, some of whom may present an infectious risk. unfortunately, definitive identification of the offending agent is generally not possible at the initial ed visit. potential respiratory agents that the 21 st century emergency physician must consider include the traditional respiratory pathogens and also emerging (eg, severe acute respiratory syndrome [sars]), highly virulent (eg, avian influenza virus), resistant, and even bioterrorism-related agents. thus, within the ed there is a potentially dangerous mixture of infections with serious possible public health consequences. threat of contagion exists for uninfected patients (in the ed, the hospital at large, and the community) and health care personnel (including ed physicians). through implementation of the most up-to-date guidelines, health care personnel can aid in minimizing respiratory infection transmission and protect patients and other hospital personnel from infection. the potential risk for respiratory infection-related morbidity and mortality is compounded in the ed because of the increasing number of immunocompromised ed patients. populations at increased risk include organ transplant patients, 1 hiv-infected patients, and postchemotherapy patients, all of whom are living longer because of improved lifesaving therapies. 2 crowded and understaffed eds further elevate risk of contagion and possible public health disasters. two potentially lethal infections that are transmitted by the respiratory route, which most emergency physicians are familiar with, are neisseria meningitis (which causes meningococcemia) and mycobacterium tuberculosis. these agents are relatively uncommon, however, in most us eds, and as recently as 2003, the centers for disease control and prevention (cdc) reported that health care facility environments are rarely implicated in respiratory pathogen transmission (except in cases of immunocompromised patients). case reports of transmission of sars among hospital workers from that year resulted in heightened awareness of the need for increased attention to respiratory precautions. for example, according to lau et al, 3 44% of sars cases (68/156 cases) at the prince of wales hospital in hong kong occurred in hospital workers who did not take special protective measures during the sars outbreak. another study found that failure of providers to recognize risk, implement strict isolation measures, and diagnose disease was responsible for the majority of nosocomial cases of sars in hong kong (with the vast majority of cases occurring among physicians and nurses). 4 internationally, health care worker infection has proven to account for up to 42% of sars cases in canada and approximately 25% of cases in hong kong. 4, 5 these findings provide compelling data that hospital workers are at significant risk of contracting respiratory infections and establish an imperative for initiating broad-scale infection control measures. the participation of emergency physicians and nurses is critical for effective responsiveness to respiratory threats in hospitals. ed personnel represent a critical link in the chain of communication and response, along the continuum from the community to the inpatient unit. policies should anticipate responses to the complex spectrum of possible respiratory illnesses, from highly transmissible and unexpected emerging global diseases such as sars to yearly influenza epidemics. lessons from the terrorist attacks on september 11, 2001 , and other recent disasters emphasize the importance of integrating the public health system with both medical and mental health services, with close attention to capacity management and surge planning. organizational systems thus require that disaster and public health planning at regional and state levels produce systems that integrate the ed (the likely focal point for patients with acute respiratory infections) with hospital and regional response plans and resources. 6, 7 the purpose of this report is to summarize, from both the peer-reviewed literature and public health sources (eg, from the cdc), information most relevant to ed respiratory infection control. specific and current recommendations and guidelines are provided, along with evidence supporting specific respiratory infection control measures, when available. the review is divided into 3 sections, addressing administrative, patientrelated, and legal issues, with some unavoidable overlap occurring. administrative topics include public health coordination, facility planning, and health care worker issues. the patient-related portion covers patient flow from out-ofhospital and triage to waiting room and ed treatment areas, with inclusion of a discussion of patient education and patient transport. the legal section summarizes federal and local laws pertinent to respiratory hygiene. because sars represents the most recent significant respiratory pathogenic threat, many of the successes and challenges about respiratory infection control reference studies from the sars outbreak. while this research is sars specific, lessons that may be generalizable about infection control are provided. further, although an all-inclusive discussion about respiratory hygiene is impossible, this summary provides the most relevant and practical information for the practicing emergency physician, with specific references provided for particular topics to allow more detailed review. according to a recent national ed-based survey, acute respiratory infections are the leading ed "illness-related" diagnosis. 8 another recent study from the pediatric literature reported that acute respiratory illnesses are the second leading category of adolescent diagnosis from ed visits among virtually every age group (except women aged 18 to 21 years). 9 the significant influx of patients expected during an outbreak (such as sars or avian influenza) would result in an even greater proportion of ed patient visits for respiratory-related complaints. the cdc has developed several specific guidelines about infection control in hospitals, with the most recent updates issued in november 2004. 10, 11 the recommendations are graded according to levels of supporting evidence, as defined in figure 1 . precautionary measures are divided into standard precautions ( figure 2 ) to be followed in care of all patients and transmission-based precautions to be used in addition to standard precautions according to the route of pathogen transmission. transmission-based precautions include contact precautions for agents with potential transmission by direct or indirect contact; droplet precautions for agents with potential transmission by coughing, sneezing, talking, or performance of procedures ( figure 3 ); and airborne precautions for agents with potential transmission by dissemination of either airborne droplet nuclei or evaporated droplets that remain suspended in the air for long periods (figure 4 ). airborne transmission is relevant for small infectious particles that are 5 m or smaller. administrative issues surrounding respiratory hygiene apply to the entire health care facility. emergency physicians should take a lead role in development and implementation of policies because the ed serves as the initial entry point for many patients. policies to address routine respiratory pathogens (eg, tuberculosis [tb] and influenza a), emerging pathogens (eg, sars or avian influenza), and bioterrorist agents are necessary. the cdc has provided detailed recommendations about health care facility response preparedness for a sars outbreak (available online at http://www.cdc.gov/ncidod/sars/guidance/ c/recommended.htm). although these may not all be generalized to every new respiratory threat, the principles described in the reference can guide institutional preparation for any large-scale respiratory pathogen threat. similar readiness plans for bioterrorism preparedness have been devised and published conjointly by the cdc and the association of professionals in infection control and epidemiology. 12 all health care facilities should have policies and procedures in place for respiratory infection control practice with specific operational plans for handling a large influx of potentially infectious patients in the event of a significant outbreak. 13 when patient influx exceeds institutional capacity, plans should designate alternative triage and treatment areas either outdoors or in other nearby large-capacity facilities. 14 although plans may designate patient care areas that exceed hospital capacity, staffing issues may limit the ability to actually use these areas in category ia. strongly recommended for implementation and strongly supported by well-designed experimental, clinical, or epidemiologic studies. category ib. strongly recommended for implementation and supported by certain experimental, clinical, or epidemiologic studies and a strong theoretic rationale. a real event. community isolation and treatment facilities may also be activated; a prototype for such a facility was developed by the cdc for sars. in general, community facilities will likely house and treat patients with milder disease, with the public health department coordinating these procedures and venues. community isolation facilities (eg, motels, hotels) should have rooms that are equipped with private bathrooms, as well as receptacles to dispose of soiled linen and contaminated waste. personnel who work at the facility should also have n-95 respirators available, as well as disposable gowns and gloves. in general, patients at these facilities will be expected to care for themselves. 15 other hospital infection-control procedures may involve cohorting potentially infectious patients (if isolation beds are not available), as well as rapidly discharging appropriate inpatients and canceling elective procedures to alleviate strain on hospital resources. preemptive planning and knowledge of health care facility (and ed) resource availability are critical components of preparedness. lack of resource planning was cited as a significant factor that contributed to the spread of sars in southeast asia and toronto. 16 unfortunately, few recent data exist describing the availability of isolation facilities in us eds. a 1995 study found that less than 20% of eds had negativepressure isolation rooms. 17 in a recent press release from november 2005, american college of emergency physicians leaders warned that there is an urgent need for increased ed and hospital planning, specifically citing lack of adequate surge capacity, isolation facilities, and staff to treat the large increase in the number of patients that may result from an influenza pandemic. 18 depending on the circumstances of the outbreak, public health officials may recommend keeping suspected noncritical infectious patients at home. the cdc's guidelines for home isolation for sars and pandemic influenza serve as prototypes. 19, 20 alternatively, specific health care facilities may be designated as referral centers for suspected cases. 15 although the public health department will ultimately be responsible for coordinating implementation of these types of large-scale overcapacity plans, emergency physicians need to understand the types of options available. ed physician participation in policy development will be critical in providing practical guidance for ed patient care and operations. policies to support rapid identification of patients with suspected respiratory infections that have serious public health consequences (eg, sars, avian influenza) should include mechanisms for definitive diagnostic testing and immediate reporting to the local health department. the hospital laboratory should be advised to take appropriate precautions with specimens and coordinate specialized testing with local or state health department laboratories. in a suspected outbreak with potential epidemic risk, procedures for contact tracing must be instituted. effective communications mechanisms between eds and health departments are required to allow contact tracing of potentially exposed patients, visitors, and health care workers who live in the community. contact tracing involves either active or passive monitoring. active monitoring consists of direct public health contact (telephone or in person), for example, once a day for exposed persons to assess for symptoms and address any needs. passive monitoring relies on the affected person's contacting the health authorities if symptoms develop. methods of monitoring depend on the exposure risk and capacity of the public health infrastructure. regardless of the type of monitoring recommended, all individuals in contact with a potentially infectious person need to be advised of symptoms and what to do if symptoms develop. additionally, persons with high-risk exposures may require activity restrictions. although the public health department would be responsible for the contact tracing process, emergency physicians need to understand these basic principles because they will likely be called on to work closely with public health departments and provide information about persons who are infected or exposed while in the ed. telephone numbers for the local health department should be readily available in all eds. policies should include clear designations of specific persons within the hospital who are responsible for communication with public health officials (eg, hospital infection control officer) and dissemination of up-to-date information to health care staff (eg, hospital chief executive officer). policies need to include processes for initiating communication with key public health officials after hours and on weekends and guidance about when communication should be initiated. potential community contacts should be identified in advance and be capable of effectively communicating needs and concerns of the public. 6 although proper patient care is the main priority within the ed, the burden of protecting uninfected individuals from communicable illnesses is critical for minimizing spread of disease and the influx of new cases. the "hierarchy of control technologies" consists of (in order of effectiveness) engineering controls, administrative and work practice controls, and use of personal protective equipment. consistent application of these principles demonstrated success in limiting tb resurgence more than a decade ago and, more recently, the spread of sars. 21 understanding the hierarchy allows comprehensive planning, clear implementation, and appropriate local adaptations. the most effective practices from each category should be implemented according to characteristics of the responsible agent. for instances in which the infectious agent is unknown, the most restrictive isolation methods available should be instituted. emergency physicians' preparedness thus requires understanding of institutional resource availability and capacity and early initiation of infectious disease or public health consultation if a new outbreak is suspected or institutional capacity is at risk of being overwhelmed. problems with limited isolation resources in the ed or inpatient setting are usually best addressed in the short term by use of cohorting strategies. engineering controls provide passive protection for health care workers, visitors, and patients. measures include use of isolation rooms (including negative pressure), filtration devices, and physical separation (eg, closing doors or cohorting). negative pressure isolation systems prevent contaminated air from traveling to other areas of the ed or hospital, which is the most efficient method for early containment of infectious respiratory pathogens because airflow from either single rooms or small units can be controlled. however, when the organism load is extremely high, negative-pressure units may not be 100% effective, because they leave live pathogen in the air or on surfaces. [22] [23] [24] [25] increased efficacy can be realized by supplementing negative-pressure isolation systems with a high efficiency particulate air (hepa) filtration system. hepa filtration systems supplement negative-pressure systems, removing fungi and bacteria greater than 0.1 m from the atmosphere. these can be installed in ventilation ducts but are also available as portable units. addition of ultraviolet lights allows killing of spores and active organisms. all hepa filters must be properly installed and maintained according to the manufacturer's instructions to ensure satisfactory decontamination. 25, 26 closing doors and cohorting of patients are recommended if no proper isolation room is available. such methods proved effective in hong kong in early 2003, when sars patients were cohorted into 3 separate observation wards, with no subsequent secondary transmission reported. 27, 28 unfortunately, if not done properly, cohorting in open wards may contribute to increased infection, as was seen in the early toronto sars experience. 27 thus, it should be recognized that although possibly beneficial as an adjunctive measure when resources are scarce, physical separation and cohorting do not guarantee protection. accordingly, health care workers should use proper infection controls when visiting patients in rooms, including droplet precautions and, if indicated, personal occupation droplet precautions (ib) are applied in addition to standard precautions for patients known or suspected to be infected with microorganisms transmitted by droplets (larger than 5 m that can be generated during coughing, sneezing, talking, or the performance of procedures). in addition to wearing a mask as outlined under "standard precautions," wear a mask when working within 3 feet of the patient. limit transport to essential purposes only. if transport is necessary, mask the patient if possible. airborne precautions (ib) are applied in addition to standard precautions for patients known or suspected to be infected with microorganisms transmitted by airborne droplet nuclei (evaporated droplets containing microorganisms that remain suspended in the air and that can be widely dispersed by air currents within a room or over a long distance). safety and health administration (osha)-approved respirators. administration of effective infection containment requires written policies and procedures and is the "second tier" in the hierarchy of infection control. operational policies should include explicit criteria for suspecting disease, restricting contact with patients suspected of having infection, controlling transport and high-risk procedures, quarantining of patients and contacts, contact tracing, implementing methods for disinfection, and monitoring of isolation procedures. 21 procedural policies should address need for supplemental staff, education and training for health care workers, medical surveillance of exposed health care workers, and communication with public health officials and the general public. 21 rapid implementation of these policies is the key to infection control in an outbreak scenario and proved critically important in controlling sars. 29 although sars specific, the cdc's checklist for sars preparedness in health care facilities 16 provides a prototype of the types of policies and procedures that should be considered in the event of any bioterrorism-related or emerging communicable respiratory outbreak. in the aftermath of an infectious outbreak, postevent analysis should be conducted to determine which management efforts were effective and which were not from the hospital's and community's perspective. representatives of all affected departments and organizations should be included, and appropriate revisions should be incorporated into policies. health care worker surveillance should also attend to posttraumatic stress assessment and treatment, as indicated. 30 personal protective equipment, including gloves, gown, masks, and respirators, provides barrier protection, preventing skin and mucous membrane exposures. although these resources offer protection to individuals by reducing likelihood of direct contact, they are categorized as the third hierarchy level because they do not eliminate the pathogen and may have limited effectiveness because of problems such as variable health care worker adherence, potential for equipment failure, and inadequate equipment availability. the 2 cdc-and oshaapproved personal air filtration systems are n95 masks and powered air-purifying respirators. n95 masks are simple and inexpensive but require individual fit-testing. powered airpurifying respirators offer the advantage of nearly universal fit but are far more expensive and cumbersome to use. both devices require medical evaluation and clearance for safe use. personal protective equipment should be used by all health care workers in outbreak settings when patients with potentially contagious respiratory infections are treated. proper education of health care workers about respiratory hygiene practice is critical for effective infection control. written policies and procedures for education and training of health care workers should be developed at the institutional level. education topics should include infection control precautions, criteria for suspecting disease at first contact and methods for restricting contact with patients suspected of having infection, limiting and controlling patient transport, and minimizing exposure during high-risk procedures. additional educational topics for ed providers and administrators include criteria and procedures for quarantining of contacts, protocols for disinfection and for monitoring isolation, and methods for maintaining medical surveillance of exposed health care workers. 21 providing adequate hospital staffing is important in any disaster, and personnel issues particular to infectious disasters must be given consideration in developing hospital and ed plans. all health care facilities should have policies and procedures for mobilizing and reassigning staff to more critical areas in the event of a disaster. because health care staff may be reluctant to come to work if they believe they are at risk of contracting an infectious illness, it is critical that the facility planning measures be reviewed in advance, with contingencies and backups in place. health care workers (particularly those working in front-line ed settings) should also be given priority for receiving vaccines or prophylactic antimicrobials, when appropriate. offering additional incentives to staff to come to work may also be required in certain situations. infectious outbreaks create the additional problem that health care workers themselves may become ill. plans for respiratory outbreaks should include regular evaluation of health care workers for infectious signs or symptoms, criteria for removing health care workers from patient care, and criteria for quarantine (either at home or in the workplace). health care airborne infection isolation rooms (aiir) workers' desire for a workplace quarantine option was demonstrated during the sars outbreak, in which individuals did not want to subject family members to an increased risk of infection. although the cdc provides recommendations for influenza vaccination among health care workers, 31 there are no uniform recommendations for health care worker vaccination for all potential respiratory pathogens. in light of this, the influenza recommendations not only serve to guide planning for annual influenza epidemics but also may provide a template for other vaccine-preventable pathogens. research has demonstrated that influenza vaccination of health care workers contributes to a substantial decrease in patient mortality, 32 which has led some experts to call for mandatory vaccination of health care workers. 33 the cdc provides specific recommendations about when to provide chemoprophylaxis for influenza, 34 which may be used as a template and adapted to other pathogens when guidelines are developed for new and emerging pathogens for which vaccines are available. vaccinations plans for certain agents (eg, anthrax and smallpox) are controversial. currently, preexposure anthrax vaccine is not recommended for health care workers. after the terrorist attacks of 2001, the us government developed a smallpox vaccination plan that included "formation of smallpox response teams" at each institution. emergency physician volunteers participated as critical members of the team. although controversy still exists in the emergency medicine community about these recommendations, 35 they remain. because the threat of a true smallpox event remains low, however, routine vaccination for all health care workers for smallpox is not recommended by the advisory committee on immunization practice. 36 facilities should create a priority list for employee smallpox vaccination in the event of an outbreak, and emergency staff should be included. concerns about the potential spread of respiratory pathogens begin at the point of entry into the health care system and continue to the inpatient setting. emergency physicians need to be aware of the potential for infection, illness, and transmissibility in a variety of potentially high-risk environments, including (1) emergency medical services (ems) and triage settings (in which historical and clinical information may be limited and risk underestimated), (2) during performance of "high-risk" invasive airway procedures, and (3) during patient transport to the various inpatient units throughout the hospital. the cdc provides specific recommendations for ems transport of sars patients. 37 although specific ems recommendations do not exist for each of the transmissible respiratory threats, the general principles outlined in the sars directives are applicable to the transport of any patient with a suspected serious and contagious life-threatening respiratory infection 37 and include the following: (1) potentially contagious patients should be transported with as few ems personnel as possible, (2) family members should not be allowed to ride with patients in the ambulance, (3) ems personnel traveling with a patient suspected of having infection should wear proper personal protective equipment, including isolation gown, double gloves, facemask, and n95 or higher-grade respirator (eg, n99, 100, a powered air-purifying respirator), (4) patients should wear a surgical mask if feasible and, if not, use tissues to cover their mouth or nose during coughing or sneezing; and (5) patients should be transported in a vehicle that has separate ventilation systems and compartments for patient and driver, whenever possible. finally, advanced ed notification is advised to facilitate prearrival planning to limit exposure of other individuals. ems personal protective equipment should be handled as medical waste, and ems vehicles should be decontaminated before transporting another patient. 38 the importance of implementing effective triage and edbased diagnostic strategies is underscored by experience with highly transmissible respiratory infections such as tb and sars. several hospital-and ed-based studies provide data that demonstrate that lack of either provider education or adherence to institutional guidelines or inadequate diagnostic evaluation of patients at risk results in increased risk of disease transmission. 3, 5, 16, 17, 39 underscoring this is the findings from one epidemiologic outbreak of sars in toronto that found that 36% of new infections in the hospital occurred in health care workers, with the highest rates in those working in eds and icus. 5 both the world health organization and the cdc provide general recommendations for handling of patients with suspected respiratory infections that include having triage staff adhere to proper hand hygiene procedures and donning face masks and eye protection. 40, 41 if sars or tb is suspected, health care workers in eds should don an n-95, 99, or 100 respirator. 42 the degree of vigilance that should be applied to screening for respiratory infections depends on the current risk level, with the most up-to-date regional risk information based on surveillance data provided on a cdc web site. 43, 44 for example, there are 3 basic risk levels that apply to sars: (1) no current sars transmission anywhere in the world, (2) active sars transmission in limited geographic areas, and (3) sars transmission within the community in which one is practicing. in the absence of person-to-person transmission of sars worldwide, the goal of domestic surveillance is to maximize early detection of cases while minimizing unnecessary laboratory testing and social disruption. in the absence of known transmission worldwide, the overall likelihood that a person in the united states with fever and respiratory symptoms will have sars is exceedingly low. 45 if sars transmission is present in limited geographic areas, screening should focus on identifying persons with possible geographic exposures. when person-toperson sars transmission is present in the community, everyone with fever or respiratory symptoms should be screened for sars. in an outbreak scenario (eg, sars, avian influenza, or tb), explicit written criteria should be provided to triage personnel to allow rapid isolation of patients who may be harboring a highly contagious infection. the cdc has issued specific screening tools to be used for rapid detection and isolation of possible sars patients, depending on the absence or presence of personto-person transmission in the world (figures 6 and 7) . various similar ed-based triage guidelines for specific agents (eg, tb, influenza and avian influenza) 13, 17, 34 that include use of early radiography have been developed, and the cdc web site (available online at http://www.cdc.gov) should be consulted for the most up-to-date recommendations, as well as the current threat level of sars. 44 from the ed perspective, development of decision guidelines may be based on the characteristics of the epidemic and may require development and modification in real time. an excellent example is provided by an ed in singapore, in which a triage tool, developed throughout a 1-year period, yielded a false-negative rate for sars case identification of 0.28%. 46, 47 the cdc recommends that tissues and masks be made readily available for all symptomatic patients who enter the ed or hospital doors (to cover their mouths and noses) and that sinks or handwashing stations be accessible for all patients in waiting rooms and triage areas. 13 during periods of increased respiratory infections (eg, influenza season), separation of symptomatic and asymptomatic patients in waiting rooms and triage areas is advised, and surgical masks should be distributed to all patients with active respiratory symptoms. when it is not feasible to set up separate waiting areas in the ed, symptomatic patients should be encouraged to sit at least 3 feet away from other patients in the waiting room. according to the cdc, this practice is supported by level ib evidence. the cdc recommends that visual education be provided at all patient entrances to eds during periods of heightened respiratory alert. visual alerts (including signs, pamphlets, and other general education measures about respiratory hygiene) are proven measures that can decrease disease transmission. 13 it is recommended that visual alerts be present in several languages (depending on the region of the country and population served) and be provided at an appropriate reading level to allow for comprehension by the majority of the population. content of educational material should include a general description of standard respiratory hygiene methods, including handwashing, use of disposable tissues for covering mouth and nose, and staying at least 3 feet away from persons with symptoms. although proper patient care is the main priority within the ed, protecting uninfected patients from communicable illnesses is also important. early isolation decreases the likelihood of person-to-person transmission. patients with a suspected but unidentified communicable respiratory infection should be placed in an environment with the highest level of protection available until definitive identification of the offending pathogen can be made or the possibility of a public health threat can be safely ruled out. laboratory diagnosis of respiratory contagious pathogens represents a critical step in decisionmaking about the need for isolation, treatment, and disposition. unfortunately, from the standpoint of the emergency physician, most current criterionstandard laboratory assays rely on serologic or culture methodologies often requiring days to weeks for definitive reporting. even when alternative nonculture-based methodologies are available (eg, acid-fast bacillus smear results for tb), reliable confirmation requires multiple sample procurement during a period of several days. for this reason, decisionmaking about patient care relies on clinical suspicion, which includes current knowledge of the community likelihood of a respiratory infectious event, risk status of the patient, and patient presenting signs and symptoms, which are often nonspecific. as described under "ed triage and waiting room," clinical guidelines may be used as well for assistance. rapid diagnostic assays for contagious respiratory pathogens hold great promise with regard to assisting ed physicians in treatment of patients with suspected respiratory contagious pathogens. although significant molecular advancements have recently been made in design and evaluation of rapid molecular-based methods, most notably using polymerase chain reaction techniques, few have reached the status of standard of care for point-of-care use. rapid diagnostic assays for influenza are available, but none has adequate sensitivity or specificity to allow recommendation for definitive care in ed settings. 48 interventional airway procedures in the ed (including use of nebulized therapy and endotracheal intubation) increase risk for airborne transmission of disease because they result in release of high pathogen loads. 49 although most procedures can be done in the ed, the us department of health and human services recommends that in outbreak settings, aerosol-generating procedures (eg, nebulized medications or bilevel positive air pressure) be avoided as much as possible. 50 when essential for patient care, health care workers involved in these procedures should use n95 respirators or powered air-purifying respirators, along with gloves and gowns. after the procedure is completed, personal protective equipment should be removed and safely discarded to avoid contaminating the health care worker or the environment. 51 specific detailed recommendations about intubation suggest that added measures be taken to reduce unnecessary exposure to health care workers, including reducing the number of health care workers present and adequately sedating or paralyzing the patient to reduce the possibility of a cough. 52 all high-risk procedures should be performed only by highly experienced staff. it is recommended that patient transport and movement from the room be limited to essential purposes only. when transport out of the room occurs, masks should be worn by the patient to reduce the opportunity for transmission to patients and staff and reduce environmental contamination. further, health care workers in the area to which the patient is to be taken should be notified in advance. 53 there is a forceful interplay between the health and wellbeing of the public in general and an individual's rights, which is set within a complex and often confusing legal field. 54 this affects emergency practitioners and health care facilities concerning respiratory infections primarily in 2 ways: (1) through the need to notify appropriate public health authorities of reportable infectious diseases, and (2) through the requirement to isolate ill patients and quarantine sick contacts. the cdc is recognized as the lead federal agency for protecting the health of the public and has various federal responsibilities in this regard, including investigations of unusual diseases and federal quarantine authority. 55 according to title 42 united states code section 264, the surgeon general, with the approval of the secretary, is authorized to make and enforce regulations to prevent the introduction, transmission, or spread of communicable diseases. however, the current legal framework of public health oversight and response in the united states is a complex mix of state and federal laws. thus, the specific requirements for any practitioner, ed, or hospital vary according to the local and state laws. 56, 57 the federal government has oversight of importation of infectious diseases and overall quarantine authority, but the individual states generally have the primary authority and responsibility of responding to public health problems within their jurisdiction, such as investigating a cluster of tb cases and isolating infectious individuals. states also have the responsibility of addressing their own public health emergencies. the interface between law, medicine, and public health requires the balancing of many potentially competing interests, especially individual human rights versus the need to protect the public's health. there is significant background and legal precedent on this topic. 58 it is best for institutions to have an existing relationship with local or state public health officials to ensure ongoing bidirectional communication in times of urgency or emergency. as in any emergency, adequate preparedness, coupled with clear communication, allows for coordinated response. the list of reportable diseases is established by each state or territory, though the cdc has recommended specific case definitions for infectious conditions that could fall under public health surveillance. 57 timeliness and mechanism for reporting also vary for different diseases. for example, a case of smallpox requires an immediate telephone call, whereas cases of gonorrhea may be reported in a weekly written report. although this reporting activity may be mandated, it raises important legal and ethical issues about the balance between the duty to report and an individual's right to privacy. the surgeon general is responsible for controlling, directing, and managing all united states quarantine stations, which includes isolation for people who are ill and quarantine for people exposed but not ill. in april 2003, sars was added to the list of diseases for which quarantine is authorized (other diseases included are cholera, diphtheria, tb, plague, smallpox, yellow fever, and viral hemorrhagic fever). a lesson from sars quarantines in singapore is the capacity of a highly contagious infection to cause a rapid pandemic. the implications of quarantining a population or individuals for the length of the incubation time (or the length of the illness if patient is infected) are numerous. quarantining a large population involves significant commitment of resources. to overcome the legal obstacles of a major quarantine, a plan must be in place well in advance of an outbreak. as part of the public health infrastructure, ed health care workers may be called on to participate in various infection containment strategies, including quarantining of individuals or vaccinating large segments of the population. one other important legal aspect relates to occupational safety. osha has a number of rules and regulations designed to protect the health and safety of health care workers. osha's jurisdiction includes all health care facilities. health care workers in eds should be aware that rules and regulations related to respiratory hygiene are legally mandated and must be implemented in hospitals in accordance with current guidelines, as described elsewhere. the increasing likelihood that a highly contagious respiratory outbreak such as pandemic influenza will be seen soon, coupled with recognition of the presence of significant gaps between experimental and theoretic advances in both technologic and methodologic approaches to infection control (versus true ed preparedness), has created the need for further research. rapid point-of-care diagnostics hold great potential for improving triage, treatment, and disposition planning. future research will need to bridge the divide between the numerous point-of-care assays that are under development and the need to have a reliable, easy-to-use test that is adequately sensitive and specific for clinical decisionmaking. although such development will likely take several years of investigation, 2 such diagnostics in early phases of development include a polymerase chain reaction-based respiratory pathogen panel 59 and a mass spectrophotometry platform that can rapidly evaluate polymerase chain reaction products to identify any potential new emerging threat. 60 there are also multiple practical issues related to ed evaluation requiring study, including development of more effective clinical decision guidelines for isolation and diagnosis and determination of the impact and best practice methods for care in ambient settings. the effectiveness of ed air filtration techniques also remains unclear, and educational research in this area is required. the numerous ethical, legal, and practical challenges associated with isolation and quarantining of patients will also require further study, with emphasis on ed-specific questions such as the role of eds in care of "routine" emergencies, development of ed surge capacity, and optimization of methods for coordination of eds with the public health sector. one other area of research that is gaining increased attention and has particular relevance for eds involves surveillance methods for tracking respiratory illnesses. current approaches that involve ed-based researchers include syndromic surveillance based on ed complaints, 61 evaluation of the efficacy of increased diagnostic testing in eds, 62 and tracking of ed prescriptions. 63 these new areas of research will likely grow rapidly as the threat of respiratory infections becomes more prevalent. this review serves as a brief synopsis of the issues surrounding respiratory hygiene as they relate to the ed. protecting patients and staff is a difficult task in the ed because cases of contagious respiratory infections are often not immediately identifiable. this report focuses on the development of appropriate policies relating to patients with potential transmissible respiratory pathogens. education of key individuals, along with rapid dissemination of accurate information, is necessary to support these policies and will be instrumental in ensuring effective implementation. emergency physicians will continue to be pivotal in the development of these policies by maintaining active administrative and leadership positions in hospitals, and advancing understanding of the critical role they play in the early identification, treatment, and containment of these potentially lethal respiratory pathogens. 25 pulmonary complications of solid organ and hematopoietic stem cell transplantation pulmonary considerations in the immunocompromised patient sars transmission among hospital workers in hong kong an outbreak of severe acute respiratory syndrome among hospital workers in a community hospital in hong kong investigation of a nosocomial outbreak of severe respiratory syndrome (sars) in toronto implications of the world trade center attack for the public health and health care infrastructures public health preparedness for mass-casualty events: a 2002 state-by-state assessment emergency department summary: advance data from vital and health statistics; no. 335. hyattsville, md: national center for health statistics emergency department utilization by adolescents in the united states guideline for infection control in health care personnel hospital infection control practices advisory committee: guideline for isolation precautions in hospitals association of professionals in infection control bioterrorism task force public health guidance for community level preparedness and response to severe acute respiratory syndrome (sars): version 2: supplement c: preparedness and response in health care facilities lessons from taiwan public health guidance for community-level preparedness and response to severe acute respiratory syndrome (sars) core document: supplement c: preparedness and response in health care facilities clinical review: sars: lessons in disaster management tuberculosis infectioncontrol practices in united states emergency departments american college of emergency physicians. emergency physicians say federal pandemic influenza plan doesn't address nation's lack of surge capacity and isolation beds questions and answers on the executive order adding potentially pandemic influenza viruses to the list of quarantinable diseases using the hierarchy of control technologies to improve healthcare facility infection control: lessons from severe acute respiratory syndrome guidelines for preventing the transmission of tuberculosis in health-care facilities environmental and occupational health response to sars an evaluation of portable high-efficiency particulate air filtration for expedient patient isolation in epidemic and emergency response public health guidance for community-level preparedness and response to severe acute respiratory syndrome (sars), version 2, supplement i: infection control in healthcare, home, and community settings core curriculum on tuberculosis: what the clinician should know sars outbreak in the greater toronto area: the emergency department experience management of inpatients exposed to an outbreak of severe acute respiratory syndrome (sars) an emergency department response to severe acute respiratory syndrome: a prototype response to bioterrorism psychiatric morbidity among emergency department doctors and nurses after the sars outbreak updated interim influenza vaccination recommendations: 2004-2005 influenza season effects of influenza vaccination of health-care workers on mortality of elderly people in long-term care: a randomised controlled trial requiring influenza vaccination for health care workers: seven truths we must accept infection control guidance for the prevention and control of influenza in acute-care facilities emergency physicians' perspectives on smallpox vaccination advisory committee on immunization practices (acip) statement on smallpox preparedness and vaccination interim guidance: ground emergency medical transport for severe acute respiratory syndrome patients toronto emergency medical services and sars emerg infect dis delayed recognition and infection control for tuberculosis patients in the emergency department public health guidance for community-level preparedness and response to severe acute respiratory syndrome (sars), version 2, supplement i, section iii: infection control in healthcare facilities world health organization. hospital infection control guidance for severe acute respiratory syndrome (sars) sars in hospital emergency room respiratory and enteric viruses branch. surveillance summaries and additional information in the absence of sars transmission worldwide: guidance for surveillance, clinical and laboratory evaluation, and reporting, version 2 evolution of an emergency department screening questionnaire for severe acute respiratory syndrome crash course in decision making performance characteristics of clinical diagnosis, a clinical decision rule, and a rapid influenza test in the detection of influenza infection in a community sample of adults cluster of severe acute respiratory syndrome cases among protected health-care workers: toronto, canada hhs pandemic influenza plan: supplement 4: infection control: personal protective equipment for special circumstances supplement 4 infection control. part iv.d. occupational health issues guideline for isolation precautions in hospitals, part ii: recommendations for isolation precautions in hospitals center for law and the public's health at johns hopkins and georgetown universities. core legal competencies for public health professionals provider disease list and reporting instructions centers for disease control and prevention. case definitions for infectious conditions under public health surveillance public health law and ethics: a reader (california, milbank books on health and the public, 4) diagnostic system for rapid and sensitive differential detection of pathogens rapid identification and strain-typing of respiratory pathogens for epidemic surveillance the frontlines of medicine project progress report: standardized communication of emergency department triage data for syndromic surveillance pcr-based diagnostics for infectious diseases: uses, limitations, and future applications in acute-care settings syndromic surveillance using emergency department data the acep public health committee is grateful to ms. margaret montgomery and ms. julie dill for their time and assistance in preparation of this manuscript. key: cord-326004-wg47sd06 authors: wilson, patrick t; baiden, frank; brooks, joshua c; giessler, katie m; apio, gavin; punguyire, damien; moresky, rachel t; sylverken, justice; nyarko-jectey, kwadwo; tagbor, harry; larussa, philip s title: respiratory pathogens in children 1 month to 5 years of age presenting with undifferentiated acute respiratory distress in 2 district-level hospitals in ghana date: 2018-09-03 journal: j pediatric infect dis soc doi: 10.1093/jpids/piy090 sha: doc_id: 326004 cord_uid: wg47sd06 ghanaian children (2176) aged <5 years who presented with undifferentiated acute respiratory distress were tested for respiratory pathogens using a biofire filmarray polymerase chain reaction assay. rhinovirus and/or enterovirus was detected in 36% of the assays, respiratory syncytial virus in 11%, and parainfluenza in 7%. respiratory syncytial virus and metapneumovirus were detected more frequently in the rainy season than in the dry season. ghanaian children (2176) aged <5 years who presented with undifferentiated acute respiratory distress were tested for respiratory pathogens using a biofire filmarray polymerase chain reaction assay. rhinovirus and/or enterovirus was detected in 36% of the assays, respiratory syncytial virus in 11%, and parainfluenza in 7%. respiratory syncytial virus and metapneumovirus were detected more frequently in the rainy season than in the dry season. keywords: pediatrics; picornaviridae infections; pneumonia; respiratory tract infections; viral diseases. despite significant improvements in the survival rates of children younger than 5 years around the world, pneumonia remains a leading killer of children globally [1] . a majority of the 1 million pneumonia deaths per year in children <5 years old occur in low-and middle-income countries; most of them are clustered in africa [1] . laboratory diagnostics, such as bacterial cultures and respiratory viral testing, are often lacking in these resource-poor settings [2] , which contributes to the dearth of accurate epidemiological data and an unnecessary use of antibiotics, especially outside of tertiary care hospitals. a broad-scope assay that provides an unbiased description of a wide array of respiratory pathogens in children in developing countries is needed. a recently conducted prospective randomized controlled trial at 2 district-level hospitals in ghana revealed that the use of continuous positive airway pressure (cpap) reduces the allcause mortality rate in children <1 year old who presented with undifferentiated acute respiratory distress [3] . in that study, nasopharyngeal swabs were collected from the children at the time of presentation and tested for common pediatric respiratory pathogens; the goal was to decrease the knowledge gap regarding the incidence of respiratory pathogens that affect children <5 years of age in low-and middle-income countries. the study sites were mampong district hospital and kintampo municipal hospital in ghana; each hospital serves as a catchment area of almost 100 000 people. both hospitals have limited access to plain radiography, and neither of them can perform bacterial cultures, respiratory virus panels, or blood gas studies or measure c-reactive protein levels. the bimodal rainy season in ghana runs from approximately april to july as the major season and september to november as the minor season; a long dry season lasts from december through march. our observational study was part of the prospective cpap survival study [3] . the aims of this observational study are to determine the percentage of children whose nasopharyngeal swab detected a common respiratory pathogen, describe the epidemiology of the pathogens detected, and report the characteristics of patients with and of those without a detectable respiratory pathogen. the study protocol was approved by and procedures were followed in accordance with the ethical standards of the columbia university medical center institutional review board and local institutional review boards at the kwame nkrumah university of science and technology and the ghana health services (this study is registered at clinicaltrials.gov under identifier nct01839474). the ghana food and drug authority provided regulatory oversight for the trial. all children who presented to 1 of the 2 hospital emergency departments with a rapid respiratory rate were screened for study eligibility according to the cpap survival study protocol [3] . specific inclusion criteria were an age of 1 month to 5 years, a respiratory rate of more than 50 breaths per minute in children aged 1 to 12 months or more than 40 breaths per minute in children older than 12 months, and use of accessory muscles or nasal flaring. patients were excluded if they had a contraindication to the use of nasal cpap. written documentation of informed consent was obtained. for the collection of nasopharyngeal swabs, a sterile cotton-tipped swab was inserted gently into 1 nostril, rolled gently in the posterior nasopharynx, and then withdrawn slowly. the swab was placed immediately into a microtest m4rt vial containing transport and storage medium (remel, lenexa, kansas). specimens were placed immediately in a −20°c freezer at each study site. approximately once per week the frozen specimens were collected and transported by land in an insulated cooler with frozen icepacks to a −80°c freezer at the kwame nkrumah university of science and technology in kumasi, ghana. every 6 months, the specimens were transported by commercial airline on dry ice to columbia university, where they were stored at −80°c until the time of testing. nasopharyngeal specimens were tested using the filmarray assay (biofire, salt lake city) [4] . the assay can detect adenovirus, coronavirus strains 229e, hku1, oc43, and nl63, metapneumovirus (mpv), rhinovirus and/or enterovirus (rv/ ent), influenza types a, a/h1, a/h1-2009, a/h3, and b, parainfluenza types 1, 2, 3, and 4, respiratory syncytial virus (rsv), bordetella pertussis, chlamydophila pneumoniae, and mycoplasma pneumoniae. data were analyzed using graphpad prism 7.00 for windows (graphpad software, la jolla, california). figures were constructed in microsoft excel. descriptive statistics were used to present the data; the t test was used to compare means, analysis of variance for multiple comparisons of means, and the χ 2 test to compare proportions. a total of 2486 children were assessed for eligibility, 2200 children were enrolled, and 2176 (99%) nasopharyngeal specimens were collected, transported, and tested for respiratory pathogens. of the 2176 specimens tested, 1 or more pathogens were detected in 1276 (59%). most patients (71%) with a positive polymerase chain reaction (pcr) assay result were younger than 2 years. fifty-six percent of the patients were male; the median weight was 9.0 kg, and the median presenting respiratory rate, heart rate, pulse oxygen saturation on room air, and temperature were 56 breaths per minute, 152 beats per minute, 98%, and 37.5°c, respectively. patients with a positive pcr result (n = 1276) were younger, weighed less, presented with a higher respiratory rate, had a higher hemoglobin level and white blood cell count, had malaria detected less often, and received less antimalarial therapy but more antibiotics than those whose pcr result was negative (p < .001). the figure 1a ). the distributions of organisms detected among the 2 study sites were similar. only 1 respiratory pathogen was detected in 1064 (83.4%) specimens, 2 pathogens in 190 (14.9%) specimens, 3 pathogens in 19 (1.5%) specimens, and 4 pathogens in 3 (0.2%) specimens. children with rsv were younger, weighed less, had a higher respiratory rate, a higher hemoglobin concentration, a lower oxygen saturation, and a lower rate of malaria than the children in whom another virus was detected (p < .0001, analysis of variance). forty-six of the patients with a positive pcr result died. rv/ ent (89%), rsv (6.5%), coronaviruses (6.5%), and parainfluenza viruses (4%) were the most common organisms detected in this group. rsv and mpv were detected more often between the months of july and december in both years of the study period, whereas parainfluenza and adenovirus were detected more often between january and june ( figure 1b ). rv/ent was detected equally throughout both years. in this report, we describe the respiratory pathogens detected in children aged 1 month to 5 years who presented to 1 of 2 district-level hospitals in ghana with undifferentiated acute respiratory distress. we found a significant burden of respiratory viruses that might have been contributing to their clinical presentation and need for medical care. a viral pathogen, most commonly rv/ent, was detected in the majority of the study participants. rvs and ents are among the most frequent infectious agents globally [5] . because of the similarities of these 2 viruses, our assay was unable to differentiate them from each other. because almost half of the patients in whom rv was detected were asymptomatic in other studies [6] , the role of rv as a lower respiratory tract pathogen remains uncertain. however, current advances in molecular diagnostic techniques have revealed the presence of rv in the lower respiratory tract, and its role in lower airway diseases is increasingly being reported [5, 7] . as in other studies [8, 9] , rsv was detected in a significant proportion of children in our study. children with rsv were found to be younger and to have more severe respiratory symptoms than children with a different viral infection, which is consistent with previous reports in the literature [9, 10] . influenza was detected in fewer than 3% of the participants, and we found no apparent seasonal peak in this 2-year study. because tropical climates usually lack identifiable fall, winter, spring, and summer seasons, the incidence of viral respiratory pathogens might not be similar to those in temperate climates. in ghana, we found a clear increase in the incidence of rsv and mpv during the rainy months and a marked decrease in the dry season, which might be an effect of more crowding during the rainy season, because it is often seen during the winter months in other settings. there were several limitations to this study. because this study was part of another study, children were excluded if they did not meet the criteria for safe use of nasal bubble cpap. no chest imaging was available, and bacterial cultures were not performed. testing on specimens obtained from the upper airway was performed and might not represent a true infection of the lower airway. also, the human immunodeficiency virus status of the study participants was not known, but other literature suggests that its incidence in ghana is low [11] . despite the limitations of this study, our results show that viral respiratory pathogens were frequent among children aged 1 month to 5 years who presented with acute respiratory distress to 1 of 2 district-level hospitals in ghana. rv/ent was detected most often, but additional studies are needed to determine the role that these viruses play in moderate-to-severe respiratory illness in children who live in a low-income country. rsv and mpv were detected more frequently in the rainy season than in the dry season, and additional studies are needed to elucidate the exact mechanism of the observed seasonal variation. global, regional, and national causes of child mortality in 2000-13, with projections to inform post-2015 priorities: an updated systematic analysis laboratory medicine in africa: a barrier to effective health care continuous positive airway pressure for children with undifferentiated respiratory distress in ghana: an open-label, cluster, crossover trial an automated nested multiplex pcr system for multi-pathogen detection: development and application to respiratory tract infection rhinoviruses and respiratory enteroviruses: not as simple as abc community surveillance of respiratory viruses among families in the utah better identification of a) frequency of organisms detected among the 1276 children with a positive pcr result; 1513 pathogens were detected. (b) percentages of participants with a specific pathogen detected each month of the 2-year study period. n = number of participants enrolled during each month. abbreviations: adeno, adenovirus; mpv, metapneumovirus; paraflu, parainfluenza; re, rhinovirus and/or enterovirus; rsv, respiratory syncytial virus. germs-longitudinal viral epidemiology (big-love) study epidemiology and clinical profile of pathogens responsible for the hospitalization of children in sousse area the burden of respiratory syncytial virus infection in young children burden of respiratory syncytial virus infection in young children the burden of respiratory syncytial virus infection in young children joint united nations program on hiv/aids (unaids) joint united nations program on hiv/aids (unaids) we express our deepest gratitude to akua amprofi, moses afafu, hamilton boateng, and memuna basiru, for their efforts to ensure consistent and timely transport of the viral samples from the study sites to the knust laboratory, and all study nurses who collected the specimens. we thank the knust laboratory for allowing use of its −80°c freezer to store viral samples. we also express significant gratitude to the columbia university medical center larussa laboratory for use of its equipment, space, and −80°c freezers for processing and storing viral samples. we acknowledge the outstanding contribution of biofire in providing filmarray testing kits and equipment at a discounted rate to the study. last, we acknowledge megan benckert, stephanie hubbard, camilla rushton, marco salerno, johane slimane, and liqun wang for their assistance and/or support in performing the pcr testing.financial support. this work was supported by the general electric foundation (grant pt-aabk1277).potential conflicts of interest. all authors: no reported conflicts of interest. all authors have submitted the icmje form for disclosure of potential conflicts of interest. conflicts that the editors consider relevant to the content of the manuscript have been disclosed. key: cord-347246-0vofftmj authors: everitt, j i; richter, c b title: infectious diseases of the upper respiratory tract: implications for toxicology studies. date: 1990-04-17 journal: environ health perspect doi: nan sha: doc_id: 347246 cord_uid: 0vofftmj the consequences of adventitious infectious agents upon the interpretation of toxicology studies performed in rats and mice are incompletely understood. several prevalent murine pathogens cause alterations of the respiratory system that can confuse the assessment of chemically induced airway injury. in some instances the pathogenesis of infection with these agents has been relatively well studied in the lower respiratory tract. however, there are few well-controlled studies that have examined the upper respiratory region, which result in interpretive problems for toxicologic pathologists. the conduct and interpretation of both short-term and chronic rodent bioassays can be compromised by both the clinical and subclinical manifestations of infectious diseases. this paper reviews several important infectious diseases of the upper airway of rats and mice and discusses the potential influence of these conditions on the results of toxicology studies. the validity and reproducibility of rodent toxicology studies depend, in part, on the interaction of numerous environmental, microbial, and genetic factors. control of important animal and extrinsic factors forms the basis of laboratory animal science programs in toxicology. despite numerous advances and sophistication in the field of laboratory animal medicine, adventitious murine microbial agents continue to pose a threat to both shortand long-term studies that use rats and mice. programs for prevention, detection, elimination, and control of rodent infectious agents are necessary in toxicology research and testing. these programs are of particular importance to both toxicologists and pathologists responsible for conducting and interpreting rodent bioassays. specific procedures and equipment used in inhalation toxicity experiments can contribute significantly to the spread of disease and the interaction of infectious agents with inhaled toxicants. many factors, such as inhalation caging; cage rotation schemes; inhalation chamber microenvironments; food and water deprivation during exposures; and stresses from crowding, frequent handling, and servicing of animals, all play important roles in the interplay between pathogen and chemical exposure. the complications of rodent infectious agents to toxicology research and testing has been the subject of increasing awareness and review (1, 2) . recent rodent serology surveys indicate that sialodacryoadenitis virus (sdav), sendai virus, and mycoplasrna pulmonis are three of the most prevalent murine pathogens (3) . all three agents cause significant rodent respiratory disease, with lesions in the upper airways, including the nasal passages. in addition to being a common site for microbial-induced disease, the upper respiratory tract is a target organ of chemically induced injury. this paper will review the respiratory pathology of several important murine pathogens and discuss their importance in toxicology and carcinogenicity studies. sialodacryoadenitis virus is a common, highly infectious coronavirus affecting rats and causing a selflimiting necrosis and inflammation of mixed or serous salivary glands, lacrimal glands, and upper airway epithelium (4, 5) . respiratory tract lesions are generally confined to the upper airway and usually precede the inflammatory changes that occur in the exocrine tissues of the head (6). gross lesions consisting of edematous and/or inflammatory changes are sometimes noted in extrarespiratory tissue in mixed or serous salivary glands, exorbital lacrimal glands, harderian glands, periglandular connective tissue, cervical lymph nodes, or the thymus. everi1t and richter microscopic lesions in experimental sdav infection begin in the nasal respiratory epithelium approximately 48 hr postinfection. initial respiratory epithelial necrosis accompanied by congestion and edema is rapidly followed by a mixed inflammatory cell infiltrate of the lamina propria. necrosis primarily occurs in the respiratory epithelium lining the ventral turbinates and lateral wall of the nasal cavity but usually spares the olfactory mucosa. the nasal meatuses may be filled with exudate composed of necrotic epithelial cells admixed with inflammatory cells and mucus. serous mucosal glands of the nasopharynx generally sustain relatively mild injury (6) , although necrosis of ducts and acini does occur. lesions similar to those in the nasopharynx, but milder and less uniform, are found in the trachea. the rhinotracheitis of sdav is usually self-limiting and it is resolved by the end of the second week of infection (6) . although not as prevalent or as severe as upper respiratory tract lesions, microscopic changes do occur in the lower respiratory tract in experimental infections of rats (7), consisting of focal nonsuppurative bronchiolitis and peribronchial lymphocytic infiltration. although sdav infection is generally a self-limiting disease without significant mortality, it can cause marked effects on toxicology studies through clinical disease or subclinical manifestations. rats with sdav infection often have reduced food consumption, weight loss, and reduced breeding performance all of which can affect toxicology studies. the interactions of sdav with other respiratory pathogens and chemical agents is not well established. recent experimental evidence suggests that sdav infection depletes salivary epidermal growth factor (egf) and may thereby affect egfdependent cell growth processes and experimental carcinogenesis studies in rats (8) . results of a 2-year inhalation toxicity study of methylene chloride were clouded by sdav infection of rats early in the study (9) . in this study, a low number of male rats exposed to the two highest doses of the chemical had an increased incidence of sarcomas in the ventral neck region. the relevance and toxicological significance of these neoplasms was questionable because the species and sex specificity of the response was inconsistent with the body of knowledge on the toxicity of the chemical. it was postulated that the tumor response may have been due to a combination of viral infection and exposure to high concentrations of methylene chloride. this case exemplifies the problems that may occur in toxicologic studies when unusual and unexpected lesions are found in animals that had infectious processes within the target organ of toxicity. sendai virus is a very common respiratory pathogen of laboratory rodents that has complicated numerous toxicology and carcinogenicity studies (10) . in mice and rats, this paramyxovirus causes clinical and subclinical changes with great strain variability in disease expression and resultant pathologic lesions. the virus has a marked tropism for the respiratory tract, including the nasal cavity. sendai virus infection has been well described for mice (11) (12) (13) , and recently the course of experimental infection in the rat has been reported (14) . sendai virus infection causes rhinitis, tracheobronchitis, bronchiolitis, and varying degrees of alveolitis in both rats and mice. there is marked strain variability in qualitative, quantitative, and chronological aspects oflesion development, which causes difficulty in making generlizations regardingthe pathology ofthe respiratory tract. although there are many excellent descriptive studies ofthe histogenesis of sendai virus-induced lesions within the lower respiratory tract, few pathology reports include a description of lesions in the nasal cavity and upper airway. nasal lesions have been well described in experimental infections of the sprague-dawley rat (14) . in this model, a rapid development of severe rhinitis with marked infiltration of the epithelium and lamina propria with lymphocytes and some neutrophils occurred at 1 day postinfection. lesions were most severe in the respiratory epithelium of the naso-and maxilloturbinates, compared to minimal lesions in the olfactory epithelium of the ethmoid region. over the next 4 days postinfection, there was a progressive accumulation of inflammatory cells, chiefly of lymphocytes, in the lamina propria. the lesions expanded to involve the middle portions of the nasal septum. respiratory epithelial cells exhibited pyknosis and karyorrhexis, particularly in the more basilar regions of the mucosa. exudate was noted in the ethmoid region, although there was little inflammation in that area. between 5 and 21 days postinfection, the severity of the nasal epithelial necrosis, inflammatory cell infiltrate, and lumenal exudate decreased. by day 28 postinfection, no discernable lesions were noted in experimentally infected rats. there are numerous systemic effects (thble 1) that toxicologists and pathologists need to consider when interpreting the impact of sendai virus infection upon study results (10, 15) . studies can be compromised through reduction in animal numbers, changes in immune function, xenobiotic metabolism, and physiology (10) . sendai virus infection has altered the course of chemically induced pulmonary carcinogenesis in strain a mice by reducing the number of resultant lung tumors (16) . in addition, the pulmonary carcinogenic responses have been altered in sendai virus infected balb/c mice following exposure to urethane (17 murine respiratory mycoplasmosis (mrm), due to mycoplasrna pulmonis is a naturally occurring, slowly progressing, chronic disease in rats and mice. numerous respiratory responses are associated with m. pulmonis infection of rodents (table 2 ) (18) . the prevalence of the pathogen and the numerous respiratory and systemic effects of infection make this disease one of the most important entities for pathologists and toxicologists during studies of the respiratory tract. respiratory mycoplasmosis is often clinically silent, although lesions of the upper respiratory tract commonly occur (19) . the principal lesions of mrm in rats include rhinitis, otitis media, laryngitis, and tracheitis (20) . gross lesions in the upper respiratory tract are generally not discernable, although rats may occasionally show mucopurulent nasal exudate and/or porphyrin-tinted oculonasal discharge. the microscopic pathology of mrm is characterized by epithelial changes including cellular hypertrophy and hyperplasia, as well as metaplastic changes. neutrophilic exudation and lymphoplasmacytic infiltrates are common throughout the upper airway. subepithelial lymphoid accumulations can occur in mrm-associated rhinitis (plates 1 and 2). ciliary loss and epithelial hyperplasias can be severe and quite extensive. the relative importance of direct mycoplasmal damage, as opposed to immune and nonimmune inflammatory reactions form. pulmonwn has yet to be completely discerned (19) . it is known that cytolysis follows attachment of m. pulmonis to upper airway epithelial cells. ciliastasis, loss of cilia, distension of intercellular spaces, cytoplasmic vacuolization, disruption of mitochondria, epithelial hyperplasia and metaplasia, and syncytial cell formation have also resulted following attachment of this organism (19) . the tympanic cavity of the ear may be completely filled with a neutrophilic exudate. frequently in mrminduced otitis media, the lining epithelium is hyperplastic and the lumenal cavity may become filled with collagenous connective tissue. thickened connective tissue lining the tympanic cavity may remain as a chronic sequellum of the infection. epithelial hyperplasias and lymphoid cell accumulations are commonly found in the certain chemical agents, including ammonia, which is commonly found in the cage environment from soiled bedding, can exacerbate mrm in rats. inhalation of the important industrial compound hexamethylphosphoramide (hmpa) can cause a synergistic enhancement of the progression and severity of mrm (21, 22) . in hmpa toxicity studies, nasal tumors, rhinitis, nasal epithelial degeneration, metaplasia, and dysplasia were noted in cornunction with an enhanced mortality from chronic pneumonia in infected rats. the increased mortality was possibly due to a chemically induced destruction of the mucociliary apparatus in the upper airway, which may have contributed to fatal lower respiratory infections. a chronic inhalation bioassay of propylene oxide revealed dose-dependent nasal epithelial proliferative lesions and two nasal adenomas in rats with intercurrent mrm (23) . the significance of the proliferative nasal lesions, which appeared to be treatment related, was difficult to interpret, since their development may have been influenced by intercurrent infectious inflammatory disease. although numerous bacteria can infect the upper airway of the rat and mouse, they are not generally prevalent in well-conducted toxicology studies begun with animals free of adventitious murine pathogens and maintained with modern methods of laboratory animal husbandry. however, the cilia-associated respiratory (car) bacillus has recently received attention. this gramnegative, filamentous, rod-shaped bacillus (24, 25) infects both rats and mice and causes lesions morphologically similar to those of m. pulmonis infections. the car bacillus was generally overlooked in the past because it often occurred as a dual infection with m. pulmonis. lesions typical of mycoplasmal chronic respiratory disease have been noted following natural and experimental infection with the car bacillus. these lesions include massive accumulations of predominantly mononuclear inflammatory cells surrounding bronchi and bronchioles (plate 3), as well as various degrees of suppurative bronchopneumonia, squamous metaplasia, and bronchiectasis. the ciliated epithelium of the airway is usually heavily colonized with filamentous bacteria, which can be readily demonstrated with warthin-starry silver impregnation techniques (plate 4). little is known regarding the prevalence of this organism as a subclinical infection. although lesions induced by the car bacillus have been described in the lungs of both rats and mice, there have been no descriptions of pathology in the upper airway where, presumably, the organism can also grow. elucidation of the significance of this pathogen for rodent toxicity awaits further studies and characterization. descriptions of naturally occurring fungal infections of the respiratory tract of rodents are extremely rare. the histological features of fungal rhinitis, which occurred in high incidence in two separate chronic carcinogenicity studies in male wistar rats (129/597 animals), have been described (26) . since these animals had titers to both sendai and sdav viruses, the authors suggested that viral-induced inflammation of the upper respiratory mucosa rendered the epithelium susceptible to the fungal infection. in addition to the 129 animals with fungal infection, 80 animals had suppurative rhinitis without evident fungal growth. an etiologic diagnosis ofaspergillusfumigatus was based upon characteristic morphology of the fruiting heads found in a small percentage of cases. aspergillus rhinitis was generally noninvasive and limited to the naso-and maxilloturbinates. although purulent inflammation was noted in the olfactory epithelium of some cases, fungal growth was rarely noted in this region. hyphal conglomerates were usually associated with foreign bodies (hair and plant material) and were surrounded by polymorphonuclear leukocytes, bacteria, debris, and nasal secretions. the underlying respiratory epithelium was usually hyperplastic, hypertrophic, or revealed squamous metaplasia. subepithelial connective tissue and submucosal glands were often infiltrated by aggregates of lymphocytes, plasma cells, and neutrophils. a relatively small number of cases had epithelial necrosis associated with fungal invasion. a review of cases of fungal rhinitis in the national tbxicology program (ntp) archives revealed the occurrence of fungal rhinitis in eight bioassays. in the affected studies that encompassed animals that were both serologically negative as well as some bearing viral titers, cases occurred in both sexes of fischer 344 rats in relatively low incidence (up to 10% of a dose group). a few scattered cases were noted in b6c3f1 mice. the histologic appearance (plates 5 and 6) was identical to that described in the literature in the wistar rat. in the affected bioassays the chemical was administered by inhalation, feed, or gavage in several different laboratories. foreign material was noted in most of the cases suggesting that particles from food or elsewhere may serve as local irritants and carriers for the fungus. one of the principal ways in which infectious diseases complicate toxicologic studies is by interference with the interpretation of the pathology data. in addition to alterations in the morphologic appearance of target organs, there can be changes in clinical signs of toxicity, food consumption, body weight gain, hematology, urinalysis, and serum chemistry parameters (27) . body weight gain depression of greater than 10% is often considered to be a sign of toxicity in treated groups of animals. the combined effects of infection and treatment influence food consumption and body weight gain to a greater extent than infection or chemical treatment alone. this synergism can confound data interpretation (27) . it has recently become apparent that infectious agents can markedly affect the metabolism of foreign compounds by impairing hepatic mixed-function monoxygenases (cytochrome p-450-dependent monoxygenase system) (28) . alteration of xenobiotic metabolism has important ramifications for toxicologists who administer compounds that are subject to bioactivation and biotransformation. the mechanistic basis for the microbialinduced inhibition of biotransformation has not yet been completely elucidated. it appears that interferon induction as a result of infection can act as a mediator in depressing p-450, and it may have a direct action on the hemoprotein itself (28) . many of the important murine viral agents including sendai virus are potent inducers of interferon (29) . infectious diseases can also modulate hepatic cytochrome p-450 by effects on the reticuloendothelial system. furthermore, microbial agents that perturb kupffer cells decrease drug biotransformation activity (28) . effects on nonhepatic xenobiotic metabolism by infectious agents have received little attention. several upper respiratory viral infections in man, including rhinoviruses and influenza viruses, are known to impair drug biotransformations by affecting the cytochrome p-450 system (28) . similar viral effects have been demonstrated in pr8 influenza-infected mice (30) . mice infected with this virus have significantly decreased pulmonary benzo[a]pyrene hydroxylase activity. nasal cytochrome p-450-dependent monooxygenases may be extremely important in the bioactivation and biotransformation of inhaled xenobiotics. in most species, the nasal olfactory concentration of these enzymes is second only to that found in the liver (31) . mouse hepatitis virus, a common mouse coronavirus infection, is reported to depress hepatic p-450 levels (32) . strains of this pathogen cause nasal cavity infections with necrotizing lesions in the olfactory mucosa (33) . it is therefore possible that extrahepatic alterations of p-450 may result and be of importance to toxicology studies. in addition to alterations of xenobiotic metabolism, infectious diseases cause other cellular effects that can modulate toxic and carcinogenic responses. the association between enhanced cell replication and cancer induction has gained the interests of many toxicologists (34) . gross and co-workers found that sites of cell replication correlated well with sites of tumor induction in the nasal cavities from rats exposed to formaldehyde in acute and chronic inhalation studies (35) . although it is uncertain how enhanced cell replication affects the carcinogenic process, such correlations suggest that a cause and effect relationship may exist. infectious diseases that cause epithelial necrosis and repair lead to significant alterations in cell turnover. microbial agents such as m. pulmonis are known to cause significant alterations in cell cycle kinetics in populations of upper airway epithelial cells (36) . this clearly has profound implications for carcinogenesis studies in which cell turnover may be an important contributing factor in the multistage process. not only would the microbial infection and degree of cell turnover be important, but the chronology of the infection with regard to the chemical administration could prove critical. a variety of important microbial pathogens including viruses, mycoplasmas, bacteria, and fungi infect the upper respiratory tract of the mouse and rat and result in significant pathologic alterations. the rodent upper respiratory tract may also be an important target organ in chronic bioassays. lbxicologists and pathologists need to understand the etiopathogenesis of common pathogen-related diseases in the murine respiratory tract, so that the effects of natural disease processes may be separated from chemically induced injury. additional studies are needed to assess the impact of several of the more prevalent adventitious pathogens on the results of rodent bioassays. the examination of the rodent nasal cavity has been overlooked in many previous experimental models of murine infectious disease. descriptive pathology studies, which carefully examine the histogenesis of upper airway injury, are warranted for many of the common pathogens that complicate toxicology studies. in addition, the effects of murine infectious diseases on xenobiotic metabolism and cytokinetics in the upper airway should be further investigated. the authors wish to thank j. griffith for slides of car bacillus. viral and mycoplasmal infections of laboratory rodents: effects on biomedical research complications of viral and mycoplasmal infections in rodents to toxicology research and testing prevalence of pathogenic murine viruses and mycoplasma that are currently a problem to research biology and diseases of rats pathogenesis of sialodacryoadenitis in gnotobiotic rats sialodacyoadenitis virus infection, upper respiratory tract, rat comparison of strain susceptibility to experimental sialodacryoadenitis in rats effects of sialodacryoadenitis virus on experimental carcinogenesis in the rat (abstract) methylene chloride: a two-year inhalation toxicity and oncogenicity study in rats and hamsters sendai virus-disease processes and research complications the pathogenesis of sendai virus infection in the mouse lung susceptibility of inbred and outbred mouse strains to sendai virus and prevalence of infection in laboratory rodents naturally occurring sendai virus infection of athymic nude mice experimental sendai virus infection in laboratory rats ii. pathology and immunohistochemistry sendai virus influence of sendai virus on carcinogenesis in strain a mice respiratory infections and the pathogenesis of lung cancer. recent results cancer res mycoplasmal infections: disease pathogenesis, implications for biomedical research, and control the pathogenic potential of mycoplasmas: mycoplasma pulmonis as a model murine respiratory mycoplasmosis, upper respiratory tract, rat enhancement of natural and experimental respiratory mycoplasmosis in rats by hexamethylphosphoramide pulmonary response to inhaled hexamethylphosphoramide in rats y carcinogenic and toxicologic effects of inhaled ethylene oxide and propylene oxide in f344 rats isolation, propagation, and characterization of a newly recognized pathogen, cilia-associated respiratory bacillus of rats, an etiological agent of chronic respiratory disease cilia-associated respiratory (car) bacillus infection of obese mice aspergillus rhinitis in wistar (crl:(wi)br) rats toxicology: complications caused by murine viruses and mycoplasmas the influence of infection on the metabolism of foreign compounds the effect of cyclophosphamide on sendai virus infection of mice effect of pr-8 viral respiratory infection on benzo possible consequences of cytochrome p-450-dependent monooxygenases in nasal tissues research complications and state of knowledge of rodent coronaviruses mouse hepatitis s in weanling mice following intranasal inoculation the value of measuring cell replication as a predictive index of tissuespecific tumorigenic potential formaldehydeinduced neoplasia, acute toxic responses and cell turnover in the nasal passages of f-344 rats (abstract) the kinetics of cell proliferation in the tracheobronchial epithelia of rats with and without chronic respiratory disease photomicrograph of the nasal cavity from a f344 rat with aspergillus rhinitis. the respiratory epithelium has early squamous metaplasia. a diffuse inflammatory infiltrate is present in the underlying lamina propria. the nasal cavity is filled with debris and polymorphonuclear leukocytes plate 6. fungal hyphae within nasal cavity debris of f344 rat with aspergillus rhinitis. gomori-methenamine silver key: cord-343390-y903mxcj authors: hoppe, ingrid bortolin affonso lux; medeiros, andréa souza ramos de; arns, clarice weis; samara, samir issa title: bovine respiratory syncytial virus seroprevalence and risk factors in non-vaccinated dairy cattle herds in brazil date: 2018-06-27 journal: bmc vet res doi: 10.1186/s12917-018-1535-8 sha: doc_id: 343390 cord_uid: y903mxcj background: the cattle industry is one of the most important brazilian agribusiness sectors and is a strong contributor to the national economy. annually about 44.6 million calves are bred, which makes the optimal management of these animals extremely important. several diseases can affect the initial stages of the bovine production chain, being the bovine respiratory syncytial virus (brsv) one of the most relevant pathogens. this study aimed to characterize the epidemiology of brsv infection in dairy cattle herds of são paulo state, brazil, using serological and risk factors analyses. for that, 1243 blood samples were collected of animals from 26 farms and a questionnaire about possible risk factors for brsv prevalence was performed. the obtained blood sera were analyzed using virus neutralization test (vnt). results: vnt results showed high brsv prevalence in dairy cattle herds, reaching 79.5% of seropositivity. the brsv seroprevalence among studied farms ranged from 40 to 100%. the analysis of risk factors indicated that the age group and the occurrence of coinfection with bovine herpesvirus 1 (bohv-1) and bovine viral diarrhea virus 1 (bvdv-1) should be associated with a higher prevalence of brsv, while natural suckling was considered a protective factor. conclusions: the study showed that adult animals over 1 year old are an important risk factor for the high seroprevalence of brsv in herds. the high brsv prevalence associated with bohv-1 and bvdv-1 suggests that biosecurity measures should be applied in order to reduce viral dissemination. additionally, the natural suckling may be an important management to protect calves from high brsv seroprevalence. bovine respiratory syncytial virus (brsv) is an economically significant pathogen in cattle production [1] , as it is one of the most important causes of lower respiratory tract infections in calves [2] . in dairy cattle, brsv infection usually occurs in young calves aged between 2 weeks and 9 months [3] . adult animals with subclinical infection are the main source of infection, since reinfections are common in the herds [1, 4, 5] . brsv, bovine herpesvirus 1 (bohv-1), bovine viral diarrhea virus (bvdv) and bovine parainfluenza type-3 (pi-3) are considered primary agents involved in the bovine respiratory complex. additionally, secondary infection by pasteurella multocida, histophilus somni and mycoplasmas contribute to the aggravation of the disease [6] . clinical signs are characterized by respiratory symptoms, initially with moderated intensity, such as nasal and ocular discharges which can be aggravated leading to pneumonia. however, mainly in calves, an acute and severe onset is also observed, due to maternal antibodies not effectively protect against brsv infection [3] . considering the high prevalence of the disease, several studies determined risk factors involved in the epidemiology of brsv. in europe, risk factors were mainly attributed to herd size, herd density, purchasing of new animals, geographic location of the farms, herd type and concomitant bvdv infection [7] [8] [9] [10] [11] . similar studies have also been performed in some latin american countries and they showed that most of the animals probably have already been exposed to the virus with consequent high brsv prevalence in cattle herds. in these countries, herd size, age group, presence of bordering farms, herd type and geographic location of the farms were the main risk factors associated with brsv infection [12] [13] [14] [15] [16] . in brazil, brsv was first diagnosed in calves in the state of rio grande do sul [17] and some studies have shown that brsv infection is widespread in southern and southeastern brazil, with high serological prevalence rates [18] [19] [20] . nevertheless, research has not been conducted in order to verify possible risk factors involved in brsv epidemiology. due to this, the current study aimed to determine antibody prevalence against brsv and investigate some risk factors associated with brsv seroprevalence in herds of an important milk producing region in são paulo state, brazil. the study was performed on 26 dairy cattle herds in 12 municipalities in the northern region of the são paulo state, southeastern brazil. this region produces about 10 million liters of milk annually [21] . the evaluated herds had between 6 and 150 animals and no animals were vaccinated against the pathogens associated with respiratory diseases. the farms were located in a region classified as aw by the climatic classification of koeppen, characterized by dry winter with average temperature higher than 18°c in the coldest month and precipitation below 60 mm in the driest month. the altitude of these areas was 440 to 617 m above sea level [22] . sampling of each farm was calculated [23] with an expected brsv prevalence of 80% [19] with acceptable error of 5% and confidence level of 95%. after setting the number of samples, bovines of all categories and age group were randomly selected. according to the management adopted in the herds, the age group was defined as ≤12 months old "calf" and > 12 months old "adult". in some farms, the number of samples collected was higher than that suggested by the mathematical calculation, once it was also used for serodiagnosis of bohv-1 and bvdv-1. on farms with a small herd size, up to 30 animals, all of them were sampled. the samples from 1243 animals were collected from april 2012 to june 2012 as shown in table 1 . blood samples were collected by jugular or coccygeal vein puncture using disposable needles and vacuum tubes. samples remained at room temperature for 1 h for coagulation and after being transported refrigerated to the laboratory, they were centrifuged at 1080×g for 10 min to obtain the sera. the sera were aliquoted in 1.5 ml identified microtubes that were stored in freezer at − 20°c until analyses. the presence of antibodies to brsv was tested by virus neutralization test (vnt). serum samples were thawed, inactivated in water bath at 56°c for 30 min, and diluted in duplicates from 1:2 to 1:1024 in 96-well microplates with 50 μl of 200 tcid 50 brsv suspended in eagle's minimum essential medium (difco e-mem®). the viral strain was previously titrated [24] . following during the sample collection, a questionnaire was administered to the owner or manager of each farm with the purpose of identifying potential risk factors related to epidemiology of brsv. thus, the questionnaire was designed according to data of risk factors described in the literature [8, 10, 15, 16] in addition to the information of the management adopted in dairy farms of são paulo state. the variables explored were: herd size (≤75, > 75 animals), herd density (≤5, > 5 animals/hectare), age group (≤12 months old "calf", > 12 months old "adult"), breed (holstein, mixed), type of reproduction (natural mating, artificial insemination), purchase of animals (last 6 months: yes, no), cleaning of facilities (often, rarely), historical of respiratory disease (last 6 months: yes, no), quarantine (yes, no), abortions (yes, no), bohv-1 infection (serodiagnosis: yes, no), bvdv-1 infection (serodiagnosis: yes, no), type of calves housing (individual, collective), type of calves feeding (natural suckling, artificial), presence of others domestic animals (yes, no), presence of wild ruminant animals (yes, no). the variables "herd size" and "herd density" were based on the average data; "age group" was defined from the management adopted on the farms; "quarantine" refers to the isolation of purchased animals before adding them to the herd. the serodiagnosis of bohv-1 and bvdv-1 was performed at the same time as brsv (unpublished data). the variables "disinfection of the umbilical cord" and "colostrum feeding" were not analyzed because these practices were applied in all dairy farms visited. additionally, the data related to climatic classification and altitude were not included in the analysis because they were similar in all 26 farms. the chi-square tests were employed to compare the brsv, bohv-1 and bvdv-1 seropositivity and also with "age group". fisher's exact test was used to compare brsv status according to the expected prevalence of 80% (≥80%, "high"; < 80%, "low") and the other variables. only the variables with p < 0,2 (two-tailed fisher) were analyzed by a logistic regression model. the analyses were performed using the epi info™ program v. 7.0. serum samples from 1243 animals belonging to 26 dairy cattle herds were taken and tested by vnt, in which 988 (79.5%) were seropositive to brsv. regarding the age group, 87% (767/ 891) of the serum samples were positives for adult animals while the prevalence rate in calves was 62.8% (352/221). antibodies to brsv were detected in all cattle herds, with prevalence rates ranging from 40 to 100%. the mean antibody titers for adult animals was 2 to 512, and for calves, 2 to 32. therefore, prevalence of brsv both in herds and animals was considered high. the chi-square test showed association of brsv seropositivity with "age group" and animals tested seropositives to bohv-1 and bvdv-1 (table 2 ). nevertheless, the fisher's exact test only detected statistical difference with the variable "type of calves feeding" (table 3 ). in this case, the relative risk (rr) value was less than one, i.e., the factor "natural suckling" was considered protective. logistic regression (values of p < 0.2, two-tailed fisher) did not show significant results, suggesting that the variables analyzed were not risk factors for the high seroprevalence of brsv in the studied population. this is the first epidemiological study to assess risk factors for brsv seroprevalence carried out in brazil. even though brsv prevalence of 79.5% in the animals sampled was similar to that estimated, the prevalence in adult animals was higher than that expected, reaching 87% of samples. in calves, the seroprevalence was lower than that found in adult animals (62.8%) and could be even lower once vnt does not allow the distinction between antibodies from colostrum and natural infection. thus, this study demonstrated that the prevalence of brsv antibodies was higher in adult animals, as previously reported in other countries [13, 16] . adult animals are associated with high seroprevalence of brsv as consequence of a repeated exposure to the virus infection throughout their life and possibility of reinfections. similarly, the highest antibody titers were associated with non-vaccinated adult cattle, probably due to the exposure to successive viral reinfections, which results in a booster effect on antibody titers [25] . other factor related to high antibody titers is recent brsv infections, which can be confirmed only by paired serology, antibody screening in calves after the period of colostral antibody detection or viral detection by direct methods. as respiratory disease was not reported in half of the herds studied, it is indicative that brsv infection can be subclinical. this is consistent with previous reports [2] . herds can remain free of clinical brsv infection for many years even in areas of high prevalence of the virus [26] . the presence of other pathogens is also associated with the prevalence of brsv [8, 11, 14, 16] . this information explains the association of brsv serological prevalence with the prevalences of bohv-1 and bvdv-1. the infection by these viral agents is also reported in brazilian herds, with high prevalences [27, 28] . bvdv infection can cause impairment of the animal's immune function and thereby decrease resistance to other infections [8] . the synergistic effects of bvdv with other respiratory pathogens have been observed [29, 30] . thus, health status of the herds may also be affected indirectly by bvdv control measures [8] . dairy cattle herds in são paulo state usually have poor biosecurity measures, such as the lack of quarantine of newly purchased animals, lack of diagnosis of respiratory diseases (particularly for brsv) and vaccination is rarely performed against these viruses. therefore, we hypothesized that risk factors for the seroprevalence of bohv-1, bvdv-1 and brsv in the studied population likely to overlap. despite the logistic regression not confirming "type of calves feeding" variable as a risk factor for high prevalence of brsv, the fisher's exact test detected "natural suckling" as a protective factor. "natural suckling" would be important as it may be able to reduce the risk of calves becoming infected by brsv. weaning can be stressful and results in impaired immune function, which may further exacerbate a brsv exposure. suckling reduces the occurrence of diarrhea, prevents the abnormal behavior of cross-suckling of other calves and improves animal health [31, 32] . prior to the current study there have been no report about "natural suckling" and its relationship with brsv seroprevalence or its role as a protective factor, therefore, based on the results presented, it has the potential to decrease seroprevalence to brsv. similarities were observed among the results found at the present study and those previously obtained by others conducted in brazil [18] [19] [20] . in latin america countries, equivalents prevalences of brsv have also been reported [12, [14] [15] [16] , as well as difficulties in detecting the risk factors involved in the dissemination of the agent, even using different forms of sampling and analyzing a considerable number of variables. thus, the dynamics of infection may differ even in a particular country or geographic area [26] . the high serological prevalence of brsv found in this study shows the importance to know more about this infection since it is not considered important in the country, mainly due to the lack of diagnosis. the awareness of the risk factors involved in the brsv dissemination can allow understanding its mechanisms, even though, as in other studies, these factors were not very clear. thereby, further studies as a complement to the current one should be performed until concrete information has been found. adult animals over 1 year old can present high seroprevalences of brsv and are an important risk factor for the virus maintenance in herds. additionally, the concomitant seroprevalence of bohv-1 and bvdv-1 leads us to suggest that biosecurity measures to reduce viral dissemination could be applied. non-stressful management, like keeping calves with their mothers promoting natural suckling, may constitute a practical management strategy to reduce the seroprevalence of brsv in brazilian dairy herds. bovine respiratory syncytial virus bovine respiratory syncytial virus (brsv): a review bovine respiratory syncytial virus) infection: its pathogenesis, diagnosis, prevention and treatment seroepizootiologic study of bovine respiratory syncytial virus in a dairy herd respiratory syncytial virus: brief review identificación de agentes virales por inmunohistoquímica en enfermedades respiratórias de bovinos en corral de engorda severe respiratory disease in dairy cows caused by infection with bovine respiratory syncytial virus bovine respiratory syncytial virus seroprevalence and risk factors in endemic dairy cattle herds risk factors for epidemic respiratory disease in norwegian cattle herds risk factors for seropositivity to bovine coronavirus and bovine respiratory syncytial virus in dairy herds seroprevalence of bovine respiratory viruses in north-western turkey bovine respiratory syncytial virus: first serological evidence in uruguay detection on antibodies and risk factors for infection with bovine respiratory syncytial virus and parainfluenza virus 3 in dual purpose farms in colima seroprevalence to bovine virus diarrhoea virus and other viruses of the bovine respiratory complex in venezuela (apure state) prevalence of and risk factors for bovine respiratory syncytial virus (brsv) infection in non-vaccinated dairy and dual-purpose cattle herds in ecuador detection of antibodies and risk factors for infections with bovine respiratory syncytial vírus and parainfluenza virus-3 in beef cattle of yucatan detection of bovine respiratory syncytial virus in calves of rio grande do sul detection of antibodies against bovine respiratory syncytial virus (brsv) in dairy cattle with different prevalences of bovine herpesvirus type 1 (bhv-1) in são paulo state vírus respiratório sincicial dos bovinos (brsv): situação no brasil serological evidence of bovine respiratory syncytial virus in brazil produção da pecuária municipal campinas: unicamp encuestas por muestreo para estudios epidemiologicos en poblaciones animales simple method of estimating 50 per cent end point dynamics of bovine respiratory syncytial virus infections: a longitudinal epidemiological study in dairy herds a longitudinal study of the dynamics of bovine corona virus and respiratory syncytial virus infections in dairy herds herpesvirus bovino tipo 1 (hvb-1): revisão e situação atual no brasil a infecção pelo vírus da diarreia viral bovina (bvdv) no brasil -histórico, situação atual e perspectivas synergistic effects of bovine respiratory syncytial virus and noncytopathic bovine viral diarrhea virus infection on selected bovine alveolar macrophage functions prevalence, outcome and health consequences associated with persistent infection with bovine viral diarrhoea virus in feedlot cattle the effects of early separation on the dairy cow and calf practical implications of increasing natural living through suckling systems in organic dairy calf rearing the authors thank coordenadoria de assistência técnica integral (cati/ saasp) for providing the dairy cattle herds for the study, dr. estevam g. lux hoppe and andressa de souza pollo for assistance with manuscript revision. this work was supported by fundação de amparo à pesquisa do estado de são paulo, fapesp (financial grant 2010/15912-7 and doctoral scholarship 2010/06950-2). the datasets analyzed within the current study are available from the corresponding author upon request.authors' contributions ibalh and asrm performed the experiment. ibalh analyzed the data and prepared the manuscript. ibalh, cwa and sis designed the study and revised the manuscript. all authors read and approved the final manuscript. the work is in accordance with ethical principles in animal experimentation, adopted by the brazilian code of experimentation (cobea) and approved by the ethics committee on animal use (ceua). protocol 027713/10. not applicable. the authors declare that they have no competing interests. key: cord-353786-284qn075 authors: chen, zhi-min; fu, jun-fen; shu, qiang; chen, ying-hu; hua, chun-zhen; li, fu-bang; lin, ru; tang, lan-fang; wang, tian-lin; wang, wei; wang, ying-shuo; xu, wei-ze; yang, zi-hao; ye, sheng; yuan, tian-ming; zhang, chen-mei; zhang, yuan-yuan title: diagnosis and treatment recommendations for pediatric respiratory infection caused by the 2019 novel coronavirus date: 2020-02-05 journal: world j pediatr doi: 10.1007/s12519-020-00345-5 sha: doc_id: 353786 cord_uid: 284qn075 since december 2019, an epidemic caused by novel coronavirus (2019-ncov) infection has occurred unexpectedly in china. as of 8 pm, 31 january 2020, more than 20 pediatric cases have been reported in china. of these cases, ten patients were identified in zhejiang province, with an age of onset ranging from 112 days to 17 years. following the latest national recommendations for diagnosis and treatment of pneumonia caused by 2019-ncov (the 4th edition) and current status of clinical practice in zhejiang province, recommendations for the diagnosis and treatment of respiratory infection caused by 2019-ncov for children were drafted by the national clinical research center for child health, the national children’s regional medical center, children’s hospital, zhejiang university school of medicine to further standardize the protocol for diagnosis and treatment of respiratory infection in children caused by 2019-ncov. since december 2019, an epidemic caused by novel coronavirus (2019-ncov) infection has occurred unexpectedly in china. at present, 2019-ncov infection has been a legal class b infectious disease of the law of the people's republic of china on the prevention and treatment of infectious diseases, and managed as a class a infectious disease for infection prevention and control practices. as of 8 pm, 31 january, more than 20 pediatric cases have been reported in china. of these cases, ten were identified in zhejiang province, with an age of onset ranging from 112 days to 17 years. following latest national recommendations for diagnosis and treatment of respiratory infections caused by 2019-ncov (the 4th edition) and current status of clinical practice in zhejiang province, recommendations for the diagnosis and treatment of respiratory infection caused by 2019-ncov for children were drafted out by the national clinical research center for child health, national children's regional medical center, children's hospital, zhejiang university school of medicine to further standardize the protocol of respiratory infection in children caused by 2019-ncov. etiology 2019-ncov is a novel human coronavirus in addition to coronavirus 229e, nl63, oc43, hku1, middle east respiratory syndrome-related coronavirus (mersr-cov) and severe acute respiratory syndrome-related coronavirus (sars-cov). 2019-ncov is enveloped single-stranded plus stranded rna virus with a diameter of 60-140 nm, spherical or elliptical in shape and pleomorphic [1] . it has been reported that the consistency of whole genome-wide nucleotide sequences of 2019-ncov with sars-like coronavirus in bats (bat-sl-covzc45) ranges from 86.9 [1] to 89% [2] . the nucleotide sequence of spines protein on the envelope of the virus is also highly consistent with that of bat-sl-covzc45 (84%) and sars-cov (78%). the physicochemical property of 2019-ncov has not been clarified clearly yet. it is thought that 2019-ncov is sensitive to ultraviolet radiation and heating. for example, according to researches on sars-cov and mers-cov, the virus can be inactivated by heating at 56 °c for 30 minutes and by using lipid solvents such as 75% ethanol, chlorinecontaining disinfectant, peroxyacetic acid and chloroform, but not by chlorhexidine, according to the researches on sars-cov and mers-cov. the main sources of the infection are patients infected by 2019-ncov with or without clinical symptoms [2] . in addition, patients in the incubation period may also have potency to transmit the virus based on the case evidence. the novel virus is spread through respiratory droplets when patients cough, talk loudly or sneeze. close contact is also a source of transmission (e.g., contact with the mouth, nose or eye conjunctiva through contaminated hand). whether transmission can occur through mother-infant vertically or breast milk has not been established yet. there are general susceptibilities in all groups, with the elderly and people with basic diseases more likely to become severe cases. children may have mild clinical symptoms after infection [3] . the incubation period of 2019-ncov infections ranges from 2 to 14 days, though most often rangs from 3 to 7 days [3] . at the onset of the disease, infected children mainly present with fever, fatigue and cough, which may be accompanied by nasal congestion, runny nose, expectoration, diarrhea, headache, etc. most of the children had low to moderate fever, even no fever. dyspnea, cyanosis and other symptoms can occur as the condition progresses usually after 1 week of the disease, accompanied by systemic toxic symptoms, such as malaise or restlessness, poor feeding, bad appetite and less activity. the condition of some children may progress rapidly and may develop into respiratory failure that cannot be corrected by conventional oxygen (nasal catheter, mask) within 1-3 days. in these severe cases, even septic shock, metabolic acidosis and irreversible bleeding and coagulation dysfunction may occur. rapid respiration rate and moist rales on auscultation usually indicate pneumonia. the criteria for rapid respiratory rate are as follows: ≥ 60 times/min for less than 2 months old; ≥ 50 times/min for 2-12 months old, ≥ 40 times/min for 1-5 years old, ≥ 30 times/min for > 5 years old (after ruling out the effects of fever and crying). with the aggravation of the disease, respiratory distress, nasal flaring, suprasternal, intercostal and subcostal retractions, grunting and cyanosis may occur. maternal hypoxemia caused by severe infection can lead to intrauterine asphyxia, premature delivery and other risks. neonates, especially preterm infants, who are more likely to present with insidious and non-specific symptoms need more close observation. according to the current conditions of the reported cases, the children mainly belong to family cluster cases. most of them have good prognosis, and in mild cases recover 1-2 weeks after disease onset. no deaths in children have been reported up to now. routine blood test white blood cell count is usually normal or reduced, with decreased lymphocyte count; progressive lymphocytopenia in severe cases. c-reactive protein (crp) normal or increased. procalcitonin (pct) normal in most cases. the level of pct > 0.5 ng/ml indicates the co-infection with bacteria. others elevation of liver enzymes, muscle enzymes and myoglobin, and increased level of d-dimer might be seen in severe cases. nucleic acid testing is the main method of laboratory diagnosis. 2019-ncov nucleic acid can be detected by rt-pcr or by viral gene sequencing of throat swabs, sputum, stool or blood samples. other methods 2019-ncov particles can be isolated from human respiratory epithelial cells through virus culture [4, 5] , but this experiment cannot be carried out in general laboratories. virus antigen or serological antibody testing kits are not available at present. chest x-ray examination in the early stage of pneumonia cases, chest images show multiple small patchy shadows and interstitial changes [4] , remarkable in the lung periphery [2] . severe cases can further develop to bilateral multiple ground-glass opacity, infiltrating shadows, and pulmonary consolidation, with infrequent pleural effusion. chest ct scan pulmonary lesions are shown more clearly by ct, including ground-glass opacity and segmental consolidation in bilateral lungs, especially in the lung periphery. in children with severe infection, multiple lobar lesions may be present in both lungs. patients should be suspected of 2019-ncov infection who if they meet any one of the criteria in the epidemiological history and any two of the criteria in clinical manifestations. city and neighboring areas, or other areas with persistent local transmission within 14 days prior to disease onset. 2. children with a history of contacting patients with fever or respiratory symptoms who have a travel or residence history in wuhan city and neighboring areas, or in other areas with persistent local transmission within 14 days prior to disease onset. 3. children with a history of contacting confirmed or suspected cases infected with 2019-ncov within 14 days prior to disease onset. 4. children who are related with a cluster outbreak: in addition to this patient, there are other patients with fever or respiratory symptoms, including suspected or confirmed cases infected with 2019-ncov. 5. newborns delivered by suspected or confirmed 2019-ncov-infected mothers. 1. fever, fatigue, dry cough; some pediatric patients may have no fever. 2. patients with the above-mentioned chest imaging findings (refer to the section of imaging features); 3. in the early phase of the disease, white blood cell counts are normal or decreased, or with decreased lymphocyte count. suspected cases who meet any one of the following criteria: 1. throat swab, sputum, stool, or blood samples tested positive for 2019-ncov nucleic acid using rt-pcr; 2. genetic sequencing of throat swab, sputum, stool, or blood samples being highly homologous with the known 2019-ncov; 3. 2019-ncov granules being isolated by culture from throat swab, sputum, stool, or blood samples. it is necessary to strengthen the awareness of the early identification of the disease. in clinic, screening was conducted mainly according to the epidemiological history and fever or respiratory symptoms, and pathogen examination should be carried out on time. furthermore, effective isolation measures and appropriate treatment need to be provided promptly. even if the common respiratory pathogen tests are positive, children who have a history of close contact with 2019-ncov-infected cases also are recommended for timely 2019-ncov pathogen testing. this type of patient includes those with asymptomatic infection, upper respiratory infection (uri) and mild pneumonia. symptoms include fever, cough, sore throat, fatigue, headache or myalgia. some patients show pneumonia signs on chest imaging. these patients do not have any of the severe or critical symptoms and complications described below. disease progresses to meet any of the following conditions [6]: 1. significantly increased respiration rate: rr ≥ 70/min (≤ 1 year), rr ≥ 50/min (> 1 year). 2. hypoxia: spo 2 ≤ 93% (< 90% in premature infants) or nasal flaring, suprasternal, intercostal and subcostal retractions, grunting and cyanosis, apnea, etc. 3. b l o o d g a s a n a ly s i s : p a o 2 < 6 0 m m h g , paco 2 > 50 mmhg. 4. consciousness disorders: restlessness, lethargy, coma, convulsion, etc. 5. poor feeding, bad appetite, and even dehydration. 6. other manifestations: coagulation disorders (prolonged prothrombin time and elevated level of d-dimer), myocardial damage (increased level of myocardial enzyme, electrocardiogram st-t changes, cardiomegaly and cardiac insufficiency in severe cases), gastrointestinal dysfunction, raised level of liver enzyme and rhabdomyolysis. disease progresses rapidly along with organ failure with any of the following conditions: 1. respiratory failure which requires mechanical ventilation patients present with acute respiratory distress syndrome (ards) and are featured by refractory hypoxemia, which cannot be alleviated by conventional oxygen therapy, such as nasal catheter or mask oxygen supplement. 2. septic shock in addition to severe pulmonary infection, 2019-ncov can cause damage and dysfunction of other organs. when dysfunction of extrapulmonary system such as circulation, blood and digestive system occurs, the possibility of sepsis and septic shock should be considered and the mortality rate increases significantly. 3. accompanied by other organ failure that needs icu monitoring and treatment. viruses such as sars virus, influenza virus, parainfluenza virus, adenovirus, respiratory syncytial virus and metapneumovirus can cause viral respiratory infections. patients commonly present with fever, cough and dyspnea, and interstitial pneumonia in some cases. most of the cases have normal or decreased white blood cell counts, while severely infected children show reduced level of lymphocyte count. similar to 2019-ncov, all of these viruses can transmit through respiratory tract or direct contact, which is characterized by clustering onset. epidemiological exposure history plays an important role in differential diagnosis, which is mainly confirmed by laboratory examinations. patients with bacterial pneumonia mostly display high fever and toxic appearance. in the early stage of the disease, cough is not obvious but moist rale can be heard. chest image may show small patchy shadows, or segmental or even lobar consolidation. blood routine examination shows increased white blood cell count with neutrophil predominating, as well as elevated crp. noninvasive pneumonia is usually accompanied by obvious cough. antibiotics are effective. blood or deep sputum culture is helpful for diagnosis of bacterial pneumonia. mycoplasmal pneumonia may occur in any season and in schools and child-care institutions with small epidemic. patients are predominantly school-age children, but the number of younger children is increasing. they usually start with high fever and cough. chest images may reveal manifestations including reticular shadows and small patchy or large consolidation. blood routine examination shows that the white blood cell count is normal or increased and crp is slightly elevated. nucleic acid detection of airway secretion and serum mycoplasma-specific igm determination are helpful for differential diagnosis. it should be noted that patients with 2019-ncov infection can have co-infection or superimposed infection with other viruses or bacteria simultaneously. the four principles of "early identification", "early isolation", "early diagnosis", and "early treatment" should be emphasized. medical isolation is supposed to be carried out once the suspected cases are identified. the confirmed cases should be admitted to the designated hospitals. avoid using broad-spectrum antibiotics and corticosteroids. antibiotics, corticosteroids, bronchoalveolar lavage, mechanical ventilation, and other more invasive intervention, such as blood purification and extracorporeal membrane oxygenation (emco) should be applied cautiously, based on cost-benefit evaluation. monitoring patient's conditions closely and adjusting the therapeutic protocols timely through multidisciplinary cooperation are of great significance. suspected case should be isolated in a single room, while confirmed cases can be arranged in the same room. during the course of treatment, pay close attention to the changes in children's conditions, regularly monitor vital signs, spo 2 , etc., and identify the severe and critical cases as early as possible. the general strategies include bed rest and supportive treatments; ensuring sufficient calorie and water intake; maintaining water electrolyte balance and homeostasis, and strengthening psychotherapy for elder children when necessary. there are no effective antiviral drugs for children at present. interferon-α2b nebulization can be applied, and the recommended usage is as follows: irrational use of antibiotics should be avoided. bacteriological monitoring should be strengthened. if there is evidence of secondary bacterial infection, appropriate antibiotics should be used timely. corticosteroids should be avoided in common type of infection. however, it can be considered in the following situations: intravenous methylprednisolone (1-2 mg/kg/day) is recommended for 3-5 days, but not for long-term use [3, 5, [9] [10] [11] . intravenous immunoglobulin can be used in severe cases when indicated, but its efficacy needs further evaluation. the recommended dose is 1.0 g/kg/day for 2 days, or 400 mg/kg/ day for 5 days [6, 9, 12, 13] . bal is not suitable for most patients, and there is an increased risk of cross-infection. the indications should be strictly controlled. bal can be considered if patients have obvious symptoms of airway obstruction, refractory massive atelectasis indicated by imaging, a significant increase in peak pressure during ventilator therapy, decrease of tidal volume, or poor oxygenation which cannot be reversed by conservative treatments. in case of circulation dysfunction, vasoactive drugs should be used to improve microcirculation on the basis of adequate liquid support [3] . patients with acute renal injury should be given continuous blood purification on time. in the meantime, paying attention to the brain function monitoring if neccessary. if intracranial hypertension and convulsion occurs in children, it is necessary to reduce the intracranial pressure and control convulsion timely [6, 12, 13] . in the case of respiratory distress that occurs despite nasal catheter or mask oxygenation, heated humidified high-flow nasal cannula (hhhfnc), non-invasive ventilation such as continuous positive airway pressure (cpap), or non-invasive high-frequency ventilation can be used. if improvement is not possible, mechanical ventilation with endotracheal intubation and a protective lung ventilation strategy should be adopted. continuous blood purification should be considered in cases of multiple organ failure (especially acute kidney injury), or capacity overload and life-threatening imbalance of water, electrolyte, and acid-base. the therapeutic model may include continuous veno-venous hemofiltration (cvvh), continuous veno-venous hemodiafiltration (cvvhdf), or heterozygosis. if combined with liver failure, plasma exchange is feasible. coronavirus investigating, and research team. a novel coronavirus from patients with pneumonia in china a familial cluster of pneumonia associated with the 2019 novel coronavirus indicating person-to-person transmission: a study of a family cluster national recommendations for diagnosis and treatment of pneumonia caused by 2019-ncov (the 4th edition). national health commission and national administrative office of chinese tradition medicine /42945 63ed3 5b432 09b31 739bd 0785e 67/files /7a930 91112 67475 a99d4 30696 2c8bf 78.pdf. access 29 clinical features of patients infected with 2019 novel coronavirus in wuhan another decade, another coronavirus diangosis and treatment guideline of community acquired pneumonia in children role of lopinavir/ritonavir in the treatment of sars: initial virological and clinical findings treatment of middle east respiratory syndrome with a combination of lopinavir-ritonavir and interferon-β1b (miracle trial): study protocol for a randomized controlled trial expert consensus on the diagnosis and treatment of viral pneumonia in children corticosteroid therapy for critically ill patients with middle east respiratory syndrome clinical management of severe acute respiratory infection when novel coronavirus (ncov) infection is suspected the editorial board, chinese journal of pediatrics. diagnosis and treatment guideline of community acquired pneumonia in children (2013 the first part) guidelines for the diagnosis and treatment of adenovirus pneumonia in children (2019 edition) pediatric branch, chinese medical association thoracic vascular anesthesiology society, extracorporeal life support committee, pediatric surgery branch, chinese medical association. consensus of extracorporeal membrane oxygenation support for acute fulminant myocarditis in children publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations ecmo therapy should be considered when mechanical ventilation, blood purification, and other means are ineffective, and cardiopulmonary failure occurs which is difficult to correct.1. ecmo indications [14] . 2. pao 2 /fio 2 < 50 mmhg or oxygen index (oi) > 40 for more than 6 h, or severe respiratory acidosis (ph < 7.15). 3. high mean airway pressure (map) during mechanical ventilation, or severe air leakage and other severe complications. 4. circulation function cannot be improved with conventional treatment, or large amounts of vasoactive drugs are required to maintain basal blood pressure, or lactic acid levels continuously rise.contraindication [14] ecmo should be contraindicated or used with caution if the duration of mechanical ventilation is more than 2 weeks, or if serious brain failure or bleeding tendency occurs. children with body temperature back to normal for at least 3 days, significant improvement in respiratory symptoms,and completion of two consecutive negative tests of respiratory pathogenic nucleic acid (sampling interval of at least 1 day) can be discharged. if necessary, home isolation for 14 days is suggested after discharge. special vehicles should be used to transfer infected patients. strict protection for staff of transportation and disinfection for the vehicle are of vital importance. enforcing close to the lines of notification of the project for transportation of pneumonia cases infected with novel coronavirus (trial version) published by the national health commission of people's republic of china is required. medical personnel are supposed to have good personal protection, hand hygiene, ward management, environmental ventilation, object surface cleaning and disinfection, medical waste management and other hospital infection control work, based on the standard prevention protocol, to minimize nosocomial infection. infectious diseases department, and isolation ward: equipped with medical overalls, caps, disposable isolation clothing, surgical masks, and goggles or face shield for daily medical activities and ward rounds; fit with goggles or face shield is stressed when collecting respiratory samples; using latex gloves in addition when contacting with blood, body fluid, secreta, or excreta; wearing surgical masks, goggles or face shield, latex gloves, medical protective clothing (disposable impermeable isolation clothing can be added), and respiratory hood when necessary, to prevent aerosol or splash during operations of endotracheal intubation, bronchoscopy, airway nursing, and sputum suction. 4. medical personnel should wear and remove personal protective equipment in strict accordance with the onoff procedure, rather than leaving ward with the contaminated equipment, so as to avoid cross-contamination in different zones. 5. patients and their accompanying family members are required to wear surgical masks. 1. channels for medical personnel and patients in the isolation ward should be separated and equipped with medical personnel channel buffer zone. 2. wearing gloves cannot replace hand hygiene. 3. perform strict visiting regulations for pediatric patients admitted in isolation, and ask visitors to have personal protection according to relevant regulations when necessary. 4. optimize medical procedures to reduce the frequency of medical personnel's contact with patients. 5. attention should be paid to the strict elimination and disinfection of the patient's secretions and excretions.author contributions all authors drafted part of the manuscript and approved the final version.funding none. ethical approval none. all authors declared no conflict of interest related to this paper. key: cord-343325-cbrly7f5 authors: denault, andré y.; delisle, stéphane; canty, david; royse, alistair; royse, colin; serra, ximena cid; gebhard, caroline e.; couture, étienne j.; girard, martin; cavayas, yiorgos alexandros; peschanski, nicolas; langevin, stéphan; ouellet, paul title: a proposed lung ultrasound and phenotypic algorithm for the care of covid-19 patients with acute respiratory failure date: 2020-05-21 journal: can j anaesth doi: 10.1007/s12630-020-01704-6 sha: doc_id: 343325 cord_uid: cbrly7f5 pulmonary complications are the most common clinical manifestations of coronavirus disease (covid-19). from recent clinical observation, two phenotypes have emerged: a low elastance or l-type and a high elastance or h-type. clinical presentation, pathophysiology, pulmonary mechanics, radiological and ultrasound findings of these two phenotypes are different. consequently, the therapeutic approach also varies between the two. we propose a management algorithm that combines the respiratory rate and oxygenation index with bedside lung ultrasound examination and monitoring that could help determine earlier the requirement for intubation and other surveillance of covid-19 patients with respiratory failure. electronic supplementary material: the online version of this article (10.1007/s12630-020-01704-6) contains supplementary material, which is available to authorized users. ou type l (low), et le phénotype à élastance élevée, ou type h (high). la présentation clinique, la physiopathologie, les mécanismes pulmonaires, ainsi que les observations radiologiques et échographiques de ces deux différents phénotypes sont différents. l'approche thérapeutique variera par conséquent selon le phénotype des patients atteints de covid-19 souffrant d'insuffisance respiratoire. keywords covid-19 á lung ultrasound á respiratory failure á respiratory rate á and oxygenation index the coronavirus disease first emerged in december 2019 in wuhan, hubei province, china. the infection, caused by severe acute respiratory syndrome coronavirus 2 quickly propagated, eventually leading to the pandemic now affecting most countries around the world. 1 the viral pneumonia caused by covid-19 was initially reported in china to be associated with a mortality rate of 1.4%. 2 higher mortality rates have been reported in other countries. 3, 4 initial reports on the pulmonary manifestation of covid-19 described abnormalities observed on computed tomography (ct) in up to 86.2% of patients who presented for acute care and in 94.6% of those with severe disease. 2, 5 these manifestations consist of groundglass opacities, local and bilateral patchy shadowing, and interstitial abnormalities. 5 abnormalities were also observed on chest radiography, such as bilateral peripheral consolidation and ground-glass opacities, 6 as well with the use of lung ultrasound. [7] [8] [9] [10] two phenotypes for one pathophysiology two phenotypes have recently been suggested to describe the clinical features of respiratory failure in covid-19-a low elastance (l-type) phenotype and a high elastance (htype) phenotype. [11] [12] [13] as the elastance is the inverse of the compliance, the l-type indicates high compliance, and the h-type the opposite. the phenotype's differing presentations are based on viral load timing, comorbidities, and likely genetic determinants of the response to hypoxemia. 13 the l-type is the most common phenotype ([ 50%). 13 those patients present with hypoxemia and hypocapnia but without dyspnea. they have normal respiratory system mechanics, hence the reported term by several emergency physicians of ''happy hypoxic covid'' because, despite significant oxygen desaturation, the patient remains alert and able to talk. while some may keep a normal respiratory drive, others may exhibit a significantly increased respiratory drive, which could induce a patient self-inflicted lung injury (p-sili). [14] [15] [16] [17] [18] hypoxemia in the l-type phenotype is thought to result from a ventilation-perfusion mismatch and loss of hypoxic vasoconstriction leading to shunt fraction up to 50%. 11 as reported in the acute respiratory system (ards), the severity seems to correlate with the difference between the end-tidal carbon dioxide and the arterial partial pressure of carbon dioxide (paco 2 ), which is an indirect measure of dead-space. 19 the ct and chest radiographs reveal well-aerated compartments, patchy disease and ground-glass opacities with some peripheral involvement. 20 the high elastance (h-type) is more consistent with a classical picture of ards from non-covid-19 settings. hypoxemia in those patients is a result of shunting through the consolidated regions of the lung, which is more important than in the l-type. as those clinical manifestations are different, the treatment will vary according to the predominant pattern. notably, a transition from l-type to h-type may evolve because of progression of covid-19 pneumonia, injurious mechanical ventilation, and/or p-sili. [14] [15] [16] [17] [18] regression from an h-type to l-type or gradual normalization has also been observed with ct during recovery (fig. 1) . 20, 21 proposed management algorithm patients with acute respiratory failure. although it will need careful prospective clinical evaluation, it can be considered an ''outside the box'' perspective in this new clinical frontier. its purpose is to help clarify the importance in ventilation strategies, which will be very different for the two ards phenotypes seen in covid-19. we propose that the algorithm be initiated when oxygen therapy is needed to correct symptomatic hypoxemia in covid-19 patients with acute respiratory failure-e.g., when hypoxemia occurs that is refractory to oxygen while in the intensive care unit, the patient experienced fever and progressive respiratory deterioration requiring intubation after 24 hr. the peripheral pulse oximetry remained at 87% despite a fraction of inspired oxygen (f i o 2 ) at 100%, positive end-expiratory pressure titration of 10 cmh 2 o, and inhaled nitric oxide of 10 ppm. blood gas revealed a ph of 7.37, partial pressure of carbon dioxide (pco 2 ) of 43.4 mmhg, and oxygen partial pressure (pao 2 ) of 63.8 mmhg. after 15 min of prone positioning, the sao 2 increased to 95% and on the following blood gas assessment, the pao 2 / f i o 2 ratio was 268 with a compliance of 53 mlácmh 2 o. b) a repeat ct scan in the same patient was done on day 4 to rule-out a pulmonary embolism. a transition to the h-type is observed. c) transition in a 49-yr-old man recovering from covid-19 respiratory failure. his phenotype changed from an h-type to an (d) l-type (courtesy of dr. emmanuel charbonney and dr. lawrence leroux) lung ultrasound in covid-19 respiratory failure therapy with a high-flow nasal cannula (hfnc) with an increased fraction of inspired oxygen (f i o 2 ). traditionally, the response to oxygen therapy is assessed with the oxygen partial pressure (pao 2 )/f i o 2 ratio 22 or alternatively, when using peripheral pulse oximetry (spo 2 ), the spo 2 /f i o 2 . 23 even though these ratios are well documented, they do not take into account the underlying respiratory workload. a pao 2 /f i o 2 b 150 mmhg is considered a severe hypoxemic state and mandates close clinical supervision and intervention. the medical community is now supporting the use of hfnc as a clinically accepted adjunct approach to treating hypoxemia in covid-19 patients. 24 the following clinical and ultrasound examination to determine the l-type (low elastance) or h-type (high elastance) pattern, oxygen therapy is initiated. there can be progression or regression from one type to the other. the respiratory rate and oxygenation (rox) index can be used, calculated, and monitored along with lung ultrasound to help in the process of deciding if and when to intubate. mechanical ventilation settings include the tidal volume (tv), the respiratory rate (rr), and the degree of positive end-expiratory pressure (peep), which will vary according to the l or h phenotype. * indicates that the decision to intubate is based on oxygenation and ventilation failure or compromised airway patency before initiating mechanical ventilation. ards = acute respiratory distress syndrome; ct = computed tomography; ecmo = extracorporeal membrane oxygenation; etco 2 = end-tidal carbon dioxide; f i o 2 = inspired oxygen; p = pressure; pao 2 = oxygen partial pressure; pap = pulmonary artery pressure; pbw = predicted body weight; pplat = plateau pressure; rox index; spo 2 = pulse oxygen saturation; v/q = ventilation perfusion. adapted in part from gattinoni et al. 11 literature from hfnc technology proposes a novel index, validated as a prognostic outcome of short-term hfnc therapy. this respiratory rate and oxygenation (rox) index is based on the traditional pao 2 /f i o 2 ratio, but is then coupled with the respiratory rate. 23, 25 for practical bedside use, pao 2 is substituted for spo 2 , so the rox index is defined as the ([spo 2 /f i o 2 ]/respiratory rate). it can be calculated using an online tool (https://qxmd.com/calculate/ calculator_724/rox-index-to-predict-risk-of-intubation). as an example, a patient with an spo 2 of 80%, an f i o 2 of 0.8, and an respiratory rate of 35/min ([80/0.8]/35) will have a rox index of 2.8. as the respiratory condition deteriorates, the rox index decreases. roca et al. have suggested various rox index cut-off values that are associated with the need for intubation. 23 values below 2.85, 3.47, and 3.85 are the cut-off values used at two, six, and twelve hour, respectively. no predictive index is perfect, and a gray zone obviously exists between 3.85 and 4.88, in which it is difficult to firmly conclude what the optimal management should be. however, monitoring the rox index over time, with emphasis from the 12th hour onwards, suggests that if the rox index is c 4.88, then the patient has a high chance of avoiding intubation. if the rox index is \ 3.85, then the risk of hfnc failure is high. in the initial study of the rox index 23 among patients in this zone, the rox index lung ultrasound in covid-19 respiratory failure could be repeated one or two hours later. if the score has increased, the patient should be considered with a higher likelihood of avoiding intubation. if it has decreased, then intubation has a higher likelihood to be necessary. if the score is unchanged, then reassessment should be performed after one or two more hours. the rox index has recently been implemented by one of the authors (n.p.) and is being considered, along with advice from other experts in mechanical ventilation, by the french ministry of health, who have arbitrarily decided to re-assess the rox index every 30 min. 26 they have chosen a change in the rox index of 0.5 to monitor the rapid onset of symptoms in the average covid-19 patient compared with the usual ards patient. therefore, a reduction of the rox index by 0.5 from baseline over 30 min is considered significant and may suggest imminent need for intubation. 23, 25, 26 indeed, such a strategy obviously requires a prospective evaluation. following the initiation of mechanical ventilation, it becomes important to discriminate between type-l vs type-h ards phenotypes for proper ventilator strategies as proposed by marini et al. 27 this is where lung ultrasound can be useful. the sensitivity and specificity of lung ultrasound findings for covid-19 remains to be determined. nevertheless, based on several case series, 7,8,28-30 editorials/ commentaries, 9,10,31-35 a recent narrative review, 36 and the authors own experience, four basic ultrasound patterns can be observed: 1) normal pattern (a lines and \ 3 b-lines); 2) mild disease: c 3 b-lines with some of them being confluent (waterfall or beam like) 34 (fig. 3a) and thickened pleura suggestive of an l-type; 3) broken pleural lines (fig. 3b) with b-lines; and 4) severe disease with subpleural consolidation (fig. 3c,3d ) and a typical ards picture. the fourth pattern is more suggestive of an h-type. in h-type, ultrasound examination will often reveal some ltype features, such as loss of aeration and overlapping areas of peripheral or subpleural consolidation (fig. 3c-f and fig. 4 ), but pathological lesions will mostly be seen in the posterior region of the lung. 7,30 management and triage would be different in patients with more extensive disease. nevertheless, it is possible that the extent of any abnormal lung ultrasound pattern might be more important than the actual pattern or type of pathology itself. a large number of abnormal regions would correlate with the severity of lung inflammation. pleural effusions have been uncommonly observed 7,32 and were present in only 4.7% of patients in a series by lomoro et al. 28 and in 10% of patients in a separate report by xing et al. 30 the reduction or absence of a vascular doppler signal in the areas of peripheral or subpleural consolidation (fig. 1d,1f) is common in covid-19 7 and could represent peripheral segmental lung infarction through a microangiopathy, as described recently by autopsies from the united states. 37 coronavirus disease could be associated with an increased incidence of thrombotic complications such as pulmonary embolism. [38] [39] [40] [41] standardization of lung ultrasound examination using a 14-region approach in covid-19 has been recently proposed by soldati et al. 33 this would allow both diagnostic and daily monitoring of the pulmonary lesions. 42 a simpler approach using six zones has also been recently proposed. 43 the relevance of serial lung ultrasound has previously been reported in a small series of patients with ards 42 unrelated to the current covid-19 pandemic but the various scoring systems would need comparing and validating in covid-19 patients. in that study, the non-resolution of pulmonary lesions was associated with worse outcome. 42 as with any patients in the intensive care unit, covid-19 patients can also develop other pulmonary complications. these include superimposed bacterial pneumonia, cardiogenic pulmonary edema related to myocardial dysfunction, right ventricular dysfunction and pulmonary hypertension due to pulmonary embolism, pleural effusions, and pneumothoraces. 44, 45 as suggested by volpicelli et al., the bilateral patchy distribution of multiform clusters, alternating with ''spared areas'' is typical of the disease. any other ultrasound signs should be considered at intermediate probability and should lead to further testing. 34 bedside ultrasound could be useful in detecting associated pulmonary complications or non-pulmonary complications such as cardiac dysfunction that can occur in such patients. 46 combining heart, lung, deep veins examination 41 and whole-body ultrasound 47, 48 can identify the mechanism, 49 risk factors, 50 and life-threatening conditions in patients with respiratory symptoms (fig. 5) . 51 lung ultrasound can also impact clinical decision-making in patients with acute respiratory failure 52, 53 and provide comprehensive monitoring of regional lung aeration changes that could be used to predict response to prone positioning with improved right (figs 6 and 7) [54] [55] [56] or higher positive end-expiratory pressure strategy. 57 newer modalities such as strain and speckle tracking could eventually be used to detect early abnormal peripheral pulmonary stress. 58, 59 the authors strongly advocate ultrasound as the key to rapid repeated progress imaging (rather than ct, which is more expensive and has logistical difficulties) and hence a decision to intubate would clearly be influenced by a rapidly deteriorating set of ultrasound images. repeated ultrasound imaging over time can allow for accurate determination of the progression of lung pathology and can strongly influence a decision to institute early mechanical ventilation. such repeated ultrasound imaging would be logistically convenient at the bedside without transporting the patient to the radiology department and would not involve any radiation dosing. nevertheless, although lung ultrasound outperforms chest radiograph, 60, 61 it cannot be as comprehensive as ct since any lung pathology not involving the surface of the lung (and therefore covered by some aerated lung) cannot be detected by ultrasound. lung ultrasound can be performed with a wide range of ultrasound machines, including handheld devices. 62 the advantage of the handheld devices is that they can be fully enclosed in a plastic sheath, thereby reducing the risk of equipment contamination and spread of infection between patients. the performance and interpretation of lung ultrasound is much simpler than other imaging modalities such as transthoracic echocardiography as the images are easy to acquire and there are a limited number of patterns to interpret. 63, 64 education can be scaled to meet the covid-19 crisis through online courses (e.g., https:// www.iteachu.com/courses/covid-19-lung-ultrasound/) simulation, and peer-to-peer mentoring. other important elements in deciding whether to intubate is the work of breathing. 27 this can also be monitored clinically by observing the use of accessory muscles or by measuring or estimating changes in pleural pressure. 13 once mechanical ventilation is initiated, tidal volume, respiratory rate, positive-pressure ventilation, prone positioning and veno-venous extracorporeal membrane oxygenation, sedation, and neuromuscular blockade will be adjusted differently depending on the phenotype and in accordance to ards management guidelines. [65] [66] [67] [68] finally, there are still several unanswered questions and uncertainties regarding covid-19 respiratory infections and the role of bedside ultrasound. we are still on an our understanding of covid-19 and its effect on the respiratory system is rapidly evolving. biochemical pathways involving cardiovascular issues have recently been described by liu et al. 46 this novel knowledge might 2) what is the sensitivity and specificity of the rox index in predicting requirement for intubation and how does it correlate with ultrasound findings? 3) what is the mechanism of hypoxia in the early phase or l-type phenotype? contribute to understanding more about the two suggested phenotypes described by gattinoni et al. 11, 13 it might also be used to confirm or challenge the aforementioned phenotypes categorization. 69, 70 bedside lung ultrasound can help identify severity of lung involvement and response to therapy, as part of evaluation for ventilation as well as during the recovery and weaning process. with the implementation of this emerging information, clinical management may be somewhat distinct from that of other evidence-based interventions. as a result, clinical trials will be required to determine the best ventilation strategies for the different lung-involvement phenotypes seen in hypoxic covid-19 patients. editorial responsibility this submission was handled by dr. hilary p. grocott, editor-in-chief, canadian journal of anesthesia. a novel coronavirus from patients with pneumonia in china clinical characteristics of coronavirus disease 2019 in china geographic differences in covid-19 cases, deaths, and incidence -united states case-fatality rate and characteristics of patients dying in relation to covid-19 in italy chest computed tomography images of early coronavirus disease (covid-19) frequency and distribution of chest radiographic findings in covid-19 positive patients a preliminary study on the ultrasonic manifestations of peripulmonary lesions of non-critical novel coronavirus pneumonia (covid-19) chinese critical care ultrasound study group. findings of lung ultrasonography of novel corona virus pneumonia during the 2019-2020 epidemic can lung us help critical care clinicians in the early diagnosis of novel coronavirus (covid-19) pneumonia? is there a role for lung ultrasound during the covid-19 pandemic? covid-19 does not lead to a ''typical'' acute respiratory distress syndrome covid-19 pneumonia: ards or not? covid-19 pneumonia: different respiratory treatment for different phenotypes? positive pressure respiration and its application to the treatment of acute pulmonary edema acute respiratory failure following pharmacologically induced hyperventilation: an experimental animal study mechanical ventilation to minimize progression of lung injury in acute respiratory failure patient selfinflicted lung injury: implications for acute hypoxemic respiratory failure and ards patients on non-invasive support patient selfinflicted lung injury and positive end-expiratory pressure for safe spontaneous breathing pulmonary dead-space fraction as a risk factor for death in the acute respiratory distress syndrome temporal changes of ct findings in 90 patients with covid-19 pneumonia: a longitudinal study time course of lung changes on chest ct during recovery from 2019 novel coronavirus (covid-19) pneumonia oxygen derived variables in acute respiratory failure predicting success of highflow nasal cannula in pneumonia patients with hypoxemic a. y. denault et al. respiratory failure: the utility of the rox index clinical consensus recommendations regarding non-invasive respiratory support in the adult patient with acute respiratory failure secondary to sars-cov-2 infection an index combining respiratory rate and oxygenation to predict outcome of nasal high-flow therapy doctrine d'usage des dispositifs de ventilation et des respirateurs pour les patients covid-19 management of covid-19 respiratory distress covid-19 pneumonia manifestations at the admission on chest ultrasound, radiographs, and ct: single-center study and comprehensive radiologic literature review a clinical study of noninvasive assessment of lung lesions in patients with coronavirus disease-19 (covid-19) by bedside ultrasound lung ultrasound findings in patients with covid-19 pneumonia point of care and intensive care lung ultrasound: a reference guide for practitioners during covid-19 thoracic ultrasound and sars-covid-19: a pictorial essay proposal for international standardization of the use of lung ultrasound for patients with covid-19: a simple, quantitative, reproducible method sonographic signs and patterns of covid-19 pneumonia our italian experience using lung ultrasound for identification, grading and serial follow-up of severity of lung involvement for management of patients with covid-19 point-of-care lung ultrasound in patients with covid-19 -a narrative review vander heide rs. pulmonary and cardiac pathology in covid-19: the first autopsy series from new orleans. medrxiv preprint acute pulmonary embolism in covid-19 patients on ct angiography and relationship to d-dimer levels incidence of thrombotic complications in critically ill icu patients with covid-19 covid-19 complicated by acute pulmonary embolism and right-sided heart failure thrombotic events in sars-cov-2 patients: an urgent call for ultrasound screening lung ultrasound for daily monitoring of ards patients on extracorporeal membrane oxygenation: preliminary experience impact of point-of-care ultrasound on the hospital length of stay for internal medicine inpatients with cardiopulmonary diagnosis at admission: study protocol of a randomized controlled trial-the imfcu-1 (internal medicine focused clinical ultrasound) study a bedside ultrasound sign ruling out pneumothorax in the critically ill. lung sliding pleural ultrasonography versus chest radiography for the diagnosis of pneumothorax: review of the literature and meta-analysis the science underlying covid-19: implications for the cardiovascular system whole-body ultrasound in the intensive care unit: a new role for an aged technique whole body ultrasound in the operating room and the intensive care unit critical care ultrasonography differentiates ards, pulmonary edema, and other causes in the early course of acute hypoxemic respiratory failure accuracy of ultrasonography performed by critical care physicians for the diagnosis of dvt focused sonography of the heart, lungs, and deep veins identifies missed lifethreatening conditions in admitted patients with acute respiratory symptoms usefulness of cardiothoracic chest ultrasound in the management of acute respiratory failure in critical care practice impact of lung ultrasound on clinical decision making in critically ill patients prone positioning unloads the right ventricle in severe ards can lung ultrasonography predict prone positioning response in acute respiratory distress syndrome patients? lung ultrasonography for assessment of oxygenation response to prone position ventilation in ards bedside ultrasound assessment of positive end-expiratory lung ultrasound in covid-19 respiratory failure pressure-induced lung recruitment ultrasound strain measurements for evaluating local pulmonary ventilation speckle tracking quantification of lung sliding for the diagnosis of pneumothorax: a multicentric observational study comparative diagnostic performances of auscultation, chest radiography, and lung ultrasonography in acute respiratory distress syndrome comparative performance of pulmonary ultrasound, chest radiograph, and ct among patients with acute respiratory failure diagnostic performance of multi-organ ultrasound with pocket-sized device in the management of acute dyspnea lung ultrasound training: curriculum implementation and learning trajectory among respiratory therapists a pilot assessment of 3 point-of-care strategies for diagnosis of perioperative lung pathology acute respiratory distress syndrome network ventilation with lower tidal volumes as compared with traditional tidal volumes for acute lung injury and the acute respiratory distress syndrome low-tidal-volume ventilation in the acute respiratory distress syndrome positive end-expiratory pressure setting in adults with acute lung injury and acute respiratory distress syndrome: a randomized controlled trial driving pressure and survival in the acute respiratory distress syndrome respiratory pathophysiology of mechanically ventilated patients with covid-19: a cohort study covid-19 in critically ill patients in the seattle region -case series quantifying systemic congestion with point-of-care ultrasound: development of the venous excess ultrasound grading system the role of point-of-care ultrasound monitoring in cardiac surgical patients with acute kidney injury brain ultrasonography: methodology, basic and advanced principles and clinical applications. a narrative review point-of-care handheld ophthalmic ultrasound in the diagnosis and evaluation of raised intracranial pressure and terson syndrome: a description of two cases key: cord-339869-euikj8fv authors: cebey-lópez, miriam; herberg, jethro; pardo-seco, jacobo; gómez-carballa, alberto; martinón-torres, nazareth; salas, antonio; martinón-sánchez, josé maría; justicia, antonio; rivero-calle, irene; sumner, edward; fink, colin; martinón-torres, federico title: does viral co-infection influence the severity of acute respiratory infection in children? date: 2016-04-20 journal: plos one doi: 10.1371/journal.pone.0152481 sha: doc_id: 339869 cord_uid: euikj8fv background: multiple viruses are often detected in children with respiratory infection but the significance of co-infection in pathogenesis, severity and outcome is unclear. objectives: to correlate the presence of viral co-infection with clinical phenotype in children admitted with acute respiratory infections (ari). methods: we collected detailed clinical information on severity for children admitted with ari as part of a spanish prospective multicenter study (gendres network) between 2011–2013. a nested polymerase chain reaction (pcr) approach was used to detect respiratory viruses in respiratory secretions. findings were compared to an independent cohort collected in the uk. results: 204 children were recruited in the main cohort and 97 in the replication cohort. the number of detected viruses did not correlate with any markers of severity. however, bacterial superinfection was associated with increased severity (or: 4.356; p-value = 0.005), picu admission (or: 3.342; p-value = 0.006), higher clinical score (1.988; p-value = 0.002) respiratory support requirement (or: 7.484; p-value < 0.001) and longer hospital length of stay (or: 1.468; p-value < 0.001). in addition, pneumococcal vaccination was found to be a protective factor in terms of degree of respiratory distress (or: 2.917; p-value = 0.035), picu admission (or: 0.301; p-value = 0.011), lower clinical score (-1.499; p-value = 0.021) respiratory support requirement (or: 0.324; p-value = 0.016) and oxygen necessity (or: 0.328; p-value = 0.001). all these findings were replicated in the uk cohort. conclusion: the presence of more than one virus in hospitalized children with ari is very frequent but it does not seem to have a major clinical impact in terms of severity. however bacterial superinfection increases the severity of the disease course. on the contrary, pneumococcal vaccination plays a protective role. to correlate the presence of viral co-infection with clinical phenotype in children admitted with acute respiratory infections (ari). we collected detailed clinical information on severity for children admitted with ari as part of a spanish prospective multicenter study (gendres network) between 2011-2013. a nested polymerase chain reaction (pcr) approach was used to detect respiratory viruses in respiratory secretions. findings were compared to an independent cohort collected in the uk. results 204 children were recruited in the main cohort and 97 in the replication cohort. the number of detected viruses did not correlate with any markers of severity. however, bacterial superinfection was associated with increased severity (or: 4.356; p-value = 0.005), picu admission (or: 3.342; p-value = 0.006), higher clinical score (1.988; p-value = 0.002) respiratory introduction molecular techniques including polymerase chain reaction (pcr) have increased the sensitivity of detection for common and emerging respiratory viruses, and often reveal the presence of more than one pathogen in respiratory patients. [1] , [2] the importance of viral co-infections in the pathogenesis, severity or course of respiratory infections is not well established. the high sensitivity of molecular techniques raises questions about the clinical relevance of positive test results. the presence of a virus does not necessarily indicate causation of clinical symptoms or disease [3] . on the contrary, bacterial co-infection is usually associated with a more severe diseases course and worse prognosis, despite the precise interaction between bacteria and viruses is not always clear. our study aims to analyze the relationship between viral or bacterial co-infection detected by molecular methods, and the clinical phenotype of children admitted to hospital with lower tract acute respiratory infections (lt-ari). two independent observational prospective patient groups were enrolled in spain (main group) and in the united kingdom (replication group). spanish children were recruited between january 2011 and january 2013 through a national hospital based research network: gendres (genetic, vitamin d and respiratory infections research network-www.gendres. org), which includes 13 spanish tertiary hospitals. uk children were recruited between october 2009 and may 2010 at st mary's hospital (uk). in both cohorts, eligible study participants were children under 14 years of age with a lower respiratory tract illness of sufficient severity to warrant admission to hospital, and a virus identified on nasopharyngeal swab or aspirate sample. all types of lt-ari were included, from bronchiolitis to pneumonia, with or without wheezing, fever, rhinorrhea and respiratory distress. a nasopharyngeal sample (aspirate or swab) was obtained during admission for viral pathogen detection. besides the diagnostic procedures performed at origin in the referring hospital, viral detection was performed in all recruited subjects using a panel of 19 viruses, by nested pcr: respiratory syncytial virus; influenza virus (a, b); parainfluenza virus types (1-4); adenovirus (a-f); rhinovirus; metapneumovirus; coronavirus (nl63, 229e, oc43) and bocavirus (methods previous described in cebey-lópez et al. [4] ). no difference was found on the molecular diagnosis yield in our series when using aspirate or swab as sample source. micropathology ltd. provided support in the form of salaries for authors es and cf, but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. the specific roles of these authors are articulated in the 'author contributions' section. competing interests: the authors have the following interests. edward sumner and colin fink are employed by micropathology ltd. there are no patents, products in development or marketed products to declare. this does not alter the authors' adherence to all the plos one policies on sharing data and materials, as detailed online in the guide for authors. all investigators were trained in the study protocol for patient recruitment, sample processing and sample storage. the study was performed according to good clinical practice. written informed consent was obtained from a parent or legal guardian for each subject before study inclusion. detailed clinical data on each patient were collected using a secured web-based platform. this included risk factors for lt-ari (prematurity, immunization status, obesity, diabetes, asthma and previous admissions to hospital), current medications, and family history of asthma or other respiratory diseases. the severity of each respiratory case was ranked as follows: 1) physician criteria (mild, moderate or severe); 2) wood-downes scale (0 to 10 points; mild < 3, moderate 4-7, severe > 8); and 3) a newly developed scale -named genvip score-(0 to 20 points) that assesses food tolerance, degree of medical intervention needed, respiratory distress, respiratory frequency, apnea, malaise and fever (see s1 table for further details on this scale). supplemental oxygen and / or mechanical ventilation requirement during admission were also recorded. bacterial superinfection diagnosis was established according to the referring physician criteria, based on clinical data, inflammatory markers, and radiological findings, and/or appropriate cultures in sterile sites (e.g blood or cerebrospinal fluid). for the study purposes, cases classified as bacterial co-infections included both cases with and without microbiological confirmation as in our series, cultures were not carried out systematically, but only when a bacterial superinfection was suspected by the referring physician. in this analysis, we compared clinical data to the results of pathogen identification in respiratory samples. we performed all the analysis using the r software, version 3.0.2 (www.rproject.org). general data were presented as means with 95% confidence intervals (ci). different statistical models were used to assess the bivariate association between the variables depending on the dependent variable. the relationship between demographic and clinical variables with mono-infection and co-infection was analysed using simple logistic regression. a binary logistic model was used for the binary variables (co-infection status, oxygen requirements, respiratory support needed and picu admission), linear model for continuous variables (wood-downes score and genvip score), negative binomial regression model for counted data (hospital stay length) and logistic multinomial model for the multinomial variable (respiratory distress status). multiple regression models were considered using the significant risk factors obtained in the bivariate analysis and sex and age variables. in order to reduce the likelihood of false significant results due to too many statistical comparisons, the bonferroni multiple test correction were considered. a χ2 test was performed to evaluate the correlation between bacterial superinfection and pneumococcal vaccine. level of statistical significance was set at 0.05. the gendres cohort included nasopharyngeal samples from 204 patients with a median age of 16.7 (95% ci: 12.7-20.6) months and a male-to-female sex ratio of 1.7. one patient was excluded due to incomplete clinical data. in the uk a cohort of 97 patients' data was analyzed, with a median of age of 36.6 (95% ci: 27.6-45.6) months and a male-to-female ratio of 0.94 (s1 fig) . comparison between both cohorts is shown in table 1 . the pcr results were preciously described in cebey-lópez et al. [4] . clinical data, family or past medical history, need of picu admission or hospital length of stay, and oxygen or respiratory support need, were equivalent in the gendres and the uk cohort (table 2 ). in the gendres cohort the presence of rhinovirus as co-pathogen was associated with a significantly increased wood-downes score by 1.289 points (95% ci: 0.387, 2.192); pvalue = 0.006. rsv infection was associated with increased oxygen requirements [or (95% ci): 3.154 (1.302, 7.966); p-value = 0.012] ( table 3) . these isolated findings were not replicated in the uk cohort (table 3) . these findings were not replicated in the uk cohort (fig 1; s1 -s7 tables). our study revealed that even though multiple viral detection is frequent in hospitalized children with lt-ari, this association is not related to either disease severity or to any other clinical features studied. picu admission, disease severity according to different scales, need for respiratory support, and length of hospital stay followed a similar pattern in viral mono-versus co-infected children. contrariwise, bacterial superinfection increased the severity of the disease course, while pneumococcal vaccination played a protective role. the detection of multiple coincident viruses in clinical settings is becoming more common since the introduction of molecular based multiplex tests, but the clinical significance of these findings remains unclear and seems to have no impact in disease severity [5] . both an increase in disease severity in relation to dual infections [6] [7] [8] [9] and the absence of this association [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] have been reported. richard et al. [8] found that co-infected children were almost three times more likely to be admitted to the picu than those with single viral infections. compared to our study richard et al. developed a retrospective and monocentric study in which they only considered dual infections, infants and bronchiolitis. there is contradictory evidence linking disease severity with specific respiratory viruses. a shorter hospital stay has been reported in children with rhinovirus bronchiolitis than with rsv [20] . rhinovirus and rsv co-infection is reported to increase the risk of severe disease [8] or the bronchiolitis relapse [21, 22] . other studies did not find significant differences in severity between co-infection and single infection [12, 18, 23, 24] . in our study we did not find increased severity of illness in children with rsv-rhinovirus dual infection. in our series, only rsv as mono-infection increased oxygen requirements, and rhinovirus as a co-infecting pathogen increased the wood-downes score in the spanish cohort, but these isolated findings arising from the multivariate analysis could not be replicated in the uk cohort. several studies have reported increased severity with bocavirus (hbov) co-infections [13, [25] [26] [27] ; this was not the case in our series (also in agreement with pen et al. [15] ). hbov was commonly detected in our patients, with no impact in the severity of the illness. as hbov was detected in alongside other respiratory viruses with an established pathogenic potential, it is possible that hbov detection reflects asymptomatic persistence or prolonged viral shedding [28] . bacterial superinfection was the only factor consistently linked to greater severity. studies of the pandemic influenza indicate that respiratory viruses predispose to bacterial complication and interaction between viruses and bacteria in respiratory infections has been extensively reported in the literature [29] , but the underlying mechanisms between viral and bacterial synergism are complex and remain unclear [30] . common respiratory viral infections, such as influenza or respiratory syncytial virus have been linked to seasonal increases in streptococcus pneumoniae disease [31] . the relationship between bacterial and viral infection is clouded by the low sensitivity of bacterial detection in sterile-site samples by traditional culture methods, and the reliance on non-specific clinical data for the for diagnosis of bacterial co-infection, including inflammatory markers, radiological findings and / or appropriate cultures, resulting in 30% of the cases in the gendres cohort and 55% in the uk cohort. bacterial superinfection increased most measures of severity in both cohorts (picu admission, respiratory support requirement, genvip score, hospital stay length and respiratory distress). interestingly, pneumococcal vaccination was revealed as an independent protective factor of disease severity in our patients. pneumococcal vaccine reduced the severity of viral lt-aris through a reduction in oxygen requirement, invasive and non-invasive ventilation, admission to picu, respiratory distress, and genvip score. a reduced incidence of viral alveolar pneumonia has been previously reported after pneumococcal vaccination [31, 32] , although there was no demonstrable reduction in the number of confirmed pneumococcal infections. this is likely to reflect the limited sensitivity of culture-proven pneumococcal disease in pneumonia. the protective effect of pneumococcal vaccines found in our study might reinforce the importance of the paradigm of viral-penumococci interaction at nasopharynx level in the pathogenesis and clinical course of the disease [33] current pneumococcal conjugate vaccines significantly decrease nasopharyngeal carriage of pneumococci [34] and thus reduces the possibility of this viral-pneumococci direct interaction. one of the limitations of the present study is that our samples were not tested for viral load by quantitative pcr and the viral load of certain viruses-like rsv-has been associated with the co-infection status and the severity [3] . also, the study did not consider milder or asymptomatic children. in addition, bacterial superinfection rate in our series might be overestimated as diagnosis was accepted as true even without microbiological confirmation, just based on referring physicians' criteria. several studies had shown that viruses can be found in children with no respiratory infections [6, 35] , and further research is needed to understand the natural history of respiratory viral carriage and infection. however, our findings were consistent in both independent cohorts very different between them, so this makes the outcomes more robust. in summary, the severity and course of an acute respiratory episode requiring hospitalization in children did not correlate with the presence of one or more viruses. in contrast, bacterial co-infection was associated with more severe disease, whereas pneumococcal vaccination decreased severity. future studies are needed to investigate whether particular viruses, or combinations of viruses, influence the risk of bacterial co-infection. supporting information s1 fig. flow chart of study population of the main cohort (gendres cohort) and replication cohort (uk-cohort). (jpg) s1 table. genvip score. (docx) s2 table. demographic characteristics, family and patient medical history, clinical course and principal virus in children with ari and disease severity, considering respiratory support and oxygen requirement the characteristics that described the severity of the illness of the main cohort. (docx) s3 table. variables analyzed in children with ari and disease severity, considering the clinical scales the characteristics that described the severity of the illness of the main cohort. (docx) s4 table. demographic characteristics, family and patient medical history, clinical course and main virus in children with ari and disease severity in gendres cohort. (docx) s5 table. children's with ari characteristics and moderate and severe respiratory distress. (docx) s6 table. comparison of virus and disease severity of the replication cohort (uk cohort) considering the virus as single pathogen or as co-infection in the sample. (docx) s7 table. demographic characteristics, clinical course and principal virus in children with ari and disease severity, considering respiratory support and oxygen requirement the characteristics that described the severity of the illness of the uk-cohort are presented. (docx) s8 table. variables analyzed in the uk-cohort children and disease severity according to hospital stay length and picu admission are shown. (docx) hospital materno infantil virgen del camino francisco giménez sánchez, miguel sánchez forte eider oñate vergara (hospital de donostia we thank stuart gormley and the clinical team at st mary's hospital for assistance in recruiting the uk cohort. we also extend our gratitude to the nursery and laboratory service from the hospital clín newly discovered respiratory viruses: significance and implications. current opinion in pharmacology comparison of multiplex pcr assays and conventional techniques for the diagnostic of respiratory virus infections in children admitted to hospital with an acute respiratory illness clinical utility of pcr for common viruses in acute respiratory illness viral co-infections in pediatric patients hospitalized with lower tract acute respiratory infections clinical disease severity of respiratory viral co-infection versus single viral infection: a systematic review and meta-analysis respiratory pathogens in children with and without respiratory symptoms frequent detection of viral coinfection in children hospitalized with acute respiratory tract infection using a real-time polymerase chain reaction the impact of dual viral infection in infants admitted to a pediatric intensive care unit associated with severe bronchiolitis dual infection of infants by human metapneumovirus and human respiratory syncytial virus is strongly associated with severe bronchiolitis viral etiology of acute febrile respiratory illnesses in hospitalized children younger than 24 months diagnostic value of respiratory virus detection in symptomatic children using real-time pcr infection with multiple viruses is not associated with increased disease severity in children with bronchiolitis. pediatr pulmonol the association of newly identified respiratory viruses with lower respiratory tract infections in korean children comparison of human metapneumovirus, respiratory syncytial virus and influenza a virus lower respiratory tract infections in hospitalized young children multipathogen infections in hospitalized children with acute respiratory infections polymicrobial acute respiratory infections in a hospital-based pediatric population prevalence and clinical characteristics of human metapneumovirus infections in hospitalized infants in spain. pediatric pulmonology single versus dual respiratory virus infections in hospitalized infants: impact on clinical course of disease and interferon-gamma response respiratory syncytial virus, its coinfection and paediatric lower respiratory infections. the european respiratory journal: official journal of the european society for clinical respiratory physiology hospital length-of-stay is associated with rhinovirus etiology of bronchiolitis multicenter study of viral etiology and relapse in hospitalized children with bronchiolitis association of rhinovirus infection with increased disease severity in acute bronchiolitis. american journal of respiratory and critical care medicine prospective multicenter study of the viral etiology of bronchiolitis in the emergency department prospective multicenter study of viral etiology and hospital length of stay in children with severe bronchiolitis. archives of pediatrics & adolescent medicine cloning of a human parvovirus by molecular screening of respiratory tract samples the incidence of human bocavirus infection among children admitted to hospital in singapore bocavirus infection in hospitalized children human bocavirus: passenger or pathogen in acute respiratory tract infections? clinical microbiology reviews viral and bacterial interactions in the upper respiratory tract influenza and rsv make a modest contribution to invasive pneumococcal disease incidence in the uk. the journal of infection influence of pneumococcal vaccines and respiratory syncytial virus on alveolar pneumonia, israel. emerging infectious diseases vaccine trialist g. a role for streptococcus pneumoniae in virus-associated pneumonia respiratory syncytial virus increases the virulence of streptococcus pneumoniae by binding to penicillin binding protein 1a. a new paradigm in respiratory infection. american journal of respiratory and critical care medicine evolving role of 13-valent pneumococcal conjugate vaccine in clinical practice detecting respiratory viruses in asymptomatic children we greatly thank all the patients that kindly contributed to this study. the authors thank the study investigators of gendres network (www. key: cord-326122-5m1727m1 authors: wishaupt, jérôme o.; versteegh, florens g.a.; hartwig, nico g. title: pcr testing for paediatric acute respiratory tract infections date: 2014-08-04 journal: paediatr respir rev doi: 10.1016/j.prrv.2014.07.002 sha: doc_id: 326122 cord_uid: 5m1727m1 acute respiratory tract infection (ari) is a frequently occurring disease in children. it is a clinical diagnosis for which no internationally accepted diagnostic test is available. the majority of ari is viral in origin, though diagnostic tests for viruses were rarely performed in the past. in the past 2 decades, new molecular techniques have been introduced in many hospitals. they are capable of generating a high yield of viral and bacterial diagnoses, but their impact upon clinical practices is still questionable. in this paper, we discuss the difficulties of diagnosing ari in children, the indications for conventional and new diagnostics and their implications. in developed countries, acute respiratory tract infection (ari) is the most common cause for hospitalization of young children [1] . a rapid and accurate diagnosis is required to start adequate therapy, minimize hospital admissions and length of hospital stay and reduce unnecessary antibiotic use. clinical decision making in ari is based on interpretation of clinical signs and symptoms, often supported with results of laboratory parameters such as c-reactive protein and white blood count combined with conventional viral and bacterial tests. in addition, new molecular tests have been introduced in many hospitals. however the role (and impact) of these molecular tests in clinical decision making is not known. in this paper, we discuss the role of molecular diagnostics in relation to patient care in paediatric ari. the anatomic approach to ari creates 2 categories: upper respiratory tract infection (urti) and lower respiratory tract infection (lrti). urti is confined to the ear, nose and throat region and is considered to be a rather harmless disease for which medical attention is generally restricted to family care. lrti on the contrary, which involves the bronchi, bronchiole and the lung tissue, is considered a more severe disease that should be seen by a paediatrician. pneumonia is thought to be more likely caused by bacteria for which prompt initiation of antibiotics is required. however, in paediatric patients this clear distinction between urti and lrti is difficult to make and appears not useful for clinical decision-making. in infants, classical lrti-symptoms like tachypnea or hypoxia are also common in paediatric urti due to nasal obstruction and mucus plugging of the anatomically small upper airways. general symptoms such as poor feeding, lowered alertness and low transcutaneous oxygen seem to influence the clinical course more strongly than the designated anatomical site of infection [2] . based on pathophysiologic mechanisms, lrti can be subdivided in pneumonia and bronchiolitis. pneumonia reflects inflammation of lung tissue (bronchi, bronchioles and alveolar tissue) in response to infection by bacterial, viral or other pathogens, while bronchiolitis primarily affects the small airways (bronchioles) and is usually of viral origin only. the clinical presentation of pneumonia overlaps substantially with that of bronchiolitis and vice versa. pneumonia and bronchiolitis are clinical diagnoses without an internationally standardized test or definition [3, 4] . though chest radiographs are often used in clinical settings, they cannot distinguish between these conditions [5] . there have been attempts to distinguish viral from bacterial ari because of its therapeutic consequences. most paediatric ari are of viral origin and the risk of concurrent (or subsequent) bacterial infection has been reported to be low in children over three months of age [6] . it is assumed that antibiotics are prescribed too often for these children [7] . this is in part explained by a high level of similarity in clinical symptoms between viral and bacterial lrti. respiratory syncytial virus (rsv) pneumonia and serologically detected pneumococcal pneumonia did have considerably overlapping clinical signs and symptoms, whereas laboratory and chest radiography findings significantly differed [8] . the majority of paediatric patients with ari are young and previously healthy children with no known risk factors for acquiring respiratory infection. assessment of disease severity in these children is based on patient's history, parental concern about the health of their child and objective findings by physical examination. different clinical scoring systems are in use, adapted to local circumstances and patient groups. examples are: the respiratory distress assessment instrument, based on wheezing and retractions [9] , the (bedside) paediatric early warning sign scores [10] [11] [12] , the preschool respiratory assessment measure, [13] the clinical scoring system described by kristjansson [14] , the silverman-anderson respiratory scale [15] and the disease severity score by gern [16] . however, none of the above scoring systems has been validated specifically for prediction of clinical outcome in paediatric ari. this diversity in scoring systems limits comparison of study data in the literature. assessment of respiratory illness in children with underlying conditions is often more difficult. known risk factors for severe bronchiolitis are best recognized for rsv. they include young age, prematurity, low birth weight, down syndrome, congenital heart disease, bronchopulmonary disease and immunodeficiency [17, 18] . other underlying conditions that lead to higher hospitalisation rates are cerebral palsy, chronic lung diseases like cystic fibrosis, asthma and recurrent respiratory tract infections. use of viral diagnostics in these patient disease categories lies outside of the scope of this article. as stated above, diagnosing paediatric ari and assessment of disease severity is primarily based on clinical grounds. for borderline cases, laboratory results are often used in clinical decision making. since real time polymerase chain reaction (rt-pcr) assays have been widely introduced in many hospitals, we will discuss its impact and role in clinical decision making for paediatric ari. the pediatric infectious disease society (pids) and the infectious diseases society of america (idsa) recommend in their guideline 'community-acquired pneumonia (cap) in infants and children' the use of sensitive and specific tests for the rapid diagnosis of influenza virus and other respiratory viruses in the evaluation of children older than three months of age with cap [19] . in the case of a positive test for influenza, they strongly recommend that no antibiotic therapy be employed in the absence of clinical, laboratory or radiographic findings suggestive for bacterial co-infection. no specific recommendation is given on antibiotic use when other viruses are detected. currently, there are no (p)idsa guidelines for paediatric ari in patients younger than three months. the american association of pediatrics (aap) recommends clinicians diagnose bronchiolitis and assess disease severity on the basis of history and physical examination without routinely ordering laboratory tests or radiography to make the diagnosis (evidence level b) [20] . clinicians should assess risk factors for severe disease (age less than 12 weeks, prematurity, underlying cardiopulmonary disease or immunodeficiency) (evidence level b). repeated observations over a short period of time will improve overall assessment and patient care. in a state of the art review in 2010, this aap recommendation is supported by evidence of a low rate of bacterial co-infection in children younger than three months of age presenting with bronchiolitis [21] . however, rates of rsv testing did not fall after publication of the 2006 guidelines [22] . the authors suggests that hospitals continue to test for rsv in order to cohort patients after admission. the guidelines of the royal college of paediatrics and child health (rcpch) and the european society of paediatric infectious diseases (espid) recognize that rapid, sensitive and specific immunofluorescence viral tests are available and that rt-pcr is increasingly replacing immunofluorescence and serology, but they do not give recommendations when to use it and what the consequences are of the results when they become available [23] . until the 1980's, viral testing was not routinely available in paediatric clinical practice. viral cultures were the gold standard, but they require specialized laboratory facilities and are timeconsuming. therefore, results are usually available too late to influence patient management. serology is more easy to implement, but requires two separate blood samples over time to show (preferably) a four-fold increase in antibody response. blood sampling is not a child-friendly procedure and the results are also not readily available. the use of serology seems restricted to epidemiologic studies in order to maximize etiologic diagnosis [24] . rapid antigen tests like direct immunofluorescent antibody tests (dfa) provide more rapidly results, but are less sensitive than viral cultures [25, 26] . they are available for rsv and influenza virus, but sensitivity ranges from 66.2% to 94.1% [27] [28] [29] . according to earlier studies, rapid viral tests contribute to reduction in hospital stay and antibiotic use [7, [30] [31] [32] . since the 1990's pcr techniques have become more widely available. conventional endpoint single-target pcrs were able to report positive and negative results of rna and dna viruses. later on, computerized quantitative rt-pcrs were able to correlate the amount of pcr product to a viral load. testing a panel of respiratory viruses within a time span of 6 to 24 hours has become routine practice in many hospitals. in paediatric populations, the sensitivity has shown to be higher than for viral culture [25] . nowadays, qualitative and even quantitative multiplex pcrs are able to detect multiple respiratory viruses in one single sample simultaneously, however sensitivity in a multiplex pcr is generally lower than in single target rt-pcr [33] . a disadvantage of commercially available multiplex pcrs is that the tests require external controls, which are difficult to perform since not all available platforms publish their targets [34] . despite the availability of commercially quantitative multiplex pcrs, in-house quantitative single target rt-pcrs are used more frequently in current laboratory practice. in a recent review by jartti et al on new molecular virus detection methods, all nasopharyngeal sampling tests, including nasopharyngeal aspirates, washes, swabs or brush appear suitable for pcr analysis. viruses in the upper airways do reflect infections of the lower airways [33] . sputum induction methods, trying to generate sputum samples from the lower respiratory tract are not recommended for routine use as the diagnostic yield will not significantly improve. introducing highly sensitive viral diagnostics has yielded a broad spectrum of viral diagnoses, but has not automatically lead to changes in patient management. in an adult population, implementation of rt-pcr in lrti increased the diagnostic yield, but did not reduce antibiotic use or costs [35] . in a multicentre paediatric study, interviewing medical doctors on fictitious ari cases, rt-pcr decreased antibiotic use. however, in real life, the same physicians did not alter their antibiotic prescriptions based on the results of rt-pcr [36] . in a cochrane review (4 rct's) that evaluated the impact of rapid viral tests in children with ari in an emergency department (ed), rapid viral testing by immunofluorescence or pcr did not lead to changes in antibiotic use, length of ed visits, blood or urine testing, but did lower the rates of chest radiographs. the authors stated that routine viral testing in the ed is promising as a means to reduce antibiotic use, but there is yet insufficient evidence to support this. large trials are needed with a focus on patient management with respect to the results [6] . in a recent retrospective study evaluating clinical differences between rsv and non-rsv patients, virological testing (enzyme linked immunoassay and / or pcr) did not help in management decisions and seemed insufficient to predict outcome at an individual level [37] . in another recent retrospective study of 177 children with ari in a general hospital, antibiotic management was not influenced after detecting a viral respiratory pathogen, although the authors state that routine testing of common respiratory pathogens could lead to a better understanding of their role in disease in children with respiratory symptoms [38] . in a 6year prospective study of children with community acquired pneumonia (cap), designed to describe the frequency of respiratory viruses, antibiotics were prescribed less frequently in viral positive versus negative children, but only when they were > 18 months old [39] . our own data from a multicentre prospective controlled clinical trial of 582 previously healthy children with respiratory symptoms showed a high diagnostic yield of rt-pcr, however the rapid communication (within 24 hours) of results to the paediatrician did not change patient care versus those who received results later [40] . these findings underlie the statement of the aap, not to recommend routine viral testing for standard ari cases, unless the physician is willing to change his patient management based on the results [20] . at present, a panel of approximately 15 respiratory rt-pcr assays is available in many hospitals. data suggest that rsv has the greatest disease burden both in hospitalized children and in outpatients, especially in children under five years of age [41] . furthermore, human rhinovirus (hrv) is generally associated with the common cold, can cause severe ari as well and is the most common pathogen in ari in young children [42, 43] . re-infections with hrv are commonly observed, and are usually caused by different virus strains. [44] as reviewed by kim, recent studies suggest that hrv subtype c may be more virulent than other hrv [45] , but not all rt-pcrs are capable of distinguishing between the different hrv strains. influenza virus (iv) is well known for its seasonal outbreaks of ari during a few weeks in winter, and also for a sepsis like syndrome without respiratory symptoms. human adenovirus (hadv) is common in respiratory disease with a severe disease course in children with underlying immunodeficiency [46] . para-influenza viruses (piv) subtypes 1 and 2 are known to cause croup and subtype 3 is known to cause bronchiolitis and pneumomia [47] . piv subtype 4 is has been reported to be a much less frequent cause of ari [48] . the incidence of polyomaviruses wu [49] and ki [50] is low and their clinical relevance remain unclear and require further evaluation [51] . the role of newly discovered viruses is of interest in many studies. some of these 'new' viruses are human metapneumovirus (hmpv), human bocavirus (hbov) and human coronavirus (hcov). hmpv was first detected in 2001 [52] , and its clinical symptoms overlap with those of other respiratory viruses [53] . disease severity seems less than for rsv [54] . another 'new' virus is hbov, discovered in 2005 [55] . in a review by brodzinsky, the incidence is found to be low (1.5-4.5%) and rates of co-infections are high (14-72%) [56] . the spectrum of disease is similar to rsv and hmpv. the group of hcov's are heterogeneous. hcov 229e [57] , oc43, [58, 59] nl63 [60, 61] and hku1 [62] are recognized frequently in young age and are correlated with less severe respiratory disease [54, 63] . sars coronavirus and the novel mers coronavirus [64] are not routinely incorporated in respiratory rt-pcr test panels. a recent swedish study compared viral pcr findings from children with ari versus asymptomatic matched controls. rsv, hmpv and pivs were highly overrepresented in symptomatic patients, suggesting that they are responsible for illness. asymptomatic controls showed high detection rates of hbov, hrv, hadv, hcov and enterovirus, suggesting that prolonged virus-shedding may occur and that pcr-results need to be interpreted with caution [65] . the incidence of mixed viral infections is reported as high as 14 -44%, depending on different populations and test panels [66] . in a multicenter study, involving 2207 children less than 2 years of age, the incidence of mixed infection in children hospitalized for bronchiolitis was 30% [67] . several circumstances can generate positive rt-pcr results. rt-pcr assays are very sensitive and can detect small amounts of viral nucleic acids, which are still present during a convalescence period. for each individual virus it is not known how long dna/rna shedding may continue during this convalescence period [68, 69] . furthermore, children with a normal immune system can asymptomatically harbor viruses in their respiratory tract. usually, a primary viral infection leads to an adaptive immune response with induction of memory t-cells, so that a second hit with the same virus usually results in less serious or even absent symptoms [70] . the relevance of finding only a single pathogen is also of interest. rt-pcr assays are sensitive tests, but detect only pathogens that are looked for. for each micro-organism a specific primer is used, therefore non-viral pathogens like bordetella pertussis, mycoplasma pneumonia and chlamydophyla pneumonia are not detected unless they are looked for. they can mimic viral disease as well, especially in young children [71] . one could hypothesize that mixed infections may lead to a more severe disease. papers on this subject show somewhat contradictory results. there are a few reports suggest that there is no relation between mixed viral ari and disease severity [72] [73] [74] [75] , while others indicate a higher disease severity in children with a mixed respiratory infection [76, 77] . further research as to how one should interpret multiple, positive rt-pcr results in one single sample is needed. quantitative studies with interpretation of viral load will possibly help to answer this question. epidemiologic surveillance programs benefit from viral rt-pcrs. examples include the worldwide search for sars and mers coronaviruses, well known for their febrile and atypical pneumonia and the dramatic course in some affected people [64, [78] [79] [80] [81] . other national and who-supported surveillance programs include seasonal influenza viruses. currently, the 'bird flu' influenza a subtype h7n9 is of special interest for its infection of humans and possible easy transmission from wild birds to humans [82] . prevention of nosocomial infections should be of high priority in any hospital and isolation or cohorting is a well-established policy. rt-pcr has shown to be a useful tool for epidemiologic studies. however, the epidemiology of many nosocomial respiratory infections is not well known. for rsv, the incidence of nosocomial spread seems low. although information about management of outbreaks is sparse and not well studied, normal prevention measurements appear to be rational [83] . for cohorting, rapid testing for rsv has shown to be a safe, cost-effective and efficient way to improve bed management [84] . although we do not doubt that isolation as strategy helps to prevent nosocomial infections, we still have concerns when cohorting is based on rt-pcr results of only a limited number of viruses. mixed infections occur in a substantial proportion of children and whether these children spread other non-detected viruses during cohorting is not yet clear. rapid antigen tests like dfa are limited useful for cohorting issues, since they may have a high proportion of false negatives. rt-pcr obviously is much more sensitive and specific, but the turnaround time is not sufficient to act in time for infection control measurements at admission. theoretically, one would expect that rt-pcr would be beneficial for infection control, however no large study has demonstrated a positive effect to date. the risk of cross-infection in children sharing a room was studied recently in a prospective observational cohort study of 48 children with bronchiolitis. room sharing between rsv-positive and rsvnegative children on the first day of admission did not influence the risk of co-infection [85] . children with respiratory failure induced by viral infection and admitted to paediatric intensive care units for mechanical ventilation frequently have concomitant bacterial infections [86] . use of an extended rt-pcr panel of respiratory pathogens seems to be justified in those conditions to determine the causing micro-organisms. however, it is unclear what the exact relevance of the rt-pcr results is, except for those viruses for which treatment is available (like influenza and adenovirus) children with underlying immunodeficiencies are susceptible to severe complications of viral infections. indications for rt-pcr diagnostics in children with hematologic malignancies, hematopoietic stem cell transplantation, solid organ transplantation, premature infants and children with cystic fibrosis lies outside the scope of this article and was well reviewed by vallieres and renaud recently [34] . an objective laboratory parameter, reflecting disease severity, would be helpful to estimate disease course in an early stage. viral load was thought to serve as such a parameter. viral load is the quantity or copy number of viral rna or dna detected per milliliter body fluid. in rt-pcr, the cycle threshold (ct) value is defined as the number of rt-pcr cycles required for a positive fluorescent amplification signal to cross the threshold. this is inversely correlated with the viral load. some authors indeed found a significant correlation between viral load and disease severity [87] [88] [89] , thereby justifying broad use of rt-pcr. however viral load does not always correlate well [90] [91] [92] [93] [94] . some viruses, like rsv, are short lived and others like hbov are long-lived, which is a complicating factor in demonstrating this relationship. the question how well disease severity is correlated with viral load is not answered yet. paediatric ari is a clinical diagnosis and most infections are caused by viruses. use of clinical scoring systems helps to assess disease severity for the individual patient and could be used for clinical management. thus far, an extended panel of rt-pcr assays contributes little to clinical decision making for the majority of children with ari; in fact rt-pcr for detecting respiratory infection is not routinely available in many hospitals. in today's paediatric practice, rt-pcr is used in patients with high-risk of complications or with an unexpected disease course. the panel of microorganisms should at least include pathogens for which a specific treatment is available, e.g. influenza virus and bordetella pertussis. the authors have no conflicts of interest to disclose. rt-pcr will help to explore the role of certain viruses in ari with mixed viral aetiology, to discover new viruses in future, and possibly to assess disease severity by repeated viral load quantification. rt-pcr is a powerful tool in public health surveillance. the role of rt-pcr in hospital hygiene is currently limited. infectious disease hospitalizations among infants in the united states clinical findings and severity of acute bronchiolitis a systematic review on the diagnosis of pediatric bacterial pneumonia: when gold is bronze the definition of pneumonia, the assessment of severity, and clinical standardization in the pneumonia etiology research for child health study evaluation of the utility of radiography in acute bronchiolitis rapid viral diagnosis for acute febrile respiratory illness in children in the emergency department the effect of rapid respiratory viral diagnostic testing on antibiotic use in a children's hospital clinical profile of serologically diagnosed pneumococcal pneumonia wheezing in infants: the response to epinephrine the pediatric early warning system score: a severity of illness score to predict urgent medical need in hospitalized children the pews score: potential calling criteria for critical care response teams in children's hospitals development and initial validation of the bedside paediatric early warning system score the preschool respiratory assessment measure (pram): a responsive index of acute asthma severity nebulised racemic adrenaline in the treatment of acute bronchiolitis in infants and toddlers a controlled clinical trial of effects of water mist on obstructive respiratory signs, death rate and necropsy findings among premature infants relationships among specific viral pathogens, virus-induced interleukin-8, and respiratory symptoms in infancy down syndrome: a novel risk factor for respiratory syncytial virus bronchiolitis-a prospective birth-cohort study risk factors for hospital admission with rsv bronchiolitis in england: a population-based birth cohort study the management of communityacquired pneumonia in infants and children older than 3 months of age: clinical practice guidelines by the pediatric infectious diseases society and the infectious diseases society of america american academy of pediatrics subcommittee on diagnosis and management of bronchiolitis. diagnosis and management of bronchiolitis bronchiolitis: recent evidence on diagnosis and management bronchiolitis management before and after the aap guidelines manual of childhood infections, the blue book additional diagnostic yield of adding serology to pcr in diagnosing viral acute respiratory infections in kenyan patients 5 years of age and older comparison of multiplex pcr assays and conventional techniques for the diagnostic of respiratory virus infections in children admitted to hospital with an acute respiratory illness currently used nucleic acid amplification tests for the detection of viruses and atypicals in acute respiratory infections evaluation of respiratory syncytial virus detection by rapid antigen tests in childhood prospective evaluation of rapid antigen tests for diagnosis of respiratory syncytial virus and human metapneumovirus infections comparison of bd directigen ez rsv and binax now rsv tests for rapid detection of respiratory syncytial virus from nasopharyngeal aspirates in a pediatric population clinical and financial benefits of rapid detection of respiratory viruses: an outcomes study a randomized, controlled trial of the impact of early and rapid diagnosis of viral infections in children brought to an emergency department with febrile respiratory tract illnesses cost-effectiveness of rapid diagnosis of viral respiratory tract infections in pediatric patients new molecular virus detection methods and their clinical value in lower respiratory tract infections in children clinical and economical impact of multiplex respiratory virus assays impact of rapid detection of viral and atypical bacterial pathogens by real-time polymerase chain reaction for patients with lower respiratory tract infection does real-time pcr for respiratory virusses change antibiotic use in children admitted at the pediatric intensive care unit with lower respiratory tract infection? thesis umcu, helms studio bv virologic testing in bronchiolitis: does it change management decisions and predict outcomes diagnostic value of respiratory virus detection in symptomatic children using real-time pcr spectrum of respiratory viruses in children with community-acquired pneumonia clinical impact of rt-pcr for pediatric acute respiratory infections: a controlled clinical trial the burden of respiratory syncytial virus infection in young children rhinovirus infections in infancy and early childhood picornavirus infections in children diagnosed by rt-pcr during longitudinal surveillance with weekly sampling: association with symptomatic illness and effect of season serial viral infections in infants with recurrent respiratory illnesses updates in the relationship between human rhinovirus and asthma adenovirus: an increasingly important pathogen in paediatric bone marrow transplant patients parainfluenza virus infection of young children: estimates of the population-based burden of hospitalization human parainfluenza virus type 4 infections: a report of 20 cases from 1998 to 2002 identification of a novel polyomavirus from patients with acute respiratory tract infections identification of a third human polyomavirus polyomaviruses ki and wu in children with respiratory tract infection a newly discovered human pneumovirus isolated from young children with respiratory tract disease human metapneumovirus: lessons learned over the first decade clinical impact and diagnosis of human metapneumovirus infection cloning of a human parvovirus by molecular screening of respiratory tract samples review of new and newly discovered respiratory tract viruses in children a new virus isolated from the human respiratory tract cultivation of a novel type of common-cold virus in organ cultures recovery in tracheal organ cultures of novel viruses from patients with respiratory disease identification of a new human coronavirus a previously undescribed coronavirus associated with respiratory disease in humans characterization and complete genome sequence of a novel coronavirus, coronavirus hku1, from patients with pneumonia human coronavirus in young children hospitalized for acute respiratory illness and asymptomatic controls isolation of a novel coronavirus from a man with pneumonia in saudi arabia clinical utility of pcr for common viruses in acute respiratory illness viral co-detection in infants hospitalized with respiratory disease: is it important to detect? prospective multicenter study of viral etiology and hospital length of stay in children with severe bronchiolitis frequent detection of respiratory viruses without symptoms: toward defining clinically relevant cutoff values respiratory pathogens in children with and without respiratory symptoms expanding roles for cd4(+) t cells in immunity to viruses bordetella pertussis and mixed infections infection with multiple viruses is not associated with increased disease severity in children with bronchiolitis multiple versus single virus respiratory infections: viral load and clinical disease severity in hospitalized children viral respiratory co-infections in pediatric patients admitted for acute respiratory infection and their impact on clinical severity clinical risk factors are more relevant than respiratory viruses in predicting bronchiolitis severity multiple simultaneous viral infections in infants with acute respiratory tract infections in spain dual infection of infants by human metapneumovirus and human respiratory syncytial virus is strongly associated with severe bronchiolitis identification of a novel coronavirus in patients with severe acute respiratory syndrome a novel coronavirus associated with severe acute respiratory syndrome sars virus: the beginning of the unraveling of a new coronavirus coronavirus as a possible cause of severe acute respiratory syndrome human infection with a novel avian-origin influenza a (h7n9) virus infection control, outbreak management rapid testing for respiratory syncytial virus in a paediatric emergency department: benefits for infection control and bed management co-infections in children hospitalised for bronchiolitis: role of roomsharing empiric antibiotics are justified for infants with respiratory syncytial virus lower respiratory tract infection presenting with respiratory failure: a prospective study and evidence review viral load drives disease in humans experimentally infected with respiratory syncytial virus disease severity and viral load are correlated in infants with primary respiratory syncytial virus infection in the community equal virulence of rhinovirus and respiratory syncytial virus in infants hospitalized for lower respiratory tract infection respiratory syncytial virus infections in hospitalized infants: association between viral load, virus subgroup, and disease severity correlation of viral load of respiratory pathogens and co-infections with disease severity in children hospitalized for lower respiratory tract infection prospective study of human metapneumovirus infection: diagnosis, typing and virus quantification in nasopharyngeal secretions from pediatric patients quantitation of respiratory viruses in relation to clinical course in children with acute respiratory tract infections illness severity, viral shedding, and antibody responses in infants hospitalized with bronchiolitis caused by respiratory syncytial virus we thank p. goswami, md for critically reviewing the manuscript in the english language. key: cord-346253-0mnsm6s4 authors: ahanchian, hamid; jones, carmen m; chen, yueh-sheng; sly, peter d title: respiratory viral infections in children with asthma: do they matter and can we prevent them? date: 2012-09-13 journal: bmc pediatr doi: 10.1186/1471-2431-12-147 sha: doc_id: 346253 cord_uid: 0mnsm6s4 background: asthma is a major public health problem with a huge social and economic burden affecting 300 million people worldwide. viral respiratory infections are the major cause of acute asthma exacerbations and may contribute to asthma inception in high risk young children with susceptible genetic background. acute exacerbations are associated with decreased lung growth or accelerated loss of lung function and, as such, add substantially to both the cost and morbidity associated with asthma. discussion: while the importance of preventing viral infection is well established, preventive strategies have not been well explored. good personal hygiene, hand-washing and avoidance of cigarette smoke are likely to reduce respiratory viral infections. eating a healthy balanced diet, active probiotic supplements and bacterial-derived products, such as om-85, may reduce recurrent infections in susceptible children. there are no practical anti-viral therapies currently available that are suitable for widespread use. summary: hand hygiene is the best measure to prevent the common cold. a healthy balanced diet, active probiotic supplements and immunostimulant om-85 may reduce recurrent infections in asthmatic children. asthma is a major public health problem with a huge social and economic burden affecting 300 million people worldwide [1] . viral respiratory infections are the major cause of acute asthma exacerbations and contribute to asthma inception in high risk young children with susceptible genetic background. a history of wheeze associated with respiratory viral infections early in life is one of the major risk factors for the later development of asthma [2] [3] [4] [5] [6] [7] , together with sensitization to aeroallergens in early life and a family history of asthma and allergies, reflecting a genetic predisposition. respiratory viral infections are also the principal cause of asthma exacerbations in children and adults [8] [9] [10] [11] [12] [13] . however, the question of whether viral infections "select" susceptible hosts or whether viral infections may induce asthma de novo by "damaging" airways is not settled. in other words, do viruses cause or simply unmask asthma? respiratory viruses first infect nasal epithelial cells which triggers an antiviral response. this response is driven by type i (α/β) and iii (λ) interferons (ifn) that are induced following recognition of viral ribonucleic acid (rna) by pattern recognition receptors (prrs). toll-like receptors (tlrs) are cell surface and endosomal prrs, whilst the rna helicase receptors (rig-i and mda-5) and nodlike receptors (nod2), detect viral rna in the cytoplasm. signalling via the prrs activates transcription factors (irf-3, irf-7, nf-κb), which lead to the production and secretion of type i and iii ifn. the ifns then bind to cell surface receptors to activate a separate pathway leading to the production of interferon stimulated genes (isgs) which encode antiviral proteins that combat infection, as well as prrs and transcriptional factors which further amplify ifn production. the respiratory syncytial virus (rsv), human meta-pneumovirus (hmpv) and human rhinovirus (hrv) are all single stranded rna viruses but engage differently with cell signalling pathways. in airway epithelial cells rsv and hmpv rna are primarily detected by rig-i in the cytoplasm [14, 15] . rsv can also be detected by nod2 [16] . hrv is endocytosed by epithelial cells, and is therefore primarily detected by tlr3 in the endosome early in the infection process and by rig-i and mda-5 later in infection following upregulation of these prrs [17] . the fusion (f) protein of rsv is recognised by tlr4 at the epithelial cell surface [18] . a successful antiviral response would see the infection limited to the upper airway, as is the case clinically with the majority of viral infections in healthy individuals. should such a response be deficient, then predominantly upper-airway viral infections, such as hrv, may spread to the lower airways, causing lower respiratory symptoms and an exacerbation of asthma in predisposed individuals. while definitive data are yet to be produced, experimental hrv infections in adult volunteers initially suggested that asthmatics were more likely to develop lower respiratory infections (lri) than healthy adults, i. e. less likely to be able to limit viral replication to the upper airways [19, 20] . subsequent in vitro infection of primary airway epithelial cells from asthmatic and healthy adults with hrv have demonstrated that asthmatic cells produce less ifn-β [21] and ifn-λ [22] making them potentially more susceptible to infection, slower to clear infection, and more susceptible to virus-induced cell cytotoxicity. deficiencies in the ifnα response of peripheral blood mononuclear cells and plasmacytoid dendritic cells from asthmatic adults and children has also been observed, in these particular studies, in response to rsv, hrv [14, 15] and influenza a [23] . it is likely that the overall impaired innate immune response of the asthmatic airway epithelium is a result of deficiencies in the antiviral response of both epithelial cells and immune cells. childhood, especially infancy, is characterized by developmentally-regulated deficiencies in innate and adaptive immunity [24] . such deficiencies are likely to increase the risk of viral lri in children, especially in those at high risk for asthma and allergies. each year, at the end of summer, parents of asthmatic children are concerned about acute asthma exacerbations following a common cold, asking how to minimize the risk during the winter viral season. it is a valid concern as up to 70% of asthmatic children have an intermittent or wheeze which is mostly symptomatic after viral infections [25, 26] . asthmatics with exacerbationprone phenotype are susceptible to acute exacerbations requiring hospitalization or an unscheduled visit for medical attention. major risk factors for acute exacerbations include previous acute exacerbation, allergy, young age, poorly controlled asthma, and, in particular, viral respiratory infections. moreover, recent data suggests an interaction between allergies and viral infections occurs to increase the risk of asthma exacerbation [27] . acute exacerbations are associated with decreased lung growth or accelerated loss of lung function and, as such, add substantially to both the cost and morbidity associated with asthma [28, 29] . viral respiratory infections are the main cause of asthma exacerbations in children (80-85%) and are a major risk factor for admission in hospital every autumn [30] [31] [32] . hrv are the most common viral agents [33] ; other respiratory tract viruses detected in children with an asthma exacerbation include rsv, influenza, coronavirus, hmpv, parainfluenza virus, adenovirus, and bocavirus [34] [35] [36] . current drugs for the prevention and treatment of virus-induced exacerbation of asthma are poorly effective and novel alternative therapies are needed. much research interest has focused on the potential role respiratory viral infections play in the inception of asthma. it is well established that hospitalization for rsv bronchiolitis is a risk factor for asthma during childhood [37, 38] . epidemiological studies have shown an increased risk of asthma with lri caused by hrv. in the childhood origin of asthma (coast) birth cohort study, wheezing with rsv (odds ratio [or], 2.6), hrv (or, 9.8), or both hrv and rsv (or, 10) was associated with increased asthma risk at age six years [7] . the childhood asthma study (cas) in perth, australia showed that wheezing with hrv or rsv in the first year of life was a risk factor (or, 2.5) for current wheeze at five years of age [4] . infant birth about four months before the winter virus peak carried the highest risk of developing asthma compared with birth 12 months before the peak [39] . the risk of asthma is increased by severe lri (slri), especially in the presence of allergic sensitization in early life [4, 25] . there appears to be a synergistic interaction between viral infection and allergic sensitization, suggesting a "two hit" model for induction of persistent asthma. these data also provide a series of novel strategies for the primary prevention of asthma by prevention of either allergic sensitization or of slri in high risk children. this strategy is also supported in a study by simoes et al. [40] , in which the use of palivizumab to prevent rsv infection decreased the risk of recurrent wheezing in nonatopic premature infants. the crucial period, with respect to asthma initiation, appears to be the first two to three years of life during which the growth and remodelling of lung and airways proceeds at maximum rates. pulmonary inflammation resulting from atopy and slri occurring during this vulnerable time is hypothesized to perturb underlying tissue differentiation programs, resulting in deleterious long term effects on respiratory functions. as a result, there is widespread belief amongst the paediatric respiratory community that intervention measures that can lower the frequency and/or intensity of slri in early life amongst the high risk atopic subgroup of children are likely to be successful at preventing asthma. if successful, these strategies would have major implications for reducing the high impact of this chronic disease on the community [17, 41, 42] . recent studies using culture-independent techniques have challenged the long-held dogma that lungs are sterile and have demonstrated that a microbiota community exists in the lung [43] [44] [45] . the implications of these new data are not clear, however new concepts and more research is required. the resident microbiome is different in the presence of respiratory disease [45, 46] ; therefore interactions between respiratory viruses and the resident pulmonary microbiome are postulated. the pulmonary and gastrointestinal microbiota influence the immune system and interventional approaches (by bacterial immunostimulants, prebiotics and/or probiotics) to create a healthy gut and respiratory microbiota are potential strategies for the prevention of viral infections [45] . children are important vectors for hrv transmission to family members particularly siblings [47, 48] . hrv shedding peaks two to four days after infection and decreases sharply thereafter, although nasal samples can be positive for rhinovirus for up to five weeks after a symptomatic infection [49] . there are three ways of common cold transmission in children. first, inhalation of small particles aerosolized by coughing; second, large particle droplets from saliva expelled while sneezing; and third, self-inoculation of one's own conjunctivae or nasal mucosa after touching a person or object contaminated with the cold viruses. the first two methods are inefficient [50] , while the third is the most important method of transmission. the mode of transmission could differ with age of the index case, duration of contact, and other factors. moreover, there is some evidence that the daily activities of infected people can lead to the contamination of environmental surfaces with hrv e.g. light switches, telephone dial buttons and handsets [48] . meticulous hand hygiene is the best measure to prevent the common cold; frequent hand washing and avoid touching one's nose and eyes [51] [52] [53] . the use of alcohol-based hand sanitizers is also effective [54, 55] . the promotion of handwashing was associated with a 12-34% reduction in respiratory-tract infections and colds in child-care centres in the usa [56] canada [57] and australia [58] and a 21% decrease in absences due to respiratory illness in the school setting [56] . hand hygiene campaigns were also successful in reducing absenteeism caused by influenza-like illnesses among schoolchildren in egypt [59] . similar programs within families would be expected to reduce transmission of hrv between family members. a recent cochrane review which included data from 67 randomised controlled trials and observational studies, investigated the effectiveness of physical interventions to reduce the spread of respiratory viruses. the authors concluded that respiratory virus spread can be reduced by hygiene measures (such as handwashing), especially around younger children and can reduce transmission from children to other family members [51] . controversy still exists and a newly published study showed that an antiviral hand treatment used by adult volunteers, recruited from a university community, did not significantly reduce rv infection or rv-related common cold illnesses [60] . asthmatic children should avoid close contact with people who have colds especially during the first three days of their illness. there is little evidence to support the effectiveness of face masks to reduce the risk of viral respiratory infections and consequently, the use of mask is generally not recommended for prevention of common cold [51, 61] . immune function and anti-viral defenses have a number of components, both specific and non-specific. asthmatic children can improve their immune function by following some simple advice including a healthy life style with regular exercise, a balanced diet, adequate sleep and avoiding environmental tobacco smoke, stress and unnecessary antibiotics. exercise has anti-inflammatory effects and in the long term can protect the development of chronic diseases and obesity [62] . regular exercise of moderate-intensity is associated with a reduced incidence of upper respiratory tract infection. however, long hours of intensive training appear to make children more susceptible to upper respiratory tract infections [63] [64] [65] . the recommended means of aerobic exercise is walking, with an optimal frequency of three to five days a week and an optimal duration of 20 to 30 minutes of continuous activity [66] . in a recent study, the iga secretion rate was negatively correlated with the incidence of infections [67] . a recent randomized trial comparing meditation and exercise with wait-list control among adults aged 50 years and older found significant reductions in ari illness [68] . malnutrition is the most common cause of immune deficiency worldwide and a balanced diet is fundamental for a healthy immune system. vitamin d deficiency has been associated with increased risk of infections, earlylife wheeze and reduced asthma control [69, 70] . vitamin a derivatives are involved in the regulation of the immune system and tissue inflammation as well as prevention of respiratory infections [71] . zinc, selenium and other trace elements are necessary for function of both innate and adaptive immune function. a high intake of fruit and vegetables ensures adequate consumption of nutrients and antioxidants and appears to be beneficial for asthma. although recent reviews have shown that zinc [72] , garlic [73] , echinacea purpurea [74] or ginseng [75] supplementation for several months may reduce cold incidence, there is insufficient evidence to recommend any vitamin or mineral supplementation in the management of asthmatic children without nutrient deficiency [76, 77] . however, a large controlled trial showed echinacea was ineffective in reducing infection rate or symptom severity of hrv infection in healthy young adult volunteers [78] . vitamin c supplementation failed to reduce the incidence of colds in the general population except in those exposed to short periods of extreme physical stress [79] . finally, it is worth remembering that infants who are not breastfed have significantly higher risk of respiratory, gastrointestinal, and other infections, as breast milk is a biologically active substance containing antimicrobial and immunomodulatory elements [80] [81] [82] . sleep and the circadian system exert a regulatory influence on immune functions. sleep deprivation can affect immune function in several ways including reduced natural killer cell activity, suppressed interleukin-2 production and increased levels of circulating proinflammatory cytokines [83, 84] . there is also evidence for an enhanced susceptibility to the common cold and pneumonia with poor sleep efficiency [85, 86] . air pollutants (nitrogen dioxide, ozone, particulate matter) and environmental tobacco smoke (ets) have long been correlated with multiple adverse effects on the immune system and susceptibility to viral respiratory tract infections in children [87] [88] [89] [90] . studies in europe and the united states have shown that 40% of children live with a smoker [34] and they have approximately twice the risk of contracting a serious respiratory tract infection in early life [91] . cigarette smoking leads to a longer duration of cough, greater frequency of abnormal auscultatory findings during acute respiratory tract illness [92, 93] and higher risk for severe exacerbations [94] . urinary leukotriene e4 levels identify children exposed to ets at high risk for asthma exacerbation [94] . there is strong evidence that some pharmacological preparations can help prevent viral infection by specific effects on immune system. these results have been promising with a hope that using these strategies can attenuate the role of viruses in asthma inception. ancient physicians of the middle east prescribed yogurt for curing disorders of the stomach, intestines and for stimulation of appetite. it is written in the old persian testament that "abraham owed his longevity to the consumption of sour milk" [95] . the popularity of probiotics and intestinal microbiota significantly increased when the nobel prize-winning russian scientist eli metchnikoff suggested in 1908 that the long life of bulgarian peasants resulted from their consumption of fermented milk products [96] . the term probiotic, meaning for life, is used for live micro-organisms (typically of the bifidobacterium and lactobacillus species) administered in adequate amounts which confer a beneficial physiological effect on the host. prebiotics are nutrients, in particular oligosaccharides, which foster the growth of probiotics in the colon. the term synbiotics is used when a product contains both probiotics and prebiotics [97] . up to 100 trillion bacteria from different species colonize the human gut [98] . this microbiota participates in: host metabolism, vitamin synthesis, control of epithelial cell growth, protection from infectious microbes, and helps proper development and function of the immune system. there is constant cross-talk between microbiota and gut-associated lymphoid tissue (the largest lymphoid tissue of the human body which contains more than 60% of all body lymphocytes) to establish mucosal immune tolerance in the gut. common mucosal immunity describes the phenomenon where immune cells, especially regulatory t-cells, traffic to and influence responses at other mucosal surfaces, including the lungs [99] . alteration in the microbiota composition (dysbiosis) results in immunological dysregulation that may underlie many human diseases such as inflammatory diseases [100] , obesity [101] , allergy [102] and autoimmunity [103] . reduced bacterial diversity in the infant's gastrointestinal tract has been associated with an increased risk of allergic sensitization and allergic rhinitis but not asthma or atopic dermatitis [102] . in the first year of life, especially the first few weeks, the microbiota of the newborn is highly variable during this critical time of post-natal maturation of the immune system. microbiota is shaped by genetic and environmental factors including: mode of delivery, neonates born by means of vaginal delivery are exposed to mothers gut, skin, and vaginal flora [104] ; breast feeding and diet [105] ; farm or urban living [106] ; vitamin d status [107] ; and antibiotic consumption [98, 108] . this knowledge stimulated interest in the use of probiotics and prebiotics as the intentional introduction or encouragement of specific microbes to shape immune system development. specifically, the microbiota can activate distinct tolerogenic dendritic cells in the gut and through this interaction can drive regulatory t-cell differentiation that modulates both th1 and th2 responses inside and outside the gut [109] [110] [111] . probiotics have been successfully used for the treatment of several gastrointestinal disorders (viral and antibiotic-associated diarrhea, inflammatory bowel disease) [112, 113] . however, attempts to prevent or treat allergic disorders such as eczema, asthma and allergic rhinitis have had inconsistent results [99, 109, [114] [115] [116] . there are a growing number of clinical trials using probiotics for the prevention and management of respiratory infections. while the precise mechanisms are largely unknown, speculations include: probiotics compete against pathogens; increase the barrier function in respiratory epithelium; immunostimulatory effects by enhancing cellular immunity with increased activity of natural killer cells and macrophages in airways [117] . probiotics reduce the frequency of gastrointestinal and respiratory tract infections in children who attend day care centres [118] . they have also been found to reduce the incidence of ventilator-associated pneumonia, respiratory infections in healthy and hospitalized children, and reduce the duration of common cold symptoms [119] [120] [121] [122] . one study demonstrated that that daily probiotic supplementation for six winter months in children three to five years of age reduced the incidence of fever, coughing and rhinorrhea by 32-43% with no notable adverse events [123] . probiotic combination with vitamins and minerals also reduced the duration and severity of common cold [124] . a recent cochrane review of 14 randomised controlled trials showed that probiotics were better than placebo in reducing the number of episodes of acute upper respiratory infections (uris) and reducing antibiotic use, while there were no differences in the mean duration of an episode and no increase in adverse events [125] . probiotic foods such as probiotic milk or yogurt (functional foods) containing well-defined probiotic strains may reduce the risk of catching the common cold and represent a simple, safe, effective, available and affordable method for preventing respiratory infections in children [112, 120, [126] [127] [128] [129] [130] [131] . although there are several clinical trials that showed the preventive effect of probiotic, prebiotic [132] or synbiotics treatments [133] on respiratory infections, not all studies are positive with some failing to show any significant preventive effect [134] . to explain the different results in clinical trials, it is of particular importance to point out that the immunomodulatory capabilities of probiotics are strain-dependent. difference in dosage, duration of intervention, population and environmental background may also affect the results. one major limitation in this field is that it is not possible to test just how "probiotic" a particular preparation is. technical advances will be required before some of the apparent discrepant results of studies can be resolved. several immunostimulants, including herbal extracts, bacterial extracts, synthetic compounds, have been promoted as increasing the immune defences of the respiratory tract. a recent cochrane review included data from 35 placebo-controlled trials including 4060 participants below the age of 18 years in which various types of "immunostimulants" were used to reduce acute respiratory tract infections, involving either upper or lower airways. the authors concluded that immunostimulants reduced the incidence of acute respiratory infections by 40% on average in susceptible children, but that trial quality was generally poor and a "high level of statistical heterogeneity was evident". a subgroup analysis focusing on bacterial immunostimulants, including om85, produced similar results with lower statistical heterogeneity [135] . om-85 bv (broncho-vaxom) is an immunostimulant extracted from eight common bacterial pathogens of the upper respiratory tract: haemophilus influenzae, diplococcus pneumoniae, klebsiella pneumoniae and ozaenae, staphylococcus aureus, streptococcus pyogenes and viridans, neisseria catarrhalis and has been used in several countries around the world for as long as 20 years [136] . recent studies showed that om-85 bv can reduce the number of acute respiratory infections by 25% to 50% compared with placebo in children with a history of recurrent infection [137] . of particular interest, razi et al. showed that children between the age one and six years with recurrent wheezing who were given om-85 bv had a 40% reduction in the rates of wheezing over the subsequent 12 months, compared to placebo (p < 0.001). in addition, the duration of each wheezing attack was two days shorter in the group given om-85 bv than in the group given placebo (p = 0.001) [138] . again, direct evidence of the mechanisms involved are lacking from human studies. however, recent data from rodents shows that baseline regulatory t lymphocyte activity in the airways can be boosted by microbe-derived stimulation of the gut [139] . bacterial immunostimulants were also shown to enhance innate immunity (i.e. intensification of phagocytosis) and adaptive immunity [140] . as discussed above, evidence exists for an impaired innate immune response to respiratory viral infections in asthmatics [141] . entry of rhinovirus into normal epithelial cells initiates a vigorous innate immune response with ifn-β secretion and apoptosis induction. in asthma, ifn-β and ifn-λ responses are impaired, resulting in viral replication, cell cytotoxicity, enhanced virion shedding and increased susceptibility to common cold [17, 142] . epithelial cells of asthmatic patients responded to exogenous treatment with ifn-β exhibiting reduced rhinovirus release (cakebread, xu et al. 2011; jackson, sykes et al. 2011). if the proposed deficiency of type i and iii contribute to asthma exacerbations [21, 22, 143] , correcting this deficiency with exogenous interferons would be a logical approach. the advantages of interferon application include the broad spectrum of activity with low risk of resistance development [47] . prophylactic intranasal recombinant ifn-α and ifn-β have been shown to be effective against rhinovirus infection in humans [144] [145] [146] . the results of these clinical trials are awaited with interest [147, 148] . however, the systemic symptoms associated with severe viral infections, e.g. influenza, are associated with interferons, so careful dosing may be required. considering the occurrence of the local side effects, neutropenia and cost, the use of long-term prophylaxis with daily, intranasal administration of interferons is not feasible [144] . however, randomized clinical trials using similar strategies are currently underway in adults with chronic respiratory disease and the results are keenly awaited. vitamin d deficiency is a common worldwide problem [149] [150] . beside importance for bone health, vitamin d plays an important role in adequate function of both the innate and adaptive immune systems including development of dendritic cells and regulatory t lymphocytes [151, 152] production of antimicrobial proteins by airway epithelium [153] , modifying the effect of intestinal flora on inflammatory disorders [107] , and modulation of the inflammatory response to viral infections [154] . recent reports suggest that vitamin d might play a role in the recent increase in allergic disease [155] [156] [157] . vitamin d insufficiency has been associated with a higher incidence of respiratory tract infection, wheezing illness in children [158] , reduced asthma control [159] , emergency department visits, severe asthma exacerbations and hospitalizations [70, 160] . in a recent study of 48 children from five to 18 years of age, with newly diagnosed asthma, vitamin d supplementation during the northern hemisphere winter months (september to july) prevented declining serum concentrations of 25(oh) d and reduced the risk of asthma exacerbation triggered by acute respiratory tract infections [161] . macrolides possess anti-inflammatory and immunomodulatory properties extending beyond their antibacterial activity [162] . indeed, they can attenuate pro-inflammatory cytokine production by bronchial epithelial cells, neutrophils and macrophages that may contribute to clinical improvement in many patients with chronic airway inflammation [163] [164] [165] . azithromycin has anti-rhinoviral activity and can reduce hrv replication and release by increasing interferon production from epithelial cells [42, 166, 167] . macrolide antibiotics inhibit rsv infection in human airway epithelial cells [168] . a three weeks treatment with clarithromycin in rsv bronchiolitis had statistically significant effects on hospital length of stay and rate of readmission to the hospital within six months after discharge [169] . however, direct evidence of macrolides preventing respiratory viral infection in children is lacking. as the majority of respiratory viral infections in young children are caused by hrv or rsv, we will briefly discuss anti-viral strategies to prevent hrv or rsv infections in asthmatic children. because there are more than 100 serotypes of hrv, antiviral drugs are considered to be more effective than vaccination. antiviral agents have been designed to inhibit rhinovirus attachment, entry to the cell, viral uncoating, and rna and protein synthesis [47] . table 1 shows how intervention strategies can be targeted to various steps in the infective process. hrv has the icosahedrally shaped capsid formed by 60 identical copies of viral capsid structural proteins vp1-4. the capsid protects the single-stranded, positive sense rna genome. while hrv-a and -b most often induce a self-limited upper respiratory infection, the recently discovered hrv-c was associated with slris in infants, bronchiolitis, and asthma exacerbations in children [170, 171] . prevention of attachment, entry and uncoating hrv deposits on nasal or conjunctival mucosa and is transported to the posterior nasopharynx by mucociliary action of epithelial cells [172] . the so-called major group of hrv uses intercellular adhesion molecule-1 (icam-1) as their receptor [173] and the minor group attach to low density lipoprotein (ldl) receptor and very-ldl (vldl) receptors on epithelial cells in the adenoid area to bind and enter cells [174, 175] . viral attachment can be prevented by specific anti-hrv neutralizing antibodies, anti-receptor antibodies and soluble receptor molecules. endothelial cells express histamine receptors and increased adhesion molecule expression, such as icam-1, was demonstrated by histamine infusion. second-generation h1-antihistamines decrease expression of icam-1 on cultured bronchial epithelial cells [176] . zinc may also act as an antiviral agent by reducing icam-1 levels [177] . the monoclonal antibody to the cellular icam-1 was not effective. cfy196 (coldsol) is a nasal spray multivalent fab fusion proteins against icam-1 with a better avidity and in vitro potency against hrv [178] . tremacamra, a soluble intercellular adhesion molecule 1 reduced the severity of experimental rhinovirus infection [179] . pleconaril, an orally administered antiviral drug, acts by binding to a hydrophobic pocket in viral protein 1, and stabilizes the protein capsid so that the virus cannot release its rna genome into the target cell. outcomes of clinical trials with pleconaril have revealed mixed results and new compounds are currently being developed [180] . despite extensive research, no agent has been approved for prevention and/or therapy of rhinovirus-induced diseases so far. ruprintrivir selectively inhibits hrv 3c protease and shows potent, broad-spectrum anti-hrv activity in vitro. ruprintrivir nasal spray (2% solution) prophylaxis reduced the proportion of subjects with positive viral culture by 26% and reduce viral titers, but did not decrease the frequency of colds [181] . hrv rna synthesis during replication can be blocked by deoxyribozymes [182] , morpholino oligomers [183] , and small interfering ribonucleic acids [184] . the novel antiviral therapies that have been discovered recently, may one day add significantly to the armamentarium of antiviral agents, against respiratory viral infections in asthmatic children. maternally-derived rsv neutralizing antibodies help to protect infants against rsv hospitalization [185] . palivizumab, a humanised monoclonal antibody against the rsv fusion protein is effective against rsv and wheezing in children and reduces hospitalization in high-risk individuals [185, 187] . rsv prophylaxis with palivizumab significantly reduced the relative risk of subsequent recurrent wheezing in nonatopic premature infants [40] . motavizumab is another monoclonal antibody against rsv, with an approximately 20-fold increase in ability to neutralize rsv and 100 fold increase in ability to reduce viral titers compared to palivizumab [188, 189] . motavizumab was also found to be superior to palivizumab in reducing outpatient medically attended lower respiratory illness by 50% [190] . vaccination against hrv and rsv have been in development for quite some time, but there are no safe and effective vaccines at present [33, 191] . high rates of exposure to viruses in early life, presence of more than 100 serotypes of hrv, the presence of maternal antibodies, the risk of vaccine induced disease and relative immaturity of the infant immune system make effective vaccination difficult [186, 192, 193] . respiratory viral infections are major contributors to the global burden imposed by asthma. in early life, they contribute to the inception of asthma and are responsible for most of the acute exacerbations for asthma in childhood. while the debate is not completely settled, children at high risk of developing asthma and those with established asthma may be at increased risk of acquiring respiratory viral infections and may be less able to contain these to the upper airway. several simple general strategies can be used to help prevent respiratory viral infections in asthmatic children (table 2) , with good personal hygiene, hand-washing and avoidance of cigarette smoke likely to reduce respiratory viral infections. general immuno-stimulatory strategies, such as eating a healthy balanced diet, active probiotic supplements and bacterial-derived products, e.g. om-85, may reduce recurrent infections in susceptible children. while research continues on specific anti-viral therapies, including vaccination, there are no currently available practical therapies that are suitable for widespread use. the role of preventative strategies in primary prevention of asthma in high risk children is of considerable academic interest and a number of studies are currently in the pipeline. the results are awaited with interest. the authors declare they have no competing interests. author's contribution ha and pds conceived and designed the review. all authors reviewed the articles, abstracted data, and participated in the data synthesis. ha, pds, ysc drafted the current manuscript, with critical review by pds and cmj. all authors contributed, read and approved the final manuscript. the global burden of asthma: executive summary of the gina dissemination committee report respiratory syncytial virus in early life and risk of wheeze and allergy by age 13 years respiratory syncytial virus bronchiolitis in infancy is an important risk factor for asthma and allergy at age 7 early-life respiratory viral infections, atopic sensitization, and risk of subsequent development of persistent asthma do early-life viral infections cause asthma? rhinovirus illnesses during infancy predict subsequent childhood wheezing wheezing rhinovirus illnesses in early life predict asthma development in high-risk children respiratory viruses and exacerbations of asthma in adults how viral infections cause exacerbation of airway diseases persistence of rhinovirus rna after asthma exacerbation in children study of modifiable risk factors for asthma exacerbations: virus infection and allergen exposure increase the risk of asthma hospital admissions in children viral infections in relation to age, atopy, and season of admission among children hospitalized for wheezing neutrophil degranulation and cell lysis is associated with clinical severity in virus-induced asthma immune pathways for translating viral infection into chronic airway disease host epithelial-viral interactions as cause and cure for asthma the epithelium takes centre stage in asthma and atopic dermatitis the sentinel role of the airway epithelium in asthma pathogenesis sensing the outside world: tslp regulates barrier immunity lower airways inflammation during rhinovirus colds in normal and in asthmatic subjects rhinovirus-induced modulation of gene expression in bronchial epithelial cells from subjects with asthma asthmatic bronchial epithelial cells have a deficient innate immune response to infection with rhinovirus role of deficient type iii interferon-lambda production in asthma exacerbations counterregulation between the fcepsilonri pathway and antiviral responses in human plasmacytoid dendritic cells tlr4 polymorphisms mediate impaired responses to respiratory syncytial virus and lipopolysaccharide interaction between adaptive and innate immune pathways in the pathogenesis of atopic asthma: operation of a lung/ bone marrow axis antibacterial antibody responses associated with the development of asthma in house dust mite-sensitised and non-sensitised children asthma exacerbations: origin, effect, and prevention severe exacerbations and decline in lung function in asthma relationship between increased airway responsiveness and asthma severity in the childhood asthma management program understanding the september asthma epidemic predicting asthma exacerbations in children role of viral respiratory infections in asthma and asthma exacerbations the role of viruses in acute exacerbations of asthma asthma exacerbations: origin, effect, and prevention the september epidemic of asthma exacerbations in children: a search for etiology viruses and asthma evidence for a causal relationship between respiratory syncytial virus infection and asthma rhinovirus and the initiation of asthma evidence of a causal role of winter virus infection during infancy in early childhood asthma palivizumab long-term respiratory outcomes study g: the effect of respiratory syncytial virus on subsequent recurrent wheezing in atopic and nonatopic children contemporaneous maturation of immunologic and respiratory functions during early childhood: implications for development of asthma prevention strategies the role of rhinovirus infections in the development of early childhood asthma the emerging relationship between the airway microbiota and chronic respiratory disease: clinical implications microbes and asthma: the missing cellular and molecular links association of bacteria and viruses with wheezy episodes in young children: prospective birth cohort study incidence of bacterial coinfection with respiratory syncytial virus bronchopulmonary infection in pediatric inpatients the human rhinovirus: human-pathological impact, mechanisms of antirhinoviral agents, and strategies for their discovery rhinovirus transmission within families with children: incidence of symptomatic and asymptomatic infections persistence of rhinovirus and enterovirus rna after acute respiratory illness in children the common cold and decongestant therapy physical interventions to interrupt or reduce the spread of respiratory viruses back to the future: rising to the semmelweis challenge in hand hygiene effect of handwashing on child health: a randomised controlled trial illness transmission in the home: a possible role for alcohol-based hand gels a randomized, controlled trial of a multifaceted intervention including alcohol-based hand sanitizer and hand-hygiene education to reduce illness transmission in the home scheduled hand washing in an elementary school population effectiveness of a training program in reducing infections in toddlers attending day care centers effect of infection control measures on the frequency of upper respiratory infection in child care: a randomized, controlled trial effects of hand hygiene campaigns on incidence of laboratoryconfirmed influenza and absenteeism in schoolchildren a randomized trial of the efficacy of hand disinfection for prevention of rhinovirus infection. clinical infectious diseases: an official publication of the infectious diseases society of america face masks to prevent transmission of influenza virus: a systematic review the antiinflammatory effects of exercise: mechanisms and implications for the prevention and treatment of disease moderate to vigorous physical activity and risk of upper-respiratory tract infection upper respiratory tract infection is reduced in physically fit and active adults mucosal iga and urti in american college football players: a year longitudinal study physical activity and exercise in asthma: relevance to etiology and treatment respiratory infection risk in athletes: association with antigen-stimulated il-10 production and salivary iga secretion meditation or exercise for preventing acute respiratory infection: a randomized controlled trial serum vitamin d levels and markers of severity of childhood asthma in costa rica serum vitamin d levels and severe asthma exacerbations in the childhood asthma management program study influence of dietary components on regulatory t cells zinc for the common cold garlic for the common cold echinacea for preventing and treating the common cold north american (panax quinquefolius) and asian ginseng (panax ginseng) preparations for prevention of the common cold in healthy adults: a systematic review a medical nutrition therapy primer for childhood asthma: current and emerging perspectives summary of the 2008 bts/sign british guideline on the management of asthma an evaluation of echinacea angustifolia in experimental rhinovirus infections vitamin c for preventing and treating the common cold breastfeeding: maintaining an irreplaceable immunological resource breastfeeding and otitis media: a review of recent evidence diet for the prevention of asthma and allergies in early childhood: much ado about something? sleep and immune function effects of sleep and circadian rhythm on the human immune system sleep habits and susceptibility to the common cold a prospective study of sleep duration and pneumonia risk in women second hand smoke exposure in children: environmental factors, physiological effects, and interventions within pediatrics smoking and the outcome of infection air pollution and respiratory viral infection the response of children with asthma to ambient particulate is modified by tobacco smoke exposure meta-analysis on the association between environmental tobacco smoke (ets) exposure and the prevalence of lower respiratory tract infection in early childhood association between cigarette smoking and acute respiratory tract illness in young adults the relationship of rhinovirus-associated asthma hospitalizations with inhaled corticosteroids and smoking urinary leukotriene e levels identify children with tobacco smoke exposure at risk for asthma exacerbation probiotics: what are they? what are their effects on gut physiology? metchnikoff and the centenary of probiotics: an update of their use in gastroenteric pathology during the age of development microbial manipulation of immune function for asthma prevention: inferences from clinical trials current concepts of the intestinal microbiota and the pathogenesis of infection gershwin me: probiotics and immunity probiotics for the treatment of allergic rhinitis and asthma: systematic review of randomized controlled trials a core gut microbiome in obese and lean twins reduced diversity of the intestinal microbiota during infancy is associated with increased risk of allergic disease at school age the gut microbiota shapes intestinal immune responses during health and disease factors influencing the composition of the intestinal microbiota in early infancy establishment of the gut microbiota in western infants exposure to environmental microorganisms and childhood asthma gut microbiota, probiotics, and vitamin d: interrelated exposures influencing allergy, asthma, and obesity? use of antibacterials in infancy: clinical implications for childhood asthma and allergies probiotics and lung diseases influence of gastrointestinal commensal bacteria on the immune responses that mediate allergy and asthma influence of dietary components on regulatory t cells a comparison between traditional yogurt and probiotic yogurt in noninflammatory acute gastroenteritis probiotics for the prevention of pediatric antibiotic-associated diarrhea a differential effect of 2 probiotics in the prevention of eczema and atopy: a double-blind, randomized, placebo-controlled trial effect of a new synbiotic mixture on atopic dermatitis in children: a randomized-controlled trial probiotics for allergic diseases: realities and myths effect of intranasal administration of lactobacillus pentosus s-pt84 on influenza virus infection in mice lactobacillus gg in the prevention of gastrointestinal and respiratory tract infections in children who attend day care centers: a randomized, double-blind, placebo-controlled trial probiotic prophylaxis of ventilatorassociated pneumonia a blinded, randomized, controlled trial effect of long term consumption of probiotic milk on infections in children attending day care centres: double blind, randomised trial lactobacillus gg in the prevention of nosocomial gastrointestinal and respiratory tract infections probiotic bacteria reduced duration and severity but not the incidence of common cold episodes in a double blind, randomized probiotic effects on cold and influenza-like symptom incidence and duration in children effect of a dietary supplement containing probiotic bacteria plus vitamins and minerals on common cold infections and cellular immune parameters probiotics for preventing acute upper respiratory tract infections reducing the risk of infection in the elderly by dietary intake of yoghurt fermented with lactobacillus delbrueckii ssp. bulgaricus oll1073r-1 immunologic effects of yogurt use of a fermented dairy probiotic drink containing lactobacillus casei (dn-114 001) to decrease the rate of illness in kids: the drink study. a patient-oriented, double-blind, cluster-randomized, placebo-controlled, clinical trial effect of long-term continuous consumption of fermented milk containing probiotic bacteria on mucosal immunity and the activity of peritoneal macrophages influence of nutritional knowledge on perceived healthiness and willingness to try functional foods taxonomy and important features of probiotic microorganisms in food and nutrition prebiotics, immune function, infection and inflammation: a review of the evidence long-term safety and impact on infection rates of postnatal probiotic and prebiotic (synbiotic) treatment: randomized, double-blind, placebo-controlled trial efficacy of probiotic lactobacillus gg on allergic sensitization and asthma in infants at risk immunostimulants for preventing respiratory tract infection in children can we prevent exacerbations of asthma caused by common cold viruses? om-85 bv, an immunostimulant in pediatric recurrent respiratory tract infections: a systematic review the immunostimulant om-85 bv prevents wheezing attacks in preschool children boosting airway t-regulatory cells by gastrointestinal stimulation as a strategy for asthma control bacterial immunostimulantsmechanism of action and clinical application in respiratory diseases epithelium dysfunction in asthma role of rhinovirus infections in asthma il-28a (ifn-lambda2) modulates lung dc function to promote th1 immune skewing and suppress allergic airway disease intranasal interferon-alpha-2 for prevention of natural rhinovirus colds interferon-beta-ser as prophylaxis against experimental rhinovirus infection in volunteers an effective dosage regimen for prophylaxis against rhinovirus infection by intranasal administration of huifn-alpha-2 a mechanistic role for type iii interferon-lambda1 in asthma exacerbations mediated by human rhinoviruses interferon-lambda as a new approach for treatment of allergic asthma? vitamin d deficiency high prevalence of vitamin d inadequacy and implications for health vitamin d and asthma vitamin d recommendations: beyond deficiency induction of cathelicidin in normal and cf bronchial epithelial cells by 1,25-dihydroxyvitamin d(3) respiratory epithelial cells convert inactive vitamin d to its active form: potential effects on host defense potential mechanisms for the hypothesized link between sunshine, vitamin d, and food allergy in children regional differences in epipen prescriptions in the united states: the potential role of vitamin d prevalence of eczema and food allergy is associated with latitude in australia vitamin d, respiratory infections, and asthma relationship between serum 25-hydroxyvitamin d and pulmonary function in the third national health and nutrition examination survey low serum 25-hydroxyvitamin d levels are associated with increased risk of viral coinfections in wheezing children vitamin d supplementation in children may prevent asthma exacerbation triggered by acute respiratory infection macrolide antibiotics in the treatment of asthma. an update anti-inflammatory effects of macrolides in lung disease macrolides in the treatment of asthma azithromycin inhibits il-5 production of t helper type 2 cells from asthmatic children macrolide antibiotics and asthma treatment azithromycin induces anti-viral responses in bronchial epithelial cells macrolide antibiotics inhibit respiratory syncytial virus infection in human airway epithelial cells clarithromycin in the treatment of rsv bronchiolitis: a double-blind, randomised, placebo-controlled trial a novel group of rhinoviruses is associated with asthma hospitalizations novel human rhinoviruses and exacerbation of asthma in children incubation periods of experimental rhinovirus infection and illness. clinical infectious diseases: an official publication of the infectious diseases society of america a cell adhesion molecule, icam-1, is the major surface receptor for rhinoviruses members of the low density lipoprotein receptor family mediate cell entry of a minor-group common cold virus multiple receptors involved in human rhinovirus attachment to live cells levocetirizine inhibits rhinovirus-induced icam-1 and cytokine expression and viral replication in airway epithelial cells duration and severity of symptoms and levels of plasma interleukin-1 receptor antagonist, soluble tumor necrosis factor receptor, and adhesion molecules in patients with common cold treated with zinc acetate viral receptor blockage by multivalent recombinant antibody fusion proteins: inhibiting human rhinovirus (hrv) infection with cfy196 efficacy of tremacamra, a soluble intercellular adhesion molecule 1, for experimental rhinovirus infection -a randomized clinical trial combating enterovirus replication: stateof-the-art on antiviral research phase ii, randomized, double-blind, placebo-controlled studies of ruprintrivir nasal spray 2-percent suspension for prevention and treatment of experimentally induced rhinovirus colds in healthy volunteers gaining target access for deoxyribozymes a morpholino oligomer targeting highly conserved internal ribosome entry site sequence is able to inhibit multiple species of picornavirus small interfering rna molecules as potential anti-human rhinovirus agents: in vitro potency, specificity, and mechanism respiratory syncytial virus neutralizing antibodies in cord blood, respiratory syncytial virus hospitalization, and recurrent wheeze understanding the mechanisms of viral induced asthma: new therapeutic directions palivizumab prophylaxis, respiratory syncytial virus, and subsequent recurrent wheezing development of motavizumab, an ultra-potent antibody for the prevention of respiratory syncytial virus infection in the upper and lower respiratory tract a review of palivizumab and emerging therapies for respiratory syncytial virus motavizumab for prophylaxis of respiratory syncytial virus in high-risk children: a noninferiority trial vaccination for asthma exacerbations viral respiratory tract infections and asthma: the course ahead association between human rhinovirus c and severity of acute asthma in children submit your next manuscript to biomed central and take full advantage of: • convenient online submission • thorough peer review • no space constraints or color figure charges • immediate publication on acceptance • inclusion in pubmed, cas, scopus and google scholar • research which is freely available for redistribution the authors wish to acknowledge dr. catherine gangell for reviewing the manuscript. key: cord-335567-ssnvr6nj authors: berry, michael; gamieldien, junaid; fielding, burtram c. title: identification of new respiratory viruses in the new millennium date: 2015-03-06 journal: viruses doi: 10.3390/v7030996 sha: doc_id: 335567 cord_uid: ssnvr6nj the rapid advancement of molecular tools in the past 15 years has allowed for the retrospective discovery of several new respiratory viruses as well as the characterization of novel emergent strains. the inability to characterize the etiological origins of respiratory conditions, particularly in children, led several researchers to pursue the discovery of the underlying etiology of disease. in 2001, this led to the discovery of human metapneumovirus (hmpv) and soon following that the outbreak of severe acute respiratory syndrome coronavirus (sars-cov) promoted an increased interest in coronavirology and the latter discovery of human coronavirus (hcov) nl63 and hcov-hku1. human bocavirus, with its four separate lineages, discovered in 2005, has been linked to acute respiratory tract infections and gastrointestinal complications. middle east respiratory syndrome coronavirus (mers-cov) represents the most recent outbreak of a completely novel respiratory virus, which occurred in saudi arabia in 2012 and presents a significant threat to human health. this review will detail the most current clinical and epidemiological findings to all respiratory viruses discovered since 2001. viral infections of the upper and lower respiratory tract are among the most common illness in humans. children and infants bear the major burden of infection, typically presenting with 5 to 6 episodes annually [1] . these infections are often associated with significant patient morbidity and related mortality. for this reason, urtis and lrtis represents the leading cause of death in children younger than five years of age worldwide [2, 3] ; this accounts for approximately 4 million deaths annually [4] . acute respiratory tract disease is the leading cause of hospitalization in children and febrile episodes in infants younger than three months of age [5, 6] . bacteria only represent approximately 10% of all upper respiratory tract infections with the subsequent 90% of infections caused by respiratory viruses [7] . despite the viral aetiological origin of most respiratory infections, antibiotics are often prescribed in the treatment of such diseases [8] , exacerbating antibiotic abuse. the morbidity and fiscal implications associated with respiratory infections are significant, with approximately 500 million cases reported in the united states alone each year with subsequent direct and indirect costs to the us economy estimated at $40 billion annually [2] . the burden of respiratory tract infections is increased in patients with chronic comorbidities or clinical risk factors including asthma [9] , chronic obstructive pulmonary disease (copd) [10] , young, elderly [11] and immunocompromised [12, 13] . the viruses primarily associated with upper respiratory tract infections commonly include rhinoviruses, enteroviruses, adenoviruses, parainfluenza viruses (piv), influenza viruses, respiratory syncytial viruses (rsv) and coronaviruses [3, 8, 14, 15] . in recent years six new human respiratory viruses have been reported including human metapneumovirus (hmpv) [16] , bocavirus and four new human coronaviruses including severe acute respiratory syndrome coronavirus (sars-cov), human coronavirus nl63 (hcov-nl63), hcov-hku1 and middle east respiratory syndrome coronavirus (mers-cov). this review will detail these newly discovered and emerging respiratory viruses. coronaviruses affect a diverse group of animal hosts, and cause a plethora of diseases in animals including progressive peritonitis, acute and chronic hepatitis, gastroenteritis, nephritis, and encephalitis [17] . in humans coronavirus infection results in respiratory tract complications with varying degree of severity and have been associated with gastroenteritis. four human coronaviruses (hcov-229e, hcov-oc43, hcov-nl63 and hcov-hku1) are endemic in the human population and are mainly associated with mild, self-limiting respiratory illnesses. another two human coronaviruses, namely sars-cov and mers-cov cause severe respiratory syndromes and present a significant threat with their high fatality rates. the first human coronaviruses were identified in the 1960s by tyrell and bynoe who passaged a virus, named b814, in human embryonic tracheal organ cultures. when this virus was inoculated intranasally into human volunteers, common cold-like symptoms were produced [18, 19] . hamre and procknow (1966) later isolated a virus, which they were able to grow in tissue culture, from subjects presenting with symptoms of the common cold. the virus was later named human coronavirus 229e (hcov-229e) [20] . seroepidemiological studies have shown that 40% of wild animal traders and 20% of people responsible for the slaughtering of animals in the region where human sars was thought to originate, were seropositive for sars-cov, although all cases were asymptomatic. this indicates that these people were previously exposed to a sars-like-cov, which resulted in asymptomatic infection [38] . sars presents as an atypical pneumonia [42, 43] , with pneumocytes being the primary target of infection. infection results in haemorrhagic inflammation in most pulmonary alveoli with alveolar thickening, diffuse alveolar damage, desquamation of pneumocytes, formation of hyaline membranes and multinucleated pneumocytes with capillary engorgement and microthrombosis [44] . approximately 60% of patients deteriorated in the second week of infection, presenting with persistent fever, dyspnoea and oxygen desaturation [29] . approximately 20%-30% of patients were subsequently admitted to intensive care, where mechanical ventilation was necessary [45] . a surprising finding with the sars outbreak was that it was not as great a threat to infants and children [33, 46] . clinical presentation was less severe in infants and no children aged between 1 and 12 required intensive care or mechanical ventilation [47, 48] . this is in sharp contrast to the age related burden of other respiratory infections and the underlying biological mechanism remains unclear [49] . a family of viruses that were previously understood to cause mild, self-limiting upper respiratory tract infections was showcased by the sars-cov outbreak to be a significant threat to global public health. the economic losses brought on by the sars pandemic was estimated to be in the region 40 billion dollars [50] with hong kong bearing a significant proportion of the losses. tourism, entertainment and restaurant industries in the area recorded up to 80% loss in business. the pressure on healthcare facilities in affected areas was substantially exacerbated by the spread to healthcare workers. several hospitals were forced to close to new admissions as large numbers of staff became infected with sars-cov. to view this as an isolated incidence would be naï ve and the potential for the emergence and re-emergence of new and existing infectious agents poses a probable risk. understanding the sars-cov outbreak has provided immense knowledge and an excellent model to replicate in the event of further outbreaks of communicable diseases. in 2003 a 7 month old child presenting with bronchiolitis and conjunctivitis was screened for several respiratory viruses to identify the causative agent, with all diagnostics yielding negative results. the group led by lia van der hoek then used a modified cdna amplified restriction fragment-length polymorphism (cdna-aflp) technique (virus-discovery-cdna-aflp or vidisca), to identify the causative agent. briefly, the technique utilizes reverse transcription-pcr of viral rna with subsequent restriction digest of the cdna using frequently cutting restriction enzymes. since the restriction sites are selected and therefore known, the resultant "sticky ends" can be ligated into anchors for amplification and sequencing with specific primers. the results showed highest sequence similarities with known coronaviruses, but with significant sequence divergence indicating the discovery of a new coronavirus species, later named human coronavirus nl63 [51] . at about the same time, two other independent groups identified essentially the same virus [52, 53] . shortly after the van der hoek paper [51] , a novel coronavirus that replicated efficiently in tertiary monkey kidney and vero cells, was retrospectively isolated from a nose swab sample collected in 1988 from an 8 month-old boy presenting with pneumonia. this virus was reported to be similar to hcov-nl63 and named hcov-nl [52] . in 2005, also reported the identification of a novel coronavirus isolated in new haven, connecticut, which was named hcov-nh. this novel virus was identified by pcr which was adapted to amplify a conserved region within the replicase 1a or pol gene [53] . subsequent sequence analysis showed that these three viruses were essentially the same virus or variants thereof [53, 54] . it is obvious that hcov-nl63 has been circulating in the human population since before 1988 [52] . in fact, molecular clock analyses have shown that hcov-nl63 and hcov-229e diverged from a most recent common ancestor, in a zoonotic event, approximately 563 to 822 years ago [55] . hcov-nl63 later diverged into two lineages with subsequent recombination of the two lineages during co-infection. this frequent recombination has given hcov-nl63 a mosaic structured genome with multiple recombination sites [56] . a 71 year old male patient from china was admitted to hospital with pneumonia in january 2004. viral cultures, rt-pcr and direct antigen detection from nasopharyngeal aspirate were all negative for respiratory viruses. a pan-coronavirus rt-pcr targeting a conserved region of the pol gene confirmed the presence of a coronavirus however attempts to culture the causative agent were all unsuccessful. sequencing the gene segment amplified by the pan-coronavirus assay indicated a high homology to other viruses of the βcov genus including hcov-oc43 but of novel origin. the human coronavirus, termed hcov-hku1, was later isolated from another female patient. efforts to culture the virus had posed complicated and the complete genome was isolated, amplified and sequenced directly from rna extracted from a nasopharyngeal aspirate [57] . successful propagation of the virus was achieved recently in human ciliated airway epithelial cells [58] but culturing of hcov-hku1 still remains a daunting task. the presence of the he gene further characterizes hcov-hku1 as belonging to the βcov genus. since the discovery of hcov-hku1, its prevalence has shown a global distribution and retrospective analysis on stored nasopharyngeal aspirates have confirmed its existence since 1995 [59] ; however, phylogenetic analysis suggests a much earlier divergence. june 2012 saw the most recent emergence of a completely novel strain of human coronavirus. a sputum sample was collected from a 60 year old male patient presenting with severe respiratory disease in the dr. soliman fakeeh hospital in jeddah, saudi arabia. viral assays frequently used could not identify any aetiological agent responsible for the disease. the sputum sample was sent to dr. ron fouchier at the erasmus medical college in rotterdam, netherlands, where the virus was identified as a novel coronavirus, provisionally termed hcov-emc (human coronavirus erasmus medical college). the patient later succumbed to the disease with acute pneumonia and subsequent renal failure [60] . a retrospective study further traced the virus back to april 2012 where an outbreak of pneumonia, resulting in two fatalities, occurred in health care workers in an intensive care unit in zarqa, jordan [61] . since the initial discovery, several new isolates were identified and described in scientific literature, databases and media under various names. this provoked the convening of the coronavirus study group to introduce a naming convention and avoid confusion within the research field, health care authorities, governments and general public. the name middle east respiratory syndrome coronavirus (mers-cov) was coined and has been widely accepted by the discoverers of the virus and pioneers of the field, the who and saudi ministry of health [61] . primary infections, to which all cases were linked directly or indirectly, occurred in middle eastern countries including saudi arabia, qatar, jordan, oman and united arab emirates and subsequently spread to united kingdom, tunisia, france, italy and germany with egypt and the united states recently reporting their first laboratory confirmed cases [62, 63] . the potential of widespread pandemic outbreak is thought to be low as human-to-human transmission is largely inefficient [64] and only reported in close, sustained contact [65] such as within families [66] , healthcare workers [67] and as nosocomial transmission [68] , especially if the patient represents with comorbidities. there has been no evidence to suggest sustained community transmission [62] . the ever increasing case numbers and countries affected by the disease however suggests mers-cov does represent potential for widespread, global outbreak [69] . from the first reported case in june 2012, until 7 february 2014, a total of 182 cases had been reported with 79 fatalities. according to the latest figures available from the who, as of 11 june 2014, the virus had resulted in a total of 699 laboratory confirmed cases with a total of 209 fatalities [70] . these statistics indicate that within a 4 month period case numbers had more than trebled suggesting the virus is far from controlled. the fatality rate of 30% is also substantially increased in patients with comorbidities, with a large number of reported cases being identified in immunocompromised patients or those with underlying disease [68, 71] and a severe threat of nosocomial transmission has been demonstrated [72] . clinical presentation is similar to sars and includes a spectrum of respiratory diseases with the most common symptoms including cough, fever and gastrointestinal symptoms [69] before progressing to pneumonia [68] . acute respiratory distress syndrome (ards), renal failure, pericarditis and disseminated intravascular coagulation have also been reported [69] . the potential of widespread pandemic outbreak is thought to be low as human-to-human transmission is largely inefficient [64] and only reported in close, sustained contact [65] such as within families [66] , healthcare workers [67] and as nosocomial transmission [68] , especially if the patient presents with comorbidities. there has been no evidence to suggest sustained community transmission [62] . the ever increasing case numbers and countries affected by the disease however suggests that mers-cov does represent potential for widespread, global outbreak [69] . the inefficient spread between infected patients strongly suggests zoonotic transmission, however over two years on from the initial discovery of mers-cov it is still unclear where it originated from and how it infects humans. during the outbreak of sars-cov the identification of the wet markets of china as the probable source greatly contributed to the control of the disease [38] . this highlights the importance in identifying the origins of disease outbreak. phylogenetic analysis places it in the same genus as sars-cov but within a new lineage, 2c. the closest detectable relatives suggests origins from bat coronavirus species with relative high sequence homology between bat-cov hku4 and hku5 in china [73] , vm314 in the netherlands [74] and a recently discovered isolate from south africa [75] . assuming that bats also form the immediate source is improbable and as with sars-cov an intermediate animal host species for transmission to humans is of greater likelihood [61] . high levels of neutralizing antibodies, viral rna and infectious viruses have been discovered in dromedary camels suggesting a potential for camels to form the source for human transmission [76] [77] [78] [79] . a recent study identified identical single nucleotide polymorphisms in a sequence of mers-cov isolated from an infected patient and camel, which was cared for by the patient. phylogenetic analysis of these two sequences supported the conclusion that transmission occurred between patient and camel, however the direction of transmission could not be confirmed [80] . it has since been proven that a patient who succumbed to mers-cov infection obtained the virus directly from his camel herd. the direction of transmission was confirmed as camel-to-human by serological analysis [81] . these facts strongly contribute to an increasingly popular hypothesis that mers-cov infections in humans are directly acquired from camels, however very few patients have had reported contact with camels. many residents of the arabian peninsula frequently consume unpasteurized camel milk and this has been suggested as a potential source as the virus has been detected and can remain viable for prolonged periods in milk [82, 83] . the who, saudi arabia and qatar have recently issued warnings and recommended against consuming unpasteurized camel milk and to be cautious when interacting with dromedary camels [84] . the outbreak of sars-cov in 2003 and mers-cov less than 10 years later highlights the significant threat of coronaviruses to humans and confirms that the sars outbreak was not an isolated incident. with the ever increasing diversity of animal coronavirus species, especially within bats, the likelihood of recombination leading to future outbreaks is high and the threat of potential pandemics is real as highly pathogenic coronaviruses continue to spill over from zoonotic sources into the human population. misdiagnosis of these outbreaks pose a further substantial threat to healthcare workers with nosocomial spread to other patients putting further pressure on an already strained healthcare system. understanding the dynamics and molecular characteristics of human coronaviruses currently in circulation and how they emerge, infect and cause disease, we can be better prepared for future pandemics allowing for improved response, management and treatment of related conditions. the clinical presentation of human coronaviruses clearly follows two distinct lines of progression. sars-cov and mers-cov present with severe respiratory complications and often multisystem involvement with renal failure and enteric symptoms. the high fatality rates associated with these infections is not reflected in the remaining four human coronaviruses, hcov-229e, hcov-oc43, hcov-nl63 and hcov-hku1. the clinical importance of sars-cov and mers-cov are evident and discussed in detail in previous sections allowing for the presentation of the involvement of the remaining non-severe human coronaviruses and there implications in human health. the clinical presentation of these four non-severe human coronaviruses is largely identical and indistinguishable symptomatically, commonly presenting with rhinorrhoea, sore throat, cough and fever [85, 86] . majority of infections are associated with self-limiting upper respiratory tract disease or "the common cold" but can also present with high morbidity outcomes of the lower respiratory tract including bronchiolitis, pneumonia, [87] [88] [89] , asthmatic exacerbations [9] acute exacerbations of chronic obstructive pulmonary disease (copd) [10] and croup in hcov-nl63 infected patients [90] . a report by esper et al. found a correlation between hcov-nl63 infections and kawasaki disease [91] , although other studies could not replicate the association [92] [93] [94] . febrile seizures have been reported for most human coronaviruses but the significance of hcov-hku1 is alarming with one study indicating that 50% of patients infected with hcov-hku1 experience febrile seizures [9] . human coronaviruses affect all age groups [85, 86] but elicit more serious disease in young, elderly and immunecompromized [87, 95, 96] , frequently resulting in hospitalization. reports on the prevalence of human coronaviruses and their association with upper and lower respiratory tract disease vary but range between 3.3% and 16% [85, 86, 95, 97] . over 70% of the general public has seroconverted towards all four non-severe human coronaviruses with primary infection shown to occur in childhood [98] and reinfection occurring throughout life [95] . given the high prevalence of respiratory infections, human coronaviruses represent a substantial disease burden which is exacerbated by the high implications of healthcare workers in coronavirus outbreaks [97] . high rates of co-infection with other respiratory viruses are commonly reported [85, 86, 95, 99] . viruses frequently associated with co-infection include enterovirus, rhinovirus and piv [100] however reports of co-infection with two human coronaviruses are limited. dijkman et al. recently demonstrated that hcov-oc43 and hcov-nl63 may elicit immunity that protects against hcov-hku1 and hcov-229e, respectively [101] . clinical progression and outcomes of disease in patients presenting with co-infection are however similar to patients presenting with mono-infection [85, 95] . there is also no substantial difference in coronaviral load between co-infected and mono-infected patients. no substantial difference in disease progression in co-infected versus mono-infected patients has been reported and therefore understood to have little impact; however, the role in facilitation of infection of one respiratory virus by another is still speculative [95] . all four human coronaviruses are endemic worldwide but the prevalence, regional distribution and pathogenicity of individual human coronaviruses is unclear and highly subjective on study conducted with parameters including population sampled, respiratory sample collected, sensitivity of diagnostic assay used, region where study was conducted and duration of the study playing a substantial role in epidemiological findings. a common finding in majority of studies conducted is the increased prevalence of human coronaviruses during winter months in temperate climates [95, 97] . however even this has shown to be geographically dependent with a spring/summer predominance in subtropical and tropical climates [9, 102] . biennial outbreaks are frequently reported [85, 95, 99, 100] for all strains of non-severe human coronaviruses. in 2001 a previously undiscovered virus was identified in 28 epidemiologically distinct patients in the netherlands. patient symptoms were similar to those infected with rsv and, several patients required hospitalization and mechanical ventilation. viral isolates were cultured in tertiary monkey kidney (tmk) cells and cytopathic effects caused by the virus were largely identical to those caused by rsv. electron microscopy of infected cell supernatants revealed paramyxovirus-like particles; however, rt-pcr assays to detect known paramyxoviruses were all negative. the low stringency of the assays used indicated a currently unknown, genetically distinct virus. a rap-pcr assay was then utilized to obtain sequence information of the unknown virus and fragments amplified by the rap-pcr allowed for further sequencing of the 3'-end of the genome. based on the sequence homology and gene organization, the unidentified virus displayed closest homology with avian pneumovirus, but to be a tentative new member of the metapneumovirus genus and the first virus in the genus to infect humans, provisionally termed human metapneumovirus (hmpv) [16] . symptomatic differentiation between hmpv and other respiratory viruses cannot be made as there is a significant overlap in clinical presentation [103, 104] . the most common presentation of hmpv in children includes complications of the upper respiratory tract with rhinorrhoea, cough and fever [105] . acute otitis media is also frequently reported [106, 107] and conjunctivitis, rash, diarrhea and vomiting are reported but infrequently [103] . bronchiolitis, pneumonia, croup and asthmatic exacerbations are the most frequently associated lower respiratory tract complications [108] and viral load is directly associated with disease severity [109] . hmpv infection in the young and elderly frequently requires hospitalization and fatalities have been reported in the elderly [110, 111] . an increased morbidity in elderly patients with a delayed clearance of symptoms has been reported and is likely related to the age related impairment of the innate and adaptive immunity [103] or an over stimulated immune response leading to inflammation [112] . elderly patients requiring hospitalization most frequently present with acute bronchitis, copd exacerbations, pneumonia and congestive heart failure [113] . in healthy adults asymptomatic infections or cold-and flu-like symptoms are the most prevalent presentation [114] . the pathogenesis of hmpv infection is strongly affected by bacterial coinfections with pneumococcus. one study has shown that administration of a conjugate pneumococcal vaccine is sufficient to reduce the incidence of hmpv infection of the lower respiratory tract and the incidence of clinical pneumonia in both hiv positive and negative patients [115] . these finding suggest that the incidence of hospitalizations in hmpv infections may be decreased by vaccination with a conjugate pneumococcal vaccine. another case report of severe respiratory failure was found to be caused by coinfection with hmpv and streptococcus pneumonia in a 64 year old patient [116] . both in vitro and in vivo studies have shown that infection with hmpv facilitates adhesion of pneumococcal bacteria, which may provide an explanation for the coinfection with pneumococcal strains and hmpv [117] . viral coinfections between hmpv and rsv have been reported, but remain a contentious issue. the typical seasonal overlap of the two viruses has been suggested to promote viral coinfection. one study reported a 10-fold increase in risk of admission to an intensive care unit in pediatric patients coinfected with rsv and hmpv and associated the dual infection as capable of augmenting severe bronchiolitis [118] . other studies do not support this finding and further report a decreased correlation between hmpv-rsv coinfections and hospitalization and additionally lists dual infection, along with breastfeeding, as having protective effects [119] . although hmpv was only discovered in 2001, it has been shown by phylogenetic analysis to have been in existence for approximately 50 years [120, 121] . soon after the discovery of hmpv, it was evident that two lineages, a and b, existed. these two lineages were further subdivided into two sublineages per lineage, a1-a2 and b1-b2 [16] . a recent report analyzing sequence divergence of the attachment and fusion surface glycoproteins indicates the presence of five sublineages, namely a1, a2a, a2b, b1 and b2 [122] . from long term retrospective studies it was evident that these lineages are not restricted to certain locations or times and that multiple lineages can exist in the same location and period [108, 123] . it has also become evident that old sublineages may be replaced by new variants [103] . disease progression or varying clinical outcomes related to different lineages of hmpv has become a contentious issue. several studies have reported that lineage a presents with more severe clinical outcomes [124] [125] [126] where the same is reported for lineage b by other groups [127, 128] . it has been further reported that there is no difference in disease outcomes related to the two lineages [108, 129, 130] . between 7% and 19% of all cases of respiratory infections in children are caused by hmpv, in both hospitalized and outpatients [108, 131, 132] and has been reported to be the second most frequently identified virus in respiratory tract infections [133] . extrapolation of consensus data suggests a total of 20 000 hospital and one million clinic visits annually in the us among children younger than 5 [131] . children hospitalized with hmpv infections are also more likely to present with pneumonia or asthma and required longer stay in intensive care units with supplemental oxygen, when compared to other respiratory viruses [131] . seroprevalence studies indicates that 100% of young adults are seropositive for hmpv with stable neutralizing titres, which further suggests that reinfection occurs throughout life [16] , with a potential for genetic variation between clades promoting reinfection [108] . hmpv has a worldwide distribution and affects all age groups but predominantly affects young, elderly and immunocompromised patients [111] , with children younger than five years of age being most susceptible to infection [134] . children and adults with underlying or chronic conditions such as asthma, chronic lung disease, congenital heart disease, cancer or copd are more likely to be hospitalized with hmpv infection [131] . infection with hmpv occurs throughout the year but seasonal prevalence in late winter and spring has been observed and coincides with the peak of rsv infection [131, [135] [136] [137] . the first human bocavirus (hbov) was discovered in 2005 from nasopharyngeal aspirates of 282 patients with unresolved lower respiratory tract infections in sweden. researchers utilized a novel technique which included steps of dnase treatment to exclude contaminating, or non-viral, nucleic acids followed by pcr amplification by nonspecific primers. the pcr-products were subsequently cloned with large-scale sequencing of the clones. bioinformatic analysis of generated sequence data yielded the discovery of a new parvovirus with a high homology to bovine and canine minute parvoviruses. the genus name bocavirus was in fact derived from the species infected by the known virus strains, namely bovine and canine. the new virus was named hbov1 and was the first virus to be discovered by molecular virus screening [138] . three additional species of hbov were later discovered in 2010 and added to the genus; these were named hbov2, hbov3 and hbov4 [139] [140] [141] . hbov1 is a respiratory pathogen affecting all regions of the globe and is associated with approximately 2%-19% of all upper and lower respiratory tract conditions [142] [143] [144] . hbov1 productively infects human airway epithelium cell cultures and leads to damage of airway epithelial cells [145] [146] [147] , which supports clinical observations that infection does result in respiratory disease. in contrast, hbov2-4, are found in the gastrointestinal tract and hbov2, and possibly hbov3, are associated with gastroenteritis [139, [148] [149] [150] . interestingly, hbov2 is the only enteric bocavirus to be isolated from nasopharygeal aspirates and may, therefore, also be associated with respiratory disease [151, 152] . hbov1 is detected in all age groups, but predominantly in young children between the ages of 6-24 months [143, 153, 154] and is rarely detected in adults [155] [156] [157] [158] [159] . transmission and infection occurs throughout the year, but predominantly during winter and spring months [158, [160] [161] [162] . seroprevalence studies suggest that maternal antibodies, which provide protection, are present in infants younger than 2 months of age [163, 164] , after which seropositivity decreases with low levels of detection until 6-12 months. virtually 100% of children aged 6 are seroconverted for hbov1 and as reinfection occurs throughout life this remains into adulthood [143, [163] [164] [165] [166] . the presence of the three enteric bocaviruses does however complicate the findings of seroconversion as cross-reactivity does exist [166] . as with many respiratory viruses, clinical differentiation with hbov1 infection is not possible by symptomatic presentation [8] . common features of infection of the upper respiratory tract include common cold-like symptoms with cough, rhinorrhoea and acute otitis media [167] . infection of the lower respiratory tract in children is associated with pneumonia, acute wheezing, asthmatic exacerbations and bronchiolitis [160, 162, [168] [169] [170] , but life-threatening complications are rare with hbov1 infection [143] . although hbov1 has been isolated from stool samples, there is no statistical evidence to associate hbov1 with gastrointestinal disease [139] . hbov1 has not only been found in the upper and lower respiratory tract and gastrointestinal specimens, but also in urine samples, serum, saliva, and tonsils [143] . rather than having a role in disease pathogenesis, this viraemia and systemic spread may be a feature common to all parvoviruses as they require proliferating host cells for replication [138] . interestingly, hbov appears to be more than just a respiratory or gastrointestinal virus. in a recent study, hbov was identified in 18.3% of lung (n = 11/60) and 20.5% of colorectal (n = 9/44) tumors screened. unfortunately, the study did not investigate whether the hbov genomes were in fact incorporated into the host genome as reported for other known parvoviruses. therefore, based on their observations as well as previous studies on other parvoviruses, the authors speculate that hbov could contribute to the development of some lung and colorectal tumors. however, they do also acknowledge that these tumors could simply be providing an optimal environment for hbov replication and more conclusive studies are required to resolve this issue [171] . hbov1 has been associated with a prolonged period of persistence in the mucosa of the respiratory tract. this prolonged presence has possibly led to a high frequency of coinfection found with hbov1 infections of the both the upper and lower respiratory tract [142, 157, 158] . the high rate of detection of multiple respiratory viruses within up to 83% of respiratory specimens, and the presence of asymptomatic hbov1 infections, does complicate the determination of the actual pathogenic role of hbov [143] . high viral load is statistically associated with symptoms [172] and this may therefore be a better indication of coinfections which are related to disease severity or symptomatic presentation. it has been further suggested that patients presenting with viraemia are better candidates to assess the symptoms of disease when compared to investigations of respiratory secretions [172] . the effects and mechanisms of latency, persistence, reactivation and reinfection are however poorly understood and therefore its effects on coinfection and its contribution to active disease cannot be accurately stated [173] . the etiological agents of 12%-39% of lower respiratory tract infections still remain to be identified [138, 158] . these results may vary significantly depending on the sensitivity of the diagnostic assay used, respiratory site sampled and even geographical location of the study but it does suggest that many uncharacterized respiratory pathogens could still remain elusive awaiting discovery. the vast improvements in molecular techniques within the past decade have however led to the discovery of four previously circulating respiratory viruses and also the rapid characterization of two completely novel viruses, namely sars-cov and mers-cov. all these viruses have varying but significant impact on human health and the potential for outbreak of completely novel, emergent respiratory viruses, seen with sars and mers, poses their own unique threats. lessons learned from these viruses, and others currently in circulation, provide health care authorities and scientists with suitable expertise and knowledge to rapidly identify and combat novel respiratory viruses and as our techniques improve we will be in a position to characterize those viruses that are currently difficult to isolate and identify. burtram c. fielding receives funding from the national research foundation, south africa. any opinion, findings and conclusions or recommendations expressed in this material are those of the author and therefore the nrf does not accept any liability in regard thereto. mb wrote the earlier drafts of the manuscript. jg edited and reviewed the early drafts of the manuscript. bcf conceptualized the paper and wrote the submission draft of the manuscript. the authors declare no conflict of interest. viral upper respiratory tract infection and otitis media complication in young children the economic burden of non-influenza-related viral respiratory tract infection in the united states the clinical impact of human respiratory virus infections the global burden of disease center for population and development studies: massachusetts serious bacterial infections in febrile infants 1 to 90 days old with and without viral infections children with multiple viral respiratory infections are older than those with single viruses systematic review of the treatment of upper respiratory tract infection viral infections of the lower respiratory tract: old viruses, new viruses, and the role of diagnosis coronavirus hku1 and other coronavirus infections in hong kong human coronavirus and acute respiratory illness in older adults with chronic obstructive pulmonary disease acute viral infections of upper respiratory tract in elderly people living in the community: comparative, prospective, population based study of disease burden respiratory viral infections in immunocompetent and immunocompromised persons community respiratory virus infections in immunocompromised patients with cancer epidemiology of respiratory viruses in bronchoalveolar lavage samples in a tertiary hospital impact of viral infections in children with community-acquired pneumonia: results of a study of 17 respiratory viruses. influenza respir. viruses a newly discovered human pneumovirus isolated from young children with respiratory tract disease coronaviruses in the limelight cultivation of a novel type of common-cold virus in organ cultures cultivation of viruses from a high proportion of patients with colds a new virus isolated from the human respiratory tract recovery in tracheal organ cultures of novel viruses from patients with respiratory disease antigenic relationships amongst coronaviruses antigenic relationships among the coronaviruses of man and between human and animal coronaviruses a major outbreak of severe acute respiratory syndrome in hong kong identification of severe acute respiratory syndrome in canada a cluster of cases of severe acute respiratory syndrome in hong kong identification of a novel coronavirus in patients with severe acute respiratory syndrome a novel coronavirus associated with severe acute respiratory syndrome coronavirus as a possible cause of severe acute respiratory syndrome the severe acute respiratory syndrome coronavirus nucleocapsid protein is phosphorylated and localizes in the cytoplasm by 14-3-3-mediated translocation the genome sequence of the sars-associated coronavirus cross-host evolution of severe acute respiratory syndrome coronavirus in palm civet and human sars molecular epidemiology: a chinese fairy tale of controlling an emerging zoonotic disease in the genomics era severe acute respiratory syndrome (sars) clinical management and infection control of sars: lessons learned the novel human coronaviruses nl63 and hku1 ecoepidemiology and complete genome comparison of different strains of severe acute respiratory syndrome-related rhinolophus bat coronavirus in china reveal bats as a reservoir for acute, self-limiting infection that allows recombination events isolation and characterization of viruses related to the sars coronavirus from animals in southern china severe acute respiratory syndrome coronavirus-like virus in chinese horseshoe bats bats are natural reservoirs of sars-like coronaviruses severe acute respiratory syndrome coronavirus as an agent of emerging and reemerging infection analysis of multimerization of the sars coronavirus nucleocapsid protein detection of antibodies against sars-coronavirus using recombinant truncated nucleocapsid proteins by elisa the sars coronavirus nucleocapsid protein induces actin reorganization and apoptosis in cos-1 cells in the absence of growth factors lung pathology of fatal severe acute respiratory syndrome history and recent advances in coronavirus discovery clinical presentations and outcome of severe acute respiratory syndrome in children severe acute respiratory syndrome among children severe acute respiratory syndrome learning from sars: preparing for the next disease outbreak-workshop summary identification of a new human coronavirus a previously undescribed coronavirus associated with respiratory disease in humans evidence of a novel human coronavirus that is associated with respiratory tract disease in infants and young children one virus, three names, three claims evidence supporting a zoonotic origin of human coronavirus strain nl63 mosaic structure of human coronavirus nl63, one thousand years of evolution characterization and complete genome sequence of a novel coronavirus, coronavirus hku1, from patients with pneumonia culturing the unculturable: human coronavirus hku1 infects, replicates, and produces progeny virions in human ciliated airway epithelial cell cultures coronavirus hku1 in children, brazil isolation of a novel coronavirus from a man with pneumonia in saudi arabia middle east respiratory syndrome coronavirus (mers-cov): announcement of the coronavirus study group advancing priority research on the middle east respiratory syndrome coronavirus world health organisation. global alert and response (gar), coronavirus infections interhuman transmissibility of middle east respiratory syndrome coronavirus: estimation of pandemic risk coronaviruses: important emerging human pathogens family cluster of middle east respiratory syndrome coronavirus infections middle east respiratory syndrome coronavirus infections in health care workers hospital outbreak of middle east respiratory syndrome coronavirus data gaps for laboratory preparedness mers-cov summary updates epidemiological, demographic, and clinical characteristics of 47 cases of middle east respiratory syndrome coronavirus disease from saudi arabia: a descriptive study first cases of middle east respiratory syndrome coronavirus (mers-cov) infections in france, investigations and implications for the prevention of human-to-human transmission full-genome deep sequencing and phylogenetic analysis of novel human betacoronavirus genomic characterization of a newly discovered coronavirus associated with acute respiratory distress syndrome in humans close relative of human middle east respiratory syndrome coronavirus in bat mers coronavirus in dromedary camel herd, saudi arabia geographic distribution of mers coronavirus among dromedary camels middle east respiratory syndrome coronavirus (mers-cov) in dromedary camels isolation of mers coronavirus from dromedary camel human infection with mers coronavirus after exposure to infected camels evidence for camel-to-human transmission of mers coronavirus stability of middle east respiratory syndrome coronavirus in milk middle east respiratory syndrome coronavirus (mers-cov) rna and neutralising antibodies in milk collected according to local customs from dromedary camels reply to "concerns about misinterpretation of recent scientific data implicating dromedary camels in epidemiology of middle east respiratory syndrome (mers characterization of human coronavirus etiology in chinese adults with acute upper respiratory tract infection by real-time rt-pcr assays clinical impact of human coronaviruses 229e and oc43 infection in diverse adult populations coronavirus 229e-related pneumonia in immunocompromised patients clinical and molecular epidemiological features of coronavirus hku1-associated community-acquired pneumonia an outbreak of coronavirus oc43 respiratory infection in normandy, france croup is associated with the novel coronavirus nl63 association between a novel human coronavirus and kawasaki disease lack of association between infection with a novel human coronavirus (hcov), hcov-nh, and kawasaki disease in taiwan kawasaki disease lacks association with human coronavirus nl63 and human bocavirus human coronavirus-nl63 infection is not associated with acute kawasaki disease epidemiology and clinical presentations of the four human coronaviruses 229e, hku1, nl63, and oc43 detected over 3 years using a novel multiplex real-time pcr method genetic variability of human coronavirus oc43-, 229e-, and nl63-like strains and their association with lower respiratory tract infections of hospitalized infants and immunocompromised patients epidemiological and clinical features of human coronavirus infections among different subsets of patients. influenza respir. viruses first infection by all four non-severe acute respiratory syndrome human coronaviruses takes place during childhood high incidence but low burden of coronaviruses and preferential associations between respiratory viruses prevalence of human coronaviruses in adults with acute respiratory tract infections in beijing the dominance of human coronavirus oc43 and nl63 infections in infants human coronaviruses hcov-nl63 and hcov-hku1 in hospitalized children with acute respiratory infections in beijing, china human metapneumovirus: lessons learned over the first decade ten years of human metapneumovirus research comparison of clinical features of pediatric respiratory syncytial virus and human metapneumovirus infections frequency of human metapneumovirus in the upper respiratory tract of children with symptoms of an acute otitis media detection of human metapneumovirus from children with acute otitis media human metapneumovirus and lower respiratory tract disease in otherwise healthy infants and children human metapneumovirus viral load is an important risk factor for disease severity in young children an outbreak of severe respiratory tract infection due to human metapneumovirus in a long-term care facility virological features and clinical manifestations associated with human metapneumovirus: a new paramyxovirus responsible for acute respiratory-tract infections in all age groups aging promotes neutrophil-induced mortality by augmenting il-17 production during viral infection human metapneumovirus infections in adults: another piece of the puzzle human metapneumovirus: a new player among respiratory viruses pneumococcal coinfection with human metapneumovirus severe respiratory failure due to co-infection with human metapneumovirus and streptococcus pneumoniae preceding human metapneumovirus infection increases adherence of streptococcus pneumoniae and severity of murine pneumococcal pneumonia human metapneumovirus in severe respiratory syncytial virus bronchiolitis comparison of risk factors for human metapneumovirus and respiratory syncytial virus disease severity in young children evolutionary dynamics of human and avian metapneumoviruses genetic diversity and evolution of human metapneumovirus fusion protein over twenty years genetic diversity and molecular evolution of the major human metapneumovirus surface glycoproteins over a decade the role of human metapneumovirus in upper respiratory tract infections in children: a 20-year experience genetic variability of human metapneumovirus amongst an all ages population in cambodia between seasonal distribution and phylogenetic analysis of human metapneumovirus among children in osaka city differences in clinical severity between genotype a and genotype b human metapneumovirus infection in children human metapneumovirus genotypes and severity of disease in young children (n = 100) during a 7-year study in dijon hospital, france a 1-year experience with human metapneumovirus in children aged <5 years genetic variability of human metapneumovirus infection: evidence of a shift in viral genotype without a change in illness comparison of human metapneumovirus genotypes from the province of bolzano in northern italy with strains from surrounding regions in italy and austria new vaccine surveillance, n. burden of human metapneumovirus infection in young children dual infection of infants by human metapneumovirus and human respiratory syncytial virus is strongly associated with severe bronchiolitis prevalence and clinical symptoms of human metapneumovirus infection in hospitalized patients clinical impact and diagnosis of human metapneumovirus infection emerging respiratory agents: new viruses for old diseases? dé ry, p. human metapneumovirus infections in hospitalized children human metapneumovirus in the community cloning of a human parvovirus by molecular screening of respiratory tract samples a novel bocavirus associated with acute gastroenteritis in australian children a newly identified bocavirus species in human stool human bocaviruses are highly diverse, dispersed, recombination prone, and prevalent in enteric infections clinical and epidemiologic characteristics of human bocavirus in danish infants: results from a prospective birth cohort study human bocavirus-the first 5 years human bocavirus, a respiratory and enteric virus establishment of a reverse genetics system for studying human bocavirus in human airway epithelia human bocavirus 1 infects commercially available primary human airway epithelium cultures productively human bocavirus can be cultured in differentiated human airway epithelial cells newly recognized bocaviruses (hbov, hbov2) in children and adults with gastrointestinal illness in the united states detection of human bocavirus-2 in children with acute gastroenteritis in south korea human bocavirus species 2 and 3 in brazil novel human bocavirus in children with acute respiratory tract infection human bocavirus 2 in children swiss paediatric respiratory research group. isolation of human bocavirus from swiss infants with respiratory infections human bocavirus: passenger or pathogen in acute respiratory tract infections? human bocavirus infection severe pneumonia and human bocavirus in adult human bocavirus epidemiological profile and clinical associations of human bocavirus and other human parvoviruses human bocavirus infections in hospitalized children and adults human bocavirus: a novel parvovirus epidemiologically associated with pneumonia requiring hospitalization in thailand human bocavirus: prevalence and clinical spectrum at a children's hospital human bocavirus infection in young children in the united states: molecular epidemiological profile and clinical characteristics of a newly emerging respiratory virus seroepidemiology of human bocavirus in hokkaido prefecture seroepidemiology of human bocavirus defined using recombinant virus-like particles correlation between bocavirus infection and humoral response, and co-infection with other respiratory viruses in children with acute respiratory infection seroepidemiology of human bocaviruses 1-4 the human bocaviruses: a review and discussion of their role in infection serodiagnosis of human bocavirus infection detection of human bocavirus in hospitalised children human bocavirus in children suffering from acute lower respiratory tract infection in beijing children's hospital the human bocavirus is associated with some lung and colorectal cancers and persists in solid tumors human bocavirus and acute wheezing in children human bocavirus infections key: cord-352222-zq9o66i4 authors: rajatonirina, soatiana; razanajatovo, norosoa harline; ratsima, elisoa hariniana; orelle, arnaud; ratovoson, rila; andrianirina, zo zafitsara; andriatahina, todisoa; ramparany, lovasoa; herindrainy, perlinot; randrianirina, frédérique; heraud, jean-michel; richard, vincent title: outcome risk factors during respiratory infections in a paediatric ward in antananarivo, madagascar 2010–2012 date: 2013-09-12 journal: plos one doi: 10.1371/journal.pone.0072839 sha: doc_id: 352222 cord_uid: zq9o66i4 background: acute respiratory infections are a leading cause of infectious disease-related morbidity, hospitalisation and mortality among children worldwide, and particularly in developing countries. in these low-income countries, most patients with acute respiratory infection (ari), whether it is mild or severe, are still treated empirically. the aim of the study was to evaluate the risk factors associated with the evolution and outcome of respiratory illnesses in patients aged under 5 years old. materials and methods: we conducted a prospective study in a paediatric ward in antananarivo from november 2010 to july 2012 including patients under 5 years old suffering from respiratory infections. we collected demographic, socio-economic, clinical and epidemiological data, and samples for laboratory analysis. deaths, rapid progression to respiratory distress during hospitalisation, and hospitalisation for more than 10 days were considered as severe outcomes. we used multivariate analysis to study the effects of co-infections. results: from november 2010 to july 2012, a total of 290 patients were enrolled. co-infection was found in 192 patients (70%). co-infections were more frequent in children under 36 months, with a significant difference for the 19–24 month-old group (or: 8.0). sixty-nine percent (230/290) of the patients recovered fully and without any severe outcome during hospitalisation; the outcome was scored as severe for 60 children and nine patients (3%) died. risk factors significantly associated with worsening evolution during hospitalisation (severe outcome) were admission at age under 6 months (or = 5.3), comorbidity (or = 4.6) and low household income (or = 4.1). conclusion: co-mordidity, low-income and age under 6 months increase the risk of severe outcome for children infected by numerous respiratory pathogens. these results highlight the need for implementation of targeted public health policy to reduce the contribution of respiratory diseases to childhood morbidity and mortality in low income countries. respiratory infections are a major cause of infectious diseaserelated morbidity, hospitalisation, and mortality among children under 5 years old worldwide, and particularly in developing countries [1] . these diseases are responsible for 4 million deaths worldwide every year [4] : they are thus among the leading causes of death and the most common infectious cause of death [2] . although many episodes of respiratory infection are mild, 7 to 13 percent are severe enough to warrant hospitalisation [3] . these diseases are responsible for at least 6% of the world's disability and death [1] . it is estimate that there are 33.7 million cases of respiratory infection annually among african children and longitudinal studies suggest that the overall mortality rate among children in developing countries is between 6.6% and 14.1% [5] . despite respiratory infections being a public health priority, data on their epidemiology in africa are sparse. diagnostic methods to identify the pathogen rapidly are not available in lowincomes settings, so most patient with respiratory infections are treated empirically with antibiotics based on local epidemiology [6] . administering appropriate therapy at the appropriate time would improve outcomes. these diseases also have an economic impact, as they lead to substantial consumption of antimicrobial agents in both community and hospital settings. furthermore, the high frequency of respiratory infections and the excessive use of antimicrobial agents are major contributors to the development of antibiotic-resistant pathogens. knowing the local epidemiology of respiratory infections is essential if appropriate empiric therapy is to be prescribed. evaluation of mixed infections and the relative importance of each potential pathogen may also contribute to improving the understanding of the pathogenesis of these infections [7, 8] . our study aimed to evaluate the risk factors associated with the evolution and outcome of respiratory illnesses in patients aged under 5 years old hospitalised in one of the four main public hospitals in antananarivo. the study was conducted at the ''centre hospitalier de soavinandriana'' (cenhosoa) in antananarivo, the capital city of madagascar, which is located in the central highlands. antananarivo consists of administrative, commercial, industrial and residential areas, with patches of agricultural land, mostly rice fields. the city is divided into six administrative districts. in madagascar, hib vaccine was introduced in 2008, and pneumococcia vaccine in 2012 cenhosoa is a national referral hospital in the third district of the urban municipality of antananarivo; hospitalization and care at this hospital is paid for by the patients. it is located near the institut pasteur of madagascar (ipm). the paediatric and neonatology ward of cenhosoa has 54 beds, and the annual number of children hospitalized for respiratory infection was estimated to be 150. a prospective, hospital-based, study was conducted at cen-hosoa from november 2010 to july 2012. the case definition and eligibility criteria used during this study are described in figure 1 . demographic, socio-economic, clinical and epidemiological data were recorded in case report forms (crfs). paediatricians completed the crfs which were then validated by a clinical research officer. instruction sheets were provided to clinical research officers to ensure accurate data collection. the data collected was managed by a senior medical epidemiologist. on admission, trained personnel using standard operating procedures had collected nasal and throat swabs, and sputum sample by aspirating airway secretion from the hypopharynx through the nose or tracheal aspirates. nasopharyngeal secretions, tracheal aspirates and sputum were obtained before start of antibiotic therapy. specimens were transported within 30 minutes of sampling to the ipm for tests of respiratory pathogens. hiv serostatus was not determined; the prevalence of hiv in madagascar is low, estimated to be around 1 per 1000 [7] . the study was approved by the national ethics committee of the ministry of health of madagascar. written informed consent was obtained from parents or legal guardians of children enrolled in the study. consenting to be enrolled in the study required consent to provide samples for diagnosis of influenza and other respiratory pathogens and to fill out a case report forms (crfs). refused consent and hospitalization within the two weeks before consultation were reasons for exclusion from the study. virological analyses were performed by national influenza centre (nic). a panel of multiplex real-time rt-pcr was used for the detection of viral pathogens in nasopharyngeal swabs. primers and probes were obtained from the authors of reported studies or were developed at the nic [8] . viral rna and dna were extracted from 140 ml of each specimen using the qiaamp viral rna mini kit and qiaampminelute virus kit (qiagen, courtaboeuf, france) according to the manufacturer's protocols. an abi 7500 fast real-time pcr system (applied biosystems) was used for amplifications. each sample was subjected to five different amplification reactions, each containing primers and probes specific for two or three viruses such that the following 12 respiratory viruses were targeted as previously described: parainfluenza virus 1-3 (piv), human coronaviruses (hcov) -oc43, -229e, -nl63 and hku1, respiratory syncytial virus (rsv), human metapneumovirus (hmpv), human rhinovirus (hrv), adenovirus (adv) and bocavirus (bov). as part of our routine influenza surveillance, screening for influenza viruses is performed by mdck cell inoculation and realtime rt-pcr according to the cdc protocols [9] . bacteriological analyses were performed by the medical analysis laboratory of ipm gram-stained sputum preparations were examined for polynuclear neutrophils (pn) and epithelial cells. the areas of maximal purulence were examined and graded as follows: class 1: more than 25 cells and fewer than 10 pn, class 2: more than 25 cells and 10-25 pn, class 3: more than 25 cells and more than 25 pn, class 4: 10-25 cells and more than 25 pn and class 5: fewer than 10 cells and more than 25 pn. only the classes 4 and 5 present a high positive predictive value. if the laboratory determined that the sample was of oral origin it had been rejected. so, classes 1 to 3 had been considered as rejection criteria. quantitative sputum cultures were performed on each specimen by a previously described method [10] : sputum specimens were homogenized with an equal volume of digest-eurh on a vortex mixer; 10 ml aliquots of the homogenate were serially diluted in saline and 10 ml aliquots of the dilutions were plated with a sterile inoculator on blood columbia agar with nalidixic acid and colistin (anc), chocolate polyvitex (pvx) agar, and drigalski agar. plates were incubated under 5% co 2 at 35uc for 24 to 48 hours. colonies growing on plates inoculated with suitable dilutions were counted to enumerate each bacterial species present. the threshold of significance was set at $10 7 /ml (for one or two species only). isolates were identified by standard laboratory methods. mono-infection was defined as the presence of only one pathogen as determined by viral and bacterial analysis. patients for which more than one pathogen was detected were considered to have co-infections. the outcome was scored as severe if the patient died, or presented a wrong clinical evolution during hospitalisation (complication) or the hospitalization was longer than 10 days (3 rd quartile). analyses were performed with r software [11] . the nonparametric wilcoxon test was used to compare means. qualitative variables were compared with the fisher exact test. statistical differences were considered significant for p-values,0.05. multivariate backward step-by-step logistic regressions were performed to take into account confounders, bias and interactions involving pathogens linked to the dependent variable. all variables with a pvalue,0.20 were included in the initial model. five age groups were defined for the analysis: [0-5] months, [6] [7] [8] [9] [10] [11] [12] months, [13] [14] [15] [16] [17] [18] months, [19] [20] [21] [22] [23] [24] months, [25] [26] [27] [28] [29] [30] [31] [32] [33] [34] [35] [36] months, .36 months. from november 2010 to july 2012, 290 children were recruited from the 301 hospitalized patients eligible. eleven eligible patients refused to participate in the study, and they did not differ from included patients for age, sex or diagnosis on admission. the baseline demographic and clinical characteristics of the patients are shown in table 1 . the sex ratio (male/female) was 1.3. the mean of age was 1.1 years [95% ci: 1.0 to 1.2], the age range was from 1 day to 5 years, and 250 (86%) patients were 2 years old or younger. the diagnoses on admission (table s1 ) were significantly different between the age groups: bronchiolitis was found more frequently among patients under 6 months old (55%, p,0.01); ltri and pneumonia were more frequent among patients aged over 12 months (respectively, 68% and 85%,p,0.01). forty-nine (17%) patients had at least one underlying disease, with asthma being the most frequent (5%). some of the children had underlying conditions, particularly atopy (52; 18%) and passive exposure to tobacco smoke (113; 39%). seventy-one (24%) had been hospitalized during the previous 12 months. patients aged under 18 months had significantly more prior hospitalisations (p = 0.01). the difference between sexes was not statistically significant. around 45% (131/290) of patients had been treated with antibiotics before hospitalisation (table s2 ). significantly more patients under than over 12 months old had received antibiotic treatment before hospitalisation (p,0.01). the difference between sex was not statistically significant. the frequency of bacterial detection was lower in the group with prior antibiotic treatment (49% versus 65%, p = 0.01). no such relationship was found for viral results. accurate information about immunisation was available on vaccination cards for 82 (26%) children. eighty-seven percent of children had completed vaccination against bcg, 77% had received one haemophilus influenzae type b vaccination. no child was vaccinated against streptococcus pneumoniae and only two were vaccinated against influenza. the average of duration of hospitalisation was 7.9 days [95% ci: 7.1 to 8.6], range 1 to 79 days. the median duration of hospitalisation was 7 days, and 61% of patients were hospitalised for more than 7 days. the duration of hospitalisation for living patients was 8.0 [7.2-8.7 ] days, and for fatal cases was 3.9 [1.4-9.2]. there were no significant differences between sex and age groups. discharge information was available for all cases: 79% (230/ 290) of patients fully recovered without any complication during their hospital stay; the condition of 60 children worsened during hospitalisation, and nine patients (3%) died. all deaths were of children under 18 months of age, and six (67%) were of children less than 6 months old. for the patients who died, streptococcus pneumoniae (56%, 5/9) was the pathogen most frequently detected, followed rsv (33%, 3/9) and influenza type a virus (22% 2/9). for multivariate analysis of factors possibly associated with coinfection, sex, age group, number of rooms in the home and antibiotic treatment before hospitalisation were included in the initial model. the multivariate analysis indicated that children under 36 months of age were more often infected by more than one pathogen; the difference was statistically significant for the 19-24 months old group (adjusted or = 8.0) ( table 1) . previous antibiotic treatment before hospitalization was a significantly protective factor against co-infection (or = 0.5). in univariate analysis, influenza a, rhinovirus, streptococcus pneumoniae, and haemophilus influenza type b were significantly associated with co-infections. haemophilus influenzae and s. pneumoniae were most often associated with co-infection (adjusted or = 20.5 and 11.1, respectively) ( table 1) . univariate analysis showed that a severe outcome was significantly more frequent for patients with comorbidity (or = 3.9), including prematurity or congenital disease (such as heart disease or cerebral malformation). also, two of the three patients presenting with malnutrition died. hospitalization during the 12 months prior to admission was also associated with a severe outcome (or = 2.1). a low household income, of less than 455 us$ (less than 182 us$ or = 4.4 and between 182 and 455 us$ or = 3.5) was associated with severe outcomes. for multivariate analysis, age group, monthly household income, admission diagnosis, comorbidities, previous hospitalisation, and infection with influenza virus type a, rsv and rhinovirus were included in the initial model. multivariate analysis (table 3 ) adjusted for these variables showed that age below 6 months (or = 5.3), living in a household with low income (or = 4.1), and the presence of comorbidities (or = 4.6) were significantly aggravating factors. infection with an influenza type a virus was a significantly protective factor against a severe outcome (or = 0.4 [95% ci = 0.2-0.9]). in the paediatric ward of cenhosoa, most children under 36 months, hospitalised for respiratory infections, were co-infected, and the most frequently encountered pathogens were h. influenza, s. pneumonia, influenza a and rhinoviruses. during hospitalisation, the evolution of respiratory infections was worse for the youngest children and for those presenting comorbidities and living in lowincome household. over all, viral infections made a large contribution to the burden of infectious respiratory disease. viral pathogens were found in 85% of the patients and bacterial pathogens in 57%, although for 45% of the patients had been treated with antibiotics before hospitalisation, and this presumably affected the observed prevalence of bacterial infections. our findings for viruses were similar to those of other studies. for example, the detection rate for viral pathogens was 72% in a study in vietnam [12] , 50% in a study in mozambique [13] , both focusing on hospitalised children. these results are consistent with those for the paediatric ward in cenhosoa. our findings are also consistent with another report from madagascar indicating that rsv (30.5%) and by influenza a (22.6%) are the most frequent viral causes of community respiratory infection [8] . few studies in developing countries have addressed the role of bacterial co-infection in severe respiratory illness. most focus on the viral aetiology because molecular diagnosis tools are available for these pathogens allowing to identify the infection in 80%-95% of cases [14, 15] . in developing countries, laboratory methods to identify bacterial pathogens are more difficult to implement, because of the lack of appropriate facilities and material in hospital laboratories and then, long transit times are prejudicial for stability of specimens. in our study the transit time was reduced because of the proximity of the cenhosoa and the ipm. unfortunately, our findings for the rate of bacterial infection may be artificially low due to prior antibiotic use. in our study, streptococcus pneumoniae was the most common bacterial pathogen as in other studies of hospitalised patients with acute respiratory illness, and haemophilus influenzae type b was the next most frequent [16] [17] [18] [19] . primary infection by a respiratory virus increases the risk of secondary bacterial pneumonia, and viral and bacterial coinfection is common in young children with pneumonia in developing countries (about 20-30% of episodes) [20, 21] . evidence of probable mixed viral-bacterial infection has been recorded in up to 45% of cases of community acquired pneumonia in children [22] [23] [24] [25] . a large proportion of our patients had coinfection (70%; 192/273) and the [0-6] months age group had the highest risk. these results are consistent with a study of patients with community respiratory infection in madagascar [8] . viral and bacterial co-infection was found in 57% of our patients; the most frequent combination was streptococcus pneumoniae with one of several respiratory viruses (88%). data from gambia and nigeria indicate that mixed bacterial and viral infections may occur in 8 -40% of cases of childhood pneumonia [26, 27] . recent data from south africa indicate that in at least 31% of cases, viral-associated pneumonia may be due to concurrent infection with streptococcus pneumoniae in the absence of vaccination with pneumococcal conjugate vaccines [28] . appropriate management of respiratory infections can have a significant impact on child mortality and it is important to target priority groups of children most at risk of dying: these groups include very young infants, children infected with hiv and malnourished children [29] . in our study, the outcome was severe for all of the children who presented malnutrition and 67% (2/3) died. chisti et al show that the combination of pneumonia with severe or moderate malnutrition is associated with a high risk of death: the rr ranges from 2.9 to 121.2 for severe malnutrition, and from 2.5 to 15.1 for moderate malnutrition. for moderate malnutrition, rr = 1.2 to 36.5. several other studies also lead to the same results [30] [31] [32] [33] . ballard et al. report that malnutrition and level of parental education both have an impact on the incidence of respiratory infections [34] . pneumonia is the leading causes of childhood mortality, and is responsible for approximately 21% of deaths of african children under five years of age each year. however, little information is available about the causes of childhood pneumonia in developing countries. in our study, only 3% of the patients died; this value is low. note that the study population consists of patients from families with a socioeconomic level that is above the average for the general population in madagascar. the patients who died were more frequently co-infected and under 6 months old. these results are consistent with other studies on this topic in developing countries [35, 36] . however, study design may have an impact on the mortality rate observed: indeed all fees for hospitalisation were paid by the study to allow follow-up to be optimised. the study identified living in a low-income household as a risk factor for severe outcome. this is important for patient management during hospitalisation and also highlights the consequences of financial barriers to access to vaccination against influenza and pneumococci. these vaccinations were not included in the panel of vaccinations in expanded programme of immunisation (epi) during this period in madagascar. they were only available in private centres and were expensive, and therefore inaccessible for much of the population. only two of the patients included in this study had received vaccination against influenza and none had received vaccination against pneumococci according to their vaccination cards. our statistical analysis identified influenza a infection as a protective factor against a fatal outcome; however, it is well-known that influenza a may have an impact on high mortality during major epidemics [37] [38] [39] . the co-circulation of influenza a virus, rsv and the high frequency of streptococcus pneumoniae in co-infections in children aged less than 5 years raised issues about the indirect impact of influenza vaccination on respiratory infections. in particular, the benefits of establishing a system of vaccination against infectious agents for which a vaccine is available should be considered to provide access to a larger proportion of the population. in conclusion, we advocate to further research in developing countries to document in more detail the impact of viral and bacterial co-infections on the prognosis of severe respiratory illness. as numerous infections found in our study could be avoided by immunisation, we recommend that appropriate vaccination must be rapidly available for all the malagasy children. this would reduce mortality among the youngest children, as this age group currently suffers the greatest burden of morbidity and mortality associated with respiratory infections. table s1 diagnosis at admission according with age groups. (docx) estimates of world-wide distribution of child deaths from acute respiratory infections community-acquired pneumonia epidemiology and etiology of childhood pneumonia study of community acquired pneumonia aetiology (scapa) in adults admitted to hospital: implications for management guidelines community-acquired pneumonia in children british thoracic society guidelines for the management of community acquired pneumonia in childhood bayesian mapping of pulmonary tuberculosis in antananarivo viral etiology of influenza-like illnesses in antananarivo who | cdc protocol of realtime rtpcr for influenza a (h1n1) (n.d.). who. available remic référentiel en microbiologie médicale r: a language and environment for statistical computing. r foundation for statistical computing viral etiologies of acute respiratory infections among hospitalized vietnamese children in ho chi minh city viral acute respiratory infections among infants visited in a rural hospital of southern mozambique intranasal fluticasone propionate does not prevent acute otitis media during viral upper respiratory infection in children human bocavirus and acute wheezing in children epidemiology and etiology of childhood pneumonia etiology of community-acquired pneumonia in hospitalized patients in northern israel etiology of severe pneumonia in children in developing countries the bacterial etiology of community-acquired pneumonia in korea: a nationwide prospective multicenter study etiology of acute lower respiratory tract infections in gambian children: ii. acute lower respiratory tract infection in children ages one to nine years presenting at the hospital diagnoses of acute lower respiratory tract infections in children in rawalpindi and islamabad induced sputum in the diagnosis of childhood community-acquired pneumonia etiology of community-acquired pneumonia in 254 hospitalized children etiology of community-acquired pneumonia in hospitalized school-age children: evidence for high prevalence of viral infections malnutrition as an underlying cause of childhood deaths associated with infectious diseases in developing countries etiology of acute lower respiratory tract infections in gambian children: ii. acute lower respiratory tract infection in children ages one to nine years presenting at the hospital the etiology of pneumonia in malnourished and well-nourished gambian children a trial of a 9-valent pneumococcal conjugate vaccine in children with and those without hiv infection challenges to improving case management of childhood pneumonia at health facilities in resource-limited settings multihospital surveillance of pneumonia burden among children aged ,5 years hospitalized for pneumonia in bangladesh bacterial aetiology and outcome in children with severe pneumonia in uganda etiologic agents and outcome determinants of community-acquired pneumonia in urban children: a hospital-based study improvements in nutritional management as a determinant of reduced mortality from community-acquired lower respiratory tract infection in hospitalized children from rural central africa the effects of malnutrition, parental literacy and household crowding on acute lower respiratory infections in young kenyan children bacterial etiology of serious infections in young infants in developing countries: results of a multicenter study. the who young infants study group neonatal pneumonia in developing countries excess mortality associated with the 2009 a(h1n1)v influenza pandemic in antananarivo this study would not have been possible without the excellent support from all the clinicians (dr emma rasoanirina, dr roland randriamirado, dr harimboahangy rakotoarison, dr lucien radozahana, dr rani razafindrakoto and dr domohina rakotovao), and all the nurses of the paediatric ward of the cenhosoa, antananarivo. we are deeply indebted to all staff who contribute, every day, to the hospital surveillance programme. we also acknowledge the tremendous work of all technicians of the virology unit and cbc.disclaimer: the views expressed in this article are those of the authors and do not necessarily reflect the official policy or position of the ministry of health. conceived and designed the experiments: sr. performed the experiments: sr jmh vr. analyzed the data: sr vr nhr. contributed reagents/ materials/analysis tools: nhr ao ehr lr rr ph fr zza ta. wrote the paper: sr nhr ehr vr. key: cord-324217-secnz2ta authors: pasdaran, a.; pasdaran, a.; sheikhi, d. title: volatile oils: potential agents for the treatment of respiratory infections date: 2016-08-05 journal: the microbiology of respiratory system infections doi: 10.1016/b978-0-12-804543-5.00016-6 sha: doc_id: 324217 cord_uid: secnz2ta due to presence of secondary bioactive metabolites, natural compounds are considered a major source of new active molecules that can be developed as new drugs. infectious diseases, and mainly the common respiratory infections, are major challenges to the current chemotherapy systems and, therefore, there is a requirement to find new compounds with therapeutic potential. the volatile natural compounds and essential oils are the main treasure agents in the natural compounds with antibiotic potential. the present chapter reviews natural traditional remedies used in the treatment of respiratory infections with the emphasis on antibacterial, antiviral, and antiinflammation activities of the volatile natural compounds (essential oils, etc.), and provides a brief view in some of structural activity relationships between antibacterial potencies and chemical structures of the essential oil’s constituents. referring to infectious disease, respiratory tract infections engage with all surfaces in the respiratory tract. based on the infected zone, respiratory infections can be categorized into upper tract infection (uri or urti) and lower tract infection (lri or lrti). each involves different parts of the respiratory tract infections, which vary in type and severity of microorganisms. although there are different types of respiratory tract infections, the acute form in the upper respiratory tract infection predominates and includes several complications, such as sinusitis, pharyngitis, epiglottitis, laryngitis, and tracheitis. on the other hand, lower respiratory tract infection (lrti) includes both acute and chorionic types, such as pneumonia and bronchitis. based on pathogenicity, bacterial and viral pathogens are the most common microorganisms in both types (ie, lrti and urti). moreover, infection distribution leads to varieties based on the patient's age; for example, acute respiratory infections pose severe problem in childhood, which mainly occur in upper respiratory tract. although the bacterial pathogens play a significant role in intensifying lrtis, the major acute respiratory infections occur in upper respiratory tract, in these cases viral pathogens are the primary common pathogens, including influenza a and b, parainfluenza (type 1 and 3), adenovirus, and respiratory syncytial virus. some of the common pathogens of the respiratory tract are listed in table 16 .1. pathogen biodiversity, complexity, and mixed infections in many cases of respiratory tract infection have generated several problems for the treatment of respiratory infections. for example, various bacterial pathogens are encountered in several cases of viral infections. therefore, the treatment of respiratory infections is a complex therapy which consists of several chemotherapy strategies. 1-3 antiviral (the same as antibacterial medication) is used to control the treatment and prevention of respiratory infections. there are several restrictive factors, such as medication resistance, recurrency, and inflammation, which will guide researchers to find new effective compounds. this will be an important field in drug development for respiratory infections. natural compounds are considered to be one of the main sources in new drug development. historically, numerous plants have been utilized as traditional medicines by people in many nations. 4 many of these plants have been investigated for their antimicrobial and antiviral properties. [5] [6] [7] [8] [9] with regard to massive variation among natural products, chemical structure diversity causes different antimicrobial potential in natural compounds. 10 besides the antimicrobial activity of the essential oils in natural products, other characteristics such as high vapor pressure, low toxicity, and antiinflammatory potential create a worthwhile theme for using of these natural compounds for new drug development in respiratory infections. parallel to the roles of the microorganisms in the pathology of respiratory infections diseases, inflammatory process also have a considerable role in the persistence and recurrence of respiratory infectious diseases. this chapter reviews the antibacterial, antiviral, and antiinflammation effects of essential oils as effective natural compounds. it will also discuss the use of these natural compounds as traditional remedies in treatment of respiratory infections. traditional medicines utilize natural sources for the treatment of the many diseases. [11] [12] [13] historically, infectious diseases have been the major human ailment. natural sources are used in a variety of forms, including water extracts, tincture or alcoholic extract and incense. 14 based on the historical uses and effective treatments that have been based on many of these traditional remedies, extensive pharmacological research of their antibacterial, antiviral, and antiinflammation activity have been performed. [15] [16] [17] abundant information about plants and active compounds in infectious diseases and inflammation related process is available. [18] [19] [20] [21] aromatic and fragrant plants are a major part of traditional therapeutic remedies, and they have shown remarkable antibacterial and antiviral activity. furthermore, many of them also have a significant antiinflammatory activity and are used as adjuvant remedies in the treatment of infection (table 16 .2 and fig. 16.1) . some of the most active extracts of traditional herbs which have been used as antibacterial and antiviral in the treatment of respiratory infections are summarized in table 16 . 3 . the use of aromatic extracts or burning plants is a common process in traditional medicine. the resultant smoke or fragrance is inhaled to treat respiratory complaints, including cough, cold, infections, and asthma. 22, 23 inhalation administration goes back to the ancient cultures and its techniques may be considered as a progressive point in respiratory complaints treatment. the direct effect of such fragrance on the respiratory tract is an advantage of this form of treatment. inhalation therapy often involves the aromatic extracts or burning of plant material, and the volatile fraction liberated during the process is inhaled to aid in the healing process. inhalation of the volatile fraction from aromatic extracts or burning plant matter is a unique method of administration and has been used traditionally to treat respiratory conditions, such as, asthma, bronchitis, and other respiratory infections including the common cold. 24 in addition, aerosol delivery of such remedies is well practiced in allopathic medicine and has the advantage of being site specific, thus enhancing the therapeutic ratio for respiratory ailments. 25 the antimicrobial effects of plants and their extracts have been recognized for a long time. essential oil is one of the most important and wide spread secondary metabolite in plants and this class of phytochemical compounds and their activities needs attention. these phytochemicals are generally isolated from plant material by distillation methods, such as, hydrodistillation and steam distillation. they contain variable mixtures of several chemical classes, such as terpenoids, specifically monoterpenes and simple phenolic compounds. some of the higher molecular structures with high molecular weight, such as sesquiterpenes and diterpenes, may be present. a variety of low molecular weight aliphatic hydrocarbons, acids, alcohols, esters or lactones, sulfur-containing compounds and other chemical groups may also be observed. among the phytochemical compounds, terpenes are responsible for many therapeutic effects in medicinal plants. [26] [27] [28] [29] [30] most terpenes are derived from the condensation of isoprene units and are categorized according to the number of these units present in the carbon skeleton. these compounds are responsible for aromaticity and fragrance in many of the plants. the antibacterial activity of volatile oils has been assessed by many researchers. [31] [32] [33] [34] this potential of essential oils has been used in many pharmaceutical, cosmeceutical, and nutraceutical applications and industrials. there are many differences between the antimicrobial effects of different essential oils. essential oils and their constituents are an attractive source in new antimicrobial compounds evaluation. 35 many of the essential oils have been tested for bactericidal and bacteriostatic effects against a wide range of microorganisms including food spoiling organisms, pathogenic bacteria, yeasts, fungi, and many others. the major differences in antimicrobial activity have been yielded of several distinctive parameters which identify antibacterial characters of the essential oils, some of the major parameters include: (1) bacterial membrane permeability, (2) the hydrophobicity/hydrophilicity of the bacterial membrane, (3) the metabolic characteristics of the microorganism, and (4) their gram-positive or negative pattern. although susceptibility of the bacteria to the essential oils is not exactly predictable, many researchers have tried to determine the relationship between the origin of the essential oils and their compounds with their antimicrobial activity. furthermore, the delivery of medications to the respiratory tract has become an increasingly important method for respiratory disease treatment. the use of inhaler medications has become an invaluable therapeutic in the treatment of different pulmonary disorders, including bronchitis, pneumonia, and others complications. 37 several studies have reported the clinical efficacy of inhalation therapy for the treatment of lung disorders. 38, 39 through the effective delivery of medication to the action site, the active compounds are delivered directly into the lungs and this can result in respiratory tract local treatment. this method achieves maximum therapeutic biebersteinii. 3 screening of the antibacterial effects of essential oils effect, small dose usage, and has fewer side-effect risks compared with those associated with larger doses. inhalation is a unique treatment with direct effects on respiratory disorder site and is based on the volatility potential of essential oils. furthermore, there is a need to develop new therapeutic agents for respiratory infections. [40] [41] [42] research has been carried out on the wide spectrum of edible plants essential oils to determine the antibacterial potential of their essential oils. the role of these plants as therapeutic agents is remarkable in many cultures. investigations have reported that thyme and oregano essential oils, based on the phenolic components [such as carvacrol (1) and thymol (2) (fig. 16. 3)] have shown a strong correlation with the inhibition of some of the pathogenic bacterial strains (eg, in escherichia coli). the correlation between the antibacterial effect of the volatile oils and their chemical compounds, including high amount of the phenolic components such as carvacrol (1) or eugenol (3), has also been confirmed. 43 other essential oils such as oregano, savory, clove, and nutmeg with high concentrations of volatile phenolic compounds inhibit gram-positive more than gram-negative pathogenic bacteria. 44 however, in some essential oils such as achillea spp. (yarrow) strong antibacterial activity was observed against the gram-negative respiratory pathogens (haemophilus influenzae, pseudomonas aeruginosa) while streptococcus pyogenes was the most resistant to the this oil. 45 the other essential oils such as peppermint and spearmint inhibit the methicillin-resistant type of staphylococcus aureus. previous reports have clarified that the essential oils containing aldehyde or phenol as a major component represent the highest antibacterial activity. these antibacterial potencies are lower in the essential oils that contain high amounts of terpene alcohols compared to the essential oils containing aldehyde or phenol as a major component. other essential oils containing terpene ketone, or ether showed much weaker activity, and oil containing terpene hydrocarbon was relatively inactive. based on these findings, essential oils such as thyme, cinnamon, lemongrass, perilla, and peppermint have demonstrated suitable effects on respiratory tract infection. 46 the tolerance of gram-negative bacteria to essential oils has been attributed to the presence of a hydrophilic outer membrane that blocked the penetration of hydrophobic essential oils to the target cell membrane because the gram-positive bacteria were more exposed to the essential oils than gram-negative bacteria, which has been reported several times. [47] [48] [49] [50] lipids are one of the principal constituents for normal cell membrane function and these compounds supply many operations, such as barrier function in the bacterial cell membrane. the external capsule of some gram-negative bacteria limits or prevents the penetration of the essential oils into the microbial cell. one of the pronounced examples of the hydrophobicity/hydrophilicity role in bacterial sensitivity to antibacterial compound is h. influenzae. it should be pointed out that the outer membrane of h. influenzae (which forms rough colonies) was more hydrophobic. hydrophobic antibiotics, such as macrolides, are more active against h. influenzae than e. coli through their shorter oligosaccharide chains than those in e. coli. the effects of the cytoplasmic membrane and/or the embedded enzymes in it have demonstrated lipophilic biocide actions. 51 it is generally recognized that the antimicrobial action of essential oils depends on their hydrophilic or lipophilic character. based on these observations, investigators are trying to indicate the relationship between structural activity of the essential oils compounds and their antibacterial activity. certain components of the essential oils can act as uncouplers, which interfere with proton translocation over a membrane vesicle and subsequently interrupt adp phosphorylation pathways (primary energy metabolism). as a member of the phytochemicals, terpenoids have been observed as a model of lipid soluble agents, which have an impact on the activities of membrane catalyzed enzymes; for 3 screening of the antibacterial effects of essential oils example, enzymes involved in respiratory pathways. particular terpenoids with functional groups, such as phenolic alcohols or aldehydes, also interfere with membrane-integrated or associated enzyme proteins, inhibiting their production or activity. a good deal of antimicrobial compounds which act on the bacterial cytoplasmic membrane cause the loss of 260 nm absorbing material. this causes an increased susceptibility to nacl, the lysosomes formation and loss of potassium ions, which results in inhibiting respiration and the loss of cytoplasmic material. investigations about the cytoplasmic membrane effects of α-pinene (4), β-pinene (5), 1,8-cineole (6) , and electron microscopy studies have shown that the essential oils containing these compounds triggered such cytoplasmic material with losing in treated bacterial cells. 31, 32 the perturbation of the lipid fraction in the plasma membrane causes antimicrobial activity of some of the phytochemicals such as α,β-unsaturated aldehydes and some of monoterpenes. although these aldehyde compounds can elicit antibacterial effects by acting on membrane functional proteins, such antibacterial effect would be achieved with modifications of membrane permeability and intracellular materials leakage. [52] [53] [54] the membrane damage leading to whole-cell lysis has been reported by oregano and rosewood essential oils which contains major components as: carvacrol (1), citronellol (7), and geraniol (8) . 26, 55 phenols such as carvacrol (1), thymol (2), eugenol (3), and other oxygenated aromatic essential oil compounds including phenol ethers [trans-anethole (9), methyl chavicol (10)] and aromatic aldehydes [cinnamaldehyde (11), cuminaldehyde (12) ] have been reported to exert both antibacterial and antifungal activity. however, this chemical class-based on the concentration used-are known as either bactericidal or bacteriostatic agents, 56 but the phenolic component's high activity may be further explained in terms of the alkyl substitution into the phenol nucleus, which is known to increase the antimicrobial activity of phenols. the alkylation has been known to change the distribution ratio between the hydrophilic and the hydrophobic phases (including bacterial phases) by the surface tension reduction or the species selectivity mutate based on the bacteria cell wall characters. 57 this does not happen with etherified or esterified isomeric molecules, it is possible by describing their relative lack of activity. 58 as a member of these compounds carvacrol (1) is one of the few components that has a break apart from effect on the outer membrane of gram-negative bacteria and causes release of lipopolysaccharide and alters cytoplasmic membrane ions transportation, similar to carvacrol (1), thymol (2) antimicrobial activity results in structural and functional alterations in the cytoplasmic membrane. 59 interestingly, eugenol (3) and isoeugenol (13) exhibit higher activity against gram-negative bacteria than gram-positive bacteria, and when cinnamaldehyde (11) is used against e. coli, its activity is similar to carvacrol (1) and thymol (2) (fig. 16.3) . these compounds alter the membrane, affect the transport of ions and atp, and change the fatty acid profile of different bacteria. 60 although in some cases alcoholic form shows better potencies compared to acetate form, the presence of an acetate moiety in the structure appeared to increase the activity of the parent compound. in the case of geraniol (8), the geranyl acetate (14) demonstrated an increase in activity against the test microorganisms. 48, 61, 62 a similar effect was also observed in the case of borneol (15) , bornyl acetate (16) , linalool (17) , and linalyl acetate (18) (fig. 16.3 ). in addition, the effectiveness of alcoholic compounds very closely depended on the bacterial cell wall, which showed different permeability to alcohol based on chain length. 44, 63 it has been suggested that an aldehyde group conjugated to a carbon double bond such as citral (19) is an extremely electronegative order, which may explain their activity, and an increase in electronegativity can raise up the antibacterial activity. 64 in addition, the under research of aldehydes potency seems to depend not only on the existence of the α,β-double bond but also on the chain length from the renal group and on microorganism tested. it seems that some electronegative compounds, mainly from the cell surface, are responsible for the inhibited growth of the microorganisms, which may interfere in biological processes involving electron transfer and respond with vital nitrogen components and alteration in the operation of membrane-associated proteins. actually, a greater electronegativity of the molecule would cause a greater encounter of intermolecular hydrogen bond formation with membrane nucleophilic groups and thus a significant irregularity in the lipidic bilayer. some studies have recommend that carbon tail length also affects the electronegativity of the aldehyde oxygen atom and thus its interaction with the nucleophilic groups of the cell membrane. 65 comparably, the similar antimicrobial activity was detected in the series of the long-chain alcohols which is demonstrated to be resulted from the alkyl chain length. 66, 67 this structural activity relationship is notable between farnesol (20) , nerolidol (21), plaunotol (22) , geranylgeraniol (23), phytol (24), geraniol (8) , and linalool (17) which act on s. aureus with damages of the cell membranes and losing of k+ ions, while similar mode of actions can be detected by the aminoglycosides such as kanamycin and streptomycin. farnesol (20) was able to damage cell membranes most effectively than other terpene alcohols. the activities of farnesol (20) , nerolidol (21) (sesquiterpenes compounds) on s. aureus were higher than that of plaunotol (22) (diterpene). the effectiveness against s. aureus are in order as follows: farnesol (19) > nerolidol (20)> plaunotol (22) > geranylgeraniol (23), phytol (24) > geraniol (8) and linalool (17) (fig. 16.3 ). it has been suggested that maximum activity against s. aureus might depend on the number of carbon atoms in the hydrophobic chain from hydrophilic hydroxyl group, which should be less than 12 but as close to 12 as possible. neither a shorter nor a longer aliphatic carbon chain, could increase such activity. 68, 69 the increased effectiveness of sesquiterpenes as enhancers of membrane permeability may stem from their structural resemblance to membrane lipids (eg, linear molecules with internal lipophilic character and a more polar terminus). 70 the bacteriostatic potential of the terpenoids was also increased when the carbonyl groups increased in structure. 63 the type of alkyl substituent incorporated into a nonphenolic ring structure is responsible for enhancement of antibacterial activity. such as an alkenyl substituent (1-methylethenyl) makes an increase in antibacterial activity, as seen in limonene [1-methyl-4-(1-methylethenyl)-cyclohexene] (25), compared to an alkyl (1-methylethyl) substituent as in p-cymene [1-methyl-4-(1-methylethyl)-benzene] (26) . furthermore, principally gram-negative were the sensitive organisms that propose alkylation control of the gram reaction sensitivity of the bacteria. an allylic side-chain seems to raise the inhibitory role of the simple phenols mainly against gram-negative organisms. this was suggested due to the majority of the antimicrobial activity of alkylated phenols in relation to phenol which has been earlier reported (fig. 16.3) . 56 it was observed that α-isomers are inactive relative to β-isomers in many compounds in stereochemistry, which is also effective in antibacterial activity observed from essential oils. 44 the (e,e)-2,4-decadienal (26) appears to be more toxic to bacterial cells than the correspondent monounsaturated aldehyde (e)-2-decenal (27) as another example of stereochemistry effectiveness in activity potential observed in the unsaturated aldehyde, but it is noticeable that two double bonds in the cis configuration in the side-chain of 2,4-decadienal (26) produce more bends and shorten the length of the carbon tail, such α,β-unsaturated aldehydes might be a good choice compared to other highly toxic sterilizers (fig. 16.3) . 53 in summary, the antimicrobial activity of essential oils depends on different amounts of specific compounds. as an example, antimicrobial properties of the essential oils with high concentrations of eugenol (3), cinnamaldehyde (11) , or citral (19) , is predictable. remarkable antimicrobial, antifungal, 3 screening of the antibacterial effects of essential oils and antiviral activity of the monoterpenes and phenols relieve from essential oils present in thyme, sage, and rosemary. due to the formation of polysaccharides that increase the resistance to essential oils there are some other essential oils, such as basil, sage, hyssop, rosemary, and oregano, which are active against e. coli, s. aureus but are less effective against pseudomonas spp. [71] [72] [73] the typical characteristic of the essential oils is hydrophobicity, which is responsible for the disruption of bacterial structures and makes an increase in permeability due to a weakness to pull apart the essential oils from the bacterial cell membrane. many cellular functions, including maintaining the energy status of the cell, membrane-coupled energy transducing processes, solute transport, and metabolic regulation result from the cell membrane permeability barrier. actually, they are responsible for leakage of the cell contents, reducing the proton motive force, reducing the intracellular atp pool via decreased atp synthesis and augmented hydrolysis are the mechanisms of action of the essential oils including the degradation of the cell wall, damaging the cytoplasmic membrane, cytoplasm coagulation, damaging the membrane proteins, increased permeability that is different from the increased membrane permeability and reducing the membrane potential via increased membrane permeability. 33, 74, 75 unfortunately, scientific investigations on the antimicrobial activity of essential oils have been retarded by the lack of appropriate susceptibility testing methods for the essential oils and because no generally approved assay method has been established for the assessment of their antimicrobial activity. many researchers have employed the disk assay method. however, the results of this method were not always in parallel with those of dilution assay methods. the differences were caused not only by differences in the solubility of the oils but also by interactions of the components in the concentrated solution used in the disk assay. 48 the dilution method could be more reliable than the disk method with regard to reproducibility and clinical relevance. when testing nonwater-soluble and highly volatile essential oils by the dilution method, it is necessary to obtain a homogeneous dispersion of the oils in the medium. chemical emulsifiers such as tween 80, tween 20 and other have been used frequently for this homogenization, but it has been reported that emulsifiers reduced the bioactivity of the oils, probably because of the formation of micelles, which inhibit adequate contact between the oil and the test organism. 48 evidence also shows that the minimum inhibitory concentration (mic) values of the essential oils under open conditions of incubation caused a two-to eight-fold rise in the mics of highly volatile oils, as compared with values obtained under sealed conditions. sealed conditions are used to examine whether essential oils showed antibacterial activity against major respiratory tract pathogens (this method was authorized by the japan society of chemotherapy to adjust for the physico-chemical properties of essential oils). the scientific information concerning the antimicrobial effectiveness of the essential oils in the vapor phase compared with direct contact case show that potential of this form of the essential oils is ambiguous, although some degree of inhibition by volatile components of the essential oils has been demonstrated in the vapor phase. in fact, more investigation into the antibacterial effectiveness of the vapor phase is required. it seems that the composition of the atmosphere generated by the essential oils is also potentially correlated with their antimicrobial behavior. this part mentioned the main antibacterial activity laboratory assay methods, as already noted. these methods do not show coordinated results in some case and this appearance is reasoned to the necessity of simultaneously running two or more methods for antibacterial activity determination of essential oils. in this method, a petri dish is commonly used as an assay chamber (5-12 cm diameter and filled with 10-20 ml of agar broth). the solidified medium in the petri dish was inoculated with an appropriate colony of the microorganisms. this inoculation was accomplished by using a solution such as physiological saline solution containing adequate colony unit (commonly 100-200 (cfu)/ml). the essential oil is incorporated into the mediums in two ways: on a paper disc or into a well (hole) which is made in the agar medium. diluted or undiluted essential oils based on the goals were added to a sterile blank filter disk (5 mm diameter) and placed on top of the cultured media in a petri dish or added to the holes. after incubation under optimal conditions such as temperature and time, two different zones were considered in view of the average diameter of changes: (1) the zone where there is no growth of the microorganism, called total inhibition; and (2), the zone where growth of the microorganisms was significantly reduced in terms of amount of colonies, compared to blank assays. the growth of the microorganisms is recognizable with instruments or can be seen visually by one of the commonly used techniques, called turbidimetry, in which the optical density changes in the growing culture (od) are measured. some commercial systems have been produced for monitoring of the microorganisms growth as well as other methods were proposed (eg, bioautography). 76 an appropriate volume (100 µl physiological saline solution) of the microorganism's colony (10-20 cfu/ml) will be inoculated to the solidified medium. each essential oil sample was diluted in suitable solvent such as ethyl ether to obtain serial dilutions (v/v). the required volume (10 µl) of each dilution was then added to sterile blank filter disks or cups and placed on the medium free cover of each petri dish. experiments were designed in two forms of petri dish positions, including direct and invert placement. the petri dishes were then sealed using sterile adhesive tape. blanks were prepared by adding the same volume (10 µl) of samples of the solvent to the filter disks or cups, and this had no effect on the viability of any of the tested organisms. after the incubation period, the minimal inhibitory concentration (mic), expressed as microliters of the essential oil per volume unit of atmosphere above the organism growing on the agar surface, that caused inhibition by comparison with control tests was measured. 77 although the modification with liquid broth is mostly practical for fungi, the serial dilution agar method is also common for bacteria and fungi. the difference is that agar broth cultures are grown in petri dishes or tubes, but liquid broth cultures are cultivated in conical flasks filled with 100 ml medium or test tubes with 2.5-5 ml medium (bacteria and moulds). the inhibitory growth index is determined for the liquid broth in conical flasks (percent changes in mould's biomass comparing to the control culture). there is an inhibitory effect of essential oil which appears in the test tube cultures and is measured turbidimetrically or with the plate count method. the estimation of essential oil activity both in agar and liquid broth would be simplified through counting the tested microorganisms that can remain in the membrane. the lowest essential oil concentration in the broth that results in the lack of visible microorganism growth changes is known as the mic. the microorganisms are then transferred from the lowest essential oil concentration medium (with no visible microorganism growth) into a new broth medium and incubation to determine the lethal activity of essential oil. 78 viruses as invasive microorganisms may cause serious respiratory illness and in some cases lifethreatening conditions, such as acute pneumonia. although acute respiratory infection rates are not very high, this condition has been steadily increasing in children and persons over 60 years of age. the rates of hospitalization and death increase substantially in these cases. multiple factors, such as decline in respiratory and immune function, likely contribute to increased morbidity. natural products in all forms including pure compounds or extracts provide massive opportunities for new antiviral-lead compounds. 4, 80 at the moment, only a few effective antiviral drugs are available for the treatment of viral diseases, especially in respiratory viral infections. therefore, finding new substances with antiviral properties is a required for medical systems. much evidence has been reported about antiviral potential of various essential oils and their constitutions on several genera of viruses. 9, 81 in some investigations the essential oils with high hydrocarbons long-chain contents such as (e,e)-2,4-decadienal (26) , showed activities against influenza virus. 82,83 on other spectrum of antiinfluenza virus compounds, sesquiterpenes and sesquiterpenes-rich essential oils showed clear effects as well as aromatic rich essential oils. [84] [85] [86] among the terpenoids compounds, terpinen-4-ol (28), terpinolene (29) , and α-terpineol (30), show inhibitory effect on influenza a/pr/8pr virus subtype h1n1. also indicated that some sesquiterpenes such as β-sesquiphellandrene (31), and tetrahydronaphthalenol (32) , showed potent antirhinoviral activity in a plaque reduction test. 87 in other researches that conducted about the antiviral essential oils and their active constitutions indicated that laurus nobilis essential oil which was characterized by the presence of β-ocimene (32), 1,8-cineole (6), α-pinene (4), and β-pinene (5), as the main constituents, showed interesting activity against sars-cov. 88 this overview caused the development of commercial therapeutics based on monoterpens compounds for viral infections especially in respiratory viral infections (fig. 16.3) . 89, 90 although, antiviral potential is an important factor for bioactivity of natural compounds, cytotoxicity effects that probably triggered host cell damaged cycles are important. therefore, this is a limiting factor for presentation of the new useful compound in treatment or control of the viral infections. viruses as invasive parasites are capable of using host cell organelles and mechanisms for reproduction, such perspicaciously reproducing process caused a creation of tenacious shield against many potent natural molecules used. therefore selective activity is an important point, as are antiviral potencies. the immune response to respiratory tract infection is a double-edged sword because many of the symptoms that accompany these infections are largely due to the microorganism's induction of cytokines and chemokines, which may result in protracted inflammatory responses. phagocytic clearance of an infecting organism by inflammatory cells is an appropriate and necessary component of the host defense system. 91 at the same time, much of the evidence points to destructive effects spectrum made by products of these inflammatory cells. 92 these products can increase mucus secretion and impair ciliary clearance, thereby setting the stage for exacerbations and recurrences of infection. primary inflammatory cell products can amplify the other steps of the inflammatory cycles and can paradoxically even impair the immune response. these observations suggest that modulating the inflammatory response may be an important aspect of definitive therapy for respiratory tract infection. the primary defense systems are the lung secretions and the mucociliary escalator, which entrap organisms and sweep them away. at the same time, the lung secretions also contain a variety of proteins that inhibit microorganisms, especially bacterial adherence for example immunoglobulin a (iga) systems, which prevents bacterial adherence to epithelial cells, inhibit bacterial growth, and attempt to neutralize bacteria. the macrophages are a secondary defense system, which not only phagocytose microorganisms but also release biochemotactic factors that recruit other defense cells such as monocytes and neutrophils that are necessary for further phagocytosis process. 93 the inflammatory response form a key part of the secondary defense system that accompanies additional proteins, such as immunoglobulins and complement factors. although neutrophil infestation is part of the natural defenses against an invading organism, it may have destructive effects on the pulmonary systems. one of the destructive factors is the proteolytic enzyme neutrophil elastase, which is preformed and stored within the neutrophil. this proteinase is released after the neutrophil is activated and migrates into the tissues and phagocytosis invasive microorganisms. it produces a condition that histologically simulates chronic bronchitis. the potential role of neutrophil elastase in the pathogenesis of pulmonary diseases is characterized by neutrophil infiltration of the airways and increased mucus secretion. 94 it has also been reported that "secretions from patients with acute bronchial infection cause a significant reduction in ciliary beat frequency and that the addition of a neutrophil elastase inhibitor can reverse this effect." 95 the increase in the mucus secretion and the decrease in ciliary beat have been found as an important feature of chronic lung disease frequently. besides the phagocytic cell immediate responses, stimulated epithelial cells and macrophages produce the potent neutrophil chemoattractant, such as interleukin-8 (il-8), which was found to be present in high concentrations in the sputum of patients with chronic inflammatory airway disease. 96 at the same time, elastase released by activated neutrophils also stimulates il-8 production by the epithelial cells. 97 therefore, when these events are initiated, they become a self-amplifying cycle. this phenomenon is observed in patients with acute pneumonia that has apparently been cured by appropriate antibiotic therapy. natural products as inflammation inhibitors have for a long time played a key role in many traditional treatment systems. several mechanisms including interaction with prostaglandin biosynthesis, interaction with other inflammatory mediators, and corticosteroid-like effects are involved in antiinflammatory action of natural products. 98 among the natural chemical compounds, significant antiinflammatory activities of plant-based essential oil have been reported by many researchers, which showed the basis for folk and traditional uses of these herbs for treatment of inflammatory diseases. the essential oils and aromatic plants have had a significant place in inflammation control in many folklore medications. this evidence supports the view that appropriate investigations about the potential of essential oils should be made and their active constituents should also be evaluated for inflammation control. monoterpene alcohols such as linalool (17) and linalyl acetate (18) and other corresponding esters which are reported as one of the major volatile components of many aromatic plants essential oil were evaluated for antiinflammatory activity. these compounds have shown promising antiinflammatory potency among the many essential oil compounds. 99 for example, 1,8-cineole (6) the major constituent of eucalyptus oil is another active essential oil constituent that is well tolerated in inhalation administrations. this compound can also be effective for airways inflammation in clinical trials, based on such finding 1.8-cineole (6) is registered as a licensed medicinal product and has been available for airways inflammation for many years. 100 similar effects were also observed in other monoterpenes especially in carvacrol (1) and thymol (2) , which are main constituents in thymus, oregano, savory, clove, and nutmeg essential oils. in other reports, similar observations have confirmed that many of the other essential oils and their constitutions can play an important role in inflammation control. emphasizing the structure-activity relationships is necessary to define different potential of natural compounds such as essential oils compositions. for instance, aliphatic aldehydes and aromatic aldehydes are said to predominantly have antiinflammatory and antimicrobial potentials. 101 the terpenes and sesquiterpenes evaluated appeared to be good inhibitors of 5-lox in vitro. there is a good correlation observed between antiinflammatory activity of the limonene (25) and the essential oils rich in limonene (25) like grapefruit, lime, and celery. other similar activity has been observed on the various inflammations pathways such as cyclooxygenase (cox) and lipoxygenases (lox) between sesquiterpenic alcohols, aliphatic aldehydes, and some phenolic esters that caused these phytochemicals considered for more investigations in respiratory inflammations during the air ways infections (fig. 16.3) . 101, 102 it has been evaluated that activity of some of the essential oils such as western red cedar (thuja plicata) to inactivate several viruses implicated in respiratory infections, and to inhibit the influenza virus-induced secretion of cytokine (il-6) in cultured human lung cells. however, the liquid essential oil phases are generally a higher irritant and possibly toxic for nasopharyngeal or oral applications, although a few reports have indicated that the vapors of some essential oils might be useful for application in inhaled vapors for respiratory infections in low concentrations. 103 because of the noteworthy role of inflammation in respiratory infectious disease persistence and recurrence, many laboratory models have been developed for inflammation and inflammation/infection-mixed respiratory complications research. some of these models, such as ovalbumin-induced respiratory allergic eosinophilia arise especially for inflammation process which responds to them, including eosinophils, neutrophils, and other immune cell infiltration to the inflammation sites. 104 in other models, in addition to this cellular pathogenesis, more attention is considered on the infection's correlation with inflammation mechanisms. 105 table 16 .5 shows some of the attributes of inflammation and inflammation/infection major models that are used in screening of the new antiinflammation compounds. respiratory infections are one of the most prevalent human health problems and they cause major difficulties for all age ranges. 106 research to find new and effective therapeutic compounds for the control and treatment of these ailments is a most attractive field in natural product screening. the massive biodiversity of natural sources, such as plants, means that the selection of the suitable starting source point is a critical step for achieving the best screening results. in particular, paying attention to the main chemical constitutions of these natural sources will help to clarify the results of this research. analysis of the relationship between the chemical structures of natural compounds has led to a better overview of the rational uses of natural compounds and natural remedies for the control and treatment of disease, and has led to the development of therapeutics. the broad diversity of the chemical makeup of essential oils has caused some uncertainty over their best selection in complementary or investigative research. therefore, a correct understanding of the relation between the potential effects and the plant's family is a useful guide for physicians and researchers. according to this finding, the volatile oils of plant families such as pinaceae have good potential for use in infectious and inflammatory respiratory diseases, in both research and treatment. these plant based essential oils have a good feasibility for presentation as therapeutics in many of the respiratory infections and inflammatory diseases. although these natural compounds have been used as complementary therapeutics, scientists must still give attention to their safety and dosage a comparison of two regimens for the treatment of mycobacterium avium complex bacteremia in aids: rifabutin, ethambutol, and clarithromycin versus rifampin, ethambutol, clofazimine, and ciprofloxacin ceftazidime alone and in combination in patients with cystic fibrosis: lack of efficacy in treatment of severe respiratory infections caused by pseudomonas cepacia antibiotic therapy of community respiratory tract infections: strategies for optimal outcomes and minimized resistance emergence natural products in drug discovery and development plant products as antimicrobial agents new antimicrobials of plant origin. perspectives on new crops and new uses antiviral properties of ent-labdene diterpenes of andrographis paniculata nees, inhibitors of herpes simplex virus type 1 antiviral screening of british columbian medicinal plants essential oils of aromatic plants with antibacterial, antifungal, antiviral, and cytotoxic properties-an overview natural products-the future scaffolds for novel antibiotics? molecular understanding and modern application of traditional medicines: triumphs and trials who. who traditional medicine strategy complementary and alternative medicine in chronic liver disease uses and abuses of plant-derived smoke: its ethnobotany as hallucinogen, perfume, incense, and medicine anti-nociceptive and anti-inflammatory effects of some jordanian medicinal plant extracts antibacterial and anti-inflammatory activities of some plants used for medicinal purposes in kenya antioxidant activity of anti-inflammatory plant extracts anti-inflammatory, anti-cholinesterase and mutagenic effects of extracts obtained from some trees used in south african traditional medicine screening of the topical anti-inflammatory activity of some central american plants screening of medicinal plants used in lesotho for anti-bacterial and antiinflammatory activity traditionally used thai medicinal plants: in vitro anti-inflammatory, anticancer and antioxidant activities medicinal plants of south africa medicinal smokes importance of novel drug delivery systems in herbal medicines antibacterial and antifungal terpenes from pilgerodendron uviferum (d. don) florin effects of two terpene alcohols on the antibacterial activity and the mode of action of farnesol against staphylococcus aureus antibacterial trichorabdal diterpenes from rabdosia trichocarpa recent developments in the bioactivity of mono-and diterpenes: anticancer and antimicrobial activity anti-inflammatory effect of kaurenoic acid, a diterpene from copaifera langsdorffii on acetic acid-induced colitis in rats antibacterial and antifungal properties of essential oil components mechanisms of antibacterial action of three monoterpenes factors that interact with the antibacterial action of thyme essential oil and its active constituents interaction of four monoterpenes contained in essential oils with model membranes: implications for their antibacterial activity chemical composition and antibacterial activity of the essential oil from agathis dammara (lamb.) rich fresh leaves chemical composition and antimicrobial activity of the essential oil from microlicia crenulata drug properties affecting aerosol behavior metabolic effects of salbutamol: comparison of aerosol and intravenous administration effect of beclomethasone dipropionate delivered by aerosol in patients with asthma effects of standardized myrtol in therapy of acute sinusitisresults of a double-blind, randomized multicenter study compared with placebo effect of a secretolytic and a combination of pinene, limonene and cineole on mucociliary clearance in patients with chronic obstructive pulmonary disease prophylactic aromatherapy for supervening infections in patients with chronic bronchitis. statistical evaluation conducted in clinics against a placebo evaluation of combined antibacterial effects of eugenol, cinnamaldehyde, thymol, and carvacrol against e. coli with an improved method antimicrobial agents from plants: antibacterial activity of plant volatile oils compositions and the in vitro antimicrobial activities of the essential oils of achillea setacea and achillea teretifolia (compositae) screening of the antibacterial effects of a variety of essential oils on respiratory tract pathogens, using a modified dilution assay method impedance measurements to study the antimicrobial activity of essential oils from lamiaceae and compositae antibacterial activity of essential oils and their major constituents against respiratory tract pathogens by gaseous contact minimum inhibitory concentrations of herbal essential oils and monolaurin for gram-positive and gram-negative bacteria antimicrobial activity of essential oils from plants against selected pathogenic and saprophytic micro-organisms mechanisms of membrane toxicity of hydrocarbons interactions of cyclic hydrocarbons with biological membranes in vitro antibacterial activity of some aliphatic aldehydes from olea europaea l study on the mechanisms of the antibacterial action of some plant α, β-unsaturated aldehydes antimicrobial effects of essential oils on streptococcus pneumoniae control of micro-organisms, the control of micro-organisms by physical agents antimicrobial properties of essential oil constituents thymol and related alkyl phenols activate the htrpa1 channel characterization of the action of selected essential oil components on gram-negative bacteria mechanisms of bactericidal action of cinnamaldehyde against listeria monocytogenes and of eugenol against l. monocytogenes and lactobacillus sakei antimicrobial activity of aroma chemicals and essential oils antimicrobial action of palmarosa oil (cymbopogon martinii) on saccharomyces cerevisiae comparison of antimicrobial properties of monoterpenes and their carbonylated products antifungal activity and molecular orbital energies of aldehyde compounds from oils of higher plants antimicrobial activity of the olive oil flavor compounds antibacterial activity of long-chain alcohols: the role of hydrophobic alkyl groups the antibacterial effects of terpene alcohols on staphylococcus aureus and their mode of action antibacterial activities against staphylococcus aureus of terpene alcohols with aliphatic carbon chains antibacterial activity of long-chain fatty alcohols against staphylococcus aureus sensitization of staphylococcus aureus and escherichia coli to antibiotics by the sesquiterpenoids nerolidol, farnesol, bisabolol, and apritone relationship between bioactivity and chemical composition of commercial essential oils chemical preservatives and natural antimicrobial compounds essential oils: their antibacterial properties and potential applications in foods-a review disruption of escherichia coli, listeria monocytogenes and lactobacillus sakei cellular membranes by plant oil aromatics the phenolic hydroxyl group of carvacrol is essential for action against the food-borne pathogen bacillus cereus techniques for evaluation of medicinal plant products as antimicrobial agent: current methods and future trends solid-and vapor phase antimicrobial activities of six essential oils: susceptibility of selected foodborne bacterial and fungal strains antimicrobial activity of essential oils and other plant extracts a sensitive and quick microplate method to determine the minimal inhibitory concentration of plant extracts for bacteria anti-infective potential of natural products: how to develop a stronger in vitro 'proof-of-concept' antiviral activity of the essential oil of melaleuca alternifolia betche) cheel (tea tree) against tobacco mosaic virus the experiment study of the anti-virus effects of zedoary oil on influenza virus and respiratory syncytial virus anti-inflammatory effects of long-chain n-3 pufa in rhinovirus-infected cultured airway epithelial cells patchouli alcohol: in vitro direct antiinfluenza virus sesquiterpene in pogostemon cablin benth chemical compositions and anti-influenza activities of essential oils from mosla dianthera chemical composition of the volatile oil from cynanchum stauntonii and its activities of anti-influenza virus chemical composition of the essential oil of elephantopus scaber from southern china phytochemical analysis and in vitro antiviral activities of the essential oils of seven lebanon species broad spectrum anti-viral herbal composition. google patents respiratory infection prevention and treatment with terpene-containing compositions. google patents the role of the alveolar macrophage in the clearance of bacteria from the lung resolution of inflammation: the beginning programs the end electron microscopic study of the phagocytosis process in lung macrophages and polymorphonuclear neutrophils in lung defense and injury role of neutrophil elastase in hypersecretion during copd exacerbations, and proposed therapies transcriptional activation of the interleukin-8 gene by respiratory syncytial virus infection in alveolar epithelial cells: nuclear translocation of the rela transcription factor as a mechanism producing airway mucosal inflammation neutrophil elastase in respiratory epithelial lining fluid of individuals with cystic fibrosis induces interleukin-8 gene expression in a human bronchial epithelial cell line modulation of neurogenic inflammation: novel approaches to inflammatory disease chemical composition and antimicrobial activity of the essential oil of cordia verbenacea anti-inflammatory activity of 1.8-cineol (eucalyptol) in bronchial asthma: a double-blind placebo-controlled trial inhibition of 5-lipoxygenase by essential oils and other natural fragrant extracts terpenoids: natural inhibitors of nf-kb signaling with anti-inflammatory and anticancer potential immune-modifying and antimicrobial effects of eucalyptus oil and simple inhalation devices depletion of murine cd4+ t lymphocytes prevents antigeninduced airway hyperreactivity and pulmonary eosinophilia ibuprofen attenuates the inflammatory response to pseudomonas aeruginosa in a rat model of chronic pulmonary infection respiratory viral infections in the elderly key: cord-315037-lmur80te authors: lin, chien-yu; hwang, david; chiu, nan-chang; weng, li-chuan; liu, hsin-fu; mu, jung-jung; liu, chang-pan; chi, hsin title: increased detection of viruses in children with respiratory tract infection using pcr date: 2020-01-15 journal: int j environ res public health doi: 10.3390/ijerph17020564 sha: doc_id: 315037 cord_uid: lmur80te respiratory viruses are a common cause of respiratory tract infection (rti), particularly in neonates and children. rapid and accurate diagnosis of viral infections could improve clinical outcomes and reduce the use of antibiotics and treatment sessions. advances in diagnostic technology contribute to the accurate detection of viruses. we performed a multiplex real-time polymerase chain reaction (pcr) to investigate the viral etiology in pediatric patients and compared the detection rates with those determined using traditional antigen tests and virus cultures. fifteen respiratory viruses were included in our investigation: respiratory syncytial virus a/b (rsv), influenza virus a (flua) and influenza virus b (flub), human metapneumovirus (mpv), enterovirus (ev), human parainfluenza virus (piv) types 1–4, human rhinovirus (rv), human coronavirus oc43, nl63, and 229e, human adenovirus (adv), and human bocavirus (boca). in total, 474 specimens were collected and tested. respiratory viruses were detected more frequently by pcr (357, 75.3%) than they were by traditional tests (229, 49.3%). the leading pathogens were rsv (113, 23.8%), rv (72, 15.2%), piv3 (53, 11.2%), flua (51, 10.8%), and adv (48, 10.1%). for children younger than 5 years, rsv and rv were most prevalent; for children older than 5 years, flua and adv were the most frequently detected. of the specimens, 25.8% (92/357) were coinfected with two or more viruses. rv, boca, piv2, flub, and piv4 had higher rates of coinfection; mpv and piv1 had the lowest rates of coinfection (9.1% and 5.3%). to conclude, the detection power of pcr was better than that of traditional antigen tests and virus cultures when considering the detection of respiratory viruses. rsv and rv were the leading viral pathogens identified in the respiratory specimens. one-quarter of the positive specimens were coinfected with two or more viruses. in the future, further application of pcr may contribute to the rapid and accurate diagnosis of respiratory viruses and could improve patient outcomes. respiratory viruses are ubiquitous and cause a large variety of clinical symptoms. respiratory tract infection (rti) is undoubtedly common, and the recognition of a causative pathogen contributes to the appropriate management [1] . in addition to the well-known respiratory viruses, such as respiratory syncytial virus (rsv) and influenza virus, human metapneumovirus (mpv) was identified in 2001, followed by the discovery of other respiratory viruses [2, 3] . currently, the disease burden of respiratory viruses is beyond our knowledge. respiratory viruses have been detected in more than two-thirds of children with radiographically confirmed community-acquired pneumonia (cap) [4] . similarly, in the united states, molecular diagnostics revealed viral infection in 43%-67% of pediatric cap cases [5] . respiratory viruses also play an important role in adult pneumonia and are detected in 15%-56% of adult cap cases [5, 6] . viruses are responsible for the majority of respiratory infectious diseases in both children and adults, causing a massive disease burden [7, 8] . furthermore, the identification of causative viruses enables the accurate diagnosis of respiratory infections and prescription of specific antiviral agents against certain viruses, such as oseltamivir for influenza viruses, and improves evaluation of the prognosis [9] [10] [11] . recognizing causative viruses can also provide information on the appropriate infection control measures, which can potentially reduce unnecessary hospital stays and allow discontinuation of unnecessary antibiotics [12] [13] [14] . in summary, respiratory virus infection is common, and testing for respiratory pathogens can improve understanding of the roles of pathogens in respiratory diseases and contribute to their better clinical management [15] . a timely and accurate diagnosis of viral infection can be challenging. rapid antigen tests are used to detect influenza virus infection worldwide, but there are some concerns regarding the sensitivity of currently available viral antigen tests [6, 15] . technological advances have improved the sensitivity, accessibility, and utility of viral diagnostic tools [16] . molecular assays have been developed and progressively multiplexed to diagnose a large number of respiratory viruses in a single assay with excellent sensitivity and specificity [10, [17] [18] [19] [20] . the importance of molecular-based diagnostic modalities is currently on the rise, and polymerase chain reaction (pcr) technology is being increasingly used in the clinic to rapidly diagnose respiratory infections [19] . this study aims to detect respiratory viruses in children using pcr and to compare the detection power of this technique against that when using traditional antigen tests and virus cultures. the clinical conditions were also investigated. this study was approved by the institutional review board of the mackay memorial hospital, taipei, taiwan (approval no. 14mmhis030). for children with respiratory symptoms and with a clinical suspicion of virus infection, a test for rsv antigen test, human parainfluenza virus (piv) type 3 antigen test, viral pcr for enterovirus, or viral cultures was prescribed by the judgment of pediatricians. a nasopharyngeal swab or aspiration was performed by pediatricians using a small swab that was inserted into the nostril. the cotton swab was then inserted and mixed in a 2.5 ml viral transport medium. after testing original tests, the residual specimens were stored at −20 • the viral nucleic acids were extracted from 200-µl of each sample using the high pure viral nucleic acid kit (roche applied science, castle hill, germany) following the manufacturer's instructions. extracted nucleic acids were eluted in 100 µl elution buffer and stored at −70 • c. reverse transcription (rt) was carried out using high-capacity complementary dna (cdna) reverse transcription kit (applied biosystems part number: 4375575 rev.c). the total volume of rt mix was 40 µl per reaction, containing 4 µl rt buffer (10×), 1.6 µl dntp mixture (25 mm of each dntp), 4 µl random primers (10×), 2 µl rnase inhibitor (20 u/µl), 2 µl multiscribe reverse transcriptase (50 u/µl), and 26.4 µl template, whereby the rt reagent mix was prepared on ice. the thermal profile of the rt program consisted of 10 min incubation at 25 • c, 120 min rt at 37 • c, 5 min rt inactivation at 85 • c, and cooling down to 4 • c and was performed in a 96-well geneamp pcr system 9700. the resulting cdna was stored at −20 • c. the following multiplex pcr assays were performed for each sample to detect rna/dna of 15 respiratory viruses, including rsv a or b, flua, flub, human enterovirus (ev), mpv, human parainfluenza virus types 1-4, human rhinovirus (rv), coronavirus oc43/nl63/229e, human adenovirus (adv), and human bocavirus (boca). in the present study, previously published primers and pcr assays were used for multiplex rt-pcr and the details of primers are summarized in table s1 [21] [22] [23] [24] [25] [26] . briefly, the pcr reaction was performed by adding 3 µl rt product to 22 µl pcr mix. the conditions of amplification were as follows: initial denaturation at 95 • c for 10 min; followed by 40 cycles of 95 • c for 1 min, 60 • c for 1 min, and 72 • c for 1 min; a final extension at 72 • c for 10 min. amplification products were visualized by 1% agarose gel electrophoresis with ethidium bromide staining and observed under ultraviolet light. for each pcr assay, a positive and negative control for each parameter was performed. internal control was also performed to detect sample inhibition and avoid false-negative results. external and internal amplification controls were designed for quality control and validation. the detection limits of the multiplex pcr assays were 10 to 100 copies of the individual virus. student's t-test and chi-square test were used to analyze and compare the categorical demographic characteristics including clinical manifestations and laboratory tests. kappa statistic was used to evaluate the consistency between pcr and original tests (categorical variables) and cohen's kappa coefficient (κ) was regarded as poor to fair consistency if κ ≤ 0.4; moderate consistency if 0.41 ≤ κ ≤ 0.60; and good consistency if 0.61 < κ. a two-sided p < 0.05 was considered statistically significant. statistical analyses were performed using the spss software version 23.0 (spss inc., chicago, il, usa). in total, 474 residual specimens for detecting respiratory viruses were obtained, including 156 specimens for rsv antigen tests, 58 for parainfluenza virus antigen tests, and 260 for viral cultures. table 1 summarizes the detection rates of viruses. the overall positive rate for traditional tests was 48.3% (229/474), and the individual positive rate was 28.8% for rsv antigen tests, 5.2% for parainfluenza virus antigen tests, and 69.6% for viral cultures. all specimens underwent present multiplex pcr for the 15 abovementioned viruses, and higher detection rates were observed; 357 (75.3%) specimens were positive for at least one virus. the leading pathogens were rsv (113, 23.8%), rv (72, 15.2%), piv3 (53, 11.2%), flua (51, 10.8%), and adv (48, 10.1%) ( figure 1 ). among these positive specimens, 25.8% (92/357) were coinfected with two or more viruses. the coinfection rates of individual virus were demonstrated in table 1 . we observed that rv, boca, piv2, flub, and piv4 were associated with higher rates of coinfection. however, mpv and piv1 had the lowest rates of coinfection (9.1% and 5.3%). the consistency of the results between virus culture and pcr was also investigated. with the exception of flub, a high consistency was observed between virus culture and pcr (coefficient k: 0.72~0.961, p < 0.01, table 1 ). were associated with higher rates of coinfection. however, mpv and piv1 had the lowest rates of coinfection (9.1% and 5.3%). the consistency of the results between virus culture and pcr was also investigated. with the exception of flub, a high consistency was observed between virus culture and pcr (coefficient k: 0.72~0.961, p < 0.01, table 1 ). the seasonality of virus detection is shown in figure 2 ; virus detection was more common in summer and autumn. the seasonal distribution of the five most commonly detected viruses (rsv, rv, piv3, flua, and adv) was also plotted. we also compared the detection rate in different age groups ( figure 3 ). for children younger than 5 years, rsv and rv were the leading pathogens; for older children, flua and adv were prevalent. the clinical manifestations and laboratory tests are summarized in table 2 (complete data available in table s2 ). except for age, no obvious differences were found between individual viruses. more than one-quarter of the specimens were coinfected with more than one virus. we further compared the clinical manifestations of patients in which either no viruses, a single infection, or coinfections were detected. the age, body weight, duration of hospitalization, intensive care unit (icu) stay, white blood cell counts (wbc), and c-reactive protein (crp) levels were not significantly different, with higher platelet counts being the only difference noted in patients with coinfections (table 3) . were found between individual viruses. more than one-quarter of the specimens were coinfected with more than one virus. we further compared the clinical manifestations of patients in which either no viruses, a single infection, or coinfections were detected. the age, body weight, duration of hospitalization, intensive care unit (icu) stay, white blood cell counts (wbc), and c-reactive protein (crp) levels were not significantly different, with higher platelet counts being the only difference noted in patients with coinfections (table 3 ). were found between individual viruses. more than one-quarter of the specimens were coinfected with more than one virus. we further compared the clinical manifestations of patients in which either no viruses, a single infection, or coinfections were detected. the age, body weight, duration of hospitalization, intensive care unit (icu) stay, white blood cell counts (wbc), and c-reactive protein (crp) levels were not significantly different, with higher platelet counts being the only difference noted in patients with coinfections (table 3 ). in this study, we found that pcr had higher detection rates compared with traditional antigen tests and viral cultures (75.3% vs. 48.3%). rsv, rv, and piv3 were the leading pathogens detected in pediatric rti patients. however, flua, adv, and ev were more prevalent in children older than 5 years. knowledge of epidemiology contributes to the awareness of pathogen, accurate diagnosis, and prompt management. we also found that approximately one-quarter of specimens were coinfected with two or more viruses. however, no obvious differences in clinical manifestations and laboratory tests were found in individual virus infection or between single infection and coinfection; the clinical significance of coinfection was not fully elucidated. a rapid and accurate diagnosis of respiratory viruses is increasingly important in clinical settings. the availability of rapid diagnostic assays is essential for optimizing the efforts of infection control teams to reduce the transmission of virulent or resistant pathogens in hospitals [27] . nucleic acid amplification tests are the new gold standard for the diagnosis of respiratory viruses. our study has shown high detectability of pcr for respiratory viruses, suggesting that pcr-based diagnostic tools may be practical for detecting a wide range of respiratory viruses. viral infection can be fatal, especially in premature infants and infants with congenital heart disease [28] . in a previous study, symptomatic and asymptomatic premature infants were prospectively screened in a neonatal icu using multiplex pcr twice weekly; respiratory viruses were identified in 52% of prematurely born infants during their birth hospitalization. their length of hospital stay was significantly longer (70 days vs. 35 days), and bronchopulmonary diseases were more frequent in infected infants [8] . in adult and pediatric patients, the major impact of respiratory viral infections with hematologic malignancies, hematopoietic stem cell transplantation, and solid organ transplantation has been recognized over the past decade [7, 28] . in the most immunocompromised populations, respiratory viruses have a high rate of progression to pneumonia (20%-40%). the mortality among those patients ranged from 30% to 50%. the application of multiplex pcr for respiratory virus detection in high-risk groups has been proved to be valuable [17] . our study showed a high detectability of pcr for respiratory viruses, suggesting that pcr-based diagnostic tools may be helpful for detecting a wider range of respiratory viruses. we also showed a high consistency of pcr with virus cultures, except for flub, suggesting the accuracy of the pcr method. virus culture is time-consuming and not feasible for clinical practice. it was even impossible to detect some viruses by virus cultures, e.g., coronaviruses (229e, oc43, nl63, and hku-1), piv4, rv, and boca. approximately just over half of the viruses could be detected after the wide application of pcr. these results re-enforce the importance of pcr-based diagnosis. viral infections are ubiquitous and may present with fever and respiratory symptoms. it is sometimes difficult to differentiate between bacterial infections and viral infections, and thus the use of unnecessary antibiotics is common. antimicrobial resistance (amr) has been increasing worldwide, resulting in poor treatment responses and deplorable clinical outcomes [29] . the problem of amr is an urgent and critical health threat and is directly associated with the overuse of antibiotics [30] . antibiotic treatment does not improve the clinical outcomes of viral infections [31] . decreasing the use of unnecessary antibiotics is the key to combating amr, and accurate and rapid diagnosis is crucial to decrease antibiotic prescriptions with a minimized risk [9, 10, 32] . the present study demonstrates that pcr has higher detectability for respiratory viruses compared to traditional antigen tests and viral cultures. pcr-based viral detection may help physicians to make appropriate decisions and decrease unnecessary antibiotic use. furthermore, the precise diagnosis of certain viruses may contribute to timely antiviral agent treatment, e.g., oseltamivir against influenza infections. we discovered that influenza is common among pediatric patients (11.6% of respiratory specimens) and is the most commonly detected pathogen in older children (27% in children aged 5-9 years and 16.7% in children older than 10 years). rapid diagnosis of influenza viruses and early treatment with oseltamivir or peramivir is crucial. in addition, prompt diagnosis of respiratory viruses also contributes to appropriate infection control measures and isolation care [8, 27] . in recent years, the cost of pcr testing has decreased, and the availability and feasibility has been largely improved. some commercialized pcr machines are increasingly available and may serve in point-of-care testing [18] . hence, the widespread use of pcr-based detection of respiratory viruses is increasing and may become more practical. the incidence of etiologic pathogens differs between adults and children. it has been reported by jain et al. that, among the hospitalized adults with cap, pathogens were detected in 38% of patients, and the leading pathogens were rv (9%) and influenza viruses (6%) [6] . by contrast, pathogens were detected in 81% of the hospitalized cap children, and the leading pathogens were rsv (28%), rv (27%), and mpv (13%) [4] . generally speaking, viral infections are more prevalent in children than in adults. the leading pathogens may also differ according to geographical region, climate, season, and year. the leading pathogens detected in our study were rsv (23.8%), rv (15.2%), and piv3 (11.2%). a previous study conducted in taiwan found that rsv was the most common pathogen (41.7%), followed by mpv (27.1%), boca (6.3%), and ev (6.3%) [33] . some important studies investigating the epidemiology of respiratory tract infection are summarized in table 4 [4, 6, 13, 15, 23, 34, 35] . rsv is always the most common pathogen in young children worldwide, but the accompanying pathogens are not always the same [4, 5, 7, 13, 36, 37] . virus detection was more common in summer and autumn in our study. taiwan is located in a subtropical zone, where there are no swift changes in temperature amplitudes. although rsv infections occur biennially, with peaks reported in the spring and autumn in taiwan, variations in rsv infections are not particularly large [38] [39] [40] . detection of respiratory viruses could enable estimation of the local epidemiology of respiratory viral infection and help pediatricians to improve their clinical judgments. one-quarter of the positive specimens were coinfected with other respiratory viruses in our study. a similar prevalence was found in previous studies [4, 41, 42] . the rates of coinfection were between 18.8% to 36.2% in previous studies (table 4 ). with the advances in diagnostic testing, the number of detectable viruses will increase. however, the clinical significance of coinfection remains unclear [43] [44] [45] [46] . some studies reported increased severity of coinfection [45, 46] , but the impact of coinfection was not particularly obvious in other studies [47, 48] . diversities in the study design, population, and detection methods may be the reason for this inconclusiveness. when we compared the clinical manifestations and laboratory tests for patients with negative detection, single infection, and coinfections, we found no statistically significant differences in age, body weight, hospitalization duration, icu stay, crp level, and complete blood cell counts; although higher platelet counts were observed in patients with coinfection. further studies are required to clarify the clinical significance of our findings. the strength of our study lies in the comprehensive detection of respiratory viruses and further comparison of the clinical manifestations and laboratory tests in single and coinfection. our study is subject to some limitations that warrant discussion. firstly, although our findings were consistent with those of previous studies, respiratory specimens were not collected in all patients with respiratory symptoms. the prevalence of ev was underestimated because the clinical diagnosis of ev infection relies mainly on the presence of oral vesicles. further virus culture might not be performed when vesicles over oropharynx were found. secondly, we did not include bacteria in our detection spectra. some bacteria such as mycoplasma pneumoniae and streptococcus pneumoniae also play an important role in respiratory infections and commonly cause coinfections with other pathogens [49] . furthermore, some respiratory viruses were not included in our testing, such as the middle east respiratory syndrome coronavirus and human polyomaviruses ki and wu [50] . the use of pcr resulted in greater detection of respiratory viruses than the use of traditional rapid antigen tests or viral cultures. more than half of the respiratory specimens that showed negative detection in the original tests were positive for the pcr-based detection method. further application of pcr has great potential for rapid and accurate diagnosis and will be beneficial for primary pediatricians. furthermore, rsv and rv were the leading pathogens identified in our pediatric respiratory specimens; in children older than 5 years, flua, adv, and ev were more prevalent. approximately one-quarter of the positive respiratory specimens were coinfected with two or more viruses, but no obvious differences in clinical manifestations and laboratory tests were observed between single infection and coinfection. further studies are warranted to investigate the accuracy, feasibility, accessibility, and cost of pcr in detecting respiratory viruses, and to clarify the clinical significance of coinfection. supplementary materials: the following are available online at http://www.mdpi.com/1660-4601/17/2/564/s1. table s1 : primers and pcr assays for multiplex pcr. table s2 a newly discovered human pneumovirus isolated from young children with respiratory tract disease identification of a new human coronavirus community-acquired pneumonia requiring hospitalization among us children viral pneumonia community-acquired pneumonia requiring hospitalization among us respiratory viral infections during the first 28 days after transplantation in pediatric hematopoietic stem cell transplant recipients unrecognized viral respiratory tract infections in premature infants during their birth hospitalization: a prospective surveillance study in two neonatal intensive care units implementation of rapid diagnostics with antimicrobial stewardship the role of multiplex pcr in respiratory tract infections in children early diagnosis of lower respiratory tract infections (point-of-care tests) etiological diagnosis reduces the use of antibiotics in infants with bronchiolitis casas, i. spectrum of respiratory viruses in children with community-acquired pneumonia resisting the use of antibiotics for viral infections diagnostic value of respiratory virus detection in symptomatic children using real-time pcr are we ready for novel detection methods to treat respiratory pathogens in hospital-acquired pneumonia? multiplex pcr system for the rapid diagnosis of respiratory virus infection: systematic review and meta-analysis the clinical significance of filmarray respiratory panel in diagnosing community-acquired pneumonia comparison of real-time pcr assays with fluorescent-antibody assays for diagnosis of respiratory virus infections in children rapid and sensitive method using multiplex real-time pcr for diagnosis of infections by influenza a and influenza b viruses, respiratory syncytial virus, and parainfluenza viruses 1, 2, 3, and 4 public health responses to reemergence of animal rabies frequent detection of viral coinfection in children hospitalized with acute respiratory tract infection using a real-time polymerase chain reaction an economical tandem multiplex real-time pcr technique for the detection of a comprehensive range of respiratory pathogens real-time reverse transcriptase pcr assay for detection of human metapneumoviruses from all known genetic lineages design and performance testing of quantitative real time pcr assays for influenza a and b viral load measurement simultaneous detection of influenza a, influenza b, and respiratory syncytial viruses and subtyping of influenza a h3n2 virus and h1n1 (2009) virus by multiplex real-time pcr rapid testing for respiratory syncytial virus in a paediatric emergency department: benefits for infection control and bed management viral respiratory tract infections in allogeneic hematopoietic stem cell transplantation recipients in the era of molecular testing the use of antimicrobial agents after diagnosis of viral respiratory tract infections in hospitalized adults: antibiotics or anxiolytics? antimicrobial drug prescription in ambulatory care settings, united states antibiotics for bronchiolitis in children reducing unnecessary chest x-rays, antibiotics and bronchodilators through implementation of the nice bronchiolitis guideline viral etiology of acute lower respiratory tract infections in hospitalized young children in northern taiwan respiratory viral infections detected by multiplex pcr among pediatric patients with lower respiratory tract infections seen at an urban hospital in delhi from etiologic spectrum and occurrence of coinfections in children hospitalized with community-acquired pneumonia respiratory virus of severe pneumonia in south korea: prevalence and clinical implications differential risk of hospitalization among single virus infections causing influenza like illnesses epidemiological study of hospitalization associated with respiratory syncytial virus infection in taiwanese children between prolonged seasonality of respiratory syncytial virus infection among preterm infants in a subtropical climate global respiratory syncytial virus-associated mortality in young children (rsv gold): a retrospective case series epidemiology and clinical characteristics of community-acquired pneumonia in hospitalized children etiology and impact of coinfections in children hospitalized with community-acquired pneumonia the role of infections and coinfections with newly identified and emerging respiratory viruses in children co-infections with influenza and other respiratory viruses respiratory syncytial virus: co-infection and paediatric lower respiratory tract infections dual infection of infants by human metapneumovirus and human respiratory syncytial virus is strongly associated with severe bronchiolitis human metapneumovirus as a causative agent of acute bronchiolitis in infants clinical features and complete genome characterization of a distinct human rhinovirus (hrv) genetic cluster, probably representing a previously undetected hrv species, hrv-c, associated with acute respiratory illness in children clinical and epidemiological characteristics in children with community-acquired mycoplasma pneumonia in taiwan: a nationwide surveillance the human polyomaviruses ki and wu: virological background and clinical implications acknowledgments: this study is supported by mackay memorial hospital, taipei, taiwan (project number: mmh-103-65). this study was approved by the institutional review board of the mackay memorial hospital, taipei, taiwan (approval no. 14mmhis030). the authors declare no conflict of interest. key: cord-325353-tx6s4ggu authors: restori, katherine h.; srinivasa, bharat t.; ward, brian j.; fixman, elizabeth d. title: neonatal immunity, respiratory virus infections, and the development of asthma date: 2018-06-04 journal: front immunol doi: 10.3389/fimmu.2018.01249 sha: doc_id: 325353 cord_uid: tx6s4ggu infants are exposed to a wide range of potential pathogens in the first months of life. although maternal antibodies acquired transplacentally protect full-term neonates from many systemic pathogens, infections at mucosal surfaces still occur with great frequency, causing significant morbidity and mortality. at least part of this elevated risk is attributable to the neonatal immune system that tends to favor t regulatory and th2 type responses when microbes are first encountered. early-life infection with respiratory viruses is of particular interest because such exposures can disrupt normal lung development and increase the risk of chronic respiratory conditions, such as asthma. the immunologic mechanisms that underlie neonatal host–virus interactions that contribute to the subsequent development of asthma have not yet been fully defined. the goals of this review are (1) to outline the differences between the neonatal and adult immune systems and (2) to present murine and human data that support the hypothesis that early-life interactions between the immune system and respiratory viruses can create a lung environment conducive to the development of asthma. are well known and include, among others, respiratory syncytial virus (rsv), influenza a/b viruses, rhinoviruses (rvs), human metapneumovirus, parainfluenzaviruses 1-4 (piv), bocaviruses, coronaviruses, and certain adenovirus strains. while infant-pattern immune responses may enhance susceptibility to some infections, mounting adult-type responses may not be optimal for overall development either. the neonatal immune system must first and foremost distinguish between self and nonself, and then rapidly proceed to distinguish between benign or even "helpful" non-self (e.g., the commensal microbiome) and potential pathogens. during this period of immune education, the developing immune system has to strike a delicate and pathogenspecific balance between the induction of potentially damaging, pro-inflammatory responses mediated by t helper (th) cell 1-type (th1) and some th17-type cells, less damaging inflammatory responses (e.g., th2-type) or even suppressive responses mediated by regulatory t cells (treg) . although the induction of suppressive responses may seem counterintuitive when dealing with potential pathogens, such responses appear to be essential to protect the fetus from reacting too vigorously to maternal antigens (3) or to colonization of the neonatal gut and other body surfaces with the normal microbial flora (4) . the "cost" to the infant of mounting an aggressive, pro-inflammatory response can be very high. early-life inflammation has been associated with a wide range of negative long-term consequences including overall development of the body and the brain, neuro-psychological reactions (e.g., to pain) and patterns of inflammatory response (5, 6) [reviewed in ref. (7, 8) ]. it is, therefore, entirely plausible that early-life experiences with respiratory pathogens can have long-lasting effects on the lung. a number of epidemiological studies have linked respiratory viral infection during infancy with the later development of asthma [reviewed in ref. (9) (10) (11) ]. it is currently unknown whether this association is a direct effect of viral replication in respiratory tissues or results from a virus-induced exacerbation of an underlying predisposition to atopy. however, there is evidence from both human and animal studies to support both hypotheses. the purpose of this review is to examine the immunology of the host-virus interaction during the neonatal period in the context of asthma development. we will first briefly describe how the neonatal immune system differs from that of the adult. because rsv, influenza, and rvs cause a large proportion of respiratorytract infections in neonates, we will focus primarily on these three pathogens as models to better understand how early-life infection and antiviral immune responses might contribute to the subsequent development of asthma. adaptive immunity in the neonate prior to the 1980s, it was commonly thought that the neonatal immune system was in a state of tolerance (12) , as demonstrated by mouse experiments in the 1950s showing the apparent lack of recall responses to antigens injected soon after birth (13) . indeed, neonates were considered by some to be "immunodeficient" (14) . upon discovery of different patterns of t cell response in the mid-1980s [e.g., th1 vs. th2 type: reviewed in ref. (15) ], it subsequently became clear that what appeared to be a deficiency in responding to recall antigens was instead a reflection of an intrinsic th2/treg bias in neonatal mice (16, 17) . a quarter of a century later, th1-and th2-type responses are two of the most well characterized aspects of cellular immunity. the classical th1 response is characterized by production of the cytokines interferon (ifn)γ and interleukin (il)-12, whereas th2 responses are typified by production of il-4 and il-13. th1-type responses tend to be pro-inflammatory in nature, with increases in cd8 + t cells, and are most often associated with challenges posed by intracellular organisms, including viruses. th2 responses typically play a central role in defending against invasive helminths, but are also important in modulating potentially damaging inflammatory responses as well as in driving allergic pathologies (e.g., atopy, asthma). other more recently recognized patterns of immune response have also been implicated in either promoting or preventing the development or evolution of asthma, including th17-, th9-, and treg. in particular, th17-and th9driven responses may promote exacerbations of severe asthma [reviewed in ref. (18) ] and/or promote allergic airways disease through interactions with th2 cells (19, 20) . tregs, on the other hand, play a central role in limiting excessive reactivity to self and innocuous allergens (21) . while th2 skewing may be a natural or default response pattern to microbial insults in the neonate, the neonatal immune system can induce adult-like, th1/th2 balanced or even th1predominant responses under certain conditions (17, 22, 23) . experimental situations in which such responses can be generated typically require potent th1 inducers such as cpg motifs found in dna vaccines (24) and oligonucleotide adjuvants (25, 26) , mycobacterial cell wall antigens either in the form of live bacille calmette-guerin (bcg) (27) or complete freund's adjuvant (17) , or lipid-based adjuvants, such as liposomes (28) and oil-in-water emulsions (29) . several of these powerful stimuli have recently been shown to act via ligation of pattern recognition receptors (prr) [e.g., toll-like receptor (tlr) 9 in the case of cpg motifs] (30) . even when an apparently "balanced" th1/th2 response has been generated in neonates, recall responses may still reveal an underlying th2 bias (31) . the mechanisms by which subsequent antigen recall results in a th2-biased response are still under active investigation. neonatal mouse t cells produce significantly higher levels of il-4 within 48 h of in vitro t cell receptor (tcr) stimulation (32) compared to adult t cells, which typically take several days to produce high levels of th2 cytokines (33) . this readiness to produce th2 cytokines has been attributed to hypomethylation of the th2 cytokine regulatory region on chromosome 11 in neonatal murine cd4 + t cells. a similar state of epigenetic control over th2 cytokine production has also been observed in human neonatal cd4 + t cells (34) . yoshimoto and colleagues have recently shown that epigenetic modifications of the th2 regulatory regions occur during fetal development in the mouse (35) . the th2 regulatory regions in the 14-day fetal thymus are hypomethylated and adult-like methylation patterns appear only 3-6 days after birth along with a decreased propensity to produce th2 cytokines. interestingly, this hypomethylation is restricted to the th2 regulatory regions, and is not seen in the regulatory regions of the ifnγ or foxp3 loci. other factors that could potentially influence th2-biased immune responses in neonates include relative antigen loads (36) , lower total numbers of t cells in neonatal organs (37) and the presence of fetal-origin t cells that are strongly th2-skewed (38) . cross-regulation between th subsets is well known and may be particularly effective in the very young. for example, there is evidence that th1-type signaling is actively suppressed by th2 cytokines in the context of both antigen challenge and recall in neonates. using tcr transgenic mice, li et al. showed not only that recall responses are primarily th2-like but that these responses are accompanied by apoptosis of antigen-specific th1 cells (39) . this apoptosis of th1 cells is driven predominantly by il-4, is enhanced by il-10, and occurs only in neonatal t cells (40) . neonatal (but not transplanted adult) th1 cells co-express il-13rα1 and il-4rα. upon exposure to il-4 after antigen challenge, th1 cells that carry this heteroreceptor undergo apoptosis (12) , resulting in a th2-biased antigen recall response. il-13rα1 upregulation on these th2 cells is due to relatively low frequencies of il-12-producing (cd8α + cd4 − ) dendritic cells (dcs) in the neonate-a phenomenon that is reversed by day 6 of life when cd8α + cd4 − dcs mature and begin to secrete il-12 (41) . by ~8 days after birth, neonatal murine t cells respond to gradual increases in il-12 by upregulating il-12rβ2 and suppressing il-13rα1, contributing to the sustained survival of antigen-specific th1-type t cells (42) . although the differences between neonatal and adult t cell responses are particularly striking, the capacity to mount competent humoral responses also varies considerably with age as different "waves" of b cell development occur through early-life (43) . for example, antibody responses to t cell independent antigens such as polysaccharide antigens can be very weak during the first years of life. compared to the adult humoral response to any given antigen, the neonatal response is characterized by generally lower levels of antibodies produced with a delayed onset, less efficient antibody affinity maturation (14) and higher levels of b cell apoptosis (44) . despite these clinical observations, major differences between neonatal and adult b cells have not yet been identified at the molecular level. as a result, the relative "defects" in antibody production for t cell independent antigens are likely attributable to other factors such as delayed appearance of b cells fully competent to handle such antigens or deficiencies in the organization of secondary response sites, such as lymphoid and follicular centers, which develop only a few weeks after birth in mice [reviewed in ref. (14) ]. innate immunity in the neonate striking differences in innate immunity between neonates and adults have also been characterized, particularly with regard to il-12, a key innate th1 cytokine. the half-life of il-12p40 mrna is decreased in cord blood mononuclear cells and these cells produce far less il-12 than adult peripheral blood mononuclear cells (pbmc) in response to lps (45) . the addition of il-12 to cord blood cultures increases th1-type responses to recall antigens in vitro (46) , increases the activity of natural killer (nk) cells and enhances ifnγ production (45) , and suppresses the induction of il-13rα1 (41) . however, the administration of supplemental il-12 to 1-week-old mice in vivo is not well tolerated, suppressing weight gain and increasing mortality compared to older mice (47) . dcs are the main producers of il-12 (12) and neonatal mice have fewer splenic dcs than adult animals (48) , as well as weaker responses to antigen stimulation in vitro and in vivo (12) . it is interesting that neonatal b cells can suppress both il-12 production and signaling in neonatal dcs in an il-10-dependent manner (49, 50). because dcs are positioned at the nexus of the innate and adaptive immune responses, the characteristics of neonatal dcs are likely central to the relatively weak th1 responses observed in neonates. monocyte-derived dcs from human cord blood have decreased markers of activation, as well as lower il-12p35 mrna expression upon stimulation with ligands such as lps, cd40 ligation, or poly i:c, and are poor inducers of ifnγ from adult t cells (51) . neonatal dcs also produce less ifnα and ifnβ than adult cells (52, 53) . recently, it has been suggested that poor production of ifn by early-life dcs is due, at least in part, to posttranscriptional downregulation of tlr7/9 signaling and increases in the regulatory mirnas, mir146 and mir155 (54) . one important outcome of decreased type 1 ifn signaling would be diminished ifnγ responses in neonates. both cell surface and cytoplasmic prrs, including tlrs, nod-like receptors, and retinoic acid induced gene i (rig-i)like receptors, play important roles in the recognition of common microbial products (e.g., lps, double stranded rna) and in triggering immune responses in key innate effector cells such as monocytes and dcs. responses of cord blood dcs to tlr ligation are both qualitatively and quantitatively different from adult dc responses, despite having similar levels of mrna (53, 55) . using agonists specific to each tlr, kollmann et al. (56) studied single cell responses of dcs and monocytes in human cord blood compared to neonatal or adult cells differentiated from pbmc. overall, the neonatal monocytes and dcs produced more il-6, il-23, and il-1β, which can induce th17 differentiation/activity, as well as il-10, a classic immunosuppressive cytokine, but less of the th1-associated cytokines: ifnα, ifnγ, tumor necrosis factor (tnf)α, and il-12p70. in another study of early-life responses, the ratio of tlr-induced il-6/tnfα production by neonatal monocytes was high compared to adult cells. newborn serum was found to have a similarly skewed ratio of these cytokines (57) . serial measurements in human infants suggest that the ratio of tlr-driven inflammatory (e.g., ifnα, il-12p70, ifnγ) vs. suppressive/regulatory cytokines (e.g., il-10) shifts slowly toward "adult" values over the first 2 years of life (58) . several factors likely contribute to the sluggish activity of tlrs in neonatal immune cells, including lower myd88 levels (59) , decreased dna and coactivator binding of ifn regulatory factor 3 (52), and decreased nuclear translocation of ifn regulatory factor 7 (53) . together, these data suggest that stimulation of innate immune cells in neonates/infants favors th2/th17 and possibly treg differentiation rather than th1 development. this age-related bias in t cell education could plausibly play a pivotal role in the subsequent development of asthma. there are still other differences between neonatal and adult innate response capabilities. for example, fetal and the neonatal invariant natural killer t cells (inkt) cells produce equal amounts of ifnγ and il-4 upon receptor stimulation in sharp contrast to adult inkt cells that produce predominantly ifnγ (60) . although nk cells are present in higher numbers in the peripheral blood of neonates compared to adults, they express more inhibitory receptors, have truncated maturation and have lower overall functionality (61) . the nk maturation defect in neonates has also recently been linked to transforming growth factor-beta (tgfβ), since murine nk cells engineered to lack tgfβ receptor were capable of fully maturing by 10 days after birth (62) . neonatal macrophages, upon stimulation with lps and polysaccharide antigens, produce more il-10 than adult macrophages, and secrete less il-1β, il-12, tnfα, probably due to weak tlr signaling (63) . neonatal macrophages are also less responsive to ifnγ due to a defect in stat1 phosphorylation (64) despite having adult levels of phagocytic function (65) . walk and colleagues studied the expression of a number of inhibitory receptors on neonatal cells, and found increased expression of lair-1, cd31, and cd200 on a wide range of innate immune cells including neutrophils, monocytes, and nk cells as well as cd4 + and cd8 + t cells (66) . at least some of the apparent "defects" in neonatal immune cell function can be overcome using specific stimuli either alone or in combination. as mentioned above, tlr8 signaling can drive a strong pro-inflammatory response from neonatal monocytes but can also induce neonatal nk cells to produce adult levels of ifnγ in an il-12-dependent fashion (67). during the postnatal period, the lung continues maturation begun in utero and undergoes alveolarization (mice, pnd4-21; humans, 36 weeks preterm to 1-2 years) and microvascular maturation (mice, pnd4-21 mice; humans, 0-3 years) (68, 69) . a strong type-2 immune cell bias characterizes the lung mucosa during alveolarization in mice as populations of innate type 2 lymphoid cells (ilc2), mast cells, eosinophils, and basophils increase (69) . furthermore, a large proportion of tissue resident alveolar macrophages, unique populations that are self-maintained throughout life and which develop from fetal liver monocytes beginning at pnd3 in mice (70) , express cd206, indicative of a type-2 alternatively activate phenotype (71) . conventional cd11b + neonatal murine lung dcs, though few in number, process antigen more efficiently and more readily express ccr7 (69, 72, 73) than adult dcs and, compared to cd103 + dcs, preferentially migrate to the draining (mediastinal) lymph node and promote th2 responses (69) . in mice, lung delivery of house dust mite (hdm) extracts at this early stage of development promotes enhanced allergic airways disease [i.e., increased eosinophil recruitment to the lung, airway hyperresponsiveness (ahr)] (69, 73, 74) , in a manner that is dependent upon il-33 (discussed in detail below) and influenced by preferential differentiation of cd4 + t cells to a th2-type phenotype expressing il-4, il-5, and il-13. in fact, additional studies in mice have investigated canonical t lymphocyte responses in the neonatal vs. adult lung (72) . in response to anti-cd3 treatment in vitro, cd3 + , cd4/cd8 double negative t cells, which are present in greater frequency during the neonatal period than in adulthood, more readily express gata-3 and produce more th2 cytokines, il-4 and il-5 (72) . interestingly, priming of neonatal or adult lung dcs with bcg induced a th2 response when cocultured with neonatal lymph node t cells, a response that switched to th1 when cocultured with lymph node t cells from adult mice (72) . altogether, these data highlight the importance the temporal window of the alveolarization stage of lung development to promote rapid, detrimental th2-type inflammatory responses in the lung. compelling new data implicate changes to the lung microbiome in differential regulation of maladaptive type-2 allergic responses between neonates and adults. the predominance of firmicutes and gammaproteobacteria in the lung is associated with allergic airways disease in both murine models of asthma (75) and human asthmatics (76) and are the major families that colonize the lung during the alveolar stage of development. these commensals change throughout ontogeny as the lung milieu adapts to harbor bacteroidetes in adulthood (73) . interestingly, helios + treg (cd4 + foxp3 + cd25 + ) cells are abundant in the neonatal lung during alveolarization, but are non-tolerogenic as repeated hdm exposure causes robust eosinophilic airway inflammation, ahr, and mucus production associated with allergic airways disease. later in life, when the lung microenvironment shifts to support bacteroidetes, helios − tregs are abundant and successfully promote regulatory, antiinflammatory responses thereby providing protection to adult mice from allergic airways disease upon exposure to hdm (73) . the neonatal lung is comprised of type 2 innate immune cells and dcs, which rapidly home to the mediastinal lns to educate naïve cd4 + t cells to develop th2 responses. furthermore, the postnatal lung supports a microbiome that promotes allergy and ineffectual treg responses. altogether, this type-2 biased neonatal mucosal lung environment has the potential to create a "perfect storm" for asthma development upon exposure to viruses or allergens. how this naturally biased state might synergize with early-life respiratory infections to create a lung microenvironment that favors the development of asthma is discussed in the next section. allergic asthma is defined as a chronic inflammatory response to inhaled allergens characterized by intermittent airway obstruction, increased th2 cytokine production, ahr and mucus production. at least 300 million people worldwide have asthma (77, 78) . despite intensive study, the etiology of asthma is still poorly understood, although a family history of atopy is consistently shown to be an important risk factor [reviewed in ref. (79, 80) ]. genetics undoubtedly play a pivotal role in the development of asthma but genotype is not fully determinative. environmental factors influence not only the overall risk of asthma but also the onset and severity of disease. as outlined above, respiratory infections are major causes of short-term morbidity and mortality in the first years of life (81) and viral infections are associated with up to 60% of all asthma exacerbations in the young (82) . in addition to this apparent "direct" association with exacerbations (83) (84) (85) , it is also possible that early-life exposure to specific viruses acts in young children (<2 years of age) with a high risk of atopy, both rsv and rv detection in nasal aspirates was associated with asthma development at 5 years of age (86) wheezing in young children (<3 years of age) at a high risk for developing asthma (one parent with asthma or respiratory allergies) that tested positive for rsv and rv, was strongly associated (or = 10) with asthma at 6 years of age (87) a greater number of respiratory infections (viral or bacterial) in young children (< 3 years of age) were associated with asthma development at 7 years of age (88) rsv in a prospective cohort study with matched controls, infants hospitalized with severe bronchiolitis and a family history of atopy/asthma had a greater prevalence of rsv-specific igg antibodies at the first year follow-up and asthma, atopy at the second year follow-up in comparison to the control group (89) in young children (<3 years of age) hospitalized with lower respiratory-tract illness, rsv was an independent risk factor for the development of wheezing at 11, but not at 13 years of age, though no association was found between rsv lower respiratory-tract illness and the development of atopy (90) severe rsv bronchiolitis during infancy was associated with increased prevalence of allergic asthma at 18 years of age (e.g., increased asthma, sensitization to perennial allergens, persistent/relapsing wheeze in association with early allergic sensitization, reduced spirometric function) wheezing in young children (<3 years of age) at a high risk for developing asthma (one parent with asthma or respiratory allergies) that tested positive for rsv was associated (or = 2.6) with asthma at 6 years of age (87) in twins, 3-9 years of age, severe rsv infection does not cause asthma, but rather indicates a genetic predisposition to asthma. hospital discharge registries and parent-completed questionnaires were fitted to genetic variance components models and direction of causation models in a prospective cohort study in twins, 3-9 years of age, hospitalization for rsv infection was associated with asthma shortly after discharge and hospitalization for asthma increased long-term susceptibility to severe rsv infection (93) hospitalization during infancy was associated with the development of childhood asthma: 59% of asthma prevalence in children hospitalized with rsv vs. 6% non-hospitalized (overall comparison estimates-systematic review of 27 articles) in young children (<2 years of age) hospitalized for wheezing respiratory illness, rv detection in nasopharyngeal aspirates was associated (or = 4.14) with asthma development 6 years later in comparison to children that were rv negative (95) wheezing in young children (<3 years of age) at a high risk for developing asthma (one parent has asthma or respiratory allergies) that tested positive for rv was strongly associated (or = 9.8) with asthma at 6 years of age in a prospective population-based surveillance of children to prime some individuals for development of asthma later in life. indeed, a growing body of epidemiological evidence suggests that early-life respiratory virus infections predispose infants to the development of asthma (see below). murine models of both the induction of ahr and the exacerbation of existing airway disease in neonatal and infant animals are beginning to provide some mechanistic understanding of these phenomena. because rsv, influenza viruses, and rvs are ubiquitous causes of respiratory infection in young infants, these agents will be our primary focus. table 1 provides an overview of studies linking viral respiratory infections and wheeze or asthma in children. background, prevention, and treatment there are two principal antigenic sub-types of rsv (a and b), each with multiple genotypes based on their surface glycoprotein g (97) . although both sub-types can infect humans, most (>85%) symptomatic disease is attributable to type a viruses. these viruses are the leading global cause of serious viral respiratory illness in infants (98, 99) . based on seroepidemiology, nearly 90% of all infants are infected at least once by the age of two. there is no vaccine for rsv and treatment options are limited. although aerosolized ribavirin may provide marginal benefit in severely ill children, this approach is cumbersome and used infrequently (100) . the monthly prophylactic use of a monoclonal antibody that targets the surface fusion (f) glycoprotein (e.g., palivuz-imab™ and others) can reduce hospitalization rates by about 50% (101) as well as the total number of wheezing days in the first year of life (102) . however, the cost of prophylaxis is so high that this approach is generally restricted to infants at greatest risk of severe disease (preterm infants, infants with immunodeficiency, etc.) [reviewed in ref. (103)]. a broad range of epidemiological data strongly supports the association between early-life rsv-related hospitalization, and the subsequent development of asthma in childhood (90) with effects lasting into early adulthood (91) . a recent meta-analysis of infants hospitalized with severe rsv during infancy found a 63% prevalence of asthma in children ≤5 years of age that increased to 92% in children between the ages 5 and 12. after 12 years of age, asthma prevalence in these children falls to 48% but all of these numbers are striking compared to background asthma rates of 1-7% in children with no history of early-life, rsv-associated hospitalization (94) . the mechanisms that underlie this strong epidemiologic association are not yet fully understood and it has been argued that hospitalization with rsv is simply a "marker" for children genetically predisposed to asthma as opposed to an environmental risk factor for asthma development (104) . in fact, it is likely that both are true to some degree. given the frequency of rsv infection in the first years of life, hospitalization due to severe infection is relatively rare regardless of genetic background, occurring only when rsv moves into the lower respiratory tract (lrt), causing pneumonia and bronchiolitis (105) . in most healthy, full-term babies and infants, rsv infection is limited to the upper respiratory tract and causes only mildmoderate symptoms. in a 10-year study in the us, the rates of rsv-related hospitalization ranged from 48.9 per 1,000 in infants less than 3 months of age to 26 per 1,000 in infants older than 1 year (106). why some children progress to lrt complications is still unknown, but risk factors include a family history of atopy, preterm birth, congenital heart disease, and low levels of maternal anti-rsv antibodies [reviewed in ref. (107)]. the tissue tropism of rsv is narrow; restricted to cells of the airway epithelia both in vitro and in vivo. rsv entry on the apical side of these cells (108) is mediated by the viral attachment (g) and fusion (f) glycoproteins (109) that utilize surface glycosaminoglycans such as heparin and nucleolin (110) , respectively, as receptors. as outlined above, a th2-biased and/or immunosuppressive microenvironment in the lungs is associated with both the development and severity of asthma. there is now considerable evidence that the major rsv surface glycoproteins can directly impact the th1/ th2 balance in the lung (111) . th2-biased airway inflammatory responses, including th2 cytokine production and influx of eosinophils into the airways, are induced upon vaccination of balb/c mice with vaccinia virus expressing the rsv g-protein (112) (113) (114) . compelling data implicate il-9, a cytokine associated with the development of asthma (19) in rsv g-protein-dependent induction of th2-biased airway inflammation in these mice (115) . the rsv f protein may also contribute to the development of a th2 microenvironment in the lung by signaling through tlr4 (116, 117) and there is strong recent data linking tlr4 genotype to severe rsv disease in humans (118) . rsv infection of airway epithelial cells (both immortalized cell lines and primary human cells) in vitro increases expression of tlr4 (119) and epithelial cell-specific tlr4 signaling is required for th2-type responses in the murine lung (120) (121) (122) . for example, allergic airways disease induced by hdm depends upon airway epithelial cell-specific tlr4 expression and is associated with secretion of several innate cytokines associated with th2-type responses [e.g., il-25, il-33, and thymic stromal lymphopoietin (tslp)], as well as the classic th2 cytokines il-4 and il-13 (122) . rsv infection of bronchial epithelial cells also induces production of tslp via activation of retinoic acid induced gene i (rig-i) and downstream activation of nuclear factor-κb (123) . importantly, rsv infection of mice sensitized to the model allergen ovalbumin (ova) leads to an increase in th2 cytokine production in the lung following ova challenge suggesting that the rsv effect on th1/th2 balance in the respiratory tract is not restricted to viral antigens (124, 125) . more recently, prior rsv infection of young mice has been shown to enhance allergic airways disease induced by hdm (126) . recognition of the powerful effects of il-25, il-33, and tslp produced by respiratory epithelial cells is an important new element in understanding the potential for respiratory viruses to cause and/or exacerbate asthma. these epithelial-origin cytokines play a pivotal role in both the initiation of th2 type responses and the progression of allergic diseases (127) . blockade of il-25 or the absence of its receptor, il-17rb, reduces th2 cytokine production, mucus production, and/or ahr in murine rsv infection and rsv-induced asthma exacerbation models (128) . moreover, in the absence of nk cells, il-25 secreted from airway epithelial cells upregulates the notch ligand, jagged1 on the surface of dcs, which induces the development of an rsv-specific th2 response (129) . treatment of murine bone marrow-derived macrophages with exogenous il-25 or il-33 can induce production of il-5 and il-13 in vitro (130) . intraperitoneal treatment of mice with il-33 results in the differentiation of macrophages from the small intestinal lamina propria into an alternatively activated (m2 or aam) phenotype in a stat6-independent fashion. such peritoneal macrophages are major producers of il-13 in vivo. furthermore, il-33 links viral infection, macrophages, and ilc2dependent production of il-13 to ahr (131-134) and induces ilc2-and il-13-dependent dc trafficking to the lymph nodes, promoting th2 adaptive immunity (135) . in the case of rsv infection models, il-33 gene expression as well as the number of leukocytes expressing the il-33 receptor (st2) increase in adult balb/c mice infected with rsv and interestingly, st2 blockade decreases lung eosinophil recruitment, il-13 levels and mucus production, without affecting ifnγ levels (136) . ilc2s are an important source of il-13 secretion post-rsv infection and evidence that they play a role in enhanced disease is provided by data showing that adoptive transfer of lung ilc2s purified from rsvinfected mice 2 h prior to rsv infection dramatically increases production of il-13 and subsequent eosinophil infiltration (137) . similarly, il-33 production in the lungs of rsv-infected neonatal mice contributes to ilc2 expansion as well as th2-biased airway inflammatory responses following rsv re-infection in adults (138) . evidence that il-33 may contribute to pathogenesis in human disease is provided by data showing that il-33 levels are increased in nasal aspirates of infants hospitalized with rsv infection (138) . similarly, il-33 mrna levels are greater in nasal aspirates of infants hospitalized with rsv bronchiolitis with a family history of atopy compared to those with no such history (139) . thymic stromal lymphopoietin likely also plays a role in the development of th2 responses in rsv infection. in rat primary airway epithelial cell cultures, infection with rsv triggers an immediate increase in tslp mrna and protein, which induces myeloid dcs to express markers associated with th2 polarization (140) . ex vivo rsv infection of human airway epithelial cells of healthy children and children with asthma results in tslp induction and has been shown to contribute to th2 inflammation (123) . infection of human primary bronchial airway epithelial cells also results in upregulation of functional tslp receptor (tslpr) on these cells, suggesting a feedback loop in which tslp binding to its receptor increases production of more tslp (141) . tslp also acts directly on dcs. lung dcs, along with alveolar macrophages and ilc2s are among the "first responders" to viral infections and allergen exposure in the lung. the relationship between lung epithelial cells and dcs and the development of asthma has been the subject of several recent reviews (142) (143) (144) and provides a framework for understanding how rsv-infected epithelial cells may program lung dcs to promote th2 immunity. tslp secreted from epithelial cells infected with rsv (123, 140) induces ox40l expression on the surface of dcs (145) that, in turn, drives t cells toward a th2 cell phenotype (146) . a further link between tslp and il-25 in rsv infection is provided by evidence that il-25 enhances the memory th2 response induced by tslp-activated dcs (147) . il-33 also upregulates ox40l surface expression on dcs, effectively promoting th2 immunity in both allergy and rsv infection models (138, 148) . tslp signaling in ilc2s is also necessary for il-13 production by these cells as tslpr ko mice and blockade of tslp signaling with anti-tslp neutralizing antibody decrease il-13 lung protein levels, mucus, ahr and weight loss during rsv infection (149) . in humans, when rsv infects primary myeloid and plasmacytoid-derived dcs (150) from healthy volunteers, the viral g-protein decreases dc activation (151) . suppression of dc maturation has also been observed by the viral non-structural (ns) proteins, ns1 and ns2 (152) . moreover, ns1-dependent activity in rsv-infected dcs suppresses the activation and proliferation of both migratory cd8 + t cells (cd103 + cd8 + ) and th17 cells while supporting the activation and proliferation of th2 cytokine-producing cd4 + t cells (153) . together, these data suggest that rsv infection of epithelial cells and dcs may act synergistically to elicit a th2-biased response in both mice and humans. this response pattern is likely accentuated by the already th2-biased nature of the neonate. yet another piece of this puzzle may be the induction by rsv of long-lived, lung-resident macrophage populations. rsv infection in adult mice induces polarization of macrophages toward an aam (m2) phenotype (126, 154) . similar polarization is observed upon rsv infection of peritoneal macrophages ex vivo (154) . the classification of macrophages is largely based on cytokine production profiles upon activation and, as is the case with th1 and th2 lymphocytes, the cytokines ifnγ and il-4 appear to play central roles in the development of m1 and m2 macrophages, respectively. upon activation, m1 macrophages (also called classically activated macrophages or cam) typically secrete inflammatory cytokines, such as ifnγ, il-12, and il-1, whereas m2 macrophages secrete il-4, il-13, and il-10 (155). m2 macrophages play an important role in tissue repair (156, 157) [reviewed in ref. (158) ] mediated, at least in part, by tgfβ and platelet-derived growth factor. studies in both human infants (159) and neonatal mice (160) have demonstrated that the production of tgfβ is increased upon rsv infection. on the one hand, this increase could reflect an adaptive m2 reparative response to the lung injury caused by rsv infection as shown by shirey and colleagues (154) . on the other hand, over-enthusiastic repair of virally damaged airways could be maladaptive with longterm changes in macrophage phenotype that promote pathologic changes in airway structure and function (131, 134, 161) . it is, therefore, interesting that rsv infection of neonatal mice is associated with long-term increases in collagen deposition and airway remodeling (125, 162) . although the precise mechanisms by which rsv infection induces m2 polarization are still unknown, it seems likely that production of tslp, il-25, and/or il-33 by rsv-infected or -exposed respiratory epithelial cells contributes to both m2 differentiation and long-term maintenance of a th2biased macrophage population in the lung (123, 130, 163, 164) . furthermore, as noted above, the ns1 and ns2 proteins of rsv inhibit the type-i ifn response in human macrophages (165) , which could also contribute to the induction of m2 macrophages. much of the evidence outlined above in support of a th2-biasing effect of rsv in the lung has been obtained using adult mice and human cells/cell lines. neither of these strategies is ideal. the limitations of cell lines and even primary cells are obvious since these reductionist models cannot reproduce the complexity of in vivo host-virus interactions. the adult mouse model of rsv infection also has important drawbacks since adult mice are not susceptible to rsv. this model requires the instillation of high titers of virus (10 6 -10 7 50% tissue culture infective dose/ml) directly into the lrt. furthermore, rsv replication in adult mice is relatively poor and occurs primarily in alveolar pneumocytes rather than the bronchiolar epithelial cells as occurs in humans (166, 167) . perhaps most importantly, rsv infection of adult mice induces a potent th1 response that rapidly clears the virus. the role of rsv-specific th2 responses in this model may be restricted to the prevention of exaggerated th1-mediated lung pathology (154) . in the case of human clinical data, it is difficult to resolve the "chicken-and-egg" problem: do children with a genetic propensity to mount th2 responses and develop asthma suffer more severe early-life rsv or does early and severe rsv infection cause the subsequent development of asthma (83, 168)? in an attempt to address these limitations, several groups, including ours, have developed models of rsv infection in neonatal and young mice with re-infection or exposure to allergens later in life (126, 160, 162, 169, 170) . these models demonstrate that early-life rsv infection (within 7 days of birth) elicits little ifnγ production in contrast to adult animals (169) and leads to a "proasthmatic" phenotype characterized by ahr, mucus production, airway remodeling, and severe disease upon subsequent allergen exposure or live rsv challenge (126, 160, 162, 170) . consistent with these th2-biased responses, il-4rα is increased on pulmonary cd4 + t cells following rsv reinfection of adults (171) . the exaggerated th2-inflammatory response upon re-infection dendritic cells (dcs), macrophages, and innate type 2 lymphoid cell (ilc2s) are among the first responders in the lung. each of these cytokines increases activation of dcs, including increased expression of ox40l. ox40l expressing dcs drive th2 differentiation in the lung mediastinal lymph nodes. these innate type-2 cytokines also promote differentiation of m2 alternatively activated macrophages (aam), which secrete type-2 cytokines il-4, il-13, and il-10 and orchestrate type-2 inflammation and tissue repair in the lung. m2 macrophage cytokine/chemokine production contributes to enhanced type-2 biased inflammation and airway remodeling. il-5 and il-13 production from ilc2s promotes eosinophil influx and responses in dcs and macrophages that further enhance type-2 inflammation, th2 differentiation, and airway hyperresponsiveness (ahr). each of these responses is enhanced in neonates upon exposure to respiratory viruses, setting the stage for enhanced type-2 inflammation upon exposure to allergens. is dependent upon the age at initial infection and production of both il-13 and il-33 (138, 170) and is enhanced by the presence of anti-rsv ige antibodies (172) . specifically targeting th2 responses in the neonate can reduce these exaggerated responses upon adult rsv reinfection. for example, delivery of antisense oligonucleotides targeting the il-4rα (173) or a cell penetrating peptide targeting the stat6 transcription factor (activated by both il-4 and il-13) (162) to neonatal mice at the time of rsv infection reduces enhanced disease upon rsv re-infection of adults. interestingly, human cord blood cd4 + t cells also upregulate il-4rα upon ex vivo rsv stimulation (171) . consistent with these data, development of enhanced disease is t cell dependent in the neonatal challenge-adult rechallenge model. however, reduced inflammatory responses are seen only if cd4 + t cells are depleted at the time of adult re-infection but not during the neonatal exposure (174) . on the other hand, depletion of cd8 + t cells during either the neonatal infection or adult re-infection significantly decreases enhanced disease in mice (174) . how cd8 + t cells exposed to rsv during neonatal infection promote disease in adult re-infection is unclear, and somewhat paradoxical, given the protective role that cd8 + t cells are believed to play in recovery from viral infections. it is also possible that cd8 + t cells play slightly different roles in mouse vs. human disease (175) . how cd4 + and cd8 + t cell populations influence the pathogenesis of rsv infection in reinfected adult mice is intriguing and deserves further evaluation. finally, repeated rsv infection of weanling mice interferes with treg-mediated tolerance and increases susceptibility to allergic asthma (176) . as described in the first section of this review, the neonatal immune system is characterized by a pre-existing th2 bias. the addition of rsv infection of airway epithelial cells with production of the type-2 innate cytokines, il-33 (138) , tslp (123) , and il-25 (128) , would, therefore, be predicted to create an even more exaggerated type-2-biased microenvironment in the lung with activation of other immune cells (e.g., m2 macrophages, dcs, and/or ilc2 cells) and the development of ahr. when neonatal mice are treated with antibodies to neutralize tslp, il-33 or their target expressed on dcs, ox40l, the ability of early rsv infection to prime pathological th2 responses upon re-infection of adults is reduced (138, 145) . as discussed above, tslp and the other innate type-2 cytokines, il-25 and il-33, all promote amplification, differentiation, and maintenance of m2 macrophages (130, 163, 164) . together, these data suggest that many different cells and cytokines, as well as other yet to be determined factors have the potential to contribute to the rsvinduced, th2 microenvironment and the subsequent development of asthma (figure 1 ). an alternative to the "too much th2" explanation for th2biased neonatal immune system would be "too little th1". ifnγ plays a key role in the induction and maintenance of th1 responses and infection of neonatal mice with recombinant rsv expressing ifnγ prevents enhanced disease upon re-infection (177) . a large proportion of the immune cells present in the neonatal mouse lung are macrophages, and these cells are particularly sensitive to stimulation by ifnγ. indeed, the work of empey and colleagues shows that ifnγ plays a central role in the balance between the inflammatory m1 macrophage phenotype and the immunosuppressive, m2 phenotype (71) . while lung macrophages from adult mice effectively induce m1 macrophage markers in response to rsv, neonatal macrophages require exogenous ifnγ to express m1 markers, produce inflammatory cytokines and drive efficient viral clearance (71) . moreover, administration of intranasal clodronate liposomes that depletes macrophages significantly reduces the ability of ifnγ to promote viral clearance (178) . furthermore, yamaguchi and colleagues (179) have shown that treatment of neonatal mice with the powerful th1 adjuvant, cpg, prior to rsv infection prevents enhanced disease upon re-infection, possibly by promoting activation of antigen-presenting cells and production of ifnγ from nk cells. remarkably, cpg treatment even 4 weeks after early-life infection provides protection (179) . these observations raise the possibility that rsv vaccines incorporating cpg or other th1-biasing adjuvants might be useful not only to prevent infection (180) but also to block the later development of asthma. whether or not the effects of "more" ifnγ are antigenspecific or simply reflect an adjuvant-modified baseline "potential to respond" in the lung is also an important question. this last possibility is strongly suggested by the work of remot and colleagues who observed that mucosal vaccination of neonatal mice with nanostructures formed by the rsv nucleoprotein (n) and a combination of th1-biasing adjuvants (e. coli enterotoxin lt and cpg) provides greater protection from immunopathology upon reinfection by rsv later in life compared to nanostructures containing the n protein + lt (181) . together, these data suggest that preventing too much th2 activity (driven by rsv or other stimuli) or promoting th1-type activity (or both) may establish a long-term more "balanced" lung microenvironment that resists subsequent development of asthma. as discussed above, rsv infection inhibits type i ifn production and plasmacytoid dendritic cell (pdc) responses (71, 150, 178) .as a result, the maintenance of pdcs during rsv infection may promote beneficial th1-type responses including the expansion of rsv-specific cd8 + t cells. in the challengerechallenge model, treatment of neonatal mice during the initial rsv infection with either ifnα or adoptively transferred adult pdcs leads to upregulation of ifnα expression and diminished th2-biased lung inflammation when these mice are reinfected as adults (182) . further support for the role of pdcs comes from experiments in which adult mice are exposed to fms-like tyrosine kinase 3 ligand (flt3-l) prior to rsv infection. in response to flt3-l, both conventional dc (cd11b + cd11c + ) and pdc (cd11b − cd11c + b220 + ) populations expand in the lung and draining lymph nodes but only the pdcs protect against airway hyperreactivity, exaggerated th2 cytokine expression, airway inflammation and mucus production (183) . these flt3-l-induced pdcs have upregulated type i ifn (ifn-α/β) expression and support the expansion of cd8 + t cell populations that decrease viral load. depletion of these pdcs reduces the protective cd8 + th1 response with greater ahr, mucus, viral titers, and th2 cytokine expression (183, 184) . similarly, flt3-l administration prior to neonatal rsv infection reduces airway mucus secretion and airway eosinophilia but promotes th1 rsv-specific cd8 + t cells upon adult re-infection (185) . although these observations suggest that flt3-l protects against rsv infection by inducing pdcs to produce type i ifn, alveolar macrophages (cd11c + siglecf + ) rather than pdcs (or epithelial cells), are thought to be the main producers of type i ifn in the adult mouse lung (186, 187) . regardless of whether pdcs or alveolar macrophages are the predominant source of type i ifn, these studies strongly suggest that promoting type i ifn production in the lung during early-life rsv infection is likely to lead to adaptive th1 responses while suppression of type 1 ifn production may favor maladaptive th2 responses. taken together, the data summarized above strongly suggest that rsv infection of the neonatal lung adds a further th2 influence to an already th2-biased respiratory microenvironment, with likely contributions from respiratory epithelial cells as well as multiple innate cell populations (e.g., macrophages, dcs, nk cells, ilc2s). the presence of multiple autocrine and paracrine amplification loops between these cells likely leads to the production of excessive il-4 and il-13 in the lung that increase both the short-and long-term risk that exposures to otherwise innocuous allergens will lead to immunopathologic responses including asthma (figure 1) . influenza viruses are far more diverse than rsv due, in large part, to their genetic organization (eight independently segregating genes in the a and b viruses that most commonly infect humans) and the enormous genetic reservoir of influenza viruses in aquatic birds and a number of mammalian species (e.g., most notably pigs but also cats, whales, elephants, skunks, among others) (188) . the influenza viruses remain a leading cause of childhood pneumonia globally (189) and early-life infections are common despite the near universal availability of influenza vaccines in resource-rich settings (190) . it has been estimated that without vaccination 10-40% of young children are infected by an influenza virus every year (191) . although a number of antivirals are available for influenza infections (e.g., m2 and neuraminidase inhibitors), these drugs have only modest efficacy and their use is further limited by resistance that is either preexisting or is rapidly induced during treatment (192) . although influenza infections are clearly associated with exacerbations of asthma [reviewed in ref. (193) ], the putative link between earlylife influenza and the subsequent development of asthma is much more tenuous. compared to the large body of work implicating rsv (above), far less work has been done to address this question for influenza viruses. at the current time, there is neither epidemiologic nor experimental evidence in humans suggesting a strong association between early-life influenza infection and asthma. although work with murine models tends to support an association, these data are not consistent from one model or laboratory to another. differences in the age of infection, both mouse and virus strain (i.e., human vs. mouse-adapted) and the infective dose may all contribute to these apparently contradictory results. for example, a study by dahl et al. (194) showed that lrt infection of adult balb/c mice with influenza a (hkx31 strain) at 6-8 weeks of age could predispose to ahr upon subsequent allergen sensitization (keyhole limpet). surprisingly, ahr in this model was associated with both th1-type (ifnγ, igg2a) and th2-type (igg1) antigenspecific responses and the allergic phenotype could be adoptively transferred with pulmonary dcs. using a similar protocol with ova as a model allergen in balb/c mice, barends et al. found that infection with a mouse-adapted influenza a strain (a/ pr/8/34) at the time of ova challenge decreased th2 cytokine production in the lungs but increased infiltration by eosinophils (195) . chang cells (i.e., ilc2s) to produce il-13 that promotes the development of ahr (132) . recently, elevated pleural il-33 and ilc2s were found to mediate the induction of asthma-like responses (e.g., ahr, production of th2 cytokines) following pdmh1n1 infection in rag1 −/− mice (on a c57bl/6 background) that lack functional t and b lymphocytes (196) . like the observations in adult mice, the reported outcomes after early-life influenza a infection are highly variable, sometimes protecting against, but sometimes promoting the development of ahr. when suckling balb/c mice (2 weeks old) are infected with influenza h3n1 (again mem71) and challenged with allergens as adults, ahr typically does not occur and the protective effect appears to be mediated by a subset of nkt cells that produce large amounts of ifnγ (197) . at high inocula, many influenza viruses are lethal for young mice (198) . using low titer h1n1 virus (pr8), lines and colleagues found a delayed t cell response with a pronounced influx of eosinophils into the lungs of c57bl/6j neonates compared to adult mice (198) . ex vivo stimulation of lung cells with phorbol 12-myristate 13-acetate/ ionomycin in these experiments showed delayed and lower levels of ifnγ in the neonatal immune t cells (198) . others have shown that infection of balb/c mouse pups with pr8 on day 7 of life can result in long-term pulmonary dysfunction, ahr and an increase in inflammatory cytokines, neutrophils and alveolar macrophages in the lungs (199) . in this model, ifnγ was not produced by the neonatal pulmonary cd8 + t cells, but adoptive transfer of adult cd8 + t cells prevented the long-term ahr (199) . although this body of work is much smaller than that dealing with rsv, the available data raise the possibility that deficient ifnγ production in the neonate during influenza virus infection may play a role in the induction of an asthmatic phenotype upon subsequent exposure to viruses or allergens. further studies, including the development of a "standard" early-life mouse model of influenza infection are required to begin to understand the potential contribution of these viruses to asthma initiation. rhinoviruses are the most important etiological agents of the "common cold". similar to rsv, rv can cause both upper and lower respiratory-tract illness at all ages. there are 100-150 strains of rv, divided into three groups: a, b, and c. groups a and b were discovered in the 1990s, while rv-c, a new group with over 50 strains, was identified only in 2006 (200, 201) . most rv infections are thought to be minimally symptomatic or completely asymptomatic. under 4 years of age, at least one rv can be found by nasal swab in 12-32% of asymptomatic children [reviewed in ref. (202) ]. although both the scientific literature and the internet offer a wide variety of therapies to prevent or cure the common cold (203, 204) , at the current time neither antivirals nor vaccines are available for rv infections. similar to rsv, the rvs, and particularly the rv-c group, have been implicated in both the development of asthma and in exacerbation of wheezing illness (87, 96, (205) (206) (207) . interestingly, rv-associated wheezing tends to be more common in older infants (>1 year old) in contrast to rsv infection in which the most serious manifestations (i.e., bronchiolitis) occur primarily in those <1 year old (208, 209) . however, both rsv and rv are frequently isolated in infants hospitalized with severe respiratory symptoms and wheezing (210) . as noted above for rsv, it is very difficult to know if this observation means that children with a genetic propensity to wheeze are more likely to be hospitalized or if rv infections are a causal factor in the onset of wheezing illness (83, 168) . until recently, animal models suffered from the absence of a receptor for human rv on mouse cells. most human rv strains bind to intracellular adhesion molecule-1 (icam-1) but not mouse icam-1 and only a minority (~10%) binds to low-density lipoprotein of both humans and mice (211) . bartlett et al. (212) have described two mouse models of rv infection, one in which balb/c mice are infected with a low-density lipoprotein-binding isolate and a second based upon transgenic expression of human icam-1. infection of adult mice in the latter model results in a strong th1 response with abundant ifnγ production as well as exacerbation of allergic responses. to date, the transgenic model has not been used to study rv infection and asthma initiation in neonatal mice. a third model was recently described by schneider et al. (213) in which 7-day-old balb/c mice are infected with rv-1b, leading to the development of ahr and mucus production 4 weeks later. surprisingly, this neonatal infection resulted in the production of ifnγ along with increases in inflammatory cytokines and chemokines, including tnfα, cxcl1, and cxcl2 in the lungs. these observations are very different from the findings in rsv-infected neonates. however, il-13 was also strongly induced by rv-1b infection in neonatal mice but not in adult mice (214) . analysis of the lungs 35 days after infection showed that a majority of cells producing il-13 were nkt cells, while cd4 t cells predominantly expressed ifnγ. there was also an increase in m2 macrophages in the lungs, cells that contribute to rv-1b-induced ahr in allergen-sensitized mice infected as adults (215) . neutralizing il-13 or il-4r decreased ahr in mice infected as neonates, suggesting that mechanisms similar to those operating in influenza (197) and rsv (138, 170, 173) may also play a role in rv infection. in hdm (dermatophagoides farina)-sensitized balb/c mice, rv infection decreases il-10 and increases production of il-13, rantes and tnfα as well as eosinophil infiltration into the lungs (216) . th2 cytokines (il-4, il-13) increase the expression of the rv receptor (icam-1) on human respiratory epithelial (h292) cells (217) . antiviral responses (ifnβ/λ) appear to be decreased in primary bronchial epithelial cells isolated from children with atopy/asthma and are more permissive of rv16 replication (218) . serum ige levels in these infants are positively correlated with viral rna levels in ex vivo-infected epithelial cells and negatively correlated with rv-induced ifnβ/λ. other investigators have found a negative correlation between ifn-induction by rv and airway th2 responses, including eosinophilia and detectable il-4, in children with a history of asthma both with and without atopy (218) . in a human challenge study with rv14, both atopic and non-atopic subjects with low ifnγ/il-5 ratios had more symptoms and prolonged viral shedding (219) . the dearth of studies on rv infection in neonatal mice makes it hard to draw conclusions about potential mechanisms of rv-mediated asthma initiation. further work on neonatal models, particularly using the group c strains associated with clinical asthma in infants (87, 96, (205) (206) (207) , would be useful. like rsv, rv primarily targets respiratory epithelial cells, although rv infections are typically "patchy" and thus may not cause the same degree of damage as rsv. recent investigations of rv infection in both animal models and human studies have focused on the innate type 2 cytokines il-25, tslp, and il-33. tslp is secreted at a low concentration from primary human bronchial epithelial cells infected ex vivo with rv, but rise dramatically with the addition of il-4 (220). tlsp and il-33 are also upregulated in bronchial epithelial cells in response to in vitro rv infection but even greater increases in il-25 are seen in cells from asthmatic patients post-rsv infection (221, 222) . rv-induced production of il-33 and/or tslp blocks ova-induced inhalational tolerance, with resultant lung eosinophilia and neutrophilia, as well as increased th2 and decreased treg cells. delivery of neutralizing antibodies that target st2 (the il-33 receptor) or rv infection of tslpr knock-out mice both disrupt the ability of rv to block tolerance in this model (222) . these data suggest that rv may increase susceptibility to allergic airways disease, by increasing tslp and/or il-33, generating a lung environment conducive to immune recognition of normally harmless antigens/allergens. in a murine model of rv-induced asthma exacerbation using ova, lung levels of il-25, expressed by epithelial cells and infiltrating immune cells are greatest in mice exposed to both ova and rv (221) . ova-rv exposed mice have increased levels of il-4 + basophils, il-4 + cd4 + t and icos + st2 + non-t (ilc2?) cells in the bronchoalveolar lavage (bal) fluid, as well as enhanced airway eosinophilia and neutrophilia, and exacerbated th2 cytokine production, all of which are reduced upon delivery of neutralizing il-25 receptor antibody (α-il-17rb) prior to rv infection. interestingly, α-il-17rb treatment also decreases lung tissue il-33 and tslp protein levels. these data suggest that targeting il-25 receptor may hold therapeutic promise through mitigation of viral-and allergen-induced inflammation promoted by innate type 2 cytokines. interleukin-33 is produced by cultured bronchial epithelial and smooth muscle cells when infected with rv (223, 224) . in response to in vivo infection with rv16, asthmatics have greater viral loads, increased bal eosinophilia, and greater respiratory symptom scores compared to non-asthmatic controls. these parameters are also associated with reductions in both peak expiratory flow and fev1. moreover, elevated levels of the th2 cytokines (il-4, il-5, il-13) as well as il-33 are present in the bal fluid of rv16-infected asthmatics. ex vivo incubation of activated (undifferentiated) cd4 + t cells (th0) or ilc2 cells with supernatants from rv-infected bronchial epithelial cells leads to production of il-5 and il-13 by both cell types in an il-33-dependent manner (223) . significantly, production of il-5 and il-13 is 100-to 200fold greater in ilc2 cells compared to cd4 + t cells suggesting that il-33-dependent activation of ilc2 cells during rv infection could play a major role in the development of asthma. taken together, these data suggest that pronounced il-33 secretion by smooth muscle cells and/or il-25, tslp, and il-33 secretion by epithelial cells in response to rv infection may skew the lung microenvironment toward th2-biased allergic airways disease through activation of both innate (e.g., macrophage, dcs, and ilc2 cells) and adaptive immune cells. at the current time, a large body of rsv data in both humans and murine models strongly suggest that there is a maladaptive and "asthma-genic" interaction between the th2-biased nature of the infant immune system and the th2-promoting effects of the virus itself. it is also very likely that host genetics play an important role in determining both the short-term (e.g., hospitalization) and long-term (e.g., asthma) consequences of this interaction (90, 93) . to date, the only factors known to increase susceptibility to rsv-induced asthma are family history of atopy, premature birth, and certain host genetic polymorphisms that are linked with severe disease (111) . whether or not any child, regardless of his/her genetic background can be "made" asthmatic by early-life rsv infection is an important question that cannot be answered at the current time. in favor of the hypothesis that asthmatics are "born" and not "made", gern and colleagues have reported bi-directional cytokine responses in cord blood mononuclear cells stimulated with mitogen or viral antigens (e.g., rsv, rv) from children who were later classified as either wheezing vs. non-wheezing at one year of age (225) . whether or not rsv is unique among early childhood respiratory virus exposures with regard to asthma development is also of great interest. the more limited data available suggest that the rv but not influenza virus infection may have effects similar to rsv. bonnelykke et al. (88) reported that the number of viral or bacterial respiratory infection episodes within the first year of life is a greater predictor of asthma development than the particular viral or bacterial type(s) (88) . in contrast to the contribution of respiratory viruses to asthma development, the role of several viruses, including rsv, rv, and influenza viruses, in asthma exacerbations is very clear (9, 84, 206, 226, 227) . of course, neonates and infants are not just exposed to respiratory viruses in the first weeks-months of life. rather, they experience a wide range of immunologic challenges with both commensal and potentially pathogenic organisms at multiple epithelial sites throughout childhood. for example, ege and colleagues have shown that children exposed to wide range of microbes early in life, including children that live on farms, have a reduced risk of developing asthma (the hygiene hypothesis) (228) and germ-free mice can more easily be made airway hyperresponsive than their non germ-free littermates (229) . a great deal of work remains to be done to fully understand how the ubiquitous respiratory viruses discussed in this review, as well as other early-life microbial exposures, contribute to the development and perpetuation of asthma. such an improved understanding is essential to develop strategies to both prevent asthma development and to mitigate the symptoms of asthma once developed. this review has focused on how a small number of viruses may also contribute to the induction of asthma. in particular, we have described the growing evidence that innate immune effector cells may orchestrate earlylife events in the lung to "set the stage" for the later development of asthma. until very recently, the potential asthma-inducing role of respiratory epithelial cells, macrophages, dcs, ilc2s, and nk/ nkt cells has been under-appreciated. although vaccination to protect the very young from early-life respiratory viruses (including maternal immunization) is one possible strategy (230), these new data suggest that modulation of innate responses during early-life viral infections may also be successful. of course, any strategy that seeks to alter either the antigen-specific or the "overall" immune response pattern of neonates/infants would have to be approached with great caution (e.g., risk of exaggerated th1 immunopathology, less effective responses to other pathogens or vaccines). as outlined above, the th2-biased nature of the neonatal immune system is a strategy that has been "proven" by evolution. nonetheless, given that asthma has now reached essentially "pandemic" proportions, very few questions in clinical medicine are of greater importance. the concepts described in this review raise the possibility of completely novel strategies for dealing with this pandemic. kr, bs, bw, and ef contributed equally to the writing of the manuscript and production of the accompanying figure. global, regional, and national causes of child mortality: an updated systematic analysis for 2010 with time trends since cost burden of viral respiratory infections: issues for formulary decision makers maternal alloantigens promote the development of tolerogenic fetal regulatory t cells in utero mir-146a mediates protective innate immune tolerance in the neonate intestine early neonatal inflammation affects adult pain reactivity and anxiety related traits in mice: genetic background counts long-term impact of infection on the preterm neonate inflammation during fetal and neonatal life: implications for neurologic and neuropsychiatric disease in children and adults role of viral respiratory infections in asthma and asthma exacerbations do early-life viral infections cause asthma? respiratory viral infections in infants: causes, clinical symptoms, virology, and immunology neonatal immunity: faulty t-helpers and the shortcomings of dendritic cells actively acquired tolerance of foreign cells neonatal adaptive immunity comes of age th1 and th2 cells: different patterns of lymphokine secretion lead to different functional properties enhanced type 2 and diminished type 1 cytokines in neonatal tolerance induction of th1 and th2 immunity in neonatal mice th17 and allergy th9 and allergic disease identification of a novel subset of human circulating memory cd4(+) t cells that produce both il-17a and il-4 regulatory t cells and asthma neonatal tolerance revisited: turning on newborn t cells with dendritic cells th1 genetic adjuvants modulate immune responses in neonates dna immunization circumvents deficient induction of t helper type 1 and cytotoxic t lymphocyte responses in neonates and during early life cpg oligodeoxynucleotides can circumvent the th2 polarization of neonatal responses to vaccines but may fail to fully redirect th2 responses established by neonatal priming cpg odn can re-direct the th bias of established th2 immune responses in adult and young mice newborns develop a th1-type immune response to mycobacterium bovis bacillus calmette-guerin vaccination a liposome-based mycobacterial vaccine induces potent adult and neonatal multifunctional t cells through the exquisite targeting of dendritic cells partial correction of the th2/th1 imbalance in neonatal murine responses to vaccine antigens through selective adjuvant effects cpg motifs in bacterial dna and their immune effects the generation of th memory in neonates versus adults: prolonged primary th2 effector function and impaired development of th1 memory effector function in murine neonates freshly isolated, murine neonatal t cells produce il-4 in response to anti-cd3 stimulation murine neonatal cd4+ cells are poised for rapid th2 effector-like function the human il-13 locus in neonatal cd4+ t cells is refractory to the acquisition of a repressive chromatin architecture the murine th2 locus undergoes epigenetic modification in the thymus during fetal and postnatal ontogeny induction of protective ctl responses in newborn mice by a murine retrovirus t cell immunity in neonates transplacental priming of the human immune system to environmental allergens: universal skewing of initial t cell responses toward the th2 cytokine profile neonatal immunity develops in a transgenic tcr transfer model and reveals a requirement for elevated cell input to achieve organ-specific responses il-4 utilizes an alternative receptor to drive apoptosis of th1 cells and skews neonatal immunity toward th2 delayed maturation of an il-12-producing dendritic cell subset explains the early th2 bias in neonatal immunity developmental expression of il-12rbeta2 on murine naive neonatal t cells counters the upregulation of il-13ralpha1 on primary th1 cells and balances immunity in the newborn b-1 b cell development in the fetus and adult low expression of the interleukin (il)-4 receptor alpha chain and reduced signalling via the il-4 receptor complex in human neonatal b cells decreased interleukin-12 (il-12) from activated cord versus adult peripheral blood mononuclear cells and upregulation of interferon-gamma, natural killer, and lymphokine-activated killer activity by il-12 in cord blood mononuclear cells neonatal tolerant immunity for vaccination against autoimmunity limitations of in vivo il-12 supplementation strategies to induce th1 early life responses to model viral and bacterial vaccine antigens phenotype and function of neonatal dc upon tlr9 signaling, cd5+ b cells control the il-12-dependent th1-priming capacity of neonatal dcs neonatal b cells suppress innate toll-like receptor immune responses and modulate alloimmunity deficient il-12(p35) gene expression by dendritic cells derived from neonatal monocytes interferon regulatory factor 3-dependent responses to lipopolysaccharide are selectively blunted in cord blood cells interferon regulatory factor 7-mediated responses are defective in cord blood plasmacytoid dendritic cells post-transcriptional down-regulation of toll-like receptor signaling pathway in umbilical cord blood plasmacytoid dendritic cells selective impairment of tlr-mediated innate immunity in human newborns: neonatal blood plasma reduces monocyte tnf-alpha induction by bacterial lipopeptides, lipopolysaccharide, and imiquimod, but preserves the response to r-848 neonatal innate tlr-mediated responses are distinct from those of adults innate immunity of the human newborn is polarized toward a high ratio of il-6/tnf-alpha production in vitro and in vivo ontogeny of toll-like receptor mediated cytokine responses of human blood mononuclear cells immaturity of infection control in preterm and term newborns is associated with impaired toll-like receptor signaling innate-like effector differentiation of human invariant nkt cells driven by il-7 natural killer cell responses to infections in early life tgf-beta is responsible for nk cell immaturity during ontogeny and increased susceptibility to infection during mouse infancy defective macrophage function in neonates and its impact on unresponsiveness of neonates to polysaccharide antigens cytokine receptor signalling in neonatal macrophages: defective stat-1 phosphorylation in response to stimulation with ifn-gamma phagocytosis-associated functions in neonatal monocyte-derived macrophages inhibitory receptor expression on neonatal immune cells potent induction of ifn-gamma production from cord blood nk cells by the stimulation with single-stranded rna evidence and structural mechanism for late lung alveolarization perinatal activation of the interleukin-33 pathway promotes type 2 immunity in the developing lung alveolar macrophages develop from fetal monocytes that differentiate into long-lived cells in the first week of life via gm-csf stimulation of immature lung macrophages with intranasal interferon gamma in a novel neonatal mouse model of respiratory syncytial virus infection neonatal lung immune responses show a shift of cytokines and transcription factors toward th2 and a deficit in conventional and plasmacytoid dendritic cells lung microbiota promotes tolerance to allergens in neonates via pd-l1 pathophysiological features of asthma develop in parallel in house dust miteexposed neonatal mice gut microbiota metabolism of dietary fiber influences allergic airway disease and hematopoiesis disordered microbial communities in asthmatic airways the global burden of asthma asthma and allergic inflammation asthma: epidemiology, etiology and risk factors risk factors for asthma: is prevention possible? pneumonia: the leading killer of children the september epidemic of asthma exacerbations in children: a search for etiology viruses in asthma asthma exacerbations: origin, effect, and prevention response to infections in patients with asthma and atopic disease: an epiphenomenon or reflection of host susceptibility? early-life respiratory viral infections, atopic sensitization, and risk of subsequent development of persistent asthma wheezing rhinovirus illnesses in early life predict asthma development in high-risk children association between respiratory infections in early life and later asthma is independent of virus type asthma and immunoglobulin e antibodies after respiratory syncytial virus bronchiolitis: a prospective cohort study with matched controls respiratory syncytial virus in early life and risk of wheeze and allergy by age 13 years asthma and allergy patterns over 18 years after severe rsv bronchiolitis in the first year of life exploring the association between severe respiratory syncytial virus infection and asthma: a registry-based twin study the causal direction in the association between respiratory syncytial virus hospitalization and asthma elevated risk of asthma after hospitalization for respiratory syncytial virus infection in infancy rhinovirus-induced wheezing in infancy -the first sign of childhood asthma? a novel group of rhinoviruses is associated with asthma hospitalizations respiratory syncytial virus: the influence of serotype and genotype variability on clinical course of infection global burden of acute lower respiratory infections due to respiratory syncytial virus in young children: a systematic review and meta-analysis global and regional burden of hospital admissions for severe acute lower respiratory infections in young children in 2010: a systematic analysis respiratory syncytial virus disease: update on treatment and prevention review of palivizumab in the prophylaxis of respiratory syncytial virus (rsv) in high-risk infants respiratory syncytial virus and recurrent wheeze in healthy preterm infants a review of palivizumab and emerging therapies for respiratory syncytial virus toward primary prevention of asthma. reviewing the evidence for early-life respiratory viral infections as modifiable risk factors to prevent childhood asthma the pathogenesis of respiratory syncytial virus disease in childhood respiratory syncytial virus-associated hospitalizations among infants and young children in the united states the risk of mortality among young children hospitalized for severe respiratory syncytial virus infection respiratory syncytial virus infection of human airway epithelial cells is polarized, specific to ciliated cells, and without obvious cytopathology the fusion glycoprotein of human respiratory syncytial virus facilitates virus attachment and infectivity via an interaction with cellular heparan sulfate identification of nucleolin as a cellular receptor for human respiratory syncytial virus immunity to rsv in early-life pulmonary eosinophilic response to respiratory syncytial virus infection in mice sensitized to the major surface glycoprotein g il-13 is required for eosinophil entry into the lung during respiratory syncytial virus vaccine-enhanced disease nonglycosylated g-protein vaccine protects against homologous and heterologous respiratory syncytial virus (rsv) challenge, while glycosylated g enhances rsv lung pathology and cytokine levels il-9 regulates pathology during primary and memory responses to respiratory syncytial virus infection pattern recognition receptors tlr4 and cd14 mediate response to respiratory syncytial virus role of human tlr4 in respiratory syncytial virusinduced nf-kappab activation, viral entry and replication tlr4 genotype and environmental lps mediate rsv bronchiolitis through th2 polarization respiratory syncytial virus up-regulates tlr4 and sensitizes airway epithelial cells to endotoxin toll-like receptor 4 is required for optimal development of th2 immune responses: role of dendritic cells lipopolysaccharide-enhanced, toll-like receptor 4-dependent t helper cell type 2 responses to inhaled antigen house dust mite allergen induces asthma via toll-like receptor 4 triggering of airway structural cells thymic stromal lymphopoietin is induced by respiratory syncytial virus-infected airway epithelial cells and promotes a type 2 response to infection timing of infection and prior immunization with respiratory syncytial virus (rsv) in rsv-enhanced allergic inflammation a role for airway remodeling during respiratory syncytial virus infection enhanced allergic responsiveness after early childhood infection with respiratory viruses: are long-lived alternatively activated macrophages the missing link? barrier epithelial cells and the control of type 2 immunity il-17e (il-25) and il-17rb promote respiratory syncytial virus-induced pulmonary disease nk cell deficiency predisposes to viral-induced th2-type allergic inflammation via epithelialderived il-25 macrophages as il-25/il-33-responsive cells play an important role in the induction of type 2 immunity persistent activation of an innate immune response translates respiratory viral infection into chronic lung disease innate lymphoid cells mediate influenza-induced airway hyperreactivity independently of adaptive immunity innate lymphoid cells responding to il-33 mediate airway hyperreactivity independently of adaptive immunity long-term il-33-producing epithelial progenitor cells in chronic obstructive lung disease group 2 innate lymphoid cells are critical for the initiation of adaptive t helper 2 cell-mediated allergic lung inflammation il-33 receptor (st2) signaling is important for regulation of th2-mediated airway inflammation in a murine model of acute respiratory syncytial virus infection natural helper cells contribute to pulmonary eosinophilia by producing il-13 via il-33/st2 pathway in a murine model of respiratory syncytial virus infection respiratory syncytial virus disease is mediated by age-variable il-33 elevated il-3 and il-12p40 levels in the lower airway of infants with rsv-induced bronchiolitis correlate with recurrent wheezing tslp from rsv-stimulated rat airway epithelial cells activates myeloid dendritic cells respiratory syncytial virus induces functional thymic stromal lymphopoietin receptor in airway epithelial cells dendritic cells and epithelial cells: linking innate and adaptive immunity in asthma the airway epithelium in asthma lung dendritic cells in respiratory viral infection and asthma: from protection to immunopathology responsiveness to respiratory syncytial virus in neonates is mediated through thymic stromal lymphopoietin and ox40 ligand tslp-activated dendritic cells induce an inflammatory t helper type 2 cell response through ox40 ligand il-25 augments type 2 immune responses by enhancing the expansion and functions of tslp-dc-activated th2 memory cells il-33, but not thymic stromal lymphopoietin or il-25, is central to mite and peanut allergic sensitization respiratory syncytial virus infection activates il-13-producing group 2 innate lymphoid cells through thymic stromal lymphopoietin primary human mdc1, mdc2, and pdc dendritic cells are differentially infected and activated by respiratory syncytial virus respiratory syncytial virus glycoprotein g interacts with dc-sign and l-sign to activate erk1 and erk2 non structural proteins 1 and 2 of respiratory syncytial virus suppress maturation of human dendritic cells respiratory syncytial virus interferon antagonist ns1 protein suppresses and skews the human t lymphocyte response control of rsv-induced lung injury by alternatively activated macrophages is il-4r alpha-, tlr4-, and ifn-beta-dependent macrophage activation and polarization macrophage diversity in renal injury and repair differential macrophage polarization promotes tissue remodeling and repair in a model of ischemic retinopathy protective and pathogenic functions of macrophage subsets transforming growth factor beta is a major regulator of human neonatal immune responses following respiratory syncytial virus infection exposure of neonates to respiratory syncytial virus is critical in determining subsequent airway response in adults sustained desensitization to bacterial toll-like receptor ligands after resolution of respiratory influenza infection stat6 inhibitory peptide given during rsv infection of neonatal mice reduces exacerbated airway responses upon adult reinfection il-33 amplifies the polarization of alternatively activated macrophages that contribute to airway inflammation thymic stromal lymphopoietin amplifies the differentiation of alternatively activated macrophages suppression of the induction of alpha, beta, and lambda interferons by the ns1 and ns2 proteins of human respiratory syncytial virus in human epithelial cells and macrophages pathogenesis of respiratory syncytial virus infection in the murine model the histopathology of fatal untreated human respiratory syncytial virus infection viruses and atopic sensitization in the first years of life age at first viral infection determines the pattern of t cell-mediated disease during reinfection in adulthood the enhancement or prevention of airway hyperresponsiveness during reinfection with respiratory syncytial virus is critically dependent on the age at first infection and il-13 production il-4ralpha on cd4+ t cells plays a pathogenic role in respiratory syncytial virus reinfection in mice infected initially as neonates virusspecific ige enhances airway responsiveness on reinfection with respiratory syncytial virus in newborn mice immunomodulation with il-4r alpha antisense oligonucleotide prevents respiratory syncytial virus-mediated pulmonary disease the role of t cells in the enhancement of respiratory syncytial virus infection severity during adult reinfection of neonatally sensitized mice host response to respiratory syncytial virus infection early infection with respiratory syncytial virus impairs regulatory t cell function and increases susceptibility to allergic asthma delivery of cytokines by recombinant virus in early life alters the immune response to adult lung infection alveolar macrophages support interferon gamma-mediated viral clearance in rsv-infected neonatal mice preexposure to cpg protects against the delayed effects of neonatal respiratory syncytial virus infection intranasal proteosome-based respiratory syncytial virus (rsv) vaccines protect balb/c mice against challenge without eosinophilia or enhanced patho logy nucleoprotein nanostructures combined with adjuvants adapted to the neonatal immune context: a candidate mucosal rsv vaccine limited type i interferons and plasmacytoid dendritic cells during neonatal respiratory syncytial virus infection permit immunopathogenesis upon reinfection the balance between plasmacytoid dc versus conventional dc determines pulmonary immunity to virus infections plasmacytoid dendritic cells inhibit pulmonary immunopathology and promote clearance of respiratory syncytial virus flt3 ligand improves the innate response to respiratory syncytial virus and limits lung disease upon rsv reexposure in neonate mice alveolar macrophages contribute to the pathogenesis of human metapneumovirus infection while protecting against respiratory syncytial virus infection alveolar macrophage-derived type i interferons orchestrate innate immunity to rsv through recruitment of antiviral monocytes influenza virus reservoirs and intermediate hosts: dogs, horses, and new possibilities for influenza virus exposure of humans epidemiology and etiology of childhood pneumonia in 2010: estimates of incidence, severe morbidity, mortality, underlying risk factors and causative pathogens for 192 countries vaccines for preventing influenza in healthy children every year is an influenza pandemic for children: can we stop them? antiviral treatments viruses and asthma viral-induced t helper type 1 responses enhance allergic disease by effects on lung dendritic cells respiratory syncytial virus, pneumonia virus of mice, and influenza a virus differently affect respiratory allergy in mice pandemic influenza virus, ph1n1, induces asthmatic symptoms via activation of innate lymphoid cells influenza infection in suckling mice expands an nkt cell subset that protects against airway hyperreactivity the migration of t cells in response to influenza virus is altered in neonatal mice inchoate cd8+ t cell responses in neonatal mice permit influenza-induced persistent pulmonary dysfunction masstag polymerase-chain-reaction detection of respiratory pathogens, including a new rhinovirus genotype, that caused influenza-like illness in new york state during the abcs of rhinoviruses, wheezing, and asthma human rhinoviruses common cold complementary and alternative medicine for prevention and treatment of the common cold a recently identified rhinovirus genotype is associated with severe respiratory-tract infection in children in germany the role of viruses in acute exacerbations of asthma rhinovirus and asthma: a storied history of incompatibility rhinovirus-associated wheezing in infancy: comparison with respiratory syncytial virus bronchiolitis bronchiolitis: age and previous wheezing episodes are linked to viral etiology and atopic characteristics equal virulence of rhinovirus and respiratory syncytial virus in infants hospitalized for lower respiratory tract infection mouse respiratory epithelial cells support efficient replication of human rhinovirus mouse models of rhinovirus-induced disease and exacerbation of allergic airway inflammation neonatal rhinovirus infection induces mucous metaplasia and airways hyperresponsiveness human rhinovirus 1b exposure induces phosphatidylinositol 3-kinase-dependent airway inflammation in mice rhinovirus infection of allergen-sensitized and -challenged mice induces eotaxin release from functionally polarized macrophages the effect of rhinovirus on airway inflammation in a murine asthma model th2 cytokines exert a dominant influence on epithelial cell expression of the major group human rhinovirus receptor, icam-1 deficient antiviral immune responses in childhood: distinct roles of atopy and asthma relationship of upper and lower airway cytokines to outcome of experimental rhinovirus infection tlr3-and th2 cytokinedependent production of thymic stromal lymphopoietin in human airway epithelial cells rhinovirus-induced il-25 in asthma exacerbation drives type 2 immunity and allergic pulmonary inflammation rhinovirus infection interferes with induction of tolerance to aeroantigens through ox40 ligand, thymic stromal lymphopoietin, and il-33 il-33-dependent type 2 inflammation during rhinovirus-induced asthma exacerbations in vivo rhinoviral stimuli, epithelial factors and atp signalling contribute to bronchial smooth muscle production of il-33 bidirectional interactions between viral respiratory illnesses and cytokine responses in the first year of life viral respiratory tract infection and exacerbations of asthma in adult patients asthma exacerbations. 3: pathogenesis exposure to environmental microorganisms and childhood asthma microbial exposure during early life has persistent effects on natural killer t cell function vaccination against rsv: is maternal vaccination a good alternative to other approaches? the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. key: cord-331288-elnwn7l5 authors: grützmacher, kim; keil, verena; leinert, vera; leguillon, floraine; henlin, arthur; couacy‐hymann, emmanuel; köndgen, sophie; lang, alexander; deschner, tobias; wittig, roman m.; leendertz, fabian h. title: human quarantine: toward reducing infectious pressure on chimpanzees at the taï chimpanzee project, côte d'ivoire date: 2017-01-17 journal: am j primatol doi: 10.1002/ajp.22619 sha: doc_id: 331288 cord_uid: elnwn7l5 due to their genetic relatedness, great apes are highly susceptible to common human respiratory pathogens. although most respiratory pathogens, such as human respiratory syncytial virus (hrsv) and human metapneumovirus (hmpv), rarely cause severe disease in healthy human adults, they are associated with considerable morbidity and mortality in wild great apes habituated to humans for research or tourism. to prevent pathogen transmission, most great ape projects have established a set of hygiene measures ranging from keeping a specific distance, to the use of surgical masks and establishment of quarantines. this study investigates the incidence of respiratory symptoms and human respiratory viruses in humans at a human‐great ape interface, the taï chimpanzee project (tcp) in côte d'ivoire, and consequently, the effectiveness of a 5‐day quarantine designed to reduce the risk of potential exposure to human respiratory pathogens. to assess the impact of quarantine as a preventative measure, we monitored the quarantine process and tested 262 throat swabs for respiratory viruses, collected during quarantine over a period of 1 year. although only 1 subject tested positive for a respiratory virus (hrsv), 17 subjects developed symptoms of infection while in quarantine and were subsequently kept from approaching the chimpanzees, preventing potential exposure in 18 cases. our results suggest that quarantine—in combination with monitoring for symptoms—is effective in reducing the risk of potential pathogen exposure. this research contributes to our understanding of how endangered great apes can be protected from human‐borne infectious disease. due to their genetic relatedness, great apes are highly susceptible to common human respiratory pathogens. although most respiratory pathogens, such as human respiratory syncytial virus (hrsv) and human metapneumovirus (hmpv), rarely cause severe disease in healthy human adults, they are associated with considerable morbidity and mortality in wild great apes habituated to humans for research or tourism. to prevent pathogen transmission, most great ape projects have established a set of hygiene measures ranging from keeping a specific distance, to the use of surgical masks and establishment of quarantines. this study investigates the incidence of respiratory symptoms and human respiratory viruses in humans at a human-great ape interface, the taï chimpanzee project (tcp) in côte d'ivoire, and consequently, the effectiveness of a 5-day quarantine designed to reduce the risk of potential exposure to human respiratory pathogens. to assess the impact of quarantine as a preventative measure, we monitored the quarantine process and tested 262 throat swabs for respiratory viruses, collected during quarantine over a period of 1 year. although only 1 subject tested positive for a respiratory virus (hrsv), 17 subjects developed symptoms of infection while in quarantine and were subsequently kept from approaching the chimpanzees, preventing potential exposure in 18 cases. our results suggest that quarantine-in combination with monitoring for symptoms-is effective in reducing the risk of potential pathogen exposure. this research contributes to our understanding of how endangered great apes can be protected from human-borne infectious disease. taï national park. they set out to habituate chimpanzees-to accustom them to human presence-in order to study their behavior (boesch & boesch-achermann, 2000) . their work has significantly contributed to our understanding of chimpanzee culture and cognition as well as human evolution. moreover, boesch and boesch-achermann made important observations on illness, on the disappearance of individual chimpanzees, and obtained biological samples that paved the way for future investigations of disease (boesch, 2008; leendertz et al., 2006) . these later investigations have led to the identification of numerous pathogens. not only do they account for pathogens occurring naturally in the chimpanzees (e.g., stlv and sfv) and in the habitat (e.g., ebola and bacillus cereus bv anthracis), but also those introduced by humans, such as the human respiratory syncytial virus (hrsv) and human metapneumovirus (hmpv) (boesch, 2008; calvignac-spencer et al., 2012; formenty et al., 1999; gogarten et al., 2014; köndgen et al., 2008; köndgen, schenk, pauli, boesch, & leendertz, 2010; le guenno et al., 1995; leendertz, boesch, junglen, since 1999 , tcp experienced at least six major respiratory disease outbreaks of human origin and with losses of up to 19% of the chimpanzee communities (köndgen et al., 2008) . other projects have also published data on human respiratory pathogens infecting and, ultimately, killing several wild, habituated great apes (grützmacher et al., 2016; kaur et al., 2008; palacios et al., 2011) . such findings emphasize the inherent risk of proximity. in all of the reported outbreaks, the viruses detected were either hrsv or hmpv: two common human paramyxoviruses that generally cause only mild symptoms in healthy human adults (falsey and walsh, 2000; webe, mulholland, & greenwood, 1998) . people can easily be unaware of their infectiousness because they may still feel physically fit enough to visit the great apes. in short, the risk of transmission is often overlooked. although the issue of whether humans should interfere with naturally occurring disease outbreaks in protected species is controversial, it is commonly understood that disease transmission from humans should be mitigated (gilardi et al., 2015) . it is the ethical responsibility of humans entering great ape habitats to help minimize the costs of habituation, including potential changes in behavior, alterations of the habitat, and the sickness and loss of individuals. indeed, since 1992, the tcp has gradually implemented preventative steps to reduce the risk of exposing chimpanzees to human pathogens. the first steps included rules not to enter the forest when ill and to remove all garbage. two years later, additional rules to bury human feces and to bring back all food remains from the forest were established and complemented by veterinary collaboration. in 1999, a minimum viewing distance of 5 m was implemented (later extended to 7 m) alongside requirements that all humans be vaccinated against yellow fever, tuberculosis, measles, and poliomyelitis. vaccination (boesch, 2008) . following the last major outbreak in 2009, the quarantine rules were adjusted to a 5-day period of quarantine in a separate quarantine camp for all humans intending to visit the chimpanzees (gilardi et al., 2015) . additionally, international travelers have to spend a minimum of 2 days in côte d'ivoire before entering quarantine. researchers and field assistants alike now enter the quarantine on day 1 (qd1). they must remain in a designated area in the quarantine camp without direct contact to people who have already cleared quarantine, anybody from the outside, or anybody in a different quarantine stage (the quarantine camp is divided into two separate sections to allow for two independent parties). after four nights, should no symptoms develop, the respective person clears quarantine on the 5th day (qd5). however, if any symptoms are detected, the person exits quarantine and starts over with qd1 after all symptoms have subsided. if anyone falls sick during quarantine, then everyone in quarantine at the same time also has to start over with qd1 (see figure 1 ). however, 5 days do not cover the longest possible incubation periods-the time between infection and symptom onsetof most human respiratory pathogens (see table 1 ). but, with the exception of measles (against which humans have to be vaccinated in order to visit habituated chimpanzees at tcp), at least 50% of this period is covered for all other relevant viruses. against this backdrop, the goal of this study is to determine the number of humans falling ill during quarantine. furthermore, the risk of potential exposure to human pathogens is assessed by testing sick humans to detect common human respiratory viruses they brought to the habituation site, and by randomly testing apparently healthy humans in the beginning and at the end of quarantine to assess the possibility of excreting hrsv and hmpv, the two most relevant viruses for wild great apes. the current study was performed at the tcp in côte d'ivoire. at the tcp, the quarantine camp is separate from the research camps with a designated kitchen, shower, and toilet. a driver supplies subjects in quarantine with food, wearing a facemask on delivery and leaving the food outside the building with no personal contact (for a more detailed description of quarantine protocols at tcp see gilardi et al., 2015) . when a subject falls ill, he or she is taken home to their village, or in case of international visitors (including researchers and film-makers), the subject is physically separated within the quarantine facilities until symptoms cease and quarantine can begin again at qd1. if symptoms are more severe, subjects are taken to the local hospital for treatment. overview of the quarantine system in the taї chimpanzee project and time from infection to observation of symptoms for hrsv and hmpv. y-axis: parametric estimates of the incubation period; p (symptomatic) = cumulative percentage of cases developing symptoms, and thus, shedding hrsv or hmpv by a given day under the estimates for the log-normal distribution (lessler et al., 2009) . the red line represents hrsv and the orange line represents hmpv to exemplify the earliest and latest possible scenario. whereas the blue dotted area illustrates the window covered through the quarantine, the red area covers the remaining risk. the staff health program of tcp includes yearly health checks at the local hospital, were a tuberculosis skin test and vaccinations against yellow fever, measles, poliomyelitis, and meningococcal disease are and qd5. in total, 262 were selected for testing. this sample selection was systematized to achieve an even seasonal distribution, wherein a minimum of 20 samples were tested for each month of the 1-year research period, and to best assess the risk of potential pathogen exposure. thus, the selection included 14 samples, collected from subjects with symptoms of illness (n = 18) plus randomly selected subjects with corresponding samples from qd1 (n = 110) and qd5 (n = 101) (when possible), and samples from subjects tested on a different day of quarantine (due to various constraints) or who had been in contact with sick individuals (n = 33). signs of illness were generally mild respiratory symptoms, including sore throat, runny nose, or cough. subjects in quarantine are expected to self-report symptoms; however, the respective camp manager and "veterinarian in charge" also oversee the quarantine process. swabs were stored and transported in liquid nitrogen and later kept in −80°c freezers. three hundred microliters of nuclease-free water were added to each sample and vortexed thoroughly before extraction. anthropogenic respiratory disease poses a serious risk to habituated wild great apes with high morbidity and considerable mortality (boesch, 2008; kaur et al., 2008; köndgen et al., 2010; leendertz et al., 2006; palacios et al., 2011) . its mitigation is therefore, an ethical responsibility for habituation projects (gruen, fultz, & pruetz, 2013) . however, preventing people from exposing wild habituated great apes to human pathogens is a challenge. in terms of potential pathogen exposure, both people from overseas who come to visit habituation sites as well as local staff on such projects routinely pose a risk to the health of wild great apes (muehlenbein & ancrenaz, 2009) . people who come to visit wild great apes from overseas are more likely to have been exposed to a variety of pathogens throughout their travel (e.g., in airports or airplanes) (mangili & gendreau, 2005) . additionally, the stress of travel and changes in climate make them more susceptible to infection, leading to disease within few days after arrival at the destination where the great apes are encountered (muehlenbein & ancrenaz, 2009 ). despite these different risk factors, eco-tourists frequently lack information about the relevance of their own health for the wildlife they intend to visit and, thus, may not take precautions or report illness (muehlenbein & ancrenaz, 2009 ). finally, local project staff can also pose an increased risk when they come into frequent contact with children in their villages-a subpopulation with high respiratory disease prevalence (walker et al., 2013) . the results show that international visitors represented 7 (39%) and local staff represented 11 (61%) of symptomatic subjects. however, only two of the seven international visitors became ill shortly after their arrival in côte d'ivoire and the remaining five rather mirror the health of permanent project staff in contact with the local diversity of pathogens. the detection of hrsv in only one out of 262 tested samples (including the 14 samples from people showing symptoms of respiratory disease) points toward a rather low prevalence of humans excreting hrsv and hmpv, when entering the tcp research area. although the possibility of false-negative results exists, it seems likely that if a person excretes these viruses in any relevant quantity-a quantity that would be sufficient for transmission to chimpanzees-it is likely to be detectable with the methods used here. of course, the negative results for asymptomatic people are not surprising. for some respiratory viruses, however, pathogen excretion might start before the first symptoms occur. for example, 1-8% of influenza infectiousness occurs prior to illness onset (lau et al., 2010) . cases of asymptomatic shedding have been reported for hrsv and hmpv-the most common causative agents of respiratory disease outbreaks in wild habituated great apesbut this is believed to be a rather rare occurrence (falsey, erdman, anderson, & walsh, 2003) . that said, hmpv has recently been reported at two different great ape field sites in 1 out of 24 and 1 out of 42 apparently healthy staff members (grützmacher et al., 2016) . although asymptomatic infections must be taken into account as a risk factor, the absence of symptoms also limits the physical spread (e.g., the absence of cough or nasal discharge limit the excretion of the pathogen). thus, strict hygiene rules should be sufficient to counteract the potential risk posed by apparently healthy people, especially those who have passed through quarantine. in this study, 13 subjects with respiratory symptoms (including cough, nasal discharge, or a sore throat) were negative for all viruses tested. that pathogens were not detected despite the observed symptoms may indicate that the symptoms were caused by viruses not included in our pcr panel or be due to the fact that symptoms do not always correlate well with viral particle excretion. these negative results can also be due to the detection limit of diagnostic methods (here pcr). furthermore, symptoms indicating respiratory disease can be caused by primary or secondary bacterial infections, such as with streptococcus pneumoniae. importantly, despite their involvement in mortalities as secondary infections (chi et al., 2007; köndgen et al., 2011; palacios et al., 2011), we did not test for potentially pathogenic bacteria. we did not do so mainly because such bacteria are often carried within the commensal flora of healthy humans, which makes it impossible to link a positive finding to the symptoms observed. this is a common problem for diagnostics in human medicine as well (bartlett, 2011; bosch, biesbroek, trzcinski, sanders, & bogaert, 2013) . beyond viruses and bacteria, the most frequent causative agents for acute respiratory illness, certain parasites, or funguses are also capable of triggering symptoms. more generally, infectious agents are not the only cause of respiratory symptoms (boutayeb, 2006) . non-infectious etiologies include allergies, asthma, chronic obstructive pulmonary disease (copd), cancer, air pollution, wildfire smoke, and smoking. while most of these causes are unlikely sources for the symptoms observed in this study-there was no particular air pollution or forest fire during the study period and neither allergies, asthma, nor any other chronic respiratory disease were reported by any of the subjects-it is impossible to entirely rule them out. from a practical point of view, however, preventing the spread of viruses is a bigger challenge because the particle size is smaller and aerosolization is more likely. therefore, hygiene measures preventing viral spread will also likely prevent the transmission of bacterial pathogens. even though a causative pathogen could only be determined in a single case, the delays through quarantine led to development of symptoms within isolation in 17 cases. without the quarantine procedure, these subjects would have already entered the forest, possibly approaching the chimpanzees and interacting with other people in the camps. notably, the one incidence in which a person developed symptoms after clearing quarantine was the very same incidence where hrsv was found. therefore, the infectious subject had not yet approached the chimpanzees and all contact persons were returned to quarantine. hrsv has a median incubation period of 4.4 days (95% ci: 3.9-4.9) and 95% of infected individuals will develop symptoms by 6.3 days (95% ci: 5.2-7.3) after infection (lessler et al., 2009) . while a 5-day quarantine does not cover the complete incubation period, it does cover 71% of the hrsv incubation period (83% for hmpv), and thus, lowers the risk considerably. quarantine has rather high costs and is a time intensive measure, so length needs to be decided carefully. extra space must also be made available to have physically separated facilities. additionally, people are very limited in the work they can do while in quarantine and lose time that could otherwise be spent doing valuable fieldwork-which is expensive for the project. yet, from a purely preventive health point of view, a 6-day quarantine would be even better as it would cover 95% of people developing symptoms of hrsv, and 100% for hmpv-the only two viruses ever found in respiratory disease outbreaks among wild great apes (kaur et al., 2008; köndgen et al., 2010; palacios et al., 2011) . in addition, the challenging process of quarantine has the positive side effect of limiting exchanges with surrounding villages, which in itself lowers the risk of project staff becoming infected. if logistics allow, then adding this one additional day to the quarantine process should be strongly considered. observations of outbreak frequency at the tcp are not high enough to statistically show that the frequency has declined since the implementation of the described quarantine procedure. that said, the patterns strongly suggest beneficial impacts from this procedure. before the implementation of quarantine, tcp experienced six major outbreaks between 1999 and 2006. since its implementation, the tcp has only experienced one further outbreak (in 2009), one that started when the quarantine was ignored (leendertz, unpublished data) . this underscores the argument that following quarantine rules is essential. it also demonstrates that all measures taken only result in risk reduction and emergency protocols must be kept in place for any outbreak intervention. our data and observations clearly suggest that quarantine does not replace monitoring for symptoms-they need to be used synergistically. in conclusion, it is important to note that single measures are insufficient for obtaining ethically acceptable risk reduction. several hygiene measures need to be combined: the implementation of quarantine and symptom monitoring with keeping a minimum distance of 7 m and wearing surgical masks (especially as chimpanzees do not follow distance rules) (gilardi et al., 2015; köndgen et al., 2008) . moreover, through monitoring and diagnostic investigation we can further characterize the real risk while assuring that the preventive measures are being followed. we thank the ivorian authorities for their long-term support, especially diagnostic tests for agents of community-acquired pneumonia the chimpanzees of the taï forest: behavioural ecology and evolution why do chimpanzees die in the forest? the challenges of understanding and controlling for wild ape health viral and bacterial interactions in the upper respiratory tract the double burden of communicable and noncommunicable diseases in developing countries origin of human t-lymphotropic virus type 1 in rural cote d'ivoire new streptococcus pneumoniae clones in deceased wild chimpanzees development of a pcr-based assay for detection, quantification, and genotyping of human adenoviruses respiratory syncytial virus infection in adults human metapneumovirus infections in young and elderly adults ebola outbreak in wild chimpanzees living in a rainforest of côte d'ivoire best practice guidelines for health monitoring and disease control in great ape populations the ecology of primate retroviruses-an assessment of 12 years of retroviral studies in the taï national park area ethical issues in african great ape field studies codetection of respiratory syncytial virus in habituated wild western lowland gorillas and humans during a respiratory disease outbreak development of rhinovirus study model using organ culture of turbinate mucosa descriptive epidemiology of fatal respiratory outbreaks and detection of a human-related metapneumovirus in wild chimpanzees pandemic human viruses cause decline of endangered great apes noninvasive monitoring of respiratory viruses in wild chimpanzees pasteurella multocida involved in respiratory disease of wild chimpanzees viral shedding and clinical illness in naturally acquired influenza virus infections characterisation of a new simian t-lymphotropic virus type 1 in a wild living chimpanzee (pan troglodytes verus) from ivory coast: evidence for a new stlv-1 group? sequence note anthrax kills wild chimpanzees in a tropical rainforest pathogens as drivers of population declines: the importance of systematic monitoring in great apes and other threatened mammals interspecies transmission of simian foamy virus in a natural predator-prey system isolation and partial characterisation of a new strain of ebola virus incubation periods of acute respiratory viral infections: a systematic review use of the p gene to genotype human metapneumovirus identifies 4 viral subtypes transmission of infectious diseases during commercial air travel. the lancet minimizing pathogen transmission at primate ecotourism destinations: the need for input from travel medicine human metapneumovirus infection in wild mountain gorillas detection of enteric viruses and bacterial indicators in german environmental waters genetic variability of group a human respiratory syncytial virus strains circulating in germany from human metapneumovirus: insights from a ten-year molecular and epidemiological analysis in germany genotyping of measles virus isolates from central europe and russia molecular epidemiology of respiratory syncytial virus infections among children with acute respiratory symptoms in a community over three seasons diagnostic approach for the differentiation of the pandemic influenza a (h1n1) v virus from recent human influenza viruses by real-time pcr sensitive and broadly reactive reverse transcription-pcr assays to detect novel paramyxoviruses global burden of childhood pneumonia and diarrhoea. the lancet respiratory syncytial virus infection in tropical and developing countries how to cite this article: grützmacher k, keil v, leinert v, et al.human quarantine: toward reducing infectious pressure on chimpanzees at the taï chimpanzee project, côte d'ivoire. am j key: cord-325611-tu1bn4hu authors: pérez-sautu, unai; wiley, michael ross; iglesias-caballero, maría; pozo, francisco; prieto, karla; chitty, joseph alex; garcía-garcía, maría luz; calvo, cristina; casas, inmaculada; palacios, gustavo title: target-independent high-throughput sequencing methods provide evidence that already known human viral pathogens play a main role in respiratory infections with unexplained etiology date: 2019-07-23 journal: emerg microbes infect doi: 10.1080/22221751.2019.1640587 sha: doc_id: 325611 cord_uid: tu1bn4hu despite the advanced pcr-based assays available, a fraction of the pediatric respiratory infections remain unexplained every epidemic season, and there is a perception that novel viruses might be present in these specimens. we systematically collected samples from a prospective cohort of pediatric patients with respiratory infections, that returned negative results by validated molecular rt–pcr assays, and studied them with a target-independent, high-throughput sequencing-based approach. we also included a matched cohort of children with no symptoms of respiratory infection, as a contrast study population. more than fifty percent of the specimens from the group of patients with unexplained respiratory infections were resolved. however, the higher rate of detection was not due to the presence of novel viruses, but to the identification of well-known viral respiratory pathogens. our results show that already known viral pathogens are responsible for the majority of cases that remain unexplained after the epidemic season. high-throughput sequencing approaches that use pathogen-specific probes are easier to standardize because they ensure reproducible library enrichment and sequencing. in consequence, these techniques might be desirable from a regulatory standpoint for diagnostic laboratories seeking to benefit from the many advantages of these sequencing technologies. the rate of discovery of new microbes and of new associations of microbes with health and disease has accelerated significantly in the last decade [1] [2] [3] [4] . many factors are contributing to this phenomenon including those that favour the true emergence of new pathogens as well as new technologies and paradigms that enable their detection and characterization [5] [6] [7] . popular media have focused attention on biodefense and emerging infectious diseases, providing a foundation for unprecedented support of basic and translational research in host, vector, and microbe biology, as well as diagnostics and surveillance. new molecular technologies based on high-throughput sequencing (hts) have facilitated discovery [7] [8] [9] [10] . appreciation that more than 75% of emerging infectious diseases represent zoonoses has also had an impact [11] . moreover, the databases needed to recognize microbial sequences improved dramatically with the development of more sophisticated algorithms for searching and identification [12, 13] . thus, the current accepted vision is that we are in a position to tackle effectively the problem of the detection of the "unknown known" (e.g. the unexpected rare pathogen) while true reliable agnostic pathogen discovery for detection of the "unknown unknown" (e.g. a true novel pathogen) is still an art. according to the latest global health estimates from the world health organization, respiratory infections are among the five leading causes of death worldwide, causing near 3 million deaths in 2016 [14] . acute respiratory infections (aris) account for 1-3% of deaths in children less than 5 years of age in industrialized countries and 10-25% of deaths in developing countries [14] . aris of viral origin are one of the main causes of hospitalization among those patients, who typically suffer more than one episode during the season [15] . despite advances, a significant proportion of the pediatric aris remain without a causative agent every epidemic season [16] . this fact is not exclusive of aris, more than 60% of the cases of viral encephalitis, and near 65% of all deaths from gastroenteritis and from foodborne disease, remain unassigned to a specific pathogen even after syndromic laboratory testing [17] [18] [19] . although there are multiple reasons that explain these results that are not related with diagnostic technology failure (e.g. non-infectious causes, inadequate, late or "investigational" sampling), the perception that there are pathogens lurking undetected in those specimens is widespread. unfortunately, no comprehensive risk assessments of the threat have been performed yet. in this report, we performed a systematic study of respiratory specimens collected from a carefully characterized and highly representative, prospective cohort of pediatric cases suffering unexplained ari, and we compared the rate of detection of pathogens by utilizing validated molecular assays, and a comprehensive sequence-independent, high-throughput sequencing-based analysis. in order to assess for the clinical relevance of the viral identifications made by hts in the specimens collected from the unexplained cases of respiratory infections, a second cohort of age-matched healthy individuals from the same epidemiologic environment was also studied with the same methodology. between september 2012 and june 2013, 1,454 children <14 years of age with a respiratory tract disease were admitted to the severo ochoa hospital (madrid, spain), and evaluated by an attending physician. clinical data recorded included age, gender, gestational age (when less than 37 weeks), clinical diagnosis, need and length for oxygen therapy, axillary temperature, duration of fever, total white blood cell count, c-reactive protein serum levels, presence of infiltrate/atelectasis in chest x-rays, administration of antibiotic therapy, hospital stay, and inclusion in pediatric intensive care unit. upper respiratory tract infection (urti) was diagnosed when rhinorrhea and/or cough were found with or without fever in the absence of wheezing, dyspnea, crackling rales, or bronchodilator use. asthma was diagnosed on the basis of the national asthma education and prevention program guidelines [naepp, 2002 retrieved from https://www.nhlbi.nih. gov/files/docs/guidelines/asthmafullrpt_archive.pdf]. acute expiratory wheezing was considered to be bronchiolitis when it occurred for the first time in children <2 years. all other episodes of acute expiratory wheezing were considered wheezing episodes. cases were considered pneumonia when both focal infiltrates and consolidation in chest x-rays were detected. cases of apparent life-threatening event (alte) were defined as an episode that is frightening to the observer and is characterized by some combination of apnea, colour change, marked change in muscle tone, choking or gagging [20] . fever without source (fws) was diagnosed when otherwise healthy child 3 to 24 months of age presented with fever of less than 7 days in duration and in whom alternative infectious etiologies were ruled out. nasopharyngeal aspirates (npas) were systematically taken from these patients and analysed by multiplex real-time rt-pcr based on previously published methods [21] [22] [23] . these assays allow for the detection of the main viral respiratory pathogens including: influenza virus a, b and c, human rhinovirus, enterovirus, human orthopneumovirus (human respiratory syncytial virus), human bocavirus, human metapneumovirus, adenovirus, human rubulavirus 1-4 (human parainfluenza virus) and human coronavirus (229e, oc46, hku1, and nl63). fiftyseven of these npas (age range <1 month to 14 years old; 45.6% female and 54.4% male) gave negative results in all the assays and were included in the present study. the clinical presentation of these patients (referred in the text as the "case" group) is described in table 1 . a second group of 70 npas taken from a prospective cohort of 21 age-matched healthy donors was included as a control (referred in the text as the "control" group). the individuals included in the control group were children visiting the hospital for other causes (e.g. food allergy testing) with no history of respiratory infection 10 days before to 10 days after their visit. these patients came from the same geographic area as the patients with respiratory tract disease and the npas were collected during the same period of time covering the epidemic season of virus circulation, and analysed with the molecular assays as described above, always resulting negative. the study was approved by the medical ethics committee of the instituto de salud carlos iii (cei pi 15_2012) and informed written consent was obtained from all participants. virus identification by target-agnostic highthroughput sequencing analysis all 127 respiratory specimens included in the study were processed and sequenced individually. from each npa, a 200 μl-aliquot was homogenized by passing the sample through 1 ml sterile syringes with 25g needles (becton dickinson, new jersey, usa) and by vortexing, centrifuged at 5,000×g for 10 min at room temperature, and filtered through a 0.45 μm pore-size filter (ultrafree-mc, millipore, massachusetts, usa) [24, 25] . each filtrate was processed with the rneasy micro kit (qiagen, maryland, usa) performing an on-column dna digestion following the manufacturer's protocol. rna extracts were then amplified by sispa rt-pcr as described previously [6, 8] . amplification products were purified with the minelute pcr purification kit (qiagen), sheared using the covaris s2 instrument ( table 2 ). data analysis was performed as follows: cutadapt v1.7 [26] , prinseq-lite v0.20.4 [27] and picard (http:// broadinstitute.github.io/picard), were used for removal of adaptors, primers, pcr duplicates, and for quality filtering of the index (<30 phred) and reads (<20 phred). removal of reads belonging to the host was performed by aligning the quality-trimmed reads to the human genome reference grch38 (https://www. ncbi.nlm.nih.gov/genome/guide/human) with bowtie2 v2.1.6 [28] . after the host removal step, reads were subjected to de novo assembly using ray v2.2 [29] . assembled contigs (109-28,945 bp) were taxonomically identified through sequence similarity to the nucleotide collection (nr/nt) database in genbank using ncbi blast v2.2.28+ (megablast and dc-megablast; e-value threshold of 1×10e−04). those contigs with no match were analysed with orffinder (https://www.ncbi.nlm.nih.gov/orffinder/) and the identified orfs were compared to the non-redundant protein sequences (nr) database in genbank by blastx analysis (e-value threshold of 100). unmapped reads were aligned to the nr database in genbank with diamond, using sensitive mode [30] . negative controls consisting on sterile rnasefree water were processed in parallel with the respiratory specimens. hits identified in the negative controls were considered as laboratory contaminants and not reported (suplemmentary methods). the results from the blastn and blastx analysis were manually curated and those contigs backed up by reads with no paired mate, as well as those that matched repeatedly to low complexity genomic regions or to endogenous and integrated viral sequences were discarded. all sequencing data generated in this study have been deposited in the ncbi sra database under the bioproject number prjna528996. all those npa specimens that contained reads of any respiratory virus were analysed by rt-pcr in order to confirm the results of the hts analysis. primer pairs were designed with the viral nucleotide sequence information obtained in the hts analysis (supplementary table 1 ). rt-pcr was performed in 5 µl of the same rna extracts that were initially used in the hts analysis. assays were performed with the agpath-id one-step rt-pcr kit (thermofisher scientific, massachusetts, usa) following the manufacturer's protocol. amplification conditions were as follows: 30 min at 45°c , 10 min at 95°c, 40 cycles of 30 s at 95°c, 30 s at 60°c and 1 min at 72°c, followed by a final extension step of 7 min at 72°c. rt-pcr products were analysed in 2% agarose gel electrophoresis, purified with the minelute pcr product purification kit (qiagen), and sequenced with the bigdye terminator v3.1 cycle sequencing kit (life technologies, california, usa). target-agnostic hts analysis of the 57 nasopharyngeal aspirates taken from the "case" group produced a total of 24.6 million reads and 35,666 contigs that were taxonomically identified by blastn analysis (figure 1 (a)). a total of 35,226 contigs (with 42.3 million reads) were identified by blastn analysis from the samples taken from the "control" group ( figure 1 (b) ). in the "case" group, contigs assigned to viruses represented 14.4% (5,117 contigs), while viruses represented 7.1% (2,486 contigs) in the "control" group (figures 1(a,b) ). among the 5,117 contigs assigned to viruses in the "case" group, 13% (667 contigs with 2.6 million reads mapped) corresponded to viruses that infect eukaryotic organisms, while 87% (4,450 contigs with 4.1 million reads mapped) corresponded to bacteriophages. among the contigs assigned to viruses that infect eukaryotic organisms 9 different families were identified namely, papillomaviridae, pneumoviridae, picornaviridae, paramyxoviridae, anelloviridae, coronaviridae, circoviridae, orthomyxoviridae, and polyomaviridae (figure 1(c) ). altogether, the number of contigs assigned to any respiratory viral pathogen accounted for 51% (340 contigs) ( table 2) . when taking into account the number of reads, the vast majority (99.7%; 2.54 million reads) mapped to any of the 340 contigs assigned to respiratory viral pathogens (table 2) . among these, human orthopneumovirus (human respiratory syncytial virus; hrsv) and human rhinovirus (hrv) were the most frequently table 2) . altogether, at least one respiratory viral pathogen could be identified by target-agnostic hts analysis in 35 out of the 57 npa specimens. other viruses identified in these samples included: sapelovirus a, human parechovirus 1 (hpev-1), human papillomavirus (hpv), human poscv5-like circular virus, torque teno virus, torque teno mini virus, and human polyomavirus 4 (wu polyomavirus) ( table 2) . from the 70 respiratory specimens taken from the "control" group, 5.9% of the contigs (147 contigs with 951,625 reads mapped) corresponded to viruses that infect eukaryotic organisms, while 94.1% (2,339 contigs with 4.4 million reads mapped) corresponded to bacteriophages. among the contigs assigned to viruses that infect eukaryotic organisms, members of the papillomaviridae, circoviridae, pneumoviridae, anelloviridae, picornaviridae, herpesviridae, totiviridae, virgaviridae, and reoviridae were identified (figure 1(d) ). viral respiratory pathogens were identified only in two specimens. one produced 4 contigs that were identified as hmpv, with the vast majority of the reads mapping to them (99.1%; 943,278 reads) ( table 2 ). in a second specimen, hrv was identified, with 2 contigs to which 5 reads mapped. the remaining 8,342 reads mapped to a total of 141 contigs identified as the following viruses: hpv, human poscv5-like circular virus, torque teno virus, red clover powdery mildew associated totivirus, tobacco mosaic virus, rotavirus a, and human betaherpesvirus 5 ( table 2) . results of the contig-specific rt-pcr assays confirmed those of the target-agnostic hts analysis. in the "case" group, hrsv and hrv were the most prevalent viruses, followed by ev and hpiv, hcov, influenza b virus, and hmpv (table 3 ). in the "control" group, the two npa where hmpv and hrv had been identified by hts analysis were confirmed to be positive for these viruses (table 3) . several specimens among the "case" group contained more than one virus (table 4 ). among these co-infections, hrsv was detected with either hpiv, ev or hrv, and hcov was detected along with hrv (table 4) . no co-infections were detected in the "control" group. in 22 out of the 57 (38.6%) npas taken from the cases of respiratory infection the target-agnostic hts analysis did not identify any known respiratory viral pathogen (table 5 ). in these samples, only bacteriophages, torque teno virus, hpv, human poscv5-like circular virus, and human polyomavirus 4 (wu polyomavirus) were identified (table 5 ). further analysis of the unidentified contigs assembled from these samples by looking for sequence homologies to known viral proteins (blastx) returned no significative results. although some hits to viral proteins were identified, all of these contigs mapped also to other protein sequences in the gen-bank database with the same e-values, and with comparable ranges of coverage and identity percentage values, and thus could not be considered viral-specific hits. representative examples of such kind of identifications are shown in table 6 . reads that did not form contigs were mapped against the nr genbank database with diamond [30] . the analysis showed that those reads matched to proteins of bacterial or human origin or belonging to bacteriophages, and no putative novel virus was identified (results not shown). we compared the bacterial hits identified by targetagnostic hts analysis in these samples with those identified in the specimens taken from the control group. several contigs were identified by blastn analysis as wellrecognized bacterial respiratory pathogens, such as streptococcus pneumoniae, haemophilus influenzae, and klebsiella pneumoniae. however, all these hits to bacterial respiratory pathogens were not exclusive of the specimens taken from the cases of respiratory infection and could be identified also in the specimens taken from the control group (results not shown). since our analysis is not designed to be quantitative, no conclusions regarding frequency could be made. between 2012 and 2013, we tested by multiplex realtime rt-pcr [21] [22] [23] a total of 1,454 specimens from pediatric respiratory infections. out of these, 57 remained negative after the analysis with different molecular assays. in order to shed light on the etiology of these infections, these 57 specimens were subjected to target-independent hts analysis, along with 70 agetable 3 . respiratory viral pathogens identified by target-agnostic hts analysis and confirmed by contig-specific molecular assays in the respiratory specimens from the cases of respiratory infection and from the control group. matched specimens from a control group. similarly to the 57 specimens from the respiratory infections, the specimens from the control group resulted negative in the aforementioned molecular assays. upon hts analysis we further identified known viral respiratory pathogens (hrsv, hrv, ev, hpiv, hcov, hmpv and influenza b virus). altogether, at least one respiratory viral pathogen could be identified in 35 out of the 57 npa specimens from the group of respiratory infection. by contrast, in the control group, only 2 samples contained contigs attributed to viral respiratory pathogens (hmpv and hrv). moreover, the hmpv contigs detected in one of these samples, hoarded the vast majority of the reads assigned to any eukaryotic virus (99.1%). the remaining viral entities were anelloviruses and papillomaviruses common to both groups. identification of respiratory viruses by hts was confirmed by contig-specific rt-pcr analysis. the hmpv and hrv identified in the control group occurred in two cases that showed no symptoms of respiratory disease. asymptomatic infections by hrv have been previously reported and are more frequent in young children [31] . although asymptomatic infections by hmpv can occur at the pediatric stage, they are more frequent in immunocompetent adult individuals [31] [32] [33] . apart from common respiratory viral pathogens, human parechovirus 1 (hpev-1) and human polyomavirus 4 (wu polyomavirus) were identified by hts analysis. both viruses were identified in specimens from the group of respiratory infection (1 sample each); none in the control group. the detection of hpev-1 is not surprising as this virus is a frequent cause of infection in childhood, where it causes mild gastrointestinal and respiratory disease [34] . human polyomavirus 4 (wu polyomavirus) was originally detected in respiratory secretions of a pediatric patient diagnosed with pneumonia of unknown origin, and from patients with acute respiratory co-infections [35] . nonetheless, a subsequent larger study found no link between wu polyomavirus and acute respiratory disease [36] . in our study, wu polyomavirus was identified in one case of respiratory infection. although no other respiratory virus was identified in this sample, wu polyomavirus presents low prevalence in cases of respiratory infection and high rates of co-infection with other common respiratory viral pathogens, and further studies are needed to ascertain its clinical significance [35] [36] [37] . thus, out of 57, 21 (36.84%) remain unexplained from a virological standpoint, as no known respiratory or novel virus was identified. analysis by blastn only returned matches to anelloviruses, papillomaviruses, table 6 . representative examples of matches in genbank identified by blastx analysis of the contigs that remained unassigned to any taxon by blastn analysis. and human poscv5-like circular virus. in spite of thorough, in-depth analysis of those contigs with no match as well as of the unmapped reads, no viral match in the genbank database was obtained. anelloviruses are frequently detected in most tissues and organs, including the respiratory tract of healthy individuals, and there is no association to any disease in humans [38] . papillomaviruses are very common worldwide, and most infections are asymptomatic and resolve spontaneously. apart from the common warts and the various types of cancer (including an oropharyngeal form) associated to infection by hpv, low-risk types 6 and 11 are the predominant cause of respiratory papillomatosis, a disease in which noncancerous tumours grow in the air passages of the respiratory tract [39] . however, other than oropharyngeal cancer and respiratory papillomatosis, there is no evidence of an association between papillomavirus infection and respiratory disease. in addition to the lack of evidence for an association with respiratory disease, in our study anelloviruses and hpv were also identified in the control group, strengthening the conclusion that their presence was not related to the respiratory disease. human poscv5-like circular virus was recently identified in respiratory secretions from an unexplained human case of febrile illness, although its association with disease was not determined [40] . small, circular, single stranded, rep-encoding, dna (cress-dna) viruses have been increasingly identified by metagenomic hts techniques [41] in environmental samples and in a variety of vertebrates as well as various invertebrates [42] [43] [44] [45] . in humans, they have been reported in feces of healthy individuals [43] , and in samples from unexplained cases of encephalitis and diarrhea [46] , pericarditis [47] , acute central nervous system infections [48] , as well as in npas from children with respiratory infections [49] . however, neither of these studies could establish a direct association with disease. thus, it is not clear what is the value of finding contigs that match this virus in the specimens from the group of respiratory infection. further studies are needed to determine their role (if any) in human respiratory disease. our findings agree with previous studies with similar design and tools [25, 50, 51] . xu et al. employed hts to analyse a set of respiratory specimens taken from children with community-acquired pneumonia that had returned negative results in a commercial respiratory viral panel detection assay [25] . they also identified hpiv, torque teno virus, torque teno minivirus, and wu polyomavirus [25] . zhou et al. studied cell-cultured supernatants with apparent cytopathic effect that had been prepared from undiagnosed respiratory specimens and identified a high prevalence of ev accompanied by hrv, hrsv, hpiv, adv, influenza c virus, herpesvirus 1 and dengue virus [51] . taboada et al. studied nasal washings from children with respiratory infections previously found negative for common bacterial and viral respiratory pathogens by pcr, where they identified at least one known respiratory virus (including hrsv, hcov, and hrv) in the vast majority of the specimens [50] . neither of these studies revealed the presence of any putative novel virus on the undiagnosed respiratory infections subject of study, and the large majority of the cases could be attributed to known viral respiratory pathogens [25, 50, 51] . these studies suggest that all respiratory viral pathogens of clinical relevance during the pediatric stage have already been identified. in our study, we analysed 57 cases that, out of a total of 1,454, were negative in all pathogen-specific pcr assays. in consequence, only 3.9% (57/1,454) of all respiratory specimens collected during the entire epidemic season remained without a known etiology of infection after using pathogen-targeted diagnostic techniques. while target-independent hts analysis allowed us to come to a specific diagnostic in more than half of the 57 undiagnosed infections, all the additionally resolved cases were produced by known respiratory pathogens. from these results, we can conclude that the current pathogen-specific techniques should be able to diagnose the vast majority of the respiratory infections. in consequence, we can conclude that the risk of overlooking a novel unknown viral respiratory pathogen in the pediatric population is very low when using target-specific diagnostic methods and that there is very little value in using target-independent assays. routine virological surveillance based on target-specific techniques, such as real-time (rt)pcr or target enrichment hts approaches (which use probes specifically designed against the viral genomic sequence of interest in order to enrich specifically for sequencing libraries derived from said virus), is appropriate and constitute a first-line diagnostic tool. the question about the etiology behind those cases of respiratory infection that remain negative for all routine diagnostic assays have been previously addressed by several groups [24, 25, [50] [51] [52] . although in our study we used an overall approach which was similar to such previous studies, we introduced several improvements to our specific study design in order to address different limitations of the previous works. the identification of viral reads in clinical specimens remains controversial because it does not necessary imply that such viruses are responsible for the symptoms observed. many viruses can cause asymptomatic or subclinical infections, or simply be present among the normal, healthy microbiota and replicate without any pathogenic consequences. this hinders the interpretation and understanding of the results unveiled by virus discovery studies based on hts in regard to their clinical significance [25, 50, 51] . in our study, we included a contrast study population (control group) formed by a prospective cohort of healthy individuals. such healthy control group was matched at all the critical levels with the cohort of patients with unexplained infection of the respiratory tract: (i) the individuals of the control group were age-matched; (ii) they came from the same geographic area; (iii) their samples were also taken systematically during the same time window covering the epidemic season of virus circulation. by including the control group, we were able to determine that the viruses detected in the clinical specimens taken from the patients with an infection of the respiratory tract were not circulating among the healthy population, providing evidence that such viruses were responsible for the respiratory disease observed in those cases. this would acquire special importance if a novel viral entity for which there is no previous information available is detected. in summary, the inclusion of the healthy control group allowed us to assess the clinical relevance of the viruses identified in the samples taken from the cases of unexplained respiratory infections. whenever possible, any future viral discovery studies should include a matched healthy control group to which the viruses identified by hts in the patients with clinical disease can be contrasted. in addition to the inclusion of a matched healthy control group, we processed and sequenced in parallel negative controls consisting on sterile nuclease-free water (see the materials and methods section) and we confirmed the viral identifications made by hts with specific rt-pcr assays. the objective of these procedures was to minimize the chances of reporting any false viral identification. it is worth highlighting also, that we performed a systematic sampling on our study groups: samples were collected from all the children arriving consecutively at the hospital during the period of the study, whose parents or legal guardians gave explicit written consent, and according to preestablished clinical and medical criteria (see the materials and methods section) with no further selection. by combining all the procedures discussed above, our study design constitutes a novel integrated approach that ensures a robust representativeness of the results, minimizes any possible bias, and provides a better understanding about the clinical implications of the viral identifications made by hts analysis. while hts was superior in the overall rate of detection of pathogens, that was not due to the presence in the cohort of unknown pathogens, but mostly on the underperformance of molecular methods (real-time rt-pcr) against targeted pathogens. all the viruses additionally identified by hts in our study were well-known respiratory pathogens, and no novel viruses were detected. altogether, our results show that already known viral respiratory pathogens play a main etiologic role behind the unexplained cases of respiratory infection in the pediatric population. this is a very significant finding, because if extrapolated to other clinical syndromes and specimens, it might allow us to quantitatively assess the risk. under that new paradigm, "agnostic" technologies would still have a role in pathogen detection under outbreak and event situations where a true "unknown unknown" is suspected, but "targeted" approaches would become desirable for next-generation sequencing-based microbial diagnostic. this paradigm might be desirable from a regulatory standpoint for those diagnostic laboratories seeking to incorporate these technologies, since "targeted" approaches allow for specific and reproducible library enrichment and thus they are easier to assess and validate. genetic detection and characterization of lujo virus, a new hemorrhagic fever-associated arenavirus from southern africa discovery of an ebolavirus-like filovirus in europe prolonged shedding of zika virus associated with congenital infection redefining the invertebrate rna virosphere a metagenomic survey of microbes in honey bee colony collapse disorder panmicrobial oligonucleotide array for diagnosis of infectious diseases using metagenomics to characterize an expanding virosphere a new arenavirus in a cluster of fatal transplant-associated diseases the new age of molecular diagnostics for microbial agents a multicomponent animal virus isolated from mosquitoes global trends in emerging infectious diseases pathogen discovery a reference viral database (rvdb) to enhance bioinformatics analysis of high-throughput sequencing for novel virus detection global health estimates 2016: deaths by cause, age, sex, by country and by region epidemiology and etiology of childhood pneumonia in 2010: estimates of incidence, severe morbidity, mortality, underlying risk factors and causative pathogens for 192 countries emerging advances in rapid diagnostics of respiratory infections mortality due to gastroenteritis of unknown etiology in the united states deaths due to unknown foodborne agents viral encephalitis of unknown cause: current perspective and recent advances national institutes of health consensus development conference on infantile apnea and home monitoring simultaneous detection of influenza a, b, and c viruses, respiratory syncytial virus, and adenoviruses in clinical samples by multiplex reverse transcription nested-pcr assay simultaneous detection of fourteen respiratory viruses in clinical specimens by two multiplex reverse transcription nested-pcr assays infections and coinfections by respiratory human bocavirus during eight seasons in hospitalized children a metagenomics study for the identification of respiratory viruses in mixed clinical specimens: an application of the iterative mapping approach characterization of the nasopharyngeal viral microbiome from children with community-acquired pneumonia but negative for luminex xtag respiratory viral panel assay detection cutadapt removes adapter sequences from high-throughput sequencing reads quality control and preprocessing of metagenomic datasets ultrafast and memory-efficient alignment of short dna sequences to the human genome ray meta: scalable de novo metagenome assembly and profiling fast and sensitive protein alignment using diamond detecting respiratory viruses in asymptomatic children human metapneumovirus infections in young and elderly adults human metapneumovirus infection in adults enterovirus and parechovirus infection in children: a brief overview identification of a novel polyomavirus from patients with acute respiratory tract infections no evidence for an association between infections with wu and ki polyomaviruses and respiratory disease wu and ki polyomavirus infections in filipino children with lower respiratory tract disease metagenomic analysis of viral genetic diversity in respiratory samples from children with severe acute respiratory infection in china human papillomavirus and diseases of the upper airway: head and neck cancer and respiratory papillomatosis identification and genetic characterization of a novel circular single-stranded dna virus in a human upper respiratory tract sample rapidly expanding genetic diversity and host range of the circoviridae viral family and other rep encoding small circular ssdna genomes diverse circovirus-like genome architectures revealed by environmental metagenomics multiple diverse circoviruses infect farm animals and are commonly found in human and chimpanzee feces dragonfly cyclovirus, a novel single-stranded dna virus discovered in dragonflies (odonata: anisoptera) genomic characterization of novel circular ssdna viruses from insectivorous bats in southern brazil small circular single stranded dna viral genomes in unexplained cases of human encephalitis, diarrhea, and in untreated sewage novel singlestranded dna circular viruses in pericardial fluid of patient with recurrent pericarditis enamel-based mark performance for marking chinese mystery snail bellamya chinensis cyclovirus in nasopharyngeal aspirates of chilean children with respiratory infections is there still room for novel viral pathogens in pediatric respiratory tract infections? metagenomics study of viral pathogens in undiagnosed respiratory specimens and identification of human enteroviruses at a thailand hospital retrospective use of next-generation sequencing reveals the presence of enteroviruses in acute influenza-like illness respiratory samples collected in south/south-east asia during 2010-2013 no potential conflict of interest was reported by the authors. key: cord-337137-0ey40gzw authors: lo, anthony wi; tang, nelson ls; to, ka‐fai title: how the sars coronavirus causes disease: host or organism? date: 2005-12-17 journal: j pathol doi: 10.1002/path.1897 sha: doc_id: 337137 cord_uid: 0ey40gzw the previous epidemic of severe acute respiratory syndrome (sars) has ended. however, many questions concerning how the aetiological agent, the novel sars coronavirus (cov), causes illness in humans remain unanswered. the pathology of fatal cases of sars is dominated by diffuse alveolar damage. specific histological changes are not detected in other organs. these contrast remarkably with the clinical picture, in which there are apparent manifestations in multiple organs. both pathogen and host factors are important in the pathogenesis of sars. the choice of specific receptors and the unique genome of the sars‐cov are important elements in understanding the biology of the pathogen. for the host cells, the outcome of sars‐cov infection, whether there are cytopathic effects or not, depends on the cell types that are infected. at the whole‐body level, immune‐mediated damage, due to activation of cytokines and/or chemokines and, perhaps, autoimmunity, may play key roles in the clinical and pathological features of sars. continued research is still required to determine the pathogenetic mechanisms involved and to combat this new emerging human infectious disease. copyright © 2006 pathological society of great britain and ireland. published by john wiley & sons, ltd. severe acute respiratory syndrome (sars) is a new viral disease caused by a novel coronavirus, sars-cov ( figure 1 ) [1, 2] . the saga of sars has officially come to an end, as no more new cases have been reported since 2004. many questions, particularly those related to how sars-cov causes disease, however, remain unanswered. the disease caused by sars-cov differs from the diseases caused by the previously known human coronaviruses, 229e and oc43. sars-cov infection results in severe and potentially fatal lung disease [1, 2] . although the majority of patients recovered after 1-2 weeks of debilitating febrile illness, a substantial proportion (up to one-third) developed severe inflammation of the lung, requiring ventilator support and intensive care. many patients in this group deteriorated into acute respiratory distress syndrome (ards). the mortality of this group of patients is high [3] . manifestations in other organ systems are characteristic. lymphopenia [4] , gastrointestinal symptoms [5] , impaired liver function [6, 7] , and impaired renal function [8] are common. the possibility of viral infection in multiple organs has been raised and viral replication in the lung, kidney, and gastrointestinal tract was reported [9, 10] . in addition, prolonged shedding of virus was found in some convalescent patients [11] . however, chronic infection by sars-cov has not, to date, been documented in humans. moreover, asymptomatic carriage of sars-cov is rare [12] . there are significant age differences in the prognosis of sars. children have a good prognosis [13] , while elderly patients with chronic illnesses fare badly. sars is predominantly a lower respiratory tract disease, yet the most consistent and powerful prognostic indicator reported so far is blood lactate dehydrogenase (ldh) concentration [1] , which is most likely a surrogate indicator and may reflect the extent of ongoing tissue damage. both pathogen and host factors are important for the progression of an infection. here, we review the pathology of sars infection. specific features of the pathogen sars-cov itself are then addressed. finally, host factors, particularly an emerging understanding of immunological and inflammatory responses to sars-cov infection, are discussed. virus particles can also be seen budding through the cytoplasmic membrane (b). each virion particle is 60-90 nm in size by transmission electron microscopy and is characterized by the numerous club-shaped projections on the outside, a ring beneath the envelope, and an electron-lucent centre. scale bars = 200 nm (a) and 50 nm (b) diffuse alveolar damage is the most characteristic pathology in sars most data on the human pathology of sars come from autopsy studies of fatal cases [14] [15] [16] [17] [18] [19] [20] [21] . these reports thus reflect the terminal stages and are likely to represent only the more severe end of the spectrum of sars. treatment and co-morbid conditions might also modify the pathological changes. diffuse alveolar damage at different stages of organization is the most consistent finding in the lungs of sars patients in the terminal stage (figures 2a-2f ). multinucleated syncytial cells ( figures 2g and 2h ) are characteristic, although these cells are rare. apart from when secondary infection occurs, the lack of a prominent inflammatory response is also distinctive. sars-cov is explicitly detected in the alveolar lining cells ( figures 2i and 2j ) [10, [22] [23] [24] [25] [26] [27] . no specific pathology is identified in the gastrointestinal tract ( figure 3) [5], urinary system [8] , or other organ systems [28] , apart from that related to end-stage multi-organ failure or those changes secondary to treatment. it is important to note that in some organs such as the liver, while definitive and distinct morphological and functional changes are observed, sars-cov may not be unequivocally demonstrable [29] . it is clear that our understanding of the pathology of sars is incomplete. an obvious large gap is the lack of information on the early pathological changes of sars. during the epidemic, very few biopsies were obtained from patients with clinically active sars. the study of animal models is important in a number of ways. it has allowed the establishment of sars-cov as the aetiological agent [30] . it also provides controlled conditions for the study of early changes in the disease. initial studies of macaque models were promising. the histology of infected lung tissue is similar to that in humans [31] [32] [33] . both acute and organized stages of diffuse alveolar damage were seen when the macaques were sacrificed on the sixth day after a heavy dose of the virus. sars-cov was detected in the alveolar epithelial cells and in the intra-alveolar syncytial cells. however, detailed morphological studies and viral distribution in other organs in these animal studies are lacking. in studies involving longer observation times, the disease in macaque models appears self-limiting and different from the genuine human disease. the usefulness of the macaque as a model of the disease remains to be established [32, 33] . civet cats, domestic cats, and ferrets are thought to have been potential reservoirs of the virus during the epidemics and subsequent smaller outbreaks in mainland china [34] . the animal coronavirus identified in civet cats shows high sequence identity with, but is distinct from, sars-cov [2, 35] . recent evidence also suggests that wide chinese horseshoe bats harbour a closely related bat-sars-cov which might also act as the animal reservoir [36] . again, details concerning the distribution of virus in different organs in these animals and the information on the pathology in the diseased or carrier animals are, surprisingly, sparse [32, 35] . other common small laboratory animal models, such as the mouse, are not particularly useful. sars-cov has a low virulence in ordinary laboratory mice and very high levels of inoculation are required to produce self-limiting diseases. these features may be although the latter finding may be related to pre-morbid lung pathology, a correlation with interstitial fibrosis and disease duration has been demonstrated [21] . diffuse alveolar damage at different stages of organization, from fibrin deposition (c, h&e, original magnification ×200), to interstitial fibrosis (d, h&e, original magnification ×100) and cellular organization (e and f, h&e, original magnification ×400), can be detected. atypical pneumocytes with enlarged nuclei and prominent nucleoli are often seen and some pneumocytes coalesce into syncytial multi-nucleated cells (g, h&e, original magnification ×600). multi-nucleated histiocytes may also be found (h, h&e, original magnification ×600). sars-cov can be detected in pneumocytes by in situ hybridization (i, using a dna probe against the m gene, original magnification ×600 [22] ). a large array of antibodies against the viral proteins including nucleocapsid n, spike s, membrane m, and sars-3a [23] , has been developed for the detection of sars-cov in formalin-fixed, paraffin-embedded tissue sections (j, showing immunohistochemical staining with an anti-peptide antibody against n, original magnification ×600) related to differences in the affinities of sars-cov for human receptors and their murine homologues [37] . sars-cov uses a protector of lung damage, angiotensin-converting enzyme 2, as a receptor characterization of the functional cellular receptor of sars-cov provides important clues to the pathogenesis of sars. angiotensin-converting enzyme 2 (ace2) interacts directly with the spike (s) proteins of the sars-cov [38] [39] [40] [41] [42] . the level of expression of ace2 correlates with the efficiency of sars-cov infection in cell culture models [42] [43] [44] . ace2 proteins are expressed by alveolar epithelial cells and by surface enterocytes of the small intestine [45] , which are the primary target cells of sars-cov. studies in the intestine cell culture model, however, suggested that, in addition to ace2, unknown co-factors or coreceptors are required to convey infectivity [46] . in addition to being a cellular receptor, ace2 may contribute to the pathogenesis of dad in sars through its role in the tissue renin-angiotensin system. in a mouse model of alveolar damage induced by acid aspiration, the balance of the renin-angiotensin system appears to affect the development of dad. ace2, which acts as a negative regulator of the local renin-angiotensin system, protects the mouse lung against experimental damage [47, 48] . sars-cov co-infection in these damaged animals downregulates ace2 in the lungs of infected mice and the severity of lung damage can be alleviated by blocking the system [49] . exciting as these findings appear, the case of a new coronavirus, nl63, immediately provides an example that other factors are acting in the overall mechanism of lung damage. nl63 utilizes the same ace2 protein as its receptor in the lung. however, infection with nl63 results in only minor cold symptoms and alveolar damage is rare [50] . the insert/deletion genotype of the ace gene was associated with dad after sars-cov infection in a small cohort of 44 patients [51] . this association was, however, not replicated subsequently in a larger series [52] . we also could not detect any association between the ace2 genotype and disease severity in sars-cov infection [53] . sars-cov may also use the c-type lectins as receptors for infecting immune cells c-type lectins, including cd209 and cd209l, are also sars-cov receptors: these were identified through the study of proteins that interact with the s (spike) protein. cd209, also known as dendritic cell-specific intercellular adhesion molecule-grabbing non-integrin (dc-sign), was shown to mediate viral entry in a lentiviral pseudo-type experimental model [54] . in chinese hamster ovary (cho) cells expressing a human lung cdna library, s protein and its fragments interacted directly with a second related cell surface glycoprotein, cd209l, also known as l-sign or dc-signr [55] . cd209l acts in conjunction with lsectin (liver and lymph node sinusoidal endothelial cell c-type lectin) and enhances viral infection [56] . tissue cultures expressing cd209 or cd209l were also susceptible to sars-cov infection [54, 55, 57] . the possible involvement of dendritic cells is particularly interesting. although sars-cov does not replicate in dendritic cells, these cells may act as a reservoir and distribute the virus to other cell types [54, 58] . this is an attractive concept and similar biological behaviours have been proposed for human immunodeficiency virus i (hiv i) [59] . no sars-cov has been detected in dendritic cells in autopsy and biopsy studies reported so far. the genome of sars-cov consists of a single 27.69 kb positive-strand rna. the genomic sequences derived from different phases of the sars epidemic revealed no association with sequence variation and virulence [60, 61] . there are two large open reading frames (orfs) and 12 potential orfs in the sars-cov genome. the two large orfs encode non-structural proteins involved in replication. these proteins have relatively higher homologies to known coronaviruses. the remaining 12 orfs are squeezed into the 3 end of the genome. these orfs include four genes encoding known structural proteins (envelope, membrane, nucleocapsid, and spike proteins, respectively). the remaining potential orfs encode hypothetical sars-cov-specific proteins which lack obvious sequence similarity to known proteins [62, 63] . the functions of these hypothetical proteins and their roles in sars pathogenesis remain obscure [64, 65] . antibodies against some of these putative proteins, notably sars3a and sars6, can be detected in the serum of sars patients [66] . there is also evidence suggesting that a number of these proteins, including sars3a, 3b, 7a, and 9b, were expressed in pneumocytes and enterocytes in deceased patients [23] . however, differential expression patterns of these proteins in cell types showing different responses to sars-cov infection have not been confirmed. by expressing the hypothetical proteins individually in tissue culture, we are beginning to see data on the cellular functions of these proteins. sars3a appears to be important in mediating apoptosis in some cell types [67] . the sars3a protein is incorporated into the viron particle and may also act as one of the structural proteins [68] [69] [70] . through an unknown mechanism, host cells overexpressing sars3a have increased expression of fibrinogen mrna [71] . sars7a has been implied in mediating apoptosis through the caspase-dependent pathways [72] . the effect of sars-cov infection varies in different cell types. apoptosis and syncytial formation are seen in infected monkey renal epithelial cells (vero e6) [67] . persistent infection with no change in cellular morphology or doubling time was detected in the colon cancer cell line lovo [46] . in clinical specimens, sars-cov was detected in the lungs and small intestine. severe cellular damage is characteristically detected in the lungs of sars patients, while no morphological changes are observed in the small intestine. the basis of these differences in cellular responses is not clear. the tissue/cellular tropism may be partly related to differential expression of membrane receptors for the sars-cov [22] . these observations highlight the importance of host cell responses in sars-cov infection. it is also clear from these observations that cytopathic damage alone cannot explain the pathogenesis of sars. the marked heterogeneity of the disease course and outcome after sars infection suggests that host responses may play an important role in pathogenesis. dad or ards appears to be a common pathway of lung parenchyma damage initiated by a variety of aetiologies, including sars-cov infection itself, systemic sepsis, shock, and direct lung contusion. once an inflammatory process reaches a certain intensity, it may self-perpetuate. the cellular inflammatory infiltrate releases toxic metabolites and proteolytic enzymes, which may cause further damage to the lung parenchyma. the surrounding inflamed capillaries launch the coagulation cascade and recruit more immune cells [73, 74] . our previous investigation in the 1997 h5n1 influenza outbreak showed that patients who died of the disease had lymphoid depletion associated with marked elevation of circulating concentrations of cytokines, including interleukin-6 (il-6), il-2 receptor, and interferongamma [75] . with the observation of characteristic lymphopenia in sars, it has been postulated that the sars-cov may similarly trigger an exaggerated hyper-cytokinemic response in patients with dad after viral infection [20] . current understanding indicates that patients with a more intense immune response are those at risk of a poor outcome, as the immune system also mounts a profound reaction to the bystander, the lung parenchyma, and causes dad [76] . sars patients have variable humoral responses to individual epitopes [66] . however, early sero-conversion and high peak total sars-cov igg levels were associated with more severe disease in a cohort of 325 patients [77] . hence, particularly strong humoral responses to sars-cov infection might not be protective but, perhaps, might be harmful to the host. the specific epitopes upon which these 'damaging' antibodies act await further characterization. there is evidence that disarray of the immune system towards the host's own antigens may play a role in the pathology of sars. in the early phase, within 1 week of sars-cov infection, igm and igg autoantibodies against antigens located in the cytoplasm of lung epithelial cells (figure 4) were detected in the sera of 36 chinese sars patients (lo, unpublished observations). in another cohort of 22 sars patients, immune activity against antigens from lung epithelial cell lines and endothelial cell lines was found in some patients' sera obtained approximately 1 month after infection [78] . moreover, high levels of these autoimmune activities in the sera were shown to be cytotoxic to lung epithelial cells and endothelial cells in culture. autoimmune antibodies may be important in mediating tissue damage at certain stages of the disease. the cause of the autoimmunity is not fully understood. these autoantibodies may be the result of humoral responses to innate antigens exposed accidentally during direct damage of the lung and, perhaps, the endothelium by sars-cov. alternatively, autoimmunity may be due to crossreactivity of antibodies against some specific epitopes of the sars-cov proteins. the chemokines are a family of small proteins that play important roles in intercellular signalling and chemotaxis. based on their protein sequences, they are broadly divided into α-chemokines with a common c-x-c (cysteine-other-cysteine) structure of amino acid residues near the amino-terminus which interacts predominantly with neutrophils, and β-chemokines with a c-c (cysteine-cysteine) structure interacting with mononuclear cells. recently, chemokines have been recognized for their roles in integrating the innate and adaptive immune responses to viral infection through a cytokine-to-chemokine-to-cytokine signalling cascade [79] [80] [81] . a global view of the spectrum of expression of the immune mediators was studied in sars by measuring the circulating concentrations of these mediators at different stages of the disease. most cytokines showed only transient and short-lived activation in patients after sars-cov infection [82] . even in patients who developed dad, most cytokine concentrations were not significantly increased [83] . in contrast, circulating concentrations of several chemokines, including cxcl9 (chemokine c-x-c motif ligand 9 or monokine induced by γ -interferon), cxcl10 (chemokine c-x-c motif ligand 10 or interferoninducible protein-10), and ccl2 (c-c motif ligand 2 or monocyte chemoattractant protein-1), were markedly increased in sars patients [82, 84, 85] . remarkably, the circulating concentration of cxcl10 measured early after infection is an independent prognostic indicator of disease outcome [86] . these chemokines therefore appear to be important elements of the pathogenesis of sars. in the lung tissues obtained from seven sars patients who died [86] , chemokines cxcl10 ( figure 5 ) and il-18 were markedly activated (25and 40-fold compared with controls, respectively). the important roles of chemokines are underscored by the findings in an experimental mouse model of sars-cov infection in which cxcl10 and a neutrophil chemokine, cxcl8 (chemokine c-x-c motif ligand 8), were also markedly activated [87] . these findings in sars compare favourably with the specific situation in hiv patients with lung allograft rejection and interstitial alveolitis, in which similar activation of the chemokine cxcl10 and its receptor cxcr3 (chemokine c-x-c motif receptor 3) was also found [88, 89] . other than pneumocytes, chemokines are also expressed and secreted by various different cell types. global gene expression profiles, generated by cdna microarray analysis of peripheral blood mononuclear cells (pbmcs) after in vitro exposure to sars-cov, also reveal the importance of chemokine activation. within 1 day after exposure to the virus, a number of chemokines (including cxcl10, cxcl9, and ccl2) were activated [90] . pbmcs and macrophages do not support productive infection as viral replication is abortive and no infectious virus is produced. the roles of these cell types in the pathogenesis of sars remain to be clarified. nonetheless, these easily obtainable cell types provide convenient experimental models and allow some insight into the patterns of host responses to the infection to be studied. similar findings were also reported in other cell types, such as dendritic cells, where the cytokine expression profiles are predominantly of inflammatory chemokines ccl3 (chemokine c-c motif ligand 3), ccl5 (chemokine c-c motif ligand 5), cxcl10, and ccl2. unlike the usual response of dendritic cells to viral infection, anti-viral cytokines, including ifn-α (interferonalpha), ifn-β, ifn-γ , and il-12b, were not activated [58] . immunogenetics of the host may affect the severity of sars other than using serum inflammatory mediators to reflect the different degree of host inflammatory reaction during an infection, the intensity of the immune response is also genetically determined. the difference in genetic makeup between individuals is mostly accounted for by single base differences (single nucleotide polymorphisms, snps). many studies have shown an association between snps and predisposition to ards, and survival after sepsis or other insults [91, 92] . in the context of predisposition to ards after trauma, among parameters such as circulating concentrations of il-1, tumour necrosis factor and plasminogen activator inhibitor-1 (pai-1), and the genotype of pai-1, insertion alleles at the promoter of pai-1 were associated with high concentrations of pai-1 in the plasma and a poor survival rate [93] . in addition to pai-1, other genetic polymorphisms, such as angiotensin-converting enzyme (ace) [94] , cd14 [95] , surfactant protein [96] , and hla genotypes [97] , are also associated with predisposition to, severity, and outcome of ards. although sasr-cov utilizes ace2 as its receptor and ace2 is known to be an important protector of lung damage in experimental ards, we and other groups found no solid association between alleles of the two ace genes (ace and ace2 ) and the severity of ards after sars infection [52, 53, 98] . several immunogenetic studies have been reported in association with sars infection. among 37 taiwanese sars patients, hla-b * 4601 was associated with both predisposition to infection and severity of infection [99] . however, the association of this allele was replicated in another chinese community of hong kong involving 90 sars patients [100] . hla-b * 0703 was found to be a predisposition allele in the latter study. it should be noted that this latter allele is rare and is found in ∼3% of the general population. hence, this allele cannot be considered a major predisposition factor for sars infection [100] . immunogenotype may play a role in determining the severity of host responses. there is considerable variability in the prevalence of immunogenotypes among different populations and the significance of detecting so-called 'predisposing' alleles in clinical practice is questionable. more studies are needed to uncover fully the real genetic determinants for both predisposition to infection and the host-pathogen interaction after infection with the virus. a considerable amount of knowledge of sars infection has accumulated as a result of almost 3 years of research since the emergence of sars. some key issues about the pathogen, sars-cov, have been addressed. these include the rapid discovery of sars-cov receptors and the actions of some of the specific viral proteins in different host cells. understanding the molecular basis of differences in host cell responses to sars-cov infection will be crucial in delineating its pathogenesis. it is also clear from clinical and experimental data that host immune responses may be the key determinant in disease progression after initial sars-cov infection. future studies aimed at characterization of the variability of host immune and inflammatory responses will be important in understanding this new emerging infectious disease. a major outbreak of severe acute respiratory syndrome in hong kong sars -beginning to understand a new virus treatment of severe acute respiratory syndrome haematological manifestations in patients with severe acute respiratory syndrome: retrospective analysis enteric involvement of severe acute respiratory syndromeassociated coronavirus infection retrospective analysis of liver function derangement in severe acute respiratory syndrome temporal patterns of hepatic dysfunction and disease severity in patients with sars acute renal impairment in coronavirus-associated severe acute respiratory syndrome fatal severe acute respiratory syndrome is associated with multiorgan involvement by coronavirus organ distribution of severe acute respiratory syndrome (sars) associated coronavirus (sars-cov) in sars patients: implications for pathogenesis and virus transmission pathways long-term sars coronavirus excretion from patient cohort sars-cov antibody prevalence in all hong kong patient contacts severe acute respiratory syndrome in children: experience in a regional hospital in hong kong the spectrum of pathological changes in severe acute respiratory syndrome (sars) the clinical pathology of severe acute respiratory syndrome (sars): a report from china lung pathology of severe acute respiratory syndrome (sars): a study of 8 autopsy cases from singapore clinicopathology of severe acute respiratory syndrome: an autopsy case report pulmonary pathology of severe acute respiratory syndrome in toronto a clinicopathological study of three cases of severe acute respiratory syndrome (sars) lung pathology of fatal severe acute respiratory syndrome pulmonary pathological features in coronavirus associated severe acute respiratory syndrome (sars) tissue and cellular tropism of the coronavirus associated with severe acute respiratory syndrome: an in-situ hybridization study of fatal cases coronaviral hypothetical and structural proteins were found in the intestinal surface enterocytes and pneumocytes of severe acute respiratory syndrome (sars) analysis of deaths during the severe acute respiratory syndrome (sars) epidemic in singapore: challenges in determining a sars diagnosis detection of severe acute respiratory syndrome-associated coronavirus in pneumocytes of the lung sars coronavirus-infected cells in lung detected by new in situ hybridization technique immunohistochemical, in situ hybridization, and ultrastructural localization of sars-associated coronavirus in lung of a fatal case of severe acute respiratory syndrome in taiwan myopathic changes associated with severe acute respiratory syndrome: a postmortem case series sars-associated viral hepatitis caused by a novel coronavirus: report of three cases koch's postulates fulfilled for sars virus newly discovered coronavirus as the primary cause of severe acute respiratory syndrome macaque model for severe acute respiratory syndrome an animal model of sars produced by infection of macaca mulatta with sars coronavirus world health organization virology: sars virus infection of cats and ferrets severe acute respiratory syndrome coronavirus-like virus in chinese horseshoe bats efficient replication of severe acute respiratory syndrome coronavirus in mouse cells is limited by murine angiotensin-converting enzyme 2 angiotensin-converting enzyme 2 is a functional receptor for the sars coronavirus a 193-amino acid fragment of the sars coronavirus s protein efficiently binds angiotensin-converting enzyme 2 dimitrov ds. the sars-cov s glycoprotein: expression and functional characterization a model of the ace2 structure and function as a sars-cov receptor susceptibility to sars coronavirus s proteindriven infection correlates with expression of angiotensin converting enzyme 2 and infection can be blocked by soluble receptor retroviruses pseudotyped with the severe acute respiratory syndrome coronavirus spike protein efficiently infect cells expressing angiotensin-converting enzyme 2 highly infectious sars-cov pseudotyped virus reveals the cell tropism and its correlation with receptor expression tissue distribution of ace2 protein, the functional receptor for sars coronavirus. a first step in understanding sars pathogenesis persistent infection of sars coronavirus in colonic cells in vitro angiotensin-converting enzyme 2 protects from severe acute lung failure ace2, a new regulator of the renin-angiotensin system a crucial role of angiotensin converting enzyme 2 (ace2) in sars coronavirus-induced lung injury human coronavirus nl63 employs the severe acute respiratory syndrome coronavirus receptor for cellular entry ace1 polymorphism and progression of sars absence of association between angiotensin converting enzyme polymorphism and development of adult respiratory distress syndrome in patients with severe acute respiratory syndrome: a case control study ace2 gene polymorphisms do not affect outcome of severe acute respiratory syndrome ph-dependent entry of severe acute respiratory syndrome coronavirus is mediated by the spike glycoprotein and enhanced by dendritic cell transfer through dc-sign cd209l (l-sign) is a receptor for severe acute respiratory syndrome coronavirus lsectin interacts with filovirus glycoproteins and the spike protein of sars coronavirus dc-sign and dc-signr interact with the glycoprotein of marburg virus and the s protein of severe acute respiratory syndrome coronavirus chemokine upregulation in sars coronavirus infected human monocyte derived dendritic cells dc-sign: escape mechanism for pathogens molecular epidemiology of sars -from amoy gardens to taiwan molecular evolution of the sars coronavirus during the course of the sars epidemic in china the genome sequence of the sarsassociated coronavirus characterization of a novel coronavirus associated with severe acute respiratory syndrome a novel severe acute respiratory syndrome coronavirus protein, u274, is transported to the cell surface and undergoes endocytosis characterization of a unique group-specific protein (u122) of the severe acute respiratory syndrome coronavirus specific epitopes of the structural and hypothetical proteins elicit variable humoral responses in sars patients the 3a protein of severe acute respiratory syndromeassociated coronavirus induces apoptosis in vero e6 cells the severe acute respiratory syndrome coronavirus 3a is a novel structural protein severe acute respiratory syndrome coronavirus 3a protein is a viral structural protein identification of a novel protein 3a from severe acute respiratory syndrome coronavirus the severe acute respiratory syndrome coronavirus 3a protein up-regulates expression of fibrinogen in lung epithelial cells overexpression of 7a, a protein specifically encoded by the severe acute respiratory syndrome coronavirus, induces apoptosis via a caspase-dependent pathway the pulmonary physician in critical care * 6: the pathogenesis of ali/ards polymorphonuclear neutrophil activation during the acute respiratory distress syndrome pathology of fatal human infection associated with avian influenza a h5n1 virus infections and the inflammatory response in acute respiratory distress syndrome anti-sars-cov igg response in relation to disease severity of severe acute respiratory syndrome autoantibodies against human epithelial cells and endothelial cells after severe acute respiratory syndrome (sars)-associated coronavirus infection chemokines in acute respiratory distress syndrome a chemokine-tocytokine-to-chemokine cascade critical in antiviral defense chemokine regulation of inflammation during acute viral infection plasma inflammatory cytokines and chemokines in severe acute respiratory syndrome dynamic changes in clinical features and cytokine/chemokine responses in sars patients treated with interferon alfacon-1 plus corticosteroids characterization of cytokine/chemokine profiles of severe acute respiratory syndrome an interferon-gamma-related cytokine storm in sars patients early enhanced expression of ip-10 (cxcl-10) and other chemokines predicts adverse outcome in severe acute respiratory syndrome (sars) mechanisms of host defense following severe acute respiratory syndromecoronavirus (sars-cov) pulmonary infection of mice cxc chemokines ip-10 and mig expression and direct migration of pulmonary cd8+/cxcr3+ t cells in the lungs of patients with hiv infection and t-cell alveolitis role of cxcl9/cxcr3 chemokine biology during pathogenesis of acute lung allograft rejection chemokine response in children with sars genetic polymorphisms associated with susceptibility and outcome in ards genetic susceptibility to acute lung injury plasminogen-activator-inhibitor-1 4g/5g promoter polymorphism and prognosis of severely injured patients angiotensin converting enzyme insertion/deletion polymorphism is associated with susceptibility and outcome in acute respiratory distress syndrome association between a genomic polymorphism within the cd14 locus and septic shock susceptibility and mortality rate polymorphisms of human sp-a, sp-b, and sp-d genes: association of sp-b thr131ile with ards common features in the onset of ards after administration of granulocyte colony-stimulating factor identification of an alternative 5 -untranslated exon and new polymorphisms of angiotensin-converting enzyme 2 gene: lack of association with sars in the vietnamese population association of hla class i with severe acute respiratory syndrome coronavirus infection association of human-leukocyte-antigen class i (b * 0703) and class ii (drb1 * 0301) genotypes with susceptibility and resistance to the development of severe acute respiratory syndrome key: cord-339852-9rq7zzqs authors: theamboonlers, apiradee; samransamruajkit, rujipat; thongme, chittima; amonsin, alongkorn; chongsrisawat, voranush; poovorawan, yong title: human coronavirus infection among children with acute lower respiratory tract infection in thailand date: 2006-11-30 journal: intervirology doi: 10.1159/000097392 sha: doc_id: 339852 cord_uid: 9rq7zzqs objective: this study was performed to further identify the previously uncharacterized human coronavirus 229e (hcov-229e) and human coronavirus oc43 (hcov-oc43) in thailand by using the rt-pcr technique. in addition, we performed this study in order to delineate the prevalence, the potential clinical impacts and evaluation of the genetic characterization of this pathogen in young children who presented with acute lower respiratory tract infections (alri). methods: we obtained nasopharyngeal secretions (nps) from 226 children <5 years of age who were either attending the outpatient department or hospitalized with alri from march 2002 to july 2003. all clinical, laboratory, rt-pcr, direct sequencing and phylogenetic analysis data were collected and analyzed. results: of the 226 nps samples from infants and young children presented with alri, 8 (3.54%) were positive for hcov-229e, 2 (0.88%) were positive for hcov-oc43, and 1 (0.44%) had co-infection. the following clinical presentations were noted: fever (100%), rhinitis (44%), acute bronchiolitis (44%), viral pneumonia (33%), viral pneumonia triggering asthma exacerbation (11%) as well as viral pneumonia causing bpd exacerbation (11%). all positive samples were subjected to direct sequencing. the amino acid sequences had 82-99% similarity to previous sequences stored in the genbank database. conclusion: the molecular technique we applied to detect human coronavirus appears justified as a valuable diagnostic approach to elucidate the prevalence, cause and clinical implications of alri among infants and young children. sionally a more severe lower respiratory tract illness in this patient group [2, 3] . due to the diffi culty in distinguishing between respiratory pathogens clinically and by laboratory-based analysis, studies on the impact of respiratory infections are still quite limited. despite improved sensitivity of diagnostic techniques, the causes of a signifi cant part of lower respiratory tract infections have as yet eluded identifi cation [4] . the development of new diagnostic techniques like rt-pcr has facilitated identification of coronavirus infection in recent years [5] . hospitalized infants with acute bronchiolitis and pneumonia have previously been shown to harbor coronavirus as one of a minor contributory factor [6] . coronavirus belongs to the coronaviridae family and contains a single molecule of linear, single-stranded rna of positive polarity, ranging from 27.6 to 31.3 kb (100-150 nm) in size, a nucleocapsid protein (n) and a lipid envelope with three major membrane proteins. coronaviruses are subdivided into three groups, human corona viruses 229e (hcov-229e) and human coronavirus oc43 (hcov-oc43) have been recognized as causes of upper respiratory tract disease including common cold and also are group 1 and 2 coronaviruses, respectively [7] . recently, another group (hcov, hcov-nl63) has been reported in the netherlands [6] . based on the structure and function of the polymerase gene, there is a valid region for phylogenetic comparisons as well as for developing a consensus polymerase chain reaction assay applicable to fi nd the differentiation of novel coronaviruses such as sars [8, 9] . this might prove very useful as these novel coronaviruses have been tentatively identifi ed by electron microscopy in association with a variety of human and animal diseases, although further characterization and definite identifi cation of these agents as coronaviruses has been extremely diffi cult and insensitive. rt-pcr is currently a rapid and sensitive method for the diagnosis of hcov [5] . coronaviruses are mostly associated with respiratory, enteric, hepatic and central nervous system diseases. nevertheless, other organs such as kidney, heart, and eye can also be infected [7] . coronavirus comprises a large family of viruses infecting a broad range of vertebrates, from mammalian to avian species. in humans and fowl, coronaviruses primarily lead to upper respiratory tract infections, whereas porcine and bovine coronaviruses cause enteric infections that result in high economic loss [7] . as for the antigenic composition of coronaviruses, research of the human strains in particular has hardly advanced because the studies have been limited to cell cul-ture or serology [10] [11] [12] and the apparently antigenic cross-reactivity between asra-cov and hcov-229e and hcov-oc43 [13] . thus, hcovs are very diffi cult to detect and the prevalence and epidemiological data are scanty, especially in asian countries. one recent report from hong kong showed that hcov infections were also found in 4.4% (hcov-nl63 1.5%, hcov-oc43 0.3%, hcov-229e 2.6%) of the children studied [14] . for isolation of suspected cases, contact tracing is only available for controlling the infection. a rapid and accurate diagnosis test is very important. since the antibody response takes (such as sars coronavirus) [14] 10-28 days after onset [19] , detection of viral rna by rt-pcr is likely to be the best option for early detection [6] . in this study we applied molecular biology techniques to identify hcov in nasopharyngeal secretions (nps) for the study on the prevalence of molecular characterization and clinical correlation of coronavirus infections in hospitalized infants and young children with acute lower respiratory tract infection (alri). the study protocol was approved by the ethics committee of the faculty of medicine, chulalongkorn university. parents were informed as to the study objective and their written consents were obtained before specimen collection. we collected np samples of 131 male and 95 female children at a mean age of 1.3 years (ranging from 24 days to 15 years) with alri attending either the outpatient clinic or admitted to king chulalongkorn memorial hospital between march 2002 and july 2003. all samples were kept at -70 ° c until further examination. patients' demographic data, clinical presentations, duration of hospitalization, basic laboratory data including complete blood count, chest radiographs and treatment were recorded for analysis. np specimens were centrifuged and the supernatant was analyzed for coronavirus rna by rt-pcr. briefl y, rna was extracted from 50 l of nps applying the guanidinium-isothiocyanate method as described elsewhere [15] and subsequently reverse transcribed into cdna using primers mf3 for hcov-oc43 and pcon4.2 for hcov-229e. the resulting cdna was amplifi ed by pcr using primer mf1 as a sense primer, and again mf3 as an antisense primer for hcov-oc43 [16] . for a replicase gene, of hcov-229e, pcon3 was used as an outer sense primer, pcon4.2 as an outer antisense primer, pcon1 as an inner sense primer, and pcon2.2 as an inner antisense primer base on the sequence accession no. x69721. the details of the primers are shown in table 1 . the amplifi cation reaction was performed in a thermocycler (9600 perkinelmer cetus, norwalk, conn., usa) applying the following conditions: initial denaturation at 95 ° c for 3 min, followed by 30 cycles each of 1 min at 95 ° c for denaturation, 1 min at 60 ° c for primer annealing, and 1 min at 72 ° c for extension and concluded by a fi nal ex-tension step at 72 ° c for 10 min. after electrophoresis in a 2% agarose gel stained with ethidium bromide on preparation, the expected 217-bp product for hcov-229e and the 334-bp product for hcov-oc43 were visualized on a uv transilluminator (gel doc 1000, bio-rad, calif., usa). all the specimens were positive for ␤ -actin which served as an internal control. for further sequencing, the pcr product bands of interest were purifi ed from residual agarose using the perfect prep gel cleanup kit (eppendorf, westbury, n.y., usa) according to the manufacturer's specifi cations and subsequently subjected to 2% agarose gel electrophoresis in order to ascertain their purity. to determine the concentration of the amplifi ed dna, we measured every sample's absorption at 260 nm in a uv spectrophotometer (shimadzu uv 160 a, tokyo, japan). the concentration was calculated according to the formula 1 od 260 = 50 g doublestranded dna. between 10 and 30 ng/ l (3-6 l) of each dna sample were subjected to cycle sequencing using 4 l of dye terminator, 2 l of dna sequencing buffer (big dye terminator v.3.1 cycle sequencing ready reaction, foster city, calif., usa) and 3.2 pmol of specifi c primer in a fi nal reaction volume of 20 l in a thermocycler (9600 perkinelmer cetus). this amplifi cation round was performed according to the manufacturer's specifi cations, using primers pcon1 and pcon2.2 for hcov-229e and mf1 and mf3 for hcov-oc43 to amplify the particular dna strand of interest for further sequencing. the results were analyzed by the sequence navigator program and submitted to the genbank database (ay751810-19, ay754762). the nucleotide sequences were also compared with the sequences published previously in genbank, applying the blast program (www.ncbi.nlm.nih.gov/blast). in order to determine the relationship between various coronaviruses isolated from the patients, we generated unrooted phylogenetic trees of nucleotide sequences comprising the polymerase gene region for hcov-229e and the m gene for hcov-oc43. ten nucleotide sequences from patients and references were multiple aligned to construct a phylogenetic tree using the clustalw package run-ning under bioedit version 5.0.9 (www.mbio.ncsu.edu/bioedit). phylogenetic trees were made with the dnaml software of the phylip version 3.6 (http://evolution.genetics.washington.edu/ phylip.html) by using 1,000 bootstraps. the obtained coronavirus sequences were aligned with those of known species stored in gen-bank and subjected to phylogenetic analysis. all isolates from this cluster grouped on the same node of the phylogenetic tree were assumed to be identical. the clinical data were analyzed and were expressed as percentage where applicable. in the present study, virus diagnosis was performed by virus identifi cation from nps obtained from the 226 infants and young children with alri attending our hospital. for hcov rt-pcr analysis, we used primer sets specifi c for the known hcov-oc43 and hcov-229e ( table 1 ). eight of 226 (3.54%) proved positive for hcov-229e and 2 of 226 (0.88%) for hcov-oc43, 1 (0.44%) showed co-infection. out of the 226 specimens, we found hcov infection in 9 patients. the clinical diagnoses of these patients with lower respiratory tract infection included acute bronchiolitis in 4 (44%), viral pneumonia in 3 (33%), viral pneumonia triggering asthma exacerbation in 2 (22%), and viral pneumonia causing bpd exacerbation in 1 (11%). their common clinical presentations were fever (100%), upper respiratory tract infection (44%), and severe cough (33%). wheezing was found in 4 (44%) and crepitations were more common in 6 (66%). their mean age was 17 8 14.9 months. the mean hospitalization required was at 6 8 2.24 days, signifi cantly more than that experienced by infants and children infected with rsv. there were 6 infants (66%) co-infected with rsv. all were treated with a bronchodilator yet only 4 (44%) responded to therapy. nobody required intensive care unit admission. all were subjected to chest radiographs, which yielded abnormal results. most chest radiographs showed perihilar infi ltrates and hyperaeration. three (33%) had patchy infi ltrates simulating bacterial infection, although 5 (55%) had been given antibiotics and all required oxygen supplement for at least 1 day after admission. a summary of the clinical data is shown in table 2 . we determined the cdna sequences of hcov-229e and hcov-oc43 by direct sequencing of the rt-pcr products and submitted them to genbank. the two samples positive for hcov-oc43, cu60th (ay751817) and cu82th (ay751818), showed 99% relative similarity to the reference strain ay382775. the following 8 samples were positive for hcov-229e: cu60th (ay751815), cu65th (ay751814), cu71th (ay754762), cu109th (ay751812), cu110th (ay751816), cu122th (ay751811), cu126th (ay751813) and cu129th (ay751810) exhibiting 82% similarity to the reference strains x69721 and ay518894 (hcov-nl). the accession numbers of references for both hcov-229e and hcov-oc43 to perform the phylogenetic analysis were: ay518894 (hcov-nl), x69721 (hcov) and af304460 (hcov-229e); af353511 (porcine epidemic area virus, pedv); aj271965 (transmissible gastroenhcov-oc43 (ay751817-18) sequences obtained in our study were aligned with those of known genotype stored in genbank and subjected to phylogenetic analysis, shown along with the hcov-229e and hcov-oc43 in different geographical areas in fi gures 1 (hcov-229e) and 2 (hcov-oc43). alris like acute bronchiolitis and viral pneumonia are major causes of morbidity in infants and young children ! 5 years of age. the majority of infections are caused by four groups of respiratory virus: rsv, parainfl uenza viruses, infl uenza viruses and adenoviruses. in the past, virus detection was markedly limited due to technical obstacles and diffi culties in virus isolation. however, owing to advances in molecular diagnostics in recent years, we are able to identify newly and deadly emerging respiratory pathogens such as sars which is also caused by coronavirus infection. thus, we realized the necessity to further explore these potential respiratory pathogens in more detail. coronaviruses have been divided into three groups or serotypes, based on their distinct genome organization and phylogenetic analyses [6, 7] . group 1 comprises hcov-229e, porcine enteric gastroenteritis virus, tgev, pedv and respiratory (prcov) coronavirus, canine coronavirus and feline coronavirus; group 2 consists of hcov-oc43, bovine coronavirus, turkey coronavirus, murine coronaviruses, porcine hemagglutinating encephalomyelitis virus, rat coronavirus, and group 3 comprises avian coronavirus including infectious bronchitis virus, turkey coronavirus, rabbit coronavirus and the sars coronavirus as a distinct entity located on a different node on the phylogenetic tree. in this study, we have identifi ed human coronavirus hcov-oc43 and hcov-229e infection in infants and young children presenting with alri. we found the human coronavirus-229e being predominant in our population. only one sample contained both. hcov-229e and hcov-oc43 have previously been proven responsible for infecting people of all age groups and causing severe lower respiratory tract infection primarily in frail patients such as young children and elderly individuals [17] [18] [19] . human coronavirus hcov causes approximately 10-20% of common colds and 6% of infl uenza-like illnesses in all age groups [20] . the clinical features associated with coronavirus infection appear to be similar to those observed with other respiratory viruses, such as rsv, parainfl uenza virus and human metapneumovirus. symptoms of upper respiratory tract infection such as rhinitis, coryza, and of lower respiratory tract infection such as tachypnea, wheezing and chest wall retraction were frequently observed in our patients. wheezing was observed in 44% compared to 9% reported from hong kong [14] . this may be explained by the difference in host response. hypoxemia was present without exception. thus, this may indicate yet another potentially severe effect of this virus infection and future impact on healthcare providers. additionally, chest hyperinfl ation, which refl ects small to medium airway obstruction caused by airway infl ammation, has proven a relatively common fi nding in our population ( 1 50%). co-infection with rsv and other pathogens has been previously described. interestingly, we found 6 (66%) patients co-infected with rsv ( table 1 ) . hence, rsv and human coronavirus might share a similar seasonal pattern. nevertheless, confi rmation of the underlying mechanism of co-infection will require further longitudinal studies conducted over several bronchiolitis seasons. the role of viral respiratory tract infections in triggering reactive airway exacerbation, especially in asthma patients, has been the subject of much research interest. rsv, parainfl uenza and rhinoviruses have previously been recognized as a primary trigger of reactive airway exacerbation. our data indicate that human coronavirus may also be important in this regard, although its role in the pathogenesis of wheezing still warrants further study. more importantly, the protection from this virus may reduce the cost of hospitalization and reduce the morbidity of these children. the hcov-229e sequences obtained from our samples showed 82% similarity to previously published data. hence, the availability of the hcov, hcov-nl and our genome sequences might contribute to the development of a variety of diagnostic assays to determine both prevalence and clinical impact of hcov infections in humans. moreover, further studies conducted on the remaining genes would be required in order to arrive at more specifi c diagnoses and elucidate the related clinical implications. acute respiratory infections: a review an outbreak of coronavirus oc43 respiratory infection in normandy, france coronavirus-related nosocomial viral respiratory infections in a neonatal and paediatric intensive care unit: a prospective study impact of respiratory virus infections on persons with chronic underlying conditions evaluation of nested polymerase chain methods for the detection of human coronaviruses 229e and oc43 osterhaus adme: a previously undescribed coronavirus associated with respiratory disease in humans coronavirus derived expression systems identifi cation of a novel coronavirus in patients with severe acute respiratory syndrome anderson lj; sars working group: a novel coronavirus associated with severe acute respiratory syndrome development and application of an enzyme immunoassay for coronavirus oc43 antibody in acute respiratory illness antigenic relationships amongst coronaviruses purifi cation and biophysical properties of human coronavirus 229e antigenic cross-reactivity between severe acute respiratory syndrome-associated coronavirus and human coronaviruses 229e and oc43 human coronavirus nl63 infection and other coronavirus infections in children hospitalized with acute respiratory disease in hong kong human metapneumovirus infection in thai children direct diagnosis of human respiratory coronaviruses 229e and oc43 by the polymerase chain reaction rhinovirus and coronavirus infection-associated hospitalizations among older adults coronavirus infection in acute lower respiratory tract disease of infants respiratory viral infections in the elderly surveillance of community-acquired viral infections due to respiratory viruses in rhône-alpes (france) during winter 1994 to 1995 we are grateful to the thailand research fund, senior research scholar and center of excellence in viral hepatitis fund, chulalongkorn university. also, we would like to express our gratitude to the entire staff of the center of excellence in viral hepatitis, chulalongkorn university, for their efforts in contributing to the present study. we would also like to thank venerable dr. mettanando bhikkhu of the foundation of king rama ix the great and ms. petra hirsch for reviewing the manuscript. intervirology 2007;50:71-77 key: cord-349956-h4i2t2cr authors: hoang, van-thuan; dao, thi-loi; ly, tran duc anh; belhouchat, khadidja; chaht, kamel larbi; gaudart, jean; mrenda, bakridine mmadi; drali, tassadit; yezli, saber; alotaibi, badriah; fournier, pierre-edouard; raoult, didier; parola, philippe; de santi, vincent pommier; gautret, philippe title: the dynamics and interactions of respiratory pathogen carriage among french pilgrims during the 2018 hajj date: 2019-11-21 journal: emerg microbes infect doi: 10.1080/22221751.2019.1693247 sha: doc_id: 349956 cord_uid: h4i2t2cr we conducted this study to describe the dynamics of the acquisition of respiratory pathogens, their potential interactions and risk factors for possible lower respiratory tract infection symptoms (lrti) among french pilgrims during the 2018 hajj. each participant underwent four successive systematic nasopharyngeal swabs before and during their stay in saudi arabia. carriage of the main respiratory pathogens was assessed by pcr. 121 pilgrims were included and 93.4% reported respiratory symptoms during the study period. the acquisition of rhinovirus, coronaviruses and staphylococcus aureus occurred soon after arrival in saudi arabia and rates decreased gradually after days 5 and 6. in contrast, streptococcus pneumoniae and klebsiella pneumoniae carriage increased progressively until the end of the stay in saudi arabia. haemophilus influenzae and moraxella catarrhalis carriage increased starting around days 12 and 13, following an initial clearance. influenza viruses were rarely isolated. we observed an independent positive mutual association between s. aureus and rhinovirus carriage and between h. influenzae and m. catarrhalis carriage. dual carriage of h. influenzae and m. catarrhalis was strongly associated with s. pneumoniae carriage (or = 6.22). finally, our model showed that m. catarrhalis carriage was negatively associated with k. pneumoniae carriage. chronic respiratory disease was associated with symptoms of lrti. k. pneumoniae, m. catarrhalis-s. aureus and h. influenzae-rhinovirus dual carriage was associated with lrti symptoms. our data suggest that rtis at the hajj are a result of complex interactions between a number of respiratory viruses and bacteria. each year, an increasing number of people travel to the kingdom of saudi arabia (ksa) for the hajj and umrah pilgrimages, which attract around 10 million pilgrims annually from more than 180 countries. more than two million pilgrims from outside saudi arabia participated in the hajj pilgrimages in 2017 [1] . each year, about 2,000 pilgrims from marseille, france, participate in the hajj [2] . the event presents major challenges for public health, including inter-human transmission of infectious diseases, notably respiratory tract infections (rtis), due the crowded conditions experienced by pilgrims [1] . in a recent study on morbidity and mortality among indian hajj pilgrims, infectious diseases represented 53% of outpatient diagnoses, with rtis and gastroenteritis being the most common [3] . between 69.8% and 86.8% of french pilgrims presented rti symptoms during the hajj [4] . a recent literature review suggested that etiology of rtis at the hajj is complex; several studies showed a significant acquisition of respiratory pathogens by pilgrims following participation in the hajj in both symptomatic and asymptomatic individuals [5] . in a systematic review of 31 studies, al-tawfiq et al. showed that human rhinovirus (hrv) and influenza viruses were the most common viral respiratory pathogens isolated from ill hajj pilgrims [6] . in addition, human non-mers coronaviruses (hcov) were also a common cause of rtis during the event [7] . on the other hand, streptococcus pneumoniae, haemophilus influenzae and staphylococcus aureus were shown to be the most commonly acquired respiratory bacteria at the hajj [5] . rtis are caused by the antagonistic and synergistic interactions between upper respiratory tract viruses and predominant bacterial pathogens [8] . pathogens are usually studied individually, although in their natural environment they often compete or coexist with multiple microbial species. similarly, the diagnosis of infections often proceeds via an approach which assumes a single agent etiology [9] . nevertheless, complex interactions occur between the different infectious microorganisms living in the same ecological niche and mixed infections are frequent [10] . a better understanding of polymicrobial interactions in the nasopharynx among hajj pilgrims is important for many reasons. carriage of more than one pathogen is common among hajj pilgrims, whether or not they present with respiratory symptoms [7] . colonization is the initial step in the disease process [11] . nasopharyngeal colonization is likely to be a reservoir for respiratory pathogens resulting in interhuman transmission between pilgrims during close contact experienced during the hajj ritual. furthermore, antibiotic use or vaccines, which target specific pathogen species, may alter polymicrobial interactions in the nasopharynx and have unanticipated consequences [12, 13] . to our knowledge, the dynamics and interaction between the main respiratory pathogens acquired during the hajj pilgrimage have not been specifically investigated, to date. risk factors for possible lower respiratory tract infection (lrti) symptoms at the hajj have not been studied. we conducted this study among french pilgrims during the 2018 hajj, to describe the dynamics of the acquisition of respiratory pathogens and their potential interactions. in addition, we investigated risk factors for possible lrti symptoms. participants and study design ( figure 1 ) pilgrims travelling to mecca, saudi arabia during the 2018 hajj from marseille, france, were recruited through a private specialized travel agency. potential adult participants were invited to participate in the study. they were included and followed-up by two bilingual (arabic and french) medical doctors who travelled with the group. all participants departed to ksa on the same date, were housed in the same accommodation during their stay and performed the rituals together. upon inclusion, before departing from france, pilgrims were interviewed using a standardized pre-hajj questionnaire that collected information about demographic characteristics, medical history and immunization status. pilgrims were considered to have been immunized against influenza when they had been vaccinated within the past year and until before 10 days of the date of travel. pilgrims were considered to be immune to invasive pneumococcal disease (ipd) when they had been vaccinated with the 13-valent conjugate pneumococcal vaccine (pcv-13) in the past five years [14, 15] . a post-hajj questionnaire was completed two days before the pilgrims' return to france. clinical data, antibiotic intake and information on compliance with face masks use as well as hand washing, the use of hand gel disinfectant and disposable handkerchiefs was collected. to evaluate the dynamic and interaction of respiratory pathogens during the hajj, all pilgrims underwent four successive systematic nasopharyngeal swabs at different times: pre-travel, five to six days post arrival, 12-13 days post arrival and just prior to leaving ksa (post-hajj). the hajj rituals took place from 19-24 august. influenza-like illness (ili) was defined as the presence of cough, sore throat and subjective fever [16] . possible lrti was defined by presence of productive cough without nasal or throat symptoms; febrile productive cough; dyspnea or febrile dyspnea [17] . based on the who classification, "underweight" was defined as having a body mass index (bmi) below 18.5, "normal" corresponded to a bmi between 18.5 and 25, "overweight" corresponded to a bmi ≥25, and "obese" referred to those with a bmi ≥30 [18]. nasopharyngeal swabs were obtained from each pilgrim, transferred to sigma-virocult® medium and stored at −80°c until processing. the sampling was done by the doctors accompanying the group, in a standardized way (3 cm in the nostril, 5 turns; post wall of the pharynx, 5 streaks). the rna and dna were extracted from the samples using the ez1 advanced xl (qiagen, hilden, german) with the virus mini kit v2.0 (qiagen) according to the manufacturer's recommendations. all quantitative real-time pcr were performed using a c1000 touch™ thermal cycle (bio-rad, hercules, ca, usa). one-step simplex real-time quantitative rt-pcr amplifications were performed using multiplex rna virus master kit (roche diagnostics, france) for influenza a, influenza b, hrv and internal controls ms2 phage [19] . hcov was detected by one-step duplex quantitative rt-pcr amplifications of hcov/ hpiv-r gene kit (ref: 71-045, biomérieux, marcy l'etoile, france), according to the manufacturer's recommendations. real-time pcr amplifications were carried out using lightcycler® 480 probes master kit (roche diagnostics, france) according to the manufacturer's recommendations. the shd gene of h. influenzae, phoe gene of klebsiella pneumoniae, nuca gene of s. aureus, lyta cdc gene of s. pneumoniae and copb gene of moraxella catarrhalis were amplified with internal dna extraction controls tiss, as previously described [20, 21] . human adenovirus, human metapneumovirus, respiratory syncytial virus, bordetella pertussis and mycoplasma pneumoniae were not tested because a low proportion (<2%) of returning french pilgrims or international pilgrims were found positive for these pathogens in previous works [7, 20, 22, 23] . negative controls (pcr mix) and positive controls (dna from bacterial strain or rna from viral strain) were included in each run. positive results of bacteria or virus amplification were defined as those with a cycle threshold (ct) value ≤35. a threshold value of 35 was used in each experimental run and we calculated the rfu cut off value recommend by cfx manager software version 3.1 (bio-rad) [24] in order to verify the positive cases. results were considered positive when the cycle threshold value of real-time pcr was greater than the cut off value. pilgrims were considered to be positive for respiratory pathogens during the hajj if they were positive at the days 5 and 6 and/or days 12 and 13 sample. stata software version 14.2 (copyright 1985-2015 statacorp llc, http://www.stata.com) was used for statistical analysis. the main outcomes of interest were the relationships between respiratory pathogens among pilgrims during the hajj. we evaluated the carriage of hrv, hcov, s. aureus, s. pneumoniae, h. influenzae, k. pneumoniae and m. catarrhalis using logistic mixed models. because each pilgrim provided four successive samples, we used a repeated measures design to take into account the variability of series samples from each pilgrim. to evaluate the effect of covariates on each respiratory pathogen carriage, we modelled carriage of hrv, hcov, s. aureus, s. pneumoniae, h. influenzae, k. pneumoniae and m. catarrhalis separately. we did not separately model the outcome of carriage of influenza a and b viruses because of the low prevalence of these viruses. only the variables with a prevalence ≥5.0% were considered for statistical analysis. unadjusted associations between respiratory pathogen carriage with multiples factors: sociodemographic characteristics (gender, ≥60 years), chronic respiratory disease, bmi classification, smoking status; individual preventive measures (vaccination against influenza, vaccination against ipd, use of a face mask, hand washing, disinfectant gel and disposable handkerchiefs); antibiotic intake 10 days before each sample; respiratory virus or bacteria and dual carriage were analysed by univariable analysis. variables with p values <0.2 in the univariable analysis were included in the multivariable analysis. a mixed model with the subject being random effect was used to estimate the relationships between respiratory pathogens and to take into account the repeated measures for pathogen carriage for each subject. regarding risk factors for lrti, the outcome was possible lrti symptoms reported during the hajj. the independent factors were sociodemographic characteristics (gender, ≥60 years), chronic respiratory disease, smoking status, bmi classification; vaccination against influenza, vaccination against ipd, respiratory virus or bacteria and dual carriage during the hajj. unadjusted associations between multiple factors and possible lrti symptoms were examined using univariable analysis. variables with p values <0.2 in the univariable analysis were included in the multivariable analysis. a logistical regression model was used to estimate factors' adjusted odds ratios regarding possible lrti. the results were presented by odds ratio (or) with a 95% confidence interval (95%ci). results with a p value ≤0.05 was considered to be statistically significant. the protocol was approved by the aix-marseille university institutional review board (23 july 2013; reference no. 2013-a00961-44). the study was performed according to the good clinical practices recommended by the declaration of helsinki and its amendments. all participants provided their written informed consent. the study included 121 pilgrims. the sex ratio of the population was 1:1.3 and the median age was 61 years with 58.7% of pilgrims aged 60 years and over. most pilgrims were born in north africa (66.9%) and sub-saharan africa (26.5%). there was a high prevalence of overweight (46.3%), obesity (28.1%), diabetes mellitus (25.6%) and hypertension (25.6%) and 13.2% participants reported that they suffered from chronic respiratory disease. in line with french recommendation, 88/121 pilgrims (72.7%) had an indication for vaccination against ipd [14, 15] ( table 1) . a total of 37/121 (30.6%) pilgrims reported that they had been vaccinated against influenza in the past year. only 17/88 (19.3%) pilgrims with an indication for ipd had been vaccinated against pneumococcal disease (pcv-13) in the past five years. regarding non-pharmaceutical preventive measures, 49/121 (40.5%) pilgrims reported using face masks during the pilgrimage. also, 67/121 (55.4%) and 70/121 (57.8%) pilgrims reported washing their hands more often than usual and using hand gel, respectively during the pilgrimage. finally, 106/121 (87.6%) reported using disposable handkerchiefs during the hajj. a total of 113/121 (93.4%) pilgrims presented at least one respiratory symptom during their stay in ksa. a cough and rhinitis were the most frequent symptoms affecting 86.8% and 69.4% of participants. over half of the pilgrims (59.5%) reported expectoration and 27.3% reported a dry cough. voice failure was reported by 37.2%, fever by 27.3% and ili by 20.7% of participants. antibiotic use for rtis was reported by 58.7% pilgrims. only one (0.8%) pilgrim was hospitalized in ksa. regarding possible lrti symptoms, 5/121 (4.1%) participants reported a productive cough without nasal or throat symptoms. in addition, 9/121 (7.4%), 16/121 (13.2%) and 25/121 (20.7%) pilgrims presented febrile dyspnoea, dyspnea and a febrile productive cough, respectively. at total of 51/113 (45.1%) pilgrims with respiratory symptoms were still symptomatic at return. the mean time between arrival in ksa and the onset of symptoms was 8.7 ± 4.6 days (range = 1-21 days) (data not shown). most ill pilgrims presented the onset of respiratory symptoms when stationed at mecca with a second minor wave in mina (figure 2 ). table 2 shows the prevalence of the carriage of respiratory pathogens according to sampling time and figure 2 show the dynamics of most prevalent pathogens over the study period. overall, 378/484 (78.1%) of all samples tested positive for at least one pathogen. s. aureus was the pathogen most frequently isolated with 33.3% of all samples testing positive. high positivity rates were also observed for h. influenzae (26.7%), k. pneumoniae (22.5%), hrv (21.1%) and m. catarrhalis (19.4%). only 9.5% of the samples were positive for coronaviruses and 7.4% for s. pneumoniae. very few samples tested positive for influenza viruses. no case was positive for hpiv. of the positive samples, the proportion that was positive for more than one pathogen was 55.6% (210/378). a total of 138/378 (36.5%) samples were positive for two pathogens, 52/378 (13.8%) for three pathogens, 16/378 (4.2%) for four pathogens and 4/378 (1.1%) for five pathogens (data not shown). in pre-travel samples, virus carriage was very low with only a few participants testing positive for hrv and hcov (<2%). bacterial carriage was higher, notably for h. influenzae (35.5%) m. catarrhalis (16.5%) and s. aureus (15.7%). k. pneumoniae and s. pneumoniae carriage were relatively low (9.1% and 2.5%, respectively). a dramatic increase in hrv carriage was observed on days 5 and 6 of the pilgrimage with prevalence 24 times higher than that of pre-travel. hrv carriage decreased progressively in subsequent samples but was still eight times higher in post-hajj samples compared to pre-hajj. a seven-fold increase of hcov carriage was observed on days 5 and 6 that persisted on into days 12 and 13 of the pilgrimage and tended to slightly decrease in post-hajj samples. regarding bacteria, carriage of s. aureus increased by a factor of three on days 5 and 6 and decreased progressively in subsequent samples but was still double in post-hajj samples compared to pre-hajj. interestingly, the carriage curves of hrv and s. aureus were strictly parallel. m. catarrhalis carriage was about 12-16% in pre-travel, days 5 and 6 and 12 and 13 samples and increased to 33% in post-hajj samples. k. pneumoniae carriage increased three-fold between pre-hajj and days 5 and 6 samples and slightly increased in subsequent samples. s. pneumoniae carriage increased constantly overtime with a seven-fold increase in post-hajj samples compared to pre-hajj. finally, h. influenzae carriage first decreased on days 5 and 6 and 12 and 13 by a factor 2.5 and then increased in post-hajj samples to a carriage rate which was higher than that of pre-hajj samples. table 3 shows the factors that were independently associated with the carriage of respiratory pathogens on 484 swabs from 121 pilgrims. a positive association was observed between males and carriage of hrv and s. pneumoniae. chronic respiratory disease was also associated with s. pneumoniae carriage. finally, the use of disposable handkerchiefs was associated with a decreased carriage of h. influenzae. antibiotic intake ten days before each sampling was positively associated with hcov and k. pneumoniae carriage. regarding interactions between pathogens, we observed that hrv carriage and s. aureus carriage were mutually positively associated. the same applied to h. influenzae and m. catarrhalis carriage. pilgrims carrying s. pneumoniae were more likely also to carry m. catarrhalis. patients with a dual carriage of h. influenzae and s. pneumoniae were six times more likely also to be carrying s. pneumoniae. by contrast, m. catarrhalis carriage was associated with a reduced carriage of k. pneumoniae. table 4 shows the results of multivariable risk factor analysis for possible lrti symptoms. chronic respiratory disease was associated with all possible lrti symptoms. obesity was associated with dyspnea. carriage of k. pneumoniae or m. catarrhalis-s. aureus or h. influenzae-rhinovirus combination was associated with a four-fold, 16-fold and eight-fold increase of dyspnoea prevalence, respectively. finally, m. catarrhalis-s. aureus dual carriage was associated with a five-fold increase in the prevalence of febrile dyspnea (table 4 ). despite the recommendation to take individual preventive measures to prevent rtis [25, 26] , these infections remain common among hajj pilgrims. overcrowding during the event is thought to increase the risk of the transmission of infectious diseases, but interaction between respiratory pathogens is probably one factor contributing towards the development of rtis. to our knowledge, no study on respiratory microbiota alteration among pilgrims during the hajj has been conducted. our results about the occurrence of rti symptoms are in line with previous results obtained regarding french pilgrims [17] and others [1, 20] . notably, we observed that rti symptoms occur soon after the pilgrims' arrival in mecca, with most symptoms starting between 4and 13 days after arrival, corresponding to the period when pilgrims are stationed in mecca hotels and are visiting the grand mosque daily, where highly crowded conditions are common [7] . we also confirmed that an overall increase in the carriage of respiratory viruses and bacteria can be seen when comparing pre-travel samples and post-hajj samples, as previously documented [7, 12, 20, [27] [28] [29] . higher acquisition rates were observed for rhinovirus with a nine-fold increase when comparing pre-travel to post-hajj carriage and for s. pneumoniae with a seven-fold increase, but an increase was observed for all pathogens tested in this study. the unique design of our study with sequential systematic sampling at regular intervals allows for a better understanding of the dynamics of pathogen carriage during the pilgrimage. carriage rates of bacteria and viruses in this study are in line with those observed during recent studies conducted on french pilgrims and in pilgrims of other nationalities, using the same methods of detection [7, 20, [27] [28] [29] . the acquisition of respiratory viruses and s. aureus occurred soon after arrival in saudi arabia and decreased gradually after days 5 and 6. by contrast, s. pneumoniae and k. pneumoniae carriage increased progressively until the end of the visit, h. influenzae and m. catarrhalis carriage increased later, after an initial clearance. we hypothesize that the brutal acquisition of respiratory viruses upon arrival was the initial step that triggered subsequent changes in the relative abundance of resident bacteria [30] that were already present in the nasopharynx of pilgrims. the apparent simultaneity of viruses and s. aureus carriage increase and the initial wave of respiratory symptoms, suggests that this pathogen association was responsible for the rtis that affected most pilgrims soon after arriving in mecca. the subsequent increase in resident bacteria that occurred during the second half of pilgrims' stays in saudi arabia appears to be contemporaneous with a second wave of respiratory symptoms, suggesting that these rtis were of bacterial origin. regarding interaction between respiratory pathogens, we observed a very clear pattern of positive association between the carriage of s. aureus and rhinovirus with acquisition curves which were strictly parallel. furthermore, an independent positive mutual association between the carriage of the two pathogens was evidenced in our multivariate model. several studies revealed a positive interaction between natural or experimental rhinovirus infection and s. aureus nasal carriers [31] [32] [33] [34] [35] . these studies also underlined that rhinovirus infection may facilitate the propagation of s. aureus from staphylococcal carriers to the environment and the transmission of the bacterium between humans. among healthy persons who were experimentally infected by rhinovirus, the relative abundance of s. aureus first increased and then returned to its baseline level after the rhinovirus infection was cleared [36] . these results suggest that changes in the composition of the respiratory microbiota following rhinovirus infection may play a role in the development of bacterial superinfection. morgene et al. proposed several potential mechanisms through which rhinovirus may increase bacterial infection [37] . rhinovirus infection promotes pro-inflammatory cytokines and ifn production mainly through the activation of nfκb. in rhinovirus infected cells, the adherence of s. aureus was significantly higher compared to uninfected cells. the inflammation due to rhinovirus infection also increased cellular patterns that facilitate the adhesion and internalization of s. aureus within host cells [37] . we also observed a parallel increase of h. influenzae and m. catarrhalis carriage in days 12 and 13 and post-hajj samples. an independent positive mutual association between the carriage of the two pathogens was evidenced in our multivariate model. dual carriage of h. influenzae and m. catarrhalis strongly associated with s. pneumoniae carriage which in turns associated with m catarrhalis carriage. these results are consistent with those of several studies conducted among children with upper rtis [38] [39] [40] . in these studies, the competitive interaction between s. pneumoniae and h. influenzae was dependent on neutrophils and complement. the additional carriage of m. catarrhalis might alter the competitive balance between h. influenzae and s. pneumoniae [39] . co-colonization of s. pneumoniae or h. influenzae with m. catarrhalis associating with increased risk of otitis media has been documented [41] . using in vivo models, mixed species biofilms play a role in increasing the persistence of ear disease [42] . other proposed mechanisms for positive associations between bacterial species include interspecies quorum sensing and passive antimicrobial resistance, which have been observed in experimental models of otitis media [43] . finally, our model showed that m. catarrhalis carriage was negatively associated with k. pneumoniae carriage which, to our knowledge, has not previously been published. additionally, we found that the male gender was independently associated with an increase in rhinovirus and s. pneumoniae carriage. we have no explanation for this unexpected observation. the carriage of s. pneumoniae was higher among pilgrims with chronic respiratory disease which support the current french recommendations that vaccination against ipd be proposed to at-risk pilgrims [44] . in one of our recent studies, pilgrims who were vaccinated against ipd were seven time less likely to harbour s. pneumoniae after the hajj compared to unvaccinated pilgrims [27] . in this study, the use of disposable handkerchiefs was associated with a significant decrease in h. influenzae carriage. non-pharmaceutical individual preventive measures such as cough etiquette, hand hygiene, use of a face mask, disinfectant gel and disposable handkerchiefs are recommended for hajj pilgrims [26] . nevertheless, the effectiveness of these measures has been poorly investigated and available results are contradictory [26] . the apparent association between antibiotic use and hcov and k. pneumoniae carriage warrants further investigation to better explore this unexpected observation. we also confirm that chronic respiratory disease is a risk factor for lrti. we also evidenced the role of respiratory bacteria including k. pneumoniae and m. catarrhalis-s. aureus association and h. influenzaerhinovirus association in the occurrence of possible lrti symptoms. this reinforces the need for antibiotic use in case of lrti symptoms [17] . our study has some limitations. the study was conducted among french pilgrims only with a relatively small sample size and cannot be generalized to all pilgrims. qpcr used to detect respiratory pathogens does not distinguish between dead and living micro-organisms. only a small number of respiratory pathogens were investigated. respiratory bacteria serotypes were not investigated. influenza viruses were not included in the model due to low carriage rates. in addition, we did not recruit a control group of individuals that did not participate to the hajj. a study addressing interactions between respiratory pathogens in the general french adult population could be of interest. nevertheless, our study is the first study on the dynamics of and interaction between the respiratory pathogens that are most frequently isolated among hajj pilgrims. our data suggest that rtis at the hajj are a result of complex interactions between a number of respiratory viruses and bacteria. further studies aimed at studying the respiratory microbiota with tools allowing the identification of larger numbers of pathogens will be necessary to better elucidate these ecological changes and their potential role in the occurrence of respiratory symptoms. mass gatherings medicine: public health issues arising from mass gathering religious and sporting events travel reported by pilgrims from marseille, france before and after the 2010 hajj morbidity and mortality amongst indian hajj pilgrims: a 3-year experience of indian hajj medical mission in massgathering medicine the inevitable hajj cough: surveillance data in french pilgrims infectious diseases and mass gatherings a systematic review of emerging respiratory viruses at the hajj and possible coinfection with streptococcus pneumoniae acquisition of respiratory viruses and presence of respiratory symptoms in french pilgrims during the 2016 hajj: a prospective cohort study associations between pathogens in the upper respiratory tract of young children: interplay between viruses and bacteria evidence from multiplex molecular assays for complex multipathogen interactions in acute respiratory infections medically important bacterial-fungal interactions nasal carriage as a source of staphylococcus aureus bacteremia. study group impact of the hajj on pneumococcal carriage and the effect of various pneumococcal vaccines effect of conjugate pneumococcal vaccine followed by polysaccharide pneumococcal vaccine on recurrent acute otitis media: a randomised study haut conseil de la santé publique. bull epidemiol hebdo. recommandations sanitaires pour les voyageurs ministère des affaires sociales, de la santé et des droits des femmes influenza and the hajj: defining influenza-like illness clinically antibiotic use for respiratory infections among hajj pilgrims: a cohort survey and review of the literature rna and dna bacteriophages as molecular diagnosis controls in clinical virology: a comprehensive study of more than 45,000 routine pcr tests mass gathering and globalization of respiratory pathogens during the 2013 hajj quantitative detection of moraxella catarrhalis in nasopharyngeal secretions by real-time pcr circulation of respiratory viruses among pilgrims during the 2012 hajj pilgrimage respiratory viruses and bacteria among pilgrims during the miniopticon system instruction manual for miniopticon real-time pcr detection system with cfx manager spfware expected immunizations and health protection for hajj and umrah 2018 -an overview non-pharmaceutical interventions for the prevention of rtis during hajj pilgrimage bacterial respiratory carriage in french hajj pilgrims and the effect of pneumococcal vaccine and other individual preventive measures: a prospective cohort survey a cohort study of the impact and acquisition of nasopharyngeal carriage of streptococcus pneumoniae during the hajj impact of the hajj on pneumococcal transmission respiratory viral infection-induced microbiome alterations and secondary bacterial pneumonia airborne dispersal as a novel transmission route of coagulase-negative staphylococci: interaction between coagulase-negative staphylococci and rhinovirus infection gesundheit!" sneezing, common colds, allergies, and staphylococcus aureus dispersion preventing the airborne spread of staphylococcus aureus by persons with the common cold: effect of surgical scrubs, gowns, and masks dispersal of staphylococcus aureus into the air associated with a rhinovirus infection coinfection can trigger multiple pandemic waves changes in microbiota during experimental human rhinovirus infection staphylococcus aureus colonization and non-influenza respiratory viruses: interactions and synergism mechanisms carriage of streptococcus pneumoniae, haemophilus influenzae, moraxella catarrhalis, and staphylococcus aureus in indonesian children: a cross-sectional study microbial interactions during upper respiratory tract infections nasopharyngeal bacterial interactions in children bacterial and viral interactions within the nasopharynx contribute to the risk of acute otitis media residence of streptococcus pneumoniae and moraxella catarrhalis within polymicrobial biofilm promotes antibiotic resistance and bacterial persistence in vivo divergent mechanisms for passive pneumococcal resistance to β-lactam antibiotics in the presence of haemophilus influenzae ministère des affaires sociales, de la santé et des droits des femmes no potential conflict of interest was reported by the authors. this study was supported by the institut hospitalo-universitaire (ihu) méditerranée infection, the french national research agency under the "investissements d'avenir" programme, reference anr-10-iahu-03, the région provence alpes côte d'azur and european feder primi funding. key: cord-320107-wels9wt7 authors: gottlieb, jens title: community-acquired respiratory viruses date: 2018-03-26 journal: semin respir crit care med doi: 10.1055/s-0037-1615799 sha: doc_id: 320107 cord_uid: wels9wt7 the incidence of community-acquired respiratory viruses (carvs) is ∼15 cases per 100 patient-years after lung transplantation (ltx). paramyxoviruses account for almost 50% of the cases of carv infection in ltx. most patients will be symptomatic with a mean decline of 15 to 20% in forced expiratory volume in 1 second. the attributable death rate is low in recent years 15 to 25% carv infected ltx patients will develop chronic lung allograft dysfunction within a year after carv infection. this risk seems to be increased in comparison to the noninfected ltx recipient. detection rate of carv dependent on clinical awareness, sampling, and diagnostic method with nucleic acid testing by polymerase chain reaction in bronchoalveolar lavage is the gold standard after ltx. there is no approved treatment for paramyxoviruses, most centers use ribavirin by various routes. toxicity of systemic ribavirin is of concern and some patients will have contraindication to this treatment modality. treatment may reduce the risk to develop chronic lung allograft dysfunction and respiratory failure. agents under development are inhibiting viral attachment and use silencing mechanisms of viral replication. the respiratory tract is a major entry through which viruses initiate infection. the respiratory tract can be divided anatomically into the upper respiratory tract and the lower respiratory tract divided by the lymphoid tissue of waldeyer's ring. community-acquired respiratory viruses (carvs) are highly developed and ubiquitous pathogens which can cause infection of both the upper and lower respiratory tract. 1 infection with these viruses usually results in a mild, self-limited disease in the nonimmunocompromised adult. some carv may result in respiratory failure even with fatal outcome (e.g., middle east respiratory syndrome coronavirus, corona virus associated severe acute respiratory syndrome). carvs have been increasingly recognized as common pathogens after lung transplantation (ltx). 2 carv are a diverse group of viruses including the paramyxoviridae: respiratory syncytial virus (rsv), parainfluenza virus (pv), human metapneumovirus (hmpv); the orthomyxoviridae: influenza a and b (flu); the picornaviridae: rhinovirus (rv) and enterovirus; the coronaviridae: coronavirus (cov); and the adenoviridae: adenovirus (av) (see ►table 1). most viruses will cause local infection in the respiratory tract first with secondary dissemination to other sites in the body, while other viruses typically remain limited to the respiratory tract and induce tissue injury locally. various defense mechanisms have evolved in the respiratory tract to prevent and control infection. advances in the pathomechanisms involved in carv infection has been made recently. 3 excessive inflammation associated with severe infection can be controlled by blocking costimulatory signals, without altering immune-mediated virus clearance. recent studies suggest that the activation of effector t cells in virus-infected lungs is generated by inflammatory cells locally. this modification of the immune response in the infected epithelium may lead to virus clearance and will regulate acute inflammation, and possibly immunological memory. resolution of respiratory virus infection requires not only the elimination of the keywords ► lung transplantation ► community-acquired respiratory viruses ► ribavirin ► bronchiolitis obliterns syndrome the incidence of community-acquired respiratory viruses (carvs) is $15 cases per 100 patient-years after lung transplantation (ltx). paramyxoviruses account for almost 50% of the cases of carv infection in ltx. most patients will be symptomatic with a mean decline of 15 to 20% in forced expiratory volume in 1 second. the attributable death rate is low in recent years 15 to 25% carv infected ltx patients will develop chronic lung allograft dysfunction within a year after carv infection. this risk seems to be increased in comparison to the noninfected ltx recipient. detection rate of carv dependent on clinical awareness, sampling, and diagnostic method with nucleic acid testing by polymerase chain reaction in bronchoalveolar lavage is the gold standard after ltx. there is no approved treatment for paramyxoviruses, most centers use ribavirin by various routes. toxicity of systemic ribavirin is of concern and some patients will have contraindication to this treatment modality. treatment may reduce the risk to develop chronic lung allograft dysfunction and respiratory failure. agents under development are inhibiting viral attachment and use silencing mechanisms of viral replication. virus but also the repair and regeneration of normal lung structures and restoration of normal pulmonary function. cells and molecules regulate processes such as epithelial cell to mesenchymal cell transitions, as well as the transformation of fibroblasts and fibrocytes into myofibroblasts. dysregulation of these processes in response to lung inflammation is associated with progressive pulmonary injury and lung fibrosis. in ltx recipients, carv infection may cause inflammatory processes mediated by both innate and adaptive immune responses that result in injury to airway epithelial cells and subepithelial structures leading to obliteration of small airways. apart from direct sequelae, carv may promote immunologically mediated lung injury resulting in the development of acute rejection (ar). a clinical term for chronic lung allograft dysfunction (clad) is bronchiolitis obliterans syndrome (bos). the clinical picture is an irreversible decline in forced expiratory volume in 1 second (fev 1 ). 4 histopathologically obliteration of terminal bronchioli by fibromyxomatous tissue is recognized. 5 bos is the most important prognosis limiting factor following ltx, and remains the major impediment to long-term graft and patient survival after ltx. 6 on average, it affects every second recipient after 5 years. 6 the annual incidence of bos after ltx is $9%. bos is a progressive disease and 75% of bos patients die from respiratory failure with a 5-year survival in affected patients of 26%. 7 in ltx recipients, symptoms of carv infections may resemble those of other clinical problems like other causes of infection (bacterial or fungal), acute rejection, and drug toxicities. bacteria (mycoplasma pneumoniae, chlamydia pneumoniae, and legionella pneumophila) also cause community outbreaks, share similar clinical features, and may present diagnostic difficulties. 8 in contrast to the nonimmunosuppressed host, carv infection usually leads to more severe illness in the lung transplanted recipient with a higher incidence of respiratory failure. in a recent clinical trial with 77 rsv-infected ltx patients, median decline in fev 1 compared with the preinfection level was 13%. 9 seventy-four percent of these patients had symptoms of a lower respiratory tract infection and 10% a significant infiltrate on chest x-ray. in a retrospective singlecenter study over a 13-year time span, 38% of 138 ltx patients presenting with carv infection (41% coxsackie and rv, 16% pv, 14% cov, 10% rsv, 8% hmpv, 6% flu) demonstrated a pulmonary infiltrate and 22% were asymptomatic. 10 acute consequences of rsv infection include bronchiolitis, pneumonia, and respiratory failure. permanent long-term effects following ltx include the development of new or progressive chronic allograft dysfunction that manifest clinically as bos. 4,5 rhinoviral infection can be persistent in lung transplant recipients with graft dysfunction. 11 no temporal association was observed between carv infection and ar. 12, 13 cumulative infection rates of carv in lung transplant recipients in earlier retrospective single-center studies of 5 to 7 years ranged from 5 to 13%. [14] [15] [16] [17] retrospective studies did not test for viruses newly recognized as important pathogens for bos, especially hmpv. therefore, apart from methodological issues, bos incidence may not be exactly estimated from these studies. there are several published prospective studies using polymerase chain reaction (pcr) techniques of carv in ltx involving between 50 and 388 patients during surveillance periods of 6 to 24 months. 12,18-21 symptoms of respiratory tract infection (rti) were frequent 50 to 64% in screened patients but incidence of carv was variable between 7.8 and 34%. the different incidences can be explained by different methodology and inclusion criteria. in the swiss study, only patients who underwent bronchoscopy were studied, making it difficult to calculate the true incidence of respiratory viral infections and comparing it with uninfected patients. 22 in a canadian study, only stable patients were included and screened for carv and matched with uninfected patients, which excluded an estimation of the true incidence of carv during the observation period. 18 in a u.s. study, ltx recipients were contacted weekly by telephone and screened for symptoms. 19 symptoms of rti were, therefore, more frequent. serology identified most of the carv in this study and hmpv was not investigated. pvs were the dominant pathogens in a german surveillance study using pcr testing in upper respiratory samples and antigen testing in sample from the lower respiratory tract followed by rsv (13-22% in other prospective studies). 21 in the german surveillance study, an annual incidence of 25% in carv-positive recipients was described, which is higher than 6 to 12% in other prospective studies using pcr techniques. 18, 19 in summary, prospective studies have described an incidence of 15 to 50 cases per 100 patientyears (►table 2). infected ltx recipients in early studies presented with respiratory failure. interestingly, in retrospective studies 2,3,5 the bos incidence in carv-positive lung transplant recipients was much more common after 1 year than in the prospective studies (32-42% versus 6-16%). 6, 10, 11 several surveillance and retrospective studies confirmed an association of carv infection (as well other carv) with the onset of bos. these studies are heterogeneous and have limitations in design, case selection, and diagnostic procedures. this association was identified mainly for paramyxoviruses, and the association with influenza and av is less well documented. according to the published literature, the incidence of bos was 6 to 40% in ltx recipients infected with carv during the following 12 months. 15, 17, 18, 21, 23 patients may present with onset of clad immediately with carv infection or later after an initial recovery of fev 1 after the viral infection. there is a lack of information on the potential role of viruses that are difficult to grow, such as rv, human cov, enteroviruses, and hmpv. some of them have recently been recognized as important pathogens in ltx. 12, 23, 24 virus isolation is still the gold standard for the laboratory detection of respiratory viruses. however, virus isolation in cell cultures is slow and not always technically successful. therefore, this method is no longer used in the transplant setting where rapid workup is crucial. rapid antigen detection tests or antibodies for immunofluorescence testing (ift) are not available for all carv. the detection of viral antigens is reported to be less sensitive and less specific than cell cultures but allows rapid detection. nucleic acid amplification tests for carv by pcr are rapid and sensitive. they are commercially available in single or multiplex format. the significance of virus detection by pcrs in asymptomatic patients is unknown 15 and virus detection by pcr may not reflect active infection. serology plays no role in detecting carv infection after ltx. upper respiratory sampling by naso-oropharyngeal swab (nos) are useful in combination with pcr techniques avoiding the need for bronchoscopy. special swabs samples should be used. 21 mouth rinses are an acceptable alternative. in ltx recipients, bronchoalveolar lavage (bal) is a widespread diagnostic tool. given the broad spectrum of other possible diagnosis in a ltx patient with symptoms of upper respiratory tract infection (urti) or lower respiratory tract infection (lrti), bronchoscopy with bal is advised in most circumstances. bal was most sensitive do detect carv in surveillance studies. 21 in a recent randomized controlled trial (rct), 6% of all rsv infections were detected by pcr. 9 the potential incidence of bos after untreated carv infections and the threat of respiratory failure in infected ltx recipients illustrate the need for effective treatment of carv infections. unfortunately, respiratory viruses are a hard target for various reasons. most of the time the virus spends in the host and will be inside the host cell. the virus is protected from the host immune system as well as from available circulating enzymes. there are a limited number of potential drug targets since viruses use the host biochemical mechanisms to multiply. viruses multiply very quickly, so antiviral drugs will often have little effect by the time symptoms appear. in addition, resistance to commonly used antivirals develops early. theoretically, viruses can be targeted by inhibiting the stage of viral attachment, internalization, fusion and uncoating, replication, assembly, and release. unfortunately, except the neuraminidase inhibitors for influenza, no registered drugs are available for most the treatment of carv infections in adults. treatment strategy may be symptomatic in most cases. it usually consists of steroids, oxygen, and antibiotics in case of concomitant bacterial infection. ribavirin is known since 1972 and is registered for use in chronic hepatitis c virus infection. ribavirin, a purine community-acquired respiratory viruses gottlieb 215 nucleoside analogue with efficacy against many rna viruses, including rsv, hmpv, and pv, represents a viable treatment option for pv infection. ribavirin has been shown to have in vitro activity against rsv and the aerosolized form has been approved for the treatment of lower respiratory tract disease due to rsv in certain at-risk populations. data for intravenous use of ribavirin remain limited in ltx recipients. 25 drawbacks with inhaled ribavirin include difficulties in administration, requiring continuous inhalation, along with associated risks of bronchoconstriction and respiratory distress, which may necessitate discontinuation. in addition, aerosolized ribavirin is considered to be potentially hazardous and teratogenic to the environment including health care workers as well as being excessively expensive. considering these limitations, widespread acceptance of the nebulized administration is missing. several centers use oral ribavirin as an off-label alternative treatment in paramyxovirus infections in ltx. 26 a retrospective study demon-strates no significant differences in 6-month outcomes between oral and inhaled ribavirin therapy for rsv infection after ltx. 27 in a recent multicenter trial involving 77 rsv-infected patients from 33 sites in australia, austria, germany, france, canada, and the united states, inhaled ribavirin was used in 24% infected individuals, oral ribavirin in 16%, intravenous ribavirin in 11%, intravenous immunoglobulin (ivig) in 5%, pulsed steroids in 38%, and palivizumab in 6%. 9 the optimal duration of ribavirin therapy is unknown, although treatment orally for 14 days led to virus elimination in 89% of patients in a retrospective study. 28 of particular note, ribavirin was contraindicated in 42% of pv-infected recipients in this study and was terminated early in another quarter of cases due to adverse effects. twice-weekly monitoring of blood cell counts (risk of hemolytic anemia) and renal function is mandatory while under ribavirin therapy. compared with other patient populations treated with ribavirin, more frequent discontinuation due to toxicity was necessary in the ltx population. the most likely explanation for this remains combined toxicity of ribavirin and that inherent to the maintenance medication required after ltx. distinction must be made between the short-and longterm effects of treatment with ribavirin. prior studies have focused on short-term effects of ribavirin treatment, such as survival of the acute phase or shorter hospital length of stay, and found no beneficial effect. retrospective single-center studies suggest that ltx patients treated with ribavirin have a better pulmonary function 6 months after paramyxovirus infection and have found that oral ribavirin reduces the number of complicated courses of paramyxovirus infection and reduces the long-term risk of bos %. recovery of pulmonary function postinfection was significantly better in ribavirin-treated patients (n ¼ 38) than in patients treated with supportive care (n ¼ 29) in a retrospective study. patients treated with oral ribavirin had a lower incidence of bos (5% of the ribavirin group versus 24% of the nonribavirin group [p ¼ 0.02]) within 6 months. 28 the latter subgroup was treated with supportive care in this study and had contraindications for ribavirin (e.g., advanced kidney disease, anemia). all ribavirin studies in ltx lack a randomized placebocontrolled design, which makes them unsuitable to make evidence-based recommendations. there is clearly the need for a rct to determine the efficacy of oral ribavirin. since unnecessary treatment with drugs should always be avoided and ribavirin has some unfavorable side effects, a firm conclusion is needed about the clinical benefits of this treatment. oral ribavirin seems to be a promising and inexpensive therapy which may have a significant impact on long-term morbidity and mortality of lung transplant recipients. anecdotal cases have reported the off-label use of pavilizumab and ivig in rsv-infected ltx recipients. several agents have been studied against influenza including das181 (fludase) and nitazoxanide, but no data are published for ltx patients. rna interference is a natural biological process whereby small interfering rnas (sirnas) can direct sequence-specific degradation of mrna, leading to reduced expression of the corresponding protein. aln-rsv01 is a sirna targeting the rsv nucleocapsid messenger rna, preventing the formation of the nucleocapsid protein and thereby reducing viral replication. intranasal aln-rsv01 administration significantly inhibited the rate of rsv infection in a phase 2 experimental infection study in healthy adults. 29 in a pivotal study in 24 rsv-infected ltx patients, nebulized aln-rsv01 treatment proved to be safe and well-tolerated and incidence of new or progressive bos was decreased at day 90 in patients treated with aln-rsv01 compared with placebo. 29 the efficacy of aln-rsv01 administration in addition to standard of care on preventing new or progressive bos in rsvinfected ltx patients was evaluated in this large randomized, double-blind, placebo-controlled trial. in this phase 2b, trial subjects were randomized to receive aerosolized aln-rsv01 or placebo daily for 5 days. 9 aln-rsv01 was found to be safe and well tolerated. at day 180 in aln-rsv01-treated patients (n ¼ 44) compared with placebo (n ¼ 33), there was a trend toward a decrease in new or progressive bos (13.6% vs. 30.3%, p ¼ 0.058), which was significant in the per-protocol cohort (p ¼ 0.025). treatment effect was enhanced when aln-rsv01 was started < 5 days from symptom onset, and the effect was independent from ribavirin treatment. presatovir (gs-5806) is an oral rsv fusion inhibitor with potent and selective anti-rsv activity in vitro. a phase 2a, randomized, double-blind, placebo-controlled study was conducted to evaluate the safety, tolerability, and efficacy of presatovir in healthy adult volunteers infected with an rsv challenge virus. 30 treatment with presatovir resulted in lower mean area under the curve (auc) viral load from initial dose through end of quarantine. viral load was assessed twice daily using nasal washes and in total symptom score during the entire quarantine period. results from a phase 2b, rct evaluating the effect of gs-5806 in ltx recipients with rsv infection (nct02534350) are expected soon. there are several unmet needs in the development of effective therapies for carv. one is a wider treatment window. most agents are reported to be less effective if the patient presents more than 48 hours of symptom onset. therapies should be cost-effective and reduce the threat of resistance by continuous antigenic drifting and antigenic shifting of viruses. alternative formulations (e.g., intravenous drugs) for hospitalized patients should be available as well as drugs for severely ill, hospitalized patients, and for pediatric patients. prevention of carv infection is of utmost importance in ltx. annual influenza vaccination is strongly recommended for all lung transplant recipients including all their household members. wearing face masks, avoiding contact with infected persons, and skin disinfection are usually recommended as preventive measures in ltx recipients, although they were not systematically evaluated in this high-risk cohort. viral pneumonia community-acquired respiratory viral infections in lung transplant recipients regulating the adaptive immune response to respiratory virus infection an international ishlt/ats/ers clinical practice guideline: diagnosis and management of bronchiolitis obliterans syndrome post-transplant bronchiolitis obliterans international society for heart and lung transplantation. the registry of the international society for heart and lung transplantation: thirty-third adult lung and heart-lung transplant report-2016; focus theme: primary diagnostic indications for transplant survival after bronchiolitis obliterans syndrome among seminars in respiratory community-acquired respiratory viruses gottlieb 217 unauthorized distribution is strictly prohibited. bilateral lung transplant recipients chlamydia pneumoniae infection after lung transplantation aln-rsv01 for prevention of bronchiolitis obliterans syndrome after respiratory syncytial virus infection in lung transplant recipients graft loss and cladonset is hastened by viral pneumonia after lung transplantation chronic rhinoviral infection in lung transplant recipients incidence and outcomes of respiratory viral infections in lung transplant recipients: a prospective study upper and lower respiratory tract viral infections and acute graft rejection in lung transplant recipients impact of human metapneumovirus and human cytomegalovirus versus other respiratory viruses on the lower respiratory tract infections of lung transplant recipients respiratory viral infections are a distinct risk for bronchiolitis obliterans syndrome and death clinical impact of community-acquired respiratory viruses on bronchiolitis obliterans after lung transplant community respiratory viral infection in adult lung transplant recipients a prospective molecular surveillance study evaluating the clinical impact of communityacquired respiratory viruses in lung transplant recipients a single-season prospective study of respiratory viral infections in lung transplant recipients the value of polymerase chain reaction for the diagnosis of viral respiratory tract infections in lung transplant recipients community-acquired respiratory viral infections in lung transplant recipients: a single season cohort study respiratory viruses and severe lower respiratory tract complications in hospitalized patients incidence and morbidity of human metapneumovirus and other communityacquired respiratory viruses in lung transplant recipients human metapneumovirus in lung transplant recipients and comparison to respiratory syncytial virus intravenous ribavirin is a safe and cost-effective treatment for respiratory syncytial virus infection after lung transplantation oral ribavirin for the treatment of noninfluenza respiratory viral infections: a systematic review oral versus inhaled ribavirin therapy for respiratory syncytial virus infection after lung transplantation single-centre experience with oral ribavirin in lung transplant recipients with paramyxovirus infections rna interference therapy in lung transplant patients infected with respiratory syncytial virus oral gs-5806 activity in a respiratory syncytial virus challenge study lower respiratory viral illnesses: improved diagnosis by molecular methods and clinical impact the impact of viral respiratory tract infections on long-term morbidity and mortality following lung transplantation: a retrospective cohort study using a multiplex pcr panel key: cord-340104-6n0sn5lk authors: fagbo, shamsudeen f.; garbati, musa a.; hasan, rami; alshahrani, dayel; al‐shehri, mohamed; alfawaz, tariq; hakawi, ahmed; wani, tariq ahmad; skakni, leila title: acute viral respiratory infections among children in mers‐endemic riyadh, saudi arabia, 2012–2013 date: 2016-07-29 journal: j med virol doi: 10.1002/jmv.24632 sha: doc_id: 340104 cord_uid: 6n0sn5lk the emergence of the middle east respiratory syndrome (mers) in saudi arabia has intensified focus on acute respiratory infections [aris]. this study sought to identify respiratory viruses (rvs) associated with aris in children presenting at a tertiary hospital. children (aged ≤13) presenting with ari between january 2012 and december 2013 tested for 15 rvs using the seeplex(r) rv15 kit were retrospectively included. epidemiological data was retrieved from patient records. of the 2235 children tested, 61.5% were ≤1 year with a male: female ratio of 3:2. viruses were detected in 1364 (61.02%) children, 233 (10.4%) having dual infections: these viruses include respiratory syncytial virus (rsv) (24%), human rhinovirus (hrv) (19.7%), adenovirus (5.7%), influenza virus (5.3%), and parainfluenzavirus‐3 (4.6%). children, aged 9–11 months, were most infected (60.9%). lower respiratory tract infections (55.4%) were significantly more than upper respiratory tract infection (45.3%) (p < 0.001). seasonal variation of rv was directly and inversely proportional to relative humidity and temperature, respectively, for non mers coronaviruses (nl63, 229e, and oc43). the study confirms community‐acquired rv associated with ari in children and suggests modulating roles for abiotic factors in rv epidemiology. however, community‐based studies are needed to elucidate how these factors locally influence rv epidemiology. j. med. virol. 89:195–201, 2017. © 2016 wiley periodicals, inc. acute respiratory illnesses (aris) remain a global burden causing nearly 4 million deaths annually. in developing countries, it is the primary cause of death in children <5 years [liu et al., 2012] . in saudi arabia, over 5.4 million cases of ari presented to emergency departments in 2013 [ministry of health, 2015] . however, much remains unknown of the proportion of these ari aetiologically linked to respiratory viruses. this situation is further complicated by the emergence of the middle east respiratory syndrome coronavirus (mers cov) in saudi arabia in 2012 [zaki et al., 2012] . the global threat posed by mers cov had led to efforts disproportionately focused on defining mers-cov burden while the overall burden (denominator) of respiratory viruses in the country remains largely uncharacterized. a broadened understanding of respiratory viruses having overlapping ecologies and transmission modes should provide more modalities for integrated public health intervention and prevention. previous studies on ari in saudi arabia have largely focused on local and foreign pilgrims visiting makkah and madeenah during the annual hajj pilgrimage [el-sheikh et al., 1998; balkhy et al., 2004; alborzi et al., 2009; mandourah et al., 2012] . respiratory infections constitute a recognized health burden during hajj [alzeer, 2009] with pneumonia in pilgrims being a major reason for hospitalization [bukhari and elhazmi, 2013] . during the 2013 hajj season, almost 10% of documented deaths of pilgrims was due to respiratory illnesses including pneumonia [ministry of health, 2015] . as the overwhelming majority of the pilgrims are adults and elderly, children are virtually excluded from these studies. giving that foreign pilgrims originate from all over the world, a major limitation with most of these hajjcentered studies is that they are not adequately powered to accurately discriminate between imported and locally acquired respiratory viruses. in 2013, almost 2 million pilgrims performed the hajj; of these, 70% were from outside the kingdom [ministry of health, 2015] . additionally, these studies often lacked seasonal or climatic data in their analyses. there have been previous studies conducted on children with ari in the kingdom; most of these studies [al-hajjar et al., 1998; ghazal et al., 1998; akhter et al., 2009; al hajjar et al., 2011; alanazi et al., 2013; amer et al., 2015] have been conducted in riyadh, the country's capital. the city of riyadh is also part of the riyadh province that reported a population of 7.5 million in 2013 [ministry of health, 2015] . the few studies reported from elsewhere in the kingdom include those done in abha, taif, and najran [al-shehri et al., 2005; abdel-moneim et al., 2013; al-ayed et al., 2014] . although, most of these studies have been plagued with small sample sizes, one large sized study of children with ari used a qualitative direct immunofluorescence test [alanazi et al., 2013] ; such tests are insensitive often giving false negative results [uyeki, 2003] . serological assays have diagnostic limitations in the clinical management of children with ari [henrickson, 2004] . the requirement of convalescent phase sera to detect a fourfold rise and thus confirm acute infections makes it irrelevant in the management of acutely ill children. however, it is a good tool in retrospective, epidemiologic studies [anderson et al., 1985; falsey et al., 2002] . additionally, it has been shown that infants with ari due to respiratory syncytial virus (rsv) usually lack detectable serologic responses [murphy et al., 1986; hall et al., 1991] . similarly, it has been the insensitivity of colorimetric or immunofluorescence based antigen assays for rsv, in comparison with molecular diagnostics, has been reported [falsey et al., 2002; casiano-colon et al., 2003] . the advent of rapid and highly sensitive multiplex molecular testing platforms overcome the drawbacks of serology and antigenic tests while permitting simultaneous detection of multiple respiratory viruses. this platform was used to detect respiratory viruses in children presenting with ari under climatic conditions prevailing in the riyadh province. the king fahad medial city (kfmc) is an advanced 1,200-bed tertiary health facility located in riyadh, in the central province of saudi arabia. typically, the country usually experiences high temperatures and low precipitation. in riyadh, average winter temperatures dips as low as 8˚c; average summer temperatures peak in july at well over 40˚c. rainfall often occur between january and may with an annual rainfall of 100 mm [unfccc, 2011] [zhang et al., 2012] . extracted cdnas were tested in a 3-tube reaction according to manufacturer's protocol. primer set a in tube1 was directed at 229e/nl63, adv and piv1-3; primer set b in tube 2 oc43/hku1, hrv, rsv a and b, and ifla; and contained primer set c tube-3 at hbov, iflb, hmpv, piv4, and hev. a reaction mix consisting of 4 ml of each 5 â rv15 multiplex primer sets (a, b, and c), 3 ml 8-methoxypsoralen (8-mop) solution, 10 ml 2 â multiplex master mix (hotstart r taq dna polymerase and dntps included), and 3 ml cdna template with a final volume of 20 ml. mixtures were subjected to a first round of denaturing at 94˚c for 15 min and a second round by 40 cycles at 94˚c for 30 sec. this was followed by annealing at 60˚c for 90 sec and extension at 72˚c for 90 sec. lastly, the mixtures were extended at 72˚c for 10 min. internal controls included a mixture of all 15 virus clones (positive) and ddh 2 o (negative) [roh et al 2008; bruijnesteijn van coppenraet et al., 2010] . the end products were visualized by electrophoresis on a 2% agarose gels with ethidium bromide staining. the seeplex rv15 has a limit of detection of 100 copies/10 ml. samples positive for iav were tested for h1n1 using a commercial kit (artus 1 infl./h1 lc/rg rt-pcr kit; qiagen, germany). children with symptoms such as runny nose, nasal or throat congestion, or itchiness of the throat were described as having upper respiratory infections while those with productive cough, shortness of breath, weakness, and fatigue were classified as having lower respiratory infections. riyadh weather data (king khalid internat ional airport) was electronically retrieved from https://weatherspark.com/history/32777/2012/riyadh-saudi-arabia. for statistical analysis, spss (version 22.0, spss inc., chicago, il) was used. specific prevalence for positive cases have been derived from total positive cases in the respective groups and the one-to-one inferences have been drawn at 95% ci using chi-square analysis and fisher's exact tests as appropriate. all tests were two-tailed and p < 0.05 was considered statistically significant. the study was approved by the kfmc institutional review board (irb). a total of 2,235 children (mean age-19.6 months; range 1 day to 13 years) were tested, 1374 (61.5%) were <1 year with a male to female ratio of 3:2. nasopharyngeal samples represented 99% of samples analyzed. respiratory viruses were detected in 1364 (61%) children, significantly higher in children (55.4%) with acute lower respiratory infection (alri) than acute upper respiratory infection (auri) (45.3%) (p < 0.001). as shown in table i , more than a third of the children were diagnosed with bronchiolitis and pneumonia; of these, over 70% had respiratory viruses detected in their tested respiratory specimens. as shown in table ii , reveal that rsvs were the most detected viruses (23%); this was followed by hrv found in almost 20% of positive samples. most respiratory viruses were detected in children 9-11 months of age. more boys had rsv and hev infections. single infections with rsv, hrv, hmpv, adv, and iflb showed a significant association with age group. half of the children had single infections, while multiple infections occurred in over 10%, mostly dual infections (233/1364; 10.4%). coinfections were significant amongst those aged 9-11 months (13.5%), 2 months (13.9%), and 6-8 months (15.0%) (p< 0.001). co-infection rates were similar in children with both alri and auri: 11.5% and 9.5% for dual infections, and 0.7% and 0.5% for triple infections, respectively. viral co-infections in ari cases were predominantly associated with, in descending order, hbov, hev, and adv (p < 0.001) (fig. 1 ). more than one virus was detected in 18.1% (247 out of 2235) of patients. children with rsv and hrv co-infection required more oxygen therapy and longer length of stay than those rsv mono-infections. the detection of co-infections with more than two viruses was not significant. children with alri had significant infections with rsvs. human coronaviruses-229e/nl63 and oc43 were detected in <4% of the children. a 45-day-old male infant had quadruple infection with adv, piv3, hrv, and rsv. the pandemic h1n1 virus was only detected in an 8month-old infant. post mers cov emergence in late 2012, children were also screened, according to prevailing ministry of health guidelines, for mer-cov infection in the regional ministry of health lab: all were negative. the frequency of detection of viruses is shown in figure 2 , with rsv, hrv, and adv being the most common in descending order. seasonal variation in the detection of respiratory viruses was observed. for both years, the detection rates of hrv, rsvs, and hmpv were lowest in june, july, and august (fig. 3a) . for hrv, increase in detection began in september for both years. as shown in figure 3b , of the two rsv genotypes, rsv a was more frequently detected throughout the 2 year study period with both peaking during the winter months of december and january. no temporal genotype shift in rsv detection pattern was recorded. though hmpv detection was low (4.2%; 94/2235) when compared to rsvs and hrv, it was mostly detected between january and march. july, the hottest month during the study period [average temperature of 44˚c and 43˚c for 2012 and 2013, respectively; fig. 4b ], recorded the least incidence for most respiratory viruses. june, the least humid month in 2012 and 2013 (fig. 4a) , was the period when rsv and hrv were least detected (fig. 1) . cumulatively, respiratory virus detection was highest in december 2013 and lowest in june 2012 ( fig. 4a and b) . this large study population determined the burden of respiratory viruses associated with ari in children over a 2-year period a multiplex molecular testing platform. with over 60% of 2235 children testing positive for respiratory viruses, more infections were detected than previous studies [akhter et al., 2009; al hajjar et al., 2011; alanazi et al., 2013; bukhari and elhazmi, 2013] . researchers from a similarly large tertiary care facility based in riyadh undertook a yearlong (between 2006 and 2007) study of 10,617 children; using direct antigen testing and shell vial assay as testing platforms, they found respiratory viruses in only 8.3% [akhter et al., 2009 ] of them. most (88%, 733/883) of these children has rsv infection. this high rate was significantly different from that obtained in the present 2-year study (23% detection for rsv in children positive for respiratory viruses), as well as others [alanazi et al., 2013; amer et al., 2015] . similarly, higher detection rates were observed for influenza and parainfluenza viruses when compared to the results from this this study. it was observed that the 4.2% hmpv detection rate in the present study was lower than the 8.3% rate obtained from another study earlier conducted in a similar tertiary health care facility in riyadh [al hajjar et al., 2011] . although, the earlier study used a lesser advanced version of the seeplex kit, the seemingly increased rate may be due to most of the children tested in the earlier study being immunocompromised. given its effectiveness as demonstrated in this study, multiplex testing platforms such as the seeplex rv15 ace can be modified to include mers-cov consensus probes. consequently, the augmented ability to simultaneously test a single specimen for mers-cov and other endemic respiratory viruses should diagnostic turnaround time and optimize utilization of limited specimen volume scenarios as may be envisaged in pediatric settings. analyzing limited specimens using monoplex assays may not only preclude detection of untested respiratory viruses, aetiologically unresolved ari quagmires will abound. additionally, such modified platforms have the potential to elicit more epidemiological data on respiratory viruses including mers-cov. in the backdrop of the incessant multidirectional human traffic of residents between countries within the arabian peninsula and as more recently demonstrated by mers-cov transmission events, it may not be impossible that the epidemiology of respiratory viruses in these countries are somewhat inter-related. more detailed research will, however, be needed to elucidate this. comparatively, a study from neighboring oman reported adv being the second most detected virus in children with ari, aged 5 years or younger [khamis et al., 2012] ; in the present riyadh study, adv infections was associated with age groups 6-8 months, 3-5 months, 1-5 years, and 9-11 months in a decreasing order. extending the findings of previous studies that linked seasonal patterns of ari in children with fewer detection rates of respiratory viruses [alanazi et al., 2013; bukhari and elhazmi, 2013] or none [ghazal et al., 1998 ], this study seasonally correlated the large rv detection rate which showed an inverse and direct relationship with average temperature and humidity, respectively (fig. 2) . however, the depicted graphical correlation was obtained while analyzing our data with externally acquired meteorological data. this study has limitations. firstly, its retrospective design may not permit capture of all ari presenting to the facility. secondly, the results obtained may not represent the true burden of ari due to respiratory viruses in children in the community, a limitation shared by other studies. usually, most mild cases of respiratory viral infections are unlikely to seek care at health care facilities with some patients engaging in self-medication. consequently, these cases remain uncaptured by hospital-based studies or surveillance systems. to address this limitation, community based respiratory virus epidemiologic studies are suggested. though such studies are presently lacking in saudi arabia, similar work done elsewhere [lambert et al., 2008b; van der zalm et al., 2011; chu et al., 2013; alsaleh et al., 2014] have yielded valuable epidemiological data. in australia, workers using community centered studies have shown the strong association between child care attendance and the detection of human metapneumovirus and human coronavirus nl63 in children [lambert et al., 2007] . they also documented evidence of significant economic impact of ari managed outside health care settings on families; such costs were greater in healthy preschool aged children with influenza infection than those infected with rsv and other respiratory viruses [lambert et al., 2008a] . in the netherlands, researchers showed that respiratory viruses regularly occur in asymptomatic children sampled in the community [van der zalm et al., 2009] . by including appropriate temporal and climatic data, such studies will further elucidate the transmission dynamics of respiratory viruses in children in community settings in riyadh. additionally, it is possible that the results of such studies may provide collateral data to help understand possible community transmission of mers cov suggested in a recent study [fagbo et al., 2015] . the technical assistance of nura al ragi, hanan al qudairy, johara al mutairy, and eman al karafi is acknowledged. we also appreciate the assistance of abdul basir pula and roderick mallete with the figures. detection of bocavirus in children suffering from acute respiratory tract infections in saudi arabia etiology of respiratory viral infections using rapid virus isolation methods at a tertiary care center in riyadh, saudi arabia viral etiology of respiratory infections in children in southwestern saudi arabia using multiplex reverse-transcriptase polymerase chain reaction respiratory viruses in children attending a major referral centre in saudi arabia bronchiolitis in abha, southwest saudi arabia: viral etiology and predictors for hospital admission human metapneumovirus and human coronavirus infection and pathogenicity in saudi children hospitalized with acute respiratory illness viruses associated with respiratory tract infections in children attending to the emergency room, king abdulaziz medical city, riyadh, saudi arabia viral etiology of acute respiratory infections among iranian hajj pilgrims nasal swab samples and real-time polymerase chain reaction assays in communitybased, longitudinal studies of respiratory viruses: the importance of sample integrity and quality control respiratory tract infection during hajj epidemiology of 11 respiratory rna viruses in a cohort of hospitalized children in riyadh, saudi arabia microneutralization test for respiratory syncytial virus based on an enzyme immunoassay influenza a common viral infection among hajj pilgrims: time for routine surveillance and vaccination comparison of two commercial molecular assays for simultaneous detection of respiratory viruses in clinical samples using two automatic electrophoresis detection systems viral agents causing acute lower respiratory tract infections in hospitalized children at a tertiary care center in saudi arabia lack of sensitivity of rapid antigen tests for the diagnosis of respiratory syncytial virus infection in adults molecular epidemiology of respiratory syncytial virus transmission in childcare bacteria and viruses that cause respiratory tract infections during the pilgrimage (haj) season in makkah, saudi arabia molecular epidemiology of hospital outbreak of middle east respiratory syndrome diagnosis of respiratory syncytial virus infection: comparison of reverse transcription-pcr to viral culture and serology in adults with respiratory illness acute respiratory tract infections: epidemiological data, guided case management and outcome in a pediatric hospital in riyadh immunity to and frequency of reinfection with respiratory syncytial virus advances in the laboratory diagnosis of viral respiratory disease epidemiology of respiratory virus infections among infants and young children admitted to hospital in oman the cost of community-managed viral respiratory illnesses in a cohort of healthy preschool-aged children community epidemiology of human metapneumovirus, human coronavirus nl63, and other respiratory viruses in healthy preschool-aged children using parent-collected specimens parent-collected respiratory specimens-a novel method for respiratory virus and vaccine efficacy research global, regional, and national causes of child mortality: an updated systematic analysis for 2010 with time trends since clinical and temporal patterns of severe pneumonia causing critical illness during hajj. bmc infec dis 12:17. ministry of health. 2015. health statistics annual book 2013 g/ 1434 h. riyadh. saudi arabia: ministry of health effect of age and preexisting antibody on serum antibody response of infants and children to the f and g glycoproteins during respiratory syncytial virus infection comparison of the seeplex reverse transcription pcr assay with the r-mix viral culture and immunofluorescence techniques for detection of eight respiratory viruses second national communication kingdom of saudi arabia influenza diagnosis and treatment in children: a review of studies on clinically useful tests and antiviral treatment for influenza isolation of a novel coronavirus from a man with pneumonia in saudi arabia high incidence of multiple viral infections identified in upper respiratory tract infected children under 3 years of age in key: cord-336562-5qmzne98 authors: auten, richard; ren, clement; yilmaz, ozge; noah, terry l. title: pediatric pulmonology year in review 2016: part 2 date: 2017-04-25 journal: pediatr pulmonol doi: 10.1002/ppul.23719 sha: doc_id: 336562 cord_uid: 5qmzne98 pediatric pulmonology continues to publish research and clinical topics related to the entire range of children's respiratory disorders. as we have done annually in recent years, we here summarize some of the past year's publications in our major topic areas, as well as selected literature in these areas from other core journals relevant to our discipline. this review (part 2) covers selected articles on neonatology, asthma, physiology and lung function testing, and infectious diseases. pediatric pulmonology continues to publish research and clinical topics related to the entire range of children's respiratory disorders. as we have done annually in recent years, we here summarize some of the past year's publications in our major topic areas, as well as selected literature in these areas from other core journals relevant to our discipline. this review (part 2) covers selected articles on neonatology, asthma, physiology and lung function testing, and infectious diseases. since mechanical ventilation inevitably exposes the developing respiratory system to unpredictable mechanical strains and stresses, clinicians have long sought safer and less invasive approaches to respiratory support, and better tools with which to monitor respiratory function during intensive care. there has been a trend to substitute high flow nasal cannulae which are able to deliver positive pressure, but this pressure is typically not regulated or monitored. in an effort to define the limitations of this approach, gerdes et al 1 identified factors affecting delivered mean airway pressure during nasal cpap delivery with the ram™ cannula, an increasingly widely used device favored by some because of its apparent toleration compared with other devices that require more complex fixation systems. using a polymer model of the upper airway and lip, ram cannulae, and corresponding standard ncpap prongs were affixed and delivered pressures measured using a pneumotachograph. as expected, complete occlusion of the nasal passage with either the ram cannula or "standard" nasal prongs delivered comparable pressures that approximated the set pressure, providing that the ram cannula was fully inserted. however, the authors point out a critical limitation to using the ram cannula, namely that nasal expiratory resistance will be quite high if the nares are occluded. for babies that must be managed with mechanical ventilation, efforts are underway to improve both delivery and accurate monitoring of therapy. the safe use of high-frequency ventilation in newborns limits the ability of the clinician to assess ventilation in realtime. minute ventilation and tidal volume cannot be measured, and capnography has been limited to transcutaneous co 2 measurements, which have been problematic in very premature newborns that may not tolerate the skin temperatures that until very recently have been required for accurate measurement. kugelman et al 2 evaluated capnography using a 2-lumen endotracheal tube and microstream capnography correlated with arterial blood gas measurements obtained from an indwelling line. although accuracy was not acceptable, capnography was able to predict very low p a co 2 < 30 torr and very high p a co 2 above 60 torr. respiratory volumes can also be assessed non-invasively using electrical impedance. van findings with their earlier reports of increased methacholine induced respiratory system resistance. 11 the evidence for human susceptibility to neonatal and early childhood pulmonary insults that affect lung function in adulthood continues to mount, as reviewed by goldizen et al. 12 impairments of small airway function were reported by verheggen et al, 13 reporting a case-control comparison of pulmonary function testing in 4-to 8-year old subjects born before 32 weeks with or without bpd. they used forced oscillometry or spirometry. they reported that pulmonary reactance, an oscillometric method used to assess small airway function, was worse in bpd subjects. although this finding is not surprising, it should be tempered by the relatively low recruitment rate they experienced from their original qualifying cohort. factors associated with bpd risk like duration of oxygen exposure and male sex were also associated with increased pulmonary reactance. the genetic basis of asthma has been investigated in many studies recently. hua et al 14 investigated the gene-gene interactions among variants of the il13, il4, il4ra, fcer1b, and adrb2 genes, which have been associated with asthma in children, and demonstrated that single nucleotide polymorphisms il13 rs20541, il4 rs2243250, adrb2 rs1042713n, and fcer1b rs569108 were associated with asthma. moreover, risk of asthma was increased more than 10 times in carriers of all four risk homozygotes (il13 rs20541 gg, il4 rs2243250 tt, adrb2 rs1042713 aa, and fcer1b rs569108 gg). these results add to the evidence for a role for epigenetic mechanisms in the relationship between environmental smoke exposure during pregnancy and pediatric asthma. gene-environment interaction is also important in the development of pediatric asthma. research done in rural and urban parts of china on 854 children revealed that physician diagnosed asthma was significantly lower in children living in rural areas. personal and family history of atopy, high consumption of milk products, and hospitalization younger than three years of age were significantly associated with asthma. similarly, being a member of a crop-farming family and dust endoxtoxin levels were negatively associated with asthma in the child. 16 lautenbacher et al 17 investigated the effects of vitamin d on pulmonary function and compared obese and non-obese asthmatic children. although the frequency of vitamin d deficiency was not different between groups, fev1 and frc were significantly worse in vitamin d deficient obese children than in normal-weight children. moreover, tnf and il-8 were higher in obese asthmatics, while the th2 cytokine il13 was higher in normal-weight asthmatics. however, these inflammatory measures were not related to the association of vitamin d deficiency with poor lung function; thus implying an independent effect of vitamin d deficiency. coexistence of obesity and poor asthma control is well known, but body mass index (bmi) may not reflect regional differences in adiposity. as part of the asthmap-2 project that is an observational study of pediatric asthma, association of neck circumference and asthma control was assessed. 18 it was demonstrated that, in boys, neck circumference combined with bmi explained the variability of asthma control test better than bmi alone. air pollution has a significant influence on respiratory health. rice et al 19 although inflammatory markers did not correlate with ahr, the degree of ahr was less in those study subjects who were receiving hyroxyurea. their results demonstrate that there is a high prevalence of ahr in scd patients and hydroxyurea may be helpful in decreasing ahr in scd. lunt et al 38 impulse oscillometry (ios) uses the forced oscillation technique to assess airway function and does not require the maximal forced expiratory maneuver needed for spirometry. 39 ios measures total respiratory impedance (z) which is a complex number that incorporates both the in and out phase elements of resistance; the former is resistance (r), and the latter is termed the reactance (x). r reflects the airway resistive properties of the respiratory system, while x reflects the visco-elastic and inertive elements of the respiratory system. potential limitations of this method include a limit on the size of the patients studied due to limitations on the volume of the washout collection bag and an inability of detect leak since there is no pnt. this system will require further study in human subjects before determining its utility in clinical research and patient care. tidal breathing analysis is a method to assess respiratory function that does not require sedation or manipulation of the infant's respiratory system. 45 respiratory inductance plethysmography has been used to obtain tidal breathing data, but it only measures chest and abdominal excursion at two points, which does not accurately reflect the true mechanics of the respiratory system. reinaux et al 46 used opto-electronic plethysmography (oep) to measure motion at 52 sites placed in the thorax and abdomen of healthy infants and compared the results obtained using this system to those obtained using a mask with pnt. they found that measurement of tidal volume using oep was in good agreement with that obtained using a pnt with a mean difference of only 0.02 ml. the ability to obtain tidal breathing measurements may lead to new insights into changes in chest and abdominal motion in pediatric respiratory disease. bronchopulmonary dysplasia (bpd) is the most common respiratory complication of preterm birth. despite advances in neonatal medicine, bpd still occurs in 30-50% of infants born before a gestational age (ga) of 29 weeks. 47 acute viral bronchiolitis, due to rsv and other pathogens, continues to have a major impact worldwide on childhood mortality and hospital admissions, 51 is associated with subsequent asthma and allergy risk, 52 and could be increasing in incidence. 53 cangiano et al 54 noted a higher incidence of acute bronchiolitis every 4 years among previously health term infants, largely due to rsv; rhinovirus was a less common cause and had less tendency to occur in seasonal clusters than rsv. while a commonly used standard definition for bronchiolitis is initial episode of wheezing in a child <12 months old, variability in how clinicians actually define bronchiolitis was highlighted in a report from fernandes et al. 55 beamer et al 56 treatment for acute bronchiolitis remains largely supportive and progress has been made in reducing widespread use of corticosteroids and bronchodilators, which have previously been shown to be ineffective. 57 flores et al 58 conducted a randomized clinical trial comparing 3% hypertonic saline to normal saline in previously healthy infants hospitalized with mild-to-moderate acute viral bronchiolitis. the median length of hospital stay, severity score, and need for supplemental oxygen did not differ significantly between groups. patients receiving hypertonic saline had significantly more cough (46% vs 20%, p = 0.025). thus, the study does not support the use of nebulized hypertonic saline over normal saline in therapy of hospitalized children with mild-to-moderate acute viral bronchiolitis. in another study with practical implications, heikkila et al 59 factors influencing delivered mean airway pressure during nasal cpap with the ram cannula diagnostic accuracy of capnography during high-frequency ventilation in neonatal intensive care units the effect of prolonged lateral positioning during routine care on regional lung volume changes in preterm infants role of electrical impedance tomography in clinical practice in pediatric respiratory medicine fgr in the setting of preterm sterile intra-uterine milieu is associated with a decrease in rds association of bnp, ntprobnp, and early postnatal pulmonary hypertension in very preterm infants bronchopulmonary dysplasia impairs l-type amino acid transporter-1 expression in human and baboon lung pulmonary ventilation and micro-structural findings in congenital diaphragmatic hernia neonatal hyperoxia increases airway reactivity and inflammation in adult mice perinatal nicotine exposure induces myogenic differentiation, but not epithelial-mesenchymal transition in rat offspring lung perinatal nicotine exposure induces asthma in second generation offspring respiratory effects of air pollution on children respiratory function and symptoms in young preterm children in the contemporary era four-locus gene interaction between il13, il4, fcer1b, and adrb2 for asthma in chinese han children dna methylation in newborns and maternal smoking in pregnancy: genome-wide consortium metaanalysis associations of early life exposures and environmental factors with asthma among children in rural and urban areas of guangdong vitamin d and pulmonary function in obese asthmatic children sex differences in the association between neck circumference and asthma lifetime exposure to ambient pollution and lung function in children association between trafficrelated air pollution and asthma in preschool children in a national japanese nested case-control study postpartum depression, a direct and mediating risk factor for preschool wheeze in girls prenatal maternal stress and atopic diseases in the child: a systematic review of observational human studies looking beyond patients: can parents' quality of life predict asthma control in children? asthma action plan receipt among children with asthma 2-17 years of age can a single dose response predict the effect of montelukast on exerciseinduced bronchoconstriction? albuterol via metered-dose inhaler in children: lower doses are effective, and higher doses are safe exhaled breath temperature measurement and asthma control in children prescribed inhaled corticosteroids: a cross sectional study infection and inflammation in induced sputum from preschool children with chronic airways diseases association between exhaled inflammatory markers and asthma control in children concordance between bronchial hyperresponsiveness, fractional exhaled nitric oxide, and asthma control in children patterns of growth and decline in lung function in persistent childhood asthma differences in pulmonary functions in various racial groups multi-ethnic reference values for spirometry for the 3-95-yr age range: the global lung function 2012 equations pulmonary diffusing capacity in healthy african-american and caucasian children spirometric reference values for hopi native american children ages 4-13 years pulmonary complications of sickle cell disease airway hyperreactivity is frequent in non-asthmatic children with sickle cell disease longitudinal assessment of lung function in children with sickle cell disease clinical application of forced oscillation respiratory impedance in patients with duchenne muscular dystrophy evaluation of children with cystic fibrosis by impulse oscillometry when stable and at exacerbation respiratory impedance in children with cystic fibrosis using forced oscillations in clinic changes in lung function measured by spirometry and the forced oscillation technique in cystic fibrosis patients undergoing treatment for respiratory tract exacerbation accurate lung volume measurements in vitro using a novel inert gas washout method suitable for infants tidal breathing analysis tidal volume measurements in infants: opto-electronic plethysmography versus pneumotachograph prematurity and respiratory outcomes program. comparisons and limitations of current definitions of bronchopulmonary dysplasia for the prematurity and respiratory outcomes program lung function gain in preterm infants with and without bronchopulmonary dysplasia mid-childhood lung function in a cohort of children with "new bronchopulmonary dysplasia global and national burden of diseases and injuries among children and adolescents between 1990 and 2013: findings from the global burden of disease viral bronchiolitis in children predictors of asthma following severe respiratory syncytial virus (rsv) bronchiolitis in early childhood admission to hospital for bronchiolitis in england: trends over five decades, geographical variation and association with perinatal characteristics and subsequent asthma bronchiolitis: analysis of 10 consecutive epidemic seasons acute viral bronchiolitis: physician perspectives on definition and clinically important outcomes spatial clusters of child lower respiratory illnesses associated with community-level risk factors value in inpatient pediatrics network quality collaborative for improving hospital compliance with aap bronchiolitis guideline (bqip). a multicenter collaborative to reduce unnecessary care in inpatient bronchiolitis a randomized trial of nebulized 3% hypertonic saline with salbutamol in the treatment of acute bronchiolitis in hospitalized infants high-flow oxygen therapy is more costeffective for bronchiolitis than standard treatment-a decision-tree analysis respiratory viral detection in children and adults: comparing asymptomatic controls and patients with community-acquired pneumonia beta-lactam versus betalactam/macrolide therapy in pediatric outpatient pneumonia paradoxical tuberculosisassociated immune reconstitution inflammatory syndrome in children protracted bacterial bronchitis: the last decade and the road ahead pediatric bronchiectasis: no longer an orphan disease coccidioidomycosis in infants: a retrospective case series interaction between 25-hydroxyvitamin d and variants at 17q12-21 on respiratory infections how to cite this article pediatric pulmonology year in review 2016: part 2 key: cord-323009-frej2qmb authors: nakouné, emmanuel; tricou, vianney; manirakiza, alexandre; komoyo, francis; selekon, benjamin; gody, jean chrysostome; victoir, kathleen; buchy, philippe; kazanji, mirdad title: first introduction of pandemic influenza a/h1n1 and detection of respiratory viruses in pediatric patients in central african republic date: 2013-02-08 journal: virol j doi: 10.1186/1743-422x-10-49 sha: doc_id: 323009 cord_uid: frej2qmb background: acute viral respiratory illnesses in children in sub-saharan africa have received relatively little attention, although they are much more frequent causes of morbidity and mortality than in developed countries. active surveillance is essential to identify the causative agents and to improve clinical management, especially in the context of possible circulation of pandemic viruses. findings: a prospective study was conducted in the central african republic (car) between january and december 2010 among infants and children aged 0–15 years attending sentinel sites for influenza-like illness or acute respiratory illness. nasopharyngeal swabs were collected, and one-step real-time and multiplex reverse transcription-polymerase chain reaction were used to detect respiratory viruses. respiratory viruses were detected in 49 of the 329 (14.9%) nasopharyngeal samples: 29 (8.8%) contained influenza viruses (5 (1.5%) had pandemic influenza a/h1n1 virus and 24 (7.3%) had influenza b viruses), 11 (3.3%) contained parainfluenza viruses types 1 and 3 and 9 (2.7%) contained human respiratory syncytial virus. most cases were detected during the rainy season in the car. analysis of the amplicon sequences confirmed the identity of each detected virus. conclusions: the influenza surveillance system in the car has provided valuable data on the seasonality of influenza and the circulation of other respiratory viruses. our network could therefore play a valuable role in the prevention and control of influenza epidemics in the car. although acute respiratory illness is a major cause of morbidity and mortality among children in sub-saharan africa, it has received relatively little attention [1] . this is unfortunate, as underlying diseases such as aids, malaria and tuberculosis, which are highly prevalent in the region, can worsen such illnesses [2] . the respiratory viruses known to cause acute illness include human respiratory syncytial virus (hrsv), human parainfluenza virus (piv), human metapneumovirus and influenza viruses [3] [4] [5] . until recently, the burden of influenza and influenza-like illness in africa was considered to be negligible [6] , mainly because of the lack of confirmation assays. reports from cameroon and senegal, however, show that influenza viruses are actively circulating and may be causing regular epidemics [7, 8] . a clear picture of the contribution of each pathogen to acute respiratory illness is needed in order to improve prevention and clinical management and consequently to reduce the burden of disease. the emergence of the novel influenza a/h1n1 of swine origin in mexico in april 2009 and its rapid spread worldwide, causing a global pandemic, led the health authorities of the central african republic (car) to collaborate with the world health organization in strengthening biological surveillance of acute respiratory illness. the aim of the study reported here was to determine the circulation of 2009 pandemic influenza a/h1n1 virus (h1n1pdm09) by molecular methods and to identify the causative viruses, the incidence and the clinical features of acute respiratory illness among infants and young children at sentinel sites in bangui and three rural areas. all infants and children aged between 0-15 years who attended sentinel sites in bangui and three rural areas ( figure 1 ) for influenza-like illness (ili) or severe acute respiratory illness between january and december 2010 were included in the study (figure 2a ). the world health organization definitions were used for ili (sudden onset of fever of > 38°c and cough or sore throat in the absence of other diagnoses) and severe acute respiratory illness (ili symptoms and shortness of breath or difficulty in breathing and requiring hospital admission). the study protocol was approved by the national ethics committee of the car. individual written informed consent was sought from the parents or guardians of all participants. nasopharyngeal samples were collected from 329 infants and children and within 48 hrs at 4°c to the national influenza centre by using rayon-budded swabs with virus transport medium pre-impregnated sponge (virocult, medical wire & equipment, uk). rna was extracted with a qiamp rna mini kit (qiagen) according to the manufacturer's instructions. influenza a viruses were detected with a previously described assay targeting the conserved matrix gene for universal detection of these viruses [9] , and h1n1pdm09 virus was identified with a specific one-step real-time reverse transcription-polymerase chain reaction (rt-pcr) assay (designed by the national influenza centre of northern france, institut pasteur, paris; primers and probe available upon request at grippe@pasteur.fr). all specimens were also tested for other respiratory viruses in two previously described multiplex semi-nested rt-pcr assays for detecting influenza a and b viruses, hrsv, human metapneumovirus and piv types 1, 2, 3 and 4 [10, 11] . all assays were performed on an abi 7500 platform (applied biosystems, foster city, california, usa) with the superscript iii platinum one-step quantitative rt-pcr system (invitrogen, carlsbad, california, usa). a specimen was considered positive if the signal curve crossed the threshold line within 40 cycles. the assay limit of detection for pandemic h1n1pdm09 influenza virus is of order of magnitude of 10 copies/μl of initial sample [9] . the assay limit of detection for influenza a and b viruses, hrsv, human metapneumovirus and piv-3 is of order of magnitude of 10 copies/μl [10] . for piv-4, the assay limit of detection is of order of magnitude of 100 copies copies/μl, and for piv-1 and −2, 1000 copies/μl [10] . after amplification, the pcr products were purified and sent to gatc biotech (konstanz, germany) for sequencing. student's t test and the pearson chi-squared test were used to assess intergroup differences. statistical analyses were performed with epiinfo software (v 3.5.1 cdc). a test was considered significant when the p value was < 0.05. the newly obtained sequences were analysed and compared with sequences available in genbank. of the 329 patients included in the study, 49 (14.8%) tested positive for respiratory viruses (table 1) . of these, five (1.5%) were positive for h1n1pdm09, 23 (6.9%) for influenza b, 10 (3.0%) for hrsv and 11 (3.3%) for piv-1 and −3 (table 1 and figure 2b ). the average age of positive patients was 41 months (range, 1 month to 9 years), with no difference in the age or gender distribution. all samples were negative for human metapneumovirus. the first case of h1n1pdm09 was detected in the car on 26 july 2010 in a child aged 36 months, followed by a second case on 11 september in an infant aged 16 months, and three cases were found in young children on 4, 11 and 22 october ( figure 2b ). of the five children, only one was admitted to intensive care for respiratory distress; no deaths were recorded. a 253-bp fragment of the haemagglutinin gene of the five h1n1pdm09 strains [accession numbers: cy092425, cy092426, cy092427, cy092428, cy092429] showed high sequence similarity (99-100%) to influenza a/california/04/2009 (h1n1) (data not shown). the 23 specimens positive for influenza b represented 82.1% of the influenza viruses detected. ten of the patients were hospitalized; one infant aged 9 months died 2 days after admission to intensive care with a clinical picture of severe acute respiratory illness. influenza b virus was implicated in respiratory infections hrsv was detected in 10 patients (table 1 ) with a median age of 28 months, mainly in november ( figure 2b ). one infant aged 10 months died 1 day after admission to intensive care with a clinical picture of fever, cough, rhinitis, bronchiolitis, dyspnoea and myalgia. fever and rhinitis were recorded in 9 of the 10 patients, and all had cough. no viral co-infections were reported. analysis of the nucleotide sequences of six isolates [accession numbers: he803082, he803083, he803084, he803085, he803086, he803087] showed sequence similarity to both genotypes a and b. piv-3 was detected in seven patients (median age, 16 months; range, 5-48 months) and piv-1 in four infants (median age, 11 months; range, 3-24 months) between april and november 2010 ( figure 2b ). respiratory viruses were found in 14.8% of the 329 collected samples, showing the presence of h1n1pdm09 infections in the car for the first time. we found that influenza b, piv-1, piv-3 and hrsv were also involved in ili in the country. most of the cases were detected during the rainy season. h1n1pdm09 infection in the car was first described in july 2010. in other african countries, the virus was shown to have been introduced by travellers [12] [13] [14] , but its source in the car has not been elucidated. all four cases detected were indigenous, with no history of travel or contact with a person returning from a country with declared cases. extensive investigations of contacts of the confirmed cases did not reveal any other cases, suggesting low dissemination of h1n1pdm09 in the country. the clinical picture of h1n1pdm09 infection was similar to that of seasonal influenza circulating before the pandemic. this is in accordance with studies showing relatively low transmissibility and severity of h1n1pdm09 [15, 16] . consequently, h1n1pdm09 did not appear to have had a significant public health impact in this area of the world. in the present study, influenza b virus was the most commonly detected respiratory virus (n=23), whereas in similar studies in africa hrsv was the most frequent causative virus of ili [17] [18] [19] . influenza b virus caused respiratory infections throughout the year, with two peaks: at the beginning of the rainy season (june-july) and at the end of the rainy season (november). antigenically and genetically distinct lineages of influenza b virus, influenza b/victoria and b/yamagata viruses have circulated in the car [20] . hrsv is a major cause of ili among infants and children worldwide [21] and is the most frequently detected respiratory virus in both developed and developing countries [17] [18] [19] [22] [23] [24] [25] [26] . in this study, hrsv was found in only 2.7% of the samples and represented 18.3% of the viruses detected. the difference from other studies might be due to a different epidemiology of hrsv in the car, a landlocked country in central africa, or to different inclusion criteria or detection techniques. most of hrsv cases detected in our study occurred between november and february, corresponding to the dry season in the car. another interesting finding was the relatively low prevalence of respiratory viruses in the children with ili, which were present in only 14.9% of samples; in similar studies, as many as 50% of samples contained respiratory viruses [17] [18] [19] [22] [23] [24] [25] [26] . in the car, vaccines against streptococcus pneumoniae and haemophilus influenzae were introduced in the enlarged programme of vaccination only recently (h. influenzae in september 2008 and s. pneumoniae in july 2011). in a study in bangui in 1995, these bacteria were found in hundreds of ill children aged less than 5 years [27] . it is therefore likely that these bacteria are still very common or even the main causes of respiratory infections in the car, as in other countries before the introduction of vaccines, when most severe respiratory infections were due to bacterial infections, and s. pneumoniae and h. influenzae were the commonest bacterial causes [28] [29] [30] . this may explain, at least partly, the differences between our results and those of other studies. it is also possible that viruses other than these we looked for were involved, such as human rhinovirus, human bocavirus, human coronavirus or adenovirus. the main limitation of our study is the limited sample size, which prevented us from investigating associations between clinical outcome and viral etiology. another weakness is a bias toward younger patients, so that we could not assess whether a particular age group is at greater risk for a specific infection. owing to the design of the study, it is also difficult to determine prevalence from the results. therefore, further studies are needed to evaluate the burden of all respiratory viruses infections in the general population of the car. our study does highlight the importance of the clinical, epidemiological and virological network for influenza surveillance in the car [31, 32] . we reported here the first data on the etiology of ili in the car. this will help central african clinicians to provide better care and treatment for patients presenting with ili, including better use of antibiotics. further studies with more patients are needed to confirm the burden of viral respiratory diseases in the car. collection of samples from healthy control children may also enable comment on virus detection and disease association. another suggestion for future studies is to collect data about underlying health status of children as risk factors such as hiv infection, malaria, malnutrition, etc. might have an impact on the acute respiratory infections [33, 34] . the temporal patterns detected should be assessed over many years in order to identify long-term seasonal patterns. abbreviations car: central african republic; ili: influenza-like illness; hrsv: human respiratory syncytial virus; piv: parainfluenza viruses. what are the implications for childhood pneumonia of successfully introducing hib and pneumococcal vaccines in developing countries childhood pneumonia-progress and challenges epidemiological and clinical study of viral respiratory tract infections in children from italy occurrence of respiratory virus: time, place and person respiratory viral infections in infants: causes, clinical symptoms, virology, and immunology influenza in africa epidemiological and virological influenza survey in dakar pandemic a(h1n1)2009 influenza virus detection by real time rt-pcr: is viral quantification useful? development of three multiplex rt-pcr assays for the detection of 12 respiratory rna viruses use of a multiplex pcr/rt-pcr approach to assess the viral causes of influenza-like illnesses in cambodia during three consecutive dry seasons epidemiology and factors associated with fatal cases cdc: introduction and transmission of 2009 pandemic influenza a (h1n1) virus-kenya pandemic influenza a (h1n1) virus outbreak and response-rwanda pandemic h1n1 influenza lessons from the southern hemisphere transmission characteristics of the 2009 h1n1 influenza pandemic: comparison of 8 southern hemisphere countries sentinel surveillance for influenza and other respiratory viruses in côte d'ivoire viral etiology of severe pneumonia among kenyan infants and children respiratory viruses in children hospitalized for acute lower respiratory tract infection in ghana burden of interpandemic influenza in children younger than 5 years: a 25-year prospective study population-based surveillance for hospitalizations associated with respiratory syncytial virus, influenza virus, and parainfluenza viruses among young children incidence and clinical characteristics of the infection by the respiratory syncytial virus in children admitted in santa casa de são paulo hospital establishing a surveillance network for severe lower respiratory tract infections in korean infants and young children virological and clinical characterization of respiratory infections in children attending an emergency department during the first autumn-winter circulation of pandemic a (h1n1) 2009 influenza virus molecular monitoring of causative viruses in child acute respiratory infection in endemo-epidemic situations in shanghai seasonal variations of 15 respiratory agents illustrated by the application of a multiplex polymerase chain reaction assay centers for disease control and prevention: antibiotic resistance among nasopharyngeal isolates of streptococcus pneumoniae and haemophilus influenzae use of nasopharyngeal isolates of streptococcus pneumoniae and haemophilus influenzae from children in pakistan for surveillance for antimicrobial resistance efficacy of nine-valent pneumococcal conjugate vaccine against pneumonia and invasive pneumococcal disease in the gambia: randomised, double-blind, placebo-controlled trial randomised trial of haemophilus influenzae type-b tetanus protein conjugate vaccine [corrected] for prevention of pneumonia and meningitis in gambian infants improving influenza surveillance in sub-saharan africa virological surveillance in africa can contribute to early detection of new genetic and antigenic lineages of influenza viruses selected major risk factors and global and regional burden of disease global and regional burden of disease and risk factors 2001: systematic analysis of population health data submit your next manuscript to biomed central and take full advantage of: • convenient online submission • thorough peer review • no space constraints or color figure charges • immediate publication on acceptance • inclusion in pubmed, cas, scopus and google scholar • research which is freely available for redistribution we would like to thank dr edgar dimbele, dr franco banawane and dr léandre mballa dimbala for assistance in sample collection. this work was supported by the french ministry of health and the office of the assistant secretary for preparedness and response within the us department of health and human services (grant number 6 idesp060001-01-01) through the international network of pasteur institutes. the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. the authors declare that they have no competing interests.authors' contributions fk and bs carried out the serological and molecular studies. am compiled the epidemiological data. en, vt and mk participated in the design of study, the analysis and the interpretation of the data and drafted the manuscript. am, jcg, kv and pb participated in the analysis and the interpretation of the data. all authors read and approved the final version of the manuscript. key: cord-324601-s3rgxtg6 authors: mhimbira, f.; hiza, h.; mbuba, e.; hella, j.; kamwela, l.; sasamalo, m.; ticlla, m.; said, k.; mhalu, g.; chiryamkubi, m.; schindler, c.; reither, k.; gagneux, s.; fenner, l. title: prevalence and clinical significance of respiratory viruses and bacteria detected in tuberculosis patients compared to household contact controls in tanzania: a cohort study date: 2019-01-31 journal: clinical microbiology and infection doi: 10.1016/j.cmi.2018.03.019 sha: doc_id: 324601 cord_uid: s3rgxtg6 abstract objectives to describe the prevalence of respiratory pathogens in tuberculosis (tb) patients and in their household contact controls, and to determine the clinical significance of respiratory pathogens in tb patients. methods we studied 489 smear-positive adult tb patients and 305 household contact controls without tb with nasopharyngeal swab samples within an ongoing prospective cohort study in dar es salaam, tanzania, between 2013 and 2015. we used multiplex real-time pcr to detect 16 respiratory viruses and seven bacterial pathogens from nasopharyngeal swabs. results the median age of the study participants was 33 years; 61% (484/794) were men, and 21% (168/794) were hiv-positive. tb patients had a higher prevalence of hiv (28.6%; 140/489) than controls (9.2%; 28/305). overall prevalence of respiratory viral pathogens was 20.4% (160/794; 95%ci 17.7–23.3%) and of bacterial pathogens 38.2% (303/794; 95%ci 34.9–41.6%). tb patients and controls did not differ in the prevalence of respiratory viruses (odds ratio [or] 1.00, 95%ci 0.71–1.44), but respiratory bacteria were less frequently detected in tb patients (or 0.70, 95%ci 0.53–0.94). tb patients with both respiratory viruses and respiratory bacteria were likely to have more severe disease (adjusted or [aor] 1.6, 95%ci 1.1–2.4; p 0.011). tb patients with respiratory viruses tended to have more frequent lung cavitations (aor 1.6, 95%ci 0.93–2.7; p 0.089). conclusions respiratory viruses are common for both tb patients and household controls. tb patients may present with more severe tb disease, particularly when they are co-infected with both bacteria and viruses. tuberculosis (tb) caused by the bacterium mycobacterium tuberculosis affected an estimated 10.4 million new cases and caused 1.7 million deaths in 2016, making tb the leading global cause of death from an infectious disease [1] . influenza pandemics have selectively caused higher mortality among persons with tb * preliminary findings of this study were presented at (i) the 9th european compared to the general population [2, 3] . for instance, the 1918 influenza pandemic brought about a sharp decline in tb burden, possibly because of the higher mortality among tb patients coinfected with influenza viruses [3, 4] . in sub-saharan africa, hiv has been the most important risk factor driving the tb epidemic in recent decades [5] . the efforts towards understanding other risk factors in tbdsuch as respiratory viruses, helminths [6] , and bacteria [7] dare becoming increasingly important [8] . evidence from experimental mouse models suggests that respiratory viruses such as influenza viruses may play a pathogenic role in individuals with tuberculous disease by negatively affecting immunity against m. tuberculosis [9] . the effect of respiratory viruses on tb may mimic the development of bacterial pneumonia immediately after an infection with respiratory viruses [10] . studies of the lung microbiota (the community of microorganisms in the airways), which have focused on respiratory bacterial populations, suggest that there are differences in respiratory bacterial species populations among patients with and without tb, among new and recurrent tb patients, as well as among those in whom tb treatment has failed [7] . the differences in airway microorganism populations could indicate that respiratory pathogens can be involved in tb pathogenesis [11] . however, little is known about the prevalence of respiratory pathogens, whether viral or bacterial, and their role in clinical presentation in tb. we therefore studied the prevalence of respiratory pathogens in tb patients and household contact controls, and assessed the associations between both respiratory viruses and bacterial pathogens and the clinical presentation of tb patients who were prospectively recruited in an area of dar es salaam (tanzania) with a high tb burden. this is a prospective cohort study conducted in the densely populated temeke district of dar es salaam, tanzania; it is a study within a previously described ongoing prospective cohort study of tb patients and household contact controls in dar es salaam (tb-dar) [6] . between november 2013 and october 2015, we recruited smear-positive tb patients diagnosed at temeke district hospital and household contact controls who lived in the same household as the index tb cases [6] . assuming (a) a prevalence of respiratory viruses of 25% in tb patients and of 12.5% in household contact controls [12] , based on the prevalence of respiratory viruses in similar settings, and assuming that respiratory viruses are more frequent in tb patients than in controls, (b) a cluster correlation of 0.2, and (c) a nonparticipation rate of 20%, we estimated that 175 caseecontrol pairs would provide 85% power to observe a statistically significant difference in the prevalence at the 5% level of significance. at the time of recruitment of study participants, we collected a single sample of nasopharyngeal swabs from tb patients and controls using flexible nasopharyngeal flocked swabs (copan diagnostics, ca, usa) [13] . for tb patients, the nasopharyngeal swabs were taken immediately after diagnosis of tb and prior to initiation of anti-tb treatment. the nasopharyngeal swab samples were then added to 1-ml enat tubes for transportation at temperatures between 2 and 8 c to the ifakara health institute (ihi) research laboratory at bagamoyo where they were stored at e80 c pending analysis. we used the validated multiplex real-time pcrs from seegene (www.seegene.com/) for detection of a broad panel of respiratory viral and bacterial pathogens in accordance with the manufacturer's instructions, as previously published [14] . the nasopharyngeal swab samples were also analysed using anyplex ii rv16 simultaneously which detects 16 respiratory viruses, and the allplex respiratory panel 4 assay which detects seven respiratory bacterial pathogens (table s1 ). sample processing and analysis to detect respiratory pathogens were all done at the ihi research laboratory in accordance with the manufacturer's instructions and standard operating procedures (sop). tb confirmation was by positive l€ owensteinejensen (lj) solid media mycobacterial culture (done at the ihi research laboratory in bagamoyo). we ruled out tb in controls with both a negative gene xpert mtb/rif result and no mycobacterial growth in lj solid media culture. hiv testing for tb patients and controls was done as per tanzania hiv testing algorithms using an alere determine hiv (alere, usa) and a uni-gold hiv (trinity biotech; wicklow, ireland) confirmatory test rapid tests [15] . cd4þ t cells and full blood-cell counts were obtained as previously described [6] . clinical severity of tb was graded as per published clinical tb score [16] , but modified to a set of 12 tb score parameters instead of 13, since tachycardia was not systematically measured as previously noted [6] . diagnosis delay was defined as a cough duration of 3 weeks as previously published from the same cohort study [17] . data were captured using electronic case report forms developed from the open source data collection software open data kit (odk, https://opendatakit.org/) on android pc tablets, and data were then managed using the emanagement tool 'odk_planner' as published previously [18] . we compared the baseline characteristics of tb patients and household contact controls using the mcnemar test, paired t-test, or wilcoxon signed rank test, as appropriate. we estimated the prevalence of any respiratory viruses and bacteria using logistic regression models adjusting for clustering at the household level. we used mixed-effects logistic regression models with random household intercepts to assess the risk factors associated with detection of respiratory pathogens in tb patients and controls. the differences in the mean ct values of respiratory bacteria detected (as a relative measure of the bacterial load) between tb patients and controls were assessed using mixed-effect linear regression models. logistic regression models adjusting for age, sex, and hiv infection were used to assess the associations between respiratory pathogens and clinical presentations of tb at the time of recruitment among tb patients, with the following outcome variables: severe tb score (score of 6) versus mild (score of 1e5), high sputum bacterial load (sputum afb smear microscopy of 2þ) versus low bacterial load, and presence versus absence of lung infiltrations and cavitations (chest x-ray findings). associations were expressed as crude odds ratios (ors) and adjusted ors (aors) with their corresponding 95% confidence intervals (95%cis). all analyses were performed in stata version 14.0 (stata corp, college station, texas, usa). the study was approved by the ihi institutional review board (ihi/irb/no: 04-2015) and the medical research coordinating committee of the national institute of medical research (nimr/ hq/r.8c/vol.i/357) in tanzania, as well as by the ethics committee of the canton of basel in switzerland (eknz ube-15/42). all study participants gave written informed consent. tb patients were managed as per national tb and leprosy programme treatment guidelines [19] . treatment and care for hiv-positive individuals were as per tanzania national hiv/aids treatment guideline [15] . between november 2013 and october 2015, 972 study participants were enrolled in the tb-dar study. a total of 794 study table 1) . the frequency distributions of the respiratory viruses detected from study participants are summarized in table 2 . the overall prevalence of any respiratory virus among tb patients and controls was 20.4% (161/794; 95%ci 17.7e23.3%), and the odds of detecting any virus was the same in tb patients and controls (or 1.00, 95%ci 0.71e1.44; p 0.98). the most common respiratory species detected was human rhinovirus a/b/c (hrv), which was found in 9.3% (74/ 794) of the study participants, followed by influenza a in 3.1% (25/ 794) and respiratory syncytial virus a (rsva) in 1.9% (15/794). we detected only minor differences between tb patients and household contact controls in the prevalence (table 2 ) and the semiquantitative detection (fig. s1 ) of respiratory viruses. we detected respiratory viruses more frequently during the months of march and april, and october to november (fig. s2 ). the prevalence of any bacterial pathogen among study participants was 38.2% (303/794; 95%ci 34.9e41.6%, table 2 ). respiratory bacteria were less likely be detected in tb patients than in controls (or 0.70, 95%ci 0.53e0.94; p 0.02). the most common bacterial species detected were haemophila influenzae, found in 26.1% of study participants (207/794), followed by streptococcus pneumoniae in 21.5% (171/794). tb patients were less likely than household contact controls to have h. influenzae (or 0.62, 95%ci 0.45e0.86; p 0.004). the mean values of cycle threshold for tb patients were slightly higher (indicating a smaller bacterial load) than those of household contact controls (greater bacterial load), but this did not reach conventional levels of statistical significance (fig. s3 ). there were 12 tb patients on a retreatment drug regimen, and in ten of them (83.3%) both respiratory bacteria and respiratory viruses were detected. the factors associated with detection of respiratory viruses include smoking and households containing three or more people. men were the more likely to have respiratory bacteria (see table s3 ). tb patients with both viral and bacterial pathogens had significantly more severe tb disease than tb-only patients (aor 1.64, 95% table 3 ). bacterial respiratory pathogens were not significantly associated with the clinical presentation of tb patients at tb diagnosis. detection of respiratory pathogens was not associated with including diagnostic delay (defined as duration of symptoms of 3 weeks or more) (see table 3 ). no association was found between detection of respiratory pathogens and chest x-ray findings among tb patients (table 4 ). in addition, the detection of respiratory pathogens was similar for hiv-positive and hivnegative tb patients (tables s2 and s3 ). both respiratory viruses and respiratory bacteria are commonly detected in a high-tb-incidence setting, and the prevalence of respiratory bacteria was lower in tb patients than in household contact controls. detection of respiratory viruses and respiratory bacteria in tb patients was associated with more severe disease. the prevalence of any respiratory viruses was the same (20%) for both tb patients and controls without tb. similar prevalence of respiratory viruses in controls as compared to tb patients could be due to controls being more active than tb patients, hence having increased social contacts. the prevalence shown in our study was lower than that reported in an indonesian study of influenza viruses that observed respective prevalences of around 46% and 41% for tb patients and controls, respectively [20] . the difference in the prevalence of influenza in that study compared to ours could be due to the different study region (asia versus sub-saharan africa) and the use of different diagnostic methods (immunological assay versus molecular detection). we detected respiratory viruses from nasopharyngeal swabs using a highly sensitive and specific molecular technique [21] , whereas the study from indonesia [20] measured influenza virus antibody titres which also detect patients with previous exposure to influenza viruses. the influenza antibody titres were higher in tb patients than in controls, suggesting recent viral infection before the clinical manifestations of tb [20] . we did not find any evidence for an association between hiv infection and detection of respiratory pathogens. in line with our results, a household study on respiratory illness surveillance and hiv testing in kenya [22] did not find any association between hiv and influenza viruses. however, household contacts of the hivinfected influenza index cases were twice as likely to develop a secondary case of influenza-like illness [22] . we also found that the presence of both viruses and bacteria could potentially alter the clinical course of tb, and present with a more severe disease as measured by the previously validated clinical tb score [16] . direct evidence of clinical effects of respiratory viruses on tb have only been demonstrated in experimental mouse models that have exhibited higher mycobacterial loads in the lungs and increased lung inflammation [9] . the immunological pathway responsible for more severe clinical presentation in tb patients may occur either via the type i interferon-receptordependent pathway [9] or via decreased mhc ii expression on dendritic cells [23] , which may result in poor clearance of m. tuberculosis from the lungs. we found smoking and living in a household with three or more persons to be risk factors for respiratory viruses. smoking has been shown, at least in animal models, to inhibit the pulmonary t-cell response to influenza viruses, thus increasing susceptibility to infection [24] , and respiratory viruses were more likely to be detected in children living with a smoker [25] . in addition, overcrowdingdwhich we defined as three or more persons in a householddis a common risk factor for most airborne pathogens such as m. tuberculosis [26] and respiratory viruses [22] . the prevalence of bacterial respiratory pathogens in our study was lower in tb patients than in household contact controls, suggesting interactions between m. tuberculosis and the bacterial populations in the airways. overall respiratory bacterial load was smaller in tb patients than in controls. this is similar to findings from a microbiota study which reported smaller bacterial loads in tb patients than in controls [27] . the authors argue that the initial phase of m. tuberculosis invasion in the lungs may prompt an immune response that could also reduce the commensal flora in the lower respiratory tract [27] . interestingly, in a mouse model, m. tuberculosis infection in the lungs appeared also to have an effect on the gut microbiota, which is part of the collective human microbiota [28] . these findings consistently suggest interactions between m. tuberculosis and the communities of microorganisms, and a role for these interactions in tb pathogenesis. we believe that this is the first study to have looked systematically at a wide range of viral and bacterial species in tb patients and controls, and using sensitive molecular techniques and clinical specimens from a well-defined compartment of the airways. a particular strength of the study is that potentially confounding and unmeasured risk factors were minimized by studying patients and controls who lived in the same households. a limitation of the study is its undifferentiated attention to respiratory viruses because of small numbers which precluded assessment of the clinical effects of individual viruses. however, we presume that all respiratory viruses have similar levels of immunomodulation, and thus we could combine all respiratory viruses together. in conclusion, respiratory pathogens are common in the high-tb setting of tanzania for both tb patients and household contact controls without tb. however, respiratory bacterial species were more frequently detected in household contact controls than in tb patients. our findings suggest that tb patients co-infected with both respiratory viruses and respiratory bacteria have severe tb disease. further research should focus on the pathogenic role of respiratory pathogens in high-tb-incidence settings and their effects on clinical and treatment outcomes. all authors have declared no conflicts of interest. this work was supported by the rudolf geigy foundation, basel, switzerland (to lf and fm), and a personal grant by the 'amt für ausbildungsbeitr€ age', basel, switzerland (to fm). world health organization testing the influenzaetuberculosis selective mortality hypothesis with union army data influenza pandemics and tuberculosis mortality in 1889 and 1918: analysis of historical data from switzerland the 1918 influenza pandemic hastened the decline of tuberculosis in the united states: an age, period, cohort analysis who. global tuberculosis report prevalence and clinical relevance of helminth co-infections among tuberculosis patients in urban tanzania sputum microbiota associated with new, recurrent and treatment failure tuberculosis tuberculosis comorbidity with communicable and non-communicable diseases: integrating health services and control efforts influenza a virus impairs control of mycobacterium tuberculosis coinfection through a type i interferon receptor-dependent pathway respiratory viruses in exacerbations of chronic obstructive pulmonary disease requiring hospitalisation: a caseecontrol study the human microbiome in the fight against tuberculosis epidemiology of respiratory syncytial virus infection in rural and urban kenya copan's patented flocked swabs evaluation of a novel real-time rt-pcr using toce technology compared with culture and seeplex rv15 for simultaneous detection of respiratory viruses national guidelines for the management of hiv and aids tb score: signs and symptoms from tuberculosis patients in a low-resource setting have predictive value and may be used to assess clinical course diagnostic delay and associated factors among patients with pulmonary tuberculosis in dar es salaam managing research and surveillance projects in real-time with a novel opensource emanagement tool designed for under-resourced countries manual for the management of tuberculosis and leprosy the influence of influenza virus infections on the development of tuberculosis respiratory viruses in patients with influenza-like illness in senegal: focus on human respiratory adenoviruses the role of hiv in the household introduction and transmission of influenza in an urban slum influenza a virus infection impairs mycobacteria-specific t cell responses and mycobacterial clearance in the lung during pulmonary coinfection exposure to cigarette smoke inhibits the pulmonary t-cell response to influenza virus and mycobacterium tuberculosis risk factors for virus-induced acute respiratory tract infections in children younger than 3 years and recurrent wheezing at 36 months follow-up after discharge prevalent infectious tuberculosis in harare, zimbabwe: burden, risk factors and implications for control complex sputum microbial composition in patients with pulmonary tuberculosis aerosol mycobacterium tuberculosis infection causes rapid loss of diversity in gut microbiota we would like to thank all the study participants whose data were used in this study. we are most grateful for the assistance and support of the district medical officer, medical officer in charge, hospital staff, the district tb coordinators in the temeke district, dar es salaam, tanzania, and the national tuberculosis and leprosy programme. we also thank the ifakara health institute study team working at the temeke district hospital for recruiting study participants. supplementary data related to this article can be found at https://doi.org/10.1016/j.cmi.2018.03.019. key: cord-342649-ysossker authors: scagnolari, carolina; midulla, fabio; selvaggi, carla; monteleone, katia; bonci, enea; papoff, paola; cangiano, giulia; marco, paola di; moretti, corrado; pierangeli, alessandra; antonelli, guido title: evaluation of viral load in infants hospitalized with bronchiolitis caused by respiratory syncytial virus date: 2012-03-10 journal: med microbiol immunol doi: 10.1007/s00430-012-0233-6 sha: doc_id: 342649 cord_uid: ysossker the relationship between viral load, disease severity and antiviral immune activation in infants suffering from respiratory syncytial virus (rsv)-associated bronchiolitis has not been well identified. the main objective of this study was to determine the existence of a correlation between rsv load and disease severity and also between different clinical markers and mrna levels of the interferon stimulated gene (isg)56 in infants hospitalized for bronchiolitis. we also evaluated whether viral load tended to be persistent over the course of the rsv infection. the levels of rsv-rna were quantified in nasopharyngeal washings, collected from 132 infants infected with rsv as a single (90.15%) or as a dual infection with other respiratory viruses (9.85%). results indicated that viral load was positively related to the clinical severity of bronchiolitis, the length of hospital stay, the levels of glycemia and the relative gene expression of isg56, whereas an inverse correlation was observed with the levels of hemoglobin. we also found that the rsv load significantly decreased between the first and second nasopharingeal washings sample in most subjects. these results suggest that infants with high rsv load on hospital admission are more likely to have both more severe bronchiolitis and a higher airway activation of antiviral immune response. bronchiolitis is an acute infection of the respiratory tract and is mainly associated with respiratory syncytial virus (rsv) [1] . the clinical spectrum of rsv bronchiolitis in previously healthy infants is extremely variable, ranging from mild upper respiratory symptoms to severe respiratory distress and, occasionally, death [2] . the factors determining severity of rsv-associated bronchiolitis have not been clearly established and are still unknown and are likely to be determined by a combination of host and viral factors. host-derived risk factors associated with severe bronchiolitis include young age (<6 months), premature birth (<35 weeks of gestation), low birth weight, immunodewciency or immunosuppression status, and congenital heart or chronic lung disease [2] [3] [4] . in addition single-nucleotide polymorphism in, and expression of, some genes codifying proteins involved especially in the control of innate immune response have been reported as being associated with the rsv disease severity [5] [6] [7] . on the other hand, the inxuence of viral factors such as the rsv-rna levels on determining the bronchiolitis severity is not fully addressed. to this regard, some studies revealed signiwcant association between disease severity and rsv load in nasopharyngeal secretion of infants with primary respiratory tract infection [8] [9] [10] [11] . in contrast, other failed to found a correlation between bronchiolitis severity and viral load during primary rsv infection [12] [13] [14] . furthermore, a recent study found that the correlation between respiratory disease severity and viral load was the highest in case of single rsv infection but was absent in rsv coinfection, suggesting that infants with rsv as the primary pathogen infection were more severely ill [15] . further, the clinical signiwcance of double infections of rsv and other respiratory viruses also appears to be unclear, and there are conxicting studies regarding the evect of coinfection on disease severity [16] [17] [18] . in the framework of a study aimed at understanding the pathogenesis of rsv infection and at further characterizing viral and host factors involved in determining the severity of bronchiolitis, we addressed whether any diverences in rsv-rna levels in the airway tracts of infants with bronchiolitis might explain the broad clinical spectrum of rsvassociated bronchiolitis. in addition, we evaluated the airway innate immune response by measuring the mrna levels of gene coding for a cytoplasmatic antiviral protein, strongly induced in the lung after interferon (ifn) production or virus challenge [19] , namely the ifn stimulated gene 56 (isg56). we, then, evaluated whether rsv load variations in bronchiolitis severity were correlated to level of isg56-mrna and whether viral load tended to be persistent over the course of the disease. a total of 132 infants with a clinical diagnosis of rsvassociated bronchiolitis, admitted over four successive winter seasons (2006) (2007) (2008) (2009) (2010) to the pediatric department of policlinico umberto i hospital, were included in this study. the institutional review board at sapienza university of rome approved the study. written informed consent was obtained from the children's parents. bronchiolitis was diagnosed from the presence of a history of upper respiratory tract infection followed by acute onset of respiratory distress with cough, tachypnea, retraction and divuse crackles on auscultation (wheezing alone was not considered suycient cause for inclusion in the study). the exclusion criteria were underlying chronic condition (such as premature birth, cystic wbrosis, chronic pulmonary disease, congenital heart disease, and immunodewciency) and recurrent (more than one) wheezing episodes [17] . disease severity and clinical evolution were evaluated using the clinical score index described by midulla et al. [17] . in particular, on admission to hospital, a clinical severity score was assigned to each infant within the range 0-8, based on respiratory rate (<45/m = 0, 45-60/m = 1, >60/m = 2) arterial oxygen saturation in room air (>95% = 0, 95-90% = 1, <90% = 2), presence of retractions (none = 0, present = 1, present + nasal xare = 2), and ability to feed (normal = 0, reduced = 1, endovenous = 2). nasopharingeal washings (npw) were collected from 132 infants suvering from bronchiolitis in the wrst 24 h after admission to hospital and an aliquot was tested for viruses as previously described [20] . a subset of 56 npw samples was centrifuged at 2,000 rpm for 10 min and each cell pellet was resuspended in 1 ml of phenol and guanidine isothiocyanate reagent (trizol, gibco brl, ny) and frozen at ¡80°c for subsequent isg56 gene expression analysis. pcr assays for respiratory viruses a panel of reverse transcription (rt) pcr or nested pcr assays, some in a multiplex format, was used for detection of fourteen respiratory viruses including: rsv, inxuenza a and b, coronaviruses, oc43, 229 e, nl63, hku1, metapneumovirus, adenovirus, rhinovirus, parainxuenza 1-3, and bocavirus as previously reported [20, 21] . taqman-based real-time rt-pcr technique for rsv detection a taqman-based real-time pcr technique for rsv-rna quantiwcation was performed on all npw specimens with positive rt-pcr results for rsv. briexy, viral rna was extracted from npw specimens that were positive for rsv using a qiaamp viral rna mini kit (qiagen spa, milan, italy). the rna was dissolved in rnase-free water and the rsv quantiwcation was performed by taqman assay after generation of cdna using a high capacity cdna archive kit (applied biosystems, monza, italy). type-speciwc primers and probes for n gene of both rsv a and b [22] were added to the universal pcr master mix (applied biosystems) at 300 and 100 nm, respectively, in a wnal volume of 50 l. the standards were obtained by cloning the 82 bp of viral n gene into the pcr2.1 plasmid using a topo ta cloning kit (in vitrogen corporation, san diego, ca, usa). a linear distribution (r = 0.99) was obtained between 10 1 and 10 8 copies of rsv-dna. viral load values were log transformed for analysis and data was expressed as the log number of rsv copies per ml of npw. the mrna copy content of isg56 was measured by a realtime 5ј exonuclease rt-pcr assay using the abi 7000 sequence detector (applied biosystems). briexy, the total cellular rna was extracted from the cells using the trizol reagent, following the manufacturer's instructions, and was retro transcribed as previously described [23] . primers and probes for isg56 gene were added to the universal pcr master mix (applied biosystems) at 300 and 200 nm, respectively, in a wnal volume of 50 l [23] . co-ampliwcation of the beta-glucuronidase gene (assay-on-demand, hs99999908_m1, applied biosystems) was used to normalize the amount of total rna present using the threshold cycle relative quantiwcation according to the supplier's guidelines. descriptive analysis was made using median (range) or frequency (percentage). rsv load values were expressed as log copy number of rsv-rna/ml and related geometric mean. diverences between infants with rsv as single or dual infection, in terms of the level of viral load, were compared using the mann-whitney test. spearman's rho coeycient was calculated in order to assess the correlation between the level of rsv load and the demographic and clinical parameters, and isg56-mrna levels measured in cells collected from npw. the diverences in the number of rsv-positive infants characterized by the presence or absence of retractions and/or nasal xare according to the rsv load levels were evaluated using the chi squared test for trend. diverences in the rsv load between the wrst and second npw sample were compared using the wilcoxon test. signiwcance was wxed at the 5% level. the analysis was performed using spss v.13.0 for windows. one hundred thirty-two infants diagnosed with bronchiolitis over a period of 4 years were included ( table 1 ). the median age of the infants was 2.20 months, of which 78.69% had <6 months, and the sex ratio was 1. a total of 119 (90.15%) infants carried a single rsv infection whereas 13 (9.85%) had a coinfection with other respiratory viruses. the median clinical score level was 4; 22 (16.7%) infants had a severe bronchiolitis with a clinical score ¸7; 48 infants (36.4%) required oxygen supplementation and 37 infants (28.0%) had retraction with nasal xaring. an rt-taqman assay was used to determine rsv load (log copy number of rsv-rna/ml) in npw collected from all 132 infants with bronchiolitis. there was a distribution of viral load values of several orders of magnitude in infants with bronchiolitis (fig. 1) . the log copies of rsv-rna per ml ranged from 1.68 to 8.93 and the median viral load values was 5.47. the distribution of rsv load showed that half of infants have a copy number of rsv-rna per ml within the range of 5.47-8.93 log. when the rsv-positive infants were divided on the basis of rsv detection as a single infection or as a coinfection, the viral load was not signiwcantly diverent between groups (p = 0.49). the relationship between patient data as independent variables and rsv-rna levels measured in npw was analyzed (table 2) . a positive correlation between log number of rsv-rna copies/ml and clinical score index was found in infants with rsv infection (r = 0.17, p = 0.024). in particular, rsv load seems to inxuence the severity of respiratory disease. indeed, when rsv loads were categorized into three groups ranked from lowest to highest values and analyzed in function of the presence or absence of retractions and/or nasal xaring, we observed that the number of infants who have both the highest rsv load values (>6.84 log) and the presence of intercostal retractions and nasal xaring was higher with respect to the other groups (p = 0.020; fig. 2 ). furthermore, we found a signiwcant correlation between rsv viral load and the length of hospital stay (r = 0.16, p = 0.038), and levels of hemoglobin (r = ¡0.18, p = 0.024) or glycemia (r = 0.24, p = 0.028). in contrast, we failed to detect any correlation between the rsv-rna levels and age or weight of tested infants, numbers of neutrophils, lymphocytes, eosinophils or platelets, and levels of sodium, or c-reactive protein ( table 2) . also no diverence was detected in viral load for male and female and between infants with fever or without fever (data not shown). in an attempt to determine whether rsv load inxuences gene expression of a well-known antiviral protein such as isg56, levels of rsv-rna were examined for any signiwcant correlation with expression of isg56-mrna. an rt-taqman assay speciwc for isg56 transcript was performed on the npw samples from a subset of infants with single rsv infection (n = 56), for which we collected cells. a sig-niwcant positive correlation (r = 0.35, p = 0.008) was observed between viral load and levels of isg56-mrna (fig. 3) . we also evaluated changes in rsv load in a subset of npw samples collected from 17 infants at the time of admission and at hospital discharge, after a median of 2 days (range 1-15 days). fourteen had single rsv infection and 3 had double infection (2 metapneumovirus; 1 rhinovirus). results indicated that rsv load decreased signiwcantly (p = 0.012) in most infants (71%) between the wrst (median log copies of rsv-rna/ml: 5.95) and second npw samples (median log copies of rsv-rna/ml: 3.22) independently of the presence of rsv as single or dual infection. in particular, a decline of rsv-rna levels of at least 2.7 log was observed in half of infants who have a viral load decrease, whereas in the remaining infants the log copy number of rsv/rna per ml decreased within the range of 1.76-0.40. in contrast no change in viral load, and an increase in copy number of rvs-rna copies/ml within the range of 0.18-4.65 log, were observed respectively in one and four infants. this study showed that level of rsv load, measured shortly after hospital admission, was highly variable among infants suvering from bronchiolitis in agreement to several other studies [9, 10, 12] . in addition we found that coinfection of rsv and other respiratory viruses did not change signiwcantly rsv load although it has been reported that rsv levels were higher in infants with rsv as the primary pathogen that in infants with rsv coinfection [15, 18] . interestingly, our results demonstrated a positive correlation between rsv load and the clinical score index used for determining the severity of bronchiolitis. in particular, we found that in infants with high levels of rsv-rna in their npw, there is a tendency to have more signs of severe respiratory distress. an association between high rsv load and the respiratory disease severity has been reported in previous papers [8] [9] [10] [11] , whereas other studies have failed to detect such an association [12] [13] [14] . studies that did not wnd this correlation may have been confounded by the presence in children of risk factors such as cardiopulmonary disease or of previous rsv infections, or by the use of diverent molecular or virological techniques for rsv level evaluation [12] [13] [14] . the conxicting results could be also explained considering the diverent bronchiolitis diagnostic criteria and/or clinical scores used in the various studies [8] [9] [10] 12] . furthermore, we demonstrated that rsv load was positively correlated with the duration of hospital stay, an indirect indicator of the severity of rsv-associated illness. in agreement, other studies have reported higher nasal rsv load as a signiwcant independent predictor of longer hospitalization [9, 24] . however, in 23% of the infants in which we could measure rsv-rna level at hospital discharge, an increase of viral load has been recorded. in addition, our results indicate that viral load correlates with glycemic levels and hemoglobin values but not with other clinical characteristics such as c-reactive protein levels or leukocytes count as reported recently by franz a and coauthors [25] . the mechanism by which rsv load avects levels of glycemia and hemoglobin is currently unknown. however it has been reported that hyperglycemia is frequent in children with rsv infection [26] . furthermore, recently anemia has been found to avect the higher rate of thrombocytosis observed in infants with rsv-associated bronchiolitis [27] . the apparent discrepancies along our data on the dynamic of rsv load in bronchiolitis, make more evident the complexity of the rsv-host interactions and suggest the need of evaluating also host factors for fully understanding the pathogenesis of rsv infection. directly related to this, it is well established that the clinical spectrum of bronchiolitis might rexect diverences in the prowle and extent of immunological and/or inxammatory mediators produced by rsv infected respiratory epithelial cells [6, 28, 29] . here, we demonstrated for the wrst time to our knowledge that a high rsv-rna level correlates with high levels of isg56-mrna in the airways of infants suvering from bronchiolitis. this protein has been implicated in the suppression of viral replication and protein translation and it seems also to be a mediator of negative-feedback regulation of virus-triggered induction of type i ifn and cellular antiviral response [30] . interestingly it has been demonstrated that the nonstructural ns1 and ns2 proteins of rsv appear to antagonize both the cellular antiviral response as well as the induction of ifn transcription [31] [32] [33] . in addition ling and coauthors have showed that ns2 inhibited isg56 promoter activity induced by rna-activated rig-i in a dosedependent manner [34] . however, although rsv has evolved accessory gene products that neutralize or inhibit various steps of the ifn pathway, in our previous study we found a strong and a coordinated induction of ifn stimulated genes in the airway tract of infants with rsv infection [28] . furthermore recently pichlmair and coauthors have proposed that isg56 protein antagonizes viruses by sequestering speciwc viral nucleic acids [35] . therefore our results suggest that quantitative diverences in the replication of rsv may inxuence the magnitude of innate immune activation which in turn can determine the clinical course of bronchiolitis in either benewcial or detrimental direction as previously suggested [5, 28] . a potential limitation of this study was that we did not perform the evaluation of known pathogenic bacteria to explore whether the presence of bacterial superinfection avects levels of rsv-rna. indeed, considering that infants hospitalized for bronchiolitis have low reported rates of serious bacterial illness [36] , we have collected only npw and not well established respiratory samples such as those from lower respiratory tract, which are likely most suitable for performing isolation and identiwcation of bacteria. in addition, although the marked elevation of c-reactive protein observed in some of the rsv-positive infants could hints to bacterial superinfection, no signiwcance diverence in leukocyte count as well as in chest x rays signs was found among infants who have elevated c-reactive protein levels and those with normal c-reactive protein values suggesting that globally there is no concomitant bacterial superinfection (data not shown). furthermore, recently it has been demonstrated that c-reactive protein levels could be avected by the presence of respiratory viral infection [25] . in conclusion, our results showed that rsv load has a signiwcant impact on the clinical and immunological parameters of hospitalized infants suvering from bronchiolitis and underline the importance of implementing diagnostic tools for a rapid, sensitive and quantitative rsv detection in order to improve the clinical management of bronchiolitis. further larger scale studies are needed to support the predictive value of measuring rsv load on the clinical course of bronchiolitis and to establish the actual "cutov value" of rsv load associated to disease severity. respiratory syncytial virus and parainxuenza virus acute viral bronchiolitis in children-a very common condition with few therapeutic options susceptibility to bronchiolitis in infants incidence and predisposing factors for severe disease in previously healthy term infants experiencing their wrst episode of bronchiolitis human genetic factors and respiratory syncytial virus disease severity viral and host factors in human respiratory syncytial virus pathogenesis genetic determinants of severe respiratory syncytial virus infection and post-bronchiolitis wheeze: a systematic review nasal quantity of respiratory syncytical virus correlates with disease severity in hospitalized infants respiratory syncytial virus load predicts disease severity in previously healthy infants respiratory syncytial virus infections in hospitalized infants: association between viral load, virus subgroup, and disease severity clinical disease and viral load in children infected with respiratory syncytial virus or human metapneumovirus illness severity, viral shedding, and antibody responses in infants hospitalized with bronchiolitis caused by respiratory syncytial virus type 1 and type 2 cytokine imbalance in acute respiratory syncytial virus bronchiolitis quantitation of respiratory viruses in relation to clinical course in children with acute respiratory tract infections disease severity and viral load are correlated in infants with primary respiratory syncytial virus infection in the community dual infection of infants by human metapneumovirus and human respiratory syncytial virus is strongly associated with severe bronchiolitis respiratory syncytial virus, human bocavirus and rhinovirus bronchiolitis in infants multiple versus single virus respiratory infections: viral load and clinical disease severity in hospitalized children tissue-speciwc and inducer-speciwc diverential induction of isg56 and isg54 in mice detection and typing by molecular techniques of respiratory viruses in children hospitalized for acute respiratory infection in human bocavirus infection in hospitalized children in italy simultaneous detection, subgrouping, and quantitation of respiratory syncytial virus a and b by real-time pcr the synergistic interaction of interferon types i and ii leads to marked reduction in severe acute respiratory syndrome-associated coronavirus replication and increase in the expression of mrnas for interferon-induced proteins correlation of viral load as determined by real-time rt-pcr and clinical characteristics of respiratory syncytial virus lower respiratory tract infections in early infancy correlation of viral load of respiratory pathogens and co-infections with disease severity in children hospitalized for lower respiratory tract infection glycemic level in mechanically ventilated children with bronchiolitis respiratory syncytial virus-positive bronchiolitis in hospitalized infants is associated with thrombocytosis upregulation of interferon-induced genes in infants with virusassociated acute bronchiolitis immunopathogenesis of respiratory syncytial virus bronchiolitis isg56 is a negative-feedback regulator of virus-triggered signalling and cellular antiviral response suppression of the induction of alpha, beta, and lambda interferons by the ns1 and ns2 proteins of human respiratory syncytial virus in human epithelial cells and macrophages respiratory syncytial virus nonstructural proteins ns1 and ns2 mediate inhibition of stat2 expression and alpha/beta interferon responsiveness respiratory syncytial virus nonstructural protein 2 speciwcally inhibits type i interferon signal transduction human respiratory syncytial virus nonstructural protein ns2 antagonizes the activation of beta interferon transcription by interacting with rig-i ifit1 is an antiviral protein that recognizes 5ј-triphosphate rna bronchiolitis: in-patient focus acknowledgments this work was supported by grants to g.a from pasteur institute (cenci bolognetti foundation; title of the project: "molecular characterization of viruses causing bronchiolitis and study of viral and host factors avecting type i ifn antiviral response induced by respiratory viruses"), and to a.p. from "sapienza" università di roma (fondi ricerche universitarie) and italian ministry of health (ricerca finalizzata conv. no. 88). key: cord-332055-lrpfzsog authors: devos, elizabeth; jacobson, lisa title: approach to adult patients with acute dyspnea date: 2015-11-27 journal: emerg med clin north am doi: 10.1016/j.emc.2015.08.008 sha: doc_id: 332055 cord_uid: lrpfzsog undifferentiated patients in respiratory distress require immediate attention in the emergency department. using a thorough history and clinical examination, clinicians can determine the most likely causes of dyspnea. understanding the pathophysiology of the most common diseases contributing to dyspnea guides rational testing and informed, expedited treatment decisions. respiratory distress is responsible for nearly 4 million emergency department (ed) visits each year and is one of the most common presenting complaints in the elderly. 1 when a patient presents with dyspnea, the primary task of the emergency physician is to assess for and ensure stability of the patient's airway, breathing, and circulation. in the case of dyspnea, presentations may range from minor symptoms to extremis. rapid assessment may necessitate the use of intubation, bipap, nebulizations, decompression, or other therapies in the immediate period following the patient's arrival, to treat dyspnea. the american thoracic society suggests that "dyspnea results from a . mismatch between central respiratory motor activity and incoming afferent information from receptors in the airways, lungs and chest wall structures." 2 this dissociation can result from increased metabolic demand, decreased compliance, increased dead-space volume, or many other disorders that are discussed later. each patient presenting short of breath uses a different set of phrases to describe the symptoms and examination reveals a different combination of disorders. the clinician's ability to interpret these varying constellations is necessary to provide appropriate treatment to these patients, who are often in serious distress. acute dyspnea, or shortness of breath, is one of the most common chief complaints in the ed. the differential diagnosis includes many disorders that can be divided based on obstructive, parenchymal, cardiac, and compensatory features. a careful history can begin to narrow this wide differential. in addition to common symptoms, consider risk factors such as past medical and family history, trauma, travel, medications, and exposures. schwartzstein and lewis 3 use the analogy of a machine to identify different causes of dyspnea based on pathophysiologic data. dysfunctions of the respiratory system may be caused by faulty controllers, ventilatory pumps, or gas exchangers ( table 1) . this table makes it easier to understand the causes of shortness of breath related to respiratory causes. cardiovascular disease manifests as dyspnea by causing disruptions of the system that pumps oxygenated blood to tissues and then transports the carbon dioxide back to the lung. decreases in cardiac output or increases in resistance limit oxygen delivery. similarly, decreased oxygen carrying capacity in anemia plays a role in its presentation with dyspnea. a detailed physical examination also provides important guidance ( table 2) . respiratory rate and oxygen saturation are obtained with vital signs. the clinician should assess the patient's work of breathing, looking for any tripoding or retractions. crepitance in the chest may indicate subcutaneous air and pneumothorax. lung sounds such as wheezing, rales, and rhonchi further guide the differential. decreased sounds, hyperresonance, or egophony may also provide additional clues. jugular venous distension, s3 gallop, and peripheral edema indicate that a patient has fluid overload. conjunctival pallor, capillary refill, and temperature of extremities can provide clues about blood volume and general circulation. pulses must also be assessed. multiple tests are available to narrow the differential diagnosis of acute dyspnea. when using tests to augment clinical decision making, be sure to weigh the information they may provide with any risks involved in performing the tests ( table 3) . ultrasonography provides valuable information about the origin of symptoms, and, often, diagnosis in the initial assessment of an acutely dyspneic patient. these images may be obtained during or shortly after initial assessment, potentially other protocols include assessments to assess for other cardiac causes of dyspnea. [4] [5] [6] focused evaluation of global left ventricular function, diastolic function, right ventricular size, and any pericardial effusion facilitates rapid assessment for massive myocardial infarction, cardiac tamponade, and massive pulmonary embolism at the bedside. in addition, inferior vena cava measurement can be used to assess for right-sided heart failure and to estimate central venous pressure. computed tomography (ct) use to evaluate acute dyspnea has increased in the last decade. 7 risks include contrast reactions and nephropathy as well as radiationinduced cancers. 8 recent american college of physicians recommendations advocate avoidance of ct as an initial test to evaluate patients at low risk for pulmonary embolism (pe). 9 further, nearly one-fourth of patients undergoing ct for pe evaluation assess for lung sliding anterior absence of lung sliding occurs with a disruption of the normal sliding of viscera on parietal pleura or separation of the two. in m mode, absence of lung slide is seen as the stratosphere sign (also known as bar-code sign) absence of lung sliding in the presence of a lines necessitates search for pneumothorax. lung point is the ultrasonography finding in which lung slide is seen in the same view with the abolished lung slide and a lines in the same location, indicating the tip of the lung have clinically significant incidental findings. although ct may provide vital diagnostic information, clinicians must not only consider the scan's necessity but also plan appropriate follow-up for any clinically important incidental findings. 10 always consider whether ct is necessary or whether less risky modalities, such as chest radiograph or ultrasonography, will answer pertinent questions. adult patients with acute dyspnea consider the 35-year-old woman discussed earlier. medics report tachypnea with very poor air movement during transport. as she rolls through the ambulance bay doors, you are already assessing her. adept clinicians can spot respiratory distress from across the room. she is diaphoretic, her shoulders are held adjacent to her ears, and she is breathing extremely rapidly with minimal air movement. you decide devos & jacobson to aggressively treat her for a severe asthma exacerbation, starting bipap ventilation with continuous nebulized albuterol and order adjunct therapies including intravenous steroids, intravenous magnesium, and intramuscular epinephrine. after 20 minutes at her bedside, she begins to breathe more comfortably with the bipap machine and repeat auscultation reveals diffuse wheezing and improved air movement. as she begins to improve, ems returns with another patient. his breath sounds are audible to everyone in the resuscitation bay. he appears diaphoretic and panicked. examination reveals stridor, periorbital edema, tachycardia, and hypotension. immediate intervention for anaphylactic shock begins and, after 2 rounds of epinephrine, fluid boluses, antihistamines, and steroids, he too begins to look better. wheezing, or musical respiratory sounds, typically result from partial airway obstruction. 11 because this obstruction can result from inflammation, secretions, or even a foreign body, patients with noisy or whistling breathing need close evaluation to devos & jacobson determine whether the noise is inspiratory or expiratory, and whether it is from the lower airways or the upper airways. stridor from a swollen airway, foreign body, or other airway obstruction is imminently dangerous. although patients in anaphylaxis may benefit from the nebulized beta-agonist treatment used to treat an asthma exacerbation, it is not sufficient to save their lives. as opposed to wheezing, which is a lower airway expiratory sound, stridor is an upper airway sound transmitted when there is obstruction to the inflow of air during inspiration. the obstruction may be fixed (food bolus; fig. 10 ) or inflammatory (anaphylaxis), but in any situation must be emergently managed. national and world organizations define asthma "by the history of respiratory symptoms such as wheeze, shortness of breath, chest tightness and cough that vary over time and in intensity, together with variable expiratory airflow limitation." 12 the reversibility of airflow obstruction is the hallmark distinguishing asthma from other obstructive respiratory disorders. in contrast, chronic obstructive pulmonary disease (copd)/ emphysema is defined as "persistent airflow limitation that is usually progressive and associated with enhanced chronic inflammatory responses in the airways and the lungs." 12 these patients also frequently wheeze, but may have a different course of acute and chronic disease. table 5 highlights the differences between these similar, at times overlapping, diseases. asthma is an obstructive disease resulting from increased airway resistance. it is a reversible but recurrent chronic inflammatory disorder that characteristically causes severe dyspnea, wheezing, and coughing. 13 there are 2 main problems in asthma: chronically inflamed airways and hyperresponsive airways. intermittent airflow obstruction in symptomatic patients results in decreased ability to expire, leading to hyperinflation, stenting open the alveoli, and increasing the work of breathing. early in an exacerbation, symptoms are bronchospastic secondary to smooth muscle contraction. as an episode progresses, inflammatory changes in the airways can cause increased airway resistance and lead to vq mismatch (fig. 11) . the severity of an exacerbation can be assessed clinically and should dictate how aggressively a patient is treated ( table 6) . adult patients with acute dyspnea copd is defined by the global initiative for chronic obstructive lung disease (gold) as "persistent airflow limitation that is usually progressive and associated with an enhanced chronic inflammatory response in the airways and the lung to noxious particles or gases." 12 the pathophysiology in each patient is typically a mix of lung parenchymal destruction, as seen in emphysema, and small airway inflammation with airway obstruction, or obstructive bronchiolitis. 11 an exacerbation of copd presents as dyspnea, cough, and increased sputum production. in the emergent devos & jacobson setting, clinicians must treat the airflow limitation. as with asthma, monitoring of pulse oximetry, degree of respiratory distress, and hemodynamic stability can help clinicians anticipate the degree of severity of a particular exacerbation. more specific testing may also have a role, because radiographs and electrocardiograms may help differentiate other causes of shortness of breath from a copd exacerbation. in addition, an increase in sputum production or the presence of purulent sputum should be treated with antibiotics, regardless of other infectious symptoms. 14 anaphylaxis is a sudden, potentially fatal, allergic reaction involving multiple organ systems. 15, 16 the second symposium on the definition and management of anaphylaxis lists the following clinical criteria for diagnosis of anaphylaxis: 1. acute onset of an illness (minutes to several hours) with involvement of the skin, mucosal tissue, or both (eg, generalized hives; pruritus or flushing; swollen lips, tongue, uvula) and at least 1 of the following: a. respiratory compromise (eg, dyspnea, wheeze-bronchospasm, stridor, reduced peak expiratory flow, hypoxemia) adult patients with acute dyspnea b. reduced blood pressure (bp) or associated symptoms of end-organ dysfunction (eg, hypotonia [collapse], syncope, incontinence) 2. two or more of the following that occur rapidly after exposure to a likely allergen for that patient: a. involvement of the skin-mucosal tissue b. respiratory compromise c. reduced bp or associated symptoms d. persistent gastrointestinal symptoms (eg, crampy abdominal pain, vomiting) 3. reduced bp after exposure to known allergen for that patient: a. infants and children: low systolic bp (age specific) or greater than 30% decrease in systolic bp b. adults: systolic bp of less than 90 mm hg or greater than 30% decrease from that person's baseline it is the respiratory compromise that is relevant to this article, and it is important to recognize that treating the allergic component of these symptoms is necessary to save the patients. now ems is at the back door with a 60-year-old patient with a history of copd and congestive heart failure (chf). he is in respiratory distress with audible wheezing and tripoding. he is diaphoretic, hypertensive, and has pitting edema to his knees. they have given albuterol with no improvement of his symptoms. acute dyspnea is the most common symptom of patients presenting with heart failure. 17 eighty percent of patients with acutely decompensated heart failure present through the ed with a chief complaint of dyspnea. 18 this symptom is related to both pulmonary and systemic fluid overload and also low cardiac output. american college of emergency physicians clinical policy makes level b recommendations that standard clinical judgment can be improved with the use of a single b-type natriuretic peptide (bnp) or n-terminal pro-b-type natriuretic peptide measurement to rule in or out the diagnosis of chf. 19 however, the true utility, may be in patients with dyspnea not expected to have a chf exacerbation, when finding a positive bnp would change management and allow a faster initiation of treatment. 20 carpenter and colleagues 20 found that the classic constellation of symptoms (jugular venous distension, peripheral edema, rales, and s3) were no more predictive of patients with both pulmonary edema on chest radiograph and an increased bnp level greater than 500 pg/dl than any individual finding alone. although rales were the most sensitive finding tested for either outcome, they had specificity of only about 50% each. jugular venous distention and s3 gallop were the individual findings most predictive for pulmonary edema on radiograph or increased bnp level. ultrasonography measurements of the inferior vena cava also improve diagnostic accuracy versus bnp and chest radiograph alone. 21 the medics are back in your department, this time with a 75-year-old man with cough and fever. his family is worried that he has been eating less and is sleepier than at hospital discharge last week. in pneumonia, the diffusion of oxygen is limited by alveolar infiltrates, leading to shortness of breath. common complaints and findings in community-acquired pneumonia include fever, cough, pleuritic chest pain, and sputum production, along with dyspnea. however, these clinical criteria may have a sensitivity as low as 50% compared with a chest radiograph. 22 on examination, many patients have crackles or evidence of consolidation. guidelines from the infectious diseases society of america and the american thoracic society, recommend chest radiograph in patients with suspected pneumonia, which may show lobular consolidation, interstitial infiltrate, or cavitation. 23 although infiltrate with suggestive symptoms makes the diagnosis, infiltrate may not be visible initially on patients with volume depletion. it is appropriate to treat empirically for 24 to 48 hours in these cases and to reimage when hydration is restored. the management of pneumonia requires history to allow classification based on the setting in which the illness was acquired. the infectious disease society of america and american thoracic society define the types of pneumonia as follows: 24 hospitalacquired pneumonia (hap) is "pneumonia that occurs 48 hours or more after admission which was not incubating at the time of admission." ventilator-associated pneumonia (vap) arises "more than 48-72 hours after endotracheal intubation." in addition, health care-associated pneumonia (hcap) is diagnosed in any patient who is "hospitalized in an acute care hospital for two or more days within 90 days of the infection, resided in a nursing home or long-term care facility, received recent iv antibiotic therapy, chemotherapy or wound care within the past 30 days of the current infection or attended a hospital or hemodialysis clinic." community-acquired pneumonia is not acquired in any of these situations. these classifications identify typical pathogens and guide appropriate initial management. important historical exposures and risk factors to refine treatment are summarized in table 7 . 23 the american thoracic society along with the infectious disease society of america's consensus statement offers 4 important principles in the initial management and evaluation of adult patients with bacterial hap, vap, or hcap; the most important to be accomplished in the ed is to promptly treat with "appropriate and adequate therapy" to decrease mortality. 24 after a brief delay, you see a 28-year-old woman with shortness of breath and chest pain. she smokes, uses hormonal birth control, and reports that her symptoms started when she came back from a business trip. pulmonary embolism (pe) interferes with both ventilation and perfusion. it ultimately causes circulatory collapse because of obstruction of right ventricular outflow eventually causing increased pulmonary artery pressure and failure of the right then left ventricles. before circulatory collapse, echocardiography can show signs of right ventricular (rv) strain, including dilatation of the right ventricle, rv hypokinesis, paradoxic septal wall motion, mcconnell sign (hypokinesis of the free rv wall with sparing of the apex), and tricuspid regurgitation. 25 dresden and colleagues 26 supported the use of ultrasonography in moderate-risk to high-risk patients to determine whether the patients were appropriate for anticoagulation while awaiting definitive imaging. early anticoagulation is recommended to improve mortality and there is evidence to support anticoagulation before diagnosis in patients with a wells score greater than 4 who will have a delay to diagnosis of more than 1 hour and 40 minutes. 27, 28 the assessment of patients with dyspnea and concern for pe requires a series of risk stratification. one common method is to use wells criteria 29 (box 1) in patients with suspicion for pe; although other stratification tools exist, none has been shown to be clearly superior. when there is low clinical suspicion for pe, perc (pulmonary embolism rule-out criteria) 30 rules or d-dimer testing may be applied. 27 if perc (box 2) is negative, or there is intermediate pretest probability for pe with negative high-sensitivity d-dimer, no further testing for pe is required. 27 when further testing is needed (positive d-dimer or high-sensitivity d-dimer not available), negative ct angiogram or low-probability vq scan may be used to rule out pe. in the next bed is a middle-aged woman with diabetes complaining of shortness of breath today. it was associated with some vague nausea and she says that she just does not feel good. angina pectoris is cardiac chest pain in which oxygen demand outweighs myocardial oxygen supply; in this case caused by occlusion of coronary arteries. although typically chest pain is a part of the presentation, dyspnea alone may be the initial complaint, termed an anginal equivalent. in one recent large series of patients undergoing stress testing, patients with dyspnea alone were at increased risk of death from cardiac causes. patients asked simply whether they experienced shortness of breath were considered dyspneic. the subset with no prior known coronary artery disease had more than 4 times the risk of sudden cardiac death versus asymptomatic patients and more than twice the risk of those with typical angina. 31 clinicians should consider past medical history and risk factors when assessing dyspnea for cardiac causes such as acute myocardial infarction and acute coronary syndrome. appropriate testing includes bedside electrocardiogram, troponin, and chest radiograph. the department eventually settles down and you are able to do some charting until a young man comes in with visible respiratory distress. he is tall and thin, smokes regularly, and reports sudden onset of severely painful breathing. pneumothorax occurs when air enters the plural space between the chest wall and the lung. typically only a thin serous layer exists between the visceral and parietal pleura. air enters this potential space only when there is damage to the lung or chest wall, or a gas-producing pleural space infection. the classic risk factors for bleb rupture causing spontaneous heart rate greater than 100 beats/min: 11.5 immobilization >3 days or surgery in last 4 weeks: 11.5 history of prior pe/deep venous thrombosis (dvt): 11.5 hemoptysis: 11 malignancy with treatment within 6 months or palliative: 11 less than or equal to 1. pneumothorax are tall men, although smoking has been suggested to increase the risk of rupture by damaging the pleural layer. pneumothoraces may be identified by ultrasonography, chest radiograph, or ct. treatment may be guided by cause, severity, comorbidities, interventions such as positive pressure ventilation, size of the pneumothorax, and patient's preference. recent studies suggest that uncomplicated spontaneous pneumothorax in patients not undergoing positive pressure ventilation may be treated as successfully with needle aspiration as with other more invasive chest drains, regardless of size. 32 tension pneumothorax is a serious event requiring immediate needle decompression to avert loss of cardiac output and arrest. however, recent review shows that the classic presentation of tension pneumothorax with hypotension, absent breath sounds, and deviated trachea may not be immediately seen in patients with spontaneous, unassisted respiration. 33 because of the slower development of the accumulation of air and pressure variations, spontaneously breathing patients may compensate much longer and present atypically, as shown in table 8 . thus, clinicians must remain vigilant. this article focuses on the cardiopulmonary system as the source of the problem in acutely dyspneic patients. it is important to also consider that the appearance of shortness of breath, tachypnea, or other typical symptoms of dyspnea may result from changing metabolic demands. these patients may appear, on the surface, to be in respiratory distress; they may be tachypneic, tachycardic, even pale or diaphoretic. in these cases, the clinician's responsibility is to identify and fix the true problem in order to improve the respiratory symptoms. severely anemic patients have limited oxygen carrying capacity. their bodies therefore experience oxygen hunger, which can manifest as shortness of breath. patients with dysfunctional hemoglobins secondary to irreversibly bound atoms or toxins may also be functionally anemic with the same symptoms. people's bodies attempt at all costs to maintain equilibrium. therefore, in metabolic acidoses (such as diabetic ketoacidosis), chemoreceptors detect acidosis and stimulate the respiratory center to hyperventilate. both the rate and the depth of ventilation often increase, leading to both tachypnea and hyperpnea, at times referred to as kussmaul respirations. this compensatory response is crucial for survival and should not be mistaken for dyspnea. it is equally important to realize that an increase in alveolar ventilation is not always a compensatory response (to acidosis or to primary pulmonary disorders) and hypocapnia may cause primary respiratory alkalosis, from central nervous system compromise, toxins (eg, salicylates), anxiety, or pain. 34 in these patients, imaging rarely reveals a source of dyspnea, but clinical suspicion based on history and examination, including signs such as the fruity breath of ketonemia, the pallor of anemia, or the cyanosis of toxic hemoglobinopathies, directs providers toward appropriate laboratory testing and treatment. in addition, sometimes dyspnea is not dyspnea. acute anxiety and panic disorder can present as shortness of breath, tachypnea, or hyperventilation. patients with panic disorder often describe symptoms similar to those of patients with true airway obstruction despite their normal pulmonary function. it has been suggested that these patients have abnormal proprioception, experiencing dyspnea without abnormal stimuli. 35 however, patients with a history of pulmonary disease can also have pure panic episodes. arterial blood gas may be useful in diagnosing anxiety-related hyperventilation. 36 severe pain can also induce abnormal respiratory patterns. like compensatory problems, pain and anxiety can be managed by managing their causes. treat pain. reduce stress and anxiety with words, behaviors, or, if necessary, medications. however, air hunger and difficulty breathing also make individuals anxious. be sure to avoid premature diagnosis of a purely anxiety-based concern without first evaluating for more dangerous disorders. acute dyspnea presents commonly to the ed and it is imperative that emergency physicians be prepared to stabilize patients' oxygenation and ventilation, which requires careful and efficient consideration of the differential diagnosis. using cues from the history and physical examination, practitioners may guide the work-up and treatment to identify a parenchymal, obstructive, circulatory, or compensatory cause of dyspnea. early use of bedside testing, including ultrasonography, may limit unnecessary tests and save time in determining the best treatment course. thus ensuring both the best care for the patient and also the physician's ability to readily respond to the next case. the epidemiology and outcome of prehospital respiratory distress dyspnea. mechanisms, assessment, and management: a consensus statement chapter 29: dyspnea rapid evaluation by lung-cardiac inferior vena cava (lci) integrated ultrasound for differentiating heart failure from pulmonary disease as the cause of acute dyspnea in the emergency setting diagnosing heart failure among acutely dyspneic patients with cardiac, inferior vena cava and lung ultrasonography diagnosing acute heart failure in patients with undifferentiated dyspnea: a lung and cardiac ultrasound (lucus) protocol s. trends in computed tomography use and diagnoses in emergency department visits by patients with symptoms suggestive of pulmonary embolism utilization and yield of chest computed tomographic angiography associated with low positive d-dimer levels appropriate use of screening and diagnostic tests to foster, high-value, cost-conscious care increased emergency department computed tomography use for common chest symptoms without clear patient benefits bates' guide to physical examination and history taking diagnosis of diseases of chronic airflow limitation: asthma, copd and asthma-copd overlap syndrome (acos) philadelphia: wb saunders antibiotics for exacerbations of chronic obstructive pulmonary disease second symposium on the definition and management of anaphylaxis: summary report -second national institute of allergy and immunology world allergy organization anaphylaxis guidelines: 2013 update of the evidence base assessment of dyspnea early in acute heart failure: patient characteristics and response differences between likert and visual analog scales the acute decompensated heart failure national registry (adhere): opportunities to improve care of patients hospitalized with acute decompensated heart failure clinical policy: critical issues in the evaluation and management of adult patients presenting to the emergency department with acute heart failure syndromes brain natriuretic peptide in the evaluation of emergency department dyspnea: is there a role? inferior vena cava assessment in the bedside diagnosis of acute heart failure testing strategies in the initial management of patients with community-acquired pneumonia infectious diseases society of america/american thoracic society consensus guidelines on the management of community-acquired pneumonia in adults infectious diseases society of america. guidelines for the management of adults with hospital-acquired, ventilator-associated, and healthcare-associated pneumonia prognostic role of echocardiography among patients with acute pulmonary embolism and a systolic arterial pressure of 90 mm hg or higher right ventricular dilatation on bedside echocardiography performed by emergency physicians aids in the diagnosis of pulmonary embolism critical issues in the evaluation and management of adult patients presenting to the emergency department with suspected pulmonary embolism usefulness of preemptive anticoagulation in patients with suspected pulmonary embolism: a decision analysis excluding pulmonary embolism at the bedside without diagnostic imaging: management of patients with suspected pulmonary embolism presenting to the emergency department by using a simple clinical model and d-dimer prospective multicenter evaluation of the pulmonary embolism rule-out criteria prognostic significance of dyspnea in patients referred for cardiac stress testing management of emergency department patients with primary spontaneous pneumothorax: needle aspiration or tube thoracostomy? clinical presentation of patients with tension pneumothorax: a systematic review metabolic acidosis in the critically ill: part 1. classification and pathophysiology panic anxiety, dyspnea, and respiratory disease value of arterial blood gas analysis in patients with acute dyspnea: an observational study high resolution computed tomography for the diagnosis of community-acquired pneumonia key: cord-340811-w4x4falm authors: frizzelli, annalisa; tuttolomondo, domenico; aiello, marina; majori, maria; bertorelli, giuseppina; chetta, alfredo title: what happens to people’s lungs when they get coronavirus disease 2019? date: 2020-05-11 journal: acta biomed doi: 10.23750/abm.v91i2.9574 sha: doc_id: 340811 cord_uid: w4x4falm the novel coronavirus sars-cov-2 was first identified in wuhan in december 2019 as cause of the consequent novel coronavirus disease 2019 (covid-19). the virus has since spread worldwide. the clinical presentation following human infection ranges from a mild upper respiratory tract infection to severe acute respiratory distress syndrome and sepsis. we reviewed literature using pubmed to identify relevant english-language articles published until april 15, 2020. search terms include novel coronavirus pneumonia, severe acute respiratory syndrome coronavirus 2, coronavirus and ventilation. we summarized what sars-cov-2 infection means for the lungs. (www.actabiomedica.it) the sars-cov-2 entry into the body sars-cov-2 belongs to the sabercovirus subgenus of the coronaviridae family, nidovirales order, and is the seventh coronavirus known to infect humans (1) . the transmission of the virus is human to human through droplets emitted by coughing and sneezing (2) . the consequences of the coronavirus disease 2019 (covid19) can be lethal, particularly in the elderly or those with comorbidities, such as hypertension, cardiac failure and diabetes (3) . sars-cov-2 uses the angiotensin converting enzyme ii (ace-2), as the cellular entry receptor (4) . ace-2 is a type i transmembrane metallocarboxypeptidase with homology to angiotensin converting enzyme (ace). in contrast to ace, which converts angiotensin i to the active vasoconstrictor, angiotensin ii, ace-2 breaks down angiotensin ii to its metabolites including angiotensin-(1-9) and angiotensin-(1-7), which are potent vasodilators, and thus may be a negative regulator of the renin-angiotensin system. ace-2 is expressed in different tissues such as upper and lower respiratory tract, myocardium and the gastrointestinal mucosa (5) . in humans, it appears to have a role in blood pressure control and cardiac function. the physiologic role of ace-2 in the airways is unknown. however, in mice, ace-2 seems to protect animals from severe lung injury related to aspiration and sepsis (6) . during sars-cov-2 infection, three stages can occur: stage i, an asymptomatic incubation period with or without detectable virus; stage ii, non-severe symptomatic period with the presence of virus; stage iii, severe respiratory symptomatic stage with high viral load (7) . during the first stage a specific adaptive immune response is crucial to eliminate the virus and avoid disease progression to severe stages. when a protective immune response is impaired, virus will propagate and a massive destruction of the infected tissues will occur, such as intestine and kidney. the damage cells induce innate inflammation in the lungs mediated by pro-inflammatory macrophages and granulocytes. lung inflammation is the main cause of life-threating respiratory disorders at the severe stage (8) . inflammatory markers are increased with sars-cov-2 infection, ranging from crp, il-6, ifn-gamma, to tnf-α (3)(9). this inflammatory response may lead to multi-organ failure and disseminated intravascular coagulation (dic) (10) . furthermore, the hypoxia seen in patients with severe pneumonia may also lead to further end-organ dysfunction/damage and death in critically-ill patients (11) . recent autopsy studies showed that the lungs are filled with clear liquid jelly, much resembling the lungs of wet drowning (8) . although the nature of the clear jelly has yet to be determined, hyaluronan (ha) may play a role. interestingly, ha is associated with acute respiratory distress syndrome (ards) (12) . the levels of inflammatory cytokines (il-1, tnf-α) are high in the lungs of covid-19 patients and these cytokines are strong inducers of ha-synthase-2 (has2) in cd31+ endothelium, epcam+ lung alveolar epithelial cells, and fibroblasts (13) . ha has the ability to absorb water up to 1000 times its molecular weight. therefore, reducing the presence or inhibiting the production of ha holds a great promise in helping covid-19 patients breathe (14) . approximately 80% of patient present with mild illness, 14% present with severe illness, and 5% present with critical illness (15) . the most common symptoms are: fever, cough, dyspnea, myalgia, fatigue. less common symptoms include anorexia, sputum production, sore throat, confusion, dizziness, headache, rhinorrhea, chest pain, hemoptysis, diarrhea, nausea/vomiting, abdominal pain, conjunctival congestion (16) . the most common laboratory abnormalities in patients hospitalized with pneumonia are lymphopenia and elevated liver transaminases. other abnormalities include neutrophilia, thrombocytopenia, decreased hemoglobin, decreased albumin, and renal impairment (3)(7) (16) . pulse oximetry may reveal low oxygen saturation (spo2 <95%), as a consequence of pneumonia. recent studies pointed out a significant relationship between troponin (tni) levels and fatal outcome in patients with confirmed covid-19 (17) . the underlying mechanism of this association is not completely understood. one case report study demonstrated the presence of coronavirus and inflammatory markers in autopsy specimens of the myocardium (18), thereby suggesting that these patients may develop myocarditis. another potential cause of myocardial injury is occlusive thrombus formation on ruptured coronary plaque, related to intense inflammatory stress on pre-existent coronary artery disease. the effect of inflammation on plaque disruption has been partially proved by cantos trial (19) . the chest imaging findings may vary with the patient's age, immunity status, disease stage, underlying diseases and drug interventions. clinical data from zhongnan hospital of wuhan university described such typical ct/x-ray imaging manifestations, multiple, patchy, sub-segmental or segmental ground-glass density shadows in both lungs. they were classified as "paving stone-like" changes by fine-grid or small honeycomb-like thickening of interlobular septa. also multiple, patchy or large patches of consolidation in both lungs, with a little grid-like or honeycombshaped interlobular septal thickening, especially in the middle and lower lobes, more common in the elderly or severe condition patients, were considered typical radiological findings (20) . respiratory failure may occur in patients with covid-19 pneumonia. in italy, up to 12% of all patients with covid-19 required icu admission following respiratory failure (21) . furthermore, ards is the most common complication in patients with respiratory failure and may occur in 60-70% of patients with covid-19 admitted to the icu (22). interestingly, patients with covid-19 pneumonia may present an atypical form of ards characterized by a dissociation between their relatively preserved lung mechanics and the severity of hypoxemia (23) . a possible explanation for such severe hypoxemia occurring in compliant lungs is the loss of lung perfusion regulation and hypoxic vasoconstriction. oxygen therapy should be considered immediately when patients affected by severe acute respiratory infection have the following conditions: hypoxemia (pao2 <60 mmhg or spo 2 <93% when breathing air); respiratory distress (respiratory frequency> 24 times/min); hypotension (systolic blood pressure <100 mmhg) (24) . it is reasonable setting the initial oxygen flow to 5 l/min, and then titrating the oxygen flow to maintain spo 2 at 94%-98%. if the patient's initial spo 2 <85%, oxygen storage masks (oxygen flow> 12 l/ min) should be preferred to correct hypoxemia. if there is no improvement or deterioration after 1 to 2 hours, other respiratory support methods should be replaced immediately. the patient should be instructed to wear a surgical mask or a simple open mask when inhaling the nasal catheter (25) . high flow nasal cannula (hfnc) is a new type of breathing support method, where the gas flow can be adjusted up to 60-70 l/min and fio 2 from 0.21 to 1.0. compared with oxygen therapy, hfnc can reduce the rate of tracheal intubation and mortality in patients with hypoxic respiratory failure. therefore, hfnc treatment could be selected when oxygen therapy cannot correct hypoxemia. in a retrospective study, wang ke at al. screened 318 patients with cov-id-19 pneumonia and showed that hfnc was the most common ventilation support for these patients, but patients with lower pao 2 /fio 2 (<200 mhg) were more likely to fail (26) . after treatment with hfnc, the efficacy response should be monitored closely (1-2 hours). if critical conditions persist, hfnc should be replaced with non-invasive ventilation (niv) in a timely manner (22) . recent data suggest that covid-19 patients who are hypoxemic respond well to peep, indicating a crucial role for niv, as a therapeutic measure to prevent intubation (27) . for patients with pao2/ fio2≤150mmhg, invasive ventilation should be implemented as soon as possible. a novel coronavirus from patients with pneumonia in china genomic characterization and epidemiology of 2019 novel coronavirus: implication for virus origins and receptor binding clinical features of patients infected with 2019 novel coronavirus in wuhan, china. lancet a pneumonia outbreak associated with a new coronavirus of probable bat origin quantitative mrna expression profiling of ace 2, a novel homologue of angiotensin converting enzyme angiotensin-converting enzyme 2 protects from severe acute lung failure clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus-infected pneumonia in wuhan, china pathological findings of cov-id-19 associated with acute respiratory distress syndrome risk factors associated with acute respiratory distress syndrome and death in patients with coronavirus disease 2019 pneumonia in wuhan, china clinical characteristics of coronavirus disease 2019 in china clinical course and outcomes of critically ill patients with sars-cov-2 pneumonia in wuhan, china: a single-centered, retrospective, observational study accumulation of hyaluronan (hyaluronic acid) in the lung in adult respiratory distress syndrome defective lung function following influenza virus is due to prolonged, reversible hyaluronan synthesis covid-19 infection: the perspectives on immune responses novel coronavirus pneumonia emergency response epidemiology team. the epidemiological characteristics of an outbreak of 2019 novel coronavirus disease (covid-19) in china epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a descriptive study cardiovascular implications of fatal outcomes of patients with coronavirus disease 2019 (covid-19) jama cardiol myocardial localization of coronavirus in covid-19 cardiogenic shock antiinflammatory therapy with canakinumab for atherosclerotic disease a rapid advice guideline for the diagnosis and treatment of 2019 novel coronavirus (2019-ncov) infected pneumonia (standard version). military covid-19 and italy: what next? intensive care management of coronavirus disease 2019 (covid-19): challenges and recommendations covid-19 does not lead to a "typical" acute respiratory distress syndrome clinical management of severe acute respiratory infection when novel ronavirus (ncov) infection is suspected: interim guidance (eb / ol) severe acute respiratory syndrome (sars): lessons learnt in hong kong( j) the experience of high-flow nasal cannula in hospitalized patients with 2019 novel coronavirus-infected pneumonia in two hospitals of chongqing efficacy and safety of early prone positioning combined with hfnc or niv in moderate to severe ards: a multi-center prospective cohort study. crit care each author declares that he or she has no commercial associations (e.g. consultancies, stock ownership, equity interest, patent/licensing arrangement etc.) that might pose a conflict of interest in connection with the submitted article key: cord-342915-r9kv67we authors: hayden, frederick g. title: advances in antivirals for non‐influenza respiratory virus infections date: 2013-11-01 journal: influenza and other respiratory viruses doi: 10.1111/irv.12173 sha: doc_id: 342915 cord_uid: r9kv67we progress in the development of antivirals for non‐influenza respiratory viruses has been slow with the result that many unmet medical needs and few approved agents currently exist. this commentary selectively reviews examples of where specific agents have provided promising clinical benefits in selected target populations and also considers potential therapeutics for emerging threats like the sars and middle east respiratory syndrome coronaviruses. recent studies have provided encouraging results in treating respiratory syncytial virus infections in lung transplant recipients, serious parainfluenza virus and adenovirus infections in immunocompromised hosts, and rhinovirus colds in outpatient asthmatics. while additional studies are needed to confirm the efficacy and safety of the specific agents tested, these observations offer the opportunity to expand therapeutic studies to other patient populations. considerable progress has been made in the development of influenza antivirals with two major classes of drugs, the adamantanes and neuraminidase inhibitors, being available for clinical use in most countries and a variety of agents in various stages of investigational development. 1, 2 the widescale use of nais in some countries during response to the 2009 a(h1n1) pandemic provided much new observational data on the effectiveness of oseltamivir treatment in reducing the risk of severe influenza outcomes like pneumonia and hospitalization and of mortality in those hospitalized. [3] [4] [5] [6] [7] such observations confirm the principle that selective inhibitors of influenza replication are associated with both symptom relief and complications reduction and suggest that timely antiviral therapy of other respiratory viral infections might provide similar benefits. unfortunately, we have only a few approved agents for other respiratory viruses and the use of these agents is typically limited to specific risk groups like infants and immunocompromised hosts. one challenge in developing both vaccines and therapeutics for non-influenza respiratory viruses is the diversity of these pathogens, representing six major virus families, primarily rna but also dna viruses, and numerous serotypes and genotypes. the pathogenesis of these infections ranges broadly depending on the virus, host, and clinical setting, and for many of these illnesses, it remains uncertain to what extent inhibition of viral replication would be associated with clinical benefits. most of the treatment data regarding antivirals for non-influenza respiratory viruses have been derived from observational studies in immunocompromised hosts, and sometimes, infants, but recent randomized, controlled trials in specific target populations have helped to address the potential value of antiviral interventions. the following commentary, based on a presentations by the author at the 2nd meeting of the international society of influenza and other respiratory viruses antiviral interest group, hanoi, november 2012, and at the xvth international symposium on respiratory viral infections, rotterdam, march 2013, is a highly selective review of antiviral treatment interventions that show particular promise for specific respiratory viruses. it focuses primarily on clinical reports but also includes some observations from pre-clinical studies. potential treatments and the lessons learned in therapeutic studies of severe acute respiratory syndrome (sars) covinfected patients. in sars patients, the role of antiviral therapy was supported by finding that viral load was positively correlated with the development of organ dysfunction and death. 12 sars was characterized by protracted virus replication, peaking during the second week of illness, prominent host pro-inflammatory responses, and histologic evidence for diffuse alveolar damage. few data regarding viral replication patterns and disease pathogenesis are currently available for mers-cov infections, although prolonged viral replication in the lower respiratory tract, extrapulmonary virus detection, lung injury, respiratory failure and often renal failure are notable features in reported cases. [8] [9] [10] [11] a wide range of agents were reported to have anti-sars-cov activity in pre-clinical studies, [12] [13] [14] [15] [16] and a considerable number of therapeutic interventions directed at inhibiting cov replication or modifying the host responses to infection were attempted in sars patients. although corticosteroids were widely used for treating those with progressive sarsrelated pneumonia in efforts to reduce excessive pro-inflammatory responses and presumed immune-mediated tissue damage, their benefits were not conclusively demonstrated, and various reports documented increases in the plasma viral loads, opportunistic infections, and both immediate and delayed (e.g., osteonecrosis) side effects. 12, [17] [18] [19] in addition, systematic reviews of the observational reports concluded that the common use of multiple agents in combination, varying dose regimens, paucity of studies with systematic data collection, complications from immunosuppressive therapy, and the lack of randomized, controlled trials meant that existing data were inconclusive with regard to putative antivirals and thus inadequate to determine appropriate management of sars infections. 12, 18, 19 the use of available agents like ribavirin and hiv protease inhibitors was not associated with proven benefit in sars patients, although retrospective studies reported that severe outcomes (ards or death) occurred less often in those receiving a combination of lopinavir/ritonavir and ribavirin with corticosteroids compared with historic controls receiving only ribavirin with corticosteroids. 20, 21 early combination treatment was also associated with fewer nosocomial infections, less use of pulse corticosteroids for progressive disease, and lower nasopharyngeal viral loads over time, perhaps related to corticosteroid-sparing effects. 21 of note, in mice, high ribavirin dosing at 75 mg/kg starting 4 hour prior to sars virus exposure and then given twice daily for 3 days was found to increase virus lung titers and prolong the duration of virus detectability, 22 and it is unclear whether ribavirin might have had similar effects in treated humans. one small pediatric study found no correlation between ribavirin administration and plasma sars rna levels. 23 in sars patients, ribavirin was associated also with significant toxicities, including hemolytic anemia and metabolic disturbances like hypocalcaemia and hypomagnesaemia. 24 one recent in vitro study found that very high ribavirin concentrations were inhibitory for mers-cov in vero and llc-mk2 cells but that when combined with recombinant human interferon-alfa2b, lower concentrations were inhibitory. 25 consequently, ribavirin's possible role in treating cov infections remains uncertain. sars-cov impairs the induction of interferons and their associated antiviral effects but is inhibited by exogenous interferon in cell culture studies. 15 injection of pegylated interferon-alfa2b was highly protective given 3 days before and reduced lung viral levels and histopathology when initiated 1 day post-infection in a cynomolgus macaque model of sars-cov, 26 and both a hybrid interferon and an interferon inducer, mismatched double-stranded poly(i:c), reduced lung viral titers in a murine model. 14 intranasal application of interferons or the interferon inducer poly(i:c) was protective and reduced virus replication in an aged mouse model of sars. 27 interferons also have immunomodulatory effects, and in an aged macaque model of sars, treatment with type i interferon reduced pathology and diminished pro-inflammatory gene expression, including interleukin-8 (il-8) levels, without affecting virus replication in the lungs. 28 one observational canadian study in sars patients with progressive illness found that synthetic interferon alfacon-1 in conjunction with corticosteroids appeared to reduce time to resolution of pulmonary infiltrates, improve oxygen saturation, reduce the duration of supplemental oxygen use, and sped resolution of serum ldh elevations compared with corticosteroids alone. 29 interferon alfacon-1 was generally well tolerated, although associated with transient reductions in neutrophil counts and elevations in transaminase levels. of note, the mers-cov has been shown to be readily inhibited by type i and iii interferons in human bronchial epithelium ex vivo. 30, 31 such observations are sufficiently encouraging to support controlled studies of systemic interferon in affected patients. in addition, one approved agent for selected parasitic infections, oral nitazoxanide, may have interferon-inducing properties, is inhibitory for various respiratory viruses including influenza and a canine cov in vitro, 32 and has shown promising dose-related activity in a phase 2, placebo-controlled, randomized trial in treating uncomplicated influenza 33 consequently, nitazoxanide would be an interesting agent to test alone and in combination with other antivirals for cov infections. the protective efficacy of sars-cov-neutralizing antibodies, including humanized and human monoclonals directed to the spike protein, has been demonstrated in various animal models. 15, 34, 35 these studies found potent antiviral effects without evidence for the immune enhancement. passive immunotherapy with convalescent plasma from sars patients was used in treating sars patients in advances in antivirals for non-influenza rvis ª 2013 blackwell publishing ltd hong kong and taiwan in open-label fashion with apparent clinical benefits and rapid decreases in plasma viral load (from up to >10e5 copies/ml to undetectable levels 24 hour after infusion). 36, 37 among 80 patients given convalescent plasma in hong kong, a higher day 22 hospital discharge rate was observed among patients who were received plasma before day 14 of illness compared with later (58á3% versus 15á6%; p < 0á001) and among those who were viral rt-pcr positive but sars-cov seronegative compared with those seropositive at the time of plasma infusion. 36 efforts to develop neutralizing human monoclonals for mers-cov are in progress, as is screening for other inhibitors. other inhibitors of the spike (s) protein and its receptor interactions have been described. for example, in a rhesus macaque sars model, sirna inhibitors targeting the sars-cov genome at s protein-and nsp12-coding regions reduced sars-cov-induced fever, numbers of infected cells, and histologic findings of diffuse alveolar damage, compared with control or a non-specific sirna, when given intranasally as prophylaxis or post-infection. 38 pre-clinical reports suggest that inhibitors of angiotensin-converting enzyme-2 (ace-2), one of the attachment sites for the sars s protein, and other s protein inhibitors like mannose-binding lectin (mbl) might provide therapeutic benefit in sars. however, the mers-cov does not use ace2 but rather dipeptidyl peptidase 4 (dpp4 or cd26) to initiate infection 39 and has a much broader spectrum of cell infectivity, including human, swine, and bats cells than sars-cov. 40 consequently, ace2 inhibitors would be unlikely to exert specific inhibition of hcov-emc. identification of the receptor used by mers-cov may provide selective inhibitors of this interaction, but available agents that inhibit the enzymatic function of dpp4 apparently do not. 39 one study of sars patients found that they had snps affecting mbl expression and reduced levels of serum mbl compared to controls without sars, 41 although serum mbl levels did not correlate with disease severity. serum mbl concentrations vary widely due to mutations of the promoter and coding regions of the human mbl gene. recombinant and plasma-derived human mbl bind to the s protein through one or more n-linked glycosylation sites and inhibit sars-cov replication in susceptible cell lines at concentrations below those observed in the serum of healthy individuals. 42 the cell entry inhibitory effect appears to be mediated in part by blocking viral binding to c-type lectins but apparently not to the ace2 receptor. 42 no in vivo therapeutic studies have been reported in sars, but high doses of recombinant human mbl showed activity in a lethal ebola virus model in mice. 43 because mbl binds to carbohydrates on the surface of a wide range of respiratory viruses, including influenza, paramyxoviruses, and coronavirus, and other important pathogens, mbl is a potential therapeutic intervention of general interest. respiratory syncytial virus is a well-recognized cause of morbidity and, in those at the extremes of age or with severe immunocompromising conditions, mortality. it is the leading cause of bronchiolitis in infants and young children, and one analysis estimated that in 2005, rsv infections were associated with approximately 34 million episodes of acute lower respiratory illness (alri), or about 22% of alri, in children below the age of 5 years. 44 these illnesses led to hospitalization in about 10% of cases and to between 66 000 and 200 000 deaths globally, with 99% of these deaths in low-and middleincome countries. 44 in contrast, in developed countries like the united states, rsv is associated with many more deaths in adults aged 65 years and older than in children. 45 the available antiviral options for rsv are quite limited. much work has been carried out on developing antibody preparations, and the anti-f protein monoclonal palivizumab is currently approved for use for prophylaxis in high-risk children. 46 while effective in reducing rsv-related hospitalization by about 50%, its very high cost limits availability to well-resourced countries. aerosolized ribavirin is approved for treating hospitalized children with serious rsv illness, but it is used very infrequently in this population due to its difficult delivery method, modest antiviral and clinical effects, cost, and concerns about healthcare worker exposure to a potential teratogen. the american academy of pediatrics recommends against its routine use. 47 however, ribavirin has been used frequently in hematopoietic stem cell transplant (hsct) recipients, because of their high mortality risk when rsv lower respiratory tract illness (lrti) develops. 48 a systematic review of observational studies across multiple of centers found that compared with no ribavirin use, ribavirin treatment, regardless of route or whether combined with an immunomodulatory regimen (intravenous immunoglobulin [ivig], rsv immunoglobulin, or palivizumab), was associated with reduced frequencies of progression to lrti. 49 less frequent progression to lrti and significantly fewer deaths in those with lrti were observed among patients treated with combinations of aerosolized ribavirin and an immunomodulatory agent than in those treated with ribavirin alone. however, a recent retrospective analysis of one center's experience concluded that the addition of palivizumab to aerosolized ribavirn did not improve outcomes in rsv-infected hsct recipients. 49a a recent randomized controlled trial comparing two aerosolized ribavirin regimens found that intermittent delivery (2 g over 2 hours every 8 hours) appeared to be more effective in preventing lower respiratory progression than a standard continuous one (6 g over 18 hoursr) and was easier to administer. 50 while data on oral ribavirin are very limited, one study suggested favorable outcomes in hsct recipients with upper respiratory rsv infection when used in combina-tion with ivig and palivizumab. 51 however, a recent retrospective analysis of hematologic malignancy and hsct patients with rsv or other paramyxovirus infections concluded that oral ribavirin was not clinically effective in preventing mortality. 52 in lung transplant recipients with rsv or human metapneumovirus infections, observational studies suggest that ribavirin given intravenously or orally may be beneficial with regard to improving clinical outcomes and recovery of lung function, 53,54 but the uncontrolled nature of these findings precludes firm conclusions. further controlled studies of oral ribavirin, which is less expensive and easier to administer than aerosolized ribavirin, are warranted in these important patient groups. a number of rsv investigational agents have received some degree of recent clinical testing, although development of several apparently has been stopped at this time. for example, a potent anti-f antibody, motavizumab, was shown to have antiviral effects in hospitalized children 55 but was not superior to palivizumab in seasonal rsv prophylaxis in atrisk children and had increased cutaneous adverse events relative to palivizumab. 56 a placebo-controlled rct of a single motavizumab injection did not find reductions in upper respiratory tract viral titers or illness measures in infants aged < 12 months and hospitalized with rsv illness [ramilo et al.,r presented at the 8th annual respiratory syncytial virus symposium, september 27-30, 2012, santa fe, nm]. more potent monoclonals to f protein are in development. among those in active clinical development, there is a polyclonal high-titer rsv immunoglobulin (ri-001; adma biologics inc., hackensack, nj, usa) being tested in immunocompromised patients to prevent progression from upper to lower respiratory tract illness, a topically applied f protein inhibitor (mdt-637; microdose therapeutx, monmouth junction, nj, usa) and an orally administered f protein inhibitor (gs-5806; gilead sciences, inc., foster city, ca, usa). the results of double-blinded, placebo-controlled studies with gs-506 to test efficacy in experimental rsv infection in adult volunteers (nct01756482) and safety in young children hospitalized for rsv illness (nct01797419) are expected to be available shortly. because the envelope glycoprotein f plays an important role in rsv fusion with and entry into the host cell, it serves as an attractive target for developing rsv entry inhibitors, although resistance emergence has been a notable limitation in earlier studies. 57 the agent that has progressed furthest in testing is a double-stranded oligonucleotide directed against the viral n gene (aln-rsv01; alnylam pharmaceuticals, cambridge, ma, usa). this sirna, complementary to the mrna that encodes the n protein, showed robust antiviral effects both in vitro and in a murine model, with over a 1000-fold reduction in lung virus titers, 58 and was active when given intranasally to volunteers experimentally infected with rsv. 59, 60 in a placebo-controlled rct of 24 lung transplant recipients with rsv infection, aerosolized aln-rsv01 (0á6 mg/kg) given daily for 3 days was generally well tolerated and significantly reduced the risk of new or progressive bronchiolitis obliterans syndrome (bos) at day 90 compared with placebo (6á3% versus 50%). 61 there were no differences in upper respiratory tract viral loads, but those in the lower could not be tested. consequently, a larger phase 2b rct was conducted at 33 sites in six countries at the same dose but extended to 5 days administration, again added to standard of care. 62 the primary endpoint of bos at day 180 among the 77 lung transplant patients in the intent-to-treat-infected population tended to be lower with inhaled aln-rsv01 (30á3% versus 13á6%; p = 0á058) and was significantly lower among the 73 patients in the per-protocol analysis (28á1% versus 9á8%). deaths occurred in 2 placebo and 1 sirna recipient that were unrelated to treatment. these encouraging results warrant further studies of this novel sirna therapeutic in other risk populations and support the study of sirna inhibitors for other respiratory viral infections. 59 parainfluenza virus (piv) illness can be very severe in immunocompromised hosts, particularly hsct recipients. the results with ribavirin have been quite mixed. aerosolized ribavirin has not resulted in reductions in viral shedding or survival benefit in hsct with piv pneumonia, while oral or intravenous ribavirin has provided apparent benefit in individual hsct recipients and hematologic malignancy patients with piv illness. 48 a recent retrospective analysis concluded that oral ribavirin did not reduce mortality in hematologic malignancy patients with paramyxovirus infections compared with supportive care alone, 52 whereas oral ribavirin seems to have been associated with benefit in lung transplant patients with paramyxovirus infections. 54 a new investigational agent for severe piv infection is das181, a fusion construct that includes a sialidase from actinomycosis viscosus that cleaves both a2,6-and a2,3-linked sialic acid receptors on host cells. 63 consequently, its antiviral spectrum includes influenza viruses and it is active when topically applied in animal influenza models, including avian h5n1 and h1n1pdm09 viruses, [63] [64] [65] and has shown antiviral effects in a phase 2 rct in uncomplicated human influenza. 66 of note, the h-n protein of piv also binds to sialic acid receptors, and das181 is inhibitory for piv in cell culture, in human airway epithelium, and given intranasally in a cotton rat model. 67 das181 has been administered on a compassionate use basis for treating hsct and lung transplant patients with severe piv illness with apparent clinical benefit and antiviral effects in some cases. [68] [69] [70] this host-directed agent is available on compassionate use from its sponsor (formerly nexbio, now ansun biopharma, inc., san diego, advances in antivirals for non-influenza rvis ª 2013 blackwell publishing ltd ca, usa), and an open-label study is being undertaken at the nih for this particular problem. adenovirus infections are often very severe in immunocompromised hosts but occasionally in those in the community as well. 71, 72 there are currently no approved antivirals for adenovirus treatment, but a number of agents have some degree of in vitro inhibitory activity. 73 ribavirin has variable in vitro activity at high concentrations 74 and proven largely ineffective in treatment of serious infections. intravenous cidofovir, a nucleoside phosphonate analog, has become the standard treatment for most immunocompromised patients with adenovirus disease, but its use is associated with nephrotoxicity in up to 50% and neutropenia in 20% of patients. 75, 76 donor leukocyte infusion and use of in vitroexpanded adenovirus-specific t cells have been used in some patients. 76 the most promising anti-adenovirus antiviral in clinical development is the orally administered, lipid ester derivative of cidofovir, designated cmx001or brincidofovir (chimerix, durham, nc, usa). in vitro cmx001 provides higher intracellular levels compared with the parent molecule and is over 50-fold more active than cidofovir for adenoviruses. 77 it was shown to be inhibitory for lethal adenovirus infection in a syrian hamster model. 78 in addition to the advantage of being orally bioavailable, cmx001 also has lower risk of nephrotoxicity compared with cidofovir during clinical use. one case series of 13 immunocompromised patients, the majority of whom were hsct recipients, described its use in severe adenovirus disease. 79 all had adenoviremia, and six had disseminated disease involving other organs. following onset of the adenoviral disease at a median of 75 days posttransplant, they were initially treated with cidofovir, but then switched to cmx001 because of either persistent viral replication or nephrotoxicity. prolonged courses of cmx001 were associated with good virologic responses, including at least 100-fold reductions in plasma adenoviral dna levels in nine patients. some delay in mortality was also observed among those with virologic responses. two current cmx001 studies will provide further insights regarding its clinical utility for adenoviral infections in high-risk populations. one is a controlled trial of pre-emptive therapy in hsct patients with adenoviremia that has finished enrollment and is under analysis (nct01241344), and the other is an open-label study of treating a variety of serious dna viral diseases, including those due to adenoviruses, in immunocompromised hosts (nct01143181). an initial press release (14 august 2013) indciated that cmx001 100 mg biw decreased levels of adenoviremia and showed potential benefits in reducing both progression to adenovirus disease and all-cause mortality, compared to subjects who received placebo or cmx001 given once weekly. phase 1 safety and pharmacology studies have been completed in healthy volunteers. 80 cmx001 is a promising agent that also warrants testing in non-immunocompromised hosts with severe adenovirus illness. human rhinovirus (hrv) infections are the most frequent infections that humans experience and are implicated in causing a wide range of respiratory tract syndromes. in addition to being the most frequent cause of colds, hrv infections are the leading cause of exacerbations of asthma and chronic obstructive pulmonary disease. past studies of the capsid-binding anti-hrv agent pleconaril showed that early treatment exerted antiviral effects and reduced the duration of uncomplicated hrv colds by about 1 day. 81 symptomatic improvement in pleconaril-treated subjects was related to the drug susceptibility of the infecting hrv, and strains with >10fold reduced susceptibility emerged in about 11% of recipients. 82 however, it was not approved for clinical use largely because of potential drug interaction concerns. recently, another capsid-binding anti-hrv agent designated bta798 (or vapendavir) (biota holdings ltd, notting hill, vic., australia) showed dose-related antiviral effects in experimentally infected volunteers 83 and beneficial effects in a phase 2 placebo-controlled rct in asthmatics with cold symptoms (nct01175226). 84 among the 300 persons enrolled, 93 had a documented hrv infection. oral vapendavir (400 mg twice daily for 6 days) was associated with significantly lower upper respiratory symptom scores early in the illness and continuing up to 2 weeks compared with placebo. vapendavir recipients also had significant improvements in secondary outcomes including higher peak expiratory flow rates on day 5, reduced overall use of asthma relief medications, and less frequent hrv rna detection on day 3 (74% versus 91%). this modest degree of antiviral effect raises the possibility that more potent inhibition might be able to provide even greater clinical benefits. intranasal recombinant interferon-alpha2b was shown to be effective in preventing hrv colds when used for postexposure prophylaxis 85, 86 but ineffective for treatment of established colds 85 and also associated with local side effects. recently, 14 days treatment with an inhaled interferon-beta designated sng001 (synairgen plc, southampton, england) was associated with therapeutic efficacy in a phase 2, placebocontrolled rct of adult asthmatics receiving inhaled corticosteroids who had a history of deterioration with colds (nct01126177). 87 among 147 enrolled, 134 met the criteria for a cold; hrvs represented 68% of the respiratory viruses detected. although the trial did not meet its primary endpoint (changes in the shortened asthma control questionnaire) in the overall population, among the subset of "difficult to treat" asthma patients representing about onehalf of those enrolled, inhaled sng001 was associated with significant improvements in asthma symptoms, 65% fewer moderate exacerbations, improved morning peak expiratory flow rates, and reduced use of relief bronchodilators. while the findings in the oral vapendavir and inhaled interferonbeta studies need to be confirmed in larger trials, the results suggest that early antiviral treatment of colds in asthmatics can moderate asthma exacerbations. in summary, currently, there are many unmet medical needs and few approved agents for the non-influenza respiratory virus infections. recent studies have provided encouraging results in serious respiratory viral infections in hospitalized immunocompromised hosts and in outpatient asthmatics with colds. while additional trials are needed to confirm the efficacy and safety of the agents discussed above, these or other selective antivirals offer the opportunity to expand therapeutic studies to other patient populations. one of the challenges in developing more effective therapeutics is the diversity of respiratory viruses and the differences in disease pathogenesis across viruses and patient groups. in addition to das181 for piv, several agents in clinical development for influenza (e.g., favipiravir, nitazoxanide) have in vitro activity against several other respiratory viruses. 32, 88 in addition, analogous to work in influenza, 89 one forward-looking strategy is understanding how respiratory viruses interact with host cellular pathways during replication and identifying potential viral-host protein interactions to target. for example, one large mrna gene expression database search identified 67 common pathways among seven different respiratory viruses; of the top five pathways, 53 had differentially expressed genes affected by at least five of the seven viruses. 90 such studies might lead to identifying existing drugs that could be repurposed to target these pathways and eventually to broader spectrum antiviral agents. newer influenza antivirals, biotherapeutics and combinations antivirals in seasonal and pandemic influenza-future perspectives antiviral therapy and outcomes of patients with pneumonia caused by influenza a pandemic (h1n1) virus antivirals for treatment of influenza: a systematic review and meta-analysis of observational studies impact of neuraminidase inhibitor treatment on outcomes of public health importance during the 2009-2010 influenza a (h1n1) pandemic: a systematic review and meta-analysis in hospitalized patients treatment with neuraminidase inhibitors for critically ill patients with influenza a(h1n1)pdm09 effectiveness of oseltamivir on disease progression and viral rna shedding in patients with mild pandemic 2009 influenza a h1n1: opportunistic retrospective study of medical charts in china isolation of a novel coronavirus from a man with pneumonia in saudi arabia a novel coronavirus capable of lethal human infections: an emerging picture the health protection agency (hpa) uk novel coronavirus investigation team. evidence of person-to-person transmission within a family cluster of novel coronavirus infections severe respiratory illness caused by a novel coronavirus severe acute respiratory syndrome and coronavirus cyclosporin a inhibits the replication of diverse coronaviruses evaluation of immunomodulators, interferons and known in vitro sars-cov inhibitors for inhibition of sars-cov replication in balb/c mice coronaviruses and their therapy potential antivirals and antiviral strategies against sars coronavirus infections pro/con clinical debate: steroids are a key component in the treatment of sars sars: systematic review of treatment effects the management of coronavirus infections with particular reference to sars treatment of severe acute respiratory syndrome with lopinavir/ritonavir: a multicentre retrospective matched cohort study role of lopinavir/ritonavir in the treatment of sars: initial virological and clinical findings enhancement of the infectivity of sars-cov in balb/c mice by imp dehydrogenase inhibitors, including ribavirin serial analysis of the plasma concentration of sars coronavirus rna in pediatric patients with severe acute respiratory syndrome common adverse events associated with the use of ribavirin for severe acute respiratory syndrome in canada inhibition of novel beta coronavirus replication by a combination of interferon-alpha2b and ribavirin pegylated interferon-alpha protects type 1 pneumocytes against sars coronavirus infection in macaques intranasal treatment with poly(i*c) protects aged mice from lethal respiratory virus infections exacerbated innate host response to sars-cov in aged non-human primates interferon alfacon-1 plus corticosteroids in severe acute respiratory syndrome: a preliminary study efficient replication of the novel human betacoronavirus emc on primary human epithelium highlights its zoonotic potential tropism and innate immune responses of the novel human betacoronavirus lineage c virus in human ex vivo respiratory organ cultures nitazoxanide, a novel potential anti-influenza drug, acting in synergism with neuraminidase inhibitors idsa annual meeting boston: new approaches to anti-viral therapy saturday a randomized, doubleblind, placebo (pcb) controlled clinical trial of nitazoxanide (ntz) in adults and adolescents with acute uncomplicated influenza idsa annual meeting human monoclonal antibody as prophylaxis for sars coronavirus infection in ferrets therapy with a severe acute respiratory syndrome-associated coronavirus-neutralizing human monoclonal antibody reduces disease severity and viral burden in golden syrian hamsters use of convalescent plasma therapy in sars patients in hong kong experience of using convalescent plasma for severe acute respiratory syndrome among healthcare workers in a taiwan hospital using sirna in prophylactic and therapeutic regimens against sars coronavirus in rhesus macaque dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-emc human coronavirus emc does not require the sars-coronavirus receptor and maintains broad replicative capability in mammalian cell lines mannose-binding lectin in severe acute respiratory syndrome coronavirus infection a single asparagine-linked glycosylation site of the severe acute respiratory syndrome coronavirus spike glycoprotein facilitates inhibition by mannose-binding lectin through multiple mechanisms high-dose mannose-binding lectin therapy for ebola virus infection global burden of acute lower respiratory infections due to respiratory syncytial virus in young children: a systematic review and meta-analysis mortality associated with influenza and respiratory syncytial virus in the united states antibodies for prevention and treatment of respiratory syncytial virus infections in children respiratory syncytial virus antiviral therapy of respiratory viruses in haematopoietic stem cell transplant recipients management of rsv infections in adult recipients of hematopoietic stem cell transplantation outcome of respiratory syncytial virus lower respiratory tract disease in hematopoietic cell transplant recipients receiving aerosolized ribavirin: significance of stem cell source and oxygen requirement an adaptive randomized trial of an intermittent dosing schedule of aerosolized ribavirin in patients with cancer and respiratory syncytial virus infection respiratory syncytial virus infection in patients with hematological diseases: single-center study and review of the literature efficacy of oral ribavirin in hematologic disease patients with paramyxovirus infection: analytic strategy using propensity scores antiviral therapies for respiratory viral infections in lung transplant patients single-centre experience with oral ribavirin in lung transplant recipients with paramyxovirus infections safety and antiviral activity of motavizumab, a respiratory syncytial virus (rsv)-specific humanized monoclonal antibody, when administered to rsv-infected children motavizumab for prophylaxis of respiratory syncytial virus in high-risk children: a noninferiority trial targeting rsv with vaccines and small molecule drugs rna interference-mediated silencing of the respiratory syncytial virus nucleocapsid defines a potent antiviral strategy the promise, pitfalls and progress of rna-interference-based antiviral therapy for respiratory viruses a randomized, double-blind, placebo-controlled study of an rnai-based therapy directed against respiratory syncytial virus rna interference therapy in lung transplant patients infected with respiratory syncytial virus alnylam presents complete results from phase iib trial with aln-rsv01, an inhaled rnai therapeutic for the treatment of respiratory syncytial virus (rsv) infection sialidase fusion protein as a novel broad-spectrum inhibitor of influenza virus infection das181, a novel sialidase fusion protein, protects mice from lethal avian influenza h5n1 virus infection novel pandemic influenza a(h1n1) viruses are potently inhibited by das181, a sialidase fusion protein a phase ii study of das181, a novel host directed antiviral for the treatment of influenza infection a recombinant sialidase fusion protein effectively inhibits human parainfluenza viral infection in vitro and in vivo clinical potential of das181 for treatment of parainfluenza-3 infections in transplant recipients treatment of parainfluenza 3 infection with das181 in a patient after allogeneic stem cell transplantation das181 treatment of severe parainfluenza type 3 pneumonia in a lung transplant recipient deaths associated with human adenovirus-14p1 infections a community-based outbreak of severe respiratory illness caused by human adenovirus serotype 14 anti-viral drugs for human adenoviruses differential susceptibility of adenovirus clinical isolates to cidofovir and ribavirin is not related to species alone safety and tolerability of cidofovir in high-risk pediatric patients how i treat adenovirus in hematopoietic stem cell transplant recipients ether lipid-ester prodrugs of acyclic nucleoside phosphonates: activity against adenovirus replication in vitro hexadecyloxypropyl-cidofovir, cmx001, prevents adenovirus-induced mortality in a permissive, immunosuppressed animal model safety and efficacy of cmx001 as salvage therapy for severe adenovirus infections in immunocompromised patients first pharmacokinetic and safety study in humans of the novel lipid antiviral conjugate cmx001, a broad-spectrum oral drug active against double-stranded dna viruses efficacy and safety of oral pleconaril for treatment of colds due to picornaviruses in adults: results of 2 double-blind, randomized, placebo-controlled trials relationship of pleconaril susceptibility and clinical outcomes in treatment of common colds caused by rhinoviruses rhinovirus both in vitro and in an experimental human infection model biota press release. hrv phase iib study achieves primary endpoint prevention of natural colds by contact prophylaxis with intranasal alpha 2-interferon prophylactic efficacy of intranasal alpha 2-interferon against rhinovirus infections in the family setting positive phase ii asthma clinical trial data t-705 (favipiravir) and related compounds: novel broad-spectrum inhibitors of rna viral infections genetic screens for the control of influenza virus replication: from meta-analysis to drug discovery identification of common biological pathways and drug targets across multiple respiratory viruses based on human host gene expression analysis acknowledgements i thank dr. michael ison for helpful comments on the draft manuscript and lisa cook for assistance with manuscript preparation. since 2008 fgh has served as an unpaid consultant to multiple companies involved in developing antivirals for respiratory viral infections including several investigational agents discussed in this article (synairgen; nexbio, now ansun). key: cord-312952-9gbb4own authors: wardzyńska, aleksandra; pawełczyk, małgorzata; głobińska, anna; makowska, joanna s.; kowalski, marek l. title: the profile of respiratory pathogens in induced sputum of elderly and non-elderly asthmatics date: 2020-01-20 journal: cent eur j immunol doi: 10.5114/ceji.2019.92790 sha: doc_id: 312952 cord_uid: 9gbb4own introduction: respiratory pathogens are thought to be involved in the pathogenesis and exacerbations of asthma at all ages; however, little is known about the airway microbiome in the elderly. aim of the study: to identify respiratory pathogens in the induced sputum (is) of elderly asthmatics, and to determine the association between pathogens and the markers of asthma activity. material and methods: twenty-nine subjects with stable asthma, 15 above 65 years of age and 14 aged 30-49 years, underwent clinical evaluation, fractional exhaled nitric oxide measurement, and sputum induction. pathogens were detected by multiplex reverse transcription polymerase chain reaction. the periostin concentration of is supernatants was measured by enzyme-linked immunosorbent assay. serum eosinophil cationic protein and total ige levels were measured by immunocap. results: elderly patients, as compared to non-elderly, had significantly higher eosinophilia in is, although other markers of eosinophilic inflammation were comparable. half of the subjects were positive for haemophilus influenzae. chlamydophila pneumoniae was found in two subjects. respiratory viruses were detected in more than 70% of patients. the detection rates and profiles of atypical bacteria and respiratory viruses were similar in both groups. only in the elderly asthmatics was influenza a positivity associated with lower predicted fvc%, rsv a positivity connected with decreased tige concentration, and rsv b positivity related to a lower percentage of lymphocytes in is. conclusions: despite the existence of differences in some clinical and inflammatory characteristics of asthma between elderly and non-elderly asthmatics, the pathogen detection rates in the is from the two groups are similar. ageing is often characterised by a progressive decline in immune response, resulting in increased susceptibility to infections and poor response to vaccination [1] . clinical observations suggest that lower respiratory tract infections may contribute to the pathogenesis of asthma and are important triggers for asthma exacerbations [2] . asthma in the elderly seems to be a distinct phenotype with higher severity and exacerbation rates [3] , which may be associated with the deterioration of immune function observed in the airways in the aged population [4] . the airway microbiome might have a profound role on the development, persistence, and clinical course of chronic inflammatory diseases like asthma and chronic obstructive pulmonary disease [5] . the presence of respiratory viruses during childhood has been identified as a significant risk factor for asthma in adolescences and adults [6] , while similar observations have been made for atypical bacteria [7] . viruses, such as rhinovirus, respiratory syncytial virus, or parainfluenza viruses, are the most common causative factors for asthma exacerbation [8] . in clinically stable periods of asthma, both viruses and bacteria are detected in the airways [9, 10] , but the significance of the persistent presence of those pathogens in the airways remains unclear. while the majority of studies indicate that the detection rate of respiratory viruses in patients with asthma and healthy subjects is similar [10] , they have been found to differ with regard to the bacterial composition of the airways [11] . information on the respiratory pathogens of the lower airways and their relationship with the nature of asthma in elderly asthmatics is limited, mostly because of the risk and inconvenience associated with direct and invasive methods of airway lumen sampling. induced sputum (is) allows for non-invasive assessment of lower airway inflammation and can be employed to detect respiratory pathogens in the lower respiratory tract. this technique was chosen for the present study, to test the hypothesis that elderly patients with asthma display a different profile of respiratory pathogens in the airways as compared to non-elderly asthmatics, and that this profile may be related to local airway and/or systemic inflammation. twenty-nine clinically stable, non-smoking patients with moderate or severe persistent asthma, 15 elderly (above 65 years of age) and 14 non-elderly (aged 30-49 years), were included to the study. none of the individuals demonstrated any symptoms of infection or had used antibiotics or oral corticosteroids for six weeks prior to the procedure. the level of asthma control was established according to global initiative for asthma (gina) 2011 guidelines [12] and assessed by the asthma control test (act) [13] . severe asthma was defined according to the american thoracic society (ats) workshop 2000 criteria [14] . spirometry was performed according to european respiratory society (ers) standards [15] , using a pneu-mo rs spirometer (abcmed artmed, kraków, poland). the panel of skin prick tests (allergopharma, reinbek, germany) included the following allergens: dermatophagoides pteronyssinus, dermatophagoides farinae, cat, dog, rabbit, hamster, guinea pig, rat, swine, birch, grass mix, mugwort, plantain, alternaria tenuis, and cladosporium herbarum. a positive result was defined as a wheal of 3 mm or more in diameter. atopy was diagnosed as the presence of at least one positive skin test. feno was measured by using the online single breath method with niox (aerocrine, solna, sweden), according to ats guidelines [16] . the measurement was performed before the sputum induction. sputum induction was performed according to pin et al. with some modifications [17, 18] . briefly, patients received 200 µg of salbutamol from a metered dose inhaler followed by inhalation of increasing doses of hypertonic saline solution (3%, 4%, 5%) using an ultrasonic nebuliser ultraneb (devilbiss health care inc., somerset, usa). patients were asked to expectorate sputum after gargling mouthwash. spirometry was performed before the procedure, 15 minutes af-ter salbutamol inhalation, and every seven minutes after the start of the saline nebulisation. when their fev1 decreased by 20% or more, the sputum induction was stopped. the sputum sample was kept on ice and processed within two hours. dithiothreitol (dtt) (sigma aldrich, st louis, usa) was added to the selected plaques of sputum and mixed for 15 min on ice. the sample was filtered using a 52-µcm nylon gauze (surtex, lodz, poland). the total cell count was measured using a haemocytometer and centrifuged. the cell pellets were resuspended with phosphate-buffered saline (pbs), and 75 µl of this cell suspension was used to perform the cytospin slides stained with rapihem solution, a variant of may-grünwald-giemsa staining (aqua-med, lodz, poland). only smears with less than 50% of non-squamous cells were considered to be adequate for the differential cell counting. on each slide, 300 cells were counted, and the macrophage, lymphocyte, neutrophil, and eosinophil values were expressed as percentages of the total number of inflammatory cells. the supernatants were stored frozen at -80° for further analysis. viral dna or rna was extracted from sputum using a qiaamp ® minelute ® virus spin kit (qiagen), according to the manufacturer's instructions. to synthesise cdna, reverse transcription was performed using a revertaid h minus first-strand cdna synthesis kit (thermo fisher scientific inc., waltham, usa). each cdna was subjected to multiplex pcr using a seeplex rv15 ace detection kit (seegene), according to the manufacturer's instructions. the multiplex pcr detected rhinoviruses (hrv) and enteroviruses (hev), parainfluenza viruses 1 (piv1), 2 (piv2), 3 (piv3), and 4 (piv4), influenza viruses a (flua) and b (flub), respiratory syncytial viruses a (rsva) and b (rsvb), coronaviruses (229e/nl63 and oc43), adenovirus (adv), human metapneumovirus (mpv), and human bocaviruses 1/2/3/4 (hbov). the multiplex pcr products were visualised by electrophoresis on 2% agarose gel. the serum concentrations of total ige (ku/l) were determined by immunocap total ige (phadia ab, uppsala, sweden), and the concentrations of ecp (µg/l) were determined using immunocap ecp (phadia ab, upsala, sweden), according to the manufacturer's instructions. periostin was measured in the is supernatants using an enzyme-linked immunoassay (uscn life science inc., wuhan, hubei, china), according to the manufacturer's instructions. comparisons between the groups were performed with the non-parametric mann-whitney u test. spearman rank correlation was used to evaluate the correlation between variables. results are expressed as median and 25 th to 75 th percentiles. the statistical analysis was performed using statistica (statsoft, usa); p values < 0.05 were accepted as statistically significant. the study was approved by the local bioethics committee, and all study subjects provided their informed consent. on average, the elderly patients with asthma had significantly lower respiratory function, as well as less current asthma control according to gina criteria and a lower act score, although both groups demonstrated similar disease severity based on the ats workshop 2000 criteria ( table 1 ). the distribution of inflammatory cells in the induced sputum varied depending on the age: older patients had a higher mean percentage of eosinophils (p < 0.05) and lymphocytes (p < 0.05), and a lower percentage of macrophages (p < 0.05) in is (table 2 ). however, the elderly and non-elderly asthmatics had similar mean feno levels and concentrations of such eosinophilic inflammation markers as periostin in is, ecp, and total ige in serum (table 1 ). there the molecular technique revealed the presence of s. pneumoniae in the is from all asthmatic patients, both elderly and non-elderly, and h. influenzae in half the patients from each group (table 3) . chlamydophila pneumoniae was detected in one subject from the elderly group and one from the non-elderly group. all sputa were negative for m. pneumoniae, legionella, and b. pertussis. respiratory viruses were detected in the induced sputum from the majority of patients in both groups: in 11 (73.3%) elderly and 10 (71.4%) non-elderly individuals. the most commonly detected virus was flu a followed by rsv a and rsv b (table 3) . no differences were observed in the detection rates of viruses between elderly and non-elderly patients. neither asthma severity according to gina or ats criteria nor current asthma control assessed by the act were found to correlate with the presence of respiratory pathogens in the is of asthmatics patients. in elderly asthmatics, but not in the non-elderly group, a few associations between the presence of specific respiratory viruses in the is and respiratory function/immunological parameters were found. flu a-positive patients had lower fvc% predicted than negative ones (81.8% pred. ±19.9 vs. 100.4 % pred. ±12,9; p < 0.05). the presence of rsv b was associated with a lower percentage of lymphocytes in the is (28.7% ±12.6 vs. 5.5 % ±9.5; p < 0.05), and the rsv a-positive patients had lower tige concentration in the serum than rsv a-negative elderly individuals (19.2 ku/l ±17.7 vs. 85.2 ku/l ±64.1; p < 0.05). this study is the first to use the is technique to compare detection rates of respiratory pathogens in the airways of elderly and non-elderly patients with asthma. the total number of positive samples and the profile of respiratory pathogens detected in the induced sputum by molecular techniques were found to be similar in elderly and non-elderly individuals. bronchial asthma, although heterogeneous, has been considered to be, on average, more severe in the elderly population and associated with increased exacerbation rates mostly related to respiratory infections [19] . our elderly patients tended to have, on average, more severe disease, as expressed by lower respiratory function, and less current asthma control over the previous four weeks, demonstrated by gina criteria and a lower mean act score. in addition, the elderly patients had higher sputum eosinophilia and lymphocytosis levels than the non-elderly patients, suggesting the presence of different types of airway inflammation in senior individuals. although a previous study documents a tendency toward neutrophilic inflammation in the is from elderly asthmatics, no correlation between neutrophilia and severity of the disease was found [20] . another study found older asthmatics with fixed obstruction to have a higher percentage of eosinophils and elevated levels of ecp in the induced sputum compared to their peers with chronic obstructive pulmonary disease (copd), despite an increased percentage of neutrophils in the sputum [21] . thus, the increased percentage of eosinophils in the is of elderly patients in our study may reflect the proposed inflammatory profile in the airway mucosa of this population of asthmatics. on the other hand, mean levels of surrogate markers of the airway eosinophilic inflammation (feno, ecp, and sputum periostin) were similar in both groups, not confirming differences in the type of inflammation between groups. ageing has been associated with several immune abnormalities in both the innate and adaptive immune systems, and consequently with deceased immune response to infections [1] . in this context, the observation of similar detection rate and profile of respiratory pathogens in the induced sputum is rather unexpected. however, our patients had stable asthma during the sputum sampling, and one cannot exclude that the difference in the microbiome between elderly and non-elderly asthmatics might be seen during the disease exacerbations although respiratory pathogens have been detected with increased frequency during asthma exacerbations, they may persist in the airways of asthmatics between exacerbation episodes, and it has been postulated that latent or persistent infection may contribute to the chronicity of the airway inflammation [22] . however, a cross-sectional study by harju et al. [9] reports similar detection rates of respiratory viruses in the is of patients with clinically stable asthma and healthy controls. similarly, in a prospective study by tucharelli et al. [23] the detection rates of 14 respiratory viruses were comparable in clinically stable asthmatics and in healthy controls at three time points. accordingly, in non-bronchoscopic bronchoalveolar lavage (bal), the percentage of viral detection did not vary between asthmatic children and controls [24] . in contrast, the presence of hrv in the lung tissue samples from stable patients was found to be higher in asthmatics than in healthy controls [25] . thus, the presence or absence of respiratory pathogens in bal or induced sputum may not reflect the persistence of infection of the airway mucosa. although the detection rate and profile of respiratory viruses in is was similar in elderly and non-elderly patients with asthma, the presence of pathogens was associated with some clinical characteristics only in older subjects. the presence of influenza a virus in the airways of stable asthma patients (without any clinical signs of infection) was associated with lower respiratory function and rsv b with a lower percentage of lymphocytes in the is. in a study by woś et al. [25] the presence of hrv was associated with lower spirometric parameters as compared to hrv-negative subjects. furthermore, in our study the presence of rsv b was associated with a lower percentage of lymphocytes in the is and the rsv a with lower total ige concentration in the serum in elderly individuals; however, the significance of this findings remains unclear. our study has several limitations. the groups were relatively small, which may have affected the variability of results. the samples were collected during late autumn and winter, which may be connected with more frequent exposure to respiratory infections. qualitative rt-pcr did not allow active replication indicating active infection to be distinguished from residuals of viral or bacterial dna or rna from past infections. the discrepancies between the positivity rates of the respiratory bacteria and viruses shown in the above-mentioned studies may be related to different methods of obtaining samples and detecting pathogens. supernatants from induced sputum were used as source material, and multiplex rt pcr assays were used for analysis. harju et al. [9] report higher detection rates of viruses in induced sputum and throat swab samples compared to gelatine-filtered expired air, ebc, or nasal swabs. however, viral identification was not consistent between these methods or during the follow-up period. in a study by falsey et al. [26] the identification of bordetella pertussis in is differed between supernatants and cells from patients with exacerbation of asthma. hence, our results should be further confirmed in studies involving larger patient populations and, if possible, more direct airway sampling techniques. in summary, our findings indicate that, despite some differences in the clinical and inflammatory characteristics of asthma between elderly and non-elderly patients, the microbial composition of is samples from stable patients is similar. inflammation & autoimmunity in human ageing: dendritic cells take a center stage respiratory infections precede adult-onset asthma asthma in the geriatric population the role of immunity and inflammation in lung senescence and susceptibility to infection in the elderly the role of the bacterial microbiome in lung disease rhinovirus illnesses during infancy predict subsequent childhood wheezing occurrence of chlamydia trachomatis and chlamydia pneumoniae in paediatric respiratory infections is asthma an infectious disease? new evidence pathogenic bacteria and viruses in induced sputum or pharyngeal secretions of adults with stable asthma asthma-associated differences in microbial composition of induced sputum disordered microbial communities in asthmatic airways global strategy for asthma management and prevention 2011 (update) development of the asthma control test: a survey for assessing asthma control proceedings of the ats workshop on refractory asthma: current understanding, recommendations, and unanswered questions standardisation of spirometry ats workshop proceedings: exhaled nitric oxide and nitric oxide oxidative metabolism in exhaled breath condensate: executive summary use of induced sputum cell counts to investigate airway inflammation in asthma wskazówki metodologiczne polskiego towarzystwa chorób płuc odnośnie do wykonywania i oceny plwociny indukowanej microbes and asthma: the missing cellular and molecular links expiratory flows and airway inflammation in elderly asthmatic patients similarity and differences in elderly patients with fixed airflow obstruction by asthma and by chronic obstructive pulmonary disease role of latent viral infections in chronic obstructive pulmonary disease and asthma repeated virus identification in the airways of patients with mild and severe asthma during prospective follow-up respiratory viral infection in lower airways of asymptomatic children the presence of rhinovirus in lower airways of patients with bronchial asthma yield of sputum for viral detection by reverse transcriptase pcr in adults hospitalized with respiratory illness the authors would like to thank mrs dorota żaromińska, mrs barbara szkudińska, and mrs marzanna jarzębska for their assistance. this study was supportthe profile of respiratory pathogens in induced sputum of elderly and non-elderly asthmatics key: cord-349287-mwj2qby4 authors: mackay, ian m.; arden, katherine e. title: mers coronavirus: diagnostics, epidemiology and transmission date: 2015-12-22 journal: virol j doi: 10.1186/s12985-015-0439-5 sha: doc_id: 349287 cord_uid: mwj2qby4 the first known cases of middle east respiratory syndrome (mers), associated with infection by a novel coronavirus (cov), occurred in 2012 in jordan but were reported retrospectively. the case first to be publicly reported was from jeddah, in the kingdom of saudi arabia (ksa). since then, mers-cov sequences have been found in a bat and in many dromedary camels (dc). mers-cov is enzootic in dc across the arabian peninsula and in parts of africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. the ksa is the focal point of mers, with the majority of human cases. in humans, mers is mostly known as a lower respiratory tract (lrt) disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. however, mers-cov has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. older males most obviously suffer severe disease and mers patients often have comorbidities. compared to severe acute respiratory syndrome (sars), another sometimesfatal zoonotic coronavirus disease that has since disappeared, mers progresses more rapidly to respiratory failure and acute kidney injury (it also has an affinity for growth in kidney cells under laboratory conditions), is more frequently reported in patients with underlying disease and is more often fatal. most human cases of mers have been linked to lapses in infection prevention and control (ipc) in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers (hcws) and higher exposures in those with occupations that bring them into close contact with camels. sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. sensitive, validated reverse transcriptase real-time polymerase chain reaction (rt-rtpcr)-based diagnostics have been available almost from the start of the emergence of mers. while the basic virology of mers-cov has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited. electronic supplementary material: the online version of this article (doi:10.1186/s12985-015-0439-5) contains supplementary material, which is available to authorized users. an email from dr ali mohamed zaki, an egyptian virologist working at the dr soliman fakeeh hospital in jeddah in the kingdom of saudi arabia (ksa) announced the first culture of a new coronavirus to the world. the email was published on the website of the professional emerging diseases (promed) network on 20 th september 2012 [1] (fig. 1) and described the first reported case, a 60 year old man from bisha in the ksa. this information led to the rapid discovery of a second case of the virus, this time in an ill patient in the united kingdom, who had been transferred from qatar for care [2] . the new virus was initially called novel coronavirus (ncov) and subsequentlty entitled the middle east respiratoy syndrome coronavirus (mers-cov). as of 2 nd of september 2015, there have been 1,493 detections of viral rna or virus-specific antibodies across 26 countries (additional file 1: figure s1 ) confirmed by the world health organization (who), with over a third of the positive people dying (at least 527, 35 %) [3] . since that first report, a slow discovery process over the following two to three years revealed a virus that had infected over 90 % of adult dromedary camels (dc; camelus dromedarius) in the ksa [4] , also dcs across the arabian peninsula and parts of africa that are a source of dc imports for the ksa [5] . to date, mers-cov has not been detected in dcs tested in zoos or herds from other parts of the world [6] [7] [8] [9] . occasionally, virus is transmitted from infected dcs to exposed humans. subsequent transmission to other humans requires relatively close and prolonged exposure [10] . the first viral isolate was patented and concerns were raised that this would restrict access to both the virus and to viral diagnostics [11, 12] . however, sensitive, validated reverse transcriptase real-time polymerase chain reaction (rt-rtpcr)-based diagnostics were quickly described and virus was made freely available subject to routine biosafety considerations [13] . subsequent epidemiology and research has identified the cell receptor as exopeptidase dipeptidyl peptidase 4 (dpp4; also called cd26); that mers-cov has a broad tropism, replicating better in some cells lines and eliciting a more proinflammatory response than sars-cov; is widespread in dcs; has the potential to infect other animals and that mers kills its human host more often than sars did (20-40 % versus 9 % for sars [14] ) [15] [16] [17] [18] [19] . in humans, overt disease was given the name middle east respiratory syndrome, with the acronym mers. from intermittent animal-to-human spill-over events, the mers-cov spreads sporadically among people, causing more severe disease among older adults, especially males, with pre-existing diseases. the spread of mers-cov among humans has often been associated with outbreaks in hospitals, with around 20 % of all cases to date involving healthcare workers (hcws). although dcs appear to suffer the equivalent of a 'common cold' from mers-cov infection, in humans, the virus can be a more serious and opportunistic pathogen associated with the death of up to 40 % of reported cases. it has yet to be established whether infections thought to have been acquired from an animal source produce a more severe outcome than those spread between humans [20] . studies have established that the mean incubation period for mers is five to six days, ranging from two to 16 days, with 13 to 14 days between when illness begins in one person and subsequently spreads to another [21] [22] [23] [24] . among those with progressive illness, the median time to death is 11 to 13 days, ranging from five to 27 days [23, 24] . fever and gastrointestinal symptoms may form a prodrome, after which symptoms decline, only to be followed by a more severe systemic and respiratory syndrome [25, 26] . the first who case definition [27] defined probable cases of mers based on the presence of febrile illness, cough and requirement for hospitalization with suspicion of lower respiratory tract (lrt) involvement. it also included roles for contact with a probable or confirmed case or for travel or residence within the arabian peninsula. if strictly adhered to, only the severe syndrome would be subject to laboratory testing, which was the paradigm early on [21] . from july 2013, the revised who case definition included the importance of seeking out and understanding the role of asymptomatic cases and from june 2014, the who definition more clearly stated that a confirmed case included any person whose sample was rt-pcr positive for mers-cov, or who produced a seroconversion, irrespective of clinical signs and symptoms. [28] [29] [30] apart from the who and the ksa ministry of health reports, asymptomatic or subclinical cases of mers-cov infection were documented in the scientific literature although not always as often as occurred early on [31, 32] . the ksa definition of a case became more strict on 13 th may 2014, relying on the presence of both clinical features and laboratory confirmation [33] . testing of asymptomatic people was recommended against from december 2014 [34] , reinforced by a case definition released by the ksa ministry of health in june 2015 [35] . the ksa has been the source of 79 % of human cases. severe mers is notable for its impact among older men with comorbid diseases including diabetes mellitus, cirrhosis and various lung, renal and cardiac conditions [36] [37] [38] . interestingly in june 2015, an outbreak in south korea followed a similar distribution [39, 40] . among laboratory confirmed cases, fever, cough and upper respiratory tract (urt) signs and symptoms usually occur first, followed within a week by progressive lrt distress and lymphopaenia [37] . patients often present to a hospital with pneumonia, or worse, and secondary bacterial infections have been reported [37, 41] . disease can progress to acute respiratory distress syndrome and multiorgan system failure [37] . mers has reportedly killed approximately 35 % of all reported cases, 42 % of cases in the ksa, yet only 19 % of cases in south korea, where mortality ranged from 7 % among younger age groups to 40 % among those aged 60 years and above [42] ; all may be inflated values with asymptomatic or mild infections sometimes not sought or not reported [34] . general supportive care is key to managing severe cases [43] . children under the age of 14 years are rarely reported to be positive for mers-cov, comprising only 1.1 % (n = 16) of total reported cases. between 1 st september 2012 and 2 nd december 2013, a study described the then tally of paediatric cases in the ksa, which stood at 11 (two to 16 years of age; median 13 years); nine were asymptomatic (72 %) and one infant died [44] . in amman, jordan, 1,005 samples from hospitalized children under the age of two years with fever and/or respiratory signs and symptoms were tested but none were positive for mers-cov rna, despite being collected at a similar time to the first known outbreak of mers-cov in the neighbouring town of al-zarqa [45] . a second trimester stillbirth occurred in a pregnant woman during an acute respiratory illness and while not rt-rtpcr positive, the mother did subsequently develop antibodies to mers-cov, suggestive of recent infection [46] . her exposure history to a mers-cov rt-rtpcr positive relative and an antibody-reactive husband, her incubation period and her symptom history met the who criteria for being a probable mers-cov case [46] . diagnostic methods were published within days of the promed email announcing the first mers case [47] , including several now gold standard in-house rt-rtpcr assays (fig. 2 ) as well as virus culture in vero and llc-mk2 cells [18, 47, 48] . a colorectal adenocarcinoma (caco-2) epithelial cell line has since been recommended for isolation of infections mers-cov [49] . we previously [18] .). open reading frames are indicated as yellow rectangles bracketed by terminal untranslated regions (utr; grey rectangles). fs-frame-shift. predicted regions encompassing recombination break-points are indicated by orange pills. created using geneious v8.1 [211] and annotated using adobe illustrator. beneath this is a schematic depicting the location of rt-pcr primers (blue arrows indicate direction) and oligoprobes (green rectangles) used in the earliest rt-rtpcr screening assays and conventional, semi-nested (three primers) rt-pcr confirmatory sequencing assays [47, 48] . publication order is noted by first [27 th september 2012; red] and second [6 th december 2012; orange] coloured rectangles; both from corman et al. [47, 48] those assays recommended by the who are highlighted underneath by yellow dots [53] . the nseq reverse primer has consistently contained one sequence mismatch with some mers-cov variants. an altered version of that from mackay im, arden ke. middle east respiratory syndrome: an emerging coronavirus infection tracked by the crowd. virus res 2015 vol 202:60-88 with permission from elsevier [5] reviewed the broad tropism of mers-cov [5] . however, as is well described, cell culture is a slow, specialised and insensitive method [50] while pcr-based techniques are the preferred method for mers-cov detection. the first open reading frames (orf 1a and 1b; fig. 2 ) have become a key diagnostic and taxonomic target for cov species identification. with less than 80 % identity between the amino acid sequence of mers orf 1ab and betacoronavirus relatives, tylonycteris bat hku4 and pipistrellus bat hku5, it can be concluded that it is a novel and distinct virus. mers-cov is predicted to encode ten open reading frames with 5' and 3' untranslated regions [51] . the structural proteins include the spike (s), envelope (e), membrane (m) and nucleocapsid (n) [52] . the products of orf1a and orf1b are predicted to encode nonstructural proteins. the majority of specimen testing to date has employed validated rt-rtpcr assays shown to be sensitive and specific [47, 48, 53] . the realstar® kit uses these whorecommended assays [54] . the target sequences of these screening assays have not changed among genomes examined until at least mid-2015 (imm observation). other rt-rtpcr assays have been developed and validated for use as laboratory-based diagnostic tools [55] [56] [57] . additionally, loop-mediated [58, 59] or recombinase polymerase [60] isothermal assays have been designed for field deployment. the detection of mers-cov antigen has not been common to date but the combination of short turnaround time from test to result, high throughput and identification of viral proteins makes this an attractive option. detection of viral proteins rather than viral rna indicates the likely presence of infectious virus. the first rapid immunochromatographic tool described could detect recombinant mers-cov nucleocapsid protein from dc nasal swabs with 94 % sensitivity and 100 % specificity compared to rt-rtpcr [61] . a different approach used a monoclonal antibody-based capture elisa targeting the mers-cov nucleocapsid protein with a sensitivity of 10 3 tcid 50 and 100 % specificity [62] . demonstration of a seroconversion to a mers-cov infection meets the current who definition of a case so optimized and thoroughly validated sero-assays employed alongside good clinical histories are useful to both identify prior mers-cov infection and help support transmission studies. because serology testing is, by its nature, retrospective, it is usual to detect a viral footprint, in the form of antibodies, in the absence of any signs or symptoms of disease and often in the absence of any viral rna [63] . strategic, widespread sero-surveys of humans using samples collected after 2012 are infrequent. much of the arabian peninsula and all of the horn of africa lack baseline data describing the proportion of the community who may have been infected by a mers-cov. however, sero-surveys have had widespread use in elucidating the role of dcs as a transmission source for mers-cov. because of the identity shared between dc and human mers-cov (see molecular epidemiology: using genomes to understand outbreaks), serological assays for dc sero-surveys should be transferrable to human screening with minimal re-configuration. also, no diagnostically relevant variation in neutralization activity have been found from among a range of circulating tested mers-cov isolates and sera, so whole virus or specific protein-based sero-assays should perform equivalently in detecting serological responses to the single mers-cov serotype [49] . the development of robust serological assays requires reliable panels of wellcharacterized animal or human sera, including those positive for antibodies specific to mers-cov, as well as to likely sources of cross-reaction [64] . obtaining these materials was problematic and slowed the development and commercialization of antibody detection assays for human testing [64] . a number of commercial elisa kits, immunofluorescent assays (ifa) kits, recombinant proteins and monoclonal antibodies have been released [31, [65] [66] [67] [68] . initially, conventional ifas were used for human sero-surveys. these relied on mers-cov-infected cell culture as an antigen source, detecting the presence of human anti-mers-cov igg, igm or neutralizing antibodies in human samples [18, 48, 69] . no sign of mers-cov antibodies was found among 2,400 sera from patients visiting hospital in jeddah, from 2010 through 2012, prior to the description of mers-cov [18] . nor did ifa methods detect any sign of prior mers-cov infection among a small sample of 130 healthy blood donors from another hospital in jeddah (collected between jan and dec 2012) [70] . of 226 slaughterhouse workers, only eight (3.5 %) were positive by ifa, and those sera could not be confirmed by virus neutralization (nt) test. the study indicated that hcov-hku1 was a likely source of crossreactive antigen in the whole virus ifa [70] . whole virus mers-cov ifa also suffered from some cross-reactivity with convalescent sars patient sera and this could not be resolved by an nt test which was also cross-reactive [71] . ifa using recombinant proteins instead of whole-virus ifa, has been shown to be a more specific tool [31] . since asymptomatic zoonoses have been posited [72] , an absence of antibodies to mers-cov among some humans who have regular and close contact with camels may reflect the rarity of actively infected animals at butcheries, a limited transmission risk associated with slaughtering dcs [70] , a pre-existing cross-protective immune status or some other factor(s) resulting in a low risk of disease and concurrent seroconversion developing after exposure in this group. ifa using recombinant proteins instead. some sero-assays have bypassed the risks of working with infectious virus by creating transfected cells expressing recombinant portions of the mers-cov nucleocapsid and spike proteins [48, 73] , or using a recombinant lentivirus expressing mers-cov spike protein and luciferase [74, 75] . a pseudo particle neutralization (ppnt) assay has seen widespread used in animal studies and was at least as sensitive as the traditional microneutralization (mnt) test. [10, 74, [76] [77] [78] ] studies using small sample numbers and ppnt found no evidence of mers-cov neutralizing antibody in sera from 158 children with lrt infections between may 2010 and may 2011, 110 sera from 19 to 52 year old male blood donors and 300 selfidentified animal workers from the jazan region of the ksa during 2012 [79, 80] . similarly, a study of four herdsmen in contact with an infected dc herd in al-ahsa, eight people who had intermittent contact with the herd, 30 veterinary surgeons and support staff who were not exposed to the herd, three unprotected abattoir workers in al-ahsa and 146 controls who were not exposed to dcs in any professional role, found none with serological evidence of past mers-cov infection using the ppnt assay [10] . a delay in the neutralizing antibody response to mers-cov infection was associated with increased disease severity in south korea cases with most responses detectable by week three of illness while others, even though disease was severe, did not respond for four or more weeks [81] . the implications for our ability to detect any response in mild or asymptomatic cases was not explored but may be a signifcant factor in understanding exposure in the wider community. a jordanian outbreak of acute lrt disease in a hospital in 2012 was retrospectively found to be associated with mers-cov infection, initially using rt-rtpcr, but subsequently, and on a larger scale, through positivity by elisa and ifa or mnt test. [46, 82, 83] this outbreak predated the first case of mers in the ksa. the elisa used a recombinant nucleocapsid protein from the group 2 betacoronavirus bat-cov hku5 to identify antibodies against the equivalent crossreactive mers-cov protein [71] . it was validated using 545 sera collected from people with prior hcov-oc43, hcov-229e, sars-cov, hcov-nl63, hrv, hmpv or influenza a(h1n1) infections but was reportedly less specific than the recombinant ifa discussed above. it was still considered an applicable tool for screening large sample numbers [82] . a protein microarray expressing the s1 protein subunit has also been validated and widely used for dc testing [5, 84] . detection of mers-cov infection using elisa or s1 subunit protein microarray [84] is usually followed by confirmatory ifa and/ or a plaque-reduction neutralization (prnt) [69, 70, 85] or mnt test. [74, 85, 86] this confirmatory process aims toensure the antibodies detected are able to specifically neutralize the intended virus and are not more broadly reactive to other coronaviruses found in dcs (bovine cov, bcov) or humans (hcov-oc43, hcov-229e, hcov-nl63, hcov-hku1, sars-cov). in the largest study of human sera, a tiered diagnostic process assigned both recombinant ifa and recombinant elisa positive sera to 'stage 1' seropositivity. a stage 2 seropositive result additionally required a suitably titred prnt result [87] . the study found 15 sera collected in 2012 to 2013 from 10,009 (0.2 %) people in 13 ksa provinces contained mers-cov antibodies, but significantly higher proportions in occurred in camel shepherds (two of 87; 2.3 %) and slaughterhouse workers (five of 140; 3.6 %) [87] . contemporary surveys are needed. mers-cov does not appear to be easily transmitted from dcs to humans, or perhaps it is [72] , but generally does not trigger a detectable immune response if only mild disease or asymptomatic infection results. serology assays are in need of further validation in this area so care is required when moving newly developed diagnostic serology algorithms from a research setting to one that informs public health decisions. this was reinforced when a false positive us case, purported to have been infected after a handshake and two face-to-face meetings, did not withstand further confirmatory analysis using a more specific, nt assay and was subsequently retracted [88, 89] . the who recommends sampling from the lrt for mers-cov rt-rtpcr testing, especially when sample collection is delayed by a week or more after onset of symptoms. [53] lrt samples are also best for attempting isolation of infectious virus, although the success of culture is reduced when disease persists [49] . recommended sample types include bronchoalveolar lavage (bal), tracheal/tracheobronchial aspirate, pleural fluid and sputum [53, 90] . fresh samples yield better diagnostic results than refrigerated material [69] and if delays in testing of ≥72 h are likely, samples (except for blood) should be frozen at −70°c [90] . if available, lung biopsy or autopsy tissues can also be tested [53] . the urt is a less invasive and more convenient sampling site however, and an oropharyngeal and throat swab or a nasopharyngeal aspirate/wash are recommended when urt sampling is to be conducted [90] . paired sera, collected two to three weeks apart are preferable for serological testing while a single sample is suggested to be sufficient if collected two weeks after onset of disease or a single serum collected during the first 10-12 days if conducting rt-rtpcr [53, 90] . human urine and stool have been found to contain mers-cov rna 12 to 26 days after symptom onset [25, 69, 91] and are listed as samples that should be considered [53, 90] . in two cases that arrived in the netherlands, urine was rt-rtpcr negative but faeces was weakly positive and sera were rt-rtpcr positive for five days or more [25] . the finding of mers-cov viral rna in serum provides an avenue for retrospective pcr-based studies if respiratory samples are unavailable [83] . rnaaemia may also correlate with disease severity; signs of virus were cleared from the serum of a recovered patient, yet lingered until the death of another [92] . clinically suspected mers cases may return negative results by rt-rtpcr. data have shown one or more negative urt samples may be contradicted by further urt sampling or the use of lrt samples, which is preferred [2, 43, 93] . higher viral loads occur in the lrt compared to the urt. [22, 69, 88, 94] this fits with the observation that the majority of disease symptoms are reported to manifest as systemic and lrt disease [21] . however, on occasion, even lrt specimens from mers cases may initially be negative, only to later become positive by rt-pcr [95] . this may be due to poor sampling when a cough is absent or non-productive or because the viral load is low [95] . despite this both the largest human mers-cov studies [32, [96] [97] [98] and smaller ones [22, 25, 99] , use samples from the urt. it is then noteworthy that one study reported an association between higher loads in the urt and worse clinical outcome including intensive care and death [94] . at writing, no human data exist to define whether the virus replicates solely or preferentially in the lrt or urt, or replicates in other human tissues in vivo although mers-cov rna has been detected from both the urt and lrt in a macaque monkey model [100] .the distribution of dpp4 in the human upper airways is also not well described. individual human case studies report long periods of viral shedding, sometimes intermittently and not necessarily linked to the presence of disease symptoms. [25, 69, 99, 101] in one instance, a hcw shed viral rna for 42 days in the absence of disease [99] . it is an area of high priority to better understand whether such cases are able to infect others. over three quarters of mers cases shed viral rna in their lrt specimens (tracheal aspirates and sputum) for at least 30 days, while only 30 % of contacts were still shedding rna in their urt specimens [91, 102] . in the only study to examine the effect of sample type on molecular analysis, 64 nasopharyngeal aspirates (npa; an urt sample), 30 tracheal aspirates, 13 sputa and three bal were examined. the tracheal aspirates and bal returned the highest viral load values followed by npa and sputum. unsurprisingly, higher viral loads generally paralleled whole genome sequencing and culture success and, in npa testing, were significantly correlated with severe disease and death [49, 94, 103] . this study demonstrated the importance of lrt sampling for whole genome sequencing. when tested, samples positive for mers-cov are often negative for other pathogens [2, 25, 93, 104] . however, many studies make no mention of additional testing for endemic human respiratory viruses [21, 23, 73, 105] . when viruses are sought, they have included human herpesvirus (hhv), rhinoviruses (hrv), enteroviruses (ev), respiratory syncytial virus (rsv), parainfluenzavirus types 1, 2 and 3 (pivs),influenzaviruses (ifvs), endemic hcovs, adenoviruses (advs) metapneumovirus (mpv) and influenza a\h1n1 virus; co-detections with mers-cov have been found on occasion [2, 22, 37, 69, 97] . bacterial testing is sometimes included (for example, for legionella and pneumococcus) but the impact of bacterial co-presence is also unclear [22, [104] [105] [106] . further testing of the lrt sample from the first mers case used ifa to screen for some viruses (negative for ifv, pivs, rsv and advs) and rt-pcr for others (negative for adv, evs, mpv and hhvs) [18] . rt-pcr also detected mers-cov. the who strongly recommends testing for other respiratory pathogens [53] but with this recommendation often discounted, there are limited data to address the occurrence and impact of co-infections or alternative viral diagnoses among both mers cases and their contacts. little is known of other causes of mers-like pneumonia in the ksa or of the general burden of disease due to the known classical respiratory viruses. testing of adult pilgrims performing the hajj in 2012 to 2014 has not detected any mers-cov. in 2012, nasal swabs from 154 pilgrims collected prior to leaving for or departing from the ksa were tested [47] . in 2013, testing was significantly scaled up with 5,235 nasopharyngeal swabs from 3,210 incoming pilgrims and 2,025 swabs from outgoing pilgrims tested [98] . it should be noted that most pilgrims arrived from mers-free countries. a further 114 swabs were taken from pilgrims with influenza-like illness [96, 107] . in earlier hajj gatherings, it was found that influenza viruses circulated widely, whilst other viruses, often rhinoviruses, circulated more selectively, interpreted as indicating their importation along with foreign pilgrims. [107] [108] [109] over time, increased influenza vaccination has been credited for a fall in the prevalence of influenza like illnesses among hajj pilgrims. [110] a lrt sample is often not collected for these studies [98, 107, 109] , so false negative findings are a possibility although little is known about the initial site of mers-cov infection and replication; it may have been assumed it was the lrt because disease was first noticed there but the urt may be the site of the earliest replication. in jeddah between march and july 2014 (hereafter called the jeddah-2014 outbreak; fig. 3 ), there was a rapid increase in mers cases, accompanied by intense screening; approximately 5,000 samples from in and around the region were tested in a month yielding around 140 mers-cov detections (~3 % prevalence) [111] . among 5,065 individuals sampled and tested across the ksa between october 2012 and september 2013,108 (2.1 %) detections were made in a hospital-centric population which included hospitalized cases (n = 2,908; 57.4 %), their families (n = 462; 9.1 %) and associated hcws (n = 1,695; 33.5 %) [32] . among the detections, 19 (17.8 %) were hcws and 10 (9.3 %) were family contacts [32] . the 2-3 % prevalence of active mers-cov infections is not dissimilar to the hospital-based prevalence of other human covs. [112] however, the proportion of deaths among those infected with mers-cov is much higher than that known for the hcovs nl63, hku1, 229e or oc43 in other countries, and even above that for sars-cov; it is not a virus that could reasonably be described as a "storm in a teacup". it is the low transmission rate that has prevented worldwide spread, despite many "opportunities". very early in the mers outbreak, some animals were highly regarded as either the reservoir or intermediate host(s) of mers-cov with three of the first five cases having contact with dcs [73, 113, 114] . today, animal mers-cov infections must be reported to the world organization for animal health as an emerging disease [115] . a summary of the first mers cases reported by the who defined animal contact with humans as being direct and within 10 days prior to symptom onset [20] . this definition made no specific allowance for acquisition from dcs through a droplet-based route, which is very likely route for acquisition of a virus that initially and predominantly causes respiratory disease [23] . camels are known to produce high levels of mers-cov rna in their urt and lungs [116] . providing support for a droplet transmission route and perhaps indicating the presence of rna in smaller, drier droplet nuclei, mers-cov rna was identified in a high volume air sample collected from a barn housing an infected dc [117] . the precise source from which humans acquire mers-cov remains poorly studied but it seems likely that animal and human behavioural factors may play roles (fig. 3) [118] . these factors may prove important for human cases who do not describe any dc contact [119] nor any contact with a confirmed case. whether the who definition of animal contact is sufficient to identify exposure to this respiratory virus remains unclear. wording focuses on consumption of dc products but does not specifically ascribe risk to a droplet route for acquisition of mers-cov from dc [120] . some mers patients are listed in who disease notices as being in proximity to dcs or farms, but the individuals have not described coming into contact with the animals. no alternative path for acquiring infection is reported in many of these instances. what constitutes a definition of "contact" during these interviews has been defined for one study [72] . despite this lack of clarity, the who consider that evidence linking mers-cov transmission between dcs to humans is irrefutable (fig. 4) [120] . the possibility that bats were an animal host of mers-cov was initially widely discussed because of the existing diversity of coronaviruses known to reside among them [121] [122] [123] [124] . conclusive evidence supporting bats as a source for human infections by mers-cov has yet to be found, but bats do appear to host ancestral representatives [53, 125] . however, these are not variants of the same virus nor always within the same phylogenetic lineage as mers-cov; they are each a genetically distinct virus. bat-to-human infection by mers-cov is a purely speculative event. the only piece of mers-cov-specific evidence pointing to bats originates from amplification of a 190 nt fragment of the rnadependent rna polymerase gene of the mers-cov genome, identified in a faecal pellet from an insectivorous emballonuridae bat, taphozous perforatus found in bisha, the ksa [121] . while very short, the sequence of the fragment defined it as a diagnostic discovery. subsequently a link to dcs was reported [85] and that link has matured into a verified association [38, 126] (fig. 4) . (see figure on previous page.) fig. 3 monthly detections of mers-cov (blue bars) and of cases who died (red bars) with some dates of interest marked for 2012 to 4 th september 2015. an approximation of when dc calving season [128] and when recently born dcs are weaned is indicated. spring (green) and summer (orange) in the arabian peninsula are also shaded. note the left-hand y-axis scale for 2014 and 2015 which is greater than for 2012/13. sources of these public data include the who, ministries of health and flutrackers [207] [208] [209] . earlier and subsequent versions of this chart are maintained on a personal blog [210] . modified and reprinted from mackay im, arden ke. middle east respiratory syndrome: an emerging coronavirus infection tracked by the crowd. virus res 2015 vol 202:60-88 with permission from elsevier [5] dcs, which make up 95 % of all camels, have a central presence in the arabian peninsula where human-dc contact ranges from little to close [119] . contact may be commonplace and could occur in variety of ways (fig. 4a) . there are several large well-attended festivals, races, sales and parades which feature dcs and dcs are also kept and bred close to populated areas in the ksa [127, 128] . dc milk and meat are widely consumed and the older dc is an animal of ritual significance after the hajj pilgrimage [129] . however, mers-cov infection frequency is reportedly much lower than is the widespread and frequent habit of eating, drinking and preparing dc products. daily ingestion of fresh unpasteurized dc milk is common among the desert bedouin and many others in the ksa. dc urine is also consumed or used for supposed health benefits. despite camel butchery being a local occupation, neither butchers nor other at-risk groups are identifiable among mers cases; this may simply be a reporting issue rather than an unexplainable absence of mers. a small case-control study published in 2015 identified direct dc contact, and not ingestion of products, to be associated with onset of mers [38] . the first sero-survey of livestock living in the middle east region was conducted during 2012-2013 [85] . dcs were sampled from a mostly canary island-born herd and from omani dcs (originally imported from the horn of africa) [85] . a neutralising antibody assay found only 10 % of strongly seropositive canary island [5] . b camel-to-human infections appear to be infrequent, while human-to-human spread of infection is regularly facilitated by poor ipc in healthcare settings where transmission is amplified, accounting for the bulk of cases. there are human mers cases that do not fall into either category of source and it is unclear if these acquired infection through some entirely separate route, or from cases that escaped diagnosis. c hypothetical ways in which subclinical (when infection may not meet a previously defined clinical threshold of signs and/or symptoms) or asymptomatic (no obvious signs or measured, noticed or recalled symptoms of illness) mers-cov infection may be implicated in transmission dc sera could neutralise mers-cov while all omani dc sera had high levels of specific mers-cov neutralizing antibody [85] . this indicated that dcs had in the past been infected by mers-cov, or a very similar virus. since this study, a host of peer-reviewed reports have looked at both dcs and other animals, and the possibility that they may host mers-cov infection. seropositive dcs have been found throughout the arabian peninsula including oman, the ksa, qatar, jordan, the united arab emirates (uae), kuwait as well as sudan, somalia, egypt, tunisia, nigeria, kenya and ethiopia in africa and the canary islands [85, [130] [131] [132] [133] [134] . other animals tested include sheep, cows, pigs, horses, donkeys, mules, birds, water buffalo, goats, bactrian camels, llamas and guanaco (south american camelids) but none had detectable neutralising antibody against mers-cov [4, 74, 78, 85, 86, 135, 136] . no virology or serology studies of human samples from areas in africa where there are camels with a history of mers-cov have been reported to date. however,an absence of unexplained pneumonia that may be attributable to mers-cov infection may not signal the absence of virus among humans in each country but simply reflect a lack of expensive epidemiology studies conducted by resource-poor countries. it is thus unclear whether mers-cov, or an antigenically related cov, is an unrecognized pathogen in these regions, perhaps circulating for even longer than it has been known in the arabian peninsula [133] . mers-cov rna has also been detected in dc samples, and recovery of infectious virus has also been achieved from dc samples [4, 77, 117, 132, [137] [138] [139] [140] [141] . from some of these, full or majority length genomes of mers-cov have been sequenced [77, 137, 138] . dc versions of mers-cov were found to be as similar to each other, as were variants detected from different humans over time and across distance. antibody screening assays have also detected crossreactive antibodies in sera. these were identified as such by screening sera against similar viruses, for example bcov or hcov-oc43 (as an antigenic facsimile for bcov). it is possible that other mers-cov-like viruses also reside within dcs, but this does not detract from the definitive finding of mers-cov genetic sequences in both dcs and humans [117, 142, 143] . screening studies have shown that juvenile dcs are more often positive for virus or viral rna while older dcs are more likely to be seropositive and rna or virus negative [76, 77, 144] . in adult dcs, mers-cov rna has been detected among animals with pre-existing antibody, suggesting re-infection is possible [77, 144] . viral loads among positive dcs can be very high [4, 76, 77, 139, 144] and dcs have been found positive both when ill with urt respiratory signs [77, 117, 142, 145] or when apparently healthy [137] . these findings indicate dcs host natural mers-cov infections. furthermore, stored dc sera have revealed signs of mers-cov in dcs which date back over three decades (the earliest collected in 1983) [4, 133, 135] . older sera have not been tested and so precisely how long dcs have been afflicted by mers-cov, whether the virus is enzootic among them, introduced to them decades or centuries ago from bats in africa or the arabian peninsula, or they are the subject of regular but short-lived viral incursions from an as yet unknown host, cannot be answered. researchers sought to determine a direction for infection; were dcs transmitting virus to humans or were humans infecting dcs? at a qatari site, a farm owner and his employee became ill in mid-october 2013 and tested positive for mers-cov rna in a sputum and throat swab sample, respectively. rt-rtpcrs found mers-cov rna in 11 of 14 positive dc nasal swabs at the farm; six (43 %) positive by two or more assays [138] . the results indicated a recent outbreak had occurred in this herd; the first indication of mers-cov rna found within dcs with a temporal association to human infections. three positive dc samples were confirmed by sequencing a 358 nt portion of the spike gene; these sequences were identical to each other, again with close homology to other human and dc mers-cov sequences [138] . the dcs and human contacts yielded orf1a and orf4b sequences differing by only a single nucleotide each, clustering closely with the hafr-al-batin_1_2013 variant [138] . subsequent case studies found evidence of a concurrent human and dc infection and the direction of that infection was inferred to be from the ill dcs and to their human owners [117, 142, 146] . partial genome sequences indicated that a human and a mers-cov rt-rtpcr positive dc had been infected by a variant of the same virus, harbouring the same distinct pattern of nucleotide polymorphisms. [142] all nine dc in the owner's herd, serially sampled, reacted in a recombinant s1 antigen elisa, with the two animals that had been rt-rtpcr positive showing a small, verifiable rise in antibody titre [142] . a rise in titre theoretically begins 10 to 21 days after dc infection [142] . the authors suggested that the rise in titre in dc sera which occurred alongside a declining rna load, while the patient was actively ill and hospitalized, indicated that the dcs were infected first followed by the owner [117, 142] . bcov antibodies were also present, and rising in one of the two rt-rtpcr positive animals but no animal's antibodies could neutralise bcov infection [142] . camel calving season occurs in the winter months (between late october and late february; fig. 3 ) and this may be a time when there is increased risk to humans of spill-over due to new infections among naïve dc populations [128] . what role maternal camel antibody might play in delaying infection of calves remains unknown [128, 142] . juvenile dcs appear to host active infection more often than adult dcs and thus the sacrificial slaughter of dcs, which must be five years of age or older (termed a thane), may not be accompanied by significant risk of exposure to infection. in contrast to earlier results, slaughterhouse workers who kill both younger and older dcs, may be an occupational group with significantly higher incidence of seropositivity to mers-cov when animals have active mers-cov infections [129, 139, [147] [148] [149] . expanded virological investigations of african dcs may lead to more seropositive animals and geographic areas in which humans may be at risk. it is possible that there are areas where humans already harbour mers-cov infections that have not been identified because of an absence of laboratory surveillance. virological investigations of bats may lead to findings of ancestral viruses and viral 'missing links' and identifying any other animal sources of zoonotic spread is important to inform options for reducing human exposures [56, 76] . infectious mers-cov added to dc, goat or cow milk and stored at 4°c could be recovered at least 72 h later and, if stored at 22°c, recovery was possible for up to 48 h [150] . mers-cov titre decreased somewhat when recovered from milk at 22°c but pasteurization completely ablated mers-cov infectivity [150] . in a subsequent study, mers-cov rna was identified in the milk, nasal secretion and faeces of dcs from qatar [151] . a single study has examined the ability of mers-cov to survive in the environment [150] . plastic or steel surfaces were inoculated with 10 6 tcid 50 of mers-cov at different temperature and relative humidity (rh) and virus recovery was attempted in cell culture. at high ambient temperature (30°c) and low rh (30 %) mers-cov remained viable for 24 h [150] . by comparison, a well known and efficently transmitted respiratory virus, influenza a virus, could not be recovered in culture beyond four hours under any conditions [150] . aerosol experiments found mers-cov viability only decreased 7 % at low rh at 20°c. in comparison, influenza a virus decreased by 95 % [150] . mers-cov survival is inferior to that previously demonstrated for sars-cov [152] . for context, pathogenic bacteria can remain viable and airborne for 45 min in a coughed aerosol and can spread 4 m. mers-cov's ability to remain viable over long time periods gives it the capacity to thoroughly contaminate a room's surfaces when occupied by an infected and symptomatic patient [153] . whether mers-cov can remain adrift and infectious for extended periods (truly airborne) remains unknown. such findings expand our understanding of the possibilities for droplets to transmit respiratory viruses in many settings, including hospital waiting rooms, emergency departments, treatment rooms, open intensive care facilities and private patient rooms. the nature and quality of air exchange, circulation and filtration are important variables in risk measurement and reduction as is the use of negative pressure rooms to contain known cases. droplet spread between humans is considered the mechanism of human-to-human transmission and the need for droplet precautions was emphasized after the al-ahsa hospital, the ksa and the south korean outbreaks [21, 23, 154, 155] . by extrapolation, aerosol-generating events involving dcs (urination, defecation, and preparation and consumption of dc products) should be factored into risk measurement and reduction efforts and messaged using appropriate context. the provision of evidence supporting the best formulation of personal protective equipment to be worn by hcws who receive, manage or conduct procedures on infectious cases remains a priority. mers-cov was found and characterized because of its apparent association with severe, and therefore more obvious, illness in humans; we were the canaries in the coal mine. sero-assays and prospective cohort studies have yet to determine the extent to which milder or asymptomatic cases contribute to mers-cov transmission chains. however, transmission of mers-cov is defined as sporadic (not sustained), intra-familial, often healthcare associated, inefficient and requiring close and prolonged contact [22, 31, 63, 93, 97, 102, 156] in a household study, 14 of 280 (5 %) contacts of 26 mers-cov positive index patients were rna or antibody positive; the rate of general transmission, even in outbreaks is around 3 % [31] . it seems that the majority of human cases of mers-cov, even when numbers appear to increase suddenly, do not readily transmit to more than one other human so to date, the localized epidemic of mers-cov has not been self-sustaining [157] [158] [159] [160] [161] . that is to say, the basic reproduction number (r 0 ) -the average number of infections caused by one infected individual in a fully susceptible populationhas been close to one throughout various clusters and outbreaks. if r 0 was greater than 1, a sustained increase in case numbers would be expected. some r o calculations may be affected by incomplete case contact tracing, limited community testing and how a case is defined. that mers has had a constant presence in the arabian peninsula since 2012 is due to ongoing, sporadic spill-over events from dcs amplified by poorly controlled hospital outbreaks. the first known mers human-to-human transmission event was one characterized by acute lrt disease in a healthcare setting in jordan. in stark contrast, a sero-survey of hcw who were sometimes in close and prolonged contact with the first, fatal mers-cov case in 2012 [162] , found none of the hcw had seroconverted four months later, despite an absence of eye protection and variable compliance with required ppe standards [162] . early on in the mers story, samples for testing were mostly collected from patients with severe illness and not those with milder acute respiratory tract infections. contacts of confirmed mers cases were often observed for clinical illness, but not tested. these omissions may have confounded our understanding of mers-cov transmission and biased early data towards higher numbers of seriously ill and hospitalized patients, inflating the apparent proportion of fatal cases. case-control studies were not a focus. as testing paradigms changed and contacts were increasingly tested, more asymptomatic and mild infections were recognized [163] . a rise in the cases termed asymptomatic (which enlarge the denominator for calculations of the proportion of fatal cases, defined in [164] ) resulted in a drop in the proportion of fatal cases during the jeddah-2014 outbreak. historically, such rises are consistent with changing definitions and laboratory responses and clinical management of a newly discovered virus infection that was first noted only among the severely ill. upon follow-up, over three-quarters of such mers-cov rna positive people did recall having one or more symptoms at the time, despite being reported as asymptomatic [165] raising some question over the reliability of other reported data. the proportion of fatal mers cases within the ksa compared to outside the ksa, as well as the age, and sex distribution change in different ways when comparing mers outbreaks. approximately 43 % of mers cases (549 of 1277) in the ksa were fatal betwen 2012 and december 2015 while 21 % (72 of 330) died among those occurring outside of the ksa. the total number of male cases always outnumber females and the proportion of male deaths is always greater than the proportion of females who die. however the proportion of male deaths from total males with mers is a similar figure to that for females. in the ksa, there is a greater proportion of younger males among cases and deaths than were observed from the 2015 south korean or the jeddah-2014 outbreaks (additional file 2: figure s2 ). why these aspects have differed may be due to differences in the time to presentation and diagnosis, the nature and quality of supportive care, the way a person became infected (habits, exposure to a human or zoonotic source, viral load, route of infection) or the extent to which different populations are burdened by underlying diseases [40] . as a group, hcws comprised 16 % of mers cases in the ksa and south korea. it is apparent that the weekly proportion of infected hcws increases alongside each steep rise in overall detections (fig. 5) . in may 2013, the who published guidelines for ipc during care of probable or confirmed cases of mers-cov infection in a healthcare setting [166] . this is explainable because to date, each case rise has been intimately associated with healthcare-facility related outbreaks [118] . these rises in mers-cov detections can decrease the average age during each event because hcws are usually younger than inpatients with mers. healthcare facilities have been a regular target for suggested improvements aimed at improving infection prevention and control (ipc) procedures [115, 118] . most of the analysis of mers-cov genetics has been performed using high throughput or "deep" sequencing methods for complete genome deduction [167] [168] [169] . mers-cov was the first subject of such widespread use of deep sequencing to study an emerging viral outbreak with global reach. the technique can produce genomic [207] [208] [209] . earlier and subsequent versions of this chart are maintained on a personal blog [210] length coverage in a single experiment with highly repetitious measurement of each nucleotide position [52, 140] . despite assays having been published early on, subgenomic sequencing, once the mainstay of viral outbreak studies, has less often been published during mers-cov characterization [48] . as more genomes from both humans and dcs have been characterized, two clades have become apparent; a and b (fig. 6) . clade a contains only human-derived mers-cov genomes from jordan, while clade b comprises the majority of human and camel genomes deduced thus far [168] . two studies during 2015, one looking at jeddah-2014 mers-cov variants and another looking at a variant exported from south korea to china, have now identified signs of genetic recombination among mers-cov variants. while human and camel whole genome sequences have retained >99 % identity with each other, members of genetically distinct lineages can and do swap genetic material when suitable conditions and coinfections co-occur [170] [171] [172] . shared identity implies that the major source for human acquisition is the dc, rather than another animal, although more testing of other animal species is needed to confirm that conclusion. over a month, a dc virus sequenced on different occasions did not change at all indicating a degree of genomic stability in its host, supporting that dcs are the natural, rather than intermediate, host for the mers-cov we know today [77] . to date, recombination has been localised to breakpoints near the boundary between orf1a and orf1b regions, within the spike gene [170] and in the orf1b region (fig. 2) [172] . it is not unexpected that recombination should occur since it is well known among other covs [124] and because the majority of mers-cov whole genomes collected from samples spanning three years (2012-2015) and from humans, camels and different countries have shown close genetic identity to each other, with just enough subtle variation to support outbreak investigations so long as whole genome sequencing is applied [52, 77, 135, 138, 168, [173] [174] [175] . changes in genome sequence may herald alterations to virus transmissibility, replication, persistence, lethality or response to future drugs. if we have prior knowledge of the impact of genetic changes because of thorough characterization studies, we can closely fig. 6 the genetic relationship between mers-cov nucleotide sequences (downloaded from genbank using the listed accession numbers and from virological.org [212] ). this neighbour joining tree was created in mega v6 using an alignment of human and dcderived mers-cov sequences (geneious v8.1 [211] ). clades are indicated next to dark (clade a) or pale (clade b) blue vertical bars. camel icons denote genomes from dcs. healthcare or community outbreaks are boxed and labelled using previously described schemes [212, 213] monitor the genomic regions and better understand any changes in transmission or disease patterns as they occur. genetic mutations noted during the largest of human outbreaks, jeddah-2014, did not impart any major replicative or immunomodulatory changes when compared to earlier viral variants in vitro [156, 176] . however, we understand very little of the phenotypic outcomes that result from subtle genetic change in mers-cov genomes. to date no clinical relevance or obvious in vivo changes to viral replication, shedding or transmission has been reported or attributed to mutations or to new recombinant viruses [156] . but vigilance and larger, more contemporary and in vivo studies are needed. genome sequence located to a distinct clade were identified from an egyptian dc that was probably imported from sudan. this does not fit into either of the current clades [125, 168, 177] . a virus sequenced from a neoromicia capensis bat was more closely related to mers-cov than other large bat-derived sequences had been to that point, but the genome of a variant of a mers-cov has yet to be discovered and deduced from any bat [125] . analyses of mers-cov genomes have shown that most single nucleotide differences among variants were located in the last third of the genome (fig. 2) , which encodes the spike protein and accessory proteins [168] . at least nine mers-cov genomes contained amino acid substitutions in the receptor binding domain (rbd) of the spike protein and codons 158 (n-terminal region), 460 (rbd), 1020 (in heptad repeat 1), 1202 and 1208 bear investigation as markers of adaptive change [140, 169] . the spike protein had not changed in the recombinant mers-cov genome identified in china in 2015 but was reported to have varied at a higher rate than that for complete mers-cov genomes, among south korean variants [172, 178] . this highlights that subgenomic regions may not always contain enough genetic diversity to prove useful for differentiating viral variants. despite this, one assay amplifying a 615 nucleotide fragment of the spike s2 domain gene for sanger sequencing agreed with the results generated by the sequencing of a some full genomes and was useful to define additional sequence groupings [177] . genomic sequence can also be used to define the geographic boundaries of a cluster or outbreak and monitor its progress, based on the similarity of the variants found among infected humans and animals when occurring together, or between different sites and times (fig. 6 ) [169] . this approach was employed when defining the geographically constrained mers hospital outbreak in al-ahsa, which occurred between 1 st april and 23 rd may 2013, as well as clusters in buraidah and a community outbreak in hafr al-batin, the ksa. genomic sequencing identified that approximately 12 mers-cov detections from a community outbreak in hafr al-batin between june and august 2013 may have been triggered by an index case becoming infected through dc contact [175] . sequencing mers-cov genomes from the 2013 al-ahsa hospital outbreak indicated that multiple viral variants contributed to the cases but that most were similar enough to each other to be consistent with human-tohuman transmission. molecular epidemiology has revealed otherwise hidden links in transmission chains encompassing a period of up to five months [179] . however, most outbreaks have not continued for longer than two to three months and so opportunities for the virus to adapt further to humans through co-infection and sustained serial passage have been rare [169] . in riyadh-2014, genetic evidence supported the likelihood of multiple external introductions of virus, implicating a range of healthcare facilities in an event that otherwise looked contiguous [23, 168, 179] . riyadh is a nexus for camel and human travel and has had more mers cases than any other region of the ksa to date but also harbours a wide range of mers-cov variants [128, 167, 179] . however the south korean outbreak originated from a single infected person, resulting in three to four generations of cases [180, 181] . studies of this apparently recombinant viral variant did not find an increased evolutionary rate and no sign of virus adaptation thus the outbreak seems to have been driven by circumstance rather than circumstance together with mutation [181] . for many mers cases detected outside the arabian peninsula, extensive contact tracing has been performed and the results described in detail. contact tracing is essential to contain the emergence and transmission of a new virus and today it is supported by molecular epidemiology. although it is an expensive and time consuming process, contact tracing can identify potential new infections and through active or passive monitoring, react more rapidly if disease does develop. results of contact tracing to date have found that onward transmission among humans is an infrequent event. for example, there were 83 contacts, both symptomatic and asymptomatic, of a case treated in germany who travelled from the uae but no sign of virus or antibody were found in any of them [73] . the very first mers case had made contact with 56 hcws and 48 others, but none developed any indication of infection [162] . in a study of 123 contacts of a case treated in france, only seven matched the definition for a possible case and were tested; one who had shared a 20 m 2 hospital room while in a bed 1.5 m away from the index case for a prolonged period was positive [26] . none of the contacts of the first two mers cases imported into the usa in 2014 contained any mers-cov footprint [182] and none of the 131 contacts of two travellers returning to the netherlands developed mers-cov antibodies or tested rna positive [25, 183] . analyses of public data reveal many likely instances of nosocomial acquisition of infection in the arabian peninsula and these data may be accompanied by some details noting contact with a known case or facility. one example identified the likely role of a patient with a subclinical infection, present in a hospital during their admission for other reasons, as the likeliest index case triggering a family cluster [93] . contact tracing was a significant factor in the termination of a 2015 outbreak involving multiple south korean hospitals [184] . such studies demonstrate the necessity of finding and understanding a role for mild and asymptomatic cases, together with restricting close contact or prolonged exposure of infected people to others, especially older family members and friends with underlying disease (fig. 4c) . the hospital-associated outbreak in jeddah in 2014 was the largest and most rapid accumulation of mers-cov detections to date. the greatest number of mers-cov detections of any month on record occurred in jeddah in april. the outbreak was mostly (>60 % of cases) associated with human-to-human spread within hospital environments and resulted from a lack of, or breakdown in, infection prevention and control [37, 185, 186] . a rise in fatalities followed the rapid increase in case numbers. in 2015 two large outbreaks occurred. south korea was the site of the first large scale outbreak outside the arabian peninsula and produced the first cases in both south korea and china, occurring between may and july 2015. this was closely followed by a distinct outbreak in ar riyad province in the ksa which appeared to come under control in early november. after staying in bahrain for two weeks, a 68 year old male (68 m) travelled home to south korea via qatar, arriving free of symptoms on the 4 th may 2015 [187] . he developed fever, myalgia and a cough nearly a week later (11 th ). he visited a clinic as an outpatient between the 12 th and 15 th of may and was admitted to hospital a on the 15 th [188] . he was discharged from hospital a on the 17 th then visited and was admitted to the emergency department of hospital b on the 18 th . during this second stay, a sputum sample was taken and tested positive for mers-cov on the 20 th [187, 188] , triggering transfer to the designated isolation treatment facility. over a period of 10 days, the index case was seen at three different hospitals, demonstrating a key feature of "hospital shopping" that shaped the south korean outbreak. approximately 34 people were infected during this time [187] . in total 186 cases were generated in this outbreak, all linked through a single transmission chain to 68 m; 37 cases died [189] . in south korea, the national health insurance system provides for relatively low cost medical care, defraying some costs by making family members responsible for a portion of the ministration of the sick, resulting in them sometimes staying for long periods in the rooms that often have more than four beds in them [24] . other factors thought to have enabled this outbreak included unfamiliarity of local clinicians with mers, ease with which the public can visit and be treated by tertiary hospitals, the custom of visiting sick friends and relatives in hospitals, the hierarchical nature of korean society, crowded emergency rooms, poor ipc measures, a lack of negative pressure isolation rooms and poor inter-hospital communication of patient disease histories [24, [190] [191] [192] . all of the reported transmission occurred across three or four generations and apart from one unknown source, were all hospital-acquired [24, 120, 181, [193] [194] [195] . few clinical details about these cases have been reported to date and detail on transmission and contact tracing is minimal. the hospitals involved were initially not identified, governmental guidance and actions produced confusing messages and there was very limited communication at all early on which resulted in unnecessary concern, distrust and a distinct economic impact [191, [196] [197] [198] . early in the outbreak, a infected traveller, the son of an identified case in south korea, passed through hong kong on his way to china where he was located, isolated and cared for in china [91, 199, 200] . no contacts became ill. the outbreak was brought under control in late july/ early august [201] after improved ipc measures were employed, strong contact tracing monitoring and quarantine, expanded laboratory testing, hospitals were better secured, specialized personnel were dispatched to manage cases and international cooperation increased [202, 203] . a review of public data showed that, as for mers in the ksa, older age and the presence of underlying disease were significantly associated with a fatal outcome in south korea. [40] even though r 0 is <1, super-spreading events facilitated by circumstances created in healthcare settings and characterized by cluster sizes over 150, such as this one, are not unexpected from mers-cov infection [204] . the dynamic of an outbreak depends on the r 0 and an individual's viral shedding patterns, contact type and frequency, hospital procedures and population structure and density [204] . in the region of ar riyad, including the capital city of riyadh, a hospital based cluster began, within a single hospital, from late june 2015 [205] . by mid-september there had been approximately170 cases reported but the outbreak appeared to been brought under control in november. it became apparent early on that mers-cov spread relatively ineffectively from human-to-human. despite ongoing and possibly seasonal introduction of virus to the human population via infected dcs and perhaps other animals yet to be identified, the vast majority of mers-cov transmission has occurred from infected to uninfected humans in close and prolonged contact through circumstances created by poor infection control in health care settings. this opportunistic virus has had its greatest impact on those with underlying diseases and such vulnerable people, sometimes suffering multiple comorbidities, have been most often associated with hospitals, creating a perfect storm of exposure, transmission and mortality. it remains unclear if this group are uniquely affected by mers-cov or if other respiratory virus infections, including those from hcovs, produce a similarly serious impact. in south korea, a single imported case created an outbreak of 185 cases and 36 deaths that had a disproportionate impact on economic performance, community behaviour and trust in government and the health care system. household human-to human transmission occurs but is also limited. educational programs will be essential tools for combatting the spread of mers-cov both within urban and regional communities and for the health care setting. vigilance remains important for containment since mers-cov is a virus with a genetic makeup that has been observed for only three years and is not stable. among all humans reported to be infected, nearly 40 % have died. continued laboratory testing, sequencing, analysis, timely data sharing and clear communication are essential for such vigilance to be effective. global alignment of case definitions would further aid accurate calculation of a case fatality ratio by including subclinical case numbers. whole genome sequencing has been used extensively to study mers-cov travel and variation and although it remains a tool for experts, it appears to be the best tool for the job. mers and sars have some clinical similarities but they also diverge significantly [206] . defining characteristics include the higher pfc among mers cases (above 50 % in 2013 and currently at 30-40 %; well above the 9 % of sars) and the higher association between fatal mers and older males with underlying comorbidities. for the viruses, mers-cov has a broader tropism, grows more rapidly in vitro, more rapidly induces cytopathogenic change, triggers distinct transcriptional responses, makes use of a different receptor, induces a more proinflammatory state and has a delayed innate antiviral response compared to sars-cov. there appears to be a 2-3 % prevalence of mers-cov in the ksa with a 5 % chance of secondary transmission within the household. there is an increased risk of infection through certain occupations at certain times and a much greater chance for spread to other humans during circumstances created by humans, which drives more effective transmission than any r 0 would predict on face value. nonetheless, despite multiple mass gatherings that have afforded the virus many millions of opportunities to spread, there have remarkably been no reported outbreaks of mers or mers-cov during or immediately after these events. there is no evidence that mers-cov is a virus of pandemic concern. nonetheless, hospital settings continue to describe mers cases and outbreaks in the arabian peninsula. as long as we facilitate the spread of mers-cov among our most vulnerable populations, the world must remain on alert for cases which may be exported more frequently when a host country with infected camel reservoirs is experiencing human clusters or outbreaks. the mers-cov appears to be an enzootic virus infecting the dc urt with evidence of recent genetic recombination. it may once have had its origins among bats, but evidence is lacking and the relevance of that to today's ongoing epidemic is academic. thanks to quick action, the sensitive and rapid molecular diagnostic tools required to achieve rapid and sensitive detection goal have been in place and made widely available since the virus was reported in 2012. rt-pcr testing of lrt samples remains the gold standard for mers-cov confirmation. serological tools continue to emerge but they are in need of further validation using samples from mild and asymptomatic infections and a densely sampled cohort study to follow contacts of new cases may address this need. similarly, the important question of whether those who do shed mers-cov rna for extended periods are infectious while appearing well, continues to go unanswered. it is even unclear just how many 'asymptomatic' infections have been described and reported correctly which in turn raises questions about the reliability of other clinical data collection to date. while the basic virology of mers-cov has advanced over the course of the past three years, understanding what is happening in, and the interplay between, camel, environment and human is still in its infancy. additional file 1: figure s1 . the severe respiratory illness caused by a novel coronavirus middle east respiratory syndrome coronavirus (mers-cov) -saudi arabia middle east respiratory syndrome coronavirus infection in dromedary camels in saudi arabia middle east respiratory syndrome: an emerging coronavirus infection tracked by the crowd absence of mers-coronavirus in bactrian camels, southern mongolia seroepidemiology of middle east respiratory syndrome (mers) coronavirus in saudi arabia (1993) and australia (2014) and characterisation of assay specificity middle east respiratory syndrome coronavirus infection not found in camels in japan absence of mers-cov antibodies in feral camels in australia: implications for the pathogen's origin and spread lack of middle east respiratory syndrome coronavirus transmission from infected camels tensions linger over discovery of coronavirus as outbreak continues middle east respiratory syndrome coronavirus (mers-cov): evidence and speculations cumulative number of reported probable cases of sars in-vitro renal epithelial cell infection reveals a viral kidney tropism as a potential mechanism for acute renal failure during middle east respiratory syndrome (mers) coronavirus infection replicative capacity of mers coronavirus in livestock cell lines human coronavirus emc does not require the sars-coronavirus receptor and maintains broad replicative capability in mammalian cell lines isolation of a novel coronavirus from a man with pneumonia in saudi arabia human cell tropism and innate immune system interactions of human respiratory coronavirus emc compared to those of severe acute respiratory syndrome coronavirus state of knowledge and data gaps of middle east respiratory syndrome coronavirus (mers-cov) in humans epidemiological, demographic, and clinical characteristics of 47 cases of middle east respiratory syndrome coronavirus disease from saudi arabia: a descriptive study family cluster of middle east respiratory syndrome coronavirus infections hospital outbreak of middle east respiratory syndrome coronavirus mers outbreak in korea: hospital-to-hospital transmission middle east respiratory syndrome coronavirus (mers-cov) infections in two returning travellers in the netherlands first cases of middle east respiratory syndrome coronavirus (mers-cov) infections in france, investigations and implications for the prevention of human-tohuman transmission case definition for case finding severe respiratory disease associated with novel coronavirus middle east respiratory syndrome coronavirus case definition for reporting to who. interim case definition as of 14 middle east respiratory syndrome coronavirus | case definition for reporting to who 14 revised interim case definition for reporting to who -middle east respiratory syndrome coronavirus (mers-cov): interim case definition as of transmission of mers-coronavirus in household contacts screening for middle east respiratory syndrome coronavirus infection in hospital patients and their healthcare worker and family contacts: a prospective descriptive study case definition and surveillance guidance for mers-cov testing in saudi arabia infection prevention/control and management guidelines for patients with middle east respitaory syndrome coronavius (mers-cov) infection infection prevention and control guidelines for middle east respiratory syndrome coronavirus (mers-cov) infection middle east respiratory syndrome coronavirus in children characteristics and outcomes of middle east respiratory syndrome coronavirus patients admitted to an intensive care unit in jeddah, saudi arabia risk factors for primary middle east respiratory syndrome coronavirus illness in humans, saudi arabia preliminary epidemiological assessment of mers-cov outbreak in south korea mortality risk factors for middle east respiratory syndrome outbreak, south korea clinical course and outcomes of critically ill patients with middle east respiratory syndrome coronavirus infection estimating the risk of middle east respiratory syndrome (mers) death during the course of the outbreak in the republic of korea invited editorial: mers-cov an emerging viral zoonotic disease: three years after and counting middle east respiratory syndrome coronavirus disease in children middle east respiratory syndrome coronavirus not detected in children hospitalized with acute respiratory illness in stillbirth during infection with middle east respiratory syndrome coronavirus detection of a novel human coronavirus by real-time reverse-transcription polymerase chain reaction assays for laboratory confirmation of novel human coronavirus (hcov-emc) infections infectious middle east respiratory syndrome coronavirus excretion and serotype variability based on live virus isolates from patients in saudi arabia development and evaluation of a 'real-time' rt-pcr for the detection of enterovirus and parechovirus rna in csf and throat swab samples mers: emergence of a novel human coronavirus genomic characterization of a newly discovered coronavirus associated with acute respiratory distress syndrome in humans laboratory testing for middle east respiratory syndrome coronavirus | interim guidance performance and clinical validation of the realstar mers-cov kit for detection of middle east respiratory syndrome coronavirus rna real-time reverse transcription-pcr assay panel for middle east respiratory syndrome coronavirus laboratory testing for middle east respiratory syndrome coronavirus development and evaluation of novel real-time reverse transcription-pcr assays with locked nucleic acid probes targeting leader sequences of human-pathogenic coronaviruses realtime sequence-validated loop-mediated isothermal amplification assays for detection of middle east respiratory syndrome coronavirus (mers-cov) a sweet spot for molecular diagnostics: coupling isothermal amplification and strand exchange circuits to reverse transcription recombinase polymerase amplification assay for the detection of middle east respiratory syndrome coronavirus development and validation of a rapid immunochromatographic assay for detection of middle east respiratory syndrome coronavirus antigen in dromedary camels a sensitive and specific antigen detection assay for middle east respiratory syndrome coronavirus investigation of an imported case of middle east respiratory syndrome coronavirus (mers-cov) infection in serological assays for emerging coronaviruses: challenges and pitfalls middle east respiratory syndrome coronavirus immunoassays test characteristics: anti-mers-cov elisa camel (igg) recombivirus anti-middle east respiratory syndrome coronavirus (mers-cov) elisa kits ncov / novel coronavirus) nucleocapsid antibody, mouse mab is mers another sars? investigation of anti-middle east respiratory syndrome antibodies in blood donors and slaughterhouse workers in jeddah and makkah, saudi arabia, fall 2012 cross-reactive antibodies in convalescent sars patients' sera against the emerging novel human coronavirus emc (2012) by both immunofluorescent and neutralizing antibody tests asymptomatic mers-cov infection in humans possibly linked to infected camels imported from oman to united arab emirates contact investigation for imported case of middle east respiratory syndrome seroepidemiology for mers coronavirus using microneutralisation and pseudoparticle virus neutralisation assays reveal a high prevalence of antibody in dromedary camels in egypt a safe and convenient pseudovirus-based inhibition assay to detect neutralizing antibodies and screen for viral entry inhibitors against the novel human coronavirus mers-cov middle east respiratory syndrome (mers) coronavirus seroprevalence in domestic livestock in saudi arabia mers coronavirus in dromedary camel herd, saudi arabia middle east respiratory syndrome coronavirus (mers-cov) serology in major livestock species in an affected region in jordan lack of mers coronavirus neutralizing antibodies in humans, eastern province, saudi arabia sparse evidence of mers-cov infection among animal workers living in southern saudi arabia during 2012. influenza other respir viruses kinetics of serologic responses to mers coronavirus infection in humans hospital-associated outbreak of middle east respiratory syndrome coronavirus: a serologic, epidemiologic, and clinical description epidemiological findings from a retrospective investigation specific serology for emerging human coronaviruses by protein microarray middle east respiratory syndrome coronavirus neutralising serum antibodies in dromedary camels: a comparative serological study antibodies against mers coronavirus in dromedary camels presence of middle east respiratory syndrome coronavirus antibodies in saudi arabia: a nationwide, cross-sectional, serological study cdc concludes indiana mers patient did not spread virus to illinois business associate middle east respiratory syndrome: what clinicians need to know interim guidelines for collecting, handling, and testing clinical specimens from patients under investigation (puis) for middle east respiratory syndrome coronavirus (mers-cov characteristics of traveler with middle east respiratory syndrome distinct immune response in two mers-cov-infected patients: can we go from bench to bedside? a family cluster of middle east respiratory syndrome coronavirus infections related to a likely unrecognized asymptomatic or mild case association of higher mers-cov virus load with severe disease and death, saudi arabia an appropriate lower respiratory tract specimen is essential for diagnosis of middle east respiratory syndrome (mers) lack of nasal carriage of novel corona virus (hcov-emc) in french hajj pilgrims returning from the hajj 2012, despite a high rate of respiratory symptoms health protection agency ukncit. evidence of person-to-person transmission within a family cluster of novel coronavirus infections prevalence of mers-cov nasal carriage and compliance with the saudi health recommendations among pilgrims attending the 2013 hajj a case of long-term excretion and subclinical infection with middle east respiratory syndrome coronavirus in a healthcare worker middle east respiratory syndrome coronavirus (mers-cov) causes transient lower respiratory tract infection in rhesus macaques virological and serological analysis of a recent middle east respiratory syndrome coronavirus infection case on a triple combination antiviral regimen middle east respiratory syndrome coronavirus (mers-cov) viral shedding in the respiratory tract: an observational analysis with infection control implications respiratory tract samples, viral load and genome fraction yield in patients with middle east respiratory syndrome ribavirin and interferon therapy in patients infected with the middle east respiratory syndrome coronavirus: an observational study laboratory-confirmed case of middle east respiratory syndrome coronavirus (mers-cov) infection in malaysia: preparedness and response a case of imported middle east respiratory syndrome coronavirus infection and public health response viral respiratory infections among hajj pilgrims in 2013 influenza a and b viruses but not mers-cov in hajj pilgrims acute respiratory infections in travelers returning from mers-cov-affected areas changes in the prevalence of influenza-like illness and influenza vaccine uptake among hajj pilgrims: a 10-year retrospective analysis of data soaring mers cases cause pandemic jitters, but causes are unclear co-circulation of four human coronaviruses (hcovs) in queensland children with acute respiratory tract illnesses in 2004 recovery from severe novel coronavirus infection human isolate 115. who statement on the fifth meeting of the ihr emergency committee concerning mers-cov | who statement mers-cov in upper respiratory tract and lungs of dromedary camels, saudi arabia evidence for camel-to-human transmission of mers coronavirus taking stock of the first 133 mers coronavirus cases globally-is the epidemic changing? human-dromedary camel interactions and the risk of acquiring zoonotic middle east respiratory syndrome coronavirus infection. zoonoses public health summary of current situation, literature update and risk assessment middle east respiratory syndrome coronavirus in bats, saudi arabia emerging infectious diseases associated with bat viruses bats and their virome: an important source of emerging viruses capable of infecting humans coronavirus diversity, phylogeny and interspecies jumping rooting the phylogenetic tree of middle east respiratory syndrome coronavirus by characterization of a conspecific virus from an african bat middle east respiratory syndrome coronavirus: another zoonotic betacoronavirus causing sars-like disease link between mers virus and camels worries breeders dromedary camels and the transmission of middle east respiratory syndrome coronavirus (mers-cov) fiqh us-sunnah antibodies against mers coronavirus in dromedary camels geographic distribution of mers coronavirus among dromedary camels acute middle east respiratory syndrome coronavirus infection in livestock dromedaries mers coronavirus neutralizing antibodies in camels serological evidence of mers-cov antibodies in dromedary camels (camelus dromedaries) in laikipia county middle east respiratory syndrome coronavirus antibody reactors among camels in dubai serologic assessment of possibility for mers-cov infection in equids mers coronaviruses in dromedary camels middle east respiratory syndrome coronavirus in dromedary camels: an outbreak investigation middle east respiratory syndrome coronavirus (mers-cov) in dromedary camels isolation of mers coronavirus from a dromedary camel prevalence of middle east respiratory syndrome coronavirus (mers-cov) in dromedary camels in abu dhabi emirate human infection with mers coronavirus after exposure to infected camels, saudi arabia detection of the middle east respiratory syndrome coronavirus genome in an air sample originating from a camel barn owned by an infected patient high proportion of mers-cov shedding dromedaries at slaughterhouse with a potential epidemiological link to human cases replication and shedding of mers-cov in upper respiratory tract of inoculated dromedary camels evidence for camel-to-human transmission of mers coronavirus the holy qur'an sacrificing an animal at mina occupational exposure to dromedaries and risk for mers-cov infection stability of middle east respiratory syndrome coronavirus (mers-cov) under different environmental conditions middle east respiratory syndrome coronavirus (mers-cov) rna and neutralising antibodies in milk collected according to local customs from dromedary camels the effects of temperature and relative humidity on the viability of the sars coronavirus viability of pseudomonas aeruginosa in cough aerosols generated by persons with cystic fibrosis middle east respiratory syndrome coronavirus: transmission and phylogenetic evolution middle east respiratory syndrome coronavirus: epidemic potential or a storm in a teacup? an observational, laboratory-based study of outbreaks of mers-coronavirus in jeddah and riyadh, kingdom of saudi arabia assessment of the mers-cov epidemic situation in the middle east region assessing the pandemic potential of mers-cov interhuman transmissibility of middle east respiratory syndrome coronavirus: estimation of pandemic risk middle east respiratory syndrome coronavirus: quantification of the extent of the epidemic, surveillance biases, and transmissibility transmission dynamics and control of ebola virus disease (evd): a review health care worker contact with mers patient, saudi arabia middle east respiratory syndrome coronavirus: epidemiology and disease control measures age-specific and sex-specific morbidity and mortality from avian influenza a(h7n9) mers-cov outbreak in jeddah-a link to health care facilities infection prevention and control during health care for probable or confirmed cases of novel coronavirus (ncov) infection full-genome deep sequencing and phylogenetic analysis of novel human betacoronavirus transmission and evolution of the middle east respiratory syndrome coronavirus in saudi arabia: a descriptive genomic study spread, circulation, and evolution of the middle east respiratory syndrome coronavirus mers-cov recombination: implications about the reservoir and potential for adaptation middle east respiratory syndrome coronavirus recombination and the evolution of science and public health in china origin and possible genetic recombination of the middle east respiratory syndrome coronavirus from the first imported case in china laboratory investigation and phylogenetic analysis of an imported middle east respiratory syndrome coronavirus case in greece middle east respiratory syndrome coronavirus quasispecies that include homologues of human isolates revealed through whole-genome analysis and virus cultured from dromedary camels in saudi arabia community case clusters of middle east respiratory syndrome coronavirus in hafr al-batin, kingdom of saudi arabia: a descriptive genomic study middle east respiratory syndrome coronavirus (mers-cov) summary and literature update-as of 9 reliable typing of mers-cov variants with a small genome fragment variations in spike glycoprotein gene of mers-cov molecular epidemiology of hospital outbreak of middle east. respiratory syndrome middle east respiratory syndrome coronavirus (mers-cov) in the republic of korea microevolution of outbreak-associated middle east respiratory syndrome coronavirus, south korea first confirmed cases of middle east respiratory syndrome coronavirus (mers-cov) infection in the united states, updated information on the epidemiology of mers-cov infection, and guidance for the public, clinicians, and public health authorities followup of contacts of middle east respiratory syndrome coronavirus-infected returning travelers, the netherlands press release -who, korea-who joint mission on mers cov call for infection control to stem mers infection control and mers-cov in health workers an outbreak of middle east respiratory syndrome coronavirus infection in south korea middle east respiratory syndrome coronavirus (mers-cov) -republic of korea samsung hospital to invest w100b in post-mers improvements why the panic? south korea's mers response questioned intensified public health measures help control mers-cov outbreak in the republic of korea south korean mers outbreak spotlights lack of research complete genome sequence of middle east respiratory syndrome coronavirus isolated in south korea complete genome sequence of middle east respiratory syndrome coronavirus (mers-cov) from the first imported mers-cov case in china urgent call for research on middle east respiratory syndrome (mers) in korea middle east respiratory syndrome (mers) in asia: lessons gleaned from the south korean outbreak no respite for korean economy even as mers patients recover middle east respiratory syndrome coronavirus (mers-cov) -china mers in south korea and china: a potential outbreak threat? objective determination of end of mers outbreak, south korea updates on mers outbreak (as of 6:00 on surveillance operation for the 141st confirmed case of middle east respiratory syndrome coronavirus in response to the patient's prior travel to jeju island the role of superspreading in middle east respiratory syndrome coronavirus (mers-cov) transmission moh holds the first press conference to update community and media on mers severe acute respiratory syndrome vs. the middle east respiratory syndrome middle east respiratory syndrome coronavirus (mers-cov) 2012-2015 case list of moh/who novel coronavirus mers ncov announced cases geneious basic: an integrated and extendable desktop software platform for the organization and analysis of sequence data recent evolution patterns of the middle east respiratory syndrome coronavirus (mers-cov) preliminary analysis of middle east respiratory syndrome coronavirus (mers-cov) sequences from korea and china any unreferenced opinions expressed herein are those of the authors and do not necessarily represent the views of any employer or institution. our thanks go to andrew rambaut, maia majumder, marion koopmans and hale abdali for helpful discussions on social media and cmam and riam for patience. the authors declare that they have no competing interests.author's contributions imm and kea contributed equally to this work and read an approved the final manuscript. key: cord-333355-ykmp4ven authors: kuchar, e.; miśkiewicz, k.; nitsch-osuch, aneta; szenborn, l. title: pathophysiology of clinical symptoms in acute viral respiratory tract infections date: 2015-03-19 journal: pulmonary infection doi: 10.1007/5584_2015_110 sha: doc_id: 333355 cord_uid: ykmp4ven in this article we discuss the pathophysiology of common symptoms of acute viral respiratory infections (e.g., sneezing, nasal discharge, sore throat, cough, muscle pains, malaise, and mood changes). since clinical symptoms are not sufficient to determine the etiology of viral respiratory tract infections, we believe that the host defense mechanisms are critical for the symptomatology. consequently, this review of literature is focused on the pathophysiology of respiratory symptoms regardless of their etiology. we assume that despite a high prevalence of symptoms of respiratory infection, their pathogenesis is not widely known. a better understanding of the symptoms’ pathogenesis could improve the quality of care for patients with respiratory tract infections. 1 introduction the literature reports that each year up to 25 million people in the us visit their doctor because of respiratory tract infections. the upper respiratory tract infections, better known as common colds, are the most common clinical presentation of said infections ). viral lower respiratory tract infections, bronchitis, bronchiolitis, and pneumonia; e.g., with respiratory syncytial virus (rsv) or influenza are generally more common in infants, young children, and in patients with chronic medical conditions, whereas older children and healthy adults usually suffer from milder upper respiratory tract infections (eccles 2005) . common cold was the third most common diagnosis made during ambulatory care visits by patients of all ages in the us (hsiao et al. 2010 ). it has been estimated that an average adult suffers from 2 to 4 colds per year and a schoolchild suffers from 6 to 10 colds per year (spector 1995; johnston and holgate 1996; winther et al. 1998; eccles 2005) . therefore, respiratory tract infections represent a significant clinical and therapeutic problem, and an economic burden (wat 2004) . upper respiratory tract infections are generally mild, selflimiting, and viral in their origin (johnston and holgate 1996; snow et al. 2001; turner 2011; kennedy et al. 2012) . in experimental studies, the infections have been defined as a short mild illness, with the early symptoms being headaches, chills, sneezing, and a sore throat, and delayed symptoms being nasal obstruction or discharge, cough, and malaise (jackson et al. 1958) . the duration of symptoms varies from 7 to 10 days, with a peak occurring on the 2-3rd day. however, some symptoms may be observed up to 3 weeks after the onset of the infection (heikkinen and järvinen 2003; eccles 2005) . common colds are triggered by rhinovirus (rv) coronavirus, influenza and parainfluenza viruses, and rsv (wat 2004; eccles 2005; kennedy et al. 2012) . the diagnosis of upper respiratory tract infections is based on symptoms and, with the exception of antivirals which may be used in influenza, treatment remains symptomatic. studies on different viruses responsible for the upper respiratory tract infections have shown that it is not possible to identify the virus based on the symptoms (johnston and holgate 1996; eccles 2002) . not only is the clinical presentation of these infections similar regardless of their etiology, but the order in which the symptoms develop is also similar. if the etiology of infections cannot be determined on the basis of clinical symptoms, the host reaction must play a major role in their pathogenesis. the similarities in the clinical presentation of viral upper respiratory tract infections stems from the same immune response pattern to different etiologic agents (eccles 2005) . furthermore, in acute respiratory tract infections a routine diagnosis to determine the etiology is not usually carried out in primary care settings. advances in molecular biology help explain the mechanisms that generate the symptoms of viral respiratory tract infections. nevertheless, the practical use of the pathophysiology of common symptoms seems relatively poor compared with the amount of scientific knowledge available. clinical manifestations of respiratory tract infections are familiar and well-known (eccles 2005; turner 2011 ). although the symptomatology depends, to some extent, on the type and location (e.g., pharyngitis, rhinitis, sinusitis, and bronchitis), the etiology of respiratory infection (viral or bacterial), patient's age, general health, co-morbidities, immunity, and on whether the infection is primary or secondary, e.g., in rsv or influenza there is a great amount of variation and overlap in both etiology and symptoms of individual infections. consequently, even defining the exact syndrome, like common cold or influenza-like morbidity, is difficult and problematic (eccles 2005) . the most significant signs of viral respiratory tract infections include sneezing, rhinorrhea (runny nose and nasal discharge), nasal congestion, cough, tachypnea, and fever. subjective symptoms include a sore throat, malaise, shivering (chills), shortness of breath, muscle aches and weakness, fatigue, and a loss of appetite and headaches (snow et al. 2001; wat 2004; eccles 2005; kennedy et al. 2012) . febrile seizures are a rare but important symptom in young children up to 6 years of age (schuchmann et al. 2011) . symptoms of upper respiratory tract infections have been traditionally classified as early or late (jackson et al. 1958; eccles 2005) . the early symptoms are those that develop quickly and resolve rapidly after 1-2 days, like headaches, sneezing, chills, sore throat, and malaise. in children a high fever may be observed, complicated by seizures in some cases (monto et al. 2000; schuchmann et al. 2011) . late symptoms include nasal discharge, nasal obstruction, and cough. the later symptoms develop over several days and are present one week after experimental infection (jackson et al. 1958; eccles 2005) . the development of sneezing before coughing in patients with a common cold may be partly explained by the involvement of the upper airways first and the infection subsequent spread to the lower respiratory tract (eccles 2005) . the aim of this review is to discuss the pathophysiology of symptoms of respiratory tract infections. we focused on the most significant symptoms of acute viral respiratory infections: sneezing, nasal discharge and obstruction, sore throat, coughing, muscle pains, malaise and mood changes, fever, and febrile seizures in children. we believe that despite a high prevalence of the symptoms, their pathogenesis is not widely known and a better understanding should have a beneficial effect on the therapeutic approach and should improve the quality of patient care. references from relevant articles were also identified. studies were included if they met the following two criteria: published in english and containing valid, consistent, and relevant data. the first two authors of the present review independently assessed all titles and abstracts to determine whether the inclusion criteria were satisfied. if either of the assessors considered the abstract potentially suitable, full-text articles were retrieved and then assessed by both assessors for their suitability for inclusion. eventually, 129 publications were selected and studied by each of the authors before the manuscript was prepared. 3 results agents of upper respiratory tract infections rhinoviruses are the most common etiologic agents of common cold. they are responsible for one-third to half of all upper respiratory tract infections reported in adults annually (proud et al. 1990; hendley 1998; heikkinen and järvinen 2003; ruuskanen et al. 2013) . improved knowledge about the structure and function of rhinoviruses has been acquired in recent years using virus culture techniques and new molecular genetics methods available. currently, more than 100 serotypes have been identified with hrv-a and hrv-b being the most important of them all (heymann et al. 2005; peltola et al. 2008; bochkov et al. 2011; kennedy et al. 2012 ). it has been demonstrated that the pathomechanism of symptoms in rhinoviral respiratory tract infections does not result from the cell damage, unlike influenza virus or rsv action (winther et al. 1990 ). rather, rhinovirus disrupts the tight junctions of the epithelial barrier, which facilitates the translocation of pathogens and stimulates the host's innate and adaptive immune responses (rezaee et al. 2011; kennedy et al. 2012) . over 90 % of rhinovirus serotypes enter the nasal epithelial cells through the host receptor with the intercellular adhesion molecule-1 (icam-1) being a glycoprotein immunoglobulin expressed on non-ciliated epithelial cells and on the basal surface of the ciliated epithelium of nasopharyngeal mucosa, while only around ten rhinovirus serotypes use a minor group of receptors, low-density lipoprotein (ldl) (bardin et al. 1994; winther et al. 1997; whiteman et al. 2003; bella and rossmann 2000; vlasak et al. 2005; kennedy et al. 2012) . newly discovered and sequenced human rhinovirus-c (hrv-c) is somehow unique as the virus isolates do not grow in typically used cell cultures, e.g., embryonic lung fibroblasts. in vitro growth of hrv-c was successfully performed using the sinus mucosal tissue as a substrate (bochkov et al. 2011; kennedy et al. 2012) . furthermore, studies on the structure and function of hrv-c have shown that the virus enters the cells using neither the icam-1 nor ldl receptor and the pathomechanism of the hrv-c infection remains unclear (arden and mackay 2010; simmonds et al. 2010; bochkov and gern 2012; lee et al. 2012; ruuskanen et al. 2013) . in otherwise healthy individuals, rhinovirus infections are generally limited to the upper respiratory tract with rhinorrhea and nasal obstruction being the most prominent symptoms (kennedy et al. 2012 ). entry of the rhinovirus triggers the release of interleukin-8 (il-8) a major cytokine in the recruitment of polymorphonuclear cells (hendley 1998) . il-8, which is produced locally, increases the production of nasal secretions and causes an influx of neutrophils (douglass et al. 1994) . oxidative stress caused by viral infection is probably responsible for the cellular mechanisms that lead to the production and release of il-8 (zhu et al. 1997) . studies of volunteers infected with a rhinovirus show a local infection in a small proportion (1-2 %) of nasopharyngeal epithelial cells (turner et al. 1982; bardin et al. 1994; arruda et al. 1995) . the higher the nasopharyngeal viral load the more severe is the disease (esposito et al. 2014) . vasoactive kinin peptides are other important mediators produced on-site in nasal mucosa and submucosa in rhinovirus-infected humans. kinins released in the nose following plasma exudation increase the symptoms of rhinoviral infection and cause an increase in vascular permeability, vasodilatation, and glandular secretion. bradykinin applied to the nasal mucosa causes symptoms that mimic the common cold, including rhinitis, nasal obstruction, and sore throat (proud et al. , 1990 . symptom scores correlate with an increase in the kinin concentration. while nasal secretion in adults with symptomatic experimentally-induced rhinovirus infection contain significantly higher concentrations of bradykinin and lysyl-bradykinin, asymptomatic infections do not result in increased kinin concentrations ). interestingly, the presence of rhinovirus has been detected by rt-pcr in 20 % of asymptomatic people who had an infected family member (jartti et al. 2008 ). the coronavirus (cov) is the second etiologic agent of upper respiratory tract infections (eccles 2005; mesel-lemoine et al. 2012) . the most common human-infecting coronaviruses include 229e, oc43, sars-cov, and the recently discovered nl63 and hku1 (arden et al. 2005; esper et al. 2005; vabret et al. 2005; pyrc et al. 2007 ). the virus is transmitted by aerosol inhalation and reinfections often occur due to a short-lived immunity (callow et al. 1990; wat 2004) . as a result, coronavirus accounts for 15-30 % of upper respiratory tract infections in humans. these infections are limited predominantly to the upper respiratory tract and rarely spread to lower airways and lungs (mesel-lemoine et al. 2012) . the coronavirus infection can occasionally involve other organs (arbour et al. 2000; collins 2002; desforges et al. 2006 ). it has been reported that the two new members of the coronavirus family, nl63 and hku1, especially the nl63, could also trigger severe lower respiratory tract infections and abdominal disorders such as pain and diarrhea (arden et al. 2005; esper et al. 2005; vabret et al. 2005; pyrc et al. 2007) . epidemics caused by cov-nl63 and cov-hku1 have been observed every 2-3 years (kahn 2006; jartti et al. 2012) . studies have confirmed that the infection with cov-nl63 is associated with croup and acute respiratory disease mostly in children, the elderly, and patients with chronic diseases, with some fatal cases being reported han et al. 2007; wu et al. 2008; oosterhof et al. 2010; sung et al. 2010; milewska et al. 2013) . the growth of coronaviruses using standard tissue cultures is poor and advanced molecular methods are needed to isolate the virus, so that most infections may remain undiagnosed (walsh et al. 2013) . human aminopeptidase n (hapn), a zinc-binding protein with endopeptidase activity, is used by cov-229e for entry into the epithelial cells, whereas cov-oc43 uses hemagglutinin-esterase (he) and spike (s) -its own surface glycoproteins -to enter the cell (tyrrell et al. 1993; künkel and herrler 1996; wat 2004) . recent studies have shown the ability of cov-229e to destroy the dendritic cells, which are essential components of the respiratory tract's immune system, which may explain multiple reinfections with the same type of cov (mesel-lemoine et al. 2012) . although the pathogenesis of infection caused by the two main groups of cov -229e and oc43 -is different, the clinical symptomatology is similar (tyrrell et al. 1993; wat 2004 ). respiratory syncytial virus (rsv) is responsible for many flu-like illnesses (zambon et al. 2001) . rsv replication starts in the nasopharynx and then the virus infects the bronchiolar epithelium presumably by cell-to-cell spread or aspiration of secretions. the virus spares the basal cells and subsequently extends to the alveolar pneumocytes. the pathologic findings of rsv are characterized by necrosis of epithelial cells, infiltration with t cells and monocytes around arterioles and with neutrophils between the vascular structures and small airways (johnson et al. 2007 ). this leads to airway obstruction, air trapping, increased airway resistance, and is also associated with neutrophilia in bronchoalveolar lavage (everard et al. 1994 ). rsv has never been isolated from blood (peebles and graham 2005) . the immune response to rsv, especially cytokines and chemokines, seems to be responsible for the symptoms and severity of bronchiolitis (garofalo et al. 2001; legg et al. 2003 ). the cytokines il-8, il-6, tnf-alpha, and il-1 beta were detected in respiratory secretions from infected children and high il-6 concentrations are associated with severe manifestations of the disease (matsuda et al. 1995; noah et al. 1995; smyth et al. 1997) . respiratory secretions from infected children contain chemokines expressed and secreted by t-cells (chemokine ligand 3 -ccl3, i.e. macrophage inflammatory protein-1 -mip-1 alpha; chemokine ligand 2 -ccl2, i.e. monocyte chemoattractant protein-1 -mcp-1; chemokine ligand 11 -ccl11 -eotaxin, and chemokine ligand 5 -ccl5, i.e. rantes; regulated on activation). mip-1 alpha, and to a lesser extent other beta-chemokines, primarily secreted by activated immune cells, are associated with severe manifestations of the disease (noah et al. 1995; welliver et al. 2002; garofalo et al. 2005) . experimental infection of explanted polarized respiratory epithelium in tissue culture generates il-8 and ccl5 (mellow et al. 2004 ). nonetheless, it is unknown whether the cytokines and chemokines are the cause of disease or are by-products of enhanced inflammatory responses (barr and graham 2014). whether respiratory tract infections caused by influenza viruses present as common colds with typical symptoms or as severe lower respiratory tract diseases depends on the type of virus, pre-existing immunity, the patient's underlying disorders, and multiple other factors (wat 2004) . the phenomena such as antigenic shift or drift have led to the formation of more recent and increasingly virulent variations of the influenza virus and, consequently, to more serious clinical manifestations (gething et al. 1980; treanor 2004) . as an example, pandemic influenza a (h1n1)pdm09 affected all age groups, but it was more prevalent in younger patients and children in whom there was the highest rate of hospitalization and pneumonia (kuchar et al. 2013 ). it has previously been shown that coughing and fever are the best predictive factors of influenza infections having a positive predictive value (ppv) of 79 % (monto et al. 2000) . however, neither symptom is sufficiently predictive in children aged 1-4 (ohmit and monto 2006) . overall, influenza viruses are generally responsible for 5-15 % of acute upper respiratory tract infections in humans (wat 2004 ). the influenza virus causes damage to the epithelial cells and its replication occurs in the airways with predilection to the lower respiratory tract (hers and mulder 1961; hers 1966; wat 2004 ). the two main glycoproteins of the influenza virus surface -hemagglutinin (ha) and neuraminidase (na)play an essential role in the infection spread. ha targets cells for infection binding to the epithelial sialylated glycans (specific for upper or lower airways) (shinya et al. 2006; de wit et al. 2010; fukuyama and kawaoka 2011) , while na is responsible for effective viral replication (pappas et al. 2008) . another viral protein, nonstructural protein 1 (ns1) is also important due to its counteracting ifn-α production in infected cells (fukuyama and kawaoka 2011) . viral replication is possible in host cells due to activation of nuclear factor kappa b (nf-κb) and the raf/mek/erk cascade, and then proinflammatory cytokines are produced with interleukin 6 (il-6) being the most important of them (kaiser et al. 2001; pinto et al. 2011; wine and alper 2012) . il-6, tumor necrosis factor-α (tnf-α), interferon-α (ifn-α), il-8, and il-1β increase significantly in response to the viral invasion resulting in the development of fever, nasopharyngeal mucous production, and respiratory and systemic symptoms. viral replication and the intensity of the main influenza symptoms are correlated with the level of cytokines, particularly with il-6 and tnf-α (hayden et al. 1998; kaiser et al. 2001 ). evidence presented in our review of the pathophysiology of signs and symptoms in the four most common viral upper respiratory tract infections suggest that the immune system's response to infection rather than the virusspecific damage to the respiratory tract is responsible for the symptomatology (turner 1997; hendley 1998; eccles 2005) . studies on different viruses responsible for upper respiratory tract infections have shown that it is not possible to identify the virus based on the symptoms (eccles 2005) . the pathology of rhinovirus infections consists of the influx of polymorphonuclear leukocytes at the beginning of the infection (winther et al. 1984) . macrophages play a key role in triggering an acute phase response with the production of cytokines (beutler 2003) , while the release of proinflammatory cytokines and other mediators cause upper respiratory tract infection symptoms (eccles 2000a, b) . cytokines are responsible for the systemic symptoms (e.g., fever) and bradykinin plays a major role in local symptoms of respiratory tract infections (e.g., sore throat and nasal congestion) shibayama et al. 1996; conti et al. 2004 ). a sore throat, irritation, and pain in the pharynx usually appear at the beginning of a respiratory tract infection. a sore throat is most likely caused by the action of prostaglandins and bradykinin on sensory nerve endings in the upper respiratory tract. intranasal administration of bradykinin causes symptoms of rhinitis and a sore throat, so it is likely to be responsible for these symptoms (rees and eccles 1994; proud 1998) . the sensation of pain is mediated by the cranial nerves supplying the nasopharynx. similar symptoms have been observed in bacterial upper respiratory tract infections, pharyngitis, and tonsillitis (georgitis 1993 ). nasal congestion is a subsequent symptom of respiratory infection that develops over the first week of symptoms (tyrrell et al. 1993) . the mechanism of nasal congestion relies on the dilation of the venous sinuses in the nasal epithelium in response to the vasodilator mediators such as bradykinin (proud et al. 1990; widdicombe 1997) . symptom scores increase as kinin concentrations rise (proud et al. 1990 ). dilatation of the sinuses in the narrow nasal valve region causes obstruction of the nasal airway (eccles 2000b) . the so-called nasal cycle (alternating congestion and decongestion of the nasal passages controlled by the sympathetic vasoconstrictor nerves) is accentuated and the asymmetry of nasal airflow is more pronounced during respiratory infection (eccles et al. 1996) . a watery nasal secretion often accompanied by sneezing is an early symptom of a respiratory tract infection. nasal discharge in respiratory infections is a complex mixture of plasma and glandular exudates with cellular elements (e.g., goblet cells, plasma cells, and neutrophils) of variable composition that changes over the course of the infection and severity of the inflammatory response (eccles 1983 ). the first phase of nasal discharge consists of a glandular secretion reflex caused by stimulation of the upper airway's trigeminal nerves. studies have demonstrated that intranasal administration of ipratropium inhibits nasal secretions in the first 4 days of a common cold (e.g., when it is caused by coronavirus) (akerlund et al. 1993; hayden et al. 1996) . the color of the nasal discharge may change from watery clear to yellow and green during the course of the respiratory tract infection and this reflects the severity of the inflammatory response rather than the etiology of the infection (stockley et al. 2001) . the green or yellow color of nasal discharge is often regarded as a clinical marker of bacterial superinfection and clinical indication to antibiotic treatment, but there is no evidence that supports this concept (murray et al. 2000) . the color change is related to the recruitment of leukocytes into the airway lumen (stockley et al. 2001) . neutrophils and activated monocytes contain chromatic, green granules (azurophil granules) containing myeloperoxidase with heme pigment. the more leukocytes present in nasal discharge the more colorful the nasal discharge appears (stockley et al. 2001) . although the literature is related to sputum color changes, the same mechanisms apply to nasal discharge. a watery nasal secretion in the infection's early stage is often accompanied by sneezing. sneezing together with a sore throat are the early symptoms of respiratory tract infections. sneezing is a reflex mediated by the trigeminal nerves which supply the nasal epithelium (leung and robson 1994; eccles 2005) . the sneeze center in the brainstem coordinates the sneeze reflex. sneezing is related to inflammatory responses in the nose and nasopharynx that stimulate the trigeminal nerves (eccles 2005) . as intranasal administration of histamine causes sneezing, sneezing is probably mediated by histamine receptors on the trigeminal nerves (mygind et al. 1983; eccles 2005) . coughing is the most common clinical symptom and the most frequent reason for visits to see a doctor (mcgarvey and morice 2006) . coughing is a protective reflex that prevents the aspiration of food and fluids into the airway and cleans the respiratory tract of mucus and other foreign bodies. the reflex is mediated exclusively by the vagus nerve (eccles 2005) . coughing is initiated in the airway through stimulation of the sensory nerves in the larynx or below (widdicombe 1995) . the airway inflammation associated with rhinitis must reach the larynx to cause coughing. the external ear and esophagus are also supplied by the vagus nerve and coughing can also be triggered by gastroesophageal reflux (morice 2002) . respiratory tract infections are often accompanied by redundant, dry, and unproductive coughing in the first days. the unproductive coughing may be caused by the inflammatory process spreading to the larynx since nasal inflammation causes sneezing rather than coughing. coughing in respiratory tract infections is believed to be mediated by hyperreactivity of the cough reflex due to the effects of inflammatory mediators on the airway's sensory nerve endings (lee et al. 2002; eccles and lee 2004) . when the larynx is inflamed and hyperreactive, coughing may occur spontaneously or in response to stimuli that would not normally cause coughing, e.g., cold air. it may persist for three weeks or longer. some coughs may be voluntary and related to airway irritation (lee et al. 2002) . productive coughing usually occurs later in the course of respiratory tract infection and is related to the mucus production associated with inflammation of the lower airways (eccles 2005) . rhinovirus and coronavirus do not usually cause significant damage to the airway cells and the common cold is typically associated with little, if any, coughing while the influenza virus may cause substantial cellular damage to the respiratory epithelium and an influenza infection is usually associated with coughing (monto et al. 2000) . respiratory tract infections are associated with impaired psychomotor function (smith et al. 1998) . mood changes and malaise may be explained by the unpleasant objective symptoms of respiratory tract infections such as nasal congestion, rhinorrhea, and coughing (eccles 2005) . these symptoms may cause discomfort and lower the patient's quality of life. however, there is increasing evidence that mood changes may also be caused by the effects of cytokines on the central nervous system (mahoney and ball 2002) . interferon alpha treatment for chronic hepatitis b and c is associated with flu-like adverse effects similar to those observed in respiratory tract infections: malaise, fever, myalgia, and mood changes (schaefer et al. 2002) . psychiatric adverse effects such as depression, irritability, impaired concentration, and psychoses have been reported with interferon alpha therapy. it has been reported that cytokines, e.g., tumor necrosis factor-α (tnf-α) and interleukins 1, 2, and 6 cause mood changes with anhedonia, cognitive dysfunction, anxiety, irritability, psychomotor slowing, fatigue, anorexia, sleep alterations, and a lower pain threshold (capuron and miller 2004) . the production of these cytokines is also associated with respiratory tract infections which may mediate mood changes associated with these infections. the exact mechanisms of cytokine action in the brain are poorly understood, but there is a growing body of evidence suggesting that anorexia associated with respiratory infections is mediated by cytokines that act directly on the feeding center in the hypothalamus (langhans 2000) . headaches are a common early symptom of respiratory tract infections. the majority (60 %) of patients with respiratory tract infections with a sore throat reported headaches in a clinical trial (eccles et al. 2003) . the mechanism of a headache associated with a respiratory tract infection is unknown but headaches may be triggered by cytokines released in response to a viral infection (smith 1992) . it has been shown that the administration of some cytokines such as tumor necrosis factor and interferons cause headaches (smith 1992; gold et al. 2005; van zonneveld et al. 2005 ). myalgia is a common symptom of respiratory tract infections. around half of patients with a common cold complain about muscle pain (eccles et al. 2003; eccles 2005) . myalgia occurs in the acute immune response to infection phase and is related to the effects of cytokines on skeletal muscles (baracos et al. 1983 ). proinflammatory cytokines including tnf-α have been implicated in the breakdown of muscle proteins (kotler 2000) . fever and myalgia associated with respiratory tract infection may be caused by the production of prostaglandin e2 in response to cytokines (baracos et al. 1983 ). the cytokine-related synthesis of prostaglandin e2 and the breakdown of skeletal muscle has been inhibited in vitro by non-steroidal anti-inflammatory agents and similarly fever and myalgia accompanying acute respiratory infection are relieved with acetylsalicylic acid, a classic anti-inflammatory agent (eccles et al. 2003) . since prostaglandin e2 is a pain mediator, its increased synthesis may explain the myalgia associated with acute respiratory tract infections. fever is a classic response to infection. it is a manifestation of cytokine release in response to a variety of stimuli. it is believed to be beneficial for the host's response to infection (cabanac 1990; eccles 2005) and is usually associated with novel or severe viral infections such as influenza (monto et al. 2000) . consequently, fever is a common symptom in infants, probably because viruses responsible for acute respiratory tract infections are new to the infant and induce a strong immune response. however, in adults who had been exposed to numerous common cold viruses in the past, subsequent infections do not elicit a strong cytokine response and fever is a rare symptom of a common cold in adults (eccles 2005) . on the contrary, some patients experience a transient fall in body temperature during the early stages of acute benign respiratory tract infection and about 1/3 of all patients experience chills (eccles et al. 2003) . chills associated with a fall in skin temperature related to vasoconstriction of the skin's blood vessels may be explained as an initial stage of fever, but chills may also be unrelated to changes in skin temperature. chills have developed after administration of exogenous pyrogens, even if the subjects maintained a neutral skin temperature (34.5 c in water) in experimental human studies (guieu and hellon 1980) . chills occur together with shivering and the latter symptom is probably related to the cerebral cortex influence over the shivering control. both chills and shivering may be caused by cytokines acting on the temperature control center of the hypothalamus. many cytokines act as endogenous pyrogens and are released from leukocytes in response to infection (conti et al. 2004 ). the proinflammatory cytokines (il-1 and il-6) are regarded as the most important cytokines for causing fever (netea et al. 2000; leon 2002) . they are believed to cross the blood-brain barrier and increase the thermal set point in the temperature control center. the hypothalamus then induces shivering, constriction of the skin's blood vessels, and chills (eccles 2005) . febrile seizures are a rare but significant symptom of acute viral respiratory infections in children. they occur in 2-5 % of all children and the majority of febrile seizures are triggered by respiratory tract infection (schuchmann et al. 2011) . febrile seizures are defined as occurring in children aged 6-60 months with a temperature 38.0 c with no central nervous system infection, metabolic disturbance, or history of afebrile seizure. they are the most common type of seizure in children under 60 months (american academy of pediatrics 2011; graves et al. 2012) . cytokines seem to play a crucial role in febrile seizures, however there is a lot of confusion about the relationship between proinflammatory and anti-inflammatory cytokines and the febrile seizure risk. is it generally accepted that the genotype il-1α-889 1/1 and il-1β-511 t/t homozygote as well as the serum concentration of il-6 are associated with an increased risk of febrile seizures (saghazadeh et al. 2014 ). the treatment of acute viral respiratory tract infections remains primarily supportive. there is evidence that medications like acetaminophen (paracetamol) and non-steroidal anti-inflammatory agents such as ibuprofen or aspirin relieve some symptoms of acute respiratory tract infections (fever, sore throat, pain, and malaise), but it is debatable whether symptomatic treatment could speed up recovery (eccles 2005) . many other common advices like drinking plenty of fluids or steam inhalation have not been scientifically proven. some new agents seem to be promising. for example in a study by asada et al. (2012) l-carbocysteine reduced the baseline and rs virus infection-induced secretion of pro-inflammatory cytokines, including il-1β, il-6, and il-8 as well as virus titers in the supernatant of human tracheal epithelial cells culture. although a virus-orientated approach and the development of anti-viral agents should be more beneficial, the diverse etiology makes the development of universal antivirals highly unlikely (passioti et al. 2014 ). since clinical symptoms are not sufficient to determine the etiology of acute viral respiratory tract infections, we believe that the host defense mechanisms are critical to the symptomatology. immune response seems to be fundamental for understanding the pathomechanisms of these infections. inflammatory mediators such as prostaglandins and bradykinin are responsible for the local symptoms of nasal congestion and rhinorrhea, while cytokines are responsible for systemic symptoms. a better understanding of the immune response including cytokines interactions will eventually allow for better treatments to be developed and should improve the quality of care for patients with acute respiratory tract infections. mucosal exudation of fibrinogen in coronavirus-induced common colds neurodiagnostic evaluation of the child with a simple febrile seizure neuroinvasion by human respiratory coronaviruses newly identified human rhinoviruses: molecular methods heat up the cold viruses new human coronavirus, hcov-nl63, associated with severe lower respiratory tract disease in australia localization of human rhinovirus replication in the upper respiratory tract by in situ hybridization l-carbocisteine inhibits respiratory syncytial virus infection in human tracheal epithelial cells stimulation of muscle protein degradation and prostaglandin e2 release by leukocyticpyrogen (interleukin-1). a mechanism for the increased degradation of muscle proteins during fever detection of rhinovirus infection of the nasal mucosa by oligonucleotide in situ hybridization respiratory syncytial virus infection: clinical features and diagnosis -virus-infection-clinical-features-and-diagnosis? source¼search_result&search¼rsv& selectedtitle¼1~138 icam-1 receptors and cold viruses science review: key inflammatory and stress pathways in critical illness -the central role of the toll-like receptors clinical and molecular features of human rhinovirus c molecular modeling, organ culture and reverse genetics for a newly identified human rhinovirus c phylogeny of fever the time course of the immune response to experimental coronavirus infection of man cytokines and psychopathology: lessons from interferon-alpha in vitro detection of apoptosis in monocytes/macrophages infected with human coronavirus molecular determinants of adaptation of highly pathogenic avian influenza h7n7 viruses to efficient replication in the human host hcov-229e infects and activates monocytes influence of interleukin-8 challenge in the nasal mucosa in atopic and nonatopic subjects physiology of nasal secretion nasal airflow in health and disease pathophysiology of nasal symptoms acute cooling of the body surface and the common cold understanding the symptoms of the common cold and influenza cough induced by airway vibration as a model of airway hyperreactivity in patients with acute upper respiratory tract infection changes in the amplitude of the nasal cycle associated with symptoms of acute upper respiratory tract infection effects of acetylsalicylic acid on sore throat pain and other pain symptoms associated with acute upper respiratory tract infection evidence of a novel human coronavirus that is associated with respiratory tract disease in infants and young children impact of rhinovirus nasopharyngeal viral load and viremia on severity of respiratory infections in children analysis of cells obtained by bronchial lavage of infants with respiratory syncytial virus infection the pathogenesis of influenza virus infections: the contributions of virus and host factors macrophage inflammatory protein-1alpha (not t helper type 2 cytokines) is associated with severe forms of respiratory syncytial virus bronchiolitis a comparison of epidemiologic and immunologic features of bronchiolitis caused by influenza virus and respiratory syncytial virus nasopharyngitis, pharyngitis, and tonsillitis cloning and dna sequence of double-stranded copies of haemagglutinin genes from h2 and h3 strains elucidates antigenic shift and drift in human influenza virus the long-term safety and tolerability of high-dose interferon beta-1a in relapsing-remitting multiple sclerosis: 4-year data from the prisms study excessive antibiotic use for acute respiratory infections in the united states febrile seizures: risks, evaluation, and prognosis the chill sensation in fever human coronavirus-nl63 infections in korean children effectiveness and safety of intranasal ipratropium bromide in common colds. a randomized, double-blind, placebo-controlled trial local and systemic cytokine responses during experimental human influenza a virus infection. relation to symptom formation and host defense the host response, not the virus, causes the symptoms of the common cold disturbances of the ciliated epithelium due to influenza virus broad aspects of the pathology and pathogenesis of human influenza role of viral infections, atopy and antiviral immunity in the etiology of wheezing exacerbations among children and young adults national ambulatory medical care survey: 2007 summary transmission of the common cold to volunteers under controlled conditions. i. the common cold as a clinical entity identification of respiratory viruses in asymptomatic subjects: asymptomatic respiratory viral infections new respiratory viral infections the histopathology of fatal untreated human respiratory syncytial virus infection epidemiology of viral respiratory infections the widening scope of coronaviruses symptom pathogenesis during acute influenza: interleukin-6 and other cytokine responses pathogenesis of rhinovirus infection pandemic influenza in the 2009/2010 season in central poland: the surveillance study of laboratory confirmed cases structural and functional analysis of the s proteins of two human coronavirus oc43 strains adapted to growth in different cells anorexia of infection: current prospects voluntary control of cough human rhinovirus species and season of infection determine illness severity type 1 and type 2 cytokine imbalance in acute respiratory syncytial virus bronchiolitis invited review: cytokine regulation of fever: studies using gene knockout mice common respiratory tract infections as psychological entities: a review of the mood and performance effects of being ill development of interleukin 6 and tumor necrosis factor alpha activity in nasopharyngeal secretions of infants and children during infection with respiratory syncytial virus clinical cough and its mechanisms the effect of respiratory synctial virus on chemokine release by differentiated airway epithelium a human coronavirus responsible for the common cold massively kills dendritic cells but not monocytes novel polymeric inhibitors of hcov-nl63 clinical signs and symptoms predicting influenza infection epidemiology of cough predictors of an antibiotic prescription by gps for respiratory tract infections: a pilot role of histamine and antihistamines in the nose kinins are generated during experimental rhinovirus colds circulating cytokines as mediators of fever nasal cytokine production in viral acute upper respiratory infection of childhood symptomatic predictors of influenza virus positivity in children during the influenza season fatal lower respiratory tract disease with human corona virus nl63 in an adult haematopoietic cell transplant recipient single gene reassortants identify a critical role for pb1, ha, and na in the high virulence of the 1918 pandemic influenza virus the common cold: potential for future prevention or cure pathogenesis of respiratory syncytial virus infection in the murine model clinical effects of rhinovirus infections inhibition of influenza virusinduced nf-kappab and raf/mek/erk activation can reduce both virus titers and cytokine expression simultaneously in vitro and in vivo the kinin system in rhinitis and asthma nasal provocation with bradykinin induces symptoms of rhinitis and a sore throat kinins are generated in nasal secretions during natural rhinovirus colds the novel human coronaviruses nl63 and hku1 sore throat following nasal and oropharyngeal bradykinin challenge polyinosinic: polycytidylic acid induces protein kinase d dependent disassembly of apical junctions and barrier dysfunction in airway epithelial cells new aspects on human rhinovirus infections proinflammatory and anti-inflammatory cytokines in febrile seizures and epilepsy: systematic review and meta-analysis interferon-alpha, cytokines and possible implications for mood disorders respiratory alkalosis in children with febrile seizures bradykinin levels during experimental nasal infection with rhinovirus and attenuated influenza virus avian flu: influenza virus receptors in the human airway proposals for the classification of human rhinovirus species c into genotypically assigned types the cytokine theory of headache effects of the common cold on mood and performance immunological responses to respiratory syncytial virus infection in infancy principles of appropriate antibiotic use for treatment of nonspecific upper respiratory tract infections in adults the common cold: current therapy and natural history assessment of airway neutrophils by sputum colour: correlation with airways inflammation role of human coronavirus nl63 in hospitalized children with croup influenza vaccine -outmaneuvering antigenic shift and drift epidemiology, pathogenesis, and treatment of the common cold goldman's cecil medicine shedding of infected ciliated epithelial cells in rhinovirus colds signs and symptoms in common colds the safety of pegylated interferon alpha-2b in the treatment of chronic hepatitis b: predictive factors for dose reduction and treatment discontinuation the minor receptor group of human rhinovirus (hrv) includes hrv23 and hrv25, but the presence of a lysine in the vp1 hi loop is not sufficient for receptor binding clinical impact of human coronaviruses 229e and oc43 infection in diverse adult populations the common cold: a review of the literature betachemokines, but neither t helper type 1 nor t helper type 2 cytokines, correlate with severity of illness during respiratory syncytial virus infection human rhinovirus selectively modulates membranous and soluble forms of its intercellular adhesion molecule-1 (icam-1) receptor to promote epithelial cell infectivity neurophysiology of the cough reflex microvascular anatomy of the nose cytokine responses in the common cold and otitis media histopathologic examination and enumeration of polymorphonuclear leukocytes in the nasal mucosa during experimental rhinovirus colds respiratory virus infection of monolayer cultures of human nasal epithelial cells surface expression of intercellular adhesion molecule 1 on epithelial cells in the human adenoid viral-induced rhinitis clinical manifestations of human coronavirus nl63 infection in children in taiwan contribution of influenza and respiratory syncytial virus to community cases of influenza-like illness: an observational study rhinovirus stimulation of interleukin-8 in vivo and in vitro: role of nf-kappab the authors have no funding or conflicts of interest to disclose. key: cord-306315-vt2e0crh authors: elabbadi, alexandre; pichon, jérémie; visseaux, benoit; schnuriger, aurélie; bouadma, lila; philippot, quentin; patrier, juliette; labbé, vincent; ruckly, stéphane; fartoukh, muriel; timsit, jean-françois; voiriot, guillaume title: respiratory virus-associated infections in hiv-infected adults admitted to the intensive care unit for acute respiratory failure: a 6-year bicenter retrospective study (hiv-vir study) date: 2020-09-14 journal: ann intensive care doi: 10.1186/s13613-020-00738-9 sha: doc_id: 306315 cord_uid: vt2e0crh introduction: acute respiratory failure is the main reason for admission to the intensive care unit (icu) in hiv-infected adults. there is little data about the epidemiology of respiratory viruses in this population. methods: hiv-infected adults admitted to two intensive care units over a 6-year period for an acute respiratory failure and explored for respiratory viruses with multiplex polymerase chain reaction (mpcr) were retrospectively selected. objectives were to describe the prevalence of respiratory viruses, coinfections with non-viral pathogens, and hospital outcome. results: a total of 123 episodes were included. an hiv infection was newly diagnosed in 9% of cases and 72% of the population were on antiretroviral therapy. real-time mpcr tests identified at least one respiratory virus in the respiratory tract of 33 (27%) patients, but with a non-viral copathogen in two-thirds of cases. rhinovirus was predominant, documented in 15 patients, followed by influenza and respiratory syncytial viruses (both n = 6). the prevalence of respiratory virus-associated infection did not vary along with the level of the cd4 t-cell deficiency, except for rhinovirus which was more prevalent in patients with a cd4 lymphocyte count below 200 cells/µl (n = 13 (20%) vs. n = 2 (4%), p < 0.01). in multivariate analysis, respiratory virus-associated infection was not associated with a worse prognosis. conclusions: viruses are frequently identified in the respiratory tract of hiv-infected patients with acute respiratory failure that requires icu admission, but with a non-viral copathogen in two-thirds of cases. rhinovirus is the predominant viral specie; its prevalence is highest in patients with a cd4 lymphocyte count below 200 cells/µl. acute respiratory failure (arf) is the leading cause of admission to the intensive care unit (icu) in hivinfected patients [1] [2] [3] . infectious causes are predominant, although the proportion of opportunistic infections has decreased significantly in the era of combination antiretroviral therapy (art) [2, 4, 5] . in contrast, the open access *correspondence: guillaume.voiriot@aphp.fr 1 assistance publique -hôpitaux de paris, service de médecine intensive réanimation, hôpital tenon, sorbonne université, paris, france full list of author information is available at the end of the article burden of non-hiv-related pulmonary events, such as bacterial pneumonia, acute bronchitis and acute exacerbation of chronic obstructive pulmonary disease (copd) has been shown increasing [2, 3, 6] . these important changes in the etiologic panel of arf in hiv-infected patients question the role of respiratory viruses. indeed, using nucleic acid amplification test such as multiplex polymerase chain reaction (mpcr), these pathogens have been shown highly prevalent (20-56%) in large cohorts of adult patients admitted to the icu for all-cause arf [7, 8] , community-acquired pneumonia [9, 10] , hospitalacquired pneumonia [11] , acute exacerbation of copd [12, 13] , and asthma [14] , compared to asymptomatic adults [15, 16] . high prevalence has also been described in specific immunocompromised populations, such as cancer and hematology patients [17, 18] . in contrast, little is known about the epidemiology of respiratory viruses in hiv-infected patients [19, 20] , especially those admitted to the icu, and the prevalence of respiratory viruses according to the cd4 t-cell deficiency. moreover, coinfections with virus and opportunistic pathogens may occur. overall, respiratory virus-associated infections may affect prognosis. therefore, we conducted a comprehensive observational study among adult hiv-infected icu patients with arf explored with respiratory mpcr. our goals were to describe the prevalence of respiratory viruses, coinfections with non-viral pathogens, and hospital outcome. we conducted a retrospective bicenter observational study in two icu of the paris area (the 26-bed icu of the bichat university hospital and the 20-bed icu of the tenon university hospital). from april 2011 to april 2017, all consecutive hiv-infected patients admitted to icu having undergone an mpcr in the respiratory tract within 72 h following their icu admission were screened. medical records were independently reviewed by two physicians (ae and gv). all patients with arf at icu admission were included. arf was defined by the presence of at least two of the following criteria: cough, expectoration, dyspnea, rales, signs of respiratory distress (tachypnea exceeding 30/min, paradoxical abdominal breathing), chest pain, hypoxemia requiring oxygen therapy, noninvasive ventilation or intubation. in case of multiple admissions over the 6-year study period, only the first admission was analyzed. at icu admission and during icu stay, data regarding demographics, comorbidity, hiv-related characteristics, clinical examinations, laboratory and radiological findings, microbiologic investigations, and therapeutic management were collected (for details, see additional file 1). mortality was defined as death from any cause within 28 days following the icu admission. respiratory mpcrs were performed either in nasopharyngeal (np) swabs or in lower respiratory tract (lrt) specimen, usually bronchoalveolar lavage (bal) fluid otherwise endotracheal aspirate. during the study period, different respiratory mpcr kits (additional file 1: table s1 ) were used (for more details about microbiological evaluation, see additional file 1). medical charts were independently reviewed by two clinicians (ae and gv). they determined the causative diagnosis of arf for each patient, using a 5-class classification. in case of an inter-reviewer discordance, a shared review of the medical charts was performed, and an agreement was found. the five mutually exclusive classes of causative diagnosis for arf were: (i) pneumocystis jirovecii pneumonia (pcp); (ii) other opportunistic lung infections; (iii) non-opportunistic acute lung infection; (iv) non-infectious lung disease, and (v) extra-pulmonary cause (for details, see additional file 1). the primary endpoint was to determine the prevalence of respiratory viruses according to the cd4 lymphocyte count. a respiratory virus documented with mpcr was always considered as a pathogen of the respiratory tract, regardless of the type of specimen (np or lrt). the cd4 lymphocyte count measured during the icu stay was used to group patients, with a cut-off of 200 cells/µl (≤ 200 cells/µl for the low-cd4 group; > 200 cells/µl for the high-cd4 group) [19, 21] . secondary endpoints were to describe the epidemiology of respiratory viruses and coinfections with non-viral pathogens, to identify risk factors for respiratory virusassociated infection, and to study outcome. a composite criterion named "complicated course" included death from any cause within 28 days following the icu admission or mechanical ventilation for more than 7 days. continuous data were expressed as median [first through third quartiles] and were compared using the pairwise mann and whitney test. categorical data were expressed as number (percentage) and were evaluated using the chi-square test or fisher exact test. p values less than 0.05 were considered significant. a univariate logistic regression with clinically relevant variables was used to identify variables associated with a respiratory virusassociated infection. a multivariate conditional logistic regression, including variables with p value less than 0.10 in the previous step, was used to identify variables independently associated with a respiratory virus-associated infection. similar statistical analyses were performed to identify variables independently associated with death from any cause within 28 days following the icu admission and mechanical ventilation for more than 7 days in survivors at day-28. quantitative variables that did not validate the log-linearity assumption were transformed into categorical variables according to their median value. missing data were imputed to the median or the more frequent value. the accuracy of the final model was tested using area under the receiver operating characteristic curve analysis and the hosmer-lemeshow chi-square test. analyses were performed using the sas software package (sas institute, cary, nc, usa). during the 6-year study period, 135 hiv-infected adult patients were admitted at least once to icu and underwent a respiratory mpcr in the first 72 h of the icu stay. among them, 12 did not fulfill criteria of arf. the final study group consisted of 123 patients. their main characteristics, stratified by the cd4 lymphocyte count at icu admission, are presented in table 1 . of these 123 patients, 2 were admitted twice during the study period and one was admitted thrice. eleven patients (9%) were newly diagnosed as having hiv infection on icu admission; the remaining 112 had been previously diagnosed, and 88 were on art but with poor adherence to the treatment in 21 patients, as mentioned by the infectiologist in the medical charts. latest available median cd4 lymphocyte count and hiv viral load were 351 cells/µl [140-600] and 0 log copies/ml [0-3.4], respectively. at least one additional factor of immunosuppression was identified in 10 (8%) patients. at icu admission, median cd4 lymphocyte count was 170 cells/µl , with 66 patients (54%) equal or below 200 cells/µl (low-cd4 group) and 57 (46%) above 200 cells/µl (high-cd4 group). both these groups did not differ regarding demographics, performance status, factors of immunosuppression other than hiv and comorbidity, except for copd which was more prevalent in the high-cd4 group (n = 12 (21%) vs. n = 4 (6%), p = 0.01). the microbiological investigations are displayed in additional file 1: table s2 . mpcr was performed in np swabs exclusively (n = 46, 37%) or in lrt specimen exclusively (n = 50, 41%), or both (n = 27, 22%). respiratory tract specimens for bacterial culture have been obtained in 110 (91%) patients. bal fluid has been obtained in 77 (63%) patients. causative diagnoses of arf are displayed in table 2 . an opportunistic lung infection was diagnosed in 38 (31%) patients. seven of the 11 patients with newly diagnosed hiv infection and 8 patients receiving art, but with a poor adherence to the treatment had pcp. non-opportunistic acute lung infections were identified as causative diagnosis of arf in 59 (48%) patients. all the bacteria-infected patients received an appropriate antimicrobial regimen within the first 24 h of icu stay. eight patients had a clinical presentation suggestive of lung infection, but without microbiological documentation. the arf was attributed to a non-infectious lung disease in 19 (15%) patients, mainly related to a decompensated chronic condition, i.e., acute exacerbation of copd and pulmonary edema. overall, 36 respiratory viruses were identified in 33 (27%) patients (table 3) . rhinovirus was predominant (n = 15), followed by influenza (n = 6), respiratory syncytial virus (n = 6) and parainfluenza virus (n = 5). only one pure virus-virus coinfection was found. the prevalence of respiratory virus-associated infection did not differ among low-and high-cd4 groups (table 1) ; therefore, the median cd4 lymphocyte count in respiratory virus-infected patients was 109 cells/µl, in comparison with 192 cells/µl in non-infected patients (fig. 1) . however, the prevalence of rhinovirus-associated infection was higher in the low-cd4 group, and three-quarters of rhinovirus-infected patients exhibited a cd4 lymphocyte count below 200 cells/µl (fig. 2) . in 22 patients, the viral documentation was accompanied by a non-viral documentation (additional file 1: figure s1), with bacteria-virus coinfection in 11 patients, bacteria-virus-virus in 2 patients, p. jirovecii-virus in 7 patients and p. jirovecii-virus-virus in one patient. the rate of viral documentation among patients explored with np swab exclusively, lrt specimen exclusively, or both, did not differ significantly (30%, 26% and 22%, respectively; p = 0.73). documentation of respiratory viruses was more frequent during the winter period (october to march) (additional file 1: figure s2 ). conversely, rhinovirus documentation did not follow a seasonal distribution, since only 7/15 were observed during the period from october to march. characteristics of the population, stratified by respiratory virus-associated infection are presented in additional file 1: table s3 . at icu admission, respiratory virus-infected patients displayed higher respiratory rate and fever. in multivariate analysis, female gender, smoking and steroid therapy were shown as independently associated with respiratory virus-associated infection ( table 4) . mortality at day-28 was 12%, and 26% of patients displayed a complicated course, without difference between high-cd4 and low-cd4 groups (table 1) . we investigated whether a respiratory virus-associated infection table 2 causative diagnosis of acute respiratory failure in 123 hiv-infected patients admitted to the icu data are presented as number (%). cd4 refers to cd4 lymphocyte count (cells/µl) a other opportunistic lung infections included cmv-associated pneumonia (n = 5) and pulmonary tuberculosis (n = 4) b non-infectious lung diseases included acute exacerbation of copd of non-infectious etiology (n = 6), cardiogenic lung edema without underlying lung agent (n = 5), cryptogenic hemoptysis (n = 1), intra-alveolar hemorrhage (n = 1); acute interstitial pneumonia (n = 2), mendelson syndrome (n = 1), sickle cell disease (acute chest syndrome) (n = 1); neoplastic pleural effusion (n = 1) and castleman disease (n = 1) c extra-pulmonary causes included histoplasmosis (n = 1), cryptococcus neoformans meningitis (n = 1), bacterial meningitis (n = 2), pyelonephritis (n = 2) and bacteremia of unknown origin (n = 1) all extra-pulmonary cause c 7 (5.7) 6 (9.1) 1 (1.8) affected prognosis. in the analysis stratified by respiratory virus-associated infection, outcome was similar between infected and non-infected patients (additional file 1: table s3 ). in multivariate analysis, a respiratory virus-associated infection was not identified as an independent factor of either a complicated course (table 5 ) or death at day-28 (additional file 1: table s4 ). this retrospective study investigated the epidemiology of respiratory viruses in hiv-infected adults admitted to the icu for arf. real-time mpcr tests identified at least one virus in the respiratory tract of 27% of patients, but with a non-viral copathogen in two-thirds of cases. the prevalence of respiratory virus-associated infection did not vary along with the level of the cd4 t-cell deficiency, except for rhinovirus which was more prevalent in patients with a cd4 lymphocyte count below 200 cells/ µl. in multivariate analysis, respiratory virus-associated infection was not associated with a worse prognosis. in this study, more than one patient out of four (27%) were infected with at least one respiratory virus. this finding illustrated the high yield of an aggressive diagnostic strategy with a broad panel mpcr on respiratory tract specimens. our results are original since prior works having described the etiological panel of arf in hivinfected icu patients were conducted before the era of real-time mpcr [2, 4, 22] . interestingly, the rate of viral documentation that we observed was similar to what had been described (27 to 29%) previously in non-hiv adults admitted to the icu for an acute respiratory disorder requiring intubation [7, 8] . we identified at least one non-viral copathogen in more than two-thirds of the patients with a viral documentation, in line with a recent report in a population with a high prevalence of tuberculosis [19, 23] . nonopportunistic acute lung infections, including pneumonia, acute bronchitis and exacerbation of copd, were the first cause of arf, consistent with previous reports in western countries [2, 4] . this finding highlights the burden of chronic respiratory conditions in aging hivinfected populations [6] . here, more than 40% of patients were smokers. synergistic effects of tobacco and hiv [24] in promoting chronic bronchitis and pro-copd changes in the lung [25] have been demonstrated. moreover, high rates of viral documentation within airways of copd patients both at stable state and during exacerbation have been reported [26] . these data may explain the high rate of viral documentation that we observed. in multivariate analysis, smoking was independently associated with respiratory virus-associated infection. this finding is in line with previous works demonstrating that tobacco exposure alters immune responses to rhinovirus [27] , influenza virus [28] and respiratory syncytial virus [29] . interestingly, female gender was associated with an increased risk of respiratory virus-associated infection on multivariate analysis. prior cohort studies in primary care described an increased risk for development of influenza-like illnesses in women compared to men [30, 31] . however, to our knowledge, no prior study has specifically explored this point in hiv-infected populations admitted for arf. in this study, we also aimed to investigate a putative role of the hiv-related cd4 t-cell deficiency in promoting respiratory virus-associated infection. previous studies explored this point in children, but with conflicting results. annamalay et al. described similar rates of viral documentation in hiv-infected and non-infected children admitted for lower respiratory tract infections [32] , whereas o'callaghan-gordo et al. observed that respiratory virus-associated infections were 6 to 16 times more prevalent among hiv-infected children admitted for pneumonia [33] . as we did not include a comparative non-hiv population, we rather examined whether or not the rate of viral documentation varied along with the level of cd4 t-cell deficiency. finally, we found no association between the cd4 lymphocyte count and the risk of respiratory virus-associated infection, in line with a previous report focusing on influenza viruses [34] . rhinovirus was the predominant virus, accounting for more than 40% of viral documentations. this high prevalence was consistent with that of previous reports in icu patients with arf [7] , community-acquired pneumonia [35] or acute exacerbation of copd [13] . surprisingly, rhinovirus was much more prevalent in low-cd4 patients. this finding is original, since no prior work has specifically explored this point in adults. in hiv-infected children, rhinovirus has been shown highly prevalent, during both pneumonia and bronchiolitis, but without data regarding a putative association with the level of cd4 t-cell deficiency [32, 36] . other data in hematology and cancer adults demonstrated high rates of rhinovirus documentation within airways during respiratory tract infections [37, 38] . to explain this high prevalence in immunocompromised patients, a mechanism of prolonged viral shedding has been proposed, rather than iterative reinfections as observed in copd patients [39] . the prolonged rhinovirus shedding may be attributable to an inefficient immunological control of a single infectious episode [40, 41] . therefore, in pediatric hematopoietic stem cell transplant recipients with a persistent rhinovirus shedding (≥ 30 days), piralla et al. demonstrated significant lower cd4, cd8 and nk lymphocyte counts at the onset of infection, as compared to children with a brief rhinovirus shedding. moreover, a decrease in rhinovirus load was associated with significant increases of the same lymphocyte counts [42] . these data would suggest an important role for the t-cell immunity in the control of rhinovirus infection, and subsequently, may explain a delayed rhinovirus clearance in low-cd4 hivinfected patients, resulting in persistent shedding and increased prevalence. we observed a high rate of dual infection, either virusbacteria or virus-opportunistic pathogen. these findings made us consider the prognostic impact of such coinfections. studies in icu adult patients with pneumonia suggested that virus-bacteria coinfection was associated with a worse prognosis [43] . in mice, the coinfection of influenza with streptococcus pneumoniae [44] , legionella pneumophila [45] or staphylococcus aureus [46] impaired the anti-influenza immune response and increased mortality. whether similar synergistic effects exist in virusopportunistic pathogen coinfection remain unknown. only one animal study has explored the couple pneumocystis jirovecii-influenza, but in a successive rather than concomitant model [47] . unfortunately, in our study, the low number of observations prevented us from analyzing the prognosis according to the presence of coinfections. our study has several limitations. first, this study included adult patients with arf that required icu admission, preventing any conclusion on other populations such as hiv-infected children or hiv-infected adults with arf that did not require icu admission. second, the study was retrospective, so we did not control the microbiological investigations. the preferred sample for mpcr test in non-intubated patients was not the sputum, but the nasopharyngeal swab [48] . several factors may have discouraged clinicians to use sputum, including the high number of patients unable to produce sputum [49] and the highly viscous nature of this sample that can make nucleic acid extraction difficult [50] . by definition, an mpcr was performed in the respiratory tract of every included patient because it was a criterion for patient screening. but some other microbiological tests were only occasionally performed, i.e., cytomegalovirus pcr. furthermore, the retrospective design prevented us from obtaining a number of data, which were rarely reported in medical records by physicians, including vaccine history, pneumocystis jirovecii prophylaxis, symptoms before hospital referral, and duration of symptoms before icu admission. third, only patients having undergone an mpcr in the respiratory tract within the 72 h following their icu admission were screened; this might suggest a confounding of indication. fourth, the choice to classify patients according to their cd4 lymphocyte count on the icu admission, instead of the latest known value, might be criticized. however, this choice was guided by the high number of missing values in the latest cd4 lymphocyte count as well as the number of newly diagnosed patients without any prior cd4 lymphocyte count. fifth, we assumed that a virus identified with pcr in nasopharyngeal or lower respiratory tract samples was always a pathogen of the respiratory tract, whatever the clinical picture and radiological features. this might be criticized since respiratory viruses might be present in asymptomatic adult subjects [15] , even if it seems rare, about 2% of asymptomatic adults seen at the emergency department [16] . sixth, to avoid overinterpreting the data, we decided to consider respiratory viruses as a homogeneous group of pathogens in the analysis stratified by respiratory virus-associated infection. this might be criticized since the pathogenicity differs from one viral species to another. viruses are frequently identified in the respiratory tract of hiv-infected patients with arf that required icu admission, but with a non-viral copathogen in two-thirds of cases. rhinovirus is the predominant viral specie; its prevalence is highest in patients with a cd4 lymphocyte count below 200 cells/µl. supplementary information accompanies this paper at https ://doi. org/10.1186/s1361 3-020-00738 -9. additional file 1. additional information on material and methods, table s1 (panels of mpcr kits used in the two participating icus over the 6-year study period), table s2 (microbiological investigations performed in 123 hiv-infected patients admitted to the icu for acute respiratory failure, according to the diagnosis of respiratory virus-associated infection), table s3 (baseline characteristics, behavior during icu stay, and outcome of 123 hiv-infected patients admitted to the icu for acute respiratory failure, according to the diagnosis of respiratory virus-associated infection), table s4 (multivariate analysis of the risk factors for death at day-28 in 123 hiv-infected patients admitted to the icu for acute respiratory failure), figure s1 (distribution of the microbiological documentations in 123 hivinfected patients admitted to the icu for acute respiratory failure), figure s2 admissions to intensive care unit of hiv-infected patients in the era of highly active antiretroviral therapy: etiology and prognostic factors etiologies and outcome of acute respiratory failure in hivinfected patients temporal trends in critical events complicating hiv infection: 1999-2010 multicentre cohort study in france survival for patients with hiv admitted to the icu continues to improve in the current era of combination antiretroviral therapy pulmonary infections in hiv-infected patients: an update in the 21st century hiv infection and risk for incident pulmonary diseases in the combination antiretroviral therapy era epidemiology and clinical outcome of virus-positive respiratory samples in ventilated patients: a prospective cohort study respiratory viruses in invasively ventilated critically ill patients-a prospective multicenter observational study viral-bacterial coinfection affects the presentation and alters the prognosis of severe community-acquired pneumonia lower respiratory tract virus findings in mechanically ventilated patients with severe community-acquired pneumonia impact of respiratory viruses in hospital-acquired pneumonia in the intensive care unit: a single-center retrospective study virus infection in exacerbations of chronic obstructive pulmonary disease requiring ventilation procalcitonin algorithm to guide initial antibiotic therapy in acute exacerbations of copd admitted to the icu: a randomized multicenter study epidemiology of respiratory viruses in patients hospitalized with near-fatal asthma, acute exacerbations of asthma, or chronic obstructive pulmonary disease community surveillance of respiratory viruses among families in the utah better identification of germs-longitudinal viral epidemiology (big-love) study community-acquired pneumonia requiring hospitalization among u.s. adults clinical significance of upper airway virus detection in critically ill hematology patients management of respiratory viral infections in hematopoietic cell transplant recipients and patients with hematologic malignancies prospective etiological investigation of community-acquired pulmonary infections in hospitalized people living with hiv. medicine (baltimore) respiratory viruses in hiv-infected patients with suspected respiratory opportunistic infection outcome of critically ill human immunodeficiency virus-infected patients in the era of highly active antiretroviral therapy survival of hiv-infected patients in the intensive care unit in the era of highly active antiretroviral therapy etiology of pulmonary infections in human immunodeficiency virus-infected inpatients using sputum multiplex real-time polymerase chain reaction impact of hiv infection and smoking on lung immunity and related disorders cigarette smoke and hiv synergistically affect lung pathology in cynomolgus macaques a prospective, observational cohort study of the seasonal dynamics of airway pathogens in the aetiology of exacerbations in copd cigarette smoke decreases innate responses of epithelial cells to rhinovirus infection cigarette smoke attenuates the rig-i-initiated innate antiviral response to influenza infection in two murine models cigarette smoke suppresses tlr-7 stimulation in response to virus infection in plasmacytoid dendritic cells recording of influenza-like illness in uk primary care 1995-2013: cohort study incidence and risk factors for influenza-like-illness in the uk: online surveillance using flusurvey respiratory viruses in young south african children with acute lower respiratory infections and interactions with hiv etiology and epidemiology of viral pneumonia among hospitalized children in rural mozambique: a malaria endemic area with high prevalence of human immunodeficiency virus pandemic (h1n1) 2009 influenza virus seroconversion rates in hiv-infected individuals incidence of respiratory viruses in patients with community-acquired pneumonia admitted to the intensive care unit: results from the severe influenza pneumonia surveillance (sips) project clinical epidemiology of bocavirus, rhinovirus, two polyomaviruses and four coronaviruses in hiv-infected and hiv-uninfected south african children respiratory virus detection in immunocompromised patients with filmarray respiratory panel compared to conventional methods virological diagnosis in community-acquired pneumonia in immunocompromised patients prolonged shedding of rhinovirus and re-infection in adults with respiratory tract illness chronic rhinoviral infection in lung transplant recipients persistent human rhinovirus type c infection of the lower respiratory tract in a pediatric cord blood transplant recipient persistent rhinovirus infection in pediatric hematopoietic stem cell transplant recipients with impaired cellular immunity viral infection in community-acquired pneumonia: a systematic review and meta-analysis coinfection with streptococcus pneumoniae negatively modulates the size and composition of the ongoing influenza-specific cd8 + t cell response role of tissue protection in lethal respiratory viral-bacterial coinfection influenza virus primes mice for pneumonia from staphylococcus aureus pneumocystis infection enhances antibodymediated resistance to a subsequent influenza infection diagnosis and management of respiratory viruses in critically ill adult patients: an international survey of knowledge and practice among intensivists diagnostic value of microscopic examination of gram-stained sputum and sputum cultures in patients with bacteremic pneumococcal pneumonia comparison of sputum and nasopharyngeal swabs for detection of respiratory viruses publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations none. gv had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis, including and especially any adverse effects. ae participated in the design of the study, participated in the data acquisition, analysis and interpretation, and the statistical analysis, and drafted the manuscript. jp, bv, as, lb, qp, jp and vl participated in the data acquisition, analysis and interpretation, and helped to revise the manuscript. rs participated in the data analysis and interpretation, and the statistical analysis. mf and jft participated in the design of the study, participated in the data analysis and interpretation, and helped to revise the manuscript. gv designed the study, participated in the data analysis and interpretation, and the statistical analysis, and revised the manuscript. all authors read and approved the final manuscript. none. data and materials supporting the findings of this study can be entirely shared if asked. not applicable. the authors have reported that no potential conflicts of interest exist with any companies/organizations whose products or services may be discussed in this article. key: cord-306480-wgl4zrnb authors: subissi, lorenzo; bossuyt, nathalie; reynders, marijke; gérard, michèle; dauby, nicolas; bourgeois, marc; delaere, bénédicte; quoilin, sophie; van gucht, steven; thomas, isabelle; barbezange, cyril title: capturing respiratory syncytial virus season in belgium using the influenza severe acute respiratory infection surveillance network, season 2018/19 date: 2020-10-01 journal: euro surveill doi: 10.2807/1560-7917.es.2020.25.39.1900627 sha: doc_id: 306480 cord_uid: wgl4zrnb background: respiratory syncytial virus (rsv) is a common cause of severe respiratory illness in young children (< 5 years old) and older adults (≥ 65 years old) leading the world health organization (who) to recommend the implementation of a dedicated surveillance in countries. aim: we tested the capacity of the severe acute respiratory infection (sari) hospital network to contribute to rsv surveillance in belgium. methods: during the 2018/19 influenza season, we started the sari surveillance for influenza in belgium in week 40, earlier than in the past, to follow rsv activity, which usually precedes influenza virus circulation. while the who sari case definition for influenza normally used by the sari hospital network was employed, flexibility over the fever criterion was allowed, so patients without fever but meeting the other case definition criteria could be included in the surveillance. results: between weeks 40 2018 and 2 2019, we received 508 samples from sari patients. we found an overall rsv detection rate of 62.4% (317/508), with rates varying depending on the age group: 77.6% in children aged < 5 years (253/326) and 34.4% in adults aged ≥ 65 years (44/128). over 90% of the rsv-positive samples also positive for another tested respiratory virus (80/85) were from children aged < 5 years. differences were also noted between age groups for symptoms, comorbidities and complications. conclusion: with only marginal modifications in the case definition and the period of surveillance, the belgian sari network would be able to substantially contribute to rsv surveillance and burden evaluation in children and older adults, the two groups of particular interest for who. respiratory syncytial virus (rsv) is an important cause of acute lower respiratory infections in children [1] . severe forms of disease caused by rsv, including pneumonia, can lead to hospitalisation resulting in several thousand deaths per year worldwide in hospitalised children under the age of 5 years [2] . rsv is also a major cause of severe acute respiratory infections (sari) in older adults (≥ 65 years old) [3] [4] [5] . currently, in europe, palivizumab is the only commercially-available antiviral designed against rsv infection, and specifically for children [6] . vaccines are in development and are expected to become available in the coming years [7] [8] [9] . to help decision-makers and to evaluate the impact of future vaccination, a dedicated surveillance is necessary. this surveillance might shed light on several aspects of rsv epidemiology (e.g. seasonality, atrisk age groups, complications) in particular why/how certain parameters vary between different areas of the world [10] . in 2016, the world health organization (who) launched a pilot study to evaluate the feasibility of using the longstanding worldwide influenza surveillance network (global influenza surveillance and response system) for a sustainable rsv surveillance [10] . the attempt to implement such a surveillance aimed at better understanding the virus, its circulation patterns and the disease it causes in the different age groups to eventually refine case definitions [10] . in addition, this study tried to look into the health burden of rsv in order to identify at-risk groups who would most benefit from vaccination [10] . the case definition recommended for countries using a hospital-based surveillance strategy was an extended who sari case definition for patients aged ≥ 6 months. for those aged < 6 months, the case definition should also include apnoea or sepsis [10] . moreover, the sampling strategy recommended ca 1,000 samples per year per country, distributed among the following age groups: < 6 months, 6 months to 4 years, 5 to 64 years, and ≥ 65 years [10] . in belgium, the national influenza centre recognised by who obtains influenza surveillance data through two networks. the first consists of general practitioners and concerns influenza-like illness (ili) surveillance (from week 40 to week 20 of the following year; i.e. influenza season surveillance period); the second is constituted by six hospitals that monitor sari (from the last week of december or first/second week of january to the third/last week of april, depending on when influenza virus circulation is detected by the ili network; i.e. the influenza activity period). historical data of the numbers of positive tests reported by all the belgian hospital laboratories show that rsv circulation generally occurs sometime between october and january, and usually precedes or slightly overlaps the circulation of influenza viruses [11] , like in neighbouring countries [12, 13] . since these data do not provide enough information to assess the rsv burden, we decided to test the existing sari network capability to contribute to rsv surveillance in belgium. during the 2018/19 influenza season (week 40 2018 to week 20 2019), three of the six hospitals of the sari network, one in each administrative region (flanders, brussels-capital, wallonia), volunteered to start the sari surveillance as early as week 40 2018, instead of at the end of december or beginning of january. our usual sari case definition, based on the who sari case definition, was used: acute respiratory infection with fever ≥ 38 °c (or history of fever reported by the patient) and cough or dyspnoea, with onset of symptoms within the past 10 days, and requiring hospitalisation (minimum overnight). however, in order to align with one of the case definitions proposed in who's rsv strategy document [14, 15] , the participating hospitals were recommended to be flexible over the fever criteria, meaning that patients without fever but meeting the other criteria could also be included. unless consent was refused, enrolment included all patients meeting the case definition. beside sampling (nasopharyngeal swab or aspirate), sari standardised questionnaires were used to collect data on age, sex, symptoms, antibiotic treatment, known comorbidities and follow-up during hospitalisation to evaluate the disease severity. respiratory samples were analysed at the national influenza centre. viral nucleic acids were extracted using biomerieux's nuclisens easymag (brussels, belgium). routine in-house multiplex reverse transcription quantitative (rt-q)pcrs were used to detect the following respiratory virus targets: adenoviruses, bocavirus, coronaviruses (cov-oc43, cov-nl63 and cov-229e separately), human metapneumoviruses, influenza virus types a and b, parainfluenzavirus types 1, 2, 3 and 4 (separately), parechovirus, picornaviruses (rhinovirus and enterovirus genera) with specifically enterovirus d68, and rsv types a and b (adapted from original protocols by united states centers for disease control and prevention; lj van elden, university medical centre utrecht, the netherlands; p overduin, the national institute for public health and the environment, the netherlands; o hungnes and k bragstad, institute of public health, norway). primer and probe sequences and rt-qpcr conditions are available upon request. a two-tailed fisher's exact test (two-by-two table) or a chi-squared test was performed for group proportion comparisons. the mann-whitney test was used to compare the distributions of the length of stay in hospital. differences were considered as statistically significant for a p value < 0.05. the sari surveillance protocol was approved by the central ethical committee (reference ak/12-02-11/4111; in 2011: centre hospitalier universitaire st-pierre, brussels, belgium; since 2014: universitair ziekenhuis brussel, brussels, belgium) and the local ethical committees of the hospitals. the amendment for the pilot study was specifically approved by the central ethical committee and the local ethical committees of the participating hospitals. informed consent was obtained from all participants or parents/guardians. from week 40 2018 until week 2 2019, the week of the official start of the influenza sari surveillance for the six hospitals, the national influenza centre received a total of 578 samples from the three hospitals participating in the study, of which 508 were eligible based on the adopted case definition (figure 1 ). the median number of samples per week was 38 (interquartile range (iqr): 18.5-37.8). the three sites each contributed to 36.0% (183/508), 39.2% (199/508) and 24.8% (126/508) of the total samples. there were slightly more samples from males (55.1%, 280/508) than females. the who's recommended four age groups were covered, but the patients aged between 5 and 64 years were less represented (table 1 ) and differences between sites were noted (data not shown). the weekly distribution of the number of rsv-positive samples among the sari patients ( figure 2a ) appeared to match the epidemiological curve reported by the national reference centre for respiratory pathogens ( figure 2b) week of sampling number of samples the 6-month-to-4-year-old children (94/137), 37.0% among the 5-to-64-year-old patients (20/54) and 34.4% among the older adults (44/128). the weekly distributions of the number of positive samples were similar for the two younger age groups, with a clear bell-shape curve starting as early as week 41 (figure 3 ). on the other hand, for the 5-to-64-and ≥ 65-year age groups, the first positive samples were detected only from week 47 and 46, respectively, and the curve resulting from the distribution did not present a clear peak. among the rsv-positive samples ( table s1 ), and less than 6% (18/326) were negative for all tested respiratory viruses. in contrast, among patients aged ≥ 5 years, more than one third (64/182) tested negative for all investigated respiratory viruses, 32% (59/182) tested positive for only rsv, nearly 30% (54/182) tested positive only for a respiratory virus other than rsv, and less than 3% (5/182) tested positive for rsv and at least one other respiratory virus of the panel (supplement table s1 ). the data for fever, cough and dyspnoea are presented in table 2 and supplement table s1 , and summarised in figure 4a . fever was more common among children aged < 5 years than among patients aged ≥ 5 years regardless of the rsv status (with fever, rsv-negative: 93.2% (68/73) for < 5 years old vs 78.8% (93/118) for ≥ 5 years old, fisher exact test p = 0.008; with fever, rsv-positive: 95.3% (241/253) for < 5 years old vs 81.3% week of sampling year. a at least one of the following: asthma, chronic cardiovascular disease, chronic respiratory disease, diabetes, exposure to tobacco, hepatic insufficiency, immunodeficiency, neuromuscular disease, obesity, prematurity, renal insufficiency. b other than death. c at least one of the following: admission in icu, detection of pneumonia based on chest radiography, development of ards, resp. assis.. d in days. (52/64) for ≥ 5 years old, fisher exact test p < 0.001) ( table 2 ). no significant difference was observed in the proportions with fever among rsv-negative and rsv-positive patients, whatever the age group. there was some evidence of a difference in the proportion of patients with cough between the rsv-positive group (90.9%, 288/317) and the rsv-negative group (84.8%, 162/191, fisher exact test p = 0.044) ( table 2 ). however, this difference was not observed when comparing within each age group. there was very good evidence that dyspnoea was more often reported for patients aged ≥ 5 years (81.9% (149/182)) than among patients aged < 5 years (45.1% (147/326)), fisher exact test p < 0.001, but without difference between rsvpositive and rsv-negative cases ( table 2 ). in addition to these clinical signs included in the case definition and stated on the questionnaire, hospital site 1 systematically reported for all patients a detailed list of other symptoms registered by the medical staff in the patients' files. some of these additional symptoms have been positively associated to rsv in another study [16] . figure 4b summarises the results of hospital site 1 for rhinitis, tachypnoea and wheezing. these three symptoms appeared to be more frequently reported for rsv-positive patients in the < 5-year-olds comorbidities were more common in patients aged ≥ 5 years than in those aged < 5 years (92.9% (169/182) vs 19.9% (65/326), two-sided fisher exact test p < 0.001), but no difference was observed between rsv-positive and rsv-negative patients within each age group (table 2) . supplement table s1 details the comorbidities per age group. among the comorbidities reported in children < 6 months (24/189), the most common were premature birth (nine patients, six of whom rsv-positive), chronic respiratory diseases (five patients, all rsv-positive), tobacco exposure (four patients, three of whom rsv-positive) and chronic cardiovascular disease (one rsv-positive patient). among the most reported comorbidities in the 6-month-to-4year-old patients (42/137), nine were classified as premature birth (seven rsv-positive), seven as chronic respiratory diseases (five rsv-positive), six as asthma (four rsv-positive), six as neuromuscular diseases (five rsv-positive), five as immunodeficiencies (four rsv-positive), three as tobacco exposure (all rsvpositive) and two as chronic cardiovascular diseases (both rsv-positive). the most frequently reported comorbidities in patients aged ≥ 5 years were chronic respiratory diseases (71/169), chronic cardiovascular diseases (62/169), immunodeficiency (41/169), diabetes (30/169), obesity (23/169) and asthma (19/169). cases were relatively evenly distributed between rsvpositive, negative for all tested viruses, and only positive for another tested respiratory virus (supplement table s1 ). there was very good evidence that the hospitalisation stay of patients aged ≥ 5 years (median: 9 days; iqr: 5-15) was longer than that of the patients aged < 5 years (median: 4 days; iqr: 2-5) (mann-whitney p < 0.001) ( table 2 ). length of stay for rsvpositive cases among patients ≥ 5 years was not different from that for the negative-for-all-testedviruses or only-positive-for-another-tested-respiratoryvirus cases (supplement table s1 ). on the contrary, in the two age groups of children aged < 5 years, hospitalisation stay was longer for rsv-positive than for rsv-negative cases. for < 6-month-olds, the median stay for rsv positive patients was 4 days (iqr: 3-6) vs a median of 3 days (iqr: 2-4) for rsv-negative patient (mann-whitney p < 0.001). for 6-month-to-4-yearolds, the median stay for rsv-positive patients was 4 days (iqr: 3-5) vs a median stay of 3 days for rsvnegative patients (iqr: 2-5; mann-whitney p = 0.055) (supplement table s1 ). twenty-two patients died during hospitalisation (four patients aged between 45 and 64 years, as well as 18 adults aged ≥ 65 years). all four patients aged between 45 and 64 years had more than one comorbidities. among the patients ≥ 65 years, two had no report of comorbidity and five had only one comorbidities. by comparison, among the 35 patients aged between 45 and 64 years who survived, one had no comorbidity, 16 had one and 18 had more than one. among the 110 surviving patients ≥ 65 years, one had no comorbidity, 40 had one and 69 had more than one. overall, other kinds of complications than death were more common among children aged ≥ 5 years (97/182) than among those aged < 5 years (128/326; two-sided fisher exact test p = 0.003). among the latter, some complications seemed more common among the rsv-positive patients: pneumonia (25/33), requirement for respiratory assistance (82/103), ards (12/15) and transfer to icu (6/6) ( table 2 ). this pilot study showed that belgian sari network built to assess the severity of influenza viruses is able to capture the rsv activity if started earlier than usual. the beginning (week 41 2018) and peak (week 49 2018) of the rsv epidemic could be observed in this investigation and may have been missed by the usual sari surveillance that started in week 2 2019 when the number of rsv positive samples had already considerably declined. the in-house rt-qpcrs allowed circulating rsvs to be typed and would allow to follow the change in type dominance if the current surveillance was extended to other years in the future [17, 18] . the targeted number of patients to be included in the rsv surveillance per year [10] would have been reached if the whole network of six hospitals had taken part. this pilot study also showed that the belgian sari network could be used to evaluate the severity of rsv without much adaptation. the sari network also has the advantage to operate based on a clear case definition and to allow the collection of data on several risk factors and severity indicators. it nonetheless appears that the two main populations of interest (children, especially those aged < 6 months, and older adults) present differences that need to be considered. having a good case definition for the different age groups represents one of the challenges in the implementation of a surveillance system for rsv, as high sensitivity is required to avoid an underestimation of the rsv burden [19] . in this pilot study, we purposefully chose to employ the sari case definition that has been used by the sari network for several years, while only being less stringent for the fever criteria. this was deliberate in order to evaluate the information that could be readily obtained from our network, but this clearly represents a limitation of our study [20] . the data obtained during one season of surveillance were moreover not sufficient to perform robust measures of association between rsv and comorbidities or complications. in addition, how the comorbidities and the complications were defined and evaluated was left to each hospital to decide. such lack of harmonisation also represents a limitation of the study that could be improved in the future. another problem was the lack of information on the patients who did not consent to participate in the study. of course, it would be impossible to obtain detailed information, but it could be useful to at least obtain the number of individuals lost to the surveillance and whether this should be taken into account in the analysis. nevertheless, the sari case definition we used, even with a less strict fever criterion, allowed to follow rsv circulation in the children population aged < 5 years and to identify comorbidities/indicators potentially associated with disease severity. wheezing, rhinitis and tachypnoea seem like notable additional clinical signs associated with rsv-positivity in children, as already described in a previous study [16] , and would need to be included in a second pilot study to fully assess their relevance in increasing the case definition sensitivity. extending the case definition to include other symptoms such as sepsis and apnoea might also need to be considered for children < 6 months, as recommended in who documents for rsv surveillance [10, 14, 15] . this would probably allow to better address the severity of disease caused by rsv in this age group, as these symptoms might be more frequently associated to severe forms [21, 22] . improving the case definition might be more challenging for older adults: the combination of clinical signs used here did not seem specific enough. dyspnoea was more often reported in this age group, but irrespective of the rsv test result. however, a specific case definition might not be the most important point for a meaningful rsv surveillance. assessment of the risk factors and rsv severity in this population would probably benefit more from a broader range of respiratory pathogens to be tested, including bacteria and fungi [23] , in order to better evaluate the exact contribution of rsv. these adaptations would have consequences on the actual sari surveillance. to maintain a simple system, the case definition should not change during the whole surveillance period. the currently used sari definition has proven effective to evaluate the severity of influenza viruses. slightly extending it to better fit rsv would lead to an increase number of sari cases being identified during the overall period of surveillance, which would then run from week 40 to week 20 of the following year. since starting at week 40 would almost double the length of the surveillance, this could potentially result in almost doubling the number of collected samples. a broader case definition, with additional possible clinical signs and without fever, would lead to even more patients meeting the criteria and being enrolled. this would, however, not prevent us to identify the subset of cases responding to the more specific influenza case definition, and thus, it should not hamper us from evaluating the specific severity associated to influenza viruses. on the contrary, a broader case definition could allow the current surveillance of influenza viruses to become a surveillance system for several respiratory viruses of public health importance, such as rsv, human metapneumoviruses, and coronaviruses including the newly emerged severe acute respiratory syndrome coronavirus 2 (sars-cov-2) virus [3, [24] [25] [26] [27] . that being said, since the current sari surveillance relies on the good will of the participating hospitals, the extra workload would require additional funding, as more personnel and more reagents would be required both at the hospital sites and at the national influenza centre. given the benefits that would be gained in terms of better understanding the burden of respiratory viruses, such evolution of the sari surveillance system might prove a more economic option on the long run. to conclude, we showed that the implementation of a rsv surveillance based on our influenza sari surveillance is feasible and would contribute to the objectives defined by who, but it would only be of interest if it can be sustained for several years. with sari surveillance being more frequently implemented in several european countries, our study provides an example on how this surveillance can be used to gain information on rsv epidemiology. respiratory syncytial virus hospitalization and mortality: systematic review and meta-analysis rsv global epidemiology network. global, regional, and national disease burden estimates of acute lower respiratory infections due to respiratory syncytial virus in young children in 2015: a systematic review and modelling study severe morbidity and mortality associated with respiratory syncytial virus versus influenza infection in hospitalized older adults respiratory syncytial virus infection in elderly and high-risk adults global disease burden estimates of respiratory syncytial virus-associated acute respiratory infection in older adults in 2015: a systematic review and meta-analysis respiratory syncytial virus clinical trials for respiratory syncytial virus (rsv) respiratory syncytial virus vaccines: are we making progress? who strategy to pilot global respiratory syncytial virus surveillance based on the global influenza surveillance and response system (gisrs) respiratory syncytial virus seasonality: a global overview european influenza surveillance network. seasonality and geographical spread of respiratory syncytial virus epidemics in 15 european countries leveraging the global influenza surveillance and response system for global respiratory syncytial virus surveillanceopportunities and challenges. influenza other respi viruses who meeting of midterm review of the rsv surveillance pilot based on the global influenza surveillance and response system can we distinguish respiratory viral infections based on clinical features? a prospective pediatric cohort compared to systematic literature review use of the moving epidemic method (mem) to assess national surveillance data for respiratory syncytial virus (rsv) in the netherlands epidemiological changes of respiratory syncytial virus (rsv) infections in israel performance of surveillance case definitions for respiratory syncytial virus infections through the sentinel influenza surveillance system performance of surveillance case definitions in detecting respiratory syncytial virus infection among young children hospitalized with severe respiratory illness-south africa risk factors for respiratory syncytial virus associated apnoea mortality due to respiratory syncytial virus burden and risk factors microorganisms associated with respiratory syncytial virus pneumonia in the adult population on behalf of inmi covid-study group and collaborating centers. differential diagnosis of illness in patients under investigation for the novel coronavirus burden and risk factors for coronavirus infections in infants in rural nepal human metapneumovirus infections are associated with severe morbidity in hospitalized children of all ages rsv global epidemiology network, resceu investigators. global patterns in monthly activity of influenza virus, respiratory syncytial virus, parainfluenza virus, and metapneumovirus: a systematic analysis the authors would like to thank jeannine weyckmans, ilham fdillate, assia hamouda and reinout van eycken from the national influenza centre for their excellent technical assistance throughout the surveillance seasons; evelyn petit from az sint-jan, françoise antoine, tiphaine mouquet and christelle nguepi from chu st-pierre, for their dedication in the follow-up of the sari cases; and all the doctors and nurses of the three hospitals who took part in the recruitment of the sari cases, accepting to start the surveillance earlier.funding: the influenza surveillance sari project received financial support from belgium federal public service 'health, food chain safety, and environment', the national insurance health care (inami/riziv), and the regional health authorities of flanders (azg, agentschap zorg en gezondheid) and of wallonia (aviq, agence pour une vie de qualité). none declared. this is an open-access article distributed under the terms of the creative commons attribution (cc by 4.0) licence. you may share and adapt the material, but must give appropriate credit to the source, provide a link to the licence and indicate if changes were made.any supplementary material referenced in the article can be found in the online version. key: cord-347255-fl9lur4h authors: may, larissa; tatro, grant; poltavskiy, eduard; mooso, benjamin; hon, simson; bang, heejung; polage, christopher title: rapid multiplex testing for upper respiratory pathogens in the emergency department: a randomized controlled trial date: 2019-11-05 journal: open forum infect dis doi: 10.1093/ofid/ofz481 sha: doc_id: 347255 cord_uid: fl9lur4h background: acute upper respiratory tract infections are a common cause of emergency department (ed) visits and often result in unnecessary antibiotic treatment. methods: we conducted a randomized clinical trial to evaluate the impact of a rapid, multipathogen respiratory panel (rp) test vs usual care (control). patients were eligible if they were ≥12 months old, had symptoms of upper respiratory infection or influenza-like illness, and were not on antibiotics. the primary outcome was antibiotic prescription; secondary outcomes included antiviral prescription, disposition, and length of stay (clinicaltrials.gov# nct02957136). results: of 191 patients enrolled, 93 (49%) received rp testing; 98 (51%) received usual care. fifty-three (57%) rp and 7 (7%) control patients had a virus detected and reported during the ed visit (p = .0001). twenty (22%) rp patients and 33 (34%) usual care patients received antibiotics during the ed visit (–12%; 95% confidence interval, –25% to 0.4%; p = .06/0.08); 9 rp patients received antibiotics despite having a virus detected. the magnitude of antibiotic reduction was greater in children (–19%) vs adults (–9%, post hoc analysis). there was no difference in antiviral use, length of stay, or disposition. conclusions: rapid rp testing was associated with a trend toward decreased antibiotic use, suggesting a potential benefit from more rapid viral tests in the ed. future studies should determine if specific groups are more likely to benefit from testing and evaluate the relative cost and effectiveness of broad testing, focused testing, and a combined diagnostic and antimicrobial stewardship approach. acute respiratory tract infection (arti) is a common cause of emergency department (ed) visits, accounting for ~12.6 million cases annually from 2001 to 2010 [1] . many of these cases are due to viral infection, and up to 30%-40% are prescribed antibiotics inappropriately [2] . this is worrisome, as overuse of antibiotics creates selective pressure for antibiotic-resistant pathogens that increases morbidity, mortality, and health care cost for affected patients [3] . in addition, antibiotic use can lead to drug-related adverse reactions that could prompt patients to return to the ed [3, 4] . factors contributing to antibiotic overprescribing include concerns regarding patient satisfaction scores, difference in patient expectation and understanding of antibiotic effectiveness for viral infections, medical liability, and diagnostic uncertainty [3, 5] . the last point is especially applicable in the ed given the patient volume and current standard of care, where microbiologic testing results are often not available to inform diagnosis and treatment. therefore, the ed remains a highpriority target for rapid microbiologic testing and antimicrobial stewardship. recent improvements in molecular diagnostics and the development of rapid, multirespiratory pathogen molecular tests provide an opportunity to diagnose viral arti with high sensitivity and specificity during the ed visit and potentially improve patient management. hence, we sought to evaluate whether having a rapid, multipathogen test result available during the ed visit would have a significant impact on management and outcomes in patients with clinical signs and symptoms of arti. we conducted a prospective, patient-oriented, pilot randomized clinical trial of rapid multiplex respiratory pathogen testing (rp test group) vs usual care (usual care or control group) in a level 1 emergency department with limited use of single-organism rapid point-of-care tests at a quaternary referral medical center. the study began in december 2016 and occurred over 2 winter respiratory seasons and 1 intervening nonrespiratory season. patients were eligible if they were age 1 or older and being evaluated for influenza-like illness (ili; fever or cough and sore throat) or upper respiratory infection (uri; nonspecific upper respiratory symptoms without fever). patients were excluded if they were already on antibiotics at the time of ed presentation, not english-or spanish-speaking, cognitively impaired with no legally authorized representative, or expected to leave before multiplex test results were available. if a patient was deemed eligible for the study, the treating provider was approached by a research team member to confirm that uri or ili was suspected, and the patient was expected to be present for at least the next 90 minutes. patients were recruited monday through friday between 8 am and 10 pm from rapid care and main adult and pediatric ed areas. screening was performed under a waiver of informed consent, and the study protocol was approved by the uc davis institutional review board. after informed consent, a patient questionnaire was administered by a trained research assistant to collect demographic information and medical history (supplementary data). information about the patient's medical history and current medications was reviewed in the electronic health record (ehr) and discussed with the patient. patients were specifically asked if they had chronic medical conditions, if they had medical conditions affecting their immune system (hiv, diabetes, cancer, liver disease, kidney disease, or dialysis, or if they were on immunosuppressive medications such as steroids), and if they were currently taking any medications. subjects were randomized to either rapid near point-of-care multirespiratory pathogen molecular testing (filmarray respiratory panel, biofire) plus clinician-directed usual care or usual care alone. this included but was not limited to no testing, targeted influenza and/or respiratory syncytial virus (rsv) point-of-care testing (quidel flu a/b or rsv before january 10, 2018, then roche liat influenza a/b or rsv), rapid point-of-care testing for streptococcal pharyngitis (acceava strep a test before january 2018, then roche liat), or multirespiratory pathogen panel testing (xtag respiratory viral panel, luminex before december 2017, then genmark eplex) performed at an off-site laboratory 3-4 times weekly in scheduled daytime batches. simple randomization was used with a computer-generated randomized list to allocate subjects to the intervention or control arms of the study, and sealed, opaque envelopes were used to blind research staff to allocation up until written consent was completed. clinicians and patients were not blinded to the testing done, and usual care proceeded in both arms. after randomization, patients in the interventional arm had a nasopharyngeal swab specimen collected by a nurse or clinician. this specimen was transported via a tube system to an onsite clinical laboratory (upstairs), where the filmarray respiratory panel was performed in real time, with the goal of results reported in the ehr within 2 hours. clinicians were informed that results of the patient's rapid molecular testing would be returned through the patient's ehr, and they received an automatic in-basket message through the ehr as well as a notification by the research coordinator or research assistant when results were back. follow-up treatment and outcome data were abstracted from each subject's ehr by a trained research staff member. abstracted data included medical history, clinical signs and symptoms, demographics, lab and radiology results, medications (including asthma, diabetes, chemotherapy, immunosuppressive, antibiotic, and antiviral medications), ed/hospital course, length of ed/hospital stay, clinician diagnoses from the index encounter, and limited review of subsequent encounters to identify deaths and 30-day revisits to the ed study site for similar complaints. up to 3 return visits were recorded, and each was categorized as either "respiratory illness" or "nonrespiratory illness" according to the primary diagnoses of the encounter. in the case of patients who returned to the ed but left before being treated, their chief complaint was used instead of their diagnosis. chest x-ray results were reviewed by a clinical investigator (l.s.m.) to identify patients with imaging consistent with bacterial pneumonia or viral pattern illness. the primary outcome was antibiotic administration or prescription in the ed by an emergency medicine clinician. secondary outcomes included the proportion of patients with a respiratory pathogen identified by the filmarray respiratory panel test or any other upper respiratory pathogen diagnostic test ordered by the physician; the proportion of patients with a laboratory-confirmed influenza diagnosis; the proportion of patients receiving appropriate anti-influenza treatment or prescription in the ed by an emergency medicine clinician (composite rate of anti-influenza treatment in positive patients and nonuse of anti-influenza treatment in negative patients); the proportion of patients discharged home from the ed vs hospital admission; the proportion of patients with all-cause or respiratory illness-related repeat ed visit, hospital or icu admission, or death within 30 days; clinician adherence to guidelines for the treatment of patients with influenza (recommendations for use of antivirals only); length of ed stay; length of hospital stay; time to influenza test results; and time to other respiratory pathogen test results. we summarized data by standard descriptive statistics: continuous variables by mean and standard deviation (sd) or median and range (= min-max), categorical variables by frequency and percentage. we analyzed the equality of the proportions for binary outcomes with the chi-square or fisher exact test, as appropriate (eg, cell count <5), and reported (2-sided) p values, unadjusted for multiple testing. we also computed the difference in event rates (ie, proportion) and the associated 95% confidence interval (ci). for continuous outcomes, the wilcoxon test was used for comparison. we also performed a post hoc stratified analysis for antibiotic use (primary outcome), antiviral use, and discharge status, based on the patient's age (eg, adult vs pediatric) for exploratory purposes. we analyzed the data based on the (modified) intent-to-treat principle; that is, we analyzed all patients as randomized, excluding early dropouts with outcomes data unavailable. we used sas, version 9.4 (sas institute, cary, nc, usa), for data analysis. the study was initiated in december 2016 with a target enrollment of 325 participants over 12 months, based on a power calculation that 304 patients (152 in each arm) would have 80% power to detect a 15% difference in antibiotic prescription between arms. the study was stopped in april 2018 (17 months) due to budgetary constraints. our study enrolled 191 patients, with 93 (48.7%) randomized to the interventional rp test group and 98 (51.3%) randomized to the usual care control group ( figure 1 ). there was no statistically significant difference in age, race, or existence of a chronic medical condition between the 2 groups. the complete baseline characteristics of the study participants are summarized in table 1 . in the rp test group, 53 (57%) patients had 1 or more viruses detected and reported during the ed visit; 8 (9%) additional rp patients had a positive virus result reported after the ed visit (table 2 ). in the control group, 7 (7%) patients had a virus detected and reported by existing single-organism tests during the ed visit, and an additional 13 (14%) patients had a virus detected via the off-site laboratory multirespiratory pathogen panel after the ed visit. the results from the rapid rp tests were available in <2 hours on average ( table 2) . the primary and secondary outcome results are shown in table 3 (table 4 ). there were no other outcome differences between groups in the post hoc stratified age analysis (table 4) . large molecular panels capable of detecting 10-20 respiratory viruses simultaneously have been available for >10 years, but the utility of detecting noninfluenza, non-rsv viruses in outpatients is debated because results are delayed, and therapy does not exist [6] . meanwhile, diagnostic advances have made it possible to get results within 1-2 hours, and reducing unnecessary antibiotic use has become a national priority [7] . the filmarray respiratory panel provides results for 20 different respiratory pathogens in approximately 1 hour with 85%-100% sensitivity and 90%-100% specificity [8] . however, (14) 17 (17) diabetes mellitus 9 (10) 9 (9) other 6 (6) 12 (12) kidney disorders 3 (3) 6 (6) immunosuppressed 17 (18) 17 (17) abbreviations: bmi, body mass index; chf, congestive heart failure; copd, chronic obstructive pulmonary disease; rp, respiratory panel. a missing bmi measurements due to lack of height measurements for some patients. these large, multipathogen tests are more expensive than targeted point-of-care tests, and the clinical utility has not been rigorously evaluated, especially in the ed. as a result, some payers are currently moving to limit utilization and reimbursement of large multipathogen panels in favor of targeted influenza and/or rsv testing [9] . this evidence gap in utility and reimbursement uncertainty create challenges for clinicians, laboratories, and hospitals in justifying the cost of instruments and tests, and there is a lack of guidance regarding optimal use. we hypothesized that rapid multirespiratory pathogen testing with results reported during the ed visit could alter treatment in the early phases of patient care and potentially impact patient and health care outcomes. thus, we conducted a randomized clinical trial of the filmarray rp vs usual care in ed patients with signs or symptoms of upper respiratory infection or influenza-like illness. the primary outcome was antibiotic prescription. the trial was stopped after 17 months before reaching the prespecified sample size due to dwindling funds and slow enrollment. however, there was a trend toward decreased antibiotic use with rp testing (-12% difference; p = .06/0.08, chi-square/ fisher exact test) that was larger in pediatric patients (-19% difference; p = .047/0.07) in an age-stratified post hoc analysis, suggesting a potential benefit of increased rapid rp testing in some ed patients. antiviral prescription (ie, oseltamivir) was not significantly affected despite a 3-fold increase in viral (66% vs 20%) and influenza detections (26% vs 8%), perhaps due to the lack of guidance regarding antiviral use in our study. other secondary outcomes, such as ed disposition, 30-day return visits, and 30-day deaths, did not differ between groups. thus, the main effects of rapid rp testing in this study were to increase the proportion of patients with a lab-confirmed viral detection for clinical decision-making by 3-fold and reduce antibiotic prescription by about one-third. it is unknown if similar benefits could be achieved with targeted testing for influenza and/or rsv or antimicrobial stewardship alone. however, it has been suggested that influenza testing alone may be sufficient and cost-effective when applied to outpatients, given that other viruses do not have any specific treatment at this time [9] . other potential benefits of rapid rp testing, such as infection prevention and patient satisfaction, were not investigated. relatively few other studies have investigated the effect of large respiratory panels on outcomes, and no prior randomized controlled trials have been performed in us ed patients to our knowledge. a recent trial in an argentinian ed found reduced antibiotic and antiviral initiation in patients receiving the filmarray assay vs standard testing; this study was better powered (432 patients) than our study but may not be generalizable to the united states as empiric treatment without testing is more common in this country [10, 11] . in a retrospective analysis by rogers et al., the biofire filmarray was associated with decreased duration of antibiotic use, length of hospital stay, and length of isolation in children admitted to the hospital with arti [12] . future investigations with more targeted use of rapid multiplex testing in the ed could yield more promising results, but additional research is needed to determine which patients would benefit most from testing. for example, xu et al. showed that the filmarray could be cost-effective when replacing off-site direct fluorescence antibody testing in pediatric ed patients [13] . a recent randomized controlled trial from the united kingdom found an increase in patients with short courses of antibiotics and a 1-day reduction in length of stay in patients with rapid rp testing but did not see a difference in the overall proportion of antibiotic treatment compared with the control arm [14] . however, this study was done outside the united states and included patients outside the ed, so the results may not be directly comparable to our study. our patients came from a heterogeneous ed patient population and included a wide range of individuals with arti anywhere on providers' differential diagnosis; in hindsight, this may not have been the ideal population, as clinicians would not necessarily have ordered rapid multiplex testing on these patients and may have been less likely to modify their behavior in response to the results. however, the high rate of chest x-ray testing in our study (~70%, both study arms) suggests that providers were at least considering a respiratory illness in most patients but may not have been in the habit of relying on viral testing for their clinical decision-making. another interpretation of our results is that while multiplex testing does well identifying influenza and other uri-causing viruses in the ed, implementation alone was not sufficient to significantly influence clinician decision-making and patient outcomes. for example, influenza testing alone may be enough for clinical decision-making during influenza season [9] . it is likely that a thoughtful and comprehensive stewardship program around rapid diagnostics could be required to lead to meaningful changes in outcomes. it is also possible that deploying rapid diagnostic tools would lead to more significant changes if applied to an area that has higher baseline antibiotic prescription rates such as an urgent care setting. shortening the 90-minute turnaround time (tat) of the biofire filmarray respiratory panel might also be necessary for this test to be effective in the ed setting. although the current test was much faster than the off-site respiratory viral panel used at our facility, it is still not fast enough to match the fast-paced workflow of many eds. andrews et al. also encountered problems with tat in their point-of-care (poc) implementation of the filmarray [6] . however, these problems were mainly attributed to staff availability to run the test after consenting subjects. the filmarray itself was reported by them to run in 65 minutes in the poc setting. if the filmarray could be implemented into the ed as a poc test in a way that would fit seamlessly into ed workflow, the tat may have been minimized for this assay, and we may have seen more promising results. this was demonstrated in the post hoc analysis by brendish et al. on their randomized controlled trial in the uk. significant improvements to outcomes, including antibiotic treatment and duration of antibiotics, were seen in patients with virus-positive rp testing that achieved a tat of ≤1.6 hours vs those of >1.6 hours [15] . in this regard, it is encouraging to see newer poc nucleic acid amplification tests for influenza and rsv coming to market with shorter tats, on the order of 10-20 minutes [16] . it is also possible that the pathogens detected do not provide sufficient information for clinicians to reduce antibiotics. the recently fda-cleared biofire filmarray pneumonia panel will identify a wider range of respiratory bacteria and viruses. depending on tat and ability to fit into ed workflow, a test such as this could help reduce diagnostic uncertainty when lower respiratory tract infections are also part of the differential diagnosis. however, detecting more pathogens may not be the answer. as a result of the recent palmetto gba decision, medicare will no longer provide coverage for large (≥6-target) multiplexed diagnostic viral panels such as the biofire filmarray for outpatients unless policy changes in the future [17] . this decision to cover only 3-5 respiratory pathogen targets and limit their use to infectious disease clinicians except in the case of urgent care, ed, and inpatient settings highlights the importance of implementation and evaluation of diagnostic tests in the context of a value-based health care system. simply providing test results without consideration of the behavioral aspects of antimicrobial prescribing may result in lack of clinical utility, when these tests could be beneficial if paired with behavioral economics strategies or as part of a comprehensive stewardship program to reduce inappropriate antibiotic use. this study was limited by a small sample size, which most likely did not provide enough power to see a significant difference in outcomes, as suggested by the wide confidence intervals we observed. the subjects themselves were selected based on symptoms, rather than a clinical decision to order multiplex testing. this in turn led to a heterogeneous subject population, many of whom might not be expected to benefit from viral testing. locating a specific target population for rapid multiplex testing in the ed may be necessary for significant differences in outcomes. finally, during the 2017-2018 influenza season, activities were undertaken to reduce inappropriate prescribing for viral respiratory infections in otherwise healthy patients using patient and provider education, provider public commitment to reducing inappropriate antibiotic use, and peer comparison data. thus, our already low prescribing rate may have been reduced, and this may have compounded our loss of power from underenrollment by decreasing the difference between the rp and usual care groups that we anticipated when we powered the study. we also did not analyze cost-effectiveness, and more research is needed to determine if multiplex testing can be economically feasible in the ed. finally, it is likely that use of comprehensive stewardship strategies and guidelines could have improved effectiveness beyond simple introduction of rapid diagnostic tests. there were also several strengths to this study, including the rigorous experimental study design, relatively rapid tat given the current state of the art, and generation of preliminary data for future more rigorous multicenter clinical trials evaluating the implementation of rapid respiratory panels in eds and other acute care settings. in summary, this randomized controlled trial aimed to evaluate the impact of rapid multiplex respiratory panel testing in the ed for patients with concern for acute respiratory tract infection. although our study lacked the power to see significant differences in outcomes, we did observe a trend in decreased antibiotic use for those who received multiplex testing vs the standard of care. further evaluation of rapid multiplex testing in the ed could see changes in outcomes if the limitations of this study were addressed. this mainly includes identifying the right patient population where testing can alter patient care (vs targeted testing or no testing) and focusing on implementing comprehensive stewardship strategies alongside diagnostic tools to ensure that testing is utilized appropriately and effectively. supplementary materials are available at open forum infectious diseases online. consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author. antibiotic utilization for acute respiratory tract infections in u.s. emergency departments unnecessary antibiotics for acute respiratory tract infections: association with care setting and patient demographics a call to action for antimicrobial stewardship in the emergency department: approaches and strategies emergency department visits for antibiotic-associated adverse events antibiotic prescriptions are associated with increased patient satisfaction with emergency department visits for acute respiratory tract infections multiplex pcr point of care testing versus routine, laboratory-based testing in the treatment of adults with respiratory tract infections: a quasi-randomised study assessing impact on length of stay and antimicrobial use clinical impact of rapid molecular detection of respiratory pathogens in patients with acute respiratory infection an automated nested multiplex pcr system for multi-pathogen detection: development and application to respiratory tract infection clinical utility of on-demand multiplex respiratory pathogen testing among adult outpatients antibiotic use in acute upper respiratory tract infections clinical diagnosis of influenza in the ed impact of a rapid respiratory panel test on patient outcomes implementation of filmarray respiratory viral panel in a core laboratory improves testing turnaround time and patient care routine molecular point-of-care testing for respiratory viruses in adults presenting to hospital with acute respiratory illness (respoc): a pragmatic, open-label, randomised controlled trial imact of turnaround time on outcome with point-of-care testing for respiratory viruses: a post hoc analysis from a randomised controlled trial clinical utility of a near patient care microarray based diagnostic test for influenza and respiratory syncytial virus infections palmetto final lcd denies coverage to large respiratory panels the authors would like to thank nicolle ocampo, m. czarina ganzon, and fatemeh memar for helping with study management and data collection. potential conflicts of interest. l.s.m. and c.r.p. have received consulting fees from biofire diagnostics, inc. all other authors report no conflicts of interest. all authors have submitted the icmje form for disclosure of potential conflicts of interest. conflicts that the editors consider relevant to the content of the manuscript have been disclosed. key: cord-296836-aizquh16 authors: brenner, hermann; holleczek, bernd; schöttker, ben title: vitamin d insufficiency and deficiency and mortality from respiratory diseases in a cohort of older adults: potential for limiting the death toll during and beyond the covid-19 pandemic? date: 2020-08-18 journal: nutrients doi: 10.3390/nu12082488 sha: doc_id: 296836 cord_uid: aizquh16 the covid-19 pandemic goes along with increased mortality from acute respiratory disease. it has been suggested that vitamin d(3) supplementation might help to reduce respiratory disease mortality. we assessed the prevalence of vitamin d insufficiency and deficiency, defined by 25-hydroxyvitamin d (25(oh)d) blood levels of 30–50 and <30 nmol/l, respectively, and their association with mortality from respiratory diseases during 15 years of follow-up in a cohort of 9548 adults aged 50–75 years from saarland, germany. vitamin d insufficiency and deficiency were common (44% and 15%, respectively). compared to those with sufficient vitamin d status, participants with vitamin d insufficiency and deficiency had strongly increased respiratory mortality, with adjusted hazard ratios (95% confidence intervals) of 2.1 (1.3–3.2) and 3.0 (1.8–5.2) overall, 4.3 (1.3–14.4) and 8.5 (2.4–30.1) among women, and 1.9 (1.1–3.2) and 2.3 (1.1–4.4) among men. overall, 41% (95% confidence interval: 20–58%) of respiratory disease mortality was statistically attributable to vitamin d insufficiency or deficiency. vitamin d insufficiency and deficiency are common and account for a large proportion of respiratory disease mortality in older adults, supporting the hypothesis that vitamin d(3) supplementation could be helpful to limit the burden of the covid-19 pandemic, particularly among women. the corona virus disease 2019 (covid-19) pandemic goes along with strongly increased respiratory disease mortality. it has been suggested that vitamin d 3 supplementation could be a potentially promising and safe approach to reduce risk of covid-19 infections and deaths [1] . meta-analyses of randomized clinical trials (rcts) have shown that vitamin d 3 supplementation reduces the risk of acute respiratory tract infections [2] . risk reduction with regular (daily or weekly) supplementation of physiological doses of vitamin d was especially strong (by 70%) among people with vitamin d deficiency, but significant risk reduction (by 25%) was also found among people with higher vitamin d levels. meta-analyses of clinical trials have demonstrated that vitamin d 3 supplementation has the potential to also reduce cancer mortality by approximately 13% [3] . people with pre-existing major diseases, such as diabetes or cancer, are at increased risk of dying from severe acute respiratory syndrome coronavirus 2 (sars-cov-2) infections. at the same time, prevention of and care for these diseases have been and keep being strongly compromised by current measures to limit the covid-19 pandemic. we previously assessed the prevalence of vitamin d insufficiency and deficiency and their association with all-cause mortality and mortality from cardiovascular, cancer and respiratory diseases during a mean follow-up of 9.5 years in a cohort of 9548 adults aged 50-75 years from saarland, germany [4] [5] [6] [7] [8] . in our previous analysis, the number of deaths from respiratory disease had still been rather small (n = 55). we aim to present considerably updated and sex-specific follow-up data of 15 years here and to calculate the proportion of respiratory disease mortality that is attributable to vitamin d insufficiency and deficiency. furthermore, we discuss potential implications for prevention in the context of the ongoing covid-19 pandemic. this investigation is based on the esther study (german name: epidemiologische studie der verhütung, früherkennung und optimierten therapie chronischer erkrankungen in der älteren bevölkerung), an ongoing statewide cohort study from saarland, germany, details of which have been reported elsewhere [4] [5] [6] [7] [8] [9] . briefly, 9940 men and women, aged 50-75 years at baseline, were recruited by their general practitioners during a routine health check-up between 2000 and 2002. blood samples were taken at baseline at the general practitioners' offices. information on socio-demographic and lifestyle characteristics and medical history were obtained by questionnaires from participants and their general practitioners, and the distribution of those characteristics was similar to the distribution in the respective age categories in the german national health survey conducted in a representative sample of the german population in 1998 [9] . the esther study was approved by the ethics committees of the university of heidelberg and the state medical board of saarland, germany. written informed consent was issued by all participants. information on socio-demographic characteristics, lifestyle and diet were obtained by a comprehensive self-administered questionnaire from the study participants at baseline. skin colour, race or ethnicity were not individually recorded. however, we assume that close to 100% of this german cohort of adults aged 50 years and older has white skin colour since 99% gave the information that they were born in germany (94%) or another european country (5%). height and weight were assessed and documented on a standardized form by the general practitioners during the health check-up. furthermore, blood and urine samples were taken during the health check-up, centrifuged, sent to the study center and stored at −80 • c until analysis. the most abundant and stable vitamin d metabolite in blood samples, 25-hydroxyvitamin d (25(oh)d) levels, was measured from stored serum samples taken at recruitment. the laboratory methods used are described in detail elsewhere [4] . in brief, 25(oh)d levels in women were measured with the diasorin-liaison analyzer (diasorin inc., stillwater, ok, usa). analyses in men were conducted in the context of a separate research project several years later (when the diasorin measurements were no longer offered) with ids-isys (immunodiagnostic systems gmbh, frankfurt main, germany). both immunoassays were standardized retrospectively to the gold standard method liquid chromatography tandem-mass-spectrometry. deaths until end of 2016 were identified by inquiry at the residents' registration offices and death certificates of deceased study participants were provided by local health authorities. the leading cause of death with an icd-10 code was available for 98.9% of deceased study participants and was coded with icd-10 codes r98-r99 "unknown cause of death" for 4.4% of deceased participants. these individuals were not excluded and censored at the time of death for cause-specific mortality outcomes. participants of the esther baseline examination (n = 9940) were excluded from this investigation if no blood sample was available or 25(oh)d could not be measured (n = 368) or if they could not be followed-up for mortality (n = 24), which resulted in a total sample size of n = 9548 subjects for this analysis. we used the institute of medicine's cut-offs to define adequate vitamin d status (>50 nmol/l), vitamin d insufficiency (30-50 nmol/l) and vitamin d deficiency (<30 nmol/l) [10] . we assessed prevalence of vitamin d insufficiency and deficiency and the distribution (median, interquartile range) of 25(oh)d values in the total study population and according to age, sex, lifestyle factors and major diseases. we provide kaplan-meier curves for mortality from respiratory diseases among participants with sufficient vitamin d status, vitamin d insufficiency and deficiency and conducted log-rank tests with comparison to the reference group "sufficient vitamin d status." in addition, dose-response relationships between 25(oh)d levels and respiratory disease mortality were estimated by restricted cubic splines [11] . furthermore, we compared all-cause, cardiovascular disease, cancer, and respiratory disease mortality between subjects with vitamin d insufficiency or deficiency and subjects with adequate 25(oh)d levels and estimated hazard ratios (hr) with 95% confidence intervals (95% ci) by multivariable cox proportional hazards models. missing values for covariates ranged from 0% to 5.8% (for fish consumption). missing covariate values were imputed with multiple imputation using the markov chain monte carlo (mcmc) method with 200 burn-in iterations. twenty data sets were generated. the imputation model consisted of all variables of the full model (modelled as used in the full model) but not the outcome data, and the imputation was carried out stratified by sex. we used an age, sex and season adjusted model and a full model that was adjusted for potential confounders, which are listed in table 1 . additional adjustment for potential intermediates (cardiovascular disease, history of cancer, diabetes mellitus, hypertension, asthma, total serum cholesterol and serum c-reactive protein levels) did not lead to substantially different results (data not shown). age and body mass index (bmi) were modelled as continuous variables and all other variables were modelled with the categories shown in table 1 . furthermore, sex-specific analyses were performed and statistical tests on interaction were carried out. finally, we estimated the population attributable fraction (paf) of respiratory disease mortality from the prevalence of vitamin d insufficiency and deficiency and their associations with respiratory disease mortality, as derived from the full model. the paf of mortality is the share of mortality in a population that is statistically attributable to a risk factor and that could be avoided by entirely eliminating that risk factor (here: vitamin d insufficiency or deficiency) [12] . all statistical tests were two-sided, and the alpha level of significance was set to 0.05, with no adjustment for multiple testing. all statistical analyses were conducted with the software package sas, version 9.4 (cary, nc, usa). the study population included 43.8% men, mean age was 62. the study population included 43.8% men, mean age was 62.1 years. among the 9548 participants included in the study, 4186 (43.8%) had vitamin d insufficiency (25(oh)d levels of 30-<50 nmol/l) and 1438 (15.1%) had vitamin d deficiency (25(oh)d levels <30 nmol/l) ( figure 1 ). furthermore, 13 (0.1%) had too high 25(oh)d levels >200 nmol/l. table 1 provides a description of the characteristics of the study population at baseline as well as prevalence of vitamin d insufficiency and deficiency and median 25(oh)d levels according to those characteristics. both vitamin d insufficiency and deficiency were more frequent among females with higher age and bmi. in subjects with low physical activity and those who consumed fish less than once per week, median 25(oh)d levels were correspondingly lower. a seasonal variation with lower 25(oh)d levels in winter than in summer months was also observed. moreover, 25(oh)d levels were particularly low among subjects with low education and current smokers. table 1 provides a description of the characteristics of the study population at baseline as well as prevalence of vitamin d insufficiency and deficiency and median 25(oh)d levels according to those characteristics. both vitamin d insufficiency and deficiency were more frequent among females with higher age and bmi. in subjects with low physical activity and those who consumed fish less than once per week, median 25(oh)d levels were correspondingly lower. a seasonal variation with lower 25(oh)d levels in winter than in summer months was also observed. moreover, 25(oh)d levels were particularly low among subjects with low education and current smokers. overall, 2363 (24.7%) study participants died during a median of 15.3 years of follow-up, of whom 815, 825 and 123 died from cardiovascular disease (cvd), cancer and respiratory disease, respectively. figure 2 shows the kaplan-meier curves for deaths from respiratory disease according to vitamin d status. mortality from respiratory diseases was consistently highest among participants with vitamin d deficiency and consistently lowest among those with sufficient vitamin d levels throughout up to 16.5 years of follow-up. the log-rank test indicated statistically significant survival differences with respect to respiratory disease mortality between the groups with vitamin d deficiency and sufficient vitamin d (p < 0.0001), as well as for the comparison of subjects with vitamin d insufficiency and sufficient vitamin d (p = 0.023). it is not statistically significant that 25(oh)d levels >75 nmol/l are associated with further decreasing respiratory disease mortality because the confidence interval is large and includes the null effect value of hr = 1. curves were assessed by using restricted cubic splines with knots at 25-hydroxyvitamin d concentrations of 30, 60, 90 and 120 nmol/l, and a 25-hydroxyvitamin d concentration of 75 nmol/l was used as the reference. the cox proportional hazards regression model was adjusted for sex, age, season of blood draw, school education, smoking, bmi, physical activity, and fish consumption. table 2 shows the associations of vitamin d status with all-cause, cvd, cancer and respiratory disease mortality after adjustment for multiple potential confounders. vitamin d insufficiency and nutrients 2020, 12, 2488 6 of 11 deficiency were associated with significantly increased all-cause mortality compared to sufficient vitamin d status (full model hrs (95%ci): 1.2 (1.1-1.3) and 1.7 (1.5-1.9), respectively) ( table 2) . vitamin d deficiency was also associated with significant increases in cvd and cancer mortality by 52% and 38%, respectively (full model results). however, vitamin d insufficiency and deficiency were particularly strongly associated with respiratory disease mortality with full model hrs of 2.1 (95%ci: 1.3-3.2) and 3.0 (95%ci: 1.8-5.2), respectively. overall, 41% (95%ci: 20-58%]) of all deaths from respiratory diseases were statistically attributable to 25(oh)d levels <50 nmol/l. figure 3 presents results on the adjusted dose-response relationship between 25(oh)d levels and respiratory disease mortality. mortality strongly increased with decreasing 25(oh)d levels below 50 nmol/l and even more so below 30 nmol/l, i.e., in the vitamin d insufficiency and deficiency range. it is not statistically significant that 25(oh)d levels >75 nmol/l are associated with further decreasing respiratory disease mortality because the confidence interval is large and includes the null effect value of hr = 1. table 3 shows the results of the sex-specific analyses. for all-cause, cardiovascular disease and cancer mortality, only modest, statistically non-significant differences were seen between women and men. although significant increases were seen for respiratory disease mortality in both women and men, they were much stronger among women, with 8.5 (95% ci 2.4-30.1) and 2.3 (95% ci 1.1-4.4)-fold increase of respiratory disease mortality in the case of vitamin d deficiency among women and men, respectively. however, the number of respiratory deaths among women was small (n = 42 overall), especially in the reference groups of those with 25(oh)d > 50 nmol/l (n = 3), which resulted in very wide confidence intervals of the estimated hazard ratios. in this large population-based cohort study from saarland, germany, the majority of participants aged 50-75 years at baseline had vitamin d insufficiency or deficiency, and these conditions were associated with increased mortality. in particular, mortality from respiratory diseases was increased by 2.1-and 3.0-fold in subjects with vitamin d insufficiency or deficiency, respectively, compared to participants with sufficient vitamin d status. significant associations with respiratory disease mortality were seen among both women and men, but they were particularly strong for women. overall, 41% of deaths from respiratory diseases were statistically attributable to vitamin d insufficiency or deficiency and could possibly be avoided by overcoming these conditions, assuming causality of the association. the assumption of causality of vitamin d 3 effects on mortality obviously requires most careful discussion. although we made the best attempts to adjust potential confounding factors, we cannot exclude the possibility of residual confounding by imperfect measurement of confounding variables, such as smoking or physical activity, or omission of unknown confounders. as addressed in detail elsewhere [6] , interpretation of the evidence is further complicated by the fact that vitamin d deficiency could be considered both a consequence of poor health as well as a risk factor for increased vulnerability to acute disease and poor outcomes of chronic diseases among people with poor health. our findings therefore require critical discussion in the light of additional criteria and evidence, such as biological mechanisms and plausibility, and, in particular, in the light of data from rcts providing vitamin d 3 supplementation. deaths from respiratory disease are mostly deaths from lower respiratory infections [13] . vitamin d 3 is thought to protect from occurrence and poor outcomes of respiratory infections by several mechanisms, including enhanced physical barriers (maintenance of junction integrity), cellular innate immunity, and adaptive immunity [1] . innate and adaptive immunity are being influenced by sex hormones [14] , which may explain the observed interaction of sex and 25(oh)d levels with respiratory disease mortality. according to data from the us-american national health and nutrition survey, women have a higher inflammation burden than men (the age range was 40-90+ years [15] ). especially postmenopausal women, like those included in the esther study, have a high inflammatory burden because a decline in estrogen levels during menopause is associated with an increased expression of pro-inflammatory cytokines, including interleukin 6 and tumor necrosis factor (tnf) α [14, 16, 17] . a cytokine storm as an adverse immune response to a sars-cov-2 infection is currently a major hypothesis for the underlying cause of a large proportion of covid-19 deaths [18] . sufficient 25(oh)d levels are suggested to contribute to prevention of the cytokine storm [1, 19, 20] . vitamin d is known to interact with the angiotensin-converting enzyme 2 (ace2), which is both used by sars-cov-2 as an entry receptor and is an important protein on an anti-inflammatory pathway [21] . while sars-cov-2 downregulates the expression of ace2, vitamin d upregulates it. in a meta-analysis of individual participant data of 25 rcts that included 11,321 participants aged 0-95 years, vitamin d 3 supplementation was shown to reduce the risk of acute respiratory tract infection (or 0.88, 95% ci 0.81-0.96) [2] . the best effects were shown for daily or weekly vitamin d 3 supplementation without additional bolus doses (or 0.81, 95% ci 0.72 to 0.91). the protective effects were particularly strong in those with baseline 25-hydroxyvitamin d levels <25 nmol/l (or 0.30, 95% ci 0.17-0.53). in a recent meta-analysis of rcts on vitamin d 3 supplementation for patients with chronic obstructive pulmonary disease (copd), the risk of acute exacerbations was estimated to be reduced by 61% (95% ci 36-77%) [22] . in remarkable consistency with our results, these meta-analyses of rcts provide strong evidence for the preventive potential of vitamin d 3 supplementation against acute respiratory infections and copd exacerbations in particular. it appears plausible to assume that the anti-inflammatory mechanisms of vitamin d would be relevant for sars-cov-2 infections in a similar manner as for other severe viral respiratory diseases, such as influenza. a first study posted on a pre-print server on 13 may 2020 suggests that the protective effects of vitamin d 3 on other acute respiratory tract infections may be translated to covid-19 infections [23] . vitamin d deficiency and vitamin d 3 treatment data were available for 499 covid-19 patients from chicago for the year prior covid-19 testing. being likely vitamin d deficient (defined as being vitamin d deficient at last available time point without increase of vitamin d treatment) at the time of covid-19 testing was associated with a 1.8-fold increased risk of being tested positive for covid-19 (p < 0.02) as compared to likely vitamin d sufficient. it is worth noting that beneficial effects of vitamin d 3 supplementation against manifestation or exacerbation of acute respiratory infection during an epidemic would be expected to go beyond individual protection of those using supplementation, as limiting such manifestation and exacerbation would also be expected to reduce the potential of spread of the disease to other persons and relieve the overload of the medical system by the epidemic. to our knowledge, no previous vitamin d 3 supplementation rcts have addressed mortality from respiratory disease as the primary endpoint, and no meta-analysis of results for this specific endpoint have been reported, which most likely reflects the relatively small share of deaths from respiratory diseases among all deaths. in our cohort of older adults, these deaths accounted for 5.2% of all deaths. even though this proportion is expected to be higher during the covid-19 pandemic, the majority of deaths still occur from other diseases, and the summary effect, benefit-harm ratio and cost-effectiveness with respect to all relevant outcomes therefore deserve most careful attention for any general prevention efforts. in that respect, vitamin d 3 supplementation appears to be a particularly promising approach, especially for population groups with high prevalence of vitamin d insufficiency or deficiency, such as the elderly and those with severe comorbidities (which essentially coincide with population groups at highest risk of severe course and death from sars-cov-2 infection [24] ). the personal, health care and societal costs of the vitamin d 3 intervention are negligibly low compared to the very high costs of currently employed "general population measures," such as extensive testing for the infection and the lockdown of large proportions of economic and social life, including the delay or omission of much of routine medical care for other relevant diseases. in fact, some of these measures are expected to severely aggravate vitamin d insufficiency or deficiency, especially in high risk groups, such as restrictions of spending time outdoors for the total population (as practiced, for example, in france and spain) or certain high risk groups, such as nursing home residents (as practiced in many countries, including germany). such restrictions dramatically reduce opportunities to maintain adequate vitamin d levels through endogenous synthesis by relevant sun exposure. avoidance of sun exposure has been shown to be associated with increased mortality in epidemiological studies [25] . vitamin d 3 supplementation has been demonstrated to be safe in numerous large-scale studies, and the risk of harm seems to be negligible compared to the risk of harmful side effects of the aforementioned and other general population measures, such as delayed diagnosis and treatment of cancer, myocardial infarction or stroke, withheld or deferred delivery of surgical or other medical services, or health risks related to unemployment and loneliness [26] [27] [28] [29] . on the contrary, one expected "side effect" would be reducing total cancer mortality by 13%, as suggested by a recent meta-analysis of rcts [3] . for germany, with currently approximately 230,000 deaths from cancer per year [30] , this would translate in prevention of approximately 30,000 cancer deaths each year, suggesting substantial additional benefit besides lowering the covid-19 burden during the covid-19 pandemic and beyond. in conclusion, our results, along with evidence from meta-analyses from rcts regarding results of vitamin d 3 supplementation on various outcomes, suggest that vitamin d 3 supplementation could contribute to lowering mortality from respiratory and other diseases during and beyond the covid-19 pandemic, in particular among women. the endocrine society recommends 1500-2000 iu vitamin d 3 /day for adults of any age at high risk for vitamin d deficiency [31] . the costs for such supplementation are in the order of 30 € per person per year, or even half that amount when sufficient vitamin d supply is ensured by carefully dosed sun exposure during the summer months. along with expected savings from prevented respiratory and other diseases, this would make vitamin d 3 supplementation a particularly cost-effective and most likely cost-saving measure, whose currently still widely neglected potential should receive increased attention in the debate on how to fight against the covid-19 pandemic. author contributions: h.b. designed the research and drafted the manuscript, b.s. conducted statistical analyses, and b.h. critically read and commented the manuscript and added aspects to the discussion. all authors contributed to the data collection for this project. all authors have read and agreed to the published version of the manuscript. evidence that vitamin d supplementation could reduce risk of influenza and covid-19 infections and deaths vitamin d supplementation to prevent acute respiratory tract infections: systematic review and meta-analysis of individual participant data vitamin d supplements and total cancer incidence and mortality: a meta-analysis of randomized controlled trials strong associations of 25-hydroxyvitamin d levels with all-cause, cardiovascular, cancer and respiratory disease mortality in a large cohort study consortium on health and ageing: network of cohorts in europe and the united states. vitamin d and mortality: meta-analysis of individual participant data from a large consortium of cohort studies from europe and the united states vitamin d as a resilience factor, helpful for survival of potentially fatal conditions: a hypothesis emerging from recent findings of the esther cohort study and the chances consortium vitamin d supplementation trials aimed at reducing mortality have much higher power when focusing on people with low serum 25-hydroxyvitamin d concentrations serum 25-hydroxyvitamin d levels as an aging marker: strong associations with age and all-cause mortality independent from telomere length, epigenetic age acceleration, and 8-isoprostane levels epidemiological investigations of the chances of preventing, recognizing early and optimally treating chronic diseases in an elderly population (esther study) dietary reference intakes for calcium and vitamin d dose-response analyses using restricted cubic spline functions in public health research world health organization health statistics and information systems global, regional, and national age-sex-specific mortality for 282 causes of death in 195 countries and territories, 1980-2017: a systematic analysis for the global burden of disease study the confluence of sex hormones and aging on immunity sex differences in age trajectories of physiological dysregulation: inflammation, metabolic syndrome, and allostatic load the complex role of estrogens in inflammation transitions in metabolic and immune systems from pre-menopause to post-menopause: implications for age-associated neurodegenerative diseases the possible pathophysiology mechanism of cytokine storm in elderly adults with covid-19 infection: the contribution of "inflame-aging" the possible role of vitamin d in suppressing cytokine storm and associated mortality in covid-19 patients vitamin d receptor stimulation to reduce acute respiratory distress syndrome (ards) in patients with coronavirus sars-cov-2 infections vitamin-d and covid-19: do deficient risk a poorer outcome? the efficacy of vitamin d therapy for patients with copd: a meta-analysis of randomized controlled trials association of vitamin d deficiency and treatment with covid-19 incidence risk factors of critical & mortal covid-19 cases: a systematic literature review and meta-analysis avoidance of sun exposure is a risk factor for all-cause mortality: results from the melanoma in southern sweden cohort decline of acute coronary syndrome admissions in austria since the outbreak of covid-19: the pandemic response causes cardiac collateral damage covid-19 pandemic will have a long-lasting impact on the quality of cirrhosis care potential impact of the covid-19 pandemic on financial toxicity in cancer survivors immediate and long-term impact of the covid-19 pandemic on delivery of surgical services krebs in deutschland für evaluation, treatment, and prevention of vitamin d deficiency: an endocrine society clinical practice guideline the esther study was funded by grants from the saarland state ministry for social affairs, health, women and family affairs (saarbrücken, germany), the baden-württemberg state ministry of science, research and arts (stuttgart, germany), the federal ministry of education and research (berlin, germany) and the federal ministry of family affairs, senior citizens, women and youth (berlin, germany). the authors declare no conflict of interest. key: cord-303322-d69o3z8d authors: chang, anne b; grimwood, keith; white, andrew v; maclennan, carolyn; sloots, theo p; sive, alan; mccallum, gabrielle b; mackay, ian m; morris, peter s title: randomized placebo-controlled trial on azithromycin to reduce the morbidity of bronchiolitis in indigenous australian infants: rationale and protocol date: 2011-04-14 journal: trials doi: 10.1186/1745-6215-12-94 sha: doc_id: 303322 cord_uid: d69o3z8d background: acute lower respiratory infections are the commonest cause of morbidity and potentially preventable mortality in indigenous infants. infancy is also a critical time for post-natal lung growth and development. severe or repeated lower airway injury in very young children likely increases the likelihood of chronic pulmonary disorders later in life. globally, bronchiolitis is the most common form of acute lower respiratory infections during infancy. compared with non-indigenous australian infants, indigenous infants have greater bacterial density in their upper airways and more severe bronchiolitis episodes. our study tests the hypothesis that the anti-microbial and anti-inflammatory properties of azithromycin, improve the clinical outcomes of indigenous australian infants hospitalised with bronchiolitis. methods: we are conducting a dual centre, randomised, double-blind, placebo-controlled, parallel group trial in northern australia. indigenous infants (aged ≤ 24-months, expected number = 200) admitted to one of two regional hospitals (darwin, northern territory and townsville, queensland) with a clinical diagnosis of bronchiolitis and fulfilling inclusion criteria are randomised (allocation concealed) to either azithromycin (30 mg/kg/dose) or placebo administered once weekly for three doses. clinical data are recorded twice daily and nasopharyngeal swab are collected at enrolment and at the time of discharge from hospital. primary outcomes are 'length of oxygen requirement' and 'duration of stay,' the latter based upon being judged as 'ready for respiratory discharge'. the main secondary outcome is readmission for a respiratory illness within 6-months of leaving hospital. descriptive virological and bacteriological (including development of antibiotic resistance) data from nasopharyngeal samples will also be reported. discussion: two published studies, both involving different patient populations and settings, as well as different macrolide antibiotics and treatment duration, have produced conflicting results. our randomised, placebo-controlled trial of azithromycin in indigenous infants hospitalised with bronchiolitis is designed to determine whether it can reduce short-term (and potentially long-term) morbidity from respiratory illness in australian indigenous infants who are at high risk of developing chronic respiratory illness. if azithromycin is efficacious in reducing the morbidly of indigenous infants hospitalised with bronchiolitis, the intervention would lead to improved short term (and possibly long term) health benefits. trial registration: australia and new zealand clinical trials register (anzctr): actrn12610000326099 we are conducting a dual centre, randomised, double-blind, placebo-controlled, parallel group trial in northern australia. indigenous infants (aged ≤ 24-months, expected number = 200) admitted to one of two regional hospitals (darwin, northern territory and townsville, queensland) with a clinical diagnosis of bronchiolitis and fulfilling inclusion criteria are randomised (allocation concealed) to either azithromycin (30 mg/kg/dose) or placebo administered once weekly for three doses. clinical data are recorded twice daily and nasopharyngeal swab are collected at enrolment and at the time of discharge from hospital. primary outcomes are 'length of oxygen requirement' and 'duration of stay,' the latter based upon being judged as 'ready for respiratory discharge'. the main secondary outcome is readmission for a respiratory illness within 6-months of leaving hospital. descriptive virological and bacteriological (including development of antibiotic resistance) data from nasopharyngeal samples will also be reported. discussion: two published studies, both involving different patient populations and settings, as well as different macrolide antibiotics and treatment duration, have produced conflicting results. our randomised, placebocontrolled trial of azithromycin in indigenous infants hospitalised with bronchiolitis is designed to determine whether it can reduce short-term (and potentially long-term) morbidity from respiratory illness in australian indigenous infants who are at high risk of developing chronic respiratory illness. if azithromycin is efficacious in reducing the morbidly of indigenous infants hospitalised with bronchiolitis, the intervention would lead to improved short term (and possibly long term) health benefits. trial registration: australia and new zealand clinical trials register (anzctr): actrn12610000326099 background worldwide, bronchiolitis is the most common acute lower respiratory tract infection (alri) in infants [1] [2] [3] . in the northern territory (nt, australia), alris are the most frequent reason for hospitalisation of young children (aged <5-years). alris are also the commonest cause of preventable deaths in indigenous infants (5 times that of non-indigenous) [4] . of alris, bronchiolitis (with or without pneumonia) is the most frequent reason for hospital admission in nt indigenous infants aged under 12-months [5] . despite this heavy burden of bronchiolitis in indigenous infants, currently no prospective studies have been published. our retrospective review of 101 infants hospitalised with bronchiolitis at the royal darwin hospital (darwin, northern territory) found that 33.7% of indigenous infants were readmitted within six-months of discharge from hospital [6] . as most were retrieved from remote communities, the impact of the illness, its costs and social dislocation were likely to have been substantial. recurrent alris are independently associated with the development of bronchiectasis [7] and reduced pulmonary function later in life [8] . low birth weight and pre-existing small lungs are important determinants of future lung function, but there is increasing evidence that events in early life are at least equally important determinants of adult pulmonary dysfunction [8] [9] [10] . the first few years of life are the most critical period [11] . thus events such as severe alris during this critical period are likely to have long term effects. australia-wide, hospitalisation rates of respiratory disorders among indigenous australians are second only to those for renal dialysis [12] . furthermore, we have previously documented that the severity of the hospitalised alri episode, as determined by oxygen requirement and duration of hospitalisation, was an independent risk factor for subsequent bronchiectasis [7] . in the northern territory (nt), bronchiectasis affects one in every 68 indigenous children, far exceeding that of cystic fibrosis (cf) in non-indigenous australian children (1 in 2857) [13, 14] . thus, any intervention that reduces bronchiolitis severity and/or risk of readmission for respiratory illness in indigenous infants may have both short term and potential long term benefits in our setting. bronchiolitis is characterised by extensive inflammation of the airways accompanied by increased mucous production and necrosis of airway epithelial cells. in paediatrics, bronchiolitis is a clinical diagnosis characterised by tachypnoea, wheeze and/or crepitations in infants following a preceding upper respiratory illness [1] . it is primarily caused by infection of the respiratory epithelial cells by a variety of viruses, especially respiratory syncytial virus (rsv). other viruses (adenovirus, influenza, parainfluenza, human metapneumovirus, rhinovirus) are also implicated and increasingly new viruses are being detected in association with this illness [15] . in addition, mycoplasma pneumoniae and chlamydia species are recognised increasingly as important contributors to the development of chronic lung disease and altered lung maturation [16] [17] [18] [19] . new treatable bacteria such as simkania negevensis (a chlamydia-like microbe) has been found in canadian inuit infants with bronchiolitis [20] . there are no published data on the nature or diversity of respiratory pathogens associated with bronchiolitis in indigenous australians infants. typically anti-microbials are not recommended in the routine management of bronchiolitis [1, 21] . while there are two rcts on macrolides for bronchiolitis, the single available rct on a non-macrolide anti-microbial was a negative study [22] . however in our setting, there are several reasons why anti-microbials may reduce the morbidity of hospitalised indigenous infants with bronchiolitis. colonisation of nasopharynx with bacteria is a known risk factor for childhood pneumonia [23] . indigenous infants not only have colonised nasopharynx very early in life (as early as aged 2-weeks), but the colonisation is also dense with common respiratory bacterial pathogens, notably streptococcus pneumoniae, haemophilus infleunzae and moraxella catarrhalis [24] . repeated micro-aspiration of nasopharyngeal secretions heavily laden with pathogenic bacteria during alri may overwhelm already compromised pulmonary defences, increasing the risk of a secondary pneumonia or other lower airway infection. indeed, indigenous infants are more likely to receive antibiotics for an episode of pneumonia diagnosed during an admission for bronchiolitis than non-indigenous infants nursed in the same paediatric unit [6] . macrolides are a class of antibiotics containing a macrocyclic lactone ring with excellent tissue penetration and antimicrobial activity against a broad range of gram positive and gram negative bacteria, including intracellular pathogens such as chlamydia. those with a 14-or 15-membered lactone ring also have several non-antimicrobial properties that have been studied extensively in-vitro and in experimental models and, to a lesser extent, in humans [25] . one of these effects is modulation of the immune response. the immune modulating properties of macrolides make them attractive candidates for treating inflammatory airways diseases. the two published [26, 27] placebo-controlled rcts on macrolides for rsv-bronchiolitis reported contradictory results. in a small turkish study of 21 hospitalised infants with moderate to severe bronchiolitis, 3-weeks of daily clarithromycin significantly reduced severity of illness (oxygen use, hospital stay) and risk of hospital readmission for respiratory illness during the next 6 months [26] . however in another larger study involving 71 infants from the netherlands, 3-days of azithromycin was not efficacious in infants hospitalised with bronchiolitis [27] . the difference in outcomes seen in the two trials may be related to the dose or length of treatment, chance, or heterogeneity of the study populations. infants in turkey are more likely to have concomitant bacterial infection compared to an affluent european group and moreover in turkey, post-infectious childhood bronchiectasis remains an important health problem [28] . the populations also differed in age. the european study included infants up to 24-months of age, whereas the turkish study involved only infants aged <7-months [26, 27] . clearly, a well designed rct on the efficacy of macrolides to reduce the burden of bronchiolitis in a population at high risk of acute and chronic respiratory disease would be beneficial. our primary research question is: amongst hospitalised indigenous infants with bronchiolitis, does azithromycin (compared to placebo) improve clinical outcomes (length of stay in hospital and duration of oxygen supplementation)? our primary hypothesis is that: the anti-microbial and anti-inflammatory properties of the macrolide, azithromycin, will improve the clinical outcomes of indigenous infants hospitalised with bronchiolitis. our secondary aims are: 2. to determine the effect of azithromycin on readmissions into hospital within 6 months of treatment; 3. to assess the short-term impact of azithromycin on macrolide resistance patterns of respiratory bacterial pathogens in the nasopharynx; and 4. to describe the point prevalence and diversity of respiratory viruses, mycoplasma pneumoniae and chlamydia species using sensitive molecular diagnostic techniques. we are conducting a parallel group, double-blind placebo rct (with concealed allocation) to assess the impact of additional treatment with azithromycin in indigenous infants admitted to two regional hospitals (darwin, northern territory and townsville, queensland) with bronchiolitis. our study plan is summarised in figure 1 . the inclusion criteria are: baseline data, nps-1 1. indigenous infants (aged ≤ 24-months) admitted to one of our hospitals (darwin and townsville) with a clinical diagnosis of bronchiolitis. in the absence of an international standardised diagnosis of bronchiolitis, [29, 30] the accepted australian clinical diagnosis is used (tachypnoea (respiratory rate ≥60/min in infants aged <2-months, ≥50/min if 2-12 months, and >40/min if 13-24 months), with wheeze or crackles); [31, 32] and 2. recruited and consented within 24-hours of presentation to the hospital for the illness. exclusion criteria: admission into intensive care, macrolide therapy contraindicated (e.g. liver dysfunction, hypersensitivity), presence of diarrhoea (stools of increased watery consistency and more than two stools above usual stooling frequency), received macrolides (in last 7-days), or clinical and radiological features consistent with a primary diagnosis of pneumonia, [33] at time of randomisation. at each site, the site-specific study nurse visits the wards twice daily to screen all newly admitted infants. a standardised collection form is used to collect clinical data (see below) and hospital outcomes associated with the bronchiolitis episode. all infants are managed according to a standardised protocol. this has been used at the royal darwin hospital since 2008. the protocol outlines when supplementary oxygen is prescribed (sp0 2 <94%) and reduced, and when nasogastric feeds or intravenous fluids are used. enrolled infants may receive additional therapies (other than macrolides) at the discretion of the attending paediatrician. if eligibility is fulfilled and after informed consent has been obtained, the infant is randomised to receive either a single, oral liquid dose of azithromycin syrup (30 mg per kg) or an equivalent volume of placebo. medication is given within 24-hours of hospitalisation. infants randomised to the intervention arm of the study will receive additional treatment doses of azithromycin syrup (30 mg per kg) on day-7 and day-14. those randomised to the control arm receive an equivalent volume of the placebo syrup. the later doses will be either supervised by study nurses or administered by families with phone support on the day the medication is due. final clinical follow up will occur in the local health clinic on day-21 (or closest available date from day 20 to 24). the randomisation sequence was computer generated and used permutated blocks (4 or 6 participants per block). the allocation sequence is concealed at all times throughout the study. the computer generation and allocation were performed by a statistician, external to the study team. upon enrolment, an infant is assigned to the next number on the appropriate stratified list. each unique number is assigned to one of the eight treatment alphabets (see below). treatment groups are stratified by age (≤6 or >6 months), site (darwin or townsville) and requirement (yes or no) for oxygen at point of randomisation. the importance of excluding older children and stratifying at the 6 month age group is well described [30, 34] . a placebo medication ensures that all children, carers, researchers, hospital staff, and clinic staff are blinded to treatment group until analyses of the data. the placebo medication has been specifically manufactured by the institute of drug technology (idt) australia limited (melbourne, vic) which has a similar taste and colour to azithromycin. the azithromycin medications were repackaged by idt. thus both the azithromycin and placebo are in identical opaque bottles and sealed with an aluminium foil. for both, equal volumes of water are added using a syringe and needle by punching the seal. eight alphabets (n, o, p, q, r, s, t, u) were used for the bottles of azithromycin and placebo medications (4 alphabets each). we used multiple alphabets rather than sequential numbers on the bottles to allow storage of extra bottles of trial medications in clinics for the day-7 and day-14 doses. this was necessary in the context of our study setting (children mainly from remote indigenous communities), to enhance availability and administration of trial medications once the child has been discharged from the hospital. all data are recorded on standardised forms. demographic information (age, gender, region, birth details, smoke exposure, breast feeding, household size, etc) and medical history are obtained from the primary caregiver and the medical charts. the primary and secondary outcome measures (see below) are monitored twice daily until the hospital admission's end-point is reached (ready for respiratory discharge, defined as >16-hours without supplemental oxygen and infant is feeding well). clinical assessment data include oxygen requirement and level, physiological measurements for clinical severity score (respiratory rate, accessory muscle use, degree of wheeze), [27] other physiological measurement (temperature, heart rate), requirement and duration of other therapies required (intravenous fluids, nutritional support, antibiotics), ear examination findings (signs of suppuration) and subsequent pneumonia (diagnosed by the independent treating 'blinded' paediatrician). in addition, the results of routine investigations (full blood count and chest radiographic findings) are recorded. on day-21, the presence of cough, wheeze and auscultatory abnormality on clinical review are documented. adverse effects (vomiting, diarrhoea, rash) are also documented. a single nasopharyngeal swab (nps) specimen for respiratory virus and other potentially important respiratory pathogen (m. pneumoniae, chlamydia spp) testing is collected from each subject at enrolment. in addition, nps is repeated before hospital discharge for bacterial culture and antibiotic susceptibility testing, as per our laboratory research protocol (see below) [35, 36] . culturing, identifying and serotyping common respiratory bacteria from nps is an established technique in our laboratory at menzies in darwin [35] . swabs are stored in smggb at -80°c before being batch processed for typical respiratory bacterial pathogens, notably h influenzae and non-typeable h influenzae, m. catarrhalis and s. pneumoniae. batches of swabs are thawed and 10 μl aliquots cultured overnight on selective media at 37°c in 5% co 2 . growth of s. pneumoniae, h. influenzae and m. catarrhalis is recorded and confirmed. four isolates each of s. pneumoniae and h. influenzae and two isolates of m. catarrhalis per positive swab are tested for anti-microbial resistance and stored [35, 37] . s. pneumoniae isolates are serotyped using the quellung method (antisera from statens serum institute, denmark). in addition to routine susceptibility testing using the calibrated dichotomous susceptibility (cds) disc diffusion method, azithromycin minimum inhibitory concentration (mic) will be determined using etest (ab biodisk, sweden) if the azithromycin disc annulus is less than 6 mm. for s. pneumoniae, the penicillin mic is determined for penicillin non-susceptible isolates (oxacillin and/or penicillin disc annulus <6 mm) and for h. influenzae, the ampicillin mic is determined for isolates if the ampicillin disc annulus is less than 6 mm. interpretive criteria (csli breakpoints) used for s. pneumoniae are penicillin non-susceptible mic > 0.12 μg/ml, azithromycin resistant mic ≥ 2 μg/ml, and for h. influenzae, ampicillin resistant mic ≥ 4 μg/ml, azithromycin resistant mic > 4 μg/ml. beta-lactamase activity will be determined for h. influenzae and m. catarrhalis isolates. our previous methods will be utilised [15, 38, 39] . nps are frozen at -80°c. upon thawing nucleic acids will be extracted from 0.2 ml of each nps specimen using the high pure viral nucleic acid kit (roche diagnostics, australia), according to the manufacturer's instructions. mono-specific pcr and reverse transcriptase pcr (rt-pcr) method will be used to detect mycoplasma pneumoniae, coronaviruses, bocavirus and human metapneumovirus (hmpv), whereas multiplex pcr and rt-pcr was used to detect adenovirus, parainfluenza (1, 2 3), influenza (a and b) , and respiratory syncytial virus (rsv). all these methods have been previously validated in our viral laboratory at the royal children's hospital, brisbane. participation is complete when day-21 outcomes have been obtained. other exit points are: intolerance to the trial medications requiring withdrawal from study (as deemed by the treating paediatrician who is not directly connected to the study team). (i) length of stay (los) for respiratory illness in hospital-defined as time from admission to time 'ready for discharge' for respiratory care. 'ready for discharge' means normoxic (sp0 2 consistently >94% in air for >16hrs) and feeding adequately; and (ii) duration of supplemental oxygen required. 'ready for discharge' for respiratory care differs from length of hospitalisation as discharge from hospital may be delayed because of social or transport factors especially in children from remote communities. the major secondary outcome is readmission for respiratory illness (within 6-months of discharge from hospital). minor outcomes during hospitalisation: clinical severity score, [27] additional use of antibiotics, and episodes of suppurative otitis media and development of pneumonia. the day-21 outcomes are: presence of cough, wheeze, abnormal auscultatory chest signs and suppurative otitis media. we will also analyse all clinical outcomes in the following pre-determined sub-groups: (i) age ≤ 6-months; and (ii) presence of bacterial respiratory pathogens that are resistant to macrolide antibiotics. (i) identification of respiratory viruses and bacterial pathogens and (ii) antibiotic resistance to penicillin and macrolides. we plan to enrol 200 indigenous infants. in our retrospective study, [6] the mean los in indigenous infants with bronchiolitis at rdh was 96 (sd 24) hours. the mean duration of supplemental oxygen requirement in indigenous infants with bronchiolitis was 36 (sd 14) hours. for a mean difference of 24-hours in los between groups (power = 90%, α = 5%) the required sample size is 23 per group. the numbers to detect a 12-hour difference in supplemental oxygen requirement is 30 per group. these are large effect sizes but more conservative estimates than seen in the turkish study [26] . in that study, [26] the difference between groups was 30-hrs for los and 31-hours for supplemental oxygen requirement. assuming similar effects, a sample size of at least 100 in each sub-age group is also sufficient for an a-priori subgroup analysis based on age (power of 90% and 2-tailed α of 5%). if the effects are smaller, we will have an 80% power to detect a difference of 10hours in los in all infants and an 80% power to detect a difference of 14-hours in los in the ≤6-months age group. we do not believe that smaller benefits than this would be sufficient to change clinical practice. for the most important secondary outcome (readmission rate for a respiratory illness within 6-months of discharge), the power of our study to detect a reduction from 30% to 10% is 95% (5% significance). this is a large effect but consistent with the reduction described in the turkish study (75% reduction) [26] . at 80% power we will be able to detect a reduction in readmission rates from 30% to 13%. for our other secondary outcomes, accurate sample size estimations are not possible given the lack of any relevant data. data will be reported and presented in accordance with the updated consort criteria [40] . children will be analysed according to allocation status (regardless of subsequent management). an interim analysis is planned and the data safety and monitoring committee will determine if the study should be ceased should superiority of azithromycin be identified after 70% of sample size is achieved. the primary outcomes (los and duration of supplemental oxygen requirement) will be compared between infants receiving placebo or azithromycin using unpaired student's t-tests or mann-whitney tests (depending on normality of distribution). although we expect randomisation to equally distribute potential confounding factors between each of the groups, we will examine the distribution of known confounders between groups (eg. birth weight, smoke exposure status in-utero and postnatal, breast feeding, etc). should baseline data differ between groups, regression will be used to check that the primary outcomes are not affected by this chance finding. an a-priori sub-analysis will compare infants aged ≤ 6months with those aged >6-months. when examining the efficacy of azithromycin at reducing readmission rate for respiratory illness (secondary aim-2), the odds ratio (or) between groups will be calculated. the or will also be used to compare additional antibiotic use between groups. the number needed to treat (nnt) (for benefit), 95% ci will be described if any significant differences are found. if significant, nnt for harm will be calculated for adverse events. for secondary aim-3 (short-term impact of azithromycin on macrolide-resistance of pathogens in nps cultures): the proportions of children with penicillinnon-susceptible s. pneumoniae and macrolide-resistant h. influenzae spp and m. catarrhalis before and after trial medications will be compared using ors and 95% ci. descriptive data will be utilised for secondary aim-4 (point prevalence of respiratory viruses and other respiratory pathogens). the acute lower respiratory tract infections are the commonest cause of hospitalisation and potentially preventable deaths in indigenous infants [4] . bronchiolitis in indigenous infants is more severe than bronchiolitis in non-indigenous infants [6] . there are higher readmission rates and an increased risk of ongoing respiratory morbidity (including chronic suppurative lung disease) in indigenous infants [7, 41] . this may be due to an increased likelihood of recurrent infections and virusbacteria interactions from an early age (as early as aged 2-wks) along with heavy bacterial colonisation of the nasopharynx [36] . despite bronchiolitis being the most common cause of alris in infants resulting in hospitalisation, there are no published prospective studies of this illness in australian indigenous infants. two small clinical trials have studied macrolides in bronchiolitis, but with contradictory results. our population setting is more closely related to the turkish study [26] where a beneficial effect for macrolides was shown. this is in contrast to the negative findings of the dutch study [27] in an affluent urban setting. our proposed double-blind rct will determine if azithromycin is an effective additional treatment in indigenous infants hospitalised with bronchiolitis. it will also determine whether in the ensuing 6 months it will prevent hospital readmissions from respiratory illnesses, which potentially reduces the likelihood of chronic lung dysfunction [7] in this high-risk population. azithromycin was chosen over other macrolides because of its prompt and potent anti-inflammatory effects as well as its 30-40 hours half-life in children, which permits once weekly dosing [42] . the possible anti-viral effects is also attractive [43] . an important safety component of the current study is to monitor for antibiotic resistance in potential respiratory bacterial pathogens colonising the nasopharynx. for the first time in this population, we will determine the nature and diversity of respiratory viruses, mycoplasma, chlamydia and chlamydia-like species in association with cases of bronchiolitis requiring hospital admission. the range of organisms includes newly discovered viruses, [44] and treatable bacteria that may contribute to chronic lung dysfunction. m. pneumoniae and chlamydia species are increasingly recognised as important contributors to development of chronic lung disease and altered lung maturation [16] [17] [18] [19] . our study addresses a large clinical research gap for an important and common cause of hospitalisation in indigenous infants. if the intervention is successful, it would lead to improved short term (and possibly long term) health benefits. conclusive results would produce changes to evidencebased standard treatment guidelines in our region and those produced for similar populations nationally and internationally. finally, the intervention would also offer the possibility of preventing (or reducing) the high rates of long-term respiratory dysfunction seen in australian indigenous children and adults. risk factors for bronchiectasis in indigenous children include recurrent hospitalisation for alris and severity of previous arlis (measured by duration of stay and requirement for oxygen supplementation during hospitalisation) [7] . in the context of the high burden of bronchiectasis in our setting, we considered that the most important outcomes are los, requirement for oxygen supplementation during hospitalisation and readmission within a 6-month period. length of hospitalisation is a common outcome measure in studies on bronchiolitis. however in our setting, hospitalised children often come from remote comunities and may have multiple co-morbidities [33] that influences their discharge. thus we used los defined in accordance with 'ready for respiratory discharge'. our study only addresses infants hospitalised for bronchiolitis. the impact of variable presentation particularly that related to the potential influence of azithromycin's acute immune modulation effect is a limitation of our study. however our study design minimises the impact of variable presentation by: (a) standardising our inclusion criteria, (b) limiting enrolment to within 24 hours of hospitalisation; (c) adopting a strategy (double blind, placebo controlled, allocation concealed methodology) that would theoretically distribute any effect equally between groups. additionally in the event that differences in baseline data between groups are found, we will use statistical methods (multivariate analysis) to adjust as required. in summary, given the very high burden of bronchiolitis in indigenous infants (the age when lung growth is most critical post-natally), and the association between alri and future lung dysfunction, our rct on azithromycin in indigenous australian infants hospitalised with bronchiolitis has the potential to have both short term gains and a long-term benefit for reducing morbidity of respiratory illness. subcommittee on diagnosis and management of bronchiolitis: diagnosis and management of bronchiolitis predicting respiratory syncytial virus hospitalisation in australian children bronchiolitis: assessment and evidence-based management from infancy to young adulthood: health status in the northern territory 2006. department of health andcommunity services hospitalisation of indigenous children in the northern territory for lower respiratory illness in the first year of life risks of severity and readmission of indigenous and non-indigenous children hospitalised for bronchiolitis a hospital-based case-control study of bronchiectasis in indigenous children in central australia lifecourse predictors of adult respiratory function: results from the newcastle thousand families study lung parenchyma at maturity is influenced by postnatal growth but not by moderate preterm birth in sheep postnatal growth rate, but not mild preterm birth, influences airway structure in adult sheep challenged with house dust mite the mammalian respiratory system and critical windows of exposure for children's health. environ health perspect australian health ministers' advisory council 2: aboriginal and torres strait islander health performance framework report. ahmac, canberra non-cf bronchiectasis-clinical and hrct evaluation bronchiectasis: so much yet to learn and to do frequent detection of human rhinoviruses, paramyxoviruses, coronaviruses, and bocavirus during acute respiratory tract infections prenatal inflammation and lung development contribution of inflammation to lung injury and development intermittent azithromycin treatment for respiratory symptoms in patients with chronic chlamydia pneumoniae infection rate of chlamydia trachomatis and chlamydia pneumoniae in paediatric respiratory infections high rate of simkania negevensis among canadian inuit infants hospitalized with lower respiratory tract infections bacteraemia and antibiotic use in respiratory syncytial virus infections antibiotics for bronchiolitis in children. cochrane database syst rev streptococcus pneumoniae colonisation: the key to pneumococcal disease bacterial colonization of the nasopharynx predicts very early onset and persistence of otitis media in australian aboriginal infants mechanisms of action and clinical application of macrolides as immunomodulatory medications clarithromycin in the treatment of rsv bronchiolitis: a double-blind, randomised, placebo-controlled trial azithromycin does not improve disease course in hospitalized infants with respiratory syncytial virus (rsv) lower respiratory tract disease: a randomized equivalence trial non-cystic-fibrosis bronchiectasis in children: a persisting problem in developing countries diagnosis and testing in bronchiolitis: a systematic review acute viral bronchiolitis: to treat or not to treat-that is the question the management of acute bronchiolitis. thoracic society of australia and new zealand respiratory syncytial virus infection and immunoprophylaxis for selected high-risk children in central australia zinc and vitamin-a supplementation in indigenous children hospitalised with episodes of lower respiratory tract infection: a randomised controlled trial how should we study responses to treatment in children with bronchiolitis? respiratory bacterial pathogens in the nasopharynx and lower airways of australian indigenous children with bronchiectasis streptococcus pneumoniae and noncapsular haemophilus influenzae nasal carriage and hand contamination in children: a comparison of two populations at risk of otitis media random colony selection versus colony morphology for detection of multiple pneumococcal serotypes in nasopharyngeal swabs the impact of viral respiratory infection on the severity and recovery from an asthma exacerbation newly identified respiratory viruses in children with asthma exacerbation not requiring admission into hospital consort 2010 statement: updated guidelines for reporting parallel group randomised trials respiratory morbidity in central australian aboriginal children with alveolar lobar abnormalities once-weekly azithromycin in cystic fibrosis with chronic pseudomonas aeruginosa infection azithromycin induces anti-viral responses in bronchial epithelial cells evidence of human coronavirus hku1 and human bocavirus in australian children randomized placebo-controlled trial on azithromycin to reduce the morbidity of bronchiolitis in indigenous australian infants: rationale and protocol we thank the research staff (lesley versteegh, clare wilson, nerida jacobsen, susan pizzutto and amber revell) for facilitating the study, joseph mcdonnell for generating the randomisation sequences and jana lai for assistance with labelling the bottles. we are also grateful to members of the indigenous reference group of the child health division at menzies for supporting this study and for over-seeing the cultural aspects. we also thank drs ka o'grady, alan isles, alan ruben and william frischman for voluntarily providing their time in their participation in the study's data safety monitoring committee. we are also very appreciative to the channel 7 children's foundation and the financial markets foundation for children for funding the preliminary study that provided us with essential pilot data for our successful nhmrc grant submission. funding study is funded by a 3-year australian national health and medical research council (nhmrc) project grant (605809). abc is supported by a nhmrc fellowship (545216). authors' contributions ac conceived the study, and participated in its design and coordination and drafted the manuscript. pm, kg, ts, aw, as, cm participated in its design, analysis plan and submission to the nhmrc. gm participated in initiating and running the project and im in the viral analysis plan. all authors read and approved the final manuscript. the authors declare that they have no competing interests. key: cord-337747-7sb03moe authors: lagare, adamou; ousmane, sani; dano, ibrahim dan; issaka, bassira; issa, idi; mainassara, halima boubacar; testa, jean; tempia, stefano; mamadou, saidou title: molecular detection of respiratory pathogens among children aged younger than 5 years hospitalized with febrile acute respiratory infections: a prospective hospital‐based observational study in niamey, niger date: 2019-10-11 journal: health sci rep doi: 10.1002/hsr2.137 sha: doc_id: 337747 cord_uid: 7sb03moe background and aims: in niger, acute respiratory infections (aris) are the second most common cause of death in children aged younger than 5 years. however, the etiology of ari is poorly understood in the country. this study aims to describe viral and bacterial infections among children aged younger than 5 years hospitalized with febrile ari at two hospitals in niamey, niger's capital city, and the reported clinical procedures. methods: we conducted a prospective study among children aged younger than 5 years hospitalized with febrile ari at two national hospitals in niamey between january and december 2015. clinical presentation and procedures during admission were documented using a standardized case investigation form. nasopharyngeal specimens collected from each patient were tested for a panel of respiratory viruses and bacteria using the fast track diagnostic 21 plus kit. results: we enrolled and tested 638 children aged younger than 5 years, of whom 411 (64.4%) were aged younger than 1 year, and 15 (2.4%) died during the study period. overall, 496/638 (77.7%) specimens tested positive for at least one respiratory virus or bacterium; of these, 195 (39.3%) tested positive for respiratory viruses, 126 (25.4%) tested positive for respiratory bacteria, and 175 (35.3%) tested positive for both respiratory viruses and bacteria. the predominant viruses detected were respiratory syncytial virus (rsv) (149/638; 23.3%), human parainfluenza virus (hpiv) types 1 to 4 (78/638; 12.2%), human rhinovirus (hrv) (62/638; 9.4%), human adenovirus (hav) (60/638; 9.4%), and influenza virus (inf) (52/638; 8.1%). streptococcus pneumoniae (249/638; 39.0%) was the most frequently detected bacterium, followed by staphylococcus aureus (112/638; 12.2%) and haemophilus influenzae type b (16/638; 2.5%). chest x‐rays were performed at the discretion of the attending physician on 301 (47.2%) case patients. of these patients, 231 (76.7%) had abnormal radiological findings. a total of 135/638 (21.2%) and 572/638 (89.7%) children received antibiotic treatment prior to admission and during admission, respectively. conclusion: a high proportion of respiratory viruses was detected among children aged younger than 5 years with febrile ari, raising concerns about excessive use of antibiotics in niger. acute respiratory infections (aris) are responsible for elevated childhood morbidity and mortality globally, 1,2 accounting for approximately four million deaths among children aged younger than 5 years in 2010 and resulting in substantial burden of healthcare systems. 3, 4 in developing countries, aris account for 19% of all deaths among children aged younger than 5 years and 8.2% of all disabilities and premature deaths. 5, 6 therefore, data on the epidemiology and seasonality of ari are important to develop control and prevention strategies. 7 in niger, the estimated mortality rate among children aged younger than 5 years was 114 per 1000 in 2012 (unicef child mortality estimate), and more than 80% of these deaths were associated with malaria, respiratory infections, and diarrhea. according to the 2012 niger health statistics yearbook, the estimated case fatality proportion associated with respiratory infections was 8.7%. in the absence of laboratory diagnosis, it is difficult to clinically differentiate between ari-related causative pathogens, due to similarities of symptoms. 8, 9 the main causative agents of ari are viruses and bacteria. 10 [11] [12] [13] in niger, there is scarcity of data on aris, although a routine influenza surveillance system exists. inf has been detected in 11% of samples from children aged younger than 5 years hospitalized with severe acute respiratory illness (sari). 14 this study aims to describe the viral and bacterial infections among children aged younger than 5 years hospitalized with febrile ari at two national hospitals of niamey, the capital city of niger, and the reported clinical procedures. we conducted a prospective study among children aged younger than 5 years hospitalized with febrile ari between january and december 2015. niger has four distinct seasons as categorized by the national directorate of meteorology: the cold season (mid-december to mid-february), the dry season (mid-february to may), the rainy season (june to september), and the hot season (october to mid-december). 15 this study was part of the "total niger infection respiratoire aiguë" (tonira) project, which was funded by the total corporate foundation in order to strengthen medical care of febrile ari among children aged younger than 5 years. the study was conducted at the pediatric wards of two national tertiary hospitals situated in niamey, namely, the hôpital national de niamey (hnn) and the hôpital national lamordé (hnl). these two hospitals provide general health care to the population of niamey estimated at 1.1 million in 2015, as well as to patients referred from across the country. laboratory detection of respiratory viruses and bacteria were conducted at the centre de recherche médicale et sanitaire (cermes), niamey, niger (the national reference laboratory for influenza). demographic and clinical data, as well as the use of antibiotics before and during admission, were collected using standard data collection forms. malaria testing was implemented on site using thick blood film microscopy. chest x-rays were performed at the discretion of the attending physician, and typical abnormal radiological findings assessed included pulmonary opacities, acute bronchiolitis, and enlargement of intercostal spaces. malnutrition was determined using the ratio of weight for height, and its level was classified as moderate or severe by the attending physician using national standards based on the 2010 technical guidelines for integrated disease surveillance and response in the african region (retrieved from https://www. afro.who.int/sites/default/files/2017-06/idsr-technical-guidelines_ final_2010_0.pdf). febrile ari in conjunction with malaria, malnutrition, and diarrhea were assessed during admission using a standardized case investigation form. in-hospital outcome (ie., discharge, referral, or death) was recorded for all enrolled patients. a febrile ari case was defined as a hospitalized child aged younger than 5 years with onset of fever 38 c or higher and cough within 10 days prior to admission and at least one of the following signs: inability to drink or breastfeed, lethargy, vomiting, convulsions, nasal flaring, chest indrawing, stridor in a calm child, or tachypnea. nasopharyngeal swabs were collected from all enrolled patients, placed in universal transport medium, stored at 4 to 8 c, and transamplification. the fluorescence was assessed at the amplification step. the positive and negative virus plasmid controls provided in the kit were included in all runs to monitor assay performance. 16 viral and/or bacterial detection was reported as percentage positive, overall and within selected categories (eg, age groups and seasons). children aged younger than 5 years were stratified into two age groups: infants (aged younger than 1 year) and young children (aged 1-4 years), for comparability with other studies from africa. [17] [18] [19] [20] in addition, the majority of enrolled children were aged younger than 1 year hindering our ability to categorize age in smaller age groups. stata version 14.2 (statacorp, college station, texas, usa) was used for the analysis. the protocol was approved by the national ethical consultative com overall, 496/638 (77.7%) specimens tested positive for at least one respiratory virus or bacterium (table 1) overall, respiratory viruses were detected in 370/638 (58.0%) specifigure 3a ). overall, respiratory bacteria were detected in 301/638 (47.2%) specimens. s pneumoniae (249/638; 39.0%) was the most frequently detected bacterium, followed by s aureus (112/638; 12.2%) and hib (16/638; 2.5%) ( table 3) . m pneumoniae and c pneumoniae were detected individually in less than 1% of specimens. of the specimens that tested positive for at least one respiratory bacteria, two or more respiratory bacteria were detected in 81/220 s pneumoniae and s aureus were mostly detected during the rainy season (june to september) ( table 3 and figure 3b ). we report the detection of respiratory viruses and bacteria among children aged younger than 5 years hospitalized with febrile ari in t a b l e 2 (continued) 21, 22 and asia (range 45%-65%). [23] [24] [25] the hpiv were also the most predominant viruses detected in children aged younger than 5 years in a previous study conducted in niger. 8 rsv is considered to be a major cause of ari in children aged younger than 5 years, 26, [28] [29] [30] and it was the most commonly detected virus also in this study (149/638; 23.3%). among the cases in which hpiv and hcov were detected, hpiv type 3 and hcov type oc43 were the predominant types, and this is consistent with previous studies. 27, 31 in this study, infs were detected in 8.5% of the cases, which is in agreement with findings from the national influenza surveillance 14 our study presents some limitations. first, the study spanned over a period of only 12 months, limiting our ability to fully assess the temporal circulation pattern of the investigated agents. due to the small sample size, we may also have been underpowered to detect small variations in the temporal distribution of the investigated agents. second, attribution of causality remains challenging due to the lack of controls in our study. the association of pathogen detection with illness could not be well assessed, although most of the viral and bacterial pathogens identified in this study have been described in previous case control studies as causative agents of ari. 10, 39 in addition, we did not collect blood samples to assess whether the detection of common bacterial colonizers of the nasopharynx, such as s pneumoniae, were associated with invasive disease. third, we did not record the number of patients with febrile ari that refused enrollment, hindering our ability to estimate underenrollment. last, given that only patients with febrile ari were enrolled, this could have resulted in an underestimation of the rsv burden since a large proportion of rsv-related illness is not associated with fever. 40 in this 1-year prospective study, both viral and bacterial pathogens were detected in high proportion among hospitalized children aged younger than 5 years with febrile ari in niamey, niger. however, further investigations should be carried out to assess risk factors and association of pathogens with illness. although national guidelines for ari treatment recommend systematic empiric antibiotic therapy, our results suggest that antibiotic use might be unnecessary in most cases, given the predominance of viral infections as potential cause of febrile ari. furthermore, other predisposing diseases such as malaria, malnutrition, and diarrhea may be contributory factors to febrile ari exacerbation among hospitalized children. the findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the us centers for disease control and prevention, usa. the authors confirm that the data supporting the findings of this study are available within the article and its supplementary materials. acute respiratory infection case definitions for young children: a systematic review of community-based epidemiologic studies in south asia estimates of world-wide distribution of child deaths from acute respiratory infections global and regional burden of hospital admissions for severe acute lower respiratory infections in young children in 2010: a systematic analysis global and regional mortality from 235 causes of death for 20 age groups in 1990 and 2010: a systematic analysis for the global burden of disease study respiratory virus infections in hospitalized children and adults in lao pdr acute respiratory infections: the forgotten pandemic epidemiology and seasonality of respiratory tract virus infections in the tropics viral and bacterial etiology of severe acute respiratory illness among children < 5 years of age without influenza in niger viral etiology of influenza-like illnesses in cameroon viral and bacterial causes of severe acute respiratory illness among children aged less than 5 years in a high malaria prevalence area of western kenya respiratory viruses seasonality in children under five years of age in buenos aires, argentina: a five-year analysis respiratory viruses identified in an urban children's hospital emergency department during the 2009 influenza a(h1n1) pandemic respiratory viral infections among pediatric inpatients and outpatients in taiwan from 1997 to 1999 influenza sentinel surveillance among patients with influenza-like-illness and severe acute respiratory illness within the framework of the national reference laboratory oukem-boyer oom: influenza sentinel surveillance among patients with influenza-like-illness and severe acute respiratory illness within the framework of the national reference laboratory detection of respiratory viruses by real-time polymerase chain reaction in outpatients with acute respiratory infection sentinel surveillance for influenza and other respiratory viruses in cote d'ivoire viral etiology of severe acute respiratory infections in hospitalized children in cameroon respiratory viral coinfections identified by a 10-plex real-time reverse-transcription polymerase chain reaction assay in patients hospitalized with severe acute respiratory illness-south africa human bocavirus, coronavirus, and polyomavirus detected among patients hospitalised with severe acute respiratory illness in south africa viral etiologies of lower respiratory tract infections among egyptian children under five years of age viral etiology of respiratory infections in children under 5 years old living in tropical rural areas of senegal: the evira project clinical and epidemiological characteristics of acute respiratory virus infections in vietnamese children epidemiology of acute respiratory infections in children in guangzhou: a three-year study bacterial and viral pathogen spectra of acute respiratory infections in under-5 children in hospital settings in dhaka city viral and atypical bacterial detection in acute respiratory infection in children under five years viral and atypical bacterial etiology of acute respiratory infections in children under 5 years old living in a rural tropical area of madagascar viral etiologies of acute respiratory infections among hospitalized vietnamese children in ho chi minh city seasonal trends of viral respiratory tract infections in the tropics investigation of respiratory syncytial virus-associated deaths among us children aged <2 years clinical and molecular epidemiology of human parainfluenza viruses 1-4 in children from viet nam viral etiology of respiratory tract infections in children at the pediatric hospital in ouagadougou (burkina faso) spatiotemporal circulation of influenza viruses in 5 african countries during 2008-2009: a collaborative study of the institut pasteur international network influenza transmission during a oneyear period (2009-2010) in a sahelian city: low temperature plays a major role the role of multiplex pcr test in identification of bacterial pathogens in lower respiratory tract infections clinico-pathological study of atypical pathogens in community-acquired pneumonia: a prospective study serotype distribution and antimicrobial sensitivity profile of streptococcus pneumoniae carried in healthy toddlers before pcv13 introduction in niamey response to conjugate haemophilus influenzae b vaccine among infants in niamey, niger etiology and factors associated with pneumonia in children under 5 years of age in mali: a prospective case-control study performance of surveillance case definitions in detecting respiratory syncytial virus infection among young children hospitalized with severe respiratory illness-south africa molecular detection of respiratory pathogens among children aged younger than 5 years hospitalized with febrile acute respiratory infections: a prospective hospital-based observational study in niamey we are thankful to the moh, cermes, and clinicians and nurses at the hnn and hnl for their contributions. this work was funded by the total corporate foundation through a partnership with pasteur institute of paris and cermes. the funder was not involved in the study design, collection, analysis, and interpretation of data, writing of the report, and decision to submit the report for publication. adamou lagare had full access to the data and takes responsibility for the integrity of the data and the accuracy of the data analysis. the lead author/manuscript guarantor (adamou lagare) affirms that this manuscript is an honest, accurate, and transparent account of the study being reported; that no important aspects of the study have been omitted; and that any discrepancies from the study as planned (and, if relevant, registered) have been explained. all authors declare that they have no commercial or other associations that may pose a conflict of interest. https://orcid.org/0000-0003-0101-9048stefano tempia https://orcid.org/0000-0003-4395-347x key: cord-346096-aml84iv1 authors: bailey, emily s.; choi, jessica y.; zemke, juliana; yondon, myagmarsukh; gray, gregory c. title: molecular surveillance of respiratory viruses with bioaerosol sampling in an airport date: 2018-09-17 journal: trop dis travel med vaccines doi: 10.1186/s40794-018-0071-7 sha: doc_id: 346096 cord_uid: aml84iv1 recognizing that crowded, high-traffic airports and airplanes have been implicated in respiratory disease transmission, we partnered with administrators of raleigh durham international airport (rdu) in conducting a pilot study of aerosol surveillance for respiratory viruses at rdu. from january to march 2018 we used niosh 2-stage samplers to collect 150 min aerosol samples in crowded areas at rdu. four (17%) of the 24 samples were positive for known respiratory pathogens including influenza d virus and adenovirus. these results suggest the feasibility of employing bioaerosol surveillance techniques in public transportation areas, such as airports, as a noninvasive way to detect and characterize novel respiratory viruses. electronic supplementary material: the online version of this article (10.1186/s40794-018-0071-7) contains supplementary material, which is available to authorized users. as the number of individuals who travel by air increases, issues regarding air quality and the potential risk of respiratory infection during travel and flight have become increasingly important. in the last 20 years alone, there have been outbreaks of several important respiratory viruses that have had a major effect on air travel. a few examples include influenza a(h1n1) pdm09 virus, severe acute respiratory syndrome coronavirus (sars-cov), middle east respiratory syndrome coronavirus (mers-cov), and emerging h7n9 avian influenza a viruses. the outbreak of sars coronavirus for example can be tracked from the hotel metropole in hong kong to areas where infected guests traveled by air after staying at the hotel [1] . this risk of the spread of pathogens by air travel has influenced the development of airport passenger screening technologies at airports. many airports use thermal scanning to screen incoming passengers for febrile illness. in general, these techniques are not entirely effective in preventing the spread of viral infectious disease because passengers, either with a fever or presenting symptoms with respiratory virus infections such as influenza virus, often shed viruses before they have fever. a notable example of the failure of screening methods occurred in 2009 during the spread of influenza a(h1n1) pdm09 virus, where passengers on a flight from cancun mexico were screened but still managed to infect other passengers during flight [2] . in response to the concern surrounding the spread of infectious disease through air travel, a 2017 national academy of science report has highlighted the need for additional research and evaluation of the exit screening procedures designed to protect public health from disease threats [3] . the united states in particular has developed a biomonitoring program that detects pathogens in air samples; however, this system (the us biowatch) is known to be error prone with numerous false positives frequently detected [4] . an alternative response to this need for public health screening is to concentrate and examine air samples, using personal or standalone air samplers. this bioaerosol sampling technique has been adapted for respiratory virus screening in swine production facilities [5] , poultry markets [6] , clinical settings [7] , and airports [8] . in this pilot study, we studied bioaerosol samples collected in raleigh durham international airport for molecular evidence of respiratory viruses. this international airport sees approximately 11.6 million passengers per year, or about 32,000 per day and uses a high throughput exhaust system (including high efficiency particulate air, hepa, filters) [9] . our overall goal was to determine if environmental air sampling is a viable method for respiratory virus detection in airport settings. ethics approval and study location "exemption from review" status was granted to this study by the institutional review board at duke university, given that the methods involved did not include contact with human subjects. permission and collaboration from the appropriate coordinating bodies at raleigh-durham international airport (rdu) was sought and obtained prior to the start of the collection period. staff members at rdu assisted in the coordination of sampling devices during the study period. sampling at rdu terminal 2 took place during a 9-week period from january 10th to march 7th, 2018. during this time, 12 sampling sessions of 150 min resulted in 24 sample collections generating 72 individual samples to be analyzed for a panel of respiratory viruses. the sampling devices were niosh bc 251 personal aerosol samplers that featured two stages of collection and a polytetrafluoroethylene (ptfe) back-up filter (.03 μm pore, 37 mm). the two stages filtered pathogens greater than 4 μm or 1-4 μm in size and the back-up filter captured pathogens less than 1 μm in size. thus, each sampler produced three samples per sampling period. an aircheck xr5000 sample pump (cat. # 210-5000, skc, inc., eighty-four, pa) was used to circulate environmental air through the samplers at a calibrated rate of 3.5 l per minute throughout the sampling period. the sampling periods were scheduled during high-traffic times of domestic and international flight arrivals and departures. the samplers were set up on tripods approximately 1.5 m above the ground (approximately breathing level), at two different locations within the rdu baggage claim areas (between baggage carousels that were the focal points of the arriving flyers) and at each end of the terminal departure gates. during each sampling period, environmental temperature, humidity, and general meteorological outlook were noted. at the end of each session, the location, time, sampler number, pump number, and run time was recorded for each sampling device. samples were transported back to the duke one health research laboratory immediately following sample collection. at this time, sterile virus collection medium (pbs with 0.5% bsa) was used to soak the filters and catchment containers for each stage. this medium was then aliquoted into 2.0 ml cryovials, which were promptly stored at − 80°c for future molecular analyses. detection of influenza a/b/c/d, human enterovirus, human adenovirus, and human coronavirus was performed using previously published real-time polymerase chain reaction (qpcr) and real-time reverse transcription polymerase chain reaction (qrt-pcr) assays with dna or cdna positive controls and negative controls of nuclease-free water (additional file 1: table s1 ). viral rna was extracted from stored samples with the qiamp viral rna mini kit (qiagen, inc., valencia, ca), and then analyzed with qrt-pcr assays utilizing superscript® iii platinum one-step qrt-pcr system with platinum® taq dna polymerase (thermo fisher scientific, inc., waltham, ma) for influenza a [10, 11] , influenza b [10, 12] , influenza c [13] , influenza d [10] , human coronavirus [14, 15] , and human enterovirus [14] . gel-based rt-pcr assays detected for pan-species coronaviruses [14] with superscript® iii platinum one-step rt-pcr system with platinum® taq dna polymerase (thermo fisher scientific, inc., waltham, ma). viral dna was extracted utilizing the qiaamp dna blood mini kit (qiagen, inc. valencia, ca), then analyzed for human adenovirus [15] by real-time pcr (qpcr) assay [16] with the sso advanced universal probes supermix (bio-rad, hercules, ca). adenovirus-positive specimens were further confirmed using previously described two-step molecular assays with focus on the hexon gene [17] . analysis for pan-species adenovirus [18] was performed on extracted viral dna using the platinum® taq dna polymerase kit (thermo fisher scientific inc., waltham, ma). sequencing of resultant amplified product from both the pan-species assay and the hexon assay was performed by eton bioscience (eton bioscience, inc., raleigh, nc, usa). sequences were then aligned, edited, and compared to the ncbi sequence database using the blast application of bioedit 7.1.9 (ibis biosciences, carlsband, ca, usa). a total of 24 niosh 2-stage samples were collected (table 1) . overall, 4 (17%) out of 24 samples indicated evidence of at least 1 respiratory pathogen, with 1 (4.1%) positive for influenza d and 3 (12.5%) positive for adenovirus. samples were most frequently positive in the 4 μm or 1-4 μm particle size ranges with one adenovirus and the influenza d sample detected at 4 μm and two adenovirus positives detected at 1-4 μm. two (67%) of the 3 adenovirus-positive specimens, samples 16 and 19, were successfully sequenced using the panadenovirus assay and were found to be human adenovirus type 1 (ncbi accession number af534906.1). adenoviruses were detected more frequently in the baggage claim 2 area, with 2 (2.8%) positive samples detected in that area. none of the 72 samples were positive for influenza a, influenza b, influenza c, or coronaviruses. although not the focus of our study, we did not detect viable viruses using culture analysis associated with positive aerosol samples at rdu airport. during sampling, air temperature ranged from 63.6°f to 78.6°f and the relative humidity (rh) ranged from 25.1 to 60.4% (table 1 ). there was no statistically significant difference between the mean air temperature or the mean rhs between the groups of samples that were positive or negative for respiratory viruses. in this pilot aerosol study, we conducted surveillance for human and zoonotic respiratory viruses in an airport setting over a period of nine weeks from january to march 2018. we detected respiratory viruses in 17% of the aerosol samples collected. through our surveillance, we observed molecular evidence of influenza d virus and adenovirus in aerosol samples through non-invasive and non-disruptive environmental sampling techniques. this sampling approach was particularly important in an airport setting, where large numbers of passengers either sat near or passed by the samplers in the terminal or baggage claim. this study of aerosols conducted in an airport is likely the first of its kind in the united states. our detection of adenoviruses in aerosol samples is comparable to similar studies conducted in an airport and other settings [5, 7] . although respiratory adenoviruses are not typically considered to have as much of an impact as other respiratory viruses, such as influenza viruses, well-documented outbreaks of novel adenovirus strains causing severe respiratory infections have occurred [19] . despite the fact that the adenoviruses detected in this study were detected using our panspecies molecular assay, upon sequencing, they were identified as human adenoviruses. we did not find evidence of novel or zoonotic viruses in aerosol samples, potentially due to the high throughput ventilation system in place at rdu airport. another limitation of this study was the inability to link aerosol results with individual passengers, or individual planes. using this detection method, we were only able to detect viruses from one source (aerosols). as the airport is a high traffic area, and the focus of this study was focused on environmental air sampling, it is not clear which people were harboring the detected viruses. additionally, as this airport uses a high throughput (hepa filtered) ventilation system it was not possible to determine the effect of high quality air circulation on our virus detection based on the methods described here and we may not have captured all virus present at the sampling locations. despite these limitations, the results of this study suggest that aerosol sampling is a useful technique for respiratory virus surveillance in high traffic and crowded areas such as airports. aerosol sampling has advantages in that it minimally interrupts walking traffic in busy places, is simple in setup and operation, and the processing procedures for isolating the viral nucleic acid is relatively simple [7] . together with our finding that 17% of collected aerosol samples showed molecular evidence for at least one respiratory pathogen and the ease of aerosol sampler use, our findings suggest that travelers may be sharing respiratory viruses in airports and other high-traffic areas. such areas may, therefore, benefit from aerosol sampling to strengthen surveillance to protect the public from respiratory viruses. this strengthened surveillance may also support public health responses to respiratory virus detection by allowing airports, or other high traffic areas employing these methods, to recommend interventions (such as respirators or masks) to at risk travelers. additional file 1: table s1 . primer and probe sequences for rpcr and rrt-pcr. update: outbreak of severe acute respiratory syndrome--worldwide international flight-related transmission of pandemic influenza a(h1n1)pdm09: an historical cohort study of the first identified cases in the united kingdom. influenza other respir viruses preparing airports for communicable diseases on arriving flights risk-based objectives for the allocation of chemical, biological, and radiological air emissions sensors bioaerosol sampling for airborne respiratory viruses in an experimental medicine pig handling facility aerosolized avian influenza a (h5n6) virus isolated from a live poultry market bioaerosol sampling in clinical settings: a promising, noninvasive approach for detecting respiratory viruses. open forum infect dis environmental sampling for respiratory pathogens in jeddah airport during the 2013 hajj season rdu air service statistics isolation of a novel swine influenza virus from oklahoma in 2011 which is distantly related to human influenza c viruses information for moleduclar diagnosis of influenza virus in humans -update evaluation of three influenza a and b real-time reverse transcription-pcr assays and a new 2009 h1n1 assay for detection of influenza viruses detection of influenza c virus by a real-time rt-pcr assay. influenza other respir viruses characterizing the picornavirus landscape among synanthropic nonhuman primates in bangladesh performance of different mono-and multiplex nucleic acid amplification tests on a multipathogen external quality assessment panel improved real-time pcr assay for detection and quantification of all 54 known types of human adenoviruses in clinical samples molecular typing of human adenoviruses by pcr and sequencing of a partial region of the hexon gene detection and analysis of six lizard adenoviruses by consensus primer pcr provides further evidence of a reptilian origin for the atadenoviruses human adenovirus associated with severe respiratory infection we thank the team at raleigh durham international airport for their support of this study, in particular michael landguth, president and ceo, william c. sandifer, senior vice president & chief operating officer, duane legan, vice president of airport operations, john a. connell, vice president of operational asset management, and kristie vanauken, communications and community affairs. we thank william g. lindsley for his support in providing the niosh 2stage air samplers. we also thank jane fieldhouse, laura borkenhagen, dingmei zhang, rick taso, and kerry mallinson in assisting with sample collection. this study was supported by duke university discretionary funding (gray pi). the data collected during the current study are available from the corresponding author on reasonable request. authors' contributions eb, jc, jz, my collected and analyzed samples. jc and jz wrote the results and materials and methods sections. eb wrote the introduction and discussion. gg conceived of the idea of the study, guided the work and manuscript development. all the authors reviewed the final version of the manuscript and agreed to its submission. this study was granted exemption from review status by the institutional review board at duke university on the grounds that the research did not directly involve contact with human subjects. not applicable. the authors declare that they have no competing interests. springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord-316727-ktrlohm9 authors: razavi, seyed mansour; mohazzab torabi, saman; salamati, payman title: treatment and prevention of acute respiratory infections among iranian hajj pilgrims: a 5-year follow up study and review of the literature date: 2014-05-10 journal: med j islam repub iran doi: nan sha: doc_id: 316727 cord_uid: ktrlohm9 background respiratory diseases/syndromes are the most common causes of referring to physicians among pilgrims in hajj. they lead to high morbidity, impose high costs on the health system and are among the major obstacles for pilgrims to perform hajj duties. the main aim of our study was to determine types, frequencies, etiologies, and epidemiologic factors of respiratory diseases among iranian hajj pilgrims and to suggest some preventive and treatment strategies. methods: to determine the types and frequencies of respiratory syndromes, we implemented a syndromic surveillance method in iranian health care system for hajj during 5 consecutive years. to achieve the etiology of these diseases, we performed 4 concurrent before and after studies. we also evaluated efficacy of the flu and pneumovax vaccines among iranian hajj pilgrims in 2 studies. to determine some other epidemiological factors, we conducted 4 additional studies. results: the most common problem was common cold like syndrome. origins of the most upper respiratory problems were infections, and allergies were less involved. among infectious agents, viruses were the most common agents and their frequencies were as follows respectively: adenoviruses 38 (36.2 %), rhinoviruses 31 (30%), influenza type b virus 21 (20%). bacteria were often the secondary causes and their frequencies were as follows respectively: intestine bacillus 69 (19.4%), chlamydia pneumonia 20(15.8%), haemophiluses 32 (9.1%) and streptococcus (a,c and g) 30 ( 8.5%). we introduced some epidemiological factors as effective in creating respiratory diseases. conclusion: in this paper, we suggested some applied points for prevention, treatment, and correction of common malpractices in the treatment of respiratory diseases of the pilgrims. every year, more than 2 million muslims travel from 140 countries in the world to saudi arabia for performing the hajj ceremonies. changes in routine activities (sleep, physiological habits and nutrition, etc.) as well as fatigue and anxiety resulting from traveling and performing pilgrimage ceremonies makes the pilgrims susceptible to diseases (1) . in addition, high density of population causes transmission of resistant respiratory organisms among pilgrims from different countries (2) (3) . consequently, respiratory diseases are very prevalent during hajj ceremony. they lead to high morbidity, impose high costs on the health system, and are among the major obstacles for pilgrims to perform hajj duties (4) (5) (6) (7) . ap-proximately, 100,000 iranian pilgrims have participated in hajj rituals each year in recent years (8) . in this regard, the following questions are raised: what forms of respiratory diseases have been emerged? what is the most common form? what are the etiological causes of these diseases? do these diseases have infectious origin? if they are infectious, what class of infectious agents (viruses, classic bacteria, atypical bacteria, and air dispersed fungal spores) is more effective on creation of the diseases? in case of infections, which organisms play a more important role? what are the roles of respiratory allergies? what are the roles of physical factors such as weather dryness, direct exposure to cold air, drinking cold liquids in sweating? does the gastroesophageal reflux play a role in causing the diseases? what are the common errors which occur in management of the diseases? to what extent chemical agents such as irritating odor of disinfectants and diffused toxins interfere? what are the ways to prevent and reduce the occurrence of these diseases? what are the best therapeutic methods? the aim of our study was to determine types, frequencies, etiologies, and some of other epidemiologic factors of respiratory diseases among iranian hajj pilgrims and to suggest some preventive and treatment strategies based on our 10 years of experience and literature review. to determine the types and frequencies of respiratory syndromes, we implemented a syndromic surveillance method, proposed by the center for control and prevention of diseases (cdc) (9) in iranian health care system for hajj. consequently, we reviewed the physicians' reports about respiratory diseases of 254,823 iranian pilgrims between 2004 and 2009 (10). to study the etiological causes of respiratory diseases in hajj, we conducted 4 concurrent before and after studies in 2004 (11) (12) (13) (14) . six educational departments (mi-crobiology, virology, immunology, mycology, social medicine, statistics and epidemiology, all affiliated to tehran university of medical sciences) cooperated with this study. we evaluated 1,018 serum samples (509 samples before and 509 samples after the journey) for serological assessments, 357 samples for studying pharyngeal classic bacteria, 105 pairs of samples for studying pharyngeal secretions in terms of viruses, 130 pairs of fresh blood samples for studying nitro blue tetrazolium test (nbt) and immunological tests, 128 pairs of serum samples for studying atypical bacteria and 146 samples for studying antibody of respiratory fungi and 500 questionnaires for evaluating the epidemiological issues. serum samples were gathered at the time of departure from the country and 3 weeks after their return to the country, and were kept at -20ºc. fourfold increase of antibodies values were considered as acceptable for confirmation of changes. laboratory tests which were used for detection of organisms were as follows: direct smear assessment, culturing the organisms in specific medias, hemagglutinin inhibition test, immune-fluorescence tests, elisa, specific medias and tests for viruses such as dmea media and dako kits, count immune electrophoresis, latex agglutination tests, and nbt for investigating fresh blood. etiological factors that were examined in this study were as follows: immunological tests include: nbt, immunoglobulin (a-g-m-e), interleukin 4, and gamma interferon. we also evaluated the efficacy of the flu and pneumovax vaccines among iranian hajj pilgrims in 2 studies (15-16). to determine some of other epidemiological factors, we conducted 4 additional studies (4-5, 8, 17) . we used the results of above mentioned studies and reviewed the literature, and offered a guideline for the treatment and prevention of acute respiratory infections (aris) in hajj. we used independent-t, paired t-tests and anova for numerical data, mann-whitney u, wilcoxon and kruskal-wallis as non-parametric tests, chi-square and fisher exact tests for nominal data and odds ratio for evaluating the efficacy of vaccines. the frequencies of symptoms and types of reported respiratory disorders through syndromic surveillance system were as follows (each patient may suffer from more than one disorder) ( the above tests were performed on 105 pairs of oropharynx gargling secretions and serums (12) . 1. fungi: seroconversion of opportunistic fungi like aspergillus-candida albicans, and cryptococcus were negative showing that fungi were not involved. laboratory tests were performed on 146 pairs of serums before and after the journey (13). immunological investigations  nbt test: the test was used for the function of neutrophils, the defensive cells in body against infections. the before and after the travel difference was statistically significant (p=0.001).  gamma interferon: this agent significantly enhances the phagocytic ability of neutrophils. the before and after the travel difference was statistically significant. (p=0.001). in the other word, these two agents were investigated as infection markers which showed statistically significant differences (14) .  interleukin 4: no statistically significant difference was observed.  ige: this immunoglobulin increases in allergies. there was not statistically significant difference between before and after titers.  iga: this immunoglobulin plays an important role in mucosal defense. no statistically significant difference was observed (p=0.49).  igg: this immunoglobulin could be a marker for chronic or previous infections. there were no statistically significant difference between the before and after tests (p= 0.93).  igm: this marker has a role in acute infections. the difference between before and after tests was statistically significant (p=0.046). the above tests were performed on 130 pairs of before and after the travel serums. other epidemiological investigations  in this part, the behavioral and environmental factors, affecting the occurrence of respiratory diseases among the pilgrims were studied. some of these factors included:  use of the flu and pneumovax vaccines (solely or combined) before the trip. based on our study published in 2004, the effectiveness of influenza vaccine was 50%. we found that injection of influenza vaccine could decrease the influenza-like illness (ili) incidence (15). we also conducted a similar study in 2005 which showed that the effectiveness of the flu vaccine was not statistically significant (16) .  we reported some epidemiological factors affecting the occurrence of respiratory diseases among iranian pilgrims in 2005 (17) . these factors were as follows:  increase in the age: the higher the age of the pilgrims, the higher the percentage of respiratory diseases was.  increase in the vulnerability of the pilgrims: respiratory diseases raised with the increase of the number of high risk pilgrims in the caravans.  score of management: we developed a new scoring system for evaluating managerial performances in each caravans. the percentage of respiratory diseases among pilgrims of related caravan decreased as the managerial performance scores in each caravan increased.  score of health management: we also developed a new scoring system for evaluating the physicians of caravans. as the health management scores of the physicians increased in each caravan, the percentage of respiratory diseases among pilgrims of the caravan decreased.  screening of the pilgrims: screening of the pilgrims before the journey was an effective way for reducing respiratory tract disorders. cdc in a paper entitled "syndromic surveillance: an applied approach to outbreak detection" has suggested a method for immediate and easy identification of diseases. in this method, by establishing a documented, telephonic or electronic reporting system, health managers can find manifestations and trend of the diseases via collecting symptoms of patients (9). the method has been used several times and has many different names such as:  early warning systems  prodromal surveillance  outbreak detection systems  information system-based sentinel surveillance  bio-surveillance systems  health indicator surveillance  symptom-based surveillance (18). using the above mentioned method, in this study the most common respiratory syndrome was common cold like syndrome. allergy just mimics the symptoms of common cold. thus, it can lead to over diagnosis or malpractice. in this condition, the main question is whether the respiratory involvements of pilgrims in hajj have infectious or allergic origin? the following points suggest that the pilgrims' respiratory problems had mostly infectious origin.  significant difference values of nbt test before and after travel showed that respiratory diseases of pilgrims might have infectious origins.  significant difference values of gamma-interferon before and after the travel, also showed that respiratory diseases of pilgrims could be infectious. (14) .  the difference between the levels of interleukin 4 in serum before and after the travel was not statistically significant. so, the respiratory diseases in hajj pilgrims could not have allergic origin.  the levels of ige in serum before and after the travel did not show a statistically significant difference. so, the respiratory diseases were not allergic.  the differences in the values of immunoglobulin a, g were no statistically significant before and after the travel, but igm levels increased after the journey, supporting the infectious origin for the diseases (5) . unfortunately, more than 90% of the physicians in caravans assumed that respiratory problems of the pilgrims were originated from allergy and they attempted to prescribe corticosteroids for their patients (4, 14) . this not only did not help the patients, but it also caused loss of immunity and deteriorated the patients' conditions. viral infections are more prevalent than bacterial infections. fungal infections do not play any role in respiratory infections (11) . therefore, considering the absence of the effect of antibiotics on viruses, irrational prescription of antibiotics has no scientific basis. prescription of antibiotics without paying attention to the indication, disturbs the normal flora of mouth and throat, which play an effective role in defending the body against other organisms. among the viruses, adenoviruses were more prevalent than the other viruses which have no special treatment (12) . transmission of this virus is perhaps one of the reasons for the prevalence of severe conjuncti-vitis in outbreaks. in our study, most frequent viruses were: adenoviruses, rhinoviruses and coronaviruses. currently there are no treatments available for these viruses. another common virus is the influenza virus which has a special treatment. however, it is not easy to confirm it in hajj ceremony. in a study by al-tawfiq et al., most common respiratory tract infection viruses were influenza and rhinoviruses (19) . in a study by alborzi et al. on 255 patients in hajj, the rates of viral causes were reported as follow: influenza 9.8%, parainfluenza 7.4%, rhinovirus 5.9%, adenovirus 5.4%, enterovirus 2%, and rsv 1.6% (20). in another study by moattari et al. it was stated that both seasonal and pandemic influenza infections occurred among the iranian hajj pilgrims; seasonal viruses were more common than the pandemic viruses even though all pilgrims were vaccinated against seasonal influenza (21) . the findings of this study are in line with our findings. among atypical bacteria, chlamydia pneumoniae plays a more prominent role in respiratory diseases of the pilgrims than the other atypical bacteria. by comparing serum values of igg and igm antibodies against chlamydia pneumonia, we found that this organism plays an evident role in respiratory infections of the pilgrims (11) . comparison of the samples before and after the travel indicated no recent affliction of the pilgrims with infections caused by legionella pneumophila. also, mycoplasma pneumoniae did not play any role in respiratory infections of the pilgrims. the results of this study showed that the most prevalent bacteria were enteric bacilli (19.4%) and chlamydia pneumonia (15.8%) (5) . therefore, in case of need for empiric antibiotic therapy, antibiotics of macrolides and fluoroquinolone groups should be used. fungi had no role in the respiratory diseases of the pilgrims (13), thus, we should consider this fact in our management. our experiences of dealing with patients with respiratory diseases during hajj period for 10 years are summarized in following suggested statements:  in most cases, affliction with severe respiratory infections occurs during or after tashriq days and it seems that the disease spread point is masharolharam (the holly place for muslims in mecca, saudi arabia). anxiety, fatigue, inevitable population density, and low hygienic standards in masharolharam compared with other locations are probably among the effective factors.  based on observations, the most severe side effects emerge at the end of travel.  according to physicians' reports, some people refer to the physicians of the caravans for more than 30 times during their travel (means every day of the journey) only for their bothering coughs which were alleviated over time. the finding suggests that the treatments were not completely effective, doctors did not spend a lot of time to explain the trend of diseases for their patients, or the expectations of pilgrims were not matched with the reality of conditions. in these conditions we need to educate pilgrims because, the duration of diseases may take up to 2 weeks (22) . a vaccine should be injected to prevent influenza. sometimes the disease is emerged from a strain other than the strain which vaccine has been made. therefore, the pilgrims may claim in this subject, particularly individuals who paid for the expenses of their own vaccines. therefore, we should educate all the pilgrims before the travel.  it seems that high risk people play some roles in increasing of the affliction rate and spread of respiratory diseases among healthy ones (17) . educating these groups is more important than the healthy pilgrims. a valuable experience in this case was pilgrimage of 2009 which iran prevented high risk people to travel due to spread of type i influenza (h1n1 type). according to the al-towfiq et al. report, despite the occurrence of pandemic of h1n1 during hajj period in 2009, the available literature did not show an increased rate of respiratory infection in hospitalized patients in 2009 (19) . it might be owing to the screening of high risk groups and the more attention paid to the personal hygiene. within 10 years (from 1999 to 2008), we have seen some therapeutic physicians' mistakes on the pilgrims:  some syndromes such as exudative pharyngitis and allergy were overdiagnosed.  antibiotics were used excessively and irrationally and were early shifted to another antibiotic due to mental pressure of the pilgrims.  corticosteroids were excessively prescribed.  antibiotics (ceftriaxone, gentamycin, etc.) were prescribed in single dose.  prescribing a combination of drugs including tramadol, dexamethasone, and penicillin l.a, named "cocktail", whereas, tramadol was excluded from iran pharmacopoeia some years ago (the drugs were prepared by physicians themselves).  prescribing antacids for the treatment of coughs by diagnosing the reflux (it is true that some coughs are caused by reflux, but we should use the medicines which block the entrance of acids to esophagus instead of prescribing the antacids).  homeotherapy and energy therapy were used, whereas, the effectiveness of them should be studied more.  one of the important and vital factors in bad therapy is continuous mental pressures of the pilgrims. correction of this problem needs long-term cultural efforts. 1. treatment of the common cold (23):  rest.  gargling warm normal saline.  drink adequate fluids.  avoid eating spicy foods.  administration of a first generation anti-histamine such as chlorpheniramine (every 12 hour, 12 mg until 5 days) for rhinorrhea (administration of ipratropium can also reduce rhinorrhea).  administration of acetaminophen or one nsaid (eg. ibuprofen, indomethacin, or naproxen)  lozenges including topical anesthetics.  administration of vitamin-c, zinc, vitamin-e, probiotics, hand-washing recommendations and exercising are useful but further investigation is needed.  administration of expectorants like guaifenesin are not effective for the elimination of cough (20). some points -new non-sedating antihistamines are not more useful than the first generation antihistamines; this may be due to their differences in their passage from brainblood barrier (bbb). -nsaids reduce headache, malaise, and cough, this may be due to their inhibitory effect on prostaglandins. -combination of prescribing first generation antihistamines and nsaids alleviate nasal congestion. so, there is no need to prescribe anti-congestions. -if it is necessary to prescribe decongestants, administration of oral products is preferred to topical drugs such as phenylephrine or oxymetazoline. -dextromethorphan and codeine are useful in reducing cough. -in a large meta-analysis, researchers demonstrated that administration of vitamin-c in prevention of common cold is beneficial (24). however some other literatures confirmed that it has a short term effect on the treatment of common cold and its preventive effect was not confirmed. benefit of echinacea and herbal medicines are not approved and they even might have suppressive effect on cd4 cells. although it was shown that they were effective in 5 of 6 trials. evidences suggest that the use of probiotics for prevention or treatment of the common cold had no more benefits (24). despite application of the above mentioned medications, we have to wait until the disease course is over. 2. treatment of influenza or influenza-like illness (ili):  rest  intake abundant of fluids  gargling warm normal saline  administration of analgesics like acetaminophen.  administration of amantadine or rimantadine for influenza type a (if confirmed)  administration of zanamivir or oseltamivir for influenza types a and b. (if confirmed). attention: oseltamivir is not in pilgrim's drug list now.  rest voice  humidification with normal saline  symptomatic treatment in case of viral laryngitis.  administration of antibiotics for viral laryngitis is not helpful. (23)  if the patient did not feel better after 3 days, it might be non-viral laryngitis and administration of an antibiotic such as azithromycin is useful. administration of dexamethasone has temporary effect. so, it is not suggested for routine use. unfortunately, physicians prescribed corticosteroids in more than 90.3 % of cases (14) . they might have temporary effects and could suppress body immune system. administration of oral or intra muscular dexamethasone (0.3 mg/kg) for the treatment of acute laryngo-tracheobronchitis (croup syndrome) is suggested. (25)  acute bronchitis does not need antibiotics administration.  * a mixture including brompheniramine, naproxen or chlorpheniramine, ibuprufen relievs the cough especially with nasal interferon alfa.  administration of oseltamivir, in case of ili or approved influenza.  administration of azithromycin or other macrolides and quinolones are not useful for bacterial bronchitis caused by mycoplasma or chlamydia.  early treatment of bronchitis caused by bordetella pertussis with tetracyclines or macrolides can prevent transmission of disease.  conservative treatments. 6. treatment of copd with super imposed infections: *amoxiciline for the treatment of haemophilus influenza and pneumococcus, doxycycline for the treatment of moraxella, macrolides for the treatment of chlamydia and fluoroquinolones for the treatment of pseudomonas (29). viral causes do not need antibiotics and are not distinguishable from bacterial infections. 7 . treatment of acute pneumonia (30) : if needed, referring to medical centers for hospitalization. pneumonia is a significant cause of hospital admission accounting for 20-50% of the admissions (19) . treatment for outpatients -in individuals with healthy condition without taking antibiotics: administration of erythromycin -in individuals with healthy condition with taking antibiotics: azithromycin and co-amoxiclav -in individuals with history of diseases such as: copd-diabetes mellitus -renal failure -cardiac failure or malignancy, without taking antibiotics: administration of azithromycin -in individuals with above mentioned diseases and history of taking antibiotics: azithromycin and cefuroxime -in individuals with pneumonia suspicious to aspiration: co-amoxiclav -in individuals with pneumonia with a history of influenza: cefuroxime. with a history of taking antibiotics: clarithromycin plus ceftriaxone preventive points:  screening of high risk groups before the journey (3).  education is necessary for pilgrims continually before and during the hajj period (2) .  reports of previous years indicated that the most prevalent cause of deaths of iranian pilgrims was cardiac diseases. in addition, about 25.6% of the pilgrims were vulnerable (high risk groups) and were mostly afflicted with critical respiratory diseases (17) . thus, we should emphasis on screening of vulnerable pilgrims for the diseases such as cardiac diseases, cancers, and respiratory disorders before the journey.  stress, fatigue, and anxiety cause immunity suppression in human beings (14) and these factors are among the reasons for the establishment and continuation of dis-eases. therefore, it is suggested to prevent all the factors which can lead to such conditions.  contaminated hands are one of the most common ways of diseases transmission (2,23). therefore, repetitive hand washing, avoiding from shaking hands with the patients and not touching eyes and respiratory mucus membranes by contaminated hands are among the effective preventive ways.  it is useful to inject influenza and pneumovax vaccines solely or jointly to reduce respiratory diseases occurrence, mortality, and hospitalization (31-32) particularly in people over 50 years of age, and patients who suffer from diabetes, renal failure, liver failure, cardiac diseases, asthma and copd at proper time (2 to 3 weeks before the trip). although a few studies doubted the effectiveness of influenza vaccine (33) , other studies strongly recommended vaccination of pilgrims (34) (35) . these results show that causal patterns of respiratory infections were different in various years and effectiveness of vaccines will also be different and the pilgrims should be educated in this regard.  to avoid excessive and unreasonable use of antibiotics and corticosteroids for therapeutics or preventive purposes.  not attending unnecessary crowded locations such as recommended tavafs (going around kabah) in high densities situations (2) .  correct and timely treatment of pneumonia and its side effects.  some specialists recommended sucking antiseptic lozenge before attending the crowds for reducing affliction with respiratory infections but this matter has not been approved by the preliminary studies and should be studied more.  garlic tablets and its medicinal derivatives have been suggested for prevention of influenza (36) .  social distancing is one of the behavioral variables which influence respiratory illnesses (2) . it is necessary for the patients to consider distance of longer than 1 meter from healthy individuals or conversely. however, it is impossible under pilgrimage conditions. therefore, the distance could be considered between beds in pilgrims' rooms.  it is unreasonable to use masks for the prevention of viral diseases (33) . however, it is useful to prevent entrance of larger particles. it is recommended to use masks especially for patients. the use of masks may reduce exposure to droplet nuclei, which is the main mode of transmission of most respiratory tract infections (19) . in a study by deris et al. on 387 malaysian hajj pilgrims it was stated that wearing masks was significantly associated with sore throat and longer duration of sore throat and fever (22) . on the contrary, in another study by al-jasser et al. on 1507 saudi arabian hajj pilgrims it was showed that occurrence of urti among the pilgrims who were wearing face mask most of the time was lower than those pilgrims who were wearing them only at some times (37) . the effects of the following factors require further investigations.  indiscriminate use of disinfectants such as chlorine compounds in caravans  population density of the rooms in caravans  distances between the beds in the rooms of the caravans  mask usage  the number of attendances at crowded locations per day  use of antiseptic lozenges before attendance at crowded locations  repeated hand washing  exposure to cold air  use of the personal prayer mat  repeated consumption of banana or the other substances producing histamine. in this paper, we reviewed types, frequencies, etiologies, and epidemiologic factors of respiratory diseases among iranian hajj pilgrims and suggested some applied points for prevention, treatment, and correction of common malpractices in the treatment of respiratory diseases of the pilgrims. pattern of diseases among visitors to mina health centers during the hajj season, 1429 h (2008 g) protective practices and respiratory illness among us travelers to the 2009 hajj the association between pre-morbid conditions and respiratory tract manifestations amongst malaysian hajj pilgrims comparison of mortality and morbidity rates among iranian pilgrims in hajj surveying respiratory infections among iranian hajj pilgrims pattern of admission to hospitals during muslim pilgrimage (hajj) pattern of medical diseases and determinants of prognosis of hospitalization during 2005 muslim pilgrimage hajj in a tertiary care hospital.a prospective cohort study trend of diseases among iranian pilgrims during five consecutive years based on a syndromic surveillance system in hajj titration of serum antibodies against mycoplasma pneumoniae, chlamydia pneumoniae and legionella pneumophila in iranian pilgrims during the fungal flora in the accommodation of iranian pilgrims and their role in respiratory diseases in hajj persian) 15. razavi sm, dabiran s, ziaee ardekani h. the incidence of influenza like illness and determination of the efficacy of flu vaccine in iranian pilgrims during hajj pilgrimage the comparison of influenza vaccine efficacy on respiratory disease among iranian pilgrims morbidity and mortality among iranian hajj pilgrims in 2003. faculty of medicine journal what is syndromic surveillance? mmwr respiratory tract infections during the annual hajj: potential risks and mitigation strategies influenza viral infections among the iranian mjiri hajj pilgrims returning to shiraz, fars province, iran. influenza other respi viruses the prevalence of acute respiratory symptoms and role of protective measures among malaysian hajj pilgrims principles and practice of infectious diseases wilder-smith a, earnest a, ravindran s, paton ni. high incidence of pertussis among hajj pilgrims chronic obstructive pulmonary disease and acute exacerbations principles and practice of infectious diseases protective effects of the 23-valent pneumococcal polysaccharide vaccine in the elderly population protective measures against acute respiratory symptoms in french pilgrims participating in the hajj of 2009 prevention of influenza at hajj: applications for mass gatherings the hajj: updated health hazards and current recommendations for 2012 preventive effect of garlic extract against influenza patterns of diseases and preventive measures among domestic hajjis from central, saudi arabia we would like to express our gratitude to the physicians of caravans, specialist physicians in hospitals of mecca and medina, researchers in departments of microbiology, virology, immunology, mycology, social medicine, statistics and epidemiology of tehran university of medical sciences and the staff of hajj medical centre for their support and participation. key: cord-299952-xvtt8fz8 authors: gao, lulu; yu, shouyi; chen, qing; duan, zhaojun; zhou, jie; mao, chen; yu, dexian; zhu, wenchang; nie, jun; hou, yunde title: a randomized controlled trial of low-dose recombinant human interferons α-2b nasal spray to prevent acute viral respiratory infections in military recruits date: 2010-06-17 journal: vaccine doi: 10.1016/j.vaccine.2010.03.062 sha: doc_id: 299952 cord_uid: xvtt8fz8 the military population has a high disease burden of acute viral respiratory infections in china. to assess the efficacy and safety of a low-dose recombinant human interferon α-2b (rifnα-2b) nasal spray in preventing acute viral respiratory infections in military population, we performed this randomized controlled trial. the results showed that application of the rifnα-2b nasal spray had the benefits in prevention of infections caused by influenza a virus, influenza b virus parainfluenza viruses 1–3 and adenovirus species b. however, no benefit was seen in preventing respiratory syncytial virus. no severe adverse events were reported. therefore, the rifnα-2b nasal spray was effective and well tolerated for preventing common viral respiratory infections in the military recruits. acute respiratory tract infections are flourishing in closed and crowded environments. military recruits are prone to outbreaks of acute respiratory tract infections because of their crowded living conditions in barracks, stressful work environments, frequent travels and exposure to novel strains of respiratory pathogens [1] [2] [3] . over 90% of acute respiratory tract infections are caused by viruses, such as influenza virus, parainfluenza virus, respiratory syncytial virus, rhinovirus, adenovirus and coronavirus. though most of viral respiratory infections usually represent mild, selflimited clinical manifestations, they are leading causes of morbidity in certain groups or populations (e.g., children, military population) and remain a heavy burden of disease [4, 5] . in history, the famous "spanish influenza" initiated from army recruits in 1916 [6] . in 1976, the novel a/new jersey/76 (hsw1n1) influenza virus caused severe respiratory illness in soldiers at fort dix [1] . adenoviruses have been the most important cause of febrile acute respiratory disease in us military recruit populations [7] . in china, a comprehensive surveillance systems for influenza in military population showed that the average incidence of influenza was 1.4 episode per person-year from 1995 to 2000 [8] . viral respiratory infections have become one of the most actual health problems in military population. therefore, it is necessary to seek reasonable and effective measures to prevent outbreaks and epidemics of acute viral respiratory infections among military population. interferons (ifns) are a family of cytokine mediators that are critically involved in alerting the cellular immune system to viral infections of host cells [9] . the previous studies have suggested that high dosage of intranasal interferons can prevent respiratory infections caused by viruses, such as influenza virus, rhinovirus, coronavirus and respiratory syncytial virus [10] [11] [12] [13] [14] . however, the major problem is higher frequency of local side effects such as mucosal irritation, dry mucous membranes, blood-tinged mucus and nasal mucosal erosion [15] [16] [17] . recently, yuance medicine company (beijing, china) has developed a low-dose recombinant human interferon ␣-2b (rifn␣-2b) nasal spray in order to reduce adverse reactions. to evaluate the efficacy and safety of this new nasal spray in preventing acute respiratory infections in military population, we performed this randomized, placebo-controlled, double-blind trial. from november 2005 to december 2005, we did a randomized, placebo-controlled, double-blind, multi-center trial in the military trainees from 12 recruit training units in three geographically distinct cities in china (guangzhou city and foshan city in guangdong province, liuzhou city in guangxi province). candidate subjects were male recruits who aged 16-23 years, and finished army physical fitness examination. they were not admitted to the study if any of the following criteria were present: (1) on regular medical treatment or took other medications within two weeks, (2) history of serious allergies (e.g., asthma, urticaria, and eczema), (3) history of autoimmune disorders, (4) psychiatric disorders, and (5) acute or chronic illnesses. all candidates were observed two weeks before enrolling into the study, in order to screen eligible subjects. a total of 1500 trainees from three areas enrolled the trial initially. after the screening had been completed, 1449 eligible recruits remained in the subject pool. all participants understood the implications of the study, and provided informed consents. the study was conducted during the basic training period of new military recruits at 12 recruit training units located in different districts. there were 120-150 new recruits in each independent training unit. all the trainees lived in barracks during a three-month training. eight to 10 recruits shared a quarters room about 30 m 2 . participants in the experimental group received the rifn␣-2b nasal sprays, the metered spray device delivered 0.1 ml (3 × 10 5 iu of rifn␣-2b) per spray into each nostril and throat, that was a total of 9 × 10 5 iu of rifn␣-2b for each administration. the spray delivered twice daily after breakfast and supper respectively for five consecutive days. the control group was given placebo in the manner identical to the experimental group. the placebo contained components which were similar to the drug except rifn␣-2b. participants were given instructions on when and how to use the sprays. each nostril and pars laryngea pharyngis should be sprayed with breathing deeply. the rifn␣-2b nasal spray and placebo were stored at 4-8 • c at health clinics of training units. all subjects were followed up and observed for clinical signs and manifestations of respiratory infections for 10 days. local symptoms (e.g., sore throat, dry pharynx, cough, nose running, sneezing, nose congestion), systemic symptoms (e.g., malaise, myalgia, headache, nausea, abdominal pain, diarrhea) and axillary temperature were recorded daily during the observed period. severe adverse reactions (such as allergy, epistaxis, and nasal mucosa erosion) or complications should be reported immediately. once the adverse reactions had occurred, subjects would stop the experiment and be given suitable treatments. assuming a 18% viral respiratory infections attack rate in the group that received placebo and a 8% attack rate in the group that received rifn␣-2b nasal spray (based on the data obtained from our preliminary studies) and assuming that sufficient data would be collected for 90% of the cases to be included in the accordingto-protocol population, we calculated that a sample of 508 recruits per group would provide more than 90% power to demonstrate the superiority of the rifn␣-2b nasal spray at a significant difference level of 0.01 (two tailed). a list of random numbers allocating to the each spray canister was determined via computer-generated randomization. the generation of randomized numbers and labeling of the spray canisters were performed by the third party (national institute for viral disease control and prevention, china center for disease control and prevention) of this study. participants were sequentially allocated to the treatments in the order in which they were recruited, i.e., the first person who was eligible for inclusion was given spray number 1, the second one spray number 2, and so on. when allocated, each participant's name was added to the label details on the spray container. the sequence was concealed until the data were analyzed. both participants and researchers were blind to group assignment. once accuracy of the data were confirmed, the database would be forwarded to the statistician who, only at this time, was supplied with the randomized list. case report forms (crf) consisted of subject demographics data, medical history, respiratory infection symptoms, adverse reactions including local reactions and systemic reactions and concomitant medications from the time that the spray was administered until 10 days later. participants were given a diary crf to record the spray administration, clinical signs and manifestations of respiratory diseases, adverse effects they might have experienced, and other medications they might have taken. observers visited the participants daily and recorded severe adverse events, including high fever (axillary temperature, ≥39.0 • c), allergy, epistaxis, nasal mucosa erosion and hemafecia. serum samples for assessment of viral respiratory infections were collected on the days 0 and day 15 after the administration. elisa (enzyme-linked immunosorbent assay) kits (shenzhen sciarray biotech co. ltd) were used to test igm antibodies against adenovirus species b (adv), respiratory syncytial virus (rsv), influenza a virus (flu-a), influenza b virus (flu-b), and parainfluenza viruses 1-3 (piv 1-3). coating antigens used in the kits were prepared from adenovirus (strain adenoid 6), respiratory syncytial virus (rsv long strain), influenza a virus (h3n2, strain a/texas 1/77), influenza b virus (strain hongkong 5/72), parainfluenza virus type 1(strain vp1), parainfluenza virus type 2 (strain greer) and parainfluenza virus type 3 (strain c243). the operating procedures of elisa were according to the manufacturer's instructions. briefly, 100 l of 1:40 diluted serum specimens was applied to each well of the microtiter plate, the positive and negative standards (provided by the manufacturer) and blank control (dilution solution) were running with each plate to ensure accuracy, then the plate was incubated for 30 min at 37 • c. the specimen was removed and the plate was washed four times by washing solution. anti-human igm (-chain specific) conjugated to horse radish peroxidase was added and incubated for 30 min at 37 • c. the plate was washed four times again. tmb (3,3 ,5,5 -tetramethylbenzidine) solution was added to each well and incubated for 15 min. the reaction was stopped by adding 100 l stop solution (2m h 2 so 4 ) to each well and the optical density (od) value at 450 nm was determined with an elisa reader. serum specific-igm antibodies were defined as positive if od 450 values were greater than two fold negative standard. we compared the positive rates of the viral igm antibodies between two groups. the subject whose serum igm against any of five viruses was positive on the initial administration (day 0) was excluded for serological analysis. the recruit whose antibody was negative on day 0, positive on day 15 after the administration, was considered as having a recent infection with the corresponding virus. serum specimens were collected from 1449 participants and detected for antibodies against three viruses (flu-a, flu-b and piv1-3), among which 548 of specimens were detected for antibodies against five viruses (adv, rsv, flu-a, flu-b and piv1-3). all study data were checked for range and consistency, and were double entered into databases. data of antibodies were entered into microsoft excel 2003, while crf and adverse events data were entered into epidata3.1. all analyses were performed by using the spss 13.0 statistical software package. a descriptive statistical analysis was carried out to compare the baseline characters between two groups. infection rates and adverse events were analyzed by 2 test. for the statistical analysis of body temperature parameters, a repeated measures anova model was used. the level of statistical significance was established as p < 0.05. meanwhile, intention-to-treat (itt) and per protocol (pp) analysis were performed as the assessment. parameters for assessment of benefits or harms of the intervention included: the rate at which events occur in the control group (the control event rate, cer), the rate at which events occur in the experimental group (the experimental event rate, eer), relative risk (rr), absolute risk reduction (arr), relative risk reduction (rrr), and number needed to treat (nnt). measures of rr, rrr, arr and nnt were determined as these following formulae: rr = eer/cer, arr = cer-eer, rrr = arr/cer, nnt = 1/arr [18] . the research protocol was followed to the tenets of the declaration of helsinki and the guidelines for good clinical practice. the clinical trial (protocol number 2003l01500) was officially approved by state food and drug administration, pr china in april 2003, and also approved by ethical committee of southern medical university. of 1500 recruits screened, 1449 recruits were eligible for the trial criteria. they were randomized to receive either treatment sprays (n = 721) or placebo control sprays (n = 728) twice daily for 5 days. during the trial, 12 recruits were lost to follow-up, among which, 6 in the experiment group and 6 in the control group. another 7 subjects dropped off the study due to they were afraid of adverse events psychologically and quit the trial by themselves. subjects who completed the study are shown in fig. 1 . all subjects were male, recruited from different provinces, such as guangdong, guangxi, hubei, liaoning, shandong. the mean age of subjects was 18.07 ± 1.05 years (range 18-23 years). the differences of age, educational level and other demographic characteristics were not statistical significance between two groups (table 1) . compliance rates between two groups were not statistically significant different, which the experimental group was 99.03% and the control group was 98.35% (p = 0.257). the positive rates of igm antibodies against the viruses in recruits whose were negative before the intervention are summarized in table 2 (pp analysis) and table 3 (itt analysis). igm positive rates of anti-adv, anti-flu-a, anti-flu-b, and anti-piv in the control group (cer) were significantly higher than that in the experimental group (eer) (p < 0.05) during the observational period. although positive rate of anti-rsv igm in the experimental group was higher than that in the control group, no significant difference was found. values of rr were less one and their 95% confidence intervals (95% ci) did not cross one except for rsv that the 95% ci included one ( table 4 ). the results indicate that the risk of developing viral respiratory infections is less in the treatment group than that in the control group. the pp analysis exhibited that rrrs (i.e., protection rates) of the rifn␣-2b against adv, rsv, flu-a, flu-b and piv1-3 were 59.4% (95% ci: 0. table 3 the detection of specific serum igm antibodies to five viruses in the intention-to-treat analysis. (95% ci: 6.6-12.1), respectively ( table 5 ). the results showed that the pp analysis was consistent with the itt analysis. to sum it up, protective efficacy of the rifn␣-2b against four viruses arranged in descending order was flu-a, piv1-3, flu-b, adv. however, there was a 95% certainty that the rifn␣-2b had no effect for rsv because the 95% confidence intervals for the rr, rrr and nnt extended from a negative number (treatment may harm) to a positive number (treatment may benefit) (tables 4 and 5). no participants withdrew from the trial due to intolerance of the spray. none of the participants were found to have allergy, high fever, nasal mucosa erosion or hemafecia during the follow-up observational period after administration. we found some flu-like symptoms including cough, sneeze, nose congestion and nose running were slightly higher in the drug group than those in the control group in the period of administrating the nasal sprays, particularly during the second to fourth days of the experiment, however, the differences were not significant (p > 0.05). the incidence rates of epistaxis in the treatment group were low, with from 1.2% (9/721) to 6.2% (45/721) during the period of administration, although the occurrences were significant higher in the treatment group than that in the control group on the third day (5.9%, 43/721, versus 2.1%, 15/728, p < 0.001) and the fourth day (6.2%, 45/721, versus 2.9%, 21/728, p = 0.003). the occurrence of dry pharynx was significantly higher in the treatment group than that in the control group during the whole drug administration period (21.3-31.9% in the treatment group, 12.1-20.7% in the control group). average incidences of dry pharynx and epistaxis were higher in the experimental group (dry pharynx 27.94%, epistaxis 4.6%) than those in the control group (dry pharynx 16.16%, epistaxis 2.58%) (p < 0.05) during the followup period ( table 6 ). the peak of these symptoms was found during the first 5 days, thereafter declined. both of the experimental group and the control group had high rates of myalgia (37.80%, 39.50%), arthralgia (21.50%, 21.44%). these symptoms happened after high intensity training and without significant difference between the two groups. viral respiratory infections are caused by a variety of viruses, among which there are approximately 200 known ones including a vast number of serotypes, and undergo frequent changes in antigenicity [19] . although several vaccines against respiratory viruses to prevent the infections have been proved useful in military populations [7, [20] [21] [22] , progress has been extremely slow. furthermore, not all kinds of viral etiological respiratory infections have been available for specific prevention and still have unknown pathogens. in the recent decade, several novel respiratory viruses which caused serious illness have been identified, such as human metapneumovirus (hmpv), new sars-coronavirus (sars-cov) that associated with severe acute respiratory syndrome (sars), h5n1 flu virus and novel h1n1 virus, which increase the difficulty of the immunological prevention for viral respiratory infections. in the past years, researchers found that ifn-␣ showed an inhibitory effect for sars-cov in vitro and in vivo [23] [24] [25] . therefore, it suggests the potential benefits of ifn-␣ in preventing and controlling viral respiratory infections in specific population groups and in epidemic period. the common viral respiratory infections possess similar clinical signs and symptoms without special clinical manifestations, explicit diagnosis only depends on pathogen examination. although isolation of virus from patients is the strongest evidence for confirming viral respiratory infections, the diagnostic procedures are complex and time-consuming because of the wide range of viruses, especially in a large sample epidemiological study. in addition, respiratory viruses are frequently detected in respiratory tract secretions samples from healthy people [26, 27] . the determinations antibodies against respiratory viruses by comparing acute and convalescent serum specimens in infective people may sometimes be helpful to confirm a specific causative infection. serum specific igm against virus is a sensitive indicator of a recent onset of viral infection, thus, we used specific serum igm as surrogate outcome variables to evaluate the preventive effects of the rifn-␣ 2b for acute viral respiratory infections in this study. the rr, rrr and arr are common parameters used in reporting randomized clinical trials and epidemiological field trails. the rr is a ratio of the probability of the event rate occurring in the exposed (or experimental) group (eer) versus a non-exposed (or control) group (cer). in interventional trials, if the treatment arm is effective in preventing disease then the rr will be less than one, and vice versa. the rrr is the percent reduction in events in the eer compared with the cer. in other word, the rrr presents the percentage of the risk that has been reduced by the intervention in the control group. the arr is the arithmetic difference in the event rate between treatment group and control group. in interventional trials, if the treatment arm is effective in preventing disease then arr will be positive quantity, on the contrary, if the treatment arm is harm, arr will be negative. rr and rrr can be used to quantify the relative magnitude of the protective (treatment) effects. the arr can be used to measure the absolute difference in event rates between two populations. therefore, arr is considered as a more intuitive measure than rr and rrr. these years, the number needed to treat (nnt) has become a widely used index for interpreting the magnitude of treatment benefits or harms. the nnt is the inverse of the arr. it represents the expected number of persons who must be treated with an intervention in order to prevent one additional adverse outcome event (or, depending on the context, to expect one additional beneficial outcome), compared to the expected event rates under the control. that is to say, the smaller the nnt, the more effective the treatment. therefore, besides rr, rrr and arr, we used the nnt to express the size of efficacy of the rifn␣-2b in the present analysis. in our study, serological detection exhibited that serum positive rates of igm antibodies against the five viruses were higher in the control group than those in the experimental group on the 15th day of the administration. in itt analysis, highly antiviral effects for flu-a, flu-b and piv1-3 were seen, all with the rr of <1 and the rrr (i.e., protective rates) of >70%. a protective effect also was seen for adv, with the rr of <1 and the rrr of 59.5%. however, there were wide range of 95% confidence intervals of the rr (0.165-0.999) and rrr (0.6-83.5%), which indicated the low reliability. no certain effect was found for rsv. although the rr was less than 1 and rrr = 72.1%, the 95% ci ranges of rr (0.057-1.356) and rrr (−35.6% to 94.3%) were very wide and included null values (rr 95% ci included 1, rrr 95% ci included negative). because of small size samples for detecting serum adv antibodies and rsv antibodies and less people of anti-adv and anti-rsv igm positive in both groups and a very wide confidence interval of protection rates in adv and rsv, we considered that the sample of this study was not enough for assessing preventive effects of the rifn-␣2b for these two viruses. in the itt analysis of the study, nnt values were 7 (95% ci: 4.8-8.4), 8 (95% ci: 6.1-11.3) and 9 (95% ci: 6.6-12.1) for flu-a, flu-b and piv1-3 infections respectively, which meant 7, 8 and 9 people need to administrate the rifn-␣2b to prevent one infection with relevant virus. the nnt for adv was 25 (95% ci: 12.5-553.9), which indicated that the benefits of preventing adv infection with the rifn-␣2b may be less than above infections with any three viruses above. the nnt for rsv was 50 (95% ci: −313.6 to 23.1), which indicated no effect in preventing rsv infection among recruits with the rifn-␣2b nasal spray. itt analysis is a method of analysis for randomized trials in which all subjects randomly assigned to one of the treatments are analyzed together, regardless of whether they completed or received that treatment or not. on the other hand, pp analysis is a method based only on those patients who complete the entire treatment protocol. in this study, similar results were observed for subjects in the itt and pp populations, which indicated no significant missingness and protocol deviation. on the whole, the intranasal rifn␣-2b with relative low dose and short term administration (1.8 ×10 6 iu daily for five days) was well tolerated. most of the clinical features reported were comparable between the control and the experimental groups. except epistaxis, no other known severe adverse events (such as allergy, high fever, nasal mucosa erosion and hemafecia) of the intranasal interferon were reported during the trial. the rrs of dry pharynx and epistaxis were 1.70 (95% ci: 1.39-2.08) and 1.85 (95% ci: 1.06-3.23), respectively, which indicated that dry pharynx and epistaxis might be linked to the interferon. however, the incidence of epistaxis was low and the clinical signs were mild and transitory in present study. the results are the same as our previous study [28] and the risk is much lower than that appraised and summarized in evidence-based medicine (or = 4.52, 95% ci = 3.78-5.41) by jefferson and tyrrell [29] . in summary, this randomized controlled trial suggested that the recombinant human interferon ␣-2b nasal spray can be used to prevent common acute viral respiratory infections caused by flu-a, flu-b, piv1-3 and adv and was generally well tolerated among military recruits. however, the limitations of this trial may be found on sampling and sample size. all subjects enrolled were healthy and young male recruits, so it may be difficult to extrapolate the conclusion to other populations. the sample was not large enough for evaluating the effects for adv and rsv infections which had relative low incidence in the army recruits. the efficacy of preventing viral respiratory infections by the rifn␣-2b nasal spray should be evaluated further in different population groups, such as children and the elderly, and more samples should be involved in the further study. swine influenza a outbreak clinical presentations for influenza and influenza-like illness in young, immunized soldiers reflections on the 1976 swine flu vaccination program current research on respiratory viral infections: fourth international symposium large epidemic of adenovirus type 4 infection among military trainees: epidemiological, clinical, and laboratory studies the social history of medicine: beyond the local a doubleblind, placebo-controlled study of the safety and immunogenicity of live, oral type 4 and type 7 adenovirus vaccines in adults influenza surveillance on the army population interferon and their application in the diseases of the lung the efficacy of intranasal interferon alpha-2a in respiratory syncytial virus infection in volunteers prevention of experimental coronavirus colds with intranasal alpha-2b interferon treatment of respiratory syncytial virus infection with recombinant interferon alfa-2a could preventive intranasal interferon lower the morbidity in children prone to respiratory illness? intranasal interferonalpha 2b for seasonal prophylaxis of respiratory infection tolerance of one-month intranasal interferon intranasal applied recombinant leukocyte a interferon in normal volunteers. ii. determination of minimal effective and tolerable dose human tolerance and histopathologic effects of long-term administration of intranasal interferon-alpha 2 glossary of evidence-based terms treatment of respiratory virus infections role of u.s. military research programs in the development of u.s.-licensed vaccines for naturally occurring infectious diseases effectiveness of the 2003-2004 influenza vaccine among u. s. military basic trainees: a year of suboptimal match between vaccine and circulating strain vaccination policies in the military: an insight on influenza severe acute respiratory syndrome-related severe coronavirus is inhibited by interferon-alpha anti-sars virus activities of different recombinant human interferons in cell culture system preventive and therapeutic effects of recombinant ifn-b nasal spray on sars-cov infection in macaca mulata a prospective, community-based study on virologic assessment among elderly people with and without symptoms of acute respiratory infection upper respiratory virus detection without parent-reported illness in children is virus-specific a field trial for evaluating the safety of recombinant human interferon ␣-2b for nasal spray vaccines for the common cold (protocol for a cochrane review) this study was funded by national high-tech r & d program (863 program), grant number 2003aa208220 from ministry of science and technology of the people's republic of china.we thank institute for viral disease control and prevention of china cdc for providing and dispensing the spray agents to be tested, and we gratefully acknowledge the cooperation and assistance of the healthcare staff members at the clinical trial sites. key: cord-301011-xbuqd0j5 authors: felten-barentsz, karin m; van oorsouw, roel; klooster, emily; koenders, niek; driehuis, femke; hulzebos, erik h j; van der schaaf, marike; hoogeboom, thomas j; van der wees, philip j title: recommendations for hospital-based physical therapists managing patients with covid-19 date: 2020-06-18 journal: phys ther doi: 10.1093/ptj/pzaa114 sha: doc_id: 301011 cord_uid: xbuqd0j5 objective: the covid-19 pandemic is rapidly evolving and has led to increased numbers of hospitalizations worldwide. hospitalized patients with covid-19 experience a variety of symptoms, including fever, muscle pain, tiredness, cough, and difficulty breathing. elderly people and those with underlying health conditions are considered to be more at risk of developing severe symptoms and have a higher risk of physical deconditioning during their hospital stay. physical therapists have an important role in supporting hospitalized patients with covid-19 but also need to be aware of challenges when treating these patients. in line with international initiatives, this article aims to provide guidance and detailed recommendations for hospital-based physical therapists managing patients hospitalized with covid-19 through a national approach in the netherlands. methods: a pragmatic approach was used. a working group conducted a purposive scan of the literature and drafted initial recommendations based on the knowledge of symptoms in patients with covid-19, and current practice for physical therapist management for patients hospitalized with lung disease and patients admitted to the intensive care unit (icu). an expert group of hospital-based physical therapists in the netherlands provided feedback on the recommendations, which were finalized when consensus was reached among the members of the working group. results: the recommendations include safety recommendations, treatment recommendations, discharge recommendations, and staffing recommendations. treatment recommendations address 2 phases of hospitalization: when patients are critically ill and admitted to the icu, and when patients are severely ill and admitted to the covid ward. physical therapist management for patients hospitalized with covid-19 comprises elements of respiratory support and active mobilization. respiratory support includes breathing control, thoracic expansion exercises, airway clearance techniques, and respiratory muscle strength training. recommendations toward active mobilization include bed mobility activities, active range-of-motion exercises, active (−assisted) limb exercises, activities-of-daily-living training, transfer training, cycle ergometer, pre-gait exercises, and ambulation. as of publication date, the number of patients with respiratory syndrome caused by coronavirus 2 (sars-cov-2), the virus that causes coronavirus disease 2019 (covid19) , is still increasing rapidly worldwide. spreading of covid-19 occurs mainly through respiratory droplets and aerosols produced when an infected person coughs or sneezes. 1 to our knowledge, there is currently no consensus on the period the virus is transmissible to other humans, however the duration and transmissibility seem to differ between patients with differing severity of illness. 2 even after resolution of symptoms, individuals might keep shedding the virus. 3 diagnosis of covid-19 requires detection of sars-cov-2 rna using a combination of nasopharynx-and throat sample. 4 ,5 sars-cov-2 rna can also be detected in stool and blood. 4 chest computed tomography (ct) images from patients with covid-19 typically demonstrate bilateral, peripheral ground glass opacities. unfortunately, this pattern is nonspecific and overlaps with other infections; therefore, the diagnostic value of chest ct imaging for covid-19 may be low. 4, 5 recent data from china and italy indicate that in 80 percent of cases covid-19 infection causes 'mild and moderate illness', approximately 15 percent of cases develop 'severe illness' leading to hospitalization, and 5 percent develop 'critical illness' requiring icu treatment. 2, [4] [5] [6] hospitalized patients with covid-19 experience a variety of symptoms, including fever, muscle pain, tiredness, cough, and difficulty breathing. 7 elderly people and those with underlying health conditions are considered to be more at risk of developing severe symptoms, 4 and have a higher risk of physical deconditioning during their hospital stay. 8, 9 physical therapists have an important role in supporting hospitalized patients through respiratory support and active mobilization. physical therapist management should be tailored to the individual patient's needs concerning frequency, intensity, type and timing of the interventions, in particular for those with severe/critical illness, >70 years of age, obesity, comorbidity and other complications. 10, 11 yet, physical therapists need to be aware of potential challenges when treating patients with covid-19. in a recent study, an international group of authors described the physical therapist management for covid-19 in the acute hospital setting, including workforce planning, screening, delivery of physical therapist interventions and personal protective equipment (ppe). 12 in line with this international study 12 and the consensus statement of italian respiratory therapists 13 we aim to provide guidance and detailed recommendations for hospital-based physical therapists managing patients hospitalized with covid-19 through a national approach in the netherlands. [h1] scope this study focuses on adult patients admitted to the (acute) hospital setting due to covid-19. in general, patients with covid-19 experience the following signs and symptoms: fever (83%-99%), cough (59%-82%), fatigue (44%-70%), weight loss (40%-84%), shortness of breath (31%-40%), secretion production (28%-33%) and myalgias (11%-35%). 4, 6 recent studies showed that illness severity can range from mild to critical: 2, [4] [5] [6]  mild to moderate (mild symptoms up to mild pneumonia): 80%  severe (dyspnea, hypoxia, or >50% lung involvement on imaging): 15%  critical (respiratory failure, shock, or multiorgan system dysfunction): 5% critical cases, needing icu treatment, may show symptoms of acute respiratory distress syndrome (ards) like lung disease, with widespread inflammation in the lungs. 5 consolidation lesions also remain at long-term and can leave fibrotic changes in the lungs. 5 furthermore, patients who are critically ill, needing icu treatment, are at risk of developing post-intensive care syndrome (pics) including icu-acquired weakness (icu-aw). [13] [14] [15] mortality among patients admitted to the icu ranges from 39% to 72%. 4 health care professionals should be aware that the clinical progression of symptoms might occur one week after illness onset. 5, 13, 14 important subgroups are elderly people (≥70 years of age) and those with underlying health conditions (eg, hypertension, diabetes, cardiovascular disease, chronic respiratory disease and cancer), who are considered to be more at risk of developing severe symptoms, 4 but also at risk of physical deconditioning during hospital stay. 8, 9 figure 1 is based on recent literature and shows the flow of patients with covid-19 with their signs and symptoms before 4,6,7 and during hospital admission; 4,5,7-9,13,15,16 the severity classification 2,4-6 and the physical therapy goals during hospital stay. [10] [11] [12] [13] 17 these recommendations focus on the physical therapist management for adult patients with covid-19 admitted to the (acute) hospital setting. recommendations contain specific physical therapy goals concerning respiratory problems and deconditioning including icu-aw and pics. the recommendations are outlined in 2 sections:  section 1: patients who are critically ill with covid-19 admitted to the icu.  section 2: patients who are severely ill with covid-19 admitted to the covid ward. we used existing international recommendations 12,13 as basis for further specification and contextualization. when our recommendations diverge from the international recommendations, we clarified this in the main text and through a separate paragraph with reflections. the recommendations are structured in the following 6 order: safety recommendations, treatment recommendations (specified for different phases of hospitalization), discharge recommendations, and staffing recommendations. due to the acute and sudden spreading of covid-19, the evidence base for optimal treatment for this group of patients is evolving rapidly and new insights are emerging at a similar pace. nevertheless, clear recommendations for hospital-based physical therapist management, either based on evidence or bestpractices, are crucial to support the recovery of patients and safety of health care professionals. these recommendations will be updated periodically based on new evidence and experience, and will be made available through the website of the royal dutch society for physical therapy and the world confederation for physical therapy. to cope with this rapidly evolving evidence base, we utilized a pragmatic approach, rather than a formal approach (such as grade), 18 respiratory droplets and aerosols may be released from patients during physical therapist interventions and may cause further spread of the virus. direct contact between physical therapists and patients with covid-19, therefore, should be minimized to avoid risk of virus transmission and reduce usage of scarce ppe. therefore, we recommend physical therapists make optimal use of telecommunication and written information material. if direct (face-to-face) contact with patients with covid-19 is required, physical therapists should use ppe. recommended ppe include a gown, gloves, eye protection and a facemask. 4  active mobilization, which may lead to coughing and secretion mobilization or disconnection of the mechanical ventilation. if one of the above procedures is performed, physical therapists are recommended to wear a facemask that filters at least 95% of airborne particles (ie, ffp2 mask, n95 facemasks). physical therapists should ensure that they are fully competent in the use of ppe. 4 safety recommendations need to be taken into account during all  recommendation: make optimal use of digital and/or written information for the instruction of patients. physical therapist management for patients hospitalized with covid-19 comprises elements of respiratory support and active mobilization. 20, 21 recommendations toward respiratory support, defined as the "proactive approach to minimize respiratory symptoms during the acute phase of a pulmonary disease," 22 are presented in detail. in the treatment of patients with covid-19, respiratory support can consist of breathing control, thoracic expansion exercises, airway clearance techniques and respiratory muscle strength training. recommendations toward active mobilization concern the "proactive approach to support any physical activity where patients assist with the activity using their own strength and control: patients may need assistance from staff or equipment, but they are actively participating in the exercise." 21 examples of active mobilization are bed mobility activities (eg, bridging, rolling, lying to sitting), active range-of-motion exercises, active (-assisted) limb exercises, adl training, transfer training, cycle ergometer, pre-gait exercises, and ambulation. [h3] phase a: patient is unconscious-respiratory support. patients with critical illness due to covid-19 may develop acute respiratory distress syndrome (ards)-like symptoms, requiring admission to the icu. 24 initially, the majority of patients are deeply sedated (rass ≤ -4) and mechanically ventilated in prone position. 25 these patients often receive neuromuscular blocking agents in order to support mechanical ventilation, as this drug application can improve chest wall compliance, eliminate ventilator dyssynchrony, and reduce intraabdominal pressures. 26 given the lack of therapeutic goals in this phase, physical therapist management concerning respiratory support is not recommended. this might be different for physical therapists outside the netherlands with other scope of practice concerning respiratory support. [h3] phase a: patient is unconscious-active mobilization. [h3] phase b: patient is conscious and able to cooperate-respiratory support. the moment sedation is reduced (rass ≥ -2) and the patient is conscious and able to cooperate (s5q ≥ 3), a new phase starts. 25 normally, this is the phase to start active mobilization and respiratory support; however, in patients with covid-19, detachment of the closed mechanical ventilation system circuit should always be avoided due to the risk of virus transmission. even in the case of weaning from mechanical ventilation, where physical therapists typically aim to ensure sufficient inspiratory muscle strength, 29,30 the risk of virus transmission via droplets or aerosols in using medical assistive testing devices is too high. therefore, we recommend to not detach the ventilation system for the purpose of respiratory function testing, respiratory muscle training, or breathing exercises. 19 to our knowledge, it remains unclear if both droplets and aerosols are filtered by disposable bacterial filters. 31 in case of prolonged weaning, patients who fail more than 3 weaning attempts or require more than seven days of weaning after the first spontaneous breathing trail, 32 respiratory muscle training should be discussed in the multidisciplinary team. 30 the team may decide that benefits of respiratory muscle training outweigh the safety risks. in the phase after prolonged (assisted) mechanical ventilation, inspiratory (imt) and expiratory muscle training (emt) can be used to counterbalance the weakness of the respiratory muscles. 29 36 however, the use of these devices is not recommended in patients with covid-19 due to the increased risk of virus transmission. in this situation, training can be started pragmatically (ie, without respiratory testing results) using a threshold training device, with low resistance (< 10 cmh2o) and can be increased based on clinical presence, experienced dyspnea and borg score for perceived exhaustion. 37 for respiratory muscle strengthening, a combination of both imt and emt is recommended, as this combination is superior to imt alone in improving respiratory muscle strength. 33 as respiratory muscle training devices could carry the virus (prolonged), the use of these devices should be discussed with hospital officers for hygiene and infection prevention. recommendation: discuss with the multidisciplinary team whether to pragmatically initiate respiratory muscle strengthening in patients with prolonged weaning. [h3] phase b: patient is conscious and able to cooperate-active mobilization. when patients become conscious and cooperative, active mobilization can be considered. active mobilization should aim to prevent icu-aw and deconditioning from immobilization and illness. the medical research council sum-score (mrc-ss) is widely used to diagnose icu-aw, which is defined as an mrc-ss < 48. 38 it is assumed that patients diagnosed with icu-aw may benefit from active mobilization also following their icu admission. physical activities for patients who are critically ill should be planned and targeted following the evidence based statement for physical therapist management in the icu as much as possible. 17 patient safety criteria according to sommers et al. 17 for active mobilization that always need to be considered at the icu, are presented in figure 3 . close monitoring of respiratory and hemodynamic functions of patients is crucial to ensure patients' safety. 17, 21 as a first step, bed mobility activities can be performed by assisting bridging, rolling, and transferring from supine to sitting. 23 medical assistive devices (eg, a bed cycle) might be used to support active mobilization. however, use of these devices should be discussed with hospital officers for hygiene and infection prevention. to evaluate and increase training intensity, frequency and/or activities, criteria of american college of sports medicine (acsm) guidelines for exercise testing and prescription, 40 ideally, the physical therapist is the leading health care professional to guide active mobilization. however, safety recommendations can also be decisive in initiating physical therapist management. if safety recommendations for health care providers do not warrant direct physical therapy contact, we recommend to instruct nurses to combine active mobilization with daily care activities. in this case the physical therapist has a coaching role. patients who are severely ill with covid-19 who require hospitalization can present with complications such as pneumonia, hypoxemic respiratory failure/ards, sepsis and septic shock, cardiomyopathy and arrhythmia, acute kidney injury, and complications from prolonged hospitalization, including secondary bacterial infections. 4 because the consequences of the infection impact the respiratory system, one of the goals of physical therapist management is to optimize respiratory function. therefore, respiratory support aims to improve breathing control, thoracic expansion, and mobilization/evacuation of secretion. active mobilization aims to increase (or maintain) physical functioning and independence in activities of daily living (adl). these recommendations also apply for patients recovering from critical illness due to covid-19. additionally, in patients recovering from critical illness respiratory muscle strength/endurance training can be continued. [h3] respiratory support. respiratory support serves several purposes: to improve vital capacity, to evacuate secretion, and to strengthen respiratory muscle. techniques and goals are briefly introduced as follows: 13 breathing control and thoracic expansion exercises, and combines these with huffing and coughing. 41, 42, 45 huffing and coughing contribute to the formation of respiratory droplets and aerosols and should be avoided in direct contact with health care professionals. therefore, these maneuvers are only recommended in case of airway obstruction due to excess secretions. the multidisciplinary team should carefully evaluate whether airway obstruction is present through medical history taking (eg, the presence of productive cough), physical examination (eg, the presence of pulmonary rhonchus), and observations. telecommunication and/or written instruction material can be used to support the use of acbt. if patients fail to effectively use acbt, teaching these techniques under direct supervision of a physical therapist can be considered.  strengthening of respiratory muscle: patients with covid-19 might have suspected respiratory muscle weakness caused by prolonged mechanical ventilation during icu stay. after transfer to the covid ward, respiratory muscle strengthening can be continued for patients recovering from critical illness according to the recommendations in section 1, phase b. training protocols typically use resistive loads ranging between 30% and 80% of mip. 46 however, the use of noninvasive handheld manometers is not recommended in patients hospitalized with covid-19 due to the increased risk of virus transmission. according to section 1, phase b, training can be started pragmatically (ie, without respiratory testing results) using a threshold training device with low resistance (< 10 cmh2o), and can be increased based on clinical presence, experienced dyspnea and borg score for perceived exhaustion. 37 one of the unique advantages of respiratory muscle training is that it can be implemented in shorter intervals (30 breaths, 2 times/day). training effects from respiratory muscle training have been observed for multiple protocols lasting only 4 weeks. 46 a telehealth or mobile app[h3] active mobilization. if patients are bedridden and suffering from covid-19, pulmonary ventilation can be stimulated by bed mobility activities through bridging, rolling, and sitting. 11 if possible, patients might assist with their own strength and control. if needed, staff and equipment can be used to support the activity. a vertical position can be obtained with less support of patients by tilting the bed or using a tilt table. in order to prevent further deconditioning, patients should be stimulated to be physically active through active mobilization as much as possible through the hospitalization period. physical therapists can provide specific exercises and training that meet the needs and preferences of patients with covid-19. maintaining or improving physical functioning should be executed following common safety recommendations, monitoring, and guidance. 17, 21 based on our expert opinion, at least patient's saturation and heart rate should be monitored before and during active mobilization, due to the low and fluctuating vital capacity of patients with covid-19. active mobilization interventions that need to be considered are bed mobility activities, active range of motion exercises, active(-assisted) limb exercises, adl training, transfer training, cycle ergometer, pre-gait exercises, and ambulation. 23 the hospital-based physical therapist should screen patients with severe illness due to covid-19 on whether physical therapist management should be continued after hospital discharge. 48 19 hospital-based physical therapists with these skills and knowledge should be tasked with training of less experienced colleagues to provide them with the necessary skills, knowledge and self-confidence for physical therapist management of patients with covid-19.  recommendation: deploy physical therapists with sufficient skills, knowledge and self-confidence in care for patients who are severely ill at a covid-19 ward or in the icu. the covid-19 outbreak presents new challenges for health care professionals. physical therapists will work intensively with patients who are severely ill, which can lead to mental health distress. it is recommended for managers to plan sufficient recovery time between work shifts of physical therapists and to let less experienced colleagues carefully be supervised by experienced peers. in these turbulent times, provision of psychosocial support should be considered.  recommendation: provide psychosocial support for hospital-based physical therapists. in this manuscript we provide detailed recommendations and intervention descriptions for hospital-based the royal dutch society for physical therapy (kngf) supported the development of the recommendations. the early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia covid-19 and italy: what next? lancet time kinetics of viral clearance and resolution of symptoms in novel coronavirus infection. american journal of respiratory and critical care medicine interim clinical guidance for management of patients with confirmed coronavirus disease (covid-19) handbook of covid-19 prevention and treatment characteristics of and important lessons from the coronavirus disease 2019 (covid-19) outbreak in china: summary of a report of 72314 cases from the chinese center for disease control and prevention corona -: situation report, 51. geneva: world health organization preventable iatrogenic disability in elderly patients during hospitalization she was probably able to ambulate, but i'm not sure response to pulmonary rehabilitation: toward personalised programmes? the european respiratory journal personalised pulmonary rehabilitation in copd physiotherapy management for covid-19 in the acute hospital setting: clinical practice recommendations joint statement on the role of respiratory rehabilitation in the covid-19 crisis: the italian position paper improving long-term outcomes after discharge from intensive care unit: report from a stakeholders' conference the incidence of intensive care unit-acquired weakness syndromes: a systematic review exercise rehabilitation following intensive care unit discharge for recovery from critical illness: executive summary of a cochrane collaboration systematic review physiotherapy in the intensive care unit: an evidence-based, expert driven, practical statement and rehabilitation recommendations grade guidelines: a new series of articles in the journal of clinical epidemiology covid-19: respiratory physiotherapy on call information and guidance physiotherapy for adult patients with critical illness: recommendations of the european respiratory society and european society of intensive care medicine task force on physiotherapy for critically ill patients expert consensus and recommendations on safety criteria for active mobilization of mechanically ventilated critically ill adults european respiratory society statement on pulmonary rehabilitation. american journal of respiratory and critical care medicine early intervention (mobilization or active exercise) for critically ill adults in the intensive care unit. the cochrane database of systematic reviews severe acute respiratory syndrome coronavirus 2 (sars-cov-2) and coronavirus disease-2019 (covid-19): the epidemic and the challenges critical care crisis and some recommendations during the covid-19 epidemic in china the use of neuromuscular blocking agents in the icu: where are we now? bewegingsbeperkingen bij patiënten die in buikligging worden verpleegd stretch for the treatment and prevention of contractures. the cochrane database of systematic reviews inspiratory muscle rehabilitation in critically ill adults. a systematic review and meta-analysis weaning failure and respiratory muscle function: what has been done and what can be improved the bacterial and viral filtration performance of breathing system filters weaning from mechanical ventilation. the european respiratory journal combination of inspiratory and expiratory muscle training in same respiratory cycle versus different cycles in copd patients: a randomized trial american journal of respiratory and critical care medicine ers statement on respiratory muscle testing at rest and during exercise. the european respiratory journal respiratory muscle rehabilitation in patients with prolonged mechanical ventilation: a targeted approach psychophysical bases of perceived exertion interobserver agreement of medical research council sum-score and handgrip strength in the intensive care unit relationship of medical research council sum-score with physical function in patients post critical illness. american journal of respiratory and critical care medicine acsm's guidelines for exercise testing and prescription bronchiectasis toolbox: the active cycle of breathing technique incentive spirometry: 2011. respir care time course of lung changes on chest ct during recovery from 2019 novel coronavirus (covid-19) pneumonia the active cycle of breathing technique: a systematic review and meta-analysis efficacy of a novel method for inspiratory muscle training in people with chronic obstructive pulmonary disease evaluation of the effectiveness of a home-based inspiratory muscle training programme in patients with chronic obstructive pulmonary disease using multiple inspiratory muscle tests role of physical therapists in reducing hospital readmissions: optimizing outcomes for older adults during care transitions from hospital to community surviving critical illness: what is next? an expert consensus statement on physical rehabilitation after hospital discharge survivors of critical illness and their relatives. a qualitative study on hospital discharge experience physiotherapy treatment approaches for survivors of critical illness: a proposal from a delphi study sars: sequelae and implications for rehabilitation key: cord-296777-6xz2aslj authors: oosterhof, l; christensen, c b; sengeløv, h title: fatal lower respiratory tract disease with human corona virus nl63 in an adult haematopoietic cell transplant recipient date: 2009-10-12 journal: bone marrow transplant doi: 10.1038/bmt.2009.292 sha: doc_id: 296777 cord_uid: 6xz2aslj nan the human corona virus nl63 (hcov nl63) was discovered in 2004 by dutch virologists subsequent to the outbreak of severe respiratory syndrome corona virus (sars). 1 hcov nl63 is present worldwide, and is considered to be a highly prevalent virus. hcov nl 63 primarily infects the upper respiratory tract, typically causing mild upper respiratory infectious symptoms such as cough, rhinorrhoea, and fever. the clinical course of hcov nl63 infection is more severe in immunocompromised patients. several inhibitors are known to reduce in vitro replication of the virus, but there is no effective antiviral treatment or vaccine at present. 2 hcov nl63 virus is transmitted by airborne droplets and infection is community acquired. community-acquired respiratory viruses are known to account for many cases of interstitial pneumonia as a common complication following allo-haematopoietic cell transplantation (hct). 3 during immune reconstitution after allo-hct, there is increased susceptibility to pulmonary viral infections, partly because of t-lymphocyte dysfunction due to the reversed cd4/cd8 ratio. 4 multiple factors such as conditioning therapy, donor type, prolonged cytopenia, use of immunosuppressive drugs and gvhd predispose to prolonged immune reconstitution after allo-hct. 5 the most commonly identified viral pathogens in the late-postengraftment phase are cmv and varicella zoster. 6 however, in many cases of suspected post transplant viral respiratory tract infection, no aetiological agent is identified. 7 we present a case of fatal hcov nl63 pulmonary infection during the late-engraftment phase, 5 months after an allo-hct with a matched unrelated donor in a patient without active gvhd and receiving only modest immune suppressive treatment. a 42-year-old man with accelerated-phase cml received an erythrocyte-depleted bm graft from a 10/10 allelematched unrelated male donor after conditioning with tbi and cy. he received cya and mtx for rejection and gvhd prophylaxis. trimethoprim and aciclovir were administered throughout the post transplant period. the patient and donor were cmv antibody negative. pcr assay for cmv in the blood was negative as well. engraftment was obtained at day þ 23. at the same time, a maculo-papular rash was noted. the patient was treated with steroids (2 mg/kg) in addition to the ongoing cya treatment for acute grade 3 skin gvhd. myeloid engraftment showed neutrophil count within normal limits, whereas the patient remained lymphocytopenic throughout the period with peripheral lymphocyte counts from 0.2 to 0.6 â 10 9 /l. after transplant, he developed secondary hypoimmunoglobulinaemia and was treated with i.v. igs twice. a bm examination at day þ 60 showed complete haematological remission, chromosomal examination showed normal male karyotype, and fish analysis showed bcr-abl negativity. pcr assay for bcr-abl taken 5 months after transplantation was negative. five months after transplantation, the patient developed an unproductive cough and influenza-like illness, leading to hospital admission. he was still on cya, valaciclovir and fluconazole treatment, but received no steroids. he reported that his 5-year-old son had shown the same symptoms a week before the disease onset. it was noted that the son spent every day at the kindergarten. at admission, the patient presented with a dry cough and intercostal pain due to coughing. he developed a fever on the day of admission. neutrophil levels were 2.5 â 10 9 /l and lymphocyte levels 0.4 â 10 9 /l; c-reactive protein level had elevated to 34 mg/l (0-10). the chest x-ray showed bilateral infiltrates that were predominantly localized to the lower right lobe. bal examination was performed twice. the bal fluid taken at the day of admission was pcr positive for hcov nl 63, whereas that taken at day 11 was negative for hcov nl63. the bal and several tracheal and nasopharyngeal fluid examinations before and during admission were negative for fungi, acid-fast bacilli, gram stain and culture, cytology, cmv, and respiratory viruses (rsv, influenzavirus a þ b, parainfluenzavirus, metapneumovirus, adenovirus, rhinovirus and pneumocystis jiroveci). on the day of admission, i.v. empirical antimicrobial therapy comprising carbapenemes, caspofungin and trimethoprim was initiated. there was no relief of symptoms. he was intubated at day 4, as his respiratory status deteriorated. at day 7, caspofungin treatment was replaced by lipid amphothericin b, and steroid treatment was initiated (80 mg/day) for idiopathic pneumonia syndrome (ips) without improvement. ct scan and lung biopsy for final diagnostic ips evaluation could not be performed because of the patient's poor respiratory status. the patient died 5 weeks after admission owing to progressive respiratory failure. post-mortem autopsy confirmed signs of chronic inflammation in the lungs, severe alveolar damage, intra-alveolar hyaline membranes and interstitial oedema. no fungi, pneumocystis, mycobacteria or other pathogens were identified. pcr analysis of the alveolar fluid was negative for cmv and hcov nl63. the bm showed cr. other organs showed no pathology. this patient initially presented with upper respiratory prodrome, which progressed to pneumonia and respiratory failure. initial blood tests and x-ray evaluation were compatible with a respiratory virus illness. in the light of the clinical features, continuous lymphocytopenia and the detection of hcov nl 63 in the bal fluid taken at the day of admission, the respiratory disease in this patient is considered significant for hcov nl63 lower respiratory tract infection. it may be noted that our case suggests a communityacquired transmission of the hcov nl63 from a symptomatic 5-year-old child to a hct patient during the lateengraftment phase 5 months after transplantation. the patient had no active gvhd and was receiving only modest immune suppressive treatment. the bal fluid at day 11 of admission and the final pcr analysis post mortem were negative for hcov nl63, suggesting that the patient had cleared the virus. the time of onset of respiratory symptoms and the radiology and autopsy findings are consistent with development of ips. as infections might be involved in the initial lung tissue injury, inflammation and cytokine release before ips development, 8 this case might suggest a possible correlation between hcov nl63 infection and subsequent ips. although evaluation for corona virus in symptomatic hct patients is not routinely performed, we believe it should be considered in the differential diagnoses of respiratory failure or as a possible causative agent before the development of ips after allo-hct for haematological disease. identification of a new human coronavirus human coronaviruses: what do they cause? emerging viruses in transplantation: there is more to infection after transplant than cmv and ebv prolonged immune deficiency following allogeneic stem cell transplantation: risk factors and complications in adult patients risk factors for fatal infectious complications developing late after allogeneic stem cell transplantation late infections after allogeneic bone marrow transplantations: comparison of incidence in related and unrelated donor transplant recipients respiratory virus infections in adult t celldepleted transplant recipients: the role of cellular immunity idiopathic pneumonia syndrome: changing spectrum of lung injury after marrow transplantation the authors declare no conflict of interest. l oosterhof 1 , cb christensen 2 and h sengelv 1 1 key: cord-312797-hohzjx74 authors: hamelin, marie-ève; abed, yacine; boivin, guy title: human metapneumovirus: a new player among respiratory viruses date: 2004-04-01 journal: clin infect dis doi: 10.1086/382536 sha: doc_id: 312797 cord_uid: hohzjx74 the human metapneumovirus (hmpv) is a newly described member of the paramyxoviridae family belonging to the metapneumovirus genus. since its initial description in 2001, hmpv has been reported in most parts of the world and isolated from the respiratory tract of subjects from all age groups. despite the fact that prospective and case-control studies have been limited, the epidemiology and clinical manifestations associated with hmpv have been found to be reminiscent of those of the human respiratory syncytial virus, with most severe respiratory tract infections occurring in infants, elderly subjects, and immunocompromised hosts. additional research is needed to define the pathogenesis of this viral infection and the host's specific immune response. part of the pneumovirinae subfamily within the paramyxoviridae family. initial electron microscopic examination of hmpv isolates revealed morphological characteristics consistent with paramyxoviruses. pleomorphic, spherical, and filamentous particles could be observed (figure 1) [6] . spherical enveloped particles vary in size, with a mean diameter of 209 nm. the nucleocapsid has a length varying from !200 to ∼1000 nm and a diameter of 17 nm. hmpv isolates were initially found to grow on tertiary monkey kidney and llc-mk2 (rhesus monkey kidney) cells, with poor or no viral growth on other cell lines, including madin darby canine kidney, vero (african green monkey kidney), human laryngeal carcinoma (hep-2), human foreskin fibroblast, human rhabdomyosarcoma, transformed human kidney (293), human lung adenocarcinoma (a-549), and human colon adenocarcinoma (ht-29) [4, 7] . in addition, hmpv isolates did not exhibit hemagglutinating activity when tested with human, turkey, chicken, or guinea pig rbcs, which is consistent with other members of the pneumovirinae subfamily [4, 7] . intranasal inoculation of ferrets and guinea pigs with hmpv isolates did not cause any clinical symptoms [4] . also, experimentally infected birds (juvenile turkeys and chickens) did not show clinical signs or virus replication over a 3-week period. on the other hand, there was evidence of efficient hmpv repfigure 1 . negative-stain electron micrographs of human metapneumovirus (hmpv). center image shows 5 pleomorphic hmpv particles; note the projections along the periphery of the viruses. upper right and lower left insets show the nucleocapsid and the filamentous rodlike particle, respectively. staining was done with 2% phosphotungstic acid. bar markers represent 100 nm. from [6] ; reproduced with permission from the university of chicago press. lication in the respiratory tract of experimentally infected monkeys (cynomolgus macaques) associated with mild upper respiratory tract signs [4] . thus, in contrast to apv, hmpv is a respiratory pathogen of primates. the hmpv genome consists of a single negative strand of rna of ∼13 kb containing genes coding presumably for a nucleoprotein (n), phosphoprotein (p), matrix (m) protein, fusion (f) protein, transcription elongation factor/rna synthesis regulatory factor (m2), small hydrophobic (sh) surface protein, major attachment (g) glycoprotein, and major polymerase (l) subunit, arranged in the order 3 -n-p-m-f-m2-sh-g-l-5 , similar to apv [8, 9] . two major differences exist between hmpv and hrsv genomes: the gene order is different, and hmpv does not contain nonstructural genes (figure 2) [10] . the possible relevance of the absence of these viral proteins, which have been associated with ifn antagonism by hrsv [11] , deserves additional study. for hrsv, the g and f surface proteins are known as the major protective antigens [11] , and this also needs to be verified for hmpv. phylogenetic analyses have demonstrated that, among members of the pneumovirinae subfamily, hmpv was most closely related to apv serotype c, the avian metapneumovirus that emerged in the united states in the late 1990s. for instance, the amino acid sequence identity between hmpv strains and apv-c and hrsv representatives varies from 66% to 89% and from 23% to 43%, respectively, when comparing the n, p, m, and f genes [4, 12] . the sh and g amino acid sequences of hmpv show important variability and share only 28% and 21% identity with their respective apv-c counterparts [8, 13, 14] . it is possible that this important divergence may account for the specific host range of these 2 related metapneumoviruses. genetic analysis on a large number of hmpv isolates has identified 2 major groups and 2 minor genetic clusters within each group [6, 7] . however, additional investigations are still required to determine whether these genotypes represent different antigenic groups. recently, the complete nucleotide sequences of hmpv isolates belonging to these 2 major groups were determined [9] . nucleotide and amino acid sequence identities between the 2 hmpv groups were found to be 80% and 90%, respectively, which is similar to what has been reported with hrsv a and b genotypes. since its initial report by the dutch researchers in 2001, hmpv has been found in most parts of the world, with reports from north america (united states and canada), europe (united kingdom, france, germany, italy, spain, and finland), asia (hong kong and japan), and australia (table 1). the virus has also been identified in hiv-infected and nonimmunocompromised children from south africa [15] . the few seroprevalence surveys from the netherlands [4] , japan [16] , and israel [17] have indicated that virtually all children are infected by 5-10 years of age. in addition, studies have shown that hmpv is not a new pathogen, with serological evidence of human infection dating from 1958 in the netherlands [4] and viral isolation for the past 10-20 years in europe and canada [4, 7] . cases of severe hmpv infection in adults [7] and of reinfection in immunocompromised subjects [18] suggest that, despite universal infection in childhood, new infections can occur throughout life due to incompletely protective immune responses and/or acquisition of new genotypes. surveys have indicated that hmpv has a seasonal distribution overlapping hrsv circulation, with most cases reported during the winter/ early spring months (table 1) . although most studies have limited their surveillance to the typical respiratory virus period, our group has shown that 87% of hmpv isolates recovered in cell culture in quebec, canada, were recovered during the period of december through may [7] . in addition, we found that outbreaks of hmpv infection tend to peak in early spring over a 4-6 week period, slightly later than outbreaks of hrsv infection, which also are more spread out in time [19] . however, additional studies over multiple years are needed to better define the seasonality of hmpv infection. the role of hmpv as the cause of arti has been evaluated in many studies, mostly using molecular detection methods (table 1) . young hospitalized children have been best studied, and hmpv has been generally found in 5%-10% of arti cases in that population. however, in one study from italy, the number of hmpv infections varied widely over a 3-year period and were associated with 7%-43% of hospitalizations for arti [20] . the relative role of hmpv in respiratory syndromes of adults has been less studied. in one study from rochester, new york, hmpv was detected by serological tests and/or rt-pcr in 4.5% of young and elderly adults with arti [21] . interestingly, evidence of hmpv infection was also found by serological testing in almost the same percentage (4.1%) in asymptomatic adults, raising the question of the causative role of hmpv in arti in adults. recent studies by our group indicate that hmpv is rarely identified by rt-pcr in npas obtained from asymptomatic young children, with a rate significantly lower than that of subjects with arti (!1% vs. 6%) [19, 22] . it is interesting to note that the rates of detection of hmpv have been generally higher in retrospective than prospective studies (table 1) , an observation consistent with some selection bias. thus, to better define the role of hmpv in various respiratory conditions, large prospective studies using appropriate controls need to be conducted. in addition, testing of all clinical samples (i.e., not only those samples that are found to be negative for other viruses) must be performed. nevertheless, most studies have so far indicated that hmpv is a frequent cause of severe arti, especially in young children. table 2 summarizes the clinical findings of the first 28 hmpvinfected canadian subjects retrospectively identified through our virology laboratory. symptoms of both upper and lower respiratory tract infections have been associated with hmpv in young children, although most reports are biased towards description of the most severe symptomatology in hospitalized subjects. in young hospitalized children, the clinical features associated with hmpv infections are very similar to those of hrsv [19, [23] [24] [25] . diagnoses of bronchiolitis, with or without pneumonitis, have been most commonly reported. a substantial proportion (up to 50%) of infected children also has concomitant otitis media [19] . acute wheezing and asthma exacerbation have been associated with hmpv in some [23, 26] but not all [27] studies. compared with hrsv infections, hmpv cases tend to occur at an older age [19, 26, 28] . the clinical outcome after hrsv infection has been more severe than that after hmpv infection when looking at the proportion of patients with hypoxemia, those with pneumonia, and those admitted to the intensive care unit in studies from canada [19] and europe [25, 28] . in contrast, researchers from hong kong found that hmpv infection was associated with a longer hospital stay and more cases of pneumonia than was hrsv infection [26] . the latter group reported an estimate of 442 hmpvassociated hospitalizations per 100,000 children aged р6 years. children with underlying medical conditions may have moresevere hmpv disease, leading to hospitalization. in studies from north america, 25%-33% of hmpv cases occurred in children with underlying conditions, such as prematurity, cardiopulmonary problems, and immunosuppression [7, 29] . hmpv has been associated with flulike illnesses and colds in healthy adults (table 2) [7] . stockton et al. [30] identified hmpv in 9 (2.2%) of 408 nasal and throat swabs obtained from subjects with flulike illnesses (i.e., patients who tested negative for influenza and hrsv rna) seen by general practitioners in england. eight of the 9 infected patients in that study were adults, and 6 of these patients also had clinical evidence of lower respiratory tract involvement. falsey et al. [21] found a higher rate of hmpv illness among young adults, although older adults experienced more dyspnea and wheezing than did younger adults, and those with cardiopulmonary conditions were ill for nearly twice as long as younger adults. among 10 retrospectively identified, infected patients aged 165 years who were hospitalized at our institution, 4 developed pneumonitis and 2 died (table 2) [7] . of note, all 10 patients had у1 underlying illness, such as leukemia/lymphoma and chronic cardiovascular, pulmonary, or neurologic diseases. collectively, available data in-dicate that the clinical presentation of hmpv is very similar to that of hrsv, with more severe outcomes in infants, elderly subjects, and persons with underlying diseases [31] . the detection of hmpv in special settings merits further discussion. preliminary data suggest that, similar to hrsv, hmpv infection may have a more fulminant course in severely immunocompromised individuals. our group reported the death of a 17-month-old girl with acute lymphoblastic leukemia due to severe pneumonitis and respiratory insufficiency [18] . although no autopsy was performed, an npa obtained before death revealed the presence of hmpv only. of interest, this child had presented with a first episode of bronchiolitis caused by hmpv 1 year earlier, and the 2 viral isolates were of different lineages (groups), highlighting the possibility of rapid reinfections in immunocompromised hosts. similarly, hmpv was the sole pathogen identified in the npa obtained from a hematopoietic stem cell transplant recipient who died of progressive respiratory failure after an upper respiratory prodrome [32] . the presence of a copathogen may also lead to more-severe hmpv infections. a report from england identified hmpv in 21 (70%) of 30 bronchoalveolar fluid specimens obtained from infants with severe hrsv bronchiolitis requiring ventilatory support, raising the possibility that hmpv might be a determinant of hrsv disease severity [33] . unfortunately, no data regarding dual infections in less severe hrsv disease were available for comparison. in contrast, the rate of bronchopneumonia was not altered by the presence of a second respiratory virus in italian infants with hmpv infection [20] . finally, the possible synergistic effect between hmpv and a new coronavirus has been recently postulated during outbreaks of severe acute respiratory syndrome (sars) in canada and hong kong [34, 35] . in a study of 48 patients with probable sars in hong kong, chan et al. [34] found evidence of hmpv infections by nested pcr in 25 cell cultures (52%) inoculated with npa samples. in fact, hmpv was more frequently isolated than the sars coronavirus in the latter study. this report is surprising, considering the results of experimental infections in macaques, in which severe multifocal pulmonary consoli-dation was induced by the sars coronavirus only, with no exacerbation after subsequent infection with hmpv [36] . hmpv growth in cell culture is fastidious; this may be one reason for its late identification. most studies have reported reliable cytopathic effects only in tertiary monkey kidney or llc-mk2 cells [4, 6, 7] . the cytopathic effect is variable, with some strains inducing hrsv-like syncytia formation and others inducing focal rounding and cell destruction ( figure 3) . typically, the cytopathic effect is displayed more than 10-14 days after inoculation (mean time was 17 days in our laboratory). confirmation of hmpv cytopathic effect is achieved by rt-pcr testing of infected supernatants in the absence of commercially available antibodies. a recent report by chan et al. [34] indicated that hmpv could replicate more efficiently in human laryngeal carcinoma (hep-2) cells, despite the absence of cytopathic effect. in that instance, presence of hmpv was confirmed by direct testing of hep-2 cell culture supernatants by rt-pcr or subsequent passage on llc-mk2 cells to observe cytopathic effect. because of the unavailability of rapid antigen detection tests and because of the slow and restrictive viral growth, rt-pcr has become the method of choice for the diagnosis of acute hmpv infection. most pcr protocols reported to date have relied on amplification of the l, n, or f gene (reviewed in [37] ), with primer sequences mainly derived from the prototype strain 001 from the netherlands (genbank accession number af371337). because of the existence of 2 hmpv lineages with significant genetic variability within each group, hmpv detection may be underestimated when inadequate primers are selected for pcr amplification. we have recently reported that rt-pcr assays targeting the n and l genes, which code for 2 internal viral proteins, are best suitable for hmpv diagnosis [37] . rapid and sensitive hmpv assays based on the real-time pcr methodology have recently been described, allowing amplification and detection of this pathogen in р2 h [37, 38] . serological testing only permits a retrospective diagnosis. because infection is almost universal in childhood, a seroconversion or a у4-fold increase in antibody titers must be demonstrated to confirm recent infection. the few serological surveys for hmpv were based on an indirect immunofluorescence assay using hmpv-infected cells [4, 16] . a home-brew elisa method has also been developed using cell lysates of hmpv [21] . clearly, simpler elisa tests using viral proteins possibly derived from the 2 main groups are needed to conduct large serological surveys in many parts of the world. no vaccines, chemotherapeutic agents, or antibody preparations are currently approved for the prevention or treatment of hmpv infection. recently, wyde et al. [39] showed that ribavirin and a polyclonal intravenous immunoglobulin (ivig) preparation had equivalent in vitro activity against hmpv and hrsv. in contrast, no activity against hmpv was conferred by palivizumab, a humanized monoclonal antibody directed against the f protein of hrsv. despite the unavailability of animal studies and the toxicity related to ribavirin administration, the combined use of ivig and ribavirin could be envisaged for treating severe hmpv infection in immunocompromised patients, as has been reported for hrsv infection [40, 41] . furthermore, until an effective vaccine is developed, another interesting strategy could consist in the development of hightitered ivig preparations with activity against hmpv. the recent identification of a presumably old viral pathogen is an exciting development in the field of respiratory viruses. available data indicate that hmpv appears to be a significant cause of both upper and lower respiratory tract infections in young children. hmpv reinfections seem to be frequent and could also lead to devastating complications in elderly subjects and immunocompromised hosts, although more studies with adequate control groups are needed to confirm this. overall, the epidemiological and clinical features of hmpv infection appear to be similar to those of hrsv, although differences have been noted. it is important to mention that many fun-damental questions related to viral pathogenesis and the host's specific immune response still remain to be answered. the tucson children's respiratory study ii: lower respiratory tract illness in the first year of life etiology of community-acquired pneumonia: impact of age, comorbidity, and severity prospective comparative study of viral, bacterial and atypical organisms identified in pneumonia and bronchiolitis in hospitalized canadian infants. pediatr a newly discovered human pneumovirus isolated from young children with respiratory tract disease metapneumoviruses in birds and humans characterization of human metapneumoviruses isolated from patients in north america virological features and clinical manifestations associated with human metapneumovirus: a new paramyxovirus responsible for acute respiratory-tract infections in all age groups analysis of the genomic sequence of a human metapneumovirus genetic diversity between human metapneumovirus subgroups cloning and sequencing of the matrix protein (m) gene of turkey rhinotracheitis virus reveal a gene order different from that of respiratory syncytial virus respiratory syncytial virus sequence analysis of the n, p, m and f genes of canadian human metapneumovirus strains subgroup c avian metapneumovirus (mpv) and the recently isolated human mpv exhibit a common organization but have extensive sequence divergence in their putative sh and g genes deduced amino acid sequence of the small hydrophobic protein of us avian pneumovirus has greater identity with that of human metapneumovirus than those of non-us avian pneumoviruses human metapneumovirus-associated lower respiratory tract infections among hospitalized human immunodeficiency virus type 1 (hiv-1)-infected and hiv-1-uninfected african infants seroprevalence of human metapneumovirus in japan high seroprevalence of human metapneumovirus among young children in israel respiratory tract reinfections by the new human metapneumovirus in an immunocompromised child human metapneumovirus infections in hospitalized children human metapneumovirus associated with respiratory tract infections in a 3-year study of nasal swabs from infants in italy human metapneumovirus infections in young and elderly adults role of human metapneumovirus and other common respiratory viruses in children's hospitalization for acute respiratory tract infection: a twoyear study using multiplex pcr metapneumovirus and acute wheezing in children presence of the new human metapneumovirus in french children with bronchiolitis high prevalence of human metapneumovirus infection in young children and genetic heterogeneity of the viral isolates children with respiratory disease associated with metapneumovirus in hong kong asthma exacerbations in children associated with rhinovirus but not human metapneumovirus infection prevalence and clinical symptoms of human metapneumovirus infection in hospitalized patients human metapneumovirus infection in the united states: clinical manifestations associated with a newly emerging respiratory infection in children human metapneumovirus as a cause of community-acquired respiratory illness human metapneumovirus infection in the canadian population human metapneumovirus in a haematopoietic stem cell transplant recipient with fatal lower respiratory tract disease human metapneumovirus in severe respiratory syncytial virus bronchiolitis human metapneumovirus detection in patients with severe acute respiratory syndrome identification of severe acute respiratory syndrome in canada koch's postulates fulfilled for sars virus comparative evaluation of real-time pcr assays for detection of the human metapneumovirus molecular assays for detection of human metapneumovirus comparison of the inhibition of human metapneumovirus and respiratory syncytial virus by ribavirin and immune serum globulin in vitro prevention and treatment of respiratory syncytial virus and parainfluenza viruses in immunocompromised patients combination therapy with aerosolized ribavirin and intravenous immunoglobulin for respiratory syncytial virus disease in adult bone marrow transplant recipients evidence of human metapneumovirus in australian children human metapneumovirus and community-acquired respiratory illness in children human metapneumovirus and lower respiratory tract disease in otherwise healthy infants and children key: cord-311275-ysr9nqun authors: chuaychoo, benjamas; ngamwongwan, sopita; kaewnaphan, bualan; athipanyasilp, niracha; horthongkham, navin; kantakamalakul, wannee; muangman, nisa title: clinical manifestations and outcomes of respiratory syncytial virus infection in adult hospitalized patients date: 2019-07-03 journal: j clin virol doi: 10.1016/j.jcv.2019.07.001 sha: doc_id: 311275 cord_uid: ysr9nqun background: respiratory syncytial virus (rsv) is an important virus found in adult hospitalized patients. objectives: to study the clinical outcomes of hospitalized patients aged ≥ 15 years and diagnosed with rsv infection. study design: both retrospective and prospective cohort studies were conducted at a university hospital between may 2014 and december 2015. results: rsv was detected in 86 of 1562(5.5%) adult hospitalized patients suspected of respiratory viral infection. sixty-nine patients were included in the study. rsv was detected by rt-pcr (82.6%), ifa (10.1%), and both rt-pcr and ifa (7.3%). most patients (87.0%) were aged ≥ 50 years. cardiovascular diseases, pulmonary diseases, immunocompromised hosts, and diabetes were the major comorbidities. the common manifestations were cough (92.8%), dyspnea (91.3%), sputum production (87.0%), tachypnea (75.4%), wheezing (73.9%), and fever (71.0%). fiftyfive patients (79.7%) were diagnosed with pneumonia. hypoxemia (spo2 ≤ 92%) was found in 53.6% patients. twenty-five of 69(36.2%) patients developed respiratory failure and required ventilatory support. cardiovascular complications were found in 24.6% of patients. congestive heart failure, acute myocardial infarction (mi), new atrial fibrillation, and supraventricular tachycardia were found in 9(13.0%), 7(10.1%), 4(5.8%), and 3(4.3%) of 69 patients, respectively. overall mortality was 15.9%. pneumonia (81.8%) and acute mi (18.2%) were the major causes of death. conclusions: most adult hospitalized patients with rsv infection were of advanced age and had comorbidities. cardiopulmonary complications were the major causes of death. management and prevention of rsv infection in these vulnerable groups are necessary. respiratory syncytial virus (rsv) is an important cause of acute respiratory infection (ari) in infants and young children [1] [2] [3] . nevertheless, it has been recognized as a cause of adult ari, especially in the elderly, those with comorbidities, and immunocompromised hosts, which leads to hospitalization and increases morbidity and mortality [1, [4] [5] [6] [7] [8] . the prevalence of rsv infection in adults varies from 3% to 13% depending on age groups, underlying diseases, diagnostic techniques, study periods, and geographic regions. [3] [4] [5] [7] [8] [9] , in addition, disease severity ranges from mild to severe acute respiratory illness [4] . a total of 4%-16% of adult patients with rsv infection required hospitalization [3, 4, 10] , with high complications including cardiopulmonary complications and mortality [4, 5, 8, 11] . there were a few data of adult hospitalized patients with rsv infection, with high complications, in thailand; [3, 12, 13] however, additional clinical data are required for planning patient management and also disease prevention in this region. the objective of the study was to determine the clinical manifestations and outcomes of rsv infection in adult hospitalized patients. immunocompromised patients were defined as patients who received chemotherapy, corticosteroids > 20 mg/day prednisolone equivalent, hematologic malignancy, or hiv infection. pneumonia was diagnosed if patients had fever, cough, and dyspnea with new pulmonary infiltrates on chest radiography. acute bronchitis was defined as an acute respiratory infection manifested predominantly by cough without pneumonia, common cold, or exacerbation of asthma or copd [14] . chest radiography was independently interpreted by two chest radiologists who did not know the clinical course of patients. if the results were discordant, the consensus was made after the discussion. hospital-acquired pneumonia (hap) was defined as pneumonia that developed 48 h or more after admission without endotracheal intubation. ventilator-acquired pneumonia (vap) was defined as pneumonia that developed more than 48 h after endotracheal intubation [15, 16] . copd exacerbation was defined as a sustained worsening of the copd patients' condition beyond normal day-to-day variations, acute onset and necessitates a change in regular medication [17] . asthma exacerbation was defined by changes in symptoms and rescue use, which were outside the patients' usual range of day-to-day variation [18] . worsening or new congestive heart failure were diagnosed by cardiologists based on clinical signs, laboratory investigation such as chest radiography and echocardiography. the pasw statistics 18.0 (spss inc., chicago, il, usa) was used for the analysis. categorical data were described as percentages. normally distributed continuous data were presented as mean and standard deviation (sd), whereas non-normally distributed data were presented as median and interquartile range (iqr). categorical and continuous variables were compared between groups using chi-square test or fisher's exact test and unpaired t-test or mann-whitney test as appropriate. multiple logistic regression analysis was performed to identify the risk factors of complications and were presented as odds ratio and 95% confidence interval. a p-value of < 0.05 was considered a statistically significant difference. respiratory specimen of 1562 adult hospitalized patients suspected of respiratory viral infection were sent to detect rsv during may 2014 and december 2015. rsv was detected in 86 (5.5%) of patients in 5 months a year between july and november during two consecutive rainy seasons (fig. 1 ). sixty-nine rsv positive patients provided written informed consent and were included in the study. rsv was detected by rt-pcr (82.6%), ifa (10.1%) and both rt-pcr and ifa (7.3%). clinical outcomes in terms of acute respiratory illnesses (ari) and mortality are demonstrated in fig. 2 . community-acquired and nosocomial-acquired rsv infections were found in 57 (82.6%) and 12 (17.4%) patients, respectively. twenty-one of 57 (36.8%) community-acquired rsv infected patients had a history of contact with persons having acute respiratory tract infection in their families. the nosocomial-acquired infections were detected during outbreak, 5 patients at hematologic ward, and 7 patients at general medical ward. pneumonia was the most common ari in both groups. mortality rates of the community-acquired and nosocomial-acquired rsv infections were 15.8% (9 of 57) and 16.7% (2 of 12), respectively. the clinical manifestations of community-acquired and nosocomial-acquired rsv infections were similar, and the data were combined for analysis. the median age of patients was 72 years (iqr 58-81 years) (table1). sixty of 69 (87.0%) patients aged ≥ 50 years with the highest prevalence (36.2%) of age 65-79 years. among patients aged < 50 years, 7 of 9 (77.8%) were immunocompromised hosts (3 hematologic malignancy, 2 hematopoietic stem cell transplant recipients, and 2 connective tissue diseases on immunosuppressive drugs). females were the predominant. all patients had at least one comorbidity. the most common comorbidities were pre-existing cardiovascular diseases (33.3%), pulmonary diseases (29.0%), immunocompromised hosts (29.0%), diabetes (29.0%), and chronic kidney diseases (26.1%). the median duration of symptoms at presentation was 3days (iqr 2-3.5days). the common presenting symptoms ( table 2) were cough (92.8%), dyspnea (91.3%), sputum production (87.0%), and history of fever (81.2%). rhinorrhea was present in 46.4%of patients. the common initial physical findings were tachypnea (75.4%), wheezing (73.9%),and fever (43.5%). wheezing was found in 69.6% (39 of 56) of patients after excluding asthma or copd. fever (bt ≥ 37.8°c) was foundin approximately 71.0% patients in the first 2 days of admission with mean temperature 38.7 ± 0.6°c. after excluding hematologic malignancy and bacterial co-infection, the median white blood counts were 7995 cells/mm 3 (iqr 5,632-10,842 cells/mm 3 ). median neutrophil and lymphocyte counts were 72.6% (iqr 60.0-84.2 %) and 16 .6% (iqr 9.0-24.5%), respectively. chest x-rays were performed for all patients. pneumonia and acute bronchitis were diagnosed in 79.7% (55/69) and 17.4% (12/69) patients, respectively (fig. 2) . chest radiography findings of pneumonia were diffuse interstitial infiltrations alone 60.0% (33/55), mixed diffused interstitial and alveolar infiltrations 30.9% (17/55), and alveolar infiltrations alone 9.1% (5/55). exacerbations were occurred in 9 of 10 asthma and all 3 copd patients. hypoxemia (peripheral oxygen saturation (spo 2 ) ≤ 92%) was found in 26 patients (37.7%) at the initial presentation and increased to 37 patients (53.6%) during the hospitalization. twenty-five of 69 (36.2%) patients developed acute respiratory failure and required ventilatory support (22 patients needed invasive mechanical ventilation and 3 patients required non-invasive ventilation). 2 . summary of clinical outcomes in terms of ari and mortality of community-acquired and nosocomial-acquired rsv infections in adult hospitalized patients. rsv, respiratory syncytial virus; ari, acute respiratory illness; copd, chronic obstructive pulmonary disease; hap, hospital-acquired pneumonia; vap, ventilator-associated pneumonia. seventeen of 69 patients (24.6%) developed cardiovascular complications and 9 of 17 (52.9%) patients had pre-existing cardiovascular diseases (cvd). congestive heart failure (chf), acute myocardial infarction (mi), new atrial fibrillation (af), and supraventricular tachycardia (svt, heart rate 130, 160 and 220 beats/min) were found in 9 (13.0%), 7 (10.1%), 4 (5.8%), and3 (4.3%) of 69 patients, respectively. four of 9 chf patients had worsening chf. among 5 new onset chf patients, 4 patients had pre-existing coronary artery disease and valvular heart disease, and the other one had no previous diagnosis of cvd but had left ventricular ejection fraction (lvef) 33.5% during rsv infection. all acute mi were diagnosed as non-st-elevation mi (nstemi). five of 7 (71.4%) acute mi had concomitant chf. three of 7 (42.8%) acute mi patients were first diagnosed, and all had the risk factors of coronary artery disease (dm, hypertension and dyslipidemia). the pre-existing coronary arterial disease (cad) was the risk factor of overall cardiovascular complications in hospitalized adult patients with rsv infection with odds ratio 6.18, (95% ci 1.18-32.5), p = 0.03, adjusted for age, sex, ht, dlp, dm, pre-existing chf, arrhythmia, and vhd. sixty-five (94.2%) and 51 (73.9%) patients received initial empirical antibiotics and antiviral drugs (oseltamivir), respectively. after confirmed rsv infection, 32 (46.4%) patients received oral ribavirin in cases of respiratory failure that needed mechanical ventilator, hematologic malignancy, immunosuppressive therapy, or hiv infection. mortality rates in patients treated with oral ribavirin compared to those without oral ribavirin were 18.8% (6/32) vs 13.5% (5/37), p-value 0.553. sputum specimens were collected in 54 (78.3%) patients, but only 18 of 54 (33.3%) specimens were acceptable for bacterial culture. bacterial co-infection with rsv identified by sputum culture were found in 6(8.7%) patients, haemophilus influenzae (1), klebsiella pneumoniae (1), pseudomonas aeruginosa (2), acinetobacter baumannii (1), and pasteurella multocida (1). hemoculture for bacteria was performed on 65 patients (94.2%). three patients had concomitant bacteremia, escherichia coli (2), and aeromonas hydrophila (1). one patient with e. coli bacteremia had urinary tract infection, whereas the source of infection including sputum culture was not identified in other two patients. the overall mortality rate was 15.9% (11 of 69 patients). two patients of nosocomial rsv infection had hematologic malignancy with agranulocytosis. they received oral ribavirin, empirical antibiotics, and antifungal drugs; however, rsv persisted and had no evidence of other organisms (bacteria or fungus). nine patients of community-acquired rsv infection were of advanced age (64-95 years) and had comorbidities. pneumonia (9 of 11, 81.8%) and acute mi (2 of 11, 18.2%) were the most and the second common causes of death. most (87.0%) adult hospitalized patients with rsv infection of age ≥ 50 years had comorbidities. rsv is an important pathogen not only in patients of age ≥ 65 years but also in the age range 50-64 years, which was similar to previous studies [9, 19, 20] . nevertheless, we found the highest prevalence in age range 65-79 years (36.2%). the major comorbidities were pre-existing cardiopulmonary diseases, hematologic malignancy, immunocompromised hosts and dm. these findings were similar to the results of previous studies [4, 6, 11, 19, 21] . rsv infection is a seasonal disease [3, 22, 23] . we found rsv infection in the hospitalized patients during rainy seasons, which was similar to the results of a previous study in another region of thailand [3] . the common manifestations were cough, dyspnea, sputum production, fever, wheezing, and tachypnea, similar to the previous studies. [6, 9, 11, 21, 24] nevertheless, nasal congestion and rhinorrhea were not the predominant symptoms in our study. these findings suggested predominant involvement of lower respiratory tract, which might be associated with disease severity. the prevalence of pneumonia and acute respiratory failure, which required ventilatory support, was higher than those in previous studies, 79.7% vs 29%-67.5% and 36.2% vs 3.4%-17.0%, respectively [6, 7, 11, [25] [26] [27] . chest radiologic findings of rsv pneumonia in our study included predominant interstitial infiltrations (60.0% diffuse interstitial infiltrations alone and 30.9% mixed diffused interstitial and alveolar infiltrations). in contrast, those of most previous studies were consolidation or ground-glass opacity in unilateral and basilar in location [6, 11, 24, 28] . however, our radiologic findings were similar to those of a previous study conducted in rural thailand by another research group [12] . cardiovascular complications were found in 24.6% of patients including both worsening and new onset chf, acute mi, new af and svt, and 52.9% of them had pre-existing cardiovascular diseases. the prevalence of cardiovascular complications in adult rsv infection had been reported in 14.3%-22.0% of patients [6, 11] . a risk of overall cardiovascular complications in our study was pre-existing coronary arterial disease (cad) with adjusted odds ratio 6.18, (95% ci 1.18-32.5), p = 0.03. in addition, we found that acute mi was the second cause of death. these supported the importance of cardiovascular complications in adult hospitalized patients with rsv infection. patients with cardiovascular disease have higher rates of health care utilization for rsv-related illness and worse outcomes [29] . most patients received initial empirical antibiotics (94.2%) and oseltamivir (73.9%), which had no effect on rsv. we might reduce the unnecessary uses of antibiotics and anti-influenza drugs, if we knew pathogen earlier. oral ribavirin was used in patients with severe respiratory failure who needed mechanical ventilator and immunocompromised hosts in our observational study; however, the benefit of treatment is not clear. the mortality rate was high (15.9%) compared to other studies (5.8%-11.9%) [4, 6, 21] . this might be because of severe rsv infection, where pneumonia was found in 79.7% of patients. in the adult hospitalized patients with rsv infection, mortality was higher in the elderly with comorbidities and young patients with hematologic malignancy. cardiopulmonary complications such as pneumonia and acute mi were the most and the second causes of death, respectively. high complications and mortality of rsv infection in our study supported the importance of rsv infection in adult hospitalized patients. rsv causing severe complications and high mortality in the elderly similar to or higher than influenza had been reported [7, 11, 30] . adult severe pneumonia can occur in other viruses including human metapneumovirus (hmpv), human rhinovirus, parainfluenza virus, adenovirus, and coronavirus [5, 10, 31] . more studies of respiratory viral infections in adult patients are needed to identify the incidence and the impact of these viruses in thailand. the mechanisms of these severe diseases in the elderly are not clear. however, the experimental studies in aged mice infected with rsv and/or hmpv demonstrated that cd4 + t lymphocytes, the cytokine response, or a defect in humoral response may be associated with the severity in this population [32, 33] . the first limitation of the study is the combination of retrospective and prospective studies, even though most of the patient data were recorded by pulmonologists on duty. the retrospective study might affect the presenting symptoms of patients; however, it should not affect the clinical outcomes including cardiopulmonary complications and mortality. the second limitation is that only the respiratory specimens of patients suspected of respiratory viral infection were sent for the detection of rsv. it may lead to bias in the prevalence and clinical presentations. the prospective study of all adult hospitalized patients with acute respiratory illness should be conducted to determine the prevalence, clinical manifestations, and outcomes of the virus. most of the adult hospitalized patients with rsv infections aged ≥ 50 years old and had pre-existing cardiopulmonary diseases, hematologic malignancy, immunocompromised hosts, and dm. pneumonia and acute mi were the major causes of death. management and preventive strategies of rsv infection in these vulnerable groups in both community-acquired and hospital-acquired infections are necessary. this research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. credit authorship contribution statement respiratory syncytial virusa comprehensive review respiratory syncytial virus-associated hospitalizations among children less than 24 months of age hospitalizations for acute lower respiratory tract infection due to respiratory syncytial virus in thailand respiratory syncytial virus infection in elderly and high-risk adults respiratory syncytial virus and other respiratory viral infections in older adults with moderate to severe influenza-like illness respiratory syncytial virus infection-associated hospitalization in adults: a retrospective cohort study respiratory viral infections among hospitalized adults: experience of a single tertiary healthcare hospital, influenza other respir risk factors for severe respiratory syncytial virus infection in elderly persons medically attended respiratory syncytial virus infections in adults aged &/= 50 years: clinical characteristics and outcomes rates of hospitalizations for respiratory syncytial virus, human metapneumovirus, and influenza virus in older adults high morbidity and mortality in adults hospitalized for respiratory syncytial virus infections the burden of hospitalized lower respiratory tract infection due to respiratory syncytial virus in rural thailand incidence of respiratory pathogens in persons hospitalized with pneumonia in two provinces in thailand diagnosis and management of cough executive summary: accp evidencebased clinical practice guidelines infectious diseases society of a. guidelines for the management of adults with hospital-acquired, ventilator-associated, and healthcareassociated pneumonia the alphabet soup of pneumonia: cap toward a consensus definition for copd exacerbations statement: asthma control and exacerbations: standardizing endpoints for clinical asthma trials and clinical practice respiratory syncytial virus hospitalization in middle-aged and older adults seasonal incidence of medically attended respiratory syncytial virus infection in a community cohort of adults &/=50 years old respiratory syncytial virus-and human metapneumovirus-associated emergency department and hospital burden in adults latitudinal variations in seasonal activity of influenza and respiratory syncytial virus (rsv): a global comparative review respiratory syncytial virus circulation in seven countries with global disease detection regional centers is clinical recognition of respiratory syncytial virus infection in hospitalized elderly and high-risk adults possible? morbidity and mortality among patients with respiratory syncytial virus infection: a 2-year retrospective review risk of mortality associated with respiratory syncytial virus and influenza infection in adults high viral load and respiratory failure in adults hospitalized for respiratory syncytial virus infections initial radiographic features as outcome predictor of adult respiratory syncytial virus respiratory tract infection respiratory syncytial virus and associations with cardiovascular disease in adults severe morbidity and mortality associated with respiratory syncytial virus versus influenza infection in hospitalized older adults pneumonia severity index in viral community acquired pneumonia in adults age-associated aggravation of clinical disease after primary metapneumovirus infection of balb/c mice respiratory infections by hmpv and rsv are clinically indistinguishable but induce different host response in aged individuals the authors thank professor khun nanta maranetra and mr. brian rochana for proof reading the article. the authors also thank miss khemajira karaketklang and miss kanokwan rattanasaengloet for review of the statistical analysis. the authors also thank miss pattranan vaidyakula for the data recording. key: cord-284858-hrljsc8l authors: cui, shu-juan; shi, wei-xian; huang, fang; pang, xing-huo; deng, ying title: co-infection cases of human common respiratory viruses in beijing, 2010–2012 date: 2013-02-27 journal: braz j infect dis doi: 10.1016/j.bjid.2012.08.026 sha: doc_id: 284858 cord_uid: hrljsc8l nan in the previous report, human co-infection cases of at least two respiratory viruses appeared as a rare event in symptomatic patients. 1, 2 however, at present the occurrence of multiple cases emerges as an increasing trend. 3, 4 in this study, from apr 2010 to mar 2012, the prevalence of human respiratory viruses every month was monitored for determining the aetiological role and epidemiological profile of every common respiratory virus among outpatients with acute respiratory tract infections (artis) in beijing. on the other hand, we put in order to include potential viral artis and to exclude typical bacterial infections, patients enrolled in the study were selected by physicians according to the following criteria: with respiratory symptoms such as cough or wheezing, acute fever (body temperature ≥ 38 • c), and normal or low leukocyte count, with or without radiological pulmonary abnormalities. a total of 1791 throat swabs were collected and tested. the information of the subject's age, gender and presence of underlying diseases was collected during a face-to-face interview. multiple pcr diagnostic kit for simultaneously detecting 12 pathogens of respiratory viruses (hangzhou neuro-hemin biotech co., ltd., china) was used to confirm the pathogen. the clinical features of the disease were closely monitored and summarized. epidata software was used to store the dataset. the overall positive rate of single respiratory virus in 1791 samples was 23.17%. among the co-infected cases, multiple (≥2) respiratory viruses were observed in 31 cases (31/1791, 1.73%). dual infections were present in 27 patients (27/1791, 1.51%), from which adenoviruses (advs) were co-detected in 9 cases, influenza viruses a (ifv a) in 7 cases, respiratory syncytial virus (rsv) in 5 cases, parainfluenza viruses (pivs) in 4 cases and human metapneumovirus (hmpv) in 2 cases, and triple infections in 4 cases (4/1791, 0.22%). all co-infection cases are summarized in table 1 . multivariable logistic-regression analysis identified independent risk factors for co-infection in relation to single viral infection: age of more than 50 years (odds ratio, 1.29; 95% confidence interval [ci], 1.08 to 1.57; p = 0.031), and having an underlying disease (odds ratio, 1.67; 95% confidence interval [ci], 1.44 to 2.01; p = 0.022). these data suggest that being more than 50 years and having and underlying disease are independent risk factors associated with co-infection among outpatients with acute respiratory tract infections (artis) in beijing. the widespread prevalence of respiratory viral co-infection in the elderly was higher than that in other age groups, and cases with underlying diseases. this survey provides some evidence for respiratory viral coinfection and the risk factors among outpatients with artis. the clinical significance of such co-detection is unclear. 5 multiple viruses add to the complexity of the clinical symptoms making the clinical diagnosis more difficult. further research is warranted among co-infected cases throughout other regions of china in order to fully understand risk factors among outpatients in china. dual infections by influenza a/h3n2 and b viruses and by influenza a/h3n2 and a/h1n1 viruses during winter 2007, corsica island respiratory viral coinfection among hospitalized patients with h1n1 2009 during the first pandemic wave in brazil prevalence of human respiratory viruses in adults with acute respiratory tract infections in beijing childhood respiratory viruses in public health care centers rate and influence of respiratory virus co-infection on pandemic (h1n1) influenza disease xing-huo pang, ying deng * institute for infectious disease and endemic disease control we would like to thank prof. jianwei wang for total guidance. this study was funded by two r e f e r e n c e s all authors declare to have no conflict of interest. key: cord-303606-ypkia5x1 authors: lee, so-lun; chiu, shui-seng susan; malik, peiris joseph s.; chan, kwok-hung; wong, hing-sang wilfred; lau, yu-lung title: is respiratory viral infection really an important trigger of asthma exacerbations in children? date: 2011-03-30 journal: eur j pediatr doi: 10.1007/s00431-011-1446-1 sha: doc_id: 303606 cord_uid: ypkia5x1 we performed a prospective cohort study from september 2003 to december 2004 to delineate attributing the effect of different respiratory viral infections including newly discovered ones to asthma exacerbations in children in hong kong. one hundred and fourteen children aged 6–14 years with chronic stable asthma and on regular inhaled steroid were monitored for respiratory symptoms over a full calendar year from recruitment. they would attend the study clinic if peak expiratory flow rate decreased to below 80% of their baselines, if they met a predefined symptom score, or if parents subjectively felt them developing a cold. virological diagnosis using virus culture, antigen detection, and polymerase chain reaction methods on nasal swab specimens would be attempted for all these visits irrespective of triggers. physician diagnosed outcome of each episode was documented. three hundred and five episodes of respiratory illnesses were captured in the cohort. nasal specimens were available in 166 episodes, 92 of which were diagnosed as asthma exacerbations, and 74 non-asthma related episodes. respiratory viruses were detected in 61 of 166 episodes (36.7%). there was no significant difference in virus detection rate between asthma exacerbations (32 out of 97 episodes, 34.8%) and non-asthma respiratory illnesses (29 out of 79 episodes, 39.2%). although newly discovered respiratory viruses were identified in these episodes, rhinovirus was the commonest organism associated with both asthma exacerbations and non-asthma related episodes. plausible explanations for much lower virus detection rate than previously reported include improved personal hygiene and precautionary measures taken during respiratory tract infections in the immediate post-severe acute respiratory syndrome period together with a significant contribution of other adverse factors like environmental air pollution. we conclude that not all viral infections in children with asthma lead to an asthma exacerbation and the attributing effect of different triggers of asthma exacerbations in children vary across different time periods and across different localities. episode was documented. three hundred and five episodes of respiratory illnesses were captured in the cohort. nasal specimens were available in 166 episodes, 92 of which were diagnosed as asthma exacerbations, and 74 nonasthma related episodes. respiratory viruses were detected in 61 of 166 episodes (36.7%). there was no significant difference in virus detection rate between asthma exacerbations (32 out of 97 episodes, 34.8%) and non-asthma respiratory illnesses (29 out of 79 episodes, 39.2%). although newly discovered respiratory viruses were identified in these episodes, rhinovirus was the commonest organism associated with both asthma exacerbations and non-asthma related episodes. plausible explanations for much lower virus detection rate than previously reported include improved personal hygiene and precautionary measures taken during respiratory tract infections in the immediate post-severe acute respiratory syndrome period together with a significant contribution of other adverse factors like environmental air pollution. we conclude that not all viral infections in children with asthma lead to an asthma exacerbation and the attributing effect of different triggers of asthma exacerbations in children vary across different time periods and across different localities. viral respiratory tract infection, with rhinovirus accounted for two thirds, was found to be associated with greater than 80% of asthma exacerbations in children in studies in the 1990s [14] . however, the prevalence of respiratory viral infection varies greatly across different places; for example, influenza is associated with more hospitalization among children in hong kong compared with temperate region [7] . recent observational studies have shown that influenza infection can be associated with asthma exacerbations. nevertheless, a metaanalysis failed to support the protective effect of influenza vaccination in asthma exacerbations [3] . newly discovered respiratory viruses such as human metapneumovirus may also play a role [20] . triggers other than respiratory tract infection, like air pollutions may become more prevalent over time and supersede respiratory tract infections as a major trigger. thus, we carried out a prospective study to delineate the current role of different viral respiratory tract infections including newly discovered respiratory viruses in asthma exacerbation in children in our locality. children aged 6-14 years who attended regular follow-up at the asthma clinic were invited to participate. children with physician-diagnosed asthma, symptoms of asthma in the preceding year, no hospital admission for exacerbation, and on regular inhaled steroid equivalent to beclomethasone ≤400 μg daily for at least 3 months prior to enrolment were recruited. exclusion criteria were those with other known chronic respiratory disease and oral steroid therapy given within 4 weeks of enrolment. the participants were followed up to cover a full calendar year to reduce potential biases associated with temporal and age-related differences in respiratory tract infections. patient's demographic data, treatment at enrolment, family history of atopy and asthma, and exposure to environmental tobacco smoke were recorded at the time of recruitment. measurement of lung function using vitalograph model 2120 was obtained according to american thoracic society recommendations in children who were able to perform spirometry [1] . skin prick test was done according to the standardized international study of asthma and allergies in childhood phase 2 protocol [12] . ten aeroallergens including dermatophagoides pteronyssinus, dermatophagoides farinae, cat, alternaria tenuis, mixed tree pollen, mixed grass pollen, dog, cockroach (american and german), and mixed moulds were tested. a wheal diameter of 3 mm greater than the diameter of the negative control was defined as positive response. children with ≥1 positive responses were defined as atopic. each child was given an asthma diary chart and a peak flow meter (mini-wright afs low range peak flow meter) during the run-in period. parents and children were taught on data entry, use of peak flow meter, and record twice daily peak expiratory flow rate (pefr) and any upper and lower respiratory symptoms for 2 weeks as baseline. [14] (appendix). the diary chart was then reviewed and the child's calculated 80% baseline pefr was recorded on a new log sheet. parents were instructed to start to record pefr twice daily and respiratory symptoms in a new log sheet when the symptoms scored >3. they were to call the research nurse if pefr fell to below 80% of the child's baseline, if total upper or lower respiratory symptom score totalled ≥4, or if parents subjectively felt the child was developing a cold even though pefr fell by <20% of baseline. an unscheduled clinic visit would be arranged within 48 h. during the unscheduled visit, upper and lower respiratory symptoms and physical signs were recorded. an asthma exacerbation was defined as a fall in morning pefr to below 80% of baseline in the absence of expiratory wheeze for ≥2 two consecutive days. the presence of wheeze detected by the attending paediatrician at the time of visit (lee sl/chiu ss), or an increase in the use of shortacting beta 2 agonists on at least two occasions per day for ≥2 consecutive days. diagnoses other than asthma exacerbation were also captured. chest radiograph were ordered if clinically indicated. respiratory secretions from children were obtained using nasal swabs. the cotton-tipped swab was inserted into the nostril for 2 to 3 cm and rotated three times against the respiratory epithelial surface of the nasal cavity. once collected, the specimen was put in a virus transport medium and immediately transported to the microbiology laboratory for processing. the child was treated as appropriate. the parents and child continued to record daily pefr and symptoms in the subsequent 2 weeks or longer until symptoms subsided completely. follow-up visits would be arranged. all participants also attended scheduled clinic visit every 3 months. at each scheduled visit, all respiratory symptoms at follow-up or any respiratory problems in between visits that were not reported would be recorded. part of the aliquot was used for routine detection of viral antigen using immunofluorescence (if) detection of viral antigens for five respiratory viruses, viz. influenza viruses types a & b, respiratory syncytial virus (rsv), parainfluenza virus, and adenovirus. it was also cultured for virus isolation. the remaining aliquot was used for polymerase chain reaction (pcr) detection of rhinovirus, human metapneumonvirus, human coronavirus nl 63, oc43, 229e, hku1, and bocavirus. [6, 8, 19, 21] ethical approval the study was approved by the institutional review board of the university of hong kong/hospital authority hong kong west cluster. the study was conducted in accordance with the declaration of helsinki. verbal consent was obtained from the participants and written consent was obtained from their parents or legal guardians. previous studies showed virus identification rate ranged from 32% to 85% of asthma exacerbation in children [14, 19] . the conservative estimation of virus detection rate of 50% would give the largest sample size estimate of 96 exacerbations with level of confidence at 95% and precision of detection rate of 10%. as the number of urgent visits due to asthma was 1.2 per person-year in children on regular inhaled steroids in another study, the number of subjects required would be around 80 [22] . we performed simple descriptive analyses of demographic data. the frequencies of presenting symptoms and physician diagnoses of unscheduled visits, virus detection rate and the distribution of different types of viruses were described. student t test (+− mann-whitney u test) was used to compare continuous variables; for example, age and pearson's chi-square test (with yates'correction/fisher's exact test) was used to compare categorical variables; for example, sex (female or male), atopic status (yes or no) between children with and without unscheduled visits. a p value less than 0.05 was considered to be statistically significant. all statistical analyses were carried out by the spss 11.0 software (spss inc., chicago, il). there were totally 122 participants recruited from the end of september to the end of december 2003 and were followed up until the end of december 2004. eight of these participants withdrew early as their parents found it inconvenient to attend unscheduled visit. one hundred and fourteen children aged 6 to 13 years completed the study. they were followed up for 12 to 15 months. their baseline characteristics were tabulated in table 1 . among these 114 children, 16 children (14.0 %) did not report any exacerbations or respiratory illnesses. children with respiratory illnesses were younger than children without respiratory illnesses (p<0.05) and there was greater proportion with normal pulmonary lung function test at the time of recruitment (p=0.02). fifteen children had reported 20 episodes of mild respiratory illness with symptoms with remaining 83 children had experienced ≥1 episode of respiratory illnesses with symptoms score >3 and the maximum number of episodes per children was seven in two children. there were a total of 211 episodes with a symptom score >3. nasal swab specimens were obtained in 166 and the interval between onset of respiratory symptoms and nasal swab collection ranged from 0.5 to 6 days. nasal swab specimens were not available in the remaining 45 episodes as the children attended general practitioner (gp) instead. there were 74 episodes of mild respiratory illnesses with symptom score ≤3 reported in these 83 children that were also managed by gp. the distribution of these episodes of respiratory illnesses among the children was illustrated in fig. 1 . thus, there were a total of 305 episodes of respiratory illnesses including asthma and non-asthma related episodes in our study cohort over the 14-month study period. the mean number of asthma exacerbations, other respiratory illnesses, and all episodes as diagnosed at unscheduled visits were 0.69, 1.6, and 2.29 per person-year, respectively. the presenting symptoms of 166 episodes of unscheduled visits with nasal swab specimens obtained are tabulated in table 2 . ninety-two episodes were diagnosed as asthma exacerbations and 74 non-asthma related. among 92 episodes of asthma exacerbations, physician also made a diagnosis of concomitant respiratory tract infection in 69 (59 with upper respiratory tract infection, 5 with lower respiratory tract infection, and 5 with sinusitis) of these episodes based on history and physical findings. respiratory viruses were detected in 61 of these 166 episodes (36.7 %) ( table 3 ). there was no significant difference in virus detection rate between asthma (32 out of 97 episodes, 34.8 %) and non-asthma related episodes (29 out of 74 episodes, 39.2 %). rhinovirus was detected in 41 episodes, influenza in 7, coronavirus in 6, parainfluenza virus in 2, rsv in 1, and mixed viruses in the remaining 4. the patterns of distribution of respiratory viruses were quite similar in asthma exacerbations and non-asthma related episodes. (table 4 ) a community study carried out in southampton, uk over a decade ago found viral infections in >80% of asthma exacerbations in 9-11-year-old children [14] . our virus detection rate was only 36.7 % of all unscheduled sick visit in children aged 6 to 14 years old and the rate was not significantly different between asthma exacerbations (34.8 %) and that of other diagnoses (39.2 %). this low detection rate was not due to inadequate power based on a priori sample size calculation. neither was it due to virus detection method as we included pcr detection of more scores ≤3 that did not warrant unscheduled visits. the recently discovered respiratory viruses including human metapneumonvirus, human coronavirus nl 63, oc43, 229e, hku1, and bocavirus in addition to the virus detection method adopted in the southampton study, i.e., using if detection of viral antigens and culture for five respiratory viruses, viz. influenza viruses types a & b, rsv, parainfluenza virus, and adenovirus and pcr for detection of rhinovirus. our previous study using pcr method in detecting rhinovirus was shown to be comparable to the global literature [5] . our result was comparable to a clinic-based prospective study [19] and more closely matched to the canadian case-control study conducted in september 2001 before the epidemics of severe acute respiratory syndrome (sars) in which 62% of asthma children attending emergency department for exacerbations had respiratory viruses isolated [13] . we offered several explanations for our findings. firstly, our study was carried out in the immediate post-sars period when the population was still highly cautious about infection control and practising good personal hygiene [15] . this could greatly reduce common viral infection. the low average number of unscheduled sick visits per person-year compared to previous epidemiology studies [18] inferred excellent general health status in our children population over the study period. the beneficial effect of improved community hygienic measures was also supported by a local study which showed significantly lower respiratory virus circulation in the community in the immediate post-sars period [17] . secondly, we used three criteria so as to capture as many as possible asthma exacerbations. the use of a symptom score that included both upper and lower airway symptoms and that parents could attend the study clinic if they subjectively felt that the child develop a cold were less stringent than the first criteria of a fall of 80% of baseline pefr. thus, 74 of 166 unscheduled visits were not asthma exacerbations. we might have overdiagnosed respiratory tract infection as a trigger in [16] . one other possibility is that all of our participants had better control of asthma symptoms that was reflected in the lower overall number of episodes of unscheduled sick visits per person time year compared to the southampton cohort. we speculated that the use of regular inhaled steroids might also have some effect. while most clinical or experimental studies failed to document the efficacy of inhaled steroids in preventing intermittent virus-induced asthma exacerbations [9, 10] , the aforementioned canadian case-control study [5] showed that children attending emergency department for asthma exacerbations were more likely to have respiratory viruses isolated but less likely to have prescription of anti-inflammatory medications. as in previous studies, rhinovirus was also the most frequent organism detected, accounting for followed by influenza among those asthma exacerbations with virus isolated in our cohort. we must address our limitations. firstly, nasal swab were not collected in 45 out of 211 of episodes that met the criteria for unscheduled visits. for the extreme case scenario whereby all these 45 episodes were asthma exacerbations with viruses isolated, the virus detection rate would have been 56.2 % but this is still much lower than southampton study. for the remaining 94 episodes of mild respiratory illnesses that did not meet the criteria for unscheduled visit, it was difficult to ascertain whether the subjects actually had very mild asthma exacerbations, leading to a possible underestimation. the second limitation was that nasal swab instead of nasopharyngeal aspirate was used for infection control reasons as the study was carried out in the immediate post-sars period. a recent study also showed that the sensitivity of nasal swabs was comparable to that of nasopharyngeal aspirates for the detection of all major respiratory viruses except rsv [11] . yet, rsv virus is not a common trigger of asthma exacerbations in school-aged children. the use of a flocked nasopharyngeal swab, which was not available at time of the study, can certainly lead to a better yield should similar study to be conducted in the future [4] . we found that viral infections accounted for about 35% of asthma exacerbations and 39% of non-asthma associated respiratory illnesses in children with stable asthma control during the immediate post-sars period. we did not negate the well-established causal relationship between respiratory viral infections and asthma exacerbations. rather, our study suggested that the improved personal hygiene and precautionary measures taken during respiratory tract infections may help to reduce the potential adverse effect at high risk groups, like children with asthma. in addition, factors like environmental air pollution may also contribute significantly to morbidity of children with asthma in locality where the problem is particularly adverse. we conclude that not all viral infections in children with asthma lead to an asthma exacerbation and the attributing effect of different triggers of asthma exacerbations in children varies across different time periods and across different localities. updated local data whenever available are preferred when planning for health care policies. standardization of spirometry, 1994 update global strategy for asthma management and prevention: gina executive summary vaccines for preventing influenza in people with asthma comparison of nasopharyngeal flocked swabs and aspirates for rapid diagnosis of respiratory viruses in children rhinovirus infection in hospitalized children in hong kong: a prospective study human coronavirus nl63 infection and other coronavirus infections in children hospitalized with acute respiratory disease in hong kong influenza-related hospitalizations among children in hong kong the association of newly identified respiratory viruses with lower respiratory tract infections in korean children effect of inhaled corticosteroids on episodes of wheezing associated with viral infection in school age children: randomised double blind placebo controlled trial rhinovirus-induced airway inflammation in asthma: effect of treatment with inhaled corticosteroids before and during experimental infection nasal swab versus nasopharyngeal aspirate for isolation of respiratory viruses phase ii modules of the international study of asthma and allergies in childhood (isaac) the september epidemic of asthma exacerbations in children: a search for etiology community study of role of viral infections in exacerbations of asthma in 9-11 year old children impacts of sars on health-seeking behaviors in general population in hong kong association between air pollution and asthma admission among children in hong kong respiratory infections during sars outbreak epidemiology of viral respiratory infections viruses as precipitants of asthma symptoms children with respiratory disease associated with metapneumovirus in hong kong early detection of acute rhinovirus infections by a rapid reverse transcription-pcr assay the childhood asthma management program research group (2000) long-term effects of budesonide or nedocromil in children with asthma acknowledgment lee sl and chiu ss participated in study design, conduct study, data analysis, and report writing. peiris js provided advice on identification of viruses in addition to study design and report writing. chan kh provided laboratory support in identification of viruses and report writing. wong whs provided statistical support. lau yl participated in study design, data analysis, and report writing. we thank our research assistants miss winnie lau and miss eunice chan, and we are also grateful to our study subjects and their parents for their support. conflict of interests all authors declare that they have no conflicts to declare.open access this article is distributed under the terms of the creative commons attribution noncommercial license which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited. what is already known on this topic viral respiratory tract infection is an important trigger of asthma exacerbations in children.rhinovirus is the most common identified virus associated with asthma exacerbations. prevalence of triggers of asthma exacerbations in children varies with geographic locations and time period.with improved vigilance in personal hygiene after the sars period, viral respiratory tract infection became less prevalent as a trigger of asthma exacerbation in children in a polluted city like hong kong.yet, rhinovirus is still the most common identified virus associated with asthma exacerbations despite the emergence of different newly discovered respiratory viruses. key: cord-295189-bz3gi15h authors: jennings, lance c.; priest, patricia c.; psutka, rebecca a.; duncan, alasdair r.; anderson, trevor; mahagamasekera, patalee; strathdee, andrew; baker, michael g. title: respiratory viruses in airline travellers with influenza symptoms: results of an airport screening study date: 2015-03-14 journal: j clin virol doi: 10.1016/j.jcv.2015.03.011 sha: doc_id: 295189 cord_uid: bz3gi15h background: there is very little known about the prevalence and distribution of respiratory viruses, other than influenza, in international air travellers and whether symptom screening would aid in the prediction of which travellers are more likely to be infected with specific respiratory viruses. objectives: in this study, we investigate whether, the use of a respiratory symptom screening tool at the border would aid in predicting which travellers are more likely to be infected with specific respiratory viruses. study design: data were collected from travellers arriving at christchurch international airport, new zealand, during the winter 2008, via a symptom questionnaire, temperature testing, and respiratory sampling. results: respiratory viruses were detected in 342 (26.0%) of 1313 samples obtained from 2714 symptomatic travellers. the most frequently identified viruses were rhinoviruses (128), enteroviruses (77) and influenza b (48). the most frequently reported symptoms were stuffy or runny nose (60%), cough (47%), sore throat (27%) and sneezing (24%). influenza b infections were associated with the highest number of symptoms (mean of 3.4) followed by rhinoviruses (mean of 2.2) and enteroviruses (mean of 1.9). the positive predictive value (ppv) of any symptom for any respiratory virus infection was low at 26%. conclusions: the high prevalence of respiratory virus infections caused by viruses other than influenza in this study, many with overlapping symptotology to influenza, has important implications for any screening strategies for the prediction of influenza in airline travellers. there is very little known about the prevalence and distribution of common respiratory viruses in air travellers. the dissemination of novel human respiratory viruses by air travellers is well established. the introduction of sars into vietnam occurred by a businessman travelling by air from china through hong kong sar [1] . subsequent dissemination from hong kong to singapore, beijing, germany, canada and other countries by air travellers led to outbreaks of infection occurring [2, 3] . since, the first cases of mers-cov were reported in september 2012, limited transmission to european and other countries has occurred by international travelers returning from the middle east [4] . the rapid global spread of the novel influenza a(h1n1) pdm09 virus after first being detected in southern california in late april 2009 was also likely to have been via air travellers [5] . the first identification of the virus in new zealand in april 2009 was in high school students returning by air from mexico [6] . similarly, studies on international travelers arriving in australia in may 2009 [7] and on medical students returning to spain in june 2009, demonstrated outbreaks among the study group and their contacts [8] . while, these and previous reports documenting seasonal influenza among air travelers [2, [9] [10] [11] have focused primarily on the in-flight transmission of influenza, clearly air travellers are responsible for the introduction of influenza viruses into countries on an ongoing basis [12] . there are few reports of the dissemination of other respiratory viruses by air travellers. a mixed outbreak of parainfluenza type 1 and influenza b viruses was reported among tourists returning to the united states [13] , while an investigation of travelers by follin et al. reported the identification of rhinovirus, coronavirus, influenza a and b, parainfluenza virus, adenovirus, metapneumovirus and enterovirus in passengers with influenza-like illness (ili) [14] . with the emergence of the mers-cov, possible introduction by hajj pilgrims with a high rate of respiratory symptoms returning to france have been investigated with no cases identified [15] . in a 2008 study, we sought to assess the prevalence of influenza infection in symptomatic and asymptomatic arriving international airline travellers and whether using a symptom-screening questionnaire and temperature measurement could reliably predict seasonal influenza infection [16] . we tested symptomatic travellers for a range of other respiratory viruses and asked them to report their symptoms. in this study, we describe the spectrum of symptoms associated with infection with respiratory viruses in arriving airline travellers. we ascertain whether, the use of symptom screening at the border would aid in predicting which travellers are more likely to be infected with specific respiratory viruses. this assessment of the prevalence of other respiratory virus infections in arriving airline travellers was carried out at christchurch international airport, new zealand, from 23 june to 12 september 2008. a questionnaire on basic demographics and symptoms was distributed on board three airlines' flights from australia to christchurch, new zealand [17] . all symptomatic travellers (defined as those reporting at least one of cough, sore throat, sneezing, fever or chills, runny or blocked nose, muscle aches or pains, feeling generally unwell, chest discomfort or breathing difficulties) who completed the questionnaire were identified as they arrived at the airport and went through immigration ( fig. 1) , and following informed consent, were asked to provide a nose and throat swab and have their temperature measured. this paper reports on specimen results from these symptomatic travelers. all combined throat and nasal swab samples (copan, italy) were analysed at canterbury health laboratories, christchurch, new zealand. influenza a and b viruses were tested using a commercial easyplex ® multiplexed tandem pcr (mt-pcr) as described by the manufacturer (easyplex ® influenza a + b kit, cat no. 3005.01, ausdiagnostics, sydney, australia). the other respiratory viruses were tested using a similar commercial mt-pcr system (easyplex, respiratory panel 12c, cat no: 6062.1 ausdiagnostics, specifically manufactured for the study). picornaviruses were confirmed as either rhinoviruses or enteroviruses using two in-house singleplex pcr assays [18] [19] [20] . data were entered into microsoft excel and all statistical tests were conducted using stata 11. chi 2 tests were used to identify significant patterns in age or nationality by virus type. influenzalike illness was defined as a measured fever ≥37.8 • c and either a cough or sore throat [21] . for each demographic characteristic and each symptom, the prevalence of infection with each virus among participants with that characteristic was calculated. this is equivalent to the positive predictive value (ppv) of that characteristic for that virus. for participants infected with each virus, the number and proportion with each symptom and the mean number of symptoms were calculated to illustrate the pattern of symptoms associated with each virus. proportions and confidence intervals around means were calculated for groups with more than 10 participants. of 2714 symptomatic travellers, 49% agreed to provide a respiratory sample, 1331 respiratory samples were obtained, of which 1313 were valid and able to be tested for respiratory viruses. table 1 characteristics of study population and infection status a among 1313 symptomatic airline travellers from whom valid respiratory samples were obtained. b three people with a virus named above plus an "other" virus appear only in the named virus column. c ili indicates patient had a temperature ≥37.8 • c plus cough or sore throat. signs and symptoms a column numbers may not add to column total due to missing information. b three people with a virus named above plus an "other" virus appear only in the named virus column. c ili indicates patient had a temperature ≥37.8 • c plus cough or sore throat. forty-nine percent of participants were male and 51% were female, with an age range 0 to 85 years and median of 34 years. most were australians (42%) or new zealanders (40%), with some british (6%) and american (2%) ( table 1) . most study participants (971; 74%) had no detectable respiratory virus. this ranged from 58% in those ≥75 years to 80% in those aged 45-54. a respiratory virus was detected in 342 (26.0%) participants. of these, influenza virus was detected in 55 (4%) and another respiratory virus in 287 (22% the range of symptoms reported and prevalence of respiratory virus infection among participants with each symptom is shown in table 1 , for the more common viruses. although 51 (4%) participants reported fever, only 14 (1%) had a measurable fever at ≥37.8 • c. the most frequently reported symptoms were stuffy or runny nose (60%), cough (47%), sore throat (27%) and sneezing (24%). for individual symptoms, the proportion of symptomatic participants who were infected with any respiratory virus was between 22% (muscle aches and pains) and 43% (self-reported fever). although based on small numbers, this proportion was higher for measured temperature ≥ 37.8 • c (8/14, 57%), and ili (8/13, 62%). of those with ili, 5/13 (38%) were infected with influenza b (table 1) . table 2 shows the pattern of symptoms associated with each identified respiratory virus (other than picornaviruses which could not be confirmed as either an enterovirus or rhinovirus). of the viruses where there were at least 10 cases, influenza b was associated with the highest number of symptoms (mean of 3.4). for study participants with enterovirus infection, the most common symptom was a stuffy or runny nose (69%). this was also the most common symptom reported among those infected with rhinovirus (76%). as well, 52% of those with rhinovirus reported cough. those infected with influenza b most frequently reported cough (85%), stuffy or runny nose (63%), and sore throat (52%). only 10% had a temperature of ≥37.8 • c and 23% reported "fever" subjectively. in this study, during the winter of 2008, 1313 samples were obtained from 2714 symptomatic travellers arriving into christchurch, new zealand on flights from australia, and tested 11 (9) 36 (28) 16 (13) 8 (6) 4 (3) 2 (2) for respiratory viruses. the most frequently identified viruses were rhinoviruses followed by enteroviruses and influenza b viruses. the respiratory symptoms reported by symptomatic travellers during on board screening were diverse with a stuffy nose (60%), cough (47%) and sore throat (27%) being the most common. an aim of this study was to determine whether the use of symptom screening at the border would aid in predicting which travellers are more likely to be infected with a respiratory virus. however, a respiratory virus was detected in only 26.0% of these symptomatic participants sampled, i.e., the positive predictive value (ppv) of 'any symptom' for the prediction of a traveller with infection by 'any respiratory virus' was low at 26%. in this group who had at least one symptom, for individual symptoms associated with the most frequently identified viruses, the ppv for any respiratory virus was low; stuffy nose (31%), cough (29%) and sore throat (33%). ili (>37.8 • c plus cough or sore throat) had the highest ppv (69%); however, infections were largely with influenza virus and the numbers were small. we have previously estimated the prevalence of influenza in all travellers (symptomatic and asymptomatic) during the 'influenza season' period of high prevalence at 1.13% [22] . the ppv for influenza infection of 'any symptom' was 5.5%, and of ili was 24.7%. this study suggests that the use of symptoms as indicators of other respiratory virus infection, as well as influenza infection, in travellers is problematic. the major strengths of this study are the novel study design involving large numbers of arriving international airline travellers and the relatively high proportion (49%) of symptomatic travellers willing to provide respiratory samples for respiratory virus testing [17] . essentially, these were a random sample of passengers, with a similar sex distribution and wide age distribution, although the numbers of samples obtained from children 0 to 15 years and elderly 75+ years was smaller than for all other age groups. this is also one of the few studies where molecular techniques have been applied to the detection of a range of 13 common respiratory viruses in airline travellers. as culture was not performed on these samples, we are unable to comment on the infectiousness of these travellers and the potential transmissibility of their viruses on entering a community. a limitation of the study was the non-testing of asymptomatic travellers which did not allow estimates of the prevalence of noninfluenza respiratory viruses to be made [22] . a further limitation was the recovery of a virus from only 26% of the symptomatic travellers, which is lower than might be expected from other studies of populations with respiratory infection symptoms. few identifications of coronavirus or metapneumovirus were made, an observation also made in a previously healthy adult population during the winter influenza season [23] . coronaviruses −nl63 and −hku1 have been found to be present in higher numbers than coronavirus −229e or -oc43, however these viruses were not tested for in this study [24] . we have also found that there is variation in analytical agreement between molecular assays and that the easyplex assay used in this study may have had a reduced sensitivity for the detection of both metapneumovirus and bocavirus [20] . the collection of nasal and throat swabs rather than nasopharyngeal swabs, although pooled together may have been suboptimal, even though sensitive fully evaluated molecular techniques were used in this study [20] . it is also likely that, the commonest reported symptom (stuffy nose) might in some cases have been caused by the airline travel itself rather than infection. a wide range of viruses were detected, however, only three virus types were detected in more than 10 travellers. consequently, we could not draw conclusions on the association of symptoms with the virus types identified other than for rhinoviruses, enteroviruses and influenza viruses. even in a study of 155 travellers meeting a who case definition of suspected or probable sars (fever plus cough or difficulty breathing), a pathogen was only detected in 43.2% of cases [25] . enrolment of a substantially larger number of symptomatic but otherwise healthy travellers would be required to identify any additional predictive potential of their symptoms. the use of symptomatic predictors to identify which respiratory infections were caused by viral infections have largely focused on influenza viruses in a number of surveillance and clinical study settings. symptomatic predictors were initially believed to be problematic because the symptoms of many illnesses were very similar. even though fever and cough were most frequently identified in association with influenza infections, surveillance data were often obtained over long periods with varying levels of influenza virus activity, resulting in a low ppv for these symptoms for influenza virus infection [26] . the use of antiviral trial data where subjects were enrolled with ili during the influenza season found that fever (temperature ≥37 • c) and cough when used as predictors during periods of influenza virus prevalence had a ppv of up to 79% in adults [27] [27] . in children ≥5 years, fever (temperature ≥38 • c) and cough resulted in a ppv of 83% [26] . interestingly, in adults it was found there was little advantage of measuring other symptoms [27] . the current study was carried out in a relatively low influenza prevalence population (4% of symptomatic travellers) resulting in a low influenza ppv for fever and ili (fever and cough or sore throat). the symptom profiles over the course of common colds, up to 50% of which are caused by rhinoviruses, have been well established in otherwise healthy adults [28] and more recently in school-children [29] . common symptoms of rhinovirus infection in children include a runny nose, nasal obstruction and cough, with 50% of children reporting these during the first 5 days of illness. in adults, only a runny nose was reported in 50% of illnesses, persisting through day 4, indicating that the symptom profiles differs between children and adults, and over the course of the illness [29] . in our study, the most common symptoms recorded were a stuffy or runny nose in 69% of travellers with an enterovirus infection and 76% with a rhinovirus infection. as well, 52% of those with rhinovirus reported cough, which was the most frequently reported symptom in those infected with influenza b (85%). these symptoms were recorded at a single time point and the stage of the illness after symptom onset of each illness was not recorded. even with rhinoviruses, the most prevalent virus detected in this study, the symptoms generated are clearly shared by different viruses suggesting that it is not possible to identify this virus on the basis of symptoms. there was a substantial overlap in the symptom profiles between the respiratory viruses found in the study participants. the mean number of symptoms reported by on board screening was highest for those with influenza b (3.4; ci 2.7-4.0) followed by rhinovirus (2.2; ci 2.0-2.5) and enterovirus (1.9; ci 1.6-2.3). it is unlikely that, symptoms alone can be used to predict infections with specific respiratory viruses. in the meantime, we should continue to learn as much as possible about potential screening tools so that their potential role, and strengths and weaknesses, are more fully understood. the high prevalence of respiratory virus infections caused by viruses other than influenza in this study, many with overlapping symptoms to influenza, has important implications for any screening strategy for the prediction of influenza in airline travellers. on the basis of clinical symptoms alone it will be very difficult to distinguish influenza from other common respiratory viral infections. this work was supported by the centers for disease control and prevention, united states [grant number 1 u01ci000445-01]. this study was approved by the new zealand health and disability multiregion ethics committee (mec/06/12/172). confronting the new challenge in travel medicine: sars transmission of infectious diseases during commercial air travel transmission of the severe acute respiratory syndrome on aircraft world health organization. mers-cov update summaries spread of a novel influenza a (h1n1) virus via global airline transportation transmission of pandemic a/h1n1 2009 influenza on passenger aircraft: retrospective cohort study transmission of influenza on international flights pandemic influenza a(h1n1) outbreak among a group of medical students who traveled to the dominican republic an outbreak of influenza aboard a commercial airliner an outbreak of influenza a/taiwan/1/86 (h1n1) infections at a naval base and its association with airplane travel outbreak of influenza-like illness [corrected] related to air travel latitudinal patterns of travel among returned travelers with influenza: results from the geosentinel surveillance network mixed outbreak of parainfluenza type 1 and influenza b associated with tourism and air travel a variety of respiratory viruses found in symptomatic travellers returning from countries with ongoing spread of the new influenza a(h1n1) v virus strain. euro surveillance: bulletin europeen sur les maladies transmissibles lack of nasal carriage of novel corona virus (hcov-emc) in french hajj pilgrims returning from the hajj 2012, despite a high rate of respiratory symptoms thermal image scanning for influenza border screening: results of an airport screening study screening for influenza infection in international airline travelers real-time rt-pcr detection of 12 respiratory viral infections in four triplex reactions incidence and characteristics of viral community-acquired pneumonia in adults comparison of four multiplex pcr assays for the detection of viral pathogens in respiratory specimens influenza activity-united states effectiveness of border screening for detecting influenza in arriving airline travelers characterization of viral agents causing acute respiratory infection in a san francisco university medical center clinic during the influenza season coronavirus hku1 and other coronavirus infections in hong kong spectrum of viruses and atypical bacteria in intercontinental air travelers with symptoms of acute respiratory infection symptomatic predictors of influenza virus positivity in children during the influenza season clinical signs and symptoms predicting influenza infection rhinovirus infections in an industrial population ii characteristics of illness and antibody response symptom profile of common colds in school-aged children we thank the christchurch international airport limited, new zealand customs service, the participating airlines and passengers for their cooperation and assistance and technical assistance of canterbury health laboratories virology staff. key: cord-287178-hu6vlc9u authors: wong-chew, rosa m; espinoza, marco a; taboada, blanca; aponte, fernando e; arias-ortiz, maría a; monge-martínez, jesús; rodríguez-vázquez, rubén; díaz-hernández, fidel; zárate-vidal, fernando; santos-preciado, josé i; lópez, susana; arias, carlos f title: prevalence of respiratory virus in symptomatic children in private physician office settings in five communities of the state of veracruz, mexico date: 2015-06-25 journal: bmc res notes doi: 10.1186/s13104-015-1239-0 sha: doc_id: 287178 cord_uid: hu6vlc9u background: acute respiratory tract infections are the leading cause of morbidity and mortality in children worldwide. many studies have described the frequency of viruses in hospitalized patients, but studies describing the prevalence of viruses in the community setting are limited, particularly in developing countries, where most of the deaths from serious respiratory diseases occur. the aim of this study was to evaluate the diversity of respiratory viruses in the community setting using molecular diagnostic tools, as well as the clinical characteristics of respiratory viral infections in the general pediatric practice in mexico. methods: children with respiratory tract infections attending private pediatric practices during a 10-month period in five cities of the state of veracruz were included. nasal swabs were taken and processed by a multiplex detection kit for 15 respiratory viruses. results: 525 children were included from july 2011 to may 2012; 44% were female, mean age was 45 months. the 3 most frequent clinical diagnosis were: rhinopharyngitis 68%, pharyngitis 18%, and 3.3% influenza-like illness. 71.5% of the samples were positive for virus. the five most frequent pathogens were respiratory syncycitial virus in 18.3% of the children, rhinovirus in 17.5%, influenza a 9.1%, adenovirus 7.2%, and enterovirus 3.4%, although all 15 viruses were detected; there were viral coinfections in 14.1%, and 28.5% of the samples were negative. conclusions: a large proportion of respiratory infections in the community setting in mexico was associated to viruses. although testing for common respiratory pathogens in children with acute respiratory tract infections may lead to a better understanding of the role of viral pathogens in, and eventually to improvement in the management of, individual patients, additional prospective studies are required to study the need of routinely using such tests in general pediatric practices in resource-limited countries. respiratory tract infections continue to be the most frequent reason for health care visits and hospitalizations and are a leading cause of morbidity and mortality, particularly in young children. according to a report of the world health organization, there were 6.6 million deaths in 2012 in children under 5 years of age worldwide [1] . the highest mortality occurred in developing countries, where 15% of the deaths were caused by acute respiratory tract infections. mexico reported more than 26 million acute respiratory tract infections in 2013 [2] . the ministry of health reports the annual incidence and prevalence, but the etiology of these pathologies is unknown. many studies have described the frequency of viruses in hospitalized patients, but studies describing the prevalence of viruses in the community setting are limited, particularly in developing countries. previous reports in mexico have evaluated the presence of at most seven viruses in a single study using different methods, such as viral culture, immunofluorescence, or single pcr. the majority of these studies have been carried out in hospital settings, which limits the available information about the pathogens causing respiratory infections in the community. newly developed commercial diagnostic assays based on real-time nucleic acid amplification, such as multiplex polymerase chain reaction (pcr) tests allow sensitive and specific detection of a broad panel of conventional and emerging viruses in respiratory tract specimens. the multiplex pcr assays are more sensitive than any other diagnostic method, including virus isolation in cell culture and antigen detection, and are being used around the world for the past few years [3] . these technological advances have changed the landscape of virus detection and provide the opportunity to better understand the epidemiology of respiratory viruses. the aim of our study was to evaluate the diversity of respiratory viruses using new diagnostic tools in children with symptomatic respiratory tract infections in private physician office settings in five communities of the state of veracruz, mexico. this information contributes to the knowledge about the etiology of one of the most frequent diseases in our country, and one of the first cause for consult in the pediatric offices. 525 children from private pediatric practices in 5 different municipalities of the state of veracruz, mexico, were included; 56% were male and the mean ± se age was 45 ± 2 months, mean ± se weight was 17 ± 0.7 kg, mean ± se height was 93 ± 1 cm. there were statistically significant differences among the municipalities (table 1) : the oldest children were from poza rica and minatitlán. the parental education was also different among the cities: the parents with the highest education were in córdoba, minatitlán, and veracruz, although córdoba had some parents with no formal education (3%). the socioeconomic level of the majority of the families was in the middle-income category, except for those attending the pediatric practice in the city of veracruz where 36% reported high income. some risk factors for respiratory tract infection were documented: asthma was present in 22%, allergic rhinitis in 25%, and incomplete vaccination scheme in 29% of the population (table 1 ). asthma was diagnosed by the attending physician according to global initiative for asthma (gina) in its report global strategy for asthma management and prevention in children 5 years and younger [4] . children under 3 years of age were not considered asthmatic. from the 525 nasal swabs tested by pcr, 71.5% were positive for the presence of viral nucleic acids of one or more respiratory virus, while 28.5% of the samples were negative. among the viruses detected, considering both single and multiple infections, rsv-a and rv showed the highest frequency, being present in 18.3 and 17.5% of the samples analyzed, respectively, followed by flua (9.1%), figure 1 ). in 73 of the 525 samples (13.9%) a viral coinfection was found. sixty-three of these samples had a dual infection, with the combination of adv/ev and rv/hucov 229/ n63 being the more frequent. eight children had triple virus infections, and two were infected simultaneously with four viruses (table 2) . seasonality is well described for several viral respiratory pathogens [5, 6] . in our study there was a seasonal predominance for rsv-a from september to december, flua was more frequent from december to february, flub from december to april, and hmpv predominated from february to may. in contrast, rv, ev, adv, and piv-3 were present during all the study period ( figure 2 ). the clinical diagnosis in order of frequency were rhinopharyngitis in 67%, pharyngitis in 18%, influenza-like illness, pharyngotonsillitis, and laringotracheitis in 3%, followed by bronchiolitis and rhinitis in 2%, rhinosinusitis in 0.6%, and pertussis-like syndrome in 0.2% (table 3) . all children were managed in an ambulatory fashion, and none required hospitalization. the most frequent signs and symptoms were cough in 85%, rhinorrea in 81%, nasal congestion in 70%, nasal discharge in 68%, fever in 68% and odynophagia in 61%. other less frequent signs and symptoms were headache in 36%, myalgia in 25%, nausea in 21%, vomiting in 22%, dysphonia in 17%, figure 1 frequencies of viral pathogens in five different municipalities of veracruz. the percentage of each virus, considering their presence in both single and multiple infections, is shown for the five cities included in the study. the percentage of viruses in the total number of samples analyzed is also shown. viral coinfection stands for the percentage of samples in which more than virus was found. respiratory distress in 13%, diarrhea in 11%, dyspnea in 11% and conjunctivitis in 8% of the children (table 3) . we found differences in the pathogens detected among single viral infections according to the clinical diagnosis. in the children with rhinopharyngitis the most frequent pathogen was rsv a and b in 20%, followed by rv in 13.5%, flua in 5.4%, adv in 3.7%, and hmpv in 3.4%. in contrast, in children with pharyngitis the most frequent pathogens were rv in 10.5%, ev in 8.4%, flua in 7.4%, hucov 229e/nl63 in 6.3%, and hmpv and piv-3 in 4.2%. in children with rhinitis the pathogens detected were rsv-a in 27.3%, rv in 9.1%. the pathogens that affected the larynx, trachea and bronchiols were rsv, rv, and flua (table 4 ). in this work viral pathogens were detected in a large proportion of children with respiratory disease (71.6%) as compared to studies previously carried out in mexico. in a study conducted by the epidemiologic reference institute (indre) of mexico, 11% of the samples collected from different regions of the country between 1995 and 2000 were positive for viruses [7] . in hospitalized children, a respiratory virus was detected in 47% of patients with lower respiratory tract infections in a large referral hospital in san luis potosi [8] , and in 14% in the hospital infantil de méxico federico gómez [9] . in other studies carried out in the community setting, up to 65% of viral detection has been described [10] [11] [12] , and the detection rate in secondary care hospitals has been reported to be between 39 and 60% [8, 11, 13] . rsv and rv were the most frequent pathogens detected in this work. other studies carried out in mexico have also reported rsv as the most frequent pathogen varying from 24 to 65% in hospitalized children with lower respiratory tract infections [8, 14] to 38% in a community setting [11] , although in these studies rv was not looked for. in contrast, a recent report from switzerland using molecular tests, described picornavirus as the most prevalent virus in upper respiratory tract infections in children (48%), followed by adv (16%) and rsv (14%), although the population included in this study was different from those carried out in mexico since it was performed in children hospitalized in a tertiary care center [15] . in another study carried out in mexico in children younger than 18 years of age with influenzalike illness, influenza was the most frequent virus found (16.7%), followed by rsv (13.2%) and rv (11.2%) [16] . in this work hmpv was not detected as frequently as in yucatán, in a study carried out in children with influenza-like illness, where 20% of the samples were positive for this virus [17] . also, 10.5% of children hospitalized in san luis potosí, which had been negative for other respiratory viruses, were reported to be positive for hmpv [12] . in our study hmpv was found in 5.3% of the tested samples. some differences in the occurrence of viruses among municipalities were observed, with the highest viral diversity found in poza rica and minatitlán, where all 15 searched viruses circulated; the frequency of the different viruses was more evenly distributed in poza rica. these two cities, located in the northern and southern part of the state of veracruz, respectively, are centers of oil extraction, and they probably have a higher traffic of people than the other municipalities, what could explain the more ample variety of viruses found. multiple viral infections were detected in 13.9% of the samples. detection of several viruses in a single sample is frequently reported in respiratory infections in which a pcr-based diagnostic method is used, however, the clinical relevance of detection of more than one virus is uncertain [18] [19] [20] [21] . in this study, however, a tendency of correlation between coinfections and the severity of disease was observed. in children with laryngotracheitis and bronchiolitis, viral coinfection was detected in 28 and 18% respectively, compared to 13.8 and 10.5% of rhinopharyngitis and pharyngitis, respectively. this suggests that a viral coinfection could predispose to a more severe disease, although the low number of children in this study with more severe disease limits this conclusion. in addition, whether the detection of virus in nasal swabs reflects the actual pathogen of the clinical disease or reflects only nasopharyngeal carriage or an asymptomatic infection is not known, particularly when highly sensitive molecular methods are employed [22] . the seasonal predominance for rsv-a in autumn and winter, influenza in winter and spring, and hmpv from february to may observed in this study is consistent with previous reports, while rv, ev, adv, and piv were detected wit similar frequency along the study. one limitation of this study is the great variability of the sample size in each city, since in orizaba 60 patients were recruited, while in poza rica 208 were studied. for this reason, this study should be considered as descriptive, in which the statistics shown analyze the differences in the proportions within each city, while comparisons between cities were not done. many studies have described the frequency of viruses in hospitalized patients with severe respiratory illness, but studies describing the prevalence of different viruses in the community setting are limited, particularly in developing countries, where most of the deaths from serious respiratory diseases occur. this is the first report to evaluate a large panel of viruses in clinical samples from children with upper respiratory tract infections in the community setting in mexico, and it provides recent information on respiratory viral pathogens not detected previously, and on the role each of them has as compared to the burden of other viruses. the availability of new molecular diagnostic methods with an increased sensitivity provides a more accurate depiction of the epidemiology of known pathogens and emerging respiratory viruses. however, although testing for common respiratory pathogens in children with acute respiratory tract infections may lead to a better understanding of the role of different viruses as etiologic agents, and eventually to improvement of individual patient management, additional prospective studies are required to study the need of routinely using such tests in general pediatric practices in resource-limited countries. a prospective clinical, epidemiological, observational, cross-sectional study was conducted in córdoba, tierra blanca, veracruz, poza rica and minatitlán, capital cities of five different municipalities of the state of veracruz, pharyngitis, n (%) 3 (5) 6 (9) 11 (17) . sample nucleic acids were tested following the protocol supplied by the manufacturer. pcr products were visualized by electrophoresis on a 2% agarose gels, staining with ethidium bromide. univariate analysis was used to determine frequencies and proportions. for bivariate analysis kruskal-wallis test was used to contrast qualitative variables and anova for quantitative variables. multivariate analysis was performed to detect correlation among clinical features, clinical diagnosis, risk factors, municipalities, and viral or bacterial detection. a p < 0.05 was considered statistically significant. written informed consent was obtained from each parent or guardian prior to enrollment in the study. world health statistics. in: wlcip d (ed) global health indicators dirección general de epidemiología detection of respiratory viruses by molecular methods global initiative for asthma seasonality of viral infections: mechanisms and unknowns epidemiology and seasonality of respiratory viral infections in hospitalized children in kuala lumpur, malaysia: a retrospective study of 27 years infecciones respiratorias agudas (iras) de etiologia viral en méxico viral etiology of lower respiratory tract infections in hospitalized children in mexico frequency of respiratory viruses and clinical characteristics in children attending a care center in mexico city frequency of viruses associated with acute respiratory infections in children younger than five years of age at a locality of mexico city comparative viral frequency in mexican children under 5 years of age with and without upper respiratory symptoms human metapneumovirus infections in mexico: epidemiological and clinical characteristics agentes etiológicos de bronquiolitis en niños impact of respiratory syncytial virus on hospital admissions in children younger than 3 years of age epidemiology of viral respiratory infections in a tertiary care centre in the era of molecular diagnosis la res ili002 study group: clinical characteristics and outcomes of influenza and other influenza-like illnesses in mexico city human metapneumovirus in children with influenza-like illness in yucatan, mexico multiple simultaneous viral infections in infants with acute respiratory tract infections in spain viral etiology of acute respiratory tract infections in children presenting to hospital: role of polymerase chain reaction and demonstration of multiple infections respiratory syncytial virus, human bocavirus and rhinovirus bronchiolitis in infants frequent detection of respiratory viruses without symptoms: toward defining clinically relevant cutoff values submit your next manuscript to biomed central and take full advantage of: • convenient online submission • thorough peer review • no space constraints or color figure charges • immediate publication on acceptance • inclusion in pubmed, cas, scopus and google scholar • research which is freely available for redistribution submit your manuscript at www we thank arely garcía botello for her valuable contribution in integrating the database used in this work. this work was partially supported by grants 87691 (to j. i. santos) and "influenza 2009" (to c. f. arias) from the national council for science and technology-mexico (conacyt). the authors declare that they have no competing interests. key: cord-303935-qdehf6rb authors: yun, heather c.; young, adam n.; caballero, manuel y.; lott, lisa; cropper, thomas l.; murray, clinton k. title: changes in clinical presentation and epidemiology of respiratory pathogens associated with acute respiratory illness in military trainees after reintroduction of adenovirus vaccine date: 2015-09-01 journal: open forum infect dis doi: 10.1093/ofid/ofv120 sha: doc_id: 303935 cord_uid: qdehf6rb background. adenovirus (ad) has long been the predominant cause of acute respiratory illness (ari) in military trainees. in 2011, live oral ad vaccines for serotypes 4 and 7 were reintroduced into us basic military training populations. this study evaluated the impact on clinical presentations and other respiratory pathogens. methods. the center for advanced molecular detection at joint base san antonio-lackland prospectively collects demographic, clinical, and polymerase chain reaction data from respiratory specimens (throat swab and nasal wash) among air force trainees presenting for care of ari. results. from june 2008 to august 2013, 2660 trainees enrolled and were tested for selected respiratory pathogens. post-vaccine introduction (vi), reported systemic symptoms were less frequent, including fever (38% vs 94%) and myalgia (37% vs 67%; p < .01). median temperature and heart rate decreased (98.4 vs 101.3°f, 81 vs 96 beats per minute; p < .01). ad detection decreased for all ad (3% vs 68%), ad4 (1% vs 70%), 7 (0% vs 8%), 14 (0% vs 5%), and 3 (0.1% vs 2%); p < .01). rhinovirus and cases with no pathogen identified increased in frequency (35% vs 18%, 51% vs 14%; p < .01). conclusions. acute respiratory illness in military trainees post-vi is associated with decreased severity of systemic symptoms and reduced fever and heart rate. marked reductions in frequency of ad serotypes are seen, including those in the vaccine, with no serotype shift. however, detection of several other respiratory pathogens, most notably rhinovirus, is observed in increasing proportions, and a majority are now undiagnosed clinical syndromes. reduction in fri due to ad [5] . however, the military was the only purchaser of vaccine, which was produced by a sole manufacturer, and vaccine production ceased in 1995 when funding requirements for updating manufacturing facilities were not met. attempts by the department of defense to find an alternative solution were unsuccessful, and existing vaccine stocks were depleted in 1999. ad quickly re-emerged as the major cause of morbidity, causing numerous outbreaks with both vaccine and nonvaccine serotypes, and directly resulting in approximately 1 death per year [6] [7] [8] [9] [10] . from 1997 to 2012, ad ( primarily serotype 4) caused 68% of all fri in the basic training environment [11] . it is estimated that associated medical care and time lost from training resulted in costs of $10-$26 million per year [12, 13] . in 2001, the us food and drug administration's (fda) newproduct approval process was initiated for resumption of production of ad4 and ad7 vaccines by a new manufacturer [14] . phase 3 vaccine trials demonstrated (1) 99% efficacy for ad4 and (2) ad7 (which was not circulating at the time) seroconversion rates of 95% [15] . in 2011, the 2 vaccines were approved by the fda and reintroduced nearly simultaneously at all 8 us military basic training locations in october/november of that year. since then, surveillance reports have consistently demonstrated great reductions in fri and ad-related illness. early data indicated a 75% decrease in fri, and proportions of collected specimens positive for ad decreased from 75% to 1% in the months surrounding vaccine introduction (vi) [11] . nearly all of this was attributable to ad4, with rare detections of serotypes 7, 3, 14, and 21, and 3 cases involving vaccine-type 4p. follow-up data published in late 2014, evaluating surveillance data from 1996 to 2013, reported reductions in ad disease burden from 5.9 to 0.02 cases/person-week [16] . the authors estimated that the current vaccines prevent 13 000 cases of fri, 1100-2700 hospitalizations, and 1 death per year. after vi, ad14 became the most prevalent circulating serotype, although actual number of cases detected decreased from approximately 610 per year to 44 in 2013. this large study reported comprehensive surveillance data for overall ad and fri, but clinical data were not captured. whether the clinical presentation of those trainees who do present for care with a respiratory illness has changed post-vi is unclear. because respiratory illness remains a leading cause of presentation for care among military trainees, understanding of trends in clinical presentation and emerging, non-ad respiratory pathogens in the post-vi era requires evaluation. the purpose of this study was to evaluate (1) changes in clinical presentations of ari pre-and post-vi, and (2) reductions in proportions of disease due to ad. we also sought to further evaluate for evidence of nonvaccine type serotype shift and to determine whether the frequencies of common non-ad respiratory pathogens have changed after vi, in trainees presenting for care of ari, which have not previously been described in the published literature. joint base san antonio (jbsa)-lackland, texas, is the sole basic military training site for the us air force. training lasts 8.5 weeks, with 6000-7000 recruits present at any given time, and approximately 43 000 training per year. units consisting of 50-60 individuals train together and live in open bay dormitories. the population is approximately 80% male. ill or injured trainees present for care at an outpatient medical clinic; those with respiratory illness and fever are then cohorted until well enough to return to training. those requiring hospitalization are admitted to the local military tertiary care hospital. vaccines, including ad vaccine beginning week 48 of 2011, and influenza vaccine seasonally, are administered during the first week. in 2009, influenza vaccine against pdm09h1n1 influenza was available and administered after december 1, 2009. oseltamivir was used for prophylaxis of close contacts of confirmed influenza cases throughout the study period. prophylaxis for streptococcus pyogenes is also administered to all trainees during the first week, consisting of benzathine penicillin, or azithromycin for penicillin allergic individuals. since 2003, the center for advanced molecular detection (59th medical wing/science and technology, air education and training command) has prospectively evaluated epidemiology of respiratory pathogens and novel technologies for detection. for the purposes of this substudy, data were evaluated from june 2008 to august 2013. ill recruits presenting for clinical care of ari at the outpatient clinic or hospital were approached by study personnel regarding participation. inclusion criteria were met if the trainee was ≥17 years of age and endorsed any symptom of respiratory infection, including cough, coryza, sore throat, or nasal or sinus congestion. for those consenting to enroll in the study, demographic information was collected, along with a symptom questionnaire, including self-reported stress levels, and clinical signs, including vital signs and physical examination findings recorded during the medical encounter. provider diagnoses given at the time of the visit were also recorded when available. provider clinical diagnosis extracted from the note, if any, associated with the visit, was also explored with reference to diagnoses of upper respiratory tract infection (urti) vs lower respiratory tract infection (lrti). the diagnoses, "rhinitis, conjunctivitis, otitis, sinusitis, pharyngitis, sore throat" were considered to be representative of urti, and the terms "bronchitis, pneumonia" were representative of lrti. terms including "common cold", "viral syndrome", "fever", or "cough" were not included in the urti vs lrti analysis given their lack of anatomical description. nasal washes and throat swabs were collected for polymerase chain reaction (pcr) assays. duplicate presentations for multiple ari-related visits were excluded; each case represents an individual subject. specimen processing was performed as previously described [17] . respiratory specimens were characterized daily by pcr on applied biosystems (abi) 7500 and 7900htfast (applied biosystems, ca) and the viia 7 real-time pcr systems. specimens were tested for ad ( panad and serotypes 4, 7, 14, 3, 11, 21, 2, and 5); rhinovirus; influenza a, including h1 and h3; influenza b, enterovirus, human coronaviruses oc43 and 229e, bocavirus, human metapneumovirus (hmpv), parainfluenza virus type 3 (hpiv), s pyogenes, streptococcus pneumoniae, mycoplasma pneumoniae, and chlamydophila pneumoniae. all primer and probe sequences used have been previously reported (association of public health laboratories guidelines for realtime reverse transcription-pcr assays of influenza respiratory viruses; non-influenza respiratory viruses from clinical respiratory specimens; respiratory bacterial pathogens) [18, 19] . influenza a and b, enterovirus and rhinovirus thermocycling conditions were 30 minutes at 50°c, 10 minutes at 95°c, followed by 15 seconds at 95°c and 35 seconds at 60°c for 45×. coronaviruses, hmpv, and hpiv thermocycling conditions were 10 minutes at 48°c, 10 minutes at 95°c, followed by 15 seconds at 95°c and 1 minute at 60°c for 45×. streptococcus pneumoniae, s pyogenes, and bocavirus thermocyling conditions were 10 minutes at 95°c followed by 15 seconds at 95°c and 45 seconds at 60°c for 45×. thermocycling conditions for m pneumoniae and c pneumoniae were 2 minutes at 50°c, 10 minutes at 95°c, followed by 15 seconds at 95°c and 1 minute at 60°c for 40×. analyses were performed using spss (spss, version 19.0, spss). dichotomous variables were compared using χ 2 or fisher's exact test as applicable. continuous variables were analyzed using mann-whitney u test for nonparametric data. all reported p values are 2-tailed with statistical significance set at <.05. subjects provided voluntary, written informed consent in the presence of ombudsmen, and the study was approved by the jbsa-lackland institutional review board. this research was conducted in compliance with all applicable international and federal regulations regarding protection of human subjects. from june 2008 to august 2013, 2660 trainees enrolled and had specimens tested for respiratory pathogens. ad vi took place week 48, 2011; 72% of this cohort enrolled pre-vi vs 28% post-vi. none of the pre-vi subjects received ad vaccine, vs 93% post-vi. overall, 86% were male, with a median age of 20 years. enrollments ranged from 437 to a peak of 757 in 2009 for years with a complete calendar-year of data. trainees reported a perceived stress level of 4 on a 10-point likert scale. post-vi, the male predominance of enrolled subjects decreased from 89% to 79% (p < .01) (see table 1 ). clinical characteristics are presented in table 2 . before vi, 91% of subjects had a recorded oral temperature >100.4°f, vs 10% afterward. one or more diagnoses representative or uri, lrti, or both, were present in 64.3% pre-vi and 74.5% post-vi. terms associated with lower respiratory tract infection (lrti) were more commonly included post-vi (26.1% vs 12.9%, p < .01) and terms associated with uri were less commonly included post-vi (79.0% vs 89.5%, p < .01). those with the diagnosis of "pneumonia" in the post-vi period were predominantly afebrile (89.5%). supporting radiographs, if performed, were unavailable. the term "allergic" or "allergy" was included in none of the diagnoses pre-vi, but was included in 8.1% post-vi (p < .01); 75% of these were listed in combination with some other diagnosis of uri or lrti. clinical signs and symptoms specifically associated with ad vs rhinovirus were also compared, with similar findings as those seen in general for pre-and post-vi (data not shown). the exceptions were the loss of statistical significance between those describing malaise and with abnormal tympanic membrane examinations. those with ad vs rhinovirus also more often described sore throat (90.8%, 83.1%, p < .01) and had more documented tonsillitis (23.5% vs 13.8%, p < .01). influenza a detections were less frequent post-vi; h1 accounted for the majority of the pre-vi detections, and these were all in 2009. in subjects with documented fever post-vi (n = 69), rhinovirus accounted for the largest proportion of abbreviations: iqr, interquartile range; vi, vaccine introduction. a abnormal examination: any of the following: "decreased breath sounds, rales, crackles, rhonchi, wheezes" for lungs, "abnormal, dull, erythematous, effusion" for tympanic membranes; "gallop, murmur, abnormal rhythm" for cardiac; "abnormal bowel sounds, distended, tender" for abdominal. these (n = 24), with influenza detected in 7 cases and any ad in 5 (4 serotype 4, 1 serotype 3). twenty-one of these had no pathogen detected the rate of detection of rhinovirus doubled in the post-vi period; an increase in raw numbers of rhinovirus positive enrollments per month was also seen despite lower enrollments in general (see table 3 ). pre-vi, there were 1.9 rhinovirus positive cases per week, and 10.5 enrolled/week; post-vi, 2.9/ week were rhinovirus positive, among 8.5 enrolled/week. reintroduction of the ad vaccine in basic training populations has again been extraordinarily successful in reducing the burden of both ad-related respiratory illness and the burden of respiratory illness with fever. 75% reductions in fri have been demonstrated by others, including at joint base san antonio-lackland where this study was conducted, against the backdrop of a 99.6% reduction in the weekly rate of ad-related illness [11, 16] . sustaining the commitment to prevention of ad-related illness in uniquely susceptible trainees will be necessary if history is not to repeat itself. however, prevention of respiratory illness in this population is a complex task. risk factors for transmission of respiratory pathogens will continue to be present in conditions inherent to basic military training. adenovirus, despite its preeminence as a pathogen of interest in this group, has never been the entire story, and large outbreaks of non-vaccine serotype ads have occurred even while vaccine serotypes were circulating [9] . influenza causes annual epidemics which, in the context of an effective vaccine program, are typically limited in this population, but which can have considerable impact when new strains emerge. in summer and fall of 2009, influenza was responsible for 20% of fri in those who were tested [20, 21] . large outbreaks of pharyngitis caused by s. pyogenes, complicated by acute rheumatic fever, pneumonia, necrotizing skin and soft tissue infections, and other suppurative and immunologic complications, have been reported throughout the past century, prompting widespread use of antimicrobial prophylaxis at training sites [22] [23] [24] . pneumococcal outbreaks have also occurred despite such prophylaxis, including pneumonia and fatal meningitis [25, 26] . others, including neisseria meningitidis, bordetella pertussis, m. pneumoniae, and c. pneumoniae, have been well-described in this population [27] . horizontal efforts at respiratory infection prevention, such as promoting hand hygiene, environmental including gas mask disinfection, cohorting of ill trainees, and respiratory etiquette, will require continued emphasis, even with near-elimination of ad-related illness. however, vertical measures targeting specific organisms have also been demonstrated to have significant impact, with ad vaccine as the prime example, and ongoing exploration into post-vi causes of illness will be necessary to direct further interventions. furthermore, although widespread efforts exist to monitor fri rates and conduct surveillance for common respiratory viruses, not all acute respiratory illness is febrile. clearly, trainees are still presenting for illness, but those without fever, which now represent >90% of those presenting for care, will not have respiratory pathogen analysis via dod-directed surveillance mechanisms. few prior data inform clinical differences between those presenting with ad vs other respiratory pathogens. recent comparisons of pdm(09)h1n1 influenza and ad, including an analysis from this cohort, corroborated a predominance of coryza and cough presentations for influenza, vs pharyngitis for ad [20, 21] . this evaluation again emphasizes a classic presentation of ad-related illness: fever, systemic complaints, and pharyngitis, distinct from the afebrile, coryza/ cough presentations of those presenting post-vi and with rhinovirus. interestingly, documentation of abnormal lung examination findings increased post-vi, as did use of clinical diagnostic terms suggesting lrti, including both bronchitis and pneumonia. it is likely that most of those labelled "pneumonia" were never confirmed with radiographs, and absence of fever with these argues against that diagnosis in this young, otherwise healthy population. the predominant organism identified among these was rhinovirus, which, while not classically associated with lower respiratory disease in healthy adults, has been described including within military training populations [28, 29] . nevertheless, the combination of increased lrti diagnoses, and the increase in cough as well as physical exam findings of the same, provide signal of an increase in lrti post-vi which should be explored with targeted research. it is also considerable that, despite a relatively broad panel of respiratory pathogens targeted with molecular methods, >50% post-vi had no pathogen detected. some of these may have been noninfectious, as suggested by the increase in clinical diagnostic terms relating to allergies, but this represents a significant research gap. the nature of respiratory illness itself in basic military trainees has changed after reintroduction of ad vaccine, transitioning from a febrile pharyngitis marked by systemic signs and symptoms, to an afebrile, cough and coryza predominant illness. the ecologic niche occupied by vaccine-serotype ad in this population was remarkable, causing approximately 70% of all fri historically and with 80% of trainees infected by the end of training [4, 11] . during ad vi in 1971, molecular methods for pathogen surveillance were not available; despite this, serotype shift was observed. initially, ad4 was the only serotype included in the vaccine program, but ad7 was later added after this emerged and replaced ad4 as the predominant cause of fri [5] . since that time, dozens of additional ad serotypes and other respiratory viral pathogens such as bocavirus and human metapneumovirus have been identified. respiratory pathogens cause outbreaks, which may come and go independently of a vaccine program's effect, so changes in frequency must be interpreted with caution. however, it is reassuring that ad14 has not yet reemerged in this population and, in fact, decreased in frequency since vi, a finding which has been corroborated by others, and potentially related to crossprotection with ad7 immunity [9, 16] . the decrease in frequency of influenza a was driven by the unusually high number of influenza a cases in 2009, which contributed 63 to the total of 76 during the entire study. the trend toward a decrease in s. pyogenes (with no changes in antibiotic prophylaxis during the study period) is potentially biologically plausible with viral coinfection increasing the likelihood of streptococcal illness, although specific associations between ad and s. pyogenes have not been established, and rates of s. pyogenes illness are not known to have changed during the first iteration of the ad vaccine program. the small increases seen in detection of m. pneumoniae, bocavirus and coronavirus oc43 may be due to chance alone or natural variation, but bear further observation. most significant was the increase seen in detections of rhinovirus, which increased as a proportion of detected pathogens, in rates of positive tests among those tested for rhinovirus, and in raw numbers despite fewer overall enrollments. rhinovirus may be associated with decreased probability of detecting other respiratory viral pathogens, including ad. an evaluation of virus pairs in a pcr-based analysis of co-detections demonstrated a negative association between ad and rhinovirus. while multiple additional respiratory viruses also demonstrated a negative association with rhinovirus, no others correlated either positively or negatively with ad [30] . a negative interaction between these 2 pathogens has previously been reported in military recruits with and without symptoms of respiratory illness, and with ad, but not rhinovirus, clearly associated with illness [31] . the most significant strength of the study is the collection of a wide array of respiratory pathogen data alongside a detailed collection of clinical and demographic data, allowing thorough evaluations for ecologic niche replacement, as well as changes in clinical illness, throughout a major change in vaccine administration, in >2600 trainees. others include the high uptake of ad vaccine, and the consistency in access to care, living and training conditions, and preventive medicine measures throughout the study period. the study also has a number of limitations. this is a single center which, although spanning several years, cannot account for natural variability of respiratory pathogens here or at other training sites, and some pathogens were tested for only from a subset of samples. these represent a convenience sample of the overall burden of trainees presenting to medical care for respiratory illness. trainees, for a number of reasons, may be reluctant to self-identify when ill and present for care. while we have no reason to believe this limitation changed over the course of the study, this limits the ability to extrapolate frequencies of detection to rates of disease. since asymptomatic subjects are not captured, it also limits the ability to determine colonization vs correlation with clinical illness. however, study procedures and approaches to enrolling trainees were unchanged over the course of the study period. there may have been increases in healthcare-seeking behavior in 2009 during the influenza pandemic, and this year saw the highest number of enrollments over the course of the study. finally, comorbidities in this group were not captured, although significant known comorbidities are typically disqualifying for military accession. in conclusion, reintroduction of ad vaccine in military trainees has markedly reduced the frequency of ad detection in trainees presenting for care of respiratory illness, and been associated with a 10-fold reduction in the proportion presenting with objective fever. acute respiratory illness has transitioned away from a febrile pharyngitis with systemic signs and symptoms to a predominantly afebrile coryza and cough illness. no evidence of ad serotype shift has been demonstrated, but rhinovirus is emerging as a potential pathogen of importance, and despite a broad array of tested viruses and bacteria, a majority now results with no pathogen identified. respiratory infections are no longer the leading cause of medical encounters among military recruits; now they are the second-leading cause [32] . their ongoing importance epidemiologically requires continued surveillance and research into additional preventive measures. surveillance snapshot: illness and injury burdens among u.s. military recruit trainees psychological stress and susceptibility to the common cold transmission dynamics and prospective environmental sampling of adenovirus in a military recruit setting epidemiology of adenovirus respiratory infections in military recruit populations control of respiratory disease in recruits with types 4 and 7 adenovirus vaccines adult adenovirus infections: loss of orphaned vaccines precipitates military respiratory disease epidemics. for the adenovirus surveillance group adenovirus transmission-worthy of our attention large epidemic of respiratory illness due to adenovirus types 7 and 3 in healthy young adults outbreak of severe respiratory disease associated with emergent human adenovirus serotype 14 at a us air force training facility in 2007 adenovirus-associated deaths in us military during post vaccination period surveillance snapshot: adenovirus among u.s. military recruit trainees prevention of adenoviral acute respiratory disease in army recruits: cost-effectiveness of a military vaccination policy cost-effectiveness analysis of reacquiring and using adenovirus types 4 and 7 vaccines in naval recruits history of the restoration of adenovirus type 4 and type 7 vaccine, live oral (adenovirus vaccine) in the context of the department of defense acquisition system a phase 3, randomized, double-blind, placebo-controlled study of the safety and efficacy of the live, oral adenovirus type 4 and type 7 vaccine dramatic decline of respiratory illness among us military recruits after the renewed use of adenovirus vaccines evaluation and validation of a real-time pcr assay for detection and quantitation of human adenovirus 14 from clinical samples pring-akerblom p. rapid and quantitative detection of human adenovirus dna by real-time pcr human coronavirus infections in rural thailand: a comprehensive study using real-time reverse-transcription polymerase chain reaction assays retrospective analysis of demographic and clinical factors associated with etiology of febrile respiratory illness among us military basic trainees pandemic influenza virus 2009 h1n1 and adenovirus in a high risk population of young adults: epidemiology, comparison of clinical presentations, and coinfection epidemic streptococcal disease among army trainees outbreak of group a streptococcal pneumonia among marine corps recruits-california acute rheumatic fever among army trainees-fort outbreak of pneumonia in the setting of fatal pneumococcal meningitis among us army trainees: potential role of chlamydia pneumoniae infection halting a pneumococcal pneumonia outbreak among united states marine corps trainees respiratory diseases among u.s. military personnel: countering emerging threats frequency of detection of respiratory viruses in the lower respiratory tract of hospitalized adults atypical pneumonia in young men with rhinovirus infections do rhinoviruses reduce the probability of viral co-detection during acute respiratory tract infections? respiratory illness post ad vaccine • ofid • 7 broad spectrum respiratory pathogen analysis of throat swabs from military recruits reveals interference between rhinoviruses and adenoviruses surveillance snapshot: illness and injury burdens among u.s. military recruit trainees we express our gratitude to dr. sandra valtier and the research coordinators at the camd for recruiting the subjects and assisting in the study logistics. we are also grateful to the trainees who participated in the study.disclaimers. the opinions or assertions contained herein are the private views of the authors and are not to be construed as official or reflecting the views of the department of the army, department of the air force, department of defense or the us government. this work was prepared as part of their official duties and, as such, there is no copyright to be transferred. financial support. the work was supported by the united states air force, 59th medical wing/science and technology, air education and training command.potential conflicts of interest. all authors: no reported conflicts. all authors have submitted the icmje form for disclosure of potential conflicts of interest. conflicts that the editors consider relevant to the content of the manuscript have been disclosed. key: cord-302111-kg0dmgq0 authors: darden, dijoia b.; hawkins, russell b.; larson, shawn d.; iovine, nicole m.; prough, donald s.; efron, philip a. title: the clinical presentation and immunology of viral pneumonia and implications for management of coronavirus disease 2019 date: 2020-04-29 journal: crit care explor doi: 10.1097/cce.0000000000000109 sha: doc_id: 302111 cord_uid: kg0dmgq0 this review will briefly examine the clinical presentation and important immunology of viral pneumonia with a focus on severe acute respiratory syndrome coronavirus 2 (coronavirus disease 2019). data sources, study selection, data extraction, and data synthesis: the most relevant, original and review literature were assessed for inclusion in this review. sources included the centers for disease control and prevention, world health organization, and pubmed. conclusions: pneumonia is a leading cause of hospitalization and death worldwide, with viral etiologies being very common. given the rapidly emerging pandemic associated with the novel severe acute respiratory syndrome coronavirus 2 causing coronavirus disease 2019, it is important to review the clinical presentation and immunologic changes associated with viral pneumonia. symptoms of viral pneumonia include common respiratory tract infection symptoms of cough, fever, and shortness of breath. immunologic changes include up-regulation of airway pro-inflammatory cytokines and pathogenand damage-associated molecular patterns contributing to cytokine and genomic changes. coronavirus disease 2019 clinical presentation is typical of viral pneumonia with an increased prevalence of early pulmonary infiltrates and lymphopenia. principles of early coronavirus disease 2019 management and isolation as well as potential therapeutic approaches to the emerging pandemic are discussed. objectives: this review will briefly examine the clinical presentation and important immunology of viral pneumonia with a focus on severe acute respiratory syndrome coronavirus 2 (coronavirus disease 2019). data sources, study selection, data extraction, and data synthesis: the most relevant, original and review literature were assessed for inclusion in this review. sources included the centers for disease control and prevention, world health organization, and pubmed. conclusions: pneumonia is a leading cause of hospitalization and death worldwide, with viral etiologies being very common. given the rapidly emerging pandemic associated with the novel severe acute respiratory syndrome coronavirus 2 causing coronavirus disease 2019, it is important to review the clinical presentation and immunologic changes associated with viral pneumonia. symptoms of viral pneumonia include common respiratory tract infection symptoms of cough, fever, and shortness of breath. immunologic changes include up-regulation of airway pro-inflammatory cytokines and pathogenand damage-associated molecular patterns contributing to cytokine and genomic changes. coronavirus disease 2019 clinical presentation is typical of viral pneumonia with an increased prevalence of early pulmonary infiltrates and lymphopenia. principles of early coronavirus disease 2019 management and isolation as well as potential therapeutic approaches to the emerging pandemic are discussed. key words: coronavirus; immunology; influenza virus; severe acute respiratory syndrome; viral pneumonia p neumonia is the leading infectious cause of hospitalization among adults and children in the united states (1) . according to the world health organization (who), lower respiratory tract infection is among the top causes of death globally (2) . the centers for disease control and prevention (cdc) etiology of pneumonia in the community study estimated prevalence of pneumonia-related hospitalizations among adults older than 50 to be 4-25 times higher than those 18 to 49 years old (3) . viral infections are the leading cause of community-acquired pneumonia (cap) and are an important source of morbidity and mortality. severe acute respiratory syndrome coronavirus 2 (sars-cov-2) is a newly discovered virus causing coronavirus disease 2019 (covid-19) that is responsible for an emerging pandemic. given the rapid spread of this virus and its association with severe pulmonary disease, the purpose of this review is to provide an overview of the presentation and immunology of viral pneumonia, principles of early management, and application to covid-19. according to the cdc, the prevalence of cap is highest among adults 65 to 79 years old (4) . hospitalization among adults is highest in elderly patients (≥ 65 yr) and those with preexisting obstructive lung disease or other cardiopulmonary disorders (4, 5) . the most common cause of community-or hospital-acquired pneumonia in adults is viral with the most frequently detected pathogen being human rhinovirus, followed by influenza (9-15% and 4-6%, respectively) (4) (5) (6) (7) (8) . other commonly detected causes of viral pneumonia include adenovirus, conventional coronaviruses, human metapneumovirus (hmpv), respiratory syncytial virus (rsv), and parainfluenza. the prevalence of viral respiratory illness is temporal in north america, with peaks of influenza, hmpv, and rsv normally seen in the winter months ( table 1 ) (1) . the clinical presentation of viral pneumonia does not differentiate between the specific viral causes of respiratory infection. the common clinical presentation of acute viral respiratory infection includes cough, dyspnea, fever, and pleuritic chest pain. viral etiologies of lower respiratory infection are less likely to cause sputum production, and if present, tends to be watery or scant. in contrast, sputum production tends to be mucopurulent when due to bacterial pneumonia (8, 9) . clinical signs of viral respiratory illness include fever, rales (crackles) on auscultation, hypoxemia, and tachycardia. these four signs together have a positive predictive value of 57.1%, with fever as the strongest clinically predictive sign of a viral respiratory infection versus that of bacterial etiology (10) . typically, patients with viral pneumonia also will present with a normal leukocyte count and bilateral pulmonary infiltrates on chest radiograph (9) . severe viral pneumonia can manifest as sepsis and respiratory distress requiring intensive care (11) . in many moderate to severe cases of pneumonia, hypoxemia occurs from impaired alveolar gas exchange (12) , often necessitating mechanical ventilation. biopsies in pneumonia are not routinely performed due to the lack of diagnostic, prognostic, and treatment value. however, since influenza has caused the most viral respiratory epidemics to date, a number of studies have examined infected patient's lung biopsy specimens (13) . biopsies obtained during influenza infection reveal a wide range of pathologies, including alveolar edema and exudate, interstitial inflammatory infiltration, and ulceration of bronchial mucosa to type ii cell metaplasia (14, 15) . in autopsy specimens from h1n1 influenza patients, the respiratory tract exhibited tracheitis, bronchitis, diffuse hemorrhagic alveolar damage, and inflammatory infiltration of alveolar ducts and alveoli (16, 17) . the host response to severe viral lung infection occurs secondary to immune dysregulation leading to lung injury and the systemic inflammatory response. there have been many studies on the immunologic changes associated with influenza a virus (iav). however, little is known about other respiratory viral illnesses in adults. therefore, much of our discussion on the immunology of viral pneumonia will focus on iav studies. during a respiratory infection, airway epithelial cells, natural killer (nk), and cd8 t-cells release interferon-gamma (inf-γ) to limit viral replication (18, 19) . there is additional release of interleukin (il)-6 and il-8, important mediators of tissue damage and associated with disease progression, respectively (20) . high levels of il-17, tumor necrosis factor (tnf)-α, inf-γ, and il-4 have been found in postmortem human lung tissue after severe iav (21) . although there seems to be a difference in cytokine response based on the cause of respiratory infection, there are mixed results in the utility of plasma cytokine levels for prediction of pneumonia etiology (22, 23) . in a recent single-center study, differences in admission plasma levels of il-6, il-10, il-17a, and inf-γ were observed between different etiologies of cap, with inf-γ most elevated in viral cap (24) . conversely, a similar study demonstrated, admission plasma cytokine levels were not statistically different based on etiology (bacterial vs viral vs mixed bacterialviral vs unknown etiology) (25) . other studies noted that serum transforming growth factor-beta (tgf-β) levels predicted viral pneumonia, as opposed to other etiologies of cap, where tgfβ had negative correlations with the sequential organ failure assessment score in patients that progressed to sepsis (26, 27) . therefore, although the specific cytokine profile elicited by particular viruses is unknown, it is clear that, as with most etiologies of sepsis, an elevation of both pro-and anti-inflammatory cytokines are responsible for the host septic and systemic inflammatory response syndrome response in all severe viral cases of pneumonia (23, (28) (29) (30) . as with many other responses to infection, it is pertinent to recognize the role of pathogen-associated molecular patterns (pamps) and damage-associated molecular patterns (damps) in viral respiratory infection. pattern recognition receptors on respiratory epithelial cells, such as toll-like receptors (tlrs), detect evolutionarily conserved microbial ligands, or pamps (31, 32) . viral pamps are typically viral envelope proteins or nucleic acids motifs within the dna or rna genomes of the virus, which are critical for structure and function (33) . the recognition of viral pamps leads to transcription and release of type i interferons (33) which effect decreased expression of viral proteins and replication, enhance antigen presentation and nk cell function, and augment adaptive immune responses. additional recognition of host cell constituents from damaged or dying cells, recognized as damps, are thought to control the magnitude of the immune response (34) (35) (36) . together, pamps and damps play a major role in the initiation of both the innate and adaptive immune response to viral lung infection (31, 35, (37) (38) (39) (40) . viruses can be the primary cause of pneumonia, present in conjunction with bacterial pneumonia, and/or contribute to increased susceptibility to secondary bacterial infection. in addition to influenza, other viruses, such as rhinovirus, can cause severe pneumonia requiring mechanical ventilation, however, this usually occurs in the elderly and immunocompromised (8, 41) . severe pneumonia associated with noninfluenza viruses is also significantly associated with bacterial coinfection (8, (42) (43) (44) , most commonly due to staphylococcus aureus, streptococcus pneumoniae, or haemophilus influenzae (45, 46) . a pattern of dysregulated inflammation caused by viral respiratory infection leads to this increased susceptibility to secondary bacterial pneumonia or coinfection. most research on viral-bacterial respiratory coinfection has been focused on elucidating the pathophysiology of influenza viruses given its high propensity to cause pandemics and higher mortality when compared with the other viruses (13) . influenza a causes a reduction in murine alveolar macrophages and dysregulation of remaining macrophages and neutrophils, one of the body's primary defense mechanisms against bacterial pathogens (47) (48) (49) . additionally, prior infection with influenza virus attenuates bacterial induced release of il-17 leading to decreased innate t cell-mediated bacterial clearance (49) . replication of iav in respiratory epithelium impairs mucociliary clearance, allowing for increased bacterial colonization (34, 50, 51) . additionally, there is evidence of sustained desensitization of tlr ligands following viral infection, leading to decreased chemokine release and nuclear factor kappa b activation in macrophages (52, 53) . this in turn results in attenuated neutrophil recruitment, further decreasing the ability to reduce bacterial load in secondary bacterial infection (52, 53) . although cap remains a significant source of morbidity and mortality, very little work has been done establishing genomic, epigenetic, or transcriptomic changes specifically associated with viral pneumonia (54) . in particular, no clear polymorphism definitively raises the risk of viral pneumonia, limiting personalized medicine for predictive models. in one of the few studies of transcriptomics in viral pneumonia, microarray analysis and ingenuity pathway analysis (qiagen, redwood city, ca) was performed on 19 critically ill patients with 2009 h1n1 influenza a pneumonia. the most severely ill group of 12 patients demonstrated impaired expression of numerous genes participating in adaptive immune responses (e.g., diminished antigen presentation, b-cell development, t-helper cell differentiation, and apoptosis), suggesting impaired adaptive immunity in severe viral pneumonia (55) . in terms of epigenetics, many postulate that longterm epigenetic changes following severe pneumonia are responsible an increased likelihood of later infections and death, although specific epigenetic changes are yet to be identified (54) . natural infection with viral causes of pneumonia does not induce long-term protective immunity due to an evolutionary advantage allowing viruses to evade host immune defenses via antigenic shift and drift. antigenic drift occurs with small point mutations in the viral genome leading to minor changes in key viral epitopes, while antigenic shift is a major change in a key gene leading to a complete exchange of a key epitope (56) . antigenic shift often leads to influenza epidemics secondary to vaccine strain-circulating strain mismatches. antigenic shift is the molecular mechanism by which novel influenza strains emerge and is the cause of pandemics such as the 2009 h1n1 pandemic (57) (58) (59) (60) (61) . influenza vaccines rely on conserved antigens such as ectodomain of influenza m2 protein, m2e, or hemagglutinin stalk domains. hemagglutinin globular head specific antibodies confer immunity since it interferes with virus attachment to host cell receptors; however, they are also one of the most variable viral antigens (56) . additional adjuvants are important in vaccine formulations to induce desired immune responses that would not be triggered with the antigen alone (62) . the need for adjuvants in vaccinations confers an additional important role for damps and pamps in viral immunity. one recent study used pamp tlr9 agonist and commonly used pharmaceutical additive to induce the release of damps to improve immunogenic response to the seasonal influenza vaccine (63) . prevention of viral pneumonia is mainly limited to influenza vaccines since the formalin-inactivated rsv vaccine in the 1960s failed secondary to adverse events (64) . however, oral adenovirus vaccination has been used in military populations with 100-fold reduction of respiratory illnesses (65, 66) . production of this vaccine was stopped in 1999 but was reintroduced in 2011, leading to a dramatic and sustained decrease of acute respiratory distress outbreaks among u.s. army trainees (67, 68) . additionally, there is still ongoing work to develop a vaccine to prevent rsv infection. recently, one study reported protective immunity against rsv with a molecularly adjuvanted adenovirus serotype 5 based rsv oral vaccine in a rat model (69) . however, two recent randomized control failed to establish an effect of anti-rsv monoclonal antibodies and recurrent wheeze of early childhood or asthma (70) (71) (72) (73) . since the covid-19 caused by the novel coronavirus known as sars-cov-2 began its rapid spread in wuhan, china, in november 2019, researchers have responded swiftly to help thwart the pandemic by quickly establishing studies to better understand the virus. sars-cov-2 is a novel beta-coronavirus that likely originated in bats. the virus uses a glycosylated spike protein to bind to and enter the human host cell predominantly via angiotensin-converting enzyme 2 receptors that are highly expressed in type 2 alveolar cells (74) . the clinical presentation of covid-19 can be indistinguishable from other viral causes of pneumonia and include fever (83-98%), dry cough (76-82%), and fatigue or myalgia (11-44%) (74, 75) . the median age of confirmed covid-19 cases is in the 6th decade of life with a slight male predominance. twenty-five percent of patients have severe symptoms requiring intensive care treatment of which 10% develop respiratory failure requiring mechanical ventilation. chest radiograph imaging of these patients reveals bilateral patchy infiltrates and ct imaging shows ground-glass infiltrates. patients typically present with laboratory findings of prolonged prothrombin time, elevated lactate dehydrogenase, and lymphopenia (70% of patients) (76) . however, it is unclear if the lymphopenia is related to direct cytotoxic effect of the virus or underlying chronic conditions (77, 78) . there are limited publications on the autopsy results of patients who have died from covid-19. however, pathologic samples show hyaline membrane formation, interstitial mononuclear inflammatory infiltrates, and multinucleated giant cells. there are also high levels of pro-inflammatory cytokines, such as il-2 and tnf-α. as with other causes of severe viral pneumonia, a "cytokine storm" occurs which also contributes to the high morbidity and mortality (79, 80) . the most important aspect of early management of viral spread has been early isolation of those presenting with concerning symptoms, history, and high likelihood of exposure to prevent spread of the disease to those in immunocompromised states, the elderly, and/or those with comorbid conditions. a chest radiograph along with throat and mid-turbinate nasal swabs for respiratory viral panel (reverse transcriptase-polymerase chain reaction) are needed for proper diagnosis of covid-19. among hospitalized patients, negative pressure rooms and airborne-droplet-contact precautions are important for prevention and further spread between patients and hospital care-workers (81) . currently, there is no approved drug or vaccination for the treatment or prevention of sars-cov-2 viral pneumonia. there are many trials underway attempting to attenuate the disease with remdesivir, il-6 receptor blockers, il-7, and antiretrovirals such as lopinavir-ritonavir (82) . the new england journal of medicine recently published a randomized controlled trial evaluating the efficacy of lopinavirritonavir versus standard care alone in the treatment of adult hospitalized patients with severe covid-19. there were no differences in hospital mortality, time to clinical improvement, or viral rna levels. although the median time to improvement was 1 day shorter with lopinavir-ritonavir on intention-to-treat analysis, 14% of patients had adverse events requiring treatment discontinuation. therefore, it was concluded that there was no benefit observed with lopinavir-ritonavir treatment versus standard treatment of severe covid-19 patients (83) . historically, hydroxychloroquine, an anti-malarial and antiinflammatory agent, has shown some promise in reducing mortality from sars and, therefore, is currently being studied for covid-19 (84) . in one very limited study from france (n = 20 per group, nonrandomized), hydroxychloroquine was associated with reduced viral load and reduced duration of viral detection which was further attenuated by the addition of azithromycin (85) . research is already underway to create a vaccine to protect against sars-cov-2. taking advantage of the similarities in structure between sars-cov (responsible for the 2003 sars epidemic) and sars-cov-2 (responsible for covid-19), studies have mapped several epitopes to be targeted for a potential vaccine (86, 87) . who estimates an approximately 18-month timeframe for covid-19 vaccine availability. until such time that effective therapies and vaccines become available, public health efforts should continue to focus on mitigating the spread of sars-cov-2 through well-established infection control strategies (88) . this can be aided in the hospital with admission of sars-cov-2 positive patients into negative pressure rooms with contact precaution protocols requiring personal protective equipment such as gowns, gloves, fit-tested n95 respirators, and face shields. additionally, rules limiting the people entering the isolation room and requiring logging of healthcare workers involved in covid-19 patient care should be followed to effectively monitor patient contact and limit spread. all equipment (monitors, etc.) in the isolation room should be designated for the case patient only. physicians should limit potential spread by recognizing any necessary aerosol-generating procedures and preparing accordingly (e.g., for intubation using controlled measures including paralytics, video laryngoscopy, n95 masks). although fomites are suspected as the main source of transmission, there is also possible fecal-oral transmission; therefore, hand washing is a mainstay of control/prevention (89) . although viral pneumonia is common, the specific inflammatory and immunosuppressive effects it has on the host is still largely unknown. covid-19 has brought viral pneumonia and subsequent host pathology to the forefront of medical care and research. sars-cov-2 spread worldwide in a matter of months to cause a pandemic not seen since influenza in 1918. our highly interconnected global society creates ample opportunity for the rapid spread of novel viruses. since these types of viral pandemics have occurred multiple times historically (e.g., influenza in 1918, middle east respiratory syndrome in 2014, and sars in 2004) and will continue to occur in the future, research into immunomodulative therapies for patients afflicted with viral pneumonia will be a key aspect to improving outcomes after viral pneumonia. a personalized approach, taking into account differences in the biology of individuals and the pathophysiology of different viruses, will also be required to make significant progress in the treatment of these patients. the authors have disclosed that they do not have any potential conflicts of interest. for information regarding this article, e-mail: philip.efron@surgery.ufl.edu most frequent conditions in u.s. hospitals, 2011: statistical brief #162. healthcare cost and utilization project (hcup) statistical briefs world health organization: the top 10 causes of death cdc epic study team: community-acquired pneumonia requiring hospitalization among u.s. adults epidemiology of viral pneumonia viruses are prevalent in nonventilated hospital-acquired pneumonia systematic review of respiratory viral pathogens identified in adults with community-acquired pneumonia in europe viral infection in community-acquired pneumonia: a systematic review and meta-analysis clinical characteristics and outcomes of severe rhinovirus-associated pneumonia identified by bronchoscopic bronchoalveolar lavage in adults: comparison with severe influenza virus-associated pneumonia viral infection in adults hospitalized with community-acquired pneumonia: prevalence, pathogens, and presentation predictors of pneumonia in lower respiratory tract infections: 3c prospective cough complication cohort study the burden of viruses in pneumonia associated with acute respiratory failure: an underappreciated issue hypoxic pulmonary vasoconstriction: from molecular mechanisms to medicine influenza virus-related critical illness: pathophysiology and epidemiology pathologic features of lung biopsy specimens from influenza pneumonia cases immunohistochemical and in situ hybridization studies of influenza a virus infection in human lungs asian influenza a in boston, 1957-1958. i. observations in thirty-two influenza-associated fatal cases activated mouse eosinophils protect against lethal respiratory virus infection antiviral instruction of bone marrow leukocytes during respiratory viral infections insight flu 002 & 003 study groups: the association between serum biomarkers and disease outcome in influenza a(h1n1)pdm09 virus infection: results of two international observational cohort studies cd206+ cell number differentiates influenza a (h1n1)pdm09 from seasonal influenza a virus in fatal cases capnetz study group: interleukin 6, lipopolysaccharide-binding protein and interleukin 10 in the prediction of risk and etiologic patterns in patients with community-acquired pneumonia: results from the german competence network capnetz systemic cytokine response in patients with community-acquired pneumonia plasma cytokine profile on admission related to aetiology in community-acquired pneumonia cytokine responses, microbial aetiology and short-term outcome in community-acquired pneumonia relevant cytokines in the management of community-acquired pneumonia tgf-β blood levels distinguish between influenza a (h1n1)pdm09 virus sepsis and sepsis due to other forms of community-acquired pneumonia local and systemic cytokine profiles in nonsevere and severe community-acquired pneumonia genims investigators: understanding the inflammatory cytokine response in pneumonia and sepsis: results of the genetic and inflammatory markers of sepsis (genims) study the association between mortality rates and decreased concentrations of interleukin-10 and interleukin-1 receptor antagonist in the lung fluids of patients with the adult respiratory distress syndrome innate immunity to virus infection nlrs and rlrs: a trinity of pathogen sensors that co-operate in innate immunity recognition of viruses by cytoplasmic sensors viral infection of the lung: host response and sequelae inflammasomes as mediators of immunity against influenza virus molecular identification of a danger signal that alerts the immune system to dying cells influenza virus activates inflammasomes via its intracellular m2 ion channel the inflammasome recognizes cytosolic microbial and host dna and triggers an innate immune response differential role of tlr-and rlrsignaling in the immune responses to influenza a virus infection and vaccination tlr signaling fine-tunes antiinfluenza b cell responses without regulating effector t cell responses incidence and characteristics of viral community-acquired pneumonia in adults influenza and bacterial coinfection in adults with community-acquired pneumonia admitted to conventional wards: risk factors, clinical features, and outcomes pneumonia with bacterial and viral coinfection seasonality of invasive pneumococcal disease: temporal relation to documented influenza and respiratory syncytial viral circulation the immunology of influenza virus-associated bacterial pneumonia bench-to-bedside review: bacterial pneumonia with influenza -pathogenesis and clinical implications marked improvement of severe lung immunopathology by influenza-associated pneumococcal superinfection requires the control of both bacterial replication and host immune responses depletion of alveolar macrophages during influenza infection facilitates bacterial superinfections influenza a inhibits th17-mediated host defense against bacterial pneumonia in mice influenza virus infection decreases tracheal mucociliary velocity and clearance of streptococcus pneumoniae adherence of type i streptococcus pneumoniae to tracheal epithelium of mice infected with influenza a/pr8 virus toll-like receptor 4 agonistic antibody promotes innate immunity against severe pneumonia induced by coinfection with influenza virus and streptococcus pneumoniae sustained desensitization to bacterial toll-like receptor ligands after resolution of respiratory influenza infection community-acquired pneumonia: genomics, epigenomics, transcriptomics, proteomics, and metabolomics host adaptive immunity deficiency in severe pandemic influenza influenza virus: dealing with a drifting and shifting pathogen changes in and shortcomings of control strategies, drug stockpiles, and vaccine development during outbreaks of avian influenza a h5n1, h1n1, and h7n9 among humans human infection with a novel avian-origin influenza a (h7n9) virus writing committee of the whococaopi: clinical aspects of pandemic 2009 influenza a (h1n1) virus infection global epidemiology of influenza: past and present influenza vaccine effectiveness in the united states during the 2015-2016 season nucleic acid sensing at the interface between innate and adaptive immunity in vaccination damp-inducing adjuvant and pamp adjuvants parallelly enhance protective type-2 and type-1 immune responses to influenza split vaccination viral pneumonia follow-up analysis of the incidence of acute respiratory infections among enlisted service members during their first year of military service before and after the 2011 resumption of adenovirus vaccination of basic trainees dramatic decline of respiratory illness among us military recruits after the renewed use of adenovirus vaccines acute respiratory disease in us army trainees 3 years after reintroduction of adenovirus vaccine (1) oral adenoviral-based vaccines: historical perspective and future opportunity orally administered adenoviral-based vaccine induces respiratory mucosal memory and protection against rsv infection in cotton rats respiratory syncytial virus prevention and asthma in healthy preterm infants: a randomised controlled trial respiratory syncytial virus and recurrent wheeze interactions between human and carp (cyprimus carpio) low density lipoproteins (ldl) and ldl receptors does respiratory syncytial virus lower respiratory illness in early life cause recurrent wheeze of early childhood and asthma? critical review of the evidence and guidance for future studies from a world health organization-sponsored meeting covid-19-new insights on a rapidly changing epidemic china medical treatment expert group for covid-19: clinical characteristics of coronavirus disease 2019 in china lymphopenic community acquired pneumonia as signature of severe covid-19 infection clinical course and outcomes of critically ill patients with sars-cov-2 pneumonia in wuhan, china: a single-centered, retrospective, observational study shared features of endothelial dysfunction between sepsis and its preceding risk factors (aging and chronic disease) immune responses in covid-19 and potential vaccines: lessons learned from sars and mers epidemic clinical features of patients infected with 2019 novel coronavirus in wuhan, china diagnosis and management of first case of covid-19 in canada: lessons applied from sars chinese clinical trial register: a randomized, controlled open-label trial to evaluate the efficacy and safety of lopinavir-ritonavir in hospitalized patients with novel coronavirus pneumonia (covid-19) a trial of lopinavir-ritonavir in adults hospitalized with severe covid-19 covid-19 -navigating the uncharted hydroxychloroquine and azithromycin as a treatment of covid-19: results of an open-label non-randomized clinical trial preliminary identification of potential vaccine targets for the covid-19 coronavirus (sars-cov-2) based on sars-cov immunological studies receptor recognition by the novel coronavirus from wuhan: an analysis based on decade-long structural studies of sars coronavirus review of the clinical characteristics of coronavirus disease 2019 (covid-19) world health organization: coronavirus disease (covid-19) technical guidance: infection prevention and control/wash. 2020 key: cord-323066-tvguutak authors: praznik, ajda; vinšek, neža; prodan, ana; erčulj, vanja; pokorn, marko; mrvič, tatjana; paro, darja; krivec, uroš; strle, franc; petrovec, miroslav; žnidaršič eržen, marta; grosek, štefan title: risk factors for bronchiolitis severity: a retrospective review of patients admitted to the university hospital from central region of slovenia date: 2018-08-09 journal: influenza other respir viruses doi: 10.1111/irv.12587 sha: doc_id: 323066 cord_uid: tvguutak aim: study's objective was to identify risk factors associated with bronchiolitis severity. methods: a retrospective chart review of all children <2 years old diagnosed with bronchiolitis at the university medical centre ljubljana between may 2014 and april 2015, who were treated as outpatients (paediatric emergency department, ped group) or as inpatients in the standard hospital setting (ward group) or in the paediatric intensive care unit (picu group). detection of respiratory viruses in nasopharyngeal swab was accomplished by rt‐pcr. severity was assessed by wang respiratory score and hospitalization longer than 24 hours. results: the study included 761 children. the three most frequently detected viruses were respiratory syncytial virus (rsv), human rhinovirus (hrv) and human bocavirus (hbov) (57.5%, 272/473; 25.6%, 121/473; 18.4%, 87/473). patient groups differed in wang respiratory score for the severity of bronchiolitis (p < 0.001). no differences regarding the causative viruses were found. there was a lower proportion of children with the presence of more than one virus in picu group compared to other two groups (p = 0.017). the three groups significantly differed in age, birthweight, comorbidities, bronchodilator treatment and antibiotic usage. however, multiple regression analysis revealed that younger age and the use of antibiotics were associated with bronchiolitis severity defined as hospitalization for >24 hours. conclusions: respiratory syncytial virus, hrv and hbov were the most frequently detected viruses. the majority of patients admitted to the picu had only one virus detected. younger age and the use of antibiotics were associated with bronchiolitis severity. bronchiolitis is a potentially life-threatening viral respiratory infection that affects children under 2 years of age. 1 it is common and occurs worldwide. the bronchiolitis mortality rate is approximately 2 per 100 000 infants and is higher in developing than in developed countries. 2 the most commonly identified causative agent is respiratory syncytial virus (rsv). 2 other viruses implicated in the aetiology of bronchiolitis include human metapneumovirus (hmpv), parainfluenza virus (piv), influenza virus a and b virus (infv), human rhinovirus (hrv), human coronavirus (hcov), adenovirus (adv), enterovirus (ev) and human bocavirus (hbov). 3 the majority of children with bronchiolitis can be managed as outpatients; approximately 2%-3% of patients younger than 1 year are hospitalized, usually during the seasonal rsv epidemics that occur in cold months. 1 treatment of bronchiolitis is symptomatic and focuses on the maintenance of hydration, oxygenation and antipyresis. in severe cases, intensive care management may be needed with continuous positive airway pressure (cpap), intubation and mechanical ventilation. some infants with secondary bacterial infection need antibiotic treatment. [4] [5] [6] in infants with pre-existing medical conditions and immunodeficiency, complications such as acute respiratory distress syndrome (ards), bronchiolitis obliterans, congestive heart failure and secondary bacterial infection may develop. [4] [5] [6] [7] [8] although there is no universally accepted measure for the assessment of bronchiolitis severity, the wang respiratory score, using clinical observation (general appearance, respiratory rate, presence of wheezing and retractions), is widely used. 9 children with bronchiolitis and wang respiratory score 3 or less can be managed on an outpatient basis, while children with scores 4-8 usually require hospitalization and those with even higher scores are admitted to the picu. 10 the objective of our study was to ascertain demographic characteristics, clinical findings and presumptive aetiologic agents (respiratory viruses demonstrated in nasopharyngeal swab) associated with bronchiolitis severity defined as length of hospitalization for >24 hours. the study was approved by the national medical ethics committee of the republic of slovenia (kme 37/09/15) (nmec rs). the study was conducted at the university medical centre ljubljana (umcl), a university hospital serving the central region of slovenia with a population of around 700 000 inhabitants (approximately one-third of all slovenian population). we performed a retrospective chart review of all patients with bronchiolitis <2 years of age, referred to the three paediatric departments, that is umcl children's hospital, department of infectious diseases and paediatric intensive care unit (picu) between 1 may 2014 and 30 april 2015. we excluded patients that had been previously diagnosed with asthma, and all previous episodes if a patient had more than one episode of bronchiolitis in studied time span. patients were identified through a query of the electronic medical record based on a bronchiolitis diagnosis (icd-10 code j21.0-9). electronic medical records of patients included in the study were reviewed, and statistical analysis was performed for the following clinical and laboratory data: gender, chronological age at admission, prematurity (defined as birth before 37 weeks of gestation), birthweight, history of allergies, number of previous bronchiolitis episodes, clinical manifestations of bronchiolitis using the wang respiratory score, 9 comorbidities (chronic lung disease, congenital heart disease, immune deficiency or neuromuscular diseases), body temperature at admission, treatment with bronchodilators, antibiotics or supplemental oxygen and respiratory virus detected in the nasopharyngeal swab. only the first swab taken from individual patient was included. swabs were routinely taken from patients in two departments, while in another one sampling was sparse. high season and low season of incidence were also included as variables. using rrt-pcr, the sensitivity of the nasopharyngeal swab is above 90% compared to a composite gold standard. 13, 14 for 288 patients, no swab analysis was performed. bronchiolitis severity was assessed by wang respiratory score and as a duration of hospitalization for >24 hours. numerical data were presented as median (range) and categorical as frequencies (percentages). the differences in categorical variables among the three groups were tested using chi-square test or likelihood ratio test; kruskal-wallis test was performed for numerical variables. the clinical and laboratory findings were compared between the three groups of patients, defined according to the site of management: patients treated as outpatients (paediatric emergency department, ped group), patients treated in the standard hospital setting (ward group) and children admitted to the paediatric intensive care unit (picu group). multiple logistic regression analysis was used to identify key variables associated with severe bronchiolitis, defined with the duration of hospitalization for >24 hours. a p-value < 0.05 was considered statistically significant. all analyses were performed using spss 23.0. the study group comprised 761 children with bronchiolitis. there was a male predominance with 468 (61.5%) boys. of 761 children, 138 were treated as outpatients (ped group), 599 were hospitalized at a regular paediatric unit (ward group), and 24 were treated in a paediatric intensive care unit (picu group). nasopharyngeal swabs were taken in 473/761 (62%) children. of 623 patients admitted to hospital, 413 (54%) were hospitalized for >24 hours. the median length of hospital stay was 12 days in picu group and 2 days in ward group (p < 0.001). the three study groups differed in wang's score for particular clinical sign parameter (p < 0.001) apart from wheezing, and in the need for supplemental oxygen treatment (p < 0.001)-only a few patients required oxygen treatment in the outpatient ped group, while almost all patients needed oxygen treatment in picu group. the three study groups differed in wang score (p < 0.001) ( figure 1 ). they also differed in several clinical and microbiological characteristics ( table 1) analysis of the presence of more than one virus in nasopharyngeal swab revealed that rsv and hbov were the two most frequently simultaneously detected viruses and that the proportion of patients with more than one of detected viruses in individual swab was statistically significantly lower in picu than in ped or ward (p = 0.017) ( table 1) . multiple logistic regression analysis showed that out of several factors only younger chronological age (p < 0.001) and treatment with antibiotics (p = 0.003) were associated with severe bronchiolitis defined as hospitalization >24 hours. (table 2 ). in the present study, we compared characteristics of children with bronchiolitis treated in the outpatient clinic, admitted to the ward or admitted to the paediatric intensive care unit. age, prematurity, comorbidity and treatment with bronchodilators and antibiotics, differed in the three groups suggesting these factors relate to disease severity. the type of virus did not correlate with treatment group (table 1 ). further analyses revealed that chronological younger age and treatment with antibiotics independently were associated with hospitalization longer than 24 hours (table 2) . chronological age is known as the most important predictor of severe bronchiolitis. similar to study, several others have found a significant association between the age of less than 6 months and a higher risk of hospitalization and severe bronchiolitis. [15] [16] [17] [18] however, grimwood et al did not find a significant correlation between children aged less than 2 months and bronchiolitis severity in a multivariate analysis. 19 hospitalization rates that are attributable to rsv bronchiolitis are usually the highest between 30 and 90 days after birth. 20 this age coincides with the declining concentration of transplacentally acquired maternal anti-rsv immunoglobulin, which protects infants against disease. 20, 21 certain studies have shown that prematurity is independently associated with more severe bronchiolitis, 15, 16, 22 while the others have not found significantly higher rates of hospitalization and severe bronchiolitis among premature compared to full-term infants. 17, 20, 23 in the present study, the proportion of preterm children was highest in the picu group and lowest in the ped group, which suggested a more severe bronchiolitis. however, multiple logistic regression model did not show a correlation between prematurity and in the present study, antibiotic treatment was significantly associated with severe bronchiolitis, which is in accordance with some previous reports. 24, 25 although the design of our study did not enable analysis of the appropriateness of antibiotic therapy, the finding that almost all patients in the picu group received antibiotic treatment compared to only 3.6% in the ped group might suggest that bacterial superinfections lead to a more severe course of bronchiolitis. another frequently mentioned risk factor for severe bronchiolitis is comorbidity. 23, [26] [27] [28] in our study, one-third of children hospitalized in picu had at least one comorbidity, while in the ped group the corresponding proportion was significantly lower. however, again, in the multiple logistic regression analysis comorbidities did not correlate with the severe course of bronchiolitis (table 2) as shown also in some other studies. 1, 16 one should take into account that our study did not analyse the association of comorbidities with specific virus types as done in mentioned studies. 5, 15, 26, 29 another limitation is that we did not analyse specific comorbidities, but only as a group determined by previous studies. 24, 30, 31 this is because there were only 31 children with comorbidity and we believe that dividing the comorbidity group into smaller groups would not give reliable information. the present study showed that rsv, hrv and hbov were the most frequently present viruses in nasopharyngeal swab of children with bronchiolitis. the predominance of rsv is in agreement with several previous reports. 3, 5, 32 the second most common pathogen is usually hrv, followed by infv or piv 5, 19, 33 while in our study hbov ranked the third. some reports indicated that hbov is rarely detected as a single agent in hospitalized children with bronchiolitis, leading to speculation that this virus is more likely to be an innocent bystander than a true pathogen. 5 however, uršič et al have recently reported the first fatal case of an extremely severe bronchiolitis caused by hbov in an immunocompetent child. 16 the absence of association between virus type and characteristics of patients is in agreement with the findings of several previous articles. 6, 17, 18 nevertheless, in some reports, differences in the severity of the disease caused by various types of viruses or viral co-infections were established. 1, 34, 35 according to our study, multiple viruses detected in the nasopharyngeal swab were moderately associated with a more severe course of the bronchiolitis, although not significant (p = 0.091). other studies proved that children with viral coinfection were less likely to be admitted to intensive care unit than children with single virus infection. 21, 22 possible explanation is that as one or more viral respiratory pathogens can be detected in the upper respiratory tract of as many as 30% of asymptomatic young children, 36, 37 it is likely that in several patients with bronchiolitis and more than one virus detected in nasopharyngeal swab, one of the viruses is responsible for the acute infection while the others are innocent bystanders, possibly as a result of prolonged shedding after an already resolved infection. it is not unusual that a child has a history of more than one episode of bronchiolitis. 38 studies suggest that repeated infections by rsv can be in part due to variability of the virus strains. 39 acute bronchiolitis can cause (transitory) anatomical and histological changes in lower respiratory tract that may prone to a more severe new episode. 29 however, in accordance with some previous reports 40 in our study, the number of previous episodes of bronchiolitis did not correlate with bronchiolitis severity. our study also showed that the proportion of children who received bronchodilators was statistically different in the ped, ward and picu group (table 1) . this is probably due to the common practice in several countries (such as united states, switzerland and belgium) that children exhibiting a moderately severe bronchiolitis are more often given bronchodilators than those with mild or severe disease. [41] [42] [43] the data on the efficiency of bronchodilators in bronchiolitis are conflicting. 41 many studies agree that bronchodilators may improve clinical symptom scores, but they do not affect disease resolution, need for hospitalization, or length of stay. 5 interpretation. in addition, children with performed viral diagnosis were younger and were treated in the high seasonal months. comparison of the three groups regarding virus repartition should be taken with precaution as the sample of children treated in the standard hospital setting with viral data available seems to be biased. data are not missing at random, but are available to higher extent for younger children and for children treated in the high season months. in conclusion, our study revealed that rsv, hrv and hbov were the most frequently detected viruses in children with bronchiolitis. chronological younger age and the use of antibiotics were associated with severe bronchiolitis defined as hospitalization longer than 1 day. further prospective studies are needed to assess the importance of various respiratory viruses and host factors in this common paediatric illness. we thank the institute of microbiology and immunology and the national institute of public health who provided us with data necessary for statistical analysis. the authors have no competing interests. ana prodan http://orcid.org/0000-0002-1116-2844 the impact of dual viral infection in infants admitted to a pediatric intensive care unit associated with severe bronchiolitis risk factors for bronchiolitis-associated deaths among infants in the united states impact of human rhinovirus types and viral load on the severity of illness in hospitalized children with lower respiratory tract infections ljubljana: združenje za infektologijo, slovensko zdravniško društvo ljubljana viral bronchiolitis in children diagnosis and management of bronchiolitis clinical findings and severity of acute bronchiolitis bronchodilators for treatment of mild bronchiolitis: a factorial randomised trial observer agreement for respiratory signs and oximetry in infants hospitalized with lower respiratory infections e-newsletter on communicable diseases and environmental health, enboz -elektronske novice s področja nalezljivih bolezni in okoljskega zdravja the role of human coronaviruses in children hospitalized for acute bronchiolitis, acute gastroenteritis, and febrile seizures: a 2-year prospective study comparing nose-throat swabs and nasopharyngeal aspirates collected from children with symptoms for respiratory virus identification using real-time polymerase chain reaction comparison of nasal and nasopharyngeal swabs for influenza detection in adults the burden of respiratory syncytial virus infection in young children incidence and risk factors of hospitalization for bronchiolitis in preterm children: a retrospective longitudinal study in italy respiratory syncytial virus infection in navajo and white mountain apache children comparison of risk factors for human metapneumovirus and respiratory syncytial virus disease severity in young children risk factors for respiratory syncytial virus bronchiolitis hospital admission in new zealand respiratory syncytial virus-associated hospitalizations among children less than 24 months of age respiratory syncytial virus transplacental antibody transfer and kinetics in mother-infant pairs in bangladesh risk factors in children hospitalized with rsv bronchiolitis versus non-rsv bronchiolitis rates of hospitalization for respiratory syncytial virus infection among children in medicaid risk factors for respiratory syncytial virus hospitalisation in children with heart disease variation in inpatient diagnostic testing and management of bronchiolitis pre-existing disease is associated with a significantly higher risk of death in severe respiratory syncytial virus infection updated guidance for palivizumab prophylaxis among infants and young children at increased risk of hospitalization for respiratory syncytial virus infection respiratory syncytial virus and parainfluenza virus evaluation of risk factors for recurrent wheezing episodes risk factors for hospital admission with rsv bronchiolitis in england: a population-based birth cohort study chronic diseases, chromosomal abnormalities, and congenital malformations as risk factors for respiratory syncytial virus hospitalization: a population-based cohort study respiratory syncytial virus, human bocavirus and rhinovirus bronchiolitis in infants the relationship of meteorological conditions to the epidemic activity of respiratory syncytial virus multicenter study of viral etiology and relapse in hospitalized children with bronchiolitis viral etiologies of infant bronchiolitis, croup and upper respiratory illness during 4 consecutive years frequent detection of respiratory viruses without symptoms: toward defining clinically relevant cutoff values respiratory viral detection in children and adults: comparing asymptomatic controls and patients with community-acquired pneumonia bronchiolitis (and rsv) in infants and children molecular epidemiology of respiratory syncytial virus infections among children with acute respiratory symptoms in a community over three seasons respiratory syncytial virus disease in preterm infants in the u.s. born at 32-35 weeks gestation not receiving immunoprophylaxis bronchiolitis management preferences and the influence of pulse oximetry and respiratory rate on the decision to admit current management of acute bronchiolitis in switzerland bronchiolitis management by the belgian paediatrician: discrepancies between evidence-based medicine and practice clinical practice guideline: the diagnosis, management, and prevention of bronchiolitis efficacy of bronchodilator therapy in bronchiolitis. a meta-analysis risk factors for bronchiolitis severity: a retrospective review of patients admitted to the university hospital from central region of slovenia key: cord-312928-ef8hqs4s authors: chavanet, pascal title: viral upper respiratory tract infection and otitis media complication in young children date: 2008-03-15 journal: clin infect dis doi: 10.1086/528686 sha: doc_id: 312928 cord_uid: ef8hqs4s nan viral infections in children are very common and raise huge problems of management. the burden of these infections is difficult to quantify because of the lack of pathognomonic features. there is a clear need to know the characteristics of these infections to give directions for care and possibly to provide specific treatments, prevention, and prophylaxis. in this issue of clinical infectious diseases, chonmaitree et al. [1] conducted a large, longitudinal observational study of children with upper respiratory infection and examined for otitis complicationeither acute otitis media or otitis media with effusion. they found that 63% of children were virus positive and concluded that the rate of otitis media complication was 61% (37% for acute otitis media and 24% for otitis media with effusion), that these risks decrease as the age increases, and that adenovirus, coronavirus, and respiratory syncitial virus represent more than two-thirds of otitis media complications. these findings confirm the findings of previous works but with differences. the main problem with this kind of study is the accuracy and sensitivity of viral investigations. indeed, in this study, the rate of respiratory syncitial virus infection was found to be low, probably as a result of the method used (conventional assays, type of molecular technique, rt-pcr vs. microarray, etc). furthermore, human metapneumovirus and bocavirus were not studied [2, 3] . thus, extensive (one-third of the children in the study were not virus positive) and "stabilized" viral investigations are still needed to capture the real facts, to create a precise hierarchy of all of the different pathogens involved [4] . however, the findings of chonamaitree et al. [1] also confirm that respiratory syncitial virus and influenza virus are 2 of the main causes of complications of upper respiratory infection. in this study, children aged !1 year and those who attended day care centers had a greater risk of acquiring upper respiratory tract infection and otitis media, compared with older children and those who were cared for at home. this finding is well known; however, the expectation that ill children can be kept out of day care centers or that home care can be provided for children until at least 1 year of age raises very difficult socioeconomic barriers. it should be noted that chonamaitree et al. [1] did not mention the rate of antibiotic treatment [5] or the number of hospitalizations [6] . management of viral infection in children is still complicated by genetics. in fact, it was recently reported that suscep-tibility to respiratory syncitial virus bronchiolitis, for example, is related in part to a genetic profile of innate immunity [7] . this article is important, especially because the rate of complications of otitis in upper respiratory tract infection was longitudinally studied. however, the results have to be integrated with other investigations that use different diagnostic tests. viral upper respiratory tract infection and otitis media complication in young children prevalence of respiratory viruses, including newly identified viruses, in hospitalised children in austria epidemiological and clinical features of hmpv, rsv and rvs infections in young children respiratory virus infections. paediatr antibiotics for acute otitis media: a meta-analysis with individual patient data rates of hospitalisation for influenza, respiratory syncytial virus and human metapneumovirus among infants and young children genetic susceptibility to respiratory syncytial virus bronchiolitis is predominantly associated with innate immune genes potential conflicts of interest. p.c.: no conflicts. key: cord-321851-ku4z34lu authors: alosaimi, bandar; hamed, maaweya e.; naeem, asif; alsharef, ali a.; alqahtani, saeed y.; aldosari, kamel m.; alamri, aref a.; al-eisa, kholoud; khojah, taghreed; assiri, abdullah m.; enani, mushira a. title: mers-cov infection is associated with downregulation of genes encoding th1 and th2 cytokines/chemokines and elevated inflammatory innate immune response in the lower respiratory tract date: 2020-02-29 journal: cytokine doi: 10.1016/j.cyto.2019.154895 sha: doc_id: 321851 cord_uid: ku4z34lu abstract mers-cov, a highly pathogenic virus in humans, is associated with high morbidity and case fatality. inflammatory responses have a significant impact on mers-cov pathogenesis and disease outcome. however, cd4+ t-cell induced immune responses during acute mers-cov infection are barely detectable, with potent inhibition of effector t cells and downregulation of antigen presentation. the local pulmonary immune response, particularly the th1 and th2-related immune response during acute severe mers-cov infection is not fully understood. in this study, we offer the first insights into the pulmonary gene expression profile of th1 and th2-related cytokines/chemokines (th1 & th2 responses) during acute mers-cov infection using rt2 profiler pcr arrays. we also quantified the expression level of primary inflammatory cytokines/chemokines. our results showed a downregulation of th2, inadequate (partial) th1 immune response and high expression levels of inflammatory cytokines il-1α and il-1β and the neutrophil chemoattractant chemokine il-8 (cxcl8) in the lower respiratory tract of mers-cov infected patients. moreover, we identified a high viral load in all included patients. we also observed a correlation between inflammatory cytokines, th1, and th2 downregulation and the case fatality rate. th1 and th2 response downregulation, high expression of inflammatory cytokines, and high viral load may contribute to lung inflammation, severe infection, the evolution of pneumonia and ards, and a higher case fatality rate. further study of the molecular mechanisms underlying the th1 and th2 regulatory pathways will be vital for active vaccine development and the identification of novel therapeutic strategies. middle east respiratory syndrome (mers) is a novel viral respiratory illness caused by the mers-cov that was first described in june 2012 in a patient who was hospitalized with signs of severe respiratory tract infection. the patient later died from respiratory and renal failure [1] [2] [3] . according to the statistics provided by the world health organization (who) on june 10, 2019, a total of 2428 laboratory-confirmed cases of human mers-cov infection have been reported, with an estimated 838 deaths in 27 countries. the accumulated evidence suggested that mers-cov infections are characterized by high levels of systemic inflammatory cytokines/chemokines as well as immunopathology [4] [5] [6] [7] [8] [9] [10] [11] . high inflammatory cytokines and chemokines levels have been strongly correlated with poor disease outcomes, immunopathology and massive infiltration of the immune inflammatory cells into the lungs [10, 12] . moreover, the stimulation of various cytokines, chemokines and innate immune cells has been associated with the appearance of cytokine storms, which occur during infections with pathogenic coronavirus (cov) and influenza virus [6, 13, 14] . in addition, infection, viral bronchiolitis pathogenesis, severe immunopathology, and disease enhancement during respiratory syncytial virus (hrsv) infection [10, 12, [15] [16] [17] . likewise, increased levels of inflammatory il-1β and il-1α cytokines have been associated with tissue damage and acute inflammatory responses leading to mortality and severe pathogenesis, as well as induction of the inflammatory loop [14, 18, 19] . mers-cov evades and antagonizes the antiviral immune response, as well as the nuclear factor-κb (nf-κb) signaling pathway and the ifn signaling cascade [20] [21] [22] . the disease pathogenesis of mers-cov infection is complex, with various factors involved in the onset of severe pulmonary damage and dissemination of the virus to other organs. th1 cells cytokines ifn-γ, tnf-α, tnf-β and il-2 play a key role in antiviral immunity by enhancing the toxic effects of cd8 t cells, stimulate nk cells and contribute to the regulation of cellular immunity [9, [23] [24] [25] [26] [27] [28] [29] . th2 cells secrete il-4, il-5, il-6, il-9, il-10, and il-13 which mediate humoral immune response and stimulate antibody production [9, [23] [24] [25] [26] [27] [28] [29] . imbalanced th1/th2 cytokines/chemokines can contribute to the pathological change, inefficient clearing infections, and host susceptibility to immune-mediated diseases. in vivo data highlights the important roles of t cells in controlling and reducing the pathogenesis and decreasing mers-cov titers [30] . there have been no reports describing the complete cellular immune response of the lower respiratory tract to mers-cov. therefore, it is still unclear whether the cd4 + t-helper (th1/th2)-induced cytokines/chemokines are beneficial or harmful for mers-cov-infected patients. moreover, the host immune responses which are activated in the lower respiratory tract during a mers-cov infection also remain unclear. in this study, we offer the first insight into the pulmonary molecular gene expression profile of th1 and th2 cytokines/chemokines (th1 & th2 responses) during acute mers-cov infection using rt 2 profiler pcr. we also describe the expression level of primary inflammatory cytokines and chemokines in the lower respiratory tract. the institutional review board at king fahad medical city reviewed and approved the study protocol (irb register number 17-182). the handling of respiratory samples, as well as aliquoting and viral and cellular rna extraction were executed using appropriate personal protective equipment in a biosafety level 3 laboratory (riyadh regional laboratory, ministry of health, riyadh, saudi arabia) the lower respiratory samples were collected from 39 mers-cov positive patients and 30 healthy non-infected individuals. to exclude effects of antiviral therapy on the expression of cytokines/chemokines understudy, the samples were collected before the initiation of any antiviral treatment for mers-cov. for the sputum sample collections, the patients were asked to rinse their mouth and gargle with water twice immediately before obtaining the sample and instructed not to expectorate saliva or postnasal discharge into the container during collection to reduce the possibility of contamination with upper respiratory tract fluids. subsequently, the patient was requested to breathe and expectorate deep cough sputum directly into a sterile sputum cup (screw cap). tracheal aspirate (ta) was collected via the endotracheal tube as reported elsewhere [31, 32] . bronchoalveolar lavage (bal) was collected by following a standardized protocol (technique, sampling, and procedure) as previously described [33] [34] [35] . the first bal fluid aliquots recovered were discarded to reduce cross-contamination with upper respiratory tract fluids. in order to exclude the effect of viral co-infection with influenza a h1n1 on cytokines/chemokines expression levels, all mers-cov positive patients were screened for h1n1 by genexpert flu (cepheid, sunnyvale, ca) according to the manufacturer′s instructions. samples were centrifuged at 1000 rpm at 4°c for 5-10 min. the cell-free supernatants were frozen at −80°c until they were used for subsequent analysis of the inflammatory cytokine and chemokine by elisarray, as well as for the mers-cov viral load test. the pellets were used for total cellular rna extraction using the rneasy mini kit (qiagen, valencia, ca) to detect mers-cov rna concentrations in the lower respiratory tract, viral rna was isolated from 140 µl of lower respiratory specimens using the qiaamp viral rna extraction mini kit (qiagen) according to the manufacturer′s instructions. the rna was dissolved in 60 µl of elution buffer and stored in separate aliquots at −80°c. an internal control (ic) was included as a control for the procedure used for sample preparation (viral rna purification). the extracted viral rna was tested by real-time rt-pcr duplex assays targeting the upe and orf1a regions of the mers-cov genome, using the realstar® mers-cov rt-pcr kit 1.0 (rt-pcr duplex assay). this system involves two independent assays, one targeting and quantifying a region upstream of the e gene (upe) and the other targeting the open reading frame regions 1a (orf1a) of the mers-cov genome. the analysis was performed using an abi prism® 7500 (applied biosystems). the main 12 human proinflammatory cytokines and chemokines (il-1α, il-1β, il-2, il-4, il-6, il-8, il-10, il-12, il-17a, ifn-γ, tnf-α, and gm-csf) were measured in lower respiratory samples from both mers-cov infected patients and the healthy non-infected control group, using multi-analyte elisarray (qiagen, germantown, md, usa) according to the manufacturer′s protocol. the absorbance of the obtained products after 30 min was measured at 450 nm, and cytokine/ chemokine levels were determined by comparing them to the negative (assay buffer) and positive control (cocktail containing all standard 12 cytokines or chemokines, respectively). the presence of a determined cytokine/chemokine was based on an absorbance value above the negative control. the mean absorbance of the samples and all standards was calculated as replicates. the concentrations were calculated using standard curves. cytokine/chemokines levels were expressed as pg/ml. pulmonary expression genes involved in the th1 and th2 response was assessed. cellular rna was isolated from lower respiratory tract samples using the rneasy mini kit (qiagen, valencia, ca). the concentration (ng/μl) and purity (a260/a280) of the rna was assessed using a nanodrop2000 (thermo-scientific). cdna synthesis and genomic dna elimination were performed using an rt 2 first-strand synthesis kit (qiagen, germantown, md, usa), using 1.5 µl (0.5 μg) of rna. mrna expression levels of th1/th2 immune genes were measured using an rt 2 -pcr profiler array kit (qiagen, germantown, md, usa) on an abi prism 7500 system (applied biosystems) with the rt 2 sybr green rox qpcr master mix (qiagen, germantown, md, usa). briefly, the cdna template was combined with the rt 2 real-time sybr green master mix and rnase-free water. a final reaction volume of 25 μl was added to each well of the rt 2 -pcr profiler array. cyclethreshold (ct) values were obtained using a constant baseline for all real-time rt-pcr runs as previously described [36] . five endogenous control genes provided by the array (actb, b2m, gapdh, gusb, and hsp90ab1) were used to calculate the arithmetic mean, which was then set as the ct value for normalization. the online rt 2 -pcr profiler data pcr array analysis software was used to identify the most appropriate reference genes to use for the normalization of the rt-pcr array gene expression data. real-time rt-pcr array results were analyzed using the online rt 2 -pcr profiler data pcr array analysis software (https://dataanalysis.qiagen.com/pcr/arrayanalysis.php). each sample was assessed for reverse transcription efficiency, pcr array reproducibility, and genomic dna contamination using the quality control function of the software. experimental gene expression was quantified as δδct and calculations were normalized to the housekeeping genes. statistical analysis was performed using spss version 16 (spss, inc., chicago, il). variables were compared between mers cov infected patients and healthy non-infected controls. the t-test and one-way anova analysis were used as appropriate. the data were presented as mean ( ± standard deviation) and counts (percentages). a p-value of < 0.05 was considered statistically significant. the demographic data, outcomes, preexisting conditions, underlying chronic diseases, clinical characteristics of mers cov infected patients and disease outcomes are presented in table 1 . the real-time rt-pcr results showed high viral loads in all mers-cov patients regardless of the type of specimen. the mean mers-cov rna ct values and standard deviations were 25.30 ± 5.1 and 26.3 ± 5.02 for upe and orf1a, respectively ( table 3) . the viral load of mers-cov and the expression levels of inflammatory cytokines/ chemokines were found to be significantly correlated with the case fatality of mers-cov infected patients (p < 0.002). furthermore, we found a significant correlation (p < 0.0001) between mers-cov viral load and expression levels of inflammatory cytokines il-1α, il-1β and neutrophils chemoattractant chemokines il-8 (cxcl8). no influenza a h1n1 co-infections were detected among mers-cov positive patients. the lower respiratory tract samples from mers-cov infected patients and healthy non-infected controls were used to quantify expression levels of the main 12 human pro-inflammatory cytokines and chemokines (il-1α, il-1β, il-2, il-4, il-6, il-8, il-10, il-12, il-17a, ifn-γ, tnf-α, and gm-csf). among the panel of 12 inflammatory cytokines/chemokines, the mean expression levels of il-1α (1148 pg/ml) (p < 0.001) and il1-β (1230 pg/ml) (p < 0.001) were significantly higher in the lower respiratory tract of mers-cov infected patients when compared to healthy non-infected controls ( table 2) . furthermore, our results showed a significant correlation between il and 1α (p < 0.035) and il-1β (p < 0.013) levels and the case fatality of mers-cov infected patients. ifn-γ expression levels were detected in only four patients and tnf-α expression levels were increased only in five patients. the expression of il-8 (cxcl8) was found to be significantly increased (1956 pg/ml) (p < 0.001) in all mers-cov patients when compared to healthy controls (table 3) . our results showed a significant correlation between the expression level of il-8 and the case fatality of mers-cov infected patients (p < 0.003). in order to study the pulmonary th1 and th2 responses in mers-cov infected patients, molecular cytokine/chemokine profiles were assessed using a rt 2 -pcr array. only genes with at least a two-fold change in expression when compared to the housekeeping gene were selected for further analysis. a side-by-side analysis of the data derived from the rt 2 -pcr profiling of pulmonary th1/th2 responses showed that genes encoding th1 and th2-related cytokines and chemokines were largely downregulated in the lower respiratory tract of mers-cov infected patients. a total of 26 genes involved in regulating the th1 and th2 immune response were downregulated in mers-cov infected patients ( table 4 ). the mrna expression levels of 10 th1-related cytokines/chemokines were downregulated. in contrast, the expression of only 6 th1 cytokine/chemokine mrnas, namely il-18, il-18r1, socs5, ccr2, cd4, and cxcr3, were upregulated (table 4) . with regards to the th2 immune response, we identified 15 cytokines/ chemokines mrnas expression levels were downregulated in mers-cov infected patients (table 4) . we did not observe any modulation or differences in th1/th2 responses and inflammatory cytokines/chemokines expression levels between mers-cov infected patients with underlying chronic medical conditions and mers-cov infected patients without underlying chronic medical conditions. the disease outcome, as well as the immunopathology and pathogenesis of mers-cov, sars, and other respiratory viral infections in humans and other animals are strongly linked to the expression levels of inflammatory cytokines and chemokines [16, 37, 38] . various cytokines, chemokines, and innate immune cells have been associated with the immunopathology during coronavirus (cov) and influenza virus infection [5, 6, 13, 14] . this study offers the first insight into the lung molecular gene expression profile of th1 and th2-related cytokines/ chemokines (th1 & th2 responses) during acute mers-cov infection. previous studies have shown that dysregulated and excessive immune responses may cause immunopathology and fatal disease [6, [39] [40] [41] . as such, direct cytopathic effects, high virus titers, and viral evasion of host immune responses are believed to play a major role in the severity of the diseases resulting from sars-cov and mers-cov infections [6, 39, 40] . our results showed high viral load (ct) values in all mers-cov infected patients regardless of the specimen type (table 3) . several pro-inflammatory cytokines, such as il-8 and il-1β, contribute to ards pathogenesis [6, 15, [42] [43] [44] . ards was shown to be the primary cause of death among patients with sars-cov or mers-cov infections [6, 45] . in this study, the majority of mers-cov infected patients progressed to ards, subsequently developing pneumonia and requiring admission to an intensive care unit (icu). in vitro studies have revealed that mers-cov induces significant but delayed modifications in the expression of both ifn and pro-inflammatory cytokines, including il-1β, il-6, il-8, and other chemokines [7, 10, 46] . our results showed high expression levels of il-1α, il-1β and il-8 (cxcl8) in the lower respiratory tract of mers-cov infected patients. evidence from studies investigating sars and respiratory syncytial virus (hrsv) has revealed that il-8 was associated with acute sars infection, bronchiolitis, immunopathology and disease enhancement during hrsv infections [10, 12, [15] [16] [17] . il-8 (cxcl8) is a critical chemokine involved in neutrophil recruitment, activation, and local neutrophil accumulation, and was shown to induce the formation of neutrophil extracellular traps (nets) [44, 47] . nets are highly immunogenic and extremely toxic to the host tissue, being able to directly cause inflammation, pathological changes, and epithelial and endothelial cell death [44, 48] . as such, lung nets can increase inflammation by stimulating il-8 expression, which leads to the recruitment of more neutrophils [44, 47] . we hypothesize that high il-8 expression levels may cause netosis, which results in severe mers-cov infection and increases immunopathology. il-1β has been associated with tissue damage, neutrophil infiltration, acute inflammatory responses, higher case fatality and severe respiratory viral infection [10, 14, 19, 49, 50] . in this study, high expression levels of il-1β were measured in the lower respiratory tracts of mers-cov infected patients. previous studies have shown that the expression of il-1β during influenza a h1n1 infection increased lung inflammation, and subsequent treatment with an il-1β antagonist significantly reduced both inflammation and lung tissue damage, suggesting that il-1β is a critical cytokine that contributes to lung inflammation [51, 52] . likewise, another study has shown that il-1β mrna levels were upregulated in calu-3 cells infected with mers-cov and/or sars-cov [10] . il-1α is an inflammatory cytokine that can be rapidly induced and quickly reaches high levels in response to a range of stimulants, such as il-1β and pathogenic agents [18, 53] . in mouse models, il-1α was shown to be a key player in triggering neutrophilic inflammation [18, 54] . likewise, our results revealed high il-1α expression levels in mers-cov infected patients. therefore, we think that high levels of il-8, il-1α, and il-1β may play a critical role in the immunopathology, severity, and case fatality of mers-cov infected patients. il-1α and il-1β establish an inflammatory loop which activates downstream signaling and enables the il-1α-il-1r1 interaction, leading to further il-1α and il-1β production. thus, a loop of continued and self-perpetuating inflammation occurs, resulting in extensive tissue damage and pathological changes [18] . as such, elevated il-1β and il-1α levels during acute mers-cov infection may cause an inflammatory loop that contributes to the extensive tissue damage and pathological changes associated with this disease, it is important to note that the il-1α and il-1β expression levels were not timely monitored at different intervals in this study. thus, the proposed inflammatory loop may not be generalizable to cases with severe disease or patients treated by antiinflammatory/antiviral drugs. it has been shown that effective control of host inflammatory response and antiviral treatment were associated with a reduction in inflammatory cytokine levels, steady improvement in disease condition, and pulmonary function [55] [56] [57] [58] [59] . consequently, studying il-1α and il-1β expression levels at different intervals could assist in a better understanding of mers-cov immunopathology. we speculate that, monitoring of il-1α and il-1β expression levels at different time points during mers-cov infection may alter the proposed inflammatory loop. tnf-α is an important innate and antiviral cytokine. high levels of tnf-α were detected in an in vitro study of sars-cov and mers-cov at both 24 and 30 h [10] . in our study, the induction of tnf-α was largely absent. similarly, in a recent study, tnf-α expression was not detected in most patients with mers-cov infections in the acute or convalescent stages [11] . this may indicate the limited early in vivo tnf-α response during mers-cov infections. one explanation for the differences between in vitro and in vivo studies could be the different kinetics of the mers-cov responses in the in vitro model, where there is a gradual increase in gene expression over time. however, a more likely explanation is that the complex interplay of target cell infection, mers-cov replication, viral load and time of sample collection. we measured cytokines/chemokines levels and viral load at a single time point in the early phase of infection; assessing cytokines/chemokines concentrations and viral load at different time points during mers-cov infection to create a kinetic profile of cytokines/chemokines might yield additional information. overall, the distinct tnf-α responses in vitro and in vivo might impact on the in vivo pathogenesis and viral amplification. cd4 + t-cell immune responses during respiratory viral infections characterized by the production of signature cytokines, ifn-γ, tnf-α, and il-2 for th1 cells and il-4, il-5, il-6, il-9, il-10, and il-13 for th2 cells [9, 28, 29] . the balance between the th1 and th2 responses is critical for the outcome of viral infections [60, 61] . to our surprise, we found that the expression of genes encoding th1 and th2 cytokines/chemokines were largely downregulated in the lower respiratory tract of mers-cov infected patients ( table 4 ). the pulmonary th1 and th2 responses showed the downregulation of 26 genes encoding th1 and th2 cytokines/chemokines in mers-cov infected patients. ifnγ plays an important role in early immunity, inducing the apoptosis of infected cells and stimulating both cd8 + and natural killer (nk) cells [62, 63] . in this study, both ifnγ and main th1associated cytokine mrna expression levels were downregulated in the lower respiratory tract of mers-cov infected patients. on the other hand, only 6 th1-related cytokines and chemokines (il-18, il-18r1, socs5, ccr2, cd4, and cxcr3) were overexpressed in the lungs of mers-cov infected patients (table 4 ). in regards to th2-induced immune responses, 15 th2 cytokines were downregulated. th2 cytokines play an important role in the humoral immune response and promote antibody production. mers-cov infection completely downregulated the th2 response in this study. we could not detect the mrnas for the specific antiviral immune response in mers-cov infected patients. reghunathan et al. also found a strong inflammatory response in acute sars despite complete downregulation of the mrna expression of genes involved in specific antiviral immune response [15] . downregulation of the major th1/th2 cytokines/chemokines can contribute to the pathological change, inefficient clearing infections, and host susceptibility to immune-mediated diseases. the upregulated th1 cytokines/chemokines (il-18, socs5, ccr2, and cxcr3) have been shown to play an important role in virus-induced immunopathology and usually associated with acute lung inflammation. il-18 was associated with acute lung inflammation [64] . in addition, il-18 was shown to enhance the severity of hrsv disease [65] . socs proteins affect antiviral signaling pathways, allowing viruses to evade the host immune response, and facilitate viral replication. moreover, socs5 has been suggested to shape the presentation of viral disease [66, 67] . previous data showed that, socs5 mrna was significantly upregulated in response to h1n1, h3n2, h5n1, and h11n9. suppressing socs signals improved influenza infection and inhibited hrsv replication [68, 69] . ccr2 mrna upregulation was correlated with hrsv-disease severity [70] . ccr2 antagonism reduced pathology, morbidity, and mortality during influenza a (h1n1) infection [71] . during hrsv infection increased cxcr3 was associated with the underlying pathology [72] . in addition, it has been shown that blocking cxcr3 reduced immunopathology during respiratory virus infections [73] . therefore, we think il-18, socs5, ccr2, and cxcr3 overexpression may play a critical role in immunopathology, severity, and case fatality of mers-cov infected patients. factors contribute to cd4 molecules upregulation during mers-cov infection in this study need further study. previous data showed that, cd4 and cd8 expression on t cells were not altered by mers-cov infection [74] . mers-cov could persistently induce the expression of pro-inflammatory cytokines/chemokines such as il-8, which would up-regulate cd4 molecules to enhance helper t cell infection. expression of high levels of il-8 and the net formation could have important effects on the cd4 expression. we propose a mechanism, which can explain the role of lower respiratory tract cytokines/ chemokines during acute mers-cov infection. il-1β and il-8 (cxcl8) recruit and activate neutrophils. subsequently, activated neutrophils undergo degranulation and netosis, which contributes to pathological changes and leads to the recruitment of more inflammatory. elevation of il-1β and il-1α during acute mers-cov infection may cause an inflammatory loop that contributes to extensive tissue damage and pathological changes. il-8 and il-1β are essential mediators in the development and pathogenesis of ards [6, 15, [42] [43] [44] . moreover, overexpression of il-1α, il-1β, il-8 (cxcl8), il-18, cxcr3, socs5, and ccr2 may play a vital role in the severity, immunopathology, and case fatality of mers-cov infected patients. it is important to note that the samples in this study were screened only for h1n1. we were not able to screen other respiratory viruses. viral co-infections could result in specific virus-virus interactions [75] . however, the inflammatory response induced by one virus may not be significantly changed by the infection of a second or third virus [76] . we did not examine the influences of co-infections with other respiratory viruses on cytokines/chemokines expression levels. any changes in immune profiles in mers-cov infected patients due to other respiratory pathogens are unknown. however, there is a possibility that other respiratory viral infections, which were not screened in our study, might skew immune profiles in mers-cov infected patients. we think that simultaneous study of other variables (ie, the interplay between mers-cov and other respiratory viruses), which could potentially skew the data is likely required to assess any alterations of the immune profiles in mers-cov infected patients. four limitations of this study should be noted. first, we measured cytokines/chemokines expression levels and viral load at a single time point in the early phase of infection; assessing cytokines/chemokines expression levels and viral replication at different time points during mers-cov infection to create a kinetic profile might yield additional information. second, in our study design, we did not include controls from patients infected with other respiratory viral pathogens. third, we have screened mers-cov positive samples only for h1n1, we did not screen our samples for other respiratory viruses. fourth, we were not able to recruit health non-infected individuals with similar mers-cov infected patients underlying diseases/pre-existing conditions. future study designs to close these gaps need to be considered. taken together, the high expression of inflammatory cytokines/ chemokines il-1 α and il-1β, il-8 (cxcl8), il-18, cxcr3, socs5, and ccr2 in the lower respiratory tracts of mers-cov infected patients confirmed the lung immunopathology [46, [77] [78] [79] [80] . therefore, the high expression of inflammatory cytokines and downregulation of the th1 and th2 immune responses in the lower respiratory tracts of mers-cov infected patients may contribute to a more severe infection, higher case fatality, lung inflammation, and immunopathology. furthermore, high levels of inflammatory cytokines/chemokines could be a key point in the subsequent development of pneumonia and ards in mers-cov infected patients. as such, the characterization of pulmonary th1/th2 response in mers-cov infection could be valuable for effective vaccine development and novel therapeutic strategies. the authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. pathogenesis of middle east respiratory syndrome coronavirus isolation of a novel coronavirus from a man with pneumonia in saudi arabia middle east respiratory syndrome coronavirus-two years into the epidemic interaction of sars and mers coronaviruses with the antiviral interferon response cytokine mers-cov infection in humans is associated with a pro-in fl ammatory th1 and th17 cytokine profile pathogenic human coronavirus infections: causes and consequences of cytokine storm and immunopathology middle east respiratory syndrome coronavirus shows poor replication but significant induction of antiviral responses in human monocyte-derived macrophages and dendritic cells haematological manifestations in patients with severe acute respiratory syndrome: retrospective analysis molecular pathology of emerging coronavirus infections delayed induction of proinflammatory cytokines and suppression of innate antiviral response by the novel middle east respiratory syndrome coronavirus: implications for pathogenesis and treatment immune responses to middle east respiratory syndrome coronavirus during the acute and convalescent phases of human infection innate immune dysfunction is associated with enhanced disease severity in infants with severe respiratory syncytial virus bronchiolitis cytokine storms in infectious diseases new fronts emerge in the influenza cytokine storm expression profile of immune response genes in patients with severe acute respiratory syndrome analysis of serum cytokines in patients with severe acute respiratory syndrome plasma inflammatory cytokines and chemokines in severe acute respiratory interleukin 1α and the inflammatory process reassessing the role of the nlrp3 inflammasome during pathogenic influenza a virus infection via temporal inhibition mers-cov accessory orfs play key role for infection and pathogenesis antagonism of dsrna-induced innate immune pathways by ns4a and ns4b accessory proteins during mers coronavirus infection mers-cov 4b protein interferes with the nf-kappab-dependent innate immune response during infection cytokines and transcription factors that regulate t helper cell differentiation: new players and new insights regulation of the t cell response t-cell subset regulation in atopy effects of various radiation doses on induced t-helper cell differentiation and related cytokine secretion mrna expression of interleukins and th1/th2 imbalance in patients with pulmonary embolism t cell-mediated immune response to respiratory coronaviruses modulation of the immune response by middle east respiratory syndrome coronavirus rapid generation of a mouse model for middle east respiratory syndrome collection of tracheal aspirate: safety and microbiological concordance between two techniques should lower respiratory tract secretions from intensive care patients be systematically screened for influenza virus during the influenza season ? a simple method of reducing complications of pediatric nonbronchoscopic bronchoalveolar lavage respiratory viruses in bronchoalveolar lavage: a hospitalbased cohort study in adults cough suppression during flexible bronchoscopy using combined sedation with midazolam and hydrocodone: a randomised, double blind, placebo controlled trial naked dna immunization with full-length attachment gene of human respiratory syncytial virus induces safe and protective immune response fatal outcome of human influenza a (h5n1) is associated with high viral load and hypercytokinemia lethal influenza virus infection in macaques is associated with early dysregulation of inflammatory related genes dysregulated type i interferon and inflammatory monocyte-macrophage responses cause lethal pneumonia in sars-cov-infected mice disease-promoting effects of type i interferons in viral, bacterial, and coinfections age-dependent dysregulation of innate immunity characterization of cytokine/chemokine profiles of seven acute respiratory syndrome human immunopathogenesis of severe acute respiratory syndrome (sars) net balancing: a problem in inflammatory lung diseases severe acute respiratory syndrome (sars) active replication of middle east respiratory syndrome coronavirus and aberrant induction of inflammatory cytokines and chemokines in human macrophages: implications for pathogenesis induction of neutrophil extracellular dna lattices by placental microparticles and il-8 and their presence in preeclampsia neutrophil extracellular traps directly induce epithelial and endothelial cell death: a predominant role of histones is il-1 a good therapeutic target in the treatment of arthritis? evidence that cytokines play a role in rheumatoid arthritis interleukin-1 is responsible for acute lung immunopathology but increases survival of respiratory influenza virus infection induction of interleukin-1 beta (il-1beta) is a critical component of lung inflammation during influenza a (h1n1) virus infection complexity of inflammatory responses in endothelial cells and vascular smooth muscle cells determined by microarray analysis cytokine endogenous il-1 a is a chromatin-associated protein in mouse macrophages cytokine inhibition in the treatment of copd nitazoxanide, a new drug candidate for the treatment of middle east respiratory syndrome coronavirus infections and the inflammatory response in acute respiratory distress syndrome dynamic changes in clinical features and cytokine/ chemokine responses in sars patients treated with interferon alfacon-1 plus corticosteroids plasma and sal cytokine response to corticosteroid rescue treatment in late ards th1/th2 balance: the hypothesis, its limitations, and implications for health and disease effects of strenuous exercise on th1/th2 gene expression from human peripheral blood mononuclear cells of marathon participants nk cells controlling virus-specific t cells: rheostats for acute vs. persistent infections distinct immune response in two mers-cov-infected patients: can we go from bench to bedside? role of il-18 in acute lung inflammation interleukin 18 coexpression during respiratory syncytial virus infection results in enhanced disease mediated by natural killer cells hsv-1-induced socs-1 expression in keratinocytes: use of a socs-1 antagonist to block a novel mechanism of viral immune evasion viral exploitation of host socs protein functions rsv replication is attenuated by counteracting expression of the suppressor of cytokine signaling (socs) molecules suppressor of cytokine signaling (socs) 5 ameliorates influenza infection via inhibition of egfr signaling chemokine-receptor upregulation and disease severity in respiratory syncytial virus infection ccr2-antagonist prophylaxis reduces pulmonary immune pathology and markedly improves survival during influenza infection production of chemokines in the lungs of infants with severe respiratory syncytial virus bronchiolitis cxcr3 directs antigen-specific effector cd4 + t cell migration to the lung during parainfluenza virus infection middle east respiratory syndrome coronavirus efficiently infects human primary t lymphocytes and activates the extrinsic and intrinsic apoptosis pathways quasispecies diversity determines pathogenesis through cooperative interactions within a viral population respiratory and gastrointestinal epithelial modulation of the immune response during viral infection, innate immun immunopathogenesis of coronavirus infections: implications for sars ultrastructural findings of a fatal case of middle east respiratory syndrome coronavirus infection in the united arab emirates comparative and kinetic analysis of viral shedding and immunological responses in mers patients representing a broad spectrum of disease severity clinical progression and cytokine profiles of middle east respiratory syndrome coronavirus infection the authors thank the research center at king fahad medical city for funding this study (grant no 017-066). we also thank the deanship of scientific research and rssu at king saud university for their technical support. key: cord-350928-vj5qlzpj authors: arnott, alicia; vong, sirenda; rith, sareth; naughtin, monica; ly, sowath; guillard, bertrand; deubel, vincent; buchy, philippe title: human bocavirus amongst an all‐ages population hospitalised with acute lower respiratory infections in cambodia date: 2012-04-25 journal: influenza other respir viruses doi: 10.1111/j.1750-2659.2012.00369.x sha: doc_id: 350928 cord_uid: vj5qlzpj please cite this paper as: arnott et al. (2013) human bocavirus amongst an all‐ages population hospitalised with acute lower respiratory infections in cambodia. influenza and other respiratory viruses 7(2) 201–210. background human bocavirus (hbov) is a novel parvovirus that is associated with respiratory and gastrointestinal tract disease. objectives to investigate the prevalence and genetic diversity of hbov amongst hospitalized patients with acute lower respiratory infection (alri) in cambodia. study design samples were collected from 2773 patients of all ages hospitalised with symptoms of alri between 2007 and 2009. all samples were screened by multiplex rt‐pcr/pcr for 18 respiratory viruses. all samples positive for hbov were sequenced and included in this study. results of the samples tested, 43 (1·5%) were positive for hbov. the incidence of hbov did not vary between the consecutive seasons investigated, and hbov infections were detected year‐round. the incidence of hbov infection was highest in patients aged <2 years, with pneumonia or bronchopneumonia the most common clinical diagnosis, regardless of age. a total of 19 patients (44%) were co‐infected with hbov and an additional respiratory pathogen. all isolates were classified as hbov type 1 (hbov‐1). high conservation between cambodian np1 and v1v2 gene sequences was observed. conclusions human bocavirus infection can result in serious illness, however is frequently detected in the context of viral co‐infection. specific studies are required to further understand the true pathogenesis of hbov in the context of severe respiratory illness. human bocavirus (hbov) is a novel parvovirus that was first identified in pools of nasopharyngeal aspirates obtained from individuals with respiratory tract infections in 2005. 1 the hbov genome has three open reading frames encoding the 2 non-structural proteins ns1 and np1, and the structural viral capsid proteins vp1 and vp2. 2, 3 there are four closely related hbov genotypes, hbov-1, hbov-2 (consisting of hbov-2a and hbov-2b strains), hbov-3 and hbov-4, all of which share the same genomic organisation. 3, 4 hbov-1, the genotype initially identified by schildgen et al. 5 in sweden, has since been detected worldwide. hbov-2-4 were mainly identified in stool samples. 6, 7 homology at the protein level between the four hbov genotypes is high, between 70 and 90%. 3 hbov-3 is thought by some to be the result of a recombination event between hbov-1 and hbov-2, owing to the similarity of the hbov-3 ns1 and v1v2 genes to those of hbov-1 and hbov-2, respectively. 6 indeed, hbov-3 is likely detected along with hbov-1 during routine diagnostic screening, as the conserved ns1 genes regularly targeted for testing are genetically similar. in patients where hbov is the only virus detected, the clinical symptoms reported are similar to those occurring as a result of infection with respiratory syncytial virus (rsv) and human metapneumovirus (hmpv), including bronchiolitis, bronchitis, pneumonia and exacerbation of asthma. 5 predominantly hbov-1, but also hbov-2 dna, has been detected in respiratory samples, 8 whereas all four genotypes have been detected in stool samples, demonstrating that hbov replication is not limited to the respiratory tract as first thought. 4, 8 indeed, up to 25% of patients with hbov infection report gastrointestinal symptoms. [8] [9] [10] [11] hence, the tropism of strains belonging to hbov genotypes 1-4 remains unknown. the seasonality of hbov infection is also unclear, with seasonal [12] [13] [14] and year-round [15] [16] [17] transmission reported. the clinical significance of hbov infection is also yet to be fully elucidated, as hbov is frequently detected in association with additional respiratory pathogens. in the absence of an established cell culture system or animal model, the pathogenicity and replication mechanisms of hbov remain unclear. 5, 18, 19 a recent report suggests that hbov is, however, a true respiratory pathogen capable of causing sometimes severe, and even life-threatening, illness. 20 here, we report the findings of the first study investigating the prevalence, seasonality, clinical characteristics and the molecular epidemiology of hbov in amongst an all-ages population of patients hospitalized for acute lower respiratory illness (alri) in cambodia over 3 consecutive years. during april 2007-december 2009, all patients admitted with symptoms of alri to takeo (southern cambodia) and kampong cham (central-north cambodia) provincial hospitals were recruited into this study. 21 the age-specific criteria used to diagnose alri cases and severe alri cases were defined previously. 21 samples were collected, transported and stored as described. 21 additional clinical specimens and data, including sputum samples for bacteria identification and chest x-rays, were collected from patients where possible. samples were screened by multiplex rt-pcr ⁄ pcr at the institut pasteur in cambodia for 18 respiratory viruses including rsv, human metapneumovirus (hmpv), hbov, influenza a and b viruses, coronaviruses oc43, 229e, hku1 and nl63, severe acute respiratory syndrome-associated coronavirus (sars-cov), parainfluenza viruses 1-4, adenoviruses, rhinovirus and enterovirus, as previously reported. 21, 22 samples that tested positive for hbov were included in this study. the national ethics committee of cambodia approved this study. all patients ⁄ parents of sick children who participated provided written informed consent. viral dna from nasopharyngeal samples was extracted using the qiaamp dna mini kit (qiagen, valencia, ca, usa) as per the manufacturer's instructions. dna was eluted in a final volume of 200 ll qiagen ae buffer and stored at )80°c until required. both hbov and human albumin dna were amplified in parallel from each sample. amplification of human albumin dna was performed as described. 23 to identify strains belonging to hbov groups 1, 2 and 3, strain-specific primers were used. to identify hbov-1 strains, a 354-bp fragment of the conserved hbov-1 np-1 gene was targeted using published primers. 1 the forward and reverse primers correspond to positions 2281 and 2634 of the np-1 gene of the hbov prototype strain st1 (genbank accession number dq000495). thermocycling was performed using go-taq flexi dna polymerase (promega, madisson, wi, usa) under the following conditions: pcr activation at 94°c for 2 minutes, followed by 40 cycles of denaturation at 94°c for 30 seconds, annealing at 59ae5°c for 30 seconds, extension at 72°c for 1 minute and a final extension cycle of 72°c for 10 minutes. all samples were also screened by pcr for the presence of hbov2 and hbov3 dna, as described. 8 for phylogenetic analysis, a 819-bp region of the variable hbov-1 v1 ⁄ v2 gene was amplified using published primers. 9 primers correspond to positions 4370-4387, and 5172-5189 of the hbov prototype strain st1 genome (genbank accession number dq000495). thermocycling was performed using go-taq flexi dna polymerase (promega) under the following conditions: pcr activation at 94°c for 2 minutes, followed by 40 cycles of denaturation at 94°c for 1 minute, annealing at 54°c for 1 minute, extension at 72°c for 2 minutes and a final extension cycle of 72°c for 10 minutes. each pcr contained the appropriate positive and negative controls. all pcr products were visualised using ethidium bromide under uv light on a 1ae5% agarose gel. when required, pcr products were purified using the qiaquick gel extraction protocol of the qiaquick pcr purification kit (qiagen) as per the manufacturer's instructions, prior to sequencing. single-pass sequencing reactions were performed at a contract sequencing facility using the abi big-dye terminator cycle sequencing kit on an abi 3730xl automatic dna analyser (macrogen, seoul, korea). consensus sequences were generated using clc main workbench software, version 5ae6ae1 (http://www.clcbio.com). nucleotide sequences of reference hbov strains were obtained from genbank and used to construct alignments and phylogeny (table 1) . cambodian and reference hbov nucleotide sequences were aligned using the clustal w alignment program of mega software, version 4ae0. 24 the average pairwise jukes-cantor distance was found to be 0ae01 for both of the hbov np1 and vp1 ⁄ vp2 alignments, indicating that the data were suitable to generate neighbour-joining trees. 25 neighbour-joining trees were constructed using the p-distance nucleotide substitution model, with 1000 bootstrap replicates, using mega 4ae0 software. pairwise nucleotide distances (p distance), the proportion of sites at which nucleotide sequences differ divided by the total number of nucleotides compared, were calculated using the pairwise distance function of mega software version 4ae0. pairwise amino acid distances (p distance), the proportion of sites at which amino acid sequences differ divided by the total number of residues compared, were calculated using the poisson model in the pairwise distance function of mega software version 4ae0. deduced partial amino acid sequences of cambodian np1 and vp1 ⁄ vp2 strains were generated by translating nucleotide sequences with the standard genetic code using mega software version 4ae0. potential n-glycosylation sites were predicted by the nxt motif, where x is not a proline; potential o-glycosylation sites were predicted by the kpx n motif (where x is any amino acid). [26] [27] [28] [29] nucleotide sequence accession numbers the nucleotide sequences of hbov strains obtained in this study were deposited in genbank under the accession numbers jq618248 to jo618263 (np1 sequences) and jq618264 to jq618278 (vp1 ⁄ vp2 sequences). a total of 2773 patients presenting with alri participated in the study (no refusals). nasopharyngeal swabs were collected from all patients, of which 43 (1ae5%) tested positive for hbov. hbov infections were detected year-round, and the annual incidence amongst the study patients did not vary significantly (1ae5-1ae7%) during 2007-2009 ( figure 1 ). the median age of the hbov-infected patients was 1 year (range, 0ae3-56 years), and 46ae5% were female. the incidence of hbov infection was highest amongst children aged <2 years ( table 2) . whereas bronchiolitis was only observed in patients aged <2 years, pneumonia or bronchopneumonia was the most common clinical diagnosis, regardless of age (37ae2%; table 2 ). only respiratory specimens were available for testing in this study, and no clinical information regarding gastrointestinal symptoms was recorded. nineteen (44%) patients were co-infected with an additional respiratory virus (table 3) . co-infection with hrhv was most frequently detected (21%), followed by parainfluenza virus type 3 (piv3, 4ae6%) and respiratory syncytial table 1 . reference human bocavirus (hbov) strains used in this study to construct phylogeny. shown are the name of the strain, genbank accession number, year of isolation, country of isolation and genotype. whether strains were included in figure 2 (hbov np1 phylogeny), figure virus (rsv, 4ae6%). one patient was co-infected with both rsv and hrhv (table 3 ). detection and distribution of hbov sequences were successfully obtained from 19 (44ae2%) samples: 16 np-1 gene and 15 vp1 ⁄ vp2 gene sequences. both np-1 and vp1 ⁄ vp2 sequences were successfully amplified from 12 (63%) samples. all cambodian hbov strains were classified as hbov-1 (figures 2 and 3 ). of the samples from which hbov-1 sequences were obtained, one was collected in 2007, nine in 2008 and nine in 2009 (figures 2 and 3) . twelve (63%) samples were collected in takeo province, and seven (37%) were collected in kampong cham province, but the patient recruitment was also higher in takeo than in kampong cham hospital (data not shown). analysis of hbov-1 np1 gene sequences pairwise distance (p distance) analysis revealed very high conservation of np-1 gene sequences amongst the cambodian hbov-1 strains. homology at the nucleotide (nt) level varied between 98ae9 and 100%. identity at the amino acid (aa) level was slightly lower, between 96ae7 and 100%. the np-1 gene is highly conserved between strains, and however, we identified 5 different non-synonymous mutations present in cambodian np-1 sequences relative to the prototype hbov-1 strain st1 ( figure 4 ). the observed nt mutations resulted in the following 5 aa substitutions: ser 92 (100% conservation amongst cambodian np-1 sequences), asn 44 (65%), asn 59 (12%), ser 47 (6%), arg 53 (6%) (figure 4) . we observed 1 potential n-glycosylation site, starting at amino acid position 86, which was conserved amongst all reference and cambodian hbov strains analysed (figure 4 ). analysis of hbov-1 vp1 ⁄ vp2 gene sequences homology at the nt level was also very high amongst the partial vp1 ⁄ vp2 gene sequences, between 97ae1 and 100%. homology at the aa level was slightly lower, between 96ae6 and 100%. present on the surface of the virion, the vp1 ⁄ vp2 protein is potentially subjected to strong selective pressure from the host immune response. relative to the reference strains st1 and st2, 8 non-synonymous nt mutations resulting in the following aa substitutions were observed amongst cambodian vp1 ⁄ vp2 gene sequences: his 546 (66%), leu 631 (26%), tyr 540 (26%), thr 650 (13%), ser 466 (6%), pro 490 (6%), gln 493 (6%) and lys 545 (6%) (figure 5 ). one additional nt mutation resulting in the aa substitution ser 590 , present in reference strain st2 but not st1, was conserved amongst all of the cambodian vp1 ⁄ vp2 sequences investigated ( figure 5) . a stop codon was present at position 672 in all reference and cambodian vp1 ⁄ vp2 sequences included in this study ( figure 5 ). potential n-and o-glycosylation sites amongst hbov-1 vp1 ⁄ vp2 gene sequences two potential n-glycosylation sites, located at amino acid positions 519 and 638, were conserved amongst the reference isolates and all of the cambodian vp1 ⁄ vp2 sequences investigated ( figure 5 ). one potential o-glycosylation site was identified, located at amino acid position 552, which was also conserved amongst the reference isolates and all of the cambodian vp1 ⁄ vp2 sequences analysed ( figure 5 ). here, we report the results of the first study to investigate the incidence and genetic diversity of hbov amongst globally, the incidence of hbov infection has been reported to range from 1ae5 to 19%. 30 overall, the incidence of hbov infection amongst the all-ages population of cambodian patients hospitalised with alri was 1ae5%, which did not vary annually and was lower than that reported regionally. [31] [32] [33] [34] amongst the same population, the incidence of hbov was similar to that of hmpv (1ae7%), 21 but lower than rsv (8ae2%). 35 a number of factors can influence incidence estimates, including disease severity cam2008-e244 ...........................................s.....r......................................s. cam2009-p280 ........................................n...............................................s. cam2008-e374 ........................................................................................s. cam2009-e532 ........................................n........................................... cam2008-e446 ........................................n...............................................s. cam2008-e473 ........................................n...............................................s. cam2008-e474 ........................................n............................................. amongst the population investigated, study design, testing protocols and sensitivity of diagnostic tests used, especially as low viral loads are common in hbov infections. 5 in this study, alri patient samples were screened for hbov infection using a highly sensitive multiplex pcr assay previously shown to have a lower limit of detection of 4 copies of hbov dna ⁄ ll of viral transport medium. 22 therefore, the low incidence of hbov infection observed amongst the cambodian alri population was not a result of limited sensitivity of the diagnostic test used. the proportion of hbov infections detected amongst hospitalised cambodian alri patients was significantly higher compared to that of cambodian outpatients with ili (1ae5% versus 0ae4%, p = 0ae02). 22 the higher incidence of hbov infection amongst alri patients was not thought to be a result of a high carriage rate within the population. in parallel, the incidence of hbov infection amongst cambodian ili outpatients and asymptomatic controls was investigated previously, with only one asymptomatic individual testing positive for hbov dna. 22 however, persistent and prolonged shedding is a characteristic of parvoviruses, and it is thought that low-level asymptomatic shedding can persist following hbov infection. 20, 36, 37 two independent stud-ies reported the hbov carriage rate to be 43% amongst asymptomatic children, 38 and that the incidence of hbov amongst asymptomatic children was higher than amongst symptomatic children. 39 however, these two studies were conducted amongst children aged <2 years. hence, the high carriage rate may have been biased towards the very young age of the sample population with hbov incidence highest amongst children aged <2 years. 1, 9, 40 amongst a population of slightly older children, up to 5 years of age, brieu et al. 36 reported that hbov dna was not detected following testing of asymptomatic children. in the absence of an established continuous culture system, it is currently unclear to what extent shedding occurs following hbov infection, whether shedding and carriage rates amongst children and adults are equivalent, and whether shedding is innocuous or infectious. however, the higher incidence of hbov infection amongst hospitalised cambodian alri patients observed in this study relative to outpatients with ili suggests that hbov infection can result in severe illness. twenty-four (56%) of the hbov-infected individuals tested positive for infection with hbov only, of which 14 patients were classified as severe respiratory infection, including three patients aged <1 year who presented with ..........n................................................................................................ cam2009-p280 .....s.......................p..q....................................................h........................ cam2009-p232 .....................................................................................h........................ cam2009-p231 .....................................................................................h........................ cam2009-e585 ...............................................................................y.............................. cam2009-e569 .....................................................................................h........................ cam2009-e532 ....................................................................................kh........................ cam2008-p125 .............................................................................................................. cam2008-p0202 .....................................................................................h........................ cam2008-e474 .....................................................................................h........................ cam2008-e473 .....................................................................................h........................ cam2008-e446 .....................................................................................h........................ cam2008-e374 .....................................................................................h........................ cam2008-e244 ...............................................................................y.............................. cam2008-e216 ...............................................................................y.............................. cam2007-e141 ...............................................................................y............................. . 43 recently, a severe case of hbov infection resulting in acute respiratory failure was reported, in which hbov was the only pathogen detected. 20 the possibility that clinical illness amongst the cambodian hbov-infected patients was exacerbated because of bacterial co-infection cannot, however, be excluded as testing for bacterial co-infection could not be performed because of a lack of suitable specimens obtained from young participants. whether hbov represents a true pathogen, or an opportunistic co-pathogen, has been the source of much debate. 2, 5 a primary reason that the role of hbov as a sole pathogenic agent is questioned is that hbov is commonly detected as a co-infection. globally, the frequency of hbov-1 co-infection with other respiratory viruses amongst children with lower respiratory tract infection is reported to be as high as 83%. 4, 5, 44 furthermore, it has been reported that hbov coinfection may be higher amongst hospitalised individuals compared with those with mild illness as shedding is potentially enhanced by airway inflammation resulting from coinfection with an additional respiratory pathogen, or that hbov may play a role in enhancing or aggravating symptoms of existing infections. 5 mechanisms proposed to explain the high rate of co-infection with other respiratory viruses include that hbov is either a helper virus, facilitating replication of other respiratory pathogens or that hbov may require a helper virus to facilitate productive infection. 45 it must also be considered that the more recent widespread use of highly sensitive multiplex assays enabling simultaneous detection of multiple pathogens in a single patient sample may also contribute to the high rate of coinfection with hbov and additional respiratory pathogens detected. in this study, 44% of patients were co-infected with hbov and an additional respiratory virus. regionally, the reported rate of hbov co-infections varies and does not appear to be particularly associated with hospitalisation: 42% amongst hospitalized children in singapore, 32 90% amongst out-patient children aged <5 years in rural thailand, 37 9% amongst hospitalised children in vietnam. 31 in this study, co-infection with hbov and hrhv was most frequently detected (table 3) , analogous to the findings of a similar study conducted amongst paediatric patients in rural thailand. 37 whether the high frequency of hbov and hrhv co-infection is a reflection of the high incidence of hrhv circulating in the general population 2 or whether hrhv co-infection is beneficial to hbov replication and pathogenesis remains unknown. in the absence of an established cell culture system or animal model, much remains unclear regarding the pathogenesis of hbov. all of the hbov strains obtained from respiratory specimens for analysis in this study were classified as hbov-1. globally, hbov-1 strains have been reported to show low nucleotide and protein diversity; mean genetic diversity <1% at nt and <0ae5% at aa levels. 44 comparatively, the diversity of hbov-2, -3 and -4 strains, all identified in stool species, is far greater. 8 the low level of diversity reported worldwide amongst hbov-1 strains has resulted in speculation that hbov-1 evolved recently from the enteric bocavirus species, acquiring tropism for cells of the respiratory tract. 8, 46 the organisation of the hbov genome is similar to that of other parvoviruses, namely that the non-structural proteins are encoded by conserved genomic regions, whereas diversity is concentrated in regions encoding the capsid proteins. 45 analysis of amino acid sequences obtained from cambodian hbov-1 strains revealed 0-2ae9% nt diversity and 0-3ae4% aa diversity amongst vp1 ⁄ vp2 sequences. greater variation amongst sequences encoding the vp1 ⁄ vp2 capsid proteins was anticipated as vp1 ⁄ vp2 is under pressure from host immune responses. the level of diversity amongst cambodian np1 sequences was 0-1ae1% at the nt level, which is similar to that recently reported by kapoor et al. 46 surprisingly, the level of diversity at the aa level amongst np1 sequences was equivalent to that observed for vp1 ⁄ vp2 sequences, at 0-3ae3%. five amino acid substitutions were observed amongst cambodian np1 aa sequences, including asn 599 that has been reported previously by ma et al., 47 following analysis of hbov-1 np1 sequences obtained from japanese children with alri. the observed diversity amongst cambodian np-1 sequences may have occurred as a result of immune pressure, as it was recently reported that the hbov non-structural proteins, including np1, are targeted by humoral responses. 30 the potential impact, if any, of non-synonymous mutations within this conserved region of the hbov-1 genome remains unclear and warrants further investigation. to the best of our knowledge, we present the results of the first study to investigate the circulation and diversity of hbov infection amongst hospitalised patients in cambodia. we demonstrate that similar to the findings of other studies, hbov infection can result in serious illness, however is frequently detected in the context of viral co-infection. ongoing studies are required to further understand the true pathogenesis of hbov-1 in the context of severe respiratory illness. cloning of a human parvovirus by molecular screening of respiratory tract samples the human bocaviruses: a review and discussion of their role in infection human bocavirus capsid structure: insights into the structural repertoire of the parvoviridae human bocavirus-the first 5 years human bocavirus: passenger or pathogen in acute respiratory tract infections? a novel bocavirus associated with acute gastroenteritis in australian children a newly identified bocavirus species in human stool newly recognized bocaviruses (hbov, hbov2) in children and adults with gastrointestinal illness in the united states human bocavirus infection in young children in the united states: molecular epidemiological profile and clinical characteristics of a newly emerging respiratory virus human bocavirus in febrile children human bocavirus: prevalence and clinical spectrum at a children's hospital clinical and molecular epidemiology of human bocavirus in respiratory and fecal samples from children in hong kong detection of new respiratory viruses in hospitalized infants with bronchiolitis: a three-year prospective study correlation between bocavirus infection and humoral response, and co-infection with other respiratory viruses in children with acute respiratory infection frequent detection of bocavirus dna in german children with respiratory tract infections viral and atypical bacterial detection in acute respiratory infection in children under five years human bocavirus infection bocavirus episome in infected human tissue contains non-identical termini detection of head-to-tail dna sequences of human bocavirus in clinical samples human bocavirus as the cause of a life-threatening infection genetic variability of human metapneumovirus amongst an all ages population in cambodia between use of a multiplex pcr ⁄ rt-pcr approach to assess the viral causes of influenza-like illnesses in cambodia during three consecutive dry seasons taqman pcr-based gene dosage assay for predictive testing in individuals from a cancer family with ink4 locus haploinsufficiency mega4: molecular evolutionary genetics analysis (mega) software version 4.0 molecular evolution and phylogenetics genetic variability of group a human respiratory syncytial virus strains circulating in from 1998 to genetic variability among group a and b respiratory syncytial viruses in mozambique: identification of a new cluster of group b isolates identification of variable domains of the attachment (g) protein of subgroup a respiratory syncytial viruses do n-glycoproteins have preference for specific sequons? antibodies against structural and nonstructural proteins of human bocavirus in human sera viral etiologies of acute respiratory infections among hospitalized vietnamese children in ho chi minh city the incidence of human bocavirus infection among children admitted to hospital in singapore detection and quantitative analysis of human bocavirus associated with respiratory tract infection in osaka city human bocavirus (hbov) in thailand: clinical manifestations in a hospitalized pediatric patient and molecular virus characterization a study of the genetic variability of human respiratory syncytial virus (hrsv) in cambodia reveals the existence of a new hrsv group b genotype human bocavirus infection in children with respiratory tract disease human bocavirus: a novel parvovirus epidemiologically associated with pneumonia requiring hospitalization in thailand frequent and prolonged shedding of bocavirus in young children attending daycare clinical and epidemiologic characteristics of human bocavirus in danish infants: results from a prospective birth cohort study evidence of human bocavirus circulating in children and adults severe pneumonia and human bocavirus in adult serologically verified human bocavirus pneumonia in children human bocavirus: a cause of severe asthma exacerbation in children human bocavirus infections in hospitalized children and adults does human bocavirus infection depend on helper viruses? a challenging case report human bocaviruses are highly diverse, dispersed, recombination prone, and prevalent in enteric infections detection of human bocavirus in japanese children with lower respiratory tract infections we would like to thank all of the technical staff in the virology and epidemiology units at the institut pasteur in cambodia for their assistance. we are also grateful to all the staff from the hospitals for their collaboration in the sisea project. key: cord-306450-sh2mrhoq authors: appak, özgür; duman, murat; belet, nurşen; sayiner, ayça arzu title: viral respiratory infections diagnosed by multiplex polymerase chain reaction in pediatric patients date: 2019-01-03 journal: j med virol doi: 10.1002/jmv.25379 sha: doc_id: 306450 cord_uid: sh2mrhoq syndromic diagnosis by multiplex nucleic acid amplification tests is the most practical approach to respiratory tract infections since the symptoms are rarely agent‐specific. the aim of this study was to investigate the respiratory viruses in children admitted to a university hospital with acute respiratory tract infection during the last 8 years by a multiplex polymerase chain reaction (pcr) assay. a total of 3162 respiratory samples collected from children between april 2011 and april 2018 tested by a multiplex real‐time pcr assay. two different commercial assays were used during the study period, "ausdiagnostics/respiratory pathogens 12 (ausdiagnostics)" used between april 2011 and december 2015, which changed to "fast track diagnostics/respiratory pathogens 21 (fast track diagnostics)" after january 2016 to cover more viruses. nucleic acid extraction was done by ez1 advanced xl platform (qiagen). respiratory pathogens detected in 1857 of the 3162 (58.7%) samples. the most prevalent viruses during the 8‐year period were rhinovirus/enterovirus (rv/ev; 36.2%), respiratory syncytial virus (rsv; 19%), and influenza virus a/b (14.7%). rhinovirus was the main contributor to the rv/ev group as shown by the assay used during the 2016‐2018 period. rv/ev and adenoviruses detected throughout the year. influenza virus was most frequently detected during january to march when both rsv and metapneumovirus were also in circulation. the coinfection percentage was 10.2%. rhinovirus was the most common virus in coinfections while rsv plus rhinovirus/enterovirus were the most frequent combination. rsv and metapneumovirus showed a similar seasonal distribution to the influenza virus, which made it necessary to use a virological diagnostic assay during the influenza season. the ausdiagnostics test is based on multiplex tandem pcr a pathogen was detected in 1857 (58.7%) of the study samples. distribution of pcr positive samples according to age groups was found as follows; 53% (978 of 1857) in less than 2 years age group, 24% (454 of 1857) in 2 to 4 years group, 15% (270 of 1857) in the 5 to 9 years group, and 8% (155 of 1857) in 10 to 17 years group. the positivity rate decreased significantly above 5 years of age (p < 0.05). the mean positivity rate was 44.4% during study period when the ausdignostics assay was used compared with 69.8% of the second part of the study using the fast track diagnostics assay. the difference is statistically significant (p < 0.001; figure 1a ). rv/ev (17.6%) and rsv (9.2%) were the most commonly detected agents followed by iav (4.8%; table 1 ). when the distribution of the agents according to years were examined, rv/ev was the most common agent for each year. rsv and coinfections took the second and third place in the frequency order except 2011 in which they are replaced by piv-3 and adv. the study covered only the first 4 months of 2018 where coinfections were the most frequent cause of the respiratory infections in the studied group and followed by rv/ev and influenza a/b, respectively ( figure 1b ). when the distribution according to age groups was determined, the first three pathogens were rv/ev, rsv, and coinfections for less than 2 years; rv/ev, coinfections, and influenza a/b for 2 to 4 years; rv/ev, influenza a/b, and coinfections for 5 to 9 and 10 to 17 years (table 2) . among the parainfluenza viruses, piv-3 was the most frequently detected subtype, followed by piv-1, -2, and -4, respectively (table 1) . table 3 . the role and importance of respiratory viruses have become more easily detectable by the use of nucleic acid-based tests in clinical laboratories. thus it became possible to obtain detailed epidemiological data and to manage the patients better. our study retrospectively assessed the respiratory tract viruses detected in children with arti symptoms of 0 to 18 years between 2011-2018. two different assays were used during the study period. ausdiagnostics assay covered a panel of 12 rv as the number one etiologic factor. 6, 12, 13 rsv (9.2%) was the second most common viral agent after rv in this study. it was particularly detected during winter and spring months with a peak between january and march. consistent with our study, rsv is observed more frequently in winter months in many studies from different regions. 14 rsv and hmpv were usually seen in children under five years of age as the cause of bronchiolitis and pneumonia. in our study, the age relation was significant for rsv and hmpv which were detected in 89% (260 of 292) and 81% (84 of 104) of children under 5 years, respectively. similar to rsv, hmpv infections were frequently seen in winter and spring months as it was the case in our study while 90% of the hmpv cases were detected between january and april. 15 iav and ibv were detected with a frequency of 4.8% and 2.4%, respectively, and peaked between january and march. the presence of rsv and hmpv in circulation during the same period was notable, which necessitates the use of virological methods for the differential there are four subtypes of pivs (1-4) which may cause croup, pharyngitis, laryngitis, tracheobronchitis, bronchiolitis, and/or pneumonia. 19 piv-3 was the most commonly detected subtype in our study, followed by piv-1, -2, and -4. piv-3 is detected more frequently in april, may, and october. it was not possible to detect the seasonal relation of other piv subtypes due to a small number of cases. in other studies, piv-3 was also reported as the most common subtype followed other pivs similar to our findings. 20, 21 they were frequently detected during spring and early summer months. bocavirus, after being identified in 2005 has been isolated from 1.5% to 19% of the respiratory tract samples of children who were admitted to the hospital due to respiratory viral disease. 22 bocavirus causes coinfections up to 90% of the cases with other viruses such as influenza, rhinovirus, parainfluenza, rsv, and metapneumovirus. 23, 24 the prevalence of bocavirus was 2.8% in our study with a coinfection frequency of 48.8%, mainly associated with rhinovirus/enterovirus, rsv, and influenza a/b. infections were seen throughout the year without a clear seasonal relation although 58% (29 of 50) of cases were between january and march which is parallel to the other reported studies. [25] [26] [27] coronaviruses were identified nearly 5% of the patients, with oc43 being the most frequent subtype followed by nl63, hku1, and 229e. coinfections with rhinovirus or rsv were seen in almost half of the samples. these infections were detected in almost all months except june and august. it was reported that coronavirus infections peaked mainly in winter without any significant difference between the four subtypes in terms of seasonality and origin (community or hospital-acquired) of the infection. 28, 29 bordetella spp is an agent that can be mistaken as viral infection since can lead to prolonged cough and severe infections in infants, and young children. there were only 28 patients with bordetella infections in our study which were seen in patients less than 3-months old, except for one. although can be seen at any age, pertussis is more frequent and cause more severe disease in children younger than 3-months-old because of lack of primary vaccination. 30 high vaccine coverage (at least 95%) is necessary to protect children against vaccine-preventable diseases with the herd immunity. the importance of herd immunity is nicely described by an italian study of three cases of b. pertussis, one 16-month old and two less than a month old children showing that reduced immunity of the childʼs family over the years increases the risk of infection especially in infants lack maternal immunity with a more severe clinical presenta human metapneumovirus: review of an important respiratory pathogen epidemiology of acute respiratory infections non-influenza viruses in acute respiratory infections among young children. high prevalence of hmpv during the h1n1v.2009 pandemic in poland molecular diagnosis of respiratory virus infections epidemiology characteristics of respiratory viruses found in children and adults with respiratory tract infections in southern china epidemiologic analysis of respiratory viral infections mainly in hospitalized children and adults in midwest university medical center after the implementation of a 14-virus multiplex nucleic acid amplification test ten year retrospective evaluation of the seasonal distribution of agent viruses in childhood respiratory tract infections rapid multiplex nested pcr for detection of respiratory viruses clinical impact of rt-pcr for pediatric acute respiratory infections: a controlled clinical trial comparison of three multiplex pcr assays for detection of respiratory viruses: anyplex ii rv16, advansure rv, and real-q rv the role of multiplex pcr in respiratory tract infections in children extensive multiplex pcr diagnostics reveal new insights into the epidemiology of viral respiratory infections analysis of respiratory viral infections detected using multiplex real-time pcr in hwaseong global burden of acute lower respiratory infections due to respiratory syncytial virus in young children: a systematic review and meta-analysis human metapneumovirus circulation in the united states influenza surveillance in five consecutive seasons during post pandemic period: results from national influenza center molecular epidemiology of adenovirus type 7 in the united states molecular and clinical characteristics of adenoviral infections in taiwanese children in 2004-2005 parainfluenza virus infection seasonal trends of human parainfluenza viral infections: united states epidemiology of parainfluenza infection in england and wales, 1998-2013: any evidence of change? human bocavirus frequent and prolonged shedding of bocavirus in young children attending daycare detection of human bocavirus type 1 infection in panamanian children with respiratory illness the association of newly identified respiratory viruses with lower respiratory tract infections in korean children detection of human bocavirus in canadian children in a 1-year study human bocavirus infection in children with respiratory tract disease epidemiology and clinical features of human coronaviruses in the pediatric population seasonality and prevalence of respiratory pathogens detected by multiplex pcr at a tertiary care medical center pertussis (bordetella pertussis and b. parapertussis) report on three cases of pertussis in the urbino area (italy) systematic review and meta-analysis of respiratory viral coinfections in children respiratory viral coinfection and disease severity in children: a systematic review and meta-analysis viral respiratory infections diagnosed by multiplex polymerase chain reaction in pediatric patients the authors declare that there are no conflicts of interest. http://orcid.org/0000-0003-1810-8346 key: cord-328501-mbwgi56x authors: pang, junxiong; jin, jing; loh, jin phang; tan, boon huan; koh, wee hong victor; ng, sock hoon; ho, zheng jie marc; gao, qiuhan; cook, alex r; hsu, li yang; lee, vernon j; chen, mark i cheng title: risk factors for febrile respiratory illness and mono-viral infections in a semi-closed military environment: a case-control study date: 2015-07-25 journal: bmc infect dis doi: 10.1186/s12879-015-1024-7 sha: doc_id: 328501 cord_uid: mbwgi56x background: febrile respiratory illness (fri) results in substantial burden in semi-closed environments. tackling risk factors may reduce transmission and infection. however, risk factors involved in one setting may not be generalizable in all settings due to differences in climate, residential environment, population genetic and cultural backgrounds. this study aims to identify risk factors of fri and mono-viral infections in a tropical military environment. methods: from year 2009 to 2012, military personnel with temperature ≥37.5 °c, cough and/or sore throat, and personnel with no fever or no respiratory symptoms were recruited as cases and controls, respectively. subjects provided nasal wash specimens and answered a standardized questionnaire. resplex assays were used to determine the viral etiologies. descriptive, univariate and multivariate analyses of the variables were performed using appropriate descriptive tests and logistic regression modelling, respectively, with r program. results: a total of 7,743 fri cases and 1,247 non-fri study controls were recruited. increasing age [adjusted odds ratio (aor) = 1.03; 95 % confidence interval (ci) = 1.01-1.05], recruit camp (aor = 4.67; 95 % ci = 3.99-5.46) and smoker (aor = 1.31; 95 % ci = 1.13-1.52) were independent risk factors of fri. malay ethnicity was positively associated with influenza a(h1n1)pdm09 (aor = 1.50; 95 % ci = 1.04-2.15) and coxsackie/echovirus (aor = 1.67; 95 % ci = 1.19-2.36) mono-infection. significant contact risk factors were stay-out personnel with ill household member (aor = 4.96; 95 % ci = 3.39-7.24), and stay-in personnel with ill bunkmate and household member (aor = 3.55; 95 % ci = 2.57-4.91). staying in camp with none ill in bunk and at home was a protective factor against fri (aor = 0.80; 95 % ci = 0.64-0.99). these contact risk factors were similarly observed for the five most common viruses detected, namely adenovirus, rhinoviruses, influenza a and b, and coxsackie/echovirus. conclusion: increasing age, smoker, recruit-camp, stay-out personnel with ill household members and stay-in personnel with ill bunkmates were independent risk factors of fri in a semi-closed military environment. early identification and isolation of ill personnel from their bunk may be effective to prevent and reduce transmission and disease burden. febrile respiratory illness (fri) results in substantial disease burden in semi-closed environments such as in the households [1] and militaries [2] [3] [4] . fri is most commonly caused by viral infections, as observed in military respiratory surveillance programmes in finland [5] , united kingdom [4, [6] [7] [8] [9] , netherlands [10] , france [11, 12] , south korea [13] [14] [15] , west africa [16] , taiwan [17] , china [18] , singapore [19] [20] [21] [22] [23] [24] , and the united states [3, [25] [26] [27] [28] [29] . identifying risk factors of infection may provide guidance on policies and strategies for the prevention and control of fri. previous documented risk factors of fri in other countries included body mass index equal or greater than 25 kg/m 2 , previous respiratory tract infections [30] , overcrowding and closed units [29, [31] [32] [33] , presence of sand and dust storms, extreme temperature changes [34, 35] , smoking [36] , female, navy service, poor latrine facilities, increasing age and higher rank [37] . however, these risk factors may not be generalizable to different environments, and may differ between specific predominant aetiological agents. the predominant viruses reported in the singapore armed forces (saf) comprised adenovirus, rhinoviruses, influenza a and b, and coxsackie/echovirus between 2009 and 2012 [21] . adenovirus-associated respiratory disease, outbreaks and death have been reported in several countries amongst military recruits [10, 13, 14, 18, 19, 21, 27, [38] [39] [40] [41] [42] [43] . males and close contact with a person with respiratory symptoms within 10 days before their own onset of illness were associated with adenovirus infection, but sleeping adjacent to someone ill with respiratory symptoms did not present higher risk to infection [39] . influenza a and b viruses have also resulted in much morbidity in outbreaks, particularly influenza a(h1n1)pdm09 virus infection [11, 12, 17, 20, 21, 26, 44] . some of the risk factors proposed were crowded living quarters defined as more than three personnel and age group less than 40 year old [45] , asthma and obesity [46] , age group less than 30 years old and the high proportion of military who had being seroconverted [47] . human rhinoviruses are known to cause common cold as well as more complicated respiratory infections [9, [48] [49] [50] [51] [52] . all known human rhinoviruses have been reported to be present in military recruits during respiratory infection [53] . association of rhinovirus with lower respiratory tract infections is well documented [54] . viral interference has also been proposed between rhinovirus and adenovirus infection [38] . stress factor due to adaptation to new and different surroundings for military recruits was also proposed as risk factor for rhinovirus infection [48] . in this study, we investigate the risk factors associated with fri and the predominant viral aetiologies of fri in a semi-closed military environment of the saf. the saf started a sentinel respiratory disease surveillance program in four major camps (including a recruit training camp) in may 2009 [21, 22] to track febrile respiratory illness (fri) cases defined as patients with temperature ≥37.5°c with cough or sore throat. patients visiting primary healthcare clinics in the camps between may 2009 and october 2012 during regular consultation hours who met the fri criteria were recruited. the sentinel respiratory disease surveillance program includes the written informed consent obtained by healthcare workers, a questionnaire, clinical specimens collection and a clinical examination of the participants. repeat consultations were excluded if the healthcare worker determined that the patient had not recovered from the first episode of illness. we also obtained samples from controls (those without respiratory symptoms or acute infections), who were recruited from the same medical center during the same week as the recruitment of cases with about 5 to 10 controls per week. this is to ensure that both cases and controls had similar health-seeking behaviour, and similar chance of exposure to a particular respiratory pathogen circulating in the same environment around the same period of the year to minimize potential misclassification bias. moreover, the controls were not matched or restricted by barrack, sex, age or symptom-onset. this is because of the fact that the aim of the study is to evaluate most of these variables as potential risk factors of fri. informed consent, the baseline questionnaire, and clinical specimens were also obtained from these controls. the questionnaire covers demographics, co-morbidities, vaccination status, stay-in camp status and contact details of ill member in bunk (for stay-in personnel) and at home (for both stay-in and stay-out personnel). stay-in personnel stay in camp on weekdays and stay outside camp only on weekends, and hence, have household members and bunkmates as their key contacts. stay-out personnel do not stay inside camp on weekdays and have to travel in and out of camp on weekdays to work. these stay-out personnel hence only have household members but no bunkmates as key contacts. before the influenza a(h1n1)pdm09 pandemic in 2009, trivalent inactivated seasonal influenza vaccine (pre-pdm tiv) was in use. then, the pandemic monovalent influenza a(h1n1)pdm09 vaccine [pdm-a(h1n1)v] was first introduced to saf and administered to all recruits only from december 2009. this was superseded by the new trivalent influenza vaccine (post-pdm tiv) which included the influenza a(h1n1)pdm09 strain, first introduced to saf in october 2010, and routinely administered to all recruits in december 2010, and then all other military personnel in november 2011 (fig. 1 ). nasal washes from both side of the nose were taken by certified medical staff and sent to the laboratory for etiological testing within 24 h. detailed laboratory methods have been described in a previous study [21, 55] . briefly, we used a multiplex pcr panel which included 18 different respiratory viruses. they are as following-adenovirus e, influenza a(h3n2), rhinovirus, coxsackie/echovirus, influenza b, influenza a(h1n1)pdm09, enterovirus (ev), human metapneumovirus (hmpv), parainfluenza 1 (hpiv-1), hpiv-2, hpiv-3 and hpiv-4, coronavirus oc43 (cov-oc43), cov-nl63, cov-229e, cov-hku1, respiratory syncytial virus a (rsv-a) and rsv-b and bocavirus (bv). additional singleplex pcr assays were then performed to determine the influenza subtype. total nucleic acids were extracted from each clinical specimen using the dna minikit (qiagen, inc, valencia, ca, usa) according to the manufacturer's instructions. a total of 20 μl of dna extract were tested with resplex i and ii (version 2.0, qiagen, inc., valencia, ca, usa) for the presence of respiratory micro-organisms on the liquichip 200 workstation, according to the manufacturer's instructions. specimens that were resplex ii positive for flu-a were further subtyped with real-time pcr for h1 or h3, or for ph1n1. briefly, 5 μl of total genetic extracts were tested using an in-house developed assay based on the one-step superscriptiii/platinum taq kit (invitrogen, carlsbad, ca, usa) following the manufacturer's instructions on either the lightcycler machine from roche or the applied biosystems real-time pcr machine (7500). we compared variables of all fri subjects, and subsets of subjects with mono-viral infection (mvi) for the five most common viral pathogens (case groups) against the non-fri patients without viral infection detected from the panel (control group). specifically, the five most common viruses were influenza b, influenza a (h1n1) pdm2009, coxsackie/echovirus, adenovirus e and rhinovirus. univariate logistic regression was conducted to identify statistically significant variables of interest. selected variables with high co-linearity were dropped, but all other variables significant at p < 0.05 were then included in a multivariable logistic regression model to determine the independent factors. the best model was determined using backward stepwise regression method. power calculation showed at least 84 % power to detect a true positive association with effect size of 1.4 with 30 % of the controls having the exposure of interest as cases. all tests were conducted at the 5 % level of significance. we report odds ratio (or) and corresponding 95 % confidence intervals (ci) where applicable. all statistical analyses were performed using an open source statistical software r 3.0.1 (r core development team). written informed consent was obtained from the study participants. this study was reviewed and approved by the singapore military's joint medical committee for research, and the national university of singapore's ethics review committee. a total of 7,743 fri cases were recruited. of these, there were 3,422 fri cases (44.2 %) with mono-viral infection (mvi). of the 3,422 mvi cases, the five most common mvi were due to influenza b with 541 cases (15.8 %), influenza a (h1n1)pdm09 with 526 cases (15.4 %), coxsackie/echovirus with 523 cases (15.3 %), adenovirus e with 467 cases (13.6 %), and rhinovirus with 378 cases (11 %); the number of cases observed in each month from may 2009 to october 2012 is shown in fig. 1 . of the 1,365 non-fri subjects recruited, 1,247 subjects (91.4 %) were confirmed to be negative for the whole panel of respiratory pathogens tested, and these served as the study controls in all subsequent analysis. the mean age of fri cases was 20.8 (±3.12) as compared to 21.0 (±2.62) years old for controls (p < 0.001; table 1 ). a significantly higher proportion of cases (71.8 %) came from the recruit camp as compared to the controls (33.7 %; p < 0.001). the proportion of fri cases who had pre-pdm tiv and post-pdm tiv were significantly lower and higher than the study controls, respectively (2.3 % vs 3.9 %, p = 0.002, and 31.0 % vs 22.4 %, p < 0.001, respectively; table 1 ). in addition, there were significant differences in the smoking status among the cases compared to the controls (p = 0.028), and there were significantly higher proportion of fri cases than the study controls (20.6 % vs 17.2 %, p = 0.004). in terms of movement history, there was a significantly lower proportion of cases who had travelled to other camp in the last 14 days before clinical presentation than that of study controls (6.1 % vs 14.2 %; p < 0.001), and there were significantly higher proportion of fri cases who stayed in camp compared to the controls (86.9 % vs 81.6 %, p < 0.001; table 1 ). increasing age was observed to be an independent risk factor for fri [adjusted odds ratio (aor) = 1.03; 95 % confidence interval (ci) = 1.01-1.05; fig. 2 (table 2) . similarly, asthma (cor = 1.25; 95 % = 1.07-1.46) was a potential risk factor of fri, but it was not independently associated with fri after adjusting for potential confounding factors (table 2) . of the five most common mvi, increasing age was positively associated with coxsackie/echovirus(aor = 1.06; 95 % ci = 1.01-1.11; fig. 2 personnel who travelled to the community in the last 14 days before clinical presentation had a significantly lower risk of adenovirus mono-infection (aor = 0.14; 95 % ci = 0.02-0.84; fig. 3 ) compared to personnel who did not. however, personnel travelling overseas in the last 14 days before clinical presentation had 2.85 times higher risk of adenovirus mono-infection (aor = 2.85; 95 % ci = 1.22-6.65) compared with personnel who did not travel overseas. compared to stay-out personnel with no ill household members in the last 14 days before clinical presentation, stay-out personnel with ill household members had 4.99 times higher risk of fri (aor = 4.95; 95 % ci = 3.39-7.293). moreover, compared to stay-out personnel with no ill household members, stay-in personnel who had neither ill bunkmates nor household members had 1.28 times lower risk of fri (aor = 0.80; 95 % ci = 0.64-0.99). however, there was a higher risk of fri for stay-in personnel with an ill member in bunk regardless of whether they had any ill household members (aor = 3.55; 95 % ci = 2.57-4.91) or not (aor = 1.75; 95 % ci = 1.39-2.20) in the last 14 days before clinical presentation. results for the analysis on each of the five most common mvi were very similar to those for all fri analysis (figs. 2 and 3 ). there was significantly higher risk of infection for all of the five most common mvi in stay-out personnel with ill household members compared with those who did not (fig. 3) . regardless of whether they had ill household members or not, stay-in personnel with no ill bunkmates were not at significantly increased risk for any of the five most common mvi compared to stay-out personnel (with no ill household members). stayin personnel with ill bunkmates but without ill household members had significantly increased risk of all the mvi except adenovirus e (aor = 1.86; 95 % ci = 0.83-4.13), where there was a non-significant increase in risk; having ill household members further increased the risk for all [52] had simultaneously document the risk of fri due to a range of specific pathogens. in this study, we had shown that the five most common viral pathogens within our military environment was strongly associated with contact history, and had fairly similar trend of the fri risk factors identified. increasing age, recruit camp, and smokers were demographic risk factors for fri. increasing age was also reported as a risk factor for ari in us military personnel in overseas deployments [37] . additional analyses showed that the risk was higher with increasing age for all the five mvi in this study, but only significantly so for coxsackie/ echovirus. in contrast, increasing age was previously reported to be a protective factor for seroconversion against influenza a(h1n1) pdm09 in the local military during the initial wave of infections from june to october 2009 [47] . these discrepant findings may be due to the changing age distribution of susceptible population towards influenza a(h1n1)pdm09 infections [57, 58] , which might have shifted to involve more older individuals over the study period presented here (up to october 2012). in addition, this may be due to the increased in herd immunity effects among the new young cohorts of conscripts, where vaccine (initially as a monovalent formulation, and then later as part of the post-pandemic trivalent inactivated vaccine) was administered to all military recruits since november 2009 [23, 24] . moreover, the lack or waning immunity against influenza a(h1n1)pdm09 in the older cohorts may have attributed to this trend, even though the individual level effects of vaccination against influenza a(h1n1)pdm09 (which was found to be significantly protective) was accounted for in the multivariate model. personnel in the recruit camp were at higher risk of fri as well as all the five most common mvi, particularly adenovirus e infection. this is likely due to the higher contact exposure rate in semi-closed environments, and increased stressors [5, 7, 19, 21, 22, 29, 31, 38, 46] . alternatively, it could be due to the fact that personnel in non-recruit camps are already protected due to the adaptive immune response developed from the previous infections in recruit camp, where recruits usually only stay on a short term basis, before posted to non-recruit camp. smoking has been shown to increase risk of upper respiratory infection among recruits [36] , hajj medical mission personnel [59] , infants and children exposed to parental smoking [60] . hence, it is not surprising to observe smoking as a risk factor of fri in our study. there are some studies that had shown that cigarette smoking impairs oral and respiratory tract immunity [61] [62] [63] . this may have predispose smokers to a higher chance of viral infection resulting in fri. however, further study is warranted to investigate mechanism behind this observation. malay ethnicity was positively associated with both influenza-a(h1n1)pdm09 and coxsackie/ echovirus monoinfections. we had previously also found malays in the community to be at higher risk of influenza a(h1n1)pdm09 infection [64] . however, a previous study in the saf found that malays conscripts actually had significantly higher score in hygiene practices and knowledge towards pandemic influenza as compared to chinese and indians [65] . hence, there may be a potential genetic basis for the higher risk of infection in malays as compared to chinese and indians, given differences in genetic backgrounds of the hla class 1 region which have been shown to result in weaker immune response against pathogen antigens [66] . nevertheless, other unmeasured sociocultural and behavioural factors might explain these observations, and further studies are needed to confirm these observations and to understand the basis for the association. the protective effects of the influenza vaccine was largely in line with expectations, with the pre-pdm tiv protecting against influenza b but not against influenza a(h1n1)pdm09, the pdm-a(h1n1)v protecting against influenza a(h1n1)pdm09 but not influenza b, and the post-pdm tiv protecting against both pdm-a(h1n1)v subtypes, as observed in our previous study [24] . however, there were also some unexpected findings. these includes a potential protective effect (aor = 0.61; 95 % ci = 0.43-0.86) of the pdm-a(h1n1)v against rhinovirus, and an increased risk (aor = 49.51; 95 % ci = 32.91-74. 48) of adenovirus e infection with the post-pdm tiv. these findings may have been due to non-specific interactions and interference between respiratory viruses which have been suggested by others [38] , but could also have been due to the periodic nature of respiratory virus outbreaks. in particular, the post-pdm tiv period included a period of heightened adenovirus e activity (see fig. 1 ) which might have been unrelated to changes in the vaccination policy, but which we could not adjust for due to co-linearity between the timing of these adenovirus fig. 3 contact risk factors for fri and the five most common mono-viral infections e outbreaks and the phased roll-out of the influenza vaccine formulations. these unexpected findings would still require more scientific and epidemiological evidence for further conclusion. travelling overseas in the last 14 days before clinical presentation was associated with a significantly increased risk for adenovirus e infection. we were not able to distinguish these as either military or personal overseas trips, but a previous outbreak of b2 human adenovirus e11a strain in a military camp in singapore was also reported to be highly similar to other asian strains involved in outbreaks, suggesting a potential import of this strain from the neighbouring regions [19] . as such, implementation of adenovirus vaccination may be useful to prevent sudden surge of cases with adenovirus e outbreak, given the high incidence of adenovirus infection in south-east asia [19, 67] . one key finding was the relatively lower risk of fri and the five most common mvi for stay-in personnel as compared with stay-out personnel. at least for influenza b and a(h1n1)pdm09, this could be due to the lower proportion of members in the households and the community who had the seasonal influenza vaccination [47] , as compared to the camps where vaccination programme was implemented for all military personnel since the end of 2009 [23] . as such, this may have resulted in a smaller pool of susceptible individuals and a larger herd immunity effects in camps as compared to within the community. the other explanation maybe that stay-in personnel have less exposure to younger household members, which was previously found to have a significant risk for seroconversion to influenza a(h1n1)pdm09, and the risk was accentuated if the household member had fri [47] . this also concurs with our findings on the effect of exposure to ill household members and bunkmates, and the effects are influenced by the domiciliary status of the soldier. for the five most common mvi, an ill household member was a major risk factor for stay-out personnel. moreover, the increase in risk for stay-in personnel from having ill household members was not as marked and mostly not significant. however, stay-in personnel with an ill bunkmate had a substantial increase in risk of infection. while our current study design does not allow us to attribute the cause of infection to contact with these ill household members or bunkmates, our findings do suggest that some of the transmission of these pathogens is mediated through close contacts, and support the use of preventive measures for fri aimed at reducing transmission from ill household members and bunkmates. this could be in the form of issuing advisories to emphasize hygiene during outbreaks, and identifying and isolating ill personnel early to break the transmission of fri. moreover, this finding also has potential applications in surveillance. we had previously reported on how it would be difficult for syndromic surveillance systems to detect outbreaks in larger military units given the high baseline rates of respiratory illness [68] . given that the relevance of ill bunkmates is consistent for the predominant viral agents of fri, outbreak detection methods could instead focus on clusters of illness in those who share the same quarters, or are from the same military subunit as a reasonable proxy. we believe such an approach to syndromic surveillance deserves a prospective validation study where such clusters of illness are systematically sampled. there are several limitations to this study. first, there was the influenza a (h1n1)pdm09 pandemic in june to september 2009 during the early part of the study period, where the force of infection for influenza a (h1n1)pdm09 is likely to be higher than usual. however, the pandemic spread was well-contained with prompt protective and preventive measures such as vaccination (fig. 1) , enhanced respiratory hygiene measures, isolation, quarantine, "ring prophylaxis" with oseltamivir during this period. as such, these measure are also likely to limit the risk of transmission of other circulating respiratory viruses during this specific period compared to other periods in the study. since different vaccines were used promptly and appropriately during the different study periods (fig. 1) , vaccine type was used as a surrogate to account for the potential bias due to the enhanced protective and preventive measures applied during the influenza a (h1n1)pdm09 pandemic. nevertheless, this bias should be minimal because the controls were also recruited in the same period and camp as the cases. second, hand washing behaviour, allergy and military rank were not evaluated as potential risk factors of fri. this is because it was very challenging to accurately assess how frequent hand washing was performed by the soldiers. moreover, the soldiers may also tend to report the expected favourable hand washing behaviour. hence, the likelihood of recall bias and information bias are likely to be high and would make any form of interpretation challenging. allergy was not evaluated because the symptoms are very broad to specifically define as an allergy, and there would be significant potential information bias as it is less likely to clinically diagnosed allergy as compared with asthma, diabetes, hypertension and heart disease. furthermore, the aim of this study is not to study clinical signs and symptoms that are associated with fri. we did not consider military rank due to fact that there is a significant number of cases that were recruited from the recruit camp, where the population is mainly made up of recruits as compared to non-recruit camp, where the population is mainly made up of higher ranks (table 1 ; p < 0.010). as such, it would be biased to include military rank as one of the variables. third, our data is limited to febrile presentations of viral respiratory infections and may not be applicable to milder acute respiratory infections. fourth, there is a lack of clinical and laboratory confirmation of the ill household members and bunkmates, and such data are hence subjected to recall bias. fifth, the prevalence for fri and mvi is about 17 % and 1 % respectively. as such, or values as proxies to rr would be similar for mvi, whereas the or of the risk factors for fri is likely an overestimation, to some extent, relative to rr. sixth, the resplex i assay (qiagen) was designed to also detect six bacterial respiratory pathogens. they were mycoplasma pneumoniae, chlamydophila pneumoniae, legionella pneumomophila, streptococcus pneumoniae, neisseria meningitides and haemophilus influenza 1, 2, 3. however, fri subjects with bacterial causes were not excluded because one of the aims of the study is to determine the potential risk factors for fri, regardless of any detected or undetected respiratory virus and/or bacteria. lastly, this study involved predominantly young adult males in a military context, and hence, the results may not be generalizable to the overall population in the community, particularly for the contact risk factors. however, during the pandemic of influenza a(h1n1)pdm09, clustering of febrile respiratory illness by classroom contact among school children [69] and ill workplace contacts among healthcare workers were also observed [47] . further studies in other settings such as nursing homes which collect contact history in a similar way should be attempted. increasing age, smokers, recruit camp, stay-out personnel with ill household members and stay-in personnel with ill bunkmates were independent risk factors of fri in a semi-closed military setting. early identification and isolation of ill bunkmates may be effective to prevent and to reduce further transmission in camp. public health campaigns and policy should take these risk factors into consideration to increase the effectiveness of interventions to reduce fri in the military environment. abbreviations fri: febrile respiratory illness; aor: adjusted odds ratio; ci: confidence interval; saf: singapore armed forces; mvi: mono-viral infection. frequency of acute respiratory illnesses and circulation of respiratory viruses in households with children over 3 surveillance seasons epidemiology of community-acquired respiratory tract infections in adults. incidence, etiology, and impact recent trends of pneumonia morbidity in us naval personnel keep your hands clean influenza c virus infection in military recruits-symptoms and clinical manifestation respiratory infections in the military symptomatic respiratory syncytial virus infection in previously healthy young adults living in a crowded military environment respiratory syncytial virus: an important cause of acute respiratory illness among young adults undergoing military training viruses associated with acute respiratory infections in royal air force personnel patterns of infections with adenovirus types 4, 7 and 21 in military recruits during a 9-year survey the french military influenza surveillance system (miss): overview of epidemiological and virological results during four influenza seasons novel influenza a(h1n1) outbreak among french armed forces in 2009: results of military influenza surveillance system outbreak of febrile respiratory illness caused by adenovirus at a south korean military training facility: clinical and radiological characteristics of adenovirus pneumonia high isolation rate of adenovirus serotype 7 from south korean military recruits with mild acute respiratory disease acute lower respiratory tract infections in soldiers building military influenza surveillance capacity in west africa outbreak of influenza a and b among military recruits: evidence from viral culture and polymerase chain reaction outbreak of acute respiratory disease caused by human adenovirus type 7 in a military training camp in shaanxi outbreak of febrile respiratory illness associated with adenovirus 11a infection in a singapore military training camp differing clinical characteristics between influenza strains among young healthy adults in the tropics respiratory viral pathogens among singapore military servicemen 2009-2012: epidemiology and clinical characteristics viral agents responsible for febrile respiratory illnesses among military recruits training in tropical singapore effectiveness of pandemic h1n1-2009 vaccination in reducing laboratory confirmed influenza infections among military recruits in tropical singapore effectiveness of seasonal influenza vaccinations against laboratory-confirmed influenza-associated infections among singapore military personnel in 2010-2013. influenza other respir viruses history of respiratory illness at the u.s. naval academy respiratory diseases among u.s. military personnel: countering emerging threats acute respiratory disease in military trainees: the adenovirus surveillance program influenza and other respiratory viruses in three central american countries respiratory infections in military recruits risk factors for acute respiratory tract illness in military conscripts association between barracks type and acute respiratory infection in a gender integrated army basic combat training population exploration of the effectiveness of social distancing on respiratory pathogen transmission implicates environmental contributions buildingassociated risk of febrile acute respiratory diseases in army trainees prevalence of acute respiratory tract diseases among soldiers deployed for military operations in iraq and afghanistan environmental factors, immune changes and respiratory diseases in troops during military activities cigarette smoking and upper respiratory infection among recruits in basic combat training self reported incidence and morbidity of acute respiratory illness among deployed u.s. military in iraq and afghanistan broad spectrum respiratory pathogen analysis of throat swabs from military recruits reveals interference between rhinoviruses and adenoviruses outbreak of severe respiratory disease associated with emergent human adenovirus serotype 14 at a us air force training facility in 2007 vaccine-preventable adenoviral respiratory illness in us military recruits transmission dynamics and prospective environmental sampling of adenovirus in a military recruit setting adenovirus-associated deaths in us military during postvaccination period retrospective analysis of demographic and clinical factors associated with etiology of febrile respiratory illness among us military basic trainees outbreak of influenza in highly vaccinated crew of u.s. navy ship retrospective investigation of an influenza a/h1n1pdm outbreak in an italian military ship cruising in the mediterranean sea pandemic (h1n1) 2009 outbreak at canadian forces cadet camp influenza a(h1n1) seroconversion rates and risk factors among distinct adult cohorts in singapore epidemiology of the common cold in military recruits with emphasis on infections by rhinovirus types 1a, 2, and two unclassified rhinoviruses patterns of illness in rhinovirus infections of military personnel relationship of rhinovirus infection to mild upper respiratory disease. ii. epidemiologic observations in male military trainees relationship of rehinovirus infection to mild upper respiratory disease. 3. further epidemiologic observations in military personnel epidemiology of pathogen-specific respiratory infections among three us populations all known human rhinovirus species are present in sputum specimens of military recruits during respiratory infection rhinovirus and the lower respiratory tract a clinical diagnostic model for predicting influenza among young adult military personnel with febrile respiratory illness in singapore surveillance for febrile respiratory infections during cobra gold influenza disease burden in adults by subtypes following the initial epidemic of pandemic h1n1 in singapore factors influencing infection by pandemic influenza a(h1n1)pdm09 over three epidemic waves in singapore acute respiratory tract infections among hajj medical mission personnel, saudi arabia the study protocol for a randomized controlled trial of a family-centred tobacco control program about environmental tobacco smoke (ets) to reduce respiratory illness in indigenous infants cigarette smoking impairs human pulmonary immunity to mycobacterium tuberculosis effect of smoking on immunity in human chronic periodontitis cigarette smoke effects on innate immune mechanisms in the nasal mucosa. potential effects on the microbiome risk factors for pandemic (h1n1) 2009 seroconversion among adults knowledge, attitudes and practices towards pandemic influenza among cases, close contacts, and healthcare workers in tropical singapore: a cross-sectional survey ramifications of hla class i polymorphism and population genetics for vaccine development dramatic decline of respiratory illness among us military recruits after the renewed use of adenovirus vaccines an assessment of electronically captured data in the patient care enhancement system (paces) for syndromic surveillance teacher led school-based surveillance can allow accurate tracking of emerging infectious diseases -evidence from serial cross-sectional surveys of febrile respiratory illness during the h1n1 2009 influenza pandemic in singapore the work was supported by a singapore ministry of defence funded operational research program and the centre for infectious disease epidemiology and research in the saw swee hock school of public health of the national university of singapore and national university health system. the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. biodefence centre, ministry of defence, singapore, singapore. 5 yale-nus college, national university of singapore, singapore, singapore. 6 program in health services and systems research, duke-nus graduate medical school, singapore, singapore. 7 department of statistics and applied probability, national university of singapore, singapore, singapore. 8 department of medicine, national university of singapore, singapore, singapore.received: 15 january 2015 accepted: 13 july 2015 the authors declare that they have no competing interests, except for vl who had previously received unrelated research grants from gsk.authors' contribution jp wrote the manuscript and analysed the data. jj analysed the data. lyh, arc and mic revised the manuscript and assisted with data analysis. vjl conceptualized the study and revised the manuscript. bht, jpl, whvk, shn were involved in the laboratory testing of the specimens. mh and qg were involved in subject recruitment and screening. all authors read and approved the final manuscript. key: cord-349606-lup6tm2s authors: biill primo, osvaldo vinícius; lourenço, edmir américo; passos, saulo duarte title: detection of respiratory viruses in nasopharyngeal swab and adenoid tissue from children submitted to adenoidectomy: preand postoperative analysis date: 2014-02-17 journal: int arch otorhinolaryngol doi: 10.1055/s-0034-1368135 sha: doc_id: 349606 cord_uid: lup6tm2s introduction the presence of respiratory viruses in lymphoid tissues of the nasopharynx and oropharynx and its impact on recurrent infections and hypertrophy of these tissues are not yet fully understood. objective to identify and determine the prevalence of major respiratory viruses in nasopharyngeal secretions and adenoid tissue preand postoperatively of children undergoing adenoidectomy. methods a prospective observational study was conducted in 36 patients under 12 years of age with upper airway lymphoid hypertrophy who were undergoing adenoidectomy, in which various respiratory viruses were investigated using real-time polymerase chain reaction in adenoid tissue and nasopharyngeal secretions collected preoperatively and 30 days postoperatively. results at least 1 viral agent was isolated in any of the samples collected in 58.3% of children and 25.9% of total samples. respiratory viruses were identified in 33.8% of preoperative nasopharyngeal specimens and in 19.8% of postoperative secretion. of the 21 patients with positive results for any respiratory virus, 6 (28.6%) had more than 1 virus. considering all 36 respiratory viruses found, the main agent isolated was rhinovirus (27.8%), followed by bocavirus (22.2%). conclusion the virus found more frequently in all samples was rhinovirus. after removal of adenoid tissue, there was a decrease in the prevalence of the virus contained in nasopharyngeal secretion 30 days after surgery. the presence of respiratory viruses in lymphoid tissues of the nasopharynx and oropharynx and its impact on recurrent infections and hypertrophy of these tissues are not yet fully understood. objective to identify and determine the prevalence of major respiratory viruses in nasopharyngeal secretions and adenoid tissue pre-and postoperatively of children undergoing adenoidectomy. methods a prospective observational study was conducted in 36 patients under 12 years of age with upper airway lymphoid hypertrophy who were undergoing adenoidectomy, in which various respiratory viruses were investigated using realtime polymerase chain reaction in adenoid tissue and nasopharyngeal secretions collected preoperatively and 30 days postoperatively. results at least 1 viral agent was isolated in any of the samples collected in 58.3% of children and 25.9% of total samples. respiratory viruses were identified in 33.8% of preoperative nasopharyngeal specimens and in 19.8% of postoperative secretion. of the 21 patients with positive results for any respiratory virus, 6 (28.6%) had more than 1 virus. considering all 36 respiratory viruses found, the main agent isolated was rhinovirus (27.8%), followed by bocavirus (22.2%). conclusion the virus found more frequently in all samples was rhinovirus. after removal of adenoid tissue, there was a decrease in the prevalence of the virus contained in nasopharyngeal secretion 30 days after surgery. for $75% of respiratory disorders in childhood, which may vary from primary snoring to severe cases of obstructive sleep apnea with cardiovascular commitment. 4, 5 the pathogenesis of inflammatory/infectious disease of tonsils and adenoids probably involves their anatomical location and its antigen-processing function. there is no certainty about what determines the onset of chronic infection. viral infection with bacterial infection may be one of the triggering mechanisms of chronic infection, but the effects of the environment, personal factors, and diet, among others, may also be involved. several studies and theories have been published based primarily on allergic mechanisms or viral and/or bacterial infectious processes. [6] [7] [8] [9] although it is a very common health problem in daily medical practice, the presence of respiratory viruses in lymphoid tissues of the nasopharynx and oropharynx and their impact on recurrent infections and hypertrophy of these tissues are not yet fully understood and are being studied by numerous researches. many authors have identified the presence of multiple viral agents in these tissues, latent in asymptomatic patients. 6, [10] [11] [12] [13] [14] [15] most of these studies in the literature are performed among symptomatic and hospitalized children with acute disease, which hampers a better understanding of this pathophysiology. this study aims to identify and determine the prevalence of major respiratory viruses in nasopharyngeal secretions collected pre-and postoperatively and adenoid tissue of children undergoing adenoidectomy. a prospective observational study was performed in 36 patients under 12 years of age with upper airway lymphoid hypertrophy undergoing adenoidectomy or adenotonsillectomy, assisted by the otorhinolaryngology clinic of a university hospital. the study was approved by the institutional research ethics committee (protocol number 363/2011). all parents or guardians signed a consent form after being informed of the objectives, procedures, and responsibilities of the research, as well as received answers to any questions regarding the study. the study included children under 12 years of age with clinical and/or radiologic criteria for adenoidectomy or adenotonsillectomy (recurrent tonsillitis and/or hypertrophy of lymphoid tissues leading to upper airway obstruction). children were excluded from the study if they had fever or any type of acute respiratory tract infection at the time of collection of biological samples, if they had craniofacial malformations, if they were immunocompromised, or if their parents did not agree with their participation in the research project. several respiratory viruses (influenza a and b; parainfluenza 1, 2, 3, and 4; rhinovirus; respiratory syncytial virus; human bocavirus; coronaviruses; and metapneumovirus) were investigated by quantitative real-time polymerase chain reaction (q-pcr) in adenoid tissue removed surgically and nasal swab specimens collected preoperatively and at 1 month postoperative follow-up visit. nasal secretion was collected with the aid of a sterile metal rod nasal swab, following the rules of asepsis. the nasal cavity was previously humidified with 1 ml of sterile sodium chloride 0.9% solution. the swab was introduced directly into the nasal cavity without contact with the patient's skin or other region. after collecting, this swab was homogenized in microtubes containing sterile ringer lactate solution. the adenoid tissue was obtained by cold adenoidectomy under general anesthesia by curettage of adenoid tissue. small fragments of adenoid tissue were stored in microtubes containing virus transport media, which is a solution for transportation and storage of viruses in tissues. after collection, the samples were transported in liquid nitrogen in microtubes to the research laboratory of virology and molecular biology, where they were frozen at à80°c, and subsequently viral genetic material in the secretion and adenoid tissue was extracted according to a standardized protocol by the laboratory. after extraction, q-pcr was performed of respiratory viruses using the handset applied biosystems 7500 real-time pcr systems (life technologies™, california, usa) following the manufacturer's specifications and protocols. the ftd-respiratory-21 kit from fast-track diagnostics™ (junglinster, luxemburg) was used to identify respiratory viruses. the database was created using microsoft excel program (microsoft, redmond, washington, united states). the data were analyzed using absolute (n) and relative (%) frequencies, average, and standard deviation, depending on the variable studied. the averages of continuous outcome variables were compared by the student t test. the association of the qualitative measures between groups was performed using the chi-square test or fisher exact test to determine statistical significance. the software used for analysis was spss (statistical package for social sciences, ibm™, armonk, new york, usa) version 13 and the assumed level of significance was 5% (p < 0.05). the study evaluated 36 children between 3 and 12 years of age undergoing adenoidectomy or adenotonsillectomy from april 2012 to january 2013, with an average age of 7.3 years and a median of 7 years. of these 36 children, 25 (69.4%) were girls and 11 (30.6%) were boys. a total of 108 pre-and postoperative nasopharyngeal specimens and adenoid tissue samples were removed surgically from these 36 patients (75 samples from girls and 33 from boys). at least 1 viral agent was isolated in any of the samples collected in 58.3% of 36 children (n ¼ 21) and in 25.9% of 108 samples (n ¼ 28), in 36.4% (n ¼ 12) of 33 samples of boys and 21.3% (n ¼ 16) samples from 75 girls. of the respiratory viruses found in 36 samples, 75% (n ¼ 27) were isolated in children under 7 years of age. of the 21 patients with positive results for any respiratory virus, 6 (28.6%) had more than 1 respiratory virus. considering the 28 positive samples, the rate of viral coinfection also was 28.6% (n ¼ respiratory viruses were found in 33.3% of preoperative nasopharyngeal specimens (n ¼ 12), with a single virus isolated in 83.3% (n ¼ 10) and coinfection by 2 viral agents in 16.7% of samples (n ¼ 2), totaling 14 respiratory viruses. in adenoid tissue samples, respiratory viruses were found in 30.6% (n ¼ 11): single viral infection in 72.8% (n ¼ 8) and coinfection in 27.2% (n ¼ 3) of these positive samples (totaling 14 viruses). of postoperative nasopharyngeal secretions, the positivity was less-in only 13.9% of samples (n ¼ 5) was a respiratory virus isolated: coinfection in 60% (n ¼ 3) and single virus infection in 40% (n ¼ 2; ►table 1). from the 14 respiratory viruses identified in preoperative nasopharyngeal secretions, 50% were rhinovirus (n ¼ 7), present in 19% of 36 samples tested. in adenoid tissue, respiratory viruses were identified in 14, and the most frequent virus found was bocavirus, present in 11% of samples (n ¼ 4), corresponding to 28.6% of the virus isolated. in nasopharyngeal secretions collected after surgery, only 8 respiratory viruses were identified, of which 25% (n ¼ 2) were adenoviruses, present in 6% of samples. considering all 36 respiratory viruses isolated in 108 samples evaluated, the main agent isolated was rhinovirus (27.8%), followed by bocavirus (22.2%). other viruses isolated with a lower frequency were adenovirus (11.1%), enterovi-ruses (8.3%), parechovirus (8.3%), coronavirus hku (5.6%), parainfluenza virus 4 (5.6%), influenza a (2.8%), coronavirus 43 (2.8%), coronavirus 229 (2.8%), and metapneumovirus a/b (2.8%). the respiratory syncytial virus was not isolated in the samples (►table 2). in general, 58.4% of the patients (n ¼ 21) and 25.9% of samples (n ¼ 28) were positive for at least 1 respiratory virus. studies in the literature show different prevalences, because some studies were virus-specific and others researched the prevalence of multiple respiratory viruses at the same time. herberhold et al investigated several respiratory viruses in adenoid tissue from 30 children. the authors found a high positivity: at least 1 respiratory virus was identified in 97% of 52 samples. 16 sato et al also searched respiratory viruses in adenoid tissue and found at least 1 respiratory virus in all samples. 17 in the study of proenca-modena et al, this positivity was 97.5%, and the highest rate of viral detection was found in the adenoid tissue (85.7%), followed by nasal secretions (78.5%), tonsils (68.6%), and peripheral blood (1%). 8 considering all viruses found in this study, rhinovirus was isolated most frequently, followed by bocavirus. in the study of herberhold et al, rhinovirus was also the most common respiratory virus, identified in 67% of samples, followed by bocavirus in 53%, similar to our study but with different prevalences. 16 in another study, adenovirus was the most common viral agent, isolated in 47.1% of patients, followed by enterovirus (40.5%), rhinovirus (38%), and bocavirus (29.8%). 8 in the study from sato et al, adenovirus was the most common viral agent, present in 80% of samples. 17 alkhalaf et al analyzed 106 palatine and pharyngeal tonsils of 57 patients undergoing routine tonsillectomy or adenoidectomy. employing the technique of real-time polymerase chain reaction, the authors identified 84 samples (72.4%) positive for adenovirus. 18 in this study, 28.6% (n ¼ 6) of patients with positive results presented coinfection with 2 viral agents, and 2 patients had viral coinfection in 2 samples, totaling 8 samples. bocavirus was isolated in 75% (n ¼ 6) of the coinfections. rhinovirus was identified in all coinfections of preoperative nasopharyngeal secretions (n ¼ 2), and bocavirus was isolated in all coinfections in adenoid tissue (n ¼ 3). in coinfections of preoperative nasopharyngeal secretions, there was no predominant viral agent. herberhold et al found multiple respiratory viruses in 83% of samples evaluated. 16 in the study of proenca-modena et al, the rate of coinfection was 69.5%. the authors evaluated 121 children who underwent adenotonsillectomy and found high rates of viral detection in adenotonsillar tissues. the authors were researching respiratory viruses on palatine tonsils, adenoid tissue, nasal secretions, and peripheral blood but did not evaluate the postoperative nasopharyngeal secretions. 8 review of the literature found no studies comparing the prevalence of the virus in nasopharyngeal secretions pre-and postoperatively. some studies revealed that the adenoid tissue may be a reservoir of viruses. comparing the prevalence of virus pre-and postoperatively, we aimed to analyze a possible influence of adenoid tissue in the maintenance of these viruses in secretions. after the analysis of preoperative nasopharyngeal samples, this study found that 33.3% of patients (n ¼ 12) were positive for at least 1 respiratory virus. of the 14 respiratory viruses isolated, 50% were rhinovirus. after surgery, the positivity of respiratory viruses in nasopharyngeal secretions of these patients decreased: in only 13.9% of patients (n ¼ 5) was at least 1 viral agent isolated in the sample. in the postoperative secretions, 8 respiratory viruses were detected, and adenoviruses were identified in 2 patients. these results suggest a decrease in the prevalence of the virus after surgery (p ¼ 0.0522). however, the number of patients in the study is relatively small, and a greater number of samples are required to confirm or reject this hypothesis. in addition to the small sample size, a seasonal effect may contribute to the discrepancies found between our results and other studies in the literature. we know that some respiratory viruses are found in certain periods of the year; however, as we have seen, apparently the virus may persist in the adenoid tissue and nasopharyngeal secretions even after the symptomatic phase of the disease. the climatic conditions of each region, air quality, and population characteristics may also contribute to a greater or lesser circulation of respiratory viruses in a given region and time of year. the present study revealed that some patients with hypertrophy of pharyngeal tonsil exhibit respiratory viruses detected on nasopharyngeal and adenoid tissue, even if asymptomatic. these data suggest that the persistence or latency of respiratory viruses on nasopharyngeal and adenoid tissue after an acute infectious process may be related to the pathogenesis of lymphoid hypertrophy of the upper airways and that these tissues may function as a reservoir of virus, with possible influence on its transmission in the community. after adenoidectomy, a decrease in respiratory viruses present in nasopharyngeal secretions was observed. from this finding, we can infer a lower chance of occurrence of infections as well as secondary superinfections and virus transmission to contacts after surgery. considering the age group studied, in which there is a greater interpersonal contact, this decrease in the circulation of respiratory viruses becomes important for public health, with positive impact on the quality of life of these children. detection of respiratory viruses in nasopharyngeal swab and adenoid tissue primo et al. 153 this document was downloaded for personal use only. unauthorized distribution is strictly prohibited. detecção do vírus epstein-barr em tonsilites recorrentes análise histopatológica de produtos de adenotonsilectomia de janeiro de 2001 a maio de chronic tonsillitis: histological and immunohistochemical aspects síndrome de apneashipopneas durante el sueño en población infantil: diferencias en su expresión entre niños con hipertrofia amigdalar y con enfermedad concomitante assessing the impact adenotonsillectomy has on the lives of children with hypertrophy of palatine and pharyngeal tonsils active replication of hiv-1 at the lymphoepithelial surface of the tonsil the risk of adenoid hypertrophy in children with allergic rhinitis high rates of detection of respiratory viruses in tonsillar tissues from children with chronic adenotonsillar disease detection of respiratory viruses and atypical bacteria in children's tonsils and adenoids prevalence and cellular reservoir of latent human herpesvirus 6 in tonsillar lymphoid tissue presence of dna of human papillomavirus 16 but no other types in tumor-free tonsillar tissue human bocavirus in tonsillar lymphocytes rhinovirus/enterovirus rna in tonsillar tissue of children with tonsillar disease growth of influenza a virus in primary, differentiated epithelial cells derived from adenoids detection of epstein-barr virus in tonsillar tissue of children and the relationship with recurrent tonsillitis frequent detection of respiratory viruses by real-time pcr in adenoid samples from asymptomatic children detection of viruses in human adenoid tissues by use of multiplex pcr prevalence and quantitation of adenovirus dna from human tonsil and adenoid tissues the rhinovirus was the most frequently found virus in all samples, followed by bocavirus. after removal of adenoid tissue, there was a decrease in the prevalence of viruses in nasopharyngeal secretions. key: cord-283604-fqc9jp0l authors: chen, meng; zhu, zhen; huang, fang; liu, donglei; zhang, tiegang; ying, deng; wu, jiang; xu, wenbo title: adenoviruses associated with acute respiratory diseases reported in beijing from 2011 to 2013 date: 2015-03-27 journal: plos one doi: 10.1371/journal.pone.0121375 sha: doc_id: 283604 cord_uid: fqc9jp0l background: adenovirus is one of the most common causes of viral acute respiratory infections. to identify the types of human adenoviruses (hadvs) causing respiratory illness in beijing, a sentinel surveillance project on the viral aetiology of acute respiratory infection was initiated in 2011. principal findings: through the surveillance project, 4617 cases of respiratory infections were identified during 2011-2013. throat swabs (pharynx and tonsil secretions) were collected from all the patients, and 15 different respiratory viruses were screened by multiplex one-step pcr method. 45 were identified as adenovirus-positive from sporadic and outbreak cases of respiratory infection by a multiplex one-step rt-pcr method, and a total of 21 adenovirus isolates were obtained. five hadv types among three species, including hadv-3 (species hadv-b), hadv-4 (species hadv-e), hadv-7 (species hadv-b), hadv-55 (species hadv-b), and an undefined hadv type (species hadv-c) were identified. the comparison results of the penton base, hexon, and fiber gene sequences of the beijing hadv-3, hadv-4, hadv-7, and hadv-55 strains in this study and those from the genbank database indicated significant spatial and temporal conservation and stability of sequences within the genome; however, the phylogenetic relationship indicated that both strain bj04 and strain bj09 isolated in 2012 and 2013, respectively, may have recombined between hadv-1 genome and hadv-2 genome within species hadv-c, indicating intraspecies recombination. conclusions: this study confirmed that at least 5 hadv types including hadv-3, hadv-4, hadv-7, hadv-55 and an undefined hadv type were co-circulating and were the causative agents of respiratory tract infections in recent years in beijing. hadv-3, hadv-4, hadv-7, and hadv-55 showed the apparent stability of the genomes, while intraspecies recombination was identified in strain bj04 and bj09. the recombinants carrying penton base gene of hadv-1 as well as hexon and fiber genes of hadv-2 might be a novel type of hadv worthy of further study. human adenoviruses (hadvs) belong to the genus mastadenovirus within the family adenoviridae [1] . adenoviruses are non-enveloped, icosahedral, double-stranded dna viruses with genomes of 26-45 kb [1] . the viral capsid is composed of two types of capsomeres: the hexon and the penton (which consists of the penton base and the fiber). antigens at the surface of the virion are mainly type-specific [2, 3] . hexons are involved in neutralization, and fibers in neutralization and haemagglutination-inhibition. a recombinant that has a unique combination of these three regions (penton base; hexon loops; fiber knob) derived from previously recognized genotypes will be assign a new genotype (http://hadvwg.gmu.edu). traditionally, the only basis for recognizing a new type of hadv is by serology, and on the basis of their biological properties, hadvs have been classified into 7 species (human mastadenovirus a to g, hadv-a to hadv-g), including 52 human hadv types, which are formally recognized by the international committee on taxonomy of viruses (ictv) [4, 5] . in addition, novel hadv genotypes (hadv-53 to hadv-68) were recently identified based on their bioinformatics and genomic analysis of the complete viral genome sequences (http://hadvwg.gmu. edu). novel hadv strains may arise from mutations or recombination among the different types of hadvs [6] . hadv can cause a variety of clinical diseases such as acute respiratory disease [7] , gastroenteritis [8] , and keratoconjunctivitis [9] , which vary depending on the cell tropism of the viruses. among the hadv-associated respiratory diseases, viruses in species hadv-b (hadv3, 7, 11, 14, 16, 21, 50, 55) , species hadv-c (hadv-1, 2, 5, 6), and species hadv-e (hadv-4) [10] [11] [12] [13] [14] are recognized as the main pathogens responsible for the respiratory tract infection. as the capital city of china, beijing covers an area of 16,800 km 2 with a large population of more than 19.72 million (chinese statistics bureau, 2011). in order to elucidate the spectrum of the viral aetiology of acute respiratory infections and provide basic data to guide local disease prevention and control measures, a sentinel surveillance project on the viral aetiology of acute respiratory infections was initiated and sponsored by the beijing municipal health bureau in 2011. adenovirus is one of the most common causes of viral acute respiratory infections. in this study, our primary aim was to identify the types of hadv causing respiratory illness in beijing since 2011, to avoid the overuse of antibiotics and to improve the level of diagnosis and treatment of respiratory viral disease especially hadv associated disease in hospitals, and to provide scientific basis for prevention and control of hadv causing respiratory illness. this study did not involve human experimentation; the only human material used in this study was throat swab specimens collected from cases with respiratory tract infection during the implementation of the surveillance project on viral aetiology of acute respiratory infection. this study was approved by the second session of the ethics review committee of the national institute for viral disease control and prevention in china cdc. written informed consent for the use of the clinical samples has been obtained from all patients involved in this study. pharynx and tonsil secretions of the patients were wiped with disinfection long cotton swabs with gently action, and after samples collection, all samples were transported under a cold chain and preserved at −80°c for further identification. a multiplex one-step reverse transcription-polymerase chain reaction (pcr) was performed to screen for 15 different respiratory viruses (respiratory syncytial virus a and b, influenza virus a and b, parainfluenza virus 1-4, human adenovirus, human enterovirus, human rhinovirus, human metapneumovirus, human bocavirus, and human coronavirus nl63-229e and oc43-hku1) simultaneously by using a commercial kit (seeplex rv 15 ace detection kit; seegene, inc., seoul, korea) [15] . adenovirus-positive specimens were cultured and further analysed. virus isolation for the adenovirus-positive specimens was performed by using hep-2 cell lines (from american type culture collection, atcc number ccl-23) following the standard protocol [13] . cells inoculated with clinical samples were incubated at 37°c for 7 days. if no cytopathic effect was observed, the culture was used to inoculate fresh cells for up to 2 additional passages; the cultures with adenovirus-like cytopathic effects were passaged again to confirm the presence of the virus. determination of the nucleotide sequences of penton base, hexon, and fiber gene the viral dna was extracted from infected cells by using a qiaamp dna mini kit (qiagen, valencia, ca, usa) according to the manufacturer's instructions. for the typing of hadv, the penton base, hexon, and fiber gene sequences were obtained for all hadv strains; pcr was performed with the platinum pcr supermix (invitrogen) following the manufacturer instructions. the primer pairs designed to amplify and sequence the penton base, hexon, and fiber gene sequences are listed in table 1 . the amplification products were analysed with capillary gel electrophoresis by using the qiaxcel dna high resolution kit (qiagen, the netherlands). after the pcr products were purified with a qia gel extraction kit (qiagen, valencia, ca, usa), the amplicons were bi-directionally sequenced using the sanger sequencing method (bigdye terminator, version 3.1, cycle sequencing kit; life technologies, grand island, ny, usa) and an abi prism 3130 genetic analyser (applied biosystems, foster city, ca, usa). sequencher software (version 5.0; gene codes corporation, usa) was used to edit and assemble the raw sequence data. the blastn program (national center for biotechnology information, bethesda, md, usa) was used to identify the homologous nucleotide sequences in the genbank database. phylogenetic trees were generated by using the neighbour-joining method and the kimura-2-parameters model implemented in the mega program (version 5.03) [16] . the reliability of the tree at each branch node was estimated by 1000 bootstrap replicates. bootstrap values greater than 80% were considered statistically significant for grouping. table 2) . all the 21 patients who got hadvs infections presented fever in between 38.2 to 40.0 degrees, seven (33.3%) of 21 patients had radiographic evidence of pneumonia, one patient (4.8%) had bronchitis, and others 13 patients (61.9%) had only upper respiratory tract infection symptoms such as cough and runny nose ( table 2) . among them, hadv-55 infections (2 cases) and hadv-7 infections (4 of 6 cases) seems led to patients of severe symptoms (pneumonia), while hadv-3 and hadv-4 infection caused minor symptoms (symptoms of upper respiratory tract infection or bronchitis), with only one hadv-3 infection causing pneumonia. it is worth noting that the two patients infected with the undefined hadv type appeared to have only mild symptoms such as fever and cough, and both patients affected by this recombinant virus are infants (below 1 years old), while patients infected with other hadvs are all teenagers or adults. in order to further identify possible recombination events, phylogenetic analyses based on the penton base, hexon, and fiber gene sequences were used to analyse the relationship of bj04 and bj09 strains in this study as well as with hadv-c strains in the genbank database. (fig. 2) . in the penton base sequences, strain bj04 and bj09 were close to the corresponding sequences of the hadv-1 strains in the genbank database, and exhibited less similarity with the hadv-2 prototype strain recorded in the genbank database (fig. 2a) . and from the nucleotide sequence alignment based on the penton base gene (fig. 2b) , strains bj04 and bj09 are a little more like hadv-1 sequences, due to hadv-2 sequences having a 9-nt deletion from nt 1099 to nt 1107. in the hexon gene and fiber gene, the sequences of strain bj04 and strain bj09 clustered with that of hadv-2 ( fig. 2c and 2d) . these findings indicated that strain bj04 and bj09 may be recombinants having penton base gene of hadv-1, and hexon gene and fiber gene of hadv-2. in addition, recombination was not found among any of the other strains of hadv-1 and hadv-2 available in the genbank database. phylogenetic analysis of other hadv-3, hadv-4, hadv-7, and hadv-55 beijing strains showed relative genome stability (figs. 3, 4) . beijing hadv strains (representing 4 types) clustered with the sequences from the different provinces of the mainland of china and other hadvs within each corresponding type, and exhibited the highest similarity between those sequences. this indicated that these 4 hadv types' genomes (hadv-3, hadv-4, hadv-7, and hadv-55) are stable. this study documents the hadv types associated with respiratory infection in beijing during 2011-2013 through the surveillance project, and viruses in species hadv-b (hadv-3, 7, 55), hadv-c (undefined hadv type), and hadv-e (hadv-4) were identified. these results are similar to those of a previous study performed between 2005 and 2010 in beijing [17] , and confirmed that at least 5 hadv types were co-circulating and were the causative agents of respiratory tract infections in recent years in beijing. in addition to beijing, these hadvs were also most frequently detected from acute respiratory tract disease cases in other cities of china such as guangzhou in central south china [18] and lanzhou city in northeast china [19] , which indicated that these hadv types are widely distributed in china. this study is also consistent with reports from argentina [20] , usa [21] , egypt [22] , and korea [23] . in this study, a multiplex one-step rt-pcr was performed to screen for 15 different respiratory viruses using a commercial kit (seeplex rv 15 ace detection kit), and hadv is one of the target viruses. the reason for the positivity rate of multiplex one-step rt-pcr (45 adenovirus positive) being higher than that of the viral isolation (21 positive), may be mainly due that this method is more sensitive than viral isolation method for hadvs, and an other possible reason may be that the specimen collection is not timely, etc. the comparison results of the penton base, hexon, and fiber gene sequences between the beijing hadv-3, hadv-4, hadv-7, and hadv-55 strains in this study and the strains from the genbank database indicated significant conservation and stability of the sequences within the genome across time and space. this genome stability of hadv-3, 4, and 7 was also reported in earlier studies, showing that hadv-3 has displayed a relatively stable genome for more than 50 years [24] ; the genomes of the hadv-4 and hadv-7 strains are also remarkably conserved, albeit only extending for at least 20 years [25] . this characteristic has also been found for other hadvs such as hadv-5, whose genome was found to be stable even beyond the 45 years of its circulation in the population [26] . limited mutations and infrequent recombination may contribute to the long-term success of hadv-3, 4, and 7 vaccines. hadv-55 infection has gained attention in the last decade. genomics and bioinformatics data indicate that hadv-55 (earlier name hadv-11a) is an emergent respiratory pathogen due to recombination between hadv-11 and hadv-14 [13, 14] . as a newly identified acute respiratory disease pathogen, hadv-55-associated outbreaks were reported to have occurred in military camps of singapore and turkey in 2005 [27] , and in a senior high school in the shaanxi province of china in 2006 [13] . since 2008, this pathogen has been isolated from cases of respiratory infection and community-acquired pneumonia among adults in beijing, hebei, shandong, chongqing, and gansu provinces of china [28] [29] [30] ; it was also isolated among military trainees in hebei province of china in 2012 [30] . this may raise a serious public health concern, because hadv-55 has the potential to spread and cause severe epidemics in china, and it may become a major etiological agent causing pneumonia among the chinese population. in this study, two patients who were affected with hadv-55 also appeared to have relatively severe symptoms (clinical diagnosis was pneumonia), highlighting the significance of hadv-55 as being an increasing cause of respiratory illness especially pneumonia. therefore, efforts should be focused on a hadv-55 vaccine because of its stable genome. in addition, the genome stability of these hadvs is also a desired property in the application of these viruses as gene delivery vectors. in contrast to the apparent stability of the genomes of hadvs in general, they are also known to undergo recombination. recombination is a well-known feature in hadv genetics and is one of the most important factors driving the evolution of hadvs [31] . in this study, the phylogenetic analysis indicated that strain bj04 and strain bj09 may have recombined between hadv-1 genome (penton base gene) and hadv-2 genome (hexon and fiber genes) within species hadv-c, which are intraspecies recombination events. intraspecies recombinations have already been identified in many emergent hadv pathogens, which were subsequently identified as novel type hadvs, such as hadv-53 to 68 [13, 31, 32] . in this study, strains bj04 and bj09 were identified as intraspecies recombinants, and this recombination pattern (p1h2f2) has not yet been found in elsewhere, which indicated that it may also be a novel hadv type. although the two patients affected with this undefined type hadv appeared to have only mild symptoms (clinical diagnosis was upper respiratory tract infection), the pathogenicity remains unclear and the virus has the potential to cause serious symptoms because of its infection of children below 1-year old. further studies on the whole genomic sequence and virulence determination including a comparison of the growth kinetics and cytopathology of the recombinant virus and the two parental strains (hadv-1 and hadv-2) will be required to elucidate the characteristics of this novel hadv type. genetic content and evolution of adenoviruses phylogenetic analysis of the main neutralization and hemagglutination determinants of all human adenovirus prototypes as a basis for molecular classification and taxonomy molecular evolution of human species d adenoviruses virus taxonomy. seventh report of the international committee on taxonomy of viruses new adenovirus species found in a patient presenting with gastroenteritis evidence of frequent recombination among human adenoviruses molecular and serological characterization of species b2 adenovirus strains isolated from children hospitalized with acute respiratory disease in genome sequence of a novel virus of the species human adenovirus d associated with acute gastroenteritis molecular epidemiology of human adenoviruses d associated with epidemic keratoconjunctivitis in rapid detection of adenovirus in throat swab specimens by pcr during respiratory disease outbreaks among military recruits abrupt emergence of diverse species b adenoviruses at us military recruit training centers multiplexed luminex xmap assay for detection and identification of five adenovirus serotypes associated with epidemics of respiratory disease in adults outbreak of acute respiratory disease in china caused by b2 species of adenovirus type 11 genomic analyses of recombinant adenovirus type 11a in china high incidence of multiple viral infections identified in upper respiratory tract infected children under three years of age in mega5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods detection of three human adenovirus species in adults with acute respiratory infection in china human adenovirus infection in children with acute respiratory tract disease in guangzhou prevalence of adenovirus in children with acute respiratory tract infection in lanzhou, china molecular typing of adenoviruses in pediatric respiratory infections in molecular epidemiology and clinical presentation of human adenovirus infections in kansas city children molecular identification of adenoviruses associated with respiratory infection in egypt from lower respiratory tract infections due to adenovirus in hospitalized korean children: epidemiology, clinical features, and prognosis natural variants of human adenovirus type 3 provide evidence for relative genome stability across time and geographic space genomic and bioinformatics analyses of hadv-4vac and hadv-7vac, two human adenovirus (hadv) strains that constituted original prophylaxis against hadv-related acute respiratory disease, a reemerging epidemic disease computational analysis of adenovirus serotype 5 (hadv-c5) from an hadv coinfection shows genome stability after 45 years of circulation outbreak of febrile respiratory illness associated with adenovirus 11a infection in a singapore military training camp emergence of community-acquired adenovirus type 55 as a cause of community-onset pneumonia severe community-acquired pneumonia caused by adenovirus type 11 in immunocompetent adults in beijing epidemiology of human adenovirus and molecular characterization of human adenovirus 55 in china evidence of molecular evolution driven by recombination events influencing tropism in a novel human adenovirus that causes epidemic keratoconjunctivitis computational analysis identifies human adenovirus type 55 as a re-emergent acute respiratory disease pathogen we thank all the laboratory staffs and the epidemiologists in beijing center for disease control and prevention and team members in the projects. key: cord-312024-qdgqif5j authors: talbot, h. keipp; falsey, ann r. title: the diagnosis of viral respiratory disease in older adults date: 2010-02-01 journal: clinical infectious diseases doi: 10.1086/650486 sha: doc_id: 312024 cord_uid: qdgqif5j viral respiratory disease in older adults has been increasingly recognized as a significant cause of hospitalizations and death. unfortunately, the recognition and diagnosis of infection due to many viral respiratory pathogens in older adults can be elusive due to atypical clinical presentations and the insensitivity of current laboratory diagnostic tests in this population. for influenza diagnosis, rapid antigen tests followed by viral culture if negative, can be useful in older adults as long as clinicians are mindful of test limitations. although specific, rapid antigen tests are insensitive in this population. erroneous negative results may lead to delays in timely administration of antiviral treatment and institution of appropriate isolation precautions. the increasing availability of new rapid and sensitive molecular diagnostics such as polymerase chain reaction testing, should provide more accurate and timely diagnoses of viral respiratory infections in older adults in the near future. the burden of illness due to a variety of viral respiratory pathogens in the elderly population is increasingly being recognized. influenza and respiratory syncytial virus (rsv), in particular, have most commonly been found to be the leading culprits of viral lower respiratory illness. however, through the use of novel diagnostic methods, other viruses have been added to the list of significant pathogens in older adults, including parainfluenza virus , human rhinoviruses, coronaviruses, and human metapneumovirus (hmpv). overall, viruses have been implicated in 13%-31% of lower respiratory illnesses in elderly adults [1, 2] . influenza and rsv alone have been estimated to cause ∼53,800 deaths [3] each year in the unites states. older adults may not present with the typical "common cold" symptoms that are associated with viral infections [1, 2, [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] . rather, the clinical picture may be dominated by lower respiratory tract symptoms or decompensation of chronic medical conditions. illness in this age group represents reinfection, because all persons were infected as children and, thus, many have partial immunity. because of preexisting mucosal antibodies, lower viral loads may present in respiratory secretions making diagnosis challenging is this age group. the identification of viral infections in older adults is of practical importance for a number of reasons. first and foremost, isolation of subjects with highly contagious viral infections, such as influenza, is crucial in the inpatient and longterm care settings to prevent transmission of disease to frail and debilitated patients, as well as to health care workers. secondly, the diagnosis of influenza can help guide antiviral treatment for individual patients. prompt diagnosis of influenza is also critical in long-term care facilities and other closed populations in the event that institutional chemoprophylaxis is needed to limit outbreaks. although antiviral treatments are not currently available for the other respiratory viruses, diagnosis of infection these agents may also be increasingly important as more efforts are made to curtail unnecessary antibiotic use [13, 14] . despite recent advances in diagnostic methods, specific viral diagnosis often remains elusive in older populations. this article summarizes what is known about the diagnosis of viral respiratory diseases in elderly adults, with the hope of increasing understanding of the utility and limitations of the currently available diagnostic tests for viral respiratory pathogens, such as culture, rapid antigen testing, polymerase chain reaction (pcr) testing, and serologic analysis. table 1 summarizes this review. children with viral respiratory diseases typically present with classic symptoms, high viral titers, and positive results of viral cultures or rapid antigen tests. however, the elderly individual may present with atypical symptoms (eg, confusion, anorexia, dizziness, and falls) [15] ; may lack fever and be unable to articulate classic symptoms of viral infection, such as sore throat or myalgias; or may experience exacerbations of underlying chronic cardiopulmonary diseases. classically, influenza presents with the acute onset of fevers, myalgias, and cough [16] , and rsv presents with nasal congestion, wheezing, and cough [17] . for research purposes, influenza-like illness has been defined by the centers for disease control and prevention as fever with either cough or sore throat [18] , providing very good sensitivity in young adults (86.8%) [19] during periods of high influenza activity. however, this has not held true for older adults, likely as a result of the lack of fever and protean manifestations of influenza infection in older adults [20, 21] . in a prospective study of patients with obstructive lung disease [22] , the presence of fever had a sensitivity of only 26%, compared with culture and serological test results, when used to diagnose influenza in older adults. in a study of hospitalized adults, babcock et al [23] reported a poor sensitivity of symptoms of influenza-like illness (43%) in adults (approximately one-half of whom were aged у65 years). specimen collection. detection of virus whether by culture, rapid antigen testing, or pcr depends on the collection of an adequate specimen. nasal washes are traditionally used in children but are not well tolerated in older adults, especially delirious patients or patients with dementia. nasopharyngeal swabs, which are frequently contained in viral culture kits, can also be difficult to perform properly [24] . we find that a sep-arate nose and throat swab that are combined in a single vial of viral transport media is more acceptable to patients and provides an acceptable sample. to collect an adequate sample, the nasal turbinates should be rubbed gently but firmly for ∼5 s. specimen collection may be difficult in the older adults because of decreased secretions and nasal dryness associated with the use of nasal cannula supplemental oxygen in hospitalized patients. culture testing. traditionally, culture has been the gold standard for the diagnosis of viral respiratory disease. viral culture usually requires specialized facilities and well-trained staff. definitive identification of a viral pathogen may take days to even weeks. as noted, older adults generally have lower viral loads in their respiratory secretions, which may affect the sensitivity of cultures. viral culture is most useful for relatively hardy viruses, such as influenza virus, which can survive transportation to a laboratory, whereas more labile viruses (eg, rsv) cannot [25] . this can be particularly problematic for off-site long-term care facilities where specimen transport to a central laboratory may be delayed. no single cell culture line can grow all medically important viruses. therefore, clinical laboratories require some knowledge of what viruses are suspected and may need to use multiple cell lines to make a diagnosis of viral infection. recently, shell viral culture techniques have been introduced into clinical laboratories to speed time to detection. this technique requires centrifugation of the specimen onto a cell monolayer with the use of antigen detection to identify pathogens [26] . to increase the number of identifiable pathogens, some shell vial cultures have incorporated mixed cells, such as the r-mix (diagnostic hybrids), which uses 2 cell types (mink lung cells and human adenocarcinoma cells) and can simultaneously detect influenza viruses a and b, parainfluenza viruses 1-4, rsv, and adenoviruses. shell vial culture can decrease the time of diagnosis from 2-5 days to 1-2 days and retains the sensitivity and specificity of conventional culture [27] . influenza can be identified by both conventional and shellvial methods. the conventional cell culture technique requires an additional step of hemadsorption, because the cytopathic effect may be subtle, and results are typically available in 3-5 days. viral culture is most useful in highly febrile patients who have been ill only 2-3 days [28] . viral culture is relatively insensitive, compared with serological tests and pcr. in a study of older adults with serologically confirmed influenza, culture only identified approximately one half (22 of 43) of the infections [1] , and studies using pcr as the gold standard have shown the sensitivity of culture to be 21%-50% [29, 30] . diagnosis of rsv infection by culture is considerably more difficult than diagnosis of influenza, with sensitivities ranging from 17% to 39%, compared with serological tests and pcr [1, 29, 31] . the poor sensitivity of culture is likely due in part to greater lability of rsv, compared with influenza virus. parainfluenza virus can also be detected by routine viral culture, but like influenza virus, isolation of parainfluenza virus often requires the additional step of hemadsorption. it is likely that, for diagnosis of parainfluenza virus infection, culture is also relatively insensitive versus pcr, although data are limited. hmpv is in the same paramyxoviridae family as rsv but is much more difficult to grow [32] . currently, only a few research laboratories have been able to successfully grow this virus; thus, culture of hmpv is not available in most clinical laboratories. coronaviruses, like hmpv, are difficult to grow regardless of the age of the patient [33] . therefore, most epidemiologic work has depended on serological tests and reverse-transcriptase (rt) pcr and only available in research settings. enteroviruses and rhinoviruses grow on a variety of fibroblast cell lines and are identified by cytopathic effect and distinguished by acid liability testing. rhinoviruses have traditionally been divided into 2 species (a and b), with 100 serotypes. recently, however, a third species (c) has been identified. a and b species of rhinovirus are culturable, but the newly described c species of rhinovirus can only be detected using molecular methods [34] . the sensitivity of viral culture in older adults for these pathogens has not been specifically compared with that of molecular techniques, but insensitivity can be inferred from recent epidemiologic studies using rt-pcr that demonstrate rhinoviruses as common pathogens in this age group [35] [36] [37] . rapid antigen testing. enzyme immunoassays (eias) are simple, straightforward tests that can be performed at the point of care, with results available in !15 minutes. eia, often referred to as rapid antigen tests in the clinical setting, have had great success in the diagnosis of influenza and rsv infection in children [30, 38] . unfortunately, similar results have not been noted in older adults, likely because most rapid antigen tests require ∼10 3 plaque-forming units of virus to generate a positive test result. as noted with culture testing, adequate collection of the clinical specimen is critical for the optimal sensitivity of these tests. the sensitivities of eia for influenza depend upon the setting used, and the gold standard is used for comparison. the sensitivity of rapid influenza antigen testing in older adults has been as high as 77% in an outbreak setting in an nursing home when compared with culture [39] but as low as 38%-43% in other settings when compared to pcr [28, 40] . in addition, steininger et al [41] found that the sensitivity of eia for the diagnosis of influenza decreases with increasing patient age and can be as low as 8%-22% in patients aged у80 years. despite the low sensitivities associated with eia, the test does has good specificity in the older adult population. therefore, a positive eia result is likely a true positive test result. however, a negative test result in older adults does not rule out influenza. the sensitivity of eia for rsv in older adults is very low; at best, it is р10% when compared with serological testing and pcr [42] . given the low overall prevalence of rsv infection (5%-10%), these tests have very poor predictive value in older adults and cannot be recommended for general use. two exceptions that can be considered are immunocompromised patients or those with respiratory failure for whom viral loads may be higher [43] . fluorescent antibody assays. fluorescent antibody staining is another rapid method of diagnosing respiratory viral diseases. this technique involves placing a pellet of cells from the sample on a microscope slide followed by staining with viral specific fluorescent antibodies. the procedure requires staff trained in the technique, but results are often available in a matter of hours. in the clinical setting, these tests are either named direct fluorescent antibody assay or immunofluorescent antibody and can be used to test for adenoviruses, influenza viruses a and b, rsv, and parainfluenza virus types 1-3. currently, a direct fluorescent antibody is being developed for hmpv [44] . the sensitivity of the direct fluorescent antibody to detect influenza in patients of all ages was 68%, compared with viral culture [45] . for rsv, immunofluorescent techniques have sensitivities of 9%-23%, compared with serological testing and pcr in older adults [25, 42] . pcr. pcr, first introduced in 1984 by kary mullis [46] , has become a popular tool in the research setting and is being introduced into clinical laboratories. because pcr can detect minute amounts of viral nucleic acid and does not require infectious organisms for detection, pcr has surmounted the problems of poor sensitivity that have plagued culture and antigen detection in older adults. extreme care to avoid contamination must be used given the extreme sensitivity of pcr. most of the common respiratory viruses are rna viruses and require an addition step of reverse transcription prior to amplification. compared with previous studies that have used viral culture for diagnosis, studies using pcr have more accurately detected the presence of viruses (including influenza virus, rsv, hmpv, parainfluenza virus, rhinoviruses, and coronaviruses) in the lower respiratory tract illness in older adults [5, 13, 31, 36, 40, 42] . the use of pcr has allowed large epidemiologic studies of wellknown pathogens, such as influenza virus [29, 30] and rsv [29] ; has allowed studies of newly discovered pathogens, such as hmpv [47] ; and has also been used successfully in the nursing home setting to identify sources of outbreaks [48] . in addition, pcr is the only currently available method for the diagnosis of disease due to coronaviruses and group c rhinoviruses. pcr can be performed to test for individual viruses (singlevirus assays) or multiple viruses simultaneously (multiplex pcr). these tests require specialized equipment and staff training. many assays have been "home brews," but the us food and drug administration has recently approved some multiplex assays, including both the xtag respiratory viral panel (luminex) and the hexaplex (prodesse), for commercial use. the xtag respiratory viral panel detects influenza viruses a, a subtype h1, a subtype h3, and b; rsv a and b, parainfluenza viruses 1-3, hmpv, rhinovirus, and adenovirus. the hexaplex can test for rsv, influenza viruses a and b, hmpv, and parainfluenza virus. some sensitivity for individual pathogens is invariably lost with multiplex assays, but in balance, they retain better sensitivity than do culture and rapid antigen testing. serological diagnosis. because viral respiratory infections in older adults represent reinfection, a single serum sample to detect viral specific immunoglobulin g is not useful for diagnosis. instead, a у4-fold increase in antibody is required to identify a recent infection. unfortunately, immunoglobulin m assays have not proven to be useful for acute diagnosis, despite several older reports suggesting promise [49] . although the aging immune system may predispose older adults to increased susceptibility to infection and severe disease, the humoral response to respiratory viral infections appears intact. although counterintuitive, older adults appear to have a more robust antibody response to natural infection than do young healthy, adults [50] . thus, if baseline serum or an acute blood samples obtained early in illness can be compared with a convalescentphase specimen, detection of a у4-fold increase in viral specific antibody is an excellent method of diagnosis. obviously, as a result of the delay in diagnosis, serological testing is not useful for clinicians and patient care decisions. however, serological testing may be useful for retrospective analysis of nursing home outbreaks of respiratory disease. clinicians should maintain a high index of suspicion for viral respiratory infection in older adults with respiratory illnesses particularly during the winter months. although diagnosis can be problematic because of atypical presentations and insensitivity of available tests, influenza testing may be important for patient care and infection control. rapid antigen tests of a properly collected nasal sample especially early in illness can be very useful. however, it is important to recognize the limitations of such tests and understand one cannot eliminate influenza from the differential diagnosis if negative and viral cultures should be performed. pcr appears to overcome the difficulties of traditional methods (better sensitivity and shorter time to diagnosis) and hopefully will be available in clinical laboratories in the near future. peetermans we. viral lower respiratory tract infection in the elderly: a prospective in-hospital study clinical analysis of community-acquired pneumonia in the elderly mortality associated with influenza and respiratory syncytial virus in the united states aetiological role of viral and bacterial infections in acute adult lower respiratory tract infection (lrti) in primary care improved diagnosis of the etiology of community-acquired pneumonia with real-time polymerase chain reaction epidemiology and clinical outcome of virus-positive respiratory samples in ventilated patients: a prospective cohort study viral community-acquired pneumonia in nonimmunocompromised adults etiology of community-acquired pneumonia in hospitalized patients in chile: the increasing prevalence of respiratory viruses among classic pathogens incidence and characteristics of viral community-acquired pneumonia in adults viral infection in adults hospitalized with community-acquired pneumonia: prevalence, pathogens, and presentation study of community acquired pneumonia aetiology (scapa) in adults admitted to hospital: implications for management guidelines frequency of detection of respiratory viruses in the lower respiratory tract of hospitalized adults impact of rapid detection of viral and atypical bacterial pathogens by real-time polymerase chain reaction for patients with lower respiratory tract infection impact of rapid diagnosis on management of adults hospitalized with influenza pitfalls in the diagnosis and therapy of infections in elderly patients-a mini-review clinical manifestations and consequences of influenza respiratory syncytial virus infection in adults: clinical, virologic, and serial pulmonary function studies does this patient have influenza? predicting influenza infections during epidemics with use of a clinical case definition the predictive value of influenza symptomatology in elderly people influence of age on symptoms and laboratory findings at presentation in patients with influenzaassociated pneumonia recognizing influenza in older patients with chronic obstructive pulmonary disease who have received influenza vaccine case-control study of clinical features of influenza in hospitalized patients identification of respiratory viruses in adults: nasopharyngeal vs. oropharyngeal sampling evaluation of four methods for the diagnosis of respiratory syncytial virus infection in older adults rapid detection of respiratory viruses by shell vial culture and direct staining by using pooled and individual monoclonal antibodies clinical features of influenza a virus infection in older hospitalized persons respiratory syncytial virus infection in elderly and high-risk adults challenges in diagnosing influenza in older adults diagnosis of respiratory syncytial virus infection: comparison of reverse transcription-pcr to viral culture and serology in adults with respiratory illness a newly discovered human pneumovirus isolated from young children with respiratory tract disease coronavirus infection in acute lower respiratory tract disease of infants distinguishing molecular features and clinical characteristics of a putative new rhinovirus species, human rhinovirus c (hrv c) acute viral infections of upper respiratory tract in elderly people living in the community: comparative, prospective, population based study of disease burden influenza-like illness in residential care homes: a study of the incidence, aetiological agents, natural history and health resource utilisation a prospective, communitybased study on virologic assessment among elderly people with and without symptoms of acute respiratory infection comparison of rapid diagnostic techniques for respiratory syncytial and influenza a virus respiratory infections in young children detection and control of influenza outbreaks in well-vaccinated nursing home populations rapid molecular detection of influenza outbreaks in nursing homes near-patient assays for diagnosis of influenza virus infection in adult patients lack of sensitivity of rapid antigen tests for the diagnosis of respiratory syncytial virus infection in adults risk factors for respiratory failure associated with respiratory syncytial virus infection in adults evaluation of a commercial direct fluorescent-antibody assay for human metapneumovirus in respiratory specimens performance of directigen flu a+b enzyme immunoassay and direct fluorescent assay for detection of influenza infection during the 2004-2005 season specific enzymatic amplification of dna in vitro: the polymerase chain reaction human metapneumovirus infections in adults: another piece of the puzzle respiratory syncytial virus outbreak in a long-term care facility detected using reverse transcriptase polymerase chain reaction: an argument for real-time detection methods detection of an immunoglobulin m response in the elderly for early diagnosis of respiratory syncytial virus infection age related differences in humoral immune response to respiratory syncytial virus infection in adults potential conflicts of interest. a.r.f. has received research funding from gsk and sanofi pasteur, has received consulting honoraria from medimmune and astra zeneca, and serves on the advisory board of quidel. h.k.t. has received research funding from protein sciences, sanofi pasteur, wyeth, and vaxxinnate. key: cord-277327-il8uaavn authors: couch, md, robert b.; englund, md, janet a. title: respiratory viral infections in immunocompetent and immunocompromised persons date: 1997-03-17 journal: am j med doi: 10.1016/s0002-9343(97)00003-x sha: doc_id: 277327 cord_uid: il8uaavn the acute respiratory illnesses are the most common type of acute illness in the united states today. the respiratory viruses—which include influenza viruses, parainfluenza viruses, respiratory syncytial virus (rsv), rhinoviruses, coronaviruses, and adenoviruses—cause the majority of these illnesses. some of these viruses cause illness throughout the year, whereas others are most common in winter. all population groups experience these infections and illnesses. as the number of elderly persons and those with underlying disease increases, awareness is growing that these common infections can have serious consequences. this has recently been emphasized for immunocompromised persons. at the m.d. anderson cancer center (mdacc), infection surveillance of mostly hospitalized adults with leukemia or a recent bone marrow transplant yielded a respiratory virus from 181 of 668 (27.1%) respiratory illness episodes. in descending order of frequency, infections with rsv, rhinoviruses, influenza viruses, parainfluenza viruses, and adenoviruses were detected in each of three surveillance years. high frequencies of nosocomial acquisition occurred, as has been noted in prior reports. similarly, persistence of infection and high frequencies of pneumonia and death among infected patients occurred, which have also been noted earlier. at mdacc, pneumonia occurred in 58–78% of infected patients, and 22–44% died. the role of the virus infection in many cases of pneumonia is uncertain, but death from pure viral pneumonia is well documented. a number of immune deficiencies in this patient population and options for control of these infections have been described that can, respectively, account for the medical problem and provide ways to approach prevention and treatment. a cute respiratory conditions are the most common acute conditions occurring among persons in the united states. 1 their incidence exceeds that of injuries, digestive conditions, and infective and parasitic conditions combined. infection with a respiratory virus is the most common cause of an acute respiratory condition. the variety and omnipresence of respiratory viruses along with their facility for spread among human populations ensure their occurrence as causes of infection and illness in all human populations. this includes persons of all ages, the healthy and the unhealthy, and the immunocompetent and the immunocompromised. the unusual severity of illnesses occurring among immunocompromised persons infected with a respiratory virus has been emphasized recently. this article is an overview of respiratory viral infections in both immunocompetent and immunocompromised persons. the respiratory viruses are listed in table i . all serotypes in each genus are significant causes of acute respiratory illness (ari), except for the adenoviruses, of which nine serotypes (1-7, 14, and 21) are important causes of ari. a large number of other viruses can cause ari or prominent respiratory symptoms; however, they are better known for other disease manifestations. these include many enteroviruses, measles virus, herpes simplex type 1 virus, and epstein-barr virus (the cause of infectious mononucleosis). in all, 20 distinct viruses plus ú100 different rhinoviruses cause ari in humans. this number of viruses-along with the facts that reinfection may occur, other viruses can produce ari, and a large portion of ari cases presumed to be viral cannot be ascribed to a specific virus using current technology-explains the high incidence of viral respiratory infections and illnesses in all human populations. the respiratory viruses exhibit a predilection for infecting the respiratory passages. localization of j the most common infections in developed countries j the cause of an extraordinary amount of morbidity, they are the most important contributor to loss of time from work or school j a major cause of severe disease, hospitalization, and death from infection, particularly among very young children and the elderly j a major predisposing cause of otitis media, sinusitis, and acute bacterial pneumonia j a major cause of acute respiratory insufficiency in persons with underlying lung disease (asthma, chronic obstructive pulmonary disease, etc.) j a major cause/contributor to severe disease and death among immunocompromised persons the infection induces symptom complexes that are well known to physicians: the common cold, pharyngitis, laryngitis, tracheobronchitis, laryngotracheobronchitis in young children (croup), bronchiolitis, and influenza. more than one symptom complex may occur, and upper respiratory illness, lower respiratory illness, or acute diffuse respiratory illness may be the appropriate designation. the respiratory viruses also cause pneumonia, particularly among infants and very young children, although most infections in immunocompetent persons do not lead to pneumonia. respiratory viral infections occur throughout the year, although they are more common in winter than in summer. they affect persons of all ages, but their incidence is higher among children than among adults. infection may be asymptomatic or induce a severe and lethal infection. a variety of complications of viral respiratory infection have been described; most notable are secondary bacterial infections causing otitis media, sinusitis, or pneumonia. in these cases, a respiratory virus infection impairs the defense mechanisms that keep these anatomic sites free from infection with bacteria. among the other described complications are post-infectious encephalitis, the guillain-barré syndrome, reyes syndrome after influenza in children, and disseminated intravascular coagulation with influenza. a summary of the major medical features of the acute viral respiratory infections appears in table ii . they are a major cause of morbidity in healthy populations; they are a major cause of severe disease, hospitalization, and death among the very young, the elderly, and persons with underlying disease, even if they are immunocompetent. severe disease is also common among immunocompromised persons with an acute respiratory viral infection, but the contribution of the virus to the severe disease and death in these populations is not always clear. results of surveillance of immunocompromised adult cancer patients at the m.d. anderson cancer center (mdacc) who were experiencing a respiratory illness are shown in table iii for a 3-year period. sampling was limited to hospitalized patients with leukemia and hospitalized or clinic patients who had recently received a bone marrow transplant. a combined nasal wash and throat swab specimen was tested for virus as described previously. 2 a total of 668 illness episodes were sampled for virus: 235 isolates were obtained from sampling of 223 illness episodes; ú1 virus was isolated for 12 episodes. thus, 33.4% of respiratory illness episodes in these mostly hospitalized patients yielded a virus. when herpes simplex virus and cytomegalovirus isolations were excluded, 181 (27.1%) episodes were virus positive; two episodes yielded two respiratory viruses. it should be emphasized that 27.1% represents a minimal estimate of the frequency of infection with a respiratory virus among these patients with an ari, since serology was not done and repeat sampling was minimal. the distribution of isolates according to type of respiratory virus is shown in table iv . as noted, each of the major respiratory viruses was detected in each year of sampling. the most commonly detected virus was rsv, followed closely by the picornaviruses. of picornaviruses that were further categorized, 90% were rhinoviruses and 10% were enteroviruses. influenza a or b virus was present each year, as were parainfluenza viruses; 92% of the parainfluenza viruses were type 3. 3 the adenovirus isolates have not been typed. the distribution of isolates by time of year is shown in figure 1 . excessive specimen contamination and limited sampling is the probable explanation for the absence of isolates during the summer of 1993. nevertheless, the overall pattern is for rhinoviruses, adenoviruses, and parainfluenza 3 virus to occur throughout the year, with periodic increases in frequency. the occurrence of rsv and influenza viruses was confined to the winter and spring, thus accounting for an overall winter increase. in summary, respiratory viral infections, which are highly prevalent among immunocompetent persons with a respiratory illness, are also highly prevalent among adult immunocompromised cancer patients experiencing a respiratory illness. the pattern of occurrences by time of year are similar to those described for these viruses among immunocompetent persons, except for the frequency of rsv infection. 4 this frequency is distinctly unusual for adult immunocompetent patients and may represent an increased susceptibility for this infection among immunocompromised patients (glezen wp, unpublished data, 1996). alternatively, increased occurrences of more severe disease that increases recognition of rsv infection or increased transmission for this virus in the social-environmental circumstance of the populations sampled might explain the finding. three major and relatively distinctive features of respiratory viral infections among immunocompromised patients are (1) high frequencies of nosocomial acquisition, (2) persistence of infection beyond the time periods reported for immunocompetent patients, and (3) high frequencies of pneumonia and death in association with the infection. published reports of frequencies of nosocomial infections among hospitalized patients are summarized in table v. 3,5 -11 infection frequencies for the different studies and the different viruses varied between 55% and 83%; there are no apparent differences in frequency for the different viruses. these infections will have been acquired from an infected person. this could be another ill patient, but most cases are probably acquired from exposure to an infected person with little or no illness who brings the virus into the hospital. hospital personnel, including doctors and nurses, have been identified as probable sources; visitors, particularly children who exhibit high infection frequencies, are an alternative source. a major contributing factor to these infection frequencies is the prolonged stay that is characteristic of hospitalized patients who are immunocompromised. reports of persistence of respiratory viral infection among immunocompromised children are shown in table vi ; no reports for adults were identified. 6,7,12 -17 the range and single-case durations are greater for immunocompromised children than for immunocompetent children in all reports. as noted, some children shed virus for months. although the durations vary, prolonged infection characterized all three virus infections. the frequencies of pneumonia and death associated with a documented infection among immunocompromised adult leukemia and bone marrow transplant patients hospitalized at mdacc are shown in table vii . 3,11,18 -26 pneumonia occurred in 58 -78% of infected persons, and 22 -44% died. although overall frequencies of pneumonia and death appear similar for each virus, analysis of patterns of occurrences suggested a greater severity for rsv infection after bone marrow transplantation or chemotherapy than for the other viruses. moreover, the apparent value of early aerosol ribavirin and intravenous immunoglobulin (iv ig) therapy for rsv reduced the overall frequencies for that virus. 8 the role of the virus infection in causing the pneumonia and death is uncertain in many of these patients, who frequently have multiple infections. however, histologic examination of lung tissue from many who died indicates that the virus infection is the primary pathogen in many cases. considerable effort has been expended in identifying the major immune correlates and mechanisms for defense against a respiratory virus infection. a variety of nonspecific defenses contribute to resistance to infection and recovery from infection. these defenses include the mucous layer covering the respiratory mucosa, fever, the inflammatory response, a and b interferon, and nonspecifically activated phagocytes. the primary defense against acquisition of a specific virus is antibody to that virus, whereas the primary mechanisms for recovery are both humoral (antibody) and cell-mediated immune responses. current information indicates that the primary immunoglobulin and antibody in the upper respiratory passage is polymeric iga that is formed locally in submucosal tissues in response to antigen and transported across epithelial cells into upper respiratory secretions. 23 with descent into the lower respiratory passage, the proportion of 7s igg in secretions increases, as does the proportion of 7s igg antibody. 24 this antibody is derived primarily from serum. thus, following infection, the major antibody protecting the nasopharynx is considered to be iga, whereas that protecting the lower respiratory passages is igg. both types of antibody can, however, be present at both sites, and both antibody types are desirable for optimal protection against a specific virus. once infection occurs, immune defenses are activated to eliminate the infection and thereby bring about recovery from illness. both a and b interferon act early in infection and are important nonspecific mechanisms for promoting elimination of infection. specific antibodies that are induced may contribute to virus elimination in a variety of ways, including aggregation for clearance, opsonization, and early lysis of infected cells; lysis may be complement mediated or represent antibody-dependent cell-mediated cytotoxicity (adcc). the major specific cell-mediated immune response promoting recovery is cd8 t lymphocyte-mediated cytotoxicity. this mechanism has been shown to be of major importance in myxovirus and paramyxovirus infections in animal models. 25, 26 a number of immune deficiencies have been described in immunocompromised persons that would be reflected in one or more deficiencies of the immune mediators summarized above. lum 27 has described deficiencies after bone marrow transplantation, a circumstance representing an extreme degree of immune deficiency. these patients exhibit b lymphocyte impairment manifested by a reduced ability of b cells to respond to stimulatory cytokines, such as il-4, reduced serum immunoglobulin levels, and depressed primary and secondary responses to antigens. deficiencies in t cell function are indicated by reduced cd4 lymphocyte and cd4/cd8 cell ratios, reduced t helper cell and increased suppressor cell activity, reduced proliferative responses, reduced delayed-type hypersensitivity responses, and reduced cd8 lymphocyte cytotoxic function. reduced neutrophil numbers and function are of major importance for the occurrence and control of bacterial infection; but, except for an ability to mediate adcc, no role for neutrophils in defense against these virus infections has been suggested. also accompanying the chemotherapy used in many transplant cases is the occurrence of mucositis with the loss of mucosal integrity and possibly iga antibody occurrence in the upper respiratory passages. 27 reduced mucosal integrity and local iga antibody would lead to increased susceptibility to upper respiratory infection, while reduced serum antibody levels would increase susceptibility to lower respiratory infection. although increased susceptibility to infection in immunocompromised patients has not been proven, kempe et al 28 reported an increased rate of influenza virus infection among immunocompromised children compared with an immunocompetent population. of interest is the finding that the increase was accounted for primarily by infection among those with preexisting serum antibody. a deficiency in new antibody synthesis induced by infection would lead to increased susceptibility to reinfection. reduced t lymphocyte numbers and function with reductions in t cell cytotoxic function would lead to reduced ability to clear virus, with a resulting increase in both magnitude and duration of infection. an increased magnitude of infection would lead to an increased likelihood of viral pneumonia as well as secondary bacterial or fungal pneumonias that occur because of impairment of antimicrobial defenses by both the immune deficiency and the viral infection. thus, the immune deficiencies described for immunocompromised persons can account for a high frequency of viral respiratory infections as well as an increased severity of illness and a significant risk of death. the described variability in frequency and severity of the infections among the various populations of immunocompromised persons should reflect the degree of immune deficiency. this appears to be the case, as persons who are less immunocompromised than others seem to have less of a problem with viral respiratory infections. [29] [30] [31] the three general categories of options for control of viral respiratory infections are (1) prevention of exposure, (2) provision of immunity, and (3) administration of antivirals. table viii lists many of the modalities that might be used. the high frequency of nosocomial acquisitions indicates that hospital infection control practices oriented toward preventing exposure would be rewarding. (early results of such practices at the mdacc are reported in the article by raad and colleagues in this supplement.) the only vaccine available for any of the respiratory viruses is inactivated influenza vaccine. although immune responsiveness is impaired in immunocompromised patients, any response should be beneficial. immunization of patients could be supplemented by immunization of contacts (as currently recommended by the committee on immunization practices of the centers for disease control) and immunization of donors for bone marrow transplant recipients, as transfer of b cell memory has been demonstrated. 32, 33 although interferon-a induces common cold-like symptoms when given intranasally for long periods, use for a shorter term or despite occurrence of side effects could be considered for prevention of rhinovirus, coronavirus, and rsv infections, for which it is effective. [34] [35] [36] enhancing immunity by passive immunization is currently done for some immunocompromised patients, but could be targeted toward a specific pathogen, such as was done for prevention of rsv infections in infants with underlying lung disease. 37 adoptive immunization with donor lymphocytes for bone marrow transplant patients has been used and reported to be effective for control of other viral infections. 38 antivirals are currently available for influenza a and rsv. amantadine and rimantadine are approved for prevention and treatment and could be superior to vaccine for prevention of influenza a in this population, although data to support this possibility are lacking. treatment should be short term, as resistance develops rapidly. 39 ribavirin by aerosol is approved for treatment of severe rsv disease in infants but may be effective in adults as well. 6,8,15,40 -43 controlled and uncontrolled studies have indicated value for ribavirin in treatment of influenza a and b and parainfluenza virus infections. 16,44 -47 comment it is becoming increasingly clear that the respiratory viruses commonly infect immunocompromised persons and that these infected persons may exhibit severe respiratory illnesses with high frequencies of pneumonia and death. although not well documented, there is surely a gradient of infection and illness severity that relates to the degree and type of impairment of immune function. it is not clear at present that impairment of immune function can lead to increased susceptibility to infection, although early data suggest this is true and described immune impairments would make this likely. on the other hand, there is little doubt that severe pure viral pneumonias and the characteristic prolonged infections that occur in immunocompromised patients are attributable to deficiencies in mechanisms of recovery. the severity of rsv infections in patients with extreme degrees of immunodeficiency is notable; an understanding of the immune basis for this severity could aid in designing immune approaches for prevention and treatment of these infections. similarly, such an understanding of the other respiratory viral infections could be of value in the care not only of immunocompromised persons but also of immunocompetent persons. most patients with pneumonia are infected or colonized with bacteria and fungi and at death yield such organisms from lung tissue; many, however, exhibit a histologic pattern of virus pneumonia only and yield evidence of a specific virus in tissues. it is not clear at present whether the most significant role of respiratory viral infection in the pneumonias is as a primary pathogen, a contributing pathogen, or a pathogen predisposing to secondary infection with bacteria or fungi that then cause pneumonia. the last of these -their role as predisposing pathogens -is considered to be the major role for these infections among immunocompetent patients who develop pneumonia. options are available for control of some of the respiratory viral infections, and efforts to recognize and apply these options are needed while other options are being identified and developed. perhaps the greatest need at present, however, is for practicing physicians to appreciate the role that respiratory viral infection can play as a cause of serious disease in immunocompromised patients. current estimates from the national health interview survey, united states efficacy of sequential annual vaccination with inactivated influenza virus vaccine respiratory disease due to parainfluenza virus in adult bone marrow transplant recipients mycoplasmal infections of the respiratory tract nosocomial transmission of respiratory syncytial virus in immunocompromised adults an outbreak of respiratory syncytial virus in a bone marrow transplant center respiratory syncytial virus infection in children with compromised immune function combination therapy with aerosolized ribavirin and intravenous immunoglobulin for respiratory syncytial virus disease in adult bone marrow transplant recipients influenza b in transplant patients influenza b virus infection in pediatric solid organ transplant recipients influenza a virus infections among hospitalized adult bone marrow transplant recipients. bone marrow transplantation medical progress: cellular response to respiratory viruses with particular reference to children with disorders of cellmediated immunity respiratory syncytial virus illnesses in human immunodeficiency, virus-infected and noninfected children. pediatr virus infections in children with acute lymphoblastic leukaemia treatment of respiratory viral infection in an immunodeficient infant with ribavirin aerosol ribavirin treatment of viral pneumonitis in severe combined immunodeficiency disease parainfluenza 3 virus and other common respiratory pathogens in children with human immunodeficiency virus infection respiratory syncytial virus (rsv) disease among hospitalized adult immunocompromised patients with leukemia community respiratory virus infections among hospitalized adult bone marrow transplant recipients epidemiology of influenza a virus infection in patients with acute or chronic leukemia influenza among hospitalized adult immunocompromised patients with leukemia community respiratory virus (crv) infections in hospitalized adult patients with leukemia host defenses at mucosal surfaces immunoglobulin a in secretions from the lower respiratory tract the specificity and function of t lymphocytes induced by influenza a viruses roles of ab and gd t cell subsets in viral immunity the kinetics of immune reconstitution after human marrow transplantation influenza in children with cancer viral pneumonia in recipients of solid organ transplants influenza a in immunocompromised patients orthomyxoviral and paramyxoviral infections in transplant patients bone marrow transplantation for malignant disease intranasal interferon as protection against experimental respiratory coronavirus infection in volunteers prevention of natural colds by contact prophylaxis with intranasal alpha2-interferon the efficacy of intranasal interferon a-2a in respiratory syncytial virus infection in volunteers. antiviral research prophylactic administration of respiratory syncytial virus immune globulin to high-risk infants and young children donor leukocyte infusion as therapy of life-threatening adenoviral infections after t-cell-depleted bone marrow transplantation emergence and apparent transmission of rimantadine-resistant influenza a virus in families respiratory syncytial virus and parainfluenza virus infections in the immunocompromised host respiratory syncytial virus pneumonia in a cardiac transplant recipient case report: severe respiratory syncytial virus pneumonia in an adult renal transplant recipient: successful treatment with ribavirin successful therapy with ribavirin of late onset respiratory syncytial virus pneumonitis complicating allogeneic bone marrow transplantation ribavirin aerosol treatment of influenza parainfluenza virus respiratory infection after heart transplantation: successful treatment with ribavirin association of parainfluenza virus type 3 infection with allograft rejection in a liver transplant recipient nebulized ribavirin for influenza b viral pneumonia in a ventilated immunocompromised adult key: cord-306411-dutbxfl4 authors: eifan, saleh a.; hanif, atif; aljohani, sameera mohammed; atif, muhammad title: respiratory tract viral infections and coinfections identified by anyplex™ ii rv16 detection kit in pediatric patients at a riyadh tertiary care hospital date: 2017-11-21 journal: biomed res int doi: 10.1155/2017/1928795 sha: doc_id: 306411 cord_uid: dutbxfl4 respiratory infections are caused by an array of viruses, and limited information is available about viral coexistence, comparative symptoms, and the burden of illness. this retrospective cohort study aimed to determine the etiological agents responsible for respiratory tract infections by anyplex ii rv16 detection kit (rv16, seegene), involving 2266 pediatric patients with respiratory infections admitted to the department of pediatrics at king abdul-aziz medical city, ministry of national guard, riyadh, from july 2014 to june 2015. the most frequent respiratory infections were recorded in the 1 to 5 year age group (44.7%). rhinovirus (32.5%), adenovirus (16.9%), and respiratory syncytial virus (rsv) b (10.4%) were most common. in single viral infections, rhinovirus (41.2%), metapneumovirus (15.3%), and bocavirus (13.7%) were most frequent. in multiple viral infections, rhinovirus (36.7%), adenovirus (35.2%), bocavirus (11.2), rsv b (7.8%), and rsv a (6.7%) were most frequent. no significant difference was observed in clinical presentations; however, rhinorrhea and hypodynamia were significantly associated with viral respiratory infections. most respiratory viral pathogens peaked during december, january, march, and april. rhinovirus, adenovirus, and bocavirus circulations were detected throughout the year. winter peaks were recorded for rhinovirus, rsv b, adenovirus, and rsv a, whereas the metapneumovirus, and the bocavirus peaked in march and april. these findings enhance understanding of viral etiology and distribution to improve respiratory infection management and treatment. respiratory tract infections lead to mortality and morbidity in children especially during early years. among children, more than 80% of respiratory infections are associated with different viral infectious agents. respiratory virus infections are a major public health problem, due to the ease of spread and considerable morbidity and mortality. the association between respiratory tract infections and different viral pathogens has been reported to vary between 40% and 90% [1] [2] [3] [4] [5] globally. different studies reported the detection of viruses like human respiratory syncytial virus a (rsv a), human respiratory syncytial virus b (rsv b), human adenovirus (adv), human metapneumovirus (hmpv), human coronavirus, and human parainfluenza virus (piv). children under the age of 5 years were detected with human coronavirus 229e (hcov-229e), human coronavirus nl63 (hcov-nl63), human coronavirus oc43 (hcov-oc43), human parainfluenza virus 1 (piv-1), human parainfluenza virus 2 (piv-2), human parainfluenza virus 3 (piv-3), human parainfluenza virus 4 (piv-4), human rhinovirus (hrv), human enterovirus (hev), and human bocavirus (hbov). coinfections with different multiple viruses were reported in 15% to 61% of patients. [6] [7] [8] [9] . molecular techniques such as multiplex polymerase chain reaction (pcr) are widely used for the detection and identification of respiratory viruses [10] [11] [12] and are helpful in the management and treatment 2 biomed research international of respiratory infections [13] . diagnosing respiratory viruses by isolation in cell cultures and serology is time consuming, laborious, expensive, and less sensitive in some cases. molecular techniques provide quick results with high sensitivity and specificity. multiplex pcr has been reported as a fast and sensitive assay for respiratory infection detection. anyplex ii rv16 (seegene, korea) is a multiplex real-time pcr based kit with tagging oligonucleotide cleavage extension (toce) technology. the pitcher and catcher are two novel components used in toce assay for unique signal generation in real time. in toce assay detection point is moved from the target sequence to the catcher so it provides the predictable melting temperature analysis for catcher duplex. this process offers the multiplex real-time pcr capability to anyplex ii rv16 kit. [14] [15] [16] [17] . respiratory infections are mostly reported in children living in developing countries. the spread of respiratory infections varies between populations and countries, depending on differences in geography, climate, and socioeconomic conditions [18] [19] [20] [21] . the central region (riyadh region) of saudi arabia has a dense population of locals and immigrants whose interaction can affect the transmission patterns of different respiratory viruses. previous studies have reported the prevalence of a small number of respiratory viruses within different regions of saudi arabia, and limited information is available on the seasonal distribution of viruses [22] [23] [24] [25] . a better understanding of the local epidemiology and risk factors is critical for the prevention and control of respiratory infections. this study aimed to determine the distribution of 16 different viruses causing respiratory infections in children, by using rv16, and to compare data on demographic characteristics, symptoms, and single infections or coinfections. design. this retrospective cohort study included 2266 patients within an age range of 0 to 14 years from july 2014 to june 2015 with suspected acute respiratory illness and respiratory infection. the patients were examined clinically and initially diagnosed by an admitting physician. nasopharyngeal aspirates, bronchoalveolar lavages, and nasopharyngeal swab specimens were sent for pcr analysis of respiratory viruses to the division of microbiology, pathology and laboratory medicine, king abdul-aziz medical city, riyadh, saudi arabia. when more than one virus was detected simultaneously from single or multiple samples of the same patient, the findings were recorded as multiple infections. patient demographic information was obtained from medical records. demographic and clinical data were recorded in a standardized performa, including age, sex, and clinical presentation. the study was approved by the ethics committee at king abdul-aziz medical city. respiratory virus samples were collected from patients presenting to hospital for the first time with symptoms of respiratory infection or within 7 days of admission. the time frame included for the sampling criteria was based on the incubation periods of these viruses [26, 27] . patient samples collected after 7 days from the date of admission were excluded from the study, as these samples were considered nosocomial infections. nucleic acids were extracted from all samples using microlab nimbus ivd (seegene inc.), and rnas were used for cdna synthesis using cdna synthesis premix (seegene inc.). the samples were tested by using anyplex ii rv16 detection kit (seegene inc.) according to the manufacturer's instructions. the assay was used to detect flu-a, flu-b, rsv a, rsvb, adv, hmpv, hcov-229e, hcov-nl63, hcov-oc43, piv-1, piv-2, piv-3, piv-4, hrv, hev, and hbov. reaction mixtures for virus detection were divided into two panels: a and b. each panel was used to detect 8 viruses with appropriate controls. two types of dna and 14 types of rna viruses were amplified and detected by using cfx 96 real-time pcr thermal cycler (bio-rad). seegene viewer software was used to analyze the amplification results. the study was approved by the research and ethical committee of king abdul-aziz medical city, riyadh. analysis. data analysis was performed by using spss (version 22.0; ibm). differences in the distribution of categorical variables were compared using chi-square or fisher's exact tests. a p value of ≤0.05 was considered significant. from among 2266 hospitalized patients, different respiratory infectious viruses were detected in 2041 (91.6%) samples (1082 male and 959 female participants). among age group of 1 to 5 years 44.7% respiratory infections were recorded and 7.8% infections were detected in age group of 11 to 14 years ( table 1) (table 2) . statistically significant relationships were found between the detection of single and multiple virus infections in the adv and hev groups ( < 0.05). in multiple viral infections coinfections were recorded among hrv (36.7%), adv (35.2%), hbov (11.2), rsv b (7.8%), and rsv (6.7%), respectively (table 3) . patients' clinical presentations with different etiological agents were compared and listed in table 4 . the monthly distribution patterns of different respiratory viruses were shown in figure 1 . the prevalence rate of sixteen respiratory viruses during spring, summer, autumn, in [29] . our findings are in line with previous studies that reported viral detection rates ranging between 47 and 95% [1, 3, 7, 30] . the wide ranging differences in viral pathogen detection rates may be attributed to heterogeneity within the study population, genetic variability, the types and numbers of viral pathogens included for testing, and the methods used for testing [1, 20, 31] . most of the respiratory infections were recorded in the 1to-5-year-old age group, followed by the age group >1 year old (table 1) , whereas a previous study [25] detected a higher ratio of respiratory pathogens among children less than 1 year of age. the higher rate of infections in infants and young children may be related to underdeveloped or weak immune systems, less healthy living conditions, greater pathogen exposure, and poorer hygiene [1, 4, 10, 31] . the detection of disease-causing viral pathogen plays an important role in patient management and treatment. in this study, the most common respiratory viruses detected were hrv (32.5%), adv (16.9%), and rsv b (10.4%). a previous study [25] reported detection rates for rsv (23.9%), hrv (14.7%), and adv (11%) in saudi arabia. in another study in china, influenza viruses (18.50%), rsv (7.86%), and adv (3.47%) were found to be the most common respiratory pathogens [32] . in multiple viral infections, hrv (36.7%), adv (35.2%), hbov (11.2), rsv b (7.8%), and rsv a (6.7%) were found most frequently. in single viral infections, hrv (41.2%), hmpv (15.3%), and hbov (13.7%) were detected most frequently (table 3) . adv and hev were found to be significantly higher in number than other viruses. this finding suggests that the occurrence of these viruses may facilitate other viral pathogens to infect the patient. the mixed viral infection rate was 42.3% for all samples in our study. hrv (36.7%), adv (35.2%), hbov (11.2), rsv b (7.8%), and rsv a (6.7%) were found among most frequent coinfected groups. another study has reported detecting multiple viral infections involving rhinovirus, adv, and hcov-oc43 groups [2] . hrv and adv have also been reported as the leading viral pathogens involved in mixed viral infectious agents among children [33] . these results further suggest that some viral groups facilitate infection or colonization by other viruses in the same patients. viral coinfection or the detection of two or more viral pathogens in a single patient may be attributed to asymptomatic persistence or the shedding of viruses [3] . the great variations in detection rates of multiple viral infections, in different studies, may be related to differences in study populations, locations, study periods, environmental factors, the number of viral pathogens tested, and differences in diagnostic techniques [2, 8, 34] . similarities in clinical presentations of patients infected with different respiratory viruses make it difficult to diagnose the precise etiological agent based on clinical signs. in our study, fever and cough were observed as common symptoms in patients infected with different respiratory viruses, and a strong association of rhinorrhea and hypodynamia was observed alongside other nonsignificant clinical symptoms. in the present study, rsv a, rsv b, hrv, and hev showed peaks of activity during december, whereas hmpv and hbov peaked in march and april. hrv, adv, and hbov circulations were observed throughout the year. winter seasonal peaks were recorded for rsv a and rsv b. an increase in the detection of viral pathogens frequencies occurred during cold and rainy seasons. seasonal variations of different respiratory viruses have been studied [35] and a significantly higher number of viruses have been detected during winter (54.7%) compared to summer (31%), with hrv being the most common pathogen in all seasons. other studies have reported detecting most influenza viruses in november and december, and rsv was detected most frequently between december and february [4, 8, 9, 28] . this study lacked further information on bacterial cultures taken from the samples used for viral pathogen detection. provision of this information would have been helpful for the assessment of concomitant infection by respiratory viruses and bacterial pathogens to help control and treat respiratory infections. this study incorporated data for a period of one year only, but the inclusion of data from the previous and following years could have provided more information on circulatory patterns of respiratory viruses. finally, this study was conducted on a single site in the riyadh region, and virus spread and circulation patterns are likely to differ in other regions of saudi arabia, such as jeddah and dammam. figure 2 : distribution of sixteen respiratory viruses during spring, summer, autumn, and winter. in conclusion, our study provides information regarding the circulatory patterns and seasonal distribution of human respiratory pathogens in the central region of saudi arabia. rhinoviruses, adenoviruses, and rsv were found to be the most common pathogens in pediatric patients. the rv16 based pcr diagnostic approach increased our understanding of viral etiology for better management, control, and treatment of respiratory infections. the authors declare that there are no conflicts of interest regarding the publication of this paper. respiratory pathogens in children with and without respiratory symptoms epidemiology of respiratory viral infection using multiplex rt-pcr in virological and clinical characterizations of respiratory infections in hospitalized children ten year retrospective evaluation of the seasonal distribution of agent viruses in childhood respiratory tract infections epidemiological and clinical characteristics of respiratory viral infections in children in viral etiology of acute respiratory tract infections in hospitalized children and adults in shandong province, china other viruses (e.g. pox, papilloma, parvo, reoviridae) viral coinfection in childhood respiratory tract infections etiology, seasonality, and clinical characterization of viral respiratory infections among hospitalized children in beirut, lebanon etiology, seasonality, and clinical characteristics of respiratory viruses in children with respiratory tract infections in eastern india (bhubaneswar, odisha) diagnostic value of respiratory virus detection in symptomatic children using real-time pcr new molecular virus detection methods and their clinical value in lower respiratory tract infections in children detection of respiratory viruses using a multiplex real-time pcr the use of multiplex pcr for the diagnosis of viral severe acute respiratory infection in children: a high rate of co-detection during the winter season detecting respiratory viruses in asymptomatic children comparative evaluation of the seegene seeplex rv15 and real-time pcr for respiratory virus detection seasonality and prevalence of respiratory pathogens detected by multiplex pcr at a tertiary care medical center comparison of anyplex ii rv16 with the xtag respiratory viral panel and seeplex rv15 for detection of respiratory viruses respiratory viruses in children hospitalized for acute lower respiratory tract infection in ghana surveillance of 16 respiratory viruses in patients with influenza-like illness in nanjing, china incidence of respiratory viruses in peruvian children with acute respiratory infections viral aetiology of acute respiratory infections among children and associated meteorological factors in southern china human metapneumovirus and human coronavirus infection and pathogenicity in saudi children hospitalized with acute respiratory illness detection of bocavirus in children suffering from acute respiratory tract infections in saudi arabia viral agents causing acute lower respiratory tract infections in hospitalized children at a tertiary care center in saudi arabia viral etiology of respiratory infections in children in southwestern saudi arabia using multiplex reverse-transcriptase polymerase chain reaction nosocomial spread of viral disease incubation periods of acute respiratory viral infections: a systematic review etiology and seasonality of viral respiratory infections in rural honduran children evaluation of viral co-infections in hospitalized and non-hospitalized children with respiratory infections using microarrays viral etiology of acute respiratory infection in gansu province viral etiology of acute febrile respiratory illnesses in hospitalized children younger than 24 months epidemiology characteristics of respiratory viruses found in children and adults with respiratory tract infections in southern china epidemiology and microbiological investigations of community-acquired pneumonia in children admitted at the emergency department of a university hospital prevalence and seasonal distribution of respiratory viruses in patients with acute respiratory tract infections seasonal variations of 15 respiratory agents illustrated by the application of a multiplex polymerase chain reaction assay the authors would like to extend their sincere appreciation to the deanship of scientific research at king saud university through the research grant no. rgp-vpp-253 and national guard hospital for funding of this work. key: cord-286924-zk7hzcla authors: haytoğlu, zeliha; canan, oğuz title: bocavirus viremia and hepatitis in an immunocompetent child date: 2017-05-15 journal: balkan med j doi: 10.4274/balkanmedj.2015.1492 sha: doc_id: 286924 cord_uid: zk7hzcla background: so far, many studies have shown that human bocavirus ( hbov) is the main pathogen of the respiratory tract. until now, there is no study that proves the association between hbov and hepatitis. hbov viremia/dnaemia has been associated closely with acute primary infection and moderate-to-severe illness but, more detailed clinical data about hbov dissemination are still unavailable. case report: here we report a 2-years-5-months-old girl suffering from respiratory distress and heptitis followed in our intensive care unit. hbov was detected in our patients nose and throat swabs concurrent with whole blood sample by positive polymerase chain reactions. after a through investigation no causative agent other than hbov viremia was found. conclusion: human bocavirus viremia with high viral loads may be associated with hepatitis. human bocavirus (hbov) is a newly described human pathogen that has been frequently associated with upper and lower respiratory tract infections. the reported prevalance of the virus has been ranging from 2% to 19% (1, 2) . hbov infections are frequently detected in <2-year-old children often with other respiratory viruses (3, 4) . the clinical manifestatons of hbov respiratory tract infection have ranged from mild upper respiratory disease (3, 4) to severe life-threatening pneumonia (2, 5) . the direct impact of hbov infection of the respiaratory tract is often difficult to assess because of its frequent detection in asymptomatic children and coinfection with other respiratory viruses in symptomatic children (4, 6) . a causal link between hbov and respiratory disease has been reported but the exact clinical characteristics await determination. in children hbov may cause more severe clininical conditions such as encephalitis and life-threatening complications (7, 8) . although previously bocavirus associated hepatitis in an immuncompromised patient was mentioned in a single case report (9) here, we confirmed hbov infection in an immunocompetent 2-years-5-months-old girl with hypoxia, cough, fever demonstrating respiratory tract infection along with hepatitis. to the best of our knowledge this is the first reported case of possible bocavirus associated hepatitis in an immunocompetent child. a previously healthy 2-year-5-month-old girl was admitted to our hospital with complaints of fever, vomiting, abdominal pain and runny nose for the previous 5 days. no diarrhoea or any rash was present. her past medical history was unremarkable. she had never been hospitalized previously. there was no history of any medication usage or toxin exposure and no travel history. because of respiratory distress, manifested by nasal flaring, intercostal, subcostal and suprasternal retractions, a respiratory background: so far, many studies have shown that human bocavirus ( hbov) is the main pathogen of the respiratory tract. until now, there is no study that proves the association between hbov and hepatitis. hbov viremia/dnaemia has been associated closely with acute primary infection and moderate-to-severe illness but, more detailed clinical data about hbov dissemination are still unavailable. case report: here we report a 2-years-5-months-old girl suffering from respiratory distress and heptitis followed in our intensive care unit. hbov was detected in our patients nose and throat swabs concurrent with whole blood sample by positive polymerase chain reactions. after a through investigation no causative agent other than hbov viremia was found. conclusion: human bocavirus viremia with high viral loads may be associated with hepatitis. bocavirus; hepatitis; viremia; immunocompetent child zeliha haytoğlu 1 , oğuz canan 2 rate of 80 breaths per minute and cyanosis, heated humidified high-flow nasal cannula (hfnc) therapy was used in our intensive care unit to reduce the work of breathing. at the time of admission, her laboratory findings were as follows; white blood cell count 13x10 9 /l, absolute neutrophil count 2500, haemoglobin 9.2 g/dl platelet counts 330x10 9 /l, c-reactive protein level 47.8 mg/dl, alanine aminotransferase level 4113 u/l, aspartate aminotransferase level 7055 u/l, total biluribin 0.8 mg/dl, direct biluribin 0.6 mg/dl, activated partial thromboplastin time 31.6 seconds, prothrombin time 24.9 seconds, international correction rate 2.38, albumin 25 g/l. blood culture was negative for bacterial infections. stool and blood specimens were negative for enteroviral infections. no spesific causative agent of hepatitis was found after a through investigation. at the time of admission, a diagnostic pcr analysis of a nasopharyngeal aspirate (npa) swab was performed (lightcyler 2.0; roche, germany). hbov, human coronavirus group 1 and group 2, human metapneumovirus, influenza virus types a and b, and respiratory syncytial virus, adenovirus, parainfluenza virus 1-4 and rhinovirus were searched for the diagnostic panel. npa was positive for only hbov, pcr analysis was negative for all other viruses. hbov viral load was detected 8x10 6 copies per ml serum in blood samples concurrent with the npa. the patient's respiratory status was improved over three days to normal. the hepatitis did not become severe and with supportive medical treatment the patient was discharged four days after admission. the written informed consent for participating in this report was obtained from the parents of the child. hbov has been determined in patients with respiratory infections but its association with hepatitis has been shown only by one report. a case report from finland in 2008 described an immunodeficient six-month-old boy without respiratory symptoms with hepatitis (9) . hbov dna was identified from the npa and blood of their patient by pcr, and diagnosis of acute primary hbov infection was confirmed by specific igm positivity in serum, and a fourfold rise of igg antibody levels in paired sera. here we described an immunocompetent child with hbov infection with clinical hepatitis and respiratory symptoms but without other concomitant viruses. the sole detected pathogen was the hbov, both in the npa and blood of our patient. respiratory infections due to hbov are systemic and can be diagnosed serologically, but in our case serological diagnosis of hbov was not done because these tests were not available at our institution. although diagnostic serology is helpful for disclosing hbov infection with disease, some studies suggest that the detection of a high viral load of hbov genomic dna in blood may be useful in establishing the diagnosis of hbov infection with disease (10) , and one study also suggested that if hbov viremia occurs it can disseminate to other parts of the body (1). when a sole viral finding in the respiratory tract is made, hbov infection usually occurs with viremia and high viral loads and is often accompanied by the evidence of specific igm and igg antibodies (1, 10) . in light of these findings we can conclude that the high hbov viral load in the blood sample of our patient can be considered as evidence of acute hbov disease and hepatitis may be the clinical result of the dissemination of hbov viremia. there is limited data about the link between hbov and hepatitis. to date, the pathogenesis of hbov infection has not been fully elucidated. hbov can be cultured only in differentiated human airway epithelial cells and no animal model is available. we detected hbov as a sole pathogen both in blood and npa samples. there are confusing data about the disease severity of single hbov infections compared with hbov co-infections in respiratory syndromes. some authors suggest a more severe clinical picture with a high blood load with mono-infection (1, 10) . although the definitive diagnosis of hbov hepatitis requires the demonstration of the virus in a liver biopsy specimen, in our case a liver biopsy was not done. the diagnosis was made based upon an hbov viremia and a compatible clinical picture with the exclusion of other causes. the findings strongly suggest the hbov as a causative agent. the true hbov pathogenesis is not yet fully understood. also, the link between hbov and hepatitits requires supporting evidence. tropism of hbov to the hepatic tissue can be studied further. to the best of our knowledge, this is the first report that demonstrates hbov viral load in both nasal and blood samples from an immunocompetent child with hepatitis and respiratory infection but without mixed infections with other viruses, thus supporting the hypothesis that hbov may cause hepatitis. human bocavirus and acute wheezing in children single detection of human bocavirus 1 with a high viral load in severe respiratory tract infections in previously healthy children human bocavirus infection in children with acute respiratory tract infection in india human bocavirus isolated from children with acute respiratory tract infections in korea high prevalance of human bocavirus detected in young children with severe acute lower respiratory tract disease by use of a standard pcr protocol and a novel real-time pcr protocol community surveillance of respiratory viruses among families in the utah better identification of germs-longitudinal viral epidemiology (big-love) study detection of human bocavirus in the cerebrospinal fluid of children with encephalitis human bocavirus as the cause of a life-threatening infection hepatitis and human bocavirus primary infection in a child with t-cell deficiency human bocavirus in children: mono-detection, high viral load and viremia are associated with respiratory tract infection no conflict of interest was declared by the authors. key: cord-281418-mvgp6qfv authors: soccal, p. m.; aubert, j.-d.; bridevaux, p.-o.; garbino, j.; y., thomas; rochat, t.; rochat, t. s.; meylan, p.; tapparel, c.; kaiser, l. title: upper and lower respiratory tract viral infections and acute graft rejection in lung transplant recipients date: 2010-07-15 journal: clin infect dis doi: 10.1086/653529 sha: doc_id: 281418 cord_uid: mvgp6qfv background. lung transplant recipients are frequently exposed to respiratory viruses and are particularly at risk for severe complications. the aim of this study was to assess the association among the presence of a respiratory virus detected by molecular assays in bronchoalveolar lavage (bal) fluid, respiratory symptoms, and acute rejection in adult lung transplant recipients. methods. upper (nasopharyngeal swab) and lower (bal) respiratory tract specimens from 77 lung transplant recipients enrolled in a cohort study and undergoing bronchoscopy with bal and transbronchial biopsies were screened using 17 different polymerase chain reaction—based assays. results. bal fluid and biopsy specimens from 343 bronchoscopic procedures performed in 77 patients were analyzed. we also compared paired nasopharyngeal and bal fluid specimens collected in a subgroup of 283 cases. the overall viral positivity rate was 29.3% in the upper respiratory tract specimens and 17.2% in the bal samples (p < .001). we observed a significant association between the presence of respiratory symptoms and positive viral detection in the lower respiratory tract (p = .012). conversely, acute rejection was not associated with the presence of viral infection (odds ratio, 0.41; 95% confidence interval, 0.20–0.88). the recovery of lung function was significantly slower when acute rejection and viral infection were both present. conclusions. a temporal relationship exists between acute respiratory symptoms and positive viral nucleic acid detection in bal fluid from lung transplant recipients. we provide evidence suggesting that respiratory viruses are not associated with acute graft rejection during the acute phase of infection. flex, and abnormal lymphatic drainage. nevertheless, our knowledge of the clinical effect of respiratory viruses is incomplete, particularly when detected by molecular assays applied to lower respiratory tract specimens. this is in part due to the retrospective design of most available studies, the use of different sampling sites (upper vs lower respiratory tract specimens), and the heterogeneity of diagnostic procedures (conventional vs molecular techniques) [1, 2, 4, [6] [7] [8] [9] [10] [11] [12] [13] . in addition to the direct consequences of any viral infection, subsequent cellular injury and altered host immunity could also initiate a cascade of immunologic events [14] [15] [16] , leading to acute and chronic allograft rejection. although several studies support the association between respiratory viruses and chronic lung rejection [1, 3, 10, 12, 14, [17] [18] [19] , the link between respiratory viruses and acute rejection has been studied mainly in small case series or by subgroup analysis [1, 2, 9-11, 20, 21] . on the basis of available evidence, the relationship between viral infection and acute rejection has not been established. the present investigation was specifically designed to assess the epidemiology of respiratory viruses in bronchoalveolar lavage (bal) fluid from lung transplant recipients and to analyze the relationship between these viruses and the presence of acute graft rejection. the most sensitive molecular assay methods were used to identify as many as 17 common respiratory viruses in not only the lower but also the upper respiratory tract. symptoms were carefully and prospectively described, and their association with either respiratory viruses or acute rejection was analyzed. during a 27-month study period from november 2003 through march 2006 (covering 3 winter seasons), lung transplant recipients were enrolled in a prospective cohort study [22] . patients were followed up at the 2 sites of a single transplantation network, including the university hospitals of geneva (geneva, switzerland) and the university hospital of lausanne (lausanne, switzerland). informed consent was required from each participant, and the study was approved by both institutional ethics committees. any lung transplant recipient who underwent bronchoscopy with bal and transbronchial biopsies was eligible, irrespective of the reasons leading to the procedure. on the basis of standardized guidelines, we perform routinely scheduled bronchoscopies at 1, 3, 6, and 12 months after transplantation and yearly thereafter. other indications for bronchoscopy with transbronchial biopsies include unexplained respiratory symptoms, functional deterioration with a у12% decrease in the forced expiratory volume in 1 s (fev 1 ), new chest radiologic infiltrate, and control procedure 1 month after treatment of any rejection of grade a3 or higher. thus, patients could undergo bronchoscopy for a variety of reasons with additional procedures during the study period. for technical reasons (eg, pathologist availability), transbronchial biopsies are not performed during weekends and holidays at our centers. for safety and ethical reasons, no additional bronchoscopy was performed only for the purpose of the study. the bal procedure and the real-time taqman reverse-transcription polymerase chain reaction (rt-pcr) assays for the detection of rna respiratory viruses (influenza viruses a, b, and c; respiratory syncytial viruses a and b; parainfluenza viruses 1, 2, 3, and 4; human rhinovirus; enterovirus; human metapneumovirus; and coronaviruses oc43, 229e, nl63, and hku1) were performed as described elsewhere [22] [23] [24] . the recently identified bocavirus was added. transbronchial biopsies were performed under fluoroscopic guidance using standard procedures [25] and analyzed according to published guidelines [26] by senior pathologists masked to the viral results. pooled nasopharyngeal and oropharyngeal swab specimens were obtained from patients who agreed to the procedure. for technical reasons and reasons of cost (110,500 rt-pcr assays were performed), the procedure (nasopharyngeal and oropharyngeal swabbing) was limited to 80% of the cases selected during the entire study period. swabs were immediately placed on appropriate transport medium and stored at ϫ80њc until further rt-pcr analysis. shortly before each bronchoscopic procedure, a specific case report form was completed and symptoms, reasons leading to the bal procedure, and the presumed diagnosis based on the available clinical data at the time were recorded. rhinopharyngitis was defined as the presence of acute respiratory symptoms with at least acute rhinorrhea with or without additional signs suggesting acute sinusitis and/or acute pharyngitis (sore throat confirmed by the presence of inflammatory signs on clinical examination). flulike illness was defined as the presence of a temperature 137.8њc plus 2 of the following 4 symptoms: cough, myalgia, sore throat, or headache. when available, lung functions measured 2-6 weeks before, at the time of, and 2-6 weeks after the bal procedure were recorded. fev 1 and maximal midexpiratory flow of 25%-75% were also recorded. impaired lung function was defined as a у12% decrease from the previous value. statistical analysis. each episode was classified into 4 categories according to the respective absence (a0 or a1 grade) or presence (a2, a3, or a4 grade) of acute rejection and/or respiratory virus. we considered only acute rejection grade a2 or higher in the analysis, because most centers would not recommend high-dose immunosuppressive treatment for a lower acute rejection grade. the fisher exact test was used to compare the frequency of respiratory symptoms in each category. generalized linear latent and mixed models (stata software, version 10; statacorp) were used for analysis of lung functions at each episode to take into account the repeated measures in each patient at the 2 study sites. odds ratios (ors) for acute rejection associated with respiratory viruses were calculated using the same statistical approach for repeated measures. to investigate a potential time-specific relationship between respiratory viruses and acute rejection, we repeated our analyses of episodes restricted to 4 key periods: 0-3 months, 4-6 months, 7-12 months, and 112 months after lung transplantation. seventy-seven lung transplant recipients underwent 343 bronchoscopies. patient characteristics are given in table 1 . all patients received induction immunosuppression with anti-interleukin 2 receptor antibodies (basiliximab) postoperatively, followed by a triple immunosuppression regimen of calcineurin or target of rapaof the 343 bronchoscopies, 229 (66.8%) were performed as routinely scheduled procedures, 85 (24.8%) were performed for a new clinical condition, and 29 (8.5%) were performed as a control biopsy after treatment for an acute rejection (grade a3 or higher) episode. some patients included for a routinely scheduled bronchoscopy presented with a new respiratory symptom with or without functional impairment and/or new radiologic infiltrate. taken together, 224 (65.3%) of 343 cases had at least 1 new respiratory symptom, and 54 (15.7%) had a new radiologic infiltrate. on the basis of the pretest evaluation, acute lung rejection and infection were suspected by the physician in charge, who was masked to microbiological results, in 176 cases (51.3%) and 99 cases (28.9%), respectively. for the remaining 68 cases (19.8%), the clinician did not suspect any particular diagnosis. rt-pcr assays. the rt-pcr viral assays performed on the 343 bal fluid specimens revealed an overall positivity rate of 17.2% ( ). eight bal specimens were discarded ben p 59 cause they had thawed during an electricity power outage. rhinovirus was the most frequently encountered virus, followed by coronaviruses and parainfluenza viruses ( table 2 ). the viral positivity rate for the 283 pooled nasopharyngeal and oropharyngeal specimens tested (82.5% of the total study population) was 29.3% ( ). the agreement between upper and n p 84 lower respiratory tract specimens according to each type of viral genus is shown in figure 1 . although influenza viruses, human metapneumovirus, and bocavirus were mostly (or even exclusively for bocavirus) found in the upper airway samples, other viruses (rhinoviruses and respiratory syncytial viruses) were found equally in the upper and lower respiratory tracts. no virus was exclusively found in the lower airways. association between clinical suspicion and final diagnosis. because bal fluid specimens were collected for a variety of clinical conditions, we were able to analyze the association among symptoms, the diagnosis suspected by the physician in charge, and the subsequent presence of a proven upper and/ or lower respiratory tract viral infection. we found a significant association between positive viral nucleic acid detection in bal fluid and the presence of at least 1 new respiratory symptom ( ), in particular cough ( ) and sputum pro-p p .012 p p .001 duction ( ). table 3 gives the clinical findings associated p p .04 with virus positivity for upper and lower tract specimens. physicians in charge suspected an infection in 20 (42.6%) of the 47 cases with virus-positive bal fluid, compared with 57 (24.2%) of the 236 cases that were virus negative. a history of rhinopharyngitis was documented in 20 (23.8%) of 84 cases with a virus-positive nasopharyngeal swab specimen, compared with 22 (11.1%) of 199 of those that were negative. of 343 biopsy specimens, 149 (43.4%) showed no evidence of acute rejection, 78 (22.7%) revealed minimal (a1) rejection, and 116 (33.8%) were graded a2 or higher. similar to the analysis given in table 3 , we first tested the accuracy of the pretest clinical evaluation performed by the physician in charge. when acute rejection was suspected by the clinician in charge, this was histologically confirmed in 64.1% of cases in the absence of respiratory virus and in 84.6% of cases when a respiratory virus was simultaneously identified in bal fluid (table 4 ). consistent with the results shown in table 3 , the presence of any respiratory symptoms, cough, or sputum was significantly associated ( for all) with the presence of a viral p ! .004 infection in bal fluid, irrespective of the presence or absence of acute rejection. impairment of lung function related to acute rejection was also significantly worsened by the presence of a viral infection (figure 2 ). in patients with simultaneous acute rejection and lower respiratory tract viral infection, the fev 1 recovery rate was significantly slower than in patients who had acute rejection without simultaneous viral infection ( ). p ! .05 we found that the or for acute rejection was !1 in the presence of a viral infection for each of the 4 periods studied (or for any period, 0.41; 95% confidence interval [ci], 0.20-0.88). the overall rate of viral infection in grade a0 and a1 cases was 25.4% (46 of 181), compared with 12.6% (13 of 103) in grade a2 or higher. thus, patients with a lower acute rejection grade were twice as likely to be positive for viral infection than those with a higher rejection grade (or, 2.0; 95% ci, 1.04-3.9). when repeating these analyses with the presence of virus in the upper respiratory tract as a predictor of acute rejection, we did not find an association between upper respiratory tract viral infection and acute rejection (or, 1.11; 95% ci, 0.6-2.05). in a supplementary analysis, we verified the occurrence of acute rejection during a 30-day and 90-day period after baseline bronchoscopy. at 30 and 90 days, the probability of acute rejection was associated with the presence of acute rejection at baseline using an extensive panel of molecular assays, we were able to show that 17.2% of prospectively collected bal fluid specimens from adult lung transplant recipients were positive for at least 1 respiratory virus. on the basis of a pretest clinical evaluation, patients testing positive were significantly more likely to present with respiratory symptoms (86.4%, compared with 60.9% with no identified virus). all analyzed individual symptoms were systematically more frequent in the presence of a respiratory virus, particularly cough and sputum, which were up to 3 times more common. consistent with these observations, the pretest clinical evaluation performed by the physicians in charge considered that an infection was more likely in those with a final positive viral detection. furthermore, lung function proved to recover significantly slower in the presence of a respiratory virus. all these findings corroborate that a positive association exists between positive viral nucleic acid detection in bal fluid and the presence of an acute respiratory illness in lung transplant patients. this is also concordant with other investigations that have linked the detection of respiratory viruses by rt-pcr to symptoms [1, 2, 4, 6, 10, 13, 27] . however, our investigation differs from these studies by its prospective design, the large panel of respiratory viruses screened, the systematic use of bal specimens, and the integration of pretest clinical evaluations performed by the physicians in charge. many of the previous studies limited the detection strategy to upper respiratory tract specimens and used an array of molecular tools that was often restricted to a subgroup of viruses (eg, rhinoviruses, coronaviruses, or bocaviruses were not systematically tested). finally, the link with pretest clinical conditions was not detailed in many of these reports. because of the availability of lung biopsy specimens in all cases, we were also able to assess the presence of acute rejection according to the presence of viral infection. a relationship between acute viral infection and subsequent acute rejection has been reported in small case series, but this possible association has not been appropriately confirmed. viral infection might trigger a chain of immunologically mediated events, leading to subsequent rejection or lung dysfunction. this is important because several studies have identified viral infection as a distinct risk factor for the development of bronchiolitis obliterans syndrome and chronic graft dysfunction [1, 3, 4, 10, 12, 14, 17, 18] . our investigation had the capacity to address this question because we were able to analyze a high number of lung biopsy specimens together with viral screening at the level of the lower respiratory tract. the biopsy analysis revealed that viral respiratory tract infections were not associated with simultaneous acute rejection. furthermore, we could show that the probability of acute rejection did not increase 30 and 90 days after a lower airways respiratory viral infection. when present, however, viral infections caused more severe lung dysfunction and significantly hampered the short-term functional recovery in the case of concomitant acute rejection. because the clinician in charge was masked to any viral result, a positive rt-pcr result did not modify the treatment of simultaneous acute rejection. thus, a less aggressive treatment of acute rejection in the presence of a virus cannot explain the slower recovery of lung function in these patients. this suggests that respiratory viruses per se do not promote acute rejection during the acute phase but could certainly worsen lung function and impair recovery from acute rejection episodes. paradoxically, we even observed a negative association between an episode of respiratory viral infection and the subsequent cumulative risk of developing acute rejection. this should not be considered a protective effect of viral infections; more likely, it is a lack of an association or a chance effect, and we refrain from drawing any other conclusions. however, this strongly suggests that when analyzing biopsy specimens according to reference guidelines [26] , pathologists should not be misguided by the presence of a viral infection. of note, our study design limited follow-up to a few weeks, and we cannot exclude the possibility that the viral infection triggered a chain of immunologically mediated events, leading to subsequent rejection or lung dysfunction. this is important because several studies have identified viral infection as a distinct risk factor for the development of bronchiolitis obliterans syndrome and chronic graft dysfunction [1, 3, 4, 10, 12, 14, 17, 18] . our investigation also provided the unique opportunity to compare viral detection in the upper (pooled nasopharyngeal and oropharyngeal swabs) and lower respiratory tract in a large number of paired specimens. the positivity rate in the upper respiratory tract was 29.6%, compared with 16.6% in the lower tract. this difference in recovery rate is similar to that observed in other smaller studies [12] , but, to the best of our knowledge, few or none of the previous investigations systematically analyzed paired specimens collected during the same procedure. an important observation is that when recovered only in the upper respiratory tract, respiratory viruses were less likely to be associated with lower respiratory symptoms. another interesting observation is that only 7.0% of negative nasopharyngeal specimens were associated with a discordant positive bal viral screening, thus suggesting a high negative predictive value. however, this value should be balanced with the relatively low prevalence of each individual respiratory virus in bal fluid specimens and the possible technical issues related to nasopharyngeal and pharyngeal swabbing that could negatively affect the recovery rate. our population was first selected on the basis of the need to perform a bal procedure; for this reason, we caution that a lower respiratory tract viral infection in lung transplant recipients cannot be definitely ruled out by a nasopharyngeal swab. yet this could be a reasonable initial screening strategy that needs to be confirmed in further studies and individually for each type of virus. in conclusion, our study demonstrates that there is a temporal relationship in lung transplant recipients between the emergence of acute respiratory symptoms and positive respiratory viral nucleic acid detection in bal fluid specimens. when detected only in the upper respiratory tract, viral infections are less likely to be associated with respiratory symptoms and graft dysfunction. we also provide solid evidence suggesting that respiratory viruses per se do not promote acute graft rejection, at least during the acute phase of infection, but that they do worsen graft function recovery when simultaneously present with acute rejection. clinical impact of communityacquired respiratory viruses on bronchiolitis obliterans after lung transplant impact of human metapneumovirus and human cytomegalovirus versus other respiratory viruses on the lower respiratory tract infections of lung transplant recipients respiratory viruses and chronic rejection in lung transplant recipients human metapneumovirus in lung transplant recipients and comparison to respiratory syncytial virus respiratory viral infections in transplant recipients a single-season prospective study of respiratory viral infections in lung transplant recipients absence of human bocavirus in bronchoalveolar lavage fluid of lung transplant patients diagnosis of human metapneumovirus infection in immunosuppressed lung transplant recipients and children evaluated for pertussis detection of severe human metapneumovirus infection by real-time polymerase chain reaction and histopathological assessment human metapneumovirus infection in lung transplant recipients: clinical presentation and epidemiology lower respiratory viral illnesses: improved diagnosis by molecular methods and clinical impact respiratory viral infections are a distinct risk for bronchiolitis obliterans syndrome and death clinical features and outcomes of paramyxoviral infection in lung transplant recipients treated with ribavirin infectious etiology of bronchiolitis obliterans: the respiratory viruses connection: myth or reality? characterization of virus-mediated inhibition of mixed chimerism and allospecific tolerance critical role for the chemokine mcp-1/ccr2 in the pathogenesis of bronchiolitis obliterans syndrome community acquired respiratory viral infections after lung transplantation: clinical features and long-term consequences parainfluenza virus infection in adult lung transplant recipients: an emergent clinical syndrome with implications on allograft function the epidemiology of parainfluenza virus infection in lung transplant recipients treatment of respiratory syncytial virus pneumonia in a lung transplant recipient: case report and review of the literature influenza virus infection in adult solid organ transplant recipients respiratory viruses in bronchoalveolar lavage: a hospital-based cohort study in adults a prospective hospital-based study of the clinical impact of non-severe acute respiratory syndrome (non-sars)-related human coronavirus infection swiss paediatric respiratory research group. viral etiology of acute respiratory infections with cough in infancy: a community-based birth cohort study persistent hepatitis c viremia predicts late relapse after sustained response to interferonalpha in chronic hepatitis c lung rejection study group. revision of the 1990 working formulation for the classification of pulmonary allograft rejection influenza and parainfluenza respiratory viral infection requiring admission in adult lung transplant recipients we thank patricia suter, sandra van belle, jean-marc fellrath, and lara turin for their excellent technical assistance and rosemary sudan for editorial assistance.financial support. swiss national science foundation (grant 3200b-101670 to l.k.).potential conflicts of interest. all authors: no conflicts. key: cord-299835-92karhpl authors: ho, khek y.; singh, kamaljit s.; habib, abdulrazaq g.; ong, benjamin k.; lim, tow k.; ooi, eng e.; sil, bijon k.; ling, ai-ee; bai, xin l.; tambyah, paul a. title: mild illness associated with severe acute respiratory syndrome coronavirus infection: lessons from a prospective seroepidemiologic study of health-care workers in a teaching hospital in singapore date: 2004-02-17 journal: j infect dis doi: 10.1086/381558 sha: doc_id: 299835 cord_uid: 92karhpl background. severe acute respiratory syndrome (sars) is a newly recognized infectious disease that has recently emerged in east asia and north america. although the clinical features of acute infection have been well described, mildly symptomatic or asymptomatic infections have not been well characterized. objective. to assess the spectrum of illness in health-care workers (hcws). methods. a prospective seroepidemiologic cohort study was conducted on 372 hcws in a large teaching hospital in singapore who were both exposed and not exposed to patients with sars. participating hcws completed a questionnaire and provided paired serum samples, which were analyzed by 2 different laboratories blinded to clinical data, by use of an enzyme-linked immunosorbent assay based on a protocol developed by the centers for disease control and prevention and a dot-blot immunoassay, with confirmation by a viral neutralization assay. results. a total of 21 patients with sars were treated at our hospital. they were associated with transmission to 14 staff members, patients, and visitors in our hospital. of the 372 hcws participating in the present study, 8 were found to have positive antibodies to the sars coronavirus in both samples by use of both test methods, and 6 had pneumonia and had been hospitalized for either probable or suspected sars infection, whereas 2 had fever but did not have changes on chest radiographs. all seropositive hcws had been exposed either directly or indirectly to patients with sars. no asymptomatic, nonexposed staff members were found to be seropositive. there was a trend towards protection for hcws who, while fully protected, had had contact with patients with sars. conclusions. although the majority of cases of sars are associated with pneumonia, a small number of mildly symptomatic individuals do seroconvert. hcws who are exposed to patients with sars can be infected with sars, regardless of the intensity of exposure. this has implications for surveillance and infection control planning, in the event that sars returns next winter. severe acute respiratory syndrome (sars) is a newly recognized coronavirus infection that recently emerged in east asia, with subsequent global spread [1] [2] [3] . in singapore, cases of sars were diagnosed in early march were in the same rooms as patients with unrecognized sars [5] . the clinical features of typical sars have been well described in large clinical studies [6] [7] [8] . atypical presentations with no fever but with changes on chest radiographs have also been reported [9] . asymptomatic or mild infections with no respiratory symptoms and no changes on chest radiographs have, however, not been previously reported to have occurred in contacts of patients with sars, with the exception of 1 case report [10] . this has been documented in other emerging viral infections and has potential implications for the transmission and control of this emerging infection [11, 12] . the national university hospital (nuh), singapore, is a 900-bed teaching hospital that employs ∼3000 doctors, nurses, allied health professionals, and clerical staff members. between 18 march and 29 april 2003, a total of 21 patients with sars were treated in the hospital wards and emergency department before being transferred to the designated sars hospital. an escalated policy on the use of more-complete personal protective equipment (ppe) and isolation of suspected patients was instituted, beginning with the required wearing of n-95 masks, gowns, and gloves for isolation-ward personnel only. later, this was extended to personnel in all areas of the hospital, with regular audits of all hcws. we conducted a seroepidemiologic study of hcws in our hospital to assess the spectrum of illness seen in hcws infected with the sars virus, in particular to document whether asymptomatic or mild infections with no respiratory symptoms and no changes on chest radiographs occur in individuals with sars. after giving written, informed consent, unselected hcws were recruited on a voluntary basis from all areas of the hospital, beginning with high-risk areas and extending to low-risk areas, including outpatient clinics and offices. they completed a simple questionnaire describing their workplaces, contact with patients with sars, use of ppe, and symptoms experienced during the preceding 4 weeks. they also provided paired serum samples, which were collected initially at the peak of the outbreak and subsequently at a median interval of 31 days (range, 17-53 days) after initial collection. samples were anonymized to ensure that the confidentiality of hcws was preserved. the study was approved by the hospital institutional review board. all policies and procedures of the ministry of health, singapore, good clinical practice were followed in the conduct of this study. serum samples were stored at ϫ80њc and subsequently were sent to 2 different external laboratories for serologic testing. the laboratory staff were unaware of the clinical details of the patients. the first laboratory used an elisa based on a protocol and using antigens provided by tom ksiazek (centers for disease control and prevention, atlanta, ga) [1] . samples found to be positive for sars by elisa were confirmed by use of an indirect immunofluorescence assay. the second test was done at the national environmental agency, singapore, and used a dot-blot immunoassay using antigens derived from virus culture supernatant. positive samples, at a titer of у1:100, were then subjected to a virus neutralization assay, in serial 2-fold dilution, starting at 1:10-1:320 and using a microneutralization format described elsewhere for human enterovirus 71 [13] . a neutralizing antibody titer of у10 was considered to be positive for sars. serum samples from volunteer patients with sars and from nonexposed laboratory staff were included in all serologic assays as positive and negative controls, respectively. thus, all the serum samples were tested by use of 2 screening tests (i.e., elisa and dot-blot immunoassay), and the positive serum samples were confirmed by indirect immunofluorescence assay and virus neutralization assay, respectively. results of the serologic testing from each laboratory were not revealed to the other laboratory until after completion of the study. definitions. a seropositive individual was defined as having provided a serum sample that received a positive confirmatory result by both the indirect immunofluorescence assay and the virus neutralization assay. an exposed hcw was defined as having worked in an area where a patient later confirmed to have sars had been cared for. direct contact was defined by use of the world health organization (who) definition of having cared for, having lived with, or having had direct contact with respiratory secretions and/or body fluids of an individual with sars [14] . hcws who worked in the same ward but did not have direct responsibility for patients with sars or did not come into physical contact with respiratory secretions and/or body fluids of an individual with sars would thus be defined as exposed-only hcws. patients with sars were defined by use of who criteria [14] for probable cases, which included fever (temperature 138њc), respiratory symptoms, and radiographic evidence of pneumonia or respiratory distress. mildly symptomatic individuals were defined as those with fever significant enough to warrant evaluation at the staff clinic or emergency department but with no evidence of pneumonia on chest radiographs and prompt resolution of symptoms (within 48-72 h of symptomatic therapy). statistical analysis. differences between groups were assessed by the x 2 test, fisher's exact test, mann-whitney u test, kruskal-wallis test, or relative risk ratios with 95% confidence intervals, as appropriate. the results of these comparisons were reported as p values. all statistics were analyzed by use of the spss (version 11.5.1; spss) and stata (version 7.0; statacorp) software. the first patient with sars seen in our hospital was a cardiology resident from ttsh who entered our emergency department on 18 march 2003. since then, a total of 21 patients with sars, including 5 hcws from our hospital, have been seen in our wards and emergency department. all of these patients had positive antibodies to the sars coronavirus. six of the 7 tested also had sars coronavirus isolated from stool samples, blood samples, and/or respiratory secretions. these 21 patients stayed in nuh a mean ‫ע‬ sd of days from admission or 3.9 ‫ע‬ 4.8 onset of symptoms to transfer to ttsh. there were 14 known nosocomial transmissions to staff members, visitors, and other patients, all of whom were eventually transferred to ttsh for treatment; 11 of these were linked to a single atypical case [15] . initial policies on the use of ppe for hcws, instituted on 17 march 2003, confined the mandatory use of gloves, gowns, and n95 masks to isolation wards only but, by 28 march, were extended to include intensive-care units and the emergency department. on 9 april, after the identification of an atypical case of sars in an open general medical ward, full use of ppe was made mandatory for all staff in contact with patients. a total of 372 staff members participated, of whom paired serum samples were obtained from 303 (81.5%). serum was drawn at 17-53 day intervals, beginning on 22 april and continuing to 5 june 2003-that is, beginning 4 weeks after the first case of sars in nuh and ending 10 weeks after the last case was transferred to ttsh. overall, mean ‫ע‬ sd age was years, and 287 (77.2%) were women, 103 (27.7%) 34.2 ‫ע‬ 9.0 were physicians, 205 (55.1%) were nurses, and the rest were allied health professionals and clerical staff members. one hundred twelve (30.1%) worked in areas where patients with sars had been cared for (i.e., the exposed group), and the rest had no exposure at all to patients with sars (i.e., the nonexposed group). the characteristics of the hcws are listed in table 1. the exposed group was younger and included more nurses, probably because of a higher representation of emergency department and intensive-care unit staff members in this group. a large number of staff members reported a variety of symptoms during the study period, including fever ( . there was a n p 69 n p 22 significant difference in the frequency of fever between exposed (28.6%) and nonexposed (11.9%) hcws ( ). twenty-p p .0001 note. mildly symptomatic subjects, health-care workers who had fever significant enough to warrant evaluation at the staff clinic or emergency department but who had no evidence of pneumonia on chest radiographs and had prompt resolution of symptoms (within 48-72 h of symptomatic therapy). one hcws (5.6%) in our study cohort were hospitalized during this period, of whom 6 were classified by clinical criteria as probably infected with sars. twenty-one (5.6%) of the participants or their spouses had traveled to other sars-affected areas during the study period. serologic data on hcws (table 2) . samples from 8 hcws (2.2%) were found to be positive for sars-associated coronavirus by elisa, and these results were confirmed by indirect immunofluorescence assay using the cdc protocol, and samples from the same hcws were found to be positive by use of the dot-blot method, and these results were confirmed by viral neutralization assay. both samples tested for each hcw were found to be positive for all 8 hcws, and no increase in titer could be demonstrated on their paired samples, since, from all 8 subjects, samples were obtained 14 weeks after the onset of symptoms. all 6 hcws hospitalized at ttsh (including 5 who were first treated in our hospital) for probable sars infection were found to be seropositive, and 2 additional individuals who had fever but did not meet sars criteria (i.e., had symptoms !4 days in duration and had no changes on chest radiographs) were also seropositive. on the basis of data submitted anonymously for the study, as well as a review of hospital epidemiology and contact-tracing data, a profile of these 2 individuals could be constructed without compromising confidentiality: both worked in the emergency department at a time when patients with sars were being treated in the department, wore full ppe, and did not have direct personal contact with any patients with sars. one was admitted to an isolation room with fever, chills, and myalgias, which resolved completely within 3 days of symptomatic treatment; she had no changes on serial chest radiographs. the other was treated as an outpatient in the staff clinic; she presented with fever and upper respiratory symptoms. again, chest radiographs were normal, and symptoms resolved within 3 days. no secondary cases resulted from any of the infected hcws. no hcw who was completely asymptomatic was found by serologic testing to have sars-associated coronaviral infection. it is interesting to note that, even after removing these 8 seropositve hcws from the exposed and febrile groups in table 1, the difference between the exposed and nonexposed groups, in relation to being febrile, remains statistically significant (23.1% vs. 11.9%; ). p p .0095 relationship between serologic test results and exposure history. when analyzed by contact history, all of the seropositive hcws worked in areas where patients with sars had been cared for (table 3) . although only 4 of 8 had had direct contact by who definition, the remaining 4 worked in the same ward where the patients were located but did not have direct responsibility for these patients or come into physical contact with these patients' body fluids. although the numbers of subjects in our study are small, there was a trend towards protection for hcws who reported use of ppe 100% of the time or who, while fully protected, had contact with patients with sars. sars is a novel coronavirus that has recently emerged in southern china and has caused widespread disruption to health-care services and international trade, especially in east asia [16] . the vast majority of infections, with a few notable exceptions, have occurred in hospitals. as with all emerging infections, the clinical picture is only beginning to be described completely. the initial descriptions included atypical pneumonia that followed a prodrome with fever and myalgia [6] [7] [8] and that progressed almost universally to a severe respiratory illness, with a variety of changes on chest radiographs [17] . in approximately one-sixth of cases, this eventually progressed to acute respiratory distress syndrome and death [18] . later reports highlighted gastrointestinal symptoms as a major element of a large community outbreak of sars that was thought to be linked to environmental contamination [19] . atypical presentations have also been reported, but all of these eventually led to the typical pattern of progressive respiratory distress with frank changes on radiographs [9] . the strengths of the present study include the prospective nature of the collection of serum samples and acquisition of clinical data, an adequate sample size with representative subjects from all areas of a large teaching hospital, use of paired serum samples, and application of 2 previously validated serologic tests, which were performed independently. the present study is the first to document sars infection in hcws with normal chest radiographs. two of our seropositive hcws had fever for !1 week and responded to symptomatic therapy for upper respiratory-tract infections. their chest radiographs were repeatedly normal. thus, they did not meet the clinical criteria for probable sars infection. the possibility that our 2 individuals with mild illness were false positives is diminished by the fact that they were found to be positive on both assays, which were done by laboratory staff who were blinded to clinnote. any exposure, working in an area where a patient later confirmed to have sars had been cared for; direct contact, having cared for or having had direct contact with respiratory secretions and/or body fluids of an individual with sars; ci, confidence interval; exposure only, working in the same ward but not having cared for and not having come into physical contact with respiratory secretions and/or body fluids of an individual with sars; no exposure, not working in an area where a patient later confirmed to have sars had been cared for; ppe, personal protection equipment; rr, relative risk. ical data. that none of the nonexposed hcws had evidence of infection and the unpublished reports that blood donors in a variety of settings had no serologic evidence of sars support our hypothesis that these were indeed mild infections. given our experience with other viral respiratory tract infections in which a spectrum of infections is the rule, the detection of mildly symptomatic infections is not surprising. we also failed to detect seroconversion in totally asymptomatic individuals. this was not the experience of other investigators screening contacts of the nipah virus or avian influenza, but those were retrospective serologic studies done some time after the outbreak, and mild clinical symptoms might not have been detected [11, 12] . given the fact that the sars coronavirus seems to be a completely novel pathogen with minimal genetic relatedness to other coronaviruses of humans and animals [20] , it is perhaps not that surprising that it produced at least some clinical disease in all infected individuals with no preexisting cross-protective immunity. this is supported by our observation that, in our hospital, individuals who were exposed to patients with sars had a significantly higher prevalence of fever. the second important finding we observed is that individuals who did not have direct contact with patients with sars also both developed clinical sars and experienced seroconversion with milder illness. in such a setting, transmission is likely to occur on either environmental surfaces or the hands of other hcws. however, our study strikingly shows the complete absence of transmission to individuals working in the same building but in different areas from locations where patients with sars had been cared for. this also has implications, since it bears out our experience with the global epidemiology of sars, in which, who global travel alerts notwithstanding, almost all transmissions in hong kong, taiwan, toronto, and singapore and the majority in mainland china could be traced back to specific household or health-care settings. this should be taken into consideration in the event that sars is detected again next winter, before widespread economic disruption is created by travel alerts and advisories. neither of the 2 individuals with mild cases of sars was associated with any secondary transmission, despite not being isolated or quarantined for any significant period of time. although the majority of cases of sars in singapore did not lead to any secondary infections [5] , it is reassuring that mildly symptomatic cases are hopefully associated with lower virus loads [19] and less likely trigger epidemics. these cases might, however, allow for a low level of transmission of the virus, which might remain "below the radar" if they are not actively sought. this has been a concern with the reemergence of sars in toronto after a period during which transmission was believed to have been halted [21] . data on sars continue to emerge. we have shown that, as with most viral infections, there is a spectrum of illness associated with sars, from mild febrile illness to severe respiratory distress. with data emerging about an animal reservoir [22] for sars, it will be critical to detect periodic human infections, even if mild. although sars seems to have disappeared in the summer months, broad surveillance using more-sensitive assays will be critical in the event that sars reappears next winter. a novel coronavirus associated with severe acute respiratory syndrome identification of a novel coronavirus in patients with severe acute respiratory syndrome update: outbreak of severe acute respiratory syndrome-worldwide severe acute respiratory syndrome (sars) in singapore: clinical features of index patient and initial contacts update: severe acute respiratory syndrome-singapore a major outbreak of severe acute respiratory syndrome in hong kong identification of severe acute respiratory syndrome in canada clinical features and short-term outcomes of 144 patients with sars in the greater toronto area atypical presentations of sars mild severe acute respiratory syndrome a survey of nipah virus infection among various risk groups in singapore risk of influenza a (h5n1) among healthcare workers exposed to patients with influenza a (h5n1), hong kong seroepidemiology of human enterovirus 71 world health organization. global surveillance for severe acute respiratory syndrome (sars) preventing local transmission of sars: lessons from singapore coronavirus as a possible cause of severe acute respiratory syndrome severe acute respiratory syndrome: radiographic and ct findings acute respiratory distress syndrome in critically ill patients with severe acute respiratory syndrome clinical progression and viral load in a community outbreak of coronavirus-associated sars pneumonia: a prospective study comparative full-length genome sequence analysis of 14 sars coronavirus isolates and common mutations associated with putative origins of infection update: severe acute respiratory syndrome-toronto, canada isolation and characterization of viruses related to the sars coronavirus from animals in southern china we are grateful to all the health-care workers who participated in this study. we would also like to thank the medical students mo-yee chau, hui-yi chia, cherylin foo, and teck-wei tan for help with data entry. key: cord-284266-tbndldhr authors: schippa, serena; frassanito, antonella; marazzato, massimiliano; nenna, raffaella; petrarca, laura; neroni, bruna; bonfiglio, giulia; guerrieri, francesca; frasca, federica; oliveto, giuseppe; pierangeli, alessandra; midulla, fabio title: nasal microbiota in rsv bronchiolitis date: 2020-05-13 journal: microorganisms doi: 10.3390/microorganisms8050731 sha: doc_id: 284266 cord_uid: tbndldhr respiratory syncytial virus (rsv) is the leading cause of bronchiolitis, and the severity may be influenced by the bacterial ecosystem. our aim was to analyze the nasal microbiota from 48 infants affected by bronchiolitis from rsv virus and 28 infants with bronchiolitis but negative for the virus. results showed a significantly lower biodiversity in the rsv-positive group with respect to the rsv-negative group, a specific microbial profile associated with the rsv-positive group different from that observed in the negative group, and significant modifications in the relative abundance of taxa in the rsv-positive group, as well as in the rsv-a group, with respect to the negative group. furthermore, microbial network analyses evidenced, in all studied groups, the presence of two predominant sub-networks characterized by peculiar interand intra-group correlation patterns as well as a general loss of connectivity among microbes in the rsv-positive group, particularly in the rsv-a group. our results indicated that infants with more severe bronchiolitis disease, caused by rsv-a infection, present significant perturbations of both the nasal microbiota structure and the microbial relationships. patients with a milder bronchiolitis course (rsv-b-infected and patients who have cleared the virus) presented less severe alterations. bronchiolitis is the most frequent acute respiratory viral infection in children less than one year of age, and it is the main cause of hospitalization in this age group. a viral etiology is detected in 80% of the patients, and respiratory syncytial virus (rsv) remains the most common etiological agent [1] . several studies have shown that bronchiolitis can be caused also by other respiratory viruses different from rsv, and in some cases a virus cannot be detected. clinical characteristics of the disease may also be influenced not only by the presence of a specific virus but also by rsv genotype [2] . recent studies have shown that the composition of the nasopharyngeal microbiome may influence the different clinical phenotypes of bronchiolitis [3, 4] . in infants with bronchiolitis, the respiratory tract microbiota undergoes a perturbation; several studies showed that the most important qualitative alteration is represented by the increase of proteobacteria and the decrease of bacteroidetes [5] . other studies underline the role of staphylococcus-dominant airway microbiota during early infancy as "risky microbiota" to enhance viral pathogenesis [6] . a recent study showed that rhinovirus significantly increased the invasive ability of staphylococcus aureus [7] . rosas et al. highlight that nasopharyngeal detection and increased abundance of the genus lactobacillus during rsv infection in infancy is associated with a reduced risk of childhood wheezing illnesses at age 2 years [8] . in 2016, a study from hasegawa et al. [9] conducted with 40 infants (median age 3 months) hospitalized with bronchiolitis identified four distinct fecal microbiota types in infants. the bacteroides-dominant profile was associated with a higher probability of bronchiolitis, while the enterobacter-/veillonella-dominant profile was associated with lower possibility of bronchiolitis. another important recent study, connecting bronchiolitis severity with nasal microbiota composition [10] , highlighted four differently dominated nasopharyngeal microbiota profiles: one dominated by haemophilus, another by moraxella, another by streptococcus, and the fourth one showed the highest bacterial richness. infants in intensive care showed principally haemophilus-dominant profiles. a more recent study, aimed to comprehend the influence of interfaces between the host transcriptome nasal microbiome and the clinical outcomes of rsv infection [11] , indicated that specific microbial taxa affect host immune responses. all these studies linked specific bacterial species with rsv bronchiolitis, but no comparisons were carried out between rsv-positive and virus-negative bronchiolitis patients. understanding the differences of nasal microbiota structures between rsv-positive and virus-negative bronchiolitis patients would give us information on rsv impact/interfere on nasal microbiota structure, and this useful information for new treatments aimed at microbiota rebuilding could promote a non-severe outcome of the disease. in our study, we analyzed nasal microbiota from a total of 76 subjects affected by bronchiolitis, of which 48 were positive for the rsv virus and 28 were negative, in order to explore the nasal microbiota composition. we also analyzed nasal microbiota compositions in infants with bronchiolitis from rsv subtypes a and b. we conducted a prospective observational study. from october 2016 to may 2017 we enrolled 122 children < 6 months, admitted to the department of paediatric emergency, "sapienza" university of rome for an acute episode of bronchiolitis, who had not been on antibiotic therapy within the last 30 days or with probiotics. infants with risk factors for severe bronchiolitis (prematurity, congenital heart malformations, chronic lung diseases, and neurological and/or neuromuscular diseases) were excluded. bronchiolitis was clinically defined as the first episode of acute lower respiratory tract infection, characterized by the acute onset of cough, tachypnea, retraction, and diffuse crackles on chest auscultation in infants younger than 12 months [12, 13] . epidemiological and demographic variables such as gender, age, gestational age, birth weight, delivery, breastfeeding history, presence of siblings, and presence of smoking cohabitants were obtained from the infant's parents with a structured questionnaire. the following clinical and laboratory data were taken from patient medical files: days of hospitalization, heart rate, respiratory rate, arterial oxygen saturation in room air, presence of retractions, o 2 therapy, intravenous fluid (iv) therapy, white blood cell count (wbc), lymphocyte count, and c-reactive protein blood concentration (crp). a clinical severity score ranging from 0 to 8, according to respiratory rate, arterial oxygen saturation in room air, presence of retractions, and iv fluid treatment, was assigned to all infants at admission and every day during hospitalization as previously described [12] . the worst score during hospitalization was used for analysis. written informed consent was obtained from parents. the study was conducted in accordance with the declaration of helsinki and it was approved by institutional review boards of the hospitals (rif.ce 5271, n. prot. 240/19, 12 march 2019). two nasopharyngeal washes (npws) were obtained during the first day of hospitalization instilling 3 ml of sterile saline in each nostril and collected with a syringe [12] (the lavages of one nostril were used for virus detection, while the lavage performed on the other nostril was employed for microbiota studies). npw samples were delivered within 2 h to the virology laboratory for virus detection and to the department of public health and infectious diseases for analysis of the nasal microbiota composition. an aliquot of nasopharyngeal washes was used for nucleic acid extraction using a total nucleic acid isolation kit (roche diagnostics, mannheim, germany). rt-pcr was used to detect the 14 respiratory viruses: influenza a and b viruses (iv-a/b), human coronavirus (hcov) 0c43 229e, nl-63, huk1, adenovirus (av), parainfluenza virus 1-3 (piv 1-3), human metapneumovirus (hmpv), human bocavirus (hbov), respiratory syncytial virus (rsv), and rhinovirus (hrv) [12] . the npws collected were stored at −20 • c or immediately transported to the microbiology lab for nasal microbiota characterization. total dna was extracted from all collect nasal samples following the instructions provided by the kit dneasy blood & tissue (qiagen, hilden, germany) starting from 200 µl of nasal wash sediment, obtained by centrifuging npws at 14,000 rpm and discarded supernatant. to ensure maximum dna extraction efficiency, even from gram-positive bacteria, the protocol was modified by adding the incubation of samples with proteinase k at 56 • c, followed by a 4 h incubation at 37 • c with 20 mg/ml (final concentration) of lysozyme (sigma-aldrich, milan, italy). nanodrop spectrophotometer determined the dna concentration. all dna samples obtained were normalized to a final dna concentration of 5 ng/µl in a final volume of 20 µl, and an aliquot was used to characterize nasal bacterial community via next-generation sequencing (ngs) on an illumina miseq platform at the italian institute of technology. to this purpose, the v3-v4 region of the bacterial 16s rrna gene was amplified by pcr with specific universal bacterial barcoded primer using total dna extracted as template, and the library obtained was sequenced. the italian institute of technology (iit) executed the amplicon sequencing by means of next-generation sequencing (ngs) on an illumina miseq platform. bioinformatic analyses, encompassing metagenomic analysis (multivariate statistics/non-parametric tests and ecological indices analysis), in order to quantify the significant differences in the structure of bacterial communities and cross-correlation of all collected data, were carried out at the department of public health and infectious diseases. briefly, raw fastq files were analyzed with mothur pipeline v. 1.39.5 for quality check and filtering (sequencing errors, chimerae) on a workstation dell t7910 (round rock, tx, usa). raw reads (on average >100k per sample) were filtered (on average >50k per sample) and clustered into operational taxonomic units (otus), followed by elimination of low-populated otus (till 5 reads) and by de novo otu picking at 97% pair-wise identity using standardized parameters and the silva rdna database v. 1.19 for alignment. sample coverage was computed with mothur and resulted to be, on average, higher than 99% for all samples, thus meaning a suitable normalization procedure for subsequent analyses. bioinformatic and statistical analyses on recognized otus were performed with python v. 2.7.11. the most representative and abundant read within each otu (as evidenced in the previous step with mothur v. 1.39.5) underwent a nucleotide blast using the national center for biotechnology information (ncbi) blast software (ncbi-blast-2.3.0) and the latest ncbi 16s microbial database accessed. a matrix of bacterial relative abundances was built at each taxon level (phylum, class, order, family, genus, species) for subsequent multivariate statistical analyses. α-diversity (within sample diversity) was evaluated, in qiime2 [14] , by computing the shannon index, the simpson index and the number of observed otus at the species level. the analysis of β-diversity (between-sample diversity) was performed by calculating both the bray-curtis dissimilarity and the weighted-unifrac distance. principal coordinate analysis (pcoa) was performed to compare the overall composition of the bacterial community between samples. the differential abundance analysis of taxa was performed at phylum, genus, and species levels by univariate statistical methods (see statistical analysis). a co-occurrence network was computed, at species level, for each group separately (virus negative, rsv positive, rsv-a and rsv-b), by using the sparcc algorithm between abundance of taxa [15] . only taxa showing a mean relative abundance ≥0.5% in almost one group were included in the network analysis, while sparcc correlations less than −0.5 and greater 0.5 with a p ≤ 0.05 based on bootstrapping of 1000 repetitions were retained. generated networks were imported and visualized in cytoscape v. 3.7.2. topological parameters of networks were calculated by using the network analyzer plugin included in cytoscape. firstly, a descriptive analysis of the samples was performed with tables and graphs corresponding to the type of qualitative or quantitative variables. statistical analysis was carried out using both parametric and nonparametric tests according to the analyzed variable. in particular, non-parametric univariate statistical tests, x 2 and fisher's exact post-hoc tests, were used for the discrete variables; non parametric mann-whitney u test and kruskal-wallis tests followed by dunn's post hoc test were performed to determine significant differences with respect to continuous variables for pairwise or multiple comparisons, respectively. in each case a p-value ≤ 0.05 was considered statistically significant. when necessary, the p values were corrected by using the benjamin-hochberg procedure to account for multiple hypothesis testing. statistical analyses were performed using spss version 25.0 (spss inc., chicago, il, usa). infants hospitalized with bronchiolitis were tested for respiratory viruses during the winter season 2016/2017. the main clinical characteristics of the studied population are provided in table 1 . out of 122 npw samples taken for nasal microbiota characterization, 67 were positive for rsv, and 28 resulted negative to the 14 respiratory viruses tested (v neg ); 27 samples positive for other respiratory viruses or co-infected were excluded to avoid bias in the analysis. after quality assessment of ngs reads (see below), the study analyses included 28 virus-negative patients and 48 patients infected with rsv. the 48 samples positive only to rsv (rsv pos ) were sequenced for subtyping as described [2] ; of these, 30 were rsv a, 17 were rsv b, and 1 could not be subtyped. the 76 children included in our study were 38 males (50.0%) and had a mean age of 72.3 days (range 23-171). significantly lower severity scores were observed in the v neg group with respect to the rsv pos (p = 0.016). consistently, the percentage of infants who received o 2 treatment and were admitted in the pediatric intensive care unit was significantly lower in the virus-negative group compared to either rsv pos groups (respectively p = 0.012 and p = 0.018). the absolute number of blood lymphocytes was significantly higher in virus-negative infants with respect to the rsv-positive infants ( table 1) . for the other clinical variables, no significant differences were found between the v neg group compared to the rsv pos . evaluation of α diversity by the shannon and simpson indexes, as well as the number of observed otus, showed a significant lower biodiversity in the rsv-positive group with respect to the virus-negative one, suggesting the presence of a microbial shifts in the nasal microbiota of rsv-positive subjects (figure 1a) . a statistically significant difference was found between the rsv-a and the virus-negative group (shannon p = 0.035, simpson p = 0.016, observed otu p = 0.007). no differences were observed between rsv-a and rsv-b groups. the β diversity analysis showed statistically significant separations between the virus-negative group and both the rsv-positive (p = 0.012) and the rsv-a (p = 0.010) groups (figure 1b) . no significant partitions were observed between infants negative for the virus and rsv-b groups. considering only taxa having a mean relative abundance ≥0.5% in at least one of the studied groups, we generated microbial profiles at taxonomy levels of phylum, genera, and species ( figure 2 ). as shown, distinctive microbial profiles were associated to the different groups (v neg , rsv pos ), but no significant differences in the relative abundance of taxa were observed at phylum level among all groups. the analysis of the relative abundance of the genera among the different groups ( figure 3 ) showed that bacteroides, pseudoflavonifractor, alistipes, kineothrix, and oscillibacter were significantly lower in abundance in the rsv pos group compared to the v neg . at species level, the relative abundance of streptococcus pneumoniae was significantly higher in the rsv pos group. the same statistically significant differences observed between rsv pos and v neg groups were kept when we matched the rsv-a and the v neg groups. no significant differences were highlighted between v neg and rsv-b groups nor between the rsv-a and rsv-b ones, although we cannot exclude that the lack of statistical significance was due to the limited number of infants positive for rsv-b. evaluation of α diversity by the shannon and simpson indexes, as well as the number of observed otus, showed a significant lower biodiversity in the rsv-positive group with respect to the virus-negative one, suggesting the presence of a microbial shifts in the nasal microbiota of rsvpositive subjects (figure 1a) . a statistically significant difference was found between the rsv-a and the virus-negative group (shannon p = 0.035, simpson p = 0.016, observed otu p = 0.007). no differences were observed between rsv-a and rsv-b groups. the β diversity analysis showed statistically significant separations between the virus-negative group and both the rsv-positive (p = 0.012) and the rsv-a (p = 0.010) groups (figure 1b) . no significant partitions were observed between infants negative for the virus and rsv-b groups. considering only taxa having a mean relative abundance ≥0.5% in at least one of the studied groups, we generated microbial profiles at taxonomy levels of phylum, genera, and species ( figure 2 ). as shown, distinctive microbial profiles were associated to the different groups (vneg, rsvpos), but no significant differences in the relative abundance of taxa were observed at phylum level among all groups. only taxa for which a mean relative abundance ≥0.5% was determined in at least one group are shown. taxa are sorted in ascending order with respect to their mean relative abundance in the vneg group. the analysis of the relative abundance of the genera among the different groups ( figure 3) showed that bacteroides, pseudoflavonifractor, alistipes, kineothrix, and oscillibacter were significantly lower in abundance in the rsvpos group compared to the vneg. at species level, the relative abundance of streptococcus pneumoniae was significantly higher in the rsvpos group. the same statistically significant differences observed between rsvpos and vneg groups were kept when we matched the rsv-a and the vneg groups. no significant differences were highlighted between vneg only taxa for which a mean relative abundance ≥0.5% was determined in at least one group are shown. taxa are sorted in ascending order with respect to their mean relative abundance in the v neg group. significantly lower in abundance in the rsvpos group compared to the vneg. at species level, the relative abundance of streptococcus pneumoniae was significantly higher in the rsvpos group. the same statistically significant differences observed between rsvpos and vneg groups were kept when we matched the rsv-a and the vneg groups. no significant differences were highlighted between vneg and rsv-b groups nor between the rsv-a and rsv-b ones, although we cannot exclude that the lack of statistical significance was due to the limited number of infants positive for rsv-b. interactions between taxa were explored by performing a network analysis based on the sparcc algorithm, which is able to reduce bias introduced by the compositionality and sparsity of microbiome data. the graphical representation of the microbial networks constitutes a sort of "correlation map" describing the positive and negative relationships between microbes drawn as edges with different colors. in this context, positive and negative correlations could reflect synergistic and antagonistic interactions between microbial groups such as metabolic interdependencies or competitions for the same ecological niches. results evidenced the presence of two predominant microbial assemblages (named assemblage 1 and 2) in analyzed networks (v neg , rsv pos ), in which taxa correlated with synergic interactions within the assemblage. differently competitive interactions between the taxa belonging to the two different assemblages were observed (figure 4) . remarkable are the taxa compositions of the two assemblages. in both cases, assemblage 1 was characterized by bacterial species resulting to be significantly more abundant within the virus-negative group, while s. pneumoniae, the unique species significantly associated with the rsv pos group, was within assemblage 2. the co-occurrence networks were evaluated for topological properties including centrality measures ( table 2) . obtained results showed a decrease in the number of nodes (taxa participating to the network) in rsv pos. similarly, results were obtained for the number of correlations (edges) indicating a network with less connection among bacteria in rsv pos with respect to the network determined for the v neg group. concerning correlation type, a decrease in both positive/synergic and negative/competitive was found in the rsv pos network with respect to v neg ; however, this could be due to the overall lower number of connections present in the rsv pos network. the decrease of relationships among bacterial species strongly evidenced that changes happened in the nasal microbial ecosystem structure, leading to an impoverished microbial network characterized by weaker interactions among microbes, a reduced interconnection among species (figure 4) , and the loss of reciprocal control between microbes. these characteristics depict a condition in which the eubiotic state has been lost, determining a state of dysbiosis. correlated with synergic interactions within the assemblage. differently competitive interactions between the taxa belonging to the two different assemblages were observed (figure 4) . remarkable are the taxa compositions of the two assemblages. in both cases, assemblage 1 was characterized by bacterial species resulting to be significantly more abundant within the virus-negative group, while s. pneumoniae, the unique species significantly associated with the rsvpos group, was within assemblage 2. rsvpos networks, computed at species level performed on taxa with a mean relative abundance ≥0.5% in at least one group. the thickness of edges represents the level of association between taxa based on the sparcc score. the size of nodes is proportional to the number of edges departing from the node, indicating its degree of interaction the co-occurrence networks were evaluated for topological properties including centrality measures (table 2) . obtained results showed a decrease in the number of nodes (taxa participating to the network) in rsvpos. similarly, results were obtained for the number of correlations (edges) figure 4 . co-occurrence network analysis. graphical representations of (a) virus-negative and (b) rsv pos networks, computed at species level performed on taxa with a mean relative abundance ≥0.5% in at least one group. the thickness of edges represents the level of association between taxa based on the sparcc score. the size of nodes is proportional to the number of edges departing from the node, indicating its degree of interaction. the main aim of the present study was to characterize the nasal microbiota in pediatric patients hospitalized for bronchiolitis from rsv and in infants affected by bronchiolitis but negative for a respiratory virus. furthermore, we attempted to characterize the nasal microbiota in infants with bronchiolitis from rsv-a and patients with bronchiolitis from rsv-b. nasal microbiota characterization was firstly carried out by evaluating the ecological indexes (α diversity). the ecological perception is fundamental to better understand the dynamic of the microbial ecosystem, and it is important to develop therapeutic approaches aimed to rebuild a balanced microbiota [16] . subsequently, we evaluated the β diversity to exploit the differences in the nasal microbial composition among the groups studied. we highlighted significant differences among the groups; in particular, we showed the following: (i) a significantly higher biodiversity in the virus-negative group with respect to the rsv-positive and to the rsv-a groups; (ii) a specific nasal microbiota composition in the virus-negative group, different from that of the rsv-positive and rsv-a groups; and (iii) a significant modification in taxa relative abundance, among rsv-positive, in particular the rsv-a, with respect to the virus-negative group. several of the bacterial species less abundant in the virus-positive group are known to be potentially beneficial species such as butyrate producers, species proposed as probiotic, or anti-inflammatory species [17] [18] [19] . the infants were negative to pcr-based tests for 14 respiratory viruses. they probably experienced a viral infection causing acute respiratory symptoms, but may have tested negative at the time of hospitalization due to viral load decline. concomitantly, they had a less severe clinical course of bronchiolitis. it can be considered a group in which the viral clearance of the upper airways is actually more efficient and faster (this could derive from a more resistant environment or from a more efficient immune response), or it could be a group of patients in which we were unable to isolate the virus because it is not part of our identification panel for the 14 respiratory viruses. interestingly, these infants' nasal microbiota are characterized by a relatively high alpha diversity, by the presence of potentially beneficial species, and by more connections in their microbial ecosystem that could be associated to a milder clinical course. on the other hand, a higher severity was observed in the rsv-a-infected group, characterized by the abundance of the unique species s. pneumoniae that is associated with several diseases such as pneumonia, otitis, and meningitis, but that can be part of the respiratory microbiota without causing any pathology. our results indicate that in rsv-a-positive group, the relative abundance of s. pneumoniae was significantly higher than that in the virus-negative group. these data support the clinical evidence that s. pneumoniae colonization is associated with increased severity during rsv infection in young children [20] . the presence of s. pneumoniae in our rsv-negative group is easily explained by the fact that our virus-negative group was affected by bronchiolitis too. furthermore, s. pneumoniae should be considered as a pathobiont bacterial species, meaning a potentially pathogenic species present in low quantity in healthy subjects that, in specific conditions under a specific selective pressure (maybe virus presence), could become predominant by establishing a dysbiosis status in the ecosystem. it has been reported that s. pneumoniae colonization and its density can enhance the risk of severed subsequent rsv infection [21] , but this association may have bidirectional interactions. rsv-a somehow promotes s. pneumoniae growth, directly [22] or through the inflammatory response induced by the viral infection, as shown for influenza virus [23, 24] . as reported in the literature for influenza virus co-infections, the nasal microbiota composition could affect individual predisposition to secondary bacterial infections, commonly triggered by gram-positive bacteria like s. pneumoniae [25] , and has a direct impact on pneumococcal attitude [26] . moreover, in host infected with influenza virus, the pro-inflammatory immune response represents a key aspect relevant to lethal pneumococcal infection, and it denotes a potential target for therapeutic intervention [23, 27] . our results corroborate results by other authors indicating the presence of streptococcus species associated with rsv bronchiolitis and its severity. a study from de steenhuijsen piters et al. [28] aimed to evaluate, in infants less than 2 years of age, if definite nasopharyngeal microbiota were associated with different host transcriptome profiles. they found rsv infection severity related to five nasopharyngeal microbiota groups, categorized by the enhancement of a specific bacterial species. children with rsv infection were associated with a microbiota dominated by h. influenzae and streptococcus. furthermore, the study showed that communications among rsv and nasopharyngeal microbiota might regulate the host immune response, hypothetically affecting clinical disease severity. another recent study [29] pointed out that streptococcus abundance was greater in samples collected during rsv infection compared with samples collected one month later. finally, we evaluated the relations among microbial community members by networks analysis, as edges that join pairs of nodes (the system components, taxa). microbial networks analyses evidenced two predominant sub-networks in all groups studied (virus negative, rsv positive); it would seem that the two sub-networks control each other. interestingly, the majority of taxa resulted to be significantly more abundant in the virus-negative group and were placed in sub-network 1, while the unique species significantly more abundant in the rsv pos group, s. pneumoniae, was in sub-network 2 and, therefore, in negative/competitive correlation with the others in both groups studied. despite the overall similarity of the two sub-networks found, we observed variations among groups in taxa interactions. a microbial ecosystem with fewer connections among microbes, and probably more susceptible to colonization by outdoor microbes, was observed in the rsv-positive group with respect to the virus-negative group. several taxa in the rsv-positive group are no longer connected and present as isolated elements. the lower connection between microbes could indicate dysbiosis, in which the mutualistic relationships among taxa are lost. enrolled infants were between 3 weeks and 6 months of age and experiencing their first severe acute respiratory infection that caused hospitalization. during this period of our life nasal microbiota composition is still very mutable, in particular in the second and third months of age [30] . the first colonizing microbes have a strong influence on what will be the following colonizers, through the expression modulation of genes coding for adhesion sites [31, 32] . notwithstanding, we found interesting differences in microbiota composition in infants when comparing, on the basis of rsv infection, groups that were similar for mean age, weight, and type of feeding, and differed for bronchiolitis severity. this is the first study that pointed out differences in nasal microbiota composition among rsv-positive and virus-negative bronchiolitis patients, and it denoted, for the first time, a more robust impact on nasal microbiota structure seemed to be related to rsv subtype a compared to rsv subtype b. although no significant differences were determined between the rvs-b group and the v neg or the rsv-a groups, we cannot exclude that the lack of statistical significance could be due to the limited number of analyzed rsv-b subjects, which represents a clear limit for this study. undoubtedly, the presence of rsv represents a disturbing factor during the first colonization phase of the nasal site, which could profile microbiota not in eubiotic equilibrium, increasing the risk of illnesses caused by microbes whose growth is favored by rsv (e.g., s. pneumoniae) as well as the risk of respiratory sequelae [33, 34] . differently, a resilient respiratory microbiota may favor a decrease in viral load and a less severe illness course. in conclusion, our results showed that two specific sub-clusters are present in the networks of the nasal microbiota in all our bronchiolitis patients, independently from rsv presence. moreover, this analysis highlighted, for the first time, a network in the rsv pos group where several taxa species have lost interaction with others and remain as bacterial species apparently disconnected from the ecosystem, shaping a state of dysbiosis ( figure 4) . accordingly, the infants affected by rsv bronchiolitis presented more perturbations in the nasal microbiota structure compared to the v neg group, thus favoring colonization of pathobiont species, such as s. pneumonia, and negatively influencing potentially beneficial species growth, defining a state of dysbiosis. all of this goes with the higher disease severity score, indicating that a dysbiosis status emphasized by rsv presence could itself act as a predisposing factor for bronchiolitis severity. longitudinal study approaches are necessary to clarify whether the observed associations are causally linked and whether the control of pathobionts overgrowth, by therapies aimed at improving the nasal microbiota composition, may contribute to prevent a severe bronchiolitis course. the study was partially funded by a sapienza university grant "ricerca 2019" n. rm11916b893fc0d0 to ap. a. for the marc-30 investigators prospective multicenter study of viral etiology and hospital length of stay in children with severe bronchiolitis how respiratory syncytial virus genotypes influence the clinical course in infants hospitalized for bronchiolitis early respiratory microbiota composition determines bacterial succession patterns and respiratory health in children the infant nasopharyngeal microbiome impacts severity of lower respiratory infection and risk of asthma development nasopharyngeal proteobacteria are associated with viral etiology and acute wheezing in children with severe bronchiolitis airway microbiota and acute respiratory infection in children rhinovirus promote internalization of s.aureus into non-fully permissive cultered pneumocytes. microbes infect nasopharyngeal lactobacillus is associated with a reduced risk of childhood wheezing illnesses following acute respiratory syncytial virus infection in infancy the fecal microbiota profile and bronchiolitis in infants association of nasopharyngeal microbiota profiles with bronchiolitis severity in infants hospitalised for bronchiolitis microbiome-transcriptome interactions related to severity of respiratory syncytial virus infection respiratory syncytial virus, human bocavirus and rhinovirus bronchiolitis in infants inferring correlation networks from genomic survey data cytoscape: a software environment for integrated models of biomolecular interaction networks interpreting infective microbiota: the importance of an ecological perspective the human gut firmicute roseburia intestinalis is a primary degrader of dietary β-mannans roseburia intestinalis inhibits interleukin-17 excretion and promotes regulatory t cells differentiation in colitis alistipes: the influence of a commensal on anxiety and depression streptococcus pneumoniae colonization of the nasopharynx is associated with increased severity during respiratory syncytial virus infection in young children streptococcus pneumoniae enhances human respiratory syncytial virus infection in vitro and in vivo respiratory syncytial virus increases the virulence of streptococcus pneumoniae by binding to penicillin binding protein 1a. a new paradigm in respiratory infection influenza and bacterial superinfection: illuminating the immunologic mechanisms of disease influenza a virus infection predisposes hosts to secondary infection with different streptococcus pneumoniae serotypes with similar outcome but serotype-specific manifestation the co-pathogenesis of influenza viruses with bacteria in the lung interaction between the nasal microbiota and s. pneumoniae in the context of live-attenuated influenza vaccine in vivo neutralization of pro-inflammatory cytokines during secondary streptococcus pneumoniae infection post influenza a virus infection nasopharyngeal microbiota, host transcriptome, and disease severity in children with respiratory syncytial virus infection airway response to respiratory syncytial virus has incidental antibacterial effects dynamics of the nasal microbiota in infancy: a prospective cohort study factors influencing the composition of the intestinal microbiota in early infancy dna isolation protocols affect the detection limit of pcrapproaches of bacteria in samples from the human gastrointestinal tract nasopharyngeal pneumococcal density during asymptomatic respiratory virus infection and risk for subsequent acute respiratory illness nasopharyngeal microbiome in respiratory syncytial virus resembles profile associated with increased childhood asthma risk this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license acknowledgments: valerio iebba, gustave roussy cancer campus (grcc) and institut national de la santé et de la recherche medicale (inserm) and equipe labellisée-ligue national contre le cancer, france. the authors declare no conflict of interest. key: cord-281754-auqh3vtr authors: nan title: emerging respiratory disease coronaviruses date: 2017-09-12 journal: dis mon doi: 10.1016/j.disamonth.2017.03.019 sha: doc_id: 281754 cord_uid: auqh3vtr nan coronaviruses -an overview coronaviruses (cov) are a diverse group of viruses capable of infecting humans, and a wide range of animals. cov affect multiple systems, and can cause respiratory, gastrointestinal, hepatic, and neurological illnesses, ranging from mild sickness to death. cov are classified into multiple genera, including alpha, beta, gamma and delta coronaviruses. [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] . of note, cov seem to be able to adapt to new hosts and changing environments; this may be related to cov ability to mutate and recombine [1, 3, 5, 7] , perhaps contributing to novel viruses with varying human pathogenicity. coronaviruses (order nidovirales, family coronaviridae, genus coronavirus) are large, enveloped, single stranded, positive-sense rna viruses, capable of infecting a variety of animals, including bats, mice, birds, dogs, pigs, cattle, and humans. identified many decades ago, coronavirus ( figure 7 [9]from the latin corona (translation "crown" or "halo") represents the appearance of cov virions as they are viewed through an electron microscope [2, [7] [8] [9] [10] . the virus appearance is created by viral spikes (s), peplomers that populate the surface and determine host tropism ( figure 7 ) [8] [9] [10] . [9] . typically cov are considered to be highly species-specific. in immunocompetent hosts, infection elicits the immune response of neutralizing antibodies and cell-mediated immune responses that attempt to kill infected cells [1, 10, 11] . coronaviruses, members of the coronaviridae family were identified and grouped based upon their serological crossreactivity, and genomic sequence homology. host ranges are diverse, and can include canines, felines, swine, mice, camels, bats, birds, and humans. [1, 2, 7, [10] [11] [12] [13] [14] across the four genera of coronaviruses are alphacoronavirus, betacoronavirus, gammacoronavirus, and deltacoronavirus [2, 5, 10, 11] there is a high frequency of recombination and rate of mutation which are believed to allow covs to adapt to new hosts and environments [3, [11] [12] [13] [14] [15] [16] . sars cov [2, 5, 6, [17] [18] [19] [20] [21] [22] [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] [33] [34] is a good illustration of this; studies revealed it originated from animalsbats as the natural reservoir [5] [6] [7] [8] 10, 11, [25] [26] [27] , and palm civet as the intermediate host [11] [12] [13] . this underscores the infection risk in human animal interactionsoccupational, or adventure or travel, as well as environmental incursion and changing habits expected with climate change, which can pose significant risks to human, as well as animal health. as an animal pathogen, coronaviruses can lead to highly virulent respiratory, enteric, and neurological diseases, in addition to hepatitis, resulting in epizootics of respiratory diseases and/or gastroenteritis. as a human virus the range of disease is broad, from cold like to severe multisystem involvement (these cov infections are associated with short incubation periods (2-7 days), such as those found in sars [2, 5, 6, 17, 18, 24, 25] . several coronaviruses are capable of causing fatal systemic diseases in animals, including feline infectious peritonitis virus (fipv), swine hemagglutinating encephalomyelitis virus (hev), some strains of avian infectious bronchitis virus (ibv) and mouse hepatitis virus (mhv). these particular cov can replicate in liver, lung, kidney, gut, spleen, brain, spinal cord, retina, as well as other tissues [2, 7, 13] . according to the cdc, the first case of sars cov was reported in asia in early 2003 [35] . further investigation notes november 2002 the first case of an atypical pneumonia emerged in china (guangdong province) where ultimately the causative agent was determined to be a newly discovered coronavirus. by 2003 an epidemic of similarly severe, atypical pneumonias, was emerging from hong kong, guangdong, and toronto, ontario. the following is the timeline of initial events that transpired referable to sars cov. on 11 february 2003 china reported to the world health organization (who) that 305 cases of atypical pneumonia of unknown etiology had been identified in guangdong province since 16 november 2002; five people had died. as of 21 february 2003 a physician from guangdong province, who was ill with an atypical pneumonia, travelled to hong kong, staying overnight in a hotel. the etiology causing his illness was identified as severe acute respiratory syndrome coronavirus (sars cov); it was likely transmitted to at least 10 additional persons. these transmissions/infections subsequently initiated outbreaks in hong kong, singapore, viet nam, and canada [20] [21] [22] [23] [24] 35, 36] symptoms characteristic of this aggressive atypical pneumonia included an onset of illness associated with high fever (temperature greater than 100.4°f [438.0°c]), headacheoften severe, an overall feeling of discomfort, and body aches, again often severe. some persons may have had respiratory symptoms at the outset. approximately 10% to 20% percent experienced diarrhea. after 2 to 7 days, sars patients may develop a dry cough. most patients develop pneumonia. given the prior outbreaks of highly pathogenic avian influenza in that same region of china, it was first considered to be an emerging flu virus. other pathogens, including members of the paramyxoviridae family, and human metapneumovirus (hmpv) were considered as causative of this new clinical illness which became known as severe acute respiratory syndrome or sars. after international collaboration among multiple research facilities, a previously unknown pathogen was ultimately determined to be causative of sars -a new coronavirus -sars cov [20] [21] [22] [23] [24] [25] [26] [27] [28] [29] . whiles sars cov is a significant pathogen capable of causing profound illness, even death, historically coronaviruses were one cause of 'the common cold.' known as endemic human betacoronaviruses hcov-oc43 and hcov-hku1. coronaviruses affecting humans (hcovs) historically were associated with mild illness. found in group 1 (hcov-229e) and group 2 (hcov-oc43) they are a widespread cause of mild respiratory illnesses [12] , although occasionally these cov cause serious infections of the lower respiratory tract in children and adults, including necrotizing enterocolitis in newborns [12] [13] [14] [15] [16] . early research into the sars co-v genomic sequence demonstrated that this new cov does not belong to any of the known groups of coronaviruses, previously described human coronaviruses hcov-oc43 and hcov-229e [20] [21] [22] [23] [24] . in fact it appears sars cov is only somewhat related to these hcov. the sars-cov genome appears to be equidistant from those of all known coronaviruses. moreover, sars cov closest relatives appear to be the murine, bovine, porcine, and human coronaviruses in group 2 and avian coronavirus ibv in group 1. research on the sars cov suggests this new virus represents a fourth group or lineage of coronavirus -group 4 [23] . genomic sequence analysis seems to support the hypothesis that of sars-cov is an animal virus for which the normal host is still unknown and that developed the ability to productively infect humans or has the ability to cross species barriers [25] . the genome shows that sars-cov is neither a mutant of a known coronavirus, nor a recombinant between known coronaviruses. as the virus passes through human beings, sars-cov maintains genotype, and is adapted to the human host [26] . testing allows genetic analysis to distinguish different strains of sars-cov, allowing epidemiological studies [28] . not surprisingly there are also economic, as well as health implications -coronaviruses cause important diseases in domestic animals, as well as in human populations. toronto during and in the aftermath of their sars outbreak saw a significant, albeit temporary decline in tourism and business related visits, as well as lost conference and trade show related commerce. recognizing the importance of animalhuman pathogen crossover, opportunities to reduce the spread of contagion, and to identify potential risks is critical to prevent or at least reduce the likelihood of sars, mers, and influenza outbreaks such as the avian influenza outbreaks of the 1990's and early2000's and the swine flu outbreak in 2009. sars co-v ( figure 8 ) [37] can be detected in extracts of lung and kidney tissue by virus isolation or pcr; bronchoalveolar lavage specimens by virus isolation, electron microscopy and pcr; and sputum or upper respiratory tract swab, aspirate, or wash specimens by pcr [20, 21, 29] . sars-associated coronavirus rna was detected in nasopharyngeal aspirates by rt-pcr in 32% at initial presentation (mean 3.2 days after onset of illness) and in 68% at day 14 [30] . in stool samples, viral rna was detected in 97% of patients two weeks after the onset of illness. 42% of urine samples were positive for viral rna [30] . viral rna was also detected at extremely low concentrations in plasma during the acute phase and in feces during the late convalescent phase, suggesting that the virus may be shed in feces for prolonged periods of time [20, 21] . the timelines of events as noted by cdc concluded towards the end of 2003 with removal of travel warnings to china and ontario. by the end of 2003, according to the cdc report of who data, reports of sars infections from 29 countries and regions revealed 8,096 persons with probable sars resulting in 774 deathswith an estimated case fatality rate just below 10% (higher in elderly, infirm patients). in the united states, eight sars infections were documented by laboratory testing and an additional 19 probable sars infections were reported. by 2004 the cdc issued a "notice of embargo of civets" as a sars-like virus had been isolated from civets (captured in areas of china where the sars outbreak originated). cdc also banned the importation of civets. the civet is a mammal with a catlike body, long legs, a long tail, and a masked face resembling a raccoon or weasel. sars cov was detected in animal handlers of civets. the ban on civets is currently still in effect. by 2012 the national select agent registry program declared sars-coronavirus a select agent. a select agent is a bacterium, virus or toxin that has the potential to pose a severe threat to public health and safety [32, 35] . in spite of significant effort to develop countermeasures for coronaviruses, three are as of yet no licensed therapeutics that have shown consistent effectiveness against mers cov or sars cov. [38] . intensive careproviding ventilator, circulatory and other organ system support to preserve renal, hepatic and neurological function, as well as prevention of secondary infection is critical. during the sars cov pandemic of 2003 immune based therapies have been tried, with equivocal results. ribavirin and interferon combinations showed some clinical improvements in non human primate studies, but unlike actual clinical experiences with mers and sars, where the illness, let alone treatment are rarely initiated rapidly after infection, the trials provided interventions soon after viral challenge [38] [39] [40] [41] [42] . during sars cov epidemics, various combinations of therapies were trialed [38] [39] [40] [41] [42] [43] [44] [45] [46] [47] [48] [49] , but no scaled, controlled approaches were conducted, making recommendations on current antivirals with or without interferon combination therapy, of concern, and questionable [38] [39] [40] [41] [42] [43] [44] [45] [46] [47] . ribavirin is a potent nucleoside analogue used with varying degrees of success against rna viruses, but there is the potential for adverse side effects including hemolytic anemia, metabolic derangements. interferon can also elicit adverse effects, although they have demonstrated value against viral infection [38] [39] [40] [41] . corticosteroids have been tried in sars cov infectionthey resulted in increased viral load, admissions to intensive care unit, and mortality [43, 44] . during sars cov, convalescent plasma, hyperimmune globulin were shown to be relatively safe, and possibly effective for reducing mortality [45] [46] [47] [48] . convalescent plasma, when administered within 14 days of illness, did decrease mortality in sars cov patients, according to one study [46] . they found this was a time critical issueadministration had to be given within a 2 week period. the challenge of course is in identifying cases and contacts rapidly and immediate utilization of such therapies for a chance at optimal effect. donor supply, technical capacity in regions where sars, mers or other emerging threats are likely to occur, safety of end product and other challenges can limit the potential of this therapeutic option. monoclonal antibodies (mabs) offer promise, and have demonstrated efficacy in the treatment of cancer and autoimmune diseases, as well as respiratory syncytial virus (rsv) [38, 47, 48] . trials are ongoing to determine the use of mabs for ebola virus disease, hiv -primary and secondary prevention [38] . unfortunately the costs, as well as research and development timelines are longer than for polyclonal antibody preparations. nevertheless, in spite of rigorous testing, regulatory and cost issues associated with mabs, their potential as therapies for mers and other potentially deadly diseases continue to drive research in this area. antiviral research into adenine analogues that can disrupt viral rna replication [50] are being developed as well as a nucleoside analogues with the potential to work against filoviruses, coronaviruses, and other rna viruses [51] . ideally an antiviral that covers a broad range of coronaviruses will be developed based upon the genetic sequence of these viruses and their life cycle. but the development of such an antimicrobial and other interventions still remains in the future. of note, extensive research is also being conducted towards developing vaccines. towards that end, not unlike vaccines directed towards other pathogens, research has focused on viral structure [51, 52] , and replication mechanisms ( figure 9 [52]/ (table 1 ) [38] . not surprisingly, as with other pathogens, such as dengue [57] [58] [59] [60] [61] where there remain unknowns, such as protective immunity, cross protection against a variety of strains, and other technical difficulties, there are a variety of challenges to overcome in developing an effective vaccine against sars or other coronaviruses. for example, in developing a live sars cov vaccine, it will be necessary to address the various coronavirus strains to recombine with each other, with the potential of attenuated parts of the genome being replaced with non-attenuated components of the genome, resulting in a pathogenic virus. one approach being considered is the use of reverse genetics; it may eliminate the risk of recombination between coronavirus strains [38, [53] [54] [55] [56] 58, 60, 62] . as suddenly as sars cov emerged, it has seemingly gone quiescent. however another significant, and previously unknown or not described coronavirus respiratory disease has emerged. in the next section the newest member of cov discovered that causes human illness will be discussed. it is the middle east respiratory syndrome coronavirus (mers-cov, mers), which is part of the beta group of coronaviruses. avian influenza a (h7n9) virus human infections with the emerging avian influenza a h7n9 virus from wet market poultry: clinical analysis and characterization of viral genome human infection with a novel avian-origin influenza a(h7n9) virus global concerns regarding novel influenza a (h7n9) virus infections a detailed epidemiological and clinical description of 6 human cases of avian-origin influenza a (h7n9) virus infection in shanghai radiological findings of chest in patients with h7n9 avian influenza from a hospital avian influenza: critical considerations for primary care physician animal -human interface h7n9 cdc emerging h7n9 influenza a (novel reassortant avian-origin) pneumonia: radiologic findings initial computed tomography findings of pneumonia in patients with human infected avian influenza (h7n9) chest x-ray and ct findings of early h7n9 avian influenza cases thoracic imaging features of 13 patients with h7n9 subtype avian influenza virus infection human infection with a novel avian-origin influenza a (h7n9) virus: serial chest radiographic and ct findings analysis of the clinical characteristics and treatment of two patients with avian influenza virus (h7n9) severe acute respiratory syndrome: radiographic review of 40 probable cases in toronto the emerging infectious disease emergence of avian influenza a (h7n9) virus causing severe human illness china chest xray and computed tomography (ct) findings of early h7n9 avian influenza cases middle east respiratory syndrome coronavirus (mers -cov_ infection pulmonary pathologic findings of fatal 2009 pandemic influenza a/h1n1 viral infections. arch patholo influenza a (h1n1) virus associated pneumonia: high resolution computed tomography -pathologic correlation cdc guidance on testing for h7n9 interim guidance on the use of antiviral agents for treatment of human infections with avian influenza a h7n9 the biology of coronaviruses coronaviruses post sars: update on replication and pathogenesis delayed induction of proinflammatory cytokines and suppression of innate antiviral response by the novel middle east respiratory syndrome coronavirus: implications for pathogenesis and treatment virus taxonomy; ninth report of the international committee on taxonomy of viruses discovery of seven novel mammalian and avian coronaviruses in the genus deltacoronavirus supports bat coronaviruses as the gene source of alphacoronavirus and betacoronavirus and avian coronaviruses as the gene source of gammacoronavirus and deltacornavirus evidence of the recombinant origin of a bat severe acute respiratory syndrome osars) like coronavirus and its implications on the direct ancestor of sars coronavirus molecular epidemiology of human coronavirus oc43 reveals evolution of different genotypes over time and recent emergence of a novel genotype due to natural recombination isolation and characterization of viruses related to the sars coronavirus from animals in southern china cdc/national institutes of health public domain coronavirus image severe acute respiratory syndrome coronavirus like virus in chinese horseshoe bats viruses and bacteria in the etiology of the common cold mandell, douglas, and bennett's principles and practice of infectious diseases spectrum of clinical illness in hospitalized patients with "common cold" virus infections coronavirus pneumonia following autologous bone marrow transplantation for breast cancer survival of human coronaviruses 229e and oc43 in suspension after drying on surfaces: a possible source of hospitalacquired infections the sars coronavirus: a postgenomic era sars coronavirus: a new challenge for prevention and therapy a newly discovered human pneumovirus isolated from young children with respiratory tract disease identification of a novel coronavirus in patients with severe acute respiratory syndrome severe acute respiratory syndrome: identification of the etiological agent coronavirus as a possible cause of severe acute respiratory syndrome the genome sequence of the sars-associated coronavirus characterization of a novel coronavirus associated with severe acute respiratory syndrome viral zoonoses -a threat under control? comparative full-length genome sequence analysis of 14 sars coronavirus isolates and common mutations associated with putative origins of infection comparative analysis of the sars coronavirus genome: a good start to a long journey chinese university of hong kong molecular sars research group. coronavirus genomic-sequence variations and the epidemiology of the severe acute respiratory syndrome a novel coronavirus associated with severe acute respiratory syndrome clinical progression and viral load in a community outbreak of coronavirus-associated sars pneumonia: a prospective study update on sars research and other possibly zoonotic coronaviruses the severe acute respiratory syndrome vaccines to prevent severe acute respiratory syndrome coronavirus-induced disease modjarrad k treatment strategies for middle east respiratory syndrome coronavirus treatment with interferon-alpha2b and ribavirin improves outcome in mers cov infected rhesus macaques ifn alpha 2a or ifn beta 1 in combination with ribavirin to treat middle east respiratory syndrome coronavirus pneumonia: a retrospective study oral ribavirin for the treatment of noninfluenza respiratory viral infections: a systematic review interferon in oncological practice e: review of interferon biology, clinical applications, and toxicities the use of corticosteroid as treatment in sars was associated with adverse outcomes: a retrospective cohort study effects of early corticosteroid treatment on plasma sars associated coronavirus rns concentrations in adult patients feasibility, safety, clinical and laboratory effects of convalescent plasma therapy for patients with middle east respiratory syndrome coronavirus infection: a study protocol the effectiveness of convalescent plasma and hyperimmune immunoglobulin for the treatment of sever acute respiratory infections of viral etiology: a systematic review and exploratory meta analysis potent neutralization of mers cov by human neutralizing monoclonal antibodies to the viral spike glycoprotein exceptionally potent neutralization of middle east respiratory syndrome coronavirus by human monoclonal antibodies rewiring the severe acute respiratory syndrome coronavirus (sars-cov) transcription circuit: engineering a recombination-resistant genome nucleotide prodrug gs 5734 is a broad spectrum filovirus inhibitor that provides complete therapeutic protection against the development of ebola virus disease evd in infected non human primates id week biocryst announces nature publication demonstrating efficacy of bbcx4430 in a non human primate model of filovirus infection zabel p molecular mechanisms of severe acute respiratory syndrome (sars) recent developments in anti-severe respiratory syndrome coronavirus chemotherapy emerging respiratory viruses: challenges and vaccine strategies safety and immunogenicity from a phase i trial of inactivated severe acute respiratory syndrome coronavirus vaccine a sars dna vaccine induces neutralizing antibody and cellular immune responses in healthy adults in a phase i clinical trial viral hemorrhagic fever viruses in novel viruses, emerging pathogens -the pandemic threat continues next generation dengue vaccines: a review of candidates in preclinical development evaluation of commercially available anti-dengue virus immunoglobulin m tests protective efficacy of the recombinant, live attenuated, cy tetravalent dengue vaccine in thai schoolchildren: a random controlled phase 2b trial cells in dengue virus infection in vivo reverse genetics of sars related coronavirus using vaccinia virus based recombination published this newly identified respiratory viral illness was caused by a novel coronavirus, which was initially designated as human betacoronavirus [2-5], but was eventually named middle east respiratory syndrome coronavirus (mers cov). reminiscent of, and worth considering as a caution for greater vigilance towards emerging pathogens, the suddenness that sars cov emerged as a new cause of severe pulmonary illness, has been replicated in this new aggressive respiratory illness mers cov. unlike sars cov, it has not caused the thousands of cases over a short period of time as with other cov, including sars cov, the exact epidemiology, including whether there are larger numbers of mild illness, remains unknown. males over 60 yrs of age seem to be at higher risk of severe disease symptoms and death among those infected. while the greatest spike in cases occurred near key: cord-285557-my16g91c authors: berger, a.; drosten, ch.; doerr, h. w.; stürmer, m.; preiser, w. title: severe acute respiratory syndrome (sars)—paradigm of an emerging viral infection date: 2004-01-31 journal: journal of clinical virology doi: 10.1016/j.jcv.2003.09.011 sha: doc_id: 285557 cord_uid: my16g91c abstract an acute and often severe respiratory illness emerged in southern china in late 2002 and rapidly spread to different areas of the far east as well as several countries around the globe. when the outbreak of this apparently novel infectious disease termed severe acute respiratory syndrome (sars) came to an end in july 2003, it had caused over 8000 probable cases worldwide and more than 700 deaths. starting in march 2003, the world health organization (who) organised an unprecedented international effort by leading laboratories working together to find the causative agent. little more than one week later, three research groups from this who-coordinated network simultaneously found evidence of a hitherto unknown coronavirus in sars patients, using different approaches. after koch’s postulates had been fulfilled, who officially declared on 16 april 2003 that this virus never before seen in humans is the cause of sars. ever since, progress around sars-associated coronavirus (sars-cov) has been swift. within weeks of the first isolate being obtained, its complete genome was sequenced. diagnostic tests based on the detection of sars-cov rna were developed and made available freely and widely; nevertheless the sars case definition still remains based on clinical and epidemiological criteria. the agent’s environmental stability, methods suitable for inactivation and disinfection, and potential antiviral compounds have been studied, and development of vaccines and immunotherapeutics is ongoing. despite its grave consequences in humanitarian, political and economic terms, sars may serve as an example of how much can be achieved through a well-coordinated international approach, combining the latest technological advances of molecular virology with more “traditional” techniques carried out to an excellent standard. severe acute respiratory syndrome (sars) is the latest in a series of emerging infectious diseases, and certainly one of the most widely publicised. this acute and often severe respiratory illness seems to have emerged in southern china in late 2002 (world health organization, 2003c) . it soon caused considerable international alarm, after several index cases had given rise to outbreaks of sometimes ଝ this review is dedicated to all those who were prepared to risk their lives to provide care to sars patients and control the first pandemic of the 21st century. * corresponding author. tel.: +49-69-6301-4303; fax: +49-69-6301-6477. e-mail address: annemarie.berger@em.uni-frankfurt.de (a. berger). enormous scales, and when the disease's ability to spread to distant areas within a very short period of time became obvious (world health organization, 2003d) . a definition was developed for suspected and probable sars cases, based on clinical and epidemiological criteria; it has since been modified on several occasions. while sars demonstrated very vividly that in the modern world with an enormous volume of intercontinental traffic, infectious agents may be spread rapidly across the globe, it also serves as an example of how modern technologyprovided there is the necessary will, determination, and coordination to make best use of it-may help in combating such threats with unprecedented speed and enormous success. sars is characterized clinically by fever followed by respiratory signs and symptoms which may lead to rapidly progressive respiratory failure. as of september 2003, 8098 people have been notified to the world health organization (who) as fulfilling the criteria for "probable sars", and of these, 774 have died from sars (http://www.who.int/csr/ sars/country/2003 08 15/en/). what made sars-in contrast, e.g. to influenza-notorious is its propensity to cause hospital outbreaks; some of these have affected over 100 people, including health care staff, other patients and visitors . in contrast to many other emerging viral infections such as ebola, hantavirus pulmonary syndrome, and nipah, sars also clearly demonstrated its ability for easy and rapid geographic spread. this is because the sars agent affected a generally rather mobile population, and because those infected normally remain well enough to travel for several days after onset of infectivity. on 17 march 2003, the who set up a worldwide network of virological laboratories investigating sars cases (world health organization, 2003a) . the investigations conducted by the members of these networks were coordinated by who's department of communicable disease surveillance and response (csr) through normally daily telephone conferences and a password-protected internet website. thus results and planned further studies were communicated and views and comments exchanged almost in "real-time" which made possible the rapid progress in elucidating the aetiological agent. in its final form, this network comprised 13 participating laboratories from ten countries (world health organization multicentre collaborative network for severe acute respiratory syndrome diagnosis, 2003). investigations had soon ruled out a novel influenza virus strain, possibly of avian origin, as the cause of sars, and then focussed on members of the paramyxoviridae family, including human metapneumovirus (hmpv), and chlamydia-like organisms, including chlamydia pneumoniae. however, further investigations did not confirm these findings; the said agents were indeed found in a number of sars patients but not in all (who multicentre collaborative networks for severe acute respiratory syndrome (sars) diagnosis, 2003). almost nobody knew at that stage that virologists in beijing had already discovered a new virus in samples from some of the earliest sars patients. however, the official line in china at the time was that the novel "atypical pneumonia" was caused by chlamydia (enserink, 2003a) . nevertheless, before the end of march, laboratories in hong kong, germany, canada, and the united states of america found evidence of a novel coronavirus in patients with sars by cell culture, electron microscopy, and by polymerase chain reaction (pcr) using primers at low stringency designed for other agents followed by sequencing (peiris et al., 2003b; drosten et al., 2003a; ksiazek et al., 2003; poutanen et al., 2003; drosten et al., 2003b) . these results could not rule out that very thorough and extensive testing had by chance led to the discovery of a novel agent that was not responsible for the new illness but rather an "innocent bystander". however, the sequences obtained in different parts of the world were shown to belong to the same, previously unrecognised, coronavirus (ruan et al., 2003) . it could also be shown that sars patients underwent seroconversion against this coronavirus, using cells infected with patient isolates as antigen for indirect immunofluorescent antibody tests (drosten et al., 2003a; ksiazek et al., 2003; fig. 1) . furthermore, no evidence of present or past infection with this agent could be detected in limited surveys of healthy control individuals not suffering from sars (ksiazek et al., 2003) . this strengthened the case for the novel coronavirus being the cause of sars, but only after it had been shown to cause a similar illness in artificially infected macaques could it be regarded as fulfilling all four of koch's postulates ; world health organisation multicentre collaborative networks for severe acute respiratory syndrome diagnosis, 2003) . on april 16, 2003, less than a month after the laboratory network had been brought into existence, who officially announced that a new coronavirus, never before seen in humans or animals and now provisionally termed sars-associated coronavirus (abbreviated as sars-cov), was the cause of sars . coronaviruses are large, enveloped, positive-stranded rna viruses with a diameter of 60-220 nm. most but not all viral particles display the characteristic appearance of surface projections, giving rise to the virus family's name (corona, latin, = crown). they have the largest genomes of all rna viruses. based on their unique transcription strategy that involves the formation of "nested" mrna molecules (cavanagh, 2000) . within the coronaviridae, the genera torovirus and coronavirus (type species: infectious bronchitis virus, ibv) are distinguished. a unique feature of coronavirus genetics is a high frequency of rna recombination as a result of discontinuous transcription and polymerase "jumping" (lai and cavanagh, 1997) . one example is the porcine respiratory coronavirus (prcov), which evolved in the early 1980s from the enteropathogenic porcine transmissible gastroenteritis coronavirus (tgev), known since the 1940s (pensaert et al., 1986) . through a large deletion in the s gene, the virus acquired an altered tissue tropism, causing mild respiratory infections. based on homologies on the amino acid sequence level, the known coronaviruses can be divided into three groups. table 1 gives an overview of coronavirus species, group assignment, host species, disease manifestation and availability of a vaccine. there are more than a dozen known coronaviruses affecting different animal species; whereas group i and ii coronaviruses affect various mammals, those in group iii infect birds. some of these cause major problems in the livestock industry or may affect companion animals; therefore, considerable efforts have been devoted to their control, including development of active immunisation. negative-stain transmission electron microscopy of respiratory samples from sars patients and of infected cell culture supernatants reveals pleomorphic, enveloped virus-like particles with diameters of between 60 and 130 nm (fig. 2) . most but not all viral particles showed the characteristic coronavirus-like surface features (ksiazek et al., 2003) . in contrast to most coronaviruses, which infect only the cells of their natural host species and a few closely related species, the sars-cov is able to infect different cell cultures, such as african green monkey (cercopithecus aethiops) kidney cells (vero) and the human colorectal adenocarcinoma cell line (caco-2), causing a massive cytopathic effect (cpe) after as little as 2 days or 3 days (fig. 3) . it should be mentioned that these cell lines were not commonly used for the isolation of human respiratory viruses. interestingly during cell culture passages of the frankfurt isolate a virus variant emerged with a nucleotide deletion of 45 bases in the orf7b . the biological significance of this finding remains to be elucidated. complete genome sequences of sars-cov were first published by a canadian laboratory and the centers for disease control (cdc), atlanta (marra et al., 2003; rota et al., 2003) . as of end-october 2003, 45 full genome sequences are available on http://www.ncbi.nlm.nih.gov/genomes/ sars/sars.html. the genomic data available so far from several sars-cov strains suggest that the novel agent does not belong to any of the known groups of coronaviruses, fig. 2 . electron microscopy image of sars-cov particle from infected cell culture supernatant after ultracentrifugation, 2% formalin fixation and negative staining with uranyl acetate (photograph by h. r. gelderblom, robert koch institute, berlin, germany). including the two human coronaviruses (hcov) oc43 and 229e (drosten et al., 2003a; marra et al., 2003; peiris et al., 2003b; rota et al., 2003) , to which it is only moderately related (fig. 4) . the sars-cov appears to be neither a mutant of a known coronavirus nor a recombinant between known coronaviruses (holmes, 2003) . it has been proposed that the new virus defines a fourth lineage of coronavirus (group iv) (marra et al., 2003) (fig. 4) . however, more recently it was suggested that sars-cov may be an early split-off from the group 2 lineage . the sequence analysis of sars-cov suggests that it is an animal virus with a still unknown natural host species that has recently developed the ability to productively infect humans. a genetically very close but not identical virus was found in wild animals (masked palm civets and a raccoon dog) from a wildlife market in guangdong . but uncertainties remain over the exact source of this virus; the animals sampled could have been infected from humans or another animal species (cyranoski and abbott, 2003; normile and enserink, 2003) . sequence analysis of different sars-cov isolates reveals two distinct genotypes. one genotype was linked with infections originating from hotel m in hong kong, the other one comprises isolates from hong kong, guangdong and beijing that had no association with hotel m (ruan et al., 2003; tsui et al., 2003) . to date, there is no information as to whether different sars-cov strains may have different degrees of virulence. there is little doubt that sars originated from guangdong province of southern china (breiman et al., 2003) . the first cases retrospectively identified as sars occurred there in november 2002. interestingly, amongst these early cases there seems to have been a significantly higher percentage of food handlers, chefs, etc. than in the general population, lending further support to a zoonotic origin. the worldwide spread of sars-cov was triggered through a single infected individual from guangdong who spent some time in hong kong before succumbing to sars (chan-yeung and yu, 2003) . during that time he unwittingly infected several others that in turn gave rise to a series of outbreaks (centers for disease control and prevention, 2003) . through sometimes several generations of transmissions, this event carried the virus to different hong kong hospitals and communities as well as to vietnam, singapore, canada, the united states of america, and beyond to a total of 30 countries and areas of the world (world health organization, 2003d). the virus travelled in infected humans and was passed on over several generations, as reflected in the genetic relatedness of isolates from these countries. although china was late in admitting it, the sars-cov had unsurprisingly also been spread within mainland china; in the end, the worst affected area was the capital, beijing, with 2521 cases in total, which surpasses the count for guangdong with 1512 by far (world health organization western pacific region, country office china: http://www.wpro.who.int/wr/chn/chn sars.asp). the incubation period of sars is short, ranging from 2 to 16 days. large studies consistently noted a median incubation period of 6 days (booth et al., 2003; lee et al., 2003; tsang et al., 2000) . however, the time from exposure to the onset of symptoms may vary considerably . the who continues to conclude that the current best estimate of the maximum incubation period is 10 days (who update 49-sars case fatality ratio, incubation period, http://www.who.int/csr/sars/archive/2003 05 07a/en/). based on the latest data, the case fatality ratio is estimated to be <1% in persons aged 24 years or younger, 6% in persons aged 25-44 years, 15% in persons aged 45-64 years, and greater than 50% in persons aged 65 years and older ; who update 49-sars case fatality ratio, incubation period, http://www.who.int/csr/sars/ archive/2003 05 07a/en/). pregnant women with sars appear to have a worse prognosis and a higher mortality. therefore, early delivery or termination of pregnancy should be considered in those who are seriously ill with sars. for women who are relatively well with sars, however, there seems to be no reason for elective preterm delivery, such as reducing the risk of materno-fetal transmission (wong et al., 2003a) . compared with adults and teenagers, sars seems to take a less aggressive clinical course in younger children (hon et al., 2003) . multivariable analysis showed that the presence of diabetes, advanced age or other comorbid conditions were independently associated with a poor outcome (booth et al., 2003; donnelly et al., 2003; fowler et al., 2003) . at the present time, with no new cases-apart from the isolated laboratory-acquired one-having been reported since 15 june 2003, sars-cov has apparently been driven out of the human population (world health organization, 2003d) . in the meantime, who has issued a consensus document on sars epidemiology (who department of communicable disease surveillance and response, 2003). the pattern of geographic spread of sars was similar across all affected areas: typically, a patient with sars arrived from a previously affected area, was not identified as such when hospitalised, and thus infected health care workers, other patients and hospital visitors; these then infected their close contacts, and then the disease moved into the larger community (hawkey et al., 2003) . the virus seems to be spread predominantly by respiratory droplets over a relatively close distance , however, at least under some circumstances direct and indirect contact with respiratory secretions, faeces or animal vectors may also lead to transmission (hong kong department of health, 2003; who environmental health team, 2003; tsang et al., 2000; ng, 2003) . shedding of sars-cov in faeces and urine also occurs but its significance is unknown. the duration of infectivity is still unclear. faecal shedding seems to last for several weeks; this however does not necessarily mean that there is sufficient excretion of infectious viral particles to infect other individuals (peiris et al., 2003a) . practising stringent droplets and contact precaution significantly reduces the risk of infection after exposure to patients with sars. therefore, the protective role of the mask suggests that the main route of transmission is by droplets . sars-cov spreads more efficiently in sophisticated hospital settings. evidence suggests that certain procedures, such as intubation under difficult circumstances and use of nebulizers increase the risk of infection . the only case of laboratory-acquired sars-cov transmission so far occurred in singapore in september 2003. it involved a postgraduate who worked in a virology laboratory. subsequent investigation showed inappropriate laboratory standards (who severe acute respiratory syndrome (sars) in singapore-update 2, http://www.who.int/csr/ don/2003 09 24/en); no secondary transmission arose from this case. it demonstrates the need for optimal biosafety precautions in laboratories working with sars-cov; these constitute the only places on earth where sars-cov is currently known to still exist and might be at the source of a re-emergence. blood transfusions or administration of blood products have not been implicated in transmission anywhere. this is despite the demonstration of viraemia during the clinical phase of the illness, albeit at low to moderate titres (drosten et al., 2003a) . nevertheless, the potential of blood-borne transmission led to the early implementation of measures such as exclusion of possibly exposed individuals from the donor pool. the sars-cov is only moderately transmissible. a single infectious case will infect about three secondary cases (lipsitch et al., 2003; riley et al., 2003) . nevertheless, the clusters of cases in hotel and apartment buildings in hong kong show that transmission of the sars-cov can be extremely efficient. attack rates in excess of 50% have been reported. one common observation in various areas was the occurrence of so-called "super-spreaders", i.e. individuals that transmit the infection to at least ten others (world health organization, 2003b). these "super-spreaders" were mostly very ill and often died from sars, and invariably serious lapses in infection control precautions had occurred during their management. so far there is no evidence that differences in virus strains may be responsible for the "super-spreader" phenomenon. there is also no firm evidence suggesting that subsequent transmissions led to clinically less severe illness, possibly through attenuation of the virus. it is also unclear why children are relatively under-represented amongst sars cases, and why on average they seem to suffer less severe sars illness. studies on the stability of the new sars-cov demonstrate the virus is more stable at room temperature than the previously known human coronaviruses (sizun et al., 2000) . the virus has been shown to survive for up to 48 hours on plastic surfaces and up to 4 days in diarrhoea. nevertheless the virus loses infectivity after exposure to different commonly used disinfectants and fixatives. heat exposure at 56 • c quickly reduces infectivity (world health organization (who): first data on stability and resistance of sars-cov compiled by members of who laboratory network available at http://www.who.int/csr/sars/ survival 2003 05 04/en/index.html). as defined by the who, a person is suspected to have sars if she has documented high fever (>38 • c), plus cough or breathing difficulty, and has been in contact with a person believed to have had sars, or has a history of travel to or stay in a geographic area where documented transmission of the illness has occurred, during the 10 days prior to onset of symptoms ("suspect case"). a suspect case with infiltrates consistent with pneumonia or respiratory distress syndrome (rds) by chest x-ray is reclassified as a probable case. the revised case definition as of 1 may 2003, (see: http://www.who.int/csr/sars/casedefinition/en/) for the first time includes virus-specific laboratory results: a suspect case that tests positive for sars-cov in one or more assays should also be reclassified as probable. while recommendations have been issued for the use of laboratory methods for sars-cov (see: http://www.who. int/csr/sars/labmethods/en/), there are, however, at present no defined criteria for negative sars-cov test results to reject a diagnosis of sars. given the rather low shedding of sars-cov from the upper respiratory tract (drosten et al., 2003a) , and the insufficient sensitivity of presently available laboratory methods, premature exclusion on the basis of negative test results may lead to tragic consequences. positive laboratory test results for other agents able to cause atypical pneumonia may serve as exclusion criteria; according to the case definition, a case should be excluded if an alternative diagnosis can fully explain the illness. nevertheless, the possibility of dual infection must not be ruled out completely. the required epidemiological linkage has repeatedly proven to be problematic. until an area is recognised as being affected, only imported cases fulfil the criteria for sars but not those who became infected locally through contact with unrecognised cases. thus, precious time may be lost until cases are recognised and appropriate measures taken. a thorough analysis showed that the existing who criteria lack sensitivity in the pre-hospital setting (rainer et al., 2003) . this again may be problematic as it may delay appropriate management of sars cases. the human coronaviruses known prior to march 2003 are difficult to propagate in cell cultures. their disease associations-generally mild respiratory illness ("common cold"), enteric and rarely possibly neurological disease-led to their widespread neglect in medical virology; only few groups worked on various scientific aspects, and very few laboratories offered routine diagnostic tests, mainly by pcr. sars-cov, on the other hand, is readily propagated in vitro and may also be detected by pcr and indirectly through antibody testing. nevertheless, and despite considerable progress in this field, much remains to be done until laboratory tests become a useful tool for the management of sars cases (world health organization multicentre collaborative network for severe acute respiratory syndrome diagnosis, 2003) . the presence of the infectious virus can be detected by inoculating suitable cell cultures (e.g., vero cells) with patient specimens (such as respiratory secretions, blood or stool) and propagating the virus in vitro. once isolated, the virus must be identified as sars-cov using further tests. according to international consensus, such work has to be performed under biosafety level (bsl) three conditions. sars-cov-specific rna can be amplified from various clinical specimens, especially in respiratory secretions and in stool, by pcr. high concentrations of viral rna of up to 100 million molecules per millilitre were found in sputum. viral rna was also detected, albeit at extremely low concentrations, in plasma during the acute phase and in faeces during the late convalescent phase, suggesting that virus may be shed in faeces for prolonged periods of time (drosten et al., 2003a) . a commercial real-time rt-pcr test kit containing primers and positive and negative controls developed by the bernhard nocht institute (http://www.bnihamburg.de/) is available (http://www.artus-biotech.de). an inactivated standard preparation is also available for diagnostic purposes through the european network for imported viral infections (enivd; http://www.enivd.de). enivd is also preparing for an international external quality assessment scheme for sars-cov assays. the existing pcr tests cannot rule out, with certainty, the presence of sars-cov in patients . on the other hand, contamination of samples in laboratories performing pcr may lead to false-positive results, unless appropriate precautions are taken. various methods were developed for the detection of antibodies produced in response to infection with sars-cov by probably virtually all patients. the first type of antibody test to be employed was the immunofluorescence assay (ifa). using cells infected with the patient's own virus isolate and an antihuman igg:fitc conjugate, we were able to demonstrate specific seroconversion in the two frankfurt sars patients (drosten et al., 2003a; fig. 1 ). an enzyme-linked immunosorbent assay (elisa) was developed that detects antibodies in the serum of sars patients and reliably yields positive results at around day 21 after the onset of illness (world health organization multicentre collaborative network for severe acute respiratory syndrome diagnosis, 2003). the neutralisation test (nt) assesses and quantifies, by means of titration, the ability of patient sera to neutralise the infectivity of sars-cov on cell culture; the nt titre may therefore be correlated to clinical immunity although this has yet to be demonstrated. however, nt is limited to institutions with bsl-3 facilities. the only antibody test commercially available so far is an ifa which yields a positive result from about day 10 after the onset of illness (euroimmun, lübeck, germany). as the diagnosis of sars is based entirely on a set of clinical and epidemiological criteria so far, reported case numbers are likely to include a substantial number of non-sars patients. therefore, recovered patients should be tested systematically for specific sars-cov antibody reactivity to confirm their diagnoses in retrospect and thus allow a better understanding of epidemiological and other features . although electron microscopy has an important role in the rapid diagnosis of infectious agents in emergent situations (hazelton and gelderblom, 2003) , it has provided only circumstantial evidence in the case of sars. when a virus-like agent was first visualised in clinical material from sars cases by electron microscopy, its classification was ambiguous; it later turned out to be human metapneumovirus (poutanen et al., 2003) . even in cases in whom coronavirus-like particles were detected, these could not be distinguished from the 'classic' human coronaviruses. no specific treatment recommendations can be made at this time. primary measures include isolation and the implementation of stringent infection control measures to effectively prevent further transmissions. empiric therapy should include coverage for organisms associated with any community-acquired pneumonia of unclear aetiology, including agents with activity against both typical and atypical respiratory pathogens. treatment choices may be influenced by severity of the illness. oxygen supplementation is often necessary, and severe cases seem to do better if intensive care including artificial respiration is commenced early (so et al., 2003) . efforts are underway at various institutions to assess potential anti-sars-cov agents in vitro. ribavirin, a "broad spectrum" agent active against various rna viruses has also been used clinically in sars patients (koren et al., 2003) , but seems to lack an in vitro effect (cinatl et al., 2003a) . corticosteroids were widely used in sars patients, particularly in china. the rationale for their administration is the observation that tissue changes suggest that part of the lung damage is due to cytokines induced by the virus (peiris et al., 2003a) . some clinical reports also underline their usefulness (zhao et al., 2003) . other therapies are being explored, such as convalescent plasma (wong et al., 2003b) or normal human immunoglobulin which may be beneficial through an immunomodulatory effect or through acting against agents causing secondary infections. preliminary clinical data suggest that protease inhibitors used for anti-hiv therapy, lopinavir and nelfinavir (yamamoto n, personal communication), might have some efficacy, both as initial therapy and in the rescue setting. hong kong researchers reported at the who global conference on sars in kuala lumpur in june 2003 that sars patients treated with kaletra (lopinavir with low-dose ritonavir) plus ribavirin experienced a 50% reduction in death rate. while efforts are underway to develop more targeted anti-sars-cov approaches, broad screening of available substances in vitro has led to some potentially important clues. recently glycyrrhizin, a compound found in liquorice roots (glycyrrhiza glabra l.), was reported to have good in vitro activity against sars-cov (cinatl et al., 2003a) . the mechanism of glycyrrhizin's activity against sars-cov is unclear. glycyrrhizin has previously been used to treat patients with hiv-1 and chronic hepatitis c virus . interestingly, this compound may be contained in some of the herbal preparations widely used in sars patients in china as part of traditional chinese medicine (lin et al., 2003) . furthermore, interferons inhibit sars-cov in vitro. in a recent study (cinatl et al., 2003b) , interferon ß was more potent than interferon ␣ or ␥. therefore, it could become a drug of choice in future, alone or in combination with other antiviral drugs. the rapid success in identifying the causative agent of sars results from a collaborative effort-rather than a competitive approach-by high-level laboratory investigators making use of all available techniques, from cell culture through electron microscopy (hazelton and gelderblom, 2003) to molecular techniques, in order to identify a novel agent. hopefully this approach, coordinated by who, will serve as a model for future instances of emerging infections that will undoubtedly take place (ludwig et al., 2003) . despite the exemplary efforts that led to the identification of the causative novel coronavirus and allowed enormous knowledge about it to be accumulated within only a few months, it is maybe surprising that this success in terminating the outbreak has to be attributed to "old-fashioned" measures such as rapid and strict isolation of suspect cases and thorough contact tracing (world health organization, 2003c); one is left wondering whether the same might also have been achieved without knowledge of the aetiology. thanks to an internationally well-coordinated and in most cases timely and determined response no new cases of sars have been notified since 15 june 2003. several countries reported sars cases imported from areas reporting outbreaks but did not experience secondary transmission; likewise, vietnam was the first country to demonstrate that-through a combination of early detection and public alert followed by decisive public health action and often heroic efforts by individuals-further transmission could be curtailed (reilley et al., 2003) . the absence of new clinical cases worldwide suggests that sars-cov no longer circulates within the human population; however, the possibility of clinically "silent" infections or of long-term virus carriers cannot be ruled out completely. furthermore, the origin of the agent remains obscure; sars-cov or a closely related virus persisting in a hitherto unidentified animal reservoir may yet again cross the species barrier and lead to human outbreaks. numerous questions relating to the epidemiology of sars have yet to be answered (normile and enserink, 2003; breiman et al., 2003) . at the time of writing (october 2003) it is completely uncertain whether sars will ever reappear. it is unclear whether seasonal recurrences may occur. in southern china, unlike europe and north america, the annual influenza peak incidence is from march to july (huang et al., 2001 ); thus, it shows a similar epidemic curve as the sars outbreak in 2003 (enserink, 2003b) . the advent of the next 'flu season will pose considerable problems, given the lack of reliable laboratory methods for the early diagnosis of sars. the case definitions, too, will need to be adjusted to a world without sars; in theory, new cases are "impossible" as the criterion of an epidemiological link cannot be fulfilled. precious time may therefore be lost before a reappearance is detected. vigilance for sars must clearly be maintained (see: alert, verification and public health management of sars in the post-outbreak period-14 august 2003-rationale for continued vigilance for sars; http://www.who.int/csr/sars/postoutbreak/ en/). for this purpose, who has defined three geographical zones according to their presumed risk for a sars recurrence: a potential zone of re-emergence, comprising guangdong and other areas where animal-to-human of sars-cov might occur; nodal areas, comprising hong kong, vietnam, singapore, canada, and taiwan, with sustained local transmission in spring 2003 or entry of numerous persons from the potential zone of re-emergence; and low risk areas. sars-related vigilance should be staged according to the zone in which a particular area is situated; for low risk areas, surveillance should be for clusters of "alert" cases among health care workers, other hospital staff, patients and visitors in the same health care unit. a sars alert is defined as two or more health care workers or hospital-acquired illness in at least three individuals (health care workers and/or other hospital staff and/or patients and/or visitors) in the same unit fulfilling the clinical case definition of sars and with onset of illness in the same 10-day period. in the other zones, this should be supplemented by enhanced surveillance, plus special studies for sars-cov infections in animal and human populations in the potential zone of re-emergence. besides improving existing detection assays-for instance, pcr methods based on the amplification of the nucleoprotein gene may be intrinsically more sensitive, due to the coronaviral transcription strategy , and thus be valuable for early diagnosis-further laboratory research needs to include detailed physico-chemical analysis of sars-cov proteins to allow the development of novel compounds based on targeted drug design (anand et al., 2003) . although an effective vaccine cannot be expected to be available soon, the relative ease with which sars-cov can be propagated in vitro is clearly helpful. a suitable animal model for sars may be available in the form of cynomolgus macaques (macaca fascicularis) . while the availability of vaccines against animal coronaviruses, such as avian infectious bronchitis virus, transmissible gastroenteritis coronavirus of pigs, and feline infectious peritonitis virus, is encouraging, the obvious lack of protective immunity in humans after infection with hcov oc43 and 229e is not. there is also currently no commercial veterinary vaccine to prevent respiratory coronavirus infections, except for infectious bronchitis virus infections in chickens. further research is also urgently needed to determine whether immune pathogenesis plays a rôle in sars or whether immune enhancement may occur, the chances of developing an effective and safe vaccine therefore remain uncertain. it is to be hoped that after such an encouraging start in an atmosphere of open collaboration and mutual trust, progress in sars-cov research will not be impeded by patent matters (gold, 2003) . coronavirus main proteinase (3clpro) structure: basis for design of anti-sars drugs clinical features and short-term outcomes of 144 patients with sars in the greater toronto area role of china in the quest to define and control severe acute respiratory syndrome update: outbreak of severe acute respiratory syndrome-worldwide outbreak of severe acute respiratory syndrome in hong kong special administrative region: case report severe acute respiratory syndrome: patients were epidemiologically linked glycyrrhizin, an active component of liquorice roots, and replication of sars-associated coronavirus treatment of sars with human interferons virus detectives seek source of sars in china's wild animals epidemiological determinants of spread of causal agent of severe acute respiratory syndrome in hong kong identification of a novel coronavirus in patients with severe acute respiratory syndrome severe acute respiratory syndrome: identification of the etiological agent identification and containment of an outbreak of sars in a community hospital china's missed chance the big question now: will it be back? koch's postulates fulfilled for sars virus critically ill patients with severe acute respiratory syndrome sars genome patent: symptom or disease? isolation and characterization of viruses related to the sars coronavirus from animals in southern china severe acute respiratory syndrome (sars): breath-taking progress electron microscopy for rapid diagnosis of infectious agents in emergent situations sars coronavirus: a new challenge for prevention and therapy clinical presentations and outcome of severe acute respiratory syndrome in children hong kong department of health. outbreak of severe acute respiratory syndrome (sars) at amoy gardens main findings of the investigation analysis of the 1991-2000 influenza epidemic in guangdong province ribavirin in the treatment of sars: a new trick for an old drug? a novel coronavirus associated with severe acute respiratory syndrome newly discovered coronavirus as the primary cause of severe acute respiratory syndrome the molecular biology of coronaviruses a major outbreak of severe acute respiratory syndrome in hong kong profile of specific antibodies to the sars-associated coronavirus clinical observation on 103 patients of severe acute respiratory syndrome treated by integrative traditional chinese and western medicine transmission dynamics and control of severe acute respiratory syndrome medicinal herbs for hepatitis c virus infection viral zoonoses-a threat under control? the genome sequence of the sars-associated coronavirus possible role of an animal vector in the sars outbreak at amoy gardens tracking the roots of a killer clinical progression and viral load in a community outbreak of coronavirus-associated sars pneumonia: a prospective study coronavirus as a possible cause of severe acute respiratory syndrome isolation of a porcine respiratory, non-enteric coronavirus related to transmissible gastroenteritis rapid diagnosis of a coronavirus associated with severe acute respiratory syndrome (sars) national microbiology laboratory, canada; canadian severe acute respiratory syndrome study team. identification of severe acute respiratory syndrome in canada evaluation of who criteria for identifying patients with severe acute respiratory syndrome out of hospital: prospective observational study transmission dynamics of the etiological agent of sars in hong kong: impact of public health interventions characterization of a novel coronavirus associated with severe acute respiratory syndrome comparative full-length genome sequence analysis of 14 sars coronavirus isolates and common mutations associated with putative origins of infection effectiveness of precautions against droplets and contact in prevention of nosocomial transmission of severe acute respiratory syndrome (sars) survival of human coronaviruses 229e and oc43 in suspension and after drying onsurfaces: a possible source of hospital-acquired infections unique and conserved features of genome and proteome of sars-coronavirus, an early split-off from the coronavirus group 2 lineage development of a standard treatment protocol for severe acute respiratory syndrome mechanisms and enzymes involved in sars coronavirus genome expression a cluster of cases of severe acute respiratory syndrome in hong kong coronavirus genomic-sequence variations and the epidemiology of the severe acute respiratory syndrome who department of communicable disease surveillance and response: consensus document on the epidemiology of severe acute respiratory syndrome (sars) world health organization regional office for the western pacific severe acute respiratory syndrome and pregnancy treatment of severe acute respiratory syndrome with convalescent plasma who multicentre collaborative networks for severe acute respiratory syndrome (sars) diagnosis severe acute respiratory syndrome-singapore severe acute respiratory syndrome (sars): over 100 days into the outbreak chronology of travel recommendations, areas with local transmission world health organization multicentre collaborative network for severe acute respiratory syndrome diagnosis. a multicentre collaboration to investigate the cause of severe acute respiratory syndrome description and clinical treatment of an early outbreak of severe acute respiratory syndrome (sars) in guangzhou, pr china key: cord-303040-ha8gufh8 authors: park, won-ju; yoo, seok-ju; lee, suk-ho; chung, jae-woo; jang, keun-ho; moon, jai-dong title: respiratory syncytial virus outbreak in the basic military training camp of the republic of korea air force date: 2015-01-14 journal: j prev med public health doi: 10.3961/jpmph.14.037 sha: doc_id: 303040 cord_uid: ha8gufh8 objectives: an outbreak of acute febrile illness occurred in the republic of korea air force boot camp from may to july 2011. an epidemiological investigation of the causative agent, which was of a highly infective nature, was conducted. methods: throat swabs were carried out and a multiplex reverse transcriptase-polymerase chain reaction (rt-pcr) assay was performed to identify possible causative factors. results: the mean age of patients who had febrile illness during the study period was 20.24 years. the multiplex rt-pcr assay identified respiratory syncytial virus (rsv) as the causative agent. the main symptoms were sore throat (76.0%), sputum (72.8%), cough (72.1%), tonsillar hypertrophy (67.9%), and rhinorrhea (55.9%). the mean temperature was 38.75°c and the attack rate among the recruits was 15.7% (588 out of 3750 recruits), while the mean duration of fever was 2.3 days. the prognosis was generally favorable with supportive care but recurrent fever occurred in 10.1% of the patients within a month. conclusions: this is the first epidemiological study of an rsv outbreak that developed in a healthy young adult group. in the event of an outbreak of an acute febrile illness of a highly infective nature in facilities used by a young adult group, rsv should be considered among the possible causative agents. respiratory syncytial virus (rsv) belongs to the family of paramyxoviridae and is classified in the genus pneumovirus. rsv is the most common cause of fatal acute respiratory tract infection pissn 1975 -8375 eissn 2233 among infants and young children [1] . virtually every infant has been afflicted with an rsv infection by the age of 4 years, and reinfection may occur throughout life [2, 3] . moreover, rsv infection has been encountered by almost all people in south korea before reaching adulthood [4] . mortality data from several studies suggest that rsv is a significant cause of death in early childhood. such child death comes from an acute lower respiratory infection, especially following pneumococcal pneumonia or haemophilus influenza type b [5, 6] . recent studies indicate that rsv is an important cause of respiratory infection in elderly patients, either those with compromised immunity or inflicted with chronic illness, as well as in adult populations in a special environment, such as military personnel [7] [8] [9] [10] [11] . rsv tends to be more variable and clinical manifestations seem less distinctive in adults than they are in children, and the viral cause of the infection is often unsuspected [12] [13] [14] [15] . acute febrile respiratory illness occurred collectively in a group of patients in a boot camp of the republic of korea air force (ro-kaf) from may 2011 to july 2011. we conducted an epidemiological investigation of the causative agent, which was of a highly infective nature. this study investigates the significance and clinical features of rsv in respiratory infections in a healthy young adult group. this study intends to describe the clinical course of this infection and to suggest strategies for treatment and prevention in cases of an rsv outbreak. a case patient was a person, among military recruits in this boot camp, who was admitted to the medical care center in the boot camp with chief complaints of fever and symptoms of upper respiratory tract illness after may 26, 2011. the hospitalization criteria include cases with an objectively confirmed fever greater than 37.3°c in the morning or greater than 37.8°c in the afternoon, in addition to cases that require hospitalization, according to the opinion of an air force physician, for the prevention or treatment of an infection on the basis of the fever management protocol of the rokaf (supplemental figure 1 ). duration of fever was defined as the period from the onset of fever to the time of objective fever disappearance, as observed with periodic body temperature measurements. tympanic temperature was measured using a digital thermometer (braun thermoscan pro 4000; welch allyn, san diego, ca, usa). the test for finding the causative pathogen was carried out on june 14, 2011, at the time when the number of new patients had peaked. twenty-two patients were admitted to the hospital with cardinal symptoms of respiratory tract infection. among them, 5 patients who underwent diagnostic tests were selected through random sampling by utilizing microsoft office excel 2003. the throat swab samples collected from these 5 patients were sent to the seoul clinical laboratories and seoul medical science institute for analysis. throat swab was carried out by smearing the posterior aspect of the pharynx with a sterile cotton swab while the tongue was pressed down with a tongue depressor. the smear was done by rotating the cotton swab 360º, three to four times. viral transport media were utilized to transport the samples in a refrigerated condition. a multiplex reverse transcriptase-polymerase chain reaction (rt-pcr) assay was performed with a seeplex rv6 detection kit (seegene inc., seoul, korea) for six types of respiratory viruses (rsv, adenovirus, influenza a, influenza b, para-influenza, and metapneumovirus). the pcr amplification was performed using a t3000 thermocycler (biometra, göttingen, germany). a retrospective investigation for all 588 patients was conducted using computerrecorded patient charts. the clinical characteristics of the disease and types of therapeutic regimens were obtained from these records. this study also examined cases with upper respiratory infection (uri) that occurred in the basic training camp of the rokaf for the last three years. this investigation also examined all the uri-associated diseases in the military. the numbers of patients with diseases recorded as acute nasopharyngitis, acute laryngopharyngitis, acute tonsillitis, tonsillitis, acute uri, or uri were acquired from the system of military medical information management. subjects were classified into four groups, depending on the type of hospital treatment they received: supportive care group, antibiotic group, antiviral group, and antibiotic and antiviral group. the kruskal-wallis test and the analysis of covariance (ancova) were used to find differences in body temperature and duration of fever among the groups. the spss version 15.0 (spss inc., chicago, il, usa) was used for data analysis. the subjects' medical records were obtained by referencing the military medical information system. this study was approved by the medical research institutional review board of the ministry of national defense (no. afmc-12-irb-014). subjects in this study included new recruits in training at the boot camp of the rokaf. all rokaf recruits are trained at this camp. together, the recruits have used the same dining and training areas for about two months, and two of their quarters are located 100 m away. contact with outsiders is prohibited during the training period ( figure 1 ). acute febrile illness occurred collectively among military recruits starting from may 26, 2011. on a suspicion of influenza a (h1n1) in the early phase of an epidemic, a rapid antigen test, an immunofluorescence assay, and a multiplex rt-pcr assay were carried out for all 11 patients with early onset of fever at the armed forces medical research institute, south korea, on june 3, 2011. however, these results were all negative. calling attention to thorough patient examination, segregation, and preventative activities to prevent further incidences did not reduce the incidence of febrile disease, owing to its clustered occurrence since june 9, 2011. preventative measures included the management of personal hygiene, such as the wearing of a mask by all recruits, thorough hand washing, and restricted sharing of personal items. further measures comprised instantaneous reporting of symptom development, medical diagnosis, treatment, and segregation. additional preventative measures for food-associated personnel were reinforced. patients largely had complaints of fever of 38°c or higher, as well as other uri symptoms. their symptoms were managed well by administration of antipyretics and antibiotics, as well as supportive care, such as fluid therapy. patients with such symptoms improved after about one to two days of hospital treatment and were discharged from the hospital. the incidence of acute febrile disease continued for approximately two months, but it ceased after july 27, 2011, which marked the end of the epidemic. training for newly arrived recruits continued at this boot camp throughout the year. of the total of 3750 newly arrived recruits at this camp, 588 developed acute febrile illness, resulting in an attack rate of 15.7% during this period. the epidemic curve depicts the number of uri patients who were cared for at the outpatient department of this boot camp, and the number of patients who were admitted to this hospital during the three-year period from 2010 to 2012 ( figure 2 ). the graph demonstrates that the number of uri patients in the period from may to july 2011 was higher than that in an ordinary year. any clustered occurrence of acute febrile illness had to be reported to the central epidemiological investigation team and was subjected to the fever management protocol. in times of clustered occurrences of acute febrile illness, as opposed to ordinary years when there is less development of acute febrile illness, as a matter of duty, such incidences must be reported to the medical division of headquarters, which should be followed up by an epidemiological investigation in accordance with the fever management protocol. until the present (april 11, 2014), there have been only two epidemiological investi a multiplex rt-pcr assay was carried out for six types of respiratory viruses (rsv, adenovirus, influenza a, influenza b, parainfluenza, and metapneumovirus). the assay result was rsv-positive for all 5 subjects, and it was negative for all other viruses. of the total of 588 case patients, 408 for whom the chart record had not been omitted were investigated. among them, 280 patients (68.6%) had a fever of 38.5°c or higher. their main symptoms were consistent with those of uri, such as sore throat, sputum, cough, tonsillar hypertrophy, rhinorrhea, chill, and/or pharyngeal injection (table 1) . their mean age was 20.24 years. these patients had a fever, with a mean body temperature of 38.75°c, and the mean duration of fever was 2.29 days. about 10.1% of those patients who had been discharged from the hospital developed a recurrent fever within a month and were treated again. they had a fever with a temperature lower than that of the first hospital admission, and a shorter fever duration, showing a satisfactory clinical course. the result of chest x-rays showed that 4 patients had pneumonia, but supportive care for all of these patients proved to be effective within a week and they were discharged from the hospital ( table 2) . this study was conducted to find any difference in the duration of fever depending on the type of treatment method. the selection of treatment method was made by air force physicians, without consultation with regulatory guidelines. the group that received only supportive care included 14 patients and had a mean fever duration of 1. h1n1, had the longest mean fever duration of 4.00 (sd, 1.55) days. 348 patients who received antibiotic therapy had a mean fever duration of 2.11 (sd, 1.21) days. forty patients, who were administered concurrently with antibiotic therapy and an antiviral regimen (oseltamivir) had a mean fever duration of 3.85 (sd, 1.93) days. there was no difference in body temperature among patients receiving the various treatment modalities (p=0.224, kruskal-wallis test), but a significant difference was found in fever duration among the patients (p<0.001). after making adjustments for the effect of body temperature and number of symptoms, the difference was still significant (p<0.001, ancova). in the south korea, hundreds of thousands of healthy young men in their early twenties join the military every year, by entering boot camps. these young recruits are confined to the barracks and restricted from contacting outsiders during the basic military training period. the compromised immune capacity, due to the dense population and high physical and psychological stresses in the boot camp, leads to numerous opportunities for respiratory infection. residential crowding is known to be a risk factor for rsv infections [16] . the results of studies up to now have revealed that the causative agents of military respiratory infectious diseases are adenoviruses, influenza a and b viruses, streptococcus pneumoniae, streptococcus pyogenes, chlamydia pneumoniae, mycoplasma pneumoniae, epstein-barr virus, coronavirus, and rhinoviruses [17] . a multiplex rt-pcr assay identified rsv as the causative agent in this study. the mean age of the febrile patients was 20.24 years. the main symptoms were sore throat (76.0%), sputum (72.8%), cough (72.1%), tonsillar hypertrophy (67.9%) and rhinorrhea (55.9%). the mean body temperature was 38.75°c and the group' s attack rate was 15.7% (588 out of 3750 recruits), while the mean duration of fever was 2.3 days. the prognosis was generally favorable with supportive care. the results of this study support existing data that not only infants but also healthy adults are readily infected, and that the disease in a healthy young adult group tends to be milder and the individuals are able to fight off the virus with relative ease [18] [19] [20] [21] [22] . the recurrence rate within a month is about 10%. accordingly, adequate patient explanation and education to encourage the patient to visit the department of medical service as rapidly as possible in recurrent cases of fever could prevent potential additional infections. the significance of this study is that it is the first investigation of an rsv outbreak that developed in a healthy young adult group. this epidemiological investigation suggests the prospect of rsv as a causative pathogen of respiratory tract infections that could intensively occur among young recruits undergoing basic military training. the rsv-caused uri would not foster immunity in patients, and re-infection might develop throughout one's life. this epidemic with rsv as the etiologic agent had the characteristic manifestations of a common cold, such as high fever and infectivity. supportive care in the acute phase for two to three days led to a favorable prognosis. none of the patients had any severe complications, and all of them returned to the barracks after the treatment and safely completed their training programme. nevertheless, this epidemic, which lasted for two months, led to a setback in the military timetable and might have caused damage to military strategy. massive antibiotic utilization in the military may increase antibiotic resistance. fever duration was significantly longer in the group of patients administered an antibiotic or antiviral drug (oseltamivir), despite the fact that there had been no difference in body temperature measured after their hospital admission. such results may be due to the fact that patients with severe symptoms and long-term hospitalization received an additional antibiotic or antiviral agent. moreover, patients who delayed seeking care might be more likely to receive an antibiotic or antiviral agent. another explanation for such results would be the fact that the antibiotic treatment or inappropriate antiviral regimen might not have been particularly effective in the rsv epidemic. this would provide the basis for a suggestion that favorable prognosis in rsv infections might be attained by supportive care in a healthy young adult group. this study verified that there could be a possibility of reoccurrences of rsv-induced epidemics, and supportive care alone would lead to an amicable prognosis in a healthy young adult group. the major limitations of this study were the fact that the pcr assay only measured samples from five patients, and also that rsv subtypes have not been investigated. the fact that patients with fever largely had similar clinical manifestations, and that five randomly selected patients, among 22 symptomatic subjects, were all rsv positive, would highly support the idea of rsv as the causative agent of this epidemic. in the test performed after 5 patients had been randomly selected from 22 inpatients, the fact that all 5 patients were positive suggested the probability that many more of the inpatients might have been rsv positive. utilizing the fever management protocol, the rokaf has monitored the collective development of acute febrile illness since 2008. however, the fact that there were only two outbreaks since that time, h1n1 outbreak in 2009 and the outbreak examined in this investigation, points to the possibility that they were caused by the same pathogen. furthermore, given that the subjects of this study inhabited their quarters, dining and training areas together for an extended period of time, this epidemiological association may be quite high. the epidemic curve also showed the typical pattern of a droplet-mediated respiratory infection with manifestations of infections inflicted by contacts among people. the symptomatic manifestation of upper respiratory tract infection in adults, unlike that of infants, concurred with that of other previous studies [20] [21] [22] . an additional limitation was the fact that some military trainees had refused medical care due to concern for their training scores. according to the fever management protocol, it is obligatory that any military trainee with a fever must receive medical treatment. in consideration of this aspect, the actual attack rate could be higher, and this might explain the cause for the extended epidemic lasting as long as two months. the introduction of an infrared thermal camera system may be necessary to promptly identify patients with a fever in the military boot camp. of the 588 cases, only 408 patients with sufficient chart records were analyzed. this may have led to the exclusion of mild cases and a depiction of the outbreak as more severe than it was. this consideration may, however, support the result of this study that healthy young adults with an rsv infection might have a good prognosis even when receiving only simple supportive care. in an attempt to prevent a military rsv epidemic, the introduction of an effective rsv vaccine, for which studies are currently in progress, would be worth consideration, if it could be made commercially available [23] . in the event of an epidemic of acute febrile respiratory illness of a highly infective nature, it is recommended to conduct a test for rsv in the young adult population in the military facilities. clinical courses can be predicted through such activity, and they may also decrease unnecessary antibiotic use. clinicians may also consider an appropriate antiviral regimen, such as ribavirin, when required [24] [25] [26] . the strong possibility that rsv was the causative pathogen for the collective development of this acute febrile illness in the military services foretells that incidences of such infection may multiply rapidly for a long time, as witnessed in this epidemiologic investigation. thus, rapid detection and segregation of febrile patients, along with installation of an infrared thermal camera system, are necessary. furthermore, personal hygiene should be promoted. rsv is unstable in the environment, surviving only a few hours on environmental surfaces, and it is readily inactivated with soap and water [18] . methods of preventing rsv infection include not sharing utensils or cups, avoiding kissing others, covering coughs and sneeze, and washing hands frequently and correctly [19] . and military movement should only be considered when patients are totally recovered and without infectivity. owing to the characteristics of frequent military mobility, there is a high tendency for propagation of respiratory viral infections to personnel in other military units. in an attempt to prevent those military trainees with high infectivity from moving to other military units during this epidemic, they were directed to move to other military facilities after they had been completely recovered and their infectivity lost. such measures could have prevented the further spread of this epidemic to other military units. military planners must focus on how best to control the spread of infectious respiratory diseases in highly mobile military populations [27] . medical microvirology genetic diversity among respiratory syncytial viruses that have caused repeated infections in children from rural india the burden of respiratory syncytial virus infection in young children seroprevalence of respiratory syncytial virus igg among healthy young adults in basic training for the republic of korea air force mortality associated with influenza and respiratory syncytial virus in the united states global burden of acute lower respiratory infections due to respiratory syncytial virus in young children: a systematic review and meta-analysis respiratory syncytial virus is an important cause of community-acquired lower respiratory infection among hospitalized adults symptomatic respiratory syncytial virus infection in previously healthy young adults living in a crowded military environment respiratory syncytial virus: an important cause of acute respiratory illness among young adults undergoing military training. influenza other respir viruses detection of respiratory syncytial virus in adults with chronic obstructive pulmonary disease humoral immunity to respiratory syncytial virus in young and elderly adults rsv infection in elderly adults respiratory syncytial virus infection in adult populations respiratory syncytial virus and parainfluenza virus clinical and epidemiological comparison of human metapneumovirus and respiratory syncytial virus in seoul yogev r. residential crowding and severe respiratory syncytial virus disease among infants and young children: a systematic literature review respiratory diseases among u.s. military personnel: countering emerging threats viruses and the lung: infections and non-infectious viral-linked lung disorders brief report: respiratory syncytial virus infection. frequently asked questions an epidemic of respiratory syncytial virus in elderly people: clinical and serological findings respiratory syncytial virus infections in previously healthy working adults prevalence and clinical characteristics of human respiratory syncytial virus in chinese adults with acute respiratory tract infection respiratory syncytial virus vaccine development respiratory syncytial virus disease: update on treatment and prevention respiratory syncytial virus prevention and therapy: past, present, and future prophylaxis and treatment of respiratory syncytial virus in adult immunocompromised patients spread of adenovirus to geographically dispersed military installations the authors have no conflicts of interest with the material presented in this paper. key: cord-282668-bs634hti authors: niang, mbayame ndiaye; diop, ndeye sokhna; fall, amary; kiori, davy e.; sarr, fatoumata diene; sy, sara; goudiaby, déborah; barry, mamadou aliou; fall, malick; dia, ndongo title: respiratory viruses in patients with influenza-like illness in senegal: focus on human respiratory adenoviruses date: 2017-03-22 journal: plos one doi: 10.1371/journal.pone.0174287 sha: doc_id: 282668 cord_uid: bs634hti background: human adenoviruses (hadvs) are highly contagious pathogens that are associated with a wide spectrum of human illnesses involving the respiratory tract. in the present study, we investigate the epidemiologic and viral molecular features of hadvs circulating in senegal after 4 consecutive years of sentinel surveillance of influenza-like illness cases. methodology and results: from january 2012 to december 2015 swabs were collected from consenting ili outpatients. adenoviral detection is performed by rrt-pcr with the anyplex(™) ii rv16 detection kit (seegene) and molecular characterization was performed using a partial hexon gene sequence. 6381 samples were collected. more than half of patients (51.7%; 3297/6381) were children of ≤ 5 years. 1967 (30.8%) were positive for hadv with 1561 (79.4%) found in co-infection with at least one another respiratory virus. the most common co-detections were with influenza viruses (53.1%; 1045/1967), rhinoviruses (30%; 591/1967), enteroviruses (18.5%; 364/1967) and rsv (13.5%; 266/1967). children under 5 were the most infected group (62.2%; 1224/1967; p <0.05). we noted that hadv was detected throughout the year at a high level with detection peaks of different amplitudes without any clear seasonality. phylogenetic analysis revealed species hadv-c in majority, species hadv-b and one hadv4 genome type. the 9 hadv-b species like strains from senegal grouped with genome types hadv-7, hadv-55 and hadv-11 as shown by a phylogenetic branch with a high bootstrap value of (88%). conclusion: in conclusion, the results of the present study suggest strong year-round hadv activity in senegal, especially in children up to 5 years of age. molecular studies revealed that the dominant species in circulation in patients with ili appears to be hadv-c and hadv-b species. the circulation of though hadv-7 and hadv-55 genome types is of note as these serotypes are recognized causes of more severe and even fatal acute respiratory infections. from january 2012 to december 2015 swabs were collected from consenting ili outpatients. adenoviral detection is performed by rrt-pcr with the anyplex™ ii rv16 detection kit (seegene) and molecular characterization was performed using a partial hexon gene sequence. 6381 samples were collected. more than half of patients (51.7%; 3297/6381) were children of 5 years. 1967 (30.8%) were positive for hadv with 1561 (79.4%) found in co-infection with at least one another respiratory virus. the most common co-detections were with influenza viruses (53.1%; 1045/1967), rhinoviruses (30%; 591/1967), enteroviruses (18.5%; 364/1967) and rsv (13.5%; 266/1967). children under 5 were the most infected group (62.2%; 1224/1967; p <0.05). we noted that hadv was detected throughout the year at a high level with detection peaks of different amplitudes without any clear seasonality. phylogenetic analysis revealed species hadv-c in majority, species hadv-b and one hadv-4 genome type. the 9 hadv-b species like strains from senegal grouped with genome types hadv-7, hadv-55 and hadv-11 as shown by a phylogenetic branch with a high bootstrap value of (88%). in conclusion, the results of the present study suggest strong year-round hadv activity in senegal, especially in children up to 5 years of age. molecular studies revealed that the dominant species in circulation in patients with ili appears to be hadv-c and hadv-b a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 species. the circulation of though hadv-7 and hadv-55 genome types is of note as these serotypes are recognized causes of more severe and even fatal acute respiratory infections. human adenoviruses (hadvs) are highly contagious pathogens that are associated with a wide spectrum of human illnesses involving the respiratory, ocular, gastrointestinal, and genitourinary systems [1] . they belong to the family adenoviridae, genus mastadenovirus with seven species (a-g), including each various types [2] . ubiquitous in the environment, hadvs are non-enveloped, double stranded dna viruses that vary in size from 70 to 100 nm [3] . hadvs are recognized as a common cause of respiratory infection in persons of all ages. the illnesses range from influenza-like fever and discomfort to pneumonia and death [4] . indeed, hadvs infections are usely mild but some groups such as very young children, elderly, immunocompromised persons, or persons with underlying pulmonary or cardiac disease, might be at higher risk degree for severe disease [5, 6, 7, 8] . the most common hadvs species that cause respiratory tract infections in children are b (hadv-b3 and b7) and c (hadv-c1, c2, and c5). serotypes b3, b7, and b21 are the most frequent strains responsible for epidemics of acute febrile respiratory disease [9] . circulating hadvs can vary temporally and geographically with possibility of emergent genomic variants which can be associated with more severe illness [10, 11] . in the present study, we investigate the epidemiologic and viral molecular features of hadvs circulating in senegal after 4 consecutive years of sentinel surveillance of influenzalike illness cases. from january 2012 to december 2015 we collected specimens (nasal-pharyngeal and oral-pharyngeal swabs) and surveillance data for influenza and other viral respiratory pathogens from outpatients presenting with influenza-like-illness (ili) at different sentinel sites in senegal. once collected, swabs are placed in 2-ml cryovials with viral transport medium (universal transport medium; copan diagnostics inc., murrieta, ca), and transported at a controlled temperature of 2˚c-8˚c to the laboratory. an ili patient was defined as a person presenting with sudden onset of fever (>38˚c) or history of sudden onset of fever in the recent past ( 3 days) and either cough or sore throat and/or rhinorrhea in the absence of other diagnosis, according to the cdc case definition. each sample is accompanied by a case report form collecting demographic and clinical data. the questions included information on date of enrollment and symptom onset, sex, age, clinical symptoms, previous treatments, travelling history, vaccination status for influenza, and whether or not the patient was hospitalized. upon arrival at the laboratory, the specimens were processed immediately for virus diagnosis. aliquots of samples were also stored at −80˚c for additional analysis (isolation and/or molecular characterization). the data obtained daily were entered into an epi info database (centers for disease control and prevention, atlanta, ga) and analyzed using epi info. total viral nucleic acid (dna and rna) was extracted from 140 μl of each clinical specimen using the purelink™ viral rna/dna mini kit (invitrogen, carlsbad ca, usa) according to the manufacturer's recommendation. dna/rna are eluted with 60 μl nuclease-free water and stored at −80˚c until use. a two-step multiplex real-time rt-pcr was performed with a bio-rad cfx-96 thermocycler (bio-rad laboratories) and the anyplex™ ii rv16 detection kit (seegene) for a simultaneous testing of influenza viruses (flua and flub), human respiratory syncytial virus (rsva and rsvb), human adenoviruses (hadv), human metapneumovirus (hmpv), human coronavirus (229e, nl63, oc43), human parainfluenza virus (piv1, -2, -3 and -4), human rhinovirus (hrv), human enterovirus (hev) and human bocavirus (hbov), as previously described [12] . in consideration with low ct-values, 80 hadv positives samples (20 per year) were selected using a random number generator on ms excel for further molecular characterization using classical pcr and sequencing. viral dna was extracted as previously described and eluted with 50 μl water nuclease-free. dnas were stored at −20˚c until pcr reactions. for hadv molecular characterization the last 300 base pairs (bp) of the hexon gene were amplified with the following specific primers: adeno3 (5'-cctttggcgcatcccattct-3') and adeno4 (5'-tgggcacctatgacaagcgc-3') previously used by garcia et al, [13] . the phusion high-fidelity pcr master mix with hf buffer (new england biolabs, ipswich ma, usa) was used for amplifications. for each sample, pcr was carried out in a total reaction volume of 50 μl consisting of 15 μl h2o rnase free, 2.5 μl of each primer (diluted at 10μm), 25 μl of 2x phusion master mix and 5 μl of dna template. cycling conditions were as follows: denaturation step of 15 min at 95˚c, 40 pcr cycles including 30 s at 95˚c, 60 s at 55˚c, 60 s at 72˚c followed by an extension step of 10 min of 72˚c. five microliters of the pcr product was then mixed with 1 μl of 10x 5prime loading dye and loaded on to a 1% agarose gel along with an appropriated molecular weight markers (100 bp ladder, new england biolabs), and gels were stained with ethidium bromide (0.5 μg/ml) before visualization under uv. for positive samples (380 bp size band), amplicons were cut and purified using the gene-jet gel extraction kit (thermo scientific). purified products are then sent for sequencing to beckman coulter services. sequencing was performed in both directions with the same pcr primers (adeno3 and adeno4) on an abi prism bigdye terminator v3.1 ready reaction cycle sequencing kit (applied biosystems) on a 96-capillary abi prism 3730-xl (applied biosystems). data in fasta format were then sent to the laboratory for analysis. sequences successfully obtained were aligned with representative genbank sequences of previously published genotypes using the bioedit sequence alignment editor [14] . the search for sequence similarities were carried out using the basic local alignment search tool (blastn) from ncbi blast web portal. phylogenetic trees were performed in mega 6 software [15] using the neighbor-joining method, and the statistical significance of the tree topology tested by bootstrapping (1,000 replicates). the evolutionary distances were derived using the tamura-nei method. bootstrap replicates with values !70 are shown on the trees. statistical analysis. regarding hadv infection comparisons between age groups were performed using the fisher's exact test. p value < 0.05 was considered statistically significant and the 0-5 year age group was used as reference group. hadv mono-infections were also compared to hadv co-infections. the r.3.0.1 tool was used to perform the analyses. this study is a component of the 4s network syndromic surveillance [12] . the principles of the 4s network were approved by the ministry of health in its guidelines for influenza surveillance policy, finalized with the support of pasteur institute in dakar and the strengthening influenza sentinel surveillance in africa (sisa) project funded by the who. the protocol and oral consent were determined as routine surveillance activity, and therefore non-research by the senegalese national ethics committee and the steering committee for 4s network, an entity representing moh, ipd, who and clinicians in compliance with all applicable national regulations governing the protection of human subjects. data were collected in an objective of surveillance and are anonymous. the information provided to participants was an informal description of the study. respiratory specimens were collected, only after informed consent was granted, verbally, to local health care workers by the patients or parents in the case of minors. oral consent was documented in the patient form with two questions about received information and about oral consent. patients could refuse to participate, no specimen will be taken. for the surveillance activities, written consent is judged not necessary by the senegalese national ethics committee, which has also previously approved the work of the national influenza center. collections of non-sensitive data or an observation from normal care in which participants remain anonymous do not require ethics committee review. the patients included in this study were of all ages and consulted the sentinel sites due to influenza-like symptoms; the patients, or parents in the case of minors, accept the tests for respiratory viruses largely because they are free and safe. of 6381 specimens tested, 1967 (30.8%) were positive for hadv (table 2) . detection rates over the study period are almost similar in the first 3 years (2012, 2013 and 2014) while in 2015 there is a marked decrease in adenoviral infections. the mean age of infected patients was 8 years 7 months and median age was 3 years. regarding the viral detection per age group, most of hadv infected cases (62.2%; 1224/1967) were under 5 years patients, a statistically significant finding (p <0.05). however, the detection rates in the other groups including the elderly (above 50 years old) remain high. no significantly gender distribution of adenoviral infection was observed. the comparison of symptoms prevalence between ili patients with adenoviral infection and patients without adenoviral infection showed that cases of myalgia (p = 0.0014), cough (p = 0.0028), diarrhea (p < 0.001), rhinitis (p < 0.001) and headache (p = 0.01) are significantly higher in patients infected by adenoviruses ( table 3 ). the fig 1 shows the temporal distribution of hadv positivity rate per month in senegal from 2012 to 2015. we noted that hadv was detected throughout the year at a high level with detection peaks of different amplitude. the highest peak, with 62% of detection rate, was recorded on december 2013. hadv circulation pattern shows no seasonality even if results suggest a higher activity of these viruses during cold periods. it should be pointed out that the cold periods (between december and february) experience some instability in senegal with possibilities of shifting. for phylogenetic analysis, we were able to obtain the partial hexon gene sequence from 54 hadv-positive samples: 8 were from samples in 2012, 13 from 2013, 11 from 2014, 16 from 2015 and 6 from 2016. unfortunately, some samples showed no amplification or poor-quality sequences. the low sensitivity of conventional pcr compared with real time pcr on samples with low viral load, and certainly non-specific amplifications could be the cause of these failures. the nucleotide sequence alignment clustered the majority of senegalese isolates into hadv-c species (44/54). 9 isolates grouped with hadv-b species and the remaining isolate, from 2012, seems close to the hadv-4 genome type belonging to the hadv-e species. in all cases bootstrap values are high (more than 85%). within the hadv-c species, 16 senegalese isolates are grouped with the type hadv-6 (36.4%); 2 isolates with hadv-2 type (4.5%), 4 with hadv-5 type (9%), and 22 isolates formed a subcluster with hadv-1 and 57 types (fig 2) . the 9 hadv-b like species from senegal grouped with genome types hadv-7, hadv-55 and hadv-11 as shown by a phylogenetic branch with a high bootstrap value of (88%). we also noted that this dominance of species c and b is confirmed over the years. in this four-year retrospective study, we characterized hadv isolates derived from an ili surveillance program conducted as collaboration between pasteur institute of dakar and the senegalese ministry of health between 2012 and 2015. it is the first nationally molecular epidemiology investigation of hadvs and even in west africa. our study suggests that hadv are strongly associated with ili syndrome in senegal with an overall detection rate of 30.8% among 6381 patients. this rate seems very higher in comparison with available data. indeed, much lower rates are reported in similar other studies conducted in other countries. a study conducted in kenya [16] on refugees from different countries (somalia, sudan, ethiopia and kenya) yielded a detection rate of 21.7%, in gabon douki et al [17] detected hadv in 16.3% of outpatients with ili. these detection rates are still lower in other geographical regions: south korea with 10.1% or 0.6% [18, 19] , china with 2.7% or 6.3% [20, 21] , philippines with 0.9% [22] , malaysia less than 2% [23], usa with 5.7% or 2.8% reported [24, 25] , canada in the ontario provence with 0.9% [26] , peru with 6.2% [27] , venezuela with 1.6% [28] , england with 6.6% [29] . the analysis of these data tends to confirm a higher prevalence of adenoviruses in the respiratory sphere in african populations. this trend was largely confirmed when we investigated the importance of hadv in children with acute respiratory infections. indeed we observed that proportions in cameroon (27.3%) [30] and senegal (29.2%) (fall et al, on submission) were considerably higher than those found in other geographical areas: nascimento-carvalho et al., [31] in brazil with 3%, moe et al., [32] in norway (1.7%), wansaula et al., [33] in usa (1%) or lu et al., [34] in china. however, these discrepancies in hadv detection rates can be also due to differences in technical approaches, virus burden geographical differences, the number of patients tested, the periods during which samples were collected and even the duration of the study. it should be also noted that adenoviral detection does not necessarily prove disease causation as coincidental upper airway infection, asymptomatic viral carrier state [35] , or prolonged shedding [36] in a previous infection could explain adenoviral detection. regarding the group age, as expected, results showed that most patients with hadv infection were younger than 5 years (62.2%), a statistically significant finding. these results are in concordance with those of other studies which findings concluded that most children are infected by adenovirus at an early age [25, 37, [38] [39] [40] [41] . indeed, it is well established that by 5 years of age, 70% to 80% of children demonstrate antibodies to at least one serotype [42] . additionally more than 80% of diagnosed hadv infections occur in children < 4 years old (due to lack of humoral immunity) [43] . although most cases exhibit low to mild symptoms are and indistinguishable from other viral causes, acute respiratory infections caused by hadv can be severe [44] , or even fatal [7, 45] , and are associated with the highest risk of long term respiratory sequelae [46] . consistent with the report from many other studies, results here showed that 79.4% of hadv infected participants were co-infected with one or more other respiratory tract viruses. the most frequently co-detected viruses were influenza viruses (53.1%), rhinoviruses (30%), enteroviruses (18.5%) and rsv (13.5%). however, we noted no significant differences in clinical characteristics and laboratory findings between patients with single hadv infection and those co-infected. the same observation was reported in studies conducted in diverse geographical contexts [27, 41] . a previous study conducted in chilean children stated that the clinical severity in patients with single hadv infection and those with mixed infections was the same [47] . the overall finding is that the clinical value of such co-infections is not clear and still requires independent investigations in order to assess the association between co-infection and severe illness or symptoms. regarding the four years of surveillance, hadv circulation pattern shows no clear seasonality even if results suggest a higher activity of these viruses during cold periods. this lack of seasonality of hadv infection has been largely reported elsewhere [27, 48, 49] . however, seasonal peaks for hadv infection were noted in summer in some china areas [50] or in spring in northern china [51] , mexico [52] and taiwan [53] . in our study, the last 300 bp region of the hexon gene were used for molecular studies of the different hadv isolates. phylogenetic analysis showed that among the 54 sequenced strains hadv-c species were the most common hadv detected (81.5%) in patients with ili in senegal from 2012 to 2016. despite some divergences, the strains from senegal were close to types 1, 2, 5, 6 and 57. this hadv-c species predominance was reported in malaysia [23], in italy [54] , in many latina america countries [27, 52, 55] in contrast with studies done in the united states of america [56] , united kingdom [37] , korea [57] , in argentina [58] and china [59] , where hadv-b species were the most commonly isolated hadv. hadv-b species were the second most common in senegal with 9 strains, and only one type belonging to hadv-e species was sequenced. hadv-b species from senegal clustered with genome types hadv-7, hadv-7d, hadv-55 and hadv-11 (88% bootstrap value). hadv-7d serotype, firstly identified in 1980 in beijing [59] , is of particular concern as it was often associated with illnesses presenting with more severe and higher levels of morbidity than other respiratory hadv pathogens, and also may result in higher levels of fatalities [60] [61] [62] . the hadv-55 genome type, formerly known as hadv-11a, is a genotype resulting from recombination between hadv-11 and hadv-14 [63] . the serotype has recently reemerged as a highly virulent pathogen, causing severe [64] and sometimes fatal pneumonia among immunocompetent adults, particularly in asia [65] [66] [67] . so the circulation of such hadv genome types in senegal emphasizes the need to reinforce hadv surveillance, especially in hospitalized patients, by including hadv genome detection and genotyping in the documentation of severe respiratory infections. the single hadv-e species strain was typed as hadv-4, the unique human type in this species, which is more commonly associated with high rates of febrile respiratory illness in us military recruits [68] though associated with viral conjunctivitis outbreak in australia [69] for example. we observed some limitations in our study. first, considering the vast number of hadv positive samples, only a small number of hadv were typed. so the sequencing results do not reflect the full spectrum of hadv strains that may be circulating in ili patients in senegal, and even for selected samples it may have a bias toward samples with a high viral load. another limitation concerned the molecular methods used for typing hadvs in this study, a method which targeted a short hexon hypervariable region that has been shown to correlate closely with serotype. this method does not provide genomic detail and might miss recombination events located in other regions of the genome. therefore, full-genome sequencing would be more informative on senegalese strains, especially for hadv-b7 and hadv-b55 types. the results of this study should also be interpreted with caution especially for hadv ili causality (carriage in healthy or asymptomatic individuals). in conclusion, the results of the present study suggest strong year-round hadv activity in senegal, especially in children up to 5 years of age. molecular studies revealed that the dominant species in circulation in patients with ili appears to be hadv-c, hadv-b species. the circulation of though hadv-7d and hadv-55 genome types is of note as these serotypes are recognized causes of more severe and even fatal acute respiratory infections. so in the interest of global public health we strongly suggest molecular surveillance and genotyping of newly detected hadv strains in senegal and even by whole genome sequencing for some especial strains. our study offers also an important perspective on the burden of adenovirus-associated respiratory illness in senegal. such a perspective, especially among children, should include asymptomatic controls, sari cases, information on disease outcome, atypical clinical signs, duration of symptoms, and treatment. data regarding viral load, shedding, and other possible etiologies (e.g., bacterial and other viruses) would also enable a more thorough assessment of the viral effective disease (or symptom) causality. adenovirus infections in immunocompetent and immunocompromised patients family adenoviridae structural studies on adenoviruses worldwide epidemiology of human adenovirus infections adenovirus infections in transplant recipients clinical severity of respiratory adenoviral infection by serotypes in korean children over 17 consecutive years (1991-2007) molecular epidemiology of human adenovirus isolated from children hospitalized with acute respiratory infection in são paulo, brazil outbreak of adenovirus type 4 infection in a long-term care facility for the elderly rapid subgenus identification of human adenovirus isolates by a general pcr adenovirus type 7 genomic-type variant emergent us adenovirus 3 strains associated with an epidemic and serious disease influenza-like illnesses in senegal: not only focus on influenza viruses molecular characterization of adenovirus circulating in central and south america during the 2006-2008 period bioedit: a user-friendly biological sequence alignment editor and analysis program for windows 95/98/nt mega6: molecular evolutionary genetics analysis version 6.0 epidemiology of respiratory viral infections in two long-term refugee camps in kenya viral etiology and seasonality of influenza-like illness in gabon epidemiology of respiratory viral infection using multiplex rt-pcr in laboratory surveillance of influenza-like illness in seven teaching hospitals the clinical and etiological characteristics of influenza-like illness (ili) in outpatients in temporal changes in respiratory adenovirus serotypes circulating in the greater toronto area adenovirus respiratory tract infections in peru sentinel surveillance of influenza-like illness in two hospitals in respiratory viral infections during the 2009-2010 winter season in central england, uk: incidence and patterns of multiple virus co-infections viral etiology of severe acute respiratory infections in hospitalized children in cameroon the role of respiratory viral infections among children hospitalized for community-acquired pneumonia in a developing country respiratory virus detection and clinical diagnosis in children attending day care surveillance for severe acute respiratory infections in southern arizona epidemiology of human respiratory viruses in children with acute respiratory tract infections in jinan, china respiratory viral infection in lower airways of asymptomatic children viral and other infections of the human respiratory tract the epidemiology of adenovirus infections in greater manchester, uk 1982-96 estimates of world-wide distribution of child deaths from acute respiratory infections viral infections of the lower respiratory tract: old viruses, new viruses, and the role of diagnosis molecular identification of adenoviruses associated with respiratory infection in egypt from adenovirus infection in children with acute lower respiratory tract infections in beijing, china feigin rd, cherry jd. philadelphia: wb saunders adenovirus: epidemiology, global spread of novel serotypes, and advances in treatment and prevention. semin respir crit care med a community-derived outbreak of adenovirus type 3 in children in taiwan between fatal pneumonia associated with adenovirus type 7 in three military trainees long term sequelae from childhood pneumonia; systematic review and meta-analysis adenovirus and respiratory syncytial virus-adenovirus mixed acute lower respiratory infections in chilean infants comparative dynamics, morbidity and mortality burden of pediatric viral respiratory infections in an equatorial city community-acquired pneumonia requiring hospitalization among u.s. children molecular identification and epidemiological features of human adenoviruses associated with acute respiratory infections in hospitalized children in southern china molecular typing and epidemiology profiles of human adenovirus infection among paediatric patients with severe acute respiratory infection in china adenoviruses c in nonhospitalized mexican children older than five years of age with acute respiratory infection molecular and clinical characteristics of adenoviral infections in taiwanese children in 2004-2005 epidemiology and clinical characteristics of respiratory infections due to adenovirus in children molecular characterization of adenoviruses from children presenting with acute respiratory disease in the seattle virus watch. iv. observations of adenovirus infections lower respiratory tract infections due to adenovirus in hospitalized korean children: epidemiology, clinical features, and prognosis molecular typing of adenoviruses in pediatric respiratory infections in molecular epidemiology of adenovirus types 3 and 7 isolated from children with pneumonia in beijing molecular epidemiology of adenovirus type 7 in the united states re-emergent human adenovirus genome type 7d caused an acute respiratory disease outbreak in southern china after a twenty-one year absence fatal community-acquired pneumonia in children caused by re-emergent human adenovirus 7d associated with higher severity of illness and fatality rate. sci rep characterizing, typing, and naming human adenovirus type 55 in the era of whole genome data severe pneumonia associated with adenovirus type 55 infection outbreak of febrile respiratory illness associated with adenovirus 11a infection in a singapore military training camp epidemiology of human adenovirus and molecular characterization of human adenovirus 55 in china fatal pneumonia cases caused by human adenovirus 55 in immunocompetent adults respiratory diseases among u.s. military personnel: countering emerging threats outbreak of adenovirus type 4 conjunctivitis in south australia this study would not have been possible without the excellent support from all the health-care workers of the 4s network who contributes, every day, to the surveillance network. we convey our special thanks to kathleen victoir from the international network of pasteur institutes for her unwavering support to the 4s network. we acknowledge the senegalese ministry of health for their help in implementing the 4s network. key: cord-300510-fhpkdqr0 authors: mojoli, francesco; mongodi, silvia; orlando, anita; arisi, eric; pozzi, marco; civardi, luca; tavazzi, guido; baldanti, fausto; bruno, raffaele; iotti, giorgio antonio title: our recommendations for acute management of covid-19 date: 2020-05-08 journal: crit care doi: 10.1186/s13054-020-02930-6 sha: doc_id: 300510 cord_uid: fhpkdqr0 nan disease, with cough, fever and flu-like syndrome, evolving to dyspnoea after 2-10 days and presenting with bilateral chest infiltrates. blood gas analysis initially shows moderate hypoxaemia, metabolic acidosis with/without respiratory compensation, normal lactates and increased anion gap; ketoacids are found in urinary sticks. blood samples show high c-reactive protein, normal procalcitonin, increased lactate dehydrogenase, creatine phosphokinase, amylases, lipases and hyperglycaemia. a nasal swab for 2019 novel coronavirus is routinely performed in any upper/lower airways disease [2] [4] . for this same purpose, add a highefficiency particulate air filter before the positive end-expiratory pressure valve or, better, connect the valve to wall gas aspiration [6] [7] [8] . 7. perform early intubation if poor response to continuous positive airway pressure in terms of oxygenation: do not trust patients' relatively good respiratory mechanics and feeling of improved dyspnoea, since these patients may have relatively normal lung compliance and the only clinical sign of fatigue may be high respiratory rate. connect ventilator expiratory valve to wall gas aspiration to limit droplets' spread. 8. once intubated, perform a closed system bronchoalveolar lavage to confirm diagnosis: minimize the use of fiberbronchoscopes to limit airways' opening; we connect a bronchoalveolar lavage test tube to the closed aspiration system-mandatory in these patients-for deep bronchial sampling. thereafter, repeat the sampling every 7 days for viral charge assessment and bacterial over-infection detection [4] . 9. after intubation, evaluate basic lung mechanics: it usually shows a respiratory system compliance of 0.5-1 ml/cmh 2 o per kilogramme of predicted body weight with high recruitability at pressure-volume curve and normal resistances. these patients usually show good response to high positive end-expiratory pressure levels; calibrated oesophageal pressure may help its setting [9] . consider neuromuscular blocking agents if deep sedation does not control the patient's trigger and ventilation is not protective; perform daily a trial of neuromuscular blocking agents stop. it is accurate in interstitial diseases and may show pathological signs before chest x-ray. a basic assessment helps deciding the ventilatory strategy: if diffuse loss of aeration, keep high positive endexpiratory pressure levels; if posterior consolidations, consider pronation. lung ultrasound may also help in limiting traditional imaging, avoiding patients' transportation to radiology department. it also allows a daily monitoring of clinical evolution, response to treatment and possible complications (pneumothorax, over-infections) [10] [11] [12] . 11. avoid positive fluid balance: perform fluid challenges and stop fluid resuscitation if no haemodynamic response; use vasoactive drugs instead to optimize tissue perfusion [4] . we accept moderate elevation of creatinine without urinary output impairment to improve the lung status. 12. fever is a frequent issue, reaching values as high as 40°c; we decided to treat it only if > 39°c, if oxygenation is acceptable. spontaneous defervescence can be the first sign of clinical improvement. 13. as soon as possible according to gas exchanges (pao 2 /fio 2 > 150 with fio2 < 50%) and lung ultrasound score (≤ 12), start assisted ventilation with a sigh while maintaining moderate to high positive end-expiratory pressure to prevent derecruitment. regularly check patient's respiratory drive (p0.1), tidal volume and plateau pressure to keep ventilation safe. dexmedetomidine may help in the weaning phase. 14. in patients having received prolonged sedation, we frequently observed prolonged awakening with altered respiratory drive and difficult patientventilator interaction; if no prompt awakening is observed, perform early tracheostomy to accelerate the weaning and discharge from icu. the number of patients requiring intensive care rapidly increases; therefore, rapid discharge is mandatory. 15. hyperinflammatory status increases the risk of thrombosis and pulmonary embolism; check for thrombotic complications systematically, mainly in correspondence of central lines [13, 14] . 16. communication with families is difficult since patients' relatives are frequently in quarantine and access to the hospital is limited; moreover, while wearing personal protective equipment, physicians' possibility to answer to relatives' phone call is limited. consider identifying each day one person in charge of phone calls to daily update relatives on clinical conditions [15] . we hope sharing our experience while facing the italian outbreak of 2019 novel coronavirus may help other units eventually facing the same threat in the future. abbreviations sars-cov-2: severe acute respiratory syndrome coronavirus 2; icu: intensive care unit critical care utilization for the covid-19 outbreak in lombardy, italy: early experience and forecast during an emergency response characteristics of and important lessons from the coronavirus disease 2019 (covid-19) outbreak in china: summary of a report of 72 314 cases from the chinese center for disease control and prevention severe sars-cov-2 infections: practical considerations and management strategy for intensivists surviving sepsis campaign guidelines on the management of critically ill adults with coronavirus disease 2019 (covid-19) rapid response to covid-19 outbreak in northern italy: how to convert a classic infectious disease ward into a covid-19 response centre effect of noninvasive ventilation delivered by helmet vs face mask on the rate of endotracheal intubation in patients with acute respiratory distress syndrome: a randomized clinical trial critical care management of adults with community-acquired severe respiratory viral infection clinical management of severe acute respiratory infection when novel coronavirus (2019-ncov) infection is suspected: interim guidance 28 esophageal and transpulmonary pressure in the clinical setting: meaning, usefulness and perspectives lung ultrasound for critically ill patients assessment of lung aeration and recruitment by ct scan and ultrasound in acute respiratory distress syndrome patients lung ultrasound for early diagnosis of ventilator-associated pneumonia risk factors associated with acute respiratory distress syndrome and death in patients with coronavirus disease 2019 pneumonia in wuhan, china thrombotic events in sars-cov 2 patients: an urgent call for ultrasound screening setup of a dedicated coronavirus intensive care unit: logistical aspects publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations we thank all the healthcare professionals involved in the management of such epidemics, in particular nurses and physicians of intensive care unit at s. matteo hospital. authors' contributions all the authors actively contributed to the conception, redaction and final revision before submission of the manuscript. the authors read and approved the final manuscript. availability of data and materials not applicable ethics approval and consent to participate not applicable key: cord-293354-55nawxos authors: kenmoe, sebastien; tchendjou, patrice; vernet, marie‐astrid; moyo‐tetang, suzie; mossus, tatiana; njankouo‐ripa, mohamadou; kenne, angeladine; penlap beng, véronique; vabret, astrid; njouom, richard title: viral etiology of severe acute respiratory infections in hospitalized children in cameroon, 2011–2013 date: 2016-05-09 journal: influenza other respir viruses doi: 10.1111/irv.12391 sha: doc_id: 293354 cord_uid: 55nawxos background: severe acute respiratory illness (sari) is recognized as an important cause of morbidity, mortality, and hospitalization among children in developing countries. little is known, however, in tropical countries like cameroon about the cause and seasonality of respiratory infections, especially in hospitalized settings. objectives: our study investigates the viral etiology and seasonality of sari in hospitalized children in yaounde, cameroon. methods: prospective clinic surveillance was conducted to identify hospitalized children aged ≤15 years presenting with respiratory symptoms ≤5‐day duration. demographic and clinical data, and respiratory specimens were collected. nasopharyngeal samples were tested for 17 respiratory viruses using a multiplex polymerase chain reaction. the viral distribution and demographic data were statistically analyzed. results: from september 2011 through september 2013, 347 children aged ≤15 years were enrolled. at least one virus was identified in each of 65·4% children, of which 29·5% were coinfections; 27·3% were positive for human adenovirus (hadv), 13·2% for human respiratory syncytial virus (hrsv), 11·5% for rhinovirus/enterovirus (rv/ev), 10·6% for human bocavirus (hbov), 9·8% for influenza virus (inf), 6·6% for human parainfluenza virus (hpiv), 5·7% for human coronavirus (hcov), and 2·3% for human metapneumovirus (hmpv). while hrsv showed seasonal patterns, hadv and rv/ev were detected throughout the year and no evident temporal patterns were observed for the remaining viruses. conclusion: respiratory viruses were associated with a high burden of hospitalizations among children in cameroon. nevertheless, additional studies evaluating asymptomatic cameroonian children will be important in understanding the relationship between viral carriage and disease. severe acute respiratory illness (sari) is the leading cause of hospitalization, morbidity, and mortality in children younger than 5 years throughout the world. the burden of these infections is particularly important in developing countries with a meta-analysis estimating that 265 000 (95% ci 160 000-450 000) in-hospital deaths took place in 2010 with 99% of those deaths occurring in developing countries. 1 although there is little information on the causes of these respiratory illnesses in developing countries, available data indicate that viral agents play an important role globally. 2 a meta-analysis estimates that hrsv causes 33á8 million infections each year among children younger than 5 years. about 3á4 million of these cases require hospital admission, and an estimated 66 000-199 000 of children die with 99% of deaths occurring in developing countries. 3 many other viruses such as influenza viruses, rhinoviruses (rv), human parainfluenza viruses (hpiv), human adenoviruses (hadv), human coronaviruses (hcov-oc43, hcov-229e, hcov-nl63, and hcov-hku1), and enteroviruses (ev) have also been implicated worldwide. 2, 4, 5 discovered recently, human metapneumoviruses (hmpvs) and human bocaviruses (hbovs) are viruses resulting in similar symptoms. other emergent viruses such as sars-cov and mers-cov are responsible for more severe symptoms like respiratory distress syndrome. [6] [7] [8] real-time reverse-transcription polymerase chain reaction (rrt-pcr) assays have been shown to be a sensitive and specific tool for the detection of these viruses. furthermore, the introduction of multiplex rrt-pcr enables the set-up of assays detecting two or more targets in single clinical specimen. 9, 10 in 2007, the centre pasteur du cameroun (cpc) began implementing sentinel surveillance for influenza in cameroon. this surveillance system has enabled a better understanding of the epidemiology of influenza in the country. 11, 12 even though our recent publication has demonstrated that those respiratory viruses play an important role in patients consulting our sentinel sites, 13 there remains a lack of information regarding patients hospitalized with sari. this study was conducted to gain new insights into the timeliness of virus circulation and viral etiology among children aged ≤15 years, hospitalized for sari. this prospective observational study was conducted in the pediatric service of 'centre hospitalier d'essos' in yaounde, cameroon. this central region has an equatorial climate with a defined moderate rainfall during march-june and intense precipitation during september-november. children aged less than or equal to 15 years hospitalized for sari were daily recruited in our study. a total of 347 patients were enrolled by experienced nurses or physicians during a 2-year period between september 2011 and september 2013. sari was defined according to a previously suggested who case definition 14 with a history of symptoms of <5 days. these criteria of hospitalized patient inclusion were as follows: fever >38°c and cough and/or sore throat. children were enrolled only after an informed written consent was obtained from their parents or legal guardians. this study was part of a global study aimed at assessing risk factors associated with severe influenza in cameroon (immi project), which was reviewed and approved by the national research ethics committee and the ministry of health of cameroon. demographic and clinical information was obtained from participants using a standardized questionnaire. nasopharyngeal swab specimens were collected from hospitalized children and transported to the cpc as previously described. 13 viral rna and dna were extracted using qiaamp viral rna mini kit and qiaamp dna mini kit (qiagen, hilden, germany), respectively, following the manufacturers' instructions. a final elution volume of 60 ll of rna and 200 ll of dna were stored at -80°c till testing by multiplex pcr/rt-pcr. testing for influenza a and b viruses were conducted, and influenza a virus-positive specimens were subtyped according to the method developed by us centers for disease control and prevention (cdc). 15 then, the detection of 14 non-influenza respiratory viruses, including hmpv, hrsv, hpiv (1-4), ev, rv, hcov-oc43, hcov-229e, hcov-nl63, hcov-hku1, hadv, and hbov were performed by 4 duplex rrt-pcr, using a commercially available respiratory multi well system r-gene tm (biom erieux, lyon, france) following the manufacturer's instructions. pcr amplifications were performed on abi 7500 fast (applied biosystems, foster city, ca, usa). a patient was considered to have a single viral infection if only one pathogen was detected in the specimens. in case of more than one viral pathogen detected, patients were considered to have viral coinfections. the data obtained and the laboratory results were anonymized and entered into a database prepared with microsoft â excel (microsoft, washington, dc, usa). the database was then checked and cleaned for abnormal wrongful entries. we analyzed the demographics of the study subjects and the positive cases, as well as the clinical characteristics of all respiratory viruses. proportions were compared using a chisquared or the fisher exact test. we analyzed the clinical and coinfection characteristics of positive according to the negative patients for respiratory viruses using a stepwise logistic regression model. uncertainty was expressed as 95% confidence intervals (cis). statistical analysis was performed with the r program version 2á15á1, and p-values of 0á05 or less were considered statistically significant. during the study period, 374 hospitalized children with sari aged 0-15 years were enrolled. as 27 patients were excluded due to insufficient material for analysis of all viruses, 347 (92,8%) subjects were analyzed. the median age of the patients was 1á5 years (iqr: 0á75-3á0 years). most patients (88á4%) were below 5 years of age, with 131 (37á8%) children ≤1 year, 100 (28á8%) children aged [1] [2] years, 76 (21á9%) children aged [2] [3] [4] [5] years, and 40 (11á5%) children aged [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] years. half of patients (180 [51á9%]) were male. among the 347 hospitalized participants with sari, the most common respiratory symptoms were cough (85á9%, 298/ 347), rhinorrhea (78á4%, 272/347), fatigue (57á9%, 201/347), and wheezing (41á8%, 145/347). demographic data and clinical characteristics of the 347 cases studied are summarized in table 1 . a majority of sari was observed among children aged ≤5 years (90á7%, 206/227), and the proportions infected were significantly negatively associated with age (odds ratio [or], 0á91; 95% ci, 0á84-0á99, p = 0á03). there were no significant differences in terms of gender between patients infected with different viruses. influenza virus were more frequently detected in the individuals over 1 year (p = 0á04). for the remaining viruses, there was no difference between rate of infection and age (table 2) . among positive samples, single viral infection accounted for 70á5% (160/227) of cases, whereas infections with multiple viruses were observed in 29á5% (67/227), including 53 double infections, 13 triple infections, and one quadruple infection. specific viruses detected in the study population of 347 hospitalized children are listed in table 3 , in descending detection rate. the mean age and age group distribution of patients with multiple viral infections were not significantly different from those of group with single infection (table 2) . hadv, hrsv, influenza and hmpv were more frequent in single infections whereas rv/ev, hbov, hpiv, and hcov were more frequent in mixed infections ( figure 2 ). in a binary logistic regression, positive samples was negatively associated only with headaches (p = 0á003, odds ratio [or], 0á08; 95% ci, 0á01-0á3). no other clinical symptoms were associated with the positive patients, nor with any other specific viruses detected or coinfections.seasonality. the study also investigated the monthly distribution of viruses. the seasonal variations of the viral respiratory infections are shown in figure 2. in the first period from september 2011 to november 2012, the average number of samples collected monthly was 18á7, whereas in the second period from december 2012 to september 2013 the number of enrolled cases was much lower, the average number of samples collected monthly being 6á6. however, hrsv showed a pronounced seasonality, with peaks during the rainy season from september to december. the circulation of influenza viruses was similar to hrsv although a few cases were detected outside the rainy season peak. rv/ev was detected almost year-round with peak activity in the rainy season between september and december corresponding to a higher number of samples collected during that period. on the other hand, hadv occurred throughout the study period with no evident temporal patterns. hbov occurred intermittently all over the study period, while the prevalence of remaining viral etiologies was low, ranging from 0 to 5 positive samples per month for each virus, numbers which appeared insufficient to allow for the description of seasonal patterns. coinfections were observed throughout the study period. the aim of this study has been to determine the viral panorama of sari in 374 hospitalized children during a 2year period in cameroon. the data presented here are unique, to the best of our knowledge. it represents viral etiologies (17 respiratory viral targets) in children aged ≤15 years hospitalized for sari in cameroon. more importantly, this study describes the incidence of adv and the recently discovered hbov in cameroon for the first time. 8 our results demonstrate the potential importance of respiratory viruses in the etiology of hospitalized children with sari in cameroon. of 347 samples tested, at least one respiratory virus was detected in 227 (64á5%). this detection rate is consistent with results of other etiologic studies of hospitalized children conducted in similar settings. [16] [17] [18] [19] [20] nonetheless, this proportion appears to vary largely between countries. kwofie et al. recorded a lower percentage of 25á7% in ghana 21 and khor et al. recorded 26á4% in malaysia. 22 higher percentages 94á2% in china, 23 91% in france, 24 and 90% in alaska 25 were reported by zhang et al., singleton et al., and huguenin et al., respectively. comparison among published studies is always difficult. possible reasons for these varying outcomes among studies include true differences in epidemiology, differences in the inclusion criteria for study population, diagnostic methods used, panel of viral agents tested, climate, study duration, and patients' ages. consider that all the above studies included hospitalized children; lower prevalence recorded by khor et al. might have been due to their 27-year study duration and laboratory methods (direct if and viral isolation) which are known to be less sensitive than molecular techniques. 25 the most prevalent virus detected in our study was adv, representing 27á3% of the total number of viruses observed. this value is higher than that (0á8-13á3%) reported in most other similar studies. 16, 17, 22, [26] [27] [28] it is nonetheless similar to what was noted in alaska 24 particularly. this finding shows that adv could play a more significant role than originally thought. accordingly, additional efforts describing our associated serotypes will help to further understand how much this virus contributes to disease severity. unlike our results, most similar studies showed that hrsv was the most prevalent viral pathogen. 22, [31] [32] [33] in our study, hrsv was the second most frequent pathogen after hadv with merely 13á2% of 347 hospitalized children testing positive. however, this proportion was similar to that reported in other studies (5á7-15%). 16, 28, 29, 34 unlike previous similar studies, rv/ev accounted for only 11á5% of positivity in our study. 17, 18, 24, 26, 29 hbov was the fourth most common virus with a prevalence of 10á6% in all cases. this result is in line with the results of others authors, who have reported a worldwide detection rate of hbov ranges between 2 and 19% in nasopharyngeal samples. 35 the remaining viral etiologies were identified in 9á8%, 6á6%, 5á7%, and 2á3% for influenza, hpiv, hcov, and hmpv, respectively. these results correspond with those reported by other authors. 23, 27, 32, 33, 36, 37 as previously shown, we also found that virus-associated hospitalization figures were significantly higher among children (p = 0á03). 27, 28, 38 according to reports, there is always a higher incidence of viral respiratory infection in children than in adults. this may be partially explained by the lower detection rate of respiratory viruses in the elderly due to reduced viral shedding in older age groups. 39 hrsv, the second most commonly virus detected in this study, has been reported to be the leading cause of sari in young children. 19, 22, 36 nevertheless, we did not find a significant age-specific prevalence in hrsv detection during this study. this was probably due to the fact that our statistical analysis was hampered by the limited size of study participants older than 5 years. headaches were negatively associated with positives samples (p = 0á003). however, as a majority of our study population was made up of children unable to report headaches, only 46 patients (13á3%) were taken into account in the analysis.multiple viral infections were observed in 29á5% of the patients with positive viral detection. this is a very similar percentage to the one found by others researchers. 22, 26, 37 bocavirus has been frequently implicated in coinfections. 17, 19, 25 our results reflect those of other studies in which hbov was associated (35á8% versus 8á1%, p < 0á001) with multiple infections. surprisingly, adv and picornaviruses were also most frequently involved in coinfections, which probably resulted from the fact that they were detected throughout the year and were among the most prevalent. in spite of our intensive surveillance strategy with daily recruitment, the number of inclusions was not stable over the study period. notwithstanding this lack of uniformity in number of samples collected during the first (september 2011 to november 2012) and second (december 2012 to september 2013) periods, we were able to detect a seasonal pattern thanks to the sufficient number of enrolled patients during two consecutive years. we found that rsv cases were detected mainly during the major rainfalls from september to november, which is consistent with the data in the literature. 22, 23, 31 the seasonality of influenza virus is very uncertain in tropical areas. the current study did not confirm that influenza circulated between september and november, as shown in our previous studies. 13 this fact might have contributed to the reduction in the inclusion rate during the second period due to a true change in the epidemiology of sari. indeed, that would have contributed to reduce the numbers eligible patients in the second period of the study. rv/ev and adv circulated year-round, a finding reported in other studies. 17, 23, 28 for the remaining viruses, no evident temporal patterns were observed. this study is ongoing, however, and any changes in the seasonal circulation will be detected. several points must be taken into consideration when looking at our findings. firstly, our study was conducted at only one site, which might have underestimated the overall detection rate for selected viruses. likewise, the enrolled cases may not be generalizable for the entire population of children in cameroon. subsequently, as discussed by broor et al., 38 there is no denial that some respiratory viruses can be detected in asymptomatic children 24, 40, 41 or shed for long periods of time after infection. 24, 42 hence, the role played by the viruses detected in these sari cases is less clear considering that we did not evaluate respiratory specimens from asymptomatic children without respiratory symptoms. otherwise, despite tests for a large panel of respiratory viruses we did not evaluate bacterial etiologies, due to the difficulty in obtaining adequate samples for culture. it has been shown that bacterial pathogens may have been responsible for sari symptoms 16 ; further studies will thus be required to specify their role in sari in cameroon. lastly, although nurses in charge of recruitment were well trained, they did not systematically record severe symptoms such as convulsion, stridor, chest pain, inability to drink or breastfeed, and outcomes of study participants. this limited our ability to assert the clinical case severity and outcome of our sari cases. this situation also added a limitation to this study regarding the representativeness of children included. notwithstanding its limits, this is the first study in cameroon to characterize 17 common respiratory viruses in pediatrics with sari during a 2-year consecutive period. using a multiplex rrt-pcr, we were able to identify a wide variety of viruses and their seasonal patterns. in our resource-limited setting, this process is particularly useful for selecting only the major contributors to sari and thereby enhancing our existing surveillance systems. additional studies evaluating asymptomatic cameroonian children will be important to understanding the relationship between viral carriage and disease. with this new method, we will also be able to evaluate the role of viral loads in respiratory infections in future research. global and regional burden of hospital admissions for severe acute lower respiratory infections in young children in 2010: a systematic analysis viruses in community-acquired pneumonia in children aged less than 3 years old: high rate of viral coinfection global burden of acute lower respiratory infections due to respiratory syncytial virus in young children: a systematic review and meta-analysis viral etiology of hospitalized acute lower respiratory infections in children under 5 years of age -a systematic review and meta-analysis viral etiology of acute lower respiratory tract infections in hospitalized young children in northern taiwan viral infections of the lower respiratory tract emerging respiratory agents: new viruses for old diseases? cloning of a human parvovirus by molecular screening of respiratory tract samples use of a multiplex pcr/rt-pcr approach to assess the viral causes of influenza-like illnesses in cambodia during three consecutive dry seasons advances in the laboratory diagnosis of viral respiratory disease circulation of human influenza viruses and emergence of oseltamivir-resistant a(h1n1) viruses in cameroon, central africa spatiotemporal circulation of influenza viruses in 5 african countries during 2008-2009: a collaborative study of the institut pasteur international network viral etiology of influenza-like illnesses in cameroon strategy to enhance influenza surveillance worldwide cdc protocol of realtime rtpcr for swine flu influenza a (h1n1) the role of respiratory viral infections among children hospitalized for community-acquired pneumonia in a developing country respiratory viruses from hospitalized children with severe pneumonia in the philippines respiratory viruses in hospitalized children with influenza-like illness during the h1n1 frequent detection of viral coinfection in children hospitalized with acute respiratory tract infection using a real-time polymerase chain reaction viral pathogens associated with acute respiratory infections in central vietnamese children respiratory viruses in children hospitalized for acute lower respiratory tract infection in ghana epidemiology and seasonality of respiratory viral infections in hospitalized children in kuala lumpur, malaysia: a retrospective study of 27 years viral etiology and clinical profiles of children with severe acute respiratory infections in china viral respiratory infections in hospitalized and community control children in alaska broad respiratory virus detection in infants hospitalized for bronchiolitis by use of a multiplex rt-pcr dna microarray system human bocavirus and other respiratory viral infections in a 2-year cohort of hospitalized children respiratory virus infections in hospitalized children and adults in lao pdr respiratory viral coinfections identified by a 10-plex real-time reverse-transcription polymerase chain reaction assay in patients hospitalized with severe acute respiratory illness-south africa etiology and epidemiology of viral pneumonia among hospitalized children in rural mozambique adenoviruses in the immunocompromised host detection of new respiratory viruses in hospitalized infants with bronchiolitis: a three-year prospective study lower respiratory tract infection in hospitalized children viral etiology of lower respiratory tract infections in hospitalized children in mexico rna viruses in young nepalese children hospitalized with severe pneumonia human bocavirus-the first 5 years viral etiologies of acute respiratory infections among hospitalized vietnamese children in ho chi minh city viral etiology of acute respiratory tract infections in children presenting to hospital rates of respiratory virusassociated hospitalization in children aged <5 years in rural northern india performance of diagnostic tests to detect respiratory viruses in older adults detecting respiratory viruses in asymptomatic children viral and bacterial causes of severe acute respiratory illness among children aged less than 5 years in a high malaria prevalence area of western kenya persistence of rhinovirus and enterovirus rna after acute respiratory illness in children viral etiology of sari in cameroon the authors. influenza and other respiratory viruses published by we are grateful to all the nurses and other healthcare workers who collected specimens, data, and information and provided their support with clinical evaluations of patients during this study. 'this work was funded by the international network of pasteur institutes (ptr 351) and the "institut de microbiologie et de maladies infectieuses (immi)" in france'. key: cord-284372-v95fzp8n authors: coyle, peter v; ong, grace m; o'neill, hugh j; mccaughey, conall; de ornellas, dennis; mitchell, frederick; mitchell, suzanne j; feeney, susan a; wyatt, dorothy e; forde, marian; stockton, joanne title: a touchdown nucleic acid amplification protocol as an alternative to culture backup for immunofluorescence in the routine diagnosis of acute viral respiratory tract infections date: 2004-10-25 journal: bmc microbiol doi: 10.1186/1471-2180-4-41 sha: doc_id: 284372 cord_uid: v95fzp8n background: immunofluorescence and virus culture are the main methods used to diagnose acute respiratory virus infections. diagnosing these infections using nucleic acid amplification presents technical challenges, one of which is facilitating the different optimal annealing temperatures needed for each virus. to overcome this problem we developed a diagnostic molecular strip which combined a generic nested touchdown protocol with in-house primer master-mixes that could recognise 12 common respiratory viruses. results: over an 18 month period a total of 222 specimens were tested by both immunofluorescence and the molecular strip. the specimens came from 103 males (median age 3.5 y), 80 females (median age 9 y) and 5 quality assurance scheme specimens. viruses were recovered from a number of specimen types including broncho-alveolar lavage, nasopharyngeal secretions, sputa, post-mortem lung tissue and combined throat and nasal swabs. viral detection by if was poor in sputa and respiratory swabs. a total of 99 viruses were detected in the study from 79 patients and 4 quality control specimens: 31 by immunofluorescence and 99 using the molecular strip. the strip consistently out-performed immunofluorescence with no loss of diagnostic specificity. conclusions: the touchdown protocol with pre-dispensed primer master-mixes was suitable for replacing virus culture for the diagnosis of respiratory viruses which were negative by immunofluorescence. results by immunofluorescence were available after an average of 4–12 hours while molecular strip results were available within 24 hours, considerably faster than viral culture. the combined strip and touchdown protocol proved to be a convenient and reliable method of testing for multiple viruses in a routine setting. acute respiratory tract infections are major causes of morbidity and mortality. in 2000, lower respiratory tract infections were globally the number one infectious cause of disability adjusted life-years [1] . the commonest respiratory viruses that cause acute upper and lower respiratory tract infections and which are routinely tested for in most virus diagnostic laboratories are: influenza a virus (fla); influenza b virus (flb); respiratory syncytial virus (rsv); parainfluenza virus type 1 (pf1); parainfluenza virus type 2 (pf2); parainfluenza virus type 3 (pf3) and adenovirus (adv). additionally, human rhinoviruses (hrv) and coronavirus 229e (cov-229e) are also linked to acute respiratory infection but less commonly included in laboratory reports; human metapneumovirus (hmpv) is not yet part of most united kingdom virus laboratory test repertoires (personal feed-back from the united kingdom clinical virology network). as part of service development it was necessary to provide an alternative to virus culture for testing immunofluorescence negative respiratory specimens. historically and indeed currently immunofluorescence [2] and virus culture [3, 4] are the main methods used to diagnose acute respiratory virus infections. culture is accepted as more sensitive than immunofluorescence but slower and therefore less useful for direct patient management decisions. using a standard culture technique [4] for the culture of respiratory viruses our median reporting times for culture positive and culture negative specimens were 6 days (based on 407 specimens) and 7 days (based on 2159 specimens) respectively; virus identification by this technique required the use of monoclonal antibody staining of the cell monolayer in addition to observation for viral cytopathic effect. we therefore wished to develop a test capable of reporting on immunofluorescence negative specimens within a 24 hour period. increasingly however, the sensitivity of nucleic acid amplification techniques for diagnosis has become recognised [5] [6] [7] [8] [9] [10] . however widespread concerns about contamination issues [11, 12] and perceived cost [13] have slowed their widespread adoption. an added problem for acute respiratory tract infections is the relatively large number of viruses that need to be accounted for, a problem which presents specific technical challenges. one such challenge is the different optimal annealing temperatures of the primer sets for each prospective virus target. the abi prism 7000 real-time facility from applied biosystems addresses this by using bundled software to design primer/probe combinations that use a common amplification protocol. however this approach is compromised by the inability of software to allow for target heterogeneity. in addition it does not allow users to adopt clinically validated primer sets from the literature. to address these problems we adopted an alternative approach through the development of a generic touchdown amplification protocol. touchdown protocols involve a pre-designed stepped reduction in the annealing temperature used for primer-to-template binding, which introduces a competitive advantage for specific base-pair priming over non-specific priming [14] . a detailed knowledge of the optimum annealing temperature is therefore not required. the study protocol was empirically constructed and proved robust when applied to a large range of respiratory viral and bacterial targets, without compromising individual test sensitivity. it was designed for use with in-house primer master-mixes that recognise 12 common respiratory viruses. before deciding on the layout of the molecular strip, as described in the methods, we undertook a wide range of preliminary validation steps for each primer set. the complexity of the strip makes it impossible to fully evaluate using the classical approach of applying an individual gold standard to each virus type. classically this approach works well where a single target is under investigation [15] . however although the strip is putatively designed to identify 12 viruses, the actual number of individual types targeted is over one hundred and sixty because of the inclusion of generic primer sets for hrv [16] and adv [17] respectively. the classical approach is further compounded for viruses (a) that cannot be grown or grown easily; (b) for which commercial if sera are not available; (c) for which specimen panels are not available. we therefore adopted a phased validation, culminating in the present study. sensitivity was ascribed by undertaking copy number determination on cloned targets and these ranged form 6 × 10 3 copies per ml for human rhinovirus type 1b to 4.2 × 10 3 copies/ml for rsv-a. specificity was ascribed through reproducibility, i.e. specimens which were repeatedly positive, following our standard clinical reporting algorithm [6] , were regarded as true positives; a similar approach was recently described for hmpv [18] . in addition amplicon sequencing was used as an initial specificity check. the primers sets were tested on clinical respiratory specimens arising from a number of ethically approved studies. these included respiratory specimens from patients: (a) with chronic obstructive pulmonary disease; (b) with acute asthma; (c) on assisted ventilation in intensive care. they were also tested on respiratory specimens collected as part of an influenza spotter program as well as on laboratory specimens of known virus reactivity. to test the feasibility of its routine use we needed to clinically validate its performance in a routine setting on specimens tested in parallel with our standard immunofluorescence protocol for the diagnosis of acute virus respiratory infections. although the routine immunofluoresence panel lacked capacity for the detection of rhinoviruses, human metapneumovirus and cov-229e, these were included on the strip for clinical reasons during the period of the study. these findings and their implications are reported. a total of 99 viruses were detected in 84/222 specimens from a total of 79/183 patients and 4/5 national external quality assurance scheme (neqas) controls; immunofluorescence did not detect the parainfluenza virus type 2 virus in one of the neqas specimens. viruses were detected in all of the specimen types processed. the molecular strip detected virus in: 16/36 (44.4%) bronchoalveolar lavages, 62/120 (51.6%) nasopharyngeal secretions, 11/35 (31.4%) sputa and 10/31 (32.2%) combined throat and nasal swabs. immunofluorescence detected virus in: 6/36 (16.6%) broncho-alveolar lavages, 23/120 (19.1%) nasopharyngeal secretions, 1/35 (2.8%) sputa and 1/31 (3.2%) combined throat and nasal swabs. the median age of male and female patients where virus was detected was 3 y (range 2 weeks -79 years) and 4 y (5 weeks -81 years) respectively. sixteen viruses were detected in 14/27 (51.8%) specimens, confirming a respiratory virus in 12 out of 24 (50%) patients investigated in general practice. seventy-nine viruses were detected in 70/ 191 (36.6%) specimens, confirming a respiratory virus in 67 out of 159 (42.1%) patients investigated in hospital. of the 16 viruses detected in specimens from the community, pcr detected all 16 in contrast to a single identification, influenza a (h3), by immunofluorescence. pcr identified one or more viruses in specimens from 84 of the 183 patients and the 4 neqas positive specimens, detecting a total of 99 viruses as shown in table 1 . the viruses detected were: influenza a (h3) virus (17); influenza a (h1) virus (4); influenza b virus (2); human rhinovirus (39); adenovirus (22) ; parainfluenza virus type 2 (1); parainfluenza virus type 3 (10); respiratory syncytial virus type a (2); respiratory syncytial virus type b (2);. no parainfluenza virus type 1, coronavirus 229e or human metapneumovirus were detected. dual infections were detected in 11/79 (13.9%) patients. the dual infections were: influenza a (h3) and adenovirus (4); influenza a (h3) virus and rhinovirus (2); influenza a (h1) and adenovirus (1); adenovirus and rhinovirus (3); respiratory syncytial virus type b and rhinovirus (1). nine patients had more than one specimen taken on the same day in which a virus was detected in at least one specimen by pcr. for 5 of the patients the same virus was detected in each of the 2 specimens. the viruses identified were rhinovirus (3), adenovirus (1) and parainfluenza type 3 (1); the latter was also immunofluorescence positive. in 2 cases a rhinovirus was detected in only one of the specimens. as part of a separate rhinovirus validation protocol one of these specimens was subjected to retesting coupled with limited sequencing of the 5' non-coding region amplicon which confirmed the presence of a rhinovirus sequence. additionally, premature twin boys admitted to intensive care on the same day with severe bronchiolitis, both had evidence of acute rhinovirus infection by pcr. limited sequencing of the 5' non-coding region of these viruses as part of the rhinovirus validation protocol confirmed the presence of an identical sequence of rhinovirus in both specimens. although touchdown pcr has been used successfully to help overcome some of the uncertainties associated with the thermal amplification of microbial nucleic acid targets [19] [20] [21] [22] , its use in this study has extended its role further and in so doing brought closer the goal of undertaking molecular diagnostics in a routine setting. previously its main impact has been seen where multiplexing [23, 24] or degenerate primers have been needed [25] [26] [27] and where the problems of choosing correct annealing temperatures are at their most difficult. in this study the large number of targets is the main problem encountered. using an empirical approach a series of amplification steps linked to a stepped reduction in annealing temperature from 55°c to 46°c was constructed. this proved extremely resilient when used with a wide range of primer sets and included the apparent anomaly of putting adenovirus through an initial reverse transcription step to stream line all of the targets on to a single strip; we have previously reported this approach for testing group f adenovirus alongside norovirus, astrovirus and rotavirus [28] . the touchdown surprisingly out-performed individual amplification protocols optimised for annealing temperature and thus proved suitable for use on the diverse range of respiratory viruses addressed in the study. where multiple viral targets are sought in clinical practice, we believe that it is only feasible to correlate the performance of the new assay in a head-to-head comparison with that already in routine use. unfortunately for many clinical laboratories there is an elusion of testing for a wider range of viruses than is the case, by the inoculation of cell lines with a theoretical ability to grow the respective viruses. the annual reports of most clinical laboratories of one of the commonest human respiratory viruses, human rhinovirus, is an example of this; using the touchdown protocol we now report approximately 450 hrv infections per annum. the under reporting of adenovirus by standard methods [17] and the paucity of hmpv reporting, further underlines this assertion. the ability to simultaneously validate the performance of multiple molecular primer sets in a routine clinical setting is a major accomplishment of the current methodological approach. the results demonstrated that a range of primers from both the medical literature and from in-house development could be amplified with a single generic touchdown protocol. it therefore confirmed the feasibility of directly incorporating primer sets into a standard operating procedure without the necessity for the individual optimisation of cycling parameters. as such the study results should facilitate primer selection and formal critical evaluation as here described. as an example of this enhanced flexibility we have recently replaced the primer sets for influenza a h1 and h3 (with respective copy number sensitivities of 8 × 10 3 and 2 × 10 3 copies per ml) with a generic matrix set (copy number sensitivity of 1 × 10 3 copies per ml). the use of strips containing pre-dispensed mastermixes facilitates their use in a routine setting where laboratory personnel have only to thaw the strip and add the specimen extract. we make and aliquot for routine use a large range of multi-reaction mastermixes which are repeatedly subjected to freeze-thaw cycles as required on a daily basis. provided the mixes are handled on ice, they remain extremely stable, over many months if so required. however the strip is designed for a single use only and thus only goes through a single freeze-thaw cycle. mix stability is not a problem and the single positive control is used only to confirm that the touchdown amplification cycle has run successfully. because the technique of using nested amplification followed by running agarose gel electrophoresis is relatively cumbersome, it was important to evaluate how the complete protocol, inclusive of report generation, would perform when introduced into a routine line-managed diagnostic setting. over the 18 months of the study the technique fitted in well to the demands of routine service. central to this was the use of pre-dispensed and quality checked primer master-mixes which allowed the molecular strip to be adapted for use in a routine laboratory. the study confirmed that a broad based molecular approach was feasible as an alternative to virus culture to support immunofluorescence in the diagnosis of respiratory viruses. the overall superior performance of the strip and the missed neqas specimen by immunofluorescence underlines the need for a more sensitive back-up for negative specimens. while nested protocols must be regarded as a pragmatic, interim solution until perfected single round systems are available, the format of the strip reduces the concern most attached to nested formats, i.e. false positive results. in our experience there is little evidence to support contamination arising from environmental sources and that the two major points of contamination in a nested system are (a) cross-contamination during manual extraction and (b) contamination of second round adjacent wells with product from a first round positive amplification. the use of the qiagen biorobot for the extraction of all specimens reduced the former while the nature of the strip prevents the latter, since all the wells have separate mixes ( table 2 ). with both nested and non-nested assays the most critical requirements for reliable results are the use of well trained, appropriately skilled and knowledgeable staff, operating in a managed environment. as with any service, test performance must stand up to both external and internal quality assurance and in this regard we welcome the new respiratory quality control panel soon to be made available from quality control for molecular diagnostics (qcmd), glasgow. the results obtained were very encouraging. although the strip was constructed to detect a wider range of viruses than immunofluorescence, over the period of validation it almost doubled (59 versus 31) the number of viruses that could have been detected by immunofluorescence, including a positive neqas specimen which was missed by immunofluorescence. of this group of viruses the detection of adenovirus showed the most dramatic increase, an observation we have also previously made in a separate study [17] and which we continue to see both in routine respiratory specimens and in a number of respiratory studies. similar to hrv viruses we believe these common infections are underdiagnosed by the standard techniques of immunofluorescence and culture. they are the second commonest virus, after hrv, that we observe in mixed infections and it is self-evident that these additional infections are at a level below the detection thresholds of standard methods. their clinical significance when detected at these lower copy numbers remains to be determined. as mentioned in the introduction a factor which often impacts negatively on a laboratory's decision to use molecular diagnostics is one of cost. it is worth considering that no matter which assay is chosen for use, it will attract the same overheads needed to provide the infrastructure of a laboratory set-up i.e. building, utilities, staff and equipment. in this regard there are no cheap tests and to use reagent costs as the sole factor in determining which assay to use is somewhat perverse. while the reagent costs of the strip are higher that commercial immunofluorescence reagents by a factor of 3, including extraction, this would undoubtedly narrow if immunofluorescence were capable of closing the pathogen gaps that currently exist e.g. hrv, hmpv. currently using this approach, we have been able to replace both immunofluorescence and viral culture and this ability makes molecular diagnostics a more cost effective method for diagnosing viral infections. taking into account the superior range, sensitivity, ability to quantify and speed of molecular techniques it is incredible how little they are used in routine laboratories. with the advent of sars and the threat of avian influenza, this deficit is now beginning to disturb health care planners at the highest level. because specimen sampling was not contiguous seasonal peaks were not detected, accounting for the small numbers of respiratory syncytial virus detected and the lack of detection of human metapneumoviruses, parainfluenza virus type 1 and coronavirus 229e; subsequent (unpublished) data from the routine use of the molecular strip support an important role for human metapneumovirus in acute respiratory infections and the sporadic nature of infections caused by parainfluenza type 1 and coronavirus 229e. several interesting observations need highlighting. first, for immunofluorescence to perform reliably it was essential that a good nasopharyngeal specimen was available. the use of throat and/or nasal swabs with immunofluorescence alone is inappropriate. second, immunofluorescence was very poor at detecting viruses from patients in the community, again almost certainly because of the universal use of swabs in that setting. third, the rapid results of immunofluorescence were complemented by the touchdown protocol which can report definitive results within 24 hours, considerably faster than culture. fourth, the molecular strip was better at detecting multiple infections. even allowing for the inability of immunofluorescence to detect rhinoviruses, it should have detected the mixed adenovirus and influenza virus infections. although immunofluorescence is capable of diagnosing dual infections, its routine use along with culture probably grossly underestimates their prevalence. the most plausible explanation is that the molecular technique detects infections where one of the viruses is below the positive control detection threshold of immunofluorescence. these low level viruses are either just starting or more likely reaching the end of an infectious episode (latency is less likely) and this raises the previously unaddressed question of their role in viral respiratory pathogenesis. fifth, the extent of rhinovirus infections was very significant. their clinical significance ranged from acting as a definitive respiratory pathogen to a less certain role when acting as the most frequently detected co-pathogen in mixed infections. in conclusion the use of the touchdown protocol with pre-dispensed and quality checked primer master-mixes was suitable for replacing virus culture for the diagnosis of respiratory viruses for immunofluorescence negative specimens. immunofluorescence results were available after an average of 4-12 hours while molecular strip results were available within 24 hours, considerably faster than viral culture. the combined strip and touchdown protocol is a convenient and reliable method of testing for multiple viruses in a routine setting. its generic nature makes it especially useful for introducing test repertoire modifications e.g. incorporating primers for the newly identified coronaviruses sars-cov and hcov-nl63. a total of 222 specimens were included in the validation between january 2002 and june 2003, including 14 from an influenza surveillance scheme. the specimens were collected from 183 patients including: 103 male, median age 3.5 y (7 m -84 y); 80 female patients, median age 9 y (7 m -84 y); both male and female ages were skewed towards the lower age ranges, and 5 national external quality assurance scheme (neqas) specimens (4 positive, 1 negative). one hundred and fifty-nine patients were in hospital and 24 were in the community at the time of sampling. specimens tested consisted of a wide range of specimens including: broncho-alveolar lavage (36), nasopharyngeal secretions (120), sputum (35) and combined throat and nasal swabs (31) . nasopharyngeal secretions, broncho-alveolar lavage and sputum specimens were received in dry sterile containers at ambient temperature. upon receipt they were re-suspended in 2 ml of virus transport medium (vtm) consisting of phosphate buffered saline ph 7.1, bovine serum albumin 7.5 µg/ml, penicillin g sodium 1000 units/ml, streptomycin sulphate 1000 µg/ml and amphotericin b 2.5 µg/ml. throat and nasal swabs were received in 2 ml of vtm and vortexed on arrival to release cells attached to the fibres of the swab. an aliquot of 410 µl was taken off for extraction after which the specimens were centrifuged at 2600 g for 5 min and the resulting cell deposits air-dried on glass multi-well slides and fixed in acetone prior to testing. immunofluorescence was set up on the respiratory specimens using commercial reagents according to the manufacturer's instructions, and was able to detect: influenza a, influenza b, respiratory syncytial virus, adenovirus, parainfluenza type 1, parainfluenza type 2 and parainfluenza type 3 (dako diagnostics, ely, uk). this protocol allows the co-extraction of both rna and dna simultaneously. simultaneous amplification of all targets was facilitated by using a standard 8 well multi-well pcr strip to which all mixes were pre-dispensed and stored frozen; this format is referred to in the paper as the "respiratory strip" because of the respiratory nature of the targets. the respiratory strip targeted the following 12 common respiratory viruses: influenza a (h3), influenza a (h1), influenza b, respiratory syncytial virus type a, respiratory syncytial virus type b, adenovirus, coronavirus 229e, parainfluenza virus type 1, parainfluenza virus type 2, parainfluenza virus type 3, human rhinovirus and human metapneumovirus. the final configuration of the single and multiplex primer mixes in the 8 well strip are shown in table 2 . the primer sets used were taken mainly from published studies [16, [29] [30] [31] but also included primer sets validated inhouse after modification or de-novo design, including those for influenza a (h1), influenza a (h3) and the generic adenovirus primers [17] . the primers, gene targets and expected product sizes following amplification are shown in table 3 . each primer master-mix was made-up and titrated against a known positive control before being aliquoted and dispensed into its respective well of the 8-well microtube strip. the strips were stored frozen at -20°c until used. a positive control was also aliquoted and stored separately at -20°c until used. for the duration of the study the positive control was the cloned target of parainfluenza virus type 1; a negative control was not deemed necessary. first round volumes were made-up in access rt-pcr buffer (promega, southampton, england, u.k) and in the final 10 µl volume contained the following reagent amounts: 2 agc aaa gct ttc agc aac tg haemagglutinin 1 591 fla 2d (h3) 2 gct tcc att tgg agt gat gc-3 fla 2e (h3) 2 agt gct gaa cgt gac tat gc 2 149 fla 2f (h3) 2 ttt gct ggc ttc tct tgg t rsv 2a gtc tta cag ccg tga tta gg nucleoprotein 1 838 rsv 2b ggg ctt tct ttg gtt act tc rsva 2c gat gtt acg gtg ggg agt ct 2 334 rsva 2d gta cac tgt agt taa tca ca rsvb 2c aat gct aag atg ggg agttc 2 183 rsvb 2d gaa att gag tta atg aca gc adv 2a 2 gccgcagtggtcttacatgcacatc hexon 1 301 adv 2b 2 cagcacgccgcggatgtcaaagt adv 2c3 2 gacgcctcggagtacctswsycc 2 185 adv 2dd 2 tacgagtacgtggtgtcctckcgrtc the first round reaction added to 9.8 µl of second round primer master-mix per well; a multi-channel pipette facilitated the transfer of the 8 volumes in one step. the positive control was run on the eighth well of each strip. the second round products were run on ethidium bromide stained 2% agarose gels and photographed. specimens were reported positive when respectively the correct size bands and the positive control bands were present. vaccine preventable disease comparison of conventional viral cultures with direct fluorescent antibody stains for diagnosis of community-acquired respiratory virus infections in hospitalized children sensitivity of respiratory virus culture when screening with r-mix fresh cells isolation of viruses from clinical specimens in microtitre plates with cells inoculated in suspension a comparison of nested polymerase chain reaction and immunofluorescence for the diagnosis of respiratory infections in children with bronchiolitis, and the implications for a cohorting strategy a comparison of virus isolation, indirect immunofluorescence and nested multiplex polymerase chain reaction for the diagnosis of primary and recurrent herpes simplex type 1 and type 2 infections nested multiplex polymerase chain reaction for the diagnosis of cutaneous herpes simplex and herpes zoster infections and a comparison with electronmicroscopy differential detection of rhinoviruses and enteroviruses rna sequences associated with classical immunofluorescence assay detection of respiratory virus antigens in nasopharyngeal swabs from infants with bronchiolitis molecular diagnosis of influenza diagnosis of acute respiratory tract infections: serology and new methods van helden pd: laboratory experience and guidelines for avoiding false positive polymerase chain reaction results evaluation of the prodesse hexaplex multiplex pcr assay for direct detection of seven respiratory viruses in clinical specimens single-step pcr optimization using touchdown and stepdown pcr programming assessment of cmv load in solid organ transplant recipients by pp65 antigenemia and real-time quantitative dna pcr assay: correlation with pp67 rna detection comparison of pcr primer pairs in the detection of human rhinoviruses in nasopharyngeal aspirates clinical assessment of a generic dna amplification assay for the identification of respiratory adenovirus infections human metapneumovirus infection among children hospitalized with acute respiratory illness a new dig-pcr-eia method for the detection of chlamydia pneumoniae dna in clinical samples diagnostic use of pcr for detection of pneumocystis carinii in oral wash samples a touchdown pcr for the differentiation of equine herpesvirus type 1 (ehv-1) field strains from the modified live vaccine strain rach improved detection of rhinoviruses in nasal and throat swabs by seminested rt-pcr combining multiplex and touchdown pcr for microsatellite analysis combining multiplex and touchdown pcr to screen murine microsatellite polymorphisms use of degenerate primers and touchdown pcr to amplify a halogenase gene fragment from streptomyces venezuelae isp5230 use of degenerate primers and touchdown pcr for construction of cdna libraries cloning and molecular characterization of cntef1 which encodes translation elongation factor 1alpha in cryptococcus neoformans clinical utility of nested multiplex rt-pcr for group f adenovirus, rotavirus and norwalk-like viruses in acute viral gastroenteritis in children and adults multiplex pcr for typing and subtyping influenza and respiratory syncytial viruses simultaneous detection and identification of human parainfluenza viruses 1, 2, and 3 from clinical samples by multiplex pcr evaluation of nested polymerase chain methods for the detection of human coronaviruses 229e and oc43 we wish to thank the northern ireland chest, heart and stroke foundation for funding that contributed to the development of the molecular strip. pvc: touchdown and molecular strip design and manuscript preparation.gmo: early application of touchdown cycling to respiratory samples. key: cord-271669-dkg6229j authors: han, seung beom; shin, ju ae; kim, seong koo; lee, jae wook; lee, dong-gun; chung, nack-gyun; cho, bin; jeong, dae chul; kang, jin han title: respiratory viral infections in children and adolescents with hematological malignancies date: 2019-01-01 journal: mediterr j hematol infect dis doi: 10.4084/mjhid.2019.006 sha: doc_id: 271669 cord_uid: dkg6229j background: despite the introduction of a polymerase chain reaction (pcr) test for the diagnosis of respiratory viral infection (rvi), guidance on the application of this test and the management of rvi in immunocompromised children is lacking. this study evaluated the clinical characteristics of rvi and established strategies for the pcr test in children and adolescents with hematological malignancies. methods: this study included children and adolescents with underlying hematological malignancies and respiratory symptoms, in whom a multiplex pcr test was performed. patients in whom rvi was identified and not identified were categorized into groups i and ii, respectively. group i was sub-divided into patients with upper and lower respiratory infections. the medical records of the enrolled patients were retrospectively reviewed. results: a total of 93 respiratory illnesses were included. group i included 46 (49.5%) cases of rvi, including 31 (67.4%) upper and 15 (32.6%) lower respiratory infections. rhinovirus (37.0%) was the most common viral pathogen. significantly more patients in group i had community-acquired respiratory illnesses (p=0.003) and complained of rhinorrhea (p<0.001) and sputum (p=0.008) than those in group ii. in group i, significantly more patients with lower respiratory infections had uncontrolled underlying malignancies (p=0.038) and received re-induction or palliative chemotherapy (p=0.006) than those with upper respiratory infections. conclusions: a multiplex pcr test should be considered for rvi diagnosis in immunocompromised children and adolescents with respiratory symptoms, especially in those with rhinorrhea or sputum prominent over a cough. the early application of the pcr test in patients with uncontrolled underlying malignancies may improve outcomes. introduction. infection is the main cause of treatmentrelated mortality in patients with hematological malignancies. 1 therefore, the early diagnosis and treatment of infection, as well as infection prevention, are essential to improve the prognosis of immune compromised patients. in these patients, neutropenic fever (nf) has been the focus, and bacterial and fungal infection is emphasized. 2 among viruses, herpesviridae, which maintains latency and reactivates during immune suppression caused by anti-cancer chemotherapy and hematopoietic cell transplantation (hct), is considered a major pathogen. however, the causative pathogens are not identified in 53-79% of patients with nf, 3, 4 and some of which may be respiratory viruses (rvs). viral culture and antigen detection methods have been used for the diagnosis of viral infection. because these conventional methods have low sensitivity for detecting rhinovirus and enterovirus, which are the most common causes of community-acquired respiratory viral infection (rvi), rvi was identified only in 6-22% of immune compromised children with respiratory symptoms in the past. [5] [6] [7] also, most previous studies were restricted to reporting rvi due to respiratory syncytial virus (rsv), parainfluenza virus, influenza virus, and adenovirus, which could be diagnosed by conventional methods. [8] [9] [10] a polymerase chain reaction (pcr) test exhibiting improved sensitivity and specificity in the diagnosis of rvi in immune compromised patients compared to those of conventional methods has been introduced and its use has been extended since the 2000s. 5, 6, 11 recent studies using pcr tests identified rvi in 33-76% of children with nf or cancer complaining of respiratory symptoms. 5,6,12-16 rsv and influenza virus were the most frequent causes of rvi in the era of conventional methods, [8] [9] [10] whereas rhinovirus was the most frequent cause in the era of pcr tests. 5, 6, [12] [13] [14] [15] [16] [17] in spite of this epidemiological change accompanied by the introduction of pcr tests, reports on the clinical characteristics and prognosis of rvi in immune compromised children and adolescents using pcr tests are lacking. accordingly, guidelines on the application of a pcr test for the diagnosis and proper management of rvi in immune compromised children and adolescents have not been established. this study was performed to evaluate the clinical characteristics and outcomes of rvi diagnosed by a multiplex pcr test for rvs and to establish strategies for performing the pcr test in children and adolescents with hematological malignancies, who comprise a major portion of immune compromised children and adolescents. children and adolescents (<20 years of age) with underlying hematological malignancies treated at the department of pediatrics, seoul st. mary's hospital, college of medicine, the catholic university of korea, were eligible for this study. among them, those who complained of respiratory symptoms such as a cough, rhinorrhea, sputum, sore throat, and dyspnea, with or without fever, and in whom a multiplex pcr test for rvs was performed between december 2016 and november 2017 were enrolled. patients in whom respiratory symptoms developed 6 months or more after the completion of anti-cancer chemotherapy or 2 years or more after hct were excluded. patients in whom hematological malignancies were newly diagnosed and anti-cancer chemotherapy had not been administered prior to the development of respiratory illnesses were also excluded. patients in whom rvs were identified and not identified were categorized into groups i and ii, respectively. the medical records of the enrolled patients were reviewed retrospectively and the clinical and laboratory characteristics were compared between groups. the patients in group i were sub-divided into those with upper respiratory tract infections (uris) and lower respiratory tract infections (lris) and the clinical and laboratory characteristics were also compared between the two subgroups. this study was approved by the institutional review board of the seoul st. mary's hospital with a waiver of informed consent (approval number: kc18resi0302). a nasopharyngeal swab was collected from patients complaining of respiratory symptoms. the samples were sent to the department of laboratory medicine where a multiplex pcr test for rvs was performed using a commercially available kit (advansure™ rv real-time pcr kit, lg life sciences ltd., seoul, republic of korea). the pcr kit tested for influenza a and b viruses, parainfluenza virus, rhinovirus, rsv, human metapneumovirus (hmpv), adenovirus, coronavirus, and human bocavirus. chest x-ray was routinely performed in patients with respiratory symptoms, and chest computed tomography was performed based on the attending physician's clinical decision. oseltamivir was administered to all patients diagnosed with influenza. based on the attending physician's decision, oral ribavirin and intravenous immunoglobulin (ivig) were administered. febrile patients received empirical antibiotic therapy. definitions. an episode of respiratory illness occurring 4 or more weeks after a previous episode was considered a separate episode if it occurred during a separate admission and the patient did not have respiratory complaints between the two episodes. uri was diagnosed when a patient had respiratory symptoms that were not accompanied by hypoxemia and abnormal findings on chest imaging studies. lri was diagnosed when abnormal findings were observed in chest imaging studies. due to the difficulty associated with obtaining sputum samples and performing bronchoscopy in children and adolescents with underlying hematological malignancies, as they are prone to bleed, nasopharyngeal samples were used to diagnose lri, although lower respiratory samples are preferred specimens. 18 community-acquired respiratory illness was defined if respiratory symptoms developed before or within 2 days of admission. hospital-acquired respiratory illness was defined if respiratory symptoms developed 2 or more days after admission. neutropenia was defined as an absolute neutrophil count <500/mm 3 . steroid use was defined if any type of glucocorticoids equivalent to 2 mg/kg/day (maximum 20 mg/day) of prednisolone or more were administered for longer than 5 days within 1 month prior to the development of respiratory illness. oxygen therapy, mechanical ventilator care, intensive care unit admission, and death that occurred within 1 month after the development of respiratory illness were considered complications. co-infections were defined when any other types of non-viral infection were identified at the same time as the patient complaint of respiratory symptoms. mortality within 1 month after the development of respiratory illness was determined. mortality due to rvi was defined when the patient died with persisting respiratory symptoms and signs and no other causes of death were identified. statistical analysis. categorical and continuous factors were compared using a chi-square and mann-whitney tests, respectively, for comparisons between the patient groups. multivariate analyses to identify the independent factors for rvi and lri were performed for significant factors in univariate analysis using a binary logistic regression analysis. ibm spss statistics for windows, version 21.0 (ibm corp., armonk, ny, usa) was used for statistical analyses, and statistical significance was defined as a p-value <0.05. during the study period, 74 children and adolescents with underlying hematological malignancies who experienced 93 episodes of respiratory illnesses were enrolled. nine and five patients each experienced two and three episodes, respectively. the median interval between recurrent episodes of respiratory illnesses was 19 weeks (range, 5-52 weeks). group i included 46 (49.5%) episodes of rvi, including 31 (67.4%) uris and 15 (32.6%) lris. eleven (73.3%) of the 15 lris were initially diagnosed with uris that progressed to lris. among the identified rvs, rhinovirus (n=17, 37.0%) was the most frequent ( table 1) , followed by parainfluenza virus (n=14, 30.4%) and rsv (n=10, 21.7%). in five (10.9%) episodes, two viruses were concurrently identified: two (4.3%) episodes of rhinovirus and parainfluenza virus, two (4.3%) episodes of rhinovirus and rsv, and one (2.2%) episode of rsv and influenza a virus. co-infections were identified in 21 (22.6%) episodes. ten (10.8%) episodes were accompanied by bacteremia (escherichia coli in two, pseudomonas aeruginosa in two, viridans streptococci in two, enterococcus faecium in two, streptococcus pneumoniae in one, and staphylococcus epidermidis in one). eight (8.6%) episodes were accompanied by invasive pulmonary aspergillosis, and two (2.2%) episodes were accompanied by herpetic gingivostomatitis. chickenpox, pneumocystis jirovecii pneumonia, and clostridium difficile infection accompanied one (1.1%) episode each. the rate of rv positivity was highest in autumn; however, no significant differences were found among the four seasons (range, 31.6-61.5%, p=0.258). seven rsv infections occurred within a one-month interval in the same ward, six (85.7%) of which were hospitalacquired infection. comparison between group i and group ii. the rates of different underlying hematological malignancies and administered chemotherapies preceding respiratory illnesses were not significantly different between the two groups. significantly more patients in group ii presented with hospital-acquired respiratory illnesses (p=0.003), and accompanied neutropenia (p=0.028) than those in group i ( table 2) . most patients overall complained of fever and cough; however, those in group i complained of rhinorrhea (p<0.001) and sputum (p=0.008) more frequently than those in group ii. in multivariate analysis, rhinorrhea, sputum, and community-acquired respiratory illness were significant factors for a diagnosis of rvi ( table 3) . significantly more complications occurred in patients in group ii compared to those in group i ( table 2) . mortality was higher in group ii than that in group i; however, the difference was not statistically significant. in the patients in group i, those with lris were more likely to have uncontrolled underlying malignancies (p=0.038) and receive reinduction or palliative chemotherapy (p=0.006) than those with uris ( table 4) . among respiratory symptoms, sputum (p=0.028) and dyspnea (p=0.001) were more frequently accompanied by lris. more patients with lris experienced co-infections than those with uris (p=0.029). among the five patients with lris and co-infections, four experienced invasive pulmonary aspergillosis: two of whom had concomitant rsv infection, another had concomitant adenovirus infection, and the other had hmpv infection. the other experienced c. difficile infection with concomitant parainfluenza virus infection. the two patients with uris and co-infections experienced chickenpox with concomitant rhinovirus infection, and s. epidermidis bacteremia with concomitant rsv infection, respectively. the rate of rhinovirus infection was significantly higher in patients with uris than in those with lris (p=0.021). other viral infections showed no significant association with lris ( table 1) . there were no independent risk factors for lri in multivariate analysis (data are not shown). of 14 table 3 . multivariate analysis to determine the independent factors for respiratory viral infection. odds ratio 95% confidence interval p value and outcomes of rvi were investigated in children and adolescents with hematological malignancies. rvi was diagnosed in about half of the enrolled patients, consistent with the results of previous studies using pcr tests to diagnose rvi. 5, [12] [13] [14] [15] rhinovirus rather than rsv and parainfluenza virus was the most frequent cause of rvi, consistent with the results of recent studies using pcr tests. 5,6,12-16 rvi investigation should be considered in immune compromised patients complaining of community-acquired respiratory symptoms, preferably in those with rhinorrhea or sputum predominant over a cough. if we consider that early termination of empirical antibiotic therapy led to a favorable outcome in children and adolescents with nf and rvi in a recent report by santolaya et al, 15 early diagnosis of rvi by a pcr test in these patients can help to avoid an unnecessary antibiotic use. neutropenia was identified in only 34.8% of the patients diagnosed with rvi in this study. therefore, the diagnosis of rvi should not be restricted to patients with nf. as a matter of fact, patients in group ii, in whom the presence of rvs was not identified, were more likely to have neutropenia, hospital-acquired and severe respiratory illnesses, and infections with nonviral pathogens. this suggests that patients in group ii underwent more aggressive anti-cancer chemotherapies, had a more severe immunosuppression, and were hospitalized for longer periods, compared to patients in group i. thus, pulmonary edema arising from hyper-hydration during anti-cancer chemotherapy, pulmonary hemorrhage due to thrombocytopenia, and bacterial and fungal pneumonias could lead to respiratory symptoms in these patients. a negative pcr result in patients with nf and hospital-acquired respiratory illnesses may suggest the presence of more severe infection or treatment-related complications. although community-onset respiratory illness was significantly associated with the diagnosis of rvi in this study, 13-80% of rvi cases were hospitalacquired infection in several studies, [6] [7] [8] [9] 19 including the present study (34.8%). outbreaks of rsv and parainfluenza virus infection have been reported in an outpatient department as well as in an inpatient ward; [20] [21] [22] we also experienced an outbreak of rsv infection in seven patients in one month in a closed hematology ward. therefore, a multiplex pcr test for rvs should be encouraged even in hospitalized patients complaining of rhinorrhea or sputum, particularly when other patients with rvi are hospitalized in the same ward or there is an rvi epidemic in the community. a timely application of the pcr test can lead early diagnosis of rvi in hospitalized patients, and to a subsequent decrease in the rvi transmission within the hospital environment. recent studies on rvi in immune compromised children showed low mortality due to rvi, 0-3%. 5, [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] 19 fortunately, there was no death due to rvi in this study. this favorable outcome may be attributed to a growing concern for rvi in physicians, increasing diagnostic rates especially of mild rvi cases using pcr tests, and improved supportive care in immune compromised patients. ribavirin-based anti-viral therapy can reduce progression from uri to lri and mortality in rsv-infected hct recipients; 23 however, it is not recommended in patients with hematological malignancies who are not receiving hct and its effect on parainfluenza virus infection has not been confirmed. 24 in this study, 80.0% of patients diagnosed with rsv infection received ribavirin-based anti-viral therapy, regardless of receiving hct, and none of them died due to rsv infection. however, the efficacy of the ribavirin-based therapy should be further evaluated as the anti-viral therapy performed in this study did not rely on currently established criteria. the risk factors for mortality due to rvi could not be determined in this study because there were no deaths attributable to rvi. most previous studies universally reported that lri is associated with the increased mortality. 7, 10, 19, 23, [25] [26] [27] even in rhinovirus infection, which causes milder respiratory illnesses compared to those of rsv, parainfluenza virus and influenza virus, mortality was significantly higher in patients with lris than that in those with uris. 26 therefore, the early detection of patients at risk of progression to lris and early application of proper management for lris are necessary to improve the outcome of rvi in immune compromised patients. low absolute lymphocyte, neutrophil, and monocyte counts; relapsed underlying malignancies; unrelated or mismatched allogeneic-hct; recent steroid use; oxygen need; and co-infections are risk factors for lri or mortality. 9,10,23,25-27 these risk factors represent the severity of immune suppression in the infected hosts. 9,25 accordingly, an immunodeficiency scoring system to predict outcomes and determine the administration of anti-viral therapy has been applied to rsv-infected hct recipients. 28 the uncontrolled state of underlying hematological malignancies and the presence of co-infections were significantly associated with the development of lri in this study, which underscores the importance of the host's overall immune status in the outcome of rvi. as a result, a multiplex pcr test for rvs should be performed preferentially in patients complaining of rhinorrhea or sputum and with relapsed or refractory underlying hematological malignancies, co-infections, or severe cytopenia. considering that 73.3% of lri cases progressed from uris in this study, the early application of a multiplex pcr test during the uri period should be emphasized. this study had some limitations, including biases arising from its retrospective study design. the number of enrolled patients may not be appropriate, and we lacked a control group including patients without respiratory symptoms. although lower respiratory samples, such as sputum and bronchial washing or bronchoalveolar lavage fluids, are preferred samples for lri diagnosis, upper respiratory samples were used in this study. to assure an improved diagnosis of lri, abnormal findings in chest imaging studies were mandatory for lri diagnosis in this study; however, lower respiratory samples could reveal clearer pathogenic profiles. the seasonal distribution and epidemics of rvi in immune compromised patients are correlated with those observed in the community. 8, 29 during the study period, the epidemics of rsv and influenza virus infection in the community were not prominent in korea compared to previous years. the inclusion of more rsv and influenza virus infection episodes may modify the outcome of rvi in this study. future studies should analyze data gathered for several years to include more cases of rvi caused by a variety of rvs. a multiplex pcr test for rvs was not routinely performed in children and adolescents with respiratory symptoms in our hospital during the study period and was hardly performed in the outpatient clinic. therefore, children and adolescents with mild respiratory symptoms and uris could not be included in this study. the outcomes of rvi in immune compromised children and adolescents may be more favorable with the inclusion of mild rvi cases. considering the confirmed rvi diagnosis in half of the immune compromised children and adolescents with respiratory symptoms in this study, the introduction of multiplex pcr tests for rv detection in this population should be encouraged, especially for patients complaining of rhinorrhea or sputum prominent over a cough. moreover, the pcr test should address patients with more severe immune suppression, e.g., those with relapsed or refractory underlying malignancies and co-infections, as they are prone to have severe rvi-related outcomes. in addition, infection control strategies to prevent rvi transmission within the hospital environment should be emphasized, considering the current scenario, in which effective anti-viral therapies have not been established for most rvi cases. thus, early rvi detection by a pcr test may open a window of opportunity for early intervention and infection control. infection-related mortality in children with acute lymphoblastic leukemia: an analysis of infectious deaths on ukall2003 infectious diseases society of america. clinical practice guideline for the use of antimicrobial agents in neutropenic patients with cancer: 2010 update by the infectious diseases society of america a prospective study on the epidemiology of febrile episodes during chemotherapy-induced neutropenia in children with cancer or after hemopoietic stem cell transplantation etiology and clinical course of febrile neutropenia in children with cancer respiratory viruses, a common microbiological finding in neutropenic children with fever respiratory viral infections in children with leukemia impact of parainfluenza virus infection in pediatric cancer patients. pediatr blood cancer respiratory tract viral infections in bone marrow transplant patients respiratory viral infections in adults with hematologic malignancies and human stem cell transplantation recipients: a retrospective study at a major cancer center respiratory virus infections after stem cell transplantation: a prospective study from the infectious diseases working party of the european group for blood and marrow transplantation detection of respiratory viruses with a multiplex polymerase chain reaction assay (multicode-plx respiratory virus panel) in patients with hematologic malignancies respiratory viral infections and coinfections in children with cancer, fever and neutropenia: clinical outcome of infections caused by different respiratory viruses prospective detection of respiratory pathogens in symptomatic children with cancer acute respiratory viral infections in pediatric cancer patients undergoing chemotherapy efficacy and safety of withholding antimicrobial treatment www.mjhid.org in children with cancer, fever and neutropenia, with a demonstrated viral respiratory infection: a randomized clinical trial nasopharyngeal detection of respiratory viruses in febrile neutropenic children human rhinovirus and coronavirus detection among allogeneic hematopoietic stem cell transplantation recipients a guide to utilization of the microbiology laboratory for diagnosis of infectious diseases: 2018 update by the infectious diseases society of america and the the impact of rsv, adenovirus, influenza, and parainfluenza infection in pediatric patients receiving stem cell transplant, solid organ transplant, or cancer chemotherapy molecular characterization of strains of respiratory syncytial virus identified in a hematopoietic stem cell transplant outpatient unit over 2 years: community or nosocomial infection? detection and control of a nosocomial respiratory syncytial virus outbreak in a stem cell transplantation unit: the role of palivizumab control of an outbreak of human parainfluenza virus 3 in hematopoietic stem cell transplant recipients impact of aerosolized ribavirin on mortality in 280 allogeneic haematopoietic stem cell transplant recipients with respiratory syncytial virus infections how i treat respiratory viral infections in the setting of intensive chemotherapy or hematopoietic cell transplantation parainfluenza virus lower respiratory tract disease after hematopoietic cell transplant: viral detection in the lung predicts outcome human rhinovirus detection in the lower respiratory tract of hematopoietic cell transplant recipients: association with mortality parainfluenza virus infections in hematopoietic cell transplant recipients and hematologic malignancy patients: a systematic review immunodeficiency scoring index to predict poor outcomes in hematopoietic cell transplant recipients with rsv infections respiratory viral infections in immunocompetent and immunocompromised persons key: cord-286449-ekvzaae2 authors: mcmanus, terence e.; marley, anne-marie; baxter, noreen; christie, sharon n.; o'neill, hugh j.; elborn, j. stuart; coyle, peter v.; kidney, joseph c. title: respiratory viral infection in exacerbations of copd date: 2008-07-30 journal: respir med doi: 10.1016/j.rmed.2008.06.006 sha: doc_id: 286449 cord_uid: ekvzaae2 background: patients with copd have frequent exacerbations. the role of respiratory viral infection is just emerging. we wished to determine prospectively the incidence of viral infection in exacerbated and stable copd patients as well as smokers who do not have airways obstruction. methods: stable and exacerbated copd patients were recruited along with a group of patients who had smoked but who did not have any airways obstruction. spirometry was performed and sputum specimens were tested for a range of 12 different respiratory viruses using pcr. results: one hundred and thirty-six patients with exacerbations of copd, 68 stable copd patients and 16 non-obstructed smokers were recruited. a respiratory virus was detected in 37% of exacerbations, 12% of stable copd patients and 12% of non-obstructed smokers, p < 0.0005. rhinovirus was most frequently detected. the symptom of fever was associated with virus detection, p < 0.05. infection with more than one virus was only found in the exacerbated copd patients. conclusion: respiratory viral infection is associated with exacerbations of copd. rhinovirus was the most common infecting agent identified and in two cases human metapneumovirus was also detected. dual infections were only seen amongst those patients admitted to hospital with acute exacerbations of copd. viruses were more commonly detected in those with more severe airways disease. patients with copd have frequent exacerbations which lead to increased airway inflammation and often subsequent hospitalization. 1 bacteria are associated with approximately 50% of exacerbations. 2, 3 a number of studies have identified viral infections of the respiratory tract as precipitating agents. studies using serology and viral culture identified respiratory viruses in 30% of patients during acute exacerbations of copd. 4 with the development of more sensitive molecular tests the role of viruses in copd has been better defined. walsh et al. identified several common respiratory viruses in a cohort of patients with copd or congestive cardiac failure. 5 viruses were identified by serology and viral culture methods. significant outcomes in terms of hospitalisation were seen for respiratory syncytial virus (rsv) and influenza a infected patients. greenberg et al., using serology and viral culture, showed that in 23% of cases of hospitalisation of copd patients a virus was detected and that the mean time to return to symptomatic baseline was 2 weeks. 6 the most common viruses identified were of the picornavirus classification (rhinoviruses). several studies have used pcr for the diagnosis of viral infection. initially, in 2000 seemungal et al. examined 33 patients with copd when stable and subsequently during an exacerbation. 7 ten of 43 exacerbations were associated with rhinovirus infection. higher symptom scores and sputum interleukin-6 levels were seen with rhinovirus exacerbations. seemungal et al. went on to show respiratory viral infection in 39% of exacerbations of copd. 8 viral infection was associated with a more severe exacerbation, and in patients who had a higher frequency of exacerbations, i.e. those with the poorest lung function. patients were reviewed in convalescence at 4e6 weeks. the majority (58%) of these viruses were rhinovirus (i.e. 23% of copd exacerbations). plasma il-6 levels were elevated in all exacerbations, and levels were significantly increased in the virus positive group when comparison was made with those in whom no virus was detected. rohde et al. used pcr to detect viruses in patients with exacerbations of copd. a respiratory virus was detected in 56% of cases in comparison to 19% of control subjects with stable copd. 9 the majority of viruses detected were picornaviruses. the most recent study from beckham et al. combined specimens obtained for two previous studies for analysis by pcr. 10 they detected a respiratory viral infection in 42% of acute respiratory illnesses. respiratory viral infection in copd patients during exacerbation has been associated with longer median symptom recovery time in comparison to exacerbations in which a viral agent was not identified (13 days versus 6 days respectively). 8 the hypothesis tested in the present study was that acute respiratory viral infection is implicated in the pathogenesis of copd exacerbations. the aims of the present study were to examine respiratory specimens for the presence of respiratory viruses in patients with copd whilst stable and also during exacerbations. this study was approved by the queen's university belfast ethics committee and all patients gave written consent. the patient groups described in this paper have been described in other related publications. patients hospitalised with exacerbations of copd were recruited within 24 h of presentation over a 2-year period. a further group of stable copd patients were also recruited. stability was defined as no change in respiratory symptoms or alteration in therapy in the previous 8 weeks. patient's symptoms were recorded in a binary format. spirometry was performed, the best of 3 reproducible readings was taken (vitalograph spirometer, vitalograph, buckingham, uk). 11 spirometry was repeated after nebulised beta agonist (salbutamol 2.5 mg). any patients with significant improvement in fev 1 (>200 ml/ 15%) were excluded. those patients with a history of bronchiectasis, neoplastic process or other serious concomitant disease were excluded. copd and assessment of severity was classified according to the gold criteria. 12 exacerbation of copd was classified according to the gold criteria with symptoms of increased dyspnoea, increased cough or increased sputum production. 13 stable copd was classified according to the gold criteria without any symptoms of exacerbation or changes in treatment within the last 8 weeks. sputum samples were obtained either by spontaneous production or following nebulised hypertonic saline. briefly, 4 ml of 3% saline was nebulised via an air driven nebuliser. every 5 min spirometry was repeated to measure fev 1 , and nebulisation continued if fev 1 had not fallen by more than 20%. this was continued up to 20 min. all sputum expectorated was collected. all samples were processed within 2 h. specimens were mixed with 4 volumes of 0.1% dithiothreitol (sigma, poole, uk) and shaken in an orbital incubator (gallenkamp, loughborough, uk) for 15 min at 37 c followed by the addition of 4 volumes of phosphate buffered saline and shaken for a further 5 min. the resulting suspension was then filtered through 50 mm nylon gauze (lockertex, warrington, uk) and spun down at 1000 â g for 10 min. after removing the supernatant the cell pellet was resuspended in lysis buffer (qiagen, crawley, uk). total nucleic acid extraction was performed on 200 ml of sputum sample suspended in lysis buffer (qiaamp dna blood mini kit). extracted samples were screened for common respiratory viruses (rhinovirus, human metapneumovirus, influenza a h1 and h3, influenza b, rsv a and b, coronavirus 229e, adenovirus and parainfluenza 1, 2 and 3) using an eight-well multiplexed, nested pcr system (fig. 1 ). 14 a positive control was used for each set of patient's tests. mastermix and pcr cycling conditions are listed in the supplementary material(appendix a1 and a2 respectively). tests results were accepted as being valid when the positive control tested positive with correct sized product(s). a positive result was noted when second (or first and second) round products were noted and confirmed as being the correct size. indeterminate test results were repeated. this method allowed rapid testing of clinical specimens for multiple viruses with sensitive and specific assays. a power calculation was performed to determine study group sizes. assuming a respiratory virus detection rate of 30% amongst copd patients during exacerbations and 10% detection when stable a power calculation was performed using epiinfoä version 6. using a confidence value of 95% with a power of 80% and a 2:1 ratio of group sizes, 111 patients with copd exacerbations and 56 stable copd subjects were required. normally distributed data were presented as mean ae standard deviation, whilst non-parametric data were expressed as median and inter quartile ranges. normally distributed continuous variables were compared by t-test; otherwise the differences were assessed by the mannewhitney utest. for discrete variables frequencies and percentages were reported and groups compared using the chi squared test. a significance level of 5% was chosen. statistical analysis was carried out using spss (chicago, il) version 10.0. a total of 220 subjects were recruited in this study (table 1) ; 136 were seen during an acute exacerbation, and 68 were recruited when stable. an additional 16 non-obstructed smokers with a fev 1 and fvc within the normal range were recruited. patient characteristics are shown in tables 1 and 2 ; the exacerbated and stable copd groups were of similar age, sex, fev 1 , tobacco use, baseline mrc score, inhaled steroid and body mass index (bmi) and pack years of tobacco use. crp and white cell count (wcc) were significantly elevated during exacerbation when compared to stable copd patients. crp levels were 14.8 mg/dl (5.0e70.2 mg/dl) at exacerbation and 5.0 mg/dl (5.0e8.4 mg/dl) in stable patients (p < 0.0001). the wcc increased from 8.7 ae 2.6 to 10.9 ae 3.7 â 10 3 /l in exacerbations of copd (p < 0.0001). similarly the neutrophil count increased from 5.4 ae 2.3 to the majority of patients described symptoms of increased dyspnoea, cough along with increased sputum volume and purulence during exacerbations ( table 3 ). the presence of fever was associated with the identification in the same patient, p < 0.05. the majority of patients were mrc score 5 during exacerbations. respiratory viruses were detected in sputum and nasal/ throat swabs in 50 of 136 (37%) patients during exacerbations of copd and 8 of 68 (12%) stable copd patients (p < 0.0001) ( table 4 ). the viruses detected (table 5) were rhinovirus (57%), adenovirus (18%), parainfluenza 3 (9%), influenza a h3 (5%), rsv b (3.5%), metapneumovirus (3.5%), coronavirus (2%) and rsv a (2%). there were dual infections in six cases however these were all confined to the exacerbated copd group. a respiratory virus was more frequently detected during exacerbations in patients with more severe airways disease, p < 0.05 (table 6 ). no associations were seen between viral infection and patient sex or medication (use of theophylline or inhaled steroid therapy). the main findings of this study were that respiratory viruses were more frequently detected during acute exacerbations of copd in patients admitted to hospital and that crp levels correlated with exacerbation and duration of hospital stay. this study was performed over a 2-year period in order to avoid bias due to seasonality. the detection rate of respiratory viruses during exacerbations of copd in this study (37%) is comparable to results obtained by seemungal 9 lower detection rates may be related to time of sampling as patients presenting to hospital had developed symptoms for a median of 5 days prior to admission. this is the first study to prospectively analyse this range of respiratory viruses in patients recruited during exacerbation of copd. the most common infecting agent was rhinovirus in keeping with previous studies, 8e10 however we also detected a high rate of adenoviral infection which has not previously been reported. this is in keeping with the findings of coyle et al. who demonstrated that adenovirus is more commonly detected using pcr than conventional immunofluorescence and virus culture techniques. 14 previous investigators have related adenoviral infection to subsequent latent infection in the form of adenovirus e1a and that it may be important in the pathogenesis of copd. 15 metapneumovirus was discovered in 2001 and initially detected in young children with a respiratory tract illness. 16 it was subsequently found to play a role in communityacquired respiratory tract illness with 2.2% of patients presenting with 'influenza-like illness' testing positive. 17 beckham et al. tested specimens from patients during exacerbations of copd and when stable for metapneumovirus but all patients were negative. 10 rohde et al. found metapneumovirus to be present in 2.3% of patients during acute exacerbations of copd and none of the stable copd patients in keeping with the findings of this study. 18 the detection of human metapneumovirus in this study confirms its role as a respiratory pathogen in adult patients with copd. the study showed relatively lower detection rates of influenza virus compared to other studies. 8e10 this may be related to improved vaccination amongst this group of at risk individuals and it may also reflect that during recruitment for this study there was no influenza epidemic. there was also a lower incidence of rsv infection (3 cases). previous investigators have suggested that it is more common. some have suggested that it is present in low copy numbers. it is possible that a more sensitive realtime pcr assay is required in order to detect it. 8 however a recent study suggests that rsv accounted for 11.4% of copd admissions in hospitalised patients. 19 this is the first study to also include a group of patients from the same community who do not have respiratory disease (non-obstructed smokers). there were similar detection rates of respiratory viruses in the stable copd (11.8%) and nos groups (12.5%) supporting previous findings which found respiratory viral infection to be present in asymptomatic individuals. 6 the frequency of detection in these groups suggests that copd patients do not have a higher carriage rate of viral infections to nos but a larger study is required in order to confirm this. one of the weaknesses of this study was that patients were recruited following admission to hospital. in some cases there may be several days between infection, development of symptoms and presentation. depending on the duration of viral infection prior to seeking medical help there may be some cases in which patients present late and thus viral replication is decreasing, resulting in a reduced detection rate. this may lead to an underestimation of the prevalence of viral infection in these patients. recent publications also suggest that rsv detection is increased with the use of real-time pcr assays. 8 all respiratory viral assays in this study utilized nested pcr technology and not real-time pcr methods. detection of respiratory viruses by pcr may in fact relate to airway colonization 20 or viral persistence. 21 however, several of the patients were seen at different time points during this study and the same virus was not detected by repeat sampling suggesting that those testing positive using the pcr screen were experiencing an acute viral infection. another possible confounding factor is that patients with copd experience exacerbations in which no precipitating agent can be identified. these episodes may be related to the progressive nature of this disease process rather than an acute event. thus the true role of respiratory viral infection in exacerbations may be underestimated. a possible area of bias was that all patients recruited were hospitalized. thus selection bias may have been imposed in that those patients admitted to hospital tended to have particular types of respiratory viral infection or that respiratory viral infection only precipitated an exacerbation and subsequent hospital admission in patients with severe copd. another potential source of bias is the seasonality of respiratory viral infection. we addressed this issue by recruiting patients during all months of the year over a 2-year period. other potential sources of bias include patient selection; those patients most ill and requiring non-invasive ventilation were too ill to participate in the study. thompson et al. have published data linking influenza and rsv infection with increased mortality in the elderly. 22 there are increased admissions in older people in winter. fleming showed that acute respiratory infections are associated with hospitalisation and increased mortality. 23 it reinforces the health service implications of winter infection and increased exacerbations of copd. it is also noteworthy that the peak death rate is in patients diagnosed with acute respiratory disease. 23 influenza has been linked with increased health care use by the elderly and there is an excess mortality particularly in the elderly. 24 this paper highlights the impact of respiratory viral infection on health care resources. it supports the use of influenza vaccination in patients with underlying respiratory disease in order to reduce exacerbations and hospital admissions. in conclusion this study supports the hypothesis that respiratory viral infection is associated with exacerbations of copd. rhinovirus was the most common infecting agent identified and in two cases human metapneumovirus was also detected. dual infections were only seen amongst those patients admitted to hospital with acute exacerbations of copd. the development of multiplexed real-time pcr assays will enable this technology to be utilized in an acute diagnostic setting. relation of sputum inflammatory markers to symptoms and lung function changes in copd exacerbations bacterial infection in chronic obstructive pulmonary disease bacterial infection and the pathogenesis of copd infectious etiology of acute exacerbations of chronic bronchitis respiratory syncytial and other virus infections in persons with chronic cardiopulmonary disease respiratory viral infections in adults with and without chronic obstructive pulmonary disease detection of rhinovirus in induced sputum at exacerbation of chronic obstructive pulmonary disease respiratory viruses, symptoms, and inflammatory markers in acute exacerbations and stable chronic obstructive pulmonary disease respiratory viruses in exacerbations of chronic obstructive pulmonary disease requiring hospitalisation: a case-control study respiratory viral infections in patients with chronic, obstructive pulmonary disease coming together: the ats/ers consensus on clinical pulmonary function testing global strategy for the diagnosis, management, and prevention of chronic obstructive pulmonary disease: national heart, lung, and blood institute and world health organization global initiative for chronic obstructive lung disease (gold): executive summary antibiotic therapy in exacerbations of chronic obstructive pulmonary disease a touchdown nucleic acid amplification protocol as an alternative to culture backup for immunofluorescence in the routine diagnosis of acute viral respiratory tract infections role of latent viral infections in chronic obstructive pulmonary disease and asthma a newly discovered human pneumovirus isolated from young children with respiratory tract disease human metapneumovirus as a cause of community-acquired respiratory illness relevance of human metapneumovirus in exacerbations of copd respiratory syncytial virus infection in elderly and high-risk adults pcr detection of viral nucleic acid in fatal asthma: is the lower respiratory tract a reservoir for common viruses? latent adenoviral infection in the pathogenesis of chronic airways obstruction mortality associated with influenza and respiratory syncytial virus in the united states the contribution of influenza to combined acute respiratory infections, hospital admissions, and deaths in winter the impact of influenza-associated respiratory illnesses on hospitalizations, physician visits, emergency room visits, and mortality staff and patients of the mater hospital belfast. this study was supported by a unrestricted grants from astrazeneca, boehringer ingelheim and the mater hospital. no conflicts of interest declared. key: cord-284889-hth8nf5b authors: tsukagoshi, hiroyuki; ishioka, taisei; noda, masahiro; kozawa, kunihisa; kimura, hirokazu title: molecular epidemiology of respiratory viruses in virus-induced asthma date: 2013-09-12 journal: front microbiol doi: 10.3389/fmicb.2013.00278 sha: doc_id: 284889 cord_uid: hth8nf5b acute respiratory illness (ari) due to various viruses is not only the most common cause of upper respiratory infection in humans but is also a major cause of morbidity and mortality, leading to diseases such as bronchiolitis and pneumonia. previous studies have shown that respiratory syncytial virus (rsv), human rhinovirus (hrv), human metapneumovirus (hmpv), human parainfluenza virus (hpiv), and human enterovirus infections may be associated with virus-induced asthma. for example, it has been suggested that hrv infection is detected in the acute exacerbation of asthma and infection is prolonged. thus it is believed that the main etiological cause of asthma is ari viruses. furthermore, the number of asthma patients in most industrial countries has greatly increased, resulting in a morbidity rate of around 10-15% of the population. however, the relationships between viral infections, host immune response, and host factors in the pathophysiology of asthma remain unclear. to gain a better understanding of the epidemiology of virus-induced asthma, it is important to assess both the characteristics of the viruses and the host defense mechanisms. molecular epidemiology enables us to understand the pathogenesis of microorganisms by identifying specific pathways, molecules, and genes that influence the risk of developing a disease. however, the epidemiology of various respiratory viruses associated with virus-induced asthma is not fully understood. therefore, in this article, we review molecular epidemiological studies of rsv, hrv, hpiv, and hmpv infection associated with virus-induced asthma. acute respiratory illness (ari) is a major cause of morbidity and mortality worldwide (williams et al., 2002; sloots et al., 2008) . ari imposes a large burden on health, particularly in children. for community-based care, ari has been estimated at a cost of over us$100 per case (ehlken et al., 2005) . the disease burden for ari is estimated at 94,037,000 disability-adjusted life years and 3.9 million deaths (world health organization, 2002) . thus, ari has a huge impact on health and society. although severe lower respiratory tract infections have been observed, ari is most often associated with mild upper respiratory infection (uri). most ari cases in early childhood are confirmed as uri, leading to symptoms of the common cold with coryza and cough. in contrast, around one-third of infants with ari develop lower respiratory tract symptoms such as tachypnea, wheezing, severe cough, breathlessness, and respiratory distress (tregoning and schwarze, 2010) . in general, viruses are the most common causative agents of ari. more than 200 different types of viruses are known to cause ari, with respiratory syncytial virus (rsv), human rhinovirus (hrv), human metapneumovirus (hmpv), and human parainfluenza virus (hpiv) most commonly identified in ari patients. indeed, together with these respiratory viruses, human enterovirus (hev), influenza virus (infv), human coronavirus (hcov), adenovirus (adv), and human bocavirus (hbov) account for around 70% of aris detected (kusel et al., 2006) . respiratory viral infections can have severe adverse outcomes in patients with established asthma and are associated with nearly 80% of asthma exacerbation episodes (nicholson et al., 1993; johnston et al., 1995; wark et al., 2002; heymann et al., 2004; grissell et al., 2005) . accumulating evidence indicates that the etiology of most cases of asthma, namely virus-induced asthma, is linked to such respiratory virus infections. in addition, rsv and hrv are the most frequently detected pathogens and may play an important role in viral induction and exacerbation of asthma. molecular biology techniques have developed rapidly over recent years. the application of molecular techniques to the study of virus-induced asthma enhances epidemiologic studies by improving our ability to classify these pathogens into meaningful groups (foxman and riley, 2001) . in this review, we focus on molecular epidemiological studies of respiratory viruses, including rsv, hrv, hmpv, and hpiv infections, associated with virus-induced asthma. in infancy, illnesses such as bronchiolitis share many clinical features with acute asthma, including wheezing, rapid breathing, prolonged expiratory phase inflammation, and respiratory www.frontiersin.org compromise. respiratory viruses are detected in the majority of asthma exacerbations in both children (80-85%) and adults (75-80%; johnston et al., 1995; grissell et al., 2005) . in addition, wheezing illnesses are also closely associated with respiratory viral infections in all age groups (gern, 2010) . fujitsuka et al. (2011) attempted to detect various respiratory viruses in japanese children with acute wheezing using pcr technology and found viruses in samples from 86.1% patients: rsv or hrv alone were detected in 40.9 and 31.3% patients, respectively and both rsv and hrv were detected in 12.2% patients. other previous reports suggested that the prevalence of rsv and hrv is similar (36 and 42%, respectively) in children less than 2 years of age, but differs (27 and 66%) in older children (johnston et al., 1995; grissell et al., 2005) . in addition, fujitsuka et al. (2011) suggested that rsv was the dominant species detected in patients with no history of wheezing and/or asthma, while hrv was dominant in patients with such a history. thus, the main causative viral agent of asthma depends on previous illness and age. around one-third of infants who have an acute wheezing illness go on to develop recurrent wheezing, indicating that viral respiratory illnesses in early life promote asthma. recently, the "two-hit" hypothesis has been proposed, whereby viral infections promote asthma mainly in predisposed children (gern, 2010) . infants who develop virus-induced wheezing episodes are at increased risk for subsequent asthma, but most acute wheezing illnesses in infancy resolve with no long-term sequelae. it has been recognized for years that rsv infections often produce the first episode of wheezing in children who go on to develop chronic asthma (lemanske, 2004) . indicators of heightened risk for developing asthma include wheezing episodes caused by hrv infections and the development of atopic features such as atopic dermatitis, allergen-specific ige for foods or aeroallergens (e.g., house dust, mites, or cat or dog dander), and blood eosinophilia (figure 1) . once asthma has been established, hrv infections are the most common cause of figure 1 | relationship between respiratory viral infections and development of asthma. host-pathogen interactions that determine the severity of respiratory illnesses, and risk for subsequent asthma was increased by respiratory virus infection, especially due to rsv, in infants. although most acute wheezing resolves within a relatively short time, a history of wheezing and host immunological conditions (e.g., atopic features) heightens the risk for asthma. once asthma is established, hrv infections are the most common causative agents of asthma in children. acute exacerbations, especially in children. as in infancy, atopy is an important risk factor for acute episodes of virus-induced wheezing (kusel et al., 2007) . many previous reports have suggested that such respiratory virus infections are deeply associated with virus-induced asthma (kusel et al., 2007; pierangeli et al., 2007; kuehni et al., 2009; fujitsuka et al., 2011; kato et al., 2011) . thus, it is entirely plausible that viral infections induction and/or exacerbation asthma in children. respiratory syncytial virus of genus pneumovirus and family paramyxoviridae causes ari in children (vardas et al., 1999; peter and james, 2006) . rsv infection may cause major problems in infants less than 1 year of age and can lead to life-threatening aris such as bronchiolitis and bronchopneumonia (shay et al., 1999; leung et al., 2005; yorita et al., 2007) . epidemiological studies suggest that around 70% of infants have experienced an rsv infection by the age of 1 year, and 100% by the age of 2 years; host response to the virus varies greatly, but includes upper respiratory tract infections, typical bronchiolitis, and rsv-induced wheezy bronchitis (cane, 2001; kuehni et al., 2009 ). long-term prospective case-control and cohort studies have also linked rsv bronchiolitis to the development of wheezing and asthma later in childhood (sigurs et al., 1995 (sigurs et al., , 2005 (sigurs et al., , 2010 henderson et al., 2005) . thus, rsv infections may be associated with the initiation and/or exacerbation of asthma. the rsv genome encodes 11 proteins (peter and james, 2006) . among these, the attachment glycoprotein (g) is a major structural protein that may be associated with both infectivity and antigenicity (johnson et al., 1987; rueda et al., 1991) . molecular epidemiological studies have shown that rsv can be classified into two phylogenetic subgroups, rsv-a and rsv-b (mufson et al., 1985) . the strains of subgroup a can be subclassified into eight genotypes (ga1-ga7 and saa1), as can those of subgroup b (ba, gb1-gb4, and sab1-3; parveen et al., 2006) . from phylogenetic analysis of the g gene of rsv, martinello et al. (2002) showed that rsv belonging to ga3 genotype may be associated with greater severity of illness in, for example, bronchiolitis and pneumonia. although ga3 genotype has been detected in the united kingdom, spain, and new zealand, it is not the most prevalent strain (cane et al., 1991; garcia et al., 1997; matheson et al., 2006) . martinello et al. (2002) therefore suggested that the association between greater severity of illness and ga3 genotype may be solely due to a transient shift in genotype-specific immune status within the community. in addition, correlations between certain strains and/or genotypes of rsv and slight differences in disease severity have been described previously (hall et al., 1990; walsh et al., 1997) . some genotypes such as subgroup a genotypes ga1, ga2, ga5, ga7, and na1 and subgroup b genotype ba have been detected throughout the world in recent years (zlateva et al., 2004; parveen et al., 2006; zhang et al., 2007; nakamura et al., 2009; rebuffo-scheer et al., 2011) . of these, na1 is a novel genotype known to be genetically close to ga2 genotype, while ga2 genotype and ba genotype are the most common genotypes of rsv subgroups a and b around the world and have persisted for many years (tran et al., 2013) . furthermore, a new genotype belonging to rsv-a, on1, has been detected in some countries, including canada, korea, malaysia, south africa, and japan (eshaghi et al., 2012; lee et al., 2012; khor et al., 2013; tsukagoshi et al., 2013; valley-omar et al., 2013) . this genotype contains a unique tandem repeat (72nt sequence duplication) in the c-terminal 3rd hypervariable region of the g gene, and may be classified as a subdivision of na1 (eshaghi et al., 2012) . some reports have suggested that the severity of illness is not linked to subgroups or genotypes, but is associated with the quantity of rsv in nasopharyngeal aspirate (sullender, 2000; campanini et al., 2007) . a larger population study is needed to identify the different rsv genotypes circulating in different areas to gain a better understanding of the relationship between disease severity and rsv genotype. the g protein is a major antigen of rsv and amino acid substitutions may be related to changes in antigenicity. there are some reports of amino acid substitutions, and some positively selected sites in the c-terminal 3rd hypervariable region of the g gene have been estimated (botosso et al., 2009; yoshida et al., 2012; kushibuchi et al., 2013) . for example, yoshida et al. (2012) estimated some sites under positive selection in the region (asn250ser, met262glu, arg297lys,and arg297glu substitutions in rsv-a strains were estimated by the rel method, and asn273tyr and leu274pro substitutions of rsv-a, as well as leu237pro substitution of rsv-b, were estimated by the ifel method). botosso et al. (2009) found 29 and 23 amino acid sites under putative positive selection in rsv-a and rsv-b, respectively. in addition, some unique positively selected sites were found in the g gene . these amino acid variations at these sites might play a key role in severe respiratory infection, such as bronchiolitis (goto-sugai et al., 2010) . furthermore, the rate of molecular evolution of the region might be high. for example, kushibuchi et al. (2013) estimated the evolutionary rate of rsv-a at 3.63 × 10 −3 substitutions/site/year, while that of rsv-b was estimated at 4.56 × 10 −3 substitutions/site/year. thus, it is suggested that this c-terminal 3rd hypervariable region in the g gene of rsv-a and -b evolved rapidly . based on host immunological conditions, it is suggested that host immunity such as tlr4 polymorphism is linked to symptomatic rsv infection (delgado et al., 2009) . thus, both the antigenicity of the viruses and host immune conditions may play important roles in the pathophysiology of severe respiratory infections such as bronchiolitis, pneumonia, and virus-induced asthma (awomoyi et al., 2007) . human rhinovirus are a group of positive-sense ssrna viruses belonging to genus enterovirus in the family picornaviridae (turner and couch, 2007) . although hrvs were previously thought to be mainly associated with the common cold causing mild respiratory symptoms, recent reports strongly suggest that hrvs may induce and/or exacerbate asthma (virus-induced asthma; chung et al., 2007; turner and couch, 2007; busse et al., 2010; gern, 2010; khadadah et al., 2010) . one report suggested that hrv wheezing illness within the first three years of life is significantly associated with the development of asthma at age 6 years (jackson et al., 2008) . another report suggested that hrvs are major agents in the induction of wheezing and exacerbation of asthma (khadadah et al., 2010) . thus, hrvs are being re-evaluated as important agents of ari in humans (imakita et al., 2000; wos et al., 2008) . the basis for these lower respiratory symptoms has been a source of controversy in terms of the mechanisms of hrv pathogenesis. there are a variety of potential barriers to hrv infection of the lungs, including temperature-sensitive replication of the virus. for this reason, it is thought that the optimum propagation temperature of hrvs may be 32-35 • c in vitro (papadopoulos et al., 1999; schroth et al., 1999) . however, a recent study suggested that hrvs can propagate in lower airway tissues and this may be an important factor in the development of airway obstruction, coughing, and wheezing that can lead to bronchiolitis and pneumonia (mosser et al., 2005) . hrv has been concomitantly isolated with bacterial pathogens in 24-54% of children and 10-18% of adults with pneumonia (juven et al., 2004; templeton et al., 2005; jennings et al., 2008) . thus, it is not clear whether hrv is ever the causative agent for the disease. human rhinovirus were previously classified into two species, hrv species a (hrv-a) and species b (hrv-b), containing over 100 serotypes (turner and couch, 2007) . however, a genetically heterogeneous third species, hrv species c (hrv-c), was discovered recently (lamson et al., 2006; mcerlean et al., 2007) . recent reports suggest that hrv-a, b, and c have a unique and wide genetic diversity simmonds et al., 2010; arakawa et al., 2012) . hrv-a and -c appear to be mainly associated with aris and virus-induced asthma, while hrv-b has been detected in a relatively small number of patients with aris (linsuwanon et al., 2009; wisdom et al., 2009; smuts et al., 2011) . our previous findings obtained from samples from children with aris in japan indicated that hrv-a and -c can be classified into many clusters in the phylogenetic tree, with 30% nucleotide divergence of the vp4/vp2 coding region (mizuta et al., 2010a; arakawa et al., 2012; kiyota et al., 2013) . in addition, kiyota et al. (2013) estimated that the rate of molecular evolution of the vp4/vp2 coding region was rapid (3.07 × 10 −3 substitutions/site/year) in hrv-c. these results suggest that hrv-a and -c detected in ari cases are the predominant strains and have varied genetic properties (wisdom et al., 2009; mizuta et al., 2010a; arakawa et al., 2012) . thus, the association between hrv type and disease severity is not fully understood. there may be important differences in the susceptibility of individuals to the replication of hrv in lower airway tissues. parry et al. (2000) and gern et al. (2000) found that weak peripheral blood mononuclear cell (pbmc) th1 (ifn-γ) response to hrv infection is associated with increased viral shedding, and decreased proliferative response of pbmcs to hrv is associated with increased severity of symptoms. in addition, it was found that weak th1 responses (ifn-γ/il-5 mrna ratio) in sputum are also associated with greater severity of illness . furthermore, weak th1 responses to viral infection in adults with asthma have been associated with decreased lung function and greater airway responsiveness (brooks et al., 2003) . these results indicate that individuals with a weak th1 response to viruses, and perhaps individuals with asthma in general, may be more susceptible to hrv illnesses, and this association may be strongest in those with more severe disease (parry et al., 2000; gern et al., 2000; brooks et al., 2003) . other epidemiological and biological factors, such as allergy, atopic dermatitis, www.frontiersin.org or a family history of allergy, may be related to virus-induced asthma (green et al., 2002; singh et al., 2007) . recently it is suggested that variants at the 17q21 locus were associated with hrv induced asthma in children who had a history wheezing illnesses, although associations of 17q21 variants with asthma were restricted to children who had a history of hrv wheezing illnesses (calışkan et al., 2013) . human metapneumovirus is a recently identified rna virus belonging to the paramyxoviridae family, of genus metapneumovirus (collins and crowe, 2007) . hmpv is a major pathogen that causes ari in all ages (collins and crowe, 2007) . the first hmpv infection appears to take place within the first six months of life, after which infections may occur repeatedly and frequently (schildgen et al., 2011) . the nosocomial impact of hmpv is estimated to be as high as that for rsv. in an hmpv outbreak in japan, 34.8% of elderly patients who shared the same day care room in a hospital were infected with hmpv (honda et al., 2006) . higher morbidity is observed in young children, the elderly, and immunocompromised adults (boivin et al., 2002; falsey et al., 2003; van den hoogen et al., 2003; sumino et al., 2005; williams et al., 2005; o'gorman et al., 2006) . hmpv is classified into two genotypes (a and b) and four subgroups (a1, a2, b1, and b2) by phylogenetic analysis, using the f and g genes (biacchesi et al., 2003; van den hoogen et al., 2004) . subgroup a2 has been subdivided into two lineages, subgroup a2a and a2b (huck et al., 2006) . it has been suggested that these genotypes circulate in variable proportions in some areas (gerna et al., 2005; mackay et al., 2006) . although the molecular epidemiological information on hmpv has gradually accumulated, the detailed epidemiology remains unclear (mizuta et al., 2010b; pitoiset et al., 2010; omura et al., 2011) . hmpv infections can occur throughout the year, but seasonality has been described in several studies, with the epidemiological peak occurring several months later than that observed for rsv epidemics (robinson et al., 2005; wilkesmann et al., 2006; madhi et al., 2007; aberle et al., 2008 aberle et al., , 2010 heininger et al., 2009 ). it remains unclear whether different hmpv subgroups are associated with differences in the clinical course of disease. several groups have suggested that hmpv subgroup a might be associated with more severe clinical disease (martinello et al., 2002; kaida et al., 2006; vicente et al., 2006; arnott et al., 2013) , while others have reported that subgroup b may cause more severe illness (esper et al., 2004; pitoiset et al., 2010) , and still other groups have found no evidence for differential severity caused by different hmpv lineages (agapov et al., 2006; manoha et al., 2007; larcher et al., 2008; xiao et al., 2010) . previous reports suggested that the substitution rates for the g gene (3.5 × 10 −3 substitution/site/year) and the f gene (7.1 × 10 −4 to 8.5 × 10 −4 substitution/site/year) are high, and some positively selected sites have been found in the latter (de graaf et al., 2008; yang et al., 2009) . it may be that there is a correlation between some positively selected epitopes and disease severity. thus, the association between hmpv subgroup and disease severity is controversial. to gain a better understanding of host responses that may contribute to differences in clinical severity between hmpv subgroups, a more detailed analysis that includes host immunological status is needed. human parainfluenza virus belong to the paramyxoviridae family. there are two genera of hpiv, respirovirus (hpiv-1 and hpiv-3) and rubulavirus (hpiv-2 and hpiv-4; karron and collins, 2007) . hpiv is classified into four serotypes (hpiv1-4) , all of which can cause various ari in humans such as uri, croup, bronchitis, asthma, and pneumonia (henrickson, 2003; karron and collins, 2007) . although hpiv type 4 (hpiv4) is rarely reported, hpiv1-3 are important causes of various ari, including the common cold, croup, bronchitis, bronchiolitis, and pneumonia in children, and they commonly re-infect both children and adults. while such infections are generally mild in healthy persons, they may cause serious diseases in children, such as asthma (henrickson, 2003; karron and collins, 2007) . although fewer hpiv strains have been detected compared with other respiratory viruses such as rsv, hrv, and hmpv, previous reports suggest that hpiv1 and 3 are the dominant viruses in children with ari (reed et al., 1997) . indeed, serological surveys indicate that at least 60% of children have been infected with hpiv3 by 2 years of age, approximately 80% have been infected by age 4, and at least 75% have been infected with hpiv1 by 5 years of age (parrott et al., 1959 (parrott et al., , 1962 . hpiv1 and 3 show high prevalence and are associated with up to 12% of acute lower respiratory tract infections in adults (azevedo et al., 2003; matsuse et al., 2005) . hpiv1 and hpiv3, may be major agents of ari throughout the world, along with other viruses such as rsv, hrv, and hmpv (laurichesse et al., 1999; iwane et al., 2004; monto, 2004; do et al., 2011) . in addition, it is suggested that hpiv is a major causative agent of virus-induced asthma (henrickson and savatski, 1997) . several previous studies have reported that hpiv1 infections demonstrate clear outbreaks in autumn, mostly in september and november, every 2 years (knott et al., 1994; hall, 2001; counihan et al., 2001) . other studies have reported that hpiv3 causes yearly outbreaks around the globe, mainly in the spring-summer season (knott et al., 1994; counihan et al., 2001; hall, 2001; mizuta et al., 2013) . a recent study suggested that four different types of hpiv cause similar clinical manifestations in patients, and the clinical presentation of hpiv infection may differ depending on patient age (liu et al., 2013) . henrickson and savatski (1996) analyzed the longitudinal evolution of the hn coding region in 13 strains of hpiv1 isolated in the usa. these results showed that the antigenic and genetic subgroups are very stable. in addition, suggested that the evolution of the hn gene in the present hpiv1 isolates was relatively slow and that the gene is highly conserved. only a few reports on the molecular epidemiology of hpiv1 are available and it appears that the molecular epidemiology of hpiv is poorly understood. larger and more detailed studies on the association of hpiv with asthma are needed. hev68 was recently detected in asthmatic patients (hasegawa et al., 2011) . hev68 was found to be relatively acid resistant and thus could be distinguished from acid-sensitive hrv87 (schieble et al., 1967; kapikian et al., 1971) . hrv87 was recently reclassified frontiers in microbiology | virology as hev68 based on phylogenetic analysis and neutralization test, and some laboratories have confirmed its acid sensitivity ishiko et al., 2002; savolainen et al., 2002) . distinguishing between hrv and hev based on the acid sensitivity of isolates is therefore not appropriate for hev68. the number of reports of an association between respiratory disease and hev68 infection has recently increased. one report of the phylogenetic analysis of hev68 based on partial vp1 gene sequences indicates wide genetic diversity (linsuwanon et al., 2012) . in addition, tokarz et al. (2012) showed the presence of multiple clades among the circulating strains, and that all strains are spreading rapidly worldwide and contributing to the prevalence rates of respiratory diseases. in addition, asthmatic individuals infected with hev68 also have the propensity to develop unstable asthma or an acute attack (hasegawa et al., 2011) . influenza virus is also a major causative agent of ari in both children and adults. furthermore, asthmatic patients were found among children and adults hospitalized with seasonal infv (dao et al., 2010; dawood et al., 2010) . although it is recognized that viral infections such as rsv or hrv may induce and/or exacerbate asthma, the effect of infv on asthma remains arguable (johnston et al., 1995) . although one study suggested that a(h1n1)pdm09 viruses impose greater risk factors on children than seasonal infv (tran et al., 2012) , infv vaccine was available before the influenza season since infv causes more severe illness than other respiratory viruses. therefore, it is suggested that infv vaccine be recommended for children with asthma (kloepfer et al., 2012) . although the level of detection of hcov, hbov, or adv is relatively low, these infections are also detected in children with acute wheezing (chung et al., 2007; jartti et al., 2007) . further studies are needed to clarify the clinical roles of hcov, hbov, or adv infections and those of other respiratory viruses. in particular, the prevalence of hcov, hbov, or adv infection in healthy control subjects, assessment of disease severity by other clinical variables, and the immunological effects should be investigated. infants with severe bronchiolitis have an increased risk of developing recurrent wheezing later in life (chung et al., 2007) . hrv may be detected concurrently with other viruses such as rsv, hmpv, infv, or hcov (richard et al., 2008; fujitsuka et al., 2011) . considering their ubiquity, it is interesting that the number of respiratory viruses detected concurrently with hrv strains is relatively low mackay, 2007) , supporting the concept that hrvs have a direct role in the clinical outcome of infection (miller et al., 2007) . in fact, hrv strains are co-detected with other pathogens in reproducible, but clinically undefined, patterns (brunstein et al., 2008) . the hrv partnership with host immunity may be a mutualistic one, inadvertently imparting an advantage to the host by protecting against more cytopathic respiratory viral pathogens while the host provides a vessel for hrv replication and transmission. respiratory viruses other than rsv and multiple viral infections may contribute to the severity of bronchiolitis and asthma. indeed, it was reported that dual infections of hmpv and rsv or hrv and rsv confer a 5-to 10-fold increase of severe disease in children admitted to pediatric intensive care units semple et al., 2005) . in contrast, other studies reported that co-infection with two respiratory viruses was not significantly associated with disease severity (van woensel et al., 2006; wolf et al., 2006) . thus, there is no consensus on the effects of co-infection on disease severity. the effect of dual infection may depend upon which viruses co-infect together. for example, although there was no increase in severity when hrv and/or adv were detected during rsv infection, co-infection with both hmpv and rsv increased the rate of intensive care unit admissions (aberle et al., 2005; semple et al., 2005) . thus, although dual infections and reinfections have been well documented in children, chronic infection with the development of quasispecies cannot be ruled out without obtaining more complete data using high performance detection methods (hall and mccarthy, 1996) . respiratory viral infections are a major cause of virus-induced asthma in early life. although antiviral therapy is not yet available for patients infected with respiratory viruses, the detection and identification of these viruses could help to explain serious respiratory illness, provide guidance for medical care, and prevent unnecessary treatment with antibiotics. based on the results of many related studies, we propose a two-step hypothesis of asthma development in children. the first step is mainly due to rsv infection: when rsv infects bronchial cells, the bronchial cells produce various cytokines and chemokines. these responses cause hyperresponsiveness in the bronchial cells. in other words, rsv infection might create a preparatory step as the first step in the development of asthma. hrv infection might then bring about the second step in the development of asthma. an infant with a history of wheezing caused by rsv infection may develop the heavy wheezing of asthma due to hrv infection followed by rsv infection. to understand the cause of asthma, we need to examine the larger complex picture of genetic susceptibility, immune components, environmental exposures, and the interactions between these elements. aberle single versus dual respiratory virus infections in hospitalized infants: impact on clinical course of disease and interferon-gamma response human metapneumovirus subgroup changes and seasonality during epidemics molecular epidemiological study of human rhinovirus species a, b and c from patients with acute respiratory illnesses in japan genetic variability of human metapneumovirus amongst an all ages population in cambodia between association of tlr4 polymorphisms with symptomatic respiratory syncytial virus infection in high-risk infants and young children detection of influenza, parainfluenza, adenovirus and respiratory syncytial virus during asthma attacks in children older than 2 years old genetic diversity between human metapneumovirus subgroups human rhinovirus 87 and enterovirus 68 represent a unique serotype with rhinovirus and enterovirus features virological features and clinical manifestations associated with human metapneumovirus: a new paramyxovirus responsible for acute respiratory-tract infections in all age groups positive selection results in frequent reversible amino acid replacements in the g protein gene of human respiratory syncytial virus association of rhinovirus induced ifn-γ with increased asthma severity evidence from multiplex molecular assays for complex multipathogen interactions in acute respiratory infections role of viral respiratory infections in asthma and asthma exacerbations rhinovirus wheezing illness and genetic risk of childhood-onset asthma human respiratory syncytial virus (hrsv) rna quantification in nasopharyngeal secretions identifies the hrsv etiologic role in acute respiratory tract infections of hospitalized infants molecular epidemiology of respiratory syncytial virus identification of variable domains of the attachment (g) protein of subgroup a respiratory syncytial viruses detection of viruses identified recently in children with acute wheezing respiratory syncytial virus and metapneumovirus human parainfluenza virus-associated hospitalizations among children less than five years of age in the united states. pediatr adult hospitalizations for laboratory-positive influenza during the burden of seasonal influenza hospitalization in children evolutionary dynamics of human and avian metapneumoviruses lack of antibody affinity maturation due to poor toll-like receptor stimulation leads to enhanced respiratory syncytial virus disease viral etiologies of acute respiratory infections among hospitalized vietnamese children in ho chi minh city economic impact of community-acquired and nosocomial lower respiratory tract infections in young children in germany genetic variability of human respiratory syncytial virus a strains circulating in ontario: a novel genotype with a 72 nucleotide g gene duplication a 1-year experience with human metapneumovirus in children aged <5 years human metapneumovirus infections in young and elderly adults molecular epidemiology: focus on infection a molecular epidemiological study of respiratory viruses detected in japanese children with acute wheezing illness nosocomial respiratory syncytial virus infections: prevention and control in bone marrow transplant patients the abcs of rhinoviruses, wheezing, and asthma relationship of upper and lower airway cytokines to outcome of experimental rhinovirus infection changing circulation rate of human metapneumovirus strains and types among hospitalized pediatric patients during three consecutive winter-spring seasons genotyping and phylogenetic analysis of major genes in respiratory syncytial virus isolated from infants with bronchiolitis in japan synergism between allergens and viruses and risk of hospital admission with asthma: case-control study il-10 gene expression in acute virus-induced asthma respiratory syncytial virus and parainfluenza virus respiratory syncytial virus," in mandell, douglas and bennett's principles and practice of infectious disease occurrence of groups a and b of respiratory syncytial virus over 15 years: associated epidemiologic and clinical characteristics in hospitalized and ambulatory children enterovirus 68 infection in children with asthma attacks: virus-induced asthma in japanese children human metapneumovirus infections -biannual epidemics and clinical findings in children in the region of basel, switzerland hospitalization for rsv bronchiolitis before 12 months of age and subsequent asthma, atopy and wheeze: a longitudinal birth cohort study parainfluenza viruses two distinct human parainfluenza virus type 1 genotypes detected during the 1991 milwaukee epidemic antigenic structure, function, and evolution of the hemagglutinin-neuraminidase protein of human parainfluenza virus type 1 viral infections in relation to age, atopy, and season of admission among children hospitalized for wheezing outbreak of human metapneumovirus infection in elderly inpatients in japan novel human metapneumovirus sublineage pneumonia caused by rhinovirus human rhinovirus 87 identified as human enterovirus 68 by vp4-based molecular diagnosis population-based surveillance for hospitalizations associated with respiratory syncytial virus, influenza virus, and parainfluenza viruses among young children wheezing rhinovirus illnesses in early life predict asthma development in high-risk children respiratory viruses and acute asthma in children incidence and characteristics of viral community-acquired pneumonia in adults the g glycoprotein of human respiratory syncytial viruses of subgroups a, and b: extensive sequence divergence between antigenically related proteins community study of role of viral infections in exacerbations of asthma in 9-11 year old children clinical response to antibiotic therapy for communityacquired pneumonia seasonal distribution and phylogenetic analysis of human metapneumovirus among children in osaka city a collaborative report: rhinoviruses -extension of the numbering system parainfluenza viruses," in fields virology different cytokine profile and eosinophil activation are involved in rhinovirus-and rs virus-induced acute exacerbation of childhood wheezing respiratory syncytial virus and human rhinoviruses are the major causes of severe lower respiratory tract infections in kuwait displacement of predominant respiratory syncytial virus genotypes in malaysia between genetic analysis of the vp4/vp2 coding region in human rhinovirus species c in patients with acute respiratory infection in japan increased h1n1 infection rate in children with asthma parainfluenza viral infections in pediatric outpatients: seasonal patterns and clinical characteristics causal links between rsv infection and asthma: no clear answers to an old question role of respiratory viruses in acute upper and lower respiratory tract illness in the first year of life: a birth cohort study early-life respiratory viral infections, atopic sensitization, and risk of subsequent development of persistent asthma molecular evolution of attachment glycoprotein (g) gene in human respiratory syncytial virus detected in japan community epidemiology of human metapneumovirus, human coronavirus nl63, and other respiratory viruses in healthy preschool-aged children using parent-collected specimens masstag polymerasechain-reaction detection of respiratory pathogens, including a new rhinovirus genotype, that caused influenza-like illness in new york state during comparison of human metapneumovirus genotypes from the province of bolzano in northern italy with strains from surrounding regions in italy and austria epidemiological features of parainfluenza virus infections: laboratory surveillance in england and wales, 1975-1997 complete genome sequence of human respiratory syncytial virus genotype a with a 72-nucleotide duplication in the attachment protein g gene viral infections and asthma inception respiratory syncytial virus bronchiolitis high prevalence of human rhinovirus c infection in thai children with acute lower respiratory tract disease molecular epidemiology and evolution of human enterovirus serotype 68 in thailand epidemiology and clinical presentation of the four human parainfluenza virus types human bocavirus; multisystem detection raises questions about infection genetic diversity of human metapneumovirus over 4 consecutive years in australia seasonality, incidence, and repeat human metapneumovirus lower respiratory tract infections in an area with a high prevalence of human immunodeficiency virus type-1 infection epidemiological and clinical features of hmpv, rsv and rvs infections in young children distinct patterns of evolution between respiratory syncytial virus subgroups a and b from new zealand isolates collected over thirty-seven years correlation between respiratory syncytial virus genotype and severity of illness naturally occurring parainfluenza virus 3 infection in adults induces mild exacerbation of asthma associated with increased sputum concentrations of cysteinyl leukotrienes characterisation of a newly identified human rhinovirus, hrv-qpm, discovered in infants with bronchiolitis analysis of genetic diversity and sites of recombination in human rhinovirus species c rhinovirusassociated hospitalizations in young children phylogenetic and cluster analysis of human rhinovirus species a (hrv-a) isolated from children with acute respiratory infections in yamagata endemicity of human metapneumovirus subgenogroups a2 and b2 in yamagata seasonal patterns of respiratory syncytial virus, influenza a virus, human metapneumovirus, and parainfluenza virus type 3 infections on the basis of virus isolation data between detailed genetic analysis of hemagglutinin-neuraminidase glycoprotein gene in human parainfluenza virus type 1 isolates from patients with acute respiratory infection between 2002 and 2009 in yamagata prefecture occurrence of respiratory virus: time, place and person quantitative and qualitative analysis of rhinovirus infection in bronchial tissues two distinct subtypes of human respiratory syncytial virus genotypic and phylogenetic analysis of the g gene of respiratory syncytial virus isolates in okinawa respiratory viruses and exacerbations of asthma in adults human metapneumovirus in adults: a short case series detection of human metapneumovirus genomes during an outbreak of bronchitis and pneumonia in a geriatric care home in shimane association of rhinovirus infection with increased disease severity in acute bronchiolitis rhinoviruses replicate effectively at lower airway temperatures acute respiratory diseases of viral etiology. iii. parainfluenza. myxoviruses clinical features of infection with hemadsorption viruses rhinovirusinduced pbmc responses and outcome of experimental infection in allergic subjects genetic variability in the g protein gene of group a and b respiratory syncytial viruses from india respiratory syncytial virus and metapneumovirus detection and typing by molecular techniques of respiratory viruses in children hospitalized for acute respiratory infection in rome, italy human metapneumovirus genotypes and severity of disease in young children (n=100) during a 7-year study in dijon hospital, france whole genome sequencing and evolutionary analysis of human respiratory syncytial virus a and b from milwaukee epidemiology and clinical impact of parainfluenza virus infections in otherwise healthy infants and young children <5 years old the impact of dual viral infection in infants admitted to a pediatric intensive care unit associated with severe bronchiolitis. pediatr seasonality and clinical features of human metapneumovirus infection in children in northern alberta premature stop codons in the g glycoprotein of human respiratory syncytial viruses resistant to neutralization by monoclonal antibodies genetic clustering of all 102 human rhinovirus prototype strains: serotype 87 is close to human enterovirus 70 a probable new human picornavirus associated with respiratory diseases human metapneumovirus: lessons learned over the first decade rhinovirus replication causes rantes production in primary bronchial epithelial cells dual infection of infants by human metapneumovirus and human respiratory syncytial virus is strongly associated with severe bronchiolitis bronchiolitis-associated hospitalizations among us children asthma and allergy patterns over 18 years after severe rsv bronchiolitis in the first year of life asthma and immunoglobulin e antibodies after respiratory syncytial virus bronchiolitis: a prospective cohort study with matched controls severe respiratory syncytial virus bronchiolitis in infancy and asthma and allergy at age 13 proposals for the classification of human rhinovirus species c into genotypically assigned types bronchiolitis to asthma: a review and call for studies of gene-virus interactions in asthma causation human rhinovirus infection in young african children with acute wheezing emerging respiratory agents: new viruses for old diseases? respiratory syncytial virus genetic and antigenic diversity detection of severe human metapneumovirus infection by real-time polymerase chain reaction and histopathological assessment improved diagnosis of the etiology of communityacquired pneumonia with real-time polymerase chain reaction worldwide emergence of multiple clades of enterovirus 68 comparison of children hospitalized with seasonal versus pandemic influenza a molecular epidemiology and disease severity of human respiratory syncytial virus in vietnam respiratory viral infections in infants: causes, clinical symptoms, virology, and immunology genetic analysis of attachment glycoprotein (g) gene in new genotype on1 of human respiratory syncytial virus detected in japan rhinovirus," in fields virology novel respiratory syncytial virus subtype on1 among children antigenic and genetic variability of human metapneumoviruses prevalence and clinical symptoms of human metapneumovirus infection in hospitalized patients absence of human metapneumovirus co-infection in cases of severe respiratory syncytial virus infection the epidemiology of respiratory syncytial virus (rsv) infections in south african children differences in clinical severity between genotype a and genotype b human metapneumovirus infection in children severity of respiratory syncytial virus infection is related to virus strain neutrophil degranulation and cell lysis is associated with clinical severity in virus-induced asthma human metapneumovirus infections cause similar symptoms and clinical severity as respiratory syncytial virus infections estimates of worldwide distribution of child deaths from acute respiratory infections human metapneumovirus infection plays an etiologic role in acute asthma exacerbations requiring hospitalization in adults genetics, recombination and clinical features of human rhinovirus species c (hrv-c) infections; interactions of hrv-c with other respiratory viruses comparison of human metapneumovirus, respiratory syncytial virus and influenza a virus lower respiratory tract infections in hospitalized young children the presence of rhinovirus in lower airways of patients with bronchial asthma prevalence and clinical and molecular characterization of human metapneumovirus in children with acute respiratory infection in china genetic diversity and evolution of human metapneumovirus fusion protein over twenty years severe bronchiolitis and respiratory syncytial virus among young children in hawaii molecular epidemiology of the attachment glycoprotein (g) gene in respiratory syncytial virus in children with acute respiratory infection in japan in 2009/2010 genetic variability of group a and b respiratory syncytial viruses isolated from 3 provinces in china molecular evolution and circulation patterns of human respiratory syncytial virus subgroup a: positively selected sites in the attachment g glycoprotein this study was supported in part by research on emerging and re-emerging infectious diseases, labour, and welfare programs from the ministry of health, labour, and welfare, japan. the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. key: cord-312615-q333qgps authors: knobbe, rebecca b; diallo, abdallah; fall, amary; gueye, aida d; dieng, assane; van immerzeel, tabitha d; ba, abou; diop, amadou; diop, abdoulaye; niang, mbayame; boye, cheikh sb title: pathogens causing respiratory tract infections in children less than 5 years of age in senegal date: 2019-12-30 journal: microbiol insights doi: 10.1177/1178636119890885 sha: doc_id: 312615 cord_uid: q333qgps introduction: while acute respiratory tract infections are the main cause of paediatric mortality and morbidity worldwide, pathogen patterns shift due to factors such as hygiene, vaccinations, and antibiotic resistance. knowledge about current cause of respiratory infections is lacking, particularly in lowand middle-income countries. the aim of this study was to identity the various respiratory pathogens causing acute respiratory tract infections in children below 5 years of age visiting a sub-urban primary care clinic in senegal. methods: a case-control study was performed in september and october 2018. oropharyngeal swabs were collected from cases; infants with fever and respiratory symptoms, and controls; children involved in the vaccination programme. viral identification was conducted by polymerase chain reaction for 21 different viruses; bacteria were identified by culture studies. associations between microorganisms, acute respiratory infection and severity of disease were calculated by multivariate regression adjusting for confounders such as age, sex, and living area. results: overall, 102 cases and 96 controls were included. microorganisms were detected in 90.1% of cases and 53.7% of controls (p < .001). influenza virus a (including h1n1), influenza virus b, respiratory syncytial virus (rsv), and streptococcus pneumoniae were independently associated with acute respiratory tract infections. co-detection of two or more pathogens was present in 49.5% of cases; 31.7% of cases had a pneumonia and 90.2% was treated with antibiotics. conclusions: this case-control study in a primary care setting in sub-saharan africa found influenza virus a and b, rsv, and s pneumoniae to be the main causes of acute respiratory tract infections in children below 5 years of age. we recommend evaluation of antibiotics prescription behaviour in this setting. worldwide, the leading cause of paediatric mortality and morbidity in the postneonatal period remains lower respiratory tract infections (lrtis). the who (world health organization) estimated that more than 800 000 of the 5.6 million total deaths in children less than 5 years old were caused by lrtis in 2016. 1 a meta-analysis found a hospital-based case-fatality ratio of 6.1% in children in lowincome countries with severe lrti, compared with 3.9% in high-income countries. 2 children with lrti can present with a highly variable package of symptoms including cough, tachypnoea, dyspnoea, fever, rhinitis, chest pain, abdominal pain, vomiting, diarrhoea, headache and lethargy. the who guidelines states tachypnoea as the main diagnostic criterion for pneumonia requiring an antibiotic. 3 however, distinction from upper airway infections and other illnesses such as asthma/bronchiolitis, anaemia or malaria can be challenging when minimal diagnostic tools are available. 4 furthermore, in many low-and middle-income countries, treatment policies are empiric or based on foreign guidelines, due to a lack of local causal studies. 5 these could be reasons why research has discovered an overdiagnosis and overprescription of antibiotics to treat pneumonia in low-income countries. 6 one of the biggest public health problems of this century is antimicrobial resistance. it threatens the effective treatment of major infections and is induced by an overuse and inappropriate usage of antibiotics. 7 this phenomena has major consequences: higher health-related costs because of the necessity of more expensive antibiotics, rising mortality rates due to pathogens causing respiratory tract infections in children less than 5 years of age in senegal 2 microbiology insights untreatable infections and an increasing danger for the immunosuppressed patient. 7, 8 moreover, viral causes of lrtis have become seemingly more important. 9 ancient studies in mostly western countries frequently described haemophilus influenza b (hib), streptococcus pneumoniae, and moraxella catarrhalis as major causative pathogens of lrtis. 10, 11 nonetheless, while in 2000, s pneumoniae was responsible for 600 000 deaths in children, in 2015, this number dropped to 294 000 children. 12 recent developments, such as the implementation of the pneumococcal conjugate vaccine and the hib vaccine, are responsible for a reduction in the burden of these respiratory bacteria but may also have induced a shift in the pathogens causing lrtis. 9, 13 recent research in senegal indicated viruses as main pathogens of all acute respiratory tract infections (artis), both upper and lower. the frequency of virus detection was even higher than those found in several other countries. 14,15 viruses most commonly found in senegal were respiratory syncytial virus (rsv ), adenovirus, rhinovirus, influenza virus a, enterovirus, human bocavirus, and parainfluenza viruses. 16 furthermore, there are indications that nowadays artis are more often caused by the sequential or concurrent interaction of different pathogens and that the presence of multiple organisms is linked with the existence of a lrti and with severity of disease. 17, 18 to reduce paediatric mortality caused by lrti, while fighting antibiotic resistance, more knowledge about the cause of respiratory infections, especially in low-and middle-income countries, is essential. case-control studies have been conducted in africa, investigating the association between different pathogens and pneumonia. these studies found strongest associations for s pneumoniae, bordetella pertussis, rsv, influenza virus a, and human metapneumovirus. [19] [20] [21] [22] [23] [24] however, these studies were conducted in hospitalized settings while most children with lrti are treated in primary care clinics. to the best of our knowledge, no case-control studies investigating artis in primary care have been conducted in sub-saharan africa. therefore, the aim of this case-control study was to investigate the prevalence of the different viruses and bacteria colonizing the airways and their association with the occurrence of artis and severity of disease in children less than 5 years of age, visiting a sub-urban primary care clinic in dakar, senegal. an unmatched case-control study was performed at the institute de pédiatrie sociale (ips), a primary care clinic in dakar, senegal, during september and october 2018. institute de pédiatrie sociale is a free government health care clinic for children and pregnant women based in pikine, a sub-urban area. children with respiratory infections were enrolled using convenience sampling at the paediatric outpatient ward. controls were enrolled during the vaccination programme, which took place once a week. exclusion criteria for controls were fever and respiratory symptoms. standardized forms were used to prospectively gather information concerning patient demographics, medical history, clinical signs, and symptoms at enrolment and treatment. oropharyngeal swabs and bloods samples were taken in cases. one week after enrolment, symptoms and signs were evaluated by phone call in all cases. every case was called a minimum of 3 times when parents did not respond. when a child had to be admitted to a hospital, daily follow-up of signs and symptoms was conducted. data were imported in the data management software, excel. databases were anonymized. this study has been approved by the ethics committee for research of the cheikh anta diop university of dakar. data were gathered after an informed consent statement had been signed by parents or a legal guardian. children aged less than 5 years old with an acute respiratory infection defined as (antecedents of ) fever and at least one respiratory symptom were found suitable for inclusion. cases were excluded if the symptoms began >10 days before enrolment and/or if antibiotics were taken within 48 hours before enrolment. exclusion criteria for controls were signs of fever or respiratory symptoms. fever was defined as a rectal or ear temperature of ⩾38.0°c or an axillary temperature of ⩾37.5°c. antecedents of fever had to occur within 24 hours before enrolment. respiratory symptoms included cough, thoracic pain, sore throat, dyspnoea, chest indrawings, tachypnoea, cyanosis, and rhinorrhoea. tachypnoea was defined using the who definition of a respiratory rate >60 breaths/min in children <2 months of age, >50 breaths/min in children 2 to 12 months of age, and >40 breaths/minute in children ⩾1 year of age. 3 tachycardia was defined as >160 beats per minute (bpm) for infants <2 years of age and >140 bpm for children 2 to 12 years of age. 25 elevated c-reactive protein (crp) suggestive for severity of disease was defined as ⩾50 mg/l. 26, 27 leucocytosis was present if leucocytes are increased to more than 11 000/mm 3 . pneumonia was defined using the who guidelines as tachypnoea with cough and/or dyspnoea. 3 alarming signs were defined as tachypnoea, chest indrawing, or hypoxemia. in all cases and controls, oropharyngeal samples were collected. all samples were analysed by the biotechnology unit of the laboratory of bacteriology of university hospital aristide le dantec and by the virology unit of pasteur institute. the samples were stocked and transported following the protocols. for bacterial samples, bacteria were isolated from appropriate culture media. morphological, cultural, and biochemical or antigenic characters were studied. strains were determined and knobbe et al 3 considered for infection if the bacterial load was at least 10 5 cfus/ml. all samples were tested using polymerase chain reaction (pcr) with a bio-ras cfx-96 thermo cycler and the ftd respiratory pathogens 33 (fast-track diagnostics), capable of detecting 21 viruses and 12 bacteria (supplement 1). in addition, blood samples were collected in cases and crp and leucocytes were determined by the laboratory of ips. c-reactive protein was determined using the crp latex kit 850a (lorne laboratories, berkshire, uk). based on g*power 3.1.9.2 version, a sample size of 182 was yielded, when using logistic regression, an alpha of .05, and a small-to-medium effect size with an odds ratio (or) of 1.60. 28 therefore, a sample size of 198 is sufficient to detect microorganisms as a predictor of acute respiratory infection. baseline characteristics were compared between cases and controls. continuous variables were expressed as medians and ranges. they were compared using the mann-whitney u test. categorical variables were expressed as frequencies and proportions and were compared using the chi-square test. to identify potential predictive factors, a univariable logistic regression was used. thereafter, established predictors of the outcomes (occurrence of respiratory infection and severity of disease) (p < .1) were included in multivariable logistic regression. data were presented as ors and 95% confidence intervals (ci). p < .05 was considered significant. epi info was used for analysis of the data. of 208 eligible children, 102 cases and 96 controls were analysed ( figure 1 ). ten cases were excluded from analysis, due to an onset of disease of more than 10 days before enrolment, missing month of birth, or an age of more than 60 months. approximately 5% of all assessed controls declined to participate in the study, mainly based on fear of discomfort for their child or possible disapproval of the other parent. none of the cases declined. overall, 96 (48.5%) children were male, and the mean age was 18.0 (±12.4) months. overall vaccination coverage was 97.7%. table 1 shows a summary of baseline characteristics of the included cases and controls at enrolment. a significant difference of means was found in mean age among case and control group, respectively, 23.5 (±13.2) and 12.2 (±8.0) with a p value of <.001. furthermore, cases more often lived in other areas in and outside of dakar, whereas more controls than cases lived in the neighbourhood near the pikine clinic, respectively, 36 (35.3%) and 63 (65.6%) with a p value of <.001. among all cases, 31.7% had a pneumonia based on who criteria. the main respiratory symptoms were rhinitis and cough, in 90 (88.2%) and 89 (87.3%) of cases, respectively. in all, 19.8% had a tachycardia and 32.7% a tachypnoea. none of the children had a hypoxemia during consultation. on physical examination, 52 (51.0%) cases had abnormal lung sounds of which rhonchi were the main type. an elevated crp was seen in 72.8% of cases with a mean crp of 47.2 (±41). in all, 46.2% had a leucocytosis with a mean number of leucocytes of 11 527.8 (±5654.5). malaria rapid diagnostic tests were performed on only 32 cases, but all were negative. data on treatment were available in 92 cases. antibiotics were prescribed in 90.2% of those. data on follow-up were only available in 51 cases. in 78.1% of those, symptoms had passed within 1 week. none of the children had to be hospitalized. table 2 shows a summary of clinical signs and symptoms and management of cases. for detection of bacteria, only culture was used, because when using pcr, equal amounts of bacteria were found in cases and controls. the reason for this is that a better differentiation between bacterial colonization and infection can be made using cultures. 29 at least 1 pathogen was detected in 90.1% of cases and 53.7% of controls. bacteria were detected in 62.8% of cases and 25% of controls, of which s pneumoniae (73.6%) and h influenzae b (24.6%) were the most prevalent. viruses were detected in 75.3% of cases and 42.1% of controls and influenza virus a (28.9%), rsv (17.8%), and influenza virus b (10.4%) were most common. in 49.5% of cases, 2 or more pathogens were detected. univariate analysis shows that s pneumoniae, rsv, influenza virus a (including h1n1) and b were significantly associated with the occurrence of arti (table 3) (continued) 3.0, ci: 1.2-7.4). respiratory syncytial virus was only significantly related to illness in children less than 2 years of age. likewise, the prevalence was higher in this age group. legionella pneumophila, b pertussis, chlamydia pneumoniae, coronavirus 229, coronavirus oc43, coronavirus hkui, piv (parainfluenza virus) 1, piv 3, piv 4, human metapneumovirus, and influenza virus c were investigated, but not detected in cases or controls. the prevalence of pathogens in cases and controls is illustrated in figure 2 . table 4 shows the prevalence of different co-detections. streptococcus pneumoniae was found in most of the co-detections (76.0%). it was most frequently found in combination with influenza virus a, in 17 cases. associations between all possible specific co-detections and illness were analysed, but no additional relationship was found, other than a cumulative association of the present pathogens. analysis of cases. treatment with antibiotics was not significantly related to alarm signs: tachypnoea (or: 3.9, ci: 0.5-33.0), withdrawals (or: 0.2. ci: 0.0-1.1), and hypoxemia (or: undefined). none of the pathogens was significantly associated with those alarm signs or an elevated crp or leucocytosis. no difference in elevated crp or leucocytosis was found between bacteria and viruses. this case-control study investigates the prevalence of the different viruses and bacteria colonizing the airways and their 6 microbiology insights association with the occurrence of artis in children visiting a sub-urban primary care clinic in senegal. dieng et al studied pathogens in children with arti in senegal and found a total of 78 bacteria in 162 children. they found a lower prevalence of s pneumoniae (18% of cases) in comparison with our study (48% of cases) and a prevalence of m cattharalis of 15% where we did not detect this pathogen. dieng et al used different swab sampling techniques (bronchoalveolar lavage, sinus fluids, and throat swab) depending on the site of infection. moraxella cattharalis causes otitis media and sinusitis rather than lrti, possibly explaining the higher detection rate. 30 furthermore, their study covered a full year, including all seasons. although our study was conducted during 2 months, it covered the rainy season, which is known for its respiratory infections. 15 streptococcus pneumoniae was the most prevalent bacteria in our sample. recent case-control studies in low-income countries found similar prevalence in cases and controls, even though those countries have lower vaccination coverages. 19, 21, 24, 31 in western countries with a similar vaccination coverage to senegal, s pneumoniae rates in children with arti are evidently lower than we detected. 32 possibly, disease burden of s pneumoniae is high in senegal due to serotype replacement following the introduction of the pneumococcal vaccine, as has occurred in many other countries. 33 staphylococcus aureus is a bacteria frequently detected in these studies. 19, 21, 24 they used nasal/nasopharyngeal swabs, but the literature does not mention major differences in detection of s aureus between nose or throat. 34 another explanation is the use of pcr for detection of bacteria. numerous studies in sub-saharan africa have been conducted studying the prevalence of viruses in children with an arti. equal total amounts of viruses and a similar prevalence of rsv and influenza viruses were detected when comparing with our results, but these studies, including some recent senegalese studies, found significantly higher percentages of adenovirus, enterovirus, and rhinovirus. 14, 15, 35, 36 results from case-control studies conducted in low-and middle-income countries show a similar prevalence of all viruses in healthy children, except that the average prevalence of rhinovirus in those studies is approximately 30%, whereas we found rhinovirus in 7.4% of controls. 19, [21] [22] [23] [24] the most obvious possible reason for the differences in virus prevalence is our short study period, given that virus prevalence is highly seasonal. 37 this study did not find an association between different pathogens and alarm signs (tachypnoea, tachycardia, hypoxemia, or an elevated crp). concordant to our findings, no association was found between alarming signs and specific pathogens in previous research. 38 both viral and bacterial infections can cause either mild or severe infections in children. other studies found an association between viral or bacterial aetiology and an elevated crp too weak to rule out bacterial infection in clinical practice. 26, 39, 40 we did not find this association, presumably because of a small number of severely ill children. colonization by more than one pathogen is believed to influence the occurrence and severity of illness, by stimulating each other's adherence and decreasing clearance. 41 co-detections were seen in 49.5% of cases and 17.7% of controls. most of the co-detections in our study were caused by s after adjusting for pathogens significantly related to illness, no relation was detected between the presence of a co-detection and illness in our study, which means that we did not detect a synergic effect in co-infection, possibly due to a small sample size. in investigating the cause of arti in low-income countries, most studies are done in hospital setting and case-control methods are rarely used. nevertheless, in these studies, similar pathogens were found with similar significant associations with illness compared with our study. [19] [20] [21] [22] [23] [24] 42, 43 this could mean that the same pathogens are responsible for both upper and lower artis. furthermore, it confirms a generally shifting pattern towards viruses of pathogens causing artis. some pathogens were more prevalent in other settings, such as human metapneumovirus, piv, and mycoplasma pneumoniae. 19, 22, 24, 42, 43 low prevalence of these pathogens was found in our study, possibly due to seasonality or their possible higher prevalence in severe infections. notable is the overprescription of antibiotics in our population. approximately 90% of cases have been treated with antibiotics, whereas only 31.7% had a pneumonia and should have been treated as such following who criteria. moreover, most of the antibiotic treatments concerned broad-spectrum antibiotics, whereas a narrow-spectrum penicillin is recommended in the primary care setting. literature confirms that practices are similar in other low-income countries. [44] [45] [46] the main strength of this study is the inclusion of all artis within a primary care clinic. this gives insight into the pathogens causing the infections. other strengths are the large number of pathogens tested for, with pcr and culture testing for bacteria, and the inclusion of treatment methods. an additional strength is that all data were gathered by one researcher, hereby decreasing the risk of variation in swabbing technique. there are also limitations to be mentioned. we could not properly adjust for some factors such as social status due to few data on these subjects. furthermore, selection bias could have occurred, because no matching was done. the average age of controls is lower than the average age of children with a respiratory infection, probably due to the young age of children enrolled in vaccination programmes. however, we did not discern significant heterogeneity regarding pathogens in different age groups (except for the mentioned differences in prevalence of rsv ) or living areas. another limitation is the use of only throat swabs. the nasopharyngeal swab is found to be superior in detecting influenza virus, s pneumoniae, and moraxella species, but oropharyngeal swabs are superior for the detection of m pneumoniae and h influenzae. [47] [48] [49] [50] it is preferable to use both techniques, but limited resources inhibited that. there was a lack of a gold standard such as chest x-ray for pneumonia. by taking a clinical definition of arti, there is possible overlap with other feverish diseases such as malaria and gastrointestinal illnesses. other limitations are the short duration of the study and a lack of follow-up data of patients due to nonresponse to phone calls. we might have missed data on severity of illness and hospitalizations. this case-control study conducted in senegal investigates the cause of artis in children less than 5 years of age in a primary care setting. in this population, illness was mainly caused by influenza virus a and b, rsv, and s pneumoniae, which confirms a generally shifting pattern towards viruses in pathogens causing artis. we suggest continuation of this case-control study for a full year, and further research on serotypes and resistance patterns of bacteria present in children with arti. these findings could lead to improvement of vaccinations against respiratory pathogens. we recommend reduction of antibiotics prescription, especially broad-spectrum antibiotics, for children with an arti visiting a primary care clinic. rbk, adg, ad and tdvi contributed to the design and implementation of the research. samples were analyzed by af, adg, amb, abd and mn. rbk analyzed the data and wrote the manuscript with consultation from all authors. adiallo supervised the project at the clinic. csbb supervised the study. rebecca b knobbe https://orcid.org/0000-0002-5562-3569 amary fall https://orcid.org/0000-0002-6890-7915 tabitha d van immerzeel https://orcid.org/0000-0001 -8809-3886 abou ba https://orcid.org/0000-0003-1216-0813 estimates developed by the un inter-agency group for child mortality estimation estimates of the global, regional, and national morbidity, mortality, and aetiologies of lower respiratory tract infections in 195 countries: a systematic analysis for the global burden of disease study world health organization. revised who classification and treatment of childhood pneumonia at health facilities -evidence summaries british thoracic society guidelines for the management of community acquired pneumonia in children: update viral etiology of hospitalized acute lower respiratory infections in children under 5 years of age -a systematic review and meta-analysis can we distinguish pneumonia from wheezy diseases in tachypnoeic children under low-resource conditions? a prospective observational study in four indian hospitals antimicrobial resistance: a global multifaceted phenomenon antibiotic resistance threats in the united states childhood pneumonia: the role of viruses burden of disease caused by haemophilus influenzae type b in children younger than 5 years: global estimates burden of disease caused by streptococcus pneumoniae in children younger than 5 years: global estimates burden of streptococcus pneumoniae and haemophilus influenzae type b disease in children in the era of conjugate vaccines: global, regional, and national estimates for 2000-15 invasive disease potential of pneumococci before and after the 13-valent pneumococcal conjugate vaccine implementation in children children under five years of age in senegal: a group highly exposed to respiratory viruses infections viral and bacterial etiologies of acute respiratory infections among children under 5 years in senegal enteroviruses and rhinoviruses: molecular epidemiology of the most influenza-like illness associated viruses in senegal community-acquired pneumonia in children -a changing spectrum of disease respiratory viruses detected in mexican children younger than 5 years old with communityacquired pneumonia: a national multicenter study microorganisms associated with pneumonia in children <5 years of age in developing and emerging countries: the gabriel pneumonia multicenter, prospective, case-control study viral and bacterial causes of severe acute respiratory illness among children aged less than 5 years in a high malaria prevalence area of western kenya aetiology of childhood pneumonia in a well vaccinated south african birth cohort: a nested case-control study of the drakenstein child health study association of respiratory viruses with outcomes of severe childhood pneumonia in botswana a preliminary study of pneumonia etiology among hospitalized children in kenya etiology and factors associated with pneumonia in children under 5 years of age in mali: a prospective case-control study national health care provider solutions. paediatric advanced life support: provider handbook the utility of serum c-reactive protein in differentiating bacterial from nonbacterial pneumonia in children: a meta-analysis of 1230 children diagnostic value of laboratory tests in identifying serious infections in febrile children: systematic review a flexible statistical power analysis program for the social, behavioral, and biomedical sciences nucleic acid amplification tests for the diagnosis of pneumonia moraxella catarrhalis, a human respiratory tract pathogen community-acquired pneumonia requiring hospitalization among u.s. children serotype replacement in disease following pneumococcal vaccination: a discussion of the evidence multi-site and nasal swabbing for carriage of staphylococcus aureus: what does a single nose swab predict detection of non-influenza viruses in acute respiratory infections in children under fie-year-old in cote d'ivoire viral etiology of respiratory tract infections in children at the paediatric hospital in ouagadougou (burkina faso) seasonal variations of respiratory viruses and etiology of human rhinovirus infection in children the value of clinical features in differentiating between viral, pneumococcal and atypical bacterial pneumonia in children differentiation of bacterial and viral pneumonia in children a three-step diagnosis of paediatric pneumonia at the emergency department using clinical predictors, c-reactive protein, and pneumococcal pcr viral bacterial co-infection of the respiratory tract during early childhood the contribution of viruses and bacteria to community-acquired pneumonia in vaccinated children: a case-control study aetiological role of common respiratory viruses in acute lower respiratory infections in children under five years: a systematic review and meta-analysis care seeking behaviour and aspects of quality of care by caregivers for children under five with and without pneumonia in ibadan the effect of rapid diagnostic testing for influenza on the reduction of antibiotic use in paediatric emergency department an under-recognized influenza epidemic identified by rapid influenza testing a comparison of nasopharyngeal and oropharyngeal swabbing for the detection of influenza virus by real-time pcr comparison of the prevalence of common bacterial pathogens in the oropharynx and nasopharynx of gambian infants comparison of four different sampling methods for detecting pharyngeal carriage of streptococcus pneumoniae and haemophilus influenzae in children comparison of oropharyngeal and nasopharyngeal swab specimens for the detection of mycoplasma pneumoniae in children with lower respiratory tract infection key: cord-308979-qhlvd2mt authors: sumino, kaharu c.; agapov, eugene; pierce, richard a.; trulock, elbert p.; pfeifer, john d.; ritter, jon h.; gaudreault-keener, monique; storch, gregory a.; holtzman, michael j. title: detection of severe human metapneumovirus infection by real-time polymerase chain reaction and histopathological assessment date: 2005-09-15 journal: j infect dis doi: 10.1086/432728 sha: doc_id: 308979 cord_uid: qhlvd2mt backgroundinfections with common respiratory tract viruses can cause high mortality, especially in immunocompromised hosts, but the impact of human metapneumovirus (hmpv) in this setting was previously unknown methodswe evaluated consecutive bronchoalveolar lavage and bronchial wash fluid samples from 688 patients—72% were immunocompromised and were predominantly lung transplant recipients—for hmpv by use of quantitative real-time polymerase chain reaction (pcr), and positive results were correlated with clinical outcome and results of viral cultures, in situ hybridization, and lung histopathological assessment resultssix cases of hmpv infection were identified, and they had a similar frequency and occurred in a similar age range as other paramyxoviral infections. four of 6 infections occurred in immunocompromised patients. infection was confirmed by in situ hybridization for the viral nucleocapsid gene. histopathological assessment of lung tissue samples showed acute and organizing injury, and smudge cell formation was distinct from findings in infections with other paramyxoviruses. each patient with high titers of hmpv exhibited a complicated clinical course requiring prolonged hospitalization conclusionsour results provide in situ evidence of hmpv infection in humans and suggest that hmpv is a cause of clinically severe lower respiratory tract infection that can be detected during bronchoscopy by use of real-time pcr and routine histopathological assessment of infection [4] . despite these aggressive approaches, an infectious agent cannot be identified in 20%-65% of patients [3] [4] [5] . missed diagnoses may be due to the poor sensitivity of the diagnostic tests as well as the occurrence of emergent pathogens. moreover, even when a pathogen is found, it may be difficult to be certain that the isolated organism is the cause of the illness. improvements in the diagnosis of viral infection may be achieved when innovative approaches are used to discover new viruses. in this regard, human metapneumovirus (hmpv) was newly discovered by random polymerase chain reaction (pcr) amplification of viral rna that was isolated from children with respiratory tract illnesses [6] . after its initial isolation in the netherlands in 2001, hmpv was identified in lower respiratory tract infections in children worldwide [7] [8] [9] [10] . analysis of stored nasopharyngeal swab samples indicated that hmpv may account for a significant portion of respiratory tract infections with previously unknown etiology [6, 10, 11] . the consequences of hmpv infection range from acute upper respiratory tract infection to severe bronchiolitis 17 (2) autoimmune disease 12 (2) none 47 (7) uncertain 46 (7) or pneumonitis, and the pattern of illness is similar to that found in respiratory syncytial virus (rsv) infection [8, 9, 11] . illness associated with hmpv develops in a broad age range, but young children, the elderly, and immunocompromised hosts appear to be at greatest risk for severe illness [8, 9, 11] . in addition, hmpv has been isolated in 51%-55% of patients with severe acute respiratory syndrome (sars), but its contribution to that illness remains uncertain [12, 13] . indeed, to our knowledge, neither a pathological characterization of hmpv infection nor a correlation with clinical findings has been reported in humans. moreover, the prevalence and other characteristics of hmpv infection in the clinical setting of patients undergoing bronchoscopy for respiratory tract infections still need to be defined. in the present study, we therefore developed and applied quantitative real-time pcr to screen for hmpv in bronchial wash (bw) and/or bal fluid samples. positive results afforded us the opportunity to further define the clinical and pathological features of illness in patients infected with hmpv and to identify a subset of patients with severe illness. a total of 806 sequential samples were collected from patients who underwent bronchoscopy from 15 december 2002 to 1 august 2003. samples obtained within 10 days from the same patient were excluded from analysis, so that a total of 688 fluid samples (576 bal and 112 bw) from 688 patients were analyzed. all samples that were screened for viruses were routinely submitted to the st. louis children's hospital clinical virology laboratory, which serves washington university medical center and acts as a referral laboratory for the st. louis metropolitan area. bronchoscopy was performed at st. louis children's hospital and barnes-jewish hospital at washington university medical center as well as at affiliated community hospitals. the sample period encompassed the peak winter-spring period for detection of hmpv in nasopharyngeal swab samples [14] . the mean age of the patients was 38.6 years; 26% of the patients were !18 years old, 62% were 18-65 years old, and 12% were 165 years old. eighty-six percent of the patients had underlying conditions, most commonly in the setting of lung transplantation (table 1) . predominantly, indications for bronchoscopy were unexplained respiratory tract symptoms or abnormalities found on chest radiographs (table 2) . the cohort also included 210 patients without acute illness who underwent routine bronchoscopy for surveillance after lung transplantation or follow-up bronchoscopy after treatment for transplant rejection. virus detection. for the detection of several non-hmpv viruses (influenza virus, parainfluenza virus, adenovirus, rsv, herpes simplex virus, and rhinovirus), samples were examined by direct immunofluorescence assay (difa) and for cytopathic effect (cpe) in cell culture, as described elsewhere [15] . cy-tomegalovirus (cmv) was detected by shell-vial culture [15] . testing for hmpv on samples stored at ϫ70њc was performed after institutional review board approval. for each sample, a 140-ml aliquot was used for the isolation of rna by use of the qiaamp viral rna kit (qiagen). a 5-ml aliquot of this solution was used for the detection of virus by use of quantitative real-time pcr with the taqman one-step rt-pcr kit, in accordance with the manufacturer's protocol (pe biosystems). primers and probes were derived from a conserved fragment of the nucleocapsid (n) gene from the nl00-1 sequence of hmpv (genbank accession no. af371337). direct and reverse primers amplified nt 993-1064 of the n gene. this system specifically detects hmpv genotype a, which was predominant in the study region during the study period; genotype a accounted for 75% of all hmpv-positive samples (e.a., k.c.s., and m.j.h., unpublished data). a synthetic rna standard for hmpv was generated from a portion of the n gene (nt 40-1172) and was cloned into the pgem plasmid (promega). rna was transcribed in vitro using the t7 mega script kit (ambion). the dna template was eliminated by trizol extraction (invitrogen), dnase treatment, rneasy mini kit preparation (qiagen), and final phenol extraction. the concentration of the rna standard was determined by measurement of the optical density and was used to calculate the copy numbers of hmpv rna. the pcr amplification cycles were as follows: initial heating at 48њc for 30 min and 95њc for 10 min, followed by 40 cycles of 95њc for 15 s and 60њc for 1 min. these conditions allowed for the detection of as few as 5 copies of the rna standard and linear amplification of the standard from 10 to 10 6 molecules. for all hmpv-positive samples, rna isolation and hmpv real-time pcr were repeated, and hmpv-negative samples were excluded from further analysis. in addition, all samples that were found to be positive for hmpv by real-time pcr were tested by a second real-time pcr, which detected genomic rna for a different gene than that used for the initial detection. the primers and probe of this second real-time pcr were designed to amplify a fragment (nt 7155-7241) of hmpv stl43-84, which was isolated in our laboratory from a nasopharyngeal swab sample [14] . all samples were also routinely processed for bacterial and fungal culture and were selectively processed for atypical pathogens (pneumocystis, legionella, and mycoplasma species). in situ hybridization. detection of hmpv was performed using in situ hybridization, because no sensitive and reproducible anti-hmpv antibody was yet available. cdna encoding the hmpv n gene (nt 40-1172; genbank accession no. af371337) was inserted into the pgem-3zf(+) plasmid (promega) and was transcribed into viral genomic rna using the t7 promoter. the antisense rna probe template was linearized by sspi digestion, and the probe was labeled with 35 s using an rna labeling kit (promega). the transcribed rna was purified by phenol extraction and ethanol precipitation and was used as a probe to detect hmpv n gene mrna by in situ hybridization, as described elsewhere [16] . because a sense control probe will also detect hmpv genomic rna, rhesus monkey kidney llc-mk2 cells inoculated with or without hmpv were used as positive and negative controls, respectively, for lung biopsy samples. the fidelity of the probe was also tested on llc-mk2 cells that were cultured in a-mem with bovine serum albumin (1 mg/ml) and trypsin (1 mg/ml) and then inoculated with or without hmpv stl43-84. before in situ hybridization, cells were checked for syncytial formation and for reactivity with a convalescent-phase serum sample from an hmpv-infected patient (patient 6). for these experiments, cultured cells were blocked with 2% gelatin, were incubated with the convalescent-phase serum sample (1:100 dilution) for 1 h and then with fluorescein isothiocyanate-conjugated anti-human igg (1:400 dilution; jackson laboratory), and finally were subjected to immunofluorescence photomicrography. histopathological assessment and clinical outcome. the histopathological appearance of the lung tissue samples and the clinical outcome were reviewed for each patient with detectable hmpv, in accordance with a protocol that was approved by the institutional review board of washington university school of medicine. lung biopsy samples were available for study in 5 of 6 patients and were subjected to hematoxylin-eosin staining and immunostaining for adenovirus, cmv, and herpes simplex virus as described elsewhere [17] . in addition, pathologists examined lung tissue samples from 4 additional patients whose bal fluid samples were positive for rsv, rhinovirus, or parainfluenza virus. pathologists reviewed all lung tissue samples in a blinded manner. clinical characteristics and outcomes were determined by a retrospective review of medical records. hmpv was detected by real-time pcr in samples from 6 patients (designated patients 1-6) at a rate that was similar to that found for other common respiratory tract viruses detected by difa and cpe (table 3). as was expected for an immunocompromised population, cmv was also detected at a high frequency in the samples (table 3) . the detection rate for influenza virus was low, probably because a rapid screening test was available and the need for proceeding to bronchoscopy was decreased. none of the 6 patients with hmpv were coinfected with other viruses, and no hmpv was detected in the 210 asymptomatic patients who underwent routine bronchoscopy for surveillance after lung transplantation or follow-up after treatment for transplant rejection. all occurrences of hmpv infection were clustered in late winter/spring, when hmpv was prevalent in the area. hmpv was the sole organism isolated from bal or bw fluid samples in 4 patients (patients 2, 3, 5, and 6). in 2 patients, we compared viral copy numbers in bw fluid samples with those in bal fluid samples and found that the bw fluid samples contained higher viral copy numbers than the bal fluid samples. for all patients with severe lower respiratory tract illness (patients 1, 2, 3, 5, and 6), the results of routine bacterial and fungal cultures were negative for significant pathogens. in addition, the results of the difa for pneumocystis species (patients 1, 2, 5, and 6), pcr for mycoplasma (patient 1), and culture or urine antigen testing for legionella species (patients 1, 2, and 5) were negative. clinical course of hmpv infection. all patients presented with nonspecific acute respiratory tract symptoms that included fever, cough, dyspnea, and wheezing. four of 6 illnesses occurred in immunocompromised patients (table 4) . patients 2 and 6 presented with acute respiratory failure that required mechanical ventilation. chest radiographs of these patients showed diffuse infiltrates that were suggestive of acute lung injury (figure 1). patients 1, 5, and 6 required prolonged hospitalization because of their acute respiratory tract illness. patient 3 presented with exacerbation of chronic obstructive pulmonary disease and required mechanical ventilation. patient 4, who had the mildest illness, coincidentally developed mild upper respiratory tract symptoms at the time of routine surveillance bronchoscopy after lung transplantation. patients with severe illness due to hmpv infection (patients 1, 2, 5, and 6) tended to have higher levels of hmpv in bal and bw fluid samples than did patients with mild illness, but the small number of patients was insufficient to establish a significant correlation. additional clinical samples were available from patients 1 and 6. for patient 1, a nasopharyngeal swab sample was negative for hmpv at 1 week after the bal fluid sample was obtained. for patient 6, tracheal aspirate and nasopharyngeal swab samples showed a low hmpv titer of 50 and 1032 copies, respectively, at 15 days after bronchoscopy. each of these findings was compatible with partial or complete clearance of the virus as well as greater sensitivity of bal and bw fluid samples for viral detection. in situ detection of hmpv infection. infection with hmpv was confirmed using immunofluorescence microscopy and in situ hybridization of samples. here, we took advantage of an hmpv isolate (designated stl43-84) that we had obtained from a nasopharyngeal swab sample and had characterized by nucleotide sequencing [14] . this isolate was used to inoculate cultured llc-mk2 cells that exhibited cpe and syncytial formation that are typical in paramyxoviral infection (figure 2). this system was then used as a substrate for testing an available convalescentphase serum sample that was obtained from a patient in the present cohort. thus, hmpv-infected llc-mk2 cells, but not control cells, showed immunofluorescent staining with the convalescent-phase serum sample from patient 6, thereby indicating the presence of anti-hmpv antibody in this patient's serum (figure 2) . in addition, we were able to demonstrate that hmpvinfected llc-mk2 cells, but not control cells, gave a positive signal by in situ hybridization with a 35 s-labeled antisense probe for hmpv n gene mrna (figure 2), thereby validating this approach for further analysis of lung biopsy samples. using the same antisense probe for hmpv, we next found a positive signal by in situ hybridization for hmpv mrna in the open lung biopsy sample from patient 1 (figure 3). the signal seemed (on the basis of morphological appearance) to localize predominantly to type ii alveolar epithelial cells and to have a heterogeneous and patchy distribution (figure 3). a lower but still significant signal by in situ hybridization was also found in bronchial epithelial cells (figure 3). signals for hmpv mrna were not detected in the few smudge cells found in the samples that were subjected to in situ hybridization. the transbronchial biopsy samples that were available for study from patients 2, 5, and 6 did not show any focal signal by in situ hybridization for hmpv. histopathological assessment of hmpv infection. lung tissue samples were available from 5 of 6 patients with hmpv infection (all but patient 3). lung tissue samples from 3 patients-from an open lung biopsy (patient 1) and 2 transbronchial biopsies (patients 2 and 5)-showed similar findings. the major finding in each case was acute and organizing lung injury; this included areas of what resembled diffuse alveolar damage with hyaline membrane formation and foci of bronchiolitis-obliterans/organizing pneumonialike reaction. each sample included enlarged type ii pneumocytes with smudged hyperchromatic nuclei that resembled the smudge cells found in adenovirus infection ( figure 4) . however, immunostaining for adenovirus, cmv, and herpes simplex virus was negative in all lung biopsy samples. a transbronchial biopsy sample from patient 4 showed changes after lung transplantation without evidence of lower respiratory tract infection. the small transbronchial biopsy sample from patient 6 showed only nonspecific acute and chronic inflammation. smudge cells were not detected in the samples from the 4 patients with lower respiratory tract infection who had rsv, rhinovirus, or parainfluenza virus detected in bal fluid samples. cultured rhesus monkey kidney llc-mk2 cells were inoculated with either hmpv (+ hmpv) or a control (ϫ hmpv) and then were incubated at 37؇c for 5 days. cells were then subjected to phase-contrast microscopy (first row) at 5 days after inoculation, immunofluorescence microscopy with patient convalescent-phase serum (1:100 dilution) and fluorescein isothiocyanate-conjugated goat anti-human igg (second row) at 2 days after inoculation, and in situ hybridization (ish) with 35 s-labeled antisense probe for viral nucleocapsid gene mrna under brightfield (third row) and darkfield microscopy (fourth row) at 2 days after inoculation. scale bars, 20 mm. ab, antibody since the discovery of hmpv, several studies have confirmed that the virus can be detected (generally by pcr) in clinical samples (generally nasopharyngeal swab samples) from children and adults with acute respiratory tract illness. its occasional detection in asymptomatic patients has also been reported [9] , which raises the question of whether there is sufficient specificity in this diagnostic approach. in the present study, we add several critical pieces to the diagnostic and clinical matrix for hmpv infection: (1) we detected hmpv at a frequency similar to that of other respiratory tract viruses in patients who underwent bronchoscopy for suspected respiratory tract infection; (2) we quantitatively detected hmpv by real-time pcr in bronchoscopy samples from patients with severe respiratory tract illness but not in those without symptoms or signs of respiratory tract infection; (3) we used a viral culture system to develop a specific antisense probe for hmpv and then demonstrated hmpv mrna in the lung tissue samples; and (4) we found organizing and acute lung injury and the prominent formation of smudge cells in the lung tissue samples of these patients, thereby suggesting that this pattern may be characteristic of hmpv infection. taken together, the detection of high copy numbers of hmpv by real-time pcr and of smudge cell formation by transbronchial biopsy can be of significant diagnostic value in defining the etiology of severe respiratory tract illness. the pattern of the present histological findings for hmpv infection is somewhat unusual. we found that, in severe illness, hmpv is capable of causing acute pneumonia and lung injury in a pattern of diffuse alveolar damage with hyaline membrane formation that is similar to the lung injury found in other types of viral respiratory tract infections. the other member of the subfamily pneumovirinae, rsv, which causes clinical disease that is similar to hmpv infection, can also cause acute lung injury with desquamated reactive pneumocytes, and this leads to accumulation of cellular debris within the alveoli and small airways. the presence of multinucleated giant cells (syncytial cells) with eosinophilic cytoplasmic inclusions are strongly suggestive of rsv infection [18, 19] , and we showed that hmpv can also cause syncytial cell formation in cell culture in vitro. by contrast, in vivo, hmpv infection appears to cause the formation of easily distinguishable smudge cells that have not been found in other paramyxoviral infections [20] . these smudge cells closely resemble the enlarged pneumocytes with darkly stained homogeneous inclusion bodies that are found in respiratory tract infection with adenovirus (a virus that causes a pattern of respiratory tract illness that is indistinguishable from that caused by hmpv). the smudge cells found in adenovirus infection have been shown to contain the virus by use of electron microscopy, in situ hybridization, and immunohistochemistry in autopsy series [21] [22] [23] . in contrast, we were able to detect hmpv mrna in the alveolar and airway epithelial cells but not in the smudge cells. this difference could be due to sampling error, because hmpv rna was detected in only a patchy distribution even in the open lung biopsy sample. it is also possible, however, that smudge cells could be reactive in hmpv infection. the present findings are also distinct from those found in experimental hmpv infection of nonhuman primates. in that situation, hmpv replication occurs mainly in ciliated epithelial cells and rarely in type i pneumocytes, and histological assessment shows that erosive and inflammatory changes are confined to the conducting airways [24] . whether these differences reflect species specificity, disease severity, host immune status, or other factors requires further study. similarly, although the results of studies of monkeys support a primary role for a new coronavirus in sars, the present findings again raise the possibility that hmpv contributes to moresevere respiratory tract illness, including sars [12, 13] . although the pattern of acute lung injury with smudge cell formation appears distinctive for hmpv infection, at least in the immunocompromised host, additional samples will be needed to more fully determine the diagnostic value of this pattern. in general, the detection of virus in bal fluid samples is not sufficient (without concomitant histopathological or clinical presentation) to establish a diagnosis of infection. nonetheless, this approach has been used to achieve prompt and accurate diagnoses in patients with possible respiratory tract infection. the highest diagnostic yield and impact of bal fluid sample analysis on therapeutic decisions has been found in immunocompromised hosts, and the combination of bal with transbronchial biopsy provides an additional diagnostic yield [25] . under these circumstances, detection of common respiratory tract viruses (influenza virus, parainfluenza virus, rsv, rhinovirus, and ad-enovirus) is similar to that in the present study (∼3% of cases by use of conventional detection methods) [1, 26] . these respiratory tract viruses have been recently recognized as an important cause of lower respiratory tract infection in immunocompromised patients, and their detection may alter the clinical course of the illness [1, 27, 28] . among these viruses, rsv in particular has been associated with high mortality (26%-100%), especially in bone marrow transplant recipients [27, 29, 30] . in our study population, which was weighted toward immunocompromised patients, hmpv (detected by pcr) was found as frequently as other common respiratory tract viruses (detected by difa and culture). although the relative insensitivity of standard lab tests may underestimate the occurrence of other viruses, this shortcoming should not detract from the importance of using the detection of hmpv to improve patient care. in particular, the detection of hmpv in bal fluid samples in a proper clinical setting may prevent these high-risk patients from undergoing additional diagnostic procedures and treatments and thereby avoid their attendant morbidity and costs. whether there is an additional benefit conferred by instituting antiviral treatment still needs to be determined. for rsv, combination treatment with ribavirin and anti-rsv antibody may improve the clinical outcome in immunocompromised patients, but no treatment has yet been tested in vivo for hmpv infection [29, 31, 32] . the results of the present study provide a further rationale for the development of anti-hmpv antibody and other possible therapies to intervene in what appears to be a severe respiratory tract illness. although we were able to identify only 6 patients with hmpv infection, the consistent clinical features and characteristic pattern of histopathological changes in these patients strongly indicate a clinical significance. in summary, hmpv is a significant cause of lower respiratory tract infection and is associated with morbidity in the immunocompromised host. we recommend that rapid assessment by pcr for hmpv in bal fluid samples be included in the evaluation of these patients. a positive result (combined with the absence of other respiratory tract viruses, such as adenovirus) can be correlated with histopathological findings of smudge cells in the background of acute and organizing lung injury to suggest the diagnosis of lower respiratory tract infection due to hmpv. conventional respiratory viruses recovered from immunocompromised patients: clinical considerations respiratory tract viral infections in bone marrow transplant patients respiratory viruses and severe lower respiratory tract complications in hospitalized patients role of flexible bronchoscopy in immunocompromised patients with lung infiltrates role of bronchoalveolar lavage in immunocompromised patients with pneumonia treated with a broad spectrum antibiotic and antifungal regimen a newly discovered human pneumovirus isolated from young children with respiratory tract disease ruuskanen o. metapneumovirus and acute wheezing in children virological features and clinical manifestations associated with human metapneumovirus: a new paramyxovirus responsible for acute respiratory-tract infections in all age groups human metapneumovirus infections in young and elderly adults human metapneumovirus and lower respiratory tract disease in otherwise healthy infants and children prevalence and clinical symptoms of human metapneumovirus infection in hospitalized patients human metapneumovirus detection in patients with severe acute respiratory syndrome identification of severe acute respiratory syndrome in canada diversity of human metapneumovirus infection in a us population (abstract w7-6) essentials of diagnostic virology retinoic acid attenuates o 2 -induced inhibition of lung septation viral induction of a chronic asthma phenotype and genetic segregation from the acute response demonstration of respiratory syncytial virus in an autopsy series fatal pneumonia in an allogeneic bone marrow transplant recipient spencer's pathology of the lung adenovirus pneumonia in lung transplant recipients expression of early and late adenoviral proteins in fatal adenovirus bronchopneumonia fatal neonatal pneumonia caused by adenovirus type 35 experimental human metapneumovirus infection of cynomolgus macaques (macaca fascicularis) results in virus replication in ciliated epithelial cells and pneumocytes with associated lesions throughout the respiratory tract pulmonary infiltrates in non-hiv immunocompromised patients: a diagnostic approach using non-invasive and bronchoscopic procedures recovery of viruses other than cytomegalovirus from bronchoalveolar lavage fluid paramyxovirus infection in lung transplant recipients community respiratory virus infections following lung transplantation combination therapy with aerosolized ribavirin and intravenous immunoglobulin for respiratory syncytial virus disease in adult bone marrow transplant recipients respiratory syncytial virusassociated infections of adult recipients of solid organ transplants respiratory syncytial virus upper respiratory tract illnesses in adult blood and marrow transplant recipients: combination therapy with aerosolized ribavirin and intravenous immunoglobulin comparison of inhibition of human metapneumovirus and respiratory syncytial virus by ribavirin and immune serum globulin in vitro key: cord-313749-f2ct57em authors: brittain-long, robin; nord, sandra; olofsson, sigvard; westin, johan; anderson, lars-magnus; lindh, magnus title: multiplex real-time pcr for detection of respiratory tract infections date: 2007-12-26 journal: j clin virol doi: 10.1016/j.jcv.2007.10.029 sha: doc_id: 313749 cord_uid: f2ct57em background: broad diagnostics of respiratory infection by molecular assays has not yet won acceptance due to technical difficulties and high costs. objectives: to evaluate clinical applicability of multiplex real-time pcr. study design: an assay targeting influenza virus a (ifa) and b (ifb), parainfluenza 1-3 (piv), human metapneumovirus (mpv), respiratory syncytial virus (rsv), rhinovirus (rv), enterovirus (ev), adenovirus (adv), human coronaviruses (229e, oc43, nl63), m. pneumoniae and ch. pneumoniae was developed and run daily on consecutive clinical nasopharyngeal swab samples. results: an etiology was identified in 48% of the 954 samples, with ifa in 25%, rv in 20%, mpv in 10% and m. pneumoniae in 10% of the positive. by a rational procedure costs could be reduced and the customer price set relatively low (€33 per sample). conclusion: streamlined testing and cost limitation is achievable and probably critical for implementation of a broad molecular diagnostics of respiratory infections. community acquired respiratory tract infections are frequent and constitute an important cause of morbidity in sweden and other european countries. a significant part of these infections are caused by viral pathogens (creer et al., 2006; diaz et al., 2007) . it is often difficult for the clinician to distinguish between viral and bacterial aetiologies, and this may result in overuse of antibiotics (lundborg et al., 2002) . diagnosis of viral respiratory tract infections using viral culture, antigen detection or serology is either too slow or too insensitive to be applicable in clinical practice (gunson et al., 2005) . the more reliable, specific and sensitive pcr methods have not yet won acceptance as the first choice for diagnosis owing to the high cost when several agents are targeted. recently, multiplex assays that detect a large number of viral agents have been described. some of these are based on traditional pcr (bellau-pujol et al., 2005; gruteke et al., 2004 ; * corresponding author. tel.: +46 73 8000 400. e-mail address: robin.bl@telia.com (r. brittain-long). liolios et al., 2001; puppe et al., 2004) , others on real-time pcr (gunson et al., 2005; kuypers et al., 2006; templeton et al., 2004; watzinger et al., 2004) or pcr combined with luminex liquid chip hybridization and identification (li et al., 2007) . we developed a real-time pcr procedure, based on automated specimen extraction and multiplex amplification, at a relatively low cost (d 33). primers and hydrolysis probes were obtained from the literature or developed in our laboratory. the following agents were analysed: influenza virus a (ifa) and b (ifb), parainfluenza 1-3 (piv), human metapneumovirus (mpv), respiratory syncytial virus (rsv), rhinovirus (rv), enterovirus (ev), adenovirus (adv), human coronaviruses 229e, oc43 and nl63. in addition, m. pneumoniae and ch. pneumoniae were included in the panel. nucleic acid from 100 l of specimen was extracted into an elution volume of 100 l by a magnapure lc robot 1386-6532/$ -see front matter © 2007 elsevier b.v. all rights reserved. doi:10. 1016/j.jcv.2007.10.029 (roche molecular systems, mannheim, germany), using the total nucleic acid protocol, and was amplified in an abi 7500 real-time pcr system (applied biosystems, foster city, ca). amplification was then carried out in 50 l reaction volumes, including 10 l of sample preparation and 25 l of one-step rt-pcr master mix from applied biosystems (this was exchanged to a master mix from invitrogen (carlsbad, ca) in the later part of the study period to improve sensitivity). after a reverse transcription step at 46 • c for 30 min followed by 10 min of denaturation at 95 • c, 45 cycles of two-step pcr was performed (15 s at 95 • c, 60 s at 58 • c). each sample was amplified in 5 parallel reactions, each containing primers and probes specific for 3 targets (mixtures a-e), as described in table 1 . multiplexing tended to hamper the performance of the amplification significantly for many combinations, and therefore the reagent mixtures were carefully evaluated: combinations were only accepted if the ct value in multiplex was no more than 2 cycles higher than when detection was carried out in separate reactions. the performance was evaluated using puc57 plasmids with inserts during the period from october 2006 through march 2007, 954 specimens were collected from patients of all ages with symptoms of respiratory tract infections who had been admitted to hospital clinics as well as health care centers in the göteborg region in western sweden (catchments area of approximately 600,000 inhabitants). both in-patients and out-patients were included. the specimens were obtained from either oropharynx or nasopharynx by rotating swabs. the specimens were submitted for diagnosis to the virology laboratory at sahlgrenska university hospital. out of the 954 specimens analysed 457 (48%) were positive for one ore more agents. the following agents were detected in order of frequency (n, % of positives): ifa (n = 114, 25%), rv (n = 93, 20%), mpv (n = 46, 10%), rsv (n = 41, 9%), oc43 (n = 33, 7%), ad (n = 33, 7%), nl63 (n = 26, 6%), piv (n = 19, 4%), ifb (n = 4, 1%), and ev (n = 1). m. pneumoniae was the most frequent bacterium (n = 44, 10%) whereas ch. pneumoniae was found only in 2 specimens. thirty-one specimens were positive for two agents, and one specimen was positive for 3 agents. the procedure was run every working day and results from samples arriving in the morning could be delivered during the same day to the clinician. the positivity rate (48%) is comparable with previous reports with similar study subjects. the rate of rsv and ifb were remarkably low, probably because these infections had appeared in extensive epidemics the preceding year. by targeting multiple agents in each reaction well, a broad range of aetiologies could be investigated in each run. the cost per sample could be kept low, since the procedure was streamlined and a reasonable number of samples arrived to the laboratory each day. the cost for was divided on reagents for nucleic acid extraction (d 3), reagents for real-time pcr (d 11), personnel (d 8), laboratory space (d 3), instrument cost (d 1), and overhead (d 5), yielding a customer price of d 33. we believe that a combination of low cost, high accuracy and prompt result delivery is the key to achieving a wide clinical use of molecular diagnostics of respiratory infections. we have also noted that inclusion of bacterial agents has promoted the willingness from clinicians to use our service, probably because mycoplasma pneumoniae or ch. pneumoniae infections may require antibiotic treatment. viral detection may support the clinician's decision to avoid antibiotics, or may prompt antiviral treatment such as oseltamivir (against influenza) to the patient or exposed family members. identifying the aetiology also allows the clinician to better inform the patient about the prognosis and ways of limiting the spread to other individuals. we therefore believe that an expanded use of molecular diagnostics of respiratory infections is of great clinical value. it should, of course, be kept in mind that viral agents detected by highly sensitive assays might reflect asymptomatic infection rather that being the cause of the present illness, and that an identified viral agent does not exclude concomitant bacterial infection. to some extent, the probability that a given agent constitutes an aetiological agent should be related to the ct value: the lower ct value, the higher the probability, and this may also be of relevance for the interpretation of double infections. further studies of respiratory infection aetiologies in different patient categories, and of the clinical utility of this and similar multiplex assays, need to be carried out. development of three multiplex rt-pcr assays for the detection of 12 respiratory rna viruses aetiological role of viral and bacterial infections in acute adult lower respiratory tract infection (lrti) in primary care etiology of community-acquired pneumonia in hospitalized patients in chile: the increasing prevalence of respiratory viruses among classic pathogens practical implementation of a multiplex pcr for acute respiratory tract infections in children real-time rt-pcr detection of 12 respiratory viral infections in four triplex reactions pring-akerblom p. rapid and quantitative detection of human adenovirus dna by real-time pcr comparison of real-time pcr assays with fluorescent-antibody assays for diagnosis of respiratory virus infections in children simultaneous detection and high-throughput identification of a panel of rna viruses causing respiratory tract infections comparison of a multiplex reverse transcription-pcr-enzyme hybridization assay with conventional viral culture and immunofluorescence techniques for the detection of seven viral respiratory pathogens antibiotic prescribing in outpatients: a 1-week diagnosis-prescribing study in 5 counties in sweden evaluation of a multiplex reverse transcriptase pcr elisa for the detection of nine respiratory tract pathogens rapid and sensitive method using multiplex real-time pcr for diagnosis of infections by influenza a and influenza b viruses, respiratory syncytial virus, and parainfluenza viruses 1, 2, 3, and 4 design and performance testing of quantitative real-time pcr assays for influenza a and b viral load measurement real-time quantitative pcr assays for detection and monitoring of pathogenic human viruses in immunosuppressed pediatric patients we are grateful to dr lars nielsen, copenhagen, for sharing experience, oligonucleotide sequences and samples. key: cord-272596-yxvg8357 authors: wu, jian jun; jin, yu; lin, na; xie, zhi ping; yu, jie mei; li, jin song; cao, chang qing; yuan, xin hui; song, jin rong; zhang, jing; zhao, yang; gao, xiao qian; duan, zhao jun title: detection of human bocavirus in children with acute respiratory tract infections in lanzhou and nanjing, china date: 2014-11-30 journal: biomedical and environmental sciences doi: 10.3967/bes2014.110 sha: doc_id: 272596 cord_uid: yxvg8357 abstract objective the aim of this study was to explore the prevalent characteristics of hbov1 and its co-infection. methods pcr was used to detect hbov1-dna (hbov1) and other viruses. a multivariate logistic regression model was used to explore possibility of co-detected for related viruses. results the positivity rates in nanjing and lanzhou were 9.38% (74/789) and 11.62% (161/1386), respectively (p<0.05). the hbov1 positive group was younger than negative group (p<0.05). seasonal differences were noted, with a higher frequency of infection in december and july. hbov1-positive children [72.34% (169/235)] were co-infected with other respiratory viruses. multifactorial analysis showed no correlations between hbov1 and the clinical classification, region, gender, age, or treatment as an outpatient or in a hospital. correlations were identified between hbov1 infections with adv (or=1.53, 95% ci 1.03-2.28), rsv (or=0.71, 95% ci 0.52-0.98), and ifva (or=1.77, 95% ci 1.00-3.13). conclusions presence of hbov1 in nasopharyngeal aspirates did not correlate with region or gender, although the prevalence of hbov1 was higher in younger children. there were no correlations between hbov1 and other variables, except for the season and adv, rsv, or ifva infections. introduction espiratory infections are often the result of combined bacterial and viral infections [1] , and severe acute respiratory infections are the leading cause of mortality among children worldwide [2] . the traditional viruses known to cause respiratory tract infections include adenovirus (adv), influenza virus (ifv) a and b, human rhinoviruses (hrv), and respiratory syncytial virus (rsv). new viruses have been discovered in recent years, including human metapneumovirus (hmpv) [3] , the coronaviruses hcov-nl63 [4] , and hcov-hku1 [5] [6] , as well as human bocavirus (hbov) which was the first virus identified by molecular virus screening in 2005 [7] . more than two-thirds of childhood pneumonia cases are associated with viral infections [8] [9] . reports of hbov in respiratory tract infections have increased rapidly in numerous countries and regions throughout the world [10] [11] [12] [13] [14] [15] [16] [17] , with hbovs detected in 1.5%-19% of respiratory tract secretions and 0.8%-9.1% of fecal samples [12] [13] . thus, increasing evidence suggests that hbov1 is an important cause of respiratory tract infections [10] [11] [12] 14] . hbov2 [18] , hbov3 [19] , and hbov4 [20] were isolated recently in stool samples from patients with diarrhea. all four hbov genotypes can be detected in stool of patients with acute gastroenteritis, while hbov1 and hbov2 were reported in respiratory tract samples [12] . however, pathogen testing of respiratory infected patient sputum found the presence of other viruses and bacteria in addition to hbov1. thus, it remains to be clarified whether high-rates of hbov co-infection in the respiratory tract lead to disease, or represents only a concomitant viral infection. there is also a lack of large-scale hbov studies determining whether there are ethnic and regional differences in hbov detection rates, since the incidence and clinical presentation of hbov vary widely. furthermore, the epidemiological characteristics of hbov1 and its relationships with other viruses need to be elucidated. from november 2007 to july 2011, nasopharyngeal aspirates (npas) were obtained from 2175 children <173 months of age with acute respiratory tract infection. all cases of children with acute respiratory tract infections were included in our study, data were collected using a standardized questionnaire, diagnostic criteria based on practical pediatrics (seventh edition), including: acute upper respiratory tract infections (including acute laryngitis, herpes angina, acute tonsillitis), acute bronchitis (including asthmatic bronchitis), pneumonia (including bronchiolitis), and acute exacerbation of bronchial asthma. inclusion criteria: (1) patients with the 'acute respiratory infection' diagnostic criteria characteristics from inpatient or outpatient, (2) patients or legal guardian signed an informed consent form, (3) age of patients were <15 years, (4) the course of disease of patients were within 48 h. exclusion criteria: (1) the patients did not meet the diagnostic criteria, (2) although within 48 h, the patients had received anti-viral or anti-inflammatory drug treatment, (3) patients associated with cardiovascular, liver, kidney, hematopoietic system, or other serious diseases, (4) the sample of patients could not be completely collected. of these samples, 1386 were from the first hospital of lanzhou university (lanzhou, china) and 789 were from nanjing children's hospital, medical school of nanjing university (nanjing, china). acute respiratory tract infections were classified according to who definitions [21] ; children with symptoms of acute upper respiratory tract infection (urti) and acute lower respiratory tract infection (lrti) were diagnosed in the presence of characteristic manifestations of rhinitis, pharyngitis, tracheitis, bronchitis, pneumonia, and severe pneumonia. the clinical diagnostic classification (i-iv) was based on previous studies [22] . it was assessed at the time of enrollment and categorized as follows. i grade: very mild (upper respiratory tract symptoms/signs only); ii grade: mild (lower respiratory tract symptoms/signs +/2 upper respiratory tract symptoms/signs but not needing hospital admission); iii grade: moderate (lower respiratory tract symptoms/signs +/2 upper respiratory tract symptoms/signs, needing hospital admission but with oxygen saturations in air >93% on pulse oximetry); iv grade: severe (lower respiratory tract symptoms/signs +/2 upper respiratory tract symptoms/signs, needing hospital admission and oxygen with saturations in air <93%). informed consent was obtained from the parents of all children who provided specimens and the study protocol was approved by the ethics committee of each hospital. demographic and clinical data were recorded from all patients and specimens were stored at -80 °c for further analysis. viral rna and dna were extracted from the npas by using a qiaamp® viral rna mini kit (qiagen, hilden, germany), which extracts viral rna and dna simultaneously according to the manufacturer's instructions. the nested pcr assay with primers (forward1 5'-a ggtaaaacaaatattgcaaa ggccat agtc-3' and reverse1 5'-tgggagttctctccgtccgtat c-3', forward2 5'-agggtttg t ctttaacgattgc agac aac-3', and reverse2 5'-tatacacagagt cgtcagcac tatgag-3') was performed using ex-taq dna polymerase (takara, beijing, china) to amplify a 455-bp fragment targeting a portion of the ns1 genes, as reported previously [23] . the amplicons from a second-round of pcr were analyzed by 1.5% agarose gel electrophoresis and sequenced with the big-dye terminator cycle sequencing kit and the automated abi prism 310 genetic analyzer (qiagen, beijing, china). sequences were edited using the dna star software package. blast (www.ncbi. nlm.nih.gov) was used to confirm gene identities in the national center for biotechnology information (ncbi) databases. all samples were tested for 11 pathogens using standard reverse transcription pcr. these pathogens included ifva, ifvb, hmpv, rsv, hrv, piv types 1-3, hcov-nl63, and hku1 [7, [24] [25] [26] . a different pcr method was used to detect adv [27] . all sample data were done using epidata analysis. categorical data and the median ages of children in different groups were compared using the chi-square (χ 2 ) test and mann-whitney test, respectively. binary logistic regression analysis and independent variables were defined by region (lanzhou=1, nanjing=2), gender (male=1, female=2), age (months), season (1-12), disease classification (i-iv), and inpatient/outpatient (inpatient=1, outpatient=2). the 11 other respiratory viruses were also defined as independent variables (positive=1, negative=0). hbov1 infection (positive=1, negative=0) was the dependent variable in multivariate analysis. eigenvalues and condition indices were used to assess multi-colinearity in the regression models. all tests were two-tailed and p≤0.05 was considered statistically significant. all analyses were performed using spss version 13.0. the 2175 children with acute respiratory infections in this study were aged 9 d to 173 months (the median age was 26 months). the majority of patients (74.11%) were aged >24 months. the ratio of males to females was 2.0:1.0 and that of inpatients to outpatients was 6.35:1. the case ratio of lanzhou to nanjing was 1.76:1.0. respiratory tract infection disease classifications at grade iii-iv accounted for 66.73% of cases. hbov1-dna was detected by nested pcr in 235 specimens, an overall frequency of 10.80%. hbov1 positive rates were 11.62% (161/1386) in lanzhou and 9.38% (74/789) in nanjing (χ 2 =2.61, p>0.05). the ratio of males to females with hbov1-dna was 1.83:1 but the hbov1 positive rate did not differ based on gender or region ( table 1 ). the positivity rates were 6.97% (53/760) for the <6 months old group, 14.55% (112/770) for the 7-24 months old group, 12.30% (53/431) for the 25-60 months old group, 13.64% (9/66) for the 61-72 months old group, 5.41% (8/148) for the >72 months old group (figure 1 ). hbov1-dna was detected in every month, but detection peaked in december (13.75%), followed by 13.16% in july, 12.70% in february, 12.50% in april, and 11.29% in may (figure 2 ). the seasonal distribution of hbov1-dna differed significantly (χ 2 =19.97, p<0.05) in the positive and negative groups. while our study used highly sensitive and low error probability nested pcr to screen hbov1, hbov1 detection rates varies widely. since our techniques result in highly precise and sensitive detection of hbov1 infection, environmental or socio-economic factors may potentiate different patient risk factors. previous studies had shown that the peak of hbov1 infection is in the summer [12, 14] or spring [37, 39] . in our study however, the peak seasons of hbov1 infection occurred in february, april, july, and december during the 48 months collecting period. thus, the seasonal frequency of hbov1 infection should be further investigated as it appears to be more common in the colder seasons [37] . regardless, the seasonal peaks of hbov1 infection have differed among counties and climate regions. previous studies have determined that hbov1 has a high co-infection rate with other respiratory viruses [12] [13] , but its pathogenic role was still debatable due to high frequency of co-infection. hbov1 may exist in the respiratory tracts as a bystander without causality to a patient's current symptoms [13] . additionally, hbov1 may have an important role in acute respiratory infections among children [43] . in patients with respiratory complaints, hbov1 can be found alone or, more often, in combination with other viruses known to cause respiratory complaints [44] . hbov1 may be a factitive with other respiratory viruses for the observed symptions, suggested by the findings that symptomatic children had higher hbov1 viral load than asymptomatic children [41] . in our study, 72.34% of hbov1-positive patients had co-infection with 10 different respiratory viruses, which is similar to previous reports [12] [13] [14] [46] [47] [48] . hbov1 co-infection has been reported with rsv [8] [9] 14, [45] [46] [47] [48] , ifva [12, 14] , and adv [8] [9] in children and adults with respiratory tract infections, and also with hmpv infections in children with community-acquired pneumonia [49] . thus, the reasons underlying the high co-infection rate of hbov1 need further study and leads to questions regarding hbov1 pathogenicity [12] . recent studies based on epidemiological evidence show that hbov1 was likely to be a major contributor to acute respiratory tract disease, particularly in infants and chlidren [13] . hbov1 co-infections with other viruses have been associated with greater disease burden (i.e., more hospitalizations and loss of school days) in children with respiratory tract infection than in those with hbov infection alone. many factors might have an impact on hbov1 infection and a larger study seemed to corroborate a synergistic relationship between hbov1 and the pathogenesis of respiratory syndromes such as influenza-like illnesses, bronchiolitis, and pneumonia [47] . however, our regression model showed that hbov1-positivity was highly correlated with season, rsv, adv, and ifva (p<0.05). hbov1 belongs to the parvoviridae family with viral head-to-tail genome sequences that use typical rolling circle replication [50] [51] . hbov1 may be triggered by helper viruses, although it had been hypothesized that picornaviruses may be a factor that promotes clinical illnesses [52] . our analysis suggested a possible synergistic effect between hbov1 and adv and ifva (or>1). the results should be confirmed by further clinical and experimental data. nevertheless, given that the or value for rsv infection in multivariate analysis was <1, it was estimated that related to the wide distribution of rsv. unfortunately, the analysis results did not show a link between age and hbov1, which may be due to the fact that infants and young children were the primary participants in our study. this connection requires further investigation before any interactions can be confirmed. deficiencies still existed in our manuscript because some patients or legal guardians did not agree to take part in our study. as such, some cases were missed, so potential sample selection bias were not absolutely avoided. however, our large sample size likely weakened any potential sample bias. samples of cases that were excluded were not collected in this study, so the frequency of infection in exclusive samples of cases needs to be revealed by more studies. in addition, distribution of hbov1 positive infections in children without respiratory tract infections remains crucial to be explored for revealing relationships between hbov1 in the health and the diseased. in summary, we examined hbov1 in children with respiratory infections but require further information to elucidate its pathogenic role. our understanding of this virus would be greatly enhanced by large-scale molecular epidemiological studies of pathogens in different regions. none of the authors has a conflict of interest. zd and yj designed the study. cc, xy, js, jz, yz, and xg collected the samples. jw, jl, zx, and xg performed the pcr tests. jw and nl analyzed the data. jw, jy, and nl wrote and finalized the manuscript. all authors read and approved the final manuscript. epidemic intelligence service investigations of respiratory illness rna viruses in young nepalese children hospitalized with severe pneumonia a newly discovered human pneumovirus isolated from young children with respiratory tract disease a previously undescribed coronavirus associated with respiratory disease in humans clinical and molecular epidemiological features of coronavirus hku1-associated community-acquired pneumonia characterization and complete genome sequence of a novel coronavirus, coronavirus hku1, from patients with pneumonia cloning of a human parvovirus by molecular screening of respiratory tract samples the role of respiratory viral infections among children hospitalized for community-acquired pneumonia in a developing country etiology of community-acquired pneumonia in hospitalized children based on who clinical guidelines detection of human bocavirus in children with upper respiratory tract infection by polymerase chain reaction detection of bocavirus in children suffering from acute respiratory tract infections in saudi arabia surveillance and genome analysis of human bocavirus in patients with respiratory infection in guangzhou, china human bocavirus-the first 5 years detection of human bocavirus from children and adults with acute respiratory tract illness in guangzhou, southern china detection of hbov dna in idiopathic lung fibrosis primary and secondary human bocavirus 1 infections in a family life-threatening respiratory tract disease with human bocavirus-1 infection in a 4-year-old child a newly identified bocavirus species in human stool a novel bocavirus associated with acute gastroenteritis in australian children human bocaviruses are highly diverse, dispersed, recombination prone, and prevalent in enteric infections integrated management of childhood illness handbook viral and atypical bacterial detection in acute respiratory infection in children under five years human bocavirus in children hospitalized for acute gastroenteritis: a case-control study development of three multiplex rt-pcr assays for the detection of 12 respiratory rna viruses human coronavirus nl63 infection in canada detection of the new human coronavirus hku1: a report of 6 cases detection of adenovirus in clinical specimens by polymerase chain reaction and liquid-phase hybridization quantitated by time-resolved fluorometry correlation between bocavirus infection and humoral response, and co-infection with other respiratory viruses in children with acute respiratory infection frequent and prolonged shedding of bocavirus in young children attending daycare human bocavirus species 2 and 3 in brazil phylogenetic and recombination analysis of human bocavirus 2 high incidence of human bocavirus infection in children in spain seroepidemiology of human bocaviruses 1-4 co-circulation of genetically distinct human metapneumovirus and human bocavirus strains in young children with respiratory tract infections in italy human bocavirus in jordan: prevalence and clinical symptoms in hospitalised paediatric patients and molecular virus characterisation human bocavirus infection human bocavirus: a novel parvovirus epidemiologically associated with pneumonia requiring hospitalization in thailand clinical relevance of human bocavirus with acute respiratory tract infection and diarrhea in children: a prospective case-control study frequent detection of bocavirus dna in german children with respiratory tract infections the association of newly identified respiratory viruses with lower respiratory tract infections in korean children high human bocavirus viral load is associated with disease severity in children under five years of age human bocavirus infection in children with acute respiratory tract infection in india human bocavirus in children with acute respiratory infections in vietnam human bocavirus, a newly discovered parvovirus of the respiratory tract complete genome sequence of a novel reassortant h11n2 avian influenza virus isolated from a live poultry market in eastern china distinct regulation of host responses by erk and jnk map kinases in swine macrophages infected with pandemic (h1n1) 2009 influenza virus human bocavirus in italian patients with respiratory diseases rapid detection of respiratory tract viral infections and coinfections in patients with influenza-like illnesses by use of reverse transcription-pcr dna microarray systems viruses and bacteria in sputum samples of children with community-acquired pneumonia detection of head-to-tail dna sequences of human bocavirus in clinical samples human bocavirus infection in young children with acute respiratory tract infection in lanzhou does human bocavirus infection depend on helper viruses? a challenging case report key: cord-351008-p0n1fdxw authors: monge, susana; duijster, janneke; kommer, geert jan; van de kassteele, jan; krafft, thomas; engelen, paul; valk, jens p.; de waard, jan; de nooij, jan; riezebos‐brilman, annelies; van der hoek, wim; van asten, liselotte title: ambulance dispatch calls attributable to influenza a and other common respiratory viruses in the netherlands (2014‐2016) date: 2020-05-14 journal: influenza other respir viruses doi: 10.1111/irv.12731 sha: doc_id: 351008 cord_uid: p0n1fdxw background: ambulance dispatches could be useful for syndromic surveillance of severe respiratory infections. we evaluated whether ambulance dispatch calls of highest urgency reflect the circulation of influenza a virus, influenza b virus, respiratory syncytial virus (rsv), rhinovirus, adenovirus, coronavirus, parainfluenzavirus and human metapneumovirus (hmpv). methods: we analysed calls from four ambulance call centres serving 25% of the population in the netherlands (2014‐2016). the chief symptom and urgency level is recorded during triage; we restricted our analysis to calls with the highest urgency and identified those compatible with a respiratory syndrome. we modelled the relation between respiratory syndrome calls (rsc) and respiratory virus trends using binomial regression with identity link function. results: we included 211 739 calls, of which 15 385 (7.3%) were rsc. proportion of rsc showed periodicity with winter peaks and smaller interseasonal increases. overall, 15% of rsc were attributable to respiratory viruses (20% in out‐of‐office hour calls). there was large variation by age group: in <15 years, only rsv was associated and explained 11% of rsc; in 15‐64 years, only influenza a (explained 3% of rsc); and in ≥65 years adenovirus explained 9% of rsc, distributed throughout the year, and hmpv (4%) and influenza a (1%) mainly during the winter peaks. additionally, rhinovirus was associated with total rsc. conclusion: high urgency ambulance dispatches reflect the burden of different respiratory viruses and might be useful to monitor the respiratory season overall. influenza plays a smaller role than other viruses: rsv is important in children while adenovirus and hmpv are the biggest contributors to emergency calls in the elderly. surveillance of respiratory viruses is mainly centred on influenza, for which robust systems have been developed in most countries, generally based on sentinel networks of general practitioners (gps, primary care). 1 comparable surveillance systems do not exist for other respiratory viruses, despite the increasing interest and leadership of the world health organization (who) in expanding surveillance for respiratory syncytial virus (rsv) now that a vaccine may become available. 2 for most viruses, surveillance is limited to laboratory-based counts, often with unknown denominator, low representativeness or lack of standard sampling criteria. who encourages surveillance of severe acute respiratory infections (sari) in the context of the pandemic influenza severity assessment program. 1 this is fundamental to determine the severity of circulating viruses, their pressure on healthcare services and the groups most at risk of severe outcomes. surveillance of severe infections requiring secondary care is much less developed than surveillance in primary care. in europe, a few countries have established hospital-based surveillance based on syndromic sari, laboratory confirmed cases or a combination of both. 3, 4 in the netherlands, a pilot involving three hospitals has been running since 2015. 3 syndromic surveillance using ready-to-use data has also been explored, mainly in emergency rooms. [5] [6] [7] few initiatives have used ambulance data 5, 8, 9, 10 or ambulance dispatch centre data. 5, 8, 10, 11 ambulance dispatch centres could be an alternative source of readily available data to monitor the occurrence of severe respiratory infections. during the triage process, information is collected and recorded in real time, including the chief symptom in very broad categories, as their objective is to rapidly assign an urgency level and prioritize resources. a recent study in the netherlands has shown how the variability in respiratory syndromes is correlated with ili from sentinel gp surveillance, 12 making it a potential source for syndromic surveillance. however, not all respiratory viruses will result in ili, and although the ili case definition focuses on detecting influenza infections, ili can be caused by a wide range of viruses. in this study, we aimed to assess to what extent ambulance dispatches reflect the activity of different respiratory viruses in order to advance our understanding of their use for the surveillance of severe acute infections by different respiratory viruses. specifically, we evaluated the association of syndromes compatible with respiratory infections in ambulance dispatches with trends in detections of influenza a, influenza b, rsv, rhinovirus, adenovirus, coronavirus, parainfluenza and human metapneumovirus (hmpv). the netherlands is divided into 25 regional ambulance services we focused our analysis to a1 urgency calls, as we previously found these to have a stronger association with ili. 12 these calls may better capture variations in acute severe infections by respiratory viruses and be a valid source for their surveillance. calls with triage codes that were potentially compatible with respiratory infections (table 1) year, and hmpv (4%) and influenza a (1%) mainly during the winter peaks. additionally, rhinovirus was associated with total rsc. high urgency ambulance dispatches reflect the burden of different respiratory viruses and might be useful to monitor the respiratory season overall. influenza plays a smaller role than other viruses: rsv is important in children while adenovirus and hmpv are the biggest contributors to emergency calls in the elderly. adenovirus, ambulance, coronavirus, influenza, respiratory syncytial virus, rhinovirus the number of respiratory virus identifications was obtained from the weekly sentinel surveillance system of the dutch working group on clinical virology. twenty-one virological laboratories voluntarily provide aggregated weekly number of diagnoses; individualized information such as age or sex is not provided. also, no distinctions are made between primary or secondary care, or different diagnostic methods, although currently the majority use molecular methods or rapid tests. 13 we included weekly reports of influenza a, influenza b, rhinovirus, rsv, adenovirus, coronavirus, parainfluenza and hmpv. we analysed weekly rsc as a proportion of the total number of calls overall, by age group and by time of the day: office hours because the trends in rsc might coincide, precede or lag behind the trends in viruses reports, we considered virus reports either in the current week, or lagged up to 4 weeks to the right, that is future in time (+lags), or 4 weeks to the left, that is backwards in time (−lags), for a total of 9 time-lagged variables of each virus. when building the models, the time lag with the lowest p-value was selected, and only one time lag per virus was allowed. because one of the viruses with the highest interest in monitoring its severity is influenza a (due to shifts, drifts and its pandemic potential), we forced it into the model, unless its coefficient was negative due to biological implausibility. subsequently, other viruses were added if statistically significant, had a positive coefficient and did not revert to negative the coefficients of viruses previously added to the model. finally, because the influenza epidemic size and severity varies by season, an interaction between influenza a and an indicator variable for the epidemiologic year (from week 27 to week 26 of the following year) was tested and retained if p < .05. the indicator variable itself was not included, as we wanted to attribute differences between years to influenza a. model assumptions and absence of remaining seasonality and autocorrelation were assessed by residuals diagnostics. we used r, version 3.4.0. table 2 ). the most frequent triage code among rsc was "abnormal breathing, troubles speaking between two breaths" (table 1) . weekly average number of rsc was 98 (range 58-138), which corresponds to 2.3 calls per 100 000 inhabitants every week. the proportion of rsc showed a periodic pattern peaking in winter, with lower interseasonal peaks (figure 1 ). the periodicity was evident in out-of-office hours and people ≥65 years, but the pattern was less clear in other groups and, in children <15 years, the peak occurred earlier. among the included respiratory viruses, the most frequently reported were rhinovirus and influenza a, followed by rsv (table 3) . most viruses had a periodic pattern similar to rsc, peaking in winter, except for rhinovirus and parainfluenza, which had a less distinct pattern with peaks in the autumn or the spring (figure 1 ). adenovirus reports showed smaller interseasonal peaks in addition to winter peaks. associations between respiratory viruses and the proportion of rsc are reported in table 4 and figure 2 . in children <15 years, only rsv was associated with rsc, explaining part of the rsc winter e adjusted by sine and a cosine term with periodicity of 1 y. f +lags mean that the rsc from the current week are best associated with viruses from x weeks in the past (ie trend in viruses precedes rsc); -lags mean that they are best associated with viruses from x weeks in the future (ie trend of rsc precedes the viruses). *calculated applying the model coefficient to the average weekly number of virus reports, and multiplied by the annual number of ambulance calls by epidemiologic year, age group, office or out-of-office hours, as appropriate; for the overall effects, this represents the average per epidemiologic year; for epidemiologic year-specific effects, the numbers for incomplete epidemiologic years are extrapolations to represent complete epidemiologic years if the average weekly ili incidence and ambulance calls were similar in non-observed weeks than in observed weeks. **calculated dividing the number of rsc attributable to each virus (from the previous column) by the number of observed rsc by age group, epidemiologic year, office or out-of-office hours, as appropriate. our results show that trends in rsc from highest urgency ambulance dispatches are associated with trends in the activity of common respiratory viruses. depending on the subgroup 0%-20% of rsc was attributable to a combination of respiratory viruses. the specific viruses contributing to rsc varied by age group, with estimates of 1%-11% of rsc being attributable per individual virus. their burden on these 4 call centres covering a 4 million population was 948 of highest urgency calls per year (22/100 000 inhabitants), although this varied by virus and age group. in emergency departments, 25% of all acute respiratory diseases are attributable to respiratory pathogens, 14 up to 80% in children. 15 in our study, the majority of rsc were incorporated into the unexplained baseline. this is not an unexpected finding, since the categories of symptoms included in ampds triage codes are very broad, resulting in high background noise. 16 nevertheless, variability in rsc above this high baseline was associated with trends of common respiratory viruses, pointing at their potential usefulness to monitor the respiratory season overall (ie irrespective of the causative pathogen), as previously shown by its association with ili. 12 the different viruses potentially involved in rsc, their individual trends, and their seasonal variation in severity would make it challenging to design indicators and models that will allow us to prospectively use rsc data for situational awareness for specific viruses separately. conversely, large or unexpected increases in a specific respiratory virus might be reflected to a certain extent in rsc. influenza a is a leading cause of acute lower respiratory tract infection, particularly in the elderly. 15, 16 by contrast, in our study its contribution to rsc was low, especially among the elderly. in children, influenza was not associated to rsc, consistently with its low to marginal role in sari in this age group. 13, [17] [18] [19] the effect of influenza a on rsc (1%-3%) is lower than what we found for ili, which was attributed 4%-34% of rsc. 12 influenza b did not show association with rsc in any group, in line with our understanding of its lower, less severe impact and lower clinical burden. lower representativeness of the laboratory data in our current study may have underestimated the association for influenza, or oppositely, its effect estimated through ili may be overestimated because ili is caused also by other viruses. the effect of influenza a on rsc was found to vary by season only for the overall sample. this is fundamental to assess whether causing severe infections, second to rsv in children, [17] [18] [19] and after influenza in adults. 15, [21] [22] [23] its presentation year-round, with peaks in autumn and winter, 24 also contributes to its high overall impact. adenovirus explained a significant proportion of rsc, especially among the elderly. adenovirus is rarely detected in cases of severe respiratory infection, 15, 22 although in a study in finland, it was the second aetiology in mechanically ventilated patients with community-acquired pneumonia. 21 hmpv had a similar relative effect as adenovirus, although its impact on number of ambulance calls was smaller, since it was less frequent. in children <15 years the peak in rsc developed earlier in the year, and our model associated this to rsv, consistent with its earlier presentation in the season. 13, 16, 25 rsv is the leading cause of sari in young children 13, [17] [18] [19] 23 and has been highly associated to sari syndromes in emergency departments 6, 14 and ambulances. 10, 18 the differences between office and out-of-office hours likely reflect that ambulance calls in these two time frames are distinct populations, probably with a different share of clinical pictures and severity. however, we cannot totally rule out a lack of statistical power during office hours, since the number of calls was smaller. ambulance dispatches are convenient for syndromic surveillance because they reflect events that are perceived as urgent (and thus potentially severe), they are recorded continuously and they have a virtually universal coverage. 8, 26 moreover, triage algorithms are increasingly standardized internationally. 5, 11 however, the true usefulness and added value of ambulance dispatches for infectious disease surveillance needs to be studied and piloted prospectively. some challenges for routinely using ambulance dispatch data prospectively include establishing data sharing routines and complying with data protection regulations. there are limitations to our data. because we did not include a2urgency level calls in our analysis, our results cannot be interpreted as the burden of respiratory viruses in ambulance services as a whole, but only in the highest urgency services. since all associations are evaluated ecologically, spurious attribution of rsc trends to respiratory viruses cannot be ruled out. sentinel laboratory surveillance has several limitations: it is passive and reported trends can include surveillance artefacts; it does not provide information on age, so overall number of virus detections was used; and while often biased to secondary care, it captures patients from both primary and secondary care, and the pathogens underlying their symptoms may differ from patients in ambulance dispatches. our study covered only 6 ravs, 25% of the population in the netherlands, but we do not believe these are fundamentally different from non-included ravs. however, because the sentinel laboratory surveillance is widespread throughout the country, it could be possible that intensity or timeliness of circulation of the different viruses nationally is different from specific regional patterns in ravs included in our study. finally, as the netherlands has a comprehensive primary care system where gps that have a strong gate-keeping role (including out-of-office services), our study results cannot be directly compared to health systems with higher use of emergency medical services. because of its ability to capture variations in respiratory virus circulation, ambulance dispatch data might be useful to signal events and to monitor the respiratory season as a whole, specifically reflecting severe infections and thus complementing existing surveillance systems. it will probably have less potential for drawing conclusions about the separate effect of specific individual viruses when not combined with information from other data sources, due to the low magnitude of some associations, the different viruses reflected in rsc and their proportional variation throughout the year. the true utility of ambulance dispatch data needs to be tested prospectively and faces potential challenges regarding timely data sharing and data protection. we appreciate the contribution of the four regional ambulance none declared. https://orcid.org/0000-0003-1412-3012 liselotte van asten https://orcid.org/0000-0002-4123-7595 wileyonlinelibrary.com/journal/irv 1. who. global epidemiological surveillance standards for influenza. world health organization world health organization. who strategy to pilot global respiratory syncytial virus surveillance based on the global influenza surveillance and response system (gisrs) severe acute respiratory infections: the missing block in the surveillance pyramid] (dutch) developing a system to estimate the severity of influenza infection in england: findings from a hospital-based surveillance system between validity and timeliness of syndromic influenza surveillance during the autumn/winter wave of a (h1n1) influenza 2009: results of emergency medical dispatch, ambulance and emergency department data from three european regions emergency department syndromic surveillance providing early warning of seasonal respiratory activity in england syndromic surveillance for influenza in the emergency department-a systematic review utility of emergency call centre, dispatch and ambulance data for syndromic surveillance of infectious diseases: a scoping review a concept for routine emergency-care data-based syndromic surveillance in europe utility of ambulance data for real-time syndromic surveillance: a pilot in the west midlands region, united kingdom assessment of ambulance dispatch data for surveillance of influenza-like illness in melbourne use of ambulance dispatch calls for surveillance of severe acute respiratory infections annual report surveillance of influenza and other respiratory infections in the netherlands: winter validation of syndromic surveillance for respiratory infections prevalence of respiratory viruses among adults, by season, age, respiratory tract region and type of medical prevalence and seasonality of six respiratory viruses during five consecutive epidemic seasons in belgium respiratory viruses in children admitted to hospital intensive care units: evaluating the clart(r) pneumovir dna array clinical evaluation of viral acute respiratory tract infections in children presenting to the emergency department of a tertiary referral hospital in the netherlands viral aetiology and clinical outcomes in hospitalised infants presenting with respiratory distress what is the real role of respiratory viruses in severe community-acquired pneumonia? lower respiratory tract virus findings in mechanically ventilated patients with severe community-acquired pneumonia clinical characteristics and outcome of respiratory syncytial virus infection among adults hospitalized with influenza-like illness in france frequency of respiratory viruses among patients admitted to 26 intensive care units in seven consecutive winter-spring seasons (2009-2016) in northern italy prevalence of rhinoviruses in young children of an unselected birth cohort from the netherlands seasonality and geographical spread of respiratory syncytial virus epidemics in 15 european countries clinical evaluation of the emergency medical services (ems) ambulance dispatch-based syndromic surveillance system ambulance dispatch calls attributable to influenza a and other common respiratory viruses in the netherlands key: cord-295074-fsbp4fky authors: broor, shobha; dawood, fatimah s.; pandey, bharti g.; saha, siddhartha; gupta, vivek; krishnan, anand; rai, sanjay; singh, pratibha; erdman, dean; lal, renu b. title: rates of respiratory virus-associated hospitalization in children aged <5 years in rural northern india date: 2013-11-21 journal: j infect doi: 10.1016/j.jinf.2013.11.005 sha: doc_id: 295074 cord_uid: fsbp4fky objectives: though respiratory viruses are thought to cause substantial morbidity globally in children aged <5 years, the incidence of severe respiratory virus infections in children is unknown in india where 20% of the world's children live. methods: during august 2009–july 2011, prospective population-based surveillance was conducted for hospitalizations of children aged <5 years in a rural community in haryana state. clinical data and respiratory specimens were collected. swabs were tested by rt-pcr for influenza and parainfluenza viruses, respiratory syncytial virus (rsv), human metapneumovirus, coronaviruses, and adenovirus. average annual hospitalization incidence was calculated using census data and adjusted for hospitalizations reported to occur at non-study hospitals according to a comunity healthcare utilization survey. results: of 245 hospitalized children, respiratory viruses were detected among 98 (40%), of whom 92 (94%) had fever or respiratory symptoms. rsv accounted for the highest virus-associated hospitalization incidence (34.6/10,000, 95% ci 26.3–44.7) and 20% of hospitalizations. there were 11.8/10,000 (95% ci 7.9–18.4) influenza-associated hospitalizations (7% of hospitalizations). rsv and influenza virus detection peaked in winter (november–february) and rainy seasons (july), respectively. conclusion: respiratory viruses were associated with a substantial proportion of hospitalizations among young children in a rural indian community. public health research and prevention in india should consider targeting rsv and influenza in young children. rates of respiratory virus-associated hospitalization in children aged <5 years in rural northern india introduction acute respiratory infections are recognized as an important cause of mortality, hospitalization, and healthcare utilization in young children globally. 1e3 respiratory virus infections are increasingly recognized as major contributors to the burden of severe acute respiratory illness in many countries due to expanding global surveillance and the advent of improved molecular diagnostic testing for respiratory viruses. 4e8 in studies of respiratory virus detection among children hospitalized with respiratory illness from different parts of the world, rsv and influenza are frequently associated with a substantial proportion of hospitalizations. 9e14 these findings are of public health importance because effective and safe influenza vaccines are available but not widely used in many countries, and development of respiratory syncytial virus (rsv) vaccines continues to be an area of intense study although no licensed rsv vaccine is available. 12, 15, 16 understanding the incidence of respiratory virus-associated severe illness and the timing of respiratory virus circulation is critical to inform research priorities and policy decisions about introduction of available respiratory virus vaccines for children. india is the second most populous country in the world with 20% of the world's population of children aged <5 years, and almost a third of global pneumonia cases among children aged <5 years are thought to occur in india. 3 public health policies that effectively address causes of severe respiratory illness among children in india would have a substantial impact on global child morbidity and mortality. however, the burden of respiratory virus-associated severe illness among young children in india is unknown. in addition, the few studies that evaluated respiratory viral etiologies of severe illness among children in india were conducted before the advent of newer and more sensitive diagnostic tests, including tests to detect more recently discovered viruses. 17e20 using data from population-based surveillance of approximately 9500 children for hospitalizations for acute medical illness in rural northern india and concomitant testing for respiratory viruses by real-time reverse transcription polymerase chain reaction (rrt-pcr), we estimate the incidence of respiratory virus-associated hospitalizations among children aged <5 years. we also describe the timing of virus circulation and clinical presentation of children hospitalized with predominant viruses. the comprehensive rural health services project (crhsp), ballabgarh study site includes a 323 square kilometer area in haryana state, about 40 km south of new delhi. the climate is temperate with a defined colder winter season during novemberemarch and rainy season during julyeseptember. as part of the crhsp, a health and demographic surveillance system (hdss) was maintained under which all residents of the crhsp study site were enrolled in a computerized database with unique identification numbers. the hdss tracked major events such as births, deaths, marriages and migrations. during the study period, the crhsp study site included a population of approximately 88,000 persons including approximately 9500 children aged <5 years in 28 villages. the main providers of inpatient care to the crhsp population are a government-funded secondary level facility with 60 beds that provides outpatient and inpatient care and serves 15e20% of the crhsp population, two other government-funded secondary level facilities, and a large number of private health facilities (ranging in size from 5 to 35 beds) that provide inpatient and outpatient health services. health facilities are largely situated outside crhsp villages within a range of 3e20 km and largely accessible by two/three wheelers. most facilities have the resources to care for patients requiring supplemental oxygen but transfer patients requiring mechanical ventilation and intensive level to tertiary care facilities outside the district. hospitalized patients were enrolled from the three secondary level facilities and 18 private facilities in ballabgarh and faridabad towns where patients from the crhsp area were likely to seek inpatient care corroborated by health utilization survey. patients were eligible for enrollment if they were residents of the crhsp area and were being hospitalized overnight with any acute medical illness, excluding hospitalizations for the following conditions assumed to be unlikely to be related to respiratory infection: trauma, diarrhea without fever, elective surgery, accidental poisonings, elective blood transfusions, or orthopedic or ophthalmologic conditions. during august 2009ejuly 2011, patients were prospectively enrolled in the study as previously described. 21 data on demographic characteristics, medical history, and clinical symptoms were obtained by interview of patients' caregivers. data on clinical signs were abstracted from the medical record using a standardized data collection form. respiratory specimen samples were collected by study doctors from enrolled patients within 24 h of admission using polyester swabs. during augustedecember 2010, study investigators visited all houses in the 28 villages of the crhsp area to conduct a healthcare utilization survey using a standardized questionnaire that was field testing in previous years; 90% of households in the area completed the survey. the survey asked whether any member of the household had been admitted to a hospital for an overnight stay during the preceding year (using a reference period of august 1, 2009ejuly 31, 2010 . for each reported hospitalization, the survey asked about the location and reason for hospitalization, and field workers attempted to validate reports with any available documentation related to the hospitalization. combined throat and nasal swabs were collected from each participant (nasal swabs alone in infants). the swabs were transported in viral transport media on ice to the all india institute of medical sciences (aiims) laboratory within 24 h. samples were then divided into aliquots for respiratory virus detection by rrt-pcr. testing for influenza viruses and influenza virus subtyping was conducted at aiims using us centers for disease control and prevention (cdc) protocols 22 and after laboratory staff received cdc-sponsored training and the laboratory underwent quality control assessment by cdc. testing for non-influenza respiratory viruses was also conducted at aiims using highly sensitive rrt-pcr assays for respiratory syncytial virus (rsv), human metapneumovirus (hmpv), human parainfluenza viruses 1e3 (piv 1e3), adenovirus, and human coronavirus 229e using cdc protocols 23e25 ; protocol are available from cdc upon request. baseline characteristics, clinical symptoms and signs, and length of hospitalization were compared between children with and without respiratory virus detection using bivariate analysis. frequencies of clinical symptoms and signs and median length of hospitalization were also calculated for children with rsv and influenza, the most commonly detected viruses. tachypnea was defined based on integrated management of childhood illness criteria for fast breathing as 60 breaths per minute in children aged <2 months, 50 in children aged 2e11 months, and 40 in children aged 1e5 years. 26 hypoxia was defined as an oxygen saturation of <92% by pulse oximetry at admission. chisquared test or fisher's exact test was used to calculate pvalues for categorical variables and the wilcoxon test was used to compare continuous variables (sas, version 9.2, sas institute inc., cary, nc). since all children hospitalized with acute medical illness were enrolled, the proportion of respiratory virus detections among children with and without fever or key respiratory signs or symptoms was evaluated. fever was defined as either measured temperature >38.0 celsius at admission or parental report of fever because antipyretic use was common in the study community. key respiratory symptoms or signs were defined as parental report of cough or fast breathing or physician exam findings of tachypnea, crepitations, wheezing, nasal flaring, chest indrawing, grunting, or stridor. inclusion of nasal discharge did not change the proportion of children meeting criteria for key respiratory symptoms or signs. all children, regardless of symptoms or signs, were included in all subsequent analyses. using data from the healthcare utilization survey, the proportion of hospitalizations among children in the study community that occurred at study facilities was calculated as reported hospitalizations at study facilities divided by total reported hospitalizations; 95% confidence limits were calculated using the wald method. average annual cumulative incidences of hospitalizations associated with detection of each respiratory virus were calculated for children aged <1 year, 1e4 years, and <5 years. incidences were also calculated for children aged <6 months for rsv and influenza since maternal immunization with rsv and among the 98 children with respiratory virus-associated illness, history of fever (82%) and cough (69%) were the most commonly reported symptoms. parental report of cough, nasal discharge or congestion, and fast breathing and exam findings of hypoxia, crepitations, wheezing, and increased work of breathing were more common in children with respiratory virus-associated hospitalizations than those without (table 2) ; the median length of hospitalization was similar in both groups (4 vs. 3 days, p z 0.05). none of the children died. a larger proportion of children with rsv detection compared to children with other respiratory viruses had a reported history of fast breathing (51% vs. 26%, p z 0.01) and exam findings of increased work of breathing (32% vs. 6%, p < 0.01), crepitations (52% vs. 21%, p < 0.01), and wheezing (40% vs. 10%, p < 0.01). of 9509 children aged <5 years residing in crhsp area, 8773 (92%) were included in the health utilization survey, and 75% (95% ci 74e76%) of all acute medical hospitalizations reported on the health utilization survey occurred at study facilities ( respiratory viruses were detected almost year-round among children hospitalized with acute medical illnesses (fig. 1) . rsv detection occurred from november through may with a longer period of detection during 2009e2010 than during 2011 (fig. 1) . in contrast, influenza virus detection peaked during the rainy season in juneejuly when influenza accounted for 20e42% of monthly acute medical illness hospitalizations. detection of other respiratory viruses occurred sporadically throughout the year. based on surveillance of approximately 9500 children, this study is the first prospective, population-based study to measure the incidence of respiratory virus-associated hospitalizations among children aged <5 years in rural india. we found that respiratory virus infections were associated with 40% of acute medical illness hospitalizations among children aged <5 years, and one in five children hospitalized for acute medical illness had rsv infection. consistent with prior studies, we also found that virus-associated hospitalization rates were highest among children aged <1 year. rsv was the predominant virus identified among children aged <1 year. among children aged 1e4 years, incidence rates of rsv and influenza viruses were similar. rsv and influenza viruses circulated with clearly defined but different seasonality and were infrequently detected among children without fever or respiratory symptoms or signs, similar to prior studies. 9, 10, 30 the incidence of rsv-associated hospitalizations in our study community was substantial, with an incidence of 35 per 10,000 child-years among children aged <5 years and 99 per 10,000 child-years among children aged <1 year. many studies have reported rsv as the most common respiratory virus resulting in hospitalization in young children, but relatively few have estimated the incidence of rsvassociated hospitalizations. rsv-associated hospitalization rates per 10,000 children ranged from 109 to 274 among children aged <1 year in studies from rural thailand, indonesia, the united kingdom, and the united states; 25e30 among children aged <5 years in hong kong and the united states; and 194 and 450 among children aged <5 years without and with hiv infection in south africa. our estimates are similar to those of prior studies from the united states, europe and asia. given the high incidence of rsv-associated hospitalization in our study, availability of an effective and accessible rsv vaccine for young children in india would profoundly impact hospitalization rates in this age group. because the incidence of rsv-associated hospitalization is highest in infants aged <6 months in whom maternal antibody interference with immune responses may make vaccination challenging, rsv vaccination 0.7 (0e3.9) 0.9 (0e5.5) a adjusted to account for hospitalizations that occurred at non-study facilities based on data from healthcare utilization survey. b children with co-detection of more than 1 respiratory virus were included in the incidence estimate of each respiratory virus detected. c among infants aged <6 months, the adjusted incidence of hospitalization for rsv was 136.8 hospitalizations/10,000 (95% ci 91.1e207.6). d among infants aged <6 months, the adjusted incidence of hospitalization for influenza was 17.8 hospitalizations/10,000 (95% ci 6.3e51.9). of pregnant women has been suggested as another potential vaccination strategy. 27e29 though licensed rsv vaccines are not currently available, candidate vaccines are under development 15, 16 and data on rsv disease burden will be important for policy decisions about rsv vaccine introduction if a licensed vaccine becomes available. although we found that influenza viruses were associated with a lower incidence of hospitalization than rsv among children aged <5 years, influenza-associated hospitalizations accounted for 7% of all acute medical hospitalizations and up to 42% of monthly hospitalizations during peak circulation. a similar population-based study conducted in western india during the same time period as ours found similar influenza-associated hospitalization rates among children aged <1 year and higher rates among children aged 1e4 years. 31 the contribution of influenza to severe respiratory illness among children in india deserves attention because influenza vaccines are not currently recommended and are not widely available in india. both traditional inactivated influenza vaccines 32e34 and live attenuated influenza vaccines 35e38 have been shown to be effective against influenza in children in high-income countries. additional studies are needed to further develop the evidence base for policy makers in india to decide whether to support influenza vaccination recommendations for children. these studies should include data on the incidence of severe influenza in other regions of india, the effectiveness of influenza vaccine among indian children, and the costs of influenza in india. a study of influenza vaccine effectiveness among children aged <5 years in rural india is underway. 39 the timing of respiratory virus circulation varies globally. rsv and influenza viruses circulate during the colder winter months in some but not all temperate countries. however, influenza viruses circulate year-round in some tropical countries and both rsv and influenza virus circulation seems to coincide with increased rainfall in some places. 40, 41 india is a large and geographically diverse country with temperate, sub-tropical and tropical climates. in our study, conducted in a temperate area, we found that rsv and influenza circulated during clearly defined but different periods. studies from western india suggest that influenza virus circulation is less defined there, with peaks during the rainy season but perennial influenza virus circulation. 20, 31 additional studies of influenza virus seasonality in other regions of india are needed to determine optimal timing for influenza vaccination and other prevention measures. the advent of highly sensitive pcr assays for respiratory viruses allows easier detection of a wider array of viruses but also presents the challenge of interpreting positive test results. prior studies have shown that some respiratory viruses are frequently detected by pcr among children without fever or respiratory symptoms. 9, 10, 30 respiratory virus detection in these children may reflect acute infection with atypical symptoms or prolonged shedding of virus from a prior infection highlighting the utility of a control group of children without acute illness to aid interpretation of test results. our study did not have a control group, but the large majority of children with respiratory virus detection had fever or respiratory findings that support the clinical diagnosis of respiratory infection. our estimates of rsv and influenza-associated hospitalization incidence are supported by prior studies of hospitalized children in which detection of these viruses was rare from control groups comprised of hospitalized children without fever or respiratory symptoms or non-hospitalized children. though adenoviruses, coronaviruses, human metapneumovirus, and parainfluenza viruses have all been previously implicated in the pathogenesis of severe illness, our hospitalization incidence estimates for these viruses should be interpreted with more caution as detection of these viruses has varied among control groups in other studies. 9, 10, 30 several points should be considered when interpreting our findings. first, our study was conducted at primary and secondary level health facilities without capacity to provide mechanical ventilation and intensive care for critically ill children. thus, we may have missed hospitalizations of children with more severe illness who were directly admitted to tertiary care hospitals outside the study area. our study community may also have been too small to evaluate the frequency of rare but severe outcomes such as death. second, although we attempted to collect the discharge diagnosis of all enrolled children, these data were largely absent from the medical record and therefore could not be used to aid interpretation of respiratory virus testing results. third, we included testing results for only a select group of respiratory viruses previously identified as being associated with a large proportion of respiratory illnesses in children. therefore, our results reflect the burden of respiratory virus-associated hospitalizations caused by this select group of viruses. we did not include test results for rhinoviruses because interpretation of rhinovirus detection in the absence of a control group is difficult, although rhinovirus was the most commonly detected virus in children with respiratory illness in some prior studies. additionally, we did not conduct testing for coronaviruses other than coronavirus 229e, and may have underestimated the incidence of coronavirus-associated hospitalization in the study community. lastly, respiratory virus circulation and incidence is known to vary by year. our study was conducted over a two year period and we present average annual cumulative incidences of virus-associated hospitalization. additional studies of respiratory virusassociated disease burden during additional years will be useful for comparison with our findings. the findings and conclusions in this report are those of the authors and do not necessarily represent the views of the centers for disease control and prevention. this study was supported in part by cooperative agreements u01 ip000206 from the centers for disease control and prevention, atlanta, us. the authors do not have any relevant conflicts of interest to declare. global, regional, and national causes of child mortality: an updated systematic analysis for 2010 with time trends since global and regional burden of hospital admissions for severe acute lower respiratory infections in young children in 2010: a systematic analysis global burden of childhood pneumonia and diarrhoea global burden of respiratory infections due to seasonal influenza in young children: a systematic review and meta-analysis viral infections of the lower respiratory tract viral pneumonia global burden of acute lower respiratory infections due to respiratory syncytial virus in young children: a systematic review and meta-analysis viral etiology of hospitalized acute lower respiratory infections in children under 5 years of age e a systematic review and meta-analysis detecting respiratory viruses in asymptomatic children viral and bacterial causes of severe acute respiratory illness among children aged less than 5 years in a high malaria prevalence area of western kenya the role of respiratory viral infections among children hospitalized for communityacquired pneumonia in a developing country challenges in developing a pediatric rsv vaccine rates of hospitalisation for influenza, respiratory syncytial virus and human metapneumovirus among infants and young children etiology and epidemiology of viral pneumonia among hospitalized children in rural mozambique: a malaria endemic area with high prevalence of human immunodeficiency virus influenza and respiratory syncytial virus (rsv) vaccines for infants: safety, immunogenicity, and efficacy an evaluation of the emerging interventions against respiratory syncytial virus (rsv)-associated acute lower respiratory infections in children etiology of acute respiratory infections in children in tropical southern india etiology of acute lower respiratory tract infection viral etiology of acute respiratory infections in south indian children respiratory viruses in acute respiratory tract infections in western india validity of clinical case definitions for influenza surveillance among hospitalized patients: results from a rural community in north india. influenza and other respiratory viruses world health organization. cdc protocol of realtime rtpcr for swine flu influenza a (h1n1) the burden of hospitalized lower respiratory tract infection due to respiratory syncytial virus in rural thailand pring-akerblom p. rapid and quantitative detection of human adenovirus dna by real-time pcr human coronavirus infections in rural thailand: a comprehensive study using real-time reverse-transcription polymerase chain reaction assays. journal of infectious diseases who guidelines approved by the guidelines review committee pocket book of hospital care for children: guidelines for the management of common childhood illnesses effectiveness of maternal influenza immunization in mothers and infants maternal immunization against viral disease immunopathology of rsv infection: prospects for developing vaccines without this complication. reviews in medical virology viral respiratory infections in hospitalized and community control children in alaska burden of seasonal and pandemic influenza-associated hospitalization during and after 2009 a(h1n1)pdm09 pandemic in a rural community in india vaccine effectiveness against laboratoryconfirmed influenza in children 6 to 59 months of age during the 2003e2004 and 2004e2005 influenza seasons vaccine effectiveness against medically attended, laboratory-confirmed influenza among children aged 6 to 59 months influenza vaccine effectiveness among children 6 to 59 months of age during 2 influenza seasons: a case-cohort study prevention of otitis media in children with live attenuated influenza vaccine given intranasally efficacy and safety of 1 and 2 doses of live attenuated influenza vaccine in vaccine-naive children efficacy and safety of a live attenuated, cold-adapted influenza vaccine, trivalent against cultureconfirmed influenza in young children in asia. pediatric infectious disease safety, efficacy, and effectiveness of coldadapted influenza vaccine-trivalent against communityacquired, culture-confirmed influenza in young children attending day care design and initiation of a study to assess the direct and indirect effects of influenza vaccine given to children in rural india seasonality, timing, and climate drivers of influenza activity worldwide epidemiology and seasonality of respiratory tract virus infections in the tropics. paediatric respiratory reviews the authors wish to acknowledge the division of viral diseases, centers for disease control and prevention for providing assay kits used for laboratory testing in this study. key: cord-331520-o9e4qqn4 authors: kistler, christine e.; jump, robin l.p.; sloane, philip d.; zimmerman, sheryl title: the winter respiratory viral season during the covid-19 pandemic date: 2020-10-26 journal: j am med dir assoc doi: 10.1016/j.jamda.2020.10.030 sha: doc_id: 331520 cord_uid: o9e4qqn4 the winter respiratory virus season always poses challenges for long-term care settings; this winter, sars-cov-2 will compound the usual viral infection challenges. this special article discusses unique considerations that covid-19 brings to the health and well-being of residents and staff in nursing homes and other long-term care settings this winter. specific topics include preventing the spread of respiratory viruses, promoting immunization, and the diagnosis and treatment of suspected respiratory infection. policy-relevant issues are discussed, including whether to mandate influenza immunization for staff, the availability and use of personal protective equipment, supporting staff if they become ill, and the distribution of a covid-19 vaccine when it becomes available. research is applicable in all of these areas, including regarding the use of emerging electronic decision support tools. if there is a positive side to this year’s winter respiratory virus season, it is that staff, residents, family members, and clinicians will be especially vigilant about potential infection. spread farther and linger longer as the temperature falls, cooler weather brings a seasonal rise of several communicable respiratory infections; in addition, heated indoor spaces encourage closer physical contact and dry mucosal surfaces, leaving individuals more susceptible to 20 increased air particles. 1 until this year, influenza has been the most feared seasonal virus, as it 21 causes 12,000 to 56,000 deaths in the u.s. each year. 2 this winter will be different, of course, due to the pandemic caused by the sars-cov-2 virus, which has already claimed the lives of 23 over 1 million people worldwide. in the u.s., more than 75% of those deaths are in people 65 24 years of age and older. 3 before the covid-19 pandemic, influenza was the most concerning viral respiratory infection 27 for nursing home (nh) residents, with outbreaks requiring both treatment and prophylaxis, and 28 even causing some buildings to close to outsiders for brief periods of time. 4 however, influenza 29 is not the only respiratory virus that abounds in the community and frequents nhs in winter. others, such as parainfluenza, rhinovirus, adenovirus, metapneumovirus, other coronaviruses, 31 and especially respiratory syncytial virus (rsv), may also cause outbreaks. 5 other than 32 influenza, there are not yet vaccines or effective antiviral therapies for these infections. making 33 matters worse, in addition to these viral infections as a cause of pneumonia, they contribute to 34 exacerbations of chronic obstructive pulmonary disease, and, in the case of influenza, may 35 predispose individuals to secondary bacterial infections and cardiac morbidity. 6 ,7 36 37 the winter respiratory virus season always poses challenges for nhs and assisted living 38 communities. overlaying the usual viral infection challenges this winter will be sars-cov-2. in 39 this special article, we discuss unique challenges that covid-19 will bring to the health and 40 well-being of residents and staff in long-term care settings this winter. specific topics include 41 preventing the spread of respiratory viruses, promoting immunization, and the diagnosis and 42 treatment of suspected respiratory infection. we also address several issues related to staff, 43 including whether or not to mandate influenza immunization, availability and use of personal 44 protective equipment (ppe), absenteeism, presenteeism (coming to work despite illness), work 45 release for illness, and paid leave. in addition, we discuss strategies to help mitigate these 46 challenges, some important differences between nhs and assisted living relevant to infection and covid-19, and conclude with a brief consideration of a future sars-cov-2 vaccine. preventing the spread of respiratory viruses 50 51 fortunately, we know more about covid-19 than we did last spring when it first appeared. we 52 know that it spreads primarily via droplets, and less commonly through fomite transmission and 53 aerosolization. 8 however, the influence of heating systems that recirculate air on increasing the 54 aerosol spread of sars-cov-2 is not yet clear. we know that masks and other ppe prevent the 55 spread of sars-cov-2, 9 and because inadequate ppe has demonstrably increased the death 56 toll in nhs, it will be critical to have access to ample supplies this winter. 10 it also will be 57 necessary to have protocols for universal screening; to require that all persons wear face 58 coverings and practice physical distancing; to test staff and residents for purposes of screening 59 and when an outbreak is identified; and to isolate persons with a viral exposure or positive test. if other respiratory viruses circulate widely as is typical in winter, nhs and assisted living 62 communities will need to have a workable plan for addressing new symptoms among residents, in addition to influenza and pneumococcal vaccines for residents, the advisory committee on 101 immunization practices recommends that all nh employees receive an annual influenza 102 vaccination, including those who do not have direct patient care responsibilities. 16 however, whereas hospitals have mandatory vaccination policies, most nhs do not. 17 consequently, only 104 two-thirds of nh staff were vaccinated in 2017-2018. mandatory vaccination policies increase 105 influenza vaccination rates to nearly 100%, 18 and amda's infection advisory committee 106 recommends that all nhs adopt a similar mandatory vaccination policy. 16 the success of these 107 policies relies on staff education, incentives, and making the vaccine readily accessible. in 108 relation to education, the centers for disease control and prevention (cdc) provide numerous 109 resources, as do state and local health departments. in terms of incentives, nh leadership 110 might consider paid leave or other benefits to workers who receive a vaccination. regarding 111 accessibility, nh leadership may need to work with their local health department and health care 112 system to provide free access to the vaccine, preferably on site and across all shifts, to promote begins with recognizing symptoms of an acute infection, followed by recognition of respiratory tract involvement. then, clinicians are alerted to a change in condition; they conduct a diagnostic evaluation, initiate supportive care, and consider whether bacterial pathogens are pneumonia. 21 further complicating matters, a study of adults with community-acquired 127 pneumonia suggested that viruses may be responsible for 23% of cases, with bacteria identified 128 in only 11% of cases; no pathogen was identified in the majority of cases. 22 among nh 129 residents, pneumonia carries with it a case fatality rate of at least 25%, 23 the specific test used to diagnose covid-19 infections may vary depending on the types of 163 tests available, the time required to obtain the results, and the sensitivity and specificity of the 164 results. 30 in general, polymerase chain reaction (pcr) based tests, which detect viral 165 ribonucleic acid from a nasopharyngeal swab, are the most sensitive and specific. test results the results. antigen-based tests are largely point-of-care tests with samples collected from the purposes, a negative antigen test should be confirmed with a negative pcr test. as nhs continue to grapple with the covid-19 pandemic, these diagnostic tests are also being tested for covid-19 at least weekly until there a two week period transpires without screening tests, whereas the frequency of staff screening depends on whether there are cases in addition to diagnostic testing for respiratory viral pathogens, several cutting-edge health 192 information technology and testing strategies may improve the diagnosis and management of 193 winter respiratory illnesses in nh residents. • for pneumonia, evidence strongly suggests that electronic decision support may improve 195 clinician decision making. 33 integrating clinical decision support in the electronic health 196 record improves evidence-based infection-related decisions. 34 the reduce trial 197 demonstrated that incorporating a pneumonia evaluation decision tool into the electronic 198 health record reduced antibiotic use for adults in outpatient care. 35 our research also found that nh residents receive an average of one prescription every three 229 months. 43 at any one time, over 10% of nh residents are taking antibiotics, and up to 75% of 230 these antibiotics are inappropriately prescribed. [44] [45] [46] because constitutional symptoms often 231 promote inappropriate antibiotic prescribing, they present an opportunity for quality of care 232 assessment. 47 consistent with cms' focus on antibiotic stewardship over the last years, 48 available; and continually monitoring the ppe supply. 60 earlier in the pandemic, approximately 20% of nhs reported having less than a one week supply of masks and gowns, and over 15% 266 reported staffing shortages; 61 recent data suggest that nhs have not closed the ppe gap. 62 shortages, as reports indicate that staff shortages persist as well. 51 plans must include preparing for potential loss of staff due to illness or exposure to covid-19. based on cdc employees or more, and provided broad exemptions to employers of emergency responders and health care workers. although the house has passed revisions of the act to remove these being mindful of challenges and implementing mitigation efforts for both residents and staff may 286 lessen the toll the winter respiratory viral season will take on long-term care residents. indeed, 287 many experts are predicting that social distancing for covid-19 will result in a mild influenza 288 season. it is unknown whether a sars-cov-2 vaccine will become available this winter. if so, important 291 questions include adverse effects of the vaccine and whether it will have immunogenicity for 292 chronically ill older adults. current evidence suggests that mrna vaccines appear safe and 293 immunogenic in older populations, 64 but whether such is the case remains a significant concern. 294 the question about cost was recently answered, as the u.s. government announced plans to 295 provide and administer covid-19 vaccines to long-term care residents across the country with 296 no out-of-pocket costs. 65 although specific plans for distribution of vaccines is unknown, nh and 297 assisted living residents should receive priority. immunizing staff will further protect this 298 vulnerable population but must affordable and accessible. due to public concerns about vaccine 299 safety and anti-vaccination resistance, public health efforts to promote the widespread uptake of 300 an effective vaccine should start in each nh and long-term care community as soon as a 301 vaccine appears imminent, to prepare staff and residents for the coming vaccination drives in 302 the spring and summer. 66 implications for practice, policy, and research the winter respiratory virus season always poses challenges for long-term care settings, and 307 those challenges will be exacerbated with the second wave of covid-19; as such, they present 308 numerous implications for practice, policy, and research. as summarized in this paper, practice 309 must focus on preventing the spread of respiratory viruses, promoting immunization, and the seasonality of respiratory viral infections centers for disease control and prevention. flu & people 65 years and older influenza in long-term care facilities. influenza other respir viruses respiratory syncytial virus and other noninfluenza respiratory viruses in older adults secondary bacterial infections associated with influenza pandemics seasonal influenza infections and cardiovascular disease mortality covid-19: transmission, prevention, and potential therapeutic opportunities physical distancing, face masks, and eye protection to prevent person-to-person transmission of sars-cov-2 and covid-19: a systematic review and meta-analysis abandoned" nursing homes continue to face critical supply and staff shortages as covid-19 toll has mounted medicare state operations manual, appendix pp: interpretive guidelines for long-term care facilities nursing home resident and facility characteristics associated with pneumococcal vaccination: national nursing home survey, 1995-1999 centers for disease control and prevention. different types of flu vaccines use of 13-valent pneumococcal conjugate vaccine and 23-valent pneumococcal polysaccharide vaccine among adults aged ≥65 years: updated recommendations of the advisory committee on immunization practices pneumococcal vaccination guidance for post-acute and long-term care settings: recommendations from amda's infection advisory committee recommendations for mandatory influenza vaccinations for health care personnel from amda's infection advisory subcommittee influenza vaccination coverage among health care personnel -united states, 2017-18 influenza season changes in influenza vaccination requirements for health care personnel in us hospitals barriers to timely care of acute infections in nursing homes: a preliminary qualitative study nursing home-associated pneumonia, part i: diagnosis defining characteristics and risk indicators for diagnosing nursing home-acquired pneumonia and aspiration pneumonia in nursing home residents, using the electronically-modified delphi method community-acquired pneumonia requiring hospitalization among u.s. adults predictors of mortality for nursing home-acquired pneumonia: a systematic review viral respiratory infections in a nursing home: a sixmonth prospective study unprecedented solutions for extraordinary times: helping long-term care settings deal with the covid-19 pandemic role of body temperature in diagnosing bacterial infection in nursing home residents temperature in nursing home residents systematically tested for sars-cov-2 clinical practice guideline for the evaluation of fever and infection in older adult residents of long-term care facilities: 2008 update by the infectious diseases society of america does universal testing for covid-19 work for everyone? centers for disease control and prevention. managing investigations during an outbreak cms-3401-ifc, updating requirements for reporting of sars-cov-2 test results by (clia) additional policy and regulatory revisions in response to the covid-19 public health emergency thinking fast and slow in pneumonia computerized clinical decision support systems and antibiotic prescribing: a systematic review and meta-analysis effectiveness and safety of electronically delivered prescribing feedback and decision support on antibiotic use for respiratory illness in primary care: reduce cluster randomised trial evaluation of viruses associated with acute respiratory infections in long-term care facilities using a novel method: wisconsin, 2016-2019 covidapp as an innovative strategy for the management and follow-up of covid-19 cases in long-term care facilities in catalonia: implementation study prognostic value of lung ultrasonography in older nursing home residents affected by covid-19 combining procalcitonin and rapid multiplex respiratory virus testing for antibiotic stewardship in older adult patients with severe acute respiratory infection efficacy of a test-retest strategy in residents and health care personnel of a nursing home facing a covid-19 outbreak bacterial and fungal co-infection in individuals with coronavirus: a rapid review to support covid-19 antimicrobial prescribing does adherence to the loeb minimum criteria reduce antibiotic prescribing rates in nursing homes? successfully reducing antibiotic prescribing in nursing homes antibiotic use in the nursing home. physician practice patterns development of minimum criteria for the initiation of antibiotics in residents of long-term-care facilities: results of a consensus conference the urine-culturing cascade: variation in nursing home urine culturing and association with antibiotic use and clostridiodes difficile infection constitutional symptoms trigger diagnostic testing before antibiotic prescribing in high-risk nursing home residents nursing home action plan: action plan for further improvement of nursing home quality infectious diseases in older adults of long-term care facilities: update on approach to diagnosis and management prioritizing prevention to combat multidrug resistance in nursing homes: a call to zction infection preventionist staffing in nursing homes long-term care providers and services users in the united states national survey of long-term care providers. long-term care providers and services users in the united states-state estimates supplement: national study of long-term care providers residential care/assisted living staff may detect undiagnosed dementia using the minimum data set cognition scale dementia prevalence and care in assisted living families filling the gap: comparing family involvement for assisted living and nursing home residents with dementia the role of physicians practicing in assisted living: time for change variability in state regulations pertaining to infection control and pandemic response in us assisted living communities the need to include assisted living in responding to the covid-19 pandemic department of labor issues alert to keep nursing home and long-term care facility workers safe during coronavirus pandemic shortages of staff in nursing homes during the covid-19 pandemic: what are the driving factors? severe staffing and personal protective equipment shortages faced by nursing homes during the covid-19 pandemic gaps in the emergency paid sick leave law for health care workers safety and immunogenicity of sars-cov-2 mrna-1273 vaccine in older adults trump administration partners with cvs and walgreens to provide covid-19 vaccine to protect vulnerable americans in long-term care facilities nationwide influences on attitudes regarding potential covid-19 vaccination in the united states. vaccines (basel) key: cord-280732-u0pncmp0 authors: nicolas, j. c.; huraux, j. m. title: entérovirus et infections respiratoires date: 1976-09-30 journal: médecine et maladies infectieuses doi: 10.1016/s0399-077x(76)80057-1 sha: doc_id: 280732 cord_uid: u0pncmp0 summary the importance of enteroviruses in upper and lower respiratory infections is reviewed. occasionaly summer epidemics of upper respiratory infections seem due to an enterovirus : on the other hand, the lower respiratory injections rarely seem due to these viruses. the relationship between the enteroviruses and respiratory infections remains to be proved. tyrrell et coll. (46) , sur une 6tude faite en angleterre chez des enfants ~g6s de quelques semaines ~ 12 ans, 6voquent le rsle des ent6rovirus dans 5 % des cas 6tudi$s. ils montrent, au cours de la m~me 6tude, que ces virus sont beaucoup plus fr6quemment isol6s chez les jeunes enfants (1 ~ 10 ans) que chez les enfants de 10 ~ 16 ans. macasaet et co11. (28) , chez 327 enfants hospi-talis6s au kansas medical center pour affection respiratoire haute, faux croup, bronchiolite ou pneumonie, ne retrouvent que 2 excr6teurs d'en-t6rovirus (2 echovirus une autre fa~on d'aborder le probl~me est de d6terminer la fr6quence des manifestations res,piratoires quelles qu'elles soient, au cours d'une 6pid6mie due tt un s6rotype particulier. hierholzer et coll. (20) l'attention du clinieien est souvent attir6e par i'association h ces manifestations respiratoires banales, de signes plus alarmants, m6ningite, p6rieardite, qui sont l'occasion de faire ]e diagnostic et justifient l'examen virologiqne. on pent conclure de ces 6tudes que l'atteinte des voles respiratoires basses est rare dans les infections ~ ent6rovirus. elle n'est signal6e que chez l'enfant. elle se volt surtout chez les nouveau-n6s oft elle n'est souvent qu'un 616ment d'une infection g6nsralis6e gravissime. dans ces cas, volontiers sporadiques, le r61e 6tiologique le diagnostic des infections rcspiratoires /t en-t6rovirus n'offre pas, en virologic, de difficuh6s rnajeures car ses conditions techniques sont celles de tout diagnostic virologique d'une infection aigu~. par contre les indications m6ritent d'en ~tre discut6es. les conditions techniques du diagnostic virologique sont simples. les ent6rovirus, comme tons lcs virus nus sans enveloppe, r6sistent relativement bien dans les pr61~vements destin6s "ales isoler : s6cr6tions pharyng6es et de selles pr6-lev6es par 6couvillonnage avec expression du coton dans quelques ml de milieu liquide en tube. i1 suffit ensuitc de transporter ces tubes dans de la glace jusqu'au laboratoire pour inoculation une batterie de cultures cellulaires diff6rentes et compl6mentaires dans leur aptitude ~ multi'plier les divers virus respiratoires. l'isolement de certains coxsackievirus type a exige, toutefois, l'inoculation aux souriceanx nouveau-n6s. a cette exception pros, l'isolement des ent6rovirus est facile, plus facile que l'isolement des myxovirus, virus parainfluenzae et virus respiratoire syncytial notamment. par contre il arrive que le typage d'un ent6rovirus demande des semaines de travail. le diagnostic recherche non pas un taux 61cv6 d'anticorps qui n'aurait en sol aucune valeur diagnostique, mais une 616vation significative du taux des anticorps ~ l'examen simultan6 de deux s6rums, 'pr6coce et tardif. les auteurs passent en revue la place des ent6rovirus dans la pathologie respiratoire haute et basse. ils concluent que cdrtaines 6pid6mies d'infections respiratoires hautes, parrot c,elles se produisant 1'6t6, sont dues h un ent6rovirus. l'atteinte des votes respiratoires basses est rare ; let r61e 6tiologique exdusif des ent6rovirus reste difficile h 6tablir en raison de la possibilit6 d'association de plusieurs agents pathog6nes. ent6rovirus -pathologie respiratoire haute et basse -diagnostic. the importance of enteroviruses in uppar and lower respiratory in~'ections is reviewed. occasionnalg su'mmer epidemics o[ upper ,respiratory injections seem due to an enterooirus : on the other hand, the lower respiratory injections rare,iy seem due to these viruses. the relationship between the enteroviruses and respiratory injections remains to be proved. -epidemics pleurodynia (bornholm disease) due to coxsackievirus b5. the in:errelationship of pleurodynia, benign pericarditis and aseptic meningitis epid6mie due au virus echo 30 recoveries of virus from premature infants during outbreaks of respiratory disease : the relation of echovirus type 22 to disease o~ the upper and lower respiratory tract in the premature infant transplacenta! infection due to echovirus type 22 inoculation of human volonteers with echovirus type 20 cystic emphysema in premature infants. a report of an outbreak with the isolation of type 19 echovirus on one case -clinical syndroms in chiidren caused by respiratory infection airborne transmission of respiratory infection with coxsackievirus a type 21 respiratory illness in six infants infected with a newly recognized echovirus. pediatrics group b coxsackievirus infection in infant with acute lower respiratory disease -an outbreak of coxsackievirus type 22 among neonates in an obstetrical ward -echovirus type 17 in the neonate histological study of two cases of coxsackie b virus pneumonia in children pneumopathies interstitielles chez l'enfant pneumopathies interstitielles d'origine infec outbreak of febrile illness associated with echovirus type 5 in a maternity unit in singapore clinical and serological observations in cases of coxsackie b3 infections in early infancy -prospective study of mixed coxsaekie virus b3 and b4 outbreak of upper respiratory illness in a children's home respiratory viral infection in childhood. a survey in general practice, roehamton 1967-1972 acute respiratory disease associated with coxsackie a21 virus infection. i : incidence in military personnel, observations in a recruit population the ro,le of enterovirus in respiratory disease -viral isolation rates during summer from children with acute upper respiratory tract disease and healthy children current status of coxsackie and echo viruses in human disease -fatal infections with echovirus types 6 and 11 in early infancy with special reference to exanthems and pneumonia the etiology of acute respiratory infections. iii : the role of viruses and bacteria epidemic pleurodynia in aden associated with infec-45 ~ nosocomial infeetio,n with echovirus type 31 in a neonatal intensive care unit -clinical syndromes associated from enterovirus and reovirus infections an epidemic of aseptic meningitis, primarily among infants, caused by echovirus 11 prime an outbreak of type 25 echovirus infection with hexanthem in an infant home near tokio eehovirus type 22 infection in a premature infant an outbreak qmong kibbutz children of a febrile illness associated with eeho~irus type 4 nonbacterial infections affecting the nasopharynx, in symp. on respiratory diso.rders. pediat -echovirus 30 infections. outbreak in new york state recovery of a cytopathogenic agent from patients with non diphteric-croup and from day nursery children. properties of the agent -respiratory entern,virus and reovirus infections observations on a newly recognized echovirus and description of an outbreak in a nursery echo 11 a respiratory virus : quantitation of infection in man e: smith a.-outbreak of coxsackie b5 virus in a children's home acute lymphonodular pharyngitis : a newly described condition due to coxsackie a virus epidemic of echovirus 11 infections in japan in 1971. japan ~ l'6tiologie des affections aigu~s des votes respiratoires en milieu militaire a collabo.rative study of the etiology of acute respiratory infections in britain key: cord-281916-v6u5mr2i authors: bonnin, paul; miszczak, fabien; kin, nathalie; resa, cecile; dina, julia; gouarin, stephanie; viron, florent; morello, remy; vabret, astrid title: study and interest of cellular load in respiratory samples for the optimization of molecular virological diagnosis in clinical practice date: 2016-08-09 journal: bmc infect dis doi: 10.1186/s12879-016-1730-9 sha: doc_id: 281916 cord_uid: v6u5mr2i background: respiratory viral diagnosis of upper respiratory tract infections has largely developed through multiplex molecular techniques. although the sensitivity of different types of upper respiratory tract samples seems to be correlated to the number of sampled cells, this link remains largely unexplored. methods: our study included 800 upper respiratory tract specimens of which 400 negative and 400 positive for viral detection in multiplex pcr. all samples were selected and matched for age in these 2 groups. for the positive group, samples were selected for the detected viral species. results: among the factors influencing the cellularity were the type of sample (p < 0.0001); patient age (p < 0.001); viral positive or negative nature of the sample (p = 0.002); and, for the positive samples, the number of viral targets detected (0.004 < p < 0.049) and viral species. conclusion: the cellular load of upper respiratory samples is multifactorial and occurs for many in the sensitivity of molecular detection. however it was not possible to determine a minimum cellularity threshold allowing molecular viral detection. the differences according to the type of virus remain to be studied on a larger scale. associated with particular diseases (respiratory syncytial virus and bronchiolitis, parainfluenza virus 3 and laryngitis, rhinovirus and common cold, influenza virus and flu syndrome), there is no evidence for a clinical specificity, and only the virological diagnosis provides an accurate identification of the ari [4, 5] . detection of respiratory viruses is of little interest in general practice, in that the infection does not present a risk of severity for the patient. however, virological confirmation of ari is needed in severe clinical presentations, requiring hospitalization in intensive care units and occurring in vulnerable subjects [4, 6] . the goal of early virological diagnosis would be an optimization of patient care, which could lead to reduction in length of hospital stay, a saving of antibiotics, and complementary examinations [7] . virological tests allow for the establishment of accurate diagnosis of infection, assessment of evolving risks (bacterial infection, acute respiratory distress syndrome), and the establishment of measures to limit its spread (isolation, wearing gloves and masks). pandemics of severe acute respiratory syndrome (sars, 2002 (sars, -2003 and influenza a-h1n1 (2009) lead to the development of molecular biological techniques applied to virological diagnosis, mainly based on pcr (polymerase chain reaction). performances of molecular methods in respiratory virology are so significant that they have replaced conventional techniques (culture, detection of viral antigens) as a reference method [8] [9] [10] [11] . multiplex pcr techniques are particularly suited to medical diagnosis because they can detect multiple viral targets in the same time, avoiding the virologist a selection of viral targets to search. there are now many commercial kits for the detection of a range of 12 to 15 respiratory viruses and some intracellular bacteria [6, 7, 12, 13] . molecular techniques (real-time pcr) also make it possible to achieve a semi quantification of the viral molecular material present in the sample, giving additional information about the respiratory viral load (interest in therapeutic monitoring and infection transmission risk) [10] . a normalized viral load can be obtained by adding a cell quantification step. the primary site for replication of respiratory viruses is the ciliated airway epithelium. the sample must be taken as soon as possible after the onset of symptoms. this is usually a nasal swab or nasopharyngeal aspiration (especially realized in children under 2) [14] . these samples are easily accessible and especially adapted to upper ari. if a rich cell collection appears to be an important prerequisite for the quality of respiratory viral diagnosis, there is currently no information on a possible cellularity threshold that would validate the result of the viral molecular detection. the main objective of this work is the study of cellularity in 800 respiratory specimens previously characterized virologically. the results should help to define the concept of "cellular richness" and determine the factors that influence it. eight hundred respiratory samples were included in this study. all were collected between september 1, 2010 and february 21, 2013 in different departments of the university hospital of caen (france), and immediately sent to the virology department for a respiratory viral diagnosis. respiratory samples were divided into 604 nasal swabs corresponding to nasopharynx sampling (posterior nares), collected on universal viral transport medium (utm) and 196 nasal aspirates. after receipt in the laboratory, each sample underwent a pre-analytical step including division into 3 aliquots: one was immediately used for the viral detection and the other two were stored frozen at -80°c. one of its two frozen fractions was used for this study. this complementary diagnostic study was then conducted on residual clinical specimens, in french law, the right to use the end of the samples is written in the code of public health : code de la santé publique -article l1211-2. these 800 aliquots were selected in the laboratory samples bank according to their results in virological diagnosis: 400 positive and 400 negative for molecular detection of respiratory panel using the respifinder ® smart_22_fast technique (pathofinder, maastricht, netherlands). this kit allows for the detection of 15 rna and 2 dna viral targets and 4 intracellular bacterial targets in respiratory specimens (tables 1 and 2). a total of 4 age groups reflecting the distribution observed in practice in the laboratory were indicated as follows: infants (age < 2; 33 %; n = 264), children (aged from 2 to 15 ; 33 %; n = 264), adults (aged from 15 to 65; 21.5 %; n = 172) and elderly (age ≥ 65; 12.5 %; n = 100). each group is composed of half positive and half negative in molecular viral detection, and so as to be matched for age. within the group of positive samples, the distribution of detected viral species was modeled on that observed in the routine activity of the laboratory (tables 1 and 2) . after thawing the samples, the extraction of nucleic acids was performed using the plc qiasymphony® (qiagen, hilden, germany). the extraction was performed with 200 μl of sample using qiasymphony_dsp_virus/ cell quantification was achieved by amplification and detection of a human household gene in real-time pcr (hypoxanthine phosphoribosyl transferase-1) [15, 16] . cell quantification was performed on a lightcycler 480 ii® platform (roche, meylan, france). the reaction mixture included 15 μl of amplification premix cell_control r-gene® (argene/biomerieux, lyon, france) and 10 μl of nucleic acid extract. each manipulation included two negative controls: one undergoing all the analytical steps, called ec (extraction control) and one introduced prior to the pcr reaction, called "negative control". cell quantification standard (qs1: 5 × 10 4 cells/pcr and qs2: 5 × 10 3 cells/pcr) was ready to use in the kit. an external standard curve was performed using these two standards and 2 additional dilutions, respectively containing 5 × 10 2 and 50 cells/pcr. the reading of the results was carried out directly from the plc software, which displays the ct values (cycle threshold) obtained and the corresponding number of cells/pcr-reaction (i.e. 10 μl of extract). this number was converted to "cells/ml" and the final results were expressed in logarithmic scale (log10/ml). the quantification kit performances were verified in our laboratory by quantifying mrc5 cells (human embryonic fibroblasts) of known concentration (rd-biothech, besancon, france). a range of 11 2-fold serial dilutions was made from initial mrc5 cell suspension in viral transport medium (utm). the nucleic acids were extracted from these 12 cell suspensions, and cell quantification reaction was carried out under the same conditions as for the respiratory samples. descriptive statistics were used to show the characteristics of the different variables. quantitative variables were described using means and standard deviation. qualitative variables were described using frequencies and percentages. the relationships between qualitative variables were studied using the chi-square test or fisher's exact tests. the anova was used to compare the means of quantitative variables in two or more independent groups with the bonferroni post hoc test. the relationship between two quantitative variables was assessed using the spearman correlation coefficient (ρ). to look for a diagnostic threshold to divide positive and negative samples, a receiver operating characteristic curve (roc curve) was used. all the tests were two-tailed and their level of significance (p) was defined as p < 0.05. ibm®-spss® 22.0 for windows® was the statistical software used. the 2-fold serial dilution range from initial mrc5 suspension had expected cellularity values between 6 log/ ml (concentration given by the manufacturer) and 2.69 log/ml (last dilution). the measured cellularity values were consistent with those expected for the concentrations between 6 and 3.59 log. the last 3 dilutions deviated more than 0.4 log of the expected value, corresponding to concentrations between 3.29 and 2.69 log ( table 3 ). the mean deviation from expected values was 0.34 log/ml. regarding our samples population, cell quantification was negative (no hprt-1 dna detection) for 8 of the 400 positive and for 16 of the the 400 negative in viral detection. comparative study of sample "swabs" and "nasal aspirates" among the 800 cell-quantified respiratory samples, 196 were nasal aspirates and 604 were nasal swabs. cellularity these results are presented in table 4 . regarding nasal swabs only, average cellularity were not significantly different between the age groups except for children compared with adults (p <0.001). comparison of cellularity among the positive (n = 400) and negative (n = 400) samples in viral detection as the subjects were matched for age, the age distribution is identical in the two groups positive and negative (p = 0.996). these two groups are comparable, as expected. the average cellularity was 5.01 (+/-1.25) log/ml for the positive group and 4.76 (+/-1.41) log/ ml for the negative group. this difference was significant (p = 0.002). the results of comparison between the age groups according to the result of the viral detection (positive or negative) are presented in fig. 1b . within a single age group (infants, children, adults, elderly), the differences between positive and negative samples were not significant (p = 0.134, p = 0.552, p = 0.074 and p = 0.098 respectively). based on the results of the comparison between positive and negative samples, a roc (receiver operating characteristic) curve was performed. no minimum cellularity threshold could be defined for molecular viral detection (fig. 2) . aspirates swabs fig. 1 average cellularity of respiratory specimens depending on the sample type and age group. a nasal aspirate was the sample type that provides, on average, more epithelial cells for patients under the age of 15 (5.70 log/ml for aspirates versus 4.96 log/ml for swabs). b although having the same shape, the average cellularity curve of positive samples was always located above the negatives among the 800 selected samples, viral detection was negative in 400, 338 were positive for 1 viral target, 58 were positive for 2 targets and 4 were positive for 3 targets. the average cellularity was 4.76 (+/-1.41) log/ml, 4.95 (+/-1.26) log/ml, 5.30 (+/-1.17) log/ml, and 6.19 (+/-0.21) log/ml for these 4 groups respectively. the average cellularity in negative samples was significantly lower than in cases of mono (p = 0.049), bi (p = 0.004) or tri-detection (p = 0.032). a significant tendency was observed between positive samples for one viral target and those positive for 2 or 3 virus (p = 0.064), this trend was confirmed by a spearman correlation (ρ = 1) indicating a strong correlation between sample cellularity and the number of viruses detected. molecular detection, including multiplex techniques, is currently the gold standard for viral respiratory diagnosis. we have very powerful molecular tools, ensuring a quality respiratory viral diagnosis, available for all clinicians supporting hospitalized patients. one factor limiting this diagnosis is represented by the collected respiratory specimens. the main objectives of this work have been to study the cellularity of these clinical respiratory specimens, to propose a possible definition of what is commonly called "cellular richness," and to measure the impact of this marker on the molecular viral diagnosis. very few published studies have been completed in this area. however, a number of facts are commonly accepted within the medical community: respiratory specimen should be "rich" to allow for "good" viral diagnosis, the "good" samples are obtained almost exclusively in infants and children. in total we identified 7 studies published in international journals between 2003 and 2014, whose objective was to compare the various upper respiratory samples, in terms of sampling equipment (flocked swabs versus rayon swab), in terms of sampling site (nasal, oropharyngeal, nasopharyngeal, combining sites), and sampling modality (swab, wash, aspiration) [17] [18] [19] [20] [21] [22] [23] . the number of target cells and/or the number of extracellular viral particles collected could define the respiratory sample quality during sampling. it is not unreasonable to consider that the majority of the viral sequences detected by molecular techniques are mainly located in the intracellular compartment. however, several factors could modulate this distribution: the viral species, the cytopathic effect induced in vivo, the inflammatory responses, and the sampling delay from the onset of the symptoms. this study is retrospective; it was therefore not possible to collect data from the time between sampling and clinical symptoms in a standardized way. this matter is nonetheless very interesting and remains to be explored. for this study, we considered cell quantification, or cellular richness, as the main marker of diagnostic efficacy for a sample. the 7 studies referenced above used indirect measurement of sample richness through the virus detected in it: 5 used molecular detection methods, and one was an antigen detection using rapid diagnostic test evaluate the number of infected and uninfected cells deposited on the slide [17] . overall, the results are consistent and show a superiority of nasopharyngeal swab versus oropharyngeal and a superiority of flocked swab versus classical ones [14, 17, 19, 20] . the superiority of the "wash" versus the "swab" is not found by all authors, and comes well counteract the widespread idea that washing is always greater than swabbing [14] . alsaleh et al. (2014) performed a molecular cell quantification using real time pcr in order to validate viral detection on nasal swabs. the cellularity of these samples was assessed using the quantification of a human endogenous retrovirus (erv3) known to be present in two copies per diploid cell [24] . the authors report their results by comparing the ct obtained upon erv3 detection and not in terms of cellular load [18] . in our study, we made the choice to use a direct detection method for assessing the number of cells in respiratory samples. to the extent that we needed a large number of samples characterized by many markers for statistical analysis (age of the sampled patient, detected viral target, etc.), a prospective study on freshly sampled respiratory specimens could not be performed. we therefore used previously characterized and stored frozen (-80°c) respiratory samples. the definition of cellular richness is not affected by freezing or thawing samples since it theoretically does not change the amount of hprt1 nucleic acid, i.e. 2 copy per haploid cell, whether or not lysed. similarly, no distinction between living and dead cells was made before the freezing process because such a distinction does not affect the quality of molecular detection by pcr (detection of viral genome into infected cells, living or not). the analysis of 800 samples, divided into 400 positive and 400 negative in viral detection, failed to establish a minimum cellularity threshold to invalidate the negative results in viral molecular detection. the roc curve shows that the cellularity is not a quantifiable predictor of the outcome of the virus detection. however, our work has yielded interesting results concerning the factors influencing the cellularity of samples and the impact of cellularity on the result of molecular detection of respiratory viruses. we have clearly demonstrated that nasal aspirates allowed us to collect more cells than with nasal swabs and this only in the age group under 15 years old. this result must be tempered by the fact that 90 % (173/196) of the nasal aspirations of the study were from children under 9 years of age, since the distribution was random at inclusion of samples in the study. this reflects a common practice of sampling methods in clinical departments: nasal aspirates are rarely performed in adults and the elderly over 65 years old. this gesture is considered invasive and unpleasant. our results support the idea that, de facto, it would not be appropriate to perform this type of sampling in this group. for all types of samples (swabs and/or nasal aspirates), the age of the sampled patient remains an important marker influencing cellularity, with a maximum average cellularity under the age of 15, a minimum average cellularity in adults group, and an intermediate average cellularity in the elderly group. the reasons for this lack of cellularity in respiratory samples from adults remain obscure. regarding the influence of cellularity on the result of viral detection, it should be noted that negative samples present, all age groups combined, an average cellularity lower than that obtained for the positive samples, even though it has not been possible to establish a predictive relationship. this difference was tenuous for the children group. this observation leads to two possible explanations: either in the positive group the infection increases the sample cellularity by promoting epithelial desquamation and, to a lesser extent, the mucus capture, containing free viral particles; or, in the negative group, there are false-negatives in virus detection, induced by a lack of cells in the sample. this result was obtained from all samples (aspirates and swabs). insofar as aspirates are evenly distributed in both positive and negative groups, we think that the result of comparison is not biased given that aspirates are richer samples. considering viral detection in its entirety, without analysis of the viral species detected, it is interesting to note that the two factors, "cellularity" and "viral codetection" (detection of 2 or 3 viruses) are associated positively. this suggests that the detection of several viruses is facilitated in the context of a rich sample. in cases of viral co-detection, the question of whether these are the same cells that are infected or not is not resolved, even if it is conventionally accepted that an already infected cell is less permissive to a second viral infection. it should also be noted that viral co-detection can be either a co-infection, or two or more sequential infections. such phenomena have already been discussed in the works of alsaleh et al. which showed that the positive samples in viral detection had, on average, a greater amount of genetic material of human origin than negative ones. similarly, samples where the gene erv3 was not detected had lower viral detection. finally, they also found that the positive samples for several viruses were also those in which cellular loads were highest [18] . the analysis of the potential impact of cellularity on the specific detection of various viruses included in the "respiratory panel" showed results that should be confirmed with larger numbers in each group. indeed, on the one hand, the highest average cellularity was obtained in the hmpv positive samples, equally detected among adults and children groups; on the other hand, the lowest average cellularity is obtained in the rsv positive samples, mostly detected in infants and children groups. these results are surprising in that the two viruses, rsv and hmpv, belong to the same virus family (paramyxoviridae) as well as to the same virus subfamily (pneumovirinae), and are genetically close. they have many similarities in the circulatory mode and in the pathophysiology of the infection they cause. yet the cellularity of hmpv positive samples is significantly greater than that of the rsv positive specimens. the quality of samples dramatically affects the quality of results provided to clinicians. it is important that a better understanding of the sample characteristics goes along with technological developments. this work is uncommon. he tries to give answers to a trivial scientific question: respiratory specimens should they be « rich in cells » to ensure optimal virological molecular diagnosis? the cellular load is multifactorial and occurs for many in the sensitivity of molecular detection. however, it was not possible to determine a minimum threshold allowing molecular viral detection. adv, adenovirus; ari, acute respiratory infection; ct, cycle threshold; dna, deoxyribonucleic acid; ec, extraction control; erv3, human endogenous retrovirus 3; flu, influenzae virus; hbov, human bocavirus; hcov, human coronavirus; hmpv, human metapneumovirus; pcr, polymerase chain reaction; piv, parainfluenza virus; rhv/ev, rhino/entero virus; rna, ribonucleic acid; roc, receiver operating characteristic; rsv, human respiratory syncytial virus; sars, severe acute respiratory syndrome; utm, universal transport medium 3 department of virology epidemiology of viral respiratory infections bronchiolitis viruses the underrecognized burden of influenza in young children viral epidemiology and severity of respiratory infections in infants in 2009: a prospective study techniques actuelles de diagnostic des infections virales respiratoires en réanimation cost analysis of multiplex pcr testing for diagnosing respiratory virus infections superiority of reverse-transcription polymerase chain reaction to conventional viral culture in the diagnosis of acute respiratory tract infections in children increased detection of respiratory syncytial virus, influenza viruses, parainfluenza viruses, and adenoviruses with real-time pcr in samples from patients with respiratory symptoms quantitation of respiratory syncytial virus rna in nasal aspirates of children by real-time rt-pcr assay molecular diagnosis of respiratory viruses comparison of multiplex pcr assays and conventional techniques for the diagnostic of respiratory virus infections in children admitted to hospital with an acute respiratory illness comparative evaluation of six commercialized multiplex pcr kits for the diagnosis of respiratory infections identification of respiratory viruses in adults: nasopharyngeal versus sampling development of an efficient qrt-pcr assay for quality control and cellular quantification of respiratory samples guideline to reference gene selection for quantitative realtime pcr comparison of flocked and rayon swabs for collection of respiratory epithelial cells from uninfected volunteers and symptomatic patients nasal swab samples and real-time polymerase chain reaction assays in community-based, longitudinal studies of respiratory viruses: the importance of sample integrity and quality control swabbing for respiratory viral infections in older patients:a comparison of rayon and nylon flocked swabs comparison among nasopharyngeal swab, nasal wash, and oropharyngeal swab for respiratory virus detection in adults with acute pharyngitis improved detection of respiratory viruses in pediatric outpatients with acute respiratory illness by real-time pcr using nasopharyngeal flocked swabs comparison of nasopharyngeal and oropharyngeal swabs for the diagnosis of eight respiratory viruses by real-time reverse transcription-pcr assays evaluation of the quidel quickvue test for detection of influenza a and b viruses in the pediatric emergency medicine setting by use of three specimen collection methods a quantification of human cells using an erv-3 real time pcr assay not applicable. this study was supported by national reference laboratory for measles and respiratory paramyxoviridae. we have no additional data to communicate and have incorporated article all data, tables and figures necessary for the understanding of the study.authors' contributions av and rm designed the study. pb and av wrote the main manuscript text and prepared figures. rm was responsible for statistics analysis and wrote the statistics part of the methods. cr provided protocoles for the use of cell quantification kit. av, fm, nk, jd, sg and fv participated in the successful conduct of experiments, construction of the methodology and the reading of the manuscript. pb conducted all experiments. all authors have read and approve of the final version of the manuscript. the authors have declared that no competing interests exist. not applicable. this is a complementary diagnostic study conducted on residual clinical specimens, in french law, the right to use the end of the samples is written in the code of public health : code de la santé publique -article l1211-2. this provision permits to work on the remaining clinical specimen sampled for diagnostic test (in our study, samples were upper respiratory specimen). it was possible to perform an additional analysis (in our study, it is the cell quantification of sampling) unless the patient, previously informed, does not express its refusal. the approval of an ethics committee was therefore not necessary for this kind of study. • we accept pre-submission inquiries • our selector tool helps you to find the most relevant journal submit your next manuscript to biomed central and we will help you at every step: key: cord-300116-r93w4jm3 authors: yi, hana; yong, dongeun; lee, kyungwon; cho, yong-joon; chun, jongsik title: profiling bacterial community in upper respiratory tracts date: 2014-11-13 journal: bmc infect dis doi: 10.1186/s12879-014-0583-3 sha: doc_id: 300116 cord_uid: r93w4jm3 background: infection by pathogenic viruses results in rapid epithelial damage and significantly impacts on the condition of the upper respiratory tract, thus the effects of viral infection may induce changes in microbiota. thus, we aimed to define the healthy microbiota and the viral pathogen-affected microbiota in the upper respiratory tract. in addition, any association between the type of viral agent and the resultant microbiota profile was assessed. methods: we analyzed the upper respiratory tract bacterial content of 57 healthy asymptomatic people (17 health-care workers and 40 community people) and 59 patients acutely infected with influenza, parainfluenza, rhino, respiratory syncytial, corona, adeno, or metapneumo viruses using culture-independent pyrosequencing. results: the healthy subjects harbored primarily streptococcus, whereas the patients showed an enrichment of haemophilus or moraxella. quantifying the similarities between bacterial populations by using fast unifrac analysis indicated that bacterial profiles were apparently divisible into 6 oropharyngeal types in the tested subjects. the oropharyngeal types were not associated with the type of viruses, but were rather linked to the age of the subjects. moraxella nonliquefaciens exhibited unprecedentedly high abundance in young subjects aged <6 years. the genome of m. nonliquefaciens was found to encode various proteins that may play roles in pathogenesis. conclusions: this study identified 6 oropharyngeal microbiome types. no virus-specific bacterial profile was discovered, but comparative analysis of healthy adults and patients identified a bacterium specific to young patients, m. nonliquefaciens. electronic supplementary material: the online version of this article (doi:10.1186/s12879-014-0583-3) contains supplementary material, which is available to authorized users. recent culture-independent community analysis performed on the human microbiome has provided an overall picture of commensal microbial communities. studies have revealed that diverse microbes occupying body habitats with strong niche specialization both within and among individuals [1] . in the case of the respiratory tract microbiome, a catalogue was initially established in 2009 [2] and then respiratory microorganisms were extensively characterized [3] [4] [5] [6] [7] [8] . collectively, studies to date have revealed that the respiratory tract harbors a homogenous microbiota that decreases in biomass from the upper to the lower tract [5] , and that the lung microbiome resembles the oral microbiome, although these microbiomes are distinguished by the overrepresentation of distinct bacterial species in the lung [7] . as with other human-body habitats, the core microbiome of nasopharynx remains undefined because it varies substantially from person to person [3] . however, existence of core microbiome was observed despite the significant inter-individual variation [8] . one study reported that the microbial composition of the upper respiratory tract is typically unique to each person and it changes little over time [4] . although the available evidence is not sufficiently strong, microbiome types are speculated to eventually affect a person's risk of disease or response to distinct drugs [9] . the human microbiota is considered to benefit the host by promoting the differentiation of the mucosal structure and function, stimulating both the innate and adaptive immune systems, and providing "colonization resistance" against pathogen invasion [10] . recently, the composition of the airway microbiota has been suggested to play roles in determining the presence and severity of diseases [11, 12] . for example, the clinical outcomes of respiratory infections caused by pseudomonas aeruginosa vary depending on the diversity of the airway microbiota [13, 14] , and a temporal loss of the diversity is linked to the development of ventilator-associated pneumonia and patient mortality [12, 13] . the importance of intact commensal microbiota was also demonstrated in viral infection, with the commensal microbiota composition critically regulating host immune response following influenza virus infection [15] . to reveal the links that exist between microbiome types and clinical traits, we have to first understand the diversity of the microbial community in target body sites. most respiratory tract infections are caused by viruses including rhinovirus, respiratory syncytial virus, parainfluenza virus, adenovirus, coronavirus, human metapneumovirus, and influenza virus. infection by pathogenic viruses significantly changes the condition of the respiratory tract as a result of the epithelial damage caused by viral invasion itself and/or by inflammatory mediators produced by the host immune response [16] . given, the pathophysiology and mechanism of local immune responses are virus specific [16] , a virus-specific bacterial profile in the respiratory tract could potentially be characterized. discovering any specific bacterial species that exhibits a tendency of opportunistic infection or coinfection in a viral species-dependent would benefit future preventive measures and current treatments. to date, no study has evaluated whether the composition of the respiratory microbiota changes in relation to the type of infectious virus. in this study, our aim was to determine whether a viral infection-related bacterial profile exists in the respiratory tract and evaluate any disparities in the microbiota structure that develops depending on the infectious virus species. we used culture-independent high-throughput sequencing to analyze the bacterial content in the upper respiratory tract of patients and healthy asymptomatic people. we also examined the presence or absence of dissimilarities in the microbiota of hospital staff and community people. this study was approved by the institutional review board of the severance hospital, yonsei university health system, seoul, korea (protocols 4-2010-0652, 4-2011-0159, and 4-2011-0862). patients and healthy adults provided written informed consent to be enrolled. de-identified demographic data and clinical measures were taken from electronic medical record system. additional file 1: table s1 presents the list and features of samples used in this study. we selected 59 patients with confirmed acute viral infections from yonsei university hospital during a 30month period (december 2010 to may 2013). the viral agents of the infections were confirmed using pcr by yonsei university hospital. the viruses included influenza (if, n = 7), parainfluenza (pi, n = 24), rhino (rh, n = 8), respiratory syncytial (rs, n = 14), corona (cr, n = 4), adeno (ad, n = 1), and metapneumo (mp, n = 1) viruses. the upper respiratory tract samples were collected from patients' oropharynx by using swabs and suspended in 1 ml of viral transport medium (vtm; becton dickinson universal viral transport, usa). sputum or nasopharyngeal aspirate was collected when available instead of swabs. sputum samples were diluted with an equal volume of suspension medium and homogenized as described [17] . the upper respiratory tract samples were also obtained from healthy adults including 17 health-care workers (9 non-icu and 8 icu staff ) and 40 community people. the 17 hospital staffs and 7 community people were recruited over the same period in yonsei university hospital (june 2011) and 33 community people were additionally recruited in the same hospital (june 2013). the oropharyngeal swabs were obtained using aseptic technique, suspended in vtm and transported to the laboratory for further processing. the samples were stored at −80°c until dna extraction. dna extraction, pcr, and pyrosequencing dna was extracted from 200 μl of samples by using a commercial microbial dna isolation kit (qiagen). the extracted dna was amplified using primers targeting the v1 to v3 regions of the prokaryotic 16s rrna gene by using methods described elsewhere [18] . dna was sequenced by chunlab inc. (seoul, korea) by using a roche/454 gs junior system according to the manufacturer's instructions. the processing of pyrosequencing data of 16s rrna gene sequences were performed as described elsewhere [18] . chimeric sequences were detected using uchime [19] and eztaxon-e database (http://eztaxon-e.ezbiocloud.net; [20] ) was used to taxonomically assign each pyrosequencing read. phylogenetic analyses of 16s rrna gene sequences were performed using the neighbor-joining [21] tree method implemented in mega program [22] . an evolutionary distance matrix was generated for the neighbor-joining tree according to the model of jukes and cantor [23] and the resultant tree topologies were evaluated using bootstrap analyses [24] . the draft genome sequence of moraxella nonliquefaciens dsm 6327 t was determined through paired-end shotgun sequencing performed by using the miseq system (illumina) with 300× coverage. the sequencing reads were assembled using clc genomics wb5 (clcbio). annotation, comparative genomic analyses and average nucleotide identity (ani) calculation were performed as described [25] . random subsampling was conducted to normalize the data size to 3,000 reads, because the total number of reads that remained after pre-processing varied depending on the samples. all statistical analyses were performed using this subset. the simpson diversity index [26] was calculated using the rrna database project's pyrosequencing pipeline (http://pyro.cme.msu.edu/). the overall phylogenetic distance between each pair of communities was estimated using the fast unifrac web interface (http://unifrac.colorado.edu/) [27] and visualized using principal coordinate analysis (pcoa) implemented in r program (http://www.r-project.org/). to compare microbiome structures based on categorical metadata, samples were pooled into binds (healthy/patient, male/female, vtm/aspirate/sputum, smoking/non-smoking, ages, causal viruses, etc.) and statistical significance tests were performed using r program. the significance of differences in bacterial profiles according to categorical metadata was determined using hotelling's t test. significant bacterial taxa based on categorical metadata were identified using q-values after multiple testing correction [28] to eliminate false discovery rates. the difference in shannon diversity index among categorical metadata was evaluated using wilcox two-sample t test. the 454 sequencing data supporting this article are available in the genbank repository, sub435282. the genome data of m. nonliquefacience is under submission to the ddbj/embl/genbank databases under accession no. prjna232737. we sequenced 57 upper respiratory tract samples from healthy adults including 17 health-care workers (9 non-icu and 8 icu staff ) and 40 community people. the 59 patients with confirmed acute viral infections with influenza (if, n = 7), parainfluenza (pi, n = 24), rhino (rh, n = 8), respiratory syncytial (rs, n = 14), corona (cr, n = 4), adeno (ad, n = 1), and metapneumo (mp, n = 1) viruses were also successfully sequenced. the pyrosequencing of 16s rrna gene amplicons resulted in 786,152 quality-filtered reads for the 116 samples. we observed an average of 823 bacterial phylotypes (97% clustering) for each samples (range 237 to 1,851). in the sample-size-normalized (3,000 nt) subsamples, the number of bacterial species ranged from 180 to 615 (average, 371), depending on samples. the genus streptococcus was identified as the core microbiome of the healthy human respiratory tract. in all healthy subjects tested in this study, members of streptococcus dominated the bacterial community, exhibiting an average abundance ratio (percentage of the taxon in the total bacterial community) of 55.8% (range 13.4%-91.1%, depending on subjects) ( table 1 ). the genera neisseria (8.0%) and gemella (5.3%) were also dominant in healthy subjects, but their abundance ratios were considerably less than that of streptococcus. the genera observed in all healthy subjects were streptococcus, prevotella, and veillonella. the genera haemophilus, gemella, rothia, and leptotrichia were detected in most subjects at abundance ratios of 2.0%-8.0%. fast unifrac analyses for the bacterial profiles in healthy samples showed that hospital staff and community people were not discriminated based on their bacterial composition (additional file 2: figure s1 ), and age, sex, year and month of sample collection, and smoking status did not discriminate the bacterial profile (data not shown). analyzing the bacterial communities of healthy-adult and patient groups revealed clear differences. we used the shannon index in which higher values represent higher diversities; the average values calculated for the healthy-adult and patient groups were 4.83 ± 0.40 and 3.77 ± 0.61, respectively ( figure 1 ). this indicated that healthy adults harbored significantly more complex and diverse microbiota than did patients (p <0.0001, wilcox test). the microbiota profiles of healthy-adult and patient groups also differed in the relative composition of the microbiome, which was highlighted in the graph showing the abundant bacterial genera observed in the tested samples ( figure 2 and additional file 3: figure s2 ). to identify the bacterial taxa that were more abundant (in a statistically significant manner) in the patient group than in the healthy-adult group, p-values were calculated for all the taxa detected. the result demonstrated that distinct bacterial genera were overrepresented in the patient and healthy-adult groups. whereas haemophilus (p = 0.010) and moraxella (p = 0.028) were identified as patient group-specific genera, streptococcus (p = 0.003), neisseria (p = 0.003), gemella (p = 0.003), aggregatibacter (p = 0.008), and actinobacillus (p = 0.001) were determined to be bacteria specific to the healthy-adult group. to hierarchically visualize the bacterial profile similarities among the samples, a upgma dendrogram was generated from the fast unifrac distance matrix. based on bacterial composition, the samples analyzed in this study were divided into 6 oropharyngeal microbiome types (additional file 3: figure s2 ), with the clusters being characterized by the dominance of several bacterial genera: type 1 (dominated by streptococcus + prevotella + veillonella), type 2 (streptococcus + haemophilus + neisseria), type 3 (streptococcus), type 4 (moraxella), type 5 (haemophilus), and type 6 (klebsiella). only 4 samples were not grouped into any of the 6 types. the healthy adults and a subset of patients harbored bacterial communities dominated by streptococcus, and to a lesser extent by haemophilus, neisseria, prevotella, veillonella, and/or gemella (types 1-3 in figure 3 ). the remaining patients carried impaired microbiota dominated by moraxella (type 4 in figure 3 ), haemophilus (type 5), or klebsiella (type 6), and this was coupled with a massive reduction in the levels of streptococcus. types 5 and 6 were dominated by well-known pathogens like h. influenzae and k. pneumonia, but type 4 was dominated by a previously unknown one, moraxella nonliquefaciens. we elucidated the differences in bacterial profiles in the context of causal agents of infections and demonstrated that virus type did not determine the structural differences in bacterial communities (additional file 4: figure s3a ). moreover, sex, sample type (swab, aspirate, or sputum), and smoking status did not influence the bacterial community structure (additional file 4: figures s3b, s3c and s3d), which was also unaffected by the year and month of sample collection (data not shown). by contrast, subjects' age was associated with the bacterial profile in a statistically significant manner (additional file 4: figure s3e ), and the samples were categorized into 2 age groups, 0-5 and 6-90 years (p <0.0001, hotelling's test). in this study, we discovered a bacterial species that was dominant in young patients (0-5 years old): m. nonliquefaciens was detected in 32.2% of the patients, with abundance ratios of 0.03%-97.0% depending on the subject, but this species was not observed in any healthy subject. most of the patients (16 out of 19 cases) harboring m. nonliquefaciens were under 6 years old. two rsv infected patients (rs1 and rs7) showed 95 and 97% of abundance of m. nonliquefaciens, indicating that the upper respiratory tracts of these patients were overwhelmed by this bacterial species. in addition, a closely related pathogenic species, m. catarrhalis, was detected in 23.7% of the patients, with abundance ratios of 0.03%-26.5%. analyzing the 16s rrna gene sequence revealed that m. nonliquefaciens and m. catarrhalis were clearly distinct species that showed 98.4% similarity between type strains (additional file 5: figure s4 ). the moraxella contigs recovered from patient samples were divided into 2 clades based on phylogenetic analysis (figure 4 ). clade i was closely related (98.5%-99.8% 16s rrna gene similarity) to the type strain of m. nonliquefaciens. the branching pattern of the contigs within the radiation of clade i indicated that m. nonliquefaciens populations in the patients encompassed 3 phylogenetic lineages. contigs belonging to clade ii were clustered together with m. catarrhalis (99.6%-100% 16s rrna gene similarity), and these contigs were further divided into 2 subpopulations, type 1 and type 2 populations [29] . to determine whether m. nonliquefaciens has a possibility to be an opportunistic pathogen, its potential pathogenicity was inferred using the genome sequence of the type strain. genome sequencing identified a 2.22-mb-sized genome of m. nonliquefaciens dsm 6327 t , featuring a 42.06% g + c ratio. the genomic relatedness calculated using ani showed that m. nonliquefaciens dsm 6327 t and m. catarrhalis rh4 shared low genomic relatedness, 75% ani, which is considerably less than the cut-off value used for species circumscription, 95%-96% ani [30] . the ani value further confirmed that the 2 species were distinct. various proteins have been reported to play pivotal roles in m. catarrhalis pathogenesis [31] . thus, we examined whether the virulence proteins in the m. catarrhalis rh4 genome were also encoded in the newly determined m. nonliquefaciens genome. the genes responsible for host-cell adhesion and invasion, evasion of host immune system, and biofilm formation were included as putative virulence factors. comparative genomic analysis revealed that most of the virulence genes identified in m. catarrhalis were encoded in the m. nonliquefaciens genome (additional file 6: table s2 ). moreover, resistance to β-lactam antibiotics was predicted based on the presence of the β-lactamase class c gene. however, bro-1 and bro-2 β-lactamases encoded by most of m. catarrhalis strains were not detected in m. nonliquefaciens. our results demonstrated that changes in bacterial profiles elicited by viral infection were not associated with the causal viral species: the microbiome compositions in samples obtained from various viral infections were not differentiated based on the causal infectious agents. regardless of the causal agents involved, the respiratory tract microbiota of patients differed substantially from the microbiota of healthy subjects in the kinds and diversities of prevalent bacteria. however, the heterogenicity of the patient group of this study (age, sampling type, and number of samples/virus type) hinders strong conclusions for this point. nevertheless, the current results from this study provide the first insight into microbiome alterations associated with viral infection in the upper respiratory tract. figure 3 dependence of microbiome structure on several key genera. principal coordinate analysis (pcoa) of the bacterial communities isolated from 57 healthy-adult and 59 patient samples was performed using the weighted pairwise unifrac distance matrix. the unifrac distance represents the distance between 2 samples in terms of the microbial community structure. samples are color-coded according to the 7 clustering groups. the diminished bacterial diversity observed in patients agreed well with previous studies reporting that the diversity in commensal airway microbiota declined following infection by specific pathogens [6, 13] . for example, in the nares of patients with s. aureus carriage, species diversity was half of that found in healthy adults' nares [6] . these results indicate that the normal microflora is depleted in respiratory tract cells impaired due to viral infection and is replaced by a few opportunistic pathogens. moreover, the dominance of streptococcus in the respiratory tracts of healthy subjects (table 1) agreed with previous culture-independent massive metagenomic sequencing studies [3, 7] . streptococcus is also known to be abundant in the oral cavity [32, 33] . thus, the oropharynx of healthy people could be characterized by high bacterial diversity and by an overwhelming abundance of the genus streptococcus. by analyzing the bacterial community, we defined 6 oropharyngeal types of bacterial populations in the upper respiratory tract. we use the word "oropharyngeal type" here based on the "enterotype" concept, which was introduced by arumugam et al. and defined as the clusters of human gut microbiome determined based on bacterial composition [34] . the concept suggests that people can be classified into several enterotypes according to the abundance of key bacterial taxa in gut microbial communities [35] . in this study, the samples included were restricted to one ethnic group, and thus the suggested 6 oropharyngeal types may be accepted only temporarily. however, because no other efforts to cluster respiratory tract microbiomes have been reported to date, our results may serve as a favorable starting point for future studies on this subject. our results revealed that haemophilus and moraxella were patient-specific genera. unlike h. influenzae, m. nonliquefaciens has not been studied for its possible role in pneumonia. although m. nonliquefaciens has been isolated from clinical cases including chronic bronchitis [36] , bronchial infection [37] , pneumonitis [38] , endophthalmitis [39] , septic arthritis [40] , thyroiditis [41] , discitis [42] , botryomycosis [43] , and endocarditis [44] , this bacterium is widely considered to be a part of the normal flora in the human upper respiratory tract and to exhibit low pathogenicity [45, 46] . by contrast, a closely related species, m. catarrhalis, is a well-studied respiratory tract pathogen that frequently colonizes the nasopharynx and is an exclusively human pathogen that displays an affinity for the human upper respiratory tract [47, 48] . long considered to be a commensal bacterium of the upper respiratory tract, m. catarrhalis has now been established as an etiological cause of otitis media and the exacerbations of chronic obstructive pulmonary disease (copd) [47, 49] . despite the distinctiveness of the 2 species at the taxonomic level, m. nonliquefaciens and m. catarrhalis share several common features. first, the age-related incidence of m. nonliquefaciens infection determined here is concordant with that of m. catarrhalis. previously, m. catarrhalis was reported to be mostly associated with upper respiratory tract infections in children [50] , and its carriage rate was shown to be high in children (up to 75%) and extremely low in healthy adults (1%-3%) [49, [51] [52] [53] [54] . moreover, the phenotype and gene incidences of m. catarrhalis isolates of children and adults presenting with respiratory disease differ substantially, possibly as a result of immune evasion in adults [48] . the age-related incidence of m. nonliquefaciens and m. catarrhalis may be indicative of the weak pathogenicity of moraxella species, which may be unable to evade the well-established immune system of adults. second, most strains of m. catarrhalis are known to produce β-lactamases and thus exhibit ampicillin resistance [49] . this antibiotic resistance was also predicted in m. nonliquefaciens based on the presence of the class c β-lactamase gene. however, although both species possess class c β-lactamase genes, the species differ with respect to the possession of bro β-lactamase; bro is unique because it shows no substantial similarity to any β-lactamase genes identified so far [55] . the absence of bro-1 and bro-2 in m. nonliquefaciens suggests that m. catarrhalis acquired the bro genes by means of lateral gene transfer after the 2 species evolved into distinct lineages. third, all but one virulence factors reported for m. catarrhalis were found to be encoded by m. nonliquefaciens (additional file 6: table s2 ), which indicates that m. nonliquefaciens has a high potential to be pathogenic even though it is currently considered to be a commensal bacterium. several reasons may account for why the overabundance of m. nonliquefaciens has not been reported. growing this organism and distinguishing it from m. catarrhalis are challenging, which may have resulted in a poor recognition of m. nonliquefaciens as a respiratory pathogen. moreover, although m. catarrhalis is focused on by clinicians, the isolation of m. catarrhalis from clinical samples is complicated by the presence of neisseria strains because these organisms share morphological similarities [49] . furthermore, m. nonliquefaciens may have been considerably underestimated because of being misidentified as m. catarrhalis or neisseria spp. [49] . lastly, the current absence of clinical interest or familiarity with m. nonliquefaciens may have resulted in under-reporting or identification of this pathogen. indeed, m. catarrhalis was previously underreported since other better recognized pathogens were also recognized and growing in the same cultures [49] . to clarify the incidence of this potential pathogen, future studies will need to differentiate between the true rates of incidence of m. catarrhalis and m. nonliquefaciens. the results of this study suggest that m. nonliquefaciens deserves considerable attention as a potential opportunistic pathogen in the respiratory tract. human microbiome project c: structure, function and diversity of the healthy human microbiome bacterial community variation in human body habitats across space and time variability and diversity of nasopharyngeal microbiota in children: a metagenomic analysis disordered microbial communities in the upper respiratory tract of cigarette smokers topographical continuity of bacterial populations in the healthy human respiratory tract the human nasal microbiota and staphylococcus aureus carriage comparison of the respiratory microbiome in healthy nonsmokers and smokers pyrosequencing analysis of the human microbiota of healthy chinese undergraduates gut microbial 'enterotypes' become less clear-cut what are the consequences of the disappearing human microbiota? airway microbiota and pathogen abundance in age-stratified cystic fibrosis patients from microbe to microbiota: considering microbial community composition in infections and airway diseases loss of bacterial diversity during antibiotic treatment of intubated patients colonized with pseudomonas aeruginosa a persistent and diverse airway microbiota present during chronic obstructive pulmonary disease exacerbations microbiota regulates immune defense against respiratory tract influenza a virus infection understanding the symptoms of the common cold and influenza metagenomic analysis of respiratory tract dna viral communities in cystic fibrosis and non-cystic fibrosis individuals effect of genetically modified poplars on soil microbial communities during the phytoremediation of waste mine tailings uchime improves sensitivity and speed of chimera detection introducing eztaxon-e: a prokaryotic 16s rrna gene sequence database with phylotypes that represent uncultured species the neighbor-joining method: a new method for reconstructing phylogenetic trees mega: a biologist-centric software for evolutionary analysis of dna and protein sequences evolution of protein molecules confidence-limits on phylogenies -an approach using the bootstrap comparative genomics of neisseria weaveri clarifies the taxonomy of this species and identifies genetic determinants that may be associated with virulence measurement of diversity fast unifrac: facilitating high-throughput phylogenetic analyses of microbial communities including analysis of pyrosequencing and phylochip data a direct approach to false discovery rates analysis of moraxella catarrhalis by dna typing: evidence for a distinct subpopulation associated with virulence traits shifting the genomic gold standard for the prokaryotic species definition molecular aspects of moraxella catarrhalis pathogenesis defining the normal bacterial flora of the oral cavity ecology of viridans streptococci in the oral cavity and pharynx enterotypes of the human gut microbiome a guide to enterotypes across the human body: meta-analysis of microbial community structures in human microbiome datasets association of mucoid encapsulated moraxella duplex var. nonliquefaciens with chronic bronchitis moraxella duplex var. nonliquefaciens as a cause of bronchial infection pneumonitis and pulmonary abscess associated with moraxella nonliquefaciens posttrabeculectomy endophthalmitis caused by moraxella nonliquefaciens moraxella nonliquefaciens septic arthritis in a patient undergoing hemodialysis acute thyroiditis caused by moraxella nonliquefaciens discitis due to moraxella nonliquefaciens botryomycosis caused by moraxella nonliquefaciens endocarditis caused by moraxella nonliquefaciens henriksen and bøvre 1968, 391 al . in bergey' s manual of systematic bacteriology the type-4 pilin of moraxella nonliquefaciens exhibits unique similarities with the pilins of neisseria gonorrhoeae and dichelobacter (bacteroides) nodosus moraxella catarrhalis, a human respiratory tract pathogen age-related genotypic and phenotypic differences in moraxella catarrhalis isolates from children and adults presenting with respiratory disease in a: moraxella catarrhalis: from emerging to established pathogen epidemiology of moraxella catarrhalis in children during the first 2 years of life: relationship to otitis media comparison of the local immune response to nontypable haemophilus influenzae (nhi) and moraxella catarrhalis (mc) during otitis media acute otitis media caused by branhamella catarrhalis: biology and therapy impact of antimicrobial therapy on nasopharyngeal carriage of streptococcus pneumoniae, haemophilus influenzae, and branhamella catarrhalis in children with respiratory tract infections respiratory-tract infections due to branhamella catarrhalis -epidemiologic data from western-australia genesis of bro beta-lactamase-producing moraxella catarrhalis: evidence for transformation-mediated horizontal transfer genome analysis of moraxella catarrhalis strain bbh18, a human respiratory tract pathogen profiling bacterial community in upper respiratory tracts we thank dr. s won for his advices on statistical analyses. this work was supported by research funds (2011-e43000-00, 2012-e43001-00, and 2013-e43001-00) of the korea centers for disease control and prevention. the pyrosequencing of bacterial community identified 6 oropharyngeal microbiome types in the upper respiratory tract, but the bacterial profile was not related to the type of causal infected viruses. the microbiota of patients differed substantially from that of healthy subjects in the kinds and diversities of prevalent bacteria. comparative analysis of healthy adults and patients identified a bacterium specific to young patients, m. nonliquefaciens. the results of whole-genome sequencing raised the possibility of m. nonliquefaciens being an opportunistic pathogen. additional file 1: table s1 . list and clinical characteristics of samples used in this study. nd, not determined.additional file 2: figure s1 . ordination diagram showing the relatedness of microbiomes in the upper respiratory tract of healthy people. principal coordinate analysis (pcoa) of bacterial communities isolated from 57 healthy adults was performed using the weighted pairwise unifrac distance matrix. the unifrac distance represents the distance between 2 samples in terms of the microbial community structure.additional file 3: figure s2 . dendrogram and circle map showing the clustering of samples into 6 groups depending on bacterial population dynamics. the unweighted pair group method with arithmetic mean (upgma) dendrogram was generated from the fast unifrac distance matrix to hierarchically visualize the manner in which samples are grouped. the relative abundance of representative microbial genera is indicated as a circle map; circle sizes represent the percentage ratio within a sample.additional file 4: figure s3 . ordination diagram showing the relatedness of microbiomes in the upper respiratory tract of people afflicted with diverse viral infections. we performed principal coordinate analysis (pcoa) on the bacterial communities isolated from 57 healthy-adult and 59 patient samples by using the weighted pairwise unifrac distance matrix. the unifrac distance represents the distance between 2 samples in terms of the microbial community structure. the structure of the bacterial community was not affected by (a) the type of virus or by (b) the sex, (c) sample type, and (d) smoking status of the subjects. however, (e) age was correlated with the microbiome structure.additional file 5: figure s4 . diagram showing the relationships among the 2 type strains and the 2 representative contigs obtained from patient samples. the average nucleotide identity (ani) value indicating genome relatedness was calculated using the complete genome sequence of m. catarrhalis rh4 strain (prjna 48809), which shows 100% 16s rrna gene sequence identity with the type strain of the species.additional file 6: table s2 . comparison of major virulence genes present in m. nonliquefaciens and m. catarrhalis. the list of virulent genes was obtained from previous reports [31, 56] . the gene locus in each genome is presented together with gene size in amino acids (in parenthesis).abbreviations upgma: unweighted pair group method with arithmetic mean; pcoa: principal coordinate analysis; ani: average nucleotide identity; vtm: viral transport medium; icu: intensive care unit. the authors declare that they have no competing interests. key: cord-307261-0a3iztns authors: hayden, randall t.; gu, zhengming; rodriguez, alicia; tanioka, lisa; ying, claire; morgenstern, markus; bankowski, matthew j. title: comparison of two broadly multiplexed pcr systems for viral detection in clinical respiratory tract specimens from immunocompromised children date: 2012-01-30 journal: j clin virol doi: 10.1016/j.jcv.2011.12.020 sha: doc_id: 307261 cord_uid: 0a3iztns background: the detection of viral respiratory tract infections has evolved greatly with the development of pcr based commercial systems capable of simultaneously detecting a wide variety of pathogens. objectives: evaluate the relative performance of two commercial broad range systems for the detection of viral agents in clinical respiratory tract specimens from immunocompromised children. study design: a total of 176 patient samples were included in the analysis, representing only the first sample collected for each patient, and excluding failed reactions. samples were de-identified and assayed in parallel using two different, broadly multiplexed pcr systems: resplex™ ii panel v2.0 (resplex), qiagen, hilden, germany and filmarray(®) respiratory panel (filmarray), idaho technology inc., salt lake city, ut. method comparison was based upon pair-wise concordance of results according to patient age, viral target and number of targets detected. results: the two systems showed an overall concordance, by patient, of 83.8% (p = 0.0001). filmarray detected at least one target in 68.8% of samples, while resplex detected at least one target in 56.8%. resplex failed to detect 20.7% of filmarray positives, and filmarray failed to detect 4% of resplex positives. the relative performance of each system (including which system detected a higher number of positive samples) varied when stratified by target viral pathogen. conclusions: broadly multiplexed pcr is an effective means of detecting large numbers of clinically relevant respiratory viral pathogens. viral respiratory tract infections can cause serious morbidity and increased mortality in immunocompromised pediatric patients. the rapid and sensitive detection of such agents has implications for treatment decisions, clinical care, and infection control practices. as in other areas of diagnostic virology, molecular diagnostic methods have shown promise in markedly improving diagnostic sensitivity when compared to culture or antigen detection assays. while such favorable performance characteristics have made molecular methods appealing, their introduction in the clinical laboratory has been slowed by issues related to cost and technical expertise required to perform this testing. early versions of such tests have focused on a limited number of pathogens, and typically detected only one or two viruses (or groups of viruses) at a time. [1] [2] [3] [4] [5] [6] [7] thus, detection of all clinically relevant entities has required running an entire panel of tests, compounding costs, and staffing requirements. the advent of broadly multiplexed assays has sought to address some of these issues. 8, 9 front-end multiplexed amplification (typically pcr) followed by detection is capable of identifying over 20 different targets. thus, a single assay utilizing the sensitivity of pcr can mimic the diagnostic spectrum of culture creating the potential for increasing routine detection beyond cultivable viruses, reducing processing costs, staffing requirements, and turn-around time. this technology also offers the possibility of increased ability to detect multi-viral infection. while the clinical importance of such capabilities is presently uncertain, this may carry important prognostic or infection control related implications, particularly among immunocompromised patients, such as those studied here. a number of different methodologies have now been proposed or marketed that use such an approach; however, limited published studies have compared different broadly multiplexed systems to one-another. in the current study, we compared two such systems to each other and to a panel of real-time pcr assays targeting individual viruses. technologies evaluated included the resplex ii panel v2.0 (resplex), qiagen, hilden, germany and the filmarray respiratory panel (filmarray), idaho technology, inc., salt lake city, ut. both products used for this study were for research use only (ruo). the filmarray product has only recently become available as an fda-cleared assay, and at the time of this submission few studies have been published examining performance of this method using clinical respiratory tract specimens. two broadly multiplexed pcr systems were compared to each other and to a panel of laboratory developed tests for the detection of respiratory viral pathogens in clinical respiratory tract specimens from pediatric immunocompromised children. samples were collected prospectively, as part of routine clinical care at st. jude children's research hospital (sjcrh) between january 13 and may 4, 2010, from children presenting with signs and symptoms of upper respiratory tract infection. results from patients 19 years of age and above were excluded from the analysis. the sjcrh institutional review board (irb) classified this study as non-human research; the study was exempted from irb approval and informed consent requirements were waived. samples consisted of bronchoalveolar lavage (bal) specimens, nasopharyngeal swabs (nps), nasopharyngeal washes (npw), and tracheal aspirates (ta). unused samples remaining after routine diagnostic testing were de-identified and blinded prior to study inclusion. unique patient and sample identifiers were assigned to each sample in order to match the results obtained from the various methods being compared. samples were divided into two 0.5 ml aliquots where one was tested by the laboratory developed test panel(ldtp), which included the pro hmpv assay kit (gen-probe, san diego, ca) for detection of hmpv, at sjrch and the other was tested using the filmarray assay. left over sample from the clinical aliquot was then tested by the resplexii assay at diagnostic laboratory services (dls), aiea, hi. extraction was performed at sjcrh and nucleic acid was sent and stored frozen to dls until testing at this remote site. this collection, transport, and storage method was used to allow preanalytical standardization between the testing methods. a 50 l nucleic acid solution was extracted from 250 l of respiratory sample together with prior addition of rna and dna controls for ldtp test; a second 50 l nucleic acid solution was extracted from 250 l aliquots of the same respiratory sample without addition of the rna and dna controls, for respplexii testing. extractions were otherwise identical and were performed using the nuclisens ® minimag magnetic extraction system (biomérieux inc., durham, nc), per manufacturer's instructions. the rna control was purchased as a ready-to-use lyophilized beadcontaining a 1 kb armored synthetic nucleic acid (cepheid). the dna control was a plasmid constructed by inserting a 357-bp dna fragment of phocid herpesvirus type 1 gb gene into vector puc57 (designed in-house at sjcrh and manufactured at genscript corp., piscataway, nj). both controls were detected only in ldtp test. a panel of real-time pcr assays was developed to detect infa and infb, rsv, adenovirus, and parainfluenza viruses 1, 2, and 3 from respiratory specimens. the pro hmpv assay kit was also run as part of this panel. the assays utilized taqman chemistry and realtime pcr technology and were carried out on the smartcycler ii platform (cepheid, sunnyvale, ca). the molecular detection targets, primer and probe sequences are listed in table s1 . the resplex tm ii panel v2.0 uses a multiplex rt-pcr analysis to amplify and detect 18 respiratory viruses ( filmarray utilizes a prefabricated pouch containing lyophilized reagents. the sample is added directly to the pouch wherein specimen preparation, amplification, and detection all take place without further offline sample manipulation. 300 l of original sample was mixed with 500 l of filmarray lysis buffer. approximately 1 ml of hydration solution was added by syringe to the filmarray pouch through hydration solution inlet port and 300 l of sample/lysis buffer mix was added to the pouch through the sample inlet port. the pouch was then loaded on the filmarray instrument, with automated extraction, amplification, and data analysis; total run time, approximately 1 h (5 min hands-on time). quality of testing was assured by the inclusion of two rna process controls(pc) in each pouch (proprietary sequences, idaho technology). the rna process controls were carried through all stages of the test process from samples lysis to pcr and dna melt analysis. both controls had to produce positive results for validation of test results. filmarray detected viral targets: adenovirus, bocavirus, coronavirus 229e, hku1, nl63, oc43, enterovirus, hmpv, human rhinovirus, influenza virus types a and b, parainfluenza viruses 1, 2, 3 and 4, and rsv. the mcnemar test was used to compare the accuracy of all three tests; comparison was made in pair-wise fashion and exact methods were used when discordant cells had less than 20 observations. to reduce bias, only the first sample of each patient with multiple samples was included in the analysis. for purposes of analysis, patient age was divided into four different age groups based on years of age: ≤2, 3-5, 6-13, and 14-18. this was a paired study, pairwise comparisons being made between performance of filmarray and resplex, filmarray and ldtp, and resplex and ldtp. when targets differed between the systems under comparison, results were collapsed into the narrowest taxonomic category that encompassed both systems of a pair. results from individual samples in which the first run failed were excluded from the analysis. however, results from second run from consecutive samples in which the entire run failed were included in the analysis as it was assumed that a systematic or technologist error was most likely the cause for the failure of the entire run for the resplex system, a positive result was determined using a cut-off mean fluorescent intensity (mfi) of 200. a total of 440 samples were collected from 210 children during the study period. after excluding samples from patients aged 19 and above, samples obtained after the first collection, and failed runs, the total number of samples analyzed was 176; only a single sample was tested per patient. while there were no failures with the ldtp, the total number of failed runs by filmarray was 11 and with resplex was 15. the mean age of children with samples included in the analysis was 6.9 years, ranging from 2 months to 18 years. the vast majority of samples were npw with 167 samples (94.9%), followed by ta with 6 samples (3.4%), bal with 2 samples (1.1%) and one nps (0.6%). over 80% of tested samples came from children who were 13 or younger: samples from children two years or less accounted for 27.3% (48) of samples, between three and five years inclusive were 24.4% (43), between six and 13 were 32.4% (57), and 15.9% (28) of samples came from children aged between 14 and 18, inclusive. overall difference in detection of targets between filmarray and resplex was found to be significant (p < 0.0001). out of the 121 samples detected positive by filmarray, resplex did not detect a target for 20.7% (25) of samples, while filmarray missed 4% (4) of samples detected positive by resplex ( table 1 ). the total number of samples for which both systems agreed was 147 samples; 96 samples testing positive and 51 samples testing negative. after stratifying by different targets and target groups, differences were observed in the detection of coronavirus 229e (exact p = 0.0313), infa (exact p = 0.0313), enterovirus (p < 0.0001), and rhinovirus (p < 0.0001). analysis stratified by patient age showed that in ages 3-5, 83.7% of samples were positive by filmarray and of these, resplex missed eight samples (22.2%), p = 0.0391; likewise, age group 6-13, 63.2% of samples were positive by filmarray but resplex missed 19.4% (7), p = 0.0156 (table s2) . when positive results were stratified by the number of targets detected by filmarray, differences in detection were found to be significant in cases where the filmarray system detected one or two targets (table s3 ). in the 76 cases where filmarray detected one target, resplex did not detect 18 of those cases, p < 0.0001. resplex did not detect any targets in six samples of the 34 samples for which filmarray detected the presence of two targets, p = 0.0313. in these six samples, the targets not detected by resplex were rhinovirus (4), coronavirus hku1 (2), rsv (2), adenovirus (1), bocavirus (1), coronavirus oc43 (1), and enterovirus (1). analysis for this comparison included only viruses targeted by both systems. these target groups were: adenovirus, hmpv, infa, parainfluenza types 1-3, and rsv. there were no differences in the number of positives detected by either system over the other, either overall or when stratified by target (table 2) . differences in detection rate were not observed when data were stratified by age groups (table s2) . after stratifying by number of targets detected by filmarray, ldtp detected viruses in six samples that filmarray called negative (p = 0.0313); but ldtp did not detect targets in seven samples in which filmarray detected one target, p = 0.0156 (table s4 ). the percentage of samples detected positive by resplex (24.4%) was lower than that for the ldtp (34.7%). among the samples detected positive by resplex, ldtp did not detect 1.1% (2); resplex did not detect targets in 20 samples (11.4%) that were detected positive by ldtp, p = 0.0001. the only target group for which significant differences were observed was influenza a; ldtp detected 12 samples as positive for influenza a but resplex did not detect 50% of these p = 0.0313 (table 3) . when results were stratified by age group, significant differences were observed in samples taken from children aged 3 to 13 (table s2 ). resplex detected 50% less positives compared to ldtp in samples from children aged between 3 and 13; and 25% less in the remaining age groups, though these differences were not significant. differences in detection between resplex and ldtp were significant in samples where resplex did not detect any targets (table s4) . the introduction of multiplex molecular amplification assays for the detection of viral respiratory pathogens has improved the sensitivity of routine viral detection methods. the range of agents detected [10] [11] [12] has been expanded to include newly discovered and emerging respiratory viruses such as coronaviruses nl63 and hku1, human metapneumovirus (hmpv), and bocavirus. [13] [14] [15] [16] [17] [18] [19] the broadly multiplexed molecular approach studied here has the capability to detect and identify more viruses simultaneously than traditional methods (i.e. culture and dfa) and shows an increased detection rate for co-infections. 8, 9, [20] [21] [22] [23] [24] in the current study, coinfections were detected in the range of 1-26%, which is similar to other investigators. sanghavi et al. investigated both adult and pediatric patients, including organ transplants and found dual infections at 10.4% and triple at 1.3%. 25 in a 2003 study, guittet et al. reported a range of 5-40% in hospitalized children. 26 the clinical significance of detecting more than one virus is not clear, but calvo et al. reported multi-viral infections more frequently in hospitalized infants with respiratory tract disease (17.4%). these infections were also linked to higher fever, longer hospital stays, and more frequent use of antibiotics than single rsv infections. 27 in another study by semper et al. co-infection with hmpv and rsv revealed a tenfold increase in the relative risk for admission into the pediatric intensive-care unit for mechanical ventilation as a result of severe bronchiolitis. 28 there is little published data beyond this on the clinical significance of multi-viral respiratory tract infection, and no work specific to the importance of detecting >2 viruses. the findings here suggest that as broadly multiplexed pcr systems become more common, a new body of literature will need to be developed to address the relevance and implications of such findings for prognosis, treatment, and infection control. the current study, to our knowledge, is the first reported that compares the filmarray with the resplex ii v2.0 for the direct detection of viral agents in clinical respiratory tract specimens from immunocompromised children. the viral targets for the two systems were similar with previously noted exceptions. overall 29 even though both systems detected 40-50% more viruses than traditional methods, the filmarray detected significantly more viruses and mixed infections than the xtag rvp. in the current study, resplex detected less rsv, influenza type a, hmpv, and adenovirus than the filmarray. increased rsv detection by the filmarray was also noted by rand et al. compared to the xtag rvp. 29 adenoviruses were not typed in this study, but the reduced rate of detection by resplex may relate to the fact that it detects only subtypes b and e, compared to the filmarray which detects all subtypes. it was noteworthy that the detection rate for enterovirus and rhinovirus were inversely related when the two systems were compared to each other. furthermore, if the filmarray and resplex enterovirus/rhinovirus were added together in each case, they would be approximately equal in the total number of detections (78 vs. 72, respectively). this could be the result of crossover detection related to differences in the nucleic acid sequences between the two assays. rhinoviruses and enteroviruses show extensive genomic similarity, but yet can be very different in their phenotypic characteristics. 30 the self-contained nature of the pouch system used by the filmarray, where sample preparation, amplification, and detection take place in a closed system markedly reduces any risk for carry-over contamination between specimens. both the resplex ii v2.0 and the xtag rvp require external nucleic acid extraction followed by multiplexed amplification and detection using a liquid-phase array technology. this approach may carry a risk of carry-over contamination due to both additional sample manipulation and the lack of a closed system. the difference in nucleic acid extraction prior to amplification and detection, along with other variables, such as nucleic acid sequence variations and chemistry may also contribute to the differences in test performance. 31 specimen collection procedures can also contribute to a difference in the overall test performance for any molecular test. the use of oropharyngeal swabs, nasopharyngeal swabs, or nasopharyngeal washings showed multiplex real-time rt pcr detection rates of 54.2%, 73.3%, and 84.9%, respectively in a study by lieberman et al. 32 other investigators have shown similar differences with the use of different swabs and collection procedures. 33, 34 the performance of both traditional and molecular-based tests varies according to the age group studied. 35 however, generally the younger children tend to show a higher viral load than older individuals. in general, this observation may account for a more favorable performance between traditional and molecular-based testing in the younger age group. in the present study, both the film-array and the resplex exhibited similar viral detection in the ≤2 years age group. however, the filmarray outperformed the resplex in the 3-5 year and 6-13 year groups. the viral load may have been lower in the >2 years group, accounting for the overall increase in sensitivity for the filmarray. the material costs for most molecular testing are greater than for dfa and culture, but the overall benefit may depend upon the protocol involved and could result in an overall cost reduction as described by mahony et al. 36 the other aspect of the cost analysis is the actual labor time involved for the filmarray and resplex. in the current study, the hands-on-time for the resplex was approximately 1 h with a total time of 4 h for testing 24 samples. the filmarray involved only 5 min for handson-time with a total time of 1 h/sample. based on the one cartridge per machine design of the filmarray instrument, the throughput for filmarray is integrally related to the number of machines available in a given laboratory. so while laboratories that process less than one sample per hour would be able to take advantage of apparently improved turnaround time with filmarray, resplex is more accommodating of larger specimen loads. it would therefore take six filmarray instruments to meet the 4-h turnaround time of a full resplex run. likewise, the apparent labor-saving benefits of the film-array system apply primarily to low volume settings. although with practice, processing time might be less than the above stated 5 min per sample, total hands-on time for more than 15-20 samples might be expected to meet or exceed that of resplex. this study demonstrates the promise of automated, broadly multiplexed pcr systems for the detection of viral respiratory pathogens. such methods combine the exquisite sensitivity of molecular amplification with a breath of targets previously attainable only through the use of culture-based techniques. these operating characteristics, together with increasingly selfcontained, automated, and easy-to-use methods, bring us closer to the mainstream adaptation of molecular diagnostic testing, no longer limited to academic centers or reference laboratory settings. the widespread use of these powerful techniques will have implications for diagnosis, treatment, infection control, and resource utilization in the healthcare setting. funding for this study was supported in part by the american lebanese syrian associated charities, alsac and the anderson charitable foundation. reagents were kindly supplied by qiagen and idaho technology, inc. instrumentation for the filmarray process was provided by idaho technology, inc. development of three multiplex rt-pcr assays for the detection of 12 respiratory rna viruses multiplex real-time pcr assay for detection of influenza and human respiratory syncytial viruses use of a multiplex real-time pcr to study the incidence of human metapneumovirus and human respiratory syncytial virus infections during two winter seasons in a belgian paediatric hospital multiplex real-time pcr for detection of respiratory tract infections evaluation of the prodesse hexaplex multiplex pcr assay for direct detection of seven respiratory viruses in clinical specimens evaluation of the hexaplex assay for detection of respiratory viruses in children rapid and sensitive method using multiplex real-time pcr for diagnosis of infections by influenza a and influenza b viruses, respiratory syncytial virus, and parainfluenza viruses 1, 2, 3, and 4 development of a respiratory virus panel test for detection of twenty human respiratory viruses by use of multiplex pcr and a fluid microbead-based assay multicode-plx system for multiplexed detection of seventeen respiratory viruses multiplex pcr and emerging technologies for the detection of respiratory pathogens detection of respiratory viruses by molecular methods nucleic acid amplification-based diagnosis of respiratory virus infections cloning of a human parvovirus by molecular screening of respiratory tract samples human metapneumovirus infections in young and elderly adults metapneumovirus and acute wheezing in children human metapneumovirus: a newly emerging respiratory pathogen a newly discovered human pneumovirus isolated from young children with respiratory tract disease identification of a new human coronavirus characterization and complete genome sequence of a novel coronavirus, coronavirus hku1, from patients with pneumonia a novel multiplex real-time rt-pcr assay with fret hybridization probes for the detection and quantitation of 13 respiratory viruses evaluation of commercial resplex ii v2.0, multicode-plx, and xtag respiratory viral panels for the diagnosis of respiratory viral infections in adults immunofluorescence versus xtag multiplex pcr for the detection of respiratory picornavirus infections in children viral and atypical bacterial detection in acute respiratory infection in children under five years comparison of the eragen multi-code respiratory virus panel with conventional viral testing and real-time multiplex pcr assays for detection of respiratory viruses clinical evaluation of multiplex real-time pcr panels for rapid detection of respiratory viral infections rhinovirus and acute respiratory infections in hospitalized children retrospective study multiple simultaneous viral infections in infants with acute respiratory tract infections in spain dual infection of infants by human metapneumovirus and human respiratory syncytial virus is strongly associated with severe bronchiolitis comparison of two multiplex methods for detection of respiratory viruses: filmarray rp and xtag rvp new complete genome sequences of human rhinoviruses shed light on their phylogeny and genomic features real-world comparison of two molecular methods for detection of respiratory viruses identification of respiratory viruses in adults: nasopharyngeal versus oropharyngeal sampling pooled nasopharyngeal and oropharyngeal samples for the identification of respiratory viruses in adults development and evaluation of a flocked nasal midturbinate swab for selfcollection in respiratory virus infection diagnostic testing performance of diagnostic tests to detect respiratory viruses in older adults cost analysis of multiplex pcr testing for diagnosing respiratory virus infections r. hayden has served as a consultant for idaho technology. other authors have no competing interests to report. the study was classified as non-human research; study was exempted from irb approval and informed consent requirements were waived. supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.jcv.2011.12.020. key: cord-282533-w6kl74c8 authors: li, jin; tao, yue; tang, mingyu; du, bailu; xia, yijun; mo, xi; cao, qing title: rapid detection of respiratory organisms with the filmarray respiratory panel in a large children’s hospital in china date: 2018-10-11 journal: bmc infect dis doi: 10.1186/s12879-018-3429-6 sha: doc_id: 282533 cord_uid: w6kl74c8 background: respiratory tract infections (rtis) are the most common illness in children, and rapid diagnosis is required for the optimal management of rtis, especially severe infections. methods: nasopharyngeal swab or sputum specimens were collected from children aged 19 days to 15 years who were admitted to a hospital in shanghai and diagnosed with rtis. the specimens were tested with the filmarray respiratory panel, a multiplex pcr assay that detects 16 viruses, mycoplasma pneumoniae (m. pneumoniae), bordetella pertussis (b. pertussis) and chlamydophila pneumoniae (c. pneumoniae). results: among the 775 children studied, 626 (80.8%, 626/775) tested positive for at least one organism, and multiple organisms were detected in 198 (25.5%). rhinoviruses/enteroviruses (25.5%, 198/775) were detected most often, followed by respiratory syncytial virus (19.5%, 151/775), parainfluenza virus 3 (14.8%, 115/775), influenza a or b (10.9%), adenovirus (10.8%), m. pneumoniae (10.6%) and b. pertussis (6.3%). the prevalence of organisms differed by age, and most of the viruses were more common in winter. of the 140 children suspected of having pertussis, 35.0% (49/140) tested positive for b. pertussis. conclusions: filmarray rp allows the rapid simultaneous detection of a wide number of respiratory organisms, with limited hands-on time, in chinese pediatric patients with rtis. electronic supplementary material: the online version of this article (10.1186/s12879-018-3429-6) contains supplementary material, which is available to authorized users. acute respiratory tract infections (rtis) are the leading causes of outpatient visits and hospitalizations in all age groups, especially during winter and spring. for children under 5 years of age, rtis are the second leading cause of death [1] . most acute rtis in children are caused by respiratory viruses, such as respiratory syncytial virus (rsv), adenovirus (adv), rhinovirus (rv) and influenza viruses. in addition to viruses, atypical pathogens are major causes of pediatric rtis. one of the most common atypical pathogens is mycoplasma pneumoniae (m. pneumoniae), accounting for 10-40% of hospitalized children with community-acquired pneumonia [2, 3] . in addition to m. pneumoniae, the incidence of pertussis in china has significantly increased since 2010. nevertheless, multiple epidemiological studies have suggested that the incidence of pertussis in china has been significantly underestimated [4, 5] . the early diagnosis of the pathogen is beneficial for the precise selection of medication, which can largely avoid the overuse or even abuse of the antibiotics and improve the clinical care of patients. more importantly, the early diagnosis of contagious pathogens, such as bordetella pertussis (b. pertussis) and influenza viruses, can enable early isolation of patients, thus reducing the spread of pathogens. at present, the routine detection methods for respiratory pathogens in china are mostly based on immunological methods, which include the detection of m. pneumoniae and several major viruses, such as rsv, adv, rv, parainfluenza virus (para), influenza a virus (flua) and influenza b virus (flub). other respiratory viruses and atypical bacteria, such as chlamydophila pneumoniae (c. pneumoniae) and b. pertussis, are typically not routinely detected. given their poor sensitivity and long turn-around time (tat), immunological methods usually lead to broad-spectrum therapy and have been gradually replaced by molecular-based methods, such as conventional and real-time polymerase chain reaction (pcr), in developed countries [6, 7] . however, most of these molecular tests are technically challenging and require independent spaces, such as pre-pcr and post-pcr rooms, to eliminate the potential risk of cross-contamination, and such requirement limits their applications in china. therefore, faster, more sensitive and easy-to-use assays for multiplex respiratory pathogen detection are urgently needed. filmarray (biofire diagnostics, utah, usa, owned by biomérieux) is a small, desktop, fully automated multiplex pcr device. the molecular system includes automated nucleic acid extraction, an initial reverse transcription step and multiplex nested pcr, followed by a melting curve analysis [8] . the filmarray respiratory panel (filmarray rp) is both fda-approved and ce ivd-marked. the current version of filmarray rp (v1.7) is able to detect 16 viral and 3 atypical respiratory organisms. the test is performed in a closed system that requires 5 min of hands-on time and 65 min of instrumentation time. several comparison studies between fil-marray and other tests for respiratory organisms showed comparable results [9] [10] [11] . the aim of this study was to evaluate the application of filmarray rp for the detection of respiratory organisms, and to provide information about the seasonality and prevalence of these organisms in pediatric patients with rtis in a large children's hospital in china. the study population was enrolled according to protocol definitions and inclusion criteria. patients with respiratory infections, with or without fever (defined as body temperature ≥ 37.5°c), were included if they had at least one of the following symptoms: (1) cough; (2) nasal obstruction; (3) tachypnoea; (4) nasal flaring; or (5) hypoxia. patients admitted to the hospital had at least one of the following conditions: (1) unabating high fever; (2) dyspnea, tachypnea or hypoxemia; (3) anorexia or dehydration; (4) radiological confirmation of lung infection; or (5) respiratory infection with underlying diseases, such as congenital heart disease, bronchopulmonary dysplasia, airway malformations, severe malnutrition. according to the chinese center for disease control and prevention (cdc), patients suspected of having pertussis should have a cough for more than 2 weeks and have at least one of the following symptoms: (1) paroxysmal cough; (2) inspiratory whoop; or (3) post-tussive vomiting. in the present study, patients suspected of having pertussis were diagnosed with pertussis when b. pertussis was positive by filmarray rp detection and were otherwise diagnosed with pertussis-like syndrome. nasopharyngeal swab (nps) or sputum specimens were obtained from patients with symptoms of rtis on the day of hospitalization at shanghai children's medical center (scmc) from december 1, 2016 to november 30, 2017. demographic data and clinical features, as well as laboratory test and imaging results, were obtained for each enrolled patient. the study was approved by the institutional review board and the ethics committee of shanghai children's medical center (scmcirb-k2017044), and written informed consent was obtained from the parents of each patient. the filmarray rp v1.7 targets 19 organisms, including adv, influenza a viruses h1, 2009h1, h3 (flua-h1, flua-2009h1, flua-h3) and flub, parainfluenza virus types 1 to 4 (para 1-4), coronaviruses 229e, hku1, oc43, and nl63 (cov-hku1, nl63, 229e, oc43), human metapneumovirus (hmpv), rsv, human rhinovirus/ enterovirus (rhino/entero), c. pneumoniae, m. pneumoniae and b. pertussis. the filmarray rp assay was performed according to the manufacturer's instructions. the principle of the assay has been previously described [8, 12] . each pouch included internal run controls for every step, and results for the assay were only provided by the software if the quality control reactions showed appropriate results. spss software package v21.0 was used for all statistical analyses. categorical variables were expressed as frequencies and percentages. the chi-square and fisher's exact tests were used to compare groups. continuous variables are expressed as the mean and standard deviation. student's t-test was used to assess the statistical significance between groups. p < 0.05 was considered to be statistically significant. a total of 775 patients diagnosed with upper or lower respiratory tract infections, aged 19 days to 15 years, were enrolled in the present study between december 1, 2016, and november 30, 2017. congenital heart disease, congenital biliary atresia, malignancy and congenital immunodeficiency were the most frequently observed underlying diseases in these patients and contributed to 50% of the deaths observed in this study. the general characteristics of the patients enrolled are presented in table 1 . overall detection rate of filmarray rp v1.7 analysis of the positive rates and prevalence in different age groups all the patients were grouped by age as follows: infants (age: < 1 year), toddlers (age: 1-2 years), preschoolers (age: 3-5 years) and school-aged children (age: 6-15 years) ( table 2 ). the highest specimen positivity rate, at 82.2% (278/338), was in the < 1-year age group, followed by 80.5% (149/185), 80.1% (117/146) and 77.4% (82/106) in the 1-2-year, 3-5-year and 6-15-year groups, respectively. there were no significant differences in the positivity rate of the different age groups. in contrast, the prevalence of organisms were different between the different age groups ( table 2) . rhino/entero, para 3, rsv and b. pertussis showed the highest prevalence in the < 1-year age group, while adv, hmpv and flua showed the highest prevalence in the 3-5-year age group. the most prevalent organism in the 6-15-year age group was m. pneumoniae. no organism showed a notably high prevalence in the 1-2-year age group. there was only one c. pneumoniae-positive patient during the study period, and this patient was in the 3-5-year age group. among the 775 specimens, 198 (25.5%, 198/775) were positive for more than one organism. the largest proportion (49.0%, 97/198) of multi-organism-positive specimens had combinations with rhino/entero. rhino/entero plus para 3 was the most common combination, making up 10.6% (21/ 198 ) of all multi-organism-positive specimens, while the combination of rhino/entero plus adv was the second most common type (6.1%, 12/198), followed by rhino/ entero plus rsv (5.6%, 11/198). the multi-organism combinations are listed in additional file 1: table s1 . seasonal prevalence of respiratory organisms from december 1, 2016 to november 30, 2017 the number of positive specimens was determined during different months of the year to demonstrate the epidemiology of the respiratory organisms. regarding the atypical bacteria, m. pneumoniae was detected throughout the year, with the highest incidence occurring in september and three minor peaks in december, january and june (fig. 1a) . the highest incidences of b. pertussis were observed in march and may. only one case of c. pneumoniae was detected in july. the seasonal prevalence of viruses with high detection rates were as follows. both flua and hmpv had two peaks that occurred in january and march, and adv showed a peak in january (fig. 1b) . the prevalence of para 3 remained high from february-august. the peaks in our study, 775 specimens were collected from pediatric patients with rtis over a period of one year and analyzed with filmarray rp v1.7. the overall results yielded a positivity rate of 80.8%, with multiple organisms detected in 25.5% of specimens, which is in accordance with litwin and piralla's reports [13, 14] . as in other studies, a notable variation in the pathogen prevalence with season and age was observed. most viruses had their highest positivity rates in winter, except that para 3 positivity rate was well distributed through the spring and summer, and the epidemiologic peaks for hmpv occurred 1 to 2 months later than those for rsv [15, 16] . the majority of respiratory viruses were observed in children younger than 5 years old. notably, rsv was the most prevalent virus in the < 1-year age group, and the prevalence decreased with age; while the incidence of m. pneumoniae increased with age [17] . multiple respiratory organisms were detected in 25.5% of the specimens in our study, the largest proportion of which included rhino/entero. other studies in adults reported lower multi-pathogen detection rates of approximately 8.7-15.9% [14, [18] [19] [20] , suggesting that pediatric patients with rtis are more likely to be infected by multiple pathogens than adults. however, the clinical significance, including disease severity and hospitalization time, of multi-pathogen infection, especially rhino/entero combination infections, is not clear. a previous report indicated that dual-positive results with rsv and rhino/entero specimens might be due to viral shedding from a previous rhino/entero infection [21] . nokso-koivisto et al. also found that rhinovirus was the most prevalent virus in asymptomatic carriers [22] . the most unexpected result in our study is the high detection rate of b. pertussis, with an overall detection rate of 6.32% in the group of 775 patients, further demonstrating the value of filmarray rp in clinical application. at present, the diagnosis of pertussis in china is based on culture and serology results. however, both the cdc and world health organization (who) use positive pcr results as the criteria for diagnosis, suggesting that filmarray rp testing, in addition to culture, can be considered for patients with suspected pertussis in order to better monitor disease outbreaks. additionally, the early diagnosis of patients with b. pertussis, which is typically difficult to distinguish from pertussis-like syndrome, can also help to reduce unnecessary macrolide treatment. the limitation of the panel is the lack of b. parapertussis, which contributes to more than 5% of pertussis cases [23] . however, it has been added to the second-generation panel, filmarray rp2 v1.1 [24] , and the prevalence of b. parapertussis in our patients is currently under investigation. as stated in the manufacturer's instructions, "filmarray respiratory panel (rp) is a multiplexed nucleic acid test intended for use with filmarray systems for the simultaneous qualitative detection and identification of multiple respiratory viral and bacterial nucleic acids in nasopharyngeal swabs (nps) obtained from individuals suspected of respiratory tract infections". therefore, nps samples are recommended for filmarray rp, but there are also studies demonstrating a comparable or even higher detection rate in sputum [25, 26] . however, the detection rate in sputum in our study was lower than that in nps samples. this might partially be attributed to the fact that most of the sputum samples (86.7%, 98/113) were from icu patients, and the sputum-providing patients showed a higher positivity rate in their sputum culture than the nps-providing patients (33.6% vs 20.5%). in addition to sputum, bronchoalveolar lavage fluid (balf) is a common type of respiratory sample, and azadah et al. showed that detection in balf by filmarray rp can provide new and useful microbiological information within 7 days after a negative nps result is obtained [27] . therefore, the choice of the most appropriate sample type and time-point for each patient, particularly in specific clinical contexts, such as undergoing fiberoptic bronchoscopy or ventilator use, may require further investigation. as with other molecular methods, distinguishing whether the microbes detected in the filmarray analysis, especially those that are also detected in asymptomatic children, such as human rhinovirus, are causative pathogens or colonizers is not feasible [28] [29] [30] . therefore, the clinicians should take caution when judging pathogens because the results are sometimes "false positive". on the other hand, despite the high detection rate of fil-marray rp, a negative result does not mean the patient is not infected; moreover, a positive result does not mean there is no other co-infecting agent, especially in critically ill patients, in whom a bacterial co-infection often occurs. for this "false-negative" limitation, biofire has a new pneumonia panel that also targets balf/sputum and covers 9 common viruses, as well as 15 bacteria, including klebsiella pneumonia, haemophilus influenza, streptococcus pneumonia and staphylococcus aureus. nevertheless, the filmarray panel only aims to rapidly provide results for potential pathogens as a reference. a more appropriate method is to comprehensively consider the results from other examinations, such as routine blood testing, c-reactive protein (crp), procalcitonin (pct), the erythrocyte sedimentation rate (esr), culture and radiography, as well as the patients' symptoms, including body temperature, breathing, blood oxygen, heart rate, and mental condition. our study also has several limitations. first, our study was performed in a single center and may not be representative of the entire chinese pediatric population. second, we did not have data from a more appropriate assay to evaluate the specificity of filmarray rp. additionally, we do not provide detailed information on the effects of filmarray rp on the use of antibiotics, clinical outcomes and health economics, which require further investigation. in conclusion, the filmarray rp assay significantly expands our ability to diagnose multiple respiratory infections caused by viruses and atypical bacteria. the array can detect 19 respiratory organisms simultaneously, with a high detection rate, in 65 min. our study provided the age groups and seasonal distributions of different organisms for pediatric rti patients. this study also provides new insights into the current status of pertussis infection in china. whether filmarray rp can enhance clinical decision-making and limit the unnecessary use of antibiotics in china as in other countries still requires further investigation. additional file 1: table s1 . combinations of multiple organisms detected with filmarray rp. (doc 99 kb) additional file 2: table s2 . overall detection rates for the nasopharyngeal swab and sputum samples from the different age groups. epidemiology of viral respiratory infections community-acquired pneumonia requiring hospitalization among u.s. children epidemiology of acute respiratory infections in children in guangzhou: a three-year study seroprevalence of pertussis in china: need to improve vaccination strategies the seroepidemiology of immunoglobulin g antibodies against pertussis toxin in china: a cross sectional study routine molecular point-of-care testing for respiratory viruses in adults presenting to hospital with acute respiratory illness (respoc): a pragmatic, open-label, randomised controlled trial multiplex pcr and emerging technologies for the detection of respiratory pathogens an automated nested multiplex pcr system for multi-pathogen detection: development and application to respiratory tract infection charnot-katsikas a. comparison of cepheid xpert flu/rsv xc and biofire filmarray for detection of influenza a, influenza b, and respiratory syncytial virus comparison of the filmarray assay and in-house real-time pcr for detection of respiratory infection comparison of filmarray respiratory panel and laboratory-developed real-time reverse transcriptionpolymerase chain reaction assays for respiratory virus detection comparison of the filmarray respiratory panel and prodesse real-time pcr assays for detection of respiratory pathogens filmarray(r) respiratory panel performance in respiratory samples from neonatal care units seasonality and prevalence of respiratory pathogens detected by multiplex pcr at a tertiary care medical center human metapneumovirus subgroup changes and seasonality during epidemics seasonality, incidence, and repeat human metapneumovirus lower respiratory tract infections in an area with a high prevalence of human immunodeficiency virus type-1 infection etiologic spectrum and occurrence of coinfections in children hospitalized with community-acquired pneumonia performance of a novel microarray multiplex pcr for the detection of 23 respiratory pathogens (symp-ari study) pcr for detection of respiratory viruses: seasonal variations of virus infections detection of respiratory viruses by molecular methods prospective evaluation of a novel multiplex real-time pcr assay for detection of fifteen respiratory pathogens-duration of symptoms significantly affects detection rate human picornavirus and coronavirus rna in nasopharynx of children without concurrent respiratory symptoms update on pertussis and pertussis immunization multicenter evaluation of biofire filmarray respiratory panel 2 for detection of viruses and bacteria in nasopharyngeal swab samples detection of respiratory viruses in sputum from adults by use of automated multiplex pcr evaluation and implementation of filmarray version 1.7 for improved detection of adenovirus respiratory tract infection filmarray respiratory panel assay: comparison of nasopharyngeal swabs and bronchoalveolar lavage samples respiratory viral detection in children and adults: comparing asymptomatic controls and patients with community-acquired pneumonia interactions of respiratory viruses and the nasal microbiota during the first year of life in healthy infants etiology of severe pneumonia in children in developing countries we would like to thank the patients and their parents for the support and cooperation in publishing this work. availability of data and materials all data described in this manuscript is available upon request via email. authors' contributions qc and xm initiated the study. jl, yt, myt and bld performed the detection of respiratory organisms. jl, yt, myt, qc and xm wrote the manuscript and analyzed the data. yjx provided technical support and assisted in the data analysis. all authors read and approved the final manuscript.ethics approval and consent to participate the study was approved by the institutional review board and the ethics committee of shanghai children's medical center (scmcirb-k2017044), and written informed consent was obtained from the parents of each patient. not applicable. yijun xia is an employee of biomérieux. he was involved in the technical support and data analysis. all the other authors declare they have no conflict of interests to disclose. all the other authors declare that they have no competing interests. key: cord-322524-bq9ok8h1 authors: belongia, edward a; king, jennifer p; kieke, burney a; pluta, joanna; al-hilli, ali; meece, jennifer k; shinde, vivek title: clinical features, severity, and incidence of rsv illness during 12 consecutive seasons in a community cohort of adults ≥60 years old date: 2018-11-27 journal: open forum infect dis doi: 10.1093/ofid/ofy316 sha: doc_id: 322524 cord_uid: bq9ok8h1 background: the epidemiology and burden of respiratory syncytial virus (rsv) illness are not well defined in older adults. methods: adults ≥60 years old seeking outpatient care for acute respiratory illness were recruited from 2004–2005 through 2015–2016 during the winter seasons. rsv was identified from respiratory swabs by multiplex polymerase chain reaction. clinical characteristics and outcomes were ascertained by interview and medical record abstraction. the incidence of medically attended rsv was estimated for each seasonal cohort. results: rsv was identified in 243 (11%) of 2257 enrollments (241 of 1832 individuals), including 121 rsv type a and 122 rsv type b. the rsv clinical outcome was serious in 47 (19%), moderate in 155 (64%), and mild in 41 (17%). serious outcomes included hospital admission (n = 29), emergency department visit (n = 13), and pneumonia (n = 23) and were associated with lower respiratory tract symptoms during the enrollment visit. moderate outcomes included receipt of a new antibiotic prescription (n = 144; 59%), bronchodilator/nebulizer (n = 45; 19%), or systemic corticosteroids (n = 28; 12%). the relative risk of a serious outcome was significantly increased in persons aged ≥75 years (vs 60–64 years) and in those with chronic obstructive pulmonary disease or congestive heart failure. the average seasonal incidence was 139 cases/10 000, and it was significantly higher in persons with cardiopulmonary disease compared with others (rate ratio, 1.89; 95% confidence interval, 1.44–2.48). conclusions: rsv causes substantial outpatient illness with lower respiratory tract involvement. serious outcomes are common in older patients and those with cardiopulmonary disease. over the past decade, respiratory syncytial virus (rsv) infection has been increasingly recognized as an important cause of acute respiratory illness in older adults. both influenza and rsv account for a substantial number of respiratory illness deaths among adults ≥65 years of age in the united states. from 1997 to 2009, influenza and rsv contributed to approximately 28 000 and 17 000 annual cardiorespiratory deaths, respectively [1] . rsv is also an important cause of adult respiratory illness in both developed and developing countries, including tropical and subtropical regions [2] [3] [4] . studies conducted in the 1980s and 1990s first identified rsv as a cause of acute respiratory illness in a variety of adult populations, including older adults, working-age adults, hospitalized patients, and residents of long-term care facilities [5] [6] [7] [8] [9] . immunocompromised adults and those with advanced age and/or cardiopulmonary disease are at especially high risk for severe complications from rsv infection [10] [11] [12] [13] [14] . the incidence and burden of rsv in adults are difficult to measure as clinical symptoms are nonspecific and physician awareness of rsv in adults is limited. most adult studies of rsv have been conducted in the hospital setting, where rsv accounts for about 4% to 12% of respiratory illness hospitalizations during the winter season [14] [15] [16] [17] . advanced age, radiographic pneumonia, ventilator support, and secondary bacterial infection are all associated with increased risk of death among hospitalized patients with rsv [18] . few studies have assessed the incidence and clinical characteristics of adult rsv infections in the community and outpatient setting. a seminal prospective cohort study of adults ≥65 years of age during 4 winter seasons (1999) (2000) (2001) (2002) (2003) found that 3%-7% developed symptomatic rsv infection each season [14] . a 6-season study of adults ≥50 years old with predominantly outpatient acute respiratory illness found that rsv was the third most common viral pathogen (after influenza and human rhinovirus) [19] . advanced age, cough, nasal congestion, wheezing, and increasing interval from symptom onset to care-seeking were independently associated with rsv detection. the latter study was conducted in a wisconsin community cohort, and the overall seasonal incidence of medically attended rsv illness was estimated as 154 episodes per 10 000 individuals m a j o r a r t i c l e ≥50 years old [20] . the incidence of medically attended rsv increased with age, and it was highest (199 episodes per 10 000) among persons ≥70 years of age. rsv vaccines and antivirals for older adults are currently in prelicensure clinical trials [21] , and more detailed knowledge of rsv incidence and burden is needed to inform policy recommendations and evaluate cost-effectiveness. the objective of this study was to describe the clinical characteristics, severity, clinical outcomes, and long-term incidence of medically attended rsv illness in a community cohort of adults ≥60 years of age from the 2004-2005 through 2015-2016 influenza seasons. this analysis significantly extends a previous report on medically attended rsv infections in adults ≥50 years of age in the community cohort from 2004-2005 through 2009-2010 [19] . the current report includes an additional 6 seasons with more detailed clinical information based on medical record abstraction for rsv cases, including physical exam findings, management, and outcomes. the marshfield clinic research institute conducted prospective seasonal studies of influenza vaccine effectiveness in a wisconsin community cohort during the 2004-2005 through 2015-2016 seasons [22] [23] [24] [25] . before each influenza season, a community cohort was defined based on residency in or near marshfield, wisconsin, and receipt of care from the marshfield clinic. individuals in the community cohort were eligible to be recruited when seeking care for acute respiratory illness in outpatient clinics (all seasons) or in the hospital setting (2004-2005 through 2009-2010) . each season, recruitment began when influenza activity was detected in the community and usually continued for 12-15 weeks. symptom eligibility criteria varied by season but included fever/feverishness or cough during most seasons. for the 2011-2012 through 2015-2016 seasons, eligibility was based on the presence of acute respiratory illness with cough. the maximum duration of illness was 10 days for the 2004-2005 through 2006-2007 seasons and 7 days for all subsequent seasons. after obtaining informed consent, a midturbinate swab was obtained for influenza detection. symptoms and onset date were assessed during the enrollment interview. realtime reverse transcription polymerase chain reaction (rt-pcr) was performed each season to identify influenza cases. after testing was complete, aliquots of samples in viral transport media were frozen at -80°c. this study was reviewed and approved by the marshfield clinic institutional review board. during each season, all study participants (or parents) provided informed consent for influenza testing. multiplex rt-pcr testing to detect additional viruses was subsequently approved by the institutional review board with a waiver of informed consent. archived samples were tested for the presence of respiratory virus nucleic acid using a multiplex respiratory virus panel (genmark dx esensor, respiratory viral panel). this is a food and drug administration (fda)-approved multiplex panel for detection of rsv a and b, human rhinovirus, human metapneumovirus, parainfluenza viruses 1-3, influenza a (h3, h1, and h1n1pdm09) , influenza b, and adenoviruses b/e and c. nucleic acid was extracted from the swabs using the roche magnapure 2.0 system and was then amplified using rt-pcr with target-specific primers. target-specific signals were determined by voltammetry, a process that generates electrical signals from ferrocene-labeled signal probes. a published comparison of 4 respiratory virus panels found that the genmark dx esensor assay had 100% sensitivity for rsv a and rsv b detection [26] . samples collected from the 2004-2005 through 2009-2010 seasons were previously tested using the genmark multiplex assay in a separate study of rsv illness in adults ≥50 years of age [19] . a subset of these was retested in 2016 for validation as the original testing was performed before fda licensure of the genmark dx esensor assay. all samples collected from 2009-2010 through 2015-2016 were tested using the fda-approved genmark assay. for the 2014-2015 season only, funding for multiplex testing of respiratory swabs was provided by the us centers for disease control and prevention (cdc). electronic diagnosis codes, procedure codes, laboratory tests, and prescribing data were extracted from the medical record for all rsv-positive patients. trained research coordinators abstracted detailed clinical data for the initial respiratory illness visit, subsequent outpatient or ed visits, and hospital admissions within 28 days for all participants with pcr-confirmed rsv illness. symptoms of acute respiratory illness and onset date were available from the original study enrollment interview. abstracted clinical data included additional symptoms not assessed during study enrollment, functional status, smoking status, physical exam findings, treatment, pulmonary function, and presence of specific comorbid chronic diseases. for each clinical encounter in the 28-day window, information was abstracted regarding diagnosis or suspicion of pneumonia, acute sinusitis, and acute otitis media. abstraction of laboratory results included hemoglobin, total leukocyte count, bun, creatinine, glomerular filtration rate, hemoglobin a1c, procalcitonin, and brain natriuretic peptide (bnp). measures of cardiac and pulmonary function were abstracted, including peak flow, fev1/fvc, oxygen saturation, and cardiac ejection fraction (from echocardiogram). additional abstractions were performed for participants who were hospitalized within 28 days after pcr-confirmed rsv illness. abstracted data included admission and discharge diagnoses, hospital course, use of supplemental oxygen or ventilation support, intensive care unit (icu) admission, antibiotic or antiviral treatment, and disposition at discharge. all chest x-ray and chest computed tomography (ct) reports for participants with rt-pcr-confirmed rsv illness were independently reviewed by 2 physicians to identify a new radiographic opacity or infiltrate within 28 days after enrollment. chest x-rays obtained >48 hours after hospital admission were excluded as they may represent nosocomial rather than community-acquired illness. discrepancies in x-ray interpretation were adjudicated by consensus. for the purposes of this analysis, pneumonia was defined as an illness meeting all of the following criteria: (1) clinical diagnosis or suspicion of pneumonia mentioned in the medical record; (2) new opacity or infiltrate identified by chest x-ray or ct scan; and (3) antimicrobial treatment. clinical outcomes for rt-pcr-confirmed rsv illness were classified as serious, moderate, or mild. a serious outcome was defined as acute care hospital admission, emergency department (ed) visit for acute illness, or pneumonia occurring within 28 days after enrollment. a moderate outcome was defined as new antibiotic or antiviral therapy, new/increased bronchodilator therapy, or new/increased systemic corticosteroid therapy during the 28-day window after enrollment. individuals were classified as having a mild outcome if they did not meet criteria for serious or moderate rsv illness outcome. a physician independently validated all serious clinical outcomes. hospital admissions were reclassified to a nonserious outcome category if the reason for admission was unrelated to cardiopulmonary disease based on review of hospital records. at the time of initial presentation, participants with rt-pcrconfirmed rsv were classified as having moderate to severe lower respiratory tract disease (rsv-mslrtd) if at least 3 of the following lower respiratory tract symptoms or signs were present: cough, wheezing (or worsening in baseline wheezing), new sputum production (or increase in baseline sputum), new (or worsening) shortness of breath, and tachypnea (≥20 breaths/minute). the same criteria were used to define rsv-mslrtd in a phase iii clinical trial for rsv vaccine in older adults (clinicaltrials.gov identifier: nct02608502). for this descriptive analysis, we compared the demographic and clinical characteristics of study participants ≥60 years of age with and without rt-pcr-confirmed rsv illness. among those with rt-pcr-confirmed rsv illness, we further assessed clinical and demographic characteristics and severity stratified by rsv subtype (a or b), as well as presence or absence of rsv-mslrtd during the initial clinical encounter. univariate comparisons were performed with the chi-square (categorical variables) or wilcoxon test (continuous variables), as appropriate; p values of <.05 were considered significant. seasonal incidence of medically attended rsv illness was estimated in the community cohort for individuals ≥60 years of age for the period 2006-2007 through 2015-2016. seasonal incidence is reported as episodes of medically attended rsv illness per 10 000 population. a detailed description of the incidence estimation procedure was published previously [27] . briefly, all estimates were standardized to a 31-week period from approximately early october through early may using wisconsin state laboratory of hygiene surveillance data. incidence calculations were based on 2 assumptions: (1) test results from enrolled patients can be extrapolated to nonenrolled cohort members with a respiratory illness visit during the enrollment period; and (2) the number of rsv cases occurring outside the enrollment period in the cohort was proportional to the number of statewide cases occurring outside the enrollment period. poisson regression with analytic weights, offsets, and robust variance estimation was used to estimate seasonal incidence with corresponding 95% confidence intervals and test for linear trends in seasonal incidence. analyses were performed using sas 9.4 (sas institute inc., cary, nc). rsv was detected in 243 (11%) of 2257 encounters (representing 241 of 1832 individuals) for acute respiratory illness. rsv cases were equally represented by rsv a (n = 121) and rsv b (n = 122). of the 243 rsv cases, 23 (9%) had an additional viral target identified. influenza was detected in 519 (23%), and 5 (<1%) were positive for both rsv and influenza. there were 2 individuals with an episode of rsv during 2 different seasons; none of the study participants had 2 rsv episodes in the same season. among 1495 encounters negative for rsv and influenza by rt-pcr, the most common viral pathogens were coronavirus oc43, nl63, hku1, 229e (n = 210; 14%), human metapneumovirus (n = 184; 12%), human rhinovirus (n = 135; 9%), and parainfluenza virus types 1-4 (n = 74; 5%); 873 (39%) encounters were negative for all viral targets in the multiplex rt-pcr assay. patients with rt-pcr-confirmed rsv were similar to those with non-rsv respiratory illness in terms of age, gender, number of ed visits, and hospital admissions in the prior 12 months ( table 1 ). the mean (median) interval from symptom onset to study enrollment and swab collection was 4.0 (4.0) days for rsv-negative patients and 4.4 (4.0) days for rsv-positive patients (p = .006). chronic obstructive pulmonary disease (copd) was present in 15% of those with rsv illness, 18% of those with other viral respiratory infection, and 27% of those with no virus detected. rsv prevalence among adults with acute respiratory illness varied from a low of 5% common symptoms of rsv illness included sore throat, sputum production, cough, fever/feverishness, dyspnea, myalgia, and wheezing ( table 2 ). the most common diagnosis codes on the date of enrollment for rsv-positive patients were cough (45%), acute upper respiratory infection (21%), acute bronchitis (13%), acute sinusitis (12%), and bronchitis, unspecified (12%). fifty-nine patients (24%) met the criteria for rsv-mslrtd during the enrollment encounter. the presence of rsv-mslrtd on enrollment was associated with a 3-fold higher risk of serious clinical outcome (relative risk, 2.99; 95% confidence interval [ci], 1.83-4.88) ( table 3) . rsv-positive participants with mslrtd during the enrollment encounter had a higher prevalence of copd and congestive heart failure (chf) and a greater number of medical encounters in the 28 days after enrollment (median, 1 vs 2; p = .05). the clinical outcome for rsv illness was serious in 47 (19%), moderate in 155 (64%), and mild in 41 (17%). nearly half of serious outcomes occurred in patients ≥75 years of age with rsv infection (table 3) . serious outcomes were not mutually exclusive (14) 2006-2007 16 (7) 144 (7) 11 (9) 5 (4) 2007-2008 23 (9) 162 (8) 10 (8) 13 (11) 2008-2009 27 (11) 132 (7) 6 (5) 21 (17) 2009-2010 29 (12) 217 (11) 24 (20) 5 (4) 2010-2011 20 (8) 134 (7) 9 (7) 11 (9) 2011-2012 22 (9) 104 (5) 15 (12) 7 (6) 2012-2013 12 (5) 190 (9) 7 (6) 5 (4) 2013-2014 13 (5) 136 (7) 2 (2) 11 (9) 2014-2015 25 (10) 252 (13) 15 (12) 10 (8) 2015-2016 21 (9) 197 (10) (17) 31 (25) .09 no. of ed visits in the prior 12 mo, median (min-max) b 0 (0-6) 0 (0-15) .5 0 (0-3) 0 (0-6) .5 no. of hospital admissions in the prior 12 mo, median (min-max) b 0 (0-6) 0 (0-13) .4 0 (0-3) 0 (0-6) .003 no. of outpatient visits w/ provider in the prior 12 mo, median (iqr) b 8 (4-13) 8 (4-15) 1.0 8 (4-12) 9 (5-15) .04 abbreviations: chf, congestive heart failure; copd, chronic obstructive pulmonary disease; ed, emergency department; iqr, interquartile range; rsv, respiratory syncytial virus. a smoking status is missing for a larger proportion of non-rsv illnesses because these cases were not abstracted. b wilcoxon test was used for differences in medians. and included hospital admission (n = 29), ed visit (n = 13), and pneumonia (n = 23). the 13 ed visits included 5 patients who were admitted and 8 who were discharged. among 155 patients with a moderate outcome, 144 (93%) received a new antibiotic prescription, 45 (29%) received bronchodilator or nebulizer treatment, and 28 (18%) received new systemic corticosteroid therapy; 5 (3%) were treated with an antiviral drug. the risk of a serious outcome was approximately double for patients with copd or chf compared with patients without these conditions (table 3) . thirty-two participants with rsv infection were admitted to a hospital within 28 days. three of these hospitalizations were for noncardiopulmonary conditions that were unlikely to be related to rsv infection, including incarcerated hernia, small bowel obstruction, and acute gastrointestinal illness. these 3 hospital admissions were excluded from the serious outcome group. fifteen of the remaining 29 hospitalized patients were enrolled in the inpatient setting. four of those were directly admitted from an outpatient clinic, whereas the remaining 11 were admitted from the emergency department. seven patients were enrolled during an outpatient encounter on the same day as their hospital admission, leaving 7 with hospital admissions 1 or more days after enrollment. the median interval from symptom onset to admission for rsv-positive hospitalized individuals was 4 days. preexisting chronic diseases were common among hospitalized patients with rsv, including copd (n = 9; 31%), chf (n = 8; 28%), asthma (n = 8; 28%), and diabetes (n = 9; 31%) ( table 4 ). twenty-one (72%) received a discharge diagnosis of respiratory tract infection (eg, pneumonia, bronchitis, exacerbation of copd). only 1 patient was recognized to have rsv at the time of hospital discharge. the mean (sd) duration of hospital admission was 3.5 (2.5) days. twenty-seven (93%) were discharged to home, and 2 (7%) were transferred to a rehabilitation or long-term care facility; there were no deaths within 28 days. the hospital course was uncomplicated for most patients. none required mechanical ventilation or icu admission. twenty-five (86%) received antimicrobials; 4 (16%) were treated with antiviral drugs. to estimate the number of hospitalized rsv cases that were not included in this analysis, we extracted hospital diagnosis codes for community cohort members during periods of study enrollment. we identified an additional 20 individuals ≥60 years of age who were hospitalized during study enrollment periods with a diagnosis code for rsv but were not enrolled in the influenza vaccine effectiveness study. the median community cohort size for adults aged ≥60 years was 13 807 (range, 12 142-13 807) for the seasons from 2006-2007 through 2015-2016. the overall seasonal incidence of medically attended rsv illness was 139 cases per 10 000 (95% ci, 122-160). the rsv incidence was 196 cases per 10 000 (95% ci, 162-236) among persons with chronic cardiopulmonary disease and 103 (95% ci, 85-125) among those without cardiopulmonary disease (incidence rate ratio [irr], 1.89; 95% ci, 1.44-2.48). there was a significant decline in the incidence of medically attended rsv from 2006-2007 through 2015-2016 for the entire cohort (p = .003, chi-square test for trend) and for those with cardiopulmonary disease (p = .014, chi-square test for trend). the incidence of medically attended rsv was also higher in women compared with men (irr, 1.46; 95% ci, 1.09-1.95). the overall incidence rates of medically attended illness caused by rsv a and rsv b were nearly identical (irr, 1.01; 95% ci, 0.75-1.35), although 1 or the other subtype was often dominant during a single season. the temporal trend in rsv incidence suggests an overall reduction among adults ≥60 years of age after the 2011-2012 season (figure 1 ). abbreviations: chf, congestive heart failure; copd, chronic obstructive pulmonary disease; ed, emergency department; iqr, interquartile range; mslrtd, moderate to severe lower respiratory tract disease; rsv, respiratory syncytial virus; rt-pcr, reverse transcription polymerase chain reaction; sob, shortness of breath. a high-risk comorbid conditions (except immune-compromised status, which was determined from electronic diagnosis codes), symptoms/exam findings from enrollment visit, and therapeutic interventions were obtained by medical record abstraction. b three additional hospital admissions occurred within 28 days for incarcerated hernia, small bowel obstruction, and acute gastrointestinal illness. these conditions were considered unrelated to the preceding rsv infection. c wilcoxon test was used for differences in medians. d p-values not shown for characteristics which contributed to the mslrtd case definition. we compared the peak month for rsv and influenza positives during each season among study participants ≥60 years old. in 7 seasons, the rsv peak and the influenza peak occurred in the same calendar month. in 2 seasons, the rsv peak occurred before the influenza peak, and in 3 seasons, the rsv peak occurred after the influenza peak. we also compared the peak month for rsv detection among study participants with the peak month based on local clinical testing of children <24 months. in 5 seasons, the peak calendar month was the same for enrolled adults ≥60 years old and children; the pediatric peak occurred 1-2 months earlier in 5 seasons and 1 month later in 2 seasons. in a longitudinal assessment over 12 seasons in a single community, we found that rsv was a common cause of outpatient respiratory illness in adults ≥60 years of age. rsv was detected in 11% of those with medically attended acute respiratory illness, and it was abbreviations: chf, congestive heart failure; ci, confidence interval; copd, chronic obstructive pulmonary disease; ed, emergency department; iqr, interquartile range; mslrtd, moderate to severe lower respiratory tract disease; rsv, respiratory syncytial virus; rt-pcr, reverse transcription polymerase chain reaction; sob, shortness of breath. a high-risk comorbid conditions (except immune-compromised status, which was determined from electronic diagnosis codes) and symptoms/exam findings from enrollment visit were obtained by medical record abstraction. the second most common viral pathogen in this age group. the number of rsv a and rsv b cases was similar overall, but 1 subtype was usually dominant during any given season. the seasonal incidence of medically attended rsv was variable but was consistently higher in persons with preexisting cardiopulmonary disease. moderate or serious outcomes, including change in therapy, hospital admission, and pneumonia, occurred in >80% of patients with laboratory-confirmed rsv infection. serious outcomes (hospital admission, ed visit, or pneumonia) occurred in nearly 1 of every 5 patients with rsv infection. patients with serious outcomes were significantly more likely to present with dyspnea and objective signs of lower respiratory tract involvement, including wheeze, rales, and rhonchi. hospital admission was the most common serious outcome, and the risk of a serious outcome increased with age. serious rsv illness was significantly associated with chronic obstructive pulmonary disease and congestive heart failure. the majority of outpatient rsv cases (64%) resulted in a moderate outcome, including a new prescription for antibiotics, antivirals, bronchodilators, or systemic corticosteroids. nearly half of individuals with rsv required a chest x-ray and measurement of oxygen saturation during the enrollment visit or follow-up period. overall, the most common therapeutic interventions included new antibiotic prescription and bronchodilator/nebulizer treatment. more than three-quarters of patients with rsv were treated with antibiotics. although this study did not evaluate bacterial coinfections, it is possible that many of these antibiotic courses were unnecessary. few studies have assessed the occurrence, clinical spectrum, and outcomes for rsv illness among older adults in the outpatient setting. a sentinel system in the united kingdom identified rsv in 15% of adults ≥65 years old with medically attended acute respiratory illness [28] . this is similar to the percent positive (11%) that we observed over 12 seasons in adults ≥60 years old. in a previous study over a shorter time period, we found that cough, nasal congestion, and wheezing were more common in adults ≥50 years old with rsv compared with those with other causes of acute respiratory illness [29] . prospective rsv illness surveillance in nearly 3000 healthy, working-age adults from 1975 to 1995 demonstrated that 84% of rsv infections were symptomatic [30] . of the latter, 22% involved the lower respiratory tract (tracheobronchitis or wheezing). additional studies are needed in diverse populations to estimate the burden of adult rsv illness and the potential impact of future licensed vaccines. rsv-associated moderate to severe lower respiratory tract illness is a composite measure of lower respiratory tract illness that has been used as a proxy for serious respiratory disease outcomes in rsv vaccine clinical trials (clinicaltrials.gov identifiers: nct02608502 and nct02266628). in this study, we found that rsv-mslrtd at the time of enrollment was significantly associated with a serious clinical outcome. in particular, patients with rsv-mslrtd at enrollment were significantly more likely to require hospital admission and were also more likely to develop pneumonia during the follow-up period. the incremental risk (absolute risk difference) for each of these outcomes exceeded 20% when rsv-mslrtd was present at enrollment. these findings suggest that rsv-mslrtd may be a useful surrogate measure to identify individuals at risk for more serious clinical end points in trials of vaccines and antivirals. the strengths of this study include consistent prospective recruitment of patients with acute respiratory illness from a defined community cohort, inclusion of 12 consecutive winter seasons, collection of standardized clinical data during the enrollment encounter, and detailed abstraction of outpatient and inpatient medical records for the 28-day follow-up period. abbreviations: bipap, bilevel positive airway pressure; chf, congestive heart failure; copd, chronic obstructive pulmonary disease; cpap, continuous positive airway pressure; icu, intensive care unit; mslrtd, moderate to severe lower respiratory tract disease; rsv, respiratory syncytial virus; rt-pcr, reverse transcription polymerase chain reaction. this observational study also has several limitations. recruitment was restricted to individuals who sought medical care for respiratory illness during periods of influenza transmission, and cough was required for enrollment during seasons after the 2009 pandemic. this study underestimated the occurrence of rsv hospital admissions as enrollment was restricted to primary care and urgent care outpatient clinics after 2010. based on diagnosis codes, we identified an additional 20 individuals ≥60 years old in our community cohort who were hospitalized with a diagnosis of rsv but not enrolled in the vaccine effectiveness study. these patients were most likely admitted through the emergency department or subspecialty clinics where study recruitment did not occur. in addition, some rsv cases may have been missed by rt-pcr testing, as serology has been shown to improve the detection of rsv infection in adults with community-acquired pneumonia [31] . finally, this study was conducted in a largely rural and racially homogenous population, and results may not reflect outpatient rsv occurrence and outcomes in urban and racially diverse settings. as new vaccines and antivirals are licensed for rsv prevention and treatment in adults, there will be a great need for data to estimate the population burden and the potential reduction in cases and serious outcomes. the potential impact on reducing antimicrobial use is another potential benefit that requires further evaluation. these data will be needed to increase awareness of rsv among adult health care providers and to inform cost-effectiveness analyses for public health planning and policy deliberations. in preparation for these decisions, additional research is urgently needed to estimate disease burden and outcomes in larger and more diverse populations. estimates of mortality attributable to influenza and rsv in the united states during 1997-2009 by influenza type or subtype, age, cause of death, and risk status respiratory viruses associated with patients older than 50 years presenting with ili in senegal all chronic cardiopulmonary disease viral etiology of influenza-like illnesses in respiratory syncytial virus infection in guatemala a study of respiratory infections in the elderly to assess the role of respiratory syncytial virus isolation of respiratory syncytial and influenza viruses from the sputum of patients hospitalized with pneumonia an epidemic of respiratory syncytial virus in elderly people: clinical and serological findings noninfluenza respiratory virus infection in long-term care facilities respiratory syncytial virus infections on an adult medical ward nosocomial transmission of respiratory syncytial virus in immunocompromised adults an outbreak of respiratory syncytial virus in a bone marrow transplant center the natural history of respiratory syncytial virus infection in cancer and transplant patients: implications for management respiratory syncytial virus is not an important community acquired pathogen in adult hematological malignancy patients respiratory syncytial virus infection in elderly and high-risk adults respiratory syncytial virus is an important cause of community-acquired lower respiratory infection among hospitalized adults respiratory syncytial virus and other respiratory viral infections in older adults with moderate to severe influenza-like illness rates of hospitalizations for respiratory syncytial virus, human metapneumovirus, and influenza virus in older adults high morbidity and mortality in adults hospitalized for respiratory syncytial virus infections medically attended respiratory syncytial virus infections in adults aged ≥50 years: clinical characteristics and outcomes seasonal incidence of medically attended respiratory syncytial virus infection in a community cohort of adults ≥50 years old immunogenicity and safety of a respiratory syncytial virus fusion protein (rsv f) nanoparticle vaccine in older adults effectiveness of inactivated influenza vaccines varied substantially with antigenic match from the 2004-2005 season to the 2006-2007 season influenza vaccine effectiveness in wisconsin during the 2007-08 season: comparison of interim and final results influenza vaccine effectiveness in the united states during 2012-2013: variable protection by age and virus type influenza vaccine effectiveness in the united states by vaccine type comparison of the biofire filmarray rp, genmark esensor rvp, luminex xtag rvpv1, and luminex xtag rvp fast multiplex assays for detection of respiratory viruses incidence of medically attended respiratory syncytial virus and influenza illnesses in children 6-59 months old during four seasons contribution of influenza and respiratory syncytial virus to community cases of influenza-like illness: an observational study medically attended respiratory syncytial virus infections in adults aged ≥50 years: clinical characteristics and outcomes respiratory syncytial virus infections in previously healthy working adults serology enhances molecular diagnosis of respiratory virus infections other than influenza in children and adults hospitalized with community-acquired pneumonia the authors appreciate the contributions of the following individuals: elizabeth key: cord-289139-5ljqnc39 authors: mengelle, c.; mansuy, j.m.; pierre, a.; claudet, i.; grouteau, e.; micheau, p.; sauné, k.; izopet, j. title: the use of a multiplex real-time pcr assay for diagnosing acute respiratory viral infections in children attending an emergency unit date: 2014-09-03 journal: j clin virol doi: 10.1016/j.jcv.2014.08.023 sha: doc_id: 289139 cord_uid: 5ljqnc39 background: the use of a multiplex molecular technique to identify the etiological pathogen of respiratory viral infections might be a support as clinical signs are not characteristic. objectives: the aim of the study was to evaluate a multiplex molecular real-time assay for the routine diagnosis of respiratory viruses, to analyze the symptoms associated with the pathogens detected and to determine the spread of virus during the period. study design: respiratory samples were collected from children presenting with respiratory symptoms and attending the emergency unit during the 2010–2011 winter seasons. samples were tested with the multiplex respifinder(®) 15 assay (pathofinder™) which potentially detects 15 viruses. results: 857 (88.7%) of the 966 samples collected from 914 children were positive for one (683 samples) or multiple viruses (174 samples). the most prevalent were the respiratory syncytial virus (39.5%) and the rhinovirus (24.4%). influenza viruses were detected in 139 (14.4%) samples. adenovirus was detected in 93 (9.6%) samples, coronaviruses in 88 (9.1%), metapneumovirus in 51 (5.3%) and parainfluenzae in 47 (4.9%). rhinovirus (40%) was the most prevalent pathogen in upper respiratory tract infections while respiratory syncytial virus (49.9%) was the most prevalent in lower respiratory tract infections. co-infections were associated with severe respiratory symptoms. conclusion: the multiplex assay detected clinically important viruses in a single genomic test and thus will be useful for detecting several viruses causing respiratory tract disorders. acute respiratory infections (aris) are more prevalent than any other form of infectious disease in children. they range from mild upper respiratory tract problems to serious lower respiratory infections such as bronchiolitis and pneumonia. viruses are the main pathogens and they account for many emergency hospital admissions [1, 2] . clinical signs and symptoms overlap between different viruses, but also between viruses and bacteria, making etiological e-mail address: mengelle.c@chu-toulouse.fr (c. mengelle). 1 both first authors equally contributed to this work. diagnosis based on clinical presentation alone difficult and sometimes leading to overuse of antibiotics. techniques involving culture, fluorescent detection of antigens or immunochromatography have been replaced by nucleic acid tests (nats). due to numerous viruses that might be involved, many monoplex nucleic acid tests are necessary to identify the pathogen(s) responsible for a respiratory disorder. this strategy is thus expensive and time consuming. the use of multiplex assays should significantly reduce hands-on time and cost, and rapidly provide reliable results. the multiplex ligation-dependent probe amplification technology (mpla)-respifinder ® respiratory assay [3] recently became commercially available. this assay is approved for in vitro diagnosis in europe and canada and can detect up to 15 respiratory viruses. this prospective study was done to evaluate this multiplex technique for use in clinical diagnosis. all the samples taken from http://dx.doi.org/10.1016/j.jcv.2014.08.023 1386-6532/© 2014 elsevier b.v. all rights reserved. children attending the emergency unit of the toulouse university hospital suffering from aris were collected prospectively and analyzed. clinical data related to the viruses detected were also analyzed, as was the spread of seasonal respiratory viruses for the winter following the influenza a h1n1pdm09 epidemic (october 2010 to march 2011). nasopharyngeal swabs (virocult ® kitnia, labarthe inard, france), aspirates or nasal washes were prospectively collected from children under 15 with symptoms of aris (see below) who attended the emergency unit of the toulouse university hospital between october 1, 2010 and march 31, 2011 and sent to the virology department for analysis. paediatricians completed a specific questionnaire related to the symptoms, including fever and upper respiratory manifestations (rhinitis, pharyngitis, otitis, sore throat, cough) and presence of symptoms of lower respiratory infections (bronchiolitis, pneumonia, acute flu and flu syndrome). whether or not a child had been vaccinated against influenza was also recorded. the collected samples were diluted in 1 ml minimum essential medium (gibco ® -life technologies, rockville, md, usa) and nucleic acids were extracted with the magna pure 96 tm instrument using the magna pure 96 dna and viral na small volume kit ® (roche diagnostics, meylan, france) according to the manufacturer's instructions (extracted volume: 200 l, elution volume: 100 l). extracts were analyzed using the respifinder ® 15 assay (pathofinder tm , maastricht, netherlands), a multiplex ligationdependent probe amplification (mlpa) technology [3] . this assay can detect 15 viruses: influenza viruses (iv) types a and b, parainfluenza viruses 1 to 4 (piv), respiratory syncytial viruses a and b (rsv), rhinovirus (rv), coronaviruses 229e, oc43 and nl63 (cov), human metapneumovirus (mpv) and adenovirus (adv). the test also includes a probe for detecting the avian influenza virus a h5n1. an internal control for pcr inhibitors detection was included in each test. a positive flu a sample and a negative sample were used as controls. samples that were positive for influenza a were subtyped with the realtime ready inf a/h1n1 detection set (roche diagnostics, meylan, france) on the light cycler 480 tm system (roche diagnostics, meylan, france). data were analyzed using stata tm v9.0 software (statacorp, texas, usa). qualitative variables were analyzed with the chisquared test. p values of less than 0.05 were considered significant. logistic regression was used to determine the odds ratios (or) of age and co-infections linked to severe respiratory symptoms. a total of 914 children, of whom 509/914 (55.7%) were male were enrolled in the study between october 1, 2010 and march 31, 2011 and provided 966 samples. the mean age was 1.6 ± 2.6 years and the median age was 7.3 months [range: 0.2-186], but 572/914 (62.6%) children were under 1 year old. the 914 children in the study group included 232/914 (25.4%) with upper respiratory tract infections (243 samples) defined as rhino-pharyngitis or sore throat, with or without otitis media. 682 children (723 samples) suffered from lower respiratory infections defined as bronchiolitis (338/914; 37%), pneumonia or bronchopneumonia (109/914; 11.9%), acute asthma (78/914; 8.5%) and flu or flu-like syndrome (158/914; 17.3%). 76 (8.3%) children were suffering from a chronic (n = 10) or a congenital disorder (n = 66): respiratory, haematological, neurological, cardiac disorders. 25/914 (2.7%) children had been vaccinated in the fall against flu, and 16/25 (64%) of them were suffering from a chronic or congenital disorder. we found co-infections in 24 (9.96%) samples from children with upper respiratory tract symptoms and in 144 (19.9%) samples from children with lower respiratory infections (p < 0.01). the most frequent co-infections in cases of severe respiratory symptoms involved rsv (associated with rv, adv, or cov) and rv associated with adv. 25% of the cases of bronchiolitis involving rsv were co-infections. rsv was more prevalent in children under 1 year old (48.2% vs 24.8%; p < 0.0001), while rv was not (26.4% vs 21.1%; ns). adv (14.8% vs 3.4%) and iv (24.4% vs 9.2%; p < 0.0001) were more frequent in children older than 6 months. co-infections were more frequent in younger children (1.3 years vs 1.7 years; p < 0.05). four parameters were included in the multivariate analysis: vrs infection, presence of co-infection, rv infection and age under 1 year. independent factors associated with severe respiratory symptoms were vrs infection (adjusted or, 3.8; 95% ci, 2.64-5.68), co-infections (adjusted or, 2.5; 95% ci, 1.55-3.94) and rv infection (adjusted or, 1.2; 95% ci, 0.85-1.83). age under 1 year was not associated with such symptoms. the frequency of positive samples varied from 71.4% (5/7 positive samples; week 40, 2010) to 98.4% (60/61 positive samples; weeks 1, 2011; p < 0.05) (fig. 3a) . all the viruses in the panel were detected in our patients, but their distributions varied (fig. 3b) . rv was the first of the three main viruses to appear and was detected throughout the study. rsv appeared in october and reached epidemic peak in week 51 of 2010; iv did not appear until december and reached a plateau between weeks 1 and 7 of 2011. iva was predominant until week 7 of 2011, while ivb was the most prevalent thereafter, decreasing slowly until the end of march. adv, mpv and cov were most frequently detected during the winter months, although piv was detected throughout the study. the availability of molecular assays has made laboratory diagnosis more efficient and has led to the improved detection of a broad spectrum of respiratory viruses [4, 5] . this study evaluates the use of a new multiplex molecular technique for detecting up to 15 respiratory viruses in routine practice. we collected respiratory samples from a group of children suffering from acute respiratory infections and the results obtained were analyzed in comparison to the clinical manifestations. the spread of the viruses was also analyzed during this winter period. a very high percentage of the samples were positive giving a virus signature in nearly 90% of cases, and all the viruses in the test panel were detected. these results are similar to those for young children and infants obtained by others (88-92%) [6] [7] [8] , whereas the rate of detection in adults was lower (43%) [9] . rv, rsv, and iv were the three most prominent pathogens detected, while adv, cov, mpv and piv were less frequent (<10% of cases). as expected, rv was the most frequently pathogen detected in upper respiratory infections [10] , but it was also found in lower respiratory infections, as was described recently by o'callaghan-gordo [11] . rsv remained however the most prominent agent in lower respiratory infections as published previously in bronchiolitis [12] and pneumonia [13] . adv, cov, mpv and piv also accounted for lower respiratory tract aris as shown previously: adv infection was shown to lead to severe chronic disease and to the increase in the mortality rate in children [14] , whereas cov [15] , mpv [16] and piv [17] can be responsible for severe lower respiratory tract infections requiring hospitalization, especially among the youngest. flu and flu syndromes were preferentially linked to influenza viruses and older children seemed to be more susceptible to these viruses. we found a relationship between an influenza virus infection and severe infection due to hyperthermia, but not with more severe respiratory symptoms, in contrast to the situation in adults [18] . the use of multiplex assays has demonstrated that infections with multiple viruses are common [4] ; the frequency of such infections can be as high as 27%, 30% or 46% [5, 19, 20] . this was exactly the case for our children: all the viruses in the panel were detected in co-infections, not only rsv, but also adv and covs. martin et al. [21] described similar results. they detected adv in association with other viruses in 52% of samples and cov in 50%. they also showed that multiple virus infections were correlated with less severe disease. the relationship between co-infections and illness severity remains uncertain and may depend upon the virus detected. dual infections involving rsv and rv [22] or rsv and mpv can lead to severe bronchiolitis [23] whereas co-infections without rsv do not [24] . we also analyzed the way viral infections spread during the winter. rsv and iv were epidemic while the frequency of the other viruses changed little. our study carried out in the winter following the influenza ah1n1pdm09 pandemia shows that iv and rv infections had returned to their pattern. this has also been described in germany by gröndahl et al. [25] who showed that the ah1n1pdm09 outbreak had a great impact on the spread of other respiratory viruses, but that the virus infections had return to their usual epidemiologic characteristics the year after. this new multiplex technique dramatically shortens hands-on time. the results for 15 viral pathogens can be obtained in about one day. it is also much simpler than conventional routine techniques, which need many and various steps and a wide range of technical competence (culture, pcr, immunofluorescence). while multiplex assays are more expensive than monoplex pcrs, laboratories can still choose to perform single pcrs in a strategic stepwise fashion. this strategy may appear to be less expensive but it needs regular revision to take into account the spread of virus(es). moreover, if several monoplex pcrs are needed, this then can become more expensive than multiplex pcrs. it must also be remembered that diagnosis of co-infections is more uncertain due to a smaller number of viruses tested. in conclusion, our results indicate that multiplex pcr techniques can be used in the routine etiological diagnosis of respiratory infections. the mlpa-respifinder ® 15 assay revealed that a high percentage of samples from children attending the emergency unit with aris during the autumn and winter of 2010/2011contained at least one virus, and that co-infections were very common. rsv and rv were the most prevalent pathogens, particularly in the youngest children, and co-infections were associated with more severe respiratory symptoms. the epidemiology of viruses responsible for aris had returned to its usual characteristics one year after the ah1n1pdm09 pandemic. none. none of the authors of this manuscript has any commercial or other association that might pose a conflict of interest (e.g., pharmaceutical stock ownership, consultancy). not required. rates of hospitalisation for influenza, respiratory syncytial virus and human metapneumovirus among infants and young children spectrum and frequency of illness presenting to a pediatric emergency department relative quantification of 40 nucleic acid sequences by multiplex ligationdependent probe amplification performance of a rapid molecular multiplex assay for the detection of influenza and picornaviruses pathogen chip for respiratory tract infections a prospective study of agents associated with acute respiratory infection among young american indian children comparison of multiplex pcr assays and conventional techniques for the diagnostic of respiratory virus infections in children admitted to hospital with an acute respiratory illness viral etiology of common cold in children prospective evaluation of a novel multiplex real-time pcr assay for detection of fifteen respiratory pathogens-duration of symptoms significantly affects detection rate do rhinoviruses cause pneumonia in children? lower respiratory tract infections associated with rhinovirus during infancy and increased risk of wheezing during childhood. a cohort study multiple viral respiratory pathogens in children with bronchiolitis viral pneumonia lower respiratory infections by adenovirus in children. clinical features and risk factors for bronchiolitis obliterans and mortality severity and outcome associated with human coronavirus oc43 infections among children burden of human metapneumovirus infection in young children viruses in community-acquired pneumonia in children aged less than 3 years old: high rate of viral coinfection adult hospitalizations for laboratory-positive influenza during the comparison of the luminex xtag respiratory viral panel with in-house nucleic acid amplification tests for diagnosis of respiratory virus infections epidemiology of viral respiratory tract infections in a prospective cohort of infants and toddlers attending daycare multiple versus single virus respiratory infections: viral load and clinical disease severity in hospitalized children severe lower respiratory tract infection in infants and toddlers from a nonaffluent population: viral etiology and co-detection as risk factors dual infection of infants by human metapneumovirus and human respiratory syncytial virus is strongly associated with severe bronchiolitis single versus dual respiratory virus infections in hospitalized infants: impact on clinical course of disease and interferon-gamma response the 2009 pandemic influenza a(h1n1) coincides with changes in the epidemiology of other viral pathogens causing acute respiratory tract infections in children the english text was checked by dr owen parkes. key: cord-315949-7id5mitl authors: sentilhes, anne‐charlotte; choumlivong, khamla; celhay, olivier; sisouk, thongchanh; phonekeo, darouny; vongphrachanh, phengta; brey, paul; buchy, philippe title: respiratory virus infections in hospitalized children and adults in lao pdr date: 2013-06-25 journal: influenza other respir viruses doi: 10.1111/irv.12135 sha: doc_id: 315949 cord_uid: 7id5mitl background: acute respiratory infections are an important cause of morbidity and mortality worldwide, with a major burden of disease in developing countries. the relative contribution of viruses in acute lower respiratory infections (alri) is, however, poorly documented in lao pdr. objective: the objective of this study is to investigate the etiology of alri in patients of all ages in two hospitals of laos. methods: multiplex pcr/rt‐pcr methods were used to target 18 major common respiratory viruses. between august 2009 and october 2010, samples from 292 patients presenting with alri were collected. results and conclusion: viruses were detected in 162 (55%) samples. in 48% (140/292) of the total alri cases, a single virus was detected while coinfections were observed in 8% (22/292) of the samples. the most frequent viruses were rhinovirus/enterovirus (35%), human respiratory syncytial virus (26%), and influenza viruses (13%). parainfluenza viruses were detected in 9%, adenovirus in 6%, human metapneumovirus in 4%, coronaviruses (229e, nl63, oc43, hku1) in 4%, and bocavirus in 3% of alri specimens. most viral infections occurred in patients below 5 years of age. the distribution of viruses varied according to age‐groups. no significant correlation was observed between the severity of the disease and the age of patients or the virus species. this study provides the description of viral etiology among patients presenting with alri in lao pdr. additional investigations are required to better understand the clinical role of the different viruses and their seasonality in laos. acute respiratory infections are a leading cause of morbidity and mortality worldwide. 1 they represent around 2 million deaths per year, especially in infants. 2 the burden of these infections is particularly important in developing countries. 3 during the last decade, south east asia received much attention from the international scientific community due to the emergence of respiratory viruses with pandemic potential (sars-cov, avian influenza a/ h5n1 virus). 4 respiratory infections can be caused by numerous viruses, including influenza viruses, parainfluenza viruses, human respiratory syncytial virus (hrsv), human metapneumovirus (hmpv), human coronaviruses (hcov), adenoviruses, human bocavirus, and human enteroviruses. molecular techniques have become more and more popular to detect these viruses. multiplex reverse transcription-polymerase chain reaction (rt-pcr) has been shown to be a sensitive tool and allows identification of a majority of respiratory viruses, as well as coinfections. [5] [6] [7] in lao pdr, the etiology of respiratory infections is still poorly documented. to improve the clinical management of the patients, limit unnecessary antibiotic use, and prevent opportunistic secondary infections, it appears important to develop surveillance and tools to assess the etiology of acute respiratory infections in this country. 8, 9 the purpose of this study was to describe during a limited period of time the viral etiology of acute lower respiratory infections (alri) in patients hospitalized in two lao hospitals by using a set of five multiplex rt-pcr/pcr targeting 18 common respiratory viruses. this study was part of the surveillance and investigation of epidemic situations in south-east asia (sisea) project. this project was implemented by the international network of pasteur institutes in asia to improve the surveillance and management of epidemic situations in the region. the study included children and adults hospitalized for alri. for patients below 5 years of age, alri was defined as cough or dyspnea on admission with a duration of symptoms <14 days and polypnea at more than 50 breaths/min for children aged below 1 year and more than 40 breaths/min for children between 1 and 5 years old. in infants and young children, fever is sometimes absent and was therefore not considered as an inclusion criteria. for the children between 5 and 15 years old, the inclusion criteria in addition to the cough, was fever >38°c on admission (or history of fever) with a history of symptoms of <14 days. for the adults (>15-years-old), alri definition consisted of cough and fever for <14 days and one of the following symptoms of lower respiratory infection: dyspnea, chest pain, and abnormal auscultatory findings. patients with known tuberculosis (tb), known acquired immunodeficiency such as hiv/aids, and cancer were excluded. severity was assessed according to the world health organization criteria. 10 the surveillance of alri was conducted in two laotian hospitals: setthathirath hospital in vientiane capital (between august 2009 and october 2010) and in the provincial hospital of luang prabang province (between january and june 2010). in setthathirath hospital, the patients enrolled in the study were from all ages and were recruited from three wards: pediatric, intensive care unit, and internal medicine. in luang prabang provincial hospital, only children below 5 years of age hospitalized in the pediatric ward were included. for each patient who met the alri case definition, nasopharyngeal and throat swabs were collected and immediately placed into a sterile tube containing viral transport medium (vtm). 11 the samples were transported at 4°c to the national center for laboratory and epidemiology. they were then aliquoted and stored at à80°c prior to testing. nucleic acids were extracted using qiagen viral rna mini kit (qiagen, ca, usa). for each sample, 140 ll of vtm was processed according to the manufacturer's instructions, eluted in 60 ll of qiagen ave buffer, and then stored at à80°c until testing by multiplex pcr/rt-pcr. five multiplex pcr/rt-pcr were used to screen for eighteen common respiratory viruses: influenza a (ia); influenza b (ib); influenza c (ic); hmpv; hrsv; parainfluenza viruses 1-4 (piv1-4); human rhinovirus (hrhv); enteroviruses; severe acute respiratory syndrome-associated coronavirus (sars-cov); hcovs oc43, 229e, hku1, and nl63; human bocavirus, and adenoviruses. these tests were performed as described by buecher et al. 6, 7, 12 influenza a viruses subtyping influenza a strains were subtyped according to the method developed by the two french national influenza centres (northern and southern france) and described in the who information for laboratory diagnosis of pandemic (h1n1) 2009 virus. 13, 14 clinical data hospital physicians filled out a standard case report form for each participant, including information on patient's medical history, clinical features, treatment, laboratory and radiological results, and status at the time of discharge. medical records and chest x-rays were retrospectively reviewed by an expert pulmonologist. severity was assessed according to the world health organization criteria. 10 clinical data were anonymized and entered into a database by two persons who did not have knowledge of virus identities. the protocol was approved by the national ethical committee of lao pdr and by the clinical research committee (corc) of institut pasteur in paris. samples were collected after an informed written consent was obtained from the patients or their guardians/parents (in children below 15 years of age). proportions were compared using a chi-squared or contingency table randomization test as appropriate. p-values < 0á05 were considered significant. the mann-whitney u-test was applied to compare continuous or ordinal measures. analyses were performed using stata/se version 11.1 (statacorp., college station, tx, usa) and r statistical software (r 2.8.1, r foundation for statistical computing, vienna, austria). for analysis purposes, the following pathogens were grouped together: hcov oc43, hcov hku1, hcov 229e, and hcov nl63 (hcovs); influenza a, influenza b, and influenza c viruses (influenza); parainfluenza viruses 1-4 between august 2009 and october 2010, 292 nasopharyngeal swabs were collected including 27 samples from luang prabang provincial hospital and 265 samples from setthathirath hospital (vientiane). of the 292 patients, 71% were hospitalized in pediatric ward, 24% in internal medicine department, and 5% in intensive care unit. the median duration of hospitalization was 4 days. of the study population, 145 (49á7%) were male. the median age of the patients was 2á2 years (range: 12 days-86 years) with 187 patients (64%) below 5 years of age. the different age-groups are described in figure 1 . to allow comparisons with statistical significance, the 5 age-groups were merged into two main groups: infants and children aged ≤5 years (n = 187, 64%), and adults and children above 5 years of age (n = 105, 36%). (table s1 ). among these multiple viral infections, the most frequent pathogens were rhinoviruses, hrsv, and adenoviruses. a majority of alri were observed among children aged ≤5 years (187/292). the positivity rate was also the highest (i.e., 70%) in children aged ≤5 (figure 1 ). human respiratory syncytial virus was found in patients aged from 15 days to 31 years but was more frequent in young children aged ≤5 years (p = 0á0097) ( figure 3 ). influenza viruses were detected in all age-groups (median age = 12á3 years; range: 3á6 months-66 years), but were more frequent in the age-group >5 years (p < 10 e-6). rhinoviruses were mainly detected in young patients and in few occasions in elderly (median age = 1á2 years; range: 12 days -86 years). bocavirus was exclusively detected in children below 5 years of age (range = 6 months -3á5 years). for the other viruses, there was no difference statistically significant between both the age-groups. the monthly distribution of the viral respiratory infections as well as of the rhinoviruses/enteroviruses and hrsv is shown in figure 4 . the average number of samples collected monthly was 19á5 (range: 8-32). we did not observe a clear seasonality for the alri associated with a respiratory virus. rhinovirus was detected all year round. human respiratory syncytial virus circulation seemed more important during the rainy season, between july and october. the prevalence of the other viruses was low during the study period, ranging from 0 to 5 positive samples per month for each virus. these numbers appeared too low to allow the description of seasonal patterns. however, pivs were detected only between march and august 2010 with a peak in may. coinfections were observed all year long, but with only one or two cases per month. as for pivs, there was a peak in may 2010, which was most probably only a bias corresponding to a higher number of samples collected during that month. the clinical classification of the 162 patients who tested positive is summarized in the table 1 . bronchitis and exacerbation of asthma were the most frequent clinical presentations recorded in patients who tested positive for respiratory viruses (59/162, 36%). bronchiolitis and pneumonia with or without pleurisy were identified in respec-tively 20% and 19% of the patients with viral infections. the expert pulmonologist could not review 39 clinical records, which were excluded from the clinical analysis and labeled as unspecified alri. there was no significant difference in clinical presentation between patients with single infections and those with multiple viral infections. a total of 64 viruses were detected in the 59 patients presenting with bronchitis, 52 39 viruses in the 33 patients who experienced a bronchiolitis, and 37 viruses in the 31 patients with pneumonia. in bronchiolitis, the most common viruses detected were hrsv (16 patients) and rhinovirus (15 patients). in patients presenting with bronchitis and pneumonia, all the respiratory viruses tested were detected with an approximately similar frequency. the proportion of patients from whom no virus was detected was higher in patient hospitalized for pneumonia (46%) than in those presenting with bronchitis or asthma (37%) or bronchiolitis (23%). bronchiolitis was diagnosed almost exclusively in patients aged <2 years (31 of 33), whereas pneumonia was more frequent in patients aged >5 years. three deaths occurred during the study period. for two of them, no virus was identified, and for one patient, a rhinovirus was detected. these patients were respectively 5, 18 and 60 years old. of the 162 patients for which respiratory viruses were detected, 39 (24%) presented symptoms of severity. among these, 45 viruses were identified. the most common virus observed was the rhinovirus (21), followed by hrsv (7), influenza viruses (6), and bocavirus (4). in this study, we report for the first time in lao pdr the viral etiologies in patients hospitalized for alris. we identified 186 respiratory viruses in 162 (55%) patients of all ages using 5 multiplex pcr/rt-pcr. rhinovirus and hrsv were the viruses the most frequently detected, representing 35% and 26% of the total number of viruses observed, respectively. these results are consistent with other studies conducted in the region previously. 6, 15, 16 the majority of the patients included in the study were aged <5 years (64%), and 48% were <2 years old. human respiratory syncytial virus is frequently defined as the predominant virus associated with hospitalizations for alri in children aged ≤5 years. [17] [18] [19] however, in our study, we detected more rhinoviruses (hrhvs) and enteroviruses (35%) than hrsv (26%) among the 292 patients included. even if hrhvs are typically associated with the common cold, 20 recent studies suggest that these viruses may also be associated with more severe illness, including lower respiratory disease and asthma exacerbations. 21, 22 in this study, hrhv was detected in respiratory specimens from 33% of patients with bronchitis or asthma, in 25% of patients with bronchiolitis, and in 16% of those presenting with pneumonia. rhinoviruses/enteroviruses were often implicated in coinfections (73% of all the coinfections detected). however, the clinical significance of the detection of a hrhv by a highly sensitive rt-pcr method has been questioned as table 1 . these viruses can also be detected in asymptomatic children. 23, 24 rhinoviruses and enteroviruses seem to circulate all year round, without clear seasonality. human respiratory syncytial virus was the second most common virus detected in this study with a total of 49 cases (26% of patients with a positive rt-pcr result). this virus is recognized as the leading cause of hospitalizations in children aged ≤5 years for respiratory illness in industrialized countries. [25] [26] [27] similarly to other countries, we demonstrated a substantial burden of hrsv-associated alri in lao pdr. the infants and children aged <5 years were significantly more frequently infected, and then the incidence of hrsv infections decreased with age, probably because of the development of anti-hrsv immunity which is boosted during each subsequent reinfection. [28] [29] [30] during the study period, the peak of hrsv activity occurred from june to october, which corresponds to the rainy season ( figure 4) . similar observations were also reported in neighboring countries. 17, 25, 31 the overall incidence of influenza viruses infection was relatively low (12%), and the majority of the cases detected (69á6%) were among patients older than 5 years with a median age of 12á3 years. these results are in line with those of a preliminary study on influenza-like illness in lao pdr in which the incidence of influenza was 10á4%. 32 the influenza a virus strains detected during the study period were exclusively 2009 pandemic h1n1 viruses. as expected and as reported in other studies, the serotypes 1 and 3 were the most frequent pivs detected in laos. 12, 17, 33 however, piv-4 was also identified only in four cases and accounted for 24á5% of all the pivs detected. piv-4 is usually uncommon, 17,34,35 but has been identified in severe respiratory illnesses, 5,36 and its role is probably more important than originally thought. 37, 38 the overall prevalence of hmpv was 4%, which is comparable to the results reported in greece, 39 the usa, 40 or thailand. 41 the detection rate of the human bocavirus, another recently discovered respiratory virus, was 3%. this prevalence appears to vary largely between countries: 0á4% in cambodia 6 where a similar study was conducted, 3á9% in thailand, 42 16% in vietnam, 17 and 24á5% in china. 16 bocavirus is often implicated in coinfections. 12, 19, 43 in this study, 66% of the bocavirus strains detected were observed during multiple infections. human coronaviruses were detected in 4% of the alri patients in lao pdr, which is comparable to other countries (china: 5%; vietnam: 8%, cambodia: 8%). 6, 16, 17 hcov-oc43, hcov-nl63, and hcov-229e were identified only in patients aged <4 years while the only case of hcov-hku1 infection was observed in a 75 years old patient hospitalized for pneumonia. of the 162 patients infected by a respiratory virus, we detected 13á6% of coinfections. the majority of these multiple infections were identified in patients <5 years of age (90%) and associated a rhinovirus (15/22) . as rhinoviruses were detected all year round, coinfection cases were also observed regularly each month. respiratory virus coinfections being frequent, 5, 19, 44 it demonstrates the usefulness of the multiplex rt-pcr approach, which allows the detection of the most important viruses in only few reactions while multiple infections are often undetected in viral culture or by direct immunofluorescence. nevertheless, the difficult question of the clinical significance of these multiple infections remains unanswered. in our study, we did not see any association between coinfection and severity of the disease, which is in line with other reports, 5, 19, 24, 45 but this has been subjected to much controversy. [46] [47] [48] we also did not find any significant association between any virus and disease severity. in this study, bronchitis and pneumonia were the most frequent clinical presentations observed among all the agegroups of patients hospitalized for alri. bronchiolitis was observed almost exclusively in patients <2 years of age (40/43 cases). the viruses that were most frequently detected in patients <2 years of age with bronchiolitis were hrsv (15) and hrhvs (14) , which is consistent with previous observations. [49] [50] [51] fifty-seven percent of pneumonia occurred in children below 5 years of age. a better understanding of the roles of the different viruses is of great importance as pneumonia is responsible for approximately 19% of all deaths in children aged <5 years, of which more than 70% take place in sub-saharan africa and south-east asia. 1 the two main viruses observed in pneumonia in lao pdr were hrhvs and influenza a viruses. even if lao pdr is significantly less populated than neighboring thailand, vietnam, and china, the viral etiologies observed in laotian patients hospitalized with alri demonstrate some similarities to those of other south-east asian countries. however, this study has several limitations. indeed, it was conducted in only two sites (vientiane capital and luang prabang), and the second site was included only during the last 6 months of the study. moreover, our sample size is limited, especially when we stratify by age and viral etiology. finally, it was difficult to collect sputum, particularly in young children. thus, identification of bacteria was not possible. this study aimed at determining the main viral etiologies of patients hospitalized in lao pdr with alri. rhinoviruses, hrsv, and influenza virus were the more common viruses detected in the patients. bronchitis and pneumonia accounted for the majority of the hospitalizations for alri. these data are consistent with those of the literature. this study demonstrated also the usefulness of multiplex pcr/ rt-pcr to detect viral infections and to expand our knowledge of respiratory infections in such country where the data are still sparse. although the low numbers of some viruses do not allow drawing clear conclusions and considering that bacterial infections cannot be dismissed, this study provides some important preliminary data that can be used for other more focused surveys in a larger population, for instance to better describe the seasonality of the respiratory viruses. the frequency of viral infection should be taken into account by pediatricians to avoid unnecessary use of antibiotics. additional supporting information may be found in the online version of this article: table s1 . multiple infections detected in 292 patients presenting with alri. epidemiology and etiology of childhood pneumonia estimates of world-wide distribution of child deaths from acute respiratory infections lung infection-a public health priority emerging infectious diseases in southeast asia: regional challenges to control development of three multiplex rt-pcr assays for the detection of 12 respiratory rna viruses use of a multiplex pcr/rt-pcr approach to assess the viral causes of influenza-like illnesses in cambodia during three consecutive dry seasons simultaneous detection of respiratory viruses in children with acute respiratory infection using two different multiplex reverse transcription-pcr assays the role of respiratory viral infections among children hospitalized for community-acquired pneumonia in a developing country pneumonia research to reduce childhood mortality in the developing world pocket book of hospital care for children: guidelines for the management of common illnesses with limited resources highly pathogenic influenza a(h5n1) virus survival in complex artificial aquatic biotopes the association of newly identified respiratory viruses with lower respiratory tract infections in korean children information for laboratory diagnosis of pandemic (h1n1) pandemic a(h1n1)2009 influenza virus detection by real time rt-pcr: is viral quantification useful? viral pathogens associated with acute respiratory infections in central vietnamese children molecular monitoring of causative viruses in child acute respiratory infection in endemo-epidemic situations in shanghai viral etiologies of acute respiratory infections among hospitalized vietnamese children in ho chi minh city incidence of respiratory pathogens in persons hospitalized with pneumonia in two provinces in thailand viral and atypical bacterial detection in acute respiratory infection in children under five years rhinovirus and the lower respiratory tract rhinovirus-associated hospitalizations in young children rhinovirus associated with severe lower respiratory tract infections in children human rhinovirus infections in rural thailand: epidemiological evidence for rhinovirus as both pathogen and bystander viral respiratory infections in hospitalized and community control children in alaska the burden of hospitalized lower respiratory tract infection due to respiratory syncytial virus in rural thailand the burden of respiratory syncytial virus infection in young children global burden of acute lower respiratory infections due to respiratory syncytial virus in young children: a systematic review and meta-analysis two distinct subtypes of human respiratory syncytial virus genetic variability of group a human respiratory syncytial virus strains circulating in germany from 1998 to seroprevalence of anti-rsv igg in thai children aged 6 months to 5 years a study of the genetic variability of human respiratory syncytial virus (hrsv) in cambodia reveals the existence of a new hrsv group b genotype an early report from newly established laboratory-based influenza surveillance in lao pdr parainfluenza virus type 3: seasonality and risk of infection and reinfection in young children specific viruses detected in nigerian children in association with acute respiratory disease progress in the development of human parainfluenza virus vaccines parainfluenza virus type 4 infections in pediatric patients human parainfluenza virus type 4 infection in chinese children with lower respiratory tract infections: a comparison study detection and identification of human parainfluenza viruses 1, 2, 3, and 4 in clinical samples of pediatric patients by multiplex reverse transcription-pcr contribution of human metapneumovirus to influenza-like infections in north greece population-based incidence of human metapneumovirus infection among hospitalized children human metapneumovirus in infants and young children in thailand with lower respiratory tract infections; molecular characteristics and clinical presentations human bocavirus: a novel parvovirus epidemiologically associated with pneumonia requiring hospitalization in thailand evidence of human coronavirus hku1 and human bocavirus in australian children detection of nine respiratory rna viruses using three multiplex rt-pcr assays incorporating a novel rna internal control transcript detection and typing by molecular techniques of respiratory viruses in children hospitalized for acute respiratory infection in rome, italy single versus dual respiratory virus infections in hospitalized infants respiratory viral infections detected by multiplex pcr among pediatric patients with lower respiratory tract infections seen at an urban hospital in delhi from impact of human metapneumovirus and respiratory syncytial virus co-infection in severe bronchiolitis viral infections of the lower respiratory tract: old viruses, new viruses, and the role of diagnosis respiratory syncytial virus and human rhinoviruses are the major causes of severe lower respiratory tract infections in kuwait role of rhinovirus in hospitalized infants with respiratory tract infections in spain vientiane climate guide to the average weather & temperatures this study was supported by surveillance and investigation of epidemic situations in south-east asia (sisea) project, a grant from the french agency for development (afd). we gratefully thank dr anne mornand for her precious expertise in pediatric pulmonology and review of the clinical data and dr jean-jacques bernatas for the coordination of the project. we thank the team of the virology unit at institut pasteur in cambodia for their assistance in implementing the multiplex pcr/rt-pcr techniques in lao pdr. key: cord-279257-a7d9a2w1 authors: puig, carme; sunyer, jordi; garcia‐algar, oscar; muñoz, laura; pacifici, roberta; pichini, simona; vall, oriol title: incidence and risk factors of lower respiratory tract illnesses during infancy in a mediterranean birth cohort date: 2008-07-09 journal: acta paediatr doi: 10.1111/j.1651-2227.2008.00939.x sha: doc_id: 279257 cord_uid: a7d9a2w1 aim: to investigate the incidence rate, viral respiratory agents and determinants of lower respiratory tract illnesses (lrtis) in infants younger than 1 year. methods: a total of 487 infants were recruited at birth for the asthma multicenter infant cohort study in barcelona (spain). cases of lrtis were ascertained through an active register including a home visit and viral test in nasal lavage specimens during the first year of life. cotinine in cord blood, household aeroallergens, indoor no(2) and maternal and neonatal ige were measured. other maternal and infants' characteristics were obtained from structured questionnaires. results: the incidence rate of at least one lrti was 38.7 infants per 100 persons‐years. the most frequently isolated viral agent was respiratory syncytial virus (44.7%). the risk of lrtis was higher in infants with a maternal history of asthma and in those with siblings (or = 2.4; 95% ci: 0.98–6.08 and or = 1.8; 95% ci: 1.04–3.21, respectively). the risk of lrtis was lower in infants who were breast fed for more than 12 weeks (or = 0.26; 95% ci: 0.26–0.86) and in those from a low socioeconomic class (or = 0.16; 95% ci: 0.06–0.42). conclusion: viral lrtis are frequent in infants younger than 1 year of age and there is an inter‐relationship between maternal asthma, siblings, breast feeding and socioeconomic status. acute illnesses of the respiratory tract are the most common diseases during childhood and most of them involve the upper respiratory tract. however, the incidence of lower respiratory tract illnesses (lrtis) could be considerable, as well. in developed countries, a lrti occurs in 20-39% of infants and children under 3 years of age (1) (2) (3) . moreover, lrtis not only constitute a burden on health care costs (4), but are also a possible risk factor for developing asthma and allergy during early childhood (5) and may lead to chronic obstructive pulmonary disease in late adulthood (6) . recent studies reporting incidence and risk factors for lr-tis in early infancy are based on hospitalized infants (7), on infants with a predisposition to allergy (8, 9) , on a particular type of lrti (with or without wheezing) (10) or on a causal agent (such as respiratory syncytial virus [rsv]) (11) . the aim of the present study was to determine the incidence rate of lrtis during infancy, identify viral respiratory abbreviations amics, asthma multicenter infant cohort study; der p1, dermatophagoides pteronyssinus 1; ets: environmental tobacco smoke; fel d1, felus domesticus 1; lrti, lower respiratory tract illness; no 2 , nitrogen dioxide; no-lrti, no lower respiratory tract illness; npa, nasopharyngeal aspirate; rsv, respiratory syncytial virus; ses, socioeconomic status. agents and ascertain the association of lrtis with different risk factors in a mediterranean population. the barcelona birth cohort consisted of 487 infants from the asthma multicenter infant cohort study (amics). the amics study was designed to investigate the effects of several pre-and postnatal environmental exposures on the inception of atopy and asthma, and included four cohorts in different european countries. mothers and their infants were recruited during the prenatal visit to the hospital del mar, barcelona, spain. participants were invited to join the study if they anticipated living in the city during the study period and had a telephone. the clinical research ethics committee (ceic-imas) approved the study protocol and mothers signed a written informed consent form. data collection on lrti was conducted between january 1996 and october 1999, when infants were recruited prenatally and followed up until they reached 1 year of age. data on the presence of lrti were obtained from two sources. diagnoses established by study paediatricians on enrolment, the mother was instructed to contact the local 24-h coordinating centre if her infant developed relevant symptoms lasting at least two consecutive days (runny nose, fever, dry cough, wet cough and difficulty breathing). within 24 h, a home visit was arranged and infant visited by a study paediatrician. the event was classified as no lrti (no-lrti) or lrti. lrti was further stratified according to the clinical features previously described (1) into laryngotracheobronchitis, bronchitis, bronchiolitis and pneumonia. a fifth diagnosis of non-specific lrti was made when the infant could not be classified within the other four diagnoses (these infants often only had wheezing). a new lrti was recorded if the infant was symptom free for at least 14 days between two episodes. a nasopharyngeal aspirate (npa) sample was obtained only in infants with a lrti diagnosed by the study paediatrician, instilling 2 ml of normal saline into the nasopharynx and aspirating with a sterile syringe. the npa sample was preserved at 4 • c in a sterile container for transport to the laboratory and was analysed within 4-8 h. during the follow-up, the mother was called every month by a nurse, who inquired whether the infant had been diagnosed with a lrti during the previous month by the family paediatrician without the 24-h coordinating centre being informed. the occurrence of a lrti was defined as a positive answer to the question, 'has a doctor told you that your son/daughter has laryngotracheobronchitis, bronchitis, bronchiolitis, pneumonia or wheezing, since we spoke with you last?' a negative answer was registered as no-lrti. npa samples were analysed at a local virology research laboratory (hospital vall d'hebron, barcelona) and were classified as positive when a respiratory virus was identified by at least one of the following procedures: • rapid detection for rsv and coronavirus with direct fluorescent monoclonal antibodies (bartels immunodiagnostics supplies, inc., bellevue, wa, usa). • virus culture in human epidermal carcinoma (hep-2 line) and madin-darby canine kidney (mdck) cells and indirect fluorescent antibody identification with a pool of monoclonal respiratory viral antibodies for rsv, adenovirus, influenza virus types a and b, and parainfluenza types 1, 2 and 3 (bartels immunodiagnostics supplies, inc.). • conventional tissue culture in hep-2, mdck and the llc-mk2 line of rhesus monkey kidney cells for examination of the characteristic cytophatic effect. at the first prenatal care visit to the hospital, usually during the third trimester of pregnancy, a detailed questionnaire including information on smoking habits, sociodemographic characteristics and parental history of asthma was completed. social class was defined by paternal occupation using the uk registrar general's 1990 classification, which groups people with similar levels of occupational skill. upon delivery, general information (gestational age, gender, weight, length and head circumference at birth) was recorded from medical files. one year after birth, a total of 381 (82.5%) mothers were contacted for a structured telephonic interview to record the approximate frequency of respiratory symptoms such as wheezing, cough and noisy breathing in the preceding months caused by airway secretions, as well as information on the duration of breast feeding (regardless of whether other foods were given), number of siblings, day care attendance and parental smoking. specific ige to dermatophagoides pteronyssinus (der p1) were determined in maternal blood samples (62.2%) obtained in the third trimester of pregnancy by the pharmacia cap system rast-feia (pharmacia, freiburg, germany) and total ige levels in umbilical cord blood (85.4%) were determined by pharmacia cap system low-range feia (pharmacia). maternal atopy was defined by detectable serum levels (>0.35 ui/ml) of specific ige to der p1 and elevated cord blood total ige levels were defined at concentrations of >0.35 ui/ml. umbilical cord serum (75.1%) was analysed to determine cotinine concentration by radioimmunoassay as a biomarker of exposure to tobacco smoke during pregnancy. a cut-off of 1 ng/ml was used for cord serum cotinine to distinguish exposed from non-exposed mothers (12) . dust samples and passive no 2 filter badges (toyo roshi no 2 filter badges, tokyo, japan) were collected 6 months after birth (78%). concentrations of household dust mite (der p1) and cat (felus domesticus [fel d1]) allergens were determined using an enzyme-linked immunoabsorbent assay according to standard procedures described elsewhere (13) and grouped in categories with potential relevance for sensitization to der p1 and fel d1 (>2 μg/g and >1 μg/g of dust, respectively) as reported in the international literature (14) . indoor no 2 concentration was measured by colorimetric reaction and spectrophotometric analysis described previously (15) . the incidence rate was estimated by incidence density and calculated as i/pt, where i is the number of infants with at least one lrti during the observation period, and pt is the amount of population time observed in the population during this period. chi-square tests were used to assess differences between infants with no-lrtis and lrtis in maternal, neonatal and postnatal characteristics. statistical significance was set at p < 0.05. multivariate logistic regression was used to evaluate associations between predictor variables and lrtis. variables were chosen for the multivariable model if they entered at a level of p ≤ 0.1; odds ratios (or) with 95% confidence intervals (95% ci) were estimated. all analyses were performed using the stata 7.0 statistical package (statacorp, college station, tx, usa). more than half of the newborns (52.7%) were male. the mean maternal age was 29 years (se: 5.4). the maternal ethnic distribution (74.9% spanish vs. 25.1% non-spanish) reflected the demographics of the population attending the hospital del mar. socioeconomic status (ses) measured by paternal profession was broadly distributed as follows: 18.5% of fathers were in professional, managerial or technical occupations (classes i and ii); 21.5% were in skilled nonmanual occupations (class iii, nm); 41.5% were in skilled manual (class iii, m) occupations and 18.5% were in partly skilled or unskilled occupations (classes iv and v). a total of 149 infants had at least one lrti during the first year of life (fig. 1 ). of these, 106 infants (71.1%) had one lrti and 43 (28.8%) had two or more other episodes of lrti. the study paediatrician directly identified at least one lrti in 99 infants (66.4%), and the monthly calls by the nurse identified retrospectively at least one lrti in 50 infants (33.6%). the incidence rate of at least one lrti was of 38.7 infants per 100 persons-years during the followup. the most frequent clinical diagnosis was bronchitis (table 1) . out of the 99 infants with an npa sample, one virus was detected in 43 infants (43.4%) and two viruses were detected in the same npa sample in 4 infants (4.0%). the most commonly detected virus was rsv in 21 infants (44.7%). adenovirus was detected in seven (14.9%), enterovirus in six (12.8%), influenza a in six (12.8%) and cytomegalovirus in five (10.6%). other viruses identified in these infants were coronavirus in three (6.4%), parainfluenza in three (6.4%) and influenza b in two (4.3%). the viral isolation rate in the 136 npa samples was 50%. the viruses recovered from different respiratory syndromes are shown in table s1 (in supplementary material online). pneumonia and bronchiolitis had a viral isolation rate of approximately 60%, rsv being the most commonly isolated virus. laryngotracheobronchitis was caused mainly by the parainfluenza virus. non-specific lrtis were mainly caused by other viruses. when examining the results of the structured telephonic interviews, no significant differences were found in the prevalence of respiratory symptoms reported by the mothers among infants with lrtis and without lrtis irrespective of the health professional making the diagnosis. there were no differences between the two groups (lrti and no-lrti) with respect to the maternal age or maternal birth country (see table s2 in supplementary material online). lrtis were less frequent in social classes iv and v, comprising skilled and unskilled occupations. a maternal history of asthma was more frequent in infants with lrti. the prevalence of mothers who smoked during pregnancy was very high not only in the no-lrti group (50%) but also in the lrti group (44.2%). indeed, more than 87% of the total number of infants had cotinine levels in cord blood above 1 ng/ml, suggesting exposure to tobacco smoke during fetal life. der p1 and fel d1 levels higher than the threshold for sensitization to allergens (>2 μg/g and >1 μg/g of dust, respectively) were less frequent among the lrti group. no differences between groups were detected in indoor no 2 levels. infants who were breast fed for more than 12 weeks showed a lower frequency of lrti. the presence of one or more siblings was more frequent in the lrti group; also, more children in this group attended day care centres compared to the no-lrti group; however, in this cohort, the difference did not reach statistical significance. in the multivariate model (see table s3 in supplementary material online), the risk of lrtis was 2.4 times higher in infants with a maternal history of asthma (or = 2.44; 95% ci: 0.98-6.08) and was 1.8 times higher (95% ci 1.04-3.21) in infants with siblings. the risk of lrtis was lower among infants who were breast fed for more than 12 weeks (or = 0.26; 95% ci: 0.26-0.86) and among those in social classes iv-v (or = 0.16; 95% ci 0.06-0.42). we present the first study in a mediterranean area of infant outpatients with a prospective record of lrtis, which includes an ad hoc register and laboratory diagnosis, and a wide record of possible risk factors. maternal asthma as a risk factor for the development of lrti in infants was found to be significant, as was the number of siblings. breast-fed infants and those from a low social class had a lower risk of lrtis. lrtis and viral agents in our study, 38.7% infants had at least one lrti during the first year of life while the incidence of lrtis reported in other community-based longitudinal studies ranges from 20 to 32.9% (1) (2) (3) . the higher percentage observed in this study may be the result of using both active follow-up of direct diagnosis and self-reporting. the viral isolation rate in the npa specimens (50%) was higher than those reported in other community-based studies (26-42.4%) (1, 3, 16) , probably due to immediate specimen collection. however, the rate was lower than those in hospital-based studies (50-66.2%.) (17) , probably because we had antigen tests only for rsv and coronaviruses and no pcr was available. when both antigen tests and pcr are used, the proportion of virus-positive cases is almost 100%. viral agents in the present cohort were identified in almost 60% of bronchiolitis and pneumonia episodes while only 47.1% of bronchitis episodes were positive. virus-negative illnesses could be caused by other viruses not isolated in this study (i.e. the recently discovered metapneumovirus (18) or rhinoviruses (19) detected by the newer pcr techniques). since both antigen tests for all respiratory viruses and pcr have been introduced, the proportion of virus identified has notably increased. at the follow-up of our cohort, new data about wheezing illnesses and its risk factors, as rsv, or other virus infections or environmental tobacco smoke exposure will be included. the association between viral isolates and clinical diagnosis in this study was not statistically significant but the distribution was similar to that in other publications (1, 2, (20) (21) (22) (23) . our finding that a maternal history of asthma was associated with lrtis in infants is in agreement with data from other birth cohort (24) . over-reporting of asthmatic mothers is not a likely explanation, because the association in the current study was evaluated with medical diagnosis and not symptoms. similar to other studies (21) we found that the presence of siblings was a significant risk factor for lrtis due to increased exposure to viral agents. however, unlike other authors (25), we found no significant differences between infants with lrtis and no-lrtis with respect to the frequency of day care attendance. this may be due to the low attendance at day care before the first year of age in our population (nearly 20% vs. more than 50% in the abovementioned study). of note, nor household dust mite (der p1) or cat (fel d1) allergens higher than the threshold for sensitization were found to be associated with lrtis. in contrast, it can be hypothesized that high der p1 and fel d1 levels were less frequent among the lrti group due to more frequent cleaning in the houses of these infants, whose families were those with higher ses. breast feeding for more than 12 weeks was significantly associated with protection against lrtis in our study, which is in agreement with other birth cohort studies (26) suggesting that specific nutritional, immunoregulatory and immunomodulatory factors in maternal milk may promote maturation of infant immune competence (27) . our finding that the prevalence of lrti was lower in infants with low ses than in those with high ses was unexpected. these results could be explained if families with low ses were less likely to call us and therefore would have diminished the frequency due to misclassification. however, the surveillance project designed a monthly call to reduce this effect. studies describing the inverse relationship between ses and lrtis provide limited information because only differences in mortality or hospitalization rates were examined (28) . recently, it has been suggested that the relationship between ses and physical health may not be constant across ages. in a cross-sectional interview about acute respiratory conditions in infants and children aged 0-18, a reverse ses gradient was found in early years (low seslow frequency of respiratory illnesses), whereas an expected ses gradient appeared during adolescence (low ses -high frequency of respiratory illnesses) (29) . interestingly, in our study cohort, no association between exposure to tobacco smoke and lrtis was demonstrated, just the fact that nearly all infants were prenatally and postnatally exposed. indeed, cord serum cotinine concentrations were higher than those in other european birth cohorts (12, 30) and the percentage of self-reported postnatal parental smoke was extremely high. in conclusion, our study highlights important risk factors such as maternal asthma, the number of siblings and ses for the development of lrtis in infants from a mediterranean area. acute lower respiratory tract infections in nonhospitalized children the tucson children's respiratory study: ii. lower respiratory tract illness in the first year of life prospective population-based study of viral lower respiratory tract infections in children under 3 years of age (the pride study) economic impact of communityacquired and nosocomial lower respiratory tract infections in young children in germany asthma and immunoglobulin e antibodies after respiratory syncitial virus bronchiolitis: a prospective cohort study with matched controls do lower respiratory tract infections in early childhood cause chronic obstructive pulmonary disease? persistence of rhinovirus and enterovirus rna after acute respiratory illness in children rhinovirus illnesses during infancy predict subsequent childhood wheezing role of respiratory viruses in acute upper and lower respiratory tract illness in the first year of life: a birth cohort study a parental history of asthma is a risk factor for wheezing and nonwheezing respiratory illnesses in infants younger than 18 months of age prevalence of respiratory syncytial virus infection in italian infants hospitalized for acute lower respiratory tract infections and association between respiratory syncytial virus infection risk factors and disease severity cord serum cotinine as a biomarker of foetal exposure to cigarette smoke at the end of pregnancy domestic aeroallergen levels in barcelona and menorca (spain) the relevance of allergen exposure to the development of asthma in childhood a badge-type personal sampler for measurements of personal exposure to no 2 and no in ambient air surveillance of respiratory viral infections among pediatric outpatients in northern taiwan viral etiology of lower respiratory tract infections in hospitalized children in mexico a newly discovered human pneumovirus isolated from young children with respiratory tract disease frequency of detection of picornaviruses and seven other respiratory pathogens in infants association of rhinovirus infection with increased disease severity in acute bronchiolitis nasal swabs versus nasopharyngeal aspirate for isolation of respiratory viruses respiratory picornaviruses and respiratory syncytial virus as causative agents of acute expiratory wheezing in children vial respiratory disease in children: classification, etiology, epidemiology and risk factors respiratory infections in infants: interaction of parental allergy, child care and siblings (the piama study) day care attendance in the first year of life and illnesses of the upper and lower respiratory tract in children with a familial history of atopy breast feeding and respiratory morbidity in infancy: a birth cohort study the role of breast-feeding in the development of allergies and asthma effects of socioeconomic status on presentation with acute lower respiratory tract disease in children in salvador, northeast brazil socioeconomic status and health: do gradients differ within childhood and adolescence? fetal exposure to tobacco smoke is common the asthma multicenter infants cohort study received the following funding: fis 95/0314, fis 96/0799, fis 00/0021, fis 03/0296, istituto superiore di sanitá, cirirt-1999sgr 00241, colt foundation. the following supplementary material is available for this article: please note: blackwell publishing is not responsible for the content or functionality of any supplementary materials supplied by the authors. any queries (other than missing material) should be directed to the corresponding author for the article. key: cord-274127-12x5cc8i authors: jeong, ji hun; kim, kyung hee; jeong, sung hwan; park, jeong woong; lee, sang min; seo, yiel hea title: comparison of sputum and nasopharyngeal swabs for detection of respiratory viruses date: 2014-05-06 journal: j med virol doi: 10.1002/jmv.23937 sha: doc_id: 274127 cord_uid: 12x5cc8i diagnostic tests for respiratory viral infections use traditionally either nasopharyngeal washes or swabs. sputum is representative of the lower respiratory tract but is used rarely for viral testing. the aim of this study was to compare the detection rates of respiratory viruses from nasopharyngeal swabs and sputum using a multiplex real‐time reverse transcription‐polymerase chain reaction (rt‐pcr). adults who were admitted or presented to the clinics of gil medical center with acute respiratory symptoms were recruited from 1 november 2012 to 31 march 2013. paired specimens of nasopharyngeal swabs and sputum were obtained from 154 subjects, and rna was extracted and tested for 16 different respiratory viruses using the anyplex ii rv16 detection kit (seegene, seoul, korea). the positive rate was 53% (81/154) for nasopharyngeal swabs and 68% (105/154) for sputum (p < 0.001). one hundred thirty‐four viruses were identified for 107 illnesses. influenza a virus, rsv a, hrv, coronavirus oc43, and adenovirus were detected more frequently in sputum samples than in nasopharyngeal swabs (p < 0.001). importantly, 12 of 44 (27%) influenza a infections and 11 of 27 (41%) rsv infections were positive in only sputum samples. the detection rates of respiratory viruses from sputum samples were significantly higher than those from nasopharyngeal swabs in adults using real‐time multiplex rt‐pcr. these findings suggest that sputum would benefit for the detection of respiratory viruses by nucleic acid amplification tests (naats) in patients who produce sputum. further studies are needed to establish standardized rna extraction methods from sputum samples. j. med. virol. 86:2122–2127, 2014. © 2014 wiley periodicals, inc. respiratory viral infections, ranging from mild upper respiratory tract infections to more severe lower respiratory infections, are the major causes of morbidity, and disease severity can be affected by host-and viral-predisposing factors. the clinical impacts of respiratory viruses increase in young children, the elderly, immunocompromised hosts, and patients with underlying diseases [monto, 2002] . rapid identification of respiratory viral infection is critical to avoid the use of unnecessary antibiotics, the use of appropriate antiviral agents, minimize the risk of nosocomial transmission, and reduce the overall costs of patient management [pérez-ruiz et al., 2012] . the diagnosis of respiratory viral infections is performed traditionally using virus culture or direct antigen assays [storch, 2000] . culture, which is often considered the gold standard, is labor-intensive and time-consuming. antigenic detection assays are simple but sometimes insufficiently sensitive. molecular techniques have been used currently for respiratory virus detection with improved sensitivity, and multiplex real-time reverse transcription-polymerase chain reaction (rt-pcr) methods can detect simultaneously a number of viruses in a single assay. sputum is used rarely for traditional viral tests due to its viscous nature, which makes sample processing difficult. however, with the advent of molecular methods, sputum has been introduced for the diagnosis of respiratory viral infection. the aim of this study was to compare the detection rates of respiratory viruses in paired nasopharyngeal swabs and sputum samples from adult patients with respiratory symptoms using multiplex real-time rt-pcr. adults over 19 years of age who were admitted or presented to the clinics of gil medical center with signs and symptoms of acute respiratory illness defined as cough, rhinorrhoea, nasal congestion, dyspnoea, sputum, or fever were recruited from 1 november 2012 to 31 march 2013. paired specimens of nasopharyngeal swabs and sputum were obtained from 154 subjects. each patient provided written informed consent, and the institutional review boards of the gil medical center approved the study. at enrolment, clinical, radiological, and laboratory information was collected. the sputum samples were expectorated spontaneously into sterile containers and delivered to the laboratory within 2 hr. the sputum samples were then diluted with an equal volume of phosphate buffered saline (pbs) and mixed by vortexing for 1 to 5 min, depending on the sputum viscosity. if the rt-pcr result was invalid, a retest was performed using a specimen diluted with twice the initial volume of pbs. nasopharyngeal swabs were obtained from each patient by physicians using flocked swabs (copan diagnostics, brescia, italy) and were transported in 3.0 ml of universal transport medium (utm; copan diagnostics). prospectively collected sputum and nasopharyngeal swab samples were tested simultaneously. rna was extracted from 300 ml of samples with 10 ml of bacteriophage ms2 as an internal control using the geneall ribospin kit (geneall biotechnology, seoul, korea). the internal control was added to each specimen as an exogenous control to check the entire process from nucleic acid extraction to rt-pcr. the final elution volume of each sample was 50 ml. an automated protocol for extraction, rt-pcr, and pcr setup was implemented using the nimbus (hamilton robotics, reno, nv) automated liquid handling workstation to maximize the workflow and accuracy. multiplex real-time rt-pcr was performed using the anyplex ii rv 16 detection kit (seegene, seoul, korea) according to the manufacturer's instructions. sputum samples and nasopharyngeal specimens from each patient were tested in parallel for the following 16 respiratory viruses: human bocavirus (hbov), human enterovirus (hev), influenza virus a and b, parainfluenza virus 1, 2, 3, and 4 (piv 1, piv 2, piv 3, and piv 4), rsv a and b (rsv a and rsv b), adenovirus (adv), metapneumovirus (mpv), coronavirus oc43 (oc43), coronavirus 229e (229e), coronavirus nl63 (nl63), and human rhinovirus a/b/c (hrv). the overall positive rates of viruses between nasopharyngeal swabs and sputum samples were compared using mcnemar's test. the clinical characteristics of patients and diagnostic yields for detection of any virus between two specimens were compared using the chi-square test or fisher's exact test. statistical significance was set at a p value of <0.05. agreement of the results between nasopharyngeal swabs and sputum specimens was assessed using kappa statistics (cohen's kappa coefficient (k): [landis and koch, 1977] . all statistical analyses were performed using spss (version 17.0; spss, inc., an ibm company, chicago, il). paired nasopharyngeal swab and sputum samples were collected from 154 patients, and the clinical characteristics of the 154 patients are shown in table i . the patients included 47 males (31%) and 107 females (69%), with a median age of 52 years (interquartile range, 35-62 years). approximately 41% of the patients had an underlying chronic pulmonary disease, and patients had been troubled with respiratory symptoms for a median of 5 days prior to sampling (table i) . a respiratory viral infection was identified in 107 (69%) of 154 patients (table ii) , and the positive rate was 53% (81/154) for nasopharyngeal swab and 68% (105/154) for sputum. sputum showed a significantly higher positive rate than nasopharyngeal swabs in detecting respiratory viruses (p < 0.001), and viral coinfections were found in nasopharyngeal swabs and sputum (table iii) . in nasopharyngeal swabs, a single virus was identified in 76 cases (94%), two viruses in four cases (5%), and three viruses in one case (1%). in sputum, a single virus was identified in 85 cases (81%), two viruses in 17 cases (16%), and three viruses in three cases (3%) ( table iii) . the overall distribution of the detected respiratory viruses is shown in table iv . one hundred thirtyfour viruses were identified in 107 illnesses. influenza a virus was the most common, followed by rsv a, hrv, and oc43. the diagnostic yields for detection of any virus were 87 (65%) of 134 for nasopharyngeal swabs and 128 (96%) of 134 for sputum. the distribution of positive results ranged from 46% to 77% of nasopharyngeal swabs tested and from 91% to 100% of sputum tested. influenza a virus, rsv a, hrv, oc43, and adv were detected more often in sputum samples than in nasopharyngeal swab (p < 0.001). other viruses numbered too few for statistical test-ing. importantly, 12 of 44 (27%) influenza a infections, 11 of 27 (41%) rsv a infection, 5 of 22 (23%) hrv infections, 5 of 13 (38%) oc43 infections, and 3 of 11 (27%) adv were positive in only sputum samples. substantial agreement between nasopharyngeal swab and sputum was observed for influenza a virus, rsv a, and oc43. hrv had nearly perfect agreement, but adv and mpv had a poor agreement (table iv) . clinical characteristics of patients with positive nasopharyngeal swab rt-pcr results were not significantly different from those of patients with only positive sputum rt-pcr results (table v) . adequate specimen collection is important for the diagnosis of respiratory viral infections. a variety of upper respiratory samples, including nasal aspirates, nasal swabs, nasopharyngeal swabs, and oropharyngeal swabs, are used commonly for the detection of respiratory viruses. early studies suggested that nasal aspirates were superior to swab specimens [lieberman et al., 2009] ; however, this might not be true for all respiratory viruses and all detection methods. in addition, several studies have shown that respiratory viruses are an increasingly recognized cause of lower respiratory tract infections. in those cases, lower respiratory specimens should be collected for the detection of respiratory viruses. sputum is the representative of lower respiratory secretions and is the most widely accepted specimen for the diagnosis of bacterial respiratory infection. however, it is used rarely for the detection of respiratory viruses using traditional methods such as the viral antigen test or viral culture. with nucleic acid amplification tests (naats), sputum samples have been used for the detection of respiratory pathogens. currently, few data comparing the diagnostic yields of nasopharyngeal swabs and sputum samples for respiratory virus detection have been published. the present study found that the overall detection rate from sputum samples in adults was significantly higher than from nasopharyngeal swabs using multiplex real-time rt-pcr. this finding is consistent with the result of a previous study that sputum samples showed higher diagnostic yields than nose-throat swabs in adults using rt-pcr [falsey et al., 2012] . the detection rates of respiratory virus from sputum and nasopharyngeal swabs were 68% and 53%, respectively. generally, it is hard to compare detection rates of respiratory viruses because of differences in testing methods, specimen types (upper or lower airways, swabs or washes, nose or throat, flocked or regular swabs), and types of transport media. our results are still relatively high compared with those of other reports [lieberman et al., 2009; falsey et al., 2012; yu et al., 2012] . these differences can be explained by several factors. first, the anyplex ii rv16 detection kit is a multiplex real-time rt-pcr assay that can detect simultaneously 16 different types of viruses. this kit offers higher sensitivity in the detection of traditionally diagnosed respiratory viruses in addition to the ability to detect newly recognized viruses, such as hbov, piv 1, and hrv c. second, nasopharyngeal samplings were obtained using flocked swabs in the present study, and it is known that flocked swabs can collect significantly more epithelial cells, thus providing higher positive rates than conventional swabs [loens et al., 2009 ]. third, this study was performed during an influenza a (h1n1) outbreak season in korea. influenza or rsv respiratory infection may manifest as severe lower respiratory tract disease in highrisk patients, and some investigators reported falsenegative results in traditional upper airway samples from patients with respiratory viral infection [bogoch et al., 2013] . a prior study compared the yields of paired nose-throat swabs and endotracheal aspirates [roa et al., 2012] . the current study also found that 32 of 44 (73%) influenza cases were detected in nasopharyngeal swabs, and 43 of 44 (98%) cases were detected in sputum samples. similarly, 11 of 27 (41%) rsv a infections were positive in only sputum samples. these findings suggest that traditional nasopharyngeal diagnostic techniques can miss cases of lower respiratory tract infections. multiple respiratory viruses have been detected in 10-30% of patients with positive viral detection, with higher rates in young children [drews et al., 2008; kim et al., 2009; gharabaghi et al., 2011; zhang et al., 2012] . in this study, co-infections in sputum samples were more common than those in nasopharyngeal swabs (19% vs. 6%). one unanticipated finding was that influenza a and rsv a were found frequently in mixed infections. these results are contrary to other reports that hbov and coronavirus were most commonly associated with multiple-agent infections, while influenza virus and rsv were least often detected in the presence of another virus [gharabaghi et al., 2011] . these differences can be explained, in part, by the differences in specimen types and applied methods. the clinical significance of co-infections is still unknown, and further evaluation is required. some problems are encountered when handling sputum for naats. first, the highly viscous nature of sputum can make nucleic acid extraction difficult. therefore, pre-treatment of sputum samples is needed, but no standardized pre-treatment procedure is available for viral detection. dithiothreitol or nalc (n-acetyl-l-cysteine)-naoh, which are used generally for the digestion of sputum in mycobacterial laboratories, may cause substantial loss of rna due to the repetitive washing and pipetting steps in the procedures [desjardin et al., 1996; xiang et al., 2001] . in this study, sputum samples were diluted with pbs and mixed by vortexing, and the volume and time depended on the sputum viscosity. this procedure was sufficient for sputum homogenization. another problem is that naat inhibitors in respiratory specimens may affect the sensitivity of the assay or lead to false-negative results. inhibition of the amplification of the internal control was noted in 15% of the sputum samples, which is a high proportion compared with that of nasopharyngeal swabs (3%). a widely applied method for the removal of pcr inhibitors is sample dilution, which may lead to decreased inhibitors. in the present study, samples that showed invalid pcr results were diluted with twice the initial volume of pbs and re-extracted manually. in the author's experience, thoroughly mixing and homogenization of sputum samples might be important to obtain valid results. however, further evaluations of nucleic acid extraction methods in automated systems for sputum samples are needed. although sputum is more sensitive than nasopharyngeal swabs for respiratory viral detection, the use of sputum can be limited because not all patients with respiratory infections produce sputum, particularly elderly patients. therefore, if a sputum sample is available, it might be the most reliable specimen for respiratory viral detection by naats. this study has a limitation. the sputum samples were not screened microscopically to assess sputum quality. therefore, contamination of sputum samples by upper respiratory secretions cannot be ruled out. in conclusion, the detection rates of respiratory viruses from sputum samples are significantly higher than those from nasopharyngeal swabs in adults using multiple real-time rt-pcr. however, the use of sputum is limited to patients who can produce sputum. these findings suggest that sputum would benefit for the detection of respiratory viruses by naats in patients who produce sputum. further studies are needed to establish standardized rna extraction methods from sputum samples. diagnosis of influenza from lower respiratory tract sampling after negative upper respiratory tract sampling alkaline decontamination of sputum specimens adversely affects stability of mycobacterial mrna use of the seeplex rv detection kit for surveillance of respiratory viral outbreaks in yield of sputum for viral detection by reverse transcriptase pcr in adults hospitalized with respiratory illness evaluation of multiple commercial molecular and conventional diagnostic assays for the detection of respiratory viruses in children rapid detection and identification of 12 respiratory viruses using a dual priming oligonucleotide system-based multiplex pcr assay the measurement of observer agreement for categorical data identification of respiratory viruses in adults: nasopharyngeal versus oropharyngeal sampling optimal sampling sites and methods for detection of pathogens possibly causing community-acquired lower respiratory tract infections epidemiology of viral respiratory infections laboratory detection of respiratory viruses by automated techniques diagnosis of influenza in intensive care units: lower respiratory tract samples are better than nose-throat swabs diagnostic virology comparison of different methods of total rna extraction for viral detection in sputum etiology and clinical characterization of respiratory virus infections in adult patients attending an emergency department in beijing high incidence of multiple viral infections identified in upper respiratory tract infected children under three years of age in we thank seegene company for providing us with anyplextm ii rv16. key: cord-310508-zgqbfmzl authors: alavi-moghaddam, mostafa title: a novel coronavirus outbreak from wuhan city in china, rapid need for emergency departments preparedness and response; a letter to editor date: 2020-02-02 journal: arch acad emerg med doi: nan sha: doc_id: 310508 cord_uid: zgqbfmzl nan on 31 december 2019, chinese authorities reported the increase in incidence of severe pneumonia in wuhan city, hubei province of china. one week later, on january 7th, they confirmed that they had identified a new coronavirus, which is a family of microrna respiratory viruses including the common cold, and viruses such as severe acute respiratory syndrome (sars) and middle east respiratory syndrome (mers). this new virus was temporarily named "2019-ncov". wuhan city is a major international transport hub. this report to world health organization (who), raised global public health concern because this is the third coronavirus âȃşassociated acute respiratory illness outbreak. currently, up to the date of submitting this letter, 4593 cases of 2019-ncov infections have been confirmed globally, both in china (4537 have been confirmed, 976 of them presented with severe disease and 106 died) and outside of china (56 confirmed in 14 countries.). who risk assessment of 2019-ncov infection is very high in china and high in other countries (1). although 2019-ncov has not been included in the who blueprint list of priority diseases yet, mers cov, and sars -cov, which are already included in this list, are both coronaviruses that have led to global outbreaks in 2003 and 2012, respectively. the specific source and the exact primary mode of transmission of 2019-ncov to humans remain unknown. the clinical features and laboratory and radiological abnormalities with 2019-ncov infections are not specific and are similar to other respiratory tract infections. adults and pediatrics who ac-quire a 2019-ncov infection can show a spectrum of respiratory illness severity, from asymptomatic to mild, moderate or severe disease. the severe disease manifests as severe acute respiratory infection (sari) or severe pneumonia, acute respiratory distress syndrome (ards), sepsis and septic shock. patients with pre-existing medical comorbidities develop a more severe disease and have higher mortality rates compared to patients who do not have any comorbidity. clinical care of patients with suspected 2019-ncov should focus on early recognition, immediate isolation (separation), implementation of appropriate infection prevention and control (ipc) measures and provision optimized supportive care. at the triage of an emergency room, early recognition of suspected patients allows for timely initiation of ipc. 2019-ncov should be considered as a possible etiology of influenza like illness (ili) under certain situations according to case definitions of who (2) . both the health care worker (hcw) and the suspected case of acute respiratory illness (ali) should wear a medical mask and the patient should better be directed to a separate area, an isolation room if available. otherwise, keep a distance of at least one meter between suspected patients and other patients. instruct all suspected patients to cover their nose and mouth during coughing or sneezing with tissue or flexed elbows for protecting others. those with mild or moderate clinical presentations of the 2019-ncov infection may not require hospitalization, unless there is concern of rapid deterioration. all patients discharged to go home directly from fast track in emergency room should be instructed to consider ipc measures and to return hospital if their symptoms worsen (3). patients with severe illness, who are admitted to the emergency ward, should be transferred to the floor and if available to the icu ward as soon as possible. as long as they stay in emergency ward, they should be placed in single rooms or grouped together with those who have the same etiological or clinical diagnosis. limit patient movement within the center and ensure that patients wear medical masks when outside their rooms. hcw should perform hand hygiene after contact with respiratory secretions. droplet and contact precautions prevent direct or indirect transmission of the disease from contact with contaminated surfaces or equipment. hcw should use personal protective equipment (ppe) including medical mask, eye protection, gloves and gown, when entering the room and remove ppe when leaving. if equipment needs to be shared among patients, they should be cleaned and disinfected after each patient's use. hcw should apply airborne precautions when performing an aerosol generating procedure (i.e. open suctioning of respiratory tract, intubation, bronchoscopy, cardiopulmonary resuscitation) (4). hcw should immediately provide supplemental oxygen therapy for patients with sari and respiratory distress, hypoxemia or shock. oxygen therapy flow rate should be aimed at spo2>=90%, spo2>=92-95% and spo2>=94%, in non-pregnant, pregnant and children, respectively. hcw should recognize severe hypoxemic respiratory failure when a patient with respiratory distress is failing standard oxygen therapy. high-flow nasal oxygen (hfno) or non-invasive ventilation (niv) should be used in selected patients with hypoxemic respiratory failure. hypoxemic respiratory failure due to ards among these patients commonly results from intrapulmonary ventilation-perfusion mismatch or shunt and usually requires mechanical ventilation. thus, rapid sequence intubation should be performed using airborne precautions. implementation of mechanical ventilation using lower tidal volumes (4-8 ml/kg predicted body weight) and higher positive end-expiratory pressure (peep) is suggested. patients with sari should be treated cautiously with intravenous fluids when there is no evidence of shock, because aggressive fluid resuscitation may worsen oxygenation. for resuscitation of septic shock in adults, at least 30 ml/kg of isotonic crystalloid should be infused in the first 3 hours of shock identification and in children rapid bolus of 20 ml/kg as loading dose and up to 40-60 ml/kg of isotonic crystalloid infusion in the first hour of shock identification is needed. vasopressor should be administered when shock persists during or after fluid resuscitation. if signs of poor perfusion persist despite reaching mean arterial pressure (map) target (i.e. >65 mmhg) with fluids and vasopressor, consider administering an inotrope such as dobutamine. empiric antimicrobials should be initiated within one hour of identification of sepsis to treat all likely pathogens causing sari. empiric antibiotic treatment should be based on the clinical diagnosis of severe pneumonia or sepsis, local epidemi-ology and susceptibility data as well as treatment guidelines. if influenza is also a concern and there is a local circulation of influenza virus, a neuraminidase inhibitor should be adjoined to empiric therapy. empiric antibiotic therapy should be de-escalated on the basis of microbiology results and clinical judgment. systemic corticosteroids should not be routinely adding to therapy unless indicated for another reason. collection of clinical specimens for laboratory diagnosis is suggested in early outbreak period and after that it is only advised for investigational purposes. if laboratory diagnosis is considered, serology is recommended only when rt-pcr is not available. otherwise, hcw should collect specimens from both the upper respiratory tract and lower respiratory tract for testing 2019-ncov via rt-pcr (5). at the time being, emergency preparedness and response for providing appropriate care to the patients suspected to coronavirus-associated acute respiratory illness (abovementioned plans) should be developed and implemented in the emergency departments, as the frontline of treating human infections of 2019-ncov in the hospitals. none. none. organization wh. novel coronavirus (2019-ncov) situation report-9 surveillance case definitions for human infection with novel coronavirus ( ncov): interim guidance v1 novel coronavirus (2019-ncov) advice for the public: interim guidance. world health organization infection prevention and control during health care when novel coronavirus ( ncov) infection is suspected: interim guidance clinical management of severe acute respiratory infection when novel coronavirus (2019-ncov) infection is suspected: interim guidance key: cord-272878-6f0q661e authors: schnepf, nathalie; resche-rigon, matthieu; chaillon, antoine; scemla, anne; gras, guillaume; semoun, oren; taboulet, pierre; molina, jean-michel; simon, françois; goudeau, alain; legoff, jérôme title: high burden of non-influenza viruses in influenza-like illness in the early weeks of h1n1v epidemic in france date: 2011-08-17 journal: plos one doi: 10.1371/journal.pone.0023514 sha: doc_id: 272878 cord_uid: 6f0q661e background: influenza-like illness (ili) may be caused by a variety of pathogens. clinical observations are of little help to recognise myxovirus infection and implement appropriate prevention measures. the limited use of molecular tools underestimates the role of other common pathogens. objectives: during the early weeks of the 2009–2010 flu pandemic, a clinical and virological survey was conducted in adult and paediatric patients with ili referred to two french university hospitals in paris and tours. aims were to investigate the different pathogens involved in ili and describe the associated symptoms. methods: h1n1v pandemic influenza diagnosis was performed with real time rt-pcr assay. other viral aetiologies were investigated by the molecular multiplex assay respifinder19®. clinical data were collected prospectively by physicians using a standard questionnaire. results: from week 35 to 44, endonasal swabs were collected in 413 patients. overall, 68 samples (16.5%) were positive for h1n1v. in 13 of them, other respiratory pathogens were also detected. among h1n1v negative samples, 213 (61.9%) were positive for various respiratory agents, 190 in single infections and 23 in mixed infections. the most prevalent viruses in h1n1v negative single infections were rhinovirus (62.6%), followed by parainfluenza viruses (24.2%) and adenovirus (5.3%). 70.6% of h1n1v cases were identified in patients under 40 years and none after 65 years. there was no difference between clinical symptoms observed in patients infected with h1n1v or with other pathogens. conclusion: our results highlight the high frequency of non-influenza viruses involved in ili during the pre-epidemic period of a flu alert and the lack of specific clinical signs associated with influenza infections. rapid diagnostic screening of a large panel of respiratory pathogens may be critical to define and survey the epidemic situation and to provide critical information for patient management. in order to monitor the spread of influenza and alert health handlers, several epidemiological tools have been developed. in france, a network of 1300 general practitioners, ''réseau sentinelles'', working throughout the country, provides real-time clinical data used to evaluate regional and national influenza spreading [1, 2] . the criteria used by this network to define clinical influenza-like illness (ili) are the occurrence of a sudden fever above 39uc with myalgia and respiratory signs. in general no formal viral diagnosis is carried out. the groupes régionaux d'observation de la grippe (grog) is a second french network that surveys the emergence and the spread of the influenza viruses [3, 4] . this network is based on clinical surveillance of acute respiratory infections and laboratory analysis of nasal specimens collected from adults and children by volunteer general practitioners and pediatricians. according to the sentinel network's criteria, french health authorities proclaimed that flu epidemic level was reached during the second week of september 2009 (week 37) [5, 6] . on the contrary, data provided by the grog showed only sporadic h1n1v activity until the last week of october (week 44) [6, 7] . thus, it became rapidly obvious that a variety of viruses were circulating in the community and that an overestimation of myxovirus infection was at stake [8, 9, 10, 11] . as a better knowledge of the epidemic status was a key feature for national healthcare organization, hospital preparedness, patient management and disease control, unambiguous viral diagnosis appeared critical. in france, data on viral aetiologies associated with ili were at best sporadic and correlations with clinical symptoms were often lacking. extensive molecular assays to screening for respiratory viruses were not available countrywide for routine diagnosis. therefore the epidemiological pattern of respiratory pathogens with overlapping seasonality was poorly known. the aim of the present study was to investigate respiratory pathogens involved in ili during the early weeks of the 2009-2010 h1n1v diffusion in france (weeks 35 through 44) and describe the associated symptoms in paediatric and adult populations. this study was a non-interventional study with no addition to usual proceedures. biological material and clinical data were obtained only for standard viral diagnostic following physicians' prescriptions (no specific sampling, no modification of the sampling protocol, no supplementary question in the national standardized questionnaire). data analyses were carried out using an anonymized database. according to the french health public law (csp art l 1121-1.1), such protocol does not require approval of an ethics committee and is exempted from informed consent application. in the two academic hospitals, saint-louis hospital (sls) in paris and tours hospital (trs), influenza-like illness (ili) was defined as a patient suffering from at least one general symptom (fever above 38uc, asthenia, myalgia, shivers or headache) and one respiratory symptom (cough, dyspnoea, rhinitis or pharyngitis), in agreement with the guidelines from the french institut de veille sanitaire (invs), a governmental institution responsible for surveillance and alert in all domains of public health [12] . criteria for severe clinical presentation were temperature below 35uc or above 39uc despite antipyretic, cardiac frequency above 120/min, respiratory frequency above 30/min, respiratory distress, systolic arterial pressure below 90 mmhg or altered consciousness. predisposing factors of critical illness were children younger than one year old, pregnant women, diabetes, chronic pre-existing disease (such as respiratory, cardiovascular, neurologic, renal, hepatic or hematologic diseases) and immunosuppression (associated with hiv infection, organ or hematopoietic stem cells transplantation, receipt of chemotherapy or corticosteroids) [13, 14] . a cluster of suspected influenza infections was defined as at least three possible cases in a week in a closed community (household, school,…) [15] . in the two institutions, the prescription of h1n1v molecular testing was recommended for patients with ili and with either a severe clinical presentation, an underlying risk factor of complications or a condition which was not improving under antiviral treatment. investigation of grouped suspected cases was also recommended. from week 35 (last week of august) to 44 (last week of october), 413 endonasal swabs were collected in 3 ml of universal transport medium (copan diagnostics inc, murrieta, ca) from adults and children seen in emergency rooms for suspected ili (table 1 ) and sent to sls and trs laboratories for h1n1v detection. the two microbiology laboratories participated in the reference laboratories network for the detection of pandemic influenza h1n1v. clinical data were collected at the time of medical attention and reported by clinicians on a national standardized questionnaire provided by invs [1, 12] . this questionnaire included the presence or absence of the main general and respiratory symptoms associated with ili (fever, asthenia, myalgia, shivers, headache, cough, rhinitis, pharyngitis, sudden onset) [12] . total nucleic acid was extracted from 400 ml of universal transport medium using the easymag system (biomérieux, marcy l'etoile, france) in sls or the ez1 advanced xl (qiagen, courtaboeuf, france) in trs, according to the manufacturers' instructions (elution volume: 100 ml in sls or 90 ml in trs). before extraction, 5 ml of an internal amplification control (iac) which contained an encephalomyocarditis virus (emc) rna transcript was added into the sample. pandemic h1n1v infection was diagnosed by real-time reverse transcription-pcr (rt-pcr) assay on a 7500 real time pcr system (applied biosystems, foster city, ca) according to the protocol of the centers for disease control (cdc) [16] . other respiratory infections were investigated by a multiplex molecular assay based on the multiplex ligation-dependent probe-amplification (mlpa) technology (respifinder19h, pathofinder, maastricht, the netherlands) that allows the detection and differentiation of 14 respiratory viruses, including influenza virus a (infa), influenza virus b (infb), rhinovirus (rhv), parainfluenza viruses 1 to 4 (piv-1 to piv-4), human metapneumovirus (hmpv), adenovirus (adv), respiratory syncytial virus a (rsva), respiratory syncytial virus b (rsvb) and human coronaviruses 229e, oc43 and nl63 (cor-229e, cor-oc43, cor-nl63) [17] . the test allows also the detection of h5n1 influenza a virus and of four bacteria: chlamydophila pneumoniae (cp), mycoplasma pneumoniae (mp), legionella pneumophila (lp) and bordetella pertussis (bp). the amplified mlpa products were analyzed on an abi 3100 genetic analyzer (applied biosystems, foster city, ca). fragment sizing analysis was performed with the genemarker software (softgenetics, llc, state college, pa). further testing for h1n1v was carried out with simplexa tm influenza a h1n1 (2009) (focus diagnostics, cypress, california) when the cdc real time rt-pcr assay was negative for h1n1 and the respifinder19h assay was positive for influenza a. if this latter assay was negative, h3n2 typing was performed as previously described [18] . data from our study are summarized as frequencies and percentages for categorical variables. quantitative variables are presented as medians, 25th and 75th percentiles. to compare those variables according to the viral infection status, fisher tests by using cdc reference assay, h1n1v was detected in 66 samples out of 413 (16.6%), more frequently in sls (38 samples) than in trs (28 samples) (p,10 24 ). overall, weekly percentage of h1n1v positive endonasal swabs remained under 10% until week 41 and increase significantly after (p trend ,0.0001) ( figure 1 ). rate of h1n1v detection reached 30% in sls at week 42 and in trs at week 44. overall, this rate was in agreement with results provided by the grog network, showing an earlier start of h1n1v epidemic in paris area [7, 19] . all 413 nucleic acid extracts were analyzed using the respifinder19h assay ( figure 2 ). sixty six patients tested h1n1v positive with cdc real time rt-pcr assay were confirmed with the multiplex assay. thirteen were also co-infected by one or two other respiratory pathogens (multiple infections) ( figure 2 ). three of the 347 h1n1v negative samples could not be studied with the multiplex assay because they contained rt-pcr inhibitors (no amplification of the internal control). two hundred and fifteen (62.5%) of the remaining 344 h1n1v negative samples were found positive for at least one respiratory pathogen ( figure 2 ). two hundred and twelve were positive for non influenza pathogens (189 single infections and 23 mixed infections with two, three or four viruses) and three additional single infections by influenza a were identified in sls, including two by pandemic h1n1v and one by seasonal h3n2, as determined after molecular typing (data not shown). overall, 68 patients (16.5%) were then positive for h1n1v, one for h3n2 and 212 for non influenza pathogens. there were 245 single infections (55 with h1n1v and 190 with other respiratory pathogens) and 36 mixed infections (13 with h1n1v and 23 without h1n1v) ( figure 2 ). among h1n1v negative single infections, the most prevalent viruses were rhinovirus (62.6%, 119 patients), followed by parainfluenza viruses 1 to 4 (24.2%, 46 patients), adenovirus (5.3%, 10 patients), human coronavirus 229e, oc43 and nl63 (3.2%, 6 patients) and respiratory syncytial virus a and b (2.6%, 5 patients) (figure 2 ). in addition, respifinder19h assay identified three patients with bacterial infection, two with mycoplasma pneumoniae (one 25 years old female in sls and one 39 years old female in trs) and one with bordetella pertussis (one 60 years old male in sls). no single infection by influenza b, hmpv, chlamydophila pneumoniae or legionella pneumophila was identified ( figure 2 to analyze if viral co-infections occurred more frequently for some viruses, we carried out a two by two comparisons, that showed a higher proportion of co-infection only for adv (p = 0.05). non-influenza respiratory viruses presented a different epidemic profile compared to h1n1v. overall, in both hospitals, weekly rate of non-h1n1v respiratory viruses whether alone or involved in co-infection increased between week 37 and 39 (from 51.4% to 81.3%) and then consistently decreased ( figure 3 ). rhv infections that represented nearly half of non-h1n1v viral infections (141 out of 213, 66.2%) were a significant contributing factor. in both hospitals, emergence of h1n1v cases was associated with a rapid decline of rhv rate of infection from 50-60% down to less than 20% with a one to two weeks gap between sls and trs. data on age ( in both institutions, 85.5% (106/124) children younger than 15 years of age were infected by at least one respiratory pathogen ( table 2 ). h1n1v infected patients were not significantly younger than h1n1v non infected patients (27 years old vs. 25 years old, p = 0.80) (figure 4) . however, 70.6% (48/68) of h1n1v cases were identified in patients under 40 years old (22 in sls and 26 in trs) and no case was observed in patients older than 65 years ( table 2) . piv infection occurred in very young patients (median (figure 4) . consequently, piv and adv were more frequently detected in the younger population of trs versus sls (p,10 24 and p,10 23 respectively). in contrast, although individuals with rhv infection were slightly younger than individuals without (median age = 24 vs. 29 for patients without rhv, p = 0.05) (figure 4) , influenza-like illness associated with rhv was more frequent in sls than in trs (p = 0.012). finally, patients with viral multiple infection were significantly younger than those with single infection (median, idr: 4, 2-18.5 vs. 25, 6-43) and rates of mixed infection at the time of medical attention, 383 (92.7%) standardized clinical questionnaires were collected out of 413 patients. four of them could not be exploited because they were too incomplete. a review of the 379 workable questionnaires showed that 90.8% (344/379) of the patients included in this study fulfilled the criteria of ili as defined above, and 52.5% had either a severe clinical presentation or an underlying risk factor of complications (45.9%, 174/379), or were in a suspected cluster of grouped cases (6.6%, 25/379). overall, most patients have fever (93.9%) and cough (86.1%) ( table 3) . other classical clinical signs associated with ili such as asthenia, myalgia, shivers, headache, rhinitis or pharyngitis were less frequent. a sudden onset was also described in 59.2% of cases. only 32.5% of the patients had a temperature above 39uc; the age of these patients ranged from zero to 86 years, with a median age of 32 years and a mean age of 34 years (data not shown). in h1n1v infected patients (including single and multiple infections), the main symptoms were also fever (98.2%) and cough (89.5%) ( we then compared clinical characteristics between patients positive for h1n1v, patients positive for other respiratory pathogens and negative for h1n1v and patients without any detection of respiratory pathogens (as detected with respifin-der19h) ( table 3 ). there was no difference between the three groups except for fever, cough, pharyngitis. however for these latter symptoms, the comparison between patients positive for h1n1v and those positive for other respiratory pathogens or between patients positive for h1n1v and those without any detection of respiratory pathogens, showed no difference except for pharyngitis, which was less frequent in patients positive for h1n1v than in patients positive for other respiratory pathogens ( table 3) . as rhv was the most frequent aetiology in ili, we also compared clinical symptoms observed in patients with a single infection by rhv or by h1n1v (data not shown). there was no difference except that rhinitis and pharyngitis were significantly more frequent in rhv infection (62.7% vs. 34.1% [p = 0.006] and 39.0% vs. 10.0% [p = 0.001], respectively). viral multiple infection (including samples with h1n1v) was not associated with a different clinical presentation. fever and cough were observed in over 90% of the patients (90.6% and 90.3%, respectively), but only 33.3% of these patients had a temperature above 39uc, which was not different from patients with single viral infection (28.6%). our results highlight the high frequency of non-influenza viruses involved in acute respiratory infections during the epidemic period of a flu alert as defined by the réseau sentinelles according to ili definition (a sudden fever above 39uc accompanied by myalgia and respiratory signs). these data extent previous observations in europe reporting high prevalence of rhv infections before seasonal influenza [4, 20] or in 2009, before h1n1v pandemic influenza [1, 8, 9, 11, 21] . we confirm that rhv represent the most frequent aetiology of acute respiratory table 2 . age of patients with respiratory samples positive for h1n1v, positive for other respiratory pathogens or negative. infections both in adult and paediatric populations and may represent more than 50% of cases. we show that other viral infections than influenza and rhv may represent up to 30% of aetiologies. we observed differences between the two hospitals, with a higher frequency of parainfluenza and adv infections in tours in contrast with a higher frequency of rhv in paris, likely explained by the higher proportion of paediatric samples collected in tours. however, despite the distance between the two institutions (about 250 km) and differences between the two populations, both presented similar patterns of high frequency of non-influenza viruses in acute respiratory infections before the flu epidemic wave and a decline when influenza reached epidemic levels. in the two cities, high frequencies of rhv were seen at the same level with a likely different evolution speed, with sudden increase and decrease in sls and more progressive variation in trs. in both institutions, there was a decrease in the proportion and number of rhv diagnoses roughly in parallel with the increase of influenza diagnoses. indeed, h1n1v exceeds 20% of positive detection's rate only when rhv dropped under 40%. these data are thus consistent with negative interaction of the two epidemics at the population level. it was previously hypothesised that rhv epidemic could interfere with the spread of pandemic influenza [20, 21, 22] . few in vitro data support this hypothesis. it has been reported that interferon and other cytokines production by rhv infected cells induced a refractory state to virus infection these data include the three patients whose respiratory samples could not be studied with the multiplex assay because of rt-pcr inhibitors. of neighbouring cells [23] . further work is needed to confirm in vitro and in vivo such negative interactions and if viral interference are really translated to a population level. analysis of rhinovirus and influenza epidemics in previous years should also help to determine if similar interferences were observed with seasonal influenza and to elaborate modelling and prediction of the spread of influenza according to respiratory viruses' circulation. systematic extensive screening of respiratory viruses at a national level should be implemented for this purpose. very few rsv infections were observed in contrast to usual epidemiology which was characterized the last four past years by a start of epidemics in weeks 44-45 [1] . it has been confirmed by other laboratories and the french invs that the 2009-10 rsv epidemic was delayed and had a lower impact compared with the previous winter season [1, 24] . delayed and reduced rsv spread may be due to viral interference between rsv and influenza. another possible explanation is better prevention behaviour about respiratory infections as recommended by a national campaign including recommendations for hands washing after sneezing and the use of mask [1] . influenza infections were mainly detected in patient under 40 years old and no case was found in patients older than 65. these results corroborate previous data suggesting that past seasonal h1n1 infections or vaccination may give partial crossed protection [10, 13, 25] . we have previously shown that the neutralizing titers against pandemic h1n1v virus correlate significantly with neutralizing titers against a seasonal h1n1 virus, and that the h1n1v pandemic influenza virus neutralizing titer was significantly higher in subjects who had recently been inoculated by a seasonal trivalent influenza vaccine [26] . viral co-infections were predominantly seen in paediatric patients, as previously described [4, 27, 28, 29] , both in influenza and non-influenza cases at a similar rate. no evidence of more pronounced respiratory impact was seen in these patients. our results showed the lack of specific clinical signs associated with proven h1n1v infections. clinical characteristics did not differ between influenza infections or other viral infections. in particular, the proportion of patients with fever above 39uc was not higher in h1n1v positive patients. in addition, the patients without any evidence of respiratory viral infections did not have different symptoms. these patients may have been infected with other virus not included in the multiplex assay (human bocavirus, coronavirus hku1) [9, 10, 11] or were seen too late at the time of viral shedding was cleared [30] . however, to determine how specific the symptoms are for influenza would require to assess also the distribution of respiratory pathogens (h1n1v and other respiratory viruses) and related symptoms in patients presented at the emergency departments in sls and trs with respiratory syndromes, but not tested for h1n1v. in addition, despite some underlying conditions that were associated with complications not previously observed in seasonal influenza, most illnesses caused by the h1n1v virus were acute and self-limited [13, 31] . the higher proportion of non influenza viruses reported in ili in 2009 was thus most likely a consequence of more frequent visits to a doctor for respiratory tract infections than usually observed for fear of the flu pandemic. the general lack of difference in symptoms in the particular context of h1n1v pandemic has therefore to be considered with caution and does not rule out that more significant differences may arise in future influenza epidemics with other influenza viruses. our data confirm that it may be virtually impossible to recognize symptoms heralding h1n1v infections and virological data should be helpful along with clinical reports to monitor influenza epidemic [10] . molecular multiplex detection has recently emerged as a potent diagnostic tool to determine acute respiratory infections' aetiologies [11, 32, 33] . these data show that sensitive molecular multiplex detection of respiratory viruses is feasible and efficient for the detection of virus involved in acute respiratory infections and provides insights into their epidemic profile. our results confirm the performance of respifinder19h assay to detecting respiratory viruses in the general population as recently shown in transplant patients with ili [34] . respifinder19h confirmed all h1n1 infections detected by the cdc reference assay and was able to identify two additional h1n1 cases suggesting a high sensitivity of this multiplex assay to detect influenza a infections. in conclusion, our results highlight that successive and mixed outbreaks of respiratory viral infections may affect influenza epidemiology and can lead to misinterpret the early development of a flu epidemic. rapid diagnostic screening of a large panel of respiratory pathogens may be critical to define and survey the epidemic situation and to provide critical information for patient management. impact of the 2009 influenza a(h1n1) pandemic wave on the pattern of hibernal respiratory virus epidemics virtual surveillance of communicable diseases: a 20-year experience in france a new influenza surveillance system in france: the ile-de-france ''grog''. 1. principles and methodology surveillance of community-acquired viral infections due to respiratory viruses in rhone-alpes (france) during winter 1994 to 1995 bulletins hebdomadaires surveillance de la grippe en france. bulletins hebdomadaires a variety of respiratory viruses found in symptomatic travellers returning from countries with ongoing spread of the new influenza a(h1n1)v virus strain frequency of detection of upper respiratory tract viruses in patients tested for pandemic h1n1/09 viral infection novel virus influenza a (h1n1sw) in south-eastern france rapid detection of respiratory tract viral infections and coinfections in patients with influenza-like illnesses by use of reverse transcription-pcr dna microarray systems surveillance de la grippe en france clinical aspects of pandemic 2009 influenza a (h1n1) virus infection severe hospitalised 2009 pandemic influenza a(h1n1) cases in france modified surveillance of influenza a(h1n1)v virus infections in france cdc protocol of real-time rt-pcr for influenza a (h1n1) respifinder: a new multiparameter test to differentially identify fifteen respiratory viruses application of a fluorogenic pcr assay for typing and subtyping of influenza viruses in respiratory samples bulletins épidémiologiques de la grippe a (h1n1) rhinoviruses, a(h1n1)v, rvs: the race for hivernal pandemics rhinoviruses delayed the circulation of the pandemic influenza a (h1n1) 2009 virus in france does viral interference affect spread of influenza? the interferon response circuit: induction and suppression by pathogenic viruses situation épidémiologique de la bronchiolite portrait of a year-old pandemic detection of extensive cross-neutralization between pandemic and seasonal a/ h1n1 influenza viruses using a pseudotype neutralization assay multiplex real-time pcr for detection of respiratory tract infections frequent detection of viral coinfection in children hospitalized with acute respiratory tract infection using a real-time polymerase chain reaction detection of multiple respiratory pathogens during primary respiratory infection: nasal swab versus nasopharyngeal aspirate using real-time polymerase chain reaction prospective evaluation of a novel multiplex real-time pcr assay for detection of fifteen respiratory pathogens-duration of symptoms significantly affects detection rate improving the clinical diagnosis of influenza-a comparative analysis of new influenza a (h1n1) cases molecular diagnosis of respiratory viruses detection of respiratory viruses by molecular methods comprehensive diagnostics for respiratory virus infections after transplantation or after potential exposure to swine flu a/h1n1: what else is out there? we gratefully acknowledge the contribution of the members of the two virology laboratories and sandrine picco for their excellent technical assistance in the detection of h1n1v pandemic virus and other respiratory viruses, and catherine scieux for her help in epidemiological data analysis. key: cord-272179-wvw5mmy3 authors: calderaro, adriana; de conto, flora; buttrini, mirko; piccolo, giovanna; montecchini, sara; maccari, clara; martinelli, monica; di maio, alan; ferraglia, francesca; pinardi, federica; montagna, paolo; arcangeletti, maria cristina; chezzi, carlo title: human respiratory viruses, including sars-cov-2, circulating in the winter season 2019-2020 in parma, northern italy date: 2020-10-02 journal: int j infect dis doi: 10.1016/j.ijid.2020.09.1473 sha: doc_id: 272179 cord_uid: wvw5mmy3 objectives: this study aimed to determine the prevalence of respiratory virus infections, including sars-cov-2, during december 2019 – march 2020, in a tertiary care hospital-based survey in parma (northern italy). methods: a total of 906 biological samples of respiratory tract were analyzed by both conventional (including culture) and molecular assays targeting sars-cov-2 and the other respiratory viruses nucleic acids. results: 474 samples (52.3%) were positive for at least one virus for a total of 583 viruses detected. single infections were detected in 380 (80.2%) samples and mixed infections were detected in 94 (19.8%). rsv (138/583: 23.7%) and rv (130/583: 22.3%) were the most common viruses identified, followed by sars-cov2 (82/583: 14.1%). rsv predominates until february with 129 detections and drastically decreases in march to 9 detections. sars-cov-2 absent in our area until february 26, in just over a month reached 82 detections. sars-cov-2 was found in mixed infections only in 3 cases all observed in children younger than one year old. conclusions: this study showed a completely different trend between sars-cov-2 and the "common" respiratory viruses that have seen children most affected without distinction of sex, as opposed to sars-cov-2 that have seen adult males the most infected. viral infections of the upper and lower respiratory tracts are among the most common illness in humans, mainly in children and infants in whom the infection can occur 5 to 6 times for year (berry et al, 2015) . briefly, the nucleic acid was extracted from 200 µl of specimens by using the nuclisens® easymag tm extraction assay (biomérieux, france). the nucleic acid amplification was carried out on the applied biosystems 7500 fast dx thermalcycler (applied biosystems, usa) at 25°c for 2 minutes for ung incubation, and then at 50°c for 15 minutes for the reverse transcription step, followed by the enzyme activation step at 95°c for 2 minutes. then, the amplification was carried out for 45 cycles (3 seconds at 95°c and 30 seconds at 55°c). in the prospective analysis, a specimen was considered negative for sars-cov-2 if markers n1 and n3 cycle threshold growth curves do not cross the threshold and the rnase p growth curve cross the line. conversely, if markers cycle threshold growth curve crosses the threshold line, the specimen was considered positive for sars-cov-2 virus. in the retrospective analysis, only n1 marker was used. table 1 ). the distribution of single and mixed infections on the basis of age and sex is shown in figure 1a and 1b, respectively. overall, a total of 583 viruses were detected, rsv (138/583: (table 1) . similarly, the 60.5% (347 out of 574) and the 14.5% (48 out of 332) of the children and adults tested, respectively, were positive (p <0.00001; or: 9.04) for at least one respiratory virus other than sars-cov-2; for this virus the 1.4% (7 out of 513) and the 24% (75 out of 313) of the children and adults tested, respectively, were positive (p <0.00001; or: 0.04) ( table 1) . table 2 (berry et al, 2015) . this suggests that many respiratory pathogens may remain undetected. all novel emergent respiratory viruses have varying but significant impact on human health and the potential to give outbreaks (berry et al, 2015) ; sars-cov-2 as seen in these months, has shown, worldwide, its own unique potential to give epidemics. in this study, we investigated the viral etiology of aris in 906 patients with acute respiratory tract infections, both outpatients and inpatients, attending the tertiary care the samples analysed in this study were sent to the university hospital of parma for routine diagnostic purposes, and the laboratory diagnosis results were reported in the medical records of the patients as answer to a clinical suspicion; ethical approval at the university hospital of parma is required only in cases in which the clinical samples are to be used for applications other than diagnosis. the authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. identification of new respiratory viruses in the new millennium virological and clinical characterizations of respiratory infections in hospitalized children sars-cov-2 infection diagnosed only by cell culture isolation before the local outbreak in an italian seven-week-old suckling baby matrix-assisted laser desorption/ionization time-of-flight (maldi-tof) mass spectrometry applied to virus identification centers for disease control and prevention. 2020. coronavirus disease genomic characterization of the 2019 novel human-pathogenic coronavirus isolated from a patient with atypical pneumonia after visiting wuhan first two months of the 2019 coronavirus disease (covid-19) epidemic in china: real-time surveillance and evaluation with a second derivative model epidemiology of human respiratory viruses in children with acute respiratory tract infection in a 3-year hospital-based survey in northern italy mammalian diaphanous-related formin-1 restricts early phases of influenza a/nws/33 virus (h1n1) infection in llc-mk2 cells by affecting cytoskeleton dynamics risk factors for sars-cov-2 among patients in the oxford royal college of general practitioners research and surveillance centre primary care network: a cross-sectional study coronaviruses and sars-cov-2 incidence and seasonality of respiratory viruses causing acute respiratory infections in the northern united arab emirates positive rate of rt-pcr detection of sars-cov-2 infection in 4880 cases from one hospital in multiple versus single virus respiratory infections: viral load and clinical disease severity in hospitalized children viruses as sole causative agents of severe acute respiratory tract infections in children mixed respiratory virus infections cresce la popolazione del parmense 2019-ncov (wuhan virus), a novel coronavirus: human-to-human transmission, travel-related cases, and vaccine readiness etiology and clinical outcomes of acute respiratory virus infection in hospitalized adults the outbreak of sars-cov-2 pneumonia calls vaccines respiratory viral infections in infants: causes, clinical symptoms, virology, and immunology respiratory virus surveillance in hospitalised pneumonia patients on the thailand-myanmar border world health organization. 2020. coronavirus disease: situation report -184 epidemiological and molecular surveillance of influenza and respiratory syncytial viruses in children with acute respiratory infections pcr for respiratory viruses other than sars-cov-2 pcr for sars key: cord-282303-idh7io9v authors: hassan, md. zakiul; sturm-ramirez, katharine; rahman, mohammad ziaur; hossain, kamal; aleem, mohammad abdul; bhuiyan, mejbah uddin; islam, md. muzahidul; rahman, mahmudur; gurley, emily s. title: contamination of hospital surfaces with respiratory pathogens in bangladesh date: 2019-10-28 journal: plos one doi: 10.1371/journal.pone.0224065 sha: doc_id: 282303 cord_uid: idh7io9v with limited infection control practices in overcrowded bangladeshi hospitals, surfaces may play an important role in the transmission of respiratory pathogens in hospital wards and pose a serious risk of infection for patients, health care workers, caregivers and visitors. in this study, we aimed to identify if surfaces near hospitalized patients with respiratory infections were contaminated with respiratory pathogens and to identify which surfaces were most commonly contaminated. between september-november 2013, we collected respiratory (nasopharyngeal and oropharyngeal) swabs from patients hospitalized with respiratory illness in adult medicine and paediatric medicine wards at two public tertiary care hospitals in bangladesh. we collected surface swabs from up to five surfaces near each case-patient including: the wall, bed rail, bed sheet, clinical file, and multipurpose towel used for care giving purposes. we tested swabs using real-time multiplex pcr for 19 viral and 12 bacterial pathogens. case-patients with at least one pathogen detected had corresponding surface swabs tested for those same pathogens. of 104 patients tested, 79 had a laboratory-confirmed respiratory pathogen. of the 287 swabs collected from surfaces near these patients, 133 (46%) had evidence of contamination with at least one pathogen. the most commonly contaminated surfaces were the bed sheet and the towel. sixty-two percent of patients with a laboratory-confirmed respiratory pathgen (49/79) had detectable viral or bacterial nucleic acid on at least one surface. klebsiella pneumoniae was the most frequently detected pathogen on both respiratory swabs (32%, 33/104) and on surfaces near patients positive for this organism (97%, 32/33). surfaces near patients hospitalized with respiratory infections were frequently contaminated by pathogens, with klebsiella pneumoniae being most common, highlighting the potential for transmission of respiratory pathogens via surfaces. efforts to introduce routine cleaning in wards may be a feasible strategy to improve infection control, given that severe space constraints prohibit cohorting patients with respiratory illness. with limited infection control practices in overcrowded bangladeshi hospitals, surfaces may play an important role in the transmission of respiratory pathogens in hospital wards and pose a serious risk of infection for patients, health care workers, caregivers and visitors. in this study, we aimed to identify if surfaces near hospitalized patients with respiratory infections were contaminated with respiratory pathogens and to identify which surfaces were most commonly contaminated. between september-november 2013, we collected respiratory (nasopharyngeal and oropharyngeal) swabs from patients hospitalized with respiratory illness in adult medicine and paediatric medicine wards at two public tertiary care hospitals in bangladesh. we collected surface swabs from up to five surfaces near each case-patient including: the wall, bed rail, bed sheet, clinical file, and multipurpose towel used for care giving purposes. we tested swabs using real-time multiplex pcr for 19 viral and 12 bacterial pathogens. case-patients with at least one pathogen detected had corresponding surface swabs tested for those same pathogens. of 104 patients tested, 79 had a laboratory-confirmed respiratory pathogen. of the 287 swabs collected from surfaces near these patients, 133 (46%) had evidence of contamination with at least one pathogen. the most commonly contaminated surfaces were the bed sheet and the towel. sixty-two percent of patients with a laboratory-confirmed respiratory pathgen (49/79) had detectable viral or bacterial nucleic acid on at least one surface. klebsiella pneumoniae was the most frequently detected pathogen on both respiratory swabs (32%, 33/104) and on surfaces near patients positive for this organism (97%, 32/33). surfaces near patients hospitalized with respiratory infections were frequently contaminated by pathogens, with klebsiella pneumoniae being most common, highlighting the potential for transmission of respiratory pathogens via surfaces. efforts to introduce routine cleaning in wards may be a feasible strategy to improve infection control, given that severe space constraints prohibit cohorting patients with respiratory illness. pathogens present in ill patients' respiratory secretions can contaminate nearby hospital surfaces, such as floors, walls, bedrails and mattresses, through coughing, sneezing and touching [1] [2] [3] [4] . respiratory viral and bacterial pathogens, including staphylococcus aureus, streptococcus pyogenes, influenza viruses, respiratory syncytial virus, adenovirus, rhinoviruses and novel coronavirus strains, can survive on hospital surfaces for days, weeks or even months. furthermore, touching contaminated surfaces may lead to nosocomial transmission of pathogens between patients, family caregivers, visitors, and healthcare workers [1, 5, 6] . patient care areas in bangladeshi hospitals are open wards with multiple beds in a room and are frequently overcrowded with patients, family caregivers, and visitors [7, 8] . a previous study by rimi et al. found a median of four people per 100 sq. feet of floor space in hospital wards in bangladesh and observed a median of five uncovered coughs or sneezes per 100 sq feet per hour [7] . due to shortage of health care workers, family caregivers (family members who provides 24 hour hands on care to sick patient, including bedside nursing and cleaning) are integral part of inpatient care in bangladeshi public hospitals, contributing crowding of hospital wards [8, 9] . the world bank estimated that in 2014 only $32 of public funds per capita were spent annually on health infrastructure in bangladesh. thus, resources for infection control are severely limited in bangladeshi hospitals [7, 10] , making it difficult to implement international infection control guidelines [11] . the lack of routine infection control practices, including no regular surface cleaning, may increase the transmission of respiratory pathogens via hospital surfaces [3, 5, 6, 8] . family caregivers, visitors, and hospital staff may acquire respiratory infections either through direct contact with infected patients or via droplets, aerosols or contaminated surfaces. contaminated hospital surfaces can pose a serious risk of infection for patients, health care workers, caregivers and visitors. within this context of scarce resources, describing the magnitude of surface contamination in bangladeshi hospitals, particularly identifying priority areas for decontamination, could influence infection control policy and practice. our objective was to assess the frequency with which patients hospitalized for respiratory illnesses in bangladeshi public hospitals contaminate nearby surfaces, to identify commonly contaminated surfaces, and to determine which pathogens are detected most frequently. we conducted the study in two public tertiary care teaching hospitals in rajshahi and jessore, bangladesh between september and november 2013. rajshahi medical college hospital contains approximately 1,200 beds with eight adult medicine wards and four pediatric wards. jessore medical college hospital is a 250-bed hospital with two adult medicine wards and one pediatric ward. paediatric wards typically admit patients <14 years of age and older patients are admitted to adult medicine wards. the mean bed occupancy proportion in these hospital wards are consistently >100% with patients being treated on the floor and in hallways when beds unavailable [8, 12] . study physicians in adult medicine and pediatric wards identified patients aged �5 years who met the severe acute respiratory illness (sari) case definition of subjective or measured fever (�38 c˚) within the past seven days with cough or sore throat [13] . in pediatric wards, physicians identified children <5 years of age who met the severe pneumonia (sp) case definition: cough or difficulty breathing and at least one danger sign (i.e. chest indrawing, stridor while calm, history of convulsions, inability to drink, lethargy or unconsciousness and/or intractable vomiting) with onset of symptoms within the last seven days [13] . since case-patients were identified immediately after admission, their illnesses were mostly community acquired. study physicians collected respiratory swabs (nasopharyngeal and oropharyngeal) from identified cases using the world health organization's laboratory safety manual protocol [14] and pooled them into a single cryovial containing viral transport media (vtm). trained research assistants in each hospital collected one swab sample from five different surfaces near each enrolled case patient: the wall next to the patient's bed, bed rail, bed sheet, clinical record files, and a multipurpose towel. the multipurpose towel is a cloth brought from home by family caregivers and used to clean patient respiratory secretions, wiping the patient's face or head, and to dry caregivers' hands and face [8] . we selected these surfaces because patients, caregivers, and healthcare workers (hcws) frequently come into contact with them in the hospital wards [6, 7, 15] . the research assistants collected surface swabs between 12-72 hours after the case-patients' admission to the hospital. this allowed for adequate time for hospital surfaces to be exposed to possible contamination by respiratory pathogens, while also making sure that surfaces were swabbed before the enrolled patients were discharged or died, as patient turnover in wards was high with a median hospital stay of three days [16] . the risk for infection from these potentially contaminated surfaces between patients within a room, between patients and healthcare workers, or between patients and family caregivers would vary based on the particular surface and how these different risk groups interacted with the surface. with one sterile rayon swab stick per surface, the research assistant swabbed the area of the wall in contact with the bed 45 cm high from the level of the bed sheet, all surfaces of the bed rail located in the area near the patients' head, half of the bed sheet where the patient's head was including underneath the patient, front and back cover of the patient file and both sides of the multi-purpose towel. not all patients had a wall or bedrail nearby as some patients were cared for on the floor, due to overcrowding. swab samples from each surface area were put into individual cryovials containing vtm and kept in a cool box for up to 30 minutes with a temperature between 2˚-8˚c. both the respiratory swabs and surface swab samples were labelled, packaged, stored in a nitrogen dry shipper (-150˚c) and sent to the icddr,b virology laboratory by batch twice a month. the swab samples were thawed and the cryovials containing the sample were vortexed. about 200 μl of the swab supernatant was used for nucleic acid extraction using invimag1 pathogen kit/kf 96 (stratec molecular, berlin, germany) and the final eluted volume of nucleic acid solution was 200 μl, as per the manufacturer's instruction [17] . the real-time multiplex pcr assay was performed as per the manufacturer's instructions using an agpath-id ™ one-step rt-pcr kit (ambion) with the fast track diagnostic (ftd) respiratory pathogens 33 kit (fast track diagnostics, luxembourg) for 19 different viruses and 12 bacterial pathogens [18] . casepatients with at least one pathogen detected in their respiratory swab had corresponding surface swabs tested for those same pathogens. detection of nucleic acid of at least one similar pathogen on respiratory swab and a nearby surface was defined as contamination of that surface. to investigate wider hospital contamination, we also tested the surface swabs collected near casepatients with no pathogens detected in their respiratory swabs for the most commonly detected pathogens identified on surfaces near patients with detected respiratory pathogens. we summarized the data using descriptive statistics. we assessed the difference in proportion of pathogen detection in respiratory swabs and surface swabs between adult and paediatric ward patients using chi-square test considering fisher exact test where appropriate. any association with a p value <0.05 was considered statistically significant. study participants (aged �18 years) or their legal guardians (if aged <18 years) provided informed written consent. the institutional review board of icddr,b reviewed and approved the study protocol. the institutional review board at the centers for disease control and prevention (atlanta, ga, usa) deferred to icddr,b's approval. we collected and tested respiratory swabs from 104 patients hospitalized with respiratory illness: 50 sari cases from adult medicine wards and 54 severe pneumonia cases from paediatric medicine wards. the median age of patients in the adult wards was 32 years (iqr 25-48) and in paediatric wards three months (iqr 2-6). the male-to-female ratio was 2.6:1 (table 1) . of the 104 patients, 79 (75%) had detectable viral and/or bacterial nucleic acid in their respiratory swabs. paediatric patients more frequently had one or more detectable pathogen in their respiratory swabs than adult patients (91% versus 60%, p = 0.001). bacterial pathogens were identified in 76% of adult respiratory swabs. klebsiella pneumoniae, streptococcus pneumoniae and human cytomegalovirus 0(0) 21(39) human parainfluenza viruses 2(4) 3(5.5) other viruses b 3(6) 6(11) 18 ( staphylococcus aureus were most commonly detected during the study period. in contrast, viral pathogens were commonly detected among paediatric patients, including human cytomegalovirus, respiratory syncytial viruses, and human rhinoviruses (table 1) . clinical features, mean duration of symptom onset to sample collection (3.2 days vs 3 days) did not vary between patients with a detectable viral and a detectable bacterial nucleic acid in respiratory swabs. two patients, one with klebsiella pneumoniae and one with streptococcus pneumoniae detected in their respiratory swabs, had been hospitalized at other facilities within two weeks prior to admission to the study hospital, suggesting that these organisms could have been hospital-acquired. both these patients had abnormal chest x-rays and were diagnosed with severe pneumonia in the study hospital. we tested the surrounding hospital surface swabs for each of the 79 patients with evidence of respiratory pathogens for the same viral/bacterial nucleic acid detected in their respiratory swabs. we collected and tested 287 surface swabs near the 79 patients as not all these patients had a wall or bedrail near them. nearly half of the hospital surface swabs (46% [133/287]) had evidence of contamination by at least one pathogen included in the testing panel. the most commonly contaminated surfaces were the bed sheet, the multipurpose towel, and the bed rail. we infrequently detected bacterial or viral nucleic acid on wall surfaces or on patients' clinical record files (fig 1) . sixty-two percent of patients (49/79) had detectable viral and/or bacterial nucleic acid on at least one (range: 1-5) nearby surface, including 60% of adults (18/30) and 63% (31/49) of pediatric patients. the most common bacterial pathogen detected on surface swabs was klebsiella pneumoniae and 97% (32/33) of patients positive for klebsiella pneumoniae had at least one surface with detectable dna. the most frequently detected viral pathogen on surfaces was human cytomegalovirus and 86% (18/21) of patients positive for human cytomegalovirus had detectable dna on nearby surfaces ( table 2) . we tested nearby surfaces for 22 patients (18 patients from adult wards and 4 from pediatric wards) without detectable viral/bacterial nucleic acid in their respiratory swabs. we tested these surfaces for six frequently identified pathogens in respiratory swabs of case-patients: klebsiella pneumoniae, streptococcus pneumoniae, staphylococcus aureus, human cytomegalovirus, respiratory syncytial viruses and human rhinoviruses. klebsiella pneumoniae was detected on at least one nearby surface in 95% (21/22) of these patients, staphylococcus aureus in 18% (4/22), and streptococcus pneumoniae in 14% (3/22) patients. viruses, including, human cytomegalovirus (4/22), respiratory syncytial viruses a and b (1/22) and human rhinoviruses (0/22), were rarely detected nearby these patients. nearly two-thirds of the patients hospitalized with laboratory-confirmed acute respiratory infection had at least one nearby contaminated surface. klebsiella pneumoniae was the most commonly detected pathogen in patients' respiratory swabs, and was detected in nearly every environmental swab testing, suggesting widespread hospital contamination from current and previously hospitalized patients. with the ability to spread rapidly in the hospital environment, klebsiella pneumoniae has been linked to several nosocomial outbreaks [19, 20] . the most frequently contaminated surfaces were the bed sheet, towel, and bed rail, further highlighting the perils of no routine surface cleaning practices in these hospitals. studies in tertiary care hospitals of bangladesh have shown that one in 20 patients with a hospital stay greater than three days developed a hospital-acquired respiratory infection, and that only 21% of those infections had a viral aetiology, suggesting a large proportion of these infections might be bacterial [21, 22] . since, future work should further investigate the role of klebsiella pneumoniae may be an important nosocomial pathogen in these hospitals. several other studies have identified multidrug resistant klebsiella pneumoniae in hospital environments and its association with severe infections, prolonged hospital stays and increased mortality rates, particularly in debilitated and immunocompromised patients [23, 24] . a 2004 study in an urban tertiary care hospital in dhaka, bangladesh, reported that 40% of the clinical specimens (sputum, pus, urine, throat, and vaginal swabs) collected from hospitalized patients were drug resistant [25] . moreover, our study findings were consistent between the two hospitals despite being located in different geographical areas and may suggest surface contamination with klebsiella pneumoniae as a wider public health problem. the predominant bacterial pathogens we identified, klebsiella pneumoniae, streptococcus pneumoniae, and staphylococcus aureus, can survive on surfaces from a few days to a few months [5, 26, 27] . bacteria, in the presence of low humidity, forms biofilms protecting microorganisms from harsh environmental influences and are difficult to eradicate [28, 29] . in bangladesh, hospital surfaces are not adequately cleaned and hospitals report insufficient supplies of cleaning agents [7, 8, 30] . to remove and prevent biofilm formation, hospital decontamination protocols should include strategies such as daily cleaning of surfaces with disinfectant (e.g. 0.3% sodium hypochlorite or 2% chlorhexidine solutions). among the most commonly detected viral pathogens, rsv was prevalent in respiratory swab of paediatric patients and nearby surfaces. rsv has been a major nosocomial hazard on pediatric wards and has been linked with hospital outbreaks [31, 32] . with reported higher case fatality among patient with nosocomial rsv infections (or 4.46, 95% ci 1.1-18), widespread surface contamination with rsv is concerning for low income hospitals (34, 35) . we identified, that towel, was frequently contaminated with respiratory secretions. [8] . islam et al. reported that family caregivers frequently used a multipurpose towel for patient secretions and for their own use, without cleaning it in between these uses [8] .this suggests that the towel may act as a potential vehicle for transmission of respiratory viral and bacterial pathogens from patient to caregiver [33] . infection control could target care giving practices associated with the use of the towel and should test feasible low-cost interventions such as the supply of low cost disinfectant by hospitals that encourage caregivers to clean the towels more frequently and to improve hand washing practices, including the use of hand sanitizer. an important limitation of our study is that we only identified the presence of viral or bacterial nucleic acid on different hospital surfaces, and cannot be sure that the pathogens we detected were viable. a second limitation is that we conducted the study in only two public hospitals, so the findings may not be representative of all public hospitals. however, our findings were consistent between the two typical tertiary care hospitals we studied, located in completely different parts of the country. a third limitation is that we only sampled surfaces once, which limits our ability to comment on duration of contamination, and did not have the ability to observe contamination from seasonal infections. influenza, for example, has a known seasonal pattern in bangladesh, circulating usually between may and september, potentially explaining the low detection rate in our study [34, 35] . lastly, we did not investigate drug resistance patterns of the bacteria we detected due to resource constraints. based on evidence from other studies, however, it is likely that many of the pathogens we detected on surfaces were drug resistant and future studies should consider including investigations about drug resistance [25] . this study identified that hospital surfaces in these bangladeshi hospitals, were frequently contaminated with respiratory pathogens and pose a potential threat for fomite-borne transmission of respiratory infections to patients, healthcare workers and family caregivers. to prevent the spread of klebsiella and other infections between patients, healthcare personnel must follow specific infection control precautions including strict adherence to hand hygiene and the use of gloves. in addition, our data clearly indicate that efforts to regularly disinfect environmental surfaces and ensure clean towels for patient caregiving could reduce risk of exposure to patients, healthcare staff and visitors. the government of bangladesh has taken a number of initiatives to improve infection control in hospitals [36] . despite this, a 2013 nationally representative survey of healthcare facilities showed that healthcare workers performed recommended hand hygiene in only 9% of 919 opportunities, suggesting low adherence to international standards [30] . barriers include awareness, training, accountability and appropriate infrastructure (among 875 health facilities, 10% handwashing locations had no water, 20% had no soap and 50-80% had no alcohol based sanitizer) to support these behaviours [30, 37, 38] this study highlighted the gaps in practice, as well as the substantial barriers to improvement that will require widespread investments to address. in 2018, the directorate of hospital infection control, directorate general of health services (dghs) at the ministry of health and family welfare in bangladesh has communicated the intention to form infection control committees in each district and tertiary care hospital across the country to improve the safe care [39] . further investigation to identify the true contribution of fomites in the transmission of respiratory pathogens within hospital settings could be useful to help these committees prioritize efforts to improve hand and surface cleaning. supporting information s1 file. dataset. (dta) significance of fomites in the spread of respiratory and enteric viral disease environmental contamination makes an important contribution to hospital infection contamination, disinfection, and cross-colonization: are hospital surfaces reservoirs for nosocomial infection? clinical infectious diseases nipah virus contamination of hospital surfaces during outbreaks how long do nosocomial pathogens persist on inanimate surfaces? a systematic review hand-touch contact assessment of high-touch and mutual-touch surfaces among healthcare workers, patients, and visitors infrastructure and contamination of the physical environment in three bangladeshi hospitals: putting infection control into context family caregivers in public tertiary care hospitals in bangladesh: risks and opportunities for infection control the health workforce crisis in bangladesh: shortage, inappropriate skill-mix and inequitable distribution universal health coverage assessment people's republic of bangladesh international nosocomial infection control consortium report, data summary of 50 countries for 2010-2015: device-associated module incidence of and risk factors for hospital-acquired diarrhea in three tertiary care public hospitals in bangladesh. the american journal of tropical medicine and hygiene who interim global epidemiological surveillance standards for influenza manual for the laboratory diagnosis and virological surveillance of influenza a quantitative approach to defining "high-touch" surfaces in hospitals economic burden of influenza-associated hospitalizations and outpatient visits in bangladesh during 2010. influenza and other respiratory viruses maternal vitamin d supplementation during pregnancy and lactation to prevent acute respiratory infections in infancy in dhaka, bangladesh (mdari trial): protocol for a prospective cohort study nested within a randomized controlled trial ftd respiratory pathogens 33 luxemburg: fast track diagnostics an outbreak of hospital-acquired klebsiella pneumoniae bacteraemia, including strains producing extended-spectrum β-lactamase outbreak of klebsiella pneumoniae producing a new carbapenem-hydrolyzing class a β-lactamase, kpc-3, in a new york medical center incidence and viral aetiology of hospital-acquired respiratory infections at three tertiary care hospitals in bangladesh rates of hospital-acquired respiratory illness in bangladeshi tertiary care hospitals: results from a low-cost pilot surveillance strategy detection and treatment options for klebsiella pneumoniae carbapenemases (kpcs): an emerging cause of multidrug-resistant infection predictors of hospital surface contamination with extended-spectrum β-lactamase-producing escherichia coli and klebsiella pneumoniae: patient and organism factors. antimicrobial resistance and infection control prevalence of extended-spectrum βlactamase-producing escherichia coli and klebsiella pneumoniae in an urban hospital in dhaka, bangladesh survival of enterococci and staphylococci on hospital fabrics and plastic survival of enterococci and staphylococci on hospital fabrics and plastic survival strategies of infectious biofilms efficacy of disinfecting solutions in removing biofilms from polyvinyl chloride tracheostomy tubes. the laryngoscope healthcare worker and family caregiver hand hygiene in bangladeshi healthcare facilities: results from the bangladesh national hygiene baseline survey risk of nosocomial respiratory syncytial virus infection and effectiveness of control measures to prevent transmission events: a systematic review. influenza and other respiratory viruses the clinical and phylogenetic investigation for a nosocomial outbreak of respiratory syncytial virus infection in an adult hemato-oncology unit nipah virus contamination of hospital surfaces during outbreaks influenza in outpatient ili case-patients in national hospital-based surveillance estimates of seasonal influenzaassociated mortality in bangladesh bangladesh: dghs, ministry of health & family welfare behaviour change intervention to reduce caregivers' exposure to patients' oral and nasal secretions in bangladesh health worker and family caregiver hand hygiene in bangladesh healthcare facilities: results from a nationally representative survey bangladesh: health economics unit, health sevices division we would like to thank authorities of participating hospitals, all the study participants, the study physicians, field staff and laboratory staff for their contribution. we acknowledge gladys leterme for reviewing and editing this manuscript.icddr,b acknowledges with gratitude the commitment of cdc to its research effort. icddr,b is also grateful to the governments of bangladesh, canada, sweden and the uk for providing core/unrestricted support. key: cord-300711-yibdumij authors: shatizadeh, somayeh; yavarian, jila; rezaie, farhad; mahmoodi, mahmood; naseri, maryam; mokhtari azad, talat title: epidemiological and clinical evaluation of children with respiratory virus infections date: 2014-09-22 journal: med j islam repub iran doi: nan sha: doc_id: 300711 cord_uid: yibdumij background: respiratory viruses are the leading cause of respiratory tract infections among children and are responsible for causing morbidity and mortality worldwide. this study was performed to detect viruses in children with respiratory infections and describe their epidemiology and clinical characteristics. methods: in this descriptive cross sectional study, throat swabs and wash specimens from 202 children younger than six years of age with diagnosis of a respiratory tract infection from a total of 897 specimens were evaluated using multiplex pcr method. results: respiratory viruses were detected in 92 children: respiratory synsytial virus, 16.8%; influenza virus, 5.4%; parainfluenza virus, 8.4%; adenovirus, 14.4% and human metapneumo virus 0.49% with male predominance and higher distribution in children younger than 1 year of age with preference in the cold months of year. the clinical presentations of all detected viruses were almost similar. conclusion: in the present study, nine different respiratory viruses were detected. rsv causes the great majority of respiratory virus infections in children. there was no significant difference in epidemiologic patterns of these viruses in comparison to other studies. among children, respiratory tract infections (rti) are a major cause of morbidity and mortality worldwide especially in developing countries (1, 2) . respiratory syncytial virus (rsv), influenza virus (ifv), parainfluenza virus (piv), adenovirus (adv) and the recently discovered human metapneumovirus (hmpv) likely account for a majority of respiratory tract illnesses (3) . the most prevalent presentations in rti include sore throat, headache and my-algia (4) . studies assessing the relationship between clinical symptoms and respiratory infections yielded conflicting results (5) . in temperate climate regions, rti display a defined seasonality occurring mainly during the winter (2) . the aim of this study was to determine, by a sensitive method, the frequency, epidemiology and clinical characteristics of respiratory viruses among young children. this might help towards better management and treatment of infected children. mjiri this cross sectional study was performed on specimens collected between 2008 and 2009 in national influenza center of iran. throat swabs and washes were collected from 897 patients with respiratory illness and transported to the national influenza center, school of public health, tehran university of medical sciences. a questionnaire was alsocompleted. two hundred and two, out of 897, specimens belonged to children less than 6 years of age were selected. nucleic acid extraction rna extraction and cdna synthesis: rna was extracted from 150 μl of samples using the neucleospin viral rna (neucleospin rna extraction kit, macherey-nagel) according to the manufactures instruction. cdna synthesis was carried out in 30 μl reaction mixture containing 6 μl of 5x rt buffer, 2.5 μl of mixed dntps (2.5μm each), 1 μl of rt-mulv enzyme (fermentase), 2.5 μl random hexamer (fermentase), 0.5 μl rnase inhibitor (fermentase) and 17.5 μl rna template, the mixture was incubated in 22° c for 10 min, 37° c for 45 min and 94° c for 5 min. dna extraction dna was extracted from 150 μl of samples using the neucleospin viral dna (neucleospin dna extraction kit, macherey-nagel) according to the manufactures instruction. primers targeted specifically the nucleocapsid gene of rsv(6), the matrix protein gene of hmpv (7), the hemagglutinin and neuraminidase gene of piv-1,2 (8) and piv-3 (9) the phosphoprotein gene of piv-4 (10) hemagglutinin (ha) gene of ifv a and b (11) and hexon gene of adv (12) . three multiplex rt-pcr methods, targeting eight respiratory viruses, were developed. multiplex pcr i for rsv and hmpv, multiplex pcr ii for piv1-4 and multiplex pcr iii for flu a and b were performed. the protocols are available upon request. uniplex hemi nested pcr was done for detection of adenovirus. briefly, 5 μl of the nucleic acid extraction was added to 45 μl of reaction mixture containing 10x pcr buffer, 4μl of mgcl2 (50mm), 2.5μl of dntp mix (10mm), 20 pmol of each degenerate primer pair and 0.4μl of taq polymerase. temperature and time profiles were as follows: 1 cycle at 94° c for 3 min, followed by 30 cycles of 94° for 1 min, 50°c for 1 min, 68° c for 1 min, and final incubation at 68°c for 50 min. then nested reaction was performed with 20 pmol of the another degenerate primer pair as above. the proportions of the patients aged <12, 12-24, 25-36, 37-48, 49-60 and 61-72 months were 16.3, 10.89, 2.97, 4.45, 3.46 and 6.93% respectively. figure 1 shows age distribution of the children according to the different viruses. among the patients there were 47(23.26%) males and 45(22.27%) females with a sex ratio of 1.04:1. female sex was more frequently associated with rsv, piv-1, ifv and hmpv in contrast adv was detected mostly in males (fig. 2) . figure 3 and figure 4 show seasonal and monthly distribution according to the different viruses, respectively. in analyses of the clinical signs and symptoms in affected children, fever (53.6%) myalgia (49.8%) and cough (38.4%) were common clinical findings. sore throat was observed in 25.3% of the patients. there were no headache and malaise found in these patients (fig. 5 ). the circulation of different respiratory viruses during the same period of the year makes it very difficult to detect their individual contribution to the global respiratory disease. moreover, the pattern of the respiratory virus activity seems to change within the different age groups with different demographic characteristics (13) . in each community, the epidemiology of respiratory viruses has to be determined prior to the implementation of novel and costly therapies. in the present study rsv was the most frequently demonstrated virus accounted for 16.8% of all viruses detected. our results were similar to those from other countries (14,15) and emphasize the role of rsv as the leading cause of arti in infants and young children. meanwhile rsv was mostly found in female infants less than one year of age with the typical seasonal pattern with a peak incidence during february (16) . fever, cough and myalgia were the common clinical signs and symptoms in these patients. one study in the united states showed rsv is responsible for 1% child hospitalization, 70% of which during the first year of life (17). adenoviruses produce lower respiratory illness in all seasons with a tendency to peak in mid-winter (18,19) . in this study, advs followed by rsv were the most frequently detected viral agents in our patients (14.4%) which occurred mostly in the summer and winter months (august, september and february) in male children in all age groups. fever, cough, sore throat and myalgia were the common clinical characteristics of them. human parainfluenza viruses are important respiratory pathogens and a major cause of croup and pneumonia in infants (20) . herein, hpiv1 and 3 were the most prevalent hpivs similar to other studies (21) and had high peak between january and february at children younger than 2 years of age which showed preference in females. the most frequently symptoms were fever and myalgia. among all respiratory virus infections because of the pandemics and annual epidemics, influenza viruses are a center of concern. in the present study influenza viruses were detected in 5.4% of the children and similar to the other studies they were in all age groups during the cold months (december to march) (22) . female dominance had been seen in these children with fever, myalgia and cough. hmpv was first identified in 2001 in children with respiratory tract disease (23). we detected 1 hmpv in 2 year old female child with rsv infection who had kawasaki syndrome and hospitalized in the pediatric ward. studies have reported high coinfection rates with these two viruses (24). similar to the catherine m. report, which showed hmpv distribution frequently in the winter months (25), we detected the only hmpv of this study on february. fever and cough were the clinical signs and symptoms of this child. rtis are common in young children, decrease in frequency with age, and predominate in boys (26, 27) . this age distribution was the same in the present study which demonstrated a higher incidence of rti in children younger than 1 year old. there was male predominance in this study but it was not significant. the relative frequency of each viral agent and the pattern of occurrence were similar to those described in previous reports (28, 29) . it is difficult to formulate a single theory to explain the epidemiology of viral infections at different times of the year. the most appropriate theory may be that, by staying indoors in cold weather, families promote the spread of virus infections which rely on the droplet transmission within confined spaces (30). susceptible children could acquire the virus from older children in whom manifestations of viral respiratory tract infections are mild (31) . in summary, respiratory virus infections (rvi) are a common cause of morbidity and mortality worldwide. viral co infections and recently discovered viruses are involved in a significant percentage of acute rvi. studies are being conducted to determine whether dual respiratory virus infections carry an increased risk for adverse outcomes or increased hospital stays for pediatric patients. better understanding of the epidemiology of rvi may be used for timely specific antiviral therapy, prophylactic and vaccination. this study described the demographic and clinical presentations of specific rvi in 202 children under the 6 years of age in iran. monitoring is necessary in a broader population and for a longer period of time in order to better delineate the clinical and epidemiological behavior of respiratory viruses in children. although routine surveillance of respiratory viruses might not seem to be mjiri, vol. 28 peres cd cde a. factors associated to hospitalization of children under five years of age epidemiology and seasonality of respiratory tract virus infections in the tropics human bocavirus infection in young children in the united states: molecular epidemiological profile and clinical characteristics of a newly emerging respiratory virus epidemiology and clinical characteristics of respiratory syncytial virus infections in jordan molecular evolution and circulation patterns of human respiratory syncytial virus subgroup a: positively selected sites in the attachment g glycoprotein development of three multiplex rt-pcr assays for the detection of 12 respiratory rnaviruses rapid and sensitive method using multiplex real-time pcr for diagnosis of infections by influenza a and influenza b viruses, respiratory syncytial virus, and parainfluenza viruses 1, 2, 3, and 4 genescan reverse transcription-pcr assay for detection of six common respiratory viruses in young children hospitalized with acute respiratory illness detection and identification of human parainfluenza viruses 1, 2, 3, and 4 in clinical samples of pediatric patients by multiplex reverse transcription-pcr comparison of multiplex nested rt-pcr with virus isolation for detection of influenza viruses a and b molecular identification of adenoviruses in clinical samples by analyzing a partial hexon genomic region simultaneous detection of fourteen respiratory viruses in clinical specimens by two multiplex reverse transcription nested-pcr assays multiple viral respiratory pathogens in children with bronchiolitis clinicalepidemiological evaluation of respiratory syncytial virus infection in children attended in a public hospital in midwestern brazil role of respiratory viruses in acute upper and lower respiratory tract illness in the first year of life: a birth cohort study molecular detection of respiratory viruses in children with acute respiratory disease in iran seasonal trends of human parainfluenza viral infections: united states prevalence of respiratory viruses, including newly identified viruses, in hospitalized children in austria epidemiological and clinical features of hmpv, rsv and rvs infections in young children respiratory viral infections among pediatric inpatients and outpatients in taiwan from 1997 to 1999 respiratory viral infections among pediatric inpatients and outpatients in taiwan from 1997 to 1999 the pathogenesis of respiratory syncytial virus disease in childhood we would like to thank the entire staff of the national influenza centre, school of public health tehran university of medical sciences. this study is funded by tehran university of medical sciences (number: 240.2694).this study is funded by tehran university of medical sciences (number: 240. 2694). key: cord-273620-gn8g6suq authors: szczawinska‐poplonyk, aleksandra; jonczyk‐potoczna, katarzyna; breborowicz, anna; bartkowska‐sniatkowska, alicja; figlerowicz, magdalena title: fatal respiratory distress syndrome due to coronavirus infection in a child with severe combined immunodeficiency date: 2012-11-30 journal: influenza other respir viruses doi: 10.1111/irv.12059 sha: doc_id: 273620 cord_uid: gn8g6suq please cite this paper as: szczawinska‐poplonyk et al. (2012) fatal respiratory distress syndrome due to coronavirus infection in a child with severe combined immunodeficiency. influenza and other respiratory viruses doi: 10.1111/irv.12059. coronaviruses have been demonstrated to contribute substantially to respiratory tract infections among the child population. though infected children commonly present mild upper airway symptoms, in high‐risk patients with underlying conditions, particularly in immunocompromised children these pathogens may lead to severe lung infection and extrapulmonary disorders. in this paper, we provide the first report of the case of a 15‐month‐old child with severe combined immunodeficiency and coronavirus hku1‐related pneumonia with fatal respiratory distress syndrome. severe combined immunodeficiency (scid) is a genetically and clinically heterogeneous group of the most severe primary immunodeficiencies, characterized by the absence of functional t lymphocytes resulting in profound impairment of the cellular and humoral adaptive immunity. depending on the genetic defect, b lymphocytes and natural killer (nk) cells may be present or absent and this feature constitutes the basis for the classical division into t-b + scid and t-b)scid, with further subdivisions into nk+ and nk) disorders. 1 respiratory tract infection is a common manifestation in children in question and may be present within the neonatal period or in early infancy. opportunistic pathogens may lead to rapidly progressive, fatal interstitial pneumonitis accompanied by hyperinflation resulting from small airway obstruction or to persistent bronchiolitic presentation. apart from pyogenic bacteria, such as pseudomonas aeruginosa, stenotrophomonas spp, burkholderia spp, as well as mycobacteria and fungi, in particular pneumocystis jiroveci, respiratory viruses like respiratory syncytial virus (rsv), adenovirus, parainfluenza virus, human metapneumovirus (hmpv) and other viruses -cytomegalovirus (cmv), varicella-zoster virus (vzv), and epstein-barr virus (ebv) are associated with severe pneumonia in scid children. 2 human coronaviruses (hcov) hcov-229e and hcov-oc43 and related new strains hcov-nl63 and hcov-hku1, identified after the epidemic outbreak of severe acquired respiratory syndrome (sars) coronavirus, are likely to be common respiratory viruses in otherwise healthy children and were not implicated in severe lung infections in immunocompromised patients thus far. 3 in this report we present the case of a child with delayed-onset scid and fatal respiratory coronavirus infection. a 15-month-old girl was referred to the university hospital due to persistent fever and interstitial pneumonitis for the purposes of diagnosis and treatment. she was the first child of young, non-consanguineous parents, born from the first pregnancy which was terminated in the 39th week of gestation using cesarian section surgery because of condylomata acuminata due to human papilloma *the institution to which the work should be attributed. virus (hpv) infection in the mother. in early infancy, mild eczema on the child's face, in the perioral and periorbital areas was observed. by the end of the first year of life the child had thrived and had not suffered from any severe infections. she received live bcg vaccine, tetanus and diphtheria toxoids, inactivated pertussis, inactivated poliomyelitis, recombinant hepatitis b and conjugated pneumococcal as well as haemophilus influenzae b vaccines without adverse reactions after the immunizations. on admission the child demonstrated paroxysmal nonproductive cough and clinical signs of respiratory insufficiency along with injected oral pharyngeal mucosa and dry erythematous lips. fine papular skin eruption affecting the face was observed. neither peripheral lymph nodes nor internal organs of the abdominal cavity were palpable. during hospitalization, lymphopenia, anemia, and thrombocytosis (the peak platelet count was 778 · 10 9 ⁄ l) as well as increased levels of inflammatory markers and coagulopathy were found in laboratory tests. dilation of the right coronary artery was revealed in echocardiography, giving rise to the suspicion of kawasaki disease and the instituting of treatment with aspirin and immunoglobulins. despite intensive pharmacotherapy with antibiotics, trimethoprime ⁄ sulfamethoxazole, acyclovir and antifungal agents, rapid deterioration of the child's clinical state and the exacerbation of respiratory insufficiency accompanied by progression in radiological features of the respiratory distress syndrome (rds) occured. on a chest x-ray, massive alveolar and interstitial infiltrations with bilaterally decreased aeration of the lung fields and blurred borders of the diaphragm and the heart shape (as shown on figure 1 ) were discernible. differential diagnostics that included examinations of the tracheal aspirate samples aimed at infectious agents were carried out. infections with viruses -rsv a and b, parainfluenza viruses 1,2, and 3, influenza a including h1n1 subtype and b, adenoviruses, cmv, ebv, mpv, rhinovirus, human immunodeficiency virus (hiv), with bacteria, such as streptococcus pneumoniae, haemophilus influenzae, legionella, bordetella pertussis, mycoplasma pneumoniae, chlamydophila pneumoniae, as well as with fungi -candida spp. and p. jiroveci were excluded based on negative pcr examinations, whereas the human coronavirus hku1 -rna proved positive. peripheral blood lymphocyte flow cytometric immunophenotyping revealed a total lack of expression of antigens characteristic for cd4 and cd8 t-cell subsets as well as nk cells along with the presence of functionally immature transitional and naïve b cells. this scid phenotype was subsequently confirmed by bone marrow flow cytometric evaluation. however, the presence of few nk cells was revealed, indicating for t-b + nk + scid. intensive therapy and mechanical ventilation conducted in the department of pediatric anesthesiology and intensive care did not contribute to either clinical or radiological improvement and the child died because of multiorgan failure. analysis of the available data concerning the effects of non-sars human coronaviruses (hcov) in children suggests that their clinical relevance in children is substantial, particularly in the hospital settings, even though the incidence of hcov airway infections are generally less frequent than with other viruses which have an established role in respiratory disease, such as rsv and influenza. 4 however, detailed epidemiological data on the prevalence of hcov infections in children are discordant, ranging from 2ae5% of nl63 strain in young children with bronchiolitis reported by ebihara et al. 5 to 18% in the study by vabret et al. 6 in different age groups of the child population. moreover, seroconversion with regard to hcov-229e and above-mentioned hcov-nl63 in young children was much higher and was estimated to 42ae9-50% and 75%, respectively. 7 the characteristics of clinical manifestation of coronavirus respiratory tract infection are predominantly reliant on case reports, and in otherwise, healthy children are comparable with bronchitis, bronchiolitis, and pneumonia due to other viral infections. the epidemiological study by kuypers et al. 8 indicated that a considerable proportion of coronavirusinfected children had underlying chronic central nervous system, cardiovascular, pulmonary, allergic, and renal or hepatic conditions and diseases. these authors also paid attention to immunocompromised pediatric patients with acute lymphocytic leukemia and organ transplant recipients as a high-risk group for the development of severe lung disfigure 1 . the chest x-ray of a 15-month-old child with severe combined immunodeficiency and respiratory distress syndrome due to coronavirus hku1 infection. note the massive alveolar and interstitial infiltrations, with bilaterally decreased aeration of the lung fields and blurred borders of the diaphragm and the heart shape. ease. however, it is worth noting that coronavirus respiratory infections have not been described in children with genetically determined immunodeficiencies thus far and this is the first report of a documented hcov-hku1-related pneumonia with the rds in a child with scid. it is also interesting to note that the preliminary clinical diagnosis in this patient was kawasaki disease, what is consistent with the hypothesis by esper et al. 9 regarding the association between kawasaki disease with hcov infection, supported by identification of the 'new heaven' coronavirus (hcov-nh) in 72ae7% of respiratory specimen from affected children. the identification of hcov-hku1 provides a novel insight into the epidemiology and clinical implications of coronavirus infections in severely immunocompromised children and indicates for consideration of this pathogen-related etiology of respiratory infection in scid. further, epidemiological studies are necessary to define the impact of hcov on lung disease in children with immunodeficiencies. the expanding clinical and immunological spectrum of severe combined immunodeficiency pneumonia in normal and immunocompromised children: an overview and update the widening scope of coronaviruses effects of coronavirus infections in children detection of human coronavirus nl63 in young children with bronchiolitis human coronavirus nl63 seroepidemiology of group 1 human coronaviruses in children clinical disease in children associated with newly described coronavirus subtypes association between a novel human coronavirus and kawasaki disease the assistance of dr husam samara from the department of immunology for the flow cytometric immunophenotyping of peripheral blood and bone marrow leukocytes is acknowledged. the authors have no competing interests. key: cord-275512-7yik78yc authors: lojkić, ivana; krešić, nina; šimić, ivana; bedeković, tomislav title: detection and molecular characterisation of bovine corona and toroviruses from croatian cattle date: 2015-08-13 journal: bmc vet res doi: 10.1186/s12917-015-0511-9 sha: doc_id: 275512 cord_uid: 7yik78yc background: bovine coronavirus (bcov) together with bovine torovirus (btov), both members of the coronaviridae family, order nidovirales are the most common viral enteric pathogens. although studied separately, their joint occurrence and the molecular diversity in cattle in croatia have not been investigated. methods: a survey is carried out on 101 fecal samples from diarrheic young and adult cattle during the 3-year period from i) one large dairy herd, ii) four small herds and iii) three nasal and paired fecal samples from calves with symptoms of respiratory disease. samples were submitted to rt-pcr and sequencing for bcov nucleocapsid gene, bcov spike gene and btov spike gene. results: bcov was detected in 78.8 % of fecal samples from symptomatic cattle and three nasal and paired fecal samples from calves with respiratory symptoms. btov was detected in 43.2 % of fecal samples from symptomatic cattle and a fecal sample from calves with respiratory symptoms. molecular characterisation of those viruses revealed some nucleotide and aminoacid differences in relation to reference strains. conclusions: btov should be regarded as a relevant pathogen for cattle that plays a synergistic role in mixed enteric infections. diarrhea is an important disease affecting cattle worldwide. together with the bovine rotavirus (brov), bovine coronavirus (bcov) and bovine torovirus (btov), both members of the coronaviridae family, order nidovirales [1] are the most common viral enteric pathogens. they both cause diarrhea and respiratory-tract infections in calves as well as in adult cattle [2] [3] [4] [5] . coronaviridae members are enveloped viruses with non-segmented positive-sense single stranded rna genome [6] . these viruses share the same basic genome organization and similar replication strategies. however, there are marked differences in genome size, host range, and virion architecture [1, 7] and there is no antigenic relationship between these two viruses. the virions of corona and toroviruses contain four and five structural proteins, respectively: the spike (s), the membrane (m), the haemagglutinin-esterase (he) protein, the nucleocapsid (n) protein and the small envelope (e) protein [8] . the latter is not present in toroviruses. another specificity of some coronaviruses is the internal nucleocapsid orf coding for i protein [9] . variations in host range and tissue tropism of coronaviruses are attributed to the spike (s) glycoprotein [10] . this protein is cleaved by an intracellular protease into two functional domains [10] . the peripheral s1 subunit is responsible for virus binding to host-cell receptors [11] , induction of neutralizing antibodies [12] , and haemagglutination activity [13] . the s1 sequence is variable, mutations in this region have been associated with altered antigenicity and virus pathogenicity [14] and this region has been exploited as a target to study the molecular epidemiology of bcov infection [15, 16] . the sequence of the s2 subunit is more conserved and this subunit is responsible for cell membrane fusion activity [17] . bcov infection has a high morbidity but a low mortality and is found worldwide among cattle of all ages [6] . outbreaks typically occur in autumn and winter [18, 19] . economic losses can be heavy due to a marked reduction in milk yield [20, 21] . btov, formerly called breda virus, was originally isolated from diarrheic calves in breda, iowa, in 1979. until today, btov was described in diarrhoeic calves in various countries [4, 2, [22] [23] [24] [25] [26] 27] . the faecal prevalence of btov in calf diarrhea ranges from 2.9 % in south korea [25] to 36.4 % in southern ontario, canada [22] . there are no available published data about the viral agents that are involved in calf diarrhea, although reports of the farmers complaining in calf diarhhea are very often. in this work we investigated fecal samples of cattle from one large dairy herd and from four small farms during the 3-year period. we also characterised nasal and paired fecal samples from calves with symptoms of respiratory disease. so for the first time, the occurrence and the molecular phylogeny of bcovs and btovs in selected herds from croatia are assessed. this statement confirms that sampling for the purpose of this research was performed as non experimental clinical work with respecting the rules of veterinary profession. all samples were collected by veterinarian nina krešić, license number 2202. sampling was performed strictly on the owner request. this statement is an annex to ethical committee permission of veterinary faculty university of zagreb; number: 251-61-01/139-11-72. fecal samples from 101 diarrheic animals were collected from march 2010 to may 2012. samples originated from calves and adult cattle from one large dairy herd (designated as "b") in eastern croatia (n = 65) and four small family farms in central croatia (designated as "k"), all mixed dairy-beef production (n = 35). three nasal swabs were collected in 2011 in east croatia from calves showing respiratory symptoms (designated as "d"). a paired pooled fecal sample was taken from the same calves. all sampled cattle were showing clinical signs of enteric infection except those designated as d71-d73 where respiratory signs were present. fecal samples were diluted 1:10 in minimal essential medium (ph 7.4; life technologies, usa) with addition of 1 % antibiotic antimycotic solution (sigma-aldrich). suspensions were centrifuged at 12,000 g for 15 min at 4°c and only the supernatants were used in the assays. all fecal samples were also tested by rt-pcr for rotavirus-a [27] . nasal samples were tested by pcr/rt-pcr for bovine herpes virus type 1 (bhv-1) [28] , bovine respiratory syncytial virus (brsv) [29] , bovine parainfluenza virus type 3 (bpiv-3) (in-house method) and bovine viral diarrhea virus (bvdv) [30] . viral rna was extracted from samples using qiaamp®-viral rna (qiagen, hilden, germany), according to manufacturer's instructions. cdna synthesis were performed with moloney-murine leukaemia virus reverse transcriptase (m-mlv rt) (invitrogen, usa) and random primers (50 ng/ll) (invitrogen) in a 20 ul final reaction volume. the cdna of each sample was screened for the bcov, btov and brov-a genome using the primers described in table 1 . pcr reactions were performed using gotaq™ green master mix (promega, usa), according to manufacturer instructions. a total of 16 samples were chosen for sequencing; all samples were sequenced for partial bcov n and s gene, and five of these samples were sequenced for btov s gene as well. eight samples were from large dairy farm in eastern croatia (b27/10, b30/10, b32/11, b37/11, b3492/11, b34649/11, b60853/11, b6075/ 12); four samples were from small family farms in central croatia (k12/10, k658/10, k5220/11, k6578/11), and four from calves with respiratory symptoms (d71-d73f). amplified pcr products were purified using exosap (usb, staufen, germany) and direct sequenced using the pcr primers in both directions by macrogen inc. (seoul, korea). nucleotide sequences generated in this study have been submitted to genbank and were assigned the following accession numbers as listed in table 2 . sequences were aligned and compared to previously published bcov n, bcov s and btov s sequences, respectively. sequence identities of nucleotides as well as estimation of the evolutionary divergence between sequences were analyzed using bioedit and mega6 [31] software, respectively. the neighbour-joining (nj) trees were obtained using mega6 program with the evolutionary model set to tamura-nei + gamma for spike gene analysis and kimura-2 parametar for n gene. estimation of best-fit model by hierarchical likelihood ratio tests (hlrts) and approximate akaike information criterion (aic) was performed with jmodeltest v.0.1.1. [32] . reliabilities of phylogenetic relationships were evaluated using nonparametric bootstrap analysis with 1000 replicates for nj analysis. bootstrap values exceeding 70 were considered well supported. the (table 2) . samples were chosen to cover three-year period. comparative analysis of the bcov n sequences (nucleotides (nt) 124-485; n protein amino acid (aa) 42-165; i protein aa showed that all croatian strains obtained in 2010-2012 shared a high identity both at the nt level (98-100 %) and at the deduced n protein aa level (98.3-100 %). sequences from eight samples from large dairy farm designated as "b" in the eastern region were mutually 100 % identical and most similar to italian strain 179/07-11 bubalus (eu019216) and irish rvlc9 (kf272913) (99,1 %). fecal samples designated as "k" and fecal and nasal samples "d" were more similar to human enteric isolates ok-0514-3 and 4408 (fj415342) (98.8-99.1 %) but phylogenetically clustered with other croatian strains and to italian 179/07-11 and irish rvlc9 (fig. 1a) . the bcov strains obtained from respiratory and enteric disease did not show any consistent nucleotide differences in the sequenced n region. the bcov strains obtained from respiratory disease showed two aa differences in the sequenced s1 region: (408 ser-ile, 470 ile-val). comparative analysis of the 975 nt bcov s sequence (nt 1258-2230; aa 387-710) showed that all croatian strains shared a high identity both at the nt level (97.7-100 %) and at the deduced aa level (97.5-100 %). "k" sequences were identical 98.6-99 %, "b" 99-100 % and "d" 99.6-100 %. our isolates were most similar to danish strain den03-2 (kf169914) (99.1-99.6 %) and italian strains bubalus 179/ 07-11, 438/06-tn and 339/06 (99.0-99.3 %) and were phylogenetically clustered with them (fig. 1b) . on aa level, our isolate k12 were 100 % identical to danish den03-2 (kf169914). analysis of the 608 nt btov s sequence (nt 65-671; aa 22-222) showed 93.2-99.8 % identity between our strains at the nt level and 83.7-99.5 % at the deduced aa level (except for the strain b60853/11 that were more divergent). croatian btov strains "b" did not show any consistent nt or aa differences in the sequenced s region. they were most similar to japanese na-7 (ab254073) (93.4-99.0 %) and were phylogenetically clustered with them (fig. 2) . isolate d71f, obtained from the feces of calf with respiratory symptoms were most identical to japanese aichi (96.5 %) and european b145 (96 %). this is the first report on the detection of torovirus infection in cattle, as well as molecular characterization of bcovs and btovs in croatia. we determined the partial n and s gene sequences of bcov and partial s gene sequences of btov from i) one large dairy herd during 3-year period ("b"), ii) four small family farms ("k") and iii) paired fecal and nasal samples from calves entered into feedlot ("d"). bcov vaccination is not practiced in croatia. all croatian bcov n and s gene sequences are clustered together and with sequences from italy, denmark and sweden, separately from the cluster with respiratory strains and the reference strain mebus (fig. 1) . in this cluster they were further grouped geographically as "b", "k" or "d" subgroup. the similar clustering is obtained when aa sequences for n and i proteins were analysed (data nor shown). all sequences from the same subgroup were mutually identical when n gene was analysed. more diversity is recognized when s gene was analysed, but sequences from large dairy herd ("b") were still 99-100 % identical. this lead to the conclusion that the same bcov strain circulated for extended period of time within one herd. in our investigation, we covered the s region from aa 387-701 which covers the previously reported hypervariable region (aa 452-593) [33, 34] but not the s cleavage site (aa 764-768). samples obtained from small family farms ("k") showed less mutual identity on s (98.6-99 %) compared with those that came from the same herd ("b"). the bcov strains obtained from respiratory disease showed two aa differences in the sequenced s region: (408 ser -ile, 470 ile -val), but compared to paired fecal sample, no aa differences and only two nt differences were found. previous researchers demonstrated that some bcov strains isolated from the respiratory tract had different biological, antigenic [35] [36] [37] and genetic [38, 39] properties compared with enteric bcov strains, whereas others did not detect any consistent differences [40, 41] and some suggested that the same strains of bcov cause natural outbreaks of respiratory and enteric disease [20, 21, 42, 43] . our findings, although based on a minimal data are in agreement with the latter fact; there were no differences between respiratory/fecal sample pair. still, the identified change in amino acid 408 could affect the aa polarity (hydrophilic to hydrophobic). regarding the herd designated as "d", outbreak of respiratory disease followed soon after entry into a feedlot. those calves were also rt-pcr positive to brsv but negative to bhv-1, bpiv-3 and bvdv. therefore, it was a typical scenario of bovine respiratory complex (brc) as a consequence of stress from transport and change in husbandry. the percentage of btov infections in the present study ( [22] . still, conclusions based on these frequencies are not reliable because this study was not originally planned as an epidemiological study. according to previous study [45] , btov alone has been shown to act as a primary enteric pathogen in cattle. in our study, btov was found only in co-infection with bcov and brv-a ( table 2) . it is noteworthy to say that co-infection with brov-a had not influence on bcov and btov sequence diversity. despite the fact that btov causes diarrhea and respiratory infections in cattle of all ages [6] our isolates from respiratory disease were negative. this also means that respiratory btov infection cannot be excluded if more samples has been analysed. croatian btovs showed moderate to high degree of nucleotide (87.1-99.8 %) and amino acid identity (83.7-99.5 %). comparison of our sequences with one available btov s gene sequence from europe (aj575373) and four sequences from japan showed high degree of sequence identity (93.4-99.0 %) between our strains from large dairy farm ("b") and japanese strain na-7 (ab254073). on contrary, sequence from feces of calves with respiratory symptoms were more similar to european strain b145 (aj575373) (96 %), and japanese strain aichi (96.5 %). phylogenetic analysis revealed that our sequences of btov s gene from symptomatic animals are more closely related to sequences from japan than to the breda 1 strain and european strain (fig. 2) . still, fecal isolate from herd with respiratory disease clustered with european and other japanese strains and was more related to breda 1 (fig. 2) . despite relatively small number of samples investigated, we can clearly conclude that bcovs and btovs (together with rotaviruses) are common enteric pathogens of cattle of all ages and production categories in croatian herds. required steps in herd management should include raising awareness of the adequate and timely colostrum intake by the calf as well as vaccination of the cows with one of the commercially available vaccine before calving. although on minimal data, we have proved that the same bcov strain circulated for extended period of time within one herd but different strains circulated in different herds. molecular characterisation of those viruses revealed some nt and aa differences in relation to reference strains, but generally, croatian sequences are clustered with other, mainly european btov and bcov isolate sequences. nidovirales: a new order comprising coronaviridae and arteriviridae association between infection of the respiratory tract attributable to bovine coronavirus and health and growth performance of cattle in feedlots isolation of respiratory bovine coronavirus, other cytocidal viruses, and pasteurella spp. from cattle involved in two natural outbreaks of shipping fever first detection and molecular diversity of brazilian bovine torovirus (btov) strains from young and adult cattle association of enteric shedding of bovine torovirus (breda virus) and other enteropathogens with diarrhea in veal calves bovine coronavirus infection the genome organization of the nidovirales: similarities and differences between arteri-, toro-, and coronaviruses the molecular biology of coronaviruses the nucleocapsid protein gene of bovine coronavirus is bicistronic coronavirus spike proteins in viral entry and pathogenesis localization of neutralizing epitopes and the receptor-binding site within the amino-terminal 330 amino acids of the murine coronavirus spike protein a single amino acid change within antigenic domain ii of the spike protein of bovine coronavirus confers resistance to virus neutralization the s protein of bovine coronavirus is a hemagglutinin recognizing 9-o-acetylated sialic acid as a receptor determinant two amino acid changes at the n-terminus of transmissible gastroenteritis coronavirus spike protein result in the loss of enteric tropism detection and isolation of winter dysentery bovine coronavirus circulated in korea during 2002-2004 molecular epidemiology of bovine coronavirus on the basis of comparative analyses of the s gene the s2 subunit of the spike glycoprotein of bovine coronavirus mediates membrane fusion in insect cells detection and characterization of bovine coronaviruses in fecal specimens of adult cattle with diarrhea during the warmer seasons respiratory disease associated with bovine coronavirus infection in cattle herds in southern italy experimental reproduction of winter dysentery in lactating cows using bcov -comparison with bcov infection in milk-fed calves bovine respiratory coronavirus the significance of bredavirus as a diarrhea agent in calf herds in lower saxony detection of bovine torovirus in fecal specimens of calves with diarrhea from ontario farms torovirus detection in faecal specimens of calves and pigs in hungary: short communication phylogenetic and evolutionary relationships among torovirus field variants: evidence for multiple intertypic recombination events molecular epidemiology of bovine toroviruses circulating in south korea epidemiological analysis of bovine torovirus in japan evaluation of a human group a rotavirus assay for on-site detection of bovine rotavirus detection of bovine herpesvirus type 1 in blood from naturally infected cattle by using a sensitive pcr that discriminates between wild-type virus and virus lacking glycoprotein restriction enzyme analysis of rt-pcr amplicons as a rapid method for detection of genetic diversity among bovine respiratory syncytial virus isolates genetic typing of croatian bovine viral diarrhea virus isolates. vet arhiv mega6: molecular evolutionary genetics analysis version 6.0 phylogenetic model averaging comparative sequence analysis of a polymorphic region of the spike glycoprotein s1 subunit of enteric bovine coronavirus isolates analysis of the s spike (peplomer) glycoprotein of bovine coronavirus synthesized in insect cells antigenic variation among bovine enteric coronaviruses (becv) and bovine respiratory coronaviruses (brcv) detected using monoclonal antibodies isolation of bovine respiratory coronaviruses from feedlot cattle and comparison of their biological and antigenic properties with bovine enteric coronaviruses antibody responses to respiratory coronavirus infections of cattle during shipping fever pathogenesis nucleotide and predicted amino acid sequences of all genes encoded by the 3' genomic portion (9.5 kb) of respiratory bovine coronaviruses and comparisons among respiratory and enteric coronaviruses bovine coronaviruses associated with enteric and respiratory diseases in canadian dairy cattle display different reactivities to anti-he monoclonal antibodies and distinct amino acid changes in their he, s and ns4.9 protein studies on the relationship between coronaviruses from the intestinal and respiratory tracts of calves comparison of the s genes and the biological properties of respiratory and enteropathogenic bovine coronaviruses failure to spread bovine virus diarrhoea virus infection from primarily infected calves despite concurrent infection with bovine coronavirus tracing the transmission of bovine coronavirus infections in cattle herds based on s gene diversity characterization of epidemic diarrhea outbreaks associated with bovine torovirus in adult cows experimental inoculation of adult dairy cows with bovine coronavirus and detection of coronavirus in feces by rt-pcr submit your next manuscript to biomed central and take full advantage of: • convenient online submission • thorough peer review • no space constraints or color figure charges • immediate publication on acceptance • inclusion in pubmed, cas, scopus and google scholar • research which is freely available for redistribution this research was supported by grant no. 048-0481186-1183 from the ministry of science, education and sports, republic of croatia. the authors declare that they have no competing interests.authors' contributions il and nk conceived the study, designed the experiments, performed all the experiments, analyzed the data and drafted the manuscript. iš participated in writing the manuscript. tb helped in study design, study implementation and manuscript revision. all the authors have read and approved the final manuscript. key: cord-284905-h8xovybl authors: musk, a.w. (bill); james, alan l.; palmer, lyle j.; ryan, gerard f.; lake, fiona; golledge, clayton l.; de klerk, nicholas h. title: respiratory infections and lung function in an australian aboriginal community date: 2008-02-05 journal: respirology doi: 10.1111/j.1440-1843.2007.01221.x sha: doc_id: 284905 cord_uid: h8xovybl background and objective: to investigate the association between serological evidence of past infections with common respiratory pathogens and lung function in members of an isolated community of aborigines from tropical coastal north‐western australia. methods: fev(1) and fvc were assessed by dry bellows spirometer. serum igg titres to 11 common respiratory pathogens were assayed. smoking history was assessed by questionnaire. reciprocal positive igg titres were taken as ≥10 for all pathogens with the exception of legionella spp. (≥40) and burkholderia pseudomallei (≥20). linear regression analysis examined associations between titres and lung function after adjustment for age, height, gender and smoking, separately for adults (age > 17 years) and children. results: an increased total number of positive igg titres was significantly associated with reduced fev(1) (p = 0.01) and fev(1)/fvc ratio (p = 0.01) suggesting the presence of airflow obstruction. this association was independent of age, gender, height, weight and smoking status. conclusions: the burden of past respiratory infections may be an important determinant of airway function in this aboriginal community. respiratory disease is a major cause of morbidity and mortality in australian aboriginal people; agestandardized hospital admission rates for chronic obstructive airways disease in western australia between 1983 and 1993 were 4.5 and 8.8 times greater for aboriginal men and women, respectively, compared with non-aboriginal western australians. 1 diseases of the respiratory system account for 12.2% of total hospital admissions for men and 8.7% for women. in 1991 the standardized mortality ratio for respiratory diseases was 5.2 for indigenous men and 6.0 for indigenous women. 2 similar observations have been made in the northern territory of australia and also indicate that this health disparity has not improved with time. 3 studies of members of an isolated coastal australian aboriginal community in the tropical kimberley region of western australia have shown that levels of lung function measured by fev1 and fvc were lower than those of australians of european descent. 4 the difference in these indices from age-and gendermatched australians of european descent was as much as 20%, even in asymptomatic individuals. [4] [5] [6] [7] airway hyper-responsiveness and the presence of respiratory symptoms were associated with lower levels of lung function, as was a history of 'asthma'. 4 the reduced levels of fev1 and fvc relative to height occurred in both children and adults. 4 therefore, reduced lung growth and/or a more rapid decline with age in adults may contribute to poor lung function. cigarette smoking, poor nutrition, overcrowding and other adverse external conditions, including respiratory infections, may be responsible for these differences between aboriginal and non-aboriginal populations. no evidence of ig deficiency or alpha-1 antitrypsin deficiency has been shown; in fact ig levels tend to be higher in aboriginal people compared with non-aboriginal australians. 8 a number of studies have shown increased frequency of bacterial upper respiratory infections and lower respiratory infections in australians of aboriginal descent with central australian aboriginal people having the highest rates of invasive pneumococcal disease, 9 as well as rates of haemophilus influenzae infection. 10 however, there is no direct evidence concerning the potential contributions of viral and other respiratory infections to reduced lung function in australian aboriginal people. studies in other populations have provided some evidence that the presence of viral respiratory infections may be associated with decreased spirometric indices or respiratory disease. in a cross-sectional community study in norway the level of serum respiratory syncytial virus (rsv) antibodies was associated with reduced fev1, suggesting that rsv infection or re-infection was an independent predictor of reduced lung function in those adults. 11 consistent with this observation, hogg showed that potential latent adenovirus infection may be associated with the presence of copd. 12 there is also evidence from experimental studies that latent adenovirus infection increases the inflammatory response to an acute exposure to cigarette smoke by increasing the numbers of macrophages and helper t cells in the airway epithelium. 13 the aim of the current study was to determine if there was an association between previous infection with common respiratory pathogens and lung function in a community-based sample of indigenous australians. study subjects were from an aboriginal community in the tropical coastal kimberley region of north-west western australia. a survey of the community was carried out over 10 days early in the dry season. seasonal effects on the traits measured could not be examined and were not considered. all individuals (n = 251) over the age of 5 years who were present in the community during the survey period were considered eligible and were invited to participate. informed personal or parental consent was obtained with the assistance of local community health workers who were employed in the project. approval for the study was granted by the human rights committee of the university of western australia and by the community council. 14 individual results were provided to each participant and the overall results have been communicated back to the community by way of written and verbal reports to its council and health staff. demographic information and histories of respiratory symptoms and smoking were assessed at interview using the british medical research council questionnaire. 15 questionnaires relating to children were administered to a parent (usually the mother). modifications to the questionnaire were made with the aid of the local medical officer by translation of questions into the local idiom as required. stated age was verified from community health records. cigarette smoking was defined as current or past use assessed by questionnaire; smokers (ever) reported smoking ն1 cigarette/day for at least a year. bronchitis was defined as cough or sputum production on most days for as much as 3 months each year. weight and standing height were measured without footwear. fev1 and fvc were measured in the sitting position using a dry wedge spirometer (vitalograph, model s, buckinghamshire, uk) according to the guidelines of the american thoracic society (ats). 16 data were excluded for five subjects who did not fulfil ats criteria for reproducibility after repeated attempts. venous blood was collected from all subjects. specific igg titres to adenovirus; influenza a; influenza b; parainfluenza 1; parainfluenza 2; parainfluenza 3; mycoplasma; rsv and psittacosis were assayed in serum by complement fixation tests. specific igg titres to legionella spp. and burkholderia pseudomallei were assayed by indirect immunofluorescence. positive reciprocal igg titres were taken as greater than 10 for all pathogens with the exception of legionella spp. (>40) and burkholderia pseudomallei (>20) as these are the conventional cut-off levels for complement fixation titres for these pathogens. 17 serology for common bacterial infections with haemophilus influenzae, streptococcus pneumoniae, and staphylococcus aureus was not performed because these organisms occur almost ubiquitously in people with chronic airway disease and are usually mucosal infections without significant humoral antibody responses. linear regression was used to model the effects of multiple covariates on the continuous spirometric outcomes. 18 two sets of models were derived separately for adults (18 years of age and over) and children, including terms for age (both linear and non-linear terms), gender, height, weight, smoking, and interactions with gender, in predicting fev1, fvc and the fev1/fvc ratio. smoking (ever smoked = '1', never smoked = '0') was considered as a binary covariate. residuals from both these sets of models (i.e. all ages combined) were then regressed on positive individual titres and then the sum of all positive (yes/no) igg titres. by using these methods, no reference was required to 'predicted normal' levels derived from external data sources that may not have been appropriate. all analyses were carried out using stata ver 9. 19 there were 113 male (49%) and 117 female participants with satisfactory lung function and serology data included in the study analysis. the mean age was 24.2 years (sem = 17.4 years). adults (subjects ն18 years) comprised 56% (n = 129) of the study population. there was a high prevalence of cough and sputum in both children and adults, particularly in men ( table 1) . the average number of positive igg titres per person was 3.5 (sd = 1.9; range = 0-8). positive titres for influenza a and b and legionella were more common among adults than children, whereas the response rates to other pathogens were comparable ( table 2 ). all those who were positive for mycoplasma (four children, seven adults) were also positive for influenza a and 87% (34 of 39) of those positive for parainfluenza 2 were also positive for rsv. using residuals from the separate models for adults and children adjusted for age, height, gender, weight to examine the robustness of these findings, analyses were repeated with the result for each pathogen in turn removed from the total positive titres. for fev1 the regression coefficients varied only 3-4 ml each way, with consistent sem and p-values. similar results were found for fev1% fvc. there was no significant separate association of lung function with rsv titres, as found in norway, although fev1 was reduced in those positive to parainfluenza 2 (coefficient -160 ml, sem = 62 ml, p = 0.01), and as described above, these subjects were nearly all positive to rsv. in this tropical aboriginal community, serological evidence of the burden of previous infections with known viral and other respiratory pathogens was associated with significant decreases in the levels of airway function, consistent with airflow obstruction. this association was independent of age, gender, height, weight and smoking status. other possible confounders such as diet, living conditions and alcohol consumption (alcohol was prohibited within this community), were not considered relevant especially as they were homogeneous in this population. influenza vaccination, offered annually to community members from the age of 15 years, partly explains why positive adult influenza titre rates were almost double those of children. it was not possible to determine which individuals took up the vaccination each year. vaccination status would tend to reduce the likelihood that an association between positive titres and lung function would be apparent, unless vaccination itself was detrimental to lung function. smoking status was included in the analysis, but measures of the amount of smoking were not (apart from age as a surrogate for duration), as the information was not considered reliable. it seems unlikely that passive smoking in childhood is important in this population as so much time is spent in the open air in this tropical community and the houses are not sealed. smoking in pregnancy by mothers could not be assessed but could be important in determining subsequent lung function; this would require a separate investigation. an association between smoking and the frequency of viral infections would be necessary for smoking to be responsible for our observations. no particular pathogen appeared to dominate the association between serological responses and lung function in this study; rather the number of positive titres was the factor that explained the observations. there was no way of determining the temporal relationship between any infection and changes in lung function. the results of the present study are consistent with the results of a cross-sectional community study in norway, 11 which found evidence that previous rsv infection is associated with lower levels of fev1. in the current study there was no separate association of rsv titre with the level of lung function, which is consistent with the concept that any effect of respiratory viral infections on lung function is not an exclusive effect of past rsv infection. the relationship of fev1 and fev1/fvc ratio with the total number of positive titres in this study suggests that repeated infections with common respiratory pathogens may have a cumulative detrimental effect on airway function or increase susceptibility to other agents such as tobacco smoke. the study does not indicate that any particular organism is likely to be more detrimental than any other; a positive titre to an organism does not indicate that it is more significant than any other. viral respiratory infections are known to be associated with subsequent bacterial infection and exacerbations of copd, 20 which may be an intermediate step in a process of accelerated rate of decline in lung function, leading to irreversible airflow obstruction. however, a role for exacerbations in causing accelerated decline in other populations has not been consistently observed, 21 and excessive rates of decline in lung function have not been directly demonstrated in aboriginal people in remote communities, although it is the subject of ongoing investigation in the community in which the present study was carried out. the previous demonstration of possible latent adenovirus infection in subjects with copd 12 is consistent with a direct effect of viral infections on the airways, but also consistent with abnormal airways such as may result from cigarette smoking giving rise to increased susceptibility to infection with viruses and other respiratory pathogens. additionally, vitalis et al. 13 showed that latent adenovirus-5 infection increased the inflammatory cell response to an acute exposure to cigarette smoke in guinea pig lungs, increasing the number of macrophages and helper t cells in the airway epithelium. recruitment of cd8 + lymphocytes in response to viral infections may cause pulmonary damage as suggested by o'shaughnessy et al. 22 and cannon et al. 23 fryer and jacoby 24 suggested that viral infections may decrease m2 muscarinic receptor function in airways, thereby increasing vagally mediated bronchoconstriction; the duration of this effect for acute viral infection is not known. viral infection with rhinovirus, coronavirus, influenza a and b, rsv and parainfluenza virus and infection with chlamydia have all been associated with exacerbations of asthma. 25 serology for h. influenzae, streptococcus pneumoniae and staphylococcus aureus was not performed in this study, nor was sputum culture available in this remote community, so that the effect of these organisms was not assessed as an a priori decision. the pathogens were selected because they are more likely to be primary pathogens. while selection bias may affect the results of crosssectional studies, all subjects over 5 years of age who were present in the community at the time of the study were assessed. full participation was therefore achieved. the age distribution of the study subjects reflects the increased mortality rates of indigenous australians 1,4,26 and reduces the power of the study to identify effects that may cause cumulative lung damage (as these would more easily be seen in older people). recall bias of questionnaire measures is also unlikely to have influenced our findings, as both the principal explanatory variables (specific antibodies to known pathogens) and the outcome variables (lung function measures) were based on objective measurements. some random misclassification of questionnaire responses may have occurred owing to language difficulties, although self-reported smoking status was validated with urinary cotinine measurements in a previous survey and showed concordance with both tobacco smoking and chewing histories (p le soeuf et al., unpubl. data, 1994) . the classification of participants in this study as previously infected or not, may have been subject to error because antibody titres decline with time following infection with these agents and because the patients were not examined for rhinoviruses and coronaviruses, which frequently cause the common cold syndrome, nor for chlamydia pneumoniae. this was because reliable antibody tests for these agents were not available. some unidentified previous infections may therefore have occurred in subjects and resulted in their being misclassified when examining the effect of total number of positive igg titres. however, this potential bias would, if anything, have tended to reduce the ability of the study to demonstrate differences between subjects with and without evidence of repeated previous respiratory pathogen infections. this study was performed over a short period (10 days) early in the dry season. therefore, seasonal effects were not examined. no seasonality of respiratory infections in isolated tropical aboriginal communities has been described. the results of this study provide some insight into the consistent observation that australian aboriginal people have lower levels of lung function than do australians of european descent. [4] [5] [6] [7] some of the observed difference may well be due to inherited factors, and therefore not amenable to intervention. however, it is likely that environmental factors are as important. 27 the prevalence of diagnosed asthma, and to a lesser extent allergy to aeroallergens, is low in aboriginal communities, 1,4 although 'asthma' admission rates are higher than for non-aboriginal people. 28 it has long been recognized that chronic respiratory disease is common in aboriginal communities. 1, 5 there is a high prevalence of bacterial infection of the sputum and invasive pneumococcal disease. 29, 30 the present study suggests that repeated episodes of respiratory infection with common respiratory viral pathogens may be directly or indirectly associated with impaired lung growth in childhood and/or excessive loss of lung function in adulthood. therefore, in addition to smoking control, targeted public health measures such as vaccination, early pharmacological (anti-biotic) intervention for (superimposed) bacterial infection, or efforts to reduce rates of transmission by the provision of better housing or nutrition could all result in reduced morbidity and mortality from respiratory disease in these people. this isolated community is similar to others in northern australia, although it is more geographically isolated than most. because alcohol is prohibited in the community the effects of aspiration pneumonias and their sequelae on lung function are less likely to confound the observations of this study. the study provides evidence of a detrimental effect of respiratory infections on lung function in individuals from any aboriginal community; this particular aboriginal community simply provided an opportunity to examine the relationship. as the relationship between infections and lung function is likely to be similar in other communities, this study provides an important public health message regarding preventive measures. the health and welfare of australia's aboriginal and torres strait islander peoples. australian bureau of statistics (cat. 4704.0), commonwealth of australia the impact of tobacco smoking and alcohol consumption on aboriginal mortality and hospitalization in wa 1983-1991 mortality in the northern territory 1979-1997. territory health services respiratory symptoms and lung function in aborigines from tropical western australia the physical health of aboriginal adults in bourke ventilatory standards for clinically well aboriginal adults respiratory function in aboriginal children the deviation of serum immunoglobulins g, a, and m in australian aboriginal infants etiology of acute lower respiratory tract infection in central australian aboriginal children the epidemiology of invasive haemophilus influenzae infections in children under five years of age in the northern territory: a three year study serum respiratory virus antibodies: predictor of reduced one-second forced expiratory volume (fev 1 ) in norwegian adults persistent and latent viral infections in the pathology of asthma the effect of latent adenovirus 5 infection on cigarette smoke-induced lung inflammation national health and medical research council. national health and medical research council guidelines on ethical matters in aboriginal and torres strait islander health research research council committee on the aetiology of chronic bronchitis. definition and classification of chronic bronchitis for clinical and epidemiological purposes standardisation of spirometry, 1987 update manual of clinical microbiology statistical methods in medical research statacorp. stata statistical software v9. stata corporation endogenous and exogenous reinfections with haemophilis influenzae in patients with chronic obstructive lung disease: the effects of antibiotic treatment on persistence the natural history of chronic airflow obstruction inflammation in bronchial biopsies of subjects with chronic bronchitis: inverse relationship of cd8+ t lymphocytes with fev 1 cytotoxic t cells clear virus but augment lung pathology in mice infected with respiratory syncytial virus effect of inflammatory cell mediators on m2 muscarinic receptors in the lungs respiratory viruses and exacerbations of asthma in adults overview of aboriginal health status in western australia. australian institute of health: aboriginal and torres strait islander health series respiratory morbidity and lung function in two aboriginal communities in western australia hospitalization of aboriginal and non-aboriginal patients for respiratory tract diseases in western australia, 1988-93 invasive pneumococcal disease in central australia invasive pneumococcal disease in the northern territory we are grateful to the members and staff of the community for willingly taking part in this study. community nurses monica frain and lex criddle (western australian department of public health) provided invaluable assistance in the collection of data. professor michael gracey and dr randolph spargo (western australian department of public health) facilitated planning and conduct of the survey. dr ian sampson (pathwest) conducted serological assays. we also thank our colleagues who assisted in the collection, analysis and interpretation of the data. this study was supported by the medical research foundation of western australia. key: cord-300019-8vxqr3mc authors: shi, ting; arnott, andrew; semogas, indre; falsey, ann r; openshaw, peter; wedzicha, jadwiga a; campbell, harry; nair, harish title: the etiological role of common respiratory viruses in acute respiratory infections in older adults: a systematic review and meta-analysis date: 2019-03-08 journal: j infect dis doi: 10.1093/infdis/jiy662 sha: doc_id: 300019 cord_uid: 8vxqr3mc acute respiratory tract infections (ari) constitute a substantial disease burden in adults and elderly individuals. we aimed to identify all case-control studies investigating the potential role of respiratory viruses in the etiology of ari in older adults aged ≥65 years. we conducted a systematic literature review (across 7 databases) of case-control studies published from 1996 to 2017 that investigated the viral profile of older adults with and those without ari. we then computed a pooled odds ratio (or) with a 95% confidence interval and virus-specific attributable fraction among the exposed (afe) for 8 common viruses: respiratory syncytial virus (rsv), influenza virus (flu), parainfluenza virus (piv), human metapneumovirus (hmpv), adenovirus (adv), rhinovirus (rv), bocavirus (bov), and coronavirus (cov). from the 16 studies included, there was strong evidence of possible causal attribution for rsv (or, 8.5 [95% ci, 3.9–18.5]; afe, 88%), flu (or, 8.3 [95% ci, 4.4–15.9]; afe, 88%), piv (or, not available; afe, approximately 100%), hmpv (or, 9.8 [95% ci, 2.3–41.0]; afe, 90%), adv (or, not available; afe, approximately 100%), rv (or, 7.1 [95% ci, 3.7–13.6]; afe, 86%) and cov (or, 2.8 [95% ci, 2.0–4.1]; afe, 65%) in older adults presenting with ari, compared with those without respiratory symptoms (ie, asymptomatic individuals) or healthy older adults. however, there was no significant difference in the detection of bov in cases and controls. this review supports rsv, flu, piv, hmpv, adv, rv, and cov as important causes of ari in older adults and provides quantitative estimates of the absolute proportion of virus-associated ari cases to which a viral cause can be attributed. disease burden estimates should take into account the appropriate afe estimates (for older adults) that we report. acute respiratory tract infections (ari), including pneumonia, constitute a substantial disease burden in adults and elderly individuals. respiratory viruses are detected more frequently than bacteria in adults with pneumonia [1] . the substantial contribution of viruses to ari hospitalizations among adults is being increasingly recognized [2, 3] . although influenza virus (flu) is the most widely recognized viral infection associated with respiratory illness, >25 viruses have been linked to pneumonia, causing a substantial disease burden in adults and elderly individuals. these include common pathogens such as rhinovirus (rv), respiratory syncytial virus (rsv), flu, human metapneumovirus (hmpv), parainfluenza viruses (piv), and human coronaviruses (covs) [2] . rsv is associated with a substantial disease burden in adults, especially among older adults (aged ≥65 years) [4] . moreover, adults hospitalized with rsv disease can develop severe respiratory complications [5] . rv has been associated with severe respiratory disease outbreaks in adults in long-term care facilities in several settings [6] . despite advances in diagnostic technology, defining the specific causes of viral pneumonia is challenging, particularly among older adults who may have lower viral loads and for whom viral diagnosis is frequently not considered and/ or testing is not performed [7] . therefore, it is necessary to measure concurrently the background prevalence of nasopharyngeal viral infection in a control (asymptomatic) group, to investigate the etiological role of viruses in older adults with ari to help inform decisions on prevention and management strategies. previously, we have conducted a systematic review to understand the etiological role of common respiratory viruses, focusing on children aged <5 years [8] . to the best of our knowledge, similar estimates for adults are lacking. therefore, we aimed to conduct a similar systematic review to identify all case-control studies since 1996 investigating the potential role of respiratory viruses in the etiology of aris in older adults aged ≥65 years. we conducted a systematic review across 7 databases (including 3 chinese databases) following the approach detailed in the prisma (preferred reporting items for systematic reviews and meta-analyses) guidelines [9] . tailored search strategies were developed and used to search the medline, embase, global health, lilacs, china national knowledge infrastructure (cnki), wanfang data, and chongqing vip databases (appendix). we further searched the reference lists of relevant articles for eligible articles. all searches were limited to between january 1996 and august 2017. no publication status criteria or language restrictions were used. we included studies that fulfilled the following selection criteria (supplementary figure 1) . three investigators (t. s., a. a., and i. s.) conducted independent searches of the english-language literature and extracted data by using standardized data extraction templates. one investigator (t. s.), whose first language is chinese, searched and extracted data from chinese-language databases (ie, cnki, wanfang, and cqvip). the protocol of this review was published in the prospero database (no. crd42017083332). the case group was defined as older adults with ari or pneumonia aged ≥65 years, adapted from world health organization integrated management of adolescent and adult illness [10] . the details of the definitions are displayed in supplementary table 1 . the control group was defined as older adults aged ≥65 years who were either healthy or did not have any respiratory symptoms. we categorized countries as either industrialized or developing, on the basis of 2015 criteria from the united nations children's fund [11] . we calculated odds ratios (ors) as the ratio of the odds of detecting each virus in older adults with ari or pneumonia to the odds of detecting each virus in healthy or asymptomatic controls, with accompanying 95% confidence intervals (cis). we used a continuity correction of 0.0005 if a virus was detected in one group but not the other [12] . this allowed calculation of an or for these instances and enabled inclusion in subsequent meta-analyses. using stata (version 13.0), we performed a meta-analysis of virus-specific ors and reported pooled meta-estimates with corresponding 95% cis, using the random effects model (ie, the dersimonian-laird method) because the included studies are heterogeneous in various aspects and are thus assumed to have different effect sizes [13] . the virus-specific attributable fraction among the exposed (afe) was used to quantify the etiological role of each virus in patients with ari. this is an estimate of the percentage of aris that can be attributed to each virus, in absolute terms [14] , and was calculated as 100 * [or − 1]/or, with a 95% ci (from the corresponding 95% ci of the or). moreover, for a specific virus, if all included studies did not report any virus detection in one group consistently (usually the control group), we assumed that a strong association indicating a possible causal role for this virus in ari could be concluded. in these circumstances, we considered that there was no need to run a meta-analysis that would only result in an extremely high or point estimate and an afe approaching 100%. we identified 4327 (239 from chinese databases) records through the literature search and 5 records from the reference lists of relevant articles. among them, only 16 studies (including 2 from chinese databases) fulfilled our inclusion and exclusion criteria ( figure 1 ) [1, [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25] [26] [27] [28] [29] . forty-three studies were excluded for a variety of reasons: no data specific to older adults ≥65 years old were available (n = 1), the case or control definitions were not fulfilled (n = 4), no applicable data for cases and controls were reported (n = 36), or serum was used as the clinical specimen (n = 2). seven studies were conducted within developing countries, while 9 were from developed countries (supplementary table 2 ). although the search was performed for articles published since 1996, all included studies were published since 2003. all included studies were case-control studies with adults who had ari or pneumonia in the case group and asymptomatic or healthy adults in the control group. methods varied among studies. among the case definitions used, 7 studies used ari or acute lower respiratory tract infection, while the others used (severe) pneumonia (n = 9). all studies investigated a control group, which had no respiratory symptoms, and in 3 studies healthy older adults (without acute illness) served as controls. of the case ascertainment methods used, 8 articles recruited the cases from inpatients; 1, from outpatients; 3, from general practices; 1, from the community; and 3, from mixed settings (outpatient settings and the emergency department, and both outpatient and inpatient settings). in 11 studies, controls were ascertained in hospital-based outpatient or clinic sites, whereas in 5 studies, controls were identified from the community. all studies collected a mixture of nasopharyngeal swab specimens, nasopharyngeal aspirates, nasopharyngeal washes, oropharyngeal swab specimens, and nasal/throat swab specimens as the clinical specimen. all studies used polymerase chain reaction analysis (pcr; in some, pcr was combined with serologic analysis or culture) as the diagnostic test. meta-analyses of virus-specific ors are reported as well as the corresponding attributable fractions among the exposed (supplementary table 3 ). rsv, flu (including flu a), hmpv, rv, and cov (also cov oc43 and 229e) were significantly more common in older adults with a diagnosis of ari or pneumonia than in asymptomatic or healthy controls (ors, 8 these viruses had statistically significant positive afes, which showed clear associations between these viruses and ari or pneumonia in older adults. moreover, piv (including piv1 and piv3; data for piv2 and piv4 were not available), flu b, and adv were only identified in cases consistently across all included studies (8, 5 , and 8 studies, respectively) and absent in control groups. thus, these viruses were all assumed to have strong associations with ari. only 2 studies had data available for bov, and although both reported virus detection in a greater proportion of cases than controls [18, 28] , the association remains a question for further research. a subgroup analysis was performed to explore the roles of viruses in ari with respect to region: developing countries and industrialized countries. the meta-estimate or was higher in industrialized countries as compared to developing countries in the case of flu (with overlapping 95% cis), while it was similar for piv, adv, and rv. there were insufficient studies to conduct a similar subgroup analysis for other viruses. a sensitivity analysis was performed to investigate the roles of these common viruses in older adults admitted to hospitals with ari or pneumonia [1, 16, 20, 22, 23, 25, 27, 28] . eight studies were included, and results are presented in supplementary table 4 . the meta-estimate or did not differ significantly from the previous estimate, in which cases from other settings (ie, outpatient and general practice settings) were also included. and cov in ari and pneumonia in older adults, thereby indicating a potential for substantive reductions in the number of ari cases if older adults were vaccinated against these viruses or treated with antivirals. for the other respiratory viruses studied, the role of bov in ari and pneumonia was uncertain because of the limited evidence available from the published literature, requiring more research to clarify its role in older adults with ari. a sensitivity analysis focusing only on older adults who were admitted to hospitals with ari or pneumonia did not differ significantly from our estimate, in which patients from all settings were considered. this might result from the limited number of studies available to provide a more robust sensitivity analysis. no studies calculated adjusted ors to account for confounding effects from age or season, which might compromise the actual association and should be considered in the study design in future research. these findings should inform the results of studies that seek to estimate the global, regional, and national burden of disease due to these viruses in older adults [30] . they show that rsv, flu, piv, hmpv, adv, rv, and cov are important causes of ari in older adults, and disease burden estimates should take into account the appropriate afe estimates (for older adults) that we report, rather than the afe estimates in children aged <5 years. there is considerable international attention on rsv-associated ari in older adults at this time, during which novel vaccine and antiviral strategies are being evaluated and prioritized [31, 32] , and more-accurate disease burden estimates (using these results) would help to inform future policies and interventions. the prevalence of virus detection from etiologic studies of pneumonia in adults is substantially lower than the detection rate in studies of children. the epic (etiology of pneumonia in the community) study team showed that the viruses were detected in 26% of adults who had been hospitalized with community-acquired pneumonia, compared with 73% of children who were admitted to the hospital [1, 33] . there are several reasons for such low levels of detection, such as the inability to obtain lower respiratory tract specimens, the use of diagnostic tests with insufficient sensitivity, the absence of appropriate diagnostic testing methods, the undetectability of the virus at the time of the study, and the presence of unknown pathogens that were not identified. the low rate of virus detection among adults who were hospitalized for pneumonia highlights the need for more-sensitive diagnostic approaches, innovative discovery of pathogens, and assessing viruses in the past history (weeks before the presence of disease) [34] . moreover, chronic obstructive pulmonary disease (copd) exacerbation is a very important cause of aris and hospital admissions [35] . only 7 of 16 studies included copd in the etiologic data, and this information was unclear in the remaining studies, which might have underestimated the role of viral infection in these patients. a previous etiological review focusing on young children aged <5 years [8] showed that rsv, flu (including flu a), piv, hmpv, and rv were significantly more common in children hospitalized with acute lower respiratory tract infection than asymptomatic controls. the associations of these viruses (except rsv) with ari and pneumonia were stronger among adults. this is in part because, in comparison to young children, the detection of viruses in the control group (ie, among individuals without respiratory symptoms or healthy controls) was less common in older adults, with the exception of rsv. several methodological issues could affect our results: sample size, age group, case ascertainment, clinical specimen, and diagnostic testing. although a thorough search has been performed across 7 databases, including 3 chinese-language databases, only 16 studies from the published literature were identified, which met our selection criteria. not every virus of interest was tested in each study. the number of studies available was even smaller when subgroup analyses and sensitivity analyses were performed. moreover, the sample size varied from 50 to 2320 adults in the case group and from 27 to 541 adults in the control group. the small sample size undoubtedly contributed to the imprecise 95% cis around the ors. thus, we may have failed to detect clinically significant ari-virus associations, owing to small sample sizes. we aimed to stratify the association between common respiratory viruses in adults with ari or pneumonia by age. however, most articles did not stratify and report data by age group. instead, they summarized the result for the entire age group, usually in adults aged >18 years. therefore, some of our meta-estimate ors may not be representative of older adults who are aged ≥65 years. since age might be a risk factor for ari in adults (the rate of severe ari increases as age advances), this could potentially affect the viral profile detected, introducing further heterogeneity [1] . fifteen of 16 studies used passive clinic or hospital based case ascertainment. among them, cases were recruited from inpatients, outpatients, emergency departments, or general practices, which might reflect different healthcare behavior and disease severity. also, since the episodes of ari and pneumonia were only diagnosed through routine care, this introduced bias, considering that testing was only done when the clinicians deemed it necessary to test. similarly, 5 studies used community-based controls, while another 11 studies recruited older adult controls from hospitals or general practices. hospital or clinical ascertained controls may not reflect the general population and may have other health conditions potentially affecting their viral carriage. moreover, recruiting controls who were selected as healthy or without respiratory symptoms could favor those who were not exposed to the respiratory virus (yielding a falsely high or). therefore, we consider that the ideal control group for these studies would be a random sample of an age-and sexmatched population of older adults who are from the same area of residence and studied at the same time as cases. all included studies obtained upper respiratory tract specimens (described as nasopharyngeal secretions). assays might have specimen-specific sensitivities and specificities for detecting viruses, which could lead to heterogeneity in the estimation of virus-specific rates. the sensitivity of using nasopharyngeal washes for detecting any virus in adults was found to be higher than that for using nasopharyngeal swab specimens, which in turn was higher than that for using oropharyngeal swab specimens (84.9%, 73.3%, and 54.2%, respectively) [36] . the limited use (due to ethical concerns and feasibility) of invasive procedures to obtain lower respiratory tract specimens directly from the lung also influenced the diagnosis of viral infection in adults with ari [37] . pcr and serology-based diagnostic testing are more sensitive for detecting respiratory viruses than other methods, such as antigen detection and culture. high sensitivity is important for accurate assessment of etiological contribution, particularly in older adults who may have a lower nasopharyngeal viral load and an atypical clinical presentation [7] . moreover, despite being uncommon, detection of viral coinfection (range across studies, 1%-10%) may tend to overstate the contribution of individual respiratory viruses (although dual or multiple infections, in which both or several viruses have etiological importance, are possible). bacterial coinfections (range across studies, 15%-26%) were also reported. with improving diagnostic methods, multiple etiological agents are increasingly identified simultaneously in older adults with ari, making the individual contribution of each agent difficult to define. viruses can be detected in individuals with no respiratory symptoms. this is often seen in volunteer challenge studies and in some community surveys [38, 39] . the detection of viruses in control groups without respiratory symptoms might be due to a nascent infection or a persisting colonization from a previous infection [40] . these factors will tend to result in true associations being attenuated. the fact that molecular detection of viruses in older adults with ari is higher than the detection rate in controls without respiratory symptoms may not necessarily indicate causation. alternative explanations should be considered first before causality can be concluded. these include the respiratory viral infection acting as a so-called innocent bystander, without a causal role, and serving only as a predisposing risk factor for ari. similarly, the absence of a positive association (and afe) does not mean that a virus is not a cause of ari. moreover, without establishing the temporal sequence of exposure and outcome, determinations of causality are less secure. therefore, the association between viruses and ari and pneumonia should be interpreted carefully. in conclusion, this review provides clear evidence that is suggestive of the potentially causal role of rsv, flu, piv, hmpv, adv, rv, and cov in older adults with ari and presents the first estimate of the proportion of ari cases that can be attributed to virus exposure. etiological studies, which simply report rates of viral identification as causal, should make attempts to interpret findings in terms of the proportion of ari cases among older adults in whom a respiratory virus is identified that can be attributed to this viral exposure. the resceu investigators are as follows national institute for public health and the environment) penta foundation) jeroen aerssens, veronique wyffels, and matthias cleenewerck (janssen) cdc epic study team. communityacquired pneumonia requiring hospitalization among u.s. adults viral pneumonia respiratory syncytial virus infection in elderly and high-risk adults modelling estimates of the burden of respiratory syncytial virus infection in adults and the elderly in the united kingdom high morbidity and mortality in adults hospitalized for respiratory syncytial virus infections rhinovirus outbreaks in longterm care facilities the diagnosis of viral respiratory disease in older adults aetiological role of common respiratory viruses in acute lower respiratory infections in children under five years: a systematic review and meta-analysis preferred reporting items for systematic reviews and meta-analyses: the prisma statement world health organization. integrated management of adolescent and adult illness (imai) the state of the world's children 2015: reimagine the future: innovation for every child what to add to nothing? use and avoidance of continuity corrections in meta-analysis of sparse data introduction to meta-analysis attributable risk percent in case-control studies a case-control study of acute respiratory tract infection in general practice patients in the netherlands respiratory viruses in adults with community-acquired pneumonia prospective evaluation of a novel multiplex real-time pcr assay for detection of fifteen respiratory pathogens-duration of symptoms significantly affects detection rate microorganisms in respiratory tract of patients diagnosed with atypical pneumonia: results of a research based on the use of reverse transcription polymerase chain reaction (rt-pcr) dna microarray method and enzyme-linked immunosorbent assay viral respiratory tract infections in adult patients attending outpatient and emergency departments respiratory viral detection in children and adults: comparing asymptomatic controls and patients with community-acquired pneumonia aetiological role of viral and bacterial infections in acute adult lower respiratory tract infection (lrti) in primary care respiratory virus is a real pathogen in immunocompetent community-acquired pneumonia: comparing to influenza like illness and volunteer controls incidence and characteristics of viral community-acquired pneumonia in adults a prospective, community-based study on virologic assessment among elderly people with and without symptoms of acute respiratory infection viral etiology of acute lower respiratory infection in adult inpatients detection on non-bacterium pathogen in 1232 cases of acute respiratory infection human coronavirus infections in rural thailand: a comprehensive study using real-time reverse-transcription polymerase chain reaction assays human bocavirus: a novel parvovirus epidemiologically associated with pneumonia requiring hospitalization in thailand detection of respiratory syncytial virus and human metapneumovirus by reverse transcription polymerase chain reaction in adults with and without respiratory illness estimates of the global, regional, and national morbidity, mortality, and aetiologies of lower respiratory tract infections in 195 countries: a systematic analysis for the global burden of disease study accessed 29 drug candidates and model systems in respiratory syncytial virus antiviral drug discovery cdc epic study team. communityacquired pneumonia requiring hospitalization among u.s. children better tests, better care: improved diagnostics for infectious diseases respiratory viruses in exacerbations of chronic obstructive pulmonary disease requiring hospitalisation: a case-control study identification of respiratory viruses in adults: nasopharyngeal versus oropharyngeal sampling lower respiratory tract virus findings in mechanically ventilated patients with severe community-acquired pneumonia time lines of infection and disease in human influenza: a review of volunteer challenge studies flu watch group. comparative community burden and severity of seasonal and pandemic influenza: results of the flu watch cohort study identification of respiratory viruses in asymptomatic subjects: asymptomatic respiratory viral infections we thank joris menten from janssen for reviewing the manuscript.financial support. this work was supported by the innovative medicines initiative 2 joint undertaking (grant 116019), which in turn receives support from the european union's horizon 2020 research and innovation programme and efpia. is the elected president of the british society for immunology, which is an unpaid appointment but provides support for travel and accommodation at some meetings. all other authors report no potential conflicts. supplementary materials are available at the journal of infectious diseases online. consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author. key: cord-276927-rxudwp2v authors: barbas, carmen sílvia valente; matos, gustavo faissol janot; amato, marcelo britto passos; carvalho, carlos roberto ribeiro title: goal-oriented respiratory management for critically ill patients with acute respiratory distress syndrome date: 2012-08-23 journal: crit care res pract doi: 10.1155/2012/952168 sha: doc_id: 276927 cord_uid: rxudwp2v this paper, based on relevant literature articles and the authors' clinical experience, presents a goal-oriented respiratory management for critically ill patients with acute respiratory distress syndrome (ards) that can help improve clinicians' ability to care for these patients. early recognition of ards modified risk factors and avoidance of aggravating factors during hospital stay such as nonprotective mechanical ventilation, multiple blood products transfusions, positive fluid balance, ventilator-associated pneumonia, and gastric aspiration can help decrease its incidence. an early extensive clinical, laboratory, and imaging evaluation of “at risk patients” allows a correct diagnosis of ards, assessment of comorbidities, and calculation of prognostic indices, so that a careful treatment can be planned. rapid administration of antibiotics and resuscitative measures in case of sepsis and septic shock associated with protective ventilatory strategies and early short-term paralysis associated with differential ventilatory techniques (recruitment maneuvers with adequate positive end-expiratory pressure titration, prone position, and new extracorporeal membrane oxygenation techniques) in severe ards can help improve its prognosis. revaluation of ards patients on the third day of evolution (sequential organ failure assessment (sofa), biomarkers and response to infection therapy) allows changes in the initial treatment plans and can help decrease ards mortality. acute respiratory distress syndrome (ards) is due to an increase in the pulmonary alveolar-capillary membrane permeability causing lung edema rich in protein and consequently acute hypoxemic respiratory failure in genetically susceptible patients exposed to determined risk factors [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] . a recent study showed that the del/del genotype (patients homozygous for the 4 base pair deletion in the promoter of nfkb1) is associated with an age-dependent increase in odds of developing ards (or 5.21, 95% ci 1. 35-20.0 ) and patients with the del/del genotype and ards also have increased hazard of 60-day mortality (hr 1.54, 95% ci 1.01-2.36) and more organ failure (p < 0.001) [15] . all age groups may be affected, although the syndrome has a higher incidence and mortality in older people [16] . the most common precipitating causes of ards are pulmonary infections, nonpulmonary sepsis, shock, gastric aspiration, thoracic trauma, fat embolism, near drowning, inhalational injury, cardiopulmonary bypass, drug overdose, acute pancreatitis, and high-risk trauma (especially traumatic brain injury) [17] . recent epidemiological studies suggested a variety of intrahospital risk factors for ards development such as multiple blood products transfusions, mechanical ventilation with high tidal volumes, excessive fluid resuscitation, and hospitalacquired pneumonia as well as high-risk surgeries (especially aortic vascular, cardiac, and acute abdomen); all risk factors are potentially preventable. chronic alcohol abuse, chronic liver disease, immunosuppression, hypoalbuminemia, and obesity are also all associated with the development of ards, whereas diabetes mellitus appears to be protective [17] . after exposure to a risk factor, there is an important activation of neutrophils and release of harmful mediators including cytokines (such as interleukins 1, 6, and 8 and soluble tumor necrosis factor-alpha receptors), proteases, reactive oxygen species, and matrix metalloproteinases leading to future damage. an overwhelming pulmonary inflammatory process is initiated leading to alveolar epithelial and vascular endothelial injury. alveolar epithelial injury of type i cells contributes to the pulmonary edema and the breakdown of this epithelial barrier exposes the underlying basement membrane, predisposing to bacteremia and sepsis. injury to type ii alveolar cells leads to an impairment of surfactant function with consequent collapse of the lungs. histopathologically there is diffuse alveolar damage with neutrophil infiltration, alveolar hemorrhage and hyaline membrane formation [18] [19] [20] [21] [22] . there are localized destruction and occlusion of the vascular bed of the lungs by intravascular thrombosis and an increment of the anatomical dead space resulting in an increase of arterial carbon dioxide associated with a poor outcome. fibrosis can be evident histologically as early as one week after the onset of ards and procollagen iii peptide, a precursor of collagen synthesis, can be elevated in bronchoalveolar lavage fluid of ards patients at the time of tracheal intubation, its increment being associated with a poor ards prognosis. vascular injury and remodeling may lead to pulmonary arterial hypertension which may compromise right ventricular function associated with a poor clinical outcome [13] . incorporation of modified risk factors such as acute increase of respiratory rate, presence of tachypnea, detection of pulse oximeter desaturation, increased necessity of oxygen supplementation, presence of low ph, acidosis, or hypoxemia in an arterial blood gas sample in clinical practice can improve the clinicians' ability to perform early diagnosis and prompt therapeutic intervention in ards [17] . the presence of these modified risk factors may alert physicians to avoid secondary hospital exposures, such as blood products transfusions, excessive fluid administration, infusion of potentially toxic drugs, high tidal volume mechanical ventilation, and gastric aspiration. implementation of ventilator associated pneumonia prevention bundles decreases the incidence of vap and can lower the incidence of ards [17] . implementation of automated ards electronic screening in usa hospitals such as "assist" (electronic alert from laboratory when the arterial blood gas analysis shows hypoxemia and the radiology department when chest x-ray shows bilateral pulmonary infiltrates) to identify intubated patients with ards in medical and surgical icus showed a sensitivity of 97.6% (95% ci, 96.8-98.4%) and specificity of 96.8% (95% ci, 96.8-98.4%) when compared to a manual screening algorithm that had a sensitivity of 57.1% (95% ci, 54.5-59.8%) and specificity of 99.7% (95% ci, 99.4-100%) in 1270 icu patients over a 21-week period during enrollment in ardsnet trials [23] . the results of this study indicated the advantages of having an in-hospital automated screening of ards over manual screening. the automated screening can increase the chances of ards diagnosis, alert the clinicians, and elicit the rapid response from the hospital team of intensivists to initiate clinical protocols and ards therapeutic interventions [24] . most hospitals and intensive care units worldwide use the standard criteria for the diagnosis of acute lung injury (ali)/ards: presence of acute hypoxemia (pao 2 /fio 2 less than 300 mmhg or 39.99 kpa for ali or less than 200 mmhg or 26.66 kpa for ards), bilateral infiltrates seen on a frontal chest radiograph that are consistent with pulmonary edema, and no clinical evidence of left atrial hypertension, or (if it is measured) a pulmonary artery wedge pressure (pawp) of less than 18 mmhg according to the 1994-1998 american-european consensus conference on ards (aecc) [25, 26] . this definition aimed to simplify and standardize the diagnosis of ards worldwide. however, in clinical practice, in order to detect and diagnose ali/ards cases, physicians must focus on patients' complaints, physical examination alterations, patients at risk of developing the disease, or patients presenting finger pulse oximeter desaturation. following the ali/ards clinical suspicion, physicians should order an arterial blood gas analysis and a chest radiograph to be able to confirm the ali/ards diagnosis. recent updates of ards definition such as the 2005 delphi consensus [8] or the berlin definition [27] were published in order to improve ards diagnosis criteria. the berlin definition reclassified ards as mild (pao 2 /fio 2 < 300 or 39.99 kpa), moderate (pao 2 /fio 2 < 200 or 26.66 kpa), and severe (pao 2 /fio 2 < 100 mmhg or 13.33 kpa) and removed the term ali and the necessity of a swan ganz catheter to access pawp. acute time frame was specified as the onset within 1 week of a known clinical insult or new or worsening respiratory symptoms chest radiography criteria were clarified and bilateral opacities consistent with pulmonary edema were maintained as the main radiological criteria of ards, but it was recognized that these findings could be demonstrated on ct scan instead of chest radiograph. the recent berlin definition of ards is a decisive step forward in refining the diagnosis of the syndrome, but pao 2 /fio 2 is influenced by ventilator settings and this fact should be considered; bilateral pulmonary infiltrates can be the result of a wide variety of acute lung diseases that should be better investigated. left and right ventricular function, pulmonary artery pressures, and volemic status could be better evaluated by bedside echocardiography and extravascular lung water can be measured using picco catheter, in order to evaluate the degree of pulmonary edema. predictors of mortality should be calculated at icu admission. with the information, the icu team can program a more careful treatment plan according to disease severity. the berlin definition shows better predictive validity for mortality compared to the aecc definition, but the absolute value of the area under the receiver operating curve is still too small (0.577), suggesting that some factors are still missing. further discussion and research are needed before we reach a comprehensive definition of ards. critical care research and practice 3 the typical findings of ards in a computer tomography reveal a heterogeneous bilateral pulmonary infiltrate predominantly in gravity-dependent regions of the lungs and more preserved lungs in nondependent lung regions. using quantitative analysis of the ct scan, the gravity-dependent pulmonary ards infiltrate is typically nonaerated lung tissue consistent with compressive atelectasis [28, 29] . lung weight assessed by ct scan is increased in ards and is correlated with the severity of the syndrome [27] . the finding of concomitant interstitial infiltrates suggests viral or mycoplasma, chlamydia or opportunistic pulmonary infections, or drug-induced lung disease. the differential diagnosis of bilateral pneumonia, alveolar hemorrhage, and acute interstitial lung disease such as acute interstitial pneumonia, hypersensitivity pneumonitis, acute eosinophilic pneumonia, and bronchiolitis obliterans with organizing pneumonia can be suggested by the characteristic ct scan findings of each specific disease [12] . the results of stepwise lung recruitment maneuvers as well as positive end-expiratory (peep) titration to keep the lungs open with minimal collapse can be assessed by computer tomography analysis [30] . this strategy is aimed at opening up the lungs and keeping the lungs open [31] as quickly and early as possible as postulated by lachmann [32] in order to have a huge improvement in lung function and avoid potential ventilator-induced lung injury. recently, our group reported the experience with maximal recruitment strategy (mrs) in 51 patients with ards. mrs consisted of 2-minute steps of tidal ventilation with pressure-controlled ventilation, fixed driving pressure of 15 cmh 2 o, respiratory rate of 10 breaths/minute, inspiratory/expiratory ratio of 1 : 1, and stepwise increments in peep levels from 10 to 45 cmh 2 o (recruitment phase). after that, peep was decreased to 25 cmh 2 o and, then, from 25 to 10 cmh 2 o (peep titration phase) in steps of 5 cmh 2 o, each one lasting 4 minutes. at each of the steps computer tomography image sequences from the carina to the diaphragm were acquired during an expiratory pause of 6-10 seconds. lung collapse was assessed online by visual inspection, for immediate clinical decision, and offline for quantitative measurements. mrs showed a statistically significant decrease in nonaerated areas of the ards lungs that was accompanied by a significant increment in oxygenation. the opening plateau pressure observed during the recruitment protocol was 59.6 (±5.9 cmh 2 o), and the mean peep titrated after mrs was 24.6 (±2.9 cmh 2 o). mean pao 2 /fio 2 ratio increased from 125 (±43) to 300 (±103; p < 0.0001) after mrs and was sustained above 300 throughout seven days. nonaerated parenchyma decreased significantly from 53.6% (interquartile range (iqr): 42.5 to 62.4) to 12.7% (iqr: 4.9 to 24.2) (p < 0.0001) after mrs. the potentially recruitable lung was estimated at 45% (iqr: 25 to 53), (figure 1 ). icu mortality was 28% and hospital mortality was 32%. the independent risk factors associated with mortality were older age and higher driving pressures (or higher delta pressure control). there were no significant clinical complications with mrs or barotrauma [33] . a better evolution of these ards patients with less necessity of oxygen supplementation in the recovery phase of the disease and a better quality of life must be tested in prospective, controlled clinical trials. a recent metaanalysis showing beneficial effects on mortality using higher peep levels compared with lower peep in ards patients corroborates the results of our clinical case series of ards patients submitted to mrs [33] . ards is a biphasic disease that progresses from an acute exudative phase, characterized by epithelial and endothelial injury, neutrophilic aggregation, formation of hyaline membranes, alveolar edema, and hemorrhage, to an organizing phase, characterized by regeneration and healing via resolution or repair with persistent intra-alveolar and interstitial fibrosis [11] . it is crucial to make the diagnosis of ards in the acute phase (preferably less than 72 hours) in order to make it possible to open up the lungs with recruitment maneuvers and keep the lungs open with sufficient peep levels to enable a more homogenous ventilation, minimizing the possible ventilator-induced lung injury (vili) triggers and allowing the recovery of the lungs [34] [35] [36] . a recent study analyzing 85 patients with ards graded into six findings according to the extent of fibroproliferation at the ct scan showed that higher ct scores were associated with statistically significant decreases in organ-failure free days as well as ventilator free days and were an independent risk factor for mortality (or = 1.2, 95% ci 1.06-1.36, p < 0.005) [37] . positron emission tomography with ( 18 f) fluorodeoxyglucose (fdg-pet) detects inflammatory cells and can assess lung inflammation in ards lungs helping in the understanding of ards physiopathology [38] [39] [40] . lung ultrasonography is a new helpful tool that can be performed at bedside without radiation exposure. thoracic ultrasound is widely used for diagnostic and therapeutic intervention in patients with pleural effusion and pneumothoraces. the assessment of lung recruitment and peep titration in ards patients at bedside using lung ultrasonography is a new promising technique [41] . currently, the two main limitations of this technique are its inability to detect lung overdistension and its operator-dependent characteristic. thoracic electrical impedance tomography (eit) is a highly promising imaging technique to apply at the bedside for peep titration in ards patients. new automated tools permit the calculation of the percentage of collapsed as well as overdistended lung tissue at decremental peep levels after lung recruitment maneuvers ( figure 3 ). the regional distribution of collapse and overdistension may provide insights about the lung pathology. this technique permits daily peep adjustments at the bedside and verification of tidal volume distribution, avoiding excessive end-expiratory collapse or tidal overdistention [42] [43] [44] [45] . one of the main sofa score during first 7 days after mrs * p < 0.005 advantages of this technique is the possibility of around the clock monitoring. further studies are needed to evaluate the clinical impact of these bedside techniques in ards patients' prognosis. randomized trials suggested that patients with acute hypoxemic respiratory failure are less likely to require endotracheal intubation when noninvasive ventilation (niv) is added to standard therapy [46] . however, most of these studies analyzed mixed causes of acute hypoxemic respiratory failure and reported the highest intubation rates for patients with ards (51 to 70%) and that the presence of ards was one factor independently associated with niv failure and higher mortalities rates (50 to 70%). recently, zhan and colleagues [47] analyzed 40 patients with ards randomly allocated to receive either noninvasive ventilation or high-concentration oxygen therapy through a venturi mask. noninvasive positive pressure ventilation decreased the respiratory rate and improved pao 2 /fio 2 with time. the proportion of patients requiring intubation and invasive mechanical ventilation was significantly lower in the noninvasive ventilation group (one of 21 versus 7 of 19; p = 0.02). therefore, noninvasive ventilation can be used as a first ventilatory support technique in selected patients with mild/moderate ards and a hemodynamic stable condition to avoid endotracheal intubation. a larger randomized trial, however, is required, with the need for intubation and mortality as the outcome of interest. a close-monitored initial trial of noninvasive ventilation should be considered in most mild/moderate ards patients, mainly the immunosuppressed ones with pulmonary infection in order to avoid intubation and invasive mechanical ventilation. however, early detection of collapse was more prominent in the right lung. after analyzing the sequence of eit images, the peep selected for this patient was 17 cmh 2 o, believed to represent the best compromise between collapse and overdistension. according to the ardsnet peep/fio 2 table, this patient had been ventilated with a peep = 24 cmh 2 o in the previous 48 hours. the patient was weaned from ventilator 3 days later. niv failure must be recognized, and a prompt intubation and mechanical ventilation must be provided in order to avoid complications. protective ards mechanical ventilation strategies with tidal volumes equal to or less than 6 ml/kg of predicted body weight have been traditionally associated with reduced mortality (when compared with 12 ml/kg of predicted body weight) [48, 49] . a recent meta-analysis, however, scrutinized the specific role of various ventilatory strategies used in randomized trials on lung protection (like plateau-pressure limitation and higher peep use) and showed that tidal volume per se is not exactly the most important parameter to prioritize. [53] demonstrated decreased lung inflammation with this protective ventilatory strategy. although these results are encouraging, the physiologic background supporting the use of p-v curves to titrate peep lacks consistency nowadays. in many different situations, investigators have reported a large dissociation between closing pressures of the lung and the calculated value for the inflection point obtained from the inspiratory p-v curve. in general, patients with high values of inflection point tend to have a more severe disease, and this may explain the relative success of this strategy. nevertheless, we will probably use better tools to titrate peep in the next few years. a more consistent use of the p-v curve has been demonstrated for the analysis of lung recruitability [54, 55] . airway pressure release ventilation is a modified form of continuous positive airway ventilation (cpap) described by stock and dows in 1987 that uses fairly high prolonged cpap levels with short and intermittent releases of the airway pressure to low cpap levels allowing ventilation and co 2 clearance. this mode of ventilatory support enhances oxygenation by augmenting alveolar recruitment and requires less sedation when used in ards patients compared to conventional mechanical ventilation [56, 57] . bipap ventilation combined with lung recruitment maneuvers can also be used in ards patients. wang and colleagues compared this modality of ventilatory support with assist/controlled volume ventilation in a prospective, randomized trial of 28 ards patients showing a better pao 2 /fio 2 ratio, pulmonary compliance, and a shorter duration of mechanical ventilation [58] . pressure support ventilation (psv) along with sufficient peep levels should be used as early as possible in ards patients to avoid respiratory muscle dystrophy and to decrease mechanical ventilation duration [32] . the reason for the improvement in oxygenation obtained with psv in ards has been challenged in the recent years [59, 60] . the apparent improvement in recruitment seems to have been overstated and there is evidence that it is related to an increased perfusion of better ventilated lung areas, but not to decreased lung collapse. growing concerns related to excessive tidal recruitment or excessive dyssynchrony during this mode of ventilation will have to be better addressed in the next years [61] . the advantages of using assist modes are to keep the respiratory muscles' activity, but sometimes it is difficult to synchronize the patients to the ventilators. recently, neurally adjust ventilation (nava) was used in ards experimental models [62] and ards patients [63] demonstrating that the ventilation cycle and the magnitude of assist breath in nava matched the patients' breath pattern better than in psv, nava improving patient-ventilator synchrony compared to psv. high frequency oscillatory ventilation (hfov) is an alternative mode of ventilatory support that can improve oxygenation by means of a higher mean airway pressure coupled with small tidal volumes generated by a piston pump oscillating at a frequency of 3-10 hz and a higher respiratory rate. however, to date there are few studies involving a small number of patients comparing hfov to conventional ventilation. a recent meta-analysis suggested a trend towards mortality benefit and more ventilator free days. however, the results of this analysis should be interpreted cautiously as the main study contributing to its results used high tidal volume in the control group rather than protective lung ventilation strategy [64] . the use of the position change (supine to prone) leads to consistent improvement in arterial oxygenation in ards patients. large randomized, controlled trials have consistently showed improvement in oxygenation without reduction in duration of mechanical ventilation or survival benefit. a recent meta-analyses suggest survival benefits in ards patients [65] or, more specifically, in a subgroup of patients with severe ards (pao 2 /fio 2 < 100 mmhg) [66] . in our experience, the prone position can be an acceptable alternative to improve oxygenation in severe ards patients with arterial pulmonary hypertension and right ventricular dysfunction, which associated with the use of inhaled nitric oxide, can minimize intrathoracic pressures to facilitate right ventricular performance. the principles of a protective ventilation with proper peep titration and minimum driving pressures should also be pursued during prone positioning protocols. clinical studies suggested that elevated pulmonary artery systolic pressure in ards patients was associated with an adverse prognosis [67] . these data have been further supported by a more recent analysis of hemodynamic data from the ardsnet fluids and catheter therapy trial (factt) [68] . the investigators assessed the transpulmonary gradient (tpg) (mean pa pressure-pulmonary capillary occlusion pressure (pcop)) and the pulmonary vascular resistance index (pvri) in a group of patients randomized to receive a pulmonary artery catheter to guide their ards management. of note, all patients received a consistent protective ventilator strategy with target tidal volume ∼6 ml/kg ideal body weight and plateau pressures maintained <30 cmh 2 o. the highest recorded daily value of tpg and pvri was used for the analysis. in the population of 475 patients randomized to receive a pulmonary artery catheter for ards management, none of the baseline measures of cardiopulmonary dysfunction, including central venous pressure, pa systolic, or diastolic pressure, pulmonary capillary occlusion pressure (paop), or cardiac index distinguished survivors from nonsurvivors. in the pulmonary artery catheter population, 73% demonstrated an elevated transpulmonary gradient (tpg > 12). patients with a tpg > 12 mmhg had a significantly greater mortality rate than patients with a tpg < 12 mmhg (30% versus 19%; p = 0.02). patients with a persistently elevated tpg through day 7 of therapy had a significantly greater mortality than patients with an elevated tpg at day 0-1 which subsequently normalized. in multivariate analysis, pulmonary vascular dysfunction as represented by an elevated tpg and pvri remained an independent predictor of an adverse outcome in the ards population. these data further support an important predictive role for pulmonary vascular disease in ards outcome [69] . in the largest published echocardiographic series of ards, 22% of patients receiving a consistent lung protective ventilation strategy (mean peep of 10 cmh 2 o and mean plateau pressure (pplat) of 23 cmh 2 o) had evidence for acute cor pulmonale. in this population, 19% demonstrated evidence of a moderate-to-large patent foramen ovale [70] . the incidence of right to left shunting increased to 34% in patients with echocardiographic evidence of acute cor pulmonale. increase of oxygenation and co 2 removal by making the ards patients' blood pass throughout a membrane oxygenator outside the body is the principle of extracorporeal membrane oxygenation that can be applied venousvenous (good for oxygenation and co 2 removal), arterialvenous (good for co 2 removal), and venous-arterial (good for cardiovascular support). early clinical trials of ecmo employed primarily an arterial-venous strategy with larger bore catheters for patients with intractable hypoxemia [71] . more modern investigations have used a safer venous-venous access approach [72, 73] . a recent uk prospective, randomized, clinical trial (cesar) showed a survival advantage in the ecmo group (63% for ecmo versus 47% for controls). nevertheless, the study was criticized as there was no standardized protocol management for the control group and some patients in the ecmo arm did not receive the proposed treatment [74] . the authors of cesar trial also recommended transferring adult patients with severe but potentially reversible respiratory failure and a ph less than 7.20 on optimal conventional management, to a center with an ecmo-based management protocol to significantly improve survival without severe disability. the authors demonstrated that this strategy is also likely to be cost effective in settings with similar services to those in the united kingdom [74] . another recent approach for application of extracorporeal carbon dioxide removal new devices (ecmo-r) in ards patients is the demonstration that in severe ards even the low tidal volume ventilation with 6 ml/kg of predicted body weight can cause tidal hyperdistension in the nondependent regions of the lungs accompanied by plateau airway pressures greater than 28 cmh 2 o and elevated plasma markers of inflammation. in this group application of ecmo-r could allow the authors to decrease the tidal volume to less than 6 ml/kg with a consequent plateau pressure less than 25 cmh 2 o that was associated with a lower radiographic index of lung injury and lower levels of lung-derived inflammatory cytokines. however, prognostic implication of this new ecmo-r devices application in clinical practice is still under investigation [75] . pumpless interventional lung assist (ila) is also used in patients with ards and is aimed at improving extracorporeal gas exchange with a membrane integrated in a passive arteriovenous shunt. ila serves as an extracorporeal assist to support mechanical ventilation by enabling low tidal volume and a reduced inspiratory plateau pressure in extremely severe ards patients. zimmermann and colleagues used ila in 51 severe ards patients and observed a decrease in paco 2 allowing the decrease in tidal volume and plateau pressure (ultraprotective ventilation) with a hospital mortality rate of 49% [76] . some authors suggest the use of combined ventilatory strategies in patients with ards. bingold and colleagues [77] successfully used superimposed high-frequency jet ventilation (shfjv) in combination with continuous positive airway pressure/assisted spontaneous breathing (cpap/asb) in five patients with h1-n1-associated ards to improve oxygenation. varpula and colleagues [78] demonstrated a significant improvement in oxygenation in 28 ards patients, when they compared apvr associated with prone ventilation to simv-pressure control/pressure support group. aprv after 24 h appears to enhance improvement in oxygenation in response to prone positioning. rival and colleagues [79] examined the effects of the prone position associated with a recruitment maneuver consisting of 45 cmh 2 o extended sigh in pressure control, in 16 ards patients. the combination of both ventilatory techniques led to the highest increase in pao 2 /fio 2 ratio without significant clinical side effects. lubnow and colleagues [80] examined the effects of 6 days of the combination of high-frequency oscillatory ventilation (hfov) and extracorporeal carbon dioxide removal with the interventional lung assist (ila) in 21 severe ards patients who failed conventional ventilation. they observed an increase in pao 2 /fio 2 ratio and ph and a decrease in paco 2 . weaning from hfov/ila was successful in 10 patients. the 30-day mortality rate was 43%, and hospital mortality rate was 57%. in conclusion, combined ventilatory strategies can be applied in severe ards patients, but the best match among all the available ventilatory techniques is still a matter of debate. pulmonary infection and sepsis are the most important triggering factors of ards. pulmonary infection has been associated with a higher risk of ards progression in comparison to nonpulmonary infection in at risk populations [81] . a wide variety of organisms can invade the respiratory tract and trigger host innate and acquired immune system initiating the inflammatory cascade of ards, sepsis, and multiple organ failure [11] . it is particularly pertinent to investigate the etiology of pulmonary infection on the first day assessing a nasal swab for a respiratory virus detection (influenza, adenovirus) lower respiratory tract secretion or a bronchoalveolar lavage fluid (balf) for bacteria (especially multiresistant species), other viruses as herpes and cytomegalovirus, coronavirus, or metapneumonic virus [82] . opportunistic agents such as pneumocystis jiroveci must be investigated in immunosuppressed patients. urinary screening for legionella species is decisive, because if positive, specific therapy must be introduced [11] . the assessment of balf on the first as well as on the third day of mechanical ventilation is of the utmost importance not only in terms of assessment of etiology of pulmonary infection but also of the assessment of proinflammatory mediators of ards (il-1, il6, il8, il 10, soluble tumor necrosis factor-alpha receptors (stnfr), and soluble intercellular adhesion molecule-1) and mediators of ventilator-induced lung injury (that can also be obtained in the plasma) such as stnfr, il6, il8, and il-10, indicators of epithelial cell injury (soluble advanced glycation end-product receptors-srage), and surfactant protein-d, components of the coagulation system (protein-c and plasminogen activator inhibitor 1) [11, 83] . elevated levels of procollagen peptide iii in lavage fluid from patients on day 3 of ards were independent risk factors for mortality [84] . procalcitonin (pct) and c-reactive protein (crp) are progressively being used in critical care setting in order to diagnose pulmonary infection and sepsis and to guide the antibiotic therapy. procalcitonin levels correlated with severe sepsis and bacteraemia [85] . a pct-based algorithm guiding initiation and duration of antibiotic therapy in critical ill patients with suspected bacterial infections was associated with a 23% relative reduction in antibiotic exposure with no significant increase in mortality [86] . the persistence of an elevated serum crp in critical ill patients with ards may alert the intensivist to a possible persistent infection or inflammatory process. at this moment, a new workup for infection and change in antibiotic therapy could help improve the patient's evolution. early and quick administration of antibiotics in sepsis and septic shock as well as early goal resuscitative measures for septic shock or early goaldirected therapy decrease mortality in this high mortality critically ill conditions [87, 88] . we also suggest that preventive measures to avoid gastric aspiration (elevated decubitus, intermittent check for residual gastric content during diet infusion) and to avoid ventilation associated pneumonia (wash hands, elevated decubitus, special endotracheal tubes) should be implemented. the resolution of pulmonary edema is central to recover from ali as it entails regression of air space inflammation and restoration of a functioning alveolar-capillary membrane. accordingly, elevated extravascular lung water measured using this technique early in the course of ali/ards, particularly if indexed to predicted body weight, was associated with a poor prognosis [89] . a study analyzing the evolution of ards patients showed that unknown-site infection (adjusted hazard ratio (hr) 3.08, 95% ci 1.37-6.90) and multiple site infection (adjusted hr 1.63, 95% ci 1.13-2.35) were associated with increased mortality [90] . in ards patients it is of considerable significance to evaluate the source of infection as well all organs and systems affected by the sepsis syndrome in order to map the organism (number of nonpulmonary organ failures), to calculate the prognostic indices (acute physiology and chronic health evaluation (apache) and simplified acute physiology score (saps)) and to plan the multiorgan system approach to treat the disease. the higher the number of multiple organ failure associated with ards, the higher the hospital mortality. trauma patients with ards are associated with lower mortality and oliguricrenal failure, while septic shock patients are associated with the highest hospital mortality rates, suggesting that during the first day of hospitalization these ards patients should be stratified and treated according to the severity of the syndrome and associated comorbidities [91] . in our case series of 51 patients with early severe ards the mean apache ii score was 20.2 ± 6.2 (predicted mortality of 40%), median sofa score (day 1) was 10 (7 to 12), median nonpulmonary organ failure was 2 (1 to 2), sepsis was present in 71% of our patients, and septic shock in 63%, vasopressors were used in 82.3% of our patients, and continuous renal replacement therapy was used in 56.8% of our patients. apache ii and day 1 sofa score were not associated with hospital mortality, but day 3 sofa score was [33] (figure 2) showing that a revaluation of the ards patients especially the ones with multiple organ failure and maintenance of sofa score higher than 8 at day 3 has to be considered in order to evaluate hidden sources of infection or to change the antibiotics according to day 1 collected cultures. in moderate-severe ards patients (pao 2 /fio 2 < 150), a phase iv randomized controlled trial comparing cisatracurium to placebo for 48 hours showed an improved critical care research and practice 9 adjusted 90-day survival rate and increased ventilator-free in the cisatracurium group without a significant increase in muscle weakness. short-term paralysis may facilitate patient-ventilator synchrony in the setting of lung protective ventilation. short-term paralysis would eliminate patient triggering and expiratory muscle activity. in combination, these effects may serve to limit regional overdistention and cyclic alveolar collapse. paralysis may also act to lower metabolism and overall ventilatory demand [92] . inhaled nitric oxide is an endogenous vasodilator that reduces v/q mismatch and improves oxygenation by pulmonary vasodilation in alveolar units that are ventilated, reducing pulmonary vascular resistance in patients with ards. a cochrane review of 14 clinical trials with 1303 patients showed only a transient improvement in oxygenation with no benefit regarding length of icu or hospital stay, ventilator-free days or survival. an increased renal impairment was observed in the inhaled nitric oxide-treated group [93] . the effects of steroids in the late-stage fibrotic phase of ards (after 7 days of onset) were tested in a phase iii study of the ards network. the study showed no mortality benefit in the treatment group, with a higher mortality in patients treated 14 days after onset [94] . recently, seam and colleagues tested the effects of methylprednisolone infusion in 55 early ards patients compared to placebo. they observed that methylprednisolone therapy was associated with greater improvement in lung injury score (p = 0.003), shorter duration of mechanical ventilation (p = 0.005), and lower intensive care unit mortality (p = 0.05) than in the control subjects. on days 3 and 7, methylprednisolone decreased interleukin-6 and increased protein-c levels (p < 0.001) compared with control subjects [95] . from the available evidence, low-dose steroids (1-2 mg/kg/methylprednisolone) may be considered in patients with severe early ards. nevertheless, it is not recommended to initiate corticosteroids beyond 14 days after the onset of ards. ketoconazole, lisofylline, sivelestat, n-acetylcysteine, and exogenous surfactant are not recommended as treatment for ards patients [12] . cumulative positive fluid balance is associated with worse clinical outcomes in patients with ards. a phase iii study conducted by the ards network (the factt study) compared liberal versus conservative fluid strategy in patients with acute lung injury. they observed an improvement in oxygenation, lung injury score (lis), and shortened duration of mechanical ventilation without any increase in other organ failure in the conservative group, despite no difference in hospital mortality [68] . beta-agonists were investigated in multicenter, prospective, randomized trials in their aerosolized presentation (the alta study) and their intravenous presentation (the balti-2 study). both studies showed no mortality benefit and betaagonists are not recommended as part of therapy for patients with ards [96] . the omega study [97] , a randomized, double-blind, placebo-controlled, multicenter trial analyzed 272 patients with early acute lung injury allocated to receive either twicedaily enteral supplementation of n-3 fatty acids, γ-linolenic acid, and antioxidants compared with an isocaloric control. enteral nutrition, directed by a protocol, was delivered separately from the study supplement. the patients that received enteral supplementation had fewer ventilator-free days (14 versus 17.2, p = 0.02), more days with diarrhea (29 versus 21%; p = 0.001), and no difference in the adjusted 60-day mortality (25.1% versus 17.6%; p = 0.11). more recently, a randomized, open-label, multicenter trial, the eden study [98] , reported 1000 patients with acute lung injury, randomized to receive either trophic or full enteral feeding for the first 6 days. initial trophic enteral feeding did not improve ventilator-free days, 60-day mortality, or infection complications but was associated with less gastrointestinal intolerance. finally, based on relevant literature articles and the authors' clinical experience, we suggest a goal-oriented management for critically ill patients with ards that can help improve clinicians' ability to care for these patients (as shown below). patients with ards. correct ards diagnosis. acute onset, increase respiratory rate, pulse oximeter desaturation and hypoxemia (pao 2 /fio 2 < 300). (i) if possible, get a computer tomography (improved diagnosis accuracy, permits differential diagnoses, and helps to set recruitment maneuvers and adequate peep levels). (ii) lung ultrasound, fdg-pet ct, electrical impedance tomography, and pressure-volume p × v curves can help assess the correct diagnosis and set protective mechanical ventilation. (iii) get nasal swab and inferior respiratory tract secretion for infection diagnosis or a bal (infection diagnosis and proinflammatory mediators and procollagen iii measurements). (iv) get hemocultures and blood for infection detection. start resuscitative measurements for septic shock and start appropriate antibiotics. critical care research and practice (v) assessment of prognostic indices (apache, saps) and sequential organ failure assessment (sofa) score. standardize initial mechanical ventilation for blood gas measurements. tidal volume: 6 ml/kg predicted body weight, peep of 5 cmh 2 o, rr = 20. classify ards severity. mild: pao 2 /fio 2 < 300, moderate: pao 2 /fio 2 < 200, and severe: pao 2 /fio 2 < 100. (i) if possible, get a doppler echocardiogram to assess left ventricular function, right ventricular function, systolic pulmonary artery pressure, and vena cava compressibility. (ii) measure extravascular lung water, if available. (a) in cases of severe ards consider recruitment maneuvers and adequate peep titration. (b) in cases of severe ards with right ventricular dysfunction and pulmonary artery hypertension consider prone position and inhaled nitric oxide. (c) in cases of excessive co 2 retention: paco 2 > 80 mmhg and ph < 7.2 consider intratracheal gas insufflation and extracorporeal co 2 removal. (i) early recognition of ards modified risk factors and avoidance of aggravating factors during hospital stay such as high tidal volume ventilation, multiple blood products transfusions, excessive fluid administration, ventilator associated pneumonia, and gastric aspiration prevention could help decrease its incidence. (ii) an early extensive clinical, laboratory, and imaging evaluation of "at risk patients" allows a correct diagnosis of ards, assessment of comorbidities, calculation of prognostic indices (apache, saps, sofa), stratification of the severity of ards, and planning a careful treatment. (iii) rapid administration of antibiotics and resuscitative measures in case of sepsis and septic shock associated with protective ventilatory strategies and early shortterm paralysis associated with differential ventilatory techniques (recruitment maneuvers with adequate peep titration, prone position, and new ecmo techniques) in severe ards can help improve its prognosis. (iv) revaluation of ards patients on the third day of evolution (sofa, biomarkers, and response to infection therapy) allows changes in the initial treatment plans and can help decrease ards mortality. (v) fibroproliferative changes on high-resolution ct in ards can predict mortality and ventilator dependency. acute respiratory distress in adults an expanded definition of the adult respiratory distress syndrome the acute respiratory distress syndrome comparison of clinical criteria for the acute respiratory distress syndrome with autopsy findings acute respiratory distress syndrome: a historical perspective acute respiratory distress syndrome: underrecognition by clinicians and diagnostic accuracy of three clinical definitions acute pulmonary edema development of a clinical definition for acute respiratory distress syndrome using the delphi technique acute lung injury and the acute respiratory distress syndrome: a clinical review acute lung injury and acute respiratory distress syndrome: diagnostic hurdles genetic variants in the angiopoietin-2 gene are associated with increased risk of ards acute respiratory distress syndrome and acute lung injury acute respiratory distress syndrome: a clinical review acute respiratory distress syndrome: pathophysiology and therapeutic options an nfkb1 promoter insertion/deletion polymorphism influences risk and outcome in acute respiratory distress syndrome among caucasians incidence and outcomes of acute lung injury early identification of patients at risk of acute lung injury: evaluation of lung injury prediction score in a multicenter cohort study diffuse alveolar damage, the pathological basis of adult respiratory distress syndrome the role of intra-alveolar fibrosis in the process of pulmonary structural remodeling in patients with diffuse alveolar damage the acute respiratory distress syndrome ards and diffuse alveolar damage: a pathologist's perspective clinical year in review iii: asthma, lung transplantation, cystic fibrosis, acute respiratory distress syndrome performance of an automated electronic acute lung injury screening system in intensive care unit patients introducing automated acute lung injury/acute respiratory distress syndrome electronic screening in intensive care unit practice: is it the future? the american-european consensus conference on ards: definitions, mechanisms, relevant outcomes, and clinical trial coordination ventilatory, pharmacologic, supportive therapy, study design strategies, and issues related to recovery and remodeling. acute respiratory distress syndrome acute respiratory distress syndrome: the berlin definition lung structure and function in different stages of severe adult respiratory distress syndrome what has computed tomography taught us about the acute respiratory distress syndrome? reversibility of lung collapse and hypoxemia in early acute respiratory distress syndrome mechanical ventilation in acute respiratory failure: recruitment and high positive end-expiratory pressure are necessary open up the lung and keep the lung open how large is the lung recruitability in early acute respiratory distress syndrome: a prospective case series of patients monitored by computed tomography state-of-the-art mechanical ventilation understanding and avoiding ventilatorinduced lung injury: lessons from an insightful experimental study best strategy to recruit primary ards: what to look for fibroproliferative changes on high-resolution ct in the acute respiratory distress syndrome predict mortality and ventilator dependency: a prospective observational cohort study imaging in acute lung injury and acute respiratory distress syndrome assessment of lung inflammation with 18 f-fdg pet during acute lung injury lungs of patients with acute respiratory distress syndrome show diffuse inflammation in normally aerated regions: a [ 18 f]-fluoro-2-deoxy-dglucose pet/ct study bedside ultrasound assessment of positive end-expiratory pressure-induced lung recruitment electrical impedance tomography bedside estimation of recruitable alveolar collapse and hyperdistension by electrical impedance tomography a unified approach for eit imaging of regional overdistension and atelectasis in acute lung injury whither lung eit: where are we, where do we want to go and what do we need to get there? does noninvasive positive pressure ventilation improve outcome in acute hypoxemic respiratory failure? a systematic review early use of noninvasive positive pressure ventilation for acute lung injury: a multicenter randomized controlled trial ventilation with lower tidal volumes as compared with traditional tidal volumes for acute 12 critical care research and practice lung injury and the acute respiratory distress syndrome pressure and volume limited ventilation for the ventilatory management of patients with acute lung injury: a systematic review and meta-analysis effects of superimposed driving-pressure on mortality in patients with acute lung injury or acute respiratory distress syndrome effect of a protective-ventilation strategy on mortality in the acute respiratory distress syndrome a high positive end-expiratory pressure, low tidal volume ventilatory strategy improves outcome in persistent acute respiratory distress syndrome: a randomized, controlled trial effect of mechanical ventilation on inflammatory mediators in patients with acute respiratory distress syndrome: a randomized controlled trial a scanographic assessment of pulmonary morphology in acute lung injury: significance of the lower inflection point detected on the lung pressure-volume curve influence of tidal volume on alveolar recruitment: respective role of peep and a recruitment maneuver airway pressure release ventilation in acute respiratory distress syndrome airway pressure release ventilation: an alternative mode of mechanical ventilation in acute respiratory distress syndrome comparison of the effects of bipap ventilation combined with lung recruitment maneuvers and low tidal volume a/c ventilation in patients with acute respiratory distress syndrome pressure support improves oxygenation and lung protection compared to pressure-controlled ventilation and is further improved by random variation of pressure support noisy pressure support ventilation: a pilot study on a new assisted ventilation mode in experimental lung injury spontaneous breathing during lung-protective ventilation in an experimental acute lung injury model: high transpulmonary pressure associated with strong spontaneous breathing effort may worsen lung injury effects of neurally adjusted ventilatory adjusted ventilatory assist on prevention of ventilator-induced diaphragmatic dysfunction in acute respiratory distress syndrome rabitts effects of neurally adjusted ventilatory assist on patientventilator synchrony in patients with acute respiratory distress syndrome high frequency oscillation in patients with acute lung injury and acute respiratory distress syndrome (ards): systematic review and meta-analysis an updated study-level meta-analysis of randomised controlled trials on proning in ards and acute lung injury prone positioning improves survival in severe ards: a pathophysiologic review and individual patient metaanalysis hemodynamic profile in severe ards: results of the european collaborative ards study comparison of two fluid-management strategies in acute lung injury pulmonary vascular dysfunction is associated with poor outcomes in patients with acute lung injury prevalence and prognosis of shunting across patent foramen ovale during acute respiratory distress syndrome extracorporeal membrane oxygenation in severe acute respiratory failure. a randomized prospective study clinical review: extracorporeal membrane oxygenation extracorporeal membrane oxygenation for ards in adults efficacy and economic assessment of conventional ventilatory support versus extracorporeal membrane oxygenation for severe adult respiratory failure (cesar): a multicentre randomised controlled trial tidal volume lower than 6 ml/kg enhances lung protection: role of extracorporeal carbon dioxide removal pumpless extracorporeal interventional lung assist in patients with acute respiratory distress syndrome: a prospective pilot study superimposed highfrequency jet ventilation combined with continuous positive airway pressure/assisted spontaneous breathing improves oxygenation in patients with h1n1-associated ards combined effects of prone positioning and airway pressure release ventilation on gas exchange in patients with acute lung injury prone position and recruitment manoeuvre: the combined effect improves oxygenation combination of high frequency oscillatory ventilation and interventional lung assist in severe acute respiratory distress syndrome clinical characteristics and outcomes of sepsis-related vs non-sepsis-related ards virus-induced acute respiratory distress syndrome: epidemiology, management and outcome biomarkers of acute lung injury: worth their salt? type iii procollagen peptide in the adult respiratory distress syndrome. association of increased peptide levels in bronchoalveolar lavage fluid with increased risk for death predictive value of procalcitonin decrease in patients with severe sepsis: a prospective observational study use of procalcitonin to reduce patients' exposure to antibiotics in intensive care units (prorata trial): a multicentre randomised controlled trial early goal-directed therapy in the treatment of severe sepsis and septic shock early interventions in severe sepsis and septic shock: a review of the evidence one decade later extravascular lung water indexed to predicted body weight is a novel predictor of intensive care unit mortality in patients with acute lung injury the influence of infection sites on development and mortality of ards predictors of hospital mortality in a population-based cohort of patients with acute lung injury neuromuscular blockers in early acute respiratory distress syndrome inhaled nitric oxide for acute respiratory distress syndrome (ards) and acute lung injury in children and adults efficacy and safety of corticosteroids for persistent acute respiratory distress syndrome effects of methylprednisolone infusion on markers of inflammation, coagulation, and angiogenesis in early acute respiratory distress syndrome randomized, placebo-controlled trial of an aerosolized beta-2 adrenergic agonist (albuterol) for the treatment of acute lung injury omega-3 (n-3) fatty acid, gamma-linoleic acid (gla) and anti-oxidant supplementation in acute lung injury (omega trial) initial trophic vs full enteral feeding in patients with acute lung injury: the eden randomized trial the authors would like to thank adriana pardini for revision of the language. key: cord-309120-05bg7rfa authors: niazi, sadegh; groth, robert; spann, kirsten; johnson, graham r. title: the role of respiratory droplet physicochemistry in limiting and promoting the airborne transmission of human coronaviruses: a critical review() date: 2020-11-06 journal: environ pollut doi: 10.1016/j.envpol.2020.115767 sha: doc_id: 309120 cord_uid: 05bg7rfa whether virulent human pathogenic coronaviruses (sars-cov, mers-cov, sars-cov-2) are effectively transmitted by aerosols remains contentious. transmission modes of the novel coronavirus have become a hot topic of research with the importance of airborne transmission controversial due to the many factors that can influence virus transmission. airborne transmission is an accepted potential route for the spread of some viral infections (measles, chickenpox); however, aerosol features and infectious inoculum vary from one respiratory virus to another. infectious virus-laden aerosols can be produced by natural human respiratory activities, and their features are vital determinants for virus carriage and transmission. physicochemical characteristics of infectious respiratory aerosols can influence the efficiency of virus transmission by droplets. this critical review identifies studies reporting instances of infected patients producing airborne human pathogenic coronaviruses, and evidence for the role of physical/chemical characteristics of human-generated droplets in altering embedded viruses’ viability. we also review studies evaluating these viruses in the air, field studies and available evidence about seasonality patterns. ultimately the literature suggests that a proportion of virulent human coronaviruses can plausibly be transmitted via the air, even though this might vary in different conditions. evidence exists for respirable-sized airborne droplet nuclei containing viral rna, although this does not necessarily imply that the virus is transmittable, capable of replicating in a recipient host, or that inoculum is sufficient to initiate infection. however, evidence suggests that coronaviruses can survive in simulated droplet nuclei for a significant time (>24 h). nevertheless, laboratory nebulized virus-laden aerosols might not accurately model the complexity of human carrier aerosols in studying airborne viral transport. in summary, there is disagreement on whether wild coronaviruses can be transmitted via an airborne path and display seasonal patterns. further studies are therefore required to provide supporting evidence for the role of airborne transmission and assumed mechanisms underlying seasonality. -airborne transmission: the spread of a pathogen embedded in an aerosol from a source to a 93 susceptible host, causing infection of the host with or without the consequent disease. (hinds, 94 1982) 95 -aerosol: a collection of particles (liquid or solid) ranging in size from 0.001µm to over 100 96 mm suspended in a gas. (wells, 1934) 97 -droplet nucleus: the airborne residue (with or without embedded pathogens) of a respiratory 98 droplet containing non-volatile solutes, from which water has evaporated to the point of 99 equilibrium with the ambient relative humidity. (wells, 1934) the smaller laryngeal and oral mode droplets are small enough to achieve equilibrium with 153 the ambient relative humidity. these droplets therefore form airborne droplet nuclei while the 154 much larger oral mode droplets settle or impact with surfaces before equilibrium can be 155 achieved. 156 the average mass concentration of these modes varies with the respiratory manoeuvre. for concentration in the exhaled aerosol decreases when the volunteer held their breath after 161 inhaling. the consistent findings of johnson and holmgren that fewer particles are observed 162 after breath-holding, this would suggest that the generated particles have adequate time to 163 deposit on the airways before being exhaled, consistent with the ffb model proposed by 164 johnson. based on previous literature, healthy subjects can produce particles between 0.01 the aerosols generated through speech, coughing, sneezing, and breathing have been 178 surveyed in several studies (table 1) 290 hygroscopic salts influence the transport of water vapor, and allow for humidity dependent 359 droplet sizes as described by köhler theory (köhler, 1936) . because of their hygroscopic 360 behavior, inorganic salts such as nacl can play an essential role in controlling the water 361 uptake and loss when present in an aerosol, and consequently limit or promote virus viability. nacl is a prominent constituent found in human respiratory aerosol. some hygroscopic salts, several studies have assessed environmental samples of wild coronaviruses collected in field 435 settings (table 5) it is predicted that covid-19 incidence will align similarly to sars-cov, potentially 576 showing seasonality. 577 in this critical review, we reviewed the ability of infected individuals to produce droplet 597 laden-virus with pandemic potential, including sars-cov, mers-cov, and sars-cov-2. there is evidence explaining the production mechanisms of airborne pathogenic bioaerosols 599 through human respiratory activities, which can travel distance over several meters in the air 600 and remain infectious. studies investigating respiratory droplets and droplet nuclei generation 601 during respiratory activities have shown that the produced droplets are of adequate size to 602 support an infectious virus. additionally, viral rna was found in the air of patients' rooms 603 under different conditions. the airborne transmission was thought to play an essential role in 604 the epidemiology of several highly transmissible coronaviruses that emerged this century. the authors state that they have no conflicts of interest to declare. 640 spatiotemporal clustering of middle east 644 respiratory syndrome coronavirus (mers-cov) incidence in saudi arabia analysis of the proteome of human airway 647 epithelial secretions effect of airway opening on production of exhaled particles airway 652 monitoring by collection and mass spectrometric analysis of exhaled particles effect of airway opening on production of exhaled particles climate factors and incidence of middle east respiratory syndrome 658 coronavirus aerosol 660 emission and superemission during human speech increase with voice loudness detection of the middle east respiratory 663 syndrome coronavirus genome in an air sample originating from a camel barn owned by an infected 664 patient susceptible supply limits 666 the role of climate in the early sars-cov-2 pandemic preliminary evidence that higher 668 temperatures are associated with lower incidence of covid-19, for cases reported globally up to 29th 669 correlation between 671 climate indicators and covid-19 pandemic some factors affecting the survival of airborne viruses weather: driving force behind the transmission of severe acute 675 respiratory syndrome in china significance of fomites in the spread of respiratory and enteric viral 677 disease detection of airborne severe acute 681 respiratory syndrome (sars) coronavirus and environmental contamination in sars outbreak units exhaled endogenous particles contain lung proteins 686 influenza virus survival in aerosols and estimates of viable virus loss resulting from aerosolization 687 and air-sampling analysis of 689 meteorological conditions and prediction of epidemic trend of 2019-ncov infection in 2020. 690 medrxiv climate effect on covid-19 spread rate: an online surveillance tool. medrxiv the effects of 696 temperature and relative humidity on the viability of the sars coronavirus characterization of expiration air 700 jets and droplet size distributions immediately at the mouth opening escalating infection control response to the rapidly 703 evolving epidemiology of the coronavirus disease 2019 (covid-19) due to sars-cov-2 in hong 704 detection of air and surface contamination by sars-cov-2 in hospital rooms 709 of infected patients who declares covid-19 a pandemic global environmental 712 drivers of influenza severe acute respiratory syndrome coronavirus on hospital 715 surfaces the size and the duration of air-carriage of respiratory droplets and droplet-nuclei airborne transmission of disease in hospitals inhaling to mitigate exhaled bioaerosols dilution of respiratory solutes in exhaled condensates dilution of respiratory solutes in exhaled condensates origin of exhaled breath 729 particles from healthy and human rhinovirus-infected subjects origin of exhaled breath 732 particles from healthy and human rhinovirus-infected subjects influenza virus in human exhaled breath: an observational study aerodynamic properties of 738 biohazardous aerosols in hospitals a field indoor air measurement of sars-cov-2 in the patient rooms of the 742 largest hospital in iran cough-744 generated aerosols of mycobacterium tuberculosis: a new method to study infectiousness collection and measurement of aerosols of viable influenza virus in liquid media in an 748 andersen cascade impactor phase separation in organic aerosol human rhinovirus infection during naturally occurring copd exacerbations the role of particle size in 754 aerosolised pathogen transmission: a review respiratory virus rna is detectable 756 in airborne and droplet particles clinical characteristics of coronavirus disease 2019 in china aerosol and 764 surface distribution of severe acute respiratory syndrome coronavirus 2 in hospital wards characterizations of particle size distribution of the 767 droplets exhaled by sneeze submicron droplet 769 formation in the human lung respiratory viral threats 772 effect of temperature and relative humidity on the stability of infectious porcine reproductive and 773 respiratory syndrome virus in aerosols impact of 775 health on particle size of exhaled respiratory aerosols: case-control study aerosol technology: properties, behavior, and measurement of airborne particles medical mask versus cotton mask for 781 preventing respiratory droplet transmission in micro environments 784 effects of breath holding at low and high lung volumes on amount of exhaled particles size distribution of 787 exhaled particles in the range from 0.01 to 2.0μm first case of 2019 novel coronavirus in the united states environmental 793 contamination by sars-cov-2 of an imported case during incubation period survival 796 characteristics of airborne human coronavirus 229e modality of human expired aerosol size distributions journal of aerosol 801 medicine and pulmonary drug delivery 22 the mechanism of breath aerosol formation modality of human expired aerosol size 806 distributions sampling and retention efficiencies of batch-808 type liquid-based bioaerosol samplers extensive viable middle east respiratory syndrome (mers) coronavirus contamination in 811 air and surrounding environment in mers isolation wards the nucleus in and the growth of hygroscopic droplets environmental persistence of influenza viruses is dependent upon virus type and host origin low 817 ambient humidity impairs barrier function and innate resistance against influenza infection transmission of influenza a in human beings role of ventilation in airborne transmission of infectious agents in the built environment-a 823 multidisciplinary systematic review electron cryotomography of 825 measles virus reveals how matrix protein coats the ribonucleocapsid within intact virions humidity-dependent decay of viruses, but not bacteria droplets follows disinfection kinetics environmental factors on the sars epidemic: 830 air temperature, passage of time and multiplicative effect of hospital infection probing the structure of the 833 sars coronavirus using scanning electron microscopy measurements of airborne influenza virus in aerosol particles 836 from human coughs measurements of airborne influenza virus in 839 aerosol particles from human coughs viable influenza a virus in airborne particles from human coughs quantity and size distribution of cough-845 generated aerosol particles produced by influenza patients during and after illness a cough aerosol 847 simulator for the study of disease transmission by human cough-generated aerosols aerodynamic analysis of sars-cov-2 in 851 two wuhan hospitals experimental air-borne influenza infection. i. 853 influence of humidity on survival of virus in air influenza virus transmission is dependent on 856 relative humidity and temperature the 858 role of absolute humidity on transmission rates of the covid-19 outbreak. medrxiv mechanistic insights into the effect 861 of humidity on airborne influenza virus survival, transmission and incidence environmental sampling for respiratory pathogens in jeddah airport during the 2013 hajj 865 season deviations in influenza seasonality: odd coincidence or 868 obscure consequence? droplets expelled during human expiratory activities and their 871 11th international conference on indoor air quality and climate change. 873 university of size distribution and sites of origin of droplets expelled from 876 the human respiratory tract during expiratory activities global seasonal 878 occurrence of middle east respiratory syndrome coronavirus (mers-cov) infection toward understanding the risk of secondary airborne 881 infection: emission of respirable pathogens toward understanding the risk of secondary 884 airborne infection: emission of respirable pathogens air surface environmental, and personal protective equipment contamination by severe acute 887 respiratory syndrome coronavirus 2 (sars-cov-2) from a symptomatic patient 890 transmission of sars and mers coronaviruses and influenza virus in healthcare settings: the 891 possible role of dry surface contamination effectiveness of commercial face masks to reduce personal pm exposure the size distribution of droplets in the exhaled breath of healthy 896 human subjects environmental factors affecting the transmission of respiratory 898 viruses environmental factors affecting the transmission of respiratory 900 viruses human pulmonary secretions in health and 902 disease human pulmonary secretions in health 904 and disease survival of the 906 enveloped virus phi6 in droplets as a function of relative humidity, absolute humidity, and 907 temperature survival of aerosolized 909 coronavirus in the ambient air sampling and detection of corona 911 viruses in air: a mini review sars-cov-2 rna detection in the air and on surfaces in the 914 covid-19 respiratory tract fluids: analysis of content and contemporary use 916 in understanding lung diseases influenza virus assembly and budding 919 diffusion through pig gastric mucin: effect of relative humidity 922 aerosol and surface transmission potential of sars-cov-2. medrxiv biochemical composition of human 924 saliva in relation to other mucosal fluids airborne sars-cov-2 is rapidly 928 inactivated by simulated sunlight atmospheric chemistry and physics: from air pollution to climate 930 change absolute humidity modulates influenza survival, transmission, and 932 seasonality absolute humidity and the 934 seasonal onset of influenza in the continental united states the impact of 936 temperature and absolute humidity on the coronavirus disease 2019 (covid-19) outbreak -evidence 937 from china. medrxiv virus survival in the environment with special attention to 939 survival in sewage droplets and other environmental media of fecal or respiratory origin. report for 940 the world health organization liquid-liquid phase separation in 942 organic particles containing one and two organic species: importance of the average 943 o&thinsp;:&thinsp;c exhalation of respiratory viruses by breathing, coughing, and talking biodefense research methodology and animal models an initial investigation of the association 949 between the sars outbreak and weather: with the view of the environmental temperature and its 950 variation review of aerosol transmission of influenza a virus numerical study of particle deposition in bends of a circular cross-954 section-laminar flow regime aerosol and surface stability of sars-cov-2 as compared with sars-cov-1 stability of middle east respiratory 960 syndrome coronavirus (mers-cov) under different environmental conditions physico-chemical characteristics of evaporating respiratory fluid 962 droplets physico-chemical characteristics of evaporating respiratory fluid 964 droplets cough-generated aerosols of pseudomonas aeruginosa and other gram-negative bacteria from 968 patients with cystic fibrosis factors affecting the viability of air-borne bacteria. iii. the role of bonded water 970 and protein structure in the death of air-borne cells far-uvc light: a new tool to control the spread of airborne-973 mediated microbial diseases on air-borne infection*: study ii. droplets and droplet 975 nuclei who guidline for natural ventilation for infection control in health-care settings,. 977 who, 2014. who guideline for infection prevention and control of epidemic-and pandemic-prone 978 acute respiratory infections in health care modes of transmission of virus causing covid-19: implications for ipc precaution 980 recommendations cluster of sars among medical students exposed to single patient association between ambient temperature and covid-19 infection in 122 985 cities from china exhaled droplets due to talking and coughing 989 effects of high temperature on pandemic and seasonal human influenza viral replication and 990 infection-induced damage in primary human tracheal epithelial cell cultures the size and concentration of droplets 992 generated by coughing in human subjects concentrations and size distributions of airborne 994 influenza a viruses measured indoors at a health centre, a day-care centre and on aeroplanes relationship between humidity and influenza a 997 viability in droplets and implications for influenza's seasonality dynamics of airborne influenza a viruses indoors and dependence on 999 humidity mechanisms by which ambient humidity may affect viruses in aerosols mechanisms by which ambient humidity may affect viruses in aerosols no association of covid-1005 19 transmission with temperature or uv radiation in chinese cities. the european respiratory journal evidence of 1008 airborne transmission of the severe acute respiratory syndrome virus a climatologic 1010 investigation of the sars-cov outbreak in beijing, china isolation of 1012 a novel coronavirus from a man with pneumonia in saudi arabia effects of 1014 temperature, relative humidity, absolute humidity, and evaporation potential on survival of airborne 1015 gumboro vaccine virus effect of hydration 1017 on structural and thermodynamic properties of pig gastric and bovine submaxillary gland mucins this study was funded by the australian research council (arc) with dp170102733 grant number. key: cord-294062-3esrg1jw authors: tam, clarence c.; offeddu, vittoria; anderson, kathryn b.; weg, alden l.; macareo, louis r.; ellison, damon w.; rangsin, ram; fernandez, stefan; gibbons, robert v.; yoon, in-kyu; simasathien, sriluck title: association between semi-quantitative microbial load and respiratory symptoms among thai military recruits: a prospective cohort study date: 2018-09-14 journal: bmc infect dis doi: 10.1186/s12879-018-3358-4 sha: doc_id: 294062 cord_uid: 3esrg1jw background: multiplex real-time polymerase chain reaction assays have improved diagnostic sensitivity for a wide range of pathogens. however, co-detection of multiple agents and bacterial colonization make it difficult to distinguish between asymptomatic infection or illness aetiology. we assessed whether semi-quantitative microbial load data can differentiate between symptomatic and asymptomatic states for common respiratory pathogens. methods: we obtained throat and nasal swab samples from military trainees at two thai army barracks. specimens were collected at the start and end of 10-week training periods (non-acute samples), and from individuals who developed upper respiratory tract infection during training (acute samples). we analysed the samples using a commercial multiplex respiratory panel comprising 33 bacterial, viral and fungal targets. we used random effects tobit models to compare cycle threshold (ct) value distributions from non-acute and acute samples. results: we analysed 341 non-acute and 145 acute swab samples from 274 participants. haemophilus influenzae type b was the most commonly detected microbe (77.4% of non-acute and 64.8% of acute samples). in acute samples, nine specific microbe pairs were detected more frequently than expected by chance. regression models indicated significantly lower microbial load in non-acute relative to acute samples for h. influenzae non-type b, streptococcus pneumoniae and rhinovirus, although it was not possible to identify a ct-value threshold indicating causal etiology for any of these organisms. conclusions: semi-quantitative measures of microbial concentration did not reliably differentiate between illness and asymptomatic colonization, suggesting that clinical symptoms may not always be directly related to microbial load for common respiratory infections. electronic supplementary material: the online version of this article (10.1186/s12879-018-3358-4) contains supplementary material, which is available to authorized users. multiplex polymerase chain reaction (pcr)-based diagnostic techniques allow rapid, simultaneous identification of a broad range of respiratory pathogens [1] . compared to classical microbiological diagnostic methods, pcr-based assays offer higher sensitivity, specificity, and reproducibility [2] . however, the high sensitivity of multiplex pcr diagnostics does not directly translate into clinical utility, because such assays do not distinguish between viable and dead organisms, or acute infection and asymptomatic colonisation [2] . in the clinical setting, the etiological agent is seldom identified and unspecific respiratory symptoms are often treated empirically [3] . although the quantification of microbial load may vary depending on the presence of co-infections, specimen type, sampling technique, or timing of sampling, quantitative or semi-quantitative microbial load data from real-time pcr assays may help define organism densities that are consistent with colonization or infection and distinguish between symptomatic and asymptomatic states [4] . in this study, we assessed whether semi-quantitative microbial load availab from real-time pcr assays can differentiate between symptomatic and asymptomatic states for common respiratory agents in a cohort of basic military trainees at two royal thai army barracks. details of the study setting and procedures have been described previously [5] . briefly, participants were recruited from six consecutive cohorts of basic military trainees at two royal thai army barracks between may 2014 and july 2015. trainees entered the camps for a 10-week training period at the start of may and november each year. individuals aged ≥18 years entering one of the two army barracks involved in the study were eligible for enrolment. suspected tuberculosis cases or individuals with immune deficiencies, such as acquired immune deficiency syndrome, leukemia or lymphoma, were excluded. throat and anterior nasal swab samples were collected using stiff synthetic swabs by trained study staff at the start and end of each training period (non-acute samples) and were placed in viral transport media (universal transport medium c330; copan diagnostics) and stored at − 20°c until time of transfer to the armed forces research institute of medical sciences for further testing. in addition, enrolled participants were asked to consult the camp's medical unit if they experienced respiratory symptoms during the training period. medical staff took a history, conducted a medical exam, and recorded symptoms of upper respiratory illness (uri) or influenza-like illness (ili). uri was defined as an illness with at least two of the following: (i) runny nose or sneezing; (ii) nasal congestion; (iii) sore throat, hoarseness or difficulty swallowing; (iv) cough; (v) swollen or tender glands in the neck; and (vi) fever (oral temperature > 38°c). ili was defined as a respiratory illness with acute onset presenting with fever and cough or sore throat. throat and nasal swab samples were collected on average 1.8 days after symptom onset from individuals who developed uri or ili during the 10-week follow-up (acute samples). specimens from two of the six cohorts (total number of individuals = 274) were tested using a commercial multiplex real-time pcr assay comprising 33 bacterial, viral and fungal targets according to the manufacturer's instructions (ftd33 kit, fast track diagnostics, esch-sur-alzette, luxembourg). these two cohorts were selected because they underwent concurrent routine environmental sampling of air and surfaces within the barracks, which were then similarly tested using the ftd33 kit (data not shown). multiplex testing of specimens from the remaining cohorts was not done due to resource constraints. a cycle threshold (ct) value below the detection limit of the assay (< 33) was considered a positive result. non-acute samples collected at the end of the training period from participants who experienced an acute episode during follow-up were excluded from the analysis, as the ct-value might reflect post-infectious shedding. we used the mcnemar test to determine whether target-specific frequencies were significantly different in non-acute baseline samples and acute samples. in addition, we computed the chi-square (χ 2 ) or fisher's exact test (for expected values < 5) to assess whether co-detection of specific microbe pairs occurred more frequently than expected by chance in non-acute baseline or acute samples. to account for data censoring at ct-value = 33, random effects tobit regression models were used to compare ct-value distributions from non-acute and acute samples, or ct-value distributions from samples containing a single or multiple organisms. in addition, we used the kruskal-wallis test to compare the median delay between illness onset and sample collection between samples containing one or multiple organisms. all analyses were conducted using stata 12 software (stata corporation). the study was approved by the institutional review boards of the royal thai army in bangkok, thailand, the walter reed army institute of research and the london school of hygiene & tropical medicine. all participants provided written informed consent. the investigators have adhered to the policies for protection of human subjects as prescribed in army regulation 70-25. we analyzed a total of 312 non-acute swab samples collected from 211 recruits at the start (n = 210) or end (n = 102) of the training period, and 145 acute specimens from 137 individuals who developed one or more uri episodes during follow-up. of 33 targets contained in the respiratory panel, 19 were detected in at least one specimen (table 1) . viruses were detected in 13.8% (43/312) and bacteria in 93.3% (291/312) of non-acute samples. among acute samples, viruses were detected in 44.1% (64/145) and bacteria in 94.5% (137/145) of specimens. haemophilus influenzae type b (hi-b) was the most commonly detected microbe (77.9% of non-acute and 64.8% of acute samples). other frequently detected bacteria included non-type b haemophilus influenzae (hi-nonb), streptococcus pneumoniae, and klebsiella pneumoniae (table 1) . rhinovirus was the most prevalent virus, detected in 6.4% of non-acute and 26.9% of acute samples. all other viruses were detected in < 10% of collected specimens (table 1) . hi-nonb, rhinovirus, and coronavirus 229 were detected significantly less frequently in non-acute samples collected at the start of the training period than acute samples (p-values < 0.05) ( table 1) . influenza b was identified in none of the non-acute, but 9.7% of acute specimens. multiple microbes were detected in 47.1% (99/210) of non-acute samples collected at the start of the training period. co-detection of multiple organisms was significantly higher in both non-acute samples taken at the end of the training period (77.5%) and acute specimens (71.7%) (p-values < 0.001; table 2 ). among acute samples, 9 specific organism pairs were co-detected more frequently than expected by chance (p-values < 0.05) ( were found in < 5% of acute specimens (table 3) . no microbe pair occurred more frequently than expected by chance among non-acute baseline samples. overall, there was a substantial overlap in ct-value distributions from non-acute samples collected at the start or end of the training period and acute samples collected from symptomatic individuals during follow-up (fig. 2) . this was the case even when considering only samples where a single organism was identified (fig. 3) . for hi-nonb and s. pneumoniae, our tobit regression models indicated significantly lower microbial load in non-acute baseline compared to acute samples (p-values < 0.05) ( table 4 ). for hi-nonb, a coefficient of 5.56 represents a 5.56 higher average ct-value in non-acute baseline samples compared to acute specimens, which corresponds to an approximately 47-fold lower microbial load in non-acute compared to acute samples. for s. pneumoniae, the average microbial load was 8.2-fold lower in non-acute baseline samples compared to acute specimens. our analysis also indicated a significantly lower average rhinovirus load in non-acute samples collected either at the start or at the end of the training period compared to acute samples (p-values < 0.05) ( table 4 ). this was in contrast with hi-b, for which regression analysis indicated a 4.7-fold higher average microbial load in non-acute baseline samples compared to acute samples (p-value < 0.001) ( table 4 ). for hi-non b and s. pneumoniae, there was a 7.7-fold or 19.4-fold increase in average microbial load in non-acute samples collected at the end of follow-up compared to acute samples collected during an uri episode, respectively (p-values ≤0.002). there was no significant difference in delay between symptom onset and specimen collection in acute samples containing one (median delay: 2 days; interquartile range (iqr): 1-3) or more (median delay: 2 days; iqr: 1-3) organisms (p-value = 0.536). six acute specimens were negative for all agents tested (median delay: 0.5 days; iqr: 0-1). thus, sampling delay is unlikely to account for any observed differences in ct-value distributions. we analyzed the patterns of infection with common respiratory agents in a well-defined population of military recruits. the use of highly sensitive multiplex pcr diagnostics allowed an accurate characterization of the spectrum of organisms contained in non-acute and acute samples. the data indicate co-circulation of several different viral agents, and high frequency of bacterial colonization in both non-acute and acute samples. up to one third of respiratory illness cases among army personnel are reportedly caused by viral or bacterial infections [6] . the gathering of individuals from diverse geographic locations and the crowded living conditions increase the risk of microbe transmission in these settings [7] . illnesses are usually self-limiting, although the emergence of highly virulent strains can lead to high morbidity and mortality [8] . streptococcus bacteria, adenoviruses, coronaviruses and influenza are among the most widely distributed microbes in the military environment, and are implicated in > 50% of febrile illness cases reported at military medical facilities [6] . we identified each of these organisms in one or more samples. for most of these microbes, overall detection frequencies were comparable in non-acute and acute samples, although influenza b and coronavirus 229 were more commonly identified among acute specimens. other infectious agents commonly circulating among military personnel include h. influenzae, rhinovirus, and, to a lesser extent, parainfluenza, rsv, and l. pneumophila, although their presence does not necessarily imply the occurrence of clinical symptoms [9] [10] [11] . h. influenzae and rhinoviruses were the most frequently detected organisms in our population in both non-acute and acute samples. we detected parainfluenza and l. pneumophila, but we did not find rsv in any of our samples. for individuals developing uri during follow-up, illness etiology could not be unequivocally determined. among acute samples, hi-b was the most frequently detected organism. it was the sole agent identified in 12% of acute specimens, while it was co-detected with other microbes in > 50% of acute samples. however, colonisation with hi-b was also common among non-acute baseline samples, where it was detected alone or in combination with other microbes in 40.5% and 43.3% of specimens, respectively. for organisms rarely detected among asymptomatic individuals but frequently found in acute samples, a causal association may be more likely. for instance, influenza b was detected in none of the non-acute, but 9.7% of acute samples. similarly, the proportion of both hi-nonb-and rhinovirus-positive samples was significantly lower among non-acute specimens collected at baseline compared to acute samples. however, > 85% of acute samples positive for hi-non b, rhinovirus or influenza b were also positive for one or more additional microbe, so that a causal table 3) association could not be determined. some agents, such as hi-non b or adenovirus, were most frequently detected in non-acute samples collected at the end of follow-up, possibly indicating post-infectious shedding or persistent infection at sub-clinical levels. in the clinical setting, overlapping clinical presentations and poor capabilities to determine the etiology of respiratory illnesses often lead to inappropriate treatment with broad-spectrum antibiotics [12] . this might occur even more frequently in the military setting, where molecular diagnostic tools are usually inaccessible [6] . since a considerable fraction of respiratory illnesses is caused by viruses, the unsubstantiated use of antibiotics is particularly problematic, because it can lead to negative health outcomes and promote the development of antimicrobial resistance [3] . studies evaluating the impact of multiplex diagnostic procedures on patient management report inconsistent results. in the outpatient setting, access to rapid molecular diagnostic tools for respiratory pathogens significantly reduced antibiotic a b fig. 2 cycle threshold value distribution in non-acute and acute samples. ct-value distribution for selected a bacteria and b viruses detected in non-acute samples collected at the start or end of the training period (orange bars) or acute samples from individuals experiencing an upper respiratory tract infection during follow-up (blue bars). a ct-value of < 33 was considered a positive result prescription rates for patients presenting with respiratory illness [13] . however, these findings were not confirmed in the hospital setting. pcr-based testing failed to reduce hospital admissions and duration of hospital stay in patients with acute respiratory infection [14, 15] . although molecular diagnostic tools may help to differentiate bacterial and viral respiratory agents, it is unlikely that antibacterial treatment would be terminated based on the mere presence of viral agents in an acute respiratory sample, especially considering the high rates of bacterial co-infection [16] . quantitative or semi-quantitative diagnostic tools can potentially help define clinically significant pathogen densities, and have proven highly valuable to understand the dynamics of diarrheal disease [17] and to improve the management of gastrointestinal illnesses [18] . among acute diarrhea patients, quantitative amplification of norovirus rna from fecal samples can help determine pathogen load thresholds that effectively distinguish between causal association and sub-pathogenic carriage [19] . similarly, rotavirus load correlates with disease severity among children with gastroenteritis [20] . because of the crucial role of microbial replication in viral pathogenesis, the value of pathogen load quantitation could be most clearly established for gastrointestinal illnesses of viral etiology, although some evidence is available for bacterial infections as well. for instance, microbial load of enteropathogenic e. coli is significantly higher among children with diarrhea compared to control subjects, especially when enteropathogenic e. coli is the sole agent identified [21] . in this study, tobit regression indicated significantly lower microbial load in non-acute relative to acute samples for rhinovirus, hi-nonb, and s. pneumoniae. however, due to a substantial overlap in ct-value distributions, it was not possible to identify a ct-value threshold indicating causality for any of these organisms. previous studies assessing the association of viral load with clinical symptoms of respiratory infections reported similar findings. mean viral load for rhinovirus and six additional viruses was significantly higher in upper respiratory tract aspirates from children with pneumonia compared to healthy controls, but the overlap in viral load distribution was substantial [22] . in pediatric patients, high rhinovirus load was associated with the presence of lower respiratory tract symptoms [23, 24] , but a threshold for clinical relevance could only be determined if rhinovirus was the sole agent identified [24] . additional studies reported a correlation between microbial load and occurrence or severity of respiratory symptoms for rsv [25] , bocavirus [26] , and human metapneumovirus (hmpv) [27, 28] , although these findings were inconsistent [29, 30] or conditional on the presence of the virus as a single microbe [31] . we did not detect any significant association between microbial load and clinical manifestations for viruses other than rhinovirus. for both h. influenzae and streptococcus species, previous studies reported a significant correlation of bacterial densities with clinical manifestations of disease [32] . in young patients with acute respiratory tract infection, s. pneumoniae load fluctuated with symptom incidence and resolution [33] . among children hospitalized with pneumonia, median nasopharyngeal s. pneumoniae load was substantially higher compared to healthy controls [32] . pneumococcal density was also associated with severity of symptoms [34] and increased duration of children's hospital stay [35] . similar associations were observed in pneumonic adults, although the correlation was not significant in this population [36] . the association between microbial load and clinical manifestations may depend on specific pathogen-host interactions. if pathogenesis is primarily related to microbial replication, a stronger correlation between microbial load and illness magnitude may be observed [37] . if clinical manifestations are largely attributable to host immune defences or bacterial toxins, the correlation with microbial load may not be obvious [37] . temporal variations in microbial load may also play an important role if the quantity of nucleic acid is significantly more abundant at the time and location of pathology [30, 33] . in acute respiratory illness patients, high bacterial colonization densities are often associated with the presence of viral co-infections [38] , and clinical manifestations may vary depending on specific co-infection patterns [39] . the ecology of respiratory pathogens is also likely to be influenced by the living conditions in military settings. mixing of individuals from diverse backgrounds living in close-quarters with high levels of inter-personal contact increases the potential for introduction and spread of multiple microbes in this population, which could account for the broad range of organisms and co-detections in this study. we analysed both non-acute and acute samples from a closely monitored population in a semi-closed, longitudinal setting. the study population was well-defined and relatively homogeneous with regards to demographics and living conditions. however, our findings may not be applicable to populations with different socio-demographic characteristics and populations outside the military environment, such as cohorts of children among whom the impact of respiratory infections may be greater. the frequent co-detection of multiple respiratory agents and the failure to distinguish between viable and dead organisms, or microbes that colonize the host at sub-pathogenic levels, may prevent the unambiguous interpretation of test results [2] . a positive result may indicate illness aetiology, asymptomatic colonisation, post-infectious shedding, or an incipient infection. therefore, ct-values may not always be a reliable surrogate for infectious load. samples from only two out of six cohorts were tested by real-time pcr. although there might be bias from seasonal effects, these are usually less pronounced in the tropics. given the relatively low frequencies of viral detection, a larger sample size and a longer follow-up may have captured a more precise picture of infection patterns in this population. this study was also limited to the detection of organisms contained in the respiratory panel. we cannot exclude the presence of additional organisms in our specimens. in addition, the data were obtained from throat and nasal swab samples, but our findings may not apply to nasopharyngeal or sputum specimens. finally, the quality and quantity of material obtained through nose and throat swabs may differ significantly among subjects, and the success of pcr-based methods also depends on the availability of intact genome sequences and the absence of random mutations. overall, the multiplex respiratory panel provided a comprehensive characterization of the microbe spectrum contained in non-acute and acute respiratory samples collected among recruits. however, semi-quantitative assessment of microbial load could not reliably distinguish between symptomatic and asymptomatic samples. more research is warranted to compare new multiplex diagnostic techniques with traditional methods and evaluate their potential with regards to diagnostic accuracy [40] and clinical utility [16, 40] in the context of respiratory infections. additional file 1: dataset. title of data: semi-quantitative microbial load in throat and nasal swab samples from thai army recruits. description of data: semi-quantitative microbial load in non-acute and acute throat and nasal swab samples from thai army recruits, determined using a commercial multiplex real-time pcr assay comprising 33 bacterial, viral and fungal targets; includes names, labels, and coding for individual variables. (xls 311 kb) abbreviations pcr: polymerase chain reactionuriupper respiratory illnessiliinfluenza-like illnessftdfast track diagnosticsctcycle thresholdhi-bhaemophilus influenzae type bhi-nonbnon-type b haemophilus influenzaeiqrinterquartile rangehmpvhuman metapneumovirus we are grateful to the participants of this study, the royal thai army, and the clinical, laboratory and administrative personnel at afrims. material has been reviewed by the walter reed army institute of research. there is no objection to its presentation and/or publication. the opinions or assertions contained herein are the private views of the authors, and are not to be construed as official, or as reflecting true views of the department of the army or the department of defense. this work was supported by the united states department of defense -global emerging infectious disease surveillance (dod -geis), protocol 989a. the datasets analysed for the current study are available as additional file 1 in this publication. author's contributions cct conceived the idea for this paper, vo conducted the analysis and wrote the manuscript. ka, aw, lm, de, rr, and ss participated in project oversight. sf, rg, rr, ss, and iy participated in the design of the study. all authors contributed to drafting the manuscript and approved the final submission. the study was approved by the institutional review boards of the royal thai army in bangkok, thailand, the walter reed army institute of research and the london school of hygiene & tropical medicine. all participants provided written informed consent. the investigators have adhered to the policies for protection of human subjects as prescribed in army regulation 70-25. not applicable. cct is associate editor for bmc infectious diseases, research area viral diseases. all other authors declare that they have no competing interests. springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. author details 1 saw swee hock school of public health, national university of singapore and national university health system, singapore 117549, singapore. advances in multiparametric molecular diagnostics technologies for respiratory tract infections the increasing application of multiplex nucleic acid detection tests to the diagnosis of syndromic infections infectious diseases society of a. an unmet medical need: rapid molecular diagnostics tests for respiratory tract infections frequent detection of respiratory viruses without symptoms: toward defining clinically relevant cutoff values elevated transmission of upper respiratory illness among new recruits in military barracks in thailand. influenza other respir viruses respiratory tract infections in the military environment environmental factors, immune changes and respiratory diseases in troops during military activities overcrowding and mortality during the influenza pandemic of 1918 broad spectrum respiratory pathogen analysis of throat swabs from military recruits reveals interference between rhinoviruses and adenoviruses respiratory diseases among u.s. military personnel: countering emerging threats serosurvey of bacterial and viral respiratory pathogens among deployed u.s. service members antiviral therapy and outcomes of influenza requiring hospitalization in ontario access to a polymerase chain reaction assay method targeting 13 respiratory viruses can reduce antibiotics: a randomised, controlled trial impact of rapid detection of viral and atypical bacterial pathogens by real-time polymerase chain reaction for patients with lower respiratory tract infection clinical impact of rt-pcr for pediatric acute respiratory infections: a controlled clinical trial impact of rapid microbiological testing on the management of lower respiratory tract infection multiplex molecular panels for diagnosis of gastrointestinal infection: performance, result interpretation, and cost-effectiveness multiplex gastrointestinal pathogen panels: implications for infection control diagnosing norovirus-associated infectious intestinal disease using viral load quantitation of group a rotavirus by real-time reverse-transcriptionpolymerase chain reaction: correlation with clinical severity in children in south india quantitative real-time polymerase chain reaction for enteropathogenic escherichia coli: a tool for investigation of asymptomatic versus symptomatic infections is higher viral load in the upper respiratory tract associated with severe pneumonia? findings from the perch study the relationship between respiratory viral loads and diagnosis in children presenting to a pediatric hospital emergency department correlation of rhinovirus load in the respiratory tract and clinical symptoms in hospitalized immunocompetent and immunocompromised patients correlation of viral load as determined by real-time rt-pcr and clinical characteristics of respiratory syncytial virus lower respiratory tract infections in early infancy clinical significance of different virus load of human bocavirus in patients with lower respiratory tract infection association between high nasopharyngeal viral load and disease severity in children with human metapneumovirus infection clinical disease and viral load in children infected with respiratory syncytial virus or human metapneumovirus clinical characteristics and viral load of respiratory syncytial virus and human metapneumovirus in children hospitaled for acute lower respiratory tract infection analysis of viral load in children infected with human metapneumovirus human bocavirus in children: mono-detection, high viral load and viraemia are associated with respiratory tract infection association between nasopharyngeal load of streptococcus pneumoniae, viral coinfection, and radiologically confirmed pneumonia in vietnamese children nasopharyngeal pneumococcal density and evolution of acute respiratory illnesses in young children dna bacterial load in children with bacteremic pneumococcal community-acquired pneumonia dna bacterial load in children and adolescents with pneumococcal pneumonia and empyema comprehensive molecular testing for respiratory pathogens in community-acquired pneumonia new concepts in diagnostics for infectious diarrhea high nasopharyngeal pneumococcal density, increased by viral coinfection, is associated with invasive pneumococcal pneumonia clinical characteristics of children with lower respiratory tract infections are dependent on the carriage of specific pathogens in the nasopharynx development of two real-time multiplex pcr assays for the detection and quantification of eight key bacterial pathogens in lower respiratory tract infections key: cord-285587-rggfg60a authors: meligy, bassant; sayed, amal; ismail, dalia kadry; kamal, dina; abdel-latif, walaa; erfan, dina m. title: detection of viral acute lower respiratory tract infection in hospitalized infants using real-time pcr date: 2015-12-30 journal: gaz egypt paediatr assoc doi: 10.1016/j.epag.2015.11.005 sha: doc_id: 285587 cord_uid: rggfg60a introduction: acute lower respiratory tract infection in children causes significant morbidity in the developing countries. documentation of virus infection using pcr and clinical characteristics of patients affected with viral pneumonia are reviewed in this study. methods: 51 children less than three years admitted to the pediatric hospital, cairo university with viral pneumonia were included. all patients had undergone nasopharyngeal aspirate for pcr viral detection. results: a total of 51 cases were enrolled in the study, of which 7 cases were negative while 44 children were positive for viruses. the most common respiratory virus was rhinovirus in 32 patients (72.2%), then parainfluenza virus (piv) in 12 (27.3%), of which subtypes piv1 were 2 (4.5%), piv3 were 5 (11.4%) and piv4 were 5 (11.4%) cases. the third common viruses were respiratory syncytial virus (rsv) in 9 (20.5%) cases of which 3 (6.8%) were rsva and 6 (13.6%) were rsvb and adenovirus in 9 cases (20.5%). boca virus was found in 8 (18.2%) patients, corona virus 2 (4.5%) patients, h1n1 2 (4.5%) patients, enterovirus 2 patients (4.5%) and human metapneumovirus in one case (2.3%). influenza b and piv2 were not detected. coinfection was found in 28 (63.7%). mortality occurred in 12 (23.5%). there was no significant relation between virus type or coinfection with disease severity. conclusions: rv was the most commonly detected virus in children under 3 years admitted with acute lower respiratory tract infections. coinfection was present in the majority of our patients; however it was not related significantly to parameters of disease severity. detection of viral acute lower respiratory tract infection in hospitalized infants using real-time pcr introduction viral pathogens account for a large proportion of community acquired pneumonia cases. 1 it is estimated to cause 30-70% of the cases of pneumonia. 2, 3 for children with acute lower respiratory tract infection (alrti) treated as outpatients, a virological diagnosis is often not sought, since the diagnosis does not lead to specific therapy. most respiratory viruses can cause alrti of variable severities. 4 the conundrum of diagnosing viral pneumonia arises from the lack of understanding of the viral etiology in clinical setting. in addition, collecting the appropriate sample from infants and young children is challenging owing to the difficulty in collecting these samples in this age group. it is still difficult to differentiate between viral and bacterial pneumonia using non-specific indirect methods such as blood culture or diagnostic serology. 5 moreover the use of conventional methods of viral detection as serology and viral culture has lead to underestimation of the role of viruses as a cause of alrti. 6 the use of pcr has gained a lot of attention recently as it permits identification of more than one virus in the same respiratory specimen that has previously been difficult to culture. in the past decade, new respiratory viruses causing alrti had been discovered, the emergence of the severe acute respiratory syndrome (sars) by coronavirus, and the avian influenza type a (h5n1) pandemics are recent examples. 6, 7 the present study aims at investigating the epidemiology of viral infection using multiplex real time pcr, and exploring the clinical spectrum of the affected children in relation to viral type, during the winter season in hospitalized children with viral pneumonia. this prospective and descriptive study was conducted at cairo university pediatric hospital. 51 children under 3 years of age from october 2013 to march 2014 were included in the study. children enrolled had clinical picture of viral alrti diagnosed as bronchiolitis or pneumonia that developed few days before the admission to the hospital. nasopharyngeal aspirates were sent to cairo university molecular laboratory. bronchiolitis was characterized by low-grade/absent fever, runny nose, progressing to cough, tachypnea, hyperinflation, and chest indrawing, though wheezes are the hallmark of the disease. 8 pneumonia diagnosis was based on clinical and radiological criteria. children with moderate to high fever, breathing difficulty, crackles, and infiltrates on lung fields on chest x-ray were diagnosed as pneumonia. 9 severe pneumonia according to the who relied on the presence of any of the following signs: working, nasal flaring, intercostal recession, grunting, difficulty during breast feeding or drinking, persistent vomiting, altered sensorium or convulsions. 10 all children enrolled in the study were subjected to full history taking and clinical examination regarding age, sex, residence, history of cough, sputum production, fever, wheezing, chronic disease, and history of repeated admission to hospital. chest x ray (cxr) was done to all cases revealing either hyperinflation in bronchiolitis or pneumonic patches in pneumonia. crp, total leucocytic count (tlc) and nasopharyngeal aspirate were done to all patients with alrti. appropriate management strategies were applied as required. all patients were followed regarding the need for intensive care admission and mechanical ventilation was used to assess the severity, while improvement or death was used to evaluate the outcome. exclusion criteria were patients who developed hospital associated pneumonia after 48 h of hospital admission, children with empyema or lung abscess, and children with history of previous hospital admission with pneumonia within the last 30 days. nasopharyngeal swabs were simply and efficiently collected using flocked swabs (copan diagnostics, italy). samples were transported in 3 ml of viral transport media (vtm), and were stored at à80 c before testing. nucleic acids were extracted from 500 ll of specimens using seeprep 12tm viral na kit (nordiag, norway) for automated purification system, according to manufacturer's instructions. ten microliter of the ic was added to each specimen before the nucleic acid extraction. 10 ll of proteinase k (20 mg/ml) was added to each sample tube. the final elution volume of each sample was 60 ll. the cdnas were synthesized from extracted rnas using the cdna synthesis premix (seegene, seoul, korea) for manual set up user, according to manufacturer's instructions. respiratory virus detection kit a and b (anyplex tm ii rv 16 seegene, korea) were used according to the manufacturers' instructions. briefly, the assay was conducted in a final volume of 20 ll containing 12 ll of pcr mastermix and 8 ll of cdna. pcr mastermix consists of 5 ll of 4xrv16 a tom or b tom, 5 ll of 4â anyplex pcr mastermix (with udg), and 2 ll of rnase free water. for negative control, use 8 ll of rnase-free water instead of sample nucleic acid. there were 2 positive control tubes: rv16 pc 1 and rv16 pc 2. for set a, one tube with rv16 pc 1 and the other with rv16 pc 2 were used. for b set, one tube with rv16 pc 1 and the other with rv16 pc 2 were used. toce assay was conducted using the cfx96 real-time pcr detection system (bio-rad, ca) under the following conditions: denaturation at 95°c for 15 min and 50 cycles at 94°c for 30 s, 60°c for 1 min, and 72°c for 30 s. the fluorophores used were fam, hex, cal red 610 and quasar 670. after reaction, catcher melting temperature analysis (cmta) was performed by cooling the reaction mixture to 55°c, holding at 55°c for 30 s, and heating from 55°c to 85°c. the fluorescence was measured continuously during the temperature rise, while the melting peaks were derived from the initial fluorescence (f) versus temperature (t) curves. the melting-temperature analysis was done by seegene viewer software. 11 interpretation of the results depended on: which tube a or b, which fluorophore (fam, hex, cal red 610 or quasar 670), and the melting temperature. (table i) the 2 isolates that tested positive for influenza a were further subjected to influenza a characterization according to cdc protocol. 12 the aim and nature of the study was explained for each parent before inclusion. an informed written consent was obtained from parents or caregivers before enrollment. the study design conformed to the requirements of latest revision of helsinki declaration of bioethics (2008). the scientific research committee of pediatrics department-faculty of medicine -cairo university revised and approved the study design. data were coded and entered using the statistical package spss version 21. data were summarized using mean, standard deviation, median, minimum and maximum for quantitative variables and frequencies (number of cases) and relative frequencies (percentages) for categorical variables. comparisons between quantitative variables were done using the nonparametrical mann-whitney test. for comparing categorical data, chi square (v 2 ) test was performed. fishers' test was used instead when the expected frequency is less than 5. p-values less than 0.05 were considered as statistically significant. a total of 51 cases were enrolled in the study meeting the inclusion criteria, of which 7 cases were negative for viruses by pcr, while 44 children were positive for virus. epidemiological data and co-morbid diseases were recorded in table ii. of the 44 cases 32 (72.7%) needed picu admission due to severe respiratory symptoms and 12 (27.3%) were admitted in hospital ward. all cases needed supplemental oxygen, while 22 (50.0%) were severe enough requiring mechanical ventilation. of the positive cases, mortality occurred in 12 cases (23.5%). (table ii) . the prevalence of respiratory viruses is shown in (fig. 1 ) 12 respiratory viruses were isolated. the most common respiratory virus detected was rhinovirus (rv) in 32 patients (72.2%), then parainfluenza virus (piv) in 12 (27.3%), of which subtypes piv1 were 2 (4.5%) piv3 were 5 (11.4%) and piv4 were 5 (11.4%) cases. the third common viruses were respiratory syncytial virus (rsv) in 9 (20.5%) cases of which 3 (6.8%) were rsva and 6 (13.6%) were rsvb and adenovirus in 9 cases (20.5%). boca virus was found in 8 (18.2%) patients, corona virus 2 (4.5%) patients, influenza a virus 2 (4.5%), enterovirus 2 patients (4.5%) and finally human metapneumovirus 1 case (2.3%). the 2 cases that tested positive for influenza a virus were further subjected to influenza a sequencing and characterization and were there was no statistical significance between virus type, coinfection and comorbidity with length of hospital stay, picu admission, mechanical ventilation or outcome of the patients. there was no significant association between coinfection (p value 0.501) and length of hospital stay, picu stay (p value 0.487), mv (p value 0.531), nor outcome (p value 1.000). again there was no significant relation between comorbidity and length of hospital stay (p value 0.644), picu admission (p value 0.457), mv (p value 0.728), nor outcome (p value 0.701). there was no correlation between each virus type and parameters of disease severity (table. iii) as length of hospital stay, picu admission, mv and death as outcome. the world health organization estimates that %2 million children die each year from alrti, and most live in developing countries. 13 the development of sensitive molecular diagnostic assays has increased the yield of virus detection in comparison to other conventional methods. multiplex pcr is used increasingly to diagnose respiratory infections. a study performed on children with acute respiratory illness analyzed the viral detection capacities of culture, direct immunofluorescence, and multiplex pcr found pcr to be the most sensitive method detecting a viral cause in 91.5% of children in their nasal samples. 14 multiplex pcr enabled laboratorians to detect a panel of viruses simultaneously while reducing hands on time and with greater analytical sensitivity. 15 there are several multiplex assays evaluated for detection of respiratory viruses. several studies have demonstrated the advantages of anyplexll rv16 detection assay. 11, 15, 16 we studied children attending our hospital and emergency department during the respiratory season for the occurrence of viral alrti. in this study 33 (75%) cases were diagnosed with pneumonia, while 11 (25%) cases were diagnosed with bronchiolitis though differentiating between those two conditions is difficult as they exhibit less specific clinical picture. 17 in the present study 7 cases were negative for viral infections that may be due to other viruses not used in the study or atypical pneumonia. the causes of alrti in children under 5 years have been studied in dissimilar terrains and population. rsv, rv, hmp, and piv have been shown to prevail, with co-existence of multiple infections with more than one viral pathogen occurring in 4-33% of children. 18 rsv infections were the most frequently encountered infections responsible for hospitalization among infants and were responsible for about 3.4 million hospital admissions of children less than 5 years worldwide. 19 although rsv is well recognized as the main agent associated with severe alrtis, recent data indicate that other viruses may play a significant role in these clinical outcomes, rv seems to be of particular interest, as the most prevalent virus in respiratory illnesses even in the first years of life, being associated with severe acute bronchiolitis. 20 the high frequency of rv co-infection with rsv has been observed previously. [21] [22] [23] rv in our study was the most commonly encountered virus accounting for 72.2% (32/44) of cases in single or mixed infection. piv virus was the second most common responsible for 12 cases (27.3%), while the third common viruses were rsv/ ad infecting 9 cases (20.5%).whether rv per se or associated with other viruses showed significant correlation with case severity cannot be assessed in this study. another one-year-prospective study conducted at cairo university children's hospital enrolled 450 children under the age of 5 years referred to outpatient and emergency department to the same hospital as ours, during a 1 year period, and viral detection was done using direct fluorescence essay, real time pcr and shell viral culture found that rsv was the most common virus responsible for hospitalization for viral alrti (63.3%) followed by piv in 38 (12.2%), hmpv in 34 (10.9%) and adenovirus in 26 (8.4%) patients, but this study did not include rv detection by pcr. 24 the results of our study were similar to a study conducted in hospitalized patients admitted with viral pneumonia in south africa where the majority of their patients were children under 5 years of age, showing that among patients aged 61 year, the most common virus was rhinovirus (985 of 3157 [31.2%]), followed by rsv (845 of 3157). 25 another study found that rhinovirus was present in 18% of asymptomatic children and in >30% of children hospitalized with severe acute respiratory infection (sari), and concluded that it might suggest a possible role in disease severity. 26 the proportional contribution of influenza viruses in alrti in children was referred to in this work. during the study period there was a major fear of swine flu infection (h1n1) that raised public health alert for fear of fatal influenza pneumonia. though influenza causes fever without localization with lack of respiratory symptoms, it makes these children not eligible for the study. in our study only 2 cases with h1n1 were detected out of 44 cases, those infected had an underlying congenital heart disease and required intensive care admission and mechanical ventilation but with no case fatality. the overall burden of influenza to alrti in other studies was in accordance with our study. a 3-year prospective study of children younger than 24 months hospitalized with a febrile respiratory illness found only 3 patients out of 201 children with influenza infection. 27 a study from australia found that the bulk of influenza-like illness in children less than 2 years during the 2009 h1n1 influenza pandemic was actually caused by rsv. 28 a meta-analysis of 9 studies in which influenza virus was lab confirmed, estimated that the burden of influenza virus results in 3.0% of hospitalized alrti in children. 29 the impact on severity of respiratory infections by pcr viral co-detections was the subject of many studies. some studies contrasting to our study have shown a positive association between viral co-detection and worse clinical outcomes. 30 in a study conducted on 749 infants less than 2 years of age found that coinfection occurred in 86 children (17.4% of positive samples) and that coinfection was associated with more fever, longer hospital stays and more frequent use of antibiotics. in this work single infection was found in 16 (36.4%) cases, while multiple infections were found in 63.7% of cases, of which dual infection occurred in 23 (52.3%) cases and triple infection in 5 (11.4%) cases, a coinfection rate in most studies is between 17% and 36% (24) (caroline et al., 2012). greensill et al. 31 studied 30 infants with bronchiolitis requiring mechanical ventilation and reported a 70% co-infection rate among hmpv and rsv. another study on hospitalized infant less than 1 year concluded that the presence of dual viral infections, (rsv and rv) increased the risk of picu admission of infant with bronchiolitis. 32 we need to perform a population based study in egypt to evaluate the viral load of arlti in children and draw epidemiologic map of the country. also we need to take into account the cost effectiveness of some vaccine preventable viral illnesses in our developing country, to redirect our financial resources to the most prevalent viruses in the region. there were some limitations in our study as the small sample of our studied patients was limited by financial issues as the study was self-funded, single institution search and not a population-based study, and also it was limited to one season. another limitation was the lack of surveillance over a prolonged period of time, which could have biased our results in case of an outbreak of a specific virus in a given year. we found that rv was the most commonly detected virus in hospitalized children under 3 years of age with alrti. we could also detect simultaneous infections in the majority of patients which did not affect short term outcome as case fatality and length of hospital stay. optimizing community case management stratifies to achieve equitable reduction of childhood pneumonia mortality: an application of equitable impact sensitive tool (equist) in five lowand middle-income countries viral etiology and symptoms of acute respiratory tract infections in children viral aetiology of acute lower respiratory tract illness in hospitalized pediatric patients of a tertiary hospital: one year prospective study respiratory viral infections in infants: causes, clinical symptoms, virology, and immunology acute lower respiratory infections in p5 year old hospitalized patients in cambodia, a low-income tropical country: clinical characteristics and pathogenic etiology viral pneumonia a newly discovered human pneumovirus isolated from young children with respiratory tract disease world health organization children: reducing mortality. world health organization factsheet no comparison of anyplex ii rv16 with the xtag respiratory viral panel and seeplex rv15 for detection of respiratory viruses world health organization: the global burden of disease: 2004 update comparison of multiplex pcr assays and conventional techniques for the diagnostic of respiratory virus infections in children admitted to hospital with an acute respiratory illness evaluation of a novel real-time rt-pcr using toce technology compared with culture and seeplex rv15 for simultaneous detection of respiratory viruses narkevicˇi ut_ e i, kucˇinskien_ e z. pcr detection of human respiratory viruses using anyplex ii rv16 1.0 (seegene inc.) diagnosis and testing in bronchiolitis: a systematic review viral and atypical bacterial detection in acute respiratory infection in children under five years global burden of acute lower respiratory infections due to respiratory syncytial virus in young children: a systematic review and meta-analysis severe lower respiratory tract infection in infants and toddlers from a non-affluent population: viral etiology and codetection as risk factors mixed respiratory virus infections correlation of rhinovirus load in the respiratory tract and clinical symptoms in hospitalized immunocompetent and immunocompromised patients the impact of dual viral infection in infants ad mitted to a pediatric intensive care unit associated with severe bronchiolitis viral etiologies of lower respiratory tract infections among egyptian children under five years of age respiratory viral coinfections identified by a 10-plex real-time reverse-transcription polymerase chain reaction assay in patients hospitalized with severe acute respiratory illness-south africa contribution of common and recently described respiratory viruses to annual hospitalisations in children in south africa viral etiology of acute febrile respiratory illnesses in hospitalized children younger than 24 months clinical and microbiological features associated with novel swine origin influenza a pandemic 2009 (h1n1) virus in children viral etiology of hospitalized acute lower respiratory infections in children under 5 years of age -a systematic review and metaanalysis multiple simultaneous viral infections in infants with acute respiratory tract infections in spain human metapneumovirus in severe respiratory syncytial virus bronchiolitis the impact of dual viral infection in infants admitted to a pediatric intensive care unit associated with severe bronchiolitis abdel latif (md) and m. erfan (md) initiated the study idea and design and helped in the analysis and interpretation of data.meligy b. (md) drafted the original manuscript and was responsible for publication.sayed a. (md), and kadry d. (md) conducted the laboratory part of the study. kamal (md) collected data and performed the clinical part of the study. all authors contributed to interpretation of data and critically revised the final manuscript. none declared. key: cord-286443-t0asknzu authors: emerson, julia; cochrane, elizabeth; mcnamara, sharon; kuypers, jane; gibson, ronald l.; campbell, angela p. title: home self-collection of nasal swabs for diagnosis of acute respiratory virus infections in children with cystic fibrosis date: 2013-07-14 journal: journal of the pediatric infectious diseases society doi: 10.1093/jpids/pit039 sha: doc_id: 286443 cord_uid: t0asknzu background: understanding the importance of respiratory viruses in children with cystic fibrosis (cf) has been limited because of challenges using clinicor hospital-based diagnostic testing. we conducted a pilot study to assess feasibility of home self(or parent-) collection of nasal swabs (ns). methods: cystic fibrosis patients aged 6–18 years with new respiratory illness participated. in clinic, a deep nasal flocked swab was collected by research staff and compared with an anterior foam ns obtained after instillation of saline spray. at home, up to 2 self-collections of paired foam ns (with and without saline) were collected and mailed for real-time polymerase chain reaction (pcr) testing. results: paired swabs were collected from 28 patients: 18 sets in clinic (deep nasal vs saline foam ns) and 43 sets at home (saline vs dry foam ns) with 9 (50%) and 35 (81%) virus detections, respectively. home-collected ns were obtained closer to illness onset, with a mean difference in symptom days of −2.3 between home and clinic collections (95% confidence interval [ci] −3.5, −1.2; p < .001). rhinovirus comprised 73% of virus detections; the difference in mean pcr cycle threshold values for rhinovirus between swabs collected at home versus clinic was −3.8 (95% ci −6.8, −0.9; p = .014), indicating significantly higher viral load for home-collected swabs. conclusions: home-collected foam ns had a higher positivity rate compared with clinic-collected swabs, likely because collection was closer to illness onset. home self-collection is feasible and well tolerated for timely respiratory virus diagnosis and provides a novel approach for clinical diagnostics and surveillance of respiratory virus infections among cf patients. background. understanding the importance of respiratory viruses in children with cystic fibrosis (cf) has been limited because of challenges using clinic-or hospital-based diagnostic testing. we conducted a pilot study to assess feasibility of home self-(or parent-) collection of nasal swabs (ns). methods. cystic fibrosis patients aged 6-18 years with new respiratory illness participated. in clinic, a deep nasal flocked swab was collected by research staff and compared with an anterior foam ns obtained after instillation of saline spray. at home, up to 2 self-collections of paired foam ns (with and without saline) were collected and mailed for real-time polymerase chain reaction (pcr) testing. results. paired swabs were collected from 28 patients: 18 sets in clinic (deep nasal vs saline foam ns) and 43 sets at home (saline vs dry foam ns) with 9 (50%) and 35 (81%) virus detections, respectively. home-collected ns were obtained closer to illness onset, with a mean difference in symptom days of −2.3 between home and clinic collections (95% confidence interval [ci] −3.5, −1.2; p < .001). rhinovirus comprised 73% of virus detections; the difference in mean pcr cycle threshold values for rhinovirus between swabs collected at home versus clinic was −3.8 (95% ci −6.8, −0.9; p = .014), indicating significantly higher viral load for homecollected swabs. conclusions. home-collected foam ns had a higher positivity rate compared with clinic-collected swabs, likely because collection was closer to illness onset. home self-collection is feasible and well tolerated for timely respiratory virus diagnosis and provides a novel approach for clinical diagnostics and surveillance of respiratory virus infections among cf patients. there is a growing body of evidence that respiratory virus infections play an important role in pulmonary morbidity and exacerbations in children with cystic fibrosis (cf) [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] . previous studies using clinic-or hospital-based testing have likely underestimated the true impact of respiratory viruses on cf pulmonary exacerbations due to delays in sample collection relative to onset of symptoms and the lack of sensitive molecular testing methods. investigations to further elucidate the impact of respiratory virus infections in cf will require timely diagnoses using molecular methods. nasal washes or invasive nasopharyngeal swabs collected by medical personnel have historically been considered "gold standard" samples for respiratory virus detection. in children, nasal swabs (ns) have been shown to have reasonable performance for polymerase chain reaction (pcr) detection of most respiratory viruses [15] [16] [17] [18] . communitybased studies have used parent-or self-collected swabs for respiratory virus research [19] [20] [21] [22] [23] [24] . although previous studies using self-collection have involved transport medium a and storage at 4°c, we have recently developed a simple, sensitive, and noninvasive method for self-collection of respiratory samples using foam swabs that does not require transport media or refrigeration [25] . furthermore, we found that swabs collected with the use of saline spray were superior to swabs collected without the use of saline spray [25] . in the current study, we investigated the feasibility of home self-collection of ns in children with cf experiencing onset of new respiratory illness, with samples mailed to a central laboratory for respiratory virus detection by real-time pcr. although the main objective was to assess feasibility, we also sought to study whether ns collected at home would compare favorably to swabs collected in clinic for detection of respiratory viruses, and whether foam ns collected with the use of saline spray would perform comparably to swabs collected without saline. the study protocol was approved by the seattle children's institutional review board. children ages 6-18 years with a diagnosis of cf were eligible to enroll if they met the following criteria: (1) attended at least 2 cf clinic visits during the previous 12 months; (2) currently experiencing a new respiratory illness or willing to return for a clinic visit or collect home samples when experiencing a new respiratory illness; (3) willing to perform self-collection of ns (collected by the child or parent); and (4) written informed consent provided. children were excluded if they had received antiviral medications during the 30 days before enrollment or if they were awaiting or had previously received a lung transplant. the period of study enrollment was february 2009 to january 2010, with up to 15 months of follow-up per patient. if respiratory illness with onset of symptoms in the previous 7 days was present at a clinic visit, subjects were first asked to blow their nose to remove mucus that might inhibit pcr, and paired swabs were obtained as follows: a deep nasal (mid-turbinate) sample was collected by research staff, by first measuring from the opening of one naris to the nasal bridge, and then inserting a standard flexible nasopharyngeal flocked nylon swab (copan diagnostics inc, murrieta, ca; catalog no. 503cs01) until mild resistance was encountered, approximately one-half to two-thirds the length of the nose. this approach to using nasopharyngeal swabs to collect deep nasal samples was previously found to be well accepted by participants in a study of respiratory viruses in cf patients at our hospital [2] . next, a polyurethane foam ns (super brush, llc, chicopee, ma; catalog no. 71-4541) was collected in the opposite naris after instillation of saline nasal spray. nasal spray was used to closely replicate a standard nasal wash. in brief, 5 sprays of saline from a polyethylene metered bottle (0.1 ml/spray) were instilled into the naris, followed by ns insertion into the anterior naris as far as comfortably possible, and rotation of the swab while exhaling through the nose for 5-7 seconds. during the clinic visit, the research nurse instructed the patient regarding self-collection of foam ns, and either the patient or parent collected the swab while observed and directed by the research nurse, or the swab was collected by the research nurse as an opportunity to demonstrate the proper technique for collection to the patient and parent. detailed written instructions regarding self-collection were reviewed at the clinic visit and were provided to the family for reference when swab collection at home was indicated (see supplementary material). also provided were kits containing all supplies necessary for collection and mailing of foam ns to be obtained at home at onset of symptoms of a new respiratory illness. subsequent collections at home consisted of foam ns collected either by the participant or parent with ("saline") and without ("dry") the use of nasal saline spray to evaluate whether the spray was essential to the procedure, as previously described [25] . dry swabs were collected by inserting a foam swab into the anterior naris as far as comfortably possible, followed by slow rotation of the swab for 5-7 seconds while exhaling through the nose. for home collections, participants were asked to collect the dry swab before the saline swab. during initial development and optimization of the selfcollection procedure, we previously evaluated virus stability over time from foam ns (for influenza a and parainfluenza virus type 3), and we found no difference in viral recovery between room temperature and 4°c, transport medium, and dry tubes, at 1, 2, or 7 days [25] . thus, for this study each foam swab was placed into an empty dry transport tube (no transport media added) and stored at room temperature. home-collected ns were mailed to the university of washington molecular virology laboratory using us postal service pre-paid priority mailers with appropriate packaging and labeling for category b infectious substances. deep ns collected in clinic were placed into lysis buffer and stored at 4°c until laboratory testing. study participants received phone or e-mail reminders every 1-2 weeks to collect samples at home at onset of new respiratory illness; home sample collection was allowed during up to 2 illnesses per participant. each participant (or parent) completed a standardized symptom survey in conjunction with each sample collection. criteria for a new respiratory illness included (1) presence of at least 1 of the 15 symptoms listed on the standardized symptom survey and (2) symptom duration of a minimum of 24 hours and 7 days. questionnaires related to tolerability of selfcollection were also completed. participants were instructed to mail home swabs and surveys within 1 day of collection. swabs were processed in the laboratory as soon as possible after receipt. specimens were tested for qualitative detection by a panel of 8 single or multiplexed real time reversetranscription (rt)-pcr (for respiratory syncytial virus, influenza virus types a and b, parainfluenza virus types 1-4, human metapneumovirus, human coronaviruses [subtypes oc43, 229e, nl63, and hku1] and rhinoviruses) and pcr (for adenovirus and bocavirus) using previously described methods [25] [26] [27] [28] [29] [30] [31] . samples were considered positive if the pcr amplification plot crossed the threshold at less than 40 cycles (cycle threshold [c t ] <40). all pcr methods were performed according to college of american pathologist standards, and the laboratory passed proficiency testing in viral diagnostics. data were summarized using counts and proportions and means and standard deviations (sd). linear regression analyses were used to compare differences in time from collection to laboratory processing, symptom duration, and number of symptoms between swab collections performed at home versus in clinic. a similar method was used to compare differences in rt-pcr c t values between swabs positive for rhinovirus alone that were collected at the same time and between swabs collected at home versus in clinic. too few swabs were positive for other virus types to perform statistical analyses. regression analyses included clustering on participant to account for repeated observations per participant, and 95% confidence interval (ci) estimates were calculated using robust variance estimates. all analyses were performed using stata version 10.1 (statacorp, college station, tx). a total of 35 children were enrolled, 28 of whom provided paired swab sets collected in clinic or at home during a new respiratory illness. baseline characteristics were similar between the total study population and those who had samples collected (table 1) . paired swabs were collected during new respiratory illnesses as follows: 18 sets (deep nasal vs foam ns with saline) collected at clinic visits and 43 sets (foam ns with and without saline) collected at home (7 participants with a single home collection and 18 with 2 home collections). study samples thus included a total of 61 swab sets collected during new respiratory illnesses, representing a total of 122 swabs available for pcr testing. for the 43 home collections, 27 (63%) swab sets were collected by the parent, 14 (33%) by the participant, and 2 by another adult individual. the mean age of participants who performed self-collection was older than that of participants who had samples collected by someone else (15.7 vs 10.3 years, respectively). viral pcr results are presented for paired swab sets collected in clinic and at home ( table 2) . among the 122 swabs tested, 81 (66.4%) had 1 or more viruses detected, and 41 (33.6%) were negative for all viruses tested. the most prevalent finding was rhinovirus, which was detected in 59 swabs (48.4%). for swabs collected in clinic, overall percent agreement was observed for 13 of 18 pairs (72.2%), including 4 pairs with both swabs positive for the same virus and 9 pairs with both swabs negative. for ns collected at home, overall percent agreement was observed for 39 of 43 pairs (90.7%), including 31 pairs with both swabs the time from collection to laboratory processing averaged 1.1 day (sd = 0.9) for swab sets collected in clinic, 5.4 days (sd = 3.6) for the first home collection, and 6.4 days (sd = 4.0) for the second home collection. there was no association between longer time from collection to laboratory processing and likelihood of negative results by viral pcr. symptom surveys were summarized according to timing of sample collection ( table 3 ). the most common symptoms reported at clinic collections were increased nasal congestion, increased cough, and increased sputum production; increased nasal congestion, sore throat, and increased cough were the most common symptoms reported at home collections. the mean difference in number of symptoms was 1.1 comparing home versus clinic collections (95% ci −0.1, 2.4; p = .08), but this result was not statistically significant. the mean difference in days with increased symptoms was −2.3 comparing home versus clinic collections (95% ci −3.5, −1.2; p < .001), indicating significantly shorter duration of symptoms at the time of collection for swabs collected at home. tolerability surveys were completed by all 28 participants on 59 occasions (16 clinic visits and 43 home collections) and indicated that self-collection of anterior nasal foam swabs was acceptable and not difficult for participants. questions were answered using a 5-point response scale (strongly agree, agree, neither, disagree, or strongly disagree). using results from the first tolerability survey completed by each participant, we found that participants regarded that collection of anterior nasal foam swabs was comfortable (71% agree or strongly agree responses, 11% neither agree nor disagree, and 18% disagree responses). among the 18% (5 subjects) who disagreed that the procedure was comfortable, specific comments included that the swab was "large and uncomfortable" and that self-collection was "hard to do" when not feeling well. subjects thought that self-collection was simple (96% agree or strongly agree responses) and that instructions were clear and easy to follow. the majority of participants indicated willingness to participate in future studies using the self-collection procedure (85% agree or strongly agree responses). restricting to responses obtained at the 16 clinic visits, participants indicated that the procedure for collection of anterior nasal foam swabs was preferred over collection of the deep nasal flocked swab (94% agree or strongly agree responses). adverse events were minimal. one episode of mild and self-limited epistaxis occurred following a clinic swab collection. in 5 home collections, 3 participants reported cough, 1 reported sneezing, and 1 subject reported "blood smear on sample." in this pilot study, we demonstrated that self-and parentcollection of foam ns at home and mailing to the laboratory for respiratory virus diagnosis was feasible for patients with cf. this method of home collection was simple, comfortable, and safe, and mailing time did not affect the likelihood of virus detection. swabs collected at home yielded a higher proportion of positive virus detections compared with swabs collected in clinic (81% vs 47%), likely explained by our finding that home-collected ns were collected significantly closer to the onset of illness. similarly, rates of virus positivity in the home-collected ns were higher when compared with other recent clinic-or hospital-based studies of respiratory virus surveillance using pcr in cf patients with respiratory symptoms or pulmonary exacerbations, with reported detection rates of 50%-60% [8, [12] [13] [14] . likewise, we found that among pcr-positive swabs for rhinovirus, the amount of virus in swabs collected at home was significantly greater than for swabs collected in clinic. in this study among children with cf, the self-collection method was feasible using foam swabs collected with saline or dry swabs with comparable rates of viral detection. a recent manuscript showed that self-collected foam ns following the use of nasal saline spray had increased sensitivity over dry swabs for detection of respiratory viruses [25] . these results were in a mostly adult population, whereas the mean age in the current study was 11.7 years. it is likely that children shed high quantities of respiratory virus, and larger studies are needed to evaluate whether the use of saline spray is essential to the self-collection procedure in children. there are few other studies that have used self-sampling and mailing to study respiratory viral pathogens, although use of self-collected swabs mailed via the postal service in the united kingdom has been reported to be a feasible method of enhancing community-based syndromic surveillance for influenza [20, 32] . in these studies, viral transport medium was required for shipping, and there were no significant differences in mean times from swabbing to laboratory analysis between positive and negative samples. our approach allows for samples to be mailed in dry tubes, simplifying the procedure and eliminating the risk of spilling the transport diluent, as previously reported [32] . because of the small sample size of our study and limited diversity of virus types identified, we are unable to make conclusions regarding the sensitivity of the various swab methods used for collection, especially for viruses other than rhinovirus. the high proportion of swabs positive for rhinovirus may simply reflect the high frequency of rhinoviruses detected in nonmedically attended acute respiratory illness. however, it is important to note that the home-collected swabs did detect a variety of important respiratory viruses, in addition to rhinovirus. although it is has been documented that children with cf shed respiratory viruses even in the absence of symptoms [2, 8, 14] , the increased detection of viruses in home-collected samples in the setting of new respiratory illness suggests that this method is useful to detect incident viral infections. because the majority of the virus detections were rhinovirus, and because the more than 100 rhinovirus serotypes do not amplify with the same efficiency using our rt-pcr assay, quantitative rt-pcr testing was not performed. using rt-pcr c t values as a semiquantitative evaluation of viral load for rhinoviruses detected, we found a difference in viral load between swabs collected in clinic versus home. the nearly 3.8 c t difference between mean c t values likely represents at least a 10-fold greater viral load in home-collected compared with clinic-collected swabs. in summary, these results provide a unique method for larger studies of respiratory virus shedding and transmission among children with cf. studies involving home self-collection of respiratory samples will provide more accurate data on the incidence of respiratory virus infections among children with cf and will help to elucidate the role of viral infections in chronic bacterial colonization and pulmonary function decline in cf lung disease. because cf patients are generally followed at quarterly clinic visits, home sample collection could provide critical interim data regarding respiratory virus infections that might otherwise go undocumented. home collection of samples could also prove useful for clinical management of patients with cf, providing more prompt recognition of these infections and potentially decreasing unnecessary or prolonged antibiotic use. thus, we recommend this approach for future studies of respiratory illness and pulmonary exacerbations in patients with cf. supplementary materials are available at the journal of the pediatric infectious diseases society online (http://jpids. oxfordjournals.org). supplementary materials consist of data provided by the author that are published to benefit the reader. the posted materials are not copyedited. the contents of all supplementary data are the sole responsibility of the authors. questions or messages regarding errors should be addressed to the author. severe viral respiratory infections in infants with cystic fibrosis respiratory viruses in children with cystic fibrosis: viral detection and clinical findings effects of upper respiratory tract infections in patients with cystic fibrosis effects of viral lower respiratory tract infection on lung function in infants with cystic fibrosis effect of respiratory virus infections including rhinovirus on clinical status in cystic fibrosis prevalence and impact of respiratory viral infections in young children with cystic fibrosis: prospective cohort study association of respiratory viral infections with pulmonary deterioration in patients with cystic fibrosis the role of respiratory viruses in cystic fibrosis importance of viruses and legionella pneumophila in respiratory exacerbations of young adults with cystic fibrosis the effect of respiratory viral infections on patients with cystic fibrosis influenza-associated cystic fibrosis pulmonary exacerbations rhinovirus c and respiratory exacerbations in children with cystic fibrosis role of respiratory viruses in pulmonary exacerbations in children with cystic fibrosis detection of viral and bacterial respiratory pathogens in patients with cystic fibrosis comparing nosethroat swabs and nasopharyngeal aspirates collected from children with symptoms for respiratory virus identification using real-time polymerase chain reaction detection of multiple respiratory pathogens during primary respiratory infection: nasal swab versus nasopharyngeal aspirate using real-time polymerase chain reaction comparative study of nasopharyngeal aspirate and nasal swab specimens for diagnosis of acute viral respiratory infection non-invasive sample collection for respiratory virus testing by multiplex pcr e-mail-based symptomatic surveillance combined with self-collection of nasal swabs: a new tool for acute respiratory infection epidemiology monitoring the emergence of community transmission of influenza a/h1n1 2009 in england: a cross sectional opportunistic survey of self sampled telephone callers to nhs direct collection by trained pediatricians or parents of mid-turbinate nasal flocked swabs for the detection of influenza viruses in childhood community epidemiology of human metapneumovirus, human coronavirus nl63, and other respiratory viruses in healthy preschool-aged children using parent-collected specimens parent-collected respiratory specimens-a novel method for respiratory virus and vaccine efficacy research self-collected midturbinate swabs for the detection of respiratory viruses in adults with acute respiratory illnesses self-collection of foam nasal swabs for respiratory virus detection by pcr among immunocompetent subjects and hematopoietic cell transplant recipients clinical disease in children associated with newly described coronavirus subtypes detection and quantification of human metapneumovirus in pediatric specimens by real-time rt-pcr comparison of realtime pcr assays with fluorescent-antibody assays for diagnosis of respiratory virus infections in children evaluation of quantitative and type-specific real-time rt-pcr assays for detection of respiratory syncytial virus in respiratory specimens from children real-time reverse transcription-pcr assay for comprehensive detection of human rhinoviruses respiratory virus infection among hematopoietic cell transplant recipients: evidence for asymptomatic parainfluenza virus infection linking syndromic surveillance with virological self-sampling self-collection of nasal swabs for viral diagnosis in cystic fibrosis 351 we thank janine jijina, libby brockman, alicia stone-zipse, and linnea brody for study coordination and assistance; and nancy wright, reggie sampoleo, and rohit shankar for laboratory expertise. potential conflicts of interest. all authors: no reported conflicts.all authors have submitted the icmje form for disclosure of potential conflicts of interest. conflicts that the editors consider relevant to the content of the manuscript have been disclosed. key: cord-314841-b5l6epy3 authors: falsey, ann regina title: respiratory viral infections date: 2019-08-15 journal: genomic and precision medicine doi: 10.1016/b978-0-12-801496-7.00009-5 sha: doc_id: 314841 cord_uid: b5l6epy3 molecular analysis of respiratory viruses and the host response to both infection and vaccination have transformed our understanding of these ubiquitous pathogens. polymerase chain reaction for the rapid and accurate diagnosis of viral infections has led to a better understanding of the epidemiology and impact of many common respiratory viruses and resulted in better patient care. over the past decade a number of new respiratory viruses including human metapneumovirus and new coronaviruses have been discovered using molecular techniques such as random primer amplification, pan-viral array and next generation sequencing. analysis of the host transcriptional response during respiratory viral infection using in-vitro, animal models and natural and experimental human challenge have furthered the understanding of the mechanisms and predictors of severe disease and may identify potential therapeutic targets to prevent and ameliorate illness. respiratory viruses are a major cause of morbidity and mortality throughout the world and affect persons of all ages [1] [2] [3] [4] . in addition to >100 million office visits for upper respiratory infections each winter, hospitals fill to capacity with admissions due to community acquired pneumonia, acute exacerbations of chronic obstructive pulmonary disease, asthma and bronchitis and many of these illnesses are due to viral infection. "pneumonia and influenza" consistently ranks as the fourth most common discharge diagnosis, and each year, 270,000 to 540,000 hospitalizations and 7600 to 72,000 deaths in the united states are attributable to influenza [3, [5] [6] [7] [8] . due to their epidemic nature influenza and rsv are widely recognized as pathogens in adults and children, respectively. however, the true burden of disease and the contributions of other viruses such parainfluenza viruses (piv), human metapneumoviruses (hmpv), coronaviruses (cov), and rhinoviruses (hrv) now being more fully recognized using modern molecular detection methods [9] [10] [11] [12] [13] . in addition to sensitive and rapid diagnostic testing, new molecular techniques allow an understanding of viral evolution, mechanisms and predictors of severe disease, interrogation of vaccine responses, improved bacterial and viral diagnostics and associations of viral infections with non-respiratory medical events. in this chapter the many ways molecular and precision medicine have impacted the field of respiratory viral disease will be reviewed. in the past defining the epidemiology and impact of viral respiratory pathogens was significantly hampered by slow and/or insensitive diagnostic techniques such as cell culture and antigen detection [13, 14] . polymerase chain reaction (pcr) has revolutionized the study of respiratory viruses and provides extremely sensitive, specific and rapid means for the detection of fastidious and non-cultivatable respiratory viruses [13] . pcr based epidemiologic studies now provide a more complete understanding of the clinical spectrum and age ranges of populations affected [15] [16] [17] [18] [19] [20] . in one study, conventional methods yielded a viral diagnosis in 14% of pneumonia cases, while use of pcr increased the yield to 56% [21] . technology has rapidly evolved from single-plex pcr and gel electrophoresis to multiplex real time assays where products are detected by luminescent signals proportional to the target amplified [14] . there are currently a variety of commercially available assays that detect from 2 to 20 viral respiratory pathogens and maintain excellent sensitivity [14] . many clinical microbiology laboratories are now moving to primarily molecular methods for viral detection and pcr formats are becoming increasingly simple so that nucleic acid extraction and pcr is fully automated with little operator input. molecular point of care assays will soon be feasible [22] . in addition to providing more sensitive means of detecting known viruses, molecular methods are extremely useful for viral discovery [23] [24] [25] . over the past several decades a number of new respiratory viruses or variants have been identified including hmpv, novel strains of coronaviruses (hku1, nl63, sars-cov, mers-cov), rhinovirus c, human boca virus, parechoviruses and new strains of avian influenza viruses. molecular methods have been critical for the rapid identification of new viruses associated with dramatic lethal outbreaks but also for pathogen discovery for routine respiratory illnesses. despite intensive investigation, in 12-62% of lower respiratory illnesses no pathogen can be identified suggesting additional agents may yet be discovered [26] [27] [28] . several different genomic approaches for pathogen discovery have been used successfully and include random primer amplification, pan-viral dna microarray and next generation sequencing ( fig. 1 ) [24] . if a viral class of an unknown pathogen or variant virus is suspected, consensus pcr using degenerate primers to detect sequences broadly conserved between members of a group can be used as was done to identify two new coronaviruses, hku1 and mers-cov [29, 30] . another technique for viral discovery is random primer amplification with conventional shotgun sequencing of pcr products [31] [32] [33] . such was the case when van den hoogen discovered a new respiratory virus in 2001, in young children with bronchiolitis who tested negative for rsv [31] . after detecting paramyxovirus like particles in cell culture, rna was subjected to random primer pcr and viral sequences were compared to all known pathogens. the new virus most closely aligned to avian pneumovirus but was determined to be a unique human pathogen and named human metapneumovirus (hmpv). similarly, in 2003 peiris identified a novel coronavirus as the cause of severe acute respiratory syndrome (sars-cov) using degenerate/random primers pcr amplification [32] . using pan viral micro array investigators at the center for disease control and prevention independently identified the same sars-cov [34] . in this technique, after random primer amplification, pcr products are hybridized to microarrays consisting of 70mer oligonucleotides derived from every fully sequenced viral genome. hybridized sequences are scraped from the microspot, amplified, cloned and finally sequenced [25] . identification of completely novel infectious agents requires unbiased and sequence independent methods for universal amplification [23, 24, 35] . conventional sanger sequencing may have poor sensitivity for genomes at low quantity. next generation sequencing (ngs) involves the analysis of millions of sequences and can detect small amounts of novel nucleic acid sequences in clinical samples. continuous sequences are assembled, host sequences are subtracted and the residual sequences are analyzed for similarity to known microbial sequences. ngs has led to the discovery a numerous novel human and animal pathogens [24] . a recent study of nasopharyngeal aspirates of thai children with respiratory illness using ngs identified a number of mammalian viral sequences belonging to newly described families of viruses such anelloviridae as well as novel strains of hrv, enteroviruses and hbov [35] . a critical step in viral discovery is the availability of bioinformatic tools to efficiently identify unique viral sequences in complex mixtures of host, bacterial and fungal sequences. new computational tools for analysis of the virome such as "virusseeker" are being developed [36] . of note, detection does equate with causation and after discovery further studies are necessary to infer more than association. the genetic and antigenic evolution of error prone rna respiratory viruses, particularly influenza, has been of interest for several decades [37, 38] . understanding the selective pressure exerted by pre-existing immunity on viral evolution may help design more effective influenza vaccines and surveillance of animal populations can be critical for early identification of emerging influenza viruses [39] . advances in deep sequencing make it possible to measure low frequency within host viral diversity and factors such as antigenic diversity, antiviral resistance, and tissue specificity can now be studied to understand the complexities of viral evolution [40] . influenza evolution at a population level has been studied years, yet, new antigenic variants are initially generated and selected at the level of the individual infected host. within a host, influenza viruses exist as a "swarm" of genetically distinct viruses [41] . sanger sequencing defines consensus sequences and cannot resolve minority variants below 20% of the viral population. deep sequencing has been used in natural infection and human challenge studies to characterize between and within host genetic diversity [41, 42] . the identification of low frequency mutations in the hemagglutinin (ha) antigenic sites or near the receptor-binding domain in vaccinated and unvaccinated influenza infected persons highlight viral evolution within a host due to selective immune pressure [41] . similarly, ngs can reveal the rapid evolution of drug resistant variants during therapy [43] . using samples collected over time, the mutational spectrum of h3n2 influenza a virus in an immunocompromised child was delineated [44] . individual resistance mutations appeared weeks before they became dominant, evolved independently on cocirculating lineages. the within host evolution of antiviral resistance reflected a combination of frequent mutation, natural selection, and a complex pattern of segment linkage and reassortment. within host sequencing diversity has also been examined in an infant with severe combined immune deficiency with persistent rsv infection [45] . ngs was performed on 26 samples obtained before and after bone marrow transplantation. the viral population appeared to diversify after engraftment with most variation occurring in the attachment protein (g). in addition, minority viral populations with palivizumab resistance mutations emerged after its administration. deep sequencing of hrv during human challenge studies has shown that hrv generates new variants rapidly during the course of infection with accumulation of changes in "hot spots" in the capsid, 2c, and 3c genes [46] . a genome-wide association study (gwas) involves rapidly scanning sets of dna, or genomes, of many people to find genetic variations associated with a particular disease. typically, the genomes of cases are compared to non-affected controls and search for single nucleotide polymorphisms (snps) or polygenic changes that are associated with risk or protection from susceptibility or severity of the condition. gwas have been useful to find genetic variations and risk for asthma, cancer, diabetes, heart disease and autoimmune illnesses with relatively limited studies relating to infectious diseases [47, 48] . recent studies examining host genetic factors conferring susceptibility to respiratory viruses such as pandemic h1n1 2009 influenza a, sars-cov and rsv now provide some insight into host genetic factors for respiratory viral infections [49] [50] [51] . previously most influenza research focused on viral genetics of novel viruses, yet experience with h1n1 2009 and h5n1 clearly indicate host factors also influence disease severity [49, 50] . a number of candidate genes influencing respiratory virus susceptibility have been identified in animal and human studies and involve host virus interactions, innate immune signaling, interferon related pathways and cytokine responses (table 1) [49] [50] [51] [69] [70] [71] [72] [73] [74] [75] . over 20 studies have evaluated genetic polymorphisms associated with severe rsv disease and none demonstrates dramatic results [51] . most focused on one or a few candidate genes resulting in only modestly increased odds ratios of severe illness. a relatively large study of almost 500 hospitalized children that examined 384 snps in 220 candidate genes demonstrated that susceptibility to rsv is complex with a several associations to a few innate immunity genes. these included a vitamin d receptor gene associated with down regulating interleukin 12 (il-12), gamma interferon (ifn-γ), nitrous oxide synthase (nos2a), the jun oncogene, an important transcriptional regulator for innate immune pathways, and ifn-α (ifna5) an antiviral cytokine [68] . the host transcriptional response can be analyzed to investigate disease pathogenesis using a variety of methods including in-vitro studies of bronchial epithelial cells (bec), animal models and infection both natural and experimental challenge [76] [77] [78] [79] . in addition, two compartments, the respiratory epithelium and blood can be sampled in human studies and interrogated using different viruses or viral strains to develop gene signatures for prognosis, as indicators of severity and to identify potential therapeutic targets. most respiratory viral mechanistic studies have been performed using influenza viruses, rsv, hrv and coronaviruses [80] [81] [82] [83] . using bec, the common and (h5n1, h7n7, h3n2 and h7n9 ) and analyzed cellular responses using microarray [83] . common proinflammatory cytokines and antigen presentation were identified although each viral response was unique and notably, h7n9 responses were most similar to h3n2. the response of different clinical isolates of rsv in a549 cells, and monocyte derived human macrophages demonstrated that the pattern of innate immune activation was both host cell and viral strain specific [85] . using rna seq, differences in il-6 and ccl5 were noted among the responses to different clinical isolates suggesting different rsv strains may vary in inherent virulence. human studies have shown significant differences in the blood transcriptional profiles which change over time and differ depending on the infecting respiratory virus. mejias and colleagues were able to differentiate rsv, hrv and influenza in young children based on the blood gene profile (fig. 3) . hrv infection exhibited the mildest innate and adaptive responses compared to rsv and influenza and neutrophil gene expression was greatest in rsv infection with marked suppression of b and t cell and lymphoid responses [79] . notably, gene expression changes persisted up to 1 month after infection. similarly, studies of h7n9 infected patients showed transcriptional profile changes persisting up to 1 month with a transition from innate to adaptive immunity [86] . because of the association of hrv and exacerbations of asthma, the host response to hrv has been of particular interest [87] [88] [89] [90] . studies using becs from asthmatic and healthy donors demonstrate different transcriptional profiles when infected with hrv [87] . hrv, similar to other picornaviruses induces gene expression down regulation by the 2a and 2c proteins. in both asthmatic and healthy control derived cells the majority of genes were down regulated after exposure to hrv. however, some significant expression differences in inflammatory, tumor suppressor, airway remodeling and metallopeptidase pathways have been noted in asthmatic derived cells. asymptomatic hrv infection is quite common and its role in asthma pathogenesis has been questioned. interestingly, heinonen et al. did not find a difference in the blood transcriptome of asymptomatic hrv infected children compared to non-infected controls [91] . whereas, wesolowska-anderson and colleagues demonstrated over 100 differentially expressed genes in the nasal epithelium of asymptomatic infected hrv patients [90] . thus, the blood transcriptome may not be as informative as the nasal epithelial transcriptional response for asymptomatic hrv infection. given the significant host response to asymptomatic infection, hrv may play a role in asthma exacerbations in the absence of clinically evident disease. lastly, it may be possible to identify patients with asthma who are prone to frequent hrv related exacerbations by examining the gene expression response of their pbmcs stimulated with hrv [89] . gene expression studies focusing on illness severity may enhance our understanding of disease pathogenesis, can identify potential therapies to modulate harmful host responses and can be used to develop biomarkers for predicting life threatening disease [79, [92] [93] [94] . a number of studies have been undertaken to understand the pathogenesis of severe rsv in young children and have identified a variety of gene expression patterns in blood including under expression of t cell cytotoxicity/nk cells and plasma cell genes, as well as upregulation of jak/stat, prolactin, il-9 signaling, cell to cell signaling, and immune activation pathways [79, 92] . using nasal epithelial gene expression analysis, van den kieboom identified 5 differentially expressed genes in 30 children with mild, moderate and severe rsv infection [81] . ubiquitin d, tetraspanin 8, mucin 13, β microseminoprotein, chemokine ligand 7 were up regulated and differentiated mild from severe illness. lastly, nasal gene expression is complicated by interactions of the nasal microbiota and host cell gene responses [95] . in nasal samples from children with rsv infection, h. influenzae and s. pneumoniae dominated microbiota, toll like receptors and neutrophil/macrophage signaling were over expressed and the presence of h. influenzae and s. pneumoniae along with age and sex were predictive of risk of hospitalization due to rsv. transcriptional profiling related to severity has been analyzed in seasonal influenza as well as emerging avian pathogens with a recognition that disease is not only due to an infection with a novel virus in a non-immune host but may also be due to an exaggerated host immune response [78, 96] . in a study of primarily seasonal influenza (h1n1, h3n2), influenza infection was associated with a significantly stronger antiviral, cytokine, attenuation of t/nk cell response compared to patients with respiratory illnesses of unknown etiology regardless of severity [96] . notably, ifn and ubiquitination was significantly down regulated in those with severe vs. mild to moderate disease. in a study of the lethality of 1918 h1n1 influenza and h5n1 vietnam influenza virus in macaques, upregulation of key components of the innate immune response and cell death pathways were noted were noted with 1918 h1n1 infection but were down regulated with h5n1 [78] . early up regulation of the inflammasome likely resulted in some of the severe tissue damage noted with the 1918 h1n1 influenza infections. in vitro, animal and human challenge studies have been used to identify new strategies control or prevent symptomatic or severe infection [82, 97] . in hrv challenge studies, virperin expression correlated with rhinorrhea and chilliness. knockdown of expression resulted in increased viral replication in becs suggesting virperin has antiviral actions and might have potential therapeutic use. influenza challenge studies clearly show a definable transcriptomic profile in the blood prior to the onset of symptoms offering the possibility of earlier and more effective oseltamivir treatment [77, 98] . lastly, host gene expression studies may allow investigation into links between respiratory viral infections and specific non-respiratory events. there is ample epidemiologic evidence that influenza epidemics are linked with increased rates of strokes and myocardial infarction (mi) [99, 100] . increased rates of falls and functional decline in nursing homes have also been associated with increased influenza activity [101, 102] . however, direct links of events to viral infection are scarce in part due the event of interest may follow the infection by several weeks when the virus is no longer detectable by traditional testing. several gene profiling studies have identified viral infection signatures that may persist up to 1-month post infection [79, 86] . thus, it might be possible to study patients with falls or cardiac events for evidence of recent viral infection using a host response viral signature. in addition, evaluating the host response can provide information on mechanisms of disease. a viral gene signature was used to evaluate patients undergoing cardiac catheterization [103] . notably, 25% vs. 12%, p = 0.04 of those with a viral gene signature present vs. those without viral signatures, suffered an mi. furthermore, h1n1 infected patients showed an increased gene platelet expression signature providing insight into how infection may induce a prothrombotic state. given the availability of rapid and accurate multiplex pcr for viral detection, host-based diagnostics might seem unnecessary. however, current pcr assays use conserved known viral sequences but can miss novel or significantly mutated viruses. this issue was seen in 2009 with pandemic h1n1 when influenza pcr assays had to be adapted to optimally detect the new influenza strain [104] . the emergence of novel respiratory viruses are a persistent threat and methods to detect a "viral signature" in the setting of clusters of severe pneumonia cases could be very useful. zaas and colleagues developed an acute respiratory viral gene signature using microarray analysis of the blood from volunteers experimentally infected with influenza a, hrv or rsv [105] . the signature was subsequently 89% sensitive and 94% specific in classifying as viral 25 influenza and 3 hrv infected patients presenting to an emergency room. additionally, a distinct blood transcriptome signature was noted in patients with severe h1n1 pneumonia [106] . upregulated genes included those related to cell cycle, dna damage, apoptosis, protein degradation, and t helper cells. down regulated genes were primarily in immune response pathways suggesting immunosuppression as a mechanism of severe influenza pneumonia. investigators developed a 29 gene classifier which predicted h1n1 influenza a regardless of concomitant bacterial infection and such a predicator could guide antiviral therapy in the face of negative pathogen detection methods. in most cases of respiratory infection, the precise microbial etiology is unknown and antibiotics are frequently administered empirically [27, 107] . although sensitive molecular diagnostics (pcr) now allow rapid diagnosis of a wide variety of respiratory viruses, their impact on patient management and antibiotic prescription has been modest primarily due to concern about bacterial co-infection [108] [109] [110] . approximately 40% of adults hospitalized with a documented viral respiratory infection have evidence of concomitant bacterial infection and thus clinician concerns are reasonable [109] . importantly, sensitive and specific diagnostic tests for bacterial lung infection are currently lacking [111, 112] . although the site of infection is the respiratory tract, blood is a convenient sample comprised of components of the innate immune system (neutrophils, natural killer cells), as well as the adaptive immune system (b and t lymphocytes) [113] . recent studies indicate that viral and bacterial infections trigger pathogen specific host transcriptional patterns in blood, yielding unique "bio-signatures" that may discriminate viral from bacterial causes of infection [114] [115] [116] [117] . in the largest study to date, tsalik et al. used gene expression in blood to discriminate bacterial from viral infection or non-infectious illness in 273 subjects with respiratory illness [118] . these investigators defined 130 predictor genes in a model with an accuracy of 87% to discriminate clinically adjudicated bacterial, viral, and non-infectious illness. most studies to date have used micro array but recently rnaseq has been used to differentiate viral and bacterial respiratory illness and in one study 141 genes were noted to be differentially expressed [119] . three pathways (lymphocyte, α-linoleic acid metabolism, igf regulation pathways) which included 11 genes as predictors for bacterial infection from non-bacterial infection (naïve auc = 0.94; nested cv-auc = 0.86). to date, a number of gene expression studies of adults and children have developed predictors with similar accuracy (auc ranging from 78% to 94%), yet there has been little overlap in classifying genes identified [105, 106, [114] [115] [116] [118] [119] [120] [121] [122] . diverse populations, types of infection, plus alternate analytic tools used, likely explain the different genes identified. more work needs to be done to refine predictive gene sets including patients with mixed viral-bacterial respiratory tract infection. most studies to date have focused on blood; however, analysis of the nasal respiratory epithelium which is the site of infection might offer advantages. although data are limited, several recent papers demonstrate that nasopharyngeal host response can also be used as a diagnostic for respiratory viruses [93, 123, 124] . immune response to influenza vaccine is variable and influenced by a variety of factors including prior vaccinations and infections, age, the presence of underlying conditions and the type of vaccine administered. yet, even among a relatively homogeneous cohort of young healthy adults, antibody responses to vaccine can be variable [125] . transcriptional profiling of whole blood provide insights into the mechanisms of variability, the effects of age, and vaccine types. the ability to predict vaccine response at baseline based on a transcriptomic signature would have significant clinical implications. to understand the biologic effects of live attenuated influenza vaccine (laiv) compared to trivalent inactivated vaccine (tiv) blood transcription profiles from 85 young children were assessed by microarray at day 7 post vaccination [126] . many more genes were differentially expressed in children receiving laiv compared to tiv (245 vs. 49, respectively) and many modulated type 1 ifn. the efficacy of laiv has been problematic in recent years and assessing stimulation of type 1 ifn genes could represent a potential biomarker for response to laiv [126] . bucasas and colleagues evaluated gene expression at multiple time points after vaccination of healthy young men with tiv [127] . they noted marked up regulation of gene expression of ifn signaling, il-6 regulation, antigen processing and presentation genes within 24 h of vaccination and were able to define a 494 gene expression signature that correlated with the magnitude of antibody response. in another study, a gene profile predictive of antibody response 28 days after influenza vaccination of young and older adults was developed [128] . notably, the predictive genes were the same in young and old as well as a subgroup of subjects with diabetes suggesting similar pathways were involved despite differences in age and underlying medical conditions. additionally, transcriptional profiling has been used to signatures in blood associated with b cell memory responses to vaccination. in a study of 150 older and middle aged adults vaccinated with tiv including an h1n12009 antigen, metabolic, cell migration/adhesion, map kinase and nf-ᵏb cell genes correlated with peak memory b cell elispots [129] . finally, in a study of over 500 subjects vaccinated over several seasons, a predictive signature of nine genes and three gene modules were significantly associated with the magnitude of the serum antibody response (fig. 4 ) [130] . interestingly and in contrast to a previous study, the signature was distinct to the younger cohort. for example, inflammatory genes were associated with better response in the young but a worse response in the elderly. in summary, gene expression studies could be used to evaluate new vaccines and develop predictors of vaccine response in different subgroups of patients based on age and disease state allowing for individualized vaccine regimens. molecular analysis of respiratory viruses and the host response to both infection and vaccination have transformed our understanding of these ubiquitous pathogens. the ability to accurately diagnosis viral infections has not only impacted patient care but also changed our perceptions of the burden of disease and populations effected. transcriptional profiling of blood and nasal epithelium may provide therapeutic targets to prevent and ameliorate illness as well as offer predictors of severe disease. (14) .) summary health statistics for u.s. children: national health interview survey global respiratory syncytial virus-associated mortality in young children (rsv gold): a retrospective case series mortality associated with influenza and respiratory syncytial virus in the united states epidemiology of viral respiratory infections epidemiology of influenza trends in hospitalizations for pneumonia among persons aged 65 years or older in the united states hospitalizations associated with influenza and respiratory syncytial virus in the united states estimating influenza-associated deaths in the united states impact of rapid detection of viral and atypical bacterial pathogens by real-time polymerase chain reaction for patients with lower respiratory tract infection human metapneumovirus infections in adults: another piece of the puzzle human coronavirus and acute respiratory illness in older adults with chronic obstructive pulmonary disease update on human rhinovirus and coronavirus infections detection of respiratory viruses by molecular methods molecular detection of respiratory viruses respiratory syncytial virus infection in elderly and high-risk adults etiology of communityacquired pneumonia: increased microbiological yield with new diagnostic methods polymerase chain reaction is more sensitive than viral culture and antigen testing for the detection of respiratory viruses in adults with hematological cancer and pneumonia incidence and characteristics of viral community-acquired pneumonia in adults molecular epidemiology of rhinovirus detections in young children human metapneumovirus in the preterm neonate: current perspectives viral infection in adults hospitalized with community-acquired pneumonia: prevalence, pathogens, and presentation profile of the alere i influenza a & b assay: a pioneering molecular point-of-care test viral surveillance and discovery viral pathogen discovery viral discovery and sequence recovery using dna microarrays population-based surveillance for hospitalizations associated with respiratory syncytial virus, influenza virus, and parainfluenza viruses among young children community-acquired pneumonia requiring hospitalization respiratory picornaviruses and respiratory syncytial virus as causative agents of acute expiratory wheezing in children characterization and complete genome sequence of a novel coronavirus, coronavirus hku1, from patients with pneumonia isolation of a novel coronavirus from a man with pneumonia in saudi arabia a newly discovered human pneumovirus isolated from young children with respiratory tract disease coronavirus as a possible cause of severe acute respiratory syndrome cloning of a human parvovirus by molecular screening of respiratory tract samples a novel coronavirus associated with severe acute respiratory syndrome exploring the potential of next-generation sequencing in detection of respiratory viruses virusseeker, a computational pipeline for virus discovery and virome composition analysis mapping the antigenic and genetic evolution of influenza virus integrating influenza antigenic dynamics with molecular evolution molecular evolution and emergence of h5n6 avian influenza virus in central china within-host evolution of human influenza virus deep sequencing reveals potential antigenic variants at low frequencies in influenza a virus-infected humans deep sequencing of influenza a virus from a human challenge study reveals a selective bottleneck and only limited intrahost genetic diversification presence of oseltamivir-resistant pandemic a/h1n1 minor variants before drug therapy with subsequent selection and transmission intrahost dynamics of antiviral resistance in influenza a virus reflect complex patterns of segment linkage, reassortment, and natural selection within-host whole-genome deep sequencing and diversity analysis of human respiratory syncytial virus infection reveals dynamics of genomic diversity in the absence and presence of immune pressure rhinovirus genome evolution during experimental human infection genome-wide association studies and susceptibility to infectious diseases host-pathogen interactions revealed by human genome-wide surveys immunogenetic factors associated with severe respiratory illness caused by zoonotic h1n1 and h5n1 influenza viruses the role of polymorphisms in host immune genes in determining the severity of respiratory illness caused by pandemic h1n1 influenza human genetic factors and respiratory syncytial virus disease severity chemokine receptor 5 big up tri, open32 allele in patients with severe pandemic (h1n1) contrasting effects of ccr5 and ccr2 deficiency in the pulmonary inflammatory response to influenza a virus enrichment of variations in kir3dl1/s1 and kir2dl2/l3 among h1n1/09 icu patients: an exploratory study ifitm3 restricts the morbidity and mortality associated with influenza the lower serum immunoglobulin g2 level in severe cases than in mild cases of pandemic h1n1 2009 influenza is associated with cytokine dysregulation genetic variants associated with severe pneumonia in a/h1n1 influenza infection associations of functional nlrp3 polymorphisms with susceptibility to food-induced anaphylaxis and aspirininduced asthma gene polymorphisms in the nalp3 inflammasome are associated with interleukin-1 production and severe inflammation: relation to common inflammatory diseases? the magnitude and specificity of influenza a virus-specific cytotoxic t-lymphocyte responses in humans is related to hla-a and -b phenotype mannose-binding lectin in severe acute respiratory syndrome coronavirus infection association between mannosebinding lectin gene polymorphisms and susceptibility to severe acute respiratory syndrome coronavirus infection significance of the myxovirus resistance a (mxa) gene -123c>a single-nucleotide polymorphism in suppressed interferon beta induction of severe acute respiratory syndrome coronavirus infection association of sars susceptibility with single nucleic acid polymorphisms of oas1 and mxa genes: a case-control study polymorphisms of interferon-inducible genes oas-1 and mxa associated with sars in the vietnamese population genetic variation in oas1 is a risk factor for initial infection with west nile virus in man a missense mutation of the toll-like receptor 3 gene in a patient with influenza-associated encephalopathy genetic susceptibility to respiratory syncytial virus bronchiolitis is predominantly associated with innate immune genes genomewide association analysis of respiratory syncytial virus infection in mice genes associated with rsv lower respiratory tract infection and asthma: the application of genetic epidemiological methods to understand causality unusual haplotypic structure of il8, a susceptibility locus for a common respiratory virus variants of the chemokine receptor ccr5 are associated with severe bronchiolitis caused by respiratory syncytial virus association of respiratory syncytial virus bronchiolitis with the interleukin 8 gene region in uk families genetic variation at the il10 gene locus is associated with severity of respiratory syncytial virus bronchiolitis a functional variation in cd55 increases the severity of 2009 pandemic h1n1 influenza a virus infection using gene expression profiles from peripheral blood to identify asymptomatic responses to acute respiratory viral infections transcriptomics in human challenge models lethal influenza virus infection in macaques is associated with early dysregulation of inflammatory related genes whole blood gene expression profiles to assess pathogenesis and disease severity in infants with respiratory syncytial virus infection gene expression patterns induced at different stages of rhinovirus infection in human alveolar epithelial cells nasopharyngeal gene expression, a novel approach to study the course of respiratory syncytial virus infection mechanisms of severe acute respiratory syndrome coronavirus-induced acute lung injury transcriptomic characterization of the novel avian-origin influenza a (h7n9) virus: specific host response and responses intermediate between avian (h5n1 and h7n7) and human (h3n2) viruses and implications for treatment options pathogenic influenza viruses and coronaviruses utilize similar and contrasting approaches to control interferon-stimulated gene responses distinct patterns of innate immune activation by clinical isolates of respiratory syncytial virus clinical correlations of transcriptional profile in patients infected with avian influenza h7n9 virus rhinovirus-induced modulation of gene expression in bronchial epithelial cells from subjects with asthma how rhinovirus infections cause exacerbations of asthma a genome-byenvironment interaction classifier for precision medicine: personal transcriptome response to rhinovirus identifies children prone to asthma exacerbations dual rna-seq reveals viral infections in asthmatic children without respiratory illness which are associated with changes in the airway transcriptome rhinovirus detection in symptomatic and asymptomatic children: value of host transcriptome analysis association of dynamic changes in the cd4 t-cell transcriptome with disease severity during primary respiratory syncytial virus infection in young infants host transcription profile in nasal epithelium and whole blood of hospitalized children under 2 years of age with respiratory syncytial virus infection gene expression signatures as a therapeutic target for severe h7n9 influenza-what do we know so far? nasopharyngeal microbiota, host transcriptome, and disease severity in children with respiratory syncytial virus infection patient-based transcriptome-wide analysis identify interferon and ubiquination pathways as potential predictors of influenza a disease severity gene expression profiles during in vivo human rhinovirus infection: insights into the host response a genomic signature of influenza infection shows potential for presymptomatic detection, guiding early therapy, and monitoring clinical responses influenza infection and risk of acute myocardial infarction in england and wales: a caliber self-controlled case series study acute myocardial infarction after laboratory-confirmed influenza infection effect of influenza on functional decline a study of the impact of influenza on the functional status of frail older people gene expression profiles link respiratory viral infection, platelet response to aspirin, and acute myocardial infarction the current epidemiology and clinical decisions surrounding acute respiratory infections gene expression signatures diagnose influenza and other symptomatic respiratory viral infections in humans a distinct influenza infection signature in the blood transcriptome of patients with severe communityacquired pneumonia appropriate antibiotic use for acute respiratory tract infection in adults treating viral respiratory tract infections with antibiotics in hospitals: no longer a case of mistaken identity bacterial complications of respiratory tract viral illness: a comprehensive evaluation routine molecular point-of-care testing for respiratory viruses in adults presenting to hospital with acute respiratory illness (respoc): a pragmatic, open-label, randomised controlled trial diagnostic and prognostic accuracy of clinical and laboratory parameters in community-acquired pneumonia developing molecular amplification methods for rapid diagnosis of respiratory tract infections caused by bacterial pathogens assessing the human immune system through blood transcriptomics superiority of transcriptional profiling over procalcitonin for distinguishing bacterial from viral lower respiratory tract infections in hospitalized adults gene expression patterns in blood leukocytes discriminate patients with acute infections gene expression profiles in febrile children with defined viral and bacterial infection robust classification of bacterial and viral infections via integrated host gene expression diagnostics host gene expression classifiers diagnose acute respiratory illness etiology transcriptomic biomarkers to discriminate bacterial from nonbacterial infection in adults hospitalized with respiratory illness diagnostic test accuracy of a 2-transcript host rna signature for discriminating bacterial vs viral infection in febrile children association of rna biosignatures with bacterial infections in febrile infants aged 60 days or younger a host-based rt-pcr gene expression signature to identify acute respiratory viral infection detection of host response to viral respiratory infection by measurement of messenger rna for mxa, trim21, and viperin in nasal swabs antiviral response in the nasopharynx identifies patients with respiratory virus infection integrative genomic analysis of the human immune response to influenza vaccination a whole genome transcriptional analysis of the early immune response induced by live attenuated and inactivated influenza vaccines in young children early patterns of gene expression correlate with the humoral immune response to influenza vaccination in humans systems biology of vaccination for seasonal influenza in humans transcriptional signatures of influenza a/h1n1-specific igg memory-like b cell response in older individuals multicohort analysis reveals baseline transcriptional predictors of influenza vaccination responses key: cord-303299-p15irs4e authors: dzien, alexander; dzien-bischinger, christine; lechleitner, monika; winner, hannes; weiss, günter title: will the covid-19 pandemic slow down in the northern hemisphere by the onset of summer? an epidemiological hypothesis date: 2020-06-23 journal: infection doi: 10.1007/s15010-020-01460-1 sha: doc_id: 303299 cord_uid: p15irs4e the covid-19 pandemic has affected most countries of the world. as corona viruses are highly prevalent in the cold season, the question remains whether or not the pandemic will improve with increasing temperatures in the northern hemisphere. we use data from a primary care registry of almost 15,000 patients over 20 years to retrieve information on viral respiratory infection outbreaks. our analysis suggests that the severity of the pandemic will be softened by the seasonal change to summer. the infection caused by the human corona virus covid-19 (sars-cov2) resulted in a worldwide pandemic affecting several million people and causing severe disease and fatality mostly based on virus mediated lung failure [1, 2] . thus far, no effective therapy is available and, therefore, contact precautions, hygiene measures, contact tracing, social distancing and quarantine are the methods of choice to control the spread of this infection [3, 4] . however, epidemics with respiratory virus such as not only influenza but also human corona viruses are prevalent in the northern hemisphere over several months during the cold season and then disappear whereas influenza remains prevalent in tropical regions throughout the whole year [5] [6] [7] . the reasons for this decline are still incompletely understood. we thus questioned whether or not the covid-19 pandemic will be in part slowed down by the change from the cold to the warm seasons in the northern hemisphere. to assess local epidemics from respiratory infections over the course of a season, we retrospectively analyzed the records of 15,336 patients (8907 women and 6429 men) who visited a primary care internal medicine practice in innsbruck, austria, between july 1, 1999, and march 15, 2020. overall, our dataset included 145,740 diagnoses, of which 16,317 are assigned to acute respiratory diseases according to the international classification of diseases (icd-10; www.icd.who.int/brows e10/2019/en). in particular, we included viral and bacterial infections classified into diagnostic groups a and b as well as infectious diseases of the respiratory tract of groups j (pulmonology), h (ear, nose and throat) and r (general symptoms consistent with infections such as dyspnea or fatigue). based on this data, we first counted the number of patients who sought medical help because of serious respiratory infections. second, we related these counts to the total number of diagnoses within each month to obtain the share of viral infections. in the denominator of this ratio, we excluded malignancies (group c), neoplasia (d), diabetes mellitus 2 (e-11.9), arterial hypertension (i-10) and ischemic heart diseases (i-20). as the mentioned practice was sometimes closed due to holiday breaks, the share of viral infections might provide a more suitable figure on seasonal patterns than the simple count measure. figure 1 plots the counts of respiratory diseases over the course of the years. it shows a strong seasonal pattern with peaks around the winter months december to march. the strongest upward deviations are observed in the years between 2008 and 2010 and for 2015. figure 2 depicts the share of respiratory diseases and pools the information from fig. 1 into one graph. the smoothed lines represent a nonlinear time trend based on year-wise regressions with the relative contributions of respiratory diseases per month as the dependent variable. in particular, we included a linear and a quadratic time trend, which allows to calculate predicted values and, in turn, the hump-shaped graphs in fig. 2 . we also used more flexible functional forms, e.g., higher order polynomials, but it turned out that the quadratic specifications performed well in terms of overall fit; a pooled regression over all seasons led to an r 2 of about 60%. all the calculations are carried out with stata (version 16). the colored lines plot the seasons in which previous pandemic respiratory infections have been recorded (02/03 sars-cov, purple line; 04/05 h5n1, green line; 09/10 pandemic flu, blue line) [8] but not showing the absolute contributions of these pandemic infections to the total number of respiratory infections. the grey lines indicate seasons where no specific pandemic respiratory infections are recorded. in line with fig. 1 , we can see that the peak of respiratory diseases (including putatively covid-19) lies between february and april 2020 followed by a prominent and sustained reduction towards summer. if covid-19 would behave similar to other respiratory viruses causing respiratory infections including human corona viruses which peak during winter time and early spring, there is hope that the covid-19 pandemic can be slowed down by this seasonal trend [7, 9] . in those seasonal infections, no herd immunity is achieved during a specific season [7] . this would go in a line with the duration of the sars-cov-1 epidemic in 2002/2003, which also started in china, peaked in february to april and was terminated in summer 2003 although strict contact precaution measures were likewise the main secret of success [9] . however, pandemics with new viruses such as the influenza h1n1v can circulate independent of typical respiratory viral seasons throughout the whole year [10] . in line with this, the pandemic "spanish flu" presented even with a summer peak in scandinavia in the year 1918 preceding the major outbreak in the cold season of 1918/1919 in this area [11] . this might be the only one published exception of an influenza summer epidemic. the next few months will provide a definitive answer to which scenario will hold true for the control of covid-19 infections and how changes of temperature and social behavior will impact on the control of this pandemic. conflict of interest none of the authors has any conflict of interest in association with this manuscript. ethical statement an approval by an ethical committee was not applicable to this study. open access this article is licensed under a creative commons attribution 4.0 international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons licence, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons licence, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/4.0/. strategic who, technical advisory group for infectious h. covid-19: towards controlling of a pandemic covid-19, sars and mers: are they closely related? novel coronavirus: where we are and what we know successful containment of covid-19: the who-report on the covid-19 outbreak in china spatiotemporal circulation of influenza viruses in 5 african countries during 2008-2009: a collaborative study of the institut pasteur international network consecutive infections with influenza a and b virus in children during the 2014-2015 seasonal influenza epidemic seasonality of respiratory viral infections pandemic influenza control in europe and the constraints resulting from incoherent public health laws sars and other coronaviruses as causes of pneumonia epidemiologic characterization of the 1918 influenza pandemic summer wave in copenhagen: implications for pandemic control strategies acknowledgements open access funding provided by university of innsbruck and medical university of innsbruck. key: cord-286930-c1zkjdgf authors: park, won-ju title: seroprevalence of respiratory syncytial virus igg among healthy young adults in basic training for the republic of korea air force date: 2014-09-02 journal: j korean med sci doi: 10.3346/jkms.2014.29.9.1325 sha: doc_id: 286930 cord_uid: c1zkjdgf this investigation enrolled 570 healthy young males gathered from all over the country for military service at the republic of korea air force boot camp. it confirmed rsv igg seroprevalence by utilizing the enzyme immunoassay method just prior to undergoing basic training. the mean age of this study was 20.25±1.34 yr old. the results of their immunoassay seroprofiles showed that 561 men (98.4%) were positive, 2 (0.4%) were negative and 7 (1.2%) were equivocal belonging to the grey zone. it was confirmed that rsv is a common respiratory virus and rsv infection was encountered by almost all people before reaching adulthood in korea. nine basic trainees belonging to the rsv igg negative and equivocal grey zone categories were prospectively observed for any particular vulnerability to respiratory infection during the training period of two months. however, these nine men completed their basic training without developing any specific respiratory illness. graphical abstract: [image: see text] respiratory infections in the military service are the most common inflictions in the military healthcare area, yet they are problems not easy to unravel. they may cause death to military personnel often through complications, such as pneumonia and meningitis. such events could induce a setback in the military schedule affecting military capability and sustaining an enormous loss. immunity is easily compromised in an ambience of high physical and psychological stress, as well as the dense population of the military training camp. they cause numerous respiratory infections. the results of studies conducted up to now revealed that primary etiologic pathogens included adenoviruses, influenza a and b viruses, epstein-barr virus, coronavirus, and rhinoviruses, as well as streptococcus pneumoniae, streptococcus pyogenes, chlamydia pneumoniae and mycoplasma pneumoniae (1) . recent studies revealed that respiratory syncytial virus (rsv) would also be a significant pathogen that causes respiratory infection and an outbreak of febrile illness in the military (2, 3) . rsv belongs to the family of paramyxoviridae, and is classified in the genus pneumovirus. rsv is the most common cause of fatal acute respiratory tract infection in infants and young children. rsv infects virtually everyone by 4 yr of age, and reinfection occurs throughout life, even among the elderly (4-6). adults infected with rsv tend to have more variable and less distinctive clinical findings than children, and the viral cause of the infection is often unsuspected (7) . this study attempted to verify the seroprevalence of rsv igg among healthy young adults in their early twenties who had just been admitted to the korea air force basic training camp. subjects with an rsv igg seronegative outcome were prospectively observed for vulnerability to respiratory infection, especially during the period of basic training. military recruits admitted to the air force boot camp were required to undergo a basic health examination which includes a blood sampling test. in this investigation, rsv igg immunoassay was added to the basic blood test. all subjects were admitted to the training camp on june 26, 2012. they underwent a blood test on june 27, 2012, which was a day after resting without training on the day of admission. the total number of recruits was 570 and all of them underwent an rsv igg test. institutional review boards of the ministry of national defense (irb no. afmc-12-irb-014). air force physicians themselves explained the purpose, method and precautions of this study, and tests were conducted after obtaining an informed consent from each subject. the mean age of subjects was 20.25 ± 1.34 yr old with a range of 18.5-28.0 yr old. the immunoassay seroprofiles showed that 561 men (98.4%) were positive, 2 (0.4%) were negative, and 7 (1.2%) were equivocal belonging to the grey zone ( table 1 , fig. 1 ). with the exception of 7 recruits in the grey zone, seropositive subjects were 99.6% (561/563). there were no significant differences in rsv igg titer among variables such as their hometown, size of city or age (p > 0.1; anova; spss ver. 15.0, chicago, il, usa). nine basic trainees belonging to the rsv igg negative or equivocal grey zone categories were prospectively observed. any particular vulnerability to respiratory infection was confirmed by checking their visits to the military medical clinic for two months during the training period. however, all of these nine recruits did not utilize the military medical facilities during the basic training period of two months. the result of this study showed that the rsv igg seropositivity of subjects in their twenties was 99.6%. it is difficult to compare the result directly because there exist the different methods or kit for detection of antibody and the criteria of each method. however, such result of seropositivity was similar to that of the us. military personnel, which was 97.8% (8) . even in several previous studies, all children older than 5 yr old were known to have experienced an rsv infection (9, 10) . this investigation also confirmed that almost all healthy young military recruits in their early twenties were rsv igg positive in the republic of korea. in an effort to verify the increased vulnerability to respiratory infection for the rsv igg negative or equivocal grey zone recruits during the training period, the medical records for all 570 subjects were verified in the basic training camp. however, the numbers of trainees with a seronegative result were small while there were totally no records of their utilization of medical facilities for symptoms relating to any respiratory infection, making a statistical analysis impossible. in the meanwhile, the outcome of not experiencing any particular respiratory infection for nine trainees with an rsv igg negative or an equivocal result during the training period of two months can lead to a notion that young adults with an rsv igg negative result may not be a risk factor for a respiratory infection in a collective group environment. nevertheless, such deduction would require further studies for verifications in the days ahead. immunity to rsv is incomplete, protective immunity against rsv infection is complex and the importance of serum antibody is controversial (11) (12) (13) . a study targeting on infants demonstrates that rsv specific maternal igg has a protective effect against severe infections. nevertheless, such studies on adult groups are inadequate (14, 15) . although there does not appear to be a defect in humoral immunity, there is evidence that the cd8+ t-cell immunity may be impaired with age (15) . deficient rsv f-specific t-cell responses contribute to susceptibility to severe rsv disease in elderly adults (16) . the significance of this study is the aspect that seroprevalence of rsv igg of healthy young adults in the republic of korea was verified for the first time. the greatest merit of this study is the fact that the subjects were collectively gathered together from all over the country at the same time and may represent healthy young male adults in the republic of korea. rsv infections occur primarily in seasonal epidemic. rsv infection occurred predominantly in the fall and winter in the republic of korea (6, 17) . therefore, our data is limited. however, as seen from the results of previous studies overseas, it was confirmed that rsv is a common respiratory virus and rsv infection was encountered by almost all people before reaching adulthood in korea. it is very important to accumulate the basic sero-epidemiological data of every infectious agent. it is anticipated that this study may provide the basic data for rsv related studies in the republic of korea in the future. frequently, an rsv infection in the military may be unsuspected. nevertheless, rsv is a common cause of respiratory illness and often causes outbreaks in particular adult groups (18, 19) . in an effort to prevent respiratory infections in the military, further rsv studies and challenges regarding diagnosis of rsv infection would be necessary. there are no financial or other issues that might lead to conflict of interests. respiratory diseases among u.s. military personnel: countering emerging threats respiratory syncytial virus: an important cause of acute respiratory illness among young adults undergoing military training symptomatic respiratory syncytial virus infection in previously healthy young adults living in a crowded military environment medical microvirology epidemiology of respiratory viral infection using multiplex rt-pcr in clinical and epidemiological comparison of human metapneumovirus and respiratory syncytial virus in seoul respiratory syncytial virus and parainfluenza virus serosurvey of bacterial and viral respiratory pathogens among deployed u.s. service members seroprevalence of anti-rsv igg in thai children aged 6 months to 5 years large-scale seroprevalence analysis of human metapneumovirus and human respiratory syncytial virus infections in beijing protective and disease-enhancing immune responses to respiratory syncytial virus relationship of serum antibody to risk of respiratory syncytial virus infection in elderly adults role of neutralizing antibodies in adults with community-acquired pneumonia by respiratory syncytial virus the level and duration of rsv-specific maternal igg in infants in kilifi kenya prevalence of respiratory syncytial virus igg antibodies in infants living in a rural area of mozambique adults 65 years old and older have reduced numbers of functional memory t cells to respiratory syncytial virus fusion protein epidemiological characterization of respiratory viruses detected from acute respiratory patients in seoul respiratory syncytial virus infection in adult populations risk factors for respiratory syncytial virus illness among patients with chronic obstructive pulmonary disease key: cord-270258-9vgpphiu authors: ko, jae-hoon; park, ga eun; lee, ji yeon; lee, ji yong; cho, sun young; ha, young eun; kang, cheol-in; kang, ji-man; kim, yae-jean; huh, hee jae; ki, chang-seok; jeong, byeong-ho; park, jinkyeong; chung, chi ryang; chung, doo ryeon; song, jae-hoon; peck, kyong ran title: predictive factors for pneumonia development and progression to respiratory failure in mers-cov infected patients date: 2016-08-09 journal: j infect doi: 10.1016/j.jinf.2016.08.005 sha: doc_id: 270258 cord_uid: 9vgpphiu background: after the 2015 middle east respiratory syndrome (mers) outbreak in korea, prediction of pneumonia development and progression to respiratory failure was emphasized in control of mers outbreak. methods: mers-cov infected patients who were managed in a tertiary care center during the 2015 korean mers outbreak were reviewed. to analyze predictive factors for pneumonia development and progression to respiratory failure, we evaluated clinical variables measured within three days from symptom onset. results: a total of 45 patients were included in the study: 13 patients (28.9%) did not develop pneumonia, 19 developed pneumonia without respiratory failure (42.2%), and 13 progressed to respiratory failures (28.9%). the identified predictive factors for pneumonia development included age ≥45 years, fever ≥37.5 °c, thrombocytopenia, lymphopenia, crp ≥ 2 mg/dl, and a threshold cycle value of pcr less than 28.5. for respiratory failure, the indicators included male, hypertension, low albumin concentration, thrombocytopenia, lymphopenia, and crp ≥ 4 mg/dl (all p < 0.05). with ≥ two predictive factors for pneumonia development, 100% of patients developed pneumonia. patients lacking the predictive factors did not progress to respiratory failure. conclusion: for successful control of mers outbreak, mers-cov infected patients with ≥ two predictive factors should be intensively managed from the initial presentation. middle east respiratory syndrome (mers) is an emerging lethal respiratory disease caused by a novel betacoronavirus (mers-cov). 1 from may to july 2015, there was a hospital-associated mers outbreak in the republic of korea reporting 186 laboratory-confirmed cases, which is the largest recorded outbreak outside the arabian peninsula. 2e6 the outbreak featured several super-spreading events with unexpectedly high human-to-human transmission rate: 136 of 186 cases (73.1%) were transmitted from only three patients. 5,7e10 as these large transmission clusters were exclusively originated from patients with pneumonia, prediction of pneumonia development has been emphasized in control of mers outbreak. 9 in addition, pneumonia progression to respiratory failure should be anticipated in advance to avoid urgent intubation or cardiopulmonary resuscitation which might break protection of healthcare workers. although several studies analyzed prognostic factors for fatal outcome, 11e16 predictive factors for pneumonia development and progression to respiratory failure have not been reported. to identify factors which can predict pneumonia development and progression to respiratory failure at the early course of the disease, we evaluated mers-cov infected patients managed in a tertiary care center during the 2015 mers outbreak in korea. to identify factors which can predict pneumonia development and progression to respiratory failure at the early course of the disease, we reviewed the electronic medical records of who were diagnosed with mers-cov infection and admitted at samsung medical center, a 1950 tertiary care university hospital which managed the largest number of mers-cov infected patients as a single center during the 2015 korean mers outbreak. as it is still unclear whether initially asymptomatic patients would develop pneumonia or not, 17 we included all the mers-cov infected patients managed at our hospital during the outbreak regardless of symptoms presence. to avoid confusion with the case definition of mers which did not included asymptomatic cases, 18 we used the term of 'mers-cov infected patient' rather than 'mers patients' throughout the present paper. mers-cov infections were confirmed on the basis of rrt-pcr assays targeting upstream of the e gene and the open-reading frame gene 1a. 18, 19 disease status of included patients was assessed at six weeks from their symptom onset and patients were divided into three groups depending on pneumonia development and progression to respiratory failure: patients without the development of pneumonia (group 1), patients who developed pneumonia without respiratory failure (group 2), and pneumonia patients who progressed to respiratory failure (group 3). for practical purposes, respiratory failure was defined as the need for mechanical ventilation (mv). the institutional review board of our hospital approved the present study. we retrospectively collected data from electronic medical records and epidemiologic investigation. to identify factors which can predict pneumonia development and progression to respiratory failure at the early course of the disease, we evaluated clinical variables measured within three days from symptom onset. during the 2015 korean mers outbreak, fever was defined as body temperature !37.5 c to increase sensitivity of screening and the same definition was used in the present analysis. 9 thrombocytopenia was defined as a platelet count lower than 150 â 10 3 cells/mm 3 , lymphopenia as an absolute lymphocyte count lower than 1,000 cells/mm 3 , and hypoalbuminemia as albumin concentration lower than 3.5 g/dl. lower respiratory tract specimens including sputum and endotracheal aspirates were used for mers-cov rrt-pcr. cycle threshold (ct) values of mers-cov rrt-pcr were used as a surrogate of viral load. pneumonia development of mers-cov infected patient was defined as presence of parenchymal infiltration on chest x-ray with respiratory symptoms. test days or events were counted from the day of symptom onset for each patient: day 1 was defined as the day of symptom onset. for asymptomatic patients, the day of diagnosis of mers-cov infection was considered as day 1. to identify predictive factors for pneumonia development and progression to respiratory failure, clinical variables measured within three days from symptom onset were compared. for evaluation of pneumonia development, patients who developed pneumonia (group 2 and 3) were compared to those who did not (group 1). for factors for respiratory failure, patients who progressed to respiratory failure (group 3) were compared to those who did not (group 1 and 2). student's t-tests or mannewhitney u tests were used to compare continuous variables, and chi-square tests or fisher's exact tests were used to compare categorical variables. statistically significant continuous variables were re-categorized into binary factors using threshold values between mean of each group, which showed lowest p value. statistically significant categorical variables and binary factors re-categorized from continuous variables were defined as predictive factors. for significant predictive factors, as a measure of association, odds ratio (or) and 95% confidence interval (ci) for or were calculated using the woolf procedure. 20 multivariate analysis was not performed due to the limited sample size. all pvalues were two-tailed, and those <0.05 were considered to be statistically significant. ibm spss statistics version 20.0 for windows (ibm, armonk, ny, usa) was used for all statistical analyses. time course of pneumonia development and progression to respiratory failure a total of 45 mers-cov infected patients were hospitalized during the outbreak with 13 patients in group 1 (including 3 asymptomatic patients), 19 patients in group 2, and 13 patients in group 3. the clinical course of symptomatic mers patients progressed serially: patients developed initial symptoms after a median 5-day incubation period (iqr 3.5e7.0), pneumonia after a median of 6 days from symptom onset (iqr 5.0e7.0), and respiratory failure after a median of 12 days from symptom onset (iqr 10.0e13.0). in group 3 patients, it took a median of 2 days from desaturation to respiratory failure (iqr 1e3 days). the development and progression of pneumonia by time sequence is depicted in fig. 1 . no one developed pneumonia before day 4 of symptom onset. to evaluate predictive factors for pneumonia development, demographics, underlying diseases, and clinical variables of patients in group 2 and 3 were compared to those of patients in group 1 (tables 1 and 2 ). identified predictive factors are summarized in table 3 with odd ratios (or). increasing age was significantly associated with pneumonia development as a continuous variable (p z 0.015), and age older than 45 years was a predictive factor for the development of pneumonia (or, 8.04; 95% ci, 1.52e42.43; p z 0.007). although proportion of male also increased with progression of pneumonia (38.5%, 57.9%, and 84.6% for group 1, 2, and 3, respectively), statistically significant association between male sex and the pneumonia development was not identified (p z 0.097). fever over 37.5 c by day 3 were more frequently detected in patients with pneumonia (18.2%, 71.4%, and 77.8% in groups 1, 2, and 3, respectively), and was identified as a predictive factor for the development of pneumonia (or, 12.75; 95% ci, 2.12e76.57; p z 0.002). thrombocytopenia (or, not applicable (na); p z 0.007), lymphopenia (or, 17.50; 95% ci, 1.88e163.02; p z 0.003), elevated c-reactive protein (crp ! 2 mg/dl; or, na; p z 0.018), and high viral load (ct value < 28.5; or, 14.00; 95% ci, 1.14e172.65; p z 0.024) were distinctly observed in pneumonia patients from the initial presentation, and identified as predictive factors for pneumonia development. to evaluate predictive factors for progression to respiratory failure, patients in group 3 were compared to those in group 1 and 2 (tables 1e3). although the mean age of patients in each group tended to increase with progression of pneumonia (37.3, 47.7, and 55.2 years in groups 1, 2, and 3, respectively) and increasing age was significantly associated with respiratory failure as a continuous variable (p z 0.036), there was no statistically significant cut-off value for prediction of respiratory failure. proportion of male also increased with progression of pneumonia, and male sex was a predictive factor for respiratory failure (or, 5.50; 95% ci, 1.05e28.88; p z 0.045). among underlying diseases, hypertension was identified as a predictive factor for respiratory failure (or, 6.04; 95% ci, 1.18e30.88; p z 0.021). initial symptoms including fever were not significantly different between patients who progressed to respiratory failure and those who did not. among initial laboratory test results, thrombocytopenia (or, 6.67; 95% ci, 1.18e37.78; p z 0.023), lymphopenia (or, 14.88; 95% ci, 1.56e142.20; p z 0.006), hypoalbuminemia (or, 14.17; 95% ci, 1.83e109.86; p z 0.005), and elevated crp (crp ! 4 mg/ dl; or, 23.00; 95% ci, 2.01e262.57; p z 0.002) were distinctly observed in group 3 patients, and identified as predictive factors for respiratory failure. sensitivity, specificity, positive predictive value (ppv) and negative predictive value (npv) by number of predictive factors were presented in table 4 . when patients presented with ! two of the predictive factors for pneumonia development, 100% of these patients developed pneumonia (sensitivity 56.3%, specificity 100.0%, ppv 100%, and npv 48.1%). patients lacking the predictive factors for respiratory failure did not progress to respiratory failure. when patients presented with ! two of these predictive factors, 50.0% of these patients progressed to respiratory failure (sensitivity 69.2%, specificity 75.0%, ppv 52.9%, and npv 85.7%). initial rapid propagation of mers-cov during the korean mers outbreak was caused by three super-spreading events responsible for 73.1% of all transmissions. 5, 7, 8 in addition to these super-spreaders, transmission of mers-cov despite application of personal protective equipment (ppe) occurred from patients with progressed pneumonia at our hospital. 9 in this regard, identifying the predictive factors for pneumonia development and progression is not only important in patient care, but also in infection control to prevent further in-hospital transmission. the present analysis of predictive factors for pneumonia development and progression to respiratory failure using variables obtained by day 3 of symptom onset could be conducted owing to the observation of entire clinical course of the disease from the exposure to mers-cov. compared to mers outbreaks in the arabian peninsula where community-acquired infections might simultaneously occur from animals, identifying epidemiologic links, exposure date, and symptom onset were relatively clear for each case. 5, 19 in our observation, the clinical course of symptomatic mers patients progressed serially and no one developed pneumonia before day 4 of symptom onset. this is the reason why we used clinical data obtained by day 3 of symptom onset. there is no other comparable data to which presented time interval from the symptom onset to the development of pneumonia. although there were no ideal cut-off scores of predictive factors with good sensitivity and specificity, it should be noted that 100% of patients with ! two predictive factors for data are expressed as the number (%) of patients or mean ae sd. as missing values were also removed from the population parameter, variables with missing values are expressed with modified population parameters. continuous variables with statistical significance were re-categorized into binary factors which are presented in italics. abbreviations: res., respiratory; wbc, white blood cell; alc, absolute lymphocyte count; ast, aspartate transaminase; alt, alanine transaminase; bun, blood urea nitrogen; crp, c-reactive protein; ld, lactate dehydrogenase; ct, threshold cycle; rrt-pcr, real-time reverse transcriptase polymerase chain reaction. a data are presented as mean value of day 1e3 ae sd. pneumonia actually progressed to pneumonia. thus, careful and intensive management should be implemented for such patients including adequate isolation of patient in an airborne infection isolation room (aiir), minimizing chance for exposure, application of ppe with hooded coverall, and consideration of experimental antiviral treatment. 9,21e24 for patients with ! two predictive factors for respiratory failure, aiirs in intensive care units should be prepared for early elective intubation. although the time interval from symptom onset to mv support was much longer than in previous reports (median 12 days versus 7 days), 1 we also experienced rapid progression of pneumonia from the moment of desaturation: 73% of group 3 patients required mv within 2 days from desaturation (median 2, iqr 1e3 days). to avoid urgent situations which might break protection of healthcare workers, elective intubation should be considered when desaturation begins to progress. in addition, sensitivities of predictive values are relatively low with cut-off value of ! two factors, clinical course of patients with any predictive factors also should be carefully monitored. of note, thrombocytopenia, lymphopenia, and increased crp level were shared predictive factor for the pneumonia development and respiratory failure. they were observed in the very early course of the illness, indicating that inflammation had already been enhanced. lymphopenia and thrombocytopenia presenting from the initial presentation of severe mers-cov infected patients were also observed in the recent report by min et al. 25 although time of measurements were not specifically described, these laboratory abnormalities were previously observed in severe mers cases and other respiratory viral illnesses including severe acute respiratory syndrome (sars) and influenza, which are caused by intense inflammatory response to the viruses. 15,26e33 this is the first report that identified these laboratory findings as predictive factors for pneumonia development and progression to respiratory failure in mers. although other predictive factors for pneumonia development and respiratory failure were different due to discordance of statistical significance, they shared the same spectrum of etiology. age increased according to pneumonia progression and was associated with both pneumonia and respiratory failure as a continuous variable (p z 0.015 and p z 0.036, respectively). these findings correlate with previous data suggesting that old age is associated with poor prognosis. 11,13e15,34,35 similarly, the proportion of males increased according to disease severity, though male sex was only significant for predicting respiratory failure. although the mean age of males was older than that of females (49.7 and 42.6 years, respectively), it was not statistically significant (p z 0.169). previous data also reported that overall proportion of male was higher and was associated with severe infection. 15, 34 it could be meaningful observation that the same finding was observed in the republic of korea where the social activity of females is not restricted, especially among healthcare workers. on the other hand, hypoalbuminemia and hypertension were predictive factors only for respiratory failure, while high viral load was predictive factor for the development of pneumonia. these factors were related with severe disease and poor prognosis of mers in previous reports. 1, 12, 13, 15, 16, 34, 35 our study has several strengths and limitations. due to its retrospective nature, there may be a bias regarding collecting medical information in retrospective manner. however, as all electronic medical records were standardized to record symptoms and signs in the same way from the beginning of the outbreak, bias was minimized. secondly, there were missing values when calculating the sensitivity and specificity of predictive factors, which is another limitation of retrospective study. lastly, the present study did not perform multivariate analysis due to limited sample size and need to be validated. prospective studies with sufficient number of patients are required for validation of the predictive factors identified in the present study. despite these limitations, our data would be suitable for identifying predictive factors because we could observe entire course of the disease from exposure and apply homogenous management to patients. in conclusion, based on 45 cases from a single tertiary care hospital during the largest mers outbreak outside of the arabian peninsula, we identified six predictive factors for the development of pneumonia and progression to respiratory failure, respectively. thrombocytopenia, lymphopenia, and high crp level were shared predictive factors. mers-cov infected patients with ! two predictive factors should be intensively managed from the initial presentation for successful control of mers outbreak. there are no potential conflicts of interest relevant to this article to report. this work was supported by the samsung biomedical research institute (sbri) grant [#smx1161321]. middle east respiratory syndrome summary of current situation, literature update and risk assessment middle east respiratory syndrome coronavirus (mers-cov) e republic of korea. world health organization an unexpected outbreak of middle east respiratory syndrome coronavirus infection in the republic of korea the characteristics of middle eastern respiratory syndrome coronavirus transmission dynamics in south korea preliminary epidemiological assessment of mers-cov outbreak in south korea identifying determinants of heterogeneous transmission dynamics of the middle east respiratory syndrome (mers) outbreak in the republic of korea, 2015: a retrospective epidemiological analysis transmission characteristics of mers and sars in the healthcare setting: a comparative study control of an outbreak of middle east respiratory syndrome in a tertiary hospital in korea mers-cov outbreak following a single patient exposure in an emergency room in south korea: an epidemiological outbreak study real-time characterization of risks of death associated with the middle east respiratory syndrome (mers) in the republic of korea viral shedding and antibody response in 37 patients with middle east respiratory syndrome coronavirus infection association of higher mers-cov virus load with severe disease and death, saudi arabia mortality risk factors for middle east respiratory syndrome outbreak, south korea clinical aspects and outcomes of 70 patients with middle east respiratory syndrome coronavirus infection: a single-center experience in saudi arabia risk factors for severity and mortality in patients with mers-cov: analysis of publicly available data from saudi arabia management of asymptomatic persons who are rtpcr positive for middle east respiratory syndrome coronavirus (mers-cov), interim guidance. world health organization case definition and management of patients with mers coronavirus in saudi arabia mers-cov outbreak in jeddah e a link to health care facilities on estimating the relation between blood group and disease treatment outcomes for patients with middle eastern respiratory syndrome coronavirus (mers cov) infection at a coronavirus referral center in the kingdom of saudi arabia treatment strategies for middle east respiratory syndrome coronavirus treatment with lopinavir/ritonavir or interferon-beta1b improves outcome of mers-cov infection in a nonhuman primate model of common marmoset interim infection prevention and control recommendations for hospitalized patients with middle east respiratory syndrome coronavirus (mers-cov), us cdc comparative and kinetic analysis of viral shedding and immunological responses in mers patients representing a broad spectrum of disease severity clinical course and outcomes of critically ill patients with middle east respiratory syndrome coronavirus infection clinical features and viral diagnosis of two cases of infection with middle east respiratory syndrome coronavirus: a report of nosocomial transmission outcomes and prognostic factors in 267 patients with severe acute respiratory syndrome in hong kong white cell differential count and influenza a thrombocytopenia in patients with severe acute respiratory syndrome (review) platelet activation and aggregation promote lung inflammation and influenza virus pathogenesis discovery of t cell infection and apoptosis by mers coronavirus middle east respiratory syndrome coronavirus efficiently infects human primary t lymphocytes and activates both the extrinsic and intrinsic apoptosis pathways epidemiological, demographic, and clinical characteristics of 47 cases of middle east respiratory syndrome coronavirus disease from saudi arabia: a descriptive study middle east respiratory syndrome coronavirus: another zoonotic betacoronavirus causing sars-like disease we would like to express our sincerest condolences to the patients and families who suffered from the mers outbreak. we also greatly appreciate the health care personnel and staff members at samsung medical center and all other hospitals who worked together to overcome the mers outbreak. key: cord-307874-0obomty2 authors: pardon, bart; buczinski, sébastien title: bovine respiratory disease diagnosis: what progress has been made in infectious diagnosis? date: 2020-05-23 journal: vet clin north am food anim pract doi: 10.1016/j.cvfa.2020.03.005 sha: doc_id: 307874 cord_uid: 0obomty2 when it is desired to identify infectious agents involved in an outbreak of bovine respiratory disease, a variety of possible sampling methods may be used. for field use, the deep nasopharyngeal swab, transtracheal wash, and nonendoscopic bronchoalveolar lavage are most feasible. at present, bacterial culture and polymerase chain reaction testing are most commonly used to identify infectious agents. interpretation of test results can be challenging, particularly for opportunistic pathogens. evidence-based guidelines for precise interpretation of microbiologic tests results are lacking; however, approaches that have been practically useful for the management of bovine respiratory disease outbreaks are presented. increasing pressure on antimicrobial use and the need for veterinarians and farmers to use antimicrobials more rationally. 2 the use of diagnostic support by laboratory analysis is one of the frequently mentioned cornerstones of antimicrobial stewardship programs. 3 however, scientifically reasoned, the evidence that systematic use of laboratory diagnostics, especially the antibiogram, would result in selection of a different first-choice therapy compared with an empiric decision preferably following (evidence-based) guidelines, is limited, especially in cattle. antimicrobial resistance in respiratory tract bacteria from cattle is present and varies highly between systems. resistance levels are generally lower in closed dairy and beef herds, substantially higher in feedlots, and most worrisome in veal calf operations, where oral mass medication is frequently used. 4, 5 although there is no doubt of the presence of resistance, and multiresistance, in respiratory bacteria from cattle, to what extent this results in therapy failure when following guidelines for antimicrobial therapy is poorly documented. in recent years, guidelines specifying first-line, second-line, and third-line antimicrobial choices for the different cattle diseases have been initiated in several european union countries, including the netherlands, belgium, denmark, sweden, and germany. 2, 6, 7 however, the amount of literature reporting the clinical benefit of every antimicrobial-bacteria combination in highly variable field settings is currently very limited. therefore, these guidelines mainly include the spectrum of the antimicrobial, pharmaceutical leaflet recommendations and follow classification of the importance of antimicrobials for human medicine of the world health organization. 8 in contrast with human medicine, to the authors' knowledge there are no extensive, sufficiently detailed, and large-scale studies available on therapy failure caused by antimicrobial resistance in cattle. regardless of the limitations mentioned earlier, diagnostics are more and more frequently used when addressing bovine respiratory disease (brd). this increased use is understandable, because antimicrobial decision making for brd still often involves a decision to use group therapy, and, in the current climate, mass medicating without any evidence of the need for this therapy will increasingly be criticized. the authors fully acknowledge the complexity of advising on the implementation and interpretation of diagnostic tests for brd, given the huge gaps in the current knowledge. 9 however, the need is urgent, and therefore this article provides a framework to assist practitioners and clinicians in their everyday decision-making process. this reasoning may not withstand time; this article does not represent a consensus of all leading experts, nor is the objective to provide a complete literature overview. this discussion reflects on the body site sampled, the test used, and the pathogen detected. the selected sampling site of the respiratory tract is of great importance for interpretation of the test result. 11 and endoscopic bronchoalveolar lavage [bal] 12 ; fig. 1 ). nasal swabs, predominantly sampling the cutaneous part of the nose, are generally considered of limited value for infectious diagnostics. in contrast, dnss sample the respiratory and associated lymphoid epithelium of the nasopharynx and return more meaningful samples. however, the biggest issue with nasopharyngeal swabs is the large number of polymicrobial samples recovered (>80%), 10 which heavily compromises clinical interpretation when only opportunistic pathogens are retrieved. contamination can be reduced by rinsing the nares (with a single-use paper towel or a gauze with alcohol) or by using a guarded dns. however, studies specifically focusing on the effect of guarded swabs to reduce nasal contamination are, to the authors' knowledge, not available in cattle. recent reports on the respiratory microbiome in cattle also put the idea of contamination at that sampling site into another perspective, given the large variation in bacterial species normally present. 13, 14 the largest disadvantage of dnss is that they do not directly sample the lower respiratory tract. despite some conflicting results, previous studies overall showed that, for most pathogens, an association between dns results and ttw or bal is present. 10, [15] [16] [17] in addition to cotton swabs, brush swabs also exist, which cause more intensive swabbing of the mucosa (although possibly also blood staining of the sample), presumably with higher detection rates. no evidence on their benefit for use in cattle is currently available. complications of dns are rare and included nasal hemorrhage and fracture of the shaft of the swab. the latter is without any harmful consequence because the animal evacuates the remaining part of the swab either by sneezing or swallowing. to overcome the issue of nasal contamination, transtracheal sampling techniques relying on perforation of the trachea with a needle or catheter after surgical preparation of the skin have been developed. historically, transtracheal swabs have been used, 18 but the transtracheal aspirate and wash are now common. although an aspirate (tta) only involves aspiration of mucus present in the respiratory tract, a wash (ttw) requires fluid instilment and immediate aspiration. despite the terminology tta being frequently used in the field, the technique usually used is a ttw. most frequently, for ttw in cattle, ttw kits (large animal trans-tracheal wash kit, mila international, inc, florence, ky), or human central venous catheters (eg. centracath 75, vygon, ecouen, france) are used, which are commercially available and sterile packed for single use. alternatively, a male dog urinary catheter can be used in combination with a 12-g catheter/needle to perforate the trachea in between 2 tracheal rings. 19 in veterinary medicine, the common thinking is that the ttw is preferred for bacteriology and bal to study inflammation (cytology). however, this recommendation generally comes from horse medicine, and seems to be expert opinion rather than supported by substantial peer-reviewed studies. 20 in humans, ttw is generally not used for ethical reasons. the general idea is that the bronchial bifurcation is the site where the efflux of the mucociliary system of the whole of the lung comes together. hence sampling there would be representative for the whole of the lung. 20 however, there are some counterarguments for this reasoning. first, the mucociliary system can be heavily impaired by pneumonia. second, microbial aspiration from the nasopharynx into the upper trachea is likely frequent. third, normal pathogenesis involves gradual descent of bacteria down the respiratory tree toward the lung. taking the second and third arguments into account, a positive ttw culture might equally represent a bacterial tracheitis or even an insignificant colonization or upper airway contamination, resulting in false positive diagnosis of infectious bronchopneumonia. advantages are that a new disposable catheter can easily be used for each animal, and sampling is theoretically achievable within a predictable time frame given that no active cooperation of the animal is required, in contrast with bal. however, sedation of the animal and local anesthesia of the puncture site can be done to improve animal comfort during the procedure. in a bal procedure, a bal catheter or flexible endoscope is introduced through the nose and trachea into the lower airways until it wedges into a larger (or smaller depending on catheter diameter) bronchus. next, while holding in this wedged position, a volume (usually 60 ml in calves, if necessary followed by a second or third injection) of sterile saline is injected and immediately aspirated. classically, as in human medicine, a bal is performed by endoscopy. the major advantage is that a specific lung lobe, previously shown to be affected on radiology or ultrasonography, can be sampled. also, protective sheets or agar plugs can be used to reduce the risk of nasal contamination. the major disadvantage of the endoscope is the high operating costs and risk for equipment damage in the farm setting. also, sampling multiple animals becomes difficult because time to resterilize the endoscope between animals is needed (15-20 minutes minimum). to overcome the cost and risks of endoscopic bal, nbal techniques have been developed. in nbal, a bal catheter is blindly introduced through the nose, larynx, and trachea until the wedged position in a large bronchus is reached. next, a volume of saline is injected and gently aspirated. the volume used varies substantially between studies (30-250 ml 16, 17 ), but a trend to reduce the volume for welfare/comfort reasons is present. 21 on average, 33.5% of the volume (12.0%-73.8%) can be recovered in nbal, which is substantially larger than in a ttw procedure. 21 it is important to realize that sedation not only suppresses the required responses (coughing, curving of the nose, and extroversion of the tongue) to ensure an intratracheal position but also causes systematic sampling of the diaphragmatic lung lobes, which are less likely to be affected. 22 good restraint of the calf with the head fixed with the nose pointing upward as much as possible is advisable to ease blind introduction of the tube into the trachea. alternatively, the calf might be surprised into allowing the tube to be advanced into the airways by placing the head in a horizontal position, and introducing the catheter on inspiration visible by the opening of the nostrils. overall, in 80% of animals, nbal sampling can be completed within minutes. for the remaining 20%, the practical advice is to select another animal to sample when undertaking group diagnosis, rather than spending excessive time and causing prolonged irritation to a reluctant animal. alternatively, a technique where a double-guarded bal catheter is orally introduced into the larynx through a pvc (polyvinyl chloride) speculum has been described for calves that are at least 3 to 4 months old, where the guarded catheter is inserted through the larynx under visual control. 23 the use of bal samples (especially nbal) for bacteriology is still highly controversial, mainly because of the risk of nasal contamination. although contamination is far less than with dns, 20.8% of nbal samples were still polymicrobial. 10 a large influence of the sampler seems to be present, 10 likely depending not only on differences in hygienic sample handling but also on skills to swiftly introduce the catheter without touching too much of the nasopharynx. however, it is important to realize that hard evidence on substantial nasal contamination by using nbal catheters is currently not available in any species. the only available study on this matter showed pure culture and negative results in 29.2% and 40.3% of the nbal samples, even though dns samples of the same animals were polymicrobial. 10 further, the currently most extensive study on sample method comparison showed very good agreement for bacteriology between dns, ttw, and nbal. 17 interestingly, in human medicine, there are growing efforts toward the use of a mini-bal procedure for bacterial diagnosis in ventilator-assisted pneumonia. 24 overall, sample contamination should be avoided and, in the case of nbal, this can be done by adequate training or visualization of the larynx by a video speculum (ivetscope, dairymac limited, hampshire, united kingdom) or endoscopic cameras intended for plumbers or auto mechanics. these devices are available at much lower prices than traditional endoscopes. next to the site of the respiratory tract sampled (upper or lower airway), the cultural perception of the effect of the sampling technique on animal welfare also plays an important role in what technique is currently preferred in a given country/region. no studies on the effect of respiratory tract sampling on stress or pain have yet been conducted in calves. a master of science thesis showed that both animals sampled by dns or nbal spent less time walking compared with the unsampled control group, whereas lying or eating were unaffected. 25 for ttw as well as nbal, the required volume of saline to be instilled is unclear; it is also unclear whether the volume instilled influences bacteriology results, as it does for cytology. 26 in summary, sampling techniques for the field need to be economically feasible both in terms of equipment/disposables cost and also invested time. the dns, ttw, and nbal best suit this profile and are currently most frequently used in the field. differences in use exist between countries, which mainly originate from historical or cultural preference. an overview of available diagnostic tests for pathogen identification in respiratory diseases in cattle is shown in table 2 . it is beyond the scope of this article to provide a complete overview of all tests possible. the focus is on the most frequently used tests and the most promising future tests likely to become widely available for practice within the next 5 years. in the current international context, the pressure to reduce antimicrobial use has become the main driver of diagnostic test performance for causal diagnosis of brd. a crucial aspect for field efficacy is a short turnaround time (tat), the time between sampling and availability of the test result. in order to be able to use the diagnostic test result to target therapy or initiate control measures tat needs to be as short as possible, ideally less than a day. however, having test results the next morning might also be workable for most outbreaks. also, the use of cow-side testing for a causal diagnosis of brd has great potential to reduce tat. however, to the authors' knowledge, no such tests are currently commercially available. hence, attention should be given to ensuring proper and timely transport to the laboratory. at refrigerator temperature (4 c-8 c), the isolation rate of mannheimia haemolytica and pasteurella multocida was not reduced for 24 hours, whereas a transport temperature of more than 30 c resulted in reduced isolation as soon as 2 hours later. 27 serology serologic tests are useful to target vaccination programs, to determine protective status, and to evaluate infection dynamics at larger scale. however, they are not suitable to direct immediate therapy because they have a tat of 3 weeks (required time for seroconversion) and only provide indirect evidence of infection. also, for the opportunistic pasteurellaceae family, maternal immunity smoothly shifts to acquired immunity, without any signs of disease or seroconversion. 28 another important issue is that sensitivity and specificity can be highly variable between different antibody enzymelinked immunosorbent assays (elisas), hampering clinical interpretation and their use for individual animal decisions (eg, culling or purchase). 29 for targeting therapy, direct identification of the pathogen is needed, and this can be achieved by microbial culture, matrix-assisted laser desorption/ionization time of flight (maldi-tof) mass spectrometry (ms), pcr, or ngs/third-generation sequencing. microbial culture is most frequently used for identification of bacteria. next to low operating costs, the possibility of antimicrobial susceptibility testing is an important advantage of culture. for mycoplasmata, specific media are required, 29 and fastidious growers, especially histophilus somni, are easily overgrown, resulting in false-negative results. 10, 30 sensitivity and specificity of microbial culture have not been determined for most of the bacteria involved in brd. a recent study using bayesian latent class analysis showed that mycoplasma bovis culture on solid medium containing tween 80 is 70.7% (95% bayesian credible intervals [bci], 52.1 to 87.1) sensitive and 93.9% (95% bci, 85.9-98.4) specific. 31 in the last decade, maldi-tof ms, which identifies bacteria by their unique protein profiles, has revolutionized routine diagnostics. it is primarily used for identification of bacteria after culturing, including mycoplasma species. 32 however, maldi-tof ms can also be applied directly on the sample after a very short period of enhanced growth in a liquid medium. relative to classic microbial culture, these culture-enriched direct maldi-tof ms techniques allow correct bacterial identification in 73% of the samples (sensitivity 5 59.1%; 95% confidence interval [ci], 47.2-71.0; specificity 5 100% [100-100]) within 6 hours. 33 the technique performed less well in polymicrobial samples and in samples with mixed infection. also for m bovis, a culture-enriched direct maldi-tof ms technique was developed, which was 86.6% (95% bci, 54-99) sensitive and 86.4% (95% bci, 80-96) specific in a bayesian latent class model including pcr and microbial culture on solid agar. 31 tat was reduced from more than 5 days to less than 3 days. 31 in addition, different maldi-tof ms methods are available for antimicrobial susceptibility testing. by means of mbt-astra (maldi biotyper antibiotic susceptibility test rapid assay), oxytetracycline resistance in p multocida could be identified with high accuracy (se 5 95.7%; 95% ci, 86.3-100.6; sp 5 100%; [95% ci, 100-100]) in as little as 3 hours, outperforming the disc diffusion antibiogram. 34 the mbt-astra technique can be designed for every bacterium-antibiotic combination, but logistical changes are needed to create a good intralaboratory workflow. the costs of maldi-tof procedures are generally low, in line with microbial culture. pcr for the causal diagnosis of brd is now very popular. the main reasons are that multiplex pcr or multiple single pcrs allow detection of multiple bacteria and viruses, providing practitioners with a more extended view of the pathogens involved and hence more options to better target therapy, control, and prevention. fastidious and metabolically active viable but unculturable viruses and bacteria can be detected, in contrast with standard microbial culture. 35 however, in contrast with sequencing techniques, specific primers are needed, and the pathogen of interest needs to be determined beforehand. in this way, the diagnostics are potentially biased and possibly lead to false-negative results. another problem is that viral genomes evolve rapidly and primers might become outdated, limiting the efficient detection of the pathogens of interest. pcr is generally not cheap, but, by pooling samples (dns, ttw, or bals), a group diagnosis can be reached and costs are decreased. in available studies, pools of samples from 5 animals were shown to improve diagnostic accuracy at the group level. 36, 37 the largest disadvantage of pcr is interpretative difficulty, because pcr can identify dead pathogens, opportunists currently not involved in infection, and contaminants, none of which signify a clinically meaningful test result. this disadvantage was shown, for example, for h somni. 19 the use of quantitative pcr is more informative because the pathogen load, especially in the respiratory disease complex, is important to consider. for this reason, quantitative pcr is increasingly used in veterinary laboratories, although interpretative questions remain. especially, when multiple pathogens are detected, determining the attributable fraction of each pathogen to the clinical presentation remains very difficult. 38 next-generation and third-generation sequencing ngs technologies are now becoming more widely available because of the democratization of the technologies, and because platforms such as minion (oxford nanopore technologies, oxford, uk) allow decentralized sequencing experiments. the first studies using ngs (metagenomics) to detect viruses involved in brd in feedlots are already reported; these detected known pathogenic viruses as well as previously unknown or incompletely understood viruses (eg, influenza d virus). 39, 40 hence, the advantage of ngs is that all pathogens can be simultaneously detected, without prior selection of which pathogens to test for. also, semiquantitative results can be reported because, for most viruses, the number of reads corresponds to the initial load of the pathogen present. 41 not only viruses but also whole genomes can be recovered at a scale that is constantly increasing. eventually, direct sequencing of bacteria will allow detection of virulence genes, phylogenetic clustering of strains during outbreaks, and ultimately prediction of antimicrobial resistance based on single nucleotide polymorphisms or resistance genes. a high total bacterial burden and low bacterial community diversity were associated with positive culture results in classic microbial culture. 35 ngs is the basis for microbiome studies, which are discussed elsewhere in this issue. 42 disadvantages are that ngs is costly and requires a long tat under the current conditions using the most accurate devices (eg, illumina, san diego, ca). however, with nanopore sequencing platforms (eg, minion) a higher throughput and shorter tat can be achieved. this long-read technology has been commercially available since 2014 and has made tremendous improvements in output and accuracy. in humans and pigs, minion has been used to characterize pathogens in different types of samples, even at the site of disease outbreaks, because data analysis can be done in the field on portable hardware. 43, 44 on human lower respiratory tract samples, within 6 hours of sampling, a result was given at a sensitivity of 96.6%. 45 however, in order to achieve wide implementation in veterinary practice, the cost, the ability to correctly interpret, and setup of an actionable logistic chain will be essential. therefore, this technology is another case in which analysis of pooled samples from multiple animals to obtain a group diagnosis of primary pathogens will be the most likely application. clinical interpretation of a diagnostic test result to determine the infectious cause of a respiratory tract disease requires information on the pathogen identified, the site of the respiratory tract sampled, the diagnostic test used, the clinical condition of the animal, and whether the sample originates from a single animal or is pooled. there is no current consensus on the way to sample the respiratory tract or to interpret diagnostic test results in humans and many other species. based on the available research, it is unlikely that an evidence-based consensus on respiratory tract sampling method, diagnostic testing, and interpretation of results in cattle can be reached. hence, this article assists readers to properly interpret results of testing by providing information not only on current recommendations but especially on the drawbacks and research gaps. the first point to consider is the nature of the pathogen retrieved, whether it is a primary or secondary pathogen. a primary or obligate pathogen (when present), per definition, induces damage to the respiratory tract, mostly followed by an inflammatory response. however, depending on the infectious dose and host immunity, infection might result in clinical disease or not. also, certain primary pathogens can chronically and even asymptomatically infect animals, resulting in carriers: for example, salmonella spp or m bovis. most primary pathogens weaken innate immunity of the airways, facilitating superinfection by opportunistic bacteria. some, such as bovine respiratory syncytial virus, bovine herpesvirus type 1 (bhv-1), and potentially also others ( table 3) , are able to induce life-threatening disease without bacterial superinfection. despite still being controversial in some scientific communities or countries, m bovis is generally considered a primary pathogen. 46 detection of a primary pathogen can, with some caution, be interpreted straightforwardly. the primary pathogen should normally not be present, and, depending on its virulence, it can, either as a sole agent or in combination with other agents, be held responsible for the clinical picture. also, detection from any site of the respiratory tract is meaningful. for animal welfare reasons and following the pathogenesis, which starts with nasal infection, dns samples might be sufficient and even most appropriate to detect primary pathogens. however, detection rates at the different sites of the respiratory tract differ between pathogens. for bovine coronavirus, dns was more frequently positive than samples from the lower respiratory tract, whereas the inverse was true for bovine respiratory syncytial virus. 17 for bhv-1, dns is recommended given that the infection most frequently remains limited to the upper airways. the true interest of any diagnostic effort lies in extrapolation of test results from the sampled animals to the whole group. detection of primary pathogens in some animals makes involvement of the same pathogen in the part of the resident flora, differences in strain virulence described possibly resulting in some primary pathogenic strains. other studies show cattle to become ill from their own resident strain on exposure to other pathogens and/or risk factors 11, 49, 52 chlamydia psittaci controversial, likely primary natural infections result in mild or subclinical disease 75 (continued on next page) moraxella bovis/ovis secondary primary eye pathogen, occasionally isolated in pure culture from animals with bronchopneumonia 68 pasteurella multocida secondary part of the resident flora. strain virulence differences exist, and some disease presentations (eg, septicemia or peritonitis) have been linked to certain strains 52, 69 salmonella spp primary primary site of infection of most salmonella spp is the gastrointestinal tract. localization in the respiratory tract is possible, most likely after septicemic spread 70 trueperella pyogenes secondary involved in purulent processes. often regarded as characteristic for chronicity. however, naturally resistant to fluoroquinolones 71 escherichia coli, gallibacterium anatis, enterobacter hormaechei, staphylococci, streptococci, fungi secondary single reports on cattle-specific strains isolated in pure culture in an outbreak of pneumonia in calves 52, [72] [73] [74] multiple other bacterial species can be detected in the bovine respiratory tract. this table is limited to either known primary pathogens or frequently isolated pathogens, currently assumed to have a pathogenic significance. data from refs. 11, 14, 19, 39, 46, 47, 49, 52, [58] [59] [60] [61] [62] [63] [64] [65] [66] [67] [68] [69] [70] [71] [72] [73] [74] pardon & buczinski cohoused animals very likely. 47 hence, to improve sensitivity of the group diagnosis, the use of pcr on a pooled sample (up to 5 animals) can be considered. 36, 37 a pitfall when working with pcr to detect primary pathogens is that vaccine antigen can be detected up to 14 days after intranasal vaccination with a live vaccine, resulting in false-positives. 48 a secondary or opportunistic pathogen can be part of the normal respiratory microbiome, without inducing inflammation. in general, breaching of innate immunity, either by another pathogen or a noninfectious cause, is needed before the opportunistic pathogen invades tissues and induces inflammation. interpretation of detection of an opportunistic pathogen is more difficult, given that they can be present in healthy animals. 10, 16, 19 therefore, simply detecting the pathogen cannot be seen as evidence of its involvement. the pasteurellaceae family and a range of other bacteria (eg, streptococcus spp and trueperella pyogenes) are generally considered secondary pathogens. although there seems to be little discussion of p multocida, scientific opinions on the potential primary role and differences in strain virulence of m haemolytica and h somni vary greatly. 11, 49 it is outside the scope of this article to review or take a position on this matter. similarly, in other species, including humans, this issue of opportunistic pathogens exists. when interpreting a positive culture result, a differentiation between contamination, colonization, and infection needs to be made. contamination is defined as the presence, usually in low numbers, of bacteria in a sample that are not expected to be present in the sampled site. colonization can be defined as the presence of a micro-organism in a host, with growth and multiplication of the organism, but without interaction between host and organisms, hence no inflammatory reaction, immune response, or clinical expression occurs. 50 similarly, infection is isolation of a high number of bacteria from a site of the respiratory tract, but in the presence of inflammation of the mucosa, presenting either clinically or subclinically. 50 hence, simply picking a suspected colony from an agar plate, to confirm the cause of the respiratory disease, and subsequently using an antibiogram based on this single colony may be misleading. more information can be derived from culture results if quantitative descriptions and at least the degree of contamination are described. a possible way to better describe culture results, previously used for research purposes, 10,33 is presented in table 4 . it is also important to realize that using selective media for pasteurellaceae, as, for example, by adding bacitracin, ensures better growth and detection of these opportunistic pathogens, but information on the amount of pathogens and degree of contamination of the sample will be lost. 5, 51 pasteurellaceae are part of the normal respiratory flora, and can even be abundantly present in the nasal cavity of healthy animals. 52 an association between the presence of a pasteurella species in the nose and its presence in the lower respiratory tract is described. 10, 15, 16 however, interpretation of dns results for opportunistic pathogens remains very difficult, especially because the composition of the nasopharyngeal microbiota seems to be heavily influenced by bioaerosols from the agricultural environment. 53 loss of biodiversity and overgrowth of opportunistic pathogens occurs in the pathogenesis of brd, resulting in higher odds that pasteurellaceae can be cultured from nasal swabs in larger quantities in ill animals. 10, 35, 54 however, with current knowledge on the interpretation of dns results at the individual or group level, samples of the lower respiratory tract are likely a better option to evaluate potential involvement of opportunistic pathogens. interpretation of detection of opportunists in lower respiratory tract samples remains difficult, even in ill animals, because, even with very strict clinical case definitions, pasteurellaceae can also be cultured from the lower airways in healthy animals. 10, 14 previously explored ways to overcome the issue of interpreting detection of opportunistic pathogens in humans and other species are the use of quantitative cultures or cytologic evidence of inflammation. quantitative culture is derived from the assumption that, in case of a severe infection, the opportunistic pathogen will be present in larger numbers. 50 cutoffs such as greater than 10 3 colony-forming units per milliliter of bal fluid have been suggested in dogs and horses. 20, 55 however, pathogen burden builds up, and sampling early in the disease process could mean that much lower numbers are detected despite the pathogen being involved in the inflammatory process. 56 another option is derived from the assumption that a bacterial infection will result in a massive airway neutrophilia. 55 however, clear cutoff percentages for neutrophilia differentiating a bacterial infection from a viral one or a strictly noninfectious airway inflammation have not been determined in calves. given that some bal techniques result in a larger contribution of the bronchial component in the total bal fluid, the neutrophil percentage is increased compared with a larger volume of mainly alveolar lavage fluid. 21 primary insights in calf bal fluid analysis show that cytologic parameters coincide poorly with clinical or ultrasonographical findings or culture results for opportunistic pathogens, at least when using nbal. 57 the presence of phagocytosis by bacteria in neutrophils or macrophages may be helpful to differentiate active infection versus simple presence of the bacteria. 57 although interpreting culture results for opportunistic pathogens is already difficult, interpretation of pcr results is even more so. not only can insignificant quantities or even dead bacteria result in a positive pcr, the nasal passages also pick up bacterial dna, making the lower respiratory tract sample positive. quantitative pcr might overcome this issue, but, to the authors' knowledge, no guidelines on how to interpret these results are currently available in the bovine species. although laboratory costs are fixed, the return on investment of an analysis greatly depends on selection of appropriate animals to sample and on the technical sampling skills of the veterinarian. an animal in the first days of the disease, not previously treated with antimicrobials and not displaying severe respiratory signs, is first choice. by sampling in the acute phase of the disease, the odds of detecting the viral component are higher. by avoiding previous antimicrobial treatment and by sampling early in the disease course, the probability that the antibiogram derived is useful for empiric therapy increases. by avoiding sampling animals in heavy respiratory distress, the odds of aggravation of disease or even mortality can be decreased. in addition, in spite of all reasoning made earlier, veterinarians still make decisions at an individual animal level. when sampling an individual, the main interest is usually to make a decision representing the group, and to judge the utility of the diagnostic test to support this group decision. different epidemiologic approaches are possible to determine an appropriate sample size. the goal is more a detection of disease approach (being 95% confident that the pathogen was detected when present) rather than determining the prevalence of the pathogen in a group of animals. in the field, sample size is currently more driven by practical reasons such as available time to sample or maximum samples allowed to pool for economic reasons. for example, performing pcr on pools of 5 animals increases sensitivity without diluting the sample too much. fig. 2 provides an overview on the risk of not finding a positive animal in 2 scenarios, 1 related to 100% prevalence of the pathogen in the diseased population and 1 scenario with a pretest probability that 70% of sick calves are affected by the pathogen (ie, where multiple pathogens are involved and can cause the same clinical disease). results of a test with 70% sensitivity (ie, detects the pathogen in 7 of 10 infected calves) and 100% specificity (no false-positive calves) are presented. using this test in a scenario with 70% of the affected animals being positive for the pathogen, after 5 calves not finding a positive, will misclassify only 3.5% of the herds (the 2 scenarios assume that the pooled tests' accuracy is the same as the individual test). in the case of opportunistic pathogens, given that they can be found in healthy or subclinical animals, at this time it might be most prudent to only sample animals with evidence of clinical bronchopneumonia by using a combination of clinical scoring and thoracic ultrasonography. 57 focusing on bacterial isolation to direct intervention strategies, without taking the clinical status into account, holds great danger for overtreatment with antimicrobials. knowledge of respiratory health is rapidly evolving in animals, following new developments in humans. in particular, better insights into the role of the respiratory microbiome and the interaction of the airway inflammatory response with different organisms and air pollutants are likely to change how the diagnostic tests discussed in this article are interpreted. the authors hope that the information, tools, and provisional advice provided can aid the large group of cattle veterinarians, already having to make rational treatment decisions today. b. pardon has received honoraria for acting as speaker or consultant for pharmaceutical (zoetis, msd, vetoquinol, dopharma, boehringer ingelheim, dechra, hipra, ceva, merial, and elanco), agricultural (algoet nutrition), and chemical (proviron) companies and nonprofit organizations (boerenbond, amcra, dgz-vlaanderen). s. buczinski has received honoraria for acting as speaker or consultant as well as research grants for pharmaceutical companies (zoetis, msd, hipra, and ceva) and companies involved in commercialization of ancillary tests used in respiratory diseases (ei medical imaging, geissler corp.). fig. 2 . risk (probability ranging between 0 and 1) of not finding a positive animal for a given pathogen according to sample size (x axis) in a scenario where 100% (solid line) and 70% (dashed line) of affected animals are positive for a given pathogen. the graph represents a test with 70% sensitivity and 100% specificity. it assumes that there are no falsepositive results (ie, when the test indicates that the pathogen is present, this is a truepositive result). in the example where the pathogen is causing the disease in 100% of affected calves, the risk of not finding an infected animal after sampling n cases is (1-se)n , where se is the test sensitivity. in the alternative scenario where only 70% of cases are caused by the pathogen (ie, in 30% of cases, this is another cause), the probability of not finding a case is (1-0.7*se)^n. laboratory test descriptions for bovine respiratory disease diagnosis and their strengths and weaknesses: gold standards for diagnosis, do they exist? fve (federation of veterinarians of europe). fve guidelines for responsible use of antibiotics strategies to enhance rational use of antibiotics in hospital: a guideline by the german society for infectious diseases effect of antimicrobial consumption and production type on antibacterial resistance in the bovine respiratory and digestive tract prevalence and antimicrobial susceptibility of mannheimia haemolytica, pasteurella multocida, and histophilus somni isolated from the lower respiratory tract of healthy feedlot cattle and those diagnosed with bovine respiratory disease classification of antimicrobials: methods procedures for the development of formularies for responsible antimicrobial use critically important antimicrobials for human medicine, 6th revision brd diagnosis: what progress have we made in clinical diagnosis? a deep nasopharyngeal swab versus nonendoscopic bronchoalveolar lavage for isolation of bacterial pathogens from preweaned calves with respiratory disease transmission dynamics of mannheimia haemolytica in newly-received beef bulls at fattening operations a sequential broncho-alveolar washing in non-anaesthetized normal bovines: method and preliminary results comparison of the nasopharyngeal bacterial microbiota of beef calves raised without the use of antimicrobials between healthy calves and those diagnosed with bovine respiratory disease distinct bacterial metacommunities inhabit the upper and lower respiratory tracts of healthy feedlot cattle and those diagnosed with bronchopneumonia use of deep nasopharyngeal swabs as a predictive diagnostic method for natural respiratory infections in calves the microbial flora of the respiratory tract in feedlot calves: associations between nasopharyngeal and bronchoalveolar lavage cultures agreement among 4 sampling methods to identify respiratory pathogens in dairy calves with acute bovine respiratory disease sample collection for diagnostics of bovine respiratory diseases respiratory disease in calves: microbiological investigations on trans-tracheally aspirated bronchoalveolar fluid and acute phase protein response collection and interpretation of tracheal wash and bronchoalveolar lavage for diagnosis of infectious and non-infectious lower airway disorders factors associated with lung cytology as obtained by non-endoscopic broncho-alveolar lavage in grouphoused calves effect of sedation on the intrapulmonary position of a bronchoalveolar lavage catheter in calves influence of tilmicosin on quantified pulmonary concentrations of three bacterial pathogens in calves with naturallyoccurring bovine respiratory disease mini-bal, blinded bronchial sampling, blinded protected specimen brush] to investigate for pulmonary infections, inflammation, and cellular and molecular markers: a narrative review comparison of deep nasopharyngeal swab, bronchoalveolar lavage and transtracheal wash for the diagnosis of infectious bronchopneumonia in calves: which one is most animal friendly? master students thesis. ghent university influence of bronchoalveolar lavage volume on cytological profiles and subsequent diagnosis of inflammatory airway disease in horses it's about time. rapid detection and susceptibility testing of pasteurellaceae causing respiratory disease in cattle by maldi-tof ms ghent university maternally and naturally acquired antibodies to mannheimia haemolytica and pasteurella multocida in beef calves a review of mycoplasma diagnostics in cattle clinical veterinary microbiology35. london: mosby international limited rapid identification of mycoplasma bovis from bovine bronchoalveolar lavage fluid with maldi-tof ms after enrichment procedure matrix-assisted laser desorption ionizationtime of flight mass spectrometry is a superior diagnostic tool for the identification and differentiation of mycoplasmas isolated from animals rapid identification of respiratory bacterial pathogens from bronchoalveolar lavage fluid in cattle by maldi-tof ms rapid detection of tetracycline resistance in bovine pasteurella multocida isolates by maldi biotyper antibiotic susceptibility test rapid assay analysis of culture-dependent versus culture-independent techniques for identification of bacteria in clinically obtained bronchoalveolar lavage fluid patterns of detection of respiratory viruses in nasal swabs from calves in ireland: a retrospective study pathogen-specific risk factors in acute outbreaks of respiratory disease in calves bayesian estimation of pneumonia etiology: epidemiologic considerations and applications to the pneumonia etiology research for child health study metagenomic characterization of the virome associated with bovine respiratory disease in feedlot cattle identified novel viruses and suggests an etiologic role for influenza d virus respiratory viruses identified in western canadian beef cattle by metagenomic sequencing and their association with bovine respiratory disease modular approach to customise sample preparation procedures for viral metagenomics: a reproducible protocol for virome analysis respiratory bacterial microbiota in cattle: from development to modulation to enhance respiratory health multiplex pcr method for minion and illumina sequencing of zika and other virus genomes directly from clinical samples nanopore sequencing as a revolutionary diagnostic tool for porcine viral enteric disease complexes identifies porcine kobuvirus as an important enteric nanopore metagenomics enables rapid clinical diagnosis of bacterial lower respiratory infection mycoplasma bovis infections in cattle transmission dynamics of mycoplasma bovis in newly received beef bulls at fattening operations detection by real-time rt-pcr of a bovine respiratory syncytial virus vaccine in calves vaccinated intranasally characterization of mannheimia haemolytica isolated from feedlot cattle that were healthy or treated for bovine respiratory disease colonization and infection variability in acquired resistance of pasteurella and mannheimia isolates from the nasopharynx of calves, with particular reference to different herd types bacterial pathogens of the bovine respiratory disease complex bioaerosols play a major role in the nasopharyngeal microbiota content in agricultural environment effects of transportation to and co-mingling at an auction market on nasopharyngeal and tracheal bacterial communities of recently weaned beef cattle quantitative bacterial cultures and cytological examination of bronchoalveolar lavage specimens in dogs differential expression of sheep betadefensin-1 and -2 and interleukin 8 during acute mannheimia haemolytica pneumonia comparison of bronchoalveolar lavage fluid bacteriology and cytology in calves classified based on combined clinical scoring and lung ultrasonography clinicopathological features of 11 suspected outbreaks of bovine adenovirus infection and development of a real-time quantitative pcr to detect bovine adenovirus type 10 prevalence of respiratory pathogens in diseased, non-vaccinated, routinely medicated veal calves bovine adenovirus type 3 pneumonia in dexamethasone-treated calves apoptosis in calf pneumonia induced by endobronchial inoculation with bovine adenovirus type 3 (bav-3) structured literature review of responses of cattle to viral and bacterial pathogens causing bovine respiratory disease complex what is the evidence that bovine coronavirus is a biologically significant respiratory pathogen in cattle? bovine viral diarrhea virus-associated disease in feedlot cattle pathogenesis of influenza d virus in cattle randomized clinical trial to evaluate the pathogenicity of bibersteinia trehalosi in respiratory disease among calves evolution of the nasopharyngeal bacterial microbiota of beef calves from spring processing to 40 days after feedlot arrival recovery of moraxella ovis from the bovine respiratory tract and differentiation of moraxella species by tdna-intergenic spacer pcr fatal peritonitis caused by pasteurella multocida capsular type f in calves salmonellosis in calves influence of systemic fluoroquinolone administration on the presence of pasteurella multocida in the upper respiratory tract of clinically healthy calves first report of enterobacter hormaechei with respiratory disease in calves septicaemic escherichia coli and experimental infection of calves isolation of drug-resistant gallibacterium anatis from calves with unresponsive bronchopneumonia infection, disease, and transmission dynamics in calves after experimental and natural challenge with a bovine chlamydia psitacci isolate key: cord-301988-cevv81h3 authors: hassoun, ali; huff, matthew d.; weisman, david; chahal, khushdeep; asis, esmeralda; stalons, don; grigorenko, elena; green, jessica; malone, leslie l.; clemmons, scott; lu, stanley title: seasonal variation of respiratory pathogen colonization in asymptomatic health care professionals: a single-center, cross-sectional, 2-season observational study date: 2015-08-01 journal: am j infect control doi: 10.1016/j.ajic.2015.04.195 sha: doc_id: 301988 cord_uid: cevv81h3 background: the purpose of this study was to determine the seasonal variance of potentially pathogenic bacterial and viral organisms in nasopharyngeal specimens obtained from asymptomatic health care professionals (hcps) during the 2014 winter and summer months. methods: nasopharyngeal specimens from 100 hcps were collected from huntsville hospital (huntsville, al) during the winter and from 100 hcps during the summer. all subjects were tested for 22 viruses and 19 bacteria using target enriched multiplex polymerase chain reaction. both seasonal cohorts were composed of students, nurses, physicians, and residents. results: of the 100 hcps tested during the winter, 34 subjects were colonized with at least 1 bacterium, and 11 tested positive for at least 1 virus. methicillin-resistant staphylococcus aureus (mrsa), moraxella catarrhalis, and coronavirus were the most frequently detected potentially infectious agents. of the 100 hcps tested during the summer, 37 tested positive for at least 1 bacterium, and 4 tested positive for a viral agent. the most prevalent bacteria were mrsa and klebsiella pneumonia. conclusion: nasopharyngeal carriage among asymptomatic hcps was common, but the frequency and presence of potential pathogens varied with each season. understanding the colonization and infection potential of upper respiratory organisms is important, particularly for viruses. although asymptomatic hcps certainly harbor a number of different potentially infectious agents, future studies are needed to determine whether colonized pathogens are transmitted or initiate infection in at-risk patient populations. within healthy subjects, the upper respiratory tract fosters a complex, commensal microbiome. 1, 2 however, introduction of a foreign bacterial or viral organism into the microbiologic community may disrupt normal intercellular relationships resulting in an imbalanced ecosystem. [1] [2] [3] the detailed mechanisms that drive these complex interactions are largely unknown; consequently, it is difficult to predict the behavior of this highly nonlinear system. downstream consequences can be influenced by activities of other organisms, use of antimicrobials, or the host's immune response. 1 in immunocompromised subjects, such as neonates, elderly adults, and patients undergoing chemotherapy, the addition of an unfamiliar pathogen can offset the delicate equilibrium and increase the likelihood of pathogenic invasion. 2 in individuals with functional immune systems, internalized microbes are eliminated, whereas noninternalized pathogens can thrive within the respiratory microbiome, thereby using their hosts as vectors. however, these asymptomatic individuals can still pose a threat of transmitting the potentially pathogenic agent to at-risk subjects. target enriched multiplex polymerase chain reaction (tem-pcr; diatherix laboratories, huntsville, al) simultaneously detects 22 viral serotypes and 19 bacterial species that often inhabit the upper respiratory tract. this multiplex assay is performed from a single patient specimen and has high levels of specificity and sensitivity (table 1) . little is known about the colonization rates of respiratory pathogens in asymptomatic adult subjects, especially for viruses. here, we describe the frequency and seasonal variation of bacterial and viral detections in asymptomatic health care professionals (hcps) during the winter and summer months of 2014. seasonal variation of infectious disease rates is a common phenomenon that is clearly manifested when temperatures are at extremes; consequently, there may be seasonality of colonization levels and commensal relationships of pathogens in asymptomatic individuals. 4 by elucidating changes in pathogen colonization rates in asymptomatic hcps during different time periods in the year, health care organizations can monitor which potentially pathogenic agents are most prevalent in carriers in a health care setting and observe correlations with infection levels in at-risk hospitalized patients. 5 new mechanisms of pathogen transmission can be hypothesized and tested, which could be potentially important in a clinical setting and have a positive effect on infection control practices. two hundred hcps, consisting of 100 during the winter and 100 during the summer, from huntsville hospital (huntsville, al) were tested for the presence of respiratory pathogens via tem-pcr. hcp categories included medical students, nurses (intensive care unit and general patient care), physicians, and residents. pertinent histories, including recent upper respiratory symptoms and antibiotic use, were obtained. all 200 hcps were asymptomatic for a minimum of 4 weeks before samples were taken. each hcp completed a questionnaire that evaluated their respiratory status. questionnaires were reviewed by physicians at huntsville hospital, and it was determined that none of the subjects likely had current infections during specimen collection intervals. none of the physicians who participated in the study reviewed the surveys. eligibility criteria required hcps be free of respiratory symptoms for at least 4 weeks prior to specimen collection and agree to provide a nasopharyngeal specimen for testing. nasopharyngeal specimens were collected in 2014 during january-february and june-july, representing the winter and summer observations, respectively. specimens were sent via courier to diatherix laboratories. results were sent to the study coordinator at huntsville hospital within 24 hours of sample receipt, and study participants were notified of the results. nucleic acid extraction for tem-pcr was performed using a kingfisher system (thermo fisher scientific, waltham, ma). the amplification steps were completed using a geneamp pcr system 9700 thermal cycler (applied biosystems, thermo fisher scientific, waltham, ma). probe fluorescence was read using a sensospot flair system (sensovation, radolfzell am bodensee, germany). a more detailed description of specimen collection and transport conditions, tem-pcr technology, and analytical validation of the assay are included in the supplemental data. all tem-pcr results were organized and extracted from a reporting database at diatherix laboratories using microsoft sql . the 100 hcps tested during the 2014 winter months consisted of 18 medical students, 33 nurses, 6 physicians, and 43 residents. the 100 hcps tested during the 2014 summer months consisted of 16 medical students, 47 nurses, 5 physicians, and 32 residents. the 2 sample groups were nonoverlapping. prior to the 4-week asymptomatic period, 34 of the 200 subjects had symptoms of a respiratory illness, and 14 of the 200 received antibiotic treatment. a more detailed description of the subject sample is shown in table 2 . figure 2 displays a summary of the respiratory pathogen detection percentage partitioned by profession and season. in the winter, residents represented the subset of hcps with the largest frequency of colonization because 26 (60.5%) of the 43 were positive for at least 1 bacterial or nonbacterial pathogen. however, the highest proportion of viruses was seen in nurses because 7 (21.2%) of the 33 tested were detected with coronavirus. in the summer, as in winter, residents had the highest frequency of detection because 14 (43.8%) of the 32 tested were positive for at least 1 pathogen. nurses exhibited a slightly lower level of colonization because 19 (46.3%) of the 41 had at least 1 pathogen detection. figure 3 characterizes all of the hcps in both seasons that had a codetection for 2 genetic targets. a larger number of subjects exhibited codetections during the winter because 12 (25.5%) of the 47 subjects were detected with 2 genetic targets. of the 19 positive detections for mrsa, 4 (21.1%) were positive for the panton-valentine leukocidin (pvl) cytotoxin gene, which represented the highest frequency of simultaneously detected genetic targets. eight (30.8%) of the 26 residents had >1 detection. in the summer, 1 resident had 3 bacterial detections, whereas no hcps in the winter exhibited >2 detections. supplemental figures s1 and s2 display the detection percentage aggregated by sex and season and age groups and season, respectively. men had a marginally higher frequency of detection because 16 (51.6%) of 31 in the winter and 19 (52.8%) of 36 in the summer had at least 1 pathogen detection. in the winter, the 30-40 years age bracket had the highest percentage of detection because 16 (55.2%) of the 29 hcps tested were positive for at least 1 bacterium or virus. in the summer, 19 (52.8%) of 36 subjects in the 20-30 years age bracket had the largest percentage of pathogen detection. figure 2 and supplemental figures s1 and s2 present data as a percentage of positive detection out of total tests given to a specific profession, sex, or age during each season. supplemental tables s1-s3 display the number of positive detections and number of tests given. during the winter, hcov was detected in 11 of the 100 asymptomatic hcps. one study reported results comparable with the 11% hcov detection frequency found in hcps at huntsville hospital. 6 taiwan and found that 25 (11.5%) were colonized with sars-cov. 6 the nosocomial spread of sars-cov in large hospitals was a major epidemic feature during early stages of the outbreak, but this problem was resolved after hospitals isolated all symptomatic patients and asymptomatic hcps detected with sars-cov. 7 all 11 detections for hcov at huntsville hospital also came from frontline asymptomatic hcps, predominantly nurses. observations from this study and the ho et al study suggest that coronavirus could possibly be spread from infected patients to frontline hcps through close contact or via contaminated droplets, but further studies are needed to substantiate this hypothesis. m catarrhalis exhibited seasonal variation because 11 hcps tested in the winter were positive, but all subjects were negative in the summer. the presence of m catarrhalis was unexpected because colonization usually only occurs in children, and only 3% of healthy adults are colonized. 8 the high winter detection frequency of m catarrhalis observed could be a result of the temperaturedependent characteristics of the organism. at lower temperatures, the speed of transcription and abundance of mrna transcripts increases, m catarrhalis grows and divides at a more rapid rate, and the organism's pathogenic behavior has a higher probability of being activated. 9,10 m catarrhalis can live commensally for extended periods; therefore, the incubation time of this organism is not well defined. however, m catarrhalis can become pathogenic in subjects with compromised immune systems or in subjects that have experienced nasopharyngeal stress, such as patients with chronic obstructive pulmonary disease. 11 as a result, it may be valuable to determine which hcps are colonized with m catarrhalis and determine whether there is a correlation with infection rates in at-risk patients. 12 s aureus and mrsa were frequently detected in both winter and summer. the high prevalence of healthy subjects colonized with s aureus and mrsa has been well described. 13 commonly, s aureus and mrsa act as commensals of the upper respiratory tract, but if a patient is immunocompromised, a potentially fatal infection can develop. as a result, many hospitals implement s aureus and mrsa infection reduction protocols and use active surveillance programs to decolonize patients before admission or surgery. 14 s aureus and mrsa also attack the lower respiratory tract where organisms are often parasitic and cause bacterial pneumonia. 15 in this study, 4 hcps in the winter and 1 hcp in the summer were colonized by pvl-positive mrsa. pvl is a potent cytotoxin produced by s aureus that causes necrotizing pneumonia with a high mortality rate. 16 many hospitals have already implemented decolonization procedures for patients colonized with s aureus and mrsa, but future studies could support the decolonization of asymptomatic hcps. k pneumoniae was the only organism that demonstrated a substantially higher frequency of colonization in the summer. summer detection was anticipated because the prevalence of k pneumoniae infections, especially bloodstream infections, increases during the summer. 17, 18 the seasonal variation in k pneumoniae colonization is not well understood. it has been reported that higher rates of k pneumoniae colonization are primarily related to the use of antibiotics. here, only 1 (14.3%) of the 7 hcps that were colonized with k pneumoniae recently received antibiotics. 19 k pneumoniae can be easily and rapidly transmitted to immunocompromised patients from the hands of contaminated hcps. 5 similar to pvl-positive s aureus and mrsa, k pneumoniae can also infect to the lower respiratory tract causing abnormalities such as bulging interlobar fissures, cavitary abscesses, and necrotizing pneumonia. 20, 21 as a result, detecting and decolonizing the carriers of k pneumoniae could have a positive impact on reducing high-risk transmission and hospital-acquired infections during the summer, particularly in patients with comorbidities. there are only a small number of studies reporting the detection frequency of viruses, particularly in asymptomatic adults, which makes it difficult to compare our results with other findings. five different viruses, including adenovirus, coronavirus, rhinovirus, coxsackievirus-echovirus, and pandemic influenza a h1n1-09, were detected at least once in either or both seasons. there are few studies that investigate the effects of viral colonization or coinfections in the upper respiratory tract. previous reports show that the prevalence of viral pathogens may increase bacterial colonization and risk of infection in the nasopharynx. 22 in addition, 1 study proposed a colonization-competition model for rna viruses and predicted that low-virulence strains initially colonize human cells and then competitive, high-virulence strains prime the nasopharynx for infection. 23 multiplex polymerase chain reaction technology can help add to these models and better define the unknown mechanisms of colonization and infection of viral pathogens. one property of tem-pcr is that it amplifies dna or rna of both viable and nonviable organisms. presumably, many of the hcps in this study had respiratory diseases long before this study and could be conveying nucleic acid remnants that were detected by tem-pcr. the 4-week clearance period (as previously described in the methods section) was intended to reduce the likelihood of this possible false detection; however, the half-life of nucleic acid fragments in this setting is not known. it is almost certain that rna remnants degrade more quickly than dna. 24 in the winter, a larger proportion of hcps had prior respiratory symptoms, which may have been responsible for the higher frequency of pathogens detected in this season. further testing methods, such as culture, would need to be conducted to discriminate between nucleic acid remnants and viable microorganisms. it is also possible that the high sensitivity of multiplex polymerase chain reaction detected recent invader cells that would later cause disease in the hcp. however, for the purposes of this study, we do not discriminate between long-standing colonies and recent invader cells because both scenarios can plausibly spread pathogens to at-risk patients. this study examined a moderately sized sample, and the proportions of professions, sexes, and ages varied during each season. in addition, medical students, physicians, and residents worked in diverse sections of the hospital, but nurses only worked in either the intensive care unit or the general population. a larger proportion of nurses tested during both seasons worked in the intensive care unit (26 [78.8%] in the winter, 29 [61.7%] in the summer), which could have been the reason for moderately high pathogen detection frequency in this subset. information was unavailable to determine the proportion of time each hcp treated patients while wearing a mask. not wearing masks would increase the likelihood of transmission from patient to hcp and vice versa. even though this study focuses on hcp colonization, the identification and management of all possible pathogen reservoirs, such as health care surfaces, equipment, and colonized visitors, are also important for preventing the spread of infection in a clinical setting. 5 nevertheless, the utilization of a sensitive and specific multiplexing nucleic acid assay is valuable because the assay detects multiple viral and bacterial pathogens simultaneously in asymptomatic subjects. colonization of viral pathogens needs substantially greater understanding, and defining the unknown colonization and infection potential of these upper respiratory organisms is important. this preliminary observational study explores the prevalence of colonization of various pathogens at 2 different points in the year, and if validated with larger follow-up studies, this information could be ultimately relevant in the clinic. determining correlations between levels of hcp colonization and at-risk patient infection rates could assist in defining novel transmission pathways. in turn, hospital protocols could be altered so that colonized hcps and at-risk patient interactions could be managed, threat of transmission could be minimized, and infection control protocols could be optimized. microbial interactions during upper respiratory tract infections bacterial interference in upper respiratory tract infections: a systematic review respiratory microbiota: addressing clinical questions, informing clinical practice seasonal infectious disease epidemiology hospital epidemiology and infection control in acute-care settings colonization of severe acute respiratory syndrome-associated coronavirus among health-care workers screened by nasopharyngeal swab fighting the sars epidemic in taiwan: a nursing perspective branamella catarrhalis in children and adults. a study of prevalence, time of colonization, and association with upper and lower respiratory tract infections moraxella catarrhalis-pathogen or commensal? physiologic cold shock increases adherence of moraxella catarrhalis to and secretion of interleukin 8 in human upper respiratory tract epithelial cells moraxella catarrhalis in chronic obstructive pulmonary disease: burden of disease and immune response outbreak of moraxella catarrhalis in a respiratory unit the management of infection and colonization due to methicillin-resistant staphylococcus aureus: a cids/camm position paper eradication or decolonization of methicillin-resistant staphylococcus aureus carriage: what are we doing and why are we doing it? pneumonia caused by methicillinresistant staphylococcus aureus panton-valentine leukocidinproducing staphylococcus aureus outbreak of multidrug-resistant klebsiella pneumoniae carrying qnrb1 and blactx-m15 in a french intensive care unit seasonal variation in klebsiella pneumoniae bloodstream infection on 4 continents outbreak of a multiresistant klebsiella pneumoniae strain in an intensive care unit: antibiotic use as risk factor for colonization and infection rapidly fatal outcome of bacteremic klebsiella pneumoniae pneumonia in alcoholics community-acquired klebsiella pneumoniae. bacteremia: global differences in clinical patterns increased pharyngeal bacterial colonization during viral illness competition-colonization dynamics in an rna virus primordial soup or vinaigrette: did the rna world evolve at acidic ph? we thank all health care professionals at huntsville hospital who participated in this study and all practitioners who collected health care professional nasopharyngeal specimens. in addition, we thank all diatherix clinical laboratory employees who conducted the 200 tem-pcr respiratory panel tests for this study. special thanks to donna hockman, who was one of the pioneer designers for diatherix's current respiratory panel. finally, we thank brint roden for generating the graphical abstract and jessica l. alleyne, blake adams, michelle hammond, and vicki caneer for proofreading manuscript drafts. supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.ajic.2015.04.195. key: cord-312741-0au4nctt authors: lin, panpan; wang, manni; wei, yuquan; kim, taewan; wei, xiawei title: coronavirus in human diseases: mechanisms and advances in clinical treatment date: 2020-10-01 journal: medcomm (beijing) doi: 10.1002/mco2.26 sha: doc_id: 312741 cord_uid: 0au4nctt coronaviruses (covs), a subfamily of coronavirinae, are a panel of single‐stranded rna virus. human coronavirus (hcov) strains (hcov‐229e, hcov‐oc43, hcov‐hku1, hcov‐nl63) usually cause mild upper respiratory diseases and are believed to be harmless. however, other hcovs, associated with severe acute respiratory syndrome, middle east respiratory syndrome, and covid‐19, have been identified as important pathogens due to their potent infectivity and lethality worldwide. moreover, currently, no effective antiviral drugs treatments are available so far. in this review, we summarize the biological characters of hcovs, their association with human diseases, and current therapeutic options for the three severe hcovs. we also highlight the discussion about novel treatment strategies for hcovs infections. f i g u r e 1 genome organization and structure of hcovs human covs generally cause only mild upper respiratory diseases, which is similar to common flu. 11, 12 a novel cov, named sars-cov-2 by the world health organization (who), has emerged again at the end of 2019, causing more infections and deaths worldwide than ever before. 13 the absence of effective antiviral treatments and serious consequences of these three covs have highlighted the urgent need for novel drug development to prevent the spread of covs. herein, this review mainly focuses on the biological characters of hcovs, their association with human diseases, and current therapeutic options for the three severe hcovs. we also highlight the discussion about novel treatment strategies for hcovs infections. covs possess a nonsegmented, positive, single-stranded rna genome of 26-32 kb. 2, 14, 15 all covs have a similar genome arrangement with a 5′-methylated cap structure along with 3′-polyadenylated tail. the replicase gene, occupying about 20 kb, two-thirds of the genome and comprising two open reading frames (orfs), orf1a and orf1b, is located at the 5′ end. 2 it encodes two large polyproteins (pp) 1a and 1ab that can be cleaved by papain-like cysteine protease (plpro) and 3c-like serine protease (3clpro) into nonstructure proteins, involving some proteases, several rna modification enzymes, as well as rna-dependent rna polymerase (rdrp) and helicase (hel) required for virus replication. 16 additionally, an untranslated region (utr) can also be identified at the 5′-end as same as the 3′terminal. structure proteins, encompassing the 3′-terminal one-third of the genome, are arranged in a certain order of hemagglutinin esterase (he) protein that is present in some beta-covs, spike protein (s), small membrane protein (e), membrane protein (m), and nucleocapsid protein (n). in brief, the arrangement of the cov genome can be shown as 5′-utr-replicase gene (orf 1a and orf 1b)-he protein (if have)-s protein-e protein-m protein-n protein-3′ utr-poly (a) 2 ( figure 1 ). cov is named for the club-shaped projections eradiating from the envelope, which forms its corona or crow-like morphology. the shape of the viral particles is roughly spherical with approximately 80-160 nm in diameters. [17] [18] [19] the nucleocapsid protein and the genome rna intertwine to form a helical structure located inside the envelope. for some covs, the spikes on the surface are not only formed by trimers of s protein, but also he proteins. m protein and e protein, two transmembrane proteins, also participate in the composition of the virus (figure 1 ). s protein, a transmembrane protein, mediates the initiation of cov infection by attaching to the specific receptors on the target cells. [20] [21] [22] for a prototypical cov, s protein is usually cleaved into an extracellular receptor binding subunit (s1) and a membrane-anchored subunit (s2), responsible for virus binding and membrane fusion, respectively. 23, 24 two heptad repeats (hr1 and hr2) and enriched alpha-helices are contained in the s2 domain, a feature typical of fusion protein. [25] [26] [27] the receptor-binding domain (rbd) of s protein specifically binds to target receptors, leading to the conformation change of s1/s2 complex that mediates virus entry. 26 furthermore, the rbd also induces potent neutralizing ab response, which turns s protein into an important antigenic determinant capable of protective immunity induction and provides a vital approach for the development of immunotherapies. [28] [29] [30] [31] [32] cov e protein is an integral membrane protein of 76-109 amino acids [33] [34] [35] and is present in small amount in virions. [36] [37] [38] it contains a short hydrophilic n-terminal followed by a single predicted hydrophobic domain and a hydrophilic c-terminal. although its membrane topology remains unclear, cov e protein is commonly referred to as a transmembrane protein with one transmembrane domain. [39] [40] [41] accordingly, the e protein mainly targets er and golgi-complex and participates in part of the life cycle of the virus, including virus assembly, budding, particles release, envelope formation, and viral pathogenesis. 33, 35, [41] [42] [43] [44] [45] [46] [47] [48] [49] [50] in addition, cov e protein may have a crucial role in virus production and maturation, because recombinant covs lacking the e protein exhibit significantly reduced viral titers and toxicity. [51] [52] [53] [54] [55] the m protein is a small transmembrane protein (25) (26) (27) (28) (29) (30) with three transmembrane segments, an n-terminal ectodomain and a c terminal-endodomain, determining the shape of the virion. 56, 57 it is considered as the most abundant structural protein and plays a pivotal role in virion assembly via interacting with other structural proteins. [58] [59] [60] binding of s protein and m protein is essential to the virus assembly and the maintenance of s protein in the er-golgi intermediate compartment (ergic)/golgi complex. [61] [62] [63] virus-like particles (vlps) are assembled when the combination of m and e proteins occurs, which suggests that they are required for the formation of the envelope. 39, [64] [65] [66] additionally, when expressed alone, it can become a homomultimeric complex, the primary driving force of envelope formation. 43, 60, 67 furthermore, as to n protein, a stable nucleocapsid and internal core of virions can be achieved when combined with m protein. 68, 69 the n protein, combined with viral genomic rna to form a helical nucleocapsid inside the viral envelope, is a multifunctional protein. 70 it contains three highly conserved domains: the n-terminal domain (ntd), responsible for rna binding; a c-terminal domain (ctd) that is a hydrophobic and helix-rich terminal, capable of dimerization and oligomerization; and an intrinsically disordered region (rna-binding domain/domain 2) that is a serine/arginine-rich domain (sr-domain) with a significant phosphorylation potential. 15, [71] [72] [73] [74] [75] [76] [77] [78] [79] [80] [81] [82] [83] [84] [85] [86] phosphorylation of the n protein can initiate structural modification leading to an increased rna-binding affinity. 72, 79, 87, 88 the n protein binds to the genomic rna through a beads-on-astring form. likewise, except for the interaction between n protein and nucleic, the ability of complex oligomerization is another pivotal activity required for the formation of the ribonucleoprotein complexes for viral assembly. 89 in addition to the role of the n protein in viral core formation and assembly, 69, [90] [91] [92] it is also involved in other critical processes of viral life cycle such as virus budding and envelope formation, 21,93-95 genomic mrna replication ,and genomic rna synthesis. 96, 97 3 although the pathogenic mechanisms of human covs have not yet been fully understood, the investigation of their unique characteristics of each cov enables to distinguish the various human covs including sars, mers, and sars-cov-2. the crucial early step of the infection of human covs into susceptible host cells is the interaction between viral s protein and cellular target receptors. one of the subunits of s protein, s1, containing rbd, is responsible for specific recognition and binding of the target receptors. the other subunit, s2, is in charge of the membrane fusion. 22, [98] [99] [100] [101] [102] the tissue tropism, as well as the susceptible host species, is mainly determined by the binding of s protein to target receptors. 102 based on lines of known evidence, human covs utilize multiple and different types of cellular receptors rather than use a common receptor. therefore, multiple cellular receptors have been identified as target receptors for the various human covs to date (table 1) . angiotensin-converting enzyme 2 (ace2), the first known human homolog of angiotensin-converting enzyme and the receptor for hcov-nl63, sars-cov, and sars-cov-2, is a vital component of the reninangiotensin system (ras). [103] [104] [105] [106] [107] [108] [109] it is a secreted enzyme with a transmembrane domain, a single metalloprotease active site and a signal peptide, 110 and predominantly expressed in heart, vascular endothelia, epithelia of the small intestine, kidney, and testis; alveolar macrophage and monocytes of the respiratory tract; and epithelial cells of the trachea, bronchi, and alveoli. 103, [110] [111] [112] in contrast to its homolog ace that contributes to the promotion of lung failure pathogenesis, induction of lung edemas, and impairment of lung function, ace2 plays a protective role in severe acute lung injury (ali). imai et al revealed that the deficiency of ace2 in the murine models of acute respiratory distress syndrome (ards) deteriorated the symptom in lung function, which could be recovered by hcov-nl63 hcov-hku1 the recombinant ace2. 113 thus, downregulation of ace2 expression in sars patients could be used as an indicator of severe clinical outcome. 114 besides lung damage caused by sars-cov infection could be attenuated by blocking the renin-angiotensin pathway. 114 overall, ace2 could serve as a novel therapeutic target for severe respiratory diseases. dipeptidyl peptidase iv (dpp4), a type ii transmembrane protein also known as cd26, is identified as a target receptor for mers. 115 it is a prolyl oligopeptidase expressed in various cells including endothelial cells, epithelial cells, and inflammatory cells in the lung and smooth muscle. [116] [117] [118] the multifunctional protein ddp4 is implicated in the activation of t-cell, the activity regulation of chemokines and growth factors, and the regulation of glucose metabolism. [119] [120] [121] [122] not only can it be embedded in the plasma membrane in the form of a homodimer, but it also presents in extracellular fluid like plasma as a soluble form. 118, 123 although ddp4 is expressed in epithelial cells of the upper respiratory tract in camels, it is principally found in alveoli but rarely in the nasal cavity or conducting airway. 115, 124 accordingly, in a murine mers model, though monocyte infiltration, alveolar edema and microvascular thrombosis were observed in the mers-cov-infected lungs, any symptoms were seldom found in the airways. 125 aminopeptidase n (apn), a type of ii metalloprotease also called cd13, is a ubiquitous enzyme expressed in various organs (lung, intestine, and kidney) and cells (epithelial cells, endothelial cells, leukocyte, and fibroblast). 126, 127 it serves as a target receptor for hcov-229e, 128 but not for hcov-oc43. hcov-oc43 shares the same specific target with hcov-hku1, namely 9-o-acetylated sialic acid. 129,130 virus entry is a finely regulated process requiring a series of interactions between the virion and host cell. [131] [132] [133] following the conjunction with the target receptor, cov fuse its envelope with the membrane of the host cell. these processes are forced by the conformational change of s protein, which is triggered by not only the target receptor binding but also ph acidification and proteolytic cleavage led by cell surface or endosomal proteases such as transmembrane protease serine 2 (tmprss2), furin, cathepsin l, elastase, and trypsin. [134] [135] [136] [137] [138] [139] [140] [141] [142] [143] cleavages of s protein are facilitated at two sites: the boundary between the s1 and s2 subunit (s1/s2) and the conserved site upstream of the fusion peptide (s2'). 138, 144 the former one is aimed at releasing rbd from the membrane fusion subunit, and the latter one is important for the exposure of the fusion peptide, hydrophobic in general, which acts as an anchor to target membrane. 138, 145, 146 then the fusion domain adopts two heptad repeats (hr1/hr2) to form a compact coiled-coil conformation called 6-helix bundle or 6hb. 144, 147 through direct interactions with lipid bilayers, the fusion domain disrupts two apposed membrane layers and fuses the viral envelope to host cell membrane. ultimately, the viral genome is successfully released into the cytosol of the target cell. in addition to the viral infection through the plasma membrane, the entry of covs into cells can be accomplished by the endocytic pathway, depending on the virus strains and host cells. 135,144 sars-cov, whose intermediary host is the palm civet, is a highly pathogenic respiratory virus that emerged in 2002, leading to global pandemic that affected more than 8000 people in 29 countries. 148, 149 patients infected with sars-cov initially present "flu-like" syndrome commonly showing high fever, headache, sore throat, myalgia, and dry cough. [150] [151] [152] during the disease progression, ali or ards is developed in a number of patients. 153 pathological manifestations can be described as three phases. the first step is the disturbance of gas exchange in the first week, owing to the extensive edema, shedding of ciliated epithelial cells, and deposition of hyaline-rich substances on the alveolar membrane. in the next step, pulmonary fibrosis occurs that is characterized as the deposition of fibrin at epithelial cells and the alveolar spaces, as well as the infiltration of inflammatory and fibroblasts. finally, fibrosis of lung tissue, collagen deposition, and proliferation of alveolar and interstitial cells represent the final step of disease, about 6-8 weeks. [154] [155] [156] [157] [158] diffuse alveolar damage (dad) accompanied by hyaline membrane formation as well as interstitial thickening is common characteristics of sars-cov inducing pulmonary damage. 159 although many investigators have devoted to inquiring into the pathogenesis of such virus, it has not yet been fully understood until now. the immune response is the earliest alert system of the host cells warning virus attacks. ironically, it also aids viral pathogenesis. pattern recognition receptors (prrs) that are retinoic acid-inducible gene i protein (rig-i, member of rlrs family) and melanoma differentiation-associated protein 5 (mda5) recognize viral pathogen-associated molecular patterns (pamps), such as viral components and replication intermediates, to initiate signaling cascades against virus infection. 160, 161 once the pamps from invaded viruses are detected, rig-i and mda5 interact with the mitochondrial antiviral signaling protein (mavs) that is a mitochondrial membrane-bound f i g u r e 2 escape mechanisms of innate immune response of sars-cov and mers-cov adaptor molecule, followed by the activation of several kinase complexes and multiple subsequent transcription factors (irf3, irf7, and nf-κb). activation of nf-κb induces the production of proinflammatory cytokines and chemokines in the nucleus that is a substantial cause of the ards. 162, 163 phosphorylation of irf3 and irf7 by the kinase complexes results in homo-or heterodimerization of irf3 and irf7. the dimerization initiates the transcription of type i interferons (ifns, ifn-α and ifn-β), activating the signal transducer and activator of transcription proteins (stats) to mediate antiviral response ( figure 2 ). [164] [165] [166] several defensive approaches are used by sars-cov to avoid the prrs defense system and, ultimately, host innate immune response. one approach is to replicate themselves within the double membrane vesicles (dmvs) that are protected from the prrs. 167, 168 the eukaryotic mrnas contain a 5′ cap that usually lacks in the viral mrnas. however, some viruses such as sars-cov are capable of building the rna cap through nsp14 and nsp16/nap10 complex, helping them bypass the recognition by prrs. [169] [170] [171] [172] in addition, a couple of more proteins encoded by the viruses participate in the suppression of the innate immune response by disrupting the ifn response. among the nonstructural proteins, nsp 1 mainly involves in the degradation of host mrnas, inactivation of host translational machinery as well as the inhibition of stat1 phosphorylating. [173] [174] [175] sars-cov plpro interferes phosphorylation of irf3 and disrupts nf-κb signaling probably via interacting with sting. [176] [177] [178] the nsp7 and nsp15 are also potential ifn antagonists though the mechanism is not clear. 177 structure proteins such as the n protein and m protein are likely to suppress the type i ifn path-ways. because it was known that n protein inhibits the ifn transcription, the n protein could have a strong potential influence on the viral rna. 179 m protein blocks the formation of signaling kinase complexes, and suppresses the irf3 and irf7 activities, suggesting the potential role of the n protein in the viral infection as well. 180 such accessory proteins as orf 3b protein are able to inhibit the rig-i activity and irf3 phosphorylation in addition to the transcription of ifn-stimulated genes (isgs) via the isre promoter, while orf6 blocks the nuclear translocation of stat1. 181, 182 in spite of the number of research findings in vitro, they have not been validated in vivo. therefore, it is urgent to examine the findings in vivo for a clear and solid understanding of the infectious process. besides the immune response of the host, ace2 also plays an essential role in the pathogenesis of sars-cov. as a negative regulator on the ras, ace2 has been closely linked to the pathogenesis of pulmonary diseases and considered as a protective factor for ali. 113 consequently, the downregulation of ace2 mediated by sars-cov binding might give an explanation for the progression of severe lung damage occurred on some sars patients. 114 a decade after sars, another novel cov was identified as the pathogen of mers that caused a higher mortality rate (30%-40%) compared with sars (around 10%). 183, 184 sars-cov and mers-cov are both emerged from bats, and are disseminated to human through palm civets and dromedary camels, respectively. [185] [186] [187] [188] mers-cov and sars-cov share some common clinical manifestations ranging from asymptomatic to severe pneumonia in multiple organs, 150, 184, 189 and pathological features including inflammatory cells infiltration and dad. 183 similar to sars-cov, mers-cov is also capable of causing immune dysregulation by attenuating the innate immune response ( figure 2 ). 190, 191 type i interferon is important for the inhibition of mers-cov replication in host cells probably via the suppression of the dmvs formation. 192, 193 capping viral mrna by nsp14 and nsp16/nap10 complex protects mers-cov, as well as sars-cov, from the prrs recognition since the structure of nsp16/nap10 complex in both viruses are analogous. 194 besides, several proteins of mers-cov are involved in immune escape mechanism by involving in the signaling cascades. it was reported that irf3 nuclear translocation and ifn promoter activation are inhibited by m protein, orf4a, orf4b, and orf5, former three of which also restrained the expression from an the isre promoter. 195 inhibition of the phosphorylation of irf3 might be the mechanism for ifr3 translocation inhibiting. 194 moreover, mers-cov orf4a can interact with ifn-inducible double-stranded dna (dsdna)dependent protein kinase activator a (prkra) and subsequently control the function of rig-i and mda5, resulting in the disruption of the ifn response. 196, 197 orf4a and orf4b are thought to participate in the nf-κb signaling by downregulating the gene stimulated by nf-κb and affecting the kinase complexes, respectively. 198 functions of plpro and nsp1 of mers-cov are analogous to the functions of those in sars-cov. 199, 200 like ace2, the entry receptor dpp4 for mers-cov has also a pivotal role in the disease pathogenesis and is considered as a key factor for the intraspecies variation shown in mers infection. 201, 202 it is usually expressed in type ii pneumocytes that cover 2% of the alveolar surface. 115, 124 approximately 95% of the surface area is occupied by the type i pneumocytes that are responsible for gas exchange. 203, 204 but the autopsy reports indicated that both type i and ii pneumocytes in patients died from mers-harbored dpp4 expression and these pneumocytes were infected by the virus. 205, 206 mers-cov infection of type i pneumocytes might lead to the damage of the cells in the alveoli subsequently causing the dad. 207 it suggests that type i pneumocytes expressing dpp4 might be included in the pathogenesis of the disease. this might explain why chronic obstructive pulmonary disease (copd) patients attacked by mers-cov had poor outcomes, since the expression of dpp4 was predominantly upregulated on type i pneumocytes in such patients. 202, 208 besides, owing to high expression of dpp4 shown in the kidney, the renal dysfunction might be caused by either the direct infection or the hypoxic damage. 116 evidence of tubular injury, such as cell debris and tubular dilation, could be observed in the late stage of the infection in mers-cov-infected mice. however, no virus could be detected in such animals in the early stage after infection, meaning such pathologic changes might be related to the hypoxia. 125 the outbreak of covid-19, whose pathogen is sars-cov-2, now poses a serious threat to the global public health. 209, 210 since the emergence of the virus, sars-cov-2 has affected more than 14 million people with more than 597 thousand deaths worldwide as of july 2020. next-generation sequencing of the novel virus has been developed soon after the outbreak, indicating that sars-cov-2 is closely related to the bat-derived sarslike covs. 107, 211 it is now believed that bats are likely to be the natural reservoir, 212, 213 and pangolins are regarded as intermediary host according to the later studies. typical clinical presentations of sars-cov-2-infected patients include fever, dyspnea, dry cough fatigue, myalgia, pneumonia, and ards symptoms, similar to those of sars and mers patients. [214] [215] [216] however, intestinal disorders such as diarrhea are less frequent in covid-19 patients than sars. 214, 215, 217 furthermore, in spite of the variation of amino acid at some residues, homology modeling informed that sars-cov-2 and sars-cov have an analogous rbd structure, and share the same target cell receptor, ace2, to mediate the viral entry. 107, 108, 216, [218] [219] [220] it is speculated that ace2 is involved in the pathogenesis of sars-cov-2. owing to the current sparsity of data on the pathological characters of sars-cov-2, it is poorly understood. a case report from an infected patient died of this disease showed that dad with hyaline membrane formation, infiltration of inflammatory cells, and pulmonary edema could be found in the samples taken from their lungs, which is notably corresponding with the symptoms of sars and mers patients. 221 additionally, lymphopenia is a common manifestation in covid-19 patients and might be an effective indicator to estimate the severity of hospitalized covid-19 patients. 222 lymphopenia is also supposed to be a vital factor related to the pathogenesis that has not been elucidated so far. 213 moreover, the concentration of some cytokines and chemokines detected in the plasma was higher in covid-19 patients compared with healthy individuals. moreover, higher plasma levels of gscf, il-2, il-7, il-10, mcp1, mip1a, ip10, and tnf-α were linked to the more severe disease. 215 all of the data reveal that immunopathology may occupy a crucial place in the development of the disease, and further researches about the pathogenesis of sars-cov-2 are urgently needed in the future. owing to the fact that no effective specific antiviral therapies are currently available for sars, mers, and covid-19, isolation and symptomatic support cares are the major management strategies for suspected and confirmed cases requiring hospital treatment including oxygen inhalation, fluid management, and rational use of antibiotics, to prevent organ failure and secondary bacterial infection and alleviate the symptoms. 189, [223] [224] [225] thus, the identification of effective agents against human covs is urgently needed in the response to the current covid-19 outbreak. all of sars-cov, mers-cov, and sars-cov-2 encode structure proteins (like s protein), nonstructure proteins (eg, plpro, 3clpro, rdrp, and helicase), and accessory proteins that are essential for the viral life cycle and that are considered as important targets for the development of antiviral agents ( figure 3 ). 226, 227 analyses of genomic sequences and protein structure indicated that the catalytic sites of four crucial enzymes and the key drug-binding pockets in viral enzymes were conserved across sars-cov, mers-cov, and sars-cov-2. 227 therefore, the therapeutic experience based on sars and mers is capable to guide the treatment of covid-19. the idea to disturb the normal life cycle of the virus provides significant insights into the clinical treatment strategy. the s protein is important for the discovery of antiviral agents due to its multifunction in virus infection. rbd located on the s1 subunit can bind to the host cell receptors (ace2 for sars-cov and sars-cov-2, dpp4 for mers-cov) initiating the conformational changes in s2 subunit to get viral and cell membranes closer and trigger membrane fusion. 228 consequently, the interaction between rbd and the host cell receptors serves as a key target for the production of neutralizing antibody followed by the vaccine development. 31, 229 monoclonal antibodies (mabs) and fusion inhibitors against s1 and s2 subunit, respectively, are potential antivirus drugs to conquer the viral infection, and the agents targeting the host receptors also play a similar role. [230] [231] [232] [233] [234] likewise, cleavage at the protease site of the s1/s2 complex by host proteases such as tmprss2 and furin is necessary for the membrane fusion and syncytium formation. 143, 235 the endosomal cysteine protease cathepsins promote the entry of covs into the host cell via the endosomal pathway. 236 inhibitors of these host proteases can potently block the cell entry, which has been proved in vitro and require further validation on animals studies. 237 once entering the host cells, covs release the nucleocapsid and genomic rna into the cytoplasm and start the translation of the replicase gene. the large replicase pp1a and pp1ab are cleaved by plpro and 3clpro to produce nonstructural proteins like rdrp and helicase, forming the replication-transcription complex. 238 numerous agents inhibiting these proteins have shown anti-cov effects in vitro. combination of the hydrophobic domains of the replication-transcription complex to the endoplasmic reticulum membrane can form the typical cov replication structures such as dmvs and convoluted membranes, protecting covs from the detection of prrs. 168, 239 viral rna synthesis produces genomic and subgenomic rnas. then the subgenomic rnas are translated to generate the structural and accessory proteins, participating in the assembly of the virion that is released into the extracellular compartment via exocytosis. 8 small interfering rnas (sirnas) disturbing these processes could be used in the treatment of covs infections. although covs are capable of disturbing the ifn response, they are still sensitive to the ifn treatment in vitro, indicating that augmented host innate ifn response can be an effective strategy to control the viral infection. 207, [240] [241] [242] in addition to the enhancement of inf response, several other cell signaling pathways are also regarded as potential anti-cov targets. these include calcineurin-nuclear factor of activated t cells (nfat) pathway and kinase signaling pathways such as erk/mapk and pi3k/akt/mtor pathways, the inhibitors of which have exhibited anti-cov activities as well. [243] [244] [245] since the discovery of new interventions may take months or even years, a more efficient approach is to repurpose existing antiviral agents approved for treating related viral infections. the followings are approved drugs or preclinical compounds that have potential antiviral abilities against sars, mers, and covid-19. virus-targeted therapeutic strategies all of the major proteases of the virus are attractive druggable targets since they are essential for viral transcription and replication ( table 2) . as a key part of replication-transcription complex, rdrp participates in the production of genomic rna and subgenomic rna. nucleoside analogues targeting rdrp is capable of inhibiting viral rna synthesis in a great variety of rna viruses including covs. [246] [247] [248] [249] [250] favipiravir (t-705), a guanine analogue approved in japan for influenza treatment, has been proven to effectively interfere the rna synthesis of rna viruses such as influenza virus, ebola virus, and other hemorrhagic fever viruses at rdrp level. [251] [252] [253] [254] [255] [256] several studies concluded that favipiravir could inhibit avian influenza a (h5n1) virus and ebola virus infection in mice. 256, 257 also, favipiravir has been proved with a notable effect increasing the survival rate of ebolainfected patients from 35.3% to 56.4% in sierra leone. 258 a recent study ended with the statement that favipiravir owns the ability against sars-cov-2 (ec50 = 61.88um, cc50 > 400um, si > 6.46). 259 covid-19 patients were enrolled in randomized trials for the evaluation of the efficacy of favipiravir plus inf-α or baloxavir marboxil (chictr2000029544 and chictr2000029600). another guanosine derivative with broad-spectrum antiviral activity, ribavirin, has been authorized for hcv and respiratory syncytial virus (rsv) treatment. 260 accurate mechanism of ribavirin against virus infection is not clear, but inhibition of mrna capping and viral rna synthesis could be pivotal to its antiviral activity. 261 although high dose of ribavirin has been applied to sars treatment, the anti-mers-cov effects were moderate at such dose in rhesus macaques infected by mers-cov and no obvious survival benefit has been observed in mers patients. 225, [262] [263] [264] [265] [266] [267] recently, an open-label, randomized phase ii clinical trial (nct04276688) has revealed that triple combination of ribavirin, interferon, and lopinavirritonavir in covid-19 treatment was safe and superior to lopinavir-ritonavir therapy alone in remission of symptoms, shortening virus shedding and promoting discharge of mild to moderate covid-19 patients. 268 remdesivir (gs-5734) is a small-molecule monophosphoramidate prodrug of an adenosine analog with the ability to interfere with the rna polymerase and the proofreading exoribonuclease and terminate the nonobligate chain. 269 on day 1 and 100 mg once-daily maintenance for 9 days. however, the first clinical trial (nct04252664) has been suspended so far and the second trial (nct04257656) with 237 covid-19 patients enrolled finally indicated that remdesivir hardly shown any statistically significant clinical benefits. 274 conversely, a research found that 36 of 53 (68%) hospitalized patients suffered from severe covid-19 and treated with compassionate-use remdesivir could achieve clinical improvement. 275 in addition, a phase iii, randomized, double-blind, placebo-controlled trial (nct04280705) conducted by beigel et al uncovered the fact that remdesivir prevailed over placebo in shortening the time to recovery in adults patients. 276 though remdesivir has been approved by the food and drug administration (fda) to treat severe covid-19 patients, further researches are urgently required to determine the efficacy and the indication of remdesivir therapy. a novel synthesized nucleoside analogue, bcx4430 (galidesivir), is designed to inhibit viral rna polymerase activity via terminating nonobligate rna chain. 277 bcx4430 exhibits a promising antiviral capability against a wide array of . 277 it is currently tested in phase i clinical trial (nct03800173) for marburg virus and can be a potential countermeasure against viral diseases that threaten the public health in the world. a recent study also concluded that penciclovir, another rdrp inhibitor that is approved for hsv, showed effects on reducing sars-cov-2 infection by high-dose administration (ec50 = 95.96 µm, cc50 > 400 µm, si > 4.17). 259 although resistance to nucleoside analogs has rarely been reported, it is worth noting that mutation in rdrp is probably responsible for the acquired resistance and should be monitored for the possible resistance. 278 covs plpro enzymes display proteolytic, deubiquitylating, and deisgylating activities. [279] [280] [281] plpro was first regarded as a druggable target for sars-cov, and then several compounds targeting sars-cov plpro were also found to be effective against mers-cov plpro, recently. 282, 283 though numerous plpro inhibitors have been identified, many of them only inhibit enzymatic activities and do not affect on the viral replication. 284, 285 a study from lin et al suggested that an fda-approved alcoholaversive drug, disulfiram could inhibit sars-cov and mers-cov plpro via different mechanisms. and the synergistic inhibition between disulfiram and known plpro inhibitors, like 6-thioguanine and mycophenolic acid, to mers-cov might offer the potential combination treatments against covs in clinical. 286 another essential protease that cleaves the viral polyproteins during viral replication is 3clpro. similar to plpro, a great many of inhibitors have been identified with the ability to target covs 3clpro. among the 3clpro inhibitors, the human immunodeficiency virus (hiv) protease inhibitors are the most comprehensively studied such as lopinavir, ritonavir, asc09f, as well as darunavir and cobicistat. lopinavir and ritonavir, applied in combination to treat hiv infection, have shown improvement in the outcome of sars patients in nonrandomized trials. 287, 288 though lopinavir alone hardly displayed antiviral activity against mers-cov in vitro, the combination of lopinavir and ritonavir ameliorated the outcome in mers-cov-infected nonhuman primates. 289, 290 therefore, the efficacy of the lopinavir-ritonavir combination in mers patients should be reappraised (nct02845843). however, no benefit was observed in lopinavir-ritonavir treatment compared to standard care in a randomized, controlled, open-label clinical trial (chictr2000029308) involving severe covid-19 patients. 291 further trials are still needed to confirm the therapeutic efficacy. in addition, several other clinical trials have been developed to confirm the efficacy of 3clpro inhibitors on covid-19 (nct04252274, nct04251871, nct04255017, chictr2000029539, nct04251871, nct04255017, and nct04261270), as well as darunavir and cobicistat (nct04252274), asc09f combined with oseltamivir (nct04261270). helicase acts on the duplex oligonucleotides and turns them into single strands in an atp-dependent manner in the cov replication cycle. that helicases in different covs are highly homologous making them potentially strong targets for the covs therapeutic options. based on the mechanism actions, the helicase inhibitors can be approximately classified into two groups. one is able to inhibit both the atpase and helicase activities represented by bananins derivatives, 5-hydroxychromone derivatives, and triazole derivatives (compound 16). 292, 293 the other group including ssya 10-001 and adks has the ability to inhibit the helicase activity with no or little effects on the atpase activity. 294 however, the toxicity of helicase inhibitors needs to be examined before being applied to human patients. the transmembrane glycoprotein, s protein, is also a promising target for antiviral agents' development ( table 2 ). one class of antiviral drugs targeting s protein mostly blocks the spike-mediated membrane fusion. a potent mers-cov inhibitor, nafamostat, has demonstrated to be inhibitive against the sars-cov-2 infection (ec50 = 22.50 µm, cc50 > 100 µm, si > 4.44). 259 griffithsin is a red-alga-derived lectin, which specially binds to oligosaccharides located on the surface of viral glycoproteins, for example, s glycoprotein of sars-cov and hiv glycoprotein 120. 295 a wide range of human covs infection could be inhibited by griffithsin, comprising hcov-229e, hcov-nl63, hcov-oc43, and sars-cov. 295, 296 but the value of griffithsin in the treatment or prevention of covid-19 is needed to be evaluated urgently. s2 subunit of s protein contains two heptad repeat (hr1 and hr2). antiviral peptides analogous derived from these regions exhibited inhibition to the spike protein-mediated cell-cell fusion and viral entry in viruses such as sars-cov, mers-cov, as well as hcov-229e. 231, 297, 298 hr2p, a peptide spanning hr2 sequences of mers-cov s protein, was capable of interacting with hr1 region to form a 6-hb complex, resulting in potent inhibition of viral fusion and replication. its analog hr2p-m2 exhibited an obvious improvement in stability, solubility, and antiviral activity after being modified with hydrophilic residues. 228 additionally, hr2p-m2 intranasal administration effectively prevented experimental mice transduced by adenoviral vectors conveying human dpp4 from mers-cov infection with a >1000-fold decrease in viral titers in the lung, and this protection was intensified via the combination of hr2p-m2 and ifn-β. 299 another newly designed fusion inhibitor from mers-cov called mers-five-helix bundle (mers-5hb), which is derived from the 6-hb, also displayed a potent suppression on s protein-mediated syncytial formation. compared with mers-6hb, mers-5hb lacks one hr2, which endows its capability to interact with viral hr2 to interrupt the membrane fusion. 300 besides, mers-5hb could effectively inhibit the entry of pseudotyped mers-cov with 50% inhibitory concentration (ic50) about 1 µm. 300 altogether, the resistance of these drugs can be overcome by combining antiviral peptides aiming at various domains of s2 subunit, which may also attain synergistic effects in vitro. as for sirnas, which displayed antiviral activities in vitro as well as in sars-cov-infected rhesus macaques, are still under preclinical development and demand further studies to seek out the reliable drug delivery methods in a human before the clinical application. [301] [302] [303] m, n, and e proteins and some accessory proteins are not only vital to the virion assembly but also involved in viral pathogenesis in which they function in the interruption of host innate immune response to assist viral infection. for instance, m protein as well as accessory proteins 4a, 4b, and 5 of mers-cov act as ifn antagonist, and n protein of sars-cov serves as an inhibitor of viral rna. researches carried out by he et al and akerstrom et al emphasized that sirnas silencing m, n, e, orf3a, and orf7a/7b play an important role in the inhibition of viral replication of the sars-cov. 304, 305 but the delivery methods still limit their application in human being. nevertheless, various agents related to these proteins are discovered via functional analysis. one example is resveratrol, a natural stilbene derivative demonstrated to reduce inflammation and exert antiviral activity against multiple viruses. [306] [307] [308] [309] [310] [311] in addition, it exhibited significant inhibition of mers-cov infection and prolonged cellular survival after virus challenge in vitro via deregulating the expression of n protein and the apoptosis induced by mers-cov. 312 alternatively, hexamethylene amiloride, a viroporin inhibitor, is capable to suppress the ion channel activity of e protein of covs such as hcov-229e and sars-cov. 313 identified as dna metabolism inhibitor, gemcitabine hydrochloride is a deoxycytidine analog inhibiting both sars-cov and mers-cov with micromolar ec50 and low toxicity, which suggests its potential antiviral capacity for other human covs. 314 lj001 and jl103, two novel lipophilic thiazolidine derivatives, could induce several changes in membrane properties including the decrease in membrane fluidity, contributing to inhibition of membrane fusion, which made them become broad-spectrum enveloped virus entry inhibitors and potential anti-cov agents. 315 host-cell-targeted therapies the host innate immune response is vital to the interruption of viral replication. recombinant interferons have been applicated in treating various viruses as well as many covs (table 3) . though the host interferon response can be inhibited by the covs, they are still proved effective against covs infection such as sars-cov and mers-cov in vitro and several animal models. 242, 264, 289, 299, 316 recombinant interferons are usually combined with other antiviral agents including ribavirin or lopinavir/ritonavir to treat sars-cov and mers-cov infections, 290, 317 and the anti-covs efficacy of interferons is enhanced when added with ribavirin. 318 a combination of interferon-α2b with ribavirin reduced virus replication and improves clinical outcomes in a rhesus macaque model of mers. 264 however, the effectiveness of combination treatments comprising interferons and ribavirin is still controversial in clinical researches. a study of five patients received interferon-α2b and ribavirin showed no survival, but the finding might be not reliable owing to the delayed administration. 266 contrarily, in another study (n = 44), mortality rates of individuals receiving interferon-α2b and ribavirin exhibited a significant reduction in day 14, compared with the individuals received standard supportive care, but no significant difference was observed in day 28. 265 moreover, no significant difference in mortality rates between interferon-α2b and ribavirin treatment group and interferon-β1a and ribavirin ta b l e 3 host-targeted agents for hcovs treating group was observed in a retrospective study. 267 on the other hand, interferon-β1b displayed stronger inhibition to the mers-cov replication in vitro compared with other interferons, and combined use of interferon-β1b with other antiviral compounds should be evaluated in further research. 289, 290 nitazoxanide, originally identified as an antiprotozoal agent, was later considered as a broad-spectrum antiviral agent able to inhibit the replication of numerous dna and rna viruses such as rsv, parainfluenza, rotavirus, hbv, hcv, hiv, yellow fever, as well as covs in vitro. 319 several clinical trials have confirmed its potential value in treating influenza, chronic hbv, and hcv. [319] [320] [321] moreover, recent research indicated that nitazoxanide was capable of inhibiting sars-cov-2 infection at a low micromolar concentration (ec50 = 2.12 µm; cc50 > 35.53 µm; si > 16. 76) , and the in vivo evaluation of this efficacy is demanded. 259 another type i interferon inducer, polyinosinic:polycytidylic acid (poly(i:c)), is an analog of dsdna with a powerful ability to reduce mers-cov load in animal models. 192 and phase ii clinical trials demonstrated that it was well tolerated by malignant gliomas patients when injected intramuscularly. 322, 323 overall, the use of interferons or interferon inducers may be a valuable strategy against covs infection and should be furtherly accessed in animal models. several host factors are considered essential to covs entry, such as the host receptors that bind to covs s protein, and host proteases that facilitated covs entry through the cell surface or endosomal pathway. thus, these factors become attractive targets for anti-cov agents' development (table 3) . antibodies, peptides, and some functional inhibitors targeting the host receptors can effectively interrupt the binding between s protein and the host cells. one example is that the n-(2-aminoethyl)-1-aziridineethanamine (naae), a small molecular inhibitor, is able to target ace2 leading to the block of s protein-mediated membrane fusion, so does the synthetic peptides analogous, p4 and p5. 324, 325 similar agents were also found in mers treatment, one of which, ys110, was confirmed to be well tolerated in patients with advance solid tumors. 326 owing to their specificity to appointed receptors, they were regarded as narrow-spectrum drugs. however, the efficacy of these receptor-targeted agents have never been evaluated in any covs-infected patients and the safeties of these agents also remain unclear. host protease such as cathepsins and tmprss2 play a key role in the cleavage of s protein and the suppression of these proteases with potent inhibitors can obstruct the virus entry through either endosomal pathway or cell surface pathway. k11777 is a cathepsin inhibitor with broad spectrum against enveloped rna virus including sars-cov and mers-cov. 237, 327, 328 however, the camostat mesylate, applied in chronic pancreatitis treating, is a tmprss2 inhibitor that interrupts the tmprss2mediated cell surface entry. 329, 330 the combination of cathepsin inhibitors and tmprss2 inhibitors is crucial to the complete suppression of mers-cov in vitro. inhibition of another host protease, furin, which is vital to furinmediated s cleavage for covs, also can block membrane fusion and the viral entry like mers-cov. 143 notably, inhibition of host proteases may result in some side effects and need further evaluation. some approved antipsychotic agents (chlorpromazine, triflupromazine, and fluphenazine) and cardiotonic steroids (ouabain and bufalin) can inhibit clathrinmediated endocytosis via affecting the assembly of clathrin-coated pits at the plasma membrane and binding sodium/potassium-transporting atpase subunit α1 (atp1a1), respectively. 314, 331, 332 thus, they are considered as clathrin-mediated endocytosis inhibitors. alternatively, endosomal acidification also has a profound impact on endocytosis. increase of endosomal ph shows an inhibitive effect on virus infection, which has been confirmed by chloroquine, a widely used autoimmune disease and antimalarial agents. 333 chloroquine proves to be active against a wide range of rna viruses including hcov-229e, hcov-oc43, sars-cov, and mers-cov. [334] [335] [336] [337] [338] recently, chloroquine is identified to function at both entry and postentry phase of the sars-cov-2 infection with the ec90 value of 6.90 µm in vero e6 cells. 259 additionally, as an immunoregulator, its antiviral activity may be synergistically intensified in vivo. 259 therefore, chloroquine is suggested as a potential option against the emerging sars-cov-2. significantly, higher dose of chloroquine should not be recommended for severe covid-19 patients owing to the its drug safety, particularly while simultaneously accepting azithromycin and oseltamivir treatment, which was presented by a randomized clinical trial (nct04323527). 339 hydroxychloroquine, a chloroquine analog with lower toxicity, was listed as a potential anti-sars-cov-2 agent and recommended to be applied in covid-19 treatment by chinese national guideline and fda. 340 however, evidence of the benefits and harms of hydroxychloroquine therapy is still insufficient and conflicting. some small studies show that hydroxychloroquine was capable of decreasing sars-cov-2 shedding that could be enhanced by the combination of azithromycin and achieving a shorter time to clinical recovery. 341, 342 but almost no clinical benefit was observed in other studies. 340, 343 therefore, therapeutic efficacy of hydroxychloroquine is still needed to be reconfirmed. moreover, there are several factors required to be reconsidered before efficacy evaluation, such as the severity of illness, definition of the endpoints, and effects of the comorbidities. except for the innate immune response, host receptors, and other factors affecting the endocytosis, some signaling pathways have also been suggested as useful approaches for discovering anti-cov reagents (table 3) . cyclophilins are peptidyl-prolyl isomerases with physiological functions showing as modulating the calcineurin/nfat pathway via reacting with covs nsp1, which is important for the immune cell activation. 244 in addition, they are also required for the replication of numerous viruses including hiv, hcv, as well as covs. 344 consequently, inhibition of cyclophilins by cyclosporines, such as cyclosporin a (csa) and its derivatives, has shown to block the replication of a wide range of covs. 244, 245, 345, 346 however, the obvious immune suppressive properties of csa limit its application in antiviral therapy. but alisporivir, a nonimmunosuppressive cyclosporin a-analog, also displayed the inhibition to the covs replication at a lowmicromolar concentration. 347 additionally, the combined use of cyclosporine and interferon or ribavirin in vitro was beneficial to inhibit sars-cov or mers-cov infection, which needed to be furtherly evaluated in animal models. 347, 348 furthermore, some antiviral agents inhibiting the cellular signaling pathways, in particular, the erk pathway and pi3k/akt pathway, also interrupt the replication of covs. 243, 314, 349 however, the efficacy and safety against covs infection of these agents still need to be reconsidered. corticosteroids, which were used in sars and mers treatment, have been linked to several complications such as psychosis, diabetes, and avascular necrosis. 350, 351 they also were pointed out to be associated with viral replication prolongation in mers patients. 351 however, an update on the efficacy of dexamethasone based on a press release publicized recently indicated that severe covid-19 patients given 6 mg dexamethasone once daily shown a lower mortality (about 8-26%) compared to patients with standard care. 352, 353 besides, another agent, methylprednisolone, also exhibited potential capacity in reducing the mortality rate and achieving better clinical outcomes in severe covid-19 patients. 354, 355 thus, it is wise to apply corticosteroids to the right patients at the right time. but physicians also need to monitor the corticosteroid-related complications. clinical trials of corticosteroid treatments are shown in table 3 . potential immunotherapeutic options s protein of sars-cov proves to be highly immunogenic during infection and responsible for eliciting a humoral immune response in the host. 356 antivirus antibodies could be detected in the plasma of convalescent patients' infected sars-cov and mers-cov. 357, 358 convalescent plasma therapy has been applicated in treating patients infected by numerous viruses involving ebola virus, junin virus, machupo virus, and lassa fever. [359] [360] [361] [362] as for sars-cov, higher day-22 discharge rate and lower mortality rate have been observed among sars patients who received convalescent plasma transfusion before day 14 of the illness. 363, 364 this is consistent with another small cohorts research concluding infected patients with severe conditions who failed to respond to current therapies but finally survived after transfused with convalescent plasma, with no obvious side effects. 358 similar results were found in mers patients. 365 additionally, plasma adoptive therapy with anti-mers-cov antibodies could block the virus adhesion and accelerate the viral elimination from mers-cov-infected animal models. 192 but the efficacy and safety of convalescent plasma therapy in covid-19 patients still need to be reevaluated. although convalescent plasma therapy proves to be a potentially effective therapeutic option for emerging covs, several factors still limit its extensive use in clinical, one of which is enough titers of serum neutralizing antibodies. the development of mabs targeting the s protein of covs is regarded as a remedial strategy (table 4 ). potent mabs against s protein of human covs can be attained via transgenic mice immunization, convalescent b cells immortalization, and cloning of small chain variable regions from naïve and convalescent patients. 366 the majority of mabs interact with the rbd of s protein leading to the interruption of rbd-receptor binding and block the viral attachment. a few mabs react with other regions of s protein besides the rbd. 366 although binding to different epitopes, these mabs exhibit capacity to reduce the viral titers. two rbd-specific neutralizing mabs, mers-4 and mers-27, which were derived from single-chain variable regions, revealed suppressive effects against both mers-cov and pseudotyped mers-cov infection at nanomolar concentrations and were recommended as promising candidates for therapeutic interventions to mers. 232 based on similar mechanisms, other human mabs for mers-cov were also capable of competing with dpp4 for rbd binding and neutralizing the virus. 233, 234, [367] [368] [369] [370] when administrated to individuals at risk, some of the mabs were capable of preventing viral replication and contributing to block the transmission of mers-cov among human. 368 thus, such antibodies could be served as prophylactic strategies in clinical and valuable tools to guild the development of effective anti-covs vaccines. 234,368 from sars-cov to sars-cov-2, the emergence of severe human covs have taught us many lessons about the importance of rapid diagnostics and effective vaccines to control the outbreak caused by these viruses. due to the persistence of zoonotic sources in endemic areas, lethal covs remain existing in human society and may lead to the epidemic at any time. thus, a priority is to develop vaccines targeting conserved alleles and providing broad-spectrum protection against varied viral strains. since the emergence of sars-cov and mers-cov, several strategies were applicated in vaccine design, including inactivated virus vaccines, live-attenuated virus vaccines, viral vector vaccines, nanoparticles, recombinant protein subunits vaccines, and dna vaccines (table 4) . 371, 372 and clinical trials have also been developed to test the efficacy of the novel vaccines (table 5) . effective vaccines are pivotal in blocking the virus spread from animals' reservoirs to human hosts. inactivated virus vaccines, preserving the viral structure and antigenicity but eliminating the infectious ability, could elicit neutralizing antibodies in animal models of sars-cov and show protection against viral replication when administrated with or without adjuvants. 372 which may be related to the disseminated infection observed in immunocompromised patients. in addition, live-attenuated virus vaccines can induce an innate and adaptive immune response and the protective value can last for a long time. 371 besides, other strategies for vaccines development are also evaluated in animal models. based on the experience of sars-cov and mers-cov, the development of novel sars-cov-2 vaccine is currently underway and requires more research. however, several concerns should be addressed about the vaccination. the first is the disease deterioration caused by vaccination. although this situation only appears in a small subset of sars vaccine studies, it is still a significant problem that needs to be properly solved. 372 second, the variability of s protein can mediate covs escape from neutralization, suggesting that recombinant protein subunits vaccines based on s protein may demand multivalent approaches. 376 last but not least, how to define ta b l e 5 important clinical trials with vaccines for sars-cov, mers-cov, and sars-cov-2 the emergence and prevalence of highly pathogenic cov severely threaten public health. a task of top priority is to make clear the viral structural and epidemiological characteristics and block the viral dissemination as well as the progression of the disease, at the first case. to date, further understanding of the life cycle and the pathogenesis of emerging human covs makes current therapeutic strategies of antiviral infection more rational. repurposing existing antiviral drugs is an effective short-term strategy to deal with emerging cov such as the ongoing sars-cov-2. various agents with different targets have been evaluated in vitro and in vivo. but not all antiviral agents are capable of achieving better efficacy than in vitro, and in vivo studies are needed to select optimal agents. suitable animal models are particularly significant. however, there are only a few effective animal models available for the studies of covs treatment, which may postpone the clinical evaluation of drugs. besides, due to the diversity of viruses and the capacity of rapidly mutating, some antiviral reagents available for existing covs may become invalid. accordingly, sequencing more natural specimens and combination therapy with two or more synergistic agents have become promising solutions. furthermore, the efficacy of current antiviral therapies needs to be estimated in larger scale, well-organized randomized clinical trial the efficacy and safety of the most practicable strategies for the ongoing covid-19 are urgently needed to be further assessed in clinical trials, involving remdesivir (gs-5734), lopinavir/ritonavir, lopinavir/ritonavir combined with ifn-β, convalescent plasma, and mabs, which prove to be potentially effective options against sars-cov-2. additionally, to control the future cov pandemics, the development of novel broad-spectrum antiviral agents covering numerous covs will become the ultimate goal. the authors declare no conflict of interest. wei https://orcid.org/0000-0002-6513-6422 coronavirus diversity, phylogeny and interspecies jumping coronavirus pathogenesis is the discovery of the novel human betacoronavirus 2c emc/2012 (hcov-emc) the beginning of another sars-like pandemic? discovery of seven novel mammalian and avian coronaviruses in the genus deltacoronavirus supports bat coronaviruses as the gene source of alphacoronavirus and betacoronavirus and avian coronaviruses as the gene source of gammacoronavirus and deltacoronavirus interspecies transmission and emergence of novel viruses: lessons from bats and birds the pdz-binding motif of severe acute respiratory syndrome coronavirus envelope protein is a determinant of viral pathogenesis epidemiology and cause of severe acute respiratory syndrome (sars) in guangdong, people's republic of china middle east respiratory syndrome coronavirus: another zoonotic betacoronavirus causing sars-like disease cultivation of viruses from a high proportion of patients with colds a new virus isolated from the human respiratory tract epidemiology, genetic recombination, and pathogenesis of coronaviruses molecular evolution of human coronavirus genomes korean society of epidemiology -nco vtft. an interim review of the epidemiological characteristics of 2019 novel coronavirus coronavirus envelope protein: a small membrane protein with multiple functions the molecular biology of coronaviruses proteolytic processing of polyproteins 1a and 1ab between non-structural proteins 10 and 11/12 of coronavirus infectious bronchitis virus is dispensable for viral replication in cultured cells supramolecular architecture of severe acute respiratory syndrome coronavirus revealed by electron cryomicroscopy cryo-electron tomography of mouse hepatitis virus: insights into the structure of the coronavirion coronavirus immunogens synthesis and characterization of a native, oligomeric form of recombinant severe acute respiratory syndrome coronavirus spike glycoprotein the m, e, and n structural proteins of the severe acute respiratory syndrome coronavirus are required for efficient assembly, trafficking, and release of viruslike particles pre-fusion structure of a human coronavirus spike protein sars coronavirus: a new challenge for prevention and therapy cellular entry of the sars coronavirus structural characterization of the fusion-active complex of severe acute respiratory syndrome (sars) coronavirus interaction between heptad repeat 1 and 2 regions in spike protein of sars-associated coronavirus: implications for virus fusogenic mechanism and identification of fusion inhibitors structural characterization of the sarscoronavirus spike s fusion protein core major receptorbinding and neutralization determinants are located within the same domain of the transmissible gastroenteritis virus (coronavirus) spike protein localization of neutralizing epitopes and the receptor-binding site within the aminoterminal 330 amino acids of the murine coronavirus spike protein identification of a receptor-binding domain of the spike glycoprotein of human coronavirus hcov-229e receptor-binding domain of sars-cov spike protein induces highly potent neutralizing antibodies: implication for developing subunit vaccine receptor-binding domain of severe acute respiratory syndrome coronavirus spike protein contains multiple conformation-dependent epitopes that induce highly potent neutralizing antibodies characterization of the coronavirus mouse hepatitis virus strain a59 small membrane protein e a highly unusual palindromic transmembrane helical hairpin formed by sars coronavirus e protein exceptional flexibility in the sequence requirements for coronavirus small envelope protein function association of the infectious bronchitis virus 3c protein with the virion envelope the 9-kda hydrophobic protein encoded at the 3' end of the porcine transmissible gastroenteritis coronavirus genome is membrane-associated mouse hepatitis virus gene 5b protein is a new virion envelope protein infectious bronchitis virus e protein is targeted to the golgi complex and directs release of virus-like particles a comprehensive comparison of transmembrane domains reveals organelle-specific properties subcellular location and topology of severe acute respiratory syndrome coronavirus envelope protein induction of apoptosis in murine coronavirus-infected cultured cells and demonstration of e protein as an apoptosis inducer assembly of the coronavirus envelope: homotypic interactions between the m proteins sars coronavirus e protein forms cation-selective ion channels differential maturation and subcellular localization of severe acute respiratory syndrome coronavirus surface proteins s, m and e bcl-xl inhibits t-cell apoptosis induced by expression of sars coronavirus e protein in the absence of growth factors the transmembrane domain of the infectious bronchitis virus e protein is required for efficient virus release biochemical evidence for the presence of mixed membrane topologies of the severe acute respiratory syndrome coronavirus envelope protein expressed in mammalian cells role of the coronavirus e viroporin protein transmembrane domain in virus assembly severe acute respiratory syndrome coronavirus envelope protein regulates cell stress response and apoptosis heterologous gene expression from transmissible gastroenteritis virus replicon particles generation of a replication-competent, propagation-deficient virus vector based on the transmissible gastroenteritis coronavirus genome the small envelope protein e is not essential for murine coronavirus replication a severe acute respiratory syndrome coronavirus that lacks the e gene is attenuated in vitro and in vivo absence of e protein arrests transmissible gastroenteritis coronavirus maturation in the secretory pathway characterization of severe acute respiratory syndrome coronavirus membrane protein sequence and topology of a model intracellular membrane protein, e1 glycoprotein, from a coronavirus genetic evidence for a structural interaction between the carboxy termini of the membrane and nucleocapsid proteins of mouse hepatitis virus the molecular biology of coronaviruses a structural analysis of m protein in coronavirus assembly and morphology envelope glycoprotein interactions in coronavirus assembly efficient assembly and release of sars coronavirus-like particles by a heterologous expression system coronaviruses: an overview of their replication and pathogenesis nucleocapsidindependent assembly of coronavirus-like particles by co-expression of viral envelope protein genes coronavirus pseudoparticles formed with recombinant m and e proteins induce alpha interferon synthesis by leukocytes the cytoplasmic tails of infectious bronchitis virus e and m proteins mediate their interaction the missing link in coronavirus assembly. retention of the avian coronavirus infectious bronchitis virus envelope protein in the pre-golgi compartments and physical interaction between the envelope and membrane proteins characterization of the coronavirus m protein and nucleocapsid interaction in infected cells the membrane m protein carboxy terminus binds to transmissible gastroenteritis coronavirus core and contributes to core stability isolation of coronavirus envelope glycoproteins and interaction with the viral nucleocapsid sequence comparison of the n genes of five strains of the coronavirus mouse hepatitis virus suggests a three domain structure for the nucleocapsid protein localization of an rna-binding domain in the nucleocapsid protein of the coronavirus mouse hepatitis virus structure of the n-terminal rna-binding domain of the sars cov nucleocapsid protein the nucleocapsid protein of the sars coronavirus is capable of selfassociation through a c-terminal 209 amino acid interaction domain the dimer interface of the sars coronavirus nucleocapsid protein adapts a porcine respiratory and reproductive syndrome virus-like structure the nucleocapsid protein of coronavirus infectious bronchitis virus: crystal structure of its n-terminal domain and multimerization properties the severe acute respiratory syndrome coronavirus nucleocapsid protein is phosphorylated and localizes in the cytoplasm by 14-3-3-mediated translocation recombinant severe acute respiratory syndrome (sars) coronavirus nucleocapsid protein forms a dimer through its c-terminal domain modular organization of sars coronavirus nucleocapsid protein characterisation of the rna binding properties of the coronavirus infectious bronchitis virus nucleocapsid protein amino-terminal region structure of the sars coronavirus nucleocapsid protein rna-binding dimerization domain suggests a mechanism for helical packaging of viral rna phosphorylation of the arginine/serine dipeptide-rich motif of the severe acute respiratory syndrome coronavirus nucleocapsid protein modulates its multimerization, translation inhibitory activity and cellular localization identification of in vivointeracting domains of the murine coronavirus nucleocapsid protein glycogen synthase kinase-3 regulates the phosphorylation of severe acute respiratory syndrome coronavirus nucleocapsid protein and viral replication oligomerization of the carboxyl terminal domain of the human coronavirus 229e nucleocapsid protein solution structure of the c-terminal dimerization domain of sars coronavirus nucleocapsid protein solved by the sail-nmr method high affinity interaction between nucleocapsid protein and leader/intergenic sequence of mouse hepatitis virus rna the nucleocapsid protein of sars coronavirus has a high binding affinity to the human cellular heterogeneous nuclear ribonucleoprotein a1 theoretical aspects of virus capsid assembly analysis of multimerization of the sars coronavirus nucleocapsid protein mass spectroscopic characterization of the coronavirus infectious bronchitis virus nucleoprotein and elucidation of the role of phosphorylation in rna binding by using surface plasmon resonance mapping of the coronavirus membrane protein domains involved in interaction with the spike protein envelope protein palmitoylations are crucial for murine coronavirus assembly the hydrophobic domain of infectious bronchitis virus e protein alters the host secretory pathway and is important for release of infectious virus the coronavirus e protein: assembly and beyond mouse hepatitis virus replicase proteins associate with two distinct populations of intracellular membranes the intracellular sites of early replication and budding of sars-coronavirus cryo-electron microscopy structure of a coronavirus spike glycoprotein trimer glycan shield and epitope masking of a coronavirus spike protein observed by cryoelectron microscopy cryo-electron microscopy structures of the sars-cov spike glycoprotein reveal a prerequisite conformational state for receptor binding immunogenicity and structures of a rationally designed prefusion mers-cov spike antigen cryo-em structures of mers-cov and sars-cov spike glycoproteins reveal the dynamic receptor binding domains a human homolog of angiotensin-converting enzyme. cloning and functional expression as a captopril-insensitive carboxypeptidase angiotensin-converting enzyme 2 is a functional receptor for the sars coronavirus human coronavirus nl63 employs the severe acute respiratory syndrome coronavirus receptor for cellular entry highly conserved regions within the spike proteins of human coronaviruses 229e and nl63 determine recognition of their respective cellular receptors genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding receptor recognition by novel coronavirus from wuhan: an analysis based on decade-long structural studies of sars angiotensin-converting enzyme 2 (ace2) in disease pathogenesis a novel angiotensinconverting enzyme-related carboxypeptidase (ace2) converts angiotensin i to angiotensin 1-9 quantitative mrna expression profiling of ace 2, a novel homologue of angiotensin converting enzyme tissue distribution of ace2 protein, the functional receptor for sars coronavirus. a first step in understanding sars pathogenesis angiotensin-converting enzyme 2 protects from severe acute lung failure a crucial role of angiotensin converting enzyme 2 (ace2) in sars coronavirus-induced lung injury dipeptidyl peptidase 4 distribution in the human respiratory tract: implications for the middle east respiratory syndrome dipeptidylpeptidase iv from bench to bedside: an update on structural properties, functions, and clinical aspects of the enzyme dpp iv physiology and pharmacology of dpp-4 in glucose homeostasis and the treatment of type 2 diabetes dipeptidyl peptidase iv (dpp-4) inhibition alleviates pulmonary arterial remodeling in experimental pulmonary hypertension inhibitors of dipeptidyl peptidase iv (dp iv, cd26) induces secretion of transforming growth factor-beta 1 (tgf-beta 1) in stimulated mouse splenocytes and thymocytes dpp-4 inhibitors and their potential role in the management of type 2 diabetes revisiting an old acquaintance: cd26 and its molecular mechanisms in t cell function cd26/dipeptidylpeptidase iv-chemokine interactions: doubleedged regulation of inflammation and tumor biology cut to the chase: a review of cd26/dipeptidyl peptidase-4's (dpp4) entanglement in the immune system differential expression of the middle east respiratory syndrome coronavirus receptor in the upper respiratory tracts of humans and dromedary camels middle east respiratory syndrome coronavirus causes multiple organ damage and lethal disease in mice transgenic for human dipeptidyl peptidase 4 the structure and main functions of aminopeptidase n human aminopeptidase n is a receptor for human coronavirus 229e molecular determinants of species specificity in the coronavirus receptor aminopeptidase n (cd13): influence of n-linked glycosylation human coronavirus hku1 spike protein uses o-acetylated sialic acid as an attachment receptor determinant and employs hemagglutininesterase protein as a receptor-destroying enzyme human coronaviruses oc43 and hku1 bind to 9-o-acetylated sialic acids via a conserved receptor-binding site in spike protein domain a virus entry: open sesame the cell biology of receptor-mediated virus entry dynamics of virus-receptor interactions in virus binding, signaling, and endocytosis characterization of severe acute respiratory syndrome-associated coronavirus (sars-cov) spike glycoprotein-mediated viral entry protease-mediated enhancement of severe acute respiratory syndrome coronavirus infection inhibitors of cathepsin l prevent severe acute respiratory syndrome coronavirus entry endosomal proteolysis by cathepsins is necessary for murine coronavirus mouse hepatitis virus type 2 spike-mediated entry activation of the sars coronavirus spike protein via sequential proteolytic cleavage at two distinct sites elastase-mediated activation of the severe acute respiratory syndrome coronavirus spike protein at discrete sites within the s2 domain cleavage and activation of the severe acute respiratory syndrome coronavirus spike protein by human airway trypsin-like protease evidence that tmprss2 activates the severe acute respiratory syndrome coronavirus spike protein for membrane fusion and reduces viral control by the humoral immune response a transmembrane serine protease is linked to the severe acute respiratory syndrome coronavirus receptor and activates virus entry host cell entry of middle east respiratory syndrome coronavirus after two-step, furin-mediated activation of the spike protein mechanisms of coronavirus cell entry mediated by the viral spike protein ready, set, fuse! the coronavirus spike protein and acquisition of fusion competence physiological and molecular triggers for sars-cov membrane fusion and entry into host cells structures and mechanisms of viral membrane fusion proteins: multiple variations on a common theme from sars to mers, thrusting coronaviruses into the spotlight isolation and characterization of viruses related to the sars coronavirus from animals in southern china sars: clinical presentation, transmission, pathogenesis and treatment options identification of a novel coronavirus in patients with severe acute respiratory syndrome a novel coronavirus associated with severe acute respiratory syndrome allelic variation in the toll-like receptor adaptor protein ticam2 contributes to sars-coronavirus pathogenesis in mice. g3 (bethesda) a clinicopathological study of three cases of severe acute respiratory syndrome (sars) lung pathology of fatal severe acute respiratory syndrome the spectrum of pathological changes in severe acute respiratory syndrome (sars) pathology and pathogenesis of severe acute respiratory syndrome evolution of pulmonary pathology in severe acute respiratory syndrome sars: prognosis, outcome and sequelae cytosolic surveillance and antiviral immunity sensing of rna viruses: a review of innate immune receptors involved in recognizing rna virus invasion biomarkers in acute respiratory distress syndrome the mercurial nature of neutrophils: still an enigma in ards? sars-cov pathogenesis is regulated by a stat1 dependent but a type i, ii and iii interferon receptor independent mechanism resolution of primary severe acute respiratory syndrome-associated coronavirus infection requires stat1 sars coronavirus pathogenesis: host innate immune responses and viral antagonism of interferon ultrastructure and origin of membrane vesicles associated with the severe acute respiratory syndrome coronavirus replication complex sars-coronavirus replication is supported by a reticulovesicular network of modified endoplasmic reticulum functional screen reveals sars coronavirus nonstructural protein nsp14 as a novel cap n7 methyltransferase in vitro reconstitution of sars-coronavirus mrna cap methylation biochemical and structural insights into the mechanisms of sars coronavirus rna ribose 2'-omethylation by nsp16/nsp10 protein complex ribose 2'-omethylation provides a molecular signature for the distinction of self and non-self mrna dependent on the rna sensor mda5 severe acute respiratory syndrome coronavirus evades antiviral signaling: role of nsp1 and rational design of an attenuated strain a two-pronged strategy to suppress host protein synthesis by sars coronavirus nsp1 protein sars coronavirus nsp1 protein induces template-dependent endonucleolytic cleavage of mrnas: viral mrnas are resistant to nsp1-induced rna cleavage regulation of irf-3-dependent innate immunity by the papain-like protease domain of the severe acute respiratory syndrome coronavirus severe acute respiratory syndrome coronavirus papain-like protease ubiquitin-like domain and catalytic domain regulate antagonism of irf3 and nf-kappab signaling coronavirus papain-like proteases negatively regulate antiviral innate immune response through disruption of sting-mediated signaling the nucleocapsid protein of coronaviruses acts as a viral suppressor of rna silencing in mammalian cells severe acute respiratory syndrome coronavirus m protein inhibits type i interferon production by impeding the formation of traf3.tank.tbk1/ikkepsilon complex molecular determinants for subcellular localization of the severe acute respiratory syndrome coronavirus open reading frame 3b protein severe acute respiratory syndrome coronavirus open reading frame (orf) 3b, orf 6, and nucleocapsid proteins function as interferon antagonists pathogenesis of middle east respiratory syndrome coronavirus middle east respiratory syndrome (mers) a review of studies on animal reservoirs of the sars coronavirus middle east respiratory syndrome coronavirus (mers-cov): announcement of the coronavirus study group middle east respiratory syndrome coronavirus neutralising serum antibodies in dromedary camels: a comparative serological study evidence for camelto-human transmission of mers coronavirus middle east respiratory syndrome tropism of and innate immune responses to the novel human betacoronavirus lineage c virus in human ex vivo respiratory organ cultures active replication of middle east respiratory syndrome coronavirus and aberrant induction of inflammatory cytokines and chemokines in human macrophages: implications for pathogenesis rapid generation of a mouse model for middle east respiratory syndrome antiviral innate immune response interferes with the formation of replication-associated membrane structures induced by a positive-strand rna virus coronavirus nonstructural protein 16: evasion, attenuation, and possible treatments the structural and accessory proteins m, orf 4a, orf 4b, and orf 5 of middle east respiratory syndrome coronavirus (mers-cov) are potent interferon antagonists middle east respiratory syndrome coronavirus accessory protein 4a is a type i interferon antagonist middle east respiratory syndrome coronavirus 4a protein is a double-stranded rnabinding protein that suppresses pact-induced activation of rig-i and mda5 in the innate antiviral response middle east respiratory syndrome coronavirus orf4b protein inhibits type i interferon production through both cytoplasmic and nuclear targets crystal structure of the middle east respiratory syndrome coronavirus (mers-cov) papain-like protease bound to ubiquitin facilitates targeted disruption of deubiquitinating activity to demonstrate its role in innate immune suppression middle east respiratory syndrome coronavirus nsp1 inhibits host gene expression by selectively targeting mrnas transcribed in the nucleus while sparing mrnas of cytoplasmic origin risk factors for primary middle east respiratory syndrome coronavirus illness in humans, saudi arabia high fatality rates and associated factors in two hospital outbreaks of mers in daejeon, the republic of korea renewal of alveolar epithelium in the rat following exposure to no2 identification of genes differentially expressed in rat alveolar type i cells clinicopathologic, immunohistochemical, and ultrastructural findings of a fatal case of middle east respiratory syndrome coronavirus infection in the united arab emirates histopathology of middle east respiratory syndrome coronovirus (mers-cov) infection -clinicopathological and ultrastructural study pegylated interferon-alpha protects type 1 pneumocytes against sars coronavirus infection in macaques dpp4, the middle east respiratory syndrome coronavirus receptor, is upregulated in lungs of smokers and chronic obstructive pulmonary disease patients first case of 2019 novel coronavirus in the united states a novel coronavirus from patients with pneumonia in china genomic characterization of the 2019 novel human-pathogenic coronavirus isolated from a patient with atypical pneumonia after visiting wuhan genomic characterization and infectivity of a novel sars-like coronavirus in chinese bats a familial cluster of pneumonia associated with the 2019 novel coronavirus indicating person-to-person transmission: a study of a family cluster epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a descriptive study clinical features of patients infected with 2019 novel coronavirus in wuhan a pneumonia outbreak associated with a new coronavirus of probable bat origin epidemiological, demographic, and clinical characteristics of 47 cases of middle east respiratory syndrome coronavirus disease from saudi arabia: a descriptive study learning from the past: possible urgent prevention and treatment options for severe acute respiratory infections caused by 2019-ncov exploring the mechanism of liver enzyme abnormalities in patients with novel coronavirus-infected pneumonia 2019-ncov: new challenges from coronavirus pathological findings of covid-19 associated with acute respiratory distress syndrome lymphopenia predicts disease severity of covid-19: a descriptive and predictive study the novel coronavirus: a bird's eye view medical treatment of viral pneumonia including sars in immunocompetent adult clinical management and infection control of sars: lessons learned coronaviruses -drug discovery and therapeutic options learning from the past: possible urgent prevention and treatment options for severe acute respiratory infections caused by 2019-ncov structure-based discovery of middle east respiratory syndrome coronavirus fusion inhibitor crossneutralization of human and palm civet severe acute respiratory syndrome coronaviruses by antibodies targeting the receptor-binding domain of spike protein suppression of sars-cov entry by peptides corresponding to heptad regions on spike glycoprotein structure of the fusion core and inhibition of fusion by a heptad repeat peptide derived from the s protein of middle east respiratory syndrome coronavirus potent neutralization of mers-cov by human neutralizing monoclonal antibodies to the viral spike glycoprotein identification of human neutralizing antibodies against mers-cov and their role in virus adaptive evolution exceptionally potent neutralization of middle east respiratory syndrome coronavirus by human monoclonal antibodies middle east respiratory syndrome coronavirus infection mediated by the transmembrane serine protease tmprss2 role of the spike glycoprotein of human middle east respiratory syndrome coronavirus (mers-cov) in virus entry and syncytia formation protease inhibitors targeting coronavirus and filovirus entry genomic characterization of a newly discovered coronavirus associated with acute respiratory distress syndrome in humans targeting membranebound viral rna synthesis reveals potent inhibition of diverse coronaviruses including the middle east respiratory syndrome virus cell host response to infection with novel human coronavirus emc predicts potential antivirals and important differences with sars coronavirus delayed induction of proinflammatory cytokines and suppression of innate antiviral response by the novel middle east respiratory syndrome coronavirus: implications for pathogenesis and treatment interferon-β and mycophenolic acid are potent inhibitors of middle east respiratory syndrome coronavirus in cell-based assays antiviral potential of erk/mapk and pi3k/akt/mtor signaling modulation for middle east respiratory syndrome coronavirus infection as identified by temporal kinome analysis the sars-coronavirus-host interactome: identification of cyclophilins as target for pancoronavirus inhibitors mers-coronavirus replication induces severe in vitro cytopathology and is strongly inhibited by cyclosporin a or interferon-α treatment inhibitors of the hepatitis c virus polymerase; mode of action and resistance approved antiviral drugs over the past 50 years new nucleoside analogues for the treatment of hemorrhagic fever virus infections design, synthesis and evaluation of a series of acyclic fleximer nucleoside analogues with anti-coronavirus activity therapeutic options for the 2019 novel coronavirus (2019-ncov) t-705 (favipiravir) induces lethal mutagenesis in influenza a h1n1 viruses in vitro in vitro and in vivo activities of t-705 against arenavirus and bunyavirus infections activity of t-705 in a hamster model of yellow fever virus infection in comparison with that of a chemically related compound, t-1106 post-exposure efficacy of oral t-705 (favipiravir) against inhalational ebola virus infection in a mouse model japanese surveillance systems and treatment for influenza successful treatment of advanced ebola virus infection with t-705 (favipiravir) in a small animal model efficacy of orally administered t-705 on lethal avian influenza a (h5n1) virus infections in mice clinical and virological characteristics of ebola virus disease patients treated with favipiravir (t-705)-sierra leone remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus (2019-ncov) in vitro broad-spectrum antiviral activity of virazole: 1-beta-d-ribofuranosyl-1,2,4-triazole-3-carboxamide depletion of gtp pool is not the predominant mechanism by which ribavirin exerts its antiviral effect on lassa virus development of a standard treatment protocol for severe acute respiratory syndrome the management of coronavirus infections with particular reference to sars treatment with interferon-α2b and ribavirin improves outcome in mers-covinfected rhesus macaques ribavirin and interferon alfa-2a for severe middle east respiratory syndrome coronavirus infection: a retrospective cohort study ribavirin and interferon therapy in patients infected with the middle east respiratory syndrome coronavirus: an observational study ifn-α2a or ifn-β1a in combination with ribavirin to treat middle east respiratory syndrome coronavirus pneumonia: a retrospective study triple combination of interferon beta-1b, lopinavir-ritonavir, and ribavirin in the treatment of patients admitted to hospital with covid-19: an open-label, randomised, phase 2 trial mechanism of inhibition of ebola virus rna-dependent rna polymerase by remdesivir therapeutic efficacy of the small molecule gs-5734 against ebola virus in rhesus monkeys coronavirus susceptibility to the antiviral remdesivir (gs-5734) is mediated by the viral polymerase and the proofreading exoribonuclease. mbio broad-spectrum antiviral gs-5734 inhibits both epidemic and zoonotic coronaviruses gs-5734 and its parent nucleoside analog inhibit filo-, pneumo-, and paramyxoviruses remdesivir in adults with severe covid-19: a randomised, double-blind, placebo-controlled, multicentre trial compassionate use of remdesivir for patients with severe covid-19 remdesivir for the treatment of covid-19 -preliminary report protection against filovirus diseases by a novel broad-spectrum nucleoside analogue bcx4430 mutations in the chikungunya virus non-structural proteins cause resistance to favipiravir (t-705), a broad-spectrum antiviral the papain-like protease of severe acute respiratory syndrome coronavirus has deubiquitinating activity nidovirus papain-like proteases: multifunctional enzymes with protease, deubiquitinating and deisgylating activities mers-cov papain-like protease has deisgylating and deubiquitinating activities thiopurine analogs and mycophenolic acid synergistically inhibit the papain-like protease of middle east respiratory syndrome coronavirus identification and design of novel small molecule inhibitors against mers-cov papainlike protease via high-throughput screening and molecular modeling the sarscoronavirus papain-like protease: structure, function and inhibition by designed antiviral compounds a noncovalent class of papain-like protease/deubiquitinase inhibitors blocks sars virus replication disulfiram can inhibit mers and sars coronavirus papain-like proteases via different modes treatment of severe acute respiratory syndrome with lopinavir/ritonavir: a multicentre retrospective matched cohort study role of lopinavir/ritonavir in the treatment of sars: initial virological and clinical findings broad-spectrum antivirals for the emerging middle east respiratory syndrome coronavirus treatment with lopinavir/ritonavir or interferon-β1b improves outcome of mers-cov infection in a nonhuman primate model of common marmoset a trial of lopinavir-ritonavir in adults hospitalized with severe covid-19 2,6-bis-arylmethyloxy-5-hydroxychromones with antiviral activity against both hepatitis c virus (hcv) and sars-associated coronavirus (scv) design, synthesis and molecular docking of novel triazole derivatives as potential cov helicase inhibitors evaluation of ssya10-001 as a replication inhibitor of severe acute respiratory syndrome, mouse hepatitis, and middle east respiratory syndrome coronaviruses broad-spectrum in vitro activity and in vivo efficacy of the antiviral protein griffithsin against emerging viruses of the family coronaviridae activity of and effect of subcutaneous treatment with the broad-spectrum antiviral lectin griffithsin in two laboratory rodent models inhibition of severe acute respiratory syndrome-associated coronavirus (sars-cov) infectivity by peptides analogous to the viral spike protein peptide-based membrane fusion inhibitors targeting hcov-229e spike protein hr1 and hr2 domains protective effect of intranasal regimens containing peptidic middle east respiratory syndrome coronavirus fusion inhibitor against mers-cov infection identification of a novel inhibitor against middle east respiratory syndrome coronavirus using sirna in prophylactic and therapeutic regimens against sars coronavirus in rhesus macaque inhibition of sars-cov replication by sirna silencing sars-cov spike protein expression in cultured cells by rna interference inhibition of sars-cov replication cycle by small interference rnas silencing specific sars proteins, 7a/7b, 3a/3b and s development of interfering rna agents to inhibit sars-associated coronavirus infection and replication resveratrol suppresses nuclear factor-kappab in herpes simplex virus infected cells resveratrol decreases nitric oxide production by hepatocytes during inflammation polygonum cuspidatum and its active components inhibit replication of the influenza virus through toll-like receptor 9-induced interferon beta expression studies on trans-resveratrol/carboxymethylated (1,3/1,6)-β-dglucan association for aerosol pharmaceutical applications resveratrol attenuates inflammatory hyperalgesia by inhibiting glial activation in mice spinal cords resveratrol-mediated gamma interferon reduction prevents airway inflammation and airway hyperresponsiveness in respiratory syncytial virus-infected immunocompromised mice effective inhibition of mers-cov infection by resveratrol hexamethylene amiloride blocks e protein ion channels and inhibits coronavirus replication repurposing of clinically developed drugs for treatment of middle east respiratory syndrome coronavirus infection singlet oxygen effects on lipid membranes: implications for the mechanism of action of broad-spectrum viral fusion inhibitors treatment of sars with human interferons inhibition of novel β coronavirus replication by a combination of interferon-α2b and ribavirin ribavirin and interferon-beta synergistically inhibit sars-associated coronavirus replication in animal and human cell lines nitazoxanide: a first-in-class broad-spectrum antiviral agent effect of nitazoxanide in adults and adolescents with acute uncomplicated influenza: a double-blind, randomised, placebo-controlled, phase 2b/3 trial clinical trial: randomized, double-blind, placebo-controlled study of nitazoxanide monotherapy for the treatment of patients with chronic hepatitis c genotype 4 induction of cd8+ tcell responses against novel glioma-associated antigen peptides and clinical activity by vaccinations with {alpha}-type 1 polarized dendritic cells and polyinosinic-polycytidylic acid stabilized by lysine and carboxymethylcellulose in patients with recurrent malignant glioma a multiinstitution phase ii study of poly-iclc and radiotherapy with concurrent and adjuvant temozolomide in adults with newly diagnosed glioblastoma structure-based discovery of a novel angiotensin-converting enzyme 2 inhibitor identification of critical determinants on ace2 for sars-cov entry and development of a potent entry inhibitor inhibition of middle east respiratory syndrome coronavirus infection by anti-cd26 monoclonal antibody a cysteine protease inhibitor rescues mice from a lethal cryptosporidium parvum infection cure of hookworm infection with a cysteine protease inhibitor efficacy of camostat mesilate against dyspepsia associated with non-alcoholic mild pancreatic disease pancreatic stellate cells: new target in the treatment of chronic pancreatitis screening of an fda-approved compound library identifies four small-molecule inhibitors of middle east respiratory syndrome coronavirus replication in cell culture atp1a1-mediated src signaling inhibits coronavirus entry into host cells anti-malaria drug chloroquine is highly effective in treating avian influenza a h5n1 virus infection in an animal model in vitro inhibition of severe acute respiratory syndrome coronavirus by chloroquine inhibition of human coronavirus 229e infection in human epithelial lung cells (l132) by chloroquine: involvement of p38 mapk and erk a systematic screen of fda-approved drugs for inhibitors of biological threat agents effects of chloroquine on viral infections: an old drug against today's diseases? chloroquine is a potent inhibitor of sars coronavirus infection and spread effect of high vs low doses of chloroquine diphosphate as adjunctive therapy for patients hospitalized with severe acute respiratory syndrome coronavirus 2 (sars-cov-2) infection: a randomized clinical trial hydroxychloroquine in patients with mainly mild to moderate coronavirus disease 2019: open label, randomised controlled trial hydroxychloroquine and azithromycin as a treatment of covid-19: results of an openlabel non-randomized clinical trial clinical efficacy of hydroxychloroquine in patients with covid-19 pneumonia who require oxygen: observational comparative study using routine care data observational study of hydroxychloroquine in hospitalized patients with covid-19 suppression of coronavirus replication by cyclophilin inhibitors human coronavirus nl63 replication is cyclophilin a-dependent and inhibited by non-immunosuppressive cyclosporine a-derivatives including alisporivir porcine epidemic diarrhea virus induces caspaseindependent apoptosis through activation of mitochondrial apoptosis-inducing factor alisporivir inhibits mers-and sars-coronavirus replication in cell culture, but not sars-coronavirus infection in a mouse model effect of interferon alpha and cyclosporine treatment separately and in combination on middle east respiratory syndrome coronavirus (mers-cov) replication in a human in-vitro and ex-vivo culture model abelson kinase inhibitors are potent inhibitors of severe acute respiratory syndrome coronavirus and middle east respiratory syndrome coronavirus fusion factors associated with psychosis among patients with severe acute respiratory syndrome: a case-control study corticosteroid therapy for critically ill patients with middle east respiratory syndrome dexamethasone in the management of covid -19 covid-19: demand for dexamethasone surges as recovery trial publishes preprint risk factors associated with acute respiratory distress syndrome and death in patients with coronavirus disease 2019 pneumonia in wuhan a retrospective cohort study of methylprednisolone therapy in severe patients with covid-19 pneumonia identification of immunodominant sites on the spike protein of severe acute respiratory syndrome (sars) coronavirus: implication for developing sars diagnostics and vaccines virological and serological analysis of a recent middle east respiratory syndrome coronavirus infection case on a triple combination antiviral regimen experience of using convalescent plasma for severe acute respiratory syndrome among healthcare workers in a taiwan hospital the use of lassa fever convalescent plasma in nigeria treatment of ebola hemorrhagic fever with blood transfusions from convalescent patients. international scientific and technical committee treatment of argentine hemorrhagic fever with convalescent's plasma. 4433 cases bolivian hemorrhagic fever. a report of four cases retrospective comparison of convalescent plasma with continuing high-dose methylprednisolone treatment in sars patients use of convalescent plasma therapy in sars patients in hong kong middle east respiratory syndrome virus pathogenesis neutralizing human monoclonal antibodies to severe acute respiratory syndrome coronavirus: target, mechanism of action, and therapeutic potential prophylactic and postexposure efficacy of a potent human monoclonal antibody against mers coronavirus a conformation-dependent neutralizing monoclonal antibody specifically targeting receptorbinding domain in middle east respiratory syndrome coronavirus spike protein a humanized neutralizing antibody against mers-cov targeting the receptor-binding domain of the spike protein pre-and postexposure efficacy of fully human antibodies against spike protein in a novel humanized mouse model of mers-cov infection a decade after sars: strategies for controlling emerging coronaviruses sars vaccines: where are we? inactivated sars-cov vaccine elicits high titers of spike protein-specific antibodies that block receptor binding and virus entry a subcutaneously injected uv-inactivated sars coronavirus vaccine elicits systemic humoral immunity in mice vaccines to prevent severe acute respiratory syndrome coronavirus-induced disease escape from human monoclonal antibody neutralization affects in vitro and in vivo fitness of severe acute respiratory syndrome coronavirus presence of middle east respiratory syndrome coronavirus antibodies in saudi arabia: a nationwide, cross-sectional, serological study reversal of the progression of fatal coronavirus infection in cats by a broad-spectrum coronavirus protease inhibitor structure-guided design of potent and permeable inhibitors of mers coronavirus 3cl protease that utilize a piperidine moiety as a novel design element synthesis and evaluation of phenylisoserine derivatives for the sars-cov 3cl protease inhibitor identification, synthesis and evaluation of sars-cov and mers-cov 3c-like protease inhibitors engineering a novel antibodypeptide bispecific fusion protein against mers-cov. antibodies (basel) identification of a peptide derived from the heptad repeat 2 region of the porcine epidemic diarrhea virus (pedv) spike glycoprotein that is capable of suppressing pedv entry and inducing neutralizing antibodies a novel peptide with potent and broad-spectrum antiviral activities against multiple respiratory viruses antiviral activity of k22 against members of the order nidovirales purified coronavirus spike protein nanoparticles induce coronavirus neutralizing antibodies in mice key: cord-293897-p30wz7b7 authors: waghmare, alpana; englund, janet a. title: respiratory viruses date: 2020-04-24 journal: pediatric transplant and oncology infectious diseases doi: 10.1016/b978-0-323-64198-2.00030-0 sha: doc_id: 293897 cord_uid: p30wz7b7 respiratory viruses are commonly detected in both healthy and immunocompromised children. in most healthy children, respiratory viruses are associated with self-limited upper respiratory tract infections and are not accompanied by significant morbidity. in immunocompromised hosts, including hematopoietic cell transplant recipients, solid organ transplant recipients, and oncology patients, respiratory viruses can be associated with significant clinical manifestations, including prolonged viral shedding, lower respiratory tract disease, the need for supplemental oxygen, late airflow obstruction, and even death. this chapter reviews the major respiratory viruses, including respiratory syncytial virus, human metapneumovirus, influenza, parainfluenza viruses, human rhinoviruses, and human coronaviruses. other viruses can manifest as pulmonary infection; however, these viruses are discussed elsewhere (see chapter 17 for discussion of cytomegalovirus and chapter 22 for discussion of adenoviruses). hct recipients. in a surveillance study of pediatric and adult hct recipients in the first year after transplant, the most common viruses detected were hrv and hcov, followed by piv, adenovirus, rsv, influenza, hmpv, and human bocavirus. 1 in a separate multicenter retrospective study of pediatric hct recipients, 16.6% of patient had at least one respiratory virus detected by pcr in the first year after hct 2 ; younger age was associated with viral detection in univariate analysis. steroid exposure, neutropenia, and lymphopenia were commonly present in the week before respiratory viral onset. in a large multicenter retrospective study of pediatric sot recipients, the highest rates of inpatient respiratory virus infection occurred in intestine/abdominal multivisceral transplant recipients, followed by thoracic (heart/lung), liver, and kidney transplants. 3 hrv was the most common detected virus (45% of respiratory virus events), followed by rsv (22%), piv (16%), hmpv (11%), and influenza (10%). lymphopenia was present in 22% of patients with respiratory virus detected, although this was not evaluated as a risk factor for acquisition. oncology patients. in a large cohort of pediatric cancer patients with fever and neutropenia, at least one respiratory virus was detected in 46% of subjects. 4 the most common respiratory viruses detected were hrv, rsv, piv, influenza, adenovirus, and hmpv. in healthy individuals, most respiratory viral infections are associated with self-limited upper respiratory tract symptoms. notable exceptions include a stronger association between rsv and bronchiolitis in young infants, piv and laryngotracheobronchitis, and hrvs and reactive airway disease exacerbations. in immunocompromised patients, respiratory viral infections can be associated with prolonged shedding, lower respiratory tract disease, the need for supplemental oxygen, late airflow obstruction, and even death. prolonged viral shedding can be associated with persistent respiratory symptoms or can be asymptomatic with durations up to 4 weeks (mean). 5 persistent shedding of piv in asymptomatic immunocompromised patients for many months has been noted using sensitive molecular detection methods, 6 and prolonged shedding has been described for hmpv, hcovs, and hrvs. [7] [8] [9] impairment of t-cell function appears to be commonly associated in children with prolonged shedding; additional risk factors in hct recipients include initial high viral load, use of steroids, and myeloablative conditioning. 9 initial clinical symptoms related to rsv infection in immunocompromised hosts are similar to those in immunocompetent persons. upper respiratory tract infection may progress to lower respiratory tract disease, with likelihood of progression probably related to immune status. over 1 to 2 weeks, lower respiratory tract involvement may become evident by increasing respiratory distress, worsening hypoxia, and potentially, the need for assisted ventilation. children or adults who acquire rsv or hmpv during or shortly after chemotherapy for malignancy may also have severe, life-threatening disease. 10 recovery after assisted ventilation for rsv and hmpv pneumonia occurs but remains uncommon despite advances in supportive care. the associated respiratory failure in severely immunocompromised patients may result in multiorgan system failure, with high mortality rates in patients requiring mechanical ventilation. 11, 12 severe and fatal infection attributed to hmpv has been reported in cancer patients, and hmpv is a relatively common cause of acute respiratory infection in children and adults with malignancy, hct recipients or organ transplant recipients. 13 risk factors for severe hmpv infection include lymphopenia. 7,10,13 quantitative bronchoalveolar lavage (bal) viral load has not been associated with mechanical ventilation or death for rsv, piv, or hmpv in adult patients after hct; however, the detection of respiratory virus rna in serum has been associated with fatal outcomes. 11 piv infection with lower respiratory tract disease in immunosuppressed patients, particularly those in the immediate posttransplantation period, may result in a similar clinical picture, although the overall mortality rate may be less than that associated with rsv. 14,15 many immunocompromised adults with piv infection first present with symptoms of mild upper respiratory tract disease, but in contrast to rsv, influenza, and hmpv, detection of piv-1 and piv-3 in asymptomatic hct recipients is relatively common, reported in 6 of 17 (35%) infection episodes in a prospective study. 6 fewer than half of piv-infected patients have a fever. in severely immunocompromised patients, such as allogeneic hct recipients less than 100 days after transplant, piv-3 is the most common piv subtype detected, reported in 80% of 544 hct recipients with piv. 16 in all piv-infected transplant recipients, infection may progress to lower respiratory tract disease with more serious disease linked to supplemental oxygen requirement, low monocyte counts, and high-dose steroid use. the detection of piv in bal or other lower respiratory tract specimens is associated with decreased survival overall in hct recipients. 16 higher pretransplant piv-3 antibody levels were not protective against severe sequelae. 15 concomitant infections with other viruses, fungi, or severe graft-versus-host disease are relatively common in adult patients with piv pneumonitis. influenza remains a significant cause of morbidity and mortality in immunocompromised children and adults. in a recent multicenter review of immunocompromised children hospitalized with influenza, immunocompromised children were more likely to present with fever and less likely to present with respiratory distress. they were also less likely to require intensive care but were hospitalized longer. 17 in a study of adult and pediatric hct and sot recipients, 22.1% of subjects presented with pneumonia and 66.5% were hospitalized. prior vaccination was associated with decreased disease severity. 18 mortality after influenza was 2.9% in adults and 0% in pediatric patients in this study, although higher mortality rates have been previously reported. with the increased use of molecular diagnostic assays, hrvs and hcovs are now the most common respiratory viruses detected in immunocompromised patients. hrv can be detected in 20% to 30% of hct recipients, with progression to lower respiratory tract infection occurring in 17% of patients. 1, 19 once hrv progresses, mortality rates can be similar to other respiratory viruses, including influenza, piv, and rsv. 20 in addition to hrvs, other members of the enterovirus genus, including enterovirus-d68, can be associated with outbreaks and severe disease. enterovirus d68 was associated with several hundred cases of severe respiratory illnesses in children in the united states in 2014 and 2018; severe disease was also seen in immunocompromised patients. 21 hcovs can also be associated with severe disease in the lower respiratory tract and can result in death. 22 specific considerations for hct, sot, and oncology patients are outlined in the following text. hct recipients of all ages may have a more fulminant course after respiratory viral infections, particularly if infection occurs around the time of transplantation. risk factors for disease progression include lack of engraftment, decreased lymphocyte count, and older age. evidence of pulmonary infiltrates on chest radiographs may be delayed or absent in patients with severe neutropenia but may become apparent after immune reconstitution or on chest computed tomography or magnetic resonance imaging. in a multicenter retrospective review of pediatric hct recipients, the all-cause and attributable case-fatality rates within 3 months of hospitalized respiratory viral infection onset were 11% and 5.4%, respectively. 2 lower respiratory tract infection was rare except in hmpv infections; fever was more common for influenza and hmpv. multivariate models indicated that onset within 60 days of hct, steroid use in the 7 days before onset, and the need for respiratory support at onset were associated with subsequent morbidity or death. several risk scores have been proposed for adult hct recipients with respiratory virus infections to predict the risk of progression to lower respiratory tract infection and mortality. 19 sot recipients. in a large multicenter retrospective review, fever was the most common clinical sign detected, although a significant proportion had signs of lower respiratory tract infection at onset (35%). 3 detection of any respiratory viral infection in sot recipients was associated with an all-cause and attributable case-fatality rate of 4% and 0%, respectively. receipt of an abdominal/intestinal multivisceral transplant was associated with increased risk of all-cause death in multivariable models. overall, approximately 50% of patients with respiratory viral infections required some form of respiratory support. oncology patients. adults with leukemia and profound chemotherapyinduced myelosuppression are also at risk of fatal outcome from respiratory viruses. in pediatric patients with fever and neutropenia, episodes caused by different types of respiratory viruses had no differences in the clinical outcome (days of hospitalization, days of fever, oxygen requirement, admission to the intensive care unit, and death) and when comparing patients with a single virus versus coinfection. 4 the mainstay of influenza prevention in immunocompromised children is vaccination. inactivated influenza vaccine (iiv) is recommended for all patients 6 months and older with hematologic malignancies by the infectious diseases society of america (idsa) guidelines. vaccination is not recommended for in patients receiving intensive chemotherapy, such as induction or consolidation chemotherapy for acute leukemia, or those who have received anti-b-cell antibodies in the past 6 months. 23 live attenuated influenza vaccine (laiv) should not be used for immunosuppressed patients except in an outbreak setting when laiv is determined to be a more effective option owing to strain type. quadrivalent vaccine should be offered when available. studies are ongoing to assess the immunogenicity and safety of high-dose influenza vaccination in immunocompromised adults and children. influenza vaccination is recommended for all family members, close contacts, and health care workers caring for immunocompromised patients. laiv is not recommended for household contacts of hct recipients (within 2 months after transplant), or those with severe immunosuppression as the result of graft-versus-host disease or severe combined immune deficiency. 23 if laiv is given to persons caring for severely immunosuppressed patients, the centers for disease control and prevention guidelines recommend avoidance of contact for 7 days after vaccination. 24 hct recipients. immune responses to influenza vaccination are likely more effective further out from hct. hct recipients 6 months or older should receive iiv annually starting 6 months after transplant or starting 4 months after transplant during a community outbreak of influenza, 23 with second doses recommended for children depending on time from transplant. in reduced intensity hct, pretransplant vaccination may be appropriate as host immunity is expected to extend into the early transplant period. high-dose vaccination is currently under study in pediatric and adult hct recipients. sot recipients. idsa guidelines recommend against administering influenza vaccine during intensified immunosuppression, including the first 2-month posttransplant period, because of the likelihood of inadequate response. however, during a community outbreak, iiv can be administered 1 month or later after transplant. 23 earlier vaccination has been supported by recent studies, and more data regarding high-dose vaccine and repeat vaccine dosing within a season are also becoming available. 25 oncology patients. iiv is recommended for all patients older than postexposure chemoprophylaxis should be considered in immunosuppressed patients who are in close contact with confirmed influenza cases or during influenza outbreaks. 27 in outbreak settings, hct recipients should receive vaccine immediately if they are 4 months or longer posttransplant. chemoprophylaxis with oseltamivir or zanamivir should also be initiated for 2 weeks after vaccination while immunity develops. 27 chemoprophylaxis is also recommended for hct recipients who are less than 24 months posttransplant or those who remain substantially immunocompromised at 24 months or longer after hct, regardless of vaccination history. for sot, similar concerns regarding vaccine response in the early posttransplant period exist and chemoprophylaxis should be considered, although recommendations regarding the timing of this strategy are lacking. 28 season-long prophylaxis has been evaluated in a randomized trial of sot and hct and showed reduction in culture-or polymerase chain reaction (pcr)confirmed influenza, although this approach is not universally used and may be targeted to periods of high influenza virus circulation. 29 antiviral resistance may emerge with widespread prophylaxis; drug resistance patterns of circulating strains should be considered when initiating prophylaxis and/or preemptive therapy. new advances in the understanding of rsv and hmpv, including the characterization of the rsv fusion (f) protein in its preinfusion and postfusion states, have contributed to the development of new rsv vaccines and monoclonal antibodies with the potential to be used for prophylaxis against rsv. vaccines stimulating antibody directed to the f protein, and specifically to the pre-f protein, are now under development. rsv-specific serum-neutralizing antibodies are efficiently transferred from the mother to the newborn, and ongoing clinical trials may offer potential protection against rsv in family members and health care workers surrounding immunocompromised patients, with the potential to protect these patients. candidate vaccines against piv and hmpv, as well as chimeric vaccines containing genes from more than one virus, have also been tested in preclinical models. the identification of serum-neutralizing antibody as a correlate of protection against serious rsv lower respiratory tract disease has been an important advance. although a humanized monoclonal antibody directed against the rsv f protein, palivizumab, has been licensed since 1998 to prevent rsv disease in young children with underlying cardiac or pulmonary disease, it is also currently used in children who are severely immunocompromised, such as infants with severe combined immune deficiency or congenital leukemia. children younger than 24 months who are profoundly immunocompromised may be considered for palivizumab prophylaxis. the development of a potent and long-acting rsv monoclonal antibody directed against the pre-f protein of rsv is undergoing clinical trials in young infants. this, or similar products, may be available in the future to prevent rsv infection or disease in transplant candidates. passive immunoprophylaxis against piv and hmpv infection has not been studied in children. the diagnosis of respiratory viral infections is critically dependent on the type and quality of the clinical specimen and proper handling of the specimen. the preferred specimens for the diagnosis of respiratory viruses in immunocompromised hosts are respiratory secretions obtained as a mid-turbinate or nasopharyngeal swab, nasal wash, nasal aspirate, or bronchoalveolar lavage. a nasal wash specimen is the classic sample used for viral diagnosis by culture and for obtaining samples to assess cytokines or antibodies. nasopharyngeal swabs and mid-turbinate swabs are not as sensitive as nasal washes for viral diagnosis using culture but have good diagnostic yields with molecular detection methods. in general, molecular methods of detection are preferred over fluorescent antigen-antibody detection or antigen detection tests in immunocompromised patients. specimens from children are often more readily diagnosed than those from adults owing to higher viral loads. other clinical specimens useful for the detection of respiratory viruses in patients of all ages include endotracheal aspirates collected from intubated patients, bronchoalveolar lavage, nasal mucosal epithelium collected by scraping, sputum, and lung tissue obtained by biopsy or at autopsy. molecular diagnostics, such as multiplex real-time pcr, have replaced cell culture, fluorescent antigen-antibody detection, and antigen detection methodologies because they have high sensitivity and specificity, excellent quality control procedures, and detect a large battery of viral and bacterial pathogens rapidly in a single sample. the use of molecular diagnostics has significantly improved the yield of detection. numerous rapid multiplex pcr assays are now commercially available that can detect up to 18 or more viruses simultaneously. these assays are sensitive and specific and can detect rsv, hmpv, influenza, hrvs, and hcovs and differentiate all four piv subtypes. the potential hypoxemia, apnea, and poor oral intake resulting from infection in young infants, both previously healthy as well as moderately or severely immunocompromised, requires close medical management. hospitalization may be required for immunocompromised children younger than 1 year, particularly if intravenous (iv) fluid replacement and oxygen therapy are necessary. systemic or inhaled steroids and bronchodilator therapies are not recommended for treatment of rsv, based on studies that failed to show decreased time in the hospital or improved outcomes. 30 children with airway obstruction or signs of hypoxia require admission to an intensive care setting for close monitoring, and children with severe disease may require intubation. supportive care for lower respiratory tract infections caused by piv and hmpv in immunocompromised hosts similarly may require hospitalization and adjunct therapy, including iv fluids and oxygen support, as well as aggressive therapy of secondary fungal, bacterial, or viral infections. respiratory syncytial virus. ribavirin, a synthetic guanosine nucleoside, has been licensed for the treatment of rsv respiratory disease in children since 1986 and for the treatment of rsv disease in patients undergoing mechanical ventilation since 1993. ribavirin is the only approved drug for lower respiratory tract disease caused by rsv, but concerns regarding efficacy, difficulties in administration, and the extremely high cost of the drug have resulted in minimal current use of the drug. 31 ribavirin is available in aerosolized, oral, and iv forms. several retrospective studies, including a pooled analysis, have shown aerosolized or oral ribavirin is protective against disease progression to lower respiratory tract infection and mortality in hct recipients; however, conclusive evidence of efficacy from randomized trials is not available. 32 in patients with leukemia, multivariate models demonstrated similar effects. 33 oral ribavirin has been retrospectively evaluated in patients receiving chemotherapy or in hct recipients, suggesting a possible effect of oral ribavirin; however, lack of randomized controls has been a limitation in all studies. 34 in adult hct and hematologic malignancy patients with rsv lower respiratory tract infection, use of aerosolized ribavirin was associated with decreased mortality. oral or iv ribavirin although not statistically significant as the effect was smaller. 11, 12 of note, some data suggest pediatric hct recipients with rsv have little morbidity and mortality even without ribavirin therapy. 35 current international guidelines recommend aerosolized or systemic (oral or iv) ribavirin with intravenous immunoglogulin (ivig) in patients with rsv upper respiratory tract infection undergoing allogeneic hct, allogeneic hct recipients with risk factors for progression to lower respiratory tract infection, and allogeneic hct patients with lower respiratory tract infection. 36 recommendations for ribavirin administration for hct and sot recipients, and oncology patients are outlined in table 21 .1. most data and guidance for use of ribavirin are in hct and oncology; few studies have clearly demonstrated efficacy in sot recipients, and most data are in lung transplant when both oral and iv ribavirin have been used, 37 for hct and oncology patients, high-risk situations include patients with lymphopenia; additional risk factors, such as smoking history and use of high-dose total body irradiation, could be used to further risk-stratify patients. in general, aerosolized ribavirin is reserved for subjects with virologically confirmed (bal positive for rsv) lower respiratory tract infection. it is administered by small-particle aerosol from a solution containing the drug at a concentration of 20 mg/ml sterile water via aerosol for 2 to 20 hours/day, or at a concentration of 60 mg/ml water over 2 hours 3 times daily. aerosol administration results in high levels of ribavirin in the secretions, with levels exceeding 1000 mm and little systemic absorption. the potential environmental release of ribavirin has caused concern in hospital personnel because of the potential teratogenicity of ribavirin and thus exposure is contraindicated in pregnant women because of its teratogenic potential. administration of ribavirin via a ventilator, using a high-dose, short-duration method of drug delivery or with a vacuum-exhausted treatment hood, results in minimal or no detectable ribavirin in the rooms of treated children. oral ribavirin can be considered in patients weighing more than 15kg; hemolytic anemia is the most important side effect to consider, and patients should be monitored carefully. systemic antibody therapy, combined antibody therapy with ribavirin, and aerosolized antibodies have been used to treat rsv disease. the combination of high-titer rsv immunoglobulin and ribavirin has been associated with therapeutic success in uncontrolled studies in severely immunocompromised adults with rsv disease but has not been confirmed in others. a small, nonrandomized unadjusted analysis in pediatric cancer patients with rsv lower respiratory tract infection suggested a beneficial effect of adjunctive palivizumab or ivig. 38 larger studies of hct recipients with rsv lower respiratory tract infection were unable to demonstrate improved outcomes with adjunctive palivizumab 11, 12 ; palivizumab is not currently recommended for treatment of rsv infection in any immunocompromised patients. the duration of ribavirin therapy in immunocompromised hosts is generally at least 3 days. initiation of antiviral therapy at the stage of upper respiratory tract disease may decrease viral load and possibly reduce the risk of respiratory failure, although most data are based on retrospective uncontrolled data. early influenza treatment is recommended for all immunocompromised individuals, although there may be benefit even with delayed treatment. 39 m2 inhibitors (amantadine and rimantadine) are currently ineffective, and neuraminidase inhibitors (nais) are now first-line therapy for prophylaxis and treatment of influenza. nais available in the united states include oral oseltamivir, inhaled zanamivir, iv peramivir, and baloxavir. clinical efficacy with oseltamivir and inhaled zanamivir has been demonstrated in patients with leukemia or hct recipients. 18 many mutations causing oseltamivir and peramivir resistance, including the common h275y mutation in a(h1n1)pdm09 influenza, do not confer resistance to zanamivir 40 and inhaled zanamivir may be used to treat these strains. iv peramivir used during the 2009 pandemic in severely ill patients was well tolerated, with evidence of recovery in most patients. 41 in a randomized trial comparing iv peramivir to oral oseltamivir in hospitalized adults, clinical outcomes were similar. 42 the optimal duration of peramivir therapy has not been determined for immunocompromised hosts. longer treatment courses (10 days), although not higher doses, with oseltamivir or zanamivir have been suggested, given the potential for recurrence and the median time for progression to lower respiratory tract infection. baloxavir was recently approved for treatment of uncomplicated influenza in adults and adolescents 43 ; studies in younger children and immunocompromised hosts are underway. multidrug-resistant influenza strains have been identified in immunocompromised patients. triple-combination antiviral therapy with amantadine, ribavirin, and oseltamivir has been proposed to treat immunocompromised patients with severe influenza a with resistance to oseltamivir. a randomized trial comparing triple-combination antiviral therapy with oseltamivir alone in high-risk adults demonstrated an effect on viral outcomes but no clinical benefit. 44 parainfluenza. in a retrospective study of patients with leukemia or hct recipients with piv infection, ribavirin had no impact on viral shedding, symptom and hospitalization length, progression to lower respiratory tract infection, or mortality. 45 a systematic review evaluated aerosolized or systemic ribavirin in 10 retrospective studies in this same population and found no difference in piv-associated mortality or in progression to lower respiratory tract infection. 46 given lack of evidence of clinical efficacy, ribavirin is not recommended for piv infections. the impact of ivig alone remains to be determined, although ivig administration for piv lower respiratory tract infection did not reduce mortality. 16 other respiratory viruses. the management of other respiratory viral infections in immunosuppressed patients is generally supportive (table 21 .2). ribavirin has shown efficacy against hmpv in vitro and in mouse models, with anecdotal reports describing its use in severe infection in immunocompromised hosts (with and without ivig). lack of controlled studies of ribavirin and the known toxicities of therapy, including hemolytic anemia, limit its recommendation for hmpv. currently, no approved antiviral agents exist for treatment of hcov, hrv, hbov, or enteroviruses, although some investigational agents are upcoming. the use of ivig has not been evaluated prospectively and is not routinely recommended for treatment of respiratory viral infections. some centers routinely check and replete immunoglobulins, but the impact on respiratory virus acquisition and severity is not known. new rsv antiviral candidates have been investigated, including presatovir (gilead sciences , foster city, ca), an oral rsv fusion inhibitor, and lumicitabine (alios/janssen, titusville, nj), an rsv nucleoside analog acting on the rsv viral polymerase. both antivirals have been tested in human challenge models and have successfully demonstrated antiviral efficacy. presatovir subsequently underwent two large placebo-controlled clinical efficacy trials for the treatment of rsv in placebo-controlled studies conducted in 185 and 60 adult hsct patients with upper and lower respiratory tract infections, respectively. clinical trial endpoints of efficacy were not met for presatovir; studies with lumicitabine have also been suspended. new studies of another nucleoside analog, jnj-53718678 (janssen), are underway in young children. nanobodies, small antibody-like molecules derived from the heavy-chain variable ig domains that occur naturally in camels, are being developed to be administered by inhalation for the treatment of rsv infection (ablynx nv, ghent, belgium). vaccines for rsv and piv are under development, as discussed earlier. a new potential antiviral therapy for piv and other sialic acidbinding viruses targets the lung epithelial sialic acid receptor for piv, thereby preventing viral entry. a novel recombinant sialidase fusion protein, das181 (ansun biopharma, san diego, ca), first developed as an antiviral agent for influenza, functions by cleaving sialic acid from the host cell surface, thereby inactivating the host cell receptor recognized by piv. successful use of this agent in pediatric and adult transplant recipients under compassionate use has been reported, and clinical trials in immunocompromised subjects are ongoing. several studies of novel influenza therapeutics are under investigation, including the nais iv zanamivir and laninamivir, monoclonal antibodies, viral polymerase inhibitors, and nitazoxanide. the performance and safety of these newer agents remain to be seen in immunocompromised children. rsv, pivs, and hmpvs are common causes of nosocomial infections contributing to significant morbidity and mortality in pediatric wards. nosocomial rsv and piv infections in adults with leukemia and bone marrow transplants are associated with a high mortality rate. 5, 14 the transmission of identical strains of rsv from the outpatient setting into the hospital has been documented, demonstrating the importance of infection control measures. nosocomial transmission of hmpv also occurs, with outbreaks in both inpatient and outpatient units. in one study, 15 patients were diagnosed with hmpv within 7 weeks in a tertiary care cancer unit. 7 molecular subtyping revealed infection with genotype a2a virus, implicating nosocomial transmission. four patients (26.6%) died of hmpv-associated pneumonia and consequent multiorgan failure. nosocomial outbreaks characteristically occur from multiple introductions of community respiratory viral strains as well as patient-topatient spread (perhaps with health care providers as intermediaries). for prevention of nosocomial transmission, contact isolation precautions are effective, provided that compliance with the policy is maintained among personnel. hospital personnel may play a role in the transmission of rsv and other respiratory viruses to susceptible patients. viral spreading can be limited by adherence to strict handwashing procedures and cohorting of infected and exposed individuals. use of gloves, masks, and goggles in the hospital setting also limits spread. such strict measures are appropriate in high-risk settings such as pediatric intensive care units or bone marrow transplant wards. restriction of visitors, including young children, in hospital wards with patients at high risk for rsv infection may be necessary during epidemic periods in the community. continued compliance through the respiratory virus season by all members of the health care team is critical. the importance of isolation based on symptoms as opposed to positive viral test results has been shown, but prolonged shedding of respiratory viruses with even minimal symptoms may complicate efforts of infection control. patients known or suspected to be infected with influenza, rsv, piv, or hmpv should be kept in contact isolation or cohorted until symptoms have resolved and repeated sensitive diagnostic test results are negative. 27 piv outbreaks may be difficult to bring under control owing to prolonged asymptomatic shedding, particularly in young children and immunocompromised hosts. 6 international guidelines recommend deferral of conditioning therapy in patients with respiratory infections prior to allogeneic hct, 36, 47 with low strength of evidence. the concern for progressive illness from respiratory viral infection needs to be balanced against concern for underlying disease progression and donor availability. in a large prospective study, respiratory viruses were detected in 116 of 458 hct patients before transplantation, and viral detection was associated with prolonged hospitalization and lower survival at day 100. 48 this risk was also present in patients with hrv alone. a recent retrospective review of hrv detected in pediatric hct recipients demonstrated that hrv detection without the presence of lower respiratory tract infection was not associated with decreased days alive and out of the hospital, suggesting the hct delay is not always warranted. 49 these data should ideally be validated in larger, multicenter studies. the impact of other factors such as viral-specific risk scores, location of infection and viral load, need to be evaluated. in pediatric patients, diagnostic pcr should be considered on all transplant candidates, regardless of symptoms, and transplant delay should be considered, if feasible, with ongoing symptoms or evidence of lower respiratory tract infection. for sot candidates, there are limited data examining clinical outcomes for pretransplant respiratory viral infections and often organ availability is a significant consideration on proceeding with transplantation. specific clinical circumstances, such as duration of infection and severity of symptoms, should be considered. donor transmission of influenza is of particular concern in lung or small bowel transplantation; however, non-lung and non-small bowel organs can be considered after donor antiviral treatment for 48 hours coupled with treatment of the recipient. 50 no clear guidance exists for other respiratory viruses, and decisions regarding organ use from infected donors should be evaluated on a case-by-case basis. keywords: hematopoietic cell transplant, immunocompromised, oncology, respiratory virus, solid organ transplant, viral pneumonia abstract: respiratory viruses are commonly detected in both healthy and immunocompromised children. in most healthy children, respiratory viruses are associated with self-limited upper respiratory tract infections and are not accompanied by significant morbidity. in immunocompromised hosts, including hematopoietic cell transplant recipients, solid organ transplant recipients, and oncology patients, respiratory viruses can be associated with significant clinical manifestations, including prolonged viral shedding, lower respiratory tract disease, the need for supplemental oxygen, late airflow obstruction, and even death. this chapter reviews the major respiratory viruses, including respiratory syncytial virus, human metapneumovirus, influenza, parainfluenza viruses, human rhinoviruses, and human coronaviruses. other viruses can manifest as pulmonary infection; however, these viruses are discussed elsewhere (see chapter 17 for discussion of cytomegalovirus and chapter 22 for discussion of adenoviruses). progression, shedding patterns, and clinical disease associated with respiratory virus infections after allogeneic hematopoietic cell transplantation a multicenter consortium to define the epidemiology and outcomes of inpatient respiratory viral infections in pediatric hematopoietic stem cell transplant recipients a multicenter consortium to define the epidemiology and outcomes of pediatric solid organ transplant recipients with inpatient respiratory virus infection respiratory viral infections and coinfections in children with cancer, fever and neutropenia: clinical outcome of infections caused by different respiratory viruses prolonged outbreak of human parainfluenza virus 3 infection in a stem cell transplant outpatient department: insights from molecular epidemiologic analysis respiratory virus infection among hematopoietic cell transplant recipients: evidence for asymptomatic parainfluenza virus infection respiratory tract infections due to human metapneumovirus in immunocompromised children prolonged shedding of human coronavirus in hematopoietic cell transplant recipients: risk factors and viral genome evolution initial high viral load is associated with prolonged shedding of human rhinovirus in allogeneic hematopoietic cell transplant recipients brief communication: fatal human metapneumovirus infection in stem-cell transplant recipients respiratory syncytial virus lower respiratory disease in hematopoietic cell transplant recipients: viral rna detection in blood, antiviral treatment, and clinical outcomes outcome of respiratory syncytial virus lower respiratory tract disease in hematopoietic cell transplant recipients receiving aerosolized ribavirin: significance of stem cell source and oxygen requirement a prospective study comparing human metapneumovirus with other respiratory viruses in adults with hematologic malignancies and respiratory tract infections respiratory syncytial virus pneumonia in hospitalized adult patients with leukemia parainfluenza virus type 3 ab in allogeneic hematopoietic cell transplant recipients: factors influencing post-transplant ab titers and associated outcomes parainfluenza virus lower respiratory tract disease after hematopoietic cell transplant: viral detection in the lung predicts outcome clinical features and outcomes of immunocompromised children hospitalized with laboratory-confirmed influenza in the united states a 5-year prospective multicenter evaluation of influenza infection in transplant recipients human rhinovirus infections in hematopoietic cell transplant recipients: risk score for progression to lower respiratory tract infection human rhinovirus detection in the lower respiratory tract of hematopoietic cell transplant recipients: association with mortality clinical disease due to enterovirus d68 in adult hematologic malignancy patients and hematopoietic cell transplant recipients clinical significance of human coronavirus in bronchoalveolar lavage samples from hematopoietic cell transplant recipients and patients with hematologic malignancies idsa clinical practice guideline for vaccination of the immunocompromised host prevention and control of influenza with vaccines: recommendations of the advisory committee on immunization practices, united states, 2015-16 influenza season influenza vaccine strategies for solid organ transplant recipients influenza vaccination in children being treated with chemotherapy for cancer guidelines for preventing infectious complications among hematopoietic cell transplantation recipients: a global perspective ast infectious diseases community of practice. rna respiratory viruses in solid organ transplantation efficacy and safety of oral oseltamivir for influenza prophylaxis in transplant recipients clinical practice guideline: the diagnosis, management, and prevention of bronchiolitis reassessment of the indications for ribavirin therapy in respiratory syncytial virus infections management of rsv infections in adult recipients of hematopoietic stem cell transplantation characteristics and outcome of respiratory syncytial virus infection in patients with leukemia. haematologica oral versus aerosolized ribavirin for the treatment of respiratory syncytial virus infections in hematopoietic cell transplantation recipients rsv infection without ribavirin treatment in pediatric hematopoietic stem cell transplantation ecil-4): guidelines for diagnosis and treatment of human respiratory syncytial virus, parainfluenza virus, metapneumovirus, rhinovirus, and coronavirus oral ribavirin for respiratory syncytial virus infection after lung transplantation: efficacy and costefficiency respiratory syncytial virus infections in children with cancer differences in clinical outcomes after 2009 influenza a/h1n1 and seasonal influenza among hematopoietic cell transplant recipients detection and management of antiviral resistance for influenza viruses. influenza other respir viruses recipients of hematopoietic stem cell transplantation parainfluenza virus infections in hematopoietic cell transplant recipients and hematologic malignancy patients: a systematic review guidelines for preventing infectious complications among hematopoietic cell transplant recipients: a global perspective clinical outcomes associated with respiratory virus detection before allogeneic hematopoietic stem cell transplant outcome of children with rhinovirus detection prior to allogeneic hematopoietic cell transplant guidance on novel influenza a/h1n1 in solid organ transplant recipients clinical experience in adults and children treated with intravenous peramivir for 2009 influenza a (h1n1) under an emergency ind program in the united states a clinical trial of intravenous peramivir compared with oral oseltamivir for the treatment of seasonal influenza in hospitalized adults baloxavir marboxil for uncomplicated influenza in adults and adolescents oseltamivir, amantadine, and ribavirin combination antiviral therapy versus oseltamivir monotherapy for the treatment of influenza: a multicentre, double-blind, randomised phase 2 trial the characteristics and outcomes of parainfluenza virus infections in 200 patients with leukemia or key: cord-276005-ifn88mjd authors: da silva filho, luiz vicente ribeiro ferreira; zerbinati, rodrigo melim; tateno, adriana fumie; boas, lucy vilas; de almeida, marina buarque; levi, josé eduardo; drexler, jan felix; drosten, christian; pannuti, cláudio sérgio title: the differential clinical impact of human coronavirus species in children with cystic fibrosis date: 2012-08-01 journal: j infect dis doi: 10.1093/infdis/jis274 sha: doc_id: 276005 cord_uid: ifn88mjd we investigated the clinical impact of human coronaviruses (hcov) oc43, 229e, hku1 and nl63 in pediatric patients with cystic fibrosis (cf) during routine and exacerbation visits. a total of 408 nasopharyngeal aspirate samples were obtained from 103 patients over a 1-year period. samples positive for hcov were submitted for nucleotide sequencing to determine the species. nineteen samples (4.65%) were positive for hcov, of which 8 were positive for nl63, 6 for oc43, 4 for hku1, and 1 for 229e. identification of hcov was not associated with an increased rate of respiratory exacerbations, but nl63-positive patients had higher exacerbation rates than patients who were positive for other hcov species. cystic fibrosis (cf) is an autosomal inherited disease characterized by recurrent and chronic respiratory infections [1] . the role of respiratory viruses in the progression of lung disease in cf is still controversial, although infection with respiratory syncytial virus (rsv) and influenza viruses was shown to carry a significant risk for respiratory exacerbations and hospital admissions [2] . while little is known of the impact of newly described respiratory viruses on lung disease in cf patients, there is evidence that some of these viruses may be more aggressive than others. for example, we recently showed that infection with human rhinovirus c is associated with an increased risk of respiratory exacerbations [3] . human coronaviruses (hcovs) were initially described in the 1960s as the causative agents of upper respiratory tract infections (urtis). although reports of lower respiratory tract infections caused by hcovs were identified thereafter [4] , the coronaviruses were still mainly considered to be the causative agents of urtis [5] until 2002, when a new coronavirus, later named sars coronavirus, was identified during an outbreak of severe acute respiratory syndrome [6] . more recently, other coronaviruses were described, with hcov-nl63 identified in the netherlands in 2004 [7] and hcov-hku1 in hong kong in 2005 [8] . these novel hcov species were hypothesized to cause lower respiratory tract infections at a higher frequency than the previously known hcovs [9] , although recent data suggest that hcov-hku1 does not cause a significantly higher proportion of lrtis than that observed with hcov-oc43 [10] . in this study, we aimed to investigate the clinical impact of all 4 circulating hcovs (oc43, 229e, hku1, and nl63) in pediatric patients with cf who attended an outpatient clinic during routine and exacerbation visits. nasopharyngeal aspirates (npas) or nasal blow (nb) specimens for viral investigation, as well as sputum or oropharyngeal samples for microbiology cultures, were collected during scheduled or unscheduled visits on 408 occasions, with a median ( ± sd) of 4 ± 1.74 visits (range, 1-9 visits) per patient. the local ethics committee approved the study protocol, and all parents/guardians gave informed consent for their children to participate in the study. clinical and functional data were obtained at all visits. exacerbation of respiratory disease was defined as the presence of ≥2 of the following symptoms and signs: fever, increase in the amount of secretion or cough intensity, change in the sputum's aspect, worsening of dyspnea, loss of appetite, a decrease of ≥10% in forced expiratory volume in 1 second (fev1), and weight loss. total nucleic acids were extracted from nasopharyngeal samples with the qiamp viral rna mini kit (qiagen, hamburg, germany), according to the manufacturer's instructions. reverse transcription was performed with a high capacity cdna archive kit (applied biosystems, foster city, ca), using 20 µl of previously extracted rna. identification of respiratory viruses was performed by individual polymerase chain reactions (pcrs) targeting rsv; influenza viruses a and b; human parainfluenza viruses 1, 2, and 3; hcov; human metapneumovirus; adenovirus; human bocavirus; enteroviruses; and the β-actin gene, as previously described [3] . the analytical sensitivity of this assay for hcov detection, determined by quantified plasmid standards, was 100 copies per reaction. all coronavirus-positive samples were confirmed by means of 1-step reverse transcription-heminested pcr, using primers cov2a-f (cttatgggttgggattatcc) and cov2a-r (taataacagacaacgccatcatc) for the first round and the inner primers cov2a-rnest a (ccatcatcactcagaat catca) and cov2a-rnest b (ccatcatcagaaagaatca tca), which generate a 404-base pair amplicon from the rna-dependent rna polymerase (rdrp) gene. nucleotide sequencing was performed using dye terminator chemistry (applied biosystems). nucleic acid alignment was conducted on the basis of the amino acid code by use of the blosum algorithm. phylogenetic classification was performed using the neighbor-joining method, with the percentage distance substitution model and bootstrap values calculated from 1000 replicates with mega 5.0 software (www.megasoftware.net). the nucleotide sequences of the partial rdrp gene of the hcov identified in this study were submitted to genbank (accession numbers: jn251784 -jn251802). the results were expressed as medians or percentages, unless stated otherwise. categorical variables were analyzed by χ 2 or fisher exact tests. the age differences between patients with and patients without hcov or within hcov species were compared by nonparametric kruskal-wallis tests. the proportion of cases with acute respiratory exacerbation among patients infected with different hcov species was compared by χ 2 or fisher exact tests. logistic regression was used to determine whether infection with hcov in general or with one particular hcov species was independently associated with respiratory exacerbation or hospital admission. to account for correlations among samples within the same subject, binomial generalized linear models were used. all analyses were performed using spss for windows (version 18; spss, chicago, il), and the level of statistical significance was set at .05. at least 1 respiratory virus was identified in 203 of 408 samples (49.7%), with rhinovirus being the main identified agent (in 139 samples [34.1%]). hcovs were identified in 19 samples (4.65%) from 17 patients (11 males and 6 females). as shown in figure 1 , 8 of the viruses were nl63, 6 were oc43, 4 were hku1, and 1 was 229e. two patients had 2 separate identifications of hcov in samples collected 2 and 6 months apart, respectively, and different species were identified on each occasion. single hcov infections were found on 13 occasions, while coinfections were observed on 6 occasions, four with the identification of 2 viruses (rhinovirus or enterovirus) and two with 3 viruses identified (rsv + rhinovirus and influenza a virus + enterovirus). overall, 142 episodes (34.8%) of respiratory exacerbations were identified, and hospital admissions occurred in 31 (7.6%) of them. significant associations were found for an increased risk of respiratory exacerbation when rhinovirus c was identified and an increased risk of hospital admission with influenza virus infections (data not shown). no significant difference was found among patients with hcov infection, with exacerbations identified on 6 of 19 occasions (31.5%) and hospital admissions on 2 of 19 occasions (10.5%). the hcov species that were found are displayed in table 1 , as are patient characteristics, including the frequency of symptoms and the clinical status at the time of the visit. the age distribution was similar among patients infected with different hcov species, although a significant difference was found among the ages of patients without viral infections and those infected with hcov or other viruses (table 1) . although no significant differences were seen concerning reported symptoms and clinical findings among patients infected with different hcov species, a significantly greater respiratory exacerbation rate was found among patients infected with hcov-nl63 as compared to those infected with other hcov species (p = .04). in the logistic regression model, however, neither the identification of any hcov nor hcov-nl63 specifically could be demonstrated as being significantly associated with a respiratory exacerbation. this is the first description of the identification of these new hcov species among patients with cf. while no significant clinical impact could be attributed to infection with these species as a whole, the identification of hcov-nl63 was associated with a significantly higher rate of respiratory exacerbation as compared to other hcov species. although hcovs were previously identified as causative agents of lower respiratory tract infections, the medical community commonly knew them as "common cold" viruses. the identification of new species of hcov [7, 8] and the emergence of sars hcov [6] highlighted the potential role of these viruses as causative agents of severe lower respiratory tract infections. following initial descriptions of these new "non-sars" hcov, several other studies describing the prevalence and clinical impact were published, although results were conflicting. while the prevalence values of these new hcov-nl63 and hcov-hku1 oscillate from 0.4% to 9% [5] , reports of severe cases of lower respiratory infections were described in australia for the hcov-nl63 [11] and in germany for the hcov-hku1 [12] . however, most of the studies on the clinical impact of different hcov species were only performed among children who were hospitalized for acute respiratory tract infections, with small sample sizes and short periods of sample collection [10] . more recent studies have included much larger samples. gaunt et al. [9] , who studied 11 600 respiratory samples obtained between 2006 and 2009 in edinburgh, united kingdom, found at least 1 coronavirus in 2.3% of the samples, with marked predominance in winter months. although they found a significant number of coinfections (mainly with rsv), single infections with hcov-oc43, hcov-nl63, and hcov-hku1 were specifically associated with lower respiratory tract disease. they also observed that hcov-229e, which was identified in a smaller proportion of samples, was predominant among immunosuppressed patients. talbot et al. [10] used molecular tools to determine the incidence and clinical features of upper and lower respiratory tract infections that are figure 1 . representative neighbor-joining phylogenetic tree of a partial rna-dependent rna polymerase region of coronavirus (cov), generated with a p-distance model on the basis of a 134-amino acid sequence by use of mega 5.0 software (available at: http://www.megasofware.net). reference cov species data were obtained from the genbank database. viruses identified in this study are indicated in boldface and labeled vfc. bootstrap values >70% in the key branches are depicted. scale bar indicates amino acid substitutions per site. the strain designations, genbank accession numbers, and corresponding hosts are indicated. the respective genera are given on the right. the highly conserved amino acid sequence fragment did not permit differentiation of the 2 nl63 cov clusters. associated with hcov-nl63, hcov-oc43, and hcov-229e during a 20-year period and showed that hcov-oc43 and hcov-nl63 were associated with a significant burden of lower respiratory tract infections in previously healthy outpatient children who manifested with clinical syndromes, such as pneumonia and bronchiolitis, that were similar to those caused by other respiratory viruses [10] . patients with cf represent a specific population that is susceptible to more severe clinical manifestations of respiratory infections overall, because of limitations in mucocilliary transport and anatomic distortions of the bronchial tree that facilitate the retention and infection of respiratory secretions. the impact of respiratory viruses in cf lung disease has been recently reassessed using molecular methods by some researchers, with conflicting results: while olesen et al [13] reported a lack of significant clinical impact of viral infections in lung function or respiratory symptoms, wat et al [2] described an association of viral infections with respiratory exacerbations in cf children, particularly when influenza a virus, influenza b virus, and rhinovirus infections were identified. one possible explanation for the differential clinical impact of respiratory viruses for cf patients is the baseline lung function, and it is possible that patients with worse lung disease would be at more risk to present an exacerbation; we cannot rule out this possibility in the present study, because of the small number of patients infected with hcov. another hypothesis is related to the mechanisms of disease of hcov. both sars coronavirus and hcov-nl63 use the angiotensinconverting enzyme 2 (ace2) as the main pathway for attachment and entry. following infection, a substantial reduction of the ace2 expression in the cell surfaces occurs [14] , but the rate and the intensity of this downregulation seems to be much lower in hcov-nl63 infections as compared to sars coronavirus infections. ace2 downregulation was previously shown to be associated with an increase of the inflammatory process in the respiratory tract and may be one of the determinants of severity in acute respiratory distress syndrome [14] . moreover, genetic polymorphisms associated with an increase of ace expression (which counterbalance ace2 action as a proinflammatory mediator) were shown to be associated with worse lung disease in cf patients [15] . we have previously shown that human rhinovirus c infections were associated with an increased risk of respiratory exacerbations in cf children [3] , and although we could not identify a significant association of hcov infections with respiratory exacerbations in the present analysis, such exacerbations were observed in a significant proportion (62.5%) of hcov-nl63-infected children. it should be mentioned that the mean age of our patients was approximately 9 years, while in the study by talbot et al. [10] , the greatest incidence of lri associated with these viruses occurred between 6 and 23 months of age. therefore, a study of infants with cf could produce very different results. in conclusion, we found all circulating species of hcov in children with cf who were enrolled in this study. symptoms were mainly related to the upper airway, and we did not find an association between hcov infections in general or infections with a given hcov species and an increased risk of respiratory exacerbation or hospital admission, although a significantly higher rate of respiratory exacerbation was observed among hcov-nl63-infected children as compared to children infected by other coronaviruses. cystic fibrosis the role of respiratory viruses in cystic fibrosis rhinovirus c and respiratory exacerbations in children with cystic fibrosis coronavirus infection in acute lower respiratory tract disease of infants human coronavirus nl63: a clinically important virus? identification of a novel coronavirus in patients with severe acute respiratory syndrome identification of a new human coronavirus characterization and complete genome sequence of a novel coronavirus, coronavirus hku1, from patients with pneumonia epidemiology and clinical presentations of the four human coronaviruses 229e, hku1, nl63, and oc43 detected over 3 years using a novel multiplex real-time pcr method the pediatric burden of human coronaviruses evaluated for twenty years new human coronavirus, hcov-nl63, associated with severe lower respiratory tract disease in australia two cases of severe obstructive pneumonia associated with an hku1-like coronavirus viral and atypical bacterial infections in the outpatient pediatric cystic fibrosis clinic renin-angiotensin system in human coronavirus pathogenesis end-organ dysfunction in cystic fibrosis: association with angiotensin i converting enzyme and cytokine gene polymorphisms financial support. this work was supported by fundação de amparo à pesquisa do estado de são paulo (grant 05/01625-8).potential conflicts of interest. key: cord-271822-ohkki0ke authors: verma, siddhartha; dhanak, manhar; frankenfield, john title: visualizing the effectiveness of face masks in obstructing respiratory jets date: 2020-06-01 journal: phys fluids (1994) doi: 10.1063/5.0016018 sha: doc_id: 271822 cord_uid: ohkki0ke the use of face masks in public settings has been widely recommended by public health officials during the current covid-19 pandemic. the masks help mitigate the risk of cross-infection via respiratory droplets; however, there are no specific guidelines on mask materials and designs that are most effective in minimizing droplet dispersal. while there have been prior studies on the performance of medical-grade masks, there are insufficient data on cloth-based coverings, which are being used by a vast majority of the general public. we use qualitative visualizations of emulated coughs and sneezes to examine how materialand design-choices impact the extent to which droplet-laden respiratory jets are blocked. loosely folded face masks and bandana-style coverings provide minimal stopping-capability for the smallest aerosolized respiratory droplets. well-fitted homemade masks with multiple layers of quilting fabric, and off-the-shelf cone style masks, proved to be the most effective in reducing droplet dispersal. these masks were able to curtail the speed and range of the respiratory jets significantly, albeit with some leakage through the mask material and from small gaps along the edges. importantly, uncovered emulated coughs were able to travel notably farther than the currently recommended 6-ft distancing guideline. we outline the procedure for setting up simple visualization experiments using easily available materials, which may help healthcare professionals, medical researchers, and manufacturers in assessing the effectiveness of face masks and other personal protective equipment qualitatively. infectious respiratory illnesses can exact a heavy socioeconomic toll on the most vulnerable members of our society, as has become evident from the current covid-19 pandemic. 1, 2 the disease has overwhelmed healthcare infrastructure worldwide, 3 and its high contagion rate and relatively long incubation period 4, 5 have made it difficult to trace and isolate infected individuals. current estimates indicate that about 35% of infected individuals do not display overt symptoms 6 and may contribute to the significant spread of the disease without their knowledge. in an effort to contain the unabated community spread of the disease, public health officials have recommended the implementation of various preventative measures, including social-distancing and the use of face masks in public settings. 7 the rationale behind the recommendation for using masks or other face coverings is to reduce the risk of cross-infection via the transmission of respiratory droplets from infected to healthy individuals. 8, 9 the pathogen responsible for covid-19 is found primarily in respiratory droplets that are expelled by infected individuals during coughing, sneezing, or even talking and breathing. [10] [11] [12] [13] [14] [15] apart from covid-19, respiratory droplets are also the primary means of transmission for various other viral and bacterial illnesses, such as the common cold, influenza, tuberculosis, sars (severe acute respiratory syndrome), and mers (middle east respiratory syndrome), to name a few. [16] [17] [18] [19] these pathogens are enveloped within respiratory droplets, which may land on healthy individuals and result in direct transmission, or on inanimate objects, which can lead to infection when a healthy individual comes in contact with them. 10, 18, 20, 21 in another mode of transmission, the droplets or their evaporated contents may remain suspended in the air for long periods of time if they are sufficiently small. this can lead to airborne several studies have investigated respiratory droplets produced by both healthy and infected individuals when performing various activities. the transport characteristics of these droplets can vary significantly depending on their diameter. [23] [24] [25] [26] [27] [28] the reported droplet diameters vary widely among studies available in the literature and usually lie within the range 1 μm-500 μm, 29 with a mean diameter of ∼10 μm. 30 the larger droplets (diameter >100 μm) are observed to follow ballistic trajectories under the effects of gravity and aerodynamic drag. 20, 31 intermediate-sized droplets 20,31,32 may get carried over considerable distances within a multiphase turbulent cloud. [33] [34] [35] the smallest droplets and particles (diameter < 5 μm-10 μm) may remain suspended in the air indefinitely, until they are carried away by a light breeze or ventilation airflow. 20, 32 after being expelled into the ambient environment, the respiratory droplets experience varying degrees of evaporation depending on their size, ambient humidity, and temperature. the smallest droplets may undergo complete evaporation, leaving behind a dried-out spherical mass consisting of the particulate contents (e.g., pathogens), which are referred to as "droplet nuclei." 36 these desiccated nuclei, in combination with the smallest droplets, are potent transmission sources on account of two factors: (1) they can remain suspended in the air for hours after the infected individual has left the area, potentially infecting unsuspecting individuals who come into contact with them and (2) they can penetrate deep into the airways of individuals who breathe them in, which increases the likelihood of infection even for low pathogen loads. at present, the role of droplet nuclei in the transmission of covid-19 is not known with certainty and the matter is the subject of ongoing studies. [37] [38] [39] in addition to generating microscopic droplets, the action of sneezing can expel sheet-like layers of respiratory fluids, 40 which may break apart into smaller droplets through a series of instabilities. the majority of the fluid contained within the sheet falls to the ground quickly within a short distance. regardless of their size, all droplets and nuclei expelled by infected individuals are potential carriers of pathogens. various studies have investigated the effectiveness of medical-grade face masks and other personal protective equipment (ppe) in reducing the possibility of cross-infection via these droplets. 13, 33, [41] [42] [43] [44] [45] [46] [47] notably, such respiratory barriers do not prove to be completely effective against extremely fine aerosolized particles, droplets, and nuclei. the main issue tends to be air leakage, which can result in aerosolized pathogens being dispersed and suspended in the ambient environment for long periods of time after a coughing/sneezing event has occurred. a few studies have considered the filtration efficiency of homemade masks made with different types of fabric; 48-51 however, there is no broad consensus regarding their effectiveness in minimizing disease transmission. 52, 53 nonetheless, the evidence suggests that masks and other face coverings are effective in stopping larger droplets, which, although fewer in number compared to the smaller droplets and nuclei, constitute a large fraction of the total volume of the ejected respiratory fluid. while detailed quantitative measurements are necessary for the comprehensive characterization of ppe, qualitative visualizations can be invaluable for rapid iteration in early design stages, as well as for demonstrating the proper use of such equipment. thus, one of the aims of this letter is to describe a simple setup for visualization experiments, which can be assembled using easily available materials. such setups may be helpful to healthcare professionals, medical researchers, and industrial manufacturers, for assessing the effectiveness of face masks and other protective equipment qualitatively. testing designs quickly and early on can prove to be crucial, especially in the current pandemic scenario where one of the central objectives is to reduce the severity of the anticipated resurgence of infections in the upcoming months. the visualization setup used in the current study is shown in fig. 1 and consists of a hollow manikin head which was padded on the inside to approximate the internal shape and volume of the nasal-and buccal-cavities in an adult. in case a more realistic representation is required, such a setup could include 3d-printed or silicone models of the internal airways. the manikin was mounted at a height of ∼5 ft and 8 in. to emulate respiratory jets expelled by an average human male. the circular opening representing the mouth is 0.75 in. in diameter. the pressure impulse that emulates a cough or a sneeze may be delivered via a manual pump, as shown in fig. 1 , or via other sources such as an air compressor or a pressurized air canister. the air capacity of the pump is 500 ml, which is comparable to the lower end of the total volume expelled during a cough. 54 we note that the setup here emulates a simplified representation of an actual cough, which is an extremely complex and dynamic problem. 55 we use a recreational fog/smoke machine to generate tracer particles for visualizing the expelled respiratory jets, using a liquid mixture of distilled water (4 parts) and glycerin (1 part). both the pressure-and smoke-sources were connected to the manikin using clear vinyl tubing and npt fittings wherever necessary. the resulting "fog" or "smoke" is visible in the right panel of fig. 1 and is composed of microscopic droplets of the vaporized liquid mixture. these are comparable in size to the smallest droplets expelled in a cough jet (∼1 μm-10 μm). we estimate that the fog droplets are less than 10 μm in diameter, based on stokes' law and our observation that they could remain suspended for up to 3 min in completely still air with no perceptible settling. the laser source used to generate the visualization sheet is an off-the-shelf 5 mw green laser pointer with 532 nm wavelength. a plane vertical sheet is created by passing the laser beam we first present visualization results from an emulation of an uncovered heavy cough. the spatial and temporal evolution of the resulting jet is shown in fig. 2 . the aerosolized microscopic droplets visible in the laser sheet act as tracer particles, revealing a twodimensional cross section of the conical turbulent jet. these tracers depict the fate of the smallest ejected droplets and any resulting nuclei that may form. we observed high variability in droplet dispersal patterns from one experimental run to another, which was caused by otherwise imperceptible changes in the ambient airflow. this highlights the importance of designing ventilation systems that specifically aim to minimize the possibility of cross-infection in a confined setting. 23, [56] [57] [58] despite high variability, we consistently observed jets that traveled farther than the 6-ft minimum distance proposed by the u.s. centers for disease control and prevention (cdc's). 7 in the images shown in fig. 2 , the ejected tracers were observed to travel up to 12 ft within ∼50 s. moreover, the tracer droplets remained suspended midair for up to 3 min in the quiescent environment. these observations, in combination with other recent studies, 35, 59 suggest that current social-distancing guidelines may need to be updated to account for the aerosol-based transmission of pathogens. we note that although the unobstructed turbulent jets were observed to travel up to 12 ft, a large majority of the ejected droplets will fall to the ground by this point. importantly, both the number and concentration of the droplets will decrease with increasing distance, 59 which is the fundamental rationale behind socialdistancing. we now discuss dispersal patterns observed when the mouth opening was blocked using three different types of face masks. for these results, we focus on masks that are readily accessible to the general public, which do not draw away from the supply of medical-grade masks and respirators for healthcare workers. figure 3 shows the impact of using a folded cotton handkerchief mask on the expelled respiratory jet. the folded mask was constructed by following the instructions recommended by the u.s. surgeon general. 60 it is evident that while the forward motion of the jet is impeded significantly, there is notable leakage of tracer droplets through the mask material. we also observe a small amount of tracers escaping from the top edge of the mask, where gaps exist between the nose and the cloth material. these droplets remained suspended in the air until they were dispersed by ambient disturbances. in addition to the folded handkerchief mask discussed here, we tested a single-layer bandana-style covering (not shown) which proved to be substantially less effective in stopping the jet and the tracer droplets. we now examine a homemade mask that was stitched using two-layers of cotton quilting fabric consisting of 70 threads/in. the mask's impact on droplet dispersal is shown in fig. 4 . we observe that the mask is able to arrest the forward motion of the tracer droplets almost completely. there is minimal forward leakage through the material, and most of the tracer-escape happens from the gap between the nose and the mask along the top edge. the forward distance covered by the leaked jet is less than 3 in. in this case. the final mask design that we tested was a non-sterile conestyle mask that is available in most pharmacies. the corresponding droplet-dispersal visualizations are shown in fig. 5 , which indicate that the flow is impeded significantly compared to figs. 2 and 3. however, there is noticeable leakage from gaps along the top edge. the forward distance covered by the leaked jet is ∼6 in. from the mouth opening, which is farther than the distance for the stitched mask in fig. 4 . a summary of the various scenarios examined in this study is provided in table i , along with details about the mask material and the average distances traveled by the respiratory jets. we observe that a single-layer bandana-style covering can reduce the range of the expelled jet to some extent, compared to an uncovered cough. importantly, both the material and construction techniques have a notable impact on the masks' stopping-capability. the stitched mask made of quilting cotton was observed to be the most effective, followed by the commercial mask, the folded handkerchief, and, finally, the bandana. importantly, our observations suggest that a higher thread count by itself is not sufficient to guarantee better stoppingcapability; the bandana covering, which has the highest thread count among all the cloth masks tested, turned out to be the least effective. we note that it is likely that healthcare professionals trained properly in the use of high-quality fitted masks will not experience leakage to the extent that we have observed in this study. however, leakage remains a likely issue for members of the general public who often rely on loose-fitting homemade masks. additionally, the masks may get saturated after prolonged use, which might also influence their filtration capability. we reiterate that although the non-medical masks tested in this study experienced varying degrees of flow leakage, they are likely to be effective in stopping larger respiratory droplets. in addition to providing an initial indication of the effectiveness of protective equipment, the visuals used in this study can help convey to the general public the rationale behind social-distancing guidelines and recommendations for using face masks. promoting widespread awareness of effective preventative measures is crucial, given the high likelihood of a resurgence of covid-19 infections in the fall and winter. the data that support the findings of this study are available within this article. a un framework for the immediate socio-economic response to covid-19 the socio-economic implications of the coronavirus pandemic (covid-19): a review fair allocation of scarce medical resources in the time of covid-19 the incubation period of coronavirus disease 2019 (covid-19) from publicly reported confirmed cases: estimation and application temporal dynamics in viral shedding and transmissibility of covid-19 covid-19 pandemic planning scenarios social distancing, quarantine, and isolation face mask use and control of respiratory virus transmission in households a rapid systematic review of the efficacy of face masks and respirators against coronaviruses and other respiratory transmissible viruses for the community, healthcare workers and sick patients droplet fate in indoor environments, or can we prevent the spread of infection? exhalation of respiratory viruses by breathing, coughing, and talking size distribution and sites of origin of droplets expelled from the human respiratory tract during expiratory activities simplified models for exhaled airflow from a cough with the mouth covered the airborne lifetime of small speech droplets and their potential importance in sars-cov-2 transmission airborne or droplet precautions for health workers treating covid-19? transmission and control of rhinovirus colds core curriculum on tuberculosis: what the clinician should know transmission routes of respiratory viruses among humans recognition of aerosol transmission of infectious agents: a commentary review of aerosol transmission of influenza a virus aerobiology and its role in the transmission of infectious diseases observing and quantifying airflows in the infection control of aerosoland airborne-transmitted diseases: an overview of approaches factors involved in the aerosol transmission of infection and control of ventilation in healthcare premises study on transport characteristics of saliva droplets produced by coughing in a calm indoor environment how far droplets can move in indoor environments-revisiting the wells evaporation-falling curve transport of airborne particles from an unobstructed cough jet a new methodology for studying dynamics of aerosol particles in sneeze and cough using a digital high-vision, high-speed video system and vector analyses the role of particle size in aerosolised pathogen transmission: a review characterizations of particle size distribution of the droplets exhaled by sneeze characterization of expiration air jets and droplet size distributions immediately at the mouth opening on air-borne infection: study ii. droplets and droplet nuclei the size and the duration of air-carriage of respiratory droplets and droplet-nuclei a schlieren optical study of the human cough with and without wearing masks for aerosol infection control violent expiratory events: on coughing and sneezing turbulent gas clouds and respiratory pathogen emissions: potential implications for reducing transmission of covid-19 toward understanding the risk of secondary airborne infection: emission of respirable pathogens aerodynamic analysis of sars-cov-2 in two wuhan hospitals air, surface environmental, and personal protective equipment contamination by severe acute respiratory syndrome coronavirus 2 (sars-cov-2) from a symptomatic patient indirect virus transmission in cluster of covid-19 cases visualization of sneeze ejecta: steps of fluid fragmentation leading to respiratory droplets the efficacy of standard surgical face masks: an investigation using "tracer particles a quantitative assessment of the efficacy of surgical and n95 masks to filter influenza virus in patients with acute influenza infection dispersion and exposure to a cough-generated aerosol in a simulated medical examination room efficacy of face shields against cough aerosol droplets from a cough simulator effectiveness of cough etiquette maneuvers in disrupting the chain of transmission of infectious respiratory diseases respiratory virus shedding in exhaled breath and efficacy of face masks assessment of a respiratory face mask for capturing air pollutants and pathogens including human influenza and rhinoviruses simple respiratory protection-evaluation of the filtration performance of cloth masks and common fabric materials against 20-1000 nm size particles testing the efficacy of homemade masks: would they protect in an influenza pandemic? effectiveness of surgical and cotton masks in blocking sars-cov-2: a controlled comparison in 4 patients aerosol filtration efficiency of common fabrics used in respiratory cloth masks rational use of face masks in the covid-19 pandemic nonpharmaceutical measures for pandemic influenza in nonhealthcare settings-personal protective and environmental measures flow dynamics and characterization of a cough coughing frequency in patients with persistent cough: assessment using a 24 hour ambulatory recorder dispersal of exhaled air and personal exposure in displacement ventilated rooms dispersion of exhaled droplet nuclei in a two-bed hospital ward with three different ventilation systems role of ventilation in airborne transmission of infectious agents in the built environment-a multidisciplinary systematic review on coughing and airborne droplet transmission to humans how to make your own face covering key: cord-275605-mbiojk39 authors: benkouiten, samir; al-tawfiq, jaffar a.; memish, ziad a.; albarrak, ali; gautret, philippe title: clinical respiratory infections and pneumonia during the hajj pilgrimage: a systematic review date: 2018-12-04 journal: travel med infect dis doi: 10.1016/j.tmaid.2018.12.002 sha: doc_id: 275605 cord_uid: mbiojk39 background: the islamic hajj pilgrimage to mecca is one of the world's largest annual mass gatherings. inevitable overcrowding during the pilgrims' stay greatly increases the risk of acquiring and spreading infectious diseases, especially respiratory diseases. method: the medline/pubmed and scopus databases were searched for all relevant papers published prior to february 2018 that evaluated the prevalence of clinical symptoms of respiratory infections, including pneumonia, among hajj pilgrims, as well as their influenza and pneumococcal vaccination status. results: a total of 61 papers were included in the review. both cohortand hospital-based studies provide complementary data, and both are therefore necessary to provide a complete picture of the total burden of respiratory diseases during the hajj. respiratory symptoms have been common among hajj pilgrims over the last 15 years. in cohorts of pilgrims, cough ranged from 1.9% to 91.5%. however, the prevalence rates of the most common symptoms (cough, sore throat, and subjective fever) of influenza-like illness (ili) varied widely across the included studies. these studies have shown variable results, with overall rates of ili ranging from 8% to 78.2%. these differences might result from differences in study design, study period, and rates of vaccination against seasonal influenza that ranged from 1.1% to 100% among study participants. moreover, the definition of ili was inconsistent across studies. in hospitalized hajj pilgrims, the prevalence of pneumonia, that remains a major concern in critically ill patients, ranged from 0.2% to 54.8%. conclusions: large multinational follow-up studies are recommended for clinic-based syndromic surveillance, in conjunction with microbiological surveillance. matched cohorts ensure better comparability across studies. however, study design and data collection procedures should be standardized to facilitate reporting and to achieve comparability between studies. furthermore, the definition of ili, and of most common symptoms used to define respiratory infections (e.g., upper respiratory tract infection), need to be precisely defined and consistently used. future studies need to address potential effect of influenza and pneumococcal vaccine in the context of the hajj pilgrimage. ksa for several weeks throughout the month-long hajj season, presenting a major public health and infection control concern, and a challenge both for the saudi authorities, as well as for the national authorities of the countries of origin of the pilgrims. in addition to physical exhaustion, sleep deprivation [3] , and heat stress [4] , inevitable overcrowding, both in housing and ritual sites, especially in mina encampment (this is approximately a 3-kilometer square area where pilgrims are accommodated in air-conditioned semi-permanent tents, some with up to 50-100 people) and inside the sacred mosque in mecca (with up to six pilgrims per square meter) [5] , greatly increases the risk of acquiring and spreading infectious diseases [6] [7] [8] , especially respiratory diseases [9, 10] . to minimize the spread of infections during the pilgrimage or in the pilgrims' home countries upon their return, vaccination and non-pharmaceutical interventions are thus recommended by national and international public health agencies [11, 12] . we carried out a systematic review of cohort and hospital studies that reported the prevalence of clinical symptoms of respiratory infections and pneumonia among pilgrims during the hajj, and both their influenza and pneumococcal vaccination status, with the aim to provide data allowing the investigation of the impact of this large mass-gathering event on public health policies and services and to identify potential targets for preventive measures. this review was performed according to preferred reporting items for systematic reviews and meta-analyses (prisma) guidelines (http://www.prismastatement.org). the medline/pubmed and scopus databases were searched for all relevant papers published prior to february 2018, using the terms: in addition, the saudi epidemiology bulletin (http://seb. drupalgardens.com/) was hand searched for additional papers for inclusion. finally, the reference lists of reviewed articles were searched for additional relevant papers. for inclusion, the article had to meet the following criteria: (1) original study involving hajj pilgrims; (2) detailed description of the study population, including influenza and pneumococcal vaccination status when available; (3) clinical or self-reported respiratory symptoms and diseases. only articles published in english were included for review. we excluded cohort studies with less than 50 participants and case reports. we also excluded studies conducted among selected groups of individuals suffering from respiratory tract infections, due to lacking denominator data. the two authors independently performed the searches, screened titles/abstracts for eligibility, selected papers that appeared to be relevant according to the review's inclusion criteria, and reviewed each of the selected manuscripts in full. the data were extracted from the included papers by one reviewer (sb) and collected in the summary table that was included in the review. the extracted data were checked by the two authors (sb and pg) for accuracy. minor discrepancies were resolved by the authors' discussion. the search strategy initially yielded 391 records, of which 143 were duplicates. twenty-nine additional papers were identified through manual searches. of the 277 papers identified 183 records were excluded after screening the title and abstract. of the 94 full text articles reviewed, 61 were deemed suitable for inclusion in this review influenza-like illness (ili) was defined according to the presence of the triad of cough, subjective fever and sore throat. c ili was defined as subjective (or proven) fever plus one respiratory symptom (e.g. dry or productive cough, runny nose, sore throat, shortness of breath). d ili was defined as subjective (or proven) fever and at least one respiratory symptom such as cough, sore throat and rhinorrhea. e ili was defined as symptoms and signs such as: sudden headache, dry cough, high grade fever, myalgia, coryza, malaise and loss of appetite with an abnormal general appearance. f upper respiratory tract infections (urti) was defined as any person who reported having developed at least one of the constitutional symptoms (fever, headache, myalgia) and one of the local symptoms (running nose, sneezing, throat pain, cough with/or without sputum) after reaching mecca for the hajj or within 2 weeks from return to riyadh. g acute febrile respiratory infection (afri) was defined as the presence of subjective fever plus at least one respiratory symptom (cough, sore throat, runny nose or breathlessness). h two travelers who reported ''bronchitis'' as a symptom were also included. i ili was defined as fever plus sore throat and/or coughing. j common cold was defined as sore throat with coryzal symptoms, and low grade fever. k ili was defined as fever > 38.5°c, myalgia, low back pain, coryzal symptoms and cough. l acute respiratory infection (ari) was defined as one of the constitutional symptoms (fever, headache, myalgia) along with one of the local symptoms (running nose, sneezing, throat pain, cough with/without sputum, difficulty breathing). m ari was defined as any person suffering from at least one of the constitutional symptoms (fever, headache, myalgia) along with one of the local symptoms (runny nose, sneezing, throat pain, cough with/without sputum, difficulty in breathing) developing after reaching makkah for the hajj. n ili was defined as cough and fever > 38°c with or without the coryzal symptoms and myalgia. o ari was defined as any person suffering from at least one of the constitutional symptoms (fever, headache, myalgia) along with one of the local symptoms (runny nose, sneezing, throat pain, cough with/without sputum, difficulty in breathing) developing after reaching mecca for the hajj. p ili was defined as sore throat with either temperature ≥38.8°c or cough. q cough or sore throat or rhinorrhea or muscle ache or headache. according to the inclusion/exclusion criteria. the results of the search strategy are shown in fig. 1 . a total of 45 publications were identified. these studies were conducted among cohorts of pilgrims from the 1999 through the 2015 hajj seasons. the results of these studies are presented in table 1 . various study designs were used, including cross-sectional studies, case-control studies, and prospective cohort studies with follow-up of pilgrims, before, during and after the hajj. participants were from different countries and continents (africa, north america, asia, europe, as well as from australia), with the majority from iran, and they were recruited from different settings, including travel medicine clinics, vaccination centers, hajj travel agencies, international airports and transit zones, mecca's city and mina encampments. their numbers varied widely in these studies, ranging from 106 to 107,074. respiratory symptoms were common during the hajj. overall, the prevalence of cough ranged from 1.9% in domestic and international pilgrims in 1999 [13] to 91.5% in malaysian pilgrims in 2007 [14, 15] ( table 3) . more recent studies, conducted in different populations of pilgrims during the 2011-2014 hajj seasons, reported prevalence of cough ranging from 46.3% to 86.8% [16] [17] [18] [19] [20] [21] [22] [23] [24] . these studies also reported a comparable prevalence of sore throat ranging from 34.7% to 91% among pilgrims [16] [17] [18] [19] [20] [21] [22] [23] . in addition, many of these studies have investigated the epidemiology of respiratory tract infections among pilgrims by estimating the common prevalence of upper respiratory tract infection (urti), acute respiratory infection (ari) or influenza-like illness (ili), which were inconsistently defined across studies by a combination of general symptoms (e.g. cough, sore throat and fever). overall prevalence of ili varied in these studies from 8% to 78.2% [14] [15] [16] [18] [19] [20] [21] [22] [23] ( [47, 48] . however, the ili syndromic case definition used in the 2003-2004 study (ili was defined as cough and fever of more than 38°c with or without the coryzal symptoms and myalgia) [41, 42] was different with that used in the 2004-2008 study (ili was defined as symptoms and signs such as sudden headache, dry cough, high grade fever, myalgia, coryza, malaise and loss of appetite with an abnormal general appearance) [47, 48] . also, it is unclear from the 2005 study [46] if the definition used was consistent with those used in the two previous studies [41, 42, 47, 48] . in a recent large study, conducted among 3364 egyptian pilgrims between 2012 and 2015, the prevalence of ili was 30.4% (ili was defined according to the world health organization definition as the presence of measured fever of ≥38 c°, and cough; with onset within the last 10 days) [45] . other studies of different sizes (from 129 to 468) and design were conducted from 2007 through 2014 among different populations of pilgrims using a common ili definition (the association of cough, sore throat, and subjective fever). these studies have shown variable results, with overall rates of ili ranging from 8% to 78.2% [14] [15] [16] 18, 19, [21] [22] [23] 25, 26, 31, 35, 37] . thus, during the 2013 hajj season, while the highest prevalence of ili was observed among malaysian pilgrims, with a prevalence estimated at 78.2% [25] , a lower prevalence was observed among french pilgrims (47.3%) [18, 21] . coverage of seasonal influenza vaccination among pilgrims was evaluated in many studies, which have yielded varying results, with reported rates of influenza vaccination ranged from 1.1% to 100% [14, 15, 18, [21] [22] [23] 25, 26, 28, 29, [31] [32] [33] [34] 36, 37, [41] [42] [43] [44] [45] [46] [47] [48] [49] [50] [51] [53] [54] [55] [56] . a variation over time in influenza vaccination coverage was observed, as exemplified by a rate of 10.5% observed in a survey of pilgrims from riyadh in 2003 [43] , but 94.4% in a similar survey in 2010 [32, 33, 36] . during the 2013 hajj season, influenza vaccination rates also varied according to pilgrims' country of origin [29] , with 20% observed among saudi pilgrims, 80% among qatari pilgrims, and 87% among australian pilgrims, while a study involving french pilgrims interestingly reported that none of them had received the 2013 influenza vaccine before departing for the hajj because the vaccine was not available at this time [18, 21] . the majority of the studies reported influenza vaccination coverage among pilgrims, but only 13 [18, 19, [21] [22] [23] 25, 27, 28, 31, [46] [47] [48] 55] reported their pneumococcal vaccination status, with rates ranging from 1.2% among a multinational cohort of 1676 pilgrims from 13 countries (from africa, asia, usa and europe) in 2013 [28] to 51.2% among a small study of 129 french pilgrims in 2013 [18, 21] . of the 61 publications that were included in this review, 16 specifically addressed ill hajj pilgrims at health care facilities from 1993 through 2014 hajj seasons. medical facilities included primary health care centers (phccs) and different specialized wards in tertiary care hospitals, including ear, nose and throat (ent) departments, intensive care units, emergency units, infectious disease units and unspecified medical units. pilgrim participants were included either as inpatients or outpatients. the results of these studies are summarized in table 2 . overall, the prevalence of upper respiratory tract infections (urti) ranged from 1.4% to 42.1% (table 3 ). this prevalence was 1.4% among 141 pakistani pilgrims who attended the king abdul aziz hospital in medina during the 1992 hajj [57] and 42.1% among 3087 saudi and non-saudi patients (47.5% of them were pilgrims) who attended the ent clinic at al-noor specialist hospital in mecca during the 2009 hajj [58] . pharyngitis was also frequently reported among ill pilgrims. thus, in this study of 3087 pilgrims during the 2009 hajj, the overall prevalence of pharyngitis was 45.7% [58] . more recently, in 2008, the prevalence of pharyngitis in a large cohort of 4136 outpatients patients from 82 nationalities who attended 13 randomly selected mina phccs (94.9% of whom were pilgrims) was found to be 23.7% [59, 60] , and 61% in a study of 1047 saudi and non-saudi patients (2.3% of them were inpatients) [61] . however, in this second study of 1047 patients, only 34.5% were pilgrims. on the contrary, lower prevalence rates of bronchitis were reported during the hajj (1.4%-9.6%) [59] [60] [61] [62] [63] . a recent retrospective cross-sectional multicenter study of 185 turkish inpatients (87.5% were pilgrims) who returned to turkey from the arabian peninsula countries between 2012 and 2014 reported a slightly higher prevalence of acute tracheobronchitis (13.6%) [64] . in addition, in this study, pneumonia was among the most common clinical diagnosis among the hospitalized hajj patients and represented about half of diagnoses [64] . as pneumonia remains a major concern in critically ill patients, most of them reported the prevalence of pneumonia among pilgrims [57, 59, 60, [62] [63] [64] [65] [66] [67] [68] [69] [70] [71] , with reported rates ranging from 0.2% in 2008 in 13 randomly selected mina primary health care centers [59, 60] to 54.8% in 2004 in two icu in mecca [68] (table 3 ). the prevalence of pneumonia was not reported in 3 papers [58, 61, 72] . pneumonia was the second most common admitting diagnosis (22%) in a study of 140 patients admitted to the icus in four hospitals in mina during the 2004 hajj [68] . this result is further confirmed by a recent study of 452 critically ill hajj patients, of over 40 nationalities, admitted to 15 hospitals in 2009 and 2010. in this study, pneumonia was defined as the primary cause of critical illness (27.2%) of all icus admissions during the hajj [65] . also, in another prospective study of pilgrims admitted in two major icus in mecca for the 2004 hajj season, community acquired pneumonia (cap) was the commonest source of sepsis, 54.8% [66] . [57] acute bronchitis: 1.4% a upper respiratory tract infection (urti) was defined as an acute infection that includes tonsillitis, pharyngitis, laryngitis, sinusitis, otitis media, and the common cold. b acute tracheobronchitis was defined as a patient with dry cough and/or low-grade of fever (< 38°c), sub-sternal pain, and fatigue in the absence of opacities on chest x-ray. c acute exacerbation of chronic obstructive pulmonary disease (copd) was defined as an association with increased frequency and severity of coughing and/or shortness of breath and wheezing, increased amount of sputum production, and/or a change in appearance of sputum in a patient with copd. d was not defined. the purpose of this review was to provide syndromic surveillance data that may be useful, in conjunction with microbiological data that will be presented in further papers, for the surveillance of respiratory infections and pneumonia during the hajj. despite the fact that some of the included studies in our review were performed among small numbers of pilgrims and cannot be extrapolated, it is clear from this work that respiratory symptoms have been common among hajj pilgrims over the last 15 years, as evidenced by the high prevalence of cough (over 90%) among malaysian pilgrims during the 2007 hajj [73] . cough is a common symptom among pilgrims [16, 74] and likely results from crowded conditions during the hajj. this close contact among such individuals may increase the risk of the transmission of respiratory pathogens, and therefore may contribute to respiratory disease outbreaks. climatic conditions and air pollution in mecca and surrounding holy sites during the hajj [75] may also play a role. recent follow-up studies thus evidenced a significant acquisition of respiratory viruses, particularly rhinovirus, influenza virus, and coronaviruses other than middle east respiratory syndrome coronavirus (mers-cov), and of bacteria, including streptococcus pneumonia, hemophilus influenza, staphylococcus aureus and klesiella pneumonia by hajj pilgrims upon their return from the hajj [76, 77] . respiratory diseases are the most common diseases observed among pilgrims attending mina primary health care centers [59] and a major cause of hospital admission during the hajj [70] , with pneumonia a leading cause of admission to intensive care units [62, 68] , where they are responsible for about half of the cases of sepsis [66] . unfortunately, while numerous articles on hajj pilgrims were retrieved from our literature search, relatively few recent articles specifically addressed ill pilgrims in the context of hospital settings. the use of cohort studies allows investigators to evaluate the actual incidence of clinical events in hajj pilgrims since it provides a denominator, but may not identify and capture the prevalence of some underlying conditions and of severe forms of respiratory tract infections, which are more likely to be evidenced in hospital patient populations. conversely, hospital studies use data that may be biased, frequently lacking denominator values, and so probably overestimating the occurrence of severe illness. moreover, a hospital-based study will, by definition, not capture some minor illness cases that do not require hospitalization. the prevalence rates of cough, sore throat and subjective fever varied widely across the included studies. these differences may result from differences in study design that may lead to potential biases (for example bias related to the method of data collection, using either selfreport questionnaires or telephone interview), study period (with regards to the seasonality of respiratory viral infections), and rates of vaccination against seasonal influenza among study participants which may widely vary from one study to another, as described in this review. thus, all data regarding the pilgrims, including demographic data, medical history, clinical data and information on vaccination status and compliance with non-pharmaceutical preventive measures, should be carefully collected by using standardized questionnaires. in addition, in the context of syndromic surveillance for respiratory pathogens, data regarding the pilgrim's symptoms should be collected prospectively during face-to-face interviews by trained medical investigators who travel with the pilgrims. one important result of this review is the finding of a lack of consistency ili syndromic case definitions across included studies. thus, in a 2003 study (that did not fulfill the inclusion criteria for this review) [78] , of 1310 malaysian pilgrims who had a clinic visit for upper respiratory tract symptoms at five clinics during the 2000 hajj, with the aim of determining influenza vaccine effectiveness against clinically defined ili, 63% had ili (defined as sore throat in combination with either temperature ≥38°c or cough) and 14% had influenza by the cdc definition (defined as measured fever [≥100°f (37.8°c)] and a cough and/or a sore throat). only one of the studies reported here used the cdc definition of ili or the who definition (an acute respiratory infection with measured fever of ≥38 c°a nd cough, with onset within the last 10 days) [45] . in his paper, rashid et al. demonstrated the low sensitivity of the cdc criteria and proposed therefore the use of the triad of 'cough, sore throat and subjective fever' to clinically define ili at the hajj or other mass gatherings, since this new simple clinical case definition is more specific and sensitive than the cdc definition [79] . this definition was used over the last years by french [16, 18, 19, [21] [22] [23] 31, 37] , malaysian [14, 15, 25] , indian [26] and afghan [35] investigators leading cohort studies among hajj pilgrims, thus allowing more reliable comparisons of findings between studies (table 1) . respiratory diseases are a major concern during the hajj. nonpharmaceutical interventions (e.g., hand hygiene, wearing face masks, social distancing) are known to reduce the spread of respiratory viruses from person to person and are therefore recommended to pilgrims by public health agencies. although hand hygiene compliance is high among pilgrims, face mask use and social distancing remain difficult challenges. data about the effectiveness of these measures for preventing acute respiratory infections at the hajj are limited, and results are contradictory, highlighting the need for future large-scale studies [80] . in addition to non-pharmaceutical interventions, vaccination against influenza is recommended for all hajj pilgrims by the ministry of health of saudi arabia [11, 12] . differences in study design and heterogeneity in the ili definition across studies make it difficult to compare findings from different studies and inhibits the drawing of conclusions regarding the potential effects of this vaccination on related clinical symptoms of influenza disease. however, recent papers by alqahtani et al. and alfelali et al. found the influenza vaccine to be effective, respectively, against both laboratory-confirmed influenza [81] and clinical influenza [82] . as influenza vaccination is generally considered effective in reducing influenza-related infections, the scientific committee for influenza and pneumococcal vaccination guidelines (scipv) thus recommends, in its recent guidelines, an influenza vaccination for all people, especially those at high risk, at least 2 weeks before the hajj [83] . it also recommends, for the next hajj seasons that will take place from june to september, the administration (prior to the hajj) of the southern hemisphere influenza vaccine for pilgrims from the southern hemisphere (where influenza positivity rates are higher during this period). furthermore, as the influenza vaccine is not expected to be available for pilgrims from the northern hemisphere before these next hajj seasons, the scipv also recommends the administration of the southern hemisphere influenza vaccine for those pilgrims from the opposite hemisphere before the hajj [83] . because of the mismatching between circulating and vaccine strains that has frequently occurred since 2003 [84] , alfelali et al. recommends, when the composition of influenza vaccines differs and whenever logistically feasible, taking into consideration the dual vaccination of hajj pilgrims with both the southern and northern hemispheres' vaccines. however, such strategy is impaired by the frequent unavailability of the southern hemisphere influenza vaccine in the northern hemisphere. the issue of influenza vaccine availability to match southern and northern hemispheres was discussed by the saudi ministry of health in consultation with the who and it was recommended to use the available hemisphere strain as long as there is a match in circulating strains [85] . despite the risk of acquisition of s. pneumoniae during the hajj, there is currently no consistent guideline on the use of pneumococcal vaccine for hajj pilgrims across pilgrim countries of origin [86, 87] . thus, and because many of the hajj pilgrims are elderly and have chronic illnesses and underlying risk conditions for which pneumococcal vaccination is recommended [86] , the scipv also recommended, in its 2016 pneumococcal vaccination guidelines, pneumococcal vaccination of the atrisk population at the appropriate time before the hajj, using the 2 types of pneumococcal vaccines that are currently available: the 23valent polysaccharide pneumococcal vaccine (ppsv23) and the 13-valent conjugate vaccine (pcv13) [88] . however, it did not recommend providing a pneumococcal vaccine routinely to healthy persons aged less than 50 years, because of lack of evidence. in addition, it has been well demonstrated that the conjugate vaccine against s. pneumoniae targets the most virulent serotypes associated with invasive pneumococcal diseases (ipd) that are also associated with antibiotic resistance [89] . these arguments reinforce the need for compliance with current recommendations for vaccinating at-risk hajj pilgrims against ipd and influenza [89] . respiratory tract infections, including influenza, continue to be a major concern during the hajj. both cohort-and hospital-based studies provide complementary data and potentially useful information, and both are therefore necessary to provide a complete picture of the total burden of respiratory diseases during this mass gathering. large multinational follow-up studies are thus recommended for clinic-based syndromic surveillance, in conjunction with microbiological surveillance. matched cohorts ensure better comparability across studies, particularly in terms of origin of pilgrims and possible travelling conditions. however, the study design and data collection procedures should be standardized, to facilitate reporting and to achieve comparability between studies. furthermore, the definition of ili, and of most common symptoms used to define respiratory infections (e.g., urti), needs to be precisely defined and consistently used. future studies need to address the potential effects of influenza and pneumococcal vaccine in the context of the hajj pilgrimage. moreover, because of the mismatching between circulating and vaccine strains that has frequently occurred since 2003 [84] , alfelali et al. recommends, when the composition of influenza vaccines differs and whenever logistically feasible, taking into consideration the dual vaccination of hajj pilgrims with both the southern and northern hemispheres' vaccines. however, such strategy is impaired by the frequent unavailability of the southern hemisphere influenza vaccine in the northern hemisphere. despite the risk of acquisition of s. pneumoniae during the hajj, there is currently no consistent guideline on the use of pneumococcal vaccine for hajj pilgrims across pilgrim countries of origin [86, 87] . thus, and because many of the hajj pilgrims are elderly and have chronic illnesses and underlying risk conditions for which pneumococcal vaccination is recommended [86] , the scipv also recommended, in its 2016 pneumococcal vaccination guidelines, pneumococcal vaccination of the at-risk population at the appropriate time before the hajj, using the 2 types of pneumococcal vaccines that are currently available: the 23-valent polysaccharide pneumococcal vaccine (ppsv23) and the 13-valent conjugate vaccine (pcv13) [88] . also, it did not recommend providing a pneumococcal vaccine routinely to healthy persons aged less than 50 years, because of lack of evidence. respiratory tract infections, including influenza, continue to be a major concern during the hajj. both cohort-and hospital-based studies provide complementary data and potentially useful information, and both are therefore necessary to provide a complete picture of the total burden of respiratory diseases during this mass gathering. large multinational follow-up studies are thus recommended for clinic-based syndromic surveillance, in conjunction with microbiological surveillance. matched cohorts ensure better comparability across studies, particularly in terms of origin of pilgrims and possible travelling conditions. however, the study design and data collection procedures should be standardized, to facilitate reporting and to achieve comparability between studies. furthermore, the definition of ili, and of most common symptoms used to define respiratory infections (e.g., urti), needs to be precisely defined and consistently used. future studies need to address the potential effects of influenza and pneumococcal vaccine in the context of the hajj pilgrimage. none. the authors have no conflicts of interest to declare. the general authority for statistics in the kingdom of saudi arabia from hajj services to mass gathering medicine: saudi arabia formalizes a novel discipline hajj: journey of a lifetime social identification moderates the effect of crowd density on safety at the hajj hajj: infectious disease surveillance and control global perspectives for prevention of infectious diseases associated with mass gatherings health risks at the hajj respiratory tract infections during the annual hajj: potential risks and mitigation strategies respiratory tract infection during hajj health conditions for travellers to saudi arabia for the pilgrimage to mecca (hajj) -2015 health conditions for travellers to saudi arabia for the pilgrimage to mecca (hajj) health status of non-organized hajjees (muftaresheen) during 1420h, hajj season the prevalence of acute respiratory symptoms and role of protective measures among malaysian hajj pilgrims the association between pre-morbid conditions and respiratory tract manifestations amongst malaysian hajj pilgrims the inevitable hajj cough: surveillance data in french pilgrims high prevalence of common respiratory viruses and no evidence of middle east respiratory syndrome coronavirus in hajj pilgrims returning to ghana respiratory viruses and bacteria among pilgrims during the acquisition of streptococcus pneumoniae carriage in pilgrims during the 2012 hajj pilot randomised controlled trial to test effectiveness of facemasks in preventing influenza-like illness transmission among australian hajj pilgrims in 2011 lack of mers coronavirus but prevalence of influenza virus circulation of respiratory viruses among pilgrims during the 2012 hajj pilgrimage lack of nasal carriage of novel corona virus (hcov-emc) in french hajj pilgrims returning from the hajj 2012, despite a high rate of respiratory symptoms serological study of bordetella pertussis, mycoplasma pneumonia and chlamydia pneumonia in iranian hajj pilgrims with prolonged cough illnesses: a follow-up study the prevalence and preventive measures of the respiratory illness among malaysian pilgrims in 2013 hajj season influenza like illness (ili): prevalence and preventive practices among indian haj pilgrims of karnataka impact of the hajj on pneumococcal transmission mass gathering and globalization of respiratory pathogens during the 2013 hajj viral respiratory infections among hajj pilgrims in 2013 acute febrile respiratory infection symptoms in australian hajjis at risk of exposure to middle east respiratory syndrome coronavirus relative risk for influenza like illness in french hajj pilgrims compared to non-hajj attending controls during the 2009 influenza pandemic patterns of diseases and preventive measures among domestic hajjis from central patterns of diseases and preventive measures among domestic hajjis from central, saudi arabia protective practices and respiratory illness among us travelers to the occurrence of acute respiratory infection, diarrhea and jaundice among afghan pilgrims pattern of diseases and preventive measures among domestic hajjis from riyadh, 1431 h protective measures against acute respiratory symptoms in french pilgrims participating in the hajj of surveying respiratory infections among iranian hajj pilgrims hajj-associated acute respiratory infection among hajjis from riyadh effect of the use of face mask on hajj related acute respiratory infection among hajjis from riyadh, a health promotion intervention study the comparison of influenza vaccine efficacy on respiratory diseases among iranian pilgrims in the 2003 and 2004 seasons the incidence of influenza like illness and determination of the efficacy of flu vaccine in iranian pilgrims during hajj pilgrimage incidence of hajj-related acute respiratory infection among hajjis from riyadh, 1423 h the incidence of vaccine preventable influenza-like illness and medication use among pakistani pilgrims to the haj in saudi arabia cross-sectional survey and surveillance for influenza viruses and mers-cov among egyptian pilgrims returning from hajj during 2012-2015 comparison of mortality and morbidity rates among iranian pilgrims in hajj treatment and prevention of acute respiratory infections among iranian hajj pilgrims: a 5-year follow up study and review of the literature trend of diseases among s iranian pilgrims during five consecutive years based on a syndromic surveillance system in hajj incidence of hajj-associated febrile cough episodes among french pilgrims: a prospective cohort study on the influence of statin use and risk factors mers-cov but positive influenza viruses in returning hajj pilgrims effect of influenza vaccination on acute respiratory symptoms in malaysian hajj pilgrims time to revisit presumptions on the essentiality of influenza vaccination for hajj pilgrims: a prospective cohort study international pandemic 2009 influenza a (h1n1) infection among 2009 hajj pilgrims from southern iran: a real-time rt-pcr-based study health related experiences among international pilgrims departing through king abdul aziz international airport effects of flu vaccine, solely or accompanied by pneumovax-23 vaccine on clinical consequences of the respiratory diseases among iranian pilgrims in hajj influenza among u.k. pilgrims to hajj medical problems of pakistani pilgrims in saudi arabia impact of ph1n1 influenza a infections on the otolaryngology, head and neck clinic during hajj pattern of diseases among visitors to mina health centers during the hajj season, 1429 h (2008 g) pattern of diseases among visitors to mina health centers during the hajj season road map of an ear, nose, and throat clinic during the 2008 hajj in makkah, saudi arabia causes of hospitalization of pilgrims in the hajj season of the islamic year pattern of surgical and medical diseases among pilgrims attending alnoor hospital makkah infections in travellers returning to turkey from the arabian peninsula: a retrospective cross-sectional multicenter study clinical and temporal patterns of severe pneumonia causing critical illness during hajj severe sepsis and septic shock at the hajj: etiologies and outcomes pattern of diseases among visitors of health care facilities in madinah during hajj season, 1428 h (2007 g) causes of admission to intensive care units in the hajj period of the islamic year 1424 pattern of medical diseases and determinants of prognosis of hospitalization during muslim pilgrimage hajj in a tertiary care hospital. a prospective cohort study pattern of admission to hospitals during muslim pilgrimage (hajj) pattern of medical problems among haj pilgrims admitted to king abdul aziz hospital, madinah al-munawarah epidemiological pattern of diseases and risk behaviors of pilgrims attending mina hospitals, hajj 1427 h (2007 g) preference of treatment facilities among malaysian hajj pilgrims for acute respiratory symptoms hajj: health lessons for mass gatherings air quality in mecca and surrounding holy places in saudi arabia during hajj: initial survey hajj-associated viral respiratory infections: a systematic review circulation of respiratory pathogens at mass gatherings, with special focus on the hajj pilgrimage a case-control study of influenza vaccine effectiveness among malaysian pilgrims attending the haj in saudi arabia influenza and the hajj: defining influenza-like illness clinically non-pharmaceutical interventions for the prevention of respiratory tract infections during hajj pilgrimage vaccinations against respiratory tract infections at hajj hajj research team. changes in the prevalence of influenza-like illness and influenza vaccine uptake among hajj pilgrims: a 10-year retrospective analysis of data the saudi thoracic society guidelines for influenza vaccinations mismatching between circulating strains and vaccine strains of influenza: effect on hajj pilgrims from both hemispheres expected immunizations and health protection for hajj and umrah 2018 -an overview prevention of pneumococcal infections during mass gathering pneumococcal infections at hajj: current knowledge gaps the saudi thoracic society pneumococcal vaccination guidelines-2016 emergence of drug resistant bacteria at the hajj: a systematic review none. supplementary data to this article can be found online at https:// doi.org/10.1016/j.tmaid.2018.12.002. key: cord-279421-rxocrgfu authors: zhang, dan; feng, zhishan; zhao, mengchuan; wang, hao; wang, le; yang, shuo; li, guixia; lu, li; ma, xuejun title: clinical evaluation of a single-tube multiple rt-pcr assay for the detection of 13 common virus types/subtypes associated with acute respiratory infection date: 2016-04-04 journal: plos one doi: 10.1371/journal.pone.0152702 sha: doc_id: 279421 cord_uid: rxocrgfu respiratory viruses are among the most important causes of human morbidity and mortality worldwide, especially for infants and young children. in the past years, a few commercial multiplex rt-pcr assays have been used to detect respiratory viruses in spite of the high cost. in the present study, an improved single-tube multiplex reverse transcription pcr assay for simultaneous detection of 13 respiratory viruses was evaluated and compared with a previously reported two-tube assay as the reference method using clinical nasopharyngeal aspirates samples. of 310 prospectively tested respiratory specimens selected from children hospitalized with acute respiratory illness, 226 (72.90%, 226/310) and 214 (69.03%, 214/310) positive for one or more viruses were identified by the single-tube and the two-tube assays, respectively, with combined test results showing good concordance (kappa value = 0.874). individually, the single-tube assay for adenovirus (adv), human metapneumovirus (hmpv), human rhinovirus (hrv), parainfluenza virus type 1 (piv1), parainfluenza virus type 3 (piv3) and parainfluenza virus type 4 (piv4) showed the significantly superior sensitivities to those of the two-tube assay. no false positives were found. in conclusion, our results demonstrates the one-tube assay revealed significant improvements over the two-tube assay in terms of the better sensitivity, more accurate quality control, less nonspecific amplification, more cost-effective and shorter turn-around time and will be a valuable tool for routine surveillance of respiratory virus infection in china. respiratory viruses are among the most common causes of human morbidity and mortality worldwide, especially for infants and young children [1, 2] . the rapid identification is important for both therapeutic and infection control purposes. diagnoses of viral respiratory tract infection have been performed generally by non-molecular approaches such as direct immunofluorescence and viral culture. they are time-consuming, labor-intensive, and often lack sensitivity or specificity. in the last few years, multiplex rt-pcr assays have been developed to detect respiratory viruses, and many of them have been commercialized, such as xtag rvp, rvp fast (luminex molecular diagnostics, toronto, canada), resplex ii (qiagen, mississauga, canada), filmarray 1 respiratory panel (idaho technology inc., salt lake city, ut, usa), anyplex™ ii rv16 and seeplex rv assays (seegene, seoul, korea), advansure™ real-time rt-pcr (lg life science, seoul, korea) [3] [4] [5] [6] [7] . however, many of these assays are costly, or require specialized laboratory equipment [8, 9] . in our previous study, the two-tube multiplex reverse transcription pcr assay (two-tube assay) to detect sixteen respiratory viruses based on the amplicon size differences using automated electrophoresis system is described. the overall detection rate of the two-tube assay for each virus was comparable to those of the luminex xtag rvp fast assay and seeplex rv15 ace assay, demonstrating the high sensitivity and specificity of the two-tube assay in the analysis of clinical samples [10] . however, two-tube assay has a few drawbacks, such as the size difference of some amplicons are not big enough leading to the difficulty in judging the results by untrained staff, the hands-on two-tube assay is till cumbersome and the internal control is not steadily detected, which limits its wide use in the routine screening in provincial and local center for diseases control and prevention (cdc) in china. in the present study, we adopted the two-tube assay as a reference, and have been progressively optimized and substantially improved the performance of simultaneous detection of thirteen respiratory viruses types/subtypes, the most frequently detected viral agents of respiratory tract infections documented by beijing monitoring network for pneumonia between 2012-2014 (unpublished data), in a single-tube assay while maintaining excellent sensitivity and specificity. the targeted 13 respiratory viruses types/subtypes including influenza a virus (flua), influenza b virus (flub), seasonal influenza a virus subtypes h1n1 2009 pandemic (09h1n1), seasonal influenza a virus subtypes h3n2 (sh3n2), parainfluenza virus type 1 (piv1), human rhinovirus (hrv), adenovirus (adv), parainfluenza virus type 2 (piv2), parainfluenza virus type 3 (piv3), parainfluenza virus type 4(piv4), respiratory syncytial virus a (rsva), respiratory syncytial virus b (rsvb) and human metapneumovirus (hmpv). the aim of this study is to provide a high throughput screening method for routine surveillance of respiratory virus infection in provincial and local centers for disease control and prevention, china. all aspects of this study were performed in accordance with national ethics regulations and approved by the institutional review boards of the center for disease control and prevention of hebei. children's parents were apprised of the study's purpose and of their right to keep information confidential. written informed consent was obtained from parents or caregivers. 2015. of those 110 (35.4%) were female and 200 (64.6%) were male. ages were ranged from 1 months to 11 years old, and 279 (90% were under three years old. trained staff collected nasopharyngeal aspirates (npa) by adding 3.5ml of transport medium and stored at −80°c. total rna/dna was extracted from195μl of clinical sample with addition of 5μl of bacteriophage ms2 as an internal control (10 5 copies) prior to the extraction using the qiaamp viral rna mini kit (qiagen, hilden, germany). the extracts were eluted into 50μl of dnaseand rnase-free water and stored at −80°c. a total 14 pairs of chimeric primers were added to a single-tube to detect 13 respiratory viruses, and one pair of internal control primer (ms2 f, ms2 r) and one universal primer (tag) was also added to the tube. the bacteriophage ms2 coat protein derived virus-like particles (vlps) was used as an internal control to monitor the extraction and detection process of each specimens [11, 12] . the primers sequences, the target genes, the amplicon sizes, and primer working concentrations are listed in table 1 . two-tube assay was performed as described [10] . briefly, one-step rt-pcr kit (qiagen, hilden, germany) was used for the amplification. a total of 25μl pcr mixture containing 2μl of extracted rna and varied primer concentrations was subjected to the following conditions: 50°c for 30min, 95°c for 15min, followed by 10 cycles of 95°c for 30s, 55°c for 30s, and 72°c 30 s; 10 cycles of 95°c for 30s, 65°c for 30s, 72°c for 30s; 25 cycles of 95°c for 30s, 48°c for 30s, and 72°c for 30s, and a final incubation at 72°c for 3min, and the products were analyzed on the qiaxcel automatic electrophoresis using qiaxcel dna high-resolution kit. single-tube assay was performed according to the protocols as described above in two-tube assay with a slight modification of the primer sequences and primer concentration (table 1 ). the x 2 -test and fisher's exact test were performed to analyze the detection agreement between the single-tube assay and the two-tube assay and resolved discordant results. all the comparative detections were double-blind tests performed by trained staff in our laboratory. all of the 310 clinical specimens were simultaneously tested by the single-tube assay and the two-tube assay. the clinical samples are considered as positive when both the targeted pathogen and ms2 are present. the samples are considered to be negative if samples are negative for pathogen but positive for ms2. if both the target and ms2 are absent, the samples are classified as invalid. no invalid sample was found in the optimal single-tube assay. a total of 226 (72.90%, 226/310) and 214 (69.03%, 214/310) samples were detected by the single-tube assay and the two-tube assay, respectively. viral coinfections were found in 57 specimens (18.38%, 57/310) by the single-tube assay, while in 48 specimens (15.48%, 48/310) by two-tube assay. in our study, piv3 and hrv were identified to be the viruses most commonly involved in multiple agent infection, while piv2 and 09h1 were not detected by either of the two methods. all failed results and discordant results were resolved with repeated tests and direct sequencing using the same primers listed in the table 1 . as shown in table 2 , there were 23 specimens with discordant results between the two-tube assay and the single-tube assay, all of them were confirmed by sequencing as true positives, a total of 24 additional viruses were detected only by the single-tube assay, including 5adv, 4hmpv, 3hrv, 2piv1, 9piv3 and 1piv4. the two-tube assay does not include the detection of piv4. the sensitivity, specificity, negative prediction value (npv), positive prediction value (ppv), the accordance rate, and the kappa value of each virus for the single-tube assay compared to two-tube assay are shown in table 3 . early detection of respiratory virus infections allows clinicians to initiate immediate therapeutic interventions that can reduce complications, antibiotic use, and unnecessary laboratory testing. in this study, we compared the performance of a single-tube multiplex reverse transcription pcr assay with the reference method, a two-tube assay, which has been confirmed to be comparable to the commercial luminex x tag rvp fast assay and seeplex rv15 ace detection kit in our previous study. the single-tube assay is also based on the qiaxcel capillary electrophoresis system, which is accessible in most of provincial centers for disease control and prevention in china. of the 310 specimens from hospitalized children, a high prevalence of infection and co-infection with the common respiratory viral pathogens were revealed. hrv was found to be most frequently, followed by piv3. the major discrepancies between the reference two-tube assay and the proposed single-tube assay were the detection rates of hrv, adv, hmpv, piv4, piv1 and piv3. the 24 viruses detected only by the singletube assay (5adv, 4hmpv, 3hrv, and 2piv1, 9piv3, 1piv4) were confirmed by sequencing as true positives. therefore, the single-tube assay is more sensitive than the two-tube assay for the detection of hrv, hmpv, adv, piv, piv3 and piv4. the overall detection rate of the single-tube assay for each virus was comparable to that of the two-tube assay (kappa>0.75) demonstrating the high sensitivity and specificity of the single-tube assay in the analysis of clinical samples. compared with the two-tube assay, the single-tube assay revealed better sensitivity, more accurate quality control, less nonspecific amplification, more cost-effective and shorter turnaround time. this improvement might be attributed to several aspects as follows. first, we have replaced human genome rnase p gene in the two-tube assay with bacteriophage ms2 as an internal control. the utility of ms2-based armored rna as an assay internal control has been documented in many clinical applications [13, 14] . in this study, the internal control ms2 was added to each specimen prior to extraction as an exogenous control to monitor the whole process from nucleic acid extraction to rt-pcr. second, we inserted a few t nucleotides when appropriate between the specific and universal sequences in the primer design to ensure each amplicon can be easily distinguished by the qiaxcel machine. third, homologous tail sequences was added to the 5' end of each chimeric primer [15] . the homo-tag assisted nondimer (hand) system introduced in this study was to prevent dimer formation and allow more, specific, sensitive, and stable detection of the viruses [16] . fourth, for detection of multiple viral targets, simultaneous presence of multiple targets in one specimen may have led to competitive inhibition of amplification of less abundant targets and may explain some loss of assay sensitivity. in this study, we avoided this problem by optimizing temperature switch pcr parameters and further fine-tuning the concentration of primers. in addition, single-tube assay is more convenient and rapidly to apply to clinical specimens, saving the cost and turn-around time. in our study, the most frequently detected pathogen was hrv, while in other study, the rsv was the most commonly detected viral pathogen [17, 18] . this difference might be caused by the following two aspects. first, the detection of rsv increased during the winter, whereas hrv was detected year-round [19, 20] . in this study, the samples collection time is not in winter (may to october). second, hrv is a well-recognized cause of common colds and associated with upper respiratory tract complications. in this study, the patients were hospitalized for both the upper respiratory tract and lower respiratory tract infections. the two-tube assay does not include piv4 while single-tube assay does. for piv4 and piv2, only one piv4 specimen was detected as positive and no piv2 were detected by the single-tube assay with acute respiratory illnesses, which is consistent with previous reports that piv2 and piv4 were not identified as frequently as piv1 and piv 3 [21] [22] [23] . the different geographic location might also lead to the different seasonal distributions of pivs. all of the flua positives detected by the single-tube assay were subsequently sequenced to be h3n2 infection. 09h1n1 were not detected by either of two methods because h3n2 is the predominant seasonal flua circulating in 2015 in china. though piv2 and 09h1n1 were not found in the samples, we detected a few stock clinical samples previously identified as 09 h1n1 or piv2 using both single-tube and two-tube assays, and the results demonstrated both assays worked very well (data not shown). moreover, no false positives were found by the single-tube assay, further suggesting the high specificity of the single-tube assay. however, the limitations of our study are that only the main causes of respiratory infection were investigated. other respiratory tract infection related viruses, such as coronaviruses 229e, hku1, nl63, and oc43, human bocavirus (hbov), enteroviruses (ev) [17, 24, 25] , are not included in this study. besides, the association of clinical outcome with the etiology information, particularly the role of co-infections in the clinical course and severity is not addressed. given the high prevalence (18.38%, 57/310) and diversity of viral co-infections in our study, further research is also needed. in summary, the improved single-tube assay in this study using automatic capillary electrophoresis is a rapid, cost-effective and reliable method with high sensitivity and specificity for the simultaneous detection of thirteen respiratory virus infections, and will be a valuable tool for routine surveillance of respiratory virus infection in china. global burden of acute lower respiratory infections due to respiratory syncytial virus in young children: a systematic review and metaanalysis mortality associated with influenza and respiratory syncytial virus in the united states comparison of the anyplex(tm) ii rv16 and seeplex((r)) rv12 ace assays for the detection of respiratory viruses. diagnostic microbiology and infectious disease comparison of the advansure real-time rt-pcr and seeplex((r)) rv12 ace assay for the detection of respiratory viruses evaluation of the advansure real-time rt-pcr compared with culture and seeplex rv15 for simultaneous detection of respiratory viruses. diagnostic microbiology and infectious disease comparative evaluation of the seegene seeplex rv15 and real-time pcr for respiratory virus detection comparison of filmarray respiratory panel and laboratory-developed real-time reverse transcription-polymerase chain reaction assays for respiratory virus detection. diagnostic microbiology and infectious disease clinical and economical impact of multiplex respiratory virus assays. diagnostic microbiology and infectious disease comparison of fast-track diagnostics respiratory pathogens multiplex real-time rt-pcr assay with in-house singleplex assays for comprehensive detection of human respiratory viruses a two-tube multiplex reverse transcription pcr assay for simultaneous detection of sixteen human respiratory virus types/subtypes. biomed research international armored long rna controls or standards for branched dna assay for detection of human immunodeficiency virus type 1 construction of armored rna containing longsize chimeric rna by increasing the number and affinity of the pac site in exogenous rna and sequence coding coat protein of the ms2 bacteriophage the use of armored rna as a multi-purpose internal control for rt-pcr armored rna technology for production of ribonuclease-resistant viral rna controls and standards multicolor combinatorial probe coding for real-time pcr the elimination of primerdimer accumulation in pcr adenovirus infection in children with acute lower respiratory tract infections in beijing, china community-acquired pneumonia requiring hospitalization among u.s. children. the new england journal of medicine viral aetiology of acute respiratory infections among children and associated meteorological factors in southern china human rhinovirus infections in hospitalized children: clinical, epidemiological and virological features epidemiology and clinical presentation of the four human parainfluenza virus types parainfluenza virus types 1, 2, and 3 in pediatric patients with acute respiratory infections in beijing during human parainfluenza virus infections in infants and young children with acute respiratory infections in beijing zhonghua er ke za zhi chinese journal of pediatrics viral etiologies of hospitalized acute lower respiratory infection patients in china three years surveillance of viral etiology of acute lower respiratory tract infection in children from zhonghua er ke za zhi chinese journal of pediatrics key: cord-289282-4oz6r7op authors: hon, kam lun; leung, karen ka yan; leung, alexander k. c.; sridhar, siddharth; qian, suyun; lee, so lun; colin, andrew a. title: overview: the history and pediatric perspectives of severe acute respiratory syndromes: novel or just like sars date: 2020-06-01 journal: pediatr pulmonol doi: 10.1002/ppul.24810 sha: doc_id: 289282 cord_uid: 4oz6r7op many respiratory viral infections such as influenza and measles result in severe acute respiratory symptoms and epidemics. in the spring of 2003, an epidemic of coronavirus pneumonia spread from guangzhou to hong kong and subsequently to the rest of the world. the who coined the acronym sars (severe acute respiratory syndrome) and subsequently the causative virus as sars‐cov. in the summer of 2012, epidemic of pneumonia occurred again in saudi arabia which was subsequently found to be caused by another novel coronavirus. who coined the term mers (middle east respiratory syndrome) to denote the middle east origin of the novel virus (mers‐cov). in the winter of 2019, another outbreak of pneumonia occurred in wuhan, china which rapidly spread globally. yet another novel coronavirus was identified as the culprit and has been named sars‐cov‐2 due to its similarities with sars‐cov, and the disease as coronavirus disease‐2019. this overview aims to compare and contrast the similarities and differences of these three major episodes of coronavirus outbreak, and conclude that they are essentially the same viral respiratory syndromes caused by similar strains of coronavirus with different names. coronaviruses have caused major epidemics and outbreaks worldwide in the last two decades. from an epidemiological perspective, they are remarkably similar in the mode of spread by droplets. special focus is placed on the pediatric aspects, which carry less morbidity and mortality in all three entities. 2.1 | origin of the virus sars is a viral respiratory disease of zoonotic origin caused by the sars coronavirus (sars-cov). the origin of sars-cov is still unsettled and remains a controversial subject to date. sars-cov is phylogenetically divergent from other coronaviruses associated with human infections such as oc43, nl63, 229e, and hku1, but is closely related to civet and bat covs. 4 however, none of the currently known bat severe acute respiratory syndrome-related coronaviruses (sars-cov) is thought to be the direct ancestor of sars-cov. 5 sars was the first severe and readily transmissible new communicable disease of the 21 st century. 6 the epidemic first started around mid-november 2002 in guangzhou where at least two patients had atypical pneumonia of unknown cause. 7 the initial cases were meat handlers who had regular contact with wild game. 4 shortly after, similar cases were reported in five cities in guangdong province, however, no escalation of public health measures was announced. by february 2003, the outbreak had unfolded and there were 305 cases reported with five mortalities. 7 8 in hong kong, 1755 people were infected and 299 died; 386 infected cases were healthcare workers and eight of them died. 8, 9 no case of sars has been reported worldwide since 2004. however, suspicious cases were reported from time to time. 3 the incubation period for sars-cov is between 2 and 10 days with a mean of 5 days and up to 13 days, symptoms usually develop 2 to 10 days after the initial infection. the immune response includes immunoglobulin m (igm) antibody to the sars-cov-this peaks during the acute or early convalescent phase (week 3), and declines by week 12. igg antibody is produced later and peaks at week 12. 10 the first symptom of sars is fever of 38°c (100.4°f) or higher, followed by nonspecific flu-like symptoms such as chills/rigor, muscle aches and pain, headaches, diarrhea, sore throat, runny nose and malaise. the symptoms usually last about 2 to 7 days. affected patients may develop a dry cough, shortness of breath, and pneumonia. in severe cases, the patient can develop respiratory failure and acute respiratory distress syndrome (ards). in the sars outbreak of 2003, about 9% of patients with confirmed sars infection died. 11 the mortality rate was much higher for those over 60 years old, with mortality rates approaching 50% for this subset of patients. 11 in the sars epidemic, there were around 135 pediatric sars cases reported worldwide, the majority of them in hong kong. [12] [13] [14] [15] [16] in pediatric cases, the presenting features can be nonspecific, hence a postive contact history and environmental exposure became very important diagnostic clues. fever was a consistent symptom in all affected children, and lasted for a median duration of 6 days, while other common symptoms included cough (60%) and nausea or vomiting (41%). 12, 13, 17 teenage patients present with symptoms of malaise, myalgia, chill, and rigor in addition to cough and respiratory symptoms similar to those of adults, while the younger children presented mainly with cough and runny nose. 13 the symptoms and clinical course were milder and shorter in young children. lymphopenia was an important laboratory finding but more severe among teenagers. 13 the most prominent radiological features included patchy infiltrates, opacities, and areas of consolidation predominantly in the lower lobes. 14 no pediatric mortalities were reported in the literature. 12 in a case series of 43 children with sars in hong kong, five required picu care and one patient required invasive ventilatory support. 17, 18 children who were 12 years of age or younger generally had a milder illness and more favorable outcome. 12 the pathophysiology of a milder disease course in children is not clear, and proposed yet putative mechanisms include relatively low dosage of ribavirin, shorter course of corticosteroids, and a less mature immune system, resulting in less autoinflammatory injury in the young. 19 follow-up of sars patients at 6 months after the illness only showed mild residual changes in exercise tolerance and pulmonary function. 12 osteonecrosis was reported in children whose sars treatment included steroids; they remained mostly asymptomatic and managed conservatively. 16 there were two reported cases of transmission from children to adults and no reports of transmission from children to children. 6 as to vertical transmission, in all reported cases of maternal sars during pregnancy, the infants survived and no perinatal transmission was detected. 6 in summary, respiratory symptoms are milder and nonrespiratory symptoms are present in pediatric patients. the mean incubation period for mers-cov is 5.2 days, and ranges from 2 to 13 days. symptoms may range from mild to severe and include fever, cough, diarrhea, and shortness of breath. spread between humans typically involves close contact with an infected person. human-to-human transmission has been limited and mainly among family members and healthcare workers. there have been clusters of cases in healthcare facilities, where infection prevention and control practices were suboptimal, however, its spread was uncommon outside of hospitals. 20, 22 overall, 17.9% of the cases reported were healthcare workers. 21 there have been no reports of sustained human-to-human transmission; thus, its risk to the global population is currently deemed to be fairly low. 20 the high morbidity associated with mers may be spurious due to bias of inadequate sampling that had missed a much larger denominator with elusive milder cases. 23 as of 2019, there was no specific vaccine or treatment for the disease; a number of antiviral medications were being studied. 20 the who recommends that those who come in contact with camels wash their hands frequently and not touch sick camels, and that camelbased food products should be appropriately cooked. in essence, symptomatic and supportive treatments constitute therapies. of the cases reported to who, 20.8% had mild to no symptoms while 46.5% had severe disease or died. 21 mers is typically more severe in those with other health problems, more than half of the reported cases had had comorbidities (eg, diabetes mellitus, hypertension, heart disease, chronic renal failure, or lung disease), which might explain the high mortality rate. 20, 21 the overall risk of death may be lower than reported as those with mild symptoms may be undiagnosed. 24 the rate of pediatric mers-cov infection is relatively low in comparison to adults. 23 a large saudi arabian study that screened for mers-cov by polymerase chain reaction (pcr) testing in a selected at-risk population, including 8032 children (<14 years old) found 0.1% who tested positive when compared with 0.7% in adults. 25 the most common source of infection was household contact and acquired infection within a healthcare facility. 26 approximately 42% of affected children are asymptomatic. 26 the most common presenting symptoms are fever (57%), vomiting (28%), diarrhea (28%), and cough (14%). 26 the severity is lower in comparison to the adult population. in one series of 31 pediatric cases, only one patient required intensive care support and there were no deaths. 26 only two fatal pediatric mers-cov cases were found in the literature, and both cases had comorbidities (infantile nephrotic syndrome and cystic fibrosis). 27 4 | covid-19 the culprit of the recent pandemic in 2020, termed by the who as 30 according to wuhan health officials, the pneumonia appeared to be viral in nature and patients were placed in isolation. potential causes including influenza, avian infleuenza, adenovirus, sars-cov, and mers-cov were ruled out. 31 since there was highly suggestive evidence that the outbreak was associated with exposure in wuhan's huanan seafood wholesale market, the market was closed on 1 january, 2020. 32 the mean incubation period for sars-cov-2 is estimated to be between 2 and 14 days, with an average of 5 days. 35 there is evidence of humanto-human transmission through droplets or direct contact; and precautions should be practiced in the healthcare settings to prevent airborne transmission, which may be underestimated, among many uncertainties about this novel virus. 36 there have been confirmed cases in healthcare workers, and the reported cases were 3.8% and 20% of the total confirmed cases in china and italy, respectively. 31, 37, 38 with the ascertainment of asymptomatic carriage, the risk of transmission within the community is very high. 39 the most common symptoms include fever, cough, dyspnea, myalgia, or fatigue. 30 in a series of 41 cases, only a few patient had prominent upper respiratory tract signs even though abnormalities in chest computed tomography (ct) images were detected among all patients. 30 ct chest scans have a higher sensitivity for diagnosis of covid-19 as compared with pcr swab samples; and the findings include bilateral, subpleural, ground-glass opacities with air bronchograms, ill-defined margins, and a slight predominance in the right lower lobe. 40, 41 indeed, ct chest abnormalities are now diagnostic criteria in the latest "novel coronavirus diagnosis and treatment plan" published by the national health commission of the people's republic of china. 42 in a report of 72 314 cases prepared by the chinese center for disease control and prevention (cdc), a majority (80.9%) of the cases were classified as mild, while 4.7% were critical cases. 31 the age group ≥80 years old had the highest case-fatality rate at 14.8%. 31 treatment is mainly supportive, as no antiviral treatment has been clinically proven to be effective against sars-cov-2 and there are no standard treatment guidelines recommended by the who. the protease inhibitors lopinavir-ritonavir can be considered, but the results of a recent clinical trial are discouraging. 43 remdesivir and chloroquine have been shown to have good inhibitory effect on sars-cov-2 in vitro. 44 remdesivir is now undergoing phase ii clinical trials in treating patients with covid-19. 45 hydroxychloroquine, sometimes in combination with azithromycin, has been widely used in both europe and the us with little evidence of efficacy, and early analyses point to increased risk and higher mortality. 46, 47 long-term outcome for covid-19 patients is yet to be dereported that about 14% of recovered patients were tested postive for sars-cov-2 in follow-up checks several weeks after discharge. this is deemed most likely due to residual rna in clinical samples rather than reinfection. 48 given that a robust test has yet to be developed, speculations about results of the testing include false negativity at the time of discharge, inadequate specimen, less desirable site of specimen acquisition (eg, specimen taken from the throat vs the nasopharynx), testing discrepancies, new tests finding the virus in the lower respiratory tract, and reinfection. most infected children had relatively milder symptoms and recovered within 1 to 2 weeks. 53 several of the patients had no overt clinical symptoms and were found by positive screening of infected close contacts. 53 a study reviewing the epidemiology of 2135 pediatric cases in china revealed the proportion of severe and critical illness to be higher in the younger age group, in particular infants. 54 in the report by the us centres for disease control and prevention, among the aged less than 1 year with known hospitalized status, 62% were hospitalized; compared to 4.1 to 14% among those aged 1 to 17 years. 50 around 2% of the pediatric cases require admission to the intensive care, which is low comparing to the adult population. 55 there were less than 10 pediatric deaths reported worldwide. 31, 50 children may play a major role in community-based viral transmission and there were evidence of covid-19 infections in children occurring early in the epidemic. 56, 57 there is no convincing evidence hon et al. | 1587 of vertical transmission to neonates so far with only a couple of studies finding igm in neonatal blood, but no viral rna in samples from the respiratory tract. 58, 59 however, early onset covid-19 disease in neonates has been reported. 60 the reason for the decreased incidence in childhood is yet to be clarified, but it has been postulated that children are less susceptible to covid-19 because of lesser maturity and function of ace2 receptor, the cell entry receptor of sars-cov-2, compared to adults. [61] [62] [63] officials and experts compared similarities and differences of the acronyms and believed that they are all novel and impactful syndromes. they may have fever, respiratory symptoms, history of contact or travel, an animal vector and a novel coronavirus, and significant mortality and morbidity. there are relatively minimal or no differences in these acronyms except for the nomenclatures ( table 1 ). the fact that a significant number of patients with covid-19 are mildly symptomatic vs patients with sars and mers, with low mortality rates, reflects that sars-cov-2 resembles other common circulating respiratory viruses. however, the lack of any population immunity against sars-cov-2 gives it the ability to cause high attack rates, which can overwhelm and cripple healthcare systems if not managed carefully. similarities and differences between sars, mers, and covid-19 are tabulated hereunder (table 1) . it is perhaps unnecessary to coin confusing and seemingly different acronyms for each of these recurring epidemics and coronaviruses. 1 the syndrome approach (as in sars) as opposed to disease approach (as in covid) has its pros and cons (table 2) • the clinical definition can be applied to any similar epidemics for surveillance without knowing the culprit pathogen. • sensitive. • the sars concept of surveillance is easy to apply in epidemics. • the acronym is used even in afebrile, asymptomatic carriers or patients with mild and extrapulmonary symptoms in some laboratory-confirmed sars patients. • stigmatizing many patients because the definition is too nonspecific or too sensitive. • may not be used for a non-coronavirus (eg, influenza or measles) or another novel coronavirus (eg, mers) even if the symptoms are severe, acute and respiratory. • may miss nonfebrile patients or patients with extrapulmonary symptoms. • contact or travel history is only good in early phase of an epidemic. • new acronym has to be coined ( | 1589 these characteristics. whether beyond the virus there might be environmental changes that have occurred over the two-decade history of these infections, that enhance its spread, such as rising temperatures, or other yet to be determined atmospheric changes. it is hoped that because of the devastation that covid-19 carries in its wake, large efforts will be invested in a search for answers to these questions, and with that, we will all be able to be wiser in dealing with the threats of the future. this will require the combined efforts of a global community, since the current events clearly manifested our interconnectedness and interdependence and hence shared vulnerability in the face of a calamity of previously unexperienced magnitude. kam lun hon http://orcid.org/0000-0002-6682-5529 karen ka yan leung http://orcid.org/0000-0002-6689-7194 personal view of sars: confusing definition, confusing diagnoses severe respiratory syndromes: travel history matters just like sars clinical management and infection control of sars: lessons learned discovery of a rich gene pool of bat sars-related coronaviruses provides new insights into the origin of sars coronavirus drosten c, editor world health organisation. consensus document on the epidemiology of severe acute respiratory syndrome (sars) changing virulence of the sars virus: the epidemiological evidence world health organization. summary of probable sars cases with onset of illness from the sars epidemic in hong kong laboratory diagnosis of sars severe acute respiratory syndrome (sars): development of diagnostics and antivirals severe acute respiratory syndrome in children clinical presentations and outcome of severe acute respiratory syndrome in children severe acute respiratory syndrome among children severe acute respiratory syndrome in children: experience in a regional hospital in hong kong ippl-s. osteonecrosis in children with severe acute respiratory syndrome clinical features, diagnosis, treatment and short-term outcome of severe acute respiratory syndrome (sars) in children severe acute respiratory syndrome (sars) in children: epidemiology, presentation and management childhood sars in hong kong middle east respiratory syndrome world health organization. who mers-cov global summary and assessment of risk middle east respiratory syndrome coronavirus (mers-cov) screening for middle east respiratory syndrome coronavirus infection in hospital patients and their healthcare worker and family contacts: a prospective descriptive study severe acute respiratory syndrome vs. the middle east respiratory syndrome surveillance and testing for middle east respiratory syndrome coronavirus, saudi arabia middle east respiratory syndrome coronavirus disease is rare in children: an update from saudi arabia middle east respiratory syndrome coronavirus in pediatrics: a report of seven cases from saudi arabia mystery deepens over animal source of coronavirus the proximal origin of sars-cov-2 clinical features of patients infected with 2019 novel coronavirus in wuhan novel coronavirus pneumonia emergency response epidemiology team. the epidemiological characteristics of an outbreak of 2019 novel coronavirus diseases (covid-19) in china world health organization. novel coronavirus -china. 2020. 33. centre for health protection. a highly suspected imported case of novel coronavirus infection covid-19 dashboard by the center for systems science and engineering at johns hopkins the next big threat to global health? turbulent gas clouds and respiratory pathogen emissions: potential implications for reducing transmission of covid-19 coronavirus: infected health workers alert. patient health risk and health maintenance covid-19 and italy: what next? the lancet transmission of 2019-ncov infection from an asymptomatic contact in germany radiological findings from 81 patients with covid-19 pneumonia in wuhan, china: a descriptive study correlation of chest ct and rt-pcr testing in coronavirus disease 2019 (covid-19) in china: a report of 1014 cases a trial of lopinavir-ritonavir in adults hospitalized with severe covid-19 remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus (2019-ncov) in vitro. springer nature adaptive covid-19 treatment trial chloroquine and hydroxychloroquine in covid-19 hydroxychloroquine and azithromycin as a treatment of covid-19: results of an open-label nonrandomized clinical trial explainer: coronavirus reappears in discharged patients, raising questions in containment fight systematic review of covid-19 in children shows milder cases and a better prognosis than adults coronavirus disease 2019 in children-united states characteristics of and important lessons from the coronavirus disease 2019 (covid-19) outbreak in china: summary of a report of 72314 cases from the chinese center for disease control and prevention diagnosis and treatment of 2019 novel coronavirus infection in children: a pressing issue epidemiology of covid-19 among children in china. pediatrics. 2020:e20200702 sars-cov-2 infection in children covid-19 in children: initial characterization of the pediatric disease detection of covid-19 in children in early possible vertical transmission of sars-cov-2 from an infected mother to her newborn antibodies in infants born to mothers with covid-19 pneumonia neonatal early-onset infection with sars-cov-2 in 33 neonates born to mothers with covid-19 in wuhan, china. jama pediatr facing the pandemic of 2019 novel coronavirus infections: the pediatric perspectives a pneumonia outbreak associated with a new coronavirus of probable bat origin pediatric characteristics of 2019 novel coronavirus: review of available published literature severe acute respiratory syndrome vs. the middle east respiratory syndrome severe acute respiratory syndrome typical or atypical pneumonia and severe acute respiratory symptoms in picu severe acute respiratory symptoms and suspected sars again 2020 comparative analysis of eleven healthcare-associated outbreaks of middle east respiratory syndrome coronavirus (mers-cov) from 2015 to 2017 the reproductive number of covid-19 is higher compared to sars coronavirus epidemiological characteristics of 2143 pediatric patients with 2019 coronavirus disease in china teen football player jaquan anderson dead from coronavirus in new orleans two covid-19 infected children, aged 12 and 13, die in belgium and uk covid-19 in children in the united states: intensive care admissions, estimated total infected, and projected numbers of severe pediatric cases in 2020 overview: the history and pediatric perspectives of severe acute respiratory syndromes: novel or just like sars key: cord-274900-s7ft1491 authors: tatarelli, p.; magnasco, l.; borghesi, m. l.; russo, c.; marra, a.; mirabella, m.; sarteschi, g.; ungaro, r.; arcuri, c.; murialdo, g.; viscoli, c.; del bono, v.; nicolini, l. a. title: prevalence and clinical impact of viral respiratory tract infections in patients hospitalized for community-acquired pneumonia: the vircap study date: 2019-11-30 journal: intern emerg med doi: 10.1007/s11739-019-02243-9 sha: doc_id: 274900 cord_uid: s7ft1491 prevalence and clinical impact of viral respiratory tract infections (vrtis) on community-acquired pneumonia (cap) has not been well defined so far. the aims of this study were to investigate the prevalence and the clinical impact of vrtis in patients with cap. prospective study involving adult patients consecutively admitted at medical wards for cap and tested for vrtis by real-time pcr on pharyngeal swab. patients’ features were evaluated with regard to the presence of vrti and aetiology of cap. clinical failure was a composite endpoint defined by worsening of signs and symptoms requiring escalation of antibiotic treatment or icu admission or death within 30 days. 91 patients were enrolled, mean age 65.7 ± 10.6 years, 50.5% female. 62 patients (68.2%) had no viral co-infection while in 29 patients (31.8%) a vrti was detected; influenza virus was the most frequently identified (41.9%). the two groups were similar in terms of baseline features. in presence of a vrti, pneumonia severity index (psi) was more frequently higher than 91 and patients had received less frequently pre-admission antibiotic therapy (adjusted or 2.689, 95% ci 1.017–7.111, p = 0.046; adjusted or 0.143, 95% ci 0.030–0.670, p = 0.014). clinical failure and antibiotic therapy duration were similar with regards to the presence of vrti and the aetiology of cap. vrtis can be detected in almost a third of adults with cap; influenza virus is the most relevant one. vrti was associated with higher psi at admission, but it does not affect patients’ outcome. community-acquired pneumonia (cap) is a common cause of hospitalisation and one of the main infectious causes of mortality worldwide [1] [2] [3] . in europe, up to 32% of patients with cap are hospitalized every year and the cap mortality rate reaches 9.1% [4, 5] . streptococcus pneumoniae is the main aetiologic agent of cap, being responsible for two thirds of cases, but other pathogens such as haemophilus influenzae, moraxella catarrhalis, chlamydophila pneumoniae, mycoplasma pneumonia, legionella spp. and staphylococcus aureus are frequently involved [2] . respiratory viruses (which include rhinovirus, metapneumovirus, adenovirus, coronavirus, respiratory syncytial virus, influenza and parainfluenza viruses) have increasingly been recognized as causes of respiratory tract infections [6] . whilst epidemiology and clinical features of viral respiratory tract infections (vrtis) were mainly explored in children and outpatients [7, 8] , a few data show increasing prevalence rates also in hospitalized patients, particularly in elderly and in presence of comorbidities such as asthma, chronic obstructive pulmonary disease or immunosuppression [9] [10] [11] . the recent development and diffusion of nucleic acid amplification tests (naats) on respiratory samples have improved the ability to diagnose multiple upper and lower vrtis, possibly affecting prevalence estimates [12] . compared to classical microbiological techniques, naats allow earlier and more sensitive diagnosis [13, 14] , providing clinical advantages in terms of length of hospitalization, infection control and antibiotics use [15, 16] . however, in non-intubated hospitalized adults with cap, lower respiratory tract sampling is not easy to obtain and vrti diagnosis usually relies on nose-throat swabs, affecting the clinical significance of identified viruses [17] . beyond viral cap, upper vrtis predispose to pulmonary bacterial infections as they cause bronchoconstriction, increase mucus production [18] [19] [20] , decrease the efficiency of muco-ciliary clearance [21] , damage respiratory mucosa and cause leukocytes dysfunction [19] . data from both in vitro animal models and clinical experiences have demonstrated that influenza and other vrtis are risk factors for pneumococcal pneumonia, both in adult and paediatric population [22] [23] [24] [25] [26] . in spite of improved viral diagnosis and well-known pathophysiology, data on the clinical impact of vrtis on cap are controversial and some authors reported higher mortality rates [17] . the primary aim of this study was to investigate the prevalence of vrtis in a prospective cohort of adult patients hospitalized at medical wards for cap; the secondary aims were to describe patient's features and outcomes according to the presence of vrti and the aetiology of cap. we prospectively enrolled all adult patients consecutively admitted for cap and tested for vrtis at two medical wards (infectious diseases and internal medicine unit) of policlinico san martino-irccs, genoa, italy, from november 2016 to february 2018. patients having nosocomial pneumonia (i.e. developed at least 48 h after hospital admission) and those diagnosed with opportunistic pulmonary infection were excluded. cap was defined as acute lower respiratory tract infection characterized by the presence of two or more signs and symptoms (among fever, cough, dyspnoea, pleuritic pain, crackles or bronchial breath at pulmonary auscultation), associated with at least one among (a) radiological findings (opacity or infiltrate at radiography or computed tomography interpreted as pneumonia by the attending physician), (b) serum levels of inflammatory markers above normal values and (c) neutrophilic leucocytosis, in patients hospitalized no longer than 48 h [1] . the definitive bacterial aetiology for cap was defined as (1) identification of an aetiological agent in the blood and/ or pleural fluid and/or bronchoalveolar lavage (with > 10 5 colony-forming units/ml; (2) detection of legionella pneumophila spp. or s. pneumoniae antigen in the urine; (3) positive pcr for legionella spp., m. pneumoniae or chlamydia spp. on pharyngeal swab and/or bronchoalveolar lavage. presumptive bacterial aetiology was defined by positive culture on sputum or positive pcr for h. influenzae, bordetella spp. or s. pneumoniae on pharyngeal swab [27] in case of vrti, cap was considered of possible viral origin (or mixed origin in the presence of concomitant bacteria identification). antibiotic treatment escalation was defined as enhancing the anti-bacterial spectrum switching to or adding another molecule after at least a full 48 h-course of an initial, inhospital treatment, with the exclusion of targeted treatment. clinical failure was defined as (1) antibiotic treatment escalation; (2) worsening of signs and symptoms of pneumonia requiring icu admission; (3) death within 30 days. in addition, the total time of antibiotic exposure (tta) was calculated. for each patient, the following information was collected: demographic data (i.e. age, gender, country of origin), comorbidities (i.e. concomitant pulmonary or extrapulmonary diseases), chronic therapies for concomitant diseases, influenza and s. pneumoniae vaccination status, antibiotic therapy, results of radiological investigations (i.e. chest x-ray or computed tomography), biochemical tests (white blood cells, c reactive protein and procalcitonin) and outcome. for every patient, the presence of vrtis was ascertained with multiplex real time-polymerase chain reaction (rt-pcr) on pharyngeal swab (allplex tm respiratory panel assay, seegene, seoul, south korea) [28] . on the same sample, rt-pcr for s. pneumoniae, h. influenzae, c. pneumoniae, m. pneumoniae and b. pertussis was performed. microbiological findings from conventional tests (i.e. blood culture, sputum and broncoalveolar fluid culture, s. pneumoniae and l. pneumophila urine antigen detection, serology for intra-cellular bacteria) were also analysed. given the non-interventional nature of the study, bronchoalveolar lavage for microbiological tests was not systematically performed, but nonetheless available results were collected. curb-65 score > 2 and pneumonia severity index (psi) class iv-v (i.e. ≥ 91) at admission [29, 30] were evaluated to assess cap severity at clinical presentation. being an observational study, treatment was decided by the physician in charge according to the usual standard of care. clinical outcome, according to the aforementioned definitions, was recorded for each patient. before analysing the study results, all the cases were reviewed by independent observers who verified their correct classification in the study definitions. statistical analysis was mainly descriptive. patients' features, pneumonia severity scores were evaluated according to the concomitant presence of a vrti and aetiology of cap. categorical variables were compared with chi-square test or fisher's exact test, when applicable. continuous variables were compared with mann-whitney and anova tests. a value of p < 0.05 was considered statistically significant. variables significantly (p < 0.05) associated with the presence of a vrti and the aetiology of cap at previously mentioned tests were evaluated by multivariate logistic regression models. the same statistical tests were used to evaluate if the presence of a vrti, the aetiology of cap and patients' features were associated with outcome variables, such as clinical failure and tta. statistical analysis was carried out using spss. the study protocol was approved by the local (ligurian region) ethic committee. upon enrolment all patients signed a dedicated informed consent. during the study period, 99 patients were admitted for cap. of them, 8 were excluded because of the diagnosis of opportunistic pulmonary infection (n = 3) and missed testing for vrtis (n = 5). thus, 91 patients with cap and tested for vrti by pharyngeal swab were finally included in our study ( fig. 1 ). patients' characteristics at baseline are reported in table 1 at least one bacterium was documented in 27 patients (30%) with cap. the most frequently identified bacterium was s. pneumoniae (n = 16, 53.3%), followed by h. influenzae (n = 4, 13.4%) ( table 2 ). bacterial diagnostic samples considered were sputum (n = 13), blood (n = 6), urine (n = 6) and bronchoalveolar lavage (n = 2). nine sputum samples were collected in patients without pre-hospital antibiotic treatment. of the 27 definitive/presumptive bacterial cap, 8 (30%) were found with a concomitant vrtis, thus they were possibly caused by mixed viral and bacterial infection. influenza virus was identified in 4 (virus a = 3, virus b = 1), rhinovirus in 2, parainfluenza virus in 1, rhinovirus and parainfluenza virus in 1 case. in 21 cases, viruses were the only pathogens identified and were thus considered as possible causes of cap. the remaining 43 cases were considered of unknown aetiology because neither bacteria nor viruses were isolated. distribution of viral and bacterial pathogens identified is summarized in table 2 . comparing patients with and without vrti, no statistically significant differences were found in terms of age, gender, origin, smoking and alcohol habits, influenza and s. pneumoniae vaccination status, number and type of comorbidities, modes of admission to hospital, curb-65 score at admission, presence of bacteraemia, pneumonia radiological features or interval between onset of symptoms and hospital arrival (table 3) . vrtis were slightly more frequently diagnosed in autumn and winter, although difference was not statistically significant (93.1% vs 77.4%, p = 0.081). in presence of vrtis, psi at admission was more frequently higher than 91 (20/29, 69%, vs 27/62, 43.5%, p = 0.027) and patients received less frequently pre-admission antibiotic therapy (2/29, 6.9%, vs 22/62, 25.5%, p = 0.004). including both these variables in a multivariate model, they remained statistically associated to the presence of vrti (adjusted or 2.689, 95% ci 1.017-7.111, p = 0.046; adjusted or 0.143, 95% ci 0.030-0.670, p = 0.014, respectively) ( table 3) . patients' features were also evaluated with regards to the aetiology of cap after exclusion of those patients with unknown aetiology. bacterial (both diagnosed with definitive and presumptive criteria), possibly viral and mixed caps were similar in terms of demographic and radiologic features, immunosuppressive status, curb and psi score at admission (p > 0.05). overall, 24 patients (27.5%) received home antibiotic therapy before hospital admission, whilst the remaining patients received first-line antibiotic treatment only thereafter. in-hospital antibiotic treatment included a beta-lactam in 78 cases (86%). of them, 26 (33%) were treated with piperacillin/tazobactam. antibiotics active against intracellular bacteria were used in 39 cases (25 macrolides, 22 of whom in association with beta-lactams, and 15 fluoroquinolones, 14 of whom as monotherapy). with regard to antiviral treatment, 9 patients received empirical treatment with oseltamivir, that was early withdrawn in 4 patients who had no confirmed influenza infection by pcr. ribavirin, after a thorough assessment of risk-benefit analysis, was not administered in any of the 5 patients (2 with underlying hematologic malignancies) with respiratory syncytial virus-b infection. overall, clinical success was recorded in 72 patients (79%). among the 19 patients who experienced clinical failure, 17 required antibiotic escalation (involving an anti-mrsa agent in 6 cases), of whom 3 required invasive ventilation and intensive care unit admission. three patients died. of them, the first was a 73-year old man with s. pneumoniae infection who died in icu, the second a 51-year old hiv-positive man with respiratory and cardiovascular disease who developed pseudomonas aeruginosa pneumonia and the third a 91-year old man with myelodysplastic syndrome and chronic kidney disease with a lobar pneumonia and rhinovirus identified on pharyngeal swab. clinical failure was not influenced by the presence of vrti and aetiology of cap. the only variable associated to clinical failure was baseline psi higher than 91 (or 3.31, 95% ci 1.08-10.16, p = 0.036). tta was similar irrespective to the presence of vrti (mean ± sd 11.3 ± 4.0 versus 12.3 ± 6.4 days, p = 0.55, in patients with and without vrti, respectively) and aetiology of cap (14.55 ± 1.61 versus 12.13 ± 1.52 versus 11.54 ± 0.83 versus 11.42 ± 0.93 days in bacterial, mixed, viral and unknown aetiology cap, respectively, p = 0.3). no other collected variables were associated to tta. in the present study, we prospectively analysed the prevalence and the clinical impact of vrtis on patients with cap hospitalized at medical wards. vrtis' prevalence rate we found was as high as 31.9% (95% ci 22.3-41.4%), but fortunately vrtis did not negatively affect clinical outcome. a recent meta-analysis reported a 22% (95% ci 17-27%) vrtis' prevalence rate in adults with cap that rose to 29% (95% ci 25-34%) including only studies where molecular assays were used [31] . our data confirm that in a real-life setting, including a mixed population of immunocompetent and immunocompromised adults hospitalized for cap, vrtis are highly prevalent. at admission, patients with vrti had more frequently psi score iv-iv than those without. three previous studies (of whom two were retrospective) found an association between vrtis and psi class iv/v, but none of them reported on curb-65 score [13, 25, 32] . contrary to psi score, we did not find any relationship between curb-65 and vrtis. reasons why higher classes for risk of severe pneumonia at psi but no at curb-65 were associated with vrti are unclear. one possible explanation might rely on the higher sensitivity of psi compared to curb-65 [33] , even if guidelines do not recommend one score over another for patient's discharge [1] . additionally, patients with vrti received pre-admission antibiotic therapy less frequently than those without. this finding may be explained in two ways: first, local and national recommendations are against the use of any antibiotic at the onset of a clinically presumed viral respiratory syndrome; second, a viral respiratory syndrome may actually favour the subsequent onset of a cap. interestingly, in our study cap radiological features were not affected by the presence of vrti. this is consistent with observations that, in some instance, viral and bacterial pneumonia may have similar radiological features [34] it is worth noting that, in spite of higher psi scores in presence of vrtis, we found no association between vrtis and need of antibiotic treatment escalation, icu admission and risk of mortality. other studies investigating clinical outcomes in the same setting found opposite results. yoon [25] found that preceding vrtis do not affect mortality in adults with pneumococcal cap, while voiriot et al. [32] reported that mixed bacterial and viral infections are associated with hospital death and/or prolonged mechanical ventilation in icu-admitted cap patients. moreover, some authors used different surrogate endpoints of clinical response, such as length of hospitalization or length of mechanical ventilation. however, these endpoints may be affected by a variety of factors, including the presence of any comorbidity and the development of superinfection, thus not sufficiently clarify the role of vrtis. on the other hand, as escalation of antibiotic treatment is usually the consequence of lack of response to previous treatment, we evaluated the impact of vrtis on the need of antibiotic escalation. the interplay between virus and bacteria, spanning from the damage of respiratory mucosa to affecting the normal immune response, has been well described, in vitro and in vivo, for either influenza virus [35] [36] [37] or other viruses, in particular rsv and metapneumovirus [38, 39] in addition, beyond favouring bacterial infection, some respiratory viruses may per se cause pneumonia [40] . thus, we stratified patients according to the supposed microbiological aetiology and we found that mixed and viral cap had pneumonia severity at admission, radiological findings and outcomes similar to bacterial cap. however, the viruses detected in the pharynx may represent only infection of the upper respiratory tract, while lower respiratory tract specimens may be more reflective of the pathogen causing pneumonia [40] [41] [42] . in our real-life cohort, bronchoalveolar lavage culture was performed only when clinically indicated. as only a few patients experienced clinical failure and required icu admission or antibiotic escalation, invasive procedures were deemed unnecessary in the majority of cases. additionally, 43% of patients included in our cohort had cap of unknown aetiology, a finding that could be at least partially explained with previous at-home antibiotic treatment before specimens collection. the limited sample size of patients diagnosed with bacterial/viral/mixed cap and the lack of lower respiratory tract sampling in almost all cases prevents any firm conclusion on the clinical presentation and outcome of cap according to the aetiology and deserves further studies. influenza virus was the most frequent viral infection we found in our cohort. while the presence of vrti did not impact on the tta, negative pcr for influenza virus was used for early antiviral treatment discontinuation. indeed, current guidelines recommend universal empirical antiviral treatment in patients with influenza-like illness, whose clinical presentation (i.e. fever plus cough started within the last 10 days) overlaps with that of cap [28] . on the other hand, vrti identification was not useful to guide antibiotic treatment, as demonstrated by the lack of association with tta. beyond the lack of low respiratory tract specimens, the other limitation of this study is that we enrolled both immunocompetent and immunosuppressed patients. as immunosuppression is a risk factor for more severe infections (depending on the type and intensity), our data may overestimate the risk of clinical failure. of note, two out of three patients who died within 30 days from admission for cap had concomitant immunosuppression. in conclusion, in spite of high prevalence rates, vrtis do not negatively affect the outcome of cap. further studies are needed to evaluate the clinical presentation and outcome of mixed and viral cap with respect to bacterial ones in hospitalized patients who do not need intensive care. funding the authors declare no funding foundation. conflict of interest the authors declare that they have no conflict of interest. ethical approval all procedures performed were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 helsinki declaration and its later amendments or comparable ethical standards. the study was approved by the regional ethics committee (regional ethics committee of liguria region, registry number 357re62016). informed consent participants signed specific informed consent. infectious diseases society of america/american thoracic society consensus guidelines on the management of community-acquired pneumonia in adults community-acquired pneumonia community-acquired pneumonia community-acquired pneumonia: the annual cost to the national health service in the uk mortality differences among hospitalized patients with community-acquired pneumonia in three world regions: results from the community-acquired pneumonia organization (capo) international cohort study a case-control study of acute respiratory tract infection in general practice patients in the netherlands respiratory viral infections in infants: causes, clinical symptoms, virology, and immunology acute viral infections of upper respiratory tract in elderly people living in the community: comparative, prospective, population based study of disease burden coronavirus hku1 and other coronavirus infections in hong kong human coronavirus and acute respiratory illness in older adults with chronic obstructive pulmonary disease respiratory viral infections in immunocompetent and immunocompromised persons identification of new respiratory viruses in the new millennium. viruses rapid and sensitive method using multiplex real-time pcr for diagnosis of infections by influenza a and influenza b viruses, respiratory syncytial virus, and parainfluenza viruses 1, 2, 3, and 4 polymerase chain reaction is more sensitive than viral culture and antigen testing for the detection of respiratory viruses in adults with hematological cancer and pneumonia effect of rapid viral diagnosis on the management of children hospitalized with lower respiratory tract infection cost-effectiveness of rapid diagnosis of viral respiratory tract infections in pediatric patients viral infection in community-acquired pneumonia: a systematic review and meta-analysis association of invasive pneumococcal disease with season, atmospheric conditions, air pollution, and the isolation of respiratory viruses influenza alone and in sequence with pneumonia due to streptococcus pneumoniae in the squirrel monkey pulmonary antibacterial defenses during mild and severe influenza virus infection acquired ciliary defects in nasal epithelium of children with acute viral upper respiratory infections insights into the interaction between influenza virus and pneumococcus respiratory viruses predisposing to bacterial infections: role of neuraminidase pneumonia and hong kong influenza: a prospective study of the 1968-1969 epidemic impact of preceding respiratory viral infections on the clinical severity of patients with pneumococcal pneumonia seasonal invasive pneumococcal disease in children: role of preceding respiratory viral infection etiologic diagnosis of adult bacterial pneumonia by culture and pcr applied to respiratory tract samples clinical practice guidelines by the infectious diseases society of america: 2018 update on diagnosis, treatment, chemoprophylaxis, and institutional outbreak management of seasonal influenzaa predicting death in patients hospitalized for community-acquired pneumonia a prediction rule to identify low-risk patients with community-acquired pneumonia systematic review of respiratory viral pathogens identified in adults with communityacquired pneumonia in europe viral-bacterial coinfection affects the presentation and alters the prognosis of severe community-acquired pneumonia prospective comparison of three validated prediction rules for prognosis in community-acquired pneumonia severe adenovirus communityacquired pneumonia in immunocompetent adults: chest radiographic and ct findings the co-pathogenesis of influenza viruses with bacteria in the lung kinetics of coinfection with influenza a virus and streptococcus pneumoniae lethal synergism of 2009 pandemic h1n1 influenza virus and streptococcus pneumoniae coinfection is associated with loss of murine lung repair responses respiratory syncytial virus increases the virulence of streptococcus pneumoniae by binding to penicillin binding protein 1a. a new paradigm in respiratory infection preceding human metapneumovirus infection increases adherence of streptococcus pneumoniae and severity of murine pneumococcal pneumonia identification of respiratory viruses in asymptomatic subjects: asymptomatic respiratory viral infections relation of diagnostic accuracy of viral respiratory polymerase chain reaction to specimen number and source in severe adenovirus pneumonia: antimicrobial stewardship implications diagnosis of influenza in intensive care units: lower respiratory tract samples are better than nose-throat swabs key: cord-286602-cdueeg47 authors: petsonk, edward l.; harber, philip title: respiratory protection for health care workers: a 2020 covid‐19 perspective date: 2020-06-04 journal: am j ind med doi: 10.1002/ajim.23144 sha: doc_id: 286602 cord_uid: cdueeg47 as the us health care system began to respond to the coronavirus disease‐2019 pandemic, demand for respiratory personal protective equipment (ppe) increased precipitously, as did the number of users. this commentary discusses ensuing deviations from accepted respiratory ppe program practices, which potentially increased risk to health care workers. such lapses included omitting user training and fit testing, provision of unapproved devices, and application of devices in settings and ways for which they were not intended. the temporary compromise of professionally accepted standards due to exigencies must not become the new normal. rather, the current attention to ppe should be leveraged to enhance practice, motivate vital research, and strengthen professional, governmental, and institutional capabilities to control health care worker exposures to infectious hazards. in the pandemic of coronavirus disease-2019 (covid19) , health care workers (hcws) have marshaled their skills and courage to care for acutely ill patients, and concurrently experienced a great burden of disease and death. 1 the burgeoning need for respiratory personal protection equipment (ppe) precipitated major challenges to supply chains as need for ppe exceeded the capacity of suppliers and strategic reserves. in the united states, shortages triggered contingency and crisis standards of practice which deviated from conventional and accepted best practices. 2 efforts are underway in the united states to overcome problems with availability and distribution of various ppe. as the role of respiratory protection becomes better defined, and the supply of respirators expands, we believe it is critically important to resume adherence to optimal protective policies and practices. this commentary briefly describes several key areas for which attention is needed. effective respiratory protection for hcws is particularly critical for emerging diseases such as covid-19, with high case fatality rates, and before modes of transmission are adequately defined and vaccines or effective treatments are available. 3 multiple reports suggest that airborne particles with viable severe acute respiratory syndrome coronavirus-2 (sars-cov-2) constitute a perilous threat to hcws. 4 transmission may follow only brief exposures and from persons without noticeable symptoms. 5 covid-19 patient care is frequently associated with aerosol generating activities such as intubation and respiratory therapies. the key principles underpinning respiratory protection are based upon an extensive foundation of professional knowledge and experience including numerous experimental and observational studies over the last century. [6] [7] [8] respiratory ppe can provide protection only when properly selected, fitted to the wearer, and correctly worn and maintained. respiratory protection programs (see table 1 ) offer users specific skills and knowledge, as well as the technical support essential for successful protection. training is particularly key for hcws assigned negative pressure devices, such as filtering facepiece or elastomeric respirators. all hcws (including support staff) with potential airborne sars-cov-2 exposure should be considered for enrollment in a complete respiratory protection program. program specifics and coverage should be adapted based upon expert assessment of personnel hazard levels for each office/facility, from small ambulatory offices to long-term care and critical care units. guidance indicates that surgical masks do not provide hcws a reliable level of protection against infectious aerosols 7 and should not be substituted for n95 respirators. surgical and procedure masks do not seal against the face, and thus permit extensive leakage of aerosols into the breathing zone. studies have shown that many more particles leak around these masks than penetrate through the filters. 9, 10 two randomized trials of surgical masks vs n95's provided conflicting information about equivalence, 11, 12 and although the quality of the evidence was rated as low, some guidelines suggest equivalence. 2 until this controversy is resolved, hcws at risk should be provided with fit tested n95 respirators. in addition, hcw's should never be encouraged to rely upon home sewn or other untested cloth masks and bandanas to protect themselves against covid-19. in the urgency to provide many hcws with respirators, fit testing procedures were relaxed both for initial assignment and annual retesting. 13 to verify respirators can perform as specified, standard fit testing protocols must be reinstituted and enforced. in the face of shortages, single use respirators have been repurposed as reusable equipment. essential considerations include uniform decontamination while maintaining integrity of the filter, strap, and face seal. when faced with critical shortages, evidence-based procedures for disinfection, followed by thorough inspection for deformation, deterioration, or soiling before each use may make limited reuse an acceptable contingency practice. an urgent priority is the documentation of feasible procedures for reliable and safe reuse for each specific respirator product, 14 which also offers potential future cost savings. guidelines are needed for safe collection, storage, and transport of masks, as well as the effective operation and maintenance of decontamination equipment. federal regulations require masks to meet multiple test specifications to be certified as an n95. in the face of initial n95 shortages, the food and drug administration issued an emergency use authorization to accept devices produced by some foreign manufacturers under specific conditions. fraudulent and unsafe devices have been reported, and many have been recalled. this loosening of respirator testing requirements should be withdrawn as the us production capacity increases and imported devices undergo the required assessments. paprs are generally very well-tolerated and are perceived by hcws to be more protective than filtering facepiece respirators (ffrs) in high-risk settings. 15 the high initial purchase cost is offset in part by a reduced ongoing need for disposable ffrs. alternative negative pressure filtering devices such as elastomeric respirators (tight sealing and reusable by design) have higher initial cost than n95s, but may be less expensive to use in the long run. furthermore, whenever respiratory protection is recommended, feasible engineering controls exposures and, therefore, warrant use of a more protective approach. 16, 17 hcw protection in these settings is more reliably assured by a reusable papr functioning within specification, or alternatively, a demonstrably effective vacuum hood or other filtered local exhaust device, in combination with an n95. in addition to the greater assigned protection factor for papr's in comparison with other devices, papr's with a shrouded hood covering the head and neck provide an additional barrier to contamination. respiratory protection programs have multiple components and are designed and managed by professionals with comprehensive technical knowledge of hazard assessment, device selection and maintenance, user ability, fit testing, and program management. occupational health professionals (eg, trained occupational nurses, physicians, and hygienists) provide unique expertise, in cooperation with infection control staff, to guide respirator selection, medical evaluation of users, fit testing, audits, and regulatory compliance. respirators or surgical masks worn continuously over an entire shift may result in noticeable discomforts. as the prominence of covid-19 decreases, hcw discomfort may deter adherence. conversely, the current crisis may provide a strong impetus for increasing support among hcws and leadership for comprehensive respiratory protection programs. hcws are demanding adequate respiratory protection, and this interest should lead to greater adherence and improved practices, as the specter of covid-19 (eventually) abates. the early us recommendations for controlling hcw exposure were proper placing (donning) and removing (doffing) of respirators are essential for reducing the risk of infection to the user and others. this requires specific training and regular supervision for both technique and compliance. it is particularly demanding since hand washing is necessary both before and after placing, fit checking, and removing the device. analyses of recent disease outbreaks (sars 2003 (sars , h1n1 2009 , and mers 2012) have highlighted the disproportionate risk for health care system workers, and the need for strengthening governmental and commercial systems, procedures, policies, and communications, to facilitate more effective protections during anticipated future outbreaks. 8, 18, 19 enhanced collaboration is desirable among the several us and international agencies responsible for testing and approval of respiratory ppe. comprehensive programmatic approaches are essential whenever respiratory protective devices are included as a part of an infectious aerosol exposure control strategy for hcws. prompt action is needed to remedy a number of recognized problems. deficient approaches and short cuts can result in harm to individuals and weaken the country's capacity to provide health care. as the united states continues to progress in managing the multiple challenges of the pandemic, hcws and their families need to be assured they are being provided the best available health protections, based upon state-of-the-art science, proven technologies, effective training, and professional program management. the authors report that there was no funding source for the work that resulted in the article or the preparation of the article. the authors declare that there are no conflicts of interest. dr. john d. meyer declares that he has no conflict of interest in the review and publication decision regarding this article. characteristics of health care personnel with covid-19 -united states infectious diseases society of america guidelines on infection prevention in patients with suspected or known covid-19. infectious diseases society of america facemasks for the prevention of infection in healthcare and community settings aerosol and surface stability of sars-cov-2 as compared with sars-cov-1 covid-19 outbreak associated with air conditioning in restaurant section viii: chapter 2, respiratory protection. osha technical manual national institute for occupational safety and health us) committee on personal protective equipment for healthcare personnel to prevent transmission of pandemic influenza and other viral respiratory infections: current research issues. preventing transmission of pandemic influenza and other viral respiratory diseases: personal protective equipment for healthcare personnel: update performance of an n95 filtering facepiece particulate respirator and a surgical mask during human breathing: two pathways for particle penetration surgical mask filter and fit performance a randomized clinical trial of three options for n95 respirators and medical masks in health workers n95 respirators vs medical masks for preventing influenza among health care personnel: a randomized clinical trial temporary enforcement guidance -healthcare respiratory protection annual fit-testing for n95 filtering facepieces during the covid-19 n95 respirator decontamination and reuse: current state of the evidence user acceptance of reusable respirators in health care california's aerosol transmissible disease standards and local health departments. richmond, ca: california department of public health are powered air purifying respirators a solution for protecting healthcare workers from emerging aerosol-transmissible diseases? ann work expo health a control banding framework for protecting the us workforce from aerosol transmissible infectious disease outbreaks with high public health consequences personal protective equipment supply chain: lessons learned from recent public health emergency responses how to cite this article: petsonk el, harber p. respiratory protection for health care workers: a 2020 covid-19 perspective key: cord-302833-6kntd89t authors: radonovich, lewis j.; bessesen, mary t.; cummings, derek a.; eagan, aaron; gaydos, charlotte; gibert, cynthia; gorse, geoffrey j.; nyquist, ann-christine; reich, nicholas g.; rodrigues-barradas, maria; savor-price, connie; shaffer, ronald e.; simberkoff, michael s.; perl, trish m. title: the respiratory protection effectiveness clinical trial (respect): a cluster-randomized comparison of respirator and medical mask effectiveness against respiratory infections in healthcare personnel date: 2016-06-02 journal: bmc infect dis doi: 10.1186/s12879-016-1494-2 sha: doc_id: 302833 cord_uid: 6kntd89t background: although n95 filtering facepiece respirators and medical masks are commonly used for protection against respiratory infections in healthcare settings, more clinical evidence is needed to understand the optimal settings and exposure circumstances for healthcare personnel to use these devices. a lack of clinically germane research has led to equivocal, and occasionally conflicting, healthcare respiratory protection recommendations from public health organizations, professional societies, and experts. methods: the respiratory protection effectiveness clinical trial (respect) is a prospective comparison of respiratory protective equipment to be conducted at multiple u.s. study sites. healthcare personnel who work in outpatient settings will be cluster-randomized to wear n95 respirators or medical masks for protection against infections during respiratory virus season. outcome measures will include laboratory-confirmed viral respiratory infections, acute respiratory illness, and influenza-like illness. participant exposures to patients, coworkers, and others with symptoms and signs of respiratory infection, both within and beyond the workplace, will be recorded in daily diaries. adherence to study protocols will be monitored by the study team. discussion: respect is designed to better understand the extent to which n95s and mms reduce clinical illness among healthcare personnel. a fully successful study would produce clinically relevant results that help clinician-leaders make reasoned decisions about protection of healthcare personnel against occupationally acquired respiratory infections and prevention of spread within healthcare systems. trial registration: the trial is registered at clinicaltrials.gov, number nct01249625 (11/29/2010). healthcare personnel (hcp) are exposed to respiratory pathogens in many clinical settings [1] . infected hcp may spread infection to their patients [2] [3] [4] [5] or coworkers [3] [4] [5] [6] , to family members [4, 7] , or to other community members [4, 8] . respiratory viral infections among healthcare workers can negatively impact delivery of healthcare services [9] [10] [11] . united states national guidelines call for modes of transmission to dictate infection control measures [3] . for most human respiratory viruses, the precise mode(s) of person-to-person transmission is incompletely understood [12, 13] . the predominant mode of transmission for some human respiratory pathogens, such as influenza virus, respiratory syncytial virus, and coronavirus is believed to be droplet transmission. airborne transmission plays a role with some human respiratory pathogens via small aerosol particles, often called droplet nuclei [3] . airborne transmission is the predominant mode of transmission for mycobacterium tuberculosis [3, 14] and recent evidence has suggested a larger role than previously thought for influenza a and b viruses [15, 16] . disposable respiratory protective devices (rpd) that fit tightly to the wearer's face, sometimes called airpurifying respirators or filtering facepiece respirators, are primarily designed to protect the wearer against infection spread by ill patients. n95 filtering facepiece respirators (commonly known as "n95 respirators") are one type of rpd capable, with proper facial fit and usage, of reducing inhalation of airborne particulates by a factor of 10 or greater [17] . medical masks (mm), typically called surgical masks in operative settings, are primarily devised to protect patients against infection spread by the wearer [18] . both types of devices also serve as a physical barrier keeping sprays and splashes of infectious materials and contaminated hands and objects away from oronasal region of wearer. although rpd and mm are capable of filtering particulates [19] , rpd are designed to filter smaller particulates that may remain airborne for long periods. a tight seal between the respirator and the wearer's face is designed to prevent leakage of particulates, a feature not provided by loose-fitting mm. the u.s. department of labor's occupational safety and health administration (osha) requires employers to ensure each hcp, who may be exposed to airbornetransmissible infections in the workplace, receives an rpd with an adequate respirator-to-face seal that is determined during a mandated annual "fit-test". however, evidence is inconclusive that rpd are better than mm at protecting hcps from respiratory infections in clinical settings [20] [21] [22] [23] [24] [25] , despite tight-fitting rpd produced by manufacturers, with higher levels of exposure reduction validated by numerous laboratory studies [19, [26] [27] [28] , and the use of a complete respiratory protection program (e.g., training, initial and annual fit test) as defined by osha to protect hcp. intuitively, rpd should better protect hcp against airborne infections than mm, but objective evidence has not validated this supposition. one possibility that may explain this discrepancy between expectations and observations is pragmatic: hcp, in general, do not tolerate n95 respirators as well as medical masks [29, 30] , perhaps prompting them to remove respirators more frequently and/or for longer periods, increasing the likelihood of exposure to infections. models have shown that 25 % or more non-wear time during exposure negates any significant differences in protective ability between types of rpds [17, 31] . given the difficulty with hcp adherence to guidelines [4] and general dissatisfaction [4, [32] [33] [34] with rpd, medical masks worn more consistently may provide similar levels of reduction in respiratory viral disease transmission as n95 respirators. this key gap in knowledge has contributed to discrepant clinical and public health recommendations about respiratory protection for hcp [35, 36] . needed are additional well-designed clinical trials conducted in patientcare settings during outbreaks of respiratory infections. the following is an abridged version of the full research protocol for the respiratory protection effectiveness clinical trial (respect). to compare the effectiveness of n95 respirators and medical masks at protecting hcp from acquiring viral respiratory illnesses in the workplace. null hypothesis: the incidence of laboratory confirmed influenza (primary), influenza-like illness (ili), acute respiratory illness (ari) and other respiratory infections will not be different between hcps who practice 2007 guidelines (medical masks) or 2009 guidelines (n95 respirators). alternative hypothesis: the incidence of laboratory confirmed influenza (primary), influenza-like illness (ili), acute respiratory illness (ari) and other respiratory infections will be different between hcps who practice the cdc's 2007 guidelines for influenza protection (medical masks) versus 2009 guidelines for influenza protection (n95 respirators). respect is a prospective comparison of respiratory protective equipment to be conducted at multiple, geographically distributed u.s. study sites. hcp who work in outpatient settings will be cluster-randomized to wear n95 respirators [37] or mm [38] for protection against infections during respiratory virus season, the "intervention" period. the null hypothesis assumes n95 and mm intervention groups will have no differences in outcomes, including (1) laboratory confirmed influenza or (2) influenza-like illness (ili), (3) acute respiratory illness (ari), and (4) laboratory confirmed respiratory illness (lcri). the alternative hypothesis asserts the incidence of at least one outcome would be different between intervention groups. because respiratory virus season varies year-to-year in onset, severity, and duration, multiple season-years of the study will be necessary to account for expected variance and optimally generalize the resulting knowledge. the beginning of each season's data collection will be independently determined for each study site using an epidemiologic predictive tool designed for respect to capture the largest possible number of respiratory infections. these data will be collected for twelve weeks each season. participant exposures to patients, coworkers, and others with symptoms and signs of respiratory infection, both within and beyond the workplace, will be recorded in daily diaries. adherence to study protocols will be measured by the study team at each site. since periodic changes in infection control guidance and practice may occur over the study years, participants will be expected to adhere to the most up-to-date guidance issued by the centers for disease control and prevention (cdc) and local policies at each study institution, at a minimum. for example, a participant randomized to the mm arm will be expected to don an n95 when participating in an aerosol-generating procedure, assuming no further changes in pertinent national guidance [39] . the study participants will be recruited from outpatient settings where patients are relatively likely to present with symptoms and signs of acute respiratory infection. participants will be eligible to enroll for multiple study seasons, yet each will be provided with informed consent and complete enrollment procedures prior to each study season. clinical study sites will be distributed geographically: participants will be cluster-randomized to one of the following n95 respirators or mm models, selected because they are commonly used in u.s. medical facilities, including the respect study sites. participants who participate in more than one of the study years will be cluster-randomized anew each year. n95 respirators: (1) precept 15320 or (2) kimberly clark technol fluidshield 47107. all subjects participating in the study will be required to pass an osha-accepted respirator fit test for the n95 respirator model(s) available at the study site. no fit testing of medical masks will be performed as these devices are not designed to be tight-fitting to the face and studies [19, 20] have shown that their fit capabilities are generally low. filter performance although medical masks are loose-fitting, they create a physical barrier that helps prevent splashes and sprays from reaching the wearer's mucous membranes. in addition to passage around the mask, some of the small particle aerosols are able to pass through the mask's filter media. therefore, in addition to rpds, filtration testing was done on medical masks prior to enrollment of subjects to ensure consistency between models across study locations. the filtration performance of the n95 respirators and medical mask models in the study were tested in a manner similar to that used by the national institute for occupational safety and health (niosh). devices were attached to a test fixture and placed in a tsi 8130 automated filter tester operated with an air flow rate of 85 liters per minute. the tsi 8130 uses a photometer to measure the flux of light scattering from aerosol particles. polydispersed particles (mass median diameter of~0.3 microns) were generated from a 2 % nacl solution and passed through each device being tested for 1 min. each test was repeated 3 times with a fresh n95 respirator or medical mask. to be certified as an n95 respirator, filter penetration needs to be less than 5 % (or 95 % efficient). as shown in table 1 , the average penetration percentages for the niosh certified n95 respirators were an order of magnitude lower than those of medical masks, which are not niosh certified. filter results between n95 respirator models and between medical mask models were comparable. filter airflow resistance was measured simultaneously using the tsi 8130. as filter airflow resistance increases, more energy expenditure is required for ventilation during device wear and the greater potential for perception of discomfort [40] . the medical mask models selected for this study have filter airflow resistance levels about half of that of the n95 respirators. however, one study [40] found that subjective and physiological responses were not different among subjects exercising while wearing devices purposely made with different filter airflow resistance levels (3 mm h 2 o, 6 mm h 2 o, and 9 mm h 2 o) in the range similar to those of the devices in this study (table 1) . participants will be instructed to don a new n95/mm with each patient interaction, every time a participant encounter occurs within 6 feet of a patient who has suspected or confirmed respiratory infection. hand hygiene will be recommended to all participants in accordance with cdc guidelines [41] and policies at each study institution. trained research assistants will observe participants during study periods to assess adherence to their assigned intervention arms and hand hygiene. a portable computer equipped with data recording software (handyaudit; toronto, canada) will be used to document adherence. participants will be expected to complete surveys about their attitudes and opinions concerning personal protective equipment before and after each seasonal study period. during the twelve week data collection period each year, participants will self-document (a) perceived occupational exposures to patients or coworkers who have symptoms or signs of respiratory infection, (b) perceived anterior nasal and pharyngeal swabs [42] [43] [44] [45] [floqswabs utm (99-08024), diagnostic hybrids; athens, oh] will be collected by research assistants when symptomatic with study defined respiratory symptoms, as well as two, randomized asymptomatic swabs during each seasonal study period. swabs will be collected when (a) participants self-report respiratory symptoms within a 24 h period, and again if participants remain symptomatic after 7 days; and (b) randomly, on all participants, twice during the active intervention period. the primary outcome measures will be the incidence of: laboratory-confirmed influenza (lci) a or b infection in participants, defined as a) detection of influenza virus by reverse-transcription polymerase chain reaction (rt-pcr) in an upper respiratory specimen swab collected within seven days of symptom onset, or b) influenza seroconversion defined as at least a 4-fold rise in hemagglutination inhibition antibody (hai) titers to influenza a or b virus from the pre-to post-season serological samples that is not deemed attributable to vaccine. the secondary outcome measures will be the incidence of: (1) acute respiratory illness (ari) defined as the occurrence of one sign or two symptoms ( the incidence rate ratios between participants randomly assigned to wear n95 respirators or medical masks will be estimated for each of the primary and secondary outcomes. investigators will be paired and provided with blinded information about clinical and laboratory data to determine if a participant meets a primary or secondary outcome. if the paired investigators do not agree, a principal investigator will adjudicate the outcome. assays will be performed at johns hopkins university. collected respiratory specimens will be stored at −80°c until analyzed using multiplex pcr (plex-id, abbott labs, chicago il). automated extraction of nucleic acid (na) from respiratory specimens will be performed utilizing nordiag's arrow instrument and the magna pure robotic system (roche indianapolis, in) per manufacturer instructions. each extraction run will include a quality control (natrol respiratory validation panel 3, zeptometrix inc., buffalo ny); runs with control failures will be repeated. purified na will be amplified via rt-pcr using a broad respiratory virus identification kit (plex-id rvs 3.0, abbott molecular, des plaines, il). desalting of rt-pcr product and electrospray mass spectrometry-based na analysis will be performed on the plex-id analyzer instrument. if funding is sufficient, samples will also be assayed by rt-pcr for bordetella pertussis, mycoplasma pneumoniae, and for chlamydophila pneumoniae. each study season, blood samples will be collected twice from each participant; one sample will be collected within two weeks of the beginning of the intervention period and a second within two weeks of the end of the intervention period. hemagglutination inhibition (hai) antibody assays will be performed on serum for influenza a and b virus strains, dependent on the antigens in each annual trivalent vaccine using standard methods [46, 47] . in brief, serial 2-fold dilutions of serum samples will be incubated with 8 hemagglutinin units of influenza antigen and a turkey red blood cell suspension. the serum hai antibody titer will be defined as the dilution factor of the highest serum dilution that completely inhibits agglutination of turkey red blood cells in the presence of type-specific hemagglutinin antigen. assays will be performed at the immunology core lab for the study at the va saint louis veterans affairs healthcare system. to optimize compliance and generalizability, a clusterrandomized design will be utilized. all participants working in the same clinical unit will be assigned to wear the same respiratory protective equipment (i.e., an n95 or mm) during patient interactions for the entire 12 week seasonal study period. clusters will be pairmatched within each study site based on the characteristics of each clinical cluster, including the (a) number of participants (b) occupational location, such as an emergency department, urgent care or primary care, (c) patient population served, such as children or adults, and (d) requirements for participants to wear additional protective equipment, such as goggles donned by dental hygienists. for each study season, the clinics in each matched pair will be randomly assigned to opposing study arms. for matched pairs participating in multiple study seasons, random sequences of arm assignments will ensure each is assigned to both study arms during the multi-year study. each study season, an individual not involved in the study implementation and data analyses will perform the randomization scheme for each study site, using a random number generator in microsoft excel. the principal investigators will be blinded to the randomization scheme prior to assignment. incidence rates of lci, ari, ili, and lcri among cluster-randomized participants will be compared. the relationships between incidence of clinically diagnosed and laboratory-confirmed illnesses will be analyzed with attention to potential confounders, such as participants' demographics, study arm compliance, attitudes and opinions about infection control, receipt of influenza vaccination, and infectious exposures within and beyond the workplace. standard statistics will describe baseline characteristics and follow-up measures, summarized by treatment arm and stratified by study site. to assess the primary outcome, a logistic regression model will be fit using a dichotomous variable to indicate whether a participant became infected with a respiratory pathogen. the odds of infection between the two treatment groups will be reported with a 95 % confidence interval. for secondary outcomes, poisson loglinear mixed effects regression models will assess the difference in seasonal respiratory infection rates between intervention groups. cluster-and individual-level random effects will be considered to account for clustered observations. additional covariates may be added to the models to adjust for confounding. participants will be encouraged to complete the study. those who withdraw from an intervention arm will be encouraged to complete follow-up laboratory specimen collection. an intent-to-treat analysis, in which all available data on all randomized participants are included, will be used for the primary comparison of interventions. a per-protocol secondary analysis will compare treatment effectiveness, accompanied by a planned sensitivity analysis that accounts for participants from whom researchers were not able to obtain a second serological sample. to detect a 25 % reduction (i.e., a relative risk of 0.75) in the incidence of laboratory confirmed influenza or laboratory confirmed respiratory illness among participants wearing an n95 respirator, compared to participants wearing a medical mask, respect will need to accumulate approximately 10,024 or 5104 personseasons of data over four seasons respectively. sample size calculations are based on several assumptions about the incidence rate and levels of withincluster correlation. the attack rate laboratory-confirmed influenza during a single study season is assumed to be 20 % among unvaccinated individuals in the medical mask group. we assume 65 % of our population will be administered a vaccine that is 65 % effective in preventing influenza infection. vaccine effectiveness at the higher end of published reports (86 % in health care workers) will lead to a reduction in the yearly attack rate to approximately 8.8 %, and effectiveness at the lower end of published reports (51 % in the general population) would lead to an increased yearly attack rate of approximately 13.4 %. importantly, the anticipated effect on the needed sample size of annual variations in influenza incidence is larger than the expected impact of variation in vaccine effectiveness. the respect study will need 157 independent clusters with a median size of 16 participants each to achieve 80 % power to detect a relative risk of 0.75 between n95 and surgical masks at preventing laboratory-confirmed influenza infection, with a type-i error rate of 0.05. the total number of individuals participating each season will need to be approximately 2506, with 10,024 total person-seasons accumulating over the multi-year study. for the secondary outcome of laboratory confirmed respiratory illness, the estimated total number of clinics will need to be 80, the total number of individuals participating each season will need to be 1276, and total person-seasons accumulated need to be 5104 (table 4) over the multi-year study. the sample size are made using the clusterpower software package for r [48] . power is estimated using an expected annual attack rate of 12 % {12 % = 0.35*0.2 + 0.65*(1-0.65)*0.2} [13] . this yearly attack rate translates into a 4-year attack rate of 39 % {39 % = 1-(1-(0.35*0.2 + 0.65*0.35*0.2)) 4 . accounting for correlation of outcomes within clusters by assuming the correlation coefficient is 0.1, leads to a design effect of 2.5. for scenarios representing the lower and higher ends of anticipated attack rates in the medical mask group, two quantities were calculated (a) the power to detect a relative-risk of 0.75 between the n95 group and the medical mask group and (b) the relative-risk that can be detected with 80 % power (table 5 ). for all of these calculations the two-sided type i error probability is 0.05. potential outcome analysis for laboratory-confirmed influenza some data on the primary outcome may be missing due to participants withdrawing from the study early and missing the second serological sample. to account for the unavoidable uncertainty posed by missing primary outcome data, due to participant withdrawal or loss of follow-up, a sensitivity analysis will be conducted that randomly assigns binary outcomes to participants who did not complete the study. a two-dimensional grid will be created that varies the influenza attack rates among participants who withdraw. withdrawal attack rates in both arms will be fixed between half and twice the observed attack rates, based on complete data. by varying these two parameters across the grid, and for each combination, the adjusted odds ratio will be calculated by averaging across n = 50 imputed datasets for each point on the grid. analysis of differential withdrawal the characteristics at the time of randomization for participants without complete follow-up will be examined. to assess the potential biases introduced by differential withdrawal among different n95 respirators, a comparison of withdrawal rates and time to withdrawal will be included as an ancillary analysis to the analyses of the primary and secondary outcomes. respect will be approved by the institutional review board at each participating study site and the centers for disease control and prevention, prior to study initiation. (an unabridged version of the respect protocol was approved by the intitutional review board at each study site and the centers for disease control and prevention). viral respiratory infections cause a wide range of illnesses, varying from mild to severe, in hcps who may spread infection to their patients, family members, and other community members. healthcare-associated infections cost $10b annually in u.s [49] . factors influencing transmission of respiratory infections in healthcare facilities include the population density of ill patients in healthcare settings, the types of exposures within healthcare settings, the administrative and physical structures of healthcare facilities, and intrinsic characteristics of virulence [3] . measures to prevent transmission within healthcare facilities include hcp vaccination, handhygiene, cleaning and disinfection of inanimate surfaces, pre-and post-exposure antiviral chemoprophylaxis, patient isolation, and personal protective equipment [3, 6] . respect is designed to better understand the extent to which ppe, specifically represented by differences in exposure reduction afforded by n95s and mms, reduces clinical illness among hcps. while it may seem that n95 respirators should better protect hcps than mm against airborne infections in the workplace, this notion has not been validated by objective clinical evidence. low tolerance to respirator wear among hcps may prompt more frequent or longer periods of removal, compared to mm, to an extent that the benefits of higher levels of filtration and lower levels of leakage around the facial seal afforded by respirators is offset or subjugated. key sources of variability in hcp health outcomes are difficult to control for, even in a rigorously designed clinical study such as respect. for example, the inability to prevent hcp community exposures to respiratory infections and the inherent year-to-year variation of viral respiratory infections provide a challenging setting in which to evaluate the effectiveness of personal protective equipment. while community-acquired infections may pose a significant source of exposure for hcps, this type of exposure, if occurring non-differentially between study arms, would bias the results from respect towards the null hypothesis. key reasons for choosing a cluster-randomized design are (a) to increase compliance by equipping all members of a healthcare team with the same equipment and (b) to capture indirect effects of the intervention at the cluster-level, such as herd immunity [50] . a fully successful study would produce clinically relevant results that help clinician-leaders make reasoned decisions about protection of hcps against occupationally acquired respiratory infections and prevention of spread within healthcare systems. abbreviations ari, acute respiratory illness; cdc, centers for disease control and prevention; dsmb, data safety monitoring board; hai, hemagglutination inhibition antibody; hcp, healthcare personnel; ili, influenza like illness; lcri, laboratory confirmed respiratory illness; mm, medical mask; n95, n95 respirator; niosh, national institute for occupational safety and health; osha, occupational safety and health administration; ppe, occupational protective equipment; respect, respiratory protection effectiveness clinical trial; rpd, respiratory protective devices; rt-pcr, reverse-transcriptase polymerase chain reaction; us, united states. occupationally acquired infections in health care workers. part i healthcare providers as sources of vaccine-preventable diseases guideline for isolation precautions: preventing transmission of infectious agents in health care settings preparing for and influenza pandemic: personal protective equipment for healthcare workers health care-associated infection outbreak investigations by the centers for disease control and prevention transmission of influenza: implications for control in health care settings risk of transmission of severe acute respiratory syndrome to household contacts by infected health care workers and patients surveillance network of catalonia spain tm. implication of health care personnel in measles transmission disruption of services in an internal medicine unit due to a nosocomial influenza outbreak the potential influence of common viral infections diagnosed during hospitalization among critically ill patients in the united states outbreak of pandemic 2009 influenza a/h1n1 infection in the hematology ward: fatal clinical outcome of hematopoietic stem cell transplant recipients and emergence of the h275y neuraminidase mutation epidemiology of viral respiratory infections how contagious are common respiratory tract infections? aerodynamics of droplet nuclei aerosol transmission is an important mode of influenza a virus spread modes of transmission of influenza b virus in households commentary: the use of respirators to reduce inhalation of airborne biological agents disposable surgical face masks for preventing surgical wound infection in clean surgery. the cochrane library surgical mask filter and fit performance surgical mask vs n95 respirator for preventing influenza among health care workers: a randomized trial a randomized clinical trial of three options for n95 respirators and medical masks in health workers efficacy of face masks and respirators in preventing upper respiratory tract bacterial colonization and co-infection in hospital healthcare workers n95 respirators or surgical masks to protect healthcare workers against respiratory infections: are we there yet? simulated workplace performance of n95 respirators respiratory protection for healthcare workers in the workplace against novel h1n1 influenza a: a letter report simulated workplace protection factors for half-facepiece respiratory protective devices performance of an n95 filtering facepiece particulate respirator and a surgical mask during human breathing: two pathways for particle penetration professional and home-made face masks reduce exposure to respiratory infections among the general population respirator tolerance in health care workers discomfort and exertion associated with prolonged wear of respiratory protection in a health care setting b95: a new respirator for health care personnel reusability of facemasks during an influenza pandemic: facing the flu health care workers' views about respirator use and features that should be included in the next generation of respirators differences in the compliance with hospital infection control practices during the 2009 influenza h1n1 pandemic in three countries institute of medicine committee on respiratory protection for healthcare workers in the workplace against novel h1n1 influenza a letter to president obama on federal ppe guidance interim recommendations for facemask and respirator use to reduce 2009 influenza a (h1n1) virus transmission guideline for isolation precautions: preventing transmission of infectious agents in healthcare settings prevention strategies for seasonal influenza in healthcare settings impact of low filter resistances on subjective and physiological responses to filtering facepiece respirators hand hygiene task force: guideline for hand hygiene in health-care settings: recommendations of the healthcare infection control practices advisory committee and the hicpac/shea/apic/idsa hand hygiene task force comparison of combined nose-throat swabs with nasopharyngeal aspirates for detection of pandemic influenza a/h1n1 2009 virus by real-time reverse transcriptase pcr nasal swab versus nasopharyngeal aspirate for isolation of respiratory viruses comparative study of nasopharyngeal aspirate and nasal swab specimens for detection of influenza comparing nose-throat swabs and nasopharyngeal aspirates collected from children with symptoms for respiratory virus identification using real-time polymerase chain reaction diagnostic procedures for viral, rickettsial, and chlamydial infections concepts and procedures for laboratory based influenza surveillance. atlanta: world health organization collaborating centers for reference and research in influenza clusterpower: power calculations for cluster-randomized and cluster-randomized crossover trials health care-associated infections: a meta-analysis of costs and financial impact on the us health care system design and analysis issues in cluster-randomized trials of interventions against infectious diseases we wish to thank the members of the respect team (complete list to be provided prior to publication). respect is jointly funded by the u.s. all authors read and approved the final manuscript. all authors meet icmje guidelines. lr and tp conceived and designed the study, coordinated and supervised the study and drafted the manuscript. mb, ae, cn, mr, cs, and ms designed the study, coordinated and supervised the study and drafted the manuscript. dc designed the study, conceived and designed the epidemiologic and statistical analyses and drafted the manuscript. gg and cg designed the study, conceived and designed laboratory analyses and drafted the manuscript. nr designed the study, conceived and designed epidemiologic and statistical analyses, coordinated and supervised the study and drafted the manuscript. rs designed the study and drafted the manuscript. the authors declare that they have no competing interests. the findings and conclusions in this manuscript are the authors' own and do not necessarily represent the views of the national institute for occupational safety and health, the u.s. department of veterans affairs, or other affiliates. mention of product names does not imply endorsement. submit your next manuscript to biomed central and we will help you at every step: key: cord-280857-0o1ikwks authors: goligher, ewan c.; jonkman, annemijn h.; dianti, jose; vaporidi, katerina; beitler, jeremy r.; patel, bhakti k.; yoshida, takeshi; jaber, samir; dres, martin; mauri, tommaso; bellani, giacomo; demoule, alexandre; brochard, laurent; heunks, leo title: clinical strategies for implementing lung and diaphragm-protective ventilation: avoiding insufficient and excessive effort date: 2020-11-02 journal: intensive care med doi: 10.1007/s00134-020-06288-9 sha: doc_id: 280857 cord_uid: 0o1ikwks mechanical ventilation may have adverse effects on both the lung and the diaphragm. injury to the lung is mediated by excessive mechanical stress and strain, whereas the diaphragm develops atrophy as a consequence of low respiratory effort and injury in case of excessive effort. the lung and diaphragm-protective mechanical ventilation approach aims to protect both organs simultaneously whenever possible. this review summarizes practical strategies for achieving lung and diaphragm-protective targets at the bedside, focusing on inspiratory and expiratory ventilator settings, monitoring of inspiratory effort or respiratory drive, management of dyssynchrony, and sedation considerations. a number of potential future adjunctive strategies including extracorporeal co(2) removal, partial neuromuscular blockade, and neuromuscular stimulation are also discussed. while clinical trials to confirm the benefit of these approaches are awaited, clinicians should become familiar with assessing and managing patients’ respiratory effort, based on existing physiological principles. to protect the lung and the diaphragm, ventilation and sedation might be applied to avoid excessively weak or very strong respiratory efforts and patient-ventilator dysynchrony. lung and diaphragm-protective mechanical ventilation is a novel approach that aims to limit side effects of mechanical ventilation in critically ill patients. this approach integrates the principles of lung-protective ventilation with the new concept of diaphragm-protective ventilation in an effort to simultaneously protect both organs. the approach centers on optimizing patient respiratory effort to avoid lung and diaphragm injury while maintaining acceptable respiratory homeostasis. ultimately, the goal of the approach is to reduce the duration of mechanical ventilation, enhance survival, accelerate recovery, and prevent long-term disability in patients with acute respiratory failure. (ventilator-induced lung injury, vili), patient breathing effort (patient self-inflicted lung injury, p-sili), or both together are generating the forces applied to the lung [2] . bedside measures of stress are available (changes in transpulmonary pressure, driving pressure), but not for measuring the resulting strain, making it challenging to appropriately individualize mechanical ventilation settings to maximize lung protection. furthermore, even if global stress can be measured quite precisely using transpulmonary pressure calculated from airway and esophageal pressure, the effect of gravity on the edematous lung makes the distribution of collapse and aeration very uneven between the dependent and non-dependent lung regions; therefore, global indices do not reflect regional stress or strain. to minimize total stress and strain, dependent regions (usually prone to atelectasis) often require recruitment while non-dependent regions (usually well ventilated) require relief of overdistension. during invasive ventilation, tidal volume (v t ) is routinely scaled to predicted body weight (pbw), which correlates with lung volume in healthy subjects. this correlation is much less accurate in patients with acute respiratory distress syndrome (ards) because of alveolar flooding and atelectasis, resulting in a "baby lung" much smaller than the predicted lung volume [3, 4] . using the driving pressure to scale tidal volume to respiratory system compliance (crs, v t /crs = airway driving pressure, δpaw) is particularly attractive because crs is affected by the aerated lung size and could, therefore, better reflect the global strain (v t / baby lung). driving pressure correlates with ards outcomes among patients with the same v t /pbw [5] and may be useful to guide tidal ventilation, although its role remains to be tested in a prospective trial. it should be acknowledged that static airway pressure is not a very reliable marker of lung stress (both at end-inspiration and end-expiration), because it reflects contributions from both the lung and chest wall (two pressures acting in series). obese patients are an example where high intrathoracic pressure (and therefore higher plateau pressure) exist because of the weight imposed by the chest wall [6] . lung stress is preferably measured as transpulmonary pressure (p l ), which allows to quantify the contribution of the lung and chest wall to changes in airway pressure. the respiratory muscle pump drives alveolar ventilation and is composed of a number of skeletal muscles acting in a highly organized fashion. the diaphragm is the primary muscle of inspiration and the lateral abdominal wall muscles are the most prominent expiratory muscles [7] . mechanical ventilation is employed to unload the respiratory muscle pump and limit the consequences of high breathing effort (e.g., dyspnea sensation, respiratory failure, possible respiratory muscle injury). however, mechanical ventilation delivered as the predominant breathing source can also lead to diaphragm atrophy and injury with a substantial deleterious impact on patient outcome [8] . clinical studies demonstrate that after 24 h of mechanical ventilation, 64% of patients exhibit diaphragm weakness [9] and at the time of weaning, diaphragm weakness is present in up to 80% of patients with weaning difficulties [10] . while many factors contribute to diaphragm weakness in the critically ill [11] , both excessive and insufficient respiratory muscle unloading rapidly result in deleterious changes in diaphragm structure and function [11] . low respiratory muscle effort, due to ventilator over-assist or sedation, may result in muscle atrophy, while high effort has been associated with load-induced injury (fig. 1) . in a landmark study, levine et al. demonstrated the development of diaphragm disuse atrophy in brain dead patients on controlled mechanical ventilation [12] and subsequent studies confirmed the presence of time-dependent fiber atrophy in the diaphragm of ventilated patients [13, 14] . in line with these findings, ultrasound studies demonstrated that low diaphragm effort during mechanical ventilation is associated with time-dependent development of atrophy [15] and that atrophy is associated with poor outcomes [8] . it may be hypothesized that patients are at risk of developing load-induced diaphragm injury, as suggested by the presence of fiber injury, sarcomeric disruption, inflammation and contractile dysfunction in biopsies [13] and acute increases in diaphragm thickness on ultrasound [15] this hypothesis requires further confirmation. taken together, these considerations suggest that the diaphragm might be protected by titrating ventilation and sedation to restore early diaphragm activity while avoiding excess respiratory effort. the various lines of physiological and clinical evidence suggesting that a respiratory effort level similar to that of resting quiet breathing is probably optimal for both lung and diaphragm protection were recently summarized elsewhere [16] . this review explains the principles of lung and diaphragm-protective mechanical ventilation. the overall aim of this approach is to limit the adverse effects of mechanical ventilation on the lung and the diaphragm at the same time. this requires understanding of the pathophysiology of ventilator-induced lung injury, critical illness-associated diaphragm weakness and especially respiratory drive. we discuss clinical applicable techniques to monitor lung and diaphragm function, and how to use these techniques to optimize ventilator settings and sedation. future techniques that allow to control respiratory drive are discussed. to implement lung and diaphragm-protective mechanical ventilation, the variables that mediate injury, principally lung stress and respiratory effort, should be monitored. the available monitoring techniques, their advantages and disadvantages, and proposed specific targets are summarized in table 1 . airway driving pressure, δpaw (i.e., plateau pressure-peep tot ), is a measure that aims to estimate global tidal lung stress [5] . δpaw can be measured either during controlled or assisted ventilation by manual or automated short end-inspiratory and end-expiratory occlusions [17] [18] [19] importantly, δpaw is determined by transpulmonary driving pressure (δp l ) and driving pressure across the chest wall (δp cw ); thus changes in chest wall elastance affect δpaw, without affecting lung stress [20] . because pendelluft and regional variations in lung stress are "dynamic" phenomena that cannot be detected under static conditions, the risk of excess regional lung stress during assisted breathing may be more accurately estimated by dynamic δp l (δp l,dyn , peak p l -end-expiratory p l ) rather than by static measures like δpaw [21, 22] . esophageal pressure (pes) monitoring, as an estimate of pleural pressure, can provide information about both the predisposition to end-expiratory collapse and atelectasis (end-expiratory p l ) and alveolar overdistension within the baby lung (elastance-derived plateau p l ) [23] . monitoring and controlling respiratory muscle effort are major challenges in implementing lung and diaphragm-protective mechanical ventilation. the gold standard to quantify global respiratory muscle effort is the esophageal pressure-time product (ptp), while the ptp of the transdiaphragmatic pressure (pdi, i.e., difference between gastric pressure (pga) and pes) during inspiration provides a measure of diaphragmatic effort [24] . the amplitude of pes or pdi during tidal breathing provides a simple estimate of the pressure generated by all respiratory muscles (pes), or the diaphragm (pdi), whereas the expiratory increase in pga reflects expiratory muscle activity. the diaphragm electrical activity (eadi) is the most precise surrogate of respiratory drive and correlates with indices of effort [25] but with considerable variability between patients. also, values for peak eadi in young healthy subjects during tidal breathing may vary between 4 and 29 μv [26] . nevertheless, changes in eadi are useful to monitor changes in patient's respiratory drive and effort, especially to identify patients at risk for ventilator over-assistance. finally, pes or eadi can complement ventilator waveform analysis to facilitate the identification of patient-ventilator dyssynchronies. other less invasive techniques are available to monitor patient breathing efforts during mechanical ventilation at the bedside. airway occlusion pressure (p 0.1 ), the deflection in paw during the first 0.1 s of an inspiratory effort against an occluded airway, is an estimate of the respiratory drive and can be used to detect both very low and high effort [27] . the maximum deflection of paw during a whole breath occlusion (δpocc) has been recently shown to accurately detect excessive respiratory muscle pressure (pmus) or δp l,dyn ; this maneuver can also be used to assess different forms of patient-ventilator dyssynchrony [28, 29] . ultrasound can be used to visualize and quantify the thickening of the diaphragm during inspiration in the zone of apposition (thickening fraction, tfdi) [30] . tfdi provides an index of diaphragmatic contractility and correlates reasonably well with inspiratory effort (δpes) and eadi [31] . in conclusion, although estimation of pleural pressure using an esophageal balloon appears to be the preferred technique to quantify lung stress and respiratory effort, the technique is currently not widely implemented; moreover, the potential impact on patient outcome remains to be determined in clinical studies. we suggest routine monitoring of tidal volume, inspiratory plateau pressures and airway driving pressure to limit lung injury, and p0.1 to monitor respiratory drive and prevent inadequate effort (table 1) . several strategies can be used to facilitate lung and diaphragm protective ventilation, including modulation of ventilator inspiratory and expiratory assist, drugs that modify respiratory drive and/or effort, extracorporeal co 2 removal (ecco 2 r) and electrical stimulation of the respiratory muscles, as shown in fig. 2 . here, we will briefly discuss these different strategies. a lung and diaphragm-protective ventilation approach aims to minimize lung stress and strain while limiting diaphragm atrophy and injury. to achieve these goals, inspiratory ventilator settings can be adjusted to (1) modulate the patient's inspiratory effort, (2) minimize the dynamic lung stress, and (3) prevent or correct patient-ventilator dyssynchrony or any form of mismatch between needs and support. titrating the inspiratory ventilator settings to optimize respiratory effort requires a thorough understanding of the control of breathing under mechanical ventilation [32, 33] , acknowledging that the control of breathing system responds to changes in ventilatory demands by modifying inspiratory effort (and thus tidal volume) to a greater extent than respiratory rate [34] . therefore, the inspiratory ventilator settings will affect the inspiratory effort by modifying the delivered tidal volume, and thus, in spontaneously breathing patients, increasing pressure or volume assist will increase the delivered tidal volume and reduce the inspiratory effort (as respiratory drive depends mainly on the chemoreflex control of arterial ph). excessive assist, resulting in a tidal volume that is higher than the patient's demands, may almost abolish the patient's the inspiratory effort, and as such promote diaphragmatic atrophy. however, increasing inspiratory support may not attenuate inspiratory effort in the presence of high respiratory drive due to stimuli other than arterial ph/paco 2 , such as pain, anxiety, or stimulation of peripheral lung receptors by lung edema or inflammation [32] . in such case, transpulmonary pressure (and hence dynamic lung stress) may progressively increase with increasing inspiratory support. increasing fio 2 to increase pao 2 and reduce the hypoxic stimulus to breathe may alleviate increased respiratory drive in some patients (hyperoxemia is not required to achieve this effect) [35] . in a volume-targeted mode, the patient's effort will be modified mainly by the set tidal volume and the flow delivery profile (flow pattern and peak flow). in pressuretargeted modes, the delivered tidal volume, and thus the patient's inspiratory effort, is influenced by the set inspiratory pressure, rise time and cycling-off criterion, and of course the mechanical properties of the respiratory system [36] . irrespective of the mode of assist, the delivered tidal volume and respiratory effort will together determine global and regional lung stress, depending on the mechanical properties of the respiratory system [37] . neurally adjusted ventilatory assist (nava) delivers inspiratory assist proportional to the electrical activity of the diaphragm [38] . increasing inspiratory assist will reduce diaphragm electrical activity (and vice versa) over a wide range of respiratory demand, and consequently tidal volume remains relatively stable over a wide range of assist [39] . in theory, pulmonary reflex mechanisms prevent patients from spontaneously inspiring large tidal volumes and nava may therefore facilitate lung-protective ventilation. also, diaphragm inactivity due to overassistance is unlikely in nava, as low diaphragm activity will immediately reduce inspiratory assist. future studies should confirm the role of nava in lung and diaphragmprotective ventilation, but recent randomized trials suggest clinical benefit of nava (reduced time on the ventilator) compared to pressure support mode [40, 41] . the expiratory ventilator setting (i.e., positive end-expiratory pressure, peep) has been traditionally adjusted to optimize oxygenation and/or lung mechanics [42, 43] . a higher peep ventilation strategy (of which there are several, generally resulting in 15 ± 4 cmh 2 o) is currently recommended over lower peep (approximately 9 ± 3 cmh 2 o) in moderate and severe ards [44] . in the presence of spontaneous breathing during mechanical ventilation, a higher peep strategy offers several additional potential advantages to facilitate lung and diaphragm-protective ventilation (fig. 1) . first, in patients with significant lung recruitability, peep reduces the amount of atelectatic 'solid-like' lung and, therefore, can achieve a more homogeneous distribution of the tidal pleural pressure swing (∆ppl) over the whole lung surface following a diaphragmatic contraction. the even distribution of inspiratory dynamic stress can diminish injurious asymmetric inflation associated with spontaneous effort (i.e., pendelluft), reducing regional lung stress in dependent lung regions [45] . second, by increasing endexpiratory lung volume, forcing the diaphragm to operate at a shorter length and thereby impairing diaphragm neuromuscular coupling [46, 47] , increased peep can attenuate the force generated by diaphragmatic contraction [48] . indeed, several clinical studies provide indirect evidence to suggest that higher peep may render spontaneous effort less injurious in patients with acute respiratory failure before intubation [49] , in patients with ards [45, 50] , and in pediatric patients with lung injury [51] . on the other hand, preliminary experimental evidence suggests that if the diaphragm is maintained at a shorter length during acute mechanical ventilation, the diaphragm muscle fibers could adapt to the reduced length by absorbing sarcomeres in series (i.e., longitudinal atrophy) [52] . this may result in fibers overstretching with the release of peep during a t-tube weaning trial or after extubation. the possibility of diaphragm weakness resulting from excess peep should therefore be borne in mind. patient-ventilator dyssynchronies may cause lung and/or diaphragm injury by increasing dynamic lung stress and/ or injurious diaphragmatic contractions, respectively. dyssynchronies may occur during inspiration (flow starvation, short cycles, prolonged insufflation and reverse triggering), during expiration (auto-triggering, ineffective effort) or both during inspiration and expiration (reverse triggering and double triggering). we will briefly discuss dyssynchronies most relevant for lung and diaphragmprotective ventilation; for more extensive discussion of dyssynchronies we refer to other reviews [53] . reverse triggering, a diaphragmatic contraction triggered by mechanical inflation, is common in fully sedated patients (in whom drive is abolished) [54] . reverse triggering can induce breath stacking resulting in excessive tidal volumes and high dynamic lung stress [55] , and it may create eccentric diaphragm loading conditions with resultant muscle injury [56] . when necessary to avoid breath stacking, reverse triggering can be abolished by neuromuscular blocking agents. alternatively, the development of reverse triggering may indicate that sedation should be stopped to allow the patient to take control of ventilation. in patients with relatively high respiratory drive and a low respiratory system time constant, the neural inspiration time may exceed the mechanical inflation (premature cycling). in such cases, the contraction of the inspiratory muscles continues during mechanical expiration and the diaphragm is forced to contract while lengthening (eccentric contraction). in volume-targeted modes, unmet high demands appear as 'flow-starvation' , a downward curvature of inspiratory paw, and the patient may experience dyspnea and distress, which can be resolved by increasing inspiratory flow rate using a decelerating flow pattern. strong inspiratory efforts may result in double-triggering, breath stacking and, therefore, delivery of high tidal volumes. a better match of mechanical and neural inspiratory time can be achieved by increasing ventilator inspiratory time and using a decelerating flow pattern in volume-assist control mode, by decreasing the cycling-off criterion in pressure support mode, or using proportional modes of assist. importantly, in patients with high respiratory drive, modification of inspiratory time may not suffice to resolve dyssynchrony. increasing the level of assist to match the patient's demands should be considered, but, if that results in an injurious high ventilation, other means to decrease the patient's respiratory drive, such as sedation, may be required. another dyssynchrony occurring in patients with absent or low respiratory drive is auto-triggering, i.e., the delivery of a ventilator-assisted breath in the absence of patient effort. auto-triggering due to strong cardiac oscillations transmitted to the paw or airflow signal is more likely to occur when the respiratory system time constant is low, such as in ards. air leaks and moisture in the ventilator circuit are also common causes of auto-triggering. ineffective triggering (or ineffective efforts) develops when a patient's effort fails to trigger a ventilator-delivered breath. ineffective triggering is generally the consequence of weak inspiratory efforts, either from low respiratory drive due to sedation, metabolic alkalosis or excessive ventilatory assist, or because of diaphragm weakness. when the respiratory system time constant is high, (i.e., obstructive lung disease), ventilator over-assistance results in delayed cycling, dynamic hyperinflation, and increased intrinsic peep, predisposing to ineffective triggering. decreasing the level of assist can therefore alleviate ineffective efforts [57] . over-assistance in assisted ventilation can also induce apneas during sleep. interestingly, several studies have demonstrated that nava improves patient-ventilator interaction, especially reducing the risks of ineffective efforts and over-assist [39, 58] . whether the reduced duration of mechanical ventilation reported in some nava trials [40, 41] results from improved patient-ventilator interaction remains to be investigated. sedation can facilitate lung and diaphragm-protective ventilation by ameliorating, when present, excessive respiratory effort. complete suppression of respiratory drive and effort with sedation can also contribute to diaphragm disuse atrophy. a judicious approach to sedation is key and monitoring of respiratory drive and effort may be helpful in selecting the sedation strategy that facilitates lung and diaphragm-protective ventilation. before administering sedation to address excessive respiratory drive or ventilator dyssynchrony, ventilator settings should be adjusted and other factors increasing respiratory drive such as metabolic acidosis or pain should be addressed. relying on sedation alone to enhance patient-ventilator interaction without addressing these issues can paradoxically exacerbate dyssynchrony, prolong mechanical ventilation, and exacerbate diaphragm dysfunction [59] . recent clinical practice guidelines have recommended an "analgesia-first approach" to minimize the risk of excessive sedation as opioids during mechanical ventilation were associated with less dyssynchrony and depressed consciousness in comparison to sedative-based approaches [60] . nevertheless, when elevated respiratory drive cannot otherwise be resolved, sedatives can attenuate the ventilatory response to hypoxemia and hypercapnia and cortical input to the respiratory centres [33] (table 2) . propofol and benzodiazepines are gamma-aminobutyric acid (gaba) agonists known to cause respiratory depression, primarily by reducing the amplitude of respiratory effort [61] [62] [63] . because benzodiazepines are associated with a high risk of delirium and prolonged mechanical ventilation [64] , propofol is the preferred sedative of choice for controlling high respiratory drive. because propofol or benzodiazepines reduce the amplitude of inspiratory effort, ineffective triggering may develop as sedation depth increases [61] . inhalational sedation offers a potential alternative for controlling respiratory effort though clinical experience is limited to date [65] . to avoid excessive sedation, strategies aimed at active titration of sedatives or daily interruption of sedation should be employed and respiratory drive and effort should be monitored closely. for patients without excessive breathing effort (table 2) , a multimodal analgesia approach that minimizes opiate use is recommended to avoid diaphragm inactivity. dexmedetomidine is a selective alpha-2 agonist which, in contrast to propofol and benzodiazepines, provides sedation, anxiolysis, and analgesia without respiratory depression [66] . this property makes it an interesting drug of choice to preserve awareness and diaphragm contractility and at the same time limiting excess delirium risk in agitated patients without elevated respiratory drive. the prone position has been used for decades in early ards to improve oxygenation and over time an appreciation for the lung-protective benefit of prone positioning has emerged [67] . as the amount of lung tissue is larger in dorsal lung regions, gravitational forces generate more dependent atelectasis in the supine position compared to prone position. therefore, ventilation-perfusion matching is improved in the prone position and, more importantly, the energy applied to the lung by mechanical ventilation is distributed among more (non-atelectatic) alveoli, reducing lung stress. this is the putative basis for the observed mortality benefit of prone positioning in patients with ards [68] . the mechanistic benefits of prone positioning may also apply under assisted ventilation with spontaneous breathing, because the lung recruitment accrued by prone positioning may attenuate 'solid-like' lung behaviour and reduce effort-dependent regional lung stress. prone positioning improves oxygenation in spontaneously breathing patients with covid-19 pneumonia [69] ; it is possible that prone positioning could also reduce the risk of patient self-inflicted lung injury [70] . thus, prone positioning might facilitate safe spontaneous breathing and diaphragm-protective ventilation as well as lung protection. eliminating co 2 is the primary purpose of alveolar ventilation. ecco 2 r reduces the ventilatory demands, decreasing the respiratory effort, and thus may ameliorate dynamic lung stress. ecco 2 r is feasible and effective in reducing tidal volume, driving pressure, and mechanical power in patients with ards [71] . in spontaneously breathing patients, ecco 2 r can dampen respiratory drive and effort [72] , theoretically reducing the requirement for ventilatory support or sedation to control respiratory effort. karagiannidis et al. showed that increasing sweep gas flow, increasing co 2 elimination, in ards patients undergoing extracorporeal membrane oxygenation (ecmo) reduced respiratory drive, estimated by eadi [73] . mauri et al. [7] also showed that higher ecco 2 r support reduced p 0.1 , respiratory muscle effort, and transpulmonary pressure in spontaneously breathing patients recovering from severe ards [74] . pilot clinical studies have explored the extreme possibility of extubating severe ards patients early after intubation by means of ecco 2 r: preliminary results were encouraging but they also recognized the need to identify the subgroup of patients with a high probability of success [75] [76] [77] . despite the appeal and physiological rationale of this strategy, there are relevant limitations. first, in some patients, non-chemoreceptive stimuli (pain, agitation, discomfort, metabolic acidosis, lung mechanical stimuli) may predominate and high respiratory drive may persist despite ecco 2 r [78] . second, ecco 2 r requires full anticoagulation and the risk of bleeding is not insubstantial [79] . third, the application of ecco 2 r may exacerbate hypoxemia by various mechanisms [80] . complete neuromuscular blockade may increase the risk for diaphragm disuse atrophy and increases sedation requirements. low-dose neuromuscular blockers ("partial neuromuscular blockade") is an interesting compromise between total paralysis and strenuous breathing efforts, particularly when respiratory effort does inadequately respond to titration of ventilatory support or sedation. the feasibility of partial neuromuscular blockade has been evaluated in a proof of concept study in patients with moderate ards and high respiratory drive on partially supported modes [81] . titration of rocuronium decreased tidal volume from approximately 9 ml/ kg to approximately 6 ml/kg while maintaining pdi at approximately 5 cmh 2 o (within the physiological range for diaphragm activity in healthy subjects). these preliminary findings suggest that partial neuromuscular blockade could be a feasible approach to achieving lung and diaphragm-protective ventilation targets in patients with high respiratory effort. importantly, partial neuromuscular blockade does not reduce respiratory drive, but only the mechanical consequences of high drive. this dissociation between central drive and respiratory muscle mechanical output may result in dyspnea [33] ; adequate relief of dyspnea and distress must be ensured by judicious application of sedatives and opioids. future clinical studies should confirm the safety and efficacy of prolonged partial neuromuscular blockade in ventilated patients. neuromuscular stimulation ("pacing") uses electrical currents to generate muscle contraction in the absence of volitional efforts, making it an attractive intervention in incapacitated critically ill patients. there is growing interest in neuromuscular stimulation as a novel strategy to preserve or restore respiratory muscle activity and, in turn, to prevent or treat icu-acquired diaphragm weakness. in addition, by inducing diaphragm contractions, neuromuscular stimulation may improve lung aeration of dependent lung regions [82] . pacing must be synchronized with the ventilator and potentially injurious inspiratory efforts must be avoided. there is as yet no clinical evidence of benefit from diaphragm pacing in icu patients. direct stimulation of the phrenic nerves by surgically implanted electrodes has been employed to restore spontaneous ventilation in patients with high-level spinal cord injury and central hypoventilation syndrome [83] . the feasibility of direct pacing using temporary implanted electrodes for the prevention of diaphragm dysfunction is currently under investigation in cardiac surgery patients identified to be at risk for prolonged mechanical ventilation (nct04309123). preclinical work showed that this technique could reduce the development of diaphragm type ii fiber atrophy [84, 85] . recently, reynolds et al. presented a first-in-human series of temporary transvenous phrenic nerve pacing in surgical patients and showed that this technology delivered safe and effective diaphragm contractions [86] . this strategy is currently being studied as potential intervention for improving diaphragm strength in difficult-to-wean patients (nct03096639). the role of transvenous phrenic nerve pacing for the prevention of diaphragm disuse atrophy remains to be investigated. neuromuscular stimulation strategies targeting the expiratory muscles of icu patients are less well studied. this is surprising, as stimulation of the expiratory abdominal wall muscles can be employed noninvasively via surface electrodes placed over the abdominal wall. feasibility of a breath-synchronized expiratory muscle stimulation technique during the early phase of mechanical ventilation was recently demonstrated with promising results [87] and its efficacy is under investigation (nct03453944). clinicians caring for mechanically ventilated patients are generally well aware of the risk of causing barotrauma, volutrauma, and atelectrauma. given the mounting evidence of clinically important diaphragm atrophy and injury, consideration must also be given to protecting the diaphragm. based on the foregoing discussion about ventilation and sedation, a basic algorithm and approach to lung and diaphragm-protective ventilation is presented in fig. 3 . clinical trials testing new ventilation algorithms and sedation strategies targeted at optimizing respiratory effort are required to confirm the benefit of the lung and diaphragm-protective approach outlined in this paper. the benefit of adjunctive strategies such as ecco 2 r, partial neuromuscular blockade and phrenic nerve stimulation requires further evaluation, in particular to identify the subpopulations of patients most likely to benefit from these more costly and invasive interventions. for the present, we encourage clinicians to incorporate routine monitoring of respiratory drive and effort in their clinical practice and to adjust the ventilator to achieve a physiological level of effort where possible while carefully attending to the effect on lung stress. this article is licensed under a creative commons attribution-noncommercial 4.0 international license, which permits any non-commercial use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons licence, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons licence, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by-nc/4.0/. springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. received: 2 september 2020 accepted: 8 october 2020 fig. 3 clinical-physiological pathway for achieving lung and diaphragm-protective ventilation targets. it should be stressed that at each step clinical evaluation of the patient, including signs of high breathing effort, agitation, and over-sedation is of major importance and should be interpreted together with clinical-physiological measurements as outlined in this pathway. δp: change in airway pressure during inspiration; p 0.1 : decrease in airway pressure during the first 100 ms of inspiratory effort against an occluded airway; paco 2 : arterial carbon dioxide tension; peep: positive endexpiratory pressure; pes: esophageal pressure; p l : transpulmonary pressure; pocc: airway pressure deflection during a whole breath occlusion; rr: respiratory rate; v t : tidal volume ventilator-induced lung injury mechanical ventilation to minimize progression of lung injury in acute respiratory failure lung stress and strain during mechanical ventilation for acute respiratory distress syndrome volume delivered during recruitment maneuver predicts lung stress in acute respiratory distress syndrome driving pressure and survival in the acute respiratory distress syndrome prevalence of complete airway closure according to body mass index in acute respiratory distress syndrome: pooled cohort analysis expiratory muscle dysfunction in critically ill patients: towards improved understanding mechanical ventilation-induced diaphragm atrophy strongly impacts clinical outcomes diaphragm dysfunction on admission to the intensive care unit. prevalence, risk factors, and prognostic impact-a prospective study coexistence and impact of limb muscle and diaphragm weakness at time of liberation from mechanical ventilation in medical intensive care unit patients critical illnessassociated diaphragm weakness rapid disuse atrophy of diaphragm fibers in mechanically ventilated humans diaphragm muscle fiber weakness and ubiquitin-proteasome activation in critically ill patients rapidly progressive diaphragmatic weakness and injury during mechanical ventilation in humans evolution of diaphragm thickness during mechanical ventilation. impact of inspiratory effort lung and diaphragm-protective ventilation driving pressure is associated with outcome during assisted ventilation in acute respiratory distress syndrome driving pressure during proportional assist ventilation: an observational study airway pressure morphology and respiratory muscle activity during end-inspiratory occlusions in pressure support ventilation esophageal and transpulmonary pressure in the clinical setting: meaning, usefulness and perspectives spontaneous effort causes occult pendelluft during mechanical ventilation understanding spontaneous vs. ventilator breaths: impact and monitoring esophageal manometry and regional transpulmonary pressure in lung injury monitoring of the respiratory muscles in the critically ill estimation of patient's inspiratory effort from the electrical activity of the diaphragm information conveyed by electrical diaphragmatic activity during unstressed, stressed and assisted spontaneous breathing: a physiological study airway occlusion pressure as an estimate of respiratory drive and inspiratory effort during assisted ventilation monitoring patient-ventilator interaction by an end-expiratory occlusion maneuver a novel non-invasive method to detect excessively high respiratory effort and dynamic transpulmonary driving pressure during mechanical ventilation respiratory muscle ultrasonography: methodology, basic and advanced principles and clinical applications in icu and ed patients-a narrative review measuring diaphragm thickness with ultrasound in mechanically ventilated patients: feasibility, reproducibility and validity physiology of the respiratory drive in icu patients: implications for diagnosis and treatment respiratory drive in critically ill patients. pathophysiology and clinical implications the injurious effects of elevated or nonelevated respiratory rate during mechanical ventilation effects of short-term oxygenation changes on acute lung injury patients undergoing pressure support ventilation effects of pressure ramp slope values on the work of breathing during pressure support ventilation in restrictive patients volume-controlled ventilation does not prevent injurious inflation during spontaneous effort neural control of mechanical ventilation in respiratory failure comparison between neurally adjusted ventilatory assist and pressure support ventilation levels in terms of respiratory effort neurally adjusted ventilatory assist versus pressure support ventilation in difficult weaning: a randomized trial neurally adjusted ventilatory assist in acute respiratory failure: a randomized controlled trial fifty years of research in ards. setting positive end-expiratory pressure in acute respiratory distress syndrome optimum end-expiratory airway pressure in patients with acute pulmonary failure an official american thoracic society/european society of intensive care medicine/society of critical care medicine clinical practice guideline: mechanical ventilation in adult patients with acute respiratory distress syndrome high positive end-expiratory pressure renders spontaneous effort noninjurious mechanics of the human diaphragm during voluntary contraction: statics diaphragmatic neuromechanical coupling and mechanisms of hypercapnia during inspiratory loading mechanics of the diaphragm effect of noninvasive ventilation delivered by helmet vs face mask on the rate of endotracheal intubation in patients with acute respiratory distress syndrome: a randomized clinical trial high positive end-expiratory pressure allows extubation of an obese patient pendelluft detection using electrical impedance tomography in an infant. keep those images in mind positive end-expiratory pressure ventilation induces longitudinal atrophy in diaphragm fibers mechanical ventilation: state of the art mechanical ventilationinduced reverse-triggered breaths: a frequently unrecognized form of neuromechanical coupling quantifying unintended exposure to high tidal volumes from breath stacking dyssynchrony in ards: the breathe criteria diaphragmatic myotrauma: a mediator of prolonged ventilation and poor patient outcomes in acute respiratory failure asynchrony consequences and management neurally adjusted ventilatory assist as an alternative to pressure support ventilation in adults: a french multicentre randomized trial observational study of patient-ventilator asynchrony and relationship to sedation level clinical practice guidelines for the prevention and management of pain, agitation/sedation, delirium, immobility, and sleep disruption in adult patients in the icu effects of propofol on patient-ventilator synchrony and interaction during pressure support ventilation and neurally adjusted ventilatory assist effects of propofol on respiratory drive and patient-ventilator synchrony during pressure support ventilation in postoperative patients: a prospective study respiratory depression by midazolam and diazepam lorazepam is an independent risk factor for transitioning to delirium in intensive care unit patients inhalational volatile-based sedation for covid-19 pneumonia and ards effects of intravenous dexmedetomidine in humans. i. sedation, ventilation, and metabolic rate prone positioning in acute respiratory distress syndrome prone positioning in severe acute respiratory distress syndrome use of prone positioning in nonintubated patients with covid-19 and hypoxemic acute respiratory failure is the prone position helpful during spontaneous breathing in patients with covid-19? determinants of the effect of extracorporeal carbon dioxide removal in the supernova trial: implications for trial design control of breathing using an extracorporeal membrane lung autoregulation of ventilation with neurally adjusted ventilatory assist on extracorporeal lung support control of respiratory drive and effort in extracorporeal membrane oxygenation patients recovering from severe acute respiratory distress syndrome spontaneous breathing during extracorporeal membrane oxygenation in acute respiratory failure extracorporeal membrane oxygenation instead of invasive mechanical ventilation in patients with acute respiratory distress syndrome extracorporeal membrane oxygenation can successfully support patients with severe acute respiratory distress syndrome in lieu of mechanical ventilation spontaneous breathing patterns during maximum extracorporeal co2 removal in subjects with early severe ards european society of intensive care medicine trials group and the "strategy of ultra-protective lung ventilation with extracorporeal co2 removal for new-onset moderate to severe ards" (supernova) investigators (2019) feasibility and safety of extracorporeal co2 removal to enhance protective ventilation in acute respiratory distress syndrome: the supernova study understanding hypoxemia on ecco2r: back to the alveolar gas equation partial neuromuscular blockade during partial ventilatory support in sedated patients with high tidal volumes effects of anesthesia and paralysis on diaphragmatic mechanics in man diaphragm pacing: the state of the art can phrenic stimulation protect the diaphragm from mechanical ventilation-induced damage? mitigation of ventilatorinduced diaphragm atrophy by transvenous phrenic nerve stimulation diaphragm activation in ventilated patients using a novel transvenous phrenic nerve pacing catheter abdominal functional electrical stimulation to assist ventilator weaning in critical illness: a double-blinded, randomised, sham-controlled pilot study key: cord-274749-ji91qq9q authors: lagare, adamou; maïnassara, halima boubacar; issaka, bassira; sidiki, ali; tempia, stefano title: viral and bacterial etiology of severe acute respiratory illness among children < 5 years of age without influenza in niger date: 2015-11-14 journal: bmc infect dis doi: 10.1186/s12879-015-1251-y sha: doc_id: 274749 cord_uid: ji91qq9q background: globally, pneumonia is the leading cause of morbidity and mortality in children, with the highest burden experienced in sub-saharan africa and asia. however, there is a dearth of information on the etiology of severe acute respiratory illness (sari) in africa, including niger. methods: we implemented a retrospective study as part of national influenza sentinel surveillance in niger. we randomly selected a sample of nasopharyngeal specimens collected from children <5 years of age hospitalized with sari from january 2010 through december 2012 in niger. the samples were selected from individuals that tested negative by real-time reverse transcription polymerase chain reaction (rrt-pcr) for influenza a and b virus. the samples were analyzed using the fast track diagnostic respiratory pathogens 21plus kit (biomérieux, luxemburg), which detects 23 respiratory pathogens including 18 viral and 5 bacterial agents. results: among the 160 samples tested, 138 (86 %) tested positive for at least one viral or bacterial pathogen; in 22 (16 %) sample, only one pathogen was detected. we detected at least one respiratory virus in 126 (78 %) samples and at least one bacterium in 102 (64 %) samples. respiratory syncytial virus (56/160; 35 %), rhinovirus (47/160; 29 %) and parainfluenza virus (39/160; 24 %) were the most common viral pathogens detected. among bacterial pathogens, streptococcus pneumoniae (90/160; 56 %) and haemophilus influenzae type b (20/160; 12 %) predominated. conclusions: the high prevalence of certain viral and bacterial pathogens among children <5 years of age with sari highlights the need for continued and expanded surveillance in niger. acute respiratory infections (aris) are responsible for substantial morbidity and mortality globally, especially in children <5 years of age, and the highest burden is observed in developing nations [1] . in 2000, approximately 2.2 million ari-associated deaths occurred among children <5 years of age of which 70 % were in africa and southeast asia [2] . both bacteria and viruses have been identified as the etiological agents of ari. the viruses most frequently detected in children with aris include respiratory syncytial virus (rsv), influenza virus (inf) types a and b, adenovirus (av), parainfluenza virus (piv), human metapneumovirus (hmpv) and rhinovirus (rv) [3] [4] [5] ; however, the clinical presentations of respiratory tract infections are similar, making it difficult to distinguish between etiologic agents without a laboratory diagnosis [6] . in addition, the interpretation of a viral detection is complicated by the fact that infections with multiple viruses are common in children with ari and many viruses are frequently found in asymptomatic children [7] . streptococcus pneumoniae and haemophilus influenzae type b (hib) are the most commonly isolated bacteria from ari cases, while, other atypical pathogens such as mycoplasma pneumoniae and chlamydophila pneumoniae are less frequently reported [8] [9] [10] . s. pneumoniae and hib are commonly identified in nasopharyngeal samples from asymptomatic children due to high rates of carriage; however, their identification from the nasopharynx is rarely indicative of invasive disease [11] . the viral and bacterial etiology of ari has been well documented in countries from the northern hemisphere [12] [13] [14] [15] ; however, few studies are available from africa [16, 17] . in niger, a sentinel surveillance system for influenza viruses was instituted in april 2009; however, no studies on the etiology of ari have been conducted in the country. we aimed to document the prevalence of selected viral and bacterial infections among children <5 years of age hospitalized with severe acute respiratory illness (sari) at selected hospitals in niger from january 2010 through december 2012. niger is a west african country with a saharan climate characterized by four distinct seasons: the cold season from mid-december to mid-february, the dry and hot season from mid-february to may, the rainy season from june to september, and the hot season from october to mid-december [18, 19] . since april 2009, influenza surveillance has been conducted among patients hospitalized with severe acute respiratory illness (sari) at 8 sentinel sites located in 5 of the 8 regions of the country by the centre de recherche médicale et sanitaire (cermes), the national reference laboratory for influenza. the influenza surveillance program in niger has previously been described [19] . briefly, all patients hospitalized at the participating sentinel sites that met the sari case definition were eligible for enrollment. verbal informed consent was obtained from all cases who were 18 years of age and older. proxy informed consent was obtained from parents or legal guardians of minors. patients who did not meet the case definition or did not provide verbal consent were not included. a sari case was defined as a hospitalized child <5 years of age with onset of cough or difficulty breathing within 7 days prior to admission, and at least one of the following danger signs: inability to drink or breastfeed, lethargy, vomiting everything, convulsions, nasal flaring, chest indrawing, stridor in a calm child or tachypnea [20] . a standardized questionnaire was administrated by clinical personnel, to record patients' demographic characteristics and medical history. the questions included information on date of enrollment and symptom onset, sex, age and clinical symptoms. nasopharyngeal (np) swabs were collected from all enrolled cases and placed in cryovials containing virus transport medium (copan kit, italy). the specimens were kept refrigerated at 4°c at the sentinel site and then transported twice weekly to cermes for testing. samples were aliquoted, screened for influenza a and b viruses by real-time reverse transcription polymerase chain reaction (rrt-pcr), and then stored at −80°c. we conducted a retrospective study on the etiology of influenza-negative sari cases among children <5 years of age enrolled in influenza surveillance during january 2010 through december 2012 in niger. we randomly selected (using random selection procedures available in stata) a sample of 160 stored np specimens from 742 sari cases which had tested influenza a and b negative. this sample represented 21 % of the total cases aged <5 years enrolled during the study period. only 5.5 % of sari cases in the <5 year old age group were tested positive for influenza. influenza-positive cases were mainly detected during the cold season [21] . nucleic acid was extracted using the qiaamp mini kit (qiagen, germany) in accordance with the manufacturer's protocol. an internal control (ic) was added to each extraction tube in order to assess the quality of the extraction at the end of the amplification. the χ 2 and the fisher's exact tests were used to assess the difference between categorical variables by comparing expected and observed frequencies across evaluated groups. in addition, we compared the characteristics of selected and non-selected children (including influenza-positive cases) using the x 2 statistics. the statistical analysis was implemented using stata version 13.1 (statacorp®, texas, usa). in 2009, the national ethics committee of niger approved the national influenza surveillance program (reference no06/2009/ccne of april 2009). in 2012, prior to the investigation of other respiratory pathogens, the ministry of health provided an extended approval. access to the study samples was granted by the director of cermes. from january 2010 through december 2012 we enrolled 785 children <5 years of age hospitalized with sari into the influenza surveillance program, of which 742 (94 %) tested negative for influenza virus. of these, 160 (21 %) were randomly selected for our study. the age, sex and symptom duration distribution did not differ significantly among selected and non-selected children (including those that tested positive for influenza). however, among the selected group there was a significantly lower proportion of specimens collected during the cold season, when the majority of influenza-positive cases were detected and excluded from randomization (table 1) . among the selected children, 56 % (90/160) were <1 year of age (median age among children age <5 years: 9 months), 46 % (73/160) were female and 78 % (125/ 160) had a duration of symptoms ≤3 days. most patients presented with a recorded temperature >38°c (58 %; 93/160), cough (96 %; 155/160), dyspnea (69 %; 111/160) and chest indrawing (76 %; 122/160). few patients presented with tachypnea or had difficulty in breastfeeding (15 %; 25/160). overall, 138/160 (86 %) of children included in the study tested positive for at least one pathogen (viral or bacterial). at least one respiratory virus was detected in 126/160 (78 %) samples and at least one bacterium was detected in 102/160 (64 %) samples. among the 138 samples positive for any pathogen, 22 (16 %) were positive for a single pathogen. of these 8 (36 %) were positive for s. pneumoniae, 4 (18 %) for rsv and 4 (18 %) for rv, while hmpv, cv, piv and hib each accounted individually for <10 % of the single organisms detected (fig. 1) . among the 116 children in whom ≥2 organisms were detected, both viral and bacterial pathogens were detected in 90 samples (78 %). among the 160 samples tested, rsv (n = 56; 35 %) was the most frequently detected virus, followed by rv (n = 47; 29 %) and piv types 1-4 (n = 39; 24 %) ( table 2) . cv, hmpv, hbv, ev and av were detected individually in <15 % of the specimens. no pv was detected in our study. of the 20 samples that tested positive for cv, 13 (65 %) were type oc43, 9 (45 %) were type 229e, 6 (30 %) were type nl63 and 4 (20 %) were type hku1. of the 39 samples that tested positive for piv, 9 (23 %) were type 1, 3 (8 %) were type 2, 31 (79 %) were type 3 and 21 (53 %) were type 4. even though we selected co-detections with different subtypes were detected in 6 (30 %) and 20 (51 %) of the cv and piv positive cases, respectively. rsv, piv and hmpv were detected more frequently in infants <1 year of age compared to children 1-4 years of age (table 2) . rsv, piv and hmpv were detected more frequently during the hot season (october to mid-december); while rv and hbv were detected more frequently during the rainy season (june to september). the other viruses were detected with similar frequencies across seasons (table 2) . among the 160 samples tested, s. pneumoniae (n = 90; 56 %) was the most frequently detected bacteria, followed by hib (n = 20; 12 %), s. aureus (n = 18; 11 %) and c. pneumoniae (n = 4; 2.5 %) ( table 3) . m. pneumoniae was not detected in our study. we report the detection rate of selected viral and bacterial pathogens among children <5 years of age hospitalized with sari in niger. we detected respiratory viruses in 78 % of our study sample. the high detection rate of viruses found in our study is consistent with results from similar studies conducted in burkina faso (73 %) [23] , kenya (68 %) [24] and brazil (85 %) [12] . however, lower rates of viral detection were reported from other studies from countries such as ghana (26 %) [25] , china (56 %) [26] and egypt (60 %) [4] . these differences can be attributed to different climatic conditions, enrollment criteria, case definitions and testing platforms. in our study, rsv was the predominant virus detected and was most commonly found in children <1 year of age. rsv has been reported to be an important pathogen in children and especially in young infants in several studies [12, [25] [26] [27] [28] [29] . in addition, rsv detection has been reported to be strongly associated with illness from studies comparing symptomatic cases to controls [30] . rhinovirus was the second most commonly detected virus (29 %) with similar rates among infants <1 year of age and children aged 1-4 years, which has been reported in previous studies [31, 32] . however, other studies reported rv as the most prevalent virus among children <5 years of age [16, 23, 28, 33] . in addition rv has been commonly detected among asymptomatic persons in several studies indicating that rv can act as both pathogen and by-stander, consequently hindering the ability to infer an association between detection and illness [34] [35] [36] . among the piv and cv detected in this study, piv type 3 and cv type oc43 were the most common virus types, which has been reported in other studies [12, 16] . we also found a high detection rate of bacterial pathogens. s. pneumoniae (56 %) and hib (12 %) were the most common bacteria detected in nasopharyngeal specimens. elevated colonization rates of these bacteria have been reported in children, but only a proportion of colonizations result in invasive disease [9, 11, 37] . the high detection rate of s. pneumoniae in our study is likely due to the fact that s. pneumoniae is a commensal of the nasopharynx [38] . it has been shown that the prevalence of s. pneumoniae carriage in healthy children <5 years of age ranges from 20 % to 93 % in low income countries [11] . the detection of s. pneumoniae from sterile sites like blood or cerebrospinal fluid, reflecting invasive pneumococcal disease, has been shown to be lower (5-9 %) [8, 39] . nonetheless, s. pneumoniae has been reported to be responsible for 12 % of meningitis cases in niger based on cerebrospinal fluid testing; 26 different serotypes were detected among cases of meningitis prior to the introduction of the pneumococcal conjugate vaccine in 2014 [40] . hib and s. aureus, the 2 nd and 3 rd most prevalent bacterial pathogens in our study, have also been shown to be commensal organisms with high nasopharyngeal carriage rates especially in young children [11] . the substantial hib nasopharyngeal colonization density found in this study should be investigated further as hib vaccine has been available in the niger expanded immunization program since 2008. nasopharyngeal specimens may be used to aid in the diagnosis of certain bacterial respiratory pathogens that do not tend to colonize the nasopharynx, such as m. pneumoniae and c. pneumoniae [38, 41] . c. pneumoniae was detected at low rates (2.5 %), and m. pneumoniae was not detected in our study. using serological methods, prevalence rates as high as 30 % have been reported for c. pneumoniae [9] ; in contrast, other studies report significant detection of m. pneumoniae (>10 %) and low detection of c. pneumoniae (<1 %) [12, 26, 42] . in our study we found an elevated prevalence (78 %) of viral-bacterial co-detections, which has been reported in other studies [12, 42] . it has been shown that viral infections may predispose to bacterial super-infection by favoring bacterial attachment sites on nasopharyngeal epithelial cells and through increased mucous production that promotes bacterial growth [38, 42] . our study has limitations that warrant discussion. first, the small sample size of our study hindered our ability to accurately assess the seasonality of the pathogens included in our study. nonetheless, our results suggest that rsv, piv and hmpv are more commonly detected during the hot season (october to december), while rv and hbv are detected more frequently during the rainy season (june to september). no difference in the detection rate of the other viruses and bacteria was noted across seasons in our study. the small sample size of our study also hindered our ability to detect patterns of co-detection and the association between specific viral and bacterial co-detections. second, we did not keep formal records of the proportion of patients consenting to participate in the sari surveillance. however, a review of the performance of the surveillance system implemented through hospital record review at sentinel sites revealed that only a few patients that met the study case definition were missed by the surveillance program. third, the lack of controls in our study limited our ability to assess the association of pathogen detection with disease. while most of the viral and bacterial pathogens identified in this study have been described by previous studies as causative agents of ari, the assignation of causality remains challenging [39, 43] . fourth, influenzapositive samples were excluded from our study. codetection of other viral and bacterial pathogens with influenza is expected and this may have resulted in an underestimation of the prevalence of the pathogens included in this study, especially during the cold season when the majority of influenza-positive cases were detected. last, we did not systematically collect information on progression of illness (including in-hospital outcome) or risk factors for severe disease, which hindered our ability to evaluate pathogen contribution to the more severe spectrum of illness or to identify groups at high risk for severe disease. this study reports the detection rate of viral and bacterial pathogens among children <5 years of age hospitalized with sari in niger. the high prevalence of certain viral and bacterial pathogens highlights the need for expanded surveillance in niger so as to inform policies and interventions. given the high rsv detection rate observed in this study and the reported association of rsv detection with illness [30] , rsv should be included in routine surveillance programs in niger. other selected pathogens could be considered for routine surveillance in the country following further assessment to determine association with illness. in addition, information on progression of illness, including in-hospital outcome and risk factors for severe disease should be collected routinely through the existing surveillance system. the authors declare that they have no competing interests. submit your manuscript at www.biomedcentral.com/submit global and regional burden of hospital admissions for severe acute lower respiratory infections in young children in 2010: a systematic analysis estimate of worldwide distribution of child deaths from acute respiratory infections respiratory viruses seasonality in children under five years of age in viral etiologies of lower respiratory tract infections among egyptian children under five years of age respiratory viral coinfections identified by a 10-plex real-time reversetranscription polymerase chain reaction assay in patients hospitalised with severe acute respiratory illness-south africa viral etiology of influenza-like illness in cameroon clinical utility of pcr common viruses in acute respiratory illness microbial aetiology of community-acquired pneumonia in hospitalised patients clinico-pathological study of atypical pathogens in community-acquired pneumonia: a prospective study epidemiology and clinical characteristics of community-acquired pneumonia in hospitalized children carriage of streptococcus pneumoniae and other respiratory bacterial pathogens in low and lower-middle income countries: a systematic review and meta-analysis viral and bacterial detection in acute respiratory infection in children under five years sentinel surveillance of influenza and other respiratory viruses, brazil viral infections of the lower respiratory tract: old viruses, new viruses, and the role of diagnosis viral etiology of acute respiratory tract infection among pediatric inpatients and outpatients from 2010 to 2012 in beijing, china viral and atypical bacterial etiology of acute respiratory infections in children under 5 years old living in rural tropical area of madagascar viral etiology of respiratory infections in children under 5 years old living in tropical rural areas of senegal: the evira project influenza transmission during a one-year period (2009-2010) in a sahelian city: low temperature plays a major role influenza sentinel surveillance among patients with influenza-likeillness and severe acute respiratory illness within the framework of the national reference laboratory strategy to enhance influenza surveillance worldwide sentinel surveillance of influenza in burkina faso: identification of circulating strains during 2010-2012. influenza other resp viruses manual for ftd resp 21plus. luxembourg: fast track diagnostics company viral etiology of respiratory tract infections in children at the pediatric hospital in ouagadougou viral and bacterial causes of severe acute respiratory illness among children aged less than 5 years in a high malaria prevalence area of western kenya respiratory viruses in children hospitalized for acute lower respiratory tract infection in ghana epidemiology of acute respiratory infections in children in guangzhou: a three-year study the impact of influenza and respiratory syncytial virus on hospitalizations for lower respiratory tract infections in young children: slovenia respiratory viral infections during the 2009-2010 winter season in central england, uk: incidence and patterns of multiple virus co-infections a novel multiplex real-time rt-pcr assay with fret hybridization probes for the detection and quantitation of 13 respiratory viruses viral etiology of severe pneumonia among kenyan infants and children survey of causative agents for acute respiratory infections among patients in khartoum-state the burden of single virus and viral coinfections on severe lower respiratory tract infections among preterm infants: a prospective birth cohort study in brazil respiratory virus infections in hospitalized children and adults in lao pdr human rhinovirus infections in rural thailand: epidemiological evidence for rhinovirus as both pathogen and bystander molecular epidemiology of human rhinovirus infections in kilifi, coastal kenya genetic diversity and molecular epidemiology of rhinoviruses in south africa the role of multiplex pcr test in identification of bacterial pathogens in lower respiratory tract infections high nasopharyngeal pneumococcal density, increased by viral coinfection, is associated with invasive pneumococcal pneumonia etiology and incidence of viral and bacterial acute respiratory illness among older children and adults in rural western kenya determination of pneumococcal serotypes in meningitis cases in niger surveillance for hospitalized acute respiratory infection in guatemala bacterial and viral etiology in hospitalized community acquired pneumonia with molecular methods and clinical evaluation huamn coronaviruses associated with upper respuratory tract infections in three rural areas of ghana we are thankful to the national ministry of public health for financing this study and our partners: the pasteur institute of paris, the world health organization, and the us centers for disease control and prevention for providing reagents, the rrt-pcr machine and technical supports for influenza surveillance in niger. key: cord-281051-i229xv0o authors: bishop-williams, katherine e.; sargeant, jan m.; berrang-ford, lea; edge, victoria l.; cunsolo, ashlee; harper, sherilee l. title: a protocol for a systematic literature review: comparing the impact of seasonal and meteorological parameters on acute respiratory infections in indigenous and non-indigenous peoples date: 2017-01-26 journal: syst rev doi: 10.1186/s13643-016-0399-x sha: doc_id: 281051 cord_uid: i229xv0o background: acute respiratory infections (ari) are a leading cause of morbidity and mortality globally, and are often linked to seasonal and/or meteorological conditions. globally, indigenous peoples may experience a different burden of ari compared to non-indigenous peoples. this protocol outlines our process for conducting a systematic review to investigate whether associations between ari and seasonal or meteorological parameters differ between indigenous and non-indigenous groups residing in the same geographical region. methodology: a search string will be used to search pubmed(®), cab abstracts/cab direct(©), and science citation index(®) aggregator databases. articles will be screened using inclusion/exclusion criteria applied first at the title and abstract level, and then at the full article level by two independent reviewers. articles maintained after full article screening will undergo risk of bias assessment and data will be extracted. heterogeneity tests, meta-analysis, and forest and funnel plots will be used to synthesize the results of eligible studies. discussion and registration: this protocol paper describes our systematic review methods to identify and analyze relevant ari, season, and meteorological literature with robust reporting. the results are intended to improve our understanding of potential associations between seasonal and meteorological parameters and ari and, if identified, whether this association varies by place, population, or other characteristics. the protocol is registered in the prospero database (#38051). electronic supplementary material: the online version of this article (doi:10.1186/s13643-016-0399-x) contains supplementary material, which is available to authorized users. acute respiratory infections (ari) contribute to a substantial global burden of morbidity and mortality [1] [2] [3] . an estimated 14.9 million children were hospitalized for ari in 2010, of which 265,000 died [3] . defined as an acute infection with coughing as a symptom [4] , ari is often associated with meteorological parameters [1] and commonly varies by season [5] . for instance, ari is commonly associated with temperature parameters, with increasing incidence during cold periods as a result of an individual's exposure, susceptibility, and the infection type [6] . furthermore, seasonal associations with ari also have been identified; in some cases, these seasonal associations have been attributed to varying meteorological parameters and in other cases attributed to the pathogen's own rhythmicity [6] . the associations between ari and meteorological parameters and season may be modified by social gradients of health [7] [8] [9] and affected by type of livelihood [8, 10] . for instance, among indigenous peoples, a strong connection to the land [11] [12] [13] , resource-based livelihoods [14, 15] , interacting social determinants of health (i.e., housing, education) [9, 14, 16] , and the legacies of colonization [9] may modify the association between infections and meteorological and seasonal parameters [14] . therefore, it is possible that there are differences in the association between weather variables and ari among indigenous and non-indigenous communities. it is important to know if these associations differ between indigenous and non-indigenous communities to aid in better planning, resource allocation, and intervention strategies. this paper outlines a protocol for conducting a systematic review to investigate whether associations between ari and seasonal or meteorological parameters differ between indigenous and non-indigenous groups residing in the same geographical region. this protocol, which outlines methods for the proposed systematic review, was designed in accordance with the preferred reporting items for systematic review and meta-analyses (prisma) guidelines [17] . the protocol is registered in the prospero database (#38051). the items of this protocol are presented in accordance with the prisma-p checklist (additional file 1) [18] . this systematic review protocol outlines the procedures for a systematic literature review that is intended to answer the question: is the association between seasonal or meteorological parameters and ari the same in indigenous and non-indigenous peoples who live in the same geographical region? the components of population, exposure, comparator, and outcome (peco) are as follows: population: communities with indigenous and non-indigenous community members; exposure: indigeneity; comparator: non-indigenous; and outcome: association between seasonal or meteorological parameters and ari. all primary epidemiological observational study designs (i.e., cross-sectional, cohort, case-control studies) are eligible for inclusion (table 1) . ecological studies will be eligible, as the population in a singular location should be equally exposed to seasonal or meteorological parameters. experimental studies (i.e., intervention studies) will not be eligible, as the exposure (i.e., indigeneity) cannot be assigned. further, reviews, commentaries, editorials, mathematical models, or other non-primary research articles will not be eligible, as these studies are not comparable with observational results. eligible study populations are those in which a portion of the population living in the same region is explicitly defined as indigenous and a portion of the population is explicitly defined as non-indigenous. this research builds from the united nations declaration of the rights of indigenous peoples [13] understanding of the term indigenous peoples, which states that an indigenous person self-identifies as indigenous; has historical continuity table 1 inclusion and exclusion criteria for a systematic literature review investigating the impact of seasonal and meteorological parameters on acute respiratory infection (ari) in indigenous and non-indigenous peoples the study is a primary epidemiological observational study design (cross-sectional, case-control, cohort, or ecological studies) the study is an experiment (i.e., interventional), review, commentary, editorial, mathematical model, or other non-primary research the research report exceeds 500 words the research report is less than 500 words the study's population includes both indigenous and non-indigenous peoples the study's population includes only indigenous or only non-indigenous peoples the study's outcome is the association between ari and seasonal or meteorological parameters the study's outcome is something other than the association between ari and seasonal or meteorological parameters the study provides sufficient epidemiological data to investigate the question of interest (i.e., two models representing the indigenous and non-indigenous strata separately with the same seasonal or meteorological parameter in both models, producing two measures of association for which the ratio of odds ratios (ror) can be calculated, or (ii) a model for an ari outcome that includes indigeneity and seasonal or meteorological parameter(s) as independent variables from which further calculations can be made) the study provides insufficient information to investigate the question of interest the study presents unique results which have not been previously published, or is the most recent and comprehensive analysis of the data. the study which duplicates the results of a previous study ari acute respiratory infection with pre-colonial society; has a strong link to territory and natural resources; has a distinct social, economic, or political system; has a distinct language, culture, and/or belief system; forms a non-dominant societal group; and/or resolves to maintain and reproduce their ancestral environments and systems as distinctive peoples and societies. indigeneity is the only eligible exposure. the comparator group is a non-indigenous population living in the same region as the indigenous population. all eligible studies will present the association between ari and weather variables. eligible studies must investigate this association among both indigenous and non-indigenous peoples living in the same region. the results can be presented in one of two eligible ways: (i) two models representing the indigenous and non-indigenous strata separately with the same seasonal or meteorological parameter in both models, producing two measures of association for which the ratio of odds ratios (ror) can be calculated (i.e., ari-exposure model for indigenous peoples and ari-exposure model for non-indigenous peoples); or (ii) a model for an ari outcome that includes indigeneity and seasonal or meteorological parameter(s) as independent variables from which further calculations can be made (fig. 1) . the case definition for ari is an acute infection (i.e., less than 14 days duration, if duration is stated) with coughing as a primary symptom, with or without any accompanying symptoms. if duration is not provided in the case definition, the word "acute" will be sufficient for inclusion. the majority of ari outcomes are anticipated to be respiratory syncytial virus, influenza, or pneumonia; however, studies reporting any ari outcome are eligible. a diagnosis of ari may include any symptomatic description that meets the case definition, a positive biological sample (e.g., swab), clinical diagnosis from a practitioner, or self-reported illness. studies of non-infectious respiratory outcomes (e.g., asthma) are not eligible. seasonal variables refers to a pattern (i.e., season) in meteorological parameters related to a predictable trend such as temperature, hours of sunlight, or total precipitation that is repeated annually [19] . meteorological parameters are defined as observable weather events, at a fig. 1 options for presentation of results in eligible studies, where option 1 represents a study presenting two models and option 2 represents a study presenting one model. e+ study exposure positive, estudy exposure negative, se standard error, β coefficient point in time, primarily consisting of temperature, precipitation, barometric pressure, humidity, sunlight, and the interactions and variability of these parameters [20] . in this review, any study that has seasonal or meteorological parameters measured at two or more points in time, and where the points are meteorologically or seasonally contrasting (i.e., measured in two different dates or in two different seasons), will be eligible. the search strategy comprises three main components: indigenous communities (population and exposure terms); and association between seasonal or meteorological parameters and ari (outcome terms; table 2 ). terms that will be used to identify indigenous peoples globally are based on a series of umbrella terms for indigenous used globally and throughout time, adapted from bartlett et al. [21] . individual group names were added to the umbrella terms from two sources. first, the international work group for indigenous affairs (iwgia; www.iwgia.org) registry provides a continental directory of indigenous peoples, further sorted as a country-bycountry list of recognized indigenous groups. these terms may be at the greater population level, rather than individual group level if the category can be expected to represent and include all of the unique peoples groups within it (i.e., the name "maori" was included in the search terms, but individual maori group names, such as ngati kuri, ngati maru, and to arawa, were not included). secondly, the united nations refugee agency (unhcr) provides a country-by-country database of minority and indigenous peoples (www.refworld.org). since this list provides both indigenous and non-indigenous peoples, only those groups explicitly listed as indigenous were collected. the names of all identified groups defined as indigenous were added to the search. when the two lists were complete, the lists were merged into one, alphabetized, and de-duplicated. the list of terms for indigenous peoples is comprehensive to the best of the ability of this strategy. searches of mesh terms for "season," "meteorology," and "weather" were performed in pubmed ® and used to compile terms for the search strategy. terms that will be used to identify ari outcomes in the literature include any pathogen known to primarily cause ari (i.e., enteric pathogens that rarely cause ari are not included) based on the medical microbiology 4th edition, chapter 93, online version [22] . any terms used for ari by the lung disease alphabetical listing generated by the american lung association (www.lung.org) were added to the search strategy. a search of mesh terms for "respiratory" and "lung infection" was performed in pubmed ® and any additional terms were added to the search strategy. it is difficult to develop a search strategy that is robust enough to represent all nuances of the terms indigenous, seasonal or meteorological parameters, and ari, and thus, the use of multiple databases is intended to increase sensitivity of the search. this review will search the following databases: pubmed ® (via ovidsp ® ), cab abstracts/cab direct © , and science citation index ® (via web of knowledge™). the search string will be appropriately adapted for each of the selected databases (tables 2, 3 , and 4). a university librarian was consulted in preparation of the search strategy for pubmed ® . the search will not be limited by language, date, or study design. search terms will be in english, although the names of indigenous groups are commonly stated in their own languages (i.e., non-english names in the roman alphabet syntax will be used). an english search string should identify all english articles and any non-english articles with an english title and abstract. if a non-english citation is collected by the search, google translate © will be used to translate the title and abstract for initial screening [23] , and if maintained for full article screening, google translate © will be used for full text screening. this will allow calculation of the total number of eligible articles to generate appropriate denominators. if after full article screening a non-english article is eligible, the article will be formally translated by a paid service, if funding is available. if it is not possible to translate non-english articles, they will be excluded from data extraction and risk of bias (i.e., after full-text screening). to minimize the risk of exclusion of relevant citations, the citation list of each included study will be searched (i.e., a snowball search). additionally, google scholar © will be used to complete a citation search on eligible studies, to identify studies that have referenced these studies. studies identified by either the hand-searches or citation searches will be screened for relevance. published and unpublished literature will be eligible. published literature can be collected by all of the proposed databases. unpublished literature can be collected by cab abstract/cab direct © and science citation index ® . unpublished primary research that exceeds 500 words in length will be eligible if it meets one of the three following criteria: (i) governmental report (i.e., produced by a regional or national government ministry); (ii) non-governmental report (i.e., produced by nongovernmental organizations); or (iii) graduate or honor undergraduate thesis or dissertation. reports that are fewer than 500 words will be excluded. if the initial search identifies any relevant government or non-governmental reports, or theses, a search will be conducted in pubmed ® to identify any relevant journal publications by the first author of the citation. this pubmed ® search will include the first author's last name "acute chest syndrome" or "acute respiratory distress syndrome" or "ards" or "bacterial pneumonia" or "black lung disease" or bronchitis or bronchiectasis or bronchiolitis or "bronchiolitis obliterans organizing pneumonia" or "boop" or "bronchopulmonary dysplasia" or bronchopneumonia or byssinosis or "chest infection*" or congest* or coccidioidomycosis or cocci or cough* or "cryptogenic organizing pneumonia" or "flu" or "hantavirus pulmonary syndrome" or histoplasmosis or "human metapneumovirus" or "hypersensitivity pneumonitis" or influenza* or "lower respiratory tract infection" or "lrti" or "lrtis" or "lung inflammation" or "mers" or "middle eastern respiratory syndrome" or "mycoplasma" or "non-tuberculosis mycobacterium" or pertussis or pleuropneumonia or pneumonconiosis or pneumocystis or and first initial, institutional affiliation, and one to three keywords from the abstract (i.e., author identified key words if available, or reviewer key words if not available). to keep the review current, if more than 12 months pass from the date the search was conducted to completion of data extraction and analysis, an update search will be conducted. if conducted, the second search will use the same search strategy as the first and will not be restricted by date. the search will be conducted in all of the original databases. thus, a recall strategy is employed [24] , which should identify all of the initial studies and all studies published since the previous search. a hand-matching method will be used to identify whether all of the original citations are included. all search result citations will be loaded into and managed in endnote™ bibliographic software and deduplicated automatically. then, citations will be uploaded from endnote™ into distillersr ® , which will be used for form generation, screening, and management of relevant screening level statistics. screening will be completed in two stages. screening processes will be piloted and tested by the reviewers on a subset of studies (5% of studies if n > 50, 10% of studies if n ≤ 50). first, title and abstract screening will be conducted on all citations identified. two reviewers with graduate-level training in epidemiology and systematic literature review processes will screen articles independently, using five evaluation questions ( table 5 ). all questions will be answered as "yes," "no," or "unsure." in this screening phase, questions will be hidden. a hidden question will not be answered if the article is excluded based on previous screening questions. articles will be excluded if both reviewers answer "no" to any of the five questions. if both reviewers answer "yes" and/or "unsure" to all questions it will be maintained for full article screening. any disagreements will be resolved by consensus. when consensus cannot be reached, a third reviewer will arbitrate. second, full article screening will be conducted on all citations remaining after title and abstract screening. two reviewers will screen articles independently, using a second form in distillersr ® . reviewers will use seven evaluation questions (table 6 ). in full article screening, reviewers will identify any studies using a duplicate dataset and will maintain the research that is most comprehensive. duplicate results will be removed. questions will not be hidden in full article screening, allowing for analysis of the reason for exclusion. studies will be excluded if both reviewers answer "no" to any question. disagreements between reviewers will be resolved by consensus and, when consensus cannot be reached, a third reviewer will arbitrate. data extraction will include study identifiers and study design; participant, exposure, and outcome information; and information about analytical methods. missing information will be noted. extracted study identifiers will be the authors' names; study title; publication type; publication date; journal, volume, issue, and page numbers of publication; place of publication (i.e., first author's institutional address); and digital object identifier. study design, time frame of study, climate zone of interest, location of study (i.e., country), and region of study (localized when reported) will also be extracted. data extracted about participants will include the definition of the target and source populations, size of the target population, and size of the source population. relevant demographic information (e.g., age, sex) at the study population level will be extracted when reported. exposure related data extracted will include the name of the indigenous population, the size of the indigenous source population, and the size of the indigenous study population. for the comparator, the size of the non-indigenous source population, and the size of the non-indigenous study population will be extracted. for all studies, the definition provided for indigenous peoples will be extracted. further, if causal mechanisms for differences in seasonal or meteorological effects on ari are provided, these will be extracted. information related to the seasonal or meteorological parameter(s) of interest, as well as the ari outcome(s) of interest will be extracted. in addition, the association(s) between ari and season or meteorological parameters will be extracted. for season and meteorological parameters, extracted information will include the name of each parameter (e.g., rainfall) and its related measure (e.g., millimeters); type of temporal pneumon* or pneumophila or "pneumocystis carnii" or pulmonary or respiratory or "respiratory distress" or "respiratory distress syndrome" or "respiratory tract infection*" or "rti" or "rsv" or "respiratory syncytial virus" or sarcoidosis or "severe acute respiratory syndrome" or streptococcus or "tuberculosis" or "tb" or "upper respiratory tract infection*" or "urti" or "urtis" or wheez* or "viral pneumonia" all terms searched as "all fields" terms. collected 1259 citations on september 27, 2016 exposure "atmospheric pressure" or barometric or cloud* or cold or "dew point" or heat* or humidity or meteorolog* or precipit* or rain* or season* or snow* or storm* or sunshine or temperature* or "uv" or "uv index" or "ultraviolet radiation" or vapor or warming or weather or wind or winds or windy cycle (e.g., daily, seasonal, annual); and number of cycles completed (e.g., years). additionally, the source of data used to evaluate the season or meteorological parameter will be collected (e.g., meteorological stations). the extracted information for each ari outcome will be the specific ari outcome (i.e., case definition), measurement of the ari outcome (e.g., self-reported), group-level metric for each population group (e.g., prevalence) and the effect size (i.e., beta) comparing the indigenous and non-indigenous peoples or strata (e.g., odds ratio). where two models are presented (e.g., ari-exposure models for indigenous and non-indigenous peoples), odds ratios will be extracted for each strata (fig. 1 ). where one model is presented (e.g., a model with indigeneity and weather parameter(s) as fixed effects), the regression coefficients for the season or meteorological parameter and indigeneity will be extracted. in the case that a study presents results for both options (i.e., two strata models and a single model with fixed effects), data will be extracted for both options. for each association, the measure(s) of precision (e.g., standard error of the mean, standard deviation, and/or confidence intervals) will be extracted when provided. if only the p value and sample size are reported, these data will be extracted and a measure of precision will be calculated from the available data for each association. finally, information will be extracted on the type of modeling or statistical approach (e.g., linear regression) used, and if and which confounders were considered. confounders considered will be extracted and a list will be generated for various ari outcomes. additionally, the unit of analysis (e.g., individual, household, or community) and spatial resolution of the climate data used for modeling will be extracted. a data extraction form will be created in distillersr ® . the extraction form will be piloted and tested by the data extractors on a subset of studies (5% of studies if n > 50, 10% of studies if n ≤ 50). following pilot testing, the form will be adapted as recommended by the extractors to improve usability and completeness. the first author and one additional extractor who each have training in epidemiology and systematic literature review processes will complete data extraction. data extraction will be completed independently and the extractors will compare the data for consensus. if the extractors cannot answer a question, consensus will confirm that the data are unavailable to answer the question. in the event that the data presented in a study are unclear, missing, or presented in a non-extractable or "acute chest syndrome" or "acute respiratory distress syndrome" or "ards" or "bacterial pneumonia" or "black lung disease" or bronchitis or bronchiectasis or bronchiolitis or "bronchiolitis obliterans organizing pneumonia" or "boop" or "bronchopulmonary dysplasia" or bronchopneumonia or byssinosis or "chest infection*" or congest* or coccidioidomycosis or cocci or cough* or "cryptogenic organizing pneumonia" or "flu" or "hantavirus pulmonary syndrome" or histoplasmosis or "human metapneumovirus" or "hypersensitivity pneumonitis" or influenza* or "lower respiratory tract infection" or "lrti" or "lrtis" or "lung inflammation" or "mers" or "middle eastern respiratory syndrome" or "mycoplasma" or "non-tuberculosis mycobacterium" or pertussis or pleuropneumonia or pneumonconiosis or pneumocystis or pneumon* or pneumophila or "pneumocystis carnii" or pulmonary or respiratory or "respiratory distress" or "respiratory distress syndrome" or "respiratory tract infection*" or "rti" or "rsv" or "respiratory syncytial virus" or sarcoidosis or "severe acute respiratory syndrome" or streptococcus or "tuberculosis" or "tb" or "upper respiratory tract infection*" or "urti" or "urtis" or wheez* or "viral pneumonia" all terms searched as "topic" terms. collected 1475 citations on september 26, 2016 bronchitis or bronchiectasis or bronchiolitis or "chest infection*" or congest* or cough* or "flu" or influenza* or "lung inflammation" or "middle eastern respiratory syndrome" or "mycoplasma" or pertussis or pneumon* or pulmonary or respiratory or "respiratory syncytial virus" or streptococcus or wheez* removed all acronyms, maintained symptoms and key diseases (more generic names only) all terms searched as "title" or "abstract" or "subject." collected 207 citations on september 27, 2016 unusable form, authors of studies published in the last 5 years (since january 1, 2011) will be contacted for clarification. authors will be contacted via email, and a follow-up email will be sent 2 weeks later. authors will be provided 4 weeks from the initial contact to respond. if data from older studies are unclear, missing, or presented in a non-extractable or unusable form, authors will not be contacted. missing data will be noted in the report. the risk of bias (rob) assessment was adjusted from existing tools (i.e., risk of bias in non-randomized studies of interventions) [25] . in particular, adjustments were needed to account for ecological studies and repeated measures. one question was added to investigate rob due to ecological studies and one question was added to investigate rob due to repeated measures. questions related to experimental unclear: it is unclear if the study design is primary research from the title and abstract. publication type: does the title and abstract come from a published study, government report, nongovernmental report, or postsecondary institutional thesis (exceeding 500 words in length)? yes: the study is a published study, or government report, nongovernmental report, or postsecondary institutional thesis (exceeding 500 words in length). no: the study is not a published study, government report, nongovernmental report, or postsecondary institutional thesis or is fewer than 500 words in length. unclear: it is unclear if the study is a published study, or government report, non-governmental report, or post-secondary institutional thesis (exceeding 500 words in length) from the title and abstract. population: does the population of interest include both an indigenous population and non-indigenous population living in the same geographical region? yes: the study population describes both indigenous and non-indigenous peoples living in the same geographical region. no: the population of interest is entirely indigenous or non-indigenous or the populations are not within the same region. unclear: it is unclear if the population of interest is both indigenous and non-indigenous from the title and abstract. outcome: does the title and/or abstract describe research on the association between ari and any exposure? yes: one or all of the outcomes of interest in the study are ari, defined by the case definition for this review (i.e. acute infection (less than 14 days duration, if duration is stated) with coughing as symptom, with or without any accompanying symptoms). no: there is no ari-related health outcome measured in the study. unclear: it is unclear if the health outcome of interest is ari from the title and abstract. exposure: is the study's exposure of interest a seasonal or meteorological factor (defined as observable weather events, primarily consisting of temperature, precipitation, barometric pressure, humidity, sunlight, and the interactions and temporal variability of these parameters)? yes: the exposure of interest is a seasonal or meteorological factor. no: the exposure of interest is not a seasonal or meteorological factor. unclear: it is unclear if the exposure of interest is a seasonal or meteorological factor. yes: the research compares associations between seasonal or meteorological parameters and ari in the context of indigeneity. no: the research does not compare associations between seasonal or meteorological parameters and ari in the context of indigeneity. duplicates: does the research use a new dataset for analysis (i.e., the dataset was not previously analyzed in another included study). yes: the research uses a new dataset for analysis. no: the research uses a dataset that has been previously analyzed by another included study. (note: when identifying the study to be included, the most comprehensive study will be used.) full article screening questions will include all screening domains and questions from title and abstract screening as well as two additional questions interventions were removed. two reviewers will conduct the rob assessment independently, in conjunction with data extraction, at the study level (i.e., one rob analysis will be conducted per study). both reviewers conducting the rob assessment will have advanced graduate training in epidemiology and bias assessment. in total, nine domains of bias will be tested according to predetermined criteria for high, low, or unclear rob. the rob will be conducted using a form in distillersr ® with a textbox to record the rationale for selecting the level of rob for each domain. since this review will focus on the association between weather parameters and ari outcomes, confounders in this study are those that affect the association between these weather parameters and ari in indigenous versus non-indigenous peoples. important confounders that could affect all or most studies are: (i) gender, (ii) age, and (iii) local wealth (e.g., regional or national). the review will analyze information for both associations (systematic review and meta-analyses) and context (e.g., via descriptive statistics, narrative, and descriptive spatial analyses). this approach is necessary to avoid comparing unlike populations, exposures, or outcomes. analyses will be completed using stata © version 13.1 and revman © version 5. prior to beginning meta-analyses, descriptive statistics will be conducted on extracted data. frequencies, proportions, and missing data will be considered for each extracted variable. the descriptive data will serve to describe the literature available on this topic and to represent the dataset under study. meta-analyses will be conducted for each ari association (i.e., each ari outcome and weather parameter identified) that has at least two studies providing data (i.e., two studies presenting the association between the same ari outcome and same weather parameter). the outcome used for meta-analyses will be the ror representing the relative effects of weather parameters on ari between indigenous and non-indigenous peoples (fig. 1) . for studies presenting two models (i.e., ari associations for each strata), odds ratios for each eligible study for each group will be extracted (i.e., indigenous, non-indigenous) to calculate a ror. in studies presenting a single model, regression coefficients will be used directly to calculate odds ratios and solve for the ror (fig. 1) . the standard error of the ror will be calculated according to golder et al. [26] for both study types. meta-analyses will be conducted using random effects models. between-study heterogeneity will be explored using the i 2 statistic (i 2 < 0.25 considered homogeneous). if heterogeneity exists, sources of heterogeneity will be explored by sub-group analyses. for eligible studies, we propose to categorize the studies by (i) population, (ii) outcome, (iii) exposure, and (iv) location. studies will be categorized by groups of indigenous peoples (e.g., all studies of maori peoples) for population; as upper respiratory, lower respiratory, or unclear/both for outcome; as seasonal or meteorological parameters for exposure; and as a high-, middle-, or low-income country and by climate zone for location. when there are at least two effect sizes for each category, we will calculate a summary effect size. descriptive statistics will be conducted on each of the domains of the rob. if enough data are available and rob profiles vary, heterogeneity will be explored using sub-group analyses on each domain as an independent variable. publication bias is the concept that significant results are more likely to be published than non-significant research results [27] . an evaluation of publication bias will be conducted using a funnel plot (if n > 10 studies). ratio measures of association will be plotted on the logarithmic scale to increase symmetry. a 95% confidence interval will be plotted, and different symbols will be used if heterogeneity is present. interpretation of the funnel plot will be done visually. a test of significance for publication bias will be conducted using egger's test [28] . finally, to summarize data about region of study and climate zone, descriptive spatial analyses will be performed. specifically, point maps will be generated. the point map will illustrate specific study locations. when point locations are not provided in text with latitudinal and longitudinal coordinates, study sites will be geolocated using google maps © and the best information available in the study. the point map will use open source shape files and will be built in r spatial © and r maptools © statistical packages. the final search strategy for each database and all ancillary searches conducted will be provided in the additional file 1 of the final report. a flow chart, following the prisma guidelines [17] , will be used to illustrate where citations were eliminated during screening and ancillary searches, including information about the rationale for exclusion in full article screening. to illustrate the potential for publication bias and small study effects, a funnel plot will plot the effect estimates (horizontal axes) against the standard error (vertical axes) for each meta-analysis with n > 10 studies [29] . the results of this review will be provided via text and characteristics of studies tables in a published journal article. the tables will describe the seasonal or meteorological exposure, the outcome(s) measured, the direction and magnitude of association, and the period of study. descriptive statistics (i.e., frequencies, proportions, missing data) will be provided as extensions of this table when appropriate or in narrative (i.e., proportion of studies with each ari outcome). individual study results will be presented in forest plots [17] . if heterogeneity exists, separate forest plots will be used to illustrate results by strata. if the data are too limited (i.e., fewer than two studies with the same population, exposure, outcome, and region) or are heterogeneous, the results will be presented in a forest plot without a summary effect size. a summary of findings table for key outcomes will be generated based on the grading of recommendations assessment, development and evaluation [30] . a priori key outcomes will include prevalence of upper ari and prevalence of lower ari. additional key outcomes identified in the systematic literature review will be documented as protocol amendments. a table will be provided to summarize the findings of the rob assessment. this table will follow the rob presentation suggested in the prisma guidelines [17] . an additional column will highlight the rationale for the study's rob level. maps indicating the specific location of studies (point map) will be generated. climate zones will be indicated on each map (e.g., tropical, temperate, or arctic). this research does not involve working directly with indigenous and non-indigenous communities, but rather with previous research conducted with these communities. in conducting this research, ethical principles will still be at the forefront, and will involve considerations for small population sizes and framing of the findings. this systematic review protocol presents the method for the synthesis of current evidence related to differences in seasonal or meteorological association with ari between indigenous and non-indigenous peoples living in the same region. this proposed review will likely be the first to summarize the potentially different associations between ari and weather parameters between indigenous and non-indigenous peoples. the results of the meta-analysis will examine whether indigenous peoples are equally susceptible to associations between weather parameters and ari, and whether this relationship varies by place, population, or other characteristics. a deeper understanding of this relationship will advance the academic literature and potentially lead to intervention strategies as climate change progresses. further, an understanding of the differences between indigenous and non-indigenous communities can aid in planning, resource allocation, and determination of appropriate interventions. global burden of acute respiratory infections in children: implications for interventions global burden of acute lower respiratory infections due to respiratory syncytial virus in young children: a systematic review and meta-analysis global and regional burden of hospital admissions for severe acute lower respiratory infections in young children in 2010: a systematic analysis the respiratory health of urban indigenous children aged less than 5 years: study protocol for a prospective cohort study what is the seasonal distribution of community acquired pneumonia over time? a systematic review are meteorological parameters associated with acute respiratory tract infections? urban as a determinant of health social status and susceptibility to respiratory infections indigenous health and climate change seasonal pattern of pneumonia mortality among under-five children in nairobi's informal settlements the land enriches the soul: on climatic and environmental change, affect, and emotional health and well-being in rigolet from this place and of this place:" climate change, sense of place, and health in nunatsiavut united nations: indigenous peoples, indigenous voices factsheet. edited by issues upfoi vulnerability of indigenous health to climate change: a case study of uganda's batwa pygmies health of indigenous people in africa social determinants of health inequalities preferred reporting items for systematic reviews and meta-analyses: the prisma statement preferred reporting items for systematic review and metaanalysis protocols (prisma-p) 2015 statement seasonality in six enterically transmitted diseases and ambient temperature descriptive meteorology identifying indigenous peoples for health research in a global context: a review of perspectives and challenges infections of the respiratory system international burden of chronic kidney disease and secondary hyperparathyroidism: a systematic review of the literature and available data precision and recall of search strategies for identifying studies on return-to-work in medline the risk of bias in nonrandomized studies -of interventions (robins-i) assessment tool meta-analyses of adverse effects data derived from randomised controlled trials as compared to observational studies: methodological overview trim and fill: a simple funnel-plot-based method of testing and adjusting for publication bias in meta-analysis bias in meta-analysis detected by a simple, graphical test the cochrane collaboration's tool for assessing risk of bias in randomised trials grading quality of evidence and strength of recommendations the authors wish to thank ali versluis, university of guelph library, for her time and expertise contributed to this protocol. funding for this protocol was provided through scholarships from the ontario veterinary college (k. bishop-williams) and the ontario graduate scholarship program (k. bishop-williams).availability of data and materials not applicable.authors' contributions kbw will serve as the first author of the protocol and review paper. she led all stages of protocol development, including development of the research question and objectives, search strategy, and extraction and analysis plans. jms, lbf, vle, ac, and slh supervised and contributed to the entirety of the development of plans for searching, screening, extracting, and writing phases. all authors have graduate training in epidemiology and/or health studies. all authors read and approved the final manuscript. the authors declare that they have no competing interests. • we accept pre-submission inquiries • our selector tool helps you to find the most relevant journal submit your next manuscript to biomed central and we will help you at every step: key: cord-269437-0pvqvhqs authors: gastañaduy, paul a. title: update: severe respiratory illness associated with middle east respiratory syndrome coronavirus (mers-cov) — worldwide, 2012–2013 date: 2013-06-14 journal: mmwr morb mortal wkly rep doi: nan sha: doc_id: 269437 cord_uid: 0pvqvhqs cdc continues to work in consultation with the world health organization (who) and other partners to better understand the public health risk posed by the middle east respiratory syndrome coronavirus (mers-cov), formerly known as novel coronavirus, which was first reported to cause human infection in september 2012. the continued reporting of new cases indicates that there is an ongoing risk for transmission to humans in the area of the arabian peninsula. new reports of cases outside the region raise concerns about importation to other geographic areas. nosocomial outbreaks with transmission to health-care personnel highlight the importance of infection control procedures. recent data suggest that mild respiratory illness might be part of the clinical spectrum of mers-cov infection, and presentations might not initially include respiratory symptoms. in addition, patients with comorbidities or immunosuppression might be at increased risk for infection, severe disease, or both. importantly, the incubation period might be longer than previously estimated. finally, lower respiratory tract specimens (e.g., sputum, bronchoalveolar lavage, bronchial wash, or tracheal aspirate) should be collected in addition to nasopharyngeal sampling for evaluation of patients under investigation. an emergency use authorization (eua) was recently issued by the food and drug administration (fda) to allow for expanded availability of diagnostic testing in the united states. on june 7, 2013 , this report was posted as an mmwr early release on the mmwr website (http://www.cdc.gov/mmwr). cdc continues to work in consultation with the world health organization (who) and other partners to better understand the public health risk posed by the middle east respiratory syndrome coronavirus (mers-cov), formerly known as novel coronavirus, which was first reported to cause human infection in september 2012 (1) (2) (3) (4) . the continued reporting of new cases indicates that there is an ongoing risk for transmission to humans in the area of the arabian peninsula. new reports of cases outside the region raise concerns about importation to other geographic areas. nosocomial outbreaks with transmission to health-care personnel highlight the importance of infection control procedures. recent data suggest that mild respiratory illness might be part of the clinical spectrum of mers-cov infection, and presentations might not initially include respiratory symptoms. in addition, patients with comorbidities or immunosuppression might be at increased risk for infection, severe disease, or both. importantly, the incubation period might be longer than previously estimated. finally, lower respiratory tract specimens (e.g., sputum, bronchoalveolar lavage, bronchial wash, or tracheal aspirate) should be collected in addition to nasopharyngeal sampling for evaluation of patients under investigation. an emergency use authorization (eua) was recently issued by the food and drug administration (fda) to allow for expanded availability of diagnostic testing in the united states. as of june 7, 2013, a total of 55 laboratory-confirmed cases have been reported to who. illness onsets have occurred during april 2012 through may 29, 2013 ( figure 1 ). all reported cases were directly or indirectly linked to one of four countries: saudi arabia, qatar, jordan, and the united arab emirates ( figure 2 ). most cases (40) were reported by saudi arabia. four countries, the united kingdom (uk), italy, france, and tunisia, have reported cases in returning travelers and their close contacts (5) (6) (7) (8) . ill patients from qatar and the united arab emirates have been transferred to hospitals in the uk and germany. to date, no cases have been reported in the united states. who and cdc have not issued any travel advisories at this time; updated information for travelers to the arabian peninsula is available at http://wwwnc.cdc.gov/travel/notices/ watch/coronavirus-arabian-peninsula. the median age of patients is 56 years (range: 2-94 years), with a male-to-female ratio of 2.6 to 1.0. all patients were aged ≥24 years, except for two children, one aged 2 years and one aged 14 years. all patients had respiratory symptoms during their illness, with the majority experiencing severe acute respiratory disease requiring hospitalization. thirty-one of the 55 patients are reported to have died (case-fatality rate: 56%) (5) (6) (7) (8) . two cases in tunisia, in siblings whose father's illness was a probable case, and a case from the uk, were in persons with mild respiratory illnesses who were not hospitalized (5, 9) . information was not available for all cases; however, several patients had accompanying gastrointestinal symptoms, including abdominal pain and diarrhea, and many cases occurred among persons with chronic underlying medical conditions or immunosuppression, as reported to who (5, 9) . the original source(s), route(s) of transmission to humans, and the mode(s) of human-to-human transmission have not been determined. eight clusters (42 cases) have been reported by six countries (france, italy, jordan, saudi arabia, tunisia, and the uk) (5) among close contacts or in health-care settings and provide clear evidence of human-to-human transmission of mers-cov. the first documented patient-to-patient nosocomial transmission in europe was confirmed recently in france (10). the first french patient, a man aged 64 years with a history of renal transplantation, became ill on april 22, 2013, within 1 week after returning from dubai. he presented with fever and diarrhea. pneumonia was diagnosed incidentally on radiographic imaging, and he subsequently died with severe respiratory disease. the secondary case is in a man aged 51 years on long-term corticosteroids who shared a room with the index patient during april 26-29 and who remains hospitalized on life support. the incubation period for the secondary case was estimated to be 9-12 days; this is longer than the previously estimated 1-9 days (10). a larger cluster, consisting of 25 cases including 14 deaths, ongoing since april 2013 in the region of al-ahsa in eastern saudi arabia, also has included cases linked to a health-care facility (5) . cases have included health-care personnel and family contacts. an additional five cases, not linked to the cluster in al-ahsa, were reported recently in another region of eastern saudi arabia (5). thus far, no evidence of sustained community transmission beyond the clusters has been reported in any country. in some instances, sampling with nasopharyngeal swabs did not detect mers-cov by polymerase chain reaction (pcr); however, mers-cov was detected by pcr in lower respiratory tract specimens from these same patients. in the two patients reported by france, nasopharyngeal specimens were weakly positive or inconclusive, whereas bronchoalveolar lavage and induced sputum were positive (10). in consultation with who, the period for considering evaluation for mers-cov infection in persons who develop severe acute lower respiratory illness days after traveling from the arabian peninsula or neighboring countries* has been extended from within 10 days to within 14 days of travel. persons who develop severe acute lower respiratory illness within 14 days after traveling from the arabian peninsula or neighboring countries should be evaluated according to current guidelines (available at http://www.cdc.gov/coronavirus/mers/case-def. html). persons whose respiratory illness remains unexplained and who meet criteria for "patient under investigation" should be reported immediately to cdc through state and local health departments. persons who develop severe acute lower respiratory illness who are close contacts † of a symptomatic traveler who developed fever and acute respiratory illness within 14 days of traveling from the arabian peninsula or neighboring countries may be considered for evaluation for mers-cov. in addition, cdc recommends that clusters of severe acute respiratory illness be investigated and, if no obvious etiology is identified, local public health officials be notified and testing for mers-cov conducted, if indicated. to increase the likelihood of detecting mers-cov, cdc recommends collection of specimens from different sites (e.g., a nasopharyngeal swab and a lower respiratory tract specimen, such as sputum, bronchoalveolar lavage, bronchial wash, or tracheal aspirate). specimens should be collected at different times after symptom onset, if possible. lower respiratory tract specimens should be a priority for collection and pcr testing; stool specimens also may be collected. specimens should be collected with appropriate infection control precautions (available at http://www.cdc.gov/coronavirus/mers/case-def.html). testing of specimens for mers-cov currently is being conducted at cdc. fda issued an eua on june 5, 2013, to authorize use of cdc's novel coronavirus 2012 real-time reverse transcription-pcr assay (ncv-2-12 rrt-pcr assay) to test for mers-cov in clinical respiratory, blood, and stool specimens. this eua is needed because, at this time, there are no fda-approved tests that identify mers-cov in clinical specimens. this assay will be deployed to laboratory response * countries considered to be on or neighboring the arabian peninsula include bahrain, iraq, iran, israel, jordan, kuwait, lebanon, oman, palestinian territories, qatar, saudi arabia, syria, the united arab emirates, and yemen. † close contacts are defined as 1) persons who provided care for the patient, including health-care personnel and family members, or who had other similarly close physical contact, or 2) persons who stayed at the same place (e.g., lived with or visited) as the patient while the patient was ill. network (lrn) laboratories in all 50 states over the coming weeks. updated information about laboratories with the capacity to conduct mers testing with the ncv-2-12 rrt-pcr assay will be provided on cdc's mers website (http://www. cdc.gov/coronavirus/mers/case-def.html). in consultation with who, the definition of a probable case of mers-cov infection has been updated to also include persons with severe acute respiratory illness with no known etiology with an epidemiologic link to a confirmed case of mers-cov infection. until the transmission characteristics of mers-cov are better understood, patients under investigation and probable and confirmed cases should be managed in health-care facilities using standard, contact, and airborne precautions. as information becomes available, these recommendations will be reevaluated and updated as needed. recommendations and guidance on case definitions, infection control (including use of personal protective equipment), case investigation, and specimen collection and testing, are available at the cdc mers website (http://www.cdc.gov/ coronavirus/mers/index.html). the mers website contains the most current information and guidance, which is subject to change. state and local health departments with questions should contact the cdc emergency operations center (770-488-7100). cases associated with a cluster cases reported in al-ahsa governorate cases not associated with a cluster travel history associated with cases occuring outside of arabian peninsula and neighboring countries * dots representing the cases are not geographically representative of the exact location of the residence of the patient. severe respiratory illness associated with a novel coronavirus-saudi arabia and qatar novel coronavirus associated with severe respiratory disease: case definition and public health measures isolation of a novel coronavirus from a man with pneumonia in saudi arabia middle east respiratory syndrome coronavirus (mers-cov); announcement of the coronavirus study group global alert and response (gar): novel coronavirus infection -update (middle east respiratory syndrome coronavirus) updated rapid risk assessment: severe respiratory disease associated with middle east respiratory syndrome coronavirus (mers-cov) european centre for disease prevention and control. epidemiological update: additional confirmed cases of middle east respiratory syndrome coronavirus (novel coronavirus) in france, saudi arabia, and tunisia world health organization. global alert and response (gar): novel coronavirus summary and literature update family cluster of middle east respiratory syndrome coronavirus infections clinical features and viral diagnosis of two cases of infection with middle east respiratory syndrome coronavirus: a report of nosocomial transmission key: cord-292828-29jbf9ik authors: alsaleh, asma n; whiley, david m; bialasiewicz, seweryn; lambert, stephen b; ware, robert s; nissen, michael d; sloots, theo p; grimwood, keith title: nasal swab samples and real-time polymerase chain reaction assays in community-based, longitudinal studies of respiratory viruses: the importance of sample integrity and quality control date: 2014-01-09 journal: bmc infect dis doi: 10.1186/1471-2334-14-15 sha: doc_id: 292828 cord_uid: 29jbf9ik background: carefully conducted, community-based, longitudinal studies are required to gain further understanding of the nature and timing of respiratory viruses causing infections in the population. however, such studies pose unique challenges for field specimen collection, including as we have observed the appearance of mould in some nasal swab specimens. we therefore investigated the impact of sample collection quality and the presence of visible mould in samples upon respiratory virus detection by real-time polymerase chain reaction (pcr) assays. methods: anterior nasal swab samples were collected from infants participating in an ongoing community-based, longitudinal, dynamic birth cohort study. the samples were first collected from each infant shortly after birth and weekly thereafter. they were then mailed to the laboratory where they were catalogued, stored at -80°c and later screened by pcr for 17 respiratory viruses. the quality of specimen collection was assessed by screening for human deoxyribonucleic acid (dna) using endogenous retrovirus 3 (erv3). the impact of erv3 load upon respiratory virus detection and the impact of visible mould observed in a subset of swabs reaching the laboratory upon both erv3 loads and respiratory virus detection was determined. results: in total, 4933 nasal swabs were received in the laboratory. erv3 load in nasal swabs was associated with respiratory virus detection. reduced respiratory virus detection (odds ratio 0.35; 95% confidence interval 0.27-0.44) was observed in samples where the erv3 could not be identified. mould was associated with increased time of samples reaching the laboratory and reduced erv3 loads and respiratory virus detection. conclusion: suboptimal sample collection and high levels of visible mould can impact negatively upon sample quality. quality control measures, including monitoring human dna loads using erv3 as a marker for epithelial cell components in samples should be undertaken to optimize the validity of real-time pcr results for respiratory virus investigations in community-based studies. acute respiratory infections (aris) caused by viruses are the most common illnesses experienced by all age groups. aris are particularly important during early life as infants have the highest infection rates and they can transmit infectious agents to other household members [1] . recently introduced molecular-based diagnostic techniques have much improved sensitivity compared with previous classical culture and phenotypic-based methods and have led to the discovery of new respiratory viruses [2] . however, contemporary studies employing these new techniques have often used convenience samples obtained from patients admitted to hospital or attending emergency department clinics [3] [4] [5] . understanding more fully the ari disease burden in the community is important for developing public health interventions, such as vaccination programs [6] , and for understanding the role respiratory viruses may play in the pathogenesis of certain chronic pulmonary disorders, such as asthma [7] [8] [9] . this has led to the instigation of community-based studies. such studies do however have some logistical challenges, particularly concerning respiratory sample collection and transport. most studies have relied upon clinic or home visits by trained healthcare workers to collect specimens during an ari episode, which imposes restrictions upon busy families and may lead to biased disease estimates and specimen availability [10] [11] [12] . cost and feasibility of using healthcare workers are also important when large longitudinal, community-based cohort studies, involving frequent specimen collections, are planned. to help address these limitations, we and others have begun testing parentcollected, anterior nasal swab specimens that have been transported to the research laboratory using the standard mail [13] [14] [15] [16] . this approach is considered to be safe, convenient and cost-effective [17] . importantly, when using highly sensitive polymerase chain reaction (pcr) assays the detection rates for respiratory viruses are similar in both anterior nasal swab specimens and samples collected by the more traditional method of nasopharyngeal aspiration [18, 19] . building on this information, later studies have also shown that pcr testing for respiratory viruses provided similar results for parent-collected anterior nasal swab specimens and either nasal swab or nasoparyngeal aspirates collected by healthcare professionals [16, 17] . other studies examining sample transport have also shown that mailing swabs at ambient temperature has limited or no impact on respiratory virus detection by pcr [14, 20, 21] , although investigating further the effects of transporting samples for extended periods and at higher temperatures was highlighted in one study [20] . the observational research in childhood infectious diseases (orchid) project is a longitudinal, communitybased, dynamic birth cohort study, which seeks to describe the nature and timing of respiratory viruses detected in australian children during the first 2-years of life [22] . the study commenced in late 2010 and involves parents collecting and mailing nasal swabs weekly to the research laboratory for pcr-based respiratory virus screening. during the first year mould was seen in some samples as they arrived in the laboratory and we became concerned about the impact of this contaminant upon sample integrity. therefore, as part of the orchid study, we undertook a broader investigation of sample quality, examining collection and transportation, and how these impact on respiratory virus detection. our objectives were first to determine the quality of specimen collection by testing for the presence of human dna (endogenous retrovirus3; erv3) and then to investigate the effects of sample quality and the presence of visible mould in samples reaching the laboratory upon pcr performance. briefly, as part of orchid, families expecting a healthy term baby were recruited antenatally at either the publically funded royal brisbane and women's hospital or the north west private hospital, in brisbane, australia, a subtropical city of more than 2 million inhabitants [22] . the human research ethics committees of the children's health queensland hospital and health service, the royal brisbane and women's hospital and the university of queensland approved the study. parents/caregivers of each baby provided written, informed consent at the time of enrolment into the study. parents were asked to record from birth a daily symptom diary and to collect anterior nasal swab samples every week until their infant's second birthday. instructions on sample collection were provided at the initial visit by research staff who also demonstrated the technique by undertaking the initial nasal swab specimen shortly after delivery of the newborn baby. in addition, parents were given written instructions on how to collect nasal swab specimens. they also received regular text messages, emails or telephone calls as means of research staff keeping in contact with participating families. regular supplies of sterile rayon swabs (virocult, mw950, medical wire & equipment, england) were provided, which were rotated against the internal anterior walls of both nostrils and then placed in the provided transport tube that contained a viral transport media-soaked foam pad in the base. parents were instructed to squeeze the foam pad to release the fluid and bathe the top of the swab. ideally within 24hours of collection, the nasal swabs were then sent by regular postal mail (in accordance with australia post regulations [23] ) at ambient temperature to our research laboratory where they were stored at −80°c until analysis. nasal swabs were vortexed in 2 ml of phosphate buffered saline from which 200 μl was spiked with 5 μl of equine herpes virus-1 (ehv1) culture supernatant, which served as an extraction and inhibition control agent, before nucleic acid was extracted using the cas1820 xtractorgene automated system (qiagen-australia) according to the manufacturer's instructions. the final volumes of specimen extracts were 150 μl/specimen eluted in 96 well racks (matrix, thermo scientific, australia). for each run (96 extracts/run), extracts were tested using a duplex real-time pcr assay for ehv1 and erv3 in the following reaction compositions; 10pmoles of each primer, 4pmoles of each probe (table 1) , 10 μl of sensimix ii probe pcr mix (bioline, australia) and 2 μl of extract in a 20 μl final reaction. cycling conditions used for amplification were: initial hold at 10 min at 95°c; followed by 45 cycles of 30 sec at 95°c and 60 sec at 60°c. the ehv1 component was performed as an extraction and inhibitor control as described previously [24] , while erv3 was used as a marker to evaluate the quality of nasal swab sample collection [25] . briefly, the samples were considered to have failed the ehv1 component (ie. failed extraction or possessed pcr inhibitors) if the ehv1 real-time pcr cycle threshold (ct) results for individual samples were more than two standard deviations from the mean value of all samples, which for this study was calculated to be approximately 30 cycles [24] . samples that passed ehv1 dna extraction quality control testing were screened for respiratory viruses using previously optimized and described pcr and reverse transcriptase pcr assays. virus testing assays included: rhinovirus (rv) [26] , influenza viruses (a and b) [27] , respiratory syncytial viruses (a and b) [28] , parainfluenza viruses (1-3) [29] , human adenoviruses [22] , human metapneumovirus [30] , human coronaviruses (oc43, hku1, 229e, and nl63) [31, 32] , human bocavirus [33] and human polyomaviruses (wupyv and kipyv) [34] . for all viruses, except rv, samples were tested in a 10 × 10 pooled format. briefly, aliquots of the sample extracts were pooled using the cas-1200 liquid handling system (qiagen-australia) and pools tested for the presence of respiratory viruses. for positive pools, individual sample extracts were then tested to confirm positivity. rv screening was performed on individual sample extracts, and not on the pooled extracts, as the number of expected positive samples was considered too high for there to be any benefits from pooling. during the initial phases of the study, mould was observed growing on a small number of nasal swabs at the time of their arrival at the laboratory. in light of this observation, before extraction all swabs were inspected visually for mould and were assigned a semi-qualitative score according to a sliding scale (0 to 3), whereby 0 = no mould observed, 1 = low, 2 = medium, and 3 = high levels of visible mould present. dna sequencing was used to identify the type of fungi present on a subset of swabs exhibiting varying degrees of visible mould growth (10 swabs where no mould was seen, and 20 each where low, medium and high levels, respectively, of mould contamination was present). pcr amplification of a fungal internal transcribed spacer (its) region was performed using 10 pmoles of forward and reverse primers (its1 forward primer tccgtaggt gaacctgcgg and its4-reverse primer tcctccgc tta ttgatatgc [35] , 25 μl of qiagen sybr master mix (qiagen, australia) and 5 μl of template in a total 50 μl reaction mix. cycling was performed using the following conditions: 95°c for 15 min, 45 cycles of 95°c for 30 sec, 50°c for 30 sec and 72°c for 60 sec and a melting step of 60-95°c at the end of the thermal cycling. pcr products were examined by gel electrophoresis using a 2% agarose gel and sent to the australian genome research facility (the university of queensland, brisbane) for automated sequencing. for this study, samples that failed ehv1 criteria or were not inspected for mould growth were excluded from the analysis (figure 1 ). the association between variables of interest and binary outcomes was investigated using mixed effects logistic regression models, with participants included as a random intercept to account for the possibly correlated outcomes within each infant. the association with continuous outcomes was investigated using mixed effects linear regression. when examining the association of mould level with sample quality and respiratory virus detection we conducted both univariate and multivariate analyses, with multivariate analyses adjusting for the potential confounders of the child's age, gender, relationship of collector to participant (e.g. father, mother or others), season specimen collected, and time from specimen collection to being frozen in the laboratory. analyses were conducted using stata statistical software v.11.0 (statacorp, college station, tx, usa). between september 2010 and july 2012, 152 infants were recruited into the study. all participants lived within the greater brisbane metropolitan area and none were from rural communities. one-hundred and twentyfive recruits remained active study participants up until the date of this analysis. of the 27 withdrawals, four had moved out of the study area, two others were later deemed ineligible, ten withdrew for personal reasons and eleven were ineligible because they could not fulfill sampling requirements. for the active families, swab return rates were >90% for almost 35,000 child-days of observation. in total, 4933 weekly nasal swab specimens (~510 nasal swabs/ month) were batched in 56 (96 well) racks, extracted and tested. the median time from collection to swab arrival in the laboratory was 2 (interquartile range 2-4) days; however 10.9% of swabs were received more than 7-days after their collection. for ehv1 extraction and inhibition testing, 42 (0.81%) dna extracts failed the ehv1 criteria. the initial 1525 samples were not inspected for mould growth during the early stages of the study and therefore were excluded from further analysis. of (figure 1 ). however, following a cluster of samples negative for erv3 (figure 1 ; batches 41, 43, 44) we contacted parents and reminded them of the optimal swab collection technique they had been shown at enrolment of their baby. after this feedback the numbers of erv3 negative samples declined. at least one respiratory virus was detected in 885 (26.2%) samples. dual or multiple virus detections were observed in 105 (2.14%) samples. rv was the most common virus detected, being present in almost 20% of specimens, followed by human bocavirus, human polyomavirus kipyv, respiratory syncytial viruses and human adenoviruses ( table 2) . of 3366 (table 3) . a diverse range of species was observed with epicoccum nigrum and cladosporium cladosporioides the most prevalent. of the 2718 samples that were erv3 positive, 810 (37.2%) had at least one respiratory virus detected by pcr. in contrast, the respiratory virus detection rate in erv3 negative samples was significantly lower (75/649, 11.5%; crude odds ratio (or) = 0.35; 95% ci 0.27-0.44) when erv3 was absent in swab specimens. we also observed that among erv3 positive swabs, the average erv3 ct value for samples positive for any respiratory virus (32.8 cycles) was significantly lower (indicating greater erv3 load) than the average ct value (35.4) in samples negative for all viruses (crude difference = 2.0, 95% ci 1.4 -2.6; figure 2 ). moreover, there was a significant difference in erv3 ct values (p = 0.001) in samples that table 4 examines the association between erv3 and respiratory virus detection and potential explanatory and confounding variables. erv3 positive sample rates increased with age, varied by season and declined with increasing mould levels and time taken for samples to reach the laboratory and to be frozen. similarly, respiratory virus detection rates increased with age, specimen collection outside the summer months, and time taken to reach the laboratory, while decreasing as visible mould levels in samples reaching the laboratory increased. the orchid project is an ongoing comprehensive community-based study using pcr assays to detect respiratory viruses in anterior nasal swab specimens taken weekly by parents from their infants throughout the first 2-years of life. this requires parents following a standardized protocol of obtaining swabs regularly and mailing them promptly to our laboratory. however, we have observed that suboptimal sample collection as determined by erv3 detection and presence of visible mould in swab samples reaching the laboratory can negatively affect sample quality and potentially respiratory virus detection. the data from the first 20-months of our longitudinal study indicate that respiratory virus detection is associated with the erv3 load in nasal swab specimens. swabs negative for erv3, presumably from sub-optimal collection, had reduced respiratory virus detection rates compared with samples containing erv3. furthermore, in those specimens positive for erv3, a higher erv3 load was associated with a higher likelihood of respiratory virus detection. overall, this shows the importance of measuring human dna as a marker for epithelial cells in swab samples, which if tested and monitored in real time during the study, can identify problems associated with collection that can be addressed quickly. this is illustrated in the current study when a sudden increase in erv3 negative samples was observed. parents were contacted and reminded about sample collection protocols following which there was a decline in erv3 negative sample rates towards baseline levels. we were also concerned at finding mould on some samples, which occurred despite the commercial swab tubes containing antifungal agents. most fungal species identified in the swabs were saprophytic, and the most common fungus found, epicoccum nigrum, is a known contaminant of clinical specimens [36] . the relationship between fungal airspora counts and meteorological conditions is complex and impacts at the species level [37] . in brisbane, cladosporium and alternaria airspora are detected commonly throughout the year, but as with epicoccum,sp their levels peak during the warmer, humid months. other factors, such as rainfall and wind speed, table. can also influence fungal airspora composition [37, 38] . in our study, mould was associated mainly with longer time intervals between taking swabs and their arrival at the laboratory. however, this was especially evident during the warm, humid spring and summer months, which leads us to speculate that fungal contamination occurred during sample collection and was influenced by the aforementioned environmental factors. unfortunately, we could not explore this further as it was beyond the scope of the present study. in addition, while mould growth proved to be an issue in the subtropical climate of brisbane, this may be less of a problem in more temperate climates with lower temperatures and humidity levels. we now remind parents regularly to mail swabs promptly after collection. of interest however, was that respiratory virus detection rates were not affected by prolonged transport times, but in fact appeared to increase with time taken to reach the laboratory. while the observed increase was unexpected and may have occurred simply by chance, it is plausible that viral nucleic acids were protected to some extent by being encapsulated within the viral capsid, and by using viral transport medium in the swabs. fungi were found to be associated with both reduced erv3 detection and, at high levels, reduced significantly respiratory virus detection. at least three points emerge from this study. first, although swabs may contain antimicrobial agents, the risk of fungal and potentially bacterial contamination may still arise. second, the times between swab collection and laboratory arrival should be monitored and feedback provided if delays occur. finally, if delays are expected swabs should be placed in the household refrigerator until mailed to the laboratory [20] . we found that erv3 as a marker for human dna and epithelial cells was also an important indicator of sample quality for our study. for community-based investigations similar to our own, real-time sample processing and erv3 detection can facilitate rapid interventions to maintain sample quality and to optimize respiratory virus detection. indeed, this may have broader implications since nasal swabs are beginning to replace the traditional, but more invasive nasopharyngeal swab or aspirate sampling techniques in hospitals and clinics, especially following the 2009 influenza pandemic [17] . thus, similar erv3 testing strategies could be used by diagnostic laboratories to improve or monitor sample collection quality for optimal respiratory virus detection. finally, the potential problem of visible mould contamination of swabs taken during community-based studies can be minimized by ensuring samples are transported promptly to the laboratory. epidemiology of viral respiratory infections molecular diagnosis of respiratory viruses detection of new respiratory viruses in hospitalized infants with bronchiolitis: a three-year prospective study evidence of human coronavirus hku1 and human bocavirus in australian children frequent detection of human rhinoviruses, paramyxoviruses, coronaviruses, and bocavirus during acute respiratory tract infections the cost of communitymanaged viral respiratory illnesses in a cohort of healthy preschool-aged children lower airway rhinovirus burden and the seasonal risk of asthma exacerbation novel human rhinoviruses and exacerbation of asthma in children role of viral respiratory infections in asthma and asthma exacerbations serial viral infections in infants with recurrent respiratory illnesses frequency of detection of picornaviruses and seven other respiratory pathogens in infants van der ent ck: viral specimen collection by parents increases response rate in population-based virus studies van der ent ck: respiratory pathogens in respiratory tract illnesses during the first year of life van der ent ck: highly frequent infections with human rhinovirus in healthy young children: a longitudinal cohort study nasal swab versus nasopharyngeal aspirate for isolation of respiratory viruses detection of multiple respiratory pathogens during primary respiratory infection: nasal swab versus nasopharyngeal aspirate using real-time polymerase chain reaction parent-collected respiratory specimens-a novel method for respiratory virus and vaccine efficacy research comparing nose-throat swabs and nasopharyngeal aspirates collected from children with symptoms for respiratory virus identification using real-time polymerase chain reaction nasal swabs for detection of respiratory syncytial virus rna successful application of a simple specimen transport method for the conduct of respiratory virus surveillance in remote indigenous communities in australia e-mail-based symptomatic surveillance combined with self-collection of nasal swabs: a new tool for acute respiratory infection epidemiology observational research in childhood infectious diseases (orchid): a dynamic birth cohort study dangerous and prohibited goods and packaging-post guide a novel gel-based method for self-collection and ambient temperature postal transport of urine for pcr detection of chlamydia trachomatis a quantification of human cells using an erv-3 real time pcr assay real-time reverse transcription-pcr assay for comprehensive detection of human rhinoviruses a 5′-nuclease real-time reverse transcriptasepolymerase chain reaction assay for the detection of a broad range of influenza a subtypes, including h5n1 applicability of a real-time quantitative pcr assay for diagnosis of respiratory syncytial virus infection in immunocompromised adults community epidemiology of human metapneumovirus, human coronavirus nl63, and other respiratory viruses in healthy preschool-aged children using parent-collected specimens evaluation of a new rapid antigen test using immunochromatography for detection of human metapneumovirus in comparison with real-time pcr assay frequent detection of human coronaviruses in clinical specimens from patients with respiratory tract infection by use of a novel real-time reverse-transcriptase polymerase chain reaction human coronavirus infections in rural thailand: a comprehensive study using real-time reverse-transcription polymerase chain reaction assays detection of human bocavirus in respiratory, fecal, and blood samples by real-time pcr infrequent detection of ki. wu and mc polyomaviruses in immunosuppressed individuals with or without progressive multifocal leukoencephalopathy rapid identification of fungal pathogens in bact/alert, bactec, and bbl mgit media using polymerase chain reaction and dna sequencing of the internal transcribed spacer regions compendium of soil fungi the air spora of brisbane submit your next manuscript to biomed central and take full advantage of: • convenient online submission • thorough peer review • no space constraints or color figure charges • immediate publication on acceptance • inclusion in pubmed, cas, scopus and google scholar • research which is freely available for redistribution we thank the orchid study team: anne cook, hannah cox, jane gaydon, rebecca holding, kevin jacob, frances maguire, lebo mahango and clair wang, the study support volunteers: lynne grimwood and patricia sloots; and especially the families who participated in the study. the authors declare that they have no competing interest. key: cord-306278-c4q4la5c authors: esposito, susanna; zampiero, alberto; bianchini, sonia; mori, alessandro; scala, alessia; tagliabue, claudia; sciarrabba, calogero sathya; fossali, emilio; piralla, antonio; principi, nicola title: epidemiology and clinical characteristics of respiratory infections due to adenovirus in children living in milan, italy, during 2013 and 2014 date: 2016-04-05 journal: plos one doi: 10.1371/journal.pone.0152375 sha: doc_id: 306278 cord_uid: c4q4la5c to evaluate the predominant human adenovirus (hadv) species and types associated with pediatric respiratory infections, nasopharyngeal swabs were collected from otherwise healthy children attending an emergency room in milan, italy, due to a respiratory tract infection from january 1 to february 28 of two subsequent years, 2013 and 2014. the hadvs were detected using a respiratory virus panel fast assay (xtag rvp fast v2) and with a hadv-specific real-time polymerase chain reaction; their nucleotides were sequenced, and they were tested for positive selection. among 307 nasopharyngeal samples, 61 (19.9%) tested positive for hadv. hadv was the only virus detected in 31/61 (50.8%) cases, whereas it was found in association with one other virus in 25 (41.0%) cases and with two or more viruses in 5 (8.2%) cases. human enterovirus/human rhinovirus and respiratory syncytial virus were the most common co-infecting viral agents and were found in 12 (19.7%) and 7 (11.5%) samples, respectively. overall, the hadv strain sequences analyzed were highly conserved. in comparison to hadv-negative children, those infected with hadv had a reduced frequency of lower respiratory tract involvement (36.1% vs 55.2%; p = 0.007), wheezing (0.0% vs 12.5%; p = 0.004), and hospitalization (27.9% vs 56.1%; p<0.001). antibiotic therapy and white blood cell counts were more frequently prescribed (91.9% vs 57.1%; p = 0.04) and higher (17,244 ± 7,737 vs 9,565 ± 3,211 cells/μl; p = 0.04), respectively, in children infected by hadv-c than among those infected by hadv-b. on the contrary, those infected by hadv-b had more frequently lower respiratory tract involvement (57.1% vs 29.7%) but difference did not reach statistical significant (p = 0.21). children with high viral load were absent from child care attendance for a longer period of time (14.5 ± 7.5 vs 5.5 ± 3.2 days; p = 0.002) and had higher c reactive protein levels (41.3 ± 78.5 vs 5.4 ± 9.6 μg/dl; p = 0.03). this study has shown that hadv infections are diagnosed more commonly than usually thought and that hadvs are stable infectious agents that do not frequently cause severe diseases. a trend toward more complex disease in cases due to hadv species c and in those with higher viral load was demonstrated. however, further studies are needed to clarify factors contributing to disease severity to understand how to develop adequate preventive and therapeutic measures. frequently cause severe diseases. a trend toward more complex disease in cases due to hadv species c and in those with higher viral load was demonstrated. however, further studies are needed to clarify factors contributing to disease severity to understand how to develop adequate preventive and therapeutic measures. human adenoviruses (hadvs) are a group of at least 68 non-enveloped viruses containing double-stranded linear dna [1] . they belong to the family adenoviridae, genus mastadenovirus and are categorized into seven species (a-g) according to their biophysical, biochemical, and genetic characteristics. moreover, in each of these species, several types have been identified [1] . species identity strongly correlates with antigenicity, epidemiologic characteristics, clinical manifestations of hadv infection, and in vitro response to some antiviral drugs [2, 3] . from a clinical point of view, species d (hadv-d8, hadv-d19, and hadv-d37) is usually associated with the development of conjunctivitis; species f (hadv-f40 and hadv-f41) is usually associated with gastroenteritis; and species b (hadv-b3 and hadv-b7), c (hadv-c1, hadv-c2, and hadv-c5), and e (hadv-e4) are usually associated with respiratory diseases [2] . recombination between members of the same species and between members of different species has been frequently described [4] . as a result, certain new types may acquire different pathogenicity and have strong potential for widespread and epidemic outbreaks. consequently, surveillance of hadv circulation with an early evaluation of the relationships between clinical manifestations and molecular characteristics of new infecting strains may be important for the development of adequate diagnostic, prophylactic, and therapeutic measures against hadv infection. hadvs play an important role in the determination of respiratory infections, particularly in children. hadvs are responsible for a number of lower respiratory tract diseases in children, including community-acquired pneumonia (cap). wo et al. reported that among 3,089 nasopharyngeal aspirates collected in children with cap in china, 186 (6.0%) tested positive for hadv [5] . although hadvs are associated with mild to moderate disease in most cases, lifethreatening disease can occur in some patients, particularly if they are immunocompromised, [2] . overall, little data on hadv circulation have been collected in europe and no recent data regarding the epidemiology, molecular characterization, and clinical features of respiratory hadv infections in children have been collected in italy. the main aim of this study was to evaluate the predominant hadv species and types associated with pediatric respiratory infections in milan, italy, during two consecutive winter seasons. clinical features related to hadv types and genetic characteristics were also studied. to evaluate the circulation of the different hadv types and the possible relationship between viral load, viral genetic characteristics, and the severity of infection, nasopharyngeal swabs were collected from otherwise healthy children consecutively attending the emergency room of the fondazione irccs ca' granda ospedale maggiore policlinico, university of milan, italy, due to a respiratory tract infection. the study was carried out during the period from january 1 to february 28 in two subsequent years, 2013 and 2014, and was approved by the ethics committee of the fondazione irccs ca' granda ospedale maggiore policlinico, milan, italy. written informed consent from a parent or legal guardian was required, and children 8 years of age were asked to give their written assent. patient demographic characteristics and medical histories were systematically recorded before the visit to the emergency room using standardized written questionnaires. the study patients were classified into disease groups (i.e., acute otitis media, rhinosinusitis, pharyngitis, croup, infectious wheezing, acute bronchitis, pneumonia) on the basis of signs and/or symptoms using well-established criteria and were subdivided into two subgroups: upper respiratory tract infections (urtis) and lower respiratory tract infections (lrtis) [6] . nasopharyngeal secretions were collected from all of the children immediately after admission to the emergency room using a paranasal flocked swab (1 swab per child), which was stored in a tube containing 1 ml of universal transport medium (kit cat. no. 360c, copan italia, brescia, italy). viral nucleic acids were extracted from the swab by means of a nuclisens easymag automated extraction system (biomeriéux, craponne, france), and the extract was tested for respiratory viruses using the respiratory virus panel (xtag rvp fast v2) (luminex molecular diagnostics, inc., toronto, canada) fast assay in accordance with the manufacturer's instructions (luminex molecular diagnostics inc.) this assay simultaneously detects the following viruses: influenza a virus (flua subtypes h1 or h3); influenza b virus (flub); respiratory syncytial virus (rsv); parainfluenzaviruses (hpiv) 1-4; adenoviruses (hadv); human metapneumovirus (hmpv); coronaviruses (hcov) -229e, -nl63, -oc43, and -hku1; enterovirus/rhinovirus (hev/hrv) and human bocavirus (hbov). moreover, considering the risk of false negative results reported for the rvp fast v2 assay [7] , the negative samples were also tested with an alternative hadvspecific real time-pcr [8] . positive results were also quantified with a hadv-specific real-time polymerase chain reaction (pcr), as previously described [9] . viral nucleic acid extracts were tested using a specific hadv plasmid by a single-plex realtime pcr using taqman universal master mix ii (applied biosystems, foster city, california, usa). amplification and detection of viral dna was performed with a 7900ht real-time pcr system instrument (applied biosystems). the real-time hadv-specific primer sequences were as follows: 5'-gccacggtggggtttctaaactt-3', adenoquant 1 (aq1) and 5'-gcccc agtggtcttacatgcacatc-3', adenoquant 2 (aq2). the sequence of the probe was 5'-tgcaccagacccgggctcaggtactccga-3' (adenoprobe) labeled with fam on the 5'end as a fluorescent dye and labeled with tamra on the 3'-end as a fluorescence quencher dye. cycling conditions were as follows: 50°c for 2 min, 95°c for 8 min and 50 cycles of 95°c for 15 sec and 59°c for 1 min. the plasmid amplified target fragment was verified by sequencing [7] . plasmid dna concentrations were detected using an nd-1000 spectrophotometer (nanodrop products, wilmington, de). real-time fluorescence quantitative pcr was carried out in a total reaction volume of 20 μl consisting of 10 μl of taqman universal master mix (applied biosystems), 0.8 μl (0.4 mm) of each primer, 0.6 μl (0.3 mm) of the probe, 5 μl of template, and 2.8 μl of double-distilled water. the real-time pcr thermal cycling reaction and quantitative measurement were performed in a stepone real-time pcr instrument (applied biosystems) using the following conditions: one cycle at 50°c for 2 min, one cycle at 95°c for 10 min, 45 cycles at 95°c for 15 s, and one cycle at 60°c for 1 min [9] . each run included plasmid and negative controls. standard precautions were taken throughout the pcr process to avoid cross-contamination. negative controls and serial dilutions of the plasmid positive control were included in every pcr assay. finally, quantitative results were reported as dna copies/ml of respiratory samples. hadv typing was performed by sequencing the hypervariable region (1-6) of loop 1 of the hexon protein using a protocol proposed by lu and erdman [10] . pcr products ranging in size from 764 to 896 bp (first pcr) and 688 to 821 bp (nested pcr). first-round amplification was carried out using primers for adhexf1 (nt 19135-19160 geneamp pcr system 9700 (applied biosystems) with the following settings: 94°c for 2 min denaturation followed by 35 cycles of 94°c for 1 min, 45°c for 1 min, and 72°c for 2 min, with a final extension of 72°c for 5 min. for the nested reaction, 0.5 μl of the first pcr product was amplified as above. amplified products were separated on 1% agarose gels and purified with the qiaquick pcr purification kit (qiagen, chatsworth, california, usa). sequencing was performed in both directions using adhexf2/adhexr2 primers and the abi prism bigdyetm terminator cycle sequencing ready reaction kit ver. 3.1 on an abi 3100 dna sequencer (applied biosystems). sequencher 3.1.1 software (gene codes, ann arbor, minnesota, usa) was used for sequence assembly and editing. all sequences were aligned using clustalx 2.1 and bioedit (version 7.1.3.0) software (ibis biosciences, carlsbad, california, usa). phylogenetic trees were generated using the maximum likelihood method with molecular evolutionary genetics analyses (mega) software, version 5.05 [11] , and adenovirus prototype strains. bootstrap probabilities for 1,000 iterations were calculated to evaluate confidence estimates. the graphs were made using graphpad prism version 5.01 for windows (graphpad software, san diego, california, usa). all the hadv sequences originated from this study were submitted to genbank (accession numbers kt963953-kt964000). descriptive statistics of the responses were generated. continuous variables were presented as the mean values and standard deviations (sds), and categorical variables were presented as numbers and percentages. for categorical data, comparisons between groups were performed using a contingency table analysis with a χ 2 or fisher's exact test when appropriate. for ordered categorical data, a cochran-armitage test for trends was used to compare the groups. continuous data were analyzed using a two-sided student's t-test after ensuring the data were normally distributed (based on the shapiro-wilk statistic) or using a two-sided wilcoxon's rank-sum test if the data were non-normal. all analyses were two tailed, and p-values of 0.05 or less were considered to be statistically significant. all analyses were conducted using sas version 9.2 (cary, nc, usa). during the two study periods, a total of 307 nasopharyngeal samples were collected in the emergency room. of these, 61 (19.9%) tested positive for hadv. the luminex xtag rvp fast v2 assay identified 30 cases, all confirmed by real-time pcr. this method revealed 31 positive cases that tested negative with the rvp fast v2 assay. among the hadv infected children, 14.8% were <1 year old, whereas 42.6% and 42.6% were 1-2 and 3 years old, respectively ( table 1) . the prevalence of hadv detection was similar in the two studied periods: 28 (45.9%) and 33 (54.1%) positive samples were collected in 2013 and 2014, respectively. hadv was the only virus detected in 31/61 (50.8%) cases, whereas it was found in association with one other virus in 25 (41.0%) cases and with two or more viruses in 5 (8.2%) cases. hev/hrv and rsv were the most common co-infecting viral agents and were found in 12 (19.7%) and 7 (11.5%) samples, respectively. molecular typing assignments were based on the identity of the closest matching sequences after both blast and phylogenetic analysis. the 61 hadvs belonged to species b in 7 cases (11.5%; all hadv-b3), species c in 37 cases (60.7%; 10 hadv-c1, 25 hadv-c2, and 2 hadv-c5), species d in 1 case (1.7%; hadv-d26), species e in 2 cases (3.4%; hadv-e4), and species f in 1 case (1.6%; hadv-f41) (fig 1) . it was not possible to identify the species and type of 13 (18.6%) samples due to inadequate sample volume. no peculiar clustering was observed among hadv strains. hadvs circulating in 2013 were closely related to strains identified in 2014. however, among hadv-c2 sequences, two distinctive branches were observed with 15 and 10 italian strains (fig 1) . similarly, in the branch of the tree corresponding to the hadv-c1 strains, two strains appeared to cluster separately from the other 8 strains. using 10 6 dna copies/ml as a cut-off, the viral load was classified as low in 37 (62.7%) and as high in 22 (37.3%) cases (2 hadv-b, 14 hadv-c, and 7 of 11 without species identification). for hadv-b, viral load varied from 1.4 x 10 2 to 3.9 x 10 8 copies/ml, whereas for hadv-c it was between 3.4 x 10 3 and 3.0 x 10 9 copies/ml. no significant difference in viral load was observed between each hadv species the sequence identity matrix of the hadv partial hexon gene for groups with at least 7 sequences (hadv-c2, -c1 and -b3) showed a minimum to maximum identity range of 97.6-100.0% for hadv-c2, 98.7-100% for hadv-c1, and 99.4-100% for hadv-b3. overall, the hadv strain sequences analyzed were highly conserved and only few amino acid changes were observed. in detail, among the hadv-c2 sequences, 15/25 strains (60.0%) had an insertion of one glutamic acid (e) in position 151 and the m305l change. in two of serotype was not available for 13 positive subjects, and viral load was not available for 2 positive subjects. viral load was categorized in two groups and was considered "low" for values <6 log(copies/ml) and "high" for values 6 log(copies/ml). no statistically significant result emerged for the relationship between adenovirus types and age or between viral load and age. doi:10.1371/journal.pone.0152375.t001 these strains, the additional change s195t was identified. among the hadv-c1 sequences, only one amino acid change (a190t) was evidenced in 2/10 strains (20.0%). finally, in 5/7 strains (71.4%) belonging to the hadv-b3 group, two changes, g205v and t254i, were observed. in table 2 , demographic, clinical, and laboratory characteristics of children infected by hadv alone or co-infected with hadv and one or more other respiratory viruses are compared with those of children with respiratory infection due to other agents. a preliminary analysis revealed that no statistically significant difference between cases infected by hadv alone or co-infected with other viruses, in particular rsv or rhinovirus could be evidenced, all the children with hadv infection were considered together. as shown, in comparison to hadv-negative children, those infected with hadv were younger (4.3 ± 3.3 vs 3.2 ± 2.5 years; p = 0.01) and had high-grade fever more frequently (56.4% vs 72.4%; p = 0.03). moreover, children infected with hadv had lower respiratory tract involvement less frequently (55.2% vs 36.1%; p = 0.007) and never suffered from wheezing unlike children with disease due to other etiologic agents. crp, c reactive protein; sd, standard deviation; spo 2 , peripheral oxygen saturation."38.0°c or more any time during the illness (before or at enrolment, or during follow-up);°39.0°c or more any time during the illness (before or at enrollment or during follow-up). a data were extracted from datasets of different studies that collected different information; therefore, the denominators vary across characteristics. however, when not indicated the reported number refers to the whole enrolled sample. children infected with other agents wheezed in 12.5% of the cases (p = 0.004) and were hospitalized more frequently (56.1% vs 27.9%; p<0.001). no other significant differences between groups were observed. in table 3 , comparisons based on demographic, clinical, and laboratory variables between subjects with hadv b and c species are shown. two significant differences were found between the groups: antibiotic therapy was more frequently prescribed (91.9% vs 57.1%; p = 0.04) and white blood cell count was higher (17,244 ± 7,737 vs 9,565 ± 3,211; p = 0.04) in children infected by hadv-c. table 4 shows data regarding characteristics of children with hadv infection according to viral load. children with high viral load were younger, had high-grade fever more frequently, were more frequently hospitalized, were absent from the community for a longer period of time, and had a higher c reactive protein (crp) level. however, differences were statistically significant only for absence from the community (14.5 ± 7.5 vs 5.5 ± 3.2 days; p = 0.002) and crp level (41.3 ± 78.5 vs 5.4 ± 9.6 μg/dl; p = 0.03). several previous epidemiological studies have shown that hadvs are considered the cause of respiratory infections in otherwise healthy children in approximately 4-10% of the cases [12] [13] [14] [15] . in this study, similarly to what has been found in asia by other authors [16] , a prevalence of approximately 20% was found, suggesting that the relevance of this infectious agent in the determination of respiratory problems could be higher than previously thought. the methods used to identify hadvs might partially explain this finding. in the past, most of the epidemiological studies of viral respiratory infections were carried out using methods that could underestimate viral presence in respiratory secretions, such as viral culture, antigen detection by immunofluorescence, and visualization by electron microscopy [17] . to overcome this problem, molecular methods were suggested. multiplex assays, including the rvp fast v2 assay, were developed to obtain the simultaneous identification of all the most common respiratory viruses and are now commonly used in routine practice. as previously reported [8] and confirmed by this study, the rvp fast v2 assay has poor sensitivity for hadv and can lead to undervaluation of the real importance of these viruses in the determination of respiratory infections. the addition of a specific real-time pcr can solve this issue. moreover, the higher than expected prevalence of hadv infection evidenced by this study could be strictly related to the period during which it was carried out. samples were collected in two winter months of two consecutive years. although hadvs circulate during the whole year, peak periods are in winter and early spring [1] . consequently, it is possible that the study was carried out during epidemics leading to the higher prevalence values reported here. in this study, the most commonly identified species were b and c, types 3, 2, and 1. this is not surprising because, despite possible temporal and regional changes in predominant type [18] , these types are more commonly reported as the cause of respiratory infection worldwide. hadv-b3 has been identified in successive outbreaks of severe acute respiratory illnesses in korea [19, 20] , brazil [21] , and taiwan [22, 23] , where this virus was the predominant type for respiratory hadv infection from 1981 to 2002. moreover, together with other hadv types, it has been the causative agent in epidemic outbreaks of respiratory diseases in europe, america, and oceania [24] [25] [26] . finally, hadv-b3 is known to be a causative agent of a characteristic syndrome of acute pharyngo-conjunctival fever in older children and adults, especially in summer camps and swimming pools [27] . types c1 and c2 have been more frequently reported as the cause of endemic or sporadic cases [28] , although there have been reports of epidemics [19] . in italy, a survey carried out approximately 10 years ago found that hadv-c1 and -c2 were the most common hadvs isolated in patients with infection due to these agents [28] . the same was shown by this study, showing that epidemics of infection due to the same hadv in a given geographic area can be prolonged compared with outbreaks of rsv, parainfluenza virus, and influenza viruses, which are well defined and have duration limited to some months [29, 30] . the severity of hadv infection varies according to age, socioeconomic status, environmental status, and above all, the immunological characteristics of the patient. detection of hadv in severely immunocompromised children has been implicated as a risk factor for poor outcome [31] . however, severe cases have been frequently described in otherwise healthy children [5, 19, 20] . in this study, most of the children with hadv infection had a mild infection and, globally, the severity of respiratory infection of children in whom hadv alone or in association with other respiratory viruses was identified was lower than that due to other infectious agents considered together. both the prevalence of lrtis and hospitalization rate were significantly lower in hadv-infected children than in children not infected by hadv. the long-term circulation of hadvs with similar genetic characteristics could partially explain the generally poor clinical relevance of infections due to the strains identified in this study. in italy, the most common hadvs identified in children during the periods of this study were of the same species and of the same types of those identified several years before. moreover, despite sequencing analyses that were focused on one of the more variable genes (hexon) [32] , no significant variation in hadv genetic characteristics was seen and no recombination between viruses was found. hadv-c2 strains were the only strains to have two slightly different clusters, suggesting the circulation of two different hadv-c2 strains with indistinct pathogenetic roles. as a result, it is possible that many of the children had previously had contact with these viruses and developed sufficiently high immunity to limit the clinical expression of subsequent infections. however, the number of non-hadv infection children included in this study is significantly higher than that of hadv infected patients and this difference could have led comparison to wrong results. a longer period and expanded surveillance may help to construct the complete picture on the hadv circulation, other infections and related clinical features. prevalence of lrti was higher in children infected by type b3, although the difference compared with type c was not statistically significant and the total number of patients with this type of infection is too small to draw definitive conclusions. potentially increased virulence of type b3 in comparison to other hadvs is not surprising because this virus has already been associated with a number of severe lrtis [5, 19, 20] and to the development of acute meningo-encephalopathy [33] . in this study, higher viral load was more common in children with some markers of more severe disease, such as higher fever, higher hospitalization rate, higher crp values, and delayed return to normal activities, independent of the infecting hadv type. however, clinical differences between patients with high or low viral load were not always significant, and it is not possible to state that hadv load can be a marker for severity of infection. by contrast, hadv load was found to be significantly higher in patients developing severe hadv infection after transplantation, especially in pediatric stem cell transplant recipients [31] . consequently, the measurement of hadv load is considered a possible method for an early diagnosis of disseminated hadv disease and for the initiation and monitoring of antiviral therapy in these subjects [34] . further studies are needed to evaluate whether high viral load could indicate which subjects might have to receive antiviral therapy to avoid negative evolution of the infection even if they are not immunocompromised. in conclusion, this study has shown that when adequately investigated, hadv infections are diagnosed more commonly than usually thought. moreover, these data seem to indicate that hadvs are stable infectious agents that do not frequently incur genetic variations and, for this reason, do not cause frequently severe diseases. it seems that there are some differences in the severity of disease outcome between types and according to viral load, with hadv type c and high viral load apparently associated with a more severe disease. however, further studies are needed to identify the potential pathogenetic role of the different species and types of hadv and the importance of viral load in the severity of infection. clarification of these unsolved problems may be useful for deciding how to develop adequate preventive and therapeutic measures for immunocompromised and otherwise healthy children who suffer from hadv infection. family adenoviridae adenovirus infections in immunocompetent and immunocompromised patients differential susceptibility of adenovirus clinical isolates to cidofovir and ribavirin is not related to species alone evidence of frequent recombination among human adenoviruses epidemical features of hadv-3 and hadv-7 in pediatric pneumonia in chongqing textbook of pediatric infectious diseases comparison of the luminex respiratory virus panel fast assay with in-house real-time pcr for respiratory viral infection diagnosis detection of a broad range of human adenoviruses in respiratory tract samples using a sensitive multiplex real-time pcr assay rapid and quantitative detection of human adenovirus dna by real-time pcr molecular typing of human adenoviruses by pcr and sequencing of a partial region of the hexon gene mega5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods impact of viral infections in children with community-acquired pneumonia: results of a study of 17 respiratory viruses rapid identification of nine microorganisms causing acute respiratory tract infections by single-tube multiplex reverse transcription-pcr: feasibility study rapid detection of respiratory viruses by centrifugation enhanced cultures from children with acute lower respiratory tract infections viral etiology of acute respiratory infections among children in porto alegre, rs, brazil high-incidence of human adenoviral co-infections in taiwan respiratory viral infections in infants: causes, clinical symptoms, virology, and immunology lower respiratory tract infections due to adenovirus in hospitalized korean children: epidemiology, clinical features, and prognosis genome type analysis of adenovirus types 3 and 7 isolated during successive outbreaks of lower respiratory tract infections in children antigenic and genomic characterization of adenovirus associated to respiratory infections in children living in northeast brazil respiratory adenoviral infections in children: a study of hospitalized cases in southern taiwan in 2001-2002 a two-decade survey of respiratory adenovirus in taiwan: the reemergence of adenovirus types 7 and 4 adenovirus type 3 pneumonia causing lung damage in childhood an outbreak of adenovirus type 3 disease at a private recreation center swimming pool on the aetiology of whooping cough molecular characterization of human adenoviruses isolated in italy epidemiology of acute lower respiratory disease in children adenovirus type 7; 1971-74 epidemic outbreaks of adenovirus 7 with special reference to the pathogenicity of adenovirus genome type 7b adenoviruses in immunocompromised hosts comparative sequence analysis of the hexon gene in the entire spectrum of human adenovirus serotypes: phylogenetic, taxonomic, and clinical implications adenovirus infection associated with central nervous system dysfunction in children adenoviral load diagnostics by quantitative polymerase chain reaction: techniques and application key: cord-266716-pghnl980 authors: wang, hai-ming; liu, tian-xin; wang, tong-yun; wang, gang; liu, yong-gang; liu, si-guo; tang, yan-dong; cai, xue-hui title: isobavachalcone inhibits post-entry stages of the porcine reproductive and respiratory syndrome virus life cycle date: 2018-02-06 journal: arch virol doi: 10.1007/s00705-018-3755-4 sha: doc_id: 266716 cord_uid: pghnl980 porcine reproductive and respiratory syndrome virus (prrsv) is a pathogen of great economic significance that impacts the swine industry globally. since the first report of a porcine reproductive and respiratory syndrome (prrs) outbreak, tremendous efforts to control this disease, including various national policies and plans incorporating the use of multiple modified live-virus vaccines, have been made. however, prrsv is still a significant threat to the swine industry, and new variants continually emerge as a result of prrsv evolution. several studies have shown that pandemic prrsv strains have enormous genetic diversity and that commercial vaccines can only provide partial protection against these strains. therefore, effective anti-prrsv drugs may be more suitable and reliable for prrsv control. in this study, we observed that isobavachalcone (ibc), which was first isolated from psoralea corylifolia, had potent anti-prrsv activity in vitro. although many biological activities of ibc have been reported, this is the first report describing the antiviral activity of ibc. furthermore, after a systematic investigation, we demonstrated that ibc inhibits prrsv replication at the post-entry stage of prrsv infection. thus, ibc may be a candidate for further evaluation as a therapeutic agent against prrsv infection of swine in vivo. porcine reproductive and respiratory syndrome virus (prrsv) is a positive-strand rna virus that is approximately 15 kb in length and belongs to the family arteriviridae [5] . prrsv is one of the most important viruses hindering the development of the swine industry globally, especially with the emergence of highly pathogenic prrsv (hp-prrsv) [5, 38] . recently, new prrsv mutant strains have become pandemic in china [1, 16, 17, 35, 37, 39] and are named nadc30-like prrsv strains for their genomic similarity to the nadc30 strain isolated in the us in 2008 [3] . nadc30-like prrsv strains feature different recombinations and have produced many mosaic isolates, enhancing their genetic diversity. currently, the most effective methods to control viral diseases are vaccines and antiviral agents. however, vaccination against prrsv infection has achieved limited success, primarily due to the high genetic diversity of the virus [9, 10, 22, 29] . in addition to genetic mutation, prrsv genetic diversity is also generated by recombination among different strains, including vaccine strains [7, 18, 23, 30, 33, 37] . importantly, commercial vaccines cannot provide complete protection against heterologous prrsv infection [1, 21] . the problems outlined above indicate that the use of vaccines may not be an ideal strategy for prrsv control; however, exploring effective anti-prrsv drugs may overcome these issues. isobavachalcone (ibc) is a prenylated chalcone of the flavonoid subclass that was first isolated from psoralea corylifolia in 1968 [14] . ibc possesses a wide spectrum of biological activities, including antibacterial, antifungal, anticancer, anti-reverse-transcriptase, antitubercular and antioxidant functions [14] . however, whether ibc has potential antiviral activity remains unclear. in the current study, for the first time, we observed that ibc has anti-prrsv activity. furthermore, after a systematic investigation, we demonstrated that ibc inhibits prrsv replication at the post-entry stage of prrsv infection. porcine alveolar macrophages (pams) were obtained from the lungs of 4-week-old specific-pathogen-free (spf) piglets, and monkey kidney cells (marc145) were cultured in dulbecco's modified eagle's medium supplemented with 10% fetal bovine serum (fbs). the highly pathogenic prrsv strain hun4 [25, 26] and the nadc30-like prrsv strain hen-l1 (isolated in our lab) were propagated and titrated in marc145 cells. ibc was purchased from shanghai tauto biotech company and was dissolved in ethanol and used at the concentrations indicated. the cytotoxicity of ibc was evaluated for pams and marc145 cells. briefly, twofold dilutions of ibc from a starting concentration of 25.6 μm were applied to 80% confluent cells in a 96-well plate. after incubation for 24 h at 37 °c, the cytotoxicity of ibc was evaluated using a cell counting kit-8 (cck8, dojindo laboratories, japan) according to manufacturer's protocol. the cc 50 was defined as the concentration of ibc that reduced the absorbance of treated cells by 50% relative to that of the cell control. a total of 3 × 10 6 marc145 cells were plated in a 6-well plate, and 18 h later, the cells were infected with hun4 (multiplicity of infection [moi] = 1) for 2 h at 37 °c. next, the culture medium was replaced with different concentrations of ibc diluted in dmem containing 2% fbs. cells were harvested at the indicated times for further western blot or immunofluorescence analysis. the total rna of all samples was extracted using an rneasy plus mini kit (qiagen, germany), and 1 μg of total rna was reverse transcribed into cdna according to the manufacturer's protocol (primescript™ rt reagent kit with gdna eraser, takara). the real-time pcr procedure was performed using an agilent mx3005p real-time pcr system. the primers and probes were described in a previous report [28] . cells were infected with hun4 for 2 h and were then treated with various concentrations of ibc. after incubating for the indicated time, cells were fixed in 95% ethanol, permeabilized with 0.5% saponin in pbs, and then blocked with 5% bovine serum albumin (bsa) for 1 h at room temperature (rt). after blocking, the cells were incubated with a mouse anti-prrsv n protein monoclonal antibody (igg2b, made in our lab) at a dilution of 1:500 for 1 h at rt, and dsrna was stained with the mouse monoclonal antibody j2 (scicons, hungary) at a dilution of 1:200 for 1 h at rt. after washing, an aliquot of 1:100-diluted fitc-labeled antimouse igg (sigma) was used as the secondary antibody. for nuclear visualization, cells were treated with dapi (sigma). immunofluorescence was observed using a leica tcs sp5 confocal microscope. cells were washed twice with pbs and then prepared with ripa lysis buffer (solarbio). the cell lysates were centrifuged at 12,000 rpm for 5 min at 4 °c. the supernatants were collected, and protein concentrations were determined by bca assay. next, proteins from each sample were separated via 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (sds-page), after which the protein bands were transferred onto a polypropylene fluoride (pvdf) membrane. the membrane was blocked with 5% nonfat milk for 2 h at rt and then incubated with mouse anti-prrsv n protein monoclonal antibody (1:3000, made in our laboratory) or anti-β-actin antibody (1:4000, sigma) for 1.5 h at rt. after being washed three times with pbst (0.05% tween 20 in pbs), the membrane was incubated for 1 h with hrp-conjugated anti-mouse igg antibody (sigma). protein bands were visualized with ecl chemiluminescence reagent (thermo scientific). western blot bands were quantified and analyzed using imagej. the relative intensity ratio of protein bands (n protein/β-actin) was calculated as an indicator of prrsv replication ability, with the value of the control group set as 1; other ibc treated groups were normalized correspondingly. to further investigate the anti-prrsv activity of ibc, the drug concentration that could inhibit prrsv infections by approximately 50% (50% inhibitory concentration, ic 50 ) was determined. marc145 cells were infected with prrsv (moi = 1) for 2 h at 37 °c and then treated with twofold dilutions of ibc. total rna was isolated from cells at 24 h postinfection (p.i.), and intracellular prrsv rna copy numbers were determined by rt-pcr. the percentage inhibition was calculated as [(rna copies in control cells -(rna copies in cells treated with ibc)] ÷ (rna copies of the control) × 100 at the indicated ibc concentration, and the value for the control group was set as 1. the other ibc-treated groups were normalized correspondingly. a virus attachment and entry assay was performed by preincubating marc145 cells at 4 °c for 30 min, after which prrsv hun4 (moi = 1) and 5 μm ibc were added, and the cells were incubated for an additional 2 h at 4 °c to allow attachment of the virus to the cells. next, the cells were washed three times with cold pbs to remove unbound virus. the cells were then collected and either used to detect prrsv rna levels via real-time pcr (to assess viral attachment) or provided fresh medium containing 5 μm ibc and incubated at 37 °c for 2 h. a temperature shift from 4 to 37 °c was performed to promote entry of the virus into the cells, and after a 2-h incubation, total rna was collected and analyzed by real-time pcr (to assess viral entry). marc145 cells were infected with prrsv hun4 (moi = 1) at 37 °c. after a 2-h incubation, cells were washed three times with pbs to remove unbound viruses and were replenished with 2% fbs. for the postinfection inhibition study, hun4-infected cells were replenished with 2% fbs supplemented with 5 or 10 μm ibc at specific time points (2, 6, 12, or 24 h). cells were harvested at 24 h after ibc treatment, and the levels of prrsv replication were estimated by western blot analysis. origin 8.0 software was used for all graphical representations. the ic 50 was calculated using non-linear regression. statistical significance was established using an independent t-test, and p-values less than 0.05 were considered significant. the structure of ibc is shown in figure 1a . to assess the anti-prrsv activity of ibc, we first evaluated the cytotoxicity of ibc on marc145 cells (fig. 1b) and pam cells (fig. 1c) , and the results indicated that cell viability was not significantly affected by ibc at concentrations up to 25.6 μm and that the cc 50 of ibc for marc145 cells was 63.09 μm (data not show). the cytotoxic effect of the ibc solvent (alcohol) was also evaluated, the results of which indicated that the alcohol had no effect on marc145 cells (fig. 1b) and pam cells (fig. 1c) . next, we evaluated the anti-prrsv capabilities of ibc in marc145 cells that were infected with prrsv hun4 (moi = 1) and then treated with different concentrations of ibc. we first determined progeny virus titers and found that ibc significantly inhibited prrsv replication in a dose-dependent manner (fig. 1d ). to confirm this finding, we also performed an indirect immunofluorescence assay by staining cells with a prrsv n protein antibody and then observed prrsv replication by fluorescence microscopy. as shown in figure 1e , prrsv replication was significantly blocked by ibc. western blot analysis revealed that prrsv n protein levels decreased markedly, indicating that increasing concentrations of ibc significantly inhibited prrsv replication (fig. 1f) . to determine whether this inhibition was strain specific, we additionally assayed a currently pandemic nadc30-like strain (hen-l1) that was isolated in our lab and observed it also to be inhibited by ibc (fig. 1g) . thus, these data indicate that ibc inhibition of prrsv is not strain specific. furthermore, the antiviral activity of ibc against prrsv hun4 was both dose dependent and successful, with an ic 50 of 3.12 μm (fig. 2 ) and a selectivity index (si; the ratio of the cc 50 to the ic 50 ) of 22.02. despite the evidence that ibc inhibits prrsv replication, the viral stage influenced by ibc remained unknown. to determine which step(s) in the viral life cycle are affected by ibc, we first performed time-of-drug-addition assays. the results indicated that ibc markedly inhibited prrsv when added early after infection and remained inhibitory when added up to 6 h p.i., whereas prrsv inhibition was limited when ibc was added late, at 12 h p.i. (fig. 3) . these data suggest that ibc targets the early phase of the viral life cycle, as it was not able to prevent viral infection after the virus entered the post-replicative stages of infection. next, we tested whether ibc inhibited prrsv by disturbing prrsv attachment or entry. the results demonstrated that prrsv attachment was not influenced by ibc (fig. 4a) . interestingly, the prrsv entry assay results were the same as those of the attachment assay, demonstrating that neither prrsv attachment nor entry was influenced by ibc (fig. 4b ). the addition of ibc was inhibitory from 2 to 6 h p.i., whereas it had no effect on prrsv attachment or entry ( fig. 4a and b) . this inhibition at post-entry stages led us to speculate that ibc inhibits prrsv replication at the stage of viral rna synthesis. to test whether ibc blocked viral rna replication, we performed inhibition experiments using pam cells and stained for dsrnas, which are intermediates in viral replication, with the j2 antibody, which recognizes dsrna. the results indicated that dsrna levels were significantly decreased in ibctreated cells (fig. 5a and b) . these data clearly suggest currently, prrsv is a significant problem for the swine industry in china and was a veritable pandora's box for the chinese pig industry during the first outbreak [17] . the current pandemic strains are highly diverse, and commercial vaccines can provide only partial protection against these strains [27, 40] . thus, anti-prrsv drugs may be more suitable for prrsv control in the future. traditional chinese medicine (tcm) has been widely used as a source of novel drugs, and many crude tcm herbal extracts have been shown to inhibit prrsv replication [6, 24, 36] . ibc is extracted from p. corylifolia, which itself is used in tcm [14] . cheng et al. systematically analyzed seventeen compounds derived from tcms and tested their prrsv antiviral activity in vitro [4] . two compounds, chlorogenic acid and scutellarin, were shown to have great anti-prrsv replication potential in their study, and the inhibition ratios of chlorogenic acid and scutellarin can reach 90.8 and 61.1%, respectively, at the maximum noncytotoxic concentrations [4] ; in contrast, the ibc inhibition ratio exceeded 90% at 15 μm, an improvement over the maximum non-cytotoxic concentrations for the other tested compounds. this result indicated that ibc is more effective than chlorogenic acid and scutellarin. ibc has multiple biological activities; it potently abrogates akt and erk signaling pathways and exerts anti-proliferative effects on several human cancer cell lines, and it also induces apoptosis associated with the mitochondrial pathway [11, 12, 14] . akt has been reported to play a positive role in prrsv randomly selected for statistical analysis. the mean rates of positive cells among the total cells in each group were calculated using imagej gene expression and entry, and prrsv replication may be dependent on the activation of pi3k/akt [19, 31, 32, 34] . the erk signaling pathway has been demonstrated to play an important role in the post-entry steps of the prrsv replication cycle and to enhance prrsv infection [15] . the erk pathway may also contribute to hp-prrsv pathogenesis [2] . apoptosis also influences prrsv replication, since the induction of apoptosis in marc145 cells increases virus replication [8] . ibc inhibition of prrsv replication may occur through modulation of the above-mentioned pathway. ibc may be applicable as an efficacious and safe drug for the treatment of some cancers, such as neuroblastoma [20] . ibc has been reported to inhibit sars-cov papain-like protease; however, whether ibc inhibits infectious virus was not tested in that study [13] . this study is the first to report that ibc has potent antiviral activity, and we demonstrate here that it blocks prrsv replication at the initiation of viral rna replication in vitro. however, there are some unanswered questions that should be further explored in the future. first, whether ibc also shows anti-prrsv activity in vivo must be investigated. second, is the blockage of viral rna formation a direct or indirect effect? third, are there other viruses that are also inhibited by ibc? in conclusion, we evaluated whether ibc has antiviral potential as a prrsv infection treatment. the in vitro study established that ibc efficiently inhibited the replication of both hp-prrsv and the current chinese epidemic nadc30-like strains without significant drug toxicity. thus, ibc may be a candidate for further evaluation as a therapeutic agent against prrsv infection of swine in vivo. commercial vaccines provide limited protection to nadc30-like prrsv infection highly pathogenic porcine reproductive and respiratory syndrome virus induces prostaglandin e2 production through cyclooxygenase 1, which is dependent on the erk1/2-p-c/ebp-beta pathway genomic sequence and virulence comparison of four type 2 porcine reproductive and respiratory syndrome virus strains in vitro screening for compounds derived from traditional chinese medicines with antiviral activities against porcine reproductive and respiratory syndrome virus the structural biology of prrsv antiviral strategies against prrsv infection observation of high recombination occurrence of porcine reproductive and respiratory syndrome virus in field condition propagation of field highly pathogenic porcine reproductive and respiratory syndrome virus in marc-145 cells is promoted by cell apoptosis porcine reproductive and respiratory syndrome virus vaccines: current status and strategies to a universal vaccine novel strategies and approaches to develop the next generation of vaccines against porcine reproductive and respiratory syndrome virus (prrsv) isobavachalcone induces the apoptosis of gastric cancer cells via inhibition of the akt and erk pathways abrogation of akt signaling by isobavachalcone contributes to its anti-proliferative effects towards human cancer cells phenolic phytochemical displaying sars-cov papain-like protease inhibition from the seeds of psoralea corylifolia isobavachalcone: an overview porcine reproductive and respiratory syndrome virus replication is suppressed by inhibition of the extracellular signal-regulated kinase (erk) signaling pathway outbreak investigation of nadc30-like prrsv in south-east china widespread of nadc30-like prrsv in china: another pandora's box for chinese pig industry as the outbreak of highly pathogenic prrsv in recombination in jxa1-r vaccine and nadc30-like strain of porcine reproductive and respiratory syndrome viruses the involvement of fak-pi3k-akt-rac1 pathway in porcine reproductive and respiratory syndrome virus entry isobavachalcone, a chalcone constituent of angelica keiskei, induces apoptosis in neuroblastoma preparation for emergence of an eastern european porcine reproductive and respiratory syndrome virus (prrsv) strain in western europe: immunization with modified live virus vaccines or a field strain confers partial protection live porcine reproductive and respiratory syndrome virus vaccines: current status and future direction recombination is associated with an outbreak of novel highly pathogenic porcine reproductive and respiratory syndrome viruses in china sodium tanshinone iia sulfonate inhibits porcine reproductive and respiratory syndrome virus via suppressing n gene expression and blocking virus-induced apoptosis open reading frames 1a and 1b of the porcine reproductive and respiratory syndrome virus (prrsv) collaboratively initiate viral minus-strand rna synthesis recombinant pseudorabies virus (prv) expressing firefly luciferase effectively screened for crispr/cas9 single guide rnas and antiviral compounds nadc30-like porcine reproductive and respiratory syndrome in china development and application of taq man-mgb fluorescence quantitative rt-pcr assay for detection of porcine reproductive and respiratory syndrome virus immune responses to modified live virus vaccines developed from classical or highly pathogenic prrsv following challenge with a highly pathogenic prrsv strain molecular epidemiology of porcine reproductive and respiratory syndrome virus in central china since 2014: the prevalence of nadc30-like prrsvs efficient porcine reproductive and respiratory syndrome virus entry in marc-145 cells requires egfr-pi3k-akt-limk1-cofilin signaling pathway role of phosphatidylinositol 3-kinase (pi3k) and akt1 kinase in porcine reproductive and respiratory syndrome virus (prrsv) replication complete genome sequence of a novel variant porcine reproductive and respiratory syndrome virus (prrsv) strain: evidence for recombination between vaccine and wild-type prrsv strains a dual effect of porcine reproductive and respiratory syndrome virus replication on the phosphatidylinositol-3-kinase-dependent akt pathway pathogenicity and antigenicity of a novel nadc30-like strain of porcine reproductive and respiratory syndrome virus emerged in china in vitro evaluation of the antiviral activity of the synthetic epigallocatechin gallate analog-epigallocatechin gallate (egcg) palmitate against porcine reproductive and respiratory syndrome virus importation and recombination are responsible for the latest emergence of highly pathogenic porcine reproductive and respiratory syndrome virus in china porcine reproductive and respiratory syndrome in china nadc30-like strain of porcine reproductive and respiratory syndrome virus efficacy evaluation of three modified-live virus vaccines against a strain of porcine reproductive and respiratory syndrome virus nadc30-like acknowledgements this study was supported by the national key r&d program (grant number 2016yfd0500100) and the national science foundation of heilongjiang (grant number zd2015006). key: cord-282420-0fcyjw7l authors: lu, cheng-wei; liu, xiu-fen; jia, zhi-fang title: 2019-ncov transmission through the ocular surface must not be ignored date: 2020-02-06 journal: lancet doi: 10.1016/s0140-6736(20)30313-5 sha: doc_id: 282420 cord_uid: 0fcyjw7l nan on jan 22, guangfa wang, a member of the national expert panel on pneumonia, reported that he was infected by 2019ncov during the inspection in wuhan. 2 he wore an n95 mask but did not wear anything to protect his eyes. several days before the onset of pneumonia, wang complained of redness of the eyes. unprotected exposure of the eyes to 2019ncov in the wuhan fever clinic might have allowed the virus to infect the body. 2 infectious droplets and body fluids can easily contaminate the human conjunctival epithelium. 3 respiratory viruses are capable of inducing ocular complications in infected patients, which then leads to respiratory infection. 4 severe acute respiratory syndrome coronavirus (sarscov) is predominantly transmitted through direct or indirect contact with mucous membranes in the eyes, mouth, or nose. 5 the fact that exposed mucous membranes and unprotected eyes increased the risk of sarscov transmission 4 suggests that exposure of unprotected eyes to 2019ncov could cause acute respiratory infection. thus, huang and colleagues 1 should have analysed conjunctival scrapings from both confirmed and suspected 2019ncov cases during the onset of symptoms. the respiratory tract is probably not the only transmission route for 2019ncov, and all ophthalmologists examining suspected cases should wear protective eyewear. we declare no competing interests. *cheng-wei lu, xiu-fen liu, zhi-fang jia clinical features of patients infected with 2019 novel coronavirus in wuhan, china peking university hospital wang guangfa disclosed treatment status on weibo and suspected infection without wearing goggles avian influenza and sialic acid receptors: more than meets the eye? ocular tropism of respiratory viruses the severe acute respiratory syndrome key: cord-281085-lqniqui0 authors: nosalova, gabriela; mokry, juraj; franova, sona title: pharmacological modulation of cough reflex date: 2006-12-31 journal: advances in phytomedicine doi: 10.1016/s1572-557x(05)02006-4 sha: doc_id: 281085 cord_uid: lqniqui0 abstract the cough reflex is an attack of powerful expiratory efforts produced by active contractions of the expiratory muscles, preceded by deep inspirations. cough is a normal physiological defensive reflex responsible for keeping the airways free of obstruction and harmful substances. however, cough is also the most frequent symptom of acute respiratory system diseases and is the most common reason why sick patients visit physicians. the largest diagnostic group associated with chronic cough is asthma or asthma-like syndromes and chronic obstructive pulmonary disease. furthermore, gastroesophageal reflux, rhinitis/postnasal drip syndrome and unpleasant side effects accompanying the therapy with angiotensin-converting enzyme inhibitors (acei) represent other causes of chronic cough. the cough reflex may be elicited by the activation of receptors of non-myelinated nociceptive aδ-fibers and c-fibers and receptors of myelinated aδ-fibers distributed throughout the respiratory tract. in recent times as proper cough receptors are thought to be rapidly adapting receptors (rars) of myelinated fibers, c-fibers and transient receptor potential vanilloid 1 (trpv1) localize on the non-myelinated fibers. as the pathological cough (especially its chronic form) has significant impact on patient's quality of life, observed either in physical activity or psychosocial domain, various treatment attitudes are used for different forms of cough (acute, subacute, chronic, productive, nonproductive, psychogenic, asthma-associated, or painful). interest of research in this field is accompanied with the fact, that many antitussive drugs (mainly from the opioid group), which have been the antitussives of choice for decades, are limited by their unpleasant and very often adverse reactions. in this chapter the authors divided the drugs used in the pharmacological treatment of cough into several groups. these include the drugs acting at the level of receptors, the drugs affecting the propagation of cough impulses in afferent nerves, the drugs modulating the central coordination of cough reflex, the drugs acting at the level of efferent nerves, and the drugs affecting the effectors. efficacy of many antitussive agents is connected with their influence on more than one level of cough reflex. another group used in the therapy of cough are mucoactive substances that change the dry, irritant and nonproductive cough into the so-called productive cough with nonirritating expectoration. the last-mentioned group in this chapter is demulcerative and hydrating drugs, which can create a defense layer protecting the mucous membranes from other irritant stimuli. exchange of gases while breathing forms the major role of the lungs. as the inspired air is markedly polluted, the airways are fit for numerous defense mechanisms. the defense of the respiratory system against inhaled particles and gases involves the integration of many complex physiological, biochemical and immunological processes that interact directly with the properties of inhaled materials (korpas and tomori, 1979) . the various defense mechanisms are integrated to provide local degradation and detoxication, as well as mechanical elimination of both exogenous substances and products of pathological processes from the airways. the most important defensive reflex of the airways is cough, which, together with the mucociliary transport system, forms the main mechanism for cleaning of the respiratory tract (korpas and nosalova, 1991; chung and chang, 2002; belvisi and geppetti, 2004) . korpas and tomori (1979) characterized the cough reactions as an attack of powerful expiratory efforts produced by active contractions of the expiratory muscles, preceded by deep inspirations. the violent expiration, which provides the high flow, helps to shear away mucus and remove foreign particles from the larynx, trachea and large bronchi. cough appears when the membrane lining of the respiratory tract produces excessive mucus or phlegm. these secretions help to protect airways from infections and irritants. coughing prevents the breathing passages from closing and also prevents infected mucus from falling into lungs and bronchial tubes, which could be very dangerous. in the absence of abnormal sputum production there is likely to be another reason for cough. the probable explanation could be an increased sensitivity of the cough reflex, which leads to an abnormal response to ''naturally'' inhaled stimuli (fuller and jackson, 1990; reynolds et al., 2004) . cough is one of the most frequent symptoms of respiratory system diseases and is the most common reason why sick patients visit physicians in the united states (bolser, 1996; ziment and o'connell, 2002) . cough prevalence in populations depends on smoking prevalence and on the level of air pollution. statistical data are in the range of 5-40% (fuller and jackson, 1990; irwin and madison, 2002; wright et al., 2004) . cough is a common complaint of patients presenting to primary care physicians. most patients with acute cough have self-limited illnesses like common cold and influenza. gwaltney (1997) showed that the viral infections represent the most frequent causes of self-limited cough. these viruses include rhinovirus, coronavirus, parainfluenza virus, respiratory syncytial virus, adenovirus and influenza virus. these viruses can cause a spectrum of diseases including common cold, acute infections bronchitis, bronchiolitis and bacterial pneumonia. according to gwaltney (1997) the highest incidence of cough occurred in patients with influenza (reaching 70% on day 3 of the illness) and rhinovirus colds (approximately 35% over the 7-day period of illness). antitussive drugs such as codeine and dextromethorphan are among the most commonly used prescription and over-the-counter (otc) drugs in the world (choundry and fuller, 1992) . the widespread use of antitussive medications for the treatment of cough associated with upper respiratory tract infection generates many millions of dollars for the pharmaceutical industry (eccles, 1996) . in the uk, the cough/cold market accounted for £350 million, whereas in the usa, well over $2 billion was spent on otc medicines (morice, 2001) . these facts demonstrate the vast socio-economic consequences of acute cough. chronic cough is one of the most common symptoms presenting to respiratory physicians and affects 10-38% of patients (hsu et al., 1994) . the largest diagnostic group is asthma or asthma-like syndromes. a second large group connected with cough is gastroesophageal reflux (plutinsky et al., 1998) . rhinitis/postnasal drip syndrome represents another cause of chronic cough. predominantly this kind of cough seems to occur (3-40%) accompanied by unpleasant side effects due to therapy using ace inhibitors (kubota et al., 1996) . cough reflex may be elicited by the activation of non-myelinated c-fibers and myelinated ad-fibers distributed throughout the respiratory tract (fox, 1996a,b; reynolds et al., 2004) . these nerve endings have strictly vagal origin. they are located underneath the airways epithelium and demonstrate high sensitivity to inhaled irritants. coughing is a reflex activity initiated by stimulation of sensory nerves in the lining of the respiratory passages, the tubes we use to breathe. coughing can be induced from the larynx and tracheobronchial tree, but not directly from structures above or below these sites (widdicombe, 1998) . in a healthy man, cough is present only when, for example, an insufficiency of mucociliar apparatus and alveolar macrophages prevents them from fulfilling their cleaning role in airways. an example of such a situation could be when solid or liquid food particles accidentally get into the airways, or through inhalation of irritant gases (widdicombe, 1995) . knowledge of structures participating in the genesis of this defense mechanism is potentially important for antitussive therapy, because the mechanism of both peripherally and centrally acting antitussive drugs may depend on the identity of the afferent pathways involved. the larynx, being the sentinel of the lungs, possesses abundant sensory innervations, which can produce violent coughing after their activation. afferent activity may be elicited from the larynx by mechanical and chemical irritants. most of the sensory traffic from the larynx is conveyed in the superior laryngeal nerves (fuller and jackson, 1990; sant'ambrogio and sant'ambrogio, 1996) . three types of receptors -pressure, drive and cold -take part in a clear respiratory modulation. pressure receptors respond to changes in trans-laryngeal pressure, drive receptors are stimulated by passive or active motion of the larynx and cold/flow receptors respond to a decrease in laryngeal temperature. although respiratory-modulated receptors play an important role in the function of the upper airways, they are not generally viewed as a primary factor in the elicitation of cough reflex. there are two types of receptors localized in laryngeal mucus, both of which are connected with the cough reflex. these are: rapidly adapting receptors (rars) or irritant receptors. the cough reflex is probably caused by the stimulation of the irregular firing irritant receptors. they are usually silent in quiet breathing and activated only by mechanical and chemical irritant stimuli (e.g. cigarette smoke, distilled water, co 2 , etc.), the prompt blocking effect of topical anesthetics are thought to have a superficial location (widdicombe et al., 1988) . laryngeal irritant receptors are stimulated by halothane (which inhibits those receptors in tracheobronchial tree) and by water solutions lacking chloride anions (anderson et al., 1990; sant'ambrogio, 1996) , but not by hyposmolal solutions in the trachea (ventresca et al., 1990) . c-fiber receptors. they are sensitive to mechanical stimulation, cooling, chemical stimulation by capsaicin and phenylbiguanide. however, we must respect the existence of considerable differences in species. the characteristic signs of the laryngopharyngeal cough are active expirations and several fast and powerful inspiratory movements. it differs from tracheobronchial cough in the presence of the inspiratory component, which is in this case as strong as, or even stronger, than the active expiratory component (figure 1 ). from the pharmacological frame of reference, this type of cough is more resistant to cough suppressant agents. from our long-standing results we can conclude that the influence of antitussive agents varies very strongly. we found that some of the substances can suppress the cough from the laryngopharyngeal area more potently than the others, which were more effective in tracheobronchial cough suppression (table 1) . there are three types of sensory nerve endings in the tracheobronchial region of the respiratory tract: (a) rapidly adapting stretch receptors (rars) or ''irritant''receptors. regarding the cough reflex this type of receptor seems to play a very important role. rars respond to mechanical and chemical stimuli (prostaglandin e 2 , histamine, cigarette smoke etc.). all of the stimuli that can induce coughing are able to stimulate rars, although most of them activate bronchial c-fiber receptor, too (karlsson, 1996; reynolds et al., 2004) . ''irritant'' receptors are situated within the mucosal surface of the major airways in high concentrations, especially at bifurcations. these places are responsible for mucus clearing and expelling foreign materials from the airways. myelinated fibers in the vagal nerves conduct the sensory information. it is interesting that some viral infections can enhance their sensitivity to stimuli, causing the cough (empey et al., 1976; chung et al., 2003) . (b) slowly adapting stretch receptors. these are located in the membranous posterior wall of conducting airways within the smooth muscle. they are supposed to be important for the act of coughing. (c) tracheobronchial c-fiber receptors. the stimulation of bronchial c-fiber receptors can cause cough, as well as apnea and bronchoconstriction, presumably in response to different varieties or concentrations of stimulants. all the stimuli used for irritation of c-fiber receptors can also activate rars. many cough-inducing chemical stimuli (like bradykinin, sulfur dioxide and capsaicin) may act directly on the rars, but in addition they can activate c-fiber receptors, which release tachykinins. these will in turn act on postcapillary venules, causing plasma extravasations and stimulation of adjacent rars. the increase in interstitial liquid volume might also affect the structure of epithelium and stimulate the branches of rars there (widdicombe, 1995) . in recent times trpv1 receptors have been identified on ad nociceptive fibers, which under normal physiological conditions do not synthesize neuropeptides, but can be activated by capsaicin. reynolds et al. (2004) suggested that c-fibers do not incite cough per se but might be involved in the sensitization of the cough reflex. (d) pulmonary c-fiber receptors. raj et al. (1995) showed that pulmonary c-fiber receptors cause cough in unanesthetized humans. however, further experiments are needed for wider acceptance of this fact. there is clear evidence that airway sensory afferents, irrespective of whether they are myelinated or non-myelinated, terminate within the brainstem, in the nucleus tractus solitarius (jordan, 1996 (jordan, , 1997 takahama, 2003) . vagal afferents have been shown to contain mediators such as glutamate and 5-hydroxytryptamine. it is also a wellknown fact that nmda receptors, opiate receptors and 5-ht receptors are able to modulate transmission through the nucleus tractus solitarius. this region is anatomically adjacent to the area postrema, where the blood-brain barrier is weak or absent, so antitussive drugs given peripherally could be acting at the central site, on the sensory side of the system. other areas, such as nucleus ambiguus and dorsal vagal nuclei, also contain these types of receptors and could be considered as additional sites of action. apart from this, activation of nmda receptors and both the opioid and serotonergic systems can modify the central respiratory network . this is also supported by our results with substances known for their ability to influence these receptors ( figure 2 ; nosalova, 1998) . the antitussive actions of these agents may occur via this respiratory action (kamei, 1996) . despite huge endeavors of experimental researchers, all of the neurochemical processes participating in the mechanism of action of antitussive agents are still not known. hedner et al. (1980) highlight an important role of gamaaminobutyric acid (gaba) in tonic modulation of respiratory activity. as the breathing and cough centers are tightly associated, we decided to verify the ability of gabaergic substances to influence the cough reflex. we obtained original priority results (nosalova et al., 1986a (nosalova et al., ,b, 1987 , which support the statement that gabaergic substances are able to suppress cough ( figure 3 ) and that gabaergic mechanisms can take part in mechanism of action of other cough-suppressing agents. cough is considered to be a pathological reflex from the moment it stops fulfilling its cleaning role and starts to burden the patient. pathological cough (especially its chronic form) has a significant impact on the patient's quality of life, observed either in physical activity or in the psychosocial domain. coughing patients suffer often from insomnia, exhaustion, nausea and emesis, worsening of performance at work or incontinence. in many of them it can lead to social problems connected to the need for changing or leaving a job or important social activities (irwin, 2001) . in addition to this, the intrathoracal pressure changes during the cough (inspirium à13 kpa and exspirium +30 kpa) can cause costal fractures, cough syncope, pneumothorax, pneumomediastinum, herniation, etc. patients with coronary lesions may experience anginal pain. cough also represents one of the ways for transmission of infections (korpas and nosalova, 1991) . the most frequent cause of abnormal cough reaction is the presence of pathological processes in the respiratory system, leading to increased irritability of afferent nerve endings or increased sensitivity to different stimuli (schuligoi et al. 1998; shinagawa et al., 2000) . these conditions can be found during inflammatory diseases of upper and lower airways of either viral or bacterial origin. inflammatory process in the airways increases mucus production, which, owing to impaired mucociliar transportation, could be one of the reasons for mechanical irritation. however, more important is the fact that accumulated phlegm rich in inflammatory cells and mediators functions as a cough-provoking chemical stimulus. many inflammatory mediators may modulate cholinergic and sensory nerves in the airways through the activation of receptors on nerve terminals (barnes, 1992; reynolds et al., 2004) . sensory nerves in the airways contain several neuropeptidesthe tachykinins. the main members of this family are substance p (sp), neurokinin a (nka) and neurokinin b (nkb), among which substance p is the best known. stimulation of these nerves causes a constellation of responses known as neurogenic inflammation. distribution of substance-p-containing nerves is close to that of the rars mediating cough in the airways (hay, 2001) . the most important enzyme for the breakdown of sp in these nerve fibers has been found (sekizawa, 1996) . neutral endopeptidase (nep) is a key enzyme in the degradation of tachykinins in the airways. this enzyme is strongly expressed in epithelial cells (barnes, 2001) . in humans, aerosol of sp did not cause cough at the concentration of up to 10 à5 m in normal subjects, but they did in patients with upper-respiratory tract infection (katsumata et al., 1989) . another factor, the attenuation of the barrier function of the airways epithelium, is involved in the inflammation. this way, the irritants can get to the nerve endings more easily. this process is aggravated by total destruction of epithelial cells, leading to loss of another important role -production of nep. during inflammation of the airways, damaged epithelium is not able to form nep, which may facilitate the penetration of substance p. fox (1996b) showed, that sp causes cough by direct stimulation of rapidly adapting pulmonary stretch receptors (rars). apart from this, tachykinins and especially sp also showed indirect activity resulting in cough reflex facilitation (advenier, 1996) . sp stimulates mucus secretion from submucosal glands and mediates the increased plasma exudation and the vasodilator response to tachykinins. tachykinins also enhance cholinergic neurotransmission by facilitating acetylcholine release at cholinergic nerve terminals and by enhancing ganglion transmission (watson et al., 1993) . a variety of species, including human tachykinins, stimulate c-fiber receptors and rapidly adapting pulmonary stretch receptors and can cause cough (widdicombe, 1996) . tachykinins may also interact with inflammatory and immune cells. besides the mechanism mentioned already, increased sensitivity of the afferent nerve fibers can also cause the genesis of pathological cough. this occurs when in an individual a dry cough is response to such concentrations of tussigen stimuli, which in normal healthy individuals do not provoke cough response. o'connell (2001) showed that the cough reflex is ''upregulated'' in persistent dry cough. this upregulation occurs at the level of the afferent cough receptors in the airways. these are the action site of the most important drugs, which lead to downregulation of the abnormally sensitive cough receptors. increased as well as decreased reactivity of afferent nerve endings, leading to the so-called ineffective type of cough, is considered to be a pathological state (laurence et al., 1997; korpas and tomori, 1979) . the other reasons for this type of cough may be central structures disorders, which take part in cough reflex regulation, or disorder of effectors mechanisms. the consequence of ineffective cough can manifest in development and intensification of pathological processes in the airways (nishino et al., 1996) . understanding the mechanisms participating in cough-reflex genesis, (especially the cough with pathological character) enables its appropriate treatment. the cough, fulfilling a physiological role and cleaning the airways of accumulated secretions, phlegm, tissue detritus, dirt and foreign bodies in the airways and the ciliary mechanisms cannot be pharmacologically influenced sada, 1997) . the best we can do is to help with expectoration (nosalova et al., 1986a (nosalova et al., ,b, 1998 . on the contrary, the pathological type of cough has to be influenced pharmacologically. we can suppress it, hold it at an acceptable level, or modify its effectiveness. the cough reflex with its characteristics is one of the strangest reflexes of all. it is one of the airway-defense reflexes and also can be evoked voluntarily (lavigne et al., 1991; lee et al., 2002) . hutching et al. (1993) showed that cough could be not only initiated but also inhibited by conscious control. there is also evidence, that cough can only occur during consciousness and does not occur during rem phase of sleep (anderson et al., 1996) . cough is the first respiratory response inhibited by general anesthesia without any prior slowing down of respiratory rate. from the clinical aspect it is particularly important that cough is present as a symptom by most respiratory system diseases (nosal and banovcin, 2001; chung et al., 2003) . cough is often registered as the only symptom of respiratory diseases (koh et al., 1999) . the particularity of cough reflex is determined also by the fact that if it is disproportionately intensive or persisting, it changes to a pathological reflex and starts to burden the patient. it disturbs daily activities like sleep, food intake, normal breathing and circulation and disables the individual in society by noise or by incontinence. this leads to a significant worsening of the quality of the patient's life. cough can induce complications, that will jeopardize human life. therefore, this pathological kind of cough has to be suppressed or held at an acceptable level (korpas and nosalova, 1991; nosalova, 1998; nosalova et al., 2004) . another particularity of cough reflex could be its adverse action on normal physiology. the increased activity of muscles involved in breathing leads to increase in intake of oxygen. when oxygen supply is impaired owing to principal respiratory system disease, the tissues are lacking it. cough is a reflex that may be initiated voluntarily without any input from afferent vagal fibers. conversely, cough induced by incoming vagal impulses may be voluntarily suppressed (o'connell, 2001) . depending on the cause and duration, cough can be divided into three categories: acute cough, lasting less than three weeks; subacute cough, lasting three to eight weeks; and chronic cough, lasting more than eight weeks. since all types of cough are acute at the outset, it is the duration of the cough at the time of presentation that determines the spectrum of likely causes. depending on the phlegm (mucus) production, the cough can be called as dry or wet (productive) (parvez et al., 1996) . painful cough, the name for the pain-eliciting cough, sometimes accompanied by symptoms of various pathological processes in the respiratory tract such as bronchial asthma, chronic bronchitis, bronchogenic carcinoma, gastroesophageal reflux, etc. it could possibly have a psychogenic basis (lavigne et al., 1991) . cough can occur as an adverse effect of drug treatments, e.g. after the administration of acei (franova and nosalova, 1998; gajdos and huttova, 2002) . acute cough is mostly mild and lasts for a very short time (up to 21 days). it occurs during acute diseases of respiratory tract caused especially by viral infections, common cold, acute bacterial sinusitis, exacerbations of chronic obstructive pulmonary disease, allergic rhinitis, rhinitis due to environmental irritants and pertussis. in the initial phase it is non-productive (dry), but it can later change into a productive one. its duration is usually several days and it weakens gradually. this kind of cough often does not need to be modulated therapeutically, as the drugs have no impact on disease outcome (irwin et al., 1993; chung et al., 2003) . the drugs are recommended if the cough attacks are too frequent and so significant that they extensively lessen the patient's comfort, or if the cough is persistent and painful. in these situations, drugs that suppress and lower the irritation of inflamed mucous membranes are used facilitating daily comfort and tranquil sleep (braga and allegra, 1989; kurz, 1989; korpas and nosalova, 1991; parvez et al., 1996) . korppi et al. (1991) recommended that the use of antitussives in children should be restricted to such situations where their efficacy has been proven, i.e. in the treatment of chronic non-productive cough. for treatment of this form of cough we recommend a combination of oldergeneration h 1 antihistamines and nasal decongestants. for patients who cannot take and tolerate this combination it is possible to use intranasal ipratropium bromide (0.06% spray). the newer generation of antihistamines is ineffective for treatment of cough caused by common cold. these agents are effective when cough is a symptom during histamine-mediated conditions such as allergic rhinitis. in this case we can also use nasal cromolyn and corticosteroids. irwin and madison (2000) recommend the usage of antibiotic therapy for patients with exacerbations of chronic obstructive pulmonary disease (if acute cough is accompanied by worsening shortness of breath, wheezing, or both), acute bacterial sinusitis, pneumonia and bordetella pertussis infection. we deem it very important to say that antibiotic treatment should not be determined generally, such as for 3 weeks, but should be individualized. the duration of the treatment depends on symptoms, remission and normalization of patient's health constitution (well-being). if the cough lasts for 3-8 weeks, it is considered to be the subacute form of cough. the treatment of this kind of cough is based on the elimination of the coughprovoking cause. practically, it does not differ from the treatment of chronic form of cough. for elimination of subacute form of cough we use older-generation antihistamines combined with nasal decongestants, ipratropium bromide, bronchodilators from the group of b-agonists, corticosteroids, antitussives and the target antibiotic treatment, if necessary. chronic cough is defined as a cough that lasts for more than three weeks (irwin et al., 1990; cap, 1999) . many physicians accept a longer limit: such as six to eight weeks. as for diagnosis, this form of cough signals the possibility of serious disease, such as chronic bronchitis, bronchial tumor, lung abscess, blood stasis in small circulation, etc. according to philp (1997) , more than 90% of cases of chronic cough are a result of five common causes: smoking, postnasal drip, asthma, gastroesophageal reflux and chronic bronchitis. we prefer specific therapy in the treatment of chronic cough ( table 2 ). the success of the specific therapy depends on the correct diagnosis of the cause of the cough mechanisms, that arouse it (korpas and nosalova, 1991; bolser, 1996; nosalova, 1998; nosalova, 2001) . symptomatic treatment of chronic cough (also called nonspecific therapy) is indicated only in cases where the cough does not fulfill its role and the complications could represent real or possible danger for the patient (korpas and nosalova, 1991; irwin et al., 1998) . we prefer the drugs from the group of non-narcotic antitussives with peripheral site of action (table 3 ). in our conditions these drugs showed lower in this case we prefer ipratropium in the therapy of cough, because it decreases mucus production, dilates the bronchi and is more effective in the therapy of cough than beta-agonists omeprazole, in a dose of 80 mg per day -anticholinergic drugs as is necessary -calcium channel blockers as is necessary -theophylline as is necessary -other muscle relaxants as is necessary -protective agent, e.g. sucralfate sucralfate may be helpful in a dose of 1 g taken one hour before meals -prokinetic agent, e.g. metoclopramide or cisapride before meals and at bedtime metoclopramide or cisapride may be added before meals and at bedtime, necessary to avoid eating or drinking for 2 h before sleeping antitussive activity than the so-called codeine-type agents (figure 4 ), but their administration was not associated with unwanted effects. from the centrally acting drugs we prefer agents from the group of synthetic morphine derivatives, or clobutinol (table 4 , groups b and c). dry, irritant, nonproductive cough significantly burdens the afflicted patient. the cough usually repeats, as rapid air expulsion irritates tracheal as well as tracheobronchial mucous membranes. in this situation the cough must be suppressed by antitussives. codeine showed especially high efficacy ( figure 5 ). adding of expectorants is also very useful. they diminish the cough by increasing the fluid content in the respiratory tract and hence calm the mucous membranes of the airway, helping the patient to breathe (korpas and nosalova, 1991) . fig. 4 . antitussive activity of peripherally acting antitussive agents compared to codeine. cod -codeine 10 mg/kg i.p., but -butamirate citrate 5 mg/kg i.p., dro -dropropizine 100 mg/kg i.p., pre -prenoxdiazine 30 mg/kg i.p. nonproductive, irritating cough characterizes psychogenic cough. its incidence is very often in young people during pubescence, often after an underlying disease subsides. it can occur without any clear cause (hutching et al., 1993) . this form of cough does not respond to antitussive or other therapy and is typically not present during eating or talking. if the patient feels that he is being observed, the cough accentuates. agents from the group of anxiolytics such as guaifenesin (guajacuran, figure 6 ), or combinations of anxiolytics, with antitussives like guaifenesin+butamirate citrate in stoptussin are used in the treatment of psychogenic cough (nosalova et al., 1986a; korpas and nosalova, 1991; parvez et al., 1996) . we found very strong cough-suppressive effect in benzodiazepine and phenotiazine derivatives ( figure 6 ). these agents could also be used in the treatment of cough induced by psychogenic factors. in addition, antitussive action could be beneficially used for its tranquilizing effect. at the same time we have to be very careful while administering these drugs in patients who need to enforce expectoration. special attention must be granted to diazepam ordination in pediatric practice, because in acute respiratory diseases associated with fever it very often used to be a part of polyvalent therapy. its application could lead to suppression of breathing, consecutive phlegm stasis and global worsening of health of the patient. psychotherapy, is very useful too. but the essential condition is to take the patient away from the surroundings in which he coughs. bronchial asthma is very often characterized by the only one symptom -irritant, nonproductive cough, which very often precedes the classical symptoms picture of asthma. in these cases the use of drugs from the group of beta 2 -sympathomimetics or corticosteroids (bush, 2002; nosal, 2003) that also suppress the cough in allergic patient with bronchial hyperresponsiveness is recommended (barnes, 1996; hupka et al., 1996) . beta 2 sympathomimetics, similar to other bronchodilators (zibolen et al., 1999) , can reduce bronchial hyperresponsiveness, stimulate mucociliary clearance and increase water and ion flow into bronchial lumen. this leads to enforcing of expectoration of viscous secretions. many authors advise a combination therapy of beta 2 -sympatomimetics with dextromethorphan in the cough therapy of asthmatics. productive cough must not be completely suppressed when the retention of secretions could occur, leading to a worsening of the global health constitution of the patient or to the development of pneumonia or atelectasis (braga et al., 1989; fox, 1996b; parvez et al., 1996) . in some cases, productive cough can be hyperactive and often repeating. it burdens the patient and disturbs the sleep. the reduction of cough frequency and intensity by expectorants or antitussive-expectorant agents, respectively, is indicated in this situation (butamirate citrate -sinecod, guaifenesin+ butamirate citrate -stoptussin, ephedrine -solutan, ipecarin, etc.). mucoactive agents are also very useful (bromhexine -bromhexin, bisolvon, ambroxol -mucosolvan, carbocysteine -mucopront, etc.), as they reduce the phlegm viscosity (table 5) . furthermore, it is advisable to add agents from the group of secretomotorics to the therapy, which act by inducing of more effective phlegm expulsion from lower parts of the respiratory tract (braga et al., 1989; korpas and nosalova, 1991; rubin, 2003) . also many plant extracts and derivatives exert their antitussive effect by expectorant and mucolytic activity (see next chapter -franova et al.: phytotherapy of the cough). there are certain kinds of cough owing to which the patients feel pain in the throat or in the chest, especially behind the sternum. painful cough is observed in pleuritis and lung cancer (homsi et al., 2001) . out of our results (figure 7 ) arises the unambiguous fact that in these patients, the group of antitussives with analgesic activity (nosalova et al., 1985) , represents the best therapy for suppression of cough. alternatively, analgesics with a cough-suppressive effect (tilidine chloride -valoron, tramadol -tramal, pentazocine chloride -fortral, and butorphanol -beforal) may be used. as in these diseases analgesic agents are often prescribed for pain relief, they can simultaneously and adequately suppress cough as well as pain (strapkova et al., , 1988 . the drugs that selectively inhibit or lessen cough reflex are called antitussive agents. the moderation of cough is a result of the reduction of number or frequency of cough efforts, a decrease in their intensity, or both (empey, 1996) . pharmacologically, it is very important to know that cough as a reflex can be modulated by agents acting at various levels of the reflex arc: at the level of receptors, afferent nerves, cough center, efferent nerves and effectors, as well as by alteration of bronchial secretion (reynolds et al., 2004) . some agents are able to influence more levels, with predominance in one of them. according to the thinking nowadays, cough reflex is triggered by the irritation of nerve endings that are present under the airway mucous membrane. they are called rapidly adapting irritant receptors and mediate the cough reflex after mechanical stimulation. in smaller bronchi there is another group of epithelial receptors with medially rapid adaptive ability. these receptors mediate the cough evoked by chemical stimuli (widdicombe, 1995) . the anatomical localization of cough receptors in the airways enables the use of local anesthetics reynolds et al., 2004) , which could tilidin 10 mg/kg i.p. fig. 7 . antitussive effect of tilidin compared to codeine. ne -number of cough efforts after mechanical stimulation of tracheobronchial area of the airways, ia + -intensity of cough attack in expirium, ia à -intensity of cough attack in inspirium, c -control, 0.5, 1, 2, 5 htime after administration of tested substance. be ideal from the pharmacological point of view, but they are not selective enough. the inhalation of anesthetizing aerosols leads to desensitization of cough receptors as well as other segments of mucous membranes in the mouth, and the upper and lower parts of the respiratory system, and increases the risk of aspiration . inhalant procaine, lidocaine and bupivacaine inhibit the cough reflex elicited by both mechanical and chemical irritation. in clinical settings, the local anesthetics are used for the elimination of cough before diagnostic procedures such as laryngoscopy, bronchoscopy, intubations, etc. in the mechanism of action of some cough-suppressing agents (benzonatate -tessalon, dropropizine -ditustat) a local anesthetic activity takes place apart from other properties korpas and nosalova, 1991) . information from cough receptors localized in the airways are spread through various branches of vagal and glossopharyngeal nerves. in cough evoked by unilateral irritation due to bronchial tumor it is possible in certain conditions to use lead anesthesia of vagal nerve stem. the drugs that suppress the cough reflex by central mechanism are listed in table 4 . in the past, alkaloids and opium derivatives were the only group of drugs the physicians used for cough suppression. the antitussive activity of these agents is associated with their ability to lower the sensitivity of the cough center to nerve impulses coming from airways receptors (fox, 1996b) . although this group can suppress the cough very potently (figure 2 ), the contemporary trend worldwide is to limit their use as they also suppress the breathing center. this unwanted effect occurs especially in children. the other negative property is the diminished activity of bronchial glands, leading to an increase in phlegm viscosity and worsening of expectoration. a very important adverse effect is their ability to induce drug dependence. dextromethorphan represents the most important member of the group of synthetic morphine derivatives (rhinotussal, romilar), which show, according to our results, excellent antitussive activity . they do not induce drug dependence and their significantly suppressive effect on cough does not suppress the breathing center. adverse effects, which occur in less than 1% of people, include drowsiness, dizziness, nausea, constipation and abdominal discomfort. dextromethorphan is contraindicated in a person taking monoamine oxidase inhibitors (maoi). another drug with central cough-suppressing effect is clobutinol (silomat). clobutinol does not have the addictive properties of codeine and it does not suppress the breathing center: rather, stimulates it. it is suitable for cough therapy to treat hemoptysis and severe pertussis and to conduct endoscopical procedures in respiratory system. efferent nerves conduct the impulses activating the expiratory skeletal musculature, laryngeal muscles, tracheobronchial smooth muscles and secreting apparatus. cough can be suppressed by pharmacodynamic elimination of any part. the most significant suppression can be achieved by myorelaxants, which block the expiratory muscles. however, this method is not recommended because of its serious adverse effects. some authors include ganglioplegics, mostly hexamethonium, in this group. lately, bronchodilatants have started to be used in the therapy of cough. bronchodilating action is one effect of dropropizine (ditustat), l-dropropizine (levopront) and butamirate citrate (sinecod), which suppress cough by relaxing the bronchial muscles and facilitating the expectoration. in addition to their bronchospasmolytic and secretomotoric activity, these drugs also have an anti-inflammatory effect. the stimulatory effect on the breathing center and the absence of drug dependence are of great importance (nosalova et al., 1984; korpas and nosalova, 1991) . another agent with significant bronchodilating activity is pentoxyverine (sedotussin), which acts at the level of the peripheral parasympathetic nerve endings, similar to atropine. mucoactive substances are representatives of a specific group of drugs. they do not affect the cough receptors directly, but modify the physical and chemical properties of bronchial secretions in order to facilitate their motion in an oral direction (rubin, 2003) . their therapeutic goal is to change dry, irritant and nonproductive cough to so-called productive cough with nonirritating expectoration (strapkova, 2000) . the effects of sirupus althaeae (nosalova et al., 1992 (nosalova et al., , 1993 and other herbal substances, mentioned in chapter 7 are especially beneficial. this group of drugs (table 5) is used in clinical conditions for the treatment of cough with hypersecretion phenomenon (braga et al., 1989) . demulcerative and hydrating drugs are water-soluble substances with high molecular weight, but without any pharmacological activity. despite this they are used for the treatment of upper airway inflammations, sore throat and other diseases associated with dryness in mouth, irritation in throat and cough. the representatives of this group are glycerin, liquorices, synthetic cellulose derivatives, sugar syrup, honey, etc. the goal of this kind of treatment is to create a defense layer protecting the mucous membranes from other irritant stimuli (braga et al., 1989; . various plants are rich in this kind of agents and are discussed in chapter 7. tachykinin antagonists and cough respiratory responses to tracheobronchial stimulation during sleep and wakefulness in the adult cat water-responsive laryngeal receptors in the dog are not specialized endings modulation of neurotransmission in airways neurogenic inflammation in the airways cough 7: current and future drugs for the treatment of chronic cough mechanisms of action of central and peripheral antitussive drugs paediatric problems of cough sensitivity of the cough reflex in patients with chronic cough therapy of cough: active agents cough: causes, mechanisms, and therapy codeine, cough and upper respiratory tract infection antitussive drugs mechanism of bronchial hyperreactivity in normal subjects after upper respiratory tract infection modulation of airway receptors and cough modulation of cough and airway sensory fibres ace inhibitors and defense reflexes of the airways herbal polysaccharides in the therapy of cough physiology and treatment of cough angiotensin ii at 1 receptor blockers and diabetes mellitus clinical and mechanistic perspectives on acute self-limited cough tachykinins and cough respiratory depression by gaba-ergic drugs in the preterm rabbit important drugs for cough in advanced cancer coughing frequency in patients with persistent cough: assessment using a 24 hour ambulatory recorder bronchial hyperresponsiveness in children after mycoplasmic pneumonia voluntary cough suppression as an indication of symptom severity in upper respiratory tract infections quality of life in coughers the diagnosis and treatment of cough the persistently troublesome cough appropriate use of antitussives and protussives chronic cough: the spectrum and frequency of causes, key components of the diagnostic evaluation, and outcome of specific therapy managing cough as a defense mechanism and a symptom central nervous mechanisms in cough central nervous control of autonomic function role of opioidergic and serotonergic mechanisms in cough and antitussives the role of capsaicin-sensitive c-fibre afferent nerves in the cough reflex inhibitory actions of procaterol, a beta-2 stimulant, on substance p-induced cough in normal subjects during upper respiratory tract infection development of wheezing in patients with cough variant asthma during an increase in airway responsiveness cough and other respiratory reflexes antitussives in the treatment of acute transient cough in children ace-inhibitor-induced cough, an adverse drug reaction unrecognized for several years: studies in prescription-event monitoring antitussiva und expektoranzien clinical pharmacology behavioral management of psychogenic cough. alternative to the ''bedsheet'' and other aversive techniques voluntary control of cough epidemiology of cough cough and other reflexes in irritation of the airway mucosa in man comparison of the bronchodilatory effects of salbutamol in children with asthma bronchiale and obstructive bronchitis selected chapters of pediatrics mechanism of action of the drugs influencing the cough reflex tussis. vademecum of pediatrics. osveta: martin cough and local anesthetic effect of various substances butamirate citrate action on cough components study of antitussive effect of analgesic tilidin antitussive activity of a rhamnogalacturonan isolated from the roots of althaea officinalis l., var. robusta objective assessment of cough suppressants under normal and pathological experimental conditions guaifenesin as a component of new czechoslovak antitussive-expectorant stoptussin antitussive action of extracts and polysaccharides of marshmallow (althaea officinalis l., var. robusta) the mechanisms of action of drugs affected the cough reflex cough and central gabaergic mechanism gaba-ergic mechanism in the central control of cough central pathways -unanswered questions evaluation of antitussive agents in man chronic cough differential diagnosis of cough and gastroesophageal reflux. abstract book, martin days of respiration sensory origin of lobeline-induced sensations: a correlative study in man and cat mucoactive agents for the treatment of cough the physiology of cough role of laryngeal afferents in cough role of laryngeal afferents in cough. sensory mechanism in cough bradykinin-evoked sensitization of neuropeptide release from afferent neurons in the guinea-pig lung role of substance p in cough pathophysiological mechanisms of meconium aspiration syndrome surfactant lung lavage in rabbits with meconium aspiration -a pilot study participation of tromboxane a 2 in the cough response in guinea-pigs: antitussive effect of ozagrel antioxidant activity of mucolytics relationship of antitussic and analgesic activity of various substances antitussive effect of tramadol mechanisms of actions of centrally acting antitussives -electrophysiological and neurochemical analysis inhaled furosemide inhibits cough induced by low chloride content solutions but not by capsaicin endogenous tachykinins facilitate transmission through parasympathetic ganglia in guinea-pig trachea neurophysiology of the cough reflex sensory mechanisms afferent receptors in the airways and cough ambrogio g editors. respiratory function of the upper airway assessment of antitussive efficacy of dextromethorphan in smoking related cough: objective versus subjective selected chapters of pediatric clinical needs for cough therapy key: cord-312613-1nl7q6cy authors: luz garcia-garcia, m.; calvo rey, cristina; del rosal rabes, teresa title: pediatric asthma and viral infection() date: 2016-03-26 journal: arch bronconeumol doi: 10.1016/j.arbr.2016.03.010 sha: doc_id: 312613 cord_uid: 1nl7q6cy respiratory viral infections, particularly respiratory syncytial virus (rsv) and rhinovirus, are the most importance risk factors for the onset of wheezing in infants and small children. bronchiolitis is the most common acute respiratory infection in children under 1 year of age, and the most common cause of hospitalization in this age group. rsv accounts for approximately 70% of all these cases, followed by rhinovirus, adenovirus, metapneumovirus and bocavirus. the association between bronchiolitis caused by rsv and the development of recurrent wheezing and/or asthma was first described more than 40 years ago, but it is still unclear whether bronchiolitis causes chronic respiratory symptoms, or if it is a marker for children with a genetic predisposition for developing asthma in the medium or long term. in any case, sufficient evidence is available to corroborate the existence of this association, which is particularly strong when the causative agent of bronchiolitis is rhinovirus. the pathogenic role of respiratory viruses as triggers for exacerbations in asthmatic patients has not been fully characterized. however, it is clear that respiratory viruses, and in particular rhinovirus, are the most common causes of exacerbation in children, and some type of respiratory virus has been identified in over 90% of children hospitalized for an episode of wheezing. changes in the immune response to viral infections in genetically predisposed individuals are very likely to be the main factors involved in the association between viral infection and asthma. r e s u m e n las infecciones por virus respiratorios, especialmente virus respiratorio sincitial (vrs) y rinovirus, suponen el mayor factor de riesgo para la aparición de episodios de sibilancias en lactantes y niños pequeños. la bronquiolitis es la infección respiratoria aguda de vías respiratorias inferiores más común en menores de un año y constituye la causa más frecuente de hospitalización en este grupo de edad. el vrs causa aproximadamente el 70% de todas ellas, seguido por rinovirus, adenovirus, metapneumovirus o bocavirus. la asociación entre bronquiolitis por vrs y desarrollo de sibilancias recurrentes y/o asma ha sido descrita hace más de 4 décadas, aunque en la actualidad se desconoce con exactitud si la bronquiolitis es la causa de los síntomas respiratorios crónicos o si, más bien, es un marcador que señala a los niños con predisposición genética a desarrollar asma a medio o largo plazo. en cualquier caso, existe evidencia suficiente como para afirmar que esta asociación existe y que es especialmente intensa si el agente asociado a la bronquiolitis es el rinovirus. el papel patogénico de los virus respiratorios como desencadenantes de exacerbaciones en el paciente asmático no está totalmente aclarado, pero sin duda los virus respiratorios, y en especial el rinovirus, son el desencadenante más frecuente de exacerbaciones asmáticas en los niños, llegando a identificarse algún virus respiratorio hasta en el 90% de los niños hospitalizados por un episodio de sibilancias. muy probablemente, las alteraciones en la respuesta inmune frente a las infecciones virales en sujetos genéticamente predispuestos sean los principales implicados en la asociación virus-asma. asthma is a chronic inflammatory disease of the airways characterized by bronchial hyperresponsiveness to a wide variety of stimuli, recurrent episodes of wheezing, respiratory distress, and cough, associated with reversible airway obstruction. asthma is one of the most prevalent chronic diseases worldwide, affecting more than 155 million individuals, so the impact of asthma is severe, and incidence is growing, particularly in developed countries. 1, 2 respiratory viruses are one of the most common causes of asthma exacerbations in both adults and children. [3] [4] [5] [6] furthermore, increasing evidence is emerging to suggest that viral respiratory infections in early life are related with the medium and long-term development of asthma. 7, 8 this article aims first to review the role of viruses as precipitating factors for asthma, and then to summarize the current state of knowledge on their role in asthma exacerbations. viral bronchiolitis is a common feature in the clinical histories of children who go on to develop wheezing and asthma during childhood. the term bronchiolitis has been in use since 1940, but it has several different interpretations, and there is no general agreement on its definition. in this review, we will use the standard criteria of mcconnochie, who describes bronchiolitis as the first acute episode of wheezing, preceded by a respiratory syndrome of rhinorrhea, cough, and tachypnea, occurring with or without fever, in children younger than 2 years of age. 9 bronchiolitis is the most common acute lower respiratory tract infection in children younger than 1 year, and accounts for 18% of all pediatric admissions. 10 respiratory syncytial virus (rsv) is the causative agent in approximately 70%-80% of cases, followed by rhinovirus, adenovirus, human metapneumovirus (hmpv), and human bocavirus (hbov). 11, 12 the most common respiratory viruses are listed in table 1 . studies reporting global analyses of all patients with a history of bronchiolitis, irrespective of the causative agent, reveal a prevalence of recurrent wheezing that ranges from 75% in the first 2 years of life, 47%-59% between the ages of 2 and 4, and 25%-43% between 4 and 6 years, [13] [14] [15] [16] showing a clear trend to diminish with age. only 2 prospective studies included a long-term follow-up of children hospitalized for bronchiolitis, irrespective of the causative virus. they found a prevalence of asthma at the age of 17-20 years of 41%-43% in patients with a history of bronchiolitis, compared to a rate of 11%-15% in controls; between 25 and 30 years of age, prevalence was 35%, with significant impact on health-related quality of life. 17, 18 these data suggest that recurrent wheezing occurs frequently in children after an episode of bronchiolitis, and also that respiratory symptoms frequently recur in young adults after a long symptom-free period during childhood and adolescence. this changes the previously held notion of a relatively good prognosis for early childhood wheezing, and indicates that the risk of asthma and lung function changes can persist until adulthood. 19, 20 rsv was the first virus to be associated with the development of asthma in children, although in recent years, other viruses, such as rhinovirus or the more recently described hmpb and hbov, have also been studied in this context. rsv is an rna virus from the paramyxoviridae family that often causes lower respiratory tract infections in infants and small children. 21 in 1959, wittig and glaser 22 first described the epidemiological association between viral bronchiolitis in childhood and the subsequent development of recurrent wheezing and/or asthma. since then, numerous studies have evaluated this relationship, although the different methodologies employed made it difficult to draw conclusions that definitively proved this association. however, more recently, several prospective studies, now considered seminal, [23] [24] [25] [26] [27] showed that a history of bronchiolitis caused by rsv is an independent risk factor for the development of recurrent wheezing and medically-diagnosed asthma. of these authors, sigurs et al. 27 performed the longest follow-up to date, with subjects reaching the age of 18 years in the last follow-up time point. the initial cohort consisted of 47 infants aged <1 year hospitalized with severe rsv bronchiolitis and 93 age-and gender-matched controls. children who had been admitted for bronchiolitis had a higher prevalence of asthma/recurrent wheezing and allergic sensitizations at the ages of 3, 7 and 13 years, compared to the control group. at the age of 18, the bronchiolitis group maintained a significantly higher prevalence of asthma (39% vs 9%), allergic rhinoconjunctivitis (43% vs 17%), and perennial allergen sensitization (41% vs 14%). moreover, at the age of 18, the bronchiolitis group had poorer lung function (fev 1 , fev 1 /fvc) than the control group, irrespective of whether they had concomitant asthma or not. they also had more prevalent bronchial hyperresponsiveness and bronchodilator response. finally, the authors reported that the only 2 risk factors independently related with the diagnosis of asthma at 18 years of age were a history of severe rsv bronchiolitis and presence of allergic rhinoconjunctivitis. these results show that severe rsv bronchiolitis in the early months of life is associated with the development of asthma, bronchial hyperresponsiveness and allergic sensitization, and suggest that this association continues until adulthood. the results of another reference follow-up study, tucson children's respiratory study, show that rsv bronchiolitis is an independent risk factor for the development of asthma up to the age of 11 years, but the association disappears after the age of 13. 24 this difference in the long-term prognosis may be related with varying severity of the acute episode, since in the sigurs study, all patients needed to be hospitalized, while the tucson cohort included mostly outpatients. the authors also observed a greater risk of asthma among children who used more healthcare resources during the acute bronchiolitis episode. 28 the rsv bronchiolitis in early life (rbel) study also supports the association between severe rsv bronchiolitis and subsequent development of asthma 29 : of the 206 infants admitted for rsv bronchiolitis, approximately 50% had been diagnosed with asthma by the age of 7 years. although rsv is without doubt the most common virus in the etiology of acute infantile bronchiolitis, the use of molecular diagnostic techniques -primarily polymerase chain reaction (pcr) -has established that other respiratory viruses, such as rhinovirus, are also associated with bronchiolitis, and probably with the development of asthma. 30, 31 indeed, several recent studies have shown that the risk of children hospitalized for bronchiolitis presenting asthma at 6 and 11 years is higher among those who were rsv-negative than those who were rsv-positive. 32, 33 rhinovirus is an rna virus from the picornaviridae family, first isolated in 1950. it comprises a large rna family of more than 100 serotypes, originally divided into 2 species, a and b, and now with the recent addition of the c type. 34 studies published in recent years suggest that rhinovirus infections involve a greater risk for the development of asthma than rsv-associated infections. in the childhood origins of asthma (coast) study, which followed a cohort of 289 newborns with high risk of developing asthma, lower respiratory tract infection associated with rhinovirus was the main risk factor for presenting recurrent wheezing at 3 and 6 years of life, with an odds ratio of 10 for rhinovirus bronchiolitis compared to 2.6 for rsv bronchiolitis. 35, 36 moreover, children with rhinovirus-associated wheezing in the first 3 years of life had worse lung function values (fev 1 , fev 0.5 , fef ) than those with other viruses or those who never presented wheezing. 37 midulla et al. 38 confirmed the role of rhinovirus bronchiolitis as one of the main risk factors for the development of asthma at the age of 6 years. the coast study also showed that, in the case of rhinovirus, the risk of developing asthma is not limited to severe infection. in fact, only 1% of children with rhinovirus bronchiolitis included in the study needed to be hospitalized, demonstrating that even mild rhinovirus infections are associated with a greater long-term risk of asthma. finally, another cohort study with a follow-up of 15-18 years showed that the risk of asthma in adolescence is greater in children hospitalized due to rhinovirus bronchiolitis, compared to rsv bronchiolitis. this study also found that the risk of asthma is greater in children whose initial episode of bronchiolitis occurred in seasons other than winter, when the predominant virus is not rsv. 39 hmpv is a paramyxovirus, discovered in 2001 and identified throughout the world as a common cause of acute respiratory infection, particularly in infants and small children. 40 the clinical features of acute hmpv infection are similar to those caused by rsv, and can manifest as mild upper respiratory tract infections, pneumonia or severe bronchiolitis requiring hospital admission. the similarity of the clinical symptoms with those of rsv has led to speculation that hmpv infections may also be associated with the development of asthma in the long term. to date, the mediumterm progress of children admitted for hmpv bronchiolitis has been examined in only 1 study that reported a similar rate of recurrent wheezing to that seen in children admitted for rsv bronchiolitis: 5-fold the rate of the control group in both cases. 41 hbov is a dna virus belonging to the parvoviridae family. it was first identified in 2005 in respiratory samples from children with lower respiratory tract infections. 42 since then, numerous studies have investigated its prevalence and its role in respiratory infections, but to date, only 1 has examined its possible role in the development of asthma: the study in question reported that 50% of children admitted for hbov bronchiolitis had asthma by the age of 5-7 years. 43 the high rate of hbov co-infection with other respiratory viruses, and its tendency to infect older children confounds the study of the real role of early hbov infections in the development of asthma. the bronchiolitis-asthma relationship: cause or coincidence? as discussed above, a wide body of evidence relates viral respiratory infections with the subsequent development of asthma, but it remains unclear if severe bronchiolitis is the real cause of asthma, or if it is a marker of susceptibility, identifying children with a predisposition for developing asthma. a prospective, multicenter study recently conducted in europe, the united states, and canada, 44 appears to support the causative role of rsv, after an 80% reduction was observed in recurrent wheezing in the medium term among premature babies with a family history of asthma and/or atopy who received prophylaxis with palivizumab, a monoclonal antibody used for preventing rsv infection. curiously, the protective effect was only observed in children with a history of atopy, suggesting that rsv may have a causative role in the pathogenesis of recurrent wheezing, but only in patients with no genetic predisposition for atopy. the possible causative role of viral bronchiolitis was questioned by an epidemiological study performed in monozygotic twins, discordant for severe rsv bronchiolitis in infancy. the authors found no difference in the frequency of asthma, lung function or nitric oxide levels at the age of 7 years between twin siblings with a history or no history of hospitalization due to bronchiolitis. 45 finally, another 2 recent studies support the hypothesis that early viral infections are markers of atopic predisposition, rather than the cause of asthma. one of these was the danish copenhagen prospective study of asthma in childhood, which followed a cohort of newborns with asthmatic mothers. investigators measured lung function and response to methacholine of infants at 1 month of life, before any respiratory symptom had been observed. they found at this time point that children who subsequently developed severe bronchiolitis already had bronchial hyperresponsiveness as a precursor to bronchiolitis. 46 these results are supported by the recent coast study, which identified allergic sensitization in the first year of life as a significant risk factor for virus-associated wheezing, while wheezing associated with respiratory infection does not increase the risk of developing allergic sensitization. 47 it seems likely that the 2 hypotheses -bronchiolitis as a cause or as a marker of asthma -are not mutually exclusive, and that the pathogenic mechanisms of rhinovirus and rsv infections differ. rsv characteristically produces a cytopathic effect in the airway, affecting children younger than 3 months, frequently requires hospitalization and occurs in epidemic outbreaks during the winter months. 48 in contrast, rhinovirus outbreaks occur throughout the year and affect older children who are generally treated as outpatients, and who often have a family history of asthma or atopy. 7, 49 these differences have led to the hypothesis of 2 different mechanisms: rhinovirus bronchiolitis may be more a marker of predisposition to asthma and atopy, while rsv bronchiolitis may have a greater causative role, particularly in severe cases requiring hospitalization. 50, 51 the role of respiratory viruses as precipitants of asthma attacks in adults and children was identified over 30 years ago. in the early studies, in which viral diagnosis was not based on molecular methods, some viral activity was detected in between 10% and 25% of asthma attacks. 52 in contrast, in recent years, the use of pcr techniques has revealed that the proportion of asthma exacerbations associated with viruses is much higher, up to 63%, according to khetsuriani et al., 53 up to 80%, according to johnston et al., 3 or even up to 95%, according to allander et al. 54 at least 1 respiratory virus was identified in 71% of patients included in a spanish study of children hospitalized for an asthma exacerbation. 5 although practically all respiratory viruses, including the newly identified hmpv and hbov, have been associated with asthma exacerbations, the agents most commonly detected in infants and schoolchildren are rhinovirus and rsv. 5, 55 in fact, a recent cohort study in 263 infants suggests that rhinovirus is the most common pathogen in the first year of life and the most important precipitant of wheezing in infants. 56 in children of school age, johnston et al. 3 found that 80% of asthma exacerbations in asthmatic children aged 9-11 years were associated with viral respiratory infection, of which two thirds were caused by rhinovirus. asthma exacerbations among pre-schooland school-age children tend to follow a seasonal pattern, and in temperate climates, the maximum incidence occurs in the month of september, coinciding with the beginning of the school year, and in spring. 57 this pattern coincides almost exactly with the peaks of maximum circulation of rhinovirus in the community, suggesting a causal relationship between this virus and asthma exacerbations. the frequency of detecting respiratory viruses in adults with asthma exacerbations ranges between 41% and 78%, according to the results of a recent meta-analysis. 58 although rhinovirus is also most common in this age group, 59 other viruses, such as rsv, hmpv, or influenza virus appear to play an important role in asthma exacerbations in adults in clinical practice. 60 moreover, viral infections can act in synergy with other stimuli, such as exposure to allergens in allergic individuals 61, 62 or exposure to high levels of environmental contaminants, 63 increasing the risk of asthma exacerbations. viral respiratory infections affect the lung in many ways, acting on epithelial cells and antigen-presenting cells. after it recognizes the viral infection, the immune system stimulates the production of cytokines, such as interleukins (il) il-25 and il-33 and thymic stromal lymphopoietin (tslp), in the epithelial cells of the airway. these cytokines induce the th2 immune response to airborne allergens in the lungs. the production of certain cytokines, such as il4, il5, and il13, by the th2 cells subsequently increases eosinophil and mast cell recruitment, causing inflammation of the airway, cell metaplasia, and bronchoconstriction. 64 however, not all individuals who contract a respiratory virus infection suffer an asthma exacerbation, so the possibility that certain risk factors increase susceptibility to present wheezing after viral infection has been explored. studies conducted by wark et al. 65 and contoli et al. 66 suggest that the absence of an efficient innate immune response, manifested by low interferon levels in the epithelial cells of asthma patients, may assist viral replication, leading to an exaggerated asthmatic response. it seems highly likely that an altered immune response to viral infections in genetically predisposed subjects are the major factors involved in the virus-asthma relationship. asthma in the united states: burden and current theories epidemiology of asthma: prevalence and burden of disease community study of role of viral infections in exacerbations of asthma in 9-11 year old children frequent detection of human rhinoviruses, paramyxoviruses, coronaviruses, and bocavirus during acute respiratory tract infections role of emerging respiratory viruses in children with severe acute wheezing hospitalizations and outpatient visits for rhinovirus-associated acute respiratory illness in adults the role of respiratory virus infections in childhood asthma inception asthma as a chronic disease of the innate and adaptive immune systems responding to viruses and allergens what's in the name? admission to hospital for bronchiolitis in england: trends over five decades, geographical variation and association with perinatal characteristics and subsequent asthma respiratory syncytial virus: the virus, the disease and the immune response prevalence and clinical characteristics of human metapneumovirus infections in hospitalized infants in spain a 2-to 3-year outcome after bronchiolitis predictors of asthma three years after hospital admission for wheezing in infancy respiratory status and allergy after bronchiolitis characteristics and prognosis of hospital-treated obstructive bronchitis in children aged less than two years increased asthma risk and impaired quality of life after bronchiolitis or pneumonia in infancy high risk of adult asthma following severe wheeze in early life asthma symptoms in early childhood -what happens then? early predictors for adult asthma and lung function abnormalities in infants hospitalized for bronchiolitis: a prospective 18-to 20-year follow-up the burden of respiratory syncytial virus infection in young children the relationship between bronchiolitis and childhood asthma: a follow-up study of 100 cases of bronchiolitis asthma and wheezing in the first six years of life. the group health medical associates respiratory syncytial virus in early life and risk of wheeze and allergy by age 13 years respiratory syncytial virus bronchiolitis in infancy is an important risk factor for asthma and allergy at age 7 severe respiratory syncytial virus bronchiolitis in infancy and asthma and allergy at age 13 asthma and allergy patterns over 18 years after severe rsv bronchiolitis in the first year of life the severity dependent relationship of infant bronchiolitis on the risk and morbidity of early childhood asthma determinants of asthma after severe respiratory syncytial virus bronchiolitis role of rhinovirus in hospitalized infants with respiratory tract infections in spain lower respiratory tract infections associated with rhinovirus during infancy and increased risk of wheezing during childhood. a cohort study preschool asthma after bronchiolitis in infancy the outcome after severe bronchiolitis is related to gender and virus a novel group of rhinoviruses is associated with asthma hospitalizations rhinovirus illnesses during infancy predict subsequent childhood wheezing wheezing rhinovirus illnesses in early life predict asthma development in highrisk children decreased lung function after preschool wheezing rhinovirus illnesses in children at risk to develop asthma rhinovirus bronchiolitis and recurrent wheezing: 1-year follow-up adolescent asthma after rhinovirus and respiratory syncytial virus bronchiolitis a newly discovered human pneumovirus isolated from young children with respiratory tract disease human metapneumovirus bronchiolitis in infancy is an important risk factor for asthma at age 5 cloning of a human parvovirus by molecular screening of respiratory tract samples recurrent wheezing and asthma after bocavirus bronchiolitis the effect of respiratory syncytial virus on subsequent recurrent wheezing in atopic and nonatopic children causal direction between respiratory syncytial virus bronchiolitis and asthma studied in monozygotic twins neonatal bronchial hyperresponsiveness precedes acute severe viral bronchiolitis in infants evidence for a causal relationship between allergic sensitization and rhinovirus wheezing in early life detection of new respiratory viruses in hospitalized infants with bronchiolitis: a three-year prospective study influence of maternal asthma on the cause and severity of infant acute respiratory tract infections the role of early life viral bronchiolitis in the inception of asthma viral infections and the development of asthma in children viruses as precipitants of asthma symptoms prevalence of viral respiratory tract infections in children with asthma human bocavirus and acute wheezing in children human metapneumovirus infection plays etiologic role in acute asthma exacerbations requiring hospitalization in adults role of respiratory viruses in acute upper and lower respiratory tract illness in the first year of life: a birth cohort study the september epidemic of asthma hospitalization: school children as disease vectors viruses and bacteria in acute asthma exacerbations -a ga2 len-dare systematic review lower airway rhinovirus burden and the seasonal risk of asthma exacerbation the importance of bacterial and viral infections associated with adult asthma exacerbations in clinical practice high titers of ige antibody to dust mite allergen and risk for wheezing among asthmatic children infected with rhinovirus study of modifiable risk factors for asthma exacerbations: virus infection and allergen exposure increase the risk of asthma hospital admissions in children synergism between rhinovirus infection and oxidant pollutant exposure enhances airway epithelial cell cytokine production viral respiratory tract infections and asthma in early life: cause and effect? asthmatic bronchial epithelial cells have a deficient innate immune response to infection with rhinovirus role of deficient type iii interferon-lambda production in asthma exacerbations the authors declare that they have no conflict of interests. key: cord-275166-qduf08kp authors: assane, dieng; makhtar, camara; abdoulaye, diop; amary, fall; djibril, boiro; amadou, diop; niokhor, diouf jean baptiste; amadou, diop; cheikh, loucoubar; ndongo, dia; mbayame, niang; lamine, fall; bouh, boye cheikh saad title: viral and bacterial etiologies of acute respiratory infections among children under 5 years in senegal date: 2018-02-13 journal: microbiol insights doi: 10.1177/1178636118758651 sha: doc_id: 275166 cord_uid: qduf08kp acute respiratory infections (aris) are the leading cause of infectious disease–related morbidity, hospitalization, and morbidity among children worldwide. this study aimed to assess the viral and bacterial causes of ari morbidity and mortality in children under 5 years in senegal. nasopharyngeal samples were collected from children under 5 years who had ari. viruses and bacteria were identified using multiplex real-time reverse transcription-polymerase chain reaction and conventional biochemical techniques, respectively. adenovirus was the most prevalent virus (50%; n = 81), followed by influenza virus (45.68%, n = 74), rhinovirus (40.12%; n = 65), enterovirus (25.31%; n = 41), and respiratory syncytial virus (16.05%; n = 26), whereas streptococcus pneumoniae (17%; n = 29), moraxella catarrhalis (15.43%; n = 25), and haemophilus influenzae (8.02%; n = 13) were the most commonly isolated bacteria. virus pathogens seem more likely to be more prevalent in our settings and were often associated with bacteria and s. pneumoniae (6%; 16) coinfection. respiratory tract infections (rtis) such as acute otitis media, sinusitis, bronchitis, and community-acquired pneumonia are a leading cause of infectious disease-related morbidity, hospitalization, and mortality among children worldwide, particularly in low-income countries. 1 according to world health organization (who), the prevalence of hospitalized children under 5 years with acute respiratory infections (aris) is estimated to be 20% and 90% of those were due to pneumonia. 2 in addition, the number of childhood deaths annually related to aris is very important and is estimated between 1.9 and 2.2 million, and 70% of the deaths took place in africa and southeast asia which are the most. 1, 3 bacteria and viruses have been reported as the main causes of aris. in children under 5 years, aris are caused mainly due to viruses; respiratory syncytial viruses (rsvs), parainfluenza viruses, influenza virus a and b, and human metapneumovirus (hmpv) are the most common viruses isolated. 4, 5 however, primary infections with viral pathogens can predispose to secondary bacterial infections, and the most frequently isolated bacteria in aris include streptococcus pneumonia and haemophilus influenzae. 6 these bacteria were increasingly resistant to the most commonly used antibiotics for ari treatment, 7 leading to increase in mortality rates, hospital durations, and health care-associated costs. 8 in resource-limited countries, bacteria have been the main cause of aris. this could be explained by the inaccessibility of molecular diagnostic tools thus leading to inadequate antibiotics prescription and consequently contributed to a rapid increase in antimicrobial resistance among bacteria causing rtis. 9, 10 in senegal, few studies on viral and bacterial etiologies of rtis are available in pediatric settings. respiratory tract infections are mainly empirically treated with antibiotics on a simple suspicion of bacterial infection. indeed, this could be one of the major causes of high morbidity and mortality rates. the aim of this study was to investigate the viral and/or bacterial infections associated with aris in children under 5 years. this study has been approved by the ethics committee for research of the cheikh anta diop university of dakar. samples and information for questionnaires have been collected after patient's informed consent. bacteria were isolated from appropriate culture media and incubation conditions prior identification following microbiological standard procedures based on morphological, cultural, and biochemical or antigenic characters. strains were identified if the bacterial load was at least 10 5 cfus/ml. data were analyzed using r statistical software version 3.3.1. 11 over a period 1 year, 162 children with aris were included in this study including 97 (59.9%) men. overall, 30 children were under 6 months of age, 26 were 6 to 12 months old, 31 were 12 to 24 months old, 53 were 24 to 60 months old, and 23 were 60 to 112 months old. table 1 shows the distribution of major viruses isolated during this study period, the frequency of single or coinfection, and the number of infections stratified by age group. among selected virus species, adenovirus was the most prevalent (50%, n = 81), followed by influenza virus (45.68%, n = 74), rhinovirus (40.12%, n = 65), enterovirus (25.31%, n = 41), and rsv (16.05%, n = 26). single infection with adenovirus was very rare (3.7%, n = 6). however, adenovirus was associated with other viruses and bacteria in 25.31% (n = 41) and 4.94% (n = 8), respectively. in addition, adenovirus/other viruses/bacteria coinfections have been detected in 16.05% (n = 26) of children. influenza virus infections were associated with single-virus coinfections (33.3%, n = 54) or virus and bacteria coinfections (12.35%, n = 20). however, influenza virus single-infected children were not detected. low prevalence of both rhinovirus and enterovirus single infections was observed (1.85%, n = 3). these infections were most often associated with single virus or virus and bacteria coinfections. adenovirus, influenza virus, and enterovirus infections were more prevalent among children aged from 24 to 60 months. the prevalence of rsv infections associated with other viruses or other viruses and bacteria was 19.14% and 1.85%, respectively; however, no rsv single infection was detected. children aged from 0 to 6 months were more likely to be more affected with rhinovirus and rsv infections. the distribution of major bacterial clinical strains is shown in table 2 . among isolated bacterial species, s. pneumoniae was the most prevalent bacteria (17%) followed by m. catarrhalis (15.43%) and h. influenzae (8.02%). bacterial single infections were also very rare: s. pneumoniae (2%), m. catarrhalis (2%), and h. influenzae (1%). no bacteria/bacteria coinfections were detected. however, coinfections with bacteria and viruses had been observed in children having aris and coinfection with s. pneumoniae (16.66%) was most frequent followed by m. catarrhalis (14, 19%) and h. influenzae (4.32%). the contribution of viral and bacterial pathogens to the clinical syndrome is depicted in tables 3 and 4 , respectively. acute respiratory infections may be caused by viruses, bacteria, or both. thus, determination of the causative agent is crucial to limit the abuse of antibiotics. in our study conducted over a period of 1 year, we showed that viruses were the most common pathogens detected in aris. similar findings have also been reported in a study conducted in united states by obasi et al 12 in 2013. several studies conducted in other countries such as burkina faso, 10 zambia, 9 and niger 13 provided evidence of the role of viruses in upper and lower airway diseases. in senegal, adenovirus was the most common pathogen detected from aris in children. this study confirms previous results reported between 2013 and 2015 in senegal by niang et al. 14 the overall detection rate of adenovirus was 50% among the 162 selected children. this adenovirus infection prevalence seems very high compared with rates documented from other countries, namely, ghana, 15 burkina, 10 and zambia. 9 however, our results are in agreement with data reported in cameroon. overall, this confirms the overall high prevalence of adenovirus ari in africa. 16 in addition to adenovirus, influenza virus, rhinovirus, and enterovirus are the most common viruses detected, as reported in other studies. 10, 17 our study revealed viral codetection is frequent in aris. similar results were found in other countries. 9,10,15 viral codetection in clinical settings is becoming more common since the introduction of molecular-based multiplex tests. although the clinical significance of these findings remains unclear, this seems to have no impact in disease severity. 18 in this study, we also found that aris were associated with bacterial pathogens; among those, s. pneumoniae (17.9%), m. catarrhalis (15.43%), and h. influenzae (8%) were the most common. in contrast with our findings, high rate (56%) of s. pneumoniae detection was observed in niger among children having aris. 13 in our study, prevalence of mono-infections with s. pneumoniae (2%), m. catarrhalis (2%), or h. influenzae (1%) was very low. thus, this low prevalence of bacterial pathogens in aris proves that antibiotics should no longer be systematically used in the treatment of aris. however, among low-prevalence bacterial isolates, codetections with viruses were more frequent and s. pneumoniae (16, 6%) coinfection was the most frequent in our findings. similar results were reported in other studies. 19, 20 there results highlight the pivotal role of viruses in aris because primary infection with viral pathogens can predispose children to subsequent bacterial infections. 21 our study has limitations; among others, the sample size which does not really allow to determine the direct association between viral or bacterial infection and disease severity. moreover, a case-control study would be more suitable to determine the exact role of viruses in aris pathogenesis. in conclusion, this study reports the profile of viral and bacterial pathogens among children under 5 years hospitalized with aris in senegal. the high prevalence rates of viral infections and its clinical impact highlight the need to implement a systematic surveillance program for a better management of aris in children, particularly in resource-limited settings. estimates of wide distribution of child deaths from acute respiratory infections world health organization. acute respiratory infections in children rapid simultaneous diagnosis of infections with respiratory syncytial viruses a and b, influenza viruses a and b, and human para influenza virus types 1, 2 and 3 by multiplex quantitative reverse transcription polymerase chain reaction-enzyme hybridization assay (hexaplex) development of three multiplex rt-pcr assays for the detection of 12 respiratory rna viruses acute bacterial sinusitis complicating viral upper respiratory tract infection in young children antibiotic resistance of bacteria responsible of acute respiratory tract infections in children attributable hospital cost and length of stay associated with health care-associated infections caused by antibiotic-resistant gram-negative bacteria identification of viral and bacterial pathogens from hospitalized children with severe acute respiratory illness in lusaka, zambia viral aetiology of respiratory tract infections in children at the paediatric hospital in ouagadougou (burkina faso) r: a language and environment for statistical computing. r foundation for statistical computing detection of viral and bacterial pathogens in acute respiratory infections viral and bacterial aetiology of severe acute respiratory illness among children <5 years of age without influenza in niger respiratory viruses in patients with influenza like illness in senegal: focus on human respiratory adenoviruses respiratory viruses in children hospitalized for acute lower respiratory tract infection in ghana viral etiology of severe acute respiratory infections in hospitalized children in cameroon viral aetiology of respiratory infections in children in south-western saudi arabia using multiplex reverse transcriptase polymerase chain reaction clinical disease severity of respiratory viral co-infection versus single viral infection: systematic review and analysis viral and bacterial detection in acute respiratory infection in children under five years bacterial and viral etiology in hospitalized community acquired pneumonia with molecular methods and clinical evaluation high nasopharyngeal pneumococcal increased by viral co-infection, is associated with invasive pneumococcal pneumonia the authors are grateful to cea-samef (dakar, senegal), micro csb system (dakar, senegal), and institute pasteur dakar (dakar, senegal) who have contributed to the success of this study. key: cord-316956-nnqi0dj1 authors: gamiño‐arroyo, ana e.; moreno‐espinosa, sarbelio; llamosas‐gallardo, beatriz; ortiz‐hernández, ana a.; guerrero, m. lourdes; galindo‐fraga, arturo; galán‐herrera, juan f.; prado‐galbarro, francisco j.; beigel, john h.; ruiz‐palacios, guillermo m.; noyola, daniel e. title: epidemiology and clinical characteristics of respiratory syncytial virus infections among children and adults in mexico date: 2016-08-18 journal: influenza other respir viruses doi: 10.1111/irv.12414 sha: doc_id: 316956 cord_uid: nnqi0dj1 background: respiratory syncytial virus (rsv) is a leading etiological agent of acute respiratory tract infections and hospitalizations in children. however, little information is available regarding rsv infections in latin american countries, particularly among adult patients. objective: to describe the epidemiology of rsv infection and to analyze the factors associated with severe infections in children and adults in mexico. methods: patients ≥1 month old, who presented with an influenza‐like illness (ili) to six hospitals in mexico, were eligible for participation in the study. multiplex reverse‐transcriptase polymerase chain reaction identified viral pathogens in nasal swabs from 5629 episodes of ili. patients in whom rsv was detected were included in this report. results: respiratory syncytial virus was detected in 399 children and 171 adults. rsv a was detected in 413 cases and rsv b in 163, including six patients who had coinfection with both subtypes; 414 (72.6%) patients required hospital admission, including 96 (16.8%) patients that required admission to the intensive care unit. coinfection with one or more respiratory pathogens other than rsv was detected in 159 cases. young age (in children) and older age (in adults) as well as the presence of some underlying conditions were associated with more severe disease. conclusions: this study confirms that rsv is an important respiratory pathogen in children in mexico. in addition, a substantial number of cases in adults were also detected highlighting the relevance of this virus in all ages. it is important to identify subjects at high risk of complications who may benefit from current or future preventive interventions. respiratory syncytial virus (rsv) is the major infectious cause of lower respiratory tract illness in infants and young children around the world. 1,2 it has also been recognized as an important etiologic agent of pneumonia and other respiratory tract infections in adults and elderly patients. 3, 4 the clinical presentation of this infection varies widely, from mild upper respiratory tract disease to bronchiolitis and pneumonia. 5 this virus is responsible for the majority of bronchiolitis cases and causes approximately 50% of pneumonia cases during the first years of life. 6 in children, host factors such as young age, prematurity, and chronic cardiopulmonary diseases have been associated with severe disease. in addition, other factors such as lower socioeconomic status, exposure to cigarette smoke, air pollution, crowded households, and the lack of breastfeeding have also been associated with severe disease. 7 viral factors associated with virulence leading to severe disease are not sufficiently understood. 8 human rsv is a member of the paramyxoviridae family. outbreaks of rsv infections occur between fall and spring in temperate climates and tend to last up to 5 months. 9,10 rsv isolates can be divided into two groups: group a and group b based on antigenic and genetic characteristics. 8 these two groups cocirculate in the human population, with group a being more prevalent. several studies have compared the severity of disease between infants infected with rsv group a and group b with mixed results. most studies have not found significant clinical differences between both subtypes. 8 despite the recognized importance of rsv as a cause of respiratory illness, the information regarding the epidemiology of this virus in latin america, particularly among adults, is limited. 11 in the present study, 570 cases of rsv infection identified during four epidemic years in mexico were evaluated to clarify the epidemiology of this infection and to assess the possible variations in demographic and clinical characteristics according to viral groups. results from patients enrolled during the first year of the study have previously been described. 6 in this report, we analyzed the characteristics of 570 patients (399 children and 171 adults) with confirmed rsv infection included in the study during a 4-year period. patients ≥1 month old who presented with an ili to any of the participating hospitals were eligible for participation in the study. ili was defined by the presence of at least one respiratory symptom (e.g., shortness of breath, nasal congestion, and cough) and one of the two following criteria: (i) fever ≥38°c on examination, or self-reported fever, or feverishness in the past 24 hours; (ii) one or more nonrespiratory symptoms (e.g., malaise, headache, myalgia, or chest pain). in order to rule out a nosocomial infection, patients who had been hospitalized for more than 48 hours at the time of symptom onset were excluded from the study. subjects were interviewed and examined at the time of enrollment, a nasopharyngeal swab was obtained for the detection of respiratory pathogens, and a blood sample for the complete blood counts and chemistry analysis were obtained. when available, the results of other tests obtained for standard clinical care were extracted from medical records. subjects were evaluated again on day 28 after enrollment, and follow-up information was also obtained by a telephone call on day 14 after the enrollment. at each follow-up visit, clinical information (symptoms, rehospitalizations, and death) was assessed. these visits allowed the ascertainment of the final disposition of the study patients (outpatient, emergency room, hospital ward, or intensive care unit [icu]). nasopharyngeal swab specimens were collected from enrolled patients, placed in a tube with viral transport media, and maintained under refrigeration until they were sent to the molecular biology verbal consent was obtained from parents/legal tutor and subjects before screening to determine eligibility. once a patient was determined to be eligible for study participation, written informed consent and assent (for children older than 8 years) were obtained. the protocol was approved by the institutional review board of each hospital. means and standard deviation were used to summarize the quantitative variables, while frequencies and percentages were used for the qualitative variables. comparisons between groups were made using student's t-test for quantitative variables and the chi-squared or fisher's exact test for qualitative variables. multivariate logistic regression analysis was used to determine the factors associated with hospitalization and icu admission. data were analyzed with the use of pspp and openepi. a p value <.05 was considered as statistically significant. between 2010 and 2014, there were 5662 subjects enrolled in the ili-002 study; 33 patients did not have respiratory samples available f i g u r e 1 respiratory syncytial virus infections in subjects included during four seasons in the ili-002 study f i g u r e 2 respiratory syncytial virus seasonality according to the viral subtype and presence of coinfection for viral testing, and therefore, the final sample size for analysis was 5629 subjects. of these subjects, there were 570 (10.7%) cases with rsv infection: 407 with rsv a infection, 157 with rsv b, and six in whom both rsv a and b were detected (fig. 1) . rsv cases were detected throughout the study period. however, a marked seasonality was observed with a high number of cases during the fall and winter and a decrease in detections during spring and summer (fig. 2) . rsv a was the predominant virus between 2010 and 2012, but in seasons between 2012 and 2014, both rsv subtypes circulated simultaneously. the characteristics of patients with rsv infection were compared to those of all other patients enrolled in the ili-002 study ( table 1) . the age group affected more frequently by rsv was that of children 0-5 years of age; 66.84% of rsv infections occurred in children in this age group, while 30% occurred in adults and 3.16% in children 6-17 years of age; 24.6% (381/1550) of respiratory tract infections in children <5 years were caused by rsv as compared to 4.7% (18/379) of children older than 5 years and 4.6% (171/3700) of adults. also, the proportion of cases seen as outpatients was lower in patients with rsv infection compared to those in which this virus was not detected, while the proportion of icu admissions was higher (table 1) . in 159 (28%) patients, another pathogen was detected in addition to rsv. in 136 of these patients, there was one additional pathogen detected, while in 22 cases there were two, and in one case, there were three; these included rhinovirus (n=61), coronaviruses (n=30), parainfluenza viruses (n=17), influenza a (n=17), influenza b (n=12), adenovirus (n=12), metapneumovirus (n=11), bordetella pertussis (n=11), mycoplasma (n=6), and bocavirus (n=6). the proportions of infections caused by each rsv subtype and the presence of coinfections, according to the age group, are shown in fig. 3 . we compared the characteristics of infections caused by rsv a and rsv b to assess whether there were significant clinical differences between them (table 2) ; the six cases with coinfection with rsv a and b were excluded from this analysis. we did not find any differences in the medical history between patients with rsv a and rsv b infections. we also compared the frequency of each symptom between patients with rsv a and rsv b infections; no significant differences were observed for any of the symptoms that were recorded (including fever, cough, sore throat, fatigue, headache, myalgias, eye symptoms, sneezing, rhinorrhea, shortness of breath, nausea, vomiting, diarrhea, confusion, malaise, and irritability) (data not shown). in addition, we compared the clinical characteristics of patients with and without coinfection with other pathogens (table 3) . differences in the age group distribution were noted between cases with and without coinfection with other pathogens: the proportion of pediatric cases was higher for those infections caused by rsv only, while a larger proportion of coinfections was noted in adults. most symptoms were as frequent in patients with infections caused by rsv only compared to those with coinfections (data not shown); however, there were differences in the frequencies of some symptoms: we present the characteristics of rsv infections detected in a prospective, multicenter study of children and adults seeking clinical care. our data obtained over four seasons provide a description of the epichildren with rsv infection in whom other pathogens were detected were hospitalized less frequently than those infected by rsv only. however, among hospitalized children, the presence of another respiratory pathogen was associated with admission to the icu. in order to interpret these results, it is important to take into account that there was a wide variety of coinfecting pathogens and that in some cases two and even three viruses, in addition to rsv, were detected. one of the main objectives of the study was to identify the factors associated with severe disease leading to rsv hospitalization in our country. approximately 40% of subjects with rsv infection that were enrolled in the study had at least one underlying disorder. the most frequent conditions included congenital malformations/congenital syndromes, cardiovascular disorders, chronic lung disease, and asthma. we observed significant differences in the factors associated with hospitalization between children and adults; while multivariate logistic regression analysis indicated that young age was the main variable associated with hospitalization in children, adults showed an association between additional factors and the requirement of hospitalization. in this age group, multivariate logistic regression analysis showed an association between rsv infection and age, the presence of any underlying condition, and asthma. in their global study of respiratory tract viral infections in elderly adults, falsey et al. 15 found that congestive heart failure, other cardiopulmonary diseases, and noninflammatory cerebrovascular/neurological disorders appeared to be associated with rsv infection; however, on multivariate analysis, no specific factors were associated with this virus. in the group of children, multivariate logistic regression analysis showed an association between the risk of icu admission and the presence of coinfection with other respiratory pathogens, cardiovasother limitations include the fact that the presence of underlying conditions (including history of prematurity and other chronic diseases) was based on patient self-report, which might not be totally reliable. also, we did not collect data on palivizumab use which might be relevant in infants; however, access to this prophylaxis is limited in mexico because of the high cost and, therefore, the number of patients that may have received it is likely to be low. on the other hand, strengths of our study include the participa instituto nacional de pediatría: beatriz llamosas-gallardo hospital infantil de méxico federico gómez juana del carmen báez national institute of allergies and infectious disease (niaid): cliff lane, mary smolskis, dean follmann, sally hunsberger la red is funded by the mexico ministry of health and the u.s. national institute of allergy and infectious diseases viral etiology of hospitalized acute lower respiratory infections in children under 5 years of age -a systematic review and meta-analysis respiratory syncytial virus is an important cause of community-acquired lower respiratory infection among hospitalized adults rates of hospitalizations for respiratory syncytial virus, human metapneumovirus and influenza virus in older adults respiratory syncytial virus infection: immune response, immunopathogenesis, and treatment clinical characteristics and outcomes of influenza and other influenza-like illnesses in mexico city performance of different mono-and multiplex nucleic acid amplification tests on a multipathogen external quality assessment panel viral and host factors in human respiratory syncytial virus pathogenesis global burden of acute lower respiratory infections due to respiratory syncytial virus in young children: a systematic review and meta-analysis respiratory syncytial virus epidemics: the ups and downs of a seasonal virus systematic review and meta-analysis of respiratory syncytial virus infection epidemiology in latin america world health organization. cdc protocol of realtime rtpcr for swine influenza a(h1n1) viral infections in adults hospitalized with community-acquired pneumonia: prevalence, pathogens, and presentation respiratory syncytial virus-and human metapneumovirus-associated emergency department and hospital burden in adults respiratory syncytial virus and other respiratory viral infections in older adults with moderate to severe influenza-like illness respiratory syncytial virus pneumonia among the elderly: an assessment of disease burden respiratory syncytial virus: how, why and what to do associations between co-detected respiratory viruses in children with acute respiratory infections viral coinfection in childhood respiratory tract infections pre-existing disease is associated with a significantly higher risk of death in severe respiratory syncytial virus infection epidemiological and clinical data of hospitalizations associated with respiratory syncytial virus infection in children under 5 years of age in spain: five multicenter study respiratory syncytial virus genotypes and disease severity among children in hospital correlation between respiratory syncytial virus genotype and severity of illness sequencing and analysis of globally obtained human respiratory syncytial virus a and b genomes epidemiology and clinical characteristics of respiratory syncytial virus infections among children and adults in mexico ili-002 study principal investigators, coprincipal investigators, study staff, niaid, leidos and westat staff include the following: instituto nacional de ciencias médicas y nutrición salvador zubirán: guillermo d. e. noyola has participated as a member of the speakers' bureau of abbvie. the rest of the authors declare that they have no relevant conflicts of interest to report. key: cord-294155-94skyx5f authors: terrosi, chiara; fabbiani, massimiliano; cellesi, carla; cusi, maria grazia title: human bocavirus detection in an atopic child affected by pneumonia associated with wheezing date: 2007-08-07 journal: j clin virol doi: 10.1016/j.jcv.2007.06.011 sha: doc_id: 294155 cord_uid: 94skyx5f background: human bocavirus (hbov) is a newly discovered human parvovirus. hbov was detected in respiratory samples by pcr, but its aetiologic role in the pathogenesis of acute respiratory infectious diseases is still unclear. results: in this report, we describe an atopic child affected by pneumonia, with a past history of wheezing. a panel of bacteria and respiratory viruses were searched in the nasopharyngeal swab, only human bocavirus was detected by pcr. conclusions: detection of hbov, as the only microbial agent, in samples from children with wheezing and acute respiratory diseases supports the assumption that this emerging virus could have an aetiologic role in the pathogenesis of respiratory diseases. screening of most of the known respiratory viruses by pcr has allowed us to diagnose many viral infections in the majority of individuals with respiratory illnesses. however, many respiratory infections still remain undiagnosed. a new parvovirus, the human bocavirus (hbov), has recently been discovered by the application of random pcr/cloning technique on respiratory samples (allender et al., 2005) . hbov is suspected to be an etiologic agent of respiratory disease in humans (manning et al., 2006; maggi et al., 2007; kesebir et al., 2007) . respiratory distress and abnormal radiographic chest findings have frequently been associated with hbov. however, its causative role is still unclear. most viruses have been identified by animal experiments or virus replication in tissue culture, but this virus was discovered by molecular screening of respiratory tract samples and it has not yet been grown in cell culture. although, koch's postulates have * corresponding author. tel.: +39 0577 233871; fax: +39 0577 233870. e-mail address: cusi@unisi.it (m.g. cusi). provided a standard for establishing a causal link between a pathogen and disease, in this case it was not possible to apply these rules. however, the frequent association of hbov infection with respiratory tract disease (anderson, 2007; schenk et al., 2007; simon et al., 2007) led us to consider this virus as a causative agent for respiratory tract diseases. some studies have shown that hbov is distributed all over the world (manning et al., 2006; maggi et al., 2007; simon et al., 2007; ma et al., 2006; bastien et al., 2007) and recent data have shown that it can also be detected in stool samples from children with gastroenteritis (vicente et al., 2007) . like other viruses, it is possible that hbov could be involved in both respiratory and enteric diseases. in this report, we describe a case of pneumonia and severe wheezing associated with hbov dna in the pharyngeal swab sample from a child. informed consent was obtained from the parents of the child who provided specimens. the child had been hospitalized at the age of 1 year due to rhinitis, airflow obstruction and acute wheezing. he was treated with corticosteroids and inhalative bronchodilatators to control bronchocostriction. no antibiotics were administered. no causative agent was found in nasopharyngeal swab or serological tests and no sign of chronic lung disease was present when he was released. during the following year, he frequently suffered from respiratory tract infections associated with bronchoconstriction. in order to understand the cause of bronchoconstriction, total and specific ige were measured and high values were reported for egg proteins. he was 3 years old when admitted for the most recent episode of respiratory distress. clinical examination revealed a weakened general condition, a body temperature of 38 • c, tachycardia (pulse rate 140 per min), tachypnea (respiratory rate 52 per min), dyspnea, subcostal retractions, wheezing and left apical fine rales upon lung auscultation. laboratory tests showed a leukocyte count of 22.0 × 10 9 l −1 , and c-reactive protein and erythrocyte sedimentation rate were normal. chest radiography, interpreted by a paediatric radiologist, revealed hyperinflation and pneumonic infiltrates of the upper left lobe. transcutaneously measured oxygen saturation was decreased to 82% while breathing ambient air and the patient required oxygen supplementation (8 l/min) and inhalative adrenaline. oxygen was given for an additional 3 days along with intravenous corticosteroids and salbutamol by inhalation for persistent bronchoconstriction. the child improved and was dismissed from hospital after 7 days. to assess the aetiology of this respiratory disease, blood and nasopharyngeal swab were drawn from the patient upon admission and tested for the presence of viral, bacterial and fungal pathogens. no infectious agent was detected by bacterial or fungal culture. the nasopharyngeal swab was also tested by pcr for chamydia pneumoniae, mycoplasma pneumoniae, bordetella pertussis and legionella pneumophila, respiratory syncytial virus (types a and b), metapneumovirus, influenza viruses (types a-c), parainfluenza viruses (piv-1, piv-2, piv-3 and piv-4), rhinovirus, enterovirus, adenovirus, coronavirus (hcov-oc43, hcov-229e, nl63 and hku11) and human bocavirus. the only positive result was obtained for hbov, using the primers described by simon et al. (2007) . briefly, 5 l of dna (extracted from 200 l of sample by using qiaamp mini kit; qiagen, milan, italy) was amplified using the forward primer 5 cccaagaaacgtcgtctaac 3 (hbov nt 2301-2320) and the reverse primer 5 gtgttgact-gaatacagtgt 3 (hbov nt 2681-2700), producing a fragment of 399 bp, partly overlapping the np1 gene. the cycling conditions were: an initial step at 94 • c for 5 min, followed by 40 cycles at 94 • c for 40 s, 48 • c for 40 s and 72 • c for 1 min; and a final incubation at 72 • c for 5 min. the sensitivity of this pcr was very high, detecting up to 10 copies of hbov genome (fig. 1) . virus detection was confirmed by sequence analysis of the pcr product. another specimen drawn from the child one month after he was discharged from the hospital was negative for hbov by pcr. the detection of hbov in the nasopharyngeal swab in a child with a clinical picture of pneumonia supports the assumption that this virus has a causative role in respiratory diseases. the fact that it has been detected in children with or without respiratory symptoms could demonstrate that this virus might become more aggressive or overgrow, causing disease in particular hosts. consequently, even the virus load could be an important parameter to be considered in the pathogenesis of the disease. we could not carry out a quantitative pcr due to the inadequacy of the sample and we did not have further samples from the child for a retrospective investigation. however, by a semi-quantitative assay, the sample had more than 10 5 genome equivalents per millilitre (fig. 1) . in this case, it should be noted that the patient was suffering from wheezing and atopy characterized by a t cell driven airway inflammatory process, making the host an excellent substrate for viral growth. no coinfection was revealed, indicating that the presence of hbov, the only agent in the sample, could not justify a pneumonia episode just as an innocent bystander. it is also possible that this virus, frequently detected in subjects with asthma or wheezing, could be reactivated by inflammatory processes and that this reactivation could cause severe respiratory diseases, particularly in children. this report is a further confirmation of other published data showing that hbov could be a causative agent of lower respiratory infections in young children. nevertheless, future studies are necessary to establish the pathological role of this virus in respiratory diseases. cloning of human parvovirus by molecular screening of respiratory tract samples human bocavirus: a new viral pathogen detection of human bocavirus in canadian children in a 1-year study human bocavirus infection in young children in the united states: molecular epidemiological profile and clinical characteristics of a newly emerging respiratory virus epidemiological profile and clinical associations of human bocavirus and other human bocavirus and other parvoviruses human bocavirus in italian patients with respiratory diseases detection of human bocavirus in japanese children with lower respiratory tract infections human bocavirus dna detected by quantitative real-time pcr in two children hospitalized for lower respiratory tract infection detection of bocavirus dna in nasopharyngeal aspirates of a child with bronchiolotis human bocavirus, a respiratory and enteric virus key: cord-296605-p67twx7a authors: lau, arthur chun-wing; yam, loretta yin-chun; so, loletta kit-ying title: management of critically ill patients with severe acute respiratory syndrome (sars) date: 2004-03-10 journal: int j med sci doi: nan sha: doc_id: 296605 cord_uid: p67twx7a severe acute respiratory syndrome (sars) is frequently complicated with acute respiratory failure. in this article, we aim to focus on the management of the subgroup of sars patients who are critically ill. most sars patients would require high flow oxygen supplementation, 20–30% required intensive care unit (icu) or high dependency care, and 13–26% developed acute respiratory distress syndrome (ards). in some of these patients, the clinical course can progress relentlessly to septic shock and/or multiple organ dysfunction syndrome (mods). the management of critically ill sars patients requires timely institution of pharmacotherapy where applicable and supportive treatment (oxygen therapy, noninvasive and invasive ventilation). superimposed bacterial and other opportunistic infections are common, especially in those treated with mechanical ventilation. subcutaneous emphysema, pneumothoraces and pneumomediastinum may arise spontaneously or as a result of positive ventilatory assistance. older age is a consistently a poor prognostic factor. appropriate use of personal protection equipment and adherence to infection control measures is mandatory for effective infection control. much of the knowledge about the clinical aspects of sars is based on retrospective observational data and randomized-controlled trials are required for confirmation. physicians and scientists all over the world should collaborate to study this condition which may potentially threaten human existence. in 2003, an outbreak of severe acute respiratory syndrome (sars) caused by the sars-associated coronavirus involved 26 countries and 8098 patients, resulted in 774 deaths [1] . thereafter, sars has re-emerged sporadically in both laboratory and community settings. its clinical spectrum varies from minimal respiratory symptoms to severe respiratory failure. we have previously contributed to an overview on the contemporary treatment of sars [2] , and the whole topic has also been reviewed elsewhere [3] . in this article, we aim to focus on the management of a subgroup of critically ill sars patients with more significant respiratory failure. critically ill sars patients frequently demonstrate the following clinical features: persistent pyrexia (occasionally from admission but often recurring after an initial period of defervescence), tachycardia (infrequently bradycardia), tachypnoea and significant oxygen desaturation. more than onethird of all the sars patients required high flow oxygen therapy [4] , 20-30% required intensive care unit (icu) admission or high dependency care, and 13-26% developed acute respiratory distress syndrome (ards) [5, 6] . the clinical course of some of these patients can progress relentlessly irrespective of all attempts at pharmacological treatment, eventually resulting in septic shock and/or multiple organ dysfunction syndrome (mods). lymphocytopaenia, neutrophilia and thrombocytopenia are frequently seen in critically ill sars patients. neutrophilia could be due to sars per se, to superimposed infection or related to corticosteroid administration. pancytopaenia, if present, could be due to haemophagocytosis syndrome [7] or reactivation of latent human parvovirus (unpublished data). prolonged activated partial thromboplastin time and picture of disseminated intravascular coagulation has been reported [8] . coinfections with other agents including chlamydia-like agents [9] , metapneumovirus [10] or influenza virus (unpublished data) have been reported. persistent and increasing elevations of creatine kinase, lactate dehydrogenase, and transaminases levels are common [11, 12, 13] . associated lung damage is believed to be the result of a virally-triggered inflammatory reaction mediated by a host of cytokines [14, 15] . in sicker patients, levels of pro-inflammatory cytokines (il-1beta, il-6, il-8, il-16, tnf-α) and tgf-β1 were higher, with slower decline on clinical recovery [14] . radiographic abnormalities in the chest usually progress upwards from initial unilateral or bilateral lower-to mid-zone peripheral ground-glass shadows, to focal, multifocal or diffuse consolidation. peak radiographic changes occurred at 8.6 days after fever onset, with 17.4% showing two peaks at 6.3 and 13.5 days, and 4% showing relentless progression [16] . cavitation is rare but may be associated with superimposed infection in patients with a prolonged illness course and who are mechanically ventilated [17] . high-resolution computer tomography (hrct) of the thorax showed focal ground-glass and scattered "crazy paving" patterns at presentation, followed by development of interstitial thickening, consolidation, pleural reaction, and scarring and fibrosis in later stages [18, 19] . small (<1 cm) pulmonary cysts may be detected even if the patient is not receiving ventilatory assistance [19] . subcutaneous emphysema, pneumothoraces or pneumomediastinum are distinct complications of severe sars [18] . hrct features of late-stage ards caused by sars are similar to those arising from other causes [19] . lung biopsy and postmortem studies [20, 21] showed acute-phase diffuse alveolar damage (dad), airspace edema, bronchiolar fibrin, increased numbers of interstitial macrophages (with focal haemophagocytosis) and alveolar macrophages in patients with shorter duration (<10 days) of illness. on the other hand, histology after >10days of illness showed organizing-phase dad with increased fibrosis, hyperplasia of type ii pneumocyte, squamous metaplasia, multinucleated giant cells, and acute bronchopneumonia [20] . in patients who died late in the course of this disease, high loads of viral rna were detectable by reverse transcriptase polymerase chain reaction (rt-pcr) in the lungs, bowel, lymph nodes, spleen, liver, and kidneys [22] . general principles anti-bacterial therapy for community-acquired pneumonia in accordance with standard guidelines [23] should always be administered before laboratory confirmation of sars-cov infection. where effective anti-viral therapy is available, it should be started as early as possible after diagnosis, and even empirically if suspicious clinical features and especially epidemiological links are present. since critically ill patients are deemed to have already progressed from the viral replicative phase to the immunopathological phase [5] , concomitant institution of an immunomodulatory therapy should also be considered [11] . since there are no consensus regarding the most optimal treatment regimen in these respects, we will thus review the more commonly used agents and discuss their relative merits based on published reports. when respiratory failure eventually sets in, oxygen supplementation, assisted ventilation and intensive supportive treatments will be required. ribavirin was the most commonly used empirical antiviral agent for sars. it is a broad-spectrum purine nucleoside analogue which inhibits both rna and dna viruses by interfering with nucleic acid synthesis. there is experimental evidence to show that it has immunomodulatory effects in the treatment of mouse coronavirus hepatitis [24] . subsequently, it was found that ribavirin has no direct in vitro activity against sars-cov [25] . higher doses given intravenously resulted in more frequent and severe adverse effects including haemolytic anaemia, elevated transaminase levels and bradycardia [13] . lopinavir-ritonavir co-formulation (kaletra ® , abbott laboratories, usa) is a protease inhibitor for the treatment of human immunodeficiency virus (hiv) infection. it can inhibit the coronaviral proteases, thus blocking the processing of viral replicase polyprotein and preventing the replication of viral rna. ritonavir inhibits lopinavir metabolism thus increasing its serum concentration, but it has no activity against sars-cov. in a retrospective analysis in hong kong [26] , 31 patients who had received kaletra as rescue therapy together with high dose corticosteroids had no difference in rates of oxygen desaturation, intubation and mortality compared with a matched cohort. however, when given as initial treatment in combination with ribavirin in another subgroup of 44 patients, there were significant reductions in the need for rescue pulsed corticosteroid therapy, intubation rate and overall mortality. in addition to the prevalence of diarrhoea among these patients which may render oral drugs more appropriate and useful, synergism between kaletra and ribavirin might have contributed to the benefits since either drug alone has only weak anti-viral activities. another hong kong study of 41 sars patients treated with a combination of lopinavir/ritonavir and ribavirin compared with 111 patients (historical controls) treated with ribavirin only showed that adverse clinical outcomes (ards or death) were significantly lower in the treatment group than in the historical controls at day 21 after symptom onset. further randomised placebo controlled trials are required [27] . interferons are a family of cytokines with important roles in the cellular immune response. interferon α has been used for sars treatment in china and canada [28, 29, 30] . in an open-label uncontrolled study [28] , nine patients treated with corticosteroids plus interferon alfacon-1 (infergen ® , intermune inc., usa) showed better oxygen saturation, faster radiographic resolution and lesser need for supplemental oxygen compared to 13 given corticosteroids alone. in vitro testing showed that interferon β was more potent than interferon α or γ, being effective even when administered after sars-cov infection in cell culture [31] . traditional chinese herbal medicine has been used concomitantly with other drugs to treat sars in mainland china with good results reported [32] . however, its value in critically ill patients has not been reported. glycyrrhizin, an active component derived from liquorice roots, is effective against sars-cov in vitro [25] . its clinical utility remains uncertain. another herbal compound, baicalin, also demonstrates anti-sars-cov activity in vitro (unpublished data). in the absence of an effective antiviral agent in the 2003 outbreak, most physicians had opted to use immunomodulatory agents, most commonly corticosteroids, in the treatment of sars [11, 12, 33, 34] it is generally agreed that corticosteroids should not be used during the early viral replicative phase, and that its administration should best coincide with the onset of the immunopathological phase [5] . clinicoradiological surrogate criteria have been used to indicate the onset of this immune hyperactive phase, thus providing a practical guide to the timing of starting corticosteroids [11] . corticosteroid dosages should be high enough, especially in the severe cases, to abort the cytokine storm, and maintained for long enough to prevent the rebound phenomenon [2, 29, 35] . this may be achieved by using a weightadjusted [11] and radiographic extent-modified dosages [29] for a period of 2-3 weeks. in one-third to half of sars patients, fever may recur while on immunomodulatory treatment due to superimposed infections, too rapid tailing of corticosteroids or persistently severe and uninhibited cytokine storm. empirical anti-pseudomonal antibiotics should then be given first. if there is no apparent clinical response, opportunistic infections like fungal infection should be excluded. if fever is accompanied by obvious respiratory deterioration in the absence of superimposed pulmonary or systemic infection, most patients can be presumed to be suffering from a severe recrudescence of the sars illness. in such critically ill sars patients, further escalation of immunomodulation is warranted. such deterioration could sometimes occur very rapidly; immediate administration of pulsed methylprednisolone therapy at 500-1000 mg per day intravenously for 2 days, followed by tapering doses in the subsequent weeks, has been associated with improved outcome [11, 34] . up to one-third to one-half of critically ill sars patients may benefit from this strategy [4, 33, 34] . because radiographic abnormalities may lag behind clinical improvement, persistent radiographic shadows per se, when accompanied by clinical improvement, do not warrant additional corticosteroids [36] . human gamma immunoglobulins have been used in selected sars patients who continued to deteriorate despite treatment [29, 33] . an igm-enriched immunoglobulin product (pentaglobin ® , biotest pharma gmbh, germany) has been used in hong kong and mainland china [29, 35, 37] . pentaglobin at 5mg/kg/day for three days given to 12 patients who deteriorated despite repeated rescue methylprednisolone and ribavirin therapy had shown some improvement in radiographic scores and oxygen requirement [38] . it has been reported that the use of combined methylprednisolone and highdose intravenous immunoglobulin (0.4g/kg) daily for three consecutive days in 15 probable sars patients with acute lung injury (ali) or ards had resulted in lower mortality and a trend towards earlier recovery [39] . randomized controlled trials in larger numbers of patients are required to confirm its efficacy. based on the assumption that the neutralizing immunoglobulins in convalescent plasma can curb increases in viral load, convalescent plasma collected from recovered sars patients has been used in hong kong to treat severely ill patients not responding to corticosteroids. some clinical benefits were reportedly observed in a small number of patients [40] . despite all efforts, at least 50% of sars patients would still develop acute hypoxemic respiratory failure, with up to 80% requiring supplemental oxygen [37] overall, 20-30% of patients had been admitted into icu, and 10-20% eventually required intubation and mechanical ventilation [4] . both non-invasive and invasive ventilatory support has been applied to critically ill sars patients. niv delivers continuous positive airway pressure (cpap) or bi-level pressure support through a tight-fitting facial or nasal mask. it was commonly employed in many chinese hospitals [29, 31, 37, 41, 42] and our own centre in hong kong [11, 43, 44] . early application may be beneficial because it could rapidly improve vital signs, oxygenation and tachypnoea [41, 43] , and may reduce the need for increasing dosages of corticosteroids for progressive respiratory failure. it could avoid intubation and invasive ventilation in up to two-thirds of critically ill sars patients [29, 32, 43] . use of niv in immunocompromised subjects of other diseases has reported similarly reduced rates of endotracheal intubation and serious complications [45] . niv in sars may be of particular benefit, since high dose corticosteroids per se would already predispose to ventilator-associated pneumonia, and risks to healthcare workers (hcw) could also be markedly reduced through obviating the need for intubation, a potentially highly infectious procedure. patients who respond to niv will usually do so within 24 hours, non-responders who will eventually need endotracheal intubation can thus be identified early [43] . niv is indicated in the presence of ali and early ards when oxygen saturation (spo 2 ) could not improve to more than 93% despite >5 litres per minute of oxygen; persistent tachypnoea of at least 30 breaths per minute; and progressive radiographic deterioration in the lungs [43] . the usual contraindications to niv apply, including impaired consciousness, uncooperative patient, high aspiration risk, and haemodynamic instability [35] . sars-related respiratory failure responds readily to niv given at low pressures. cpap of 4-10 cm h 2 o, or bi-level pressure support with inspiratory positive airway pressure (ipap) of <10 cm h 2 o and expiratory positive airway pressure (epap) of 4-6 cm h 2 o are reasonable starting pressures [43] . higher pressures should be avoided whenever possible, because it may increase the risk of pneumothorax and pneumomediastinum, which are frequently spontaneous complications of sars even without assisted positive pressure ventilation [5] . when patients do not improve within one to two days of niv or continue to deteriorate, or if niv is contraindicated, endotracheal intubation and mechanical ventilation should be considered. most centres [64] adopted a ventilatory strategy similar to that recommended for ards from other causes [46] . both pressure and volume control ventilation may be employed [64] . the tidal volume should be kept low (e.g. 5-6 ml/kg predicted body weight), and plateau pressures maintained below 30 cm h 2 o. because of a higher risk of barotraumas in sars, the lowest positive end-expiratory pressure (peep) which could achieve satisfactory alveolar recruitment and oxygenation, usually 5-6 cm water, should be employed. other adjunctive measures employed in the usual ards cases had been tried in sars, including: prone positioning [64, 47] , high frequency oscillatory ventilation [64, 47] , nitric oxide [47] , high peep and regular lung recruitment [64] , but their efficacy is uncertain. tracheostomy is required in patients requiring prolonged mechanical ventilation and icu stay. strict adherence to infection control guidelines is mandatory in performing tracheostomy in the icu or operating room, as well as during subsequent changes of the tracheostomy tube [48, 49] . critically ill sars patients on high dose corticosteroids and mechanical ventilation are particularly susceptible to superimposed bacterial and opportunistic infections. their peripheral blood cd3+, cd4+ and cd8+ were also lower than normal [8, 24] . ventilator-associated infection with organisms like pseudomonas aeruginosa, methicillin-resistant staphylococcus aureus, acinetobacter baumanii, as well as invasive mucor sp [50] and aspergillosis [50, 51] have been reported. strict control of hyperglycaemia during corticosteroid administration is essential to reduce the chance of septic complications [52] . spontaneous subcutaneous emphysema, pneumothoraces and pneumomediastinum are common complications that are potentially aggravated by noninvasive or invasive ventilation [5] . while chest drain insertion is useful to relieve pneumothoraces, prolonged air leak may sometimes occur. by itself, sars predominantly results in single organ failure of the lungs. other complications reported are more likely the result of sepsis and its attending problems, including acute renal failure (6%), acute liver failure (1%), rhadomyolysis, cardiovascular dysfunction, or of prolonged immobilization and underlying co-morbidities, including deep vein thrombosis, pulmonary embolism, ischaemic strokes, etc [53] . the case-fatality ratio (cfr) of sars has been estimated to range from 0% to >50% depending on the age group affected. the overall cfr is approximately 15% [54] . variability may be due to different host and viral factors as well as treatment strategies. cfr may also be significantly affected by the duration of follow-up and inclusion of different mixes of suspected, probable and laboratory confirmed cases in different series [55] . based on the treatment principles presented above, we have developed a standard treatment protocol early on in the outbreak, comprising initially high (but not pulsed) dose methylprednisolone with tapering over three weeks [11] . this protocol was eventually applied to 88 consecutively admitted sars patients [56] . their mean age was 42 years, with 97% having laboratory-confirmed sars. a low overall mortality of 3.4% (3/88) was obtained, with all three deaths occurring in patients over the age of 65 years. twenty four percent required icu admission: 14% received niv (bi-level pressure support) alone and 10% had both niv and invasive mechanical ventilation. hrct thorax in all survivors taken 50 days after commencement of treatment showed most did not have clinically significant lung scarring. another multi-centered study comparing four treatment regimens in guangzhou, china, also found that a regimen of high dose corticosteroids adjusted according to clinical and radiological severity, coupled with nasal cpap ventilation, produced the best result: zero mortality in all 60 clinically-defined sars patients, mean age 30.5 years. with 40% treated with cpap and none requiring mechanical ventilation. subsequently, very low mortality was again recorded among a further 160 patients treated with the same regimen [29] . many prognostic factors have been reported to independently predict adverse outcome in sars. they include advanced age [4, 57, 58, 59] , diabetes [5, 13, 59] , heart disease [5, 59] , other significant coexisting conditions [53, 59, 60] , shortness of breath on admission [60] , degree of hypoxaemia [58] , high total leukocyte count on admission [4, 12, 60] , high initial lactate dehydrogenase [4, 57, 58] , low platelet counts [58] , and use of pulsed doses of corticosteroid [4, 60] . compared to patients with nasopharyngeal aspirates negative for sars-cov by rt-pcr, pcr-positive ones are more likely to require icu care and mechanical ventilation, develop acute renal failure and die [60] . in particular, mortality was high among icu patients: 28-day icu mortality was variously reported to be 26-37% [42, 64, 65] . older age, severity of illness, lymphocyte count, decreased steroid dose, positive fluid balance, chronic disease or immunosuppression, and nosocomial sepsis were associated with poor icu outcome [65] . patients who had diarrhoea were more likely to require ventilatory support and icu care [60] . higher serum sars-cov concentration in the early stage of the disease was a prognostic indicator for later icu admission [62] . patients presenting with more extensive radiographic involvement also predicted the need for icu care or death [63] . age alone is a consistent and strong prognostic factor in all series. age-stratified death rates were estimated to be <1% in patients below 24 years of age, 6% between 25 and 44 years, 15% between 45 and 64 years, and >50% in elderly patients over 65 years old [66] . corresponding estimates in hong kong were 13% in those below 60 years of age, and 43% in those over 60 years [67] . the cause of death in sars is usually progressive respiratory failure with or without concomitant sepsis. sudden cardiac arrest is also possible, and has been hypothesized to be due to hypoxemia (which would worsen during activities including defaecation), direct viral myocardial injury and extreme anxiety, all of which may lead to electrical instability in the myocardium and induction of arrhythmia [68] . sars is primarily transmitted by direct or indirect contact of mucous membranes (eyes, nose, or mouth) with infectious respiratory droplets or fomites [12, 69] . transmission risks increase with duration and proximity of contact. infection control precautions in the icu are shown in appendix [70] . endotracheal intubation should be considered earlier and in anticipation of impending deterioration, so that ample time is available for preparation. it should be performed by the most skilful airway practitioner in a negative-pressure room behind closed doors. should the operator choose to wear additional personal protective equipment like the airmate hepa powered air purifying respirator system (3m, mn, usa), he/she must be familiar with its mode of operation and the precautions required for gowning and degowning, and must be assisted by a colleague with similar knowledge [11] . a "modified awake" intubation technique has been suggested as the best possible compromise between patient and operator safety by administration of a combination of midazolam, fentanyl and lidocaine until the patient reaches the desired level of sedation [71] . the patient is then paralysed after intubation to minimize coughing. alternatively, the "rapid sequence induction" technique with intravenous administration of midazolam and suxamethonium can also minimize patient coughing. it should however be emphasized that, unless there is prior preparation for a surgical airway, neuromuscular paralysis should be avoided in anticipated difficult intubation in order to maintain spontaneous respiration [71] . both bronchoscopy and niv should be performed in a negative pressure room. although there is widespread fear of infective risk by niv [6, 55] , centres with such experience, including ours, have found that its use is safe if the necessary precautions are taken [11, 29, 42, 43] . finally, strict adherence to infection control measures in the form of strict isolation and effective cohorting, early diagnosis and contact tracing, timely reporting and institution of public health measures, as well as enhancement of environmental ventilation is key components in the effective management of infectious diseases. managing critically ill sars patients is a challenging task. most, if not all, knowledge about the clinical aspects of sars are based on retrospective observational data, and randomized-controlled trials are required for confirmation. physicians and scientists all over the world should collaborate to study this condition which may potentially threaten human existence. summary of probable sars cases with onset of illness from 1 sars reference 3 rd ed. flying publisher the severe acute respiratory syndrome severe acute respiratory syndrome: clinical outcome and prognostic correlates clinical progression and viral load in a community outbreak of coronavirus-associated sars pneumonia: a prospective study icu management of severe acute respiratory syndrome microbiologic characteristics, serologic responses, and clinical manifestations in severe acute respiratory syndrome haematological manifestations in patients with severe acute respiratory syndrome: retrospective analysis chlamydia-like and coronavirus-like agents found in dead cases of atypical pneumonia by electron microscopy human metapneumovirus detection in patients with severe acute respiratory syndrome development of a standard treatment protocol for severe acute respiratory syndrome a major outbreak of severe acute respiratory syndrome in hong kong clinical features and short-term outcomes of 144 patients with sars in the greater toronto area inflammatory cytokine profile in children with severe acute respiratory syndrome dynamic changes in blood cytokine levels as clinical indicators in severe acute respiratory syndrome sars: radiographical appearance and pattern of progression in 138 patients sars: radiological features high-resolution ct findings in patients with severe acute respiratory syndrome: a patternbased approach late-stage adult respiratory distress syndrome caused by severe acute respiratory syndrome: abnormal findings at thin-section ct lung pathology of severe acute respiratory syndrome (sars): a study of 8 autopsy cases from singapore pathological study on severe acute respiratory syndrome fatal sars is associated with multiorgan involvement by coronavirus (sars-cov) guidelines for the management of adults with community-acquired pneumonia: diagnosis, assessment of severity, antimicrobial therapy, and prevention ribavirin inhibits viral-induced macrophage production of tng, il-1, the procoagulant fgl2 prothrombinase and preserves th1 cytokine production but inhibits th2 cytokine response glycyrrhizin, an active component of liquorice roots, and replication of sars-associated coronavirus treatment of severe acute respiratory syndrome with lopinavir/ritonavir: a multicentre retrospective matched cohort study role of lopinavir/ritonavir in the treatment of sars: initial virological and clinical findings interferon alfacon-1 plus corticosteroids in severe acute respiratory syndrome: a preliminary study description and clinical treatment of an early outbreak of severe acute respiratory syndrome (sars) in guangzhou, pr china clinical investigation of outbreak of nosocomial severe acute respiratory syndrome treatment of sars with human interferons our strategies for fighting severe acute respiratory failure management of severe acute respiratory syndrome: the hong kong university experience high dose pulse versus non-pulse corticosteroid regimens in severe acute respiratory syndrome severe acute respiratory syndrome treatment: present status and future strategy chest x-ray changes after discontinuation of glucocorticoids treatment on severe acute respiratory syndrome (5 cases report) a hospital outbreak of severe acute respiratory syndrome in guangzhou, china acute respiratory distress syndrome in critically ill patients with severe acute respiratory syndrome treatment of severe acute respiratory syndrome with convalescent plasma evaluation of non-invasive positive pressure ventilation in treatment for patients with severe acute respiratory syndrome clinical observation of non-invasive positive pressure ventilation (nippv) in the treatment of severe acute respiratory syndrome (sars) effectiveness of non-invasive positive pressure ventilation in the treatment of acute respiratory failure in severe acute respiratory syndrome management of critical severe acute respiratory syndrome and risk factors for death noninvasive ventilation in immunosuppressed patients with pulmonary infiltrates, fever, and acute respiratory failure the acute respiratory distress syndrome network. ventilation with lower tidal volumes as compared with traditional tidal volumes for acute lung injury and acute respiratory distress syndrome critically ill patients with severe acute respiratory syndrome tracheostomy in a patient with severe acute respiratory syndrome safe tracheostomy for patients with severe acute respiratory syndrome lung pathology of severe acute respiratory syndrome (sars): a study of 8 autopsy cases from singapore fatal aspergillosis in a patient with sars who has treated with corticosteroids intensive insulin therapy in critically ill patients outcomes and prognostic factors in 267 patients with severe acute respiratory syndrome in hong kong department of communicable disease surveillance and response. consensus document on the epidemiology of severe acute respiratory syndrome severe acute respiratory distress syndrome (sars): a critical care perspective outcome of coronavirus-associated severe acute respiratory syndrome using a standard treatment protocol outcomes and prognostic factors in 267 patients with severe acute respiratory syndrome in hong kong prognostic factors for severe acute respiratory syndrome: a clinical analysis of 165 cases short term outcome and risk factors for adverse clinical outcomes in adults with severe acute respiratory syndrome (sars) coronavirus-positive nasopharyngeal aspirate as predictor for severe acute respiratory syndrome mortality enteric involvement of severe acute respiratory syndrome-associated coronavirus infection quantitative analysis and prognostic implication of sars coronavirus rna in the plasma and serum of patients with severe acute respiratory syndrome prognostic significance of the radiographic pattern of disease in patients with severe acute respiratory syndrome acute respiratory syndrome in critically-ill patients with severe acute respiratory syndrome short-term outcome of critically ill patients with severe acute respiratory syndrome update 49 -sars case fatality ratio epidemiological determinants of spread of causal agent of severe acute respiratory syndrome in hong kong cardiac arrest in severe acute respiratory syndrome: analysis of 15 cases effectiveness of precautions against droplets and contact in prevention of nosocomial transmission of severe acute respiratory syndrome (sars) sars: ventilatory and intensive care a practical approach to airway management in patients with sars infection control precautions in the icu effective staff education in infection control, emphasizing on • precautions to be used in high-risk procedures and alternative procedures to reduce risks • limit opportunities for exposure, e.g., avoid aerosol generating procedures & limit number of health care workers (hcws) present, alternative nursing practices to limit number of hcws exposed to each patient • effective use of time during patient contact • "gowning" and "degowning" without contamination precautions: disposable gloves, gown, cap • eye protection with non-reusable goggles and face-shield • powered air purification respirators (papr) are optional ppe when performing high-risk procedures • pens, paper, personal items and medical records should where feasible, increase to ≥ 12 ach + re-circulate air through hepa filter • preferred: negative pressure isolation rooms with antechambers • a viral/bacterial filter should be placed at the expiratory port of bag-valve mask • place two filters per ventilator: between expiratory port and the ventilator, and another on the exhalation outlet of the ventilator • use closed-system in-line suctioning for endotracheal/tracheostomy tubes • handle contaminated heat and moisture exchangers (hme) and heated humidifiers carefully • scavenger system for exhalation port of ventilator is optional if negative pressure with high air change (> 12/hour) is achieved • preoxygenate patient and temporarily switch off machine whenever ventilator circuit disconnection is required the authors have declared that no conflict of interest exists. key: cord-268490-e8jub01m authors: moscona, anne title: csi microbiology: emerging pathogens and a staged strategy for detection and discovery date: 2007-12-15 journal: j infect dis doi: 10.1086/524313 sha: doc_id: 268490 cord_uid: e8jub01m nan disease, both by enabling target selection for vaccine and drug development and by guiding patient care. in recent years, there has been substantial progress in applying molecular biologic advances to respiratory virus diagnosis [1] ; nonetheless, a major gap has been the lack of a costeffective, systematic way to identify the causes of respiratory diseases in populations. the article by renwick et al. [2] in this issue of the journal makes 2 unique contributions to this field: it demonstrates the need to consider and identify rhinoviruses as a cause of serious acute respiratory disease in children, and it establishes the masstag polymerase-chainreaction (pcr) multiplex platform as a practical tool for microbial surveillance. the masstag pcr method, developed by renwick et al. [2] and used in their study investigating the etiology of respiratory disease in hospitalized children, provides a paradigm for new detection strategies for early recognition and containment of a wide range of respiratory pathogens. this multiplex technology, in its first evaluation in 2005, was shown to be effective in identifying all the main respiratory viral pathogens, including respiratory syncytial virus, human parainfluenza viruses 1-3, metapneumovirus, influenza, and sars-cov as well as s. pneumoniae and h. influenzae [3] . in 2006, lamson et al. [4] , in the same group, went on to report the use of the method to investigate both undiagnosed influenza-like illness in new york state and the discovery of a novel genetic clade within the picornaviruses; "human rhinovirus new york" was the first new agent to be detected by use of masstag pcr. rhinoviruses have long been a relatively underappreciated cause of acute respiratory infection, a view that is beginning to change [5] . in their study in this issue of the journal, performed in collaboration with the robert koch institute (berlin, germany), renwick et al. [2] report clear evidence that links these viruses to severe respiratory disease: 75% of viruses detected among 97 nasopharyngeal aspirates from children hospitalized with acute respiratory infection-with no pathogen identified by routine methods-were rhinoviruses. independently, in publications following that by lamson et al. [4] , other investigators found similar evidence that associates severe respiratory disease with rhinovirus infection, implicating known serotypes of rhinovirus groups a and b as well as the same, novel viruses found in new york in pediatric lower-respiratory-tract infection [6] or asthma exacerbations [7] . with work now reported by 3 research groups employing different methods, the likely contribution of rhinoviruses to acute respiratory disease cannot be ignored and deserves further study. the diagnostic strength of highly multiplexed systems, such as the masstag pcr platform applied by renwick et al., in the lipkin group, builds on a series of advances by this group [2] . in 1987, the first application of subtractive cloning in microbiology led to the identification of a novel pathogen, borna disease virus, the prototype of a new family in the order mononegavirales [8, 9] . another new strategy-namely, domain-specific differential display pcr-ultimately led to the recognition of west nile virus as the cause of the outbreak of encephalitis in new york in 1999 [10, 11] . the recent development of pathogen microarrays, combined with a comprehensive panmicrobial sequence database, has added to the repertoire of methods for sensitive and broad differential diagnosis of infectious disease [12, 13] and has facilitated differential diagnosis of filovirus versus malaria in samples obtained during an outbreak of hemorrhagic fever. in early 2007, the same research group [14] pioneered the use of unbiased, high-throughput sequencing to assemble a comprehensive inventory of microflora from honeybee colonies affected by colony-collapse disorder [14] . the transition of cutting-edge pathogendiscovery technologies from research to clinical laboratories will not occur immediately. nonetheless, it is not premature to plan for this in the near future. key to implementation will be the development of a staged strategy for pathogen detection that enables low-cost and efficient differential diagnosis of infectious diseases [15] . one approach might be to begin with a multiplex pcr screen, such as the masstag pcrbased assays described here. if the initial screen fails, microarrays could be em-ployed. more labor-and resource-intensive unbiased, high-throughput sequencing would be reserved for the most difficult challenges. considerable emphasis has been placed on ensuring global access to vaccines and drugs for treatment of infectious diseases. less attention has been focused on the importance of understanding microbiological data in the context of public health. to this end, efforts such as those undertaken by the lipkin group, directed at training and equipping an international cast of investigators in state-of-theart methods for molecular diagnostics, are critical. it is no surprise that the list of investigators collaborating on papers from their laboratory often reads like a united nations of science. such worldwide capability in global infectiousdisease surveillance is important to local as well as international security. commitment to technology transfer and global collaboration is essential if we are to have the agility required to keep pace with emerging infectious diseases. pathogen surveillance and discovery can promote global interaction via collaborations on matters that know no national or political boundaries but simply reflect our common humanity. the cell biology of acute childhood respiratory disease: therapeutic implications a recently identified rhinovirus genotype is associated with severe respiratory-tract infection in children in germany diagnostic system for rapid and sensitive differential detection of pathogens masstag polymerase-chain-reaction detection of respiratory pathogens, including a new rhinovirus genotype, that caused influenza-like illness in new york state during rhinovirusassociated hospitalizations in young children characterisation of a newly identified human rhinovirus, hrv-qpm, discovered in infants with bronchiolitis pan-viral screening of respiratory tract infections in adults with and without asthma reveals unexpected human coronavirus and human rhinovirus diversity isolation and characterization of borna disease agent cdna clones genomic organization of borna disease virus identification of a kunjin/west nile-like flavivirus in brains of patients with new york encephalitis genetic analysis of west nile new york 1999 encephalitis virus panmicrobial oligonucleotide array for diagnosis of infectious diseases detection of respiratory viruses and subtype identification of influenza a viruses by greene-chipresp oligonucleotide microarray a metagenomic survey of microbes in honey bee colony collapse disorder pathogen surveillance and discovery key: cord-268324-86a0n0dc authors: charitos, ioannis a; ballini, andrea; bottalico, lucrezia; cantore, stefania; passarelli, pier carmine; inchingolo, francesco; d'addona, antonio; santacroce, luigi title: special features of sars-cov-2 in daily practice date: 2020-09-26 journal: world j clin cases doi: 10.12998/wjcc.v8.i18.3920 sha: doc_id: 268324 cord_uid: 86a0n0dc the severe acute respiratory syndrome-coronavirus-2 (commonly known as sars-cov-2) is a novel coronavirus (designated as 2019-ncov), which was isolated for the first time after the chinese health authorities reported a cluster of pneumonia cases in wuhan, china in december 2019. optimal management of the coronavirus disease-2019 disease is evolving quickly and treatment guidelines, based on scientific evidence and experts’ opinions with clinical experience, are constantly being updated. on january 30, 2020, the world health organization declared the sars-cov-2 outbreak as a "public health emergency of international concern". the total lack of immune protection brought about a severe spread of the contagion all over the world. for this reason, diagnostic tools, patient management and therapeutic approaches have been tested along the way, in the desperate race to break free from the widespread infection and its fatal respiratory complications. current medical knowledge and research on severe and critical patients’ management and experimental treatments are still evolving, but several protocols on minimizing risk of infection among the general population, patients and healthcare workers have been approved and diffused by international health authorities. the severe acute respiratory syndrome-(sars-) coronavirus 2 (cov-2), officially named as "coronavirus disease-2019 (covid-19)" by the world health organization (who) on february 11, 2020, is actually the novel coronavirus responsible for one of the most severe worldwide pandemics in recent history [1] . on january 9, 2020, the chinese center for disease control and prevention (cdc) reported and confirmed that a cluster of cases of acute pneumonia in people associated with the huanan seafood wholesale market in wuhan, a city in the hubei province of china, were caused by a novel coronavirus, 2019-ncov [1] [2] [3] . from that day, the epidemic spread throughout china, being followed by a rapidly increasing number of cases in other countries throughout the world, with a high morbidity and mortality ratio [4, 5] . on 11 march, 2020, the director-general of the who declared covid-19 a global pandemic [6] . the coronavirus subfamily, nidovirales order, includes four genera: alpha, beta, gamma, and delta coronaviruses. they are medium-sized, enveloped, positivestranded rna viruses which replicate using a nested set of mrnas [7, 8] . their genetic material represents the largest known viral rna genomes (between 27-32 kb). the host-derived membrane surrounds the genome, encased in a nucleocapsid, and contains glycoprotein spikes. viral rna replication by rna polymerase occurs in the host cytoplasm. the coronaviruses genome encodes four or five structural proteins [8] . coronaviruses are very common among birds and mammals, especially in bats, pigs and feline, which represent the major hosts [7, 9] . the human coronaviruses (hcovs) number seven. there are five non-sars september 26, 2020 volume 8 issue 18 coronavirus serotypes (two beta coronaviruses -hcov-oc43 and hcov-hku1and two alpha coronaviruses -hcov-229e and hcov-nl63) [8] and a novel coronavirus, the middle east respiratory syndrome coronavirus (mers-cov), that emerged in 2012 [9] . they are community-acquired viruses that continually circulate through the human population, causing asymptomatic infections, accounting for 5% to 10% of overall colds and upper respiratory tract infections during winter in adults and some proportion of lower respiratory illness in children [10, 11] . in contrast, the last two beta coronaviruses -sars-cov and sars-cov-2 -jumped to the human population in 2002 and 2020 respectively, causing acute pneumonia, with a higher mortality rate. respiratory coronaviruses probably spread in a fashion similar to that of the rhinoviruses, via direct contact with infected secretions or large aerosol droplets [12] [13] [14] [15] . protein-protein binding assays have confirmed that angiotensin-converting enzyme 2 (ace2) is most likely to be the cell receptor through which the virus invades the host cell [16, 17] . the scientific community is currently trying to identify the source of the infection, which is still uncertain. according to recent lines of evidence, in late 2019, someone at the huanan seafood market in wuhan was infected with sars-cov-2, but specific animal associations have not been confirmed. the viral infectious disease then spread from that first cluster in the capital of china's hubei province to a pandemic. some argue that the involved animals would be bats and pangolins [18] . the spread of covid-19 caused by the sars-cov-2 outbreak has been growing since its first identification in december 2019. on may 17, 2020, the who's coronavirus disease situation report counted 4525497 confirmed cases globally since the beginning of the global pandemic and a total of 307395 deaths all over the world [19] . actually, the case fatality rate of the ongoing covid19 pandemic (the ratio between confirmed deaths and confirmed cases) is 6.78% for the world and 14.3% for italy [20, 21] . as a comparison for this global value with the case fatality rate of other coronavirus outbreaks, it was 10% for sars-cov and 34% for mers-cov [24] , instead and approximately between 0.1%-0.2% for the seasonal flu [21] [22] [23] [24] . covid-19 is a new disease and there is limited information regarding risk factors for developing a severe case. what we know is that the covid-19 pandemic has shown an opposite behaviour than that of other global infectious diseases. indeed, for many other viral and bacterial diseases, such as the previous 'spanish flu' pandemic in 1918 and malaria (which is still endemic in many areas of the world), the majority of deaths were young and children; for covid-19 cases, the elderly are at the greatest risk of dying if infected with the virus. yet, old age is not an isolated risk factor for developing a severe acute viral pneumonia by covid-19. based on currently available information, older adults, but also people of any age, who have serious underlying medical conditions might be at higher risk for severe illness. in fact, analysing the case fatality rate of each condition shows that those with an underlying health condition have a higher percentage than those without. more than 10% of people with a cardiovascular disease, more than 7% of people with diabetes, 6% with chronic respiratory disease or moderate to severe asthma, 6% with hypertension, and more than 5% with cancer who were diagnosed with covid-19 have died [21] . other important risk factors for developing severe complications of covid-19 disease are related to immunocompromised status due to congenital and acquired immune-deficiencies (i.e., cancer treatment, bone marrow or organ transplantation, autoimmune deficiency syndrome, prolonged use of corticosteroids and immunosuppressive drugs), severe obesity (body mass index of 40 or higher), hypertension, liver disease, chronic kidney disease undergoing dialysis, and cerebrovascular diseases [21, [25] [26] [27] . a possible reason why the elderly are most at risk of dying from covid-19 might be the fact that they are also most likely to have underlying health conditions. italy and other european countries emerged early on as the countries with the largest outbreaks of the novel sars-cov-2 outside asia. several prevention and containment measures have been applied worldwide to contain the covid-19 disease. in italy, the particular seriousness of covid-19 disease regarding morbidity and mortality and the enormous overload of intensive care units (icus) brought about the italian government's establishment of a series of decree laws [28] , from february to may 2020, to apply strict and extensive containment measures in all of the italian territory. most non-essential commercial activities were temporarily stopped. the decrees also restricted movements within the regional territory and beyond it, except for proven work reasons, absolute urgency, or for health reasons. but, first of all, to limit the spread of the contagion, careful personal hygiene measures were highly recommended, such as frequent hand-washing, interpersonal safety distancing of at least 1 m to avoid close contacts with potentially infected people, the mandatory use of masks in public closed spaces, and the constant sanitization of public spaces. thanks to all these measures and practices, on may 4, 2020, the italian government declared initiation of the so-called "phase two" [28] [29] [30] , a period of greater freedom but always with strong recommendations of respecting constant hygiene measures and being on alert for eventual rise in new cases. social distancing is fundamental to prevent the inter-human spread of covid-19 though flügge's drops, produced as a result breathing, talking, sneezing, or coughing and able to contaminate surfaces. according to the latest release from the national health commission, known as the prc, sars-cov-2 is believed to be transmitted mostly through respiratory droplets and close contacts. prolonged exposure to high concentrations of aerosols may facilitate transmission. spread is also possible through the conjunctiva. it will be very important during the summer of 2021 to better investigate the possible role of air conditioning systems in increasing the virus circulation. finally, an observational study found that sars-cov-2 does not seem to be present in breast milk and its transmission may take place through respiratory droplets rather than the milk; for this reason, it is not recommended to interrupt breastfeeding [10, 11] . sars-cov-2 persistence (and infectivity) on different surfaces (liquid, solid, or gaseous) is still debated. the 50% tissue culture infective dose (tcid 50 ) is the measure of infectious virus titre. this endpoint dilution assay quantifies the amount of virus required to kill 50% of infected hosts or to produce a cytopathic effect in 50% of inoculated tissue culture cells. a recent experiment performed using aerosols (< 5 µm) containing sars-cov-2 (105.25 tcid 50 per ml) showed a reduction from 10 3.5 to 10 2.7 tcid 50 per l of air after 3 h, a reduction from 10 3.7 to 10 0.6 tcid 50 per ml on plastic after 72 h, and the same reduction after 48 h on stainless steel [31] . these results show a persistence of sars-cov-2 for many hours on surfaces in experimental conditions, but further peer-reviewed investigation on this topic is needed because it represents an environmental and public health problem concerning hospitals (especially in covid departments), schools, offices, and every day public places. furthermore, previous studies have found that air pollution is a risk factor for respiratory infection, by carrying microorganisms, but it also causes oxidative, proinflammatory and immunological damage to the lungs. various recent studies have explored the relationship between ambient air pollutants and covid-19 infection. most of these have shown a relationship between long-term exposure to air pollution in cities and risk of infection [32] [33] [34] [35] . another study investigated in the united states whether long-term average exposure to fine particulate matter (pm 2.5 ) is associated with an increased risk of covid-19 death. it was found that an increase of only 1 μg/m 3 in pm 2.5 increases vulnerability to experiencing the most severe covid-19 outcomes, with statistically significant evidence that this exposure is associated with a 15% increase in the covid-19 mortality rate [36] . however, caution should be used in translating high values of conventional aerosol metrics, such as pm 2.5 and pm 10 concentrations, in a mortality predictive factor, because many biases may interfere in a real-life situation, relating to different temperature, humidity, and ultraviolet radiation. we can interpret these data considering air pollution as an additional risk factor for covid-19 disease, which might contribute to increasing the vulnerability and the clinical outcome, probably also through a previous increase in heart diseases, lung problems and cancer. this could also partially explain the prevalence of the infection in the most industrialized cities of the world, northern italy included, and the effect of national lockdown. finally, it could provide implications for the control and prevention of this novel disease and underscores the importance of continuing to enforce existing air pollution regulations to protect human health both during and after the covid-19 crisis. as the clinical spectrum of covid-19 ranges widely from asymptomatic cases to severe pneumonia with a high risk of mortality, there is a need for more research to identify the earliest markers of disease severity. the incubation period for covid-19 is currently estimated to be between 1 d and 14 d. most infected people develop mild to moderate illness and recover without hospitalization. in the early phases of the disease, clinical manifestations are very unspecific, so differential diagnosis should include other infectious viral diseases that appear with the same symptoms, such as influenza and parainfluenza, the common cold caused by rhinovirus, and those caused by human metapneumovirus, human respiratory syncytial virus and adenoviruses, but also with non-infectious (e.g., vasculitis, dermatomyositis) common respiratory disorders. the most common symptoms of covid-19 infection are fever (not very responsive to antipyretics), dry cough, dyspnoea and increased respiratory rate (in more fragile patients, the dyspnoea may appear at the onset of symptoms, while in younger subjects without other comorbidities it may appear later), myalgia, and intense fatigue [37] [38] [39] [40] . clinical studies have shown an incidence rate of diarrhoea ranging from 2% to 50%. it may precede or trail the respiratory symptoms [40] . increasing evidence indicates possible faecal-oral transmission [41] [42] [43] . other gastrointestinal symptoms are nausea and vomiting, abdominal pain, and loss of appetite [37] [38] [39] [40] [41] [42] [43] [44] (figure 1 ). less common symptoms include sore throat, headache, nasal congestion, hyposmia/anosmia, ageusia, diffuse aches and pains, and conjunctivitis [45] . the most common skin manifestations associated with covid-19 infection include a maculopapular or papulovesicular rash, urticarious lesions, painful acral red purple papules, livedo reticularis, and petechial lesions. the most common areas involved are the trunk, hands and feet, with little itching experienced. these symptoms usually present before the onset of respiratory symptoms and spontaneously disappear within 10 d in all patients. there is no demonstrated correlation, in the majority of the studies, between skin lesions and covid-19 severity [46] [47] [48] . an italian paper reported skin manifestations of covid-19 in 3 young patients, 2 of whom were asymptomatic and potentially contagious. these lesions began as erythematous-violaceous patches in the acral sites and slowly evolved to purpuric and then to ulcero-necrotic lesions, followed by a complete "restitutio ad integrum" of tissues. burning and itching were present with some of the lesions [49] . data provided by the who health emergency dashboard (may 21, 2020, 10.00 am cest) indicated 4893186 confirmed cases worldwide since the beginning of the epidemic, and 103981 in the last 24 h. in total, 323256 cases were fatal, with 4467 in the last 24 h [50] . looking at these data, the global situation still appears to be in a dramatic evolution. trying to investigate the reasons why, in some patients, the covid-19 infection rapidly evolves into a severe acute respiratory syndrome, has led to multiple organ dysfunction and even death being a focus of primary importance. the largest cohort of patients with covid-19 from china (more than 44,000) showed that illness severity can range from mild (81%) to severe and critical (14% and 5% respectively), as shown in table 1 [51] . mild covid-19 illness is characterized by a lower respiratory disease, evidenced by clinical assessment or imaging, and with blood o 2 saturation level of > 93% on room air at sea level. these patients should be admitted to a healthcare facility for close observation. covid-19 severe illness is defined by a blood o 2 saturation level of ≤ 93% on room air at sea level, respiratory rate of > 30, arterial partial pressure of o 2 /fraction of inspiration o 2 of ≤ 300 mmhg, or lung infiltrates of > 50%. these patients may experience rapid clinical deterioration into a critical disease state. the clinical picture of critical patients with severe inflammatory-induced lung disease and with sepsis or septic shock needing intensive care support and mechanical ventilation is characterized by a wide range of signs and symptoms of life-threatening multiorgan dysfunction or failure, including dyspnoea, tachypnoea (respiratory rate of > 30/min), tachycardia, chest pain or tightness, hypoxemia, virus-induced distributive shock, cardiac dysfunction, elevations in multiple inflammatory cytokines, renal impairment with oliguria, altered mental status, functional alterations of organs expressed as laboratory data of hyperbilirubinemia, acidosis [serum lactate level > 2 mmol/l (18 mg/dl)], coagulopathy, and thrombocytopenia. moreover, an exacerbation of underlying comorbidities is often possible [51] [52] [53] [54] [55] [56] [57] . old-age patients with pre-existing comorbidities or dyspnoea should be hospitalized and closely monitored, especially at 1-2 wk after symptom onset. in fact, as already mentioned, in patients with other pre-existing diseases, covid-19 may be fatal [21, [25] [26] [27] . the sequential organ failure assessment (commonly known as sofa) score is used for the evaluation of multiorgan damage and to predict icu mortality risk based on lab results and clinical data [52, 53] , as well as for validation in a paediatric version [57] . a kawasaki-like disease -a vasculitis for which diagnosis is based on the presence of persistent fever, polymorphic rash, lymphadenopathy, conjunctival injection, changes to the mucosa, swollen extremities and with coronary artery aneurysms as its main complication -has been described in children infected with covid-19, with a monthly incidence much more higher than observed for kawasaki disease across the previous 5 years. there was also a high proportion of shock in those children presenting with hypotension and requiring fluid resuscitation and some needing inotropic support. it is still uncertain, however, if this emerging phenomenon is a kawasaki disease type, with sars-cov-2 as the triggering agent, or if it represents an emerging kawasaki-like disease characterized by multisystem inflammation [58] [59] [60] . current studies are investigating the relationship between different variables and the risk of death of covid-19 patients hospitalized for pneumonia. the pulmonary imaging techniques for diagnosis of covid-19 lung damage include an initial evaluation with chest x-ray, ultrasound, and, if indicated, computed tomography. electrocardiogram should be performed if indicated, especially in patients with september 26, 2020 volume 8 issue 18 cardiovascular risk factors. laboratory testing includes a complete blood count with differential and a metabolic profile, including liver and renal function tests. a chinese sex-, age-and comorbid illness-matched case-control study identified lymphopenia (cd3 + cd8 + tcells ≤ 75 cells· μl -1 ) and cardiac troponin i value of ≥ 0.05 ng· ml -1 as negative prognostic factors associated with an increase in risk of mortality from covid-19 pneumonia [61] . many studies have designed different increased laboratory results as early predictors of critical illness, such as leucocytosis with agranulocytosis, elevated lactate dehydrogenase, alanine aminotransferase, aspartate aminotransferase, bilirubin, creatine phosphokinase, myoglobin and cytokines [i.e., interleukin (il)-2, il-7, il-10, granulocyte colony-stimulating factor, interferon gamma-induced protein 10, monocyte chemoattractant protein-1, macrophage inflammatory protein-1 alpha, and tumour necrosis factor-alpha]. although, these findings remain to be validated by further studies. measurements of inflammatory markers such as c-reactive protein, ddimer, and ferritin, while not part of standard care, may have prognostic value [62] [63] [64] . during infectious disease outbreaks, triage is particularly important to separate patients likely to be infected with the pathogen of concern. the cdc has released comprehensive guidelines for management of patients with covid-19, including those who are critically ill [65] . it would be very important to make hotlines available that patients can call to notify the facility that they are seeking care and which can be used as telephone consultation for patients to determine the need to visit a healthcare facility; through this, patients could be informed, before arriving for triage, of preventive measures to take as they come to the facility (e.g., wearing a mask and having tissues to cover cough or sneeze). moreover, healthcare facilities should consider telemedicine (e.g., cell phone videoconference or teleconference) to provide clinical support without direct contact with the patient [65] . emergency departments should have a "respiratory waiting area" for patients coming in with respiratory symptoms (suspected covid-19 patients), with clear signage at the entrance, physical barriers (e.g., glass or plastic screens) installed to limit close contact between registration desk personnel and potentially infectious patients. a facemask should be given to patients with respiratory symptoms as soon as possible, if they do not already have one. the number of accompanying family members in the waiting area should be limited. dedicated clinical staff (e.g., physicians or nurses) should be assigned for physical evaluation of patients presenting with respiratory symptoms at triage. these staff should be trained on triage procedures, covid-19 case definition, and appropriate personal protective equipment (commonly referred to as ppe) use (e.g., mask, eye protection, gown, and gloves) [65] . all the hospital staff (healthcare workers, lab technician, cleaners) and visitors must protect themselves and others by correctly using the ppe and respecting standard precautions, which will include performing hand hygiene frequently (with an alcohol-based hand rub if your hands are not visibly dirty or with soap and water if hands are dirty), contact and droplet precautions, selection of ppe-based risk assessment (e.g., n95 respirators or powered, air-purifying respirators rather than a surgical mask, eye protection such as face shield or goggles, gowns, simple gloves or heavy-duty gloves, and boots or closed work-shoes), cleaning, disinfection and injection safety practices, and single-patient dedicated medical equipment (e.g., stethoscopes, blood pressure cuffs, and thermometers) [65] [66] [67] [68] [69] [70] [71] . the confirmed cases must be hospitalized, possibly in single, isolated rooms with negative air pressure as well as a dedicated bathroom and anteroom. if not possible, the confirmed cases must be, in any case, hospitalized in a single room with a dedicated bathroom and transferred as soon as possible to a safe structure. confirmed covid-19 patients may be hosted in the same room. if available, airborne infection isolation rooms should be used. covid-19 patients are often very complex and require a multidisciplinary medical team which includes at least the following specialists: emergency doctor, pulmonologist, infectious disease specialist, critical care physician, and medical laboratory technician [72] [73] [74] . a first approach during triage is using the quick-sofa (commonly referred to as the qsofa) for a rapid identification of high-risk septic covid-19 patients [51, 52] . the american disease cdc recommends some criteria priorities for testing patients with suspected covid-19, as shown in september 26, 2020 volume 8 issue 18 table 2 . for initial diagnostic testing for sars-cov-2, the cdc recommends collecting and testing an upper respiratory specimen (nasopharyngeal/oropharyngeal swab collected by a healthcare professional) for a rapid molecular in vitro diagnostic test, utilizing an isothermal nucleic acid amplification technology intended for the qualitative detection of nucleic acid from the sars-cov-2 viral rna (real-time reverse transcriptasepolymerase chain reaction) [72, 73] . testing lower respiratory tract specimens is also an option. for patients who develop a productive cough, sputum should be collected and tested for sars-cov-2. the induction of sputum is not recommended. when under certain clinical circumstances (e.g., those receiving invasive mechanical ventilation), a lower respiratory tract aspirate or bronco-alveolar lavage sample should be collected and tested as a lower respiratory tract specimen [72, 73] . plasma cells take at least 5-10 d to develop antibodies against covid-19. for this reason, serological tests are not sensitive enough to accurately diagnose a recent infection, even in symptomatic patients. clinical recovery has been correlated with the detection of igm and igg antibodies, which signal the development of immunity. actually, there are no data concerning the possibility of reinfection after recovery from covid-19. viral rna shedding declines with resolution of symptoms and may continue for days to weeks. however, the detection of rna during convalescence does not necessarily indicate the presence of viable infectious virus [71] [72] [73] . optimal management of covid-19 is evolving quickly and treatment guidelines based on scientific evidence and experts' opinions with clinical experience are updated frequently. until now, there are no food and drug administration (fda)-approved drugs for covid-19 and no vaccine is currently available (even if there are many experimental trials, such as the vaccine promoted by the united states' biotech firm moderna); hence, infected people primarily rely on symptomatic treatment and supportive care [74] . meanwhile, an array of drugs approved for other indications as well as multiple investigational agents are being studied for the treatment of covid-19 in several hundred clinical trials all over the world. patients with severe infection are currently being treated with o 2 therapy. patients with viral pneumonia, hypoxemic respiratory failure/acute respiratory distress syndrome, sepsis and septic shock, cardiomyopathy and arrhythmia, and/or acute kidney injury often require noninvasive or mechanical ventilation and support in the icu. hemodynamic support is essential for managing septic shock. hospitalization is also fundamental for the management of complications from prolonged hospitalization itself, including secondary bacterial infections, thromboembolism, gastrointestinal bleeding, and critical illness polyneuropathy/myopathy [74] [75] [76] [77] . at present, the national institutes of health covid-19 treatment guidelines do not recommend the use of any agents for pre-exposure prophylaxis and post-exposure prophylaxis against sars-cov-2 outside of the setting of a clinical trial, because no drug has actually been proven to be safe and effective for treating covid-19 [74] [75] . moreover, no specific treatment is also recommended for persons with suspected or confirmed asymptomatic or pre-symptomatic covid-19 infection. to help reduce fever and diffuse aches related to covid-19 infection, either acetaminophen or ibuprofen can be prudently used, without exceeding the recommended dose per day of 3000 mg [74, 75] . most of the recommendations for the management of severely and critically ill patients with covid-19 are extrapolated from experience with other lifethreatening infections and they do not deviate substantially from the management of other patients with severe diseases; although, special precautions in this infectious disease are required. these measures include high-flow nasal oxygen and noninvasive ventilation in non-severe forms of respiratory failure, while intubation and protective mechanical ventilation are required in severe forms. prone position ventilation and extracorporeal membrane oxygenation have been used many times for very acute patients with refractory hypoxemia despite lung-protective ventilation [76] . systemic corticosteroids and inappropriate administration of antibiotics are not recommended for the viral pneumonia's treatment, although some centres recommend it but only in case of evidence of bacterial infection [52, 53, 69] . although no antiviral treatments have been approved, several approaches have been proposed to limit viral reproduction, particularly drugs that have been used to treat malaria and autoimmune diseases and already used against past outbreaks, including those of sars-cov and mers-cov. these include antiviral drugs, such as lopinavir, ritonavir, nelfinavir, and remdesivir. the last one is an inhibitor of rna polymerase with in vitro activity against multiple rna viruses [77] . alpha-interferon ( e.g., 5 million units by aerosol inhalation twice per day), chloroquine (500 mg every 12 h), and hydroxychloroquine (200 mg every 12 h) are also used. chloroquine was introduced in clinical practice in 1946 to treat malaria, while hydroxychloroquine was introduced in 1955 and prescribed for the treatment of systemic lupus erythematosus [78] . the efficacy for systemic lupus erythematosus is based on the capability of this drug to inhibit toll-like receptor signalling and to reduce cytokine production, especially that of il-1 and il-6. starting from this consideration, hydroxychloroquine, and thereafter chloroquine, has been proposed as a helpful treatment for covid-19 patients, in which some reports showed a direct antiviral effect in vitro due to an interference with ace-2 receptors [79] . chloroquine and hydroxychloroquine, which are not fda approved for covid-19, are available from the strategic national stockpile for hospitalized adults and adolescents (weighing ≥ 50 kg) under an emergency use authorization [74, 78, 79] . there has been supposition (never proven) that azithromycin may help to reduce the overactive immune response to the sars-cov-2 infection that otherwise causes inflammatory damage. unfortunately, the most recent human studies suggest no benefit and a strong statement was released, advising against the use of the combination of hydroxychloroquine and azithromycin, underlying the higher risk of death due to lethal heart arrhythmias with both hydroxychloroquine and azithromycin are used alone and especially when used in combination. in fact, it is well known that hydroxychloroquine and, moreover, chloroquine may have several side effects on the extrapyramidal, cardiovascular and digestive systems, which are more severe if associated with other medications (i.e. haemolysis with dapsone, severe hypoglycaemic effects with anti-diabetics, qt elongation, or torsades de pointes with ciprofloxacin and other antimicrobials, etc.) [80] [81] [82] . however, to date, this issue remains controversial [83] . tocilizumab, a humanized igg1 monoclonal antibody directed against the il-6 receptor and commonly used in the treatment of rheumatoid arthritis, has already been used in a rhesus macaque model of mers-cov infection and is currently being used in experimentations against covid-19. two chinese large randomized clinical trials, which enrolled over 700 patients and other studies which are also underway in european countries and in the united states are likely to definitively answer the question of whether the drug is effective in treating covid-19, and so it could be approved for use and produced in large amounts [84] . september 26, 2020 volume 8 issue 18 on 24 march, 2020, the fda allowed convalescent plasma from recovered patients to be used in patients with serious or immediately life-threatening covid-19 infections. these antibody-containing plasma, in many cases, showed the intended effect in fighting the illness, shortening the length or reducing the severity of the disease, but this treatment is still considered experimental and more randomized, controlled studies must be done to test its efficacy and safety. it has been estimated that herd immunity against covid-19 (that is, an indirect protection given by recovered patients to those who are not immune to the disease) is around 50% to 66.66% [85] . moreover, covid-19 patients show a contemporary hypercoagulation and hypofibrinolytic state due to dysregulation of the coagulation and fibrinolytic systems, with elevated d-dimer and fibrinogen and deposition of fibrin in the air spaces and lung parenchyma caused by the activated tissue factor exposure on damaged alveolar endothelial cells and on the surface of leucocytes. the patients also show significantly elevated levels of plasminogen activator inhibitor 1 released from lung epithelium and endothelial cells. prophylaxis treatment with low molecular weight heparin is considered important to limit covid-19 patients' coagulopathy, but, at the same time, it is fundamental to degrade pre-existing fibrin in the lung by promoting local fibrinolysis with tissue-type plasminogen activator as intravenous thrombolytic treatment. its nebulizer form is currently in phase ii clinical trial and may provide a targeted approach in covid-19 patients to degrade fibrin and improve oxygenation in critically ill patients [86] . new research fields are concerned with the use of immuno-enhancers (interferons, thymosin α-1, thymopentin, levamisole, cyclosporine a), vitamins (a, b, c, d, e), alipoic acids, minerals (selenium, zinc, iron), omega-6 polyunsaturated fatty acids, nacetylcysteine and d-ribose-l-cysteine, probiotics, and the intravenous infusion of allogeneic expanded umbilical cord mesenchymal stem cells that show antiviral and antimicrobial properties, and which must be deepened. the italian college of anesthesia, analgesia, resuscitation and intensive care have reported guidelines to use these cells in covid-19 patients, in the hope of decreasing the number of patients going to the icu, and also getting them out of icu relatively quickly [87, 88] . despite this, there are insufficient data to recommend either for or against the use of any antiviral or immunomodulatory therapy in patients with covid-19 who have mild, moderate, severe, or critical illness [89] . researchers are carrying out incessant efforts towards understanding these topics, including on translational regenerative approaches, such as mesenchymal stem cells [90, 91] . the sars-cov-2 pandemic is a serious health problem and a challenge of global concern. during these months, we've had to learn, step-by-step, all about this novel coronavirus, ranging from its origins (which are still uncertain) to its mode of transmission, identifying people most at risk, and searching for old and new strategies which could help patients in fighting against this invisible enemy. still, despite this, even more studies are needed to provide more effective preventive measures and treatment policies and to determine what is the proper social behaviour in public places and the rules of conduct for healthcare professionals' management and resource planning; ultimately, the collective knowledge will lay a solid foundation for winning the battle against this epidemic. world health organization. pneumonia of unknown cause -china rapid progression to acute respiratory distress syndrome: review of current understanding of critical illness from covid-19 infection what we know so far: covid-19 current clinical knowledge and research diarrhea during covid-19 infection: pathogenesis, epidemiology, prevention, and management the occurrence of diarrhea in covid-19 patients covid-19 and the digestive system coronavirus disease 2019 (covid-19) pandemic burst and its relevant consequences in dental practice comment to the article "olfactory and gustatory dysfunctions as a clinical presentation of mild-to-moderate forms of the coronavirus disease (covid-19): a multicenter european study calzavara-pinton p. varicella-like exanthem as a specific covid-19-associated skin manifestation: multicenter case series of 22 patients cutaneous manifestations of covid-19: report of three cases and a review of literature cutaneous manifestations in covid-19: a first perspective silent covid-19: what your skin can reveal world health organization. coronavirus disease (covid-19) situation report-122 characteristics of and important lessons from the coronavirus disease 2019 (covid-19) outbreak in china: summary of a report of 72 314 cases from the chinese center for disease control and prevention covid-19 -what should anaethesiologists and intensivists know about it? current issues and perspectives in patients with possible sepsis at emergency departments new criteria for sepsis-induced coagulopathy (sic) following the revised sepsis definition: a retrospective analysis of a nationwide survey derivation, validation, and potential treatment implications of novel clinical phenotypes for sepsis the preoperative inflammatory status affects the clinical outcome in cardiac surgery adaptation and validation of a pediatric sequential organ failure assessment score and evaluation of the sepsis-3 definitions in critically ill children kawasaki-like disease: emerging complication during the covid-19 pandemic an outbreak of severe kawasaki-like disease at the italian epicentre of the sars-cov-2 epidemic: an observational cohort study hyperinflammatory shock in children during covid-19 pandemic predictors of mortality for patients with covid-19 pneumonia caused by sars-cov-2: a prospective cohort study analysis of heart injury laboratory parameters in 273 covid-19 patients in one hospital in wuhan approved respirator standards positive rate of rt-pcr detection of sars-cov-2 infection in 4880 cases from one hospital in standard operating procedure (sop) for triage of suspected covid-19 patients in non-us healthcare settings: early identification and prevention of transmission during triage world health organization. water, sanitation, hygiene, and waste management for the covid-19 virus: interim guidance overview of testing for sars-cov-2 rational use of personal protective equipment for coronavirus disease 2019 (covid-19) enhancing immunity in viral infections, with special emphasis on covid-19: a review interim guidelines for collecting, handling, and testing clinical specimens for covid-19 rapid and sensitive diagnostic procedure for multiple detection of pandemic coronaviridae family members sars-cov-2, sars-cov, mers-cov and hcov: a translational research and cooperation between the phan chau trinh university in vietnam and university of bari "aldo moro" in italy national institutes of health. management of persons with covid-19, in: covid-19 treatment guidelines national institutes of health. persons at risk for infection with sars-cov-2, in: covid-19 treatment guidelines successful treatment of covid-19 using extracorporeal membrane oxygenation, a case report the antiviral compound remdesivir potently inhibits rna-dependent rna polymerase from middle east respiratory syndrome coronavirus breakthrough: chloroquine phosphate has shown apparent efficacy in treatment of covid-19 associated pneumonia in clinical studies treatment of covid-19: old tricks for new challenges response to the editorial "covid-19 in patients with cardiovascular diseases": covid-19 treatment with hydroxychloroquine or chloroquine and azithromycin: a potential risk of torsades de pointes off-label" use of hydroxychloroquine, azithromycin lopinavir-ritonavir and chloroquine in covid-19: a survey of cardiac adverse drug reactions by the french network of pharmacovigilance centers qt prolongation, torsades de pointes, and sudden death with short courses of chloroquine or hydroxychloroquine as used in covid-19: a systematic review covid-19: 146 researchers raise concerns over chloroquine study that halted who trial prophylactic and therapeutic remdesivir (gs-5734) treatment in the rhesus macaque model of mers-cov infection covid-19: herd immunity and convalescent plasma transfer therapy fibrinolytic abnormalities in acute respiratory distress syndrome (ards) and versatility of thrombolytic drugs to treat covid-19 n-acetylcysteine as an antioxidant and disulphide breaking agent: the reasons why a successful history: probiotics and their potential as antimicrobials current status of potential therapeutic candidates for the covid-19 crisis mesenchymal stem cells as promoters, enhancers, and playmakers of the translational regenerative medicine expanded umbilical cord mesenchymal stem cells (uc-mscs) as a therapeutic strategy in managing critically ill covid-19 patients: the case for compassionate use key: cord-285096-g9y3au1a authors: mitchell, judy a.; brooks, harriet w.; szladovits, balázs; erles, kerstin; gibbons, rachel; shields, shelly; brownlie, joe title: tropism and pathological findings associated with canine respiratory coronavirus (crcov) date: 2013-03-23 journal: vet microbiol doi: 10.1016/j.vetmic.2012.11.025 sha: doc_id: 285096 cord_uid: g9y3au1a canine infectious respiratory disease (cird) occurs frequently in densely housed dog populations. one of the common pathogens involved is canine respiratory coronavirus (crcov), however little is known regarding its pathogenesis and the role it plays in the development of cird. the pathogenesis of five geographically unrelated canine respiratory coronavirus (crcov) isolates was investigated. following experimental infection in dogs, all five crcov isolates gave rise to clinical signs of respiratory disease consistent with that observed during natural infection. the presence of crcov was associated with marked histopathological changes in the nares and trachea, with loss and damage to tracheal cilia, accompanied by inflammation. viral shedding was readily detected from the oropharynx up to 10 days post infection, but there was little or no evidence of rectal shedding. the successful re-isolation of crcov from a wide range of respiratory and mucosal associated lymphoid tissues, and lung lavage fluids demonstrates a clear tropism of crcov for respiratory tissues and fulfils the final requirement for koch's postulates. by study day 14 dogs had seroconverted to crcov and the antibodies raised were neutralising against both homologous and heterologous strains of crcov in vitro, thus demonstrating antigenic homogeneity among crcov strains from the two continents. defining the role that crcov and other agents play in cird is a considerable, but important, challenge if the disease is to be managed, treated and prevented more successfully. here we have successfully developed a model for studying the pathogenicity and the role of crcov in cird. tropism and pathological findings associated with canine respiratory coronavirus (crcov) a b s t r a c t canine infectious respiratory disease (cird) occurs frequently in densely housed dog populations. one of the common pathogens involved is canine respiratory coronavirus (crcov), however little is known regarding its pathogenesis and the role it plays in the development of cird. the pathogenesis of five geographically unrelated canine respiratory coronavirus (crcov) isolates was investigated. following experimental infection in dogs, all five crcov isolates gave rise to clinical signs of respiratory disease consistent with that observed during natural infection. the presence of crcov was associated with marked histopathological changes in the nares and trachea, with loss and damage to tracheal cilia, accompanied by inflammation. viral shedding was readily detected from the oropharynx up to 10 days post infection, but there was little or no evidence of rectal shedding. the successful re-isolation of crcov from a wide range of respiratory and mucosal associated lymphoid tissues, and lung lavage fluids demonstrates a clear tropism of crcov for respiratory tissues and fulfils the final requirement for koch's postulates. by study day 14 dogs had seroconverted to crcov and the antibodies raised were neutralising against both homologous and heterologous strains of crcov in vitro, thus demonstrating antigenic homogeneity among crcov strains from the two continents. defining the role that crcov and other agents play in cird is a considerable, but important, challenge if the disease is to be managed, treated and prevented more successfully. here we have successfully developed a model for studying the pathogenicity and the role of crcov in cird. ß 2012 elsevier b.v. all rights reserved. since its initial discovery in 2003 (erles et al., 2003) ; canine respiratory coronavirus (crcov) is now considered to be a significant cird pathogen, most frequently detected in dogs with mild respiratory clinical signs during the early stages of cird onset (erles et al., 2003) . although crcov has been found worldwide (decaro et al., 2007; erles and brownlie, 2008; kaneshima et al., 2006; priestnall et al., 2006 priestnall et al., , 2007 yachi and mochizuki, 2006; knesl et al., 2009) ; little is known regarding its pathogenesis, tissue tropism or virulence differences among global isolates in the canine host. it is postulated that crcov plays an important role during the early stages of cird by predisposing the dog to more severe clinical disease from secondary infections. through the use of an in vitro tracheal explant culture system, a moderate reduction in ciliary function and a down-regulation of pro-inflammatory cytokine mrna levels (tnf-a, il-6 and the chemokine il-8) was observed in response to crcov exposure (priestnall et al., 2009) . such alterations in the mucociliary and innate immune systems could be linked to increased susceptibility to secondary infection and is consistent with the proposed role for crcov in cird. however, the limitation of this in vitro model precludes the understanding of the clinical relevance and pathogenesis of a crcov infection in the dog. furthermore, given the global presence of this virus, insight into crcov pathogenesis among isolates originating from geographically distinct locations would be valuable to determine the need for a global vaccine. recently our group has collected findings from a preliminary in vivo challenge study of crcov. in that study we demonstrated that young dogs were susceptible to experimental infection with both crcov isolates, which gave rise to clinical signs of respiratory disease consistent with naturally occurring infection. crcov was detected in the oropharynx of infected dogs and spread rapidly to sentinel dogs which also displayed clinical signs of disease (mitchell et al., unpublished data) . here we extend this study to gain a better understanding of crcov pathogenesis in vivo. analyses specifically focused on the histopathological changes in the canine upper and lower respiratory tissues, virulence differences among crcov isolates derived from cird cases representing wide geographical locations; uk and usa [mo, ne, ut and mi] , and the demonstration of koch's postulates. the information obtained from this study vastly enhances our understanding of crcov pathogenicity and its involvement in the cird complex. five crcov isolates originating from geographically distinct regions of the uk and usa were used in this study (table 1) . crcov isolates uk 4182, np631, np787, and np742 were propagated in culture on human rectal tumour cells (hrt-18g; american type culture collection cell line, manassas, va, usa), and used at 10 6 tcid 50 /ml for intranasal challenge. crcov lu298 was not was not propagated or expanded in vitro, instead 0.2 mm filtered viral fluids obtained from the tissues of a crcov infected dog in the usa were used to challenge animals in to1. prior to challenge each of the crcov virus and control hrt-18g cell culture fluids were satisfactorily tested for sterility and canine extraneous agents (including canine distemper virus, measles, canine adenovirus-2, canine parainfluenza virus, canine rotavirus, rabies, canine parvovirus and canine enteric coronavirus). thirty-six, 12-16-week old specific pathogen free (spf) purpose bred beagle dogs were used in this study. all dogs were demonstrated as crcov negative and seronegative for crcov and b. bronchiseptica prior to the study. dogs were housed in temperature controlled isolation rooms with dedicated shower in and out procedures, disinfection and sterilisation of all items prior to entry and a pasteurised diet were used to maintain bio-security. colony dogs are screened quarterly to determine the spf status. throughout the study dogs from the same treatment group were housed in pairs to minimise stress. dogs were randomly divided into six treatment groups (t1-t6) (table 1) , each consisting of six dogs. dogs in groups t1-t5 inclusive were challenged with the crcov isolates as described in table 1 . dogs in t6 were mock challenged with uninfected hrt-18g cell culture supernatant to serve as negative controls. intranasal inoculations took place over two consecutive days (study day zero and one). on each challenge day dogs were sedated prior to intranasal administration with 1.0 ml of virus or control material (0.5 ml/nostril). dogs were monitored throughout the trial for clinical signs of disease. on study days 3, 6, and 14, as detailed in table 1 , dogs were humanely euthanatized within each treatment group as determined by randomisation completed prior to the start of the trial, and necropsies were performed immediately. gross pathological changes were documented throughout each necropsy and the lungs of each animal were photographed. all experiments involving animals were carried out at a contract research organisation, in compliance with national legislation, and subject to local ethical review. general health observations were performed on each dog, twice daily, and scored for general appearance, breathing, sneezing, coughing, and ocular and nasal discharge as detailed in table 2 . body temperatures were recorded twice daily via implanted microchips, and dogs were weighed on study days à7, à1 and on the day of euthanasia. appetite was recorded according to the quantity of food eaten per room. oropharyngeal viral swabs for rt-pcr analysis (sterilin, uk) and virus isolation (dacron swabs, puritan, in 3 ml virus transport medium (pah)) were collected on study days à1 (pre challenge) 2, 3, 4, 5, 6, 8, 10, 12, and 14 from all dogs on the study. rectal swabs were collected from each dog at necropsy. after sampling, viral swabs were frozen and stored at à70 8c until processed. swab tips for rt-pcr analyses were immersed in 1 ml of rpmi medium (sigma, dorset, uk) and mixed prior to analysis. samples from the following tissues were harvested at necropsy and stored at either à70 8c or fixed in 10% buffered formalin: nasal cavity (included ciliated area), nasal tonsil, palatine tonsil, trachea, apical lung lobe, diaphragmatic lung lobe, and bronchial lymph node. each tissue sample was taken using a new set of sterile instruments. lung lavage fluid samples were collected in dmem cell culture medium and stored at à70 8c. formalin-fixed tissues were processed and stained for histology and were examined by a veterinary pathologist as described in section 2.9. on study days à1, 3, 6, and 14, serum and edta whole blood samples were collected for serological and haematological analysis as described below. swabs and tissue samples were tested for the presence of crcov by rt-pcr and virus isolation. prior to inoculation on to hrt-18 g cells, swab and lung lavage samples were clarified by centrifugation, and filtered (0.2 mm filter). similarly, approximately 1 g of each tissue sample was homogenised in 3.0 ml of virus transport medium (vtm), clarified by centrifugation and filtered (0.2 mm filter). filtered samples were inoculated onto confluent t25 or t150 monolayers of hrt-18 g cells. inoculated cultures were maintained in dmem supplemented with final concentrations of 200 mm l-glutamine, 22.5 mg/ml gentamicin, and 1% fbs. inoculation media was also supplemented with trypsin. trypsin concentrations were determined for each batch (circa 1 mg/ml) based of cell toxicity. cell culture fluids were sampled weekly for 2 weeks to test for the presence of crcov by immunofluorescence assay (ifa) (section 2.5.3). rna was extracted using the rneasy mini kit (qiagen, crawley, uk) as recommended by the manufacturer from 200 ml of the swab fluid or a 0.5 cm 2 piece of tissue. rna was transcribed into cdna using random hexameres (ge healthcare, little chalfont, uk) and improm ii reverse transcriptase (promega, southampton, uk) according to the manufacturer's protocol. all samples were tested for the presence of the house keeping gene glyceraldehyde-3-phosphate dehydrogenase (gapdh) by pcr as described previously (grone et al., 1996) . results were expressed as a positive or negative outcome to ensure successful nucleic acid extraction. samples were analysed for crcov using the nested spike gene pcr as described previously (erles et al., 2003) . formalin-fixed tissues were processed for histology and sections were stained using haematoxylin and eosin. the histological sections were examined by a veterinary pathologist who was blinded to the study groups. each tissue was ascribed a histological and cilia score as described in table 3 . a single overall histological ''weighted'' score was assigned to each dog, in which scores assigned to the lower respiratory tract (trachea, lungs, bronchial lymph nodes) were considered more noteworthy than those in the upper respiratory tract (nares, tonsils). this weighting of the overall score was used to take into account that histological changes (especially inflammatory reactions or lymphoid hyperplasia) in the upper respiratory tract are not uncommon, especially in young animals, and unless considerable, are considered part of the normal defences of the upper respiratory tract. conversely, such changes in the lower respiratory tract are more likely to be clinically significant. thus moderate to marked lymphoid hyperplasia or neutrophilic inflammation of the palatine tonsil (score 4) would have less effect on the overall histology score for that individual dog than would a similar grading of lymphoplasmacytic or neutrophilic aggregation in the tracheal mucosa or lungs. paraffin-embedded formalin-fixed tissues (4 mm) were mounted on superfrost plus microscope slides (menzel-glä ser, braunschweig, germany). slides were heated at 60 8c for 1 h, deparaffinised and dehydrated. endogenous peroxidase was blocked with 3% h 2 o 2 for 10 min then slides were washed in dh 2 o, then incubated in prewarmed (37 8c) protease xiv 0.05% (sigma) in tbs for 15 min. slides were rinsed in dh 2 o then incubated with blocking serum (2% normal goat serum [vector laboratories, peterborough] in tbs). staining for crcov was performed as described previously (priestnall et al., 2009) . positive cells were identified microscopically by the presence of staining. haematological analysis of edta blood samples was performed using a cell-dyn 3500 automated haematology machine. a 100 cell differential count of the white blood cells was performed on blood smears stained with a hematek 1 automated stainer using modified wright's stain, by a board certified veterinary clinical pathologist blinded to the treatment groupings. the results for day à1 were analysed for normality and reference intervals were estimated (n = 36). total neutrophil, band neutrophil, lymphocyte and monocyte concentrations were calculated using the automated total wbc concentration, and the observed percentages of each leucocyte type determined by the differential count. the results within each treatment group for days 3, 6 and 14 were compared with each other and with those at day à1 to evaluate trends or significant differences. the paired t-test or wilcoxon signed ranks test with repeat measures were used if distribution was normal or non-normal, respectively. statistical significance was set at p = 0.05. hrt-18g cells were cultured to confluency in 96 well plates. monolayers were inoculated with a usa crcov isolate, incubated for 5 days, and fixed with 80% acetone. canine serum samples were diluted 1:40 in pbs supplemented with 1% bovine serum albumin (w/v) (steris corporation, mentor, oh, usa) and 0.09% sodium azide (mallinkrodt chemicals, hazelwood, mo, usa) followed by two-fold serial dilutions to 1:1280. diluted serum was dispensed at 100 ml/well onto the fixed cells, incubated for 1 h, and then washed twice with water. bound antibody was detected with 50 ml/well of fluorescein isothiocyanate-labelled secondary antibody (rabbit anti-dog igg) (sigma-aldrich, jerusalem, israel) diluted 1:250 in pbs supplemented with 1% bovine serum albumin (w/v) and 0.09% sodium azide (w/v), incubated and washed as before. endpoint crcov titres were observed using fluorescence microscopy and defined as the inverse of the last dilution of serum exhibiting definite crcov fluorescence. in instances where no virus-specific fluorescence at the 1:40 dilution was observed, dogs were considered seronegative or non-exposed to crcov. dog sera collected on study day 14 were tested for serum neutralising antibody titres against 1 uk, and 10 usa crcov isolates. hrt-18g cells were grown to confluency in a 96 well plate. cells were rinsed once in serum-free dmem and then pre-treated for 1 hour with 100 ml/well of serum-free dmem containing c1 mg/ ml trypsin. heat inactivated serum samples were diluted 2-fold, 1:10 through 1:1280, in dmem containing 1% fbs. a further 1:2 dilution of the serum was made in 50-300 tcid 50 of virus per 100 ml, to obtain final serum dilutions of 1:20-1:2560. virus:serum mixtures were incubated for 1 h at room temperature. cells were inoculated in quadruplicate with 200 ml/well of the virus: serum mixture, incubated for 5-7 days, and fixed with 80% acetone. virus growth was detected by ifa as described previously (section 2.5.3). the fifty percent neutralisation endpoint for each serum sample was calculated by the statistical methods of spearman-karber. oropharyngeal amies swabs (sterilin, uk) and lung lavage fluid samples were collected from all dogs at necropsy in order to screen for the presence of bacterial pathogens. briefly each swab was plated onto 1â chocolate agar, 1â macconkey agar, and 2â blood agar (1â aerobic and 1â anaerobic) and submitted to the clinical services division at the royal veterinary college for analysis. samples were also plated onto mycoplasma experience agar (mycoplasma experience ltd. surrey, uk) and incubated at 37 8c with 5% co 2 for 3 days. dogs from all six treatment groups were healthy prior to challenge. dogs in t6 (negative control) remained healthy for the duration of the study, the only exception being one dog which had some serous ocular discharge prior to challenge, and throughout the study. all 36 dogs completed the study on the pre-assigned day. following challenge, a number of dogs from t1 to t5 inclusive displayed mild clinical signs of respiratory disease which included nasal discharge, sneezing and coughing. table 4 shows the total scores for each observation by treatment group. there were no significant changes in body temperature all dogs had normal or fair appetites and gained weight over the course of the study (data not shown). no consistent differences between dogs treated with different strains of the virus were observed, although respiratory signs were recorded most frequently and most severely in one dog from t3 and one from t5 (table 4 ). it is worth noting that in both instances these dogs were euthanized on study day 14 and displayed the clinical signs of respiratory disease recorded throughout the 14-day study period, although the scores were highest in these two dogs on study days 10-13. oropharyngeal swabs were analysed for crcov by both rt-pcr and virus isolation (fig. 1) . all dogs from all six treatment groups were negative for crcov on study day à1 (pre-challenge) using both techniques. all dogs in t6 (negative control) remained negative for the duration of the study. shedding of crcov in t1-t5 was consistently detected for up to 6 days post challenge by both rt-pcr and vi. all rectal swabs, collected at necropsy, were positive for the internal pcr control gapdh. all rectal swabs collected from dogs in t6 (negative control) were negative for crcov by both rt-pcr and vi for the duration of the study, as was the case for dogs in groups t3, t4 and t5. in t1, two dogs euthanized on study day 3, and one dog euthanized on study day 6 were positive for crcov, as was one t2 dog euthanized on study day 3. the remaining dogs in these groups were negative. all rectal swabs were negative for crcov by vi (data not shown). tissues collected at necropsy were tested for the presence of crcov by rt-pcr and vi (fig. 2) . crcov was detected in at least five of the eight tissues [diaphragmatic lung lobe, apical lung lobe, trachea, nasal cavity, bronchial lymph node, nasal and palatine tonsil and lung lavage fluid] from groups t1-t4 by rt-pcr. in t5 crcov was detected in all the tissues tested. vi was successful in seven of eight tissue types from groups t1-t5 including the diaphragmatic lung lobe, apical lung lobe, trachea, nasal cavity, nasal and palatine tonsil as well as the lung lavage fluid. only bronchial lymph nodes were negative for crcov by vi. overall, crcov was detected most frequently from the trachea, followed by the lung lavage fluid, nasal cavity and nasal tonsil, in all five treatment groups. histological examination was carried out on the palatine tonsil, external nares, nasal tonsil, trachea, apical and diaphragmatic lung lobes, and bronchial lymph node. the most significant findings were seen in the trachea and nares where inflammation, with notable changes in the length and distribution of the cilia were observed as described below and shown in figs. 3 and 4. in t6 the overall weighted histology score was consistently minimal (average 1.1), with minimal changes in all the tissues examined. in t1 the weighted score indicated minimal to modest abnormalities (average 1.7), including inflammatory aggregates in the nares and inflammation in the trachea which was associated with loss of cilia from the mucosal surface. one dog in particular on day 3 showed marked changes (scoring 3 or 4) in all of the tissues examined, with the exception of the diaphragmatic lung lobe and nasal tonsil where only minimal to modest changes were observed. in t2 and t3 the overall weighted score showed modest to marked histological abnormalities with average scores of 2 and 2.5, respectively. in the nares modest to marked inflammation was seen, which in t3 trailed off in the later stages of the trial period, whilst inflammation in the trachea of dogs from both groups was mild but with moderate shortening and irregularity of the cilia. in t4, with the exception of two dogs (one each on days 3 and 6), marked histological changes in the nares were observed throughout the trial period. in addition marked changes were also observed in the trachea and cilia. one dog on day 6 also had marked changes in the bronchial lymph node and diaphragmatic lung lobe (data not shown). the overall weighted scores indicated consistently marked abnormal histological changes in all dogs in this group with an average score of 3.3. in t5 modest to marked inflammation was observed in the trachea of dogs in t5 which was reflected in the condition of the cilia, whilst only modest changes were observed in the nares. on study day 14 marked changes were also seen in the bronchial lymph nodes. the overall weighted score showed consistently marked abnormal histological changes (average 2.8), with the exception of one of the dogs at the first time point which had a score of 1. in t1-t5 inclusive histological abnormalities in the lungs, although not marked, were variable and tended to be lymphoid aggregates adjacent to airways or blood vessels, an observation which was not seen in the broth control which yielded consistently mild scores. in all groups, including the broth control, the palatine tonsils had medium to high histological scores (data not shown). coronavirus antigen-positive cells were detected within the epithelium of the trachea and bronchioles of the infected dogs (fig. 5) . positive staining was present in the cytoplasm of ciliated columnar epithelial and goblet cells. positive cells were widespread but often found in focal clusters, and often associated with small aggregates on the luminal surface of the trachea. positive cells were surrounded by areas of vacuolation within the epithelium, possibly representing a cytopathic effect of the virus. seroconversion to crcov was measured by ifa. all dogs were seronegative (ifa < 40) for crcov on study days à1, 3 and 6, and all dogs in the t6 control group remained seronegative throughout the study whilst all dogs in the crcov challenge groups seroconverted by study day 14. crcov serum cross-neutralisation antibodies from serum collected on study day 14 were tested against 1 uk and 10 usa crcov isolates (table 5 ). all serum samples tested from dogs in crcov treatment groups were shown to be serum neutralisation positive against all the crcov isolates tested. serum collected from t6 control dogs showed no neutralising activity against any of the crcov strains. the mean concentrations of lymphocytes, neutrophils and monocytes, for each group on study days à1, 3, 6 and 14 were determined (data not shown). in t6, and t3 no statistical differences in the lymphocyte, neutrophil or monocyte concentrations were detected. in t1, t2, t4 and t5 increases in total white blood cell concentrations were observed as a result of lymphocytosis which was significant in t1 (p = 0.002) on day 6 and t5 (p = 0.027) on day 3. in t2 a marked lymphocytosis was seen on day 14. in t4 there was moderate lymphocytosis accompanied by significant changes in monocyte concentrations which peaked on day 6 [>day 3 (p = 0.003), >day 14 (p = 0.006)]. in contrast there was a significant decrease in neutrophil concentration on day 3 in t1, t4 and t5 (p = 0.004, p = 0.001 and p = 0.005, respectively) compared to day à1, which in t1 resulted in 3 of the dogs becoming neutropenic. across the groups seven dogs had rare to occasional toxic neutrophils exhibiting foamy cytoplasm, mild cytoplasmic basophilia or rare dohle bodies, and detectable levels of band neutrophils. in group t1, all dogs had detectable levels of band neutrophils on day 3, which was a significant increase when compared to day à1 (p = 0.031). on day 3 one dog in each of t2, t4, and t5 had band concentrations higher than the estimated upper reference limit; however this did not result in statistically significant changes at a group level and no other significant changes could be detected. oropharyngeal amies swabs collected from all dogs on day à1 and at euthanasia yielded growth of normal respiratory flora. a majority of dogs also yielded scanty growth of either streptococcus canis or staphylococcus intermedius. in the vast majority of dogs the lungs were sterile for bacterial growth, with the exception of the growth of mycoplasma spp. in four dogs, one from each treatment group t2-t5 inclusive (data not shown). previous publications have collectively demonstrated the global distribution of crcov and its association with respiratory disease in dogs under field conditions (erles et al., 2003; decaro et al., 2007; kaneshima et al., 2006; priestnall et al., 2006 priestnall et al., , 2007 yachi and mochizuki, 2006; knesl et al., 2009) . this is the first publication to report experimental infection of dogs using crcov, and the comparison of different crcov isolates from wide geographic origins. each crcov isolate was derived from dogs with respiratory disease in mo, mi, ut and ne in the usa, and one isolate from london, uk. this study builds upon a preliminary experimental challenge study (mitchell et al., unpublished data) . in that study we demonstrated rapid shed-spread of crcov from experimentally infected dogs to sentinel dogs within 4 days of exposure. peak viral loads were detected in oropharyngeal swabs at 4 and 6 days post infection in experimentally infected, and sentinel dogs, respectively; with shedding lasting for 8-10 days in both groups. both inoculated and sentinel dogs displayed clinical signs of mild respiratory disease, and seroconverted to the virus. in the current study prolific shedding of crcov from the oropharynx of dogs in all treatment groups (detected by rt-pcr and vi from oral swabs) was seen by day 2. in a majority of dogs viral shedding ceased after day 6, although crcov was detected up to 10 days post infection by rt-pcr in one dog. small differences in the duration of viral shedding between the two studies are most likely to be explained by differences in the age of the dogs used in each of the studies (preliminary study: 1-3 weeks old, current study: 12-16 weeks old). despite differences, both studies clearly illustrate that crcov is a quick-hit respiratory pathogen, and supports field data in which the rapid spread of crcov throughout a large rehoming centre was observed (erles et al., 2003) . consistent with observations made during naturally occurring infection, dogs in this study also displayed clinical signs of mild respiratory disease following viral challenge (nasal discharge, sneezing, and coughing); whilst the control group remained healthy. there appeared to be no profound difference in the clinical observations made between groups of dogs challenged with the different strains. two dogs however (one each in t3 and t5) showed more severe and prolonged clinical signs compared to the others. this included increased respiratory noise, and more frequent and prolonged coughing and sneezing. the reason for this was unclear. disease in these two dogs did not appear to be associated with any secondary bacterial infections in the lung, nor with notably different histopathological changes when table 5 study day 14 cross neutralisation titres. two dogs remained on the study at day 14 in each treatment group. samples with sn values of 14 were considered negative at the 1:20 starting dilution. serum samples collected at earlier time points were negative for crcov antibody. treatment group (isolate) lu131 lu172 lu189 lu295 lu317 np599 np604 np617 np631 np634 uk4182 t1 (lu295) 320 80 95 113 113 190 135 135 95 113 80 381 135 80 160 160 226 160 135 135 135 113 t2 (uk4182) 369 80 48 57 113 113 95 113 40 135 57 67 34 28 40 40 28 28 17 40 57 57 t3 (np631) 40 28 48 28 28 28 34 17 34 48 28 95 95 80 95 67 48 95 48 67 113 compared to other challenge dogs in the same group (data not shown). given its multifactorial aetiology; modelling cird under controlled conditions, to mimic disease as it appears in the field is difficult to achieve. when studied in isolation dogs infected with other viruses involved in the cird complex, such as cpiv and cav-2, also cause only mild respiratory disease such as a dry cough and nasal discharge (appel and binn, 1987a,b; castleman, 1985; buonavoglia and martella, 2007) . more severe clinical signs, representative of the disease complex, are likely to occur when other viruses or bacterial respiratory pathogens are also present, and when environmental stresses such as those encountered in rehoming facilities are experienced (appel and binn, 1987a,b; buonavoglia and martella, 2007) . overall there appeared to be little difference in tissue tropism, when comparing the different crcov isolates investigated. crcov was detected in at least five of the eight necropsy samples examined (lung lavage, diaphragmatic lung lobes, trachea, nasal tonsils and nasal cavity) in dogs from all five challenge groups. crucially, isolation of crcov was achieved from six of the seven tissues collected (the bronchial lymph node remained negative) as well as the lung lavage fluid. re-isolation of virus from experimentally infected dogs displaying clinical signs of disease, signifies a causal relationship between crcov and respiratory disease, which until now has been best demonstrated through epidemiological surveys (erles et al., 2004 (erles et al., , 2003 decaro et al., 2007; kaneshima et al., 2006; priestnall et al., 2006 priestnall et al., , 2007 knesl et al., 2009) . in all treatment groups crcov was detected most frequently in the trachea, nasal tonsil and lung lavage fluid, and these same tissues also exhibited the highest viral titres (data not shown). this is consistent with previous findings from a small cohort of naturally infected dogs in which the highest viral loads, detected by quantitative rt-pcr, were seen in the trachea and nasal tonsil . importantly, histological analyses were consistent with our molecular and virological findings. ihc revealed coronavirus positive staining in the cytoplasm of ciliated epithelial and goblet cells of the trachea and bronchioles of crcov infected dogs. this pattern of staining is consistent with previous work which identified coronavirus antigenpositive epithelial cells within the trachea of dogs naturally infected with crcov (ellis et al., 2005; priestnall, 2007) , and in crcov infected tracheal sections maintained in culture (priestnall et al., 2009; priestnall, 2007) . furthermore, histopathological analysis showed a clear association between exposure to crcov and inflammation in the nares and trachea, with loss or damage to cilia in the latter. these changes were particularly marked in dogs belonging to t4 and t5, which may indicate a higher degree of pathogenicity for those crcov strains; although, with the exception of one dog in t5 this was not apparent from the clinical signs. ciliary clearance is a key strategy for the removal of pathogens from the respiratory tract. reductions in the efficiency of ciliary clearance would potentiate infection with secondary respiratory pathogens, leading to increased severity and duration of disease. in addition to the trachea, the nasal tonsil may also represent an important site for crcov infection and pathogenicity; given the consistency and duration of crcov isolation and detection in this tissue. preliminary ihc analysis has shown that crcov infection in the nasal tonsil is associated with the epithelium, and with large mononuclear cells which have the appearance of macrophages (priestnall, 2007) . a clear association of crcov with macrophages is yet to be confirmed, but this could present another interesting area given the importance of macrophages in the pathogenesis of disease associated with other coronaviruses, such as feline infectious peritonitis virus (fipv), severe acute respiratory syndrome coronavirus (sars-cov) and human coronavirus 229e (hcov-229e) (desforges et al., 2007; rottier et al., 2005; shieh et al., 2005) . the detection and re-isolation of crcov from the lungs indicates the virus can also establish an infection in the lower airways. gross and histopathological analysis showed that both the control and challenged dogs had focal reddening of the lungs at necropsy, however no significant histological abnormalities were associated specifically with this reddening, and such reddening is assumed to be an artefact of barbiturate euthanasia (lopez, 2001) . nonetheless, in most cases there were microscopic differences between the lungs of these dogs, which could not be appreciated grossly, and therefore the gross appearance of the lungs was not considered an accurate predictor of histopathological changes in these experimental conditions. pulmonary inflammation in crcov challenge dogs was associated with lymphoid aggregates adjacent to the airways or blood vessels. the significance of this is uncertain, but notably these aggregates were not a striking feature of the control dogs. histological changes in the lungs and bronchial lymph nodes of dogs in t6 tended to be more consistent and mild; whilst scores for dogs in crcov challenge groups, although not marked, were more variable throughout the study. histological changes in the lymphoid tissues of most dogs in the study (t1-t6 inclusive) included hyperplasia. lymphoid hyperplasia is not uncommon in dogs at this age, particularly in the palatine tonsil. acute inflammatory responses were evident; however in some dogs this may have been associated with the presence of fragments of foreign material (e.g. hair) in the tonsillar crypts. where marked lymphoid hyperplasia was seen, this was reflected in the histological scores, which were more variable in the challenge dogs compared to those in the broth control animals throughout the trial period. the close genetic relationship between crcov and bovine coronavirus (bcov) (erles et al., 2003 , raised the question as to whether crcov may have an extended tropism which involves the gastrointestinal tract, as seen with some bcov strains (park et al., 2007) . the molecular detection of crcov in the rectal swabs of some dogs from t1 is interesting, and consistent with findings from a number of previous reports of dogs naturally infected with crcov (decaro et al., 2007; yachi and mochizuki, 2006; mitchell et al., 2009) . dogs in the t1 challenge group were unique in that they were the only dogs to be challenged with virus that had not been grown in vitro. the inability to detect crcov from dogs in other challenge groups may therefore be a result of cell culture adaptation; alternatively, this may be a strain specific phenomenon. at present there is no conclusive evidence that crcov displays a true dual tropism for respiratory and enteric tissues. the failure to isolate crcov from enteric samples collected during the peak oropharyngeal shedding period, suggests that crcov may pass through the canine gut as a bystander, without infection. nevertheless, enteric shedding could have implications for managing the spread of disease, and therefore further investigation is warranted. in addition to the histological changes observed in the tissues, significant changes in the leukogram can also be detected in association with crcov infection. this is summarised by a statistically and also clinically relevant lymphocytosis between days 6 and 14; most clearly seen in groups t1, t2, and t5. this observation is not unexpected due to the viral antigenic stimulation, and the lymphocytic reactions found in various tissues. what was somewhat less expected is the high number of dogs with decreased neutrophil concentrations, including a number which developed neutropenia, mild left shift and toxicity, best seen in groups t1, t2, t4, and t5. these changes suggest an acute inflammatory reaction with a high demand for neutrophils and accelerated production in the marrow. transient neutropenia is not uncommon during infections with some viruses; however, there is currently limited data relating to the leukogram profile following infections with other beta coronaviruses. in one report detailing the experimental infection of cows with bcov, significant reductions in neutrophil concentrations were observed at 2 days post infection, followed by neutrophilia at 14 days post infection (traven et al., 2001) . in sars coronavirus infected patients the picture is mixed. neutropenia has been reported in some cases, however, in most cases high blood neutrophil concentrations were observed, and this is often associated with a poor clinical outcome manocha et al., 2003; wong et al., 2003) . neutrophil infiltration of tissues infected with different coronaviruses such as sars, mhv and rat cov has also been described (bhatt and jacoby, 1977; iacono et al., 2006; ding et al., 2003) ; however, their role in viral clearance and possible immune pathology is largely unknown. considering the presence of mostly lymphoid aggregates in crcov infected tissues in this study, the observed changes are intriguing, and a direct effect of crcov on the myeloid series in the bone marrow cannot be ruled out. seroconversion to crcov and the acquisition of neutralising antibodies to heterologous strains from distinct geographical locations within the usa and uk occurred in all challenge dogs remaining on the study at day 14. this degree of serological cross reactivity is not unexpected given the high degree of amino acid similarity seen in the spike protein of different isolates published to date. whilst the correlation between neutralising titres and protection in vivo is yet to be determined, this finding supports published data which demonstrated that the presence of crcov specific antibodies in dogs significantly decreased the risk of developing respiratory disease upon entry to the kennel (erles et al., 2003) . these findings support a possible role for vaccinating against crcov as part of a preventative strategy for respiratory disease in kennelled dogs. moreover, based on the high degree of cross neutralisation and high degree of amino acid identity among the crcov spike proteins described to date, it is anticipated that a single crcov isolate as a vaccine antigen will be sufficient to provide protection against crcov induced respiratory disease. in summary, this is the first study to describe the development of a model for studying crcov pathogenesis; and to fully demonstrate koch's postulates through the successful re-isolation of crcov from experimentally infected dogs with clinical signs of respiratory disease. isolation of crcov was achieved from a wide variety of respiratory and mucosal associated lymphoid tissues, lung lavage fluids and swabs collected over the 2-week period, thus providing clear evidence of tropism for the canine respiratory tract, accompanied by respiratory shedding. moreover, we have shown crcov infection is associated with marked histopathological changes in the nares and trachea, with loss and damage to tracheal cilia alongside inflammatory responses. significant effects on the leukogram, in the form of clinically relevant lymphocytosis and neutrophil changes were also documented. strong serological and cross neutralising reactivity between heterologous crcov isolates demonstrates antigenic homogeneity among crcov from the two continents. this study provides vital evidence supporting a role for crcov in the cird complex. defining the role that crcov and other agents play in cird is a considerable, but important, challenge if the disease is to be managed, treated and prevented more successfully. canine infectious tracheobronchitis short review: kennel cough canine infectious tracheobronchitis short review: kennel cough sv-5-like parainfluenza virus in dogs bordetella and mycoplasma respiratory infections in dogs and cats pathogenesis of canine bordetellosis experimental infection of adult axenic rats with parker's rat coronavirus canine respiratory viruses bronchiolitis obliterans and pneumonia induced in young dogs by experimental adenovirus infection mycoplasmas associated with canine infectious respiratory disease serological and molecular evidence that canine respiratory coronavirus is circulating in italy activation of human monocytes after infection by human coronavirus 229e the clinical pathology of severe acute respiratory syndrome (sars): a report from association of canine adenovirus (toronto a 26/61) with an outbreak of laryngotracheitis (''kennel cough''): a preliminary report detection of coronavirus in cases of tracheobronchitis in dogs: a retrospective study from 1971 to investigation into the causes of canine infectious respiratory disease: antibody responses to canine respiratory coronavirus and canine herpesvirus in two kennelled dog populations canine respiratory coronavirus: an emerging pathogen in the canine infectious respiratory disease complex detection of a group 2 coronavirus in dogs with canine infectious respiratory disease longitudinal study of viruses associated with canine infectious respiratory disease isolation and sequence analysis of canine respiratory coronavirus canine glyceraldehyde-3-phosphate dehydrogenase complementary dna: polymerase chain reaction amplification, cloning, partial sequence analysis, and use as loading control in ribonuclease protection assays both spike and background genes contribute to murine coronavirus neurovirulence the prevalence of a group 2 coronavirus in dogs in japan canine tracheobronchitis: isolation and characterization of the agent with experimental reproduction of the disease role of bordetella bronchiseptica in infectious tracheobronchitis in dogs the seroprevalence of canine respiratory coronavirus and canine influenza virus in dogs in new zealand a major outbreak of severe acute respiratory syndrome in hong kong respiratory system, thoracic cavity and pleura in thomson's special veterinary pathology severe acute respiratory distress syndrome (sars): a critical care perspective development of a quantitative real-time pcr for the detection of canine respiratory coronavirus dual enteric and respiratory tropisms of winter dysentery bovine coronavirus in calves the role of a novel coronavirus in canine infectious respiratory disease. royal veterinary college serological prevalence of canine respiratory coronavirus serological prevalence of canine respiratory coronavirus in southern italy and epidemiological relationship with canine enteric coronavirus quantification of mrna encoding cytokines and chemokines and assessment of ciliary function in canine tracheal epithelium during infection with canine respiratory coronavirus (crcov) acquisition of macrophage tropism during the pathogenesis of feline infectious peritonitis is determined by mutations in the feline coronavirus spike protein immunohistochemical, in situ hybridization, and ultrastructural localization of sars-associated coronavirus in lung of a fatal case of severe acute respiratory syndrome in taiwan experimental reproduction of winter dysentery in lactating cows using bcv -comparison with bcv infection in milk-fed calves haematological manifestations in patients with severe acute respiratory syndrome: retrospective analysis survey of dogs in japan for group 2 canine coronavirus infection this study was funded by pfizer animal health. the authors would like to thank m. ikeh for technical assistance. rvc manuscript id number: pid_00399. key: cord-274007-zndtddty authors: rasmussen, sonja a.; smulian, john c.; lednicky, john a.; wen, tony s.; jamieson, denise j. title: coronavirus disease 2019 (covid-19) and pregnancy: what obstetricians need to know date: 2020-02-24 journal: am j obstet gynecol doi: 10.1016/j.ajog.2020.02.017 sha: doc_id: 274007 cord_uid: zndtddty coronavirus disease 2019 is an emerging disease with a rapid increase in cases and deaths since its first identification in wuhan, china, in december 2019. limited data are available about coronavirus disease 2019 during pregnancy; however, information on illnesses associated with other highly pathogenic coronaviruses (ie, severe acute respiratory syndrome and the middle east respiratory syndrome) might provide insights into coronavirus disease 2019’s effects during pregnancy. coronaviruses cause illness ranging in severity from the common cold to severe respiratory illness and death. currently the primary epidemiologic risk factors for coronavirus disease 2019 include travel from mainland china (especially hubei province) or close contact with infected individuals within 14 days of symptom onset. data suggest an incubation period of ∼5 days (range, 2–14 days). average age of hospitalized patients has been 49–56 years, with a third to half with an underlying illness. children have been rarely reported. men were more frequent among hospitalized cases (54–73%). frequent manifestations include fever, cough, myalgia, headache, and diarrhea. abnormal testing includes abnormalities on chest radiographic imaging, lymphopenia, leukopenia, and thrombocytopenia. initial reports suggest that acute respiratory distress syndrome develops in 17–29% of hospitalized patients. overall case fatality rate appears to be ∼1%; however, early data may overestimate this rate. in 2 reports describing 18 pregnancies with coronavirus disease 2019, all were infected in the third trimester, and clinical findings were similar to those in nonpregnant adults. fetal distress and preterm delivery were seen in some cases. all but 2 pregnancies were cesarean deliveries and no evidence of in utero transmission was seen. data on severe acute respiratory syndrome and middle east respiratory syndrome in pregnancy are sparse. for severe acute respiratory syndrome, the largest series of 12 pregnancies had a case-fatality rate of 25%. complications included acute respiratory distress syndrome in 4, disseminated intravascular coagulopathy in 3, renal failure in 3, secondary bacterial pneumonia in 2, and sepsis in 2 patients. mechanical ventilation was 3 times more likely among pregnant compared with nonpregnant women. among 7 first-trimester infections, 4 ended in spontaneous abortion. four of 5 women with severe acute respiratory syndrome after 24 weeks’ gestation delivered preterm. for middle east respiratory syndrome, there were 13 case reports in pregnant women, of which 2 were asymptomatic, identified as part of a contact investigation; 3 patients (23%) died. two pregnancies ended in fetal demise and 2 were born preterm. no evidence of in utero transmission was seen in severe acute respiratory syndrome or middle east respiratory syndrome. currently no coronavirus-specific treatments have been approved by the us food and drug administration. because coronavirus disease 2019 might increase the risk for pregnancy complications, management should optimally be in a health care facility with close maternal and fetal monitoring. principles of management of coronavirus disease 2019 in pregnancy include early isolation, aggressive infection control procedures, oxygen therapy, avoidance of fluid overload, consideration of empiric antibiotics (secondary to bacterial infection risk), laboratory testing for the virus and coinfection, fetal and uterine contraction monitoring, early mechanical ventilation for progressive respiratory failure, individualized delivery planning, and a team-based approach with multispecialty consultations. information on coronavirus disease 2019 is increasing rapidly. clinicians should continue to follow the centers for disease control and prevention website to stay up to date with the latest information (https://www.cdc.gov/coronavirus/2019-ncov/hcp/index.html). e merging infections have been shown to have an important impact on pregnant women and their fetuses, 1 with the increased risk of complications in pregnant women with the 2009 pandemic h1n1 influenza virus 2 and the severe fetal effects of zika virus as recent examples. 3, 4 the emergence of a coronavirus not previously seen in humans, first reported in wuhan, china, on dec. 31, 2019, has attracted much interest throughout the world. since then, the number of reported cases has increased rapidly, with more than 51,800 laboratory-confirmed cases and 1600 deaths as of feb. 16, 2020. in addition to china, cases have spread to 25 other countries ( figure 1 ) including 15 cases in the united states. initial outbreak data from china show a near exponential growth of reported cases. 5 reported numbers are likely underestimates of the true numbers because milder cases are less likely to be reported. on jan. 30, 2020, the world health organization declared the outbreak as a public health emergency of international concern; on jan. 31, 2020, the united states declared a public health emergency, and the centers for disease control and prevention issued a federal quarantine for 195 americans who traveled from wuhan, china, its first federal quarantine in more than 50 years. on feb. 11, the new coronavirus disease (previously referred to as 2019 novel coronavirus (2019-ncov)) received an official name from the world health organization (who), coronavirus disease 19 (covid-19) (figure 2 ). 6 the international committee on taxonomy of viruses has proposed severe acute respiratory syndrome coronavirus 2 (sars-cov-2) as the name of the virus that causes covid-19. 7 coronaviruses are single-stranded rna, nonsegmented, enveloped viruses, which cause illness ranging in severity from the common cold to severe and fatal illness. the term coronavirus derives from the latin word corona, which means crown or halo; that designation arises from the appearance of coronavirus virions viewed by electron microscopy, in which the virus particles display a crown-like fringe typically referred to as spikes ( figure 3 ). in the past 2 decades, 2 other coronaviruses that cause severe respiratory illness in humans have emerged: severe acute respiratory syndrome coronavirus (sars-cov) and the middle east respiratory syndrome coronavirus (mers-cov). with the emergence of sars-cov-2, a third coronavirus that can cause severe respiratory illness has been identified. in a short period of time, this novel coronavirus has caused more cases of illness than were reported for mers and sars combined. here we summarize what is currently known about covid-19 and what this means for practicing obstetricians and their pregnant patients. because so little is currently known about covid-19 in pregnancy, we also review available information on the effects of sars and mers during pregnancy to inform care of pregnant women with covid-19 until additional data on pregnant women and their fetuses become available. severe acute respiratory syndrome (sars) is caused by the sars-cov. reports of the emergence of sars-cov appeared in february 2003, with the first cases observed in guangdong province in china. the virus spread to nearly 30 countries throughout the world, resulting in more than 8000 cases and 770 deaths. 8 the outbreak was brought under control after public health control measures to reduce contact with infected persons were put into place, and no cases have been seen since 2004. manifestations of sars consist of fever, chills, headache, malaise, and myalgia. diarrhea was seen in some patients. pneumonia was nearly always seen in patients diagnosed with sars, with mechanical ventilation being required in 10e20% of cases. case fatality rate was estimated at 9e10% (table) . the natural reservoir for sars-cov is believed to be bats; however, some evidence supported civet cats or raccoon dogs as possible intermediate sources of these illnesses. 8 sars is transmitted by close person-to-person contact through contact of the mucus membranes of the respiratory tract with respiratory droplets formed when an infected person coughs or sneezes. fecal-oral transmission and transmission via fomites have also been reported. 8 airborne spread because of inhalation of small particle aerosols may also be possible. transmission in health care settings was frequently seen during the 2003 outbreak, with superspreading (when a single patient transmits infection to a disproportionate number of contacts) reported. 9 the incubation period was estimated at a mean of 4.6 days, with a range of 2e14 days. transmission appeared to occur most often during the second week of illness when viral excretion is highest; there is no evidence that a person with sars is contagious before symptom onset. the largest case series of pregnant women with sars was from the 2003 outbreak in hong kong, in which 12 pregnant women were identified. 10 the case-fatality rate was 25% (3 deaths). clinical and laboratory findings were similar to those seen in the nonpregnant population. pneumonia on chest radiograph or computed tomography was seen in all patients. major medical complications included adult respiratory distress syndrome in 4, disseminated intravascular coagulopathy (dic) in 3, renal failure in 3, secondary bacterial pneumonia in 2, and sepsis in 2 patients. pregnancy outcomes varied by trimester of presentation. 10 among the 7 women who became ill in the first trimester, 4 had a spontaneous abortion, 2 had pregnancy terminations for social reasons after recovery from sars, and 1 delivered a full-term healthy infant. among the 5 women who presented after 24 weeks' gestation, 4 delivered preterm. three women delivered by cesarean delivery because of deteriorating maternal condition from their sars illness at 26, 28, and 32 weeks' gestation. 11 these babies had birthweights appropriate for gestational age. two of the infants had respiratory distress syndrome requiring surfactant (born at 26 and 28 weeks' gestation), with one later developing bronchopulmonary dysplasia. gastrointestinal complications were observed in 2 infants, including a jejunal perforation in an infant delivered at 26 weeks and necrotizing enterocolitis with ileal perforation in an infant delivered at 28 weeks' gestation. whether these gastrointestinal complications were related to complications from sars or its treatment or whether they were secondary to preterm delivery is unknown. 11 the two infants who were delivered after their mothers' recovery from sars had intrauterine growth restriction. no clinical, radiologic, or laboratory evidence for transmission from mother to fetus was observed, despite laboratory testing of different specimens. 12, 13 a matched case-control study 14 compared 10 of the 12 pregnant women noted in the previous text (2 were excluded because they were unable to be matched) with 40 nonpregnant women with sars. women were matched on sex, age, timing of contracting sars, health care worker status, underlying illness, and whether the woman resided in a housing area in which there was a large outbreak. pregnancy appeared to have no effect on clinical symptoms or time to presentation after symptom onset. however, complications and adverse outcomes were more common among pregnant women: women who were pregnant had a longer hospital stay, were statistically significantly more likely to develop renal failure, sepsis, and dic, and were more likely to require intensive care unit admission. forty percent of pregnant women required mechanical ventilation, compared with 13% of nonpregnant patients (p ¼.07). pregnant women were also significantly more likely to die (p ¼ .01). we identified 5 reports of additional cases of sars during pregnancy treated in hong kong (n ¼ 2), the united states (n ¼ 2), and canada (n ¼ 1). 15e19 two of the 5 women required mechanical ventilation, 1 required hemodialysis for acute renal failure, and 1 had seizures and positive cerebrospinal fluid for sars-cov, suggestive of a central nervous system infection. all patients recovered from their illness. in 1 case, the pregnancy was terminated at the mother's request; the remaining pregnancies ended in liveborn infants (2 at term and 2 preterm). testing of neonatal specimens for sars-cov rna was negative. several hospitals in toronto and hong kong reported measures instituted on obstetrics services during the sars outbreak to decrease transmission to pregnant women, their families, community members, and health care workers. 20, 21 for example, all hospital staff, patients, and visitors were screened for symptoms at the hospital entrance and wore n95 respirators. visitors were limited to 1 per patient on labor and delivery, with no visitors allowed in the postpartum ward. global map of confirmed covid-19 cases (as of feb. 14, 2020) (from https://www.cdc.gov/ coronavirus/2019-ncov/locations-confirmed-cases.html). rasmussen. 2019 novel coronavirus and pregnancy. am j obstet gynecol 2020. postpartum stays were reduced in length with a postpartum nurse home visit added. postpartum patients were asked to observe a 10 day home quarantine. health care workers were asked to observe a work quarantine in which they were asked to go directly from home to work and vice versa to minimize interaction in the community. obstetric services considered to be nonessential such as routine ultrasound and prenatal diagnosis were suspended. although the impact of these interventions was not evaluated, there may be some relevant lessons learned from these experiences during sars that could help inform the approach to covid-19. middle east respiratory syndrome (mers) is a respiratory illness caused by mers-cov. the illness was first identified in saudi arabia in 2012, with spread to other countries in the arabian peninsula and eventually to countries outside the arabian peninsula, including the united states. 22, 23 the largest outbreak outside the arabian peninsula was in the republic of korea in 2015. nearly 2500 cases of mers-cov illness and more than 860 deaths have been reported with continuing reports into the present. the manifestations of mers include severe respiratory illness characterized by fever, cough, and shortness of breath. some patients also have diarrhea. the case fatality rate is estimated to be 35e40%. patients who developed mers were more likely to be older (median age is 50 years) with about two thirds of patients being male. patients with mers were also more likely to have an underlying illness. some patients with mers-cov infection have been asymptomatic (identified through contact investigations). the mean incubation period is 5.2 days, with a range of 2e13 days. as with sars, mers is mainly spread person to person through close contact, with transmission in health care settings, and superspreading events have been observed. however, since 2016, the number of cases of mers-cov has been dramatically reduced after public health efforts to prevent mers-cov transmission were put into place. 24 information on mers among pregnant women is limited. we identified reports of 13 cases of pregnant women with mers from several countries, including saudi arabia (n ¼ 8), korea (n ¼ 2), jordan (n ¼ 1), united arab emirates (n ¼ 1), and philippines (n ¼ 1). 13,25e31 two women were asymptomatic, identified as part of a contact investigation. among the 11 symptomatic women, manifestations were similar to those seen in nonpregnant patients with mers. timeline showing key events in the covid-19 outbreak expert review ajog.org seven of 13 patients were admitted to an intensive care unit for respiratory deterioration or acute respiratory distress syndrome, 5 required ventilator support, 3 died, and 8 recovered. among the 3 deaths, the mothers died 8e25 days after delivery. both babies born to asymptomatic women were born healthy at term; among those who were symptomatic, there was 1 intrauterine fetal demise, 1 stillbirth, 1 baby delivered at 25 weeks who died 4 hours after birth, 2 healthy preterm infants, and 5 healthy term infants (infant status was not mentioned for 1). early data suggested an association between the huanan seafood wholesale market and covid-19 with 27 of 41 cases in 1 report 32 and 26 of 47 in another report 33 with epidemiologic links to the market, leading to closure of the market on jan. 1, 2020. given that the earliest case reported (illness onset on dec. 1, 2019) 32 did not have exposure to the market raises the possibility that the initial emergence into humans occurred elsewhere. however, sampling of the market's environment supports the market's importance in early transmission of the virus. later cases were much less likely to have visited the market, supporting the role of personto-person transmission in later cases. the sars-cov-2 is a betacoronavirus similar to sars-cov and mers-cov (table) . sequencing data show that the sars-cov-2 is most closely related to coronaviruses found in bats, with more than 85% nucleotide identity with a bat sars-like cov. 34, 35 the virus has 79% nucleotide identity to sars-cov and about 50% to mers-cov. 35 bats appear to be the natural reservoirs of both sars-cov and mers-cov. the emergence of these viruses in humans has been attributed to host switching: the virus jumped from an intermediary host species (eg, civet cats for sars-cov and dromedary camels for mers-cov) to humans. an intermediary host species is thought to be likely for sars-cov-2, 35 although it has been yet to be identified. sequence data show a high degree (>99.98%) of similarity of the virus among different patients, suggesting a recent emergence in humans. clinical manifestations of covid-19 are similar to those with sars and mers (table) . studies of hospitalized patients with covid-19 show that patients commonly develop severe pneumonia, with 23e32% admitted to the intensive care unit and 17e29% of cases progressing to acute respiratory distress syndrome. 32, 36, 37 among hospitalized patients, 4e15% have died. 32, 36, 37 overall case fatality ratio estimates (including asymptomatic and symptomatic infections) appear to be in the range of 1% (95% confidence interval, 0.5e4%), 38 although these estimates should be considered preliminary. average age of hospitalized patients was 49e56 years, with 32e51% having an underlying illness. most patients (54e73%) were men. children with covid-19 appear to be rarely identified, with only 28 children reported as of jan. 30, 2020 (<1% of total), and most of those identified had mild symptoms. 39 no pregnant women were reported in any of these initial cohorts. common manifestations among hospitalized patients were fever (83e100%), cough (59e82%), myalgia (11e35%), headache (7e8%), and diarrhea (2e10%). all patients had abnormalities on radiographic imaging of the chest. person-to-person transmission of sars-cov-2 is thought to be similar to transmission of influenza and other respiratory pathogens; respiratory droplets are formed when an infected person coughs or sneezes and these droplets are inhaled by close contacts, generally within 6 feet. it is unclear whether infection can be transmitted from fomites. fecal-oral transmission might be possible, given that sars-cov-2 has been identified in stool specimens 40 and sars-cov might have been transmitted in this manner. 41 the basic reproduction number, r0 (the average number of people who will become infected from a single infected person in a population in which all persons are susceptible) is affected by factors such as the duration of infectivity, the transmissibility of the pathogen, and the number of susceptible contacts. measles, which is highly infective, has an r0 of 12e18, while 2009 h1n1 influenza and sars have an r0 of 1.2e1.6 and 2e5, respectively. 42 current estimates of r0 for sars-cov-2 places it at 2.2 (95% confidence interval, 1.4e3.9) 33 as with sars and mers, nosocomial transmission is playing a key role in transmission, presumed to be responsible for infection of 29% of affected health professionals and 12% of hospitalized patients in a recent study. 37 in the midst of a rapidly evolving outbreak that could have significant effects on our public health and medical infrastructure, the unique needs of pregnant women should be included in preparedness and response plans. in previous outbreaks, clinicians have at times been reluctant to treat or vaccinate pregnant women because of concerns for fetal safety. 43 it is critical that pregnant women not be denied potentially lifesaving interventions in the context of a serious infectious disease threat unless there is a compelling reason to exclude them. as with all decisions regarding treatment during pregnancy, carefully weighing of the benefits of interventions for the mother and fetus with potential risks is necessary. as surveillance systems 32, 33, 36, 37 this observed gender difference could be due to differences in reporting, susceptibility, exposure, or recognition and diagnosis of infection. there are no data to inform whether pregnancy increases susceptibility to covid-19. previous data on sars and mers suggest that clinical findings during pregnancy can range from no symptoms to severe disease and death. the most common symptoms of covid-19 are fever and cough, with more than 80% of hospitalized patients presenting with these symptoms. 36 in a recent study by chen et al, 44 9 women diagnosed with covid-19 during the third trimester of pregnancy were reported. in this small series, clinical presentation was similar to that seen in nonpregnant adults, with fever in 7, cough in 4, myalgia in 3, and sore throat and malaise each in 2 women. five had lymphopenia. all had pneumonia, but none required mechanical ventilation, and none died. all women had a cesarean delivery, and apgar scores were 8e9 at 1 minute and 9e10 at 5 minutes. in a second series of 9 pregnancies with 10 infants (1 set of twins) reported by zhu et al., 45 symptom onset was before delivery (1e6 days) in 4, on the day of delivery in 2, and after delivery (1e3 days) in 3 cases. clinical presentation of covid-19 was similar to that seen in nonpregnant patients. among the 9 pregnancies, intrauterine fetal distress was noted in 6, 7 were cesarean deliveries, and 6 infants were born preterm. based on these limited reports and the available data from other respiratory pathogens such as sars and influenza, it is unknown whether pregnant women with covid-19 will experience more severe disease. travel guidance for pregnant women. travel recommendations have been instituted to limit exposure to persons in the united states. all persons, including pregnant women, should not travel to china. on feb. 2, 2020, the us state department upgraded their travel advisory to level 4, the highest level of travel advisory. obstetric providers should obtain a detailed travel history for all patients and should specifically ask about travel in the past 14 days to areas experiencing widespread transmission of sars-cov-2. currently this is limited to china, but this situation is rapidly evolving and obstetricians should stay alert to the global situation by consulting the centers for disease control and prevention website and following media coverage. there is currently no vaccine to prevent covid-19. since posting of a sars-cov-2 virus genetic sequence online on jan. 10, 2020, multiple organizations, including the national institutes of health, have been working to rapidly develop a covid-19 vaccine. development of this vaccine builds on and benefits from work on sars and mers vaccines. 46 however, it is not known how quickly a safe and effective vaccine may be readily available. infection control measures and diagnostic testing. all patients, including pregnant women, should be evaluated for fever and signs and symptoms of a respiratory infection. ideally, screening procedures begin before arrival on a labor and delivery unit or prenatal care clinic. for example, when those patients with respiratory symptoms should be separated from other waiting patients and a facemask should be placed on them. patients who meet criteria for a person under investigation (box 1) should be immediately placed in an airborne infection isolation room (single-patient rooms at negative pressure). once in isolation, the patient's facemask may be removed. health care personnel should adhere to standard, contact, and airborne precautions. infection control personnel and local/ state health departments should be notified immediately; local/state health departments can help to arrange testing of relevant specimens (upper and lower respiratory specimens and serum are currently recommended; other specimens [stool and urine] may also be sent). general principles regarding management of covid-10 during pregnancy include early isolation, aggressive infection control procedures, testing for sars-cov-2 and coinfection, oxygen therapy as needed, avoidance of fluid overload, empiric antibiotics (because of secondary bacterial infection risk), fetal and uterine contraction monitoring, early mechanical ventilation for progressive respiratory failure, individualized delivery planning, and a team-based approach with multispecialty consultations (box 2). team-based management is recommended for pregnancies managed in a health care facility and should include a determination of the optimal clinical unit on which to provide care. ability to provide surveillance for early detection of a worsening maternal course of illness, as well as an ability to monitor for evidence of obstetric complications (eg, preterm labor or fetal compromise), are needed. changes in fetal heart rate pattern may be an early indicator of maternal respiratory deterioration. based on experience with sars and mers, severe respiratory failure might occur in box 2 principles for management of pregnant women with confirmed or suspected covid-19 48,53,54,a patients with respiratory symptoms should adhere to respiratory hygiene, cough etiquette, and hand hygiene. ensure rapid triage of pregnant patients with respiratory symptoms. patients with respiratory symptoms should wear a facemask and wait in a separate, wellventilated waiting area at least 6 feet from other people. confirmed and suspected cases of covid-19 should be isolated as soon as possible in an aiir. if an aiir is not available, consider transfer to a hospital with an aiir. implement cdc infection prevention and control procedures for health care providers including standard, contact, and airborne precautions. eye protection and properly fitted n95 respirators should be used. provide additional staff training in correct use of personal protective equipment including correct donning, doffing, and disposal of personal protective equipment. contact hospital infection personnel. in coordination with local/state health department, collect and send relevant specimens for diagnostic sars-cov-2 testing. limit visitor and health care personnel access to patient rooms with a confirmed or suspected case. pregnancy should be considered a potentially increased risk condition and monitored closely including fetal heart rate and contraction monitoring. consider early oxygen therapy (target o 2 saturations 95% and/or po 2 70 mm hg). consider early mechanical ventilation with evidence of advancing respiratory failure. noninvasive ventilation techniques may have a small increased risk of aspiration in pregnancy. use intravenous fluids conservatively unless cardiovascular instability is present. screen for other viral respiratory infections and bacterial infections (because of risk of coinfections). consider empiric antimicrobial therapy (because of risk for superimposed bacterial infections). consider empiric treatment for influenza, pending diagnostic testing. do not routinely use corticosteroids. use of steroids to promote fetal maturity with anticipated preterm delivery can be considered on individual basis. if septic shock is suspected, institute prompt, targeted management. delivery and pregnancy termination decisions should be based on gestational age, maternal condition, and fetal stability, and maternal wishes. consult with specialists in obstetrics, maternal-fetal medicine, neonatology, intensive care, anesthesia, and nursing. communicate with patients and families regarding diagnosis, clinical status, and management wishes. ajog.org expert review pregnant women, and in the most severe cases, mechanical ventilation might not be sufficient to support adequate oxygenation. if that occurs, limited literature suggests a potential role of extracorporeal membrane oxygenation in pregnancy; use should be considered only in centers that have experience with this technique. 47 whether delivery provides benefit to a critically ill mother is unknown; decisions regarding delivery should consider the gestational age of the fetus and should be made in conjunction with the neonatologist. 48 there are currently no antiviral medications approved by the us food and drug administration for treatment of covid-19, although broad-spectrum antivirals used in animal models of mers are being evaluated for activity against sars-cov-2. 46 corticosteroids for the treatment of coronavirusassociated pneumonia should be avoided unless other indications are present because they were not shown to be beneficial in mers and could lead to delayed mers-cov clearance. 49 therefore, decisions about the use of corticosteroids for fetal lung maturity should be made in consultation with infectious disease specialists and maternal-fetal medicine consultants. all guidance should be considered subject to revision as additional data on pregnant women with covid-19 become available. care of infants born to mothers with covid-19. although the limited experience with newborn evaluations after delivery with sars and mers has not identified cases of maternalto-fetal transmission, reports have appeared in the media of a 30 hour infant who was diagnosed with covid-19, suggesting the possibility of in utero transmission. 50 however, insufficient information is included in media reports to rule out perinatal or postnatal modes of transmission. data from the recent case series published by chen et al 44 and zhu et al 45 of 18 women (19 infants) infected in the third trimester of pregnancy with sars-cov-2 identified no laboratory evidence of vertical transmission. testing of amniotic fluid, cord blood, and neonatal throat swab samples was negative for sars-cov-2 in the 6 patients reported by chen et al. 44 in the report by zhu et al., 45 some infants were symptomatic (shortness of breath in 6, cyanosis in 3, gastric bleeding in 2, and 1 baby died of multiple organ failure and dic); however, throat swab testing of all infants was negative for sars-cov-2 , suggesting that these neonatal complications might not be related intrauterine transmission. thus, at this time, it is unknown whether sars-cov-2 can be transmitted from mother-to-fetus. given the current lack of information, it seems reasonable to assume that a newborn born to a mother with covid-19 at delivery could possibly be infected, either in utero or perinatally, and thus should be placed in isolation to avoid exposure to other newborns. although the ideal setting for a healthy infant is within a healthy mother's room, temporary separation of an ill mother and her infant, as was recommended during pandemic h1n1, 51 seems prudent. whether covid-19 can be transmitted through breastmilk is unknown. we are aware of a single report of sars-cov testing of breastmilk in a mother who had recovered from sars and no viral rna was detected; however, the specimen was collected w130 days after illness onset. 15 sars-cov antibodies were seen in breastmilk of that patient 15 but not in another patient who was infected at 7 weeks' gestation with breastmilk tested at postpartum days 12 and 30. 16 breastmilk was tested for sars-cov-2 in 6 of the mothers reported by chen et al 44 ; all specimens were negative. until additional data are available, mothers who intend to breastfeed and are well enough to express breastmilk should be encouraged to do so; breastfeeding can be instituted after she is no longer considered infectious. no data are available to guide length of separation and will need to be decided on a case-by-case basis after discussion between infection control experts and neonatologists. the covid-19 outbreak is rapidly increasing in the number of cases, deaths, and countries affected. much is unknown about the virus and its effects, including its modes of transmission, the basic reproduction number, risk factors for illness, and case fatality rate. although cases are primarily in china, it is highly likely that there will be additional global spread of the virus. at the present time, limited data are available on pregnant women with covid-19 on which to base recommendations for pregnancy-specific care; however, early reports and lessons from sars, mers, and other respiratory infections suggest that pregnant women could have a severe clinical course. surveillance systems for cases of covid-19 need to include information on pregnancy status as well as maternal and fetal outcomes. it is important to be vigilant about the spread of the disease and be able to provide rapid implementation of outbreak control and management measures once the virus reaches a community. standard interventions to manage any severe respiratory infection is the foundation of care for any pregnant woman with covid-19 and should be implemented aggressively in a team-based care model. public health approach to emerging infections among pregnant women pandemic 2009 influenza a (h1n1) virus illness among pregnant women in the united states characterizing the pattern of anomalies in congenital zika syndrome for pediatric clinicians zika vrus and birth defectsreviewing the evidence for causality preliminary estimation of the basic reproduction number of novel coronavirus (2019-ncov) in china, from 2019 to 2020: a data-driven analysis in the early phase of the outbreak world health organization. coronavirus disease (covid-19) outbreak. available at: https:// expert review ajog.org www.who.int/emergencies/diseases/novelcoronavirus-2019 severe acute respiratory syndrome-related coronavirus: the species and its viruses-a statement of the coronavirus study group severe acute respiratory syndrome: historical, epidemiologic, and clinical features the role of superspreaders in infectious disease pregnancy and perinatal outcomes of women with severe acute respiratory syndrome infants born to mothers with severe acute respiratory syndrome sars in newborns and children emergency cesarean section in an epidemic of the middle east respiratory syndrome: a case report a casecontrolled study comparing clinical course and outcomes of pregnant and non-pregnant women with severe acute respiratory syndrome sars and pregnancy: a case report sars during pregnancy, united states severe acute respiratory syndrome in pregnancy specific immunoglobulin g antibody detected in umbilical blood and amniotic fluid from a pregnant woman infected by the coronavirus associated with severe acute respiratory syndrome possible central nervous system infection by sars coronavirus the effect of severe acute respiratory syndrome on a hospital obstetrics and gynaecology service managing obstetrical patients during severe acute respiratory syndrome outbreak first confirmed cases of middle east respiratory syndrome coronavirus (mers-cov) infection in the united states, updated information on the epidemiology of mers-cov infection, and guidance for the public, clinicians, and public health authorities middle east respiratory syndrome (mers) worldwide reduction in mers cases and deaths since 2016 middle east respiratory syndrome coronavirus (mers-cov) infection during pregnancy: report of two cases and review of the literature impact of middle east respiratory syndrome coronavirus (mers-cov) on pregnancy and perinatal outcome middle east respiratory syndrome coronavirus infection during pregnancy: a report of 5 cases from saudi arabia middle east respiratory syndrome coronavirus during pregnancy stillbirth during infection with middle east respiratory syndrome coronavirus contact tracing the first middle east respiratory syndrome case in the philippines mers-cov infection in a pregnant woman in korea clinical features of patients infected with 2019 novel coronavirus in wuhan, china early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia a novel coronavirus from patients with pneumonia in china genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a descriptive study clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus-infected pneumonia in wuhan, china who collaborating centre for infectious disease modelling, mrc centre for global infectious disease analysis diagnosis and treatment of 2019 novel coronavirus infection in children: a pressing issue first case of 2019 novel coronavirus in the united states evidence of airborne transmission of the severe acute respiratory syndrome virus pathogenicity and transmissibility of 2019-ncov-a quick overview and comparison with other emerging viruses pregnant women and the ebola crisis clinical characteristics and intrauterine vertical transmission potential of covid-19 infection in nine pregnant women: a retrospective review of medical records clinical analysis of 10 neonates born to mothers with 2019-ncov pneumonia coronavirus infections-more than just the common cold extracorporeal membrane oxygenation (ecmo) during pregnancy and postpartum management of acute respiratory failure in pregnancy corticosteroid therapy for critically ill patients with middle east respiratory syndrome can coronavirus pass from mother to baby? maybe, but experts need more research preparing for influenza after 2009 h1n1: special considerations for pregnant women and newborns receptor recognition by novel coronavirus from wuhan: an analysis based on decade-long structural studies of sars. j virol, in press. for disease control and prevention. interim infection prevention and control recommendations for patients with confirmed 2019 novel coronavirus (2019-ncov) or patients under investigation for 2019-ncov in healthcare settings centers for disease control and prevention. interim guidance for implementing home care of people not requiring hospitalization for 2019 novel coronavirus (2019-ncov) ajog.org key: cord-278935-3lgud7l8 authors: chen, zheng‐rong; mize, maximillion; wang, yu‐qing; yan, yong‐dong; zhu, can‐hong; wang, yunji; ji, wei title: clinical and epidemiological profiles of lower respiratory tract infection in hospitalized children due to human bocavirus in a subtropical area of china date: 2014-04-30 journal: j med virol doi: 10.1002/jmv.23952 sha: doc_id: 278935 cord_uid: 3lgud7l8 lower respiratory tract infection is a major cause of morbidity and mortality in children. human bocavirus (hbov) is confirmed to have an association with pediatric lower respiratory tract infection. seasonal and meteorological factors may play a key role in the epidemiology of hbov. the purpose of this study was to ascertain the frequency, season, and clinical characteristics of hospitalized children with hbov infection. in addition, an evaluation of the effects of meteorological factors on the incidence of hbov in a subtropical area in china will be conducted. children were <14 years in age and hospitalized for lower respiratory tract infection between january 1, 2009 and december 31, 2012 in the respiratory disease department at the children's hospital affiliated to soochow university. multi‐pathogens were detected in nasopharyngeal aspirate samples. the association between hbov activity and regional meteorological conditions was analyzed. the average incidence of hbov infection was 6.6% (502/7,626). of the 502 hbov positive children, the median age was 13 months (range 1–156 months). the hbov infection rate was highest among the 7–12 months groups (12.9%, 163/1,267). seasonal distribution of hbov was noted during june to november, especially during the summer season (june to august). hbov activity was associated with temperature and humidity although the lag effect between temperature and hbov activity observed. hbov is one of the most common viral pathogens in children with lower respiratory tract infection. hbov infection occurs throughout the year with a peak during the summer. temperature and humidity may affect the incidence of hbov. j. med. virol. 86:2154–2162, 2014. © 2014 wiley periodicals, inc. acute respiratory tract infection is a major cause of morbidity and mortality, especially for infants and young children under 5 years old [united nations children's fund, 2011] . the incidence of communityacquired childhood pneumonia in low-and middleincome countries in the year 2010, using world health organization's definition, was about 0.2 (interquartile range [iqr] 0.1-0.5) episodes per childyear, with 11.5% (iqr 8.0-33.0%) of cases progressing to severe episodes [rudan et al., 2013] . a variety of viruses and atypical pathogens, including influenza viruses (iv), respiratory syncytial virus (rsv), parainfluenza virus (piv), adenovirus (adv), human metapneumovirus (hmpv), and mycoplasma pneumoniae have all been associated with acute respiratory tract infection in children. human bocavirus (hbov), belonging to the family parvoviridae, subfamily parvovirinae, and genus bocavirus, was first identified from pooled nasopharyngeal aspirate specimens by large-scale molecular virus screening [allander et al., 2005] . evidence supporting hbov role as an etiologic agent in upper and lower respiratory tract infection in children under 5 years has begun to emerge [manning et al., 2006; weissbrich et al., 2006; allander et al., 2007; canducci et al., 2008; huang et al., 2008; deng et al., 2012] . global hbov activity varies substantially from year-to-year among different populations and regions. furthermore, epidemiological data shows that hbov is present year-round with different incidence rates from 2.2% to 19% in children with lower respiratory tract infection [manning et al., 2006; weissbrich et al., 2006; allander et al., 2007; canducci et al., 2008] . recent studies conducted in several countries have reported that the incidence of hbov fluctuates based on seasonal attributes with infection being higher during winter months [allander et al., 2005; manning et al., 2006; weissbrich et al., 2006] . however, in japan the infectious rate of hbov increases during the spring and summer seasons leading to the belief that regional, in addition to seasonal, factors play a role in the rate of disease transmission [moriyama et al., 2010] . while this suggests that seasonal changes may influence the development of hbov infection [naghipour et al., 2007] , meteorological factors (temperature, humidity, and precipitation) may play the greatest role in the epidemics of hbov based on research conducted in brazil [do amaral de leon et al., 2013] . although few studies have investigated the epidemiological files of hbov infection in children with lower respiratory tract infection in subtropical area in china, a possible relationship between meteorological factors and incidence of hbov is currently unknown. the purpose of this study was to ascertain the frequency, seasonal, and clinical characteristics in hospitalized children with lower respiratory tract infection and evaluate the effects of meteorological factors on the incidence of hbov in a subtropical region of china. in this retrospective study, subjects were <14 years in age and hospitalized for lower respiratory tract infection between january 1, 2009 and december 31, 2012 in the respiratory department at soochow university affiliated children's hospital. the demographic and clinical characteristics of all subjects were collected for analysis. this tertiary teaching hospital in suzhou is the only hospital that provides special care to pediatric patients and the participation rate was $60%. the city of suzhou is located in east of china and has a subtropical climate. informed consent was obtained from parents or legal guardians. this study was conducted with the approval of the institutional human ethical committee of soochow university. patients were considered eligible for enrollment if they had a clinical and radiological diagnosis of community-acquired lower respiratory tract infection including acute bronchiolitis, bronchitis, and pneumonia. chest radiography was performed using standard equipment and radiographic techniques, and reviewed by the radiologists in digital format. before hospital discharge, an attending physician recorded information such as age, gender, clinical diagnosis, underlying chronic diseases, clinical manifestation, and peripheral blood routine test. children with a history of chronic lung disease, underlying immunodeficiency, or preexisting cardiac, renal, neurologic, or hepatic dysfunction, or bronchopulmonary malformation were excluded from the study. lower respiratory tract infection was defined as the presence of wheezing, tachypnea, chest retractions, abnormal auscultatory findings (wheezing and crackles), the presence of fever and radiologic evidence indicative of a lower respiratory tract infection. pneumonia was defined as the presence of focal infiltration (bronchopneumonia) or consolidation (lobar pneumonia) in the lung by chest radiography. very severe pneumonia was defined as the presence of (i) central cyanosis (ii) inability to breastfeed or drink without vomiting (iii) convulsions, lethargy, or unconsciousness (iv) severe respiratory distress as defined by the world health organization. nasopharyngeal aspirate samples were obtained from all patients within 24 hr of admission. this involved passing a suction catheter through the nose with the intent of passing it into the lower part of the pharynx. the depth of penetration for the nasopharyngeal aspirate catheter was set at 5-10 cm. a total 2 ml nasopharyngeal aspirate sample was obtained and centrifuged at 500âg for 10 min and resuspended in 2 ml saline and divided into two aliquots for pathogen detection using direct immunofluorescence assay (dfa) and pcrs as described previously [chen et al., 2013b] . one of the equally divided samples of nasopharyngeal aspirate was centrifuged at 12,000âg for 5 min, followed by extraction of dna and rna from a 400-ml sample using dna-ez reagents (sangon biotech, shanghai, china) or trizol reagent (life technologies, carlsbad, ca) in accordance with the manufacturer's instructions. a final 200 ml of dna or rna was eluted and dna sample was divided into two aliquots for hbov and mycoplasma pneumoniae gene amplification via pcr. rna sample was used for hmpv gene detection. real-time pcr primers were designed using sequence information from the np1 gene sequence available from the genbank database. primers and probe were synthesized using the following sequences: hbov-f:5 0 -tga-cattcaactaccaacaacctg-3 0 ;hbov-r:5 0 -cagat-ccttttcctcctccaatac-3 0 ;hbov-probe:agcac-cacaaaacacctcagggg-tamra (sangon biotech). pcr was performed in a volume of 25 ml using iq5 tm bio-rad icycler (bio-rad, carlsbad, ca). the 25 ml amplification reaction contained 3 ml of sample dna, 0.25 ml of taqman (promega, madison, wv), depc treated water 14.75 ml, buffer solution 2.5 ml, 25 mm mgso 4 2 ml, dntp 1 ml, forward, reverse primers and probe 0.5 ml, respectively. amplification was performed with the following settings: 94˚c for 30 sec, 56˚c for 30 sec, 72˚c for 30 sec for 40 cycles. positive and no-template controls were included in each run. positive samples containing the target genes were used as positive controls for all four hbov subtypes and were constructed by shanghai sangon biotech co., ltd. the concentration of each detected sample was then calculated automatically according to standard curve. mycoplasma pneumoniae samples were also analyzed for seven common viruses, including rsv, iv-a and iv-b, piv-1, 2, 3, and adv using dfa with virus-specific fluorescencelabeled monoclonal antibodies (diagnostic hybrids, athens, oh) and ultraviolet light microscopy. hmpv and mycoplasma pneumoniae were detected by reverse transcription pcr and real-time pcr, respectively. briefly, for hmpv detection, primers were designed to specifically amplify the n gene (213 bps). the forward and reverse primers were 5 0 -aaccgtgtactaagt-gatgcactc-3 0 and 5 0 -cattgtttgaccggcccca-taa-3 0 , respectively. reverse transcription reactions were performed with m-mlv reverse transcriptase (promega) and random hexamers for cdna synthesis according to the manufacturer's specifications. pcr was performed in a volume of 25 ml and the pcr conditions were as follows: denaturation at 95˚c for 5 min, then 45 cycles of denaturation at 94˚c for 30 s, annealing at 55˚c for 30 s, and extension at 68˚c for 30 s, followed by a final extension at 68˚c for 7 min as described previously . for mycoplasma pneumoniae detection, another fluorescent real-time pcr was performed to identify the p1 adhesion protein gene of mycoplasma pneumoniae as described previously [chen et al., 2013a,b] . briefly, a 21 ml pcr master mixture (daan gene, guangzhou, china) containing the primers and probes was combined with 3 ml of the sample dna and 1 ml of the gotaq 1 dna polymerase (promega) for the pcr reactions. real-time pcr was performed using the iq5 tm bio-icycler (bio-rad), and the cycling conditions were as follows: 2 min at 37˚c; 10 min at 94˚c, and 40 cycles of 10 s at 94˚c, 30 s at 55˚c, and 40 s at 72˚c. the quantitation curves were plotted using several concentrations of standard control samples, which were purchased from daan gene co. positive samples were defined with a concentration of dna >2.5 â 10 3 copies/ml in case of mycoplasma pneumoniae colonization. meteorological data for suzhou, including daily mean temperature (˚c), mean relative humidity (%), total monthly rainfall (mm), sum of sunshine (h), and mean wind velocity (m/s), were obtained from suzhou weather bureau at longitude 120˚6 0 east and latitude 31˚3 0 north, which is located 8 km away from the hospital. meteorological data were obtained hourly, and average daily values were calculated. monthly means were calculated using the daily means for temperature, relative humidity, and wind velocity. total rainfall and hours of sunshine were calculated as a total measurement for the month. values were expressed as percentages for discrete variables, as mean and standard deviation for continuous variables. the continuous variables were compared using the student t-test or mann-whitney u test if the data were abnormal in distribution. categorical data were analyzed using the mentel-haenszel, chi-squared (x 2 ), or fisher's exact tests. correlations of incidence of hbov with meteorological factors were evaluated using pearson's or spearman rank correlation. because of colinearity between meteorological factors, associations and lag effects between meteorological factors and hbov incidence were also analyzed using linear regression. for the final goal of predicting the incidence of hbov on the basis of meteorological data and season, a time series analysis utilizing a seasonal model was established for the time period between january 2009 and december 2011 (estimation period). it was evaluated by comparing the predicted versus the observed incidence of hbov during the period between january 2012 and december 2012 (evaluation period). the r 2 autoregression coefficient was calculated to judge the fitness of the model. from january 2009 to december 2012, a total of 8,288 children with acute respiratory tract infection were admitted to our hospital. nasopharyngeal aspirate samples were not taken from 510 hospitalized children due to a refusal to participate from their parent or guardian. one hundred fifty-two hospitalized children were excluded on account of congenital heart disease, pulmonary tuberculosis, down's syndrome or bronchopulmonary malformation. finally, a total of 7,626 nasopharyngeal aspirate samples were collected, 3,491(45.8%) of which were positive for at least one virus or mycoplasma pneumoniae. the most commonly identified pathogen was rsv (15.7%, 1, 197/7, 626) , followed by mycoplasma pneumoniae (14.3%, 1,094/7,626), hbov (6.6%, 502/ 7,626), piv-3 (6.1%, 464/7,626), hmpv (4.2%, 318/ 7,626), iv-a (2.8%, 166/7,626), adv (1.3%, 97/7,626), iv-b (0.8, 62/7,626), piv-2 (0.1%, 8/7,626), and piv-1 (0.03%, 2/7,626). a total of 388 nasopharyngeal aspirate samples (5.1%, 388/7,626) were detected as containing at least two viruses or co-infection with mycoplasma pneumoniae and hbov. the latter combination was one of the most common co-infections accounting for 37.6% (146/388) of total co-infected samples as well as 29.1% (146/502) of total hbov positive samples. a total of 356 samples (4.7%, 356/7,626) were detected single hbov infection and rsv (27.4%, 40/146) and mycoplasma pneumoniae (25.3%, 37/146) were most common co-infection pathogens with hbov (table i) . of the 502 hbov positive children, the median age was 13 months (range 1-156 months). hbov infection incidence of different age groups are as follows: 1-6 months (3.5%, 71/2,005), 7-12 months (12.9%, 163/ 1,267), 13-36 months (8.1%, 170/2,109), 37-60 months (5.6%, 65/1,155), and >60 months (3.0%, 33/ 1,090), respectively. the infectious rate of hbov was highest among the 7-12 months old group compared with the other four groups (all p < 0.05) and lowest among the 1-6 and >60 months old group when compared to the other three groups (all p < 0.05) as shown in figure 1 . the ratio of male to female children with hbov infection was 1.67:1 and no gender difference was observed in the incidence of hbov infection compared with total children with lower respiratory tract infection (male to female, hbov infected children 1.7 vs. total children 1.7, p > 0.05). the demographics and clinical information of hospitalized children with single hbov infection or coinfection are summarized in table ii . no significant differences were observed in the demographic characteristics, clinical presentations, and laboratory tests between single hbov infection and co-infection groups except for diagnosis of lobar pneumonia. children with co-infection presented with a higher rate of lobar pneumonia compared to single hbov infection group (x 2 ¼ 5.2, p ¼ 0.02). to determine the seasonal distribution of hbov infection, we assessed incidence over a 4-year period from january 2009 to december 2012. the incidence of infection for 2012 (4.2%, 93/2,240) was significantly lower than that of 2009 (8.1%, 144/1,779), 2010 (7.6%, 122/1,599), and 2011 (7.1, 143/2,008) (all p < 0.05). hbov could be detected every year-round except for december 2010. a seasonal distribution of hbov was noted during june to november, especially during the months of summer (june to august) which accounted for 37.8% (190/502) of the total hbov cases. however, variations in this trend were observed from 1 year to another. interestingly, in 2009 two peaks of hbov activity occurred, one in june (11.8%) and the other in november (15.7%). this was also seen in 2010, however, the peaks occurred during april (11.8%) and july (14.0%) as shown in figure 2 . the suzhou area has a typical subtropical monsoon climate. from 2009 to 2012, the monthly mean temperature was 17.1 ae 9.0 (mean ae standard deviation)˚c, relative humidity was 68.4 ae 6.3%, total rainfall was 83.6 ae 69.7 mm, sum of sunshine was 148.4 ae 47.3 h, and wind velocity was 1.8 ae 0.4 m/s. the monthly mean data for these meteorological variables over the course of this study are shown in figure 2 . the associations of hbov activity with meteorological factors were performed using pearson's or spearman correlations. as shown in table iii , hbov activity was associated with mean temperature and relative humidity. because of colinearity between meteorological factors (data not shown), associations between meteorological factors and hbov incidence were also analyzed using linear regression based on a stepwise procedure. in model 0, which had no lag time for analyzing hbov activity and meteorological factors, hbov activity is positively associated with mean temperature and relative humidity. when the lag time was taken into consideration, the m1 model was a better fit, with a higher r 2 value of 0.370 when compared to the m0 and m2 models that had r 2 values of 0.308 and 0.230, respectively. these indicated hbov activity could be better explained according the meteorological factors. specifically, this study shows that hbov activity is associated with mean temperature and a lag time effect (supplementary table si) . time series analysis was also performed to predict the hbov activity in hospitalized children with lower respiratory tract infection. the r 2 and p were 0.8 and 0.006, respectively, which showed a good fitness for this simple seasonal model. taken together, hbov activity in hospitalized children with lower respiratory tract infection could be predicted based on periodicity and season (fig. 3) . the findings of this study confirmed the hypothesis that hbov is a common pathogen in children with lower respiratory tract infection. in addition, coinfection frequently occurs with rsv and mycoplasma pneumoniae. children with co-infection were prone to lobar pneumonia compared to children infected with hbov only. to our knowledge, this is the first time that an association between hbov activity and meteorological factors were taken into consideration by a 4-year respiratory virus surveillance in a subtropical region of china. the present study shows that mean temperature was the main meteorological factor associated with hbov activity and this influence also had a month lag time effect. based on the data from this study, hbov was the third most common pathogen after rsv and mycoplasma pneumoniae with an incidence of 6.6% in all hospitalized children with lower respiratory tract infection in suzhou area. this is consistent with recent reports in shanghai area (7.0%, 39/554) [zhao et al., 2013] and lanzhou area (7.1%, 29/406) [zheng et al., 2010] . although this data does differ when looking at the gansu area (2.2%) [huang et al., 2013] and beijing area (33.8%) in china . both serology and pcr methods could be applied to detect the hbov infection in children. certainly, pcr methods have a higher sensitivity when compared to the igm detection method using elisa (100% vs. 81.1%) [zaghloul, 2011] . most studies have shown that the incidence of infection with hbov is highest among young infants less than 3 years of age [nascimento-carvalho et al., 2012; zhao et al., 2013; abdel-moneim et al., 2013] . in the present study, the median age was 13 months, and the age group of 7-12 months and 13-36 months accounted for 66.3% of the total cases. furthermore, a higher incidence rate (12.9% and 8.1%, respectively) compared to that of the other age groups was observed. this parallels the findings from studies conducted in tokyo, japan and salvador, brazil [moriyama et al., 2010; nascimento-carvalho et al., 2012] . several previous studies have reported that rsv, hmpv, rhinovirus, and human coronavirus are principal causes of co-infection with hbov. presumably, this is because the seasonal distribution of hbov and these other viruses appear to overlap [allander et al., 2007; koseki et al., 2012; xu et al., 2012] . indeed, the reported prevalence of co-infections with other respiratory viruses varies from 18.3% to 71.1% [weissbrich et al., 2006; garcía-garcía et al., 2007; brieu et al., 2008; kim et al., 2011] . co-infection was also common in this study and accounted for 29.1% (146/502) of the total hbov positive samples. interestingly, mycoplasma pneumoniae was a major pathogen that co-infected with hbov after rsv and accounted for 25.3% (37/146) of total hbov co-infection cases. the major presenting clinical picture of lower respiratory tract infection due to hbov was similar to other common respiratory virus infections like rsv and hmpv including cough, fever, wheezing and tachypnea and no characteristic symptom was found. the present study indicated pneumonia and bronchiolitis were the most frequent diagnoses associated with hbov infected children, which is consistent with previous study [allander et al., 2007; garcía-garcía et al., 2007; moriyama et al., 2010] . there was no difference in clinical manifestation, laboratory test and proportion of severe pneumonia between single hbov infection and co-infection groups. coinfections were similar to simple infections in children with hbov except that hypoxia was slightly more frequent (p ¼ 0.038) in children with co-infection [garcía-garcía et al., 2007] . on the contrary, another study suggested that the association between hbov and other respiratory viruses may be of clinical importance, and that infection with hbov alone appears to produce no major symptoms in infants seen in emergency departments [esposito et al., 2008] . it might have contributed to a lower severity of this present study because of the exclusion of children with underlying diseases. however, a recent study showed that children with single hbov infection had a higher viral load compared to children with co-infection and there was a direct correlation of high viral load (>10 6 copies/ml) with increasing disease severity in children co-infected with hbov but not in children with single hbov infection [zhao et al., 2013] . meanwhile, high viral load (>10 4 copies/ ml) of hbov correlates with the duration of wheezing in children with severe lower respiratory tract infection [deng et al., 2012] . nevertheless, a study reported that there was no apparent association between the viral load of hbov and co-infection or disease severity [zheng et al., 2010] . taken together, the role of co-infection with hbov is still not clear and further studies need to be taken to explore these clinical observations. interestingly, children with coinfection had a higher rate of lobar pneumonia compared to children with single hbov infection. it is hypothesized that co-infection with mycoplasma pneumoniae maybe play a role in such phenomenon due to it being the most common cause of lobar pneumonia in children according our 8 year surveillance (data not shown). it has been described in earlier studies that hbov infection seems to have a seasonal distribution. hbov positive patients were most prevalent during january to may and the peak occurred during april to may in japan [ma et al., 2006] while the seasonal distribution is november to june with peaks occurring during march to april in france [jacques et al., 2008] . in hong kong, the peak seasons were in fall and winter [chieochansin et al., 2008] . in the guangzhou area of china with similar subtropical climate, hbov activity was year-round and the peak season was june [xu et al., 2012] , similar to what is seen in the present study. in india, however, which has a tropical climate, hbov appeared to have no seasonal distribution [bharaj et al., 2010] . these studies suggest that different areas with different climates have their own seasonal distribution of hbov incidence. however, how a seasonal epidemic of hbov starts is currently unknown. factors such as climate may impact the survival and spread of infectious disease indicating that the environment may contribute to an epidemic and the seasonal outbreaks of respiratory viruses [du prel et al., 2009 ]. in the present study, hbov activity was positively associated with mean temperature and relative humidity, which was consistent with another recent report [do amaral de leon et al., 2013] . the temperature and relative humidity during the summer was significantly higher than the other three seasons (shown in fig. 2 ). to our interest, a lag time effect existed between hbov activity and mean temperature especially for 1 month lag and no lag effect was found between hbov activity and relative humidity. taking into account that hbov causing lower respiratory tract infection possesses an incubation period, several days of medical consultation, and an average of 5-7 days medical treatment before admission to our hospital (data not show), we presume that the lag time between climate parameters and hbov, as confirmed by real-time pcr, is 2-3 weeks. this is consistent with the results of 1 month lag effect analyzed using linear regression in present study. this explains why the m1 model including mean temperature has a higher r2 value than the m0 or m2 models. taken together, this study suggests that high temperature plays a more important role than high relative humidity in hbov seasonal activity. the data on predicting hbov activity could help in geographical areas where detection of hbov infection may be difficult. some limitations of this study should be noted. first of all, it is difficult to confirm hbov infection depending solely upon pcr because of certain bacterial colonization in healthy children although positive samples were defined with a concentration of dna >2.5 â 10 3 copies/ml. secondly, the data analysis alone may not serve as a conclusive interpretation, since any associations with meteorological factors may be an indication of other social or environmental factors that also vary with the seasons. what's more, our study was based on a single center for data, which might have potential biases. despite these limitations, this study indicates that hbov is a common cause of lower respiratory tract infection in children less than 3 years. understanding the impact of meteorological factors, especially temperature, on hbov activity can be useful and important in predicting seasonal outbreaks of lower respiratory tract infection. detection of bocavirus in children suffering from acute respiratory tract infections in saudi arabia cloning of a human parvovirus by molecular screening of respiratory tract samples human bocavirus and acute wheezing in children human bocavirus infection in children with acute respiratory tract infection in india human bocavirus infection in children with respiratory tract disease two-year prospective study of single infections and coinfections by respiratory syncytial virus and viruses identified recently in infants with acute respiratory disease epidemiology and associations with climatic conditions of mycoplasma pneumoniae and chlamydophila pneumoniae infections among chinese children hospitalized with acute respiratory infections etiology of acute bronchiolitis and the relationship with meteorological conditions in hospitalized infants in china human bocavirus (hbov) in thailand: clinical manifestations in a hospitalized pediatric patient and molecular virus characterization high viral load of human bocavirus correlates with duration of wheezing in children with severe lower respiratory tract infection clinical and epidemiologic profile of lower respiratory tract infections associated with human bocavirus are meteorological parameters associated with acute respiratory tract infections? impact of human bocavirus on children and their families human bocavirus infections in spanish 0-14 year-old: clinical and epidemiological characteristics of an emerging respiratory virus establishment of a reverse genetics system for studying human bocavirus in human airway epithelia viral etiology of acute respiratory infection in gansu province human bocavirus quantitative dna detection in french children hospitalized for acute bronchitis human bocavirus in patients with respiratory tract infection detection of human bocaviruses 1 to 4 from nasopharyngeal swab samples collected from patients with respiratory tract infections detection of human bocavirus in japanese children with lower respiratory tract infections epidemiological profile and clinical associations of human bocavirus and other human parvoviruses distinctive clinical features of human bocavirus in children younger than 2 years infections with the novel human bocavirus human bocavirus infection diagnosed serologically among children admitted to hospital with community-acquired pneumonia in a tropical region epidemiology and etiology of childhood pneumonia in 2010: estimates of incidence, severe morbidity, mortality, underlying risk factors and causative pathogens for 192 countries estimate developed by the un interagency group seasonal distribution and epidemiological characteristics of human metapneumovirus infections in pediatric inpatients in southeast china frequent detection of bocavirus dna in german children with respiratory tract infections surveillance and genome analysis of human bocavirus in patients with respiratory infection in guangzhou human bocavirus (hbov) in children with respiratory tract infection by enzyme linked immunosorbent assay (elisa) and qualitative polymerase chain reaction (pcr) viral etiology and clinical profiles of children with severe acute respiratory infections in china high human bocavirus viral load is associated with disease severity in children under five years of age human bocavirus infection in young children with acute respiratory tract infection in lanzhou we thank the members of the research team, laboratory staff and all cooperating institutes for their dedication to the project. most of all, we are indebted to all participating children and their families. additional supporting information may be found in the online version of this article at the publisher's web-site. key: cord-286472-pqtem19t authors: mcfee, r.b. title: middle east respiratory syndrome (mers) coronavirus date: 2020-07-28 journal: dis mon doi: 10.1016/j.disamonth.2020.101053 sha: doc_id: 286472 cord_uid: pqtem19t nan ) (1) was isolated from a patient who died from severe pneumonia and multi-organ failure in saudi arabia (2, 3) . this newly identified respiratory viral illness was caused by a novel coronavirus, which was initially designated as human betacoronavirus (2) (3) (4) (5) , but was eventually named middle east respiratory syndrome coronavirus (mers cov). reminiscent of, and worth considering as a caution for greater vigilance towards emerging pathogens, the suddenness that sars cov emerged as a new cause of severe pulmonary illness, has been replicated in this new aggressive respiratory illness mers cov. unlike sars cov, it has not caused the thousands of cases over a short period of time. but it also differs from sars cov in that it carries a much higher case fatality rate, and causes more severe illness (6, 7) . unlike sars which seems to have gone quiescent, new mers cases have been reported well after the initial outbreak, including december 2019 (7b) . since september 2012 there have been cases reported in 27 countries across 4 continents, with most human cases occurring in saudi arabia (map 1 and 2) (6) . while the greatest spike in cases occurred near 2014, new cases to date continue to be reported. consider by the end of 12/19 the number of mers cases 2492, with 858 deaths, resulting in a case fatality rate of 34.43% (7b, 7c) . earlier data from world health organization (who) as of 12/05/16 there have been 1917 laboratory confirmed cases of mers cov since 09/12, reported from 27 countries (maps 1 and 2), resulting in 677 deaths, yielding a significant case fatality rate (~35%) (6); a fatality rate for a coronavirus that is significantly greater than that associated with sars cov delta (~9%), at least among confirmed cases (6) (7) (8) . these data demonstrate mers continues to cause illness, though thankfully not at pandemic rates in the same manner as covid-19. as with other cov, including sars cov and covid-19, the exact epidemiology, including the magnitude of mild or asymptomatic illness, remains unknown. that said, among those presenting with mers, males over 60 yrs of age seem to be at higher risk of severe disease symptoms and death among those infected. the risk associated with age seems consistent with covid-19 findings as well. abnormalities associated with mers cov include thrombocytopenia, lymphopenia, leukopenia, elevated serum lactate dehydrogenase (ldh), elevated aspartate aminotransferase (ast), elevated alanine aminotransferase (alt), along with abnormal renal function tests (5, 29, 30) . superinfection from viral or bacterial causes has been found in some patients and may complicate the course of the disease. severe cases of mers may require intensive care and mechanical ventilation, with a relatively large number of patients progressing to respiratory or renal failure. a retrospective radiology review of ct findings in laboratory confirmed cases of mers cov noted that cough, fever, and dyspnea were the most common presenting symptoms. three of the seven subjects died (31) . the high case fatality rate associated with mers cov has sparked interest in the role of virus on the immune system. one area of interest is the potential for cytokine storm, which is also found in covid-19 patients, and currently being studied extensively (31b) . some studies have shown mers cov is associated with an attenuated interferon (ifn) response, and does not induce inflammatory cytokines (7, 32, 33 chest xrays should be obtained early in the course of progressive pulmonary illness ( figure 2 ) (31). radiographic findings may include unilateral or bilateral patchy densities or opacities, interstitial infiltrates, consolidation, and pleural effusions. rapid progression to acute respiratory failure, acute respiratory distress syndrome (ards), refractory hypoxemia, and extrapulmonary complications (acute kidney injury requiring renal replacement therapy, hypotension requiring vasopressors, hepatic inflammation, septic shock) has been reported. key to appropriate testing is a thorough history and physical. it is important to consider multisystem function as well as pulmonary status in patients with severe respiratory illness, including suspected mers cov, especially those returning from regions where aggressive pathogens are noted. involving infectious disease and pulmonologist specialists early on, as well as good critical care management are essential. as noted earlier, laboratory findings at admission may include leukopenia, lymphopenia, thrombocytopenia, and elevated lactate dehydrogenase levels (ldh). renal and hepatic function tests, at least for baseline information are worth obtaining. in the ajlan study (31) , all seven patients had lymphopenia. elevated creatinine developed after admission in all patients. ast also were elevated in all patients. of note, co-infection with other respiratory viruses and a few cases of co-infection with communityacquired bacteria at admission has been reported in mers cov patients. not surprisingly, nosocomial bacterial and fungal infections have also been reported in mechanically-ventilated patients. according to clinical experience reported to cdc, mers-cov virus can be detected with higher viral load and longer duration in the lower respiratory tract compared to the upper respiratory tract, and has been detected in feces, serum, and urine. however, very limited data are available on the duration of respiratory and extrapulmonary mers-cov shedding. as mentioned earlier, aggressive testing, isolation, and management are critical in treating highly pathogenic coronaviruses, especially mers, which among patients who present for medical care, has a high case fatality rate, extrapulmonary involvement in some cases, and rapid deterioration. this includes early radiographic studies (7b). according to the aljan radiology study (31) , chest xray findings were most notable for airspace and interstitial opacities with mers cov. radiographic findings such as airspace opacities are nonspecific, whereas the interstitial changes were described as reticular or reticulonodular. total lung opacification and thickening of the bronchovascular markings have been reported as well. previous chest ct findings with mers have been reported as bilateral patchy or extensive opacities (5, 7b) . not unexpected, imaging that had features consistent with acute respiratory distress syndrome were typically identified in sicker patients, and can be found in covid-19 patients as well (table 1 ). in aljan (31) mers cov the most common findings on ct were shows that airspace opacities on ct are common in patients hospitalized with mers-cov infection. airspace opacities are suggestive of an organizing pneumonia pattern. it was also described in h1n1 influenza a viral infections. in most of our patients, ground-glass opacities were more extensive than consolidation. septal thickening and pleural effusions also were demonstrated. importantly, tree-in-bud pattern, cavitation, and lymph node enlargement were not seen in our cohort. chest xrays and ct scanning ( figure 2 ) are recommended depending upon the presentation, and clinical course. in the aljan study (31) the most common ct finding in hospitalized patients with mers -cov infection reveals predominantly subpleural and basilar airspace changes, with more ground glass opacities than consolidation. it is important to note this is a small study. nevertheless this pattern is consistent with organizing pneumonia. patients recently returning from the middle east, presenting with significant respiratory illness, with ct findings of peribronchial region abnormalities, organizing pneumonia, should be considered for mers cov infection, and if possible, queried about international travel and occupational exposures. 27-year-old man with middle east respiratory syndrome patient was a smoker who was healthy otherwise. ct was performed 8 days after admission, and 20 days after onset of symptoms. patient was eventually discharged. lower lung ct image show large right lower lobe and small focal left lower lobe subpleural consolidations the cdc provides case definitions. the reader is requested to review periodically this site for updates as the mers cov situation can change (8, 34) . although emergency testing can be possible for suspected asymptomatic cases, the cdc guidelines for sending samples for laboratory confirmation are predicated upon a combination of clinical and epidemiological factors to identify persons under investigation (pui) table 1 . the other two categories per cdc are: a confirmed case is a person with laboratory confirmation of mers-cov infection. confirmatory laboratory testing requires a positive pcr on at least two specific genomic targets or a single positive target with sequencing on a second. laboratory confirmation of mers cov can be obtained by real time polymerase chain reaction (rt pcr) a probable case is a pui with absent or inconclusive laboratory results for mers-cov infection who is a close contact 3 of a laboratory-confirmed mers-cov case. examples of laboratory results that may be considered inconclusive include a positive test on a single pcr target, a positive test with an assay that has limited performance data available, or a negative test on an inadequate specimen. nb the reader is advised to check for updated testing procedures with the cdc (35) . this is an updated version of the interim guidance document issued by the centers for disease control and prevention (cdc) january 2014 based on input from public health partners, healthcare providers, professional organizations, and others. cdc will continue to update the document as necessary to incorporate new information that increases our understanding of mers-cov. updates: minor changes were made to clarify specimen type and collection procedures. of note -respiratory specimens should be collected as soon as possible after symptoms beginideally within 7 days. however, if more than a week has passed since symptom onset and the patient is still symptomatic, respiratory samples should still be collected, especially lower respiratory specimens since respiratory viruses can still be detected by rrt-pcr. for example -if symptom onset for a pui with respiratory symptoms was less than 14 days ago, a single serum specimen, an np/op specimen and lower respiratory specimen should be collected for cdc mers rrt-pcr testing. 1. if symptom onset for a pui with an ongoing respiratory tract infection, especially lower, was 14 or more days ago, a single serum specimen for serologic testing in addition to a lower respiratory specimen and an np/op specimen are recommended. 2. if symptom onset for a pui with an ongoing respiratory tract infection, especially lower, was 14 or more days ago, a single serum specimen for serologic testing in addition to a lower respiratory specimen and an np/op specimen are recommended. for short periods (≤ 72 hours), most specimens should be held at 2-8°c rather than frozen. for delays exceeding 72 hours, freeze specimens at -70°c as soon as possible after collection (with exceptions noted below). label each specimen container with the patient's id number, specimen type and the date the sample was collected. broncheoalveolar lavage, tracheal aspirate, pleural fluid collect 2-3 ml into a sterile, leak-proof, screw-cap sputum collection cup or sterile dry container. refrigerate specimen at 2-8°c up to 72 hours; if exceeding 72 hours, freeze at -70°c and ship on dry ice. have the patient rinse the mouth with water and then expectorate deep cough sputum directly into a sterile, leak-proof, screw-cap sputum collection cup or sterile dry container. refrigerate specimen at 2-8°c up to 72 hours; if exceeding 72 hours, freeze at -70°c and ship on dry ice. nasopharyngeal swab and oropharyngeal swab (np/op swab) use only synthetic fiber swabs with plastic shafts. do not use calcium alginate swabs or swabs with wooden shafts, as they may contain substances that inactivate some viruses and inhibit pcr testing. place swabs immediately into sterile tubes containing 2-3 ml of viral transport media. np/op specimens can be combined, placing both swabs in the same vial. refrigerate specimen at 2-8°c up to 72 hours; if exceeding 72 hours, freeze at -70°c and ship on dry ice. nasopharyngeal swab -insert a swab into the nostril parallel to the palate. leave the swab in place for a few seconds to absorb secretions. swab both nasopharyngeal areas. oropharyngeal swab (e.g., throat swab) -swab the posterior pharynx, avoiding the tongue. nasopharyngeal wash/aspirate or nasal aspirate collect 2-3 ml into a sterile, leak-proof, screw-cap sputum collection cup or sterile dry container. refrigerate specimen at 2-8°c up to 72 hours; if exceeding 72 hours, freeze at -70°c and ship on dry ice. for serum antibody testing: because we do not want to delay detection of mers infection and since the prevalence of mers in the us is low, serologic testing on a single serum sample collected 14 or more days after symptom onset may be beneficial. this is in contrast to serologic testing for many other respiratory pathogens which require collection and testing of acute and convalescent serum specimens. serologic testing is currently available at cdc upon request and approval. please be aware that the mers-cov serologic test is for research/surveillance purposes and not for diagnostic purposes -it is a tool developed in response to the mers-cov outbreak. contact cdc's emergency operations center (eoc) (770-488-7100) for consultation and approval if serologic testing is being considered . a single serum specimen collected optimally during the first 10-12 days after symptom onset is recommended. note: the kinetics of mers-cov are not well understood. once additional data become available, these recommendations will be updated as needed. the minimum amount of serum required for mers-cov testing (either serologic or rrt-pcr) is 200 µl. if both mers-cov serology and rrt-pcr tests are planned, the minimum amount of serum required is 400 µl (200 µl for each test). serum separator tubes should be stored upright for at least 30 minutes, and then centrifuged at 1000-1300 relative centrifugal force (rcf) for 10 minutes before removing the serum and placing it in a separate sterile tube for shipping (such as a cryovial). refrigerate the serum specimen at 2-8°c and ship on ice-pack; freezing and shipment of serum on dry ice is permissible. children and adults: collect 1 tube (5-10 ml) of whole blood in a serum separator tube. a minimum of 1 ml of whole blood is needed for testing pediatric patients. if possible, collect 1 ml in a serum separator tube. specimens from suspected mers cases must be packaged, shipped, and transported according to the current edition of the international air transport association (iata) dangerous goods regulations. shipments from outside of the united states may require an importation permit that can be obtained from cdc. specimens should be stored and shipped at the temperatures indicated above. if samples are unable to be shipped within 72 hours of collection, they should be stored at -70°c and shipped on dry ice. when shipping frozen specimen from long distances or from international locations, it is best to use a combination of dry ice and frozen gel ice-packs. the gel ice-packs will remain frozen for a day or two after the dry ice has dissipated. all specimens must be pre-packed to prevent breakage and spillage. specimen containers should be sealed with parafilm® and placed in ziplock bags. place enough absorbent material to absorb the entire contents of the secondary container (containing primary container) and separate the primary containers (containing specimen) to prevent breakage. send specimens with cold packs or other refrigerant blocks that are self-contained, not actual wet ice. this prevents leaking and the appearance of a spill. when large numbers of specimens are being shipped, they should be organized in a sequential manner in boxes with separate compartments for each specimen. saturday delivery is planned, special arrangements must be made with the shipping company. there are no commercially available or fda approved therapeutics specifically designated as mers cov antivirals. as of 2020 there remain a variety of candidate therapeutics in development against covid019 specifically, highly pathogenic coronaviruses such as sars and mers, and coronaviruses in general, including those that cause "common cold-like" symptoms, but none have emerged as fully capable of treating these viruses with the same consistency as neuraminidase antivirals have against influenza. and even in that context there are some clinical considerations, and limitations. there continues to be ongoing research into repurposing antivirals that are approved for other clinical indications such as hiv, influenza, and other illnesses, as possible treatments -monotherapy or in combination, against covid-19 and other coronaviruses. brief updates are provided below. covid-19 has reenergized research into greater study of the pathogenicity of coronaviruses, and newly or better described characteristics of highly pathogenic coronaviruses has led to increased insights into additional antiviral and vaccine targets. significant research into a wide array of therapeutic approaches is undergoing at unprecedented levels internationally. as will be seen in the covid-19 therapeutics section of this article, multiple options are being clinically tested that warrant consideration for the clinician faced with treating covid-19 patients. the mainstay of therapy for mers cov remains supportive care, especially respiratory care, while managing circulatory, renal, hepatic and neurological function, as well as protecting against secondary infections. although attempted in both the sars cov and early mers cov outbreaks, immune based interventions -interferon -resulting in equivocal outcomes. non human primate studies using ifn a2b and ribavirin against mers cov demonstrated improved outcomes but it is worth noting treatment was initiated soon after viral infection initiated; this is unlikely going to be the case with human infection, where a delay in presentation to health care, or delayed diagnosis will likely preclude the same rapidity of treatment that occurred in the study (6 -8, 32, 33) . the role for interferon in mers cov remains unclear; clinical trials are needed to better characterize successful strategies. but the limited number of cases make such studies difficult. in addition to various interferon treatment protocols, ribavirin -a potent nucleoside analog -has been utilized against rna viruses with varying degrees of success (7,32,33, 33b, 33c) . unfortunately ribavirin presents a risk for adverse effects including hemolytic anemia. interferon is not without side effect risk either. the early use of corticosteroids in sars cov infected patients resulted in increased viral load, critical care/intensive care unit admission, and death (7, 33c). other approaches to cov include neutralizing antibodies from convalescent plasma or hyperimmune globulin; these may hold some promise in reducing the cfr (33c, [36] [37] [38] . convalescent plasma was shown to decrease mortality in sars cov patients if provided to patients within 14 days of illness (36) . however in order for this to be effective, rapid diagnosis of patients, and survivors is required in order to obtain, and utilize these interventions. towards that end, a network for convalescent plasma is being assembled, allowing safety, effectiveness and logistic feasibility testing. (37) . that said, to date no host derived interventions have been shown to consistently confer significant benefit to severely ill mers cov patients via controlled study. while to date there are no antivirals currently licensed for use against mers cov, some candidate drugs are in development. two categories of candidates showed early promise. an adenine analogue that can be incorporated into viral rna, which can disrupt virus replication, and has shown in vitro activity against mers cov, as well as benefit against ebola in non human primates (nhps). also there is a nucleoside analogue for treatment of filoviruses, cov, and other rna viruses. researchers are working on other molecular approaches to treat cov (7, 39, 40) . ribavirin is a guanine, oral nucleoside analogue, which inhibits viral rna-dependent rna polymerase. in trials exploring the use of ribavirin against mers, it was often in combination with other therapeutics, including interferon. evidence revealed it did not confer significant clinical benefit on outcomes or viral clearance (33c, 41, 42) . it has shown some in vitro activity against sars, where high concentrations/high doses were required to inhibit viral replication (1.2 g to 2.4 g po every 8 hours), and combination therapy via intravenous or enteral administration (33b, 33c, 43) . studies on the use of ribavirin as a treatment for sars were either inconclusive in terms of clinical benefit, or suggested possible harm referable to adverse events, which included hepatic and hematologic deleterious effects (33c, 43). ribaviran as a potent nucleoside analogue has been, and continues to be used with varying degrees of success against rna viruses, but there is the potential for adverse side effects including hemolytic anemia, metabolic derangements. interferon can also elicit adverse effects, although they have demonstrated value against viral infection (44) (45) (46) (47) . another repurposing of antivirals involved lopinavir-ritonavir combination therapy trialed against mers. the medication is an oral protease inhibitor (33b, 33c). early success with treating sars in 2003, using lopinavir/ritonavir and ribavirin was suggested (33b, 48) . mortality and need for intensive respiratory support was noted (33b, 48) . in an animal study involving marmosets, lopinavir-ritonavir or interferon beta -1b reduced viral load and improved lung pathology (33b, 49) . combination antiviral therapy for patients hospitalized with severe influenza was noted in some cases to confer greater results, for those with high viral loads at presentation (33b, 50, 51) . it is worth noting that studies have shown both sars coronavirus and mers coronavirus their viral loads peak at ~7 -10 days after symptoms begin compared to covid-19 coronavirus where viral loads seem to peak at the time of clinical presentation (33b, 52,53) . this adds another level of clinical challenge in terms of managing this new, highly pathogenic coronavirus, and underscores the importance of time sensitivity in the administration of potentially lifesaving medications. another promising approach includes monoclonal antibodies (mabs). mabs have already demonstrated benefit against certain cancers and autoimmune disorder management (7, 38) . to date the only pathogen for which there is a licensed mab is respiratory syncytial virus (rsv). who recommends standard precautions with all patients, especially those displaying early signs of respiratory illness, as the diagnosis -whether common cold, or mers cov or rsv or pertusis or other pathogen related infection. also droplet precautions should be added to standard precautions when providing care to such patients, including those with acute respiratory illness. although eye protection is recommended for probable or confirmed cases of mers cov, and airborne precautions for aerosol generating procedures, this would seem prudent even in cases clearly not mers cov or sars cov. from an infection control perspective, among the known human coronaviruses they are capable of surviving on environmental surfaces for up to 3 hours (11) . the sars and mers experiences, as well as previous hcov, underscore the threat of transmissibility; coronaviruses may be transmitted from person-to-person by droplets, hand contamination, fomites, and small particle aerosols. avoiding camels, especially dromedary camels, farms, as well as not consuming raw milk, urine, or meat are also worth considering. camels infected with mers cov may not appear ill. if contact with animals, especially dromedary camels cannot be avoided, strict hand washing is recommended. personal protection, including face/eye if working with camels or other animals. patients who must travel to the region are encouraged to present to a health care facility especially if fever and respiratory symptoms develop. according to a who update, multiple cases from the united arab emirates, qatar, oman, kuwait, and bahrain have links to dromedary camels. these patients were thought to be infected via contact with infected dromedaries or close primary cases (uae). a good practice for anyone who works with potential contaminants -whether in the us or middle east farm -remove work clothes that may carry materials from farms and animal contact (poster 1) (53). to date few cases have occurred in europe or the us. however with the emergence of medical tourism -cosmetic and advanced surgeries -patients who reside in the middle east may bring with them diseases endemic to the region, including mers cov. the astute clinician will be aware of potential importation of illness. as of 2020 there are sporadic cases of mers-cov, which continues to cause illness since its emergence in 2012, and with a mortality rate estimated at greater than 30%. mers still remains a potential outbreak threat, and is a dangerous disease causing pathogen. as with sars cov and other viral threats of public health concern, there is a great need for both prophylactic measuresvaccines, and more targeted therapies. there is significant research effort underway internationally to develop an effective vaccine. as with the sars cov, there are a variety of approaches. one attempt which shows early animal efficacy is by a vaccine candidate consisting of chimeric virus-like particles (vlp) expressing the receptor binding domain (rbd) of mers-cov. the researchers have fused canine parvovirus (cpv) vp2 structural protein gene with the rbd of mers-cov; it self-assembles into chimeric, spherical vlp (svlp). this svlp retained certain parvovirus characteristics, such as the ability to agglutinate pig erythrocytes, and structural morphology similar to cpv virions. immunization with svlp induced rbd-specific humoral and cellular immune responses in mice. svlp-specific antisera from these animals were able to prevent pseudotyped mers-cov entry into susceptible cells, with neutralizing antibody titers reaching 1: 320. of note, interferon (ifn) -ifn-γ, il-4 and il-2 secreting cells induced by the rbd were detected in the splenocytes of vaccinated mice by elispot. mice given svlp or an adjuvanted svlp vaccine elicited t-helper 1 (th1) and t-helper 2 (th2) cell-mediated immunity. although early stage, resarch svlp displaying the rbd of mers-cov may become, or lead to an effective vaccine against mers-cov. human research is needed (55) . of note, several of these approaches will be applied to developing vaccines against covid-19, which will be discussed in the next section (33c). -or -a member of a cluster of patients with severe acute respiratory illness (e.g., fever 1 and pneumonia requiring hospitalization) of unknown etiology in which mers-cov is being evaluated, in consultation with state and local health departments in the us. and close contact 3 with a confirmed mers case while the case was ill. fever may not be present in some patients, such as those who are very young, elderly, immunosuppressed, or taking certain medications. clinical judgement should be used to guide testing of patients in such situations countries considered in the arabian peninsula and neighboring include: bahrain; iraq; iran saudi arabia; syria; the united arab emirates meters), or within the room or care area, of a confirmed mers case for a prolonged period of time (such as caring for, living with, visiting, or sharing a healthcare waiting area or room with, a confirmed mers case) while not wearing recommended personal protective equipment or ppe (e.g., gowns, gloves, nioshcertified disposable n95 respirator, eye protection); or b) having direct contact with infectious secretions of a confirmed mers case (e.g., being coughed on) while not wearing recommended personal protective equipment. see cdc's interim infection prevention and control recommendations for hospitalized patients with mers for detailed information regarding healthcare personnel (hcp) please review cdc interim u.s. guidance for monitoring and movement of persons with potential middle east respiratory syndrome (mers-cov) exposure for more information: call 800-cdc-info (232-4636) or visit www.cdc.gov/travel poster 1 (54) mers references 1. image of middle east respiratory syndrome (mers) coronavirus as isolated by the national institute of allergy and infectious diseases isolation of a novel coronavirus from a man with pneumonia in saudi arabia severe respiratory illness caused by a novel coronavirus in a patient transferred to the united kingdom from the middle east middle east respiratory syndrome coronavirus (mers -cov) announcement of the cornoavirus study group epidemiological, demographic, and clnical characteristics of 47 cases of midle east respiratory syndrome coronavirus disease from saudi arabia: a descriptive study world health organization (who) mers update and global summary treatment strategies for middle east respiratory syndrome coronavirus radiology perspective of coronavirus disease 2019 (covid-19): lessons from severe acute respiratory syndrome and middle east respiratory syndrome ajr middle east respiratory syndrome coronavirus (mers-cov) differential cell line susceptibility to the emerging novel human betacoronavirus 2c emc/2012: implications for disease pathogenesis and clinical manifestation world health organization middle east respiratory syndrome coronavirus emergency preparedness response survival of human coronaviruses 229e and oc43 in suspension after drying on surfaces: a possible source of hospital-acquired infections middle east respiratory syndrome coronavirus (mers cov): what lessons can we learn? assessment of the middle east respiratory syndrome coronavirus (mers -cov) epidemic in the middle east and risk of international spread using a novel maximum likelihood analysis approach estimation of mers -coronavirus reproductive number and case fatality rate for the spring 2014 saud arabia outbreak insights from publicly available data hadi mohamed rae. outbreak of middle east respiratory syndrome coronavirus in saudia arabia: a retrospective study middle east respiratory syndrome coronavirus infection in dromedary camels in saudi arabia serological evidence of mers cov antibodies in dromedary camesl (camelus dromedaries) in laikipia county, keny isolation of mers coronavirus from a dromedary camel risk factors for primary middle east respiratory syndrome coronavirus illness in humans, saudi arabia a more detailed picture of the epidemiology of middle east respiratory syndrome coronavirus presence of middle east respiratory syndrome coronavirus antibodies in saudi arabia: a nationwide cross sectional serological study disocvery of seven novel mammalian and avian coronaviruses in the genus deltacoronavirus supports bat coronaviruess as the gene source of alphacoronavirus and betacoronavirus and avian cornnaviruses as the gene source of gammacoronavirus and deltacornavirus evidence of the recombinant origin of a bat severe acute respiratory synddrome osars) like coronavirus and its implications on the direct ancestor of sars coronavirus delayed induction of proinflammatory cytokines and suppression of innate antiviral response by the novel middle easst respiratory syndrome coronavirus: implications for pathogenesis and treatment middle east respiratory syndrome: an emerging coronavirus infection tracked by the crowd middle east respiratory syndrome coronavirus (mers cov) infection family cluster of middle east respiratory syndrome coronavirus infections hospital outbreak of middlle east respiratory syndrome coronavirus middle east respiratory syndrome coronavirus (mers cov): evidence and speculation middle east respiratory syndrome coronavirus the pathogenesis and treatment of the 'cytokine storm' in covid-19 treatment with interferon-alpha2b and ribavirin improves outcome in mers cov infected rhesus macaques ifn alpha 2a or ifn beta 1 in combination with ribavirin to treat middle east respiratory syndrome coronavirus pneumonia: a retrospective study triple combination of interferon beta-1b, lopinavirritonavir, and ribavirin in the treatment of patients admitted to hospital with covid-19: an open label randomized, phase 2 trial pharmacologic treatments for coronavirus disease 2019 (covid-19); a review centers for disease control (cdc) mers clinical laboratory testing recommendations the effectiveness of vonvalescent plasma and hyperimmune immunoglobulin for the treatment of severe acute respiratory infections of viral etiology: a systematic review and exploratory meta-analysis feasibility, safety, clinical and laboratory effects of convalescent plasma therapy for patients with middle east respiratory syndrome coronavirus infection: a study protocol a conformation dependent neutralizing monoclonal antibody specifically targeting receptor binding domain in middle east respiratory syndrome coronavirus spike protein nucloeotide prodrug gs-5734 is a broad spectrum filovirus inhibitor that provides complete therapeutic protection against the development of ebola virus disease (evd) in infected non-human primates id week biocryst announces nature publication demonstrating efficacy of bcx4430 in a non-human primate model of filovirus infecton clinical outcomes of current medical approaches for middle east respiratory syndrome: a systematic review and metaaanalysis ribavirin and interferon therapy for critically ill patients with mers: a multicenter observational study sars: systematic review of treatment effects modjarrad k treatment strategies for middle east respiratory syndrome coronavirus treatment with interferon-alpha2b and ribavirin improves outcome in mers cov infected rhesus macaques ifn alpha 2a or ifn beta 1 in combination with ribavirin to treat middle east respiratory syndrome coronavirus pneumonia: a retrospective study oral ribavirin for the treatment of noninfluenza respiratory viral infections: a systematic review role of lopinavir/ritonavir in the treatment of sars: initial virological and clinical findings treatment withlopinavir/ritonavir or interferon b1b improves outcome of mers-cov infection in a nonhuman primate model of common marmoset antiviral combinations for severe influenza efficacy of clarithromycin-naproxen-oseltamivir combination in the treatment of patients hospitalized for influenza a (h3n2) infection: an open-label randomized, controlled, phase iib/iii trial temporal profiles of viral load inposterior oropharyngeal saliva sample and serum antibody responses during infection by sars cov-2: an observational cohort study medical treatment of viral pneumonmia including sars in immunocompetent adult novel chimeric virus-like particles vaccine displaying mers-cov antiviral res key: cord-306266-8qdrshz3 authors: scully, crispian title: respiratory medicine date: 2014-06-25 journal: scully's medical problems in dentistry doi: 10.1016/b978-0-7020-5401-3.00015-1 sha: doc_id: 306266 cord_uid: 8qdrshz3 ●. upper respiratory infections are commonplace, especially in young people, and are often contagious; ●. lower respiratory infections are often contagious and some are potentially fatal; ●. asthma is common and may be life-threatening; ●. chronic obstructive pulmonary disease is common and disabling; ●. tuberculosis worldwide is an important infection, affecting people with hiv/aids or malnutrition particularly; ●. lung cancer is common and usually has a poor prognosis. • upper respiratory infections are commonplace, especially in young people, and are often contagious the respiratory tract consists of the upper respiratory tract (urtnose, paranasal sinuses, pharynx and larynx; discussed in ch. 14) and the lower respiratory tract (lrt): the respiratory airways (trachea, bronchi and bronchioles) and lungs (respiratory bronchioles, alveolar ducts, alveolar sacs and alveoli), discussed in this chapter. protective mechanisms in the respiratory tracts include a mucociliary lining. particles or pathogens are trapped in the mucus and driven by ciliary action (the ciliary elevator) to the pharynx. mucociliary trans port declines with age but any effect on clinical infection has not been proved. lymphoid tissues of the waldeyer ring (adenoids, palatine and lingual tonsils) are important in developing an immune response to pathogens. however, the best respiratory defence mechanism is the cough reflex, the components of which include cough receptors, affer ent nerves, the cough centre, and efferent nerves and effector muscles. impairment of any of these -as may be seen in older patients or those with conditions associated with lowered consciousness (e.g. sedative use and neurological disease) -can weaken protection. dysphagia or impaired oesophageal motility may exacerbate the tendency to aspi rate foreign material. the alveolar defence mechanisms include mac rophages, immunocytes, surfactant, phospholipids, immunoglobulin g (igg), ige, secretory iga, complement components and factor b; many immune defects manifest with recurrent respiratory infections. lung function is vital to gas exchange -the blood absorbs oxygen and releases carbon dioxide. normal gas exchange requires adequate alveolar ventilation, normal ventilation/blood flow relationships and adequate alveolar-capillary membrane surface area. breathing (ven tilation) depends on respiratory drive, which reacts to the respiratory load. this process requires work and results in gas exchange. oxygen is transported in combination with haemoglobin in erythro cytes and a small amount dissolved in plasma. the oxyhaemoglobin dissociation curve is sigmoidal; once the oxygen saturation falls below 95%, the amount of o 2 transported to the tissues and brain falls rapidly. high temperatures, acidosis, raised co 2 and raised 2,3 diphosphoglycerate (2,3dpg) levels encourage oxygen offloading, whereas fetal haemoglobin and carboxyhaemoglobin have the con trary effect. chronic hypoxaemia (e.g. at high altitudes) stimulates release of erythropoietin from the kidneys, with a rise in red cell pro duction, and raised 2,3dpg. athletes have abused erythropoietin to gain competitive advantage (ch. 33). the most common lrt disorders are asthma and chronic obstructive pulmonary disease (copd). respiratory disorders are common, and are often caused or aggravated by tobacco smoking. they may significantly affect general anaesthesia (ga) and conscious sedation (cs), since they are often a contraindica tion to use of benzodiazepines, opioids, ga agents and other respira tory depressants. impaired gas exchange leads to laboured breathing and can cause significant incapacity. features include cough, sputum production, wheeze, dyspnoea, chest pain, cyanosis, fingerclubbing ( fig. 15.1) , use of accessory muscles of respiration with indrawing of the intercostal spaces (hyperinflation), and abnormalities in chest shape, movements, respiratory rate and breath sounds. cough may be a feature of any respiratory problem but, if chronic, may herald serious disease -for example, copd, cancer or infec tion such as tuberculosis. mucoid or mucopurulent sputum is often a feature ( fig. 15 .2); purulent sputum indicates acute bronchitis, bronchiectasis or lung abscess. blood (haemoptysis) or bloodstained sputum, though common in acute infections (especially in preexisting copd), bronchiectasis and pulmonary embolism, may herald an even more serious condition -for example, possibly one due to carcinoma or tuberculosis. wheezing is caused by airways obstruction and is a typical sign of asthma or copd. breathlessness (dyspnoea) is distress ing, and may be caused by respiratory or cardiovascular disease, or by anaemia, and is particularly ominous if it persists at rest. excessive resistive load, such as in asthma, copd and cystic fibro sis, impairs airflow. elastic load increases because of, for example, interstitial fibrosis, muscle paralysis and obesity. diagnosis of respiratory disorders is from the clinical features sup ported by imaging (especially chest radiography). spiral computed tomography (ct) can now scan the lungs in a quick 20-30second breathhold and therefore, instead of producing a stack of individual ct slices, which may be misaligned due to patient movement or breathing in between slices, provides highresolution three dimensional images. respiratory function tests can measure individual components of the respiratory process. spirometry is the basic screening test for assess ing mechanical load problems, the quantification involving determi nation of the vital capacity (vc) -slow vital capacity (svc) and/or forced vital capacity (fvc) -and the speed of maximal expiratory flow (mef; fig. 15 .3). in health, about 75% of a normalsized vc is expelled in 1 second (fev 1 ). the peak flow meter, which measures the peak expiratory flow rate (pefr; the earliest portion of forced expiration), is a simple measure of airflow obstruction, when the fev 1 is a much smaller fraction of the vc. in lung restriction, the diminished vc can be mostly expelled in about 1 second. serial meas urements (e.g. in asthma) provide valuable information about disease progress. the reversibility of airways obstruction is usually assessed by spirometry before and after use of a bronchodilator agent. arterial blood gas analysis yields considerable information about gas exchange efficiency. arterial hypoxaemia in adults is defined as pao 2 below 10.7 kpa breathing room air, although it is not usually treated as clinically important unless below 8 kpa, when oxygen saturation will be 90% or less (table 15 .1). arterial carbon dioxide tension (paco 2 ) is used as an inversely pro portional index of 'effective' alveolar ventilation. hence, a high paco 2 is taken to indicate poor alveolar ventilation. alveolar hypoventila tion (raised paco 2 ) with a normal ph probably represents a primary ventilatory change present long enough for renal mechanisms to compensate, as in chronic ventilatory failure. ventilation/blood flow relationships are most simply assessed by considering the size of the difference between the amounts of oxygen and carbon dioxide in the blood and in the air; the differences are small if the lungs are work ing efficiently. disparity between ventilation/blood flow ratios results in abnormally wide differences -and then alveolar-arterial po 2 and arterial-alveolar pco 2 gradients will be abnormal. alveolar capillary surface area is assessed by measuring the uptake of carbon monoxide -usually abnormal in diffuse interstitial inflam matory and fibrotic processes and in emphysema. assessing bronchial reactivity and the exercise response can help evaluate breathlessness. simple exercise testing provides information about overall fitness and the appropriateness of cardiorespiratory responses. radionuclide lung scanning, blood gas analysis and sputum cytology or culture are sometimes needed in addition. management can include oxygen administration by mask or nasal cannula (figs 15.4 and 15.5) . lrt disorders can cause significant incapacity and are often a con traindication to ga, and even to cs. asthma is common, affecting 2-5% of the overall population; it is on the increase, particularly in childhood, with a frequency of up to 20% in some highincome countries. asthma usually begins in childhood or early adult life; about half the patients with asthma develop it before age 10 years. bronchial hyperreactivity causes reversible airway obstruction from smooth muscle constriction (bronchospasm), mucosal oedema and mucus hypersecretion. there are two main types, extrinsic (allergic) and intrinsic asthma (table 15 .2). extrinsic (allergic) asthma, the main childhood type, may be pre cipitated by allergens in animal dander, feathers or hair, drugs (e.g. nonsteroidal antiinflammatory drugs [nsaids] and some antibiot ics), food (e.g. eggs, fish, fruit, milk, nuts), house dust (mite allergens) or moulds. patients frequently have or develop other allergic diseases, such as eczema, hay fever and drug sensitivities. extrinsic asthma is associated with ige overproduction on allergen exposure, and release of mast cell mediators (histamine, leukotrienes, prostaglandins, bradykinin and platelet activating factor), which cause bronchospasm and oedema. about 75% of asthmatic children lose their asthma or improve by adulthood. intrinsic asthma is usually of adult onset and not aller gic, but appears rather to be related to mast cell instability and airway hyperresponsivity. triggers include emotional stress, gastro oesophageal reflux or vagally mediated responses. either type of asthma can be triggered by: infections (especially viral, mycoplasmal or fungal); irritating fumes (e.g. traffic or cigarette smoke); exercise (possibly due to cold air); weather changes; emotional stress; foods (e.g. nuts, shellfish, strawberries or milk) or additives (such as tartrazine); and drugs (e.g. aspirin and other nsaids, beta blockers and angiotensinconverting enzyme inhibitors [aceis]). in wellcontrolled patients with asthma, clinical features may be absent. during an asthmatic episode, symptoms may include dysp noea, cough and paroxysmal expiratory wheeziness with laboured expiration. the frequency and severity of attacks vary widely between individuals (table 15 .3). patients may become distressed, anxious and tachycardic, have reduced chest expansion and be using accessory respiratory muscles to increase their ventilatory effort. nasal polyps are common, especially in aspirinsensitive asthmatics. children with asthma initially suffer from repeated 'colds' with cough, malaise and fever, often at night. asthma is typically diagnosed when the patient has more than one of the following -wheeze, cough, difficulty breathing and chest tightness -particularly if these are frequent and recurrent; are worse at night and in the early morning; occur in response to, or are worse after, exercise or other triggers, such as exposure to pets, cold or damp air, or with emotions or laughter; or occur without an association with colds. there is often: ■ a personal history of atopic disorder ■ a family history of atopic disorder and/or asthma ■ widespread wheeze, heard on chest auscultation ■ a history of improvement in symptoms or lung function in response to adequate therapy. a prolonged asthmatic attack, which is refractory to treatment, may lead to lifethreatening status asthmaticus (persisting for more than 24 hours). failure of the patient to complete a sentence, indrawing of the intercostal muscles, a rapid pulse, a silent chest and signs of exhaustion are suggestive of impending respiratory arrest. diagnosis of asthma is from the clinical history and presentation, based on recognizing a characteristic pattern of episodic symptoms in the absence of an alternative explanation. investigations include a chest radiograph (to exclude other diagnoses, such as a pneumo thorax), spirometry (serial pefr), skin tests and blood examination (usually eosinophilia, raised total ige and specific ige antibody concentrations, which may help identify allergens). occasionally, a histamine or methacholine challenge is used if the diagnosis is unclear. in children with an intermediate probability of asthma, who can perform spirometry and have evidence of airways obstruction, assess the change in fev 1 or pefr in response to an inhaled bronchodilator (reversibility) and/or the response to a trial of treatment for a speci fied period; if there is significant reversibility, or if a treatment trial is beneficial, a diagnosis of asthma is probable. management includes patient education, smoking cessation advice, avoidance of identifiable irritants and allergens, and use of drugs. home use of peak flow meters allows patients to monitor progress and detect any deterioration that may require urgent modification of treatment. treatment should be based on the amount by which peak flow is reduced (a pefr diary should be kept). drugs used for asthma management (table 15 .4) include oxygen, shortacting β 2 agonists (sabas; such as salbutamol), corticosteroids, leukotriene receptor antagonists and omalizumab (a recombinant humanized monoclonal antiige antibody that reduces the antigen specific ige). inhaled longacting β 2 agonists (labas) may be needed ( fig. 15.6 ). deaths from asthma are usually a result of failure to recognize dete rioration or reluctance to use corticosteroids. other factors that have been studied include: ■ air pollution -there is an association between air pollution and aggravation of existing asthma ■ allergen avoidance -there is no consistent evidence of benefit ■ breast-feeding -there is evidence of a protective effect in relation to early asthma ■ electrolytes -there is no consistent evidence of benefit ■ fish oils and fatty acid -there is no consistent evidence of benefit ■ house dust mites -measures to reduce the numbers of house dust mites do not affect asthma severity ■ immunotherapy -allergenspecific immunotherapy is beneficial in allergic asthma ■ microbial exposure -there is insufficient evidence to indicate that the use of probiotics in pregnancy reduces the incidence of childhood asthma ■ modified milk formulae -there is no consistent evidence of benefit pets -there are no controlled trials on the benefits of removing pets from the home ■ tobacco -exposure to cigarette smoke adversely affects quality of life, lung function, need for rescue medications and longterm control with inhaled steroids. there is an association between maternal smoking and an increased risk of infant wheeze ■ weight reduction -there is an association between increasing body mass index and symptoms of asthma. elective dental care should be deferred in severe asthmatics until they are in a better phase; this can be advised by the patient's general practitioner. asthmatic patients should be asked to bring their usual medica tion with them when coming for dental treatment. local anaesthe sia (la) is best used; occasional patients may react to the sulphites present as preservatives in vasoconstrictorcontaining la, so it may be better, where possible, to avoid solutions containing vasoconstric tor. adrenaline (epinephrine) may theoretically enhance the risk of arrhythmias with betaagonists and is contraindicated in patients using theophylline, as it may precipitate arrhythmias. relative analgesia with nitrous oxide and oxygen is preferable to intravenous sedation and gives more immediate control. sedatives in general are better avoided as, in an acute asthmatic attack, even ben zodiazepines can precipitate respiratory failure. ga is best avoided, as it may be complicated by hypoxia and hyper capnia, which can cause pulmonary oedema even if cardiac function is normal, and cardiac failure if there is cardiac disease. the risk of post operative lung collapse or pneumothorax is also increased. halothane or, better, enflurane, isoflurane, desflurane and sevoflurane are the preferred anaesthetics, but ketamine may be useful in children. allergy to penicillin may be more frequent in asthmatics. drugs to be avoided, since they may precipitate an asthmatic attack (see later), include those listed in box 15.1. acute asthmatic attacks may also occasionally be precipitated by anxiety; it is important to attempt to lessen fear of dental treatment by gentle handling and reassurance. even routine dental treatment can trigger a clinically significant decline in lung function in approximately 15% of asthmatics. acute asthmatic attacks are usually selflimiting or respond to the patient's usual medication, such as a betaagonist inhaler, but status asthmaticus is a potentially fatal emergency (ch. 1). there may be complications caused by the antiasthmatic drugs (table 15 .5). gastrooesophageal reflux is not uncommon, with occasional tooth erosion. periodontal inflammation is greater in asthmatics than in those without respiratory disease. persons using steroid inhalers may develop oropharyngeal candidosis or, occasionally, angina bullosa haemorrhagica. guidelines on the management of asthma may be found at: http://www.sign.ac.uk/guidelines/fulltext/101/index.html, http:// www.nice.org.uk/guidance/qualitystandards/indevelopment/asthma. jsp and http://www.britthoracic.org.uk/portals/0/guidelines/ asthmaguidelines/qrg101%202011.pdf (all accessed 30 september 2013). churg-strauss syndrome (css) is a rare, potentially fatal, systemic vasculitis similar to polyarteritis nodosa (pan), characterized by severe asthmalike attacks with peripheral eosinophilia, and intravas cular and extravascular granuloma formation with eosinophil infiltra tion and skin lesions in 70%. cardiopulmonary involvement is the main cause of death. css is diagnosed if at least 4 of the 6 criteria listed in box 15.2 are positive. the 5year survival of untreated css is 25%. combination treatment with cyclophosphamide and prednisolone (prednisone) provides a 5year survival of 50%. management problems relating to patients with css may include res piratory impairment and corticosteroid treatment (ch. 6). chronic obstructive pulmonary disease (copd; chronic obstructive airways disease, coad) is a common, chronic, slowly progressive, irre versible disease (most frequently a combination of chronic bronchitis and emphysema), characterized by breathlessness and wheeze (airways obstruction), cough and sputum. chronic bronchitis is defined as the excessive production of mucus and persistent cough with sputum production, daily for more than 3 months in a year over more than 2 consecutive years. it leads to production of excessive, viscous mucus, which is ineffectively cleared from the airway, obstructs and stag nates, and becomes infected, usually with streptococcus pneumoniae, moraxella catarrhalis and haemophilus influenzae. patchy areas of alveolar collapse can result. emphysema is dilatation of air spaces dis tal to the terminal bronchioles with destruction of alveoli, reducing the alveolar surface area available for respiratory exchange. copd is now the preferred term for conditions with airflow obstruction because of a combination of airway and parenchymal damage; patients were previ ously diagnosed as having chronic bronchitis or emphysema. copd is characterized by airflow obstruction -defined as an fev 1 / fvc ratio reduced to less than 0.7. if fev 1 is 80% or more, a diagno sis of copd should only be made if there are respiratory symptoms (e.g. dyspnoea or cough). the airflow obstruction is not fully revers ible, does not change significantly over months, and is usually progres sive in the long term. the most important causes of copd include cigarette smoking, environmental pollution, dusts, chemicals or occupational exposures to various substances. exposure to smoke from home cooking or heating fuels may contribute. deficiency of the antiproteolytic enzyme alpha1antitrypsin is a rare cause of emphysema. there is often significant airflow obstruction before the person is aware of it and so copd typically remains undiagnosed until patients are in their fifties. differentiation from asthma is important (table 15 .6). a diagnosis of copd should be considered in patients over the age of 35 who have a risk factor (e.g. smoking) and exertional breath lessness, chronic cough, regular sputum production, frequent winter 'bronchitis' or wheeze. clinical judgment is based on history, physical examination, confirmation of airflow obstruction using spirometry (postbronchodilator spirometry) and assessment of the severity of dyspnoea (tables 15.7 and 15.8). copd is characterized by breathlessness and wheeze (airways obstruction), cough and an early morning mucoid sputum production. to investigate symptoms that seem disproportionate to spirometric impairment progressive dyspnoea, low oxygen saturation, carbon dioxide accumu lation (hypercapnia) and metabolic acidosis mean that patients may ultimately become dyspnoeic at rest ('respiratory cripples'), especially when recumbent (orthopnoea), and eventually develop respiratory failure, pulmonary hypertension, right ventricular hypertrophy and rightsided heart failure (cor pulmonale). two clinical patterns of copd are recognized: ■ 'pink puffers' -patients with emphysema who manage to maintain normal blood gases by hyperventilation, and are always breathless but not cyanosed; rather they are pink from vasodilatation ■ 'blue bloaters' -patients with chronic bronchitis who lose their co 2 drive, fail to maintain adequate ventilation and become both hypercapnic and hypoxic with central cyanosis, cor pulmonale and oedema (for these patients, the respiratory drive is from the low po 2 and thus oxygen administration is contraindicated) (table 15 .9). the diagnosis of copd is based upon clinical history and presen tation. investigations include a chest radiograph (which may show hyperinflated lung fields with loss of vascular markings); arterial blood gases (which should be measured if pulse oximetry shows oxygen satu ration less than 92%); spirometry; and lung function tests. fev1 is reduced in all cases (fev 1 of less than 40% signifies severe copd) and the flow-volume curve shows a typical pattern, with reduced flow rates at mid and lowerlung volumes. a ratio of fev 1 :fvc of less than 70% confirms airways obstruction. patients with copd and their family should be educated about the disease, and about required lifestyle changes and medication. nondrug therapy includes: stopping smoking (nicotine replacement therapy or bupropion may help); exercise by pulmonary rehabilitationof proven benefit; weight loss (improves exercise tolerance); and vaccination (pneumococcal and influenza vaccines). drug therapy includes shortacting bronchodilators (anticholinergic drugs [ipra tropium bromide]) and β 2 agonists (salbutamol) to treat the reversible component of airway disease; corticosteroids (inhaled or systemic); and antibiotics (amoxicillin, trimethoprim or tetracycline). mucolytics, such as carbocisteine, reduce acute exacerbations by almost onethird. longterm oxygen therapy (ltot) reduces mortality. people with stable copd who remain breathless or have exacerba tions, despite using shortacting bronchodilators, should be offered the following as maintenance therapy: ■ if fev 1 is 50% of predicted or more: use either a longacting β 2 agonist (laba) or longacting muscarinic antagonist (lama). ■ if fev 1 is less than 50% predicted: either a laba with an inhaled corticosteroid (ics) in a combination inhaler, or a lama. offer a lama in addition to a laba plus ics to people with copd who remain breathless or have exacerbations, despite taking laba plus ics, irrespective of their fev 1 . provide pulmonary rehabilitation for all who need it; noninvasive ventilation (niv) is the treatment of choice for persistent hyper capnic ventilatory failure during exacerbations not responding to medical therapy. the frequency of exacerbations should be reduced by appropriate use of inhaled corticosteroids and bronchodilators, and vaccinations. bronchodilators (shortacting β 2 agonists [saba] and shortacting muscarinic antagonists [sama]) should be the initial empirical treat ment for the relief of breathlessness and exercise limitation. ics have potential adverse effects (including nonfatal pneumonia) in people with copd. offer a oncedaily lama in preference to fourtimes daily sama to people with stable copd who remain breathless or have exacerbations, despite using shortacting bronchodilators as required, and in whom a decision has been made to commence regular maintenance bronchodilator therapy with a muscarinic antagonist (see above). most patients -whatever their age -are able to acquire and main tain an adequate inhaler technique. bronchodilators are usually best administered using a handheld inhaler device (including a spacer device if appropriate). patients with distressing or disabling dyspnoea, despite maximal therapy using inhalers, should be considered for nebulizer therapy. they should be offered a choice between a face mask and a mouth piece to administer their nebulized therapy, unless the drug specifically requires a mouthpiece (for example, anticholinergic drugs). some patients with advanced copd may require maintenance oral corticosteroids when these cannot be withdrawn following an exacer bation. these individuals should be monitored for the development of osteoporosis and given appropriate prophylaxis. theophylline should only be used after a trial of saba and laba, and only to those who are unable to use inhaled therapy, as there is a need to monitor plasma levels and interactions. the dose of theo phylline prescribed should be reduced at the time of an exacerbation if macrolide or fluoroquinolone antibiotics (or other drugs known to interact) are given. there is insufficient evidence to recommend prophylactic antibiotic therapy in the management of stable copd. mucolytic drug therapy should be considered in patients with a chronic cough productive of sputum. if patients remain symptomatic on monotherapy, their treatment should be intensified by combining therapies from different drug classes, such as: ■ β 2 agonist and theophylline ■ anticholinergic and theophylline. inappropriate oxygen therapy in people with copd may depress respiration. ltot is indicated in patients with copd who have a pao 2 of less than 7.3 kpa when sta ble, or a pao 2 greater than 7.3 kpa and less than 8 kpa when stable, and one of: secondary polycythaemia, nocturnal hypoxaemia (oxygen saturation of arterial blood [sao 2 ] of less than 90% for more than 30% of the time), peripheral oedema or pulmonary hypertension. to reap the benefits of ltot, patients should breathe supplemental oxygen for at least 15 hours per day. to ensure that all those eligible for ltot are identified, pulse oximetry should be available in all health care settings. the assessment of patients for ltot should comprise the measurement of arterial blood gases on two occasions at least 3 weeks apart in patients who have a confident diagnosis of copd, who are receiving optimum medical management and whose copd is stable. patients should be warned about the risks of fire and explosion and told not to smoke when using oxygen. ambulatory oxygen therapy should be considered in patients on ltot who wish to continue oxygen therapy outside the home, and who have exercise desaturation, are shown to have an improvement in exercise capacity and/or dyspnoea with oxy gen, and are motivated to use oxygen. adequately treated patients with chronic hypercapnic respiratory failure who have required assisted ventilation during an exacerbation, or who are hypercapnic or acidotic on ltot, should be referred to a specialist centre for consideration of longterm niv. advanced emphysema is occasionally treated with sur gery -excision of large acquired bullae or, rarely, lung transplantation. patients with copd who need dental care can be classified as follows: ■ patients at low risk -experience dyspnoea on effort but have normal blood gas levels. these patients can receive all dental treatment with minor modifications. ■ patients at moderate risk -experience dyspnoea on effort, are chronically treated with bronchodilators or recently with corticosteroids, and pao 2 lowered. a medical consultation is advised to determine the level of control of the disease before any dental treatment. ■ patients at high risk -have symptomatic copd that may be end stage and poorly responsive to treatment. with these patients, a medical consultation is essential before any dental treatment is carried out. patients with copd are best treated in an upright position at midmorning or early afternoon, since they may become increasingly dyspnoeic if laid supine. it may be difficult to use a rubber dam, as some patients are mouthbreathers and not able to tolerate the additional obstruction. la is preferred for dental treatment, but bilateral mandibular or palatal injections should be avoided. patients with copd should be given relative analgesia only if absolutely necessary, and only in hospital after full preoperative assessment. cs with diazepam and midazolam should not be used, as benzodiazepines are respiratory depressants. patients should be given ga only if absolutely necessary, and intravenous barbiturates are contraindicated. secretions reduce airway patency and, if lightly anaesthetized, the patient may cough and contaminate other areas of the lung. postoperative respiratory complications are more prevalent in patients with preexisting lung diseases, especially after prolonged operations and if there has been no preoperative preparation. the most important single factor in preoperative care is cessation of smok ing for at least 1 week preoperatively. respiratory infections must also be eradicated; sputum should first be sent for culture and sensitivity, but antimicrobials such as amoxicillin should be started without await ing results. the medical management of copd should be optimized prior to surgery. the ultimate clinical decision about whether or not to proceed with surgery should rest with a consultant anaesthetist and consultant surgeon, taking account of comorbidities, functional status of the patient and necessity for the surgery. composite assessment tools, such as the american society of anesthesiologists (asa) scoring system, and not just lung function, are the best criteria for the assessment of patients with copd before surgery. those taking corticosteroids should be treated with appropri ate precautions (ch. 6). interactions of theophylline with other drugs, such as adrenaline (epinephrine), erythromycin, clindamycin, azithro mycin, clarithromycin or ciprofloxacin, may result in dangerously high levels of theophylline. ipratropium can cause dry mouth. guidelines for the management of copd may be found at: http:// publications.nice.org.uk/chronicobstructivepulmonarydisease cg101 (accessed 30 september 2013). respiratory viruses usually spread by touch or airborne transmission and the very small particles (2-0.2 micrometres) can avoid the upper respiratory tract defences and the mucociliary elevator to reach the lung alveoli. a range of viruses can cause lower respiratory tract infections (lrtis ; table 15 .10). some viruses (e.g. influenza and respiratory syncytial) can spread from the upper to the lower respira tory tract via infection of the respiratory epithelium and can lead to bacterial superinfection and pneumonitis (pneumonia). mycoplasmal (atypical) pneumonia and tuberculosis (tb) may be direct infections. epidemics of a potentially fatal severe acute respiratory syndrome (sars) have been caused by a coronavirus that originated in china and spread worldwide; h5n1 bird influenza also arose as an epidemic; and a similar epidemic, but of swine influenza (h1n1), emanated from mexico (see later). bacterial infections, such as pneumonia or lung abscess, can also result from material aspirated into the lungs, and are usually unilat eral. those who aspirate more than others have, as a result, more frequent lrti and this is seen in alcohol and other drug abusers, as well as comatose patients. exogenous penetration and contamination of the lung can result from trauma (e.g. a stab wound or road traffic accident) or surgery. entamoeba histolytica can occasionally cause pneumonia -by direct extension from an amoebic liver abscess (table 15 .11). patients with endocarditis, or septic pelvic or jugular thrombo phlebitis, may experience lrti acquired haematogenously and then it is often bilateral. immunocompromised persons (e.g. those with human immunode ficiency virus/acquired immune deficiency syndrome [hiv/aids] and transplant recipients) and people with bronchiectasis or cystic fibrosis are also susceptible to respiratory infections by a range of opportun istic microbes. pneumocystis jiroveci (p. carinii), for example, is a com mon cause of potentially fatal pneumonia in immunocompromised patients -especially those with hiv/aids (chs 20 and 21). clinical features of lrti vary according to the part of the respiratory tract mainly affected: ■ bronchiolitis causes rapid respiration, wheezing, fever and dyspnoea -but is restricted mainly to infants. ■ bronchitis causes cough, wheezing and sometimes dyspnoea. ■ pneumonia causes cough, fever, rapid respiration, breathlessness, chest pain, dyspnoea and shivering. antimicrobial therapy is indicated, particularly for pneumonia. antivirals have not been highly effective. oxygen may be needed. pneumococcal vaccine is indicated for older people. the majority of lrtis are severe illnesses, and are contraindications to all but emergency dental treatment. ga is hazardous and absolutely contraindicated. dental treatment should be deferred until recovery, or be limited to pain relief. influenza is mainly a communitybased infection transmitted in house holds and communities. healthcareassociated influenza infections can arise in any healthcare setting, most commonly when influenza is also circulating in the community. influenza is a contagious disease caused by influenza virus types a, b or c. type a has two main subtypes (h1n1 and h3n2); it causes most of the widespread influenza epidemics and can occasionally be fatal. type b viruses generally cause regional outbreaks of moderate severity, and type c viruses are of minor significance. a person can spread influenza starting 1 day before they feel sick and for another 3-7 days after symptoms start. influenza can be pre vented or ameliorated by vaccination each autumn; this is especially indicated for older people and those with cardiorespiratory disease. influenza attacks virtually the whole respiratory tract; symptoms appear suddenly after 1-4 days and include fever, sore throat, nasal congestion, headache, tiredness, dry cough and muscle pains (myalgia). most people recover in 1-2 weeks but infection can be lifethreatening, mainly because primary influenzal viral pneumonia can lead to sec ondary bacterial pneumonia or can exacerbate underlying conditions (e.g. pulmonary or cardiac disease). the old and very young, and those with chronic disorders, are more likely to suffer complications, such as pneumonia, bronchitis, sinusitis or otitis media. influenza has also been followed by depression, encephalopathy, myocarditis, myositis, pericarditis, reye syndrome and transverse myelitis. rest, maintenance of fluid intake, analgesics, antipyretics, and avoid ance of alcohol and tobacco help relieve symptoms. aspirin must never be given to children under the age of 16 years who have 'flulike symptoms, and particularly fever, as this can cause reye syndrome. zanamivir (an antiviral that works against influenza types a and b) can shorten the symptoms by approximately 1 day, if treatment is started during the first 2 days of illness. other antiviral drugs include amantadine, oseltamivir and rimantadine; they may be helpful but their use is restricted mainly to immunocompromised persons, since they can cause adverse effects. influenza can be a severe contagious illness so all but emergency den tal treatment should be deferred until recovery. ga is hazardous and absolutely contraindicated. influenza type a subtype h5n1 can cause an illness known as 'avian influenza' or 'bird 'flu' in birds, humans and many other animal spe cies. hpai a(h5n1) -'highly pathogenic avian influenza virus of type a of subtype h5n1' -is the causative agent and is enzootic in many bird populations, especially in southeast asia. it has spread globally and resulted in the deaths of over 100 people and the slaughter of mil lions of chickens. a vaccine that could provide protection (prepandrix) has been cleared for use in the european union. h5n7 is a more recent emergent infection, similar in many respects. swine influenza is common in pigs in the midwestern united states, mexico, canada, south america, europe (including the uk, sweden and italy), kenya, china, taiwan, japan and other parts of eastern asia. transmission of swine influenza virus from pigs to humans is not com mon, but can produce symptoms similar to those of influenza. a 2009 outbreak in humans ('swine 'flu') was due to an apparently new strain of h1n1 arising from a reassortment produced from strains of human, avian and swine viruses. it can pass from human to human. antiviral agents such as oseltamivir may help. vaccines are now available. an outbreak of a lifethreatening febrile respiratory infection appeared in 2003, originating from guangdong, china, and was named severe acute respiratory syndrome (sars). caused by a newly recognized coronavirus (sarsassociated coronavirus, sarscov), sars spread via close contact to many countries across the world. according to the world health organization, 8437 people worldwide became sick with sars during the course of the first recognized outbreak and 813 died. the incubation period of 2-7 days is followed by a high fever (above 38.0°c), malaise, headache and myalgia. some people also experience mild upper respiratory symptoms and, after 2-7 days, lower respiratory signs -a dry cough and dyspnoea, potentially progressing to hypox aemia. sars can cause a pneumonia with a mortality approaching 10%, particularly in older or immunocompromised people. artificial ventilation has been needed in 10-20% of cases. antiviral agents, such as oseltamivir or ribavirin, may help. inactivated vaccines, virally and bacterially vectored vaccines, recombinant protein and dna vaccines, as well as attenuated vaccines, are under development. sars is a severe illness, and all but emergency dental treatment should be deferred until recovery. ga is hazardous and absolutely contraindicated. for all contact with suspect sars patients, careful hand hygiene is important, including handwashing with soap and water; if hands are not visibly soiled, alcoholbased handrubs may be used as an alternative to handwashing. if a suspected sars patient is admitted to hospital, infection control personnel should be notified immediately. infection control measures (www.cdc.gov/ncidod/hip/iso lat/isolat.htm; accessed 30 september 2013) should include standard precautions (e.g. hand hygiene): healthcare personnel should wear eye protection for all patient contact; contact precautions (e.g. gown and gloves for contact with the patient or their environment); and airborne precautions (e.g. an isolation room with negative pressure relative to the surrounding area and use of an n95 filtering disposable respirator for persons entering the room). pneumonia is classed as 'primary' if it occurs in a previously healthy individual, and is usually lobar; it is called 'secondary' if it follows some other disorder, such as previous viral respiratory infections, aspir ation of foreign material, lung disease (bronchiectasis or carcinoma), depressed immunity (e.g. alcoholism or immunosuppression), or aspir ation of oral bacteria ( pneumonia causes cough, fever, rapid respiration, breathlessness, chest pain, dyspnoea and shivering. complications can include lung abscess or empyema (pus in pleural cavity). it is important to avoid alcohol and tobacco, but use analgesics and antipyretics to relieve the symptoms. broadspectrum antimicrobi als given promptly and empirically usually include a macrolide (azithromycin, clarithromycin or erythromycin), quinolone (moxiflox acin, gatifloxacin or levofloxacin), or doxycycline for outpatients. for in patients, cefuroxime or ceftriaxone plus a macrolide is used. prophylaxis includes immunization against influenza and pneumococci. pneumonia is a severe illness and all but emergency dental treatment should be deferred until recovery. ga is hazardous and absolutely contraindicated. ventilatorassociated pneumonia (vap) is discussed later. legionellosis is a bacterial respiratory infection caused by one of the family legionellaceae, gramnegative aerobic bacilli, ubiquitous in water and soil but particularly preferring warm aquatic environments. the term legionnaire's disease was coined as a result of an outbreak of the previously unrecognized respiratory disease in an american legion meeting in philadelphia in 1976, but it is now recognized worldwide, many infections being contracted during travel abroad, particularly to spain, turkey and some other mediterranean areas. legionella bacteria can be found in natural freshwater environments, usually in insufficient numbers to cause disease. legionella grow best in warm water, as in hot tubs, cooling towers, hot water tanks, large plumbing systems, or the airconditioning systems of large buildings. though there are over 30 legionellaceae, most infections are caused by legionella pneumophila. disease is contracted by inhalation of contaminated mist or vapour, mainly (approximately 46%) through aerosolization of infected water in airconditioning systems, hotwater systems, humidifiers, nebulizers, showers and spa pools. outbreaks have mostly been linked to aerosol sources in the community, cruise ships and hotels, with the most likely sources being whirlpool spas, air conditioning units in large buildings, potable (drinking) water systems, and water used for bathing. risk factors include: ■ exposure to: recent travel with an overnight stay outside of the home (outbreaks of travelassociated legionellosis are infrequently identified but more than 20% of cases are thought to be associated with recent travel) whirlpool spas recent repairs or maintenance work on domestic plumbing ■ systemic illhealth: alcohol use chronic kidney disease diabetes immune defects liver disease malignancy smoking. illness mainly affects males over 45, smokers, heavy drinkers, older people and the immunocompromised. also vulnerable are travellers, especially middleaged and older tourists, and conference or business groups, possibly because of tiredness or age. many young people have been exposed to infection and become seropositive, but remained healthy. there is no evidence of persontoperson transmission of legionellosis. legionellosis manifests as one of two clinical syndromes (table 15 .14). legionnaire's disease is typically a lobular type of pneumonia, which can be fatal but is fortunately rare; infection can range from discrete patches of inflammation and consolidation to involvement of whole lobes. pontiac fever is milder and usually subsides rapidly, often with out treatment. people who should be tested for legionnaire's disease include those with pneumonia in the following groups: because legionella is commonly found in the environment, clinical isolates are necessary to interpret the findings of an environmental investigation. diagnosis can be by rapid urine molecular testing for l. pneumophila antigen, and culture of respiratory secretions on selective media. sensitivity and specificity of the diagnostic tests are shown in table 15 .15. pontiac fever is a selflimited illness; most cases recover within 1 week and few benefit from antibiotic treatment. overall mortality in legionnaire's disease may be as high as 10%, and over 25% in older people and up to 80% in the immunocompromised. erythromycin is standard treatment; cephalosporin is an alternative. legionella species are present in roughly twothirds of potable water samples collected from domestic and institutional taps and drinking fountains, and from a similar percentage of dental units, but water from these dental units often has higher bacterial concentrations (ch. 31). there are reports of legionella infections in dental unit water lines, and antibodies and occasionally frank infection demonstrated in dental staff; at least one patient appears to have contracted and died from infection emanating from a dental practice. prevention is crucial, involving (ch. general aspects tuberculosis (tb) , an infection caused by mycobacteria, affects approxi mately onethird of the world's population (1.5 billion people); it is a major global health problem, some 2 million people dying from it annu ally. tb disproportionately affects the poorest persons in both high income and developing countries. in highincome countries, most human tb arises from mycobacterium tuberculosis, transmitted from person to person through the air. tb usually affects the lungs initially (pulmonary tb) but can also involve brain, kidneys, spine and other parts. from victorian times to about the second world war, mycobacterium bovis infection from infected cows' milk (bovine or btb) was a major cause of morbidity and mortality; it was clinically and pathologically indistin guishable from infection caused by m. tuberculosis. cattletesting and a slaughter programme became compulsory in 1950 and, by the 1980s, the incidence of tb in cattle had been substantially reduced. tuberculosis from m. bovis in cows' milk was virtually eliminated in highincome countries by the tuberculin testing of cattle and pasteurization of milk. in the developing world, many cattle still have tb, and btb is still seen. btb has also increased in highincome countries over the last two dec ades and an infection rate of up to 38% in badgers -and transmission to cattle -may explain this. tb is not spread by touch or by drinking glasses, dishes, sheets or clothing. it is usually transmitted by infected sputum, typically from close contacts such as family members, but is unlikely to be transmit ted between normal social contacts. tb can present an occupational risk to healthcare professionals, including dental staff. one outbreak of drugresistant tb in new york involved at least 357 patients, most of whom contracted tb in one of 11 hospitals; nearly 90% of the patients were also hivpositive, and most were young males of hispanic or african heritage. tb has been transmitted between pas sengers during longhaul airline flights. the risk of transmitting tb though air circulation is now low because the highefficiency particu late air (hepa) filters on newer commercial aircraft are of the same type as those used in hospital respiratory isolation rooms; indeed, the number of times air is cleaned each hour exceeds the recommendation for hospital isolation rooms. subsaharan africa has the highest rates of active tb per capita, driven primarily by the hiv epidemic. the absolute number of cases is highest in asia, with india and china having the great est burden of disease globally. in the usa and most western european countries, the majority of cases occur in foreignborn residents and recent immigrants from countries in which tubercu losis is endemic. immunocompromised people -such as diabetics and severely immuno deficient patients, like those with hiv/aids (about 30% of south africans with hiv/aids also have tb) -and patients in prisons or institutions are at risk. tb also mainly affects medically neglected persons, such as vagrants, alcoholics, intravenous drug abusers or older homeless people. the main groups at increased risk for infection therefore include people who are resourcepoor or immunoincompetent, especially: tb in developing countries is particularly widespread and is increasing, the highest rises in incidence being in southeast asia, subsaharan africa and eastern europe. in highincome countries, the incidence is also rising, probably because of worsening social deprivation, homelessness, immigration, hiv infection and intra venous drug abuse. it is now as common in london as in the devel oping world, and is seen especially in immigrants, such as those from the indian subcontinent, africa and south asia. this increase appears to be a result of the development of tb disease in individu als who may have been infected for some time and of new infections acquired in the uk, or as a result of travel to other countries where tb is common. london accounted for the highest proportion of cases in the uk in 2011 (39%), followed by the west midlands region (11%); 74% of these were born outside the uk and mainly originated from south asia and subsaharan africa. in 2011, there was a rise in the number of tb cases compared to 2010, as well as an increase in drug resistance. more information on tb, including statistics, can be found at: http:// www.hpa.org.uk/publications/infectiousdiseases/tuberculosis/ and http://www.tbfacts.org/tbstatistics.html (both accessed 30 september 2013). initial infection with tb is usually subclinical. about 10% of those infected develop overt disease; of these, half will manifest within 5 years (primary tb), while the remainder will develop postprimary disease. inhaled mycobacteria may cause subpleural lesions (primary lesion) and lesions in the regional lymph nodes (primary complex). body defences usually localize the mycobacteria, though these remain viable; infected persons are not obviously ill and are unlikely to know they are infected (latent ; table 15 .16). latent tb infection (ltbi) usu ally becomes active only after many years, if body defences become weakened (box 15.3). however, active tb can develop shortly after mycobacteria enter the body, if body defences are impaired such as in ageing, drug or alcohol abuse, or hiv/aids. also, in massive infec tions, acute active tb can result, typically causing a chronic productive cough, haemoptysis, weight loss, night sweats and fever. erythema nodosum may be associated. extrapulmonary tb is less common; it may appear as glandular involvement in the neck or elsewhere, and is less infectious than pulmonary tb. lymph node tb may lead to lymphadenopathy, caseation of the nodes and pressure symptoms -for example, on the bronchi. postprimary tb follows reactivation of an old primary pulmonary lesion and results in features ranging from a chronic fibrotic lesion to fulminating tuberculous pneumonia. the pulmonary lesions may extend and lead to a pleural effusion. reactivation or progression of primary tb may also result in widespread haematogenous dissemina tion of mycobacteria -'miliary tb'. multiple lesions may involve the central nervous system, bones, joints, and cardiovascular, gastrointes tinal and genitourinary systems. clinical presentation in tb is thus variable, depending on the extent of spread and the organs involved. as it frequently passes unrecog nized for so long, the mortality is high. similar illnesses to tb may also be caused by atypical (nontuber culous) mycobacteria, such as m. avium complex (mac; see below). the diagnosis of tb is suggested by the history and confirmed by physical examination, a massively raised erythrocyte sedimentation rate (esr), positive tuberculin skin tests (tsts; mantoux or heaf test for a delayed hypersensitivity reaction to protein from m. tuberculosis [purified protein derivative; ppd]) and chest imaging. hypersensitivity develops with 2-8 weeks of infection and can be detected by conversion of the tst from negative to positive, but tsts are neither 100% sensi tive nor specific. a positive mantoux reaction indicates previous immu nization (bcg; bacille calmette-guérin -live attenuated m. bovis) or current infection -not necessarily disease. chest radiography may show scarring and hilar lymphadenopathy. computed tomography (ct) may show areas of calcification or highlight a tuberculous abscess. smears and culture of sputum, blood, laryngeal swabs, bronchoalveolar lavage, gastric aspirates or pleural fluid may be tested for mycobacteria. polymerase chain reaction (pcr) techniques have greatly acceler ated the diagnosis and speciation, though ziehl-neelsen, auramine or rhodamine microbial stains are still used. the mycobacteria growth indicator tube (mgit) system gives results as early as 3-14 days. blood assay for m. tuberculosis (bamt) may be positive by interferongamma release assay (igra). some 15% of people over 65 years have a positive igra. the igra can be used in place of (but not in addition to) tst. igras measure the immune reactivity to m. tuberculosis. white blood cells from most persons that have been infected with m. tuberculosis will release interferongamma (ifnγ) when mixed with m. tuberculosis antigens. a positive test result sug gests that m. tuberculosis infection is likely; a negative result suggests that infection is unlikely. latent infection (ltbi) can be diagnosed with either a tuberculin skin test or an igra (more specific). igra gives a result within 24 hours and should be used biological therapy is given, such as for rheumatoid arthritis or inflammatory bowel disease. prior bcg vacci nation does not cause a falsepositive igra test result. more informa tion on the igra is available at: http://www.cdc.gov/tb/publications/ factsheets/testing/igra.htm (accessed 30 september 2013). active tb is diagnosed by sputum microscopy and culture in liquid medium with subsequent drugsusceptibility testing. nucleic acid people who should be tested for tb include those who have symp toms, those who have had close daytoday contact with active tb disease (family member, friend or coworker), those who have hiv infection or aids, those with lowered immunity, those who are required to for employment or school, and those about to be treated with biological agents. the top priority of tb control programmes is to identify and give complete treatment to all patients with active disease. tb is a notifi able disease and contact tracing is an important aspect of limiting spread. treatment with antibiotics is indicated for people who are sick with tb, those infected but not sick, and those who are close contacts of infectious tb cases. treatment for 'symptomatic sputumpositive' patients, which should be instituted as soon as possible, is combination chemotherapy, usually isoniazid plus rifampicin plus pyrazinamide or ethambutol for 2 months, with continuation of daily isoniazid and rifampicin for a further 4 months. treatment for 'asymptomatic' patients who are believed to have been infected by contacts, but are not unwell, includes isoniazid for 6 months or isoniazid and rifampicin for 3 months. rifapentine is a longacting rifampicin used once weekly. fluoroquinolones (moxifloxacin) may also act against tb. there may be resistance to one or more than one antibiotic. currently, given the potential risk of drugresistant tb being present, treatment is usually started with isoniazid, rifampicin, pyrazinamide and ethambutol (or a quinolone such as gatifloxacin or moxifloxacin) for 2 months, then isoniazid and rifampicin for 4 months. all antituberculous drugs (table 15 .17) have potentially serious adverse effects and require careful monitoring. if patient compliance is considered to be poor, directly observed therapy (dot), where drugs are dispensed by and taken in the presence of a healthcare profes sional, may be indicated. new drugs are on the horizon. immunization using bcg is advocated for schoolchildren, highrisk individuals and healthcare professionals -although its efficacy has been questioned. new vaccines are in development. chemoprophylaxis with isoniazid and rifampicin is indicated in a number of situations (box 15.4) . tb can become resistant to the drugs used to treat it particularly when the drugs are misused or mismanaged. this may occur, for example, when: in some developing countries, approximately 10% of cases are multi ple antibioticresistant; this is termed multidrugresistant tuberculosis (mdrtb); in the uk, only a small minority currently fall into this category but the number of cases is increasing. mdrtb is defined as resistance to rifampicin and isoniazid; it may be atypical in presenta tion and the infection disseminates. more than 4% of people with tb worldwide have mdrtb, and eastern europe has a high prevalence. mdrtb is seen mainly in people with hiv/aids and in hiv/aids and in africans. bedaquiline, is a new antitubercular agent the first active agent against tuberculosis to be registered since 1963. extensively drugresistant tuberculosis (xdrtb) is a rare type of mdrtb, not only resistant to isoniazid and rifampin, but also to any fluoroquinolone and at least one of three injectable secondline drugs (i.e. amikacin, kanamycin, or capreomycin). xdrtb is of special concern for immunocompromised people (e.g. with hiv/aids), who are more likely to develop tb, and have a higher risk of death if they do develop it. xdrtb is most often encountered in people from eastern europe, russia and africa. it has been transmitted in healthcare facilities and is now seen worldwide. it is essentially untreatable, though capreomycin has been used effectively to treat mdrtb in hivpositive individuals. totally drugresistant tb was reported initially in 2007-2009 in india, iran and italy; it is spreading, despite denials, and is most disquieting. chronic ulcers, usually on the tongue dorsum, are the main oral manifestation of tb. they result from coughing of infected sputum from pulmonary tb, including in hivinfected persons with tb, but are rare and such cases (usually middleaged males) may result from neglect of symptoms or default from treatment. occasionally, the diagnosis is made from biopsy of an ulcer after granulomas are seen microscopically. acidfast bacilli are rarely seen in oral biopsies, even with the help of special stains, so unfixed material should also be sent for culture if possible. tuberculous cervical lymphadenopathy is the next most common form of the infection and is particularly com mon among those from south asia. most tb lymphadenitis is pain less, with several enlarged, matted nodes, but systemic symptoms are present only in a minority and only about 15% have pulmonary mani festations on radiography (fig. 15.7) . diagnosis relies on tuberculin testing, which can be positive in both tuberculous and non tuberculous mycobacterial cervical lymphadenitis. any person with lymphadenop athy and recent conversion from a negative to positive tuberculin test should be suspected of having mycobacterial infection, and this should prompt biopsy (e.g. fineneedle aspiration biopsy) for culture or histo logical confirmation. pcr will improve diagnosis, as culture must wait 4-8 weeks for a result. oral complications of antitubercular therapy are rare, but rifabutin and rifampicin can cause red saliva. pulmonary tb is of high infectivity, as shown by cases of tuber culous infection of extraction sockets and cervical lymphadenitis in 15 patients treated by an infected member of staff at a dental clinic. dental staff who themselves were hivpositive, working in a dental clinic for hivinfected persons in new york, have died from tb con tracted occupationally. transmission of mdrtb between two dental workers may have occurred in an hiv dental clinic. infection control is thus important, so staff with tb are usually precluded from their occupation until treated. management of a patient with tb depends upon the level of poten tial infectivity (table 15.18) . patients with open pulmonary tb are con tagious, and dental treatment is thus best deferred until the infection has been treated. treatment with appropriate drugs for 2 weeks drasti cally reduces the infectivity of patients with pulmonary tb. if patients with open pulmonary tb must be given dental treatment, special pre cautions should be used to prevent the release of mycobacteria into the air, to remove any that are present and to stop their inhalation by other persons. reduction of splatter and aerosols, by minimizing cough ing and avoiding ultrasonic instruments, and use of a rubber dam, are important. improved ventilation, ultraviolet germicidal light, new masks and personal respirators, and other personal protective devices, such as hepa filters, are indicated ( fig. 15.8) . mycobacteria are very resistant to disinfectants, so that heat sterilization must be used. la is safe and satisfactory. relative analgesia is contraindicated because of the risk of contamination of the apparatus. ga is also contraindicated for dental treatment because of the risk of contamina tion of the anaesthetic apparatus and because of impaired pulmonary function. aminoglycosides, such as streptomycin, enhance the activity of some neuromuscular blocking drugs and in large doses may alone cause a myasthenic syndrome. possible drug interactions are shown in table 15 .19. other factors, such as alcoholism or intravenous drug use (ch. 34), hepatitis (ch. 9) or hiv disease (ch. 20), may also influence dental management. mycobacteria other than tuberculosis (mott) are widely distributed in water, soil, animals and humans, and rarely cause disease. severe mott infections have been seen, however, in individuals predisposed because of defects in the interleukin12 (il12) and interferongamma (ifngamma) pathways. mycobacterium abscessus, a bacterium found in water, soil and dust, has been known to contaminate medications and products, including medical devices. healthcareassociated m. abscessus can cause a vari ety of infections, usually of the skin, but it can also cause lung infec tions in persons with various chronic lung diseases and is increasingly recognized as an opportunistic pathogen in cystic fibrosis (cf) patients persontoperson transmission of atypical mycobacteria is not important in acquisition of infection, except for skin infections. on rare occasions, mott skin infections have followed tattooing with contaminated tattoo inks. many people become infected with and har bour mott in their respiratory secretions without any symptoms or evidence of disease. individuals with respiratory disease from mott do not readily infect others and, therefore, do not need to be isolated. mott are generally not infectious to others. infection with m. abscessus is usually caused by injections of con taminated substances or by invasive medical procedures employing contaminated equipment or material. infection can also occur after accidental injury where the wound is contaminated by soil. there is very little risk of transmission from person to person. mac complex, m. scrofulaceum and m. kansasii are possible causes of tuberculous cervical lymphadenitis. mac may also infect the lungs (similar to tb), skin or lymph nodes. lung disease is also caused occasionally by m. kansasii, mainly in middleaged and older persons with underlying chronic lung conditions. m. fortuitum and m. chelonae may cause skin and wound infections and abscesses, frequently associated with trauma or surgery. m. marinum may cause 'swimming pool granuloma', a nodular lesion that may ulcerate, usually on an extremity. m. ulcerans may produce chronic ulcerative skin lesions, usually of an extremity. m. abscessus skin infections present with swollen and/ or painful areas that are usually red, warm and tender to the touch, and which can also develop into boils or pustules. other features of m. abscessus infection are fever, chills, muscle aches and malaise. cervical lymphadenitis due to mac, m. scrofulaceum and m. kansasii may affect otherwise healthy young children, most commonly pre school females who have unilateral cervical lymphadenopathy, typically in the submandibular or jugulodigastric nodes, and they may form a 'cold abscess'. mott is the usual cause in children under 12 years but tb is more common in older patients. absence of fever or tuber culosis, a positive tuberculin test and failed response to conventional antimicrobials are highly suggestive of mott, but definitive diagnosis is by smear, culture or pcr of biopsy material obtained by fineneedle aspiration or removal of nodes. treatment is based on results of laboratory testing, which should identify the appropriate antibiotic. preventive treatment of close contacts of persons with disease caused by mott is not needed. most mott are resistant to standard antitubercular medication and, though it is possible that clarithromycin or clofazimine may have some effect, excision of affected nodes is the usual recommended therapy. water from dental units may contain mott species; mycobacterial proliferation in biofilms may explain the extent of this contamination (ch. 31). aspiration syndromes are conditions in which foreign substances are inhaled into the lungs and which can have consequences ranging from asphyxia to infection and lung abscess. dental restorations or frag ments of teeth, plaque, gastric contents and other materials may be aspirated, especially if material enters the pharynx, and particularly if the cough reflex is impaired for any reason. most commonly, aspiration syndromes involve oral or gastric contents associated with gastrooesophageal reflux disease (gord), swallowing dysfunction (ch. 7), neurological disorders and structural abnormalities, such as a pharyngeal pouch. cricopharyngeal dys function involves cricopharyngeal muscle spasm or achalasia of the superior oesophageal sphincter, and can be seen in infants who have a normal sucking reflex but have incoordination during swallowing, pos sibly secondary to delayed development or cerebral palsy. anatomical disorders, such as cleft palate, pharyngeal pouch, oesophageal atresia, tracheooesophageal fistula, duodenal obstruction or malrotation, and motility disorders, such as achalasia, may have an aspiration risk. infirm older patients are also at risk of aspiration, especially if they are bedbound or have neurological disorders. isolated superior laryngeal nerve damage, vocal cord paralysis, cerebral palsy, muscular dystrophy and riley-day syndrome (familial dysautonomia) are all associated with increased risk of aspiration. ventilatorassociated pneumonia (vap), as defined by the centers for disease control and prevention (cdc), is present when the chest radiograph shows new or progressive infiltrate, consolidation, cavitation or pleural effusion in conjunction with either new onset of purulent sputum or change in character of sputum, and an organism isolated from blood, or the isolation of an aetiological agent from a specimen obtained via suction aspiration through an endotracheal or tracheostomy tube. the major route for acquiring endemic vap is oropharyngeal colo nization by endogenous flora or by exogenously acquired pathogens from intensive care units. vap is the most commonly reported health careacquired infection in patients receiving mechanical ventilation, with prevalence rates consistently in the 10-20% range. mortality rates in vap are at least double those in patients without vap, ranging from 24% to 85% when the infection is caused by a multidrugresistant gramnegative pathogen. the healthcare infection control practices advisory committee of the cdc has developed guidelines for the prevention of vap. these include strategies aimed at preventing aspiration of contaminated oral or gastric material (e.g. raising the head of the bed and draining subglottic secretions), and interventions to alter bacterial coloniza tion of stomach (e.g. stress ulcer prophylaxis and selective digestive decontamination) and mouth. oral hygiene, suctioning and the provi sion of moisture to lips and oral mucosa, plus toothbrushing, may be important in prevention of vap. there are also strategies for manag ing ventilator circuits (e.g. replacement of ventilator circuits, use of closed rather than open suction, and use of heat moisture exchange as opposed to heated circuit technology). lung abscess is a localized infection leading to cavitation and necro sis. while some cases result from aspiration of foreign material, most develop from pneumonia caused by infection with staph. aureus or klebsiella pneumoniae. bronchial obstruction by carcinoma is another important cause. symptoms resemble those of suppurative pneumonia. there is a risk of infection spreading locally or leading, via septicaemia, to a brain abscess. diagnosis rests mainly on the chest radiograph, which may sometimes show cavitation or a fluid level. antimicrobial chemotherapy, postural drainage and relief by bronchoscopy of any obstruction are indicated. a wellrecognized cause of lung abscess is inhalation of a tooth or fragment, a restoration or rarely, an endodontic instrument. when undertaking endodontics or cementing restorations, such as inlays or crowns, a rubber dam or other protective device should always be used to avoid the danger of inhalation. lung abscesses may also result from aspiration of oral bacteria, particularly anaerobes, especially in infirm older patients or those who are intubated. the other main dangers in dentistry are with ga, particularly if an inadequate throat pack has been used. patients who inhale tooth frag ments or dental instruments must have chest radiographs (lateral and posteroanterior) and, if necessary, bronchoscopy. loeffler syndrome appears to be an allergic reaction, usually to the parasitic worm ascaris lumbricoides, or drugs such as sulphonamides. it manifests with pulmonary infiltrates (and abnormal chest radio graph) and eosinophilia (eosinophilic pneumonia). the disease usually clears spontaneously. sarcoidosis, so named because skin lesions resembled a sarcoma, is a multisystem granulomatous disorder, seen most commonly in young adult females in northern europe, especially in people of african heritage. the aetiology is unclear but propionibacterium acnes and p. granulosum have been implicated and associations have been reported with exposure to inorganic particles, insecticides, moulds and occupations such as firefighting and metalworking. serum sam ples contain antibodies directed against mycobacterium tuberculosis antigens. sarcoidosis is associated with hladrb1 and dqb1, and a butyrophilinlike 2 (btnl2) gene on chromosome 6. thelper 1 (th1) cells release il2 and ifnγ, and augment macrophage tumour necrosis factor alpha (tnfα) release. cd25 regulatory t cells cause a limited impairment of cellmediated immune responses (partial anergy) but no obvious special susceptibility to infection. sarcoidosis affects the thorax in 90%, but has protean manifestations and can involve virtually any tissue (table 15 .20). sarcoid most typi cally causes löfgren syndrome (fever, bilateral hilar lymphadenopathy, arthralgia and erythema nodosum, especially around the ankles; figs 15.9 and 15.10). other common presentations may include pulmonary infiltration and impaired respiratory efficiency, with cough and dyspnoea in severe cases, or acute uveitis, which can progress to blindness. susceptibility to lymphomas has been suggested but not confirmed. because of its vague and protean manifestations, sarcoidosis is under diagnosed. in the presence of suggestive clinical features, helpful investigations include: chest radiography (enlarged hilar lymph nodes); raised serum angiotensinconverting enzyme (sace ; table 15 .21) in acute disease (this is insensitive, nonspecific and a poor guide to therapy); positive gallium67 citrate or gadolinium or positron emis sion tomography (pet) scans; labial salivary gland or transbronchial biopsy (for histological evidence of noncaseating epithelioid cell granulomas) -except in löfgren syndrome, which is a classical clini cal diagnosis. 18 fdeoxyglucose pet is helpful in identifying sites for biopsy. nonspecific findings may include mild anaemia, leukopenia, eosinophilia, hypergammaglobulinaemia, raised esr and low serum albumin. hypercalcaemia is common because of extrarenal produc tion of active vitamin d and can result in renal damage. alkaline phosphatase, 5'nucleotidase, lysozyme and adenosine deaminase levels are raised in hepatic sarcoidosis. evidence of impaired delayed hypersensitivity reactions to some antigens may be useful. kveim skin tests are not now used. half the patients with sarcoidosis remit within 3 years and about 66% remit by 10 years. patients with only minor symptoms usually need no treatment but corticosteroids, sometimes with azathioprine, methotrexate, tetracyclines, hydroxychloroquine, infliximab or etaner cept, are given if there is active organ disease (ocular disease, progres sive lung disease, hypercalcaemia or cerebral involvement). biopsy of the minor salivary glands frequently shows noncaseating granulomas and association with other features of sarcoidosis, par ticularly hilar lymphadenopathy. this is an important diagnostic find ing that may obviate more invasive procedures. sarcoidosis can involve any of the oral tissues but has a predilection for salivary glands. asymptomatic swelling of the parotid glands or cervical nodes, and less frequently the lips, may accompany systemic disease. superficial or deepseated red submucosal nodules may develop intraorally and on the lips. nontender, wellcircumscribed, brownishred or violaceous nodules with superficial ulceration have also been reported. the oral and lip lesions may occasionally precede systemic involvement. there is enlargement of the major salivary glands in about 6% of cases; some have xerostomia, and the association of salivary and lacri mal gland enlargement with fever and uveitis is known as uveoparotid fever (heerfordt syndrome). salivary swelling may also be seen with out other features of heerfordt syndrome. the salivary gland swellings usually resolve on treatment of sarcoidosis but this may take up to 3 years. facial palsy and other cranial neuropathies may be seen. there is also an association with sjögren syndrome, when ssa and ssb serum autoantibodies are found. rarely there is an association of thyroiditis with addison disease, sjögren syndrome and sarcoidosis (tass syndrome). there is a group of patients who have histologi cal features of sarcoid in one or more sites in the mouth, such as the gingivae, but no systemic manifestations. a few of these patients may ultimately develop other more or less systematized disease but the majority probably have isolated lesions. such cases, where no exog enous cause for the granulomatous reaction can be found, are regarded as having 'sarcoidlike' reactions (orofacial granulomatosis) and treat ment is unnecessary. however, patients should be kept under observa tion for as long as possible. management of patients with systemic sarcoidosis may include con sideration of respiratory impairment, uveitis and visual impairment, renal disease, jaundice or corticosteroid treatment. la is safe and satisfactory. cs is contraindicated if there is any res piratory impairment. ga should only be given in hospital. lung cancer is the most common cancer in highincome countries in males and most frequently affects adult urban cigarettesmokers. bronchogenic carcinoma accounts for 95% of all primary lung cancer and has also become increasingly common in women (because of increased tobacco use), to the extent that the mortality rate for the two sexes has become almost equal. metastases from cancers elsewhere are also frequently found in the lungs. recurrent cough, haemoptysis, dyspnoea, chest pain and recurrent chest infections are the predominant features. local infiltration may cause pleural effusion, lesions of the cervical sympathetic chain (horner syndrome), brachial neuritis, recurrent laryngeal nerve palsy or obstruction of the superior vena cava with facial cyanosis and oedema (superior vena cava syndrome). there are many nonmetastatic extrapulmonary effects of bron chogenic (or other) carcinomas -for example, weight loss, anorexia, fingerclubbing, neuromyopathies, thromboses (thrombophlebitis migrans), muscle weakness, various skin manifestations and ectopic hormone production (of antidiuretic hormone, adrenocorticotropic hormone, parathyroid hormone and thyroidstimulating hormone). metastases from bronchogenic cancer are common and typically form in the brain (which may manifest with headache, epilepsy, hemi plegia or visual disturbances), liver (hepatomegaly, jaundice or ascites) or bone (pain, swelling or pathological fracture). the diagnosis is based on history and physical examination, supported by radiography, ct and magnetic resonance imaging (mri), sputum cytology, bronchoscopy and biopsy. spiral ct appears to detect tumours at an early stage. the overall 5year survival rate is only 8%. radiotherapy is the most common treatment. only some 25% of patients are suitable for surgery but, even then, the 5year survival is only about 25%. chemotherapy has been disappointing, except in smallcell carcinomas. dental treatment under la should be uncomplicated. cs should preferably be avoided. ga is a matter for specialist management in hospital, as patients often have impaired respiratory function, espe cially after lobectomy or pneumonectomy. this, along with any muscle weakness (myasthenic syndrome, eaton-lambert syndrome) that can make the patient unduly sensitive to the action of muscle relaxants, makes ga hazardous. oral cancer may be associated with lung cancer, and vice versa, or develop at a later stage (ch. 22). such synchronous or metachronous primary tumours must always be ruled out. metastases can occasionally affect the orofacial region and cause enlargement of the lower cervical lymph nodes, epulislike softtissue swellings or labial hypoaesthesia or paraesthesia in the jaw. soft palate pigmentation is a rare early oral manifestation. lung cancer is a fairly common cause of death in dental techni cians, but it is unknown whether this is due to smoking alone or to dust inhalation. cystic fibrosis (cf) is one of the most common fatal hereditary dis orders. inherited as an autosomal recessive trait, with an incidence of about 1 in 2000 births, it is the most common inherited error of metabolism and is seen mainly in people of european descent. the gene responsible is on chromosome 7q. cf is caused by defects in the cystic fibrosis transmembrane conductance regulator (cftr), a protein that appears to be part of a cyclic adenosine monophosphate (camp)regulated chloride channel, regulating cl − and na + transport across epithelial membranes, and ion channels and intracellular fluid flow in sweat, digestive and mucus glands. the basic defect in cf is abnormal chloride ion transport across the cell membrane of nearly all exocrine glands. the blockage of salt and water movement into and out of cells results in the cells that line the lungs, pancreas and other organs producing abnormally thick, sticky mucus that can obstruct the airways and various glands, especially in the respiratory tract and pancreas. involved glands (lungs, pancreas, intestinal glands, intrahepatic bile ducts, gallbladder, submaxillary and sweat glands) may become obstructed by this viscid or solid eosino philic material. recurrent respiratory infections result in a persistent productive cough and bronchiectasis, with the lungs becoming infected with a variety of organisms including staph. aureus, haemophilus influenzae, pseudomonas aeruginosa, strep. pneumoniae, burkholderia cepacia, and sometimes mycoses or mycobacteria. mycobacterium abscessus is a nontuberculous mycobacterium increasingly recognized as an opportunistic pathogen in cf patients. viral infections, such as mea sles, can have severe sequelae. pancreatic duct obstruction leads to pancreatic insufficiency, with malabsorption and bulky, frequent, foulsmelling, fatty stools. gallstones, diabetes, cirrhosis and pancreatitis may result. sinusitis is very common. growth is frequently stunted. the mutations can also cause con genital bilateral absence of the vas deferens, so fertility is impaired in most males with cf. in women, fertility may be impaired by viscid cervical secretions, but many women have carried pregnancies to term. most patients have a high concentration of sodium in their sweat (also reflected in the saliva); a sweat test showing sodium and chloride values of more than 60 mmol/l is considered positive, between 40 and 60 mmol/l equivocal, and less than 40 mmol/l negative. physiotherapy and postural drainage are crucially important. clearance of sputum is helped by water aerosols and bronchodila tors (terbutaline or salbutamol), but mucolytics such as carbocisteine, methyl cysteine and dornase alfa are of questionable effectiveness. treatment with ivacaftor, a cftr potentiator, improves chloride transport through the ion channel. amoxicillin and flucloxacillin are effective prophylactic antimicrobi als and may be given by aerosol. vaccination against measles, whoop ing cough and influenza is important. a low fat intake, adequate vitamins and oral pancreatic enzyme replacement (pancreatin) are also necessary. doublelung or heart-lung transplantation may eventually become necessary. sinusitis is very common; most cf patients have recurrent sinusitis and nasal polyps. the major salivary glands may enlarge and hyposali vation sometimes occurs. the lowfat, highcarbohydrate diet and dry mouth may predispose to caries. enamel hypoplasia and black stain may be seen, and both dental development and eruption are delayed. tetracycline staining of the teeth was common but should rarely be seen now. pancreatin may cause oral ulceration if held in the mouth. la is satisfactory but cs is usually contraindicated because of poor respiratory function. ga is contraindicated if respiratory function is poor. lung disease, such as bronchiectasis, liver disease and diabetes, may complicate treatment. bronchiectasis is dilatation and distortion of the bronchi. causes include: ■ congenital defects, which should be considered in all patients include cystic fibrosis, kartagener syndrome, alpha1antitrypsin deficiency, collagen defects (e.g. marfan syndrome) there is no identifiable underlying cause in about 50% of adults and 25% of children. the damaged and dilated bronchi lose their ciliated epithelium and therefore mucus tends to pool, causing recurrent lrtis, typically with strep. pneumoniae, haemophilus influenzae or pseudomonas aeruginosa. overproduction of sputum, which is purulent during exacerbations, a cough (especially during exercise or when lying down) and finger clubbing are typical features, with recurrent episodes of bronchitis, pneumonia and pleurisy. haemoptysis is not uncommon. in advanced bronchiectasis, chest pain, dyspnoea, cyanosis and respiratory failure may develop. complications may include cerebral abscess and amyloid disease. chest radiography and pulmonary function tests are required. high resolution ct (hrct) is useful. postural drainage is important. antimicrobials, such as amoxicillin, cephalosporins or ciprofloxacin, are given for acute exacerbations and for longterm maintenance treatment. ga should be avoided where possible and is contraindicated in acute phases. workers exposed to airborne particles may develop pulmonary disease (pneumoconiosis), which ranges from benign (e.g. siderosis) to malig nant, as in mesothelioma from asbestosis (see appendix 15.1), but any pneumoconiosis can cause significant incapacity. ga may be contraindicated; the physician should be contacted before treatment. berylliosis may be a hazard in some dental technical laboratories, when lung cancer is more frequent. respiratory complications following surgical operations under ga include segmental or lobar pulmonary collapse and infection. they are more common after abdominal surgery or if there is preexistent respiratory disease or smoking (see also ch. 3), and can be signifi cantly reduced by smoking cessation, preoperative physiotherapy and bronchodilators, such as salbutamol. if postoperative pulmonary infection develops, sputum should be sent for culture, and physiotherapy and antibiotics should be given. the common microbial causes are strep. pneumoniae and haemophilus influenza; in this case, suitable antibiotics include amoxicillin and erythromycin. hospital infections may include other microorganisms, such as mrsa, klebsiella, pseudomonas and other gramnegative bacteria. inhalation (aspiration) of gastric contents can cause pulmonary oedema and may be fatal (mendelson syndrome); it is most likely if a ga is given to a patient who has a stomach that is not empty, has a hiatus hernia or is in the last trimester of pregnancy. prevention is by ensuring the stomach is empty preoperatively; if it is not, an anaes thetist should pass an endotracheal tube. antacids or an h2receptor blocker, such as cimetidine or ranitidine, may be given by mouth pre operatively to lower gastric acidity. if gastric contents are aspirated, the pharynx and larynx must be carefully sucked out. systemic corticosteroids have been recommended but probably do not reduce the mortality. respiratory distress in premature infants may be caused by immaturity of surfactantproducing cells, when the alveoli fail to expand fully; this necessitates endotracheal intubation for many weeks. it may, in turn, result in midface hypoplasia, palatal grooving or clefting, or defects in the primary dentition. the same oral effects may be seen with prolonged use of orogastric feeding tubes. the degree to which subsequent growth corrects these deformations is currently unknown, though the palatal grooves typically regress by the age of 2 years. using soft endotracheal tubes does not obviate this problem and, at present, the best means of avoiding palatal grooving appears to be the use of an intraoral acrylic plate to stabilize the tube and protect the palate. acute respiratory distress syndrome (ards) is a sequel to several types of pulmonary injury and some infections, including those with oral viridans streptococci. patients with endstage pulmonary disease are considered for potential transplantation, usually using a lung from a braindead organ donor. a combination of ciclosporin, azathioprine and glucocorticoids is usu ally given for lifelong immunosuppression to prevent a tcell, alloim mune rejection response. inhaled nitric oxide modulates pulmonary vascular tone via smooth muscle relaxation and can improve ventilation/perfusion matching and oxygenation in diseased lungs. early graft failure following lung transplantation has been described by various investi gators as reimplantation oedema, reperfusion oedema, primary graft failure or allograft dysfunction. pathologically, this entity is diffuse alveolar damage. see also chapter 35. a meticulous presurgery oral assessment is required and dental treatment must be undertaken with particular attention to establishing optimal oral hygiene and eradicating sources of potential infection. dental treatment should be completed before surgery. for 6 months after surgery, elective dental care is best deferred. if surgical treat ment is needed during that period, antibiotic prophylaxis is probably warranted. cardiopulmonary transplantation (heart and lung transplantation) is the simultaneous surgical replacement of the heart and lungs in patients with endstage cardiac and pulmonary disease, with organs from a cadaveric donor. all transplant recipients require lifelong immunosuppression to pre vent a tcell, alloimmune rejection response. see also chapter 35. a meticulous presurgery oral assessment is required and dental treatment must be undertaken with particular attention to establishing optimal oral hygiene and eradicating sources of potential infection. dental treatment should be completed before surgery. for 6 months after surgery, elective dental care is best deferred. if surgical treat ment is needed during that period, antibiotic prophylaxis is probably warranted. national institutes of health: national institute of allergy and infectious diseases healthcare infection control practices advisory committee guideline for the prevention of healthcare associated pneumonia nosocomial pneumonia: state of the science extensively drugresistant tuberculosis as a cause of death in patients coinfected with tuberculosis and hiv in a rural area of south africa a review of the possible role of oral and dental colonization on the occurrence of health careassociated pneumonia: underappreciated risk and a call for interventions reducing ventilatorassociated pneumonia through advanced oraldental care: a 48month study apic infection control and applied epidemiology: principles and practice sepp. ventilatorassociated pneumonia and oral care: a successful quality improvement project guidelines for preventing the transmission of mycobacterium tuberculosis in healthcare settings a randomized trial of dental brushing for preventing ventilatorassociated pneumonia pneumonia associated with a dental unit waterline the pathogenesis of ventilatorassociated pneumonia: its relevance to developing effective strategies for prevention aspects of human disease 32 chronic obstructive pulmonary disease (copd) aspects of human disease 31 in vitro antibacterial activities of oral care products against ventilatorassociated pneumonia pathogens the impact of a simple, lowcost oral care protocol on ventilatorassociated pneumonia rates in a surgical intensive care unit intermittent suction of oral secretions before each positional change may reduce ventilatorassociated pneumonia: a pilot study current trends and newer concepts on diagnosis, management and prevention of respiratory tract infections key: cord-278031-vpq4yghn authors: zumla, alimuddin; gant, vanya; bates, matthew; mwaba, peter; maeurer, markus; memish, ziad a title: rapid diagnostics urgently needed for killer infections date: 2013-06-30 journal: the lancet respiratory medicine doi: 10.1016/s2213-2600(13)70099-7 sha: doc_id: 278031 cord_uid: vpq4yghn nan respiratory tract infections (rtis) cause millions of deaths worldwide. they are caused by disparate bacteria, viruses, fungi, and parasites that have similar presentations and thus cannot be easily distinguished clinically. there is presently no standardised, rapid, accurate, sensitive, and specifi c point-of-care diagnostic test available that can identify causative pathogens within a few hours of consultation. therefore, patients presenting with rtis at all points of health care are empirically treated with broad-spectrum antibiotics designed to cover many possible pathogens. the management of patients with rtis is further complicated by the growing global threat posed by the emergence and spread of antibiotic-resistant bacteria 1 (including multidrug-resistant strains of mycobacterium tuberculosis 2 ) and azole-resistant fungi. 3 clinicians are often unaware of the presence of more than one pathogen, although co-infections of bacteria 4 or other respiratory viruses 5 and infl uenza are well described, as are bacterial, viral, or parasitic infections with pulmonary tuberculosis. 6 autopsy studies of patients who die because of rtis suggest that, in many cases, the empirical antibiotic treatment prescribed was inappropriate for the causative pathogens, and that coinfections were not suspected before death. 7 new and emerging pathogens with epidemic potential also pose diagnostic challenges. global scientifi c and political attention is currently focused on two new viruses associated with severe rtis and high mortality: the avian infl uenza a h7n9 virus from china and novel coronavirus from the arabian peninsula. both were rapidly reported by the program for monitoring emerging diseases (promed). on march 31, 2013, the china health and planning commission reported three cases of avian infl uenza a h7n9-a virus originating from poultry and wild birds. 8 as of may 14, a total of 132 confi rmed cases of avian infl uenza a h7n9 (with 35 deaths; 27% case fatality) had been recorded by promed. the fi rst case of novel coronavirus was reported in september, 2012, from saudi arabia. as of may 15, 2013, a total of 40 cases (with 20 deaths; 50% case fatality) have been reported to who according to promed: 30 in saudi arabia (15 deaths); two in jordan (two deaths); four in the uk (two deaths); two in germany (one death), and two in france (no deaths). most cases were linked with the middle east. the source of the novel coronavirus remains unknown. personto-person transmission has been recorded, but only in individuals with comorbidities, according to promed. transmission of rtis-particularly imported infl uenza viruses-at mass gatherings is well documented, 9 and poses a threat to global health security. about 10 million pilgrims from 183 countries are expected to visit mecca and medina in saudi arabia for the umrah and hajj pilgrimages in 2013, and will be in close proximity to each other, thus posing a major public health concern. rapid serological diagnostic tests for the novel coronavirus are in development and will be useful for surveillance and cross-sectional and longitudinal cohort studies. advances in molecular diagnostics have been applied to simultaneous detection of pathogens and their antibiotic resistance from one sputum sample. the absence of rapid accurate diagnostic tests for pulmonary tuberculosis was further compounded by the widespread inability to screen for drug-resistant bacteria. the introduction of the genexpert mtb/rif assay, which detects mycobacterium tuberculosis dna and rifampicin resistance from sputum samples in 2 h, could revolutionise tuber culosis diagnostics. 10 a lateral fl ow immunoassay for detection of meticillin-resistant staphylococcus aureus in sputum is also now available. 11 although several new rapid molecular diagnostic plat forms capable of simultaneously identifying both pathogens and the genetic determinants of anti microbial resistance are emerging, 12 they do not seem to be well suited to clinical presentations or downstream manage ment strategies for patients with rtis. furthermore, none can identify new emerging organisms for which genetic sequences are unavailable. however, rapid sequencing technologies linked to bio informatics have provided proof of diagnostic principle in a short time. 13 until new diagnostics are developed to serve clinical need, the mainstay of clinical management for any rti will remain dependent on imperfect diagnostics and linked to empirical broad-spectrum antibiotics. thus, clinicians have to maintain clinical awareness of other possible causes of rtis, whether they are opportunistic or not. an ideal diagnostic test for rtis should be rapid (a result within 1 h of consultation), cheap, easy to use, sensitive, and specifi c, and should screen for many microorganisms and their antibiotic resistance. furthermore, the diagnostics platform should be transportable, robust, and ideally run on solar power for use in the remote health-care settings in developing countries. whether or not an ideal diagnostic platform for rtis can be delivered, advances in biotechnology give hope. such a device would improve the health and lives of patients with rtis worldwide, and would also contribute to global health surveillance and security. to achieve this ideal, physicians, scientists, biotechnology companies, funding agencies, and governments need to work together to drive the development of improved diagnostic tests for both developed and developing countries. the clinical eff ect was larger in children with more severe asthma exacerbation (p=0·03) and those with symptoms present for less than 6 h (p=0·049)". in table 1, the age range for both mgso 4 and placebo groups should have been 2-15. and in table 3, the footnote p=0·143 for interaction between attack duration and mgso 4 should have been added to the legend. this correction has been made to the online version as of may 30, 2013, and the printed article is correct. antimicrobial resistance and virulence: a successful or deleterious association in the bacterial world? surveillance of anti-tuberculosis drug resistance in the world: an updated analysis frequency, diagnosis and management of fungal respiratory infections bacterial coinfection in infl uenza: a grand rounds review co-infections with infl uenza and other respiratory viruses tuberculosis comorbidity with communicable and non-communicable diseases: integrating health services and control eff orts deaths due to respiratory tract infections in africa: a review of autopsy studies epidemiological characteristics of cases for infl uenza a (h7n9) virus infections in china respiratory tract infections during the annual hajj: potential risks and mitigation strategies advances in tuberculosis diagnostics: the xpert mtb/rif assay and future prospects for a point-of-care test evaluation of a rapid lateral fl ow immunoassay for staphylococcus aureus detection in respiratory samples are we ready for novel detection methods to treat respiratory pathogens in hospital-acquired pneumonia? a metagenomic analysis of pandemic infl uenza a (2009 h1n1) infection in patients from north america key: cord-267274-3ygl3stc authors: britton, philip n; hu, nan; saravanos, gemma; shrapnel, jane; davis, jake; snelling, tom; dalby-payne, jacqui; kesson, alison m; wood, nicholas; macartney, kristine; mccullagh, cheryl; lingam, raghu title: covid-19 public health measures and respiratory syncytial virus date: 2020-09-18 journal: lancet child adolesc health doi: 10.1016/s2352-4642(20)30307-2 sha: doc_id: 267274 cord_uid: 3ygl3stc nan in new south wales (nsw), australia, the public health response was highly effective in controlling the early phase of the covid-19 pandemic. 1 during this time, clinicians reported fewer than expected presentations and admissions with acute respiratory illness to the sydney children's hospitals network (schn). respiratory syncytial virus is among the most common viruses that cause hospitalisation in children and has predictable winter seasonality. 2 we aimed to quantify the change in frequency and burden of acute respiratory syncytial virus-associated illness presenting to schn, the largest provider of tertiary paediatric services in australia, in 2020 compared with previous years. we analysed three separate datasets from the schn electronic records from jan 1, 2015, to june 30, 2020, in children younger than 16 years: (1) laboratory tests for respiratory syncytial virus by pcr; (2) hospital admissions for bronchiolitis coded by the icd-10 australian modification (j21.0, j21.1, j21.8, and j21.9); and (3) emergency department attendances for acute respiratory illness coded by the systematised nomenclature of medicine clinical terminology (appendix p 4). for each dataset, we plotted counts by month and did a time series analysis comparing the frequencies in the peak respiratory syncytial virus epidemic months (april-june) in 2020 with those in 2015-19. we observed concurrent lower frequencies of respiratory syncytial virus (a and b) detection, admission to hospital for bronchiolitis, and emergency department attendance for acute respiratory illnesses (appendix p 1) in 2020 compared with preceding years. the observed mean frequency of respiratory syncytial virus detections from april to june, 2020, was 94·3% (se 22·8) lower than predicted on the basis of the underlying trend of 2015-19 data (absolute reduced frequency per epidemic month [arf] 99 [se 24]; p=0·026). the observed mean frequency of bronchiolitis admissions was 85·9% (se 15·2) lower than predicted (arf 130 [se 23]; p=0·011), and that of emergency department attendance was 70·8% (se 16·3) lower than predicted (arf 915 [se 211]; p=0·023; appendix p 2, 3). we also observed an 89·1% (se 32·7) reduction in bronchiolitis admissions to the intensive care unit (arf 16 [se 6]; p=0·074). the reduction in respiratory syncytial virus detections cannot be accounted for by reduced testing because the number of tests done in 2020 was double the number done in previous years (data not shown). the aggressive public health interventions aimed at preventing severe acute respiratory syndrome coronavirus 2 (sars-cov-2) transmission has created a natural experiment of their effect on respiratory syncytial virus-associated illness and other communicable diseases. here, we show a strong association between the implementation of these measures and the burden of respiratory syncytial virus disease among children in sydney, nsw. given that handwashing and isolation are known to affect nosocomial respiratory syncytial virus transmission, some effect might have been expected, 3 but the size of the apparent impact at a population level is startling. respiratory syncytial virus is one of the most burdensome viruses globally, and bronchiolitis (up to 80% of which is caused by respiratory syncytial virus) is a leading cause of hospital admission in young children. 4,5 efforts to develop vaccines and other preventive measures to address this considerable burden remain unfulfilled. australians reported a very high uptake (>84%) of enhanced hygiene and physical distancing measures in march, 2020. 6 handwashing damages the lipid envelope that surrounds respiratory syncytial virus, thereby impairing its ability to infect host cells. population lockdowns are justifiable to contain transmission of high lethality pandemics but are undesirable due to their wider negative impacts on society. the observation we report here should prompt deeper analysis to identify which components of the public health intervention were most effective for preventing respiratory syncytial virus infection in 2020, and prompt a discussion about which interventions, such as those described by dalton and colleagues, 7 might be sustainable for future primary prevention of seasonal respiratory disease in children. school closures in nsw occurred for a brief period (march 23-april 29, 2020; appendix p 1), and early childhood education centres remained open throughout this period, although attendance rates decreased. mask wearing was neither recommended, nor practised widely in the community before july, 2020. our findings might be limited by idiosyncrasies in both the social and pandemic contexts in nsw. furthermore, the relative effects of hygiene measures, physical distancing, and reduced population movement could not be directly assessed. an important caveat is that the period we studied was brief, and it remains to be seen whether community transmission of respiratory syncytial virus has been averted in 2020 or merely delayed, especially as restrictions are relaxed. the small uptick in emergency department attendances and bronchiolitis admissions in june, 2020 (appendix p 1) was not associated with increased respiratory syncytial virus detections. our laboratory reported almost exclusively rhinovirus detections in june, 2020 (results not shown). www rhinoviruses are easily transmitted between children in close contact and are non-enveloped so might be inherently less susceptible to inactivation by handwashing. there are legitimate concerns about a range of potential negative effects of lockdowns; it will be crucial to assess and quantify these consequences, and we support efforts to actively mitigate them. 8 nonetheless, our results suggest that the beneficial effect of lockdown on transmission of respiratory syncytial virus in nsw has been impressive. suppressing the epidemic in new south wales respiratory syncytial virus-associated hospitalisations in australia risk of nosocomial respiratory syncytial virus infection and effectiveness of control measures to prevent transmission events: a systematic review we declare no competing interests. pnb, gs, ts, jd-p, amk, nw, km, cm, and rl conceptualised the study. nh, js, and jd analysed the data. nh and pnb produced the figures. pnb, nh, js, jd, ts, amk, km, and rl interpreted the data. pnb wrote the first draft. all authors reviewed and revised the manuscript. the corresponding author had full access to all of the data and the final responsibility to submit for publication. key: cord-274763-i6e3g3te authors: liu, wen-kuan; liu, qian; chen, de-hui; tan, wei-ping; cai, yong; qiu, shu-yan; xu, duo; li, chi; li, xiao; lin, zheng-shi; zhou, rong title: epidemiology of hbov1 infection and relationship with meteorological conditions in hospitalized pediatric patients with acute respiratory illness: a 7-year study in a subtropical region date: 2018-07-16 journal: bmc infect dis doi: 10.1186/s12879-018-3225-3 sha: doc_id: 274763 cord_uid: i6e3g3te background: human bocavirus 1 (hbov1) is an important cause of acute respiratory illness (ari), yet the epidemiology and effect of meteorological conditions on infection is not fully understood. to investigate the distribution of hbov1 and determine the effect of meteorological conditions, hospitalized pediatric patients were studied in a subtropical region of china. methods: samples from 11,399 hospitalized pediatric patients (≤14 years old), with ari were tested for hbov1 and other common respiratory pathogens using real-time pcr, between july 2009 and june 2016. in addition, local meteorological data were collected. results: of the 11,399 patients tested, 5606 (49.2%) were positive for at least one respiratory pathogen. two hundred forty-eight of 11,399 (2.2%) were positive for hbov1 infection. co-infection was common in hbov1-positive patients (45.2%, 112/248). a significant difference in the prevalence of hbov1 was found in patients in different age groups (p < 0.001), and the peak prevalence was found in patients aged 7–12 months (4.7%, 56/1203). two hbov1 prevalence peaks were found in summer (between june and september) and winter (between november and december). the prevalence of hbov1 was significantly positively correlated with mean temperature and negatively correlated with mean relative humidity, and the mean temperature in the preceding month had better explanatory power than the current monthly temperature. conclusions: this study provides a better understanding of the characteristics of hbov1 infection in children in subtropical regions. data from this study provide useful information for the future control and prevention of hbov1 infections. human bocavirus 1 (hbov1), which belongs to family parvoviridae, was firstly identified in respiratory secretions of children with respiratory tract disease in 2005 [1, 2] . hbov1 has been confirmed as an important respiratory pathogen and is found in respiratory infections in children and adults worldwide. the prevalence of hbov1 nucleic acid detection varies from 1.5 to 33% in patients with acute respiratory illness (ari), according to different studies [3] [4] [5] [6] [7] . serological and nucleic acid test results are generally consistent [8] [9] [10] [11] , showing hbov1 infection is very common. hbov1 can cause both upper respiratory illness (uri) and lower respiratory illness (lri) [12] [13] [14] [15] [16] [17] [18] . infection with hbov1 can lead to development of a cough, rhinitis, fever and other common clinical symptoms [15, 19] . in some cases, it can cause respiratory distress, hypoxia, wheezing and other severe respiratory symptoms [18, 20] . clinical diagnosis is mainly pneumonia, bronchitis, pneumothorax, mediastinal emphysema and otitis media and other complications [18] [19] [20] [21] [22] . in some cases, patients develop severe respiratory injury symptoms, which can be fatal [21, 23] . hbov1 can be detected in fecal samples [24] , blood samples [25, 26] , urine [27, 28] , cerebrospinal fluid [29] [30] [31] , river water [32] and sewage [33, 34] , indicating that hbov1 may be associate with a variety of diseases. current in vitro studies modeling tissue-like airway epithelial cells cultures show hbov1 infection can lead to disruption of the tight-junction barrier, loss of cilia and epithelial cell hypertrophy [35] [36] [37] , similar to lung injury tissue changes in vivo. there is currently no vaccine or specific treatment for this virus; prevention and treatment of hbov1-related diseases still require further research. the prevalence of respiratory viruses is associated with many factors, including local climate, which may impact the survival and spread of the viruses [38] . studying the epidemiology of hbov1 and its relationship with meteorological conditions will improve diagnosis, treatment, control and prevention of this virus. in this study, we investigated the epidemiology of hbov1 infection in children (≤14 years old) hospitalized with ari in a subtropical region in china over a 7-year period. in addition, we collected climate data to determine if there was a relationship between hbov1 prevalence and meteorological conditions. this study will add to existing epidemiological data on hbov1 and its relationship with climate conditions in subtropical regions and will play a positive role in hbov1 control and prevention. the study sites were three tertiary hospitals in guangzhou, southern china (longitude: e112°57′ to e114 03′; latitude n22°26′ to n23°56′). inclusion criteria were pediatric patients (≤14 years old) who presented with at least two of the following symptoms: cough, pharyngeal discomfort, nasal obstruction, rhinitis, dyspnea or who were diagnosed with pneumonia by chest radiography during the previous week. chest radiography was conducted according to the clinical situation of the patient. throat swab samples were collected from the enrolled patients between july 2009 and june 2016 for routine screening for respiratory viruses, mycoplasma pneumoniae (mp), and chlamydophila pneumoniae (cp). the samples were refrigerated at 2-8°c in viral transport medium, transported on ice and analyzed immediately or stored at − 80°c before analysis, as described previously [15, 39] . meteorological data for guangzhou, were collected from july 2009 to june 2016, from the china meteorological administration, including the monthly mean temperature (°c), mean relative humidity (%), rainfall (mm), mean wind speed (m/s), mean air pressure (hpa), mean vapor pressure (hpa), sunshine duration (h). real-time pcr for hbov1 and common respiratory pathogen detection dna and rna were extracted from the respiratory samples using the qiaamp dna mini kit and qiaamp viral rna mini kit (qiagen, shanghai, china), respectively, in accordance with the manufacturer's protocols. taqman real-time pcr for hbov1 was designed based on the conserved region of the np1 gene, as described previously [15] . common respiratory pathogens, including respiratory syncytial virus (rsv), influenza a virus (infa), influenza b virus (infb), four types of parainfluenza (piv1-4), adenovirus (adv), enterovirus (ev), human metapneumovirus (hmpv), four strains of human coronavirus (hcov-229e, oc43, nl63 and hku1), human rhinovirus (hrv), mp and cp were detected simultaneously as previously reported [40] . data were analyzed using chi-squared test and fisher's exact test in spss 19.0 (spss inc., chicago, il, usa). correlation with climate data was analyzed using multiple linear regression analysis. all tests were two-tailed and a p value < 0.05 was considered as statistically significant. eleven thousand three hundred ninety-nine pediatric patients (≤14 years old) hospitalized with ari were enrolled in the study between july 2009 and june 2016. the male-to-female ratio was 1.82:1 (7361:4038) and the median age was 1.75 years (interquartile range 0.75-3.83). overall, 86.5% (9857/11399) of patients were under the age of 5 years. all the 11,399 patients were tested for all 18 pathogens mentioned, and 5606 (49.2%) were positive for one or more of those pathogens (table 1) , and had a median age of 1.50 years (interquartile range 0.67-3.00). the male-to-female ratioes were 1.94: 1 (3698:1908) in pathogen-positive patients and 1.72: 1 (3663:2130) in pathogen-negative patients (p = 0.002). two hundred forty-eight of 11,399 patients (2.2%) tested positive for hbov1 infection. of the hbov1-positive patients, 112 (45.2%) were co-infected with other pathogens, most frequently with rsv (11.7%, 29/248) ( table 1 ). the median age was 1 year (interquartile range 0.75-1.83). the male-to-female ratio was 2.54:1 (178:70) in hbov1-positive patients and 1.81:1 (7183:3968) in hbov1-negative patients (p = 0.019). to clarify the age distribution of hbov1, patients were divided into seven age groups; 0-3 months, 4-6 months, 7-12 months, 1-2 years, 3-5 years, 6-10 years and 11-14 years old. there was a significant difference in the prevalence of hbov1 in patients in different age groups (p < 0.001) and the peak prevalence was found in patients aged 7-12 months (4.7%, 56/1203) (fig. 1) . in this study, we monitored the prevalence of hbov1 in patients (≤14 years old) hospitalized with ari from july we collected meteorological data for guangzhou, including monthly mean temperature, mean relative humidity, rainfall, mean wind speed, mean air pressure, mean vapor pressure and sunshine duration for a 7-year period, to explore the correlation between meteorological conditions and prevalence of hbov1. guangzhou, which is located in southern china (longitude 112°57′ to 114°3′, latitude 22°26′ to 23°56′), has a maritime subtropical monsoon climate. between july 2009 and june 2016, the mean temperature was 21.8 ± 5.8°c (mean ± standard deviation), humidity was 77.2 ± 7.3%, sunshine duration was 132.7 ± 59.5 h, wind speed was 2.2 ± 0.6 m/s, rainfall was 175.2 ± 165.9 mm, air pressure was 1005.6 ± 6.0 hpa and vapor pressure was 21.3 h ± 7.4 hpa. between 2009 and 2016, the mean temperature from may to september was greater than 25°c (fig. 3) . for multiple linear regression analysis of hbov1 prevalence and meteorological conditions correlation, independent variables of mean air pressure (adjusted r 2 = 0.793, p < 0.001) and mean vapor pressure (adjusted r 2 = 0.929, p < 0.001), which linearly associated with mean temperature, and rainfall (adjusted r 2 = 0.278, p < 0.001), which strongly correlated with mean relative humidity, were excluded. the independent variables for the final multiple linear regression analysis included mean temperature, mean relative humidity, mean wind speed and sunshine hours. the effect of temperature had a delay therefore mean temperature in the preceding month (mean temperature 1 month before) was also included as an independent variable in the analysis ( table 2) . both regression models were established (p < 0.001) and the adjusted r 2 values were 0.373 and 0.231 in the mean temperature in the preceding month model and the current monthly temperature model, respectively. hbov1 prevalence was positively correlated with temperature (coefficient = 0.259 in the current temperature model (p = 0.002), coefficient = 0.328 in mean temperature in the preceding month model (p < 0.001)). conversely, hbov1 prevalence was negatively correlated with relative humidity (coefficient = − 0.126 in the current temperature model (p = 0.024), coefficient = − 0.083 in the temperature delay model (p = 0.039)) ( table 2 ). ari is one of the most common human diseases, predominantly caused by different respiratory viruses [41, 42] . one of these viruses, hbov1 infection, causes global epidemics, has a high public health burden and circulates with different patterns in different areas [3] [4] [5] [6] [7] 43] . in general, the prevalence of viruses varies because of factors such as multiple linear regression analysis was performed using hbov1 monthly prevalence as the dependent variable, monthly mean temperature (or mean temperature in the preceding month), mean relative humidity, mean wind speed and sunshine duration as the independent variables data captured in bold are highly significant geographical location, climatic conditions, population and social activity [38] . epidemiology of hbov1 in temperate regions has been described in more detail and a high incidence of infection has been observed in children under the age of 2 years in winter and spring [15, 16, 39, 44] . to describe the epidemiology of hbov1 in guangzhou, we collected throat swabs from 11,399 children (≤14 years old), hospitalized with ari and monitored hbov1 and other common respiratory pathogens over a 7-year period (table 1 ). in the current study, 86.5% (9857/11399) of patients were under the age of 5 years, with a median age of 1.75 years, indicating that infants and young children were most at risk of ari, consistent with previous reports [45, 46] . overall, 49.2% (5606/11399) of patients tested positive for one or more respiratory pathogens, 2.2% (248/11399) of patients were tested with hbov1 infection (table 1) . a higher prevalence of hbov1 was detected in male patients compared with female patients (p = 0.019), consistent with previous reports [15, 16, 39, 44] . co-infection with hbov1 and other pathogens is common [14, 15] . in our study, 45.2% (112/248) of hbov1-positive patients also tested positive for other pathogens (table 1 ). this may be partly caused by coinciding epidemics of hbov1 and other pathogens. in our study, the hbov1 seasonal distribution and total positive pathogen distribution were consistent, confirming this inference (fig. 2) . current research shows that hbov1 infection can lead to the collapse of the first line of defense of airway epithelium [35] [36] [37] , which may lead to a higher susceptibility to other pathogens, explaining the high rate of co-infection. whether co-infection leads to more severe disease is currently unknown and more research is needed to determine this. the characteristics of the hbov1 infection are likely to be a good model for studying the effects of co-infections. in this study, there was a significant difference in prevalence of hbov1 in patients of different ages (p < 0.001). the majority of hbov1 infections occurred in patients under 2 years old and the peak frequency of hbov1 infection occurred in patients aged 7-12 months (fig. 1) , consistent with previous serological and epidemiological reports on the virus [8-11, 15, 16, 39, 44] . this might be because children's immune systems are still under development and maternal antibodies gradually disappear in this age group. the distribution of hbov1 in patients of different ages will provide important reference for future vaccines and new drug research and development, as well as providing important data for disease prevention and control. many factors affect the epidemiology of pathogens, such as geographical location and local climate. guangzhou, a central city and main transport hub in southern china, is located in a subtropical region. guangzhou is hot and has high annual rainfall, long summers, short winters and the annual precipitation and high temperature are almost in the same period (fig. 3) . in this study, two hbov1 peaks were observed (fig. 2) . the large prevalence peaks of hbov1 infection occurred between june and september of each year, which are the summer months in guangzhou, with mean temperatures of higher than 25°c (fig. 3) . small peaks of hbov1 infection occurred in winter, between november and december of each year. this seasonal distribution is similar to the prevalence in subtropical regions reported previously [47] , but different from the hbov1 epidemics in temperate regions, which mostly occur in winter and spring [15, 16, 39, 44] , as well as from tropical regions, such as india, where no obvious epidemic season has been found [48] . to analyze the correlation between hbov1 prevalence and meteorological conditions, multiple linear regression analysis was performed, with hbov1 monthly prevalence as the dependent variable and mean temperature (or mean temperature in the preceding month), mean relative humidity, mean wind speed and sunshine duration as the independent variables (table 2) . both regression models were established (p < 0.001) and the adjusted r 2 value (0.373) of the temperature dorp 1 month model was greater than the adjusted r 2 value (0.231) of the current monthly temperature model, indicating that the temperature dorp 1 month model had better explanatory power than the current monthly temperature model. both of the models showed that the prevalence of hbov1 was significantly correlated with temperature and relative humidity ( table 2 ). in detail, hbov1 prevalence was positively correlated with temperature, that is consistent with previous reports [47, 49] . conversely, hbov1 prevalence was negatively correlated with relative humidity, this was different from a previous report in suzhou [47] , which may be related to guangzhou high humidity (mean monthly relative humidity was 77.2 ± 7.3%) (fig. 3) . it is common for pathogen prevalence to fluctuate over time because of a variety factors. in this study, hbov1 prevalence was relatively low in 2013 to 2014. it might be partly related to the relatively higher mean relative humidity during this period (fig. 3) . climate conditions may impact the survival and spread of respiratory viruses, however no significant linear relationship between hbov1 infection and wind speed or sunshine duration were found in this study (p > 0.05) ( table 2) . some limitations of this study should be noted. first, because our study mainly focused on hbov1 circulation in hospitalized patients with ari, hbov1 in outpatients and the asymptomatic population were not included. second, many factors can affect virus epidemics, meteorological data analysis alone may not serve as a final conclusive interpretation. third, the study was only conducted in three hospitals and may not be representative of the overall population. our study has provided a better understanding of the epidemiology of hbov1 in subtropical regions, specifically correlations with climate data; these data will be helpful for future control and prevention of hbov1 infections. cloning of a human parvovirus by molecular screening of respiratory tract samples human parvoviruses virological and clinical characterizations of respiratory infections in hospitalized children detection of human bocavirus type 1 infection in panamanian children with respiratory illness detection of human bocavirus in nasopharyngeal aspirates versus in broncho-alveolar lavage fluids in children with lower respiratory tract infections human bocavirus detection in nasopharyngeal aspirates of children without clinical symptoms of respiratory infection detection of bocavirus in saliva of children with and without respiratory illness b-cell responses to human bocaviruses 1-4: new insights from a childhood follow-up study seroepidemiology of human bocavirus defined using recombinant viruslike particles seroepidemiology of human bocaviruses 1-4 the genomic and seroprevalence of human bocavirus in healthy chinese plasma donors and plasma derivatives human bocavirus: current knowledge and future challenges spontaneous pneumomediastinum as a complication in human bocavirus infection human bocavirus-the first 5 years detection of human bocavirus from children and adults with acute respiratory tract illness in guangzhou, southern china comorbidity and high viral load linked to clinical presentation of respiratory human bocavirus infection epidemic and molecular evolution of human bocavirus in hospitalized children with acute respiratory tract infection human bocavirus amongst an all-ages population hospitalised with acute lower respiratory infections in cambodia. influenza other respir viruses human bocavirus in chile: clinical characteristics and epidemiological profile in children with acute respiratory tract infections single detection of human bocavirus 1 with a high viral load in severe respiratory tract infections in previously healthy children human bocavirus as the cause of a life-threatening infection clinical and microbiological impact of human bocavirus on children with acute otitis media severe human bocavirus infection clinical epidemiology and molecular profiling of human bocavirus in faecal samples from children with diarrhoea in guangzhou human bocavirus and acute wheezing in children prevalence and clinical aspects of human bocavirus infection in children high incidence of human bocavirus infection in children in spain correlation between bocavirus infection and humoral response, and co-infection with other respiratory viruses in children with acute respiratory infection human bocavirus in patients with encephalitis identification of human bocaviruses in the cerebrospinal fluid of children hospitalized with encephalitis in china detection of human bocavirus in the cerebrospinal fluid of children with encephalitis detection and quantification of human bocavirus in river water mixed viral infections causing acute gastroenteritis in children in a waterborne outbreak frequent detection of highly diverse variants of cardiovirus, cosavirus, bocavirus, and circovirus in sewage samples collected in the united states in vitro modeling of human bocavirus 1 infection of polarized primary human airway epithelia establishment of a reverse genetics system for studying human bocavirus in human airway epithelia human bocavirus 1 infects commercially available primary human airway epithelium cultures productively are meteorological parameters associated with acute respiratory tract infections? epidemiology of acute respiratory infections in children in guangzhou: a three-year study epidemiology and clinical presentation of the four human parainfluenza virus types global burden of childhood pneumonia and diarrhoea viral etiology of hospitalized acute lower respiratory infections in children under 5 years of age -a systematic review and meta-analysis human bocavirus capsid messenger rna detection in children with pneumonia human bocavirus-1 primary infection and shedding in infants respiratory virus surveillance and outbreak investigation respiratory viral infections in infants: causes, clinical symptoms, virology, and immunology clinical and epidemiological profiles of lower respiratory tract infection in hospitalized children due to human bocavirus in a subtropical area of china human bocavirus infection in children with acute respiratory tract infection in india clinical and epidemiologic profile of lower respiratory tract infections associated with human bocavirus we thank the study volunteers for their generous participation. we thank yinghua zhou, haiping huang, jing zhang and jing ma for their technical assistance. . the sponsors of the study had no role in the study design, data collection, data analysis, data interpretation, or writing of the report. the corresponding author had full access to all the data in the study and had final responsibility for the decision to submit for publication. the datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. key: cord-283399-iz4l9i0d authors: o’gorman, c.; mchenry, e.; coyle, p. v. title: human metapneumovirus in adults: a short case series date: 2006-03-14 journal: eur j clin microbiol infect dis doi: 10.1007/s10096-006-0116-0 sha: doc_id: 283399 cord_uid: iz4l9i0d this study was carried out to further the available information on adult cases of human metapneumovirus (hmpv), a recently described cause of respiratory infection. among a cohort of 741 symptomatic patients tested since 2003, the virus was diagnosed in six adults using reverse transcriptase polymerase chain reaction. of the six, two were from the community, two were hospital inpatients with chronic obstructive pulmonary disease and two were immunocompromised patients, both of whom required ventilation and later died. this report discusses the clinical features, epidemiology and diagnosis of hmpv, highlighting that this infection may be associated with death in high-risk adults. for adults presenting with respiratory symptoms and a background of pre-existing respiratory disease or who are immunocompromised, nucleic acid-based techniques are a cost-effective means of making the viral diagnosis in a clinically relevant time frame. acute respiratory tract infections are a major cause of morbidity and mortality worldwide, and they account for a high percentage of hospital admissions. however, up to 50% of cases of community-acquired pneumonia in adults and 15-35% of cases of bronchiolitis and pneumonia in children are of uncertain aetiology [1] [2] [3] . it is clear that respiratory viruses are the principal pathogens in a proportion of such cases [4] . hmpv is a recently described respiratory pathogen of the subfamily pneumovirinae, which is divided into the pneumovirus genus, containing respiratory syncytial virus, and the metapneumovirus genus, containing hmpv. in young children and elderly patients hmpv is most commonly associated with a clinical diagnosis of bronchiolitis or bronchitis, respectively, whereas in middle-aged adults, it may produce an influenza-like illness, which can be complicated by pneumonitis in the presence of immunocompromising factors [5] . hmpv has been detected as the sole pathogen in the nasopharyngeal aspirate of a haematopoietic stem cell transplant recipient who subsequently died of respiratory failure following an upper respiratory prodrome [6] . the regional virus laboratory in belfast carries out viral diagnostics for northern ireland, a region with a population of 1,700,000. it began routine use of a multiplex reverse-transcriptase polymerase chain reaction (rt-pcr) method for virus detection from respiratory samples in july of 2003 [7] . the samples were from three sources: adult hospital inpatients (25.57% of total processed), paediatric hospital inpatients (64.48%) and general practitioners participating in a sentinel influenza surveillance program (9.96%). the laboratory tested any specimens of respiratory tract material including bronchoalveolar lavage, nasopharyngeal and tracheal aspirates, and sputum, nose and throat swabs. this retrospective observational study reviewed all cases of hmpv detected in patients over 18 years of age, from the time the rt-pcr method was adopted in july 2003 through to january 2005. we selected adult cases for review as there is less data in the literature pertaining to hmpv infection in this age group. the routine assay used to detect respiratory viruses in the regional virus laboratory is a multiplex, nested rt-pcr for 12 common respiratory pathogens: influenza a (h3 and h1) and b; parainfluenza virus types 1, 2 and 3; respiratory syncytial virus a and b; adenovirus; human rhinovirus; coronavirus 229e; and hmpv. the pcr target for hmpv was the fusion gene. sensitivity was determined at 3×10 3 copies per ml using an hmpv amplicon cloned from a clinical specimen during the preliminary development of the c. o'gorman (*) . e. mchenry . p. v. coyle regional virus laboratory, royal group of hospitals trust, belfast bt126ba, northern ireland e-mail: ciaran.ogorman@bll.n-i.nhs.uk tel.: +44-28-90634117 fax: +44-28-90634803 molecular strip assay. direct comparison of the assay with an alternative was not possible because of the lack of specimens containing hmpvor alternative tests; virus identification was confirmed by selected amplicon sequencing [7] . following ethics committee approval, we obtained written consent from the living patients in whom hmpv was detected. we reviewed their clinical case notes, noting presenting symptoms, significant medical and drug histories, results of other infectious investigations, chest radiograph findings, and clinical outcomes. a total of 741 respiratory samples from adults were tested over the winters of 2003/04 and 2004/05. six were positive for hmpv, accounting for a period prevalence in the population assayed of 0.81%. in all cases, hmpv was the only virus detected. unless otherwise stated, bacterial culture and serology for atypical respiratory pathogens were negative. the clinical characteristics of these six patients are summarised in table 1 . of the six cases, five were female and one male. their ages ranged from 51 to 83 years (mean 71.5 years). two cases presented to sentinel general practitioners with selflimiting influenza-like illness (cases 1 and 2). neither had a background respiratory disease or any other significant aspect of their medical history. two cases were diagnosed while admitted for acute exacerbations of chronic obstructive pulmonary disease (cases 3 and 4). they presented with dyspnoea, decreased exercise tolerance, cough and lowgrade pyrexia with a background of respiratory disease requiring home oxygen and nebulised corticosteroids. both had been smokers, had received prednisolone as inpatients, and were discharged on maintenance oral steroids. sputum culture of case 3 revealed serratia liquefaciens, which was treated with intravenous ertapenem; for case 4, hmpv was the sole pathogen isolated. two cases (5 and 6) were fatal. the fatalities occurred in individuals receiving immunosuppressive therapy for rheumatological conditions. case 5 had rheumatoid arthritis for which she received prednisolone and methotrexate. she presented in a collapsed and unresponsive condition following a 1-week history of cough, nausea and increasing dyspnoea. within 6 h of admission she was intubated, ventilated and admitted to the intensive care unit (icu). hmpv was the sole pathogen isolated. she died with multiorgan failure 4 days after admission. case six was admitted in an acute confusional state attributed to the oral steroids she was taking for polymyalgia rheumatica. she had been an inpatient for 14 days when she became acutely dyspnoeic with hypoxia, hypothermia and hypotension requiring intubation and admission to the icu. she was found to have bacteremia caused by escherichia coli and was treated with piperacillintazobactam, fluconazole and pulsed methylprednisolone. on the first day of her icu stay, having been an inpatient for 15 days, she was found to have hmpv. this patient made no significant progress while receiving maximal inotropic and ventilatory support and she died after 2 weeks in the icu. whilst our detection of hmpv in symptomatic patients does not establish causation, it is possible that the virus may be associated with serious sequelae in high-risk adults as reported previously [8] and supported by the fact that two of our six cases required icu admission and ventilation. we cannot be sure that case 6 represented nosocomial acquisition as our assay may detect hmpv up to 2 weeks after clinical infection. the clinical syndrome associated with this virus is probably too indistinct to be of diagnostic value. treatment is principally supportive; at present there are no antiviral agents active against hmpv, nor any available vaccine. effective diagnosis of viral respiratory pathogens is necessary to elucidate their epidemiology and assess their clinical impact. moreover, such data is crucial for effective infection control in hospitals and may ultimately allow more rational prescribing of antibiotics and corticosteroids [9] . defining the assay sensitivity by comparison with a gold-standard detection system is a growing problem for viruses such as hmpvor human rhinovirus, for which pcr is becoming adopted as the diagnostic test of choice [8] . we recommend the routine use of rt-pcr or other nucleic acid-based techniques for adults presenting with respiratory symptoms and a background of pre-existing respiratory disease or immunocompromise. the tucson children's respiratory study ii. lower respiratory tract illness in the first year of life etiology of community-acquired pneumonia: impact of age, comorbidity, and severity prospective comparative study of viral, bacterial and atypical organisms identified in pneumonia and bronchiolitis in hospitalized canadian infants virological features and clinical manifestations associated with human metapneumovirus: a new paramyxovirus responsible for acute respiratorytract infections in all age groups a newly discovered human pneumovirus isolated from young children with respiratory tract disease human metapneumovirus in a haematopoietic stem cell transplant recipient with fatal lower respiratory tract disease a touchdown nucleic acid amplification protocol as an alternative to culture backup for immunofluorescence in the routine diagnosis of acute viral respiratory tract infections prevalence and clinical symptoms of human metapneumovirus infection in hospitalized patients acknowledgements the royal group of hospitals trust (royal research office, grosvenor road, belfast) has agreed to act as sponsor for this study. ethical approval for this study was obtained on 21 january 2005 from the office for research ethics committees northern ireland (orecni); rec reference number: 04/nir01/75. written consent was obtained from case patients as required by the research ethics committee. key: cord-276348-vr5fit8r authors: ogra, pearay l. title: respiratory syncytial virus: the virus, the disease and the immune response date: 2004-01-31 journal: paediatric respiratory reviews doi: 10.1016/s1526-0542(04)90023-1 sha: doc_id: 276348 cord_uid: vr5fit8r abstract rsv is the primary cause of hospitalisation in the first year of life for children in most parts of the world, and nearly 100% of children in the usa are infected with the virus by 2 to 3 years of age. the agent is an enveloped rna virus with a non-segmented single-stranded negative-sense genome. the viral genome encodes 8 structural and 2 non-structural proteins. important structural proteins include the fusion (f) protein and the attachment (g) protein which are essential for viral penetration and attachment to the host cells. both proteins are important in development of immune responses. the virus is estimated to cause 3000 to 4000 deaths annually. primary infections are as a rule symptomatic. the spectrum of clinical manifestations ranges from mild upper tract illness, infection in middle ear which progresses to acute otitis media, croup, to apnoea in premature infants, pneumonia and bronchiolitis. premature babies born at 30–35 weeks of gestation, infants with cyanotic congenital heart disease, hiv-infected subjects, and patients on intensive immunosuppressive therapy especially after bone marrow transplant are considered to be at risk for increased mortality and morbidity during rsv infection. the virus does not normally replicate outside of the bronchopulmonary tree and the infection is exquisitely restricted to the respiratory mucosa. however, development of extrapulmonary disease has been observed in certain t and b cell immunodeficiency states. the association of rsv with asthma and reversible reactive airway disease in early childhood has attracted significant attention. recurrent wheezing for up to 5 to 7 years of age and established airway disease has been observed in a significant number of children with a strong family history of allergy, after primary infection or reinfection with rsv. immune response to primary infection is relatively small but on reinfection, a significant booster effect with sustained immunologic reactivity is observed in serum and respiratory mucosa. both cd4and cd8-specific as well as th1and th2-cell specific immune responses have been observed during human infection. in addition, proinflammatory as well as immunoregulatory cytokines and chemokines are induced in the respiratory tract after natural and induced (in vitro) infection. significant progress has been made in understanding the role of th1 vs. th2, ige, viral induced cytokines and chemokines in the mechanisms of pathogenesis of the disease, development of wheezing and in the prevention and treatment of the infection and its sequelae. respiratory syncytial virus (rsv) is one of the commonest human viral infections, and virtually every child is infected by the third birthday. because of its restricted mucosal immunopathology, and frequent association with bronchial hyperreactivity and development of wheezing, rsv has served as an important model to investigate mechanisms of mucosal immune responses and development of mucosal disease following infection. the importance of rsv in bronchopulmonary disease and development of bronchial hyperreactivity has been the focus of several recent symposia [kimpen jl, simoes eaf. am j respir crit care med 2001; 163:s1–s6]. this brief report will only summarise, based on selected references, the historical landmarks of its discovery and current understanding of the mechanisms of immunity, and their possible role in the pathogenesis of bronchopulmonary disease. s120 p.l. ogra rsv was discovered in 1956, when a group of chimpanzees in a colony outside of washington, dc (usa) were noted to have developed cold-like illness. morris and colleagues recovered a cytopathic agent from one of these chimpanzees, who had an upper respiratory tract illness with coryza, runny nose, and malaise. they named this agent "chimpanzee coryza agent"(cca). 2 these investigators examined the entire colony, and nearly 100% of the chimpanzees were found to be infected. an interesting observation was that the human contacts working with the chimpanzees were also infected and exhibited mild upper respiratory tract illness and coryza, somewhat less severe than observed in the chimpanzees. subsequent studies identified two major isolates of the virus recovered from other patients with upper respiratory tract illnesses. the long strain, commonly used in laboratory studies, was recovered from the bronchopulmonary washings of a child with bronchopneumonia, and the schneider strain was recovered from a patient with croup. 3, 4 on the basis of the cytopathology of this agent in tissue culture with formation of syncytia, and the similarities between the isolates recovered from the monkeys and the long and the schneider strains recovered in humans, chanock and colleagues coined the term "respiratory syncytial virus" to incorporate all available isolates, and provided a classic description of the disease in children. 3, 4 shortly thereafter, beem and colleagues described in detail the epidemiology of rsv infection during community outbreaks. 5 the agent is a pleomorphic, enveloped, cytoplasmic virus containing single-stranded, negativesense rna. the rna is associated with viral proteins, consisting of a nucleocapsid core that is packaged within a lipid envelope. rsv is classified in the genus pneumovirus, which belongs to the family paramyxoviridae. the paramyxoviridae family also includes two other genera, paramyxovirus (containing, e.g., parainfluenza virus types 1, 2 and 3 and mumps virus) and morbillivirus. the genera are differentiated by the diameter of the helix, the number of genes, and the nature of their surface glycoproteins. the surface g glycoprotein of the virus lacks neuraminidase and hemagglutinin. complementary dna (cdna) cloning has identified ten different viral genes, each coding for a single protein. the sequences of each gene have been described. the characteristics of these genes differentiate rsv from the other members of paramyxoviridae as reviewed previously. 6 eight of the ten rsv proteins are present in infected cells and in the virions, and therefore are structural proteins. the disulfide-bonded glycoprotein (f, fusion protein) and the large glycoprotein (g, attachment protein) are surface proteins and are the major antigenic determinants of the virus. these proteins induce protective antibodies. the g protein mediates viral attachment. the f protein mediates viral penetration and syncytium formation. the small hydrophobic protein (sh), the matrix protein (m), and the m2 protein are envelope-associated proteins. the nucleoprotein (n), the phosphoprotein (p), and the large nucleoprotein (l) are present in the rsv nucleocapsid. ns1 and ns2 are non-structural proteins; they are found only in infected cells but not in virions. 6 rsv displays minimal antigenic heterogeneity. however, two major groups a and b, with antigenic differences on the g, f, n, and p proteins, have now been identified. the g protein is the most variable protein, with only 53% homology in the amino-acid sequences between the proteins of the a and b groups. in contrast, the f and n proteins have a high degree of genetic and antigenic homology between the two groups. both f and g proteins have several distinct antigenic sites. 6 recent data have shown considerable genetic diversity among groups a and b. the g-protein sequences may differ 20% in different group-a lineages and 9% in different group-b lineages. the replication of the virus includes the following specific events. the virus attaches the cell through g protein. the viral envelope fuses with the plasma membrane of the host cell through the f protein. after penetration, the nucleocapsid of the virus is released into the cellular cytoplasm, where the replication takes place. the viral rna serves as a template for messenger rna. the messenger rna serves as a template for translation of viral proteins, and complementary rna serves as a template for transcription of virion rna. the viral antigens can be detected in 9 hours in cell culture, and the infectious virus in 11 to 13 hours. human rsv replicates in several animal species, including mice, cotton rats and chimpanzees, and to a smaller extent in guinea pigs and ferrets. 6 the clinical picture of rsv infection varies according to age. the primary infection at 6 weeks to 2 years of age is usually symptomatic and involves the lower respiratory tract. asymptomatic primary rsv infection in children is rare. repeated infections in older children are usually less severe. respiratory tract infections are frequently associated with expiratory wheezing (variously referred to as bronchiolitis or wheezy bronchitis, asthma), pneumonia and acute otitis media. rsv infections in neonates differ from those in older children. such neonates do not often exhibit wheezing, and apnoea may be the only symptom of infection. the mortality in healthy children is extremely low, but life-threatening infections are common in immunocompromised patients and in patients with cardiac abnormalities. pneumonia is the most common manifestation in elderly subjects. isolated upper respiratory tract infections associated with rsv have been noted, especially in older children and adults during re-exposure. the common symptoms are rhinorrhea, nasal congestion, pharyngitis, and cough. some studies suggest that common colds induced by rsv may be more prolonged and severe than those induced by other viruses. 6, 7 the term bronchiolitis has been used as a diagnosis of a specific clinical symptom complex since 1940. the diagnostic criteria vary between different centers in the usa and in other countries. in general, bronchiolitis is a clinical presentation in infants less than 12 months old in whom a first attack of an acute illness, after a brief prodrome of upper respiratory symptoms, is characterised by wheezing, dyspnea, respiratory distress, poor feeding, tachypnea (>50/min), and radiologic evidence of hyperaeration of the lung. fine crepitation can usually be heard by auscultation of the chest. 6, 7 in the majority of patients, the symptoms and signs resolve within a few days to a week after the onset of illness. in sicker infants, the duration of hospitalisation may last up to 7 days. in the past infants under 6 weeks old and those with underlying illnesses often needed longer hospitalisation. although rsv is the most common etiologic agent of bronchiolitis and virtually the only agent that induces epidemics, other respiratory viruses can also induce bronchiolitis. these include parainfluenza type 1 and 3 virus, rhinoviruses, adenoviruses, coronaviruses, and influenza a virus. adenoviruses can induce very severe bronchiolitis and with high mortality. numerous follow-up studies have shown that up to 75% of the patients with rsv bronchiolitis exhibit recurrent wheezing or pulmonary function abnormalities years later. the clinical symptoms gradually decrease and disappear usually during the following 10 years. in some of these patients, the symptoms continue and they are classified as having asthma. a diagnosis of asthma has been established in up to 92% of patients followed prospectively for 5−10 years after bronchiolitis. even after milder lower respiratory tract rsv disease, increased morbidity was documented through the third and fourth year of life. however, normal pulmonary function was found between the ages of 8 and 12 years. 6, 7 it is not clear whether rsv can induce long-lasting or permanent damage to the small airways and in the growing lung. it is possible that development of bronchiolitis during primary infection may be restricted to subjects already genetically and anatomically at risk for pulmonary hyperreactivity. this possibility is strongly supported by the findings that pre-existing diminished lung function measured very early in life (before any respiratory illness) was found to be a risk factor for recurrent wheezing. 7, 8 although rsv infection is rare in the first 4 weeks of life, epidemics in neonates have been described. rsv infection has been observed in 20−30% of babies studied in a neonatal unit and in 35% of those hospitalised for 6 days or longer. 9 in this study, 61% of the babies had respiratory illness, and of these, approximately half had upper respiratory tract infection, the other half had pneumonia. when pneumonia during the first month of life was studied, rsv was found to make up 55% of all isolates. 9 it is now well-documented that rsv infection occurs commonly in adults as well as children. in a family study, 17% of the adults living with infected children also became infected. 8 in adults, rsv infection can be asymptomatic or can induce mild to moderate upper respiratory tract symptoms. in healthy adults, the infection is rarely severe or fatal. the symptoms include fever for 1 to 4 days, nasal congestion, rhinorrhea, sore throat, ear pain, and cough lasting 10 days or longer. the average duration of virus shedding is 5 days. based on clinical features, rsv infection cannot be differentiated from other agents associated with cold-like symptoms. adults who are immunocompromised, institutionalised, or aged, or who have some underlying illness (especially chronic pulmonary disease) are at risk of severe rsv pneumonia. the occurrence of pneumonia in long-term care facilities varies from 5% to 67%, with mortality rates ranging from 0 to 53%. the chest radiograph usually reveals patchy changes, diffuse consolidation or interstitial infiltrates. during outbreaks, rsv must be included in the differential diagnosis of fever with evidence of pulmonary infiltrates in immunocompromised adults. children at increased risk from rsv infection include young infants with prematurity, bronchopulmonary dysplasia, congenital heart disease, congenital or acquired immunodeficiency, subjects with hematologic malignancies, patients with bone-marrow or organ transplants, and cystic fibrosis. as mentioned earlier, premature infants are more likely to have apnoeic spells, atelectasis/infiltrates, and hyperinflation as seen on the chest radiograph, and may require oxygen therapy and mechanical ventilation. consequently, these patients need longer hospitalisation. 10 some studies suggest that intubation increases the risk for fatal illness. rsv infection is a major reason for re-hospitalisation of children with bronchopulmonary dysplasia. in these patients, a large number of siblings, parental smoking and recent need for home oxygen therapy are major risk factors. 6 clinicians have long been aware that rsv infection may be particularly severe, long lasting, and sometimes fatal in children with congenital immunodeficiency diseases, especially those with both t-and b-cell defects. animal studies have suggested that t-cell mediated cellular immunity is responsible for terminating rsv infection. no reports are available of possible increased severity of rsv disease in children with isolated hypogammaglobulinemia. increased morbidity and mortality have, however, been documented in children undergoing chemotherapy. severe rsv infection has been reported in children who underwent liver transplantation. the risk factors appeared to be acquisition of infection soon after transplantation and pre-existing lung disease. 10 recently, rsv infection has been studied in human immunodeficiency virus (hiv)-infected children who experienced pneumonia and prolonged viral carriage. up to 25% of such children may develop fatal illness. 10, 11 congenital heart disease is another well-established risk factor for severe rsv infection. cardiac function is not depressed in patients with normal hearts who have rsv infection. infants with heart disease and rsv infection often need more treatment in the intensive care unit and more ventilator therapy than those without congenital heart disease. the mortality in infants with heart disease has been estimated to be about 37%. even higher mortality (73%) has been reported in patients with pulmonary hypertension. 6,10,11 the mortality associated with primary rsv infection in otherwise healthy children is estimated to be 0.005% to 0.020%. 6 in hospitalised children, mortality rates are estimated to range from 1% to 3%. 3 however, considerably higher mortality rates have been observed in children with cardiopulmonary abnormalities and in immunosuppressed subjects. due to the ubiquitous nature of rsv infection, even a low mortality rate may have marked impact on the total mortality of young children. the temporal patterns of respiratory viral isolations from ten laboratories in the usa with that of deaths of children has suggested that rsv isolations were clearly associated with the respiratory deaths of children 1 to 11 months old and with influenza in children 24 to 59 months of age. a significant correlation has been shown to exist between the occurrence of sudden infants death syndrome (sids) and rsv infections. 12, 13 rsv has been demonstrated in the lungs of up to 25% of infants who died from sids. 13 prolonged apnoea, which is a major sign of newborn rsv infection, may explain some of these deaths. at present, however, the role of rsv in sids in not fully understood. community-acquired respiratory viruses are important causes of potentially serious acute respiratory illnesses in hospitalised bone-marrow transplant (bmt) recipients. approximately one-third of such patients are infected with one of these viruses, although multiple viruses have been isolated continuously during these winter study periods. 10 the morbidity and mortality associated with these infections were substantial. more than half (58%) of these infections may be complicated by pneumonia, with an associated mortality of over 50%. in immunocompromised patients with rsv infection, the pneumonias are almost exclusively viral in origin and may be associated with a mortality of 100% if not treated promptly. many of the pneumonias in patients infected with other viruses, such as influenza virus, appeared to be either self-limited or appeared to have a substantial bacterial component, as judged by their favourable response to antibacterial therapy. however, fatal viral pneumonias occur as well. it is noteworthy that only a few infections due to parainfluenza virus and adenovirus are observed during these winter seasons and that their potential to cause fatal viral pneumonia has not been appreciated. 10, 11 in bmt recipients with an acute respiratory illness, community-acquired respiratory viruses must be considered seriously. other recent studies have also suggested extremely high morbidity and mortality with rsv infection in patients with leukemia and hematologic malignancies. 10 rsv infection is followed by the development of both serum and mucosal igm, iga and igg antibodies however in a few studies, igm antibodies against rsv were found to remain detectable for at least 1 year. 6 rsv-specific igg antibody response can be detected in most patients; it reaches maximum values in 20 to 30 days after the onset of symptoms. again, lower responses in young infants have been reported. igg responses occur mainly in igg1 and igg3 subclasses, indicating the antigenic nature of the protein moieties of the f and g proteins of rsv. one year after the primary infection occurs, rsv-specific igg levels appear to decline to low levels. after re-infection, a booster effect is noted, with high titers of igg detectable within 5 to 7 days. 14 the serum iga response occurs several days later than igm and igg responses. 15 interestingly, iga can be found free and cell-bound in nasopharyngeal secretions of patients with rsv infections. free anti-rsv iga appears within 2 to 5 days after infection, and peak titers are obtained between 8 and 13 days. the nasopharyngeal iga response is greater in children older than 6 months. a mucosal immune response to rsv has also been demonstrated by rsv-induced antibody response in vitro in tonsillar lymphocytes. 16 furthermore, nasal secretions contain free rsv-specific ige and cell-bound ige during rsv infection. 14 several studies have demonstrated specific antibody responses to major rsv structural proteins. antibody responses to the f protein of rsv are often cross-reactive with both rsv strains tested, whereas antibody responses to the g protein are subgroup specific. similar findings were reported for group-specific antibody responses to primary and secondary rsv infections. these observations suggest that primary and secondary infection with group-a viruses can induce cross-reactive neutralising antibody responses to group-b viruses. rsv infection induces specific cell-mediated immune responses, including lymphocyte transformation, cytotoxic t-cell responses, and antibodydependent cellular cytotoxicity responses. 1 a number of mechanisms have been proposed to explain the association of rsv infection with increased bronchial reactivity and wheezing (table 1) . these include anatomic restrictions of the neonatal bronchial tree, and tissue damage produced by the infection itself as a result of direct cytopathology of the mucosa. during the course of acute infection, there is sloughing of the respiratory epithelium with exposure and activation of irritant receptors, which induce neurogenic stimulation of bronchial smooth muscle and development of bronchial spasm. there is loss of inhibitory mediators and cholinergic neural activity as observed during parainfluenza and influenza virus infections. recent in vitro studies have demonstrated that rsv significantly augments the proinflammatory effects of substance p by upregulating the expression and density of its specific receptor nk1 on target cells. substance p is a neuropeptide which has been shown to exhibit significant bronchoconstrictor effects in experimental animal studies. 17 because of the anatomy of airway in the neonate, these neurogenic factors may be more important in the induction of bronchial hyperreactivity in early infancy and childhood than later in life. 17 there is good evidence from both clinical studies and experimental models with rsv, that early respiratory infections may contribute to early systemic sensitisation to other antigens or allergens in a genetically prone or atopic child (table 2) . such children may start building up homocytotropic immune responses to environmental allergens very early on in life. 18, 19 antibody-mediated immune responses are to a large extent protective. igg, iga and igm do not seem to contribute to the development of disease. in fact, they seem to be important in protection. high levels of maternal antibody are indeed protective against disease. however, the magnitude of such immune response in the early years is low. it has been observed that the primary immune responses against rsv are relatively ineffective, s124 p.l. ogra table 2 characteristics of cellular immune reactivity in children and adults relative to atopic susceptibility susceptibility response atopic th 2 response to common inhalant allergens non-atopic low levels of th 1 response cord blood low levels of th 1 response to food and inhalant allergens (evidence of in utero sensitization) atopic early childhood boosted th2 non-atopic early childhood deviated to th 1 but when such children get reinfected, they develop significant boosting, particularly for the igg and iga responses. 6 it is important to recognise that virtually all children who get infected with rsv develop virus-specific ige homocytotropic antibody in the respiratory tract. such ige activity is predominantly cell-bound to the mucosal epithelial of the respiratory tract. 20 in general, there is not much free ige detectable in respiratory excretions, unless children are wheezing or they have bronchiolitis or pneumonia. it seems that most children produce ige to rsv early in life, but it is the amount, the persistence and the duration of this response which is critical in determining which patients are going to develop bronchiolitis and wheezing. it has been observed that persistent virus-specific ige response in respiratory mucosa is an important element in the development of immunopathology for both rsv and parainfluenza viruses. 20 in the last decade there has been considerable interest in examining the role of cell-mediated immune responses in the mechanism of immunopathology in the respiratory tract. both cd4+ and cd8+ cells and th 1 and th 2 types of cd4+ cells have been implicated in the development of disease especially during rsv, rhinovirus and influenza virus infections. 1, 18, 19 in addition to ige antibodies and their interaction with mast cells and the subsequent release of inflammatory mediators, the interaction between rsv and the respiratory epithelium also results in the release of several other mediators. these proteins are very important in mobilising other cells to the site of disease. studies conducted by our group during the past decade have demonstrated the release of leukotrienes, eosinophil degranulation byproducts and epithelial cell-derived cytokines and chemokines during the course of rsv infection in in vitro and/or in vivo settings. 20−23 there is a wide spectrum of cytokines, chemokines and arachidonic acid metabolites, which are generated during the course of rsv infection. many of these products are critical in recruiting cells to the site of disease. furthermore, during the course of infection, there is induction of several cell-adhesion molecules and homing ligands (cd11b, icam-1, e-selectin) necessary for inflammatory and immune cells to be mobilised to the site of disease, to rollover, bind, and stick to the virus-infected tissues. they also induce expression of antigen-presenting molecules like hla class i and ii. thus, there is a complex array of events set in motion by rsv infection of respiratory epithelium, which mobilise inflammatory and possibly immunoregulatory cells to the site of disease in the mucosal epithelium. 24 considering the large number of mechanisms proposed as explanations for the disease caused by the virus it is important to attempt to separate the primary cause and the subsequent effects of specific cell-virus interactions in mucosal tissues. is rsv disease a true consequence of all these different pathogenic mechanisms proposed, or are they set in motion as a result of the activation of different immunoregulatory pathways during the replication of the virus in the mucosa? studies to date have identified a number of other viral-or host-induced events during rsv-associated clinical disease. clearly, all these events cannot be the cause of the disease seen after infection with rsv. in order to provide a unifying base to the multiplicity of phenomena observed, it has been proposed that rsv infection may trigger the activation of a "master switch" of genetic control, regulating the expression of one or more cellular functions identified above. previous studies in molecular biology have described in detail the role of transcription factors in activating certain genes or groups of genes. there are a number of nuclear factors, which mobilise gene activation processes and activate many cellular functions. rsv has been shown to induce activation of several such transcription factors. recently garafalo et al. and others have shown that rsv activates the gene promoters for nf-il-6 and nf-úb. such transcription factors regulate the synthesis of a variety of cytokines and chemokines and other important immunomodulating proteins. these include tnf-a, il-1b, il-2, il-6, gm-csf, and g-csf; adhesion molecules icam-1, vicam-1 and e-selectin; and chemokines, il-8, mip-1a, mcp-1, and eotaxin. these molecules are important in initiating the inflammatory cascade and in mobilising cells to the site of disease. 24, 25 it appears that rsv, during the course of replication at the level of the cell, initiates activation of certain transcription processes which may have a profound effect on expression of many mediators of immunoregulation and inflammation. other viruses such as influenza, rhinoviruses and adenoviruses have been shown to induce a similar activity. recent studies suggest that the ultimate expression of allergy involves a multiplicity of genetic and environmental factors. failure to switch off expected th2 phenotype may be a key factor in eventual development of allergic sensitisation. a number of other environmental factors in early infancy and childhood may also determine the outcome of allergic sensitisation by shifting cellular immune response towards immunologic hyperactivity (table 3). it is thus proposed that a balance between the expression of different pro-inflammatory cytokines and chemokines, and the development of th 1 vs th 2 or cd 4 vs cd 8 cellular response following rsv infection may ultimately determine the degree of pathology or the level of protection against bronchopulmonary disease. respiratory syncytial virus and reactive airway disease recovery of cytopathogenic agent from chimpanzees with coryza recovery from infants with respiratory illness of virus related to chimpanzee coryza agent (cca): i. isolation properties and characterization recovery from infants with respiratory illness of a virus related to chimpanzee coryza agent (cca): ii. epidemiologic aspects of infection in infants and young children respiratory syncytial virus neutralizing antibodies in persons residing in chicago respiratory syncytial virus respiratory syncytial virus and parainfluenza virus respiratory syncytial virus infections within families neonatal respiratory syncytial virus infection respiratory syncytial virus infections in immunocompromised adults respiratory syncytial virus pneumonia in hospitalized adult patients with leukemia respiratory viruses and sudden infant death secretory component and sudden-infant death syndrome the antibody response to primary and secondary infection with respiratory syncytial virus: kinetics of classspecific responses immunoglobulin class-specific antibody response in respiratory syncytial virus infection measured by enzyme immunoassay characteristics of in vitro production of mucosal antibody to respiratory syncytial virus in tonsillar tissue lymphocytes neural mechanisms of respiratory syncytial virus-induced inflammation and prevention of respiratory syncytial virus sequelae th1 and th2 cd4+ cells in the pathogenesis of allergic diseases the development of respiratory syncytial virusspecific ige and the release of histamine in nasopharyngeal secretions after infection interleukin-1a mediates the enhanced expression of intercellular adhesion molecule-1 in pulmonary epithelial cells infected with respiratory syncytial virus inducible translational regulation of the nf-il6 transcription factor by respiratory syncytial virus infection in pulmonary epithelial cells characteristics of il-6 and tnf-a production by respiratory syncytial virus-infected macrophages in the neonate clinical aspects of bronchial reactivity and cell-virus interaction transcriptional activation of the interleukin-8 gene by respiratory syncytial virus infection in alveolar epithelial cells: nuclear translocation of the rela transcription factor as a mechanism producing airway mucosal inflamation key: cord-277276-j2qzhvzi authors: al-ayed, mohamed s.; asaad, ahmed m.; qureshi, mohamed a.; ameen, mohammed s. title: viral etiology of respiratory infections in children in southwestern saudi arabia using multiplex reverse-transcriptase polymerase chain reaction date: 2014 journal: saudi med j doi: nan sha: doc_id: 277276 cord_uid: j2qzhvzi objectives: to investigate 15 respiratory viruses in children with acute respiratory tract infections (artis) using multiplex reverse-transcriptase polymerase chain reaction (rt-pcr), and to analyze the clinical and epidemiological features of these viruses. methods: in a cross-sectional study, 135 children, ≤5 years of age who presented with artis in najran maternity and children hospital, najran, saudi arabia between october 2012 and july 2013 were included. the clinical and sociodemographic data, and the laboratory results were recorded using a standardized questionnaire. two nasopharyngeal swabs were collected from each child: one for bacteriological examination, and the second for viral detection using multiplex rt-pcr. results: a single viral pathogen was detected in 76 patients, viral coinfections in 9, and mixed viral and bacterial pathogens in 15. respiratory syncytial virus was isolated in 33 patients, human rhinovirus (hrv) in 22, adenovirus (adv) in 19, human metapneumovirus in 13, influenza virus in 10, parainfluenza virus in 7, human corona virus (hcov) in 4, and human bocavirus in one. conclusion: respiratory syncytial virus, hrv, and adv were the most frequent viruses, accounting for more than two-thirds of the cases. other viruses, such as mpv, hcov nl63, and hcov oc43, may play a role in pediatric artis. of significance is the potential use of multiplex rt-pcr to provide epidemiological and virological data for early detection of the emergence of novel respiratory viruses in the era of the middle east respiratory syndrome coronavirus. a cute respiratory tract infections (arti's) are a leading cause of infectious disease-related morbidity and mortality among children under 5 years of age worldwide, particularly in the developing countries. 1 the arti's can be caused by bacteria and fungi, but viral infections are the most common causes. in developing countries, viruses represent a considerable proportion of the pathogens associated with arti's, varying from 40-90% across studies. [2] [3] [4] the most frequently implicated viruses among children are respiratory syncytial virus (rsv), influenza a and b viruses (iav and ibv), parainfluenza viruses (pivs), adenoviruses (adv), human rhinoviruses (hrv), and human enteroviruses (hev). several recently discovered viruses, such as human metapneumovirus (hmpv), human bocavirus (hbov), and the human coronaviruses (hcovs) (nl63 and oc43), have been identified as potential respiratory pathogens. in addition, 2 novel human polyomaviruses (washington university virus [wu] and karolinska institute virus [ki]) have been detected in patients with respiratory infections. [5] [6] [7] lower respiratory tract infections (lrti's) are the most severe forms of infection and cause of approximately 1.9 million annual deaths among children, 8 while upper respiratory tract infections (urti's) are the most frequent presentation of respiratory infections. an estimated 50% of all illnesses in all age groups, and approximately 75% of illnesses in young children are viral urti's. 9 proper diagnosis of viral arti's has been shown to reduce the misuse of antibiotics and shorten the length of hospital stay. however, current challenges in detecting respiratory viruses include inconsistent clinical manifestations of viral rti's and variable sensitivity and specificity of available diagnostic assays. the traditional diagnostic methods for the respiratory viruses including cell culture and direct immunofluorescence assays are time consuming and technically demanding. 10 several types of molecular biological methods, including reverse transcription pcr (rt-pcr), pcr-hybridization, and real time pcr, have been introduced as more rapid and sensitive detection methods for respiratory infections. 3, 5, [11] [12] [13] most studies in saudi arabia focused on a small number of viral pathogens, and information on the relative contribution of each pathogen to clinical forms of arti's is incomplete. [14] [15] [16] hence, the aims of our study were to investigate the distribution of 15 respiratory viruses in children with arti's using multiplex rt-pcr, and to evaluate the presenting clinical, demographic, and epidemiological features of these different viral infections. methods. this study was conducted in najran maternity and children hospital, a 200-bed, tertiarycare hospital in najran, southwestern saudi arabia between october 2012 and july 2013. included in the study were children ≤5 years of age who presented with signs and symptoms of upper and/or lower respiratory tract infection seen in the pediatric emergency room, or admitted to the pediatric ward, or the pediatric intensive care unit (picu). written consent was obtained from their parents. inclusion criteria were as follows: a temperature of ≥38 0 c, and at least one of the following: rhinitis, pharyngitis, cough, earache, hoarseness of voice, rhonchi, crepitations, or wheezy chest. the diagnosis for each patient was performed by the attending pediatrician based on the world health organization (who) standard clinical criteria. 17 the clinical and socio-demographic data and routine laboratory tests results were recorded using a standardized questionnaire, including the age, gender, underlying medical conditions, signs and symptoms at presentation, findings on physical examination, laboratory findings (complete blood count [cbc], c-reactive protein [crp] , and bacterial cultures), and definitive clinical diagnosis. sampling. two nasopharyngeal swaps were collected for each patient under strict infection control guidance using rayon-budded swabs with 2 ml of virus transport medium (virocult, wiltshire, uk); one for bacteriological examination and the other for respiratory virus detection using rt-pcr multiplex. the specimens were transported in an icebox to the microbiology department, college of medicine, najran university for further processing. bacteriological examination. each nasopharyngeal sample from each case was inoculated on blood and macconkey agar media (difco laboratories, detroit, mi, usa). the plates were incubated at 37⁰c for 24-48 hours. suspected colonies were identified as pathogenic organisms using standard laboratory procedures according to the biochemical and serological techniques recommended by the who. 18 multiplex rt-pcr analysis. viral nucleic acids were extracted from the nasopharyngeal samples using qiaamp viral mini kit (qiagen, maryland, usa) disclosure. this work was supported by a grant from prince meshaal bin abdullah bin abdulaziz chair for endemic diseases, najran university, najran, saudi arabia (nu500/13). authors have no conflict of interests, and the work was not supported or funded by any drug company. according to the manufacturers' instructions. the total rnas extracted from the clinical samples were used for the synthesis of the first-strand cdnas (revert aidkit, fermentas, canada) according to the manufacturer's instructions. the sample cdnas were tested in a 3-tube reaction following the protocol supplied by the manufacturer (qiagen, maryland, usa). tube-1 contained primers set a targeting adv, hcov 229e/ nl63, and piv1-3. tube-2 contained primers set b targeting hcov oc43/hku1, hrv a/b/c, rsv a/b, and iav. tube-3 contained primers set c targeting hbov 1/2/3/4, ibv, hmpv, piv4, and hev. positive controls included a mixture of all 15 virus clones. the negative control contained only double distilled water. the pcr amplification was performed using 10 μl of seeplex rv master mix (seegene, seoul, korea), 4 μl of 5x multiplex primer sets, 3 μl of 8-mop solution, and 3 μl of newly synthesized first-strand cdna (1 μg/3 μl). the seeplex rv contained a, b, and c sets of primers designed from highly conserved regions of genetic sequences for the 15 respiratory viruses. an initial pre-pcr step of 94°c for 15 minutes was performed in a thermocycler 9600 (perkin-elmer, ohio, usa), followed by a total of 35 pcr cycles under the following conditions: 94°c for 30 sec, 60°c for 1.5 min, and 72°c for 1.5 min. the final cycle was followed by an extension step at 72°c for 10 min. the amplified pcr products were separated on 2% agarose gel and stained with ethidium bromide. the type of respiratory virus was identified by comparison with the reference band size provided by the manufacturer. statistical analysis. values were expressed as percentages for discrete variables, or as mean and standard deviation for continuous variables. clinical characteristics and laboratory variables were compared using the chi-square or the student t-test or fisher's exact test, as appropriate. statistical significance was defined as a p-value less than 0.05. all analyses were performed with the statistical package for the social sciences (spss), version 15.0 (spss inc., chicago, il, usa). a total of 135 patients were included in the study period. the median age of patients was 12 months, and the number of males was 80 (63.3%). a single viral pathogen was detected in 76 (56.3%); viral co-infections in 9 (6.7%); and mixed viral and bacterial pathogen in 15 (11.1%) patients. the specific pathogens were rsv in 33 patients, hrv in 22, adv in 19, hmpv in 13, ifv in 10, piv's in 7, hcov in 4, and hbov in one) ( table 1) . children were divided into 5 age-groups: ≤12 months (n=73; 54.1%), 13-24 months (n=32; 23.7%), 25-36 months (n=9; 6.7%), 37-48 months (n=9; 6.7%), and 49-60 months (n=12; 8.9%). the infection rate in children ≤12-month age was significantly higher than in the other age groups. interestingly, we observed a significantly larger number (figure 1) . demographic data and clinical presentation of patients were compared to general pathogens identified ( table 2 ) and the individual viral pathogens (table 3) . generally, there were no significant differences in the clinical presentation, as well as, the gender of patients for the various pathogens isolated. however, the median age of children with hmpv was significantly higher than that of children infected with the other viruses (p=0.036). wheeze was more frequently associated with viral infections (p=0.005) mainly rsv (p=0.01). discussion. viral infection is a major cause of respiratory illness among children. the frequency of respiratory virus detection in 135 children with arti's, in this study, was 80.7%. this finding is in support of previous studies, which reported viral detection rates of 47-95% in children. [2] [3] [4] [5] 19 possible explanations for the wide differences in detection rates in the literature include heterogeneity in study populations, differences in presenting respiratory symptoms, number of respiratory pathogens tested, method used for detection and genetic variability between populations. 2, 20 overall, in 6.7% of the patients, viral co-infections were found. previous studies using rt pcr techniques reported viral co-infection rates of 5-10%, with rsv, hrv, pivs, and hmpv being the most commonly implicated viruses in cases of mixed infections. 12, [19] [20] [21] in this study, the high detection rate of rsv with hmpv (4/9; 44.4%) and hrv with hcov (2/9, 22.2%) in viral co-infections was an interesting finding. huijskens et al 3 found that in 67% of the hrv-positive patients and 42% of the rsv cases, hrv and rsv were co-detected with another virus. it has been reported that the unique characteristic of rsv facilitates infection with a second respiratory virus. 22, 23 previous studies reported that hrv in urti's could serve as a clinical illness promotion factor, functioning additively or synergistically in the pathogenesis of lower respiratory syndromes such as bronchiolitis. 9, 23, 24 however, identification of 2 or more viruses in a patient may be due to prolonged viral shedding or asymptomatic persistence of viruses. 5 further, work would be needed to clarify this situation. quantitative identification of the viral genome may help in explaining co-infections. as expected, most of the respiratory pathogens in this study were detected in children <1 year. the higher detection rate of respiratory pathogens among infants and young children has been ascribed to a higher infection rate, lower viral clearance rate due to underdeveloped immune system, and higher load of the infectious agent associated with living conditions such as crowding. 2, 3, 20 furthermore, the parents of younger children may seek healthcare earlier in the course of disease due to parental anxiety. our results confirm that rsv and hrv play a key role in rti's in children; rsv was the most frequent respiratory virus detected, followed by hrv. the predominance of rsv in this study is in accordance with the assertion that this virus is the single most frequent lower respiratory tract pathogen in infants and young children in saudi arabia and worldwide. 4, 5, 15, 20 it would be important for local pediatricians to use antibiotics cautiously when children are hospitalized with arti's. the hrv has been known to be responsible for urti's and some lrti's in children. 19, 24 our findings showed that the presence of hrv in all age groups. most hrvs (17/22; 77.3%) were associated with urti's and 22.7% of hrv were detected in bronchiolitis (4 cases), or pneumonia (one case). adenovirus was reported to be responsible for 5-10% of arti's in children, and has been largely associated with bronchiolitis obliterans and acute wheezing episodes. [25] [26] [27] in our study, advs were found in 17.4% of all patients, being the third most frequent viral pathogen, and most advs (16/19; 84.2%) were associated with urti's. the hmpv is one of the causes of upper and lower rti's, especially in preschool and older children. in our study, hmpv was the fourth most frequent viral pathogen accounting for 11.9% of all cases. in a previous saudi study, 14 hmpv was identified in 8.3% of 489 children with arti's. in contrast to some studies, 12, 26, 28 all hmpv-positive cases in this study were below 2 years of age and almost half of the cases (7/13; 54%) were co-infected with other viruses. the detection rates for ivs and pivs in our study were comparable with other studies, reporting rates of 0.8-12.6% for iv's and 2.8-19.4% for piv's. 3, 5, 13, 19, 20, 23, 26 in our study, hcovs were detected in 3.7% of all patients including 2 cases with bronchiolitis and 2 cases with pneumonia. in a previous saudi study, 14 hcovnl63 type was detected in 2.8% of 489 children. 14 however, in our study 2 hcov types (hcov nl63 and hcov oc43) were reported. in september 2012, a novel human coronavirus, called the middle east respiratory syndrome coronavirus (mers-cov), was first identified in samples obtained from a saudi arabian businessman who died from acute respiratory failure. 29 as of may 2013, a total of 49 confirmed cases of mers-cov infection with 26 deaths have been reported to the who, including 37 in saudi arabia with 21 deaths. 30 these figures highlight the characteristic distribution of hcovs emerging types circulating in saudi arabia. the similar clinical presentations of patients infected by various respiratory viruses and some bacterial pathogens make etiological diagnoses difficult when decisions are based only on clinical presentation. [3] [4] [5] 12 in our study, except the strong association between rsv infection and chest wheeze, the clinical manifestations of other viral infections were largely nonspecific with fever and cough as the main symptoms. one limitation of this study is the inability to describe the seasonal distribution of respiratory viruses due to the short duration of the study. further, molecular-based studies of longer surveillance duration are necessary to elucidate the seasonal patterns and disease burden associated with respiratory viral pathogens. in conclusion, our study provided background information concerning the respiratory viral etiology in najran, southwestern saudi arabia. the rsv, hrv and adv were the most frequent pathogens, accounting for over than two-thirds of cases with arti's. however, other emerging viruses as hmpv, hcov nl63 and hcov oc43 seem to play an important role in pediatric lrti's. moreover, the use of multiplex rt-pcr is needed to provide not only epidemiological and virological data, but also the opportunity to understand the emergence of novel respiratory viral pathogens ahead of time. the burden of acute respiratory infections in crisisaffected populations: a systematic review respiratory pathogens in children with and without respiratory symptoms diagnostic value of respiratory virus detection in symptomatic children using real-time pcr respiratory viruses in children hospitalized for acute lower respiratory tract infection in ghana virological and clinical characterizations of respiratory infections in hospitalized children emerging respiratory agents: new viruses for old diseases? novel respiratory virus infections in children epidemiology and etiology of childhood pneumonia treatment of the common cold in children and adults detecting respiratory viruses in asymptomatic children comparative evaluation of the seegene seeplex rv15 and real-time pcr for respiratory virus detection rapid detection of respiratory tract viral infections and coinfections in patients with influenza-like illnesses by use of reverse transcription-pcr dna microarray systems detection and genotyping of human respiratory viruses in clinical specimens from children with acute respiratory tract infections human metapneumovirus and human coronavirus infection and pathogenicity in saudi children hospitalized with acute respiratory illness detection of bocavirus in children suffering from acute respiratory tract infections in saudi arabia prevalence of respiratory syncytial virus infection in riyadh during the winter season 2007-2008 and different risk factors impact world health organization. who integrated management of childhood illness hand book. geneva (ch): world health organization basic laboratory procedures in clinical microbiology viral etiology of acute respiratory infection in gansu province viral etiology of acute febrile respiratory illnesses in hospitalized children younger than 24 months aetiology of influenza-like illness in adults includes parainfluenzavirus type 4 multiple versus single virus respiratory infections: viral load and clinical disease severity in hospitalized children epidemiology of respiratory viral infection using multiplex rt-pcr in update on rhinovirus and coronavirus infections frequent detection of respiratory viruses without symptoms: toward defining clinically relevant cutoff values role of emerging respiratory viruses in children with severe acute wheezing respiratory viral infections in infants: causes, clinical symptoms, virology, and immunology human metapneumovirus: review of an important respiratory pathogen isolation of a novel coronavirus from a man with pneumonia in saudi arabia brookline (ma): promed, international society for infectious diseases key: cord-282867-kbyxdegu authors: shah, sayed zulfiqar ali; nasb, mohammad; lu, min; huang, liangjiang; wang, yizhao; chen, hong title: scaling the need, benefits, and risks associated with covid-19 acute and postacute care rehabilitation: a review date: 2020-08-26 journal: rehabil res pract doi: 10.1155/2020/3642143 sha: doc_id: 282867 cord_uid: kbyxdegu coronavirus is an rna virus, which attacks the respiratory system causing complications including severe respiratory distress and pneumonia and many other symptoms. recently, a novel coronavirus (covid-19) outbreak emerged in wuhan, which caused a significant number of infections in china and resulted in a global pandemic. the main aim of this study is to review and summarize the evidence regarding the supportive role of physical rehabilitation techniques in managing covid-19-associated pneumonia. in this review, we also emphasize the use of rehabilitation techniques in the management of pneumonia in covid-19-infected patients. based on the evidence presented, we conclude that certain physical rehabilitation techniques and modalities could be of great support in the management of covid-19-associated pneumonia. the safety of staff and patients when applying rehabilitation intervention requires attention. the combination of physical rehabilitation and medical treatment would result in improved treatment outcomes, faster recovery, and shorter hospital stay. many rehabilitation techniques are safe and feasible and can be easily incorporated into the management protocol of covid-19 victims. decisions of early rehabilitation induction should be based on the patient's medical condition and tolerability. coronaviruses are crown-like enveloped viruses which belong to the coronaviridae family [1, 2] . so far, six coronaviruses (229e, oc43, mers-cov, sars-cov, hku1, and nl63) have been successfully identified [3] and can cause deadly infections in humans. the coronavirus severe acute respiratory syndrome (sars) outbreak was first identified in [2002] [2003] in guangdong province of china, which infected 8477 people in many asian and european countries, as well as the united states. the disease was quickly transmitted from person to person via contact in households and healthcare settings, and later, the outbreak spiked massively in communities [4] . in 2012, mers-cov had caused an outbreak in the middle east and was therefore named middle east respiratory syndrome (mers) [5] . coronaviruses are widely distributed around the globe. the cov genetic variations, frequent genetic recombination events, multiple animal hosts, and animal-to-human interface are increasing the chances of the emergence of novel and more virulent coronaviruses [6] . a novel and more virulent coronavirus termed sars-cov-2 was identified in wuhan, central china, in january 2020 when an outbreak of pneumonia of an unknown aetiology emerged [7] . the outbreak of this novel coronavirus disease, covid-19, has resulted in a global pandemic, and so far, 3,939,119 cases have been reported worldwide, with 274,917 global deaths (5/10/2020) [8] . the purpose of this study was to review the evidence regarding the supportive role of treatment options available in physical rehabilitation to manage covid-19 pneumonia effectively. we are also aiming to present our rehabilitation experience from china, the potential of local covid-19 transmission, staff safety, and infection control during rehabilitation interventions. evidence strongly supports that many rehabilitation techniques including chest physiotherapy and physical therapy modalities can be of great support to manage covid-19-associated pneumonia [9, 10] . combined medical and rehabilitation therapy would result in improved treatment outcomes in coronavirus-infected patients. potential relevant articles were identified through a pubmed, ovid, and google scholar search. english and chinese language studies of mixed designs were reviewed, focusing on different aspects of rehabilitation of coronavirus-infected patients. sars-cov-2 can be isolated through rt-pcr and generation sequencing of the nucleic acid extracted from the sam-ples collected from the respiratory system of the infected patients. investigations for the diagnosis should include routine complete blood count, serum analysis, and computed tomographic (ct) scans for assessing pulmonary changes. clinical signs and symptoms, chest radiography, and ruling out of other pathogens play an important role in the diagnosis of coronavirus-associated pneumonia [11, 12] . in figures 1-3 , we have shown ct images of three cases of pneumonia taken at the onset of symptoms featuring different stages of the disease. it is clear that sars-cov-2 is more transmissible than sars-cov, but transmission dynamics, mode, and route of transmission remain unclear [13] . along with the zoonotic transmission to humans due to sustained human-to-animal contact, human-to-human spread of coronaviruses (sars and mers-covs) was also confirmed. human-to-human transmission was mostly seen when there was sustained contact with infected individuals, for example, in healthcare rehabilitation research and practice settings and households [14] . it is also suggested that for human coronavirus infection, an intermediate host may not be required [15] . coronavirus transmission through respiratory droplets, human contact, or contaminated objects like clothes and utensils was confirmed by many studies [16, 17] . [12] . chest physiotherapy and faecal management have been declared as a high-risk procedure [18] . the exhaled or leaked air during noninvasive ventilation (niv) can propagate virus dispersion, thus increasing the facility-based infection rate [19] . simonds [18] . a sealed plastic bag should be used to cover the mouth during expectoration to prevent the spread of infection [20] . since the virus spreads through respiratory droplets or aerosol particles, for staff safety, a positive air-purifying respirator (papr) is highly recommended. papr is a battery-operated device, which expels filtered air and helps in the prevention of inhaling the virus, which is necessary while performing suctioning or other techniques generating such particles and droplets. heat generation, movement restrictions, device weight, and non-air-conditioned environments make wearing papr difficult for staff [21] . care should be taken while performing cpt and postural drainage as these techniques produce coughing and assist in expectorating secretions. therefore, the above techniques and use of nebulizers should be carried out in closed rooms, with staff wearing ppe and papr. faecal management and the disposal of diapers should be done while wearing ppe [22] . the n95 mask increases the breathing rate, inducing early fatigue and even dizziness in some staff members. the ppe use was especially troublesome in pregnant staff and in tropical regions [18] . healthcare workers with respiratory problems who found wearing ppe such as n95 difficult, or staff suffering from wet eczema who found that it could become worse by following some of the protective measures including frequent hand washing and gloves, should not be compelled to work in areas requiring high ppe protective measures [23] . research has shown that the virus can transmit locally and through surfaces of physiotherapy equipment including modality probes, pads, therapy balls, and hot packs. it is recommended to clean surfaces with 62-71% ethanol which inactivates the virus within a minute; otherwise, it could remain on the surface for a longer period of time [24] . longitudinal studies have reported that due to long periods of bed rest, some pathological changes including pulmonary fibrosis, alveolitis, atelectasis, muscular weakness, and other neuromuscular problems may persist [25] . the aforementioned problems in sars patients impair the quality of life, cause dyspnea on exertion, and lead to residual pulmonary dysfunction [26, 27] . we speculate that extensive lung damage, multiple organ failure in some covid-19 patients, and longer bed rest may cause residual pathological problems, physical dysfunction, and functional limitations which provide strong evidence for the incorporation of rehabilitation into covid-19 management. evidence. common problems identified in covid-19 patients that could be managed by rehabilitation specialists in the postacute phase include musculoskeletal pain, joint pain, reduced range of motion, muscular weakness, neuropathy and myopathy, pulmonary dysfunction, dysphagia, dyspnea, confusion, and impaired activities of daily living. it remains unclear whether the impact of the problems mentioned would persist in the chronic phase [28] . long-term exposure to corticosteroid therapy can cause many musculoskeletal problems. after the sars outbreak in china at the end of 2002, chinese and international literature reported many cases of avascular osteonecrosis (avn) [29] [30] [31] . drug therapy represents an essential treatment of pneumonia, whereas some other treatments are mainly supportive, including but not limited to intravenous hydration, supplementary oxygen, and chest physiotherapy [32] . chest physiotherapy is commonly used as an adjuvant treatment for pneumonia [33] . chest physiotherapy aids in the treatment of symptoms of several respiratory disorders, which are the most familiar cause of both morbidity and mortality in icu patients [34] . in such cases, physiotherapy is aimed at improving the patient's respiratory system in addition to accelerating recovery by enhancing airway clearance and decreasing airway resistance in lung conditions accompanied by hypersecretion and increased airway resistance. chest physiotherapy is a key treatment for individuals with copious secretions (>30 ml per day) and decreased ability to cough [35] . a six-week pulmonary rehab program of discharged 133 sars patients included aerobic exercise and upper and lower extremity exercise. the experimental group was given pulmonary rehabilitation including aerobic exercise, upper and lower extremity exercises, while the control group received only conventional care, the study reported significant 3 rehabilitation research and practice improvement in oxygen consumption, 6-minute walk test and muscle strength in experimental group [36] . experience from china. chinese experts and frontline covid-19 doctors developed pulmonary rehabilitation guidelines with short-term goals of relieving dyspnea and anxiety and long-term goals of functional improvement, enhancing quality of life, and bringing the patient to a normal societal level. the chinese rehabilitation protocol broadly included aerobic exercises and strength, balance, respiratory, and healthcare training, using traditional chinese medicine. according to chinese experts, the consensus rehabilitation program should be stopped if the patient has peripheral capillary oxygen saturation ðspo 2 þ < 88% or reports chest tightness, palpitations, sweating, and dyspnea, which are considered inappropriate for rehabilitation [37] . group therapies should be discontinued, and practice should be one-on-one mostly at the bedside [21] . according to consensus-based acute care physiotherapy recommendations developed in qatar, rehabilitation patients could be categorized into the following six categories: category a (paralyzed, ventilated, and sedated), category b (ventilated and minimally sedated), category c (no mechanical ventilation), category c.1 (bedridden but minimally conscious), category c.2 (conscious and active but dependent), and category c.3 (independent, active, and conscious). category-wise, rehabilitation interventions were recommended as follows: a: passive range of motion (prom), positioning; b: positioning, prom, and graded mobility; and c: c.1: positioning, prom, and mobilization; c.2: rom, progressive strengthening, and coordination exercises; c.3: rom, breathing exercises, and graded ambulation [38] . the weaning phase and referral to rehabilitation should be staged and gradual: a gap of three weeks each is suggested for moving icu patients to an acute care ward and finally rehabilitation. covid-19 pneumonia patients have to remain immobilized and stay in prone positions for longer hours, due to which early rehabilitation induction may not be tolerable. a sudden drop in saturation, fatigue, and worsening of respiratory symptoms could occur [28] . acute care rehabilitation 5.5.1. active cycle of breathing technique (acbt). it refers to the technique, which is used to mobilize and clear pulmonary secretions in addition to improving lung function. it includes two phases: the first phase is aimed at relaxing the airways by thoracic expansion exercises and the second phase is the forced expiratory technique (fet), which involves a huff and cough breathing exercise, which is used to help clear secretions from the airways (figure 4 ) [39] . note: as these techniques create droplets, the benefits and risks should be weighed up before applying them [40] . it is the usage of force of gravity combined with percussion (clapping on the chest and/or back) to release the thick, sticky mucus from the lung peripheries to remove it easily by coughing. lots of studies have shown its effectiveness in treating pneumonia cases [41, 42] . pd includes comfortably lying in the proper gravity-assisted drainage position depending on the affected lung zones. in each position, the chest wall should be percussed for three to five minutes, followed by deep breathing exercises accompanied by vibrations on expiration and then two to three huffs. the patient should then be encouraged to cough and expectorate the dislodged secretions. this is followed by a short rest interval of controlled breathing between two cycles. this cycle is repeated for each position, respectively. this treatment is usually done twice daily or according to the patient's situation. each cycle takes approximately 30 minutes [43] . note: scale the expected improvement first; some positions are difficult to achieve, and also, changing position is associated with droplet generation. . niv therapy through cpap and hfno can manage hypoxia, improve respiratory failure, and help in delaying or avoiding endotracheal intubation [44] . invasive ventilation techniques should be adopted if the patient did not respond to niv quickly [45] . the application of niv in a properly ventilated ward with negative pressure could be helpful [46] . it is a device used to offer positive expiratory pressure during expiration ranging from 10 to 25 cmh 2 o. pep can help in keeping the airways open during expiration which could enable airway clearance. it is a treatment technique that is aimed at improving the oxygen saturation of patients using a high-pressure gas to provide a constant pressure in the airways in both inspiration and expiration in patients breathing spontaneously. cpap masks could be placed over the nose or mouth [47] . brambilla et al. revealed that cpap could decrease the risk of meeting endotracheal intubation criteria in patients with severe hypoxemic acute respiratory failure caused by pneumonia [48] . recommendation: it is recommended that if an hour treatment with cpap/niv did not bring significant improvement in symptoms, patients should proceed to invasive ventilation [44] . a single-tube cpap with an exhalation port, antiviral filter, and nonvented mask is highly recommended [45] . it delivers two different levels of pressure throughout the respiratory cycle: a higher pressure with inspiration which is called inspiratory positive airway pressure (ipap) and a lower one during the expiration which is called expiratory positive airway pressure (epap). when the patient begins to inhale or exhale, the device automatically shifts between epap and ipap settings. bi-pap could enhance comfort for patients who suffer from breathing out difficulty [49] . a prone position is suggested for 12-16 hours daily. when the pao 2 /fio 2 ratio (p/f) reaches ≥150 mmhg with peep ≤ 10 cmh 2 o and fio 2 ≤ 0:60 for at least four hours after supine positioning, prone positioning should be stopped [51] . note: changing position may arouse cough and produce respiratory droplets, so unnecessarily changing posture is not recommended. 5.5.9. mobility. passive rom exercises are recommended to delay muscular weakness, cure reduced rom due to immobility, and prevent integumentary problems [45] . note: active mobility is not recommended in acute care, due to the chances of sudden desaturation and respiratory droplet generation. postural drainage (pd) is relatively contraindicated in patients with raised intracranial pressure (icp), fresh spinal injury, recent eye surgery, hemodynamic instability, hernia of the diaphragm, and patients who went through esophageal anastomosis recently. additionally, precautions should be taken while performing pd in pleural effusion, hemoptysis, pleural edema, and ascites patients. chest percussion and vibration are relatively contradicted in hemoptysis, neglected tension pneumothorax, pulmonary embolism, open wounds, low platelet count, hemodynamic instability, and fresh thoracic skin graft patients. while performing percussion and vibration alone or together with pd, precaution is required in patients with osteoporosis, rib fractures, and bronchospasm, as well as in recent pacemaker transplant patients [52] . positive pressure therapies need tremendous caution in unstable hemodynamic conditions and patients with cranial, facial, or oral surgeries [50] . cpap is contraindicated in severe facial injuries, skull fractures, undrained pneumothorax, paralytic ileus, and surgical emphysema [53] . there is a variety of contraindications to endotracheal suctioning differing from case to case, but the most common are bronchospasm and raised icp. in cases reporting bleeding disorders, epiglottitis, skull or facial injuries, and laryngospasm, suctioning is contraindicated [54] . chinese literature has prudently proven that uswt has anti-inflammatory and analgesic effects in pulmonary infection. the application of curapulse 970™ shortwave diathermy machines and two radarmed 950+™ microwave machines was performed, and the results were promising for covid-19 treatment. the treatment session involved the application of the uswt electrode on the anterior and posterior parts of the trunk for 15 m/d till patient discharge [55] . a review of the biological effects of shortwave concluded that based sw has fewer side effects and more benefits, but they also require further research on the side effects [56] . chinese uswt literature in pulmonary conditions is prevailing because of the wide use of uswt in such conditions. chinese studies had shown significant results on the use of uswt for different pulmonary conditions in humans and animals. some of the studies are zhang et al. [57] on therapeutic effects of uswt on sars, zhang et al. [58] on uswt in type 1i respiratory failure, zhou et al. [59] on uswt in interstitial fibrosis in preclinical models, and hou et al. who utilized swd in bactericidal and virucidal problems [60] . some of these studies claimed to be randomized controlled trials (rcts), but they were lacking the features of an rct and also did not provide important details regarding uswt therapy. it has previously been shown that raising the temperature decreases the activity and viability of viruses [61] . thus, and depending on earlier studies, the utilization of shortwave diathermy could aid in such infectious conditions [60, 62] . shortwave diathermy (swd) is among the first physiotherapy modalities, and it remains in use in rehabilitation departments. it has been also used to treat a wide range of conditions [63] . moreover, pulsed swd was the third most commonly used physiotherapy in the uk [64] . the therapeutic effects of swd on the body parts include the production of efficient deep heating in human body structures within the physiological limits [65] . if applied to a specific muscle, swd roughly raises its temperature to approximately 15.14°c, while the application of swd on lungs would not cause it to rise to that temperature, as the lungs own an efficient water cooling system in addition to the pulmonary circulation which prevents any substantial rising of local heating. this feature supports the safety of swd and prevents tissue damage [65] . moreover, the practical application of swd showed promising effects on relieving thoracic pain, dyspnea, and cyanosis. 5.7.1. swd contraindications. the contraindications for shortwave diathermy include the presence of internal metallic objects in the treatment field, such as an intrauterine device, pacemakers, and metal implants. moreover, swd is contraindicated to be applied on the following body parts and conditions: malignancy, pregnancy, menstruation, the eyes, gonads and growing epiphyses, ischemic tissues, hemorrhagic areas, venous thrombosis/phlebitis, cardiac conditions, blood pressure abnormalities, impaired sensation, wet dressings or adhesive tape, and open wounds. in addition, use on a pregnant uterus should be avoided. due to the risk of tissue burns and necrosis, precautions need to be taken before proceeding with treatment because sweating could cause tissue burn [66] . moreover, patients should not move during the treatment, as it could result in burning the subcutaneous fat necrosis [67, 68] . although chinese literature and some other studies have proven the effectiveness of uswt in pulmonary infection and reported fewer or no side effects, uswt is still debated around the globe. to address this ambiguous situation about the therapeutic efficacy of uswt, we conducted a rigorously designed rct in tongji hospital, wuhan. the results of the study are promising regarding different aspects of covid-19 treatment. the study will be published soon. postacute care rehabilitation. the postacute care phase has been considered as more appropriate for commencing rehabilitation in covid-19 patients as most of the patients appear to be medically stable at this stage and can tolerate individual rehabilitation programs. rom, strengthening, balance and coordination exercises, mild treadmill use, and respiratory therapy if needed have been recommended [36, 37] . moreover, telerehabilitation can be introduced in this phase, offering comparative effectiveness and more safety [37] . there is a lack of evidence to support the use of telerehabilitation during a pandemic. however, telerehabilitation offers online assessment and treatment sessions without exposure of the practitioner to an infection-prone environment. on the other hand, telerehabilitation is a better choice for patients at higher risk of covid-19 mortality, such as immunocompromised individuals and diabetes patients. e-physio, online assessment, and therapy sessions on webcams could be helpful [23] . with advancement in technology and portable devices, intelligent practice and digital rehabilitation offer more safety and comparable effectiveness is possible today. downloadable applications are available with video and textual trainings for respiratory exercises. such devices are effective in offering assessment, feedback, therapy reminders, and treatment progression [37] . another study also recommended the use of telerehabilitation, training videos, and brochures in order to save ppes and reduce the spread of infection [20] . not to be applied in the acute phase. covid-19 patients have reduced respiratory efficiency and less strength of respiratory muscles, so techniques that exhaust such patients should not be applied. the following respiratory therapy procedures are not recommended in the covid-19 acute phase: pursed lip and diaphragmatic breathing, reexpansion techniques for lungs, use of incentive spirometer, and rib mobilization [45] . based on the evidence presented, we conclude that certain physical rehabilitation techniques and modalities could be of great support in managing covid-19-associated pneumonia. 6 rehabilitation research and practice a combination of physical rehabilitation and medical treatment would result in better treatment outcomes, quick recovery, and shorter hospital stays. early rehabilitation is not recommended because of less tolerability; however if the patient's condition permits, and the benefits outweigh the risks, rehabilitation should be commenced early. telerehabilitation should be considered in covid-19 patients, which could offer less staff and patient exposure and more safety, as well as save ppe. this manuscript is our own work. the manuscript is not under consideration for publication in any other journal. the authors declare that they have no conflicts of interest. author hc contributed to the conception of the study, supervision of the manuscript writing, guidelines for rehabilitation techniques, and proofreading and drafting of the manuscript. szas and mn equally contributed to reviewing the literature and writing the manuscript. authors ml, lh, and yw contributed to identifying the relevant literature and daily updating the data regarding the number of coronavirus infections in mainland china. all authors have read and finalized this manuscript for publishing. sayed zulfiqar ali shah and mohammad nasb made equal contributions to this study and are the first authors. coronavirus pathogenesis and the emerging pathogen severe acute respiratory syndrome coronavirus coronaviridae epidemiology, genetic recombination, and pathogenesis of coronaviruses update: outbreak of severe acute respiratory syndrome-worldwide mers: emergence of a novel human coronavirus origin and evolution of pathogenic coronaviruses discovery of a novel coronavirus associated with the recent pneumonia outbreak in humans and its potential bat origin covid-19 dashboard by the center for systems science and engineering shortwave diathermy (s.w.d.) in the treatment of unresolved pneumonia effects of hospital-based physical therapy on hospital discharge outcomes among hospitalized older adults with community-acquired pneumonia and decliningphysical function clinical features of patients infected with 2019 novel coronavirus in wuhan, china clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus-infected pneumonia in wuhan, china transmission dynamics of 2019 novel coronavirus coronaviruses: important emerging human pathogens isolation and characterization of a bat sars-like coronavirus that uses the ace2 receptor temporal-spatial analysis of severe acute respiratory syndrome among hospital inpatients hospital outbreak of middle east respiratory syndrome coronavirus cluster of severe acute respiratory syndrome cases among protected health-care workers-toronto evaluation of droplet dispersion during non-invasive ventilation, oxygen therapy, nebuliser treatment and chest physiotherapy in clinical practice: implications for management of pandemic influenza and other airborne infections randomised controlled trial of adjunctive inspiratory muscle training for patients with copd development of a standard treatment protocol for severe acute respiratory syndrome impact of a viral respiratory epidemic on the practice of medicine and rehabilitation: severe acute respiratory syndrome how should the rehabilitation community prepare for 2019-ncov? persistence of coronaviruses on inanimate surfaces and their inactivation with biocidal agents rehabilitation research and practice impact of severe acute respiratory syndrome (sars) on pulmonary function, functional capacity and quality of life in a cohort of survivors six month radiological and physiological outcomes in severe acute respiratory syndrome (sars) survivors a major outbreak of severe acute respiratory syndrome in hong kong rehabilitation and respiratory management in the acute and early post-acute phase pathogenesis and natural history of osteonecrosis avascular necrosis of bone in severe acute respiratory syndrome steroidinduced osteonecrosis in severe acute respiratory syndrome: a retrospective analysis of biochemical markers of bone metabolism and corticosteroid therapy chest physiotherapy for pneumonia in adults chest physiotherapy in primary pneumonia epidemiology and outcome of acute respiratory failure in intensive care unit patients effects of sputum on pulmonary function a randomised controlled trial of the effectiveness of an exercise training program in patients recovering from severe acute respiratory syndrome pulmonary rehabilitation for patients with coronavirus disease 2019 (covid-19) acute care physiotherapy management of covid-19 patients in qatar: consensus-based recommendations a randomized evaluation of the acute efficacy, acceptability and tolerability of flutter and active cycle of breathing with and without postural drainage in non-cystic fibrosis bronchiectasis endotracheal suction in intubated critically ill adult patients undergoing mechanical ventilation: a systematic review chest physiotherapy in mechanically ventilated patients without pneumonia-a narrative review chest physical therapy for children hospitalised with acute pneumonia: a randomised controlled trial long-term comparative trial of two different physiotherapy techniques; postural drainage with percussion and autogenic drainage, in the treatment of cystic fibrosis efficacy and safety of early prone positioning combined with hfnc or niv in moderate to severe ards: a multi-center prospective cohort study respiratory physiotherapy in patients with covid-19 infection in acute setting: a position paper of the italian association of respiratory physiotherapists (arir) more awareness is needed for severe acute respiratory syndrome coronavirus 2019 transmission through exhaled air during noninvasive respiratory support: experience from china continuous positive airway pressure (cpap) during the postoperative period for prevention of postoperative morbidity and mortality following major abdominal surgery helmet cpap vs. oxygen therapy in severe hypoxemic respiratory failure due to pneumonia the effect of bi-level positive airway pressure on postoperative pulmonary function following gastric surgery for obesity respiratory physiotherapy in the critical care unit prone positioning in severe acute respiratory distress syndrome essentials of cardiopulmonary physical therapy -e-book continuous positive airway pressure ventilation. part two: indications and contraindications suctioning techniques for the removal of respiratory secretions ultra-shortwave diathermy-a new purported treatment for 8 rehabilitation research and practice management of patients with covid-19 biological effects and mechanisms of shortwave radiation: a review application of ultrashort wave diathermy in treatment of severe acute respiratory syndrome effects of ultrashort wave therapy on the clinical efficacy of noninvasive ventilation in patients with type ii respiratory failure caused by chronic obstructive pulmonary disease effect of ultrashortwave diathermy on experimental pulmonary interstitial fibrosis the effect of short wave diathermy on dendritic cells in hepatitis b virus transgenic mice temperature, humidity, and ultraviolet b radiation predict community respiratory syncytial virus activity application of short wave in comprehensive treatment of infectious atypical pneumonia a survey of electrotherapeutic modalities: ownership and use in the nhs in england short-wave diathermy: a review of existing clinical trials medical diathermy in pneumonia contraindications to continuous and pulsed short-wave diathermy therapeutic modalities, f.a. davis company diathermy and pulsed electromagnetic fields rehabilitation research and practice key: cord-263141-n200x6z1 authors: zelaya, hortensia; alvarez, susana; kitazawa, haruki; villena, julio title: respiratory antiviral immunity and immunobiotics: beneficial effects on inflammation-coagulation interaction during influenza virus infection date: 2016-12-23 journal: front immunol doi: 10.3389/fimmu.2016.00633 sha: doc_id: 263141 cord_uid: n200x6z1 influenza virus (ifv) is a major respiratory pathogen of global importance, and the cause of a high degree of morbidity and mortality, especially in high-risk populations such as infants, elderly, and immunocompromised hosts. given its high capacity to change antigenically, acquired immunity is often not effective to limit ifv infection and therefore vaccination must be constantly redesigned to achieve effective protection. improvement of respiratory and systemic innate immune mechanisms has been proposed to reduce the incidence and severity of ifv disease. in the last decade, several research works have demonstrated that microbes with the capacity to modulate the mucosal immune system (immunobiotics) are a potential alternative to beneficially modulate the outcome of ifv infection. this review provides an update of the current status on the modulation of respiratory immunity by orally and nasally administered immunobiotics, and their beneficial impact on ifv clearance and inflammatory-mediated lung tissue damage. in particular, we describe the research of our group that investigated the influence of immunobiotics on inflammation–coagulation interactions during ifv infection. studies have clearly demonstrated that hostile inflammation is accompanied by dysfunctional coagulation in respiratory ifv disease, and our investigations have proved that some immunobiotic strains are able to reduce viral disease severity through their capacity to modulate the immune-coagulative responses in the respiratory tract. influenza virus (ifv) is a major respiratory pathogen of global importance, and the cause of a high degree of morbidity and mortality, especially in high-risk populations such as infants, elderly, and immunocompromised hosts. given its high capacity to change antigenically, acquired immunity is often not effective to limit ifv infection and therefore vaccination must be constantly redesigned to achieve effective protection. improvement of respiratory and systemic innate immune mechanisms has been proposed to reduce the incidence and severity of ifv disease. in the last decade, several research works have demonstrated that microbes with the capacity to modulate the mucosal immune system (immunobiotics) are a potential alternative to beneficially modulate the outcome of ifv infection. this review provides an update of the current status on the modulation of respiratory immunity by orally and nasally administered immunobiotics, and their beneficial impact on ifv clearance and inflammatory-mediated lung tissue damage. in particular, we describe the research of our group that investigated the influence of immunobiotics on inflammation-coagulation interactions during ifv infection. studies have clearly demonstrated that hostile inflammation is accompanied by dysfunctional coagulation in respiratory ifv disease, and our investigations have proved that some immunobiotic strains are able to reduce viral disease severity through their capacity to modulate the immune-coagulative responses in the respiratory tract. keywords: immunobiotics, influenza virus, inflammation, coagulation, respiratory immunity introduction influenza virus (ifv) is a member of the orthomyxoviridae family that contains a negative-sense, single-stranded, segmented rna genome protected by a capsid of viral ribonucleoproteins. this virus is categorized into subtypes based on the expression of hemagglutinin (ha) and neuraminidase on the surface of the viral envelope. influenza is a highly contagious viral infection that has a substantial impact on global health and ifv is a major respiratory pathogen that causes a high degree of morbidity and mortality, especially in high-risk populations such as infants, elderly, and immunocompromised hosts. given the high capacity of ifv to change antigenically, acquired immunity is often not effective to limit infection and therefore vaccination must be constantly redesigned to achieve protection. improvement of respiratory and systemic innate immune mechanisms has been proposed to reduce the incidence and severity of ifv disease. in the last decade, several research works have demonstrated that microbes with the capacity to modulate the mucosal immune system (immunobiotics) are a potential alternative to beneficially modulate the outcome of ifv infection. this review provides an update of the current status on the modulation of respiratory immunity by orally and nasally administered immunobiotics, and their beneficial impact on ifv clearance and inflammatorymediated lung tissue damage. in particular, we describe the research of our group that investigated the influence of immunobiotics on inflammation-coagulation interactions during ifv infection. studies have clearly demonstrated that hostile inflammation is accompanied by dysfunctional coagulation in respiratory ifv disease, and our investigations have proved that some immunobiotic strains are able to reduce viral disease severity through their capacity to modulate the immune-coagulative responses in the respiratory tract. the first barrier that protects the host against ifv infection is the respiratory epithelium through its capacity to recognize the viral attack. when ifv successfully overcomes the respiratory barrier constituted by the mucus layer and the ciliar movement, it mediates its attachment and internalization into respiratory epithelial cells to start its replication (1) . during the viral attack, several pathogen-associated molecular patterns (pamps) are exposed and recognized by pattern-recognition receptors (prrs) expressed in respiratory cells (figure 1 ). it is now well established that the most important prrs involved in the recognition of ifv are the toll-like receptor (tlr)-3 and tlr7 and the rna recognition protein rig-1 (2) . tlr3 is expressed in endosomes and is able to recognize viral double-stranded rna (dsrna) that is produced during viral replication; while endosomal tlr7 and cytoplasmic rig-i recognize single-stranded rna (ssrna). rig-i signals through mitochondrial antiviral signaling protein. the pamps-pprs interaction leads to the activation of several signaling pathways that induce the activation of nuclear factor κb (nf-κb) and interferon (ifn) regulatory factor 3 (irf3) and the production of type i and iii ifns and inflammatory cytokines (2) . type i ifns, especially ifn-β, produced and released during the earlier stages of ifv infection are key to develop an antiviral state in the respiratory tract. it was reported that human bronchial epithelial cells release preformed ifn-β in response to ifv challenge inducing a protective role (3) . ifns produced by infected cells are able to act in a paracrine or autocrine manner activating their receptors (ifnar) and increasing the expression of hundreds of genes that counteract viral replication. functional genomic studies have identified several of the ifn-induced factors that have important roles in controlling ifv replication (2) including the ifn-inducible transmembrane proteins 1, 2, and 3 (4), mx1 proteins (5) , and 2′,5′-oligoadenylate synthetase (oas)-rnaasel system (6) . proinflammatory cytokines and chemokines produced as a result of tlr3 and rig-i activation during ifv infection are also important for the generation of the respiratory antiviral innate immune response. infection of epithelial cells by ifv increases the expression of tnf-α, il-6, il-8, ccl2 (mip-1), ccl5 (rantes), ccl3 (mip-1α), and cxcl10 (ip-10) (7). the production of these cytokines is complemented by activity of inflammasomes that induce the activation of caspase-1 and promote the generation of the active forms of il-1β and il-18 (figure 1) . ifv has been shown to activate mainly the nlrp3 inflammasome which is essential for the protection against the virus since several studies demonstrated that mice lacking nlrp3 or caspase-1 have decreased il-1β and il-18 secretion and increased mortality after ifv challenge (8) (9) (10) . the proinflammatory cytokines and chemokines are responsible for the activation of resident immune cells such as innate lymphoid cells, alveolar macrophages, and dendritic cells (dcs) as well as for the recruitment of neutrophils, macrophages, and lymphocytes into the respiratory tract (2, 7) (figure 2) . respiratory cells infected with ifv express ha on their surface that is important for its recognition by nk cells (11) . it was established that ha expressed by the infected cells is recognized by nkp44 and pkp46 receptors of nk cells that then mediated the lysis of ifv-infected cells (12) . macrophages activated during ifv infection produce ifns, il-6, tnf-α, and nitric oxide synthase that amplify the inflammatory response. in addition, macrophages limit the viral spread by the elimination of apoptotic-infected cells and through phagocyte-mediated opsonophagocytosis of ifv (7) . the production of proinflammatory cytokines during the generation of the respiratory innate immune response against ifv also conditions the adaptive immune response, which includes the production of virusspecific systemic and mucosal antibodies as well as the induction of specific t cell responses (13) . after exposure to ifv there is an activation of antibody responses in the respiratory tract. upper airway exposure results primarily in an iga response while the contact of ifv with the deep lung induces an increased production of pathogen-specific igg (14) . following exposure to ifv in the airways there is an antigen uptake and processing by dcs, activation of cd4 + th cells, and generation of iga-producing plasma cells that populate airway lamina propria. secretory iga has a noninflammatory protective function since these antibodies can bind to virus without activating complement or stimulating the release of inflammatory mediators by innate immune cells (14, 15) . iga prevents ifv from adhering to the epithelial surface by inducing viral agglutination, and masking adhesion epitopes. in the deep lung, when ifv reach the alveolar space, there is a differentiation and expansion of antibody-secreting plasma cells that are committed to the production of igg. induction of neutralizing respiratory and serum igg antibodies is a key event in the defense against influenza infection since igg prevents systemic spread (16) . influenza infection in the lungs also activates the cellular adaptive immune response by stimulating the production of ifn-γ by th1 cells that effectively activate cd8 + t cells and macrophages, which clear virus and infected cells from the lungs (17) . therefore, during uncomplicated influenza, adaptive immune response ultimately results in clearance of ifv from the lungs through the activity of virus-specific antibodies and cd4 + and cd8 + t cells. the gut microbiome, which is defined as the collective group of microorganisms and their associated genes within the intestinal tract, is considered as a key player in the modulation of host intestinal immune responses (18, 19) . in fact, the impact of gut commensal bacteria on the innate and adaptive immune responses to enteric pathogens has been recognized conclusively (20) (21) (22) . however, the effect of gut microbiome on the immune responses in distal mucosal sites and its impact in the outcome of respiratory infections has recently been exposed. in this regard, some studies have demonstrated an important role for intestinal microbiota in maintaining respiratory antiviral immunity against ifv (23, 24) . iwasaki and colleagues observed that commensal bacteria within the gut, especially gram-positive bacterial populations, had an important role in supporting an appropriate immune response to ifv infection in the respiratory tract (23) . the work demonstrated that oral antibiotic treatments impaired the resistance of mice to the intranasal infection with ifv as noted by the elevated lung viral titers when compared to non-antibiotictreated animals. results indicated that gut gram-positive bacteria provided protection by triggering an adequate inflammatory response through inflammasomes activation. in antibiotictreated mice, synthesis of pro-il-1β, pro-il-18, and nlrp3 was impaired even at the steady state. in addition, depletion of grampositive bacterial populations in the gut resulted in an alteration of the distribution and activation of respiratory dcs at steady state as well as in a diminished dcs migration from the lung to the draining lymph nodes, resulting in reduced activation of cd8 + and cd4 + t cells after influenza challenge (23) . alteration of respiratory dcs activities also correlated with impaired expansion of influenza-specific b cells and reduced influenza-specific antibodies. by using germ-free and antibiotic-treated mice challenged with ifv, abt et al. (24) showed that the absence or the alteration of intestinal microbiota induced an exacerbated weight loss, a greater drop in blood oxygen saturation, increased mortality, and elevated lung viral titers indicating a weaker ability to resist influenza. even more, germ-free and antibiotic-treated mice infected with ifv experienced higher epithelial cell necrosis, peribronchiolar inflammation, severe bronchiole epithelial degeneration, and epithelial hyperplasia when compared to conventional animals (24) . interestingly, those effects were observed when both the pr8 strain and the x31-gp33 virus, a less pathogenic strain of ifv that causes minimal mortality and morbidity in conventional mice, were used. consistent with the work by ichinohe et al. (23) , germ-free and antibiotic-treated mice challenged with ifv had an impaired adaptive immune response as shown by the lower influenza-specific antibodies (serum igm and igg), fewer number of ifv-specific t cells present in lungs, as well as a reduced capacity of specific t cells to produce effector cytokines such as tnf-α, mip-1α, il-2, and ifn-γ (24) . moreover, authors demonstrated that the alterations of adaptive immune responses were related to defects in the early innate immune response mediated by macrophages. in fact, transcriptional profiling and computational analyses of macrophages from antibiotic-treated mice indicated a reduced expression of antiviral genes including ifnb, tnfa, il1b, irf7, mx1, and oas1a when compared to immunobiotics for influenza virus infection frontiers in immunology | www.frontiersin.org december 2016 | volume 7 | article 633 conventional mice. in addition, functional assays of macrophages from antibiotic-treated mice demonstrated that those cells had a defective response to type i ifns and an impaired capacity to limit ifv replication (24) . the cellular and molecular mechanisms through which the gut microbiome and their derived signals maintain and modulate immune responses in distal mucosal sites are poorly understood. two possible mechanisms that are not mutually exclusive have been proposed to explain this beneficial effect of the gut microbiome. one possibility is that distal mucosal and peripheral immune cells are directly exposed to bacterial products that activate prrs in the steady state and help to maintain the normal immune tone. there is evidence that bacterial products from gut commensals such as peptidoglycan can be absorbed and circulate throughout the host and help to modulate the normal development of immune cells (25) . in line with this hypothesis, iwasaki and colleagues speculated that bacterial products from gut commensals trigger prrs to stimulate immune cells systemically and that factors released by those cells supported steady-state production of pro-il-1β, pro-il-18, and nlr proteins. this idea was sustained by their observation that intestinal injection of tlr ligands restored immune responses to ifv in antibiotic-treated mice (23) . another possibility is that commensal bacteria may indirectly influence systemic and distal mucosal immune responses through immune factors released from the intestinal mucosa including cytokines, chemokines, and grow factors. these research works demonstrated that the gut microbiome provides signals to sustain antiviral innate immune defense mechanisms in the respiratory tract allowing robust and efficient effector responses upon challenge by viral pathogens such as ifv. therefore, the role of the gut microbiome in regulating respiratory antiviral immunity represents an exciting area of research that could help to provide the scientific basis for the development of novel prevention strategies for lung infectious diseases. however, several questions need to be answered to identify new alternatives to improve antiviral respiratory defenses by modulating the microbiota. how the different microbial species from the gut microbiota influence the common mucosal immune system? which prrs are activated by the gut microbiota to functionally modulate antiviral immunity locally and in distal mucosal sites? which cellular functions are modulated by the microbiota after prr activation? has the microbiota the ability to influence immune responses to other respiratory viruses? are similar immune mechanisms activated by the microbiota in high-risk populations (infants, elderly, immunocompromised hosts) in which respiratory viral infections are more frequent and severe? is it possible to beneficially modulate antiviral respiratory defenses by orally administering selected microorganisms with immunomodulatory capacities? research from the last years has provided some answers for the last question. the first studies that assessed the capacity of immunobiotics to favorably modulate the immune response against ifv focused on the humoral immunity ( table 1) . yasui et al. (26) reported that the oral administration of bifidobacterium breve yit4064 improved the production of anti-ifv igg antibodies in serum of ifv-infected mice. the yit4064 strain reduced viral titers, improved the survival rate, and decreased the severity of the symptoms associated to the influenza infection. similarly, it was shown that orally administered non-viable lactobacillus pentosus b240 (27) or viable lactobacillus brevis kb290 (28) were able to improve the levels of respiratory specific iga and igg antibodies of mice challenged with ifv. moreover, the improved humoral response induced by these strains correlated with significant reduction of viral titers, body weight loss, and a decrease of the alterations of physical conditions induced by ifv. more recently, kikuchi et al. (29) demonstrated a beneficial effect on the outcome to ifv infection related to an improved respiratory humoral response in lactobacillus plantarum ayatreated mice. in addition, the work proposed a mechanism for the distal immunomodulatory activity induced by orally administered immunobiotics. authors showed that l. plantarum aya fed to mice impacted in peyer's patches (pps) inducing an activation of antigen presenting cells (mainly cd11b + dcs) and increasing the production of il-6. those changes promoted an igm-to-iga class switch recombination, the differentiation of iga + b cells into plasma cells, and improved the production of mucosal iga in both the intestine and the respiratory tract. those studies show that immunobiotics are capable to modulate the production of systemic and mucosal antibodies against influenza and therefore, to enhance the humoral immune response (figure 2) . however, the precise mechanism by which orally administered immunobiotics induce iga production in distant mucosal sites remains unclear. it was also demonstrated that immunobiotics are able to improve cellular immune response against ifv (figure 2) . in this regard, it was reported that orally administered lactobacillus casei shirota improved the outcomes of ifv infection of aged (30) and infant mice (31) by increasing systemic and respiratory nk cell activity and improving the production of ifn-γ and tnf-α by respiratory lymphocytes. both studies also demonstrated that ifv titers were significantly reduced in aged and infant mice treated with the shirota strain (30, 31) . similar to the mechanism proposed to explain the improvement of humoral response, it was postulated that immunobiotic l. casei shirota stimulated th1 cells and nk cells in pps and induced a mobilization of those cells to lungs and respiratory-associated lymphoid tissues where they produced ifn-γ and enhanced the antiviral defenses. several other studies corroborated these findings by showing similar effects for orally administered lactobacilli (32, 33) . immunobiotic lactobacillus strains (l. gasseri tmc0356, l. rhamnosus gg, or l. plantarum 06cc2) beneficially modulated nk cells activity and th1 response against ifv, diminished virus titers and reduced lung pathological changes (32, 33) ( table 1) . more recently, kawahara et al. (34) described the improvement of respiratory antiviral response by an orally administered bifidobacteria strain. it was shown that bifidobacterium longum mm-2 increased respiratory nk cell activity and ifn-γ production resulting in improved clinical symptoms, reduced mortality, and decreased virus titers after ifv challenge. research work has also demonstrated that nasal administration of immunobiotics is an interesting alternative to improve cellular response against influenza infection (35-37) ( table 1) . hori et al. (35) observed that balb/c mice nasally treated with non-viable l. casei shirota had increased levels of il-12, ifnγ, and tnf-α in mediastinal lymphoid nodes and lungs. this improved cellular respiratory immunity correlated with a beneficial clinical outcome to ifv challenge. similar observations were performed with nasally administered l. pentosus s-pt84 (36) and l. rhamnosus gg (37). other recent studies have also demonstrated the ability of immunobiotics to improve respiratory innate antiviral defenses in the respiratory tract (table 1; figure 2) . it was reported that orally administered non-viable l. plantarum l-137 improved protection against ifv by increasing type i ifn production (39). the work clearly demonstrated that the increased production of ifn-β induced by the immunobiotic strain correlated with the reduction of viral loads in lungs as well as the improved survival of infected mice. more recently, it was shown that l. gasseri sbt2055 enhanced survival rates and reduced lung viral titers in mice infected with ifv (40). interestingly, authors observed that the lung expression of the antiviral genes mx1 and oas1a was enhanced in l. gasseri sbt2055-treated mice and that the inflammatory response triggered by ifv was differentially regulated inducing a lower inflammatory damage (40). our group has also reported a beneficial regulation of the ifv-triggered inflammatory response by immunobiotics. lung damage induced by ifv is known to be produced by virus replication as well as the uncontrolled inflammatory response that is characterized by a hypersecretion of proinflammatory cytokines, especially tnf-α, il-1β, and il-6 (45). the adequate production of inflammatory factors is necessary to protect against ifv infection together with an appropriate regulation with anti-inflammatory cytokines to prevent the damage of lung tissue. thus, the proper balance of cytokines is a key factor in determining the outcome of ifv infection. in this regard, we observed that orally (41) or nasally (42) administered immunobiotic l. rhamnosus crl1505 differentially regulated the levels and kinetics of inflammatory cells and cytokines in mice after ifv challenge. in our experimental model, we observed increased levels of respiratory tnf-α, il-6, neutrophils, and macrophages in crl1505-treated mice early after the challenge with ifv. later, proinflammatory cytokines and infiltrated cells started to decrease in immunobiotic-treated animals in contrast to control mice, in which those parameters continued increasing. the trend toward lower inflammatory factors and cells registered later during ifv infection in l. rhamnosus crl1505-treated mice correlated with a reduced severity of pulmonary damage when compared to control mice (41, 42). chen et al. (43) also investigated the ability of orally administered enterococcus faecalis kh2 to beneficially modulate the innate immune response to influenza infection. authors observed that kh2 strain protected c57bl/6 mice against ifv as observed by the reduced mortality, weight loss, and lung viral titers. as expected, ifv enhanced the levels of proinflammatory mediators in the respiratory tract including il-6, tnf-α, ifn-γ, il-1β, il-17, and mcp-1 while the treatment with e. it is not clear how immunobiotics initiates the cross-talk with the immune system in order to modulate the respiratory antiviral immunity. it is not known exactly which prrs are activated by immunobiotics in the intestinal or respiratory mucosa to functionally modulate antiviral immunity locally and in distal mucosal sites, respectively. neither it has been determined with exactitude which cellular functions are modulated by immunobiotics immediately after prr activation. research from the last decade has demonstrated that the immunomodulatory effects of probiotic bacteria are the consequence of complex interactions between several bacterial molecules and host receptors located in different immune and non-immune cells (46, 47). it has also been shown that the immunomodulatory properties of immunobiotics are dependent on the strains. therefore, studies carried out with certain strains cannot be easily extrapolated to other bacteria, even those of the same genus and species (48, 49). consequently, it is still necessary to carry out deeper studies to find out the molecular mechanisms by which immunobiotics beneficially influence the respiratory antiviral immunity. the studies mentioned before showed the potential of immunobiotics to be used for the reduction of the incidence and severity of ifv infections. however, in addition to deepening the knowledge of their mechanisms of action, several other points should be considered for the efficient application of immunobiotics in humans. for example, it is necessary to determine the best time as well as the most appropriate route for their administration. immunobiotics used as components of functional foods can be included in diets on a regular basis and thus help to improve respiratory defenses, especially in high-risk populations and during the seasons with the highest incidence of respiratory infections occurs. in this sense, in a randomized controlled trial we demonstrated that l. rhamnosus crl1505 (administered in a yogurt formulation) improved mucosal immunity and reduced the incidence and severity of intestinal and respiratory infection in children (50). hence, the incidence of infectious events was reduced from 66% in the placebo group to 34% in the group that received the probiotic yogurt. furthermore, there was also a significant reduction in the occurrence of indicators of disease severity such as fever and the need for antibiotic treatment in children receiving the probiotic yogurt. this immunobiotic yogurt (yogurito ® ) has been included into official national nutritional programs in argentina and is given daily to children at schools in several provinces thanks to the government actions. epidemiological studies in the schools receiving the immunobiotic product have shown a reduction in the incidence of infections and in the associated school absenteeism (alvarez et al., unpublished results). on the other hand, as mentioned earlier the nasal administration of immunobiotics is more efficient than the oral administration to enhance respiratory immunity. this route of administration poses a practical disadvantage considering that the treatments with immunobiotics showed favorable results when they were used before the infectious challenges. in this way, it would be necessary to predict the exact moment in which the viral pathogen will be in contact with the host in order to carry out the prophylactic immunobiotic treatment. this option could be used for example during a school or work outbreak in which cases of respiratory infections occur and it is desired to prevent or reduce the severity of infections in asymptomatic individuals. for an intervention of these characteristics, it would be also important to determine the exact time after the contact with the virus in which it is possible to administer immunobiotics to achieve the beneficial effect. in a recent study, percopo et al. (51) have defined this as "the window of opportunity. " the work evaluated the effect of the nasal administration of live or inactivated l. plantarum ncimb 8826 in a mice model of severe respiratory infection with the pneumonia virus of mice (pvm) and found that immunobiotic treatment promoted full survival from acute pvm infection when administered within 1 day after virus challenge (51) . similar studies would be of value in ifv infection models. another point of interest is related to the duration of the improvement of respiratory defenses after the last immunobiotic administration. our studies have showed that the immunomodulatory effect of some nasally administered immunobiotics persisted for at least 15 days (villena et al., unpublished results) . other studies have also reported short-term protection after nasal treatment with different immunobiotic strains (43). interestingly, garcia-crespo et al. (52) found that adult mice primed nasally with l. plantarum ncimb 8826 or lactobacillus reuteri f275 were completely protected against lethal pvm infection and that protection persisted for at least 5 months after the initial priming. these findings open an interesting challenge in the study of immunobiotics to improve the defenses against ifv, since it would be very useful to establish the duration of the protective effect for each strain and treatment, since in the majority of cases these long-term studies were not taken into account. ifv infections often result in mild to moderate lung infection; however, life-threatening disease can occur. it has been demonstrated that the most severe disease outcomes are associated with secondary bacterial pneumonia caused primarily by staphylococcus aureus or streptococcus pneumoniae (53) . taking into account the high incidence of viral infections and the frequency of associated secondary bacterial infections which contribute to aggravate the health status of the host and reduce its chance of recovery, various approaches for preventing and treating influenza and secondary bacterial pneumonia are been investigated. a wide range of antibiotics and anti-inflammatory drugs has been tested in mice [reviewed in ref. (54) ]. it would be of interest to evaluate the potential beneficial effect of immunobiotics on these circumstances. in this regard, preliminary studies from our laboratory showed that nasally administered l. rhamnosus crl1505 is able to improve survival, reduce bacterial cell counts in lung and blood, and limit lung inflammatory damage caused by s. pneumoniae infection in mice produced after the infection with ifv or respiratory syncytial virus (rsv) (villena et al., unpublished results) . these results opened an interesting topic for future investigations. finally, it would be also of interest to investigate whether immunobiotic treatments may influence other physiological systems involved in the defenses against viral respiratory infections such as the coagulation system. our group has made some progress in this regard, as mentioned below. coagulation is an extremely ordered process that involves the interaction of three key components: endothelial cells (ecs), platelets, and coagulation factors. tissue injury that activates ecs typically initiates coagulation that is characterized by the binding of platelets to activated ecs and the formation of the platelet plug. almost simultaneously, tissue factor (tf) released by ecs result in factor x activation, which induces thrombin and the generation of fibrin strands to strengthen the platelet plug leading to a stable platelet-fibrin clot. all these processes are tightly regulated by anticoagulant and fibrinolytic mechanisms to avoid thrombotic and/or haemorrhagic complications. a key role has been attributed to ecs in the temporal and special regulation of coagulation activation. resting ecs avoid the inappropriate plug formation by controlling platelet adhesion and activation and generating several anticoagulant factors providing a non-thrombogenic barrier (55, 56) . once activated or injured, ecs expose collagen to blood, increase platelet binding and aggregation, reduce the expression physiological anticoagulant factors, increase the expression of tf and von willebrand factor, and suppress the fibrinolytic activity (57, 58) . all these changes in the hemostatic system facilitate thrombosis in the infected or inflammated tissue. both hemorrhagic and thrombotic complications have been described during ifv infection. influenza is able to cause pulmonary hemorrhage and edema related to coagulopathy or induce uncontrolled thrombosis through an over-activated coagulation (figure 3 ) (55, 58) . animal models have helped to explain the mechanisms by which ifv infection activates coagulation and key role has been attributed to tf. it was described that ifv activates coagulation by enhancing tf production, thrombin generation and fibrin deposition in c57bl/6 mice (59) . in a mice model of ifv infection, it was recently shown that wild-type animals increased lung tf expression and activation of coagulation but presented alveolar hemorrhage (60) . moreover, selective deletion of tf in epithelial cells from lung significantly reduced tf expression after ifv infection and had higher alveolar hemorrhage and immunobiotics for influenza virus infection frontiers in immunology | www.frontiersin.org december 2016 | volume 7 | article 633 reduced survival than controls. on the contrary, deficiency of tf in either respiratory myeloid cells or ecs did not enhanced alveolar hemorrhage or modified survival of ifv-infected mice (60) . these results indicate that an appropriate modulation in the production of tf in the lung during ifv infection is necessary to maintain tissue hemostasis avoiding hemorrhage and excessive fibrin deposition. production of tf by lung epithelial cells will be required to maintain alveolar hemostasis during ifv infection, while excessive release of tf by macrophages and ecs would contribute to pathology and lung tissue injury (59, 60) . it is considered that ecs may play an important role in the pathogenesis of ifv. influenza infection is able to induce alveolar edema and pulmonary hemorrhage through the alteration of ecs via several mechanisms, including direct damage and loss of tight junctions and apoptosis (61) . in addition, recognition of damageassociated molecular patterns such as hmgb1 or oxidized phospholipids through tlr4 activates ecs to drive lung injury (62) . direct stimulation of tlr3 by viral rna also results in the upregulation of tf and the downregulation of thrombomodulin (tm) in ecs (63) . at the same time, the inflammatory activation of ecs leads to the activation of the coagulation cascade. inflammation caused by ifv infection increases various proinflammatory cytokines such as tnf-α, il-1β, and il-6 that induce the secretion of tf by ecs and monocytes (58) . in addition to their roles in coagulation, activated proteins such as thrombin, fxa, and fviia also enhance the inflammatory response. the inflammatory potentiating abilities of coagulation factors are mediated through their activation of protease-activated receptors (pars) that are expressed in platelets, ecs, macrophages, and respiratory epithelial cells (58) . the tf/thrombin/par-1 pathway has been associated to the promotion of a deleterious innate inflammatory response to ifv infection in mice (64, 65) . therefore, both the hyper-inflammatory response and the aberrant activation of coagulation, which are potentiated with each other, are involved in severe influenza pneumonia and are key events that have to be controlled in order to reach a favorable resolution of the infectious process. considering the importance of the coagulative response in the outcome of influenza infection and the ability of immunobiotics to beneficially influence the immune response to this respiratory pathogen, we wonder whether some immunobiotic strains would be able to beneficially modulate the immuno-coagulative response triggered by ifv. for this purpose, we performed challenge-infection experiments in mice and evaluated the influence of viable and non-viable immunobiotic l. rhamnosus crl1505 strain on the respiratory immuno-coagulative response induced by ifv (41, 42) . our data demonstrated that oral administration of l. rham nosus crl1505 to mice significantly reduced lung viral titers and tissue damage after the challenge with ifv (41). we later explored the capacity of nasally administered l. rhamnosus crl1505, alive or heat killed, to reduce the influenza burden of disease (42). those treatments induced a significant decrease in ifv titers in lungs, lessened pulmonary damage, and increased survival. interestingly, a similar effect was achieved with the nasal administration of viable and non-viable crl1505 strain. moreover, the nasal route was more efficient than the oral administration to protect mice against ifv infection (41, 42). the protective effect achieved by the immunobiotic strain was related to its ability to modulate the respiratory antiviral immune response, particularly to its capacity to improve the levels of ifn-γ and ifn-β in the respiratory tract (figure 2) . type i ifns trigger the activation of the jak-stat pathway and increase the expression of antiviral genes. in addition, ifn-γ is produced by immune cells, especially th1 cells, and it further improves antiviral immune response by inducing activation of nk cells and macrophages. therefore, the modulation of type i ifns and ifn-γ would be responsible of the reduction of viral loads in ifv-infected mice previously treated with the crl1505 strain, similarly to other immunobiotic strains as mentioned before ( table 1) . we demonstrated that the crl1505 strain increased the levels of gut cd3 + cd4 + ifn-γ + t cells, induce a mobilization of these lymphocytes into the lung and enhanced the respiratory production of ifn-γ and the activity of local antigen presenting cells (41, 66, 67) . it was also noted that nasal administration was more effective than the oral route to increase pulmonary cd3 + cd4 + ifn-γ + t cells (41, 42). the mechanism by which nasally administered viable or heat-killed l. rhamnosus crl1505 improves ifn-γ + t cells population is not clear. however, our studies support the possibility that the immunobiotic strain l. rhamnosus crl1505 impact in the nasalassociated lymphoid tissue or bronchial-associated lymphoid tissue producing an innate imprinting in antigen presenting cells that contribute to the enhanced number and activity of cd3 + cd4 + ifn-γ + t cells. our studies also showed that immunobiotic treatments were able to beneficially modulate the activation of coagulation during respiratory viral infection, an effect that was not reported before (41, 42). then, our studies were the first in demonstrating a beneficial modulation of the immune-coagulative response during respiratory trl3 activation and ifv infection induced by immunobiotic microorganisms (figure 3) . although ifv is an ssrna virus, it generates dsrna replication intermediates that activate tlr3 and contribute to the initiation of the antiviral respiratory immune response. in fact, ifv triggers type i ifn secretion through tlr3 recognition in immune (myeloid dcs or macrophages) and non-immune (fibroblasts or pneumocytes) cells (68) . challenge-infection experiments with respiratory viruses in tlr3 −/− mice showed that tlr3 does not modify the clearance of viral pathogens but it is relevant for the modulation of the lung inflammatory response (69, 70) . it was showed that wild-type mice mount a robust inflammatory response in the lung after ifv infection and that this process is significantly diminished in tlr3 −/− animals (70) . tlr3 −/− mice showed a longer survival when compared wild-type animals and this effect was associated with a reduction of inflammatory cells recruitment and lower levels of inflammatory factors in the respiratory tract. other in vivo studies also demonstrated that tlr3 activation by poly(i:c) enhanced proinflammatory cytokines and immunobiotics for influenza virus infection frontiers in immunology | www.frontiersin.org december 2016 | volume 7 | article 633 antiviral factors expression (71), altered vascular permeability (72) , and incremented the levels of d-dimers indicating that coagulation and fibrinolysis were triggered. in line with these findings, it was observed that the levels of d-dimers in tlr3 −/− mice were significantly lower than in wild-type animals after poly(i:c) administration (63 (neutrophils and macrophages) and proinflammatory mediators (il-1β, tnf-α, il-8, and il-6) in the respiratory tract. moreover, tlr3 activation also induced an increase in tf expression and thrombin-antithrombin complex (tatc) levels in the lung while it reduced tm expression. these inflammatory-coagulative modifications were accompanied by respiratory tissue alterations and impairment of lung function (41, 42) . of interest, we demonstrated that orally (41) or nasally (42) administered immunobiotics before the challenge with poly(i:c) differentially modulated the inflammatory-coagulative response. l. rhamnosus crl1505 was able to reduce and increase the expression of tf and tm, respectively, after the respiratory activation of tlr3. thus, the crl1505 strain significantly diminished coagulation activation in blood and in the respiratory tract after the nasal stimulation with poly(i:c). we also evaluated pulmonary coagulation during ifv infection (41, 42). the respiratory virus induced activation of coagulation in the lungs of infected mice as demonstrated by the increased levels of respiratory tatc. these procoagulant changes were related to alterations in the expression of tm and tf in lungs. our findings are in line with previous studies in humans and animal models of influenza infection demonstrating increased lung fibrin deposition and enhanced numbers of intravascular thrombi in the respiratory tract (59, 73, 74) . we demonstrated that immunobiotic treatment is able to significantly diminish the activation of coagulation in ifv-challenged mice. in fact, lower levels of respiratory tatc and a reduced expression of tf was observed in l. rhamnosus crl1505-treated mice infected with ifv when compared to controls (41, 42). as mentioned before, ifv promote a procoagulant state directly through its capacity to infect ecs and monocytes stimulating the expression of tf (75, 76) . in addition, ifv induce activation of coagulation indirectly by the enhancement of proinflammatory factors such as il-6 (75, 76) . therefore, the ability of immunobiotics to modulate the ifv-triggered immune-coagulative response could be explained by their direct influence on viral replication related to the enhancement of the antiviral state in the respiratory mucosa, and indirectly through the modulation of the inflammatory response. considering this last point, we performed experiments using anti-il-10r blocking antibodies in order to evaluate the role of the regulation of the inflammatory response in the reduction of coagulation activation. results showed that il-10 is important for the regulation of coagulation induced by the immunobiotic l. rhamnosus crl1505 (41). blocking of il-10r abolished the capacity of the crl1505 strain to change the expression of tm and tf in the lungs. this was in line with our previous studies evaluating the ability of l. rhamnosus crl1505 to confer protection against inflammatory damage induced by tlr3 activation or rsv infection, which showed that il-10 is a key factor for the reduction of lung injury (67) . additionally, it was demonstrated that lethal disease caused by ifv infection is prevented by il-10 administration through the reduction of lung immunopathology (77) . moreover, tf expression and procoagulant activity of macrophages and ecs are reduced by il-10 (78, 79) . therefore, we demonstrated that immunobiotic administration induce an early increase in the levels of tnf and il-6 in the respiratory tract after poly(i:c), rsv, or ifv challenge, while the levels of those proinflammatory factors are significantly reduced later during infection (41, 42, 67) . the early increase of proinflammatory mediators and the augmented levels of ifn-γ explain the ability of l. rhamnosus crl1505 to diminish viral replication while the improved production of il-10 would lead to a beneficial modulation of the immune-coagulative response which results in a reduced severity of lung damage. it has been suggested that respiratory viral infections increase the risk of venous thromboembolism and ischemic heart disease through ecs perturbation, coagulation activation, reduction of anticoagulant factors, and inhibition of fibrinolysis (80) (81) (82) . then, our studies suggest that immunobiotics could be an interesting alternative not only to reduce the incidence and/or severity of respiratory viral infections, but in addition to reduce the risk of atherothrombotic alterations associated to respiratory viral infections. research from the last decade has clearly demonstrated that beneficial microorganisms are able to modulate respiratory tract immunity and promote the resolution and lessen the severity of respiratory infections caused by pathogens such as ifv. studies in animal models have demonstrated that orally or nasally administered immunobiotics are able to improve protection against ifv by three main mechanisms. first, immunobiotics increase the respiratory antiviral state by their capacity to improve levels of type i ifns, the number and activity of antigen presenting cells, nk cells, cd4 + ifn-γ + t, and iga + b lymphocytes, as well as the levels of systemic and mucosal specific antibodies. second, immunobiotics beneficially modulate the ifv-triggered respiratory inflammatory response by inducing changes in the levels and kinetics of proinflammatory factors and immunoregulatory cytokines such as il-10 that allow the clearance of virus with a minimal inflammatory lung tissue damage. finally, as demonstrated by our recent research works, immunobiotics modulate lung immune-coagulative response triggered by tlr3 activation or ifv infection, mainly by downregulating lung tf and restoring tm levels. studies in animal models suggest that immunobiotics would influence principally the innate immune response, modulating in that way the early antiviral inflammatory response and the subsequent cellular and humoral immune responses. therefore, immunobiotics would have mainly an adjuvant effect. however, the exact molecular mechanisms by which immunobiotics differentially modulate the innate antiviral immune response against ifv remain to be elucidated. additionally, a growing number of studies in humans have examined the effect of immunobiotics on the incidence and severity of ifv infection. considering the impact of immunobiotics in the innate immune response clinical studies have evaluated principally their potential adjuvant effects on ifv vaccination ( table 2) . although mechanistic studies have not been addressed in depth, there is promising evidence for beneficial effects of immunobiotics on human respiratory health and resistance against ifv. these observations might be helpful to propose new influenza a penetrates host mucus by cleaving sialic acids with neuraminidase the amazing innate immune response to influenza a virus infection critical role of constitutive type i interferon response in bronchial epithelial cell to influenza infection ifitm3 restricts the morbidity and mortality associated with influenza molecular signatures associated with mx1-mediated resistance to highly pathogenic influenza virus infection: mechanisms of survival regulation of ifn-alpha/beta, mxa, 2ʹ,5ʹ-oligoadenylate synthetase, and hla gene expression in influenza a-infected human lung epithelial cells induction of innate immunity and its perturbation by influenza viruses the intracellular sensor nlrp3 mediates key innate and healing responses to influenza a virus via the regulation of caspase-1 the nlrp3 inflammasome mediates in vivo innate immunity to influenza a virus through recognition of viral rna inflammasome recognition of influenza virus is essential for adaptive immune responses nkp46 o-glycan sequences that are involved in the interaction with hemagglutinin type 1 of influenza virus recognition of viral hemagglutinins by nkp44 but not by nkp30 immune responses to influenza virus infection exploiting mucosal immunity for antiviral vaccines secretory iga possesses intrinsic modulatory properties stimulating mucosal and systemic immune responses b cell responses to influenza infection and vaccination regulating the adaptive immune response to respiratory virus infection innate sensing of the gut microbiota: modulation of inflammatory and autoimmune diseases the commensal microbiota drives immune homeostasis interactions between the microbiota and pathogenic bacteria in the gut resurrecting the intestinal microbiota to combat antibiotic-resistant pathogens antibiotics and the intestinal microbiome: individual responses, resilience of the ecosystem, and the susceptibility to infections microbiota regulates immune defense against respiratory tract influenza a virus infection commensal bacteria calibrate the activation threshold of innate antiviral immunity recognition of peptidoglycan from the microbiota by nod1 enhances systemic innate immunity protection against influenza virus infection of mice fed bifidobacterium breve yit4064 oral administration of heat-killed lactobacillus pentosus strain b240 augments protection against influenza virus infection in mice oral administration of lactobacillus brevis kb290 to mice alleviates clinical symptoms following influenza virus infection oral administration of lactobacillus plantarum strain aya enhances iga secretion and provides survival protection against influenza virus infection in mice augmentation of cellular immunity and reduction of influenza virus titer in aged mice fed lactobacillus casei strain shirota reduction of influenza virus titer and protection against influenza virus infection in infant mice fed lactobacillus casei shirota oral administration of lactobacilli from human intestinal tract protects mice against influenza virus infection efficacy of oral administration of heat-killed probiotics from mongolian dairy products against influenza infection in mice: alleviation of influenza infection by its immunomodulatory activity through intestinal immunity immunobiotic lactobacillus administered post-exposure averts the lethal sequelae of respiratory virus infection lactobacillus priming of the respiratory tract: heterologous immunity and protection against lethal pneumovirus infection influenza and bacterial superinfection: illuminating the immunologic mechanisms of disease the immunology of influenza virus-associated bacterial pneumonia influenza virus and endothelial cells: a species specific relationship endothelium -role in regulation of coagulation and inflammation endothelial activation and dysfunction in the pathogenesis of influenza a virus infection aberrant coagulation causes a hyper-inflammatory response in severe influenza pneumonia effects on coagulation and fibrinolysis induced by influenza in mice with a reduced capacity to generate activated protein c and a deficiency in plasminogen activator inhibitor type 1 tissue factor deficiency increases alveolar hemorrhage and death in influenza a virus-infected mice endothelial cells are central orchestrators of cytokine amplification during influenza virus infection damp molecule s100a9 acts as a molecular pattern to enhance inflammation during influenza a virus infection: role of ddx21-trif-tlr4-myd88 pathway a key role for toll-like receptor-3 in disrupting the hemostasis balance on endothelial cells par1 contributes to influenza a virus pathogenicity in mice par-1 contributes to the innate immune response during viral infection orally administered lactobacillus rhamnosus modulates the respiratory immune response triggered by the viral pathogen-associated molecular pattern poly(i:c) immunobiotic lactobacillus rhamnosus improves resistance of infant mice against respiratory syncytial virus infection toll-like receptor and rig-i-like receptor signaling deletion of tlr3 alters the pulmonary immune environment and mucus production during respiratory syncytial virus infection frontiers in immunology | www.frontiersin.org december detrimental contribution of the toll-like receptor (tlr)3 to influenza a virus-induced acute pneumonia essential role of mda-5 in type i ifn responses to polyriboinosinic:polyribocytidylic acid and encephalomyocarditis picornavirus toll-like receptor 3 mediates west nile virus entry into the brain causing lethal encephalitis pulmonary pathology of severe acute respiratory syndrome in toronto highly pathogenic h5n1 influenza virus causes coagulopathy in chickens procoagulant activity of endothelial cells after infection with respiratory viruses procoagulant and inflammatory response of virus-infected monocytes autocrine regulation of pulmonary inflammation by effector t-cell derived il-10 during infection with respiratory syncytial virus monocyte il-10 produced in response to lipopolysaccharide modulates thrombin generation by inhibiting tissue factor expression and release of active tissue factor-bound microparticles anticoagulant properties of the anti-inflammatory cytokine il-10 in a factor xa-activated human monocyte model risk of myocardial infarction and stroke after acute infection or vaccination risk of deep vein thrombosis and pulmonary embolism after acute infection in a community setting crosstalk between inflammation and thrombosis oral intake of lactobacillus fermentum cect5716 enhances the effects of influenza vaccination a probiotic fermented dairy drink improves antibody response to influenza vaccination in the elderly in two randomised controlled trials lactobacillus gg as an immune adjuvant for live-attenuated influenza vaccine in healthy adults: a randomized double-blind placebo-controlled trial daily intake of heat-killed lactobacillus plantarum l-137 enhances type i interferon production in healthy humans and pigs evaluation of the immune benefits of two probiotic strains bifidobacterium animalis ssp. lactis, bb-12(r) and lactobacillus paracasei ssp. paracasei, l. casei 431(r) in an influenza vaccination model: a randomised, double-blind, placebo-controlled study nonpathogenic lactobacillus rhamnosus activates the inflammasome and antiviral responses in human macrophages immunoprotective effects of oral intake of heat-killed lactobacillus pentosus strain b240 in elderly adults: a randomised, double-blind, placebo-controlled trial lactobacillus in jelly enhances the effect of influenza vaccination in elderly individuals the use of the probiotic lactobacillus rhamnosus gg and viral findings in the nasopharynx of children attending day care effects of probiotic lactobacillus brevis kb290 on incidence of influenza infection among schoolchildren: an open-label pilot study prebiotic and probiotic supplementation prevents rhinovirus infections in preterm infants: a randomized, placebo-controlled trial effects of oral intake of plasmacytoid dendritic cells-stimulative lactic acid bacterial strain on pathogenesis of influenza-like illness and immunological response to influenza virus key: cord-269508-d82qjyyr authors: xu, min; qin, xuan; astion, michael l.; rutledge, joe c.; simpson, joanne; jerome, keith r.; englund, janet a.; zerr, danielle m.; migita, russell t.; rich, shannon; childs, john c.; cent, anne; del beccaro, mark a. title: implementation of filmarray respiratory viral panel in a core laboratory improves testing turnaround time and patient care date: 2013-01-01 journal: am j clin pathol doi: 10.1309/ajcph7x3nlyzphbw sha: doc_id: 269508 cord_uid: d82qjyyr the filmarray respiratory virus panel detects 15 viral agents in respiratory specimens using polymerase chain reaction. we performed filmarray respiratory viral testing in a core laboratory at a regional children’s hospital that provides service 24 hours a day 7 days a week. the average and median turnaround time were 1.6 and 1.4 hours, respectively, in contrast to 7 and 6.5 hours documented 1 year previously at an on-site reference laboratory using a direct fluorescence assay (dfa) that detected 8 viral agents. during the study period, rhinovirus was detected in 20% and coronavirus in 6% of samples using filmarray; these viruses would not have been detected with dfa. we followed 97 patients with influenza a or influenza b who received care at the emergency department (ed). overall, 79 patients (81%) were given oseltamivir in a timely manner defined as receiving the drug in the ed, a prescription in the ed, or a prescription within 3 hours of ed discharge. our results demonstrate that molecular technology can be successfully deployed in a nonspecialty, high-volume, multidisciplinary core laboratory. the filmarray respiratory virus panel detects 15 viral agents in respiratory specimens using polymerase chain reaction. we performed filmarray respiratory viral testing in a core laboratory at a regional children's hospital that provides service 24 hours a day, 7 days a week. the average and median turnaround time were 1.6 and 1.4 hours, respectively, in contrast to 7 and 6.5 hours documented 1 year previously at an on-site reference laboratory using a direct fluorescence assay (dfa) that detected 8 viral agents. during the study period, rhinovirus was detected in 20% and coronavirus in 6% of samples using filmarray; these viruses would not have been detected with dfa. we followed 97 patients with influenza a or influenza b who received care at the emergency department (ed). overall, 79 patients (81%) were given oseltamivir in a timely manner defined as receiving the drug in the ed, a prescription in the ed, or a prescription within 3 hours of ed discharge. our results demonstrate that molecular technology can be successfully deployed in a nonspecialty, high-volume, multidisciplinary core laboratory. acute respiratory infection is a leading cause of outpatient visits and hospitalization in young children, especially in winter and spring. 1 most of the acute respiratory infections are caused by viral agents, with primary or secondary bacterial infections occurring less frequently. without definitive diagnosis, patients with viral infection are more likely to receive unnecessary antibacterial agents. 2 therefore, laboratory tests providing accurate, timely determination of the infectious agents associated with viral respiratory disease are important. a broad array of tests is available to detect viral respiratory agents. rapid antigen tests are available for individual respiratory viruses such as influenza a, influenza b, and respiratory syncytial virus (rsv). however, these tests have low sensitivity and specificity. [3] [4] [5] [6] several molecular tests have been developed to detect viral rna or dna using the polymerase chain reaction (pcr) method. [7] [8] [9] these tests show high sensitivity and specificity, but most of these molecular tests are technically challenging and time consuming and require experienced, specialized medical technologists. they are usually performed in large medical centers in highly specialized molecular or virology laboratories with limited hours of operation. filmarray (idaho technologies, salt lake city, ut) is a small desktop closed single-piece flow real-time pcr system. this end-to-end molecular system includes automation of nucleic acid extraction, an initial reverse transcription and multiplex pcr, followed by singleplex second-stage pcr reactions for detection of specific viral agents in a single-use cartridge. the respiratory virus panel performed on filmarray is able to detect 15 viral agents from respiratory specimens. 10 the test requires 5 minutes hands-on time and 65 minutes of instrumentation time. comparison studies between filmarray and other molecular tests for respiratory viral agents showed comparable results. [11] [12] [13] [14] [15] as part of our preimplementation planning, we surveyed 10 other clinical laboratories in the united states. only 1 laboratory performed the test in the general laboratory, with the rest performing the test in the microbiology, virology, or molecular laboratories. we reasoned that a general medical technologist with proper training would be able to perform the test. to provide 24 hours per day, 7 days per week (24/7) service to our emergency department (ed) and urgent care center, we decided to perform the test in the core laboratory. our core laboratory is a rapid response facility staffed by approximately 35 full-time equivalent (fte) employees providing analysis encompassing automated chemistry, hematology, coagulation, urinalysis, blood gas analysis, and selected therapeutic drug monitoring. in addition, individual manual tests such as pregnancy tests, cardiac markers, drug abuse screening, sickle cell screening, occult blood, and heterophil antibodies are also performed in the core laboratory. we do not currently have a 24/7 microbiology laboratory, and during the night shift the core technologists perform culture setup and gram staining. our core specimen processing and testing are designed based on lean, single-piece flow principles without batching. 16 because the core laboratory was already involved in such work flow, the institution of filmarray should be relatively simple. in this study we describe the implementation of filmarray in the core laboratory to provide rapid 24/7 service for respiratory virus diagnosis. pediatric patients up to 21 years of age who were cared for in our hospital or urgent care clinics and who underwent respiratory viral pcr testing between december 14, 2011, and april 19, 2012, were included in the study. for filmarray assay, the midturbinate nasal swab was collected using a nylon flocked swab (copan diagnostics, murrieta, ca) that was immediately placed in universal transport media (utm) (copan diagnostics). nursing staff collected all samples and were trained in the technique before the december initiation of the filmarray assay. specimens in utm were tested as soon as they were received in the laboratory. before the institution of the filmarray panel, direct fluorescence assay (dfa) testing was used for respiratory specimens, with the nasal wash the preferred specimen type. more than 95% of the specimens submitted for dfa by the ed the previous year were nasal washes. viruses present in respiratory samples were tested using the filmarray respiratory panel, which detects 15 viral agents including adenovirus, coronavirus hku1, coronavirus nl63, human metapneumovirus, rhinovirus/enterovirus, influenza a, influenza a h1, influenza a h1 2009, influenza a h3, influenza b, parainfluenza 1, 2, 3, 4, and rsv. samples were loaded into the testing cartridge under a ventilation hood. after hydrating the pouch with 1.0 ml of hydration solution, 300 ml of respiratory sample was diluted in 0.5 ml of sample buffer, of which 300 ml was injected into the sample pouch, which was then loaded onto the instrument. after entering the sample identification, the instrument was started and the result was available in approximately 1 hour. the testing pouch contains all the reagents for nucleic acid extraction, reverse transcription, first-step multiplex pcr amplification, and second-step real-time pcr amplification with single specific primer pair. for each viral agent, the second-step pcr was performed in triplicate. the software automatically analyzes the melting curve of the second-step pcr to report the results as positive or negative. this was the first molecular test implemented in the core laboratory, so microbiology staff assisted in the initial test validation and personnel training. multiple continuing education sessions were given to core laboratory staff on all shifts regarding technical procedures as well as the principles underlying the technology. all core staff competency evaluations were fully completed before the test was made available clinically. dfa was performed on nasal wash samples as described. 17 the procedure detected 8 viral agents including adenovirus, rsv, human metapneumovirus, influenza a, influenza b, and parainfluenza 1, 2, 3. however, it could not differentiate among parainfluenza 1, 2, and 3. turnaround time (tat) data for filmarray and dfa were collected using the pathnet laboratory clinical information system (cerner, kansas city, ks). the tat was the interval from when the sample was logged into the laboratory information system (inside the laboratory) to when the result was verified. the information on administration of antiviral therapy was obtained from chart review in the clinical information system. the positive results of viral agents were tabulated daily. the study was approved by the institutional review board of seattle children's hospital, seattle, wa. respiratory virus testing with filmarray was initiated on december 14, 2011. during the 4-month period ending april 19, 2012, a total of 2,537 specimens were tested. seventy-one percent of the samples were from the ed or urgent care. the daily testing volume varied from 3 to 44 ❚figure 1❚. the average daily volume for the first 2 months and second 2 months were 15 and 25, respectively. because the filmarray instrument is designed for single-specimen throughput and requires 65 minutes of instrument time, we purchased 3 instruments that could run simultaneously 24 hours a day. the average and median tat was 1.6 and 1.4 hours, respectively, with 82% completed in less than 2 hours and 95% in less than 3 hours ❚table 1❚. before this molecular test was available, we sent respiratory viral testing to an on-site reference laboratory for dfa. dfa testing involved multiple handoff steps in our laboratory and specimen transport to the reference laboratory. dfa specimens were run in batches 3 or 4 times per day depending on the volume during respiratory virus season. the testing time was approximately 3 hours. during the same period 1 year previously, 1,399 dfas for respiratory viruses were performed. the average and median tat for dfa was 7 and 6.5 hours, respectively, with 2% completed in less than 3 hours (table 1 ). the test volume of respiratory virus during the study period was almost doubled compared with the same period last year. during the study period, the instrument failed 4 times. the rate of failed testing, which included quality control failures, insufficient vacuum in the pouch, and pouch leaking, was 1.3%. the tat was significantly longer when instruments or reagent pouches failed. during the study period, 63% of all samples tested positive for viral agents. rhinovirus/enterovirus and rsv were j a n u a r y 4 j a n u a r y 7 j a n u a r y 1 0 j a n u a r y 1 3 j a n u a r y 1 6 j a n u a r y 1 9 j a n u a r y 2 2 j a n u a r y 2 5 j a n u a r y 2 8 j a n u a r y 3 1 j a n u a r y 3 f e b r u a r y 6 f e b r u a r y 9 f e b r u a r y 1 2 f e b r u a r y 1 5 f e b r u a r y 1 8 f e b r u a r y 2 1 f e b r u a r y 2 4 f e b r u a r y current guidelines for treating patients with influenza a or b infection with oseltamivir indicate that the medication should be given within 48 hours of symptom onset to be most effective. in general, children with respiratory symptoms may not seek or be brought to medical attention immediately. a delay in laboratory testing may further prolong the interval between symptom onset and the administration of medication. for treatment benefits, we specifically followed patients (n = 97) who tested positive for influenza a or influenza b admitted to the ed during march 2012. the mean and median length of stay in the ed during the study period was 3.1 and 2.8 hours, respectively. in these 97 patients, respiratory viral results provided by filmarray were available for 44 patients (45%) before they were discharged from the ed. in 50 patients (52%), positive results were called within 3 hours after discharge. the remaining 3 patients (3%) were notified of the results in more than 3 hours. of the 97 patients, 22 patients (23%) were treated with oseltamivir in the ed, and 30 patients (31%) were given the prescription while they were in the ed. the prescription was called in to another 27 patients (28%) within 3 hours after discharge from the ed. the remaining 18 (19%) patients received no prescription for oseltamivir. overall, 79 patients (81%) with influenza virus were given oseltamivir in a timely manner-defined as receiving the drug in the ed, a prescription in the ed, or a prescription within 3 hours of ed discharge. among the 18 patients who did not receive prescriptions, 6 reported having symptoms for more than 48 hours before the ed visit, 4 patients showed improvement of symptoms according to parents, 4 had a follow-up appointment, 2 received physicians' decisions not to treat, and no information was available for 2 patients. the daily tally of all positive results during the respiratory virus season clearly demonstrated the epidemiology of the multiple respiratory viral agents present in symptomatic children receiving care at our institution ❚figure 2❚ and contributed to both hospital-wide as well as community-wide public health information. unusually, influenza a and b did not emerge until later in the season at the end of february and peaked in april. before the emergence of influenza viruses, rsv was the major infectious agent, with a peak noted in mid-february. we also found that 1 patient who received live attenuated influenza vaccine less than 7 days before testing was positive for both influenza a and b; in retrospect, we would not recommend viral testing in patients recently immunized with live vaccine. molecular testing for respiratory viruses has become widely available and rapidly deployed in diagnostic laboratories during the past few years. therefore, the critical operational decision involves which system to choose to meet patient care needs while integrating smoothly into a laboratory's infrastructure. in general, molecular tests for respiratory viruses have to be conducted in batches in specialized molecular, microbiology, or virology laboratories that rarely offer testing during evening and night shifts. the tat for most molecular tests is also relatively long, ranging from 6 to 8 hours. filmarray is a recent us food and drug administration (fda)-approved pcr method for detecting 15 respiratory viral agents. recently, idaho technology received fda approval for 5 additional respiratory viral and bacterial agents including coronavirus 229e, coronavirus oc43, bordetella pertussis, chlamydophila pneumoniae, and mycoplasma pneumoniae. it is simple and fully automated (sample in, result out), with a 65-minute tat and a single-piece throughput platform. we implemented filmarray in our core laboratory and ran the test 24/7 during this respiratory viral season. we started with 2 filmarray instruments. as the volume of tests increased, a third instrument was added and then a fourth one was made available to us as a backup. the redundancy of our instrumentation allowed the laboratory to continue to perform testing if 1 or more instruments were not in operation. implementing filmarray in the core laboratory significantly reduced the tat of respiratory virus test reporting. in addition, the filmarray respiratory viral panel includes more viral agents that were not included in previous testing using dfa. although current treatment for respiratory viral infection is limited to influenza a and b, detection of other viral agents is valuable because clinical suspicion of viral respiratory tract infections can be confirmed, additional workup and therapy can be avoided, and clinicians and parents can be reassured. in addition to early treatment of patients with influenza virus, early detection of the infectious agent is important to place the patients into appropriate isolation cohorts. because of its rapid tat, filmarray testing for respiratory viruses was also used to place patients into cohorts effectively. in fact, 20% of the respiratory viral tests we performed were primarily for the purpose of patient isolation and forming cohorts. in 2 cases in which patients were admitted for urgent surgery, the rapid respiratory viral testing quickly ruled out influenza infection so that the surgery did not have to be performed under strict isolation procedures using masks and negative pressure in the operating room. the purchase price of a filmarray respiratory viral pcr panel was $109 per reagent pouch after discount (list price, $129). adding 1% to 2% of pouch failure rate and additional quality control testing, the cost was approximately $115 per test. the test took approximately 5 minutes to perform, which was about the same amount of time required for packaging a sample to be sent to a reference laboratory. we did not add any fte in the core laboratory with the addition of the test. the cost of sending a sample to the reference laboratory for respiratory viral testing by dfa was $98.26. the actual price of filmarray was only slightly higher than that previous spent on respiratory viral testing. for the dfa testing performed in previous years, nasal wash was the preferred specimen type. the collection of a nasal wash sample resulted in the generation of aerosols, and therefore procedure rooms had to be closed for 30 minutes before the rooms could be used again. replacing the nasal wash with a midturbinate swab increased efficiency by eliminating the 30-minute room closures. because 71% of the respiratory viral testing samples were collected from the ed, we potentially saved 900 hours of ed room for the 2,537 respiratory viral tests performed. the cost savings could not be accurately calculated. however, in 1 report it was found that a 1-hour reduction in ed boarding time would result in $9,693 to $13,298 of additional daily revenue. 18 in this report the calculation included patients who left without being seen, which represented 7% of all ed visits. the rate of patients who left without being seen at our institution is 0.6%, so opportunity cost savings in our institution are probably lower. the major drawbacks of filmarray are single-sample throughput, the relatively low sensitivity of detecting adenovirus, 11 and the inability to separate rhinovirus and enterovirus. furthermore, we were unable to quantify viral load, which may also be important in some patients. in addition, the high detection rate of rhinovirus cannot be necessarily interpreted as high rates of acute rhinovirus infection because prolonged rates of rhinovirus shedding have been reported. finally, because there is no interface with our computerized data entry system currently, we required 2 technologists to verify the reported results for clerical error reduction. with this peer review verification reporting process we had only 1 data entry error, which was corrected without affecting patient care. the error rate related to data entry was less than 0.04%. in summary, the implementation of the filmarray respiratory panel in our core laboratory significantly decreased the time required to detect and report respiratory viruses. patients with influenza a and b were treated rapidly and appropriately, which had not been possible using dfa. detection of other viral agents assisted physicians in the differential diagnosis of respiratory syndromes and isolation of patients admitted to the hospital. overall, we implemented a molecular-based efficient diagnostic test in our core laboratory for the first time, marking a new era in pediatric clinical laboratory medicine. multiplex pcr for detection of respiratory viruses: can the laboratory performing a respiratory viral panel (rvp) assay trigger better patient care and clinical outcomes? the effect of rapid respiratory viral diagnostic testing on antibiotic use in a children's hospital performance of a rapid antigen test (binax now® rsv) for diagnosis of respiratory syncytial virus compared with real-time polymerase chain reaction in a pediatric population comparison of the performance of direct fluorescent antibody staining, a point-of-care rapid antigen test and virus isolation with that of rt-pcr for the detection of novel 2009 influenza a (h1n1) virus in respiratory specimens diagnostic tests for influenza and other respiratory viruses: determining performance specifications based on clinical setting performance of influenza rapid point-of-care tests in the detection of swine lineage a(h1n1) influenza viruses. influenza other respir viruses multicode-plx system for multiplexed detection of seventeen respiratory viruses diagnostic assays for respiratory syncytial virus disease comparison of the luminex xtag respiratory viral panel with xtag respiratory viral panel fast for diagnosis of respiratory virus infections an automated nested multiplex pcr system for multi-pathogen detection: development and application to respiratory tract infection comparison of the filmarray respiratory panel and prodesse real-time pcr assays for detection of respiratory pathogens comparison of two multiplex methods for detection of respiratory viruses: filmarray rp and xtag rvp comparison of the luminex xtag rvp fast and the idaho technology filmarray rp assays for the detection of respiratory viruses in pediatric patients at a cancer hospital comparison of two broadly multiplexed pcr systems for viral detection in clinical respiratory tract specimens from immunocompromised children comparison of the idaho technology filmarray system to real-time pcr for detection of respiratory pathogens in children application of the toyota production system improves core laboratory operations comparison of real-time pcr assays with fluorescent-antibody assays for diagnosis of respiratory virus infections in children the financial consequences of lost demand and reducing boarding in hospital emergency departments key: cord-256827-tht5h1tu authors: jain, neemisha; lodha, r.; kabra, s. k. title: upper respiratory tract infections date: 2001 journal: indian j pediatr doi: 10.1007/bf02722930 sha: doc_id: 256827 cord_uid: tht5h1tu acute respiratory infections accounts for 20–40% of outpatient and 12–35% of inpatient attendance in a general hospital. upper respiratory tract infections including nasopharyngitis, pharyngitis, tonsillitis and otitis media constitute 87.5% of the total episodes of respiratory infections. the vast majority of acute upper respiratory tract infections are caused by viruses. common cold is caused by viruses in most circumstances and does not require antimicrobial agent unless it is complicated by acute otitis media with effusion, tonsillitis, sinusitis, and lower respiratory tract infection. sinusitis is commonly associated with common cold. most instances of rhinosinusitis are viral and therefore, resolve spontaneously without antimicrobial therapy. the most common bacterial agents causing sinusitis ares. pneumoniae, h. influenzae, m. catarrhalis,s. aureus ands. pyogenes. amoxycillin is antibacterial of choice. the alternative drugs are cefaclor or cephalexin. the latter becomes first line if sinusitis is recurrent or chronic. acute pharyngitis is commonly caused by viruses and does not need antibiotics. about 15% of the episodes may be due to group a beta hemolytic streptococcus (gabs). early initiation of antibiotics in pharyngitis due to gabs can prevent complications such as acute rheumatic fever. the drug of choice is penicillin for 10–14 days. the alternative medications include oral cephalosporins (cefaclor, cephalexin), amoxicillin or macrolides. acute respiratory infections are a major cause of morbidity and mortality in children and of particular significance in developing countries like india. outpatient attendance attributed to acute respiratory infections is as high as 20-40% of all outpatients and 12-35% of in patients. 1 in a study in rural zone of delhi with an estimated population of 3700, the prevalence of acute respiratory infection was 12.1% for the under-fives as a whole. 2 the overall incidence of acute respiratory infection in the under-fives was observed to be 2.5 episodes per child per year, of which, 2.2 episodes per year per child were upper respiratory tract infections. upper respiratory tract infections including nasopharyngitis, pharyngitis, tonsillitis and otitis media constituted 87.5% of the total episodes of infection. lower respiratory tract infections including pneumonia and bronchiolitis constituted the remaining 12.5%. 2 in a survey of acute respiratory infection in a rural area of south india, the prevalence of acute respiratory infection was 7.6 % in a total of 10,951 children below 5 years. majority of acute respiratory infection was mild (defined by cough + respiratory rate <50) only 1.7% episodes were severe in nature (cough + respiratory rate >50 + chest indrawing/inability to drink). 3 thus, acute respiratory infection constitutes a significant problem in childhood and the majority of these infections are upper respiratory infection. bacterial this bring forth the fact that, majority of respiratory infections on children are due to a viral etiology which do not need antibiotics for management and judicious use of antibiotics is most imperative in upper respiratory tract infections. the principles of judicious use of antibiotics in upper respiratory tract infections outlined below are based on recent reviews. most children have 3-8 episodes of common cold per year. 9-1~ common cold is a viral illness, the most common cause being rhinovirus; others being parainfluenza, respiratory syncytial, corona virus, adenovirus, echovirus, coxsackievirus and parainfluenza virus, n rhinovirus accounts for upto 60% of infections. 12 the etiological agents vary with host age, and time of the year. symptoms include nasal discharge, nasal obstruction, and throat irritation. associated with this there usually is lowgrade fever, malaise, sneezing and nasal secretions can become purulent. symptoms are non-specific and not characteristic for any agent. the usual duration of symptoms is about 7 days. common cold in children resolves on its own and no specific therapy is indicated in majority of cases. use of antibiotics does not change either the course or the outcome. 6 on the contrary antibiotic use is potentially harmful, because it increases the risk of colonization with resistant organisms. this may result in subsequent bacterial infection being unresponsive to standard antibiotics. 13 common cold almost always has an excellent outcome with complete recovery, but at times complications can occur. these include acute otitis media with effusion, tonsillitis, sinusitis, and lower respiratory tract infection. '~ unless specific diagnostic criteria for the diagnosis of these complications are met, antibiotics should not be used for the signs and symptoms of cold alone? studies have shown that use of antibiotics for the prevention of bacterial complication of common cold has no use. a meta-analysis of five randomized clinical trials of efficacy of antimicrobial treatment of cold to prevent lower respiratory infection found that it had no protective effect. it also did not shorten the duration of the upper respiratory illness. 14 recognizing the signs and symptoms of the usual course of common cold will help to prevent the overprescription of antibiotics. unfamiliarity with them can lead to the mistaken diagnosis of secondary bacterial infection and the inappropriate use of antibiotics. mucopurulent nasal dicharge, which is thick, opaque at times greenish or yellowish frequently accompanies common cold. n antimicrobial treatment is not indicated for such a discharge unless it persists for more than 10-14 days. 6 in a study on 142 children with mucopurulent nasopharyngitis, a comparison of cephalexin treatment with placebo was done. five to six days after initiation of treatment, cephalexin and placebo groups did not differ in the presence of nasal discharge, incidence of complications, or parental opinion of benefit of medicationy cough occurs in 60-80% of common cold and does not suggest a bacterial etiology. tm duration of symptoms is usually 2-7 days. patients usually improve within 10 days, but at times cough and nasal discharge can persist for 2 weeks or morey to summarize 1. antibiotics should not be given for common cold. 2. mucopurulent nasal discharge is feature of common cold and is alone not an indication of antibiotics unless it persists for more than 10-14 days. sinusitis is defined as inflammation of the mucosal lining of one or more of the paranasal sinusesy it can occur from infection or non-infectious (allergic) causes. in children sinusitis almost always occurs as a complication 1136 of common cold. n even in uncomplicated viral upper respiratory infection, as occurs in common cold, there is inflammation and congestion of both the nasal and the sinus mucosa. these infections should, therefore, be considered as rhinosinusitis. 19 this inflammation undergoes a spontaneous resolution in vast majority of cases. about 0.5 to10 percent of upper respiratory infections are complicated by development of acute sinusitis. tm since only a small percentage of children with symptoms of rhinosinusitis have a bacterial etiology, the use of appropriate diagnostic criteria is essential, as this will avoid the overuse of antibiotics. this is all the more important because viral rhinosinusitis is 20 -200 times more common than bacterial sinusitis. 7 sinusitis is classified as per the duration of symptoms into acute (upto 3 weeks), subacute (3-10 weeks), and chronic (>10 weeks). sinusitis is almost always precipitated when a viral upper respiratory infection produces mucosal inflammation leading to obstruction of the sinus ostia. this in turn leads to fluid trapping in the sinus cavities and the normal upper respiratory bacterial flora proliferates. the most common organisms causing sinusitis are s. pneumoniae, h. influenzae, m. catarrhalis, s. aureus and s. pyogenesj ~ but it must be emphasized that majority of rhinosinusitis is viral and therefore, approximately 60% resolve spontaneously without antimicrobial therapy. 2~ the major problem in the diagnosis of sinusitis in children is to distinguish simple upper respiratory tract viral infections and allergic inflammation from bacterial infection of the sinuses. the signs and symptoms in older children can be classical i.e. sinus tenderness, tooth pain, headache, and high fever. however, in young children these classical sign and symptoms are almost never present. persistence of signs and symptoms of upper respiratory infection i.e. rhinorrhea or cough for more than 10-14 days is a pointer towards sinusitis. the rhinorrhea mostly is purulent but can be serous or watery. thus, the character of the discharge is not helpful in distinguishing infected sinus fluid, from uninfected fluid. 21 other indicators of sinusitis are the presence of severe signs and symptoms during an episode of upper respiratory infection. these include high fever > 39 ~ c, persistent fever, periorbital swelling and facial pain. diagnosis of sinusitis is clinical and rarely are investigations warranted. radiographic evaluations are indicated when episodes of sinusitis are recurrent, when complications are suspected or when the diagnosis is not clear. 7 the plain radiograph shows air fluid level, opacification, or mucosal thickeningy ct scan or mri can be done for a more detailed examination and show the same findings. these features can also be present in viral rhinosinusitis and therefore, x-ray should only be done when indicated. in a study evaluating ct findings in 31 adults during first 48-96 hours of uncomplicated viral respiratory illnesses, 90% had abnormality of the upper respiratory tract infection maxillary sinus cavity. after two week these findings resolved in 79% even though none received any antibiotics? 9 when the child is younger than six months of age, opacification of the ethmoid air cells is normal. when the child is under the age of one year, opacification or thickening of the mucosa of the maxillary sinusitis may be normal. 11 this should be kept in mind when evaluating xray findings. the primary treatment of sinusitis is antibiotics once the diagnostic criteria have been met. amoxycillin (40 mg/kg/day) is successfij1 for the initial treatment of acute uncomplicated sinusitis in most children. 2~ about 35% of nontypable h. influenzae and 85% of m. catarrhalis strains produce beta-lactamases and are resistant to amoxicillin. 21 therefore, if there is no response within 48 hours a beta-lactamase-stable antibiotic like amoxycillinclavulanate or cephalexin or cetactor should be considered. these drugs should be considered first line if sinusitis is recurrent, fails to improve, or is a severe infection with high fever and facial swelling. the duration of therapy should be minimum of 10 days. 22 to summarize, 1. diagnosis of sinusitis should be made when symptoms of nonspecific upper respiratory infection are present for more than 10-14.days or there are severe upper respiratory signs and symptoms in the form of facial pain, facial swelling and high fever. 2. since radiological evidence of sinusitis may be seen even in common cold, radiographs should be interpreted with caution and done when indicated. 3. antimicrobial for sinusitis should be narrow spectrum against the likely pathogen. pharyngitis is an inflammatory illness of the mucous membranes and underlying structures of the throat. the clinical diagnostic category includes tonsillitis, tonsillopharyngitis and nasopharyngitis. pharyngitis is subdivided into two categories: illness with nasal symptomatology (nasopharyngitis) and illness without nasal involvement (pharyngitis or tonsillopharyngitis). nasopharyngitis nearly always is a viral etiology, whereas pharyngitis without nasal signs has diverse etiologic possibilities including bacteria, viruses, fungi and other infectious agents. adenoviruses are the most common cause of nasopharyngitis, other viruses being influenza, parainfluenza and enteroviruses. the bacterial agents causing pharyngitis include streptococcus pyogenes, h. influenzae, c. diphtheriae, and n. meningitides. 23 group a streptococci accounts for approximately 15% of bacterial pharyngitis and about 1-2 patients out of 10 with sore throat. infection in most of the others is attributable to viruses, u early diagnosis and treatment of group a streptococcal pharyngitis is important, as antibiotics initiated with nine days of onset are effective in preventing acute rheumatic fevery but since most sore throats are viral in etiology antibiotic have to be given only when diagnosis of group a streptococcal pharyngitis is certain. this will prevent the unnecessary overuse of antibiotics. symptoms of sore throat accompany pharyngitis but its presence alone should not be used for diagnosis as these complain can be present even in common cold. the objective evidence of inflammation in the form of erythema, exudate or ulceration in the pharynx is necessary for the diagnosis. but, these signs can also be present in some viral infections like adenovirus or enterovirus. the classical features of group a streptococcal pharyngitis include acute onset of pharyngeal pain, dysphagia and fever. rhinorrhea, cough, hoarseness, conjunctivitis and diarrhea suggest a viral etiology. presence of patchy exudate on the posterior pharynx, palatal petechie and enlarged and tender anterior cervical lymph nodes favour group a streptococci pharyngitis. but in an individual child the clinical distinction between streptococcal pharyngitis and viral pharyngitis is unreliable. therefore, in all cases of acute pharyngitis, streptococcal disease must be considered. diagnosis of group a streptococcal pharyngitis should be based on throat swab culture, which is the standard for diagnosis. antigen-detection tests alone can miss some cases; therefore the recommendation is to do a culture if antigen-detection test is negative in a child with suspected streptococcal pharyngitis. 26 furthermore, if antibiotics have been started in a child with suspected streptococcal pharyngitis and subsequently, the cultures are negative the treating physician should inform the parents to stop the antibiotic therapy. some studies have shown that early initiation of antibiotic on clinical suspicion can result in decreased desirable antibody response, thus allowing reinfection with type specific organisms. 27 bacteria other than group a streptococci are rare causes of pharyngitis. moreover, sequelae such as acute rheumatic fever do not occur. also, as already stated most pharyngitis in children is viral in etiology. therefore, antibiotics should not be given to a child with pharyngitis in the absence of diagnosed group a streptococcal pharyngitis or other bacterial infection. the recommendation is to use 10-day course of penicillin for the treatment of group a streptococcal pharyngitis. this is because it has a narrow spectrum of activity, low cost and high efficacy. group a streptococcal resistance to macrolide antibiotics has been observed, whereas, resistance to beta-lactam antibiotics has not been detected to date. resistance to macrolides like azithromycin or clarithromycin would be similar to that for erythromycin. a meta-analysis of 19 studies favoured cephalosporins over penicillin in terms of higher eradication of gabs from oropharynx. 28,29 these agents have a wide spectrum of activity against bacteria and thus encourage the emergence of resistance over a broad range of bacterial pathogens. alternative treatments must be used in patients with penicillin allergy, compliance issues or penicillin treatment failure. patients who do not respond to initial treatment should be given an antimicrobial that is not inactivated by penicillinaseproducing organisms (e.g., amoxycillin-clavulanate potassium, cefaclor, cephalexin or a macrolides). to summarize 1. group a streptococcal pharyngitis should be diagnosed using laboratory tests with clinical and epidemiological findings. 2. antibiotics should be given to a child with pharyngitis only when group a streptococcal or other bacterial infection has been identified. 3. penicillin is the drug of choice for group a streptococcal pharyngitis. acute respiratory infection magnitude of acute respiratory infection in under five acute respiratory infection in children-a survey in the rural community an indian hospital study of viral causes of acute respiratory infections in children resistant pneumococci: protecting patients through judicious antibiotic use the common coldprinciples of judicious use of antimicrobial agents principles of judicious use of antimicrobial agents for pediatric upper respiratory infections acute sinusitisprinciples of judicious use of antimicrobial agents pharyngitis-principles of judicious use of antimicrobial agents frequency and severity of infections in daycare the epidemiology, pathogenesis, and treatment of the common cold infections of the upper respiratory tract respiratory viral infection in childhood. a survey in general practice, rohanpton. 1967-1972 potential intervention for preventing pneumonia among young children : lack of effect of antibiotic treatment for upper resiratory infections bacteriology and treatment of purulent nasopharyngitis: a double blind, placebo controlled evaluation present concepts of the common cold the coughing child. etiology and treatment of a common symptom textbook of pediatric infectious diseases computed tomographic study of the common cold childhood sinusitis; pathophysiology, diagnosis and treatment resistance among problem respiratory pathogens in pediatrics sinusitis in children pharyngitis (pharyngitis, tonsillitis, tonsillopharyngirls, and nasopharyngitis) diagnosis and treatment of group a streptococcal pharyngitis rheumatic heart disease-a challenge red book report of the committee on infectious diseases adverse and beneficial effects of immediate treatment of group a-betahemolytic streptococcal pharyngitis with penicillin comparison of cephalosporin and penicillins in the treatment of group a beta-hemolytic streptococcal pharyngitis: a metaanylysis supporting the concept of microbial copathogenicity judicious use of antibiotics for common pediatric respiratory infections key: cord-266516-0ure8256 authors: lim, tow keang; siow, wen ting title: pneumonia in the tropics date: 2017-08-01 journal: respirology doi: 10.1111/resp.13137 sha: doc_id: 266516 cord_uid: 0ure8256 pneumonia in the tropics poses a heavy disease burden. the complex interplay of climate change, human migration influences and socio‐economic factors lead to changing patterns of respiratory infections in tropical climate but also increasingly in temperate countries. tropical and poorer countries, especially south east asia, also bear the brunt of the global tuberculosis (tb) pandemic, accounting for almost one‐third of the burden. but, as human migration patterns evolve, we expect to see more tb cases in higher income as well as temperate countries, and rise in infections like scrub typhus from ecotourism activities. fuelled by the ease of air travel, novel zoonotic infections originating from the tropics have led to global respiratory pandemics. as such, clinicians worldwide should be aware of these new conditions as well as classical tropical bacterial pneumonias such as melioidosis. rarer entities such as co‐infections of leptospirosis and chikungunya or dengue will need careful consideration as well. in this review, we highlight aetiologies of pneumonia seen more commonly in the tropics compared with temperate regions, their disease burden, variable clinical presentations as well as impact on healthcare delivery. pneumonia continues to be an important cause of death and accounts for 16% of all deaths in children under the age of 5. 1 the disease burden, especially in children, is heaviest in south asia and sub-saharan africa. 1 these regions have tropical climates, which are characterized by a hot climate present all year round, whilst large volumes of rainfall each year result in the spread of zoonotic diseases. besides environmental influences, socio-economic factors also impact heavily on the epidemiology of tropical diseases. the tropics, which is the geographical region of the earth centred on the equator and limited by the tropic of cancer on the north and the tropic of capricorn on the south, is also home to some of the poorest nations. together with the movement of people triggered by globalization, mass migrations and climate change, 2 these dynamics have a profound effect upon the patterns of respiratory tract infections in the tropics. global temperatures have risen through the past decades due to greenhouse gas emissions, and we have also seen a rise in a number of extreme meteorological events such as tropical super typhoon haiyan in 2013. 3 warmer temperatures and altered rainfall patterns are anticipated to promote outbreaks of infectious diseases due to more hospitable environments for pathogens and lack of ready access to health care. 4 prevalence and mortality from pneumonia in children are the highest during rainy months in tropical and subtropical regions of asia and africa, which again highlights the pattern of pneumonia against climate. 5 global warming and climate change also result in an increase in average as well as nadir temperatures, even in temperate regions. 2, 6 hence, areas that were previously free from tropical diseases may now encounter such entities. this is shown by an emergence of tropical diseases in temperate regions, such as an outbreak of leptospirosis in 1998 in springfield, illinois in the united states, 7 and an outbreak of human pneumonic plague in colorado in 2014. 8 natural disasters have been linked to disease outbreaks including pneumonia, due to an increased risk of water-, air-and vector-borne diseases. in the post-disaster period, phase 1 is the impact phase (0-4 days) where victims are rescued and immediate treatment is provided for disaster-related injuries. phase 2 which is the postimpact phase (4 days to 4 weeks post-disaster) is the period when the first surge of infectious diseases may surface, and phase 3 the recovery phase (after 4 weeks) when symptoms of diseases with longer incubation periods may declare. 9 examples of such diseases are influenza and leptospirosis. 9 a tertiary hospital reported an increase in patient admissions for pneumonia and tuberculosis (tb) in the aftermath of typhoon haiyan especially in the impact and post-impact phases, 3 and the entity 'tsunami lung' 10 has been described in victims who have pneumonia following a near-drowning episode after surviving a tsunami event. victims described by inoue et al. 10 were rescued immediately after the tsunami and were in respiratory distress. the term tsunami lung describes both pneumonia from various organisms (such as stenotrophomonas maltophilia, burkholderia cepacia and pseudomonas aeruginosa), 10 as well as severe systemic after-effects such as disseminated aspergillosis. 11 there are no clear differences in viral aetiologies of pneumonia when comparing tropical with temperate climates. [12] [13] [14] [15] [16] different authors have found varying impacts of precipitation on viral pneumonias. [17] [18] [19] respiratory syncytial virus respiratory syncytial virus (rsv) is a common causative pathogen in respiratory tract infections and was the most commonly isolated virus in children with respiratory tract infections in a malaysian study, accounting for 70.6% of patients. 20 in adults, most manifestations are in the upper respiratory tract, although about 10% will develop pneumonia. 21 the virus is detected using polymerase chain reaction methods, immunofluorescent or immunoassay antigen detection, cultures or serology analysis. treatment is mainly supportive. aerosolized ribavirin can be used as a specific antiviral therapy for rsv, but this is less well studied in adults compared with infants. 22 influenza a influenza, which remains a global health burden, displays seasonality. a recent study of viral infectious patterns according to time of the year in a singaporean medical intensive care unit by siow et al. 16 showed peaking of influenza cases around the start and middle of the year. in this study, the most common viral isolate was the influenza a h1n1/2009 virus, followed by human rhinovirus. the seasonal distribution of influenza a in this study echoes the results of tang et al. who found the incidence of influenza a in singapore to peak during january and june to july period, 23 as well as chew et al. 24 who noted two peaks during the november to january and june to july period. in singapore, months with heaviest rainfall are clustered around year-end (october to december), and drier months are clustered around mid-year, in june and july. pharmacological treatments include antivirals such as oseltamivir and zanamivir to help shorten the duration of illness if administered within 48 h of onset of illness. 25 the seasonality of influenza therefore cannot be fully explained by rainfall alone. multiple contributory reasons have been hypothesized, including host behaviour such as spending more time indoors during adverse weather conditions, and altered host defences, but uncertainties remain. 26 we must consider the complex relationship of climate and human behaviour when determining patterns, not just for influenza, but for other respiratory viruses as well. the h5n1 strain of avian influenza was the causative agent of an influenza pandemic in asia in 1997. prevalent in poultry and wild birds, animal-to-human transmission occurs to cause a spectrum of pneumonia/ pneumonitis, culminating in acute respiratory distress syndrome (ards). as of 20 april 2017, the world health organization (who) recorded a total of 858 confirmed human cases of h5n1 with 453 deaths with a 53% mortality rate. 27 as recently as october 2016, who was notified of the influenza a (h7n9) virus outbreak in china. 28 since then, it has been noted to have an increased number of cases in december and january. 29, 30 the major risk factor of infection was live poultry exposure. as live poultry markets are commonplace in china, and with chinese new year festivities the consumption of poultry in the populace will increase, there will be a higher risk of continued exposure leading to sporadic infections or small clusters of human cases. 30 hantavirus is associated with the hantavirus pulmonary syndrome (hps). its manifestation is as a rapidly progressing non-cardiogenic pulmonary oedema and can mimic that of a severe pneumonia clinically and radiologically. 31 hps was first discovered in 1993 in the southwestern united states, and since then has been described in latin america as well. 32 risk factors are exposure to rural activities and rodents, and treatment is largely supportive. other viruses associated with pneumonias are the corona viruses, such as the middle eastern respiratory syndrome corona virus (mers-cov) and severe acute respiratory syndrome corona virus (sars-cov). these entities are beyond the scope of discussion of this paper, but suffice to say their impact on global health has been daunting given 690 confirmed deaths out of 1936 confirmed mers-cov infections 33 and more than 8000 cases of sars-cov, of which a large proportion were concentrated within asia (in particular, china, hong kong and taiwan). 34 there are differences between causative organisms encountered in the tropics compared with temperate climates. due to both environmental and socioeconomic factors, diseases such as melioidosis, leptospirosis and tb are more widespread in the tropics-these will be discussed later. observation of the aetiologies of community-acquired pneumonia (cap) in an asian outpatient setting showed that the most common isolates were chlamydophila pneumoniae, followed by mycoplasma pneumoniae and streptococcus pneumoniae. 35 that same systematic review by peto et al. 35 identified s. pneumoniae as the most common pathogen in hospitalized patients with cap. although there was great variation in terms of proportion between countries in this study, frequency was similar to the trends observed in european studies quoted in the review and also comparable to findings by siow et al. 16 interestingly, the most common gram-negative bacillus isolated in the studies was klebsiella, with higher proportions being reported in india and southeast asia. the authors found that asian patients with cap requiring hospitalization yielded a larger proportion of gramnegative bacilli (9.0% vs 2.7%) and staphylococcus aureus (4.0% vs 1.4%) isolates compared with referenced european studies. 36 however, there was no comparison made between the asian countries, as certain countries such as korea and japan experience a temperate climate compared with the tropical climates of thailand and malaysia. 35 siow et al. found the top two causative pathogens to be s. pneumoniae and klebsiella pneumoniae in a recent study looking at bacterial isolates from severe cap patients in a singaporean medical intensive care unit. 16 streptococcus pneumoniae was the main grampositive bacterium isolate, and s. aureus was the next common gram-positive organism. otherwise, gramnegative organisms such as k. pneumoniae, escherichia coli and p. aeruginosa represented the majority of cases detected. similarly, lin et al. 37 described k. pneumoniae not only as a prevailing cause of cap with bacteraemia in a taiwanese tertiary hospital, but also showed that it was associated with a more fulminant clinical course and worse prognosis when compared with patients with s. pneumoniae cap with bacteraemia. in a series of severe cap cases in singapore, patients who had gram-negative organisms isolated tended to have a worse outcome including a higher mortality, especially for patients with pseudomonas and burkholderia pseudomallei infections. 38 this has changed the way local clinicians initiate their empiric treatment for patients admitted for severe cap, with antibiotics deliberately chosen to cover gram-negative organisms, melioidosis as well as gram-positive pathogens. a systematic review by goyet et al. 39 looking at resistance patterns of cap pathogens in cambodia and neighbouring countries showed that up to 14% of s. pneumoniae and 26.5% of k. pneumoniae were resistant to amoxicillin-clavulanic acid. streptococcus pneumoniae also displayed a high resistance to trimethoprim/sulfamethoxazole (average of 78.2%) and wide range of resistance patterns to cephalosporins: between 5.7% and 33.3% to ceftriaxone, and up to 47.4% to cefuroxime. there was also a mean high-level resistance rate to penicillin g of 25.2%. burkholderia pseudomallei did not show resistance to first-line treatments ceftazidime, carbapenems and trimethoprim/sulfamethoxazole. as a result of this study, the authors have advocated the preservation of fluoroquinolones as they are not warranted as first-line therapy, and they are also used to treat tb, which importantly is endemic in this region. 39 this highlights the importance of continued surveillance of regional resistance patterns and revision of therapeutic guidelines. melioidosis was first described by krishnaswami and whitmore in 1911, when they noticed a 'glanders-like' disease afflicting opiate addicts in rangoon. 40 today, melioidosis still poses a threat to public health due to mortality rates up to 40% 41 if early treatment is not instituted. in an endemic country like thailand, it can account for up to 32% of the pathogens identified in adult patients with pneumonia. 42 currie and kaestli 41 estimated global mortality from melioidosis in 2015 to be 89 000, comparable with deaths from measles, and higher than those from dengue and leptospiral disease. the causative pathogen, b. pseudomallei, is a gramnegative rod that is found in soil and fresh water. it occurs mainly in northern australia, southeast asia, china and south asia with increased incidences during rainy seasons. this is in contrast to temperate countries where melioidosis is extremely rare and almost always encountered in migrants or travellers. 43 presentation of melioidosis can be either acute or subacute. acute illnesses usually present with pneumonia which can be associated with ards and shock. subacute presentations may take a more insidious course, mimicking tb. there is also a propensity for the pathogen to spread haematogenously, and patients may present with extrapulmonary manifestations such as solid organ and skin abscesses, and even septic arthritis and encephalomyelitis. diagnosis of melioidosis is confirmed on positive cultures. with potential mortality rates approaching 40%, 41 a clinician's suspicion must be high when faced with a patient with severe cap coupled with an appropriate travel history. in the local context, because of being in an endemic region, intensive care units including those in singapore have adopted empirical antibiotic treatment to include specific coverage for melioidosis (ceftazidime and meropenem would be appropriate) for patients admitted with severe cap. 38, 44 indeed, in a prospective study over 20 years in darwin, mortality rates have improved over time (22% described by the authors) with better recognition of the disease as well as early treatment with appropriate antibiotics. 45 leptospirosis is a zoonotic disease prevalent in the tropics, with a much higher incidence than in temperate regions. within the asia pacific region, high-incidence countries include thailand, bangladesh and cambodia. there are also certain regions such as korea and china where leptospirosis incidences, although low, are increasing. 46 leptospira are aerobic spirochetes. both feral and domesticated animals can host the diseasecommonly, dogs, cattle, rodents, swine, but interestingly, and rarely in cats. human infection typically occurs after contact with contaminated urine, animal tissue, water or soil. in the tropics, it especially affects low-income regions with poor sanitation, low education and poor housing, where outbreaks are common and morbidity is high. even in higher income regions, heavy rainfall leading to flooding increase the risk of both humans and livestock exposure to contaminated water. for example, there was an outbreak in anuradhapura, sri lanka. anuradhapura is a region with abundant paddy fields for rice farming and was not previously known as an endemic area, so the diagnosis and outbreak of leptospirosis which followed flooding was initially challenged by local clinician. 47 recreational events such as caving, canoeing and freshwater swimming could expose humans to contaminated sources. an example highlighting the impact of these activities would be the 1998 leptospirosis outbreak in springfield, illinois, involving triathletes who were exposed to lake water. 7 clinical features are variable. it can take a subclinical, self-limited course, or can progress to severe and potentially life-threatening illness complicated by jaundice, renal failure and ards, with reported mortality rates up to 30%. 48 typical presenting complaints include fever, myalgia, headaches and conjunctival insufflation. cough, nausea, vomiting and diarrhoea are common. dall'antonia et al. described cough and haemoptysis in patients with serologically proven leptospirosis. 49 severe forms of the disease with multiorgan dysfunction and ards-like syndromes may be fatal. 50 chest roentgenogram findings are non-specific; they commonly show non-segmental patchy or even nodular infiltrates with poorly defined margins usually in the lower lobes, which can be unilateral or bilateral. 51 interestingly, leptospirosis and chikungunya coinfection can potentially lead to a delayed diagnosis and subsequent deleterious outcomes. nhan et al. 52 described a fatal case of leptospirosis and chikungunya co-infection in a french-polynesian outbreak during the rainy season, where diagnosis was delayed due to overlapping symptoms. co-infections with dengue have also been described by pan et al., where three cases of co-infection were detected during a dengue outbreak. 53 again, diagnosis was challenging because of nonspecific symptoms such as fever, chills and myalgia. adding on to the diagnostic challenge, sathiyakumar et al. described a case of haemorrhagic pneumonitis secondary to leptospirosis, 54 which showcases the spectrum of clinical presentation. the diagnosis of leptospirosis is both clinical and microbiological, but the gold standard is the microscopic agglutination test (mat). cumberland et al. found the mat to have a sensitivity of between 30% and 76% (depending on when samples were taken in the disease's time course), and 97% specificity. 55 leptospira can be grown in vitro from blood, cerebrospinal fluid and urine from infected hosts. however, the laboratory needs to be notified if leptospira needs to be isolated as it requires specialized culture media, and time to positive cultures can take between 1 week and 3 months. should the clinician strongly suspect leptospirosis clinically, empiric antibiotics such as doxycycline or ceftriaxone should be started. the strain of rickettsial illness encountered in the tropics is scrub typhus-a mite-borne disease caused by orientia tsutsugamushi, a gram-negative coccobacillus. it is predominantly found in the asia pacific rim, with larval mites ('chiggers', from the genus leptotrombidium) that live on vegetation and rodents. wu et al. have described a rise in the incidence of scrub typhus in mainland china between 2006 and 2014 with a 12.8 times increase. 56 on top of seasonal peaks, the authors postulated that the increase of popularity in ecotourism have exposed more people to vector habitats. 56 clinical manifestations typically include myalgia, high fevers, headaches, as well as a rash and eschar at the chigger bite. scrub typhus is usually self-limiting over 2-3 weeks but is sometimes associated with severe illness and multiorgan failure leading to death, although this is rare. [57] [58] [59] pneumonia can occur in the late phase of the disease 57,58,60 as well as in an ardslike picture. 58 pulmonary involvement is well described. chest roentgenograms may be abnormal in 59-72% of patients, and may show bilateral diffuse reticulonodular opacities, septal lines and hilar lymphadenopathy. consolidation is not common, and would tend to appear in the lower zones. pleural effusions can be found in up to 42.6% of patients. 60, 61 the diagnosis is confirmed on serological testing or eschar biopsy, but there should be a suspicion of scrub typhus infection if there is an appropriate exposure history. patients who have been started on appropriate antibiotics (such as doxycycline, chloramphenicol and azithromycin) usually have defervescence of the fever within 48 h. countries in the tropics bear the brunt of tb, 62 and south east asia holds approximately one-third of the global burden of tb. 63 peto et al. found more than 10% of cases of cap in asia were attributable to mycobacterium tuberculosis. 35 however, with the rise in tourism and immigration, tb is now seen in higher income countries, with a substantial proportion of cases diagnosed in immigrants in the united states and england. 64, 65 people at risk include those with poor nutrition, immunocompromised and those living in poorly ventilated and overcrowded environments. the american thoracic society and infectious diseases society of america recommends repeat examinations of expectorated sputum for acid fast bacilli (afb) augmented by a nucleic acid amplification test such as the xpert mtb/rif assay (cepheid, sunnyvale, ca, usa) in the rapid diagnosis of pulmonary tb. 66 in smear-negative cases, they suggest the testing of induced sputum instead of proceeding to bronchoscopy and lavage which seems to be a very popular option. 66 sputum induction is more cost effective than bronchoscopic examination and should be the test of choice if smear-negative pulmonary tb is the most likely diagnosis. 66 68 we must also consider that in certain settings, it is common to lack access to high-quality chest roentgenograms and people who can reliably interpret them, and there may not be access to the xpert mtb/rif assay. who has an alternative algorithm to reference in settings where chest roentgenograms and/or xpert mtb/rif assays are not available, and it is largely based upon careful history taking, clinical examination and sputum smear analysis. 69 treatment regimens using first-line drugs include rifampicin, isoniazid, ethambutol and pyrazinamide. directly observed therapy (dot) has been utilized in some countries to ensure compliance as this is the crux of treatment success, but a cochrane review of 11 trials in 2015 found no significant differences in cure rates, treatment completion when comparing dot and selfadministered therapy. 70 the authors have stated that given the costs and personnel involved in dot, policymakers may wish to have alternative strategies to help improve adherence to treatment. 70 multidrug-resistant tb and extensively drug-resistant strains are beyond the scope of this review and will not be examined. helminthic and protozoal parasitic diseases are common in the tropics. pulmonary disease typically presents as an eosinophilic lung disease, with or without peripheral blood eosinophilia. lung infiltrates may be fleeting on radiology-this was famously described by loffler in 1932. 71 lymphatic filariasis lymphatic filariasis can manifest as a syndrome known as tropical pulmonary eosinophilia (tpe). the disease is seen mainly in south asia, southeast asia and the south pacific islands. three species of filarial nematodes cause tpe: wuchereria bancrofti, brugia malayi and brugia timori. 72, 73 mosquitoes transmit the disease and humans are the definitive hosts. microfilariae trapped in the lungs lead to an immune hyper-responsiveness, leading to symptoms of cough, fever, night sweats, wheezing and weight loss. pulmonary radiology can appear miliary or nodular, mimicking tb. 73 however, imaging can be normal in up to 20% of patients. 73 spirometry tends to demonstrate a restrictive pattern with airways obstruction, and airway obstruction is usually reversed by bronchodilators. treatment with diethylcarbamazine is associated with rapid improvement in signs and symptoms, as well as a gradual trend towards normal in spirometric values, although permanent impairment in lung function can occur. 74 the causative pathogen, paragonimus westermani, is endemic in much of asia and south america. a foodborne trematode, infection is cause by ingestion of raw of improperly cooked freshwater crabs. patients may be asymptomatic although can also experience a chronic cough, chest pain and haemoptysis which can be recurrent. radiological findings include pleural effusions, pneumothorax, ring shadows and consolidation on chest roentgenograms. 75, 76 computer tomography scanning can reveal cysts within the consolidated lung. 76 paragonimiasis is treated with triclabendazole or praziquantel. strongyloides infection is common in the tropics, sub tropics and warmer temperate climates. nematode larva spread haematogenously as well as via the lymphatics to the heart and lungs. patients can present with loeffler's syndrome and peripheral eosinophilia during larval migration through the lungs. respiratory signs and symptoms include cough, bronchospasm and in some cases haemoptysis. chest roentography can be normal. during larval migration, miliary nodules or ill-defined patchy consolidation may be present. in an overwhelming infection especially in the immunocompromised host, a marked bilateral alveolar pattern similar to that of pulmonary oedema can be seen, and clinically the patient would be in ards. 75, 76, 78 diagnosis of strongyloides can be strengthened with examination of several stool samples on several days. larvae may also be demonstrated on duodenal aspirates, sputum and bronchoalveolar lavage fluid. 77 ivermectin and albendazole can be used for effective treatment. 77, 78 additionally, clinicians need to be aware of gut translocation of enteric organisms especially in immunocompromised hosts, leading to further complications of sepsis. 78 malaria is caused by the intraerythrocytic protozoa plasmodium. it is transmitted to humans by the bite of the female anopheles mosquito, and falciparum malariae is the most severe of all malarial infections. the symptoms leading to a suspicion of malarial infection are fever which can be cyclical, breathing difficulties and anaemia. once the disease is suspected, light microscopy is the standard tool used to detect parasites on blood smears. rapid diagnostic tests utilize antigen detection technology as an alternative when reliable light microscopy is unavailable, and the who is recommending its use as a field alternative when rapid diagnosis is paramount. 79 there is a wide range of pulmonary manifestations, from a non-productive cough to ards, occurring in up to 25% of adults with severe falciparum malaria infection although any strain of plasmodium can lead to ards. 80 the development of ards portends an extremely grave prognosis 81,82 -gachot et al. described a 33% mortality rate in patients with malaria and acute lung injury despite admission to an intensive care unit. 82 chest roentgenogram findings are non-specific and can range from confluent nodules to basal and/or bilateral pulmonary infiltrates, mimicking pulmonary oedema, although this is usually non-cardiogenic. 83 resistance to antimalarial drugs especially chloroquine and sulfadoxine-pyrimethamine has become widespread. who now recommends artemisinin-based combination therapy (act) as the firstline treatment in uncomplicated falciparum malaria. for uncomplicated, non-falciparum malaria in regions with low choroquine resistance, chloroquine can be used. 84 we have summarized the common pathogens causing pneumonia in tropical regions in table 1 . other differentials of pneumonia in the tropics would be tpe, pulmonary plague, histoplasmosis, cryptococcosis, thoracic actinomycosis, nocardiosis and pulmonary anthrax. however, these are beyond the scope of this review, and we would urge clinicians to practice careful history taking including a travel and exposure history, and conscientious examination to lead them towards the correct diagnosis. the burdens of pneumonia in tropical and subtropical regions remain high, especially when coupled with global warming and climate change. with the advances in air travel, immigration patterns and international tourism would mean tropical diseases including pneumonias would be encountered in the temperate countries as well. it is important for clinicians to recognize these relations and conditions so that correct treatment can be instituted early, as some of the tropical diseases such as leptospirosis, melioidosis and malaria with ards herald a poor prognosis if treatment is delayed. clinicians will need to be cognizant of co-infections with overlapping symptoms such as chikungunya or dengue co-infection with leptospirosis, as late diagnosis would potentially lead to deadly consequences. t.k.l. is professor of medicine and senior consultant in the department of medicine, national university hospital singapore. his research interests include effective implementation of clinical evidence and improving clinical reasoning expertise by deliberate practice and structured reflection. w.t.s. is an associate consultant in the department of respiratory and critical care medicine, national university hospital singapore. her clinical interests include critical care medicine and biomedical technology. world health organization. pneumonia factsheet climate change and respiratory infections characterizing hospital admissions to a tertiary care hospital after typhoon haiyan human health: impacts, adaptation, and co-benefits childhood pneumonia: a neglected, climate-sensitive disease? diurnal temperature range and daily mortality in shanghai leptospirosis working group. outbreak of leptospirosis among triathlon participants and community residents in outbreak of human pneumonic plague with dog-to-human and possible human-to-human transmission -colorado infectious diseases following natural disasters: prevention and control measures tsunami lung disseminated aspergillosis associated with tsunami lung epidemiology, co-infections, and outcomes of viral pneumonia in adults: an observational cohort study viral pneumonia: epidemiological, clinical, pathophysiological and therapeutic aspects viral infection in patients with severe pneumonia requiring intensive care unit admission viral pneumonia in older adults the use of polymerase chain reaction amplification for the detection of viruses and bacteria in severe community-acquired pneumonia respiratory syncytial virus infection: denominator-based studies in indonesia respiratory syncytial virus infection in tropical and developing countries epidemiology and seasonality of respiratory tract virus infections in the tropics epidemiology and seasonality of respiratory viral infections in hospitalized children in kuala lumpur, malaysia: a retrospective study of 27 years acute lower respiratory illnesses during the first three years of life: potential roles for various etiologic agents. the group health medical associates treatment of rsv pneumonia in adults: evidence of ribavirin effectiveness? comparison of the incidence of influenza in relation to climate factors during 2000-2007 in five countries seasonal trends of viral respiratory tract infections in the tropics community acquired pneumonia in the tropics environmental factors affecting the transmission of respiratory viruses cumulative number of confirmed human cases for avian influenza a (h5n1) reported to who world health organization. monthly risk assessment summary: influenza at the human-animal interface sudden increase in human infection with avian influenza a(h7n9) virus in china hantavirus pulmonary syndrome: a clinical description of 17 patients with a newly recognized disease hantavirus pulmonary syndrome in latin america who data. who middle east respiratory syndrome coronavirus the bacterial aetiology of adult communityacquired pneumonia in asia: a systematic review community-acquired pneumonia in europe: causative pathogens and resistance patterns bacteremic communityacquired pneumonia due to klebsiella pneumoniae: clinical and microbiological characteristics in taiwan severe community-acquired pneumonia in singapore etiologies and resistance profiles of bacterial community-acquired pneumonia in cambodian and neighboring countries' health care settings: a systematic review an account of the discovery of a hitherto undescribed infective disease occurring among the population of rangoon epidemiology: a global picture of melioidosis community-acquired pneumonia in adults at srinagarind hospital melioidosis in travelers: review of the literature severe community-acquired pneumonia requiring intensive care: a study of 80 cases from singapore the epidemiology and clinical spectrum of melioidosis: 540 cases from the 20 year darwin prospective study leptospirosis in the asia pacific region regional differences of leptospirosis in sri lanka: observations from a flood-associated outbreak in 2011 world health organization. human leptospirosis: guidance for diagnosis leptospirosis pulmonary haemorrhage: a diagnostic challenge pulmonary manifestations of leptospirosis pulmonary manifestations of leptospirosis fatal leptospirosis and chikungunya co-infection: do not forget leptospirosis during chikungunya outbreaks leptospirosis and dengue coinfection: report of three cases with review of literature snowflakes in august: leptospirosis hemorrhagic pneumonitis assessment of the efficacy of an igm-elisa and microscopic agglutination test (mat) in the diagnosis of acute leptospirosis rapid increase in scrub typhus incidence in mainland china serious complications in scrub typhus scrub typhus complicated by acute respiratory distress syndrome and multiorgan failure; an unrecognized alarming entity in central india: a report of two cases a case series of possibly recrudescent orientia tsutsugamushi infection presenting as pneumonia scrub typhus: clinical, pathologic and imaging findings clinical role of interstitial pneumonia in patients with scrub typhus: a possible marker of disease severity tuberculosis in tropical areas and immigrants tuberculosis in the who south-east asia region reported tuberculosis in the united states, department of health and human services tuberculosis in england: 2016. public health england official american thoracic society/infectious diseases society of america/centers for disease control and prevention clinical practice guidelines: diagnosis of tuberculosis in adults and children induced sputum and bronchoscopy in the diagnosis of pulmonary tuberculosis the clinical utility of xpert ® mtb/rif testing in induced sputum who tb screening guidelines directly observed therapy for treating tuberculosis zur differential-diagnose der lungeninfiltrieurgen: 11 tropical parasitic lung diseases tropical eosinophilia. in: herzog h (ed) pulmonary eosinophilia: progress in respiration research pulmonary function in tropical eosinophilia before and after treatment with diethylcarbamazine acute tropical pneumonias respiratory infections unique to asia strongyloides -resources for health professionals. centers for disease control and prevention strongyloides stercoralis in the immunocompromised population world health organization. malaria -malaria rapid diagnostic tests respiratory manifestations of malaria acute lung injury and acute respiratory distress syndrome in malaria acute lung injury complicating imported plasmodium falciparum malaria clinical review: severe malaria guidelines for the treatment of malaria key: cord-260630-vvpzp73r authors: mandell, lionel a. title: etiologies of acute respiratory tract infections date: 2005-08-15 journal: clin infect dis doi: 10.1086/432019 sha: doc_id: 260630 cord_uid: vvpzp73r nan in this issue of clinical infectious diseases, there are 2 articles that provide us with some insight into the various etiologic agents that can cause acute respiratory tract infection (arti) in general practice patients in the netherlands [1] and into the significance of the human metapneumovirus (hmpv) in patients with community-acquired pneumonia (cap) and exacerbations of chronic obstructive pulmonary disease (copd) in quebec, canada [2] . if we examine the broad spectrum of infectious and noninfectious illnesses experienced by people of all ages worldwide, it is clear that artis are, without question, the most common maladies encountered, regardless of age or sex [3] . the objectives of the dutch study were to estimate the incidence of influenza-like illnesses (ilis) and of other artis in patients visiting their general practitioners (to determine the etiologic agents) and to test the hypothesis that asymptomatic persons with subclinical infection may act as sources of transmission [1] . the objective of the canadian study was to examine the role of hmpv infection in adults with cap and adults with exacerbations of copd [2] . a case-control method was used in the dutch trial, and ili was defined as an "acute onset of illness" (duration of prodromal stage, у3-4 days) and the presence of at least 1 of the following symptoms: cough, rhinitis, sore throat, frontal headache, retrosternal pain, or myalgia. arti was defined as an acute respiratory illness other than ili with at least 1 of the following symptoms: cough, rhinitis, or sore throat. nose and throat swab specimens were obtained from case patients and control subjects, and viral cultures and pcr tests were performed for detection of adenovirus, coronavirus, enterovirus, hmpv, influenza virus, parainfluenza virus, rhinovirus, and respiratory syncytial virus (rsv), as well as for mycoplasma pneumoniae, chlamydophila pneumoniae, and chlamydophila psittaci. bacterial cultures of throat swab specimens were also performed to detect all bacterial pathogens that are known to potentially cause "community-acquired" respiratory infection. the findings were then semiquantitatively classified into 5 groups: no colonies, sporadic colonies, few colonies, several colonies, or many colonies. the spectre of respiratory illnesses and of epidemic respiratory illnesses in particular, such as can be seen with influenza, was heightened recently by the epidemic of severe acute respiratory syndrome (sars) during 2002-2003 and widespread publicity about concerns regarding a possible epidemic or even pandemic attrib-utable to avian influenza [4, 5] . the more we learn and understand about the epidemiology and etiology of artis, the better position we will be in to prevent and treat them. essentially, the article by van gageldonk-lafeber et al. [1] shows us a number of things. the overall incidences of consultations for ilis and other artis are 132 and 413 consultations per 10,000 personyears, respectively. the diagnoses made for case patients with artis other than ilis were primarily the common cold (36% of patients), acute pharyngitis (30%), and acute tonsillitis (20%). the main symptom for case patients with ili was fever (90% of patients), and for those with other artis, it was sore throat (76%). for the control subjects, the most common symptoms were joint-muscle complaints (21% of patients) and skin disorders (14%). approximately 20% of control subjects consulted their family physicians for other reasons, such as to pick up prescriptions, to undergo a routine physical examination, and to accompany relatives. not surprisingly, no pathogens were detected in 35% of case patients; in contrast, pathogens were found in 31% of control subjects without airway complaints. in the case patients, the pathogens were viruses (58% of patients), group a b-hemolytic streptococci (11%), and mixed pathogens (virus plus group a b-hemolytic streptococci; 3%). case patients were defined as persons with ili or an arti other than ili who had not used antimicrobials in the previous 2 weeks, and the findings reported above are certainly in keeping with the general supposition that artis are primarily caused by viruses. a breakdown of the most common pathogens in case patients and control subjects revealed that influenza a viruses were the most common pathogens in case patients with ili (42% of patients), and rhinovirus was the most common pathogen in case patients with arti (25%). in control subjects, rsv (17% of subjects) was the most common pathogen. the main conclusions of van gageldonk-lafeber et al. [1] are that most artis are viral, rhinovirus was the most common pathogen in both case patients and control subjects, and patients without symptoms suggestive of artis may harbor pathogens and could be a potential source of transmission of respiratory pathogens. the majority of respiratory tract infections in general and of viral infections in particular are caused by rhinoviruses and coronaviruses [3] . in the category of respiratory infections, influenza was reported more frequently than the common cold, despite the fact that colds occur more frequently [3] . this is further supported by data on viruses associated with the common cold in which influenza virus was reported to occur in 25%-30% of cases [6, 7] . in another community-based study, the tecumseh study [8] , the viruses that caused all respiratory infections were reported. here too, as in the dutch study, rhinoviruses were the most frequently found pathogens. the pathogens capable of causing viral respiratory disease vary in their ability to both initiate and transmit infection. if one examines the relative role or impact of different respiratory viruses in producing artis, the top 3 etiologic agents (expressed as estimated percentages of all respiratory illnesses) are as follows: rhinovirus, 34%; coronaviruses, 14%; and influenza viruses, 9% [3] . the viral pathogens also vary in their ability to transmit infection. coronavi-ruses are generally transmitted by large droplet spread, whereas influenza viruses are spread by airborne methods, including both aerosol and droplet spread [9] . the exact mode of transmission of rhinoviruses, however, is not clear, and it is still debated whether rhinovirus is transmitted primarily by direct contact (e.g., droplet nuclei) or by indirect contact. for example, it has been shown that these viruses can survive on surfaces and can be spread by inoculation of nose or eyes with one's fingers, and others have shown that spread by droplet transmission can take place as well [10] . one of the most recent outbreaks that drew worldwide attention to the potential of a pandemic was the outbreak of the sars. this began in the guandong province of china in november 2002 and subsequently spread to hong kong and, ultimately, to far reaches of the world. the etiologic pathogen was determined to be a new coronavirus (sars-cov) that presumably spread from animals that are sometimes eaten as part of exotic banquets by humans. as happens with other coronaviruses, the sars-cov probably first mutated and then spread to new and susceptible species (e.g., humans). it is likely that spread among humans happened primarily by close contact that probably included droplet spread, direct contact, or fomite transmission. there were also several instances that may have involved small particle airborne or fecal/oral transmission. usually, 1 case would only result in spread of the virus to a few other individuals, but there were several well-documented cases of "super transmitters," in which 1 person could infect у10 other persons [11, 12] . one of the interesting findings of the dutch study was the fact that the researchers found pathogens in 30% of control subjects. this was higher for the youngest age groups and might act as a potential source of infection of others. they drew attention to the fact that only 2 other studies have addressed this issue, one that involved children and adults and the other that examined only adults [13, 14] . the former showed that results of virologic assessments of asymptomatic subjects were positive for 12% of children and 4% of adults for rhinovirus and enterovirus, respectively [13] . the latter study showed that only 4% of asymptomatic subjects aged у60 years tested positive for rhinovirus or coronavirus [14] . these figures are significantly lower than those reported by the dutch study [1] . the frequencies of subclinical infection and of asymptomatic subjects, by age group, were as follows: children (age, 0-15 years), 68%; adults, 55%; and persons aged у65 years, 51%. a number of possible explanations are offered, but the important point still remains that asymptomatic persons, whether young or old, may act as unsuspected sources of infection. the article by hamelin et al. [2] is an attempt to assess the role that hmpv plays in adults with cap and exacerbations of copd. hmpv is a newly discovered viral respiratory pathogen for which, thus far, there are limited epidemiologic data to describe it. it was first isolated in the netherlands from nasopharyngeal secretions of 28 children collected over a 20-year period. the virus itself is a single-stranded negative-sense rna virus, and sequence studies of isolates have identified 2 main lineages. it belongs to the order mononegavirales, family paramyxoviridae, and genus metapneumovirus [15, 16] . as mentioned in the study by van gageldonk-lafeber et al. [1] , a significant percentage of patients with artis never have an etiologic pathogen identified. this is true not only for ilis and other artis but also in serious cases of cap that require admission to the hospital and in nosocomial and ventilator-associated pneumonia that is managed in medical or surgical wards and the intensive care units in tertiary care university hospitals, despite the availability of the best equipment and access to excellent diagnostic laboratories. the article by hamelin et al. [2] assesses 2 patient populations: patients with exacerbations of copd aged у40 years (with or without cap) who sought help at one of the emergency departments at study hospitals, and patients aged у18 years without copd who had cap that required admission to the hospital. three hospitals were involved for the periods of 17 january 2002 through 6 may 2002 and 6 january 2003 through 6 may 2003. statistical analysis involving regression modeling showed an association between artis and excess winter deaths, particularly for influenza and rsv, but it was also implied that other agents may have been involved as well [17] . the large number of negative results of culture, pcr, and serologic testing to identify a causative agent certainly bears this out as well. identification of the different pathogens that contribute to the various respiratory illnesses is very important not only from an epidemiological point of view but also from a treatment and prevention point of view, because such information may also help in the development of specific treatments aimed at these pathogens, as well as the development of preventive measures through the use of chemoprophylaxis or immunoprophylaxis (i.e., vaccines). hamelin et al. [2] studied nasopharyngeal aspirate specimens for the presence of influenza viruses a and b, rsv, and hmpv by pcr, and they studied paired blood samples (obtained at a 3-4-week interval) for antibody to hmpv (by elisa) and antibody to rsv and influenza (both by complement fixation), using serologic testing. all of the patients with cap and 81% of the patients with exacerbations of copd were admitted to the hospital. among the 145 patients from the study period, the incidence of hmpv infection was 4.1%, the incidence of influenza a was 6.2%, the incidence of influenza b was 0%, and the incidence of rsv infection was 9%, as determined by pcr and/or serologic testing. of the 6 patients in whom hmpv was identified, 4 had cap (2 of whom also had copd), and 2 had acute exacerbations of copd (but not cap). other features of note are that all 6 patients pre-sented to the emergency department with cough and sore throat, 5 had a mean elevated oral temperature of 38.3њc, 5 of 6 were dyspneic, 4 of 6 had nasal congestion and/or rhinorrhea, 3 of 6 had purulent sputum, 3 of 6 had wheezing, and 2 of 6 had myalgias. one of the largest studies of patients with hmpv infection is that by williams et al. [18] published in the new england journal of medicine. nasal wash specimens were obtained over a 25-year period from otherwise healthy children who presented to the pediatric division of infectious diseases at vanderbilt university medical center (nashville, tn) with acute respiratory tract illness. the samples were studied for the presence of hmpv, which was found in 49 of 248 specimens. it is difficult to compare the vanderbilt cohort and the quebec cohort, because the former consisted exclusively of otherwise healthy infants and children, whereas the latter included adults only, many of whom had comorbid illnesses. also, the sample sizes were significantly different: the quebec study had a total of only 145 subjects, 6 (4.2%) of whom were hmpv positive. in the vanderbilt study, the authors concluded that 12% of all lower respiratory tract infections were most likely caused by hmpv, whereas the figure is only 4.1% in the quebec study. the symptoms of diarrhea and vomiting were seen in 17% and 10% of the children in the vanderbilt study, respectively, whereas these symptoms were not even mentioned in the canadian study, and although fever occurred in 5 (83.3%) of the 6 subjects in the quebec study, it was only found in 52% of children in the vanderbilt study. hamelin et al. [2] claimed that hmpv is associated with a significant number of cases of cap and exacerbations of copd in adults during the early spring. i would take issue with this claim, unless we interpret the statement literally (i.e., if we consider the etiology to be specifically in the early spring [all 6 cases occurred in april]). otherwise, it is hard to imagine that 4% is a particularly significant figure. the study, however, is an important one, and it is definitely a step in the right direction. to date, we have few data on hmpv, and anything that sheds light on its role as a pathogen is welcome. what is very important is the fact that the quebec data show that, in the elderly population-particularly for persons with underlying lung disease-hmpv infection can lead to severe infection that requires hospitalization (mean duration, 10 days). hamelin and colleagues' finding that ∼85% of the adult population in their study had preexisting hmpv antibody is consistent with the premise that hmpv infection is almost universal by the age of 5 years. a case-control study of acute respiratory tract infection in general practice patients in the netherlands human metapneumovirus infection in adults with community-acquired pneumonia and exacerbation of chronic obstructive pulmonary disease epidemiology of viral respiratory infections coronavirus as a possible cause of severe acute respiratory syndrome a novel coronavirus associated with severe acute respiratory syndrome virologic studies of acute respiratory disease in young adults. iv. virus isolations during four years of surveillance rhinovirus infections in tecumseh, michigan: frequency of illness and number of serotypes the tecumseh study of illness. xiv. occurrence of respiratory viruses, 1976-1981 rhinovirus transmission: one if by air, two if by hand aerosol transmission of rhinovirus colds severe acute respiratory syndrome-singapore transmission dynamics and control of severe acute respiratory syndrome use of polymerase chain reaction for diagnosis of picornavirus infection in subjects with and without respiratory symptoms a prospective, community-based study on virologic assessment among elderly people with and without symptoms of acute respiratory infection human metapneumovirus infections in young and elderly adults a newly discovered human pneumovirus isolated from young children with respiratory tract disease impact of influenza and respiratory syncytial virus on mortality in england and wales for january 1975 to december 1990 human metapneumovirus and lower respiratory tract disease in otherwise healthy infants and children potential conflicts of interest. l.a.m.: no conflicts. key: cord-308201-lavcsqov authors: desforges, marc; le coupanec, alain; dubeau, philippe; bourgouin, andréanne; lajoie, louise; dubé, mathieu; talbot, pierre j. title: human coronaviruses and other respiratory viruses: underestimated opportunistic pathogens of the central nervous system? date: 2019-12-20 journal: viruses doi: 10.3390/v12010014 sha: doc_id: 308201 cord_uid: lavcsqov respiratory viruses infect the human upper respiratory tract, mostly causing mild diseases. however, in vulnerable populations, such as newborns, infants, the elderly and immune-compromised individuals, these opportunistic pathogens can also affect the lower respiratory tract, causing a more severe disease (e.g., pneumonia). respiratory viruses can also exacerbate asthma and lead to various types of respiratory distress syndromes. furthermore, as they can adapt fast and cross the species barrier, some of these pathogens, like influenza a and sars-cov, have occasionally caused epidemics or pandemics, and were associated with more serious clinical diseases and even mortality. for a few decades now, data reported in the scientific literature has also demonstrated that several respiratory viruses have neuroinvasive capacities, since they can spread from the respiratory tract to the central nervous system (cns). viruses infecting human cns cells could then cause different types of encephalopathy, including encephalitis, and long-term neurological diseases. like other well-recognized neuroinvasive human viruses, respiratory viruses may damage the cns as a result of misdirected host immune responses that could be associated with autoimmunity in susceptible individuals (virus-induced neuro-immunopathology) and/or viral replication, which directly causes damage to cns cells (virus-induced neuropathology). the etiological agent of several neurological disorders remains unidentified. opportunistic human respiratory pathogens could be associated with the triggering or the exacerbation of these disorders whose etiology remains poorly understood. herein, we present a global portrait of some of the most prevalent or emerging human respiratory viruses that have been associated with possible pathogenic processes in cns infection, with a special emphasis on human coronaviruses. the central nervous system (cns), a marvel of intricate cellular and molecular interactions, maintains life and orchestrates homeostasis. unfortunately, the cns is not immune to alterations that lead to neurological disease, some resulting from acute, persistent or latent viral infections. several viruses have the ability to invade the cns, where they can infect resident cells, including the neurons [1] . although rare, viral infections of the cns do occur [2] . however, their incidence in clinical practice common virus, is associated with febrile illness, fever, cough and congestion [97, 98] , as well as a characteristic rash and koplik's spots [99] . in rare circumstances, significant long-term cns diseases, such as [99] post-infectious encephalomyelitis (pie) or acute disseminated encephalomyelitis (adem), occur in children and adolescents. other examples of rare but devastating neurological disorders are measles inclusion body encephalitis (mibe), mostly observed in immune-compromised patients, and subacute sclerosing panencephalitis (sspe) that appears 6-10 years after infection [100] . yet, with the exception of hiv, no specific virus has been constantly associated with specific human neurodegenerative disease. on the other hand, different human herpes viruses have been associated with alzheimer's disease (ad), multiple sclerosis (ms) and other types of long-term cns disorders [101] [102] [103] . as accurately stated by majde [104] , long-term neurodegenerative disorders may represent a "hit-and-run" type of pathology, since some symptoms are triggered by innate immunity associated with glial cell activation. different forms of long-term sequelae (cognitive deficits and behavior changes, decreased memory/learning, hearing loss, neuromuscular outcomes/muscular weakness) were also observed following arboviral infections [83, 103, [105] [106] [107] . including the few examples listed above, more than one hundred infectious agents (much of them being viruses) have been described as potentially encephalitogenic and an increasing number of positive viral identifications are now made with the help of modern molecular diagnostic methods [8, 70, [108] [109] [110] . however, even after almost two decades into the 21st century and despite tremendous advances in clinical microbiology, the precise cause of cns viral infections often remains unknown. indeed, even though very important technical improvements were made in the capacity to detect the etiological agent, identification is still not possible in at least half of the cases [110, 111] . among all the reported cases of encephalitis and other encephalopathies and even neurodegenerative processes, respiratory viruses could represent an underestimated part of etiological agents [104] . respiratory syncytial virus (rsv), a member of the orthopneumovirus genus [112] , infects approximately 70% of infants before the age of 1 and almost 100% by the age of 2 years old [113] , making it the most common pathogen to cause lower respiratory tract infection such as bronchiolitis and pneumonia in infants worldwide [32, 114] . recent evidence also indicates that severe respiratory diseases related to rsv are also frequent in immunocompromised adult patients [8, 115] and that the virus can also present neuroinvasive properties [8] . over the last five decades, a number of clinical cases have potentially associated the virus with cns pathologies. rsv has been detected in the cerebrospinal fluid (csf) of patients (mainly infants) and was associated with convulsions, febrile seizures and different types of encephalopathy, including clinical signs of ataxia and hormonal problems [116] [117] [118] [119] [120] [121] [122] [123] [124] [125] [126] . furthermore, rsv is now known to be able to infect sensory neurons in the lungs and to spread from the airways to the cns in mice after intranasal inoculation, and to induce long-term sequelae such as behavioral and cognitive impairments [127] . an additional highly prevalent human respiratory pathogen with neuroinvasive and neurovirulent potential is the human metapneumovirus (hmpv). discovered at the beginning of the 21st century in the netherlands [128] , it mainly causes respiratory diseases in newborns, infants and immunocompromised individuals [129] . during the last two decades, sporadic cases of febrile seizures, encephalitis and encephalopathies (associated with epileptic symptoms) have been described. viral material was detected within the cns in some clinical cases of encephalitis/encephalopathy [130] [131] [132] [133] [134] but, at present, no experimental data from any animal model exist that would help to understand the underlying mechanism associated with hmpv neuroinvasion and potential neurovirulence. hendra virus (hev) and nipah virus (niv) are both highly pathogenic zoonotic members of the henipavirus genus and represent important emerging viruses discovered in the late 1990s in australia and southern asia. they are the etiological agents of acute and severe respiratory disease in humans, including pneumonia, pulmonary edema and necrotizing alveolitis with hemorrhage [135] [136] [137] [138] . although very similar at the genomic level, both viruses infect different intermediate animal reservoirs: the horse for hev and the pig for niv as a first step before crossing the barrier species towards humans [135] . in humans, it can lead to different types of encephalitis, as several types of cns resident cells (including neurons) can be infected [139, 140] . the neurological signs can include confusion, motor deficits, seizures, febrile encephalitic syndrome and a reduced level of consciousness. even neuropsychiatric sequelae have been reported but it remains unclear whether a post-infectious encephalo-myelitis occurs following infection [141] [142] [143] . the use of animal models showed that the main route of entry into the cns is the olfactory nerve [144] and that the nipah virus may persist in different regions of the brain of grivets/green monkeys [145] , reminiscent of relapsing and late-onset encephalitis observed in humans [146] . influenza viruses are classified in four types: a, b, c and d. all are endemic viruses with types a and b being the most prevalent and causing the flu syndrome, characterized by chills, fever, headache, sore throat and muscle pain. they are responsible for seasonal epidemics that affect 3 to 5 million humans, among which 500,000 to 1 million cases are lethal each year [147, 148] . associated with all major pandemics since the beginning of the 20th century, circulating influenza a presents the greatest threat to human health. most influenza virus infections remain confined to the upper respiratory tract, although some can lead to severe cases and may result in pneumonia, acute respiratory distress syndrome (ards) [30, 149] and complications involving the cns [150] [151] [152] . several studies have shown that influenza a can be associated with encephalitis, reye's syndrome, febrile seizure, guillain-barré syndrome, acute necrotizing encephalopathy and possibly acute disseminated encephalomyelitis (adem) [153] [154] [155] [156] [157] [158] . animal models have shown that, using either the olfactory route or vagus nerve, influenza a virus may have access to the cns and alter the hippocampus and the regulation of neurotransmission, while affecting cognition and behavior as long-term sequelae [8, 69, [159] [160] [161] [162] . the influenza a virus has also been associated with the risk of developing parkinson's disease (pd) [151] and has recently been shown to exacerbate experimental autoimmune encephalomyelitis (eae), which is reminiscent of the observation that multiple sclerosis (ms) relapses have been associated with viral infections (including influenza a) of the upper respiratory tract [163] [164] [165] . another source of concern when considering human respiratory pathogens associated with potential neuroinvasion and neurovirulence is the enterovirus genus, which comprises hundreds of different serotypes, including polioviruses (pv), coxsackieviruses (cv), echoviruses, human rhinoviruses (hrv) and enteroviruses (ev). this genus constitutes one of the most common cause of respiratory infections (going from common cold to more severe illnesses) and some members (pv, ev-a71 and -d68, and to a lesser extent hrv) can invade and infect the cns, with detrimental consequences [166] [167] [168] [169] . even though extremely rare, hrv-induced meningitis and cerebellitis have been described [170] . although ev infections are mostly asymptomatic, outbreaks of ev-a71 and d68 have also been reported in different parts of the world during the last decade. ev-a71 is an etiological agent of the hand-foot-mouth disease (hfmd) and has occasionally been associated with upper respiratory tract infections. ev-d68 causes different types of upper and lower respiratory tract infections, including severe respiratory syndromes [171] . both serotypes have been associated with neurological disorders like acute flaccid paralysis (afp), myelitis (afm), meningitis and encephalitis [166, [172] [173] [174] [175] . last but not least, human coronaviruses (hcov) are another group of respiratory viruses that can naturally reach the cns in humans and could potentially be associated with neurological symptoms. these ubiquitous human pathogens are molecularly related in structure and mode of replication with neuroinvasive animal coronaviruses [176] like phev (porcine hemagglutinating encephalitis virus) [177] , fcov (feline coronavirus) [178, 179] and the mhv (mouse hepatitis virus) strains of mucov [180] , which can all reach the cns and induce different types of neuropathologies. mhv represents the best described coronavirus involved in short-and long-term neurological disorders (a model for demyelinating ms-like diseases) [181] [182] [183] . taken together, all these data bring us to consider a plausible involvement of hcov in neurological diseases. the first strains of hcov were isolated in the mid-60s from patients presenting an upper respiratory tract disease [184] [185] [186] [187] . before the severe acute respiratory syndrome (sars) appeared in 2002 and was associated with sars-cov [188] [189] [190] , only two groups of hcov, namely hcov-229e (previous group 1, now classified as alphacoronavirus) and hcov-oc43 (previous group 2, now classified as betacoronavirus) were known. several new coronaviruses have now been identified, including three that infect humans: alphacoronavirus hcov-nl63 [191] and betacoronaviruses hcov-hku1 and mers-cov [192, 193] . the hcov-229e, -oc43, -nl63 and -hku1 strains are endemic worldwide [31, 184, [194] [195] [196] [197] [198] [199] and exist in different genotypes [200] [201] [202] [203] [204] [205] [206] [207] . in immunocompetent individuals they usually infect the upper respiratory tract, where they are mainly associated with 15-30% of upper respiratory tract infections (uri): rhinitis, laryngitis/pharyngitis as well as otitis. being highly opportunistic pathogens [14] , hcov can reach the lower respiratory tract and be associated with more severe illnesses, such as bronchitis, bronchiolitis, pneumonia, exacerbations of asthma and respiratory distress syndrome [17, 31, [208] [209] [210] [211] [212] [213] [214] . the 2002-2003 sars pandemic was caused by a coronavirus that emerged from bats (first reservoir) [25] to infect palm civets (intermediary reservoir) and then humans [215] . a total of 8096 probable cases were reported and almost 10% (774 cases in more than 30 countries) of these resulted in death [216] [217] [218] . the clinical portrait was described as an initial flu-like syndrome, followed by a respiratory syndrome associated with cough and dyspnea, complicated with the "real" severe acute respiratory syndrome (sars) in about 20% of the patients [31,219]. in addition, multiple organ failure was observed in several sars-cov-infected patients [220] . in the fall of 2012, individuals travelling from the arabian peninsula to the united kingdom were affected by the middle-east respiratory syndrome (mers), a severe lower respiratory tract infection that resembled sars, leading also to gastrointestinal symptoms and renal failure among some patients [221] . molecular sequencing rapidly showed that the new epidemic was caused by a new coronavirus: the mers-cov [193, 222, 223] . mers-cov most probably originated from bats before infecting an intermediary reservoir (the dromedary camel), and also represented a zoonotic transmission to humans. phylogenetic analyses suggest that there have been multiple independent zoonotic introductions of the virus in the human population. moreover, nosocomial transmission was observed in multiple hospitals in saudi arabia [221, [224] [225] [226] [227] [228] [229] [230] . although possible, human-to-human mers-cov transmission appears inefficient as it requires extended close contact with an infected individual. consequently, most transmission have occurred among patients' families and healthcare workers (clusters of transmission). a more efficient human-to-human transmission was observed in south korea, during the 2015 outbreak of mers-cov [231, 232] . even though it has propagated to a few thousand people and possesses a high degree of virulence, mers-cov seems mostly restricted to the arabic peninsula and is not currently considered an important pandemic threat. however, virus surveillance and better characterization are warranted, in order to be prompt to respond to any change in that matter [23, [233] [234] [235] . as of october 8, 2019, the world health organization (who) reported that mers-cov had spread to at least 27 different countries, where 2468 laboratory-confirmed human cases have been identified with 851 being fatal (https://www.who.int/emergencies/mers-cov/en/). as observed for the four circulating strains of hcov [31, 194] , both sars-cov and mers-cov usually induce more [228, 236] severe illnesses, and strike stronger in vulnerable populations such as the elderly, infants, immune-compromised individuals or patients with comorbidities [31,237]. over the years, like sars-and mers-cov, the four endemic hcov have also been identified as possible etiological agents for pathologies outside the respiratory tract. indeed, myocarditis, meningitis, severe diarrhea (and other gastrointestinal problems) and multi-organ failure [220, 221, [238] [239] [240] [241] have been reported, especially in children. recent investigations on hcov as enteric pathogens demonstrated that all hcov strains can be found in stool samples of children with acute gastroenteritis; however, no evidence of association could yet be clearly demonstrated with disease etiology [242, 243] . different reports also presented a possible link between the presence of hcov within the human central nervous system (cns) and some neurological disorders among patients examined [244] [245] [246] [247] [248] [249] . like all viruses, hcov may enter the cns through the hematogenous or neuronal retrograde route. in the human airways, hcov infection may lead to the disruption of the nasal epithelium [250] and, although they bud and are released mostly on the apical side of the epithelial cells, a significant amount of viruses is also released from the basolateral side [251] . thus, although hcov infections are, most of the time, restricted to the airways, they may under poorly understood conditions pass through the epithelium barrier and reach the bloodstream or lymph and propagate towards other tissues, including the cns [33, 38, 208, 252] ; this was also suggested for other respiratory viruses that can reach the human cns, namely, rsv [8, 53] [257, 258] . moreover, persistently-infected leukocytes [252] may serve as a reservoir and vector for neuroinvasive hcov [245] . therefore, neuroinvasive hcov could use the hematogenous route to penetrate into the cns. the second form of any viral spread towards the cns is through neuronal dissemination, where a given virus infects neurons in the periphery and uses the machinery of active transport within those cells in order to gain access to the cns [35, 36] . although the olfactory bulb is highly efficient at controlling neuroinvasion, several viruses have been shown to enter cns through the olfactory route [259, 260] . after an intranasal infection, both hcov-oc43 and sars-cov were shown to infect the respiratory tract in mice and to be neuroinvasive [261] [262] [263] [264] [265] . over the years, we and others have gathered data showing that hcov-oc43 is naturally neuroinvasive in both mice and humans [244, 245, 261, 263, 266] . experimental intranasal infections of susceptible mice also indicate that, once it has invaded the cns, the virus disseminated to several regions of the brain and the brainstem before it eventually reaches the spinal cord [266] [267] [268] . furthermore, based on more recent work [269] , figure 1 illustrates the olfactory route, which is clearly the main route of neuroinvasion used by hcov-oc43, as well as the early steps of subsequent neuropropagation within the cns in susceptible mice and recapitulates the suggested equivalent pathway in humans. nevertheless, our data suggest that hcov-oc43 may also invade the cns from the external environment through other pathways involving other cranial peripheral nerves [269] , reminiscent of what was shown for other human respiratory viruses such as rsv and influenza virus [8] . therefore, on the one hand, an apparently innocuous human respiratory pathogen such as the hcov may reach the cns by different routes and induce short-term illnesses, such as encephalitis. on the other hand, it may persist in resident cells of the human cns and may become a factor or co-factor of neuropathogenesis associated with long-term neurological sequelae in genetically or otherwise predisposed individuals. because of their natural neuroinvasive potential in humans and animals, a possible association between the presence of ubiquitous human coronaviruses in the triggering or exacerbation of neurological human pathologies has often been suggested over the years. it is now accepted that hcov are not always confined to the upper respiratory tract and that they can invade the cns [220, 245, 248, 270] . as other viruses listed herein, hcov are neurotropic and potentially neurovirulent. even though no clear cause and effect link has ever been made with the onset of human neurological diseases, their neuropathogenicity is being increasingly recognized in humans, as several recent reports associated cases of encephalitis [244] , acute flaccid paralysis [271] and other neurological symptoms, including possible complications of hcov infection such as guillain-barré syndrome or adem [249, [272] [273] [274] [275] [276] [277] [278] [279] . the presence and persistence of hcov in human brains was proposed to cause long-term sequelae related to the development or aggravation of chronic neurological diseases [245] [246] [247] [248] [280] [281] [282] . given their high prevalence [31, 283] , long-term persistence and newly recognized neuropathogenesis, hcov disease burden could currently be underestimated. this suggest that better surveillance, diagnoses and deepened virus-host interactions studies are warranted in order to gather more knowledge that will make possible the development of therapeutic strategies to prevent or treat occurrences. potential short-term neuropathologies sars-cov, hcov-oc43 and -229e are naturally neuroinvasive and neurotropic in humans and therefore potentially neurovirulent [220, 244, 245, 249, 270, 271] . furthermore, animal models showed that sars-cov could invade the cns primarily through the olfactory route [265] or even after an intra-peritoneal infection [284] , and induce neuronal cell death [265, 284] . to our knowledge, no reports on the presence of the three other coronaviruses that infect humans in the cns have been published. however, neurological symptoms have been described in patients infected by all three viruses [276, 277, 285] . making use of our in vivo model of hcov neuropathogenesis, relying on the natural susceptibility of mice to hcov-oc43-the most prevalent strain among endemic hcov [210, 286] -encephalitis and transient flaccid paralysis associated with propagation towards the spinal cord and demyelination and long-term persistence in surviving mice were observed [261, [266] [267] [268] [287] [288] [289] ; thus, recapitulating the neurological afflictions reported in some patients infected by hcov [244, 249, 271, 272, [275] [276] [277] 290] . although we must interpret data obtained in rodents with all the caution dictated by the use of a non-human host, it is likely that the underlying mechanisms described will have relevance to the human situation or at least provide leads to investigate neurotropic hcov in humans. in susceptible mice, hcov-oc43 has a selective tropism for neurons in which it is able to use axonal transport as a way of neuron-to-neuron propagation [269] . these results, together with data harvested with the use of microfluidic devices (xona microfluidic), helped to elaborate a putative model of propagation adapted from tomishima and enquist [291] , in which infectious hcov-oc43 could either be assembled in the cell body or at different points along the axon using the anterograde axonal transport to propagate between neurons or from neurons to glial cells surrounding neurons in the cns (figure 2 ). furthermore, based on previous data using different mutant recombinant viruses harboring mutation in the s protein [266, 268, 269] and making use of a luciferase expressing recombinant hcov-oc43 [292] [293] [294] , we are now showing that the rate and success of virus propagation towards the spinal cord, in part through the neuron-to-neuron pathway, correlates with the exacerbation of neurovirulence ( figure 3 ). [269] and adapted from tomishima and enquist [291] . in this model, solid arrows represent fully assembled virus transport and dashed arrows represent subvirion assemblies [291] . schematic representations were assembled with the motifolio neuroscience toolkit, 2007. [292] was injected intra-nasally (i.n.) into mice. virus spread was assessed by bioluminescence imaging (bli) with the xenogen vivo vision ivis 100 imaging system (perkin-elmer) in infected anaesthetized mice placed in a light proof specimen chamber after intraperitoneal injection of d-luciferin. images were taken with a ccd camera mounted in a light-tight imaging chamber, using the acquisition software living image version 4.3.1 (caliper-lifesciences). evaluation of associated clinical scores: (levels 0 to 4: 0 is asymptomatic; 1 is mice with early hunched back; 2 is mice presenting slight social isolation, weight loss and abnormal gait; 3 is mice presenting total social isolation, ruffled fur, hunched back, weight loss and almost no movement; and 4 is mice moribund or dead (presented elsewhere; [266] ), indicate that only mice with a positive signal at both the level of the brain and spinal cord were evaluated to be at level 2 to 3. hcov-oc43 structural and accessory proteins are important for infection and some clearly represent virulence factors [38, [266] [267] [268] [269] 289, 295, 296] . using neuronal cell cultures and our murine model, we gathered data indicating that some of these proteins also play a significant role in viral dissemination [269, 296] and now aim to exploit these promising leads to fully understand the course and determinants of propagation to and through the cns and complete the neurologic portrait of short term hcov neuropathogenesis. the presence of hcov rna in the human cns establishes the natural neuroinvasive properties of these respiratory viral agents. moreover, it also suggests that they persist in human cns [245] as they do in human neural cells [297, 298] and in the cns of mice that survive acute encephalitis. these surviving mice exhibited long-term sequelae associated with decreased activity in an open field test and a reduced hippocampus with neuronal loss in the ca1 and ca3 layers [287] , reminiscent of what was observed after infection by the influenza a virus and rsv [127, 162] and to the significant loss of synapses within the ca3 region after infection by wnv [299, 300] . the precise and complete etiology of several long-term neurological pathologies still represent a conundrum. multiple sclerosis (ms) represents one such neurological disease for which an infectious agent or agents may play a triggering role, with viruses the most likely culprit in genetically predisposed individuals [301] . it has been suggested that several neurotropic viruses could be involved in ms pathogenesis but that they may do so through similar direct and/or indirect mechanisms [302] [303] [304] [305] [306] . however, although research has not yet led to a direct link to any specific virus, association of coronaviruses with ms has been suggested [307] . even though hcov-oc43 and -229e were detected in some control brains and in some brains coming from patients with different neurological diseases, there was a significantly higher prevalence of hcov-oc43 in brains of ms patients [245] . moreover, autoreactive t cells were able to recognize both viral and myelin antigens in ms patients but not in controls during infection by hcov-oc43 and hcov-229e [308, 309] . thus, the immune response may participate in the induction or exacerbation of long-term neuropathologies such as ms in genetically or otherwise susceptible individuals. furthermore, it was shown that in recombination activation gene (rag) knock-out mice, hcov-oc43-induced encephalitis could be partially mediated by the t-cell response to infection [263] . this underlines the possibility that, like its murine counterpart mhv, long term infection of the cns by hcov [245] may participate in the induction of demyelinating ms-like lesions. immune cell infiltration and cytokine production were observed in the mouse cns after infection by hcov-oc43. this immune response was significantly increased after infection by viral variants, which harbor mutations in the viral glycoprotein (s) [267] . these variants also induced glutamate excitotoxicity [268, 289] , thus increasing damage to neurons [310] and/or disturbing glutamate homeostasis [311] and thereby contributing to neuronal degeneration and hind-limb paralysis and possible demyelination [266] [267] [268] [269] . the degeneration of neurons may eventually lead to death of these essential cells by directly generating a cytotoxic insult related to viral replication and/or to the induction of different regulated cell death (rcd) pathways [312] [313] [314] . our results indicate that the underlying mechanisms appear to involve different cellular factors and pathways of rcd, described and reviewed elsewhere [38, 315] . virus-cell interactions are always important in the regulation of cell response to infection. for hcov-oc43, we clearly showed that the viral s and e proteins are important factors of neurovirulence, neuropropagation and neurodegeneration of infected cells [267] [268] [269] 296, 312] . we have also demonstrated that the he protein is important for the production of infectious hcov-oc43 and for efficient spreading between neuronal cells, suggesting an attenuation of the eventual spread into the cns of viruses made deficient in fully active he protein, potentially associated with a reduced neurovirulence [269, 295] . coronavirus accessory proteins have been extensively studied and are now considered as important viral factors of virulence implicated in pathogenesis while counteracting innate immunity [316] [317] [318] [319] . two of these accessory proteins (ns2 and ns5) produced during infection by hcov-oc43 play a significant role in virulence and pathogenesis in the mouse cns [38] . like for several other respiratory viruses, accumulating evidence now indicate that hcov are neuroinvasive in humans and we hypothesize that these recognized respiratory pathogens are potentially neurovirulent as well, as they could participate in short-and long-term neurological disorders either as a result of inadequate host immune responses and/or viral propagation in the cns, which directly induces damage to resident cells. with that in mind, one can envisage that, under the right circumstances, hcov may successfully reach and colonize the cns, an issue largely deserted and possibly underestimated by the scientific community that has impacted or will impact the life of several unknowing individuals. in acute encephalitis, viral replication occurs in the brain tissue itself, possibly causing destructive lesions of the nervous tissue with different outcomes depending on the infected regions [320] . as previously mentioned, hcov may persist in the human cns as it does in mice [245, 287] and potentially be associated with different types of long-term sequelae and chronic human neurological diseases. in their famous review on cns viral infection, published a few years ago, koyuncu et al. [35] insisted that, under the right conditions, all viruses can have access to the cns. what "under the right conditions" means certainly represents a subject of debate among virologist and physicians. nevertheless, as stated in the introduction of this review, viral factors (mutations in specific virulence genes), host factors (immunodepression, age) or a mixture of both (underlining the importance of virus-host interactions), are all good candidates to refer to if one intends to find the beginning of an explanation. a fast and accurate diagnosis would certainly improve prognosis for patients with a suspected cns infection. identification of a specific virus provides relevant information on how to treat a patient; therefore, the development of modern technologies, such as high throughput sequencing (next generation sequencing) are warranted as it represents a potentially unbiased marvelous tool for rapid and robust diagnosis of unexplained encephalitis or other types of encephalopathies or neuronal manifestations, especially in the context where more traditional techniques have failed to identify the etiological agent [21, 108, 111, 244, 321, 322] . therefore, although our attention is mainly on a few different viruses such as hsv, arboviruses and enteroviruses, it may now be the time to look at cns viral infection from another perspective. these viruses truly represent an important proportion of cns viral infection associated with encephalitis, meningitis, myelitis and long-term neurological disorders. nevertheless, accumulating evidence in the scientific literature strongly suggest that many other viral candidates could be underestimated in that matter. several human respiratory viruses are neuroinvasive and neurotropic, with potential neuropathological consequences in vulnerable populations. understanding the underpinning mechanisms of neuroinvasion and interaction of respiratory viruses (including hcov) with the nervous system is essential to evaluate potentially pathological short-and long-term consequences. however, viral infections related to diseases that are rare manifestations of an infection (like long term chronic neurological diseases), represent situations where koch's postulates [323] need to be modified. a series of new criteria, adapted from sir austin bradford hill, for causation [324, 325] was elaborated by giovannoni and collaborators concerning the plausible viral hypothesis in ms [326] . these criteria certainly represent a pertinent tool to evaluate the involvement of human respiratory viruses as a factor that could influence long-term human neurological diseases. to continue the gathering of epidemiological data is justified to evaluate the clear cause and effect link between neuroinvasive respiratory viruses and short-and long-term human neurological diseases. understanding mechanisms of virus neuroinvasion and interactions with the central nervous system is essential for different reasons. first, to help better understand potentially pathologically relevant consequences of infection, and second in the design of novel diagnostic and intervention strategies that will help uncover potential "druggable" molecular virus-host interfaces highly relevant to symptoms of various neurological diseases with a viral involvement. virus and immune-mediated encephalitides: epidemiology, diagnosis, treatment, and prevention update and new insights in encephalitis viral infections of the central nervous system: a case-based review acute viral encephalitis emergence and re-emergence of viral diseases of the central nervous system global research priorities for infections that affect the nervous system the airway epithelium: soldier in the fight against respiratory viruses neurologic alterations due to respiratory virus infections. front viruses associated with pneumonia in adults viral infections in immunocompromised patients: what's new with respiratory viruses? new respiratory viral infections emerging respiratory agents: new viruses for old diseases? epidemiology of acute upper and lower respiratory tract infections in children time course and cellular localization of sars-cov nucleoprotein and rna in lungs from fatal cases of sars the diagnosis of viral respiratory disease in older adults respiratory viral infections in infants: causes, clinical symptoms, virology, and immunology respiratory virus infections in hematopoietic beyond respiratory syncytial virus and rhinovirus in the pathogenesis and exacerbation of asthma: the role of metapneumovirus, bocavirus and influenza virus respiratory viral infection: a potential "missing link" in the pathogenesis of copd respiratory viruses other than influenza virus: impact and therapeutic advances transolfactory neuroinvasion by viruses threatens the human brain respiratory syncytial virus tropism for olfactory sensory neurons in mice a new model for hendra virus encephalitis in the mouse distribution of insulin in trigeminal nerve and brain after intranasal administration intranasal delivery of biologics to the central nervous system multiple neural circuits mediating airway sensations: recent advances in the neurobiology of the urge-to-cough the nervous system of airways and its remodeling in inflammatory lung diseases the invasion routes of neurovirulent a/hong kong/483/97 (h5n1) influenza virus into the central nervous system after respiratory infection in mice the vagus nerve is one route of transneural invasion for intranasally inoculated influenza a virus in mice management of viral central nervous system infections: a primer for clinicians viral encephalitis: causes, differential diagnosis, and management viral encephalitis viral encephalitis: a practical review on diagnostic approach and treatment inflammation in neuroviral diseases viral encephalitis: familiar infections and emerging pathogens epidemiology of encephalitis in children: a 20-year survey overview, prevention, and treatment of rabies herpes simplex virus encephalitis update manifestations of herpes virus infections in the nervous system herpes simplex encephalitis hsv-induced apoptosis in herpes encephalitis arbovirus infections. continuum (minneap minn) zika virus: an emerging flavivirus zika virus: pathology from the pandemic neuroinvasive flavivirus infections emerging flaviviruses: the spread and resurgence of japanese encephalitis, west nile and dengue viruses poliovirus and poliomyelitis: a tale of guts, brains, and an accidental event cell death in hiv dementia friends turn foe-astrocytes contribute to neuronal damage in neuroaids hiv-1 persistence in the central nervous system: viral and host determinants during antiretroviral therapy human polyomavirus jcv and expression of myelin genes progressive multifocal leukoencephalopathy persistence and pathogenesis of the neurotropic polyomavirus jc the risk of development of htlv-i-associated myelopathy/tropical spastic paraparesis among persons infected with htlv-i pulmonary measles disease: old and new imaging tools blue moon neurovirology: the merits of studying rare cns diseases of viral origin human paramyxoviruses and infections of the central nervous system viruses in chronic progressive neurologic disease herpes simplex virus type 1 and alzheimer's disease neurotropic virus infections as the cause of immediate and delayed neuropathology neuroinflammation resulting from covert brain invasion by common viruses-a potential role in local and global neurodegeneration review: evidence of neurological sequelae in children with acquired zika virus infection long-term neurological outcomes in west nile virus-infected patients: an observational study long-term neuromuscular outcomes of west nile virus infection: a clinical and electromyographic evaluation of patients with a history of infection causality in acute encephalitis: defining aetiologies challenge of the unknown. a systematic review of acute encephalitis in non-outbreak situations viral sequences detection by high-throughput sequencing in cerebrospinal fluid of individuals with and without central nervous system disease changes to taxonomy and the international code of virus classification and nomenclature ratified by the international committee on taxonomy of viruses global burden of acute lower respiratory infections due to respiratory syncytial virus in young children: a systematic review and meta-analysis respiratory syncytial virus epidemics: the ups and downs of a seasonal virus respiratory syncytial virus infection in adults neonatal encephalopathy and siadh during rsv infection central nervous system alterations caused by infection with the human respiratory syncytial virus classification of acute encephalopathy in respiratory syncytial virus infection cerebrospinal fluid analysis in children with seizures from respiratory syncytial virus infection. scand detection of subgroup b respiratory syncytial virus in the cerebrospinal fluid of a patient with respiratory syncytial virus pneumonia neurological complications of respiratory syncytial virus infection: case series and review of literature encephalopathy associated with respiratory syncytial virus bronchiolitis sequential mri, spect and pet in respiratory syncytial virus encephalitis respiratory syncytial virus infection causing neurological disorder in neonates viral antibodies in the csf after acute cns infections neurological, electroencephalographic, and virological findings in febrile cheldren impaired learning resulting from respiratory syncytial virus infection a newly discovered human pneumovirus isolated from young children with respiratory tract disease burden of human metapneumovirus infection in young children sommerstein, r. cerebrospinal fluid findings in an adult with human metapneumovirus-associated encephalitis encephalitis-associated human metapneumovirus pneumonia in adult adult human metapneumovirus encephalitis: a case report highlighting challenges in clinical management and functional outcome human metapneumovirus rna in encephalitis patient human metapneumovirus in the cerebrospinal fluid of a patient with acute encephalitis henipavirus infection of the central nervous system henipavirus pathogenesis in human respiratory epithelial cells nipah virus-the rising epidemic: a review nipah virus infection human hendra virus infection causes acute and relapsing encephalitis nipah virus infection: pathology and pathogenesis of an emerging paramyxoviral zoonosis long-term neurological and functional outcome in nipah virus infection neuropsychiatric sequelae of nipah virus encephalitis clinical and pathological manifestations of human henipavirus infection rapid nipah virus entry into the central nervous system of hamsters via the olfactory route nipah virus persists in the brains of nonhuman primate survivors relapsed and late-onset nipah encephalitis pathogenesis of influenza virus infections: the good, the bad and the ugly emerging influenza d virus threat: what we know so far! neurologic complications of influenza b virus infection in adults highly pathogenic h5n1 influenza virus can enter the central nervous system and induce neuroinflammation and neurodegeneration pathology of human influenza revisited guillain-barre syndrome and influenza virus infection role of viral infections in the etiology of febrile seizures acute disseminated encephalomyelitis associated with influenza a h1n1 infection influenza-associated central nervous system dysfunction: a literature review acute encephalopathy and encephalitis caused by influenza virus infection clinical and mri features of neurological complications after influenza a (h1n1) infection in critically ill children subclinical brain injury caused by h5n1 influenza virus infection long-term neuroinflammation induced by influenza a virus infection and the impact on hippocampal neuron morphology and function influenza a virus infection causes alterations in expression of synaptic regulatory genes combined with changes in cognitive and emotional behaviors in mice influenza infection induces neuroinflammation, alters hippocampal neuron morphology, and impairs cognition in adult mice influenza virus infection exacerbates experimental autoimmune encephalomyelitis disease by promoting type i t cells infiltration into central nervous system temporal relationship between environmental influenza a and epstein-barr viral infections and high multiple sclerosis relapse occurrence clinical relapses and disease activity on magnetic resonance imaging associated with viral upper respiratory tract infections in multiple sclerosis surveillance of enteroviruses from paediatric patients attended at a tertiary hospital in catalonia from enterovirus d68 in critically ill children: a comparison with pandemic h1n1 influenza innate immunity evasion by enteroviruses: insights into virus-host interaction enterovirus infections in neonates rhinovirus-associated acute encephalitis/encephalopathy and cerebellitis human picornaviruses associated with neurological diseases and their neutralization by antibodies acute flaccid myelitis and enterovirus d68: lessons from the past and present acute flaccid myelitis associated with enterovirus d68: a review enterovirus d68: acute respiratory illness and the 2014 outbreak the epidemiology of non-polio enteroviruses: recent advances and outstanding questions coronavirus genome structure and replication a hemagglutinating virus producing encephalomyelitis in baby pigs diagnostic features of clinical neurologic feline infectious peritonitis inflammation and changes in cytokine levels in neurological feline infectious peritonitis mechanism of demyelination in jhm virus encephalomyelitis. electron microscopic studies pathogenesis of murine coronavirus in the central nervous system murine coronavirus neuropathogenesis: determinants of virulence the pathogenesis of murine coronavirus infection of the central nervous system human coronavirus infections cultivation of a novel type of common-cold virus in organ cultures a new virus isolated from the human respiratory tract growth in suckling-mouse brain of "ibv-like" viruses from patients with upper respiratory tract disease identification of a novel coronavirus in patients with severe acute respiratory syndrome aetiology: koch's postulates fulfilled for sars virus a novel coronavirus associated with severe acute respiratory syndrome identification of a new human coronavirus characterization and complete genome sequence of a novel coronavirus, coronavirus hku1, from patients with pneumonia isolation of a novel coronavirus from a man with pneumonia in saudi arabia epidemiological and clinical features of human coronavirus infections among different subsets of patients. influenza other respir. viruses epidemiology and clinical presentations of the four human coronaviruses 229e, hku1, nl63, and oc43 detected over 3 years using a novel multiplex real-time pcr method isolation of rhinoviruses and coronaviruses from 38 colds in adults human coronavirus nl63 infection and other coronavirus infections in children hospitalized with acute respiratory disease in hong kong co-circulation of four human coronaviruses (hcovs) in queensland children with acute respiratory tract illnesses in 2004 human coronavirus infection among children with acute lower respiratory tract infection in thailand identification and evolutionary dynamics of two novel human coronavirus oc43 genotypes associated with acute respiratory infections: phylogenetic, spatiotemporal and transmission network analyses genotype shift in human coronavirus oc43 and emergence of a novel genotype by natural recombination genomic analysis of 16 colorado human nl63 coronaviruses identifies a new genotype, high sequence diversity in the n-terminal domain of the spike gene and evidence of recombination genetic variability of human coronavirus oc43-, 229e-, and nl63-like strains and their association with lower respiratory tract infections of hospitalized infants and immunocompromised patients molecular epidemiology of human coronavirus oc43 reveals evolution of different genotypes over time and recent emergence of a novel genotype due to natural recombination inter-and intra-variant genetic heterogeneity of human coronavirus oc43 strains in france genetic variability of human respiratory coronavirus oc43 comparative analysis of 22 coronavirus hku1 genomes reveals a novel genotype and evidence of natural recombination in coronavirus hku1 pathogenesis of human coronaviruses other than severe acute respiratory syndrome coronavirus update on human rhinovirus and coronavirus infections characterization of human coronavirus oc43 and human coronavirus nl63 infections among hospitalized children <5 years of age respiratory viral detection in children and adults: comparing asymptomatic controls and patients with community-acquired pneumonia characteristics and outcomes of coronavirus infection in children: the role of viral factors and an immunocompromised state prolonged shedding of human coronavirus in hematopoietic cell transplant recipients: risk factors and viral genome evolution clinical significance of human coronavirus in bronchoalveolar lavage samples from hematopoietic cell transplant recipients and patients with hematologic malignancies isolation and characterization of viruses related to the sars coronavirus from animals in southern china progress in global surveillance and response capacity 10 years after severe acute respiratory syndrome the chronology of the 2002-2003 sars mini pandemic the first pandemic of the 21st century the pathology and pathogenesis of experimental severe acute respiratory syndrome and influenza in animal models multiple organ infection and the pathogenesis of sars mers: emergence of a novel human coronavirus emergence of the middle east respiratory syndrome coronavirus announcement of the coronavirus study group transmission and evolution of the middle east respiratory syndrome coronavirus in saudi arabia: a descriptive genomic study community case clusters of middle east respiratory syndrome coronavirus in hafr al-batin, kingdom of saudi arabia: a descriptive genomic study middle east respiratory syndrome novel corona mers-cov infection. epidemiology and outcome update epidemiological, demographic, and clinical characteristics of 47 cases of middle east respiratory syndrome coronavirus disease from saudi arabia: a descriptive study hospital outbreak of middle east respiratory syndrome coronavirus neutralizing the mers coronavirus threat mers-cov outbreak following a single patient exposure in an emergency room in south korea: an epidemiological outbreak study epidemiological investigation of mers-cov spread in a single hospital in south korea middle east respiratory syndrome coronavirus and the one health concept mers coronavirus outbreak: implications for emerging viral infections an opportunistic pathogen afforded ample opportunities: middle east respiratory syndrome coronavirus severe acute respiratory syndrome vs. the middle east respiratory syndrome severe acute respiratory syndrome the role of human coronaviruses in children hospitalized for acute bronchiolitis, acute gastroenteritis, and febrile seizures: a 2-year prospective study coronavirus infections of man associated with diseases other than the common cold human enteric coronaviruses: antigenic relatedness to human coronavirus oc43 and possible etiologic role in viral gastroenteritis isolation and propagation of a human enteric coronavirus human coronaviruses are uncommon in patients with gastrointestinal illness detection of human coronaviruses in children with acute gastroenteritis human coronavirus oc43 associated with fatal encephalitis neuroinvasion by human respiratory coronaviruses human coronavirus polyadenylated rna sequences in cerebrospinal fluid from multiple sclerosis patients cerebrospinal fluid antibodies to coronavirus in patients with parkinson's disease human coronavirus gene expression in the brains of multiple sclerosis patients detection of coronavirus in the central nervous system of a child with acute disseminated encephalomyelitis the effects of coronavirus on human nasal ciliated respiratory epithelium isolation and characterization of current human coronavirus strains in primary human epithelial cell cultures reveal differences in target cell tropism activation of human monocytes after infection by human coronavirus 229e in vitro detection of apoptosis in monocytes/macrophages infected with human coronavirus a human coronavirus responsible for the common cold massively kills dendritic cells but not monocytes cells of human aminopeptidase n (cd13) transgenic mice are infected by human coronavirus-229e in vitro, but not in vivo inhibition of cytokine gene expression and induction of chemokine genes in non-lymphatic cells infected with sars coronavirus systemic immune deficiency necessary for cytomegalovirus invasion of the mature brain development of a transgenic mouse model susceptible to human coronavirus 229e olfactory transmission of neurotropic viruses the olfactory bulb: an immunosensory effector organ during neurotropic viral infections vacuolating encephalitis in mice infected by human coronavirus oc43 lethal infection of k18-hace2 mice infected with severe acute respiratory syndrome coronavirus murine encephalitis caused by hcov-oc43, a human coronavirus with broad species specificity, is partly immune-mediated human respiratory coronavirus oc43: genetic stability and neuroinvasion severe acute respiratory syndrome coronavirus infection causes neuronal death in the absence of encephalitis in mice transgenic for human ace2 cleavage of a neuroinvasive human respiratory virus spike glycoprotein by proprotein convertases modulates neurovirulence and virus spread within the central nervous system mutations in the spike glycoprotein of human coronavirus oc43 modulate disease in balb/c mice from encephalitis to flaccid paralysis and demyelination glutamate excitotoxicity is involved in the induction of paralysis in mice after infection by a human coronavirus with a single point mutation in its spike protein axonal transport enables neuron-to-neuron propagation of human coronavirus oc43 detection of severe acute respiratory syndrome coronavirus in the brain: potential role of the chemokine mig in pathogenesis a rare cause of acute flaccid paralysis: human coronaviruses possible central nervous system infection by sars coronavirus guillain-barre syndrome with unilateral peripheral facial and bulbar palsy in a child: a case report effects of coronavirus infections in children neurological manifestations in severe acute respiratory syndrome neurological complications of middle east respiratory syndrome coronavirus: a report of two cases and review of the literature severe neurologic syndrome associated with middle east respiratory syndrome corona virus (mers-cov) middle east respiratory syndrome coronavirus (mers-cov) outbreak in south korea, 2015: epidemiology, characteristics and public health implications coronavirus infections in the central nervous system and respiratory tract show distinct features in hospitalized children detection of coronavirus rna and antigen in multiple sclerosis brain clinical viral infections and multiple sclerosis coronavirus and multiple sclerosis: results of a case/control longitudinal serological study an overview of their replication and pathogenesis bioterror: infection control awareness for the optometrist coronavirus immunoreactivity in individuals with a recent onset of psychotic symptoms severity and outcome associated with human coronavirus oc43 infections among children human coronavirus oc43 infection induces chronic encephalitis leading to disabilities in balb/c mice neuroprotective effect of apolipoprotein d against human coronavirus oc43-induced encephalitis in mice novel treatment with neuroprotective and antiviral properties against a neuroinvasive human respiratory virus neurological complications during treatment of middle east respiratory syndrome in vivo egress of an alphaherpesvirus from axons safe and sensitive antiviral screening platform based on recombinant human coronavirus oc43 expressing the luciferase reporter gene high-throughput screening and identification of potent broad-spectrum inhibitors of coronaviruses non-invasive bioluminescence imaging of hcov-oc43 infection and therapy in the central nervous system of live mice the acetyl-esterase activity of the hemagglutinin-esterase protein of human coronavirus oc43 strongly enhances the production of infectious virus the oc43 human coronavirus envelope protein is critical for infectious virus production and propagation in neuronal cells and is a determinant of neurovirulence and cns pathology acute and persistent infection of human neural cell lines by human coronavirus oc43 persistent infection of human oligodendrocytic and neuroglial cell lines by human coronavirus 229e a complement-microglial axis drives synapse loss during virus-induced memory impairment viruses have multiple paths to central nervous system pathology epidemiologic evidence for multiple sclerosis as an infection multiple sclerosis: autoimmunity and viruses infectious causes of multiple sclerosis viral triggers of multiple sclerosis infectious agents and different course of multiple sclerosis: a systematic review viruses and autoimmunity: a review on the potential interaction and molecular mechanisms human coronaviruses. respiratory pathogens revisited as infectious neuroinvasive, neurtropic, and neurovirulent agents long-term human coronavirus-myelin cross-reactive t-cell clones derived from multiple sclerosis patients myelin basic protein and human coronavirus 229e cross-reactive t cells in multiple sclerosis inflammation in neurodegenerative diseases tumor necrosis factor-alpha modulates glutamate transport in the cns and is a critical determinant of outcome from viral encephalomyelitis a human coronavirus oc43 variant harboring persistence-associated mutations in the s glycoprotein differentially induces the unfolded protein response in human neurons as compared to wild-type virus human coronavirus-induced neuronal programmed cell death is cyclophilin d dependent and potentially caspase dispensable pivotal role of receptor-interacting protein kinase 1 and mixed lineage kinase domain-like in neuronal cell death induced by the human neuroinvasive coronavirus oc43 molecular definitions of cell death subroutines: recommendations of the nomenclature committee on cell death coronavirus gene 7 counteracts host defenses and modulates virus virulence cell-type-specific activation of the oligoadenylate synthetase-rnase l pathway by a murine coronavirus antagonism of the interferon-induced oas-rnase l pathway by murine coronavirus ns2 protein is required for virus replication and liver pathology cell-type-specific type i interferon antagonism influences organ tropism of murine coronavirus coronaviruses as encephalitis-inducing infectious agents clinical metagenomic sequencing for diagnosis of meningitis and encephalitis human virome in nasopharynx and tracheal secretion samples the aetiology of tuberculosis (translation of die aetiologie der tuberculose (1882) sequence-based identification of microbial pathogens: a reconsideration of koch's postulates the environment and disease: association or causation? infectious causes of multiple sclerosis this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license we thank jessy tremblay for his excellent work in confocal microscopy images. the authors declare no conflict of interest. the funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. key: cord-261908-olcuq6tm authors: lai, ka-man; bottomley, christian; mcnerney, ruth title: propagation of respiratory aerosols by the vuvuzela date: 2011-05-23 journal: plos one doi: 10.1371/journal.pone.0020086 sha: doc_id: 261908 cord_uid: olcuq6tm vuvuzelas, the plastic blowing horns used by sports fans, recently achieved international recognition during the fifa world cup soccer tournament in south africa. we hypothesised that vuvuzelas might facilitate the generation and dissemination of respiratory aerosols. to investigate the quantity and size of aerosols emitted when the instrument is played, eight healthy volunteers were asked to blow a vuvuzela. for each individual the concentration of particles in expelled air was measured using a six channel laser particle counter and the duration of blowing and velocity of air leaving the vuvuzela were recorded. to allow comparison with other activities undertaken at sports events each individual was also asked to shout and the measurements were repeated while using a paper cone to confine the exhaled air. triplicate measurements were taken for each individual. the mean peak particle counts were 658×10(3) per litre for the vuvuzela and 3.7×10(3) per litre for shouting, representing a mean log(10) difference of 2.20 (95% ci: 2.03,2.36; p<0.001). the majority (>97%) of particles captured from either the vuvuzela or shouting were between 0.5 and 5 microns in diameter. mean peak airflows recorded for the vuvuzela and shouting were 6.1 and 1.8 litres per second respectively. we conclude that plastic blowing horns (vuvuzelas) have the capacity to propel extremely large numbers of aerosols into the atmosphere of a size able to penetrate the lower lung. some respiratory pathogens are spread via contaminated aerosols emitted by infected persons. further investigation is required to assess the potential of the vuvuzela to contribute to the transmission of aerosol borne diseases. we recommend, as a precautionary measure, that people with respiratory infections should be advised not to blow their vuvuzela in enclosed spaces and where there is a risk of infecting others. aerosols play an important role in the spread of communicable diseases [1, 2] . aerosol transmission can be airborne, where contaminated droplet nuclei exhaled by an infected individual are inhaled by a susceptible individual. a second route of infection is when deposited droplets are carried to the mouth or nose through physical contact, often by hand [3] . airborne aerosol transmission is believed to make a major contribution to the spread of diseases such as tuberculosis and measles [4, 5] . aerosols have also been implicated in the transmission of diseases such as the common cold, chickenpox, rubella, influenza, pneumococcal disease and severe acute respiratory syndrome (sars) [3, 6, 7, 8, 9, 10] , although their contribution is less clear cut as non aerosolized respiratory secretions also contribute to the spread of these diseases. some airborne pathogens are extremely contagious; in the usa an outbreak of measles was traced to a sporting event where transmission was found to have occurred between an athlete in the arena and spectators in the stadium, with no evidence of close contact [11] . spread of respiratory disease is of particular concern in large crowds and at international gatherings [12, 13] . this includes the annual muslim pilgrimage to mecca and associated sites, during which respiratory infections are the most common cause of hospitalization [14] . the reported infections include tuberculosis and influenza and the saudi ministry of health recommends wearing of protective face masks by those attending the hajj [13, 15] . the emergence of epidemic strains of flu have also caused concern; in 2009 fears around the spread of influenza h1n1 resulted in a temporary ban on public events in some countries [16] . aerosols are created and expelled into the atmosphere during coughing, sneezing, singing or talking. if the person has a respiratory infection a proportion of the aerosols may carry pathogenic particles [17, 18, 19] . the size of a contaminated aerosol droplet is crucial in determining its ability to transmit disease. whereas large drops (.100 microns diameter) will rapidly fall to the ground smaller droplets may remain suspended in the air where evaporation can occur resulting in the formation of tiny 'droplet nuclei' that can stay airborne for hours or days [20, 21] . these particles can be breathed in by susceptible individuals who may then become infected. the fate of the droplet nuclei on inhalation also depends on their size; particles greater than five microns are likely to remain in the upper airways but smaller particles are more likely to deposit in the alveoli and so may transmit infections of the lower respiratory tract such as tuberculosis [22, 23] . the vuvuzela is a plastic blowing horn that has been adopted by sports fans to provide audible support for their team. it is used in several countries in asia and africa and is particularly popular in south africa where it figured prominently during the recent fédération internationale de football association (fifa) world cup. the instrument is typically 60 cm in length, tapering from a bell end 11.5 cm in diameter to a mouthpiece of 2.5 cm. vuvuzela playing requires forceful and sustained blowing. air from the lungs is expelled from the mouth through vibrating lips held against the plastic mouthpiece and out through the instrument. we speculated that the mode of action may facilitate the propagation and dissemination of aerosols from the respiratory tract of the person blowing the instrument. the instrument is frequently used in crowded situations and it was therefore important to determine the extent of aerosol production to assess whether blowing the vuvuzela might assist in the spread of aerosol borne diseases. to assess the number of aerosols propagated during blowing the vuvuzela eight volunteers (4 male and 4 female) were each given an instrument and asked to blow enthusiastically, as if they were attending a football match. to enable comparison with other activities undertaken at sporting events each volunteer also shouted into a paper cone constructed to have the same 115 mm diameter exhale opening as the vuvuzela ( figure 1 ). particles exiting the vuvuzela or shouting cone were assessed using a laser particle counter and enumerated in six categories according to their diameter. the velocity of air as it exited the devices was measured with a hot-wire anemometer and peak airflows were recorded. the duration of playing was recorded with a stopwatch. triplicate experiments were undertaken for each individual tested. airborne particles exiting the instrument were measured every second throughout the experiment and reported as particles per litre. the mean concentration of particles recorded from playing the vuvuzela and shouting were 658610 3 and 3.7610 3 per litre respectively. to compare the number of particles emitted by an individual when shouting to the number emitted when playing the vuvuzela, the data were log 10 transformed and the difference was calculated for each individual. the mean log 10 difference was 2.20 (95% ci: 2.03,2.36; p,0.001). men expelled particles at a higher mean concentration than the women when playing the vuvuzela (741610 3 vs 575610 3 per litre) although this was not statistically significant (p = 0.69).when shouting there was no difference in the numbers of particles captured (male:female; 3.4610 3 vs 3.9610 3 per litre, p = 0.89). aerosols were enumerated in six size categories according to the diameter of the particle: 0.5-0.7 mm; 0.7-1.0 mm; 1.0-3.0 mm; 3.0-5.0 mm; 5.0-10.0 mm and .10.0 mm. the distribution of particles by size category is presented in figure 2 . the great majority (97%) of particles captured from both the vuvuzela and the shouting cone were between 0.5 and 5 microns in diameter and small enough to enter the lower respiratory tract. the geometric mean (gm) particle diameter was calculated for each experiment and is presented in table 1 . slightly larger particles were emitted when playing the vuvuzela compared to shouting the mean duration for vuvuzela playing events was 2.1 sec (range: 1.25-3.90 sec) and the shouting lasted for an average of 2.2 sec (range: 0.96-4.72 sec). the peak velocity of air exiting the vuvuzelas was higher than from the shouting cone with a mean of 0.59 ms 21 (range:0.12-1.80 ms 21 ;) compared to a shouting mean of 0.18 ms 21 (range: 0.07-0.32 ms 21 ) and this difference was statistically significant (p = 0.03). this was equivalent to airflow of 6.1 and 1.8 ls 21 respectively, for the vuvuzela and shouting. although the duration in playing vuvuzelas between females and males were similar (2.1 sec), the mean peak airflow was nearly double in males compared to females, 7.9 compared to 4.3 ls 21 (this difference was not statistically significant p = 0.19). the difference between females and males in shouting was not as apparent, although males also had a higher peak airflow compared to females, 2.1 compared to 1.6 ls 21 (p = 0.37). we have estimated the numbers of aerosols exiting the vuvuzela when blown by male and female adults. in triplicate experiments from eight individuals the mean concentration of particles exiting the vuvuzela was 658,000 per litre. the mean peak volume of air exiting the instrument was 6.1 litres per second. thus we estimate that approximately 4 million particles per second were being disseminated from the vuvuzela at peak blowing times. for shouting we estimated a peak aerosol concentration of 3,700 per litre or 7,000 particles per second (assuming peak flow volume of 1.8 ls 21 ). the data we obtained for shouting is in broad agreement with a recent study of particles exhaled by healthy adults during normal to deep breathing (tidal volume range: 20-80%) where between 5 and 5,000 droplets per litre were recorded [24] . the differences we observed between male and female volunteers might be explained by differences in their lung capacities, however this was not measured [24] . our results suggest that the vuvuzela is an efficient means of propagating large numbers of aerosols. the great majority of particles measured were of a size that could remain suspended in the air as droplet nuclei and would be capable of entering the alveolar airspaces of the lung. during normal (resting) breathing an adult inhales approximately 7 litres of air each minute, of which 5 litres reaches the respiratory bronchioles [25] . when attending a sporting event and surrounded by vuvuzela players a spectator could expect to inhale large numbers of respiratory aerosols over the course of the event. actual exposure would be affected by the proximity of the vuvuzelas and ambient ventilation which would serve to dilute the stream of particles. the large number of aerosols emitted by the vuvuzela raises the possibility that, if used by persons with an infection of the respiratory tract, they could act a conduit for the spread of infectious particles. for ethical and safety reasons we only examined healthy volunteers during this study; assessment of pathogenicity of aerosols disseminated by the vuvuzela will require further study using patients with known respiratory infections. aerosols can be created at various locations within the respiratory tract [26] and carriage of pathogens by exhaled aerosols depends on the site of infection and the quantity pathogenic particles in the airways [18] . we speculate that aerosols propagated while blowing the vuvuzela may originate in either the lower or upper respiratory tract, or the mouth. to obtain the desired trumpet sound when blowing the vuvuzela air is forced through the lips into the opening of the instrument which may serve to create further aerosols, or alter the size of droplets produced elsewhere in the respiratory tract. in addition to the manner in which the instrument was blown the number of contaminated particles expelled will vary according to the pathogen, the site of infection and the extent of disease. some infections may result in inflammation and physiological changes within the respiratory tract that would affect the person's capacity to blow the vuvuzela [27] . in addition, some conditions are associated with changes in the rheology of respiratory secretions that might affect aerosol formation [28, 29] . studies of cough aerosols from pulmonary tuberculosis patients and cystic fibrosis patients with bacterial infections found that the concentration of infectious particles varied widely between patients [30, 31] . to attain an accurate assessment of the vuvuzela's potential to disseminate infected aerosols, sample sizes will need to be increased to include individuals having a range of upper and lower respiratory tract infections. symptomatic and non symptomatic carriers should be assessed. in addition to counting the number and size of particles, the viability of infectious particles should also be assessed. for bacterial infections this might be achieved by modification of a cough aerosol sampling system previously used to assess tuberculosis patients [30] . coughing, sneezing, singing and talking can all produce aerosols capable of transmitting airborne respiratory diseases [17, 18, 30, 32] . reports from earlier investigators suggest that coughs may produce up to 5,000 droplet nuclei and a sneeze may generate as many as 900,000 particles [21, 33] . the data we present suggests that blowing the vuvuzela for even a short time period has the potential to create more droplet particles than either coughing or sneezing. there were some limitations to this study that may have had an impact on the results. the particle counter used to assess the concentration of particles recorded measurements at one second intervals and it is possible that the peak values recorded were not the maximum level of particle produced. as it was not possible to assess variation in flow rates over the blowing period the total number of particles expelled during a blowing or shouting event could not be estimated. the performance of individuals and production of aerosols may have been influenced by their respective lung capacities [24] , this factor was not assessed in the experiment. the use of a paper cone to assess the droplets from shouting was not ideal as the surface areas and shape of the paper cone may increase the chance that particles attach to the surface rather than remain in the airstream, affecting the number and size of particles reaching the counter. as exhaled air cools and mixes with ambient air condensation droplets may form. although ambient air temperature and humidity remained similar in all experiments, the difference in shape between the cone and the vuvuzela may have affected the mixing and rate of formation of table 1 . exhale duration, peak air velocity, particle concentration and mean particle diameter recorded during playing the vuvuzela and shouting by four male and four female volunteers. these transient droplets. a further consideration is that only healthy individuals were recruited for this study, and as described above, it is possible that people with respiratory illness with impaired lung function would perform differently when blowing the vuvuzela. nonetheless we have demonstrated that these plastic trumpets provide an excellent means of propagating respiratory aerosols, exceeding both sneezing and coughing as a means of disseminating droplet nuclei and we conclude that their potential to spread respiratory diseases requires further investigation. the frequency, duration, and vigor of vuvuzela playing will vary considerably from person to person, depending on the occasion and their expertise at blowing and we are unable to comment on the number of aerosols produced during an entire sporting event. a further factor is the environment in which they are used; open situations with a strong draft or breeze will serve to rapidly dilute the aerosols produced but transmission risks may be considerably higher in enclosed arenas. a further risk factor for disease transmission will be the density of vuvuzela players and the prevalence of respiratory infections in the population. as far as we are aware this is the first report in the scientific press regarding the issue of aerosol dissemination by the vuvuzela and no epidemiological data regarding impact of the instrument on disease transmission have been reported. similarly there have been no reports of disease transmission from sharing vuvuzelas, or from transfer of non aerosolized respiratory secretions that collect inside the instruments. the vuvuzela has become popular in south africa, a country with the highest urban prevalence of tuberculosis in the world and that recently experienced a measles epidemic [34] . it has been used at domestic soccer games for the past decade and was adopted by many visiting fans during the 2010 fifa world cup competition. the tournament was held during late june and early july and coincided with the annual flu season. surveillance reports show an increase in the proportion of influenza b compared to previous years, but evidence to link this to the presence of visiting spectators is not presented [35] . similarly a number of measles cases were confirmed amongst visitors from other countries but evidence as to the source of their infections is not available [36] . the plastic vuvuzela is believed to have emerged as a child's toy, before being adopted by sports fans in africa and parts of asia, where it is a multi-million dollar industry. in africa it has become a symbol of the soccer industry but vuvuzelas are also blown by fans of cricket and rugby football. they have been banned from a number of sports grounds due to the volume of noise emitted and safety concerns arising from their ability to nullify public address systems. studies have previously suggested that vuvuzela playing poses a risk of noise induced hearing loss [37, 38] . we recommend that consideration is taken of their propensity to disseminate respiratory aerosols and that persons with respiratory infections be advised not to blow their vuvuzela in places where they risk infecting others. this should include enclosed spaces and crowded venues such as large sporting events. we also recommend that research be commissioned to determine the risks to public health posed by the vuvuzela. this study was undertaken at the healthy infrastructure research centre at university college london. ethical approval was obtained from university college london research ethics committee and informed consent was obtained in writing from all participants. eight healthy volunteers, 4 males and 4 females working in the research centre participated. the experiments were conducted in a closed room free from drafts. the study subjects were in the age range 20 to 45 years and all self reported as being free from illness. to avoid cross contamination a new vuvuzela instrument was provided for each participant (boogie blast co, johannesburg, sa). the velocity of air leaving the instrument was measured using a hot-wire anemometer (testo, ukflow) and duration of playing recorded with a stopwatch. our initial measurements showed that the average time of playing the vuvuzela was about 2 sec. to enable comparison with other activities undertaken at sporting events each individual tested was requested to also shout into a paper cone constructed to have the same diameter exhale opening as the vuvuzela (figure 1 ). subjects were requested to hold their shout for about 2 sec (not compulsory) and to shout the word ''go''. particles exiting the vuvuzela or the shouting cone were measured using a six channel laser particle counter (lighthouse 5016, uk). particles were enumerated in six categories according to their diameter: 0.5-0.7 mm; 0.7-1.0 mm; 1.0-3.0 mm; 3.0-5.0 mm; 5.0-10.0 mm and .10.0 mm. a 0.047 litre sample of air was tested every second and the number of particles recorded. analyses are based on either the average or peak concentration observed during the vuvuzela or shouting event. triplicate experiments were undertaken for each individual tested. volume of the airflow was estimated by multiplying the peak air velocity recorded in the anemometer with the duration of playing and shouting and the surface area of the exhale opening of the vuvuzela and paper cone. in each experiment (in which an individual either shouted or blew on a vuvuzela) data were collected on particle concentration every second. these data were summarized in one of two ways: i) as the concentration observed in the 2 nd second after the start of the experiment, which usually corresponded to the peak concentration or ii) as the average concentration over the length of the shout or vuvuzela blow. all analyses were carried out using both peak and average concentrations. however, since both yielded similar results, we restrict our presentation to the analysis of peak concentrations. the geometric mean size (gm) of particles was calculated for each experiment by fitting a log normal distribution to the particle size data at peak concentration. estimates were obtained by maximum likelihood, allowing for interval-censoring (particle sizes were recorded using the categories 0.5-0.7 mm; 0.7-1.0 mm; 1.0-3.0 mm; 3.0-5.0 mm; 5.0 -10.0 mm and .10.0 mm). each individual shouted and blew the vuvuzela three times. statistical analysis of particle concentrations and gm particle size were based on the means of these triplicate measurements; this was done to eliminate dependence in the data arising from repeat measurements on the same individual. to compare the number of particles emitted when shouting to the number emitted when playing the vuvuzela, we logtransformed each individual's average concentration (averaged over the three measurements) when shouting and when playing the vuvuzela and calculated the difference between these logtransformed values. the confidence interval for the mean difference and p-value were based on a paired (one-sample) t-test. differences in the average (gm) particle size between vuvuzela and shouting were similarly assessed using a paired t-test (although the data were not log-transformed in this instance). comparisons between men and women of particle concentration and airflow were made using permutation tests (based on the wicoxon rank sum statistic) rather than t-tests owing to the small numbers (4 men and 4 women). the mechanism of transmission of pathogenic organisms affecting the respiratory tract factors involved in the aerosol transmission of infection and control of ventilation in healthcare premises mechanisms of transmission of rhinovirus infections strategies for minimizing nosocomial measles transmission aerial dissemination of pulmonary tuberculosis. a two-year study of contagion in a tuberculosis ward viruses as agents of airborne contagion airborne transmission of communicable infectionthe elusive pathway review of aerosol transmission of influenza a virus an epidemic of pneumococcal disease in an overcrowded, inadequately ventilated jail airborne transmission of chickenpox in a hospital an outbreak of measles at an international sporting event with airborne transmission in a domed stadium pandemic influenza: mass gatherings and mass infection health risks at the hajj pattern of admission to hospitals during muslim pilgrimage (hajj) bacteria and viruses that cause respiratory tract infections during the pilgrimage (haj) season in makkah, saudi arabia questions raised over response to influenza a outbreak singing and the dissemination of tuberculosis expulsion of pathogenic organisms from respiratory tract airborne infection on air-borne infection study ii. droplets and droplet nuclei the size and the duration of air-carriage of respiratory droplets and droplet-nuclei distribution and deposition of inhaled particles in respiratory tract on the mechanics of droplet nuclei infection. ii quantitative experimental air-borne tuberculosis in rabbits characterization of exhaled particles from the healthy human lung-a systematic analysis in relation to pulmonary function variables respiratory physiology upate in anaethesia: world federation of societies of anaesthesiologists size distribution and sites of origin of droplets expelled from the human respiratory tract during expiratory activities pulmonary function during and after common respiratory infections macrorheology of cystic fibrosis, chronic obstructive pulmonary disease & normal sputum airborne infectious disease and the suppression of pulmonary bioaerosols cough-generated aerosols of mycobacterium tuberculosis: a new method to study infectiousness cough-generated aerosols of pseudomonas aeruginosa and other gram-negative bacteria from patients with cystic fibrosis epidemiology of primary tuberculosis in an industrial school characterization of expiration air jets and droplet size distributions immediately at the mouth opening global tuberculosis control:who severe acute respiratory illness (sari) surveillance: influenza report fifa soccer would cup, south africa: communicable disease risks and surveillance. pretoria: national institute for communicable diseases vuvuzela sound measurements vuvuzela -good for your team, bad for your ears we would like to thank hector altamirano-medina for providing technical support. we are grateful to the volunteers who gave their breath in this study. key: cord-259422-5ex12eun authors: graat, judith m; schouten, evert g; heijnen, marie-louise a; kok, frans j; pallast, esther g.m; de greeff, sabine c; dorigo-zetsma, j.wendelien title: a prospective, community-based study on virologic assessment among elderly people with and without symptoms of acute respiratory infection date: 2003-12-11 journal: j clin epidemiol doi: 10.1016/s0895-4356(03)00171-9 sha: doc_id: 259422 cord_uid: 5ex12eun background and objective: community-based elderly studies concerning microbiology of acute respiratory infections are scarce. data on subclinical infections are even totally absent, although asymptomatic persons might act as a source of respiratory infections. methods: in a 1-year community-based study, we prospectively investigated the possible virologic cause of acute respiratory infections in 107 symptomatic case episodes and 91 symptom-free control periods. participants, persons ⩾60 years, reported daily the presence of respiratory symptoms in a diary. virologic assessment was performed by polymerase chain reaction (pcr) and serology. results: in 58% of the case episodes a pathogen was demonstrated, the most common being rhinoviruses (32%), coronaviruses (17%), and influenzaviruses (7%). the odds ratio for demonstrating a virus in cases with symptoms vs. controls without symptoms was 30.0 (95% confidence interval 10.2–87.6). in 4% of the symptom-free control periods a virus was detected. conclusion: this study supports the importance of rhinovirus infections in community-dwelling elderly persons, whereas asymptomatic elderly persons can also harbor pathogens as detected by pcr, and thus might be a source of infection for their environment. elderly people have an increased susceptibility for respiratory infections and related complications [1] . on average, community-dwelling elderly people suffer from 1.2-1.6 acute respiratory infections per year [2, 3] . medical consultation and hospitalization because of such an infection has been reported in 40 and 0.8% of community-dwelling elderly people, respectively, during the winters of 1992-1993 and 1993-1994 in england [2] . viruses play a crucial role in acute upper respiratory tract infections, the most common being rhinoviruses, coronaviruses, influenzaviruses, and respiratory syncytial viruses [4] . however, laboratory diagnosis of acute respiratory infections in symptomatic elderly people so far focussed on institutionalized elderly persons [5] [6] [7] , on patients reporting for medical consultation [8, 9] , and to a far less degree on community-dwelling elderly persons [2] . besides, no data are available on the presence of respiratory pathogens in asymptomatic elderly persons. asymptomatic people with a subclinical infection might, however, transmit the pathogen to other persons and act as an unrecognized source of respiratory infections. therefore, in this prospective, community-based study, we investigated the presence of known respiratory viruses in elderly persons both with and without symptoms of an acute upper respiratory tract infection. second, we compared the clinical characteristics of the persons suffering from an acute respiratory infection, during episodes with positive and negative virologic laboratory diagnosis. persons with and without symptoms of an acute respiratory infection, hereafter referred to as cases and controls, were recruited from october 1, 1998, until october 1, 1999 , from an intervention trial investigating the effect of micronutrient supplementation on acute respiratory infections in community-dwelling elderly persons (у60 years) [3] . during the 1-year study period, a diary was used daily by all participants for reporting symptoms that indicated an acute respiratory infection. participants were requested to report the onset of symptoms of a possible infection to the study nurse. a subject was identified as case if (1) he/she had respiratory symptoms with a sudden onset; (2) rhinorrhoea/ sneezing, sore throat/hoarseness, or dry cough were present for at least 2 days; and (3) the symptoms had a pattern that differed from any usual symptoms [10, 11] . apart from a check by telephone, the study nurse evaluated the symptoms of cases during home visits. from those cases that reported their symptoms within 3 days to the study nurse every other case, with a maximum of five cases per week, was selected for virologic assessment. cases who reported their symptoms after 3 days to the study nurse were excluded for virologic assessment to overcome false negative test results. each case episode, i.e., the period during which a case had respiratory symptoms, had to have been preceded by a 7-day symptom-free period. during the 1-year study period, 624 incident case episodes were reported by 346 cases. in total 107 (17%) case episodes-reported by 97 cases-were selected for virologic assessment. for each case episode an asymptomatic control was selected as follows. participant numbers, including all cases and controls, ranged from 1-652. if the participant number of the case was 325 or lower, a closest eligible control was selected on participant number by counting back on these numbers. if the participant number of the case was 326 or higher, a closest eligible control was selected by counting forward on these numbers. controls were subjects without symptoms of a respiratory infection within a time window of 8 weeks before and 8 weeks after the symptomatic period of the index case. the study nurse checked the absence of symptoms at the time of recruitment of the control and the diary was checked for absence of symptoms in the previous eight weeks. in total, 99 controls were selected. cases and controls were matched on age (ϯ5 years) and sex, and they were not living in the same house or apartment, did not have chronic obstructive pulmonary disease, asthma, or cancer, and did not use severe immunosuppressive medication. afterwards, we excluded 8 (8%) of the 99 originally enrolled controls because they developed symptoms after a median duration of 21 days (range 9-54). for six excluded controls serologic testing was negative, while for two it was missing. with pcr two times a rhinovirus and two times a coronavirus oc43 was detected in the eight excluded controls. results presented are therefore based on the 107 case episodes and 91 control periods. this study was approved by the medical ethics committee of the wageningen university, the netherlands, and written informed consent was obtained from all participants prior to the study. all participants filled out a questionnaire concerning relevant subject characteristics at baseline. a diary was used daily for self-report of symptoms that indicated an acute respiratory infection. apart from the symptoms that had to be present because of our case definition (rhinorrhoea/ sneezing, sore throat/hoarseness, dry cough), also accompanying symptoms were recorded in the diary: (1) symptoms of a lower respiratory tract infection (sputum production, wheezing, pain on respiration), (2) systemic symptoms (fever-self-assessed by a supplied thermometer-malaise, headache, rigors, muscular pain, perspiration), (3) other symptoms (tearful eyes, pain in facial sinuses or ear), (4) restriction of activity (staying in bed, not being able to do daily activities, staying at home), (5) episode-related medication, including antibiotic use, (6) medical consultation, and (7) hospitalization [2] . if the study nurse judged during a home visit the case's symptoms as an acute respiratory infection, in both the case and the matched control an acute phase serum sample and one swab from the nose and one from the throat were taken within 3 days and a convalescent serum sample was taken within 2-4 weeks after onset of the first symptoms of the case. samples in cases and controls were taken on the same day to exclude seasonal differences. pcr or serology was used to diagnose infection with the eight most common respiratory viruses and mycoplasma pneumoniae (m. pneumoniae). pcr was performed for those viruses for which either no or only aspecific serology was available and for which validated pcr tests were available in our lab. infections with rhinovirus, enterovirus, coronavirus oc43 and 229e, and respiratory syncytial virus were diagnosed by pcr. serology was performed for those viruses for which either no pcr was available, or the nucleic acid extraction method had to be changed for dna isolation (in the case of m. pneumoniae). infections with influenzavirus a and b, parainfluenzavirus 1, 2, and 3, adenovirus and m. pneumoniae were diagnosed by serology. swabs from the nose and from the throat, hereafter referred to as "nose/throat samples," were placed together in 4-ml hanks' balanced salt solution containing gelatin, lactalbumin, yeast, and antibiotics. upon receipt of the nose/ throat samples at the laboratory, the swabs were twirled in the transport medium and removed. an aliquot of 200 µl of the sample was used for nucleic acid extraction by using the high pure rna isolation kit (boehringer, mannheim, germany). five microliters of the eluted rna preparation was used in a 25 µl single-tube rt-pcr followed by a nested-pcr using primer pairs as described previously for rhino-/enterovirus [12] . another 5 µl of extracted rna was used in a single 25 µl single-tube rt-pcr followed by a nested-pcr using primer pairs as described previously for respiratory syncytial virus (rsv) and coronavirus oc43 and 229e [13, 14] in a multiplex format. in the rna isolation procedure and pcr-method for rsv detection, sensitivity for rsv a was about one virus particle and for rsv b about 70 virus particles. the virus particle count was determined by quantitative em (advanced biotechnologies incorporated, columbia, md). positive controls from culture were used in each pcr test for the respective viruses. to prevent carryover contamination within the laboratory, preparation of the patient samples and pcr mixtures was performed in safety hoods in separate dedicated positive pressure laboratories. to check for carryover contamination of samples and for amplicon contamination during the procedure, negative controls, consisting of transport medium, were included after every fifth patient sample. subjects with a positive pcr result were considered to be infected by a known virus, which was interpreted as a laboratory-confirmed infection. paired sera from all cases and controls were analyzed for igg antibodies against influenzavirus a and b, adenovirus, and m. pneumoniae. for para-influenzavirus 1, 2, and 3, iga antibodies, combining the three antigens in one assay, were detected. analyses were performed using commercially available elisa (serion immunodiagnostica gmbh, würzburg, germany), and quantitative results, expressed in units/milliliter, were calculated using a lot-specific standard curve and calculation table as supplied in the test kit. results were interpreted as negative, indeterminate, or positive according to the manufacturer instructions. in the case of indeterminate results for the parainfluenza iga assay on paired sera, detection of total antibodies against separate parainfluenza 1, 2, and 3 antigens was repeated in a complement fixation assay (cfa), using commercially available parainfluenza 1, 2, and 3 antigens (virion, ruschlikon, switzerland). in elisas, a change from negative to positive result and in the cfa a fourfold rise in antibody titer between the paired sera was interpreted to be a laboratory-confirmed respiratory infection. data analysis concerning virologic (including m. pneumoniae) assessment was performed with the 107 case episodes and the 91 control periods. differences in the distributions for continuous data, i.e., age, self-perceived health, and illness duration were compared with independent sample student's t-test. illness duration was not normally distributed, and was log transformed to obtain normality. a chi-square test or a fisher's exact test was used to test the correlation between discrete variables, i.e., sex, influenza vaccination, smoking habits, allergy, sharing an apartment, presence of micro-organisms, symptoms of a lower respiratory tract infection, systemic and other symptoms, restriction of activity, fever, medical consultation, hospitalization, episode-related medication, and episode-related antibiotic use. a fisher's exact test was used to calculate the odds ratio for demonstrating a virus in cases with symptoms of acute respiratory infection vs. controls without symptoms of such an infection. alpha was taken as 0.05 in all analyses. the matching procedure on sex and age resulted in wellbalanced groups of cases and controls with respect to these and other relevant variables (table 1) . micronutrient supplementation related to the intervention trial was also similar between cases and controls [3] . the 97 symptomatic cases had 107 case episodes of respiratory infection, during which virologic (including m. pneumoniae) tests were performed. in 62 (58%) of these case episodes at least one micro-organism was demonstrated, whereas in two of these 62 two different micro-organisms were demonstrated. in 45 (42%) case episodes none of the applied tests was positive. of 10 cases, two case episodes were included. for seven out of the mentioned 10 cases, test results were different, i.e., different pathogens, or negative in one and positive virology in the other episode. in two cases, both episodes had negative virology. only in one case, in both episodes rhinovirus was detected. the most common viruses demonstrated were rhinoviruses (32%) and coronaviruses (17%) followed by influenzaviruses (7%), enteroviruses (2%), parainfluenzaviruses (2%) and m. pneumoniae (1%). respiratory syncytial virus and adenovirus were not detected. three of the seven cases diagnosed with an influenzavirus infection had been vaccinated against influenza. none of the titer rises on which the influenzavirus infection was diagnosed, was related to vaccination, as 2 to 4 months passed between vaccination and the diagnosis of an influenzavirus infection. presence of rhinovirus infections was almost five times higher compared to influenzavirus infections in this community-dwelling elderly population (table 2 ). in 4 out of 91 control periods (4%) a virus was demonstrated, i.e., two times a rhinovirus and two times a coronavirus. two out of these four controls with positive virology never showed symptoms of a respiratory infection during the 1-year study period. the two remaining controls with positive virology did not have any symptoms at least 3.5 and 4 months before and 8 and 4 months after sample collection, respectively. overall, the odds ratio for demonstrating a virus (or m. pneumoniae) in cases with symptoms vs. controls without symptoms of acute respiratory infection was 30.0 (95% confidence interval 10.2-87.6). despite small numbers (n ϭ 5) significantly more influenzavirus a infections were identified during symptomatic periods in winter (october-march) compared to summer (p ϭ .02). enteroviruses, parainfluenzaviruses and m. pneumoniae were only detected in summer (april-september). clinical characteristics of the persons suffering from an acute respiratory infection, during episodes with positive and negative virologic laboratory diagnosis, are described in table 3 . influenzavirus infection was associated with significantly longer illness duration and more systemic symptoms than the other infections with positive and negative virology. restriction of activity, presence of fever, medical consultation, and antibiotic use were also more frequently reported during influenzavirus infections, although not significantly different from the other infections with positive and negative virology. this study shows that subclinical respiratory infections occur in a minor part (4%) of asymptomatic elderly persons. besides, we showed the importance of rhinovirus infections in community-dwelling elderly people because of its high frequency. to our knowledge, this is the first study to investigate several common respiratory pathogens in community-dwelling elderly persons both with and without symptoms of an acute respiratory infection. so far, only two studies reported on microbiologic evidence of respiratory infection in community-dwelling healthy subjects with and without symptoms of such an infection. one study focussed on detection of rhinoviruses and enteroviruses by pcr in children and adults [15] . in 12 and 4% of the asymptomatic children and adults, respectively, virologic assessment was positive. although johnston et al. [15] tested only for rhinoviruses and enteroviruses, the frequency of subclinical respiratory infections in those healthy adults is similar to what we observed in our older population. preliminary results of a dutch study being performed in persons consulting their general practitioner for signs and symptoms of an acute respiratory infection, showed a positive virologic assessment in 19% of the controls [16] . this percentage is higher than observed in our study. however, that study population existed of participants from all age categories, including babies and children. as showed by johnston et al. [15] the percentage of asymptomatic persons with positive virologic assessment is clearly higher in children, which might explain the discrepancy. common viral pathogens demonstrated during symptomatic periods in children and adults [4, 17] , in institutionalized elderly patients [7, 11] , in patients with medical consultation [8] , and in community-dwelling elderly persons [2] are rhinoviruses, coronaviruses, influenzavirus a and b, and rsv, which is in line with our results. the frequency of the most common viruses varies between the different subpopulations. corresponding to one previously performed communitybased elderly study, we also showed that rhinovirus infections are highly prevalent, and can cause a great overall disease burden in this population [2] . corresponding to the results of nicholson et al. in community-dwelling elderly persons [2] , but in contrast to studies in more frail elderly persons as those living institutionalized and to studies with a general practitioner-based setting [7, 11, 18] , we also observed that influenzavirus infections and rsv infections seem to occur less frequent in free-living elderly people. a severe morbidity is caused by viruses such as influenzavirus and rsv [19] , which corresponds to our results on influenzavirus infections. this might explain the higher frequency of rsv and influenzavirus infections demonstrated in studies with general practitioner-based or institutionalized setting [19] . in total, 4 out of 7 patients with influenzavirus infection were not vaccinated against influenza. this might indicate the need for preventive vaccination in elderly persons. in agreement with other studies in institutionalized [7, 11] and in community-dwelling elderly subjects [2] , we found that infections with parainfluenzavirus, enterovirus, adenovirus, and m. pneumoniae are of minor importance in causing acute respiratory infections in elderly persons. we obtained a microbiologic diagnosis in 58% of the case episodes. the diagnostic deficit of 42% is relatively low, as in most studies a micro-organism was demonstrated in at maximum 50% of the case episodes [2, 7, 8] . other, partly new or unknown viruses, bacteria, and atypical microorganisms other than m. pneumoniae may be responsible for some of the clinical and possible additional subclinical infections with negative microbiology. bacterial, atypical and viral micro-organisms in adult patients consulting for respiratory infection have been shown in 12, 20, and 50% of the patients, respectively [20] . also, chlamydia species are reported to cause acute respiratory infections in communitydwelling elderly persons [2] , although the proportion of bacterial infections is reported to be rare in adult patients with common cold [4] . besides, chlamydia infections occurred in 1% only of the community-dwelling elderly people, and were mainly analyzed in patients with copd and asthma, while we excluded those patients [2, 21] . however, we cannot exclude that part of the diagnostic deficit in our study might be explained by such bacterial and atypical micro-organisms. little is known about the time period after infection during which pcr-based tests are positive [12, 14, 22] . this issue is especially crucial in interpreting pcr positive results in nose/throat samples obtained from subjects both with and without symptoms of a respiratory infection. andeweg et al. [12] demonstrated that rhinoviruses were no longer detected by pcr in patients who had recovered from disease. in our study, all cases and controls were followed day to day by using a self-reporting diary system. it was therefore possible to include only controls not having any symptoms 2 months before and 2 months after sampling. thus, it is very unlikely that the controls, in which a virus was detected, were in the postinfectious or incubation period of a symptomatic infection. moreover, in nose/throat samples of four of the eight excluded controls a respiratory virus was detected, and those controls apparently were in the incubation period. detection of rhinovirus, enterovirus, rsv, and coronavirus infections by the pcr method has been used before and is widely accepted [12] [13] [14] . although pcr-based tests are highly sensitive and specific, false positives due to contamination of negative samples with pcr product in the laboratory might have occurred [23] . however, given the strict conditions under which pcr was performed [24] , this is very unlikely. negative controls included after each fifth test sample were pcr-negative in all samples, indicating that contamination was effectively prevented. underreporting could have occurred if cases were admitted to the hospital when having an acute respiratory infection. because none of the cases reported having been admitted to a hospital because of acute respiratory infection or its complications, underreporting because of hospitalization is no issue in this study. the subjects who participated in this study were recruited from an intervention trial studying the effect of micronutrient supplementation on acute respiratory infections. random selection of participants of this double-blind intervention trial resulted in a similar distribution of supplementation between cases and controls. there was no significant correlation between positive microbiologic testing and (type of) supplementation [3] . therefore, confounding by the supplementation is likely to be negligible in this study. in conclusion, rhinovirus infections cause substantial morbidity among community-dwelling elderly persons because of its high prevalence in this population. also, although definitely more respiratory micro-organisms were demonstrated among persons with symptoms of an acute respiratory infection, asymptomatic elderly persons can also harbor respiratory pathogens, and thus might be a source of infection for their environment. the aging immune system: primer and prospectus acute viral infections of upper respiratory tract in elderly people living in the community: comparative, prospective, population based study of disease burden effect of daily vitamin e and multivitamin-mineral supplementation on acute respiratory tract infections in elderly persons-a randomized controlled trial viruses and bacteria in the etiology of the common cold meeting the nutritional needs of the elderly in the institutional setting non-influenza respiratory viruses may overlap and obscure influenza activity acute respiratory tract infection in daycare centers for older persons prospective study of aetiology and outcome of adult lower-respiratorytract infections in the community evaluation of clinical case definitions of influenza: detailed investigation of patients during the 1995-1996 epidemic in france the tecumseh study of respiratory illness. i. plan of study and observations on syndromes of acute respiratory disease acute upper respiratory tract viral illness and influenza immunization in homes for the elderly improved detection of rhinoviruses in clinical samples by using a newly developed nested reverse transcription-pcr assay detection of respiratory syncytial virus in acute bronchiolitis in infants evaluation of nested polymerase chain methods for the detection of human coronaviruses 229e and oc43 use of polymerase chain reaction for diagnosis of picornavirus infection in subjects with and without respiratory symptoms ari-el studie: acute respiratoire infecties in de eerste lijn acute respiratory illness in the community. frequency of illness and the agents involved a brief clinical instrument to classify frailty in elderly people respiratory syncytial virus or influenza? aetiology of respiratory tract infections: clinical assessment versus serological tests assessment of polymerase chain reaction and serology for detection of chlamydia pneumoniae in patients with acute respiratory tract infection low prevalence of chlamydia pneumoniae and mycoplasma pneumoniae among patients with symptoms of respiratory tract infections in dutch general practices polymerase chain reaction demonstration by a nested pcr for mycoplasma pneumoniae that m-pneumoniae load in the throat is higher in patients hospitalised for m-pneumoniae infection than in non-hospitalised subjects this work was funded by grant 28-2639 from the netherlands organization for health research and development (zon-mw), the hague. we are grateful to francis van rooij, division of human nutrition and epidemiology, wageningen university, the netherlands for her assistance in data and specimen collection. we want to thank berry wilbrink, henk boswijk, hans van der nat, and henk hooft, diagnostic laboratory for infectious diseases and perinatal screening, national institute of public health and the environment, the netherlands, for their technical help. key: cord-258366-fu9b446y authors: couto, carla r.; pannuti, cláudio s.; paz, josé p.; fink, maria c. d.; machado, alessandra a.; de marchi, michela; machado, clarisse m. title: fighting misconceptions to improve compliance with influenza vaccination among health care workers: an educational project date: 2012-02-06 journal: plos one doi: 10.1371/journal.pone.0030670 sha: doc_id: 258366 cord_uid: fu9b446y the compliance with influenza vaccination is poor among health care workers (hcws) due to misconceptions about safety and effectiveness of influenza vaccine. we proposed an educational prospective study to demonstrate to hcws that influenza vaccine is safe and that other respiratory viruses (rv) are the cause of respiratory symptoms in the months following influenza vaccination. 398 hcws were surveyed for adverse events (ae) occurring within 48 h of vaccination. ae were reported by 30% of the hcws. no severe ae was observed. a subset of 337 hcws was followed up during four months, twice a week, for the detection of respiratory symptoms. rv was diagnosed by direct immunofluorescent assay (dfa) and real time pcr in symptomatic hcws. influenza a was detected in five episodes of respiratory symptoms (5.3%) and other rv in 26 (27.9%) episodes. the incidence density of influenza and other rv was 4.3 and 10.8 episodes per 100 hcw-month, respectively. the educational nature of the present study may persuade hcws to develop a more positive attitude to influenza vaccination. the compliance with influenza vaccination has been historically poor among health care workers (hcws) varying from 2 to 36% around the world [1] [2] [3] [4] . a recent review of relevant predictor studies of self-reported reasons for accepting or rejecting influenza vaccination showed that the two major reasons for rejecting are misconceptions or lack of knowledge about influenza infection; and a lack of convenient access to vaccine. on the other hand, hcws compliant to seasonal vaccination are generally older, believe in vaccine efficacy, take the vaccine for self-protection, and have been previously vaccinated [5] . at hospital das clinicas, university of sã o paulo school of medical sciences, a previous study showed a 34% compliance with influenza vaccination among hcws. in the mentioned study, the main reasons for non-compliance were the perception of vaccine inefficacy and the fear of adverse reactions [4] . respiratory symptoms occurring after vaccination are frequently misinterpreted as vaccine failure which reinforces the hcw's skepticism on vaccine efficacy. to overcome these false beliefs, we proposed a prospective study in a cohort of hcws to demonstrate that influenza vaccine is safe and other respiratory viruses (but not influenza) are generally the cause of respiratory symptoms in the months following influenza vaccination. this study was conducted at hospital das clinicas, university of são paulo school of medical sciences (hc-fmusp) from may to october 2006. the hospital das clinicas is a 2,000-bed tertiary teaching hospital consisting of 5 buildings attached to the university of são paulo. the main building has approximately 900 beds and contains most of the surgical and clinical wards and 12 intensive care units. hospital das clinicas has an estimated 15,000 hcws, including permanent and casual staff, employees, students, and volunteers. since 1999, annual influenza vaccination has been offered free of charge to all hcws. vaccination usually takes place at the hospital's immunization center during working hours, from monday to friday. in 2006, as a strategy to increase compliance with influenza vaccination, vaccine was offered at places of easy access during expanded hours, as suggested by 61% of the interviewed in previous survey [4] . in addition, an educational campaign was carried out emphasizing the safety and importance of influenza vaccination. detailed information about the 2006 educational and vaccination campaign have been published elsewhere [6] . during vaccination campaign, hcws were invited to participate in the present study which had two steps: 1) evaluation of vaccine safety and, 2) cohort study to evaluate which respiratory viruses were more frequently detected in hcws presenting respiratory symptoms in the four-month period following vaccination. sample size was estimated taking into account an expected frequency of 10% of adverse events in adult population. considering an acceptable frequency rate of up to 13%, we estimated to enroll at least 377 hcws (epiinfo version 6). three hundred and ninety eight vaccinated hcws were surveyed for adverse events occurring within the first 48 h after influenza vaccination. a subset of 337 hcws participated in the follow-up phase of the study. to assure that all hospital sectors were represented, the cohort was defined during the assessment of adverse events which was performed by a hospital epidemiologist nurse, through visits in all hospital floors and sectors. afterward, these hcws were actively surveyed twice a week, at work place, during four months, to check for the occurrence of respiratory symptoms and nasal wash sampling which was done in 93 of the participants. figure 1 shows the algorithm of the study. a followup time of four months was proposed taking into account the period when serum antibodies elicited by influenza vaccine are expected to maintain protective levels. the following adverse events were actively surveyed: fever, headache, malaise, myalgia, local pain, local edema and allergic reaction. other adverse events spontaneously reported were also registered. during follow-up visits, participants were asked about the presence of the following symptoms: fever, coryza, blocked nose, sneeze, cough, watery eyes, headache, myalgia, sore throat, hoarseness, sibilance, and dyspnea. allergy was ruled out in those with sneezing as the only symptom. influenza like illness (ili) was defined by the presence of fever and cough and/or sore throat according to the cdc definition [7] . in the presence of any of the above mentioned symptoms, a nasal wash sample was taken according to englund et al, kept at 4uc to 8uc and processed at the virology laboratory within four hours from sampling [8] . nasal washes were taken from hcws who consent with sampling and whose duration of symptoms did not exceed three days. respiratory syncytial virus (rsv), influenza (inf) a and b, adenovirus (adv) and parainfluenza virus (piv) were diagnosed by direct immunofluorescent assay (dfa) according to the manufacturer's instructions (imagenh dako, cambridgeshire, uk). aliquots of nw samples were stored at 280uc for later pcr and real time pcr processing. pcr was used to detect coronavirus and picornavirus. rt-pcr products for picornavirus were subsequently sequenced to differentiate rhinovirus from enteroviruses. real time pcr (taqman assay) was used to diagnose human metapneumovirus (hmpv). to increase the sensitivity of influenza diagnosis, a real time pcr (taqman assay), was added to the diagnostic tools. similarly, a nested adenovirus pcr was used along with dfa due to the low sensitivity of the latter in diagnosing adv. the pcr protocols used in the present study have been published elsewhere [9] [10] [11] [12] . hcws were informed about the results of the dfa up to 48 h after sampling. spss 15 .0 (spss inc., chicago, il) was used with the x 2 test or fisher's exact test for discrete variables, and student's t test or the mann-whitney u test for continuous variables. tests of significance were two sided, and p,0.05 was considered to be statistically significant. incidence density (id) of respiratory symptoms, influenza virus infections and other respiratory virus infections were calculated by the formula described below. id results were expressed per 100 hcw-month [13] . the interviews for adverse events occurring within 48 h of vaccination were made from one to 10 days after vaccination. the majority of the participants (81.4%) were surveyed within the first week of vaccination. all hospital sectors were represented (table 1) . one hundred and twenty of the 398 hcws (30.2%) reported at least one adverse event (ae). table 1 shows the occurrence of adverse events according to the demographic characteristics of the vaccinees. sector of work was the only variable associated with the presence of adverse events (p = 0.017). the sectors with highest frequency of adverse events were the virology laboratory (87.5% of the subjects), burn unit (54.5%), nephrology (52.4%) and pneumology (45.5%). in the remaining sectors, ae were reported by less than 40% of the subjects. those surveyed in the first five days after vaccination were more likely to report such events [95 (79.2%) versus 25 (20.8%); p,0.0001]. local ae were reported by 18.3% of the participants, systemic ae by 71.6%, and 10% of the participants reported both local and systemic aes. headache, myalgia and malaise were more frequently reported (50%, 45.8% and 45%, respectively). local pain and local edema was reported by 17.5% and 5% of the hcws. no severe adverse event was observed. during the 4-month period following influenza vaccination, respiratory symptoms were evaluated in 337 hcws. a total of 4,182 follow-up visits were performed (median 12 per hcw, ranging from one to 25 visits). one hundred and twenty-one hcws (36%) developed 192 episodes of respiratory symptoms. coryza, cough, sore throat and myalgia were reported by 36.3%, 25%, 17.4% and 13.7% of the participants, respectively. seventy-one of them (58.7%) presented more than one episode suggestive of upper respiratory infection (uri). ili was observed in 17 of the 192 episodes (8.8%). mean time to the occurrence of respiratory symptoms was 2.9 (0.7 to 5.2) months. the incidence density of respiratory symptoms was 12.4 episodes per 100 hcw-month. nasal washes were taken in 93 of the 192 episodes of uri. in 61 episodes (66.3%) no respiratory virus was found, even though 82% had coryza, 53% had cough and 49% had sore throat. the frequency of ili was similar among hcws who agreed with sampling and those who did not agree (58.8% versus 41.2%, p = 0.37) ( misconceptions about influenza vaccine, be it about its safety or its effectiveness, have been identified in all studies included in a recent review of attitudes and predictors of influenza vaccination among hcws, highlighting the importance of education efforts [5] . initial symptoms of rv infections are often unspecific such as fever, malaise, or myalgia. as influenza vaccine is offered when other rv are circulating (e.g., rsv), vaccinated hcws developing symptoms within 48 h of vaccination misinterpret those signs as vaccine adverse events. in addition, the occurrence of respiratory symptoms in the months following vaccination is mistaken as vaccine failure. other respiratory infections as the cause of such symptoms are hardly ever considered. to diminish the arguments of fear of adverse events or perception of vaccine inefficacy, this prospective study was conducted to demonstrate to a subset of hcws from our hospital, that severe adverse events following influenza vaccination are rare and the episodes of respiratory symptoms occurring in the first four months after vaccination are generally caused by other respiratory viruses and not by influenza virus. as expected, no severe adverse event was observed in the present study, and the events more frequently reported, such as headache, myalgia and malaise could be related to influenza vaccine itself as well as to other causes, given their unspecificity. in adults, the adverse event more frequently reported after intramuscular administration of inactivate vaccines is local pain, affecting 10% to 64% of the vaccinated [14] [15] [16] . in the present study, 17.5% of the participants reported local pain. systemic reactions like fever, malaise and myalgia can also occur after inactivate vaccines. in the present study, the frequency of systemic aes (over 70%) was higher than reported in previous studies. recent publications have shown rates of systemic adverse events ranging from 30% to 59% in hwcs [17, 18] . ideally, the subjects should have been surveyed within the first four days of vaccination. as we preferred to apply the questionnaire personally, rather than by mail or phone calls, only 49.7% of the participants were interrogated up to the fourth day, due to the great number of interviews. thus, the high rate of systemic adverse events observed in the present series may be either an overestimation by the subjects or a consequence of the survey method applied. a recent study evaluating vaccine coverage in korea has demonstrated that interview surveys provide more reliable information than telephone surveys, showing lower missing rates and 100% of agreement with the immunization registry record [18] . anaphylaxis and neurological reactions are rare [15, 19] . the frequency of adverse events observed in the present study may be overestimated taking into account the subjectiveness of selfreported unspecific symptoms. as hcws are aware of vaccine adverse events and fear its consequences, it is comprehensible that these events will be more frequently reported by them than by general population. another study conducted in the same hospital demonstrated that hcws reported significantly more adverse events (52.9%) than the elderly (25.3%) [20] . the higher frequency of adverse events reported by hcws surveyed in the first five days of vaccination, as compared with those surveyed after the fifth day, may suggest that people may be more predisposed to remember any symptom possibly associated with the vaccine if inquired within the first days of vaccination. on the other hand, we believe that if the adverse events were severe or important, they would not be missed if inquired after 6 to 10 days. interestingly, we observed that some sectors showed significantly higher rates of ae than others, supporting the subjectivity of the information. also, this data may suggest a mouth to mouth effect among sector coworkers influencing the self-report of ae. among hcws, the belief that coworkers take influenza vaccine influences the vaccine uptake. thus, it is possible that the same occurs concerning to adverse events. continued education of health professionals is essential to highlight not only the epidemiological importance of the vaccine, but also its safety and the low risk of severe adverse events. our study also demonstrated that the respiratory symptoms occurring in the months following influenza vaccination were more frequently caused by other respiratory viruses and generally do not mean vaccine failures. one limitation of our study is that in only 93 of the 192 episodes of respiratory symptoms (48.4%) the subjects agreed with nw sampling. nw sampling is a simple but uncomfortable procedure and this fact may explain why some hcws preferred not to get tested during working hours. one could argue that influenza cases could be missed among those not tested. however, we believe that this loss has not affected our results as the frequency of ili was similar between those who agreed with sampling and those who did not ( table 2 , p = 0.37). the incidence density of other respiratory viruses was 2.4 times greater than incidence density of influenza. probably, this difference would be even greater if real time pcr was also performed to increase the sensitivity of the diagnosis of other respiratory viruses as well. in addition, more cases of other rv infections would be diagnosed if a larger number of professionals were tested, increasing the difference between the incidence density of influenza and other rv. influenza infection is characterized by the abrupt occurrence of fever, headache, myalgia, and dry cough. during influenza season, the presence of these symptoms is highly predictive of influenza infection and summarizes the case definition of influenza-like illness (ili), which has been used worldwide for influenza surveillance purposes. however, the sensitivity and positive predictive value of such definition can vary greatly depending on the co-circulation of other respiratory viruses in the community [21] . indeed, bellei et al. have recently reported that 70% of ili cases in the city of são paulo were caused by other agents, mainly rhinovirus, which peaks along with influenza [22] . similar results have been previously published by other authors [21] . in our series, influenza cases in vaccinated hcws were mild and occurred significantly earlier following vaccination in comparison to other respiratory viruses. this finding may be explained by the marked seasonality of influenza in são paulo city as reported previously [23, 24] , peaking in early winter and coinciding with the initial period of the study. the effectiveness of influenza vaccines is related predominantly to the age and immune competence of the vaccinee and the degree of similarity between the viruses in the vaccine and those in circulation. vaccine effectiveness in preventing laboratory-confirmed influenza illness when the vaccine strains are well matched to circulating strains is 70-90% in randomized, placebo-controlled trials conducted among children and young healthy adults, but is lower among elderly or immunocompromised persons [25] . in adults $65 years old, the efficacy of influenza inactivate vaccine varies from 30% to 40% [26] . trials that measure laboratory-confirmed influenza virus infections as the outcome are the most persuasive evidence of vaccine efficacy [25] . in the present study, only five of the 337 vaccinated hcws (1.5%) acquired influenza. in accordance with the educational nature of our study, we considered all cases of influenza as vaccine failures, since vaccinated health personnel look forward to be protected against influenza. molecular characterization of influenza cases was not performed to check for possible mismatches between circulating viruses and vaccine strains, which could possibly justify those failures. our study demonstrated that the fear of severe adverse events seems unjustified as well as the perception of vaccine inefficacy. uri following influenza vaccination were generally caused by other respiratory viruses and not by influenza. in times of pandemic influenza a h1n1 and widespread vaccination, healthcare and emergency medical services personnel are among the priority groups recommended to receive the h1n1 influenza vaccine. it is time to overcome definitively the misconceptions about the vaccine as well as the fear of adverse events. so far, the vast majority (93%) of adverse events reported to vaers after receiving the trivalent 2010-2011 influenza vaccine, were classified as ''non serious'', e.g., soreness at the vaccine injection site [27] . we believe that the educational nature of the present study may persuade hcws to develop a more positive attitude to influenza vaccination. influenza vaccination among medical residents in a teaching hospital influenza vaccination rates and motivators among healthcare worker groups influenza immunisation: attitudes and beliefs of uk healthcare workers attitudes of health care workers to influenza vaccination: why are they not vaccinated? influenza vaccination of health care workers in hospitals-a review of studies on attitudes and predictors intervention to increase influenza vaccination rates among healthcare workers in a tertiary teaching hospital in brazil * update: influenza activity-united states rapid diagnosis of respiratory syncytial virus infections in immunocompromised adults detection of human rhinovirus rna in nasal washings by pcr polymerase chain reaction for rapid diagnosis of respiratory adenovirus infection frequency of human metapneumovirus infection in hematopoietic sct recipients during 3 consecutive years simultaneous detection of influenza viruses a and b using real-time quantitative pcr measures of disease frequency effectiveness and cost-benefit of influenza vaccination of healthy working adults: a randomized controlled trial frequency of adverse reactions after influenza vaccination the effectiveness of vaccination against influenza in healthy, working adults immunogenicity of a monovalent pandemic influenza a h1n1 vaccine in health-care workers of a university hospital in japan adverse events associated with the 2009 h1n1 influenza vaccination and the vaccination coverage rate in health care workers adverse reactions to influenza vaccine in the elderly occurrence of early adverse events after vaccination against influenza at a brazilian reference center predicting influenza infections during epidemics with use of a clinical case definition acute respiratory infection and influenza-like illness viral etiologies in brazilian adults low mortality rates related to respiratory virus infections after bone marrow transplantation use of oseltamivir to control influenza complications after bone marrow transplantation prevention and control of seasonal influenza with vaccines: recommendations of the advisory committee on immunization practices (acip) the efficacy of influenza vaccine in elderly persons. a meta-analysis and review of the literature summary of 2010-2011 trivalent influenza vaccine data from the u.s. vaccine adverse event reporting system the authors thank the infection control group of hospital das clínicas, school of medical sciences, university of são paulo. key: cord-301254-093yih5n authors: brittain-long, robin; westin, johan; olofsson, sigvard; lindh, magnus; andersson, lars-magnus title: prospective evaluation of a novel multiplex real-time pcr assay for detection of fifteen respiratory pathogens—duration of symptoms significantly affects detection rate date: 2010-01-18 journal: j clin virol doi: 10.1016/j.jcv.2009.12.010 sha: doc_id: 301254 cord_uid: 093yih5n background: nucleic acid amplification techniques have improved the diagnostic possibilities in respiratory tract infections, although their clinical applicability is not yet fully defined. we have evaluated a multiplex real-time pcr method for the detection of 13 respiratory viruses and 2 bacteria (mycoplasma and chlamydophila) in a clinical setting. objectives: the aim of the present study was to evaluate the diagnostic performance and clinical use of a novel multiplex pcr method in adults with community-acquired respiratory viral infection, and the impact of duration of symptoms on detection rates. study design: nasopharyngeal swab samples were prospectively collected from 209 adult outpatients with respiratory infections and 100 asymptomatic controls. results: an infectious agent was identified in 43% of samples from patients and 2% of asymptomatic controls. the detection rate was significantly higher in samples from patients with a duration of symptoms of 6 days or less (51%) than in samples from patients with a duration of symptoms of 7 days or more (30%, p < 0.01). for human corona viruses, and influenza virus a and b there was a correlation between the amount of virus in each patient sample as measured ct values and duration of symptoms. conclusions: duration of symptoms significantly affects the detection rate of respiratory pathogens by multiplex real-time pcr in nasopharyngeal swab samples from adult patients with respiratory infections. our finding should be taken into account when using these tests in clinical practise. real-time polymerase chain reaction (pcr) techniques are gaining increasing acceptance for diagnosis of viral respiratory tract infections (rti). high sensitivity and short analysis time along with the ability to detect several pathogens in a single sample are advantages compared with serology, viral culture and antigen detection. 1-3 pcr assays may enhance identification of viral respiratory pathogens by at least 50% compared with traditional diagnostic methods, 4-6 partly by detecting viruses for which no conventional methods exist, such as human rhinovirus (hrv), human coronavirus (hcov) and human metapneumovirus (hmpv). kaye et al. 7 found a respiratory virus as the only causative agent in 15% of adult patients admitted to hospital with acute rti. in patients with severe pneumonia, requiring intensive care treatment, who had negative bacterial cultures from bronchoalveolar lavage (bal) samples, legoff et al. 8 reported detection of a respiratory virus in as much as 63% of cases. viruses believed to cause only mild upper respiratory tract infections (urti), for example hrv and hcov have been detected in the lower respiratory tract in patients with severe disease. [9] [10] [11] on the other hand, it is not known if asymptomatic shedding occurs following clinical recovery or during subclinical infection, and results of molecular methods with high sensitivity must be assessed with caution regarding the clinical relevance of the finding. detection of viral agents in respiratory tract samples using multiplex real-time pcr is both quick and sensitive 12, 13 and will likely replace traditional means of diagnosing rti. the diagnostic performance of these methods in adult immunocompetent individuals and the optimal time point for specimen collection is, however, poorly defined. the aim of the present study was to evaluate the diagnostic performance and clinical use of a novel multiplex pcr method in adults with community-acquired respiratory viral infection, and the impact of duration of symptoms on detection rates. we conducted a prospective study in western sweden during two consecutive winter seasons, october through april 2006-2008. adult patients (≥18 years old) with symptoms of acute rti with duration of less than 2 weeks were included in the study, by the treating physician. an acute rti was defined as having at least two of the following symptoms; coryza, congestion, sneezing, soar throat, odynophagia, cough, chest pain, shortness of breath or fever, for which the physician found no other explanation. patients with suspected or confirmed bacterial infection were excluded. patients were asked to return for a follow-up visit 10 days (±2 days) after the initial visit. signs and symptoms were recorded in a web-based form. the same protocol was used at initial and follow-up visit. a control group of 100 healthy adults without history of fever or symptoms of rti during the preceding 14 days were also included. control subjects were contacted for a telephone interview 2 days after testing. individuals who had developed symptoms of rti were excluded, to avoid detecting virus that might be shed in high levels prior to onset of symptoms. nasopharyngeal and throat swab specimens were collected from each patient and control subject. the swabs were jointly placed in a sterile container with 1 ml of sodium-chloride solution, and sent to the virology laboratory the same day. at the laboratory, specimens were either analysed directly or frozen at −70 • c for later analysis. the research ethics committee at gothenburg university, gothenburg, sweden, approved the study and all patients gave their informed consent. we have focused on the analysis of viruses found with our pcr assay, although two bacteria were also included in the panel. we utilized a real-time pcr procedure, based on automated specimen extraction and multiplex amplification. primers and hydrolysis probes were obtained from the literature or developed in our laboratory. nucleic acid from 100 l of specimen was extracted into an elution volume of 100 l by a magnapure lc robot (roche molecular systems, mannheim, germany), using the total nucleic acid protocol, and was amplified in an abi 7500 real-time pcr system (applied biosystems, foster city, ca). amplification was then carried out in 50 l reaction volumes. after a reverse transcription step, 45 cycles of two-step pcr was performed. each sample was amplified in 5 parallel reactions, each containing primers and probes specific for 3 targets, as previously described, 14 ct values (cycle threshold) for each patient sample positive by real-time pcr were recorded for semi-quantitative estimation of the amount of dna/rna in each specimen. chi-square test was used to compare proportions and spearman's rank correlation coefficient to analyse correlations. a p-value of <0.05 was considered as significant. no of samples positive for one microbial agent or more, n (%) 94 (43) 2 (2) a one patient was excluded prospectively due to bacterial tonsillitis and one patient was excluded retrospectively due to pneumococcal sepsis. two hundred and nineteen patients with symptoms of rti and 100 asymptomatic controls were included in the study. ten patients were excluded (table 1) , leaving 209 for the final analysis. an infectious agent was identified in 94 patients (43%) and in 2 controls (2%). demographic characteristics and the agents detected are presented in table 1 . no cases of enteroviruses or chlamydia pneumoniae were identified. in 7% of positive outcomes (n = 7) more than one agent was identified (data not shown). the duration of symptoms ranged from 0 to 14 days. the majority of patients (63%) were included within the first 7 days of disease. the detection rate was significantly higher in samples from patients with a duration of symptoms of 6 days or less (51%) than in samples from patients with a duration of symptoms of 7 days or more (30%, p < 0.01) (fig. 1) . for hcov, ifa and ifb there was a correlation between ct values and duration of symptoms (n = 13, rs = 0.33, p < 0.05; n = 24, rs = 0.17, p < 0.05; n = 10, rs = 0.65, p < 0.01) for hrv no such correlation was found (fig. 2) . two patients with influenza a were omitted because of results incongruent with patient history (data not shown). in total, 63% (n = 138) of the patients returned for a follow-up visit 10 ± 2 days after the initial visit. nasopharynx/throat swab samples were collected. out of 94 patients with a positive sample at initial visit, 57 patients (61%) showed up for follow-up testing. the same pathogen was detected in 13 of these patients (23%) (see table 2 ). eight of 13 (62%) of samples positive for the same virus at initial and follow-up visit were positive for hrv. a new microbial agent was found in five subjects, all of who still had respiratory tract symptoms at follow-up. all patients still positive for the same agent on follow-up had a higher ct-value (corresponding to a lower table 2 follow-up (10 ± 2 days after initial visit) test result from analysis with real-time pcr of nasopharyngeal/throat swab specimens. amount of viral rna in the specimen) 10 days later except one patient with hrv on both occasions (data not shown). we have shown that the diagnostic yield increases significantly if a multiplex pcr assay for respiratory viruses is used within the initial 6 days of symptoms, in immunocompetent adults with rti. this is in concordance with reports on single-plex pcr assays for influenza where the detection rate correlates to duration of symptoms. 15 we detected an infectious agent in 43% of patients. previously, detection rates ranging between 43% and 63% have been described, 6, 12, 16, 17 with exceptions for very high rates among young children and infants. 18, 19 in asymptomatic children, higher frequencies of positive results have been reported. 20, 21 in particular detection of hrv and ev may represent a previous infection dating back 4-5 weeks, due to prolonged shedding of virus. 22 lack of pre-existing immunity, greater viral exposure as well as an immature immune system may cause higher levels of viral replication and prolonged viral shedding in children. templeton et al. reported a detection rate of only 24%, but hrv, hcov, adv or hmpv were not included in their panel. 23 studies performed retrospectively on clinical samples will select for a short duration of symptoms as will studies including children, who tend to seek medical care early and who may shed more virus during a longer time period. thus, the prospective approach and the adult population may explain the relatively low detection rate found in our sample. as the study period included the influenza season, we detected influenza a and b virus in a relatively high proportion of patients (41%), which is comparable to other studies of adults. 9 we detected a pathogen in only 2% of control subjects, both hrv. although most studies do not include controls, creer et al. 16 found a respiratory virus in 12% of controls, who reported no signs of respiratory tract infection during 2 months before sampling, predominantly hrv. asymptomatic shedding of hrv, rsv and piv has also been reported in immunocompromised patients. 24, 25 our results suggest that viral shedding exceeding 14 days is rare in immunocompetent adults. the rate of asymptomatic carriage of virus might be affected by epidemiological factors that vary over time. our control samples were, however, collected throughout the entire study period. for several viruses analysed in our study, the amount of viral dna/rna decreased with duration of symptoms, suggesting a gradual reduction of viral shedding from the epithelial surface over time. this is in concordance with the gradual reduction of rsv levels in nasopharyngeal aspirates described by gerna et al. 26 and campanini et al. 27 dual viral infections are previously described in approximately 5-20% of infected patients, with a higher frequency among young children. 17, 28, 29 in accordance with previous studies we found two viruses in 7% of the samples. the clinical impact of multiple infections remains to be determined. in one study of children with rsv infection, higher fever, longer hospital stays and more frequent use of antibiotics was associated with multiple infections. 30 semiquantification of viral dna, as measured by ct values, may be of use when determining the clinical relevance of a positive test, particularly in multiple infections. we found a correlation between ct values and duration of symptoms for hcov, ifa, and ifb but not for hrv. the high genetic variability of hrvs, which increase the probability of probe-target mismatch, may explain this. for other viruses, such as influenza b virus, with less genetic variability viral detection will be more stable. the lack of association between duration of illness and ct values in hrv infection may also reflect variations in viral shedding and differences in pre-existing immunity between individuals as well as limited sample size. interestingly, the majority of patients, 7/13 (62%) positive for any agent at the follow-up visit (day 10 ± 2 days) had hrv. whether this translates into a longer period of infectivity remains to be determined. however, the semi-quantitative estimation of viral loads by ct values must be interpreted with caution since relative viral loads were not compared to standard amounts of virus. the clinical relevance of the outcome of multiplex pcr tests for respiratory viruses is not yet fully determined. for some agents, such as influenza a and b virus, a positive test may provide the basis for antiviral treatment. it has been suggested that a rapid etiologic diagnosis of viral rti could reduce unnecessary prescription of antibiotics, but this remains to be shown. we conclude that duration of symptoms affects detection rate by real-time multiplex pcr in adult patients with rti. duration of symptoms should be taken into account when using these tests in clinical practise. for some viruses, with relatively low genetic variability, semi-quantification might be of value when interpreting results where more than one respiratory virus is found. none declared. development of three multiplex rt-pcr assays for the detection of 12 respiratory rna viruses development and implementation of a molecular diagnostic platform for daily rapid detection of 15 respiratory viruses practical experience of high throughput real time pcr in the routine diagnostic virology setting respiratory viruses and severe lower respiratory tract complications in hospitalized patients enhanced identification of viral and atypical bacterial pathogens in lower respiratory tract samples with nucleic acid amplification tests impact of rapid detection of viral and atypical bacterial pathogens by real-time polymerase chain reaction for patients with lower respiratory tract infection surveillance of respiratory virus infections in adult hospital admissions using rapid methods high prevalence of respiratory viral infections in patients hospitalized in an intensive care unit for acute respiratory infections as detected by nucleic acid-based assays detection of respiratory viruses by molecular methods a recently identified rhinovirus genotype is associated with severe respiratory-tract infection in children in germany rhinovirus associated with severe lower respiratory tract infections in children rapid multiplex nested pcr for detection of respiratory viruses influenza diagnosis: from dark isolation into the molecular light west of scotland respiratory virus study group multiplex real-time pcr for detection of respiratory tract infections duration of influenza a virus shedding in hospitalized patients and implications for infection control aetiological role of viral and bacterial infections in acute adult lower respiratory tract infection (lrti) in primary care development of a respiratory virus panel test for detection of twenty human respiratory viruses by use of multiplex pcr and a fluid microbead-based assay viral etiology of common cold in children comparison of multiplex pcr assays and conventional techniques for the diagnostic of respiratory virus infections in children admitted to hospital with an acute respiratory illness human picornavirus and coronavirus rna in nasopharynx of children without concurrent respiratory symptoms predominance of rhinovirus in the nose of symptomatic and asymptomatic infants persistence of rhinovirus and enterovirus rna after acute respiratory illness in children rapid and sensitive method using multiplex real-time pcr for diagnosis of infections by influenza a and influenza b viruses, respiratory syncytial virus, and parainfluenza viruses 1, 2, 3, and 4 respiratory viral infections in transplant recipients respiratory virus infection among hematopoietic cell transplant recipients: evidence for asymptomatic parainfluenza virus infection correlation of viral load as determined by real-time rt-pcr and clinical characteristics of respiratory syncytial virus lower respiratory tract infections in early infancy human respiratory syncytial virus (hrsv) rna quantification in nasopharyngeal secretions identifies the hrsv etiologic role in acute respiratory tract infections of hospitalized infants respiratory picornavirus infections in korean children with lower respiratory tract infections detection and typing by molecular techniques of respiratory viruses in children hospitalized for acute respiratory infection in rome, italy multiple simultaneous viral infections in infants with acute respiratory tract infections in spain the authors would like to acknowledge the staff at the clinical virology laboratory, department of virus detection, at sahlgrenska university hospital for technical expertise and patients and staff at the following centres in the västra götaland region; the infectious disease clinics in uddevalla, skövde, borås, and göteborg, and primary health care centres in stenungsund, sollebrunn, floda, kungshöjd, kungsten, olskroken, carlanderska, and capio axess.funding: the study was partly funded by the swedish strategic programme against antibiotic resistance (strama), capio research foundation, grant # 2006-1166 and the västra götaland region research funds, grant # vgfoureg-8402.ethical approval: granted by the research ethics committee at gothenburg university, # 403-06. key: cord-267003-k7eo2c26 authors: hendaus, mohamed a; jomha, fatima a; alhammadi, ahmed h title: virus-induced secondary bacterial infection: a concise review date: 2015-08-24 journal: ther clin risk manag doi: 10.2147/tcrm.s87789 sha: doc_id: 267003 cord_uid: k7eo2c26 respiratory diseases are a very common source of morbidity and mortality among children. health care providers often face a dilemma when encountering a febrile infant or child with respiratory tract infection. the reason expressed by many clinicians is the trouble to confirm whether the fever is caused by a virus or a bacterium. the aim of this review is to update the current evidence on the virus-induced bacterial infection. we present several clinical as well in vitro studies that support the correlation between virus and secondary bacterial infections. in addition, we discuss the pathophysiology and prevention modes of the virus–bacterium coexistence. a search of the pubmed and medline databases was carried out for published articles covering bacterial infections associated with respiratory viruses. this review should provide clinicians with a comprehensive idea of the range of bacterial and viral coinfections or secondary infections that could present with viral respiratory illness. viral respiratory tract infections (vrtis) are very common in children and their presentations vary from simple colds to life-threatening infections. [1] [2] [3] [4] [5] the detection of a respiratory virus does not necessarily infer that the child has only a viral infection, 6 since outbreaks of vrtis are being linked to increased incidence of bacterial coinfections. 7 the human body is usually capable of eliminating respiratory viral infections with no sequelae; however, in some cases, viruses bypass the immune response of the airways, causing conceivable severe respiratory diseases. 8 robust mechanical and immunosuppressive processes protect the lungs against external infections, but a single respiratory tract infection might change immunity and pathology. 9 health care providers often face a dilemma when encountering a febrile infant or child with respiratory tract infection. the reason expressed by many clinicians is the challenge to confirm whether the fever is caused by a virus or bacterium. 10 acute otitis media (aom) is a usual bacterial coinfection that occurs in 20%-60% of cases of vrtis. [11] [12] [13] [14] in addition, almost 60% of children with vrti have changes in the maxillary, ethmoidal, and frontal sinuses. 11, 12 moreover, in the year 1918, it was estimated that 40-50 million individuals died from the influenza pandemic, many of which were due to secondary bacterial pneumonia with streptococcus pneumoniae. 15 a search of the pubmed database and google was carried out, using different combinations of the following terms: virus, induced, bacteria, pathogenesis, prevention, vaccine, and children. in addition, we searched the references of the identified articles for additional articles. we then reviewed abstracts and titles and included studies that were submit your manuscript | www.dovepress.com hendaus et al relevant to the topic of interest. finally, the search was limited to studies of disease in humans that were published in english and spanish from 1918 to the end of 2014 ( figure 1 ). the epithelium ( figure 2) is usually covered by a layer of mucus that functions as a boundary. 16 mucins, which are charged glycoproteins, are the main components of mucus. 17, 18 muc5ac and muc5b are the most common mucins in the human sputum, and they assist the innate immune system through their anti-inflammatory and antiviral properties. 19, 20 in addition, they facilitate trapping and clearance of viruses; however, overproduction of those mucins might have a paradoxical effect. 18, 19 the airway epithelium not only functions as a physical barrier but also recognizes microorganisms through pattern recognition receptors such as toll-like receptors (tlrs), 18 nucleotide-binding oligomerization domain (nod)-like receptors (nlrs), and retinoic acid-inducible gene (rig)like helicases. 21, 22 tlrs are single, noncatalytic, membrane-spanning receptor proteins used by the innate immune system. 23 respiratory viruses collaborate with tlr lanes, leading to extended bacterial load in the lungs. 21, 24 in comparison, nlrs and rig-like helicases activate innate immune responses through cytosolic sensing of viral and bacterial components. 22, 25 nod1 and nod2, which are family members of nlrs, are induced by molecules synthesized during the production and/or degradation of bacterial peptidoglycan. [26] [27] [28] [29] in addition, many epithelial cells express the classical antiviral interferons (infs), especially ifn-α and ifn-β. 30, 31 moreover, the respiratory virus-infected epithelia facilitates the attraction of inflammatory cells, including natural killer cells, neutrophils, macrophages, and eosinophils from the bloodstream into the infected site. 32 finally, the airway epithelium consists of many molecules including intercellular adhesion molecule 1 (icam-1), carcinoembryonic antigen-related cellular adhesion 1 (ceacam-1), and platelet-activating factor receptor (paf-r). 33 viruses have an effect in modulating these receptors, leading to an increase risk of bacterial adherence; for example, rhinovirus upregulates the expression of paf-r, leading to the binding of s. pneumoniae to bronchial epithelial cells. 34 different mechanisms might contribute to the debilitation in host defense of the respiratory tract against bacteria following viral infection. some of the mechanisms have been extrapolated from studies conducted in animal models of sequential infections by respiratory viruses and several bacterial pathogens. mammalian cells are prone to bacterial attachment during a viral illness. 8, 35 viruses can debilitate the mucociliary clearance structure, leading to the increased attachment of bacteria to mucins and colonization; moreover, the condensed mucus will impede the penetration of antibacterial material and immune cells. 36 viruses like the respiratory syncytial virus (rsv) can damage ciliated cells, resulting in ciliostasis and, therefore, deterioration of mucociliary clearance. 37 the same concept applies to an influenza virus infection, leading to decreased tracheal mucociliary velocity and clearance of s. pneumoniae. 35, 38 moreover, virus-induced cell death debilitates the mechanical elimination of the attached pathogens and displays novice receptors for bacterial adherence. 39 studies have shown that the rsv virus induces the adherence of s. pneumoniae, pseudomonas aeruginosa, and haemophilus influenza to airway epithelial cells. [40] [41] [42] [43] in addition, adenovirus and rhinovirus play the same role in the adherence of s. pneumoniae to the airway epithelial cells; 8, 44 however, the measles virus decreases the risk of adherence of streptococcal bacteria, implying that every virus has a specific mode of changing the host cell membrane. 44 moreover, bacterial adhesion might also be a result of the upregulation of surface receptors including paf-r, which is involved in pneumococcal invasion. 45, 46 in patients with cystic fibrosis, bacterial adherence forms a biofilm, creating permanent airway colonization with p. aeruginosa. 47 viruses such as rsv, rhinovirus, and influenza virus also lead to pneumococcal biofilm formation on the airway lining. 48 furthermore, rsv increases the risk of adherence of staphylococcus aureus and bordetella pertussis to hep-2 (human epidermoid cancer) epithelial cells. 49, 50 virus effect on the immune system post-viral sustained desensitization of lung sentinel cells to tlr signals may be one possible contributor to the common secondary bacterial pneumonia associated with viral infection. for instance, tlr4 and tlr5 pathways are altered after influenza virus infection, resulting in decreased neutrophil attraction, thereby leading to increased attachment of s. pneumonia and p. aeruginosa to the airway epithelial cells. 25 the interrelation between host cells and microorganisms during an infection induces immune responses that include the generation of proinflammatory molecules. despite their crucial role as a bactericidal, proinflammatory cytokines such as tnf-α produced in response to infection could be detrimental to the host cells. 51 during a viral infection, tlr and rig-i-like receptor activation induces production of type i ifns, which can augment the inflammatory response to tlr ligands including lipopolysaccharide (lps). 52, 53 in addition, certain bacteria such as s. aureus integrate into the a549 respiratory epithelial cells (adeno-carcinomic α β hendaus et al human-alveolar basal-epithelial cells) during a respiratory viral infection by increasing the expression of icam-1. 54 rsv differs from influenza virus in that the former upregulates cellular receptors including ceacam-1 and icam-1, which eventually leads to bacterial infection. 45 finally, interaction between type i ifns and nod1/nod2 signaling leads to bacterial recognition, but indicts harmful effects in the virally infected host. 55 another study showed that the rates of bacteremia and om were 18% and 44%, respectively, in children with viral-induced bronchiolitis. 11 the highest incidence of aom is usually 2-5 days after an upper respiratory infection. 64, 65 isolation of viruses alone from sinus aspirates or in concomitance with bacteria proposes the role of viruses in the induction of bacterial sinusitis, 62 with rhinovirus and parainfluenza viruses being the culprits. 66 the rate of bacteremia in children with acute bronchiolitis ranges from 0.2% to 1.4%. [67] [68] [69] [70] [71] [72] [73] [74] [75] in addition, the rate of bacterial urinary tract infection (uti) in children with bronchiolitis can be as high as 11.4%. 67 in a recent study, hendaus et al 76 the human myxovirus resistance protein 1 (mxa) is an important intermediary of the ifn-induced antiviral response against a variety of viruses. mxa expression is firmly modified by type i and type iii ifns, which also requires signal transducer and activator of transcription 1 signaling. additionally, mxa has many characteristics similar to the superfamily of large guanosine triphosphatases. 78 mxa analysis could be beneficial to differentiate between bacterial and viral infections. engelmann et al 79 conducted a prospective, multicenter cohort study in different pediatric emergency departments in france on the role of mxa in the diagnosis of viral infections. mxa blood values were calculated in infants and children with verified bacterial or viral infections, uninfected controls, and infections of unknown origin. a receiver operating characteristic analysis was used to verify the diagnostic performance of mxa. the study, which included 553 children, showed that mxa was significantly higher in children with viral versus bacterial infections and uninfected controls (p0.0001). additionally, mxa levels were significantly higher in children with clinically diagnosed viral infections than in those with clinically diagnosed bacterial infections (p0.001). 79 other authors have also reported the usefulness of blood mxa testing in patients with viral infections. 80, 81 the use mxa in diagnosing viral infection is very promising, especially in patients who are at risk of infectious complications. two separate studies have shown that blood mxa is beneficial in differentiating between viral illness and acute graft-versushost disease after allogenic stem cell transplantation. 82, 83 it has been recommended that treatment or prevention of a viral disease may be a superior method for diminishing 62 it has also been published that live attenuated influenza vaccine is effective in reducing the incidence of all-cause aom [86] [87] [88] and pneumonia 89 compared to placebo in children. in addition, the intranasal influenza vaccine can reduce om by 44%. 90 moreover, studies have shown that a combined influenza/pneumococcal vaccine is efficient in the prevention of om in children and pneumonia. 91, 92 however, the credit of protection was awarded to the influenza vaccine since studies have shown that pneumococcal vaccine has no benefit in the reduction of aom. 93, 94 in addition, the pneumococcal polysaccharide vaccine showed no efficacy in the prevention of pneumonia in adults. 95 treatment of viral infection is anticipated to prevent bacterial superinfections. currently, the only respiratory virus that is pharmacologically treatable is the influenza viruses (type a and b). 62 neuraminidase inhibitors can potentially diminish the morbidity related to influenza. 96 oseltamivir can reduce the incidence of aom in preschool children, 97 and the reduction rate can be up to 44%. 98 a meta-analysis review showed that oral oseltamivir reduces the rate of hospitalization by 25% and morbidity by 75%. 99 in addition, its use can reduce the use of antibiotics by up to 50%, 100, 101 the same concept of protection applies to vaccines that prevent against rsv infections. 62 the vaccine available for rsv is palivizumab (medimmune, gaithersburg, md, usa), a humanized monoclonal antibody that perceives the fusion protein of rsv. the other monoclonal antibody that is under clinical trials is motavizumab (medimmune), which has a higher affinity for rsv fusion protein than palivizumab and can prevent against medically attended lower respiratory tract infection. 102 the rate of concurrent serious bacterial infections with viral illness is appreciable. similar emphasis must be given to the prevention and treatment of viral illnesses, especially in young children. furthermore, health care providers should emphasize to parents on the importance of clinical follow-up of infants and young children diagnosed with vrti. moreover, the introduction of mxa in the diagnosis of viral illnesses in children is promising. the authors declare no conflicts of interest in this work. respiratory viral infections in infants: causes, clinical symptoms, virology, and immunology paediatric respiratory research group. viral etiology of acute respiratory infections with cough in infancy: a community-based birth cohort study respiratory viral infections in adults emerging respiratory agents: new viruses for old diseases? respiratory pathogens in children with and without respiratory symptoms asthma exacerbations in children associated with rhinovirus but not human metapneumovirus infection how do viral infections predispose patients to bacterial infections? the airway epithelium: soldier in the fight against respiratory viruses influenza virus lung infection protects from respiratory syncytial virus-induced immunopathology occult serious bacterial infection in infants younger than 60 to 90 days with bronchiolitis: a systematic review bacterial coinfections in children with viral wheezing epidemiology of documented viral respiratory infections and acute otitis media in a cohort of children followed from two to twenty-four months of age acute otitis media and respiratory viruses detection of respiratory viruses in nasopharyngeal secretions and middle ear fluid from children with acute otitis media influenza infection and copd mucins, mucus, and sputum structure and function of the polymeric mucins in airways mucus respiratory tract mucin genes and mucin glycoproteins in health and disease regulation of mucin genes in chronic inflammatory airway diseases airway mucus obstruction: mucin glycoproteins, muc gene regulation and goblet cell hyperplasia pathogen recognition by innate immunity and its signaling pathogen recognition and innate immunity intracellular nod-like receptors in host defense and disease toll to be paid at the gateway to the vessel wall sustained desensitization to bacterial toll-like receptor ligands after resolution of respiratory influenza infection caterpiller: a novel gene family important in immunity, cell death, and diseases an essential role for nod1 in host recognition of bacterial peptidoglycan containing diaminopimelic acid nod1 detects a unique muropeptide from gram-negative bacterial peptidoglycan nod2 is a general sensor of peptidoglycan through muramyl dipeptide (mdp) detection host recognition of bacterial muramyl dipeptide mediated through nod2. implications for crohn's disease viruses and the type i interferon antiviral system: induction and evasion viruses and interferon: a fight for supremacy host defense function of the airway epithelium in health and disease: clinical background effects of rhinovirus infection on the adherence of streptococcus pneumoniae to cultured human airway epithelial cells influenza virus infection decrease stracheal mucociliary velocity and clearance of streptococcus pneumoniae the biology of bacterialcolonization and invasion of the respiratory mucosa respiratory syncytial virus and human bronchial epithelium the platelet activating factor receptor is not required for exacerbation of bacterial pneumonia following influenza airway epithelial cell-pathogen interactions non typeable haemophilus influenzae and streptococcus pneumoniae bind respiratory syncytial virus glycoprotein the effects of disodium cromoglycate on enhanced adherence of haemophilus influenzae to a549 cells infected with respiratory syncytial virus direct binding of respiratory syncytial virus to pneumococci: a phenomenon that enhances both pneumococcal adherence to human epithelial cells and pneumococcal invasiveness in a murine model rsv mediates pseudomonas aeruginosa binding to cystic fibrosis and normal epithelial cells rhinovirus enhances various bacterial adhesions to nasal epithelial cells simultaneously respiratory viruses augment the adhesion of bacterial pathogens to respiratory epithelium in a viral species-and cell type-dependent manner lethal synergism between influenza virus and streptococcus pneumoniae: characterization of a mouse model and the role of platelet-activating factor receptor pseudomonas aeruginosa biofilm infections in cystic fibrosis: insights into pathogenic processes and treatment strategies bacterial biofilm in upper respiratory tract infections developmental and environmental factors that enhance binding of bordetella pertussis to human epithelial cells in relation to sudden infant death syndrome (sids) factors enhancing adherence of toxigenic staphylococcus aureus to epithelial cells and their possible role in sudden infant death syndrome toll-like receptors in the pathogenesis of human disease viral infection causes rapid sensitization to lipopolysaccharide: central role of ifn-alpha beta a role for ifn-alpha beta in virus infection-induced sensitization to endotoxin rhinoviruses promote internalisation of staphylococcus aureus into non-fully permissive cultured pneumocytes viral infection augments nod1/2 signaling to potentiate lethality severe pneumococcal pneumonia in previously healthy children: the role of preceding influenza infection the role of respiratory viral infections among children hospitalized for communityacquired pneumonia in a developing country etiology of community-acquired pneumonia in 254 hospitalized children community acquired pneumonia and influenza in children nasopharyngeal carriage of streptococcus pneumoniae in finnish children younger than 2 years old a longitudinal study of respiratory viruses and bacteria in the etiology of acute otitis media with effusion respiratory viruses predisposing to bacterial infections: role of neuraminidase acute otitis media and respiratory virus infections time to development of acute otitis media during an upper respiratory tract infection in children temporal development of acute otitis media during upper respiratory tract infection mandell, douglas and bennett's principles and practices of infectious diseases concurrent serious bacterial infections in 2396 infants and children hospitalized with respiratory syncytial virus lower respiratory tract infections low risk of bacteremia in febrile children with recognizable viral syndromes a prospective study of the risk for serious bacterial infections in hospitalized febrile infants with or without bronchiolitis office-based treatment and outcomes for febrile infants with clinically diagnosed bronchiolitis diagnostic testing for serious bacterial infections in infants aged 90 days or younger with bronchiolitis sepsis evaluations in hospitalized infants with bronchiolitis multicenter rsv-sbi study group of the pediatric emergency medicine collaborative research committee of the american academy of pediatrics sepsis workup in febrile infants 0-90 days of age with respiratory syncytial virus infection risks for bacteremia and urinary tract infections in young febrile children with bronchiolitis risk of urinary tract infection in infants and children with acute bronchiolitis diagnosis and outcomes of enterovirus infections in young infants human mxa protein: an interferon-induced dynamin-like gtpase with broad antiviral activity diagnosis of viral infections using myxovirus resistance protein a (mxa) mxa-based recognition of viral illness in febrile children by a whole blood assay new sandwich-type enzymelinked immunosorbent assay for human mxa protein in a whole blood using monoclonal antibodies against gtp-binding domain for recognition of viral infection mxa expression in patients with viral infection after allogeneic stem cell transplantation mxa rna quantification in febrile patients who underwent hematopoietic cell transplantation for primary immunodeficiency current strategies for management of influenza in the elderly population role of neuraminidase in lethal synergism between influenza virus and streptococcus pneumoniae effectiveness of intranasal live attenuated influenza vaccine against all-cause acute otitis media in children the role of new vaccines in the prevention of otitis media prevention of otitis media by vaccination benefits of influenza vaccination for low-, intermediate-, and high-risk senior citizens efficacy of intranasal virosomal influenza vaccine in the prevention of recurrent acute otitis media in children additive preventive effect of influenza and pneumococcal vaccines in elderly persons effects of a largescale intervention with influenza and 23-valent pneumococcal vaccines in elderly people: a 1-year follow-up effect of conjugate pneumococcal vaccine followed by polysaccharide pneumococcal vaccine on recurrent acute otitis media: a randomised study pneumococcal conjugate vaccines for preventing otitis media vaccine safety datalink. effectiveness of pneumococcal polysaccharide vaccine in older adults neuraminidase inhibitors: zanamivir and oseltamivir neuraminidase inhibitors for preventing and treating influenza in children oral oseltamivir treatment of influenza in children impact of neuraminidase inhibitor treatment on outcomes of public health importance during the 2009-2010 influenza a(h1n1) pandemic: a systematic review and meta-analysis in hospitalized patients impact of oseltamivir treatment on influenza-related lower respiratory tract complications and hospitalizations efficacy and safety of the oral neuraminidase inhibitor oseltamivir in treating acute influenza: a randomized controlled trial: us oral neuraminidase study group motavizumab for prophylaxis of respiratory syncytial virus in high-risk children: a noninferiority trial publish your work in this journal submit your manuscript here: http://www.dovepress.com/therapeutics-and-clinical-risk-management-journal therapeutics and clinical risk management is an international, peerreviewed journal of clinical therapeutics and risk management, focusing on concise rapid reporting of clinical studies in all therapeutic areas, outcomes, safety, and programs for the effective, safe, and sustained use of medicines. this journal is indexed on pubmed central, cas, embase, scopus and the elsevier bibliographic databases. the manuscript management system is completely online and includes a very quick and fair peer-review system, which is all easy to use. visit http://www.dovepress.com/testimonials.php to read real quotes from published authors. key: cord-260472-xvvfguht authors: papadopoulos, nikolaos g.; konstantinou, george n. title: antimicrobial strategies: an option to treat allergy? date: 2007-01-31 journal: biomedicine & pharmacotherapy doi: 10.1016/j.biopha.2006.10.004 sha: doc_id: 260472 cord_uid: xvvfguht abstract respiratory infections by bacteria and viruses often trigger symptoms of asthma in both adults and children. this observation and subsequent mechanistic studies have demonstrated important interactions among allergens, microbes and the atopic host. the mechanisms responsible for microbe-induced asthma exacerbations are only incompletely understood. a focal point of current research is the inflammatory response of the host following an encounter with a pathogenic microbe, including variations in chemokine and cytokine production and resulting in changes in bronchial hyper-responsiveness and lung function. direct bronchial infection, exposure of nerves with resulting neurogenic inflammation and a deviated host immune response are among the mechanisms underlying these functional disorders. lately, suboptimal innate immune responses, expressed as defective interferon production, have gained attention as they might be amenable to intervention. this review describes the suggested mechanisms involved in the complex interactions between ‘asthmagenic’ microbes, the immune system and atopy, based on in-vitro and in-vivo experimental models and epidemiological evidence. in addition, it provides a synopsis of potential therapeutic strategies either directly against the microorganisms or in respect to the associated inflammation. the prevalence of allergies and asthma has been increasing, especially in children, for the last several decades. modern lifestyle and various environmental factors significantly influence the onset of these complex, chronic disorders. considerable research effort has focused on the potential effects of exposure to pollutants, aeroallergens and infectious agents that could adversely affect lung development or function but also precipitate asthma exacerbations [1] . it is widely recognized that respiratory viral infections are among the most important triggers of asthma exacerbations, both in children and adults [2e4] . the association between upper respiratory viral infections and asthma exacerbations in children was demonstrated almost three decades ago using virus cultures and serological techniques [5] . these findings were subsequently confirmed and expanded using more sensitive techniques for virus detection, such as reverse transcription polymerase chain reaction (rtepcr) assays in well-designed longitudinal studies [6e9]. after implementation of these techniques more than 80% of reported episodes of wheeze or drop in lung function could be attributed to respiratory viral infections [7] , rhinovirus (rv) being the most prevalent virus. similar studies in adults implicate respiratory pathogens in almost half of the exacerbations, rhinovirus being once again the prevailing virus [8, 10, 11] . abbreviations: rtepcr, reverse transcriptionepolymerase chain reaction; rv, rhinovirus; piv, parainfluenza virus; rsv, respiratory syncytial virus; mpv, human metapneumovirus; icam-1, intracellular adhesion molecule-1; ifn-b, interferon-beta; ngf, nerve growth factor; sp, substance p; nk1, neurokinin 1 receptor; mbl, mannose-binding lectin; laba, long-acting b 2 agonists. in addition, there is the evidence for an association between 'atypical' bacterial respiratory pathogens and the pathogenesis of asthma, with mycoplasma pneumoniae and chlamydophila pneumoniae most commonly implicated. however, many studies investigating such a link have been uncontrolled and have provided controversial evidence, mainly reflecting the difficulty in accurately diagnosing infection with these pathogens [12, 13] . taking into account these strong associations, it is conceivable that antimicrobial agents and/or strategies may have the potential to reduce the burden of asthma-associated morbidity. the majority of viruses implicated in the pathogenesis of asthma exacerbations are single-stranded rna viruses, including rv, influenza and parainfluenza (piv) viruses, respiratory syncytial virus (rsv) [3, 7, 14] , coronaviruses [15] and the newly described human metapneumovirus (mpv) [16] and bocavirus [17e19]. among the double-stranded dna viruses adenoviruses have also been involved [20] . in the human respiratory tract, all the above agents are able to produce a spectrum of clinical acute infection phenotypes, ranging from the common cold, croup and acute bronchiolitis, to pneumonia, although each virus has increased propensity for a particular clinical disease (e.g. parainfluenza for croup, rsv for severe bronchiolitis, influenza for pneumonia) [21, 22] . there is some evidence that adenovirus can also cause a latent infection in the human lung [23] . the spectrum of viral-triggered asthma exacerbations and reported viral prevalence may vary according to various factors. the age of the subjects is important [24] , since, overall, the frequency of viral respiratory illness is highest in children up to 4 years of age, gradually declines in teenagers, rises again in young parents exposed to children, re-declines in older adults, while the elderly are again more susceptible [25] . in addition respiratory viral infections have strong seasonal patterns although sporadic cases or nosocomial outbreaks can occur [6,26e30] . the presence or absence of a lipid-containing envelope affects viral survival in the environment [31, 32] . in temperate areas, the enveloped viruses, (e.g. influenza virus, rsv and coronavirus), are, characteristically, prevalent during middle-winter periods, whereas nonenveloped ones, such as rvs, are found most often in spring and fall. rv characteristically produces epidemics soon after children return to school. september epidemics of asthma exacerbations coincide with such increase in the rate of respiratory track infections [28, 29] . there is evidence that different infectious agents may induced asthma exacerbations of varying severity, however, stronger evidence is needed in this respect [6,33e35] . finally, the type of diagnostic test used to identify infection may considerably affect epidemiological results [7, 36] . although rsv remains the agent associated with the majority of cases of bronchiolitis requiring hospitalization, recent evidence suggests that in the community rv is the most prevalent virus at all ages [35] . furthermore, with a few exceptions, studies assessing virus-induced exacerbations of asthma have shown that rvs are the prevalent agents, attributing for 50e80% of virus-confirmed cases or around half of all exacerbations studied [7, 8] . there is also evidence that rv may be more 'asthmagenic' than other viruses (papadopoulos et al, unpublished) . rvs belong to picornaviridae family and probably represent the most abundant pathogenic microorganisms universally. these viruses have small rna genomes are nonenveloped and are capable of surviving on surfaces for several hours under ambient conditions [37] . more than 100 serotypes of rvs are identified and numbered. they are divided into major (90%) and minor (10%) groups depending on their receptor specificity. major rvs attach to the intracellular adhesion molecule-1 (icam-1) while minor group rv binds the low-density lipoprotein receptor. in vitro and in vivo, rv infects the bronchial epithelium and upregulates a range of pro-inflammatory cytokines, chemokines, adhesion molecules, mucins and growth factors, all of which are thought to contribute to lower airway inflammation and consequent effects on lung function [38, 39] . a large number of these mediators are upregulated partly or solely through the transcription factor nf-kb [40e42]. several mechanisms have been suggested as part of the complicated pathways leading from a viral infection to an acute asthma exacerbation. these include direct infection of the lower respiratory tract [43, 44] , induction of local inflammation [38, 41, 45] , increase in bronchial reactivity [43, 44, 46, 47] and induction of bronchial obstruction [48] . local inflammation produced after bronchial epithelial cell infection, neurogenic inflammation induced directly or indirectly through the epithelium, and the immune response of the host are probably the most important. there is increasing evidence that the epithelium of the lower airway does not simply act as a physical barrier. not only it has important regulatory role on the immune response inasmuch it may act as an antigen presenting structure [49] but also contributes to the inflammatory response following a viral infection through the production of cytokines and chemokines (e.g. il-6, il-8, il-11, tnf-a, rantes, gm-scf, eotaxin 1 and eotaxin 2) that attract inflammatory cells involved in asthma exacerbations [50, 51] . the epithelial cells' structure and function is altered after the infiltration with inflammatory cell and the oedema of the airway wall. it has been recently suggested that epithelial cells from asthmatic subjects may have an abnormal innate response to infection by rvs, resulting in increased virus replication and cell lysis compared with cells from healthy normal controls. cells from asthmatic individuals did not produce enough interferon-beta (ifn-b) in response to infection, leading to a reduced apoptosis rate, a consequent increase in viral replication within the cells and finally increased cytotoxicity because of the increased viral load [52] . another proposed mechanism by which acute infections might enhance airway narrowing and hyper-responsiveness is the stimulation of the airway neural network which may lead to neurogenic inflammation [39, 44, 53, 54] . virus-mediated damage to the epithelial layer can expose the dense subepithelial nerve endings, increasing stimulation of sensory nerves by inhaled particles or pro-inflammatory mediators. sensory nerves can directly release neuropeptides which may trigger reflex bronchoconstriction. among the mediators of neurogenic inflammation, nerve growth factor (ngf) may have an important role in the pathogenesis of hyper-responsiveness induced by respiratory viruses. it has been documented that ngf induces a selective up-regulation of the high-affinity neurokinin 1 receptor (nk1) for the tachykinin substance p (sp) [55] . sp is a neuropeptide released from sensory nerves with both bronchoconstrictive effects and immunomodulatory properties which regulates the functions of all white blood cells by affecting their migration and response to various mitogens and allergens [56] . one recent study [57] focused on neural development in the lungs during early life and has proposed that this process is under the control of ngf and its corresponding receptor trka. these factors control the branching of nerves into the developing lungs and are downregulated with age. ngf is strongly upregulated during rsv infections, especially in infants and such overexpression may result in prolonged viral clearance from the infected epithelial cells. finally, another interesting pathway attributes to protracted inflammation, associated with an imbalance in t h 1/t h 2 immune responses. in the lower airways of atopic asthmatic individuals a t h 2 environment predominates. although ifng, and il-12 (t h1 1 cytokines) are produced both in normal and atopic asthmatic subjects, the ratio between ifn/il-4 is considerably reduced in asthmatics compared with normal subjects [58] . furthermore, atopic individuals may have impaired antiviral responses concerning ifn-a [59, 60] or/ and ifn-b [52] or/and ifn-g [50, 61, 62] reduced secretion, something that may result in prolonged bronchial inflammation and increased asthma severity. it has been also demonstrated that this impairment is extended to cell recruitment, since in asthmatic patients there seems to be an increased number of eosinophils, compared with normal individuals which also indicates a difference in the immune response to viral infections [54, 63] . in the natural history of asthma exacerbations, interactions between viral infections and other environmental stimuli are often noted. in several occasions synergistic effects have been shown. this is important as it implies that therapeutic results may occur with treatment of only one of such factors. an association between upper respiratory tract infection and air pollution, especially no 2 , and tobacco smoke exposure, has been observed in children [64e66]. there are several potential mechanisms by which pollutants can exacerbate asthma interacting with respiratory viral infections. direct effects of the pollutant on the airways include epithelial damage and an acquired ciliary dyskinesia; release of pro-inflammatory mediators and increases in ige concentration may follow. indirectly no 2 can also impair local antiviral immunity in the airways [67e69]. recent studies have suggested that viruses and allergens may have a synergistic effect on individuals with asthma [43, 70] . this has been shown for both clinical outcomes [71] (symptoms severity) and in experimental models, where there is evidence that viral infections enhance allergen induced inflammatory responses, eosinophil recruitment, histamine release and late phase airway response [72] . although there is increasing evidence from controlled studies to support an association between atypical bacterial infection and both chronic stable asthma and acute exacerbations of asthma, it is still unclear whether such association is causal or patients with asthma are just more susceptible to colonization and/or infection with atypical bacteria. nevertheless, case reports, but also controlled trials during several decades have suggested that postinfectious wheezing may respond to antibiotic therapy, in particular to macrolides [73, 74] . problems with diagnostic techniques for acute and chronic infections with mycoplasma pneumoniae and chlamydophila pneumoniae have made difficult to conduct and interpret epidemiologic studies of the potential relation between these microorganisms and asthma. to add to this complexity, macrolides and ketolides have been shown to have anti-inflammatory properties [75, 76] , making it difficult to assess the true role of infection. in asthmatic patients, exacerbations can be associated with an increase in antibody titres to chlamydophila pneumoniae or mycoplasma pneumoniae [13, 74, 77, 78] . it has recently been proposed that c. pneumoniae might modulate epithelial cell apoptosis by upregulating both pro-apoptosis and anti-apoptosis genes. it has been suggested that c. pneumoniae-induced inhibition of apoptosis increases the longevity of the host cell, enhancing the survival of c. pneumoniae in patients with chronic asthma [79] . consequently, bronchial infection with atypical bacteria is likely to be associated with increased airway inflammation and thus possibly increasing asthma severity and airway remodelling. although, these organisms are common causes of infection, not all infected patients develop or exacerbate their asthma. this suggests that certain individuals may be genetically predisposed to the chronic effects of atypical bacteria, or be genetically susceptible to infection [52] , rendering them more likely to be persistently infected. only a few studies have investigated the possibility of such susceptibility. in one of them [80] , isotype-specific serologic tests have been performed for c. pneumoniae, and the results have been compared with variations in mannose-binding lectin (mbl), a complement component that is important in clearance of respiratory pathogens. the presence of variant alleles in mbl was associated with increased susceptibility to other types of respiratory infections, significantly increasing the risk of asthma development among children infected with c. pneumoniae. on the other hand, a recent study, detailed below, showed improved outcomes when a ketolide was used in patients with asthma exacerbations [81] . although there has been much progress in understanding the mechanisms of microbe-induced asthma exacerbations, there is a need for the development of new therapeutic agents as well as preventive strategies. both antimicrobial and immune modulators could have therapeutic benefits in this respect. rhinovirus is the key virus accounting for the majority of exacerbations both in children and adults and thus the effective treatment or prevention of that infection would be a major asset in asthma therapy. unfortunately, there is currently no available antiviral therapy of clinical value and vaccination seems to be far away because of the large number of rvs' serotypes (more than 100). although this genetic diversity has hampered vaccine development, modern vaccination strategies (such as recombinant proteins, reverse genetics, replication defective particles and other techniques) may make it feasible to induce cross-reactive neutralizing antibodies to the majority of serotypes and produce an effective vaccine [82] . in contrast to rv, rsv has gained more attention because of its association with severe bronchiolitis in infancy. ribavirin, although initially promising, did not find a place in majority of cases, although still included among possible choices for severe bronchiolitis. passive immunoprophylaxis by monthly administration of anti-f monoclonal antibody (palivizumab) reduces the risk of lower respiratory tract rsv disease and hospitalization in high-risk infants and children [83] . however, it cannot be used widely or in an outpatient basis. no vaccine against rsv is available yet, but studies of intranasal live-attenuated vaccine in children and injected subunit vaccine in elderly persons are ongoing [84] . influenza viruses a and b cause annual outbreaks of illness worldwide. a variety of antiviral agents are available for treatment of influenza. the previous generation of agents, amantadine and rimantadine, have demonstrated clinical efficacy, however, potential side effects and most importantly resistance considerably reduced its usefulness [85e87]. the more recent neuraminidase inhibitors, zanamivir and oseltamivir, are active against both a and b viruses, including the avian influenza a/h5n1 strain [87, 88] , and are promising as important tools against a pandemic. more possibilities for anti-influenza agents are being explored [89] . influenza vaccination is available in two forms: an intramuscular preparation containing formalin-inactivated virus and purified surface antigen and an intranasal spray containing live attenuated viruses [90] . the efficacy of these vaccines is approximately 70e90% in young adults, especially when the vaccine antigen and the circulating strain are closely matched. immunization in healthy working adults is associated with fewer upper respiratory illnesses and fewer visits to physicians' offices [90e93]. however, the use of influenza vaccines in reducing virus-induced exacerbations remain controversial [94, 95] . concerning the other asthmagenic viruses (coronaviruses, adenoviruses, human metapneumovirus, bocavirus), clinically available therapeutic or prophylactic agents are still awaited. as mentioned above, a causal link between deficient interferon-impaired apoptosis and increased virus replication has been demonstrated, suggesting that type i interferons might be useful in the treatment or prevention of virus-induced asthma exacerbations. type i ifns include the numerous ifn-as, ifn-b and the newly identified ifn-ls [96] . in the past, ifn-a2 was shown to be effective when given prior to experimental rv infection [97e99], or as a prophylactic therapy [100, 101] , in a context of natural rv infections, however cost and side-effects have prevented its exploitation in the common cold and/or asthma exacerbation fields. ifn-b has not been very effective in preventing experimental or natural rv colds [52,102e104] , but its effects on asthma exacerbations have not been investigated. promising data have been recently published about ifn-ls [105] . it should be noted that in addition to the antiviral approach, combinations of ifn-a with intranasal ipratropium, or oral naproxen, or chlorpheniramine, or ibuprofen have been tested with promising results, but these were also not commercialized [106, 107] . although quite active in vitro [108] , glucocorticosteroids (gcs) so far have been disappointing in their ability to control symptoms in models of experimental rv challenge of asthmatics [109, 110] and high-dose steroids remain only partially effective at controlling virus-induced exacerbations of asthma [111, 112] . a synergistic effect of gcs with long-acting b 2 agonists (laba) has been shown both in in vitro studies and clinically. labas act via a g protein coupled receptor, activate adenylate cyclase and through the second messenger camp, induce intracellular signalling events affecting a broad range of physiological processes, providing by this way an extra potentiality to enhance the anti-inflammatory properties of gcs when acting together in a combination therapy [113] . studies have confirmed clinical benefit in exacerbations, although the viral origin of these events has not been confirmed [114, 115] . evidence suggests that leukotrienes play a key role in viralinduced respiratory illness [116, 117] . the leukotriene receptor antagonist, montelukast, has proven efficacy in the control of asthma exacerbations in adults [118] , but also in preschool and school children with persistent [119, 120] and intermittent asthma [121] . in addition, montelukast significantly reduced symptoms and exacerbations from respiratory syncytial virus postbronchiolitis in infants without asthma [122] . other agents, including antihistamines [123] and antioxidants [124] can block pro-inflammatory mechanisms induced by virus infections in airway epithelial cells, although in vitro evidence has not been paralleled by convincing clinical data. although viral infections are of major interest, the potential role of antibacterial therapy should also be discussed. a number of different antibacterial agents, namely tetracyclines, macrolides, quinolones, azalides and the ketolide telithromycin have in vitro and in vivo activity against the common atypical bacteria c. pneumoniae and m. pneumoniae [125e128]. clarithromycin, roxithromycin, azithromycin [73, 74, 129, 130] and recently telithromycin [81] , have shown some clinical benefit in patients with chronic stable asthma or acute exacerbations. in the most recently published double-blind, randomized, placebo-controlled study [81] telithromycin (at a daily dose of 800 mg for 10 days) provided improvement in symptoms and lung function among adult patients with acute exacerbations of asthma. the study design did not incorporate an analysis of the mechanism by which telithromycin was associated with improvement, but the data imply a benefit not solely attributable to an antimicrobial effect. it should be noted that resistance need also to be considered before initiation of long term therapy in order to control or to prevent probable bacteria-induced asthma attacks. although promising, many questions should be answered before antibacterial therapies can be proposed for the treatment or prevention of asthma exacerbations. certainly, a combination of the antimicrobial with anti-inflammatory approaches seems reasonable. there is evidence suggesting that a window of opportunity exists between appearance of a viral infection and the initiation of an exacerbation [131] . on the other hand, more controlled studies with macrolides, especially in children, are urgently needed as management of exacerbations is an important unmet need in this age group. global initiative for asthma (gina) and its objectives virusinduced asthma rhinovirus respiratory infections and asthma viruses as precipitants of asthmatic attacks in children the relationship between upper respiratory infections and hospital admissions for asthma: a timetrend analysis community study of role of viral infections in exacerbations of asthma in 9-11 year old children respiratory viruses and exacerbations of asthma in adults rhinovirus and respiratory syncytial virus in wheezing children requiring emergency care. ige and eosinophil analyses the incidence of respiratory tract infection in adults requiring hospitalization for asthma epidemiology of respiratory viruses in patients hospitalized with near-fatal asthma, acute exacerbations of asthma, or chronic obstructive pulmonary disease is asthma an infectious disease?: thomas a. neff lecture chlamydophila pneumoniae and mycoplasma pneumoniae: a role in asthma pathogenesis? respiratory viruses and asthma human coronavirus nl63 infection and other coronavirus infections in children hospitalized with acute respiratory disease in hong kong human metapneumovirus infection plays an etiologic role in acute asthma exacerbations requiring hospitalization in adults human bocavirus: prevalence and clinical spectrum at a children's hospital the association of newly identified respiratory viruses with lower respiratory tract infections in korean children detection of human bocavirus in japanese children with lower respiratory tract infections persistent adenoviral infection and chronic airway obstruction in children viral infections of the respiratory tract virus infections, wheeze and asthma latent adenoviral infection in the pathogenesis of chronic airways obstruction viral respiratory infection and the link to asthma studies of the community and family: acute respiratory illness and infection a population based time series analysis of asthma hospitalisations in using seasonal variations in asthma hospitalizations in children to predict hospitalization frequency the september epidemic of asthma exacerbations in children: a search for etiology the september epidemic of asthma hospitalization: school children as disease vectors seasonality in pediatric asthma admissions: the role of climate and environmental factors the influence of environment on the survival of airborne virus particles in the laboratory virus survival in the environment rhinovirus viremia in children with respiratory infections role of respiratory viruses in acute upper and lower respiratory tract illness in the first year of life: a birth cohort study predominance of rhinovirus in the nose of symptomatic and asymptomatic infants use of induced sputum for the diagnosis of influenza and infections in asthma: a comparison of diagnostic techniques chemical disinfection to interrupt transfer of rhinovirus type 14 from environmental surfaces to hands experimental rhinovirus 16 infection increases intercellular adhesion molecule-1 expression in bronchial epithelium of asthmatics regardless of inhaled steroid treatment experimental rhinovirus 16 infection. effects on cell differentials and soluble markers in sputum in asthmatic subjects rhinovirus replication in human macrophages induces nf-kappab-dependent tumor necrosis factor alpha production rhinovirus infection induces expression of its own receptor intercellular adhesion molecule 1 (icam-1) via increased nf-kappab-mediated transcription respiratory epithelial cell expression of vascular cell adhesion molecule-1 and its up-regulation by rhinovirus infection via nf-kappab and gata transcription factors rhinovirus infection preferentially increases lower airway responsiveness in allergic subjects duration of postviral airway hyperresponsiveness in children with asthma: effect of atopy the role of intercellular adhesion molecule-1 (icam-1), vascular cell adhesion molecule-1 (vcam-1), and regulated on activation, normal t-cell expressed and secreted (rantes) in the relationship between air pollution and asthma among children effect of experimental rhinovirus 16 colds on airway hyperresponsiveness to histamine and interleukin-8 in nasal lavage in asthmatic subjects in vivo rhinovirus inhalation causes long-lasting excessive airway narrowing in response to methacholine in asthmatic subjects in vivo experimental rhinovirus 16 infection causes variable airway obstruction in subjects with atopic asthma rhinovirus infection induces major histocompatibility complex class i and costimulatory molecule upregulation on respiratory epithelial cells relationship of upper and lower airway cytokines to outcome of experimental rhinovirus infection cytokines in asthma asthmatic bronchial epithelial cells have a deficient innate immune response to infection with rhinovirus expression of costimulatory molecules in peripheral blood mononuclear cells of atopic asthmatic children during virus-induced asthma exacerbations lower airways inflammation during rhinovirus colds in normal and in asthmatic subjects nerve growth factor and nerve growth factor receptors in respiratory syncytial virus-infected lungs the role of neural inflammation in asthma and chronic obstructive pulmonary disease neurotrophin overexpression in lower airways of infants with respiratory syncytial virus infection a defective type 1 response to rhinovirus in atopic asthma atopic phenotype in children is associated with decreased virus-induced interferon-alpha release impaired virus-induced interferon-alpha2 release in adult asthmatic patients il-4 and interferon-gamma production in children with atopic disease rhinovirus-induced interferon-gamma and airway responsiveness in asthma antigen-nonspecific recruitment of th2 cells to the lung as a mechanism for viral infection-induced allergic asthma personal exposure to nitrogen dioxide (no2) and the severity of virus-induced asthma in children epidemiology of childhood asthma are non-allergenic environmental factors important in asthma? air pollution and infection in respiratory illness nitrogen dioxide exposure: effects on airway and blood cells synergism between rhinovirus infection and oxidant pollutant exposure enhances airway epithelial cell cytokine production study of modifiable risk factors for asthma exacerbations: virus infection and allergen exposure increase the risk of asthma hospital admissions in children synergism between allergens and viruses and risk of hospital admission with asthma: case-control study presence of asthma risk factors and environmental exposures related to upper respiratory infection-triggered wheezing in middle school-age children trial of roxithromycin in subjects with asthma and serological evidence of infection with chlamydia pneumoniae mycoplasma pneumoniae and chlamydia pneumoniae in asthma: effect of clarithromycin immunomodulatory activity and effectiveness of macrolides in chronic airway disease clinical implications of the immunomodulatory effects of macrolides chlamydia pneumoniae immunoglobulin a reactivation and airway inflammation in acute asthma chlamydia pneumoniae infection and asthma chlamydia and apoptosis: life and death decisions of an intracellular pathogen the development of asthma in children infected with chlamydia pneumoniae is dependent on the modifying effect of mannose-binding lectin the effect of telithromycin in acute exacerbations of asthma vaccines, vaccination, and vaccinology prophylaxis with palivizumab against respiratory syncytial virus infection in infants with congenital heart diseasedwho should receive it? immunopathology of rsv infection: prospects for developing vaccines without this complication incidence of adamantane resistance among influenza a (h3n2) viruses isolated worldwide from 1994 to 2005: a cause for concern adamantane resistance among influenza a viruses isolated early during the 2005e2006 influenza season in the united states antiviral resistance in influenza viruseseimplications for management and pandemic response neuraminidase inhibitor susceptibility network position statement: antiviral resistance in influenza a/h5n1 viruses the structure of h5n1 avian influenza neuraminidase suggests new opportunities for drug design comparison of the efficacy and safety of live attenuated cold-adapted influenza vaccine, trivalent, with trivalent inactivated influenza virus vaccine in children and adolescents with asthma estimating efficacy of trivalent, cold-adapted, influenza virus vaccine (caiv-t) against influenza a (h1n1) and b using surveillance cultures safety and efficacy of trivalent inactivated influenza vaccine in young children: a summary for the new era of routine vaccination direct and total effectiveness of the intranasal, live-attenuated, trivalent cold-adapted influenza virus vaccine against the 2000e2001 influenza a(h1n1) and b epidemic in healthy children randomised placebo-controlled crossover trial on effect of inactivated influenza vaccine on pulmonary function in asthma influenza vaccination in children with asthma: randomized double-blind placebo-controlled trial ifn-lambdas mediate antiviral protection through a distinct class ii cytokine receptor complex prevention of rhinovirus colds by human interferon alpha-2 from escherichia coli efficacy and tolerance of intranasally applied recombinant leukocyte a interferon in normal volunteers intranasal interferon alpha 2 for prevention of rhinovirus infection and illness intranasal interferon-alpha 2 prophylaxis of natural respiratory virus infection intranasal interferonalpha 2b for seasonal prophylaxis of respiratory infection interferon-beta ser as prophylaxis against experimental rhinovirus infection in volunteers tolerance and efficacy of intranasal administration of recombinant beta serine interferon in healthy adults ineffectiveness of recombinant interferon-beta serine nasal drops for prophylaxis of natural colds role of deficient type iii interferon-lambda production in asthma exacerbations comparative susceptibility of respiratory viruses to recombinant interferons-alpha 2b and -beta combined antiviral and antimediator treatment of rhinovirus colds corticosteroids inhibit rhinovirus-induced intercellular adhesion molecule-1 up-regulation and promoter activation on respiratory epithelial cells rhinovirus-induced airway inflammation in asthma: effect of treatment with inhaled corticosteroids before and during experimental infection a randomized controlled trial of glucocorticoid prophylaxis against experimental rhinovirus infection doubling the dose of inhaled corticosteroid to prevent asthma exacerbations: randomised controlled trial doubling the dose of budesonide versus maintenance treatment in asthma exacerbations combination therapy: synergistic suppression of virus-induced chemokines in airway epithelial cells meta-analysis of increased dose of inhaled steroid or addition of salmeterol in symptomatic asthma (miasma) low dose inhaled budesonide and formoterol in mild persistent asthma: the optima randomized trial increased production of ifn-gamma and cysteinyl leukotrienes in virus-induced wheezing the release of leukotrienes in the respiratory tract during infection with respiratory syncytial virus: role in obstructive airway disease montelukast, a once-daily leukotriene receptor antagonist, in the treatment of chronic asthma: a multicenter, randomized, doubleblind trial. montelukast clinical research study group montelukast once daily inhibits exercise-induced bronchoconstriction in 6-to 14-year-old children with asthma montelukast for chronic asthma in 6-to 14-year-old children: a randomized, double-blind trial. pediatric montelukast study group montelukast reduces asthma exacerbations in 2-to 5-year-old children with intermittent asthma a randomized trial of montelukast in respiratory syncytial virus postbronchiolitis effect of desloratadine and loratadine on rhinovirusinduced intercellular adhesion molecule 1 upregulation and promoter activation in respiratory epithelial cells oxidants in asthma and in chronic obstructive pulmonary disease (copd) activity of gemifloxacin and other new quinolones against chlamydia pneumoniae: a review effects of respiratory mycoplasma pneumoniae infection on allergen-induced bronchial hyperresponsiveness and lung inflammation in mice activity of telithromycin, a new ketolide antibacterial, against atypical and intracellular respiratory tract pathogens review of macrolides and ketolides: focus on respiratory tract infections macrolides for chronic asthma macrolides for chronic asthma effect of inhaled formoterol and budesonide on exacerbations of asthma. formoterol and corticosteroids establishing therapy (facet) international study group key: cord-272538-gclrtie7 authors: li, xuechao; li, juansheng; meng, lei; zhu, wanqi; liu, xinfeng; yang, mei; yu, deshan; niu, lixia; shen, xiping title: viral etiologies and epidemiology of patients with acute respiratory infections based on sentinel hospitals in gansu province, northwest china, 2011‐2015 date: 2018-02-22 journal: j med virol doi: 10.1002/jmv.25040 sha: doc_id: 272538 cord_uid: gclrtie7 understanding etiological role and epidemiological profile is needed to improve clinical management and prevention of acute respiratory infections (aris). a 5‐year prospective study about active surveillance for outpatients and inpatients with aris was conducted in gansu province, china, from january 2011 to november 2015. respiratory specimens were collected from patients and tested for eight respiratory viruses using polymerase chain reaction (pcr) or reverse transcription polymerase chain reaction (rt‐pcr). in this study, 2768 eligible patients with median age of 43 years were enrolled including pneumonia (1368, 49.2%), bronchitis (435, 15.7%), upper respiratory tract infection or urti (250, 9.0%), and unclassified ari (715, 25.8%). overall, 29.2% (808/2768) were positive for any one of eight viruses, of whom 130 cases were identified with two or more viruses. human rhinovirus (hrv) showed the highest detection rate (8.6%), followed by influenza virus (flu, 7.3%), respiratory syncytial virus (rsv, 6.1%), human coronavirus (hcov, 4.3%), human parainfluenza (piv, 4.0%), adenovirus (adv, 2.1%), human metapneumovirus (hmpv, 1.6%), and human bocavirus (hbov, 0.7%). compared with urti, rsv was more likely identified in pneumonia (χ(2) = 12.720, p < 0.001) and hcov was more commonly associated with bronchitis than pneumonia (χ(2) = 15.019, p < 0.001). in patients aged less than 5 years, rsv showed the highest detection rate and hcov was the most frequent virus detected in adults and elderly. the clear epidemical seasons were observed in hrv, flu, and hcov infections. these findings could serve as a reference for local health authorities in drawing up further plans to prevent and control aris associated with viral etiologies. distribution, 70% of children who died from aris were in southeast asia and africa. 2 in china, despite the mortality of live births reduced significantly in past decades, aris were still the main cause of children mortality. 3 several studies have reported the prevalence of respiratory viruses causing aris in china [4] [5] [6] [7] [8] ; however most of these studies were conducted in developed regions of eastern coastal china. thus, this paper presents data on the epidemiology of viral etiologies associated with aris in gansu province, which located in a relatively undeveloped area of china and aims to provide basic data of viral etiologies of aris to direct local disease prevention and control. this study was approved by the ethics review committee of chinese center for disease control and prevention (cdc) and all participants were informed of the study objectives, and written consent was obtained from patients or guardians. inpatients and outpatients were first screened by physicians of sentinel hospitals for aris and if they met inclusion criteria as follows, these patients would be enrolled into our study. a patient was considered to be having aris if they had: (1) at least one of listed manifestation of acute infection: measured fever (≥38°c), abnormal white blood cell (wbc) differential, leukocytosis (a wbc count more than 10 000/µl) or leucopenia (a wbc count less than 4000/µl), and chill; (2) at least one of listed signs/symptoms: cough, sputum, shortness of breath, lung auscultation abnormality (rale or wheeze), tachypnoea, and chest pain. among aris patients, those with a chest radiograph demonstrating punctuate, patchy or uniform density opacity were defined as having radiographic evidence of pneumonia. 5 the diagnosis for each patient admitted in this study was made by attending physicians and based on standard clinical criteria. thus, pneumonia was diagnosed with fever, tachypnoea, chest pain, and respiratory distress where focal or diffuse crackles or decreased vesicular sounds were present on auscultation. chest radiograph was used to distinguish pneumonia and other aris, but some diagnosis of pneumonia were based on clinical criteria alone. bronchitis was diagnosed in whom upper respiratory symptoms preceded lower respiratory symptoms of wheeze, dyspnea, and signs of respiratory distress. upper respiratory tract infections were diagnosed based on symptoms such as cough, runny nose, sore throat, and coryza. aris other than pneumonia, bronchitis, and upper respiratory tract infections were defined as unclassified aris. respiratory specimens (nasopharyngeal swab or aspirate, sputum, bronchoalveolar lavage, or lung puncture aspirate) were collected in aris patients and placed immediately in viral transport media (vtm). collected specimens were stored at 4-8°c at the local hospital and were transferred to the sentinel laboratories for diagnostic testing. viral molecular tests were completed within 24 h after collection; otherwise specimens in vtm should be stored at −70°c. every specimen from patients was detected for eight viruses. the viral nucleic acid was directly extracted from specimens by commercial kits (qiampminielute virus spin kit, qiaamp viral rna mini kit or rneasy mini kit, qiagen, valencia, ca) recommended by surveillance protocol. adv and hbov were determined by polymerase chain reaction (pcr). 9,10 reverse transcription-polymerase chain reaction (rt-pcr) was performed to detect the other six viruses. [11] [12] [13] [14] the primer sequences of pcr or rt-pcr were shown in table 1 . if any one of the targeted viruses was detected in the specimens, the patient was considered to be positive for that viral etiology. the cases where only one virus identified were labelled as single infection, two etiologies were co-infection, and three or more were multiple-infection. demographic characteristics, clinical symptoms were collected by staff of sentinel hospitals through a standardized questionnaire of protocol. data were analyzed using spss (v20.0, spss, chicago, il). two tailed mann-whitney test was used to compare median of two groups and comparison of median in more than two groups used kruskal-wallis test. categorical data was performed using chi-square test or fisher exact test. p-value < 0.05 was considered to be statistically significant. of all 2768 aris patients tested for eight viruses, 808 (29.2%) were positive for at least one virus. the median age of these patients was lower than patients who were negative for any respiratory viruses (p < 0.05, mann-whitney test). hrv showed the highest detection rate (table 3) . of 2336 inpatients, 29.8% tested positive for at least one virus. this rate was similar to that of outpatients (χ 2 = 3.027, p > 0.05). the most common virus was hrv, with 8.3% (195/2336) detected in inpatients and 9.7% (42/432) in outpatients, whereas the detection rate of hrv had no difference between two groups (χ 2 = 0.880, p > 0.05). compared with outpatients, only rsv and piv were more likely detected in inpatients (rsv: χ 2 = 4.206, p < 0.05; piv: χ 2 = 4.849, all of aris patients were divided into six age groups. the overall detection rate between age groups had significant difference apart from hmpv and hbov, each of else respiratory viruses incidence differed among age groups (table 5 ). over the 59 months study period, there were clear seasonal peaks for hrv, flu, and hcov infections. hrv infections were occurred each month throughout the year during 5 years with an annual a peak in september-october (figure 1, panel a) . flu and hcov infections showed a similarly circulation of one peak annually, with the peak of flu infections was observed during december-january and hcov during july to august (figure 1, panels b and d) . rsv and piv did not show the clear epidemic season (figure 1 , panels c and e). due to the infections of adv, hmpv, and hbov appeared sporadically among our study period, we did not present seasonal distributions of these viruses in figure 1 . before this study there was a similar report about prevalence of children infected with respiratory viruses in gansu province. 15 however, that study was only based on children aged less than of patients but also in each of diagnosis groups. highest detection rate (45.3%) in patients aged 1-5 years indicated that aris still was a risk factor for younger children's health, although the mortality due to respiratory tract infections decreased more than 35% in this age population. 17 pneumonia, a leading cause of mortality of children less than 5 years especially in developing country, 18 was commercially available, such as inactivated vaccine, which was administered intramuscularly or intranasally. [29] [30] [31] however, it had an extremely low coverage rate in china. 32 we considered influenza vaccine should be used in widespread areas to reduce the incidence of influenza disease in gansu. it should be noted that piv was a another major virus for children aged 1-5 years besides rsv, which was in agreement with studies conducted before. [33] [34] [35] this finding indicated that local pediatricians not only took priority over rsv infections but global mortality, disability, and the contribution of risk factors: global burden of disease study estimates of world-wide distribution of child deaths from acute respiratory infections causes of deaths in children younger than 5 years in china study on the viral etiology of acute respiratory tract infections in shanghai area during 2009-2010 prevalence of human respiratory viruses in adults with acute respiratory tract infections in beijing identification of viral and atypical bacterial pathogens in children hospitalized with acute respiratory infections in hong kong by multiplex pcr assays viral etiology of acute respiratory tract infections in hospitalized children and adults in shandong province prevalence of respiratory viruses among children hospitalized from respiratory infections in shenzhen, china polymerase chain reaction for detection of adenoviruses in stool samples bocavirus infection in hospitalized children simultaneous detection of influenza a, b, and c viruses, respiratory syncytial virus, and adenoviruses in clinical samples by multiplex reverse transcription nested-pcr assay simultaneous detection of fourteen respiratory viruses in clinical specimens by two multiplex reverse transcription nested-pcr assays children with respiratory disease associated with metapneumovirus in hong kong characterization and complete genome sequence of a novel coronavirus, coronavirus hku1, from patients with pneumonia viral etiology of acute respiratory infection in gansu province, china viral etiologies of hospitalized acute lower respiratory infection patients in china estimates of the global, regional, and national morbidity, mortality, and aetiologies of lower respiratory tract infections in 195 countries: a systematic analysis for the global burden of disease study acute lower respiratory infections in developing countries respiratory syncytial virus and other respiratory viral infections in older adults with moderate to severe influenza-like illness viral infection in adults hospitalized with community-acquired pneumonia: prevalence, pathogens, and presentation human coronaviruses: clinical features and phylogenetic analysis molecular characterization of human respiratory syncytial virus in the philippines impact of respiratory syncytial virus infection as a cause of lower respiratory tract infection in children younger than 3 years of age in japan global burden of acute lower respiratory infections due to respiratory syncytial virus in young children: a systematic review and meta-analysis respiratory syncytial virus hospitalization and mortality: systematic review and meta-analysis detection and typing by molecular techniques of respiratory viruses in children hospitalized for acute respiratory infection in rome, italy respiratory consequences of rhinovirus infection the common cold: a review of the literature the common cold review of intranasal influenza vaccine pre-clinical and clinical investigation of the safety of a novel adjuvant for intranasal immunization seasonal influenza vaccine supply and target vaccinated population in china viral and atypical bacterial detection in acute respiratory infection in children under five years viral etiology of severe pneumonia among kenyan young infants and children acute viral lower respiratory tract infections in cambodian children: clinical and epidemiologic characteristics frequency and natural history of rhinovirus infections in adults during autumn prevalence of human coronaviruses in adults with acute respiratory tract infections in beijing epidemiology and clinical presentations of the four human coronaviruses 229e, hku1, nl63, and oc43 detected over 3 years using a novel multiplex real-time pcr method viral etiologies and epidemiology of patients with acute respiratory infections based on sentinel hospitals in gansu province the authors have declared that no competing interests exist. http://orcid.org/0000-0002-6485-6970 key: cord-260871-dtn5t8ka authors: silva, marcus tulius t.; lima, marco; araujo, abelardo q.-c. title: sars-cov-2: should we be concerned about the nervous system? date: 2020-07-17 journal: am j trop med hyg doi: 10.4269/ajtmh.20-0447 sha: doc_id: 260871 cord_uid: dtn5t8ka the covid-19 pandemic has proved to be an enormous challenge to the health of the world population with tremendous consequences for the world economy. new knowledge about covid-19 is being acquired continuously. although the main manifestation of covid-19 is sars, dysfunction in other organs has been described in the last months. neurological aspects of covid-19 are still an underreported subject. however, a plethora of previous studies has shown that human covs might be neurotropic, neuroinvasive, and neurovirulent, highlighting the importance of this knowledge by physicians. besides, several neurological manifestations had been described as complications of two other previous outbreaks of cov diseases (sars ad middle east respiratory syndrome). therefore, we should be watchful, searching for early evidence of neurological insults and promoting clinical protocols to investigate them. our objectives are to review the potential neuropathogenesis of this new cov and the neurological profile of covid-19 patients described so far. the world has been facing a pandemic for less than 6 months that has already resulted in thousands of deaths and is paralyzing the world economy. in december 2019, severe pneumonia cases of unknown origin were seen in wuhan, china. 1, 2 the pathogen was rapidly identified as a novel enveloped rna β-cov, named sars-cov-2. sars-cov-2 quickly spread to other parts of china and later to all continents. thenceforth, in march 2020, the who declared covid-19 a new pandemic. several neurological manifestations were described as complications of two other previous outbreaks of cov diseases, namely, sars and the middle east respiratory syndrome (mers). [3] [4] [5] [6] [7] furthermore, recent clinical observations had stressed the possibility of neurological diseases also in the context of covid-19. [8] [9] [10] [11] therefore, we should be vigilant, searching for early evidence of neurological insults and promoting clinical protocols to investigate them. for instance, considering that encephalitis is associated with high mortality and morbidity, early diagnosis and management may contribute to better outcomes. several clinical and laboratory studies have shown that human coronaviruses (hcovs) might be neurotropic, neuroinvasive, and neurovirulent. [12] [13] [14] [15] the objectives of this article are to review the potential neuropathogenesis of this new cov and the neurological profile of covid-19 patients described worldwide. viral respiratory infections in humans are generally secondary to the human respiratory syncytial virus, influenza virus, hcov, measles virus, rhinovirus, adenovirus, and human metapneumovirus. 16 transmission of these agents occurs mainly by contact with fomites or suspension droplets. 17 all these viruses can produce bronchiolitis and pneumonia, being responsible for a large number of hospitalizations every winter season. 16, 18, 19 in some cases, central nervous system (cns) diseases can also be seen. it is well known that viral respiratory infection can result in several neurological disorders such as seizures, status epilepticus, encephalopathy, and encephalitis (table 1) . respiratory viruses, in general, can invade the cns through three main routes: the hematogenous route, through the infection of the endothelium or by transendothelial mechanisms; the "trojan horse" mechanism, by which viruses in the bloodstream infect leukocytes that can transmigrate across the permeable blood-brain barrier; and through the olfactory nerves by axonal transport via olfactory neurons. this last example is an elegant mechanism to access the cns for a virus that enters the body intranasally, such as most of the respiratory viruses highlighted before. 20, 21 furthermore, it is tempting to associate this route with anosmia, a frequent symptom of covid-19. 22, 23 however, in experimental work, the probable route for brain infection in macaques was the hematogenous one. 24 cov rna and cov antigen were detected in nonhuman primates' brain after intranasal, intraocular, or intravenous inoculation of murine cov jhm omp1. in this animal model, both the lack of detection of virus products in the trigeminal ganglia or olfactory bulbs and the presence of viral antigen in vessels and perivascular regions suggest that cov entered the cns through vascular endothelium. covs are widespread and infect different species, generally causing mild, uncomplicated respiratory and enteric diseases. usually, they infect the upper respiratory tract, being mainly associated with the common cold. however, in some patients, they can reach the lower respiratory tract, causing interstitial pneumonia, exacerbations of asthma, respiratory distress syndrome, or even sars. 25 covs are enveloped positive-sense rna viruses characterized by club-like spikes that project from their surface, an unusually large rna genome, and a unique replication strategy. covs are classified into four different groups; α-, β-, γ-, and δ-cov. 26 sars-cov-2 is an rna β-cov with a characteristic crown-shaped appearance, grouped within the family coronaviridae, order nidovirales. sars-cov-2 shares significant genetic homology with sars-cov, a virus associated with the pandemic of sars that occurred in 2003. similar to sars-cov, angiotensinconverting enzyme 2 (ace2), an enzyme that physiologically counters renin-angiotensin-aldosterone system activation, is the functional receptor to sars-cov-2. 27 it is known that the ace2 receptor is also expressed in the brain. 28 several hcovs are pathogenic to humans, such as hcov-oc43, hcov-229e, mers-cov, and sars-cov, all of them having different genotypes. [29] [30] [31] neurotropic and neuroinvasive abilities of hcov have been described both in animals and humans, and is implicated in conditions such as multiple sclerosis and encephalomyelitis. 5, 18 interestingly, the first detection of hcov in the human brain was made at autopsy cases of multiple sclerosis in the early eighties. 32 more recently, arbour et al. 13 detected the presence of the hcov-oc43 in brain parenchyma samples of 35.9% patients with multiple sclerosis, compared with 13.7% of controls. also, murine hepatitis virus, another cov, has been linked to chronic inflammation and demyelination of the cns in animal models. 33 the viral glycoprotein s (spike) has an essential role for the neurovirulence, especially for the hcov-oc4. 34 sars-cov. sars was a novel zoonotic infectious disorder associated with sars-cov. it was first diagnosed in china, in november 2002. the comparison of sars-cov sequences isolated from civets and patients supported the concept of transmission from these animals to humans. 35, 36 phylogenetic analysis showed that sars-cov was not a novel cov, but a branch of the β-cov. [37] [38] [39] typically, sars patients exhibited a triphasic pattern of disease, initially presenting with fever, a nonproductive cough, sore throat, and myalgia. 40 generally, dyspnea does not become a prominent feature until the second week of illness. in the second phase, dyspnea and hypoxia with fever become more prominent. some patients progress to acute respiratory distress by the third week, often requiring mechanical ventilation. the severity of the disease was correlated with increasing age, and the mortality can reach 50% for patients older than 60 years. 40, 41 during the sars-cov outbreak, several neurological diseases were reported. peripheral nervous system manifestations associated with sars were described in four patients, including both axonal polyneuropathy and myopathy. 4 the neuromuscular disorders developed approximately 3 weeks after the onset of sars, and the prognosis was good. interestingly, olfactory neuropathy was described during the sars outbreak. 42 this finding is relevant because, nowadays, many patients with sars-cov-2 infection have reported anosmia. 23 a neuroinvasive behavior of sars-cov could be found in some other reports. sars-cov rna was present in both the serum and cerebrospinal fluid (csf) from a patient with status epilepticus and sars during the 2003 outbreak. 43 in another report, sars-cov rna was recovered from a csf sample of an infected patient admitted because of generalized seizures. 5 in addition, a 39-year-old patient died of sars after a severe chronic progressive viral cerebritis. 44 neuropathological examination showed gliocyte hyperplasia, neuron denaturation, and necrosis coupled with striated encephalomalacia. neuroinvasion by sars-cov was confirmed by the typical viral morphology observed under electron microscopy, by genetic identification, and by the detection of the viral antigen (n protein) in the brain. in a neuropathological study of patients with sars, performed during the 2003 chinese outbreak, sequences of sars-cov genome were detected in all brain samples, but in none of the control cases. 45 the authors stressed that the infection of neurons occurred in selected areas such as the hypothalamus and cortex. the degeneration seen in these cases was probably secondary to neuronal hypoxia/ischemia. it is hypothesized that the infection of neurons may explain a higher than usual percentage of neurological and psychological abnormalities observed in patients with late-stage sars. 46 therefore, those neuropsychiatric symptoms could not be merely attributed to negative social pressure during the epidemic. interestingly, it is not uncommon for survivors of sars to report neuropsychiatric symptoms, such as lethargy, malaise, orthostatic dizziness, apathy, depression, and anxiety. recently, studies have linked neuroendocrine aberrations to some neuropsychiatric conditions. dysfunction in the hypothalamic-pituitary-adrenal axis was reported in survivors of sars by leow et al. 47 in a cohort of 61 sars patients, 39.3% had a central hypocortisolism. the authors speculated that either a hypophysitis or a direct viral hypothalamic infection could be the cause of these extrapulmonary symptoms. all these clinical, laboratory, and neuropathological evidence suggest that cns infection is possible in patients with sars. most hcovs share similar viral structures and mechanisms of infection and have documented neurotropism. therefore, infectious mechanisms previously found in other hcovs may also apply to sars-cov-2. 48, 49 taking these observations into account, a proactive search for neurological symptoms and signs could elucidate if the same occurs in sars-cov-2 infection. sars-cov-2. sars-cov-2 is a new virus that shares almost 80% genomic homology with sars-cov. however, the highest level of similarity is with a horseshoe bat cov. 50 therefore, it is believed that sars-cov-2 is a recombinant virus, transferred from bats to human hosts via an intermediate host. 51 because it is an rna virus with an rna-dependent rna polymerase-based replication, mutation and recombination are not uncommon events. sars-cov-2 has been recently associated with severe interstitial pneumonia, called covid-19. 1,2 covid-19 is an acute viral pneumonia, potentially lethal in many cases. characteristics of patients with severe evolution are the rapid progression to respiratory failure; it is estimated that among those with respiratory difficulties, 50% have to be admitted to intensive care units, and, of these, 46-65% worsen in a short period and may die in a few days. although sars-cov and sars-cov-2 use the same receptor to enter human cells, the ace2 binding affinity of the sars-cov-2 spike protein is 10-to 20-fold higher than that of the sars-cov spike protein. 52 once within the cns environment, its interaction with ace2 receptors expressed in neurons can initiate a cycle of viral budding accompanied by neuronal damage, without substantial inflammation, as seen in the past in cases of sars-cov. 53 it is also important to mention that long before the neuronal damages occur, endothelial tears in cerebral capillaries, followed by bleeding within the cerebral tissue, can have fatal consequences in patients with covid-19. 54 experimental studies with hcov and other viruses have shown the presence of viral particles in the brain, especially in the brainstem. viral antigens of influenza and pseudorabies virus have been detected in the nucleus of the solitary tract and the nucleus ambiguous. 55, 56 the nucleus of the solitary tract receives sensory information from the mechanoreceptors and chemoreceptors of the lung and upper and lower airways, whereas the efferent fibers from the nucleus ambiguous and the nucleus of the solitary tract provide innervation to airway smooth muscle, glands, and blood vessels. such neuroanatomical interconnections suggest that the death of many infected animals and even patients may be due to a dysfunction of the cardiorespiratory center in the brainstem. 53, 57 likewise, experimental studies using transgenic mice have demonstrated brainstem infection by sars-cov and mers-cov. 53, 57, 58 whether the neuroinvasion of sars-cov-2 has a role in the development of respiratory failure in covid-19 patients is still a matter of speculation. this information is essential for the prevention and better treatment of sars-cov-2-induced respiratory failure. several reports discussed neurological complications of covid-19. in a clinical series, giacomelli et al. 23 described olfactory and taste disorders in 20 of 59 sars-cov-2-infected individuals. lechien et al. 59 studied 417 patients and found olfactory dysfunction in 85.6% and gustatory dysfunction in 88%. in 11.8% of patients, smell loss was the first symptom of covid-19. because the ace2 receptor is widely expressed on the epithelial cells of the mucosa of the oral cavity and sars-cov exhibits a transneural penetration into the olfactory bulb, the pathogenetic mechanism of taste and olfactory disorders in sars-cov-2 infection could be justified. 53, 60 a case of a 56-year-old patient with covid-19 and encephalitis was recently described; csf tested positive for sars-cov-2 by gene sequencing. 61 in another report, a woman with covid-19 and acute necrotizing encephalopathy (ane) was described. 9 acute necrotizing encephalopathy is a rare and potentially severe neurological complication of some viral infections, such as influenza. it has been associated with an exaggerated inflammatory response in the cns named cytokine storm, responsible for a blood-brain barrier breakdown. characteristic imaging features of ane include symmetrical, multifocal lesions with invariable thalamic involvement. lesions appear hypodense on computador tomography images, and magnetic resonance imaging (mri) demonstrates t2/fluid attenuation inversion recovery hyperintense signal with internal hemorrhages. as in other severe viral infections, covid-19 has been associated with cytokine storms. 62 a case of guillain-barre syndrome (gbs) was recently published by zhao et al. 11 the authors described a 61-yearold woman infected by sars-cov-2 who developed acute weakness in both legs and severe fatigue, progressing within 1 day. although it is not possible to exclude an epiphenomenon between sars-cov-2 infection and gbs, considering the temporal association, the authors speculate that sars-cov-2 might have been responsible for the development of gbs in this patient. in this report, the disease followed a parainfectious profile, instead of the classic postinfectious pattern, reported in gbs associated with other pathogens. more recently, toscano et al. 63 described a clinical series of five patients with gbs in italy. the interval between the onset of symptoms of covid-19 and the first neurological symptoms ranged from 5 to 10 days. csf samples of all patients were negative to sars-cov-2. in three patients, the findings were consistent with an axonal variant of gbs and with a demyelinating process in two. all patients were treated with intravenous immune globulin. in addition, two patients with sars-cov-2 infection were reported with miller-fisher syndrome and polyneuritis cranialis, respectively. 64 in the first patient, vertical diplopia, perioral paresthesia, and gait ataxia were noted 7 days following covid-19 symptoms (fever, anosmia, ageusia, low back pain, and malaise). this patient was treated with intravenous immune globulin, with the resolution of the neurological symptoms, except for anosmia and ageusia. goh et al. 65 recently described a 27-year-old man who developed isolated peripheral facial palsy on the sixth day of sars-cov-2 infection. mao et al. 8 have published a clinical series on neurological aspects of covid-19. among 214 covid-19 patients, 37% had some neurological manifestation, with nearly 50% having severe covid-19 disease ( table 2 ). the authors drew attention to the fact that in patients with severe infection, the neurological involvement was more frequent, and included acute cerebrovascular diseases, disturbances of consciousness, and skeletal muscle injury. in this seminal series of cases, the nervous system involvement was associated with a poorer prognosis. the authors described mental status alterations in 15% of severe cases and nonspecific symptoms, including headache and dizziness, in nearly 20%. the authors referred to another common finding: "skeletal muscle injury" (creatine kinase > 200 iu/ml) that was seen in approximately 20% of severe cases. unfortunately, the authors did not describe whether there were clinical manifestations suggesting myositis or myopathy, or even signs of acute motor neuron injury. in another clinical series of neurological patients with covid-19, helms et al. 66 described the following neurological disturbances: agitation in 40 of 58 patients (69%), corticospinal tract signs in 39/58 (67%), and dysexecutive syndrome in 14/39 (36%). brain mri findings could be summarized as leptomeningeal enhancement in eight of 13 patients (62%), perfusion abnormalities in 11/11 (100%), and ischemic stroke in 3/13 (23%). these data should be interpreted with caution because they did not allow the determination of which of these clinical and radiological characteristics were due to critical illness-related encephalopathy, brain cytokine effects, or the effect of withdrawal of medication, and which features were directly associated with sars-cov-2 infection. stroke is one of the most frequent neurological diseases associated with sars-cov-2 infection, 8 and large-vessel stroke in younger patients was recently reported in five patients. 67 the mean national institutes of health stroke scale score in these patients was 17 (scores range from 0 to 42; the higher the score, the greater the severity of stroke). intravascular coagulation is the most likely factor in causing ischemic stroke in these patients; a high d-dimer is associated with increased risk for thrombosis and with a poor prognosis in covid-19 patients. 68, 69 finally, the presence of sars-cov-2 in the human brain was recently documented in the frontal lobe. 70 a patient with parkinson's disease died 11 days after sars-cov-2 infection because of cardiac and pulmonary complications. transmission electron microscopy showed viral particles in frontal lobe sections. noteworthy, virus-like particles were also found in the capillary endothelium and actively budding across endothelial cells, which could suggest a hematogenous route for sars-cov-2 entry into the cns. the real spectrum of neurological manifestations of covid-19 is an ongoing story. as the virus spreads to all continents, we may observe different manifestations in populations with diverse genetic and environmental backgrounds. besides, rna viruses such as sars-cov-2 suffer frequent mutations, which can be associated with new and unidentified neurological manifestations. close epidemiological surveillance is necessary to follow gbs's frequency and acute demyelinating encephalomyelitis, autoimmune conditions that usually follow viral infections. another critical aspect of the covid-19 pandemic is the collapse of many national health services worldwide. many neurological diseases require continuous follow-up and regular outpatient visits, and patients with stroke and epilepsy, among other neurological conditions, frequently present to the emergency every day. the impact of the pandemic in the care of patients with other neurological diseases has already been observed in many countries, raising the fear of additional load over an already overburdened health system. neurological societies are urged to devise guidelines and recommendations based on the best current information available on how neurologists should manage patients with neurological conditions that can be directly affected by covid-19, such as multiple sclerosis, epilepsy, or myasthenia gravis. another exciting field of research is about the long-term neurological consequences of sars-cov-2 infection. because a vast number of people worldwide will be infected, the inflammatory response elicited by sars-cov-2 may trigger or accelerate via impaired blood-brain barrier function some subclinical mechanisms that underlie the earliest stages of some neurodegenerative disorders. 71 prospective studies on this topic could answer this question in the future. although covid-19 does not appear to have exuberant neurological manifestations like the recent epidemics of the zika virus, hcovs have previously demonstrated the potential to invade and damage the nervous system. therefore, physicians should be alert to new clinical syndromes related to infection and be prepared to face worsening clinical control of preexisting neurological diseases in patients with covid-19. clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus-infected pneumonia in wuhan, china clinical characteristics of coronavirus disease 2019 in china neurological complications during treatment of middle east respiratory syndrome neuromuscular disorders in severe acute respiratory syndrome possible central nervous system infection by sars coronavirus severe neurologic syndrome associated with middle east respiratory syndrome corona virus (mers-cov) spontaneous intracranial hemorrhage in a patient with middle east respiratory syndrome corona virus neurologic manifestations of hospitalized patients with coronavirus disease 2019 in wuhan, china covid-19-associated acute hemorrhagic necrotizing encephalopathy: ct and mri features novel coronavirus and central nervous system guillain-barré syndrome associated with sars-cov-2 infection: causality or coincidence? neuroinvasive and neurotropic human respiratory coronaviruses: potential neurovirulent agents in humans neuroinvasion by human respiratory coronaviruses murine encephalitis caused by hcov-oc43, a human coronavirus with broad species specificity, is partly immune-mediated human respiratory neurotropic coronaviruses: an ambiguous relationship between neurovirulence and protein cleavage respiratory viruses other than influenza virus: impact and therapeutic advances transmission routes of respiratory viruses among humans the diagnosis of viral respiratory disease in older adults respiratory viral infections in infants: causes, clinical symptoms, virology, and immunology viral diseases of the central nervous system virus infections in the nervous system isolated sudden onset anosmia in covid-19 infection. a novel syndrome self-reported olfactory and taste disorders in sars-cov-2 patients: a cross-sectional study entry of coronavirus into primate cns following peripheral infection sars-associated coronavirus coronavirus pathogenesis renin-angiotensin-aldosterone system inhibitors in patients with covid-19 angiotensin-converting enzyme 2 in the brain: properties and future directions epidemiological and clinical features of human coronavirus infections among different subsets of patients epidemiology and clinical presentations of the four human coronaviruses 229e, hku1, nl63, and oc43 detected over 3 years using a novel multiplex real-time pcr method detection of the human coronavirus 229e, hku1, nl63, and oc43 between 2010 and 2013 in yamagata two coronaviruses isolated from central nervous system tissue of two multiple sclerosis patients murine hepatitis virus-a model for virus-induced cns demyelination human coronaviruses: viral and cellular factors involved in neuroinvasiveness and neuropathogenesis genomic characterization and infectivity of a novel sars-like coronavirus in chinese bats cross-host evolution of severe acute respiratory syndrome coronavirus in palm civet and human phylogeny of the sars coronavirus the genome sequence of the sarsassociated coronavirus characterization of a novel coronavirus associated with severe acute respiratory syndrome the severe acute respiratory syndrome severe acute respiratory syndrome and critical care medicine: the toronto experience olfactory neuropathy in severe acute respiratory syndrome: report of a case detection of sars coronavirus rna in the cerebrospinal fluid of a patient with severe acute respiratory syndrome pathological findings of covid-19 associated with acute respiratory distress syndrome multiple organ infection and the pathogenesis of sars persistence of physical symptoms in and abnormal laboratory findings for survivors of severe acute respiratory syndrome hypocortisolism in survivors of severe acute respiratory syndrome (sars) mechanisms of host defense following severe acute respiratory syndrome-coronavirus (sars-cov) pulmonary infection of mice coronavirus infection of rat dorsal root ganglia: ultrastructural characterization of viral replication, transfer, and the early response of satellite cells genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding a pneumonia outbreak associated with a new coronavirus of probable bat origin cryo-em structure of the 2019-ncov spike in the prefusion conformation severe acute respiratory syndrome coronavirus infection causes neuronal death in the absence of encephalitis in mice transgenic for human ace2 evidence of the covid-19 virus targeting the cns: tissue distribution, host-virus interaction, and proposed neurotropic mechanisms the vagus nerve is one route of transneural invasion for intranasally inoculated influenza a virus in mice cns innervation of vagal preganglionic neurons controlling peripheral airways: a transneuronal labeling study using pseudorabies virus middle east respiratory syndrome coronavirus causes multiple organ damage and lethal disease in mice transgenic for human dipeptidyl peptidase 4 lethal infection of k18-hace2 mice infected with severe acute respiratory syndrome coronavirus olfactory and gustatory dysfunctions as a clinical presentation of mild-to-moderate forms of the coronavirus disease (covid-19): a multicenter european study detection of severe acute respiratory syndrome coronavirus in the brain: potential role of the chemokine mig in pathogenesis a first case of meningitis/encephalitis associated with sars-coronavirus-2 covid-19: consider cytokine storm syndromes and immunosuppression guillain-barré syndrome associated with sars-cov-2 miller fisher syndrome and polyneuritis cranialis in covid-19 pearls and oysters: facial nerve palsy as a neurological manifestation of covid-19 infection neurologic features in severe sars-cov-2 infection large-vessel stroke as a presenting feature of covid-19 in the young covid-19 and its implications for thrombosis and anticoagulation d-dimer levels on admission to predict in-hospital mortality in patients with covid-19 central nervous system involvement by severe acute respiratory syndrome coronavirus-2 (sars-cov-2) severe acute respiratory syndrome coronavirus 2 (sars-cov-2) and the central nervous system neurologic alterations due to respiratory virus infections key: cord-265380-2gs34xcw authors: leist, sarah r.; cockrell, adam s. title: genetically engineering a susceptible mouse model for mers-cov-induced acute respiratory distress syndrome date: 2019-09-14 journal: mers coronavirus doi: 10.1007/978-1-0716-0211-9_12 sha: doc_id: 265380 cord_uid: 2gs34xcw since 2012, monthly cases of middle east respiratory syndrome coronavirus (mers-cov) continue to cause severe respiratory disease that is fatal in ~35% of diagnosed individuals. the ongoing threat to global public health and the need for novel therapeutic countermeasures have driven the development of animal models that can reproducibly replicate the pathology associated with mers-cov in human infections. the inability of mers-cov to replicate in the respiratory tracts of mice, hamsters, and ferrets stymied initial attempts to generate small animal models. identification of human dipeptidyl peptidase iv (hdpp4) as the receptor for mers-cov infection opened the door for genetic engineering of mice. precise molecular engineering of mouse dpp4 (mdpp4) with clustered regularly interspaced short palindromic repeats (crispr)/cas9 technology maintained inherent expression profiles, and limited mers-cov susceptibility to tissues that naturally express mdpp4, notably the lower respiratory tract wherein mers-cov elicits severe pulmonary pathology. here, we describe the generation of the 288–330(+/+) mers-cov mouse model in which mice were made susceptible to mers-cov by modifying two amino acids on mdpp4 (a288 and t330), and the use of adaptive evolution to generate novel mers-cov isolates that cause fatal respiratory disease. the 288–330(+/+) mice are currently being used to evaluate novel drug, antibody, and vaccine therapeutic countermeasures for mers-cov. the chapter starts with a historical perspective on the emergence of mers-cov and animal models evaluated for mers-cov pathogenesis, and then outlines the development of the 288–330(+/+) mouse model, assays for assessing a mers-cov pulmonary infection in a mouse model, and describes some of the challenges associated with using genetically engineered mice. in february of 2018 mers-cov was listed as a priority on the r&d blueprint for the global strategy and preparedness plan outlined by the world health organization (who) [1] . the r&d blueprint includes viruses that pose a global public health risk, and for which there are no available therapeutic countermeasures [1] . twenty-seven countries have reported cases of mers-cov with most cases confined to the arabian peninsula. diagnosed cases of mers-cov in countries outside the arabian peninsula are primarily traveler associated. the potential for global spread of mers-cov was realized in 2015 when a single traveler returning to south korea initiated an outbreak that infected 186 people resulting in 20% fatality and caused widespread fear that crippled the economy for nearly 6 months [2] [3] [4] . human-to-human transmission is often associated with close contact in the health care setting, but can also occur between family members within a household [5] . asymptomatic individuals pose a particular risk of transmission due to their unknown carrier status as demonstrated in the health care setting [6] . despite the high percent of fatalities associated with mers-cov outbreaks on the arabian peninsula most epidemiological studies suggest r 0 values <1, indicative of a low risk of sustainable human-to-human transmission, whereas epidemiological studies from the south korean outbreak describe r 0 values (>1) akin to more sustainable human-to-human transmission [7] . recurring spillover events from dromedary camels (zoonotic reservoir for mers-cov on the arabian peninsula) likely contribute to newly diagnosed cases in humans [8] [9] [10] . the potential for continuous reintroduction to humans increases the risk of mers-cov adapting in humans to acquire enhanced human-tohuman transmission profiles, a scenario suspected to have initiated the sars-cov pandemic in 2002-2003 [11] . effective public health measures and culling of civet cats, the zoonotic host for sars-cov, brought the sars-cov pandemic to a rapid end [11] . eliminating mers-cov through culling of infected camel herds is not a practical solution. furthermore, detection of pre-emergent mers-cov-like, and sars-cov-like, strains circulating in bat species indicate that the natural environment is ripe for future human exposures to potentially pathogenic coronaviruses [12] [13] [14] . therefore, the development of therapeutic countermeasures that can interfere with mers-cov pathogenesis is critical to break zoonotic-to-human and human-to-human transmission cycles that may instigate global spread. evaluating the toxicity and efficacy of novel mers-cov therapeutics require the availability of animal models that effectively recapitulate mers-cov pathogenesis during fatal cases of human infections. therefore, the first question in generating a mers-cov animal model would be: what are the pathological features of a human infection? limited histopathological findings from human autopsies indicate that fatal cases of mers-cov results from pneumonia initiated by infection of bronchiolar and alveolar epithelia of the lower respiratory tract (lrt) [15, 16] . pneumonia in the lrt is also the prominent finding on radiographs from x-rays and cts of diagnosed human cases [17] . high viral loads in tracheal aspirates from patients are also associated with severe pulmonary disease [18] , which is indicative of actively replicating mers-cov in the lrt. initial evaluation of the human mers-cov emc/2012 isolate in rhesus macaques demonstrated replication in the lrt with mild pneumonia-like disease ( fig. 1 ) [28] . achieving respiratory pathology reflecting a lethal human disease proved to be more complicated in nonhuman primates. severe respiratory disease in the marmoset produced clinical endpoints consistent with fatal disease that required euthanasia ( fig. 1) [29, 30] . evaluation of two human isolates, jordan and emc/2012, and a tissue cultureadapted mers-cov strain (mers-0) in nonhuman primates resulted in mild disease in rhesus macaques or marmosets ( fig. 1 ) [31] [32] [33] , confounding the reproducibility of near-lethal disease in nhps. nonhuman primates are central to late-stage preclinical evaluation of therapeutic countermeasures, but may be impractical for initial preclinical studies. a small animal model may be applicable if there is limited therapeutic available for toxicity and efficacy testing, especially if large animal numbers are needed to determine confidence and reproducibility. early studies in mouse, hamster, and ferret revealed that conventional small animal models were fully resistant to mers-cov infection and replication (fig. 1) [19, 20, 36] . a seminal study identifying the mers-cov receptor as human dipeptidyl peptidase iv (hdpp4) [49] , and publication of the crystal structure of hdpp4 interacting with the receptor binding domain (rbd) of the mers-cov spike protein [50] , exposed tropism determinants critical for susceptibility. dipeptidyl peptidase iv contact amino acids at the hdpp4/rbd interface are highly conserved among mers-cov-susceptible mammalian species (human, camel, and specific events since the emergence of mers-cov in 2012 are emphasized above the timeline. references to mammalian models evaluated for mers-cov pathogenesis comprise hamster [19] , ferret [20] , rabbit [21] [22] [23] [24] , camel [25] [26] [27] , nonhuman primates [28] [29] [30] [31] [32] [33] [34] [35] , and mouse [36] [37] [38] [39] [40] [41] [42] [43] [44] [45] [46] [47] [48] bat) (fig. 2) [51] . although mouse, hamster, ferret, and guinea pig dpp4 orthologs exhibit high overall similarity to hdpp4, specific amino acid differences at the dpp4/rbd interface account for the inability of these species to support infection [51] [52] [53] [54] [55] [56] . overexpression of a mouse dpp4 (mdpp4) with changes in the contact residues at the dpp4/rbd altered cellular profiles from resistant to susceptible to mers-cov infection [52, 53, 56] . the dependence on dpp4-specific contact was further substantiated by similar studies evaluating modified dpp4 orthologs from the hamster, ferret, and guinea pig [55] . dipeptidyl peptidase iv was identified as the major determinant of mers-cov tropism. researchers rapidly leveraged knowledge of the dpp4 receptor to generate susceptible small animal models ( fig. 1 ) [12] . zhao et al. utilized a unique approach for producing susceptible mice that could replicate human isolates of mers-cov in the lungs by infecting mouse lungs with an adenovirus that constitutively expresses the full-length hdpp4 gene ( fig. 1) [37] . transient expression of hdpp4 supported infection and replication with human strains of mers-cov in the lungs and indicated that this technology may be an effective rapid response platform for initial (c) zoomed-in view of the human dpp4 structure (light gray) with highlighted mers-cov rbd contact residues (dark gray). species-specific contact residues that differ from human are highlighted in red evaluation of emergent and pre-emergent viruses. however, pathology associated with a fatal mers-cov infection was not observed in the ad-hdpp4 model [37] , which limited the capacity to evaluate the efficacy of therapeutic countermeasures. genetic engineering of mice would be necessary to develop preclinical mers-cov mouse models with respiratory phenotypes that reflected clinical outcomes in patients. knock-in of full-length hdpp4 rendered mice susceptible to human isolates of mers-cov at low infection doses ( fig. 1 ) [38] [39] [40] . knock-in mice exhibited severe pulmonary pathology and increased mortality; however, widespread constitutive expression of full-length hdpp4 resulted in high levels of mers-cov infection and replication in extrapulmonary tissues [38] [39] [40] . in some studies, higher viral loads could be detected in the brain compared to the lungs [39, 40] . mice with infections of the central nervous system (cns) exhibited encephalitis that corresponded with the kinetics of mortality [39] . currently, there is no evidence to support a cns component associated with mers-cov pathogenesis in humans. attempts to restrict hdpp4 expression to epithelial cells of the lungs using constitutive tissue specific promoters (e.g., cytokeratin k18) yielded outcomes similar to those observed with sars-cov mouse models, wherein high levels of mers-cov infection/replication were detected in the brains (fig. 1 ) [39] . to circumvent confounding problems associated with global bio-distribution of overexpressed hdpp4 receptor, researchers engineered mouse models using sophisticated molecular approaches. pascal et al. employed regeneron's velocigene technology to replace sequences encoding nearly the entire mdpp4 genomic region with those encoding the exons/introns from the hdpp4 genetic region ( fig. 1 ) [41] . retaining the mdpp4 5 0 and 3 0 genetic elements that regulate expression maintained inherent expression profiles of full-length hdpp4 in mice [41] . importantly, mers-cov infection/replication was readily detected in the lungs with little involvement of extrapulmonary tissues [41] . infection with human isolates of mers-cov caused moderate respiratory pathology with mortality determined by euthanasia of mice at 20% weight loss [41] . unfortunately, commercial restrictions limit the availability and use of this model to the broader scientific community. in addition to the concerns raised above, the first generation of mouse models was developed with the full-length hdpp4, which may alter the inherent physiological properties of the mouse. the multifaceted involvement of dpp4 in maintaining immune homeostasis is of significant importance regarding susceptibility to infectious disease [57] . dpp4 exists in two forms: (1) a membrane anchored form on the surface of multiple cells types (e.g., b cells, t cells, nk cells, and epithelial cells to mention a few) and (2) a secreted form that can be identified in human serum [57] . dpp4 interacts with and modifies heterologous protein molecules involved in nociception, neuroendocrine function, metabolism, cardiovascular function, immune regulation, and infection [57] . modification of heterologous protein function can proceed through cleavage of n-terminal amino acids through the enzymatic activity of the α/b-hydroxylase domain, or allosteric interaction/ signal transduction [57] . the species specificity of dpp4 is exemplified by the interaction of hdpp4 with adenosine deaminase (ada), a well-recognized binding partner of hdpp4, which modulates downstream t cell functions [58] [59] [60] . the hdpp4/ada interaction evolved in higher mammalian species (human, nhp, bovine, rabbit), but not in mouse or rat [58] [59] [60] . interestingly, in one study ada was demonstrated to block infection of mers-cov in tissue culture [20] , indicating that the binding site on hdpp4 for ada, and the mers-cov rbd, may overlap. consequently, introducing full-length hdpp4 into mice may skew innate immune mechanisms that could influence responses to therapeutic countermeasures. in the second generation of mers-cov-susceptible mouse models amino acid residues predicted to function at the mdpp4/ mers-cov rbd interface were modified to avoid the introduction of full-length hdpp4 ( fig. 1) [42, 43] . li et al. recently developed a mouse model wherein the mdpp4 genomic region encompassing exons 10-12 were replaced with the respective genomic region from hdpp4, referred to as an hdpp4 knock-in model (hdpp4-ki) [43]. exons 10-12 encode contact amino acids at the hdpp4/mers-cov rbd interface that were able to support replication of human mers-cov isolates in the lungs, but did not elicit a mortality phenotype [43] . adaptive evolution of human mers-cov in the hdpp4-ki mouse resulted in mouseadapted viruses that evoked a lethal respiratory phenotype with little involvement of extrapulmonary tissues. the lethal respiratory phenotype is a consequence of novel mutations acquired during adaptive evolution. a combination of mutations in both the s1 and s2 regions of the mers-cov spike protein facilitated a lethal respiratory phenotype [43] . results in the hdpp4-ki model substantiate an earlier mouse model referred to as the 288-330 +/+ model, which was designed with only two amino acid changes in mdpp4 to generate mers-cov susceptible mice. genetic engineering and implementation of the 288-330 +/+ mouse model, combined with mers-cov adaptive evolution, is the subject of this chapter. initial studies in tissue culture revealed that human and rodent cell types were resistant to mers-cov infection upon overexpression of mdpp4; however, overexpression of hdpp4 conferred permissivity to infection/replication [53] . comparative structural modeling of hdpp4 and mdpp4 revealed putative contact residues in mdpp4 amenable to modification at the dpp4/rbd interface. modification of two amino acids (a288l and t330r) was sufficient to endow mdpp4 with the capacity to mediate mers-cov infection/replication [53] . shortly after the emergence of mers-cov into humans in 2012, the crispr/cas9 genome editing technology became available for applications to modify mammalian genomes in vitro and in vivo [61] [62] [63] . recognizing our unique situation, we designed crispr/cas9 targets to modify the mouse genome encoding amino acids a288 and t330 in exons 10 and 11 of the mdpp4 gene (fig. 3) [12, 42] . concomitant with mouse development, in vitro studies were initiated to adapt mers-cov to the modified mdpp4 [42] . tissue culture adaption resulted in mers-0 virus, which contained an rmr insertion and s885l mutation in the s2 region of the mers-cov spike protein [42] . a mers-0 molecular clone exhibited enhanced replication kinetics and higher titers compared to human mers-cov isolates. additionally, the mers-0 virus replicated to higher levels in the lungs of 288-330 +/+ mice, compared to human and camel mers-cov isolates [42] . based on these data the mers-0 virus was used to initiate passaging in mice heterozygous for mdpp4 with a288l and t330r mutations, 288-330 +/à (fig. 4) . we reasoned that adaptation around one expressed copy of the mdpp4 with 288-330 mutations, and a wild-type mdpp4 expressed copy, might cultivate generation of a mouse-adapted mers-cov that could utilize wild-type mdpp4 as the primary receptor. after 15 passages we obtained a mouse-adapted mers-cov (mers15c2) exhibiting a lethal respiratory phenotype in the 288-330 +/+ mice [42] . our mers-cov reverse genetic system was used to generate an infectious clone of the mouse-adapted virus, icmersma1 [42] . lethal respiratory pathology with icmersma1 required high infectious doses (5 â 10 6 pfu). an additional 20 passages of icmersma1 in 288-330 +/à mice bore a novel mouse-adapted mers-cov that produced lethal respiratory disease at doses of 5 â 10 5 pfu, and lung pathology associated with severe respiratory disease at 5 â 10 4 to 5 â 10 5 pfu [44] (fig. 1) . this mers-cov model system (288-330 +/+ mice and mouseadapted mers-cov viruses) is now being employed to: (1) understand complex virus-host interactions [12, 31, 42, [64] [65] [66] [67] , (2) evaluate antibody-based therapeutics [42] , (3) evaluate drug-based therapeutic countermeasures [68] , and (4) evaluate anti-mers-cov vaccines [42, 66] . the goal of this chapter is to provide an outline of how to rationally design a mouse with altered susceptibility to mers-cov. for additional information there are a number of detailed reviews and book chapters describing the design and utilization of the crispr/cas9 technology for generating mouse models [69, 70] . . agarose gel separation based on size allows for discrimination between target dna, cas9 digested targets, and guide rnas. (b) schematic utilizing crispr/cas9 technology to genetically engineer mice. fertilized c57bl/6 j zygotes are collected and injected with rna encoding cas9, dpp4 single guide rna, and oligos to facilitate homology-directed repair (hdr). microinjected zygotes are implanted into pseudopregnant recipient female c57bl/6 j mice. offspring are screened by sequencing for the intended change at positions 288 and 330. mice identified as having the appropriate changes are backcrossed to c57bl/6 j mice to maintain the pure c57bl/6 j background, or may be crossed to any desired strain (e.g., balb/cj or 129s1/svimj). (c) table describing sequences of cas9 guide rnas and oligos for hdr to genetically engineer amino acid changes at position 288 (ala to leu) and 330 (thr to arg). (d) sequencing chromatograms highlighting how the f0 offspring from embryo implantation can be a mosaic of insertion/ deletions (indel's) generated by random non-homologous end joining from cas9 cutting at the genomic alleles, and the hdr repair that incorporates the intended changes encoding amino acids at positions 288 and 330. pure homozygous 288-330 +/+ lines were obtained by backcrossing onto c57bl/6 j mice. the highlighted mutations caa (ttg in the reverse orientation) and aga encode the novel 288 l and 330r amino acids 9. after a predetermined number of passages the region encoding the spike protein of mers-cov is sequenced using rt-pcr to amplify the region of interest followed by standard sanger sequencing (see note 6). 10. after 10 passages viruses were plaque purified by diluting the heterogenous stock of virus 10 à1 to 10 à6 , and infecting a monolayer of vero ccl81 cells similar to a standard plaque assay. 11. single plaques are isolated using a pipet tip and the virus expanded on a freshly seeded monolayer of vero ccl81 cells. 12. virus stocks are generated, viral rna is isolated using standard trizol purification, and the region encoding the mers-cov spike protein is amplified by standard rt-pcr techniques and sequenced using standard sanger sequencing. 13 . mutations identified by sequencing must be confirmed using a reverse genetic system to generate an infectious clone encoding the identified mutations [72] . cockrell (fig. 3) the details for generating and using the crispr/cas9 system to generate mutations are outlined in the materials and methods by cockrell et al. [42] . notably, the 288-330 +/+ mice were initially generated in the animal models core facility at the university of north carolina at chapel hill. the extensive technical expertise required for genetic engineering of mice is the subject of many expert reviews and book chapters that will not be covered here. nevertheless, we provide a conceptual overview of the steps to generate the 288-330 +/+ mice. 1. design guide rnas to target each of the a288 and t330 alleles. cockrell et al. designed the guide rnas to direct the cas9 to cut as near the mutation site as possible (fig. 3 ) (see note 7 for helpful resources to design and genetically engineer mouse knockouts). 2. test guide rnas in vitro for the capacity to cut a target sequence (fig. 3) . (a) generate double-stranded odns or a plasmid containing the target sequence with the correct pam site. (b) assemble ribonucleoprotein (rnp) complexes according to manufacturer's instructions (see note 8). (c) subject double-stranded dna with target sequence to rnp complexes and assess digestion pattern on an agarose gel (fig. 3 ). 3. two separate oligos are also designed to introduce the novel mutations on exons 10 (a288l) and 11 (t330r) of mdpp4, through homology-directed repair (fig. 3 ). 4. fertilized zygotes are collected from c57bl/6j female mice that were superovulated and mated to male c57bl/6j mice. cas9 endonuclease, combined with odns encoding the replacement alleles for 288 and 330 in mdpp4, were all pronuclear injected into the fertilized zygote [42] (fig. 3) . the fertilized zygotes were from c57bl/6j mice. 6. the injected embryos were implanted into pseudopregnant recipient females. 7. newly born pups were screened for the presence of the correct change at the 288 and 330 alleles by standard pcr amplification and sanger sequencing (fig. 3 ) (see note 9). 3. 288-330 +/+ mice are brought into the bsl3 laboratory 7 days before start of the experiment to allow for environmental acclimation. 4. mice are anesthetized via intraperitoneal injection of 50-100 μl of a ketamine (50 mg/kg)/xylazine (15 mg/kg) mixture (see note 10). level of anesthesia is assessed by pedal reflex. 6. measure initial weight (day 0 weight) while waiting for mouse to be anesthetized (see note 11). 7. once a pedal reflex is no longer triggered, mice should be immediately infected by the intranasal route. holding the animal vertically, apply 50 μl of virus solution by pipetting onto their nostrils and allow them to inhale. to ensure that all of the 50 μl reach the lower respiratory tract hold the mouse upright for an additional 30 s (see note 12). 8. note any inconsistencies during infection, including: (1) presence of bubbles of inoculum from nasal cavity, (2) occurrence of inoculum in mouth, or (3) failure to inhale entire dose of inoculum. notes will help to explain potential inconsistencies in readout parameters and may be used as exclusion criteria for inefficient infections. 9. mice are put back into the cage and placed on their back to ensure virus solution will stay in the lungs. note: cages are returned to cage rack, but the respiration of mice is continuously monitored by observing breathing. 10. place mice next to each other to keep body temperature as close to normal as possible. 11. check cage after 30-45 min to confirm that all mice wake up from anesthesia and infection. adaptation of mers-0 in 288-330 +/à mice (fig. 4) 1. mouse adaptation was initiated in heterozygous 288-330 +/à mice by infecting with 50 μl of the mers-0 infectious clone (see note 13). 2. at 3 days post-infection the mouse is euthanized by extended exposure to isoflurane.~2 ml of isoflurane is added to the bottom of a jar that can be firmly sealed (see note 14). a thoracotomy is performed to expose the lungs. 4. lungs are removed and placed in a 2 ml gasket sealed skirted screw cap tubes. tubes are previously prepared with 1 ml of 1â pbs containing~5-10 mm of glass beads. lungs are homogenized for 60 s in a bead homogenizer. 6. lung lysates are centrifuged in a microcentrifuge for 5 min at max speed to pellet debris. 7. this is considered passage 1 (p1) and 50 μl of lung homogenate is used to infect a naïve 288-330 +/à mouse. 8. the process is repeated for a desired number of cycles. 1. after infection mice are monitored daily for weight loss for the entire duration of the experiment. 2. to record daily weights, pick up mice by the tail, identify by ear notch, and place into cup on a scale. record weight and calculate percentage of starting body weight (see note 15). 3. mice can also be monitored to determine if they are moribund using a clinical scoring scale whereby: 0 ¼ no clinical signs; 1 ¼ ruffled fur; 2 ¼ ruffled fur with hunched posture (only slight with no signs of dehydration); 3 ¼ as defined in number 2 with more severe signs of dehydration and some loss of body strength; 4 ¼ pronounced dehydration and prominent loss of mobility; 5 ¼ unresponsive to stimuli and prominent eye squinting. 4. it is important to note that weight loss might not always be the most appropriate parameter and animals should be euthanized at the discretion of the researcher even if animals have not reached 80% of their starting weight. mice that approach 80% of their starting body weight (20% weight loss) are euthanized via isoflurane overdose followed by a secondary euthanasia method (thoracotomy or cervical dislocation). depending on the experimental circumstances an institutionally approved exception may be implemented to allow continuation of the experiment (increasing the frequency with which the mice are monitored will likely need to be implemented) (see note 16). 3. respiratory function can be performed every day over the entire course of infection, or on single selected days. investigating a novel respiratory virus may require the investigator to perform a time course to determine the most effective time points for measurement. the largest differences between groups typically correlate with peak viral replication. 4. at each time point measured the mice need to be randomized into different chambers to avoid technical artifacts (e.g., a mouse measured at day 1 in chamber 1 should be evaluated in a different chamber on day 2 measurement). practical considerations dictate that 8-12 animals can be measured at any one time, and each group of 8-12 mice may take an hour for a proficient technician. therefore, experiments should be carefully planned to limit the number of mice to be evaluated. 5 . mice that are difficult to handle can be slightly anesthetized by applying isoflurane to the chamber in order to remove them from the chamber and return to their cage (see note 18). 6. open thorax, paying attention not to cut into lung tissue. 7. assess lung tissue for reddish discoloration and record severity by applying a number 0 (no hemorrhaging) to 4 (severe hemorrhaging in all lobes of the lungs). 8. harvest lung tissue and place in tubes prefilled with sample type specific solution. 9. put scissors and forceps first into cidecon to remove any residual blood and then into 70% ethanol in order to not crosscontaminate samples. 10. whole lung can be used for one assay or different lobes can be used for different assays. steps 1-6 from subheading 3.7. 2. cut into superior vena cava and collect blood with a pipette. 3. blood is typically transferred to a serum/plasma separation tube that allows for separation of serum/plasma from cells. 4. in the event that it is necessary to harvest cells for flow cytometry analysis or vetscan hm5 analysis, the blood sample can be transferred to a tube containing edta. note: vetscan hm5 is a veterinary diagnostic machine that analyzes basic immune cell counts and additional hematological parameters within 2 min. 5. all vetscan assays are performed under bsl3 conditions. removal of samples for downstream analysis outside of bsl3 conditions require specific inactivation procedures that must be pre-approved by the institutional biosafety committee. 3. this can also be achieved using a readily transfectable human embryonic fibroblast cell line such as hek293t cells and selecting for stably transfected cells. 4. cells can be counted by seeding an extra well, but it is safe to assume that the cell number is approximately doubled after 24 h. 5. this can also be achieved using a microscope to assess plaque size if plaques can be readily detected. 6. since the major determinant of mers-cov tropism is the spike protein, it would be anticipated that mutations having the most significant impact on infection might occur within the gene encoding the spike protein. 7. at the time that these mice were being generated in early 2014, crispr/cas9 reagents were not readily available. additionally, there were few bioinformatics tools available to facilitate guide rna design and off-target potential. in the current research environment crispr/cas9 reagents can be sourced from multiple commercial entities and there are a number of bioinformatics tools to assist with design. addgene is a nonprofit plasmid repository where crispr reagents and resources can be readily obtained (https://www.addgene. org/). additional guidance for generating mice using crispr/cas9 technology can be found in more comprehensive protocols [69, 70] . 8. all relevant reagents and protocols can now be obtained from commercial sources as readily synthesized rnas and purified proteins (e.g., integrated dna technologies). 9 . although a number of pups may be identified to have the correct mutation, many will likely be mosaic for random mutations including insertions/deletions due to the higher efficiency of non-homologous end joining (nhej) after cas9 digestion compared to the desired hdr employed to mediate allele modification. 10. the administered dose will depend on the weight of the animal which should be predetermined the day prior to initiating the experiment. 11. it is not necessary to anesthetize mice for measuring daily weights. 12. it is important that the inoculum reaches the lower respiratory tract for a successful mers-cov infection. 13 . mouse adaptation can be initiated with any mers-cov strain that exhibits some pulmonary replication. 14. mice should never come into contact with the isoflurane. to prevent direct contact, a layer of aluminum foil is placed at the bottom of the jar and this is covered with two additional layers of paper towel. 15 . the percent body weight is typically calculated after leaving the bsl3 environment. therefore, the weight sheets should have the anticipated weights of each animal at 20% weight loss. this will provide a real-time indication of when the mice are approaching the criteria established for humane euthanasia. 16. institutional approval is required for animals to be placed under exception. it cannot be emphasized enough that all animal work should be pre-approved by appropriate university iacuc and ibc committees and should be in accordance with the recommendations for the care and use of animals by the office of laboratory animal welfare at nih. 17. assessing respiratory function using plethysmography under bsl3 conditions requires costly equipment and extensive training prior to use. 18. anesthesia should be avoided prior to measuring lung function to prevent interference with lung function measurements. the who r&d blueprint: 2018 review of emerging infectious diseases requiring urgent research and development efforts comparative analysis of eleven healthcare-associated outbreaks of middle east respiratory syndrome coronavirus (mers-cov) from economic impact of the 2015 mers outbreak on the republic of korea's tourism-related industries costly lessons from the 2015 middle east respiratory syndrome coronavirus outbreak in korea middle east respiratory syndrome coronavirus: risk factors and determinants of primary, household, and nosocomial transmission middle east respiratory syndrome coronavirus transmission among health care workers: implication for infection control mers transmission and risk factors: a systematic review high prevalence of mers-cov infection in camel workers in saudi arabia reported direct and indirect contact with dromedary camels among laboratory-confirmed mers-cov cases dromedary camels and the transmission of middle east respiratory syndrome coronavirus (mers-cov) severe acute respiratory syndrome modeling pathogenesis of emergent and pre-emergent human coronaviruses in mice origin and evolution of pathogenic coronaviruses jumping species-a mechanism for coronavirus persistence and survival histopathology of middle east respiratory syndrome coronovirus (mers-cov) infection-clinicopathological and ultrastructural study clinicopathologic, immunohistochemical, and ultrastructural findings of a fatal case of middle east respiratory syndrome coronavirus infection in the united arab emirates middle east respiratory syndrome viral load kinetics of mers coronavirus infection the middle east respiratory syndrome coronavirus (mers-cov) does not replicate in syrian hamsters adenosine deaminase acts as a natural antagonist for dipeptidyl peptidase 4-mediated entry of the middle east respiratory syndrome coronavirus asymptomatic middle east respiratory syndrome coronavirus infection in rabbits enhanced inflammation in new zealand white rabbits when mers-cov reinfection occurs in the absence of neutralizing antibody prophylaxis with a middle east respiratory syndrome coronavirus (mers-cov)-specific human monoclonal antibody protects rabbits from mers-cov infection lack of middle east respiratory syndrome coronavirus transmission in rabbits bactrian camels shed large quantities of middle east respiratory syndrome coronavirus (mers-cov) after experimental infection replication and shedding of mers-cov in upper respiratory tract of inoculated dromedary camels an orthopoxvirus-based vaccine reduces virus excretion after mers-cov infection in dromedary camels pneumonia from human coronavirus in a macaque model treatment with lopinavir/ritonavir or interferon-beta1b improves outcome of mers-cov infection in a nonhuman primate model of common marmoset infection with mers-cov causes lethal pneumonia in the common marmoset a spike-modified middle east respiratory syndrome coronavirus (mers-cov) infectious clone elicits mild respiratory disease in infected rhesus macaques 3b11-n, a monoclonal antibody against mers-cov, reduces lung pathology in rhesus monkeys following intratracheal inoculation of mers-cov jordan-n3/2012 intratracheal exposure of common marmosets to mers-cov jordan-n3/2012 or mers-cov emc/2012 isolates does not result in lethal disease middle east respiratory syndrome coronavirus (mers-cov) causes transient lower respiratory tract infection in rhesus macaques an animal model of mers produced by infection of rhesus macaques with mers coronavirus wild-type and innate immune-deficient mice are not susceptible to the middle east respiratory syndrome coronavirus rapid generation of a mouse model for middle east respiratory syndrome generation of a transgenic mouse model of middle east respiratory syndrome coronavirus infection and disease middle east respiratory syndrome coronavirus causes multiple organ damage and lethal disease in mice transgenic for human dipeptidyl peptidase 4 multi-organ damage in human dipeptidyl peptidase 4 transgenic mice infected with middle east respiratory syndrome-coronavirus pre-and postexposure efficacy of fully human antibodies against spike protein in a novel humanized mouse model of mers-cov infection mouse-adapted mers coronavirus causes lethal lung disease in human dpp4 knockin mice adaptive evolution influences the infectious dose of mers-cov necessary to achieve severe respiratory disease elevated human dipeptidyl peptidase 4 expression reduces the susceptibility of hdpp4 transgenic mice to middle east respiratory syndrome coronavirus infection and disease cd8+ t cells and macrophages regulate pathogenesis in a mouse model of middle east respiratory syndrome a human dpp4-knockin mouse's susceptibility to infection by authentic and pseudotyped mers-cov acute respiratory infection in human dipeptidyl peptidase 4-transgenic mice infected with middle east respiratory syndrome coronavirus haagmans bl (2013) dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-emc structure of mers-cov spike receptorbinding domain complexed with human receptor dpp4 coronavirus host range expansion and middle east respiratory syndrome coronavirus emergence: biochemical mechanisms and evolutionary perspectives receptor variation and susceptibility to middle east respiratory syndrome coronavirus infection mouse dipeptidyl peptidase 4 is not a functional receptor for middle east respiratory syndrome coronavirus infection glycosylation of mouse dpp4 plays a role in inhibiting middle east respiratory syndrome coronavirus infection permissivity of dipeptidyl peptidase 4 orthologs to middle east respiratory syndrome coronavirus is governed by glycosylation and other complex determinants host species restriction of middle east respiratory syndrome coronavirus through its receptor, dipeptidyl peptidase 4 cut to the chase: a review of cd26/dipeptidyl peptidase-4's (dpp4) entanglement in the immune system direct association of adenosine deaminase with a t cell activation antigen revisiting an old acquaintance: cd26 and its molecular mechanisms in t cell function crystal structure of cd26/ dipeptidyl-peptidase iv in complex with adenosine deaminase reveals a highly amphiphilic interface multiplex genome engineering using crispr/cas systems rna-guided human genome engineering via cas9 one-step generation of mice carrying reporter and conditional alleles by crispr/cas-mediated genome engineering the human sodium iodide symporter as a reporter gene for studying middle east respiratory syndrome coronavirus pathogenesis giving the genes a shuffle: using natural variation to understand host genetic contributions to viral infections middle east respiratory syndrome coronavirus nonstructural protein 16 is necessary for interferon resistance and viral pathogenesis mers-cov accessory orfs play key role for infection and pathogenesis broad-spectrum antiviral gs-5734 inhibits both epidemic and zoonotic coronaviruses generating genetically modified mice: a decision guide genome editing in mouse embryos with crispr/cas9 reverse genetics with a full-length infectious cdna of the middle east respiratory syndrome coronavirus efficient reverse genetic systems for rapid genetic manipulation of emergent and preemergent infectious coronaviruses new metrics for evaluating viral respiratory pathogenesis key: cord-267973-uvz7kavu authors: do, lien anh ha; bryant, juliet e.; tran, anh tuan; nguyen, bach hue; tran, thi thu loan; tran, quynh huong; vo, quoc bao; tran dac, nguyen anh; trinh, hong nhien; nguyen, thi thanh hai; le binh, bao tinh; le, khanh; nguyen, minh tien; thai, quang tung; vo, thanh vu; ngo, ngoc quang minh; dang, thi kim huyen; cao, ngoc huong; tran, thu van; ho, lu viet; farrar, jeremy; de jong, menno; van doorn, h. rogier title: respiratory syncytial virus and other viral infections among children under two years old in southern vietnam 2009-2010: clinical characteristics and disease severity date: 2016-08-08 journal: plos one doi: 10.1371/journal.pone.0160606 sha: doc_id: 267973 cord_uid: uvz7kavu background: despite a high burden of respiratory syncytial virus (rsv) infections among children, data on demographic and clinical characteristics of rsv are scarce in low and middle income countries. this study aims to describe the viral etiologies, the demographic, epidemiological, and clinical characteristics of children under two years of age who were hospitalized with a lower respiratory tract infections (lrti), focusing on rsv (prevalence, seasonality, subgroups, viral load) and its association with disease severity. methods: a prospective study among children under two years of age, hospitalized with lrti was conducted in two referral pediatric hospitals in ho chi minh city, vietnam, from may 2009 to december 2010. socio-demographic, clinical data and nasopharyngeal swabs were collected on enrolment and discharge. multiplex real-time rt-pcr (13 viruses) and quantitative rsv rt-pcr were used to identify viral pathogens, rsv load and subgroups. results: among 632 cases, 48% were rsv positive. rsv infections occurred at younger age than three other leading viral infections i.e rhinovirus (rv), metapneumovirus (mpv), parainfluenza virus (piv-3) and were significantly more frequent in the first 6 months of life. clinical severity score of rsv infection was significantly higher than piv-3 but not for rv or mpv. in multivariate analysis, rv infection was significantly associated with severity while rsv infection was not. among rsv infections, neither viral load nor viral co-infections were significantly associated with severity. young age and having fever at admission were significantly associated with both rsv and lrti severity. a shift in rsv subgroup predominance was observed during two consecutive rainy seasons but was not associated with severity. conclusion: we report etiologies, the epidemiological and clinical characteristics of lrti among hospitalized children under two years of age and risk factors of rsv and lrti severity. respiratory syncytial virus (rsv) is the leading cause of lower respiratory tract infections (lrtis) in young children. 50% of children are infected by rsv during their first year of life, and by 3 years of age, 100% have experienced at least one infection [1] . previous studies [2] [3] [4] have shown the importance of rsv in hospitalized children in vietnam. hospital records from the two main paediatric referral centers in ho chi minh city show that 77% of hospitalized children with lrti are under 2 years old (unpublished data). in addition, severe disease from rsv infection seems to exclusively occur in this population [3, 5] . however, information on detailed clinical, epidemiological features and virological characteristics of rsv infections (e.g. disease burden, demographics, seasonal variations of rsv and other viral infections, circulating genotypes and subgroups, viral load) or on the frequency / impact of other respiratory viruses among vietnamese children under two years old are limited [6] . here, we aimed to describe the viral etiologies and the demographic, epidemiological, and clinical characteristics of children under two years of age who were hospitalized with a lrti, focusing on rsv (prevalence, seasonality, subgroups, viral load) and its association with disease severity. the study was conducted from may 2009 to december 2010 (to cover two rsv seasons, that normally coincide with the rainy season) at children's hospital 1 (ch1) and children's hospital 2 (ch2), the two largest pediatric referral centers for southern vietnam, located in ho chi minh city. children from the respiratory wards (rw), emergency care units (ecu) and intensive care units (icu) were eligible for inclusion in the study. patients admitted to the rw are typically those that initially present to the outpatient clinics and were subsequently indicated for admission, while those admitted to ecu or icu typically presented with more severe symptoms. inclusion criteria were age between 1 month to 2 years, cough, a clinical diagnosis of lrti based on who criteria [7] , and onset of symptoms 4 days prior to hospital admission. exclusion criteria were patients with known non-respiratory or non-infectious respiratory diseases such as asthma. were collected on the day of enrolment (day 1) and on day 7 or discharge (if patients were discharged before day 7). edta blood were used for whole blood cells counting, and liver enzyme measurement by ch1 and ch2 laboratory. swabs were placed in viral transport medium [8] and kept at 4°c for a maximum of 24h, then aliquoted and stored at -80°c until further processing for molecular diagnostics [9, 10] . the study was approved by the institutional review board of children's hospitals 1 and 2, the scientific and ethics committee of the hospital for tropical diseases, ho chi minh city, vietnam and by the oxford university tropical research ethics committee (oxtrec), oxford, uk. written informed consent was obtained from parents or legal guardians of children prior to enrolment into the study. rna extraction from nasopharyngeal swabs was done as described previously [11] . rna was analyzed by multiplex real-time rt-pcr on a roche light cycler 480 ii thermocycler (roche diagnostics, penzberg, germany) [9, 10] and rsv locked nucleic acid (lna) real-time rt-pcr (lna assay) on a dna engine peltier thermocycler and chromo 4 real-time pcr system detector (bio-rad, hercules (ca), usa) [11] . the multiplex real-time pcr detects 13 other human respiratory viruses: influenza virus a (flu a); influenza virus b (flu b); adenovirus (adv), enterovirus (env), human metapneumovirus (mpv), human coronaviruses (cov-229e, oc43, hku1, sars cov & nl63), human rhinovirus (rv a, b and c), parainfluenza virus 1, 2 and 3, 4 (piv1, 2, 3, 4), parechovirus (pev) and human bocavirus (bov) [9, 10] . the lna assay assesses viral load (log10 copies/ml) of rsv subgroups a and b (rsv a, rsv b) [11] . for every rsv positive patient, the second sample (at discharge or day 7) was also assessed by lna assay. definitions. severe disease on admission was defined as having an oxygen saturation of spo2<92 or requiring supplemental oxygen or ventilatory support (by nasal cannula, nasal continuous positive airway pressure (ncpap), mask cpap or mechanical ventilation/intubation) or clinical cyanosis. a clinical severity score (adapted from [12] [13] [14] [15] ) was introduced to grade the clinical status of patients at enrolment (table 1) . only patients from whom all components in the score table were available were given a clinical severity score. long hospitalization was defined as longer than 7 days. statistical analysis. associations between categorical and continuous variables were analyzed using the mann-whitney-u test or kruskal-wallis test for continuous variables, and the fisher exact test for dichotomous variables. spearman's rank correlation coefficient was used to assess a general monotonic trend between two continuous variables. a simple linear regression model was used to measure linear associations between rsv viral load and age or day of illness or number of leucocytes in blood. multivariable logistic regression analysis was performed to assess risk factors for severe disease or long hospitalization. the following variables were considered for the models: age, sex, premature birth, previous hospitalization for respiratory illness, other household members sick at home, living with smoker(s), number of members at home, fever, rsv infection (rsv positivity, viral load, and subgroup) and rv infection (rv positivity as single infection or co-infection with rsv). the models' predictive ability was investigated by calculating the area under the receiver operating characteristic (roc) curve of the model (auc), i.e the higher the auc the better prediction power the model has. the hosmer-lemeshow goodness-of-fit test was done to assess model adequacy. the hosmer-lemeshow test of goodness-of-fit tests the null hypothesis that there is no difference between the observed and predicted values of the response variables. therefore, when the test is not significant (p>0.05) the null hypothesis cannot be rejected and this means that the model fits the data well. risk factors for disease severity or long hospitalization were further assessed using odds ratios (or) and 95% confidence intervals (95% cis). all statistical tests were performed as two-tailed tests at 5% significance in either r version 2.13.1 (r foundation for statistical computing, vienna, austria) or intercooled stata version 9.2 (college station, tx, usa). a total of 632 children aged 1-24 months (median 7, iqr 4-12 months) were enrolled into the study between may 2009 and december 2010. 10/632 (2%) patients were admitted to icu, 36/ 632 (6%) to the ecu and 586/632 (92%) to the respiratory ward. the monthly distribution of children hospitalized for lrti and enrolled in this study during the study period are shown in fig 1. diagnoses on admission were based on clinical symptoms, routine laboratory tests and chest radiography; physicians were unaware of viral diagnostic laboratory results. 438/632 (69%) patients were diagnosed with bronchiolitis; 164/632 (26%) with pneumonia, 22/632 (3%) with combined bronchiolitis and pneumonia, 3/632 (0.4%) with laryngotracheitis, and 5/ 632 (0.8%) with undifferentiated lrti. in addition to respiratory symptoms, 21/632 (3%) patients had diarrhoea on admission, and 4/632(0.6%) had congenital heart disease (ventricular or atrial septum defects). only 18/598 (3%) had blood culture test which was part of standard clinical care at hospitals and only 5/18 were positive (1 staphylococcus aureus, 1 streptococcus pneumoniae, 1 haemophilus influenzae, 1 pseudomonas aeruginosa, 1 klebsiella spp). 535/632 (85%) children received antibiotics, 100% children with clinical diagnosis of pneumonia received antibiotics. high alt** 1 high ast** 1 discharge information was available for 596/632 (94%) cases: 363/596 (61%) of patients fully recovered; 226/596 (38%) had incomplete recovery at the time of discharge; 4/596 (1%) went home without permission (mostly due to economic reasons, patients could not pay hospitalization fee) or formal hospital discharge; and 3 patients (1%) died in the hospital. one fatal case (8 months old) was diagnosed with septicemia and severe pneumonia (s. pneumoniae was recovered from blood culture; no respiratory viruses were detected in nasopharyngeal swabs) and died on the second day of admission. the two other fatal cases were 6 and 3 months old and did not have any severe indications on admission but deteriorated quickly after 5 days of hospitalization. no organisms were recovered from blood culture and virology results yielded a single rv infection and a triple infection (rv, piv-3 and bov), respectively. viral etiologies were identified in the vast majority (91%, 574/632) of patients; single viral infections accounted for 375/632 (59%) of cases and co-infections with multiple viruses were found in 199/632 (31%) ( table 2) . rsv was the most frequently detected: overall (302/632, 48%) and in single infections, while rv was most frequently detected in co-infections (table 2) . a significantly higher proportion of rv, env, bov, adv, piv-2, piv-3, cov and pev were detected among co-infections when compared to the single infections (table 2 ). for mpv, rv, piv-2, piv-3 and piv-4, the relative viral load in single infections was significantly higher than in co-infections (table 2) . rsv-rv co-infection was the most common double infection, identified in 147/632 (23%) cases. triple infections were identified in 47/632 (7%) and in 5/632 cases, 4 different viruses were detected (rsv-rv-piv3-cov, env-rv-cov-bov, adv-env-rv-piv2; rsv-adv-env-rv). (2) median log copies/ml rsva (iqr) 7.5 (6.7-8.2) 7.7 (6.7-8.1) 7.4 (6.3-8.2) 0.2 (1) rsv b, n(%) 139 (22) 93 (25) 46 (23) 0.7 (2) median log copies/ml rsvb (iqr) 7.6 (6.9-8.2) 7.7 (7.1-8.2) 7.5 (6.6-8.0) 0.2 (1) flu, n(%) median cp-value flu b (iqr) 31 (29-33) 32 (29) (30) (31) (32) (33) 30 (20) (21) (22) (23) (24) (25) (26) (27) (28) (29) (30) (31) (32) (33) (34) 0.8 (1) adv, n(%) 37 (6) 3 (1) 34 (17) 0.001 (2) median mpv, n(%) 30 (5) 17 (5) 13 (7) 0.3 (2) median (1) piv-3, n(%) 47 (7) 18 (5) (1) pev, n(%) 12 (2) 2 (1) 10 (5) 0.001 (2) median cp-value pev (iqr) 28 (26) (27) (28) (29) 28 (26) (27) (28) (29) (30) (31) 28 (27) (28) (29) 0.7 (1) bov, n(%) 42 (7) 7 (2) 35 (18) 0.001 (2) median cp-value bov (iqr) 29 (25-33) 23 (22) (23) (24) (25) (26) (27) (28) (29) (30) 30 (25) (26) (27) (28) (29) (30) (31) (32) (33) (34) (35) 0.1 (1) p-value was calculated between single infections and co-infections groups, (1) mann-whitney test, in the univariate analysis, lrti severity was associated with younger age (median age in months [iqr] = 5 [3] [4] [5] [6] [7] [8] [9] [10] in severe cases versus 7 [4] [5] [6] [7] [8] [9] [10] [11] [12] in non-severe cases, mann-whitney test pvalue = 0.001 -s1 fig) , presence of fever (72%, 76/106 in severe cases versus 47%, 247/526 in non-severe cases, fisher exact p-value = 0.001), living in a household with a high number of people (median household members [iqr] = 5 [4] [5] [6] in severe cases versus 4 [3] [4] [5] in nonsevere cases, mann-whitney test p-value = 0.03), or having other household members sick at home (50%, 52/103 in severe cases versus 37%, 193/528 in non-severe cases, fisher exact pvalue = 0.001). co-infections were more frequent among the non-severe cases, and the clinical severity score was inversely correlated to the number of viruses detected (spearman cor = -0.09, p = 0.04). among the single infections, viral load was not associated with severity. for rv, single infections were significantly more common (21%, 22/106) in severe cases versus in non-severe cases (12%, 65/526) (fisher's exact test p-value = 0.02). the relationship between rv and severity was also observed in increased odds ratios for elevated (worse) clinical score (or = 1.86, 95% ci (1.08-3.17), p = 0.02). similar relationships were not observed for rsv single infection cases. in multivariate analyses, significant predictors of severity within the entire study population were younger age, presence of fever, and rv single infection ( table 3 ). the hosmerlemeshow goodness-of-fit test result was χ 2 = 5.94, p-value = 0.65) and the auc for the prediction model was 0.74. similarly, predictors of long hospitalization were younger age, presence of fever, and previous hospitalization with respiratory illness were significant ( table 3 ). the hosmer-lemeshow goodness-of-fit test result was χ 2 = 7.38, p-value = 0.5) and the area under the roc curve for the prediction model of long hospitalization was 0.66. rsv infection was not correlated to disease severity, or to duration of hospitalization (table 3) . rsv infections occurred at a younger age than the 3 other leading single viral infections, i.e rv, mpv, piv-3 (tables 4 and 5) , and were significantly more frequent in the first 6 months of life (fisher exact test p-value = 0.001). in contrast, none of the other viruses exhibited significant age group distributions (tables 4 and 5). a significant inverse correlation was observed between rsv load at enrolment and patient age. indeed, on average rsv viral loads decreased by 0.03 log copies/ml per month increase in age (t = -2.37, p-value = 0.02). a higher clinical severity score was found only among rsv single infection cases compared to those of piv-3 single infection cases (mann-withney test p-value = 0.02, table 5 ), but not in any of the other pair-wise comparisons. while rsv single infection had a significantly shorter duration of hospitalization, a higher prevalence of elevated ast and a lower leucocyte count than rv single infection cases (table 5 , mann-withney test p-value = 0.02, 0.02 and 0.001, respectively). we also observed a significantly lower number of leucocytes in rsv single infection versus those in rsv co-infection (mann-withney p-value = 0.001), and an inverse (2) 12 (71) 0.9 (2) 10 (56) 0.3 (2) median number of household members (iqr) 4 (4-6) 4 (4-6) 0.7 (1) 4 (3) (4) (5) 0.3 (1) 4 (3-6) 0.4 (1) living with smokers, n (%) 102/196 (52) 51/86 (59) 0.3 (2) 5 (29) 0.07 (2) 12 (67) 0.2 (2) medical story (2) 16 (94) 0.07 (2) 13 (72) 0.8 (2) premature birth, n(%) 20/199 (10) 14/85 (16) 0.1 (2) 0/16 (0) n.a 1/17 (6) 0.5 (2) daycare, n (%) 25/197 (13) 10/84 (12) 0.9 (2) 2/16 (13) 1.0 (2) 5/18 (28) 0.08 (2) previous hospitalization with respiratory illness, n (%) 22 (11) 23 (26) 0.001 (2) 2 (12) 0.9 (2) 3 (17) 0.5 (2) other household members sick at home, n (%) 76/197 (39) 36/85 (42) 0.6 (2) 5 (29) 0.5 (2) 5 (28) 0.4 (2) clinical characteristics fast breathing, n(%) 166 (83) 75 (86) 0.4 (2) 14 (82) 1.0 (2) 14 (78) 0.6 (2) cyanosis, n(%) 6 (3) chest indrawings, n(%) 193 (96) 81 (94) 0.3 (2) 15 (88) 0.1 (2) 15 (83) 0.01 (2) stridor, n(%) wheezing, n(%) 192 (96) 76 (88) 0.01 (2) 16 (94) 0.8 (2) 17 (94) 0.8 (2) fever (>37.5°c), n(%) 112 (56) 50 (57) 0.8 (2) 7 (41) 0.2 (2) 6 (33) 0.07 (2) fever ( 38.5°c), n(%) 46 (23) 13 (15) 0.1 (2) 5 (29) 0.5 (2) 2 (11) 0.2 (2) rash, n(%) 2 (1) 3 (3) 0.1 (2) 0 (0) n.a 1 (6) 0.1 (2) runny nose, n(%) 181 (91) 70 (81) 0.03 (2) 15 (88) 0.8 (2) 17 (94) 0.5 (2) low spo 2 , n(%) 19/140 (14) 6/60 (10) 0.5 (2) 1/14 (7) 0.5 (2) 1/17 (6) 0.4 (2) median of duration of hospitalization (iqr) 6 (5-7) 6 (5-8) 0.02 (1) 6 (4-7) 0.9 (1) 6 (5-7) 0.9 (1) duration of hospitalization 7 days, n(%) 77 (38) 43 (49) 0.08 (2) 7 (41) 0.8 (2) 7 (39) 1.0 (2) high alt, n(%) 54 (27) 16 (18) 0.1 (2) 2 (12) 0.2 (2) 3 (17) 0.3 (2) high ast, n(%) 44 (22) 9 (10) 0.02 (2) 4 (23) 0.9 (2) 3 (17) 0.6 (2) median of number of white cells in blood (k/mm 3 ) (iqr) 10 (8-14) 13 (11) (12) (13) (14) (15) (16) (17) 0.001 (1) 11 (9-13) 0.8 (1) 11 (8) (9) (10) (11) (12) (13) (14) (15) (16) (17) 0.6 (1) severe cases, n(%) 36 (18) 22 (25) 0.2 (2) 3 (18) 1.0 (2) 3 (17) 1.0 (2) median of clinical score (iqr) 6 (4-7) (n = 186) 5 (4-7) (n = 80) 0.5 (1) 5 (4-6) 0.4 (1) 4.5 (4-6) 0.02 (1) diagnosis bronchiolitis, n(%) 162 (81) 65 (75) 0.3 (2) 10 (59) 0.06 (2) 9 (50) 0.005 (2) treatments antimicrobial agents, n(%) 169 (84) 62 (71) 0.01 (2) 17 (100) 0.07 (2) 17 (94) 0.2 (2) corticosteroids, n(%) 9 (4) 6 (7) 0.4 (2) 1 (6) 0.8 (2) bronchodilators, n(%) 168 (84) 67 (77) 0.2 (2) 9 (52) 0.002 (2) 10 (56) 0.004 (2) supplemental oxygen, n(%) 31 (15) 21 (24) 0.08 (2) 2 (12) 0.7 (2) 2 (11) 0.6 (2) outcomes full recovery, n(%) 119/195 (61) 46/81 (57) 0.5 (2) 11 (73) 0.8 (2) 11 (65) 0.9 (2) severe cases, n(%) 36 (18) 22 (25) 0.2 (2) 3 (18) 1.0 (2) 3 (17) 0.9 (2) death, n(%) p-values were based on comparison with children having rsv single infections. (1) mann-withney test, (2) fisher's exact test, n.a: not applicable correlation between rsv load in nasopharyngeal swabs and leucocyte count in blood collected at enrolment (coeff = -0.56, 95%ci = -0.98 to -0.14, p-value = 0.009). among 302 rsv positive cases, 156/302 (52%) were rsv a, 139/302 (46%) were rsv b, and 7/302 (2%) were both rsv a and b. there was no difference in proportion of a and b subgroups between single and co-infections, and no difference in viral load among subgroups at enrolment (table 2) . strong seasonal variations of rsv prevalence with peaks during the rainy season from may to october were observed over the two seasons of the study. rsv b was dominant during the first season and rsv a during the second season (fig 2) . overall case numbers for mpv and influenza were insufficient to detect patterns of seasonality. the most frequently detected viruses in rsv co-infection were rv, env and adv (s1 table) . in the univariate analysis, the number of viruses co-detected with rsv or the rsv viral load was not associated with rsv severity, or with clinical severity score (cor = 0.06, p = 0.1 and cor = -0.3, p = 0.2, respectively). rsv patients with long duration of hospitalization had a significantly higher rsv viral load than others (median of rsv load (iqr) 8 (7-8) versus 7 (7) (8) mann-withney test p-value = 0.004, respectively). no associations with rsv subgroups and severity or duration of hospitalization were found. in multivariate analyses for the rsv-infected population, the logistic regression analysis of ten selected predictors identified age (or = 0.88, 95%ci:0.81-0.95, p-value = 0.001), fever (or = 4.80, 95%ci:2.26-10.19, p-value = 0.001), and having another household member sick at home (or = 2.04, 95%ci: 1.03-4.02, p-value = 0.04) as independent variables associated with disease severity in rsv-infected children ( table 3 ). the hosmer-lemeshow goodnessof-fit test result was χ 2 = 5.73, p-value = 0.68) and the area under the roc curve for the prediction model was 0.76. for risk factors of long hospitalization among rsv patients, only fever (or = 2.67, 95%ci: 1.59-4.48, p-value = 0.001) was a significant independent predictor ( table 3 ). the hosmer-lemeshow goodness-of-fit test result was χ 2 = 9.35, p-value = 0.31 and the auc for the prediction model of long duration hospitalization was 0.67. again, rsv subgroups or rsv viral load were not associated with disease severity or long hospitalization in multivariate analyses. over the last decade, multiplex molecular diagnostics have revolutionized the diagnostics of respiratory infections and greatly expanded the available data on viral etiologies and coinfection [6, [16] [17] [18] [19] [20] [21] [22] . here, using a previously described multiplex assay to detect 13 different respiratory viruses, viruses were identified in 91% of enrolled patients, and viral co-infection was found in 31%. the high prevalence of viral infections among lrti cases, the predominance of rsv and rv infections, and the co-infection rates between rsv and rv, env and adv in our study were similar to other published work in comparable populations [21, 23] . in these previous studies, among hospitalized children, the proportion of viral causes ranged from 93-97%; rsv and rv were the leading causes, ranging from 64 to 73% and from 30-34%, respectively [21, 23] . our study confirms the importance of rsv infection in children under two as shown by many other studies [17, 21, [23] [24] [25] and is the first study to examine the demographic, clinical and virological characteristics of rsv infections in south vietnam. our results confirm and extend previous observations regarding associations of rsv infections with young age compared to the 3 other leading viral infections (rhinovirus (rv), metapneumovirus, parainfluenza virus (piv-3), wheezing, runny nose, leucopenia (among rsv single versus rsv co-infection or rv single infection) and risk factors (premature birth) [17, 21, [23] [24] [25] [26] . in addition, we observed a significant negative correlation between rsv load in nasopharyngeal swabs and leucocyte count in blood collected at enrolment (coeff = -0.56, 95%ci = -0.98 to -0.14, pvalue = 0.009). these findings are consistent with previous reports [26, 27] and it could be hypothesized that leukocytes in rsv infection are being recruited at specific sites away from the circulating blood [28, 29] . one of our aims was to determine risk factors for severity and long hospitalization among all study patients and among rsv-infected patients. we observed that rsv load at enrolment was significantly related to long hospitalization in univariate analysis, but this was not confirmed in the multivariate analysis; and rsv infection, rsv viral load, rsv subgroups and rsv slope (i.e rsv load dynamic between two time points: admission and discharge, it was calculated by dividing the difference of viral load at admission and at discharge by the number of days between these two time points-data not shown), did not correlate to disease severity or long hospitalization. only young age and fever were independent predictors for disease severity in both populations (study population and rsv population). reports about the relation between rsv infection [30, 31] or rsv load [12, 26, 32, 33] or rsv subgroups [34] [35] [36] [37] [38] and disease severity have been contradicting. marguet et al. reported that rsv infection caused more severe disease than rv infection among children under 1 year of age. in a study by tran et al. conducted in children hospital 2, ho chi minh city, targeting hospitalized children under 15 years, rsv positive children had significantly higher clinical severity scores compared to rsvnegatives. however, this comparison was based on univariate analysis without considering other confounding factors such as age [6] . papadopoulos et al. found, among children less than 18 months, that the presence of rv increases the risk for severe disease significantly, by approximately five-fold. rsv infection was also correlated with severity, however, this did not reach significance [30, 31] . rsv loads at the second collection had a significant negative correlation (correlation = -0.33, p-value = 0.009) with day of illness, while this correlation was not found for the enrolment samples which were collected on any day during the first days of illness (s2 fig). this rsv load trend during rsv infection was also observed in other studies [39] and confirmed the study samples were collected on the early day of rsv illness. thus far, observations regarding the relation between viral co-infections and disease severity have been contradictory and biased by different study designs and viral diagnostic tools used [24, 40] . similar to other studies, we observed a significantly lower proportion of co-infections in severe cases compared to non-severe cases [21, 41] . however, among rsv infected cases, we did not observe significant differences in disease severity between single and co-infected cases. in addition, there were significant differences between rsv-rv coinfections (3/45, 6.6%) versus rsv-env (9/27, 75%)-fisher's exact p-value = 0.006 but no significant versus rsv-adv co-infections (2/18, 11%). interestingly, and in agreement with papadopoulos et al. [31] , although rv was detected mostly as a coinfection, rv single infection was identified as an independent risk factor of severe disease. this finding should be interpreted with caution because the role of rv in the pathogenesis of severe lrti remains a topic of debate [30, 31, [42] [43] [44] [45] [46] [47] . cross-reactivity in the pcr detection of env and rv using 5'utr primers is a known problem, particularly for ev-d68 which has a rhinoviral 5' utr sequence, and this complicates ascertainment of env and rv diagnoses [48] . despite the fact that the study population from our current study and our previous study [3] and from other studies [2, 6] consisted of hospitalized children and is therefore not representative of vietnamese children in general, a consistent rsv seasonal pattern with peaks during the rainy season (between may and october) was observed from 2005 to 2010. the clear shift of rsv subgroups was not observed during the period 2005 to 2007 [3] but we observed this in our current study and tran et al. also confirmed the dominance of rsv a during the same period [6] . in many studies, rsv has been reported as a highly seasonal infection and rsv outbreaks are frequently associated with the rainy season in areas with tropical and subtropical climate [5, 49] . possible explanations for this include meteorological effects such as humidity and uvb radiation on the environmental stability of rsv viruses. the stability of rsv in aerosols was shown to increase with higher humidity [50] . moreover, population behaviors as staying indoors during cold or rainy seasons may facilitate transmission of rsv. from this cohort, we have recently reported analysis of rsv whole genomes [51] and are currently analysing host expression profiles of blood and nasopharyngeal swabs at two time points (manuscript in preparation). there are a number of limitations to our study. firstly, few bacterial diagnostic results were obtained from patients. the role of bacteria as a cause of lrti or as cause of superinfection, especially in rsv and influenza virus infection [52] , is important for antibiotic intervention strategies. lower airway secretions (sputum for example) are considered the optimal specimen type for detecting bacterial (co-) infection, yet are often difficult to obtain from young children, and can only be readily obtained from intubated children. secondly, only a limited number of patients were enrolled as compared to the total number of lrti hospitalizations in two hospitals during the study period (632/45134, 1%) although the number of enrolled patients followed a similar pattern as the total number of patients (fig 1) . in summary, our study has contributed detailed clinical and virological data on rsv and other viruses in respiratory infections among children under 2 years old, the most vulnerable age group, in a lower middle income setting in asia: vietnam. the data on seasonality of viruses are crucial for health care management, such as preparedness for the annual epidemics in terms of hospital capacity and rsv prevention in high-risk children using palivizumab. respiratory syncytial virus infections: old challenges and new opportunities viral pathogens associated with acute respiratory infections in central vietnamese children viral etiologies of acute respiratory infections among hospitalized vietnamese children characterization of hospital and community-acquired respiratory syncytial virus in children with severe lower respiratory tract infections in respiratory syncytial virus infection in tropical and developing countries molecular epidemiology and disease severity of human respiratory syncytial virus in vietnam generic protocol to examine the incidence of lower respiratory infection due to respiratory syncytial virus in children less than five years of age collecting, preserving and shipping specimens for the diagnosis of avian influenza a(h5n1) virus infection simultaneous detection of five different dna targets by real-time taqman pcr using the roche lightcycler480: application in viral molecular diagnostics development and evaluation of a four-tube real time multiplex pcr assay covering fourteen respiratory viruses, and comparison to its corresponding single target counterparts a sensitive real-time pcr for detection and subgrouping of human respiratory syncytial virus disease severity and viral load are correlated in infants with primary respiratory syncytial virus infection in the community respiratory syncytial virus immune globulin treatment of rsv lower respiratory tract infection in previously healthy children relationships among specific viral pathogens, virus-induced interleukin-8, and respiratory symptoms in infancy a multicenter, randomized, double-blind, controlled trial of nebulized epinephrine in infants with acute bronchiolitis analysis of respiratory viral coinfection and cytomegalovirus coisolation in pediatric inpatients two-year prospective study of single infections and co-infections by respiratory syncytial virus and viruses identified recently in infants with acute respiratory disease frequent detection of viral coinfection in children hospitalized with acute respiratory tract infection using a real-time polymerase chain reaction epidemiology of respiratory syncytial virus infection in northern taiwan clinical presentation and severity of viral community-acquired pneumonia in young nepalese children infection with multiple viruses is not associated with increased disease severity in children with bronchiolitis. pediatr pulmonol clinical evaluation of multiplex real-time pcr panels for rapid detection of respiratory viral infections high rate of viral identification and coinfections in infants with acute bronchiolitis multiple simultaneous viral infections in infants with acute respiratory tract infections in spain viral respiratory infections in hospitalized and community control children in alaska correlation of viral load of respiratory pathogens and co-infections with disease severity in children hospitalized for lower respiratory tract infection incidence and predisposing factors for severe disease in previously healthy term infants experiencing their first episode of bronchiolitis analysis of cells obtained by bronchial lavage of infants with respiratory syncytial virus infection peripheral blood lymphopenia and neutrophilia in children with severe respiratory syncytial virus disease in very young infants severity of acute bronchiolitis depends on carried viruses association of rhinovirus infection with increased disease severity in acute bronchiolitis nasal quantity of respiratory syncytical virus correlates with disease severity in hospitalized infants respiratory syncytial virus infections in hospitalized infants: association between viral load, virus subgroup, and disease severity correlation of viral load as determined by real-time rt-pcr and clinical characteristics of respiratory syncytial virus lower respiratory tract infections in early infancy human respiratory syncytial virus (hrsv) rna quantification in nasopharyngeal secretions identifies the hrsv etiologic role in acute respiratory tract infections of hospitalized infants evaluation of quantitative and type-specific real-time rt-pcr assays for detection of respiratory syncytial virus in respiratory specimens from children comparison of a realtime reverse transcriptase pcr assay and a culture technique for quantitative assessment of viral load in children naturally infected with respiratory syncytial virus natural infection of infants with respiratory syncytial virus subgroups a and b: a study of frequency, disease severity, and viral load respiratory syncytial virus infections in infants: quantitation and duration of shedding viruses in communityacquired pneumonia in children aged less than 3 years old: high rate of viral coinfection th17 cytokines are critical for respiratory syncytial virus-associated airway hyperreponsiveness through regulation by complement c3a and tachykinins role of rhinovirus in hospitalized infants with respiratory tract infections in spain correlation of rhinovirus load in the respiratory tract and clinical symptoms in hospitalized immunocompetent and immunocompromised patients rhinovirus associated with severe lower respiratory tract infections in children respiratory syncytial virus, human bocavirus and rhinovirus bronchiolitis in infants role of real-time reverse transcription polymerase chain reaction for detection of respiratory viruses in critically ill children with respiratory disease: is it time for a change in algorithm? pediatr crit care med equal virulence of rhinovirus and respiratory syncytial virus in infants hospitalized for lower respiratory tract infection viral aetiology of acute respiratory infections among children and associated meteorological factors in southern china the epidemiology of respiratory syncytial virus lower respiratory tract infections in children less than 5 years of age in indonesia the relationship of meteorological conditions to the epidemic activity of respiratory syncytial virus direct whole-genome deepsequencing of human respiratory syncytial virus a and b from vietnamese children identifies distinct patterns of inter-and intra-host evolution increased susceptibility to bacterial superinfection as a consequence of innate antiviral responses we acknowledge study nurses hoang thi minh tu, nguyen thi hong ngoc, nguyen viet truong, nguyen thi ngoc ha, nguyen thi thanh nha, le thi kim loan, ho huynh nhu, tran thi tuyet nhung, huynh thi phuong thao, for sample collection; staff in data management at oucru for entering data; vo nhi ha and nguyen thi thanh thuy and the clinical trials unit at oucru and children's hospital 1 and 2 for coordinating the trial.this study was funded by the wellcome trust of great britain (077078/z/05/z). the funder had no role in study design, data collection or analysis, the decision to publish, or preparation of the manuscript. wrote the paper: lahd jeb hrvd mdj. key: cord-283910-k10j5dzd authors: fretzayas, andrew; moustaki, maria title: etiology and clinical features of viral bronchiolitis in infancy date: 2017-05-04 journal: world j pediatr doi: 10.1007/s12519-017-0031-8 sha: doc_id: 283910 cord_uid: k10j5dzd background: bronchiolitis is a common lower respiratory tract infection in infancy. the aim of this review is to present the clinical profile of viral bronchiolitis, the different culprit viruses and the disease severity in relation to the viral etiology. data sources: databases including pubmed and google scholar were searched for articles about the clinical features of bronchiolitis and its viral etiology. the most relevant articles to the scope of this review were analyzed. results: currently there are two main definitions for bronchiolitis which are not identical, the european definition and the american one. the most common viral pathogen that causes bronchiolitis is respiratory syncytial virus which was identified in 1955; now many other viruses have been implicated in the etiology of bronchiolitis such as rhinovirus, adenovirus, metapneumovirus, and bocavirus. several studies have attempted to investigate the correlation of bronchiolitis severity with the type of detected virus or viruses. however, the results were not consitent. conclusions: for the time being, the diagnosis of bronchiolitis remains clinical. the isolation of the responsible respiratory pathogens does not seem to confer to the prognosis of the disease severity. v iral bronchiolitis is a common lower respiratory tract infection in infancy. although the term "bronchiolitis" implies the inflammation of bronchioles, the diagnosis of the disease is clinical and there is still debate regarding the most appropriate definition for the disease. recent advances in laboratory diagnostic methods confer to the identifi cation of different viruses as etiologic pathogens of bronchiolitis, although respiratory syncytial virus (rsv) remains the main culprit the aim of this review was to present the clinical picture of bronchiolitis as a whole entity irrespective of the virology as well as in relation to the particular viral parthogens that are incriminated as etiological factors. databases including pubmed and google scholar were searched for articles about the clinical features of bronchiolitis and its viral etiology. we searched the databases with the following key words: viral bronchiolitis, clinical features, definition, epidemiology, etiology, guidelines, severity score, adenovirus, bocavirus, coronavirus, influenza, metapneumovirus, parainfluenza, respiratory synchytial virus, and rhinovirus. manual searching through cross-references was also performed to identify additional relevant studies. there was no chronological restriction. the most relevant articles to the scope of this review were analyzed. the diagnosis of bronchiolitis is based on the history and the clinical findings. in 1973 it was shown by court [1] that there was not sufficient agreement and reproducibility for the definitions that were used for the classification of acute respiratory illness including bronchiolitis in childhood by the clinicians even in the same country. based on the clinical symptoms and signs he defined acute bronchiolitis as follows: "illness mainly affecting infants, especially in the first 6 months of life. rapid respiration, dyspnoea, wheezing, chest recession, cough, rhonchi and rales are very frequent. visible distention of the chest and increased pulmonary translucency on the chest radiograph are frequent and of high diagnostic significance. upper respiratory features, especially nasal discharge and a red pharynx are frequent. fever is very frequent but high fever uncommon". as it is evident in this definition there was not a specific upper limit for the age, the wheezing episode was not necessarily the first one in the infant's life and radiographic findings were also included in the definition. ten years later in 1983, mcconnochie [2] summarized the clinical criteria for the case definition of bronchiolitis. bronchiolitis was considered as the first episode of wheezing of acute onset in children younger than 24 months of age with preceding signs of viral respiratory illness such as coryza, otitis or fever. respiratory distress symptoms, pneumonia and atopy signs may or may not be present. according to this definition wheezing was a sufficient auscultatory finding and crackles were not mentioned in this classification as a necessary finding on auscultation. in 2006 american academy of pediatrics (aap) published a clinical practice guideline for bronchiolitis, [3] that was updated in 2014 [4] according to which bronchiolitis is "a constellation of signs and symptoms including children younger than 2 years including a viral upper respiratory tract prodrome followed by increased respiratory effort and wheezing". this definition is in accordance with the abovementioned of mcconnochie [2] regarding the upper limit of age and the presence of wheezing as a constitutional finding for the diagnosis of bronchiolitis. according to aap, rales are among the possible signs of bronhiolitis on auscultation. [3] however, in europe [5, 6] and australia, [7, 8] till recently it was considered that the hallmark for the diagnosis was fine crackles on auscultation and that wheezing is not a prerequisite for the diagnosis. the most recent guidelines from the united kingdom [9, 10] state that the presence of fi ne inspiratory crackles and/or high pitched expiratory wheeze on auscultation lead to a diagnosis of bronchiolitis for children under two years of age. the discrepancy of these definitions regarding the hallmark auscultatory finding for the diagnosis of bronchiolitis has been highlighted by other reviews, [11] [12] [13] and it had as a result different definitions to be used by the investigators. some researchers sometimes limit their population to the age of 12 months, in an attempt to minimize the possibility to include in their studies children with early onset virus induced wheezing instead of bronchiolitis. however, these different approaches do not facilitate the comparison of the results from different studies as well as the generability of their results. rsv is the most common viral agent that causes bronchiolitis in infancy. rsv was first identified in 1955 as causing chimpanzee coryza [14] and one year later it was recovered from infants with respiratory illness in the usa. [15] since then, with the evolution of different laboratory techniques, rsv was found to be a culprit viral agent for the majority of bronchiolitis cases in infancy. [16] epidemiological studies revealed that the average annual rate of rsv-associated hospitalizations was 32 per 1000 infants in the usa [17] for the period 1997-2006 indicating the high burden of morbidity due to rsv. during the same period (1953), adenovirus was also first isolated from human adenoids. [18] it affects respiratory system as well as other organs. in different series of children with upper and/or lower respiratory illness due to adenovirus, 5%-24% had bronchiolitis. [19] [20] [21] the prevalence of adenovirus infection in children with bronchiolitis ranges from 1% to 9%. [22] [23] [24] [25] rhinovirus which is known as the common cold virus, also first isolated in 1956 [25] was found to be implicated as a viral pathogen in bronchiolitis cases in 8%-29% of infants with bronchiolitis. [22, [26] [27] [28] [29] [30] [31] [32] it is of interest that children with a history of bronchiolitis caused by rhinovirus are at increased risk for developing recurrent wheezing later [33, 34] without its precise role having being clarified. however, it is beyond the scope of this paper to analyze further this issue. influenza virus was first obtained and identified from patients with infl uenza disease in 1933. this virus can also cause bronchiolitis, less frequently however, as it was identifi ed as the primary pathogen or as a copathogen in 0.5%-5% of children with bronchiolitis in different cohorts. [24, 26, 29, 30, 32] parainflueza virus was first isolated in patients with respiratory disease in 1956. in different bronchiolitis cohorts it was identifi ed as the causative agent either primary or as a co-pathogen in 2.6%-6% of children. [24, 28, 30, 32] the availability of modern molecular techniques and the identification of new viruses such as human metapneymovirus (hmpv) and human bocavirus (hbov) made possible the characterization of the viral culprit agent in about one third of bronchiolitis which used to be simply considered in the past as non rsv bronchiolitis. hmpv was characterized for the first time in 2001 in the netherlands. [35] however, serological studies confi rmed the circulation of the virus in humans for at least since 1950s. [35] the frequency of its detection in infants with bronchiolitis ranged from 1.7% to 16.8%. [27] [28] [29] 32, [36] [37] [38] hbov was first described by allander et al [39] in 2005 in children with respiratory tract infection. since then, its prevalence in children with respiratory infections worldwide was corroborated by many studies. [28, 29, 40] however, high rates of co-infections have been described that question to some extent the role of hbov alone as a causative agent of lower respiratory tract infections. [37] it was isolated in 0.6%-27% of infants [22, 24, 26, [28] [29] [30] 38, 41, 42] with bronchiolitis, with most studies identifying it in about 10% of children. [22, 26, 29] recently, human coronaviruses (hcov) nl63 and hku1 have been linked to bronchiolitis. hcov nl63 was fi rst isolated from a child with bronchiolitis and conjunctivitis in 2004. [43] and the presence of this virus worldwide in individuals with respiratory illness was confirmed by different investigators. [44] [45] [46] it was isolated from children with bronchiolitis from several patient cohorts, [26, 38, 45, 46] but in low percentages either as the primary pathogen or as a co-pathogen. hcov hku1 was fi rst detected in 2005 in a adult with pneumonia [47] and it was also isolated from children with bronchiolitis. [48, 49] it is of interest to mention that in infants with bronchiolitis not fully vaccinated for pertussis, bordetella pertussis was isolated along with rsv or other viruses in 8%-16% of infants, indicating the need to consider the possibility of pertussis in young infants with clinical signs of bronchiolitis and even positive virology tests for rsv or other respiratory viruses. [50, 51] furthermore, atypical bacteria such as mycoplasma pneumoniae, chlamydophila pneumoniae and chlamydophila trachomatis were isolated in infants with viral bronchiolitis in a cohort from thailand [52] indicating that the identification of a virus in infants with viral bronchiolitis does not necessarily precludes the possibility of bacterial co-infection at least with atypical bacteria. viral prodrome symptoms of upper respiratory tract such as nasal obstruction and/or coryza and cough usually precede bronchiolitis findings, lasting 1-3 days. [3, 4, 10] the main symptoms/signs of bronchiolitis are wheezing and/ or crackles on auscultation and increased respiratory effort characterized by tachypnoea and/or chest wall retractions and/or nasal flaring. it is important for the clinicians to determine the clinical severity of the disease so that the affected infant is given the appropriate care as outpatient or inpatient. although the diagnosis of the disease and the assessment of the disease severity is clinical, there are no universally used validated clinical scores of disease severity. according to the aap definition [3, 4] wheezing is a prerequisite for the characterization of bronchiolitis, a finding that is not considered obligatory according to the national institute for health and care excellence (nice) guidelines. [10] wheezing may be present at the end or throughout expiration or it may be also present during inspiration. however, hypoventilation may occur due to severe obstruction when wheezing is not audible. crackles may be present over one or more lung fields and their presence was associated with the severity of the disease using as a surrogate for the disease severity the requirements for oxygen; [53] in this study, [53] wheezing was considered as a prerequisite for the diagnosis of bronchiolitis, signs of respiratory distress such as tachypnoea and chest wall retractions were not related to the disease severity. however, according to nice guidelines, [10] respiratory rate over 60 breaths/ minute is considered as an indicator for referral to the hospital. marlais et al [54] suggested that that respiratory rate as well as heart rate were among the clinical predictors of admission in infants with bronchiolitis. respiratory effort should be also evaluated assessing the degree of chest wall retractions and nasal flaring. according to the nice guidelines [10] marked chest wall retractions is an indicator for admission to the hospital. a rather uncommon but alarming symptom of bronchiolitis, which is an indicator for admission to the hospital, is apnoea. recent prospective studies estimated that the rate of apnea among the hospitalized infants with bronchiolitis is about 5%. [30, 55, 56] there is not an excess risk for apnea for any type of isolated viral pathogen, whereas prematurity and younger corrected age seem to confer to an excess risk. [56] a systematic review [57] of apnea in hospitalized infants with rsv bronchiolitis found the rates ranged widely from 1.2% to 23.8%; but the majority of the studies were retrospective and included infants with serious underlying conditions. infants with bronchiolitis may also have hypoxemia and sao 2 is considered an indicator of disease severity. [10, 54, 58, 59] an sao 2 lower than 92% was considered an indication for admission to the hospital. [10] the general condition of the affected infant should be also evaluated. the behavior may be normal indicating mild disease, or the infant may be irritable or lethargic in cases of moderate or severe disease. if the general appearance is toxic, the infant should be carefully evaluated for other causes that could possibly explain the situation. it should be noted that infants with bronchiolitis may have fever or history of fever although very high temperature is rather uncommon. in a series of 90 infants with bronchiolitis, fever was identified in about 1/3 of them, however temperature >40ºc was recorded in only 2/90 children of the study. [60] the investigators [60] found that infants with fever had a more severe clinical course compared to the afebrile counterparts, a finding that was not corroborated by other investigators. [30, 54] it was also shown that the risk of sepsis was extremely low even in febrile infants with bronchiolitis younger that 3 months of age, whereas the risk of urinary tract infection was low but not negligible. [61, 62] the type of isolated virus was not associated with the presence and duration of fever, as it was shown by studies that compared the clinical characteristics of bronchiolitis in relation to the detected viruses. [26, 30, 38, 52] another indicator of infant's general condition is its ability for feeding and the degree of hydration. poor oral fluid intake, inability or indifference for eating due to breathlessness are indicators of moderate or severe disease and the affected infant should be referred to the hospital. [10] the clinicians should make the diagnosis of bronchiolitis on the basis of the history and physical examinations and evaluate the clinical severity of the disease in order to decide outpatient or inpatient care for the patient. although there are not universally used validated clinical scores of disease severity, different scores have been used for the assessment of bronchiolitis severity in clinical studies. in 1972, wood et al [63] introduced a clinical scoring system for the diagnosis of respiratory failure that has been also used for bronchiolitis since then. the score includes the following parameters: arterial oxygen tension or cyanosis, type of inspiratory breath sounds, usage of accessory muscles, presence of expiratory wheezing and level of cerebral function. in 1983, tal et al [64] used a score in order to evaluate the clinical changes in bronchiolitis severity in relation to the type of treatment. this score has been also used in other clinical studies since then and it includes the following parameters: respiratory rate, accessory muscle use, degree of wheezing and cyanosis. in 1987 the respiratory distress assessment instrument (rdai) was first used by lowell et al [65] to evaluate the response of bronchiolitis to treatment. rdai includes the following items: presence of wheezing on expiration, inspiration, location of wheezing, presence of retractions (supraclavicular, intercostals, subcostal). in 1992, wang et al [66] used another score (respiratory rate, retractions, wheezing, general appearance) for the evaluation of lower respiratory infection severity in infants. in 2004, liu et al [67] evaluate another clinical score with the following components: respiratory rate, presence and location of retractions, presence and degree of dyspnea, presence and type of wheezing. there are several limitations for the usage of severity scores in everyday practice but it is beyond the scope of this paper to analyze further this issue. many studies attempted to investigate the relation of the disease severity with the number and the type of detected viruses, although only a few of these studies were initially designed to answer this question. their results are controversial. this would not be unexpected as the studies differ regarding the eligibility criteria of patients with bronchiolitis and the indicators of the disease severity. although viruses other than rsv have been implicated as etiologic agents of viral bronchiolitis, it is not clarifi ed whether the type of carried virus confers to the disease severity. when wheezing alone was not considered sufficient for the characterization of bronchiolitis, the clinical severity score on admission was higher among infants with rsv bronchiolitis compared to those with rhinovirus or hbov bronchiolitis. [29] bamberger et al [22] also showed that patients infected solely with rhinovirus had a lower severity score on admission as well as during hospitalization compared to rsv bronchiolitis. additionally, they found that only rsv bronchiolitis patients had a signifi cantly higher severity scores on admission and during hospitalization compared to any type of non-rsv bronchiolitis. this observation is of interest as bronchiolitis is a dynamic situation and disease severity on admission can not necessarily predict the severity during hospitalization. when the length of hospitalization was used as a surrogate for the disease severity it was also found that rhinovirus only or rhinovirus plus any other non-rsv pathogen had a significantly shorter length of stay at the hospital. [32] similarly, other researchers [27, 28] found that rsv bronchiolitis was associated with a longer length of hospital stay, whereas rhinovirus as well as hmpv bronchiolitis with a shorter one. [26] however, when admission to pediatric intensive care unit (picu) was used as a surrogate for bronchiolitis severity richard et al [23] did not find any difference in rsv or rhinovirus prevalence among infants hospitalized either in the short term unit or in the picu. in contrast to these observations, it was also shown that rhinovirus bronchiolitis indicated an excess risk for having a significantly higher clinical severity score on admission. [30, 31] it is obvious that all these findings are not necessarily comparable as different indicators of severity were used by the investigators as well as different bronchiolitis defi nitions with the main difference in the inclusion criteria the age up to 12 or up to 24 months, sufficiency of wheezing alone or not as a hallmark for bronchiolitis. as co-infection with two or even more than two viruses was found in a substantial proportion of children with viral bronchiolitis in different series, researchers studied the possible impact, if any, of coinfection on disease severity. using as a surrogate for disease severity the need of admission to picu, richard et al [23] found that infants with viral co-infection were about three times more likely to be admitted to picu even after adjusting for other factors that in univariate analyses increased the likelihood of admission to picu. similarly, another research group [68] also showed that children with dual infection with hmpv and rsv have a ten-fold increased relative risk of admission to picu for mechanical ventilation. it should be noted, however, that in this study the term bronchiolitis was restricted to children younger than 2 years of age world j pediatr, vol 13 no 4 . august 15, 2017 . www.wjpch.com with bilateral inspiratory crackles on auscultation. in the ward settings it was shown [32] that longer stay at the hospital was more likely among children with rsv/rhinovirus co-infection, whereas children with rhinovirus in combination with other than rsv viruses had a significantly shorter stay at the hospital. therefore, it seems that it is not only the number of the viruses but also their type that confers or not to the severity of the bronchiolitis course. these fi ndings were not corroborated by marguet et al [27] who found that rsv infants had longer duration of hospitalization even in comparison with rsv/rhinovirus infants with bronchiolitis. as these results do not point to the same direction it can be speculated that in some cases of coinfections there may be a cumulative pathogenic effect but this is not always the case as in some infants coinfection may be a result of successive or concomitant infections whereas some infants may be simply carriers of one or more of the co-pathogens. another issue that was raised in the literature is whether viral load is related to the disease severity. a few studies were designed to address this issue. scagnolari et al [69] indicated that there was a correlation of rsv viral load with duration of hospitalization in children with rsv bronchiolitis as well as with the clinical score severity index. in this study, the term "bronchiolitis" was restricted to children with diffuse crackles on auscultation whereas infants with wheezing alone were excluded. however, similar results were found by other investigators, relating rsv load with disease severity upon presentation at the emergency department [70] or with the need for mechanical ventilation. [71] nevertheless, there are also results that support the view that viral load does not predict the severity of the disease either on admission or during hospitalization. [72] however, the population of the latter study included a signifi cant proportion of infants with serious underlying diseases. a positive correlation of disease severity score with rsv load was also found in non-hospitalized infants with bronchiolitis [73] a fi nding that was not corroborated in the rhinovirus bronchiolitis group. it seems therefore that heterogeneity of the population of different bronchiolitis cohorts as well as heterogeneity of the parameters that depict the disease severity among the studies do not allow the extraction of reliable conclusions regarding the correlation of the type of the virus or the viral load with the severity of viral bronchiolitis. although there is controversy regarding the management of bronchiolitis there are certain guidelines of aap (2014) [4] and nice (2015) [10] that address this issue thoroughly. management of bronchiolitis is not within the scope of this paper, however it should be mentioned that there are no official guidelines that correlate the management of bronchiolitis with the type of the viral pathogen. the type of management depends on the disease severity but not on the disease viral etiology, at least till now. many viruses are implicated in the etiology of viral bronchiolitis in infancy. however, it is not clear whether and to what extent different viruses confer to the degree of the disease severity or to the disease phenotype. the identification of the carried viruses in infants with bronchiolitis is not therefore necessary in the every day clinical practice. however, for research purposes it is important, using a universal definition for bronchiolitis and introducing a standardized severity score to clarify further whether the type of the culprit viruses confer to the bronchiolitis prognosis. the definition of acute respiratory illnesses in children what's in the name? american academy of pediatrics subcommittee on diagnosis and management of bronchiolitis. diagnosis and management of bronchiolitis clinical practice guideline: the diagnosis, management, and prevention of bronchiolitis viral bronchiolitis in children: a common condition with few therapeutic options acute bronchiolitis bronchiolitis: assessment and evidence-based management viral bronchiolitis for the clinician scottish intercollegiate guidelines network (sign) they said it was bronchiolitis; is it going to turn into asthma doctor? acute bronchiolitis in infants, a review viral bronchiolitis in children recovery of cytopathogenic agent from chimpanzees with coryza recovery from infants with respiratory illness of a virus related to chimpanzee coryza agent (cca). i. isolation, properties and characterization diagnosis and testing in bronchiolitis: a systematic review respiratory syncytial virus-associated hospitalizations among infants and young children in the united states isolation of a cytopathogenic agent from human adenoids undergoing spontaneous degeneration in tissue culture clinical features, adenovirus types, and local production of inflammatory mediators in adenovirus infections adenoviral infections in children: the impact of rapid diagnosis adenovirus respiratory infection in hospitalized children in hong kong: serotype-clinical syndrome association and risk factors for lower respiratory tract infection what is the clinical relevance of respiratory syncytial virus bronchiolitis?: findings from a multi-center, prospective study the impact of dual viral infection in infants admitted to a pediatric intensive care unit associated with severe bronchiolitis infection with multiple viruses is not associated with increased disease severity in children with bronchiolitis the isolation of a new virus associated with respiratory clinical disease in humans detection of new respiratory viruses in hospitalized infants with bronchiolitis: a three-year prospective study in very young infants severity of acute bronchiolitis depends on carried viruses broad respiratory virus detection in infants hospitalized for bronchiolitis by use of a multiplex rt-pcr dna microarray system respiratory syncytial virus, human bocavirus and rhinovirus bronchiolitis in infants clinical risk factors are more relevant than respiratory viruses in predicting bronchiolitis severity association of rhinovirus infection with increased disease severity in acute bronchiolitis prospective multicenter study of viral etiology and hospital length of stay in children with severe bronchiolitis rhinovirus bronchiolitis and recurrent wheezing: 1-year follow-up rhinovirus-induced bronchiolitis and asthma development a newly discovered human pneumovirus isolated from young children with respiratory tract disease human metapneumovirus as a causative agent of acute bronchiolitis in infants detection of human metapneumovirus rna sequences in nasopharyngeal aspirates of young frenchchildren with acute bronchiolitis by real-time reverse transcriptase pcr and phylogenetic analysis viral etiology of bronchiolitis among pediatric inpatients in northern taiwan with emphasis on newly identified respiratory viruses cloning of a human parvovirus by molecular screening of respiratory tract samples human bocavirus infection in hospitalized children in italy human metapneumovirus and human bocavirus in children sole pathogen in acute bronchiolitis: is there a role for other organisms apart from respiratory syncytial virus? identifi cation of a new human coronavirus characterization of human coronavirus oc43 and human coronavirus nl63 infections among hospitalized children <5 years of age human coronavirus-nl63 infections in korean children infections by human coronavirus-nl in hospitalized children characterization and complete genome sequence of a novel coronavirus, coronavirus hku1, from patients with pneumonia coronavirus hku1 and other coronavirus infections in hong kong coronavirus hku1 in an italian pre-term infant with bronchiolitis pertussis and respiratory syncytial virus infections bordetella pertussis infection is common in nonvaccinated infants admitted for bronchiolitis atypical bacterial pathogen infection in children with acute bronchiolitis in northeast thailand clinical findings and severity of acute bronchiolitis clinical predictors of admission in infants with acute bronchiolitis frequency of apnea and respiratory viruses in infants with bronchiolitis apnea in children hospitalized with bronchiolitis incidence of apnea in infants hospitalized with respiratory syncytial virus bronchiolitis: a systematic review outpatient assessment of infants with bronchiolitis bronchiolitis: clinical characteristics associated with hospitalization and length of stay association of fever and severe clinical course in bronchiolitis utility of sepsis evaluation in infants 90 days of age or younger with fever and clinical bronchiolitis occult serious bacterial infection in infants younger than 60 to 90 days with bronchiolitis: a systematic review a clinical scoring system for the diagnosis of respiratory failure. preliminary report on childhood status asthmaticus dexamethasone and salbutamol in the treatment of acute wheezing in infants wheezing in infants: the response to epinephrine observer agreement for respiratory signs and oximetry in infants hospitalized with lower respiratory infections use of a respiratory clinical score among different providers dual infection of infants by human metapneumovirus and human respiratory syncytial virus is strongly associated with severe bronchiolitis evaluation of viral load in infants hospitalized with bronchiolitis caused by respiratory syncytial virus respiratory syncytial virus infections in hospitalized infants: association between viral load, virus subgroup, and disease severity nasal quantity of respiratory syncytical virus correlates with disease severity in hospitalized infants illness severity, viral shedding, and antibody responses in infants hospitalized with bronchiolitis caused by respiratory syncytial virus disease severity and viral load are correlated in infants with primary respiratory syncytial virus infection in the community key: cord-257299-z9u12yqb authors: mansi, n.; de maio, v.; della volpe, a.; ripa, g.; malafronte, l.; de filippis, c. title: ear, nose and throat manifestation of viral systemic infections in pediatric patients date: 2009-12-31 journal: international journal of pediatric otorhinolaryngology doi: 10.1016/s0165-5876(09)70006-0 sha: doc_id: 257299 cord_uid: z9u12yqb abstract objective/methods an exhaustive review of literature was performed to investigate available data and evidences regarding pediatric otolaryngologic manifestations of viral systemic infections. results/conclusions modern otolaryngologists should be familiar with viral systemic infections since many have head and neck manifestations. cooperation between otolaryngologist, paediatrician and virologist can be considered and excellent tool in diagnosis and treatment of these diseases in particular when complications occur. there are multiple systematic viral infections that can manifest themselves in orl related organs. their actions can work directly or indirectly causing an alteration in the human immune system and a consequent secondary bacterial invasion. notable advances in the diagnosis and treatment of viral infections have been mitigated by the appearance of new pathological processes, for example aids, which often has its initial manifestations in orl regions. table 1 is a list of illnesses affecting different anatomical sites and the viral etiologies that commonly strike each particular location. considering the vast nature of the subject, we subdivided our treatment into three parts corresponding to the same groups of interrelated viral illnesses: -viruses that can cause deafness. -viruses that can cause inflammation in the upper respiratory tract. -viruses particularly relevant to ent (infectious mononucleosis, papillomatosis, herpes infections). ascertaining specific viral causes of most infections is neither necessary nor cost-effective, and should be reserved only for specific cases. clinical and epidemiological acumen remain the basis for a presumptive diagnosis. when a specific diagnosis is necessary, diagnostic procedures based on biochemical and molecular biological processes provide sensitive, specific and rapid results [1] . in most viral infections, immunity to re-infection generally lasts a short period of time due to the host's limited immunological response or, rather for an antigenic change in the virus. viral pathologies that can cause deafness can be congenital, appear in either the pre-natal or postnatal period and can also be acquired upon contact with the pathogen [2] [3] [4] (table 2 ). in particular, the hearing damage caused by congenital infections can be part of a severe syndrome (such as "congenital rubella syndrome") but more frequently it is the first and only manifestation of intrauterine infection. common childhood viral infections, such as measles and mumps are probably an unrecognized cause of acute or progressive damage to hearing [5] . in prenatal deafness, a pathogen introduced during pregnancy can provoke an arrest or alteration of the normal development of the ear, even causing lesions on the already-formed hearing mechanism [6] . the most serious lesions manifest themselves in the first three months of pregnancy, especially between the seventh and tenth week, when the cochlea is developing; this would be considered a case of embriopathy. fetopathy refers instead to lesions that form between the fourth month of pregnancy and birth. since the hearing organ has already formed in these cases, patients do not generally suffer serious alterations although the inner ear is certainly sensitive. the viruses that most frequently cause prenatal deafness are rubella and citomegalovirus (cmv). rubella is caused by an rna virus of the togaviradae family of the rubivirus genus. congenital rubella is typically passed on to the fetus from a primary infection in the mother. the virus invades the upper airways of the mother causing viremia and spreading into different sites including the placenta. it has been hypothesized that in the first gestational phases, the rubella virus provokes a chronic intrauterine infection. fetal infection in the first trimester, particularly in the first 8-10 weeks, has an extremely high risk of malformations such as hypoacusia, cardiac and ocular defects (gregg triad); however, if the rubellum infection is contracted in the second or third trimester, it results in hypoacusia and pigmented retinas. thus, the more precocious the maternal rubellum, the greater the risk of fetal infections and the more serious the fetal malformations (100% in the first month, 80% in the first trimester, 70% in the second trimester and 30% in the third). this reduction is likely due to either a maturation of the placenta after the first trimester which limits the transfer of the virus, or the greater resistance of the differentiated cells [5] . the deriving hypoacusia is generally sensorineural and bilateral and at birth can already be progressive or it can manifest itself later. the hearing damage seems to be caused by a "teratogenic" effect of interference with the normal development of the organ at the cochlear level [6, 7] . unlike other congenital infections, rubella is easily prevented. between 12 and 15 months of age, a livevirus rubellum vaccine is administered along with a measles and mumps vaccination, giving the patient immunity to rubellum for about 15 years (mmr); a booster vaccination is administered before elementary or middle school. women of child-bearing age who are not immune to rubella must undergo vaccination and not get pregnant in the following three months. vaccination immediately after giving birth is advisable for mothers at risk of being infected. citomegalovirus (cmv) is a dna virus that belongs to the herpesviridae family. it can go into latency and then reactivate and has been isolated in various sites including saliva, urine, breast milk, sperm, brain fluid, and amniotic fluid. congential cmv infection is thought to be derived from transplacental infection from a primary or recurring maternal infection occurring in the first half of pregnancy. prenatal cmv infection is contracted by contact with infected cervical secretions, breast milk, or blood derivatives. it is believed that maternal antibodies have a protective function and that most of these newborns are either born asymptomatic or are not infected by the virus in the case of contact. many women who are infected by cmv during pregnancy are asymptomatic, but occasionally develop an illness similar to mononucleosis. it is still unclear if more serious lesions are a consequence of a precocious maternal infection or of a later one during the course of gestation. nearly 10% of children with congenital cmv infection are symptomatic at birth. manifestations include delayed intrauterine growth, premature birth, microcephalus, jaundice, petechia, hepatosplenomegalia, periventricular calcification, corioretinitis e pneumonia. the virus causes deafness by infecting the inner ear and altering the organ of corti. moreover, it can cause malformations of the labyrinth of ethmoid and at the same time also lesions on the auditory tract due to secondary toxicity. the hypoacusia that establishes itself is sensorineural, almost always bilateral, and profound, generally regarding acute tones. symptomatic newborns have a mortality rate of up to 30% and 70-90% of those who survive have neurological deficits such as hearing loss, mental retardations and visual disturbances [8] [9] [10] . a vaccine for cmv is still under research. exposure to the disease in non-immunized pregnant women must be controlled, despite the fact that cmv is ubiquitous everywhere. since it is frequent in children who attend preschools, pregnant women must observe all the common norms of good hygiene after contact with or being exposed to the urine or expectorate of such children [11, 12] . in post-natal deafness, numerous infective illnesses can be responsible for serious damage to the viii nerve and the cochlear apparatus. a large part of hearing defects arising in childhood can be traced back to the intrauterine period. the most frequent forms are: viral meningoencephalitis (arbovirus, herpesvirus, mixovirus, poxvirus, etc.), mumps, chickenpox and measles. meningoencephalitis can be primitive or constitute the secondary complication of a viral infection. the forms of primitive meningoencephalitis can be both epidemic (arbovirus, poliovirus, echovirus, coxsackievirus illnesses), and sporadic (herpes simplex, varicella zoster, parotite) [13] . secondary encephalitis, such as complications of a viral infection, probably have an immunological, pathgenic mechanism. secondary encephalitis to rubella, mumps, measles, smallpox, cow's pox and other less defined illnesses are all examples. currently, deafness caused by meningoencephalitis is not infrequent among the post-natal causes of deafness; in some cases it can be caused by a virus that, before birth, reach the cochlea via the external hearing conduct by way of the vascular system, causing a relatively symmetrical bilateral sensorineural hypoacusia which is either mild-serious or profound. in other cases, deafness is due to a meningoencephalital localization of liquor infection in the first four weeks of life as a complication of neaonatal sepsis (25%). deafness caused by meningoencephalitis occurs more often in males and is found in 2/10,000 neonates born at full term and in 2/1,000 low-weight neonates. no vaccines exist for the described viral forms. mumps (parotitis) is instead caused by a paramixovirus, spread through drops of infected saliva or through direct contact with material contaminated by infected saliva. the virus probably penetrates the body through the mouth. it can be found in saliva 1-6 days before the appearance of the swelling of the salivary glands and lasts throughout the duration of the illness (usually 5-9 days). an infection usually results in permanent immunity, even when there is unilateral swelling of the salivary glands. although the illness can occur at any age, most cases occur in children between 5 and 10 years of age; it does not usually occur in children under 2 years old. breast-fed children less than one year old are usually immune. the incubation period is 14-24 days. deafness can be a complication in 5/10,000 cases of mumps. in 80% of cases, deafness is sudden and unilateral in the context of an acute infection in association with aseptic meningitis and often accompanied by tinnitus, ataxia, and vomiting. hearing loss is profound and permanent for high frequencies and can go unrecognized. damage is confined to the cochlear duct and consists in the degeneration of the vascular strip of the corti organ, the degeneration of the upper membrane, usually more serious on the basal curve of the cochlea. active immunization is obtained through a single-dose, live-virus inoculation between 12 and 15 months of age with a mmr vaccine. a booster dose is administered before starting elementary or middle school. measles, caused by a paramixovirus, is extremely contagious and is spread primarily through either the nasal and oral excretions of an individual in the prodrome or precocious eruptive stage of the disease or through the nuclei of drops dispersed in the air. the contagious stage of the illness extends from 2 to 4 days before the appearance of the eruptions until 2-5 days after their appearance. the virus disappears from the nose and pharynx secretions as soon as the eruption on the skin has cleared up. the incubation period is 7-14 days. measles virus causes permenant, bilateral deafness in 1/1000 cases. deafness appears suddenly at the same time as the cutaneous rash. the viral infection of the inner ear spreads through the vascular strip and destroys the structures of the cochlea as well as most of the nervous and ganglionic and fibers [14] . measles infection can be avoided by administering a reduced, live-virus vaccine to children between the ages of 12 and 15 months (mmr). the vaccine confers long-term immunity and provokes an antibody response similar to that of natural measles. in some cases, the vaccine can provoke a light or asymptomatic infection that is not contagious. sensitive subjects at risk of contracting measles can be protected if the live-vaccine is administered within 2 days of exposure. in other cases, such as pregnant women, or children under one year of age, an immunoglobuline specific measles vaccine (mig) or a 0,25 ml/kg im dose of serum immunoglobulin may be administered. chickenpox is caused by the varicella-zoster virus (herpesvirus) and represents its acute, invasive phase, while the reactivation from its latent phase causes the herpes zoster illness. it is believed that chickenpox, which is extremely contagious, is transmitted through drops of saliva which are infected and even more infective during the brief prodromic period and the first phase of eruption. incubation period is 14-16 days and transmission is considered possible 10-21 days after exposure. the deafness it causes can lead to the destruction of the nervous and sensorial cells of the neuroepithelia through a process of neurolabyrinthitis which can result in severe bilateral, sensorineural hypoacusia [15] . chickenpox can be prevented by inoculating with a reduced, live-virus vaccine in all healthy children between 12 and 18 months of age, after which children who lack immunity to chickenpox may be vaccinated at any time. subjects over 13 years old who have not been immunized must receive two doses of the vaccine, with a period of 4-8 weeks between doses. the severity of the disease can be lessened by administering an immunoglobuline anti-zoster (zig) or anti-varicella-zoster (vzig) within 96 hours after exposure. its use, however, is restricted to subjects at risk, like those affected by leukemia, immunodeficiency syndromes or other serious pathologies, and pregnant women. neonates with mothers who were infected by chickenpox five days before giving birth or two days after are also candidates for such treatment [16, 17] . viral respiratory infection is almost always a benign pathology. its beginning is connected with the socialization of the child, and as such is most frequent during preschool. it noticeably interferes with the child's wellbeing and provokes significant medical-social costs.unfavorable environmental factors (atmospheric pollution, passive smoking, etc.), precocious socialization, and predisposing immunological factors with immunological immaturity could all be predisposing factors. viral respiratory infections are characterized by a series of acute episodes that can involve the entire respiratory system or a single sector (pharyngotonsillitis, otitis, rhinosinusitis, laryngitis, bronchitis, pneumonia) [18] [19] [20] [21] . the damage that a viral infection can inflict on the mucous membranes of the upper respiratory tract are influenced by the reduction of the mucous flux and phagocytes as well as the increase in the bacteria's adhesiveness to mucus cells [22, 23] . an upper-resipiratory viral infection is characterized by multiple processes. first, the virus replicates itself in the epithelium, spreading fragments of the disintegrated cells into respiratory secretions and demonstrating the presence of the virus, viral peptides, or viral nucleic acids [24, 25] . the host then responds to the infection by producing a range of cellular products. some, such as alpha-interferon, are specifically anti-viral. others, such as interleukin, are aspecific. specific antibodies are produced in sequence, for example igm followed by igg and iga; the comparison of a high level of igm without an increase in igg is an index of recent infection (the comparison of blood examinations confirms the diagnosis but is only clinically useful for epidemiologic purposes. acute rhino, pharengeal and tonsillar inflammation, caused by viral infections [26] are some of the most common deseases found in pediatric populations. less frequently, the pharangeal-tonsillar forms are accompanied by an involvement of the oral and or respiratory tract mucosa [27] [28] [29] . such infections, in anglo-saxon countries, the term, upper respiratory tract infection (urti) or "common cold" is used to describe an inflammation of the upper airways [30] . the episodes, which often reoccur, effect mostly preschool-age children and have socioeconomic repercussions, which are not related to the gravity of the pathology, but mostly to the increase in the requests for visits to the doctor, the costs incurred for treatment and the days of school and work lost by the children and the parents who must look after them. the etiology of the acute forms in the respiratory airways is, initially, of a viral nature in most patients, with later, secondary bacterial infections on the mucous lesions caused by the viral agents [31] . the transmission of the pathogens responsible for the urti frequently occurs in public locations by direct or indirect contact with the nasal secretions or plugge drops from infected subjects. seasonability, which could increase or reduce sensitivity to such infections, constitutional factors, and an incomplete maturation of the immune system of the child are hypothesized to be pathogenic mechanisms. multiple viruses are responsible for these infections as described in table 1 . the course of acute, unspecific viruses is quite variable but almost always concludes with a recovery within 2-5 days as long as other complications do not develop. the most common complications are infections such as otitis media (mostly in younger children), rhinosinusitis, satellite lymphoadenitis, spreading of the infection into the lower respiratory tract and obstructed respiration, and bronchial spasms in subjects with bronchial hyperactivity. occasionally respiratory viruses are responsible for clinical pictures described as the common cold [32] , caused by picornavirus (rhinovirus, echovirus and coxackievirus), and influenza syndrome [8, [33] [34] [35] , caused by influenza viruses that initially strike the epithelium of the respiratory mucous membranes which other viral strains attach themselves to, aggravating and complicating the original clinical portrait [36, 37] . both herpangina and laryngotracheitis have a unique clinical picture. herpangina is an extremely contagious illness caused by a coxackievirus characterized by a presence of a vesicular exanthema at the velopharyngeal mucous level and acute or croup laryngotracheitis [38] [39] [40] [41] when viral infections are associated. the infections are caused by diverse viruses but more frequently by type 1 and type 2 parainfluenza viruses [42] [43] [44] , which cause an inflammation of the tracheal and subglottic muscous membrane with a charateristic symptomology (inspiratory stridor and barking cough) occasionally relapsing with serious, obstructing respiratory complications [45] [46] [47] . the varicella-zoster virus (vzv) belongs to the herpesvirus group. it is a dna virus that gives rise to chickenpox as a primary infection and to herpes zoster (hz) as a localized relapse due to modifications of the pathogenic power of the virus and/or alterations of cellular immunity [38, 48, 49] . hz is an acute cutaneous-nervous illness that is locally circumscribed and provoked by a resurgence of the vzv acquired during infancy and latent in one or more of the more sensitive ganglia of the dorsal roots of the spinal marrow and/or cranial nerves for a prolonged period of time (often decades); during the latency period the virus does not replicate itself or give any sign or symptom of its presence [50] [51] [52] . in particular, at the auricular level, the illness takes on the name herpes zoster oticus but it is also described in the auricular zone as herpes zoster auris or ramsay hunt illness, in honor of the author who, in 1907, described its characteristics and its correlation with the geniculate ganglion [53, 54] . the illness is caused by a reactivation of the vzv in the geniculate ganglion of the facial nerve; through the sensitive nervous fiber, the virus reaches the skin and causes a characteristic centrifugal root vesicular eruption. we can clinically define four stages according to the involvement of the vii n.c. the diffusion of the virus from cell to cell must be impeded with the use of antiviral drugs (5 mg/kg acyclovir administered intravenously per day in three daily doses for 7-10 days followed by an oral administration for another 7 days); results of treatment with more recent antiviral drugs (such as famciclovir and valaciclovir) are promising, while a significant inflammatory reaction reactive in the nerve must be treated with cortisonebased anti-inflammatory medication (1 mg/kg/daily for ten days) and sometimes with surgical decompression [13] . analgesics and local antiseptics should be added to treament with cortisones and antiviral medication. laryngeal papillomatosis (lp) [55, 56] is caused by subtypes of the human papilloma virus (hpv) which is a member of the papova family of viruses [57] . seventy subtypes have been described, but only hpv 11, hpv 6, and more rarely hpv 16 are specifically associated with laryngeal papillomas [58] [59] [60] . typical of such a pathology is the multifocal nature of the lesion (>85% of cases), localized on the vocal cords in 60% of cases but also involving the upper-glottic plain (35%), the oropharynx, the bronchial tree and, rarely, the cervical esophagus. in the united states, the annual incidence rate of laryngeal papillomatosis is 4.3/100,000 in children and 1.8/100,000 in adults, with a prevelance of 5.7/100,000 [61, 62] . this data is substantially analogous to that revealed in a 1991 danish study [63] which registered an annual incidence in children of 3.6/100,000. histologically, papilloma cosists of a cartilaginous fiber scaffolding with the presence of connective vascular tissue its surface surrounded by squamous epithelium. keratinizing aspects are not observed on its surface. the course of this pathology is characterized by frequent relapses and aggravations that require frequent and repeated laryngoscopy and bronchoscopy to ablate the rapidly forming pappillose formations and to avoid obstruction of the airways [64] . even if the illness is usually resolved by a spontaneous recovery, some cases can move toward an unfavorable prognosis at any time for no recognizable reason and involve the trachea, the bronchi and the lungs [65] . it is believed that hpv 11 has a greater propensity for a distal, pulmonary diffusion and that, moreover, therapeutic action, such as tracheotomy or repeated endolaryngeal ablation can favor the distal insemination of papillomas. the tracheal, bronchial, and pulmonary involvement occurs, according to various authors, in 2-4% of cases. malignant degeneration of the laryngeal papillomatosis, is a rare, but serious event and leads to an unfavorable prognosis. most described cases involve adults who have other risk factors, such as the use of tobacco, long-term illnesses, and previous exposure to radiation due to radiation therapy for papillomatosis. there is a greater probability of malignant transformation with hpv 16 but also hpv 6 and 11 are capable of oncologically transforming the nature of a cell culture [66] . in surgical treatment of laryngeal papillomatisis, many techniques are used: asportation with tweezers via indirect laryngoscopy, electro-cauterization, cryosurgery, direct asportation in larynfissure, endoscopic asportation with microlaryngoscopy in suspension. currently, most surgeons prefer endoscopic laser surgery for its high precision and the haemostatic control which the technique permits [10] . more recent techniques include argon laser photo sensitization and hematoporphyrin derivatives, known as photodynamic therapy [67, 68] , and the technique with scaples and rf ("coblation") which seems to avoid the modest heat damage and the edema which results from the use of the laser [69, 70] ; the same advantage is shared by ablation with microdebrider which is also more rapid than excision by laser. that is why it is coming ever closer to replacing the co 2 laser as the ablative method of choice for use on children [71] . despite the radical nature of the treatment, relapse is the norm. this makes reiterated procedures necessary with the possibility, occasionally, of having to resort to a tracheotomy. such a procedure should be avoided for as long as is possible because of the recognized possibility of colonization on the part of the papillomas in the region of the tracheostomy and tracheobronchial tree. the risk of the tracheobronchial tree being colonized is also believed to be a consequence of the repeated intubations. the need to lengthen the amount of time between surgical asporation of the papillomas and the possibility of complete medical resolution have spurred many researchers to find adjuvant or resolving treatments. recent studies have supplied a way to identify adjuvant therapies to control papillomatosis and its relapses such as interferon-alpha [56] , acyclovir [72] , indolo-3-carbinolo [73] , retinic acid [74] , metotressato, cidofovir [75] [76] [77] [78] . even if the above therapies have sometimes significantly reduced relapses of papillomas, we believe the most effective to be intralesional injection of cidofovir associated with surgical treatment [76] . however, none of them are able to eradicate the hpv genome from the mucous cells of the respiratory tract [79] . the most promising therapies for pl are based on both therapeutic and prophylactic hpv vaccines that are currently in experiemental phases [56, 80, 81] . the epstein-barr virus (ebv) or human herpesvirus 4 is a ubiquitous gammaherpesvirus that infects more than 95% of the world's population. the most common manifestation of the primary infection of this organism is infective mononucleosis (im), a sometimes acute, but often asymptomatic clinical syndrome which more often strikes children, adolescents, and young adults [82] . it is a self-limiting lymphoproliferative illness connected to a first contact with the epstein-barr virus. the virus generally comes into contact with the mucous membranes of the oropharanx where it causes a localized primary infection from which it can circulate through the bloodstream.the period of incubation is not known, and to be the source of infection, is often misunderstood, even if it is known that it is mainly spread orally. in particular, the cells which host cells are mainly the b-lymphocytes and the cells of the human nasalpharanx are where the virus replicates itself. the b-lymphocytes transformed by ebv are the target of a multiform immune response. the immune response (production of antibodies) documents a primary ebv infection. the cellular immune response, consisting in part of the induction of an activated, postive t-lymphocyte cd8, is mostly responsible for atypical lymphocytes which is the consequence of a primary ebv infection. the virus can be found in the oropharangeal secretions of 15-25% of healthy adults who test positive for ebv. the reactivation of ebv is generally asymptomatic, the opposite of that of the herpes simplex and varicella-zoster virus. ebv is relatively labile. it has not been isolated from environmental sources and it is not very contagious. in the majority of cases, it is believed that the incubation period is 30-50 days. the virus can be spread by the transfusion of blood derivatives but is more frequently passed on by oropharangeal contact (kissing) between a non-infected subject and a healthy carrier that asymptomatically secretes the virus from the oropharynx. during early childhood, infection occurs more frequently in lower socioeconomic classes and in conditions of overcrowding [83] . ebv has also been associated with african burkitt lymphoma and some b-cell neoplasias in immunodepressed patients (especially transplant recipients, hiv or ataxia-teleangectasia patients) and to nasalpharangeal carcinoma [29, 84, 85] . these associations are based on seriologic evidence of an increased ebv activity and on proof of nuclear antigens (epstein-barr nuclear antigens, ebna) and of ebv dna found in tumor biopsies. it has been postulated that ebv places a role in some b-cell lymphomas, polyclonally stimulating and transforming the b-lymphocytes, making them more susceptible to a successive chromosome transfer to an evolution toward an oligoclonal or monoclonal lymphoproliferation. the classic symptomology of mi includes fatigue, fever, inflammation, lyphoproliferation, however, patients can also present all or only some of these symptoms. ebv infection in children is usually asymptomatic or with a light symptomology. usually patients present with an illness that has lasted several days to a week, followed by fever, inflammation, and adenopathy. fatigue is usually highest in the first 2-3 weeks. fever reaches its peak in the afternoon or early evening with a temperature of around 39.5â°c, but can even reach 40.5â°.when fatigue and fever are the dominant signs (the so-called typhoid form), the beginning and the resolution can take longer. inflammation can be serious, painful and sedating and can resemble streptococcica inflammation. lymphoadenopathy can involve almost any group of lymphnodes but is usually asymmetrical; anterior and posterior cervical adenopathy is often relevant. the enlargement of only one lymphnode or a group of lymphnodes can be the only manifestation; in these cases, studies of the heterophiles can forgo lymph nodal biopsy or help the interpretation of alarming histopathological aspects. splenomegaly, present in around 50% of cases, is at its maximum during the second and third weeks, manifesting itself through pain the upper left quadrant. slight hepatomegaly can also be present as well as a pain on the hepatic percussion. less frequent signs are malcularpapular eruptions, jaundice, periorbital edema, and palatal exanthema [40] . infective mononucleosis is usually self-limiting. the duration of the illness is variable, usually about 2 weeks, but generally 20% of patients can go back to school or work after a week, and 50% after two weeks. patients can usually begin their normal activites again after this period but sometimes the complete resolution of asthenia requires several weeks. only in 1-2% of patients does asthenia last months. the decline happens in less than 1% of all cases and is generally caused by complications of the primary ebv infection (encephalitis, rupture of the spleen, airway obstruction). generally, the diagnosis is clinical but it must always be confirmed by laboratory testing, and, in particular, identification of ebv. it should be mentioned that the tendency of a late positive score and the elevated possibility of false negatives. treatment of mi is generally supportive and consists of antipyretic and/or analgesic drugs. the use of antibiotics is controversial while therapy is underway with cortisones, which is considered by some to be routine, but considered by other aa to be exclusively reserved for the most serious cases or complications. mi is generally considered to be a benign and self-limiting illness. however, in some, rare cases, complications can arise putting the life of the young patient at risk. serious hepatic complications are those which progress toward cirrhotic forms, reye's syndrome or in extreme cases, toward duncan's syndrome, a syndrome characterized by massive hepatitis linked to the x chromosome and caused by a defect in the immune response to ebv. respiratory complications are, in general, obstructive and linked to adenotonsillar hypertrophy or to serious interstitial pneumonia. hematologic complications are particularly alarming and can lead to the bursting of the spleen as well as neurological complcations where encephalitis is the leading cause of death. in this regard, particular attention should be paid to guillain-barrã©'s syndrome which is an inflammatory, demyelinating form that can complicate infective mononucleosis and cause a progressive paralysis of the respiratory muscles or rather a more or less diffused involvement of the cranial nerves. none declared. evaluation of recurrent respiratory tract infection in children genetic evaluation guidelines for the etiologic diagnosis of congenital hearing loss temporal relationship between human parechovirus 1 infection and otitis media in young children immune-mediated inner ear desease and parvovirus b19 the patology of ribella deafness concurrent influenza a and group a beta-hemolytic streptococcal pharyngotonsillitis role of cytomegalovirus in sensorineural hearing loss of children: a case-control study tehran, iran laserchirurgia in otorinolaringoiatria syntomatic congenital cytomegalovirus infection: neonatal morbilitã  and mortalitã  the 4 ,4 -difluoro analog of 5 -noraristeromycin: a new structural prototype for possible antiviral drug development toward orthopoxvirus and cytomegalovirus sensorineural hearing loss in postmeningitic children studying the etiology of deafness in the "deaf" schools of alexandria varicella-zoster virus infections. 1: chickenpox and shingles. treatment and prevention utility of dna microarrays for detection of viruses in acute respiratory tract infections in children premorbid factors and outcome associated with respiratory virus infections in a pediatric intensive care unit. premorbid factors and outcome associated with respiratory virus infections in a pediatric intensive care unit inner ear and facial nerve complications of acute otitis media with focus on bacteriology and virology malattie delle tonsille palatine e loro trattamento upper respiratory virus detection without parent-reported illness in children is virus-specific le basi patogenetiche delle infezioni respiratorie ricorrenti. il bambino con infezioni ricorrenti inflammatory diseases of the upper and lower airways. epidemiology and pathophysiology over-the-counter cough and cold medication use in young children infezioni delle alte vie respiratorie, faringotonsilliti e rinosinusiti acute e croniche etiologia delle faringo-tonsilliti: nuovi dati interessanti. novitã  faringo-tonsilliti the epidemiology of peritonsillar abscess disease in northern ireland principles and practice of pediatric infectious diseases the influenza season has started in a number of european countries human rhinoviruses: the cold wars resume vaccines for preventing influenza in healthy children global epidemiology of human infections with highly pathogenic avian influenza a (h5n1) viruses influenza virus and acute asthma in children optimal allocation of pandemic influenza vaccine depends on age, risk and timing ramsay hunt syndrome in a 3-month-old infant clinical practice. croup viral croup in children out-of-hospital cardiac arrest in children with bacterial tracheitis respiratory viruses in laryngeal croup of young children an update on inflammatory disorders of the pediatric airway: epiglottis,croup and tracheitis use of throat swab or saliva specimens for detection of respiratory viruses in children viral etiology and epidemiology of acute lower respiratory tract infections in children varicellazoster virus reactivation is an important cause of acute peripheral facial paralysis in children varicella-zoster virus load and cochleovestibular symptoms in ramsay hunt syndrome secondary intermedius neuralgia-like pain in a young child ramsay-hunt syndrome with vesicular stomatitis in a 4-year-old infant. oral surg oral med oral pathol oral radiol endod infective causes of facial nerve paralysis on herpetic inflammations of the geniculate ganglion: a new syndrome and its complications task force on recurrent respiratory papillomas. a preliminary report recurrent respiratory papillomatosis: a review recurrent respiratory papillomatosis quaderni monografici di aggiornamento aoico prevalence of human papillomavirus in the oral cavity/oropharynx in a large population of children and adolescents hspe7 treatment of pediatric recurrent respiratory papillomatosis (rrp): interim results utilizing a laryngeal staging and severity scale (lsss). sentac (society for ear, nose, and throat advances in children) meeting recurrent respiratory papillomatosis laryngeal papillomas: the epidemiology in a danish subpopulation 1965-1984 recurrent respiratory papillomatosis: juvenile versus adult forms clinical and radiological features in three cases of pulmonary involvement from recurrent respiratory papillomatosis malignant transformation of recurrent respiratory papillomatosis associated with integrated human papillomavirus type 11 dna and mutation of p53 variable light-dose effect on photodynamic therapy for laryngeal papillomas treatment of recurrent respiratory papillomatosis with argon plasma coagulation radiofrequency coblation for the treatment of laryngotracheal papillomas soft-tissue complications of laser surgery for recurrent respiratory papillomatosis american society of pediatric otolaryngology members' experience with recurrent respiratory papillomatosis and the use of adjuvant therapy acyclovir in the treatment of recurrent respiratory papillomatosis. a pilot study treatment of recurrent respiratory papillomatosis with indole-3-carbinol regression of aggressive laryngeal papillomatosis with 13-cis-retinoic acid (accutane) the histopathologic effects of cidofovir on cartilage intralesional cidofovir and surgical excision for laryngeal papillomatosis treatment of severe laryngeal papillomatosis with intralesional injections of cidofovir systemic cidofovir in papillomatosis adjuvant drug strategies in the treatment of recurrent respiratory papillomatosis can mumps vaccine induce remission in recurrent respiratory papilloma? world health organization initiative for vaccine research: state of the art of new vaccines -research & development. world health organization clinical manifestations and quantitative analysis of virus load in taiwanese children with epstein-barr virus-associated infectious mononucleosis the merck manual of diagnosis and therapy nasopharyngeal carcinoma in tunisian children: retrospective epidemiological, clinical and biological study about 48 cases unilateral tonsillar lymphoepithelioma with ipsilateral parapharyngeal space involvement: a case report key: cord-267436-mivxm8oh authors: groneberg, david a; poutanen, susan m; low, donald e; lode, hartmut; welte, tobias; zabel, peter title: treatment and vaccines for severe acute respiratory syndrome date: 2005-03-10 journal: lancet infect dis doi: 10.1016/s1473-3099(05)01307-1 sha: doc_id: 267436 cord_uid: mivxm8oh the causative agent of severe acute respiratory syndrome (sars), which affected over 8000 individuals worldwide and was responsible for over 700 deaths in the 2002–2003 outbreak, is a coronavirus that was unknown before the outbreak. although many different treatments were used during the outbreak, none were implemented in a controlled fashion. thus, the optimal treatment for sars is unknown. since the outbreak, much work has been done testing new agents against sars using in-vitro methods and animal models. in addition, global research efforts have focused on the development of vaccines against sars. efforts should be made to evaluate the most promising treatments and vaccines in controlled clinical trials, should another sars outbreak occur. severe acute respiratory syndrome (sars) is an infectious disease characterised by substantial morbidity and mortality, first recognised after an outbreak in 2002-2003. 1, 2 the who issued a global alert on sars on march 12, 2003 after receiving reports from china's guangdong province, hong kong, and vietnam regarding clusters of respiratory illness of unknown aetiology. one of the first reports was made by who scientist carlo urbani, who was called to investigate cases of pneumonia of unclear aetiology in a hospital in hanoi; he later died of sars. 3 following the who alert, probable sars cases were also reported from other regions in china, and other asian countries including singapore, taiwan, indonesia, thailand, and the philippines. other countries, including canada, the usa, and germany also identified cases. in retrospect, sars originated in guangdong at the end of 2002. it first spread to other regions in asia and then international travel facilitated its spread to other continents. a cumulative total of 8096 probable cases of sars were recorded during the period from november 1, 2002 to july 31, 2003 , with 774 deaths in 29 countries. 4 soon after sars was identified as a new disease, the who initiated a collaborative global network striving to work together to identify the aetiological agent of sars. 5 in unprecedented time, a novel coronavirus-sars coronavirus-was identified as the probable causative agent of sars 6-8 (figure 1) and koch's principles were demonstrated to be fulfilled by this agent. 10, 11 this virus belongs to the coronavirus family-enveloped, positive-sense rna viruses 12 associated with respiratory disease in human beings and animals. 13, 14 evidence suggests that sars coronavirus originated from sars-like viruses in animals in the southern chinese province of guangdong; the most frequently implicated animal is the himalayan palm civet, an animal found in food markets and eaten as a delicacy. 15, 16 sars coronavirus is organised into 13-15 open reading frames (orfs) containing approximately 30 000 nucleotides. 8, 17, 18 in total, 61 sars-coronavirus sequences derived from different sars epidemic phases have been analysed and genotypes characteristic of each phase have been identified. 19, 20 the different sars-coronavirus orfs represent typical viral genes such as protease and replicase, spike, envelope, membrane, and nucleocapsid, all of which may represent potential therapeutic targets (figure 2). in common with all infections caused by coronaviruses, after infection sars coronavirus induces the synthesis of 3ј coterminal sets of subgenomic mrnas in target the causative agent of severe acute respiratory syndrome (sars), which affected over 8000 individuals worldwide and was responsible for over 700 deaths in the 2002-2003 outbreak, is a coronavirus that was unknown before the outbreak. although many different treatments were used during the outbreak, none were implemented in a controlled fashion. thus, the optimal treatment for sars is unknown. since the outbreak, much work has been done testing new agents against sars using in-vitro methods and animal models. in addition, global research efforts have focused on the development of vaccines against sars. efforts should be made to evaluate the most promising treatments and vaccines in controlled clinical trials, should another sars outbreak occur. cells. 21 in laboratory settings, sars coronavirus is able to infect macaque monkeys, 11 mice, 22 ferrets, and domestic cats. 23 clinically, sars is characterised by systemic symptoms such as fever and myalgia, followed by respiratory symptoms including a non-productive cough and dyspnea. laboratory findings include lymphopenia, and chest radiographs commonly exhibit unilateral or bilateral infiltrates. approximately 15% of cases deteriorate, requiring intubation and mechanical ventilation. 24 the overall mortality rate has been reported to be about 10%. however, sars mortality rates in those over 60 years old have been reported to be as high as 50%. 25 affected children seemed to have milder symptoms with no reports of death. 26 at the time of the 2002-2003 outbreak, physicians shared their personal experiences supporting or rejecting various treatments for sars. because of the rapid progression of the outbreak, multicentre, randomised, controlled interventional trials were not possible, and the success of various treatments remains largely anecdotal. thus, a consensus on therapeutic strategies has not yet been reached. since the outbreak, global research efforts have focused on testing new agents against sars with in-vitro methods and animal models. in addition, much effort has been placed on developing effective vaccines against sars. this review summarises the clinical experience of the use of various treatments during the outbreak and provides an overview of the data, both in vitro and in vivo, supporting, or otherwise, the effectiveness of these treatments and those that have been proposed since the outbreak. in addition, we summarise the progress made to date regarding sars vaccines. a summary of the pharmacological agents that have been used or proposed for the treatment of sars is shown in figure 3 . during the 2002-2003 outbreak, suspected sars cases were usually treated initially with broad-spectrum antibacterial drugs effective against typical bacterial causes of acute community-acquired pneumonia. the administration of broad-spectrum antibiotics-eg, respiratory fluoroquinolones, second-generation cephalosporins, or third-generation cephalosporins-plus a macrolide is recommended at the first signs of the sars, because the initial features of the disease are nonspecific. however, after sars coronavirus is identified as the causative agent, antibiotics may be withdrawn, because there is no evidence that antibiotics are clinically beneficial in the treatment of sars. even before the causative agent of sars was discovered, treatment with ribavirin was used empirically to treat patients with sars. 27 ribavirin is a synthetic nucleoside with broad-spectrum antiviral activity. 28 clinical studies that have assessed the effectiveness of ribavirin in sars range from anecdotal case reports and retrospective case series to one randomised clinical trial with multiple treatment arms. however, none of these studies definitively determine whether or not ribavirin is effective against sars. case reports and case series suggest that combined treatments including ribavirin may be beneficial to some extent; however, in all of these studies, the effect of ribavirin is confounded by the concomitant use of other agents. for example, studies describe clinical and radiological improvements in patients treated with ribavirin and steroids, 29,30 but, without a control group, it is difficult to determine whether the improvements result from ribavirin, steroids, the combination of both, or the natural course of the illness. one study showed that the delayed initiation of combined therapy with ribavirin and steroids was among the risk factors associated with severe complicated disease, suggesting that ribavirin might be beneficial, but it is difficult to delineate the role of delaying the use of ribavirin from the delay in the use of steroids. 31 the results of a randomised clinical study in guangdong, involving multiple different treatment arms, suggest that ribavirin given at a low dose (400-600 mg/day) was less effective compared with an early and aggressive use of steroids with interferon alfa. 32 however, the lack of a control arm in this study does not allow for one to make definitive conclusions about whether or not ribavirin has any positive effect on sars compared with no treatment. invitro testing showed that ribavirin was not able to inhibit sars-coronavirus replication at clinically achievable concentrations. 33, 34 this finding, combined with postmortem findings demonstrating high viral loads in most patients despite treatment with ribavirin, 35 suggests that if ribavirin has any effect against sars coronavirus, it is likely to have only a small beneficial effect at best. this is important when the side-effects that have been associated with ribavirin use are considered. knowles and co-workers reported common adverse events in 110 people with suspected or probable sars who were treated with ribavirin. 36 61% of these people had evidence of haemolytic anaemia; hypocalcaemia and hypomagnesaemia were reported in 58% and 46% of the people, respectively. the combination of the protease inhibitors lopinavir and ritonavir was used less frequently during the sars outbreak compared with ribavirin. the lopinavir/ ritonavir combination was first considered a potentially useful treatment after in-vitro studies showed it had antiviral activity against sars coronavirus. 37, 38 chan and colleagues 38 compared outcomes in people who received lopinavir/ritonavir as initial treatment, and as rescue therapy, with matched controls; all patients were given ribavirin and steroids according to a standardised protocol. the addition of lopinavir/ritonavir as initial treatment was associated with a statistically significant reduction in the overall death rate and intubation rate compared with matched controls (pͻ0·05). however, the subgroup that received lopinavir/ritonavir as rescue therapy did not show a significant difference in these endpoints. chu and co-workers 37 also assessed treatment with lopinavir/ritonavir compared with historic controls; all patients were also treated with ribavirin and steroids in a similar protocol to that of chan and collegues. adverse events (development of acute respiratory distress syndrome [ards] or death within 21 days) were significantly lower in the lopinavir/ritonavir group than in the historic controls (pͻ0·001). in addition, a significant reduction in the need for rescue pulsed steroids for severe respiratory deterioration (pͻ0·001) and significantly lower nosocomial infections were also noted in those treated with lopinavir/ritonavir, compared with controls (pͻ0·043). by multivariate analysis, it was demonstrated that the lack of treatment with lopinavir/ritonavir, age 60 years old or greater, and positive hepatitis b carrier status were independent predictors of an adverse outcome including death or the development of ards requiring intensive care within 21 days of onset of illness. 37 based on these studies, lopinavir/ritonavir appears to be a promising anti-sarscoronavirus agent. other protease inhibitors have been studied in vitro for potential antiviral effects in sars. for example, yamamoto and colleagues 39 screened a set of compounds that included antiviral drugs already widely used, and found that nelfinavir strongly inhibited sarscoronavirus replication. nelfinavir inhibited the cytopathic effect induced by sars-coronavirus infection, and the expression of viral antigens was much lower in infected cells treated with nelfinavir than in untreated, infected cells. in addition, barnard and colleagues 40 found that two protease inhibitors-calpain inhibitor vi (val-leu-cho) and calpain inhibitor iii (z-val-phe-ala-cho)-inhibited sars coronavirus, suggesting that other protease inhibitors may also be useful in the treatment of sars. the membrane-associated carboxypeptidase angiotensinconverting enzyme 2 (ace2), is a cellular receptor for sars coronavirus, interacting with the s1 domain of the spike protein. 41 thus, peptides and small compounds that bind to ace2 42, 43 are possible agents for the treatment and prevention of sars. in addition, a soluble form of the receptor, antibodies to it, or the receptorbinding domain of the spike protein, may be candidate treatments. indeed, sui and co-workers 44 searched a nonimmune human antibody library and successfully identified an anti-s1 human monoclonal antibody, 80r, that potently neutralises sars-coronavirus infection and efficiently inhibits syncytium formation by blocking binding to ace2. 80r was shown to compete with soluble ace2 for association with the s1 domain of the spike protein and bound to it with high affinity. theoretical reasoning and in-vitro evidence suggest that fusion inhibitors are promising treatment candidates for sars. 45, 46 peptides derived from the heptad repeat regions 1 and 2 of hiv-1 gp41-a transmembrane protein involved in the fusion of hiv and target cellsare the basis for anti-hiv fusion inhibitors. based on similarities between the heptad repeat regions of gp41 in hiv-1 and the heptad regions in the spike protein of sars coronavirus, a common mechanism mediating fusion between each virus and target-cell membranes was postulated. 45, 46 liu and colleagues 45 tested two sets of peptides corresponding to the heptad regions in the spike protein for inhibitory activity against sars coronavirus, and found that one peptide-cp1inhibited sars-coronavirus infection in vitro. it has been postulated that cp1 binds to heptad region 1 of the spike protein and interferes with the conformational changes needed to allow fusion with target cells. rna interference (rnai) treatment is a process by which small interfering rnas (sirna) are administered, leading to degradation of mrna with identical sequence specificity. 47 this technology has been used to silence genes in cultured cells and in animals, and to target hiv, hepatitis b, and hepatitis c viral infections. [48] [49] [50] to explore the possibility of interrupting sars-coronavirus replication with sirnas, specific sirnas targeting the spike gene in sars coronavirus were synthesised. these sirnas effectively and specifically inhibited gene expression of the spike protein in sars-coronavirusinfected cells. 51 another study assessed the in-vitro efficiacy of six sirna molecules targeting different sites of the replicase 1a region of the sars-coronavirus genome. 52 judged by morphological changes, three of the molecules markedly inhibited the cytopathic effects caused by viral infection and replication. the three sirnas also inhibited the infection and replication of different strains of sars coronavirus, indicating that sirnas targeting the replicase 1a region may be an option for future clinical use. 52 in-vitro studies have shown that glycyrrhizin, a component of liquorice roots, is able to inhibit sarscoronavirus replication. 34 glycyrrhizin inhibits hiv replication in vitro 53 and has been used clinically in the treatment of hepatitis c 54 and hepatitis b 55 with some success. the mechanism of glycyrrhizin-induced inhibition of viral replication-and specifically sarscoronavirus replication-is unclear, but possibly involves inhibition of replication through an antiviral effect of nitric oxide (no). glycyrrhizin upregulates expression of inducible no synthase and production of no in mouse macrophages. 56 in addition, preliminary results by cinatl and colleagues 34 show that glycyrrhizin induces no synthase in vero cells used to cultivate sars coronavirus. cinatl and colleagues 34 showed that sars-coronavirus replication is inhibited when deta nonoate-a no donor compound-is added to the culture medium. this finding has been further corroborated by keyaerts and co-workers 57 using a different no donor compound, s-nitroso-n-acetyl-penicillamine. keyaerts and colleagues also report their findings on the use of inhaled no gas to treat a number of people with sars. their results suggest an associated immediate improvement in oxygenation and a lasting effect after termination of inhalation of no, which is known to be a potent mediator of airway inflammation. 58, 59 niclosamide wu and colleagues 60 screened a set of marketed drugs that were not registered for antiviral use to determine if any had in-vitro activity against sars coronavirus. they found that niclosamide, an existing antihelmintic drug, was able to inhibit replication of sars coronavirus. the underlying mechanism by which the drug exerts this effect is unclear, but the study shows that niclosamide does not interfere with the virion's attachment to, or entry into, cells, nor does it appear to inhibit the protease activity. new compounds continue to be tested, with the goal of finding more potential candidate treatments for sars. for example, from over 10 000 agents tested, wu and colleagues 61 found 15 compounds with potent anti-sars-coronavirus activity. more compounds are likely to be discovered in the future. during the 2002-2003 sars outbreak, systemic steroids became a mainstay of sars therapy in many centres. 62 the rationale for their use was based on the paradoxical finding that, despite a fall in sars-coronavirus viral load and a rise in sars-specific igg typically seen during the 3rd week of illness, a clinical deterioration was observed in some people. 31 in addition, pathological findings consistent with bronchiolitis obliterans organising pneumonia and ards led to the hypothesis that immune hyperactivity resulting from cytokine dysregulation may be a component of sars that could be reduced by steroid treatment. 63 in most cases, steroids were administered as adjunctive therapy to ribavirin treatment. if the patient's review respiratory condition worsened clinically, pulsed, highdose steroids were added. however, most studies were confounded by the concomitant use of other agents, and none of the studies contained a control group. thus, whether or not steroids have a beneficial effect in the treatment of sars cannot be readily determined. in some studies, treatment regimens containing steroids seemed to be associated with chest radiographic improvements, fever defervescence, and improvement in oxygenation rates earlier than patients not treated with steroids. 32, 64, 65 however, in a study by hsu and colleagues, 65 adding steroids was not associated with clinical improvement, although the dose of steroids in this study was lower than in those where benefit was seen. ho and co-workers 66 retrospectively compared the clinical and radiographic outcomes of people with probable sars who received ribavirin, 17 of whom initially received pulsed, highdose steroids and 55 of whom initially received lowdose steroids. pulsed, high-dose steroids were also given to any patient as rescue therapy in the presence of deteriorating respiratory status. the cumulative steroid dose, intensive care unit admission rate, need for mechanical ventilation, and mortality rates were similar in both groups after 21 days. however, those people initially given pulsed steroids required less oxygen and had earlier radiographic improvement. in addition, they required substantially less rescue pulsed steroids. this study suggests that early initiation of pulsed steroids may have a role in the treatment of sars. however, definitive studies are needed and the potential benefits of steroids must be compared with the associated risks, such as the development of avascular necrosis, secondary sepsis, and fatal aspergillosis, some of which have been described in people with sars. 67, 68 in beijing, hong and du 69 evaluated 67 people with sars who had received steroids and ribavirin, and who presented with largejoint pain, potentially caused by avascular necrosis, between march and may 2003. both plain radiographs and magnetic resonance imaging examination were completed on the same day. 28 people were identified with avascular necrosis. the mean time to diagnosis of avascular necrosis was 119 days after the onset of sars, or 116 days after steroid use. interferons type 1 interferons have been shown to inhibit sarscoronavirus replication in in-vitro studies. 33, [70] [71] [72] because of initial reports describing these in-vitro results, interferons were used clinically during the latter part of the outbreak. loutfy and colleagues 73 described their clinical experience with interferon alfacon 1-a recombinant, non-naturally occurring type 1 interferon containing common aminoacids from several natural interferon alfa subtypes-in 22 people with probable sars treated in an open-label study in toronto. 13 people with sars who received treatment with steroids alone were compared with nine people who received steroids plus interferon alfacon 1. the group treated with interferon alfacon 1 had significantly improved oxygen saturation levels (p=0·02) and a more rapid resolution of radiographic lesions. in addition, this group exhibited substantially less elevation in creatine kinase levels and a trend towards a more rapid normalisation of lactate dehydrogenase levels. 73 however, this group also received higher doses of steroids, so it is difficult to determine whether or not the beneficial effects were due to the interferon alfacon 1. haagmans and co-workers 74 investigated the prophylactic use of interferons in a macaque model. 3 days before inoculation with sars coronavirus, macaques were given pegylated interferon alfa. substantially reduced viral replication, viral excretion, viral antigen expression by type 1 pneumocytes, and pulmonary damage were noted in the treated macaques compared with untreated macaques. post-exposure treatment with pegylated interferon alfa yielded intermediate results. these results suggest that interferons have a role in the treatment of sars. because most patients develop antibodies against sars coronavirus and survive the disease, passive and active immunisation are viewed as possible effective means to prevent and/or treat sars. 75 indeed, the development of various vaccines is one of the most important goals of ongoing sars research. one of the initial proposals to treat sars was to use sera from people convalescing from sars as passive immunotherapy. this passive immunisation was attempted with anecdotal success. 76 since then, prior infection and passive transfer of murine neutralising antibodies have been shown to prevent replication of sars coronavirus in the respiratory tract in mice. 77 technological advances enabling the development and purification of human monoclonal antibodies can be exploited to create specific monoclonal antibodies in large-scale production. indeed, monoclonal antibodies obtained from immortalised b lymphocytes isolated during convalescence from people with sars have been shown to neutralise virus infection in vitro and to prevent virus replication in a mouse model of sarscoronavirus infection. 78 in addition, ter meulen and colleagues 79 showed that prophylactic administration of a human igg monoclonal antibody reactive with whole inactivated sars coronavirus was able to reduce replication of sars coronavirus in the lungs of infected ferrets, completely prevent the development of sars-coronavirus-induced macroscopic lung pathology, and stop the shedding of virus in pharyngeal secretions. although passive immunisation strategies appear promising, the ideal approach to ensure rapid control of future outbreaks of sars is to generate an effective and safe vaccine. there are numerous teams worldwide working on the creation of vaccines using inactivated sars coronavirus, recombinant subunits, recombinant dna, and viral vectors. 80 given the potential for antibody-directed viral enhancement and disease exacerbation, as reported for vaccines directed against another coronavirus (feline infectious peritonitis coronavirus), 81 it is important that all vaccines created be carefully evaluated before being used clinically. of all of the vaccines in development, most work relates to viralvectored vaccines and dna vaccines. to date, three different viral-vectored vaccines have been described with successful results reported in animal models. [82] [83] [84] gao and colleagues 82 reported using three adenoviralbased vectors expressing codon-optimised sarscoronavirus spike, membrane, and nucleocapsid proteins. intramuscular vaccination with all three vaccines at day 0 and day 28 was shown to induce broad, virus-specific immunity in rhesus macaques. all six vaccinated macaques had antibody responses against the spike protein and t-cell responses against the nucleocapsid protein. in addition, all vaccinated animals showed strong neutralising-antibody responses to sars-coronavirus infection in vitro. challenge tests to determine whether or not this immune response was able to prevent, or reduce the severity of, infection with sars coronavirus were not completed. bisht and co-workers 84 constructed recombinant forms of the highly attenuated modified vaccinia virus ankara (mva) containing the gene encoding the full-length sars-coronavirus spike protein and assessed whether expression of the spike protein alone in mva could raise neutralising antibodies and protectively immunise mice. 84 both intranasal and intramuscular administration of the vaccine to balb/c mice at 0 and 4 weeks led to the production of serum antibodies against the spike protein that neutralised sars coronavirus in vitro. 4 weeks after the second immunisation, vaccinated animals and control animals were challenged with sars coronavirus. those given the vaccine had reduced titres of sars coronavirus in the respiratory tract. likewise, the passive transfer of serum from mice immunised with the vaccine to naive mice led to a reduction in sars-coronavirus replication. these findings suggest that this mva-based vaccine is a promising sars-coronavirus vaccine candidate. bukreyev and colleagues 83 reported their successful experience with the mucosal immunisation of african green monkeys with an attenuated parainfluenza virus expressing the sars-coronavirus spike protein. the complete sars-coronavirus spike protein gene was incorporated into a recombinant attenuated parainfluenza virus that is being developed as a live attenuated, intranasal paediatric vaccine against human parainfluenza virus type 3. four african green monkeys were vaccinated with a single dose of the vaccine, administered via the respiratory tract, and four other monkeys were vaccinated with a control. all monkeys were challenged with sars coronavirus 28 days after immunisation. neutralising serum antibodies were noted in all of the vaccinated animals. after sarscoronavirus challenge, viral shedding was documented in all of the control animals but not in any of the vaccinated animals. 83 the authors concluded that a vectored mucosal vaccine expressing the sarscoronavirus spike protein alone may be highly effective for the prevention of sars in a single-dose format. dna vaccines are also an attractive option for sars vaccines. 85 thus far, three experimental studies have been published addressing dna vaccination in sars. [86] [87] [88] yang and colleagues 86 showed that giving mice a sars-coronavirus dna vaccine encoding the spike glycoprotein induced t-cell responses, neutralisingantibody responses, and protective immunity. alternative forms of the spike protein were assessed and all were found to induce substantial neutralisingantibody titres and strong immune responses mediated by cd8 and cd4 cells. in addition, a reduction in viral replication in the lungs by more than six orders of magnitude was noted after sars-coronavirus challenge; the protection was shown to be mediated by a humoral, but not a t-cell-dependent, immune mechanism. these findings show that dna vaccines based on the spike glycoprotein may lead to effective immune responses with protective immunity in animal models. kim and co-workers 87 reported the generation and characterisation of dna vaccines targeting the nucleocapsid protein of sars coronavirus by antigen linkage to calreticulin, which has been shown to enhance mhc class i presentation to cd8(+) t cells. with a murine model, it was shown that the vaccination with this dna vaccine leads to the generation of a more potent nucleocapsid-specific humoral and t-cellmediated immune responses, compared with nucleocapsid dna alone. in addition, mice vaccinated with the dna vaccine were capable of substantially reducing the titre of challenging vaccinia virus expressing sars-coronavirus nucleocapsid protein. in a similar study by zhu and colleagues, 88 immunisation of mice with a nucleocapsid-based dna vaccine led to nucleocapsid-specific antibodies and specific cytotoxic t-cell activity. challenge tests were not completed. together, the data presented on potential vaccines reflect enormous international efforts. because a vaccine usually takes 6-8 years of clinical development after review entering phase i clinical trials before being licensed, it is not expected that any of these vaccines will be available for clinical use in the near future. however, given the pace and amount of progress to date, the period of time before clinical production of a sars vaccine may be substantially shortened compared with other vaccines. whether or not sars will re-emerge is a matter of debate. 89, 90 however, in the event that sars does recur, the most promising-and immediately available-agents for the treatment of the syndrome seem to be type 1 interferons, steroids, and lopinavir/ritonavir, based on the available data on agents already clinically approved. however, by the time another outbreak arrives, many of the other promising agents-eg, sars-coronavirusspecific receptor-binding inhibitors, fusion inhibitors, and sirnas-may have been approved for clinical use. the choice of agents will need to be determined based on the available data at that time. ideally, the most promising agents would be given in a controlled clinical trial. the difficulties in designing and implementing controlled clinical trials-which limited the ability of researchers to do such trials during the past sars outbreak, and which will continue to pose problems in the event of future outbreaks of sars or other novel pathogens-have been summarised. [91] [92] [93] the best solution to facilitate the implementation of clinical trials in future outbreaks would be the establishment of an international collaborative clinical-trials group with access to appropriate contingency funds, and an internationally accepted ethics review board. until then, research based on in-vitro studies and in-vivo animal models should be continued to determine the best agent, or combination of agents, worthy of further clinical consideration. we declare that we have no conflicts of interest. data for this review were identified by searches of medline, current contents, and references from relevant articles; numerous articles were identified through searches of the extensive files of the authors. search terms were "severe acute respiratory syndrome", "sars", "treatment", "coronavirus", "infection", "sars coronavirus", "vaccination", and "antiviral". english language papers were reviewed. severe acute respiratory syndrome: global initiatives for disease diagnosis the aetiology, origins, and diagnosis of severe acute respiratory syndrome summary of probable sars cases with onset of illness from 1 world health organization multicentre collaborative network for severe acute respiratory syndrome diagnosis. a multicentre collaboration to investigate the cause of severe acute respiratory syndrome coronavirus as a possible cause of severe acute respiratory syndrome identification of a novel coronavirus in patients with severe acute respiratory syndrome a novel coronavirus associated with severe acute respiratory syndrome morphology and morphogenesis of severe acute respiratory syndrome (sars)-associated virus newly discovered coronavirus as the primary cause of severe acute respiratory syndrome koch's postulates fulfilled for sars virus nidovirales: a new order comprising coronaviridae and arteriviridae the structure and replication of coronaviruses the biology and pathogenesis of coronaviruses isolation and characterization of viruses related to the sars coronavirus from animals in southern china wild animals could be source of sars the genome sequence of the sars-associated coronavirus characterization of a novel coronavirus associated with severe acute respiratory syndrome molecular evolution of the sars coronavirus during the course of the sars epidemic in china epidemiological and genetic analysis of severe acute respiratory syndrome the molecular biology of coronaviruses mice susceptible to sars coronavirus virology: sars virus infection of cats and ferrets severe acute respiratory syndrome: clinical outcome and prognostic correlates epidemiological determinants of spread of causal agent of severe acute respiratory syndrome in hong kong children hospitalized with severe acute respiratory syndrome-related illness in toronto antiviral treatment of sars: can we draw any conclusions? the broad-spectrum antiviral ribonucleoside ribavirin is an rna virus mutagen a cluster of cases of severe acute respiratory syndrome in hong kong a major outbreak of severe acute respiratory syndrome in hong kong clinical progression and viral load in a community outbreak of coronavirus-associated sars pneumonia: a prospective study description and clinical treatment of an early outbreak of severe acute respiratory syndrome (sars) in guangzhou, pr china severe acute respiratory syndromerelated coronavirus is inhibited by interferon-alpha glycyrrhizin, an active component of liquorice roots, and replication of sars-associated coronavirus severe acute respiratory syndrome-associated coronavirus in lung tissue common adverse events associated with the use of ribavirin for severe acute respiratory syndrome in canada role of lopinavir/ritonavir in the treatment of sars: initial virological and clinical findings treatment of severe acute respiratory syndrome with lopinavir/ritonavir: a multicentre retrospective matched cohort study hiv protease inhibitor nelfinavir inhibits replication of sars-associated coronavirus inhibition of severe acute respiratory syndrome-associated coronavirus (sarscov) by calpain inhibitors and beta-d-n4-hydroxycytidine angiotensin-converting enzyme 2 is a functional receptor for the sars coronavirus novel peptide inhibitors of angiotensin-converting enzyme 2 substrate-based design of the first class of angiotensin-converting enzyme-related carboxypeptidase (ace2) inhibitors potent neutralization of severe acute respiratory syndrome (sars) coronavirus by a human mab to s1 protein that blocks receptor association interaction between heptad repeat 1 and 2 regions in spike protein of sars-associated coronavirus: implications for virus fusogenic mechanism and identification of fusion inhibitors cloaked similarity between hiv-1 and sars-cov suggests an anti-sars strategy inhibiting severe acute respiratory syndrome-associated coronavirus by small interfering rna modulation of hiv-1 replication by rna interference interference of hepatitis c virus rna replication by short interfering rnas short interfering rna-directed inhibition of hepatitis b virus replication silencing sars-cov spike protein expression in cultured cells by rna interference inhibition of sars-associated coronavirus infection and replication by rna interference effect of glycyrrhizin, an active component of licorice roots, on hiv replication in cultures of peripheral blood mononuclear cells from hiv-seropositive patients long-term treatment of chronic hepatitis c with glycyrrhizin [stronger neo-minophagen c (snmc)] for preventing liver cirrhosis and hepatocellular carcinoma lamivudine and glycyrrhizin for treatment of chemotherapy-induced hepatitis b virus (hbv) hepatitis in a chronic hbv carrier with non-hodgkin lymphoma induction of inducible nitric oxide synthase and proinflammatory cytokines expression by o,pј-ddt in macrophages inhibition of sars-cov infection in vitro by s-nitroso-nacetylpenicillamine, a nitric oxide donor compound role of nitric oxide in allergic inflammation and bronchial hyperresponsiveness role of nitric oxide in chronic allergen-induced airway cell proliferation and inflammation inhibition of severe acute respiratory syndrome coronavirus replication by niclosamide small molecules targeting severe acute respiratory syndrome human coronavirus pro/con clinical debate: steroids are a key component in the treatment of sars lung pathology of fatal severe acute respiratory syndrome clinical features and short-term outcomes of 144 patients with sars in the greater toronto area severe acute respiratory syndrome (sars) in singapore: clinical features of index patient and initial contacts high-dose pulse versus nonpulse corticosteroid regimens in severe acute respiratory syndrome fatal aspergillosis in a patient with sars who was treated with corticosteroids sars: prognosis, outcome and sequelae avascular necrosis of bone in severe acute respiratory syndrome role of interferons in the treatment of severe acute respiratory syndrome inhibition of sars coronavirus infection in vitro with clinically approved antiviral drugs interferon-beta 1a and sars coronavirus replication interferon alfacon-1 plus corticosteroids in severe acute respiratory syndrome: a preliminary study pegylated interferon-alpha protects type 1 pneumocytes against sars coronavirus infection in macaques how the sars vaccine effort can learn from hivspeeding towards the future, learning from the past treatment of severe acute respiratory syndrome with convalescent plasma prior infection and passive transfer of neutralizing antibody prevent replication of severe acute respiratory syndrome coronavirus in the respiratory tract of mice an efficient method to make human monoclonal antibodies from memory b cells: potent neutralization of sars coronavirus human monoclonal antibody as prophylaxis for sars coronavirus infection in ferrets caution urged on sars vaccines a review of feline infectious peritonitis virus: molecular biology, immunopathogenesis, clinical aspects, and vaccination effects of a sars-associated coronavirus vaccine in monkeys mucosal immunisation of african green monkeys (cercopithecus aethiops) with an attenuated parainfluenza virus expressing the sars coronavirus spike protein for the prevention of sars severe acute respiratory syndrome coronavirus spike protein expressed by attenuated vaccinia virus protectively immunizes mice nucleic acid vaccines: an overview a dna vaccine induces sars coronavirus neutralization and protective immunity in mice generation and characterization of dna vaccines targeting the nucleocapsid protein of severe acute respiratory syndrome coronavirus induction of sars-nucleoproteinspecific immune response by use of dna vaccine sars-one year later seasonality of infectious diseases and severe acute respiratory syndrome-what we don't know can hurt us clinical trials and novel pathogens: lessons learned from sars preparing to prevent severe acute respiratory syndrome and other respiratory infections collateral damage: the unforeseen effects of emergency outbreak policies support from the deutsche atemwegsliga and the german research foundation (dfg gr2014/2-1) to dag is gratefully acknowledged. key: cord-315339-dcui85lw authors: broadbent, andrew j.; boonnak, kobporn; subbarao, kanta title: respiratory virus vaccines date: 2015-03-13 journal: mucosal immunology doi: 10.1016/b978-0-12-415847-4.00059-8 sha: doc_id: 315339 cord_uid: dcui85lw this chapter reviews the main viral pathogens of the respiratory tract, the immune responses they induce, currently available vaccines, and vaccines that are in development to control them. the main viruses responsible for acute respiratory infection in people include respiratory syncytial, influenza, human parainfluenza, human metapneumo-, human rhino-, corona-, and adenoviruses. licensed vaccines are available only for influenza virus, with vaccines against the other pathogens either in clinical trials or in preclinical stages of development. the majority of studies evaluating respiratory virus vaccines measure serum antibody responses, because, although both cellular and humoral responses contribute to the clearance of a primary infection, neutralizing antibodies are known to protect against secondary infection. humoral responses can be readily detected after vaccination with inactivated or subunit vaccines; however, fewer individuals seroconvert after vaccination with live vaccines. alternative immune mechanisms such as mucosal antibody responses are probably responsible for protection by live attenuated vaccines, and immune correlates of protection are under investigation. as we breathe, we sample an estimated 10 l of air per minute (kohlmeier and woodland, 2009 ). the mucosa of our respiratory system is, therefore, in direct and continual contact with the environment and, as such, is highly exposed to microorganisms, some of which may be pathogenic. respiratory infections are among the leading causes of acute illness and mortality worldwide, being responsible for nearly 4 million deaths annually, the majority of which occur in infants and children in developing countries (girard et al., 2005) . the main viruses responsible for acute respiratory infection include respiratory syncytial virus (rsv), influenza virus, human parainfluenza virus (hpiv), human metapneumovirus (hmpv), human rhinovirus (hrv), coronaviruses, and adenoviruses. however, despite the public health importance of these infections, licensed vaccines are currently available only for influenza viruses. protective immunity against respiratory virus infection is a complex interplay between systemic and mucosal responses. however, immune responses generated during a natural infection may not provide complete protection from reinfection and may actually contribute to the pathogenesis of disease (reviewed in sections pathogenesis and immune responses to respiratory virus infection). vaccine-induced immune responses must, therefore, aim to be more protective and less pathogenic than those induced naturally. in addition, our understanding of the relative contribution of mucosal and systemic immunity to protection remains incomplete. for example, it is well known that inactivated vaccines against influenza given intramuscularly (i.m.) are protective owing to the induction of systemic humoral immunity in the absence of a robust mucosal immune response, and current guidelines for vaccine licensure require influenza vaccines to induce systemic immune responses. however, it is also evident that some intranasal vaccines are protective owing to the induction of mucosal immunity, despite less impressive systemic immune responses (reviewed in section respiratory virus vaccines). unfortunately, standardized methods of measuring mucosal immune responses are lacking, and reliable correlates of protection for vaccines that protect through mucosal immunity have not been identified. in this chapter, we review the main viral pathogens of the respiratory tract, the immune responses they induce, current vaccines, and vaccines that are in development to control them. the viruses that infect the respiratory tract belong to various families and vary in their genome composition, the presence or absence of an envelope, and their replicative cycles. the majority of respiratory viruses that are responsible for acute respiratory infection belong to the paramyxoviridae family and include rsv, hpiv, and hmpv. these viruses infect cells lining the respiratory tract by first attaching to the cell through the interaction of viral envelope glycoproteins, with one or more cellular receptors in the host cell plasma membrane. for example, the hpiv envelope protein hn binds sialic acid residues extending from host cells (schomacker et al., 2012) , and the g protein of rsv and hmpv binds to glycosaminoglycans (gags) that comprise long chains of disaccharides that form part of the cellular glycocalyx (feldman et al., 1999) . the rsv and hmpv f protein are also known to bind gags, and findings indicate that the f protein of these viruses is involved in attachment by interacting with the cellular proteins nucleolin and integrin αvβ1, respectively (tayyari et al., 2011; cseke et al., 2009) . upon binding to the host cell, the f protein undergoes a conformational change that exposes a hydrophobic fusion peptide, which is responsible for the fusion of the paramyxovirus envelope and the host cell plasma membrane. after viral fusion, the genome is released into the cytoplasm, viral genes are transcribed, and viral genomes are replicated (collins and crowe, 2007; collins and melero, 2011) . the paramyxovirus genome comprises single-stranded, negative-sense, nonsegmented rna. the viral rna (vrna) must first be transcribed into positive-sense messenger rna (mrna) before viral proteins can be translated by the host cell machinery. this is achieved by a viral rna-dependent rna polymerase (the large, l, protein) that is packaged into the virion and enters the host cell upon infection. the l protein is also responsible for genome replication, in which positive-sense complementary rna (crna) serves as an intermediate template for the production of vrna. an essential cofactor for the l protein is the phospho (p) protein, which tethers the polymerase so it can reach the bases in the vrna and also binds the n protein, which encapsidates the vrna and crna. there is also evidence that transcription is enhanced by the m2-1 protein and that the switch from transcription to replication is mediated by the m2-2 protein (collins and crowe, 2007; collins and melero, 2011) . once transcribed, viral structural proteins assemble and newly synthesized viral genomes are packaged into virions that bud from the plasma membrane. the matrix, or m protein, lines the inner surface of the viral envelope and may play a role in budding (henderson et al., 2002; teng and collins, 1998) . in addition, the hn protein of hpiv is also involved in budding and in clearing sialic acid residues from the plasma membrane (karron and collins, 2007) . to complete the replication cycle, paramyxoviruses have evolved multiple mechanisms to prevent the activation of cellular defenses in response to infection, such as the nonstructural (ns) proteins 1 and 2 of rsv (collins and crowe, 2007) and the c or v proteins of hpiv (karron and collins, 2007) . one additional protein found in paramyxoviruses is the short transmembrane glycoprotein (sh) that is anchored to the envelope and shares structural features with viroporins, a group of hydrophobic molecules that insert into the membrane of infected cells and increase their permeability to small molecules and ions (gonzalez and carrasco, 2003) . the orthomyxoviridae family includes influenza viruses, which bind to terminal sialic acid-galactose linkages by the hemagglutinin (ha) envelope glycoprotein. orthomyxovirus attachment to the host cell initiates receptor-mediated endocytosis and endosome acidification. protons are permitted to enter the influenza virion via the m2 ion channel, and acidification results in a conformational change in the ha protein, revealing the fusion peptide that initiates membrane fusion between the viral envelope and the endosome membrane (reviewed by palese and shaw (2007) ). the ha is synthesized as a precursor (ha0) that is cleaved into its active form (ha1 and ha2) by cellular proteases, and the amino acid sequence at the cleavage site determines the type of protease that is able to activate the ha. if trypsin-like proteases are required for cleavage, the virus is limited in its tissue tropism to the respiratory tract of humans and the gastrointestinal tract of birds; however, the presence of multiple basic residues at the cleavage site extends the range of proteases that can cleave the ha, resulting in a disseminated, often lethal, infection in poultry (wright et al., 2007b) . once the virus envelope has fused with the endosome, the influenza genome enters the cytoplasm. the orthomyxovirus genome comprises seven or eight segments of singlestranded, negative-sense rna, and each segment encodes one or more proteins. each segment is encapsidated in nucleoprotein (np) and forms a panhandle comprising the 5′ and 3′ ends, to which a polymerase complex is attached. together, these are known as the viral ribonucleoprotein complex. in the nucleus, orthomyxovirus vrna is either transcribed into mrna or replicated by means of a positive-sense crna intermediate. viral mrna molecules exit the nucleus and are translated in the cytoplasm by the host cell machinery. structural proteins assemble at the plasma membrane, where newly synthesized viral genomes are packaged and virions bud (palese and shaw, 2007) . how the individual segments traffic to the plasma membrane and are packaged remain active areas of research. the matrix (m1) protein lines the virion beneath the envelope and may be important for morphology and viral assembly at the plasma membrane. in addition, the neuraminidase (na) protein permits budding by cleaving sialic acid residues from the host cell plasma membrane (palese and shaw, 2007) . to complete the replication cycle, influenza viruses inhibit interferon (ifn) production and signaling. this is achieved by the ns protein 1 (palese and shaw, 2007) (section adenoviruses). the genomes of coronaviruses and rhinoviruses comprise positive-sense, single-stranded rna that can be translated by the host cell machinery in the cytoplasm (kennedy et al., 2012) . coronaviruses, which belong to the coronaviridae family, are enveloped and attach to host epithelial cells by the spike (s) envelope proteins (blau and holmes, 2001) . fusion occurs at the plasma membrane, or after endocytosis, and the genome is translated into a polyprotein, which is then posttranslationally processed into structural proteins that form viral particles and nonstructural proteins that aid in viral genome replication (lai et al., 2007) . rhinoviruses, which belong to the picornaviridae family, are not enveloped and instead have a capsid of icosahedral symmetry comprising four proteins, vp1-4 (reviewed by greenberg (2011) , kennedy et al. (2012) ). the majority of rhinoviruses bind to intercellular adhesion molecule-1 (icam-1) (greve et al., 1989) , and binding leads to a conformational change in the capsid that creates a pore, through which the genome enters the cytoplasm to be translated and replicated (bella and rossmann, 2000) . adenoviruses are nonenveloped and possess a capsid of icosahedral symmetry. at each of the 12 corners, a fiber protrudes from the capsid that makes contact with the host cell receptor to initiate receptor-mediated endocytosis. acidification of the endosome results in conformational changes in the capsid that lead to viral uncoating and the release of the double-stranded dna genome into the cell. the genome is transported into the nucleus, where it is transcribed into rna, which is alternatively spliced into monocistronic mrnas that are translated by the host cell machinery into early gene products. early gene products remodel the intracellular environment to favor viral replication and are responsible for viral replication. the late phase of the viral life cycle is concerned with the production of structural proteins in sufficient quantities to ensure adequate packaging of the newly synthesized genomes and maximizing the production of progeny virions, which are released by cell lysis (berk, 2007) . respiratory viruses can infect various parts of the respiratory tract and cause a range of illness. mild upper respiratory tract (urt) infection (urti) can be complicated by sinusitis or otitis media, and a lower respiratory tract (lrt) infection (lrti) can lead to bronchiolitis or pneumonia and possible postinfectious respiratory complications such as sensitization to asthma. the major public health impact is from lrtis, and rsv is responsible for the majority of cases in infants. hmpv, hpiv, and influenza can also lead to lrti, with hpiv3 and hmpv affecting infants almost as young as those afflicted with rsv, whereas hpiv1, hpiv2, and influenza are often diagnosed in children 6 months of age or older. rsv and influenza are also recognized as an important cause of lrtis in the elderly and in those with cardiopulmonary disease or immunosuppression (schmidt, 2011) . moreover, influenza pandemics occur at irregular and unpredictable intervals with widespread morbidity and mortality and economic consequences. in addition, although there have been no cases of severe acute respiratory syndrome (sars) since 2004, several novel coronaviruses have been identified, including the virus responsible for middle east respiratory syndrome (mers) (zaki et al., 2012) . given the clinical significance of these infections, and the fact that licensed vaccines are available only for influenza viruses, there is an unmet need for vaccines. humans are the only natural host for rsv, with infections occurring in annual epidemics during winter and spring months in temperate climates and the rainy season in the tropics (girard et al., 2005) . the virus is highly contagious, with most children being infected in the first year of life. the peak of severe disease usually occurs before 6 months of age, with the peak incidence of hospitalization in 2-to 3-month-old infants (collins and melero, 2011) . reinfection is also common. in one study, among children who had been infected in their first year of life, 47% were reinfected in their second year, and 45% in their third year of life (glezen et al., 1986) . moreover, reinfection is independent of antigenic changes in the virus, implying that the protective immunity mounted during an infection does not protect against subsequent reinfection (collins and melero, 2011) . this is of note when attempting to induce protective immune responses by vaccination. globally, there were an estimated 34 million cases of lrti caused by rsv in children under 5 years of age in 2005, with 10% requiring hospitalization (martinez et al., 1997) . in the united states, one study estimated that 2.1 million children under 5 years of age require medical attention each year owing to rsv (botosso et al., 2009) , and another study estimated that rsv was responsible for 18,000-75,000 hospitalizations per year (girard et al., 2005) . in the united kingdom, the total annual incidence of hospitalization attributed to rsv was 28.3 per 1000 children under 1 year of age and 1.3 per 1000 children between 1 and 4 years of age (waris, 1991) . more than half of the hospitalizations for rsv occur in previously healthy, fullterm infants, and children who experienced severe lrti caused by rsv were at increased risk of wheezing and asthma later in life (girard et al., 2005) . pediatric mortality from rsv in the united states was estimated to be between 5.3 per 100,000 per year in infants under 1 year of age and 0.9 per 100,000 per year in children 1 to 4 years of age in one study (cooney et al., 1975) , and in another u.s. study, rsv was estimated to be responsible for 90-1900 deaths per year (girard et al., 2005) . however, an estimated 99% of rsv-associated deaths occur in developing countries, possibly because of limited health-care resources (martinez et al., 1997) . in healthy adults, reinfection rates are approximately 5-10% per year, though hospitalizations are rare. morbidity and mortality are more pronounced in the elderly and it has been estimated that rsv causes an average of 17,358 deaths annually in the united states, with 78% occurring in individuals over 65 years of age (cooney et al., 1975) . in addition, the cost of caring for patients with severe lrti from rsv and its sequelae are substantial (girard et al., 2005) . rsv infection induces antibodies against the two main antigens, the f and g envelope glycoproteins. the g protein is the most variable protein in rsv and is the basis for the separation of strains into two antigenic groups (a and b). moreover, sites of positive selection that partially coincide with epitopes recognized by anti-gprotein monoclonal antibodies (mabs) suggest immunedriven rsv evolution (botosso et al., 2009) . however, most anti-g mabs do not neutralize infectivity (martinez et al., 1997) and the selection pressure is, therefore, weak. this favors a slow coevolution of several rsv lineages, and multiple genotypes within each group can cocirculate within the same season, with shifts in the predominance of groups a and b occurring in 1-to 2-year cycles (waris, 1991) . in contrast, the sequence of the f gene is highly conserved among rsv isolates, despite the identification of a number of neutralizing mabs against the protein that should impart a selection pressure for mutation (collins and melero, 2011) . this implies that the function of the f protein confers structural restrictions that may limit antigenic diversity. human parainfluenza viruses are also a common cause of acute respiratory infection, with 80% of children seropositive by 5 years of age (cooney et al., 1975) . as with rsv, reinfection is common (schomacker et al., 2012) . there are four serotypes of hpiv (hpiv 1-4), with each serotype associated with a broad spectrum of upper and lower respiratory symptoms. hpiv1 and 2 are, however, more frequently associated with croup (laryngotracheobronchitis), and hpiv3 is more likely to cause bronchiolitis, pneumonia, and lrti resembling disease caused by rsv (schomacker et al., 2012) . hpiv4 is a less frequent cause of clinically significant disease, though a study found hpiv4 in 10% of hpiv-positive samples in a day-care setting (fairchok et al., 2011) . hpiv lrti is a major cause of hospitalization in children under 5 years of age, second only to rsv, though infection is usually self-limiting and rarely fatal, unless an individual is immunosuppressed. severe infection may have long-term effects on lung function, but this remains unclear (schomacker et al., 2012) . hmpv belongs to the same subfamily as rsv, and two major groups (a and b) and four minor subgroups (a1, a2, b1, and b2) have been identified, based on sequence variability in the g and f glycoproteins (kroll and weinberg, 2011) . as with rsv, and hpiv, by 5 years of age, most children will have been infected with hmpv, and reinfections are common (kroll and weinberg, 2011) . the virus also has a seasonal distribution, with the main occurrence in winter and spring (kahn, 2006) . hmpv typically leads to flu-like symptoms in otherwise healthy adults, but is responsible for 5-15% of hospitalizations for lrti in children and can lead to severe disease in the elderly or immunocompromised hosts (papenburg and boivin, 2010; boivin et al., 2007) . influenza viruses are divided into types a, b, and c based on antigenic differences in the np and m genes. influenza a viruses are the most clinically significant and are divided into subtypes based on antigenic differences in the ha and na genes. to date, 16 ha and 9 na subtypes have been identified from waterfowl (palese and shaw, 2007) and 17th and 18th subtypes of ha have been identified from bats in guatemala and peru (tong et al., 2012 (tong et al., , 2013 . influenza viruses cause a spectrum of clinical illness associated with infection of the upper and lower respiratory tract, with more severe disease associated with lrti. the viruses are spread by respiratory droplets or direct contact. annual influenza epidemics have a seasonal distribution, with the main occurrence in winter months (seasonal influenza) in temperate climates (girard et al., 2005) . however, unlike rsv, hpiv, and hmpv, influenza a viruses have a broad host range that includes birds, pigs, dogs, horses, marine mammals, and humans, with the main reservoir for infection being aquatic birds (palese and shaw, 2007; wright et al., 2007b) . this broad host range, together with the segmented nature of the influenza virus genome, makes the epidemiology of influenza complex and gives rise to zoonotic infections and pandemics. pandemic influenza can arise if a novel virus emerges that readily transmits from person to person and if the majority of the population is susceptible to infection. if an avian or animal virus crosses the species barrier to circulate in humans, the population will probably be immunologically naïve and, therefore, susceptible to infection. however, the virus must be able to transmit efficiently from person to person for a pandemic to occur. as the influenza virus genome is segmented, if a host is infected with two or more influenza viruses, the potential exists for the gene segments to reassort, such that a progeny virus containing genes from each parent virus can be produced (wright et al., 2007b) . if a virus that circulates within the human population reassorts with one that is novel for humans, the resultant virus may possess genes that allow it to replicate efficiently in humans, but with glycoproteins to which the population is immunologically naïve, and a pandemic could occur. the introduction of a virus with a novel ha subtype into the human population is known as "antigenic shift" (wright et al., 2007b) . three global influenza pandemics were recorded in the twentieth century from viruses of the subtypes h1n1, h2n2, and h3n2, respectively. in 2009, another pandemic h1n1 (ph1n1) influenza virus emerged in mexico (girard et al., 2010) . this h1n1 virus was antigenically unrelated to previously circulating seasonal h1n1 viruses, and molecular studies revealed that it was a reassortant with genes derived from viruses that had been circulating in pigs: the north american h3n2 triple-reassortant, the classical swine h1n1, and the eurasian "avian-like" swine h1n1 viruses. in most countries, the median age of infection during the 2009 pandemic was estimated to be 12-17 years, and in most individuals, infection led to a mild, self-limiting urti. however, 2-5% of confirmed cases in the united states and canada required hospitalization, and the case-fatality rate was 0.15-0.25%. moreover, nearly one-third of the fatalities among hospitalized patients occurred in previously healthy individuals (girard et al., 2010) . after each pandemic, the newly emerged subtype became established and caused annual seasonal influenza epidemics. in the united states, it has been estimated that 25-50 million cases of influenza occur annually, with approximately 225,000 requiring hospitalization (lambert and fauci, 2010) . current vaccines are aimed at the circulating h1n1 and h3n2 subtypes of influenza a and the predominant circulating strain of influenza b and are therefore trivalent vaccines. however, two antigenically distinct lineages of influenza b viruses (victoria and yamagata) cocirculate, and the world health organization recommended that influenza vaccines should contain both of these lineages. clinical trials of quadrivalent vaccines containing the h1n1 and h3n2 influenza a viruses and the victoria and yamagata influenza b viruses have been conducted, and their use in the united states received an interim recommendation of the advisory committee on immunization practices for the 2013-2014 influenza season (dolin, 2013) . owing to the low fidelity of the rna-dependent rna polymerase of influenza, and immune selection pressure on the ha protein, viral replication can yield a quasi-species that may differ antigenically from the parent virus. therefore, each season, the predominant circulating strain may be antigenically distinct from the previous year. this phenomenon is known as "antigenic drift" and leads to a need to update the influenza vaccine annually (wright et al., 2007b) . sporadic infections by h5n1, h9n2, h7n7, and h7n9 subtypes of influenza have been reported in humans who were in close direct contact with infected animals. additionally, h3n2 variant viruses have infected humans, the majority of whom were in close contact with pigs (epperson et al., 2013) . so far, there has been limited transmission of these viruses between people, though there is concern that they may acquire mutations or gene segments that allow efficient spread from person to person. coronaviruses are frequent causes of the common cold, causing urtis throughout the world, in all age groups, leading to millions of days of work and school absence, physician visits, and frequent inappropriate antibiotic use (greenberg, 2011) . coronaviruses are transmitted by respiratory droplets and are reported to cause 7-30% of common colds, with a peak prevalence in late fall, winter, and early spring. the first human coronaviruses (hcov) to be recognized as significant respiratory pathogens, hcov-229e and oc43, were identified in the 1960s (greenberg, 2011) . whereas infection with the majority of coronaviruses is associated with self-limiting urt symptoms in otherwise healthy individuals, a coronavirus was identified as the agent responsible for sars in 2003 (drosten et al., 2003; ksiazek et al., 2003) . the sars coronavirus (sars-cov) emerged in the guangdong province of china in november 2002 and spread to 32 countries, leading to 8096 cases and 774 deaths worldwide by the time the outbreak was brought under control in june 2003 (who, 2004) . subsequently, heightened international surveillance for coronaviruses led to the identification of the strains hcov-nl63, nh, and hku1 in 2004 (greenberg, 2011 previously, hrvs were classified into two species, hrv-a (containing 75 serotypes) and hrv-b (containing 25 serotypes). in 2009, a novel species, hrv-c, was identified, which contains at least 50 serotypes (jacobs et al., 2013) . hrvs are spread by direct contact, hand-to-hand contact or aerosols. traditionally, they have been associated with a urti, causing between 25 and 50% of common colds (makela et al., 1998) . however, they are increasingly recognized as a cause of lrti, particularly in patients with asthma and in infants, the elderly, and immunocompromised individuals (jacobs et al., 2013) . bronchiolitis is a common clinical manifestation in hospitalized children infected with hrv, and hrv is also a common pathogen in viral community-acquired pneumonia. hrv has also been associated with exacerbations of asthma and chronic obstructive pulmonary disease (jacobs et al., 2013) . as of this writing, 52 serotypes and seven species of adenovirus have been identified. tissue tropism and clinical manifestations vary between the serotypes, and the viruses are responsible for both febrile respiratory disease and gastrointestinal illness (reviewed by lynch et al. (2011) ). adenoviruses are estimated to be responsible for 5-10% of pediatric and 1-7% of adult respiratory tract infections (ison, 2006; lee et al., 2010) . they are spread primarily via respiratory droplets, direct contact, or fomites, and more than 80% of cases are in children under 4 years of age (lynch et al., 2011) . however, epidemics have also been described in children and adults, especially in military recruits in closed or crowded settings. most individuals develop a self-limiting urt infection that may be asymptomatic, but conjunctivitis, tonsillitis, otitis media, or croup can occur. infection can also spread to cause bronchiolitis or pneumonia or disseminate to cause viral meningitis or encephalitis that can be fatal (lynch et al., 2011) . there are many factors that determine the pathogenesis of disease and clinical outcome, including factors pertaining to the virus, host genetics, host immune responses, and the environment. the site of viral replication may influence the pathogenesis of disease and outcome of infection. for example, seasonal influenza viruses usually infect the epithelium of the urt, which is consistent with the most common clinical manifestations of seasonal influenza, whereas highly pathogenic avian influenza viruses of the h5n1 subtype show a stronger tropism for the lrt than for the urt (kuiken et al., 2012) . h5n1 viruses attach abundantly to "clara" or club cells lining the bronchioles, type ii pneumocytes lining the alveoli, and alveolar macrophages in the alveoli, consistent with the clinical manifestation of diffuse alveolar damage (kuiken et al., 2012) . rsv also targets both type i alveolar and nonbasilar epithelial cells and possibly alveolar macrophages, which may contribute to lrti (van drunen littelvan den hurk and watkiss, 2012). tissue tropism is determined, in part, by the receptor preference of the virus. for example, cells lining the urt of humans predominantly possess sialic acid residues with a terminal α2, 6 linkage to galactose, whereas cells lining the human lrt have both α2, 3 and α2, 6 linkages (shinya et al., 2006) . it is believed that the ability of an influenza virus ha to preferentially bind α2, 6-or α2, 3-linked sialosaccharides therefore partly determines the tissue tropism and hence the clinical outcome. when host cells are infected, type i ifn and proinflammatory cytokines are expressed, cellular translation is suppressed, and an antiviral state is induced (discussed in section immune responses to respiratory virus infection). however, most viruses that infect the respiratory tract modulate the host response to infection by blocking ifn activation and/or signaling and inhibiting apoptosis. this prevents the host from effectively clearing virally infected cells and inducing an antiviral state in neighboring cells, thereby promoting viral replication in infected tissues that may contribute to the observed pathology. rsv is the most effective paramyxovirus at subverting host cell responses, inhibiting apoptosis and type i ifn production and signaling by means of the ns1 and 2 proteins, inhibiting nuclear factor-κb (nf-κb) activation through the binding and sequestration of cellular protein kinase r (pkr) by the viral n protein, and inhibiting the production of stress granules that can restrict viral replication (collins and melero, 2011) . influenza a viruses encode the ns1 protein that downregulates ifn production (palese and shaw, 2007) , and hpiv encodes either a c protein or a v protein that suppresses ifn induction and signaling (karron and collins, 2007) , whereas sars-cov encodes eight proteins that antagonize the ifn response by a variety of mechanisms (totura and baric, 2012) . in addition to blocking ifn activation and signaling, viruses employ other means of ensuring their replication in the face of host immunity. the rsv g protein is highly glycosylated, a feature that may inhibit the binding of antibodies, and the protein is highly variable, enabling a substantial population of immune-escape mutants to be produced during infection. additionally, a truncated, soluble form of the g protein is produced during infection, which acts as a decoy antigen that can bind rsv-specific antibodies, thus reducing their availability to neutralize virus. rsv also infects antigen-presenting cells (apcs), such as dendritic cells (dcs), and can affect their maturation and antigen-presentation functions and can lead to dysregulation of adaptive immune responses (van drunen littel-van den hurk and watkiss, 2012). a number of virulence determinants have also been identified in influenza viruses, including the ha and the polymerase complex. for example, the presence of a multibasic site in the ha gene renders viruses highly pathogenic in poultry, as they are able to replicate systemically (bosch et al., 1981; kawaoka and webster, 1988) . in addition, substitution of a glutamic acid (e) residue for a lysine (k) residue at amino acid position 627 in the pb2 protein (e627k) of the polymerase is associated with altered host range (subbarao et al., 1993) and virulence in humans and in mice that are experimentally infected with avian h5n1 and h7n7 viruses (hatta et al., 2001; munster et al., 2007; subbarao et al., 1993) . in the absence of the e627k mutation, a pb2 d701n mutation is also correlated with increased virulence (li et al., 2005) , and in the absence of both the pb2 e627k and the d701n mutations, pb2 590s and 590r were found to contribute to 2009 ph1n1 influenza virus replication and virulence (mehle and doudna, 2009 ). in addition, an n66s mutation in the pb1 f2 protein has been shown to contribute to the virulence of h5n1 and 1918 pandemic h1n1 viruses (conenello et al., 2007) , and several virulence determinants have been identified in the ns1 protein, including d92e, p42s, l103f, and i106m, reviewed by kuiken et al. (2012) . there are several host factors that are known to contribute to the severity of respiratory virus disease and the outcome of infection. for example, it is known that the more severe rsv lrti in infants is associated with prematurity, chronic lung disease, congenital heart disease, and t cell immunodeficiency. other risk factors include low birth weight, multiple births, male gender, and low titers of maternally derived anti-rsv antibodies (groothuis et al., 2011; van drunen littel-van den hurk and watkiss, 2012) . additionally, low levels of vitamin d in cord-blood of healthy neonates is correlated with an increased risk of rsv lrti in the first year of life (belderbos et al., 2011) . infants are also at a greater risk of lrt disease from hpiv infection than older children; this has been attributed to smaller airways that are more susceptible to obstruction, immature immune responses, and the presence of anti-hpiv maternal antibodies that can suppress primary antibody responses (crowe and williams, 2003; karron and collins, 2007) . in adults, immunodeficiency, immunosuppression, or old age may lead to more severe illness (collins and melero, 2011) . in addition, a number of genetic polymorphisms have also been described in host genes that may affect the outcome of respiratory virus infection and disease severity. single-nucleotide polymorphisms have been identified in genes that encode surfactant proteins, such as surfactant proteins a, b, c, and d; pattern recognition receptors (prrs), such as toll-like receptors (tlrs); chemokines and cytokines, such as rantes, il-4, -5, -6, -8, -9, -10, -13, and -18, tnf-α, tgf-β, and ifn-γ; chemokine and cytokine receptors, such as ccr5, il-4ra, and il-8ra; adhesion molecules, such as icam-1, vcam-1, and e-selectin; and hla molecules such as hla-a and -b, among others (miyairi and devincenzo, 2008; poland et al., 2008) . however, few consistent results have been obtained between studies, probably a result of differences in study design, sample size, etc., in addition to true variability. clearly the contribution of genetic polymorphisms to disease outcome is complex and remains an active area of research. as well as being responsible for viral clearance and protection against reinfection, the host innate and adaptive immune responses to respiratory viruses can lead to pathology and enhanced disease. this is especially important to bear in mind in vaccine development, as vaccine-induced immune responses must protect against infection without leading to immunopathology. excessive inflammation is an important component in the pathogenesis of respiratory virus infections. upregulation of il-8 leads to the recruitment of neutrophils to the site of infection, and although these cells may participate in virus elimination, in high numbers they can also cause pathology. upregulation of il-8 is known to correlate with rsv disease severity (goetghebuer et al., 2004) and, during an influenza infection, overproduction of cytokines such as tnf-α, il-6, il-8, and type i and ii ifns and chemokines can also result in the recruitment of immune cells to the site of infection and result in damage to lung tissue (cheung et al., 2002; de jong et al., 2006) . elevated il-8/cxcl8, mip1α and β/ccl3 and 4, rantes/ccl5, and cxcl9 have also been described in children with hpiv disease, with an association of more severe hpiv disease with high concentrations of il-8 and ip-10 (reviewed by schomacker et al. (2012) ). additionally, the rsv soluble g protein can lead to leukocyte recruitment by mimicking the chemokine fractalkine (tripp et al., 2001) and this can further exacerbate inflammation. pathogenesis can also be enhanced by an insufficiency of anti-inflammatory immune responses in the lung, such as the cytokines il-10 and tgf-β (carlson et al., 2010; lebouder et al., 2009; sun et al., 2009) , or insufficient numbers of immunosuppressive resident alveolar macrophages (rygiel et al., 2009; snelgrove et al., 2008) . dysregulation of adaptive immune responses can also lead to increased pathology, and a th2-biased cellular immune response has been implicated in the immunopathogenesis of rsv disease (van drunen littel-van den hurk et al., 2007) . various defense mechanisms have evolved in the respiratory tract to prevent and control infection. currently, there is considerable effort to develop or improve vaccines against respiratory viruses. however, achieving this goal has been complicated by an incomplete knowledge of how the immune system recognizes, contains, and eliminates respiratory viruses. this section discusses the immune responses against respiratory virus infections, from the initiation of innate and adaptive responses following primary virus infection to the recall of immune responses during a secondary infection. in addition, advances in our understanding of respiratory mucosal immunity are discussed. a common feature of respiratory virus infections is that the initial infection is established in epithelial cells lining the respiratory tract. epithelial cells, as well as alveolar macrophages and dcs, are exposed to the contents of the airway lumen and detect the presence of an invading virus through prrs (holt et al., 2008) . the recognition of pathogen-associated molecular patterns (pamps) by these receptors initiates a cascade of signals that results in the production of cytokines and chemokines. the release of these inflammatory mediators into the surrounding environment establishes a local antiviral state. in addition, chemokines provide the necessary signals for the recruitment of leukocytes to the site of infection. finally, the combination of inflammatory cytokines and prrs initiates the process of dc maturation and trafficking that is required for the induction of adaptive immune responses (holt et al., 2008) . the best described of the prrs are those of the tlr family. with respect to respiratory viruses, tlr3, 7, and 9 recognize various products of viral replication (doublestranded rna, single-stranded rna, and unmethylated cpg dna, respectively) (alexopoulou et al., 2001; diebold et al., 2004; hagglund et al., 2004; lund et al., 2003) , and tlr4 recognizes the f protein of rsv (kurtjones et al., 2000) . tlrs that recognize nucleic acids are located in late endosomes. this location optimizes the ability of tlrs to interact with viral nucleic acids while limiting their access to host-derived nucleic acids (heil et al., 2003; matsumoto et al., 2003) . although tlrs expressed on the cell surface or within the cell utilize different signaling pathways, each of these receptors can activate the transcription of ifninducing genes . viral rna is also recognized by cytoplasmic sensors such as rna helicases. the retinoic acid-inducible gene i protein interacts with 5′-triphosphate rna and is important for early cytokine production in response to numerous rna viruses (hornung et al., 2006; pichlmair et al., 2006; yoneyama et al., 2004; kato et al., 2005; pothlichet et al., 2013; graham et al., 2013) . the melanoma differentiationassociated gene 5 protein is a related helicase that recognizes polyinosinic polycytidylic acid and is crucial for innate recognition for picornaviruses and human metapneumovirus infection (banos-lara mdel et al., 2013) . similar to signaling through tlrs, the pathways utilized by rna helicases ultimately trigger ifn-regulatory factor and nf-κb activation (le goffic et al., 2007) . the key difference between these molecules and tlrs is that the rna helicases are localized throughout the cytosol, rather than being restricted to intracellular compartments. thus, pathogens such as paramyxoviruses that do not enter endosomes can trigger innate immune responses via rna helicases. the innate recognition of viral components through prrs described above leads to a program of gene expression that promotes a localized antiviral state and elicits the recruitment of inflammatory cells to the site of infection. type i interferons, including ifn-α and ifn-β, are most commonly associated with early antiviral responses in the lung, and numerous studies in mice and humans have shown that plasmacytoid dcs (pdc's) are the primary source of these cytokines after infection with viruses (asselin-paturel et al., 2001; mcgill et al., 2009) . however, there is a level of redundancy with respect to ifn production, with alveolar macrophages or pdcs predominating depending on the type of viral infection (pribul et al., 2008) . the precise contribution of pdcs to lung antiviral immunity is also controversial; several in vitro studies show that respiratory viruses, including influenza viruses, can infect pdcs and pdcs can activate virus-specific cd4 + t cells (wikstrom and stumbles, 2007; geurtsvankessel and lambrecht, 2008) , but evidence for a major role of pdcs in controlling influenza virus in vivo is absent. type i ifns produced after respiratory virus infection act in concert with prr signaling to form a feedback loop, by signaling through the ifn-α/β receptor to promote sustained production of proinflammatory cytokines such as tnf-α, il-1, and il-6 by lung-resident innate immune cells (chan et al., 2005; nakajima et al., 2013; almansa et al., 2012) . these proinflammatory cytokines and prrmediated signals also prompt alveolar macrophages, dcs, and epithelial cells to initiate a coordinated program of chemokine production after viral infection. for example, dcs secrete successive waves of chemokines after influenza virus infection, beginning with those capable of recruiting inflammatory cells such as neutrophils and natural killer (nk) cells to the lung, followed by chemokines associated with the recruitment of monocytes and t cells (piqueras et al., 2006; marois et al., 2012; teijaro et al., 2010) . the cytokines il-1β, il-18, and il-33 activate monocytes, macrophages, and neutrophils and drive the development of cd4 + t cell adaptive responses in both mice and humans. cd4 + t cell differentiation in the presence of il-1β, il-18, or il-33 results in th17, th1, or th2 effector cells, respectively (chung et al., 2009; dinarello, 1999; lasiglie et al., 2011; ohno et al., 2009 ). these cytokines are processed as a result of caspase-1 activation, and activation of caspase-1 is regulated by the inflammasome, a large mutimeric structure (martinon et al., 2002) . a subgroup of the nucleotide-binding domain, leucine-rich repeat-containing proteins (nlrps) are key mediators of the inflammasome. activation of the inflammasome can be divided into two categories: activation driven by host-and environmentderived molecules and activation driven by pathogen-associated activators, including pamps derived from bacteria, virus, fungus, and protozoa. paramyxoviruses such as rsv and orthomyxoviruses such as influenza viruses can activate the nlrp3 inflammasome (kanneganti et al., 2006; segovia et al., 2012; komune et al., 2011) , which has been shown to play a critical antiviral role in influenza virus-infected mice (thomas et al., 2009; ichinohe et al., 2009; allen et al., 2009 ). after infection of the lrt, antigen-bearing mature dcs enter the lymph nodes draining the lung, where they form stable interactions with naïve t cells specific for that antigen through t cell receptors (tcrs). signals delivered by antigen recognition, in addition to accessory signals delivered through costimulatory molecules, result in t cell priming and the clonal expansion of antigen-specific effector t cells (moon et al., 2007; obar et al., 2008) . the instructions delivered by dcs during the initial expansion phase can have a dramatic impact on the survival and function of the responding t cells. for instance, expression of fasl on dcs after influenza infection has been shown to regulate the magnitude of the cd8 + t cell response (legge and braciale, 2005) . resident cd8α + conventional dcs in the mediastinal lymph node also mediate the induction of protective immunity to influenza virus, and these cells have been found to cross-present viral antigens to cd8 + t cells without being directly infected by the virus (belz et al., 2004) . after activation and clonal expansion of antigen-specific effector t cells in the draining lymph nodes, the cells lose their preference for the lymphoid tissue and migrate via the bloodstream to the site of infection, where the antiviral mechanisms described below are exerted. analysis of chemokine expression in the lung during the adaptive phase of the immune response has shown elevated expression of numerous molecules associated with effector t cell trafficking (monick et al., 2007) . for instance, the trafficking of effector t cells during rsv infection is partially dependent on cxcr1, and this chemokine receptor may play a role in other paramyxovirus infections (harcourt et al., 2006) . the appearance of antigen-specific effector t cells at the site of virus infection is first observed around 6-7 days after infection with influenza and parainfluenza viruses in mice (pommerenke et al., 2012; kohlmeier and woodland, 2009; lawrence and braciale, 2004; roman et al., 2002) . the continual migration of effector t cells from lymphoid tissues during acute infection results in a massive increase in the number of antigen-specific cells in the lung airways and parenchyma from 7 to 10 days after influenza virus infection (flynn et al., 1998) , and the arrival of effector t cells has an immediate and dramatic impact on the viral load (kohlmeier et al., 2010) . the adaptive immune responses induced after a respiratory virus infection are shown in figures 1-3 . upon arrival at the effector site (figure 1 ), antigen-specific t cells first interact with apcs such as dcs (shen et al., 2010) . moreover, a subset of dcs, classed as cd11c hi , present a crucial t cell survival factor (il-15) to antiviral cd8 + t cells in trans (mcgill et al., 2010) . immune responses are determined by the cytokine milieu in the respiratory tract, as well as the type and level of costimulatory molecules expressed by apcs. for instance, lung dcs are biased to promote th2 responses, most likely via the production of il-6 in the absence of the th1-prone il12p70 cytokine or through the production of leukotriene ltc4 (dodge et al., 2003; barrett et al., 2011) . effector t cells employ one of the following three antiviral mechanisms ( figure 2 ). first, t cells can promote the lysis of infected cells by exocytosis of granules containing perforin and granzyme (trapani and smyth, 2002; hou and doherty, 1995) . second, t cells can induce apoptosis of infected cells by expressing cd95 (fas) ligand (fasl) hou and doherty, 1995) or tnf-related apoptosis-inducing ligand (trail) (brincks et al., 2011) . third, t cells can produce proinflammatory and regulatory mediators, such as ifn-γ, after an encounter with virally infected cells (hamada et al., 2013) . several studies suggest a crucial role for the cytolytic functions of cd8 effector t cells in influenza virus infection. the direct lysis of infected cells requires tcr-mediated recognition of processed viral antigens on the infected target cell (brincks et al., 2008; topham and doherty, 1997) . in contrast, the release of proinflammatory mediators such as ifn-γ by cd8 + t cells has only a modest impact on virus clearance and recovery. there is also evidence from models of influenza infection that infected alveolar epithelial cells may be eliminated by the host response through the action of macrophages capable of triggering apoptosis through a trail-dependent mechanism (herold et al., 2008) . effector cd4 + t cells have also been found to exhibit cytotoxic activity in vitro, but the contribution of this mechanism to virus clearance in vivo is modest (agrewala et al., 2007; graham et al., 1994) and is restricted to the cytolysis of cells that bear viral antigens presented by major histocompatibility complex (mhc) class ii molecules. such cells include cd45 + mononuclear phagocytic cells, and a few cd45 − lung parenchymal cells, such as type ii alveolar epithelial cells, that express mhc class ii molecules in either a constitutive or an inducible manner (debbabi et al., 2005) . the primary role of antiviral cd4 + t cells is to support the activation and differentiation of b cells, which leads to antibody production (topham et al., 1996; topham and doherty, 1998) as discussed below. another aspect of effector t cells is their potential to exert cytotoxic activity and simultaneously produce cytokines such as ifn-γ. for example, during an influenza infection, the interaction of primed cd8 + t cells with lung dcs elicits both cytokine production and cytotoxic phenotypes (hufford et al., 2011) . to summarize the cellular immune responses during a primary influenza infection, specific cd4 + and cd8 + effector t cells in the lung predominantly produce ifn-γ and figure 1 upon virus infection, viral antigen from dying respiratory epithelial cells is transported from the lung to the lymph node by migratory antigen-presenting cells such as mouse cd103 + dendritic cells (dcs) or human monocyte-derived dcs that can stimulate both cd4 + and cd8 + t cell responses. viral antigen can also be transferred to lymph node-resident cd8α + dcs and presented to naïve cd8 + t cells. in addition, plasmacytoid dendritic cells (pdcs) are an early source of antiviral type i interferon via recognition of viral rna. tnf-α, and cd4 + effector t cells also produce il-2 and il-10 (carding et al., 1993; pipeling et al., 2008; mayer et al., 2005) . cd8 + effector t cells localize to the respiratory epithelium and induce apoptosis of infected epithelial cells through fas-fasl interactions or the exocytosis of cytolytic granules containing perforin and granzyme tripp et al., 1995) . b cells are found interspersed in the lung interstitium and in the cervical and mediastinal/bronchial lymph nodes that drain the upper and lower respiratory tract, respectively. during a respiratory virus infection, a tertiary lymphoid structure also forms along the branching point of the bronchial tree, called the bronchus-associated lymphoid tissue (balt) (brandtzaeg, 2010) . the balt contains organized b cell areas, germinal centers, and antibody-forming cells (randall, 2010) . b cell responses can be classified into three categories: innate-like b cell responses, t-dependent b cell responses, and t-independent b cell responses. innate-like b cell responses consist of antibodies produced almost exclusively from b-1 cells, a small subset of b cells characterized by a unique developmental origin, phenotype, tissue distribution, and regulation, compared with conventional b cells (baumgarth et al., 1999; baumgarth, 2011) . t-dependent b cell responses are b cell responses that are facilitated by cd4 t cells, whereas t-independent b cell responses are not. it has been shown that cd4 t cell deficiency results in a drastically reduced humoral response to influenza virus infection in mice (mozdzanowska et al., 2005) . however, mice lacking cd4 and cd8 t cells are still protected from lethal infection mozdzanowska et al., 2005) , highlighting the importance of t-independent b cell responses. b cell responses are critical for viral clearance in primary respiratory virus infections, such as influenza (gerhard, 2001) . although control of early infection (3-6 days postinfection) is not impaired in b-cell-deficient mice, the mice fail to clear the virus and ultimately succumb to infection (graham and braciale, 1997; lee et al., 2005) . studies in influenza virusinfected mice also show that serum antibody titers are first detected around 6-7 days postinfection, at least 3 days later than responses are detected in the respiratory tract. they steadily increase for about a month, after which relatively high figure 2 during the effector phase of the response, influenza-specific cd8 + t cells elicit a cytotoxic response. tnf-α and nitric oxide (no) produced from a subset of dcs and neutrophils contribute to both viral clearance and immunopathology. lung cd11c hi dcs present the crucial t cell survival factor il-15 to antiviral cd8 + t cells in trans and promote the production of chemokines. in draining lymph nodes, cd11b + dcs contribute to the expansion of specific cd8 + t cells, and conventional dcs (cdcs) induce the th2 response. plasmablasts initially produce igm, and effector cd4 + t cells provide help for virus-specific b cell/plasmablast differentiation and proliferation. pdcs also play a detrimental role by eliminating virus-specific cd8 t cells in a process involving fasl. antibody titers are maintained for life. virus-specific antibodyforming cells reside transiently in the spleen, from around 6 or 7 days postinfection, and persist for the long term in the bone marrow (jones and ada, 1987; hyland et al., 1994) . after recovery from an infection (figure 3 ), a state of immunological "memory" ensues, in which the individual is better able to control a subsequent infection with the same pathogen (ahmed and gray, 1996) . immunological memory is maintained by both t and b cell subsets, and there are profound differences in the generation, trafficking, and maintenance of t and b cell memory. antigen-specific memory t cells persist at increased frequencies, have a reduced requirement for costimulatory signals in comparison to naïve t cells, and respond quickly to antigenic restimulation (woodland et al., 2002) . in the case of influenza and parainfluenza (piv) virus infections, it has been clearly established that both cd4 + and cd8 + memory t cell subsets respond to and control secondary infection (woodland and dutton, 2003) . b cell memory is characterized by two distinct populations: long-lived plasma cells that continually secrete antibody and memory b cells that persist in a quiescent state (bachmann et al., 1994; slifka and ahmed, 1998) . the generation of long-lived plasma cells is dependent on cognate t-b cell interaction and cd40 signaling that occurs in the germinal center (noelle et al., 1992; lee et al., 2003) . antigen-specific igg and iga antibodies are maintained long after infection and may be protective against heterologous strains of virus. for example, up to 96% of people born between 1909 and 1919 in finland had preexisting antibodies to the 2009 ph1n1 influenza virus, probably because of its relationship to the 1918 h1n1 pandemic influenza virus that circulated in the first part of the twentieth century (ikonen et al., 2010; yu et al., 2008) . the presence of crossreactive antibodies contributed to the unexpectedly low numbers of the elderly with severe illness during the 2009 pandemic, compared with seasonal influenza virus strains (monsalvo et al., 2011; o'donnell et al., 2012) . although neutralizing antibodies directed against the ha globular head are highly efficient at preventing and clearing influenza virus infection, they can also figure 3 in the memory phase, migratory lung dcs capture viral antigen retained on follicular dcs (fdcs) in tertiary lymphoid organs and present it to specific t cells in the respiratory draining lymph nodes. these stimulated t cells upregulate the expression of cd69, causing them to be retained in the lymph nodes that drain the site of primary infection. resident dcs are able to reactivate memory responses in the lymph nodes. lung dcs can promote the production of iga in a process that depends on tgf-β. the generation of b cell memory, particularly the generation of long-lived plasma cells, is dependent on cognate t-b cell interaction and cd40 signaling that occurs in the germinal center. provide a selective pressure for viral immune evasion. crossprotection against various influenza a subtypes, termed heterosubtypic immunity, requires the immune system to recognize epitopes that are conserved between subtypes. such epitopes can be found in the membrane-proximal stalk region of ha (han and marasco, 2011) or in internal proteins such as nucleoprotein or the m protein. anti-stalk antibodies do not inhibit virion binding to mammalian host cells, but inhibit fusion between the viral envelope and the endosomal membrane. they have broadly neutralizing activity and passively protect mice from lethal challenge in vivo (okuno et al., 1994; throsby et al., 2008; sui et al., 2009 ). interestingly, cross-reactive anti-ha stalk monoclonal antibodies have been generated from the acute response to 2009 h1n1 pandemic virus and also from healthy subjects vaccinated with inactivated virus (corti et al., 2011; sui et al., 2009; wrammert et al., 2008) . how to induce high-titers of anti-ha stalk antibodies in humans remains an active area of universal influenza vaccine research. cellular immune responses to cross-reactive epitopes (often expressed on internal viral proteins) also provide a substantial degree of protection against serologically distinct viruses (yewdell et al., 1985; rimmelzwaan and osterhaus, 1995) , and although these heterosubtypic cellular responses are not able to prevent reinfection, they can ameliorate disease by reducing the maximal viral load, mediating faster viral clearance, and providing a substantial degree of protection against challenge with a lethal dose of virus in animal models (hillaire et al., 2011) . there is also some epidemiological evidence that heterosubtypic cellular immunity plays a role in the response to infection with novel influenza viruses in humans; however, the protective effect appears to be weak and may wane over time (epstein, 2006; epstein and price, 2010) . it has, therefore, been suggested that protective cellular immunity could be sustained by reinfection or annual immunization. the effector mechanisms of heterosubtypic immunity remain ambiguous . in murine models of influenza a virus infection, heterosubtypic immunity is observed in the absence of antibodies that recognize influenza envelope glycoproteins and is thought to be mediated primarily by cd8 + t cells, with a relatively small contribution by cd4 + t cells mckinstry et al., 2012) . heterotypic influenza-specific cd8 + t cells have also been shown to lyse influenza virus-infected cells (nguyen et al., 1998) . however, heterosubtypic immunity has been observed in cd8 + t-cell-deficient mice, but not in mice lacking b cells , indicating that there is redundancy in the system. the same investigators found that the heterosubtypic immunity does not require ifn-γ (nguyen et al., 2000) , but does require a properly diversified antibody repertoire (nguyen et al., 2007) . the mucosal immune system has been described in detail elsewhere in this book; however, there are some features unique to the respiratory tract that are worth noting. like the immune system in general, the mucosal immune system of the respiratory tract uses innate and specific mechanisms to prevent and limit infection. innate defenses include physical and chemical factors such as the secretion of mucus, which traps microorganisms and antigens and facilitates their transport out of the body by mucociliary motion. mucosal secretions also contain chemically active substances, including acids, lactoferrin, and lysozyme, which inhibit the growth of microbes. in addition, the luminal side of the respiratory tract is physically protected by layers of epithelial cells that adhere to each other at tight junctions, using occludin and various members of the claudin family, and at adherent junctions using e-cadherin (tsukita et al., 2008) . in humans, the respiratory tract can be divided anatomically into the urt, which comprises the nose, mouth, and pharynx, and the lrt, which includes the trachea, bronchi, and lungs, with the lymphoid tissue of waldeyer's ring representing the line of demarcation. the unpaired nasopharyngeal tonsils (also called the adenoids) and the palatine and lingual tonsils constitute most of waldeyer's ring, with the tubal tonsils and lateral pharyngeal bands as less prominent components (dolen et al., 1990) . this lymphoid tissue is functionally comparable to the nasal or nose-associated lymphoid tissue (nalt) in rodents, which is composed of two paired lymphoepithelial structures beside the nasopharyngeal duct, dorsal to the cartilaginous soft palate (kuper et al., 1992; fukuyama et al., 2002) . however, rodents do not have tonsils. waldeyer's ring is more strategically situated than the nalt to generate mucosal immunity, because its elements are exposed to both airborne and alimentary antigens. in addition, human tonsils have deep antigen-retaining crypts, and tonsils express germinal centers shortly after birth, whereas the rodent nalt has a plain surface and requires an external stimulus to induce the expression of germinal centers (brandtzaeg, 2010) . tissue equivalent to waldeyer's ring has also been found in nonhuman primates (loo and chin, 1974; harkema et al., 1987) and horses (mair et al., 1987 (mair et al., , 1988 , but functional studies have not been performed in these species. the mucosa-associated lymphoid tissues (malt), including the nalt and balt, consist of follicle-associated epithelium and t-cell-and b-cell-enriched areas. the initiation of antigen-specific immune responses occurs at special gateways, which comprise microfold (m) cells located in the epithelium overlying the malt follicles. the cilia of the apical side of the m cells are shorter than those of conventional epithelial cells, and on the basal side, there is a large pocket-like structure that can hold immunocompetent cells required for the generation of immune responses such as t cells, b cells, and apcs. as lysosome development in m cells is poor, in most cases antigens pass through the cells unmodified and are taken up by dcs in the pocket (sato and kiyono, 2012) . upon encountering antigen, dcs migrate to the t cell region of the malt and present peptide antigen via mhc molecules to naïve t cells. antigen-specific t cells become primed, clonally expand, and leave the follicle to enter the circulation. they then home to effector sites to elicit mechanisms involved in viral clearance as described above. in the b cell region, a germinal center forms and antibody class switching occurs (cerutti, 2008) . a class switch to iga predominantly occurs in the malt owing to the action of the iga-associated cytokine family of tgf-β, il-2, il-4, il-5, il-6, and il-10 (mcghee et al., 1989; mestecky and mcghee, 1987; cerutti, 2008) . postswitched iga + b cells leave the malt through efferent lymph vessels under the control of the lipid mediator, sphingosine-1-phosphate, and the cells then enter the circulatory system (gohda et al., 2008; lazarus et al., 2003) and home to effector sites found in unorganized lymphatic tissue spread over the lamina propria that underlies the mucosal epithelium. in the lamina propria, b cells differentiate into plasma cells and secrete iga, igd, igm, and igg antibodies, although iga is the major mucosal antibody isotype (shikina et al., 2004; shimoda et al., 2001) . surgical removal of the murine nalt or cervical lymph nodes does not, however, abrogate cellular or antibody immune responses to experimental influenza infection, suggesting that there may be additional inductive sites other than the nalt and demonstrating that dissecting the relative contributions of anti-influenza immunity is difficult. in the urt, iga is the major mediator of immunity to influenza. in mice that had recovered from an influenza infection, immunity to reinfection was abrogated by the intranasal instillation of anti-iga antibodies, but not anti-igg or igm (renegar and small, 1991a) , and intravenous passive transfer of iga resulted in iga in nasal secretions that protected mice from intranasal challenge with influenza (renegar and small, 1991b) . after passive transfer, nasal iga titers that conferred protection were at a concentration equivalent to that seen in convalescent mice, whereas igg transudation into the urt could be detected only after 2.5 times the normal convalescent serum titer had been passively transferred (renegar et al., 2004) . moreover, recombinant iga is sufficient to prevent influenza transmission in a guinea pig model (seibert et al., 2013) . in humans, iga is present in monomeric and dimeric forms. during transcytosis through mucosal epithelial cells, an extra polypeptide secretory component is added to dimeric iga and the resulting molecule is known as secretory iga (s-iga). dimeric and s-iga are 7-10 times more efficient than monomeric iga at neutralizing influenza viruses (renegar et al., 1998) . dimeric and s-iga are represented by two subclasses, iga1 and iga2, with covalently or noncovalently joined dimers, respectively. both subclasses are detected in nasal secretions after an influenza infection; however, ha preferentially stimulates an iga1 response (brown et al., 1985) . s-iga is not considered to be inflammatory because the fc region is not available to activate immune cells or bind complement. s-iga is also resistant to proteolysis and can neutralize viruses inside epithelial cells and transport viruses that have passed the epithelial barrier to the lamina propria back to the lumen (sato and kiyono, 2012) . s-iga may therefore be useful in preventing viruses from breaching the mucosal barrier, while avoiding immunopathology by not activating inflammatory responses directly and by limiting the number of antigen-antibody complexes in the lamina propria that can trigger inflammation. it is currently thought that plasma igg serves as a backup for s-iga in the urt, whereas in the lrt, igg is the dominant antibody involved in protection (renegar et al., 2004) . the fc receptor for igg mediates transport of igg across epithelial barriers by transcytosis, permitting the transudation of igg from the serum into the lung where it is able to neutralize viruses (spiekermann et al., 2002) . this explains why passively transferred igg is effective at preventing severe disease from respiratory infections in experimental animals and why serum igg antibodies are the main correlate of protection for parentally administered inactivated influenza vaccines in humans (section respiratory virus vaccines). viruses that can cause repeated infection are typically characterized either by a failure to induce robust immunity or by significant antigenic diversity in the face of protective immune responses. influenza viruses can reinfect hosts because the antigenic sites evolve and drift to avoid neutralization by prior immunity. infection induces a strong homosubtypic neutralizing antibody response in healthy individuals that contributes to recovery and protection from repeat influenza virus infection with homologous virus or an antigenically similar virus (wrammert et al., 2008) . moreover, natural infection can lead to long-lasting immunity to the infecting virus. for example, when the influenza a h1n1 subtype reemerged in 1977, the most susceptible members of the population were those born after the time when similar h1n1 viruses had previously circulated, the 1950s (shortridge et al., 1979) . however, because influenza viruses undergo antigenic drift and shift, the effective period of protection may last only until an antigenic variant emerges. in contrast to the robust strain-specific protection after influenza virus infection, primary infection with rsv, hpiv, and hmpv provides only partial protection from reinfection. rsv commonly reinfects the host even though genetic diversity in the virus is not extreme. in healthy adults challenged every few months with the same strain of rsv, about 25% were infected each time and about half of those became symptomatic (hall et al., 1991) . these studies are now being reproduced with experimental human challenge infection (devincenzo et al., 2010) , and access to modern immunological techniques may provide some insight into the mechanism of immune evasion. most reinfections are limited to the upper respiratory tract, unless subjects are immunocompromised. reinfections may be the consequence of a highly prevalent and contagious virus, effective evasion of local and innate immunity, or a steep gradient for transudation of antibody from the serum to the nasal epithelium (graham, 2011) . alternatively, rsv infection may alter the characteristics of the adaptive immune effectors and memory. although rsv infection provides a sufficient antigenic stimulus to induce both antibody (shinoff et al., 2008) and t cell responses (heidema et al., 2008) , the durability of the antibody is poor (handforth et al., 2000; dakhama et al., 1997; collarini et al., 2009 ). the most efficient means of preventing respiratory virus infections is vaccination. however, among respiratory viruses, licensed vaccines are available only for influenza. it seems logical to consider live attenuated vaccines delivered intranasally for protection against respiratory viruses, as they would induce a mucosal immune response. however, a systemic immune response can be protective if it is sufficiently robust, such as that induced by inactivated influenza vaccines administered by the i.m. route. moreover, achieving an appropriate balance between sufficient attenuation and immunogenicity, especially in young infants who must be vaccinated in the face of maternal antibody, is a challenge. this section describes licensed vaccines as well as vaccines that are currently in development. vaccination remains the primary strategy for the prevention and control of influenza (lambert and fauci, 2010) . as described in section immune responses to respiratory virus infection, after an influenza infection, both cellmediated immunity and systemic and mucosal neutralizing antibodies are produced. whereas cell-mediated immunity contributes significantly to the clearance of a primary influenza virus infection, and can ameliorate disease caused by reinfection, neutralizing antibodies play an important role in preventing reinfection. the goal of vaccination is to prime the immune response to limit viral replication upon subsequent infection, and inactivated, recombinant hemagglutinin and live attenuated influenza vaccines (laivs) are licensed for use, with novel vaccines in varying stages of development. this section focuses on licensed vaccines and the immune responses they elicit, as determined from clinical trial data. owing to antigenic drift in circulating viruses (discussed in section virology), an influenza vaccine from one season may not be effective in subsequent seasons. each year, the strains that are to be included in the vaccine for the next influenza season are chosen and vaccine seed viruses are generated (lambert and fauci, 2010) . for inactivated vaccines, the influenza a seed viruses are reassortant viruses, with the ha and na gene segments derived from the circulating virus and the internal protein genes derived from a vaccine donor strain that is adapted for high yield in eggs (a/puerto rico/8/34; pr8) (kilbourne, 1969) . licensed laivs also contain the ha and na from the circulating virus, combined with the internal protein genes from temperature-sensitive, cold-adapted, attenuated master donor viruses that limit replication of the vaccine viruses to the cooler upper respiratory tract (maassab, 1967) . if the yield of the vaccine virus in eggs is poor, they may be "egg-adapted" through serial passage. vaccine viruses are then amplified in hundreds of millions of eggs and purified. the inactivated vaccine viruses are treated with formalin or β-propriolactone and "split" with detergents before being formulated for clinical use, with or without thimerosal as a preservative (fiore et al., 2013) . until 2013, seasonal influenza vaccines were trivalent, containing two subtypes of influenza a viruses (h1n1 and h3n2) and one influenza b virus. however, from the 2013 winter season in the northern hemisphere, quadrivalent vaccines, containing two subtypes of influenza a viruses and two strains of influenza b viruses, have become available. it takes several months from the generation of a seed virus to the manufacture and distribution of a vaccine. typically, for seasonal influenza in the northern hemisphere, manufacturers amplify vaccine viruses and inactivate or purify them between february and late summer and formulate and distribute them for administration in the fall before the anticipated peak of the influenza season (lambert and fauci, 2010) . in 2009, the h1n1 pandemic virus emerged in april, when the manufacture of seasonal trivalent vaccines was already under way. a monovalent h1n1 vaccine was produced as quickly as possible in addition to the seasonal vaccines, but the monovalent vaccine was not available for widespread use until after the pandemic had peaked in the northern hemisphere and was not available at all during the 2009 winter season in the southern hemisphere (broadbent and subbarao, 2011; skowronski et al., 2011) . in addition, current global vaccine production capacity does not cover the high-risk population around the world (girard et al., 2005) . one means of increasing capacity is to move toward using cell culture instead of eggs for vaccine production, and several companies are investigating this. additionally, the amount of ha in each vaccine dose could be reduced and used with an adjuvant such as mf59 or as03. however, adjuvanted seasonal influenza vaccines are not yet licensed in the united states. trivalent inactivated vaccines (tivs) against seasonal influenza are administered i.m. and have an efficacy ranging from 60 to 100% in preventing influenza morbidity and mortality in healthy adolescents and adults (osterholm et al., 2012) . a trivalent laiv consisting of an a or b ann arbor cold-adapted backbone together with the ha and na of the target viruses (flumist ® , medimmune), administered by a nasal spray, is licensed in several countries, including north america and europe. in the united states, the seasonal laiv is licensed for healthy individuals between 2 and 49 years of age, but it is currently not approved for use in children under 2 years of age, and it is not approved for use in the elderly or in immunocompromised individuals (ambrose et al., 2011) . there is a substantial body of evidence from largescale randomized clinical trials demonstrating the effectiveness of laivs, and a number of clinical studies have also shown that the efficacy of laivs is equivalent or superior to that induced by i.m. vaccination with tivs in children (reviewed by luke et al. (2013) ). in adults, however, the majority of double-blind, randomized, placebo-controlled trials have shown tivs to have a greater efficacy than laivs monto et al., 2009; ohmit et al., 2006 ohmit et al., , 2008 . in the military, vaccine effectiveness was also found to be higher for tivs than for laivs, except in new recruits (eick-cost et al., 2012; wang et al., 2009 ). tivs primarily induce serum antibodies against the influenza ha glycoprotein, which are typically measured by hemagglutinin inhibition (hi) assays, which serve as a surrogate for virus-neutralization assays. in healthy individuals who are immunologically primed by previous infection or vaccination, influenza-specific antibodysecreting cells in the peripheral blood peak 1 week after vaccination and serum antibody levels peak 2 to 4 weeks postvaccination. however, in unprimed individuals, for example, children, it may take 4 weeks or longer for serum antibody levels to peak after vaccination (brokstad et al., 1995; cox et al., 1994; el-madhun et al., 1998) . the rise in serum antibody titer after tiv administration has been documented for multiple isotypes, including igm, iga, and igg, with a more pronounced rise in igg titers (moldoveanu et al., 1995) . in contrast, vaccination with an laiv leads to seroconversion more frequently in immunologically naïve individuals than in those who are immunologically primed. for example, in children, after a single dose of trivalent laiv, seroconversion rates of 16-58%, 92-100%, and 88-100% have been reported against influenza a h1n1, h3n2, and b, respectively, which increased to 77% and 61% for h1n1 after a second dose at day 28 or 60, respectively (belshe et al., 1998; lee et al., 2004) . however, in healthy adults, serum antibody titers are lower after laiv than tiv vaccination (moldoveanu et al., 1995) , and in one study only 59% of laiv recipients had an increase in serum igg titer compared to 94% of tiv recipients . protection mediated by inactivated vaccines therefore correlates with serum neutralizing antibody titers, whereas other immune mechanisms contribute to protection mediated by laiv. a higher percentage of laiv recipients have mucosal antibodies and antibody-secreting cells than those receiving tiv. one study recorded that 83% of laiv recipients had increased influenza-specific iga in the mucosa, compared to only 38% of tiv recipients . moreover, levels of iga in nasal wash specimens correlated with protection against challenge with wild-type influenza viruses . mucosal iga in adults vaccinated with laiv declined 6 months after vaccination . in addition to iga, igg has also been found in nasal secretions after laiv (moldoveanu et al., 1995) . as described (section immune responses to respiratory virus infection), iga is the major mediator of immunity to influenza infection in the urt, with igg serving as a backup. this also appears to be the case after vaccination with laiv. the majority of antibodies induced by influenza vaccines that are associated with protection are directed against the globular head of the ha. recently, neutralizing antibodies have also been identified that bind to a conserved epitope in the ha stem. the ha stem antibodies have been found to be broadly neutralizing, and there is much effort to generate vaccines that elicit these antibodies to produce a more broadly cross-protective vaccine (corti et al., 2010; ekiert et al., 2009; kashyap et al., 2008; okuno et al., 1993; sui et al., 2009 ). in addition, antibodies directed against the na protein are also generated after vaccination with both tiv and laiv (murphy et al., 1972) . anti-na antibodies restrict virus release from infected cells and reduce the severity of disease by limiting spread (murphy et al., 1972) . however, currently licensed inactivated vaccines are standardized to ha, but not na protein content. the amount of na protein varies from vaccine to vaccine, and the contribution of vaccine-induced anti-na antibodies to protection against influenza is not well understood (hassantoufighi et al., 2010) . in addition to humoral immunity, influenza-specific cd8 + cytotoxic t lymphocytes (ctls) are associated with accelerated clearance of virus and recovery from infection. however, the extent to which the cellular immune response is protective against infection is unknown because the recall response is likely to occur after the peak of viral replication (subbarao et al., 2006) , and cell-mediated immunity induced after vaccination has been less well studied than the humoral response, and the results are variable. studies have shown that immunization of healthy adults with whole-virus inactivated vaccine resulted in enhanced ctl responses in peripheral blood, whereas immunization with a subunit vaccine resulted in poor ctl responses, the duration of which varied from several months to years (ennis et al., 1977; mcmichael et al., 1981; powers and belshe, 1993) . in addition, an increase in ifn-γ-producing t cells was seen in children ages 6 months to 9 years of age who were vaccinated with inactivated vaccine; however, similar responses were not induced in adults (he et al., 2008) . h5n1-specific cd4 + t cells were also detected after a single dose of as03-adjuvanted inactivated vaccine and was amplified by a second dose of vaccine (moris et al., 2011) . in addition, numerous studies have found a significant increase in ifn-γ-producing cd4 + and cd8 + t cells after vaccination with laiv in both adults and children (basha et al., 2011; forrest et al., 2008; he et al., 2006a; hoft et al., 2011; lanthier et al., 2011) ; however, the role of cellular immune responses in laiv-mediated protection needs further investigation. more than 90% of annual influenza-related deaths in the united states occur in individuals 65 years of age or older (thompson et al., 2003) . vaccinating elderly individuals is therefore a public health priority. however, a randomized placebo-controlled trial estimated the efficacy of the tiv to be 50% for the prevention of influenza in older adults (govaert et al., 1994) . elderly individuals often have a significantly reduced antibody response to influenza vaccination (goodwin et al., 2006) . in a quantitative review of 4492 elderly subjects, 42%, 51%, and 35% seroconverted to h1n1, h3n2, and influenza b vaccination, respectively, compared to 60%, 62%, and 58% in younger subjects (goodwin et al., 2006) . the impaired ability of the elderly to mount an adequate immune response to influenza vaccines has been attributed to immunosenescence. immunosenescence is the decline in the body's ability to fight infection, mount novel immune responses, and recall responses (targonski et al., 2007) , and both innate and adaptive responses are implicated. impaired function of costimulatory molecules, altered secretion of inflammatory cytokines, and diminished function of natural killer cells, macrophages, and neutrophils have been observed in the elderly, as well as a decreased proliferative capacity of b cells and impaired t cell memory recall (sullivan et al., 2010) . in addition, thymic involution and a decline in t cell output are features of advancing age. this, together with a lifetime of exposure to a variety of pathogens, leads to a reduction in the naïve t cell pool and a relative increase in the proportion of memory t cells in the elderly compared with young adults. the most pronounced functional changes are in the cd8 + t cell subset, in which progressive exhaustion occurs (pawelec et al., 2005) , whereas the cd4 + t cell subset is less affected by replicative senescence (czesnikiewicz-guzik et al., 2008) . the tiv is standardized on the basis of the amount of ha protein, with one dose of 15 μg per strain being recommended in healthy, previously primed individuals. increasing the dose increases serum antibody response to the vaccine, and in an attempt to enhance immune responses to influenza vaccines in the elderly, a high-dose tiv (60 μg ha protein per strain) was licensed in 2009 for use in persons ages 65 years and older. postlicensure studies have shown enhanced immune responses in this age group, compared to the standard dose (sullivan et al., 2010) , and vaccine effectiveness studies are under way. in addition, an as03-adjuvanted tiv (discussed below) containing 15 μg ha protein per strain showed a 12% higher efficacy than a nonadjuvanted tiv in a phase 3 randomized clinical trial in the elderly, but the difference was not statistically significant (mcelhaney et al., 2013) . laivs are not licensed for use in the elderly at present; however, they have been evaluated in clinical studies in persons 50 years of age and older and are safe and well tolerated. in clinical trials, laivs were administered in addition to tivs, and coadministration was reported to enhance local ha-specific iga antibody responses. however, the efficacy of the combination was not greater than that of tiv alone (gorse et al., 2004; powers et al., 1989 powers et al., , 1991 treanor et al., 1996) . in individuals with chronic or immunocompromising conditions, serological responses to tiv vaccination are typically lower than in healthy adults. antibody responses against influenza were adequate in hiv-seropositive individuals who had no or minimal immunodeficiency or had responded well to antiretroviral therapy (chadwick et al., 1994; huang et al., 1987; madhi et al., 2011; staprans et al., 1995) . however, in individuals with advanced hiv disease and low cd4 t cell counts, tivs may not induce protective titers even after two doses (kroon et al., 2000; miotti et al., 1989) . laivs are not licensed for use in immunocompromised individuals. although there is a large amount of data available on the immune responses to seasonal influenza vaccines in humans, improved seasonal and pandemic influenza vaccines are evaluated first in animal models. the most commonly used models are mice and ferrets. mice immunized with inactivated influenza vaccines develop serum hi and neutralizing antibodies, the titers of which correlate with protection from subsequent challenge (luke and subbarao, 2011) . although cellular immune responses are mounted during a secondary influenza infection (woodland et al., 2002) , passively transferred antibodies protected immunosuppressed mice, suggesting that cell-mediated immunity is not essential for protection if sufficient antibody is present (virelizier, 1975) . the goal of parenteral immunization with inactivated influenza vaccines is to induce sufficient serum antibody titers to limit influenza disease. this protection is mediated by serum igg that transudes into the lower respiratory tract, neutralizing virus. passive transfer of immune serum to naïve mice reduced the replication of influenza virus in the lungs and protected recipient mice from lethal influenza pneumonitis, but did not prevent tracheitis or replication of the influenza virus in the urt (ramphal et al., 1979) . during a natural influenza infection of the urt, mucosal immune responses, including secretory iga antibodies, play an important role in controlling disease (section immune responses to respiratory virus infection). several studies have documented that higher levels of serum antibodies are required to provide protection against respiratory viruses in the urt compared to the lrt (prince et al., 1985; ramphal et al., 1979; takiguchi et al., 1992) . additionally, influenza in ferrets is primarily a urt infection and vaccination with killed or inactivated influenza viruses does not protect against influenza infection unless administered with an adjuvant (potter et al., 1972a,b) . adjuvants are probably required for parenterally administered inactivated vaccines to elicit the higher levels of serum igg antibody that are needed to restrict viral replication in the urt, in the absence of robust mucosal immune responses. in mice, laivs induce a range of systemic and pulmonary immune effectors and protect animals against challenge virus replication (chen et al., 2011; lau et al., 2011) . the magnitude of the pulmonary iga and memory cd8 + t lymphocyte responses depends on the replication efficiency of the vaccine virus in the respiratory tract, but systemic immunity, such as serum antibody titers and memory cd8 + t lymphocytes in the spleen, does not . after one dose of an h5n1 laiv that replicated in the lungs of mice and induced local immunity, influenza-specific lymphocytes in the lung contributed to the clearance of challenge virus from the lungs, whereas the contribution of serum antibody and splenic cd8 + t cells was negligible. after two doses, complete protection was achieved and was associated with maturation of the antibody response (lau et al., 2012) . taken together, the data suggest that laiv protects animals by inducing multiple arms of the immune response, including mucosal immunity and pulmonary and systemic antibody and cellular immune responses, in a manner similar to natural infection. however, inactivated vaccines aim to induce serum antibody alone. although this does not mimic a natural infection, if antibody titers are sufficiently high, the inactivated vaccine will protect against disease caused by influenza viruses. for inactivated influenza vaccines, serum anti-ha antibody titers correlate well with resistance to influenza infection in people as well as in animal models. lower antibody titers are associated with an increased risk of illness, though a specific antibody titer that can guarantee protection from infection has not been identified. an hi titer of 1:40 represents the level at which it is anticipated that approximately 50% of persons will be protected (hobson et al., 1972) , and this benchmark forms the basis of the licensing criteria for inactivated vaccines (cber, 2009) . although several papers refer to "seroprotection," there is insufficient evidence to support the use of this term for vaccines. the benchmark of an hi titer of 1:40 was defined in healthy adults who were experimentally challenged with an influenza virus (hobson et al., 1972) . however, antibody titers that correlate with protection in healthy adults may not translate to clinical improvements in influenza outcomes in the elderly (gorse et al., 2004) . in addition, laivs are effective despite inducing variable serum hi antibody titers. therefore, alternative correlates of protection are needed. as iga and cellular immune responses are generated in the lungs of mice vaccinated with laiv, in addition to systemic antibody and cellular responses, the extent to which the different arms of the immune response contribute to laiv-induced protection are beginning to be evaluated (chen et al., 2011; lau et al., 2011 lau et al., , 2012 . laiv-induced nasal wash igg and iga correlated with protection from virus replication, and in human challenge studies, either serum antibody or nasal wash iga was a predictor of protection (belshe et al., 2000b) . cellular immune responses have also been investigated as a correlate of laiv-induced protection, and one study found a correlation between ifnγ-producing t cells (measured by elispot) and protection from culture-confirmed influenza illness in young children (forrest et al., 2008) . in addition, it has been shown that laivs alter the expression of ifn-related genes, whereas tivs do not, indicating that the innate immune response plays an important role in protection mediated by laivs (nakaya et al., 2011) . adjuvants are added to vaccine formulations to enhance immune responses to the antigen in the vaccine. aluminum salts (alum) are the most commonly and historically used adjuvants worldwide. they act by capturing antigens at the injection site, so the antigen is slowly processed and presented by the immune system (the so-called depot effect), and they cause mild cell damage and inflammation that promotes a th2 immune enhancement (tetsutani and ishii, 2012) . moreover, alum particles enter host cells and bind dna, introducing it into the cytoplasm of antigen-bearing dcs, where it engages receptors that promote both mhc class ii presentation and dc-t cell interactions (mckee et al., 2013) . oil-in-water adjuvants, such as mf59 (novartis) and as03 (glaxosmithkline), are more effective at inducing high-titer antigen-specific serum antibody responses than alum and have been used with inactivated split-virion influenza vaccines in europe. these adjuvants induce broad, cross-clade humoral responses and permit dose sparing, in which comparable immune responses are induced with a reduced amount of ha in the vaccine (galli et al., 2009; leroux-roels et al., 2007) . our understanding of the mechanism of action of mf59 and as03 remains incomplete. neither appears to act via a depot effect; instead they induce a local and transient proinflammatory cytokine and chemokine response at the injection site and draining lymph nodes that recruit immune cells from the circulation. in mice, as03 induced the cytokine il-6 and chemokine cxcl1, which peaked locally by 6 h postvaccination. the neutrophil-mobilizing cytokine csf3 and lymphocyte-mobilizing cytokines ccl2, 3, and 5 were induced by 24 h postvaccination. in addition, the eosinophil-recruiting chemokine ccl11 and the cytokine il-1β were induced at low levels, and ifn-γ, csf2 (gm-csf), and tnf-α were induced at levels that were only marginally above background. ifn-α and -β were not induced. the local cytokine response was paralleled by an enhanced recruitment of monocytes and granulocytes in the draining lymph node (morel et al., 2011) . mf59 also induces local upregulation of cytokines, chemokines, and other innate immunity genes, promoting the recruitment of immune cells such as monocytes, dendritic cells, and granulocytes. however, the mechanism of action of mf59 was found to be independent of the nlrp3 inflammasome, but required myd88 for a tlr-independent signaling pathway (seubert et al., 2011) . whereas the inactivated vaccine viruses are typically disrupted with detergents to make split-virion (subunit) vaccines, inactivated whole influenza virion (wiv) vaccines have also been developed, in which the virions are left intact. these are less widely used because of increased reactogenicity and adverse events (fiore et al., 2013) . however, mice immunized with wiv vaccines consistently showed higher hi titers and virus-neutralizing antibody titers than subunit vaccines, as well as an increased production of proinflammatory cytokines by dendritic cells and ifn-α by plasmacytoid cells, resulting in a desired th1 response (geeraedts et al., 2008; koyama et al., 2010) . the approach of using inactivated whole influenza vaccines is being revisited with pandemic influenza vaccines. whole-virion inactivated h1n1 and h5n1 vaccines administered with alum are immunogenic in humans (kulkarni et al., 2012; lagler et al., 2012; lin et al., 2006; nakayama et al., 2012) . dna vaccines encoding one or several proteins of influenza viruses induce an immune response targeting the encoded proteins (fynan et al., 1993; ulmer et al., 1993; wolff et al., 1990) . dna vaccines can be produced rapidly and at low cost; however, designing dna vaccines is complex. over the years, it has been shown that numerous factors play roles in the efficiency of expression, such as the promoter, the g/c content, supercoiling, polyadenylation, and codon optimization (laddy and weiner, 2006) . in addition, safety remains a concern, as there might be a risk of integration into the host genome (klinman et al., 1997) . numerous studies have evaluated dna vaccines expressing np, m1, or ha proteins in animal models (fu et al., 1999; saha et al., 2006; tao et al., 2009; ulmer et al., 1998 ulmer et al., , 1996a . in mice, the administration of dna vaccines encoding the np protein of influenza induces a strong ctl response, which correlates with protection against challenge with homologous or heterologous viruses (ulmer et al., 1993) . in addition, one study showed that delivering the vaccine by in vivo electroporation instead of the classical epidermal route also induces protective humoral and cellular immune responses in mice, ferrets, and nonhuman primates (laddy et al., 2008) . recently, a phase 1 clinical trial with an adjuvanted plasmid dna vaccine encoding influenza h5, ha, np, and m2 elicited t cell responses against ha in the majority of the subjects and against np and m2 in some (smith et al., 2010) . licensed influenza vaccines primarily elicit an immune response to the globular "head" region of the ha glycoprotein. however, immune selection pressure leads to antigenic drift (discussed in section clinical features and epidermology), so new influenza vaccines need to be selected for each season as well as when a pandemic emerges. it remains difficult to predict which strains will circulate in the upcoming influenza season, and rates of morbidity and mortality are greater in influenza epidemics when the virus and vaccine are "mismatched" (pica and palese, 2013) . the expectation is that vaccines capable of protecting against a broad(er) spectrum of influenza viruses would result in less frequent updating of seasonal influenza vaccines and would provide a degree of preexisting immunity if a novel strain emerges. vaccines that aim to provide broad cross-protection against multiple subtypes of influenza are known as universal vaccines, and several platforms are in development, including those that target the ha, m2, np, m1, and na proteins (subbarao and matsuoka, 2013) . unlike the head region of ha, the "stalk" domain has a high degree of sequence conservation between influenza viruses, and broadly neutralizing stalk-reactive antibodies have been identified. these antibodies may inhibit phinduced conformational changes in the ha required for cellular entry, prevent the cleavage of ha0 into ha1 and ha2 (discussed in section virology), or act via antibodydependent cell-mediated cytotoxicity (adcc) or through the activation of complement . however, the ha stalk is less immunogenic than the head, and several techniques have been employed to elicit stalk-reactive antibodies, including removing the head, novel prime-boost strategies, and sequential vaccination with chimeric ha molecules that contain the same stalk region but "exotic" head domains that aim to boost antibodies to the conserved epitopes within the stalk . these strategies have been tested in mice with varying degrees of success in generating protective immune responses against heterologous influenza viruses (subbarao and matsuoka, 2013) . taken together, ha stalkbased strategies show promise as universal influenza vaccine candidates. the influenza m2 protein extends beyond the viral envelope, and antibodies against the m2 extracellular domain (m2e) may act via adcc, nk cell activity, or complementmediated lysis. in addition, the sequence of the m2e is conserved among human influenza a viruses, making it an attractive target for universal vaccines. however, the peptide is poorly immunogenic, and techniques to improve immunogenicity are under investigation in mice, including fusing the peptide to an immunogenic carrier protein or delivering the antigen in a virus-like particle (vlp) (reviewed by subbarao and matsuoka (2013) ). the influenza np and m1 proteins are also conserved among influenza a viruses and have potential for use in a universal vaccine, but as they are not exposed on the surface of virions or infected cells, they mainly induce cellular immune responses. phase 1 and 2 clinical trials with modified vaccinia virus ankara (mva) expressing np and m1 proteins report a 60% reduction in laboratory-confirmed influenza infection after experimental challenge (lillie et al., 2012) ; however, larger studies are required to confirm this. immune responses directed against the influenza na protein do not protect against infection, but rather limit the spread of infection and reduce the severity of disease. recently, baculovirus-expressed vlps containing n1 na proteins were found to induce heterosubtypic na antibodies in mice that protected them against homologous and heterologous challenge (quan et al., 2012) . a universal vaccine may therefore benefit from anti-na immune responses. it may be useful to combine several components into a universal vaccine, for example, np and m1 to induce cellular responses and ha and m2e to induce humoral responses. however, there are several challenges to the development of a universal vaccine. as outlined above, most of the viral proteins that potentially induce broad heterosubtypic immunity are poorly immunogenic and require a large dose of antigen, multiple doses, addition of adjuvants, fusion to immunogenic carriers, or the use of vectors or vlps. additionally, regulatory challenges include how to determine and define the potency of the vaccine and the need to identify immune correlates of protection and develop validated assays to measure them. in addition, clinical trials will be challenging because it is likely that efficacy will be measured in terms of amelioration of disease rather than preventing infection (subbarao and matsuoka, 2013) . given these hurdles, achieving a truly universal vaccine that protects against all types or subtypes of influenza will probably proceed in a stepwise manner, with the first step being a vaccine that is more broadly cross-protective than currently licensed vaccines. the primary target populations for rsv vaccination are young infants and the elderly, because hospitalization rates are the highest in these age groups and 78% of rsv-related deaths occur in individuals over 65 years of age (thompson et al., 2003) . there is a large body of evidence that protection against infection is conferred mainly by neutralizing antibodies (collins and melero, 2011) ; however, multiple doses of vaccine might be necessary in young infants, because of the immature immune system and the presence of maternal antibodies. in addition, protective immunity mounted during infection does not protect against subsequent reinfection. reinfections are common and are independent of antigenic changes in the virus (collins and melero, 2011) . these factors present challenges to rsv vaccination and, as yet, vaccines have not been licensed. this section discusses clinical trial data from various approaches to rsv vaccination. the first approach to vaccination was the development of a formalin-inactivated rsv vaccine (f1-rsv) that was evaluated in the 1960s in infants and young children. a concentrated f1-rsv vaccine, given i.m. with alum, was well tolerated and moderately immunogenic, but was poorly protective against infection. in fact, vaccinees who were exposed naturally to infection during the subsequent rsv season had immune-mediated enhancement of disease, with 80% of individuals requiring hospitalization and two fatalities (fulginiti et al., 1969; kapikian et al., 1969; kim et al., 1969) . subsequent studies revealed that vaccine-induced immune responses were different from those elicited after natural infection, with poor induction of neutralizing antibodies (murphy and walsh, 1988) and an exaggerated cd4 + t-cell response (kim et al., 1976) . vaccine-mediated enhancement of disease also occurred in murine models, with poor induction of neutralizing antibodies probably due to denaturation of antigen in the vaccine or a lack of antibody affinity maturation after poor tlr stimulation (delgado et al., 2009 ). in addition, an exaggerated th2 response and a loss of regulatory t cells contributed to disease (connors et al., 1992 (connors et al., , 1994 de swart et al., 2007; loebbermann et al., 2013; waris et al., 1996) . therefore, it is clear that in addition to a neutralizing antibody response, a balanced cd4 and cd8 t cell response is also desirable. after this experience, inactivated rsv vaccines were considered unsuitable for pediatric use, and alternative approaches were sought. in the 1960s, a series of live attenuated vaccines (lavs) was developed by serial passage of rsv at suboptimal temperatures (cold passage, cp) or by growth in the presence of mutagens to produce temperature-sensitive (ts) mutants. the lav did not cause disease enhancement in animal models or in clinical trials (wright et al., 2007a) . the most promising vaccine generated by this method, cpts248/404, was well tolerated and immunogenic in seronegative infants and children greater than 6 months of age, but caused mild congestion in younger infants and was deemed to be insufficiently attenuated (wright et al., 2000) . several lavs have also been developed using reverse genetics techniques and were evaluated in clinical trials. the most promising, ra2cp248/404/1030δsh (medi-559), was strongly ts and was well tolerated in infants . the first dose provided substantial reduction in replication of a second dose of vaccine given 2 months later. however, the majority of individuals did not have increases in serum antibody titers, indicating that other immune mechanisms might play a role in the protection from replication of the second dose of vaccine. as with live attenuated influenza vaccines, establishing correlates of protection for live rsv vaccines is an active area of research, made more challenging by the weak immune responses in infants and limitations on sampling. as of this writing, clinical trials are under way to further monitor tolerability and immunogenicity and the ability of candidate vaccines to induce protection against natural rsv exposure in the community (collins and melero, 2011) . genetic changes have been employed to generate candidate lavs, including deletion of the m2-2 coding sequence, which increases transcription and antigen synthesis at the expense of viral replication (bermingham and collins, 1999) , and deletion of the ns1 and ns2 genes, which increase ifn production and signaling and might limit viral replication and pathology, but increase immunogenicity (teng et al., 2000) . the use of attenuated parainfluenza virus as a vector to express rsv f and/or g protein has also been considered as a pediatric vaccine against both hpiv and rsv, as piv has the advantage of improved in vitro growth and stability compared with rsv. bovine piv3 is attenuated in humans and has been used as a vector into which the f and hn genes from human piv3 and the f and/or g protein of rsv are incorporated to make a bivalent vaccine against both hpiv3 and rsv (schmidt et al., 2002; tang et al., 2008) . one example of this approach is in clinical trials in children older than 6 months of age and who are seronegative to rsv and hpiv3 (collins and melero, 2011) . the vaccine in this study comprises a bovine piv3 vector into which the human piv3 f and hn genes and the rsv f gene have been added (tang et al., 2004) . as an alternative to bovine piv3, murine piv1 (sendai virus) is also being evaluated as a vaccine backbone into which rsv antigens are inserted (jones et al., 2009) . there is substantial antigenic crossreactivity between sendai virus and hpiv1, and the virus may be attenuated in humans. other approaches to live vaccines for rsv include the use of alphaviruses (elliott et al., 2007) or replicationdefective adenoviruses (shao et al., 2009) as vectors for rsv antigens. in addition to an rsv vaccine in infants, there is also a need for rsv vaccines in older children, adults, and the elderly. however, these individuals are likely to have been previously infected with rsv, and preexisting immunity may restrict the replication of lavs. in addition, vectored vaccine approaches should not be based on common human pathogens, as preexisting immunity to the vector may limit vaccine virus replication and interfere with the development of immunity. the use of protein-based vaccines in these populations is currently being evaluated. disease enhancement has not been documented in adults who were previously exposed to rsv, and candidate rsv f protein-based vaccines are well tolerated in healthy adults, children over 12 months of age, pregnant women, and the elderly (girard et al., 2005) . in a phase 3 clinical trial involving 289 children 1-12 years of age with cystic fibrosis, a purified f protein-3 (pfp-3) vaccine induced a fourfold rise in serum antibody titer, but was not associated with significant protection against lrti episodes compared to placebo . in another approach, a subunit vaccine of copurified f, g, and m proteins from rsv-a was tested in adults and found to induce neutralizing antibodies in 76-93% of vaccinees, but titers waned after 1 year, suggesting that annual immunization might be necessary with this vaccine (girard et al., 2005) . interestingly, in a phase 1 clinical study of 35 pregnant women vaccinated with pfp-2, anti-f antibodies were persistently elevated in newborns of the vaccinated mothers, without enhancement of disease. maternal immunization could, therefore, be a strategy to protect infants under 6 months of age, for whom the development of an rsv vaccine has been such a challenge (durbin and karron, 2003) . a recombinant postfusion form of the f protein with a deletion of the major hydrophobic regions was also produced. this subunit vaccine forms stable trimers that are recognized by neutralizing mabs. high levels of neutralizing antibodies were induced in the sera of vaccinated rodents, and the animals were protected from challenge. this may represent an improved subunit vaccine (mclellan et al., 2011; swanson et al., 2011) . another protein-based vaccine, bbg2na, has been evaluated in clinical trials. bbg2na consists of a fragment of the rsv g protein that contains a central conserved domain fused to the albumin-binding domain of streptococcal g protein, expressed in bacteria. however, this vaccine was not very immunogenic in clinical trials and was associated with hypersensitivity reactions (purpura) in some individuals (power et al., 2001) . vlps have also been evaluated for use as an rsv vaccine, and one such formulation (rsv-f particle vaccine) is currently in clinical trials (collins and melero, 2011) . it is the hope that improved methods of producing rsv antigens or using vlps will result in an effective vaccine that could be given to adults periodically, possibly with the annual influenza vaccine. however, in children, it remains to be seen whether a vaccine can be developed that is sufficiently attenuated, yet immunogenic enough to provide protection. however, even if complete protection is unrealistic, immunity that results in substantial reduction of virus replication may be sufficient to prevent severe disease. as 80% of children are seropositive for hpiv by 5 years of age, and hpiv lrti is a major cause of hospitalization in this age group (schomacker et al., 2012) , young infants and children are the target population for hpiv vaccination. protective immunity against infection is mediated primarily by mucosal and serum neutralizing antibodies; however, reinfection is common owing to a difficulty in maintaining protective titers of s-iga and igg in the respiratory lumen, thus representing a challenge to vaccine development (glezen et al., 1984) . this section reviews data from clinical trials of inactivated, live, and vectored hpiv vaccines. the first approach taken to hpiv vaccination was the use of formalin-inactivated piv3 (fi-piv3) in an intramuscular vaccine. although vaccine-induced disease enhancement seen with formalin-inactivated rsv was not observed with fi-piv3, there was no evidence of protection either (kim et al., 1969) . subsequent vaccine efforts, therefore, focused on the generation of live attenuated vaccines, and two approaches were investigated: the use of a ts hpiv3 strain and the use of a bovine piv strain. successive passage at lower temperatures has been shown to attenuate hpiv. the most promising vaccine candidate using this approach, cp45, is designated by the number of times the virus was passaged at low temperature in african green monkey kidney cells (belshe and hissom, 1982; ray et al., 1995) . the vaccine was safe and immunogenic in adults, seropositive and seronegative children, and infants 1 month of age or older (belshe et al., 2004b) . in a phase 2 clinical trial in 6-to 18-month-old children, the vaccine was well tolerated and 84% of previously seronegative vaccinees had a fourfold or greater rise in serum geometric mean antibody titer (belshe et al., 2004b) . moreover, coadministration of an rsv and cp45 piv vaccine was successful, with little evidence of interference (belshe et al., 2004a) . however, the cp45 piv vaccine was overattenuated in seropositive children and adults. bovine piv3 makes an attractive lav as it is known to be antigenically related to human piv, is attenuated in humans, and protects monkeys against challenge with hpiv3. clinical trials were conducted in adults, seropositive children, seronegative infants, and children 2-6 months of age with residual maternal antibodies. in seropositive individuals, the vaccine was overattenuated, but in seronegative children and infants, the vaccine virus was highly infectious. despite replication of the vaccine virus, serum antibody responses to hpiv3 were low, consistent with an incomplete antigenic relatedness between the human and the bovine piv hn genes (greenberg et al., 2005; karron et al., 1995) . a phase 2 clinical trial of 192 children showed that seroconversion to bovine piv3 occurred in 57-67% of vaccinated children after three doses, but that seroconversion to hpiv3 occurred in only 21-25% of vaccinees (greenberg et al., 2005) . additionally, during the study, 47% of the placebo group experienced piv3 infection, highlighting the ubiquitous nature of the pathogen. after these studies, it was concluded that bovine piv3 might better serve as a backbone for the insertion of hpiv3 genes. as described above, at this writing, one such vaccine, with hpiv3 hn and f genes and the rsv f gene inserted into a bovine piv3 backbone, is being evaluated in clinical trials in seronegative children. this vaccine candidate was highly attenuated in seropositive children and adults. additional vaccine candidates include those generated by reverse genetics. one such vaccine contains mutations in the p/c gene, which reduces the virus's ability to inhibit type i ifn induction and signaling, and another contains ts and attenuating mutations from other paramyxoviruses (sato and wright, 2008) . by the age of 5 years, most children will have been infected with hmpv. reinfections are common, and hmpv is responsible for 5-15% of childhood hospitalizations for lrti. in addition, severe disease can also be seen in the elderly or immunocompromised individuals (feuillet et al., 2012) . despite this clinical need, hmpv vaccines have not yet entered clinical trials, and the data reviewed in this section are from studies in animal models. as with rsv, vaccination of macaques and mice with inactivated hmpv led to disease enhancement after challenge and a strong th2-type immune response associated with a lack of neutralizing antibodies (herfst et al., 2008a; hamelin et al., 2007) . therefore, alternative approaches have been evaluated, including soluble protein-based subunit vaccines, live attenuated vaccines, or dna vaccines encoding viral proteins. soluble f-protein subunit vaccines have been shown to generate high titers of neutralizing antibody in serum in golden syrian hamsters and cotton rats, associated with protection against subsequent infection (cseke et al., 2007; herfst et al., 2007) . however, stable and long-term immunity was not induced in monkeys (herfst et al., 2008b) . live attenuated vaccines have been generated either by cold passage or by reverse genetics. temperature-sensitive viruses conferred complete protection against heterologous hmpv challenge in golden syrian hamsters (herfst et al., 2008a) , and a recombinant hmpv lacking a region of the sh (δ 101−103 sh), the g, or the m2-2 protein induced high titers of neutralizing antibody and protected hamsters and african green monkeys against piv and hmpv challenge (biacchesi et al., 2004; buchholz et al., 2005 buchholz et al., , 2006 . a chimeric vaccine has also been developed in which the f2 subunit of the hmpv fusion protein is inserted into a backbone of bovine piv that also contains the hpiv3 f and hn genes (tang et al., 2003) . this b/hpiv3/hmpv f2 vaccine induced neutralizing antibodies and protected hamsters and african green monkeys against piv and hmpv challenge (tang et al., 2003 . coronaviruses such as hcov-229e and oc43 are frequent causes of respiratory illness throughout the world, and sars-cov and mers-cov represent significant public health threats. however, at present there are no licensed vaccines for human coronavirus infections, though a number of vaccines are licensed against animal coronaviruses, and several vaccine platforms have been developed for sars-cov that show great promise in preclinical studies. whole sars-cov particles inactivated with β-propiolactone, formalin, uv light, or a combination of two techniques have been evaluated in mice, ferrets, rabbits, and nonhuman primates. such vaccines have been administered by a variety of routes and tested in the presence or absence of various adjuvants. in general, vaccination elicited a serum igg response in animal models, with higher doses of vaccine eliciting higher igg antibody titers ( subbarao, 2010) . moreover, some studies demonstrated that inactivated sars-cov vaccines were efficacious in protecting mice (see et al., 2006; spruth et al., 2006; stadler et al., 2005) and nonhuman primates (qin et al., 2006; zhou et al., 2005) , although many studies did not investigate protective efficacy. in addition, an inactivated sars vaccine that was evaluated in clinical trials was safe and well tolerated and induced neutralizing antibodies (lin et al., 2007) . many subunit vaccines comprise purified, recombinant sars-cov s protein, as this protein is the target for protective neutralizing antibodies elicited by sars-cov infection. a truncated soluble form of the s protein that lacks the transmembrane domain also neutralized infectivity of sars-cov (bisht et al., 2005; he et al., 2006b; zhou et al., 2006) and substantially reduced the titer of challenge virus replication in the respiratory tract of mice (bisht et al., 2005) . a trimeric spike protein vaccine (trispike) was also immunogenic in mice and hamsters and provided protection against challenge in hamsters, with reduced occurrence and severity of pneumonitis and no evidence of pulmonary consolidation or sars-cov-associated hepatic cellular necrosis (kam et al., 2007) . however, sera from animals immunized with trispike were associated with a 100-to 1000-fold increase in virus entry into fcγrii-positive (ace2-negative) human b cells, which has led to concerns about antibody-dependent enhancement of disease (kam et al., 2007) . vectored vaccines have been developed against sars-cov. in this approach, sars-cov proteins are expressed by venezuelan equine encephalitis virus replicon particles (vrps), chimeric bovine/human piv3 vectors, recombinant replication-defective human adenovirus-5 (ad-5), poxvirus mva, attenuated rabies viruses, or attenuated vesicular stomatitis virus (subbarao, 2010) . when animals were immunized with vectored vaccines expressing the s protein of sars-cov, neutralizing antibodies were induced, and vaccinated animals were protected from challenge. however, if other sars-cov proteins were expressed, animals were not protected from challenge. for example vrps expressing the sars-cov s protein provided protection from challenge in mice; however, vrp expressing the sars-cov n protein (vrp-n) did not (deming et al., 2006) . moreover, vrp-n vaccination resulted in a marked bronchiolitis, alveolitis, and interstitial accumulation of eosinophils and mononuclear leukocytes (deming et al., 2006) . in addition, chimeric bovine/ human (b/h) piv3 vectors expressing the sars-cov s protein or the s, m, and e proteins together also generated neutralizing antibodies in hamsters and protected animals from challenge, whereas b/h parainfluenza viruses expressing m, n, or e proteins were not protective . the route of administration is also important in determining protective efficacy of a vaccine. an ad-5 vector expressing the sars-cov s and n genes given i.m. had a limited effect in reducing pulmonary replication of challenge virus, despite eliciting higher serum igg titers and a greater cellular immunity than when the vaccine was given intranasally (i.n.). however, the vaccine administered i.n. induced higher mucosal iga responses than when given i.m. and provided protection from pulmonary replication, suggesting that mucosal immunity is important in mediating protection (see et al., 2006) . mice, rabbits, and monkeys immunized with a modified vaccinia virus mva-s vaccine were protected from replication of challenge virus (bisht et al., 2004; chen et al., 2005) . however, in ferrets, mva-s antibodies declined rapidly after vaccination, and the vaccine did not protect the animals from replication or spread of the challenge virus (weingartl et al., 2004) . a recombinant live attenuated sars-cov vaccine virus in which expression of the e protein was abrogated by point mutations and a deletion in the nucleotide sequence was restricted in vitro and attenuated in hamsters (dediego et al., 2007) . in addition, inactivation of the 3′-5′ exonuclease attenuated sars-cov, and the resulting virus was protective in an aged immunocompromised mouse model of sars infection (graham et al., 2012) . dna vaccines expressing sars-cov s protein (either without the cytoplasmic domain or without the cytoplasmic and transmembrane domains) also induced humoral and cellular immunity in mice and protected animals from replication of challenge virus . in addition, priming with a dna vaccine and boosting with an inactivated vaccine resulted in an increase in cd4 + t cells and a stronger antibody response (zakhartchouk et al., 2005) . the dna vaccine was well tolerated and produced cellular immune responses and neutralizing antibody in a phase 1 clinical trial (martin et al., 2008) . despite these successes in preclinical vaccine development, as the sars outbreak was declared over in 2003, the impetus was not sustained long enough for many of these products to be evaluated in clinical trials. a cautionary note about sars-cov vaccines is the association of a variety of vaccine platforms with pulmonary immunopathology in mice after challenge with the virus, despite immunogenicity and protective efficacy (tseng et al., 2012) . the relevance of the rodent model to vaccines in humans remains uncertain. adenovirus epidemics have been described in adults, especially in military recruits in closed or crowded settings (lynch et al., 2011) . live attenuated, orally administered adenovirus vaccines against serotypes 4 and 7, which cause respiratory disease, were used by the u.s. military starting in 1971 (top, 1975) . during this time the incidence of disease fell substantially. however, when the manufacturer ceased vaccine production in 1995, epidemics reemerged in military facilities (lynch et al., 2011) . in 2011, a new live attenuated adenovirus vaccine was issued to military recruits during basic training, and there has since been a reduction in the rate of febrile respiratory illness (armed forces health surveillance, 2013). the vaccine is not available to children or the civilian adult population at present, though adenoviruses are estimated to account for 5-10% of pediatric and 1-7% of adult respiratory tract infections (lynch et al., 2011) . in summary, the majority of studies evaluating respiratory virus vaccines measure serum antibody responses, because a large body of evidence indicates that, although both cellular and humoral responses contribute to the clearance of a primary infection, neutralizing antibodies protect against secondary infection. humoral responses can be readily detected after vaccination with inactivated or subunit vaccines; however, fewer individuals seroconvert after vaccination with live vaccines. alternative immune mechanisms such as mucosal antibody responses are probably responsible for protection by live attenuated vaccines, and immune correlates of protection are under investigation. because respiratory pathogens infect the host at mucosal surfaces, the induction of mucosal immunity by vaccination is desirable. vaccines that are administered by intramuscular or subcutaneous injection induce protective immunity in the systemic immune compartments, but are generally poor at inducing mucosal immune responses. mucosally delivered vaccines, in contrast, induce both mucosal and systemic immunity and are also more readily accepted because they do not require needles or syringes (levine and dougan, 1998; yuki and kiyono, 2009) . ongoing research into the molecular and cellular mechanisms of surface immunological barrier systems has provided practical strategies for the development of a new mucosal vaccine for the control of respiratory viruses. m cells, located in the follicle-associated epithelium of the malt, play a pivotal role in the uptake of luminal antigens in the respiratory tract and the induction of antigen-specific immune responses in both the systemic and the mucosal compartments . it is, therefore, logical and attractive to develop m-cell-targeted mucosal vaccines. several molecules have been found to bind preferentially to m cells, such as ulex europaeus agglutinin-1 (uea-1), which has specificity for α(1,2)-fructose (sharma et al., 1998) , and rho-protein 1 derived from reovirus, which binds to a carbohydrate structure containing α2, 3-linked sialic acids on the plasma membranes of m cells (helander et al., 2003) . moreover, intranasal vaccines conjugated to either uea-1 (manocha et al., 2005) or rho-protein induced not only strong antigen-specific plasma igg and mucosal iga responses, but also ctl immunity (wu et al., 2001) . in addition, a novel m-cell-specific monoclonal antibody has recently been identified that selectively recognizes m cells, but not goblet cells or epithelial cells. this has been used as a carrier for an m-cell-targeted mucosal vaccine (wu et al., 2001; nochi et al., 2007) . concerns have been raised about the potential induction of unwanted mucosal inflammation associated with m cell targeting (kuolee and chen, 2008) , and additional research is needed to better understand how m cells sample antigens and transcytose them to the basolateral membrane. however, most microparticles administered orally become trapped in the mucus, and only a small fraction of them enter mucosal inductive sites. new approaches that are being developed to transitionally or conditionally enhance the number and function of m cells (neutra and kozlowski, 2006) are likely to reduce these concerns. there is substantial interest in the exploitation of nanoparticle technology for drug and vaccine delivery. nanoparticles are solid particles ranging in size from 10 to 1000 nm in diameter that are often made from biodegradable materials. an antigen payload can be dissolved, entrapped, adsorbed, attached, or encapsulated into the matrix of the particle and released as the particle degrades over a period of time, which may vary from days to weeks, depending on the formulation (adair, 2009) . several types of nanoparticles have been investigated for vaccine delivery and have proven to be safe, nontoxic, and suitable for loading with antigens. these include the polyesters (poly(lactic acid), poly(glycolic acid), and their copolymers), polyorthoesters, polyanhydrides, and polycarbonates (reddy et al., 2006) . these materials can protect antigens from degradation, and the particles can be prepared in a chemically reproducible manner within a narrow size limit. in addition, some biopolymers exhibit a natural adjuvant behavior, for example, poly(lactide co-glycolide) (plga) appears to activate the maturation of dcs, possibly by providing a necessary stimulatory signal, though the exact mechanism is not fully elucidated (bennewitz and babensee, 2005; yoshida and babensee, 2004) . surface modifications of nanoparticles that change their overall charge and hydrophobicity are aimed at improving mucoadherence properties (des rieux et al., 2006) . this can be achieved by coating with stabilizers, other polymers, or surfactants. polyethylene glycol has been used for its stabilizing properties and because it can enhance the affinity of the nanoparticles for mucosal surfaces. molecules such as lectins and chitosan can also increase interaction with and transport across the mucosal surface. interestingly, chitosan is reported to be able to open tight junctions between epithelial cells, facilitating the transport of encapsulated macromolecules across the epithelial layer . nanoparticles coated with mannose (mannan) have also been produced with the aim of targeting mannose receptors on apcs, thus improving cell adhesion and uptake (cui and mumper, 2003) . a plga-coated nanoparticle vaccine encapsulating influenza virus proteins has been developed. the influenza ha protein retained its antigenicity after encapsulation, and mice vaccinated with plga particles of 2.2-10.8 μm in diameter mounted both systemic and mucosal responses, which were protective against intranasal challenge with an h3n2 influenza virus (amidi et al., 2007; moldoveanu et al., 1993) . chitosan-coated nanoparticles containing purified influenza virus have also been tested in human volunteers, in which a fourfold or greater increase in anti-ha antibodies was observed in >40% of the volunteers . moreover, a single dose of the intranasal vaccine resulted in high titers of nasal iga antibody and strong systemic antigen-specific responses, which were greater than those induced after intramuscular inoculation with soluble influenza antigen (amidi et al., 2007) . nanoparticleconjugated rsv proteins have also been shown to generate strong systemic th1 immunity in mice, associated with protection against wild-type rsv challenge (kalkanidis et al., 2006; xiang et al., 2006) . in addition, plgacoated nanoparticles incorporating hpiv hn and f glycoproteins have been shown to induce virus-neutralizing antibodies, which were protective against challenge infection in hamsters (ray et al., 1993) . studies in animal models and humans have shown that the choice of adjuvant can dramatically affect the immediate immune response and long-term protective effect of a vaccine (ogra et al., 2001; galli et al., 2009) . in addition, the quality of the immune response, especially the development of high-affinity b cells, long-lived memory b cells, and plasma cells can be influenced by the choice of adjuvant (galli et al., 2009) . although the mechanisms have not yet been fully elucidated, mucosal adjuvants can be broadly classified into two categories: those that act as immunostimulatory molecules and those that facilitate vaccine delivery (o'hagan, 2001) . the former include adjuvants that are toxin-based or cytokine-based and molecules associated with innate immunity, for example, pamps. the latter contain immune-stimulating complexes, liposomes, live attenuated vectors, and chitosan (discussed above). from a mechanistic point of view, mucosal adjuvants modulate innate immune responses in the same way as parenterally administered vaccines (lambrecht et al., 2009) , and tlr agonists constitute a major category of mucosal adjuvants. these adjuvants are based on pamps and are often formulated as oil-inwater emulsions. virosomes are virus-like particles that have been investigated for their potential as vaccines bungener et al., 2005) . they closely resemble native virus; however, they are nonreplicating and consist of reconstituted viral envelopes, generated by treatment with a detergent. they have a diameter of 100-200 nm and, as such, fall into the size range of small particles. virosomal influenza vaccines are available commercially and have been shown to induce ha-specific antibodies after i.m. administration. phase 1 clinical trials revealed the tolerability and immunogenicity of a matrix tm -adjuvanted virosomal h5n1 dna vaccine (cox et al., 2011) . influenza virosomes that incorporate the rsv-fusion protein have also been constructed and have been shown to generate virus-specific iga in the urt and lrt after i.n. administration with adjuvant in mice (cusi et al., 2002) . immune stimulating complexes (iscoms) are nonreplicating particles of ∼40 nm diameter, comprising viral glycoproteins complexed with saponin derived from the bark of the tree quillaia saponaria (gregory et al., 2013) . iscom antigens from a number of microorganisms, including viruses, bacteria, and parasites, have been shown to induce humoral and cell-mediated immunity; th1 immune responses appear to predominate . extensive studies have been carried out with influenza virus iscoms in several species, including mice and monkeys, and an equine influenza iscom vaccine is available commercially (mumford et al., 1994 ). an influenza iscom vaccine (sundquist et al., 1988) stimulated high levels of virus-specific igm and igg serum antibodies and proliferative t cell responses in macaques, and the animals were completely protected from intratracheal challenge with the virus (rimmelzwaan et al., 1997) . intranasal immunization of mice with rsv iscoms induced very high levels of long-lasting rsv-specific iga in both the urt and the lrt (hu et al., 1998) . bovine rsv iscoms inoculated subcutaneously into calves were completely protective against challenge with virulent bovine rsv, whereas calves immunized with a conventional bovine rsv vaccine developed moderate to severe respiratory disease after challenge (hagglund et al., 2004) . the iscom-vaccinated animals developed high titers of nasal and serum virus-specific igg as well as serum iga, which correlated with protection. detoxified derivatives of cholera toxin (ct) and heat-labile enterotoxin (lt) produced by enterotoxigenic escherichia coli are effective mucosal adjuvants that promote mucosal and systemic immunity to coadministered protein antigens via oral or nasal routes (freytag and clements, 1999; mestecky et al., 1999) . the results of murine studies demonstrated that both ct and lt induced the expression of b7-1 and/or b7-2 on apcs that deliver costimulatory signals to cd4 + t cells (freytag and clements, 1999; mestecky et al., 1999) , but ct acts in the presence of il-4 to induce predominantly th2 responses, whereas lt supports th1 responses with ifn-γ production. recent vaccine development efforts have focused on the nasal administration of antigen with ct and lt derivatives for the induction of mucosal iga (byun et al., 2001; fujihashi et al., 2002) . both ct and lt are ab5-type molecules, consisting of one a subunit and five b subunits. chimeric molecules, made by the spontaneous association of the a subunit of ct with the b subunit of the lt, or vice versa, are effective mucosal adjuvants for protein vaccines. the type of t helper responses induced is dictated by the origin of the b subunit (takahashi et al., 2009) . one such example is the nasal administration of an influenza ha subunit vaccine together with a chimeric mutant (m) ct-a/lt-b adjuvant. in murine studies, this adjuvanted vaccine protected animals from influenza virus challenge (kweon et al., 2002) . however, there are some concerns about the safety profile of toxin-derived adjuvants. nasal administration of mct-a/lt-b targeted neuronal tissues, though it did not affect trafficking of coadministered vaccine antigens into the neuronal tissues (kweon et al., 2002) . taken together, these findings suggest that although recombinant chimeric toxin-based molecules show promise as a new generation of mucosal adjuvants, the safety of the ct adjuvant may need to be improved. the inactivated lt-adjuvanted intranasal influenza vaccine (nasalflu) was withdrawn because of concomitant facial nerve paralysis (bell's palsy) that was noted as a potential adverse event caused by the ct adjuvant kunzi et al., 2009; garner-spitzer et al., 2009 ). several cytokines associated with innate immunity and inflammation support the generation of antigen-specific s-iga and serum igg/iga and may be of use as mucosal adjuvants. mice nasally immunized with soluble antigens, including ovalbumin or tetanus toxoid (tt) plus il-1α and il-1β developed antigen-specific antibody responses that were similar to those induced by coadministered ct adjuvant. the cytokines il-1α and il-1β promoted antigen-specific igg1 antibody responses initially, followed by igg2b, with minimal igg2a antibody responses, a pattern associated with a predominantly th2-type response (staats and ennis, 1999; thompson and staats, 2011) . furthermore, levels of antigen-specific s-iga antibody similar to those induced by ct were found in mucosal secretions of mice that received nasal il-1. these results indicate that il-1 could be a useful mucosal adjuvant. apcs are known to contribute to the creation of an appropriate cytokine environment for the growth and development of th1 or th2 responses. two well-known cytokines secreted by apcs, il-6 and il-12, can influence the outcome of th1 and th2 cell subset-mediated immune responses (vajdy et al., 1995; rincon et al., 1997) . when tt was used as an antigen, nasal coadministration of il-6 or il-12 induced antigen-specific serum igg antibody responses and promoted protective immunity against lethal challenge with tetanus toxin. interestingly, whereas nasal treatment with il-12 promoted mixed th1/th2-type responses, il-6 supported predominantly th2-type responses. in addition, il-12 but not il-6 can induce antigen-specific mucosal iga antibody (boyaka et al., 1999) . these findings suggest that il-12 could be a potent mucosal cytokine for the upregulation of antigen-specific mucosal immune responses. type i ifn affects the differentiation and function of immune cells, including t cells and dcs, and efficiently enhances a primary antibody response (marrack et al., 1999; luft et al., 1998) . it was reported that type i ifn was effective as both a systemic and a mucosal adjuvant, promoting th1-type immune responses (proietti et al., 2002) . nasal administration of influenza vaccine with type i ifn was effective at inducing serum antigen-specific igg2a and mucosal iga antibody responses and at providing full protection against influenza virus challenge (proietti et al., 2002) . it is well known that i.n. vaccination stimulates immune responses in the nalt and is effective at inducing mucosal immunity in the respiratory tract (brandtzaeg, 2011; jabbal-gill, 2010) . intranasal administration of the live attenuated influenza vaccine flumist (medimmune) has proven effective in protection against seasonal influenza virus infection and protects children against drifted influenza virus strains (belshe et al., 2000a; mendelman et al., 2004) . furthermore, owing to the development of novel technologies, both aerosol spray and droplet delivery of vaccines are attractive possibilities (jabbal-gill, 2010) . in addition to the i.n. route of vaccine inoculation, the sublingual route has been explored (shim et al., 2011; czerkinsky et al., 2011) . the sublingual epithelium harbors a dense lattice of dcs, and vaccine delivered via this route stimulates broad and disseminated mucosal and systemic immune responses, similar to intranasal inoculation . sublingual vaccination with soluble or particulate antigens promotes strong mucosal iga and systemic igg antibody responses as well as cd8 + t cell responses. overall, sublingual immunization was comparable to nasal immunization in the magnitude, breadth, and anatomic dissemination of the induced immune responses. importantly, sublingual administration did not redirect antigens and/or adjuvants to the brain . sublingual vaccination against influenza has been shown to protect mice against lung infection (song et al., 2008) . in addition, sublingual administration of inactivated influenza a/pr8 (h1n1) vaccine virus together with a mucosal adjuvant such as ct (cuburu et al., 2007) or a nontoxic mct-a/lt-b adjuvant induced both systemic and mucosal virus-specific antibody responses as well as ctl responses with protective immunity after respiratory challenge with the a/pr8 virus (kweon et al., 2002; song et al., 2008) . these studies demonstrated that sublingual administration of an inactivated influenza virus with a toxin adjuvant such as ct did not migrate to or replicate in the central nervous system. moreover, using mucosal adjuvant such as ct mobilizes dcs within the sublingual epithelium. these observations suggest that sublingual immunization may be another attractive and safe mucosal route for the administration of respiratory virus vaccines. given the global burden of respiratory tract infection, there remains an unmet need for effective methods of intervention. the most efficient means of preventing respiratory virus infections is vaccination. however, among respiratory viruses, licensed vaccines are available only for influenza. systemic immune responses can be protective in the absence of mucosal immunity if they are sufficiently robust, such as that induced by i.m. inactivated influenza vaccines; however, many vaccines in development for respiratory viruses are live attenuated viruses that are mucosally administered. although live vaccines show promise, our understanding of the mechanism of protection at mucosal surfaces is incomplete, and there is currently a lack of adequate immune correlates of protection for these vaccines. in addition, immune responses mounted to a natural infection are often not sufficient to prevent reinfection and may also be involved in the pathogenesis of disease. achieving an appropriate balance between sufficient attenuation and immunogenicity, especially in young infants who must be vaccinated in the face of an immature immune system and maternal antibody, is a challenge. nanoparticle vaccines against respiratory viruses unique ability of activated cd4 + t cells but not rested effectors to migrate to non-lymphoid sites in the absence of inflammation immunological memory and protective immunity: understanding their relation recognition of double-stranded rna and activation of nf-kappab by tolllike receptor 3 the nlrp3 inflammasome mediates in vivo innate immunity to influenza a virus through recognition of viral rna viral infection is associated with an increased proinflammatory response in chronic obstructive pulmonary disease an integrated, multistudy analysis of the safety of ann arbor strain live attenuated influenza vaccine in children aged 2-17 years n-trimethyl chitosan (tmc) nanoparticles loaded with influenza subunit antigen for intranasal vaccination: biological properties and immunogenicity in a mouse model incidence of acute respiratory illnesses among enlisted service members during their first year of military service: did the 2011 resumption of adenovirus vaccination of basic trainees have an effect? mouse type i ifn-producing cells are immature apcs with plasmacytoid morphology free recirculation of memory b cells versus antigen-dependent differentiation to antibody-forming cells critical role of mda5 in the interferon response induced by human metapneumovirus infection in dendritic cells and in vivo dectin-2 mediates th2 immunity through the generation of cysteinyl leukotrienes comparison of antibody and t-cell responses elicited by licensed inactivatedand live-attenuated influenza vaccines against h3n2 hemagglutinin the double life of a b-1 cell: self-reactivity selects for protective effector functions innate and acquired humoral immunities to influenza virus are mediated by distinct arms of the immune system cord blood vitamin d deficiency is associated with respiratory syncytial virus bronchiolitis icam-1 receptors and cold viruses cold adaptation of parainfluenza virus type 3: induction of three phenotypic markers the efficacy of live attenuated, cold-adapted, trivalent, intranasal influenzavirus vaccine in children efficacy of vaccination with live attenuated, cold-adapted, trivalent, intranasal influenza virus vaccine against a variant (a/sydney) not contained in the vaccine correlates of immune protection induced by live, attenuated, cold-adapted, trivalent, intranasal influenza virus vaccine evaluation of combined live, attenuated respiratory syncytial virus and parainfluenza 3 virus vaccines in infants and young children phase 2 evaluation of parainfluenza type 3 cold passage mutant 45 live attenuated vaccine in healthy children 6-18 months old distinct migrating and nonmigrating dendritic cell populations are involved in mhc class i-restricted antigen presentation after lung infection with virus the effect of the physical form of poly(lactic-co-glycolic acid) carriers on the humoral immune response to co-delivered antigen adenoviridae: the viruses and their replication the m2-2 protein of human respiratory syncytial virus is a regulatory factor involved in the balance between rna replication and transcription recombinant human metapneumovirus lacking the small hydrophobic sh and/or attachment g glycoprotein: deletion of g yields a promising vaccine candidate severe acute respiratory syndrome coronavirus spike protein expressed by attenuated vaccinia virus protectively immunizes mice neutralizing antibody and protective immunity to sars coronavirus infection of mice induced by a soluble recombinant polypeptide containing an n-terminal segment of the spike glycoprotein human coronavirus hcov-229e enters susceptible cells via the endocytic pathway an outbreak of severe respiratory tract infection due to human metapneumovirus in a long-term care facility proteolytic cleavage of influenza virus hemagglutinins: primary structure of the connecting peptide between ha1 and ha2 determines proteolytic cleavability and pathogenicity of avian influenza viruses positive selection results in frequent reversible amino acid replacements in the g protein gene of human respiratory syncytial virus il-12 is an effective adjuvant for induction of mucosal immunity function of mucosa-associated lymphoid tissue in antibody formation potential of nasopharynx-associated lymphoid tissue for vaccine responses in the airways cd8 t cells utilize trail to control influenza virus infection the magnitude of the t cell response to a clinically significant dose of influenza virus is regulated by trail influenza virus vaccines: lessons from the 2009 h1n1 pandemic parenteral influenza vaccination induces a rapid systemic and local immune response subclass distribution and molecular form of immunoglobulin a hemagglutinin antibodies in sera and nasal secretions after experimental secondary infection with influenza a virus in humans contributions of the structural proteins of severe acute respiratory syndrome coronavirus to protective immunity deletion of m2 gene open reading frames 1 and 2 of human metapneumovirus: effects on rna synthesis, attenuation, and immunogenicity live vaccines for human metapneumovirus designed by reverse genetics virosome-mediated delivery of protein antigens in vivo: efficient induction of class i mhc-restricted cytotoxic t lymphocyte activity nasal immunization with e. coli verotoxin 1 (vt1)-b subunit and a nontoxic mutant of cholera toxin elicits serum neutralizing antibodies activation of cytokine genes in t cells during primary and secondary murine influenza pneumonia transforming growth factor-beta: activation by neuraminidase and role in highly pathogenic h5n1 influenza pathogenesis guidance for industry: clinical data needed to support the licensure of seasonal inactivated influenza vaccines the regulation of iga class switching serologic response to standard inactivated influenza vaccine in human immunodeficiency virus-infected children proinflammatory cytokine responses induced by influenza a (h5n1) viruses in primary human alveolar and bronchial epithelial cells recombinant modified vaccinia virus ankara expressing the spike glycoprotein of severe acute respiratory syndrome coronavirus induces protective neutralizing antibodies primarily targeting the receptor binding region seasonal influenza infection and live vaccine prime for a response to the 2009 pandemic h1n1 vaccine induction of proinflammatory cytokines in human macrophages by influenza a (h5n1) viruses: a mechanism for the unusual severity of human disease? critical regulation of early th17 cell differentiation by interleukin-1 signaling development and persistence of local and systemic antibody responses in adults given live attenuated or inactivated influenza a virus vaccine serum and nasal wash antibodies associated with resistance to experimental challenge with influenza a wild-type virus potent high-affinity antibodies for treatment and prophylaxis of respiratory syncytial virus derived from b cells of infected patients respiratory syncytial virus and metapneumovirus progress in understanding and controlling respiratory syncytial virus: still crazy after all these years a single mutation in the pb1-f2 of h5n1 (hk/97) and 1918 influenza a viruses contributes to increased virulence pulmonary histopathology induced by respiratory syncytial virus (rsv) challenge of formalin-inactivated rsv-immunized balb/c mice is abrogated by depletion of cd4 + t cells enhanced pulmonary histopathology induced by respiratory syncytial virus (rsv) challenge of formalininactivated rsv-immunized balb/c mice is abrogated by depletion of interleukin-4 (il-4) and il-10 the seattle virus watch. vi. observations of infections with and illness due to parainfluenza, mumps and respiratory syncytial viruses and mycoplasma pneumoniae heterosubtypic neutralizing antibodies are produced by individuals immunized with a seasonal influenza vaccine a neutralizing antibody selected from plasma cells that binds to group 1 and group 2 influenza a hemagglutinins an early humoral immune response in peripheral blood following parenteral inactivated influenza vaccination evaluation of a virosomal h5n1 vaccine formulated with matrix m adjuvant in a phase i clinical trial immunology of viral respiratory tract infection in infancy human metapneumovirus fusion protein vaccines that are immunogenic and protective in cotton rats integrin alpha-beta1 promotes infection by human metapneumovirus sublingual immunization induces broad-based systemic and mucosal immune responses in mice the effect of co-administration of adjuvants with a nanoparticle-based genetic vaccine delivery system on the resulting immune responses influenza virosomes are an efficient delivery system for respiratory syncytial virus-f antigen inducing humoral and cell-mediated immunity sublingual vaccination t cell subset-specific susceptibility to aging persistence of respiratory syncytial virus (rsv) infection and development of rsv-specific igg1 response in a guinea-pig model of acute bronchiolitis fatal outcome of human influenza a (h5n1) is associated with high viral load and hypercytokinemia immunization of macaques with formalin-inactivated human metapneumovirus induces hypersensitivity to hmpv infection primary type ii alveolar epithelial cells present microbial antigens to antigen-specific cd4 + t cells a severe acute respiratory syndrome coronavirus that lacks the e gene is attenuated in vitro and in vivo lack of antibody affinity maturation due to poor toll-like receptor stimulation leads to enhanced respiratory syncytial virus disease vaccine efficacy in senescent mice challenged with recombinant sars-cov bearing epidemic and zoonotic spike variants nanoparticles as potential oral delivery systems of proteins and vaccines: a mechanistic approach a randomized, double-blind, placebo-controlled study of an rnaibased therapy directed against respiratory syncytial virus innate antiviral responses by means of tlr7-mediated recognition of single-stranded rna il-18: a th1-inducing, proinflammatory cytokine and new member of the il-1 family il-6 production by pulmonary dendritic cells impedes th1 immune responses effector cd4 + and cd8 + t-cell mechanisms in the control of respiratory virus infections the hidden tonsils of waldeyer's ring the quadrivalent approach to influenza vaccination identification of a novel coronavirus in patients with severe acute respiratory syndrome progress in the development of respiratory syncytial virus and parainfluenza virus vaccines a randomized controlled trial of cold-adapted and inactivated vaccines for the prevention of influenza a disease effectiveness of seasonal influenza vaccines against influenza-associated illnesses among us military personnel in 2010-11: a case-control approach antibody recognition of a highly conserved influenza virus epitope systemic and mucosal immune responses in young children and adults after parenteral influenza vaccination alphavirus replicon particles encoding the fusion or attachment glycoproteins of respiratory syncytial virus elicit protective immune responses in balb/c mice and functional serum antibodies in rhesus macaques correlation of laboratory studies with clinical responses to a/new jersey influenza vaccines human infections with influenza a(h3n2) variant virus in the united states prior h1n1 influenza infection and susceptibility of cleveland family study participants during the h2n2 pandemic of 1957: an experiment of nature cross-protective immunity to influenza a viruses adjuvants in influenza vaccines a prospective study of parainfluenza virus type 4 infections in children attending daycare identification of a linear heparin binding domain for human respiratory syncytial virus attachment glycoprotein g ten years of human metapneumovirus research inactivated influenza vaccines virus-specific cd8 + t cells in primary and secondary influenza pneumonia correlation of cellular immune responses with protection against culture-confirmed influenza virus in young children bacterial toxins as mucosal adjuvants dose dependence of ctl precursor frequency induced by a dna vaccine and correlation with protective immunity against influenza virus challenge a dilemma for mucosal vaccination: efficacy versus toxicity using enterotoxin-based adjuvants initiation of nalt organogenesis is independent of the il-7r, ltbetar, and nik signaling pathways but requires the id2 gene and cd3(−)cd4(+)cd45(+) cells respiratory virus immunization. i. a field trial of two inactivated respiratory virus vaccines; an aqueous trivalent parainfluenza virus vaccine and an alum-precipitated respiratory syncytial virus vaccine dna vaccines: protective immunizations by parenteral, mucosal, and gene-gun inoculations fast rise of broadly cross-reactive antibodies after boosting long-lived human memory b cells primed by an mf59 adjuvanted prepandemic vaccine correlation between humoral and cellular immune responses and the expression of the hepatitis a receptor havcr-1 on t cells after hepatitis a re-vaccination in high and low-responder vaccinees whole inactivated virus influenza vaccine is superior to subunit vaccine in inducing immune responses and secretion of proinflammatory cytokines by dcs the role of the antibody response in influenza virus infection division of labor between dendritic cell subsets of the lung a review of vaccine research and development: human acute respiratory infections the 2009 a (h1n1) influenza virus pandemic: a review parainfluenza virus type 3: seasonality and risk of infection and reinfection in young children risk of primary infection and reinfection with respiratory syncytial virus genetic predisposition to wheeze following respiratory syncytial virus bronchiolitis sphingosine 1-phosphate regulates the egress of iga plasmablasts from peyer's patches for intestinal iga responses antibody response to influenza vaccination in the elderly: a quantitative review immunity to influenza in older adults with chronic obstructive pulmonary disease the efficacy of influenza vaccination in elderly individuals. a randomized double-blind placebo-controlled trial biological challenges and technological opportunities for respiratory syncytial virus vaccine development resistance to and recovery from lethal influenza virus infection in b lymphocyte-deficient mice influenza virus-specific cd4 + t helper type 2 t lymphocytes do not promote recovery from experimental virus infection a live, impaired-fidelity coronavirus vaccine protects in an aged, immunocompromised mouse model of lethal disease inflammatory response of mast cells during influenza a virus infection is mediated by active infection and rig-i signaling update on rhinovirus and coronavirus infections a bovine parainfluenza virus type 3 vaccine is safe and immunogenic in early infancy vaccine delivery using nanoparticles the major human rhinovirus receptor is icam-1 prevention of serious respiratory syncytial virus-related illness. i: disease pathogenesis and early attempts at prevention bovine respiratory syncytial virus iscoms-protection in the presence of maternal antibodies immunity to and frequency of reinfection with respiratory syncytial virus multiple redundant effector mechanisms of cd8 + t cells protect against influenza infection enhanced lung disease and th2 response following human metapneumovirus infection in mice immunized with the inactivated virus structural basis of influenza virus neutralization basic epidemiology and immunopathology of rsv in children respiratory syncytial virus g protein and g protein cx3c motif adversely affect cx3cr1 + t cell responses nonolfactory surface epithelium of the nasal cavity of the bonnet monkey: a morphologic and morphometric study of the transitional and respiratory epithelium a practical influenza neutralization assay to simultaneously quantify hemagglutinin and neuraminidase-inhibiting antibody responses molecular basis for high virulence of hong kong h5n1 influenza a viruses cellular immune responses in children and adults receiving inactivated or live attenuated influenza vaccines antigenic and immunogenic characterization of recombinant baculovirus-expressed severe acute respiratory syndrome coronavirus spike protein: implication for vaccine design phenotypic changes in influenzaspecific cd8 + t cells after immunization of children and adults with influenza vaccines generation of temperature-sensitive human metapneumovirus strains that provide protective immunity in hamsters dynamics of human respiratory virus-specific cd8 + t cell responses in blood and airways during episodes of common cold the toll-like receptor 7 (tlr7)-specific stimulus loxoribine uncovers a strong relationship within the tlr7, 8 and 9 subfamily the viral sigma1 protein and glycoconjugates containing alpha2-3-linked sialic acid are involved in type 1 reovirus adherence to m cell apical surfaces sorting of the respiratory syncytial virus matrix protein into detergent-resistant structures is dependent on cell-surface expression of the glycoproteins immunization of syrian golden hamsters with f subunit vaccine of human metapneumovirus induces protection against challenge with homologous or heterologous strains immunogenicity and efficacy of two candidate human metapneumovirus vaccines in cynomolgus macaques lung epithelial apoptosis in influenza virus pneumonia: the role of macrophage-expressed tnf-related apoptosis-inducing ligand eleven years of inflexal v-a virosomal adjuvanted influenza vaccine crossprotective immunity against influenza ph1n1 2009 viruses induced by seasonal influenza a (h3n2) virus is mediated by virus-specific t-cells the role of serum haemagglutination-inhibiting antibody in protection against challenge infection with influenza a2 and b viruses live and inactivated influenza vaccines induce similar humoral responses, but only live vaccines induce diverse t-cell responses in young children regulation of immunological homeostasis in the respiratory tract clearance of sendai virus by cd8 + t cells requires direct targeting to virus-infected epithelium the immunostimulating complex (iscom) is an efficient mucosal delivery system for respiratory syncytial virus (rsv) envelope antigens inducing high local and systemic antibody responses antibody responses after influenza and pneumococcal immunization in hiv-infected homosexual men the virosome concept for influenza vaccines antiviral cd8 + t cell effector activities in situ are regulated by target cell type respiratory virus infection of mice provokes a permanent humoral immune response inflammasome recognition of influenza virus is essential for adaptive immune responses high frequency of cross-reacting antibodies against 2009 pandemic influenza a(h1n1) virus among the elderly in finland nasal vaccination: a non-invasive vaccine delivery method that holds great promise for the future chitosan as a novel nasal delivery system for vaccines adenovirus infections in transplant recipients nasal vaccine innovation human rhinoviruses persistence of influenza virus-specific antibody-secreting cells and b-cell memory after primary murine influenza virus infection human piv-2 recombinant sendai virus (rsev) elicits durable immunity and combines with two additional rsevs to protect against hpiv-1, hpiv-2, hpiv-3, and rsv epidemiology of human metapneumovirus methods for nano-particle based vaccine formulation and evaluation of their immunogenicity antibodies against trimeric s glycoprotein protect hamsters against sars-cov challenge despite their capacity to mediate fcgammarii-dependent entry into b cells in vitro critical role for cryopyrin/nalp3 in activation of caspase-1 in response to viral infection and double-stranded rna an epidemiologic study of altered clinical reactivity to respiratory syncytial (rs) virus infection in children previously vaccinated with an inactivated rs virus vaccine parainfluenza viruses a live attenuated bovine parainfluenza virus type 3 vaccine is safe, infectious, immunogenic, and phenotypically stable in infants and children identification of a recombinant live attenuated respiratory syncytial virus vaccine candidate that is highly attenuated in infants combinatorial antibody libraries from survivors of the turkish h5n1 avian influenza outbreak reveal virus neutralization strategies cell type-specific involvement of rig-i in antiviral response differential roles of mda5 and rig-i helicases in the recognition of rna viruses sequence requirements for cleavage activation of influenza virus hemagglutinin expressed in mammalian cells pathogenesis of rhinovirus infection future influenza vaccines and the use of genetic recombinants respiratory syncytial virus disease in infants despite prior administration of antigenic inactivated vaccine cell-mediated immunity to respiratory syncytial virus induced by inactivated vaccine or by infection nalt-versus peyer's-patch-mediated mucosal immunity dna vaccines: safety and efficacy issues immunity to respiratory viruses type i interferons regulate cytolytic activity of memory cd8(+) t cells in the lung airways during respiratory virus challenge measles virus v protein inhibits nlrp3 inflammasome-mediated interleukin-1beta secretion plasmacytoid dendritic cells delineate immunogenicity of influenza vaccine subtypes influenza virus hemagglutinin stalk-based antibodies and vaccines human metapneumovirus antibody response after influenza vaccination in hiv-infected individuals: a consecutive 3-year study a novel coronavirus associated with severe acute respiratory syndrome pathogenesis of influenza virus infections: the good, the bad and the ugly safety and immunogenicity of an adjuvanted whole virion, inactivated a (h1n1) 2009 influenza vaccine in young and elderly adults, and children plasma-cell homing ccr10 expression is a common feature of circulating and mucosal epithelial tissue iga ab-secreting cells immunogenicity and safety of low dose virosomal adjuvanted influenza vaccine administered intradermally compared to intramuscular full dose administration m cell-targeted delivery of vaccines and therapeutics the role of nasopharyngeal lymphoid tissue pattern recognition receptors tlr4 and cd14 mediate response to respiratory syncytial virus a nontoxic chimeric enterotoxin adjuvant induces protective immunity in both mucosal and systemic compartments with reduced ige antibodies from plasmids to protection: a review of dna vaccines against infectious diseases heterosubtypic protection against pathogenic human and avian influenza viruses via in vivo electroporation of synthetic consensus dna antigens pandemic whole-virion, vero-cell-derived, adjuvant-free influenza a h1n1 vaccine in patients with solid tumors and hematologic malignancies receiving concurrent anticancer treatment: immunogenicity, tolerability, and acceptability during the pandemic situation coronaviridae influenza vaccines for the future mechanism of action of clinically approved adjuvants live attenuated influenza vaccine (laiv) impacts innate and adaptive immune responses role of il-1 beta in the development of human t(h)17 cells: lesson from nlpr3 mutated patients the magnitude of local immunity in the lungs of mice induced by live attenuated influenza vaccines is determined by local viral replication and induction of cytokines the contribution of systemic and pulmonary immune effectors to vaccine-induced protection from h5n1 influenza virus infection activation, differentiation, and migration of naive virus-specific cd8 + t cells during pulmonary influenza virus infection a common mucosal chemokine (mucosaeassociated epithelial chemokine/ccl28) selectively attracts iga plasmablasts cutting edge: influenza a virus activates tlr3-dependent inflammatory and rig-i-dependent antiviral responses in human lung epithelial cells immunosuppressive hla-g molecule is upregulated in alveolar epithelial cells after influenza a virus infection cd40, but not cd154, expression on b cells is necessary for optimal primary b cell responses measuring antibody responses to a live attenuated influenza vaccine in children cd4 t cell-independent antibody response promotes resolution of primary influenza infection and helps to prevent reinfection comprehensive serotyping and epidemiology of human adenovirus isolated from the respiratory tract of korean children over 17 consecutive years (1991-2007) lymph node dendritic cells control cd8 + t cell responses through regulated fasl expression antigen sparing and cross-reactive immunity with an adjuvanted rh5n1 prototype pandemic influenza vaccine: a randomised controlled trial optimism over vaccines administered via mucosal surfaces molecular basis of replication of duck h5n1 influenza viruses in a mammalian mouse model preliminary assessment of the efficacy of a t-cell-based influenza vaccine, mva-np+m1, in humans safety and immunogenicity of an inactivated adjuvanted whole-virion influenza a (h5n1) vaccine: a phase i randomised controlled trial safety and immunogenicity from a phase i trial of inactivated severe acute respiratory syndrome coronavirus vaccine defective immunoregulation in rsv vaccine-augmented viral lung disease restored by selective chemoattraction of regulatory t cells lymphoid tissue in the nasal mucosa of primates, with particular reference to intraepithelial lymphocytes type i ifns enhance the terminal differentiation of dendritic cells the role of animal models in influenza vaccine research influenza vaccine-live toll-like receptor 9-mediated recognition of herpes simplex virus-2 by plasmacytoid dendritic cells adaptation and growth characteristics of influenza virus at 25 °c trivalent inactivated influenza vaccine in african adults infected with human immunodeficient virus: double blind, randomized clinical trial of efficacy, immunogenicity, and safety the histological features of the immune system of the equine respiratory tract the distribution of mucosal lymphoid nodules in the equine respiratory tract viruses and bacteria in the etiology of the common cold enhanced mucosal and systemic immune response with intranasal immunization of mice with hiv peptides entrapped in plg microparticles in combination with ulex europaeus-i lectin as m cell target initial infectious dose dictates the innate, adaptive, and memory responses to influenza in the respiratory tract type i interferons keep activated t cells alive a sars dna vaccine induces neutralizing antibody and cellular immune responses in healthy adults in a phase i clinical trial antigenic structure of the human respiratory syncytial virus g glycoprotein and relevance of hypermutation events for the generation of antigenic variants the inflammasome: a molecular platform triggering activation of inflammatory caspases and processing of proil-beta subcellular localization of toll-like receptor 3 in human dendritic cells the functional heterogeneity of type 1 effector t cells in response to infection is related to the potential for ifngamma production as03-adjuvanted versus non-adjuvanted inactivated trivalent influenza vaccine against seasonal influenza in elderly people regulation of iga synthesis and immune response by t cells and interleukins innate immune control and regulation of influenza virus infections il-15 trans-presentation by pulmonary dendritic cells promotes effector cd8 t cell survival during influenza virus infection host dna released in response to aluminum adjuvant enhances mhc class ii-mediated antigen presentation and prolongs cd4 t-cell interactions with dendritic cells memory cd4 + t cells protect against influenza through multiple synergizing mechanisms structure of respiratory syncytial virus fusion glycoprotein in the postfusion conformation reveals preservation of neutralizing epitopes the human cytotoxic t cell response to influenza a vaccination adaptive strategies of the influenza virus polymerase for replication in humans live attenuated influenza vaccine induces cross-reactive antibody responses in children against an a/fujian/411/2002-like h3n2 antigenic variant strain immunoglobulin a (iga): molecular and cellular interactions involved in iga biosynthesis and immune response intestinal iga: novel views on its function in the defence of the largest mucosal surface the influence of hiv infection on antibody responses to a two-dose regimen of influenza vaccine human genetic factors and respiratory syncytial virus disease severity oral immunization with influenza virus in biodegradable microspheres human immune responses to influenza virus vaccines administered by systemic or mucosal routes respiratory syncytial virus synergizes with th2 cytokines to induce optimal levels of tarc/ccl17 comparative efficacy of inactivated and live attenuated influenza vaccines naive cd4(+) t cell frequency varies for different epitopes and predicts repertoire diversity and response magnitude iscom, a delivery system for parenteral and mucosal vaccination adjuvant system as03 containing alpha-tocopherol modulates innate immune response and leads to improved adaptive immunity h5n1 influenza vaccine formulated with as03 a induces strong crossreactive and polyfunctional cd4 t-cell responses roles of cd4 + t-cell-independent and -dependent antibody responses in the control of influenza virus infection: evidence for noncognate cd4 + t-cell activities that enhance the therapeutic activity of antiviral antibodies antigenicity and immunogenicity of equine influenza vaccines containing a carbomer adjuvant the molecular basis of the pathogenicity of the dutch highly pathogenic human influenza a h7n7 viruses formalin-inactivated respiratory syncytial virus vaccine induces antibodies to the fusion glycoprotein that are deficient in fusion-inhibiting activity association of serum anti-neuraminidase antibody with resistance to influenza in man pathological study of archival lung tissues from five fatal cases of avian h5n1 influenza in vietnam systems biology of vaccination for seasonal influenza in humans alum-adjuvanted h5n1 whole virion inactivated vaccine (wiv) induced igg1 and igg4 antibody responses in young children mucosal vaccines: the promise and the challenge influenza virus-infected epithelial cells present viral antigens to antigen-specific cd8 + cytotoxic t lymphocytes gamma interferon is not required for mucosal cytotoxic t-lymphocyte responses or heterosubtypic immunity to influenza a virus infection in mice heterosubtypic immunity to influenza a virus infection requires b cells but not cd8 + cytotoxic t lymphocytes heterosubtypic immunity to influenza a virus infection requires a properly diversified antibody repertoire a novel m cell-specific carbohydrate-targeted mucosal vaccine effectively induces antigenspecific immune responses a 39-kda protein on activated helper t cells binds cd40 and transduces the signal for cognate activation of b cells endogenous naive cd8 + t cell precursor frequency regulates primary and memory responses to infection vaccination strategies for mucosal immune responses recent developments in adjuvants for vaccines against infectious diseases prevention of antigenically drifted influenza by inactivated and live attenuated vaccines prevention of symptomatic seasonal influenza in 2005-2006 by inactivated and live attenuated vaccines caspase-1, caspase-8, and calpain are dispensable for il-33 release by macrophages a common neutralizing epitope conserved between the hemagglutinins of influenza a virus h1 and h2 strains protection against the mouse-adapted a/fm/1/47 strain of influenza a virus in mice by a monoclonal antibody with cross-neutralizing activity among h1 and h2 strains efficacy and effectiveness of influenza vaccines: a systematic review and metaanalysis effect of priming with h1n1 influenza viruses of variable antigenic distances on challenge with 2009 pandemic h1n1 virus orthomyxoviridae: the viruses and their replication the distinguishing features of human metapneumovirus and respiratory syncytial virus human immunosenescence: is it infectious? immunol toward a universal influenza virus vaccine: prospects and challenges rig-i-mediated antiviral responses to single-stranded rna bearing 5'-phosphates sequential annual administration of purified fusion protein vaccine against respiratory syncytial virus in children with cystic fibrosis differential cmv-specific cd8 + effector t cell responses in the lung allograft predominate over the blood during human primary infection upon viral exposure, myeloid and plasmacytoid dendritic cells produce 3 waves of distinct chemokines to recruit immune effectors immunogenetics of seasonal influenza vaccine response global transcriptome analysis in influenza-infected mouse lungs reveals the kinetics of innate and adaptive host immune responses type i ifn triggers rig-i/tlr3/ nlrp3-dependent inflammasome activation in influenza a virus infected cells immunity to influenza in ferrets. i. response to live and killed virus immunity to influenza in ferrets. ii. influence of adjuvants on immunization safety and immunogenicity of a novel recombinant subunit respiratory syncytial virus vaccine (bbg2na) in healthy young adults effect of age on cytotoxic t lymphocyte memory as well as serum and local antibody responses elicited by inactivated influenza virus vaccine systemic and local antibody responses in elderly subjects given live or inactivated influenza a virus vaccines in elderly persons live attenuated influenza a virus vaccines do not offer an advantage over inactivated virus vaccine in inducing serum or secretory antibodies or local immunologic memory alveolar macrophages are a major determinant of early responses to viral lung infection but do not influence subsequent disease development quantitative aspects of passive immunity to respiratory syncytial virus infection in infant cotton rats type i ifn as a natural adjuvant for a protective immune response: lessons from the influenza vaccine model influenza m1 vlps containing neuraminidase induce heterosubtypic cross-protection serum antibody prevents lethal murine influenza pneumonitis but not tracheitis bronchus-associated lymphoid tissue (balt) structure and function microencapsulated human parainfluenza virus induces a protective immune response characterization of a live, attenuated human parainfluenza type 3 virus candidate vaccine strain targeting dendritic cells with biomaterials: developing the next generation of vaccines immunoglobulin a mediation of murine nasal anti-influenza virus immunity passive transfer of local immunity to influenza virus infection by iga antibody in vitro comparison of the biologic activities of monoclonal monomeric iga, polymeric iga, and secretory iga role of iga versus igg in the control of influenza viral infection in the murine respiratory tract cytotoxic t lymphocyte memory: role in cross-protective immunity against influenza? induction of protective immunity against influenza virus in a macaque model: comparison of conventional and iscom vaccines interleukin (il)-6 directs the differentiation of il-4-producing cd4 + t cells cd4 effector t cell subsets in the response to influenza: heterogeneity, migration, and function lack of cd200 enhances pathological t cell responses during influenza infection a fused gene of nucleoprotein (np) and herpes simplex virus genes (vp22) induces highly protective immunity against different subtypes of influenza virus the mucosal immune system of the respiratory tract current status of vaccines for parainfluenza virus infections progress in respiratory virus vaccine development mucosal immunization of rhesus monkeys against respiratory syncytial virus subgroups a and b and human parainfluenza virus type 3 by using a live cdna-derived vaccine based on a host range-attenuated bovine parainfluenza virus type 3 vector backbone pathogenesis of acute respiratory illness caused by human parainfluenza viruses comparative evaluation of two severe acute respiratory syndrome (sars) vaccine candidates in mice challenged with sars coronavirus tlr2/ myd88/nf-kappab pathway, reactive oxygen species, potassium efflux activates nlrp3/asc inflammasome during respiratory syncytial virus infection recombinant iga is sufficient to prevent influenza virus transmission in guinea pigs adjuvanticity of the oil-in-water emulsion mf59 is independent of nlrp3 inflammasome but requires the adaptor protein myd88 immunogenic properties of rsv-b1 fusion (f) protein gene-encoding recombinant adenoviruses lectin histochemistry reveals the appearance of m-cells in peyer's patches of scid mice after syngeneic normal bone marrow transplantation antigen-bearing dendritic cells regulate the diverse pattern of memory cd8 t-cell development in different tissues iga class switch occurs in the organized nasopharynx-and gut-associated lymphoid tissue, but not in the diffuse lamina propria of airways and gut sublingual immunization with m2-based vaccine induces broad protective immunity against influenza isotype-specific selection of high affinity memory b cells in nasalassociated lymphoid tissue young infants can develop protective levels of neutralizing antibody after infection with respiratory syncytial virus avian flu: influenza virus receptors in the human airway reappearance of h1n1 influenza virus in man: evidence for the persistence of the virus in domestic chickens effectiveness of as03 adjuvanted pandemic h1n1 vaccine: case-control evaluation based on sentinel surveillance system in canada long-lived plasma cells: a mechanism for maintaining persistent antibody production phase 1 clinical trials of the safety and immunogenicity of adjuvanted plasmid dna vaccines encoding influenza a virus h5 hemagglutinin a critical function for cd200 in lung immune homeostasis and the severity of influenza infection sublingual vaccination with influenza virus protects mice against lethal viral infection receptor-mediated immunoglobulin g transport across mucosal barriers in adult life: functional expression of fcrn in the mammalian lung a double-inactivated whole virus candidate sars coronavirus vaccine stimulates neutralising and protective antibody responses il-1 is an effective adjuvant for mucosal and systemic immune responses when coadministered with protein immunogens sars vaccine protective in mice activation of virus replication after vaccination of hiv-1-infected individuals sars vaccines the prospects and challenges of universal vaccines for influenza a single amino acid in the pb2 gene of influenza a virus is a determinant of host range development of effective vaccines against pandemic influenza structural and functional bases for broad-spectrum neutralization of avian and human influenza a viruses advances in the vaccination of the elderly against influenza: role of a high-dose vaccine effector t cells control lung inflammation during acute influenza virus infection by producing il-10 influenza virus iscoms: antibody response in animals structural basis for immunization with postfusion respiratory syncytial virus fusion f glycoprotein (rsv f) to elicit high neutralizing antibody titers new horizon of mucosal immunity and vaccines protective effect of serum antibody on respiratory infection of influenza c virus in rats effects of human metapneumovirus and respiratory syncytial virus antigen insertion in two 3' proximal genome positions of bovine/ human parainfluenza virus type 3 on virus replication and immunogenicity parainfluenza virus type 3 expressing the native or soluble fusion (f) protein of respiratory syncytial virus (rsv) confers protection from rsv infection in african green monkeys a host-range restricted parainfluenza virus type 3 (piv3) expressing the human metapneumovirus (hmpv) fusion protein elicits protective immunity in african green monkeys development of a piv-vectored rsv vaccine: preclinical evaluation of safety, toxicity, and enhanced disease and initial clinical testing in healthy adults enhanced protective immunity against h5n1 influenza virus challenge by vaccination with dna expressing a chimeric hemagglutinin in combination with an mhc class i-restricted epitope of nucleoprotein in mice immunosenescence: role and measurement in influenza vaccine response among the elderly identification of nucleolin as a cellular receptor for human respiratory syncytial virus memory cd4 t cells direct protective responses to influenza virus in the lungs through helper-independent mechanisms identification of the respiratory syncytial virus proteins required for formation and passage of helper-dependent infectious particles recombinant respiratory syncytial virus that does not express the ns1 or m2-2 protein is highly attenuated and immunogenic in chimpanzees the intracellular sensor nlrp3 mediates key innate and healing responses to influenza a virus via the regulation of caspase-1 cytokines: the future of intranasal vaccine adjuvants mortality associated with influenza and respiratory syncytial virus in the united states heterosubtypic neutralizing monoclonal antibodies cross-protective against h5n1 and h1n1 recovered from human igm + memory b cells a distinct lineage of influenza a virus from bats new world bats harbor diverse influenza a viruses. plos pathog. 9 control of adenovirus acute respiratory disease in u.s. army trainees cd8 + t cells clear influenza virus by perforin or fas-dependent processes clearance of an influenza a virus by cd4 + t cells is inefficient in the absence of b cells quantitative analysis of the influenza virus-specific cd4 + t cell memory in the absence of b cells and ig sars coronavirus pathogenesis: host innate immune responses and viral antagonism of interferon functional significance of the perforin/ granzyme cell death pathway evaluation of a recombinant hemagglutinin expressed in insect cells as an influenza vaccine in young and elderly adults recruitment and proliferation of cd8 + t cells in respiratory virus infections cx3c chemokine mimicry by respiratory syncytial virus g glycoprotein immunization with sars coronavirus vaccines leads to pulmonary immunopathology on challenge with the sars virus tight junctionbased epithelial microenvironment and cell proliferation heterologous protection against influenza by injection of dna encoding a viral protein generation of mhc class i-restricted cytotoxic t lymphocytes by expression of a viral protein in muscle cells: antigen presentation by non-muscle cells toward the development of dna vaccines protective cd4 + and cd8 + t cells against influenza virus induced by vaccination with nucleoprotein dna impaired mucosal immune responses in interleukin 4-targeted mice pathogenesis of respiratory syncytial virus immunopathology of rsv infection: prospects for developing vaccines without this complication host defenses against influenza virus: the role of anti-hemagglutinin antibody innate immunity to respiratory viruses live attenuated or inactivated influenza vaccines and medical encounters for respiratory illnesses among us military personnel pattern of respiratory syncytial virus epidemics in finland: two-year cycles with alternating prevalence of groups a and b respiratory synctial virus infection in balb/c mice previously immunized with formalin-inactivated virus induces enhanced pulmonary inflammatory response with a predominant th2-like cytokine pattern immunization with modified vaccinia virus ankara-based recombinant vaccine against severe acute respiratory syndrome is associated with enhanced hepatitis in ferrets summary of probable sars cases with onset of illness from 1 middle east respiratory syndrome coronavirus (mers-cov) summary and literature update -as of 11 mouse respiratory tract dendritic cell subsets and the immunological fate of inhaled antigens direct gene transfer into mouse muscle in vivo heterogeneity of cd4(+) and cd8(+) t cells antiviral memory t-cell responses in the lung rapid cloning of high-affinity human monoclonal antibodies against influenza virus evaluation of a live, cold-passaged, temperature-sensitive, respiratory syncytial virus vaccine candidate in infancy the absence of enhanced disease with wild type respiratory syncytial virus infection occurring after receipt of live, attenuated, respiratory syncytial virus vaccines orthomyxoviruses m cell-targeted dna vaccination pathogen recognition and development of particulate vaccines: does size matter? a dna vaccine induces sars coronavirus neutralization and protective immunity in mice influenza a virus nucleoprotein is a major target antigen for cross-reactive antiinfluenza a virus cytotoxic t lymphocytes the rna helicase rig-i has an essential function in double-stranded rna-induced innate antiviral responses poly(lactic-co-glycolic acid) enhances maturation of human monocyte-derived dendritic cells neutralizing antibodies derived from the b cells of 1918 influenza pandemic survivors mucosal vaccines: novel advances in technology and delivery augmentation of immune responses to sars coronavirus by a combination of dna and whole killed virus vaccines isolation of a novel coronavirus from a man with pneumonia in saudi arabia immunogenicity, safety, and protective efficacy of an inactivated sarsassociated coronavirus vaccine in rhesus monkeys a recombinant baculovirus-expressed s glycoprotein vaccine elicits high titers of sars-associated coronavirus (sars-cov) neutralizing antibodies in mice key: cord-254766-585iu5ey authors: tauro, sharyn; su, yung-chang; thomas, sandra; schwarze, jürgen; matthaei, klaus i.; townsend, dijana; simson, ljubov; tripp, ralph a.; mahalingam, suresh title: molecular and cellular mechanisms in the viral exacerbation of asthma date: 2008-08-13 journal: microbes infect doi: 10.1016/j.micinf.2008.07.037 sha: doc_id: 254766 cord_uid: 585iu5ey the aetiology of asthma associated with viral infection is complex. the dynamics that contribute to disease pathogenesis are multifactorial and involve overlapping molecular and cellular mechanisms, particularly the immune response to respiratory virus infection or allergen sensitization. this review summarizes the evidence associated with factors that may contribute to the development or exacerbation of asthma including age, host factors, genetic polymorphisms, altered immune responses, and aspects of viral antigen expression. this review also provides an important perspective of key events linked to the development of asthmatic disease and related pulmonary inflammation from human and animal studies, and discusses their relationship as targets for disease intervention strategies. asthma is a chronic, reversible inflammatory disorder of the airways characterized by laboured breathing due to variable and reversible airway flow limitation. asthmatics have sensitive airways that react to stimuli with inflammation, swelling of the airway lining, muscle tightening and mucus production; all of which make breathing more difficult. the symptoms can be one or more of the following: wheezing, coughing, tightness in the chest and shortness of breath. the prevalence or disease burden of asthma varies from country to country. it is estimated that there are approximately 300 million affected individuals worldwide [1] . annual worldwide deaths from asthma have been estimated at 250,000. the highest prevalence of asthma is in the uk, australia, new zealand and ireland [1] . in australia, 12% of children and young adults aged 0e24 years have asthma [2] and expenditure on asthma in 2000 was estimated at aus$693 million [3] . in the usa, 20 million people are affected by asthma and the total direct and indirect costs of asthma exceeded us$11.3 billion in 1998 [4] . the underlying causes of asthma are not well understood but many factors are recognized as contributing to the disease, including an atopic (allergic) disposition (often a family history of allergies and asthma), certain genetic mutations, exertion, irritants (including smoking, indoor and outdoor air pollutants), both inhaled and food allergens (e.g. house dust mites, mould spores, pollens, peanuts, egg), and viral infections [3] . in people with underlying asthma, respiratory viruses frequently trigger exacerbation of the disease, with 80e90% of exacerbations in children associated with respiratory infections [5] and up to two thirds of cases in adults [5] . respiratory viruses are also thought to have a role in people becoming sensitive to allergens and developing asthma [9, 10] . asthma is a multifactorial disease, with the genetics of the host interacting with a variety of environmental triggers. viral infections are one of the environmental triggers, having been implicated both in the induction of the disease and, more frequently, in its exacerbation. a number of viruses are associated with the exacerbation of asthma in children and adults. the most common viruses detected in connection with the symptoms of asthma are rhinoviruses (rv); however, human respiratory syncytial virus (rsv), human metapneumovirus (hmpv), parainfluenza and influenza viruses, adenoviruses and coronaviruses are also thought to contribute to the exacerbation of asthma [5, 6] . the characteristics of the main viruses are listed in table 1 . for some of these viruses, there is evidence from both epidemiological and experimental studies to confirm the role of a viral infection in exacerbating asthma, while for other viruses the evidence is less clear. the immune responses triggered by viral infections and allergens are well established (fig. 1 ). this review focuses on the association between viruses and the exacerbation of asthma and outlines current understanding of the molecular and cellular mechanisms involved. the inflammatory cascade associated with asthma involves mast cells, dc, t cells, eosinophils, macrophages, fibroblasts and neutrophils (table 2) . when the inflammation intensifies, the airways become very sensitive to provoking stimuli and airway hyper-responsiveness (ahr) develops. the mast cells degranulate and they, and other cells, release chemical mediators into the lower respiratory tract that prolong the response and cause contraction of the bronchial smooth muscles (table 2) . these actions, and airway swelling, mucus secretion and inflammation, contribute to the bronchoconstriction and airway obstruction seen in asthma attacks. a number of factors are known to affect the likelihood of asthma being exacerbated in response to a viral infection (fig. 2 ). these factors include the age at which the first viral infection occurs, the gender, genetic makeup of the host, the ability of the virus to persistently infect or to become latent in the host and viral antigens. viral infections that occur early in life appear to have an important role in shaping immunological development. if initial infection occurs early in life, the t helper 1 (th1) response may be weak, allowing the development of stronger th2 responses to challenges later in life. mice initially infected with rsv as neonates were more likely to have a th2 type response upon secondary infection, whereas those first infected when older were more likely to have a th1 type response [7] . one interpretation of these findings is that rsv infection in neonates suppresses the development of the th1 immune response and a th2 response occurs. in contrast, if the initial infection is delayed for several weeks, a th1 immune response is mounted which lacks the pathogenic th2 component associated with asthma. there is some evidence that infection at one stage in life may lead to a reduction in atopy while, at another stage, it toll like receptor tnf-a tumour necrosis factor-alpha may potentiate the emergence of allergies [8] and that early severe rsv infections in infancy leads to allergic asthma later in life [9, 10] . however, this issue is controversial. boys are more likely to have severe asthma than girls; however at puberty (about age 13e14) the ratios reverse and many more adult women are admitted to hospital for asthma than adult men [11] . the reasons for this switch in susceptibility are not known but this observation suggests a potential role for sex hormones in the development of asthma. a predisposition towards developing asthma runs in families, indicating a genetic component to the disease that has been confirmed by studies in twins [12] . specific genes have been associated with asthma in nearly 200 studies, with 64 different genes associated with asthma or atopy identified in one study [13] . some of these genes have repeatedly been found to be associated with asthma, including the th2 cytokines il-4 and il-13, human leukocyte antigen drb1 (hla-drb1), tnf-a, lymphotoxin-alpha (la-a), high-affinity ige receptor (fc3r1a) and the il-4 receptor (il-4r). the gene b2 adrenergic receptor (adrb2) has been associated with asthma severity [13] . other genes associated with atopy and asthma include, for atopy, pattern recognition receptor cd14 (receptor for endotoxins such as lipopolysaccharide (lps)) and granulocytemacrophage colony stimulating factor (gmcsf); for asthma, clara cell secretory protein (cc16), cd14, acyloxyacyl hydroxylase (aoah), tnf-a, leukotriene c4 (ltc 4 ) synthase, il-10 and il-8; and, for asthma severity, adrb2 [14, 15] . host genetics is also thought to affect the severity of viral infections and the type of immune response to the infection. for example, specific alleles of pulmonary surfactant genes have been associated with more severe rsv infections in infants [16] . surfactant proteins line the alveolar surface of the lungs and are essential for normal respiratory function. other genetic variations have been shown to affect the expression of cytokines in response to viral infections. a variation causing an increase in il-8 transcription was found to lead to enhanced susceptibility to rsv induced bronchiolitis [17] and a variant affecting transcription of il-4 was found to be associated with severe rsv disease and elevated levels of ige [18] . similarly, variations in genes for il-10 have been identified, with those people heterozygous for the gene found to be less likely to develop rsv bronchiolitis [19] , and two distinct mutations in the gene for toll like receptor 4 (tlr-4) have been found to be associated with severe rsv bronchiolitis [20] . an association between differences in il-13 genes and severe rsv infection has also been found [21] . environmental factors, such as viral infections, may impact on genetically predisposed individuals to affect the development of asthma and the viral exacerbation of asthma. a number of respiratory viruses have been found to persist in the host, despite the host mounting an immune response. for example, hmpv can persist in the lungs of mice for up to 60 days, despite the presence of neutralizing antibody [22] . in mice, rsv was found to persist in the lungs for more than 100 days, despite normal cytotoxic t-cell responses and normal rsv specific antibodies [23] and viral proteins have been found in guinea pigs 6 weeks after rsv infection, despite the presence of neutralizing antibodies [24] . the evidence about respiratory viral persistence in humans is less clear, with several recent studies finding directly opposing results. a study by wilkinson et al. found that rsv persisted in patients with chronic obstructive pulmonary disease (copd) [25] , while falsey et al. found no rsv persistence associated with copd [26] . studies on viral persistence with the other main virus associated with the exacerbation of asthma, rv, have also produced opposing results. in one study, 95% of adults with an exacerbation of asthma were found to be clear of rv 6 weeks after the exacerbation [27] , whereas a study in children found that more than 40% still had detectable rv after 6 weeks and this viral persistence was associated with more severe acute exacerbations of asthma [28] . a possible mechanism for immune evasion is persistence at a site that avoids immune system surveillance. with respiratory viruses, such a site may be the pulmonary neurons and rsv infection of nerve cells has recently been demonstrated [29] . in addition, a role for cytokine in viral persistence was demonstrated in mice by alvarez and tripp. they showed that the persistent presence of hmpv is associated with increased il-10 expression and weak ctl activity [30] . it is currently not clear whether respiratory viruses persist in the host, despite a functional immune response. further characterization is required to understand the possible relationships between respiratory viral latency, the host's immune responses and the exacerbation of asthma. it is well established that rsv g protein can influence the immune response in murine models. the g protein seems to elicit pre-dominantly a th2 driven response characterized by lung eosinophilia and local production of il-4 and il-5 [31] . prior sensitization with a recombinant vaccinia virus expressing rsv g protein leads to a th2 driven augmented disease, contrasting with the usual th1 response seen in primary viral infections [31] . studies have also shown that the non-structural proteins of rsv and influenza virus are capable of modulating the immune response through their ability to inhibit ifn responses in the infected host [31] . released mediators derived from neural signalling pathways contribute to the bronchoconstriction associated with asthma. studies in humans and animals have shown that an intact nervous system contributes to virus-induced ahr. in a guinea pig model, cutting the vagus nerve reduced airway responsiveness to histamines in animals previously infected with parainfluenza virus [32] . in humans, virus induced ahr was also vagally mediated [33] . in guinea pigs, rats and cats, the efferent, parasympathetic bronchoconstrictor limb of the ). the production of il-12 and the binding of antigen-mhc molecules commit the differentiation of na€ ıve t cells to the th1 cell subset that secretes th1 cytokines including ifn-g. the cytokine contributes to the activation of macrophage (make more il-12), b cells (make igg2a) and cytotoxic t cell (kill infected cells). an ifn-g dominated microenvironment inhibits the development of a th2 cell subset. together, these responses result in the resolution of the infection in the airways. (b) in the early phase of allergen exposure, cross linking of antigen-specific ige on the surface of mast cells results in the activation and release of mediators that cause bronchoconstriction and inflammation. activated mast cells also produce il-4 that commits na€ ıve t cells to the th2 subset as well as b cell isotype class switching to ige production. in addition, antigenic peptide is presented to na€ ıve t cells by apcs in the context of mhc class ii and co-stimulatory signals. in an il-4 dominated microenvironment, this triggers the differentiation of na€ ıve t cells to th2 cell subset that generates th2 cytokines (il-4, il-5, il-10 and il-13). these cytokines are responsible for orchestrating the late phase of the allergic response. il-4 and il-13 contribute to mast cell activation and the synthesis of ige. il-5 is implicated in eosinophilia and is known to stimulate these cells, resulting in degranulation and release of toxic basic proteins (e.g. ecp, mbp). il-10 inhibits apcs, therefore preventing il-12 from initiating a th1 immune response. cough and bronchoconstriction reflexes was enhanced during acute viral illness [32, 34] . acetylcholine is released from the nerve fibres that innervate smooth muscle in the airways and binds to m 3 muscarinic receptors on the smooth muscle and causes bronchoconstriction. this is normally limited because acetylcholine also binds to m 2 muscarinic receptors on neurons that inhibit further release of acetylcholine [35] . dysfunction of these m 2 receptors is thought to contribute to ahr during viral infections [34, 36] . a number of factors are thought to cause this receptor dysfunction including viral neuraminidase [37] , endogenous tachykinins [35] , induced inflammatory cell products, such as eosinophil cationic proteins (ecp) [37] , eosinophil major basic protein (mbp) [38] , ifns from macrophages or macrophage-stimulated t cells or nitric oxide (no) released from virus-activated macrophages [39] . the effects on the m 2 receptor function are transient and normal functions are restored several weeks after resolution of the acute infection [34] . stimulation of the nerve fibres during viral infection augments the release of neuropeptides, including somatostatin, calcitonin gene-related peptide (cgrp) and the tachykinins substance p and neurokinin a. a number of these neuropeptides are associated with symptoms of the exacerbation of asthma. for example, cgrp can cause eosinophilia in rat lungs [40] , substance p and neurokinin a are associated with an increase in eosinophil numbers in the airways [41] , and substance p activates eosinophils and mast cells and enhances the degranulation of eosinophils and the release of histamines from mast cells [42] . cgrp also inhibits the production of th1 cytokines, therefore unbalancing the th1/th2 equation in favour of a th2 response [43] . a role for the neurotrophin nerve growth factor (ngf) in viral induced asthma is suggested by results finding that ngf is increased in rsv infected rats, compared to control animals [44] . ngf up-regulates the expression of the high affinity receptor for substance p (the nk1 receptor) and substance p contributes to the neurogenic inflammation seen in rsvinfected airways [45] . ngf expression in the lungs normally declines as animals age but rsv infection interferes with this decline [44] . ngf may lead to short-and long-term changes in the distribution and development of nerves and changes in the reactivity of sensory nerves in the respiratory tract [44] and may also contribute to ahr, inflammation and the activation of recruited eosinophils and mast cells [46] . in understanding the mechanisms underlying viral exacerbation of asthma, it is helpful to observe models of primary virus infection. respiratory viruses including parainfluenza, influenza and rsv have been shown to induce airway inflammation and lung function changes in rodent models. in rsv infection in mice, airway inflammation and the development of ahr are dependent on the presence of the th2 cytokines il-5 [47] and il-13 [48] and not on ifn-g, the most abundant cytokine produced during rsv infection. in addition, inflammation and lung function changes in this model depend on cd8 þ t cells [49] . taken together this suggests that type 2 cytokine producing cd8 þ t cells may drive virus induced reactive airway disease. further, respiratory viral infections lead to a marked expansion of mature dc in the lung [50] . these mature lung dc have a strong capacity to activate both na€ ıve and memory t cells and to induce their proliferation. in a model of influenza infection, maturation of lung dc resulted in strong immunogenicity of an otherwise non-immunogenic antigen [50] . increases in numbers of lung dc, which arise from local precursors in the lung, outlast the resolution of disease in rsv infection [51] . importantly, pulmonary dc can stimulate t-cell responses in the lung in situ without migration to the regional lymph nodes, favouring th2 and tc2 responses [52] . these observations suggest that respiratory viral infections lead to a prolonged period of increased antigen presentation in the airways resulting in de novo and memory t-cell responses not only to the virus but also to unrelated antigens including allergens. in addition to studies of primary infections, models studying the interactions between respiratory viral infections and allergen sensitization are essential in understanding the mechanisms of virus induced asthma exacerbations. many small animal models were designed to reveal the pathogenic mechanisms behind the enhancement of allergic sensitization by respiratory virus infections; the increased airway inflammation and responsiveness resulting from allergic airway sensitization following respiratory viral infection and, in those with established allergic airway sensitization, the increased airway inflammation and responsiveness due to respiratory viral infections. these studies show that the immune responses to allergen sensitization and respiratory viral infections interact to cause persistent inflammation and ahr, symptomatic of the asthmatic response (fig. 2) [53] . many studies have been done in animals sensitized to different allergens and then infected with respiratory viruses, a model that mimics viral exacerbation of asthma in sensitized individuals. in one study, guinea pigs were sensitized with ovalbumin (ova) and challenged with rsv and those that underwent both treatments were found to have more severe symptoms of ahr and inflammation (fig. 3) [53] . similar results were also found in mice sensitized to ova and then infected with rsv [54] , mice sensitized to ova and challenged with murine cytomegalovirus [55] , and mice sensitized to the house dust mite dermatophagoides farinae then infected with rsv [56] . experiments have been performed to determine the contribution of released inflammatory mediators on ahr following viral infection. il-4 is important in the exacerbation of asthma but the response is not solely dependent on il-4 and il-5 has been observed to increase in virally infected sensitized mice and has been directly associated with the viral exacerbation of asthma [57] . il-10 is thought to be necessary for the development of allergic ahr [58] , however, its role in viral exacerbation of ahr is less certain. the role of il-10 is complex, inhibiting il-5 production, eosinophilic inflammation and chemotaxis but potentially inducing a decrease in th1 cytokine production [59] . mice deficient in il-10 that were both sensitized (to ova) and infected with rsv did develop ahr including eosinophilic inflammation, th2 cytokine production and goblet cell hyperplasia. inhibition of il-13 has been shown to significantly reduce ahr in sensitized and infected mice, to the level of that seen in mice only infected or only sensitized [60] . these factors all appear to contribute in varying extents to the viral exacerbation of asthma. the cysteinyl leukotrienes (cyslts) such as ltc 4 , ltd 4 and lte 4 are products of activated eosinophils, basophils, mast cells and macrophages. these potent inflammatory mediators have a diverse range of biological activities, including the ability to exacerbate asthma by inducing the contraction of bronchial smooth muscle [61] . ltc 4 has been found at elevated levels in the nasopharyngeal secretions of children during the acute phase of rsv infection, with higher levels in patients with lower respiratory tract involvement than in those with upper respiratory illness alone [62] . lte 4 has been found to be elevated in urine samples collected from patients with asthma exacerbations [63] . cyslt levels have also been found to be increased in the lower airways during rsv bronchiolitis, although their concentrations are lower than those in acute asthma [64] . in mice, rsv infection has been found to produce a marked increase in cyslts in the balf and lung tissue, resulting in the recruitment of neutrophils and lymphocytes into the airways and ahr [65] . cyslts have been found to have multiple effects in animals, including the induction of th2 responses in the lungs through effects on dcs and cytokine generation, the recruitment and activation of cells such as eosinophils and mast cells, and inflammation [63] . interestingly, an lt synthesis blocker prevents the development of rsv induced lung function changes in mice [66] and montelukast, a cyslt antagonist, seems to reduce post-bronchiolitis wheeze in small children [67] . bronchial reactivity to stimulation by histamine has been found to be increased in asthmatic patients following infection with influenza a virus [68] . human rv infection in adults with mild asthma often has little effect on the lower respiratory tract or lung function [69] ; however, when infection in asthmatics is followed by allergen challenge, ahr and eosinophilia in balf have been observed for up to a month following challenge [70] . deliberate infection with rv has found that asthmatic symptoms tended to peak later during infection and were more pronounced and persistent during the later stages of infection [71] . although nasal and upper respiratory symptoms were similar between asthmatics with high ige levels, asthmatics with low ige levels and control subjects before infection, asthmatics with higher ige levels had more symptoms of lower respiratory inflammation before and during infection. asthmatics with high ige levels also had lower lung function results before infection, while the results for the asthmatics with low ige levels were similar to the controls. however, the lung function tests results did not vary significantly during the course of rv infection [71] . a similar study of people with allergic rhinitis (hay fever) found that rv infection increased ahr and the inflammatory reaction to allergen challenge. before rv infection, most subjects developed only short-term responses to the allergen; however, during rv infection nearly all subjects developed late asthmatic reactions [70] . these late asthmatic reactions were independent of changes in airway reactivity or the size of the immediate response to allergen challenge, and the propensity to develop them persisted for up to 4 weeks after virus infection [70] . a similar study found that the allergic response changed from nearly all subjects having only an immediate response before infection to a majority of subjects having both an immediate and late asthmatic reaction during or following rv infection [72] . changes associated with rv infection and subsequent allergen challenge include an immediate increase in the release of histamine and the recruitment of eosinophils into the airways within a 48 h period [73] . a study of peripheral blood mononuclear cells from patients with atopic asthma and normal controls found that exposure to rv induced the production of ifn-g, il-12, il-10 and il-13 in both groups, with significantly lower levels of ifn-g and il-12 and higher levels of il-10 in the cells from asthmatics than the cells from normal subjects. il-4 was induced only in the asthmatic group and the ifn-g/il-4 ratio was more than three times lower in this group. this suggests that the normal type 1 immune response to rv infection is defective in atopic asthmatic individuals, with infection inducing a th2-immune response similar to their response to allergens [74] . vaccines and therapeutics have the potential for eliminating or reducing the synergy between viral infections and asthma, as well as decreasing unrecognized but associated disease burden and health care costs. vaccination is the mainstay of prophylaxis for respiratory viruses; however, for some viruses such as rsv and rv, developing safe and effective vaccines has been difficult. despite the need for anti-viral drugs as an adjunct to vaccines, few are available that are effective. therefore, novel and new approaches are highly desirable. recently, mahalingam and tindle have demonstrated the efficacy of an hmpv ctl epitope vaccine that triggers a strong th1 immune response which was associated with accelerated viral clearance in a murine model [75] . in addition, rna interference (rnai), a naturally occurring process for controlling gene expression that occurs through a process mediated by short interfering rna (sirna) molecules, has been shown to have the remarkable ability to silence rsv replication both in vitro and in vivo (tripp et al., unpublished data). these approaches can be used to target host cell genes to silence aspects of the inflammatory pathway that include th1 or th2 cytokines, chemokines or proteins that regulate their expression. thus, disease intervention strategies that target the virus and/or host response are rational approaches for breaking the link between the synergy of virus infection and asthma. wright and nhmrc peter doherty fellowships respectively. s.m. acknowledges support from the nhmrc for research on respiratory viral infections (project grant #399701). k.m. acknowledges support from nhmrc programme grant #224207. r.a.t. acknowledges the georgia research alliance for their continued support. j.s. acknowledges support from the wellcome trust (senior fellowship grant #067454). we thank mr timothy lewis for excellent editorial assistance. the international study of asthma and allergies in childhood (isaac) steering committee, worldwide variation in prevalence of symptoms of asthma, allergic rhino conjunctivitis and atopic eczema national health survey: summary of results australian centre for asthma monitoring, in: asthma series 2, woolcock institute of medical research/australian institute of health and welfare data fact sheet: asthma statistics, national institutes of health: national heart, lung, and blood institute community study of role of viral infections in exacerbations of asthma in 9e11 year old children respiratory viral infections as promoters of allergic sensitization and asthma in animal models age at first viral infection determines the pattern of t-cell mediated disease during reinfect ion in adulthood paediatric basis of adult lung disease severe respiratory syncytial virus bronchiolitis in infancy and asthma and allergy at age 13 asthma and immunoglobulin e antibodies after respiratory syncytial virus: a prospective cohort study with matched controls asthma exacerbations 1: epidemiology genetics of asthma and hay fever in australian twins association studies for asthma and atopic diseases: a comprehensive review of the literature impact of il-8 and il-8-receptor alpha polymorphisms on the genetics of bronchial asthma and severe rsv infections polymorphisms in the novel gene acyloxyacyl hydroxylase (aoah) are associated with asthma and associated phenotypes association between surfactant protein a gene locus and severe respiratory syncytial infection in infants increased in vivo transcription of an il-8 haplotype associated with respiratory syncytial virus diseasesusceptibility a common haplotype of interleukin 4 gene il-4 is associated with severe respiratory syncytial virus disease in korean children influence of promoter variants of il-10, il-9 and tumour necrosis factor-alpha genes on respiratory syncytial virus bronchiolitis association between common toll-like receptor 4 mutations and severe respiratory syncytial virus disease association between severe respiratory syncytial virus infection and il-13/il-4 haplotypes human metapneumovirus persists in balb/c mice despite the presence of neutralizing antibodies latency and persistence of respiratory syncytial virus despite t cell immunity persistence of airway hyperresponsiveness and viral antigen following respiratory syncytial virus bronchiolitis in young guinea pigs respiratory syncytial virus, airway inflammation, and fev1 decline in patients with chronic obstructive pulmonary disease detection of respiratory syncytial virus in adults with chronic obstructive pulmonary disease rhinovirus is associated with severe asthma exacerbations and raised nasal interleukin-12 respiration persistence of rhinovirus rna after asthma exacerbation in children respiratory syncytial virus infects neuronal cells and processes that innervate the lung by a process involving rsv g protein the immune response to human metapneumovirus is associated with aberrant immunity and impaired virus clearance in balb/c mice perspective on the host response to human metapneumovirus infection: what can we learn from respiratory syncytial virus infections? in vivo and in vitro studies on the use of the guinea pig as a model for virus-provoked airway hyperreactivity mechanisms of bronchial hyperreactivity in normal subjects following upper respiratory tract infection virus-induced airway obstruction and parasympathetic hyperresponsiveness in adult rats virus-induced asthma attacks parainfluenza virus infection damages inhibitory m2 muscarinic receptors on pulmonary parasympathetic nerves in the guinea pig function of pulmonary m2 muscarinic receptors in antigen-challenged guinea pigs is restored by heparin and poly-lglutamate pretreatment with antibody to eosinophil major basic protein prevents hyperresponsiveness by protecting neuronal ms muscarinic receptor function role of macrophages in virus-induced airway hyperresponsiveness and neuronal m2 muscarinic receptor dysfunction the effect of human calcitonin gene-related peptide on eosinophil chemotaxis in the rat airway clinical and inflammatory responses to exogenous tachykinins in allergic rhinitis substance, p induces histamine release from human pulmonary mast cells differential effects of neuropeptides on cytokine production by mouse helper t cell subsets nerve growth factor and nerve growth factor receptors in respiratory syncytial virus-infected lungs respiratory syncytial virus upregulates expression of the substance p receptor in rat lungs nerve growth factor: the central hub in the development of allergic asthma? il-5 and eosinophils are essential for the development of airway hyperresponsiveness following acute respiratory syncytial virus infection il-13-induced airway hyperreactivity during respiratory syncytial virus infection is stat6 dependent cd8 t cells are essential in the development of respiratory syncytial virus-induced lung eosinophilia and airway hyperresponsiveness influenza virusinduced dendritic cell maturation is associated with the induction of strong t cell immunity to a coadministered, normally nonimmunogenic protein local cd11cþ mhc ii precursors give rise to pulmonary myeloid dendritic cells and are depleted in the process after rsv infection resident lung antigen-presenting cells have the capacity to promote th2 t cell differentiation in situ allergic sensitization increases airway reactivity in guinea pigs with respiratory syncytial virus bronchiolitis prior airway exposure to allergen increases virus-induced airway hyperresponsiveness murine cytomegalovirus infection alters th1/th2 cytokine expression, decreases airway eosinophilia and enhances mucus production in allergic airway disease recurrent respiratory syncytial virus infections in allergen sensitized mice lead to persistent airway inflammation and hyperresponsiveness timing of infection and prior immunization with respiratory syncytial virus in rsv-enhanced allergic inflammation il-10 is necessary for the expression of airway hyperresponsiveness but not pulmonary inflammation after allergic sensitization the failure of il-10 deficient mice to develop airway hyperresponsiveness is overcome by respiratory syncytial virus infection in allergen sensitized/challenged mice respiratory syncytial virus induced airway hyperresponsiveness is independent of il-13 compared with that induced by allergen cysteinyl leukotrienes and their receptors: cellular distribution and function in immune and inflammatory responses the release of leukotrienes in the respiratory tract during infection with respiratory syncytial virus: role in obstructive airway disease recovery of leukotriene e4 from the urine of patients with airway obstruction increased levels of bal cysteinyl leukotrienes in acute rsv bronchiolitis role of cysteinyl leukotrienes in airway inflammation and responsiveness following rsv infection in balb/c mice zileuton reduces respiratory illness and lung inflammation, during respiratory syncytial virus infection, in mice study group on montelukast and respiratory syncytial virus. a randomized trial of montelukast in respiratory syncytial virus postbronchiolitis bronchial reactivity following uncomplicated influenza a infection in healthy subjects and in asthmatic patients rhinovirus-16 colds in healthy and in asthmatic subjects. similar changes in upper and lower airways rhinovirus upper respiratory infection increases airway hyperreactivity and late asthmatic reactions experimental rhinovirus challenges in adults with mild asthma: response to infection in relation to ige experimental rhinovirus 16 infection potentiates histamine release after antigen bronchoprovocation in allergic subjects a common cold virus, rhinovirus 16, potentiates airway inflammation after segmental antigen bronchoprovocation in allergic subjects a defective type 1 response to rhinovirus in atopic asthma cytotoxic t-lymphocyte epitope vaccination protects against human metapneumovirus infection and disease in mice key: cord-268038-9jjpc1a2 authors: boukhvalova, m; blanco, j c g; falsey, a r; mond, j title: treatment with novel rsv ig ri-002 controls viral replication and reduces pulmonary damage in immunocompromised sigmodon hispidus date: 2015-09-14 journal: bone marrow transplant doi: 10.1038/bmt.2015.212 sha: doc_id: 268038 cord_uid: 9jjpc1a2 respiratory syncytial virus (rsv) is a significant cause of bronchiolitis and pneumonia in several high health risk populations, including infants, elderly and immunocompromised individuals. mortality in hematopoietic stem cell transplant recipients with lower respiratory tract rsv infection can exceed 80%. it has been shown that rsv replication in immunosuppressed individuals is significantly prolonged, but the contribution of pulmonary damage, if any, to the pathogenesis of rsv disease in this susceptible population is not known. in this work, we tested ri-002, a novel standardized ig formulation containing a high level of rsv-neutralizing ab, for its ability to control rsv infection in immunocompromised cotton rats sigmodon hispidus. animals immunosuppressed by repeat cyclophosphamide injections were infected with rsv and treated with ri-002. prolonged rsv replication, characteristic of immunosuppressed cotton rats, was inhibited by ri-002 administration. ab treatment reduced detection of systemic dissemination of viral rna. importantly, pulmonary interstitial inflammation and epithelial hyperplasia that were significantly elevated in immunosuppressed animals were reduced by ri-002 administration. these results indicate the potential of ri-002 to improve outcome of rsv infection in immunocompromised subjects not only by controlling viral replication, but also by reducing damage to lung parenchyma and epithelial airway lining, but further studies are needed. respiratory syncytial virus (rsv) is a significant cause of bronchiolitis and pneumonia in high-risk infants and children, elderly and immunocompromised individuals. among hematopoietic stem cell transplant recipients (hsct), rsv affects between 2 and 17% of patients, [1] [2] [3] [4] [5] with infection progressing to lower respiratory tract infection in 17-84% of cases. 1, [6] [7] [8] [9] the mortality rate of rsv lower respiratory tract infection in hsct patients, if left untreated, can reach 83% or higher. [10] [11] [12] [13] studies in patients with hematologic disorders and in recipients of hsct show that they maintain dramatically prolonged rsv replication, with a reported median duration of 30.5 days. 14 early intervention with ribavirin (aerosolized or oral) can reduce mortality associated with rsv infection of patients with hematologic malignancies and transplant recipients, highlighting the importance of controlling viral replication in this cohort. however, a significantly higher dose of ribavirin is required to achieve the therapeutic effect. 14, 15 efficacy of ribavirin therapy may be improved by combining it with the administration of intravenous immunoglobulin (ivig) or intravenous rsv immunoglobulin (rsv-ivig, a preparation containing 5-10 times the amount of rsv-neutralizing ab found in standard ivig preparations). 16, 17 rsv-ivig respigam approved for prophylaxis of rsv infection is no longer manufactured (http://www.aetna.com/cpb/medical/data/ 300_399/0318.html) and the humanized monoclonal ab palivizumab is the only currently available drug for the prevention of rsv in high-risk infants and children. 18 palivizumab use in adult patients is restricted due to cost considerations; nevertheless, it has been tested in hsct patients as a part of the combined therapy with ribavirin. the treatment was found to be safe and well-tolerated, however, its efficacy was not conclusive. 8, 19 in the normal immunocompetent subject, antivirals administered after rsv infection reduce viral replication but do not ameliorate the disease. [20] [21] [22] in contrast, antivirals given before infection reduce both viral replication and disease, suggesting that once triggered, the inflammatory response leads to cytokine response and subsequent pulmonary pathology that remains even after the viral load is decreased. 23 as cellular infiltration of the lung is a large component of rsv-induced pulmonary damage, it has been shown using an animal model that a combination of antiviral (for example, palivizumab) and anti-inflammatory (for example, corticosteroids) treatment may represent an effective therapeutic regime. [22] [23] [24] however, whether the same approach would be required for immunosuppressed individuals is not known. pulmonary pathology in hsct patients with rsv infection may be mediated by the lung damage caused by prolonged viral replication. in this case, treatment with a drug that has strong antiviral activity may have a higher therapeutic efficacy in immunosuppressed patients compared with immunocompetent individuals infected with rsv because of the reduction in pulmonary damage caused by excessive viral replication. in this work, we tested a novel rsv-ivig formulation ri-002, manufactured in compliance with the food and drug administration's standards for treating patients with immune deficiency diseases, for its ability to reduce rsv infection in normal and immunocompromised cotton rats s. hispidus. the cotton rat model has accurately reproduced significantly prolonged rsv replication under conditions of immunosuppression. 25 cotton rats were instrumental in proving efficacy of ab immunoprohylaxis, 26 which led to the development of both respigam and palivizumab. 27, 28 the model also confirmed that anti-rsv ab was less effective when given after the onset of rsv infection, [20] [21] [22] [23] indicating the important contribution of host inflammatory response to infection in the pathogenesis of rsv disease. the goal of this particular work was to determine whether ri-002 can mitigate prolonged rsv replication and reduce pathology of rsv disease in immunosuppressed cotton rats. ri-002 intravenous immune globulin (human): 10% human igg, prepared to contain standardized amounts of rsv-neutralizing antibodies was isolated from source plasma obtained from more than 1000 donors who were selected to have high titers of naturally occurring neutralizing ab to rsv. reference ab respigam (liquid sd lot livrsv-82a reference rsv-ivig, 5%) and cyclophosphamide (cy) for injection (20 mg/ml usp, baxter, deerfield, il, usa) were obtained from adma biologics, ramsey, nj, usa and blue door pharma (rockville, md, usa), respectively. the prototype a/long strain of rsv was obtained from the american type culture collection (manassas, va, usa). virus was propagated in hep-2 cells and serially plaque-purified to reduce defective-interfering particles. 29 a single pool of rsv a/long (3 × 10 7 plaque-forming unit (pfu)/ml) was used for the studies described herein. rsv viral titers in the lungs of infected animals were determined by plaque assay. 30 animals and animal studies inbred s. hispidus cotton rats were obtained from a colony maintained at the sigmovir biosystems, inc. six-to eight-week-old inbred female cotton rats were used for the studies without the need for additional randomization. animals were housed in large polycarbonate cages and were fed a standard diet of rodent chow and water. the colony was monitored for ab to adventitious respiratory viruses and other common rodent pathogens and no such ab were found. all studies were conducted under applicable laws and guidelines and after approval from the institutional animal care and use committee of the sigmovir biosystems, inc. efficacy of ri-002 therapy and prophylaxis (each) was verified in two consecutive experiments conducted first in normal and then in immunosuppressed animals. sample size of five animals per group was chosen based on results of previous experiments, as allowing detection of statistically significant differences between groups. comparison between groups was run using student's t-test for unpaired data with unequal variance (kaleidagraph, synergy software, reading, pa, usa). unless indicated, samples were not blinded before analysis. for studies on ri-002 prophylaxis in normal cotton rats, animals were inoculated i.p. under isoflurane anesthesia with ri-002 and 24 h later challenged with rsv a/long (10 5 pfu per animal) administered in 100 μl intranasally. control animals were inoculated i.p. with saline or respigam solution 24 h before rsv infection. animals were killed by co 2 asphyxiation on day 4 post infection and lung and nose samples were collected for viral quantification by plaque assay. blood was collected before infection and before killing the animal for quantification of rsv-neutralizing ab via microneutralization assay. for studies on ri-002 therapy of rsv infection, animals were treated 24 h after rsv infection with ri-002 and killed on day 4 post infection. control animals were inoculated i.p. with saline. immunosuppression was induced in cotton rats by repeated treatment with cy based on the method described before 25 with some modifications. cy dose of 50 mg/kg was administered i.m. for therapy study or i.p. for prophylaxis study under isoflurane anesthesia as 250 μl per 100 g animal for 18 days. at the end of this period, whole blood and serum samples were collected to verify the decline in total white blood cell and lymphocyte counts. cy treatment was continued until the end of the study. twenty-one days after the start of cy treatment, animals were infected with rsv a/long (10 5 pfu per animal). on days 1, 4 and 7 post infection, animals were treated i.p. with ri-002. control immunosuppressed rsv-infected animals were inoculated i.p. with saline. as an internal control for rsv infection without immunosuppression, age-matched normal animals were infected with rsv and treated with saline i.p. on day 1 post infection. groups of five animals were killed on days 4 and 10 post infection for collection of lungs and noses for viral quantification by plaque assay. on day 10 post infection, lungs were collected for histopathology analysis and fragments of the liver and kidney were snap-frozen for quantitative pcr analysis. blood samples were collected from all animals before each animal was killed for wbc, lymphocyte count analyses and microneutralization assay. for ri-002 prophylaxis studies, immunosuppressed cotton rats were given ri-002 i.p. one day before infection with rsv, followed by ri-002 treatments on days 4 and 8 post infection. samples were collected for analysis of viral replication on days 4 and 10 post infection. lungs were prepared for histopathology analysis as previously described and scored blindly for peribronchiolitis (inflammatory cells around small airways), perivasculitis (inflammatory cells around small blood vessels), alveolitis (inflammatory cells within alveolar spaces) and interstitial pneumonitis (inflammatory cell infiltration and thickening of alveolar walls). 31 each parameter was scored on a 0-4 scale. epithelial damage was evaluated as a hyperplasia of airway epithelial cell lining, multilayered cell arrangement and small, papillae-like structures projecting into the airway lumens and was scored on a 0-3 scale (0 = absent, 1 = minimal, 2 = mild, 3 = moderate). expression of rsv ns1 mrna was analyzed in rna extracted from different organs by real-time quantitative sybr green pcr as previously described. 32, 33 neutralizing ab assay anti-rsv-neutralizing titers in the cotton rat serum samples were determined by microneutralization assay as previously described. 34, 35 in brief, serum dilutions were incubated with ∼ 50 pfu of rsv a2 strain for 30 min at room temperature, followed by the addition of 1.5 × 10 4 hep-2 cells in 96-well culture plates. after 3 days, the quantity of rsv ag was determined by rsv enzyme immunoassay using a monoclonal ab to the rsv f protein. the neutralizing titer was defined as the serum dilution that results in a 50% reduction in color development. to verify the antiviral potency of ri-002 prophylaxis, normal cotton rats were inoculated i.p. with 500, 750 or 1000 mg/kg of ri-002 and infected with 10 5 pfu of rsv a/long per animal 24 h later. lungs and noses were harvested for viral titration 4 days after infection. viral load in rsv-infected, ri-002-treated animals was compared with that in rsv-infected animals treated with saline or inoculated with positive control reference rsv-ivig formulation respigam (750 mg/kg). saline-treated, rsv-infected animals had 4.7 and 5 log 10 pfu/g of rsv in the lung and nose tissue, respectively (figure 1a ). ri-002 completely protected the lung against rsv replication at all doses tested. significant reduction of viral replication in the nose was also achieved. in the lungs, protection afforded by ri-002 was improved as compared with that induced by the reference respigam formulation. levels of rsv-neutralizing ab were quantified by a microneutralization assay of serum collected immediately before infection and again at the time the animal was killed ( table 1 ). the data show doseresponse serum rsv-neutralizing titers, which correlated with the protection evidenced by the lung and nose rsv viral titers in the cotton rat tissues. to determine the ability of ri-002 to reduce rsv load when administered after infection (ri-002 therapy), animals were infected with rsv and treated one day later with ri-002. treatment with 1500 mg/kg ri-002 completely suppressed viral load in the lungs of infected animals on day 4 post infection ( figure 1b) . treatment with 750 mg/kg ri-002 abolished replication of rsv in the lungs of two out of five animals, with barely detectable virus isolated from the remaining three animals in the group. rsv replication in the nose was also reduced in ri-002-treated animals in a dose-dependent manner. efficacy of ri-002 against rsv infection in immunosuppressed cotton rats cotton rats were immunosuppressed via repeat cyclophosphamide treatment administered over a 3-week period (figure 2a) . blood samples collected before each animal was killed were used to verify the drop in white blood cell (wbc) and lymphocyte counts in all immunosuppressed cotton rats, and a rise in serum rsv-neutralizing ab titer in ri-002-treated animals (figure 2b ). significantly delayed viral clearance was seen in immunosuppressed cotton rats infected with rsv ( figure 3 ). although normal cotton rats treated with saline cleared rsv by day 10 post infection, immunosuppressed saline-treated animals had~6 log 10 pfu of virus in their lungs and noses at that time. immunosuppressed cotton rats inoculated with ri-002 after rsv challenge showed~99.9% reduction in viral load in the lung and nose at days 4 and 10 post infection when compared with saline-treated, immunosuppressed cotton rats. both h/l/l and h/h/h regimes of treatment were effective in suppressing viral replication. to determine whether ri-002 has an effect on pulmonary pathology associated with prolonged rsv replication in immunosuppressed animals, lung tissue was collected for histopathology analysis on day 10 post infection. immunosuppressed animals had more severe inflammation in the lung parenchyma (interstitial pneumonia) than non-immunosuppressed animals (figures 4a and b) . immunosuppression and rsv infection were also associated with significant hyperplasia of the mucosal epithelium of the conducting airways, which was absent in non-immunosuppressed, rsv-infected animals (figures 4a and b , compare top and bottom panels). immunosuppressed, rsv-infected animals treated with a high-dose antiviral regime had little to no inflammation in the parenchyma and a significantly reduced epithelial hyperplasia (figure 4b , compare top and middle panels), whereas intermediate-dose treatment was slightly less effective at reducing epithelial damage. these results suggest that the high-dose antiviral regimen reduces both pneumonia and airway epithelial hyperplasia in immunosuppressed animals infected with rsv. as the hyperplasia is a likely response to airway epithelial injury, these findings suggest that the high-dose antiviral regime may have either protected the animals from past or ongoing bronchiolar injury or may have enhanced recovery of the airway epithelium of immunosuppressed animals after the viral infection. rsv gene expression in organs outside of the respiratory tract was evaluated in immunosuppressed cotton rats infected with rsv. samples of the liver and kidney were collected on day 10 post infection and analyzed by real-time pcr. liver and kidney of immunosuppressed rsv-infected animals treated with saline had elevated expression of the rsv ns1 gene compared with the liver and kidney samples from normal animals infected with rsv and treated with saline ( figure 5 ). although the differences failed to reach statistical significance, ri-002 treatment of immunosuppressed, rsv-infected animals appeared to reduce rsv ns1 expression in the liver and kidney to the basal level detected in normal cotton rats infected with rsv. ri-002 ivig was also used to evaluate prophylactic efficacy of rsv-ivig against rsv disease in immunosuppressed animals. cotton rats were immunosuppressed with cy and treated with ri-002 one day before rsv infection, and on days 4 and 8 post rsv infection in a total of three repeated doses of 1500 mg/kg or 750 mg/kg. lungs of immunosuppressed animals treated with 1500 mg/kg were completely protected against rsv replication on both days 4 and 10 post infection ( figure 6 ). animals treated with 750 mg/kg rsvig had no detectable viral replication in the lungs on day 4 post infection, and minimal detectable replication (only two out of five animals in a group had a few plaques) recorded for day 10 samples. the studies described here demonstrate that ri-002 treatment can significantly improve outcome of rsv disease in immunocompromised cotton rats. the treatment was found to accelerate viral clearance and to reduce damage to pulmonary tissue and airway results represent the mean ± s.e. for 5 animals per group. *p o0.05 when compared to rsv-infected immunosuppressed animals treated with saline and killed on the same day. lining. repeat administration of ri-002 was used to achieve the desired effect, based on the earlier observation that an insufficient number of treatments can lead to rebound rsv replication in immunosuppressed animals undergoing ab therapy. 36 a regime consisting of three administrations of 1500 mg/kg ri-002 was largely comparable in antiviral efficacy to a combination treatment that included one dose of 1500 mg/kg followed by two doses of 750 mg/kg. however, the high-dose treatment of repeat 1500 mg/ kg ri-002 doses appeared to be more effective in restoring the pulmonary histology to the level similar to that seen in normal cotton rats infected with rsv. in normal cotton rats, a single administration of 750 or 1000 mg/kg ri-002 given prophylactically inhibited rsv replication in the lower respiratory tract, reducing viral titers to undetectable level. stronger antiviral effects of ri-002 correlated with higher serum rsv-neutralizing ab titers achieved one day after ab inoculation, consistent with the previously established correlation between serum rsv-neutralizing ab titers and rsv load. 26 ri-002 infusion ameliorated pulmonary pathology associated with prolonged rsv replication in immunosuppressed cotton rats. both elevated interstitial pneumonia and epithelial hyperplasia, features characteristic of immunosuppressed rsv-infected animals, were strongly reduced by ri-002. antiviral therapy, therefore, appears more effective as a stand-alone treatment against pulmonary disease in immunosuppressed compared with normal cotton rats infected with rsv. 22, 23 this suggests that excessive viral replication may indeed be a driving factor of rsv pathogenesis in immunosuppressed subjects and that rsv antivirals may have a higher therapeutic potential as a stand-alone treatment in immunosuppressed patients, but future studies in the human patient population are needed to confirm this hypothesis. in addition to antiviral activity, an anti-inflammatory activity, which is present in ivig, 37-39 could be beneficial and could have contributed to the disease-ameliorating effect seen in this work. dissemination of microbial pathogens outside of primary target organs is also a significant problem for bone marrow transplant recipients. 40 dissemination of rsv in immunocompromised patients is not well documented, but rsv rna has been detected in the blood and found to correlate with poor outcome of rsv lower respiratory tract disease in hsct recipients. 41, 42 our results show that detection of rsv rna outside of respiratory tract of immunosuppressed cotton rats was reduced by ri-002 treatment. there is a well-defined segment of patients with immune deficiency diseases that require regular infusions of ig to reconstitute their deficient humoral ab compartment and who are also extremely susceptible to rsv disease. [43] [44] [45] with the withdrawal of respigam from the market, an important ivig product in the arsenal for controlling rsv disease in vulnerable populations is no longer available. the improved efficacy of ivig vs palivizumab against rsv in hsct patients 17, 19 suggested that broader spectrum ab may be required. a pivotal phase iii ri-002 clinical trial in patients with immune deficiency disease has recently been completed. 46 the favorable secondary outcomes that were achieved suggest that the unique ab profile of ri-002 may have contributed to the positive results. ri-002 not only has higher titer ab to rsv, but also contains significantly higher titers of ab to other respiratory viruses including coronavirus, parainfluenza virus, metapneumovirus and influenza virus (manuscript submitted for publication). multiple respiratory viruses, including adenoviruses, rhinoviruses and coronaviruses have higher infection rates in hsct recipients and may have prolonged viral replication in these subjects. lower respiratory tract infection complication rates are high for rsv, influenza, parainfluenza, adenoviruses and human metapneumovirus (reviewed in shah et al. 47 ), suggesting that the polyvalent and broad antiviral characteristics of ivig may be important in this patient population. figure 6 . effect of ri-002 prophylaxis on rsv replication in immunosuppressed cotton rats. cotton rats were immunosuppressed with cyclophosphamide and inoculated with ri-002 at 750 (low dose) or 1500 mg/kg (high dose). twenty-four hours later, animals were infected with 10 5 pfu of rsv a/long per animal. on days 4 and 8 post infection, ri-002 treatment was repeated in the same dose as the original one. groups of animals were killed on days 4 and 10 post infection and lungs were collected for viral titration (vt). control animals were immunosuppressed, infected with rsv and inoculated with saline one day before and on days 4 and 8 post infection. results represent the mean ± s.e. for 4-5 animals per group. *po0.05 when compared with rsv-infected animals treated with saline killed on the same day. community respiratory virus infections among hospitalized adult bone marrow transplant recipients respiratory syncytial virus infection in patients with hematological diseases: single-center study and review of the literature respiratory tract viral infections in bone marrow transplant patients prospective study of the incidence, clinical features, and outcome of symptomatic upper and lower respiratory tract infections by respiratory viruses in adult recipients of hematopoietic stem cell transplants for hematologic malignancies timing and severity of community acquired respiratory virus infections after myeloablative versus non-myeloablative hematopoietic stem cell transplantation respiratory virus infection among hematopoietic cell transplant recipients: evidence for asymptomatic parainfluenza virus infection respiratory virus infections after stem cell transplantation: a prospective study from the infectious diseases working party of the european group for blood and marrow transplantation phase 1 evaluation of the respiratory syncytial virus-specific monoclonal antibody palivizumab in recipients of hematopoietic stem cell transplants randomized controlled multicenter trial of aerosolized ribavirin for respiratory syncytial virus upper respiratory tract infection in hematopoietic cell transplant recipients community respiratory virus infections in bone marrow transplant recipients: the m.d. anderson cancer center experience community-acquired respiratory syncytial virus and parainfluenza virus infections after hematopoietic stem cell transplantation: the fred hutchinson cancer research center experience supportive care immunodeficiency scoring index to predict poor outcomes in hematopoietic cell transplant recipients with rsv infections risk factors and containment of respiratory syncytial virus outbreak in a hematology and transplant unit oral ribavirin for treatment of respiratory syncitial virus and parainfluenza 3 virus infections post allogeneic haematopoietic stem cell transplantation combination therapy with aerosolized ribavirin and intravenous immunoglobulin for respiratory syncytial virus disease in adult bone marrow transplant recipients management of rsv infections in adult recipients of hematopoietic stem cell transplantation the impact-rsv study group. palivizumab, a humanized respiratory syncytial virus monoclonal antibody, reduces hospitalization from respiratory syncytial virus infection in high-risk infants palivizumab treatment of respiratory syncytial virus infection after allogeneic hematopoietic stem cell transplantation respiratory syncytial virus (rsv) immune globulin intravenous therapy for rsv lower respiratory tract infection in infants and young children at high risk for severe rsv infections: respiratory syncytial virus immune globulin study group respiratory syncytial virus immune globulin treatment of rsv lower respiratory tract infection in previously healthy children treatment of respiratory syncytial virus bronchiolitis and pneumonia in a cotton rat model with systemically administered monoclonal antibody (palivizumab) and glucocorticosteroid age-related differences in pulmonary cytokine response to respiratory syncytial virus infection: modulation by anti-inflammatory and antiviral treatment comparison of corticosteroids for treatment of respiratory syncytial virus bronchiolitis and pneumonia in cotton rats respiratory syncytial virus infection in cyclophosphamide-treated cotton rats immunoprophylaxis and immunotherapy of respiratory syncytial virus infection in the cotton rat prophylactic administration of respiratory syncytial virus immune globulin to high-risk infants and young children. the respiratory syncytial virus immune globulin study group reduction of respiratory syncytial virus hospitalization among premature infants and infants with bronchopulmonary dysplasia using respiratory syncytial virus immune globulin prophylaxis stabilization of respiratory syncytial virus (rsv) against thermal inactivation and freeze-thaw cycles for development and control of rsv vaccines and immune globulin the pathogenesis of respiratory syncytial virus infection in cotton rats vaccine-enhanced respiratory syncytial virus disease in cotton rats following immunization with lot 100 or a newly prepared reference vaccine respiratory syncytial virus infects and abortively replicates in the lungs in spite of preexisting immunity methods for monitoring dynamics of pulmonary rsv replication by viral culture and by real-time reverse transcription-pcr in vivo: detection of abortive viral replication relationship of serum antibody to risk of respiratory syncytial virus infection in elderly adults risk factors for severe respiratory syncytial virus infection in elderly persons effectiveness of rsvig prophylaxis and therapy of respiratory syncytial virus in an immunosuppressed animal model the antiinflammatory activity of igg: the intravenous igg paradox identification of a receptor required for the anti-inflammatory activity of ivig intravenous immunoglobulin: exploiting the potential of natural antibodies infection in the bone marrow transplant recipient and role of the microbiology laboratory in clinical transplantation respiratory virus pneumonia after hematopoietic cell transplantation (hct): associations between viral load in bronchoalveolar lavage samples, viral rna detection in serum samples, and clinical outcomes of hct respiratory syncytial virus lower respiratory disease in hematopoietic cell transplant recipients: viral rna detection in blood, antiviral treatment, and clinical outcomes risk factors and outcomes for respiratory syncytial virus-related infections in immunocompromised children pulmonary disease in primary immunodeficiency disorders respiratory syncytial virus infections in infants affected by primary immunodeficiency pharmacokinetics of ri-002, an investigational ivig preparation management of respiratory viral infections in hematopoietic cell transplant recipients we thank hannah sanford-crane, lauren hippler, julio canas, charles smith and martha malache for help with the animal studies. we also thank tom march for help with evaluating pulmonary histopathology and maryanne formica for technical assistance with the neutralizing assays. sigmovir biosystems was funded by adma to test ri-002 in the cotton rat model. key: cord-268388-kkhuzf3p authors: sharif-yakan, ahmad; kanj, souha s. title: emergence of mers-cov in the middle east: origins, transmission, treatment, and perspectives date: 2014-12-04 journal: plos pathog doi: 10.1371/journal.ppat.1004457 sha: doc_id: 268388 cord_uid: kkhuzf3p nan two years have passed since the initial description of the middle east respiratory syndrome coronavirus (mers-cov), yet the epidemic is far from being controlled. the high case fatality rate, the recent steep increase in reported cases, and the potential to cause a global pandemic during the upcoming hajj season are serious concerns. although a wealth of information about the pathophysiology, proposed animal reservoir, and intermediate host has been revealed, many questions remain unanswered. we herein review mers-cov, covering its proposed origins, route of transmission, treatment options, and future perspectives. first reported in 2012 [1] , middle east respiratory syndrome coronavirus (mers-cov) is a novel coronavirus and the first lineage 2c betacoronavirus known to infect humans [2] . with a case fatality rate of 35%, an urgent response is needed to prevent a global pandemic [3] . prior to 2003, coronaviruses were not considered serious human pathogens since they only caused mild upper respiratory tract infections (urtis) [4] . the first zoonotic introduction of a coronavirus into the human population occurred with the severe acute respiratory syndrome coronavirus (sars-cov) in 2002. sars-cov caused a global pandemic, with 8,400 recorded cases and 800 deaths [5] . mers-cov marks the second known zoonotic introduction of a highly pathogenic coronavirus, probably originating from bats. three lines of evidence currently support this theory: (1) the very close phylogenetic similarity with the bat betacoronaviruses: btcov-hku4 and btcov-hku5 [6] ; (2) closely related coronavirus sequences have been recovered from bats in africa, asia, the americas, and eurasia; and (3) mers-cov uses the evolutionary conserved dipeptidyl peptidase-4 (dpp4) protein in pipistrellus pipistrellus bats for cell entry [7] . since human-bat contact is limited, camels have been implicated as probable intermediate hosts. mers-cov appears to have been circulating in dromedary camels for over 20 years [8] . mers-cov uses the dpp4 (cd26) receptor to gain entry and effectively replicate in camel cell lines [9] . neutralizing antibodies for mers-cov have been detected in dromedary camels from oman, canary islands, egypt, jordan, united arab emirates, and saudi arabia [10, 11] . the exact mode of transmission from camels to humans remains to be confirmed [12] . camel milk was investigated as a possible route of transmission, given the common practice of consuming camel milk in the arabian peninsula. the first reported case of mers-cov in yemen occurred in a yemeni pilot who consumed raw camel milk [13] , and reusken et al. reported the finding of mers-cov in camel milk in qatar [14] . however, respiratory transmission is currently considered as the most likely route of transmission [15] . mers-cov has been detected by reverse transcription pcr (rt-pcr) from the nasal swabs of three camels in qatar and was linked to two confirmed human cases with high similarity upon sequencing, suggesting a possible respiratory mode of transmission [16] . several clusters of mers-cov cases have been reported, mainly among household members and health care workers (hcws), suggesting that transmission is through close contact. the largest cluster reported so far has been in 23 hcws in a hospital in al hasa, kingdom of saudi arabia (ksa), while the largest family cluster has been in three infected brothers from riyadh, ksa [17, 18] . the basic reproductive rate for mers-cov has still not been determined with certainty [11] . using two transmission scenarios, breban et al. reported an r 0 of 0.60 and 0.69 [18] . cauchemez et al. reported a similar r 0 at 0.63, but warned that in the absence of infection control measures, r 0 may range from 0.8-1.3 and could lead to a self-sustaining transmission [19] . propensity for the mers-cov to replicate in the lower respiratory tract may account for the observed limited transmission [20] . the united states centers for disease control and prevention (cdc) recommends standard contact and airborne precautions with the use of an n-95 mask when caring for an infected patient [21] . the cdc defines a laboratory-confirmed case of mers-cov as a patient with a positive pcr from a respiratory sample, and a probable case as a patient who had close contact with a confirmed case but inconclusive laboratory evidence [22] . the incubation period for the presentation of mers-cov symptoms is 2-14 days and it remains unknown whether patients are infectious during the incubation period [23] . the average age of presentation is 50 years, with a male predominance [24] . clinically, mers-cov causes symptoms of upper and lower rtis [23] . the severity of symptoms varies widely. most asymptomatic cases have been discovered through screening after contact with a known case [2] . presenting signs and symptoms may include highgrade fever, non-productive cough, dyspnea, headache, myalgia, nausea, vomiting, and diarrhea that may precede the respiratory symptoms [25, 26] . renal failure has been frequently reported, yet no conclusive evidence of a direct viral invasion of renal tissues exists [11, 27, 28] . notably, most patients who developed complications had coexisting medical co-morbidities [11] . laboratory findings on admission may include leukopenia, lymphopenia, thrombocytopenia, and elevated lactate dehydrogenase levels [25] . mers-cov can also cause severe pneumonia with acute respiratory distress syndrome (ards), requiring mechanical ventilation and intensive care admission [24] . to date, there is still a lack of surgical and pathological information from patients infected with mers-cov, which hampers full understanding of the pathogenesis. lastly, coinfection with other respiratory viruses and with community-acquired bacteria has been also reported in mers-cov patients [11, 29, 30] . as of june 26, 2014, who officially reported 707 affected patients in 21 countries in three continents. two-hundred and fifty-two patients have died of mers-cov, setting the case fatality rate at 35% [3] . the cases so far have been acquired either directly through a probable zoonotic source, or as a result of human-human transmission via close contact. retrospective analysis tracked the first outbreak to a hospital in the city of al-zarqa in jordan in april 2012 [31] . an unexplained observation has been the seasonal variation in reported numbers, with a peak between april and june of each year. the number of cases reported during april 2014 alone was alarming, because it was greater than the cumulative number of cases reported since the outbreak began [32] . the recent increase in the number of infected patients may arguably be attributed to better case detection and active surveillance programs. yet other factors may have contributed to the observed surge, including suboptimal infection control practices in affected hospitals in saudi arabia, as documented in a recent report of the who mission to jeddah [33] . another explanation for the seasonal variation may be that it coincides with camel birthing season, and younger camels seem to be more often infected than their older counterparts [34] . the distribution of the total reported cases by country is as follows: 85.8% in the ksa, 8.1% in the united arab emirates, 1.7% in jordan, and 1% in qatar [32] . cases have also been reported in kuwait, yemen, oman, iran, lebanon, tunisia, algeria, bangladesh, malaysia, france, italy, germany, the netherlands, united kingdom, greece, italy, and the united states [32] . furthermore, seropositive camels for mers-cov were detected in egypt, kenya, nigeria, tunisia, and ethiopia, suggesting that there may be mers-cov cases that are undetected in africa [8, 35, 36] . in 2012, 3,161,573 muslims from 188 countries gathered in mecca to perform the annual hajj, the largest gathering of muslims in the world. the identification of mers-cov in saudi arabia has generated international concern of a global pandemic. as a response, the saudi government requested that elderly and chronically ill muslims avoid hajj in 2013 and restricted the number of pilgrims to 2,061,573. consequently, no cases were reported during the pilgrimage of that year [37] . nasopharyngeal specimens collected from 5,235 pilgrims revealed no cases of mers-cov nasal carriage [38] . a prospective cohort study of 129 french pilgrims did not reveal any mers-cov cases [39] . nevertheless, the potential for spreading of mers-cov during the 2014 hajj season (october 2-6) remains possible, especially since documented transmission occurred this year in patients from iran and malaysia after their return from umrah in mecca. it is worth noting that the two most frequently visited cities during the hajj, mecca and medina, have so far reported 32 and 35 cases respectively [32] . mers-cov binds to the dpp4 (cd26) surface receptor using the spike (s) surface protein with subsequent cell entry [40] . the exact mechanism of entry after receptor binding is still unknown. the s surface protein is composed of a core subdomain that shares similarity with that of sars-cov and a receptor binding subdomain (rbsd) that exhibits significant variation from the sars-cov rbsd. the development of vaccines targeting the rbsd of mers-cov is currently under investigation because they are thought to be safer and more effective than vaccines based on inactivated virus, dna, or viral vectors [40] . another potential therapeutic approach is the inhibition of the papain-like and/or 3c-like protease of mers-cov [41] . to date, no effective therapy or prophylaxis for mers-cov exists. supportive therapy remains the cornerstone of management. current treatment is based on previous experience with the sars-cov, in-vitro studies, and case series. various agents have been tried, including those that block virus entry, inhibit viral replication, or interfere with host immune response [42] . the international severe acute respiratory and emerging infection consortium (isaric) suggested therapeutic options for treatment of mers-cov infection with various agents alongside continuous evaluation of efficacy, and in the setting of clinical trials [43] . based on experience with sars-cov, the use of convalescent plasma, hyper-immune globulin, or human monoclonal antibodies that contain neutralizing antibodies may be efficacious and is recommended as first-line treatment when available [43] . ribavirin and interferon alpha-2b both showed promising results, especially when used in combination, both in vitro and in animal studies using rhesus macaques monkeys [44] . however, these positive results did not translate clinically in an observational study in five patients, all of whom succumbed to the infection [45, 46] . repurposing of currently available agents may be an efficient approach. dyall et al. screened various agents with potential therapeutic efficacy [46] . cyclosporin a, mycophenolic acid, interferon-beta, homoharringtonine, cycloheximide, anisomycin, and emetine dihydrochloride hydrate were found to have the most potent in vitro activity against mers-cov. despite the progress in our understanding of mers-cov, many questions remain unanswered. the definitive origin, exact mechanism of transmission, and the reason behind seasonal variability are still unclear. although most cases have been described in countries of the arabian peninsula, the increasing travel to the region and the hajj season in ksa pose a threat of a potential global pandemic. extensive efforts are required to speed up the development of an effective therapy and vaccine. repurposing of currently available pharmaceutical agents is highly desirable for a more rapid drug development. meanwhile, hcws who encounter patients with respiratory symptoms who have lived or traveled to areas with mers-cov should have a low threshold to consider a diagnosis of mers-cov, with testing and immediate implementation of proper infection control practices to prevent further spread. finally, given the important role that camels may play in transmission of the virus, the common practices in the arabian peninsula of herding and consuming unpasteurized camels' milk should be discouraged until conclusive evidence is obtained that such practices do not contribute to infection. isolation of a novel coronavirus from a man with pneumonia in saudi arabia mers: emergence of a novel human coronavirus middle east respiratory syndrome coronavirus (mers-cov) summary and literature update as of 26 identification of a novel coronavirus in patients with severe acute respiratory syndrome consensus document on the epidemiology of severe acute respiratory syndrome (sars) genomic characterization of a newly discovered coronavirus associated with acute respiratory distress syndrome in humans dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-emc antibodies against mers coronavirus in dromedary camels replicative capacity of mers coronavirus in livestock cell lines middle east respiratory syndrome coronavirus neutralising serum antibodies in dromedary camels: a comparative serological study the emergence of the middle east respiratory syndrome coronavirus (mers-cov) middle east respiratory syndrome coronavirus: epidemic potential or a storm in a teacup? global alert and response: middle east respiratory syndrome coronavirus (mers-cov) -update middle east respiratory syndrome coronavirus (mers-cov) rna and neutralising antibodies in milk collected according to local customs from dromedary camels, qatar middle east respiratory syndrome coronavirus (mers-cov) in dromedary camels middle east respiratory syndrome coronavirus in dromedary camels: an outbreak investigation hospital outbreak of middle east respiratory syndrome coronavirus interhuman transmissibility of middle east respiratory syndrome coronavirus: estimation of pandemic risk middle east respiratory syndrome coronavirus: quantification of the extent of the epidemic, surveillance biases, and transmissibility middle east respiratory syndrome coronavirus (mers-cov) causes transient lower respiratory tract infection in rhesus macaques interim infection control and prevention recommendations for hospitalized patients with mers-cov mers case definition mers clinical update from the idsa center for disease control and prevention cdc epidemiological, demographic, and clinical characteristics of 47 cases of middle east respiratory syndrome coronavirus disease from saudi arabia: a descriptive study clinical features and viral diagnosis of two cases of infection with middle east respiratory syndrome coronavirus: a report of nosocomial transmission family cluster of middle east respiratory syndrome coronavirus infections a patient with severe respiratory failure caused by novel human coronavirus a family cluster of middle east respiratory syndrome coronavirus infections related to a likely unrecognized asymptomatic or mild case clinical features and virological analysis of a case of middle east respiratory syndrome coronavirus infection epidemiological findings from a retrospective investigation who concludes mers-cov mission in saudi arabia mers-cov enigma deepens as reported cases surge geographic distribution of mers coronavirus among dromedary camels mers coronaviruses in dromedary camels middle east respiratory syndrome coronavirus (mers-cov) summary and literature update as of 20 prevalence of mers-cov nasal carriage and compliance with the saudi health recommendations among pilgrims attending the 2013 hajj lack of mers coronavirus but prevalence of influenza virus in french pilgrims after 2013 hajj the receptor binding domain of mers-cov: the dawn of vaccine and treatment development proteolytic processing, deubiquitinase and interferon antagonist activities of middle east respiratory syndrome coronavirus papain-like protease what are our pharmacotherapeutic options for mers-cov? international severe acute respiratory & emerging infection consortium (isaric)-clinical decision making tool for treatment of mers-cov v.1.1 an animal model of mers produced by infection of rhesus macaques with mers coronavirus ribavirin and interferon therapy in patients infected with the middle east respiratory syndrome coronavirus: an observational study repurposing of clinically developed drugs for treatment of middle east respiratory coronavirus infection the authors would like to acknowledge sima l. sharara for editing the manuscript. key: cord-268729-n7slf5tx authors: wissinger, e l; saldana, j; didierlaurent, a; hussell, t title: manipulation of acute inflammatory lung disease date: 2008-05-07 journal: mucosal immunol doi: 10.1038/mi.2008.16 sha: doc_id: 268729 cord_uid: n7slf5tx inflammatory lung disease to innocuous antigens or infectious pathogens is a common occurrence and in some cases, life threatening. often, the inflammatory infiltrate that accompanies these events contributes to pathology by deleterious effects on otherwise healthy tissue and by compromising lung function by consolidating (blocking) the airspaces. a fine balance, therefore, exists between a lung immune response and immune-mediated damage, and in some the “threshold of ignorance” may be set too low. in most cases, the contributing, potentially offending, cell population or immune pathway is known, as are factors that regulate them. why then are targeted therapeutic strategies to manipulate them not more commonplace in clinical medicine? this review highlights immune homeostasis in the lung, how and why this is lost during acute lung infection, and strategies showing promise as future immune therapeutics. supplementary information: the online version of this article (doi:10.1038/mi.2008.16) contains supplementary material, which is available to authorized users. the mucosal immune system must maintain composure in the presence of an onslaught of antigenic and potentially pathogenic material. exposed to the outside world with, in most cases, only a single epithelial cell barrier protecting them, our mucosal surfaces have developed a sophisticated system of immune exclusion, ignorance and tolerance. the best characterized of these are described in the gastrointestinal tract. an understanding of immunity in the respiratory tract has lagged behind that of the gut, and although numerous key components have emerged, the sequence of events from initial inhalation to immune pathology in the lower respiratory tract is still unclear. despite best efforts to maintain immune homeostasis, respiratory inflammatory disease is common and significantly life threatening. this review will highlight mechanisms that maintain lung immune homeostasis and current therapeutic efforts to contain infection-induced exaggerated acute inflammation once it occurs. the respiratory tract includes the nasopharyngeal cavity, trachea and larynx, bronchi, bronchioles, and finally the alveoli. organized lymphoid tissue is embedded in some, but importantly not all, of these stages in the respiratory tree. similarly, draining lymph nodes are associated with only a few of these sites. the cellular composition, requirements for activation, and expansion dynamics of respiratory tract associated lymph nodes are virtually similar to any other lymph node and will therefore not be discussed in detail here. we will focus on the regulation (or de-regulation) of immune cells embedded in the respiratory tract itself. considering the total surface area of the respiratory tract constitutive, embedded organized lymphoid tissue is actually quite rare ( figure 1 ). organized structured lymphoid tissue exists in the nasal cavity of rodents (nasal associated lymphoid tissue, nalt) as paired lymphoid structures at the entrance to the pharyngeal duct, but identical structures in man remain elusive (for a review, see reference bienenstock and mcdermott 1 ). organized lymphoid follicles are observed in post-mortem specimens extracted from 150 children that contain occasional germinal centers, which are associated with lymphocytes in the overlying nasal epithelium and the presence of high endothelial venules. however, in adults such lymphoid tissue is disseminated across the whole nasal mucosa, 2 and is analogous to the less well-organized diffuse lymphoid tissue (termed d-nalt) lining the nasal passages of mice. 3 in man, diffuse nalt develops after birth, likely in response to antigen, and b-and t-cell responses parallel those that occur in lymph nodes. the waldeyer ' s ring comprising the nasopharyngeal (upper midline in nasopharynx, adenoids), paired tubal (around openings of auditory tube), paired palatine (either side of the oropharynx), and lingual (under the mucosa of the posterior third of the tongue) tonsil(s) are thought of as analogous structures to nalt, but are located outside of the respiratory tract and probably also contribute to gastrointestinal immunity. experiments with mice show that, unlike peripheral lymphoid organs, nalt develops independently of lymphotoxin-. however, its structure and function are perturbed in lymphotoxin--knockout mice, possibly due manipulation of acute inflammatory lung disease el wissinger 1 , j saldana 1 , a didierlaurent 1 , 2 and t hussell 1 inflammatory lung disease to innocuous antigens or infectious pathogens is a common occurrence and in some cases, life threatening. often, the inflammatory infiltrate that accompanies these events contributes to pathology by deleterious effects on otherwise healthy tissue and by compromising lung function by consolidating (blocking) the airspaces. a fine balance, therefore, exists between a lung immune response and immune-mediated damage, and in some the " threshold of ignorance " may be set too low. in most cases, the contributing, potentially offending, cell population or immune pathway is known, as are factors that regulate them. why then are targeted therapeutic strategies to manipulate them not more commonplace in clinical medicine? this review highlights immune homeostasis in the lung, how and why this is lost during acute lung infection, and strategies showing promise as future immune therapeutics. to impaired expression of cxcl13, c -c chemokine ligand19 (ccl19), and ccl21, which are crucial for the recruitment and placement of lymphocytes and dendritic cells (dcs). 4 the only other organized lymphoid structure described to date located within the respiratory tract is bronchus-associated lymphoid tissue (balt) (reviewed by bienenstock and mcdermott 1 ). whether it routinely contributes to primary immune responses or maintenance of t-and b-cell memory in the respiratory tract is not known. 5,6 however, a recent study in mice lacking peripheral lymphoid organs suggests that balt can initiate anti-influenza immunity and provide sufficient t cells to mediate protection against a second infection. 7 humoral immune responses elicited by balt are primarily mediated by immunoglobulin a (iga) and igg produced both locally and by balt-derived b cells that traffic to distant mucosal sites. 8, 9 similarly located t-cell responses have been noted. on the basis of these findings, balt can be thought of as functionally analogous to mucosal lymphoid aggregates in the intestine. present in up to 40 % of children and adolescents (to age 20), balt is rare in the lungs of healthy adults. 10,11 although originally described at the bifurcations of the bronchi, immediately beneath the epithelium, 12,13 in the absence of antigen balt is rare 14 and may be controlled or limited by regulatory t cells. 15 inflammation in the lung is associated with balt neogenesis and is described in a variety of pulmonary (moyron-quiroz et al 7 and references therein) and non-pulmonary 16 inflammatory conditions. homeostatic chemokines, including ccl19 and ccl21, in mice are required for development of such inducible balt (ibalt). 4 the extent of ibalt appears to depend on the level of inflammation in the local microenvironment, and suggests that it is developed when required. mice lacking oxidoreductases, that protect from oxidative stress, display heightened cellularity and inflammatory cytokines and ibalt is more prevalent. 17 whether it remains associated with larger airways and persists long after resolution of inflammation is still uncertain. intriguingly, mice lacking peripheral lymph nodes and spleen, but retaining ibalt, clear influenza infection (albeit slower) and survive higher doses of virus than do immune-competent mice. such lymphotoxin-knockout mice show slower generation of influenza-specific t cells that eventually reach wild-type levels, similar to antibody isotype switching to igg and t-cell cytokine production and effector function. this indicates that immune responses generated in ibalt although slower are protective and potentially less pathologenic. 7 this may represent a qualitative difference between local and peripherally derived immune cells or simply reflect the reduced magnitude of immunity when ibalt is the only inducing immune compartment. regardless, in the case of lung immunity, secondary lymphoid tissues are not essential for the maintenance of immunological memory, since a pulmonary infection with influenza virus is handled equally as efficiently in their absence. 18 to date no studies have shown that organized embedded lymphoid tissue such as ibalt and nalt contribute directly to inflammatory pathology in the lung. they may initiate production of immune t and b cells that then track to less organized lung immune compartments, 7,19 -22 but their neogenesis (ibalt) or continued presence (nalt), per se , is not associated with pathology. instead, pathological lung inflammation is attributed to those compartments that lack organized lymphoid structures, the airways and lung parenchyma. this could also be said of inflammation in the gut; peyer ' s patches and mesenteric lymph nodes may not be directly associated with pathology, whereas the lamina propria is. what the lamina propria and lung parenchyma have in common is a loose scattering of non-organized immune cells and a vast surface area of potentially non-professional antigen-presenting cells (apcs); the epithelium. the epithelium expresses constitutive mhc class i and, when inflamed, mhc class ii and b7 molecules. 23, 24 it can, therefore, process and present antigen and activate t cells, but can it turn them off? we believe this non-professionalism and lack of immune-cell organization leads to immune dysregulation. during inflammation, the mediastinal lymph nodes and ibalt expand in an organized and precisely compartmentalized manner. although low frequencies of antigen-specific cells can be observed in these sites, 25 the ratio of immune-cell subsets does not significantly change. contraction of lung-associated lymph nodes is also well controlled; again cell proportions are retained. in lung compartments devoid of organized lymphoid tissue however, immune cells are recruited in droves by a chemotactic gradient 26 -28 from infected epithelium and / or tissue resident and alveolar macrophages. once in the lung parenchyma or the airways, they do not form structures analogous to ibalt or lymph nodes, and it is here that pathological damage occurs, toll-like receptors ligands dominate, and inflammatory cytokines are produced by the infiltrate in abundance. unlike the lung-associated lymph nodes, the airways and lung parenchyma, therefore, experience dramatic shifts in their cellular composition. this is illustrated graphically in figure 2 . the approximate cellular composition in nalt, airways, and lung is shown in homeostasis and at the peak of respiratory infections by three very different pathogens, influenza virus, the bacterium streptococcus pneumoniae , and the fungus cryptococcus neoformans . obviously at other stages of the infection, slightly different cells will dominate, but only the peak of inflammation is presented for clarity. for example, natural killer cells dominate in the airways at days 3 -4 of a viral infection. 29 at its peak of activity, the influenzainfected airway and lung is dominated by cd8 + and cd4 + t cells, 25,30 -32 whereas during s. pneumoniae infection, macrophages, neutrophils, and t cells are more abundant. 33, 34 c. neoformans in c57bl / 6 mice induces an eosinophil-dominated response in the lung and airways. 35 -37 this infiltrate in the air spaces and lung for all pathogens is dramatically different to the same sites in homeostasis that contain few lymphoid cells but a prominent macrophage population. 38 -40 note that the nalt, despite being infected with all three pathogens, does not substantially alter the proportion of immune-cell subsets present, and the same is also true for the relatively non-infected lung draining lymph nodes. this lack of control and excessive response is only observed in a minority, but when it occurs it is life threatening. for most of us, respiratory pathogens are cleared by non-inflammatory means, including iga that does not fix complement well, but in dimeric form agglutinates and physically excludes antigen by a process known as immune exclusion. 41, 42 what, therefore, goes wrong in a minority? to address some of these questions, we need to understand how immune homeostasis is maintained in health in these non-organized lung compartments (reviewed by holt 43 ) and what pathways contribute to immune pathology. epithelial cells contribute a multitude of strategies to maintain lung immune homeostasis (for a review, see reference holt 43 ). in addition to barrier function, they secrete a variety of antimicrobial substances (surfactant protein c, mucins, and antimicrobial peptides), affect airway smooth-muscle, dc, and memory t-cell activation via nitric oxide production; 44, 45 assist in cell recruitment via production of cytokines and chemokines; 46, 47 and prolong cell survival by secreting stimulating factors such as granulocyte -macrophage colony-stimulating factor. 48, 49 raz and co-workers 50,51 highlight an interesting pathway critical for maintaining alveolar macrophage homeostasis, involving integrin v 6 that localizes these cells next to epithelial expressed transforming growth factor-(tgf-). this may explain why these cells are refractory to migration to the draining lymph nodes. 52 for inflammation to proceed, this inhibitory pathway must be overcome, which is mediated by a toll-like receptor-induced conformational change of macrophages, disruption of tgf-signaling, and reduced integrin expression. we often assume that innate immunity is inactive in the absence of antigen. however, the work of raz et al . clearly shows that active suppression is required for homeostasis. this is also observed in mice lacking components of nadph oxidase 53 that have heightened basal levels of airway macrophage activation due to loss of feedback inhibition. active suppressive mechanisms, therefore, set a " threshold of ignorance " . those that succumb continually to inflammatory lung disease may, therefore, have dysregulated homeostatic pathways or the threshold, which antigen must exceed to induce inflammation, set too low. in the cases of tgf--mediated suppression of alveolar macrophages, homeostasis is overcome by cleavage of the integrin tethering it to the respiratory epithelium. homeostasis is restored when macrophage-released matrix metalloproteinases transform latent tgf-into its active form. 51 it may, therefore, be possible to harness these pathways artificially to dampen inflammatory lung disease with the caveat that pathogen clearance may be affected by such a global antiinflammatory strategy. 54 macrophages, particularly in the airways, have long been known to have an immune-suppressive phenotype. renewal is achieved primarily via local cell proliferation; recruitment via ccl2:ccr2 does occur, although such cells may take days to mature into the classical immune-suppressive phenotype. 55 in addition to shielding the immune system from inhaled antigens, 56 they display poor phagocytic activity 57 and tend not to migrate well to draining lymph nodes. macrophages held in homeostasis also affect other cell types that may otherwise be proinflammatory within the respiratory tract (for a review, see reference holt 43, 58 ) . dc migration to the draining lymph nodes is enhanced upon macrophage depletion, 52 and t-cell-mediated inflammatory disease ensues to antigens that would otherwise have been ignored, 59 most likely due to the usual direct suppressive influence that alveolar macrophages have on dc function. 39 both myeloid and plasmacytoid dcs (pdcs) are present within the lung, both increase and are recruited rapidly during inflammation, and are attracted by chemokines and cytokines produced by epithelial cells and alveolar macrophages. 43,60 -62 myeloid dc responses are similar to counterparts found elsewhere in the body. pdcs, however, also appear to play a tolerogenic role in the respiratory tract. they have poor apc activity 63 (but once activated enhance cd8 + t-cell responses in vivo 64 and possibly cd4 + t cells at distant sites 65 ), promote inhalation tolerance, 66 and can protect from development of allergic airway disease (reviewed by de heer et al , 66 hammad and lambrecht, 67 and lambrecht 68 ). during acute respiratory viral infections, pdcs perform dual functions of promoting viral clearance by secretion of type i interferon (ifn), and limit inflammation by induction of interleukin-10 (il-10) (reviewed by grayson and holtzman 69 ). their role in limiting lung inflammation can be clearly seen in respiratory syncytial virus (rsv)infected mice where pdc depletion leads to increased viral replication and enhanced immunopathology in the lungs 70, 71 . dcs encounter antigen predominantly in the lung parenchyma, although microbial sampling, via dendrite projections through the epithelial cells into the airway lumen, may induce activation, maturation, and migration, arming them to support potent t-cell responses. 72 quite what returns these cells to homeostasis is unknown but may involve the level of toll-like receptor signals and / or the influence of surface expressed inhibitory receptors such as cd200r. 73 airway epithelial cells may also control dc activity via tgf-analogous to alveolar macrophages, 51 although this has not been proven. whether dcs actually transmigrate into the airspaces is still controversial. 74 -76 ccr2 assists their transit across the endothelium and ccr6 their subsequent migration into the airway. 74 the recently described cd103 + ( e 7), cd207 + , cd11b lo dcs express all of the requisite chemokine receptors to draw them into the airways, 77 but their presence in this compartment has not been proven. clearly, harnessing the homeostatic pathways described above may help to resolve ongoing inflammation. however, it is equally likely that immune cell types and pathways brought into the lung with inflammatory cells during inflammation also provide a therapeutic opportunity. it would not be possible to cover all of the pathways attempted to limit lung inflammation. we will, therefore, restrict our analysis to acute infectious events (i.e., not asthma) and to targeted therapeutic strategies (i.e., not global anti-inflammatories such as corticosteroids or experiments in gene-deleted animals). immune-mediated pathology can be manipulated at any stage of its generation. from a clinical perspective, however, after onset of identifiable symptoms would be the most beneficial. for this reason it is often resolution of inflammation that is targeted, which can include modulation of cell survival, successive waves of recruitment, and ongoing innate immunity. it is still unclear whether immune excess in the respiratory tract stems from over-exuberant recruitment, proliferation within the airspaces, and / or accumulation in the absence of clearance of innate and adaptive immune cells. evidence suggests that the airspaces may not support efficient t-cell proliferation 78 -80 despite memory t cells acquiring bromodeoxyuridine staining at the same rate as secondary lymphoid organs. 25, 80 it is likely that the inflammatory cytokine and chemokine cascade that ensues upon infection 81 -86 will prolong immunecell survival, but at the same time enhance their recruitment, either directly or by altering vascular or epithelial permeability. 87, 88 an inflammatory environment is also associated with the highest expression of " late co-stimulatory molecules " on t cells that prevent activation-induced cell death and rely on cognate ligands expressed on apcs for signalling. ox40 (cd134) is one such late co-stimulator that is restricted to recently activated t cells, whereas the ligand (ox40l) is expressed on a number of cell types, predominantly apcs. 89 stimulation through ox40 promotes cd4 and cd8 t-cell survival, clonal expansion, 90 inhibition of regulatory t cells, 91 and enhanced immunity to a variety of pathogens. 36,92 -94 however, during acute influenza virus infection of mice, transient blockade of ox40 is more beneficial; alleviating illness and pathology to influenza, without review compromising pathogen clearance or immunological memory. 95 a permanent absence of multiple late co-stimulators, however, can compromise immunological memory. 96 -98 manipulation of one late co-stimulatory pathway, therefore, leaves others intact to seed the memory t-cell pool. 97 this suggests that a full compliment of late co-stimulators may actually be prolonging t-cell survival and contributing to pathology. inflammatory cytokines, such as those abundantly expressed in the airways during infection, are known to increase ox40l on apcs. 99 due to lack of immune organization in the airways, late co-stimulatory molecules and their ligands may be downregulated too late to avoid bystander tissue damage. the observation of ox40l on inflamed endothelium highlights the difficulty in separating the effects of immune modulators on cell survival vs. cell recruitment, 100, 101 especially when ligation of ox40l on endothelial cells induces secretion of chemokines. 101,102 4-1bb, like ox40, is restricted to late-effector t cells and its absence or blockade impairs cd8 + t-cell responses to influenza, 103 although the effect on associated respiratory pathology is not yet known. care must be taken, however, since not all inducible co-stimulators result in a beneficial outcome when blocked. during lung influenza infection, icos blockade impairs respiratory t cells to such an extent that the virus escapes clearance. 32 this is similar to treatment of influenza-infected mice with a ctla4-ig fusion protein (that blocks cd28 binding to b7 molecules) 104 and cd40l-knockout mice infected with c. neoformans 105 (where macrophage antimicrobial strategies are impaired). it is likely that targeting cd27 may also produce untoward side effects, since it is expressed on resting na ï ve and memory t cells and is crucial for the formation cd8 + t-cell responses. 96, 106 perhaps the defining line for therapeutic potential should be placed between those late co-stimulators that absolutely depend on t-cell receptor and constitutive cd28 signaling for their expression and those that can appear in a bystander fashion in the presence of inflammatory cytokines. the likely effect of co-stimulatory blockade and the site where manipulation may have the most effect is shown in figure 3 . in addition to late co-stimulatory molecules, a number of other pathways affect the longevity of lung inflammation during acute infection. whether apoptosis is beneficial or harmful depends on the specific infection and the dominant cell type that mediates its clearance. 107 as a general rule, apoptosis favors the host in chronic and acute intracellular bacterial and viral infections (as the process clears the pathogen), but is detrimental for extracellular bacteria. 107 for example, during infection of rats with pneumocystis , alveolar macrophage apoptosis delays clearance of the organism that can be improved by administering caspase-9 inhibitors. 108 similarly, apoptosis of airway epithelial cells via fas / fas ligand is essential to prevent dissemination of pseudomonas aeruginosa . 109 however, during influenza infection it may be more advantageous to reduce cell survival, especially in the case of tnf producing cd8 + t cells. tnf receptor-ii and very late antigen--1 synergize to protect cd8 t cells in the influenza virus infected airways from apoptosis, 110 whereas engagement of qa-1b by cd94 / nkg2a transmits a negative signal that limits immune pathology. 111 it may, therefore, be possible to resolve t-cell inflammation before bystander tissue damage occurs by blocking or enhancing these surface receptors once the viral load has reduced. this would only be useful, however, if the strategy specifically targeted a defined cell population, since apoptosis of airway epithelial cells and leukocytes may be linked to the pathology observed in those infected with highly pathogenic avian influenza. 112, 113 furthermore, apoptosis leading to systemic lymphopenia, 114 observed during influenza infection, may assist virus propagation and survival. other respiratory pathogens, such as cytomegalovirus and parainfluenza virus, use antiapoptotic strategies to prolong survival of the cells they infect. 115 chlamydia blocks apoptosis by affecting the release of cytochrome c from mitochondria. 116 the extracellular pathogens pseudomonas cepacia figure 3 the location of the dominant site of action of co-stimulatory molecule blockade during acute lung infection. this model assumes that antigen specific t cells are primed in the lung-associated lymph nodes (e.g., mediastinal). as such blockade of cd28 on t cells using the b7 competitor ctla4:ig will affect t-cell priming in organized lymphoid tissue ( a ). the same is also likely to be true for icos that is induced rapidly after t-cell receptor and cd28 signalling. by contrast, ox40 and 4-1bb are expressed at very low levels in the lung-associated lymph nodes but upregulated in the lung parenchyma and airways, most likely due to re-recognition of antigen in situ and / or the inflammatory cytokine environment. blockade will, therefore, impact these sites rather than the lymph nodes ( b ). note that ox40 and 4-1bb blockade may also affect primed t-cell migration, as their respective ligands are present on inflamed endothelium ( c ). in the airways, the dominant effect will be to allow activation-induced cell death to progress; in the parenchyma, however, it may be a combination of activation-induced cell death ( d ) and migration. and s. pneumoniae cause apoptosis of neutrophils and airway epithelial cells ( table 1 ) , 117 respectively, to aid survival. modulation of apoptosis is, therefore, complicated; what may benefit the host for one infection would compromise it to another. the recruitment and trafficking of leukocytes in response to inflammation is a tightly regulated process that can tip the balance between protection from infection and immune mediated damage in the lung. in brief, it involves slow rolling (concomitant to the activation of leukocytes), an increase in integrin expression and avidity to regulate the rolling arrest, adhesion strengthening as well as spreading, and intravas-cular crawling culminating in paracellular or trans-cellular migration. 118, 119 since excessive cell recruitment is a feature of many acute lung infections, targeting specific molecules involved in any of the above-mentioned events may be beneficial. blocking monoclonal antibodies against the integrin very late antigen-4 (natalizumab) 120 and lymphocyte function-associated antigen-1 (efalizumab) 121 are currently used to treat inflammatory autoimmune disorders and crohn ' s disease, but are also immunosuppressive and have not been studied in the context of acute respiratory infection. 122 the potential benefit of blocking chemokines or their receptors using competitive blockers or antagonistic compounds is well described (reviewed by glass et al 123 ransohoff 124 ). secreted chemokines bind glucosaminoglycans on endothelial cells, forming chemoattractant gradients that direct cells to inflammatory sites, 125 and are classified into constitutive homeostatic and inflammatory (requiring a proinflammatory stimulus such as ifn-, tnf, or microbial products) chemokines. 126 chemokine receptors are transmembrane g-protein-coupled molecules that trigger a signal transduction event resulting in activation and firm adhesion of the migrating cell. 127 the interactions between chemokines and their receptors are functionally redundant; many chemokines bind the same receptor and one chemokine can bind several receptors. as such it can be difficult to design reliable therapeutics that disrupt the interaction of one particular chemokine with its receptor. 128 therapeutic administration of antibodies that block macrophage inflammatory protein-2 during influenza infection reduces neutrophil recruitment by 49 % and improves lung pathology without altering viral clearance. 129 however, this strategy requires testing in co-infection models, since neutrophils are critical for clearance of most respiratory bacteria that commonly cause secondary pneumonia in the presence of influenza virus. 130, 131 rantes (ccl5) is another potential target produced by respiratory epithelial cells during a variety of viral infections. rantes induces ccr1-, ccr3-, and ccr5-expressing t cell, eosinophil, and monocyte recruitment to the lung. 132 during rsv infection, ccl5 is expressed by infected epithelia and resident macrophages and secreted into the airway during the first 48 h of infection. production is then taken over by newly recruited t cells. blocking this chemokine with a competitive inhibitor (met-rantes) 133 during a primary rsv infection reduces lung immunopathology. however, heightened cell recruitment occurs during homologous rsv re-challenge, suggesting that manipulation during the first infection severely compromises immunological memory. 134 the same strategy has been tested in a mouse model of pneumonia virus infection. this highly lethal mouse pathogen induces a disease closely resembling severe human rsv infection in man, that is abrogated by co-administration of the antiviral agent rivabirin and met-rantes. 135 in contrast to inhibition of chemokines during virus-induced lung inflammation, some infections may require their administration. p. aeruginosa lung infection causes airway neutrophil infiltration that rapidly apoptose and become toxic. administration of recombinant monocyte chemoattractant protein-1 / ccl2 recruits and activates lung macrophages that clear apoptotic neutrophils before they cause pathology. 136 ifninducible protein 10 (cxcl10) is critical not only for clearance of klebsiella pneumoniae , 137 but is also produced during lung viral infection where neutralization may be beneficial. 138 many chemokine receptors are also increased during respiratory viral infection; cxcr3, for example, is upregulated during murine gammaherpes virus b8 infection, and its absence delays viral clearance. 139 antagonism of ccr1 reduces mortality of pneumovirus infection of mice, 140 and absence of ccr1 also prevents the rsv-induced exacerbation of asthma. 141 relatively little has been accomplished in this area, especially therapeutically, however, due to the paucity of reagents available for chemokine-receptor blockade. another method for altering lymphocyte migration is to manipulate sphingosine 1-phosphate receptors that are required for egress of lymphocytes from the thymus and peripheral lymph nodes, and impact on vascular permeability. fty720 is a novel synthetic immunosuppressive drug that inhibits lymphocyte emigration from lymphoid organs by binding and activating sphingosine 1-phosphate receptors. 142 sphingosine 1-phosphate is known to play a role in endotoxin-induced lung injury by affecting endothelial barrier function, 143 and is currently in trial for a variety of inflammatory disorders, including transplantation, 144 but is yet to be tested in acute lung infection. modulation of lung innate immunity represents another potential therapeutic target. however, once again, few studies have tested manipulating it after the onset of symptoms during acute respiratory infection; most examine the impact of an innate immunity-associated molecule using gene-depleted animals or prior to infection. manipulation of innate immunity may seem to be akin to " shutting the stable door after the horse has bolted, " since innate immunity is activated first and drives subsequent adaptive immune responses. however, each epithelial cell or macrophage infected sends off the next wave of an immune response. at the time of clinical presentation, therefore, both innate and adaptive immunity will be in full swing. manipulating innate immunity at this stage will assist the resolution process, but whether inhibition or activation is required will depend on the pathogen. one way to temper innate immunity is to mature the lung microenvironment or instill probiotic microbes that would compete with the survival of pathogenic microorganisms. oral administration of lactobacillus casei during lung s. pneumoniae infection is protective, resulting in more rapid clearance, a shorter period of septicemia, and decreased s. pneumoniae load in the lungs. this benefit is attributed to increased neutrophils, myeloperoxidase, and il-10 that limits lung tissue damage and is likely mediated by migration of mature apcs from the gut to the lung that are better equipped for bacterial clearance. 145 prior infection in the lung also has a beneficial effect on some subsequent acute respiratory infections through modification or maturation of the microenvironment. 146 -151 prior influenza infection, for example, reduces subsequent toll-like receptor responsiveness of alveolar macrophages for prolonged periods of time. 152 administration of microbial products, such as cpg dna or a modified bacterial labile toxin (ltk63), also protects against an array of subsequent respiratory pathogens, 153,154 as do chronic or acute infections in distant sites. 155 -157 the ability of pathogen-derived proteins to modify acute respiratory infections, however, is yet to be tested therapeutically. resveratrol, a polyphenolic compound found in red wine, inhibits nuclear factor-b activation, decreases mortality and pro-inflammatory cytokines (tnf, il-1 , and il-6) to serratia marcescens pneumonia in rats. though this strategy increases neutrophil numbers, they resolve more rapidly. 158 similarly, review an acidic polysaccharide compound from cordyceps militaris , an insect-borne fungus, has anti-viral properties in a murine influenza infection model. 159 intranasal administration of the fungal polysaccharide decreases mortality and influenza viral titers, while increasing lung pro-inflammatory cytokines. in vitro , influenza infected macrophage cell lines treated with resveratrol display enhanced inducible nitric oxide synthase and nitric oxide (no), suggesting that this compound may function through non-specific stimulation of alveolar macrophages in vivo . 159 enhanced innate and adaptive immunity and reduced microbial load is also observed upon therapeutic administration of retinoic acid in mice infected with mycobacterium tuberculosis . 160 the increased numbers of macrophages, natural killer and t cells, and increased expression of ifn-, tnf, inducible nitric oxide synthase, il-10, and cxcl10 may also benefit other acute respiratory bacteria or fungi, though during viral infection it would be predicted to be detrimental. acute respiratory infections are yet to be examined. a critical pathway for clearance of pathogens and infected lung epithelial cells is via no and reactive oxygen and nitrogen species. no contributes to host defence and mediates both proand anti-inflammatory effects (reviewed in 161, 162 ) . as with many of the therapeutic treatments discussed, the timing and extent of modulation of no and associated free radicals is critical. while there is no clear consensus in the literature, there are a few examples of successful intervention in this pathway. in rats with p. aeruginosa pneumonia, treatment with inhaled no post-infection improves bacterial clearance through direct bactericidal effects, increased recruitment of neutrophils to the airways 163 or enhanced endothelial permeability. 164 during k. pneumoniae infection of rats, inhaled no also suppresses bacterial replication and decreases lung intercellular adhesion molecule-1 expression, myeloperoxidase activity, tnf levels, and nuclear factor b activity. thus, no has many paracrine effects on inflammatory cells, but also promotes bacterial clearance. 165 during viral infection, the impact of no manipulation is less clear. inhibition of no during rsv infection reduces pulmonary inflammation and bystander tissue damage, but viral replication increases. 166 similar therapeutic strategies have been employed to reduce local concentration of reactive oxygen species and reactive nitrogen species, which are produced by infected lung epithelium and macrophages. during rsv infection of mice, administration of the antioxidant, butylated hydroxyanisole, decreases illness scores, weight loss, and lung neutrophil recruitment. again a multi-factorial attenuation of inflammation occurs. 167 the same is observed in influenza-infected mice treated with a free-radical scavenger, manganese superoxide dismutase, within 48 -96 h of infection. this enzyme has potent anti-inflammatory properties, resulting in less lung consolidation and improved arterial oxygen saturation, presumably as a result of decreased tissue damage. 168 reduced tissue damage also occurs in mice lacking superoxide dismutase or treated therapeutically with a manganic porphyrin that scavenges reactive oxygen species. 53 therefore, inhibition of reactive oxygen species / reactive nitrogen species and no is beneficial for acute respiratory viral infection, but likely to be detrimental for concurrent respiratory bacteria. modulation of immune-receptor signaling is in its infancy with regards to acute respiratory infection and may be limited by (a) the toxicity / safety profile of available drugs, (b) formulation challenges for in vivo delivery, (c) a lack of specificity due to shared receptor associations, and (d) problematic pharmacokinetics (sustained blockage will be detrimental for protection against infection and drugs may have to be delivered locally to prevent systemic effects). the only truly therapeutic manipulation, to date, is abrogation of airway fluid clearance during rsv infection, caused by interaction of uridine triphosphate with purinergic receptors, by post-infection administration of an active metabolite of leflunomide, a77-1726. leflunomide restores airway fluid clearance, providing symptomatic relief and reduced lung inflammation and hypoxemia, without impairing viral replication or clearance. 169 although signaling molecules associated with pattern recognition and cytokine receptors are well described, little is known about the complexity of innate pathways induced by a whole pathogen, especially in the lungs. 170 controlling the signaling pathways leading to exuberant inflammation is of major interest, but a balance needs to be struck to maintain host defence against infection. a feasible approach might be by treating patients at the peak of inflammation where immune mediators are in excess and using drugs with a short half-life. this is a promising strategy, but, to our knowledge, no drugs targeting signaling components are reported efficacious in human lung infection, although they are under development. 171 a comprehensive review of relevant inhibitors of inflammatory signaling pathways can be found elsewhere. 172 receptors recognizing pathogens such as toll-like receptors or nod-like receptors, but also tnf and il-1 , which are often involved in amplification of the inflammatory response, are potent activators of nf-b. 173 nf-b induction is a key factor triggering inflammation in the influenza-infected epithelium. 174 mice treated with a cell-permeable peptide that reduces nf-b levels, via an effect on i-kappa-b kinase beta, reduces pulmonary rsv-induced inflammation. 175 again, this strategy may negatively affect respiratory bacteria, since intra-tracheal adenovirus delivery of a dominant nf-b inhibitor impairs clearance of respiratory p. aeruginosa , 176 despite the reduced inflammation observed with purified bacterial products. 177, 178 peroxisome proliferator-activated receptor-is a nuclear receptor involved in the stress response during lung injury and attenuates inflammatory responses by inhibiting nf-b. 179, 180 many steroids target peroxisome proliferator-activated receptor-and show encouraging results in models of lung inflammation. 172 a series of natural and synthetic ligands for these receptors have been developed and rsv-specific responses in human lung epithelial cell lines are reduced by some of these agonists. 181 mitogen-activated protein kinase p38 and c-jun-n-terminal kinase pathways may also provide future suitable targets. activation of the p38 pathway is often associated with induction of nf-b (in toll-like receptor responses, for example) and is thought to maintain inflammatory responses by stabilizing cytokine mrna. 182 inhibiting the mitogen-activated protein kinase pathway may, therefore, favor termination of inflammation. inhibitors of p38 reduce the epithelial disruption caused by rsv and bordetella pertussis . 183, 184 again, inhibition of this pathway, to our knowledge, although tested in asthma, 185 has not been tested in lung infection models. another approach to reduce pathogen-induced immunopathology during acute infection is to target signaling molecules involved in cell migration. downstream of chemokine receptors is the phosphatidylinositol 3-kinase, which activates protein kinase c and rho gtpases. 186 phosphatidylinositol 3-kinase inhibitors reduce the recruitment of neutrophils and t cells in vivo . 187 -189 although this treatment is efficient in alleviating chronic inflammation such as asthma, 190 it is not validated for acute microbial infection. in addition, use of phosphatidylinositol 3-kinase inhibitors may interfere with development of the innate response to bacteria. 191 with the identification of each new cytokine, a series of papers describing their manipulation in models of acute infection has followed. all of them cannot be detailed here due to space constraint, but our discussion can be limited to those that have been tested therapeutically in acute lung infection models. the first, type-i ifn, plays such an important role in limiting viral replication that they have developed strategies to avoid it. during rna virus lung infection ifn-is produced predominantly by alveolar macrophages (or, to a lesser extent, pdcs) whose depletion impairs viral clearance. 192 dosing with ifn-and a double-stranded rna ifn-inducer, 4 h after sars coronaviruas infection, reduces lung viral titers. 193 similarly, rsv or human metapneumovirus-infected balb / c mice, treated intranasally with recombinant ifn-, have reduced lung viral titers and inflammatory disease as compared with untreated controls. 194 prophylactic treatment of sars coronavirus-infected macaques with pegylated ifn-significantly reduces viral load and pulmonary damage; post-exposure treatment is effective, although producing intermediate results. 195 recombinant ifn-, therefore, appears beneficial for reducing viral replication and associated pathology when administered early after infection. the influence of concomitant bacterial infection requires examination. other early innate cytokines important in respiratory viral and bacterial infections include il-1, il-12, and tnf. their blockade or promotion, however, is complicated by the fact that respiratory bacteria tend to require them for clearance. for example, rhinovirus induces il-1-receptor antagonist, il-1ra, from airway epithelial cells, which facilitates resolution of inflammation. 196 however, il-1ra enhances bacterial outgrowth in the lungs of mice with pneumococcal pneumonia, without the benefit of reducing the host response. 197 therefore, this pathway is predicted to be good for one lung infection but bad for another. blockade of il-12 is another example where neutralization benefits the severity of lung viral infection, 198, 199 but impairs clearance of lung histoplasma capsulatum 200 and legionella pneumophila 201 infections. the list continues with neutralization of tnf, which benefits immune pathology induced by influenza and rsv infection, 81, 202 but not respiratory bacteria or fungi, 203, 204 especially if treatment is prolonged (although this will depend on the precise strategy used 205 ). webster and coworkers reported recently that absence of tnf and il-6 is of no benefit during murine influenza h5n1 infection. 206 however, up to 50 % survival was observed in some experiments and the gene-depleted animals used may harbor other developmental abnormalities. viruses induce local production of ifn-by t and non-t cells in the respiratory tract, and its neutralization not only reduces local lung cellularity and systemic humoral responses to influenza virus infection in mice, 207 but may also delay viral clearance. 208 ifn-is also required for clearance of s. pneumoniae . 209 it would appear that the new cytokine on the block, il-17, may also present opposing effects in viral and bacterial lung infection. suitable reagents are yet to be developed for il-17 neutralization, but in vivo blockade of il-23p19 alone, or in combination with il-23 / il-12p40 (required for th17 development), significantly reduces mycoplasma pneumoniae -induced il-17 and subsequent bacterial clearance, possibly via reduced neutrophil activity. 210 k. pneumoniae clearance also depends on il-17, 211 but the influence of il-17 or il-23 neutralization on respiratory viral infection is unknown. administration of immune suppressive cytokines has been considered for infection induced lung inflammatory disease and encountered similar problems. il-10 neutralization increases survival of mice infected with k. pneumoniae . 212 in contrast, influenza induces indoleamine 2,3-dioxygenase and il-10 production, which may limit lung inflammation. however, treatment with an indoleamine 2,3-dioxygenase inhibitor (that would reduce il-10) induces a 20-fold reduction in lung s. pneumoniae load. 213 intranasal il-10 treatment of rsv-infected mice reduces lung nuclear factor b dna-binding activity, chemokine gene expression, and airway inflammation. 175 similarly, administration of tgf--encoding plasmid reduces inflammation to viral and fungal lung pathogens, but, without exception, prevents their clearance. 54 although a plethora of strategies have been used to modulate lung inflammation during acute infection, few are tested therapeutically after the onset of clinical symptoms, and even less are tested in models of common co-existing lung pathogens. hundreds of immune modulators are, therefore, beneficial during influenza infection, but what of the bacteria that sometimes accompany them? would immune therapeutics work best in combination with antibiotics? equally, selection of immune modulators requires precision in determining exactly what the patient is infected with. in the absence of this knowledge, we may apply a beneficial strategy to one supposed infection, but create an altogether different type of problem. several gaps remain in our knowledge of how immune homeostasis is maintained in the respiratory tract, inflammatory pathways that overcome review them, and the precise effector / memory phenotype of immune cells within the airways and lung parenchyma. controversy also still surrounds the potential of " acute " respiratory infections to persist, since detection of pathogen genome is common, but few studies have been able to demonstrate classical reactivation long after the primary infection. should persistence exist then, depending on the nature of the persisting organism, immune modulators may cause their reactivation. the development of sensitive tools for pathogen detection, and elucidation of specific gene expression patterns in patients with acute infection, 214 mean that future use of targeted immune modulators is not impossible as long as we are able to strike a balance between immune pathology and immune defence. inducible bronchus-associated lymphoid tissue (ibalt) in patients with pulmonary complications of rheumatoid arthritis balt development and augmentation of hyperoxic lung injury in mice defi cient in nqo1 and nqo2 . free radic persistence and responsiveness of immunologic memory in the absence of secondary lymphoid organs role of cxc chemokine ligand 13, cc chemokine ligand (ccl) 19, and ccl21 in the organization and function of nasal-associated lymphoid tissue antigenspecifi c cd8(+) t cells persist in the upper respiratory tract following infl uenza virus infection antibody-forming cells in the nasal-associated lymphoid tissue during primary infl uenza virus infection anti-tnp-forming cells in bronchus-associated lymphoid tissue (balt) and paratracheal lymph node (ptln) of the rat after intratracheal priming and boosting with tnp-klh induction of mhc class ii antigens on rat bronchial epithelial cells by interferon-gamma and its effect on antigen presentation luminal antigens access late endosomes of intestinal epithelial cells enriched in mhc i and mhc ii molecules: in vivo study in crohn's ileitis frequency, specifi city, and sites of expansion of cd8+ t cells during primary pulmonary infl uenza virus infection memory t cell recruitment to the lung airways chemokines in acute respiratory distress syndrome chemokines and their receptors guiding t lymphocyte recruitment in lung infl ammation intracellular interferon-gamma expression in natural killer cells precedes lung cd8+ t cell recruitment during respiratory syncytial virus infection t cell responses to infl uenza virus infection: effector and memory cells effector cd4+ and cd8+ t-cell mechanisms in the control of respiratory virus infections a critical role for icos co-stimulation in immune containment of pulmonary infl uenza virus infection accumulation of gamma/delta t cells in the lungs and their roles in neutrophil-mediated host defense against pneumococcal infection evidence for the involvement of lung-specifi c gammadelta t cell subsets in local responses to streptococcus pneumoniae infection immunity to a pulmonary cryptococcus neoformans infection requires both cd4+ and cd8+ t cells ox40 ligation on activated t cells enhances the control of cryptococcus neoformans and reduces pulmonary eosinophilia il-5 is required for eosinophil recruitment, crystal deposition, and mononuclear cell recruitment during a pulmonary cryptococcus neoformans infection in genetically susceptible mice (c57bl/6) the prolonged life-span of alveolar macrophages downregulation of the antigen presenting cell function(s) of pulmonary dendritic cells in vivo by resident alveolar macrophages th1 and th2 cytokine induction in pulmonary t-cells during infection with respiratory syncytial virus induction of secretory immunity and memory at mucosal surfaces iga receptors in health and disease regulation of immunological homeostasis in the respiratory tract inhibition of the immunosuppressive activity of resident pulmonary alveolar macrophages by granulocyte/macrophage colony-stimulating factor human bronchial epithelium controls th2 responses by th1-induced, nitric oxide-mediated stat5 dephosphorylation: implications for the pathogenesis of asthma differential recognition of tlr-dependent microbial ligands in human bronchial epithelial cells impact of bronchial epithelium on dendritic cell migration and function: modulation by the bacterial motif kpompa bronchial epithelial cell-derived cytokines (g-csf and gm-csf) promote the survival of peripheral blood neutrophils in vitro diesel exhaust particle-exposed human bronchial epithelial cells induce dendritic cell maturation organ-specifi c regulation of innate immunity induction of a homeostatic circuit in lung tissue by microbial compounds modulation of dendritic cell traffi cking to and from the airways an absence of reactive oxygen species improves the resolution of lung infl uenza infection tgf-beta prevents eosinophilic lung disease but impairs pathogen clearance cytokine modulation of the immunosuppressive phenotype of pulmonary alveolar macrophage populations sequestration of inhaled particulate antigens by lung phagocytes. a mechanism for the effective inhibition of pulmonary cell-mediated immunity inhibitory activity of unstimulated alveolar macrophages on t-lymphocyte blastogenic response alveolar macrophage in the driver's seat alveolar macrophage elimination in vivo is associated with an increase in pulmonary immune response in mice identifi cation and characterization of human pulmonary dendritic cells mouse respiratory tract dendritic cell subsets and the immunological fate of inhaled antigens origin and steady-state turnover of class ii mhc-bearing dendritic cells in the epithelium of the conducting airways different roles for human lung dendritic cell subsets in pulmonary immune defense mechanisms murine plasmacytoid dendritic cells induce effector/ memory cd8+ t-cell responses in vivo after viral stimulation organ-dependent in vivo priming of naive cd4+, but not cd8+, t cells by plasmacytoid dendritic cells dendritic cell subsets and immune regulation in the lung recent progress in the biology of airway dendritic cells and implications for understanding the regulation of asthmatic infl ammation dendritic cells and the regulation of the allergic immune response emerging role of dendritic cells in respiratory viral infection plasmacytoid dendritic cells inhibit pulmonary immunopathology and promote clearance of respiratory syncytial virus plasmacytoid dendritic cells limit viral replication, pulmonary infl ammation, and airway hyperresponsiveness in respiratory syncytial virus infection lung dendritic cell migration cd200 and membrane protein interactions in the control of myeloid cells ccr2 and ccr6, but not endothelial selectins, mediate the accumulation of immature dendritic cells within the lungs of mice in response to particulate antigen chemokine receptor ccr2 but not ccr5 or ccr6 mediates the increase in pulmonary dendritic cells during allergic airway infl ammation specifi c migratory dendritic cells rapidly transport antigen from the airways to the thoracic lymph nodes a major lung cd103 (alphae)-beta7 integrin-positive epithelial dendritic cell population expressing langerin and tight junction proteins long-term maintenance of virus-specifi c effector memory cd8+ t cells in the lung airways depends on proliferation differential t cell function and fate in lymph node and nonlymphoid tissues memory t cell populations in the lung airways are maintained by continual recruitment tumor necrosis factor as a mediator of infl ammation in infl uenza a viral pneumonia cascade of fever production in mice infected with infl uenza virus tnf soluble receptor and antiserum against tnf enhance lipopolysaccharide fever in mice sickness behavior in mice defi cient in interleukin-6 during turpentine abscess and infl uenza pneumonitis the role of mip-1 alpha in infl ammation and hematopoiesis prolonged production of tnf-alpha exacerbates illness during respiratory syncytial virus infection effects of infl ammatory cytokines on the permeability of human lung microvascular endothelial cell monolayers and differential eosinophil transmigration role of tnf-alpha in lung tight junction alteration in mouse model of acute lung infl ammation tnf/tnfr family members in costimulation of t cell responses costimulation of cd8 t cell responses by ox40 ox40 ligand shuts down il-10-producing regulatory t cells evaluation of ox40 ligand as a costimulator of human antiviral memory cd8 t cell responses: comparison with b7.1 and 4-1bbl immunotherapy with ox40l-fc or anti-ctla-4 enhances local tissue responses and killing of leishmania donovani 4-1bb and ox40 stimulation enhance cd8 and cd4 t-cell responses to a dna prime, poxvirus boost vaccine a critical role for ox40 in t cell-mediated immunopathology during lung viral infection during viral infection of the respiratory tract, cd27, 4-1bb, and ox40 collectively determine formation of cd8+ memory t cells and their capacity for secondary expansion 4-1bb and ox40 act independently to facilitate robust cd8 and cd4 recall responses cooperation between 4-1bb and icos in the immune response to infl uenza virus revealed by studies of cd28/icos-defi cient mice ox40 ligand expressed by dcs costimulates nkt and cd4+ th cell antitumor immunity in mice role of ox40 signals in coordinating cd4 t cell selection, migration, and cytokine differentiation in t helper (th)1 and th2 cells signaling of gp34 (ox40 ligand) induces vascular endothelial cells to produce a cc chemokine rantes/ccl5 therapeutic targeting of the effector t-cell co-stimulatory molecule ox40 temporal segregation of 4-1bb versus cd28-mediated costimulation: 4-1bb ligand infl uences t cell numbers late in the primary response and regulates the size of the t cell memory response following infl uenza infection differential requirement for cd80 and cd80/cd86-dependent costimulation in the lung immune response to an infl uenza virus infection disruption of cd40/cd40l interaction infl uences the course of cryptococcus neoformans infection cd27 promotes survival of activated t cells and complements cd28 in generation and establishment of the effector t cell pool lung infections: role of apoptosis in host defense and pathogenesis of disease suppression of alveolar macrophage apoptosis prolongs survival of rats and mice with pneumocystis pneumonia cd95/cd95 ligand interactions on epithelial cells in host defense to pseudomonas aeruginosa the alpha1beta1 integrin and tnf receptor ii protect airway cd8+ effector t cells from apoptosis during infl uenza infection cutting edge: engagement of nkg2a on cd8+ effector t cells limits immunopathology in infl uenza pneumonia apoptosis and pathogenesis of avian infl uenza a (h5n1) virus in humans avian infl uenza a/hk/483/97(h5n1) ns1 protein induces apoptosis in human airway epithelial cells depletion of lymphocytes and diminished cytokine production in mice infected with a highly virulent infl uenza a (h5n1) virus isolated from humans the role of interleukin-10 in the inhibition of t-cell proliferation and apoptosis mediated by parainfl uenza virus type 3 inhibition of apoptosis in chlamydia-infected cells: blockade of mitochondrial cytochrome c release and caspase activation aspects on the interaction of streptococcus pneumoniae and haemophilus infl uenzae with human respiratory tract mucosa getting to the site of infl ammation: the leukocyte adhesion cascade updated lymphocyte homing and homeostasis pharmacological properties, toxicology and scientifi c rationale for the use of natalizumab (tysabri) in infl ammatory diseases anti-adhesion antibodies efalizumab, a humanized anti-cd11a monoclonal antibody fatalities in natalizumab treatment -a ' no go ' for leukocyte recirculation approaches? chemokine regulation of infl ammation during acute viral infection the many roles of chemokines and chemokine receptors in infl ammation strategies for chemokine antagonists as therapeutics chemokines: role in immune cell traffi c intracellular signalling controlling integrin activation in lymphocytes multiple chemotactic factors: fi ne control or redundancy? therapeutic effect of anti-macrophage infl ammatory protein 2 antibody on infl uenza virus-induced pneumonia in mice interactions between infl uenza and bacterial respiratory pathogens: implications for pandemic preparedness how do viral infections predispose patients to bacterial infections? selective attraction of monocytes and t lymphocytes of the memory phenotype by cytokine rantes extension of recombinant human rantes by the retention of the initiating methionine produces a potent antagonist role of ccl5 (rantes) in viral lung disease the pneumonia virus of mice infection model for severe respiratory syncytial virus infection: identifying novel targets for therapeutic intervention essential contribution of monocyte chemoattractant protein-1/c -c chemokine ligand-2 to resolution and repair processes in acute bacterial pneumonia interferon-inducible protein 10, but not monokine induced by gamma interferon, promotes protective type 1 immunity in murine klebsiella pneumoniae pneumonia differential chemokine expression following respiratory virus infection refl ects th1-or th2-biased immunopathology role of cxcr3 in the immune response to murine gammaherpesvirus 68 functional antagonism of chemokine receptor ccr1 reduces mortality in acute pneumovirus infection in vivo respiratory syncytial virus-induced exaggeration of allergic airway disease is dependent upon ccr1-associated immune responses lymphocyte egress from thymus and peripheral lymphoid organs is dependent on s1p receptor 1 novel therapies for microvascular permeability in sepsis phenotypic and functional differences between lymphocytes from nalt and nasal passages of mice lactobacillus casei administration reduces lung injuries in a streptococcus pneumoniae infection in mice memory of mice and men: cd8+ t-cell cross-reactivity and heterologous immunity induction, exacerbation and inhibition of allergic and autoimmune diseases by infection infl uenza virus lung infection protects from respiratory syncytial virus-induced immunopathology memory cd8+ t cells in heterologous antiviral immunity and immunopathology in the lung specifi c history of heterologous virus infections determines antiviral immunity and immunopathology in the lung prior exposure to live mycobacterium bovis bcg decreases cryptococcus neoformans -induced lung eosinophilia in a gamma interferon-dependent manner sustained desensitization to bacterial toll-like receptor ligands after resolution of respiratory infl uenza infection stimulation via toll-like receptor 9 reduces cryptococcus neoformans -induced pulmonary infl ammation in an il-12-dependent manner innate imprinting by the modifi ed heat-labile toxin of escherichia coli (ltk63) provides generic protection against lung infectious disease infections and allergy -helminths, hygiene and host immune regulation amelioration of infl uenza-induced pathology in mice by coinfection with trichinella spiralis colonic bacterial infection abrogates eosinophilic pulmonary disease resveratrol ameliorates serratia marcescens -induced acute pneumonia in rats in vivo anti-infl uenza virus activity of an immunomodulatory acidic polysaccharide isolated from cordyceps militaris grown on germinated soybeans retinoic acid therapy attenuates the severity of tuberculosis while altering lymphocyte and macrophage numbers and cytokine expression in rats infected with mycobacterium tuberculosis nonspecifi c defence mechanism: the role of nitric oxide infl ammatory cells and oxygen radicals benefi cial effects of nitric oxide inhalation on pulmonary bacterial clearance inhaled nitric oxide increases endothelial permeability in pseudomonas aeruginosa pneumonia anti-infl ammatory effects of inhaled nitric oxide are optimized at lower oxygen concentration in experimental klebsiella pneumoniae pneumonia immune and functional role of nitric oxide in a mouse model of respiratory syncytial virus infection antioxidant treatment ameliorates respiratory syncytial virus-induced disease and lung infl ammation inhibitory effects of recombinant manganese superoxide dismutase on infl uenza virus infections in mice post-infection a77-1726 blocks pathophysiologic sequelae of respiratory syncytial virus infection complexities of targeting innate immunity to treat infection nuclear factor kappa b is a promising therapeutic target in infl ammatory lung disease targeting signal transduction as a strategy to treat infl ammatory diseases signaling pathways downstream of patternrecognition receptors and their cross talk the ikappab kinase is a key factor in triggering infl uenza a virus-induced infl ammatory cytokine production in airway epithelial cells ikappab kinase is a critical regulator of chemokine expression and lung infl ammation in respiratory syncytial virus infection targeted immunomodulation of the nf-kappab pathway in airway epithelium impacts host defense against pseudomonas aeruginosa airway epithelium controls lung infl ammation and injury through the nf-kappa b pathway duration and intensity of nf-kappab activity determine the severity of endotoxin-induced acute lung injury peroxisome proliferator-activated receptor-gamma is a new therapeutic target in sepsis and infl ammation involvement of ppar nuclear receptors in tissue injury and wound repair peroxisome proliferator-activated receptor-gamma agonists inhibit respiratory syncytial virus-induced review expression of intercellular adhesion molecule-1 in human lung epithelial cells the many paths to p38 mitogen-activated protein kinase activation in the immune system mapk and heat shock protein 27 activation are associated with respiratory syncytial virus induction of human bronchial epithelial monolayer disruption critical involvement of p38 map kinase in pertussis toxin-induced cytoskeletal reorganization and lung permeability inhaled p38alpha mitogen-activated protein kinase antisense oligonucleotide attenuates asthma in mice chemoattractant receptor signaling and the control of lymphocyte migration airway infl ammation: chemokine-induced neutrophilia and the class i phosphoinositide 3-kinases tissue-and stimulus-dependent role of phosphatidylinositol 3-kinase isoforms for neutrophil recruitment induced by chemoattractants in vivo regulation of phosphatidylinositol 3-kinase by polyisoprenyl phosphates in neutrophil-mediated tissue injury blockade of infl ammation and airway hyperresponsiveness in immune-sensitized mice by dominant-negative phosphoinositide 3-kinase-tat importance of phosphoinositide 3-kinase gamma in the host defense against pneumococcal infection alveolar macrophages are the primary interferon-alpha producer in pulmonary infection with rna viruses evaluation of immunomodulators, interferons and known in vitro sars-cov inhibitors for inhibition of sars-cov replication in balb/c mice activity and regulation of alpha interferon in respiratory syncytial virus and human metapneumovirus experimental infections pegylated interferon-alpha protects type 1 pneumocytes against sars coronavirus infection in macaques rhinovirus regulation of il-1 receptor antagonist in vivo and in vitro : a potential mechanism of symptom resolution interleukin-1 receptor antagonist transiently impairs antibacterial defense but not survival in murine pneumococcal pneumonia interleukin 12 administration enhances th1 activity but delays recovery from infl uenza a virus infection in mice il-12 treatment attenuates th2 and b cell responses but does not improve vaccine-enhanced lung illness jr interleukin-12 neutralization alters lung infl ammation and leukocyte expression of cd80, cd86, and major histocompatibility complex class ii in mice infected with histoplasma capsulatum in vivo regulation of replicative legionella pneumophila lung infection by endogenous interleukin-12 inhibition of tumor necrosis factor reduces the severity of virus-specifi c lung immunopathology the role played by tumor necrosis factor during localized and systemic infection with streptococcus pneumoniae transient neutralization of tumor necrosis factor alpha can produce a chronic fungal infection in an immunocompetent host: potential role of immature dendritic cells neutralization of tumor necrosis factor (tnf) by antibody but not tnf receptor fusion molecule exacerbates chronic murine tuberculosis inhibition of the cytokine response does not protect against lethal h5n1 infl uenza infection in vivo blockade of gamma interferon affects the infl uenza virus-induced humoral and the local cellular immune response in lung tissue administration of anti-ifn-gamma antibody to b2-microglobulindefi cient mice delays infl uenza virus clearance but does not switch the response to a t helper cell 2 phenotype role of interferon-gamma in valpha14+ natural killer t cell-mediated host defense against streptococcus pneumoniae infection in murine lungs il-23-dependent il-17 production is essential in neutrophil recruitment and activity in mouse lung defense against respiratory mycoplasma pneumoniae infection cutting edge: roles of toll-like receptor 4 and il-23 in il-17 expression in response to klebsiella pneumoniae infection neutralization of il-10 increases survival in a murine model of klebsiella pneumonia infl uenza-induced expression of indoleamine 2,3-dioxygenase enhances interleukin-10 production and bacterial outgrowth during secondary pneumococcal pneumonia gene expression patterns in blood leukocytes discriminate patients with acute infections comparison of murine nasal-associated lymphoid tissue and peyer's patches isolation and characterization of mouse nasalassociated lymphoid tissue role of type 1 t helper cells in the resolution of acute streptococcus pneumoniae sinusitis: a mouse model key: cord-257805-pcp3qgn0 authors: mehta, harsh; ivanovic, sasa; cronin, amanda; vanbrunt, lindsey; mistry, nirav; miller, richard; yodice, paul; rezai, fariborz title: novel coronavirus-related acute respiratory distress syndrome in a patient with twin pregnancy: a case report date: 2020-05-16 journal: case rep womens health doi: 10.1016/j.crwh.2020.e00220 sha: doc_id: 257805 cord_uid: pcp3qgn0 we present the case of a 39-year-old woman, g1p0, who had conceived twins via in-vitro fertilization, who presented at 27 weeks of gestation with nasal congestion and dry cough for 7 days. on presentation, her physical examination was benign, except for sinus tachycardia, and she was oxygenating adequately on room air. laboratory studies were unremarkable, except a pcr test positive for sars-cov2, and a ct scan of her chest showed bilateral multi-focal ground-glass opacities. a fetal non-stress test was reassuring. she was treated with intravenous fluids, ceftriaxone, azithromycin, and hydroxychloroquine. during her hospital stay, she developed progressively worsening respiratory failure, initially requiring non-invasive ventilation, and subsequently progressed to acute respiratory distress syndrome requiring mechanical ventilation. she then suffered from sudden hypoxemia and hemodynamic collapse, on maximal ventilatory support, prompting an emergency cesarean section at bedside. this led to rapid stabilization of hemodynamic parameters, and of her overall respiratory status. both the twins were born prematurely, and one of them tested positive for sars-cov2. vitro fertilization, who presented at 27 weeks of gestation with nasal congestion and dry cough for 7 days. on presentation, her physical examination was benign, except for sinus tachycardia, and she was oxygenating adequately on room air. laboratory studies were unremarkable, except a pcr test positive for sars-cov2, and a ct scan of her chest showed bilateral multi-focal ground-glass opacities. a fetal non-stress test was reassuring. she was treated with intravenous fluids, ceftriaxone, azithromycin, and hydroxychloroquine. during her hospital stay, she developed progressively worsening respiratory failure, initially requiring non-invasive ventilation, and subsequently progressed to acute respiratory distress syndrome requiring mechanical ventilation. she then suffered from sudden hypoxemia and hemodynamic collapse, on maximal ventilatory support, prompting an emergency cesarean section at bedside. this led to rapid stabilization of hemodynamic parameters, and of her overall respiratory status. since its first reported case in china, more than 4 coronaviruses are known pathogens and have previously been responsible for epidemics caused by severe acute respiratory syndrome coronavirus (sars-cov1) and middle eastern respiratory syndrome coronavirus (mers-cov) [2] . pregnancy is a unique immunologic state, where the maternal immune system is modulated to allow tolerance to paternally derived fetal antigens, thereby leading to an increased susceptibility to infections, especially pneumonia [3] . in one study, up to 25% of pregnant women with pneumonia required critical care services, including mechanical ventilation [4] . overall, pregnant women are more susceptible to respiratory pathogens, and might be at an increased risk for covid-19 [5] . during the previous sars and mers outbreaks, pregnant women were reported to have poorer outcomes than the general population [4] . we report a case of a high-risk pregnant woman who developed respiratory failure associated with covid-19, and had a favorable outcome postdelivery. we also report possible vertical transmission of covid-19 infection in this case. a 39-year-old african american pregnant woman (g1p0) presented to the emergency department with a 7-day history of nasal congestion and dry cough. she was at 27 j o u r n a l p r e -p r o o f journal pre-proof weeks of ultrasound-confirmed in-utero gestation with diamniotic-dichorionic twins, achieved via in-vitro fertilization. two days prior to presentation, she started experiencing chest discomfort and shortness of breath. she eventually developed fever, which prompted her visit to the emergency department. she had no known medical problems, and her current pregnancy had been uneventful. she denied recent travel. her only medications included prenatal vitamins and aspirin. on arrival at the emergency department, she had a temperature of 98.3°f (36.8°c), a heart rate of 121 beats/minute, blood pressure of 135/105 mmhg, respiratory rate of 20 breaths/minute, and an oxygen saturation of 96% on room air. on physical examination, her lungs were clear to auscultation bilaterally, while a cardiac exam revealed tachycardia with a regular rhythm and normal heart sounds. abdominal exam was consistent with 27 weeks of gestation. bilateral lower extremities were symmetric without cyanosis or edema. a fetal non-stress test (nst) showed moderate heart rate variability and no decelerations for both the twins. laboratory tests were mostly unremarkable, as shown in table 1. tests for influenza and other respiratory pathogens were negative. a nasopharyngeal swab for covid-19 rt-pcr was obtained. a ct angiogram of her chest was negative for pulmonary embolism (pe), but revealed multifocal patchy ground-glass opacities in both her lungs, consistent with multi-lobar pneumonia. she was started on intravenous fluids, ceftriaxone and azithromycin. her covid-19 test came back as positive after 27 hours. hydroxychloroquine was added, and betamethasone was administered for fetal lung maturity. over the next two days, her j o u r n a l p r e -p r o o f respiratory status worsened, leading to a requirement for supplemental oxygen. on day 4, she became tachypneic and started requiring increasing amounts of oxygen, for which the fio2 was increased to 80%, delivered at 35l/min via a high-flow nasal cannula. she was then transferred to the intensive care unit (icu) and required endotracheal intubation and mechanical ventilation. settings were adjusted to a tidal volume of 500 ml/breath, respiratory rate of 18 breaths/minute, positive end-expiratory pressure (peep) of 10 cm h2o and fio2 of 70%. a repeat chest x-ray showed worsening bilateral pulmonary infiltrates, raising concern for development of acute respiratory distress syndrome (ards). over the next two days, she required maximal ventilator support, with a fio2 of 100%. her blood pressure also dropped to 92/52 mmhg, for which was started on norepinephrine. fetal heart tracings were recorded daily, which continued to be reassuring up to this point. on day 7, her condition rapidly deteriorated, with oxygen saturation dropping to 80%; hence peep was increased to 14 cm h2o, while maintaining an fio2 of 100%. chest x-ray showed worsening bilateral alveolar infiltrates, consistent with progressing ards. during this time, her vasopressor requirements also increased significantly, and the norepinephrine was increased to 30 mcg/min to achieve a mean arterial pressure (map) of 65 mmhg. due to rapid maternal decompensation, we decided to deliver the fetuses. a classical c-section was performed at the patient's bedside, and two fetuses were delivered successfully. baby a was female, weighed 925 g, and had apgar scores of 1, 3 and 6 at 1, 5 and 10 minutes respectively. baby b was male, weighed 1050 g, and had apgar scores of 5, 6 and 6 at 1, 5 and 10 minutes respectively. the neonates were immediately transferred to the neonatal intensive care unit (nicu). journal pre-proof immediately after uterine decompression, hemodynamic parameters improved significantly. norepinephrine was titrated down to 6 mcg/min and the patient gradually weaned off. fio2 was decreased to 40% within an hour, while peep was gradually decreased to 10 cm h2o. details of her vasopressor and ventilator requirements are outlined in figure 1. on day 9, she was successfully extubated and eventually transferred out of the icu. she was weaned off supplemental oxygen and was soon discharged home. the neonates were tested for covid-19. twin a tested positive 72 hours after birth; however, she did not exhibit any infectious symptoms. twin b tested negative at 72 hours after birth. his course was complicated by development of alveolar hemorrhage, which was attributed to preterm delivery. he required invasive mechanical ventilation for the same. our patient suffered from a progressively worsening respiratory failure, culminating in ards. high circulating volume, low albumin levels, increased capillary leak and development of a pro-inflammatory state have all been proposed as mechanisms predisposing pregnant women to ards [3] . following the american thoracic society's guidelines for management of ards, our patient was mechanically ventilated while a low tidal volume was delivered along with a high peep to maintain an oxygen saturation of more than 92% [11] . of note, on the seventh day of hospitalization, our patient's condition rapidly j o u r n a l p r e -p r o o f journal pre-proof deteriorated, and she required increasing ventilatory and vasopressor support. this deterioration was attributed to rapidly worsening ards. however, it is also important to identify that the high peep, which was originally applied to combat ards, could have led to increased intrathoracic pressure, ultimately leading to decreased venous return and cardiac output [11] . furthermore, in pregnancy, despite hypervolemia, the effect of the gravid uterus on inferior vena cava can compromise venous return, which, in combination with a high peep, can precipitate hypotension [12] . and hence, the fact that after delivery, her hemodynamic parameters rapidly improved can be attributed to the reduction in oxygen consumption and effective deliverance of preload, thereby leading to an improvement in cardiac output, and ultimately, in gas exchange. while prone positioning has been shown to reduce mortality in patients with severe ards by means of improved lung recruitment, data regarding its utility in pregnancy is sparse [13, 14, 15] . due to this reason, we decided against pronating the patient at the time of her rapid deterioration. in the data available in pregnant patients from the previous coronavirus outbreaks, no vertical transmission was reported [4] . in a recently published review of 37 pregnant mothers with covid-19 and their 38 newborns, no vertical transmission was reported. of these, all neonates with confirmed covid-19 had been infected after birth, via droplet or contact transmission [10]. in our case, the patient was intubated at the time of delivery, making droplet transmission unlikely. the babies were delivered via c-section, eliminating the possibility of fetal contact with maternal feces, which has been reported as a mode of transmission. during delivery, appropriate sterilization techniques and precautions were utilized, and any maternal contact with the neonates was avoided. both the neonates were transferred to the nicu immediately after birth. they were not breastfed, and appropriate aerosol and contact precautions were utilized during their handling in the nicu. twin a tested positive and twin b tested negative for covid-19 at 72 hours. due to the reasons above, we believe that twin a tested positive due to vertical transmission. we acknowledge that there are limitations to our theory. firstly, the placenta and umbilical cord blood were not tested for covid-19 and, secondly, vertical transmission would have affected both twins, but twin b, in our case, tested negative. in conclusion, our knowledge regarding the disease course of covid-19 during pregnancy is limited to the data available from previous epidemics of sars-cov1 and mers-cov infections. covid-19 has so far proven to be a highly contagious and lethal infection that can lead to rapidly worsening respiratory failure and death in the general population. our case reflects that pregnant women are certainly as susceptible, if not more, and, hence, these patients should be closely followed by a multidisciplinary team of physicians. prearranged plans for urgent delivery and neonatal resuscitation should be put in place, in anticipation of spontaneous labor or sudden maternal or fetal decline. while the vertical transmission of infection has not been reported until now, our case prompts us to think that it could certainly be possible and further research is warranted on the subject. harsh mehta contributed to project conceptualization, and writing and editing of the manuscript. sasa ivanovic contributed to editing of the manuscript. amanda cronin contributed to editing of the manuscript. lindsay vanbrunt contributed to editing of the manuscript. nirav mistry contributed to critical review of the manuscript. richard miller contributed to critical review of the manuscript. paul yodice contributed to critical review of the manuscript. fariborz rezai contributed to editing and critical review of the manuscript. the authors declare that they have no conflict of interest regarding the publication of this case report. the authors declare that they did not receive any funding for their work in the production of this manuscript. obtained. this case report was peer reviewed. a novel coronavirus from patients with pneumonia in china acute respiratory failure in pregnancy. obstetric medicine potential maternal and infant outcomes from coronavirus 2019-ncov (sars-cov-2) infecting pregnant women: lessons from sars, mers, and other human coronavirus infections. viruses why are pregnant women susceptible to covid-19? an immunological viewpoint a familial cluster of infection associated with the 2019 novel coronavirus indicating possible person-to-person transmission during the incubation period incubation period and other epidemiological characteristics of 2019 novel coronavirus infections with right truncation: a statistical analysis of publicly available case data clinical features of patients infected with 2019 novel coronavirus in wuhan, china. lancet clinical characteristics of 138 hospitalized patients with novel coronavirus-infected pneumonia in wuhan risks of novel coronavirus disease an official american thoracic society/european society of intensive care medicine/society of critical care medicine clinical practice guideline: mechanical ventilation in adult patients with acute respiratory distress syndrome physiological changes in pregnancy prone positioning in severe acute respiratory distress syndrome lung recruitability in sars-cov-2 associated acute respiratory distress syndrome: a single-center how safe is the prone position in acute respiratory distress key: cord-252012-hdjbxah8 authors: mcerlean, peter; greiman, alyssa; favoreto, silvio; avila, pedro c. title: viral diversity in asthma: immunology and allergy clinics of north america: asthma and infectious disease date: 2010-11-01 journal: immunology and allergy clinics of north america doi: 10.1016/j.iac.2010.08.001 sha: doc_id: 252012 cord_uid: hdjbxah8 asthma exacerbations are precipitated primarily by respiratory virus infection and frequently require immediate medical intervention. studies of childhood and adult asthma have implicated a wide variety of respiratory viruses in exacerbations. by focusing on both rna and dna respiratory viruses and some newly identified viruses, this review illustrates the diversity and highlights some of the uncertainties that exist in our understanding of virus-related asthma exacerbations. peter mcerlean, phd a, *, alyssa greiman, bsc a , silvio favoreto jr, phd, dds b , pedro c. avila, md c asthma is a heterogeneous inflammatory disease of the airways characterized by reversible airway obstruction, airway hyperresponsiveness, inflammatory cell infiltration, thickening of the lamina reticularis, and the accompanying symptoms of chest tightness, wheezing, coughing, and shortness of breath. 1, 2 asthma now affects an estimated 16.4 million adults and 7.0 million children (7.3% and 9.4% of the population, respectively) within the united states 3,4 and is additionally indicated as a "contributing factor" in nearly 7000 deaths each year. 5 whereas chronic asthma results from the daily inhalation and response to allergen (eg, dust, pollen, animal dander) and is by and large successfully managed by the individual, acute asthma attacks or exacerbations are precipitated primarily by respiratory viral infections and frequently require immediate medical intervention. in the united states alone, severe asthma exacerbations lead to over 400,000 hospitalizations each year, at a cost of one-third of the total $11.5 billion in annual asthma-related health care expenditures. 6 although the exact mechanism(s) by which respiratory viral infection causes asthma exacerbation remains to be determined, the respiratory viruses implicated in exacerbations have themselves been largely identified and well characterized ( table 1) . traditionally associated with acute respiratory illness (ari) or symptoms of the "common cold," the respiratory viruses implicated in asthma exacerbations predominantly possess rna genomes with a distinct genome organization (positive [1] or negative [à] sense), virus particle (virion) morphology (enveloped or nonenveloped), host cell receptor interaction, and well-defined annual or seasonal prevalence. the full extent of respiratory virus involvement in exacerbation has recently been revealed in asthma studies with the implementation of molecular methods of viral detection, specifically the reverse transcriptase polymerase chain reaction (rt-pcr). 7, 8 molecular methods of viral detection have superior sensitivity and specificity compared with cell culture-based methods and additionally allow for the improved identification of multiple viruses (and other pathogens), revealing the role dual or multiple infections play in asthma exacerbations. indeed, molecular methods have also been invaluable in identifying new respiratory viruses, the majority of which were described after the emergence of the severe acute respiratory syndrome-associated coronavirus (sars-cov) that prompted research into the etiology of ari. these "newly identified viruses" (nivs) including human metapneumovirus (hmpv; described pre-sars), the human rhinovirus (hrv) species c (hrv-cs), human coronaviruses (hcovs)-nl63 and -hku1, human bocavirus (hbov), and the ki and wu polyomaviruses (kipyv and wupyv) are now the focus of intense research, and their involvement in asthma exacerbations is slowly beginning to be determined. because those respiratory viruses most associated with exacerbations-the hrvs, respiratory syncytial virus (rsv), and hmpv-are reviewed elsewhere in this issue by miller and colleagues, this review discusses some of the other respiratory viruses implicated in childhood and adult asthma exacerbations, including additional rna viruses and those with dna genomes. by also encompassing some of the recently described nivs, this article illustrates the diversity that exists in virus-related asthma exacerbations. influenza viruses (ifvs) are probably the best known of all the respiratory viruses, due to their ability to cause annual epidemics and potential pandemics of serious respiratory disease. 9 ifvs pose the greatest risk of morbidity and mortality to young children and the elderly, and as such have been the focus of repeated public health and vaccination campaigns. however, despite their potential to cause serious respiratory illness in otherwise healthy individuals, most asthma studies describe relatively low levels of ifvs in exacerbations, accounting for approximately 1% to 9% of all virus-related asthma exacerbations (see table 1 ). 7, [10] [11] [12] [13] [14] [15] [16] ifvs constitute the family orthomyxoviridae and are segmented àsingle stranded (ss) rna viruses. the ifv virion has a pleomorphic envelope derived from host cell membranes and incorporates 3 viral encoded surface proteins: hemagglutinin (ha), neuraminidase (na), and matrix protein 2 (m2). under the envelope and encased within the matrix protein (m1), the core of the ifv virion contains the ribonucleoprotein complex, consisting of the viral rna segments, polymerase proteins (pb1, pb2, and pa), and the nucleoprotein (np). 17 there are 3 types of ifvs, -a (ifav), -b (ifbv), and -c (ifcv), which are divided further in subtypes and strains based on the combination of ha and na proteins (eg, h1n1, h3n2). ifav and ifcv have subtypes known to infect both animals (eg, birds, pigs) and humans, whereas ifbv is predominately a human virus. although all types of ifvs can cause ari in humans, ifav and ifbv subtypes and strains are of the most prominent in the annual epidemics or "flu season" that occurs during the winter months. ifvs initiate infection via the ha protein attaching to sialic acid (sa) linked to galactose (gal) sugars on the terminal ends of host cell surface glycans. it has been determined that the ha interaction is dependent on the sialic acid-galactose linkages, with ha from subtypes that infect humans recognizing a2,6 linkages (saa2,6gal), whereas ha from subtypes infecting other animals mostly recognize a2,3 linkages (saa2,3gal). 17, 18 although ifvs are implicated in 1.9% to 6.6% of wheezing illnesses in children 7,10,12,13 and have been detected in 9.8% of asthmatic adults during emergency department (ed) visits, 15 uncertainty remains as to whether ifvs are specifically responsible for the production of asthma exacerbations. 19, 20 the main contention arises from the role that vaccination has played in preventing exacerbations. because, unlike other respiratory viruses, vaccines against ifv subtypes and strains are available and asthmatics frequently have increased vaccination rates due to their at-risk status during flu season, ifv-related exacerbations should be reduced or indeed eliminated among asthmatic children and adults. 21, 22 however, some studies have shown that despite their three-to fourfold greater odds of having an influenza vaccination (as reported by parents), asthmatic children have higher rates of ifv-related hospital visits 23 and that vaccination does not affect the number, duration, or severity of ifvrelated asthma exacerbations compared with placebo. 24 other similar studies have shown that influenza vaccination also fails to reduce severe and fatal complications in adults with asthma and chronic obstructive pulmonary disease (copd). 20 the association of ifvs with exacerbations in vaccinated asthmatics questions the efficacy of seasonal influenza vaccines in this group and also suggests a role for other respiratory viruses in ifv-related exacerbations. if influenza vaccinations are effective, then it is likely that ifv-related exacerbations are caused by infection with another respiratory virus, such as those that peak in prevalence during the same time as ifvs (eg, rsv and hmpv). however, if influenza vaccination of asthmatics fails to reduce ifv-proven exacerbations (ie, by rt-pcr during symptomatic illness) then alternative vaccination strategies may be required to increase efficacy. ensuring that comprehensive respiratory virus testing is employed during both influenza vaccination and asthma studies and that vaccination rates among asthmatics remain high, the contention over vaccination and ifv-related exacerbations will be better clarified. some recent studies focusing on ari suggest that infection with other respiratory viruses, particularly hrvs, may actually provide "protection" against ifv infection. 25, 26 although such claims remain to be fully substantiated, viral competition may be a contributing factor in the relatively low rates of ifv-related asthma exacerbations being reported. it remains to be determined whether the asthmatic phenotype preferentially facilitates infection with respiratory viruses other than ifvs or whether, owing to vaccination in the wider community, the overall prevalence of ifvs is reduced making infection with another "uncontrolled" respiratory virus more likely. the association of ifvs with asthma exacerbation is complicated by the emergence of more virulent pandemic strains, such as the novel 2009 swine-origin ifav h1n1 (s-oiv), which may pose a greater risk to asthmatics than seasonal strains. 27 although the full impact of s-oiv remains to be determined, initial studies have indicated that among both children and adults hospitalized with rt-pcr-identified s-oiv, asthma accounted for the largest percentage (28%) of all underlying medical conditions. 27 however, it is unclear whether s-oiv evokes a specific immune response among asthmatics or whether, owing to increased awareness of this particular strain, asthmatics were more likely to seek medical care when respiratory symptoms initially began. hcovs were initially described in the 1960s in studies aiming to determine the etiologic agent responsible for ari, but gained notoriety after the emergence of sars-cov in 2003. since that time, 2 additional hcovs have been described in ari studies, viral diversity in asthma indicating that there are several hcovs potentially associated with respiratory illness in humans. because of the severity of associated illness, this review excludes sars-cov and focuses solely on the 4 hcovs associated with ari and currently implicated in 1% to 4% of virus-related asthma exacerbations: hcov-oc43, hcov-229e, and the nivs, hcov-nl63 and hcov-hku1 (see table 1 ). 10, 12, 28, 29 hcovs are classified within the family coronaviridae, subfamily coronavirinae, with the genus alphacoronavirus containing hcov-229e and hcov-nl63 and the genus betacoronavirus containing hcov-oc43 and hcov-hku1. 30 hcovs possess a 1ssrna genome, which is associated with nucleocapsid (n) phosphoprotein within the core of a host cell-derived enveloped virion. the hcov virion also comprises 3 viral encoded proteins (s, e, and m), and characterization has indicated that binding to host cell receptors is mediated predominately via the spike (s) glycoprotein, with each hcov employing a specific host cell receptor during infection (see table 1 ). the virion of betacoronavirus also contains a hemagglutinin (he) protein, which is thought to be involved in either host-receptor interactions or release of virus from infected cells. [31] [32] [33] the molecular and receptor interaction differences existing between hcovs are reflected in their seasonal prevalences. studies of ari have indicated that hcov-nl63 can be detected in the spring, summer, or winter whereas hcovs -hku1, -oc43, and -229e infections mainly occur during the autumn and winter months. [34] [35] [36] [37] a recent study of hcovs -nl63, -oc43, and -229e also found fluctuations occurring between their yearly prevalence. 37 the clinical impact of each hcov also appears to vary, but all present the greatest disease burden within the childhood population. 34 in a retrospective study of clinical samples taken over a 20-year period from young children (median age 14.5 months), the percentage of lower respiratory tract illness (lrti; including asthma exacerbations and bronchiolitis) associated with any hcov, hcov-nl63, or hcov-oc43 was estimated to be 4.6%, 2.6%, and 1.9%, respectively. 37 although this study failed to include hcov-hku1 in the testing panels, other studies have associated hcov-hku1 with wheezing illness in children. 34, 38 among asthmatic adults, hcovs have been detected during both ed visits and welldefined episodes of exacerbation prompted by ari. atmar and colleagues 15 reported that hcovs were detected in 21 of 148 ed visits during a 2-year study of 122 asthmatic adults, and kistler and colleagues 39 reported detections of hcovs, oc43, hku1, and nl63 in asthmatic adults with ari, of which hcov-nl63 occurred most in episodes of exacerbations. despite being associated with ari, hcov-229e appears to be the hcov least associated with asthma exacerbations. recent studies using rt-pcr have failed to find hcov-229e in either adult or childhood episodes of asthma exacerbation. 34, 37, 39 although some previous studies detected hcov-229e in asthmatic children, detection occurred with hcov-oc43 and individual hcov detection rates were not reported, despite the investigators using hcov-oc43-and hcov-229e-specific primers and antibodies. 16 another early study of asthmatic adults detected hcov-229e through analysis of paired sera in 12 instances where exacerbation could be objectively measured. however, hcov-299e detections could not be associated with a peak expiratory flow rate (pefr) mean decrease of greater than 50 l/min. 40 the reason for the lack of hcov-229e detections in asthma exacerbations remains unclear. parainfluenza viruses (pivs) are primarily associated with bronchiolitis and laryngotracheobronchitis or croup, in children younger than 4 years of age. 41, 42 pivs also pose a serious risk to immunocompromised individuals, and outbreaks of viral pneumonia among transplant recipients and patients undergoing chemotherapy have occurred. 41, 42 the pivs consist of 4 serotypes (1-4) and 2 subtypes (4a and 4b), and as a group are responsible for 1% to 8% of virus-related asthma exacerbations (see table 1 ). 7, 10, 12, 13, 29, 43 like rsv and hmpv, pivs are nonsegmented àssrna viruses belonging to the family paramyxoviridae. 44 pivs 1 to 3, 4a, and 4b are classified within 2 of the 5 genera of the subfamily paramyxovirinae, genus respirovirus (piv-1 and piv-3) and genus rubulavirus (piv2 and piv-4a and -4b). 45 in contrast to the g glycoprotein encoded by rsv and hmpv, pivs encode a hemagglutinin-neuraminidase (hn) glycoprotein, which interacts with gangliosides (sialic acid-containing oligosaccharides) on target host cells during infection. 46, 47 seasonal and yearly prevalence of pivs have been shown to vary among the serotypes. piv-1, -2, and -4 have the highest prevalence in the autumn and winter months whereas piv-3 occurs mostly in the spring and summer. peaks in piv-1 prevalence have been shown to occur biennially. piv-2 prevalence exhibits some yearly variation and piv-3 is consistently detected each year. piv-4 is the least prevalent of all pivs. 42 although each piv has the potential to cause respiratory illness in any age group, pivs 1 to 3 appear to be more associated with a particular clinical presentation at certain ages. piv-1 is associated with croup in children 1 to 4years old, piv-2 with bronchiolitis in the age group younger than 1 year, and piv-3 is more associated with viral pneumonia in individuals older than 15 years. 42 studies of asthmatic children have revealed that each piv type plays a different role in the production of asthma symptoms. in studies where the incidence of each individual piv is described, piv-1 is detected in 1.4% to 2.9%, piv-2 in 1.4%, piv-3 in 2.9% to 6%, and piv-4 in up to 1.9% of wheezing illnesses. [12] [13] [14] 43 although pivs are largely considered as a single group in adult asthma studies, pivs are still detected in episodes of exacerbation. atmar and colleagues 15 detected pivs 1 to 3 in 16 of 138 episodes of ari in asthmatic adults, with at least one infection each of piv-2 and piv-3 resulting in an ed visit, and nicholson and colleagues 40 detected 5 cases of piv1-3 in asthmatic adults, 3 of which had instances of a pefr mean decrease of greater than 50 l/min. adenoviruses (advs) comprise a large group of dna viruses that are known to cause a wide variety of clinical syndromes including diarrhea, keratoconjunctivitis, and hemorrhagic cystitis. 48, 49 however, advs are best known as a primary cause of ari and, particularly in young children, have been implicated in the production of more severe lrti. fifty-one adv serotypes have been identified, and as a group advs are associated with up to 7% of virus-related asthma exacerbations (see table 1 ). 7, 10, 13, 29, 43 advs are classified within the family adenoviridae, genus mastadenovirus, and divided into species a through g based on biochemical and biophysical properties, hemagglutination reaction, and sequence identity. the adv virion is a nonenveloped icosahedral capsid consisting of 240 hexon and 12 penton polyproteins or capsomers. a long fiber protein protrudes from each of the 12 penton capsomers and contains a terminal "knob" domain, which interacts with host cell receptors. 49 advs have been shown to employ a diverse variety of cell receptors, including coxsackie-adenovirus receptor (car), heparan sulfate glycosaminoglycans, cd86, and an array of cell surface integrins. 50 although each serotype exhibits some variation, advs are detected primarily during the winter and spring months. 10, 48 of all the serotypes, adv-1, -2, -5, and -6 are the viral diversity in asthma most common cause of ari. 51, 52 however, similar to other respiratory viruses (eg, rsv subtype detections reported by lee and colleagues 12 versus matthew and colleagues 14 ) , location can affect the predominance of a given serotype, with adv-4, -7, and -5 being the most frequently detected in ari studies conducted in the united states. 52, 53 as with most respiratory viruses, adv infections occur primarily in infants and children, and it is within these populations specifically that advs have been associated with more serious respiratory illness. 48, 52, [54] [55] [56] in an 8-year study of children younger than 2 years hospitalized with lrti, larranaga and colleagues 55 detected adv at a rate of 8.6% (sole detections), second only to rsv (26.3%), and predominately in patients with pneumonia and wheezing bronchitis (69.8%). the investigators also typed the adv isolates and determined that the species b and c were most frequently detected among their population and that serotype-7h was particularly associated with a longer duration of hospitalization. although this study employed culture-based methods of detection, more recent studies employing pcr for adv detection describe comparable detection rates (0.4%-7%) in episodes of childhood wheeze and asthma exacerbations. 7, 10, 13, 29, 43 an interesting aspect of the association of adv with asthma was described in a study of asymptomatic asthmatic children conducted by marin and colleagues. 56 these investigators reported that adv dna was detected in 78.4% of the asthmatic group but in only 5% of the nonasthmatic control group. this study also described hrv and rsv detection rates in the asthmatic group of 32.4% and 2.7%, respectively, despite none of the participants experiencing respiratory symptoms for the duration of the study (3 weeks). however, a confounding aspect of this study is that the subjects were classified as having mild asthma that was well controlled (fluticasone 100-250 mg daily) in the 6 months before the start of the study. nevertheless, the disparity in viral detection between the asthmatic and control groups may imply some mechanism of either persistence in asthmatics or an association with the long-term compliance of glucocorticoid therapy and inhibition of symptoms during respiratory virus infection. human bocaviruses (hbov) were discovered in 2005 by allander and colleagues 57 in pooled nasopharyngeal aspirates obtained from children with lrti. further characterization in ari-focused studies has revealed that hbov is frequently associated with ari in both children and adults, with detection rates of 1.5% to 19%. [58] [59] [60] [61] in addition, hbov has been associated with wheezing and gastrointestinal illness predominately in children younger than 2 years. 57,61-75 however, the exact role of hbov in respiratory and other illnesses remains ambiguous, with a high overall codetection rate with other respiratory viruses being described (median 42.5%) and the detection of hbov dna in serum, urine, and lymph node samples. 60, 61, 66, 76, 77 hbov is a parvovirus, a single-stranded dna virus of the family parvoviridae, genus bocavirus. 65 the genome, which requires host cell dna polymerases for replication, encodes 2 structural (vp1 and vp2) and 2 nonstructural (ns1 and np1) proteins. 65 although hbov has only recently been propagated in cultured cells and its host cell interactions remain to be determined, 78 initial studies have found that hbov shares similar virion morphology with other parvoviruses, a nonenveloped icosahedral capsid consisting of 60 copies of each structural protein. 79 recently, 2 viruses related to hbov have been identified, namely hbov-2 and hbov-3, but it is unknown whether these are unique viral entities or closely related genotypes of the same virus. 80, 81 while the epidemiology of hbov is still being determined, ari studies have shown that hbov infection occurs primarily during the winter, with a smaller peak in spring. 65, 74, 82 several studies have indicated that hbov is associated with severe respiratory disease in children, particularly in those hospitalized with asthma and other wheezing illnesses. 29, 65 a study conducted by vallet and colleagues 83 of children aged 2 to 15 years hospitalized for asthma detected hbov in 13% of children with asthma exacerbations compared with only 2% in children with stable asthma, suggesting hbov plays a causative role in the development of exacerbations. similar findings were reported by nadji and colleagues, 84 who detected hbov at a rate of 6% in children younger than 10 years with asthma exacerbations, and garcia and colleagues, 69 who reported that wheezing was seen in more than 50% of children in whom hbov was the sole virus detected. although hbov is more often detected in symptomatic than healthy individuals, high levels of codetection with other respiratory viruses, particularly picornaviruses and ifvs, confounds hbov's role in respiratory illness. 25, 65 although a study of infants younger than 12 months hospitalized with bronchiolitis found that dual infections of rsv and hbov were associated with higher clinical severity scores and longer length of hospitalization than infants with a single hbov or hrv and hbov dual infections, 85 most studies have found that coinfection of hbov with another respiratory virus does not alter the severity or duration of associated illness. in 2007, 2 novel human polyomaviruses (pyvs) were described, the ki polyomavirus (kipyv) by allander and colleagues 86 and the wu polyomavirus (wupyv) by gaynor and colleagues. 87 initially identified in nasopharyngeal aspirates obtained from individuals with respiratory illness, further characterization has revealed kipyv and wupyv are detected in 2.6% and 6.2% of ari cases, respectively, suggesting these nivs have a causative role in respiratory illnesses. [88] [89] [90] [91] however, similar to hbov, a high rate of codetection (>80%) with other respiratory viruses and detection in other tissue types currently confounds any direct association of kipyv and wupyv with ari and other illnesses. 92 pyvs are classified in the single genus polyomavirus of the family polyomaviridae, and contain histone-associated circular dsdna genomes encoding 3 structural (vp1, vp2, vp3) and 2 nonstructural (large-t and small-t antigens) proteins. 87, 93 as with some other nivs (eg, hrv-c, hcov-hku1), kipyv and wupyv cannot be grown using current cell culture systems. this drawback has postponed identification of host cell receptor interactions and determination of virion morphology, although the latter is believed to be similar to the nonenveloped icosahedral configuration of other pyvs. 93 kipyv and wupyv characterization in retrospective ari-focused studies has determined that they exhibit a year-round prevalence, with the greatest peaks in detections occurring in the spring, autumn, and winter months. 90, [94] [95] [96] although no studies have yet looked specifically for kipyv and wupyv during episodes of asthma exacerbations, wheezing illness and other more serious lrti have been reported in patients positive for kipyv and wupyv. bialasiewicz and colleagues 90 showed that 15% of kipyv-positive and 23% of wupyv-positive patients had symptoms of bronchiolitis, and payungporn and colleagues 96 found that 11 of 15 combined kipyv and wupyv sole detections originated in children younger than 1 year with pneumonia. however, the overall high codetection rates (35%-80%) described in these studies again indicates the need for further clarification of the role of kipyv and wupyv in respiratory illness. an interesting aspect of the previously described human pyvs is their ability to establish a form of latency (ie, low-level persistent infection) and undergo reactivation when an infected individual's immune system experiences stress (eg, infection, inflammation, or immune suppression). 93, 97 while characterization of the host-virus interactions of kipyv and wupyv is still continuing, sharp and colleagues 98 have proposed that they may also reactivate during illness, as evidenced by mutations in the transcriptional control region of kipyv and wupyv genomes detected in autopsy tissue from immunosuppressed individuals. given our incomplete knowledge about the role of kipyv and wupyv in respiratory illness, it may be plausible that the high codetection rates observed in ari studies are not the result of bona fide dual infections, but rather may be caused by infection with another respiratory virus evoking an immune response that reactivates latent kipyv and wupyv. although sharp and colleagues 98 did not observe the high frequency of transcriptional control region mutations in kipyv and wupyv genomes detected from respiratory samples, a seroepidemiology study by nguyen and colleagues 99 may provide evidence of kipyv and wupyv reactivation during ari. these investigators observed that the proportion of kipyv-and wupyv-seropositive individuals increased at around 4 years of age, a finding that they speculated was the result of kipyv and wupyv exposure during school attendance. however, they conceded that the serum obtained for their study originated from 2 tertiary referral hospitals and was likely from individuals with underlying medical conditions. given that ari represents the most common reason for hospital visits and admissions among school-aged children, and that the incidence of other respiratory virus infections also increases among this age group, the kipyv and wupyv antibody detected may not be that produced in response to initial exposure, but rather antibody produced during kipyv and wupyv reactivation evoked by infection with another ari-causing respiratory virus. indeed, nguyen and colleagues 99 reported that a high proportion of adults were kipyv-and wupyv-seropositive which, considering the nature of the study cohort (ie, hospital-based), may not indicate preexisting immunity but again kipyv and wupyv reactivation during illness. future communitybased studies will no doubt determine when initial kipyv and wupyv exposure occurs and will better elucidate the relationships that exist between ari, viral codetections, and kipyv and wupyv reactivation. this review focuses on some of the less common respiratory viruses currently implicated in both childhood and adult asthma exacerbations. although the respiratory viruses covered here have a lesser, or in the case of nivs, an as yet uncharacterized involvement in the development of exacerbation compared with the "usual suspects" (eg, hrv, rsv, hmpv), they exemplify the diversity that exists in virus-related exacerbations. the implementation of more sensitive methods of virus detection and the identification and characterization of nivs in future asthma studies will further expand our understanding of viral diversity and the mechanisms underlying development of asthma exacerbations during respiratory virus infection. experimental rhinovirus 16 infection causes variable airway obstruction in subjects with atopic asthma the state of childhood asthma summary health statistics for u.s. adults: national health interview survey mortality in patients hospitalized for asthma exacerbations in the united states london: informa healthcare respiratory picornaviruses and respiratory syncytial virus as causative agents of acute expiratory wheezing in children respiratory viruses and acute asthma in children philadelphia: lippincott-raven rhinovirus infection in hospitalized children in hong kong: a prospective study human metapneumovirus infection plays an etiologic role in acute asthma exacerbations requiring hospitalization in adults high-throughput, sensitive, and accurate multiplex pcr-microsphere flow cytometry system for large-scale comprehensive detection of respiratory viruses asthma exacerbations in children are associated with rhinovirus but not human metapneumovirus infection distribution and seasonality of rhinovirus and other respiratory viruses in a cross-section of asthmatic children in trinidad, west indies respiratory tract viral infections in innercity asthmatic adults community study of role of viral infections in exacerbations of asthma in 9-11 year old children orthomyxoviridae: the viruses and their replication sialic acid species as a determinant of the host range of influenza a viruses influenza virus and acute asthma in children is influenza vaccination in asthma helpful? identification and recall of children with chronic medical conditions for influenza vaccination asthma and influenza vaccination influenza burden for children with asthma influenza vaccination in children with asthma: randomized double-blind placebo-controlled trial do rhinoviruses reduce the probability of viral co-detection during acute respiratory tract infections? rhinoviruses delayed the circulation of the pandemic influenza a (h1n1) 2009 virus in france hospitalized patients with 2009 h1n1 influenza in the united states role of human metapneumovirus, human coronavirus nl63 and human bocavirus in infants and young children with acute wheezing human bocavirus and acute wheezing in children release. international committee on taxonomy of viruses human coronavirus nl63 employs the severe acute respiratory syndrome coronavirus receptor for cellular entry human aminopeptidase n is a receptor for human coronavirus 229e coronaviridae: the viruses and their replication coronavirus hku1 and other coronavirus infections in hong kong spectrum of clinical illness in hospitalized patients with "common cold" virus infections human coronavirus nl63 infection and other coronavirus infections in children hospitalized with acute respiratory disease in hong kong the pediatric burden of human coronaviruses evaluated for twenty years evidence of human coronavirus hku1 and human bocavirus in australian children pan-viral screening of respiratory tract infections in adults with and without asthma reveals unexpected human coronavirus and human rhinovirus diversity respiratory viruses and exacerbations of asthma in adults respiratory syncytial virus and parainfluenza virus epidemiological features of parainfluenza virus infections: laboratory surveillance in england and wales, 1975-1997 identification of human metapneumovirus and chlamydophila pneumoniae in children with asthma and wheeze in singapore paramyxoviridae: the viruses and their replication philadelphia: lippincott-raven role of sialic acid-containing molecules in paramyxovirus entry into the host cell: a minireview receptor specificities of human respiroviruses review of new and newly discovered respiratory tract viruses in children adenoviridae: the viruses and their replication adenovirus receptors philadelphia: lippincott-raven clinical features, adenovirus types, and local production of inflammatory mediators in adenovirus infections respiratory adenoviral infections in children: a study of hospitalized cases in southern taiwan in 2001-2002 adenovirus surveillance on children hospitalized for acute lower respiratory infections in chile (1988-1996) persistence of viruses in upper respiratory tract of children with asthma cloning of a human parvovirus by molecular screening of respiratory tract samples human bocavirus clinical and epidemiological aspects of human bocavirus infection detection of human bocavirus in respiratory, fecal, and blood samples by real-time pcr correlation between bocavirus infection and humoral response, and co-infection with other respiratory viruses in children with acute respiratory infection human bocavirus: prevalence and clinical spectrum at a children's hospital human bocavirus infection two-year prospective study of single infections and co-infections by respiratory syncytial virus and viruses identified recently in infants with acute respiratory disease the human bocaviruses: a review and discussion of their role in infection evidence of human bocavirus circulating in children and adults bocavirus infection in hospitalized children human bocavirus: a novel parvovirus epidemiologically associated with pneumonia requiring hospitalization in thailand clinical characteristics of human bocavirus infections compared with other respiratory viruses in spanish children human bocavirus detection in nasopharyngeal aspirates of children without clinical symptoms of respiratory infection human bocavirus infection in young children in the united states: molecular epidemiological profile and clinical characteristics of a newly emerging respiratory virus human bocavirus in italian patients with respiratory diseases epidemiological profile and clinical associations of human bocavirus and other human parvoviruses sole pathogen in acute bronchiolitis: is there a role for other organisms apart from respiratory syncytial virus? novel human bocavirus in children with acute respiratory tract infection human bocavirus in tonsillar lymphocytes disseminated bocavirus infection after stem cell transplant human bocavirus can be cultured in differentiated human airway epithelial cells electron microscopy observation of human bocavirus (hbov) in nasopharyngeal samples from hbov-infected children a novel bocavirus associated with acute gastroenteritis in australian children a newly identified bocavirus species in human stool detection of new respiratory viruses in hospitalized infants with bronchiolitis: a three-year prospective study human bocavirus: a cause of severe asthma exacerbation in children phylogenetic analysis of human bocavirus isolated from children with acute respiratory illnesses and gastroenteritis in iran respiratory syncytial virus, human bocavirus and rhinovirus bronchiolitis in infants identification of a third human polyomavirus identification of a novel polyomavirus from patients with acute respiratory tract infections ki polyomavirus detected in respiratory tract specimens from patients in polyomaviruses ki and wu in children with respiratory tract infection presence of the newly discovered human polyomaviruses ki and wu in australian patients with acute respiratory tract infection identification of the novel ki and wu polyomaviruses in human tonsils no evidence for an association between infections with wu and ki polyomaviruses and respiratory disease philadelphia: lippincott-raven identification of wu polyomavirus from pediatric patients with acute respiratory infections in beijing clinical and epidemiologic characterization of wu polyomavirus infection prevalence and molecular characterization of wu/ki polyomaviruses isolated from pediatric patients with respiratory disease in thailand the role of polyomaviruses in human disease reactivation and mutation of newly discovered wu, ki, and merkel cell carcinoma polyomaviruses in immunosuppressed individuals serologic evidence of frequent human infection with wu and ki polyomaviruses key: cord-257073-dm80bxnd authors: akmatov, manas k.; krebs, stephan; preusse, matthias; gatzemeier, anja; frischmann, ursula; schughart, klaus; pessler, frank title: e-mail-based symptomatic surveillance combined with self-collection of nasal swabs: a new tool for acute respiratory infection epidemiology date: 2011-08-17 journal: int j infect dis doi: 10.1016/j.ijid.2011.07.005 sha: doc_id: 257073 cord_uid: dm80bxnd objective: we examined the feasibility of combining communication by e-mail and self-collection of nasal swabs for the prospective detection of acute respiratory infections in a non-medical setting. methods: the study was conducted among a convenience sample of employees (n = 53) at a research institution (december 2009–april 2010). real-time data on the occurrence of acute respiratory symptoms and a nasal self-swab were collected prospectively, with automated weekly e-mails as a reminder mechanism. reverse transcription polymerase chain reaction (rt-pcr) was used to detect respiratory viral pathogens in the swabs. results: fifty-one out of 53 participants completed the study. the study design was well accepted. thirty (∼57%) participants reported at least one episode of acute respiratory infection and returned the nasal swab during the study period (eight participants reported two episodes). the majority had no difficulties taking the self-swab and preferred this to swabbing by study personnel. most participants obtained and returned the swabs within the recommended time. viral respiratory pathogens were detected in 19 of 38 swabs (50%), with coronaviruses 229e/nl63 and oc43 and rhinoviruses a and b constituting 17 positive swabs (89%). conclusions: combining e-mail-based symptomatic surveillance with nasal self-swabbing promises to be a powerful tool for the real-time identification of incident cases of acute respiratory infections and the associated pathogens in population-based studies. research on acute respiratory infections in human populations is limited by certain methodological difficulties. first, their acute nature makes a timely diagnosis difficult. second, symptoms are usually not unique for specific pathogens. these difficulties impede collecting epidemiologic (e.g., risk factors for acute respiratory infections) and clinical (e.g., the course and severity of infections) data, as well as biosamples for pathogen identification. in particular, the real-time collection of diagnostic specimens such as nasal or nasopharyngeal swabs during an acute respiratory infection season is necessary to link symptomatic data with specific pathogens. 1 therefore, there is an urgent need to develop epidemiologic research tools that ensure the timely detection of incident acute respiratory infections and the collection of diagnostic biosamples during the episode. most epidemiologic studies on acute respiratory infections have been based in medical settings or have been conducted in specific target populations such as trained medical personnel. data on the occurrence and severity of symptoms have been collected in a few studies only, either retrospectively, 2 usually at the end of an acute respiratory infection season, or prospectively, for instance by using diary-based questionnaires. 3, 4 recently, modern communication tools such as weekly e-mails 5 and internet-based questionnaires 6, 7 have been introduced in population-based studies to collect real-time data on respiratory infections. in a recent study of influenza infection, short message service (sms) was used in addition to e-mail. 8 the main limitation of these studies is the lack of pathogen identification during specific episodes of acute respiratory infections. nasal swabs for pathogen detection are usually collected by study personnel at the study site or in hospital. however, due to logistic problems and higher costs, this is difficult to organize in population-based studies with their inherently larger sample sizes. in several recent studies subjects were asked to obtain swabs from their own nares ('self-swabbing') to detect viral respiratory pathogens, but most of these studies were performed s u m m a r y objective: we examined the feasibility of combining communication by e-mail and self-collection of nasal swabs for the prospective detection of acute respiratory infections in a non-medical setting. methods: the study was conducted among a convenience sample of employees (n = 53) at a research institution (december 2009-april 2010). real-time data on the occurrence of acute respiratory symptoms and a nasal self-swab were collected prospectively, with automated weekly e-mails as a reminder mechanism. reverse transcription polymerase chain reaction (rt-pcr) was used to detect respiratory viral pathogens in the swabs. results: fifty-one out of 53 participants completed the study. the study design was well accepted. thirty ($57%) participants reported at least one episode of acute respiratory infection and returned the nasal swab during the study period (eight participants reported two episodes). the majority had no difficulties taking the self-swab and preferred this to swabbing by study personnel. most participants obtained and returned the swabs within the recommended time. viral respiratory pathogens were detected in 19 of 38 swabs (50%), with coronaviruses 229e/nl63 and oc43 and rhinoviruses a and b constituting 17 positive swabs (89%). conclusions: combining e-mail-based symptomatic surveillance with nasal self-swabbing promises to be a powerful tool for the real-time identification of incident cases of acute respiratory infections and the associated pathogens in population-based studies. ß 2011 international society for infectious diseases. published by elsevier ltd. all rights reserved. in healthcare environments. for instance, parents collected swabs from their symptomatic children when presenting for pediatric medical evaluation, 9 or nurses self-swabbed during symptomatic episodes in a study comparing two measures for the prevention of influenza transmission. 10 in the uk, swabs for the collection of nasal specimens were sent to individuals who contacted the health advice and information service ('nhs direct') because of influenzalike symptoms. 11 the only population-based study was conducted among parents who collected nasal swabs from their children at home. 12 thus, the feasibility of nasal self-swabbing for the detection of respiratory pathogens in population-based studies of adults remains to be demonstrated. moreover, little is known about whether any added benefit results when active symptomatic surveillance is conducted to ensure the timely self-collection of swabs during the time window in which causative pathogens are detectable. we therefore examined the feasibility of combining email-based active symptomatic surveillance with nasal selfswabbing for the detection of viral respiratory pathogens in a prospective study spanning one acute respiratory infection season. we conducted a prospective study among employees of the helmholtz centre for infection research (hzi) in braunschweig, germany, from december 2009 to april/may 2010. in december 2009, invitations to participate in the study were sent to all employees (age 18-69 years) through the internal e-mail system. this invitation contained a link to the institutional intranet where information about the study was made available. subjects not eligible for study participation were those vaccinated against seasonal influenza in the season 2009/2010, staff of the department of infection genetics (due to ethical considerations), and those who planned to leave braunschweig during the study period. the study was approved by the ethics committee of the state board of physicians of the german federal state of lower saxony. fifty-three participants responded to the invitation e-mail, corresponding to a response rate of approximately 12% ( figure 1 ). all subjects gave written informed consent before entering the study. at baseline (december 2009), information on sociodemographics (sex, age, education, profession, country of birth, number of individuals living in the household, etc.), contacts with animals, history of vaccination against influenza, and general health status was collected through a self-administered questionnaire. the pre-season blood sample was also obtained at this time. in april/may 2010 the study participants were reinvited to give the post-season blood sample and fill in a short questionnaire. every participant who completed the post-study questionnaire and blood sampling received a remuneration of 20 s. serum samples were stored at à70 8c. however, at the conclusion of the study it was decided to forgo the originally planned influenza hemagglutination inhibition assays because of the unexpectedly low incidence of influenza infection in germany during the study period. during january-march 2010 the participants were asked to take a swab from one of the anterior nares and return it to the study site as soon as possible if they had at least one of the following acute respiratory symptoms: sudden onset of stuffy or running nose, cough, sore throat, or fever >38 8c. they received instruction by a physician (s.k.) on how to perform the nasal swab. briefly, the swab was to be inserted into one nostril to a depth of 1-2 cm, rotated three times, and then placed into transport medium. two kits for nasal swabbing containing a regular flocked swab with molded breakpoint (copan, brescia, italy; product number 359c) and viral universal transport medium were given to the participants. during symptomatic surveillance, weekly automated e-mail messages were sent to the participants containing (1) a reminder to take a swab at the onset of at least one of the abovementioned symptoms and (2) instructions on how to collect, store, and return the swab. visual instructions on how to collect the swab were also available on the package. participants were instructed to store the self-collected swab in the refrigerator (+4 8c) until returning it, as soon as possible, to the study team. upon receipt at the study site, swabs were held at à70 8c until analysis. because of the low response rate, we conducted a non-responder survey in april 2010 ( figure 1 ). the survey was done through a selfadministered questionnaire, which was sent via the internal e-mail system. to maintain anonymity, responding individuals (n = 142) were asked to return the completed questionnaire by in-house mail. among other items, information was collected regarding reasons for not participating in the study and the occurrence of respiratory infections during the study period. data were described as percentages for categorical variables and medians with range for continuous variables. differences between groups were tested with the chi-square test (for categorical variables) and the mann-whitney u-test (for continuous variables). overall, there were only minor differences between the participants and the non-responders (table 1 ). participants were slightly younger than non-responders (p = 0.03); approximately 17% of the participants and 9% of the non-responders were born outside germany (p = 0.09). about half of the subjects in both groups had a university degree (including universities of applied sciences). there was a slightly higher proportion of smokers among the responders (p = 0.02). reasons for not participating were: 'did not meet inclusion criteria' ($31%), 'no time' ($27%), 'absent because of illness or vacation' ($16%), 'did not read the invitation e-mail' ($12%), 'fear of blood draw' ($7%), 'concern of inadequate data protection', 'no interest' ($6% each), and 'information about the study was unclear' ($5%) (subjects were allowed to give more than one reason). out of 53 participants, 30 provided 38 nasal swabs during the symptomatic follow-up period (eight individuals had two symptomatic episodes). thus, the clinical attack rate of acute respiratory infection, based on at least one reported symptom, was 53.6% (table 2 ). about 38% of the participants reported at least two, 18% at least three, and 9% at least four symptoms. similar proportions of non-responders reported three or more symptoms. however, the proportion of individuals who reported mild infections (one or two symptoms) was significantly lower among the non-responders than the study participants. fifty-one out of 53 participants completed all aspects of the study. one participant had to leave the study because he moved away and one participant was lost to follow-up. nearly all study participants found the study design acceptable (table 3) , and the vast majority of participants would participate again in such a study. only six percent found a weekly e-mail reminder to take the swab unacceptable. the majority of those who collected a nasal swab reported no difficulties in self-swabbing. only one participant reported difficulties opening the swab tube. about 15% felt discomfort while performing the swab. all participants who reported acute respiratory symptoms during the period of symptomatic surveillance (n = 30) selfcollected the nasal swab. eight subjects reported two episodes of respiratory infections, and all eight returned two swabs. at the end of the study period one person reported that he/she took the swab but did not bring it to the study center. the reason stated was ''i forgot to bring the swab to the study center''. of these participants with symptoms of an acute respiratory infection, 85.7% collected the swab within the first 3 days of the onset of symptoms. one person collected the swab on the sixth day. half of the participants brought the swab to the study center on the day of taking it. the maximum time between swab collection and delivery was 2 weeks. respiratory pathogenic viruses were detected by reverse transcription polymerase chain reaction (rt-pcr) in about 50% of the swabs (table 4) , and the most frequently recorded ones were human coronaviruses 229e/nl63 (8/38 swabs, $21%) and oc43 (5/ 38 swabs, $13%). co-infections were detected in two specimens. influenza viruses were not detected. there were no differences in the proportion of positive (for any viruses) and negative swabs in terms of participant sex, age, and level of education. also, there were no differences in the time elapsed between the onset of symptoms and self-swabbing (figure 2a) or the time between selfswabbing and arrival of the swab at the study center ( figure 2b ). likewise, we did not detect any effects on viral detection when we controlled for potential effects of each variable (time (change per one day) between symptoms and swabbing, adjusted odds ratio (aor) 0.98, 95% confidence interval (ci) 0.50-1.93; time between swabbing and delivery, aor 0.96, 95% ci 0.76-1.21). we tested the feasibility of combining real-time symptomatic surveillance with nasal self-swabbing for the prospective collection of epidemiologic and virological data on acute respiratory infections. in the pre-analytical phase, this novel approach turned out to be highly feasible in that acceptance, satisfaction, compliance, and timeliness of logistics were high. notably, more than 90% of the participants who self-swabbed reported that the swab was easy to obtain and that they preferred self-collection to collection by study personnel. the reason for this high degree of satisfaction may be that self-swabbing reduces duration and frequency of contact with study personnel as well as travel to a study site. the resulting greater convenience would very likely impact positively on compliance in any largescale prospective study. another important finding of the presented study is that neither the time between onset of symptoms and self-swabbing nor the time between selfswabbing and specimen arrival at the laboratory influenced the viral detection rate. this agrees well with results from a study in the uk, 11 and is noteworthy, since in population-based studies employing self-swabbing, shipping time needs to be included in the time between swab collection and expected arrival of the specimen at the study center. factors other than time that could not be addressed in this study may influence the viral detection rate. one obvious candidate is the swabbing technique. the study physician instructed the participants in a standardized manner in the proper application of the technique. we do not believe that an inadequate technique impacted negatively on the rate of pathogen detection, since the detection rate of 50% recorded by us corresponds to what has been reported in other studies employing staff-collected swabs and similar detection technology. 13 also, two recent studies showed that there were no differences in pathogen detection between self-and staff-collected swabs. 14, 15 another factor that may influence the detection rate is the type of nasal swab used for specimen collection. recently, smieja et al. developed a flocked nasal mid-turbinate swab and compared it with the gold standard (e.g., rayon nasal and nasopharyngeal swabs). the mid-turbinate swab turned out to provide better results (based on epithelial cell counts) than the gold standard. 16 we used a regular flocked swab. thus, using the mid-turbinate flocked swab might have resulted in a higher detection rate in our study. the non-responder survey allowed us to compare prospectively (participants) and retrospectively (non-responders) collected data on acute respiratory infection symptoms, and it revealed that the prospective approach resulted in a higher rate of detection of mild infections. thus, one immediate strength of using active symptomatic surveillance in population-based studies on acute respiratory infections would be the more efficient identification of individuals with reduced susceptibility to infection, which would constitute an invaluable asset for large-scale studies on genetic determinants of infection susceptibility and resistance in humans. in such a study, using self-swabbing instead of swabbing by study personnel to detect specific pathogens would be immensely attractive due to its anticipated lower cost. indeed, expenses for personnel and logistics were estimated to be 80% less if self-swabbing was used instead of swabbing by study personnel. 17 previous methods of active symptomatic surveillance have been, for example, weekly telephone calls 18 or daily symptom diaries. 19 however, these methods might be costly and have lower compliance rates. limitations not addressed above include the representativeness of the study population. for instance, we had higher-thanexpected proportions of female ($75%) and highly educated subjects ($50%). moreover, due to working in a research institution, the participants could be expected to be more receptive to the study design than the general population. since e-mail has become the primary tool of communication in professional work environments, including our institution, the population sampled for the present study may have a higher acceptance of modern communication tools than the general population. further studies are needed regarding the use of electronic communication methods in population-based studies, particularly those targeting the less educated and the elderly. lastly, due to the unexpected near absence of influenza infection during the study period, we could not evaluate the usefulness of the study design for the detection of influenza infection. indeed, considering that upper respiratory symptoms occur only in about 60% and fever in about 35% of episodes of influenza infection, 20 inclusion of surveillance questions about other influenza-associated symptoms (e.g., myalgia or headache) would likely increase the efficiency of screening for influenza infection with e-mail-based surveillance. combining e-mail-based active symptomatic surveillance with self-collection of nasal swabs ensured prospective, accurate collection of data on incident episodes of acute respiratory infections and timely sample collection for the detection of respiratory pathogens. it promises to be an efficient and cost-effective approach in population-based studies on the epidemiology of respiratory infections. beyond the influenza-like illness surveillance: the need for real-time virological data incidence and recall of influenza in a cohort of glasgow healthcare workers during the 1993-4 epidemic: results of serum testing and questionnaire a children's acute respiratory illness scale (carifs) predicted functional severity and family burden the effect of giving influenza vaccination to general practitioners: a controlled trial internet-based monitoring of influenza-like illness (ili) in the general population of the netherlands during the 2003-2004 influenza season internet-based monitoring of influenza-like illness in the general population: experience of five influenza seasons in the netherlands internet-based surveillance of influenza-like-illness in the uk during the 2009 h1n1 influenza pandemic seasonal influenza risk in hospital healthcare workers is more strongly associated with household than occupational exposures: results from a prospective cohort collection by trained pediatricians or parents of mid-turbinate nasal flocked swabs for the detection of influenza viruses in childhood surgical mask vs n95 respirator for preventing influenza among health care workers: a randomized trial monitoring the emergence of community transmission of influenza a/h1n1 2009 in england: a cross sectional opportunistic survey of self sampled telephone callers to nhs direct parent-collected respiratory specimens-a novel method for respiratory virus and vaccine efficacy research multiplex real-time pcr for detection of respiratory tract infections evaluation and clinical validation of an alcohol-based transport medium for preservation and inactivation of respiratory viruses selfcollected mid-turbinate swabs for the detection of respiratory viruses in adults with acute respiratory illnesses development and evaluation of a flocked nasal mid-turbinate swab for self-collected respiratory virus diagnostic testing self-collected nasal swabs to detect infection and colonization: a useful tool for population-based epidemiological studies? effectiveness of influenza vaccine in health care professionals: a randomized trial community epidemiology of human metapneumovirus, human coronavirus nl63, and other respiratory viruses in healthy preschool-aged children using parentcollected specimens time lines of infection and disease in human influenza: a review of volunteer challenge studies we would like to thank the participants for their kind participation in the study. we thank prof. udo buchholz (robert koch institute, berlin, germany) for helpful comments on the study design and a critical reading of the manuscript. this work was supported with intramural funds from the helmholtz association (program infection and immunity).conflict of interest: the authors declare that they have no competing interests. key: cord-260457-m1jbpo5l authors: allander, tobias; jartti, tuomas; gupta, shawon; niesters, hubert g. m.; lehtinen, pasi; üsterback, riikka; vuorinen, tytti; waris, matti; bjerkner, annelie; tiveljung-lindell, annika; van den hoogen, bernadette g.; hyypiä, timo; ruuskanen, olli title: human bocavirus and acute wheezing in children date: 2007-04-01 journal: clin infect dis doi: 10.1086/512196 sha: doc_id: 260457 cord_uid: m1jbpo5l background. human bocavirus is a newly discovered parvovirus. it has been detected primarily in children with acute lower respiratory tract infection, but its occurrence, clinical profile, and role as a causative agent of respiratory tract disease are not clear. methods. we investigated the presence of human bocavirus by quantitative polymerase chain reaction of nasopharyngeal aspirate specimens and selected serum samples obtained from 259 children (median age, 1.6 years) who had been hospitalized for acute expiratory wheezing. the samples were analyzed for 16 respiratory viruses by polymerase chain reaction, virus culture, antigen detection, and serological assays. results. at least 1 potential etiologic agent was detected in 95% of children, and >1 agent was detected in 34% of children. human bocavirus was detected in 49 children (19%). a large proportion of the cases were mixed infections with other viruses, but human bocavirus was the only virus detected in 12 children (5%). high viral loads of human bocavirus were noted mainly in the absence of other viral agents, suggesting a causative role for acute wheezing. in addition, infections that had uncertain clinical relevance and low viral loads were prevalent. human bocavirus dna was frequently detected in serum specimens obtained from patients with acute wheezing, suggesting systemic infection. conclusions. human bocavirus is prevalent among children with acute wheezing and can cause systemic infection. results suggest a model for bocavirus infection in which high viral loads are potentially associated with respiratory symptoms and low viral loads indicate asymptomatic shedding. therefore, quantitative polymerase chain reaction analysis may be important for additional studies of human bocavirus. acute viral respiratory tract infection is the leading cause of hospitalization for infants and young children in developed countries and is a major cause of death in developing countries [1, 2] . human bocavirus (hbov) is a newly discovered parvovirus that was first identified in sweden from pooled nasopharyngeal aspirate specimens by large-scale molecular virus screening [3] . in the original study, 540 nasopharyngeal aspirate specimens obtained from hospitalized patients were analyzed for hbov, and 3.1% yielded positive results. all hbov-positive samples had been obtained from young children with respiratory distress (mainly acute expiratory wheezing). many of the children had pneumonia, with interstitial infiltrates noted by chest radiography [3] . recent studies have detected hbov in 1.5%-11.3% of investigated respiratory tract samples in north america, europe, asia, and australia, suggesting that the virus has a global distribution [4] [5] [6] [7] [8] [9] [10] . respiratory distress and abnormal chest radiography findings have been frequent findings associated with hbov in these studies. however, the causative role of hbov remains unclear. in fact, hbov was detected concurrently with other potential pathogens in 33%-56% of cases in which it was studied [5, 7, [9] [10] [11] . these findings have raised the question of whether hbov is at all a causative agent of respiratory tract disease. we investigated the prevalence of hbov and the genome hbov load in the respiratory tract and blood specimens obtained from children who had been hospitalized for acute expiratory wheezing-the most common manifestation of lower respiratory tract infection in children-to investigate the association between hbov infection and acute respiratory tract illness. importantly, the samples had earlier been investigated for evidence of different respiratory virus infections, and a potential causative agent had been detected in 88% of cases [12] . in the present, expanded study, we searched for a total of 16 respiratory viruses. as part of a randomized, placebo-controlled clinical trial evaluating the efficacy of systemic corticosteroids for the treatment of acute expiratory wheezing in children, we performed extensive diagnostic evaluations for respiratory virus infection. the study included 293 children who presented to the department of pediatrics, turku university hospital (turku, finland), during the period from september 2000 through may 2002 [12, 13] . acute expiratory wheezing was considered to be bronchiolitis when it occurred for the first time in children aged !3 years. asthma was diagnosed on the basis of the national asthma education and prevention program guidelines [14] . all other episodes of acute expiratory wheezing were considered to be recurrent wheezing. all patients were examined by 1 of the 2 study physicians (t.j. and p.l.) twice daily during hospitalization. signs and symptoms were recorded on a standardized form. of the 293 children who were randomized, 259 children (median age, 1.6 years; range, 3 months to 15 years) who had sufficient sample material available for complete virus diagnostic evaluation (nasopharyngeal aspirate specimens were used for pcr [for 16 viruses], virus culture [for 9 viruses], and antigen detection [for 7 viruses]; acute-and convalescent-phase serum samples were used for serologic testing [for 7 viruses]) were included in the present study. sixty-four asymptomatic children (median age, 4.1 years; range, 5 months to 14 years) who had been admitted to the division of pediatric surgery, turku university hospital, during the period from september 2000 through october 2002 and who did not have respiratory symptoms during the preceding 4 weeks were also investigated for the presence of hbov [15] . the study protocol was approved by the ethics committee of the turku university hospital. at hospital admission, a nasopharyngeal aspirate sample was obtained using a standardized procedure, as described elsewhere [12] . antigen detection and virus culture were performed immediately, and the samples obtained for pcr assays were stored in tubes at ϫ70њc before processing. blood samples were collected at the time of the patient's admission and 2-3 weeks after discharge from the hospital and were stored at ϫ20њc. nasal swab specimens were obtained from the asymptomatic children. nasopharyngeal aspirate specimens could not be obtained from healthy children, because they did not have mucus in the nasopharynx. real-time pcr for hbov. nucleic acids were extracted from 400 ml of the nasopharyngeal aspirate samples using the magattract virus mini m48 kit and the biorobot m48 instrument (qiagen), and samples were eluted in 100 ml of rnasefree water. for serum samples, 100 ml of serum was processed using the qiaamp blood mini kit (qiagen), and dna was eluted in 50 ml. the pcr assay targeted the np-1 gene of hbov. the 20-ml amplification reaction contained 5 ml of sample dna, 10 ml of taqman universal pcr master mix (pe applied biosystems), 0.1 ml of bovine serum albumin (20 mg/ ml), 300 nmol/l each primer (boca-forward, gga aga gac act ggc aga caa; and boca-reverse, ggg tgt tcc tga tga tat gag c), and 150 nmol/l boca probe (fam-ctg cgg ctc ctg ctc ctg tga t-tamra). amplification was performed using a lightcycler 1.2 instrument (roche) with the following instrument settings: 95њc for 10 min, 50 cycles of 95њc for 0 s, and 60њc for 1 min. for standardizing the quantification, a plasmid (npsc3.1) containing the hbov np-1 gene was used in serial dilutions covering a range of 6 logs. the criteria for a positive reaction were a cycle threshold !40 cycles and fluorescence count 10.5. the minimum genome viral load that would allow reproducible quantification was 10 copies per reaction, corresponding to 500 copies/ml for nasopharyngeal aspirate specimens or 1000 copies/ml for serum specimens. the analyses were performed at a diagnostic laboratory, with rigorous measures to prevent contamination. the realtime pcr assay was compared with the previously published single end-point pcr assay targeting the np-1 gene [3] by parallel analysis of 74 samples. fifteen samples yielded positive results with both assays, 5 yielded positive results with realtime pcr only, and the remaining 54 yielded negative results with both assays. virus culture was performed for adenovirus, influenza a and b viruses, parainfluenza virus types 1-3 (piv 1-3), respiratory syncytial virus (rsv), enteroviruses, and rhinoviruses, in accordance with routine diagnostic protocols, in a549, hela, and llc-mk 2 and human foreskin fibroblast cells, as well as for human metapneumovirus in tertiary monkey kidney cells [16] . viral antigens were detected for adenovirus, influenza a and b viruses, piv 1-3, and rsv by time-resolved fluoroimmunoassay [17] . levels of igg antibodies specific for the same viruses (except for piv 2 and human metapneumovirus) were analyzed in paired serum samples, in addition to igm antibodies for enteroviruses. rt-pcr was used for the detection of enteroviruses and rhinoviruses, rsv, coronaviruses 229e and oc43, and human metapneumovirus. these results were reported elsewhere [12] . in addition to hbov diagnostic evaluations, the present study included extended diagnostic evaluations by real-time pcr for influenza a and b viruses, adenovirus, piv 1-4, and coronavirus nl63 (in rotterdam) and coronavirus hku-1 (in stockholm). total nucleic acids for these analyses were isolated using a magnapure lc isolation station (roche). amplification was performed with an abi7700 instrument, using commercially available master mixes (applied biosystems) and standard protocols. the primer sequences for piv 1-4 and coronavirus hku-1 are available from the authors. otherwise, the primer and probe sequences, pcr protocols, and other viral detection methods were described elsewhere [12, 16, [18] [19] [20] [21] . the sequencing of the 12 previously reported, nontypable rhinoviruses and enteroviruses [12] identified them as rhinoviruses. a patient was considered to be positive for the virus if results of at least 1 of the tests used were positive. a potential viral pathogen was identified in 245 children (95%) ( tected in 11% of wheezing patients. the nasal swab samples obtained from the 64 asymptomatic children were hbov negative. hbov was found significantly more frequently among children with acute wheezing than among asymptomatic children (19% vs. 0%; , by fisher's exact test). however, p ! .001 because age distributions and sampling techniques differed between the 2 groups, this comparison should be interpreted with caution. we searched for additional evidence of causality by studying whether detection of hbov was unrelated or inversely correlated to the occurrence of other viruses in children with acute wheezing [3] . hbov was indeed more prevalent among children with symptoms of an otherwise unexplained etiology than among those in which other viruses were detected (46% vs. 16%; ) (table 2) . stratification of the patients on p ! .001 the basis of a nasopharynx hbov load of !10 4 copies/ml versus 110 4 copies/ml revealed that only the patients with a high hbov load had this association (table 2). this suggests that the presence of hbov at high-but not at low-viral loads is associated with previously unexplained acute wheezing. detection of hbov dna in serum specimens. acute-and convalescent-phase serum samples were available in sufficient quantity from 43 of the 49 patients who tested positive for hbov in the nasopharynx. hbov dna was detected in 23 (53%) of the 43 acute-phase serum samples and in 8 (19%) of the 43 convalescent-phase serum specimens ( , by p ! .001 fisher's exact test). detection of hbov in serum was mainly, although not exclusively, associated with a high viral load in the nasopharynx (table 3) . viral loads in serum samples ranged from ! to copies /ml and did not correlate 3 5 1.0 ϫ 10 5.9 ϫ 10 with genome viral loads in the corresponding nasopharyngeal aspirate specimens ( ; data not shown). we also an-r p ϫ0.24 alyzed 45 randomly selected serum samples obtained from the 196 wheezing patients who tested negative for hbov in the nasopharynx but who had demonstrated infection with some other virus. three tested positive for hbov dna (2 had low viral loads, and 1 had a viral load of copies/ml). 5 hbov was the only virus detected in 12 children (table 4). the median age of these children was 1.3 years. eight children received a diagnosis of bronchiolitis, 3 had recurrent wheezing, and 1 had acute exacerbation of asthma. acute otitis media was diagnosed in 5 (42%) of the children, and chest radiographs taken from 9 patients all showed interstitial infiltrates. one child had leukocytosis ( cells/l), and 2 children had 9 16.3 ϫ 10 increased levels of serum c-reactive protein (73 and 78 mg/l). in all other cases, total wbc counts and serum c-reactive protein levels were within normal limits. the reported duration of symptoms varied from 2 days to 120 days. when characteristics of the children positive only for hbov were compared with those of children with other demonstrated virus infections, no clinically significant differences were recorded (table 4). we found hbov in the nasopharynx of 19% of children with acute wheezing illness, and it was the fourth most frequent virus detected after rhinoviruses, enteroviruses, and rsv. these findings show that hbov is a common virus in the community. the prevalence of hbov was higher in our study than in previous reports [3] [4] [5] [6] [7] [8] [9] [10] [11] ; this may have been the result of technical factors and study design rather than true differences in virus prevalence. a large number of the samples had low hbov loads, making evaluation of prevalence highly dependent on assay sensitivity. differences in pcr assay sensitivity may, of course, similarly have affected the relative prevalence estimates for all 16 of the viruses studied. it should also be recognized that nasopharyngeal sampling is not a standardized procedure. finally, the present study included only hospitalized patients with acute wheezing illness, which may be the major manifestation of hbov infection, whereas previous studies have included a broader selection of samples. despite the large number of mixed hbov infections, our findings are still consistent with a potential etiologic role for hbov in respiratory tract disease in young children. in the absence of in vitro virus propagation systems and animal models, final proof of causality will remain difficult to obtain. fredricks and relman [22] proposed molecular diagnostic criteria for causality for use in this situation, and the present study provides data relevant for many of these criteria: 1. hbov was present at the site of the symptoms (i.e., in the respiratory tract). genome viral loads were generally higher in the respiratory tract specimens than in the serum specimens. 2. hbov was more prevalent in wheezing patients than in asymptomatic children. the significance of this observation alone should not be exaggerated. first, the age distribution differed between symptomatic and asymptomatic children. second, sampling techniques were not matched. however, these results are consistent with the recent findings of kesebir et al. [23] , who reported that nasal wash samples obtained from 96 asymptomatic children were negative for hbov. nevertheless, the comparison of symptomatic and asymptomatic individuals remains problematic. the large number of mixed infections observed with hbov may indicate that hbov is reactivatedor its detection enhanced-by other infections. this would result in a higher prevalence among symptomatic patients than among control subjects, even in the absence of an etiologic role. respiratory secretions recovered during acute infection are also inherently very different from those recovered from asymptomatic subjects (e.g., with regard to cell counts). therefore, evidence of a virus-disease association must rely also on criteria other than differences in the prevalence between case patients and control subjects. 3. hbov was more prevalent among patients with previously unexplained wheezing than among patients who tested positive for other viruses. 4. this association was seen only for patients with a high viral load (i.e., there was a dose-response relationship). 5. occurrence of hbov in serum specimens was linked in time with an episode of acute wheezing, because hbov became less prevalent in the same patients after clinical recovery. taken together, these findings suggest an association between high hbov load and acute wheezing of otherwise unknown etiology, but they do not prove a causal relationship. at the same time, shedding of hbov (continuous or secondary to other infections) also appears to be common. this model for hbov biology may prove to be valuable for additional studies of the possible causative role of hbov in respiratory tract disease. applying this model to the present study would suggest that hbov is a potential etiologic agent in 28 patients (11%) with a high viral load, of whom 10 (3.9%) had hbov infection alone. however, some studies have reported that dual viral infections are associated with more-severe disease than are infections with a single agent, so the clinical relevance of hbov infection with a low viral load will also require additional study [24] [25] [26] . detection of hbov dna in serum suggests that hbov is a systemic infection, like most parvovirus infections, including animal bocavirus infection [27, 28] . it is possible that children with high-titer hbov in both the respiratory tract and the blood had a primary hbov infection, and this hypothesis is supported by the finding that serum viral loads generally became low or undetectable after clinical recovery. however, confirmation of a primary infection will have to await the development of an hbov antibody assay. an hbov infection with a low viral load may reflect long-term virus persistence after clinical recovery, as has been reported for parvovirus b19 [29, 30] . the findings of hbov in a few convalescent-phase serum samples and in serum samples obtained from some patients whose respiratory tract specimens tested negative for hbov are also consistent with virus persistence. cases of possibly symptomatic primary hbov infection (in the patients with hbov infection alone) seemed to be particularly common among children aged ∼1 year (i.e., soon after the loss of maternal antibodies). such cases emerged throughout the year (table 4) . these findings, together with the high prevalence rate, suggest that hbov is an endemic virus in the study area. we only studied children with bronchiolitis, recurrent wheezing, and acute asthma. thus, possible other manifestations of hbov infection were not addressed. isolated hbov positivity was associated with acute otitis media in nearly one-half of the subjects, and interstitial infiltrates were seen in all chest radiographs, suggesting that hbov could be a frequent causative agent of acute otitis media and childhood community-acquired pneumonia. hbov-associated acute wheezing did not differ clinically from that induced by rhinoviruses, enteroviruses, or rsv. our results indicate that acute wheezing is almost invariably associated with virus infection-and often with multiple-virus infection. many new respiratory viruses, such as human metapneumovirus and 3 new types of coronaviruses, have been identified over the past few years [3, 16, 18, 31, 32] . our results suggest that 5% of suspected respiratory virus infections are still unaccounted for, but that estimate is, of course, highly uncertain. on one hand, 5% of patients may not experience virus infections or may be infected by a known virus not detected because of assay limitations. on the other hand, the large number of mixed infections that we observed indicates that additional respiratory agents may also be found in other patients in addition to the 5% who still had negative results. in conclusion, we found evidence of virus infection in nearly all children with acute wheezing. hbov was detected in onefifth of patients, and it was the fourth most prevalent virus detected. results suggest that hbov at a high viral load could be an etiologic agent of respiratory tract disease, whereas little support was found for the etiologic role of hbov at a low viral load. quantitative analysis may, therefore, be important for future studies of hbov infection. like other parvoviruses, hbov can cause a systemic infection and may persist after resolution of symptoms. global and regional burden of disease and risk factors, 2001: systematic analysis of population health data bronchiolitis-associated hospitalizations among us children cloning of a human parvovirus by molecular screening of respiratory tract samples human bocavirus infection human bocavirus in children detection of human bocavirus in japanese children with lower respiratory tract infections evidence of human coronavirus hku1 and human bocavirus in australian children human bocavirus: prevalence and clinical spectrum at a children's hospital the association of newly identified respiratory viruses with lower respiratory tract infections in korean children frequent detection of bocavirus dna in german children with respiratory tract infections frequent detection of human rhinoviruses, paramyxoviruses, coronaviruses, and bocavirus during acute respiratory tract infections respiratory picornaviruses and respiratory syncytial virus as causative agents of acute expiratory wheezing in children evaluation of the efficacy of prednisolone in early wheezing induced by rhinovirus or respiratory syncytial virus expert panel report: guidelines for the diagnosis and management of asthma update on selected topics-2002 persistence of rhinovirus and enterovirus rna after acute respiratory illness in children a newly discovered human pneumovirus isolated from young children with respiratory tract disease viruses and bacteria in the etiology of the common cold a previously undescribed coronavirus associated with respiratory disease in humans pring-akerblom p. rapid and quantitative detection of human adenovirus dna by real-time pcr diagnosing herpesvirus infections by real-time amplification and rapid culture design and performance testing of quantitative real-time pcr assays for influenza a and b viral load measurement sequence-based identification of microbial pathogens: a reconsideration of koch's postulates human bocavirus infection in young children in the united states: molecular epidemiological profile and clinical characteristics of a newly emerging respiratory virus human metapneumovirus in severe respiratory syncytial virus bronchiolitis dual infection of infants by human metapneumovirus and human respiratory syncytial virus is strongly associated with severe bronchiolitis association of rhinovirus infection with increased disease severity in acute bronchiolitis minute virus of canines (mvc, canine parvovirus type-1): pathogenicity for pups and seroprevalence estimate pathological and virological studies of experimental parvoviral enteritis in calves persistent b19 infection in immunocompetent individuals: implications for transfusion safety slow clearance of human parvovirus b19 viremia following acute infection identification of a new human coronavirus characterization and complete genome sequence of a novel coronavirus, coronavirus hku1, from patients with pneumonia we thank kaisu kaistinen for traveling with the samples between turku and stockholm.financial support. academy of finland (to t.j.), the pediatric research foundation (to o.r. and t.j.), the torsten and ragnar söderberg foundation, the swedish cancer foundation, nana svartz' fund, and the swedish society for clinical microbiology (to t.a.). t.a. is a fellow of the swedish research council.potential conflicts of interest. t.a. is a coinventor on a patent application related to hbov and assigned to karolinska institutet innovations. all other authors: no conflicts. key: cord-022431-f6caajxy authors: wellemans, g. title: bovine respiratory syncytial virus date: 2013-11-17 journal: virus infections of ruminants doi: 10.1016/b978-0-444-87312-5.50051-5 sha: doc_id: 22431 cord_uid: f6caajxy nan some differences, e.g. the spectrum of susceptible cells and an antigenic dissimilarity, suggest that the two viruses have different natural hosts (inaba et al., 1970) . moreover, virus could not be recovered from the respiratory tract of mice inoculated with bovine strains . however, jacobs and edington (1975) succeeded in an experimental infection of a calf with a human strain. this experiment was successfully repeated by thomas et al. (1984) . the protein composition of human and bovine rsv strains is very similar, with only minor differences in mol. wt. (cash et al., 1977) . two subgroups of human rsv can be distinguished; the major antigenic differences are located on the g protein (mufson et al., 1985) . brsv can be classified as a separate group, sharing epitopes with most proteins of the two human subgroups, but being distinct with respect to the epitopes on the g protein (orvell et al., 1987) . monoclonal antibodies to the f protein showed neutralizing activity and prevented spread of the virus in vitro (walsh et al., 1985) . passive transfer of monoclonal antibodies to the f and g proteins offered protection against challenge with rsv, while antibodies to internal proteins did not (walsh et al., 1984) . in tissue culture, brsv shows a wide range of susceptible host cells. it replicates in all cell types (kidney, testicle, thyroid, thymus, duodenum, rectum) of bovine origin as well as in cells from swine (embryonic kidney), hamster (lung, kidney), monkey (vero) and human (embryonic lung and kidney, hela, hep-2). better growth is observed in bovine than in human cell cultures (matumoto et al., 1974) . this is in contrast to the observations of paccaud and jacquier (1970) , who did not succeed in cultivating their brsv strains in a bhk-21 cell line and in various human cells. larger amounts of virus are obtained in calf kidney and testicle cell culture, and higher titers are scored in secondary than in primary cultures of the same cells (mohanty, 1978; wellemans, 1977) . the direct if test in which the immunoglobulins of brsv antiserum are conjugated with fluorescein isothiocyanate detects the antigens as soon as 16-18 h after inoculation. fluorescence is always confined to the cytoplasm. within 24 h, fine fibrils appear, usually parallel to the long axis of the cell, and cytoplasmic granules are formed around the nucleus (rossi and kiesel, 1977b) . after 24 h, coincident with rounding of the cells, fluorescence slowly moves to the periphery of the cytoplasm. by if and as determined by the release of brsv into the supernatant fluid, the minimal time for a single cycle of infection was between 24 and 26 h (rossi and kiesel, 1977b) . brsv grows in organ cultures of bovine fetal trachea expiants at 37°c and ph 7.2. it reaches maximum titers of 10 5 pfu/ml between 11 and 21 days after inoculation. virus growth does not affect ciliary activity of the cultured cells (thomas et al., 1976; rossi and kiesel, 1977a) . brsv has been isolated in most european countries, north america, australia, japan and more recently in north africa (mahin and wellemans, 1982) . the virus generally appears in a brsv-free country after the introduction of an infected animal. although the presence of virus carriers has not yet been proven (probably because of the difficult isolation of the virus), one should be aware of this possibility. humans, especially veterinarians and animal handlers, could play a role in the transmission of the virus. considering the success-fui infection of a calf with a human strain (jacobs and edington, 1975) and the similarities between human and bovine strains, humans cannot be excluded as a virus reservoir (berthiaume et al., 1973) . however, inaba et al. (1970) and paccaud and jacquier (1970) do not endorse this view. once a herd in a region is affected, the disease rapidly spreads from farm to farm. in an already infected region, the disease becomes endemical and affects the same herds almost every year. the animals, especially beef cattle, are most susceptible from 3 up to 9 months of age, though older animals are not always resistant (paccaud and jacquier, 1970; wellemans et al., 1970; van bekkum et al., 1977) . on the other hand, younger calves of only a few weeks of age can also be affected, and their protection by vaccination is still questionable (wellemans, 1982) . differences in housing and other aspects of management probably have some effect; in some herds the virus is present without causing any disease, as shown by occasional seroconversions, in others one or even two outbreaks occur every year (van bekkum et al., 1977) . however, severe disorders can be observed also in animals at pasture. most of the severe cases appear from october to january (van bekkum et al., 1977; wellemans, 1977) , but recently brsv outbreaks have also occurred in spring and summer. therefore, disease caused by brsv can be expected during the whole year, as in the case of ibr. the weather, particularly a fall in atmospheric pressure, plays an important role in the outbreak of the disease (wellemans, 1982) . the disease symptoms observed after experimental inoculation are not as severe as in cases of natural infection (inaba et al., 1972; jacobs and edington, 1975; smith et al., 1975; mohanty, 1978; elazhary et al., 1979; thomas et al., 1984) and the infection frequently remains inapparent. stott (1985) suggests that virus unpassaged in tissue culture may have a greater virulence for the natural host. treatment with dexamethasone enhances lung lesions produced by bovine strains, extends the period of virus shedding and increases peak titers . the concomitant presence of bvdv as well as a sudden fall in atmospheric pressure or a drop of the minimum temperature seem to aggravate the disease (verhoeff and van nieuwstadt, 1984) . the first symptoms are observed 2-8 days after inoculation. the virus can be reisolated from nasal secretions for 4-10 days after inoculation, and from the nasal, tracheal and bronchial mucosae for 7-13 days after infection (jacobs and edington, 1975; thomas et al., 1984) . it appears that brsv infection can occur in the presence of circulating antibodies (mohanty, 1978; mcnulty et al., 1983) . there is no evidence, however, that preexisting serum antibody causes exacerbation of the disease in young calves, as was reported in infants (kim et al., 1969) ; other conclusions have been drawn by smith et al. (1975) . however, the protective effect of nasal neutralizing antibodies against infection with brsv has been shown by mohanty (1978) . this author found that young calves with traces of neutralizing antibodies in the nasal secretions remained solidly immune to challenge. the quick appearance and the pathogenic action of brsv on the nasal mucosa would indicate that the nasal cavity might be the point of departure to the target cells; baskerville (1981) disagrees with this hypothesis. brsv is unable to suppress ciliary activity, even though the virus replicates within ciliated epithelial cells of tracheal rings. it was therefore suggested that the tracheal epithelium may not be important in the pathogenesis of brsv infection (thomas et al., 1976; rossi and kiesel, 1977a) . the accumulation of cellular debris and exudate favors bacterial proliferation, which may lead to extensive purulent pneumonia chiefly in young calves. however, the sudden onset of pulmonary emphysema is not explained by brsv infection. immunological hypersensitivity reactions probably are involved as complicating factors, as has been shown in humans (gardner et al., 1970) . mcintosh and fishaut (1980) have reviewed several theories about the pathogenesis of rsv disease in infants: serum antibodies reacting with the virus, prior sensitizing infection, cmi, ige-mediated reactions-no firm conclusions are possible about immunopathologic mechanisms in bronchiolitis. in typical outbreaks, the disease develops in two distinct episodes. suddenly 80-90% of a certain age group may show symptoms. affected calves are 3-9, sometimes up to 15 months old. the animals cough and there is nasal discharge and a conjunctivitis with lachrymation. the body temperature is about 40°c. about 2 or 3 days later, when everything seems to be back to normal, the second episode begins and signs of lung emphysema appear. some animals have difficulty in breathing, accompanied by bouts of dry coughing. body temperature at the time these symptoms are first manifest is close to normal. the calves lose weight and have a rough hair coat. respiration rates can go up to > 100. the breathing of the sick animals becomes increasingly rapid and shallow, and the condition is aggravated by bouts of coughing. there is little or no discharge from the nostrils. frequently there is froth at the commissure of the lips. ill calves may stand with a stretched neck and extended forelimbs (fig. 131) . symptoms of abdominal breathing can develop. the animals can neither lie down nor eat and make desperate efforts to breath through the open mouth. constipation is common and there is complete loss of appetite. the mucosae become cyanotic. on auscultation, some harshness in the breathing can be detected. there can be up to 20% mortality in the herd. farms specializing in babybeef tend to have the highest losses. mortality often occurs within a few hours. surviving calves recover after a few days: breathing becomes easier and appetite returns (holzhauer and van nieuwstadt, 1976; van bekkum et al., 1977; wellemans, 1977; verhoeff and van nieuwstadt, 1984) . a slightly different symptomatology can be observed in 6-week-old calves, especially in fattening farms. few or no emphysema lesions are encountered, but bacterial superinfections are frequently found. cough, high fever and serous to mucopurulent nasal discharge are the most common symptoms. paccaud and jacquier (1970) noted that all animals aged less than 7 years showed signs of acute respiratory disease. pregnant animals or good milk producers tend to be affected (inaba et al., 1972) . in belgium, respiratory disorders are seldom observed in cattle more than 2 years old. in animals that have died from the disease, pathological changes are restricted to the respiratory tract; postmortem lesions are not really pathognomonic: they can also be found in cases of bvd and pi3 infection. the lungs are voluminous and emphysematous. the apical and cranial lobes show lobular bronchopneumonia. numerous large ecchymotic hemorrhages are present in emphysematous bullae and under the pleura (figs. 132, 133) ; often foamy exudate covers the injected mucosae of the bronchi. the diaphragmatic lobes are edematous and interlobular septa are distended. subpleural emphysema is present in all lobes but is more severe in the diaphragmatic lobe, where numerous large dissecting bullae are present. in some cases the pleural wall is broken, resulting in pneumothorax. in most cases there is also marked mediastinal emphysema and in some calves emphysema extends into the subcutaneous tissues of the shoulder, back and neck. the animals then show crepitating, asymétrie swellings. petechia are frequently seen, particularly on the larynx. in the heart, petechia are often found on the myocard (holzhauer and van nieuwstadt, 1976; wellemans, 1977; bryson et al., 1983) . calves of a few weeks of age present hepatization in the anterior lobes; emphysema is rare. histological lesions, e.g. mononuclear infiltration in the hyperplastic alveolar walls, bronchiolitis or lobular interstitial pneumonia with edema, are not pathognomonic of brsv infection. however, in some cases, mainly in young calves, syncytia with eosinophilic inclusions are observed in the alveoles (mohanty, 1978; bryson et al., 1983) . two types of lesions can be found in ultrathin sections of apical and cranial lobes, with the aid of a fluorescein isothiocyanate (fitc) conjugated anti-brsv serum. the lesions are referred to as types a and b. in type a lesions the antigen is distributed homogenously in the cytoplasm of the affected cells. these cells look undamaged, and syncytium formation is frequently seen. this type of lesions is found in calves of a few weeks of age and sometimes in older animals of the charolais or blonde d'aquitaine breed (wellemans, 1982) . in type β lesions the antigen is clustered in packs. the cells are disrupted and antigen masses are spread i the alveolar or bronchial lumen. rarely giant cells are found in these lesions (wellemans, 1977) . the type β lesions are encountered in emphysematous lungs of older calves of the belgian blue white breed. epidemiological studies have shown that there is no solid protection in humans against nasal reinfection. however, cotton rats infected with rsv develop complete resistance to pulmonary infection lasting for at least 18 months. nasal resistance was of shorter duration and decreased from 8 months on. immunity to rsv infection is therefore more long lasting in the lungs than in the upper tract and the level of immunity to rsv in the upper respiratory tract does not necessarily reflect resistance in the lungs (prince et al., 1983) . preexisting maternal antibodies to brsv did not protect calves from infection (mohanty, 1978; mcnulty et al., 1983) . on the other hand, maternal immunity was effective but transient in the lungs of young cotton rats (prince et al., 1983) . immune factors other than neutralizing antibodies may play a role in the maternal passive immunity (prince et al., 1983) . in experimentally infected calves, high interferon titers are detected during the early stage of infection. this is followed by a period of at least 1 week during which interferon is not detectable. after this, moderate to low interferon titers reappear in most animals and persist for a number of weeks (elazhary et al., 1981) . in contrast, in children no interferon or only low levels are found (hall et al., 1978; mcintosh, 1978) , and mean levels do not fluctuate significantly in relation to disease and recovery. these discordant results are probably due to the fact that the first blood sample in children is taken in the course of the disease, whereas in calves the first sample is taken at an early beginning stage. antibodies have been demonstrated by vn, cf, indirect if, elisa and precipitation tests in sera from cattle infected with brsv. neutralizing antibodies appear in serum of experimentally infected calves after 7 days at extremely low titers (4-8) (mohanty et al., 1975) . in natural infections, however, the sn titers score much higher (64: rosenquist, 1974; 256: inaba et al., 1972) and the maximum level is reached after 3 weeks. using indirect if, antibodies may be evidenced as early as 3 days p.i., with maximum titers at about 10 days p.i. (elazhary et al., 1981) . in natural infections, it is not uncommon to find highly seropositive calves in the acute stage of the disease. experimentally infected animals can react in different ways: in some calves, antibodies develop very early, as described by elazhary et al. (1981) ; in others their appearance is delayed (7-9 days). in nasal mucus antibodies evidenced by indirect if are excreted from the first week on, and they are present for at least 3 months (wellemans, 1977) . results of leukocyte migration-inhibition tests under agarose indicate that a cmi response is elicited after infection of calves with brsv. the calves also develop a delayed hypersensitivity skin response (field and smith, 1984) . recovery of 6-7-month-old calves from severe brsv-associated disease was accompanied by an antibody response that was mainly directed to the f and ν proteins. calves 2-3 weeks of age with moderate levels of maternal antibodies to brsv particularly directed to the f and ν proteins became seriously ill after infection. the antibody response in these calves was severely suppressed. in sera of 4-9-month-old calves that had died in the course of infections, high anti-f and anti-n antibody levels were found. apparently, the presence or development of antibodies to the f and ν proteins is not sufficient for protection against or recovery from infections with brsv (westenbrink et al., 1989) . a clinical diagnosis based on symptomatology is impossible. only the laboratory can help the practitioner who suspects a brsv outbreak in a farm. the laboratory diagnosis will be based on either brsv isolation, the detection of viral antigen in suspected organs, or by evidencing a seroconversion in diseased animals. the nasal mucus is taken with sterile cotton swabs during the initial stage of the disease (serous discharge, fever, conjunctivitis). the swabs are then put into a tube with a protein-rich medium (maintenance medium for cell cultures) and forwarded very rapidly in a cool box to the laboratory. postmortem samples of lung tissue from very young calves should also reach the laboratory rapidly. in contrast to other viruses, e.g. ibrv, viral presence in the nasal mucous is of short duration and limited to the first stage of the disease, which often passes unnoticed. the isolation of the virus in cell culture is difficult because of the late appearance of cpe; the time of incubation may be extremely long: 20 days (inaba et al., 1970) , 30 days (paccaud and jacquier, 1970) , 45 days (wellemans et al., 1970) or even 50 days (smith et al., 1975) . secondary fetal calf kidney or testicle cells are most susceptible. virus growth depends on the age of the cell culture, and the medium has to be replaced frequently. the first changes noted are small areas where four or five cells become ballooned with shrinkage of cytoplasm. in the following days, syncytia develop in these cultures. they become opaque while contracting, and holes appear in the cell monolayer. the aspect becomes more and more granular, resembling a mycoplasma-infected cell culture (fig. 134) . in preparations fixed with bouin solution and stained with h & e , homogenous eosinophilic inclusion bodies are observed in the cytoplasm of syncytial cells. these inclusions are less polymorphic than in cells infected with pi3 virus. the cells grown on glass slide of leighton tubes are fixed in aceton at -20°c and stained with an antiserum to brsv conjugated with fluorescein isothiocyanate. plaques of fluorescent cells confirm the presence of the virus (fig. 135) . isolation of brsv is difficult and of long duration; it is not recommended as a routine procedure (edwards et al., 1984) . viral antigen can be detected in nasal mucus and in lung tissue. nasal mucus can be taken with a swab. though the most characteristic emphysema lesions are situated in the mediastinal lobes of the lungs, antigen has to be searched for mainly in the apical and cardiac lobes. the samples have to be forwarded quickly and under refrigeration to the laboratory. for the direct if test, monospecific hyperimmune serum is required that is marked with fluorescein isothiocyanate using the classical technique. typical small, round cells in which fluorescence is confined to the cytoplasm are detected in the nasopharyngeal specimens . fluorescence is not seen in ciliated columnar epithelial cells, although many are present in most specimens (thomas and stott, 1981) . problems of nonspecific fluorescence are encountered with the method when applied to nasopharyngeal material but not in the examination of lung material (thomas and stott, 1981) . evans blue (1:10.000) can be used to reduce nonspecific fluorescence. ultrathin sections of lung fragments, preferably from the apical and cardiac lobes, are stained with conjugated serum. fluorescent antigen is detected in this material for up to 48 h post mortem, even after freezing. however, it is noteworthy that the viral antigen disappears after longer freezing periods, even at low temperatures ( -100°c). in most of the lung specimens, antigen is found located in the alveoli and sometimes in the bronchioles. two types of fluorescence are distinguished (fig. 136) : type a, in which the antigen is spread homogenously throughout the cytoplasm. the cells look intact and syncytia are frequently observed (fig. 137) ; and type b, in which the antigen is aggregated in granular packs. the cells are disrupted and antigenic masses are spread in the alveolar and bronchial lumen. rarely giant cells are found in these lesions (fig. 138) . type a lesions are encountered in 2-3-week-old calves and also in older cattle of the charolais and blonde d'aquitaine breed; type β lesions are found in emphysematic lungs of older animals of the belgian blue white breed. elisa is applied more and more for the detection of viral antigens. hornsleth et al. (1981) described its use for rsv diagnosis and confirmed its reliability. an increase of anti-brsv antibodies can be evidenced in paired sera taken from the same animals in the acute stage and 15 days to 3 weeks later. neutralizing antibodies appear as a consequence of a brsv infection. sn titers in convalescent animals rarely exceed 4-16 according to bartha (1976) , whereas rosenquist (1974) and inaba et al. (1970) report titers of 256. despite the improved micromethods the sn test remains a time-consuming technique with difficult reading of the results; it is used in research work rather than for routine diagnosis. numerous authors have used the cf test (e.g. takahashi et al., 1975; holzhauer, 1978) . untreated infected cell culture fluid, fluorocarbon-treated and ether-treated material are equally suitable as antigen. human rsv antigen is commercially available. best specific reactions are obtained with 5% fresh normal calf serum added to the diluent of complement (wellemans et al., 1970; takahashi et al., 1975) . neutralizing and complement fixing antibody titers are closely related. the cf test is very reliable for the diagnosis of brsv infection (takahashi et al., 1975) . seroconversion can be detected by agid. after infection, more than 50% of the animals have immunodiffusion antibodies (zygraich and wellemans, 1981) . however, this test is only seldom used because of the difficulty in obtaining precipitating antigen and the 3-day delay before the test can be read. espinasse et al. (1978) have adapted passive hemagglutination (pha) to the detection of brsv antibodies. the preliminary results are encouraging and appear equivalent to the sn scores. anti-brsv antibodies in serum, nasal mucus and organ extracts can be evidenced rapidly and specifically using the indirect if test (wellemans, 1977; potgieter and aldridge, 1977) . the production of a batch of slides covered with antigen-containing cells, which can be stored in a freezer for a long time, makes this test highly reproducible. after experimental inoculation, the antibodies appear generally from the 7th day on and the titers rise quickly to levels of ^ 1280 at the end of the second week. titers exceeding 5000 are frequently found. in other cases, however, a very slow titer evolution is noted, with a maximum score of 135 -although a brsv infection could be confirmed on postmortem examination of the lungs. no valid diagnosis can be made without paired sera. however, very high indirect if or cf antibody levels in single samples are indicative of a brsv infection in severe respiratory distress. the increase of anti-brsv antibodies reflects an infection but no conclusions can be drawn about the pathogenic role of the virus. striking seroconversions have been observed in animals vaccinated 2-3 months before, although no respiratory problems were noticed (wellemans, 1982) . serological examination is useful but the possibility of atypical seroconversions exists. brsv carriers have not yet been proven to exist but it seems possible they will, as was shown for other bovine pathogens, e.g. ibrv (bitsch, 1973) , coronavirus (van opdenbosch et al., 1979) and bvdv (coria and mcclurkin, 1978) . the fact that the disease becomes enzootic after its introduction in a brsv-free area supports this hypothesis. therefore it is advisable to forbid the introduction of diseased animals into a brsv-free region. modification of breeding and fattening conditions (habitat, environment, feeding) has to be taken into consideration, although it is our experience that the most severely affected herds are not always the most badly managed ones. the vaccination of young children with an inactivated rsv vaccine caused severe hypersensitivity reactions (kim et al., 1969) . in cattle, mohanty et al. (1981) used a formalin-inactivated and freund-adjuvanted vaccine. there was no evidence after challenge that vaccinal serum antibodies caused exacerbation of disease in young calves. the vaccine did not induce a nasal antibody response. all but one of the five vaccinated calves appeared to be protected against the disease after challenge exposure. stott et al. (1984) used a vaccine containing cells persistently infected with brsv and fixed with glutaraldehyde to retain viral antigens on the cell surface. the glutaraldehyde-fixed cells were combined with incomplete freund's adjuvant or saponine quil-a. after challenge, virus was recovered from all control calves but from only one out of twelve vaccinated calves. a brsv strain (poum 2) at the 94th passage level in bfk cell cultures is presently used in belgium (wellemans, 1982) and in the netherlands (holzhauer, 1982) for immunization of young cattle. the animals (at least 3 months old) are vaccinated intramuscularly in august-september and boostered 3 weeks later. some vaccination problems that are not attributable to the vaccine virus strain have been described (wellemans, 1982) . this strain has been shown to be safe in over a million vaccinated animals (zygraich, 1982) and the results of protection are satisfactory (holzhauer, 1982; wellemans, 1982) . isolation of respiratory syncytial virus from cattle with a respiratory disease mechanisms of infection in the respiratory tract serological evidence of respiratory syncytial virus infection in sheep infectious bovine rhinotracheitis virus infection in bulls, with special reference to preputial infection respiratory syncytial virus pneumonia in young calves: clinical and pathologic findings a comparison of the polypeptides of human and bovine respiratory syncytial viruses and murine pneumonia virus specific immune tolerance in an apparently healthy bull persistently infected with bovine viral diarrhea virus respiratory syncytial virus diagnosis experimental infection of calves with bovine respiratory syncytial virus (quebec strain) interferon, fluorescent antibody and neutralizing antibody responses in sera of calves inoculated with bovine respiratory syncytial virus hemagglutination passive: application de la méthode au diagnostic sérologique des affections respiratoires virales des jeunes bovins cell-mediated immune response in cattle to bovine respiratory syncytial virus speculation on pathogenesis in death from respiratory syncytial virus infection interferon production in children with respiratory syncytial, influenza and parainfluenza virus infections bronchopneumonia of yearling cattle prevention of pinkengriep by vaccination, a field trial the etiological role of bovine respiratory syncytial virus in "pinkengriep detection of respiratory syncytial virus in nasopharyngeal secretions by inhibition of enzyme-linked immunosorbent assay respiratory syncytial virus mrna coding assignments nomi virus, a virus isolated from an apparently new epizootic respiratory disease of cattle bovine respiratory syncytial virus. studies on an outbreak in japan structure of bovine respiratory syncytial virus experimental infection of calves with respiratory syncytial virus respiratory syncytial virus disease in infants despite prior administration of antigenic inactivated vaccine demonstration that glycoprotein g is the attachment protein of respiratory syncytial virus interferon in nasal secretions from infants with viral respiratory tract infections immunopathologic mechanisms in lower respiratory tract disease of infants due to respiratory syncytial virus experimental respiratory syncytial virus pneumonia in young calves: microbiologic and immunofluorescent findings serological evidence for the intervention of bovine respiratory syncytial virus in a respiratory disease outbreak in moroccan cattle bovine respiratory syncytial virus: host range in laboratory animals and cell cultures bovine respiratory viruses experimentally induced respiratory syncytial viral infection in calves effect of vaccinal serum antibodies on bovine respiratory syncytial viral infection in calves two distinct subtypes of human respiratory syncytial virus preparation and characterization of monoclonal antibodies directed against five structural components of human respiratory syncytial virus subgroup b a respiratory syncytial virus of bovine origin use of indirect fluorescent antibody test in the detection of bovine respiratory syncytial virus antibodies in bovine serum mechanisms of immunity to respiratory syncytial virus in cotton rats isolation of respiratory syncytial virus from calves with acute respiratory disease susceptibility of bovine macrophage and tracheal-ring cultures to bovine viruses bovine respiratory syncytial virus infection of bovine embryonic lung cultures: a kinetic study by the fluorescent antibody technique isolation, characterization and pathogenicity studies of a bovine respiratory syncytial virus respiratory syncytial virus from cattle with a respiratory disease a comparison of three vaccines against respiratory syncytial virus in calves diagnosis of bovine respiratory syncytial virus infection by complement fixation test respiratory syncytial virus infection in mice diagnosis of respiratory syncytial virus infection in the bovine respiratory tract by immunofluorescence the growth of respiratory syncytial virus in organ cultures of bovine foetal trachea infection of gnotobiotic calves with a bovine and human isolate of respiratory syncytial virus. modification of the response by dexamethasone viral infections of the respiratory tract in cattle in the netherlands neonatal calf diarrheoa: complex viral etiology brs-virus, pi3 virus and bhv 1 infections of young stock on self-contained dairy farms: epidemiological and clinical findings monoclonal antibodies to respiratory syncytial virus proteins: identification of the fusion protein protection from respiratory syncytial virus infection in cotton rats by passive transfer of monoclonal antibodies purification and characterization of the respiratory syncytial virus fusion protein laboratory diagnosis methods for bovine respiratory syncytial virus evaluation du programme de vaccination anti-virus respiratoire syncytial bovin en belgique isolement d'un virus (220/69) sérologiquement semblable au virus respiratoire syncytial (rs) humain analysis of the antibody response to bovine respiratory syncytial virus protein in calves biological characteristics of respiratory syncytial virus vaccination against rs virus: a five year experience with rispoval immunological markers of an attenuated bovine respiratory syncytial virus vaccine key: cord-268251-mcg1v24t authors: martins, ronaldo bragança; carney, sharon; goldemberg, daniel; bonine, lucas; spano, liliana cruz; siqueira, marilda; checon, rita elizabeth title: detection of respiratory viruses by real-time polymerase chain reaction in outpatients with acute respiratory infection date: 2014-09-17 journal: mem inst oswaldo cruz doi: 10.1590/0074-0276140046 sha: doc_id: 268251 cord_uid: mcg1v24t viruses are the major contributors to the morbidity and mortality of upper and lower acute respiratory infections (aris) for all age groups. the aim of this study was to determine the frequencies for a large range of respiratory viruses using a sensitive molecular detection technique in specimens from outpatients of all ages with aris. nasopharyngeal aspirates were obtained from 162 individuals between august 2007-august 2009. twenty-three pathogenic respiratory agents, 18 respiratory viruses and five bacteria were investigated using multiplex real-time reverse transcriptase polymerase chain reaction (rt-pcr) and indirect immunofluorescence assay (iif). through iif, 33 (20.4%) specimens with respiratory virus were recognised, with influenza virus representing over half of the positive samples. through a multiplex real-time rt-pcr assay, 88 (54.3%) positive samples were detected; the most prevalent respiratory viral pathogens were influenza, human rhinovirus and respiratory syncytial virus (rsv). six cases of viral co-detection were observed, mainly involving rsv. the use of multiplex real-time rt-pcr increased the viral detection by 33.9% and revealed a larger number of respiratory viruses implicated in ari cases, including the most recently described respiratory viruses [human bocavirus, human metapneumovirus, influenza a (h1n1) pdm09 virus, human coronavirus (hcov) nl63 and hcov hku1]. acute respiratory infections (aris) are the main causes of morbidity and mortality worldwide, especially in children during the first years of life (osterhaus 2008) . the viruses most commonly associated with aris are influenza viruses a and b (inf a and inf b), respiratory syncytial virus (rsv), parainfluenza viruses 1-4 (piv 1-4), human adenovirus (hadv), human rhinovirus (hrv), human coronavirus (hcov) and enterovirus (ev) (arruda et al. 2006) . since 2001, new viruses have been detected in ari cases, such as human metapneumovirus (hmpv) (van den hoogen et al. 2001) ; two new types of hcov, hku1 and nl63 (van der hoek et al. 2004, woo et al. 2005) and human bocavirus (hbov) (allander et al. 2005) . the diagnosis of respiratory viruses based on virus isolation, detection of antigens or serology is too time-consuming and, in some cases, has low sensitivity (gunson et al. 2005) . therefore, molecular diagnostic methods using "in-house" or commercially available techniques are alternatives to obtain faster results and higher sensitivity and specificity. these molecular methods can potentially reduce the length of hospitalisation and unnecessary treatment costs; furthermore, they can also contribute to nosocomial infection control programs and can help to guide therapy (barenfanger et al. 2000 , mahony 2008 , brittain-long et al. 2010 . the aim of the present study was to determine the frequencies of a range of respiratory pathogens using the fast-track diagnostics respiratory pathogens 21 plus (ftdrp 21 plus) multiplex reverse transcriptase polymerase chain reaction (rt-pcr) assay in patients of all ages with aris treated in emergency rooms or primary care units. this descriptive study was developed in vitória, southeast brazil, from a set of biological samples obtained over a two-year period (august 2007-august 2009) as a part of the respiratory virus surveillance program of the ministry of health, brazil. nasopharyngeal aspirates (npas) were collected from patients of all ages who were attended at either emergency rooms or primary care units. the patients presented with fever (temperature equal to or greater than 38ºc) and runny nose, in addition to one or more of the following symptoms: cough, myalgia, nasal congestion, headache, sore throat and earache, within five days of the symptom onset. the nasopharyngeal aspiration procedure is a part of the routine of the respiratory virus surveillance program. all samples were tested by indirect immunofluorescence assay (iif) and ftdrp 21 plus multiplex realtime rt-pcr assay. seven respiratory viruses (rsv, piv 1-3, inf a, inf b and hadv) were screened in all specimens by iif using the respiratory panel 1 viral screening & identification kit tm (chemicon international, millipore, usa) according to the manufacturer's instructions. twenty-three respiratory pathogens, including 18 human respiratory viruses and five bacterial species, were tested using the multiplex protocol. total nucleic acid was extracted using the magna pure lc automated extraction system (roche, switzerland). nucleic acid was extracted from 200 µl of each npa sample and was eluted to a final volume of 100 µl following the magna pure lc total nucleic acid isolation v.12.0 (roche) protocol. an internal rna virus control, brome mosaic virus (fast-track diagnostics, luxembourg), was introduced into the lysis buffer for each specimen to monitor the sample extraction and reverse transcription. multiplex real-time rt-pcr was performed using the ftdrp 21 plus according to the manufacturer's instructions (fast-track diagnostics). the reaction volume for each test was 25 µl, made up of 10 µl of nucleic acid and 15 µl of buffer/enzyme mix from the agpath-id™ onestep rt-pcr kit (ambion, life technologies, usa). amplification was performed in the abi 7500 real-time pcr system thermocycler (applied biosystems, usa) and the following cycling conditions were used: 15 min at 50ºc, 10 min at 95ºc and 40 cycles of 8 s at 95ºc and 34 s at 60ºc. the fluorescence reading was taken in the 60ºc/34 s step in each cycle and the threshold cycle (c t ) values were determined by manual adjustment. each sample was amplified in six parallel reactions, which contained primers and probes for four different targets, detecting viruses and pathogenic bacteria, in addition to the internal control of the reaction. the positive and negative virus plasmid controls provided in the kit were included in all runs to monitor the assay performance. exploratory analysis of the data was performed using the spss software package v.17.01 (spss inc, usa). ethics -the research ethical committee of the centre for health sciences of federal university of espírito santo approved this study, with the registration cep-093/07. here, we present the results of patients who met all inclusion criteria and whose samples were analysed by both methods proposed in this study. one hundred sixty-two outpatients (79 male and 83 female) were included. the study consisted of both adult (n = 46) and paediatric (n = 116) individuals. the paediatric age group consisted of individuals zero-19 years of age, including children and adolescents, according to the world health organization criteria. the median age of the entire cohort was seven years (range 1 month to 75 years). children under nine years of age accounted for 66.7% (108/162) of the samples analysed. all patients had recent (≤ 5 days) symptoms. iif detected 33 positive specimens (20.4%). the influenza virus was the most common etiologic agent detected (23/33). inf a and inf b corresponded to 8% (13/162) and 6.2% (10/162) of the samples, respectively. the iif technique detected only single infections. eighty-eight (54.3%) specimens were positive for one or more respiratory viruses by multiplex real-time rt-pcr (table i ). the influenza virus (15.4%), hrv (8%) and rsv (7.4%) were the viruses most frequently detected and accounted for one-third of the positive samples. regarding the patient age groups, the influenza viruses were more frequently identified in young adults (20-39 years of age). all viruses were identified in infants (0-2 years old), with the exception of piv 2, piv 4 and ev. rsv and hrv were the most prevalent. most of the samples positive for rsv (91.6%) and hrv (77%) were identified in infants. no virus was identified in subjects above 40 years old (figure) . the following bacteria were detected: streptococcus pneumoniae [n = 43 (26.3%)], staphylococcus aureus [n = 20 (12.3%)] and haemophilus influenzae [n = 2 (1.2%)]; mycoplasma pneumoniae and chlamydophila pneumoniae were not detected (table i) . six specimens (3.7%) were positive for two viruses. all co-detections occurred in children under two years of age and rsv accounted for half of those cases (table ii) . no co-detections involved three or more respiratory viruses. bacterial pathogens were identified in 27 codetection cases, with at least one infectious agent from the viral or bacterial pathogen groups (table ii) . the laboratory diagnosis of viral respiratory tract infections is usually accomplished through conventional techniques, such as culture or antigen detection tests, with limitations related to the delay of the results and the availability of monoclonal antibodies for newly identified viruses (mahony et al. 2007 mercial multiplex ftdrp real-time rt-pcr assay has enhanced the diagnosis of aris, with simultaneous detection of 16 respiratory viruses (sakthivel et al. 2012) . the new version of the ftdrp assay used in this study has expanded the capacity for detecting pathogens (18 viral and 5 bacterial species) and has thus increased the diagnostic potential of the test. more than half (66.7%) of the study population were children under nine years of age, which is representative of the occurrence of aris in the community. the paediatric age group is the main group affected by aris, which occur three-eight times a year in infants and young children, with incidences varying inversely to age (bryce et al. 2005 , bezerra et al. 2011 . the viral agents most frequently identified were influenza viruses, including inf a (h1n1) pdm09 (2 cases in patients with uncomplicated respiratory infection), followed by hrv and rsv. since april 2009, the inf a (h1n1) pdm09 virus has spread worldwide. given that the country's health authorities only declared sustainable transmission of the new influenza virus in brazil (ms 2009) on 16 july 2009, we assume that the observed low frequency of inf a (h1n1) pdm09 is related to the endpoint of the present study, in august 2009. the frequency of influenza virus infection observed in this study (15.4%) is consistent with previously published rates varying from 2-26% of ari cases in studies in south, southeast and northeast brazil (arruda et al. 1991 , bellei et al. 2008 . although wide inter-regional variations should be expected in a large country with different climates, the variation may also be due to study design differences. all viruses screened in this study were 6 (3.7) 0 (0) 12 (7.4) 0 (0) more frequently identified in children, with the exception of influenza viruses, which were more often identified in adults (8.6%) than in paediatric patients (6.8%), as has been documented in a recent study (pelat et al. 2013 ). the second most prevalent viral agent was hrv (8%): 77% of hrv-positive samples were identified in children under two years of age. this finding highlights the importance of this virus in infants, as hrv has been associated with recurrent respiratory illnesses and wheezing in this age group (jartti et al. 2008) . other picornaviruses may be etiologic agents of aris; however, hrv is the main virus detected in cases of the common cold (pitkäranta & hayden 1998) . in a multicentre study, rhinoviruses were often associated with aris, even during the peak influenza season (caruso et al. 2007 ). in brazil, according to arruda et al. (1991) , hrv was the most frequent viral agent (46%) detected in children with aris. the frequency of rsv detection (7.4%) was similar to that detected by raboni et al. (2011) , who reported a rate of 8% using multiplex rt-pcr for 14 respiratory viruses. eleven (91.6%) of the rsv cases in this study were detected in children under two years of age. rsv antibodies are found in virtually all adults and children older than three years of age. at one year of age, 25-50% of children have antibodies against rsv, demonstrating the high frequency of this infection at a young age (domachowske & rosenberg 1999) . in keeping with this observation, the frequency of rsv in the youngest age group in our study was the highest among all ages (91.6%). however, the study was conducted with patients of widely variable ages: only two-thirds were children under nine and only 40% of the studied patients were children under two years of age. hence, it is reasonable to consider that the observed relatively low frequency of rsv could be attributed, at least in part, to the underrepresentation of patients of very young ages. the frequency of hbov was 3.1% and all cases occurred in children under two years of age. this virus has been frequently identified in this age group, although its pathogenicity is not yet well defined (longtin et al. 2008 , lüsebrink et al. 2009 ). recently, a higher hbov-positive rate has been documented in inpatients when compared with outpatients or patients attended in emergency departments, suggesting a significant role of this virus in the pathogenesis of aris (xu et al. 2012) . the use of multiplex real-time rt-pcr increased the overall viral detection rate to 54.3%, compared to 20.4% in the iff test. this difference is likely due to the high sensitivity of virus genome detection (lassaunière et al. 2010 ). in addition, 47.9% of the samples that were positive by multiplex real-time rt-pcr corresponded to respiratory viruses that were not tested for using the iif kit, such as hrv, hcov, hmpv, hbov and ev. this finding confirms the importance of using panels that identify multiple viral agents causing aris. in this study, there were six cases (3.7%) of viral codetection, most commonly with rsv and hrv. the real clinical significance of these infections has not yet been fully elucidated. in a recent brazilian study, co-detection between rsv and hrv increased both the length of hospitalisation and the time of supplemental oxygen use in children with lower respiratory tract infections, suggesting the possibility of co-infection among these viruses and not only co-detection (da silva et al. 2013 ). all cases of co-detection observed in our study occurred in children under two years old, the age group in which ari cases are more frequent. it is quite possible that residual nucleic acids from sequential viral infections are detected simultaneously by multiplex pcr with high sensitivity (lesley 2012). additionally, lymphoid tissues may serve as a reservoir of respiratory viruses in asymptomatic individuals and may be involved in the transmission of these viruses to the community (proencamodena et al. 2012) . the likelihood of a pathogen to be the aetiologic agent of a given infection could be related to the c t value in the real-time pcr reaction. lower c t values suggest higher loads of the pathogen detected, which may in turn suggest aetiology (brittain-long et al. 2008) . in this study, the c t values were well below the cut-off value for positivity proposed by the manufacturer. the overall bacterial detection rate was 40% and s. pneumoniae was the most frequent agent identified. despite the vaccination program, s. pneumoniae is one of the main causes of pneumonia mortality (responsible for at least 18% of severe episodes and 33% of deaths worldwide) (walker et al. 2013) . bacteria were co-detected with viruses in 25 specimens. the potentially pathogenic bacteria detected in this study could reflect transient microbiota or a nasopharyngeal flora and may not be associated with aris; however, it is well known that viral infections within the respiratory tract predispose the individual to bacterial infections, notably through the disruption of the respiratory mucosal epithelium (bakaletz 1995) . atypical bacteria (m. pneumoniae and c. pneumonia) were not detected in the studied population. in brazil and other countries, the respiratory virus surveillance programs use conventional diagnostic techniques, such as iif. a large proportion of biological samples remain negative, even with clinical evidence of respiratory infection. a molecular technique such as real-time pcr can be an important tool, increasing the detection capacity of a large number of respiratory pathogens. moreover, this type of technique can contribute to the correct indication of antiviral medications, can avoid unnecessary use of antibiotics and can promote the adoption of appropriate hospital-control measures. the findings of this study show that the application of pcr assays in a real-time multiplex format for respiratory pathogens considerably increases the pathogen detection rate when compared to conventional methods, highlighting the role of influenza virus in ari cases in patients of all ages, in addition to reporting the circulation of recently described respiratory viruses (inf a h1n1 pdm09, hmpv, hbov, hcov-nl63 and hcov-hku1). cloning of a human parvovirus by molecular screening of respiratory tract samples respiratory tract viral infections acute respiratory viral infections in ambulatory children of urban northeast brazil viral potentiation of bacterial superinfection of the respiratory tract clinical and financial benefits of rapid detection of respiratory viruses: an outcomes study acute respiratory infection and influenza-like illness viral etiologies in brazilian adults viral and atypical bacterial detection in acute respiratory infection in children under five years multiplex real-time pcr for detection of respiratory tract infections prospective evaluation of a novel multiplex real-time pcr assay for detection of fifteen respiratory pathogens -duration of symptoms significantly affects detection rate who estimates of the causes of death in children treatment of naturally acquired common colds with zinc: a structures review severe lower respiratory tract infection in infants and toddlers from a non-affluent population: viral etiology and co-detection as risk factors comparison of three multiplex pcr assays for the detection of respiratory viral infections: evaluation of xtag respiratory virus panel fast assay, respifinder 19 assay and respifinder smart 22 assay respiratory syncytial virus infection: immune response, immunopathogenesis and treatment real-time rt-pcr detection of 12 respiratory viral infections in four triplex reactions serial viral infections in infants with recurrent respiratory illnesses a novel multiplex realtime rt-pcr assay with fret hybridization probes for the detection and quantitation of 13 respiratory viruses the complication of coinfection human bocavirus infections in hospitalized children and adults human bocavirus -insights into a newly identified respiratory virus development of a respiratory virus panel test for detection of twenty human respiratory viruses by use of multiplex pcr and a fluid microbead-based assay detection of respiratory viruses by molecular methods informe epidemiológico: influenza pandêmica (h1n1) new respiratory viruses of humans hospitalization of influenza-like illness patients recommended by general practitioners in france between rhinoviruses: important respiratory pathogens high rates of detection of respiratory viruses in tonsillar tissues from children with chronic adenotonsillar disease laboratory diagnosis, epidemiology and clinical outcomes of pandemic influenza a and community respiratory viral infections in southern brazil comparison of fast-track diagnostics respiratory pathogens multiplex real-time rt-pcr assay with in-house singleplex assay for comprehensive detection of human respiratory viruses clinical evaluation of multiplex real-time pcr panels for rapid detection of respiratory viral infections osterhaus ad 2001. a newly discovered human pneumovirus isolated from young children with respiratory tract disease identification of a new human coronavirus global burden of childhood pneumonia and diarrhoea characterization and complete genome sequence of a novel coronavirus, coronavirus hku1, from patients with pneumonia surveillance and genome analysis of human bocavirus in patients with respiratory infection in guangzhou key: cord-261756-4lybl57r authors: dubert, marie; visseaux, benoit; birgy, andré; mordant, pierre; metivier, anne-cécile; dauriat, gaelle; fidouh, nadhira; yazdanpanah, yazdan; grall, nathalie; castier, yves; mal, hervé; thabut, gabriel; lescure, françois-xavier title: late viral or bacterial respiratory infections in lung transplanted patients: impact on respiratory function date: 2020-02-24 journal: bmc infect dis doi: 10.1186/s12879-020-4877-3 sha: doc_id: 261756 cord_uid: 4lybl57r background: respiratory infections are a major threat for lung recipients. we aimed to compare with a monocentric study the impact of late viral and bacterial respiratory infections on the graft function. methods: patients, who survived 6 months or more following lung transplantation that took place between 2009 and 2014, were classified into three groups: a viral infection group (vig) (without any respiratory bacteria), a bacterial infection group (big) (with or without any respiratory viruses), and a control group (cg) (no documented infection). chronic lung allograft dysfunction (clad) and acute rejection were analysed 6 months after the inclusion in the study. results: among 99 included lung recipients, 57 (58%) had at least one positive virological respiratory sample during the study period. patients were classified as follows: 38 in the vig, 25 in the big (among which 19 co-infections with a virus) and 36 in the cg. the big presented a higher initial deterioration in lung function (p = 0.05) than the vig. but 6 months after the infection, only the vig presented a median decrease of forced expiratory volume in 1 s; − 35 ml (iqr; − 340; + 80) in the vig, + 140 ml (+ 60;+ 330) in the big and + 10 (− 84;+ 160) in the cg, p < 0.01. acute rejection was more frequent in the vig (n = 12 (32%)), than the big (n = 6 (24%)) and cg (n = 3 (8%)), p < 0.05, despite presenting no more clad (p = 0.21). conclusions: despite a less severe initial presentation, single viral respiratory infections seem to lead to a greater deterioration in lung function, and to more acute rejection, than bacterial infections. thanks to a better selection of recipients and donors, to an improvement in surgical / anaesthetic procedures and to better management of immunosuppressive therapies, early post-operative survival of lung transplant recipients (ltrs) has improved since the advent of lung transplantation. whereas most early deaths are related to primary graft dysfunction or acute rejection (ar), the long-term prognosis is threatened by chronic lung allograft dysfunction (clad), usually in the form of a bronchiolitis obliterans syndrome (bos), that affects 45 to 75% of lung recipients within 5 years after transplantation [1] [2] [3] [4] . clad represents the leading cause of death 1 year after lung transplantation [3, 5, 6] . infectious respiratory complications are also a major cause of morbidity and mortality for ltrs and are responsible for a third of deaths occurring in the first year post transplant, and half of all deaths during longterm follow-up [7, 8] . the severity of these infections results from several factors including induced immunosuppression, direct exposure of the graft to microorganisms, and finally less effective mucociliary function and lymphatic drainage and cough reflex following denervation of the graft [9] [10] [11] . moreover, stenosis and ischemic processes occurring at the surgical anastomosis decrease the clearance of secretions, and promote their colonization and invasion by microorganisms [12] . bacterial infections are the leading cause of respiratory infections in ltrs [7, 11, 13] . their association with the occurrence of clad is well established [2, 14] . many authors admit that viral respiratory tract infections (vrti) may be associated with clad [3, [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] , but this remains controversial, depending on the definition of respiratory infection, the virus panel studied, the time limit between vrti and spirometric analysis, and the consideration of intercurrent events possibly influencing the respiratory function [25] . similarly, the association between vrti and ar continues to be debated in the literature. while some studies have identified an association between these two events [17, 26, 27] some others, including a recent meta-analysis, have not found any link between them [21, 25, 28] . however, to our knowledge, the impact of viralbacterial co-infections on graft survival has not been specifically studied, and was not compared to single vrti or patients without any respiratory infections. the development of rapid antigenic tests and molecular biology techniques has facilitated the detection and diagnosis of several viruses. the new multiplex pcr methods (polymerase chain reaction) are fast, sensitive andable to detect an enlarged number of viruses not easily detected before (e.g. metapneumovirus, coronavirus nl63 and hku1, bocavirus, rhinovirus c) [29, 30] . thus, we hypothesis that the impact of the viral infection is at least as severe as bacterial respiratory infections on lung graft function among ltx. the objectives of our study were to assess, in a cohort study, the occurrence of late viral and bacterial respiratory infections in ltrs and to compare their respective impact on graft function with those without any respiratory infections. we retrospectively screened all individuals who underwent lung transplantation between september 2009 and september 2014 at the bichat-claude bernard teaching hospital, paris, france. in this cohort we evaluated the occurrence of late viral and bacterial respiratory infections and graft function overtime. exclusion criteria were: (1) death during the first 6 months after transplantation, (2) no available pulmonary function assessment at the time of documented infection or 6 months after, and (3) herpetic or cytomegalovirus pneumonia. in order to include patients at a steady state, we studied only the late respiratory infections, i.e. to censure the first 6 months after the lung transplantation, a period during which postoperative complications are frequent and usually intertwined. these patients were regularly followed up. at each routine follow-up visits with patients, symptoms were recorded. spirometric measurements, blood tests, radiological explorations, bronchoscopic procedures with bronchoalveolar fluid and transbronchial biopsies were systematically performed and tested for both bacterial and viral pathogens according to local guidelines. all respiratory samples, bronchial aspirates, bronchoalveolar fluids and nasopharyngeal samples were reviewed. several [31] [32] [33] , as confirmed by our internal method validations and the similar viral diversity observed over time [34] . their reliability was also assessed throughout the study period by regular qcmd controls (glasgow, uk). a sample was defined as bacteriologically positive if the bacteria were present at 10 7 cfu/ml or more for sputum, 10 6 cfu/ml or more for bronchial aspirates and 10 4 cfu/ml or more for bronchoalveolar fluid. according to virological and bacteriological results from respiratory samples, patients were divided into three groups, as follows: -patients presenting a viral respiratory infection: viral infection group (vig), i.e. -patients with at least one positive virological respiratory sampling, symptomatic or not. nasopharyngeal swabs that tested positive only for rhinovirus were not considered to be positive for a significant virus, and were ignored. -patients without any respiratory infection: control group (cg), i.e. patients with no virological or bacteriological positive respiratory samples during the study, or those with nasopharyngeal swabs positive for rhinovirus only; or with a positive bacteriological respiratory sample not followed by antibiotic treatment (i.e. considered as a simple bacterial carriage). the date of inclusion was defined as the date of the respiratory sample of interest for the two infected groups (vig and big), and the date of the first outpatient consultation for the cg. the primary endpoint was the occurrence of bos 6 months after inclusion. secondary endpoints were the occurrence of ar and the quantitative change of forced expiratory volume in 1 s (fev-1) at 6 months after inclusion, as well as death during the whole study period. the following definitions were used to assess the outcomes: biopsy that demonstrated at least a grade a1 of acute rejection as defined by the ishlt for cellular rejection. in patients in whom a biopsy could not be performed, acute rejection was defined by deterioration in lung function with no other identifiable aetiology and that positively responded to a high-dose corticosteroid therapy. 4. fev-1 delta was defined as 6-month-fev-1last fev-1 before inclusion. quantitative variables were presented using the median (quartile 25-75) and were compared by t-test or variance analysis. qualitative variables were compared using a chi2 test or a fisher-exact test. outcomes were compared using a logistic regression multivariable model, with adjustment for time since transplantation, age and sex. a sensitivity analysis was carried out excluding patients who had had a bos before inclusion. all of the statistical analysis was done using r software, version (3.1.1). this study was approved by the cepro (comité d'evaluation des protocoles de recherche observationnelle) ethical committee, number cepro 2016-027. between september 2009 and september 2014, 154 patients received lung transplants at bichat hospital. fiftyfive patients were excluded from the analysis: 47 (31%) because of death within 6 months following transplantation, six (4%) due to a lack of spirometry assessment at inclusion or 6 months afterwards, and two because of pathological, proven herpetic pneumonia. among the 99 remaining ltrs, 57 (57%) had at least one positive virological respiratory sample. thus, patients were divided into three groups according to the criteria described above: 38 (38%) in the vig, 25 (25%) in the big (6 patients mono-infected by bacteria and 19 co-infected patients) and 36 (36%) in the cg. the corresponding flow chart is presented in fig. 1 . nineteen 4 % of the respiratory samples were broncho-alveolar lavages or bronchial aspirations. general characteristics, type of immunosuppression and prior viral infections were similar across the three groups with the exception of renal dysfunction (egfr < 60 ml/min/1.73m 3 ), more prevalent in the vig (table 1) . previous ar requiring treatment in the 3 months prior to inclusion occurred for 9 (24%) patients in the vig, 3 (12%) in the big, and none of the cg (p < 0.01). among 13 patients presenting a bos at inclusion, 9 were classified in vig, compared to 3 in the big, and 1 in the cg (p = 0.02). clinical presentation (table 2) patients were included at a median time of 303 (iqr, 213-560) days after transplantation. both infected groups displayed similar respiratory symptoms. likewise, the biological presentations of both infected groups were close, and inflammatory surrogates (leucocytes, creactive protein, platelets) did not differ statistically. big patients were more prone to be hospitalized (64 vs. 34%, p = 0.04), and to present a more severe decrease of fev-1 at inclusion (− 260 ml (− 410-0) vs. -50 ml (− 170-60)), compared to the vig. among patients examined with a thoracic tomodensitometry, seven patients (11%) presented a new infiltrate. picornavirus was the most frequently detected virus in the vig (n = 21), followed by parainfluenzae virus (n = 13), and syncytial respiratory virus (n = 10) ( table 3) . pseudomonas aeruginosa was the main detected bacteria; found in 11 (52%) patients in the big. in the cg, the neglected bacteria were pseudomonas aeruginosa (n = 4), corynebacteriae striatum (n = 2) and others (n = 2). three patients had more than one detected virus and eight patients had more than one detected bacteria. the overall rate of worsening 6-month-bos was 13% with no significant differences between the three groups (table 4 ). sixteen patients (16%) died during the study period (6 bos, 2 strokes, 2 neoplasia, 2 severe infections, 4 from unknown cause), with no significant differences between the three groups ( in the two infected groups, these outcomes were not different between the symptomatic patients (n = 41) and the asymptomatic patients (n = 15). thirteen patients had a prior bos at inclusion: 1 (2.8%) in the cg, 9 (23.7%) in the vig and 3 (12.0%) in the big. there was a trend toward more viral respiratory investigation among patients with a bos at inclusion (median of 14.0 (9.0-21.0) samples/patients) than for patients without bos at inclusion (8.0 (4.0-15.0), p = 0.06). however, the sensitivity analysis performed on the remaining 86 patients without bos at inclusion found similar results in terms of significance and association effect with the development of a new bos or with ar (table s1 ). in the same way, infected patients were significantly more investigated than those in the cg: this study shows that, after the first 6 months following transplantation, more than half of ltrs were affected by vrti. clinical presentations for late viral and/or bacterial infections at baseline were very similar, albeit with additional signs of severity for the bacterial infections. single late vrti strongly impacted the patients' prognosis by leading to an increased risk of ar, a trend to an increased risk of bos (without significant association), and a more severe secondary decline in respiratory function compared to the late bacterial respiratory infection. the consequences of these different infections were similar whether or not the infection was symptomatic at the time of viral or bacterial detection. in this study, 57 patients (57%) exhibited at least one positive viral respiratory sampling during follow-up. this rate varies among studies as do screening techniques and reasons for withdrawal. while studies using cell cultures in their screening method report virus detection rates in respiratory samples at around 8% [2, 15] , the use of molecular biology tests significantly increases this prevalence from 17 to 52% [17, 28, 35] . we decided to exclude infections with cmv, especially because cmv diseases respond to different triggers than those infections, were prevented by different protocol evolutions during the study period, and can induce the death or graft rejection in both of our patient groups. with regard to microbiological aetiology, we confirmed that the most frequent viruses detected with the pcr test were those corresponding to the picornavirus group, followed by parainfluenzae viruses and coronaviruses, as already described [5, 6, 18, 23, 24, 27] . it is worthy of note that, in the big, picornavirus were the most frequently detected viruses in co-infections (52%). picornavirus are often considered as a contaminant with a controversial clinical impact. indeed, a recent prospective study demonstrated that rhinoviruses were frequent in ltrs, even in those patients who were asymptomatic [35] . influenza were rarely identified among lung graft patients, especially when compared to the non-lung graft patients in our hospital using the same mpcr assays (5 vs 27%) [34] . this is explained by the high vaccination rates and specific prevention measures compliance among lung graft patients and their relatives. among bacterial infections, pseudomonas aeruginosa and corynebacteriae striatum were more often detected. both bacteria are known to be responsible for serious infections in ltrs. as already shown, the symptomatic feature of the initial infection did not impact on graft survival [15, 17, 25] , suggesting that, for these patients, the symptomatic nature of the infection should not be taken into account. concerning ar, the impact of late respiratory viral and/ or bacterial infections on the graft function was significantly different with three times more ar within 6 months for both the vig and big compared to the cg. while some studies supported this association [16, 36] , other studies, including a meta-analysis, did not find any significant link [17, 25, 28, 30] . this difference could be explained by the variety of criteria used to define ar. we chose to identify ar when the histological pattern showed a stage of at least a1. indeed, previous studies demonstrated that minimal rejection (≥a1) was associated with an increased risk for bos development and progression that was comparable to a2 rejection [37] . on top on that, we noticed a significantly longer delay in ar in the vig than in the big, suggesting that the impact of viral infection on lung graft function must be screen even after several weeks. especially in asymptomatic ltrs and the lack of specific management, the morbidity of viral infection could be attributed to a trivialization of viral colonization, leading to a neglected and chronic cause of inflammation and, thus, to potential rejection. therefore, it seems important to assess the impact of respiratory viral infections on the graft function: to emphasize the prevention of viral infections for immunocompromised with more frequent sampling of patients including wide respiratory virus detection by molecular techniques and to strengthen spirometric controls after viral infections. the all-cause mortality was evaluated to 31% at 6 months in this study. this rate in consistent with other studies [8, 15] . it is explained by the high rate of comorbidity among our patients (more than half with arterial hypertension and diabetes mellitus), the deep immunosuppression required and the numerous complications of lung grafting. to our knowledge, this study is the first to allow a direct comparison of the impact of late viral and bacterial respiratory infections in ltrs. we were able to analyze all the spirometry data both at inclusion and 6 months after, and to present results of an extensive panel of viral pcr tests. this study has several limitations. firstly, the small number of patients could restrict the power of the conclusions, especially for patients classified only on nasopharyngeal swabs (n = 4). aors present wide ranges and must be interpreted accordingly. however, this study remains one of the largest available cohort on the topic to date. despite this small sample size we were able to illustrate that late vrti strongly impacted the patients' prognosis by leading to an increased risk of ar. in addition, although not significant there was a trend to higher risk of occurrence of other outcomes such as bos and death with vrti. we believe that these findings are important and should lead to the design of larger studies. because of the small number of patients among the group with bacterial infections and the group with bacterial and viral infections, we decided to merge these two groups and this could be also debated. however, this was done first because of the similar presentation of patients in these groups and second on the basis of the hypothesis that: (i) bacterial infections were responsible of the major acute part of the lung graft malfunction, and (ii) these groups were both subject to an intervention with the use of antibiotics. secondly, the retrospective design leads to several biases: an indication bias leading to higher infection detection in patients presenting ar 3 months prior to inclusion or a bos prior to infection because of more frequent followup visits and a trend to more respiratory sampling streptocoque, n (%) 1 0 (0) 1 (4) other bacteria, n (%) 2 2 (5) 0 (0) abbreviations: big bacterial infections group; vig viral infections group a among 25 patients with bacterial infections, 7 had no bacteria identification, but samples were examined after antibiotic therapy (n = 3) and/or presented a localized chest x-ray condensation (n = 3) and/or patients had purulent sputum (n = 2) for these patients. although treatment for ar 3 months prior to inclusion could be considered as a biais, this rate was not significantly different between the infected groups. because of the small number of patients, we could not perform a sensitivity analysis among these patients who had ar 3 months prior to inclusion. nevertheless, we performed a sensitivity analysis to address the hypothesis of indication bias. we repeated the multivariable model without considering patients with a bos at inclusion and found similar results, suggesting that this bias, if it existed, did not alter our conclusions. we also assume a survival bias that may explain why despite higher lung function deterioration at 6 months after inclusion in the viral infections group, the rate of bos was not significantly different. the relatively short 6 months follow-up period used in our study may not be sufficient to identify a potential difference in the progression of bos between groups. indeed, the incidence of bos development 6 months after the first infection episode was at 13% in our study, while previous studies described an incidence rate of 50% over a five-year period [2] . to conclude, viral respiratory infections and virusbacteria co-infections were frequent in lung graft recipients and led to similar clinical and biological presentations. bacterial infections strongly diminished initial lung function and led to more hospitalizations whereas single viral respiratory infections seem to lead to a greater deterioration in lung function, and to more acute rejection, than bacterial infections. supplementary information accompanies this paper at https://doi.org/10. 1186/s12879-020-4877-3. additional file 1: table s1 . multivariate analysis of association with development of bos. without patients with bos at inclusion service de maladies infectieuses et tropicales, 46 rue henri huchard, f-75018 greffe cardio-pulmonaire et pulmonaire effect of etiology and timing of respiratory tract infections on development of bronchiolitis obliterans syndrome an international ishlt/ats/ers clinical practice guideline: diagnosis and management of bronchiolitis obliterans syndrome lung transplantation the registry of the international society for heart and lung transplantation: twenty-sixth official adult lung and heart-lung transplantation report-2009 post-transplant bronchiolitis obliterans bronchiolitis obliterans syndrome development in lung transplantation patients the registry of the international society for heart and lung transplantation: twenty-eighth adult lung and heart-lung transplant report--2011 lung transplant infection pulmonary infection defense after lung transplantation: does airway ischemia play a role? epidemiology and management of infections after lung transplantation anastomotic airway complications after lung transplantation infection in lung transplantation pseudomonas aeruginosa colonization of the allograft after lung transplantation and the risk of bronchiolitis obliterans syndrome respiratory viral infections are a distinct risk for bronchiolitis obliterans syndrome and death clinical impact of community-acquired respiratory viruses on bronchiolitis obliterans after lung transplant a prospective molecular surveillance study evaluating the clinical impact of community-acquired respiratory viruses in lung transplant recipients respiratory viruses and chronic rejection in lung transplant recipients the pathogenesis and management of influenza virus infection in organ transplant recipients incidence and morbidity of human metapneumovirus and other community-acquired respiratory viruses in lung transplant recipients rhinovirus and other respiratory viruses exert different effects on lung allograft function that are not mediated through acute rejection respiratory virus infections and chronic lung allograft dysfunction: assessment of virology determinants symptomatic respiratory virus infection and chronic lung allograft dysfunction viral respiratory tract infection during the first postoperative year is a risk factor for chronic rejection after lung transplantation respiratory viruses in lung transplant recipients: a critical review and pooled analysis of clinical studies the epidemiology of parainfluenza virus infection in lung transplant recipients human metapneumovirus infection in lung transplant recipients: clinical presentation and epidemiology upper and lower respiratory tract viral infections and acute graft rejection in lung transplant recipients development of a respiratory virus panel test for detection of twenty human respiratory viruses by use of multiplex pcr and a fluid microbeadbased assay incidence and outcomes of respiratory viral infections in lung transplant recipients: a prospective study comparison of the luminex xtag rvp fast assay and the idaho technology filmarray rp assay for detection of respiratory viruses in pediatric patients at a cancer hospital comparison of anyplex ii rv16 with the xtag respiratory viral panel and seeplex rv15 for detection of respiratory viruses comparison of three multiplex pcr assays for the detection of respiratory viral infections: evaluation of xtag respiratory virus panel fast assay, respifinder 19 assay and respifinder smart 22 assay prevalence of respiratory viruses among adults, by season, age, respiratory tract region and type of medical unit role of rhinovirus load in the upper respiratory tract and severity of symptoms in lung transplant recipients respiratory metapneumoviral infection without co-infection in association with acute and chronic lung allograft dysfunction association of minimal rejection in lung transplant recipients with obliterative bronchiolitis springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations not applicable. authors' contributions md: designed the research, monitored the database, analyzed and interpreted the patient data, contributed to the writting of the manuscript. bv: performed the virological study, interpreted the patient data and contributed to the writting of the manuscript. ab: monitored the database, analyzed and verified the patient data, contributed to the writting of the manuscript. pm: took care of the lung transplants reciepients, contributed to the writting of the manuscript. acm: took care of the lung transplants reciepients, contributed to the writting of the manuscript. gd: took care of the lung transplants reciepients, contributed to the writting of the manuscript. nf: performed the virological study, interpreted the patient data and contributed to the writting of the manuscript. yy: designed the research, monitored the database, interpreted the patient data, wrotte the manuscript. ng: performed the bacteriological study, interpreted the patient data and contributed to the writting of the manuscript. yc: took care of the lung transplants reciepients, contributed to the writting of the manuscript. hv: took care of the lung transplants reciepients, contributed to the writting of the manuscript. gt: took care of the lung transplants reciepients, contributed to the writting of the manuscript. fxl: designed the research, analyzed and interpreted the patient data, contributed to the writting of the manuscript. the author(s) read and approved the final manuscript. no funding was obtained for this study. the datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. data used in this study was anonymised before its use. the authors declare that they have no competing interests. key: cord-264255-q5izs39f authors: chieochansin, thaweesak; samransamruajkit, rujipat; chutinimitkul, salin; payungporn, sunchai; hiranras, thitikul; theamboonlers, apiradee; poovorawan, yong title: human bocavirus (hbov) in thailand: clinical manifestations in a hospitalized pediatric patient and molecular virus characterization date: 2007-12-31 journal: j infect doi: 10.1016/j.jinf.2007.11.006 sha: doc_id: 264255 cord_uid: q5izs39f objective: human bocavirus (hbov), a novel virus, which based on molecular analysis has been associated with respiratory tract diseases in infants and children have recently been studied worldwide. to determine prevalence, clinical features and perform phylogenetic analysis in hbov infected thai pediatric patients. methods: hbov was detected from 302 nasopharyngeal (np) suctions of pediatric patients with acute lower respiratory tract illness and sequenced applying molecular techniques. results: the incidence of hbov infection in pediatric patients amounted to 6.62% with 40% co-infected with other respiratory viruses. there were no clinical specific manifestations for hbov; however, fever and productive cough were commonly found. generalized rales and wheezing were detected in most of the patients as well as perihilar infiltrates. the alignment and phylogenetic analysis of partial vp1 genes showed minor variations. conclusion: our results indicated that hbov can be detected in nasopharyngeal aspirate specimens from infants and children with acute lower respiratory tract illness. acute respiratory tract infection is a major cause of pediatric morbidity and mortality worldwide. in most cases, viruses including influenza a and b, parainfluenza viruses, adenoviruses, respiratory syncytial virus (rsv), and human metapneumovirus (hmpv) are the causative agents. recently, a new respiratory tract virus of the parvoviridae family, human bocavirus (hbov), has been discovered applying molecular analysis on pooled respiratory tract aspirations taken from children in sweden. this virus is closely related to the bovine parvovirus and canine minute virus, which have been classified as members of the genus bocavirus. the virus comprises two major open reading frames (orfs) encoding a nonstructural protein (ns1) and at least two capsid proteins (vp1 and vp2), respectively. moreover, the hbov genome also contains a third middle orf encoding a nonstructural protein (np1) of unknown function. 1 the most conserved region of this virus is the ns1 and np1 gene whereas the vp1/vp2 gene constitutes the variable region. 2 the induction of respiratory illness by hbov is not clearly defined due to lack of propagation techniques in cell culture or animal models. 1 however, many studies have reported this virus infection to be associated with acute respiratory illness. 1, 3, 4 upon discovery of hbov in respiratory pools, its global prevalence has been reported to range from 1.5% to 19% and co-infection with other viruses was commonly found. 1,3e24 moreover, in few studies were shown negative results for hbov infection in nasopharyngeal (np) swabs from healthy volunteers. 20, 25, 26 additional epidemiological and clinical investigation will be essential in order to elucidate what exactly engenders hbov related illness. therefore, in the present study we applied polymerase chain reaction to detect hbov from np suctions collected from infants or children who had been admitted with respiratory tract illness. nasopharyngeal suction specimens were collected from 302 individual infants or children (age range: 5 days to 14 years) who were admitted and diagnosed as having acute lower respiratory illness during the period february 14, 2006 to february 28, 2007 . all of the clinical samples were provided by the department of pediatrics, king chulalongkorn memorial hospital, thailand. nasopharyngeal suction samples were collected in transport medium with antibiotics (0.5% bsa, penicillin g (2 â 10 6 u/l), streptomycin 200 mg/l) and stored at à70 c until tested. the study was conducted after receiving approval by the ethics committee of the faculty of medicines, chulalongkorn university. prior to enrolment, all participating parent gave their written informed consent. dna and rna were extracted from 150 ml of np suction using tri reagent â ls (molecular research center inc., cincinnati, oh) and solubilized in 20 ml of 8 mm naoh or 12 ml of depc treated water for dna or rna, respectively. for hbov detection we amplified the np1 gene by conventional pcr modified from a previous study. 1 the reaction mixture contained 2 ml dna, 0.5 mm 188f primer and 0.5 mm 542r primer, 10 ml 2.5â eppendorf mastermix (eppendorf, hamburg, germany), and nuclease-free water to a final volume of 25 ml. the amplification reaction was performed in a thermocycler (eppendorf) under the following conditions: initial denaturation at 94 c for 3 min, followed by 35 amplification cycles consisting of 94 c for 30 s (denaturation), 55 c for 30 s (primer annealing), and 72 c for 1 min (extension), and concluded by a final extension step at 72 c for 7 min. another set of primers specific for the vp1 gene, vpf2 forward primer (5 0 -ttcagaatggt cacctctaca-3 0 : nt 3639e3659) and vpr2 reverse primer (5 0 -ctgtgcttccgttttgtctta-3 0 : nt 4286e4266), were used in a separate pcr reaction to exclude false positives. after 2% agarose gel electrophoresis stained with ethidium bromide, the expected products of 354 and 648 bp representing the np1 and vp1 gene, respectively, were visualized on a uv transilluminator. influenza a virus detection was performed by conventional pcr using 1 ml of cdna, 0.5 mm of flua_m_f: 5 0 -rggccccctcaaagccga-3 0 (nt 76e93), 0.5 mm of flua_m_r: 5 0 -actgggcacggtgagygt-3 0 (nt 235e218), 10 ml of 2.5â eppendorf mastermix, and nuclease-free water to a final volume of 25 ml. the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (gapdh) of each sample was amplified in one additional reaction mixture of identical volume with primers gapdh_f: 5 0 -gtg aaggtcggagtcaacgg-3 0 (nt 112e131) and gapdh_r: 5 0 -gttgtcatggatgaccttggc-3 0 (nt 603e583) at a 0.5 mm concentration, each. the amplification reaction was performed in a thermocycler (eppendorf) under the following conditions: initial denaturation at 94 c for 3 min, followed by 40 amplification cycles consisting of 94 c for 30 s (denaturation), 55 c for 30 s (primer annealing), and 72 c for 1 min (extension), and concluded by a final extension step at 72 c for 7 min. after 2% agarose gel electrophoresis, the expected products were influenza a virus (160 bp) and the housekeeping gene (492 bp). parainfluenza virus was detected by real-time pcr using sybr green i carried out in a rotor-gene 3000 (corbett research, new south wales, australia). the reaction mixture contained 1 ml of dna sample, 10 ml 2.5â eppendorf mastermix, 0.5 mm paraf: 5 0 -gctaaatactgtcttmah tggagat-3 0 (nt 11,254e11,278), 0.5 mm parar: 5 0 -gtaagg atcaccwacatadawtgta-3 0 (nt 11,392e11,370), 2 ml 1â sybr green and nuclease-free water to a final volume of 20 ml. the amplification reaction consisted of a preincubation step at 95 c for 3 min followed by 35 cycles of amplification including 95 c for 15 s, 55 c for 15 s and 72 c for 30 s. the fluorescent signal was detected once per cycle upon completion of the extension step. after amplification, melting curve analysis was performed by heating to 95 c then cooling to 60 c for 15 s, followed by a temperature increase to 95 c, while continuously collecting the fluorescent signal data. adenovirus, influenza b virus, respiratory syncytial virus (rsv) and human metapneumovirus (hmpv) were detected by the method described by krafft et al., 27 chi et al., 28 samransamruajkit et al. 29 and thanasugarn et al., 30 respectively. all hbov positive samples were subjected to vp1 gene sequencing. the partial vp1 gene at the 5 0 end of hbov was amplified into two segments with two primer sets, vpf1 (5 0 -gataactgacgaggaaatgct-3 0 : nt 3009e3029) and vpr1 (5 0 -agtatgtccatggagttgtga-3 0 : nt 3731e3711) for the first segment and vpf2 and vpr2 for the second. the expected product sizes after 2% agarose electrophoresis were 723 and 648 bp, respectively. pcr conditions have been described elsewhere. 2 the pcr products were purified using the perfectprep gel cleanup kit (eppendorf). dna sequencing was performed using the gene amp pcr system 9600 (perkineelmer, boston, ma). the sequencing products were subjected to a perkin elmer 310 sequencer (perkine elmer) for subsequent sequence analysis. the dna sequences were analyzed with the blast (http://www. ncbi.nlm.gov/blast) program and the phylogenetic analyses and genetic comparisons between hbov strains were performed using the molecular evolutionary genetics analysis (mega) version 3.1 program. we applied pcr and rtepcr to detect hbov, other respiratory viruses and gapdh. of 302 specimens, 20 (6.62%) were positive for hbov. all specimens had been collected throughout the year and plotting of seasonal hbov detection was shown in fig. 1 . among the 302 specimens we also detected co-infection with other respiratory viruses such as rsv in 48 (15.89%) samples, influenza a virus in 33 (10.92%) samples, hmpv in 28 (9.27%) samples, adenovirus in 18 (5.9%) samples, parainfluenza in 14 (4.63%) samples, and influenza b virus in 1 (0.33%) sample. hbov-positive samples co-infected with other respiratory viruses comprised altogether 40% (table 1) . the clinical manifestations of hbov infected patients are summarized in table 1 . the majority of infants positive for hbov were male (14/20) , between 4 and 36 months old (median age 12 months). on average, they were hospitalized for 6.15 days (range 2e22 days). the most common clinical symptom in all hbov infected infants was fever with a mean duration of 3.47 days and other common symptoms such as runny nose (55%) and productive cough (50%) were found. generalized rales were the most common (70%) and wheezing the second most common lung signs (35%). acute bronchiolitis was diagnosed in 9 out of 20, and 11 samples had viral pneumonia. perihilar infiltration was ubiquitously present on the chest x-ray of hbov positive patients (except for cu6 and cu15 where no chest x-ray result was available) (data not shown). additional diseases of some patients with hbov infection included congenital heart disease (cu33 and cu74), chronic lung disease (cu 31 and cu171), asthma (cu71) and cow milk protein sensitive enteropathy (cu157). the nucleotide sequences obtained from this study have been submitted to the genbank database under accession numbers: ef690641eef690657 (table 1 ) except for cu6 (ef203920), cu49 (ef203921) and cu74 (ef203922) that were subjected to analysis for complete coding sequences as described elsewhere. 2 the 5 0 terminal 1182 base pairs (bp) of the vp1 gene were sequenced in all hbov positive samples for phylogenetic analysis. other vp1 sequences from several areas were available at the genbank database and used for phylogenetic analysis including sequences from china: dq778300, ef441262, ef584447, dq457413, dq987595, dq987596, dq677523, dq494203, dq457415, dq988933, dq988934, dq987597, dq987598 and dq494201, japan: ef035488, usa: dq340570, and sweden: dq000495 and nc_007455. the constructed phylogenetic trees of the vp gene (nt 2986e4167) are shown in fig. 2 . alignment of these partial vp1 genes showed minor variations in that the percent identity with the st1 swedish prototype strain ranged from 98.6% to 99.5%. a phylogenetic tree of the hbov vp gene was constructed based on neighbor-joining (nj) trees. confidence values for the tree topologies were evaluated by bootstrap analysis of 1000 pseudo-replicate datasets that showed the low genetic diversity. in the past few years, applying molecular techniques has led to the discovery of hbov as a novel virus apparently associated with respiratory tract illness in humans. 1 however, due to lack of suitable culture systems and animal models to propagate the virus previous studies could not meet with koch's postulates in order to clarify that hbov can cause respiratory illness. it can be concluded from epidemiology data that hbov has been distributed throughout every continent with a different incidence rate (1.5% to 19%). in this study, the np suctions had been collected from pediatric patients hospitalized with acute lower respiratory tract illness. hbov has been detected in 20 (6.62%) out of 302 np suction samples, whereas the previous study in the rural area of thailand had the prevalence of 4.5%. 18 co-infection with other respiratory viruses was found in 40% of the samples tested. rsv and parainfluenza virus were frequently detected as co-infecting viruses. however, some of the respiratory viruses were not included in this study, for examples; rhinoviruses and coronaviruses. therefore, the percentage of co-infection may be higher than this report. thailand has a tropical climate country and seasonal detection of hbov can not be deduced from the few (1e3) hbov-positive samples found in each month. rsv and hmpv were shown the seasonal distribution that peaked in july to september which correlated to the number of samples collection (data not shown). moreover, the study of mannig et al. reported a similarity in seasonal appearance between hbov and rsv 12 whereas weissbrich et al. did not. 16 the clinical features commonly found in hbov positive patients were fever and productive cough. bilateral rales and wheezing were among the most common abnormal lung signs observed and almost equally found in these patients (table 1) . these findings might indicate both lung parenchyma and airway involvement by this pathogen. furthermore, the detection of this virus in specimens aspirated from the nasopharynx together with the significant lower respiratory tract illness indicated the lung pathology in these patients. in conclusion, we detected hbov infection in 6.62% and co-infection with other respiratory viruses in 40% of the np suctions obtained from infants and children with acute lower respiratory tract illness with non-specific clinical features and age distribution. seasonal appearance of hbov was not significant. based on phylogenetic analysis of the vp1 gene, the low genetic diversity was defined. the present study has investigated associations of hbov with clinical manifestations and performed genome analysis but in order to completely describe this novel virus, further studies on serology, tissue and animal culture systems or clinical manifestation inductions will be required. cloning of a human parvovirus by molecular screening of respiratory tract samples complete coding sequence and phylogenetic analysis of human bocavirus (hbov) human bocavirus: prevalence and clinical spectrum at a children's hospital detection of human bocavirus in cannadian children in a 1 year study frequent detection of human rhinoviruses, paramyxoviruses, coronaviruses, and bocavirus during acute respiratory tract infection human bocavirus infection the association of newly identified respiratory viruses with lower respiratory tract infections in korean children bocavirus infection in hospitalized children human bocavirus in french children human bocavirus infection among children detection of human bocavirus in japanese children with lower respiratory tract infection epidermiology profile and clinical associations of human bocavirus and other human parvovirus real-time pcr assays for detection of bocavirus in human specimens evidence of human coronavirus hku1 and human bocavirus in australian children human bocavirus in hospitalized children frequent detection of bocavirus dna in german children with respiratory tract infections human bocavirus and acute wheezing in children human bocavirus: a novel parvovirus epidemiologically associated with pneumonia requiring hospitalization in thailand high prevalence of human bocavirus detected in young children with severe acute lower respiratory tract disease by use of a standard pcr protocol and a novel real-time pcr protocol human bocavirus in italian patients with respiratory diseases human bocavirus in febrile children, the netherlands human bocavirus in iranian children with acute respiratory infections human bocavirus infection, people's republic of china human bocavirus dna detected by quantitative real-time pcr in two children hospitalized for lower respiratory tract infection human bocavirus infection in young children in the united states: molecular epidemiological profile and clinical characteristics of a newly emerging respiratory virus detection of bocavirus dna in nasopharyngeal aspirates of a child with bronchiolitis evaluation of pcr testing of ethanol-fixed nasal swab specimens as an augmented surveillance strategy for influenza virus and adenovirus identification detection and characterization of new influenza b virus variants in 2002 plasma endothelin-1 in infants and young children with acute bronchiolitis and viral pneumonia human metapneumovirus infection in thai children this study was supported by the thailand research fund (senior research scholar), royal golden jubilee ph.d. program, the thailand research fund and the center of excellence in clinical virology, chulalongkorn university. we would like to thank the entire staff of the pulmonary unit, department of pediatrics, faculty of medicine, chulalongkrong university for their assistance in collecting the specimens. we also would like to thank ms petra hirsch for reviewing the manuscript. key: cord-259997-8f8di4eu authors: botti, chiara; micillo, alberto; ricci, giuseppe; russo, adolfo; denisco, alberto; cantile, monica; scognamiglio, giosuè; de rosa, antonio; botti, gerardo title: characterization of respiratory infection viruses in hospitalized children from naples province in southern italy date: 2018-04-13 journal: exp ther med doi: 10.3892/etm.2018.6061 sha: doc_id: 259997 cord_uid: 8f8di4eu most acute respiratory infections (aris) in children are due to viral etiology, and represent an important cause of mortality and morbidity in children <5 years old in developing countries. the pathogens that cause aris vary geographically and by season, and viruses serve a major role. in the present study, the distribution of the seven respiratory viruses that are more prevalent in southern european countries were retrospectively analyzed in a southern italy hospital, that centralizes pediatric diseases from the naples province. viruses were categorized by a filmarray respiratory panel, and demonstrated no substantial differences in sex, age and seasonal viruses distribution. however, all the investigated viruses had a higher detection rate in the surrounding municipalities than in the metropolitan area of naples. in recent years, the association between air pollution and respiratory infections has become an increasing public health concern. the data in this study support this association in the surrounding areas of naples extensively contaminated by environmental toxic agents. in these areas, characterization of the epidemiology of aris is required to implement a prevention and control program. acute respiratory infections (aris) represent a health issue of great importance, leading cause of mortality in children worldwide, particularly in developing countries (1) . these represent about 50% of all diseases in children aged <5 years (2) . the major viral agents of ari include influenza a, b, and c viruses (flu), respiratory syncytial virus (rsv), parainfluenza virus (piv), adenovirus (adv), human metapneu-movirus (hmpv), human coronavirus (hcov), and rhinovirus (hrv). the etiology of respiratory diseases is multifactorial and includes, among others, interactions between genetic predisposition and environmental factors (3) . numerous studies have confirmed that short-and long-term exposures to ambient air pollutants can be associated with a wide range of pathologies, in particular respiratory diseases and cancer. although air pollution has not been shown as the sole cause of respiratory infections, it has been reported that several air pollutants were correlated with increased morbidity of respiratory infections (4) . in urban areas, irrespective of seasonal frequencies, this correlation increases due to the high incidence of polluting factors. several epidemiological studies have documented a positive association between exposure to particulate air pollution and respiratory symptoms especially among children (5) (6) (7) . it is profusely reported that during the past three decades, large areas of naples county in the southern italy campania region have been extensively contaminated by environmental toxic agents, in particular for the presence of many landfills of industrial wastes. this region was already defined as one of the geographical areas most at risk of neoplastic and respiratory diseases for environmental factors in the report of who (world health organization) in 1997 (8) . although, the epidemiological studies for the association between air pollution and the incidence of respiratory infection in this geographical area are relatively few, virus types identification and their molecular characterization is fundamental not only for surveillance, for diagnostic and therapeutic purposes, but also for highlight the relationships between respiratory diseases in children and concentrations of environment pollutions (9) . in the present study we characterized the viral spectrum and pattern of aris in children from children's hospital 'santobono pausillipon' which centralizes pediatric diseases of the entire naples province. the aim of this study was to determine the association between respiratory viruses types, patients features (sex, age, season of disease occurrence) and, in particular, geographical origin. patients selection. we retrospectively reviewed the electronic medical records of 356 patients between 11 days and 14 years, with suspected respiratory infection, evaluated at the santobono hospital of naples, between 1 january 2016 and 31 january 2017, with filmarray ® respiratory panel (farp) testing on nasopharyngeal swab (nps). from patient electronic medical records the following information was obtained: demographics (age and sex), month of hospitalization, and geographical origin. we have divided the patients into three age groups (early childhood, 0-2 years; preschool age, 2-5 years; third childhood: 5-11 years), in the season of hospitalization (winter: december, january, february; spring: march, april, may; summer: june, july, august; autumn: september, october, november) and geographical origin (metropolitan area of naples and extra-urban areas). filmarray testing. npss were collected according to a standard procedure, kept in viral transport medium, and stored at -20˚c prior to analysis. farp (filmarray ® respiratory panel biofire diagnostics llc 390; wakara way salt lake city, ut, usa) is a test based on multiplex pcr. the filmarray rp cartridge is designed for the simultaneous detection and identification of following viruses and bacteria of the upper respiratory tract: influenza a virus (h1n1, h1n1 2009, and h3n2), influenza b virus, rsv, pivs 1-4, adv, hrv/enterovirus (the assay does not distinguish between these two pathogens), hmpv, hcov (229e, hku1, oc43, and nl63), mycoplasma pneumoniae, chlamydophila pneumoniae, and bordetella pertussis. the filmarray instrument and pouch system have been described in detail elsewhere (10) . the research use only version of the filmarray rp system reported a cycle threshold for each positive pcr assay (11) . statistical analysis. statistical analysis was performed using spss 13.0 (spss inc., chicago, il, usa). viral prevalence were compared using the chi-square test for categorical variables, and the cartogram was drawn using excel software (microsoft corporation, redmond, wa, usa). p<0.05 was considered to indicate a statistically significant difference. demographic characteristics. a total of 356 nasopharyngeal swabs were collected and analyzed. in detail, 163 patients were female (45.78%) and 193 were male (54.21%). most of the patients (319) were <5 years old while 36 patients were >5 years old. patients are heterogeneously distributed in different seasons, while regarding geographical origin, 123 (37.9%) patients are of naples metropolitan area, 201 (62.03%) originated from neighboring municipalities, and for 32 patients this information is lacking. in our study we considered only viral infections, but we detected also other etiologic agents (bordetella pertussis, chla myd ophila pneu m oniae, m ycopla s m a pneumoniae), highlighted the presence of these infections in 24 patients (6.7%). respiratory infection viruses distribution. the total rate of detection of all seven viruses was 78% (278/356) of patients. in detail, hrv viruses were detected in 44% of patients, followed by adv viruses (18%), rsv viruses (13%), and piv (12%). a lower incidence has been instead highlighted for flu (7%), hcov (4%) and hmpv (2%) (fig. 1) . in a significant proportion of individuals co-infections were also highlighted. in detail, double co-infections were detected in 69/256 (26.9%) patients, triple in 17/256 (6.6%) and quadruple infections only in 1 patient (table i) . the more frequent association was detected between hrv and piv viruses 19/87 (21%), followed by hrv and adv viruses 15/87 (17.24%), and hrv and rsv viruses 9/87 (10.3%) ( table i) . sex and age distribution. viruses appear heterogeneously distributed between sex, with the exception of piv. in detail, adv was slightly higher in males (18/34, 52.9%), while flu (10/14, 71.4%), hmpv (3/4, 75%) and hrv (50/86, 58.13%) were strongly higher in males children (see detection rate fig. 2 ). on the contrary hcov (5/7, 71.4%) and rsv (18/25, 72%) are better represented in female patients (see fig. 2 ). regarding age distribution a decline in the incidence of viral infections with age was observed for respiratory viruses, except for flu (fig. 3) . in fact the detection rate for flu viruses was lowest in 2-5 years patients. the detection rate for adv gradually decrease from 0-2 years patients to >5 years patients. the same trend was highlighted for hcov, piv, hrv and rsv (with a consistently increasing of detection rate in 0-2 year-old patients). hmpv was detected prevalently in 0-2 year-old children (fig. 3) . seasonal distribution. the total detection rate for all respiratory viruses in spring, summer, autumn and winter was calculated. the rates of detection was more heterogeneous during the different seasons. in detail, adv infection was prevalent in summer (20/34, 58.8%) and spring (10/34, 29.4%) seasons, flu was prevalent in spring (7/14, 50%) and winter (4/14, 28.5%), hmpv in winter (2/4, 50%), hcov in autumn/winter (3/7, 42.8%), hrv in autumn (34/86, 39.5%) and summer (30/86, 34.8%), piv in summer (13/24, 34.8%) and rsv in winter (24/25, 96%) (fig. 4) . geographical distribution. the pediatric patients were divided into two main groups according to origin from the metropolitan of naples and neighboring municipalities. the detection rates for the two areas appear different with a prevalent distribution in extra-urban areas. this appears more evident especially for adv, hrv and rsv viral infections (fig. 5) . who reported that ari can represent among the leading causes of mortality in children under 5 years of age. many studies in the literature have described in detail the viruses mainly associated with ari and also their distribution, but showing much conflicting data on populations from different countries (1) . these variations may be due to socio-economic factors, geographical and climatic differences and efficiency of local health care systems. in recent years, advances in pcr techniques have aided in the rapid and accurate detection of common respiratory pathogens from patient specimens. multiplex pcr can identify and differentiate a large panel of viral and bacterial targets simultaneously and are more rapid and more sensitive methods than cultures or antigen detection (12, 13) . in the present report we analyzed the distribution of seven respiratory viruses in a case series of 356 hospitalized patients in metropolitan area of naples and in the neighboring municipalities. the incidence of all analyzed virus is prevalent in early childhood. the distribution of viruses, considered individually, appears to be very heterogeneous, with the largest percentage of hrv in line with other studies on different populations (14, 15) . hrv (including rhinovirus and enterovirus) are rna viruses related to picornavirus family (16) . hrv is associated with the common cold, but can also be implicated in exacerbating asthma attacks and severe complications (15) . the enteroviruses are categorized into four species, which include a total of 89 serotypes associated with different clinical manifestations (16, 17) . in our case series hrv appears more expressed in the male population and its seasonality is mainly associated with summer and autumn, in line with the data present in the literature (16, 18) . rsv and adv are, after hrv, the most common virus in our case series, with rsv prevalent among the female children and adv among the male population. rsv is a member of the paramyxoviridae rna viruses family related to human metapneumovirus and pivs. rsv is the most common cause of severe respiratory illness in infants with acute bronchiolitis as a leading cause of hospitalization (19) . in our case series the winter seasonality of rsv viruses corresponds to the other reported data (19) . advs are dna viruses uncoated consists of seven species (a-g) and classified by hemagglutination with about 55 serotypes. the species of adv b, c and e cause acute respiratory disease with the main risk factor the long stay indoors (18) while the adv (species a, d, f and g) can cause cystitis, gastroenteritis and conjunctivitis (20) . in our study the seasonal distribution of adv is prevalent in summer in line with the literature (21) . while there are no substantial differences in piv distribution, flu seems prevalent in the male population and their seasonality is consistent with data reported in the literature (22) . finally, hcov and hmpv viruses are less frequent in our population, with the first more present in male pediatric population and hmpv in female children. coronavirus hcov are characterized 4 serological variants (229e, hku1, nl63 and oc43), and are most commonly associated with infections of the upper respiratory tract (23) . hmpv belong to the paramyxoviridae family and the infection in newborns and young children is commonly associated with bronchiolitis (24). it is also widely documented the occurrence of co-infections in some cases. in our case series the more frequent co-infection are between hrv and piv viruses and hrv and adv viruses. these data, in some cases, contrast with other reports (25) . however, this might be closely related to the geographical location, climate and different social and socio-cultural conditions. co-infections were more common in pediatric patients than in adults as documented by other studies (26) (27) (28) . however, multiple viral infections can be linked to hospital stay, abuse of antibiotic and social conditions, but there are not proves that co-infections can worsen the disease course. our hospital centralizes the majority of patients coming from the metropolitan area of naples but also from neighboring municipalities. our data are interesting for the geographic distribution of patients. in fact all the investigated viruses have a detection rate higher in surrounding municipalities than in the metropolitan area of naples. this is in contrast with most of the data present in the literature, where the prevalence is just in urban areas due to the high presence of pollutants (29) (30) (31) . however, during the past three decades, surrounding areas of naples have been extensively contaminated by environmental toxic agents, in particular for the presence of many landfills of industrial wastes. the most contaminated areas were defined as 'land of fire' (32) . the relationship between air pollution and respiratory infections has become an increased public health concern in recent years (33) (34) (35) . in fact, the etiology of respiratory diseases is multifactorial and includes, among others, interactions between genetic predisposition and environmental factors as climate change, chemical air pollution and airborne pollens. the short-term respiratory effects of air pollution include decreases in pulmonary function (36) , increases in inflammatory biomarkers and respiratory symptoms (37) , infections (38) , and respiratory mortality (39) . the environmental risk factors may have an impact on children's respiratory health, above all in urban areas, especially because children inhale more pollutants per kilogram of body weight than adults (40) . in conclusion, the risk factors between populations can be extremely different, suggesting the need to adequately characterize epidemiology of aris to implement prevention and control program. acute respiratory infections in children aetiological role of common respiratory viruses in acute lower respiratory infections in children under five years: a systematic review and meta-analysis chronic obstructive pulmonary disease pulmonary health effects of air pollution locally generated particulate pollution and respiratory symptoms in young children using air pollution based community clusters to explore air pollution health effects in children acute respiratory infections among under-five age group children at urban slums of gulbarga city: a longitudinal study the world health report 1997-conquering suffering, enriching humanity detection and characterization of respiratory viruses causing acute respiratory illness and asthma exacerbation in children during three different seasons filmarray, an automated nested multiplex pcr system for multi-pathogen detection: development and application to respiratory tract infection respiratory virus detection in immunocompromised patients with filmarray respiratory panel compared to conventional methods multiplex pcr: optimization and application in diagnostic virology biofire filmarray respiratory panel for detection of enterovirus d68 rhinovirus-associated hospitalizations in young children clinical severity of rhinovirus/enterovirus compared to other respiratory viruses in children diagnosis and treatment of rhinovirus respiratory infections epidemiological, molecular, and clinical features of enterovirus respiratory infections in french children between 1999 and manual of clinical microbiology epidemiologic, experimental, and clinical links between respiratory syncytial virus infection and asthma abrupt emergence of diverse species b adenoviruses at us military recruit training centers division of viral diseases (dvd) web site prevention and control of seasonal influenza with vaccines; recommendations of the advisory committee on immunization practices--united states clinical disease in children associated with newly described coronavirus subtypes epidemiology characteristics of respiratory viruses found in children and adults with respiratory tract infections in southern china development of three multiplex rt-pcr assays for the detection of 12 respiratory rna viruses viral and atypical bacterial detection in acute respiratory infection in children under five years detection of nine respiratory rna viruses using three multiplex rt-pcr assays incorporating a novel rna internal control transcript air pollution and children's health the role of air pollution in asthma and other pediatric morbidities air pollution, aeroallergens and admissions to pediatric emergency room for respiratory reasons in turin, northwestern italy cancer prevalence in the city of naples: contribution of the gp database analyses to the cancer registries network long-term associations of morbidity with air pollution: a catalogue and synthesis urban air pollution and meteorological factors affect emergency department visits of elderly patients with chronic obstructive pulmonary disease in taiwan the effects of particulate matter on inflammation of respiratory system: differences between male and female air pollution and lung function among susceptible adult subjects: a panel study urban air and tobacco smoke as conditions that increase the risk of oxidative stress and respiratory response in youth fine particulate air pollution and hospital admission for cardiovascular and respiratory diseases revised analyses of the national morbidity, mortality, and air pollution study: mortality among residents of 90 cities oxidative stress in adolescent passive smokers living in urban and rural environments key: cord-225826-bwghyhqx authors: jiang, zheng; hu, menghan; fan, lei; pan, yaling; tang, wei; zhai, guangtao; lu, yong title: combining visible light and infrared imaging for efficient detection of respiratory infections such as covid-19 on portable device date: 2020-04-15 journal: nan doi: nan sha: doc_id: 225826 cord_uid: bwghyhqx coronavirus disease 2019 (covid-19) has become a serious global epidemic in the past few months and caused huge loss to human society worldwide. for such a large-scale epidemic, early detection and isolation of potential virus carriers is essential to curb the spread of the epidemic. recent studies have shown that one important feature of covid-19 is the abnormal respiratory status caused by viral infections. during the epidemic, many people tend to wear masks to reduce the risk of getting sick. therefore, in this paper, we propose a portable non-contact method to screen the health condition of people wearing masks through analysis of the respiratory characteristics. the device mainly consists of a flir one thermal camera and an android phone. this may help identify those potential patients of covid-19 under practical scenarios such as pre-inspection in schools and hospitals. in this work, we perform the health screening through the combination of the rgb and thermal videos obtained from the dual-mode camera and deep learning architecture.we first accomplish a respiratory data capture technique for people wearing masks by using face recognition. then, a bidirectional gru neural network with attention mechanism is applied to the respiratory data to obtain the health screening result. the results of validation experiments show that our model can identify the health status on respiratory with the accuracy of 83.7% on the real-world dataset. the abnormal respiratory data and part of normal respiratory data are collected from ruijin hospital affiliated to the shanghai jiao tong university medical school. other normal respiratory data are obtained from healthy people around our researchers. this work demonstrates that the proposed portable and intelligent health screening device can be used as a pre-scan method for respiratory infections, which may help fight the current covid-19 epidemic. during the outbreak of covid-19 epidemic, early control is essential. among all the control measures, efficient and safe identification of potential patients is the most important part. existing researches show that human physiological state can be perceived through breathing [1] , which means respiratory signals are vital signs that can reflect human health condition to a certain extent [2] . many clinical literature suggests that abnormal respiratory symptoms may be important factors for diagnosis of some specific diseases [3] . recent studies have found that covid-19 patients will have obvious respiratory symptoms such as shortness of breath fever, tiredness, and dry cough [4, 5] . among those symptoms, atypical or irregular breathing is considered as one of the early signs. for many people, early mild respiratory symptoms are difficult to be recognized. therefore, through the measurement of respiration condition, potential covid-19 patients can be screened to some extent. this may play an auxiliary diagnostic role, thus helping to find potential patients as early as possible. traditional respiration measurement requires attachments of sensors to the patient's body [6] . the monitor of respiration is measured through the movement of the chest or abdomen. contact measurement equipment is bulky, expensive, and time-consuming. the most important thing is that the contact during measurement may increase the risk of spreading infectious diseases such as covid-19. therefore, the non-contact measurement is more suitable for the current situation. in recent years, many non-contact respiration measurement methods have been developed based on imaging sensors, doppler radar [7] , depth camera [8] and thermal cam-era [9] . considering factors such as safety, stability and price, the measurement technology of thermal imaging is the most suitable for extensive promotion. so far, thermal imaging has been used as a monitoring technology in a wide range of medical fields such as estimations of heart rate [10] and breathing rate [11] [12] [13] . another important thing is that many existing respiration measurement devices are large and immovable. given the worldwide epdemic, the partable and intelligent screening equipment is required to meet the needs of largescale screening and other application scenarios in a real-time manner. for thermal imaging based respiration measurement, nostril regions and mouth regions are the only focused regions since only these two parts have periodic heat exchange between the body and the outside environment. however, until now, researchers seldom considered measuring thermal respiration data for people wearing masks. during the epidemic of infectious diseases, masks may effectively suppress the spread of the virus according to recent studies [14, 15] . therefore, developing the respiration measurement method for people wearing masks becomes quite practical. in this study, we develop a portable and intelligent health screening device that uses thermal imaging to extract respiration data from masked people which is then used to do the health screening classification via deep learning architecture. in classification tasks, deep learning has achieved the state-of-the-art performance in most research areas. compared with traditional classifiers, classifiers based on deep learning can automatically identify the corresponding features and their correlations rather than extracting features manually. for breathing tasks, algorithms based deep learning can also better extract the corresponding features such as breathing rate and breath-to-exhale ratio, and make more accurate predictions [16] [17] [18] [19] . recently, many researchers made use of deep learning to analyze the respiratory process. cho et al. used a convolutional neural network (cnn) to analyze human breathing parameters to determine the degree of nervousness through thermal imaging [20] . romero et al. applied a language model to detect acoustic events in sleepdisordered breathing through related sounds [21] . wang et al. utilized deep learning and depth camera to classify abnormal respiratory patterns in real time and achieved excellent results [8] . the disadvantage of this research may be that the equipment is not portable. in this paper, we propose a remote, potable and intelligent health screening system based on respiratory data for pre-screening and auxiliary diagnosis of respiratory diseases like covid-19. in order to be more practical in a situation where people often choose to wear masks, the breathing data capture method for people wearing masks is introduced. after extracting breathing data from the video obtained from the thermal camera, a deep learning neural network is performed to work on the classification between healthy and abnormal respiration conditions. to verify the robustness of our algorithm and the effectiveness of the proposed equipment, we an-alyze the influence of mask type, measurement distance and measurement angle on breathing data. the main contributions of this paper are threefold. first, we combine the face recognition technology with dual-mode imaging to accomplish a respiratory data extraction method for people wearing masks, which is quite essential for current situation. based on our dual-camera algorithm, the respiration data is successfully obtained from masked facial thermal videos. subsequently, we propose a classification method to judge abnormal respiratory state with deep learning framework. finally, based on the two contributions mentioned above, we have implemented a non-contact and efficient health screening system for respiratory infections using the actual measured data from hospital, which may contribute to finding the possible cases of covid-19 and keeping the control of the secondary spread of sarscov-2. a brief introduction to the proposed respiration condition screening method is shown below. we first use the portable and intelligent screening device to get the thermal and the corresponding rgb videos. during the data collection, we also perform a simple real-time screening result. after getting the thermal videos, the first step is to extract respiration data from faces in thermal videos. during the extraction process, we use the face detection method to capture people's masked areas. then a region of interest (roi) selection algorithm is proposed to get the region from the mask that stands for the characteristic of breath most. finally, we use a bidirectional gru neural network with attention mechanism (bigru-at) model to work on the classification task with the input respiration data. our data collection is achieved by the system shown in fig. 1 . the whole screening system includes a flir one thermal camera, an android smartphone and the corresponding application we have written, which is used for data acquisition and simple instant analysis. our screening equipment, whose main advantage is portable, can be easily applied to measure abnormal breathing in many occasions of instant detection. as shown in fig. 1 , the flir one thermal camera consists of two canmeras, an rgb camera and a thermal camera. we collect the face videos from both cameras and use face recogition method to get the nostril area and forehead area. the temperatures of the two regions are calculated in time series and is shown in the screening result page in fig. 1(b) . the red line stands for the body temprature and the blue line stands for breathing data. from the breathing data, we can predict the respiratory pattern of the testcase. then, a simple real-time screening result is given directly in the application fig. 1 : overview of portable and intelligent health screening system for respiratory infections: a) device appearance; b) analysis result of the application. (notice that the system can simultaneously collect body temperature signals. in the current work, this body temperature signal is not considered in the model and is only used as a reference for the users. ) according to the extracted features shown in fig. 1 . we use the the raw face videos collected from both rgb camera and thermal camera as the data for further study to ensure accuracy and higher performance. when continuous breathing activities performs, there is a fact that periodic temperature fluctuations occur around the nostril due to the inspiration and expiration cycles. therefore, respiration data can be obtained by analyzing the temperature data around the nostril based on thermal image sequence. however, when people wear masks, many facial features are blocked because of this. merely recognizing the face through thermal image will lose a lot of geometric and textural facial details, resulting in recognition errors of the face and mask parts. in order to solve this problem, we adopt the method based on two parallel located rgb and infrared cameras for face and mask region recognition. the masked region of face is first captured in the rgb camera, then such region is mapped to the thermal image with a related mapping function. the algorithm for masked face detection is based on pyramidbox model created by tang et al. [22] . the main idea is to apply tricks like gaussian pyramidbox in deep learning to get the context correlations as further characteristics. the face image is first used to extract features of different scales using gaussian pyramid algorithm. for those high-level contextual features, a feature pyramid network is proposed to further excavate high-level contextual features. then, the output together with those low-level features are combined in lowlevel feature pyramid layers. finally, the result is obtained after another two layers of deep neural network. for faces that a lot of features are lost due to the cover of mask, such a context-sensitive structure can obtain more feature correlations and thus improve the accuracy of face detection. in our experiment, we use the open source model from paddlehub to detect the face area on rgb videos. the next step is to extract the masked area and map the area from rgb video to thermal video. since the position of the mask on the human face is fixed, after obtaining the position coordinates of the human face, we obtain the mask area of the face by scaling down in equal proportions. for a detected face with width w, and height h, the loaction of left-up corner is defined as (0, 0), the loaction of right-bottom corner is then (w, h). the corresponding coordinate of the two corners of mask region is decalred as (w/4, h/2) and (3w/4, 4h/5). considering that the background to the boundary of the mask will produce a large contrast with the movement, which is easy to cause errors, we choose the center area of the mask through this division. then the selected area is mapped from the rgb image to thermal image to obtain the masked region in thermal videos. after getting the masked region in thermal videos, we need to get the region of interest (roi) that represents breathing features. recent studies often characterize breathing data through temperature changes around the nostril [10, 23] . however when people wear masks, there exists another problem that the nostrils are also blocked by the masks, and when people wearing different masks, the roi may be different. therefore, we perform a roi tracking method based on maximizing the variance of thermal image sequence to extract a certain area on the masked region of the thermal video which stands for the breath signals most. due to the lack of texture features in masked regions compared to human faces, we judge the roi by the temperature change of thermal image sequence. the main idea is to traverse the masked region in the thermal images and find a small block with the largest temperature change as the selected roi. the position of a certain block is fixed in the masked region among all the frames since the nostril area is fixed on the face region. we do not need to consider the movement of the block since our face recognition algorithm can detect the mask position in each frame's thermal image. for a certain block with height m, and width n, we define the average pixel intensity at frame t as: for thermal images,s(t) represents the temperature value at frame t. for every block we obtained, we calculate their s(t) on time line. then, for each block n, the total variance of the list of average pixel intensity with t frames σ 2 s (n) is calculated as shown in eq. 2, where µ stands for the mean value ofs(t). since respiration is a periodic data spread out from the nostril area, we can consider that the block with the largest variance is the position where the heat changes most in both frequency and value within the mask, which stands for the breath data most in the masked region. we adjust the corresponding block size according to the size of the masked region. for a masked region with n blocks, the final roi is selected by: for each thermal video, we traverse all possible blocks in the mask regions of each frame and find the rois for each frame by the method above. the respiration data is then defined ass(t)(0 < t < t ), which is the pixel intensities of rois in all the frames. we apply a bigru-at neural network to do the classification task on judging whether the respiration condition is healthy or not as shown in fig. 3 . the input of the network is the respiration data obtained by our extraction method. since the respiratory data is time series, it can be regarded as a time series classification problem. therefore, we choose the gate recurrent unit (gru) network with bidirection and attention layer to work on the sequence prediction task. among all the deep learning structures, recurrent neural network (rnn) is a type of neural network which is specially used to process time series data samples [24] . for a time step t, the rnn model can be represented by: where x (t) , h (t) and o (t) stands for the current input state, hidden state and output at time step t respectively. v, w, u are parameters obtained by training procedure. b is the bias and σ and φ are activation functions. the final prediction is y (t) . long-short term memory network is developed on the basis of rnn [25] . compared to rnn, which can only memorize and analyze short-term information, it can process relatively long-term information, and is suitable for problems with short-term delays or long time intervals. based on lstm, many related structures are proposed in recent years [26] . gru is a simplified lstm which merges three doors of lstm (forget, input and output) into two doors (update and reset) [27] . for tasks with a few data, gru may achieve a better result than lstm since it includes less parameters. in our task, since the input of the neural network is only the respiration data in time sequence, the gru network may perform a better result than lstm network. the structure of gru can be expressed by the following equations: where r t is the reset gate that controls the amount of information being passed to the new state from the previous states. z t stands for the update gate which determines the amount of information being forgotten and added. w r , w z and w h are trained parameters that vary in the training procedure.h t is the candidate hidden layer which can be regarded as a summary of the above information h t−1 and the input information x t at time step t. h t is the output layer at time step t which will be sent to the next unit. the bidirectional rnn has been widely used in natural language processing [28] . the advantage of such network structure is that it can strengthen the correlation between context of the sequence. as the respiratory data is a periodic sequence, we use bidirectional gru to obtain more information from the periodic sequence. the difference between bidirectional gru and normal gru is that backfoward sequence of data is spliced to the original forward sequence of data. in this way, the hidden layer of the original h(t) is changed to: where − → h t is the original hidden layer and ← − h t is the backfoward sequence of − → h t . during the analysis of respiratory data, the entire waveform in time sequence should be taken into consideration. for some specic breathing pattern such as sphyxia, several paticular features such as sudden acceleration may occur only at a certain point in the entire process. however, if we only use the bigru network, these features may be weakened as the time sequence data is input step by step which may cause a larger error in prediction. therefore, we add an attention layer to the network, which can ensure that certain key point features in the breathing process can be maximized. attention mechanism is a choice to focus only on those important points among the total data [29] . it is often combined with neural networks like rnn. before the rnn model summarizes the hidden states for the output, an attention layer can make an estimation of all outputs and find the most important ones. this mechanism has been widely used in many research areas. the structure of attention layer is: where h t represents the bigru layer output at time step t, which is bidirectional. w u and b w are also parameters that vary in the training process. a t performs a softmax fucntion on u t to get the weight of each step t. finally, the output of the attention layer s is a combination of all the steps from bigru with different weights. by applying another softmax function to the output s, we get the final prediction of the classification task. the structure of the whole network is shown in fig. 3 . our goal is to distinguish whether there is an epidemic infectious disease such as covid-19 according to the abnormal breathing in the respiratory system. our dataset is not collected from patients with covid-19. these data were obtained from the inpatients of the respiratory disease department and cardiology department in ruijin hospital. most of the patients we collected data from only caught with basic or chronic respiratory disease. they did not have fever which is the typical respiratory symptoms of infectious diseases. therefore, the body temperature is not taken into consideration in our current screening system. in ruijin hospital wards, we use a flir one thermal camera connected to an android phone to work on the data collection. we collected data from 73 people. for each person, we collect two 20-second infrared and rgb camera data with a sampling frequency of 10 hz. through data cutting and oversampling, we finally obtained 1,925 healthy breathing data and 2,292 abnormal breathing data, a total of 4,217 data. each piece of data consists of 100 frames of infrared and rgb videos in 10 seconds. in the bigru-at network, the hidden cells in bigru layer and attention layers are 32 and 8 respectively. the breathing data is normalized before input into the neural network and we use cross-entropy as the loss function. during the training process, we separate the dataset into two parts. the training set includes 1,427 healthy breathing data and 1,780 abnormal breathing data. and the test set contains 498 healthy breathing data and 512 abnormal breathing data. once this paper is accepted, we will release the dataset used in the current work for non-commercial users. among the whole dataset, we choose 4 typical respiratory data examples as shown in fig. 4. fig. 4(a) and fig. 4 (b) stand for the abnormal respiratory patterns extracted from patients. fig. 4 (c) and fig. 4(d) represent the normal respiratory pattern called eupnea from healthy participants. by comparison, we can find that the respiratory of normal people is in strong periodic and evenly distributed while abnormal respiratory data tend to be more irregular. generally speaking, most abnormal breathing data from respiratory infections have faster frequency and irregular amplitude. the experimental results are shown in table. 1. we consider four evaluation metrics viz. accuracy, precision, recall and f1. to measure the performance of our model, we compare the result of our model with three other models which are gru-at, bilstm-at and lstm respectively. the result shows that our method performs better than any other networks in all evaluation metrics despite the precision value of gru-at. by comparison, the experimental result demonstrates that attention mechanism is well-performed in keeping important node features in the time series of breathing data since the networks with attention layer all perform a better result than lstm. another point is that gru based networks achieve better result than lstm based networks. this may beacuse our data set is relatively small which can't fill the demand of the lstm based networks. gru based networks require less data than lstm and perform better result in our respiration condition classification task. to figure out the detailed information about the classification of respiratory state, we plotted the confusion matrixs of the four models as demonstrated in fig. 5 . as can be seen from the results, the performance improvement of bigru-at compared to lstm is mainly in the accuracy rate of the negative class. this is because many scatter-like abnormalities in the time series of abnormal breathing are better recognized by the attention mechanism. besides, the misclassification rate of the four networks are relatively high to some extent which may because many positive samples do not have typical respiratory infections characteristics since part of the patients caught other lung-related diseases. in the analysis section, we give 3 comparasions from different aspects to prove the robustness of our algorithm and device. in order to measure the robustness of our breathing data acquisition algorithm and the effectiveness of the proposed portable device, we analyze the breathing data of the same person wearing different masks. we design 3 mask wearing scenarios that cover most situations: wearing one surgical mask (blue line); wearing one kn95 (n95) mask (red line) and wearing two surgical masks (green line). the results are shown in fig. 6 . it can be seen from the experimental results that no matter what kind of mask is worn, or even two masks, the respiratory data can be well recognized. this proves the stability of our algorithm and device. however, since different masks have different thermal insulation capabilities, the average breathing temperature may vary as the mask changes. to minimize this error, respiratory data are normalized before input into the neural network. in order to verify the robustness of our algorithm and device in different scenarios, we design experiments to collect respiratory data at different distances. considering the limitations of handheld devices, we test the collection of facial respiration data from a distance of 0 to 2 meters. the result is demonstrated in fig. 7 . the signal tends to be periodic from the position of 10 centimeters, and it does not start to lose regularity until about 1.8 meters. at a distance of about 10 centimeters, the complete face begins to appear in the camera video. when the distance comes to 1.8 meters, our face detection algorithm begins to fail gradually due to the distance and pixel limitation. this experiment verifies that our algorithm and device can guarantee relatively accurate measurement results in the distance range of 0.1 meters to 1.8 meters. considering that breath detection will be applied in different scenarios, we design this experiment to show the actual situation under different shooting angles. we define the camera directly towards the face to be 0 degree, and design an experiment in which the shooting angle gradually changed from 45 degrees to 0 degree. we consider the transformation of two angles: horizontal and vertical, which respectively represent left and right turning and nodding. the results in the two cases are quite different as shown in fig. 8 . our algorithm and device maintain good results in horizontal rotation, but it is difficult to obtain precise respiratory data in vertical rotation. this means participants can trun left or turn right during the measurement but can't nod or head up since this may impact the measurement result. in this paper, we propose a abnormal breathing detection method based on a portable dual-mode camera which can reocrd both rgb and thermal videos. in our detection method, we first accomplished an accurate and robust respiratory data detecion algorithm which can precisely extract breathing data from people wearing masks. then, a bigru-at network is applied to work on the screening of respiratory infections. in validation experiments, the obtained bigru-at network achieves a realtively good result with the accuracy of 83.7% on the real-world dataset. it is foreseeable that in patients with covid-19 who have more clinical respiratory symptoms, this classification method may yield better results. during the current outbreak of covid-19, our research can work as prescan method for abnomral breathing in many scenarios such as community, campus and hospital which may contribute to distuigishing the possible cases , and then keep the control of the virus spread. in future research, on the basis of ensuring portability, we plan to use a more stable algorithm to minimize the effect of different masks on measurement of breathing condition. besides, temperature may be taken into consideration to achieve a higher detection accuracy on respiratory infections. respiratory rate: the neglected vital sign non-contact respiratory rate measurement validation for hospitalized patients dysfunctional breathing: a review of the literature and proposal for classification pathological findings of covid-19 associated with acute respiratory distress syndrome world health organization declares global emergency: a review of the 2019 novel coronavirus (covid-19) respiration rate monitoring methods: a review novel methods for noncontact heart rate measurement: a feasibility study abnormal respiratory patterns classifier may contribute to large-scale screening of people infected with covid-19 in an accurate and unobtrusive manner synergetic use of thermal and visible imaging techniques for contactless and unobtrusive breathing measurement combination of near-infrared and thermal imaging techniques for the remote and simultaneous measurements of breathing and heart rates under sleep situation remote monitoring of breathing dynamics using infrared thermography a novel method for extracting respiration rate and relative tidal volume from infrared thermography rgb-thermal imaging system collaborated with marker tracking for remote breathing rate measurement rational use of face masks in the covid-19 pandemic mass masking in the covid-19 epidemic: people need guidance performance characterization of deep learning models for breathing-based authentication on resource-constrained devices deep learning versus professional healthcare equipment: a fine-grained breathing rate monitoring model respiration-based emotion recognition with deep learning a deep learning framework using passive wifi sensing for respiration monitoring deepbreath: deep learning of breathing patterns for automatic stress recognition using low-cost thermal imaging in unconstrained settings deep learning features for robust detection of acoustic events in sleep-disordered breathing pyramidbox: a context-assisted single shot face detector robust tracking of respiratory rate in high-dynamic range scenes using mobile thermal imaging finding structure in time long short-term memory lstm: a search space odyssey learning phrase representations using rnn encoder-decoder for statistical machine translation neural machine translation by jointly learning to align and translate attention is all you need key: cord-048197-9785vg6d authors: domachowske, joseph b; bonville, cynthia a; rosenberg, helene f title: gene expression in epithelial cells in response to pneumovirus infection date: 2001-05-11 journal: respir res doi: 10.1186/rr61 sha: doc_id: 48197 cord_uid: 9785vg6d respiratory syncytial virus (rsv) and pneumonia virus of mice (pvm) are viruses of the family paramyxoviridae, subfamily pneumovirus, which cause clinically important respiratory infections in humans and rodents, respectively. the respiratory epithelial target cells respond to viral infection with specific alterations in gene expression, including production of chemoattractant cytokines, adhesion molecules, elements that are related to the apoptosis response, and others that remain incompletely understood. here we review our current understanding of these mucosal responses and discuss several genomic approaches, including differential display reverse transcription-polymerase chain reaction (pcr) and gene array strategies, that will permit us to unravel the nature of these responses in a more complete and systematic manner. rsv and pvm are viruses of the family paramyxoviridae, subfamily pneumovirus; they are enveloped, singlestranded, nonsegmented rna viruses that can cause intense viral bronchiolitis in humans and mice, respectively. in its most severe form, the lower respiratory tract infection caused by pneumoviruses is associated with the development of peribronchiolar infiltrates that are accompanied by submucosal edema and bronchorrhea, and ultimately leads to bronchiolar obstruction and compromised oxygen transfer. as the infection is confined to the respiratory epithelium, the responses of these cells are clearly of primary importance in determining the nature and extent of the resulting inflammatory process. most of our understanding of responses to pneumovirus infection has emerged from studies of rsv infection of human epithelial target cells in vitro; a list of genes and/or gene products produced by epithelial cells in response to rsv infection in vitro is provided in table 1 . at the cellular level, epithelial cells initially respond to rsv infection by reducing their ciliary beat frequency. production and release of chemoattractant cytokines (chemokines) can be observed as early as 12 h after infection, leading to the recruitment of specific leukocyte subsets to the lung tissue. rsv-infected epithelial cells become resistant to tumor necrosis factor (tnf)-α-induced apoptosis, but later fuse to form giant-cell syncytia and die by cellular necrosis. we review the molecular bases (to the extent that they are understood) of these specific responses, and discuss several novel strategies that may permit us to study the responses to rsv and pvm infection in a more coherent and systematic manner. tristram et al [1] observed that explanted respiratory epithelial cells slow their ciliary beat frequency almost immediately after exposure to rsv, with complete ciliostasis seen as early as 6 h after the initial infection. the molecular basis of ciliostasis remains completely unknown. the chemokines and cytokines with production and release that has been associated with rsv infection of human epithelial cells are listed in table 1 . much of this work was also recently reviewed elsewhere [2, 3] . we focus here on the three chemokines whose molecular mechanisms and physiologic implications are best understood. the earliest reports on this subject described production of the neutrophil chemoattractant il-8 from tissue culture supernatants from rsv-infected cells [4] [5] [6] and in nasal secretions from patients with viral rhinitis [7] . il-8 has since been detected in lower airway secretions from patients with severe rsv bronchiolitis [8] , and the neutrophil influx observed in response to this infection is probably due, at least in part, to the activity of this chemokine. at the cellular level il-8 production can be observed in response to inactivated rsv virions, whereas il-8 production in response to active infection was inhibited by ribavarin, amiloride, and antioxidants [9, 10] . several groups have demonstrated activation of the transcription factor nuclear factor-κb (nf-κb) in response to rsv infection, and nf-κb is recognized for its central role in eliciting the production of il-8 [9, 11, 12] . the transcription factor nf-il-6 is also produced in response to rsv infection [13] , and participates in a co-operative manner with nf-κb in the regulation of il-8 gene expression [11] , although later studies suggest that activator protein-1 may function preferentially in this role [14] . interestingly, the nf-κb regulator iκbα, which functions by inhibiting nf-κb activation in response to tnf-α, was produced with different kinetics and does not promote a reversal of nf-κb activation in response to rsv infection as it does in response to tnf-α [15] . most recently, casola et al [16] demonstrated that the il-8 promoter contains independent response elements, with nucleotides -162 to -132 representing a unique rsv response element that is distinct from elements necessary for il-8 production in response to tnf-α. this concept of a stimulus-specific response will probably make an important contribution toward our understanding of how pneumoviruses promote transcription of unique and specific sets of independent gene products. the pleiotropic chemokine regulated upon activation, normal t-cell expressed and secreted (rantes) has also been detected in supernatants from rsv-infected epithelial cells in culture [17, 18] , as well as in upper and lower airway secretions from patients infected with this virus [7, 8] . rantes acts as a chemoattractant for eosinophils and monocytes in vitro, although its role in vivo is somewhat less clear. similar to il-8, rantes can be produced in vitro in response to inactivated virions [8] , and involves nf-κb activation, binding, and nuclear translocation [19] . however, koga et al [20] demonstrated that stabilization of rantes mrna, a response to rsv infection mediated in part by nucleotides 11-389 of the rantes gene, is probably the primary mechanism underlying increased production and secretion of rantes protein. further studies will determine whether a similar mechanism is also in place for il-8 and other rsv-mediated responses. several groups have recently shown that macrophage inflammatory protein (mip)-1α is released from rsvinfected cells in culture [7, 21] ; mip-1α was also detected in upper and lower airway secretions from rsv-infected patients [7, 8] . interestingly, of the three aforementioned chemokines, mip-1α is the one that is most closely correlated with the presence of eosinophil degranulation products; this, together with data from our pvm model of pneumovirus infection [22] , has suggested to us that mip-1α plays a pivotal role in eosinophil recruitment in response to primary pneumovirus infection. interestingly, production of mip-1α in cell culture requires active viral replication [8] , which suggests that this response may proceed by a mechanism that is completely distinct from that which elicits production of rantes and il-8. however, no reports to date have addressed the molecular mechanism that underlies the rsv-mediated mip-1α response. a list of cell-surface molecules that have been reported as expressed in response to rsv infection is shown in table 1 . we focus here on the expression of intercellular adhesion molecule (icam)-1 (cd54) and the leukocyte integrin cd18. increased expression of this cell-surface adhesion protein was observed in both respiratory epithelial cell lines [23, 24] and in human nasal epithelial cells [25] in response to infection with rsv in vitro. chini et al [26] demonstrated that the expression of icam-1 mrna, similar to il-8 and rantes, was dependent on an intact nf-κb site in the gene promoter, and demonstrated a role for the consensus binding site for the factor ccaat/ enhancer-binding protein. stark et al [27] demonstrated that icam-1 and cd18 expressed in response to rsv serve to enhance neutrophil and eosinophil binding to epithelial cells. cd18 is a polypeptide of the integrin family that functions in mediating cell-cell interactions. several groups have observed expression of cd18 on epithelial cells in response to rsv infection [27, 28] , with cd18 shown to enhance the degranulation of eosinophils in this specific setting [28] . of particular interest are the recent findings relating expression of cd18 (along with cd14) to earlier literature on bacterial superinfections in the setting of viral infections. earlier studies [29, 30] reported enhanced binding of bacteria to respiratory epithelial cells that were infected with rsv, findings that had clinical implications relating to acute bacterial otitis media in infants. two more recent studies addressed the question of binding sites. saadi et al [31] determined that two strains of the pathogen bordetella pertussis bound more efficiently to rsv-infected cells, and that the binding was reduced upon pretreatment of the cells with anti-cd14 or anti-cd18 antibodies. similarly, raza et al [32] reported that both cd14 and cd18 on rsv-infected epithelial cells contributed to the binding of nonpilate neisseria meningitidis. in vivo testing is required before the clinical significance of these intriguing findings can be appreciated. rsv-infected epithelial cells in culture do not show features that are suggestive of apoptosis (ie no evidence of membrane blebbing, fragmentation of chromosomal dna, or characteristic changes in nuclear morphology). takeuchi et al [33] showed that, although rsv-infected epithelial cells express a number of apoptosis-associated genes, including interferon regulatory factor-1, il-1β-converting enzyme and caspase 3, they do not undergo formal apoptosis. as part of our attempts to understand mucosal responses in a more systematic manner (see below), we discovered that rsv-infected epithelial cells express the recently described antiapoptosis gene iex-1l [34] . in our studies, we found that expression of iex-1l is a response to active virus; no gene expression was observed in response to irradiated, replication-incompetent virus. moreover, expression of iex-1l is not observed in response to adenoviral infection, suggesting that expression of this gene is not a universal response to cellular perturbation, or indeed to all viral infections. functionally, we also demonstrated that rsv infection protects epithelial cells from tnf-α-induced apoptosis, an effect that is temporally associated with the expression of iex-1l. apoptosis is generally considered to be a highly efficient self-defense mechanism employed by host target cells, because it permits the infected host to dispose of viral proteins and nucleic acids on a single-cell basis without inducing an inflammatory response. it is thus not surprising that many viruses have evolved strategies to circumvent this response. of interest, krilov et al [35] demonstrated that monocytes and cord blood mononuclear cells are similarly protected from apoptosis when infected with rsv. although virus-induced protection from apoptosis appears advantageous to the virus alone, another interpretation may be considered. because respiratory epithelial cells are now recognized as a major source of leukocyte chemoattractants, and because leukocyte recruitment to the lung has been associated with enhanced viral clearance and prolonged survival in pneumovirus infection [22] , the ability to maintain chemoattractant production from viable cells may ultimately benefit the host organism as well. available online http://respiratory-research.com/content/2/4/225 in tissue culture, rsv-infection is characterized by the formation of giant-cell syncytia. the mechanisms for the formation of these fused masses of cells depend in part on the expression of the rsv-specific fusion (f) protein on the surface of infected host cells, and in part on virusmediated changes in cytoskeletal architecture. it is important to note that rsv-induced changes in cytoskeletal architecture are not restricted to cell lines grown in vitro, as giant-cell syncytia have also been found in pathologic lung specimens obtained from both humans and animals that were infected with rsv. again, as part of our systematic study of gene expression in response to pneumovirus infection, we found that human respiratory epithelial cells respond to rsv infection with increased expression of the cytoskeletal protein cytokeratin-17 [36] . cytokeratin-17 is a 46-kda cytoskeletal protein that belongs to the class i acidic cytokeratin family. in the lung, expression of cytokeratin-17 is normally restricted to basal epithelial cells of the larynx, trachea, and bronchi. in rsv-infected cells, we found expression of ck-17 predominantly at sites of syncytia formation, and thus provided the first description of a unique component of these pathognomonic structures at the molecular level. similar to what has been reported for the production of il-8, expression of ck-17 is dependent on the activity of the transcription factor nf-κb, and future studies will determine the role of the nf-κb consensus site (-200 to -208 of the cytokeratin-17 promoter) in mediating this response. to date, efforts to study pneumovirus-induced alterations in gene expression have relied heavily on in vitro models of virus-infected cells and cell lines. the intrinsic value of characterizing the genes identified in this artificial system is by definition limited, and the clinical and physiologic sig-nificance of any findings must ultimately be tested in vivo. to some extent, the study of gene products in clinical specimens is possible, but this approach is limited, cumbersome, and dictated by sample availability. it is clear that an appropriate animal model of inflammatory bronchiolitis is required to characterize the alterations in gene expression discovered using the available in vitro models. although rsv has been used for the study of specific allergic reactions to viral antigens, it is not a natural pathogen of mice, and intranasal inoculation of virus at high titer results in, at best, a minimal primary infection with a correspondingly minimal inflammatory response. in order to study gene expression in response to primary pneumovirus infection in vivo, we developed a novel mouse model of inflammatory bronchiolitis using the natural rodent pneumovirus pathogen and closest phylogenetic relative of rsv [37] -pvm. we presented our initial findings on pvm infection in mice in three recent publications [22, 38, 39] . a summary of these findings is presented in table 2 and fig. 1 . to begin, we described the cellular and biochemical pathology observed in response to pvm infection in mice [38] . we found that infection could be established with as few as 30 plaque-forming units (pfu) of pvm in the inoculum, with infection resulting in significant morbidity and mortality, and viral recoveries in the order of 10 8 pfu/g lung tissue. we also noted inflammatory bronchiolitis as among the immediate responses to this infection, with bronchoalveolar lavage fluid containing virtually 100% neutrophils and eosinophils obtained as early as 3 days after inoculation. furthermore, we found that infection was accompanied by the production of the proinflammatory chemokine mip-1α, which was previously shown by cook et al [40] to be an important component of the inflammatory response to the orthomyxovirus influenza virus. we also described the role played by mip-1α in the pathogenesis of pvm-induced bronchiolitis [22] . specifically, we explored the responses of gene-deleted mice to infection with pvm, and found no inflammatory response in mice deficient in mip-1α expression (mip-1α -/-) and only minimal virus-induced inflammation in mice that lacked the major mip-1α receptor on granulocytes chemokine receptor (ccr)1 (ccr1 -/-). although the inflammatory response is often considered to be unnecessary and indeed detrimental, we demonstrated that the absence of granulocytic inflammation was associated with enhanced recovery of infectious virions, as well as with accelerated mortality. these results suggest that the mip-1α/ccr1-mediated acute inflammatory response protects mice by delaying the lethal sequelae of viral infection. our most recent report on this subject [39] presents a direct comparison between the responses of mice to challenge with pvm and rsv. although rsv is not a natural pathogen of mice, it has been used extensively in mouse models of human infection because a limited, or 'semipermissive' infection can be established via intranasal inocula-tion of virus at very high titers. in this regard, we found (as have others) that rsv infection did not result in any measurable degree of morbidity, and that viral recovery was significantly lower than that found in the inoculum; these results suggested that there was no significant viral replication in mouse lung tissue. we further demonstrated that the inflammatory response to rsv challenge was minimal, as few leukocytes were recruited to the lungs (fig. 1) . taken together, our results suggest that infection of mice with pvm provides a superior model for the study of acute inflammatory bronchiolitis in response to pneumovirus infection in vivo. the advantages of this model include the following: clinical parameters -morbidity and mortalitythat can be measured clearly and specifically; clear evidence of viral replication in lung tissue, with incremental recoveries that, at peak, are in excess of 10 8 pfu/g in response to as few as 30 pfu in the inoculum; and a dramatic granulocytic response that is modulated at least in part by the proinflammatory chemokine mip-1α and its receptor ccr1. traditionally, analysis of gene expression through measurement of steady-state levels of individual mrnas could be conducted only one gene at a time using northern blotting, dot blots, or quantitative reverse transcription-pcr. differential display, serial analysis of gene expression, and total gene analysis offer great promise, because they are multiplex technologies that provide simultaneous analysis of multiple mrnas isolated under conditions of interest via pcr amplification techniques. dna hybridization arrays are theoretically the most efficient of the gene expression analysis techniques. although many skeptics have described these genome-based approaches as expensive, nonhypothesis-driven 'fishing expeditions', we view them as broad-based screening techniques that will enable us to identify patterns of gene expression that can then be subjected to careful characterization and analysis. differential display is a semiquantitative, reverse transcription-pcr-based technique that is used to compare mrnas from two or more conditions of interest. both increased and decreased expression of specific amplicons will be evident -an obvious advantage to this approach. total rna can be isolated from virus infected versus uninfected cells or mouse lungs both before and during infection, and differential display is performed using degenerate t11(xy) anchoring primers and random upstream oligomers, as described elsewhere [34, 36] . the resulting pcr products are separated by electrophoresis, and the gel is dried and exposed to film. an example of our results comparing cdna amplicons from rna extracted from rsv-infected epithelial cells daily for 4 days is shown in fig. 2 . differentially expressed bands are cut from the gel, eluted and reamplified using the same primers that generated the original signal, and northern blots generated from rna extracted from pneumovirus-infected cells or tissue over time and probed with the differentially expressed amplicons serve to confirm differential expression of the identified sequence. the dna sequences of the newly identified differentially expressed amplicons are compared with sequences present in the genbank database. viral sequences are expected to be upregulated over time and can be identified immediately, because the entire genomes of both pvm and rsv are present in genbank. in cases in which the amplicon represents a newly discovered gene, potential openreading frames are compared with sequences that are present in the swiss protein database; motifs that share homologies with known proteins represent important clues to the identity of the differentially expressed gene. with the help of differential display, we have identified and characterized several genes that are upregulated in rsv-infected respiratory epithelial cells. two specific examples of genes that were found to be induced during rsv infection, and later characterized as playing independent roles in the pathophysiology of rsv infection, are the antiapoptosis gene iex-1l [34] and the gene that encodes the cytoskeletal protein cytokeratin-17 [36] . unlike dna viruses, which are known to encode virus-specific antiapoptosis genes, rsv -an rna virus with a small (approximately 15.2 kb) viral genome -was shown to alter host cell expression of the apoptosis inhibitor iex-1l. after demonstrating that iex-1l mrna was present at sevenfold higher concentrations in rsv-infected respiratory epithelial cells when compared with uninfected cells, we concluded that this cellular response protected against tnf-α-induced programmed cell death during viral infection. further efforts to determine which of the 11 rsv proteins participate in the trans-activation of the iex-1l gene (either directly or indirectly) are ongoing. a second example of a gene that is specifically upregulated in rsv-infected respiratory epithelium, as identified by differential display, is that which encodes cytokeratin-17 [36] . upon characterizing the molecular events that are important for cytokeratin-17 induction, we demonstrated a link to an nf-κb signaling pathway. above, we discussed the importance of this transcription factor in the regulation of proinflammatory cytokine gene expression, and because of this involvement we were not surprised to discover its role in virus-induced cytokeratin-17 gene regulation. perhaps the most interesting observation made during these experiments was the in situ localization of cytokeratin-17 protein to areas of cytopathic syncytia formation, suggesting a role for this cytoskeletal protein in their formation. of note, we observed a dramatic decrease in rsv replication and in syncytia formation when we blocked cytokeratin-17 expression, suggesting that blocking syncytia formation, at least in part, impairs the direct cell-cell spread and productive replication of virus. although there are several companies that market these systems and components, the cytokine gene macroarray systems recently developed by r&d systems (sigma genosys ® ; minneapolis, mn, usa) and clontech (atlas ® ; palo alto, ca, usa) represent some of the newer opportunities available that have a focus on gene products that are known to be involved in inflammation. these arrays consist of different cdnas printed as pcr products onto charged nylon membranes. an example of our experience with the sigma genosys array is shown in fig. 3 . for this example, total rna was extracted from rsv-infected hep-2 cells and uninfected controls at day 3 after infection. three micrograms of total rna was used in a cdna synthesis reaction, using a proprietary mixture of 378 primer pairs and trace amounts of 32 p-radiolabeled dctp. the resulting radiolabeled products were hybridized to the macroarrays overnight at 65°c, and then washed and exposed to film. the arrow highlights one of the most obviously upregulated sequences from this experiment, which was identified as the gene encoding human mip-1α. the physiologic importance of mip-1α upregulation during human rsv infection and during rodent pvm bronchiolitis has already been described. microarrays can be differentiated from macroarrays in several ways. among these differences, the microarray matrix is a glass or plastic slide, probes are labeled with fluorescent dye rather than via radioisotopes, and, most significantly, microarrays generally include a larger number and a higher density of imbedded sequences than do macroarrays. although this may seem to be highly appealing at first, the massive amounts of data generated by microarray technology poses new challenges with respect to data normalization, management, and development of mathematical models to assist in data interpretation. the pneumoviruses rsv and pvm enter respiratory epithelial cells via a receptor-mediated event. during hostcell attachment and internalization, the target cell begins to alter its gene expression, which, among other events, involves the transcriptional upregulation of cytokine and chemokine genes. as rsv replication progresses, additional changes in cellular gene expression can be observed, including induction of the potent antiapoptosis gene iex-1l and increased expression of the otherwise quiescent gene that encodes cytokeratin-17. what we know regarding the physiologic importance of these genes and their gene products has been described, but there is more to be learned. as the available technologies evolve, we can continue to capitalize on the use of display of amplicons generated from rna extracted from rsv-infected cells at daily intervals following infection (days 0-4) using a single anchoring primer, t11gc (downstream primer 8) and (a-h) eight random 10mers. two differentially expressed sequences are highlighted by arrows (the black arrow shows an upregulated amplicon, and the white arrow highlights a downregulated amplicon). several other differentially expressed signals are also seen. genomic approaches as large-scale screening tools to identify genes that play important roles in the pathophysiology of pneumovirus infection. these elegant and simple tools will provide us with the means for thorough and systematic exploration of gene expression, both in the estabcytokine macroarray probed with radiolabelled cdna generated from total rna extracted from epithelial cells 48 h after rsv infection (upper panel) or 48 h after exposure to conditioned medium (lower panel). signal intensity of each gene under each condition is compared. the arrow highlights the signal for human mip-1α present at 12-fold higher concentration in infected epithelial cells compared with the uninfected controls. respiratory syncytial virus and human bronchial epithelium host responses to respiratory virus infection and immunization epithelial regulation of innate immunity to respiratory syncytial virus interleukin-8 expression in normal nasal epithelium and its modulation by infection with respiratory syncytial virus and cytokines tumor necrosis factor, interleukin-1, and interleukin-6 respiratory syncytial virus increases il-8 gene expression and protein release in a549 cells interleukin-8, interleukin-6, and soluble tumour necrosis factor receptor type i release from a human pulmonary epithelial cell line (a549) exposed to respiratory syncytial virus macrophage inflammatory protein-1α α and rantes are present in nasal secretions during ongoing upper respiratory tract infection respiratory syncytial virus-induced chemokine expression in the lower airways: eosinophil recruitment and degranulation inhibiton of viral replication reverses respiratory syncytial virus-induced nf-κ κb activation and interleukin-8 gene expression in a549 cells oxidant tone regulates il-8 production in epithelium infected with respiratory syncytial virus induction of interlukin (il)-8 gene expression by respiratory syncytial virus involved activation of nuclear factor (nf)-κ κb and nf-il-6 transcriptional activation of the interleukin-8 gene by respiratory syncytial virus infection in alveolar epithelial cells: nuclear translocation of the rela transcription factor as a mechanism producing airway mucosal inflammation inducible translational regulation of the nf-il6 transcription factor by respiratory syncytial virus infection in pulmonary epithelial cells activator protein-1 is the preferred transcription factor for cooperative interaction with nuclear factor-κ κb in respiratory syncytial virus-induced interleukin-8 gene expression in airway epithelium incomplete regulation of nf-κ κb by iκ κbα α during respiratory syncytial virus infection in a549 cells requirement of a novel upstream response element in respiratory syncytial virus-induced il-8 gene expression virus-inducible expression of a host chemokine gene relies on replication-linked mrna stabilization cell-specific expression of rantes, mcp-1, and mip-1α α by lower airway epithelial cells and eosinophils infected with respiratory syncytial virus the chemokine mip-1α α and its receptor ccr1 control pulmonary inflammation and anti-viral host defense in paramyxovirus infection adhesion molecule expression on epithelial cells infected with respiratory syncytial virus icam-1 expression and low-molecularweight g-protein activation of human bronchial epithelial cells (a549) infected with rsv induction of intercellular adhesion molecule-1 in human nasal epithelial cells during respiratory syncytial virus infection essential roles of nf-κ κb and c/ebp in the regulation of intercellular adhesion molecule-1 after respiratory syncytial virus infection of human respiratory epithelial cell cultures respiratory syncytial virus infection enhances neutrophil and eosinophil adhesion to cultured respiratory epithelial cells. roles of cd18 and intercellular adhesion molecule-1 respiratory syncytial virus-infected pulmonary epithelial cells induced eosinophil degranulation by a cd18-mediated mechanism effect of respiratory syncytial virus infection on binding of neisseria meningitidis and haemophilus influenzae type b to a human epithelial cell line (hep-2) effect of respiratory syncytial virus on adherence, colonization and immunity of nontypable haemophilus influenzae: implications for otitis media developmental and environmental factors that enhance binding of bordetella pertussis to human epithelial cells in relation to sudden infant death syndrome (sids) infection with respiratory syncytial virus enhances expression of native receptors for non-pilate neisseria meningitidis on hep-2 cells respiratory syncytial virus infection of human alveolar epithelial cells enhances interferon regulatory factor 1 and interleukin-1β β-converting enzyme gene expression but does not cause apoptosis respiratory syncytial virus infection induces expression of the anti-apoptosis gene iex-1l in human respiratory epithelial cells responses of monocytes in peripheral blood mononuclear leukocytes (pbmls) exposed to respiratory syncytial virus (rsv) in vitro cytokeratin 17 is expressed in cells infected with respiratory syncytial virus via nf-κ κb activations and is associated with the formation of cytopathic syncytia reverse genetics of the paramyxoviridae pulmonary eosinophilia and production of mip-1α α are prominent responses to infection with pneumonia virus of mice mip-1α α is produced but it does not control pulmonary inflammation in response to respiratory syncytial virus infection in mice smithies o: requirement of mip-1alpha for an inflammatory response to viral infection we thank dr andrew easton for his assistance with the pvm model. we thank drs leonard weiner, harry malech, and john gallin for their support for the ongoing research in our laboratories. this work was funded in part by an american heart association scientist development grant to jbd, the sinsheimer foundation and the children's miracle network of central new york. key: cord-260750-utbuj5iz authors: dear, jonathan d. title: bacterial pneumonia in dogs and cats date: 2013-11-21 journal: vet clin north am small anim pract doi: 10.1016/j.cvsm.2013.09.003 sha: doc_id: 260750 cord_uid: utbuj5iz bacterial pneumonia is a common clinical diagnosis in dogs but seems to occur less commonly in cats. underlying causes include viral infection, aspiration injury, and foreign body inhalation. identification of the organisms involved in disease, appropriate use of antibiotics and adjunct therapy, and control of risk factors for pneumonia improve management. bacterial pneumonia remains one of the most common clinical diagnoses in dogs with either acute or chronic respiratory disease. new research suggests a complex relationship between viral respiratory diseases and development of bacterial pneumonia in dogs. over the past decade, much has been discovered about the convoluted interplay between host and environmental factors that leads to this complex of diseases. in cats, bacterial pneumonia is less commonly identified than inflammatory feline bronchial disease. aspiration pneumonia results from the inadvertent inhalation of gastric acid and/or ingesta and remains a common cause of bacterial pneumonia, accounting for roughly 23% of clinical diagnoses in a study of human patients admitted to the intensive care unit. 1 although inhalation of gastroesophageal material is a common theme, different factors lead to the development of this phenomenon. risk factors that have been identified for the development of aspiration pneumonia include esophageal disease, refractory vomiting, seizures, prolonged anesthesia, and laryngeal dysfunction ( table 1) . 2 in a healthy animal, physiologic and anatomic features reduce the chance of aspiration. during a normal swallow, fluid and food are propelled caudally in the oropharynx and through the upper esophageal sphincter by contraction of the oral cavity and tongue. at the same time, the epiglottis retracts to cover the laryngeal aditus and protect the trachea from particulate inhalation. adduction of the arytenoid cartilages then contributes to further occlusion of the upper airways. any process impeding these primary defenses or inhibiting the normal swallowing reflexes increases the likelihood of aspiration. aspiration injury results from inhalation of either sterile, acidic gastric contents (resulting from vomiting or gastric regurgitation) or of septic material from gastric or oral secretions. irritation induced by acid inhalation promotes a local environment in which bacterial colonization can develop and lead to bacterial pneumonia. the severity of disease varies depending on the quantity and nature of the material aspirated as well as the length of time between the event and its diagnosis. conscious patients with intact airway reflexes tend to cough and prevent massive aspiration injury. animals under anesthesia or with reduced airway reflexes because of neurologic disorders are less likely to cough in response to the aspiration event and are, therefore, more likely to develop diffuse pulmonary infiltrates and acute lung injury. in many instances aspiration injuries occur under general anesthesia and the presence of a cuffed endotracheal tube does not prevent inadvertent aspiration. infectious, or community-acquired, pneumonias in dogs commonly begin with viral colonization and infection of the upper respiratory tract (canine respiratory coronavirus, herpesvirus, pneumovirus, and parainfluenza virus, among others). 3 often, such diseases are acute and self-limiting, but in a subset of dogs inflammation associated with these organisms immobilizes the host's immune defenses and predisposes infection with other (often bacterial) respiratory pathogens. 4 many bacteria have been implicated in canine infectious respiratory disease (cird), although special focus has been directed toward streptococcus (specifically streptococcus equi subsp zooepidemicus and s canis), mycoplasma cynos, and bordetella bronchiseptica. cird is especially prevalent in dogs naive to the pathogens and exposed in overcrowded, stressful environments such as animal shelters, boarding kennels, and treatment facilities. the pathophysiology associated with this disease and infectious lower respiratory tract disease in cats is discussed later in this article (boxes 1 and 2). inhaled foreign bodies carry mixed bacterial and fungal organisms into the lung and are associated with focal pneumonias that are often initially responsive to antimicrobial medications but relapse shortly after discontinuation of therapy. 5, 6 foreign bodies reported in the veterinary literature include grass awns, plant materials, or plastic materials. 6 organisms associated with grass awn inhalation include pasteurella, streptococcus, nocardia, actinomyces, and anaerobic bacteria. 6, 7 most often, foreign material remains at the carina or enters caudodorsal principal bronchi (accessory, right and left caudal lobar bronchi). features associated with pulmonary foreign bodies include: young, sporting breeds environmental exposure to grass awns focal, recurrent radiographic alveolar pattern history of other cutaneous or visceral foreign bodies spontaneous pneumothorax or pyothorax box 1 cird complex: changing the nature of kennel cough cird complex (formerly known as kennel cough) is a syndrome in which multiple pathogens, both viral and bacterial, coinfect either naive, immunocompromised dogs or previously vaccinated dogs. this complex is multifactorial and it is likely that both host and environmental factors play a role in the development of illness. 27 organisms associated with this disease are ubiquitous, especially in overcrowded housing facilities such as animal shelters and training facilities. it is likely that stress induced by the new environment and exposure to novel pathogens both play a role in development of disease. in most cases, respiratory signs are present for days to weeks and most animals show mild to moderate clinical signs. viral infections typically cause either a bronchopneumonia or bronchointerstitial pneumonia because of their propensity to infect and damage type i pneumocytes. 28 as the condition progresses, desquamation of the respiratory epithelium and aggregation of inflammatory cells further reduce the lungs' natural defenses, increasing the potential for secondary bacterial colonization and infection. previous studies have implicated viral organisms such as canine adenovirus or canine parainfluenza 29 as major participants in cird, although recent studies have proposed novel respiratory pathogens such as canine respiratory coronavirus, 3, 20, 30, 31 canine influenza virus, 20 and canine herpesvirus 32 as additional important pathogens associated with cird. b bronchiseptica, 33 streptococcus canis, s equi subsp zooepidemicus, 29 and m cynos 3, 34 have been implicated as secondary bacterial infections associated with cird. s equi subsp zooepidemicus infections, in particular, have been associated with a rapidly progressive and often fatal hemorrhagic pneumonia. 27, 35 some strains identified in outbreaks of this pathogen have been identified as resistant to tetracycline antibiotics, which are often the drug of choice prescribed for other bacterial pathogens associated with this complex. normal thoracic radiographs do not rule out the possibility of an airway foreign body 8 and even computed tomography (ct) can fail to identify an affected bronchus. chronic pulmonary foreign bodies are associated with marked inflammation that can lead to massive airway remodeling and bronchiectasis that, when seen on radiographs, should raise the degree of suspicion for foreign body. 5 ventilator-associated pneumonia (vap) is a common cause of hospital-acquired pneumonia in people, although there are few veterinary reports in the literature. colonization of the oropharynx by pathogenic and multidrug-resistant bacteria occurs and the endotracheal tube acts as a conduit to transmit pathogens into the airways, which leads to tracheobronchitis and potentially pneumonia. in addition, any animal with a compromised respiratory tract or serious systemic disease is particularly prone to development of infectious airway disease while hospitalized. the use of mechanical ventilation in human patients raises the risk of nosocomial infection by 6-fold to 20-fold. 9 no published studies assess the risk in ventilated veterinary patients, although a study investigating differences in bacterial sensitivity between ventilated and nonventilated patients suggested that patients requiring mechanical ventilation were more likely to be infected with bacteria resistant to the antimicrobials most commonly used in veterinary practice. 8 this finding parallels the increase in incidence of multidrug-resistant vap in human medicine. 9 both the innate and adaptive immune systems protect against the development of infectious airway disease, and a breakdown in either increases the likelihood of opportunistic infection ( table 2 ). congenital immunodeficiencies have been recognized that make an animal particularly sensitive to infectious disease. young animals are especially prone to the development of bacterial pneumonia because of their naive immune systems, and when coupled with alterations to the innate immune system, such as primary ciliary dyskinesia (pcd) or complement deficiency, the risk of life-threatening infection increases greatly (see veterinary clinics of north america 2007;37(5):845-60 for a comprehensive review of respiratory defenses in health and disease). any cause of systemic immunocompromise increases the risk for bacterial pneumonia, and any additional alterations to the body's natural defense mechanisms increase the risk. medications such as chemotherapy, immunosuppressive therapy, or antitussive therapy significantly increase the likelihood of bacterial pneumonia. organisms that have been reported as lower respiratory pathogens of cats include pasteurella spp, escherichia coli, staphylococcus spp, streptococcus spp, pseudomonas spp, b bronchiseptica, and mycoplasma spp, 36 and specific attention has been paid to mycoplasma spp because of a possible association with the induction and exacerbation of asthma in adult and pediatric human patients. 37 however, the association between lower respiratory infection and chronic inflammatory lower airway disease in cats is unclear and is a topic of ongoing interest. mycoplasma species are considered normal flora in the upper respiratory tract and their role is controversial in lower respiratory tract infection. because they are rarely identified cytologically, and specific culture or polymerase chain reaction is needed to document the presence of these organisms, the role of mycoplasma in cats (as well as in dogs) remains difficult to define. underlying respiratory viruses or systemic viruses such as feline leukemia virus and feline immunodeficiency virus have the potential to enhance the severity of respiratory illness. clinical signs of bacterial pneumonia vary depending on its cause, severity, and chronicity of disease. they can be acute or peracute in onset or can display an insidious onset, resulting in chronic illness. early in disease, mild signs such as an intermittent, soft cough might be the only evidence of disease. as infection spreads, clinical signs worsen and often include a refractory, productive cough, exercise intolerance, anorexia, and severe lethargy. owners can note a change in the respiratory pattern, with increased panting or rapid breathing and, in cases of severe infection, cyanosis and orthopnea can be observed. in general, these systemic signs are more obviously displayed in dogs than in cats. cats with pneumonia can display similar clinical signs, although the cough can be misinterpreted as a wretch or vomit by owners. clinical signs and radiographic findings can also be considered consistent with inflammatory airway disease. as disease worsens, cats can become tachypneic with short, shallow breaths and nasal flaring. 10 owners rarely notice exercise intolerance associated with bacterial pneumonia. as with the history and clinical signs of bacterial pneumonia, physical examination findings vary with the state and severity of disease. dogs or cats with mild disease can have no abnormalities detected on physical examination. an early clue to the diagnosis might be a change in the respiratory pattern, with an increase in rate and effort. the clinician needs to pay close attention to thoracic auscultation because adventitious lung sounds (crackles and wheezes) can be subtle, focal, or intermittent. in many cases, only harsh or increased lung sounds are detected rather than crackles. 5 the examination should also include a thorough auscultation of the trachea and upper airway for evidence of upper airway signs (eg, nasal congestion or discharge) that can result from lower airway infection, either as an extension of epithelial infection or from nasopharyngeal regurgitation of lower airway secretions. animals with bacterial pneumonia generally present with mixed inspiratory and expiratory signs, similar to those seen with other diseases of the pulmonary parenchyma. fever is detected in 16% to 50% of cases, so it is not a reliable indicator of disease. 4, [11] [12] [13] diagnosis bacterial pneumonia implies sepsis of the lower airway and lungs, so the diagnosis is confirmed by showing septic suppurative inflammation on airway cytology obtained through bronchoalveolar lavage (bal) or tracheal wash, along with a positive microbiology culture. in some cases, this is completed easily and yields results consistent with clinical suspicion. however, financial limitations or patient concerns can inhibit the ability to collect samples needed to document specifically a bacterial infection, and in those cases a clinical diagnosis of bacterial pneumonia might be presumed based on available information. a clinical diagnosis of bacterial pneumonia should be reached after obtaining compelling evidence to suggest a bacterial cause for the animal's clinical signs (after excluding other causes), with appropriate resolution of signs following suitable antimicrobial therapy. acute bacterial pneumonia is a common diagnosis in the small animal clinic and can often be easily identified; however, early and chronic pneumonias are more challenging to recognize because clinical signs can be subtle. the complete blood count is a useful diagnostic test in animals with respiratory signs. bacterial pneumonias are typically associated with an inflammatory leukogram, characterized primarily by a neutrophilia, with or without a left shift and variable evidence of toxic changes, 7,14 although the absence of inflammatory change does not exclude the possibility of pneumonia. 4, 11 furthermore, the leukogram and differential can provide clues to suggest that bacterial pneumonia is less likely. for example, eosinophilia in an animal with respiratory signs would suggest eosinophilic bronchopneumopathy or parasitic lung diseases as an underlying cause rather than a bacterial cause. the erythrogram and platelet evaluation are generally not helpful in determining a bacterial cause of respiratory disease. a biochemistry panel, urinalysis, and fecal flotation do not always contribute to the diagnosis of bacterial pneumonia but can provide clues to the presence of metabolic or endocrine diseases that could make the development of bacterial pneumonia more likely. thoracic radiographs are crucial diagnostic tests in the evaluation of lower airway and pulmonary parenchymal disease. radiographic evidence of bacterial pneumonia can appear as a focal, multifocal, or diffuse alveolar pattern, although early in the disease process infiltrates might be primarily interstitial (figs. 1 and 2) . ventral lung lobes are most commonly affected in aspiration pneumonia, and a caudodorsal pattern is expected with inhaled foreign bodies or hematogenous bacterial spread. a lobar sign can be seen in cases of aspiration pneumonia in which the right middle lung lobe is most often affected ( table 3) . three-view thoracic radiographs (left lateral, right lateral, and either dorsoventral or ventrodorsal views) should be obtained when screening for pneumonia because differential aeration associated with positional atelectasis can either mask or highlight pulmonary changes. for example, a radiograph taken in left lateral recumbency is preferred when aspiration is suspected because it increases aeration of the right middle lung lobe, the most commonly affected lobe. diffuse radiographic involvement is expected to suggest more severe disease, although radiographic changes lag behind clinical disease. consequently, bacterial pneumonia cannot be ruled out in patients with acute onset of clinical signs and unremarkable radiographs. 7 advanced imaging is rarely necessary in the diagnosis of uncomplicated bacterial pneumonia, although it can be helpful in more complicated cases. thoracic ultrasound can be used to characterize peripheral areas of consolidation and to obtain fine-needle aspirates for cytology. cytology is often helpful in distinguishing inflammation from neoplastic infiltration. in addition, sonographic evaluation is particularly fig. 1 . dorsoventral (a) and right lateral (b) thoracic radiographs from a dog with an alveolar pattern in the cranioventral lung lobes, suggesting aspiration. in this case, the left cranial lobes were most affected which are most easily examined on the right lateral view. in many cases the right middle lung lobe is most affected, necessitating a left lateral orthogonal view. useful in the detection of superficial foxtail foreign bodies when they remain in the periphery of the lobe (fig. 3) . 10 ct provides greater detail and resolution of lesions within the pulmonary parenchyma and gives the clinician better spatial information regarding the severity and extent of pulmonary involvement (fig. 4) . in some cases, ct can be useful to identify migration tracts associated with inhaled foreign bodies. 7 however, in most cases, general anesthesia is required for ct acquisition and prolonged recumbency can dear lead to atelectasis, which is difficult to differentiate radiographically from infiltrates. repeating the ct in a different position after providing several maximal inspirations can alleviate atelectasis. nuclear scintigraphy can be useful for the evaluation of ciliary dyskinesia, although secondary causes of mucociliary stasis (ie, infection with mycoplasma or bordetella, as well as exposure to smoke) must be excluded before assuming the diagnosis of pcd. because of the time necessary for image acquisition, magnetic resonance imaging is not commonly used for the diagnosis of most respiratory diseases. examination of the trachea and bronchial tree should be performed systematically. the endoscopist should note the color and character of the mucosa and any airway sections, making sure to evaluate all branches of the lower airways for evidence of foreign bodies, bronchiectasis, or collapse (diffuse or focal changes). airway mucosa in a normal animal should be pale pink with visible mucosal and pulmonary vessels. airway bifurcations should appear as narrow, crisp mucosal margins. animals with pneumonia can have hyperemia of the epithelium, prominent mucosal vessels, and evidence of airway inflammation, appearing as rounded, thickened airway bifurcations and airway nodules. airway secretions are usually opaque, viscous, and discolored (brown, yellow-green, or red tinged). when available, bal is preferred for collection of a lower airway sample rather than tracheal wash because the trachea and carina are not sterile, even in healthy dogs. 15 in addition, the sensitivity for detecting cytologic features of sepsis is greater with bal than tracheal wash. 12 however, when only a tracheal wash specimen can be obtained, because of the lack of equipment for bal or because of patient instability, collection of a lower airway sample is desirable to identify infecting bacteria and to determine appropriate antibiotic therapy through susceptibility testing. fig. 4 . ct image of a dog with severe, diffuse pneumonia resulting from a chronic foxtail foreign body (see fig. 3 ). the foreign body was not visible on thoracic radiographs, but is clearly evident in the left principal bronchus on this image. bal cell counts in animals with bacterial pneumonia are markedly higher than in patients with chronic bronchitis or other respiratory disease. 14 septic, suppurative inflammation is a reliable indicator of bacterial pneumonia in dogs 14 and is likely to indicate bacterial pneumonia in cats. in those cases that lack evidence of airway sepsis (intracellular bacteria), bal cytology generally reveals suppurative or mixed inflammation. 13 in animals with suspected or confirmed foreign bodies, a bal sample should always be obtained from the affected airway and submitted individually for cytologic analysis. airway bacteria are more likely to be found in the cytologic sample from the site of the foreign body than from an alternate site. 8 furthermore, cytology of bal samples obtained from multiple lobes can reveal different findings, even in cases of sterile inflammatory diseases like feline bronchial disease, thus reliance on a single-segment bal cytology could lessen the chance of yielding diagnostic results. 16 diagnosis of bacterial pneumonia relies on identification of septic inflammation in conjunction with a positive bacterial culture. aerobic and mycoplasma culture and sensitivity are typically requested, and, in cases with markedly purulent secretions or a history of known aspiration or foreign bodies, anaerobic cultures should also be requested. samples should be refrigerated in sterile containers until submitted. if multiple alveolar segments are sampled during bal, these are usually are pooled for culture submission. cultures should always be performed when possible in order to guide appropriate antimicrobial therapy. with the liberal use of antibiotics, increasing populations of resistant microbes are being identified, particularly in patients with hospitalacquired pneumonia. 17, 18 however, airway samples cannot be collected in all animals and, in those instances, judicious use of antibiotics must be followed. common bacteria cultured from lung washes of cats or dogs with bacterial pneumonia include enteric organisms (escherichia coli, klebsiella spp), pasteurella spp, coagulase-positive staphylococcus spp, beta-hemolytic streptococcus spp, mycoplasma spp, and b bronchiseptica ( table 4) . 4, 13, 27 pulmonary function testing arterial blood gas analysis is a useful test to measure the lung's ability to oxygenate. for patients with significant respiratory compromise, arterial blood samples ideally should be collected and analyzed to determine the severity of pulmonary disease. furthermore, trends in arterial oxygen partial pressures can be used to track progression or resolution of disease. in many cases, blood gas analysis is not available or patient factors preclude the acquisition of samples. pulse oximetry is a quick, noninvasive evaluation of oxygen delivery to body tissues that measures percentage of hemoglobin saturation with oxygen. it provides only a crude assessment of oxygenation and is subject to variability; however, trends in hemoglobin saturation can provide additional clinical support to progression or resolution of disease. treatment treatment of bacterial pneumonia varies considerably with the severity of disease, and appropriate antibiotic therapy is essential. the international society for companion animal disease is currently constructing guidelines for antibiotic therapy for respiratory infections. pending those guidelines, antibiotic recommendations from previous literature should be considered ( table 5) . for stable animals with mild disease, outpatient therapy consisting of administration of a single, oral antibiotic is often all that is necessary. antimicrobial choices should ideally be based on culture and sensitivity results from airway lavage samples, although sometimes empiric therapy is more practical. regardless, in cases of severe pneumonia, initial empiric therapy should be instituted while awaiting culture results. antibiotics are typically administered for 3 to 6 weeks, and at least 1 to 2 weeks beyond the resolution of clinical and/or radiographs signs of disease. animals with more advanced disease require more intensive care, including hospitalization with intravenous fluids to maintain hydration. adequate hydration is essential to facilitate clearance of respiratory exudates. nebulization to create particles that enter the lower airways (<5 mm) can also enhance clearance of secretions. nebulizer types include ultrasonic devices, compressed air nebulizers, and mesh nebulizers. nebulization with sterile saline can be achieved by directing the hosing from the pneumonia in dogs and cats nebulizer into a cage or animal carrier covered in plastic. depending on how viscous secretions are, therapy can be provided for 15 to 20 minutes 2 to 4 times daily. in many cases, nebulization coupled with coupage helps the animal expectorate airway secretions. coupage is performed by cupping the hands and gently and rhythmically pounding on the lateral thoracic walls in dorsal to ventral and caudal to cranial directions. coupage should not be performed in animals with regurgitation because any increase in intrathoracic pressure could exacerbate regurgitation and subsequent reaspiration. supplemental oxygen is necessary for animals with moderate to marked hypoxemia (documented by a pao 2 less than 80 mm hg or oxygen saturation via pulse oximetry less than 94% on room air) in conjunction with increased respiratory effort. oxygen supplementation at 40% to 60% is provided until respiratory difficulty lessens and the animal can be weaned to room air. animals with refractory pneumonia that fail to improve on supplemental oxygen can succumb to ventilatory fatigue and need to be referred to an intensive care facility for mechanical ventilation. administration of an oral mucolytic agent such as n-acetylcysteine can be useful for animals with moderate to severe bronchiectasis that are prone to recurrent pneumonia. decreasing the viscosity of airway secretions might improve expectoration of fluid and debris that accumulates in dependent airways, although no published information is available on use of mucolytics in animals. n-acetylcysteine is typically not used via nebulization because of the risk of bronchoconstriction and epithelial toxicity. under no circumstances are cough suppressants (such as butorphanol or hydrocodone) appropriate for use in the management of bacterial pneumonia, particularly when it is complicated by bronchiectasis. by decreasing the cough reflex, these drugs perpetuate retention of mucus, debris, and other material in the airways and therefore hinder clearance of infection. also, furosemide should not be used because drying of secretions traps material in the lower airway and perpetuates infection. in cases in which aspiration pneumonia is suspected, strategies should be used to reduce the chance of reaspirating through appropriate treatment of the underlying condition. with disorders of esophageal motility, upright feedings of either slurry or meatballs can enhance esophageal transit. furthermore, diets low in fat can increase gastric emptying. in patients with refractory vomiting, antiemetic and prokinetic agents can be used to reduce the episodes of vomiting. drugs like maropitant (cerenia; 1 mg/kg subcutaneously once daily) or ondansetron (zofran; 0.3-1 mg/kg intravenously or subcutaneously once to twice daily) act peripherally and centrally to decrease the urge to vomit and are safe to use in both cats and dogs. the role of antacids in management of aspiration pneumonia remains controversial. by neutralizing the ph of gastric secretions, animals with refractory vomiting or regurgitation are less likely to succumb to chemical injury related to aspiration. however, in cases treated with acid suppression, the aspirant is likely to contain a greater concentration of bacteria that can colonize the lower airways and lead to bacterial pneumonia. no controlled studies have assessed the severity of aspiration pneumonia or the relative risk of using antacid therapy in dogs or cats. because radiographic findings lag behind clinical disease, recheck radiographs are not helpful early into the disease process, although they are useful to document resolution of disease and should be obtained within a week of discontinuation of antimicrobial therapy. in cases of refractory pneumonia, recheck radiographs midway through therapy can be used to assess resolution or progression of disease and help to guide further therapy. in animals suspected of having contagious or multidrug-resistant pathogens, appropriate contact precautions should be used. isolation gowns, examination dear gloves, and good hand washing technique along with appropriate quarantine facilities are essential to preventing transmission of disease to other patients or members of the health care team. prognosis for animals with bacterial pneumonia varies depending on the severity of disease, the animal's immunocompetence, and the virulence of the infectious agent. in general, between 77% and 94% of patients diagnosed with pneumonia are discharged from the hospital. 4, 19 no long-term studies assess the overall prognosis of patients with multidrug-resistant bacteria or recurrent pneumonia. the outcome associated with these cases presumably will be worse. an 8-year-old female spayed chihuahua mix presented for a wet cough. cough had been present for 4 months and there had been minimal response to antibiotics combined with a cough suppressant and no response to heart failure medication (furosemide, enalapril, and pimobendan). temperature (38.9 c [102 f]), pulse (140 beats per minute [bpm]), and respiratory rate (30 breaths per minute) were normal. no murmur was auscultated and lung sounds were normal. chronic cough in a small-breed dog is often associated with airway collapse or chronic bronchitis; however, infectious and neoplastic disease must remain on the differential list. congestive heart failure is unlikely given the lack of a heart murmur and the lack of response to diuretic therapy. a white blood cell count was normal (5800 cells/ml) with 4400 neutrophils. thoracic radiographs revealed scattered bronchial markings in the caudal thorax (fig. 5) . fluoroscopic examination did not reveal evidence of tracheal or airway collapse. laryngoscopy indicated lack of abduction of the arytenoid cartilages consistent with bilateral laryngeal paralysis. secretions were evident throughout the upper and lower airways. diffuse airway hyperemia and irregularities of the mucosa were apparent. bal cytology was remarkable for septic suppurative inflammation, and bacterial cultures were positive for pasteurella, mycoplasma spp, and anaerobic bacteria, consistent with an aspiration cause. a 5-year-old male castrated domestic medium hair cat was presented for evaluation of acute respiratory distress. lethargy and anorexia had been noted 3 days before the onset of respiratory signs. temperature (38.7 c [101.6 f]) and pulse (210 bpm) were normal. tachypnea was noted (respiratory rate, 60 breaths per minute) with increased respiratory effort on inspiration and expiration. diffuse expiratory wheezes were auscultated. acute onset of respiratory difficulty in a cat is most commonly related to inflammatory airway disease. the physical examination is consistent with this diagnosis, although it is uncommon for affected cats to show lethargy and anorexia. infectious and neoplastic diseases were also on the differential diagnosis list, along with aspiration and foreign body pneumonia. thoracic radiographs revealed a focal opacity in the left caudal lung lobe and a diffuse bronchial pattern (fig. 6) . complete blood count revealed a normal white blood cell count (8500/ml) with a left shift (6800/ml neutrophils, 1000/ml bands). bronchoscopy with lavage was performed. a moderate amount of airway hyperemia and edema was noted along with purulent material obstructing several airways. bal cytology showed increased cellularity (1500, normal 500 cells/ml) with neutrophilic inflammation (84%, normal 5%-8%). neutrophils contained dark blue granular debris, suspicious for sepsis. aerobic and anaerobic cultures were negative but a pure culture of mycoplasma was isolated on special medium. a diagnosis of mycoplasma bronchopneumonia was made. community-acquired aspiration pneumonia in intensive care units. epidemiological and prognosis data potential risks, prognostic indicators, and diagnostic and treatment modalities affecting survival in dogs with presumptive aspiration pneumonia: 125 cases mycoplasmas and novel viral pathogens in canine infectious respiratory disease community-acquired infectious pneumonia in puppies: 65 cases capnocytophaga cynodegmi in a rottweiler dog with severe bronchitis and foreign-body pneumonia the role of bronchoscopy in foreign body removal in dogs and cats: 37 cases radiographic, computed tomographic, and ultrasonographic findings with migrating intrathoracic grass awns in dogs and cats airway microbial culture and susceptibility patterns in dogs and cats with respiratory disease of varying severity ventilator-associated tracheobronchitis and pneumonia: thinking outside the box bordetella bronchiseptica infection in cats. abcd guidelines on prevention and management clinical, clinicopathologic, and radiographic findings in dogs with aspiration pneumonia: 88 cases cytological analysis of bronchoalveolar lavage fluid in the diagnosis of spontaneous respiratory tract disease in dogs: a retrospective study microbiologic and cytologic assessment of bronchoalveolar lavage fluid from dogs with lower respiratory tract infection: 105 cases quantitative bacterial cultures and cytological examination of bronchoalveolar lavage specimens in dogs bacterial isolates from the lower trachea of clinically healthy dogs interpretation of multisegment bronchoalveolar lavage in cats genetic diversity of streptococcus equi subsp. zooepidemicus and doxycycline resistance in kennelled dogs molecular epidemiology of feline bordetellosis in two animal shelters in california, usa etiology and clinical outcome in dogs with aspiration pneumonia: 88 cases a serological survey of canine respiratory coronavirus and canine influenza virus in korean dogs clinical factors associated with death before discharge and overall survival time in dogs with generalized megaesophagus age-related thoracic radiographic changes in golden and labrador retriever muscular dystrophy primary ciliary dyskinesia in newfoundland dogs immunoglobulin deficiency in cavalier king charles spaniels with pneumocystis pneumonia x-linked severe combined immunodeficiency in the dog comparison of clinical signs, diagnostic findings, organisms isolated, and clinical outcome in dogs with bacterial pneumonia: 93 cases (1986-1991) a clonal outbreak of acute fatal hemorrhagic pneumonia in intensively housed (shelter) dogs caused by streptococcus equi subsp. zooepidemicus textbook of respiratory disease in dogs and cats. st louis (mi): saunders the association of streptococcus equi subsp zooepidemicus with canine infectious respiratory disease the seroprevalence of canine respiratory coronavirus and canine influenza virus in dogs in new zealand tropism and pathological findings associated with canine respiratory coronavirus (crcov) nosocomial outbreak of serious canine infectious tracheobronchitis (kennel cough) caused by canine herpesvirus infection recent advances in canine infectious disease. international veterinary information service mycoplasmas associated with canine infectious respiratory disease streptococcus zooepidemicus: an emerging canine pathogen lower respiratory tract infections in cats: 21 cases (1995-2000) mycoplasma pneumoniae in children with acute and refractory asthma key: cord-304876-txaoz7oh authors: jordan, paul c; stevens, sarah k; deval, jerome title: nucleosides for the treatment of respiratory rna virus infections date: 2018-03-21 journal: antivir chem chemother doi: 10.1177/2040206618764483 sha: doc_id: 304876 cord_uid: txaoz7oh influenza virus, respiratory syncytial virus, human metapneumovirus, parainfluenza virus, coronaviruses, and rhinoviruses are among the most common viruses causing mild seasonal colds. these rna viruses can also cause lower respiratory tract infections leading to bronchiolitis and pneumonia. young children, the elderly, and patients with compromised cardiac, pulmonary, or immune systems are at greatest risk for serious disease associated with these rna virus respiratory infections. in addition, swine and avian influenza viruses, together with severe acute respiratory syndrome-associated and middle eastern respiratory syndrome coronaviruses, represent significant pandemic threats to the general population. in this review, we describe the current medical need resulting from respiratory infections caused by rna viruses, which justifies drug discovery efforts to identify new therapeutic agents. the rna polymerase of respiratory viruses represents an attractive target for nucleoside and nucleotide analogs acting as inhibitors of rna chain synthesis. here, we present the molecular, biochemical, and structural fundamentals of the polymerase of the four major families of rna respiratory viruses: orthomyxoviridae, pneumoviridae/paramyxoviridae, coronaviridae, and picornaviridae. we summarize past and current efforts to develop nucleoside and nucleotide analogs as antiviral agents against respiratory virus infections. this includes molecules with very broad antiviral spectrum such as ribavirin and t-705 (favipiravir), and others targeting more specifically one or a few virus families. recent advances in our understanding of the structure(s) and function(s) of respiratory virus polymerases will likely support the discovery and development of novel nucleoside analogs. respiratory viral infections are a global health concern caused by dozens of different types of viruses. the respiratory diseases resulting from these viral infections represent one of the main causes of death in developing countries. 1 a more thorough understanding of respiratory viruses, their epidemiology, as well as medical impact on the communities they affect will delineate the path toward eventual treatments and future abatement of the illnesses. while symptoms of many respiratory viruses are similar, the viruses themselves are characteristically unique. categorically, viruses are grouped based on similarities such as the nature of their nucleic acid genome, envelope presence, overall size, and even capsid uniformity. 2 this review focuses on the following families of rna viruses: orthomyxoviridae, paramyxoviridae and pneumoviridae, picornaviridae, and coronaviridae. orthomyxoviridae comprise negative (-) sense singlestranded (ss) rna viruses that are segmented, enveloped, and includes the influenza viruses (see table 1 ). paramyxoviridae and pneumoviridae are also (-)ssrna viruses, but are non-segmented and enveloped, and include parainfluenza virus (piv), human respiratory syncytial virus (rsv), and human metapneumovirus (hmpv). the picornaviridae family, which contains positive (þ)ssrna viruses are non-enveloped; the key members include the rhinoviruses and enteroviruses. lastly, the coronaviridae are (þ)ssrna enveloped viruses, which include, chiefly, human coronavirus (hcov), and severe acute respiratory syndrome (sars)-associated and middle eastern respiratory syndrome (mers) cov. 3 young children, the elderly, and patients with compromised cardiac, pulmonary, or immune systems are at greatest risk for serious disease associated with these rna virus respiratory infections. in a 10-year study, over 85% of acute respiratory viral infections in critically ill children admitted to a pediatric intensive care unit were caused by either a picornavirus, rsv, piv, or hmpv (see figure 1 ). 4 other dna viruses such as adenovirus can be the source of respiratory infections but will not be discussed here. in addition to their wide variation in viral characteristics, respiratory rna viruses are also remarkable in their epidemiological variety. they differ in (1) their outbreak calendar, where some are seasonal and others are year-round, (2) their patient profile, whether infant, geriatric, or otherwise healthy adults, and (3) the morbidity and/or mortality associated with infection. influenza virus is a (-)ssrna virus and a member of the orthomyxoviridae family. 5 there are four influenza genera within this family, called a, b, c, or d. influenza a and b contain hemagglutinin and neuraminidase envelope glycoproteins. influenza c and d have a single surface glycoprotein called the hemagglutinin-esterase fusion protein. 6, 7 antigenic variation in these glycoproteins results in limited vaccine protection. influenza, or the flu, presents with symptoms such as headache, cough, fever, sore throat, malaise, and chills. 8 generally, the flu lasts from 5 days to 2 weeks and the severity of infection is determined by the host. the highest incidence of influenza infection occurs in younger patients (<25 years old) where a shorter infection is typical, while those at risk for longer and more severe illness and complications associated with infection are the pediatric (<2 years old) and geriatric populations (>65 years old), pregnant women, and immunocompromised individuals. 9, 10 it is estimated that 3-5 million cases of the flu occur annually around the globe, with a quarter to half million deaths resulting from these illnesses. 11 piv (paramyxoviridae family), rsv, and hmpv (pneumoviridae family) until recently, piv, hmpv, and rsv were all categorized in the paramyxoviridae family due to their phylogenetic proximity in the order mononegavirales, the non-segmented negative-strand rna viruses. more recently, rsv and hmpv have been assigned as members of the newly formed pneumoviridae family. 12 while influenza outbreaks are most prevalent in the winter, some viruses such as piv persist year-round. human piv has four types (1 to 4) and was known historically to induce respiratory complications mainly in children and the immunocompromised; however, more recently, it has been identified as a concern in the adult population as well. 13 symptoms of piv include upper and lower respiratory tract infection, middle ear inflammation, bronchitis, pneumonia, and croup, the last of which results in the most hospitalizations in the pediatric patients infected by this virus. 14, 15 up to one-third of the nearly 5 million annual cases of lower respiratory tract infection in children is at least partially due to the presence of pivs. 16 rsv and piv infections are among the most common reason for hospitalization of young children. 17, 18 the two strains of rsv, a and b, are distinguished by genetic variations in the g surface glycoprotein. 19 dissimilar to piv, rsv occurs mostly in the winter months in its target pediatric population. symptoms include runny nose, nasal inflammation, cough, sore throat, low-grade fever, wheezing, bronchiolitis, and pneumonia. 20 current estimates in developing and industrialized countries suggest as many as 33 million cases of rsv worldwide in the pediatric population less than 5 years old, 10% of which require hospitalization, and 2% to 18% of hospitalized cases result in mortality. this amounts to between 66,000 and 600,000 deaths in young children annually. 18, 21 hmpv, like rsv and influenza, tends to have greatest prevalence in the winter and studies have shown that by the age of 5 years, nearly all children have been infected with this virus. 22 the clinical manifestations of infection with this virus are upper and lower respiratory tract infections, bronchiolitis, middle ear inflammation, fever, chills, pneumonitis, and wheezing. 23 of note, hmpv tends to occur in populations with seasonal inconsistency as studies done on italian populations shortly after its discovery from 2000-2002, showed a range of infection from 7% to 40% depending on the year. patterns of seasonal irregularity like this have been noted with other respiratory viruses, particularly rsv and influenza. 24 rhinoviruses are thought to be the cause of up to twothirds of what is termed the common cold, worldwide. children tend to experience up to 12 of these infections, or colds, per year, while this incidence drops in adults to just 2-3 per year. 25 there are three distinct species of rhinovirus, rv-a, rv-b, and rv-c, each of which infects humans at different periods throughout the year. 26 symptoms include cough, fever, sneezing, nasal congestion, sore throat, fever, and headache and usually last 7-10 days after an initial 48-h viral incubation. 27 in addition to the three rhinoviruses, four enterovirus species result in disease in humans, ev-a, ev-b, ev-c, and ev-d, while ev-e through ev-h, and ev-j affect non-human hosts. 28, 29 enteroviruses differ from rhinoviruses in that while rhinoviruses are limited to the respiratory airways, enteroviruses infect a wide range of cell types. they result in a large array of complications associated with the respiratory, gastrointestinal, and central nervous systems. manifestations of enterovirus infection range from a febrile cold to encephalitis, pneumonia, viral meningitis, and even death. 30 although ev-c and ev-d are the principal enteroviruses that cause respiratory illness, ev-a also includes ev71. ev71 causes hand-foot-and-mouth disease, a highly contagious pathogen in children that mainly results in a maculopapular rash, blisters on the limbs, and ulcers in the mouth. 31 ev71 is most prevalent in the summer months and tends to be more ubiquitous in tropical zones of the globe. in rare cases of severe ev71 infection, respiratory illness can lead to pulmonary edema, hemorrhage, and lung failure. 32 presently, six hcov are recognized: hcov-229e, hcov-nl63, hcov-oc43, hcov-hku1, and the well-known sars and mers cov. 33 these cov can be further characterized based on genera of alpha, beta, gamma, or delta; the alpha and beta cov comprise the six viruses mentioned above, and are those that infect humans. cov 229e and oc43 are both pathogens associated with the common cold, but can cause pneumonia as well. 34 hcov-nl63 and hcov-hku1 infection show similar clinical features to those in patients with 229e and oc43, but clinicians have also reported bronchiolitis, croup, and pneumonia in infected individuals. 35, 36 the first cov recognized as pandemic threat is sars-cov. sars was discovered in 2002-2003 after a perplexing epidemic of pneumonia among hospital workers in china. 37 by the end of its global epidemic, sars disseminated to 29 countries, infecting over 8000 individuals, and killing roughly 10% of those infected. 38 roughly a decade later, a similar pattern occurred with mers, which began in 2012 in jordan with an outbreak of a respiratory illness among hospital workers, one of whom died of the infection. later that year, a man with pneumonia and multiple organ failure in saudi arabia was found to have the mers pathogen. 39 adults are the target population for both sars and mers with a median age range of 39-50 years; mers occurs predominantly in men whereas sars does not. the clinical features of both sars and mers range from mild to severe respiratory illness, fever, chills, cough, shortness of breath, vomiting, and diarrhea, with the latter displaying a more lethal pneumonia and renal failure. 40, 41 even worse than sars mortality, retrospective analysis has shown that of the 2040 confirmed cases of mers, 35% were fatal. 42 viral polymerase: an important molecular target for antiviral therapy nucleoside analogs represent one of the dominant classes of antiviral agents due to their widespread use against the common chronic infections caused by human immunodeficiency virus (hiv), hepatitis b virus, and herpesviruses. in the past 15 years, multiple nucleoside and nucleotide analogs have been developed as direct-acting agents against rna virus infections such as hepatitis c virus (hcv), but have not yet been successfully applied to acute infections caused by respiratory viruses. only a handful of nonnucleoside drugs have been developed for the treatment and prevention of these viruses. such drugs include the fda approved oseltamivir, zanamivir, and peramivir for influenza virus infection, palivizumab for rsv prevention, as well as the two discontinued clinical candidates targeting rhinovirus, pleconaril and rupintrivir. these molecules possess limitations preventing their widespread use, such as short therapeutic window and risk of resistance selection for the neuraminidase inhibitors, and only partial protection associated with prophylactic use for palivizumab. this has provided the impetus for the approval of new drugs with a broader therapeutic use. the recognized advantages of direct-acting agent nucleosides over other classes of antiviral agents are (1) their propensity to cover a broad-spectrum of virus strains and sometimes species and (2) their high barrier to antiviral resistance. both properties are best explained by the mechanism of action common to most antiviral nucleosides: targeting viral polymerases. 43 after being metabolized by host kinases to their triphosphate form, antiviral nucleotides compete with natural nucleoside triphosphates (ntps) to bind to the active site of viral polymerases and alter dna or rna synthesis. the nucleotide binding site of these proteins is usually well conserved among virus families and any changes in neighboring amino acids often comes at a cost for the enzyme and the virus. the functional and structural features of rna polymerases of respiratory viruses targeted by antiviral nucleosides are described in the following paragraphs. influenza virus is a (-)ssrna virus and a member of the orthomyxoviridae family. 5 the viral genome has eight segments in influenza a and b and seven segments in influenza c and d. in influenza a, these encode for 11 or 12 proteins. these are non-structural protein 1 (ns1), non-structural protein 2 (ns2), matrix protein m1 and ion channel protein m2, polymerase acidic (pa) protein, polymerase basic protein (pb1), polymerase basic protein 1-f2, polymerase basic protein 2 (pb2), nucleoprotein (np), hemagglutinin (ha), and neuraminidase (na). 44 some viruses express the pb1-n40 protein. all four species of influenza adopt similar arrangements with the viral genomic segments forming viral ribonucleoprotein complexes associated with one heterotrimeric polymerase. influenza a polymerase is composed of three subunits that yield a 270 kda heterotrimeric complex. the longest viral rna segments encode for the pa protein, pb1, and pb2, which assemble to form the influenza polymerase complex (see figure 2 ). 45 the three subunits interact noncovalently to exert their polymerase activity. the polymerase transcribes viral rna into messenger rna (mrna) and then replicates it using a complementary rna intermediate. 5 the process of transcription includes cap snatching, where short-capped cellular rna are bound by the pb2 subunit, cleaved from the pa endonuclease domain, and then utilized for priming mrna synthesis by the pb1 domain. [46] [47] [48] [49] [50] [51] the pb2 subunit has an n-terminal domain (pb2-n) from residues 1 to 247 and a c-terminal domain (pb2-c) from residues 248 to 760. 52 pb2-n, including a lid domain, interacts with the c-terminal extension and thumb of pb1. 52 the pb2-c includes several notable structural features and subdomains such as the c-terminal nuclear localization signal, the pb2 627-domain, the pb2 cap-627 linker, the mid domain, and a cap binding domain. 46 based on structural biology, the pb2 domain has a key exterior, positively charged residue at the 627 position within a flexible loop that partially wraps around an alpha helix to form what is known as a phi-loop. 53 importantly, this residue is in the middle of a set of highly conserved, basic residues forming a net positive charge. a signature structural element is the conserved p[f/p]aaapp motif on the n-terminal side of the 627 residue that is part of the alpha helix previously described. 53 mutation of the p620 or f621 residue significantly decreased the ability of the virus to replicate, presumably by causing a slight kink in the alpha helix that alters the polymerase function. the exact role of the pb2 627 domain remains unclear, but recent evidence suggests it is not necessary for in vitro binding and transcription of viral rna; this has not been proven true in a cell-based format. 54 the pb1 domain is at the center of the polymerase complex and within its center is a classic right-handed shape with the signature fingers, palm, and thumb subdomains (see figure 2 ). 5 these subdomains are described as conserved rna-dependent rna polymerase (rdrp) motifs pre-a/f and a-e. 52 the pre-a/f motif describes the fingertips and a small loop, which spreads over to the thumb domain; the tip of this loop is stabilized by an alpha helix within the pa domain. residues within the pre-a/f may guide and bind ntps and the incoming template. in addition to these subdomains, n-terminal and c-terminal extensions interact with pa and pb2 domains. the fingers and palm are covered by a linker connecting the two subdomains of pa. the pb1 possesses a b-hairpin loop within motif e from residues 641 to 657 in the thumb subdomain of influenza a. 46 in de novo initiation, it has been shown with other related polymerases that this priming loop may serve as a platform for the initial ntp on the 3 0 end of the template and ensure against double-stranded rna. biochemical investigations have shown that the proline found within the loop tip motif of 648-ala-his-gly-pro is necessary for in vitro and cell-based rna synthesis. this loop may also be necessary during replication mode for terminal de novo initiation but unnecessary for internal initiation and transcription. 55 the pa endonuclease domain or the pa subunit, as it will be named here, has little homology to other proteins and its exact enzymatic function was discovered only recently. the subunit was expressed in insect cells to reveal an n-terminal third (pa-nter) and a c-terminal two-thirds (pa-cter) subdomains. they have molecular weights of 25 and 55 kda, respectively. these two subdomains are connected by a flexible linker. the endonuclease activity was originally thought to occur through the pb1 or pb2 domains but the structure of the pa-nter was solved by two groups to reveal that the catalytic residues for endonuclease activity reside in the pa domain, not in the pb1 subunit as originally thought. 50, 51 the pa endonuclease domain contains a signature (p)dxn(d/e)xk motif that is conserved among influenza viruses and coordinate divalent cations such as magnesium or manganese. 56 although the exact quantity and identity of ions present in the native influenza enzyme are unclear, the endonuclease activity is strongly activated by metal ion binding through hydrolysis of ssdna and ssrna substrates. human metapneumovirus and human rsv are nonsegmented, negative strand rna viruses from the pneumoviridae family of the order mononegavirales. the polymerases from this class of viruses are multifaceted with multiple enzymatic functions contained within a single protein. it exists as part of a ribonucleoprotein complex, or replicase, composed of l, n, p, and m2-1 proteins in complex with rna. these include rna synthesis activities carried out by an rdrp domain but also include capping and methylation functions. the rdrp carries out transcription and replication of its genome in response to cis-acting elements within the genome. 57 the genome of rsv is approximately 15 kilobases long and is transcribed into capped and poly-adenylated mrnas. 58 the hmpv genome is 13 kilobases long. current understanding of hmpv transcription is based on knowledge gained from the more extensive characterization of rsv transcription, which is the focus here. at the beginning and end of each viral gene lies a conserved region (cr) of 9-10 nucleotides and 12-13 nucleotides, respectively. these are termed the gene start (gs) and gene end (ge) signals with an intergenic, non-transcribed region between these genes. at the 3 0 side of the genome, prior to the first gene, is the leader (le) extragenic region and at the 5 0 end is the trailer (tr) extragenic region. the lengths of these extragenic regions can vary based on the virus but in rsv the le region is 44 nucleotides long. to transcribe its rna, the polymerase initiates at the third nucleotide to transcribe a short uncapped transcript of about 25 nucleotides. 59 this rna is released but the polymerase remains affixed to the template where it proceeds along until it encounters the gs signal for the first gene and subsequently begins rna synthesis. these mrnas are modified with a 5 0 methyl cap and when the ge signal is reached, a 3 0 polyadenylated tail is added, and the mrna is released. the polymerase then scans for the next gs region. the genome is replicated starting at the leader promoter in a processive manner to yield a positive sense antigenome rna. the 3 0 and 5 0 ends of the antigenome contain the trailer and leader complement. the trailer complement ultimately directs genome rna synthesis. the core rsv polymerase consists of a 250 kda large (l) protein of approximately 2000 amino acids and a 27 kda phosphoprotein (p) that synthesizes an rna product upon the addition of an rna template. [59] [60] [61] [62] the p protein is thought to act as a chaperone to aid in the stability and expression of the polymerase. during rna synthesis, the p protein anchors the l protein to the n protein and also binds to the m2-1 transcription antitermination factor. [63] [64] [65] [66] this matrix protein, m2-1, serves as an elongation factor and is necessary for the polymerase to be fully processive in producing long mrna products. 67 no structure is available for any l protein from the paramyxoviruses and pneumoviruses, largely due to the size, solubility, and complexities with yielding enough highly pure protein. however, the cryo-electron microscopic (em) structure of the l-p complex from a highly similar virus, vesicular stomatitis virus (vsv), has recently been solved. 68 vsv is a non-segmented, negative strand virus from the rhabdoviridae family and given the high sequence conservation of the l protein, the structure of vsv l has provided important structural insights. the l proteins of rsv, vsv, and other related negative sense rna polymerases can be divided into six cr. 69 the first three regions (cri-iii) of vsv generate a doughnut-like structure in negative stain em and cryo-em analysis. 68, 70 the remaining crs appear as globular appendages on this doughnut. the doughnut structure adopts a classic right-handed configuration in the cryo-em reconstruction composed of fingers, palm, and thumb domains, like other polymerases (see figure 3 ). the criv and crv contain conserved residues and catalytic motifs necessary for enzymatic function. among these is the gxxt[n]hr motif, a highly conserved set of residues necessary for ntp binding and the hr motif, which forms covalent histidine rna intermediate. 73 within crvi there is a motif with sequence similarity to the 2 0 -o-ribose methyltransferase (mtase) domain, which has been characterized in the vsv l system. 74 while it is unknown exactly how similar the capping mechanism of vsv is to rsv and hmpv, the detailed mechanism of the vsv capping biochemistry provides insights. the capping of vsv is unique in that it is accomplished through an unconventional rna:gdp polyribonucleotidyltransferase (prntase) rather than a guanyltransferase. [75] [76] [77] the vsv capping occurs in two parts starting with conversion of gtp to gdp through a gtpase followed by the covalent attachment of a histidine to prna to form a phosphamide bond. this gdp then serves as a nucleophile to attack the prna and results in the release of the gppprna. 58 the second part of the reaction consists of the mtase reaction, which uses s-adenosylmethionine to methylate nitrogen 7 and the 2 0 -oxygen of the cap. a recent crystal structure of the mtase domain from hmpv has provided additional clues into this reaction (see figure 3 ). 72 a 406-residue fragment was expressed, consisting of the cr-iv containing the putative mtase with an additional c-terminal k-k-g motif (termed cr-viþ). with the exception of the k-k-g motif, the fold of the hmpv mtase domain indicates a conserved fold compared to vsv. 72 while the cr-viþ was active, the reaction rate was very slow and structural and biochemical results did not clearly identify active site residues. this suggests that mtase requires other co-factors or additional parts of the l protein to be catalytic. human rhinovirus (hrv), enterovirus 71 (ev71), and poliovirus (pv) are nonenveloped, positive strand rna viruses and are all members of the picornaviridae family. picornaviruses replicate their genome using an rdrp, called 3d pol . 78 replication takes place in one of two forms: primer-dependent format or de novo rna synthesis. 79 de novo rna synthesis, which uses a single initiation nucleotide, gives the 3 0 -hydroxyl group for adding the sequential nucleotide whereas a primer-dependent format uses a protein-based primer or an oligonucleotide for the hydroxyl group donor. 80 the polymerases from the picornavirus family only use a protein-primed mechanism of initiation. 80, 81 the 3d pol uses a small viral peptide (vpg) to initiate both plus and minus rna synthesis in vivo, making picornaviridae unique in their initiation mechanism. 78 rna synthesis is initiated using a highly conserved tyrosine residue within vpg using cis-acting replication element as a template whose position varies depending on the genus. 78, 82 the 3d pol aids in the binding of two ump molecules to the tyrosine hydroxyl group of vpg. [83] [84] [85] the product of this reaction, vpg-pu is then extended by an additional uridine to form vpg-pupu. 86 the 3d pol is located at the c-terminal end of a longer viral polyprotein of approximately 250 kda and the structures of 3d pol have been solved for ev71, hrv16, and pv, among many others. [86] [87] [88] the structures of these polymerases are largely similar and have sequence domains a-g indicating this conservation. 89 the 460-residue polymerase domain adopts a right-handed configuration with fingers, palm, and thumb subdomains providing an optimal arrangement for substrate and metal cation access during the catalytic cycle. the palm, fingers, and thumb subdomains contain these sequence motifs (motifs a-e in the palm subdomain shown in figure 4) . 90, 91 these motifs have specialized roles in catalysis including nucleotide binding and overall structural integrity of the active site. 80 the palm contains the active site of all the rdrps and its structure consists of an antiparallel beta-sheet surrounded by three alpha helices. 90 additional substructures within the fingers domain are referred to as the index, middle, ring, and pinky fingers. 82, 92 all but the pinky fingers build an extended beta-sheet that seems to be conserved. the index finger within the fingers subdomain makes an important contact with the thumb subdomain and pushes the ring finger down to trap it. this conformation results in the ring finger being the roof for ntp entry and making important interactions with the triphosphate. an additional structural feature of motif f is a positively charged tunnel that modulates the interactions of ntp. 93 this tunnel aides in the diffusion of nucleotides and is conserved among this family of viruses. proteins and enzymes rarely exist in a monomeric state in nature but are energetically driven to higher order, oligomeric states through polar and hydrophobic interactions and disulfide bond formation. the development of increasingly sophisticated structural biology techniques, including high-resolution x-ray crystal structures and cryo-em, has provided a snapshot into how polymerases may adopt such oligomeric states. the understanding and characterization of the oligomeric states place these multifunctional enzymes in a greater biological context. an example of such oligomeric states and how this impacts the catalytic function is the polymerase from pv. pv rdrp is described as having macromolecular contacts at two polymerase interfaces (interface i and interface ii). 94 interface i is defined by the interaction of the thumb domain from one polymerase with the palm of another polymerase. interface ii describes interactions of the n-terminus of one polymerase with the thumb of a neighboring polymerase within the crystal lattice. additionally, pv can form tube-like structures suggesting it is a highly dynamic structure able to undergo and adopt multiple conformational arrangements. 95 the oligomeric state of pv polymerase is required for membrane-associated rna replication in infected cells, as demonstrated by mutating residues involved in protein-protein interactions. 96, 97 the polymerase of cov the cov are part of the larger nidovirus family and have exceptionally large genomes of up to 32 kilobases in length. 98 cov are positive sense rna viruses, with a notable example being sars as one of the most pathogenic member of this viral family. 99 the cov genome has a 5 0 -cap, is polyadenylated on the 3 0 end, and generates a total of 16 non-structural proteins (nsp1 to nsp16). the 5 0 two-thirds of its genome encodes for non-structural proteins that combine to form a replication and transcription complex that completes viral rna synthesis. 100 the nsp12 protein is the rdrp and is typically composed of a n-terminal domain composed of a nidovirusspecific rdrp-associated nucleotidyltransferase (niran) and a c-terminal containing the rdrp domain, which contains a set of six conserved motifs (motifs a-f) responsible for recognizing substrates and template. 98, 101 the niran domain has only been identified in nidoviruses and is approximately 300 residues long in cov. 101 in sars-cov, a reverse genetic system identified this motif as a requirement for replication of its viral genome. while niran activity has not been directly observed outside of a reverse genetic system for cov, based on the nucleotidylation activity of eav nsp9, the niran domain is hypothesized to play a role in protein priming, capping, or as a potential universal ligation mechanism. 98 the active site of the polymerase is located within motif c and is composed of conserved (within the nidovirus family) ser-asp-asp residues. importantly, conserved aspartates found in motif a of sars-cov, which combined with those found in motif c, contribute to the polymerase and rna synthesis activities. this is different from other positive strand rna viruses which contain a gdd active site. motif a along with motif c aid in coordinating the metal ions necessary for catalysis. the sars-cov harbors a signature sequence in motif g necessary for primer-dependent rna synthesis. 102, 103 due to difficulties in obtaining large enough amounts of highly pure protein, the structures of cov nsp12 have not been solved either by x-ray crystallography or cryo-em. therefore, the structural information currently available is based solely on structural models obtained via sequence alignment and homology modeling techniques. these models indicate a right-handed fold composed of fingers, palm, and thumb subdomains with clearly defined entry and exit channels, consistent with rdrp domains for other structurally characterized positive sense rna polymerases (for example, foot-and-mouth disease virus) but clearly distinct from the known molecular topology of negative-stranded rdrps. no structural models predict the presence of a priming loop. these data combined with biochemical data indicating no de novo initiation of rna synthesis may account for the functionality of motif g. a complete characterization of the in vitro nsp12 rdrp activity has demonstrated overall weak activity. initial evidence suggested that a previously uncharacterized n-terminal domain may have been required for polymerase activity. 104 however, the addition of this domain using a c-terminally tagged protein still yielded protein with poor activity and processivity. based on these results and to increase the in vitro activity of the nsp12, two other non-structural proteins, nsp7 and nsp8, were added to the nsp12 protein in a primer extension mode. 100 the addition of nsp7 and nsp8 to nsp12 appear to activate and increase the processivity of the polymerase allowing it to produce an rna synthesis product of 340 nucleotides in the presence of mg 2þ . linking the nsp7 and nsp8 subunits together also increased the polymerase reaction efficiency suggesting that nsp7-nsp8 complex formation may influence the reaction rate. importantly, nsp14 can interact with an nsp7-nsp8-nsp12 complex without influencing rna synthesis activities. 100 nsp14 contains an exoribonuclease domain that has been shown to decrease nucleotide mismatch, in many ways similar to the proofreading exoribonuclease activities correlated with a polymerase. 105 in this section, we aim to answer the following questions that are important to medicinal chemists, biologists, and drug developers working in the field of virology. are there clinically relevant nucleoside analogs that are potent against one or multiple respiratory viruses? how were these molecules first identified, and which ones have been approved for commercial use? why aren't molecules like ribavirin and its analogs more widely used to treat respiratory viral infections? what are the current approaches to develop new nucleoside analogs against respiratory rna viruses? the broad-spectrum antiviral effect of ribavirin, a guanosine analog, was first reported in the 1970s. 106, 107 it was found at the time that ribavirin inhibits 16 dna and rna viruses, including herpesviruses, vaccinia, vsv, as well as respiratory infections caused by influenza a and b viruses and parainfluenza 1 virus. ribavirin is currently approved for the treatment of chronic hcv infection in combination therapy and against severe rsv infection in monotherapy. in the case of rsv infection, ribavirin is administered as a small-particle aerosol that requires use of a mask and a tent. ribavirin was originally developed against influenza based on its efficacy in a mouse model of influenza, 108,109 but its effect in human clinical trials was less clear, so it was not approved for the treatment of influenza. 110 its clinical use for the treatment of rsv infection via aerosol delivery remains limited due the inconvenient route of administration, lack of clear evidence for efficacy, and safety concerns associated with anemia and risk of teratogenicity. studies evaluating the mechanism of action of ribavirin have produced contradictory results. it is usually acknowledged that ribavirin exerts its main effect through its monophosphate metabolite by inhibiting the host inosine monophosphate dehydrogenase (impdh) enzyme, leading to depletion of intracellular gtp pools, which results in indirect inhibition of rna synthesis during viral replication (for review 111 ). the nucleoside form of ribavirin is also believed to enhance t-cell-mediated immune response through increased expression of interferon-gamma and tumor necrosis factor-alpha. 111 in addition, it has been proposed that prolonged replication of pv in the presence of ribavirin increases the viral mutation frequency and decreases infectivity. 112 one hypothesis is that the mutagenic effect of ribavirin is caused by its triphosphate form that is recognized by the viral rna polymerase. 112 once incorporated into the viral genome, ribavirin monophosphate could equally base pair with cytidine and uridine, therefore causing random mutations throughout the viral genome. ribo-cytidine and adenosine analogs containing a methyl group at the 2 0 -position on the ribose are known inhibitors of hcv and other related members of the flaviviridae family. [113] [114] [115] [116] [117] [118] [119] one of the most potent molecules of this series, 7-deaza-2 0 -c-methyladenosine (7dma, mk-0608), was once a development candidate for the treatment of hcv infection ( figure 5) . 117, 120 the adenosine analog 7dma also inhibits hrv type a infection in vitro, with ec 50 values ranging from 2 to 12 mm. 121 a subgenomic replicon assay was used in transient transfection experiments to demonstrate that 7dma is equipotent against multiple strains of hrv type c. this important proof-of-concept experiment demonstrated that 2 0 -methyl nucleosides prevent picornavirus replication, most likely by inhibiting the viral rna polymerase function. this class effect was confirmed with nitd008, another adenosine analog containing a 2 0 -c-ethynyl on its 2 0 -ribose ( figure 5 ). just like 7dma, nitd008 was previously known to inhibit flaviviruses, and was once a development candidate for the treatment of dengue infection. 122 nitd008 blocks the replication of dengue virus in cell culture and in mice by inhibiting the rdrp activity of the ns5 protein. 122, 123 therefore, the possibility that nitd008 would inhibit other (þ)ssrna viruses is not unexpected. indeed, nitd008 blocks the replication of ev71, another enterovirus-related to rhinovirus. 124 this in vitro antiviral effect was confirmed in a separate study that also demonstrated in vivo efficacy. 125 investigators in the latter study infected 2-week-old ag129 immunocompromised mice with ev71 by intraperitoneal inoculation. treatment of the infected animals with nitd008 given orally at 5 mg/kg twice a day for 4 days resulted in 100% survival at the end of the study, compared to 0% survival for the vehicle control group. nitd008 cannot be developed in the clinic due to severe toxicity seen in 14-day studies in rats and dogs. 122 however, the results summarized here indicate that nucleoside analogs targeting the viral rna polymerase of rhinovirus, ev71, and other enteroviruses have the potential to be efficacious in preclinical animal models, providing a rationale to conduct human studies with safer molecules sharing the same mode of action. 2 0 -deoxy-2 0 -fluoro nucleosides for influenza fluorinated nucleosides are well known for their antiviral and anticancer properties (for review 126 ). in particular, 2 0 -deoxy-2 0 -fluoro guanosine (2 0 fdg) was at one time considered a potential candidate for influenza treatment ( figure 5 ). in vitro, 2 0 fdg inhibits influenza a virus replication with an ec 50 of about 20 mm, without causing apparent cell toxicity. 127 in ferrets, treatment with 2 0 fdg at 20 mg/kg starting 1 h postinfection significantly reduced h3n2 influenza a virus titers in nasal washes, associated with reduction in fever and inflammation. 128 although time-of-addition experiments suggested that the molecule inhibits an early step of virus replication, more direct evidence for the mechanism of action came from enzyme inhibition studies. 129 in cell-free transcription experiments, 2 0 fdg triphosphate inhibited influenza a virus rna polymerase activity by competing with natural gtp. the inhibition of the enzyme was caused by the incorporation of 2 0 fdg monophosphate into the viral rna. 129 more recently, the related nucleoside analog 2 0 -deoxy-2 0 -fluoro cytidine (2 0 fdc) was evaluated against the highly pathogenic h5n1 and the pandemic h1n1 strains. 130 when administered intraperitoneally, 2 0 fdc significantly enhanced survival of balb/c mice infected with a lethal dose of either h5n1 or h1n1 viruses. although these studies show compelling evidence of in vivo efficacy in preclinical species, 2 0 fdg and 2 0 fdc are not suitable candidates for clinical development. one of the main limitations of these molecules is their lack of specificity for influenza virus polymerase. the ability of a nucleotide to inhibit distant molecular targets is not detrimental per se. as such, 2 0 -deoxy-2 0 -fluoro nucleotides and their derivatives interact with the rna polymerase of hcv. [131] [132] [133] [134] but the substitution of the 2 0 -hydroxy by a fluoro group also makes the resulting nucleotides broad substrates for viral 135 and human 136 dna polymerases. in the latter study, the authors have shown that the monophosphate form of both 2 0 fdc and 2 0 fdg can be incorporated into dna by human dna polymerase alpha and gamma. this might explain the changes in cell cycle distribution and cytostatic effect caused by prolonged in vitro incubation with 2 0 fdc. 133 paradoxically, the same molecule was well tolerated when administered intravenously to rats and woodchucks for up to 90 days. 137 one hypothesis for this discrepancy is that a low organ exposure of the phosphorylated metabolite(s) of 2 0 fdc could limit the toxic effect on dividing cells in these animals. the antiviral 6-fluoro-3-hydroxy-2-pyrazinecarboxamide (t-705, favipiravir, avigan) has been approved in japan for the treatment of influenza infection since 2014. t-705 is a nucleoside precursor inhibiting influenza virus with broad-strain coverage 137,139 ( figure 5 ). it is often proposed that t-705 exerts its antiviral activity through its ntp form (t-705 rtp) by directly inhibiting the rdrp activity of influenza a virus polymerase, 140 but the exact mode of action and precise molecular interaction between the nucleotide and the viral polymerase has been elusive. in vitro, t-705 is efficiently converted to its ribofuranosyl 5 0 -triphosphate (t-705 rtp) form by cellular enzymes. 141 treatment of influenza a virus-infected cells with t-705 results in a significant increase of lethal mutations within the viral genome, a phenomenon also described as error catastrophe. 142 the lethal mutagenesis hypothesis is supported by enzymatic assays showing that t-705 rtp is efficiently recognized by influenza a virus polymerase both as a guanosine and an adenosine analog. 143 in addition, single events of t-705 rmp incorporation into rna by influenza a virus polymerase delayed but did not block the extension of the rna primer strand. the antiviral potency of t-705 covers other virus families well beyond orthomyxoviruses. t-705 has been shown to inhibit a number of diverse rna viruses unrelated to influenza, including representatives of noroviruses, bunyaviruses, arenaviruses, flaviviruses, and filoviruses. [144] [145] [146] [147] [148] [149] [150] [151] [152] it is interesting to point out that the mutagenic effect of t-705 has also been documented for hcv. 153 at the biochemical level, we showed that t-705 is recognized as substrate for rna synthesis not only by viral polymerases, but also by human mitochondrial rna polymerase. 154, 155 this host-based interaction did not result in any measurable in vitro mitochondrial toxicity, but it raised more questions about the mechanism of action of the compound. recently, the possibility that t-705 exerts its main antiviral effect without converting to its triphosphate form came from the observation that t-705 ribonucleoside is chemically unstable under biological conditions. 156 even though t-705 does not seem to potently inhibit the human impdh enzyme, 157 its very broad antiviral spectrum and its capacity to induce lethal mutagenesis are somewhat reminiscent of ribavirin, another nucleoside that inhibits hcv replication through host-based mechanisms. therefore, the possibility that t-705 exerts its inhibition through interactions with host proteins cannot be ruled out and remains to be further explored. considering its similarities with ribavirin in terms of antiviral spectrum and mode of action, it will be interesting to see if t-705 becomes more widely used in patients suffering from respiratory viral infections, or if it will remain limited to stockpiling for potential influenza pandemic in japan. the discovery of als-8112, the parent molecule of the prodrug als-8176 (lumicitabine), was the result of a screening campaign using a focused library of structurally diverse nucleoside and nucleotide analogs tested against rsv in an in vitro infectious assay. 158 the main scaffold identified from this screen was 2 0 difluoro-4 0 azido-cytidine. further modifications at the 2 0 -and 4 0 -positions to improve anti-rsv potency and selectivity, led to the identification of als-8112 (2 0 fluoro-4 0 chloromethyl-cytidine) ( figure 5 ). in vitro, als-8112 inhibits a broad panel of rsv a and b subtypes, as well as related pneumo-, paramyxo-, and rhabdoviruses. 159 in particular, we recently reported that als-8112 inhibits rsv and hmpv with similar in vitro potency. 160 the molecular target of als-8112 was determined by two independent methods. the polymerization function of the rsv l protein was identified as the target of als-8112 inhibition, first, by selecting and characterizing drug resistanceassociated mutations located in the l gene. when introduced into a wild-type rsv genome, four amino acid mutations (m628l, a789v, l795i, and i796v) were phenotypically associated with resistance to als-8112. 159 enzymatic assays using purified recombinant rsv polymerase were critical to validate the mode of action of als-8112. in these assays, the 5 0 -triphosphate form of als-8112 (als-8112-tp) caused immediate chain termination of rna synthesis and inhibition of the viral polymerization activity. this inhibitory effect was specific to rsv polymerase, since als-8112-tp did not inhibit polymerases from host or viruses unrelated to rsv such as hcv. the lack of inhibition against hcv was rationalized by molecular modeling, predicting steric clashing of als-8112-tp inside the active site of hcv polymerase. because of the low oral bioavailability of als-8112, a series of 2 0 ,3 0 -diester prodrugs was evaluated for improved pharmacokinetic properties. one prodrug, als-8176, formed high levels of monophosphate and triphosphate in the lungs when administered orally to nonhuman primates. because of its high oral bioavailability, als-8176 was evaluated for in vivo efficacy in african green monkeys infected with rsv. at the end of treatment, rsv rna was undetectable in bronchoalveolar lavage samples from all four als-8176-treated animals. 159 subsequently, a randomized, double-blind, clinical trial evaluated als-8176 given for 5 days to healthy adults inoculated with rsv. 161 the reduction in viral load in nasal washes associated with als-8176 treatment varied from 73% to 88% depending on the dose regimen. rsv rna was undetectable 1.3 to 2.3 days after the start of als-8176 treatment compared with 7.2 days for placebo. assessment of symptom scores and quantity of mucus produced also showed a clear effect on rsv-induced disease. this important result represents the first proofof-concept validation that an rsv replication inhibitor can be efficacious in humans. als-8176 is currently in clinical development for the treatment of rsv infection in hospitalized infants and adults (clinicaltrials.gov identifier: nct02202356, nct02935673). the recent ebola virus outbreak of 2013-2016 in west africa triggered increased efforts to identify new antivirals targeting filoviruses. as a result, the development of a new series of c-linked nucleoside analogs with anti-ebola properties was soon reported. 162 in a cellbased infectious assay, the 1 0 -cyano c-linked adenosine derivative (gs-441524, or compound 4) was moderately active against ebola replication with ec 50 values around 1.5 mm, whereas the 1 0 -methyl and -ethynyl counterparts were completely inactive. gs-441524 is also a broad-spectrum inhibitor of a variety of rna viruses from four families (filoviridae, flaviviridae, paramyxoviridae, and pneumoviridae), including hcv and rsv. 163, 164 however, the addition of a 2 0 -c-methyl group, as in the case of the gs-6620, 165, 166 significantly reduces the antiviral spectrum to hcv only. the relatively weak antiviral activity of gs-441524 across all viruses (0.5-50 mm ec 50 ) was attributed to its inefficient intracellular phosphorylation, which could be improved by adding a monophosphate prodrug to the parent nucleoside. the resulting compound, gs-5734 ( figure 5) inhibits the zaire and sudan species of ebola virus and marburg virus with ec 50 values ranging from 0.01 to 0.20 mm, and exhibits moderate cytotoxicity (cc 50 ¼ 2 to >20 mm) in multiple human cell types. gs-5734 exhibits the same broad antiviral spectrum as its parent molecule. 163 the triphosphate form of gs-5734 is recognized as substrate by rsv polymerase, but its incorporation into rna does not lead to immediate chain termination. 162 the favorable in vitro data led to further evaluation of gs-5734 in a macaque lethal model of ebola virus disease. complete protection was achieved when gs-5734 was administered at a daily intravenous dose of 10 mg/kg, beginning on day 3 post-infection. 162 following phase i safety testing in healthy human volunteers, gs-5724 was first given as a 14-day course for compassionate use to an ebola-infected nurse who had survived the disease and developed a recurrence in the central nervous system. 167 soon after, a neonate who had congenital ebola virus infection received three different experimental therapies, including a 12-day treatment with gs-5734. 168 in both cases, patients cleared the virus and survived the infection. the characterization of the broad antiviral spectrum of gs-5734 was further expanded to another (þ) ssrna virus family: coronaviridae. it was shown that gs-5734 inhibits sars-cov and mers-cov replication in multiple in vitro systems, including primary human airway epithelial cell cultures with sub-micromolar ec 50 values. 169 gs-5734 was also effective against other human and bat cov. in a mouse model of sars-cov infection, prophylactic and early therapeutic administration of gs-5734 reduced lung viral load and improved clinical signs of disease as well as respiratory function. although there is limited data to confirm the proposed mechanism of action of gs-5734 against each virus, it is generally assumed that the molecule targets the rdrp function of the viral polymerase. in the case of cov, this is supported by the identification of two mutations (f476l and v553l) within the predicted fingers subdomain of the rdrp protein nsp12 from murine hepatitis virus. 170 these mutations emerged over 23 passages and confer 4-to 6-fold resistance to gs-5734, combined with overall reduced replication fitness. at this point, the precise mechanism of action of gs-5734 against cov remains elusive. it is possible that gs-5734 triphosphate is not excised by the proofreading activity of nsp14 because of lack of immediate chain termination, as observed for rsv polymerase. in this case, could the resistance mutations identified in nsp12 alter the chain termination profile of gs-5734, and make it more susceptible to excision? such studies are needed, not only to understand how gs-5734 works but also to design new molecules against cov polymerases. in this review, we summarized the exciting advances in discovery and development of novel nucleoside analogs as potential new treatments for respiratory rna virus infections. the medical need is high because very few drugs have been approved for the treatment of respiratory viral infections despite worldwide health impacts attributed to them. the approved drugs include zanamivir, oseltamivir, peramivir, and favipiravir (japan only) for influenza virus and palivizumab for rsv, all of which have limitations that prevent their widespread use in a therapeutic setting. drug candidates intended for use against rhinovirus infections, such as the capsid inhibitor pleconaril and the protease inhibitor rupintrivir, have been tested in the clinic without success. the first nucleoside analog developed for respiratory viral infection was ribavirin, but despite its approval for use in rsv, its utility for treating severe viral infections remains low. therefore, the concept of nucleoside analogs against respiratory viruses remains relatively new and needs to be further explored. what are the molecular determinants of polymerase selectivity against nucleotide analogs? we currently do not understand well how specific changes made in nucleotide analogs alter their recognition as substrates for rna synthesis, and how substrate selectivity differs among positive and negative strand rna virus polymerases. for example, many 2 0 -modified nucleotide analogs are known to inhibit hcv polymerase, often with an antiviral spectrum extended to flaviviruses and picornaviruses. however, there is no clear mechanistic basis to explain why none of these compounds inhibit (-)ssrna viruses, or even other (þ)ssrna viruses such as cov. could the exonuclease/proofreading activity of cov polymerases excise chain terminators and resume rna synthesis? are there specific amino acid within the active site of (-)ssrna virus polymerases responsible for the discrimination of 2 0 -c-methyl nucleotides? these hypotheses have not been tested, in part, due to the difficulty to conduct biochemical and structural studies on viral polymerases from respiratory viruses. until recently, the production of soluble, pure viral protein targets has been limiting, especially in the case of large protein complexes. as mentioned earlier in this review, the development of robust expression systems for influenza polymerase trimer, as an example, have made it possible to use x-ray crystallography and potentially cryo-electron microscopy to provide molecular visualization of binding pockets for small molecule inhibitors, entry and exit channels for substrate(s), and potential new ways to disrupt domain interactions. these structural insights will tremendously aid the development of new drugs as well as to further elucidate the mechanisms of action and binding of existing drugs to their protein targets. molecular modeling is also a useful approach that we and others have used to rationalize the differences in selectivity of lumicitabine against rsv and hcv polymerase. 159 more studies such as these ones will be needed to rationally design new nucleotide analogs targeting respiratory virus polymerases. in the past, many nucleoside analogs failed during development for safety/toxicity reasons, especially molecules with suboptimal specificity for their viral polymerase target and those used for chronic treatment of infections such as hiv and hcv. in the context of acute respiratory infections, evaluation of safety must be based on both the intended duration of treatment and the targeted patients, which sometimes include vulnerable populations such as children and the elderly. other considerations to ensure successful future development of nucleoside analogs directed against respiratory infections will be to optimize delivery to the lung by evaluating different routes of administrations, including aerosol formulations, and developing lung-targeting nucleoside prodrugs. despite these challenges, the prospect of developing nucleoside analogs directed against respiratory rna virus infections represents an exciting new avenue in antiviral research. the global burden of respiratory disease structure and classification of viruses the classification of viruses infecting the respiratory tract virus detection in critically ill children with acute respiratory disease: a new profile in view of new technology influenza virus rna polymerase: insights into the mechanisms of viral rna synthesis selective inactivation of influenza c esterase: a probe for detecting 9-o-acetylated sialic acids an open receptor-binding cavity of hemagglutinin-esterase-fusion glycoprotein from newly-identified influenza d virus: basis for its broad cell tropism clinical review: primary influenza viral pneumonia a narrative review of influenza: a seasonal and pandemic disease incidence of medically attended influenza during pandemic and postpandemic seasons through the influenza incidence surveillance project a review of vaccine research and development: human acute respiratory infections taxonomy of the order mononegavirales: update 2016 respiratory syncytial virus and parainfluenza virus pediatric hospitalizations for croup (laryngotracheobronchitis): biennial increases associated with human parainfluenza virus 1 epidemics glide: a new approach for rapid, accurate docking and scoring. 1. method and assessment of docking accuracy epidemiology and clinical presentation of the four human parainfluenza virus types population-based surveillance for hospitalizations associated with respiratory syncytial virus, influenza virus, and parainfluenza viruses among young children global burden of acute lower respiratory infections due to respiratory syncytial virus in young children: a systematic review and meta-analysis respiratory syncytial virus, an ongoing medical dilemma: an expert commentary on respiratory syncytial virus prophylactic and therapeutic pharmaceuticals currently in clinical trials the pathogenesis of respiratory syncytial virus disease in childhood respiratory syncytial virus disease: update on treatment and prevention human metapneumovirus infection in adults human metapneumovirus: a new player among respiratory viruses human metapneumovirus associated with respiratory tract infections in a 3-year study of nasal swabs from infants in italy epidemiology, pathogenesis, and treatment of the common cold picornavirus and enterovirus diversity with associated human diseases symptom profile of common colds in school-aged children enterovirus and parechovirus infection in children: a brief overview recombination in the evolution of enterovirus c species sub-group that contains types cva-21 rhinoviruses and respiratory enteroviruses: not as simple as abc enterovirus 71 infection and vaccines enterovirus 71 infection and neurological complications human coronaviruses: a review of virus-host interactions coronavirus 229e-related pneumonia in immunocompromised patients understanding human coronavirus hcov-nl63 coronavirus hku1 infection in the united states description and clinical treatment of an early outbreak of severe acute respiratory syndrome (sars) in guangzhou, pr china severe acute respiratory syndrome coronavirus as an agent of emerging and reemerging infection epidemiological findings from a retrospective investigation middle east respiratory syndrome coronavirusesdrug discovery and therapeutic options infection with middle east respiratory syndrome coronavirus antimicrobial strategies: inhibition of viral polymerases by 3'-hydroxyl nucleosides influenza a viruses: new research developments the rna polymerase of influenza a virus: mechanisms of viral transcription and replication structural insight into cap-snatching and rna synthesis by influenza polymerase role of two of the influenza virus core p proteins in recognizing cap 1 structures (m7gpppnm) on rnas and in initiating viral rna transcription identification of the cap binding protein of influenza virus the structural basis for cap binding by influenza virus polymerase subunit pb2 the cap-snatching endonuclease of influenza virus polymerase resides in the pa subunit crystal structure of an avian influenza polymerase pa n reveals an endonuclease active site structure of influenza a polymerase bound to the viral rna promoter conserved features of the pb2 627 domain impact influenza virus polymerase function and replication role of the pb2 627 domain in influenza a virus polymerase function the role of the priming loop in influenza a virus rna synthesis the influenza virus polymerase complex: an update on its structure, functions, and significance for antiviral drug design initiation and regulation of paramyxovirus transcription and replication polymerases of paramyxoviruses and pneumoviruses new antiviral approaches for respiratory syncytial virus and other mononegaviruses: inhibiting the rna polymerase transcription elongation factor of respiratory syncytial virus, a nonsegmented negative-strand rna virus production of infectious human respiratory syncytial virus from cloned cdna confirms an essential role for the transcription elongation factor from the 5' proximal open reading frame of the m2 mrna in gene expression and provides a capability for vaccine development bacterial expression of human respiratory syncytial viral phosphoprotein p and identification of ser237 as the site of phosphorylation by cellular casein kinase ii new insights into structural disorder in human respiratory syncytial virus phosphoprotein and implications for binding of protein partners interaction between human respiratory syncytial virus (rsv) m2-1 and p proteins is required for reconstitution of m2-1-dependent rsv minigenome activity structure and functional analysis of the rna-and viral phosphoprotein-binding domain of respiratory syncytial virus m2-1 protein characterization of a viral phosphoprotein binding site on the surface of the respiratory syncytial nucleoprotein central role of the respiratory syncytial virus matrix protein in infection structure of the l protein of vesicular stomatitis virus from electron cryomicroscopy sequence comparison of five polymerases (l proteins) of unsegmented negative-strand rna viruses: theoretical assignment of functional domains molecular architecture of the vesicular stomatitis virus rna polymerase template-based protein structure modeling using the raptorx web server x-ray structure and activities of an essential mononegavirales l-protein domain a conserved motif in region v of the large polymerase proteins of nonsegmented negative-sense rna viruses that is essential for mrna capping in silico identification, structure prediction and phylogenetic analysis of the 2 0 -o-ribose (cap 1) methyltransferase domain in the large structural protein of ssrna negative-strand viruses unconventional mechanism of mrna capping by the rna-dependent rna polymerase of vesicular stomatitis virus in vitro capping and transcription of rhabdoviruses conventional and unconventional mechanisms for capping viral mrna rnadependent rna polymerases of picornaviruses: from the structure to regulatory mechanisms initiation of viral rna-dependent rna polymerization a comparison of viral rna-dependent rna polymerases possible unifying mechanism of picornavirus genome replication picornaviral polymerase structure, function, and fidelity modulation biochemical and genetic studies of the vpg uridylylation reaction catalyzed by the rna polymerase of poliovirus a "slide-back" mechanism for the initiation of protein-primed rna synthesis by the rna polymerase of poliovirus genetic and biochemical studies of polioviruscis-acting replication element cre in relation to vpg uridylylation crystal structure of complete rhinovirus rna polymerase suggests front loading of protein primer crystal structure of enterovirus 71 rna-dependent rna polymerase complexed with its protein primer vpg: implication for a trans mechanism of vpg uridylylation structural basis for active site closure by the poliovirus rna-dependent rna polymerase evolution of tertiary structure of viral rna dependent polymerases structure of the rna-dependent rna polymerase of poliovirus identification of four conserved motifs among the rna-dependent polymerase encoding elements structural basis for proteolysis-dependent activation of the poliovirus rna-dependent rna polymerase poliovirus rna-dependent rna polymerase (3dpol): structural, biochemical, and biological analysis of conserved structural motifs a and b visualization and functional analysis of rna-dependent rna polymerase lattices surface for catalysis by poliovirus rna-dependent rna polymerase oligomeric structures of poliovirus polymerase are important for function structurefunction relationships of the rna-dependent rna polymerase from poliovirus (3dpol) a surface of the primary oligomerization domain functions in capsid precursor processing and vpg uridylylation nidovirus rna polymerases: complex enzymes handling exceptional rna genomes coronaviruses post-sars: update on replication and pathogenesis one severe acute respiratory syndrome coronavirus protein complex integrates processive rna polymerase and exonuclease activities discovery of an essential nucleotidylating activity associated with a newly delineated conserved domain in the rna polymerase-containing protein of all nidoviruses molecular model of sars coronavirus polymerase: implications for biochemical functions and drug design the palm subdomain-based active site is internally permuted in viral rna-dependent rna polymerases of an ancient lineage expression, purification, and characterization of sars coronavirus rna polymerase structural basis and functional analysis of the sars coronavirus nsp14-nsp10 complex design, synthesis, and broad spectrum antiviral activity of 1-b-d-ribofuranosyl-1,2,4-triazole-3-carboxamide and related nucleosides broad-spectrum antiviral activity of virazole: 1-beta-d-ribofuranosyl-1,2,4-triazole-3-carboxamide amantadine and ribavirin aerosol treatment of influenza a and b infection in mice protection of mice from lethal influenza virus infection with high dose-short duration ribavirin aerosol ribavirin-current status of a broad spectrum antiviral agent mechanism of action of ribavirin in the combination treatment of chronic hcv infection the broad-spectrum antiviral ribonucleoside ribavirin is an rna virus mutagen 2'-c-methyl branched pyrimidine ribonucleoside analogues: potent inhibitors of rna virus replication inhibition of hepatitis c virus rna replication by 2'-modified nucleoside analogs structure-activity relationship of heterobase-modified 2'-c-methyl ribonucleosides as inhibitors of hepatitis c virus rna replication efficacy of 2'-c-methylcytidine against yellow fever virus in cell culture and in a hamster model a 7-deazaadenosine analog is a potent and selective inhibitor of hepatitis c virus replication with excellent pharmacokinetic properties a dengue fever viremia model in mice shows reduction in viral replication and suppression of the inflammatory response after treatment with antiviral drugs synthesis and pharmacokinetics of valopicitabine (nm283), an efficient prodrug of the potent anti-hcv agent 2 0 -c-methylcytidine robust antiviral efficacy upon administration of a nucleoside analog to hepatitis c virus-infected chimpanzees multiple classes of antiviral agents exhibit in vitro activity against human rhinovirus type c an adenosine nucleoside inhibitor of dengue virus biochemical characterization of the inhibition of the dengue virus rna polymerase by beta-d-2'-ethynyl-7-deazaadenosine triphosphate an adenosine nucleoside analogue nitd008 inhibits ev71 proliferation inhibition of enterovirus 71 by adenosine analog nitd008 fluorinated nucleosides as antiviral and antitumor agents purine 2'-deoxy-2'-fluororibosides as antiinfluenza virus agents efficacy of 2'-deoxy-2'-fluororibosides against influenza a and b viruses in ferrets inhibition of influenza virus transcription by 2'-deoxy-2'-fluoroguanosine in vitro and in vivo efficacy of fluorodeoxycytidine analogs against highly pathogenic avian influenza h5n1, seasonal, and pandemic h1n1 virus infections phosphorylation of ribavirin and viramidine by adenosine kinase and cytosolic 5'-nucleotidase ii: implications for ribavirin metabolism in erythrocytes synthesis and anti-viral activity of a series of d-and l-2'-deoxy-2'-fluororibonucleosides in the subgenomic hcv replicon system inhibition of the subgenomic hepatitis c virus replicon in huh-7 cells by 2'-deoxy-2'-fluorocytidine efficiency of incorporation and chain termination determines the inhibition potency of 2'-modified nucleotide analogs against hepatitis c virus polymerase the effect of 2'-fluoro-2'-deoxycytidine on herpes virus growth polymerization of 2'-fluoro-and 2'-o-methyl-dntps by human dna polymerase alpha, polymerase gamma, and primase an evaluation of the toxicities of 2'-fluorouridine and 2'-fluorocytidine-hcl in f344 rats and woodchucks (marmota monax) in vitro and in vivo activities of anti-influenza virus compound t-705 efficacy of orally administered t-705 on lethal avian influenza a (h5n1) virus infections in mice mechanism of action of t-705 against influenza virus t-705 (favipiravir) and related compounds: novel broad-spectrum inhibitors of rna viral infections t-705 (favipiravir) induces lethal mutagenesis in influenza a h1n1 viruses in vitro the ambiguous base-pairing and high substrate efficiency of t-705 (favipiravir) ribofuranosyl 5'-triphosphate towards influenza a virus polymerase successful treatment of advanced ebola virus infection with t-705 (favipiravir) in a small animal model post-exposure efficacy of oral t-705 (favipiravir) against inhalational ebola virus infection in a mouse model treatment of late stage disease in a model of arenaviral hemorrhagic fever: t-705 efficacy and reduced toxicity suggests an alternative to ribavirin in vitro and in vivo activities of t-705 against arenavirus and bunyavirus infections efficacy of favipiravir (t-705) and t-1106 pyrazine derivatives in phlebovirus disease models activity of t-705 in a hamster model of yellow fever virus infection in comparison with that of a chemically related compound, t-1106 effect of t-705 treatment on western equine encephalitis in a mouse model t-705 (favipiravir) inhibition of arenavirus replication in cell culture t-705) inhibits in vitro norovirus replication lethal mutagenesis of hepatitis c virus induced by favipiravir structure-activity relationship analysis of mitochondrial toxicity caused by antiviral ribonucleoside analogs biochemical evaluation of the inhibition properties of favipiravir and 2'-c-methylcytidine triphosphates against human and mouse norovirus rna polymerases synthesis of t-705-ribonucleoside and t-705-ribonucleotide and studies of chemical stability distinct effects of t-705 (favipiravir) and ribavirin on influenza virus replication and viral rna synthesis discovery of 4'-chloromethyl-2'-deoxy-3',5'-di-o-isobutyryl-2'-fluorocytidine (als-8176), a first-in-class rsv polymerase inhibitor for treatment of human respiratory syncytial virus infection molecular basis for the selective inhibition of respiratory syncytial virus rna polymerase by 2'-fluoro-4'-chloromethyl-cytidine triphosphate development of als-8112/als-8176 as an effective replication inhibitor of human metapneumovirus activity of oral als-008176 in a respiratory syncytial virus challenge study therapeutic efficacy of the small molecule gs-5734 against ebola virus in rhesus monkeys gs-5734 and its parent nucleoside analog inhibit filo-, pneumo-, and paramyxoviruses discovery and synthesis of a phosphoramidate prodrug of a pyrrolo [2,1-f][triazin-4-amino] adenine c-nucleoside (gs-5734) for the treatment of ebola and emerging viruses inhibition of hepatitis c virus replication by gs-6620, a potent c-nucleoside monophosphate prodrug discovery of the first c-nucleoside hcv polymerase inhibitor (gs-6620) with demonstrated antiviral response in hcv infected patients late ebola virus relapse causing meningoencephalitis: a case report first newborn baby to receive experimental therapies survives ebola virus disease broad-spectrum antiviral gs-5734 inhibits both epidemic and zoonotic coronaviruses the nucleoside prodrug gs-5734 inhibits multiple coronaviruses and selects for resistance mutations in the rna-dependent rna polymerase that are associated with a decrease in viral replication fitness we wish to thank leo beigelman, julian symons, andreas jekle, and peggy korn for their review of the manuscript. the author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. janssen research & development provided support in the form of salaries for all authors but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. key: cord-261025-y49su5uc authors: sampathkumar, priya; temesgen, zelalem; smith, thomas f.; thompson, rodney l. title: sars: epidemiology, clinical presentation, management, and infection control measures date: 2003-07-31 journal: mayo clinic proceedings doi: 10.4065/78.7.882 sha: doc_id: 261025 cord_uid: y49su5uc severe acute respiratory syndrome (sars) is a recently recognized febrile respiratory illness that first appeared in southern china in november 2002, has since spread to several countries, and has resulted in more than 8000 cases and more than 750 deaths. the disease has been etiologically linked to a novel coronavirus that has been named the sars-associated coronavirus. it appears to be spread primarily by large droplet transmission. there is no specific therapy, and management consists of supportive care. this article summarizes currently available information regarding the epidemiology, clinical features, etiologic agent, and modes of transmission of the disease, as well as infection control measures appropriate to contain sars. it is now believed that the disease is caused by a novel coronavirus (sars-cov). sars was first recognized as a distinct entity in february 2003 by dr carlo urbani, an epidemiologist with the world health organization who was investigating the outbreak in hanoi, vietnam. unfortunately, he himself contracted the disease and died. since then and up to , the disease has spread to affect more than 8000 worldwide. 1 although the medical community at large has been aware of this disease for only a few months, considerable progress has been made in understanding the epidemiology and clinical features, and the etiologic agent has been identified. a highly contagious atypical pneumonia first appeared in the guangdong province, people's republic of china, in november 2002. this was not widely publicized, and the condition remained isolated to china for the next 3 months. on february 21, 2003, a chinese physician from the guangdong province (patient a in figure 1 ) who cared for patients with pneumonia and had himself developed symp-toms traveled to hong kong to visit relatives. he stayed on the ninth floor of hotel m for a day. on february 22, 2003, he was admitted to hospital 2 with fever and respiratory symptoms and died of respiratory failure on march 4, 2003 . he infected 2 of his family members and 4 health care workers (hcws) in hospital 2. in addition, 12 other hotel guests at hotel m developed sars, 10 of whom (patients b through k) were in the hotel the same day as patient a; the other 2 patients (patients l and m) stayed in the hotel during the time that 3 other symptomatic patients were guests at the hotel. because of international travel and transmission to hcws before the institution of protective measures, these patients were responsible for subsequent clusters of sars around the globe. patient b was the index patient for the outbreak in hanoi involving hcws and close contacts, including dr urbani. patients c, d, and e were the index cases in singapore. patients f and g traveled back to toronto, canada, which resulted in the cluster of cases in toronto. patients h and j caused outbreaks among hcws in other hospitals in hong kong. patient l appears to have become infected during his stay at hotel m, with subsequent transmission to his wife, patient m in the united states ( figure 1 ). aggressive and unprecedented measures that included quarantine of thousands of people, travel restrictions, and temperature checks at airports have been successful to a large extent in containing the disease. vietnam reported its last case more than 30 days ago. singapore used its military forces to assist in contact tracing and enforcement of home for personal use. mass reproduce only with permission from mayo clinic proceedings. and lower respiratory tract symptoms within 10 days of either travel to an area with documented transmission of sars or close contact with a person believed to have sars. a probable case is a suspect case who also has chest radiographic findings of pneumonia, acute respiratory distress syndrome (ards), or an unexplained respiratory illness resulting in death, with autopsy findings of ards without identifiable cause. 3 suspect and probable cases are further classified based on laboratory findings as laboratory positive, laboratory negative, or indeterminate ( table 1) . it is important to understand the meaning of close contact. it is defined as having cared for or lived with a person known to have sars or unprotected contact with body secretions from such a person. this includes kissing, embracing, sharing utensils or bedding, performing a physical examination, or other direct physical contact between persons. it does not include sitting across a waiting room for a brief period, walking by a person with sars, or other casual contact. the incubation period is 2 to 10 days. early manifestations include influenza-like symptoms, such as fever, myalgias, and headache. fever occurs in virtually all patients and is often the presenting symptom. often, fever is high and sometimes associated with chills and rigors. fever may occasionally be absent in elderly persons or may have resolved by the time respiratory symptoms occur. typically, rash and neurologic findings are absent. diarrhea has been reported in up to 25% of patients. [5] [6] [7] the respiratory phase starts within 2 to 4 days of onset of fever with a dry, nonproductive cough. this may progress to shortness of breath, usually in the second week of the illness, and might be accompanied by or progress to hypoxemia. in 10% to 20% of patients, the respiratory illness is severe enough to require tracheal intubation and mechanical ventilation. the case fatality rate is approximately 3% to 12% overall. the mortality rate may be as high as 45% in patients older than 60 years, particularly those with preexisting comorbidity (eg, diabetes, renal failure, and other chronic medical conditions). 8 a concerning feature of this disease has been that young, previously healthy persons, many of them health care professionals, have also died of sars. the reason for this is unclear but may be due to exposure to patients with higher viral loads or due to their host response. in contrast, sars has affected relatively few children and appears to be milder in the pediatric age group. 9 a biphasic course has been described in many patients, with an initial illness followed by improvement and then subsequent deterioration. this worsening can present as recurrent fever 4 to 7 days after initial defervescence, new chest infiltrates, respiratory failure, or watery diarrhea. in a cohort of 75 patients in hong kong, 85% had recurrent symptoms after initial improvement. 8 the authors described a triphasic course with fever, myalgia early in week 1, and recurrent fever, hypoxemia, diarrhea, and shifting chest infiltrates in week 2. twenty percent of patients progressed to ards during the third week of the illness. quantitative reverse-transcriptase polymerase chain reaction (rt-pcr) of nasopharyngeal aspirate in 14 patients with relapse showed peak viral loads occurred at day 10 after onset of symptoms, suggesting that the late deterioration may be due to the host immune response rather than to uncontrolled viral replication. chest radiographs of patients with sars show patchy focal infiltrates or consolidation often with a peripheral distribution, which may progress to diffuse infiltrates. pleural effusions have not been reported. early findings may be subtle, and initial findings on chest radiographs may be normal in up to 25% of patients. 6 computed tomography (ct) may be more sensitive than plain films. highresolution ct scans have shown abnormalities in patients with suspected sars who have normal findings on plain films. 10 the characteristic ct finding is bilateral peripheral airspace ground-glass consolidation mimicking that seen in bronchiolitis obliterans with organizing pneumonia. laboratory findings in patients with sars include thrombocytopenia and leukopenia (in particular lymphopenia). elevated creatine kinase, lactate dehydrogenase, and transaminase levels have been noted. a high peak lactate dehydrogenase level and an initial elevated white blood cell count may carry a poor prognosis. 7 the etiologic link of a coronavirus with the sars epidemic was established by peiris et al 11 in hong kong. several diagnostic laboratory methods were used to initially recover and then characterize coronavirus infection in various specimens obtained from patients with sars. these included inoculation and recovery of the virus in cell cultures, characterization of morphologic features by electron microscopy, serologic antibody determination, and molecular amplification and sequencing of the target rna of the agent. overall, 45 of the 50 patients these investigators studied had 1 or more laboratory tests (serology, 32; rt-pcr, 22; culture, 2) that supported a coronavirus etiology. the results of this initial report were quickly confirmed and expanded by collaborative studies in several major public health centers and medical institutions throughout the world coordinated by the world health organization. 12, 13 molecular sequencing analyses have indicated that the virus is only distantly related to previously sequenced coronaviruses. based on serologic studies, it appears that this virus has not previously circulated in humans and is now referred to as sars-cov. 14, 15 coronaviruses are enveloped rna viruses that cause disease in humans and animals. the previously known human coronaviruses are a major cause of the common cold and can occasionally cause pneumonia. research teams in hong kong and shenzhen, china, recently detected several coronaviruses closely related genetically to the sars-cov in 2 animal species (masked palm civet and raccoon-dog) and antibodies against the sars-cov in 1 additional species (chinese ferret badger). these and other wild animals are traditionally considered delicacies and are sold for human consumption in markets throughout southern china. this study provides the first indication that the sars-cov exists outside a human host. studies are needed to determine how widespread the sars virus might be in animals in guangdong and elsewhere and if these animals can excrete the virus in an amount sufficient to infect humans and through what route such transmission occurs. the sars-cov appears to be transmitted primarily by large droplet spread, although surface contamination and possibly airborne spread may play a role. recent data suggest that the virus may remain viable for considerable periods on a dry surface (up to 24 hours); hence, transmission through fomites may occur. the outbreak in an apartment complex in hong kong that accounted for more than 300 cases has been attributed to fecal spread. a patient with sars who had diarrhea stayed with his brother in this building. it is thought that infection spread from him to other residents in the building through a leaking sewage drain, which allowed aerosolization of virus-containing material. sewage also backed into bathroom floor drains in some apartments and may have accounted for some of the transmission. the sars-cov is stable in feces (and urine) at room temperature for at least 1 to 2 days. the virus is more stable (up to 4 days) in stool from patients with diarrhea (which has a higher ph than normal stool). 16 it is presently unclear at what stage of the disease viral shedding occurs or whether someone who is infected but asymptomatic can infect others. as our knowledge of sars and the etiologic coronavirus evolves, we will be able to address these important issues. some close contacts have reported a mild febrile illness without respiratory signs or symptoms, suggesting the illness might not always progress to the respiratory phase; others have not become ill at all. in contrast, "super spreaders" have been described who have infected 10 or more contacts, including hcws, family and social contacts, or visitors to the health care facilities where patients were hospitalized. a similar phenomenon has been described with some other diseases, such as rubella, laryngeal tuberculosis, and ebola virus, and might be the result of a combination of host, environment, and virus interactions. in singapore, 5 super spreaders were responsible for a total of 170 suspect and probable cases of sars. 17 additional data on the natural history of infection are needed to understand factors that might be associated with this phenomenon. regardless of whether it is the result of other transmission routes, inadequate infection-control measures, or more viral shedding by certain patients, the fact remains that transmission of the sars virus is highly efficient in some circumstances. the initial diagnosis of sars is one of exclusion. hence, common causes of respiratory illnesses should be sought. initial diagnostic testing for patients with suspected sars should include chest radiography, pulse oximetry, blood cultures, sputum gram stain and culture, and testing for other respiratory pathogens, notably influenza a and b, respiratory syncytial virus, and legionella (table 2 ). clinicians should save any available clinical specimens (respiratory, blood, serum, and stool) for additional testing until a specific diagnosis is made. acute and convalescent (>21 days after onset of symptoms) serum samples should be collected from each patient who meets the sars case definition. paired sera and other clinical specimens can be forwarded through state and local health departments for testing at the cdc. specific instructions for collecting specimens from suspected sars patients are available from the cdc. 18 laboratory diagnostic tests used at the cdc to test clinical specimens for evidence of sars-cov include serology, pcr testing, and viral cultures. serologic testing for coronavirus antibody consists of indirect fluorescent antibody testing and enzyme-linked immunosorbent assays that are specific for antibody produced after infection. patients seem to seroconvert at a mean of 10 days after onset of symptoms. the cdc has made reagents for sars antibody testing available to state public health laboratories. an rt-pcr test specific for rna from the sars-cov has been positive within the first 10 days after fever onset in respiratory specimens from most patients considered probable cases of sars who have been tested and in stool samples in the second week of illness. the duration of detectable viremia or viral shedding needs further study. 12, 13 despite the fact that several thousand specimens from patients with sars have been processed in laboratories worldwide, to date there have been no reported clusters of illness in laboratory workers. nevertheless, reasonable precautions should be taken in handling these specimens. the cdc recommends that specimens from patients with sars be labeled accordingly and that the laboratory be alerted before the samples are sent. laboratory workers who handle these specimens should use standard precautions in a biosafety level 2 laboratory. any procedure with the potential to generate fine particulate aerosols should be performed in a biological safety cabinet. 19 treatment there is no specific treatment currently available for sars. management consists of supportive care and appropriate infection control measures to prevent spread. because the diagnosis is uncertain, empirical therapy for community-acquired pneumonia should be administered by using antibiotics with activity against both typical and atypical respiratory pathogens including influenza when appropriate. in all series of sars cases described to date, therapy has involved broad-spectrum antibiotics, including a fluoroquinolone or macrolide. the antiviral drug ribavirin has been used in most patients treated in hong kong and in toronto, without evidence of efficacy. 7,10 the adverse effects of ribavirin are significant, particularly hemolytic anemia and electrolyte disturbances such as hypokalemia and hypomagnesemia; hence, empirical therapy with ribavirin is not warranted. anecdotal evidence suggests that corticosteroids may be beneficial, particularly in patients with progressive pulmonary infiltrates and hypoxemia. various regimens have been used in different centers, with dosages of methylprednisolone ranging from 40 mg twice daily (similar to therapy for pneumocystis carinii pneumonia) to 2 mg/kg per day (similar to late-phase therapy for ards) to pulse doses of 500 mg intravenously per day. 20 because cortico-steroids have potential adverse effects, clinicians should carefully assess the risk vs the benefit on a case-by-case basis. early recognition and isolation of patients with sars is key to limiting spread of the disease. based on our current understanding of disease transmission, infection control measures for suspected sars cases should include the following. early identification of patients with sars in health care facilities and appropriate isolation are essential in preventing large outbreaks. the cdc has recommended that in health care settings, patients should be screened with targeted questions as soon as possible after arrival or while the patient is on the telephone making an appointment. we have been using a simple screening tool have you been asked about your travel history today? have you traveled to china, hong kong, singapore, taiwan, or vietnam* in the past 2 weeks or had close contact with someone who has traveled and is ill with fever or cough or has difficulty breathing? have patient put on surgical mask and put patient in the room immediately physician to evaluate further all health care workers must wear an niosh-approved (tb) mask and gloves before entry to patient's room (figure 2 ) to screen all patients in our facility. patients who have positive findings on the initial screen are given a mask, separated from other patients, placed in an examination room (a negative-air room if available), and evaluated further by a physician with airborne (n-95 respirator) and contact (gown, gloves) precautions. if, during this evaluation, the patient fits the cdc case definition for sars and needs admission, the patient is admitted to a negative-air room. if admission is unnecessary but further testing is required, the patient is escorted to appropriate areas (for laboratory tests, radiological studies, etc) and testing expedited so that exposure of other patients is minimized. diagnosis of sars does not automatically necessitate admission. patients who do not otherwise need to be admitted to a hospital should be managed as outpatients as long as they can limit interactions outside the home. persons being managed as outpatients should be instructed to wear a surgical mask when in the presence of household members, and they should contain respiratory secretions in facial tissue and place these in lined containers for disposal with household waste. they should wash their hands frequently with soap and water or an alcoholbased hand sanitizer, especially after touching respiratory secretions and other body fluids. sharing of eating utensils, towels, and bedding between patients with sars and others should be avoided until these items have been washed with soap and hot water. such patients should stay away from work, school, or other activities in a public place for 10 days after the resolution of fever provided that cough and other respiratory symptoms have resolved or have improved. a plan will need to be formulated to decide how these outpatients will obtain food and other supplies such as surgical masks during the period of isolation and how arrangements will be made for travel to and from necessary medical appointments. household members or other close contacts of patients with sars should be advised to wear disposable gloves for any direct contact with body fluids from the patient and to practice good hand hygiene. such people should be vigilant for the development of fever or respiratory symptoms, and if these occur, they should seek health care evaluation. they should inform their health care provider in advance of arriving for the evaluation that they have had close contact with a patient with sars so that arrangements can be made, as necessary, to prevent transmission to others in the health care setting. in the absence of fever or respiratory symptoms, they need not limit their activities outside the home. patients who require hospitalization should be admitted to a negative pressure, specially ventilated room. visitors should be discouraged; the number of hcws involved in the patient's care should be limited. all hcws should use personal protective equipment appropriate for standard, contact, and airborne precautions (ie, hand hygiene, gown, gloves, and n-95 respirator) in addition to eye protection while caring for these patients. procedures that result in coughing and aerosolization of respiratory secretions should be avoided. if bronchoscopy is considered essential for patient care, it should be performed by experienced personnel using maximum infection control precautions (table 3) . an important feature of the sars outbreaks in canada, singapore, and hong kong has been the fact that several hcws have developed sars after caring for patients with sars. transmission to hcws appears to have occurred after close, unprotected contact with symptomatic individuals. this has placed additional strain on health care systems that have already been stressed by outbreaks of sars. active surveillance for fever and respiratory symptoms (eg, daily screening) should be conducted in hcws with unprotected exposure for 10 days after the exposure, including checking the temperature when the employee reports to work and taking a history of respiratory symptoms. workers with unprotected exposure who develop fever and/or respiratory symptoms should not come to work; they should stay home and report symptoms to infection control or the employee health service immediately. health care workers with unprotected exposure during aerosolgenerating procedures (intubation, suctioning, bronchoscopy, etc) should be quarantined for a 10-day period at home because these procedures pose a higher risk of disease transmission. case definitions of sars are currently based on the presence of epidemiological risk factors (close contact with patients with sars or travel to sars-affected areas) and a combination of fever and respiratory symptoms, with or without chest radiographic changes. however, if sars spreads into the general population, our ability to distinguish it from other community-acquired pneumonias based on such epidemiological linkages will fail. if this happens, sars will need to be considered in the differential diagnosis of any community-acquired or nosocomial pneumonia. a "typical" history, suggestive laboratory values, and failure to respond to conventional antibiotics should raise suspicion. diagnostic tests will be crucial in the future both to ensure that patients are isolated rapidly and to provide appropriate therapy. sars has resulted in important challenges for the medical community. in affected areas, policies are changing daily as more information about the virus and the disease is obtained. the resurgence of cases in toronto in early may 2003 shows the difficulty of maintaining control over a new disease characterized by many puzzling epidemiological and clinical features. the continued alertness of singapore, which most recently broke the chain of sars transmission, shows the importance of maintaining a high level of vigilance and preparedness to ensure that a single imported case does not reignite an outbreak. currently, control depends on prompt detection and isolation of cases, good infection control in hospitals, and the tracing and quarantine of contacts. world health organization. cumulative number of reported probable cases of severe acute respiratory syndrome (sars) preliminary clinical description of severe acute respiratory syndrome updated interim u.s. case definition of severe acute respiratory syndrome (sars) updated interim surveillance case definition for severe acute respiratory syndrome (sars)-united states identification of severe acute respiratory syndrome in canada severe acute respiratory syndrome (sars) in singapore: clinical features of index patient and initial contacts clinical features and short-term outcomes of 144 patients with sars in the greater toronto area clinical progression and viral load in a community outbreak of coronavirus-associated sars pneumonia: a prospective study clinical presentations and outcome of severe acute respiratory syndrome in children a major outbreak of severe acute respiratory syndrome in hong kong coronavirus as a possible cause of severe acute respiratory syndrome identification of a novel coronavirus in patients with severe acute respiratory syndrome a novel coronavirus associated with severe acute respiratory syndrome the genome sequence of the sars-associated coronavirus characterization of a novel coronavirus associated with severe acute respiratory syndrome. science mag [serial online world health organization. first data on stability and resistance of sars coronavirus compiled by members of who laboratory network. available at: www.who.int/csr/sars/survival_2003_05_04 /en severe acute respiratory syndrome-singapore guidelines for collection of specimens from potential cases of sars. available at: www.cdc.gov/ncidod/sars/specimen_collection_sars2.htm. accessibility verified interim laboratory biosafety guidelines for handling and processing specimens associated with severe acute respiratory syndrome (sars) development of a standard treatment protocol for severe acute respiratory syndrome key: cord-270947-6e5cw2q9 authors: huang, h.-s.; tsai, c.-l.; chang, j.; hsu, t.-c.; lin, s.; lee, c.-c. title: multiplex pcr system for the rapid diagnosis of respiratory virus infection: systematic review and meta-analysis date: 2017-12-05 journal: clin microbiol infect doi: 10.1016/j.cmi.2017.11.018 sha: doc_id: 270947 cord_uid: 6e5cw2q9 objectives: to provide a summary of evidence for the diagnostic accuracies of three multiplex pcr systems (mpcrs)—biofire filmarray rp (filmarray), nanosphere verigene rv+ test (verigene rv+) and hologic gen-probe prodesse assays—on the detection of viral respiratory infections. methods: a comprehensive search up to 1 july 2017 was conducted on medline and embase for studies that utilized filmarray, verigene rv+ and prodesse for diagnosis of viral respiratory infections. a summary of diagnostic accuracies for the following five viruses were calculated: influenza a virus (flua), influenza b virus, respiratory syncytial virus, human metapneumovirus and adenovirus. hierarchical summary receiver operating curves were used for estimating the viral detection performance per assay. results: twenty studies of 5510 patient samples were eligible for analysis. multiplex pcrs demonstrated high diagnostic accuracy, with area under the receiver operating characteristic curve (auroc) equal to or more than 0.98 for all the above viruses except for adenovirus (auroc 0.89). filmarray, verigene rv+ and proflu+ (the only prodesse assay with enough data) demonstrated a summary sensitivity for flua of 0.911 (95% confidence interval, 0.848–0.949), 0.949 (95% confidence interval, 0.882–0.979) and 0.954 (95% confidence interval, 0.871–0.985), respectively. the three mpcrs were comparable in terms of detection of flua. conclusions: point estimates calculated from eligible studies showed that the three mpcrs (filmarray, verigene rv+ and proflu+) are highly accurate and may provide important diagnostic information for early identification of respiratory virus infections. in patients with low pretest probability for flua, these three mpcrs can predict a low possibility of infection and may justify withholding empirical antiviral treatments. acute respiratory tract infections (ari) cause high morbidity and mortality [1] . among them, viral aris are one of the leading causes for paediatric and geriatric hospitalization and clinic visits [2, 3] . each year, seasonal influenza causes >200 000 hospitalizations and more than $10 billion direct medical costs in the united states. in specific populations (e.g. immunocompromised patients, neonates, and chronic pulmonary disease patients), the high complication and mortality rates from viral aris is a major concern [4] . moreover, empirical antibiotics are commonly prescribed to patients with viral aris because of the lack of rapid and sensitive diagnostic methods and nonspecific symptoms, which delay proper treatments and precipitate antibiotic resistance [4e6] . traditional diagnostic techniques (e.g. virus culture, haemagglutination inhibition assay, enzyme immunoassay and direct fluorescent antibody) were once the mainstays for pathogen detection. however, these methods were either insensitive, time consuming, labor intensive or operator dependent [7e9] . new technologies have emerged as a result of massive clinical demands, such as melting curve analysis, microfluidic device and nucleic acid amplification technologies [5,6,10e13] . these molecular diagnostic tools have shorter turnaround times and higher sensitivity for viral pathogens [14, 15] . in addition, they allow for detection of a broader panel of viruses and coinfection [15, 16] , and they thus have become more widely used than the conventional virologic assays [8,17e19] . in particular, multiplex pcr (mpcr) is a validated strategy for the rapid detection and precise identification of a large number of respiratory viruses [19e22] by incorporating several primers within one reaction tube to amplify genomic fragments of many pathogens [22, 23] . with the use of a mpcr panel, one study demonstrated a 30% to 50% increase in the diagnostic yield of respiratory viruses compared to direct fluorescent antibody and culture [24] . there are a number of us food and drug association (fda)cleared mpcrs available today for detecting respiratory pathogens, each with pros and cons. the characteristics of the three fdaapproved mpcr systems included in our study are listed in table 1 . the biofire filmarray rp (filmarray) respiratory panel [24] , which utilizes melting curve analysis, is a random-access molecular test using principles of real-time pcr. the verigene rvþ test is based on gold nanoparticle technology and silver signal amplification. lastly, hologic gen-probe prodesse launches several assays with variable run sizes that also utilize melting curve analysis but with limited multiplexing ability. although each prodesse assay can only detect two to three viruses at a time, the prodesse assays are still viewed as mpcr [25] . these three mpcrs were chosen because they have shorter turnaround times and have more available studies for analysis among a number of fda-approved mpcrs. there is also one original study that provided direct comparison of these three mpcrs [25] . to gain insight into the optimal diagnostic tool for routine clinical use, we here provide a summary of evidence comparing the diagnostic accuracies of filmarray, verigene rvþ and hologic gen-probe prodesse assays for the detection of viral respiratory infections. the protocol of our study was based on the prisma (preferred reporting items for systematic review and meta-analysis) statement [26] and the standard guideline for systematic reviews of diagnostic tests by the cochrane collaboration [27] . a comprehensive search of literature was conducted using two databases: pubmed (from inception to april 2015) and embase (from inception to april 2015). the search term combination was: (multiplex and pcr or (multiplex and polymerase and chain and reaction) or filmarray or verigene or prodesse or proflu or profast or proadeno or proparaflu or (pro hmpv)) and ((respiratory and tract and infection) or (respiratory and infection) or (respiratory and virus) or (respiratory and tract and disease) or (respiratory and disease) or (common and cold) or influenza or pneumonia or bronchitis or bronchiolitis or rhinosinusitis or pharyngitis or laryngitis or (otitis and media) or tonsillitis or asthma or copd or (chronic and obstructive and lung and disease)). the detailed search strategy is provided in supplementary materials s1. no language restrictions were applied to the search. the search was then supplemented by bibliographies of retrieved full-text articles and the latest narrative reviews. we also contacted the authors of publications that did not provide required data. an updated search to 1 july 2017 was performed before starting the statistical analysis. studies that evaluated the performance of fda-approved mpcr systems for the detection of viral respiratory infection were included, as follow: (a) they assessed the accuracy of one or more the following systems: filmarray, nanosphere verigene rvþ and hologic gen-probe prodesse assays (profluþ, profast, proparafluþ, proadenoþ and pro hmpvþ) against reference standards and (b) they provided sufficient information to calculate sensitivity and standard to validate other mpcr systems and (c) they used an mpcr assay not approved by fda as reference. in addition, we excluded reviews, guidelines, case reports, editorials, panel discussions, letters, notes and comments. for multiple publications, only the latest available publications with complete data of the same patient group were included. studies that compared more than one fda-approved mpcr systems with the same reference standard, used different reference methods for different viruses, used different samples for different viruses or reported prospective and retrospective samples independently have separate data sets for each comparison. three reviewers independently screened the titles and abstracts from pubmed and embase. disagreements or uncertainties were resolved by discussion. a data extraction form was used in 20 included studies by two reviewers before being finalized. each data set was extracted by two authors independently to avoid bias. the form consisted of the following characteristics: study type, study design, patient age, patient inclusion criteria, specimen type, mpcr systems used, reference standard and 2 â 2 tables. the 2 â 2 tables were further used to calculate sensitivities and specificities of the target assays. for the reference methods, virus culture and direct fluorescent antibody were grouped together because they are universally recognized as the reference standard [28, 29] . reverse transcription (rt) pcr referred to both commercial rt-pcrs and in-house rt-pcrs. composite reference standard was defined as a standard that used more than one comparator assays. discrepant analyses encompassed studies that resolved discrepancies between target assay and comparator assay by bidirectional sequencing, rt-pcr, repeated testing or other methods. studies were also grouped on the basis of whether they incorporated filmarray, verigene rvþ and hologic gen-probe prodesse assays as part of a composite reference standard. studies that had <5% of the samples taken from the lower respiratory tract were categorized as using upper respiratory specimens. children were defined as patients younger than 18 years. studies that included both children and adult population were categorized as mixed. the quality of the eligible studies was independently assessed by two reviewers using the quality assessment of diagnostic accuracy studies 2 tool (quadas-2) [30] . for each diagnostic study, we determined the risk for bias and general applicability in all four domains of quadas-2 and reported them separately. those with low risk of bias or low concern regarding applicability were judged as low. a study would be judged as unclear if there were insufficient data for interpretation. a bivariate model was applied to estimate summary sensitivity and specificity. the positive likelihood ratios (lrþ) and negative likelihood ratios (lrà) were then calculated from summary sensitivity and specificity. the bivariate model approach modelled the logit-transformed sensitivity and specificity simultaneously to account for the inherent negative correlation between sensitivity and specificity that may arise due to different thresholds in different studies [30] . in addition, the bivariate model could also account for between-study heterogeneity. all analyses except for the summary receiver operating characteristic curve (roc) were performed by the 'mada' package in r software (r foundation for statistical computing, vienna, austria; http://www.r-project.org/). the summary roc and area under the roc was calculated by the 'midas' package in stata (stata inc, college station, texas). a twosided p value of <0.05 indicated statistical significance for all tests. our comprehensive search yielded 1900 studies from embase and 1350 studies from pubmed. after inclusion and exclusion following the protocol and an updated search to 1 july 2017 (fig. 1) , a total of 20 studies (25 data sets) were eligible for analysis, encompassing a total of 5510 patient samples. profluþ was the only prodesse assay with enough data sets for quantitative analysis. parainfluenza virus was not analysed because of insufficient data. the laboratory characteristics of filmarray rp, verigene rvþ and prodesse profluþ are illustrated in table 1 . a simplified summary of the characteristics of the included studies is provided in table 2 . details of the characteristics and key results of each individual study are provided in supplementary materials s2. none of the studies specifically recruited adults only, and approximately 30% of the studies obtained their samples from children. discrepant analysis and composite reference standard were the two most commonly applied reference standards. the studies varied in quality. quality assessment by the quadas-2 tool is demonstrated in fig. 2 . for the 'patient selection' domain, some studies were not clear about how the patients were recruited. some studies used refrigerated samples that were precollected without specifying their original sources; others did not avoid a caseecontrol design. for the 'reference standard' domain, most studies did not specify the use of blinding for reference standard. some studies that were of low quality in these regards used the index test as part of the reference method. others did not use the same reference method on all their samples. however, most studies were identified as high quality for flow and timing. overall, the majority of the studies had low concern regarding applicability. the overall lrà were all below 0.1, which suggested a high rule-out value. table 3 provides point estimates for flub, rsv, hmpv and adenovirus. fig. 3 shows the forest plot of sensitivity and specificity of the mpcrs for each virus. fig. 4 illustrates the receiver operating characteristic curve and area under the receiver operating characteristic curve for the detection of each virus. overall, in our analyses including 5510 patient samples, mpcr systems demonstrated high diagnostic accuracy (auroc !0.98) for flua, flub, rsv and hmpv, with the exception of adenovirus (auroc 0.89). filmarray rp, verigene rvþ and prodesse profluþ demonstrated a summary sensitivity for flua of 0.911 (95% confidence interval (ci), 0.848e0.949), 0.949 (95% ci, 0.882e0.979) and 0.954 (95% ci, 0.871e0.985), respectively. the three mpcrs were comparable in terms of detection of flua, and filmarray rp and verigene rvþ were comparable for rsv detection. although prodesse profluþ was found to have a statistically significant higher sensitivity than filmarray rp for flub detection, they demonstrated comparable auroc and thus comparable accuracy. of the five respiratory viruses that these systems detect, the diagnosis of influenza virus infection may have the greatest clinical impact [31] . on the basis of our study, overall, the mpcrs exhibited reasonable sensitivity (0.940; 95% ci, 0.902e0.964) and high specificity (0.987; 95% ci, 0.979,0.992) for flua. in current clinical practice, the immunoassay-based rapid influenza diagnostic test is most widely used for screening influenza infections [8] . although the rapid influenza diagnostic test can detect flua and flub in respiratory specimens in approximately 15 to 30 minutes, its sensitivity is limited, as reported by a previous meta-analysis [32] . this may give false-negative results, which prevents its use as a reliable excluding diagnostic tool in clinical practice. furthermore, commonly used reference standards such as rt-pcr or virus culture may take several days to yield results and have little value for treatment decision. our meta-analysis showed that mpcrs provide highly accurate results in a clinically relevant time frame and may potentially change the current diagnostic and treatment practice for flua infection. as a result of the low sensitivity of the rapid influenza diagnostic tests, the decision of antiviral treatment for flua infection is largely based on clinical grounds alone [5] , which may lead to overprescription of anti-influenza drugs and increasing drug resistance [33, 34] . a study by van wesenbeeck et al. [25] of 171 clinical samples concluded that filmarray rp and prodesse profluþ have better sensitivity for flua than verigene rvþ. this is contrary to our results, which found the three mpcrs to be comparable for flua detection. our meta-analysis synthesized data across different studies encompassing 5510 patient samples and may present a more accurate estimate of the real situation. although these three systems have comparable accuracy for flua, filmarray rp has the shortest hands-on time (2 minutes) and run time (1 hour) among the three mpcrs and includes sample preparation in its panel. furthermore, its reagents can be stored at room temperature, and its assay detects the largest number of targets. therefore, filmarray rp may be the best choice in emergency rooms or during the influenza season. there is less interest in rapid diagnostic tests for rsv, hmpv and adenovirus as a result of a lack of specific treatments available against these viruses and the common practice of supportive care in clinical settings. nevertheless, rsv is one of the leading causes of aris in children [35] and immunocompromised patients, and it may cause severe complications; it therefore requires definitive diagnosis. our results revealed that mpcrs are highly sensitive and specific for rsv and hmpv, and they offer a more rapid and accurate alternative to traditional methods [7, 36] . the current treatment is ribavirin for high-risk infants and young children [37] , with several clinical trials for novel rsv treatments underway [38e41]. in contrast, for adenovirus, filmarray rp had a moderate to low sensitivity (<70%), but its superior specificity (>0.99) makes it a reliable rule-in tool. the literature has demonstrated that commercial filmarray rp (v1.6) has low sensitivity for adenovirus [24,42e44] , but its sensitivity greatly improved in the commercial filmarray 1.7 [42] , developed later. of note, none of our included studies specifically reported using filmarray 1.7. to our knowledge, this is the first report to perform a comparative meta-analysis on different mpcr platforms and different viruses. not only did we provide detailed comparison on the characteristics of the three systems but we also provided quantitative accuracy measure for the different viruses by different systems. there are several limitations to our study, however. firstly, some included studies did not provide sufficient details regarding the version of filmarray rp used (premarket or commercial). we traced their publication dates and contacted the filmarray rp manufacturers as well as the authors of the original study at an attempt to confirm the version of filmarray rp. however, sensitivity analysis showed no clinically significant difference between the sensitivities and specificities of precommercial and fdaapproved commercial assays (data not published). secondly, some included studies have a retrospective study design; they failed to specify their inclusion criteria, and they used only previously stored samples from patients with unknown characteristics. there is also concern regarding diagnostic review bias, in which the interpretation of the result of reference test is made with knowledge of the index test result [45] . however, a test review bias is highly unlikely because the interpretation of the index tests (mpcrs) was objective. of note, five of the included studies adopted the caseecontrol design, which tends to give an overestimation of the accuracy of the index test [45] . in addition, results of this study only showed the accuracy of three commercial mpcr systems for diagnosis of respiratory virus infection. whether they have equal strength in therapeutic guidance remains to be validated. lastly, our study does not provide an answer to whether mpcr systems improve the outcome of patients with respiratory virus infection compared to clinical diagnosis alone. analysis of eligible studies showed that three commercial mpcr systemsdfilmarray, verigene rvþ and genprobe prodesse pro-fluþdmay provide important diagnostic information to help early identification of influenza virus, respiratory syncytial virus, adenovirus and hmpv. the great improvement of the sensitivity of the rapid diagnosis of influenza may have the potential to change current modes of diagnosis and management. however, not all of these systems are equally useful in terms of inclusion or exclusion diagnosis. clinicians should interpret the results on the basis of likelihood ratio and pretest probability. the next step would be to assess whether provision of information from these mpcr tests can modify clinical outcomes. estimates of world-wide distribution of child deaths from acute respiratory infections prevalence and correlation of infectious agents in hospitalized children with acute respiratory tract infections in central china community-acquired pneumonia requiring hospitalization: 5-year prospective study the effect of rapid respiratory viral diagnostic testing on antibiotic use in a children's hospital cost-effective use of rapid diagnostic techniques in the treatment and prevention of viral respiratory infections excessive antibiotic use for acute respiratory infections in the united states detection of respiratory viruses by molecular methods current approaches for diagnosis of influenza virus infections in humans current best practices for respiratory virus testing cost-effectiveness of rapid diagnosis of viral respiratory tract infections in pediatric patients cost analysis of multiplex pcr testing for diagnosing respiratory virus infections thermally multiplexed polymerase chain reaction genotyping of respiratory syncytial virus (rsv) group a in stockholm, sweden, using pcr and two-dimensional melting curve analysis nucleic acid amplificationebased diagnosis of respiratory virus infections evaluation of multiple test methods for the detection of the novel 2009 influenza a (h1n1) during the new york city outbreak emerging molecular assays for detection and characterization of respiratory viruses development of a respiratory virus panel test for detection of twenty human respiratory viruses by use of multiplex pcr and a fluid microbead-based assay evaluation of a multiplexed pcr assay for detection of respiratory viral pathogens in a public health laboratory setting respifinder: a new multiparameter test to differentially identify fifteen respiratory viruses respiratory viruses in children admitted to hospital intensive care units: evaluating the clart® pneumovir dna array broad respiratory virus detection in infants hospitalized for bronchiolitis by use of a multiplex rt-pcr dna microarray system simultaneous detection and high-throughput identification of a panel of rna viruses causing respiratory tract infections rapid detection of respiratory tract viral infections and coinfections in patients with influenza-like illnesses by use of reverse transcriptionepcr dna microarray systems the filmarray® respiratory panel: an automated, broadly multiplexed molecular test for the rapid and accurate detection of respiratory pathogens comparison of the filmarray rp, verigene rvþ, and prodesse profluþ/fastþ multiplex platforms for detection of influenza viruses in clinical samples from the 2011e2012 influenza season in belgium the prisma statement for reporting systematic reviews and meta-analyses of studies that evaluate healthcare interventions: explanation and elaboration cochrane diagnostic test accuracy working group. systematic reviews of diagnostic test accuracy diagnostic virology: from animals to automation role of cell culture for virus detection in the age of technology cochrane diagnostic test accuracy reviews antivirals for treatment of influenzada systematic review and meta-analysis of observational studies accuracy of rapid influenza diagnostic tests: a meta-analysis emerging influenza antiviral resistance threats influenza drug resistance. seminars in respiratory and critical care medicine risk of primary infection and reinfection with respiratory syncytial virus respiratory syncytial virus infection ribavirin for respiratory syncytial virus infection of the lower respiratory tract in infants and young children oral gs-5806 activity in a respiratory syncytial virus challenge study discovery of an oral respiratory syncytial virus (rsv) fusion inhibitor (gs-5806) and clinical proof of concept in a human rsv challenge study activity of oral als-008176 in a respiratory syncytial virus challenge study coadministration of the broad-spectrum antiviral, brincidofovir (cmx001), with smallpox vaccine does not compromise vaccine protection in mice challenged with ectromelia virus evaluation and implementation of fil-marray version 1.7 for improved detection of adenovirus respiratory tract infection comparison of the idaho technology filmarray system to real-time pcr for detection of respiratory pathogens in children comparison of the filmarray respiratory panel and prodesse real-time pcr assays for detection of respiratory pathogens evaluating bias and variability in diagnostic test reports comparison of the focus diagnostics simplexa flu a/b & rsv direct assay with the prodesse profluþ assay for detection of influenza a virus (iav), influenza b virus (ibv), and respiratory syncytial virus (rsv) in clinical specimens detection of influenza a, b and rsv in respiratory specimens by the profluþtm kit and roche lc 480 performance analysis of pandemic influenza detection methods simultaneous detection of influenza a and its subtypes (h1, h3, 2009 h1n1), influenza b, and rsv a and b in respiratory specimens on an automated, random access, molecular platform comparative evaluation of the nanosphere verigene rvþ assay and the simplexa flu a/b & rsv kit for detection of influenza and respiratory syncytial viruses comparison of the biofire filmarray rp, genmark esensor rvp, luminex xtag rvpv1, and luminex xtag rvp fast multiplex assays for detection of respiratory viruses comparison of the luminex xtag rvp fast assay and the idaho technology filmarray rp assay for detection of respiratory viruses in pediatric patients at a cancer hospital comparison of two multiplex methods for detection of respiratory viruses: filmarray rp and xtag rvp comparison of xtag respiratory virus panel and verigene respiratory virus plus for detecting influenza virus and respiratory syncytial virus fil-marray, an automated nested multiplex pcr system for multi-pathogen detection: development and application to respiratory tract infection rapid multiplex pcr assay to identify respiratory viral pathogens: moving forward diagnosing the common cold the clinical utility of a near patient care rapid microarray-based diagnostic test for influenza and respiratory syncytial virus infections in the pediatric setting evaluation of three influenza a and b real-time reverse transcriptionepcr assays and a new 2009 h1n1 assay for detection of influenza viruses development of a rapid automated influenza a, influenza b, and respiratory syncytial virus a/b multiplex real-time rt-pcr assay and its use during the 2009 h1n1 swine-origin influenza virus epidemic in performance of the cobas® influenza a/b assay for rapid pcr-based detection of influenza compared to prodesse profluþ and viral culture surveillance of upper respiratory infections using a new multiplex pcr assay compared to conventional methods during the influenza season in taiwan effect of genomic drift of influenza pcr tests we thank the staff of core labs, department of medical research, national taiwan university hospital, for technical support; and we thank our medical librarian, h.-p. chiu, for consultation in formulating the search strategy. research grant ntuh106-p04. all authors report no conflicts of interest relevant to this article. supplementary data related to this article can be found at https://doi.org/10.1016/j.cmi.2017.11.018. key: cord-255623-qdpdsye9 authors: pham, hien t.; nguyen, phuc t. t.; tran, sinh t.; phung, thuy t. b. title: clinical and pathogenic characteristics of lower respiratory tract infection treated at the vietnam national children's hospital date: 2020-03-11 journal: can j infect dis med microbiol doi: 10.1155/2020/7931950 sha: doc_id: 255623 cord_uid: qdpdsye9 lower respiratory tract infections are commonly caused by viruses and cause significant morbidity and mortality among children. early identification of the pathological agent causing these infections is essential to avoid unnecessary antibiotic use and improve patient management. multiplex pcr techniques were recently developed to detect multiple viral pathogens using a single pcr reaction. in this study, we identify viral pathogens in children with respiratory infections. we collected 194 nasopharyngeal aspirates from infants (2–24 months old) with lower respiratory tract infections treated at the vietnam national children's hospital between november 2014 and june 2015 and assessed the presence of 16 virus types and subtypes by multiplex pcr using the xtag respiratory viral panel (rvp) assay. overall, 73.7% of the samples were positive for at least one virus, and 24.2% corresponded to infections with multiple viruses. the most common viruses were respiratory syncytial virus and enterovirus/rhinovirus. these viruses were more frequent among younger patients (2–5 months old) and caused symptoms similar to those of bronchiolitis and pneumonia. the most common clinical manifestation caused by respiratory tract infection was bronchiolitis. elevated neutrophils levels were associated with adenovirus infection. our results showed that the xtag respiratory viral panel (rvp) can effectively detect multiple viruses causing respiratory infections in children and that the nasopharyngeal aspirates are a good sample choice to detect respiratory viruses in children. applying this approach in the clinical setting would improve patient management and allow early diagnosis, thus avoiding the unnecessary use of antibiotics. acute respiratory infections (aris) are common among children worldwide. although they have similar incidence rate in developed and developing countries, the mortality rate is higher in developing countries [1] [2] [3] . aris are responsible for approximately 20% of all deaths in children under 5 years in 2006; about 70% of these deaths occur in sub-saharan africa and the southern regions of asia. viruses are the main causative agents of respiratory infection, but bacteria, fungi, and parasites can cause them as well. e leading ari-causing viruses are rhinovirus and respiratory syncytial virus (rsv) [4] , with rsv affecting mainly infants under 1 year of age [5, 6] . viruses primarily infect and replicate in the airway epithelium, causing injuries in the proximal (conducting) and distal airways (alveoli and parenchyma), where gas exchange occurs. viral infection can have multiple clinical manifestations, such as pneumonia, defined as an inflammation of the lung parenchyma. is condition is often associated with visible changes on chest x-rays, ctscanning, or gallium scanning and with abnormalities in alveolar gas exchange. e presentations of viral pneumonia vary considerably depending on the age and immunological competence of the host, as well as the viral pathogen. viral pneumonia is an important cause of morbidity and mortality in immune compromised individuals and children [7] . e clinical presentation depends on the specific causative agent but typically includes fever and lower respiratory tract symptoms, such as tachypnoea, nonproductive cough, wheeze, and increased breath sound [7, 8] . inappropriate antibiotic use during acute respiratory infection has led to an increase in antimicrobial resistance [9] . erefore, early detection of the pathogen causing respiratory infection may prevent antibiotic issue and help improve patient management [10] . however, the lack of rapid, affordable, sensitive, and specific diagnostic tools is one of the main reasons for inefficient diagnosis. pcr has revolutionized the field of infectious disease diagnosis. multiplex pcr, a variant that uses several pairs of primers to amplify more than one target sequence in a single tube, was developed to detect different viruses in a single sample [11] . previous studies showed that multiplex assays using a limited amount of sample had similar performance to that of traditional methods when used for clinical diagnosis [12] . additionally, they have the benefit of detecting a large number of viruses in a short time. e xtag respiratory viral panel fast (rvp fast) is a qualitative nucleic acid multiplex test that simultaneously detects and identifies the presence of nucleic acids from multiple respiratory viruses in a single tube. it works with samples such as nasopharyngeal swabs, nasal aspirates, nasopharyngeal aspirates, and bronchoalveolar lavages from individuals suspected of respiratory tract infections. e xtag rvp fast test has shown superior sensitivity when compared to single real-time pcr and conventional pcr assays [11, 13, 14] . in the present study, we used the xtag rvp fast assay to identify the viruses causing ri in children and the relationship between specific viruses and clinical outcome. is cross-sectional study was held between november 2014 and june 2015. we enrolled 194 pediatric infants (2-24 months old) who had lower respiratory tract infections and were treated at the vietnam national children's hospital. we collected nasopharyngeal aspirates from each participant. patients were divided into 3 age groups. e first group included infants between 2 and 5 months (n � 71) who were breastfed and likely received passive immunity from their mothers. e second group included infants between 6 and 11 months old (n � 66) who spent most of their time at home. e third groups included infants between 12 and 24 months old (n � 57) who attended different nurseries. is study was approved by the vnch ethics committee (approval number: 521b/nch-rich). written informed consent was obtained from the parents or legal guardians of the children enrolled in the study. we collected 2 ml of nasopharyngeal aspirate from each participant and stored the samples at −70°c until analysis. npa collection was performed by trained nurses. e catheter was inserted into the nostril to a depth of 5 to 7 cm and drawn back while applying gentle suction with a syringe. npa sample (200 μl) from the patient was subjected to total nucleic extraction after addition of internal control bacteriophage ms2 (20 μl) using the magna pure lc total nucleic acid isolation kit (roche diagnostics, germany) on magna pure lc 2.0 platform, following the manufacturer's instructions [15] . e method is based on magnetic-bead technology. e procedure included cellular destruction, nucleic acid binding on beads, and washing steps to remove cellular and purified nucleic acid elution. extracted nucleic acids were eluted in 50 µl of elution buffer and stored at −80°c for rvp fast assay. cluding subtypes h1n1 (1977), h1n1pdm09, h3n2, respiratory syncytial virus a and b (rsva, rsvb), enteroviruses including rhinoviruses (ev/rhi), human parainfluenza viruses 1-4 (piv1-4), human metapneumovirus (hmpv), adenovirus (adv), human coronavirus nl63 (hcov nl63), hcov hku1, hcov 229e, hcov oc43, and human bocavirus (hbov). e assay was performed according to the manufacturer's instructions. assay performance was controlled using bacteriophage lambda included in every run. e assay comprised two steps: a multiplex pcr amplification step and hybridization step. 2.6. multiplex rt-pcr. pcr amplification was performed on the applied biosystems gene amp 9700 pcr (applied biosystems, usa) with the following program: preheating at 50°c for 20 min; template denaturation at 95°c for 15 min; 34 cycles of 95°c for 30 s, 59°c for 30 s, and 72°c for 30 s; and final extension at 72°c for 2 min and kept at 4°c until ready tousle. e hybridization reaction was performed on luminex 100/200 system (lmd, toronto, canada). e hybridization assay includes 20 µl xtag rvp fast bead mix, 2 µl amplified nucleic acid, and 75 µl streptavidin, r-phycoerythrin conjugate (sa-pe) reporter. all reagents were incubated at 45°c for 20 minutes. signal acquisition was done on a luminex 100/200 instrument. data were analyzed and reported by xtag data analysis software (tdas). e infections were classified as pneumonia, bronchiolitis, or bronchitis based on clinical findings and chest x-rays (cxr). pneumonia was defined as ari characterized by infiltrates on the cxr. bronchiolitis was defined as ari in children under 2 years who presented wheezing and hyperaeration and atelectasis or peribronchial thickening in the cxr. bronchitis was defined as ari in children over 2 years old who presented wheezing, hyperaeration, and peribronchial thickening on the cxr. analysis. statistical analysis was performed using fisher's exact test. p < 0.05 was considered significant (spss 13). e xtag rvp fast assay system detected one or more respiratory viruses in 73.7% (143/194) of the samples (table 1) . rsv was the most common virus (51%, 73/143), followed by enterovirus/rhinovirus (ev/rhi), parainfluenza (piv), adenovirus (adv), influenza a/b, and human bocavirus (hbov). overall, 96 samples were positive for a single virus, 37 for 2 viruses, 9 for 3 viruses, and 1 for 4 viruses. groups. rsv and ev/rhi were detected more frequently in children under 6 months (figure 1 ). for other viruses, there was no difference among age groups. children with viral infection in the upper respiratory tract showed symptoms such as runny nose, cough, and hoarseness. some of them also present lower respiratory tract symptoms such as wheezing, severe cough, breathlessness, and respiratory distress, which may be due to bronchiolitis or pneumonia. we divided the patients according to three clinical manifestations: pneumonia, bronchiolitis, and bronchitis, and investigated whether the detected viruses were associated with a specific clinical manifestation. our analysis showed that in most cases, rsv infections induced bronchiolitis (n � 45), followed by pneumonia (n � 22, p � 0.004) and bronchitis (n � 6, p � 0.0015). ev/rhi infections more often induced pneumonia or bronchiolitis (n � 19 for both) instead of bronchitis (n � 6, p � 0.03) (figure 2 ). other total positive specimens 143 73 62 28 15 6 9 3 3 single infection 96 44 28 12 3 3 3 1 1 double infection 37 22 26 11 7 3 2 1 1 triple infection 9 6 7 in the respiratory syncytial virus-(rsv-) infected group, the prevalence of pneumonia symptoms was significantly lower than that of bronchiolitis symptoms and significantly higher than that of bronchitis symptoms. in the enterovirus (ev)/rhinovirus (rhi) infected group, the prevalence of pneumonia symptoms was significantly higher than that of bronchitis symptoms. other viruses showed a similar prevalence of each manifestation. * p < 0.05. viruses showed a similar prevalence of each clinical manifestation. virus infections. all respiratory viruses may cause symptoms such as nasal congestion, runny nose, wheezing, and cough. we found no significant association between the viruses and a specific symptom. we studied whether the presence of specific viral agents was associated with clinical parameters such as respiratory rate (breaths/min), heart rate (beats/min), and peripheral capillary oxygen saturation (spo 2 , %) or with blood test parameters such as white blood count, neutrophil count, or c-reactive protein (crp). elevated neutrophils were associated with adenovirus infection (or � 1.4, p � 0.006, figure 3 ). ere were no other noticeable associations. crp was slightly higher in the adenovirus and enterovirus/rhinovirus infection groups than in the negative group (p � 0.29 and 0.32, respectively). neutrophils were significantly higher in the adenovirus infection group (48.7%) than in the negative group (35.6%, p � 0.0058). in this study, we calculated the frequency of different respiratory viruses present in children with lower respiratory tract infection at the vietnam national children's hospital. e high proportion of viruses detected in pediatric ari patients agreed with previous studies done in vietnam [5] . with the severity of children with respiratory infections, while those viruses were detected rarely in the north of vietnam due to the difference of the location and climate between the south and north of vietnam. [16] . rsv and ev/ rhi were the most frequent viruses, which was similar to the findings of a study done in southern vietnam [6] . rsv, rhinoviruses, influenza viruses, parainfluenza viruses, enteroviruses, coronaviruses, and certain strains of adenovirus are the leading causes of viral respiratory infections in children. e nasal or respiratory secretions from children with viral respiratory tract infections contain more viruses than those from infected adults. e increased output of viruses, together with an overall reduced attention to hygiene, makes children more likely to spread their infection to others. when viruses invade the cells of the respiratory tract, they trigger inflammation and mucus production, which causes nasal congestion, runny nose, scratchy throat, and cough. e small airways of young children can be significantly narrowed by inflammation and mucus, making breathing difficult. airway problems are most common in infections caused by parainfluenza viruses, rsv, and human metapneumovirus [1, 8, 17] . bronchiolitis occurs predominantly in the first year of life and with decreasing frequency in the second and third years. it is characterized by inflammatory obstruction of the small airways and hyperinflation of the lungs and typically presents along with breathing problems and wheezing. rsv is the primary causative agent of bronchiolitis worldwide, causing between 70 or 80 percent of aris during the high season [18] [19] [20] . rsv was the only virus associated with bronchiolitis in this study. neutrophils are immune cells that are present in many lung diseases associated with acute respiratory distress syndrome (ards) and may contribute to acute lung injury. neutrophils are poorly studied with respect to viral infection. we observed an association between elevated neutrophil count and adenovirus infection, which might indicate an association between neutrophil count and damage to the alveolar epithelium. finding biomarkers to diagnose specific viral infections is essential to improve patient care [21] . crp is an acute phase protein synthesized by the liver in response to il-6 increase, which is used as a biomarker of inflammation [22] and to distinguish between bacterial and viral infections. it is not well known if crp levels differ between different viral respiratory infections. in this study, we found no significant association between crp and a specific virus. bronchiolitis was the most common clinical characteristic of lower respiratory tract infection at the vietnam national children's hospital. xtag rvp fast can effectively detect different virus from specimens with low viral loads. e assay should be applied in the clinic for the screening of multiple respiratory viral infections. e data used to support the findings of this study are included within the article. is study has been approved by the vnch ethics committee (approval number: 521b/nch-rich). written informed consent was obtained from all patients enrolled in the study or their legal guardians. e authors declare that there are no conflicts of interest regarding the publication of this article. e changing face of pediatric respiratory tract infections: how human metapneumovirus and human bocavirus fit into the overall etiology of respiratory tract infections in young children respiratory viral infections and host responses; insights from genomics multiplex pcr: optimization and application in diagnostic virology fatal respiratory infections associated with rhinovirus outbreak clinical and epidemiological characteristics of acute respiratory virus infections in vietnamese children human rhinovirus infections in hospitalized children: clinical, epidemiological and virological features comparison of the luminex xtag respiratory viral panel with xtag respiratory viral panel fast for diagnosis of respiratory virus infections mini-symposium: microbiological diagnostic procedures in respiratory infection respiratory virus infection access to a polymerase chain reaction assay method targeting 13 respiratory viruses can reduce antibiotics: a randomised, controlled trial diagnostic errors that lead to inappropriate antimicrobial use multiplex pcr and emerging technologies for the detection of respiratory pathogens assessment of the usefulness of multiplex real-time pcr tests in the diagnostic and therapeutic process of pneumonia in hospitalized children: a single-center experience comparison of the luminex xtag rvp fast assay and the idaho technology filmarray rp assay for detection of respiratory viruses in pediatric patients at a cancer hospital comparison of the luminex xtag respiratory viral panel with in-house nucleic acid amplification tests for diagnosis of respiratory virus infections fully automated nucleic acid extraction: magna pure lc human bocavirus in children with acute respiratory infections in vietnam cellular immunity and lung injury in respiratory virus infection respiratory syncytial virus infection pathogen screening and prognostic factors in children with severe ards of pulmonary origin respiratory syncytial virus infections: recent prospects for control association between c-reactive protein and metabolic syndrome in korean adults c-reactive protein: ligands, receptors and role in inflammation key: cord-129086-ra2njvcz authors: kumar, sanjay; lee, heow pueh title: the perspective of fluid flow behavior of respiratory droplets and aerosols through the facemasks in context of sars-cov-2 date: 2020-10-10 journal: nan doi: nan sha: doc_id: 129086 cord_uid: ra2njvcz in the unfortunate event of current ongoing pandemic covid-19, where vaccination development is still at the initial stage, several preventive control measures such as social distancing, hand-hygiene, and personal protective equipment have been recommended by health professionals and organizations. among them, the safe wearing of facemasks has played a vital role in reducing the likelihood and severity of infectious respiratory disease transmission. the reported research in facemasks has covered many of their material types, fabrication techniques, mechanism characterization, and application aspects. however, in more recent times, the focus has shifted towards the theoretical investigations of fluid flow mechanisms involved in the virus-laden particles prevention by facemasks. this exciting research domain aims to address the complex fluid transport that led to designing a facemask with a better performance. this review paper discusses the recent updates on fluid flow dynamics through the facemasks. key design aspects such as thermal comfort and flow resistance are discussed. furthermore, the recent progress in the investigations on the efficacy of facemasks for prevention of covid 19 spread and the impact of wearing facemasks are presented. finally, the potential research directions for analyzing the fluid flow behavior are highlighted. the person-to-person transmission of infectious respiratory diseases occurs primarily due to the transportation of virus-laden fluid particles from the infected person. the contagious fluid particles originate from the respiratory tract of the person and are expelled from the nose and the mouth during breathing, talking, singing, sneezing, and coughing. [1] [2] [3] these particles have been broadly classified into two types: aerosols (aerodynamic particle size < 5 µm) and droplets (aerodynamic particle size ≥ 5 µm-10 µm). [4] [5] [6] the finding indicated that the transmission phenomena of these expelled virus particles by patients would be dependent on droplet sizes. larger respiratory droplets, once expelled from the mouth or nose, undergo gravitational settling before evaporation; in contrast, the smaller droplets particles evaporate faster than they settle, subsequently forming of the aerosolized droplet nuclei that can be suspended in the air for prolonged periods and travel in air over long distances. the research studies have revealed that the severe acute respiratory syndrome (sars) epidemic in 2003 and the current global pandemic of coronavirus disease 2019 (covid-19) predominantly transmitted by contact or through the airborne route. [7] [8] [9] [10] several preventive strategies such as safe distancing, contact tracing, isolation of the infected person, hand hygiene, and facemasks have been widely employed against the rapid spread of these diseases. [11] [12] [13] [14] among them, the use of the facemasks have proven to be one of the most effective protective measures against the airborne virus transmission. [15] [16] [17] [18] [19] [20] the research suggested that face coverings could essentially reduce the forward distance traveled by a virus-laden droplet, and thus has a great potential to provide personal protection against airborne infection. 21, 22 recently, the world health organization who has recommended using facemasks for initial control of covid-19 spread. 23 in general, facemasks fall in the category of respiratory protection equipment (rpe) whose primary function is to protect the wearer from airborne viruses and contaminated fluids. there are various rpe types, ranging from simple homemade reusable cloth-based masks to surgical facemasks and n95 respirators to self-contained breathing apparatus. 18, [24] [25] [26] [27] different types of masks provide different levels of protection to the wearer. surgical facemasks are loose-fitting, fluid-resistant, single-time use, and disposable, designed to cover the mouth and nose. these masks are fluid resistant and intended for reducing the emission of large respiratory droplets released during coughing and sneezing. 28, 29 however, there is a possibility of leakage around these facemask's edge during the inhaling and exhaling process. such a dynamic leakage allows the direct contact of fluid droplets from the outside air to the wearer and vice-versa. such respiratory masks may also not provide adequate protection against extremely fine aerosolized particles, droplets, and nuclei. 30 for efficient trapping of droplets, the facemask filters should contain microscopic pores; however, the minute-sized pores prevent air ventilation, which creates an uncomfortable situation for the wearer. hence, a better tradeoff between the pore sizes and the breathability is desirable for the suitable facemasks. some mask types come with inbuilt respirators such as filtering facepiece respirator, p100 respirator/gas mask, self-contained breathing apparatus, full face respirator, and kn95 respirators provide better breathability of the users. the name designation 'n95' in the n95 respirators refers to the filtration of 0.3 μm sized particles with 95% efficiency. 31 the filtration mechanism of n95 facemasks operates on three possible principles: diffusion, inertial impaction, and electrostatic attraction. the smaller particles (<1 micron) usually get diffused and stuck on the filter's fibrous layers. whereas particles of typically 1 micron or larger realize the inertia effect, preventing them from flowing across the fibers in the filtration layers slam into the mask layers and filtered. n95 masks are designed for single-use because of potential contamination of filter layers, resulting in rapid degradation of their filtration efficiency. however, several innovative techniques have been demonstrated for decontaminating and reusing n95 masks. 32, 33 some recent n95 respirator masks are fabricated using the electrocharged polymers or electrospun nanofibers. 34, 35 these materials have intrinsic electrostatic properties to attract the small-tolarge oppositely charged particles, which help in better filtration of small-size particle transmission. 36, 37 because of the ongoing covid-19 pandemic, a significant demand for facemasks has been reported worldwide, while stimulating research about their efficacy for filtering expelled droplets from the mouth and nose of the infected person. in this regard, considerable efforts have been made in the past for the evaluation of facemasks performance. the quantitative performance of the facemasks has been typically characterized by evaluating the filtration efficiency (fe) and the total inward leakage (til). [38] [39] [40] [41] the filtration efficiency refers to the percentage of blocked particles by the tightly-fitted facemasks. the filtration efficiency can be calculated as = (1 − ( ⁄ )) × 100%, where 0 , are the particle count in the upstream feed prior to filtration and in the downstream filtrate, respectively. til is defined as the percentage of particles entering the mask through both the filter and the leakage between mask and face. the total inward leakage is calculated by dividing the particle concentrations on the outside and inside the facemasks. the protection factor of the facemasks can be determined from the expression; pf = 1/til. higher pf value of the masks perform better in virus transmission control. 16 furthermore, the fluid penetration resistance performance of the facemasks have been evaluated as per the astm f1862 /f1862m -17 standards. 42, 43 however, this test method does not evaluate facemasks' performance for airborne exposure pathways or in the prevention of the penetration of aerosolized fluids deposited on the facemask. in recent times, some qualitative analysis has been demonstrated for the rapid design characterization of facemasks. 44 while these experimental studies are essential for the broad characterization and design evaluation of respiratory facemasks, further theoretical and numerical methods and algorithm-based investigations provide a better insight into the facemask's fluid flow dynamics and the droplet leakage through the facemask openings. if the facemask is donned for a prolonged period, the captured fluid vapor on the filter surface may reduce the filtration efficiency. this saturation effect of the facemasks has been usually neglected in the experimental studies. to involve these factors, an alternative approach, the computational fluid dynamics (cfd) method, can be invaluable for understanding the fluid-particle flow behavior through the facemasks. the fluid dynamics based numerical techniques have gained momentum in the field of the facemask research domain. the computational fluid flow models have shown their potentials in an improved prediction of the spreading of respiratory virus-laden droplets and aerosols, sensitive to the ambient environment, and crucial to the public health responses. this review paper focuses on the fluid flow aspects of the facemasks and their efficacy in virus transmission control. following a brief introduction to the respiratory infectious diseases and their control strategies (section i), the respiratory droplet transportation mechanisms in conjunction with the possible governing equations required for estimating the transport phenomena have been presented in section ii. then, the droplet transport behavior through the facemasks has been described in section iii. key design aspects for the facemasks have been explained in section iv. section v covered the recent progress in investigating the efficacy of facemasks for preventing virus spread. the impact of using the facemasks have been discussed in section vi. the concluding remarks and a brief outlook for future research directions are summarized in section vii. during the sneezing or coughing process, the dispersion of saliva droplets or aerosols from the mouth to the ambient, and eventually on the floor accomplish in several stages. the complete transmission cycle involves complex flow phenomena, ranging from air-mucous interaction, breaking of droplets, turbulent conical jets, droplet evaporation and deposition, flow-induced particle dispersion, and sedimentation. 45 after exhalation from the mouth or nose, the saliva droplet movement is initially led by the inertia force, followed by the formation of a conical jet (vortical flow) near the mouth. once the droplets are expelled from the mouth, the inertia force gradually decreases, and other forces like gravity control the dispersion of larger size droplets, while drag and brownian forces control the smaller size droplets. after traveling up to a particular distance, these virus-laden droplets settle down on the floor. 46 thus, there are two major possible pathways for the respiratory virus transmission: airborne inhalation of smaller droplets, which are suspended in ambient air for a more extended period and carrying to the longer distance, and contact (direct or indirect between people and with contaminated surfaces) of large size droplets. 47 the fluid flow behavior of these droplets has been modeled using two different phases: continuous phase for the small size droplet nuclei and discrete phase for large size droplets. the fluid flow is governed by the navier-stokes and mass transfer equations which are as follows. continuity: momentum: where , , ⃗ , , , denotes the density ( −3 ), time ( ), flow velocity ( −1 ), pressure ( ), diffusion coefficient and kinetic viscosity, respectively. the conservation laws can be written in tensor form as: here, ⃗ represents the flow velocity (m/s) and is the source term that represents other forces such as gravity, lorentz force, etc. which also leads to momentum accumulation. for the newtonian fluids, there is a linear relationship between shear stress and velocity gradient. so, the viscous stress tensor can be defined by: in the overall vector form of the constitutive equation, where t denotes the transpose of the second velocity gradient outer product. for a newtonian fluid with constant µ and ρ, the momentum equation can be rewritten as: also, the fluctuation velocity component for the laminar-to-turbulent airflow field can be predicted by the reynolds-averaged navier-stokes equations (rans) model. where are the damping factors to reflect the anisotropic magnitude of the fluctuation velocity in the near-wall region. these are the random numbers from the standard normal distribution. the cough spreading phenomena can be predicted by solving the diffusion equation (3) in conjunction with some source and sink terms. vuorinen et al. 49 developed diffusion-based monte-carlo models to realize a transmission phenomenon via inhalation of aerosols in the ambient flow field. the source and sink terms have been included in conjunction with eqn. (3) . the source term represented the transient location of the infected persons while the sink term has been used for the ventilation surface. the developed models were capable of predicting the aerosol dispersions at more realistic locations like generic public place and supermarkets where cough may release from the walking person. for the droplets with the high droplet-to-air density ratio, the droplet trajectories have been predicted by solving a series of translation equations (lagrangian approach) of the discrete phase with the assumptions of stationary droplets and limited thermophoresis. continuous dispersion of saliva droplets throughout the computational domain has been considered in the computations. also, some basic parameters like velocity, mass, and position of each droplet have been computed at every time step. the translational equation for the saliva micro-droplet trajectory is given by, 46 where , , , are the stokes drag force, gravity, lift or buoyancy force, and brownian motioninduced force, respectively. also, , , , , ⃗ are the mass, radius, volume, density, and velocity vector of the saliva droplets, respectively. , ⃗ are the fluid density and the fluid velocity vector, respectively. the drag coefficient values depend on the droplet's reynolds number and can be calculated from, here, = 2 |⃗ ⃗ −⃗ ⃗ | . in above expressions, the droplet distribution is an important factor as their size decides the travel path distance, and eventually the infection risk. 50 so, for coughing simulation the droplet breakup approach is used. pendar and páscoa 46 used rosin-rammler breakup approach in their coughing simulation work which is expressed as: where and are the exponential factors and average radius of the droplet, respectively. these parameters are based on the saliva flow rate. recently, several studies have attempted to understand the dynamics of droplet formation and transport. cummins et al. 51 investigated the dispersion of spherical droplets in the presence of a sourcesink pair flow field. the maxey-riley equation was used to describe the finite-sized spherical particle motion in an ambient fluid flow. the presented non-dimensional mathematical models were based on the newton's second law of motion in which the forces acting on the particle involved the gravity force, the drag force, an added mass force, the force due to the undisturbed flow, and a basset-boussinesq history term. the analytical results suggested that droplets with a smaller size (<75 μm) moved a greater distance because of gravity's smaller impact. in comparison, the larger size droplets (>400 μm) traveled a relatively long distance before getting pulled into the sink by their more considerable inertia. however, the dispersion of intermediate size droplets (75 μm -400 μm) was found to be complicated under the influence of both drag and gravity forces. busco et al. 52 used the computational fluid dynamics approach to predict droplets and aerosols spread. the biomechanics of a human sneeze, including complex muscle contractions and relaxations, were included in the simulation by imposing a momentum source term to the coupled eulerian-lagrangian momentum equations (13) . the instantaneous magnitude of the sneezing momentum source term has been defined as | ( )| = ( )⁄ , where p(t) is the experimental pressure signal, and l is the characteristic equivalent length of the human upper-respiratory system ducts. the experimental results validated the developed model for the estimation of droplets and aerosols spreads. das et al. 53 investigated the airborne virus transmission through sneezed and coughed droplets and aerosols. the ejected droplet motions were estimated both for still and flowing air conditions by solving the langevin differential equation using monte-carlo numerical method. the langevin equations for the transport of the droplets of mass (m) in the still air is given as, and are the coordinate and velocity shift in each discrete time step , respectively, and stands for the cartesian components of the position and velocity vectors. the first term in the righthand side of eq.(17) represents the dissipative force. the second term stands for the diffusive (stochastic) force where ξ(t) that is regulated by the diffusion coefficient d. is the gravitation force term acting on a droplet of mass m. in the expression, the value of the drag coefficients is obtained using the stokes formula, = 6 , here is the droplet radius and is viscosity. the diffusion coefficient d is obtained from the einstein relation, = , where = 1.38 × 10 −23 ⁄ is the boltzmann constant and t is the temperature in kelvin. as shown, the langevin differential equations contain a stochastic source term (diffusive force), which is usually ignored in the eulerian-lagrangian approach. also, environmental factors such as temperature, humidity, and airflow rate, which could influence the air droplet dynamics, were included. the results revealed that the small droplets travel a larger distance and remain suspended in the air for a longer time under the influence of airflow, supporting the mandatory use of facemasks to prevent the virus. vadivukkarasan et al. 54 experimentally investigated the breakup morphology of expelled respiratory liquid. it was revealed that the droplet formation from the ejected fluid during coughing or sneezing occurred due to three possible mechanisms: kelvin-helmholtz (k-h) instability, rayleigh-taylor (r-t) instability, and plateau-rayleigh (p-r) instability in sequence. the flapping of the expelled liquid sheet was the result of the k-h mechanism, and the ligaments formed on the edge of the rim appeared due to the r-t mechanism, and finally, the hanging droplet fragmentation was the result of the p-r instability. droplet evaporation is one of the crucial factors that affect transmission phenomena. the evaporation rate of the droplets depends on the difference between the saturated vapor pressure of the fluid droplet surface and the vapor pressure of the surrounding air (ambient temperature and humidity). 55 the other factors, such as the mass-diffusion coefficient and the relative velocity between the droplet and surrounding gas, influence the evaporation rate. the non-dimensional parameters such as reynolds, nusselt, and sherwood numbers govern the droplet evaporation phenomena. 56 moreover, the condensation and evaporation effects between the ambient water vapors and the water liquid in cough droplets can be considered by solving the mass and energy balance for each droplet. 57 mass balance: energy balance: where is the average mass flux of evaporable component on the surface that can be expressed as: where is the density of the ambient air, , and ,∞ are the mass fractions of evaporable component on the droplet surface and in the gas phase far from the droplets, respectively. ℎ is the sherwood number. 58 several other researchers have studied the flow behavior of evaporating droplets. recently, weiss et al. 59 investigated the clustering and evaporation of droplets using the gas phase and droplet coupling equations. the evaporation of droplets and spreading of vapors into the ambient condition were mostly governed by few parameters: the reynolds number, which is related to the shear rate, the stokes number, and the mass loading, which is the ratio between the mass of the liquid to the gas phase. 60 the results suggested that the clustering and evaporation of droplets are primarily affected by the mass loading and stokes number while the taylor-scale reynolds number was small. when the mass loadings decreased, and the stokes number increased, the droplets dispersed more evenly with a faster evaporation rate. chaudhuri et al. 61 presented a chemical reaction mechanism based collision rate model for prediction of the growth rate of the infected population for the early phases of a covid-19 like pandemic. besides, they developed a theoretical model for the aerodynamics of respiratory droplets by considering the evaporation characteristics of levitated droplets. the evolution of the droplets was characterized by a complex interaction of aerodynamics, evaporation thermodynamics, and crystallization kinetics. the fidelity of proposed model was further confirmed by the experimentation. respiratory droplet transmission is considered critical for the rapid spread and continued circulation of viruses in humans. in recent years, the respiratory droplets flow behavior through the facemasks has typically well-predicted using the computational fluid dynamics (cfd) techniques. 21 the navier-stokes equations have been used as basic governing equations to solve the velocity field in a multi-dimensional computational domain. these equations have been used for the analytical assessment of the respiratory performance of the facemasks and other respirators. dbouk and drikakis 21 performed the fluid dynamics analysis of the respiratory droplets transmission through and around a facemask filter. the compressible reynolds-averaged navier-stokes equations and the k-ω turbulence model were employed. zhang etl. 62 analytically investigated the carbon dioxide co 2 transportation performance inside the ventilator mask. the 3d model of the ventilator mask is shown in fig. 1a. classical navier-stokes theorem and masstransport equations were used to estimate the co 2 residual concentrations below the nostrils. the governing equations were solved using the finite element solver ansys fluent 15.0 software. the following governing equations were used in the simulation; (i) at the entrance of the ventilator mask, the inlet pressure = 0.98 × 10 3 , the average concentration of co 2 = 0.03%. (ii) at the exhaust holes: outlet pressure = 0 , (iii) inlet boundary condition at the nostrils: the averaged velocity = 6 × sin ( 2 ), expiratory phase time t = 0~2.0 s, inspiratory phase time t = 2.0~4.0 s, and the averaged concentration of co 2 excreted from the nostrils was set as 4%. the airflow inside the ventilator mask was considered to be turbulent flow. fig. 1b shows the distribution of the average residual co 2 concentration inside the ventilator mask varying with time during a complete respiratory cycle. as shown from the curve, initially, the co 2 concentration increased with the increasing exhaled air and reaches the peak value of 3.65 %, and then it declined gradually with the decrease of the exhaled air and reaches down to the value of 1.8% at the end time of expiratory cycle. based on these results, the ventilator mask was redesigned by changing the exhaust hole to the bottom side and the local residual co 2 concentration was decrease to 0.7%. bates et al. 63 performed computational fluid dynamics simulations to access the respiratory airflow in the human upper oral airway with airway wall movement. the breathing flow rate data was acquired by imaging the breathing cycle of the participant while wearing of a size-5 anesthesia facemask (fig. 1c) . the air pressure drop and flow velocity were estimated by solving the navier-stokes equations for the moving mesh vertices in the finite volume domain. the governing equations for moving mesh of the finite volume form is given by: momentum equation: where is time, is the volume of each cell in the mesh, is the air density, ⃗ is the air flow rate, ⃗⃗⃗⃗ is the mesh velocity as calculated from the mesh displacement for each control points, is a vector representing the surface of each mesh cell, is the identity matrix, and is the viscous stress tensor. these equations were solved using the large eddy simulation (les) techniques. the instantaneous air flow resistance was calculated as the pressure loss between two locations divided by the air flow rate through them. fig. 1d shows the estimated airflow resistance through several different regions of the extra thoracic airway during the complete breathing cycle. the aerosol droplets transmission phenomena through the facemasks have also been investigated analytically. the facemask leakage factor has been considered in the analytical models. lei et al. 64 predicted the fluid leakage between an n95 filtering facepiece respirator (ffr) and a headform using the computational fluid dynamics (cfd) simulation approach. the mass flow rate at the faceseal and through the filter medium was calculated under three different boundary conditions: varying breathing velocity, varying viscous resistance coefficients of the filter, and the freestream air flows. the filter-to-faceseal leakage (ftfl) ratio for the respirator was obtained by dividing the mass flow rate through the filter medium and the faceseal leakage. a higher ftfl ratio refers to the higher percentage of airflow passing through the filter medium than the faceseal leakage. the results revealed the nonlinear increase in the ftfl ratio with increasing breathing velocity values and decreasing the filter viscous resistance coefficient values. furthermore, the freestream flow had limited influence on the airflow inside the respirator resulting in nonsignificant variations on the ftfl ratio. perić et al. 65 thermal comfort is an essential aspect of a facemask as it may affect the compliance of the use of facemask during summer or in tropical countries. there were reported incidence of skin rashes, increased heat stress, sweating, and discomfort due to prolonged wearing of a facemask in hot and humid conditions. 66 to improve the thermal comfort level of facemasks, researchers have developed some unique facemasks by using the nanocomposites. polymer-based nanofibers with large surface area-to volume ratio have shown great potential for use in facemasks to achieve both high filtration efficiency and sufficient air permeability. 67 . 3(a-c) show the schematic, photographs, and scanning electron micrographs of the proposed hybrid nanofiber-based facemask. the comparative pm capture efficiency and air permeability results have demonstrated the superiority of presented facemask over the commercial masks (fig. 3d, e) . moreover, the thermal image revealed that the fiber/nanope facemasks had high transparency to the human body radiation (cooling effect). in contrast, the commercial facemasks blocked a large portion of it. they further modified the nanope substrate with ag coating and demonstrated that fiber/ag/nanope had a warming effect. zhang et al. 71 reported the use of an active ventilation fan to reduce the dead space temperature and co2 level. an infrared camera (irc) method was used to elucidate the temperature distribution on the prototype ffr's outside surface and the wearer's face, surface temperature was found to be lowered notably. both inside and outside temperature resulted from the simulation were found to be in good agreement with experimental results. however, the inward blowing fans may compromise the filtering effectiveness of the facemask. there are commercially available facemasks fitted with one-way valve for facilitating the removal of humidity and expired air within the space between the facemask and the face. however, during the covid-19 pandemic, one of the main reasons for wearing the mask is not only to protect the inhalation of virus, but also to prevent the spread of virus into the air if the wearer happens to be a carrier of the virus. if the wearer is a healthy subject, the use of a one-way valve and ventilation fan would indeed mitigate the buildup of humidity and carbon dioxide within the dead space. zhu et al. 72 reported a three-dimensional model of normal human nasal cavity to simulate the volume of fraction of both fresh air and respired air within the nasal cavity. the model consisted of large rectangular domain outside the nasal cavity representing ambient air, human nasal cavity and partial of the pharynx. this was the first reported piece of work that modelled the details of nasal cavity instead of just the nostrils as openings for the flow simulations. the advantage for this simulation was that the flow field within the space between the nostrils and facemask could be more accurately simulated as the boundary condition could be specified away from the nostril at the pharyngeal area. two cases were simulated. case i refers to a human face with a n95 respirator onto human face, and case ii refers to a human face without a respirator. the results showed that above 60% of inspired air was respired air in case i compared to less than 1.2% in case ii. during expiration, the volume of fraction (vof) of respired air in both cases was above 95%. the streamlines at peak inspiration were relatively smooth while entering the cavity in both cases; while at peak expiration large vortex was observed within the air space between human face and respirator in case i. for future studies, one could explore the in vivo experimental studies with the use of miniaturized and wireless sensors for monitoring not just the temperature, but also the humidity and carbon dioxide content within the space between the nostrils and the facemask. the sensors need to be small so as not to disrupt the flow fields. if a single sensor cannot be small enough for the measurement of all the three parameters, one may need to have separate sensors and repeat the experiment for the same human subject. another important parameter affecting the comfort of the wears is the flow resistance of the facemask. in principle, if the flow resistance is lower while maintaining the same filtering efficiency, the comfort level will be enhanced. however, the facemask's flow resistance is just an indicator and does not specify the wearer's breathing resistance. while the flow resistance could be measured using a typical setup for correlating the fluid flow rate to the pressure drop across the facemasks, the breathing resistance could only be measured using a human subject or a replica of the nasal pharyngeal system. lee and wang 73 presented the pioneering work of measuring the nasal airflow resistance during inspiration and expiration using a standard rhinomanometry and nasal spirometry. a modified full-facemask was produced in-house to measure nasal resistance using n95 (3m 8210) respirators. the results showed a mean increment of 126 % and 122% in inspiratory and expiratory flow resistances, respectively, with n95 respirators. there was also an average reduction of 37% in air exchange volume with the use of n95 respirators. the same group did a follow-up study investigating the change of human nasal functions after wearing an n95 respirator and surgical facemask. 74 the human subject study involved 87 healthy healthcare workers. each of the volunteers attended two sessions and wore an n95 respirator in session 1 (s1) and surgical facemask in session 2 (s2) for 3 hours. the mean minimum cross-sectional area (mmca) of the two nasal airways via acoustic rhinometry and nasal resistance via rhinomanometry was measured before and immediately after the mask. the equipment could not perform in vivo measurement with the facemask on. rhinomanometry was repeated every 30 minutes for 1.5 hours after the removal of masks. a questionnaire was distributed to each of the volunteers during the 3 hours mask-wearing period to report subjective feelings on the discomfort level of breathing activity. among 77 volunteers who completed both the two sessions, the mean nasal resistance immediately increased upon removing the surgical facemask and n95 respirator. the mean nasal resistance was significantly higher in s1 than s2 at 0.5 hours and 1.5hours after removing the masks (p<0.01). there was an increase of nasal resistance upon removal of the n95 respirator and surgical facemask, potentially due to nasal physiological changes. n95 respirator caused higher post-wearing nasal resistance than surgical facemask with different recovering routines. this was the first time that the effect of long duration wearing a facemask was objectively monitored. however, the during of three hours for the wearing of a facemask was deemed to be too short under the current covid-19 simulations, and human subject study for a longer duration of wearing facemask should be attempted. the research could also be enhanced using miniaturized pressure, temperature, humidity, and gas sensors for in vivo monitoring of the air condition within the space between the nostrils and the facemask. such experimental data would be useful for validating numerical models for assessing the comfort level for wearing different types of facemask. another potential approach is to develop a replica for replacing human subject for such long duration study, similar to the use of an acoustic head for replacing human subjects in the more extended duration noise exposure study. zhu et al. 75 reported another investigations on effect of long duration wearing of n95 and surgical facemasks on upper airway functions. a total of 47 volunteers of national university hospital singapore were participated for the study. each of the volunteers wore both n95 respirator and surgical facemask for 3 hours on two different days. during the period of mask wearing, relative airflow rates were recorded. the study revealed that the increased level of discomfort to the user with time while wearing the masks. moreover, n95 respirator caused higher post-wearing nasal resistance than the surgical facemask with different recovering routines. the current studies recognized that the airborne transmission of aerosols produced by asymptomatic individuals during speaking and breathing as a key factor leading to the spread of infectious respiratory diseases such as covid 19. 58, [76] [77] [78] however, the spread of these airborne diseases has been successfully controlled up to a certain extent by using the facemasks. 11, 19, 47, [79] [80] [81] in the ongoing global pandemic of the covid 19, where vaccine developments still at a phase trial stage, the respiratory protective equipment such as facemasks has proven to be a complementary countermeasure against the spread of the novel coronavirus. in this regard, several researchers have performed theoretical and experimental investigations of virus transmissibility through the facemasks and alternatives. stutt et al. 82 developed the holistic mathematical frameworks for assessing the potential impact of facemasks in covid 19 pandemic management. the results revealed that professional and home-made facemasks were highly efficacious to reduce exposure to respiratory infections among the public. also, when people wear the facemasks alltime at the public places, the certain epidemiological threshold, known as the effective reproduction number, could be decreased below 1, leading to the prevention of epidemic spread. ngonghala et al. 83 developed a parametric model for providing deeper insights into the transmission dynamics and control of covid-19 in a community. they used the covid 19 data from new york state and the entire us to assess the population-level impact of the various intervention strategies. the results suggested that the consistent use of facemasks could significantly reduce the effective reproduction number. the highly efficacious facemask, like surgical masks with estimated efficacy of around 70%, could lead to the eradication of the pandemic if at least 70% of the residents use such masks in public consistently. the use of low efficacy masks, such as cloth masks with an estimated efficacy of 30%, could also lead to a significant reduction of covid-19 burden. yan et al. 84 evaluated the effectiveness of different respiratory protective equipment in controlling infection rates in an influenza outbreak. they used a previously developed risk assessment model 85 to show n95 respirators' efficacy, low-filtration surgical mask (adult), high-filtration surgical mask (adult), high filtration pediatric mask, and low filtration pediatric mask. the study revealed that donning these masks with a 50% compliance rate resulted in a significant reduction in transmission risk, and with 80% compliance rate nearly eradicated the influenza outbreak. prasanna simha and rao 86 quantitatively investigated the distance of travel of typical human coughs with and without different masks: disposable three-ply surgical masks and n95 masks. in their study, the schlieren method, a highly sensitive, non-intrusive flow imagining technique, was used to visualize the human cough flow features. the experimental statistics showed that the propagation of a viscous vortex ring mainly governed cough flow behavior. while wearing regular face masks, the cough droplets traveled approximately half the distance traveled by expelled droplets without a mask. however, n95 was found to be most effective in limiting the spread of cough droplets. leung et al. 87 performed experimental studies to investigate the efficacy of surgical facemasks to prevent respiratory virus shedding. the surgical facemasks' efficiency was measured against the coronavirus, influenza virus and rhinovirus of two broad particle sizes, respiratory droplets (≥5µm) and aerosols (droplet nuclei with aerodynamic diameter ≤5µm). the results indicated that surgical facemasks could efficaciously prevent transmission of human coronaviruses and influenza viruses into the environment in respiratory droplets, but no significant reduction in aerosols. moreover, the steep rise in demand for medical facemasks during the current pandemic covid 19 has resulted in a subsequent breakdown of the global supply chain that led to an acute shortage in the market. to mitigate this discontinuous supply chain system, scientists have put much effort into exploring alternative fabrics with sufficient filtering capacity that are readily available and affordable. kähler and hain 88 performed a detailed analysis of the efficacy of facemasks to prevent virus spread. in the first step, the transmission of droplets released by the mouth when breathing, speaking, and coughing was characterized. then, the filtering capacity of the various facemasks was analyzed. the experimental results have shown that most household materials tested do not provide much protection against the virus transmission via droplets and, therefore, unsuitable as materials for protective masks. however, filtering facepiece respirators (ffr) performance-based masks such as ffp2 (europe en 149-2001), n95 (united states niosh-42cfr84), ds2 (japan jmhlw-notification 214, 2018), and kn95 (china gb2626-2006) offer adequate protection, as they are only permeable to a tiny fraction of few micron-sized droplets. konda et al. 89 evaluated the filtration efficiency of various commonly available fabrics, including cotton, silk, chiffon, flannel, various synthetics, and their combinations, which were used in the fabrication of cloth masks. the filtration performance of these fabrics was conducted by generating the aerosol particles at the cloth sample's upstream side. the aerosol particulates ranging from ~10 nm to ~ ten μm scale sizes, particularly relevant for respiratory virus transmission, were produced by commercial sodium chloride (nacl) aerosol generator. also, the air with a controlled airflow rate was drawn through the sample using a blower fan. the filtration efficiency of each sample was computed by measuring the particles' concentration upstream and downstream as = − × 100, where and are the mean particle concentrations per bin upstream and downstream, respectively. moreover, the pressure drop across the facemasks and the air velocities were measured using a digital manometer and a hot wire anemometer. the experimental investigations revealed that the materials such as natural silk, a chiffon weave (90% polyester−10% spandex fabric), and flannel (65% cotton−35% polyester blend) provided good electrostatic filtering of particles. also, fabric with tighter weaves and low porosity, such as cotton sheets with high thread count, have resulted in better filtration efficiencies. for instance, a 600 tpi (thread per inch) cotton sheet can provide average filtration efficiencies of 79 ± 23% (in the 10 nm to 300 nm range) and 98.4 ± 0.2% (in the 300 nm to 6 μm range). a cotton quilt with batting provides 96 ± 2% (10 nm to 300 nm) and 96.1 ± 0.3% (300 nm to 6 μm). surprisingly, four-layer silk (e.g., scarf) was found to be effective with an average filtration efficiency of >85% across the ten nm−6 μm particle size range. moreover, the hybrid masks made by combinations of two or more fabric types, leveraging mechanical and electrostatic filtering, could be an effective approach for better filtration (fig. 4a) . verma et al. 44 performed the qualitative investigations for assessing the effectiveness of easily available facemasks such as bandana (elastic t-shirt material, 85 threads/inch), folded handkerchief (cotton, 55 threads/inch ), stitched mask (quilting cotton, 70 threads/inch) and other commercial masks. they observed that a stitched mask made of quilting cotton was most effective, followed by the commercial mask, the folded handkerchief, and, finally, the bandana. their observations also suggested that a higher thread count by itself is not sufficient to provide a better droplet filtration capability. the material types and fabrication techniques have a significant impact on the performance of facemasks. davies et al. 39 examined the efficacy of homemade masks as an alternative to commercial surgical masks. various household materials such as 100% cotton t-shirt, scarf, tea towel, pillowcase, antimicrobial pillowcase, vacuum cleaner bag, cotton mix, linen and silk were evaluated for the capacity to prevent bacterial and viral aerosols transmission. the performance of these household facemasks was compared with the standard surgical mask. the experimental outcomes showed that these homemade masks could reduce the likelihood of infection, but not efficient for the complete elimination of risks. a similar conclusion has been made in a previously published review article by rossettie et al. 90 and loupa et al. 91 recently, ho et al. 92 investigated the droplet filtration efficiency of the self-designed triple-layer cotton masks, their performance was compared with the standard medical mask. all tests were performed in two different locations; in a regular bedroom and a car with air conditioning. the particles with a size range of 20-1000 nm were taken into consideration, and the filtration efficiency was measured. other factors like environmental conditions (temperature and relative humidity) and cough/sneeze counts per hour were measured for each measurement. the results revealed that cotton and surgical masks could significantly reduce the number of microorganisms expelled by participants with the filtration efficiency of 86.4 % and 99.9 %, respectively (fig. 4b) . however, the surgical mask was three times more effective in blocking transmission than the cotton mask. in a recent study, fischer et al. 93 performed testing of 14 different facemasks or mask alternatives ranging from the kind worn by healthcare professionals to neck fleeces and knitted masks. fig. 4c shows the photographs of the facemasks and alternatives considered in the investigation. a comparison was made on the dispersal of droplets from a mask wearer's breath while wearing one of the face coverings to the results of a controlled trial where their mouth was fully exposed. the study revealed that some mask types matched standard surgical masks' performance, while some mask alternatives, such as neck fleece or bandanas, offered little protection against infection. the neck fleece was found to increase the risk of disease by having a "droplet transmission fraction" of 110% (fig. 4d) . besides, they demonstrated a simple optical measurement method to evaluate the efficacy of facemasks to reduce respiratory droplets transmission during regular speech. fig. 4e shows the schematic of developed setup. the proposed optical system is inexpensive and easy-to-operate, even by non-experts. furthermore, the use of face shields has widely been used along with standard face masks. face shields are generally made of transparent plastic sheets. they offer several advantages: comfortable to wear, easy-to-clean, clear conversations between the speakers with visible facial expressions, and reduce autoinoculation by preventing the wearer from touching their face. 94 also, face shields prevent the user's face from the direct contact of liquid droplets. more recently, verma et al. 95 investigated the effectiveness of the face shields and exhalation valves in the respiratory droplets transport context. they performed experimentation in an emulated coughing and sneezing environment for qualitative visualizations analysis. the results indicated that although face shields block the initial forward motion of the fluid jet, the expelled droplets can move around the visor with relative ease and spread out over a large area depending on environmental conditions. also, for the facemasks equipped with an exhalation port, the droplets pass through the exhalation valves. based on the observations, they opined that high-quality cloth or surgical masks perform better than the face shields and exhalation valves. in the past few decades, especially post-outbreak of the severe acute respiratory syndrome (sars) in 2003, wearing the facemasks has grown extensively. the people from asian countries like china, singapore, thailand, japan, etc. can be easily seen donning facemasks in public places. there are well proven-studies about the prevention of airborne pathogens transmission by covering the mouth and nose using the facemasks. the recently published article by gandhi and rutherford, 96 claimed that the universal facial masking might help reduce the severity of disease and enhance the wearer's immunity. however, prolonged use of facemasks has some side effects on human respiratory health, such as carbon dioxide builds up, drowsiness, and breathing problems because of restricted fresh airflow, and unusual heart rate. 97, 98 if a facemask is donned for a longer period, the filter gets wet because of facial sweat, and vapor is formed inside the facemasks due to the breathing, resulting in clogging of particulates. also, wearers get a false sense of security, encouraging them to spend more time in public places. 99 other potential side-effects of facemasks wearing include skin irritation, uncomfortable feeling due to the arrival of exhaled air into the eye, comprised quality and the volume of the speech during the conversations. 19, 100, 101 moreover, there are some environmental concerns associated with the use of single-use facemasks. some of these facemasks are made from layers of plastics, which may not bio-degrade easily, thus creating a massive burden on the environment. a recent analysis has reported that if every person in the uk used one single-use facemask each day for a year, it would create 66,000 tonnes of contaminated plastic waste, roughly ten times higher than that of using reusable masks. the new coronavirus is continuously evolving and spread all over the world. researchers from all disciplines, especially the medical professionals and engineers, are continuously working on the facemasks design improvement for a better performance against the virus transmission. zhou et. al. 102 presented an electrospun polyetherimide (pei) electret nonwoven material based bi-functional smart facemask to remove the sub-micron particulate matter and generate electricity. the facemask could harvest sufficient energy from the airflow to supply power to the inbuilt lcd panel. the lcd screen was used to display the measured breathing rate. hossain et al. 103 developed a rechargeable n95 facemask that composed of a charged polypropylene electret fiber made an intermediate layer for capturing the foreign particles. these particles are trapped through the electrostatic or electrophoretic effects of the polypropylene terephthalate (pet) layer. the mask has a provision for the in-situ recharging of the polypropylene electret for maintaining its filtration performance. williams et al. 104 proposed a facemask used for the sample collection of respiratory sars-cov-2 virus. they have successfully presented a facemask prototype the detects exhaled mycobacterium tuberculosis, a deadly lung infection, and now working for sampling for the sars-cov-2 virus. the facemask consisted of four 3d printed polyvinyl alcohol (pva) sampling strips attached inside it. the sampling matrices trapped the particulates during exhalation and was further post-processed for the virus diagnosis. face-mask sampling offered a highly efficient and non-invasive method for respiratory disease diagnosis. the presented approach showed great potential for diagnosis and screening, particularly in resource-limited settings. moreover, several innovative facemask prototypes with better filtration performance are available in the market. recently, korean electronics and appliance company lg® ltd. has developed an air purifier wearable mask (puricare™) 105 equipped with battery-operated miniature fans that draw in the fresh air and help reduce stuffiness. the massachusetts institute of technology and brigham and women hospital, boston's researchers have developed the, a silicone-based transparent reusable facemask with a comparable performance level with n95 respirators. 106 the facemasks have shown their potentials for preventing the spread of respiratory disease. a variety of facemasks ranging from a simple homemade cloth mask to the ventilated respirators, have played their role in the current covid-19 pandemic. in general, the facemasks have been experimentally characterized by determining the filtration efficiency and total inward leakage ratio. also, the fluid flow dynamics-based numerical methods have gained much attention to investigating the facemask performances. the present article has also highlighted the insufficiencies of assessing the breathing resistance of the wearers with the facemask by just examining the flow resistance of the facemask. in the longer term, there may be a need for a more elaborate system approach including the study and modeling of how the human lung would respond to the increase in breathing resistance due to the use of facemask, drawing the analogy of modeling the behavior of the heart for the blood circulation system. this article summarizes the perspective of the fluid dynamics of the facemask filtration performance, including droplet and aerosol transports, droplet evaporation, and facemask aerodynamics. furthermore, recent investigations for the efficacy of the facemasks in the context of respiratory virus transmission have been discussed. proc. natl. acad. sci infection prevention and control of epidemic-and pandemic-prone acute respiratory infections in health care national center for immunization and respiratory diseases (ncird), division of viral diseases face masks and coverings for the general public: behavioural knowledge, effectiveness of cloth coverings and public messaging advice on the use of masks in the context of covid-19: interim guidance standard test method for evaluating the bacterial filtration efficiency (bfe) of medical face mask materials using a biological aerosol of staphylococcus aureus, 42 astm f1862/f1862m -17, standard test method for resistance of medical face masks to penetration by synthetic blood (horizontal projection of fixed flow analyses to validate sars-cov-2 protective masks the southwest respiratory and critical care chronicles lg revolutionizes personal clean air with puricare™ wearable air purifier engineers design a reusable, silicone rubber face mask the first author would like to acknowledge the financial support from the ministry of education rsb research fellowship singapore. the data that support the findings of this study are available from the corresponding author upon reasonable request. key: cord-270647-vn4kirrx authors: romero-espinoza, jose a.; moreno-valencia, yazmin; coronel-tellez, rodrigo h.; castillejos-lopez, manuel; hernandez, andres; dominguez, aaron; miliar-garcia, angel; barbachano-guerrero, arturo; perez-padilla, rogelio; alejandre-garcia, alejandro; vazquez-perez, joel a. title: virome and bacteriome characterization of children with pneumonia and asthma in mexico city during winter seasons 2014 and 2015 date: 2018-02-15 journal: plos one doi: 10.1371/journal.pone.0192878 sha: doc_id: 270647 cord_uid: vn4kirrx background: acute asthma exacerbations and pneumonia are important causes of morbidity and mortality in children and may coexist in the same children, although symptom overlap may lead to difficulties in diagnosis. microbial and viral diversity and differential abundance of either may play an important role in infection susceptibility and the development of acute and chronic respiratory diseases. objectives: to describe the virome and bacteriome present in the upper respiratory tract of hospitalized children with a clinical diagnosis of asthma and pneumonia during an acute exacerbation and an acute respiratory illness ari episode respectively. methods: during the winter seasons of 2013–2014 and 2014–2015, 134 nasopharyngeal swabs samples of children <15 years of age with ari hospitalized at a referral hospital for respiratory diseases were selected based on clinical diagnosis of asthma or pneumonia. the virome and bacteriome were characterized using whole genome sequencing (wgs) and in-house bioinformatics analysis pipeline. results: the asthma group was represented mainly by rv-c, bov-1 and rsv-b and the pneumonia group by bacteriophage ej-1 and ttmv. ttv was found in both groups with a similar amount of reads. about bacterial composition moraxella catarrhalis, propionibacterium acnes and acinetobacter were present in asthma and veillonella parvula and mycoplasma pneumoniae in pneumonia. streptococcus pneumoniae and haemophilus influenzae were mostly found with both asthma and pneumonia. conclusions: our results show a complex viral and bacterial composition in asthma and pneumonia groups with a strong association of rv-c presence in asthmatic children. we observed streptococcus pneumoniae and haemophilus influenzae concurrently in both groups. acute respiratory infection (ari), both from the upper and lower tract (urti/lrti), represent a major public health problem worldwide especially in developing countries [1, 2] . besides being one of the most common diseases, it is a leading cause for health care use, and mortality, particularly due to the presence of pneumonia [3] [4] [5] . in the clinical practice, acute asthma exacerbations and pneumonia have an overlap of symptoms, and may also coexist, leading to misdiagnosis. for example over-diagnosis of pneumonia and under-diagnosis of asthma may contribute to significantly untreated respiratory morbidity and mortality in children less than five years old in low-income countries [6] . both groups differ with respect to the associated virus and bacteria: while asthma exacerbations have been associated to a specific rhinovirus infection, pneumonia can be related to a wide range of bacterial, fungal and viral agents, with a high prevalence of respiratory syncytial virus (rsv) [2, 7] . it has been proposed that microbial composition plays an important role in infection susceptibility and the development of acute and chronic respiratory diseases [8] . recent studies describe the community of microorganisms that reside in the respiratory tract, referred to also as the microbiome, by using next generation sequencing (ngs), and in most cases by evaluating bacterial components based only for specific gene (16s) [9, 10] . on the other hand, the whole genome shotgun (wgs) approach provides enough information to identify all viral and bacterial species not only for a specific gene [11] . while bacterial composition of the microbiome is called bacteriome, the virome (or viral metagenome) refers to the viral fraction comprising viruses responsible for acute, persistent, or latent infections, and viruses integrated into the host genome such as endogenous retroviruses [12] . a limited number of studies have attempted to characterize the virome (yang et. al. [13] ; zoll et.al. [14] ; moesker et.al. [15] ; wang et.al. [16] ; bal et.al. [17] ) and bacteriome (hilty et. al. [10] ; taboada et.al. [18] ; teo et.al. [19] ) of pediatric patients with aris. a few others studies have used well-defined clinical parameters of asthma exacerbation and pneumonia (moreno-valencia et.al. [2] ; vazquez-perez et.al. [20] ). here we describe the virome and bacteriome present in the upper respiratory tract of hospitalized children clinically diagnosed with asthma and pneumonia, during an acute exacerbation and an ari episode respectively, at the national institute of respiratory diseases (iner, mexico city) during 2014 and 2015 winter seasons. the science and bioethics committee of the national institute of respiratory diseases revised and approved the protocol and the consent procedure (b2613). for all children, the corresponding legal guardians provided written informed consent. nasopharyngeal swabs (ns) were collected from children less than 15 years of age with clinical signs of aris hospitalized at the national institute of respiratory diseases (iner) in mexico city, a referral center for respiratory diseases, which primarily provided services for uninsured individuals, during the winter season 2013-2014 and 2014-2015. all subjects enrolled were classified with an asthmatic crisis (characterized by wheezing and difficult breathing especially in previously known asthmatics or with atopic symptoms) or pneumonia (with the presence of new lung opacities, with changes in the white blood count). maximum sample number for each group was defined after each season, resulting in 42 for asthma, and 78 for pneumonia (table 1) . for all samples, automated nucleic acids isolation was performed on magna pure lc 2.0 using the total nucleic acids kit (roche diagnostics). an in-house rt-qpcr respiratory viral panel (moreno-valencia et al., 2015) was used as previous screening and for virus identification (s1 table and s1 appendix). nucleic acids were isolated directly from stored samples. for each one, 250 μl of transport media was centrifuged at 5,000xg for 5min to remove cellular debris. the supernatant was filtered through 0.2 μm-pore-size disc filters (syringe filter, corning). samples were pooled in a 15ml concentrator tube (amicon ultra -15, merck millipore) and centrifuged at 4,000xg until volume was reduced to 500 μl. nucleic acids were isolated from the concentrated sample pool using the purelink viral rna/dna kit according to the manufacturer's instructions (invitrogen, waltham, ma). nucleic acids were eluted in 60 μl of nuclease free water and stored in aliquots at -80˚c. to improve viral detection, one aliquot of 20 μl for each extraction was processed for rna viruses and another aliquot for dna viruses. the aliquot for rna viruses was treated with rnase-free dnase i, (thermo scientific) for 30 min at 37˚c and immediately chilled on ice. to reduce the amount of human dna, the aliquot for dna viruses was treated with nebnext microbiome dna enrichment kit, according to the manufacturer's instructions (new england biolabs inc.). for rna viruses, reverse transcription was performed on 10 μl de rna using a m13-random hexamer primer with transcriptor first strand cdna synthesis kit (roche diagnostics) next double strand cdna (dscdna) was generated by two rounds of synthesis using m13-random hexamer primer with klenow fragment (thermo scientific). for dna viruses and bacteria, dna previously enriched was labeled using m13-random hexamer primer. dna from each process was amplified with platinum taq dna polymerase high fidelity (thermo scientific) using m13 primers and the following program: 2 min 95˚c, 30 cycles of 15 sec at 95˚c, 30 sec at 50˚c, 3 min at 68˚c and a final step of 5 min at 68˚c. dna was quantified by qubit dsdna hs assay kit (thermo scientific), and length fragment was evaluated with agilent high sensitivity dna kit (agilent technologies). a whole genome approach (shotgun) was used for both viral and bacterial genomes. libraries were built with 1 ng of amplified dna from each sample of rna viruses and dna viruses/ bacteria, and processed with nextera xt dna library preparation kit according to the manufacturer's instructions (illumina). libraries were labeled with nextera xt index kit (illumina) according the s1 table and pooled into one. the pooled library was loaded in a flow cell and sequencing was performed in a miseq desktop sequencer (illumina) to obtain paired-end reads of 250 bp in length (2x250). an in-house pipeline was developed to perform the bioinformatics analysis for all files (fig 1 step 3) . datasets of human, bacteria and virus sequences (downloaded from the ncbi ftp server in march 2015) were downloaded to build smalt, bwa index, local blastn and blastx databases. all data from miseq instrument were trimmed through a phred-like q20 < 20 using fastqc software. reads were aligned to databases described above using a standalone blastn for direct assignment of each read with an evalue of 1e-30. for viruses, blastn with an e-value of 1e-30 and blastx with an e-value of 1e-01, on trimmed reads and after human, bacterial and viral mapping, were used respectively. velvet algorithm was used for contig assembly. to reduce data complexity for bacterial detection, human sequences were removed by mapping with human smalt index before the pathogen identification process (fig 1, step 3 ). bacterial species were identified using local blastn with an e-value of 1e-30 and the first 10 hits were obtained for each sequence. megan [21] 5.2.2 software was used to assign reads to the most appropriate taxonomic level, by assigning a read to the lowest common taxonomic ancestor of the organisms corresponding to the set of significant hits. for the rhinovirus analysis, full-length genome of human rhinovirus a, b c (rv-a, rv-b, rv-c), enterovirus 68 and 71 (ev68, ev71) were retrieved from the genbank database. for phylogenetic analysis of parvovirus b19 (pvb19), bocavirus (bov) and respiratory syncytial virus b (rsv-b), full-length genomes of representative sequences of human strains were used. analyses also were performed for three different anellovirus, torque teno virus (ttv), torque teno midi virus (ttmdv) and torque teno mini virus (ttmv). alignments were created and manually edited with mega [22] 6.0. unrooted maximum likelihood tree with 1,000 bootstrap replicates was constructed using the tajima-nei model with 5-parameter gamma distributed rates. one hundred and twenty respiratory samples were grouped into seven pools: asthma 2014 (a2014), asthma 2015 (a2015), pneumonia 2014 (p2014) and pneumonia 2015 (p2015) each one with qpcr positive (qpcr+) and qpcr negative (qpcr-) results, except qpcr-for a2015 season since we did not have enough samples to analyze (s1 table) . the average amount of sequences obtained from each group after quality filtering was 2,247193 within a range of 3,634,710 to 519,470 reads. from those reads, 0.18 to 0.35% belonged to viral or bacterial sequences, and as much as 28.08% belonged to endogenous retrovirus and sequences without certain identification. to describe the virome of the two groups of children, we grouped reads from both seasons 2014 and 2015 into one. the most represented viruses were in order of number of reads rhinovirus c (rv-c), bocavirus 1 (bov-1), rsv-b and parvovirus b19 (pvb19) in patients with asthma while bacteriophage ej-1, ttmv, streptococcus phage, rsv b and rv a were in subjects with pneumonia. ttv, influenza virus and staphylococcus phage were present in similar levels for both groups of patients ( table 2 ). most of the viral reads were classified into six virus families picornaviridae, parvoviridae, paramyxoviridae, anelloviridae, orthomyxoviridae and herpesviridae (fig 2a and 2b) . bacteriopaghes also had high abundance of reads ( fig 2b) . the viral composition in samples was complex and dominated by the most common respiratory four representative viruses were used to compare the genome coverage and depth. contigs were ensembled to generate partial consensus genomes of rv-c (97% coverage, 6,824 bp) (s1 the bacterial composition in samples was also complex. streptococcus pneumoniae and haemophilus influenzae were the species with more reads in both groups. staphylococcus aureus, mycoplasma pneumoniae veillonella parvula and different streptococcus spp had more reads in the pneumonia group (fig 3a and 3b) . while moraxella catarrhalis, acinetobacter and propionibacterium acnes had more reads in the asthma group. other bacteria genus detected of clinical interest with low frequency were: pseudomonas, neisseria and prevotella. we conducted a metagenomic analysis of the virome and bacteriome of children with asthma and pneumonia. this is the first report to describe both communities in a well-defined group of pediatric patients. previously, our group reported the role of specific viral infections on asthma exacerbations in hospitalized children using rt-qpcr detection [2, 20] . although rt-qpcr is the gold standard for viral and bacterial diagnosis, it is still limited to specific target assays. by contrast, ngs is a platform that generates millions of reads without the need of previous knowledge of the sequences present in a sample. it is also a powerful tool to confirm and discover microbial etiologies in clinical samples. whole genome shotgun sequencing (wgs) provides enough information to identify microbes to the species level as a result of a more accurate alignment with genome reference sequences, not just in particular for specific genes such as 16s in bacteria [11] . in our analyses, most of the bacterial reads were classified into a species level, implying that wgs can be a useful tool to describe the microbiome in a clinical sample. in our study, 90% of reads were classified as q30, and only 5% were discarded due to low sequencing quality. after being analyzed, human reads ranged from 71 to 91% in each group despite the use of an experimental strategy to reduce human dna. these values are found in the same range for equivalent sample types previously reported by yang et al. [13] and nakamura et al. [23] : 76-95% and 90-94.6%, respectively. however, the percentage range obtained for bacterial and viral reads (0.10-0.32%) was lower than the quantities previously reported by yang et al.(1.03%) [13] , nakamura et al. (1.33-3 .48%) [23] and taboada et al. (9.4%) [18] . all viruses detected by the qpcr panel were identified through ngs except coronavirus 229e in the asthma group and coronavirus 229e and adenovirus in the pneumonia group, probably owing to a low number of reads and the dilution effect of sample-pooling. however, the ngs method combined with the bioinformatics workflow showed greater sensitivity for detection of infa h1n1, piv-1 and bov-2 compared with qpcr detection, probably as a result of the identification of less conserved sequences. moreover, other viruses such as ttv, ttmv, ttmdv and different phages like propionibacterium phage, streptococcus phage, staphylococcus phage and bacteriopage ej-1 were also detected without having knowledge of the viruses. a diverse viral community was found in the respiratory tract of children with asthma and pneumonia; rv-c and rsv-b were the predominant species. viruses found in these respiratory samples belonged to different families, mainly picornaviridae, orthomyxoviridae and paramyxoviridae. members of the anelloviridae family were found in both groups of pediatric patients; although members of this family are usually considered ubiquitous and benign, a potential role of ttmv in pneumonia pathogenesis has been suggested [24] , other studies have implicated ttv as contributing elements in lung impairment and it has been associated with asthma [25] and chronic obstructive pulmonary disease (copd) [26] . even so, more studies are needed to elucidate the role of respiratory pathogenicity of the torque teno viruses. evd68 has been associated with asthma exacerbation and pneumonia in children [20] , in this study, ev-d68 was detected in the 2014-2015 winter seasons, confirming the presence of this virus in mexico city. with respect to cmv, some studies have found evidence of its implications in asthma pathogenesis and asthmatic inflammation [27] . infa h1n1pdm09 was found in most of the studied groups, its participation and association with bacterial pneumonia [7] are well known, but a strong association with asthma exacerbation has been reported as well [28] . among other of the found viruses, adenovirus (adv) has been associated with pneumonia [29] , and bov has been deemed as a cause of sari in the absence of viral and bacterial co-infections [15] . finally, wiersbitzky et al. [30] suggested that pvb19 can cause acute respiratory diseases with obstructive ventilatory disturbances of the upper or lower airways in children with a specific endogenous predisposition. other components detected in the viruses were bacteriophages, viruses commonly excluded in the final analysis of the viral community, yet, these highly suggest the presence of their specific bacterial hosts in the sample. we compared our results to previous reports that have used metagenomics in children [17] , and shown strategies that use pooling of samples (lysholm et.al. [31] and wang et.al. [16] ), where greater diversity has been found. in our study, we detected 25 different species of viruses using this same strategy. this is most likely due to a greater ability of analyzing more samples in the same library, thereby increasing the probability to detect more viruses. in our study, we found the preferred virus associated with respiratory diseases, rhinovirus, and consistent with others reports (lysholm et.al. [31] and wylie et.al. [12] ), however in contrast with a report from wang et.al. [16] that found a higher prevalence of rsv-b. furthermore, few reports included healthy individuals as a control group (wang et.al [16] ), detecting lesser viral diversity with a high prevalence of viruses belonging to the anelloviridae family. according to this report, the torque teno virus and torque teno mini virus that we found in children with asthma and pneumonia in our study, may be otherwise normal residents in children free of respiratory disease. further data is lacking concerning the prevalence of viruses in the respiratory tracts of healthy children, which may be a limitation of our study. in performing the bacteriome analysis, we found streptococcus pneumoniae and haemophilus influenzae had the greater amount of identified reads in both groups with a significant presence of moraxella and staphylococcus aureus in asthma and pneumonia groups respectively (fig 3a and 3b ). these findings are consistent with data reported by bisgaard et al. [32] and hilty et al. [10] for children with asthma. before rendering judgment, is important to remember that the individual presence of any virus or bacteria does not determine a direct cause for disease. interactions between invaders and host commensals may offer a better understanding about respiratory diseases [33] . identifying coinfections between different microorganisms either between viruses or viruses and bacteria is essential in determining the etiology of the respiratory disease. identifying the etiologic agents present on each disease may assist in developing better treatments and thereby improving patient care. metagenomic and bioinformatic analyses are very powerful tools to describe the microbiome, which allow discerning the etiologic agents involved, and eventually may lead to the discovery of new respiratory viruses. molecular detection of human rhinoviruses in respiratory samples: a comparison of taqman probe-, sybr green i-and boxtobased real-time pcr assays detection and characterization of respiratory viruses causing acute respiratory illness and asthma exacerbation in children during three different season (2011-2014) in mexico city. influenza other respir viruses respiratory severity score separates upper versus lower respiratory tract infections and predicts measures of disease severity characterization of acute respiratory infections among 340 infants in wuxi human pharyngeal microbiome may play a protective role in respiratory tract infections simple predictors to differentiate acute asthma from ari in children: implications for refining case management in the ari control programme the role of respiratory viruses in the etiology of bacterial pneumonia: an ecological perspective host-microbiome interactions in acute and chronic respiratory infections the lung microbiome and exacerbations of copd disordered microbial communities in asthmatic airways strengths and limitations of 16s rrna gene amplicon sequencing in revealing temporal microbial community dynamics sequence analysis of the human virome in febrile and afebrile children unbiased parallel detection of viral pathogens in clinical samples by use of a metagenomic approach direct multiplexed whole genome sequencing of respiratory tract samples reveals full viral genomic information human bocavirus infection as a cause of severe acute respiratory tract infection in children metagenomic analysis of viral genetic diversity in respiratory samples from children with severe acute respiratory infection in china metagenomic analysis of the respiratory virome associated with acute respiratory illness of unknown etiology in infants is there still room for novel viral pathogens in pediatric respiratory tract infections? the infant nasopharyngeal microbiome impacts severity of lower respiratory infection and risk of asthma development ev-d68 infection in children with asthma exacerbation and pneumonia in mexico city during 2014 autumn. influenza other respir viruses microbial community analysis using megan mega: molecular evolutionary genetics analysis software for microcomputers direct metagenomic detection of viral pathogens in nasal and fecal specimens using an unbiased high-throughput sequencing approach potential implication of new torque teno mini viruses in parapneumonic empyema in children associations between nasal torquetenovirus load and spirometric indices in children with asthma the presence of torque teno virus in chronic obstructive pulmonary disease cytomegalovirus dna is highly prevalent in the blood of patients with asthma and is associated with age and asthma traits prevalence of respiratory viral infection in children hospitalized for acute lower respiratory tract diseases, and association of rhinovirus and influenza virus with asthma exacerbations characteristics of adenovirus pneumonia in korean military personnel characterization of the viral microbiome in patients with severe lower respiratory tract infections, using metagenomic sequencing childhood asthma after bacterial colonization of the airway in neonates viral and bacterial interactions in the upper respiratory tract key: cord-017140-k4lzwfge authors: andersen, bjørg marit title: protection of upper respiratory tract, mouth and eyes date: 2018-09-25 journal: prevention and control of infections in hospitals doi: 10.1007/978-3-319-99921-0_13 sha: doc_id: 17140 cord_uid: k4lzwfge pathogenic bacteria and viruses may invade via upper and lower respiratory tract and via eye mucosa. when an infected person coughs or sneezes heavily, small, invisible droplets with the infective agent may reach a good distance from the source. by using the right form of protection at the right time, infection and disease are prevented. the present chapter is focused on the protection against airborne infections. • contact with infectious patients, according to the isolation procedures. • when performing sterile procedures. • when in close contact with patients who are in an infection-prone situation, for example, during operations and patients with compromised immune system. • contact with wounds and tissues and/or in direct contact with sterile equipment used invasively or when present during ongoing invasive procedures. • self-protection against splashes/aerosol of biological material (trachea suction, vomiting, cough, diarrhoea, secretions, diathermy, etc.). • patient with suspected contagious respiratory infection-during transport, examination, treatment, etc.; use a face mask-also on the patient-to protect others and the environment from contamination. • when cleaning and disinfecting contaminated rooms like isolates and when handling used patient equipment/machinery with organic material (ventilator, cpap, etc.) and used patient textiles, infectious waste and bio-organic waste. • during work with plumbing and construction with increased risk of soil, splatter, aerosols or dust particle clouds, such as working with instrument channels in the patient rooms and surgical departments, ventilation systems, sinks, sewer, water leakages with fungal growth, etc. • caps should always be used when putting on masks to protect hair. hospital management should ensure an infection control programme that informs all employees about the standards of hygiene and infection control at the hospital. furthermore, to provide resources to the acquisition, stock reserves and logistics of adequate personal protective equipment (ppe), also for emergency situations [5, 8] . department management is responsible for training, use and control of face masks, respirators and eye protection and that the equipment and written guidelines are available [5] . each user is responsible for the proper use of ppe at the right time and in accordance with current guidelines. the following are available in all relevant departments/posts: • guidelines-written-for the use of a face mask/respirator/eye protection. • surgical masks: put a date on the box when it opens. only surgical masks of good quality are used. other face masks-thin and of poor quality-fastened behind ears should never be used in the healthcare system because of no protective effect. • respiratory protection-p3 mask-with and without valve, separately packed. put the date on the box. • surgical face masks with visor. • face shield. • goggles. single-use or multi-use goggles that can be disinfected and autoclaved between uses. • cap/hood/head or neck protection (phantom hood, operation caps)-always when using masks. • access to good hand hygiene-hand disinfectant-at the place where the ppe is used. when airways, mouth and eyes are protected, the hair and head should also be covered simultaneously. many people have long hair that can come in contact with the patient, bedding or equipment which can lead to transmission of infection to other patients, in addition to themselves becoming a carrier. nb! always use cap/hood/head protection when using surgical mask or other ppe. these may be single-use or multi-use. single-use devices are thrown away immediately after use. surgical mask with visor may protect against direct spills and splashes. a complete face shield protects against direct splashing. the multi-use equipment (check with infection control personnel) is soaked in chloramine 5% 1 h (or household chlorine) before laundered in soapy water or washed in the instrument washing machine by more than 85 °c or autoclaved. it is stated on the package if the goggles may be autoclaved. quality-controlled surgical face mask is disposable and used for: putting on • disinfect your hands. • put on the cap-thin operating hood-that collects all hair. pull it over your ears. • disinfect your hands. • take the mask from the surgical mask box, and take only one mask. the box must have the date of opening. all boxes that have been exposed to infection should be discarded afterwards. • put the face mask over your mouth and nose with a drawstring at the back of the head/top and the other on the neck. • adapt the face mask that has metal string over the nose-so that it fits tightly and comfortably around the mouth and nose. • replace the face mask between each patient/situation/procedure or after 2-6 h if you are with the same patient or when it is wet on the inside. avoid changing the mask if this can cause more contamination and risk, for instance, during surgery. surgical masks are usually not changed during surgery. • never go with a face mask under the chin or around your neck! it is usually heavily contaminated by mouth and nose secretion after use and by splatter from the patient. • perform hand hygiene. • first take hold of the string in the neck and loose this while you bend forward. the lower part of the mask then falls away from the face. then gently loosen the string on the head, and gently put the mask into the waste container. • face masks should not come in contact with hair or clothes. • dispose in regular waste during normal use or infectious waste if infection. • perform hand hygiene afterwards. • grip the cap back and carefully pull it off while bending forward, and put it into the waste container. • perform hand hygiene afterwards. filtering half masks and guidelines for use of respiratory protection should be available at relevant clinical departments. p3 mask is used by the surgical team and during all sterile procedures: in the case of operative treatment of patients with special types of airborne infection such as tuberculosis, etc., see above. if there is an open breathing valve on the p3 mask, surgical mask must be used outside the p3 mask. p3 mask with covered breathing valve can be used instead. p2 mask or surgical mask is put on the patient with defined or suspected airborne infection (e.g. tuberculosis, varicella, etc.) during transport, and stay outside isolation units. putting on • disinfect your hands. • put on the cap-thin operating hood-that collects all hair. pull it over your ears. • disinfect your hands. • take the p3 mask from the surgical mask box, and take only one mask. each mask is usually separately wrapped. the box must have the date of opening. all boxes that have been exposed for infection should be discarded afterwards. • put the respirator over your mouth and nose with a drawstring at the back of the head/top and the other on the neck. the cap hood underneath makes it easier to put the mask on-it does not slip. • adapt the face mask that has metal string over the nose-so that it fits tightly and comfortably around the mouth and nose. • test tightness by blowing vigorously or breathing in; leaks are then sensed on the sides of the mask. • change the p3 mask after 3-6 h or longer or if it is wet on the inside. • avoid change if this may lead to risk of infection. take it off very carefully, as the p3 mask may be used in a serious contagious situation and may be contaminated on the outside. • disinfect your hands. • grasp the band/string posteriorly, bend forwards and remove the mask gently without coming into contact with the clothing, skin or hair. do not touch the mask directly. • loosen the band in the neck first so that the mask falls forwardly away from the face. then carefully loosen the band on the head. • put the mask gently into infectious waste bin. • perform hand hygiene. • grip the cap back, bend forwards and carefully pull it off without coming into contact with the skin or clothes and place the mask in infectious waste bin. • perform hand hygiene afterwards. eye protection is always used when there is a risk of splashing of human biological material to the eyes and for protection against highly infectious diseases. in the event of a risk of severe airborne disease, wear tight protective goggles where you can use regular glasses on the inside. multi-use goggles may be reused after 1 h of treatment in 5% chloramine bath (or household chlorine 10,000 ppm) with subsequent soapy water and rinsing. putting on • perform hand hygiene. • put on the cap-thin operating hood-that collects all hair. pull it over your ears. • in case of severe, dangerous infection, put goggles on outside of a phantom cap that is sitting outside a surgery cap and a p3 mask; see strict isolation. • disinfect your hands. • goggles are retrieved from the box, usually separately wrapped. the box must have the date of opening. all boxes that have been exposed for infection should be discarded afterwards. • put the glasses or shield over the eyes with a rubber band on the occiput. the operation hood under prevents it from slipping. • adapt the goggles-so that it fits tightly and comfortably all around the eyes. • they can be used as long as they are needed. take it off very carefully, as the goggles/face shields may be contaminated on the outside. • disinfect your hands. • grasp the band/string posteriorly, bend forwards and remove the goggles/face shield gently without coming into contact with the clothing, skin or hair. do not touch the devices directly. • if multi-use: put it carefully into the container with 5% chloramine. if single-use: put it into the infectious waste bin. • perform hand hygiene. • grip the cap back, bend forwards and carefully pull it off without coming into contact with the skin or clothes, and place it in infectious waste bin. • perform hand hygiene afterwards. "the employer must ensure that protective equipment made available to the worker, meets requirements of regulations on construction, design and manufacture of personal protective equipment" [5] . this should be in accordance with official regulations for the use of personal protective equipment [5, 9] . already in roman times, it was pointed out by doctor galen that "when many get sick and die at once, we must look for a common cause, the air we breathe". [1, [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] . most cases of these serious, life-threatening diseases may be transmitted via air, droplets and re-aerosols, from patients, carriers and the environment. an adult breathes in at rest 5-8 litres of air per minute, at medium heavy work 30-40 l and at great exertion 70-100 litres of air per minute. in small rooms and with a high air contamination of infectious agents, some contaminants will be drawn into the respiratory tract. it has been demonstrated that when a person coughs or sneezes, drops, droplets and droplet nuclei from the mouth and nose may reach up to 9 m from the source [2] . surgical masks and respiratory protection (filter masks) are defined as "equipment that can help prevent the spread of microbes from one person to another" [2, 3] . the difference between surgical mask and respiratory protection is that surgical masks primarily protect the patient and sterile area/equipment against mouth and nose secretion from healthcare professionals, while respiratory protection protects healthcare workers and others from airborne infections from nearby sources of infection [3-5, 14, 23] . however, surgical masks are not approved as protection against airborne infections: [5, 14, 24, 25] "harmful microorganisms (bacteria, viruses, fungi) or components of microorganisms (e.g. endotoxins) may occur in air, either in dust, smoke or aerosols, or even finer distributed as droplet nuclei where all liquid has dried in. surgical masks only protects against splash and drop, not against airborne infection. therefore, to protect against airborne infection, wear respiratory protection. in most situations, a filtering half mask will provide a good protection. particle filter class p2 protects against most spores of fungi. at risk of exposure to airborne viruses and bacteria, especially tuberculosis, particle filter p3 must be used. in particularly dangerous situations, during prolonged work or if carrying a beard, should special breathing systems be used" [5] (fig. 13.1 ). many human pathogenic bacteria and viruses invade via upper and lower respiratory tract [1] . some viruses may also invade via eye mucosa, such as influenza, hepatitis b and c and hiv. carl schiøtz, a norwegian professor in hygiene and infection control, wrote in the textbook of hygiene in 1937 (80 years ago) that the most important measures against communicable diseases are the following: (1) isolation of the sources of infection (home or hospital), (2) disinfection of the exposed rooms and equipment, (3) vaccination if vaccine-preventable disease, (4) quarantine, (5) food hygiene and (6) insect eradication [26] . he noticed that airborne infection was considered to be "the most important form of transmission of infections at our latitude" [26] . schiøtz meant that drops from the respiratory tracts were large and heavy and went 1.5-2 m away, while saliva droplets went in "up to 20 m distance from high-speaking people and they could stay floating in the air for several hours" [26] . this was important for the spread of "flu, colds, pneumonia, pest-pneumonia, tuberculosis, pertussis, measles, small-pox, chickenpox, scarlet fever, rubella, diphtheria, poliomyelitis, epidemic cerebrospinal meningitis and several other diseases" [26] . he also focused on inhalation of dust containing microbes [26] . the introduction of antibiotics in post-war times and the sharp reduction of lung tuberculosis caused many to forget that it was something that was infected via air. tuberculosis has been "recognized" as an airborne infection at least in 100 years, although some still believe that short-term exposure to the patient does not lead to infection. this perception has led to multiple outbreaks of multiresistant tuberculosis, including in the united states [27] . airborne transmission is most often downgraded by the health authorities to contact and droplet transmission-traditionally within 1 m from the patient. this applies to healthcare professionals who are going to treat patients with severe, deadly infections where surgical masks are estimated "good enough" [10-16, 23-25, 28-31] . droplet transmission is a definition that may put healthcare personnel at risk during dangerous situations since it is still a "form of contact transmission" [17] . the cdc definition from 2007 is upgraded since 2004 but is still very vague and controversial; see the following quotations [17] : droplet transmission is generated when "an infected person coughs, sneezes, or talks -or during procedures such as suctioning, endotracheal intubation, cough induction by chest physiotherapy and cardiopulmonary resuscitation. ---the maximum distance for droplet transmission is currently unresolved,---historically, the area of defined risk has been a distance of ≤3 feet around the patient----investigations during the global sars outbreaks of 2003 suggest that droplets --could reach persons located 6 feet or more from their source. ----thus, a distance of ≤3 feet around the patient is best viewed as an example of what is meant by 'a short distance from a patient' ----it may be prudent to don a mask when within 6 to 10 feet of the patient or upon entry into the patient's room, especially when exposure to emerging or highly virulent pathogens is likely--" [17] . "droplet size is another variable under discussion ---defined as being >5 μm in size. droplet nuclei, particles arising from desiccation of suspended droplets, have been associated with airborne transmission and defined as ≤5 μm in size-----particle dynamics have demonstrated that a range of droplet sizes, including those with diameters of 30μm or greater, can remain suspended in the air" [17] . the worst example until now is the recommendation of "contact and droplet transmission within 1 m" with the use of surgical masks (within 1 m from the patient), by who, the first phase (3 months) of the sars epidemic in 2003, where 90% of those who became ill during this first phase were health professionals [28] . the same was done during the avian influenza epidemic in 2005 and mostly throughout the influenza pandemic in 2009 [29] . who and cdc recommended both "contact and droplet transmission" measures during the first 6 months of the ebola epidemic in 2014 [1, 30] . during some of the most serious global epidemics that have happened in the last 80 years, healthcare personnel were exposed to infection without proper protective equipment and had the highest death rate associated with work-related infection [10-17, 30, 31] . during the sars outbreak in toronto, canada, 169 health personnel were infected with nosocomial sars, and 3 of these died [14, 31] . infection control personnel in toronto insisted that sars was primarily transmitted through large droplets-within 1 m-and that there was lack of evidence-based documentation for airborne infection! [31] despite the fact that health professionals requested it, the respiratory protection (n95) was not handed over to the staff, and the outbreak continued for a long time [31] . a state investigation commission came to the following bottom line conclusion: safety comes first and reasonable measures to reduce the risk of healthcare professionals do not have to wait for scientific evidence [31] . in the event of outbreaks of less severe airborne infections such as common flu, rsv, mycoplasma, adenovirus, metapneumovirus, etc., it is important to protect healthcare professionals from infection. the purpose is that infected personnel should not be "vectors" of infections in the hospital. in addition, a large outbreak among the staff may cause that patients with life-threatening diseases like heart attack, trauma, etc. do not get the necessary treatment. despite all the controversies surrounding airborne transmission, a number of international guidelines for the use of respiratory and face protection equipment have been developed under varying epidemiological conditions and experiences over the past 100 years. recent surveys and experiences show that schiøtz and other researchers had correct facts and guidelines according to airborne infections. secrets from the respiratory tract can go far off 9 m or more, for example, when coughing and sneezing, and pathogenic viruses and bacteria are detected in the air in rooms with infected patients [2, 14, 17, 18, 20, 21, 26, [32] [33] [34] [35] [36] [37] [38] [39] [40] [41] [42] [43] . airborne mrsa and other multiresistant bacteria may be a greater problem than previously thought [17, 18, 20, 42] . even clostridium difficile spores may become airborne under certain conditions [43] . today, it is also known that most microbes are strong, surviving organisms outside the body [1, 44] . they can also survive on the outside of the protective equipment and even re-aerosol from these and even penetrate a wet mask [1, 4, [44] [45] [46] [47] [48] . respirators (half masks) are more expensive (about 4 usd) than surgical mask (<0.1 usd). because of a shortage during large outbreaks, it has been attempted to decontaminate respirators for reuse. however, this is not effective, is not recommended and cannot be done with disposable equipment [49] . most infectious agents are spread through contact, blood, drops, droplets, drop nuclei (aerosol), airborne on dust particles and often in several ways simultaneously [12, 13, 17, 23, [33] [34] [35] [36] [37] [38] [39] [40] [41] [42] [43] [50] [51] [52] . transmission of microbes from the respiratory tract to the air occurs by breathing, speech, cough, sneezing, laughing, singing or other aerosol-generating procedures. sneezing usually leads to greater amount of microbes transferred to air than coughing [51] . how long the contaminant stays alive in the air depends on the agent, temperature, humidity, virus dose response, particle size and presence of other material [50] [51] [52] [53] [54] [55] [56] . some larger drops are deposited close to the patient, and smaller dropsdroplet nuclei-pass into a floating phase in the air and are falling slowly over time, such as influenza a virus [2, [50] [51] [52] [53] [54] [55] [56] . transmission of microbes via small particles and droplet nuclei from influenza patients is not adequately controlled by the use of surgical mask [50] [51] [52] . airborne transmission always includes contact transmission and often re-aerosols from the environment [18-21, 39, 42-44, 55-61] . transmission to the air from the skin occurs via the release of millions of skin particles where approximately 10% carry bacteria or virus from the skin or wound. re-aerosols from infectious particles whirled up in the air, for instance, during bed-making, dry mopping of contaminated floor surfaces, from contaminated areas in the room during air currents or when the door is opened or closed quickly [62] . re-aerosols of pathogenic microbes may be important because of a long survival in the environment, for days, months and years [1, 44] . influenza a virus survives on paper, textiles and equipment for more than 3 weeks and can become airborne [55] [56] [57] [58] [59] [60] [61] . influenza a virus is very contagious via aerosols and small airborne droplets [56] . microbial load in the air depends on cleaning and air exchange [5, [10] [11] [12] [13] [14] 63] . proper cleaning and ventilation with a positive pressure of filtered air reduce the load in the operating units or during protective isolation. decontamination and cleaning of rooms, textiles and surfaces and a good air exchange with a negative air pressure are important during isolation of patients with infections. air exchange it has been demonstrated that after aerosol-generating procedures such as bronchoscopy, airway suction and intubation, five fresh air changes in the room can greatly reduce air contamination, provided that the source of infection is gone [13] . respiratory protection prevents people from touching their nose or mouth in an infectious situation and coughing on their own uniforms and thereby exposing themselves to others and to infection [6, 7] . correct use of respiratory protection most microbes survive for hours to days on the outside of a respiratory protection as shown for bacteriophage, influenza virus and coronavirus [45] [46] [47] . this is the reason why respiratory protection should not be reused and why it is not advisable to touch the outside of the mask. during the sars outbreak in canada in 2003, the staff reinfected themselves by handling the mask incorrectly and reusing equipment without decontamination [28] . among 1441 hospital personnel in china, there was a significantly lower incidence of respiratory tract pathogenic microbes among personnel who used respiratory protection with filter n95 (p = 0.02) than among personnel using face mask (p < 0.01) compared to controls [64] . face mask, covering the nose and mouth, has been used since the spanish flu in 1919. it was actually developed to protect patients from postoperative infections from the airway flora of the operating team and protect the team against blood and tissue splash from the patient. there is no standard definition of surgical masks, adaptation, leak test or quality of filter [5, 13, 14] . surgical mask is not approved by the occupational health authorities [3-5, 24, 25] . nevertheless, there are extensive use and purchase of face masks, especially in the national emergency preparedness plans. the combination of good hand hygiene and the use of surgical mask within 36 h after the onset of influenza in an index case may however reduce spread of the flu in the household [65] [66] [67] . volunteer subjects infected with seasonal flu breathed out nearly nine times more virus in small particles, <5 μm, than in larger one, >5 μm, after the onset of the flu [50] . the use of surgical masks reduced particularly the larger particles and showed a 3.4-fold reduction of virus released to the air [50] . using surgical face masks can reduce infection and also had some suppressing effect on the spread of infection by sars [10] . surgical face mask protects the nose and mouth from splashing and saliva particles but does not protect effectively against bacteria and viruses that are, respectively, from 0.2 to 8 μm (0.0002-0.008 mm) and from 20 to 300 nm (0.02-0.3 μm) in diameter. there is no safe protection against aerosol or droplet nuclei, either through the mask or from the sides of the mask [5, 13, 24, 25, 68] . it is not certain that a mask may stop microparticles of the blood that can be formed by certain procedures, like centrifugation accidents, spray from dental treatment or surgery, etc. [36, 68] . in such cases, also eye protection should be used. persons close to sterile areas can contaminate the area by direct drops of saliva and aerosols that are spilled by speech, coughing and sneezing [2, 13] . a good face mask captures these droplets from the respiratory tract and prevents deposition thereof. it catches drops both ways to some extent, but when the surgical mask is wet, it can become less effective, with some growth of bacteria. it is usually effective for at least 2-3 h. face masks with visors protect to some extent both nasal/oral and eye mucosa against infected blood and tissue particles/droplets. airborne microparticles of aerosol, drop nuclei and blood drop-bearing bacteria and viruses can be formed during activities of or around the patient (coughing, sneezing, speech, excessive bodily activity, bed-making, dust cleaning etc.). respiratory protection such as p3 mask or n95 has shown protective effect in highly severe infections such as sars, tuberculosis and pandemic influenza [5, 9-16, 20-23, 56, 64] . respirators are not designed for children or people with beards, and therefore does not provide full protection for these [5, 23] . respiratory protection is filtering half mask, looks almost like surgical mask and is often called filtering face piece (ffp) respirators or dust filter [5] . they are of different quality (ffp1-ffp3) and capture microbes in both ways. the filter is hepafilter/polypropylene filter with static charge to increase the filter power. ffp2 is equivalent to the us n95 masks that are widely used around the world. the european standard is en149: 2001 which is equivalent to ffp3 and has a somewhat higher level of protection [13] . procedures involving large load on the environment around a patient with airborne infection are, for example, bronchoscopy, trachea suction, diathermy aerosol, suction and drilling (orthopaedics, dental treatment, etc.). in such cases, both respirators and eye protection should be used. the p3 mask protects the user and can also be used on patients with specific respiratory infection and then without exhalation valve. protection factor is calculated by reducing the degree of pollution in the air. if there is a pollution of 1000 mg/m 3 air, a respirator with a protection factor of 500 may reduce the pollution to a residue of 2 mg/m 3 . that includes inhalation of air through or on the sides of the respiratory protection, which will almost always happen [69] [70] [71] [72] . "protection factors for filtering half masks ffp1, ffp2 and ffp3 matches the protection factors for half mask with respectively filter classes p1, p2 and p3. motor-assisted filtering air purifying respirators (equipment with a turbo unit) have varying protection factor depending on the equipment design" [5] . a wet filtering mask may be permeable for viruses and bacteria. the norwegian directorate of labour inspection indicates protection factors for filtering respirators as follows [5] : lower protection is expected, because it is often leaked due to poor adaptation; factor 10 is for filtering half mask classes 2 and 3, and factor 100 is for full face masks. "protection factor for both supplied air and air purifying respirators with half and full face masks also require a good fit, and this cannot be expected if beard, glasses or the like, in squeeze along the edge of the mask" [5] . requirements for filtering half masks [5, 12, 13, [69] [70] [71] [72] . p3 has the highest protection factor with 50 times cleaner air inside the mask than outside. the mask's overall efficiency depends on the filter quality of the mask, fitting the face shape (leakage) and leakage through any exhalation [5, 12, 13, [69] [70] [71] [72] . fit test is the control and training using special odour tests that the person perceives within the mask if it is not tight enough [5, 12, 13] . in a study of five models of the n95 mask (3m 1860s cup, 3m 1870 flat fold, kimberly-clark pfr95 duckbill, safelife t5000 cup added with iodine and glaxosmithkline actiprotect cup, added with lemon acid), all five types had more than 95% efficiency: −0.8 μm particles of h1n1 influenza virus and corresponding inert particle sizes [69] . filtration efficiency was largely based on particle size [69] . so-called elastomeric masks appear to be more effective than regular filter masks [70] . studies show considerable variation with regard to protection, and for some dangerous, microbial agents, 95% protection may be too low [71, 72] . • turbo equipment (papr = -powered air-purifying respirators) with p3 filter (protection factor 500-2000) -may have problems concerning the disinfection of battery-operated, recycling systems, and is dependent on a good training [5, 12, 13] . • fresh air/pneumatic equipment (protection factor, 100-100 000) is used for particularly risky situations of special personnel in laboratories and when working in areas with severe epidemics. however, there may be serious infection problems regarding disinfection of reuse systems (filters, mechanical/electrical appliances). during non-epidemic times and with low-virulent microbes, respiratory protection (n95 or p3 masks) are not used for other than special types of respiratory infections and pulmonary tuberculosis. this may influence on the state of readiness for major outbreaks such as pandemic influenza. consumption of masks can be very large as during the sars outbreak in 2003, where a canada-based hospital with sars outbreak used up to 18,000 n95 masks each day [13] . the durability of respiratory protection equipment is good-up to 10 years or more in storage. items made with rubber or rubber parts should be checked, and there should be some systemic rotation in the warehouse [49] . at ouh, ullevål, there has been a strategic stockpile system of such equipment with replacement as required [8] . the experience from norway is that the national health authorities have been unprepared for stockpiling of emergency response requirements for ppe, also during the influenza pandemic of 2009 [29] . the market may soon be empty for respiratory protection masks during serious outbreaks such as sars and ebola virus. using several surgical face masks superposed did not have enough protective effect [73] . during the sars epidemic, the chinese healthcare personnel made their own masks of 12 layers of gauze that supposedly would work well. this is used when there is risk in soil and splatter of biological materials and to protect against highly dangerous infection. if there is a risk of transmission of severe respiratory infection, tight-fitting goggles are used. the goggles can be reused after 1 h of treatment in 5% chloramine, followed by washing with soap and water, and they may also be reprocessed and decontaminated in a washing machine at >85 °c. controversial issues concerning airborne infection and protection against "droplet transmission" should be further studied. particle studies; ventilation variations; kinetics; the effect of humidity, temperature and filter types; re-aerosols from dust; environment and ppe equipment; and the survival of microbes in the environment should be better studied. this is the case for most important microbial agents: bacteria, viruses and fungi. respirators should be preferred over surgical masks when there is suspected droplet or airborne transmission. this choice is essentially a misunderstood price and attitude problem. microbiology and infection control. handbook of hygiene and infection control in hospitals violent expiratory events: on coughing and sneezing protection of workers from the risks when working with biological factors on the protection of workers from the risks related to exposure to biological agents at work (seventh individual directive according to article 16 norwegian directorate of labour inspection. respiratory protection controlling the novel a (h1n1) influenza virus: do not touch your face! european centre for disease prevention and control -swine flu guidelines: "cough hygienically" into your sleeve? serious, communicable disease -personal protective equipment-ppe. in: handbook of hygiene and infection control in hospitals. oslo: ullevaal university hospital respiratory protection. breathing equipment with fresh air hose connected to a full face mask, half mask or mouthpiece means. requirements, testing, mark. ns-en 138 non-pharmaceutical interventions for pandemic influenza, national and community measures interim guidance on infection control measures for 2009 h1n1 influenza in healthcare settings, including protection of healthcare personnel guidance on the use of respiratory and facial protection equipment respiratory and facial protection: a critical review of recent literature controversy: respiratory protection for healthcare workers ecdc health information. personal protective measures for reducing the risk of acquiring or transmitting human influenza respiratory protection for healthcare workers in the workplace against novel h1n1 influenza a guideline for isolation precautions: preventing transmission of infectious agents in healthcare settings air contamination around patients colonized with multi drug-resistant organisms the exposure to hospital room is fed as a risk factor for healthcare-associated infection aerial dispersal of methicillin-resistant staphylococcus aureus in hospital rooms by infected or colonised patients evaluation of bedmaking-related airborne and surface methicillin-resistant staphylococcus aureus contamination guidelines for preventing the transmission of mycobacterium tuberculosis in health-care facilities food and drug administration osha. respiratory protection standard work place safety and health topics: respirator the infectious diseases. textbook in hygiene. oslo: fabritius & sønners forlag mycobacterium tuberculosis. in: bacteria and disease. epidemiology, infection and immunity. oslo: gyldendal academic in: microbiology and infection control. handbook of hygiene and infection control in hospitals. part 1. bergen: fagbokforlaget pandemic influenza. in: microbiology and infection control. handbook of hygiene and infection control in hospitals. part 1. bergen: fagbokforlaget international infection control guidelines may not protect against ebola. hospital healthcare ontario ministery of health and long-term care. sars commission-spring of fear: final report role of ventilation in airborne transmission of infectious agents in the built environment-a multi-disiplinary systematic review virus diffusion in isolation rooms review of aerosol transmission of influenza a virus aiborne dispersal as a novel transmission route of coagulase-negative staphylococci, interaction between coagulase-negative staphylococci and rhinovirus infection the risk of blood splash contamination during angiography aerosol transmission of influenza a virus: a review of new studies transmission of influenza a in human beings detection of bordetella pertussis and respiratory syncytial virus in air samples from hospital rooms an outbreak of influenza abroad a commercial airliner droplet fate in indoor environments, or can we prevent the spread of infection? indoor air air and surface contamination patterns of methicillinresistant staphylococcus aureus on eight acute hospital wards the potential for airborne dispersal of clostridium difficile from symptomatic patients virus survival in the environment corona virus survival on healthcare personnel protective equipment survival of surrogate viruses on n95 respirator material persistence of the 2009 pandemic influenza a (h1n1) virus on n95 respirators re-aerosolization of ms2 bacteriophage from an n95 filtering facepiece respirator by simulated coughing evaluation of five decontamination methods for filtering face-piece respirators influenza virus aerosols in human exhaled breath: particle size, culturability, and effect of surgical masks viral kinetics and exhaled droplet size affect indoor transmission dynamics of influenza infection aerosol transmission is an important mode of influenza a virus spread dynamics of airborne influenza a viruses indoors and dependence on humidity mechanisms by ambient humidity mayaffect viruses in aerosols inactivation of influenza a viruses in the environment and modes of transmission: a critical review high infectivity and pathogenity of influenza a virus via aerosol and droplet transmission survival of influenza viruses on environmental surfaces survival of influenza viruses on banknotes association of private isolation rooms with ventilatorassociated acinetobacter baumannii pneumonia in surgical intensive-care unit an evaluation of hospital special-ventilation-room pressures survival of influenza a (h1n1) on materials found in householders: implications for infection control door-opening motion can potentially lead to a transient breakdown in negative-pressure isolation conditions: the importance of vorticity and buoyancy airflows mopping up hospital infection efficacy of face masks and respirators in preventing upper respiratory tract bacterial colonization and co-infection in hospital healthcare workers face masks and hand hygiene to prevent influenza transmission in households: a cluster randomized trial preliminary findings of a randomized trial of nonpharmaceutical interventions to prevent influenza transmission in households face mask use and control of respiratory virus transmission in households aerosol penetration and leakage characteristics of masks used in the healthcare industry challenge of n95 filtering face-piece respirators with viable h1n1 influenza aerosols comparison of performance of three different types of respiratory protection devices do n95 respirators not provide 95% protection level against airborne viruses, and how adequate are surgical masks? performace of an n95 filtering face-piece particulate respirator and a surgical mask during human breathing: two pathways for particle penetration protecting staff against airborne viral particles: in vivo efficiency of laser masks key: cord-260238-2p209g2p authors: peiris, j s m; guan, y; yuen, k y title: severe acute respiratory syndrome date: 2004-11-30 journal: nat med doi: 10.1038/nm1143 sha: doc_id: 260238 cord_uid: 2p209g2p severe acute respiratory syndrome (sars) was caused by a previously unrecognized animal coronavirus that exploited opportunities provided by 'wet markets' in southern china to adapt to become a virus readily transmissible between humans. hospitals and international travel proved to be 'amplifiers' that permitted a local outbreak to achieve global dimensions. in this review we will discuss the substantial scientific progress that has been made towards understanding the virus—sars coronavirus (sars-cov)—and the disease. we will also highlight the progress that has been made towards developing vaccines and therapies the concerted and coordinated response that contained sars is a triumph for global public health and provides a new paradigm for the detection and control of future emerging infectious disease threats. of these deletions, however, is not clear. similarly, sars-cov in individuals before february 2003 was genetically more diverse than the later isolates 26, 34, 35 . the spike protein (the viral surface glycoprotein which mediates viral attachment and entry into the cell; fig. 3 ) of early isolates contained higher rates of nonsynonymous mutations, probably reflecting the ongoing adaptation to the new host. the relative genetic homogeneity of sars-cov isolates from later in the outbreak 34-37 may reflect a virus better adapted to the new host. the fact that much of the global spread arose from one index case in hotel m in hong kong 3, 35 may also contribute to this genetic homogeneity. a ban on the sale of wildlife in wet markets in guangdong imposed during the later period of the sars outbreak was lifted in september 2003. between 16 december and 30 january 2004, there were four new cases of sars, the first nonlaboratory-associated cases diagnosed in humans since the end of the sars outbreak in july 2003. epidemiological linkage and phylogenetic data suggest that the associated viruses were new introductions from animals (y. guan, unpublished observations) 34, 38, 39 . these human cases were relatively mild and did not lead to secondary transmission, reflecting that the animal precursor virus is probably not well adapted to efficient human-tohuman transmission. this is probably a recapitulation of events in late 2002 in the run-up to the sars outbreak in 2003. this time, the findings led to the reintroduction of the ban on wild-game animal markets and there have been no further naturally acquired human cases since. it is likely that the precursor of sars-cov has repeatedly crossed the species barrier but only occasionally has it succeeded in adapting to human-human transmission. this adaptation clearly occurred in late 2002 and it may happen again in the future. but given the present understanding and awareness about sars, we expect that such reemergence is unlikely to lead to a global outbreak on the scale of 2003. the major routes of transmission of sars are droplet infection, aerosolization and fomites (refs. 40,41 and world health organization, http://www.who.int/csr/sars/en/whoconsensus.pdf ) . deposition of infected droplets or aerosols on the respiratory mucosal epithelium probably initiates viral infection. whether infection can occur through the oral or conjunctival routes is unknown, but sars-cov has been detected in tears 42 . although exposure to the animal precursor of figure 1 the global spread of sars. the number of probable cases of sars and the date of onset of the first case in each country (or group of countries) is denoted. the countries denoted in red are those where substantial local transmission occurred. the data are based on world health organization, http://www.who.int/csr/sars/country/table2004_04_21/en_21/en/print.html and the figure is adapted from ref. 15 . wet markets in guangdong: 'wet markets' selling live poultry, fish, reptiles and other mammals are commonplace across southeast asia and southern china to service the cultural demand for freshly killed meat and fish produce. in some regions (e.g., guangdong province, china), increasing affluence has led to the proliferation of markets housing a range of live 'wild' animal species, such as civet cats, pictured, linked to the restaurant trade servicing the demand for these exotic foods. 29, 30 , once the virus had adapted to human-to-human transmission in the later part of the outbreak, asymptomatic infection seemed to be rare 43 . other peculiarities about sars-cov transmission were also evident. transmission was infrequent during the first five days of illness 44 and, unlike transmission of influenza, was relatively inefficient in the household setting 45 . despite sars's fearsome reputation and global spread, the average number of secondary infectious cases generated by one case (r 0 ) was low (2.2-3.7); in contrast, the r 0 of influenza ranges from 5 to 25 (ref. 22) . although not unique to sars, 'superspreading events' (in which a few affected individuals disproportionately contribute to transmission) were characteristic of the outbreak 22, 46 . the factors underlying the superspreading phenomenon of sars are poorly understood but may include coinfection with other viruses and host factors, as well as behavioral and environmental factors. the clinical symptoms of sars-cov infection are those of lower respiratory tract disease [4] [5] [6] [7] 14 . besides fever, malaise and lymphopenia, affected individuals have slightly decreased platelet counts, prolonged coagulation profiles and mildly elevated serum hepatic enzymes. chest radiography reveals infiltrates with subpleural consolidation or 'ground glass' changes compatible with viral pneumonitis. but although the main clinical symptoms are those of severe respiratory illness, sars-cov actually causes infection of other organs: some affected individuals have watery diarrhea, and virus can be cultured from the feces and urine, as well as the respiratory tract [47] [48] [49] . in addition, rt-pcr has identified the virus in the serum, plasma and peripheral blood leucocytes 50, 51 . individuals with sars also have a pronounced peripheral t-cell lymphocytopenia: numbers of cd4 + and cd8 + cells are both reduced, and more than one-third of individuals have a cd4 + t-cell count of less than 200 cells/mm 3 (refs. 52,53) , suggesting increased susceptibility to secondary infections. the mechanisms underlying the t-cell lymphopenia remain to be elucidated. around 20-30% of individuals with sars require management in intensive care units 14 and the overall fatality rate is ∼15% (world health organization, http://www.who.int/csr/sars/en/whoconsensus.pdf). the age dependence of disease severity and mortality is notable; during the outbreak, mortality rates of affected individuals in hong kong who were 0-24, 25-44, 45-64 and >65-year old were 0%, 6%, 15% and 52%, respectively (world health organization, http://www.who.int/ csr/sars/en/whoconsensus.pdf). none of the 1-12-year-olds infected with sars-cov in hong kong had disease severe enough to require intensive care or mechanical ventilation 54, 55 . this progressive age dependence in mortality is not totally explained by comorbid factors and the underlying biological basis remains unclear. quantitative studies of viral load have provided insights into the pathogenesis of sars. viral load is higher in the lower respiratory tract than in the upper airways 56, 57 . viral load in the upper respiratory tract 47 and feces 57 is low during the first 4 days and peaks at around day 10 of illness. in marked contrast, viral load in influenza peaks soon after onset of clinical symptoms 58 . this unusual feature of sars-cov infection explains its low transmissibility early in the illness. it also explains the poor diagnostic sensitivity of the first-generation rt-pcr diagnostic tests on upper respiratory tract and fecal specimens collected early in the illness (reviewed in ref. 21) . affected individuals with high serum viral loads have a poor prognosis 59 . between days 10-15 of illness, high viral load in nasopharyngeal aspirates, feces and serum, as well as detection of virus in multiple anatomic sites, are independently predictive of adverse clinical outcome 60 . serial studies of viral load throughout illness also reflect clinical outcome 61 . taken together, these findings suggest that poor clinical outcome is associated with continued uncontrolled viral replication. sars-cov rna can be invariably detected in the lungs of individuals dying of sars, but viral load is higher in those dying earlier in the course of the illness (<21 days) 62 . the respiratory tracts of affected individuals who die during the first ten days of illness show diffuse alveolar damage with a mixed alveolar infiltrate, lung edema and hyaline membrane formation. macrophages are a prominent component of the cellular exudates in the alveoli and lung interstitium 63, 64 . multinucleate syncytia of macrophage or epithelial cell origin are sometimes seen later in the disease. immunohistochemistry, in situ hybridization and electron microscopy on autopsy or tissue biopsy have unequivocally demonstrated sars-cov replication in pneumocytes in the lung and enterocytes in the intestine [65] [66] [67] [68] . individual reports of virus detection by in situ hybridization or immunohistochemistry in other tissues 69 await confirmation by electron microscopy 70 . in the large and small intestines, the virus replicates in enterocytes 71 . viral particles primarily are seen on the apical surface of enterocytes and rarely in the glandular epithelial cells. but there is no villous atrophy or cellular infiltrate in the intestinal epithelium and the pathogenic mechanisms responsible for watery diarrhea in individuals with sars is unclear. some human intestinal epithelial cell lines support productive replication of sars-cov 72 and gene expression arrays have shown that virus replication is associated with the expression of an antiapoptotic host cellular response, perhaps explaining the lack of enterocyte destruction in vivo 73 . studies using pseudotyped lentiviruses, carrying the spike, membrane and envelope surface glycoproteins of sars-cov (fig. 3 ) separately and in combination demonstrated that the spike protein is both necessary and sufficient for virus attachment on susceptible cells [74] [75] [76] [77] . the sars-cov spike protein uses a mechanism similar to that of class 1 fusion proteins in mediating membrane fusion 78, 79 . there is no consensus as to whether the virus entry occurs through a ph-dependent receptor-mediated endocytosis or through direct membrane fusion at the cell surface 74, 77, 80 . the receptor for sars-cov was identified as the metallopeptidase ace-2 (refs. 81, 82) . the soluble ace-2 ectodomain blocks sars-cov infection 76 , and amino acids 270-510 of the spike protein are required for interaction with ace-2 (ref. 83) . other coronaviruses use different cell receptors and enter cells either by means of fusion at the plasma membrane or through receptor-mediated endocytosis 84 . immunostaining techniques have identified ace-2 on the surface of type 1 and 2 pneumocytes, the enterocytes of all parts of the small intestine and the proximal tubular cells of the kidney. this localization explains the documented tissue tropism of sars-cov for the lung and gastrointestinal tract and its isolation from the urine. but it is notable that colonic enterocytes lack ace-2 protein expression although sars-cov replication does occur in colonic epithelium 71, 85 . in contrast, whereas ace-2 is strongly expressed on the endothelial cells of small and large arteries and veins of all tissues studied and the smooth muscle cells of the intestinal tract, there is no evidence of virus infection at any of these sites. this lack of virus infection in tissues that express the putative receptor prompts the question of whether a coreceptor is required for successful virus infection 70 . vasculitis is known to occur in individuals with sars but its relation to infection of endothelial cells is unknown. because only the basal layer of the nonkeratinized squamous epithelium of the upper respiratory tract expresses ace-2 (ref. 85) , undamaged epithelium of the nasopharynx is unlikely to support sars-cov replication. other receptors for virus entry that are independent of ace-2 expression may exist. pseudotyped virus containing the spike protein has also been shown to bind to dendritic cell-specific intercellular adhesion molecule 3grabbing nonintegrin (dc-sign) 74 . dc-sign is a type-ii transmembrane adhesion molecule found on dendritic cells consisting of a c-type lectin domain that recognizes carbohydrate residues on a variety of pathogens. unlike the ace-2 receptor on pneumocytes and enterocytes, dc-sign does not permit sars-cov infection of the dendritic cells. instead, binding of sars-cov to dc-sign allows dendritic cells to transfer infectious sars-cov to susceptible target cells 74 . a similar mechanism has been described for dengue virus, human immune deficiency virus (hiv) and cytomegalovirus, and may be relevant in sars pathogenesis. many details of sars-cov pathogenesis remain to be elucidated, but the development of a full-length infectious cdna clone of sars-cov should permit precise manipulation of the virus genome and will help our understanding of the viral determinants of pathogenesis 86 . several inflammatory cytokines (il-1β, il-6 and il-12) and chemokines chemotactic for monocytes (mcp-1) and neutrophils (ip-10) are elevated in adults and children with sars [87] [88] [89] [90] . the increased levels of monocyte-tropic chemokines may contribute to the prominently monocytic macrophagic infiltrate observed in the lung 63 . but increases of these same chemokines occur in other viral diseases (e.g., influenza) 91 and are not a unique feature of sars. in addition, elispot assays of peripheral blood leukocytes have revealed prolonged immunological dysregulation in individuals with sars 92 . it is difficult to evaluate the overall pathogenic significance of these findings because immunological markers in the peripheral blood do not always reflect the local microenvironment of the lung 93 . genetic factors associated with susceptibility to, or severity of, sars are under investigation. hla-b*4601 has been associated with severe sars disease in taiwan 94 but not hong kong 95 . hla-b*0703 has also been associated with disease susceptibility and hla-drb1*0301 with resistance to sars. the coinheritance of b*0703 and b60 was significantly higher in individuals with sars than in the general population 95 . the mechanisms underlying these disease associations remain to be elucidated. key to the development of effective antiviral drugs and vaccines against sars-cov was the development of animal models of sars ( table 1) . sars-cov seems to cause infection in cynomolgous macaques following intratracheal inoculation [96] [97] [98] . but whereas some researchers find evidence of disease pathology reminiscent of that seen in individuals dying of sars and can show sars-cov antigen and viral particles in the pneumocytes of infected macaques 96, 97 , others only find evidence of a mild upper-airway disease and low levels of virus by rt-pcr 98 . these differences in outcome may reflect differences in the viral strain, pre-exposure history and age of the animals, route of inoculation, stage of infection at which necropsy was performed or other factors. other animal models include ferrets, cats, golden syrian hamsters, mice and african green monkeys ( table 1) [99] [100] [101] [102] [103] . these animal models support viral replication in the upper and lower respiratory tracts [96] [97] [98] [99] [100] [101] [102] [103] . ferrets and hamsters also develop notable lung pathology. infected cats and ferrets transmit sars-cov to noninfected animals held in the same cage 99 . natural asymptomatic infection in cats was documented during the community outbreak at amoy gardens, hong kong (world health organization, http://www.who.int/csr/sars/en/ whoconsensus.pdf). these animal models of sars differ from natural human disease in that the period between infection and peak disease pathology or peak viral load is shorter than is found in human disease and because the disease pathology, when present, is self-limited and rarely progresses to a fatal outcome as occurs with sars. they also do not accurately reproduce the intestinal component of the human disease. but these models provide the only options presently available for addressing questions relevant to therapeutics and vaccine development. they can provide useful information providing their limitations are recognized. several potential antiviral agents have been evaluated in vitro, and a few have been tested in animal models. screening of currently available antiviral drugs and chemical libraries reveals that interferons, glycyrrhizin, baicalin, reserpine, niclosamide, luteolin, tetra-o-galloylβ-d-glucose and the protease inhibitors have in vitro activity against sars-cov 104-108 . differences in in vitro susceptibility of sars-cov to interferon (ifn)-β1b, ifn-α2 and ribavirin 106,109-111 probably relate to differences in the testing methods used. overall, ifn-αn1/n3, leukocytic ifn-α, ifn-β and the hiv protease inhibitors (especially nelfinavir) are consistently active in vitro and should be considered for animal studies and randomized placebocontrolled clinical trials. type 1 interferons render uninfected cells refractory to sars-cov replication through a mxa-independent mechanism 112 , whereas the hiv protease inhibitors may block the activity of the main sars-cov proteinase 113 . so far, only interferons have been tested in animal models: in cynomolgous macaques, pegylated ifn-αn2 provided prophylaxis but was only marginally effective for early treatment 114 . no randomized placebo-controlled trials have been performed for any of these antiviral drugs, although treatment studies using historical controls have suggested clinical benefit from ifn-α (infacon-1) 115 and the combination of a protease inhibitor with ribavirin 61 . the rapidity with which the sars-cov genome was sequenced, the determination of the structure of potential drug targets 116 and the prediction of functional properties of sars-cov proteins based on prior knowledge of homologs from other coronaviruses 117 have allowed identification of potential new drug targets. peptides derived from the heptad-repeat-2 region of the spike protein have been shown to block virus infection, albeit at much higher molar concentrations than similar inhibitors needed to prevent hiv entry 78, 79 . short interfering rnas also seems to be effective in decreasing viral replication in cell lines [118] [119] [120] , but this remains an experimental strategy rather than one immediately amenable to clinical application. screening of combinatorial chemical libraries has identified inhibitors of sars protease, helicase and spike-protein-mediated cell entry 121 . for successful treatment of influenza, antiviral drugs must be administered within 48 hours of disease onset to obtain substantial clinical effect. but because the sars-cov load increases until day 10 of illness 47 , and in light of the correlation of high viral load in the second week of illness with adverse outcome 60 , the window of opportunity for antiviral therapy may be wider. much scientific effort has been focused on developing a vaccine to protect against future outbreaks of sars-cov. the commercial viability of developing a vaccine for sars-cov will ultimately depend on whether the virus re-emerges in the near future. as discussed above, it is unlikely that future outbreaks will reach global proportions, but nevertheless, vaccines or passive immunization would be relevant in the context of protecting high-risk individuals such as laboratory and health-care workers. a vaccine could also be considered in the setting of the farmed-game-animal trade, if farming of civets for human consumption continues. in the short time since the virus was identified, substantial progress has been made toward developing a vaccine. immunodominant b-and t-cell epitopes of sars-cov are being defined [122] [123] [124] . natural human infection with sars-cov leads to a long-lived neutralizing antibody response and immune sera crossneutralize diverse human sars-cov 125 , suggesting that active immu(in the press) (103) nization against sars may be a feasible proposition. but so far there has been no known instance of human re-exposure to sars-cov to confirm that the naturally acquired immune response confers protection from reinfection. when sars-cov spike, envelope, membrane and nucleocapsid proteins were individually expressed in an attenuated parainfluenza type 3 vector, only the recombinants expressing the spike protein induced neutralizing antibody and protected from challenge in hamsters 102 ( table 2) . mucosal immunization of african green monkeys with this parainfluenza-spike protein chimeric virus led to neutralizing antibody and protection from viral replication in the upper and lower respiratory tracts after challenge with live sars-cov 100 , and spike protein-encoding dna vaccines stimulated neutralizing antibody production and protection from live-virus challenge in mice 126 . these studies confirm the assumption that the spike protein is the dominant protective antigen for sars. experiments using adoptive transfer and t-cell depletion showed that humoral immunity alone can confer protection 126 . other vaccine strategies have included the use of naked dna 127-129 , adenoviral vectors 130 or modified vaccinia (ankara) 131 and inactivated whole virus 132, 133 . many investigators have optimized the codon usage of the gene target to improve expression. in summary, all vaccines based on the spike protein seem to induce neutralizing antibody responses, and those carrying nucleoprotein can induce nucleoprotein-specific cellmediated immunity. but thus far only four studies have used live sars-cov to challenge immunized animals ( table 2 ). an inactivated vaccine with alum adjuvant, which induces neutralizing antibody in mice, is entering phase 1 human clinical trials in china 13 . experience with coronavirus vaccines for animals is relevant for sars vaccine development 134 . one problem facing animal coronavirus vaccines has been strain variation among field isolates, leading to variable vaccine efficacy. a further concern is the experience with feline infectious peritonitis coronavirus, in which prior immunization led to enhanced disease rather than protection 135 . in the case of sars-cov, neither vaccination nor passive transfer of antibody has yet been reported to lead to disease enhancement, but challenge with live sars-cov has occurred soon after immunization. whether waning immunity or low titers of antibody lead to sars disease enhancement remains unclear; the recent suggestion that immunized ferrets became more ill after challenge clearly needs to be confirmed or refuted 13 . passive transfer of immune serum protects naive mice from sars-cov infection 101 , and hyperimmune globulin with sufficient neutralizing activity for use in humans could be prepared from pooled convalescent human plasma or from horses immunized with inactivated vaccine. alternatively, monoclonal antibodies with sufficient neutralizing antibody activity have been developed by screening phage-display antibody libraries and by immortalizing b-cell repertoires of convalescent sars individuals with epstein-barr virus ( table 3) [136] [137] [138] . one of these (80r) blocks the virus-ace-2 receptor interaction through binding to the spike protein s1 domain 136 . passive immunization of ferrets and mice was effective in suppressing viral replication in lungs, but less so in the nasopharynx 137, 138 . no randomized placebo control trial evaluated antibody therapy for pre-or post-exposure prophylaxis in at-risk groups during the sars outbreak. retrospective analysis of outcome in a limited human study using human sars convalescent plasma suggested that passive immunization had no obvious adverse effects 139 . the antigenic diversity of sars-cov-like precursor viruses in the wild-animal reservoir is undefined. in the event of a new interspecies transmission event prompting another sars outbreak, the crossprotection afforded by current vaccine constructs based on the human sars-cov of 2003 is unknown and is likely to influence the efficacy of both passive and active immunization strategies. sars provided a painful reminder of the global impact of emerging infectious diseases. it illustrated how microbes, with their evolutionary drive to preserve and propagate their genes, exploit new opportunities and niches created by modern society 140 . interspecies transmission of viruses to humans clearly has occurred throughout human history. but recent developments allowed sars-cov increased opportunity to adapt to human-to-human transmission and, subsequently, to spread globally. in particular, large centralized wet markets and hospitals proved to be venues for amplification of transmission to humans, and the burgeoning increase of international travel (currently ∼700 million travelers annually) exploded the local outbreak of an emerging infection into a potential pandemic. because most recent emerging infectious disease threats have a zoonotic origin, we need to better understand the microbial ecology of livestock and wildlife. in the context of increased attention and research funding directed at preparedness to combat bioterrorism threats, it is relevant to note that nature remains the greatest 'bioterrorist.' although microbes that cause commercially important diseases in livestock are well studied, organisms that pose threats to human health are not necessarily ones known to cause disease in livestock, or for that matter, in wildlife. nipah virus, hendra virus and sars-cov all have a wildlife reservoir. furthermore, at present there is concern over the possible role played by wild birds and ducks in the maintenance and spread of avian influenza a (h5n1) in asia 141 . greater understanding of the viral ecology of apparently healthy domestic animals and wildlife is therefore important. for example, the attention on ecological studies arising from the nipah virus and sars outbreaks have already led to the identification of a number of new viruses, including tioman, menangle, australian bat lyssavirus and a novel group 1 coronavirus 142, 143 . some of these are now known to be associated with human or livestock disease. but prioritizing such research efforts and assessing the public health relevance-if any-of such findings, poses challenges. three incidents of laboratory-acquired sars have arisen from biohazard level 3 and 4 laboratories, with community transmission arising from one (world health organization, http://www.wpro.who.int/ sars/docs/update/update_07022004.asp). these incidents were associated with lapses in biohazard level 3 and 4 practices. sars-cov can be safely handled in biohazard level 3 laboratories provided that biohazard level 3 practices are rigorously complied with. but as hospital health-care workers learned to their cost, sars-cov is an unforgiving virus; one lapse may be one too many, and it is irrelevant whether the lapse occurs in a biohazard level 3 or 4 laboratory. despite the impressive speed of scientific understanding of the disease, the global success in containing sars owed much to traditional public health methods of clinical case identification, contact investigation, infection control at healthcare facilities, patient isolation and community containment (that is, quarantine) 25 . but the application of such measures in modern society during the control of sars highlighted several ethical and medical dilemmas, many of which arose from the need to balance individual freedoms against the common good 144, 145 . sars signaled a paradigm shift in international public health. it highlighted the need for rapid information exchange regarding unusual infectious disease outbreaks and the possibility of 146 , and the need for 147 , a coordinated global response to emerging infectious disease threats. during the early stages of the outbreak, the who acted independently, issuing travel alerts and geographically specific travel advisories, without the express consent of the countries affected. the need for such measures was acknowledged post hoc by member states at the s94 volume 10 | number 12 | december 2004 nature medicine supplement world health assembly meeting in may 2003 where the who was formally empowered to take such actions, as necessary, in the future. although future emerging pandemics (e.g., influenza because of its transmissibility during early illness) may not be quelled through similar measures, the success of containing sars remains a triumph for global public health. an influenza epicentre? update: outbreak of severe acute respiratory syndrome-worldwide a cluster of cases of severe acute respiratory syndrome in hong kong a major outbreak of severe acute respiratory syndrome in hong kong identification of severe acute respiratory syndrome in canada coronavirus as a possible cause of severe acute respiratory syndrome a novel coronavirus associated with severe acute respiratory syndrome identification of a novel coronavirus in patients with severe acute respiratory syndrome rapid diagnosis of a coronavirus associated with severe acute respiratory syndrome (sars) the genome sequence of the sars associated coronavirus characterization of a novel coronavirus associated with severe acute respiratory syndrome one year after outbreak. sars virus yields some secrets the severe acute respiratory syndrome severe acute respiratory syndrome severe acute respiratory syndrome: clinical features, diagnosis and management severe acute respiratory syndrome: identification of the aetiologic agent sars coronavirus: a new challenge for prevention and therapy potential for antiviral treatment of severe acute respiratory syndrome sars-beginning to understand a new virus the aetiology, origins and diagnosis of sars epidemiology, transmission dynamics, and control of sars. the 2002-2003 epidemic severe acute respiratory syndrome: an update transmission and control of severe acute respiratory syndrome planning for epidemics-the lessons of sars epidemiology and cause of severe acute respiratory syndrome (sars) in guangdong, people's republic of china epidemiologic clues to sars origin in china our strategies for fighting severe acute respiratory syndrome (sars) isolation and characterization of viruses related to sars coronavirus from animals in southern china prevalence of igg antibody to sarsassociated coronavirus in animal traders crossing the species barrier-one small step to man, one giant leap to mankind unique and conserved features of genomes and proteome of sars-coronavirus, an early split-off from the coronavirus group 2 lineage severe acute respiratory syndrome coronavirus phylogeny: toward consensus the chinese sars molecular epidemiology consortium. molecular evolution of the sars coronavirus during the course of the sars epidemic in china molecular epidemiology of sars coronavirus in hong kong comparative full-length genome sequence analysis of 14 sars coronavirus isolates and common mutations associated with putative origins of infection characterization of severe acute respiratory syndrome coronavirus genomes in taiwan: molecular epidemiology and genome evolution management and prevention of sars in china laboratory diagnosis of four recent sporadic cases of communityacquired sars cluster of sars among medical students exposed to single patient effectiveness of precautions against droplets and contact in prevention of nosocomial transmission of severe acute respiratory syndrome (sars) the severe acute respiratory syndrome coronavirus in tears sars cov antibody prevalence in all hong kong patient contacts transmission dynamics and control of severe acute respiratory syndrome secondary household transmission of sars super-spreading sars events in beijing clinical progression and viral load in a community outbreak of coronavirus-associated sars pneumonia: a prospective study detection of sars coronavirus (scov) by rt-pcr, culture, and serology in patients with acute respiratory syndrome (sars) sars-coronavirus replicates in mononuclear cells of peripheral blood (pbmcs) from sars patients serial analysis of the plasma concentration of sars coronavirus rna in pediatric patients with severe acute respiratory syndrome haematological manifestations in patients with severe acute respiratory syndrome: retrospective analysis significant changes of peripheral t lymphocyte subsets in patients with severe acute respiratory syndrome severe acute respiratory syndrome in children clinical presentation and outcome of severe acute respiratory syndrome in children evaluation of advanced reverse transcription pcr assays and an alternative pcr target region for detection of severe acute respiratory syndromeassociated coronavirus viral shedding patterns of coronavirus in patients with probable severe acute respiratory syndrome performance of virus isolation and directigen flu a to detect influenza a virus in experimental human infection quantitation analysis and prognostic implication of sars coronavirus rna in the plasma and serum of patients with severe acute respiratory syndrome viral loads in clinical specimens and sars manifestations the role of lopinavir/ritonavir in treatment of sars: initial virological and clinical findings severe acute respiratory syndrome-associated coronavirus in lung tissue lung pathology of fatal severe acute respiratory syndrome lung pathology of severe acute respiratory syndrome (sars): a study of 8 autopsy cases from singapore detection of severe acute respiratory syndrome-associated coronavirus in pneumocytes of the lung tissue and cellular tropism of the coronavirus associated with severe acute respiratory syndrome: an in situ hybridization study of fatal cases sars coronavirus-infected cells in lung detected by new in situ hybridization technique analysis of deaths during the severe acute respiratory syndrome (sars) epidemic in singapore organ distribution of severe acute respiratory syndrome (sars) associated coronavirus (sars cov) in sars patients: implications for pathogenesis and virus transmission pathways exploring the pathogenesis of severe acute respiratory syndrome (sars): the tissue distribution of the coronavirus (sars cov) and its putative receptor, angiotensin-converting-enzyme 2 (ace-2) enteric involvement of severe acute respiratory syndrome-associated coronavirus infection persistent infection of sars coronavirus in colonic cells in vitro infection of cultured intestinal epithelial cells with severe acute respiratory syndrome coronavirus ph-dependent entry of severe acute respiratory syndrome coronavirus is mediated by the spike glycoprotein and enhanced by dendritic cell transfer through dc sign characterization of severe acute respiratory syndrome-associated coronavirus (sars cov) spike glycoprotein-mediated viral entry susceptibility to sars coronavirus s protein-derived infection correlates with expression of angiotensin converting enzyme 2 and infection can be blocked by soluble receptor development of a safe neutralization assay for sars cov and characterization of s-glycoprotein severe acute respiratory syndrome associated coronavirus (sars cov) infection inhibition using spike protein heptad repeat-derived peptides interaction between heptad repeat 1 and 2 regions in spike protein of sars-associated coronavirus: implications for virus fusogenic mechnism and identification of fusion inhibitors the life cycle of sars coronavirus in vero e6 cells angiotensin-converting enzyme 2 is a functional receptor for the sars coronavirus expression cloning of functional receptor used by sars coronavirus amino acids 270 to 510 of the severe acute respiratory syndrome spike protein are required for interaction with receptor coronaviridae and their replication tissue distribution of ace-2 protein, the functional receptor for sars coronavirus. a first step in understanding sars pathogenesis reverse genetics with a full-length infectious cdna of severe acute respiratory syndrome coronavirus plasma inflammatory cytokines and chemokines in severe acute respiratory syndrome analysis of serum cytokines in patients with severe acute respiratory syndrome inflammatory cytokine profile in children with severe acute respiratory syndrome altered p38 mitogen-activated protein kinase expression in different leukocytes with increment of immunosuppressive mediators in patients with severe acute respiratory syndrome re-emergence of fatal human influenza a subtype h5n1 disease prolonged disturbance of in vitro cytokine production inpatients with severe acute respiratory syndrome (sars) treated with ribavirin and steroids what does the peripheral blood tell you in sars? association of hla class 1 with severe acute respiratory syndrome coronavirus infection association of human-leukocyte-antigen class 1 (b*0703) and class ii (drb1*0301) genotypes with susceptibility and resistance to the development of severe acute respiratory syndrome koch's postulates fulfilled for sars virus newly discovered coronavirus as the primary cause of severe acute respiratory syndrome macaque model for severe acute respiratory syndrome virology: sars virus infection of cats and ferrets mucosal immunisation of african green monkeys (cercopithicus aethiops) with an attenuated parainfluenza virus expressing the sars coronavirus spike protein for the prevention of sars prior infection and passive transfer of neutralizing antibody prevent replication of severe acute respiratory syndrome coronavirus in the respiratory tract of mice contributions of the structural proteins of severe acute respiratory syndrome coronavirus to protective immunity sars coronavirus infection in golden syrian hamsters small molecules targeting severe acute respiratory syndrome human coronavirus inhibition of severe acute respiratory syndrome coronavirus replication by niclosamide in vitro susceptibility of 10 clinical isolates of sars coronavirus to selected antiviral compounds small molecules blocking the entry of severe acute respiratory syndrome coronavirus into host cells hiv protease inhibitor nelfinavir inhibits replication of sarsassociated coronavirus inhibition of sars coronavirus infection in vitro with clinically approved antiviral drugs interferon-β 1a and sars coronavirus replication severe acute respiratory syndrome-related coronavirus is inhibited by interferon-α the antiviral effect of interferon-β against sars coronavirus is not mediated by mxa protein old drugs as lead compounds for a new disease? binding analysis of sars coronavirus main proteinase with hiv, psychotic, and parasitic drugs pegylated interferon-α protects type 1 pneumocytes against sars coronavirus infection in macaques interferon alfacon-1 plus corticosteroids in severe acute respiratory syndrome: a preliminary study coronavirus main protease (3clpro) structure: basis for design of antiviral drugs multiple enzymatic activities associated with severe acute respiratory syndrome coronavirus helicase inhibition of sars-associated coronavirus infection and replication by rna interference inhibition of severe acute respiratory syndrome virus replication by small interfering rna in mammalian cells silencing sars cov spike protein expression in cultured cells by rna interference identification of novel small molecule inhibitors of severe acute respiratory syndrome associated coronavirus by chemical genetics immunological characterization of the spike protein of the severe acute respiratory syndrome coronavirus identification of an antigenic determinant on the s2 domain of the severe acute respiratory syndrome coronavirus spike protein capable of inducing neutralizing antibodies t cell epitopes in severe acute respiratory syndrome (sars) coronavirus spike protein elicit a specific t-cell immune response in patients who recover from sars neutralizing antibodies in patients with severe acute respiratory syndrome-associated coronavirus infection a dna vaccine induces sars coronavirus neutralization and protective immunity in mice generation and characterization of dna vaccines targeting the nucleocapsid protein of severe acute respiratory syndrome coronavirus characterization of humoral responses in mice immunized with plasmid dnas encoding sars-cov spike gene fragments induction of sars-nucleoprotein-specific immune response by use of dna vaccine effects of a sars-associated coronavirus vaccine in monkeys severe acute respiratory syndrome coronavirus spike protein expressed by attenuated vaccinia virus protectively immunizes mice a subcutaneously injected uv-inactivated sars coronavirus vaccine elicits systemic humoral immunity in mice inactivated sars-cov vaccine prepared from whole virus induces a high level of neutralizing antibodies in balb/c mice severe acute respiratory syndrome vaccine development: experiences of vaccination against avian infectious bronchitis coronavirus a review of feline infectious peritonitis virus: molecular biology, immunopathogenesis, clinical aspects, and vaccination potent neutralization of severe acute respiratory syndrome (sars) coronavirus by a human mab to s1 protein that blocks receptor association human monoclonal antibody as prophylaxis for sars coronavirus infection in ferrets an efficient method to make human monoclonal antibodies from memory b cells: potent neutralization of sars coronavirus retrospective comparison of convalescent plasma with continuing high dose methyl prednisolone treatment in sars patients microbial threats to health: emergence, detection and response genesis of a highly pathogenic and potentially pandemic h5n1 influenza virus in eastern asia managing emerging diseases borne by fruit bats (flying foxes), with particular reference to henipaviruses and australian bat lyssavirus identification of a novel coronavirus in bats ethics and sars: lessons from toronto informed consent and public health world health organization. a multicentre collaboration to investigate the cause of severe acute respiratory syndrome germs, governance, and global public health in the wake of sars the authors declare that they have no competing financial interests. key: cord-253279-8j7esl8j authors: wasserman, richard l.; greener, benjamin n.; mond, james title: ri-002, an intravenous immunoglobulin containing high titer neutralizing antibody to rsv and other respiratory viruses for use in primary immunodeficiency disease and other immune compromised populations date: 2017-10-16 journal: expert rev clin immunol doi: 10.1080/1744666x.2017.1389647 sha: doc_id: 253279 cord_uid: 8j7esl8j introduction: novel immune globulin (ig) products (ri-002, ri-001) have been designed to provide protection against respiratory syncytial virus (rsv) mediated respiratory illness while at the same time meeting the manufacturing requirements established by fda for antibody supplementation in immunocompromised subjects. areas covered: this review covers the manufacture and development of both ri-001 and ri-002, including the selection of plasma donors for ig preparation with high-titers of anti-rsv antibody, in vitro, and preclinical data in the cotton rat model s. hispidus, and clinical trials including phase ii and compassionate use studies of ri-001 and a multi-center, pivotal phase iii study of ri-002 in pidd patients. expert commentary: the data demonstrate that ri-002 is efficacious in the prevention and treatment of rsv in preclinical normal and immune suppressed animal models and is safe and efficacious in the treatment of patients with various forms of primary immunodeficiency disease (pidd). this product offers potential advantages over other available ig’s for prophylaxis in immunocompromised patients requiring polyclonal immunoglobulin supplementation because of its unique antibody composition. in addition to its enhanced neutralizing anti-rsv activity and its polyclonal ig composition, there is preclinical data to support the use of ri-002 for humoral protection against other respiratory pathogens. supplementation with immunoglobulins (ig) has been considered the standard of care for the treatment of patients with a variety of antibody production disorders due to b cell or b and t cell abnormalities, collectively referred to as primary immunodeficiency diseases (pidds) [1] . while regular infusions with ig prevent the vast majority of serious bacterial infections within this population, there is no single prophylactic regimen that provides protection against the wide range of viral pathogens to which these patients are susceptible [2] [3] [4] [5] [6] [7] [8] . in the usa, there are several commercially available ig products that meet minimum standards of concentrations of antibodies to diphtheria toxoid (≥1.21 u/ml), measles (≥0.60 cber reference), and polio (≥0.28 cber reference) viruses in conformance with the us fda guidance 21cfr 640 subpart j [9] . although the fda defines minimum concentrations of these antibodies, standards for antibodies against pathogens that are the most important to pidd patients are not required. titers of these antibodies in commercial polyclonal ig vary widely [10, 11] . while the optimal dose of therapeutic polyclonal intravenous ig appears to be specific to the individual patient [4, 12] , increasing ig dosage is associated with better outcomes and a decrease in the incidence of infection [3, 13] . even with appropriate ig therapy, pidd patients remain at an increased risk of infection, including those caused by upper and lower respiratory viral pathogens including rsv and influenza. pidd patients and patients with secondary immunodeficiencies (e.g. transplantation, cancer, etc.) are at high risk for respiratory syncytial virus (rsv) infections that often result in significant morbidity and mortality from bronchiolitis and pneumonia [3, 14] . at present, the only prophylactic treatment available for rsv infection is palivizumab (synagis ® , medimmune, gaithersburg, md), an anti-rsv monoclonal antibody that is used to confer seasonal passive immunity and has been shown to be effective in preventing rsv-related hospitalizations, particularly in low birthweight infants [15] [16] [17] . the american academy of pediatrics (aap) recently revised its recommendation for use guidelines and available evidence suggest that use of this monoclonal antibody for rsv should be reserved only for patients born prematurely under 29 weeks gestation and under 1 year of age. there currently is no approved, recommended treatment for older age groups for the prevention of rsv disease. although treatment of established disease is primarily supportive care, ribavirin (virazole ® ) is the only fda-approved treatment for rsv pulmonary infection in pediatric patients. presently, the efficacy of ribavirin in rsv treatment is controversial and there is the potential for significant side effects in both patients and caregivers who administer it [18] [19] [20] [21] [22] [23] . because ribavirin is costly and must be administered in a hospital-based setting adding to the costs in the context of uncertain efficacy, an alternative therapy is needed. a polyclonal rsv-ivig product (respigam ® , medimmune inc., gaithersburg, md) enriched for antibodies against rsv derived from plasma donors with high-titers of anti-rsv antibodies was previously available and approved for the same indication as palivizumab, but was voluntarily withdrawn from the us market following approval of palivizumab. ri-002 was developed to meet an unmet medical need to provide both rsv protection and simultaneously supplement humoral immunity in patients with pidds or other immune compromised states. respigam was marketed from 1996 to 2003 for prevention of rsv disease in infants and children younger than 24 months with chronic lung disease or a history of premature birth [24] . this hyperimmune globulin product was derived from a plasma pool of only a few hundred donors who had high neutralizing titers of anti-rsv antibody. the product was not developed for, nor was ever intended to be used in pidd and as such, its manufacturing specifications did not adhere to fda standards for collection of plasma from at least one thousand donors and was not tested for standard ig potency requirements. the product was never evaluated for efficacy in pidd populations. several randomized controlled trials demonstrated the efficacy of this rsv-ivig product in preventing rsv infection. in a study of 249 infants and children with bronchopulmonary dysplasia due to congenital heart disease, or prematurity, 750 mg/kg of respigam reduced the incidence of lower respiratory tract infections (7 vs. 20 in the control group, p = 0.01), hospitalizations, hospital days, and days in intensive care [25] . in the prevent study, a randomized, double-blind, placebo-controlled, multicenter study conducted during the 1994-1995 rsv season, administration of 750 mg/ kg respigam every 30 days reduced the incidence of hospitalizations due to rsv infection by 41% compared to placebo in 510 children with bronchopulmonary dysplasia and/or a history of prematurity [26] . in addition, administration of respigam in the prevent study also reduced hospitalization for respiratory illness of any cause by 38% suggesting a protective effect for other, non-rsv-related respiratory tract infections. additionally, a post hoc analysis of 109 of the 249 children in the groothuis study demonstrated a statistically significant decrease in episodes of acute otitis media compared to the placebo group (0 vs. 5, p = 0.047) following immunoprophylaxis with 750 mg/kg of respigam, suggesting that the protective effects of rsv enriched hyperimmune globulins may not be limited to rsv alone [27] . when respigam was commercially available, the aap recommendations stated that, 'palivizumab or rsv-igiv immunoprophylaxis has not been evaluated in randomized trials in immunocompromised children. although specific recommendations for immunocompromised patients cannot be made, children with severe immunodeficiencies (e.g. severe combined immunodeficiency or severe acquired immunodeficiency syndrome) may benefit from immunoprophylaxis. if these infants and children are receiving standard immune globulin intravenous (igiv) monthly, physicians may consider substituting rsv-igiv during the rsv season' [28] . this recommendation and subsequent clinical practice failed to consider that respigam did not meet 21cfr640 subpart j requirements that are intended to ensure appropriate immunoprophylaxis in this high-risk population. while the product was available, there were numerous published reports regarding the successful use of respigam for treatment and prevention of rsv in various at-risk immunocompromised patient populations [25, 27, [29] [30] [31] . there have also been reports that the efficacy of combination therapy of ribavirin + rsv-ivig may lead to better outcomes than either product used alone [32] . respigam was voluntarily withdrawn from the market in 2004, at least in part because the newly approved anti-rsv monoclonal antibody, palivizumab, did not require iv access. the discontinuation of respigam left a void in the options for rsv immunoprophylaxis, particularly for simultaneous anti-rsv activity and ig supplementation. ri-001 was developed to address the therapeutic void following respigam discontinuation. ri-001 is an aqueous intravenous immune globulin preparation manufactured from the pooled plasma of healthy adult donors with high titers of neutralizing anti-rsv antibodies (>1.5 times the mean of commercial ivig) as measured by a microneutralization assay. product activity between respigam and ri-001 was shown to be comparable both in the cotton rat model and in vitro testing [unpublished observations]. it is prepared using a process comparable to respigam employing cohn oncley cold-alcohol fractionation with a solvent/detergent treatment viral reduction step to eliminate enveloped viruses. an additional virus nano-filtration step to increase product safety and further reduce the probability of transmission of viral and prion contaminants (not part of the original respigam manufacturing process) was also included in ri-001 manufacture. ri-001 efficacy was studied in a phase ii randomized, double-blind, placebo controlled trial in immunosuppressed patients with rsv upper respiratory tract infection (urti) confirmed by rt-pcr at the time of enrollment [33] . patients between 2 and 65 years of age who had received a hematopoietic stem cell transplant or solid organ transplant within 2 years of enrollment were treated with study drug. patients were treated concurrently with immunosuppressive treatment but were excluded if they had lower respiratory tract infection (lrti) or required extracorporeal membrane oxygenation, cpap, or other mechanical respiratory or cardiac support. participants were randomly assigned 1:1:1 to each of the 3 treatment arms: ri-001 1500 mg/kg, followed by 750 mg/kg 2 days after first administration (high-dose group), ri-001 750 mg/kg followed by 750 mg/kg 2 days after first administration (low-dose group), or placebo. a total of 21 patients (7 patients per arm) with a mean age of 38.04 years (sd 25.03) were enrolled in the usa, canada, australia, and new zealand. the primary objective of this phase ii study was to define the dose that produced a >fourfold rise in anti rsv neutralizing titer at day 18 compared to baseline. in the lowdose group, the mean fold change was a 4.85 increase with 3/ 7 (43%) patients achieving a rise in titer ≥fourfold. a mean fold change of 1.42 was observed in the placebo group. the highdose group met the primary end point, with a mean fold change in circulating titer of ri-001 of 9.24 and 6/7 (86%) patients achieving a rise ≥fourfold. ri-001 was well tolerated with no study drug-related saes and a low incidence of aes. the most frequently reported aes in the active or placebo groups were cough, nausea, pyrexia, sinus congestion, diarrhea, platelet count decrease, and back pain. there was not a significant difference in the ae rate between the two groups. no significant risks associated with vital signs, laboratory assessments, or physical exams were identified. ri-001 has also been evaluated in a nonrandomized, openlabel compassionate use study in the usa, australia, and new zealand that collected data from december 2008 to february 2011 [33, 34] . compassionate use requests were accepted from patients with rsv lrti considered at high risk of mortality who had either failed standard-of-care therapy (e.g. ribivarin, standard ivig, steroids, etc.) or had no alternative therapy available for their infection. patients were diagnosed with rsv via rapid antigen test, rt-pcr, dfa, ifa, or viral culture. all patients received aerosolized ribavirin and some patients received infusions of ivig, palivizumab, or steroids. data were collected from 15 patients who received compassionate use (5 in australia, 9 in the usa, and 1 in new zealand). they ranged in age from 2 months old to 71 years with a median age of 15 years. 53.3% of patients were less than <18 years of age. nine patients were male and six were female. ri-001 was administered at a dose of 1500 mg/kg followed by an additional dose of 750 mg/kg after 2 days. all patients received at least 2 doses of ri-001. 11 patients (73.3%) survived and were discharged from the hospital, while four patients (26.7%) died. no deaths were related to administration of ri-001. causes of death included respiratory failure attributed to rsv infection, rsv pneumonitis and acute respiratory distress, asystole following chest tube placement after development of left pneumothorax and hypoxic respiratory failure following withdrawal of care in a 71-year-old female with a dual infection of rsv complicated by acute influenza. infusion with ri-001 was generally well tolerated in the compassionate use experience with three infusion reactions noted that resolved upon completion of the infusion or after decreasing the infusion rate. two patients experienced nonserious aes of respiratory distress and there was a single nonserious ae of herpes simplex reactivation. all three aes were considered to be not related or unlikely to be related to the ri-001. four saes were documented including 3 of the deaths that were previously discussed and a single incident of dyspnea that resolved prior to discharge and was considered by the investigator to be not related to ri-001. the data collected from the ri-001 trials is limited and represents a small sample size with heterogeneous demographics and disease characteristics. although inconclusive due to limited data, several trends were observed in the compassionate use study. possible seasonal variability was observed with all deaths occurring within the usa during the 2009-2010 and 2010-2011 rsv seasons. no apparent gender bias was observed, nor did survival appear to be impacted by underlying condition including oncological status. survival appeared to be influenced by age with higher mortality in the youngest and oldest patients, consistent with the potential for reduced immune response within these populations, as well as their generally more fragile health. importantly, there was a notable effect of early drug intervention on survival. first, patients that did not require ventilator support all survived (10 patients), while only one of five of those who required ventilator support survived. further, the adult (≥18 years old) survivors were treated with ri-001, on average, 13.4 days after the onset of respiratory symptoms while the nonsurvivors were not treated until 29 days after symptom onset. a similar trend was observed in pediatric patients (<18 years old) with survivors receiving ri-001 treatment earlier (19.8 days) than non-survivors (39.5 days) . similarly, the time from positive rsv test to first treatment with ri-001 was also shorter in survivors (adults, 3.6 days; children, 4.7 days) versus non-survivors (adults, 12.5 days; children, 8.5 days). of note, ri-001 was associated with significant increases in rsv serum neutralizing antibody titer in a subset of 7 immunocompromised adults with good clinical outcomes that were enrolled in the compassionate use study [35] . these subjects experienced a 3-151-fold rise in anti-rsv antibody titers compared to baseline for up to 30 days posttreatment. taken together, these data suggest that early intervention with ri-001 is likely to be important in improving survival. trends observed in this compassionate use study are consistent with a previous report as factors associated with a more severe clinical course and increased mortality in rsv disease [36] . although ri-001 was considered a promising candidate to replace respigam in patients with rsv infection requiring immune supplementation, it was subject to the same limitations as respigam in pidd and other patients who derive benefits from ig use. ri-002 (adma biologics, inc.) was developed to address the inconsistency in neutralizing anti-rsv titers found in commercial lots of ig while simultaneously meeting the fda guidance requirements for treatment of pidd patients defined in 21cfr640 subpart j. in addition to standardization with respect to minimum concentrations of antibodies required in the fda guidance, ri-002 is manufactured according to a patented process to yield consistent and predictable levels of neutralizing anti-rsv antibodies obtained from a plasma pool of donors screened via a validated microneutralization assay for rsv a/2 and found to be hyperimmune to rsv [unpublished observations]. the principal difference between ri-001 and ri-002 is that ri-001 was derived from a pool of plasma donors all of whom had elevated titers to rsv. ri-002 is derived from pooling plasma from high-titer donors with plasma from normal source donors to meet the fda guidance stating that for treatment of patients with pidd, the plasma pool needs to be derived from a minimum of 1000 unique donors. a study was conducted to compare rsv neutralizing antibody titers in multiple lots of ri-002 to titers from a single available lot of ri-001. the overall average ratio of ri-002 to ri-001 titer was 1.1 ± 0.2 with individual lots ranging from 0.7 to 1.5. overall, anti-rsv titers observed in the study were considered to be equivalent between both ig products [unpublished observations]. the cotton rat (sigmodon hispidus) model of rsv disease was selected for preclinical evaluation of ri-002 as it is a widely used animal model that is accepted as an appropriate surrogate to predict efficacy of anti-rsv interventions as well as interventions for other respiratory pathogens including parainfluenza, metapneumovirus, and influenza [37] [38] [39] . importantly, rsv infection in the s. hispidus model closely mirrors the course of natural infection in humans with respect to immunological response, time course, pulmonary inflammatory component, and clinical symptoms [36] . further, s. hispidus exhibits susceptibility to infection across its lifespan and has been used to successfully model rsv infection in young, elderly, and immunosuppressed populations [37, [40] [41] [42] [43] . the s. hispidus model has previously demonstrated that anti-rsv antibody titers provided by standard ig prophylaxis are not sufficient to protect against rsv disease, while serum titers between 1:200 and 1:400 that can be provided by hightiter anti-rsv ig provide protection [44, 45] . this finding is consistent with results in human infants that indicate that patients with high titers (1:400) of maternally acquired anti-rsv antibodies were less susceptible to rsv disease than infants with anti-rsv serum antibody titers from 1:100 to 1:200 [46] . this correlation suggests that the model can be used to predict the efficacy of immunoprophylaxis in addition to the dose required for immunoprophylaxis in human infants. both respigam and palivizumab were advanced to clinical trials without the need for intermediate studies in nonhuman primates based on data from studies in s. hispidus [38, 39] . to establish the relationship between protective titers in the cotton rat and in vitro microneutralization assays, human ivig with elevated anti-rsv titers has been tested in animals with a strong correlation observed between neutralizing antibody titers and protection seen in the animal model. human plasma donors were successfully identified as donors containing high-titer neutralizing anti rsv ig using this microneutralization assay [45] . the same assay is currently used for screening donors for the manufacture of ri-002. the antibody composition of ri-002 has been characterized and compared to commercial lots of ig in several in vitro studies. following rsv infection, viral f protein (the target of palivizumab) mediates viral fusion and entry while g protein displays anti-inflammatory effects that dampen the host immune response to infection [47] . as neutralizing antibodies to both viral proteins may contribute to the therapeutic efficacy of ri-002, microneutralization assays were conducted to quantify anti-g protein and anti-f protein titers in 3 lots of ri-002 and 9 lots of commercial ig [unpublished observations]. enhanced levels of antibody to f protein and g protein (1.5-fold or greater compared to commercial ivig lots) were observed in ri-002 lots compared to commercial ig, suggesting that selection of donors based on high anti-rsv titers in microneutralization assays enhances anti-rsv antibodies for multiple rsv surface proteins ( table 1) . as previously reported, respigam administration in the prevent study decreased the incidence of hospitalization for lower respiratory illness of any cause [26] . although the donor pool for ri-002 is only screened for high anti-rsv antibody titers, enriching for rsv antibodies may be correlated with increases in antibody titers to other respiratory pathogens. orange et al. performed elisa to evaluate virus-specific igg titers for 9 respiratory viruses in ri-002 produced from 3 plasma pools and 10 commercially available ivig lots representing 7 brands (table 2 ) [11] . respiratory viruses tested include influenza a and b, rsv, parainfluenza virus serotypes 1, 2, and 3, hmpv, and coronavirus 229e and oc43. aggregate mean titers across all viruses for ri-002 were 1.5-fold higher than those observed in commercial ig. the difference in titers for all viruses excluding hmpv achieved statistical significance with geometric mean ratios of ri-002/commercial ivig ranging from 1.3 to 1.9. titers to respiratory viruses were consistent across ri-002 lots for all viruses tested but varied widely among ivig products. a direct correlation was observed between rsv titers and titers to the other tested respiratory viruses with pearson linear correlation coefficients ranging 0.29-0.67 log 2 scale (p < 0.05). this positive correlation suggests that elevated titers to respiratory viruses may be a consistent feature of each lot of ri-002. the protective efficacy of these elevated titers against respiratory pathogens other than rsv remains to be determined. while there is no data to support a hypothesis that underlies the enhanced antibody titers to other non-rsv viruses, there are a number of possibilities to consider. it is possible that the donors who have elevated titers to rsv may have been living in an area or workplace where they were exposed over long periods of time to many other respiratory viruses and thus have elevated titers to multiple respiratory viruses. it may also be possible that those individuals with high-titer neutralizing antibody to rsv have an immune response gene that encodes for elevated antibody responses to respiratory viruses where immune response is regulated by a common histocompatibility locus. ri-002 has also undergone extensive in vitro and preclinical testing in the cotton rat model and a summary of these studies is provided in tables 3 and 4 . first, a prevention study was performed in this model with either saline, ri-002 500 mg/kg, ri-002 750 mg/kg, ri-002 1000 mg/kg, or respigam administered intraperitoneally to five animals in each of the groups (day 1) as prophylaxis [50] . one day following dosing, animals were challenged with 10 5 pfu of rsv-a long strain via the intranasal route. serum was collected on day 4 and animals were euthanized for harvesting of lung and nasal tissue. rsv was undetectable in the lungs of animals that had received ri-002 at all doses and in 4/5 animals that received respigam. virus was undetectable in nasal tissue of all animals receiving ri-002 1000 mg/kg, below quantifiable levels in all animals at the other doses, and detectable in 2/5 animals receiving respigam. anti-rsv microneutralization assays established that sera from all groups reached neutralizing titers that would be considered protective in humans (10log 2 ) through day 4 [50] , unpublished observations. this experiment established the efficacy of ri-002 in the cotton rat model and showed that the higher dose of 1000 mg/kg is required to sterilize the nasal tissue of animals challenged with rsv as compared to lower doses required for sterilization of pulmonary tissue. further, a dose-response relationship was apparent for both nasal infection and neutralizing titers in s. hispidus sera. because ri-002 is intended to be utilized in an immunocompromised population, cotton rats were treated with cyclophosphamide 50 mg/kg for 18 days resulting in approximately 80% suppression of white cell count and circulating ig compared to untreated animals [50, 52] . following intranasal infection with rsv a-long strain (10 5 pfu/100g body weight), 10 animals in each group were treated with 3 doses (days 1, 4, and 7 ) of either saline, ri-002 1500 mg/kg for all doses, or ri-002 1500 mg/kg followed by 750 mg/kg for subsequent doses. control animals were also infected and received a single dose of either saline, ri-002 1500 mg/kg, or ri-002 750 mg/kg. cyclophosphamide treated animals were euthanized on day 10 and untreated animals were euthanized on day 4. ri-002 was effective in reducing rsv viral titer by about 2-3 logs compared to saline in both the lungs and nasal tissue of treated normal and immunosuppressed animals, with a greater reduction in viral titer observed in the cyclophosphamide treated animals that received the higher dose. rsv gene expression measured by qpcr confirmed systemic dissemination of rsv in lung, liver, and kidney as well as undetectable rsv rna in lungs and diminished rna levels in liver and kidney following treatment with ri-002. in the same study, pulmonary histopathology of immunosuppressed cotton rats treated with high-dose ri-002 was approximately equivalent to that observed in normal uninfected rats unlike the extensive inflammatory infiltration observed in lungs of saline-treated animals ( figure 1 ). rsv neutralizing titers were achieved with all ri-002 dose regimens that would be considered protective in humans. an equivalent prophylaxis study, in which immunosuppressed cotton rats were treated with ri-002 1500 mg/kg or 750 mg/kg 24 h prior to rsv challenge, showed a 3-4 log 10 reduction in rsv viral titers from lung homogenates at both doses demonstrating that a single dose of ri-002 prior to exposure is sufficient to protect cotton rats from infection. ri-002 was also tested against a wild-type rsv/a tracy strain and a palivizumab-resistant rsv/a tracy strain with a singleamino acid mutation at position 262 [51, 52] . since a polyclonal antibody contains thousands of antibodies of different antigenic specificities, the concentration of each specificity can never be as high as that of a monoclonal which is of a single specificity. for this reason, higher doses of a polyclonal antibody would have to be administered compared to a monoclonal antibody to achieve comparable final concentrations of a given specificity. palivizumab 15 mg/kg reduced wild-type rsv/a tracy lung titers by greater than 2 logs but this decrease was not observed with the palivizumab-resistant strain. ri-002 1500 mg/kg reduced lung titers by greater than 2 logs in both wild-type and palivizumab-resistant strains compared to palivizumab (p ≤ 0.00001). an independently validated microneutralization assay established that mean rsv serum neutralizing titers from s. hispidus were increased by 1.5-1.9 log 2 following prophylaxis with ri-002 versus palivizumab. these results were confirmed by a third laboratory that found an approximate 2 log reduction in viral load following prophylaxis with ri-002 1000 mg/kg and elimination of detectable virus with ri-002 1500 mg/kg. in the same study, virus was also eliminated from lung tissue by palivizumab 15 mg/kg ( figure 2 ). reduction of rsv/a long strain viral titer in the nasal tissue of normal cotton rats was also observed with both ri-002 doses and palivizumab at 15 mg/ kg, with the greatest decrease achieved with ri-002 at 1500 mg/kg [38] . ri-002 activity against influenza is supported by in vitro studies demonstrating neutralizing activity against influenza virus and in vivo data in the s. hispidus model showing significant reduction in influenza virus load in the lung of influenza-infected cotton rats. the protective activity of ri-002 against multiple influenza strains has also been investigated in hemagglutination inhibition assays (hai) and in the s hispidus model [49] . it is well established that protective unpublished observations [49] activity against influenza correlates with hai inhibition titers greater than 1:40 [53, 54] . hai inhibition titers from six lots of ri-002 were measured against 10 influenza strains. [49] . lung histology showed a significant reduction in perivasculitis and alveolitis four days after infection in animals treated with ri-002 compared to saline. influenza mrna in the pulmonary tissue of ri-002 pretreated animals was significantly reduced on both day 1 and day 4. further, chemotactic chemokine mrna including ifn-gamma-inducible protein 10 (cxcl10) and rantes (ccl5) were reduced in the pulmonary tissue of ri-002 treated animals suggesting that inflammation inducing infection was prevented or markedly attenuated. an open-label phase clinical trial was conducted to evaluate efficacy, safety, and pharmacokinetics of ri-002 in subjects with pidd [55] . subjects were given intravenous infusions of ri-002 at doses of 300-800 mg/kg every 3-4 weeks based on their prestudy infusion schedule for approximately 1 year. infusion rates started at 0.5 mg/kg/min and were incrementally increased every 15 min up to a maximum of 8 mg/kg/min. dose adjustment to maintain trough igg concentrations >500 mg/dl was permitted throughout the study. the mean dose administered was 505.2 (4.84) mg/kg with a range of 284-1008 mg/kg with 95.8% of infusions administered at the maximum rate. subjects were 3-74 years of age, inclusive, with a confirmed diagnosis of pidd who had been receiving a stable dose of ivig (no change >50% of mean mg/kg dose) every immunosuppressed/ri-002 normal/saline 3-4 weeks for ≥3 months. subjects were excluded for a history of adverse reactions to blood-derived products, selective iga deficiency with ige anti-iga, abnormal liver function, a history of deep vein thrombosis, hemolysis, positive coombs test, or current pregnancy or lactation. 59 subjects (28 males, 31 females) with a mean of 8.66 years since diagnosis and an average age of 41.8 years were enrolled. all subjects who received at least one dose of ri-002 were included in the safety population. demographic data was comparable for the three and four week dosing schedule subjects. the primary efficacy end point of the study was the acute serious bacterial infection (asbi) rate over the 12-month treatment period. consistent with fda recommendations, an asbi rate of less than 1.0 infection per person-year was considered efficacious. over 55.99 subject years, zero asbis were observed. secondary end points included the incidence of all infections of any kind (3.436 infections per subject per year), number of days lost from work/school/usual activity due to infection (1.66 days per subject per year), unscheduled visits to a physician or er due to infections (0.97 visits per subject per year), hospitalizations due to infection (0.018 hospitalizations per subject per year), number of days of antibiotic therapy (32.9 days per subject per year). a summary of clinical efficacy data is provided in table 5 . taken together, these data attest to the efficacy of ri-002 in preventing serious infections and minimizing unscheduled medical visits and hospitalizations in this pidd population. overall, ri-002 was well tolerated and met the target (<0.40) for proportion of study infusions with a temporally associated adverse event (taae, 0.142). overall, 43 (72.9%) subjects experienced a taae in 113/793 (14.2%) infusions. 618 treatment emergent aes were documented in 58 subjects during the study of which 55 teaes in 26 (44.1%) subjects were recorded as related to study drug. the most frequently reported aes unrelated to the study drug, were headache, sinusitis, diarrhea, viral gastroenteritis, nasopharyngitis, and urti with headache being the most frequent. no study drug-related, serious adverse events (saes) were reported although two saes (postoperative wound infection and migraine) were documented. 31 adverse infusion reactions in 18 (30.5%) subjects in 29 (3.7%) infusions were reported with headache and myalgia being most frequent. four infusion site reactions in two subjects were also documented (infusion site extravasation and pain). a summary of clinical safety is presented in table 6 . the relationship between dose, trough level, and risk of serious and nonserious infections as well as total trough igg and specific antibody levels was also investigated. pharmacokinetic sampling was performed in a subset of patients beginning at infusion 7 or 9 to ensure washout of previous ig products. administration of ri-002 for both treatment intervals resulted in increases in specific antibodies to cmv, tetanus, hib, measles, rsv, and s. pneumonia. figure 3 shows the profile of anti-rsv antibodies following infusion that are maintained throughout the dosing interval. as expected, the greatest increase was observed in rsv neutralizing antibodies with a post-infusion mean (95% ci) fold change from baseline (c max /baseline) of 5.47 (4.37, 6.56) overall and fold changes of 4.23 (2.95, 5.51) and 6.79 (5.11, 8.47) for doses <500 mg/kg and >500 mg/kg, respectively. no apparent differences were observed between 3 and 4-week dosing intervals for mean igg concentrations or c max . there was no sex or age effect on igg pharmacokinetics. a biologics license application (bla) for ri-002 was filed with fda's center for biologics evaluation and research (cber) in 2015. adma biologics has also filed united states patent 9,107,906 entitled 'compositions and methods for the treatment of immunodeficiency' [55] . the patent covers adma's method for producing pooled plasma containing elevated rsv neutralizing antibodies and antibodies directed against one or more respiratory pathogens. the process includes assays to screen donors for high rsv neutralizing titers, selection of the highest assayed donors where donors have an ig fraction neutralizing titer of 1800 or above, the pooling of plasma from 1000 or more donors selected in the previous steps, and the cold alcohol fractionation process for purification of the finished product. as previously discussed, the process outlined in this patent ensures consistency from product lot-to-lot, anti-rsv neutralizing titers at least twofold higher than control ig products, and enhanced binding activity against rsv and other respiratory viruses. ri-002, and its predecessor ri-001, are novel immune globulins manufactured using a unique approach that meets specific criteria for plasma donor selection and plasma pool formulation. respigam, the only previous hyperimmune globulin product enriched for anti-rsv activity, is no longer marketed for several reasons including the approval of the monoclonal anti-rsv antibody palivizumab. although it demonstrated efficacy in preventing rsv infection, it had a number of limitations on administration and was not indicated as a sole therapy for immune supplementation in the pidd population. although palivizumab is a current commercially available therapeutic option for patients that require prophylaxis against rsv, a monoclonal antibody, cannot provide immune reconstitution in the pidd population-like polyclonal products, nor does it have the potential to provide protection against other respiratory viruses or palivizumab-resistant strains of rsv which are becoming more prevalent. in addition, polyclonal antibodies have the advantage of providing anti-inflammatory activity that is triggered by rsv while the monoclonal does not [56] . ri-001 and ri-002 were developed by adma biologics to fill the void left by the market withdrawal of respigam. ri-001 was well tolerated in a phase ii trial of immunocompromised patients with rsv urti and achieved the primary efficacy end point, with a mean fold change in circulating rsv neutralized titer ≥fourfold in 6/7 (86%) patients in the high-dose arm [35] . an open-label, compassionate use treatment study was conducted in 15 patients with rsv lrti at high risk of mortality with 73.3% survival [35] . although no conclusions can be drawn due to limits in sample size, several encouraging trends toward improved clinical outcomes were observed. factors associated with survival included age, early intervention, absence of need for ventilator support (10/10 patients), fewer average days from onset of respiratory symptoms to first treatment with ri-001, and shorter time from positive rsv test to first treatment with ri-001. ri-001 was also associated with significant increases in rsv serum neutralizing antibody titer in a subset of 7 immunocompromised adults with favorable clinical outcomes that were enrolled in the compassionate use study [35] . although promising, from an efficacy and safety perspective, ri-001 was subject to the same limitations as respigam and did not meet fda requirements for supplementation in the pidd population [9] . ri-002 is currently under regulatory review and is manufactured using the novel approach of pooling plasma from more than 1,000 donors who exhibit anti-rsv neutralizing antibody titers [57] . in comparison to ri-001 titers [unpublished observations], ri-002 meets fda ig supplementation requirements for minimum titers of antibodies to diphtheria toxoid, measles, and polio viruses in immunocompromised populations including pidd patients [9] . the company also has anti-rsv potency testing manufacturing standards that are performed for each batch of ri-002. although it has not yet been approved, comprehensive evaluation of the preclinical and clinical data collected suggests that ri-002 may provide an important option for patients benefitting from ig supplementation who are at risk of viral-induced respiratory illness. microneutralization assays conducted to quantify igg titers against rsv surface proteins (f protein and g protein) demonstrated that the manufacturing process for ri-002 selects for substantially higher anti-rsv antibody titers against multiple rsv antigens compared to commercially available ig products [11] . further, this enrichment is not limited to rsv but also yields consistently higher (1.5-fold higher aggregate mean) neutralizing titers against a host of other respiratory pathogens including influenza a and b, parainfluenza virus serotypes 1, 2, and 3, hmpv, and coronavirus 229e and oc43 [11] . although prophylactic efficacy in human populations against these additional pathogens has yet to be established, it is probable that ri-002 may impact the incidence of respiratory infections of any cause within immunocompromised populations with a similar trend to that observed in the respigam prevent study [26] . serum from s. hispidus ri-002-treated animals was tested against 10 strains of influenza by hai inhibition assays and was considered protective against 4 prevalent circulating strains, with titers ranging from 1:160 to 1:640 [51] . similar protection was not observed for noncirculating influenza strains, suggesting that the donor selection process used to manufacture ri-002 only enriches humoral antibodies against common respiratory pathogens when donors have been exposed. s. hispidus challenged with influenza/a california 07/2009 (h1n1) one day following prophylactic administration of ri-002 1500 mg/kg had significantly improved lung histology, reduced viral mrna in pulmonary tissue, and reduced chemotactic chemokine (cxcl10 and rantes) mrna compared to saline-treated animals suggesting that the observed titers may be adequate to provide improved protection against circulating influenza strains [49] . in the s. hispidus model, ri-002 administered intraperitoneally at a dose of 500 mg/kg, 750 mg/kg, or 1000 mg/kg prior to challenge prevented rsv disease in healthy young animals. rsv was undetectable in the lungs of animals at all doses and in nasal tissue of all animals receiving 1000 mg/kg with neutralizing titers that would be considered protective in humans. in a treatment setting, s. hispidus that were immunosuppressed with cyclophosphamide had approximately 2-3 log reductions in rsv viral titer in lungs and nasal tissue compared to saline-treated animals [50] . importantly, pulmonary histopathology of immunosuppressed animals treated with ri-002 1500 mg/kg was similar to the histopathology of normal uninfected animals. in a prophylaxis study of immunosuppressed animals, a single dose of ri-002 was sufficient to protect animals from infection [38] . the open-label phase iii clinical trial conducted to evaluate efficacy, safety, and pharmacokinetics of ri-002 in subjects with pidd provides strong evidence regarding the efficacy of this product against viral respiratory pathogens and serious bacterial infection in pidd patients [49, 55, 58] . overall, ri-002 was well tolerated in these patients with few adverse events and no saes related to the study drug. ri-002 met the primary efficacy end point of the study, achieving an asbi rate of less than 1.0 infection per person-year with no asbis observed during approximately 56 subjectyears. the secondary end points also supported the efficacy of ri-002 against infection, with a low incidence of infection of any kind and few days lost from usual activity, unscheduled medical visits, hospitalizations due to infection, and days of antibiotic therapy. these clinical outcomes correlated with elevations in igg and antibodies to cmv, tetanus, measles, and s. pneumonia. as anticipated from the donor screening methods using to manufacture ri-002, the greatest increase was observed in post-infusion mean titers for rsv neutralizing antibodies, with the magnitude of the change dependent on dose. while it is clear that ri-002 confers passive immunity against bacterial infections and some respiratory viruses including rsv, the prophylactic efficacy of ri-002 for other respiratory viruses in humans is yet to be established. there is a gap in therapeutic options for immune globulin supplementation in pidd patients who are at high risk of viral respiratory infections. the preclinical and clinical data collected for ri-002 supports the use of this product in a population that has few available options. commercial ig products are standardized against pathogens that are not common within the population and are approved based on reduction in the incidence of asbi. ri-002 extends the coverage of these products to include rsv and possibly other respiratory viruses that are commonly acquired in patients at high risk of rsv disease. the broad range of protection provided by ri-002 suggests that it will be an important agent to supplement the physician's arsenal for immunocompromised patients during rsv season and for patients that might benefit from the broad coverage of this product. the previous aap recommendation for use of respigam as a replacement for commercial ig products in immunocompromised children during rsv season did not take standardization of respigam into account [28] . while respigam was not appropriately indicated for use in this population without additional supplementation, ri-002 may be uniquely poised to fill this therapeutic role. data collected in the s. hispidus model for multiple agents has proven its reliability in predicting the efficacy of new therapeutic agents against rsv and, importantly, this model can be extended to young, elderly, and immunosuppressed populations. ri-002 achieved protective titers in s. hispidus, protected animals from infection, and was successful in maintaining normal lung histology and in eliminating infection in treated animals [11, 33, 34, 50] . these findings extended to both immunocompromised animals and animals infected with palivizumab-resistant rsv [48, [50] [51] [52] . data collected in s. hispidus are similar to the trends observed in the phase ii and compassionate use study of ri-001, which provided equivalent anti-rsv neutralizing titers compared to ri-002. the neutralizing titers that were reached in the animal model were on average twofold higher than that achieved with the clinical dosing regimen of palivizumab. although limited in size, these studies demonstrate that ri-002 achieves high rsv neutralizing antibody titers that can be considered protective in humans and could result in improved outcomes in rsv-infected patients when treatment begins soon after the onset of symptoms. importantly, ri-002 meets ig antibody standardization requirements and its phase iii primary efficacy end point, with zero serious bacterial infections observed over 56 person-years, and is therefore suitable for use in supplementation of pidd patients [9, 11, 55] . in addition to reduction in the incidence of serious bacterial infection and rsv, neutralizing antibody titers against other respiratory pathogens are also elevated [11] . in s. hispidus, ri-002 achieved protective titers against four strains of commonly circulating influenza, improved lung histology postinfection, reduced or eliminated influenza viral mrna, and suppressed chemotactic signaling [49, 50] . taken together, these data suggest that a comparable effect is possible in humans although this remains to be determined. the process of enriching for anti-rsv antibodies within the donor pool is strongly correlated with increased antibody titers against other respiratory viruses and this finding is reproducible in all lots tested [10] . the impact of increased humoral protection against a range of commonly circulating respiratory viruses that pose a risk to pidd patient is not yet known, although consistent supplementation with ig against respiratory illness is likely to be of clinical benefit based on previous data generated by the prevent study group. ri-002 has potential to redefine treatment within immunocompromised populations where vaccines are often ineffective. while commonly used prophylactic agents provide protection against a limited spectrum of specific pathogens, the potential for broad-spectrum protection against respiratory viruses that have previously required additional prophylactic agents or for which no protective agents are available will be an invaluable to physicians seeking to optimize protection of these patients with enhanced passive immunity to the range of commonly encountered pathogenic viruses. in addition to these patients, it could be used in patients who have been hospitalized with severe rsv respiratory infections who have not responded to conventional therapy. the observed correlation between selection for donors with high-titer rsv neutralizing antibodies and elevated antibody titers against commonly circulating viruses may pave the way for additional ig products with expanded coverage. while a single prophylactic option for rsv is currently marketed, no product has been previously developed that provides both rsv prophylactic activity and meets minimum fda requirements for antibody standardization for use as an ig supplement in pidd patients. • ri-001, an immune globulin product manufactured from pooled plasma of donors with elevated rsv neutralizing titers, was well tolerated in a phase ii study and achieved a fold-change in circulating rsv neutralizing titer ≥ 4-fold in 6/7 (86%) patients at a dose of 1500 mg/kg. an open-label compassionate use study was also conducted that suggested that ri-001 may provide a survival benefit to subjects, particularly when treatment is initiated early. • ri-002 is currently under development and is manufactured using the pooled plasma of more than 1000 donors that had the highest titers of anti-rsv neutralizing antibodies. in comparison to ri-001, ri-002 is standardized with equivalent anti-rsv titers using a proprietary standard. ri-002 meets fda immunoglobulin supplementation requirements for minimum titers of antibodies to diphtheria toxoid and measles and polio viruses in immunocompromised populations including pidd patients. • the manufacturing process for ri-002 selects for higher titers of anti-rsv antibodies against multiple rsv antigens compared to commercially available ig products. further, this selection also yields consistently higher (1.5-fold higher aggregate mean) neutralizing antibody titers against other respiratory pathogens. • evidence from s. hispidus preclinical testing demonstrates that ri-002 is effective for rsv treatment and prophylaxis in both normal and immunocompromised hosts and also shows some efficacy against commonly circulating strains of influenza. further, ri-002 is protective against palivizumab-resistant rsv, probably because ri-002 contains antibodies targeting multiple rsv surface proteins. • ri-002 was well tolerated in a phase iii clinical trial and met the primary efficacy endpoint of <1 serious bacterial infection per person year, with no serious bacterial infections documented over 56 person-years. additional information regarding the status of ri-002 can be found on the adma biologics, inc. website, http://www.admabiologics.com/ this article was funded by adma biologics, inc. bn greener is an employee of ppd inc. adma biologics inc contracted with ppd inc to provide editorial assistance in the preparation of this manuscript. j mond is the chief medical and scientific officer of adma biologics inc. the authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. peer reviewers on this manuscript have no relevant financial or other relationships to disclose. practice parameter for the diagnosis and management of primary immunodeficiency optimizing immunoglobulin treatment for patients with primary immunodeficiency disease to prevent pneumonia and infection incidence: review of the current data impact of trough igg on pneumonia incidence in primary immunodeficiency: a meta-analysis of clinical studies infection outcomes in patients with common variable immunodeficiency disorders: relationship to immunoglobulin therapy over 22 years recurrent and persistent respiratory tract viral infections in patients with primary hypogammaglobulinemia subclinical infection and dosing in primary immunodeficiencies severe respiratory syncytial virus (rsv) infection in infants with neuromuscular diseases and immune deficiency syndromes respiratory syncytial virus infections in infants affected by primary immunodeficiency characterization of antibodies to capsular polysaccharide antigens of haemophilus influenzae type b and streptococcus pneumoniae in human immunoglobulin intravenous preparations therapeutic immunoglobulin selected for high antibody titer to rsv also contains high antibody titers to other respiratory viruses this article describes the unique antibody composition of ri-002 and provides titers against common respiratory viruses biologic igg level in primary immunodeficiency disease: the igg level that protects against recurrent infection evaluation of correlation between dose and clinical outcomes in subcutaneous immunoglobulin replacement therapy pulmonary manifestations of primary immunodeficiency disorders in children the impact rsv study group. palivizumab, a humanized respiratory syncytial virus monoclonal antibody, reduces hospitalization from respiratory syncytial virus infection in high-risk infants palivizumab outcomes registry study. palivizumab prophylaxis of respiratory syncytial virus disease in 2000-2001: results from the palivizumab outcomes registry prevention of hospitalization due to respiratory syncytial virus: results from the palivizumab outcomes registry ribavirin utilization and clinical effectiveness in children hospitalized with respiratory syncytial virus infection prevention and treatment of respiratory syncytial virus and parainfluenza viruses in immunocompromised patients respiratory syncytial virusassociated infections in adult recipients of solid organ transplants respiratory syncytial virus infections in autologous blood and marrow transplant recipients with breast cancer: combination therapy with aerosolized ribavirin and parenteral immunoglobulins respiratory syncytial virus: current and emerging treatment options phase i study of intravenous ribavirin treatment of respiratory syncytial virus pneumonia after marrow transplantation prophylactic administation of respiratory syncytial virus immune globulin to high risk infants and young children reduction of respiratory syncytial virus hospitalization among premature infants and infants with bronchopulmonary dysplasia using respiratory syncytial virus immune globulin prophylaxis • the prevent study demonstrated the efficacy of respigam in prophylaxis of rsv and also provided evidence that anti-rsv enriched immunoglobulin products may demonstrate efficacy against a range of respiratory viruses respiratory syncytial virus-enriched globulin for the prevention of acute otitis media in high-risk children prevention of respiratory syncytial virus infections: indications for the use of palivizumab and update on the use of rsv-igiv respiratory syncytial virus immune globulin treatment of lower respiratory tract infection in pediatric patients undergoing bone marrow transplantation -a compassionate use experience respiratory syncytial virus (rsv) immune globulin and palivizumab for prevention of rsv infection respiratory syncytial virus immune globulin for prophylaxis against respiratory syncytial virus disease in infants and children with congenital heart disease management of rsv infection in adult recipients of hematopoietic stem cell transplantation polyclonal human intravenous immune globulin (igiv) with high-levels of rsv neutralizing antibodies: a summary of animal and human studies polyclonal human intravenous immune globulin (igiv) with high-levels of rsv neutralizing antibodies: a summary of animal and human studies use of high titer rsv immunoglobulin (ri-001-rsv ivig) in immunocompromised adults. poster session presented at: infectious disease week respiratory syncytial virus infections in pediatric liver transplant recipients the pathogenesis of respiratory syncytial virus infection in cotton rats the cotton rat model of respiratory viral infections diversifying animal models: the use of hispid cotton rats in infectious diseases age-related differences in pulmonary cytokine response to respiratory syncytial virus infection: modulation by anti-inflammatory and antiviral treatment age-dependent replication of respiratory syncytial virus in the cotton rat respiratory syncytial virus infection in cyclophosphamide-treated cotton rats effectiveness of rsvig prophylaxis and therapy of respiratory syncytial virus in an immunosuppressed animal model immunoprophylaxis and immunotherapy of rsv in the cotton rat comparison of antibody concentrations and protective activity of respiratory syncytial virus immune globulin and conventional immune globulin quantitative aspects of passive immunity to respiratory syncytial virus infection in infant cotton rats respiratory syncytial virus-a comprehensive review high titer anti-rsv polyclonal antibody (ri-002) prevents infection with palivizumab resistant rsv in cotton rats and achieves greater neutralizing anti-rsv activity as compared to palivizumab protective levels of neutralizing antibodies to influenza are present in an ivig (ri-002) containing standardized and elevated levels of neutralizing antibodies to rsv and can protect influenza infected cotton rats. poster session presented at treatment with novel rsv ig ri-002 controls viral replication and reduces pulmonary damage in immunocompromised sigmodon hispidus this article outlines many of the s. hispidus preclinical studies performed for r-002 treatment and prophylaxis in both normal and immunocompromised animals and the pulmonary impact of ri-002 administration to infected animals gilbert be protection against palivizumab resistant rsv with an ivig containing high titer anti-rsv neutralizing antibodies. poster session presented at: 2nd international primary immunodeficiencies congress treatment of normal and immune suppressed cotton rats with ivig containing high neutralizing titer anti-rsv antibody. poster session presented at: 9th international respiratory syncytial virus symposium relationship between haemagglutination-inhibiting antibody titres and clinical protection against influenza: development and application of a bayesian random-effects model the role of serum haemagglutination-inhibiting antibody in protection against challenge infection with influenza a2 and b viruses efficacy, safety, and pharmacokinetics of a new 10 % liquid intravenous immunoglobulin containing high titer neutralizing antibody to rsv and other respiratory viruses in subjects with primary immunodeficiency disease this article reports efficacy, safety, and pharmacokinetic data from the pivotal phase iii clinical trial of ri-002 in a pidd population anti-inflammatory activity of ivig mediated through the inhibitory fc receptor adma biologics, assignee. composition and methods for the treatment of immunodeficiency. united states patent us 9 results of a phase iii trial in patients with pidd using an ivig containing high titer neutralizing antibody to respiratory syncytial virus (rsv) key: cord-017126-7ebo3cy3 authors: nan title: lungenversagen date: 2007 journal: chirurgische intensivmedizin doi: 10.1007/978-3-211-29682-0_10 sha: doc_id: 17126 cord_uid: 7ebo3cy3 das akute lungenversagen ist eine schwere diffuse entzündliche erkrankung der lunge. nach der „american-european consensus conference“ (bernard et al., 1994) wird zwischen einem ards — acute respiratory distress syndrom und einem ali — acute lung injury unterschieden. das ausmaß der respiratorischen insuffizienz wird nach der höhe des scores definiert. score ist die gesamtsumme der einzelwerte dividiert durch 4 score: 0 kein lungenversagen 0,1-2,5 leichtes lungenverasgen > 2,5 schweres lungenversagen multiple pathomechanismen (tabelle 10) sind verantwortlich für schwerwiegende morphologische (lorraine et al., 2000) und funktionelle lungenveränderungen, sodass eine ausreichende oxygenierung der patienten mittels konventioneller beatmung oft nicht mehr möglich ist. in der frühphase des ards findet sich eine massive ansammlung neutrophiler granulozyten in der lunge sowie deren migration durch die gefäßwand. sie setzen eine reihe von endothelschädigenden und den pulmonal-vasku-lären gefäßwiderstand erhöhende substanzen frei (abb. 42), die den ausgangspunkt für eine weitere kaskadenartige aktivierung verschiedener mediatorsysteme darstellen. (freisetzung freier radikale, elastase, aktivierung der phospholipase, aktivierung des kallikreinsystems, freisetzung von zytokinen: tnf, interleukin -1, 4, -6, -8, -10, -13; platelet activating factor (paf), komplement-komponente (c5a), adhäsionsmoleküle. des weiteren spielt jedoch auch die freisetzung chemotaktischer moleküle, der chemokine ein wichtige rolle (puneet et al., 2005) . es kommt zunächst einerseits zu einer erhöhung der permeabilität der gefäßwand mit interstitieller ödembildung und anderseits zu einer permeabilitätsstörung der alveolarwand, hervorgerufen durch membranfragmentationen der endothelzellen und eine degeneration der alveolären epithelzellen (typ-i-pneumozyten) mit ablösung von der basalmembran. sie machen 90 % der alveolären fläche aus und sind vulnerabel als die typ ii zellen, die 10 % der alveolarfläche ausmachen. dadurch wird die entstehung eines proteinreichen interstitiellen und intraalveolären ödems (patroniti et al., 2005) ausgelöst, wodurch wiederum der gasaustausch erheblich erschwert wird. das zunehmende gewicht der lunge, bedingt durch das zunehmende ödem, führt zu einem kollaps von lungenabschnitten entlang eines vertikalen gradienten verursacht durch hydrostatische kräfte, die kompressionsatelektasen erzeugen. diese veränderungen treten bevorzugt in abhängigen lungenabschnitten auf. die in der computertomographie sichtbaren dichteänderungen stellen eine kombination von atelektasen, ödem, konsolidation oder eine kombination dieser drei formen dar. die schädigung der alveolarepithelien führt weiters zu einer abnahme der surfaktantproduktion und fördert damit die bildung von atelektasen. so kommt es zu einer zunahme des intrapulmonalen shunts und des totraumquotienten. der zusätzliche funktionsverlust des surfactant ist auf die anwesenheit funktioneller inhibitoren, seine abnorme zusammensetzung und die proteolytische störung der einzelkomponenten zurückzuführen. bei längerem bestehen des krankheitsbildes kommt es zur fibrosierung und proliferation der typ-ii-pneumozyten. durch die abnahme der compliance der lunge steigt das risiko für die entstehung eines barotraumas. im gegensatz zu der noch bis vor einiger zeit bestehenden meinung, dass die rückbildung eines alveolaren ödems alleinig als ergebnis aus der druckdif-ferenz zwischen hydrostatischem und osmotischem druck (starling-kräfte) resultiert, zeigen nun neuere studien, dass die beseitigung eines alveolären ödems durch einen aktiven natrium-chlorid transport durch das pulmonale epithel, einschließlich der alveolären epithelzellen vom typ i und typ ii als auch der distalen epithelzellen des atemweges reguliert wird , verghese et al., 1999 , ware et al., 2001 . so erfolgt durch eine na + , k + -at-pase eine aktive förderung von na + von der basolateralen oberfläche in das interstitium (matthay et al., 2005) . so zeigt es sich, dass bei einer akuten lungenschädigung die alveoläre flüssigkeitsclearance geringer ist als bei einem kardial bedingten hydrostatischem lungenödem. erste medikamentöse therapieansätze zeigen, dass unter einer -adrenergen agonisten (z. b. isoproterenol) bzw. epithelialer wachstumsfaktoren-applikation möglicherweise die alveoläre flüssigkeitsclearance gesteigert werden kann (sartori et al., 2002 bei patienten mit ali/ards kann das auftreten apoptotischer vorgänge an pulmonalen epithelialen zellen (song y et al., 1999 , li et al., 2004 , martin et al., 2005 (abraham, 2003) derselben, sodass es zur aufrechterhaltung eines von leukozyten geführten inflammatorischen prozesses kommt, der typisch für eine akute lungenschädigung ist (wang et al., 1999 , yum et al., 2001 die verminderte apoptose der neutrophilen ist bedingt durch: 1. verhinderung einer zytochrom c freisetzung aus den mitochondrien, 2. hemmung der aktivierung des proapoptotischen protein bad, 3. gesteigerte transkription des antiapoptotischen protein mci-1 und bci-2, welches die protease caspase-9 hemmt und unter aktivierung von nf-kb eine gesteigerte transkription antiapoptotischer gene induziert. anschließend an diese zone findet sich meist ein dorsobasal lokalisierter erguss unterschiedlichen ausmaßes. computertomographischen untersuchungen (puybasset et al., 1998 , peseti et al., 2001 , pelosi et al., 1996 der lunge an patienten mit ali zeigen, dass hyperdensitäten entlang eines anteriorposterior gradienten als auch gleichzeitig entlang eines zephalokaudalen gradienten feststellbar sind. diese hyperdensitäten entsprechen nonaerated-nichtbelüfteten lungenarealen, welche sich von aerated lungenarealen unterscheiden lassen. nonaerated lungenareale sind bevorzugt in zwerchfellnahen lungenabschnitten zu finden. bei anwendung eines peep findet sich ein alveoläres rekruitment mehr in den nondependend als in den dependend lungenregionen und mehr in den zephalen als in den kaudalen regionen. wenn jedoch der zur verwendung kommende peep ausreichend hoch ist um in den dorsalen regionen und den unteren lungenabschnitten alveolen zu rekrutieren, dann kommt es zu einer überdehnung der oberen lungenabschnitte. überdehnung der lunge (quadri et al., 2003) auch als atelektrauma (uhlig et al., 2002) (ranieri et al., 1999 , lorraine et al., 1998 . konzepte der beatmung von patienten mit ards beinhalten die anwendung von spontanatmungsverfahren (putensen et al., 2001) (kuhlen et al., 2003 , luce 1998 , wobei keiner beatmungstechnik ein entscheidender vorteil eingeräumt werden kann, jedoch müssen gleichzeitig respiratoreinstellungen angestrebt werden, welche eine protektive beatmung ermöglichen (malarkkan et al., 2003 , eisner et al., 2001 . (gattinoni et al., 2005 , amato et al., 1995 , adams et al., 2003 , malarkkan et al., 2003 , bhattacharya 1998 die anwendung eines peep ist eine unumstrittene maßnahme bei jeder form einer respiratorischen insuffizienz (gattinoni et al., 1993 , medoff et al., 2000 , durante et al., 2002 . diskussionspunkte sind die höhe des verwendenen peep und die klinische umsetzung eines best-peep. ist das lungenvolumen, bei dem es während der exspiration zu einem verschluss der kleinen atemwege kommt. ist die summe aus verschlussvolumen und residualvolumen. überschreitet die verschlusskapazität die frc, dann tritt endexspiratorisch ein verschluss basaler luftwege auf. der verschluss der kleinen atemwege tritt vor allem in den abhängigen dorsobasalen lungenabschnitten auf, wo der extraluminale gravitations-bedingte gewebsdruck größer ist als der endobronchiale intrapulmonale atemwegsdruck. (neumann et al., 1998) darauf hin, dass zur vermeidung eines kollapses von alveolen (ohne extrinsischen peep) eine exspirationszeit von weniger als 0,6 sec notwendig wäre. bei länger bestehender exspirationszeit kommt es trotz peep-anwendung bis zu einer höhe von 15 cm h₂o zur ausbildung von atelektasen. erst bei einem peep von 20 cm h₂o kann der exspiratorische alveolenkollaps vermieden werden. resultierend aus den experimentellen ergebnissen bedeutet die wertigkeit der zeitkonstante in der klinischen anwendung, dass prinzipiell eine regelmäßige adaptation des respirators an die gegebene lungensituation (compliance, resistance) notwendig ist um z. b. durch adaptation der exspirationszeit des respirators an die exspiratorische zeitkonstante die entwicklung eines inadvertent peep vermeiden zu können. bei übertragung der experimentellen ergebnisse auf patienten mit schwerer respiratorischer insuffizienz wäre jedoch bezogen auf die respiratortherapie unter anwendung einer sehr kurzen exspirationszeit mit der entwicklung eines erhöhten intrinsischen peep und auch des mittleren atemwegsdruckes zu rechnen. hingegen konnte gezeigt werden, dass bei patienten mit ards eine kinetische therapie in form der bauchlage eine signifikante reduktion der exspiratorischen zeitkonstante (vieillardbaron et al., 2005) in der akutphase des lungenversagens sind ein beträchtlicher teil von lungenarealen bedingt durch einen kollaps von alveolen nicht belüftet. ziel eines open-lung-konzeptes (lachmann, 1992; blanch et al., 2002 , lim et al., 2003 , haitsma, 2003 ist es eine eröffnung von kollabierten alveolen bzw. rasche rekrutierung von konsolidierten lungenarealen unter kurzfristiger anwendung hoher inspirationsdrücke (50-60 mbar) zu erzielen und dann durch entsprechend hohe peep-werte ein offenhalten der alveolen zu garantieren (abb. 46). dazu ist es notwendig, dass der inspiratorische plateaudruck den alveolar-öffnungsdruck übersteigt und dann der peep über dem alveolarverschlussdruck liegt. ausgehend von dem hier beschriebenen grundkonzept gibt es inzwischen unterschiedlichst angewandte modifikationen (methoden) von rekrutierungsverfahren. wird der peep schrittweise auf 15-25 cm h₂o erhöht. damit sollen alle während der sich anschließenden inspiratorischen druckerhöhung rekrutierten alveolen offengehalten werden. 2. nun wird schrittweise der atemwegsspitzendruck auf 40-60-80 mbar über 5 atemzüge erhöht, womit ein kritischer wert des öffnungsdrucks erreicht wird. diese hohen inspiratorischen druckwerte werden als notwendig angesehen, um einerseits die adhäsivkräfte kollabierter alveolen (böhm et al., 1999) , anderseits auch die kapillarkräfte in den flüssigkeitsgefüllten atemwegen überwinden zu können. als parameter der erreichung des öffnungsdrucks ist das arterielle po₂ anzusehen, welches sich in einer deutlichen erhöhung zeigt. bei optimaler rekrutierung von alveolen führen nun weitere drucksteigerungen zu keiner erhöhung des pao₂. sind alle rekrutierten alveolen offen, dann ist ein derart hoher atemwegsspitzendruck für deren beatmung nicht mehr notwendig. resultierend aus dem zusammenwirken von surfactant und dem "la place gesetz" ist der notwendige innendruck in der alveole nach ihrer eröffnung geringer als vor ihrer eröffnung. es muss nun der verschlussdruck der lunge ermittelt werden, indem der atemwegsspitzendruck schrittweise gesenkt wird. kommt es zu einem pao₂-abfall, dann ist er durch einen kollaps von alveolen bedingt und somit ist der kritische verschlussdruck der alveolen festgestellt. die sich nun entwickelnden atelektatischen lungenareale müssen erneut eröffnet werden. die zuvor bestimmten eröffnungsdrucke sind bekannt. durch eine kurze beatmungsdauer mit diesen bekannten drücken, über einen zeitraum von 30 sekunden, wird die lunge erneut eröffnet. anschließend kann der atemwegsspitzendruck soweit gesenkt werden, dass ein druck verwendet wird, der 2 cm h₂o über dem bekannten verschlussdruck liegt. eine permissive hyperkapnie wird als teil einer lungenprotektiven maßnahme bei einem akuten lungenversagen angesehen (carvalho et al., 1997 nachdem no lange zeit als schadstoff angesehen wurde, gelang erst 1987 und 1988 im rahmen dreier studien der nachweis, dass der endothelium-derived relaxin factor (edrf), entdeckt von furchgott und zawadzki, ident mit no ist (ignarro et al., 1987; palmer et al., 1987; furchgott, 1988) die hochfrequenzbatmung ist eine form der künstlichen beatmung, bei der kleine tidalvolumina mit einer supraphysiologischen frequenz appliziert werden. verschiedenste typen der hochfrequenzbeatmung sind in den letzten 30 jahren entwickelt und angewendet worden worden (froese et al., 1987) . von den zahlreichen möglichen anwendbaren hochfrequenzbeatmungsformen wie: hfp-high-frequency pulsation, fdv-forcierte diffusionsventilation (baum et al., 1980) , hfjv-high frequency jet oscillation haben sich im klinisch anwendbaren bereich jedoch techniken wie die hfpp-high-frequency positive-pressure ventilation, hfj-high frequency jetchang (1984) beschreibt in einer übersichtsarbeit mehrere mechanismen des gasaustausches, die vor allem bei der hochfrequenzoszillation von bedeutung sind. (shimaoku et al., 1998) . erste klinische ergebnisse zeigen, dass es möglicherweise unter hochfrequenter beatmung zu einem rascheren rekruitment von dependend lungenarealen kommt, ohne dass es gleichzeitig zu einer massiven überdehnung von non-dependend lungenarealen kommt. denkbar ist, dass der unter hochfrequenzbeatmung oft zu beobachtende bessere gasaustausch nicht so sehr über mechanismen einer gesteigerten diffusion zu erklären ist, sondern durch pulsatile mechanismen, die zu einer rascheren rekrutierung von lungengewebe führen. . des weiteren kann diese jet-ventilationstechnik mit no-applikationsgeräten kombiniert werden, wobei eine exakte no-dosierung gewährleistet ist . meist kommt sie erst dann zu einsatz, wenn eine konventionelle beatmung versagt. in der literatur handelt es sich daher meist um fallberichte oder nicht randomisierte kleinstudien. jedoch zeigt es sich, dass die angewandten techniken, wenn oft auch aufwendig, sicher in der anwendung sind und oft eine verbesserung der oxygenierung ermöglichen. durch die verwendung eines konventionellen beatmungsteiles (abb. 49) mit niedriger beatmungsfrequenz mit konventionellem peep, jedoch mit größerem vtid, ist eine ausreichende co₂-elimination gewährleistet. überlagert wird dem konventionellen teil eine hochfrequente beatmungsform (tabelle 11). die oxygenierung wird bevorzugt durch den hochfrequenten pulsatilen teil der beatmung verbessert. so wurden konventionelle beatmungstechniken mit unterschiedlichen hochfrequenten beatmungstechniken kombiniert. die jet-gasapplikation erfolgt bei einer kombinierten jet-ventilation (chfjv) (abb. 50): abb. 48. darstellung des hochfrequenten jet-gas-impuls-volumens unter variabler jetfrequenz bei konstantem trachealdruck von 10 mbar. bei anwendung einer hohen jet-frequenz (links) ist das tidalvolumen des jet-gasimpulses niedrig. je niedriger die jet-frequenz (rechts), desto höher ist das jet-impuls-gasvolumen ein suffiziente anfeuchtung und erwärmung des atemgases ist für eine maschinelle beatmung von wesentlicher bedeutung. als zielwerte werden eine temperatur von 32 grad und eine relative feuchtigkeit von 80 % angesehen (roth, 1993) . als optimaler feuchtigkeitsgehalt der inspirationsluft werden werte bis 44 mg/l angesehen (williams et al., 1996 , christiansen et al., 1998 . (perkins gd et al., 2004) im entzündungsgeschehen bei einem ards (verminderung der sequestration von neutrophilen, beschleunigung der alveolären flüssigkeitsclearance, erhöhung der sekretion von surfactant) zugeschrieben. dennoch liegt auch eine beschreibung über die entwicklung eines lungenödems (russi et al., 1988) unter der kontinuierlichen therapie einer tokolyse mit -mimetica vor. bei patienten mit ards konnte abnahme von atemwegsspitzendruck, plateaudruck sowie des atemwegswiderstandes erzielt werden (morina et al., 1997) . antiphlogistica ist ein inhalatives glukokortikoid, lokaler entzündungshemmender effekt dosierung 4 × 1 atemhub bei der jet-ventilation sollte nach 24 stunden eine kontrolle der trachealschleimhaut durchgeführt werden, um frühzeitig schleimhautveränderungen, die durch eine zu geringe befeuchtung bedingt sind, zu verhindern. danach weitere kontrollen der befeuchtung durch bronchoskopien in größeren zeitabständen. bei der jet-ventilation werden kleine gasvolumina mit hoher frequenz von einer düse über den endotrachealtubus in die trachea appliziert. wird nur --ein einzelimpuls des jet-gas-volumens abgegeben, dann gelangt das gasvolumen höchstens einige trachealdurchmesser in die trachea; aber durch die periodische kontinuierliche abgabe eine jet-impulses mit hoher frequenz wird das gasvolumen kontinuierlich bis in die alveolen transportiert. dieser gastransport ist begleitet von einer interaktion zwischen konvektion und molekularer diffusion innerhalb der atemwege. der konvektive teil des gasbewegung setzt sich aus zwei teilen zusammen: so besteht erstens ein turbulent konvektiver gasaustausch mit der atmosphäre verursacht durch den jet in der trachea und zweitens eine konvektive strömungsbewegung entlang der atemwege gesteuert durch eine interaktion zwischen dem jet und einer expandierenden und kontrahierenden bewegung der atemwege, verursacht durch die compliance der lunge (scherer et al., 1989) . in den peripheren atemwegen besteht ein pulsierender bidirektionaler koaxialer gasfluss sowie eine verstärkte konvektive dispersion die so genannte "augmented diffusion", die um ein vielfaches größer ist als die normale molekulare diffusion. unter anwendung der superponierten hochfrequenz-jet-ventilation dürfte neben gesteigerten gasaustauschmechanismen jedoch ein rasches rekruitment von minderbelüfteten abhängigen lungeanarealen ursächlich an der oft feststellbaren raschen verbesserung des pulmonalen gasaustausches beteiligt sein, wie es mittels einer computertomographischer studie (kraincuk, 2003) gezeigt werden konnte. wobei der pulsatilen gasbewegung offensichtlich eine wichtige rolle zuzukommen scheint. die hochfrequenzoszillation unterscheidet sich von der hochfrequenz-jet-ventilation, dass sowohl neben der inspiration auch die exspiration aktiv ist, die tidalvolumina kleiner sind als bei einer combined hfjv (meist unter 150 ml), der möglich anwendbare frequenzbereich erstreckt sich bis 15 hz (meist liegt er bei 10 hz ähnlich der jet-ventilation). kolla et al., 1997; lewandowski et al., 1997) größere patientenzahlen mit einer überlebensrate von über 50 % aufwiesen, des weiteren schwere bakterielle als auch virale pneumonien (vida et al., 2005) als auch sekundäre lungenschädigungen, die nach einem trauma, einer pankreatitis, nach systemischer erkrankung mit lungenbeteiligung (loscar et al., 1997) nach einer chemischen pneumonitis sowie einer sepsis auftreten können. zur anwendung kommt sie auch bei transplantations-empfängern unmittelbar postoperativ nach einer lungentransplantation als auch nach herzchirurgischen eingriffen. ebenso ist der frühzeitige beginn mit anschließenden mobilem inter-hospitalen transfer an ein ecmo zentrum beschrieben linden, 2001) . die anwendung extrakoproraler oxygenierungsverfahren stellt eine notwendige therapieoption (kopp et al., 2004 , henzler et al., 2004 bei schwerster respiratorischer insuffizienz dar, die mit der laufenden weiterentwicklung (bartlett, 2005 , bensberg et al., 2005 einschließlich der entwicklung kleinerer und einfacherer systeme den einsatzbereich erweitert. betreffend die gesundheitsbezogene lebensqualität (stoll et al., 1998) bei einer zunahme des herzzeitvolumens steigt der mikrovaskuläre druck, gleichzeitig kommt es zu einer rekrutierung von bis dahin nicht oder kaum perfundierten gefäßen. damit kommt es zu einer zunahme von kapillaroberfläche über die flüssigkeit filtriert wird. das so genannte permeabilitäts-oberflächenprodukt steigt. bei einem anstieg des hzv kommt es somit durch rekrutierung von gefäßen und der damit einhergehenden vergrößerung des gefäßquerschnittes zu einem abfall des postkapillären gefäßwiderstandes der gesamten lunge, wobei dennoch die transvaskuläre flüssigkeitsfiltration zunehmen kann. akutes nierenversagen durch eine extreme hypovolämie splanchnikusischämie vermeidung einer extremen hypovolämie: akutes nierenversagen splanchnikusischämie hypovolämie bedingt eine freisetzung vasoaktiver mediatoren. im tierexperiment führt volumenmangel zu translokation von bakterien und endotoxinfreisetzung aus dem darmlumen in den intravasalraum und zu gesteigerter freisetzung von tnf-. die kinetische therapie ist eine lagerungstherapie mit dem ziel einer verbesserung der lungenfunktion und damit des pulmonalen gasaustausches. zahlreiche arbeiten beweisen die effektivität dieser maßnahmen (langer et al., 1988 , hörman et al., 1993 , lamm et al., 1994 , stiletto et al., 2000 , koutsoukou, 2005 (spragg rg et al., 2004) nur eine kurzfristige besserung des gasaustausches innerhalb der ersten 24 stunden nach seiner verabreichung sowie keine verbesserung des outcome. die problematik liegt in der notwendigen schwierigen zerstäubung einer fetthaltigen lösung, der platzierung in der gesunden lunge, der inaktivierung in geschädigten -----alveolen (baudouin sv 2004) , der hohen dosierung beim erwachsenen und der damit verbundenen hohen kosten der jeweiligen präparate, die eine routinemäßge applikation derzeit nicht rechtfertigen, obwohl kleine studien auf die positiven effekte hinweisen (seeger et al., 1997 , günther et al., 2002 , von kaam et al., 2004 , calkovska et al., 2005 . (seear et al., 1990 , kudoh et al., 1995 die endotheliale permeabilität und das lungenödem. in klinischen studien (the ards clinical trials network 2002) konnten jedoch keine entsprechenden ergebnisse erzielt werden, die eine gezielte applikation von pentoxyphyllin rechtfertigen würden. acetylcystein -antioxidans wurde als antioxydans in verschiedenen studien verwendet. die tierexperimentell günstigen ergebnisse (bernard et al., 1984) konnten jedoch in klinischen studien (bernard et al., 1997) cepkova, 2006) dieser substanzgruppe bei ali oder ards. es fand sich keine verbesserung des gasaustausches, keine reduktion der der mortalität. -adrenoceptor agonisten es ist bekannt, dass -agonisten über alveolar type ii zellen die surfactantproduktion stimulieren. ₂-agonisten erhöhen in der lunge jedoch auch den transepithelialen flüssigkeitstransport einer klinischen studie zeigen, dass eine applikation von -ago neutrophils and acute lung injury ventilator-induced lung injury beneficial effects of the "open lung approach" with low distending pressures in acute respiratory distress syndrom; a prospective randomized study on mechanical ventilation effect of a protective-ventilation strategy on mortality in the acute respiratory di stress syndrome incidence, risk factors and outcome of barotrauma in mechanically ventilated patients -the ards clinical trials network. randomized, placebo-controlled trial of lisofylline for early treatment of acute lung injury and acute respiratory distress syndrome extracorporeal life support: history and new directions forced diffusion ventilation (fdv), bases and clinical application artificial lung and extracorporeal gas exchange the american-european consensus conference on ards. definitions, mechanisms, relevant outcomes, and clinical trial coordination effect of a protective-ventilation strategy on mortality in the acute respiratory distress syndrome recruitment maneuvers in acute lung injury exogenous surfactant replacement in ards -one day, someday, or never? effect of nacetylcystein on the pulmonary response to endotoxin in the awake sheep and upon in vitro granulocyte function a trial of antioxidans n-acetylcysteine and procysteine in ards. the antioxidant in ards study group chest das konzept der offenen lunge exogenous surfactant administration by asymmetric high-frequency jet ventilation in experimental respiratory distress syndrome temporal hemodynamic effects of permissive hyperkapnia associated with ideal peep in ards pharmacotherapy of acute lung injury and the acute respiratory distress syndrome mechanisms of gas transport during ventilation by high-frequency oscillation messungen des feuchtigkeitsgehaltes der inspirationsluft bei beatmeten patienten bei verwendung verschiedener befeuchtungssysteme high frequency percussive ventilation in pediatric patients with inhalation injury granton j and the multicenter oscillatory ventilation for acute respiratory distress syndrome trial (moat) study investigators, high frequency oscillatory ventilation for acute respiratory distress syndrome in adults high frequency oscillatory ventilation in the management of a high output bronchopleural fistula: a case report ardsnet lower tidal volume ventilatory strategy may generate intrinsic positive end-expiratory pressure in patients with acute respiratory distress syndrome the acute respiratory distress syndrom network: efficacy of low tidal volume ventilation in patients with different clinical risk factors for acute lung injury and the acute respiratory distress syndrome combined high-frequency ventilation for management of terminal respiratory failure: a new technique combining high-frequency oscillatory ventilation and recruitment maneuvers in adults with early acute respiratory distress syndrome: the treatment with oscillation and an open lung strategy (tools) trial pilot study high frequency ventilation -state of art dramatic beneficial effects of sildenafil in recurrent massive pulmonary embolism the concept of "baby lung regional effects and mechanism of positive end-expiratory pressure in early adult respiratory distress syndrome effects of positive end-expiratory pressure on regional distribution of tidal volume and recruitment in adult respiratory distress syndrome inhaled nitric oxide therapy in adults bronchoskopic administration of bovine natural surfactant in ards and septic shock: impact on biophysical and biochemical surfactant properties hemodynamic effects of high-frequency oscillatory ventilation in severe pediatric respiratory failure open lung in ards biphasic positive airway pressure (bipap)-a new mode of ventilatory support extracorporeal life support for 100 adult patients with severe respiratory failure turn the ards patient prone to improve oxygenation and decrease risk of lung injury alveolar recruitment of atelektasis under combined high-frequency jet-ventilation: a computed tomography study hypercapnic acidosis and mortality in acute lung injury inhaled nitric oxide and inhaled prostaglandin e 1: effect on left ventricular contractility when used for treatment of experimental pulmonary hypertension high-frequency ventilation for acute lung injury and ards the effect of pentoxifylline on acid-induced alveolar epithelial injury die intensivtherapie bei akutem lungenversagen open up the lung and keep the lung open mechanism by wich the prone position improves oxygenation in acute lung injury the prone position in ards patients extracorporeal membrane oxygenation for severe acute respiratory failure high survival rate in 122 ards patients managed according to a clinical algorithm including extracorporeal membrane oxygenation effect of alveolar recruitment maneuver in early acute respiratory distress syndrome according to antiderecruitment strategy, etiological category of diffuse lung injury, and body position of the patient inter-hospital transportation of patients with severe acute respiratory failure on extracorporeal membrane oxygenation -national and international experience ventilator-induced injury: from barotrauma to biotrauma acute lung injury and the acute respiratory distress syndrome hochfrequenzventilation (hfo) bei akuter lungenschädigung und ards application of transtracheal pressure oscillations as a modification of "diffusing respiration new aspects of ventilation in acute lung injury-review article temporal dynamics of lung aeration determined by dynamic ct in a porcine model of ards apoptosis and epithelial injury in the lungs lung epithelial fluid transport and the resolution of pulmonary edema alveolar epithelium, role in lung fluid balance and acute lung injury alveolar fluid clearance in patients with ards -does it make a difference science review: apoptosis in acute lung injury high-frequency oscillation in acute respiratory distress syndrome (ards) use of recruitment maneuvers and high positive end-expiratory pressure in a patient with acute respiratory distress syndrome prospective trial of highfrequency oscillation in adults with acute repiratory distress syndrome effects of nebulized salbutamol on respiratory mechanics in adult respiratory distress syndrome an expanded definition of the adult respiratory distress syndrome hedenstierna g, effect of different pressure levels on the dynamics of lung collapse and recruitment in oleic-acidinduced lung injury pesenti a, measurement of pulmonary edema in patients with acute respiratory distress sindrome computed tomography in adult respiratory distress syndrome: what has it taught us benchto-bedside review: beta2-agonists and the acute respiratory distress syndrome computerised tomography scan imaging in distress sindrome acute and chronic effects of sildenafil in patients with pulmonary arterial hypertension chemokines in acute respiratory distress syndrome long-term effects of spontaneous breathing during ventilatory support in patients with acut lung injury a computed tomography scan assessment of regional lung volume in acute lung injury high tidal volume ventilation induces proinflammatory signaling in rat lung endothelium administration of albumin to patients with sepsis syndrome: a possible beneficial role in plasma thiol repletion albumin influences total plasma antioxidant capacity favourably in patients with acute lung injury effect of mechanical ventilation on inflammatory mediators in patients with acute respiratory distress syndrome -a randomized contolled trial a prospective comparison of atrio-femoral and femoro-atrial flow in adult venovenous extracorporeal life support extracorporeal membrane oxygenation for transport of hypoxaemic patients with severe ards intensivmedizinisches seminar band 5 beatmung high permeability pulmonary edema (ards) during tocolytic therapy -a case report beta-adrenergic agonist stimulated alveolar fluid clearance in ex vivo human und rat lungs effect of pentoxifilylline on hemodynamics, alveolar fluid reabsorbation, and pulmonary edema in a model of acute lung injury alveolar epithelial fluid transport in acute lung injury: new insights convective mixing in high frequency intermittend jet ventilation erste erfahrungen mit der superponierten hochfrequenz-jetventilation in der intensivmedizin superponierte hochfrequenz jetventilation (shfjv) unter verwendung von no, technische grundlagen und erster klinischer einsatz surfactanttherapie des ards, hintergrund und erste klinische erfahrungen high frequency oscillatory ventilation attenuates the activation of alveolar macrophages and neutrophils in lung injury conventional ventilation versus high-frequency oscillation: hemodynamic effects in newborn babies ventilation with small tidal volumes effect oof recombinant surfanctant protein c-based surfactant on the acute respiratory distress syndrome national heart, lung, and blood instituete acute respiratory distress syndrome (ards) clinical trials network. efficacy and safety of corticosteroids for persistent acute respiratory distress syndrome kinetische therapie zur therapie und prophylaxe der posttraumatischen lungeninsuffizienz. ergebnisse einer prospektiven studie an 111 polytraumatisierten gesundheitsbezogene lebensqualität; langzeitüberleben der erwachsenen patienten mit ards nach extracorporaler mmbranoxygenation (ecmo) mechanotransduction in the lung ventilation-induced lung injury and mechanotransduction: stretching it too far high-frequency percussive ventilation improves oxygenation in patients with ards alveolar epithelial fluid transport and the resolution of clinically severe hydrostatic pulmonary edema prolonged ecmo support for virus-induced cardiorespiratory failure early after cardiac surgery prone position improves mechanics and alveolar ventilation in acute respiratory distress syndrome response to exogenous surfactant is different during open lung and conventional ventilation alveolar fluid clearance is impaired in the majority of patients with acute lung injury and the acute respiratory distress syndrome the acute respiratory distress syndrome fas-induced apoptosis epithelial cells requires ang ii generation and receptor interaction pathophysiologie und aktuelle medikamentöse therapiekonzepte relationship between the humidity and temperature of inspired gas and the function of the airway mucosa high-frequency jet ventilation produces auto-peep involvement of phosphoinositide 3-kinases in neurtophil activation and the development of acute lung injury arteriovenous carbon dioxide removal: development and impact on ventilator management and survival during severe respiratory failure key: cord-265445-bazcczdj authors: arias-bravo, guisselle; valderrama, gustavo; inostroza, jaime; reyes-farías, marjorie; garcia-diaz, diego f.; zorondo-rodríguez, francisco; fuenzalida, loreto f. title: overnutrition in infants is associated with high level of leptin, viral coinfection and increased severity of respiratory infections: a cross-sectional study date: 2020-02-18 journal: front pediatr doi: 10.3389/fped.2020.00044 sha: doc_id: 265445 cord_uid: bazcczdj objective: to investigate the relationship of overnutrition (obese and overweight) with severity of illness in children hospitalized with acute lower respiratory infections (alris), frequency of viral coinfections and leptin levels. methods: we studied 124 children <2 years old that were hospitalized for alri. nutritional status was calculated by z-scores according to weight-for-age z-scores, length or height-for-age z-scores, and weight-for-height z-scores. nasopharyngeal aspirates (npas) were obtained and viral respiratory pathogens were identified using reverse transcription polymerase chain reactions (rt-pcr). respiratory syncytial virus (rsv) load was assessed using quantitative rt-pcr. npa and plasma leptin level were measured. clinical data and nutritional status were recorded, and patients were followed up until hospital discharge. viral coinfection was defined as the presence of two or more viruses detected in the same respiratory sample. severity of illness was determined by length of hospitalization and duration of oxygen therapy. results: children with overnutrition showed a greater frequency of viral coinfection than those with normal weight (71% obese vs. 37% normal weight p = 0.013; 68% overweight vs. 37% normal weight p = 0.004). a lower rsv load was found in obese (5.91 log(10) copies/ml) and overweight children (6.49 log(10) copies/ml) compared to normal weight children (8.06 log(10) copies/ml; p = 0.021 in both cases). in multivariate analysis, obese, and overweight infants <6 months old were associated with longer hospital stays (rr = 1.68; ci: 1.30–2.15 and obese: rr = 1.68; ci: 1.01–2.71, respectively) as well as a greater duration of oxygen therapy (rr = 1.80; ic: 1.41–2.29 and obese: rr = 1.91; ci: 1.15–3.15, respectively). obese children <6 months showed higher plasma leptin level than normal weight children (7.58 vs. 5.12 ng/μl; p <0.046). conclusions: in infants younger than 6 months, overnutrition condition was related to increased severity of infections and high plasma leptin level. also, children with overnutrition showed a greater frequency of viral coinfection and low rsv viral load compared to normal weights children. these findings further contribute to the already existent evidence supporting the importance of overnutrition prevention in pediatric populations. acute lower respiratory tract infections (alris) are a common illness in children <5 years old, with significant morbidity and mortality in infants and young children under the age of two (1) (2) (3) . respiratory syncytial virus (rsv) is the most important viral pathogen causing alris in young children (4) . there are many risk factors related to alris. some of these are relative to pathogens, such as viral load (5) , while others are directly associated with the host, such as prematurity, inadequate breastfeeding, and undernutrition (6) . the influenza ah1n1 2009 pandemic showed, for the first-time, that obesity is a risk factor for the severity of alris in adults (7, 8) . pediatric obesity has also been described as a risk factor for lower respiratory tract infections, as well as the severity and morbidity of these infections (9) . however, the empirical evidence needed to estimate the impact of overnutrition (including overweight and obese conditions) on the severity of viral respiratory infections in children is still lacking (10) . overnutrition causes an excessive accumulation of body fat. it is a highly prevalent condition and has been ranked as the foremost epidemic of the twenty-first century (11) . obesity results in a chronic state of inflammation with systemic implications for immunity (12) . leptin, cytokine-like hormone that is positively correlated with the body mass index, mediates upregulation of suppressor of cytokine signaling (socs) proteins (13) . socs proteins are involved in the negative regulation of janus-activated kinase-signal transducer and activator of transcription (jak-stat) signaling and the induction of type i and type ii ifns and pro-inflammatory cytokines, suggesting a potential mechanism by which respiratory viruses response in obesity may be attenuated (8, 14, 15) . in addition to immune modulation of obesity, alterations in membrane lipid composition have been observed in erythrocyte from obese children. the membrane composition has also revealed an increase in cholesterol content as has the cholesterol to phospholipid molar ratio, both of which have been positively correlated with a decrease in membrane fluidity, body-mass index, and plasma cholesterol levels (16) . cholesterol is a critical structural component of lipid rafts, key structures in binding and endocytosis of respiratory viruses (17) . in accordance with this, it could be expected that the membrane of obese children's respiratory cells may undergo some type of modification that could benefit the entry of respiratory viruses. hence, the objective of this study was to estimate the relationship of overnutrition on severity of illness in infants (aged between 0 and 5 months) and children (aged between 6 and 24 months) hospitalized with alris. moreover, frequency of viral coinfection, rsv viral load and levels of leptin according to nutritional status were evaluated. a cross-sectional study was conducted for 2 consecutive epidemic periods (from may to august) in 2016 and 2017. patients were selected based on following inclusion criteria: children <24 months old that were hospitalized for alri at two medical centers in santiago, chile: urgencia materno-infantil at clínica dávila and dr. exequiel gonzález cortés hospital. patients were classified as having bronchiolitis, bronchitis or pneumonia. diagnoses were made in patients with dyspnea, signs of lower respiratory tract infections (wheezing, retractions) and/or a positive chest x-ray (infiltrates, atelectasis and air trapping). the clinical and demographic characteristics of each patient were also recorded. the exclusion criteria were: children with nutritional risk and/or undernourished children, newborns <28 days old, patients with a diagnosis of recurrent wheezing, previous hospitalization for any cause, primary or secondary immunodeficiency, gestational age of <37 weeks, bronchopulmonary dysplasia, previous mechanical ventilation, congenital heart disease, respiratory infection during the previous 2 weeks, corticosteroid intake 72 h before sample was taken, and children with negative samples for respiratory viruses. figure s1 shows exclusion criteria. children were classified into three groups according to their nutritional status (ns) using who anthro 2011 v.3.2.2 program: normal weight, overweight, and obese. ns was determined by z-scores according to the following anthropometric indicators: weight-for-age z-scores, length or height-for-age z-scores, and weight-for-height z-scores. normal weight was defined as −0.9 to 0.9 sd, overweight as 1.0 to ≤2.0 sd and obese as >2 sd. undernourished children and those with nutritional risk were defined with a weight-forage z-score and weight-for-height z-score of <1 sd, below the mean, and were therefore excluded from the study. the weight of each patient was measured at hospital admission. disease severity was assessed by using the standard criteria of length of hospitalization and duration of treatment with supplemental oxygen (18, 19) . approximately 3 ml of nasopharyngeal aspirate (npa) were collected from each patient, usually during the admission process (<3 h) and always within the first 24 h of hospitalization. secretions from both nostrils were aspirated without flushing using a soft catheter placed in a collection trap with 3 ml of sterile saline solution and immediately transported on ice to the laboratory. aliquots of 2 ml were stored at −80 • c for viral analysis. aliquots of 1 ml were centrifuged at 1,000 g for 15 min at room temperature and the supernatants obtained were stored at −80 • c until determination of leptin level. viral rna and dna were simultaneously extracted from 150 µl of the npa samples using the viral rna isolation kit nucleospin (macherey-nagel r , düren, germany) following the manufacturer's instructions, and stored at −80 • c, until use. viral infection was detected with real-time pcr kit argene r (biomérieux, marcy-i ′ étoile, france), for rsv, human metapneumovirus (hmpv), parainfluenza virus 1-4 (hpiv), human coronavirus (hcov), adenovirus (adv), bocavirus (hbov), influenza a (flua), influenza b (flub), and rhinovirus/enterovirus (hrv/hev), following the manufacturer's instructions. viral co-infection was defined as the presence of two or more viruses detected in the same respiratory sample. forty-five patients that tested positive for rsv were selected using the argene r real-time pcr kit. a new aliquot was used with 150 µl of npa. nucleic acids were extracted using the viral rna isolation kit nucleospin (macherey-nagel r , düren, germany). the qrt-pcr was performed using the takyon tm rox probe mastermix dttp blue kit (eurogentec, usa), using primers with n1 (5 ′ -ggaacaagttgttgaggt ttatgaatatgc3 ′ ) and n2 probes (cttctgctgtcaa gtctagtacactgtagt-3 ′ ) (20) . the conditions of the pcr reaction were: preincubation at 95 • c for 3 min; 40 cycles at 95 • c for 10 s and 60 • c for 30 s. threshold cycles of positive samples for rsv were compared with the standard of curves generated by the amplification of known plasmid copy numbers of the pgem-t dna (promega) containing primer targets. in order to determine the effective amount of copies of viral dna molecules contained in each sample of the endogenous rnase p gene, the taqman r rnase p kit (applied biosystems, woolston, uk) was used. the cq values were interpolated and normalized according to the following formula: cq normalized sample = (cq viral sample) × (cq rnase p of the sample)/average value of cq of all the rnase p samples) (21) . rsv quantification was reported as log 10 copies/ml. about 2 ml of blood were collected in sodium heparin collection tubes (bd vacutainer) usually during the admission process. the sample was centrifuged at 1,000 g for 15 min at room temperature. the plasma obtained was divided into aliquots and stored at −80 • c. leptin concentration in plasma and npa was measured using the magnetic luminex r assay (r&d), according to manufacturer protocol. descriptive analyses, medians (ranges), and frequency distributions were used to summarize the demographic and baseline attributes of patients. associations among the dependent and independent variables were assessed by either the χ 2 , fisher's exact or mann-whitney tests. spearman's test was used to estimate the association between viral load and severity. multivariate poisson regression models were used to determine the factors that predict severity of infection (length of stay and duration of oxygen therapy). multicollinearity analyses among the independent variables were performed and none were observed. the cutoff point for multicollinearity was determined as variance inflation factor <5 and tolerance >0.2. we did not include birth weight in the multivariate regression model because it increases the multicollinearity in the model (variance inflation factors from 2.32 to 6.47). the relative risk (rr) for increased length of stay and oxygen therapy was calculated by taking the estimated poisson regression coefficient (β) for each variable and transforming it by eβ [exp * confidence interval] of each independent variable for the model. statistical significance was set at p < 0.05 for all analyses, along with a 95% confidence interval (ci). all analyses were performed with stata 14.1 software (statacorp, texas, usa). a total of 160 children were screened, and 124 patients were eligible for this study ( figure s1 ). the clinical and demographic characteristics of patients are presented in table 1 . obesity was found in 13.7% of the patients, 25.8% were overweight, and 60.5% presented normal weights. most of the children had both breastfeeding and complete vaccination schedules. the principal diagnosis was pneumonia (55.6%), followed by bronchiolitis and bronchitis ( table 1) . a total of 63 (50.8%) patients tested positive for a single respiratory virus, while another 61 patients (49.2%) tested positive for more than one respiratory virus. of these, 50 (82.0%) tested positive for two viruses and 11 (18.0%) for three viruses. rsv was the most frequently detected virus in 88 cases (70.9%), followed by hrv/hev in 33 (26.6%) and hbov in 27 (21.7%). most viruses were found in coinfection (figure 1 ). no differences were found between ns and type of virus detected ( figure s2) . nutritional status and coinfection (≥2 viruses) were evaluated to assess the effect of overnutrition in viral coinfection. obese and overweight infants showed a greater frequency of coinfection (obese 71% and overweight 68%) than those with normal weights (37%) (p = 0.013 and p = 0.004 respectively) (figure 2a) . no significant differences were found between overweight and obese infants (p = 0.839). the effect of obesity and overweight conditions on the rsv load was assessed in 46 infants (10 obese, 14 overweight, and 22 normal weight) with available nasopharyngeal samples. the median of rsv loads in obese infants was 5.91 log 10 copies/ml (min: 3.22 log 10 copies/ml and max: 7.86 log 10 copies/ml) and 6.49 log 10 copies/ml (min: 3.71 log 10 copies/ml and max: 8.11 log 10 copies/ml) for overweight infants, while those with normal weights had a median of 8.06 log 10 copies/ml (min: 4.15 log 10 copies/ml and max: 9.17 log 10 copies/ml). the difference between obese and overweight infants compared to those with normal weights was significant (obese p = 0.0205 and overweight p = 0.0212, respectively) ( figure 2b) . no significant differences were found between overweight and obese infants (p = 0.6395). no correlation was evidenced between rsv load and length of hospital stay (r = −0.124; p = 0.411) or days of oxygen therapy (r = −0.097; p = 0.517). tables 2, 3 present the results of the multiple poisson regression by transforming beta coefficients from the regression into relative risk calculations, which allowed us to test and evaluate the hypothesis that overnutrition is associated with the severity of viral respiratory infections, by adjusting covariates. when studying all of the patients, we found that only an overweight condition proved to be associated with a greater severity of infection both in length of stay ( also, we analyzed whether assisted ventilation is related to nutritional status and our results suggest that assisted ventilation, as a proxy of severity, was not statistically associated with overnutrition (fisher's exact, p = 0.862). no significant differences were seen between leptin level and nutritional status in both plasma and npa samples (figure 3) . on the other hand, we analyzed npa and plasma leptin level in children according to nutritional status separating by age group analyzed above (less or greater than 6 months) (figure 4) . we found a higher plasma leptin level in obese children <6 months than normal weight children <6 months (figure 4c ; 7.58 vs. 5.12 ng/µl; p < 0.046). obesity and alris constitute two important morbidity factors in the world, and its relationship began to be studied after the influenza h1n1 pandemic (7, 8) . in a multivariate analysis, we found that obese and overweight infants <6 months old were associated with disease severity as defined by longer hospitalization and oxygen therapy treatment. some previous studies have examined the impact of nutritional status on respiratory infections in children. similar to our results, rivera-claros et al. concluded that obesity is a risk factor for worse clinical course of acute lower respiratory tract infections in chilean infants with rsv infections and without chronic disease (9) . a study of 1,129 polish children found that overweight children had a higher risk for acute upper respiratory tract infections (22) . another observational study of 1,116 children hospitalized with community-acquired pneumonia in the united states, found that overweight or obese children were more likely to be admitted to the icu (23). however, it is still unclear why children with overnutrition have proven to be more prone to have severe respiratory infections. we found that in the group of children <6 months, plasma leptin level of obese patients was higher than normal weight patients. it is described that the adipose tissue acts as an endocrine organ, producing adipokines that exert immunomodulatory effects. leptin is a pro-inflammatory adipokine produced primarily in adipose tissue that increases in proportion to the body adiposity. the primary function of leptin is the control of appetite via the hypothalamus, and it has been established as a key hormone in deregulation of immune responses in obese patients (24) . teran-cabanillas et al. (13) showed that leptin induces socs3 overexpression which leads to reduced type i ifn production in obese patients. downstream in the route of type i ifns, the antiviral 2 ′ -5 ′ -oligoadenylate synthetase 1 (oas1) gene is also activated via jak-stat. we analyzed a small number of samples available for expression of oas1 gene. despite not finding significant differences, we found a tendency to decrease the expression of the oas1 gene in overweight and obese patients compared to normal weight children (data not shown). some studies have related leptin level with hepatitis virus infection. in the study by caner et al. (25) leptin levels were found unaltered in children with acute hepatitis a. however, tóth et al. (26) showed an association between the changes of serum leptin levels in children and the severity of hepatitis disease. it remains to be determined whether the infection by respiratory viruses affects leptin levels. we analyzed plasma and npa leptin level according to age (data not shown). we found that children <6 months showed higher plasma leptin level than older children, regardless of nutritional status (5.26 vs. 1.29 ng/µl; p < 0.0001). most of children <6 months (69%) were exclusive breastfeeding at the time of sampling. savino et al. (27) observed a positive association of breast milk leptin values not only with maternal body mass index but also with maternal and infant serum leptin values. on the other hand, it has been described that infants have an increased risk of obesity later in life if they have an overweight or obese mother (28) which can be reduced with the use of lowprotein feedings after 3 months (29) . chile has one of the highest prevalence rates of overweight and obesity in latin america (30) . overweight has 39% prevalence and obesity has 29%; 51% of women in reproductive age have a state of overnutrition (31). according to this, we speculate that the excess weight of mothers causes an increase of leptin in children under 6 months who receive breastfeeding, and this could have an impact on the severity of viral infections. therefore, further studies are needed to study the effect of breast milk leptin in infants. here, we have shown for the first time to our knowledge, that obesity and overweight conditions are associated with viral coinfections in children hospitalized for lrtis. although it is still unclear why this is so, there is some evidence that can start to shed light on this association. it has been reported that the erythrocyte membrane responds very early to modifications of plasma lipoproteins and suggest that in childhood obesity a modified transfer of cholesterol from plasma to erythrocyte membrane may take place (16) . several viruses use cholesterolrich microdomains (lipid rafts) to infect host cells (32) (33) (34) , as has been shown in influenza virus infections. influenza a virus rafts serve as a site for concentrating viral proteins and promoting the re-production of infectious viruses (35) . moreover, it has been reported that rsv uses lipid rafts in the plasma membrane as attachment platforms to enter normal human bronchial epithelial cells (36) . according to this, it is possible that the membrane composition of the respiratory tract cells of children with overnutrition is modified in a way that favors the entry of respiratory viruses. it has long been known that coinfections exhibit a phenomenon called viral interference, where one virus blocks the growth of another (37), thus the high frequency of simultaneous respiratory infections in obese and overweight patients is somewhat surprising and needs further study. consequently, we believe that overnutrition in children could alter the composition of cholesterol in the membrane of the respiratory epithelial tissue, facilitating the entry of viruses in different cells, which could explain the results obtained in this study. an inadequate immune response might also allow a greater entry of these pathogens. obesity provokes an imbalance in the immune system, including an aberrant type i interferon response, which are the key cytokines involved in the early immune response to viral infections (38) . the increase in coinfection related to obesity and overweight conditions may have additional health effects. it was recently reported that asthma in children between 6 and 8 years old is more frequent and severe in those that have been previously hospitalized with viral coinfection-bronchiolitis, compared to those with single infections (39) . the long-term effects of metabolic changes caused by early onset obesity must be explored in followup studies. obese and overweight patients, in addition to exhibiting high levels of viral coinfections as well as more severe infections, also presented lower viral loads than infants with standard weights. through a mathematical model, hrv, the fastestgrowing virus, reduces the replication of the remaining viruses during a coinfection, while piv, the slowest-growing virus is suppressed in the presence of other viruses (40). according to this, coinfections could limit the increase of rsv load because of competition for resources with other viruses. these results are in agreement with those previously described by garcia-mauriño (41) , where high viral load was associated with less severe rsv in children. a possible limitation of our study is that we analyzed npa samples using the rt-pcr method. it is unknown if this highly sensitive molecular technology detects only pathogens that cause lrtis or if some of the viruses detected could be commensal pathogens (42) . some of these detections might represent post infectious shedding. we addressed and controlled this problem by excluding children that had suffered from respiratory infections 2 weeks prior to the study. effective interventions are required to reduce childhood obesity. meanwhile, further research is needed to determine the role of leptin and other immunological markers in the severity of viral infection and to understand the pathophysiology of viral coinfection as it relates to nutritional status as well as to explore the epithelial-specific response to respiratory viruses in primary cells of lean and obese child. the datasets generated for this study are available on request to the corresponding author. the studies involving human participants were reviewed and approved by scientific ethics committee, universidad autónoma de chile. written informed consent to participate in this study was provided by the participants' legal guardian/next of kin. the concept and design of the study was carried out by lf, ga-b, ji, dg-d, and gv. lf, ga-b, and gv obtained the clinical data. ga-b and mr-f conducted the experimental analysis. lf and fz-r carried out the statistical analysis. lf, ga-b, and fz-r wrote the manuscript. all of the authors contributed to the interpretation of the data and critically revised the manuscript, providing important intellectual content and approving the final report. respiratory syncytial virusassociated mortality in hospitalized infants and young children defining the epidemiology and burden of severe respiratory syncytial virus infection among infants and children in western countries respiratory syncytial virus hospitalization and mortality: systematic review and metaanalysis global burden of acute lower respiratory infections due to respiratory syncytial virus in young children: a systematic review and meta-analysis respiratory syncytial virus genomic load and disease severity among children hospitalized with bronchiolitis: multicenter cohort studies in the united states and finland epidemiology and etiology of childhood pneumonia in 2010: estimates of incidence, severe morbidity, mortality, underlying risk factors and causative pathogens for 192 countries a novel risk factor for a novel virus: obesity and 2009 pandemic influenza a (h1n1) obesity and susceptibility to severe outcomes following respiratory viral infection nutritional status and clinical evolution of hospitalized chilean infants with infection by respiratory syncytial virus (rsv) obesity as a risk factor for complications during acute respiratory infections in children financial impact of obesity and bariatric surgery impact of obesity and metabolic syndrome on immunity role of leptin and socs3 in inhibiting the type i interferon response during obesity cutting edge: innate immune response triggered by influenza a virus is negatively regulated by socs1 and socs3 through a rig-i/ifnar1-dependent pathway socs1 is an inducible negative regulator of interferon lambda (ifn-lambda)-induced gene expression in vivo changes of erythrocyte membrane fluidity associated with childhood obesity: a molecular study using fluorescence spectroscopy cholesterol is required for stability and infectivity of influenza a and respiratory syncytial viruses single versus dual respiratory virus infections in hospitalized infants: impact on clinical course of disease and interferon-gamma response impaired immune response in severe human lower tract respiratory infection by respiratory syncytial virus comparison of virological profiles of respiratory syncytial virus and rhinovirus in acute lower tract respiratory infections in very young chilean infants, according to their clinical outcome pandemic a(h1n1)2009 influenza virus detection by real time rt-pcr: is viral quantification useful? predisposition to acute respiratory infections among overweight preadolescent children: an epidemiologic study in poland relationship between body mass index and outcomes among hospitalized patients with community-acquired pneumonia obesity, fat mass and immune system: role for leptin serum leptin levels in children with acute viral hepatitis a serum ghrelin, adipokine and insulin levels in children with acute hepatitis mother and infant body mass index, breast milk leptin and their serum leptin values systematic review and meta-analyses of risk factors for childhood overweight identifiable during infancy lowprotein formula slows weight gain in infants of overweight mothers organización de las naciones unidas para la alimentación y la agricultura américa latina y el caribe. panorama de la seguridad alimentaria y nutricional en américa latina y el caribe measles virus assembly within membrane rafts epstein-barr virus coopts lipid rafts to block the signaling and antigen transport functions of the bcr plasma membrane rafts play a critical role in hiv-1 assembly and release influenza virus assembly and lipid raft microdomains: a role for the cytoplasmic tails of the spike glycoproteins cholesterol-rich microdomains as docking platforms for respiratory syncytial virus in normal human bronchial epithelial cells interference phenomena between animal viruses; a review decreased interferon-alpha and interferon-beta production in obesity and expression of suppressor of cytokine signaling role of viral coinfections in asthma development coinfections of the respiratory tract: viral competition for resources viral load dynamics and clinical disease severity in infants with respiratory syncytial virus infection frequent detection of respiratory viruses without symptoms: toward defining clinically relevant cutoff values we thank the children and their parents, the clinicians and staff of dr. exequiel gonzález cortés hospital and urgencia materno-infantil, clínica dávila for participating in this study. the supplementary material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fped. 2020.00044/full#supplementary-material conflict of interest: the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © 2020 arias-bravo, valderrama, inostroza, reyes-farías, garcia-diaz, zorondo-rodríguez and fuenzalida. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord-257244-gryp0khc authors: edwards, m. r.; walton, r. p.; jackson, d. j.; feleszko, w.; skevaki, c.; jartti, t.; makrinoti, h.; nikonova, a.; shilovskiy, i. p.; schwarze, j.; johnston, s. l.; khaitov, m. r. title: the potential of anti‐infectives and immunomodulators as therapies for asthma and asthma exacerbations date: 2017-08-10 journal: allergy doi: 10.1111/all.13257 sha: doc_id: 257244 cord_uid: gryp0khc asthma is responsible for approximately 25,000 deaths annually in europe despite available medicines that maintain asthma control and reduce asthma exacerbations. better treatments are urgently needed for the control of chronic asthma and reduction in asthma exacerbations, the major cause of asthma mortality. much research spanning >20 years shows a strong association between microorganisms including pathogens in asthma onset, severity and exacerbation, yet with the exception of antibiotics, few treatments are available that specifically target the offending pathogens. recent insights into the microbiome suggest that modulating commensal organisms within the gut or lung may also be a possible way to treat/prevent asthma. the european academy of allergy & clinical immunology task force on anti‐infectives in asthma was initiated to investigate the potential of anti‐infectives and immunomodulators in asthma. this review provides a concise summary of the current literature and aimed to identify and address key questions that concern the use of anti‐infectives and both microbe‐ and host‐based immunomodulators and their feasibility for use in asthma. research in asthma over the last 20 years or more repeatedly shows an overwhelming link between the actions of microorganisms and asthma. 1, 2 from acting as direct pathogens, to educating the immune system and preventing opportunistic pathogen colonization through occupying specific niches, it is now well accepted that viruses, bacteria and fungi are positively associated with asthma onset, 3,4 asthma severity, 5,6 asthma exacerbation (ae) 7, 8 and asthma management 9 and even secondary prevention strategies of asthma (summarized in table 1 ). despite these important associations, the use of antiinfectives (antibiotics, antivirals, antifungals, vaccines) that specifically target known pathogens, or drugs that are based on or exploit microbe-host receptor interactions (toll-like receptor agonists, bacterial lysates) or are immunomodulators (vitamin d), and/or may work in part by altering our associated microbiology (probiotics) are, with the exception of severe asthma, seldom considered in asthma treatment, prevention and guidelines. these treatment options are summarized in figure 1 . task force on anti-infectives in asthma was initiated in 2014 to ask open questions about the potential use of anti-infectives and immunomodulators as treatments for asthma and ae. we thus provide a thorough review of the field ( table 2 for search methods and terms), and specifically, have considered several important points in relation to the use and implementation of anti-infectives in asthma, thus identifying important challenges for the field. in our investigation, we chose to include any such studies of treatment that is based on microorganisms or host molecules, or exploits microbe-host interactions and thus alters the host response or microbiome. in this review, we offer our findings on the above points and consider the role of anti-infectives, immunomodulators and alteration of host microbiology in both asthma development and ae. we include findings from recent clinical trials and discuss the relative merits of these approaches in the light of the many challenges facing asthma research and the state of the art of the field. this review thus provides important insights aimed at young researchers and clinicians and also experienced researchers in the field to inform and stimulate scientific discussion of this important topic. overall, antibiotic use is associated with asthma risk rather than protection at most stages of human development, including pregnancy, 10, 11 early life 12 and childhood, 13 although why this is so is a subject widely debated. in denmark, antibiotic use in pregnancy use was shown to increase risk of ae in five-year-old children by twofold if used in the third trimester. 10 antibiotic exposure in foetal life was associated with an increased risk of asthma in cohort analyses, and this association more than tripled if antibiotics were used to treat respiratory tract infections rather than antibiotics used for either urinary tract or skin infections. these associations decreased, however, when sibling analysis was included (when nonaffected siblings are used as controls). 11 early antibiotic use is also believed to increase asthma risk by two-to threefold in seven-to eight-year-olds. 13 t a b l e 1 common respiratory tract pathogens linked with asthma and ae. this aspect of the field has been thoroughly reviewed elsewhere 2 can produce aerosolized allergen common in asthma epidemics associated with thunder storms, involved in severe asthma with fungal sensitization (safs) the potential negative impact of antibiotics was explored in a birth-cohort study at age 11 from manchester, united kingdom. 12 there was a significantly higher risk of physician-confirmed wheezing after antibiotic prescription and a twofold increase in severe wheeze or ae after antibiotic prescription. in children who wheezed, the risk of ae and admissions to hospital were also significantly increased in the 2 years after the first antibiotic prescription. children who received antibiotics in infancy had significantly lower induction of cytokines from pbmcs (taken at age 11) stimulated ex vivo with viruses, but surprisingly, not bacteria. the authors concluded that an increased susceptibility to viral infections is associ the microbiome describes the bacteria, fungi and other microorganisms that are present in the environment and co-exist within our bodies. the respiratory microbiome can now be studied by 16s rrna sequencing, and certain microbial phyla or genera are thought to be harmful or protective. 5, 16 antibiotics may negatively affect bacterial ecology in early life and this in turn affects asthma development. 10 in retrospective studies, the association between antibiotic use and increased risk of asthma or wheezing in children is further confused due to the potential of reverse causation. 12, 17 in experimental animals, the negative impact of antibiotics has already been shown, and long-term oral antibiotic treatment affects the gut microbiome, which in turn affects lung immunity to influenza virus. 18 indeed, how diet and the gut microbiome affect mucosal immunity including respiratory immunology is now a subject of wide interest; 19, 20 hence antibiotics through regulating the gut microbiota thus directly affect f i g u r e 1 summary of anti-infectives, immunomodulators and microbiome modulators and their methods of action. anti-infectives (such as antibiotics, antifungals and antivirals) directly act on the pathogen or its receptor limiting infection or replication. vaccines work by boosting both innate and importantly, adaptive immune responses (provided by dendritic cells, t and b lymphocytes) to the pathogen providing longlasting protection. immunomodulators including tlr agonists, bl, vitamin d act on the immune system, boosting innate immune responses providing short-term protection. vitamin d may act on underlying immune responses, such as inflammatory responses or allergic inflammation, reducing pathogen-driven inflammation or pathogen-driven enhanced allergic inflammation. microbiome modulators such as probiotics may alter the microbiome of the gut. this may have important downstream effects on the immune system, such as those that affect respiratory immunology and asthma the development of the immune system. the potential of antibiotic-induced changes in the developing microbiome to be directly responsible for asthma or ae risk is yet to be formally proven, however. macrolide antibiotics are a class of antibiotic commonly prescribed for respiratory tract infections or inflammatory respiratory disorders. macrolides may have additional properties to their bacteriostatic function, such as anti-inflammatory 21 and even antiviral activity. 22, 23 despite their ability to inhibit both bacteria and virus infections, only a few studies have tested macrolides as therapies for asthma or ae. guidelines state very little on macrolide use in asthma, and studies have shown positive effects on severe and neutrophilic asthma, yet the evidence supporting this is conflicting. these studies in both stable asthma and ae have been recently reviewed, 24 and the data suggest macrolide-responsive subgroups (eg neutrophilic asthma) may exist. 25, 26 additionally, recent studies also report a reduction in severe lrti rates 27 and asthma-like symptoms 28 antibiotics are associated with asthma risk and their use should be discouraged for asthma or wheeze-like illnesses. a possible explanation for this association is that antibiotics affect the microbiome in a negative way and thus increase susceptibility to disease. with the realization that the microbiome is key in controlling host immunity early in life, and the design of supportive animal studies that have modelled this association and identified protective genera, 31 this idea has merit but the overall hypothesis remains to be thoroughly tested in human clinical models. antibiotics may also be associated with asthma via other, as yet to be identified mechanisms, and likely involve reverse causation. the narrower antibacterial spectrum of some macrolide antibiotics, combined with their other advantageous properties, suggests that these may have use in ae but their use remains controversial. the prophylaxis with intramuscular/intravenous antibody palivizumab has been shown to effectively decrease respiratory syncytial virus (rsv)-induced bronchiolitis-related hospitalization, need for mechanical ventilation and recurrent wheezing. 32 interestingly, it has also decreased recurrent wheezing induced by other viruses postbronchiolitis. the second-generation monoclonal antibody motavizumab has also markedly reduced hospitalization with rsv. 33 but the development programme has been discontinued. 38 only a few patients benefit from the m2 ion channel inhibitors, amantadine and rimantadine, drug resistance is high, and they have significant adverse events. 39 neutralizing antibodies against rsv, coronaviruses and influenza viruses are being developed. inhibiting virus replication through interfering with viral enzymes active within cells poses additional problems in drug discovery; however, several useful inhibitors for respiratory tract viruses have found their way into phase i/ii clinical trials. few, however, have been specifically tested in asthma. favipiravir is a novel antiviral compound that selectively and potently inhibits the rna-dependent rna polymerase of many rna viruses including influenza virus, enteroviruses and paramyxoviruses. 40 a phase iii clinical trial of favipiravir for influenza therapy has been just completed in japan and the united states. rupintrivir is a potent, irreversible inhibitor of rv 3c protease. it has a broad antipicornaviral spectrum, but the development programme has been discontinued. 41 the nucleoside inhibitor ribavirin is a synthetic purine nucleoside analogue exhibiting antiviral activity against a broad range of both dna and rna viruses in vitro. 42 it is the only antiviral agent currently available against rsv infection, but its use has many concerns. als-008176 is an oral rsv replication inhibitor (a cytidine nucleoside analogue). in randomized, double-blind, clinical trial in healthy adults inoculated with rsv, more rapid rsv clearance and a greater reduction in viral load were observed in the groups of patients treated with als-008176 than in the placebo group. 43 small interfering rna (sirna) 44 infected with wild-type rsv. 46 it was well tolerated, effective as well as decreased the incidence and progression of bronchiolitis obliterans syndrome in lung transplant recipients with naturally occurring rsv infection. 47 the development of cidofovir and its derivative, brincidofovir, broad-spectrum antivirals active against five families of dsdna viruses (including adenoviruses), are currently in phase iii clinical trials. 48 the neuraminidase inhibitors prevent influenza virus release from infected cells and infection of adjacent cells. oral oseltamivir and inhaled zanamivir are recommended for the treatment and chemoprophylaxis of influenza in children and adults. 49 inhaled laninamivir has shown good safety and efficacy profiles in the treatment for influenza in patients with chronic respiratory tract diseases and has been approved in japan. 50 single dose of intravenous peramivir has been shown to be noninferior to oseltamivir and to have good safety profile also in patients with chronic respiratory tract diseases. 51 biologically, interferons (ifns) are induced within hours of infection 52 and consequently induce the expression of hundreds of antiviral effecter molecules blocking virus replication. 53 the effectiveness of itraconazole in the treatment for abpa has been confirmed in two randomized, placebo-controlled trials 60, 61 leading to the recommendation for azole use in asthma-abpa by the cochrane collaboration. 62 pooled data from these studies suggest itraconazole is effective in around 60% of asthma-abpa patients. 63 newer triazoles including voriconazole and posaconazole have also been studied in abpa with promising results. however, the greater incidence of drug toxicity with voriconazole, and substantial financial costs of both voriconazole and posaconazole limit their current widespread use. more recently, the use of azoles has also been studied in safs. in the first such study by denning et al, 64 subjects with over half having to discontinue treatment due to bronchospasm. 66 consequently, the prospect of amphotericin b use in asthma remains unlikely. the majority of reports to date have investigated the use of antifungal agents in adult asthmatics; however, the potential for their use in children has also been highlighted in a recent paediatric study describing sensitization to fungal allergens in 59% of severe asthmatic children. 67 unfortunately, reports of azole use in asthmatic children are limited to a few subjects only leading to the current joint ers/ats guideline recommending the consideration of treatment only after detailed evaluation in a specialist severe asthma centre. 68 in summary, to date, there have only been a handful of placebo-conheterogeneity of asthma continues to develop, it is possible that subgroups within abpa/safs will emerge in whom treatment with antifungal therapy will be predictably beneficial. however, we still remain some distance away from this goal at present. influenza infections can precipitate acute aes and may be more severe among asthma patients 69 more studies are needed to validate these findings. a comparison of laiv with tiv also failed to show significant differences in ae numbers in adults or children (>6 years). 70 thus, the potential role of influenza vaccines in ae prevention requires further in-depth study, in virologically confirmed influenza and with improved definition and characterization of asthmatic subgroups. aes. three large cross-over trials show no evidence of increases in aes in the two weeks following tiv in adults or children. 71 concerns that laiv could increase aes, wheezing episodes and hospitalizations in children 70 were allayed the absence of an association of laiv with ae in high-risk children. 72 f i g u r e 3 challenges and unknowns facing new treatments for asthma exacerbations that target respiratory pathogens. challenges facing the design and implementation of therapies that directly target pathogens or their biology (in blue) include site of infection and site of drug delivery, route of delivery, specific mechanism of action, the drug's pharmacodynamics (pd) and pharmacokinetics (pk) and also the patient demographic (pt), subset or specific endophenotype of asthma concerned. these challenges can at least in part be addressed by preclinical studies and are often taken into account during drug design. unknowns are also identified (in red) and can be model or pathogen specific. these include, but are not limited to, the window of therapeutic opportunity (dt) for therapeutic treatments, which defines the time between infection and onset of clinical disease (lrti symptoms for asthma), and importantly, this variable thus describes the window in which suppressing replication in theory will suppress symptoms and/or clinical disease. how pathogen load affects clinical disease is also controlled by a second variable (dl), which defines the quantity of pathogen that has to be affected to observe a quantitative change in clinical disease or symptoms. the unknown c defines a comparison, between a new drug (eg antiviral) versus the standard treatment (eg gc). this unknown is important as regulatory authorities will not approve a new drug if does not show improvements or a better safety profile compared with the standard treatment already available. the unknown d represents duration of treatment; this takes into account other variables that are often difficult to predict and include the possible effects of secondary infections (eg bacterial co-infection), drug resistance (eg as seen with macrolides), plasticity of endophenotype treated and other complexities that can impact on clinical disease after pathogen load is decreased as defined by dl. theoretical relationships between pathogen and load and clinical disease are based on human challenge studies with rv, in asthmatic individuals 57,58 the presence of streptococcus pneumoniae (pneumococcus) has been linked to ae, and in children, early-life pneumococcal colonization is associated with an increased risk of wheezing and asthma later in childhood. 4 furthermore, asthma is a risk factor for invasive pneumococcal disease (ipd), 75 warranting pneumococcal vaccination, which is recommended for all people with asthma over 6 years of age if they did not receive routine childhood pneumococcal immunization. 76 the there has been concern over potential pro-allergic and pro-asthmatic effects of childhood immunizations, in particular regarding measles, mumps and rubella vaccine (mmr). however, the suspected association between mmr and asthma (since been discredited) was based on a small study comparing anthroposophic to nonanthroposophic children and was not confirmed in a subsequent multinational study in these populations. 82 a wealth of studies has also failed to show evidence of pro-asthmatic effects of vaccination against poliovirus, pertussis, tetanus, hepatitis b or haemophilus influenzae. in contrast, significant evidence suggests antiasthmatic effects of childhood vaccinations. inverse associations between childhood asthma prevalence and high cumulative vaccine doses, 83 pertussis vaccination 84 and mmr 85 have been found and reduced asthma hospitalization rates and medication use in mmr-vaccinated children. 86 the immunogenic potential of different vaccine formulations in asthma has been questioned, but normal antibody responses to pertussis, varicella, hepatitis b, measles and rubella vaccination have been reported. mumps antibody titres after mmr were lower and measles antibodies waned more quickly from 9 years of age in asthmatics. treatment with inhaled glucocorticoids (gcs) did not affect the antibody response to hepatitis b or varicella immunization, but these were impaired by oral gc therapy. the vaccinations discussed are safe and effective in asthma, may help prevent asthma development, and pneumococcal and annual influenza vaccination in particular should be offered to asthmatics. future vaccines against rv and rsv, which are the main triggers for aes and have been linked to asthma inception, should help reduce asthma morbidity and mortality. host responses bacterial lysates (bl) have been extensively used in europe to effectively reduce the number of seasonal acute respiratory illnesses (ari). bl are microbial products that when given orally, may exhibit certain immunostimulatory and immunomodulatory effects. 87 their effectiveness was confirmed in numerous interventional trials (reviewed in ref. 88) . bl have been successfully tested in preventing wheezing attacks provoked by aris in preschool children demonstrating a 38% reduction in symptomatic wheezing and a decrease in the number of urti. 89 there is only one interventional clinical study to date, demonstrating a promising antiallergic potential of an orally applied bl. 90 therefore, one may speculate that bacteria-derived preparations may become an interesting class of immune modulators for the future. recognition of invading microbes is controlled by a range of innate pattern recognition receptors including tlrs which are directed at highly conserved molecular motifs expressed on the invading pathogen. this ancient surveillance network provides a form of first line of defence in the airway. 91 signalling through tlrs produces a broad range of pro-inflammatory cytokines, chemokines and importantly, antimicrobial proteins. therefore, precise targeting of individual tlr pathways could provide a mechanism of promoting specific immunity, allowing for tailored immunomodulation, an approach that is highly attractive in chronic diseases such as asthma where numerous immunological disparities are evident. withdrawal of inhaled gcs whilst asthma conditions remain stable or even improved. 101 the proposed mechanism is the restoration of the th1/th2 balance. however, supporting evidence is yet to be provided. a follow-up, double-blind, placebo-controlled study carried out in poorly controlled, moderate-to-severe asthmatics where cyt003a was administered as an add-on to current gcs and b 2agonist therapy showed no significant advantage over placebo in relation to the primary outcome of change from baseline in asthma control questionnaire (acq) or in secondary outcomes of change from baseline in prebronchodilator forced expiratory volume (fev 1 ). 102 these data suggest that whilst cyt003a may have a use in initial control of disease in gc-sensitive patients, it has no efficacy as an add-on therapy for more severe patients. in addition, more prolonged administration of the tlr9 agonist, assessing its ability to readdress the supposed th2 skewing, was not conducted. additional concerns stem from recent reports of potential impairment of tlr9 function in pbmcs in severe asthmatic patients. 103 evidence is accumulating on the potential of vitamin d in immunoregulation, particularly in lymphocyte function and cytokine production, suggesting its potential in modifying asthma incidence and severity. 104 the association between low levels of vitamin d and asthma has been supported by many observational and epidemiologic studies. 105 a recent meta-analysis of epidemiological studies demonstrated a positive association of vitamin d deficiency and asthma (rr 1.68 95% ci 1.3-2.2). 106 parallel-conducted, prospective, observational studies on vitamin d supplementation in infancy, however, showed rather conflicting data and do not support these implications. 107 other studies in pregnant woman, including two large clinical trials, did not find protective effects on offspring wheeze. 108, 109 interpretations of existing evidence argue an advantage of vitamin d supplementation. 107 115 however, interventional trials in infants receiving both pre-and probiotics in allergy and/or asthma prevention provided equivocal results. 116 whilst moderate positive effects in infant eczema were found, a lack of evidence that probiotics prevent any other allergy including asthma in three other similar meta-analyses remains a matter of concern. [117] [118] [119] in view of two well-conducted meta-analyses, an opportunity of asthma/wheezing prevention with probiotics seemed to not be plausible. 118, 119 one may speculate, however, that the studies to date may not have used the right probiotic, the right dose, the right timing or duration and/or population. therefore, more studies are still needed. evidence to date suggested that modulation of the gut microbiome may represent an interesting therapeutic or preventative opportunity for the prevention of allergic asthma and ae, whilst clinical trials do not confirm initial enthusiastic expectations. in view of the recent systematic reviews, probiotics cannot be recommended as adjunctive therapy for asthma or asthma prevention. though, recent technological developments that permit identification of the most promising microbial strains and their products that may exhibit more profound positive effects will keep this area active and interesting to follow. with the exception of antibiotics and antifungals, current guidelines do not take into account the potential of specific or broader-spectrum anti-infectives or immunomodulatory agents in asthma or ae. despite wide interest and active research in this area, the basis for this is likely due to a lack of clinical studies that allow robust or clear conclusions to be made regarding efficacy. for the clinical studies that have been performed with available anti-infectives or immunomodulatory agents, conclusions are often contradictory or show a lack of robust evidence supporting an effect. reasons for these outcomes include a small number of studies, differences in study design making direct comparisons difficult or studies that are underpowered for primary or secondary endpoints, or subgroup analysis. as shown in table 3 there is also a substantial body of literature investigating preclinical development of anti-infectives and immunomodulatory drugs and preparations in animal models, with many studies endeavouring to better understand the scientific principles behind their mechanism (s) of action. this is an absolute necessity in the anti-infective drug discovery pipeline; however, there is some confusion regarding how best to progress these anti-infectives in these studies in clinical trials, or alternatively, how interpretations of preclinical work can best inform on future clinical trials is a subject of wide debate. much consideration still needs to be given to how future drug targets related to anti-infectives are progressed, and how drug discovery programmes are best implemented to exploit these. this manuscript is the result of a task microbes and mucosal immune responses in asthma the microbiology of asthma short-and long-term efficacy of prednisolone for first acute rhinovirus-induced wheezing episode childhood asthma after bacterial colonization of the airway in neonates disordered microbial communities in asthmatic airways airway microbiota and bronchial hyperresponsiveness in patients with suboptimally controlled asthma community study of role of viral infections in exacerbations of asthma in 9-11 year old children the september epidemic of asthma exacerbations in children: a search for etiology reduced clinic, emergency room, and hospital utilization after home environmental assessment and case management use of antibiotics during pregnancy increases the risk of asthma in early childhood antibiotics in fetal and early life and subsequent childhood asthma: nationwide population based study with sibling analysis assessing the association of early life antibiotic prescription with asthma exacerbations, impaired antiviral immunity, and genetic variants in 17q21: a population-based birth cohort study does the use of antibiotics in early childhood increase the risk of asthma and allergic disease? genetic variants regulating ormdl3 expression contribute to the risk of childhood asthma rhinovirus wheezing illness and genetic risk of childhood-onset asthma exposure to environmental microorganisms and childhood asthma a longitudinal analysis on the association between antibiotic use, intestinal microflora, and wheezing during the first year of life microbiota regulates immune defense against respiratory tract influenza a virus infection a brave new world: the lung microbiota in an era of change lung microbiota promotes tolerance to allergens in neonates via pd-l1 azithromycin distinctively modulates classical activation of human monocytes in vitro azithromycin induces anti-viral responses in bronchial epithelial cells novel antiviral properties of azithromycin in cystic fibrosis airway epithelial cells the role of macrolides in asthma: current evidence and future directions a trial of clarithromycin for the treatment of suboptimally controlled asthma azithromycin for prevention of exacerbations in severe asthma (azisast): a multicentre randomised double-blind placebo-controlled trial early administration of azithromycin and prevention of severe lower respiratory tract illnesses in preschool children with a history of such illnesses: a randomized clinical trial azithromycin for episodes with asthma-like symptoms in young children aged 1-3 years: a randomised, double-blind, placebo-controlled trial metabolic and metagenomic outcomes from early-life pulsed antibiotic treatment short-term antibiotic treatment has differing long-term impacts on the human throat and gut microbiome maternal tlr signaling is required for prenatal asthma protection by the nonpathogenic microbe acinetobacter lwoffii f78 respiratory syncytial virus and recurrent wheeze in healthy preterm infants efficacy of motavizumab for the prevention of respiratory syncytial virus disease in healthy native american infants: a phase 3 randomised double-blind placebo-controlled trial oral gs-5806 activity in a respiratory syncytial virus challenge study generation and characterization of alx-0171, a potent novel therapeutic nanobody for the treatment of respiratory syncytial virus infection oral pleconaril treatment of picornavirus-associated viral respiratory illness in adults: efficacy and tolerability in phase ii clinical trials antiviral activity of broad-spectrum and enterovirus-specific inhibitors against clinical isolates of enterovirus d68 efficacy of tremacamra, a soluble intercellular adhesion molecule 1, for experimental rhinovirus infection: a randomized clinical trial amantadine and rimantadine for influenza a in children and the elderly favipiravir (t-705), a novel viral rna polymerase inhibitor phase ii, randomized, double-blind, placebo-controlled studies of ruprintrivir nasal spray 2-percent suspension for prevention and treatment of experimentally induced rhinovirus colds in healthy volunteers mechanisms of action of ribavirin against distinct viruses activity of oral als-008176 in a respiratory syncytial virus challenge study rna interference-mediated silencing of the respiratory syncytial virus nucleocapsid defines a potent antiviral strategy small interfering rnas targeted to interleukin-4 and respiratory syncytial virus reduce airway inflammation in a mouse model of virus-induced asthma exacerbation a randomized, double-blind, placebo-controlled study of an rnai-based therapy directed against respiratory syncytial virus rna interference therapy in lung transplant patients infected with respiratory syncytial virus new drug on the horizon for treating adenovirus neuraminidase inhibitors in patients with underlying airways disease a randomized double-blind controlled study of laninamivir compared with oseltamivir for the treatment of influenza in patients with chronic respiratory diseases intravenous peramivir for treatment of influenza a and b virus infection in high-risk patients respiratory virus induction of alpha-, beta-and lambda-interferons in bronchial epithelial cells and peripheral blood mononuclear cells a diverse range of gene products are effectors of the type i interferon antiviral response the effect of inhaled ifn-beta on worsening of asthma symptoms caused by viral infections. a randomized trial role of the interleukin (il)-28 receptor tyrosine residues for antiviral and antiproliferative activity of il-29/interferonlambda 1: similarities with type i interferon signaling il-28a (ifn-lambda2) modulates lung dc function to promote th1 immune skewing and suppress allergic airway disease il-33-dependent type 2 inflammation during rhinovirus-induced asthma exacerbations in vivo rhinovirus-induced lower respiratory illness is increased in asthma and related to virus load and th1/2 cytokine and il-10 production the ambitious '95-95 by 2025' roadmap for the diagnosis and management of fungal diseases a randomized trial of itraconazole in allergic bronchopulmonary aspergillosis anti-inflammatory effect of itraconazole in stable allergic bronchopulmonary aspergillosis: a randomized controlled trial azoles for allergic bronchopulmonary aspergillosis associated with asthma treatment options in severe fungal asthma and allergic bronchopulmonary aspergillosis randomized controlled trial of oral antifungal treatment for severe asthma with fungal sensitization: the fungal asthma sensitization trial (fast) study effectiveness of voriconazole in the treatment of aspergillus fumigatus-associated asthma (evita3 study) efficacy and safety of nebulised amphotericin b (nab) in severe asthma with fungal sensitisation (safs) and allergic bronchopulmonary aspergillosis (abpa) fungal sensitization in childhood persistent asthma is associated with disease severity international ers/ats guidelines on definition, evaluation and treatment of severe asthma influenza burden for children with asthma. pediatrics vaccines for preventing influenza in people with asthma the safety of inactivated influenza vaccine in adults and children with asthma vaccinating high-risk children with the intranasal liveattenuated influenza vaccine: the quebec experience safety of live attenuated influenza vaccine in young people with egg allergy: multicentre prospective cohort study induction of broadly reactive anti-hemagglutinin stalk antibodies by an h5n1 vaccine in humans asthma as a risk factor for invasive pneumococcal disease prevention of pneumococcal disease among infants and children -use of 13-valent pneumococcal conjugate vaccine and 23-valent pneumococcal polysaccharide vaccinerecommendations of the advisory committee on immunization practices (acip) paediatric asthma and pneumococcal vaccination pneumococcal vaccine for asthma impact of pneumococcal vaccination on pneumonia rates in patients with copd and asthma respiratory syncytial virus disease in infants despite prior administration of antigenic inactivated vaccine cross-serotype immunity induced by immunization with a conserved rhinovirus capsid protein allergic disease and sensitization in steiner school children transient suppression of atopy in early childhood is associated with high vaccination coverage early bcg and pertussis vaccination and atopic diseases in 5-to 7-year-old preschool children from augsburg, germany: results from the miriam study asthma and allergy in children with and without prior measles, mumps, and rubella vaccination measles-mumps-rubella vaccination and asthma-like disease in early childhood immunoregulatory and immunostimulatory responses of bacterial lysates in respiratory infections and asthma immunostimulants for preventing respiratory tract infection in children the immunostimulant om-85 bv prevents wheezing attacks in preschool children oral application of bacterial lysate in infancy decreases the risk of atopic dermatitis in children with 1 atopic parent in a randomized, placebo-controlled trial the airway epithelium in asthma safety of the intranasal toll-like receptor 4 agonist crx-675 in allergic rhinitis repeated intranasal tlr7 stimulation reduces allergen responsiveness in allergic rhinitis early clinical evaluation of the intranasal tlr7 agonist gsk2245035: use of translational biomarkers to guide dosing and confirm target engagement vtx-1463, a novel tlr8 agonist for the treatment of allergic rhinitis immunostimulatory sequences regulate interferon-inducible genes but not allergic airway responses anti-inflammatory effects of pre-seasonal th1-adjuvant vaccine to parietaria judaica in asthmatics long-lasting effect of a monophosphoryl lipid-adjuvanted immunotherapy to parietaria. a controlled field study use of a-type cpg oligodeoxynucleotides as an adjuvant in allergen-specific immunotherapy in humans: a phase i/iia clinical trial assessment of clinical efficacy of cyt003-qbg10 in patients with allergic rhinoconjunctivitis: a phase iib study the novel tlr-9 agonist qbg10 shows clinical efficacy in persistent allergic asthma cyt003, a tlr9 agonist, in persistent allergic asthma -a randomized placebo-controlled phase 2b study toll-like receptor 9 dependent interferon-alpha release is impaired in severe asthma but is not associated with exacerbation frequency mechanisms underlying the regulation of innate and adaptive immunity by vitamin d cord serum 25-hydroxyvitamin d correlates with early childhood viral-induced wheezing association between vitamin d deficiency and insufficiency and the risk of childhood asthma: evidence from a meta-analysis vitamin d as an adjunctive therapy in asthma. part 2: a review of human studies effect of prenatal supplementation with vitamin d on asthma or recurrent wheezing in offspring by age 3 years: the vdaart randomized clinical trial effect of vitamin d3 supplementation during pregnancy on risk of persistent wheeze in the offspring: a randomized clinical trial efficacy of high-dose vitamin d in pediatric asthma: a systematic review and meta-analysis double-blind randomised placebo-controlled trial of bolus-dose vitamin d3 supplementation in adults with asthma (vidias) vitamin d supplementation for the prevention of childhood acute respiratory infections: a systematic review of randomised controlled trials probiotics for preventing acute upper respiratory tract infections probiotics for the prevention of pediatric upper respiratory tract infections: a systematic review probiotic-induced suppression of allergic sensitization and airway inflammation is associated with an increase of t regulatory-dependent mechanisms in a murine model of asthma probiotics for prevention of atopic diseases in infants: systematic review and meta-analysis probiotics for the prevention of allergy: a systematic review and meta-analysis of randomized controlled trials probiotic supplementation during pregnancy or infancy for the prevention of asthma and wheeze: systematic review and meta-analysis probiotic administration in early life, atopy, and asthma: a meta-analysis of clinical trials viruses and bacteria in acute asthma exacerbationsa ga(2) len-dare systematic review association of azithromycin with mortality and cardiovascular events among older patients hospitalized with pneumonia the health and economic benefits associated with pneumococcal vaccination of elderly persons with chronic lung disease asthma in the elderly: a study of the role of vitamin d eaaci anti-infectives in asthma and asthma exacerbations task force. the potential of anti-infectives and immunomodulators as therapies for asthma and asthma exacerbations interests in respect to this publication. all authors participated in the discussion and approved the final version of this position paper. key: cord-317619-o7qfugjw authors: nye, steven; whitley, richard j.; kong, michele title: viral infection in the development and progression of pediatric acute respiratory distress syndrome date: 2016-11-24 journal: front pediatr doi: 10.3389/fped.2016.00128 sha: doc_id: 317619 cord_uid: o7qfugjw viral infections are an important cause of pediatric acute respiratory distress syndrome (ards). numerous viruses, including respiratory syncytial virus (rsv) and influenza a (h1n1) virus, have been implicated in the progression of pneumonia to ards; yet the incidence of progression is unknown. despite acute and chronic morbidity associated with respiratory viral infections, particularly in “at risk” populations, treatment options are limited. thus, with few exceptions, care is symptomatic. in addition, mortality rates for viral-related ards have yet to be determined. this review outlines what is known about ards secondary to viral infections including the epidemiology, the pathophysiology, and diagnosis. in addition, emerging treatment options to prevent infection, and to decrease disease burden will be outlined. we focused on rsv and influenza a (h1n1) viral-induced ards, as these are the most common viruses leading to pediatric ards, and have specific prophylactic and definitive treatment options. acute respiratory distress syndrome (ards) was first described in 1967 in adults presenting with tachypnea, hypoxemia, and decreased pulmonary compliance (1). since then, the understanding, diagnosis, and management of ards has advanced greatly, with most research performed in adults. for more than two decades, pediatric health-care providers have relied heavily on adult-derived diagnostic criteria (2, 3). in recent years, the pediatric acute lung injury consensus conference (palicc) has provided age-specific diagnostic and management guidelines for pediatric ards (4, 5). with these new pediatric-specific recommendations, future research in pediatric ards looks promising, as new areas for investigation have been identified (5). one area of interest warranting further exploration is the role of viral infection in the development and progression of pediatric ards. while some information is available specific to pediatrics, much of our understanding continues to be derivative from adult data. this review outlines what is known about ards attributable to viral infections, specifically respiratory syncytial virus (rsv) and influenza a (h1n1) virus, as well as viral-specific treatment options. epidemiology of rsv and influenza-induced ards accurate incidence outcomes reporting for pediatric ards are limited by the changing definition for ards. studies using the 1994 american european consensus conference (aecc) definition (2) outlined epidemiology and outcomes for both acute lung injury (ali) and ards as separate viral infection and pediatric ards frontiers in pediatrics | www.frontiersin.org november 2016 | volume 4 | article 128 entities (6). however, both the berlin definition (3) and the palicc recommendations (5) no longer identify ali as a separate entity, instead categorizing ards as mild, moderate, or severe. since these guidelines are recent, few studies have been conducted to determine the incidence of ards utilizing these new criteria (7, 8) . in addition, management for ards has also changed over the years, with improved outcomes demonstrated with lung-protective mechanical ventilation strategies (9, 10). pediatric studies conducted using the aecc definition define an incidence ranging from 2.96 to 12.8 per 100,000 children for all causes of ali/ards, with the predominant etiology reported to be secondary to pneumonia, with or without systemic infection (11). in a more recent study, using the berlin definition, barreira et al. reported that ards accounts for 10% of all picu admissions, and was associated with a high mortality rate of 24.5% (7). while the overall incidence of respiratory virus infection, in particular rsv and influenza a (h1n1) virus, leading to lower respiratory tract disease is widely studied (12, 13), the frequency of progression to pediatric ards has yet to be clearly determined. respiratory syncytial virus infection has been recognized as an important cause of lower lung disease. in an earlier study, dahlem et al. reported 15.9% of ards cases due to rsv-related infection, but specific mortality for this group was not reported (14) . in a more recent study by 19 .8% of patients admitted to the picu with ards tested positive for rsv, with a reported mortality of 13.7% (15). a 12-year study in the netherlands by schene et al. analyzed 155 patients mechanically ventilated for rsv with 129 (83%) patients progressing to ards (16). of those with ards, 38% were found to have a bacterial coinfection. the mortality rate was not used as a measure of outcome and therefore not reported. since the beginning of the influenza a (h1n1) virus pandemic of 2009, influenza-related respiratory failure has become a notable cause of ards (17). during the pandemic and postpandemic era, it is clear that children are particularly vulnerable to disease even if they had been previously healthy (18). in 2009 alone, more than 43,000 cases were reported, with an estimated 73% of cases occurring in patients less than 24 years of age (13). in previous years, influenza-related pediatric deaths averaged 82 annually but increased to 317 during the 2009 pandemic (19). while post-pandemic studies suggest a decrease in influenza a (h1n1) virus disease severity and burden (20, 21), it continues to be a significant cause of severe illness and pediatric ards (22). in a retrospective analysis of adult patients within the german ards network, investigators reported that 32% of ards patients were influenza a (h1n1) virus positive (23). in another pediatric study in india, kinikar et al. reported that 18% of patients hospitalized with confirmed influenza a (h1n1) virus developed ards (24). in this study, 9 of the 15 children who died were found to have histologic pulmonary findings reflective of ards at autopsy. in argentina, farias et al. studied 147 patients admitted with respiratory failure due to influenza a (h1n1) virus and found 118 (80%) met criteria for pediatric ards, 45% of whom died within 28 days after picu admission (25). the second, less common novel influenza virus, avian influenza a (h5n1) virus, was first identified in 1998 (26) and remains a common cause of severe respiratory disease (27). kawachi et al. reviewed pediatric patients with ards over a 4½-year period in vietnam and found 12 (32.4%) of the 37 patients to have confirmed infection with the highly pathogenic influenza a (h5n1) virus (28). they described rapid progression of disease to ards with nine (75%) resulting in death. further investigation has led to improved understanding of transmission, predominantly direct avian-to-human transmission with significant risk in handling sick or dead poultry. type 2 pneumocytes and macrophages are the primary lung target (29). together, these studies demonstrate that rsv and influenza virus infection play a role in the development of pediatric ards. however, to better understand the disease burden, future studies should seek to more clearly identify the rate of occurrence of primary viral-induced ards, as well as incidence of secondary viral-induced lung injury. furthermore, in patients who have a coinfection with a bacterial pathogen, it may be hard to determine whether the virus or bacteria played the inciting role in the development and progression of pediatric ards. taken together, the overall mortality attributable to either rsv or influenza is relatively similar; thus, it is more likely the syndrome of ards and associated pathology that is responsible for outcome. while rsv and influenza a (h1n1) virus are the most commonly reported viruses leading to pediatric ards, other viral pathogens are worth mentioning. typically viral infections leading to respiratory failure in the icu are separated as community acquired and nosocomial (30). community acquired viral infections include both seasonal and pandemic pathogens (31). seasonal viruses most commonly include rsv, non-pandemic influenza, rhinoviruses, parainfluenza, adenovirus, coronaviruses, and human metapneumovirus (hmpv). seasonal viruses remain the most frequent cause of childhood community acquired pneumonia (32). the most common etiology of pediatric ards is primary pneumonia, with or without systemic infection (15). it can then be assumed that viral infections may play an important role in development of pediatric ards. however, determining an accurate estimate of the disease burden of viral-induced pediatric ards will be difficult, as many simple viral infections can progress to coinfection with the second virus or a bacterial pathogen. as will be detailed below, with the development of multiplex pcr diagnostic platforms that identify multiple viral agents, further insight into coinfections will develop. a single-center adult study reported seven patients developing ards from adenovirus, four of whom died (33). hung and lin described a case of a 9-month-old male with adenoviral ards requiring extracorporeal membrane oxygenation (ecmo) (34 cases of severe disease in immunocompetent patients are becoming increasingly reported (38). at an international level, community acquired, novel pathogens have been recognized as a significant cause of ards in the last 15-20 years (31). in 2015, the world health organization (who) developed a panel of experts to prioritize emerging pathogens to likely cause severe outbreaks in the near future, and for which little or no preventative or curative treatments are available (39). the list includes two novel coronaviruses, severe acute respiratory syndrome (sars)-cov and mers-cov, which are widely recognized as noteworthy causes of ards. in 2002, sars-cov led to the development of sars in china (40). affecting patients of all ages, sars led to significant mortality worldwide within a few months (41). a large number of infected patients developed severe complications, with 20% developing ards (42). however, reported sars cases have ceased since 2004 as the spread of infection has subsided (43). more recently, the second novel coronavirus, mers-cov, led to the middle east respiratory syndrome (44). clinical symptoms range from mild upper respiratory symptoms to severe pneumonia and ards, septic shock, and multi-organ failure (45) and carry an estimated mortality of 40% (46) . this virus continues to be a substantial etiology of ards with high mortality as no definitive prevention or treatment other than supportive care has been identified (47) . with the unpredictable nature of epidemics and pandemics, these novel viruses illustrate the need to improve our understanding of viral progression to ards in order to advance management and reduce mortality. aside from community acquired viral infections, nosocomial infections are an important cause of respiratory illness, and can lead to ards in both adults and children. in mechanically ventilated adults, reactivation of latent herpes simplex virus (hsv) in the oropharynx can potentially lead to lower respiratory tract infection and ards (48, 49) . however, the pathogenicity of reactivated hsv lower respiratory tract infection may not be that straightforward as it remains unclear whether hsv contributes to worsening illness or whether reactivation occurs due to the underlying critical illness (50). schuller et al. found higher levels of clinical severity and mortality in critically ill immunocompetent adults with hsv-1 infection compared to immunocompromised patients with hsv-1 (51). the true extent of hsv reactivation in critically ill children leading to respiratory illness has yet to be studied. hennus et al. described two previously healthy children presenting with respiratory failure due to human herpes virus 6 (hhv-6), and later workup revealed an immunodeficiency in both patients (52) . a separate pediatric case reported a child with hsv ards resulting in need of extracorporeal support (53) . these cases illustrate the rare, but possible severe infection and progression to ards from hsv-1. finally, many seasonal and pandemic viruses are a potential nosocomial infectious risk secondary to either a health-care provider or air-ventilation transmission. in a study over two influenza seasons in germany, huzly et al. reported a rate of nosocomial transmission of 24% (2012-2013) and 20% (2013-2014) (54) . specific guidelines are available to help prevent transmission of infectious pathogens through isolation precautions (55) . however, dhar et al. found that an increased number in patients placed on contact isolation led to a decrease in compliance with isolation precautions (56) . decreasing nosocomial transmission within care areas for critically ill patients is an important area for improvement. the palicc has recently provided guidelines for diagnosing pediatric ards (5). the new guidelines define important diagnostic criteria, including age, timing, origin of edema, imaging, and oxygenation. patients with perinatal lung disease are excluded, and pards criteria must be met within 7 days of a clinical insult. the cause of respiratory failure must not be explained by heart failure or fluid overload, and must be evidenced by new pulmonary infiltrate(s) on chest radiograph consistent with parenchymal disease. finally, the use of oxygenation index is preferred over the pao2:fio2 (pf) ratio in determining the severity of pards in mechanically ventilated patients, while the pf ratio or spo2:fio2 (sf) ratio may be used in patients requiring non-invasive ventilation (5). the same clinical guideline is used in the diagnosis of viral-induced pediatric ards. currently, there are several different types of laboratory tests that are commercially available for diagnosis. most clinical laboratories utilize antigen detection tests, which consist of multiple steps to accurately identify a single virus (57) , with or without cell culture. it is worth noting that over the past 20 years, the development and refinement of real-time reverse transcriptase polymerase chain reaction (rt-pcr) has enhanced the clinician's ability to diagnose an array of viruses rapidly and accurately. multiplex rt-pcr testing analyzes a single sample for multiple viral agents and subtypes simultaneously, producing sensitive and specific results in a short period of time (58) . even with the 2009 influenza a (h1n1) virus pandemic, the centers for disease control and prevention (cdc) quickly modified standard pcr assays to detect the new virus (59) . a challenge to routine rt-pcr testing in all patients who present with viral symptoms is the prohibitive cost, need for specialized equipment, and the relatively longer time between sampling and availability of results (60) . furthermore, pcrs detect viral genes that are used as a surrogate measurement of whole virions. in some instances, viral gene detection may actually reflect non-replicating, non-infectious virions. newer rapid point-of-care pcrs are currently being developed, but their implications for clinical decision making remain uncertain (61) . in addition, rapid antigen detection tests (radt) are also available commercially for detection of both rsv and influenza virus infection in the outpatient and emergency department settings (62, 63) . however, in a recent study by moesker et al., radts were found to have relatively low sensitivity compared to rt-pcr testing which limits their use for clinical decision making (64) . nonetheless, radt maybe a valuable tool, especially during an outbreak, because it is a point-of-care test that is easy to use with a rapid turnaround time (65) . since clinical symptoms for different viral respiratory infections are often the same, and with the limitations of our current testing methods, it is critical that clinicians obtain microbiology data early, especially in the risk population (66) (67) (68) (69) . there is also a large variability of disease severity in children infected with rsv or influenza a (h1n1) virus. in rsv infection, development of lower respiratory track disease in premature infants, with or without chronic neonatal lung disease is associated with a significantly higher risk of hospitalization, intensive care unit admission, need for mechanical ventilation, and death (12, [70] [71] [72] [73] . in a study of 2,147 children with lower respiratory infection due to rsv, rodriguez et al. reported age less than 6 months, history of prematurity, chronic respiratory disease or congenital heart disease, and coinfection with adenovirus were significant predictors of increased disease severity (74) . similar predictors exist for children infected with influenza a (h1n1) virus, including age less than 5 years, a history of chronic lung disease, congenital heart disease, and immune compromise ( table 1 ) (75) . it is therefore prudent that clinicians should conduct laboratory evaluations early in the illness for viral infections in these at-risk populations presenting with respiratory failure and ards. in contrast to clinical predictors of disease severity, the contribution of viral factors to disease burden remains unclear. in rsv infection, although earlier studies suggested no correlation between viral load and disease severity (76, 77) newer findings suggest otherwise. studies by both devincenzo et al. and houben et al. reported a direct correlation between viral load and disease severity in infants with primary rsv infection (78, 79) . el saleeby et al. also reported that viral load is independently associated with increased risk of patients with rsv requiring prolonged hospitalization or intensive care, or to develop respiratory failure (80) . the relevance of viral load in influenza a (h1n1) virus infection is unclear. launes et al. found that in children who had more than 5 days of symptoms, a higher influenza a (h1n1) viral load at diagnosis correlated with an increased risk of requiring mechanical ventilation (81) . similarly others have found that patients with systemic symptoms and pneumonia had higher viral load when compared to those with uncomplicated upper respiratory tract infections alone (82) . as would be expected children have a higher influenza a (h1n1) viral load compared to adults because of less exposure to influenza antigens. however, this finding did not correlate with the occurrence of disease complications (83) . both rsv and influenza a (h1n1) virus result in a broad spectrum of disease, ranging from mild upper respiratory symptoms to fulminant respiratory failure and ards (59, 84) . this high degree of variability may be due to the pathogenicity of the viral pathogen, host immune response, or a combination of both (85) . human rsv consists of subgroups a and b and primarily infects humans. the rsv genome encodes 11 different proteins involved in transmission, infection, evasion of host response, and replication (86) . infection is typically restricted to respiratory epithelial cells, including both type i and type ii alveolar pneumocytes, from the trachea to the level of bronchioles. infection leads to epithelial and interstitial inflammation with progression to inflammatory infiltrates and epithelial sloughing (87) . after infection and viral replication, rsv causes epithelial cells to fuse, forming a syncytium from which the virus spreads from cell to cell (88) . those infected epithelial cells are then destroyed, releasing inflammatory cytokines and chemokines that ultimately attract additional inflammatory cells and degrade capillary integrity (89) . disruption of the alveolar-capillary barrier results in leakage of plasma proteins into interstitial tissue and within the alveoli, finally interfering with surfactant function (90) . with an understanding of the pathophysiologic process of rsv, it is no surprise that progression to ards is a potential end point. aside from viral pathogenicity, host immune-mediated factors also contribute to disease severity. a rapidly progressive area of research is in understanding the role of biomarkers not only in the diagnosis and prognosis of ards, but also in potential therapeutic options to alter such biomarkers (91) . inflammatory proteins in the matrix metalloproteinase (mmp) family have been shown to be elevated in pediatric ali (92) with a specific increase in mmp-9 production in rsv infection. blocking mmp-9 in vitro and in vivo resulted in decrease in viral load (93) . in addition, activation of several chemokine and interleukin subtypes, as well as tumor necrosis factors, has been shown to positively correlate with severity of illness in children with rsv (94) . in their study, fernandez et al. discovered that higher levels of soluble interleukin-10 (il-10) positively correlated with both disease severity and duration of supplemental oxygen in infants with acute rsv infection (95) . a separate study confirmed this associate with increased levels of il-10 in nasopharyngeal secretions (96) , but the pathogenicity of this correlation has yet to be determined. antigenic variability exists with both influenza a (h1n1) virus and the resultant immune-mediated response. like influenza b and c, influenza a is made up of structural proteins and two groups of surface glycoproteins, hemagglutinin (ha), and neuraminidase (na). these glycoproteins are responsible for attachment and entry into cells, viral spread throughout the respiratory tract, and are capable of a large degree of variability (97) . waterfowl serve as the largest natural reservoir for influenza a subtypes (98) . the avian-to-human leap can occur through direct transmission (99) or, alternatively, through pigs (100). although transmission from pigs to humans is a rare event, it occurred in the 1918 pandemic (101) a small outbreak in new jersey in 1976 (102) and the most recent pandemic of influenza a (h1n1) starting in 2009 (103) . influenza a (h1n1) virus primarily targets alveolar epithelial cells that serve as first-line defense against respiratory infections (104) . histological evaluation of 100 fatal cases of influenza a (h1n1) virus infection revealed diffuse alveolar damage with inflammation, fibrosis and edema, disruption of surfactant production, and detection of viral antigens throughout the lung parenchyma (105) . like rsv, this pathogenic can process rapidly progresses to refractory hypoxemia and ards (106) . in addition to viral pathogenicity, disease severity of influenza a (h1n1) virus infection is also closely associated with host response (107) . in both healthy patients and those with comorbidities, influenza a (h1n1) virus can lead to an exaggerated inflammatory response with dysregulation of local and systemic chemokine and cytokine production (108, 109) . in a study specifically in the pediatric population, takano et al. found a positive correlation with disease severity and elevated levels of serum interferon gamma, several interleukin types, and monocyte chemoattractant protein-1 (mcp-1) (110) . some studies suggest that these elevations may be adequate immune response to infection (111) , but further exploration of the inflammatory response in influenza a (h1n1) virus infection is likely to yield development of potential interventions to prevent disease progression to ards. the treatment of ards has significantly evolved over the past several decades. perhaps the greatest improvement in management developed from studies involving lung-protective ventilation (9). other interventions such as inhaled nitric oxide (112) , fluid management (113), use of steroids (114) , and prone positioning (115) are being further investigated and validated. however, specific treatment for rsv infection remains lacking. despite the substantial short and long-term morbidity and mortality associated with rsv disease in children, the current management for rsv infection consists of supportive care, in the form of oxygen supplementation, adequate hydration, and mechanical ventilation for those who develop respiratory failure. multiple therapeutic strategies have been explored with very limited success, and a vital need remains for an effective disease treatment. interventions for either virus can be separated into primary prevention of infection, typically through vaccination, and reduction of infectious burden once transmission has already taken place. primary prevention through vaccination continues to be a major area of research and potential advancement for both rsv and influenza a (h1n1) virus. to date, no rsv vaccine has proven efficacious. while significant research has been devoted to vaccine development, major obstacles specific to rsv, such as young age of infection, lack of persistent immunity, and poorly validated animal models make it difficult to find an effective and safe solution (116) . one current prospect, medi-534, a liveattenuated, intranasal vaccine providing protection against both rsv and parainfluenza 3, although safe in children ages 1-9, has yet to show a beneficial immunogenic response in infants (117) . other more recent advances continue to focus on live-attenuated vaccines, as well as chimeric live vectors (118) , with varying antibody response between children (119, 120) . while vaccine development continues, prophylactic use of polyclonal rsv intravenous immunoglobulin (respigam) or human anti-f monoclonal antibodies (palivizumab and motavizumab -which is not yet licensed for use) in high-risk infants has been shown to reduce the risk of rsv-associated acute lower respiratory tract infections and disease severity (121) . palivizumab is a human, monoclonal antibody targeted to block viral infected cells from fusing with adjacent cells (122) . palivizumab has been shown to be most effective in high-risk populations, specifically premature infants and those with chronic lung disease or congenital heart disease (123) . the use of palivizumab as treatment for rsv infection in mechanically ventilated pediatric patients has not been shown to be effective (124) . furthermore, studies have also shown that palivizumab prophylaxis in these patients has a limited effect on the total disease burden of rsv infection, including overall rsv-related hospital admissions and resource utilization (12, 125). although not approved for use in the united states, motavizumab, the second-generation derivative of palivizumab, decreased viral load compared with placebo (126) . however, in a more recent study of hospitalized rsv infected infants treated with motavizumab or placebo, no antiviral effect was demonstrated (127) . furthermore, both therapies produce only temporary, passive immunity (128) . the 2009 influenza a (h1n1) virus was a novel strain, leaving children and young adults with little if any preexisting antibodies and without adequate protection with the seasonal influenza vaccine alone (129) . a new influenza a (h1n1) virus vaccine was rapidly developed and has subsequently been shown to be safe and effective at providing adequate immunological response (130, 131) . one post-pandemic study showed a correlation with higher rates of influenza a (h1n1) virus infection, compared with other influenza types, along with increased icu admissions for countries with limited numbers of the population having received influenza a (h1n1) virus vaccination (132) . in addition to general supportive care, the second goal of therapy in viral infection focuses on reducing the infectious burden and, theoretically, subsequent viral sequelae. currently, inhaled ribavirin is the only approved antiviral treatment for rsv infection in children (133) but its use is associated with potential teratogenicity, and its efficacy remains uncertain (134) . ribavirin directly and indirectly inhibits replication of both dna and rna viruses, including rsv (135) . studies in infants found a decrease in mortality and respiratory deterioration, and a decrease in days of hospitalization and days of mechanical ventilation in ventilated infants (136) . luo et al. reported an adult case of severe rsv infection progressing to ards that was successfully treated with inhaled ribavirin (137) , but overall effectiveness in treatment of viral pediatric ards has yet to be determined. in addition to its use in rsv, ribavirin has also been used in treatment of severe influenza a (h1n1) virus infection (138) . ribavirin can be given orally but is typically aerosolized when used for respiratory viral infections. however, safety considerations regarding potential teratogenicity and exposure to health-care workers during administration (134) limit its use. the american academy of pediatrics does not recommend the routine use of ribavirin to treat rsv infection, reserving its use for patients with potentially life-threatening disease (139) . several small molecule inhibitors that interfere with rsv f protein (mdt-637 and jnj-2408068) (140) including the gs-5806 that was recently evaluated in a challenge safety study of healthy adults (141) . in this study by devincenzo et al., treatment resulted in decreased viral burden and severity of clinical disease. the use of these small molecule inhibitors in the context of pediatric subjects who develop ards remains untested at this point. neuraminidase inhibitors (which prevent the release of influenza virions), including oral oseltamivir, inhaled zanamivir and laninamivir, and parenteral peramivir, remain first-line interventions for influenza. only oseltamivir, peramivir, and zanamivir are available in the united states. perhaps the most widely used, oseltamivir results in a significant decrease in duration of symptoms as well as severity of illness with early treatment (<48 h of symptoms) (142) . use of oseltamivir in severe cases of influenza a (h1n1) has become standard practice (24, 143), although some studies have shown increased resistance (144) . randolph et al. conducted a retrospective study of 838 children admitted to the picu with confirmed influenza a (h1n1) infection (145) . overall, 564 (67.3%) required mechanical ventilation, but the rate of progression to ards was not reported. although 88% were treated with oseltamivir, there was no association with improved mortality. farias et al. found a reduced mortality in patients with pards from influenza a (h1n1) if oseltamivir was administered within the first 24 h (25). while further studies are needed to look at the effectiveness of antiviral medications in the treatment of viral-induced ards, in recent years, investigators are also focusing on the potential benefits of immune modulation. with enhancement in research surrounding viral pathogenicity and host immune response, potential targets of intervention will hopefully be identified. aside from viral-specific therapies, ecmo has been utilized as rescue therapy for severe respiratory failure in pediatrics for more than 20 years, with more than 50% survival (146) . the overall use of ecmo for treatment of ards has increased with improvement in mortality (147) . the recent palicc recommendations conclude that ecmo should be considered for treatment of pards when lung-protective strategies have failed, when the cause of respiratory failure is thought to be reversible, or when the child may be suitable for lung transplantation (148) . the use of ecmo in pediatric respiratory failure due to rsv is well reported (149, 150) . in their retrospective review of 151 children requiring mechanical ventilation for rsv bronchiolitis, flamant et al. reported the use of ecmo in 14 patients (151) . in this study, the median duration of ecmo was 12.5 (5-18) days with a survival rate of 71.4%. on the other hand, the use of ecmo in pards due to influenza a (h1n1) virus is sparsely reported (152, 153) and most of our understanding stems from adult studies. in a study in australia and new zealand during the 2009 influenza a (h1n1) pandemic, 68 adult patients with influenza-induced ards were treated with ecmo (154) . the median duration of ecmo was 10 (7-15) days. when the report was submitted, 48 (71%) patients had survived to icu discharge, with 14 deaths and 6 patients remaining in the icu, 2 of whom remained on ecmo. in their study in the united kingdom during the same pandemic, noah et al. discovered a decrease in mortality for patients with ards due to influenza a (h1n1) who were referred and transferred to an ecmo center compared with matched non-ecmo-referred patients (155) . in this study, 69 patients received ecmo with a mortality rate of 14.4%. expanded use of ecmo within the pediatric population for influenza a (h1n1) virus induced ards has yet to be investigated. however, the use of ecmo in refractory cases or rsv or influenza a (h1n1) virus induced ards should be considered when applicable. while it is clear that viral infections are an important cause of pediatric ards, the exact disease burden remains unknown. with more definitive diagnostic criteria, clinicians now have a wide array of research possibilities regarding pediatric ards, both retrospective and prospective. further studies to expand our understanding of viral-induced pediatric ards will be of great benefit, both in understanding the epidemiology and viral-specific treatment options available. in addition, an improved comprehension of viral transmission, pathogenicity, and host response will be particularly important in times of pandemics, either from known or novel viruses. finally, continued efforts in prevention and treatment of viral infections will likely be of greatest advantage to decrease viral progression to pediatric ards. sn, rw, and mk contributed to the conception, writing, and final edits of this manuscript. this work was funded by the national institutes of health (nichd 5k12hd047349 and 5k08hl119359-02) to mk. middle east respiratory syndrome acute management and long-term survival among subjects with severe middle east respiratory syndrome coronavirus pneumonia and ards herpes simplex virus in the respiratory tract of critical care patients: a prospective study herpes simplex virus lung infection in patients undergoing prolonged mechanical ventilation vandenbroucke-grauls cm. herpes simplex virus type 1 and respiratory disease in critically-ill patients: real pathogen or innocent bystander? herpes simplex virus from respiratory tract secretions: epidemiology, clinical characteristics, and outcome in immunocompromised and nonimmunocompromised hosts life-threatening human herpes virus-6 infection in early childhood: presenting symptom of a primary immunodeficiency? treatment of acute pulmonary failure with extracorporeal support: 100% survival in a pediatric population characterisation of nosocomial and community-acquired influenza in a large university hospital during two consecutive influenza seasons guideline for isolation precautions: preventing transmission of infectious agents in health care settings contact precautions: more is not necessarily better multiplex pcr for typing and subtyping influenza and respiratory syncytial viruses multiplex real-time pcr assay for detection of influenza and human respiratory syncytial viruses emergence of a novel swine-origin influenza a (h1n1) virus in humans the influenza virus: disease, diagnostics, and treatment evaluation of novel second-generation rsv and influenza rapid tests at the point of care accuracy of rapid influenza diagnostic tests: a meta-analysis antigen-based assays for the identification of influenza virus and respiratory syncytial virus: why and how to use them in pediatric practice diagnostic performance of influenza viruses and rsv rapid antigen detection tests in children in tertiary care world health organization. who recommendations on the use of rapid testing for influenza diagnosis comparison of human metapneumovirus, respiratory syncytial virus and rhinovirus respiratory tract infections in young children admitted to hospital role of respiratory viruses in acute upper and lower respiratory tract illness in the first year of life: a birth cohort study predictors of rsv lrti hospitalization in infants born at 33 to 35 weeks gestational age: a large multinational study (poni) populations at risk for developing respiratory syncytial virus and risk factors for respiratory syncytial virus severity: infants with predisposing conditions children's hospital respiratory syncytial virus database: risk factors, treatment and hospital course in 3308 infants and young children birth weight and hospital readmission of infants born prematurely respiratory syncytial virus and premature infants born at 32 weeks' gestation or earlier: hospitalization and economic implications of prophylaxis readmission with respiratory syncytial virus (rsv) infection among graduates from a neonatal intensive care unit predictors of severity and mortality in children hospitalized with respiratory syncytial virus infection in a tropical region clinical features of severe influenza a (h1n1) virus infection illness severity, viral shedding, and antibody responses in infants hospitalized with bronchiolitis caused by respiratory syncytial virus type 1 and type 2 cytokine imbalance in acute respiratory syncytial virus bronchiolitis respiratory syncytial virus load predicts disease severity in previously healthy infants disease severity and viral load are correlated in infants with primary respiratory syncytial virus infection in the community respiratory syncytial virus load, viral dynamics, and disease severity in previously healthy naturally infected children viral load at diagnosis and influenza a h1n1 (2009) disease severity in children correlation of pandemic (h1n1) 2009 viral load with disease severity and prolonged viral shedding in children distinct differences in clinical manifestation and viral laboratory parameters between children and adults with influenza a(h1n1) pdm09 infection -a retrospective comparative analysis acute respiratory distress syndrome caused by respiratory syncytial virus viral and host factors in human respiratory syncytial virus pathogenesis respiratory syncytial virus: virology, reverse genetics, and pathogenesis of disease the histopathology of fatal untreated human respiratory syncytial virus infection in vitro modeling of respiratory syncytial virus infection of pediatric bronchial epithelium, the primary target of infection in vivo novel pathways in the pathogenesis of respiratory syncytial virus disease surfactant abnormalities in infants with severe viral bronchiolitis biomarkers in pediatric ards: future directions matrix metalloproteinase activity in pediatric acute lung injury matrix metalloproteinase-9 mediates rsv infection in vitro and in vivo novel inflammatory markers, clinical risk factors and virus type associated with severe respiratory syncytial virus infection plasma interferon-gamma, interleukin-10 and soluble markers of immune activation in infants with primary adenovirus (adv) and respiratory syncytial virus (rsv) infection correlation between inflammatory mediators in the nasopharyngeal secretion and in the serum of children with lower respiratory tract infection caused by respiratory syncytial virus and disease severity molecular character of influenza a/h1n1 2009: implications for spread and control evolution and ecology of influenza a viruses adaptation of avian influenza a virus polymerase in mammals to overcome the host species barrier the pig as a mixing vessel for influenza viruses: human and veterinary implications updating the accounts: global mortality of the 1918-1920 "spanish" influenza pandemic swine influenza a outbreak swine influenza a (h1n1) infection in two children -southern california differential susceptibilities of human lung primary cells to h1n1 influenza viruses pandemic influenza a (h1n1): pathology and pathogenesis of 100 fatal cases in the united states pneumonia and respiratory failure from swine-origin influenza a (h1n1) in mexico influenza virus pathogenicity regulated by host cellular proteases, cytokines and metabolites, and its therapeutic options three fatal cases of pandemic 2009 influenza a virus infection in shenzhen are associated with cytokine storm antibody and inflammatory response-mediated severity of pandemic 2009 (ph1n1) influenza virus cytokine and chemokine response in children with the 2009 pandemic influenza a (h1n1) virus infection monocyte chemoattractant protein 1 contributes to an adequate immune response in influenza pneumonia role of inhaled nitric oxide in the management of severe acute respiratory distress syndrome comparison of two fluid-management strategies in acute lung injury efficacy and safety of corticosteroids for persistent acute respiratory distress syndrome prone positioning in severe acute respiratory distress syndrome biological challenges and technological opportunities for respiratory syncytial virus vaccine development phase-i study medi-534, of a live, attenuated intranasal vaccine against respiratory syncytial virus and parainfluenza-3 virus in seropositive children live-attenuated respiratory syncytial virus vaccines a gene deletion that up-regulates viral gene expression yields an attenuated rsv vaccine with improved antibody responses in children enhanced neutralizing antibody response induced by respiratory syncytial virus prefusion f protein expressed by a vaccine candidate antibodies for prevention and treatment of respiratory syncytial virus infections in children development of a humanized monoclonal antibody (medi-493) with potent in vitro and in vivo activity against respiratory syncytial virus effectiveness of palivizumab in preventing rsv hospitalization in high risk children: a real-world perspective comparison of intravenous palivizumab and standard of care for treatment of respiratory syncytial virus infection in mechanically ventilated pediatric patients impact of palivizumab on admission to the icu for respiratory syncytial virus bronchiolitis: a national survey safety and antiviral activity of motavizumab, a respiratory syncytial virus (rsv)-specific humanized monoclonal antibody, when administered to rsv-infected children motavizumab treatment of infants hospitalized with respiratory syncytial virus infection does not decrease viral load or severity of illness safety and effectiveness of palivizumab in children at high risk of serious disease due to respiratory syncytial virus infection: a systematic review serum cross-reactive antibody response to a novel influenza a (h1n1) virus after vaccination with seasonal influenza vaccine safety and immunogenicity following administration of a live, attenuated monovalent 2009 h1n1 influenza vaccine to children and adults in two randomized controlled trials a novel influenza a (h1n1) vaccine in various age groups pandemic vaccination strategies and influenza severe outcomes during the influenza a(h1n1)pdm09 pandemic and the post-pandemic influenza season: the nordic experience a controlled trial of aerosolized ribavirin in infants receiving mechanical ventilation for severe respiratory syncytial virus infection safety issues related to the administration of ribavirin broad-spectrum antiviral activity of virazole: 1-beta-d-ribofuranosyl-1,2,4-triazole-3-carboxamide ribavirin for respiratory syncytial virus infection of the lower respiratory tract in infants and young children inhaled ribavirin therapy in adult respiratory syncytial virus-induced acute respiratory distress syndrome virological clearance rate of high-dose oseltamivir or triple-combination antiviral therapy in complicated 2009 pandemic influenza a (h1n1) infection respiratory syncytial virus small molecules vp-14637 and jnj-2408068 inhibit respiratory syncytial virus fusion by similar mechanisms oral gs-5806 activity in a respiratory syncytial virus challenge study use of the oral neuraminidase inhibitor oseltamivir in experimental human influenza: randomized controlled trials for prevention and treatment h1n1 influenza and experience in three critical care units systematic review of influenza resistance to the neuraminidase inhibitors critically ill children during the 2009-2010 influenza pandemic in the united states probability of survival after prolonged extracorporeal membrane oxygenation in pediatric patients with acute respiratory failure. extracorporeal life support organization extracorporeal membrane oxygenation for ards: national trends in the united states extracorporeal support in children with pediatric acute respiratory distress syndrome: proceedings from the pediatric acute lung injury consensus conference role of ecmo in the treatment of respiratory syncytial virus bronchiolitis: a collaborative report use of extracorporeal membrane oxygenation in the treatment of respiratory syncytial virus bronchiolitis: the national experience, 1983 to 1988 severe respiratory syncytial virus bronchiolitis in children: from short mechanical ventilation to extracorporeal membrane oxygenation continued high incidence of children with severe influenza a(h1n1)pdm09 admitted to paediatric intensive care units in germany during the first three post-pandemic influenza seasons mechanically ventilated children with 2009 pandemic influenza a/h1n1: results from the national pediatric intensive care registry in japan extracorporeal membrane oxygenation for 2009 influenza a(h1n1) acute respiratory distress syndrome referral to an extracorporeal membrane oxygenation center and mortality among patients with severe 2009 influenza a(h1n1) the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord-258336-zs04l3s0 authors: leotte, jaqueline; trombetta, hygor; faggion, heloisa z.; almeida, bernardo m.; nogueira, meri b.; vidal, luine r.; raboni, sonia m. title: impact and seasonality of human rhinovirus infection in hospitalized patients for two consecutive years date: 2017-06-30 journal: jornal de pediatria doi: 10.1016/j.jped.2016.07.004 sha: doc_id: 258336 cord_uid: zs04l3s0 abstract objectives to report epidemiological features, clinical characteristics, and outcomes of human rhinovirus (hrv) infections in comparison with other community acquired respiratory virus (crv) infections in patients hospitalized for two consecutive years. methods this was a cross-sectional study. clinical, epidemiological, and laboratory data of patients hospitalized with acute respiratory syndrome in a tertiary care hospital from 2012 to 2013 were reviewed. results hrv was the most common crv observed (36%, 162/444) and was present in the majority of viral co-detections (69%, 88/128), mainly in association with human enterovirus (45%). most hrv-infected patients were younger than 2 years (57%). overall, patients infected with hrv had a lower frequency of severe acute respiratory infection than those infected with other crvs (60% and 84%, respectively, p =0.006), but had more comorbidities (40% and 27%, respectively; p =0.043). however, in the adjusted analysis this association was not significant. the mortality rate within the hrv group was 3%. detection of hrv was more prevalent during autumn and winter, with a moderately negative correlation between viral infection frequency and temperature (r =−0.636, p <0.001) but no correlation with rainfall (r =−0.036, p =0.866). conclusion hrv is usually detected in hospitalized children with respiratory infections and is often present in viral co-detections. comorbidities are closely associated with hrv infections. these infections show seasonal variation, with predominance during colder seasons. human rhinoviruses (hrvs) belong to picornaviridae family, genus enterovirus, and are divided in three species (hrv-a, hrv-b, and hrv-c) with about 100 serotypes within these species. 1, 2 the development of highly-sensitive molecular techniques for characterization of the hrv genome has recently allowed recognition of the hrv-c species. 3 there is already evidence that this new species may be more virulent and more strongly associated with lower respiratory tract infections than hrv-a and hrv-b. 4 hrv is the most common cause of upper respiratory tract infections, being responsible for at least 50% of cases of the common cold. this leads to considerable economic burden in terms of medical visits and both school and work absenteeism. 2, 4 hrvs have also been linked to lower airway effects that result in significant morbidity and mortality, 1 such as exacerbations of chronic pulmonary disease, severe bronchiolitis in infants and children, as well as fatal pneumonia in elderly and immunocompromised adults. 4, 5 in general, hrv infections occur during spring and autumn, 6 and manifest differently depending on whether the lower or upper respiratory tract is infected. infections of the upper respiratory tract ordinarily include symptoms of the common cold, but can present as acute otitis media or rhinosinusitis. on the other hand, infections of the lower respiratory tract can cause severe symptoms and result in bronchiolitis and pneumonia. 4 in brazil, since the influenza a pandemic of 2009, referral hospitals have been conducting active surveillance to detect respiratory viruses. such surveillance includes notification and laboratory investigation of cases meeting the diagnostic criteria of severe acute respiratory infection (sari). this viral respiratory infection monitoring has resulted in important information about the circulation of other community-acquired respiratory viruses (crv). the present study reports the epidemiological features, clinical characteristics, and outcomes of hrv infections in comparison with other crv infections in patients hospitalized in a referral hospital in southern brazil, for two consecutive years. patients hospitalized at an academic tertiary care center in southern brazil from whom respiratory samples were collected and sent for investigation, or who were diagnosed with sari in 2012 or 2013, were included in the study. respiratory samples (nasal swab or nasopharyngeal aspirate, or bronchoalveolar lavage) were collected in different periods of hospitalization according to medical recommendation. individuals with more than one sample collected during the same symptomatic period were considered as a single case and only the first result was evaluated, so that the number of respiratory viral detections was not overestimated. the medical records and influenza notification forms of patients with detectable respiratory virus were reviewed, focusing on epidemiology, clinical manifestation, outcome, laboratory findings, and diagnosis of sari. sari was defined as a flu-like syndrome with signs of severity (dyspnea or oxygen saturation below 95%). during the study, a total of 1002 cases were identified in both databases. of these, five cases were excluded because the researchers could not access the medical records and a further 242 were excluded as no samples had been sent for virus detection. thus, 755 patients with respiratory samples investigated for respiratory virus detection were included. the institutional ethics review board approved the study (no. #18714013.4.0000.0096). rvs were detected using a multiplex reverse transcription polymerase chain reaction (rt-pcr) technique. the viral genome was extracted using a high pure viral rna kit (roche inc., mannheim, germany) in accordance with the manufacturer's instructions. first strand cdna synthesis was achieved using random primers and an improm-ii reverse transcription system (promega inc., wi, usa). the resulting cdna was then subject to pcr by using a seeplex ® rv15 ace detection kit (seegene inc., korea), in accordance with the manufacturer's protocol. this multiplex pcr technology enables a simultaneous detection of 15 respiratory viruses: human adenovirus (adv), human metapneumovirus (mpv), parainfluenza virus type 1, 2, 3, and 4 (piv-1, piv-2, piv-3, and piv-4), influenza a (flua), influenza b (flub), respiratory syncytial virus type a and b (rsv-a, rsv-b), human rhinovirus types a/b/c (hrv), human enterovirus (ev), human bocavirus (bov), and human coronavirus (cov) types 229e/nl63 (alpha coronaviruses) and oc43/hku1 (beta coronaviruses). data were compiled using jmp version 5.2.1 (sas institute inc., cary, nc, usa) and analyzed using graphpad prism version 5.03 (graphpad software inc., ca, usa). parametric and non-parametric tests were used as appropriate. the nonparametric spearman correlation coefficient was used to analyze meteorological data. variables with an associated pvalue < 0.05 in the univariate analysis and those considered as confounding factors (age and length of hospitalization) were subjected to multivariate logistic regression to identify independent predictors for severe disease. all p-values were two-tailed and a value of <0.05 was considered significant. curitiba is located in southern brazil and has a temperate climate. data on monthly measures of temperature and rainfall were supplied by the meteorological system of paraná (sistema meteorológico do paraná [simepar]). were compared with those with other crv infections, groups 1 and 2, respectively) ( table 1) . for this analysis, only cases with single infections were included, excluding nosocomial infections, since given that patients with nosocomial infections were already in the hospital, it would be expected that they would have some underlying illness, which could overestimate the severity of infections. most hrv infected patients were younger than 2 years (57%), 53% were younger than 1 year, and 37% were younger than 6 months of age. the groups showed no statistically significant difference in median age. gender was unequally distributed between the groups. the main clinical manifestations observed in both groups included fever, cough, and dyspnea. however, the hrv positive group had a significantly lower proportion of cases with fever (62%, p = 0.001), and more comorbidities than the other group (40% vs. 27%, p = 0.043). although immunosuppression, chronic lung disease, and heart disease were the major medical conditions present in both groups, chronic lung disease was more frequent in the hrv group (17%), but without significant difference. concerning the diagnosis of sari, there were fewer patients diagnosed with this syndrome in the hrv group (60%, p = 0.006) than in the other crv group. in the multivariate analysis of the relationship between clinical characteristics significantly different between the hrv-and other crv-groups, only the higher prevalence of previous comorbidities in the hrv group was significant. patients with no comorbidities were 1.6 (95% ci: 1.04---2.34) times more likely to be infected crvs other than hrv, in comparison to patients with comorbidities. despite the fact that most of chest x-rays of hrv infected cases were missed (53%), radiologic alterations were described in 83% of the performed exams. most of the findings observed were interstitial infiltrate (33%) and pulmonary consolidation (28%), with no significant difference between both groups. severe disease was defined as that requiring mechanical ventilatory support, or intensive care unit (icu) admission, or death during hospitalization; no significant difference was found between the groups. two hrv infected patients died during hospitalization, both from respiratory infection complications. hrv had the highest rates of coinfection (69%) of the 128 samples with more than one crv detected. the viruses most co-detected with hrv were ev (40/88; 45%) and rsv (29/88; 33%), as shown in fig. 1 . in 52% of cases, the co-detection involved hrv plus another virus, and in 16% of cases, hrv was associated with two or more viruses. in 65 cases, hrv was the only virus detected. a comparison of the clinical characteristics of monoinfected hrv patients with co-infected hrv patients was performed, and no relation between viral co-detection and disease severity (p = 0.717) was found. the clinical features significantly associated with hrv co-detection were as follows: age 6---48 months (or = 3.2; 95% ci: 1.3---8.1), length of hospitalization more than 30 days (or = 9.7; 95% ci: 2.5---36.8), and no sari diagnosis (or = 4.0; 95% ci: 1.7---9.3). as co-infection with hrv alone was not related with severe disease, a second analysis was performed comparing disease severity between hrv co-infected patients and patients infected with other crvs. neither the presence nor absence of hrv co-detection was associated with disease severity (p = 0.196). cases of hrv infection occurred throughout the study period (fig. 2) . the months of may to august demonstrated the highest number of hrv infections during 2012, peaking in august (17 cases; 10%). in 2013, the seasonality of hrv infection was from march to may, peaking in may (19 cases; 12%). comparing the monthly distribution of hrv cases with average temperature ( • c) and rainfall (mm), a negative correlation between the number of hrv cases and the average temperature (rs = −0.636, p < 0.001) was demonstrated, but there was no significant correlation with rainfall (rs = −0.036, p = 0.866). since the implementation of systematic investigation into respiratory viruses in hospitalized patients with sari, hrv has been found with high frequency, either alone or codetected with other respiratory viruses. since some of these infections may have a poor prognosis, including death, it is critical to know and better characterize this infection to be prepared for its early diagnosis, appropriate clinical management, and prevention of nosocomial spread. the high frequency of hrv found in clinical samples from patients with sari was similar to that reported by kim et al. 7 in a tertiary hospital in korea. he et al. 8 analyzed a hospitalized pediatric population and found a prevalence of 48%, while walker and ison 9 found a prevalence of 14% in a hospitalized adult population. this difference between age groups was observed in this study, emphasizing the greater vulnerability of children to this infection, probably as consequence of a more intense inflammatory process triggered by primary infection or by favorable anatomical conditions of the respiratory tract in younger children. rsv was the first pathogen identified to be associated with the severe respiratory disease in children. however, since the development of molecular techniques to detect hrv, this virus has been the focus of the most recent studies in this field. nowadays, hrv infections have been linked with exacerbations of chronic lung diseases and fatal pneumonia in patients at the extremes of age or with pre-existing comorbidities. 4, 5 the importance of one study that evaluated the characteristics of hrv infections in a tertiary hospital is based on these new concerns. marcone et al. 10 reported a higher number of hrv infections in hospitalized children in argentina when compared with pediatric outpatients with acute respiratory infection, demonstrating a scenario that contributed to the increased concern about hrv infection in the hospital setting. previous studies have reported that the hrv infection rate in hospitals varies from 21% to 41%. 8, 11, 12 however, this variation is probably accounted for by the differences in age and clinical characteristics of patients analyzed in each study. although this study included all age groups, a predominance of pediatric patients infected with hrv was observed, mostly younger than 1 year old. in addition, the hrv-positive group had a greater proportion of patients with pre-existing comorbidities than the group with other identified crvs. 6,7,11---14 among the comorbidities observed in this study, chronic lung diseases were frequent in the hrv group, a correlation that has already been demonstrated in another similar study, 14 which corroborates the close association of hrv infection and exacerbations of chronic lung diseases, such as chronic obstructive pulmonary disease (copd), asthma, and cystic fibrosis. 4 of 444 samples positive for crvs, 128 samples (29%) had at least two co-detected viruses, in keeping with the expected 10---31% encountered in hospitalized individuals. 7, 8, 15 the viruses most frequently co-detected with hrv were ev (45%), rsv (33%), and adv (18%); consistent with previously reported patterns. 14, 15 however, the high frequency of ev and hrv co-detection may be as a result of using the 5 nrt region of the viral genome to identify both pathogens, which may have lowered the specificity of the rt-pcr test. 16 a more appropriate means to discern between these species would be to carry out the rt-pcr followed by nucleotide sequencing of amplicons. 17 the high level of dual infection involving hrv and rsv is often explained both by the coexistence of these viruses throughout the year and their similar seasonal variation, 15 but this hypothesis is not unanimous. 12 in contrast to findings reported by goka et al., 15 this study did not show an association between disease severity and viral co-detections. furthermore, hrv was not found to have a protective influence in cases where it was involved in codetection, as there was no significant difference in disease severity when comparing the groups with or without hrv co-detection. likewise, in contradiction to the present findings, asner et al. 18 observed an increased disease severity in children infected with hrv alone. among the probable factors associated with these contrasting findings, the following may be cited: (i) analysis in different groups of patients (outpatient and hospitalized), (ii) assessing a small number of patients, and (iii) adoption of different severity criteria. fever was less frequent in patients infected with hrv compared to other crvs. 19, 20 chest x-ray findings were normal in 17%, showed interstitial infiltrate in 33%, and demonstrated pulmonary consolidation in 28% of cases, with no significant difference between the hrv and other crv groups. these patterns are similar to those reported by fica et al. 11 in hospitalized adults in chile. there were significantly fewer cases of sari diagnosed in the patients infected with hrv than other crvs. overall, 14% (110/770) of patients with sari were infected with hrv. this concurs with a previous study. 20 reports about the seasonality of hrv infection, including a study from argentina, 10 which is in close geographical proximity of southern brazil, show that it circulates mainly in autumn and spring. 7, 8, 19 although hrv occurred in almost all months of the study period, different peaks were observed in 2012 and 2013, and neither peak included spring. in 2013, the highest prevalence of hrv infection was in autumn, followed by winter, and vice versa in 2012. the analysis of meteorological data found a negative correlation between the number of hrv cases and the average temperature, but no significant correlation with rainfall. however, in order to more accurately establish the seasonality of hrv infections, the analysis should include additional years. this research had some limitations: (i) it was a retrospective study and some medical records were incomplete; (ii) hrv species were not identified, which would have yielded important data since the genotypes reportedly have different virulence; (iii) this analysis was carried out only with hospitalized patients, which may have overestimated the impact of hrv infection; and (iv) it was not possible to evaluate the frequency of nosocomial hrv infection, data critical to guide preventive measures in the most affected settings. however, this is the first report about hrv infections in the region and a critical analysis of the data is important to obtain greater awareness of the dynamics of dispersion and impact of these respiratory viruses in the community. in conclusion, hrv has a high prevalence in the hospitalized children and was present in cases of severe disease, including death. however, a dependent relationship between the presence of hrv in viral co-detections and severity of disease was not observed in this study. conflicts in the literature demand a closer analysis of cases of co-detection involving hrv with review of the data to determine the impact of this, since the lack of standardization between studies probably contributes to the divergent data. hrv infection is closely associated with comorbidities, mainly chronic lung diseases, and is an important factor in exacerbations of these underlying lung diseases. the colder seasons were the period with higher frequency of hrv infections in southern brazil, and therefore must be a period to warn clinicians about young age children affected by respiratory infections, especially those with comorbidities such as chronic lung disease. patients with this profile should have respiratory samples collected to identify possible viral infection, and if hrv is detected, the medical staff should be ready for adequate management, taking into consideration the possible poor outcomes. smr is sponsored by a cnpq fellowship. the authors declare no conflicts of interest. analysis of the complete genome sequences of human rhinovirus the abcs of rhinoviruses, wheezing, and asthma clinical and molecular features of human rhinovirus c human rhinoviruses wheezing rhinovirus illnesses in early life predict asthma development in high-risk children seasonality and prevalence of respiratory pathogens detected by multiplex pcr at a tertiary care medical center epidemiology of respiratory viral infection using multiplex rt-pcr in a 3-year prospective study of the epidemiology of acute respiratory viral infections in hospitalized children in shenzhen, china. influenza other respir viruses respiratory viral infections among hospitalized adults: experience of a single tertiary healthcare hospital. influenza other respir viruses genetic diversity and clinical impact of human rhinoviruses in hospitalized and outpatient children with acute respiratory infection, argentina clinical relevance of rhinovirus infections among adult hospitalized patients do rhinoviruses reduce the probability of viral codetection during acute respiratory tract infections? respiratory viral coinfections identified by a 10-plex real-time reverse-transcription polymerase chain reaction assay in patients hospitalized with severe acute respiratory illness the significance of rhinovirus detection in hospitalized children: clinical, epidemiological and virological features single, dual and multiple respiratory virus infections and risk of hospitalization and mortality evaluation of multiple commercial molecular and conventional diagnostic assays for the detection of respiratory viruses in children rhinovirus detection using different pcr-based strategies is virus coinfection a predictor of severity in children with viral respiratory infections? rhinovirus is an important pathogen in upper and lower respiratory tract infections in mexican children epidemiology of pathogen-specific respiratory infections among three us populations key: cord-027693-a4up394g authors: sampol, júlia; gonzález-viejo, miguel ángel; gómez, alba; martí, sergi; pallero, mercedes; rodríguez, esther; launois, patricia; sampol, gabriel; ferrer, jaume title: predictors of respiratory complications in patients with c5–t5 spinal cord injuries date: 2020-06-24 journal: spinal cord doi: 10.1038/s41393-020-0506-7 sha: doc_id: 27693 cord_uid: a4up394g study design: retrospective chart audit. objectives: describing the respiratory complications and their predictive factors in patients with acute traumatic spinal cord injuries at c5–t5 level during the initial hospitalization. setting: hospital vall d’hebron, barcelona. methods: data from patients admitted in a reference unit with acute traumatic injuries involving levels c5–t5. respiratory complications were defined as: acute respiratory failure, respiratory infection, atelectasis, non-hemothorax pleural effusion, pulmonary embolism or haemoptysis. candidate predictors of these complications were demographic data, comorbidity, smoking, history of respiratory disease, the spinal cord injury characteristics (level and asia impairment scale) and thoracic trauma. a logistic regression model was created to determine associations between potential predictors and respiratory complications. results: we studied 174 patients with an age of 47.9 (19.7) years, mostly men (87%), with low comorbidity. coexistent thoracic trauma was found in 24 (19%) patients with cervical and 35 (75%) with thoracic injuries (p < 0.001). respiratory complications were frequent (53%) and were associated to longer hospital stay: 83.1 (61.3) and 45.3 (28.1) days in patients with and without respiratory complications (p < 0.001). the strongest predictors of respiratory complications were: previous respiratory disease (or 5.4, 95% ci: 1.5–19.2), complete motor function impairment (ais a–b) (or 4.7, 95% ci: 2.4–9.5) and concurrent chest trauma (or 3.73, 95% ci: 1.8–7.9). conclusions: respiratory complications are common in traumatic spinal cord injuries between c5–t5. we identified previous respiratory disease, complete motor function impairment and the coexistence of thoracic trauma as predictors of respiratory complications. identification of patients at risk might help clinicians to implement preventive strategies. traumatic spinal cord injuries are medical emergencies associated with substantial morbidity and mortality [1] . respiratory complications are common in patients with spinal cord injuries and their presence predicts worse prognosis and increases health care costs [2, 3] . the level of the injury and its degree, complete or incomplete, are key factors for predicting the severity of respiratory events in these patients. it is known that injuries above c5 seriously compromise the respiratory pump and frequently cause long-term full or partial dependence on mechanical ventilation or diaphragm pacing [4] [5] [6] [7] . acute respiratory complications associated to spinal cord injuries below c4 are less well known. a reduction in the number of respiratory events has been reported as the level of the lesion descends: while respiratory complications are still frequent in patients with injuries above t1 [2] , respiratory function is usually normal with lesions at t12 level or lower [4, 5] . historical series have shown that these complications are frequent in patients with high thoracic spinal cord injuries [8, 9] , who experience mortality rates of 10%, nearly 27 times higher than in patients without respiratory complications [10] . consequently, it is of great interest to investigate the factors that may predict the development of respiratory complications in these patients during their initial hospitalization. previous research has found ais impairment scales a and b are predictors of respiratory complications [9] . however, to our knowledge, at least two other potential predictive factors have not been evaluated. first, the coexistence of previous comorbidities, especially respiratory diseases. second, the frequent presence of thoracic trauma in patients with spinal cord lesions between c5-t5, which may itself lead to respiratory complications. in recent years significant advances in the respiratory care of spinal cord injuries, especially non-invasive ventilation and assisted coughing techniques, have been made. the identification of predictive factors of respiratory complications might provide clinicians with useful information in order to identify those patients at risk of developing respiratory complications and lead to a better prevention and management of these complications. based on these considerations, the aims of our study were: (1) to describe the respiratory complications in patients with acute traumatic spinal cord injuries at c5-t5 level during the initial hospitalization and (2) to analyze the predictors of these respiratory complications. the research protocol was approved by the ethics and clinical research committee of our institution. the vall d'hebron university hospital spinal cord injury unit is a reference service in our region's public health system and provides care to the population of catalonia, balearic islands and andorra. we retrospectively reviewed all patients with an acute thsci and a level of injury between c5 and t5 admitted during the period 2010-2015. patient information is systematically recorded at our unit during admission. the only exclusion criteria was the coexistence of cranioencephalic trauma with significant head injury (glasgow coma scale of <8 at first assessment). for this study the following variables were assessed: (1) demographic data (age, gender), etiology of the traumatic spinal cord injury, and duration of hospital stay from initial admission to discharge. (2) smoking: yes (current or previous) or not. (3) presence of a history of respiratory disease. patients were asked for a previous diagnosis of asthma, copd, sleep apnea or other chronic respiratory disease. (4) comorbidity was assessed by the charlson index [11] that takes into account the number and the severity of comorbid diseases and results in a single score, with lower values indicating less comorbidity: no comorbidity, with score 0; mild, with scores of 1-2; moderate, with scores of 3-4; and severe, with scores ≥5. (1) an arterial oxygen saturation of <90% or a partial pressure of arterial oxygen of <60 mm while breathing room air, and/or (2) respiratory acidosis (defined as an arterial ph below 7.35 with a partial pressure of arterial carbon dioxide >45 mm hg). (b) respiratory infection according to centre for disease control criteria [13] . (1) non-pneumonia lower respiratory infection was diagnosed if the patient had no clinical or radiographic evidence of pneumonia and had two of the following: fever (>38°c), cough, new or increased sputum production, rhonchus, wheezing and either of the following: a. organism isolated from culture obtained by deep tracheal aspirate or bronchoscopy, b. positive antigen test on respiratory secretions. (2) pneumonia was diagnosed if the patient met one of the following criteria: 1. rales or dullness to percussion on physical examination of chest, and any of the following: a. new onset of purulent sputum or change in character of sputum, b. organism isolated from blood culture, c. isolation of pathogen from specimen obtained by transtracheal aspirate, bronchial brushing, or biopsy; 2. chest radiographic examination with new or progressive infiltrate, consolidation, cavitation, or pleural effusion, and any of the following: a. new onset of purulent sputum or change in character of sputum; b. organism isolated from blood culture; c. isolation of pathogen from specimen obtained by transtracheal aspirate, bronchial brushing or biopsy; d. isolation of virus or detection of viral antigen in respiratory secretions; e. diagnostic single antibody titre (igm) or fourfold increase in paired serum samples (igg) for pathogen; f. histopathologic evidence of pneumonia. descriptive data are presented as percentages, means (sd) or medians (range). comparisons between patients with and without respiratory complications were performed with unpaired t test for quantitative variables or pearson chisquared test for categorical data. subsequently, a logistic regression model was created to determine the predictors of respiratory complications. in this model, the presence of respiratory complications was the dependent variable and age, gender, smoking and the variables that were significant in the univariate study were included as predictors of the model. analyses were conducted using stata ic 14 (stata-corp. 2015. stata statistical software:release 14. college station, tx: statacorp lp). the characteristics of the 174 patients studied are shown in table 1 . the majority were young men with low comorbidity. the hospital stay (median, p25-75) was 56 days (34-82). fifty-nine patients (34%) had thoracic trauma associated with the spinal cord injury, most of them with some kind of fracture as detailed in table 1 . thoracic trauma was more frequent in patients with a lesion level of t1-t5: 24 patients (19%) with cervical lesions and 35 patients (75%) with thoracic spine injuries (p < 0.001). ninety-two patients (53%) had some respiratory complication during the admission ( table 2) . regarding ventilatory assistance, 52 patients (30%) required mechanical ventilatory support during admission: invasive in 45 cases and non-invasive in seven. at the time of discharge, two patients required home ventilatory support, one invasive and the other non-invasive. two patients died during their stay, both due to respiratory failure. patients' characteristics according to the presence or absence of respiratory complications during admission are displayed in table 3 . patients who had respiratory complications during their admission required a longer hospital and intensive care unit stay. respiratory complications were more frequent in patients with thoracic injury, complete motor impairment (ais a-b), chest trauma or a history of previous respiratory disease. these variables were introduced in the multivariate analysis along with age, gender and smoking. the best model for predicting the onset of respiratory complications in our patients included the presence of previous respiratory disease, motor impairment and the concurrence of chest trauma (hosmer-lesmeshow asthma (n = 4), chronic obstructive pulmonary disease (n = 9), previous pulmonary thromboembolism (n = 2) and sleep apnoea syndrome (n = 2). c icu: intensive care unit. goodnes of fit test = 0.877). table 4 shows the or values for this model. the results of our study in a relatively large series of patients with traumatic low cervical or high thoracic spinal cord injuries showed a high prevalence of respiratory complications during the initial hospitalization just after the injury. as predictors of these complications we identified the presence of previous respiratory disease, complete motor impairment (ais a-b) and coexistent thoracic trauma. hospital stay was almost twice as long in patients with respiratory complications than in those without. only two patients died due to respiratory complications during admission, and two more required home ventilatory support at discharge. these data contrast with the 11% mortality and 6.5% ventilator-dependance rates previously reported in patients with high spinal cord injuries [14] . in our patients, the presence of a complete motor lesion was independently associated with a fourfold risk of respiratory complications. in contrast in the multivariate analysis the level of injury was not identified as an independent predictive factor. these findings are consistent with a predictive model previously described, that only found ais impairment scales a and b as predictors of respiratory complications [9] . we know that the lesions in the segments considered in the present study affect intercostal musculature, as well as accessory inspiratory muscles and abdominal muscles. a decrease in the respiratory activity of the inspiratory motor neurons in the caudal direction has been demonstrated [15] [16] [17] , and the presence of interneurons modulating the activity of these motoneurons has been found in segments of the thoracic medulla [18] . however, we do not have precise knowledge of the interactions between the different muscle groups. the integration of information from respiratory centres into each level of the lower cervical and upper thoracic spinal cord and its transmission to respiratory muscles is not yet clearly understood either. the non-explanatory design of our study do not allow us a causal interpretation of our findings and studies addressing these causal mechanisms and their relationship with the alterations of respiratory mechanics described in the first phase of spinal shock [4, 19] and their posterior improvement linked to the progressive increase in muscle tone [20] and potencial plasticity of the involved neural network [21, 22] are needed. as in previous series of traumatic spinal cord injuries [23, 24] , our patients were predominantly men with little associated comorbidity and charlson comorbidity index was not predictive of respiratory complications. however, asthma (n = 4), chronic obstructive pulmonary disease (n = 9), previous pulmonary thromboembolism (n = 2) and sleep apnoea syndrome (n = 2). c icu: intensive care unit. the presence of previous respiratory disease was associated with a very high risk of respiratory complications. this finding has been previously found in chronic spinal cord injuries [25] but has not been analysed until now in acute injuries. we think that it is clinically relevant since it could identify a group of patients in need of specific preventive protocols. early introduction of intensive physiotherapy and assisted coughing techniques together with the early use of mucolytics and bronchodilators should be considered [26] . the presence of thoracic trauma was also an independent risk factor for respiratory complications. as expected, it was more frequent in patients with thoracic injury but it was also detected in 18.9% of cervical injuries. in previous series focused on high cervical injuries its presence was not considered. our data underline the need to assess the coexistence of thoracic trauma when considering the study of respiratory complications in these patients and to be aware of the likelihood of respiratory complications. our study has several strengths. it tests a model to predict respiratory complications in a wide series of patients seen at a reference unit of spinal cord injuries and focuses on levels of spinal cord injuries on which little previous information is available. however, its retrospective nature is a limitation. as spinal cord injuries are rare events, this limitation is a common characteristic in the literature; we believe that it is counteracted in our study by the systematic approach applied to spinal cord injury and the information collected during the admission at our unit, which is a specialised reference centre. however, even though assisted coughing techniques have been progressively introduced during the years of study, precise data on its use or other physiotherapy techniques were not available. it would also be desirable to have information on lung function data and breathing during sleep in our patients to be able to assess periods of hypoventilation or the coexistence of sleep apnoeas, which are frequent in spinal cord injuries [27] [28] [29] . for purposes of the predictive model analysis, we clumped several respiratory complications all together. the predictor factors for each respiratory complications might be different. lastly, although in this study we focused on c5-t5 spinal cord injuries, it would have been interesting to compare the rates of respiratory complications in people with high and low cervical injuries. in summary, spinal cord injuries are acknowledged as catastrophic clinical events requiring complex clinical management at specialised units. our study draws attention to the frequent presence of respiratory complications during the hospital stay in patients admitted after and acute traumatic spinal cord injury between c5-t5. we have identified former respiratory disease, motor involvement according to ais grades a and b and the coexistence of thoracic trauma as predictive factors of respiratory complications in these patients. early identification of patients at risk of respiratory complications might help clinicians to implement preventive strategies to decrease these complications. the datasets generated and/or analysed during the current study are available from the corresponding author on reasonable request. author contributions js: responsible for the conceptualization of the study, data curation, methodology and formal analysis and paper development. magv: assisted with conceiving and designing the study and major revisions. ag, sm and mp: contributed with data curation, writing and overall editing. er and pl: assisted with writing of results and overall editing. gs: responsible for conceiving and designing the study, writing introduction and editing. also provided input on methodology, analysis, results and discussion section. jf: contributed with the conceptualization of the study and assisted with writing of introduction, results and discussion sections and editing. conflict of interest the authors declare that they have no conflict of interest. ethical approval we certify that all applicable institutional and governmental regulations concerning the ethical use of human volunteers were followed during the course of this research. publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. life expectancy after spinal cord injury: a 50-year study impact of respiratory complications on length of stay and hospital costs in acute cervical spine injury survival after spinal cord injury: a systematic review respiratory dysfunction and management in spinal cord injury forced vital capacity in two large outpatient populations with chronic spinal cord injury predictors of ventilator weaning in individuals with high cervical spinal cord injury multicenter analysis of diaphragm pacing in tetraplegics predictors of respiratory complications in patients with c5-t5 spinal cord injuries with cardiac pacemakers: positive implications for ventilator weaning in intensive care units incidence of respiratory complications following spinal cord injury predictors of pulmonary complications in blunt traumatic spinal cord injury respiratory complications and mortality risk associated with thoracic spine injury a systematic review identifies valid comorbidity indices derived from administrative health data international standards for neurological and functional classification of spinal cord injury cdc definitions for nosocomial infections what are the perspectives for ventilated tetraplegics? a french retrospective study of 108 patients with cervical spinal cord injury distribution of inspiratory drive to the external intercostal muscle in humans respiratory action of the intercostal muscles spatial distribution of inspiratory drive to the parasternal intercostal muscles in humans the respiratory control mechanisms in the brainstem and spinal cord: integrative views of the neuroanatomy and neurophysiology spinal shock revisited: a four-phase model repeated measurements of pulmonary function following spinal cord injury light-induced rescue of breathing after spinal cord injury functional regeneration of respiratory pathways after spinal cord injury risk of death after hospital discharge with traumatic spinal cord injury: a population-based analysis incidence, prevalence and epidemiology of spinal cord injury: what learns a worldwide literature survey? risk factors for chest illness in chronic spinal cord injury cough augmentation with mechanical insufflation/exsufflation in patients with neuromuscular weakness identification and treatment of sleep-disordered breathing in chronic spinal cord injury sleepdisordered breathing in spinal cord-injured patients: a short term longitudinal study sleep-disordered breathing and spinal cord injury: challenges and opportunities acknowledgements we gratefully acknowledge the spinal cord injury unit and the respiratory department in our centre, hospital universitari vall d'hebron, for their generous support during the development of this study. key: cord-255901-nl9k8uwd authors: barasheed, osamah; alfelali, mohammad; mushta, sami; bokhary, hamid; alshehri, jassir; attar, ammar a.; booy, robert; rashid, harunor title: uptake and effectiveness of facemask against respiratory infections at mass gatherings: a systematic review date: 2016-03-29 journal: int j infect dis doi: 10.1016/j.ijid.2016.03.023 sha: doc_id: 255901 cord_uid: nl9k8uwd objectives: the risk of acquisition and transmission of respiratory infections is high among attendees of mass gatherings (mgs). currently used interventions have limitations yet the role of facemask in preventing those infections at mg has not been systematically reviewed. we have conducted a systematic review to synthesise evidence about the uptake and effectiveness of facemask against respiratory infections in mgs. methods: a comprehensive literature search was conducted according to the preferred reporting items for systematic reviews and meta-analyses (prisma) guidelines using major electronic databases such as, medline, embase, scopus and cinahl. results: of 25 studies included, the pooled sample size was 12710 participants from 55 countries aged 11 to 89 years, 37% were female. the overall uptake of facemask ranged from 0.02% to 92.8% with an average of about 50%. only 13 studies examined the effectiveness of facemask, and their pooled estimate revealed significant protectiveness against respiratory infections (relative risk [rr] = 0.89, 95% ci: 0.84-0.94, p < 0.01), but the study end points varied widely. conclusion: a modest proportion of attendees of mgs use facemask, the practice is more widespread among health care workers. facemask use seems to be beneficial against certain respiratory infections at mgs but its effectiveness against specific infection remains unproven. the risk of acquisition and transmission of respiratory infections amplifies at mass gatherings (mgs) straining healthcare of the host country. for instance, in hajj, one of the largest annual mg events in the world, more than 2 million people attend each year in makkah, and over 90% suffer from at least one respiratory symptom, the risk of viral respiratory infections increases several folds and more severe respiratory infections such as pneumonia are the leading causes of hospital admission. [1] [2] [3] likewise, a number of influenza outbreaks were reported during the world youth day 2008, a large catholic gathering in sydney. 4 mgs are also linked to globalisation of various infections. for instance, the iztapalapa play passion, a religious festival in mexico, was believed to spark the outbreak of swine flu leading to its accelerated dissemination across the world. 5 therefore, international public health agencies, including world health organization (who), have issued guidelines on mass gathering preparedness to minimise the possible risks. 6 from a public health perspective, one of the key concerns is to prevent global spread of respiratory infections during mgs. interventions like vaccinations against viral and bacterial respiratory infections, anti-influenza prophylaxis and hand hygiene are considered as preventive measures but the measures have limitations. for instance, vaccinations against respiratory infections, such as influenza, are recommended for travellers to mgs such as hajj, 7 and even though a recent systematic review generally supports its effectiveness against laboratory-confirmed influenza at hajj, 8 frequent mismatch between vaccine strains and circulating strains is an important concern. 9 soaring antiviral resistance against both adamantanes and neuraminidase inhibitors is an issue that limits their widespread use in mgs. 4, 10 similarly, while hand hygiene has been recommended as a protective measure for attendees of mgs, its effectiveness is not fully evaluated in a mass gathering setting and the efficacy is debatable. 11 therefore, the role of another protective measure, facemask, should be explored in the prevention of respiratory infections. 12 facemask is believed to have a protective role in preventing nosocomial infections since the time of spanish influenza. 13 several studies have assessed the usefulness of facemask in household, community and healthcare settings, the findings of which have been summarised in a few reviews. [14] [15] [16] noticeable disparities of facemask effectiveness between these studies were observed. studies conducted in community or health care settings found facemasks to be generally effective against influenza-like illness (ili) or even against severe acute respiratory syndrome (sars) but its effectiveness against respiratory infections at mgs remains unknown. 15, 17 a review of non-pharmaceutical interventions against respiratory tract infections among hajj pilgrims presented data on the uptake of facemask and acknowledged that compliance was generally poor, but did not evaluate its effectiveness during hajj. 11 subsequently, further data on the uptake and effectiveness have become available, especially from a pilot randomised controlled trial (rct). 18 the aim of this systematic review is to explore the uptake and effectiveness of facemask against respiratory infections in mgs. studies were identified through searching electronic databases including; medline (pubmed and ovid), embase, scopus and cinahl from database inception to february 8, 2016. we used a combination of mesh terms and text words including: 'crowding' or 'mass gathering' or 'large event' or 'group assembly' or 'holiday' or 'travel' or 'sport' or 'olympic' or 'fifa' or 'festival' or 'hajj' (also alternative spelling 'hadj' or 'haj') or 'pilgrimage' and 'mask' or 'facemask' or 'surgical mask' or 'medical mask' or 'simple mask' and 'infection' or 'respiratory tract diseases' or 'disease outbreaks' or 'infectious disease' or 'respiratory tract infections' or 'influenza' or 'pneumonia'. additionally, an online search of pertinent epidemiology journals, including those not indexed in the mentioned databases (e.g. saudi epidemiology bulletin) was carried out through free hand google engine search. finally, manual search was performed reviewing reference lists of included studies to identify additional potentially relevant studies. the search result was presented according to the preferred reporting items for systematic reviews and meta-analyses (prisma) guidelines ( figure 1 ). 19 in the first phase, three authors (ob, sm and hb) identified the potential titles, and sifted the titles and abstracts against the inclusion criteria. titles of all studies published in english language and reported the use or effectiveness of facemask against respiratory infections in mgs were preliminarily included. studies that dealt with attendees of mgs of any age, gender and country were considered for inclusion. at the end of the screening phase, full texts of potentially relevant studies were retrieved for detailed study. finally studies that met the inclusion criteria were included for data synthesis. duplicates were excluded. five authors (ob, ma, hb, sm and ja) independently extracted the data from each study into a data extraction sheet which was divided in two sections, 'facemask uptake' and 'facemask effectiveness' and five authors subsequently cross-checked the entries (ob, aa, hb, sm and ja),while a sixth author (hr) arbitrated when a discrepancy occurred. the following data were abstracted in each extraction sheet: study design, year of conducting the study, sample size, country of origin, age, gender, diagnostic method used, definitions of study end point, and history of participants' chronic diseases, if available. the quality of the included studies were categorised according to a modified ranking criteria based on oxford evidence based medicine (http://www.cebm.net/) into groups (e.g., a, b, c, d) where a was for rcts of adequate sample size, b for observational studies of adequate sample size with good quality or pilot rcts or non-randomised trial, c for observational studies of inadequate sample size or of poor quality, and d for cases series, such as focus groups or qualitative surveys. the search results are summarised in figure 1 the study sample sizes varied widely ranging from 10 to 1717 participants. the included studies contained the pooled data of 12710 participants aged between 11 and 89 years (mean age ranged from 33.5 to 61.7 years in individual studies). about 37% of the pooled samples were females, in individual studies the proportion of females ranged from 10% to 63%. excluding three studies, which involved hcws deployed at hajj, [23] [24] [25] all other included studies involved hajj pilgrims. the origin of the participants varied depending on the study, seven studies included multinational participants, while the other 18 were exclusive to participants from a single country of origin; seven out of 18 (38.9%) were from saudi arabia, 20,23-28 according to study types 11 out of 25 were cohort studies, 1,2,22-24,28-33 another 11 cross-sectional studies, 21,25-27,34-40 two trials (not necessarily rcts) 18, 20 and one case-series conducted as a qualitative study 41 (table 1) . the median uptake of facemask in pooled sample was 53.5%. the lowest reported uptake was 0.02% by elachola et al. among pilgrims in a unique study that involved quantification of facemasks through photo frames from surveillance camera during the hajj in 2013, therefor it is considered as an outlier. 21 the highest uptake was 92.8% observed by al-asmary et al. among health care workers during hajj in 2005. 24 excluding these two studies (elachola et al 21 and al-asmary et al 24 ) , uptake rate among pilgrims has remained generally steady with gradual increase from 24% in 1999 to 64% in 2014 with minor fluctuations (figure 2 ). studies involving hcws reported an uptake from 50% in 2009 to 92.8% in 2005. according to the pilgrims' country of origin, malaysian pilgrims were noticed to be most compliant to using facemasks (70.9%), 37,40 followed by french (60.5%) 1,2,32,33 and iranians (60%) 31 (table 1) . only three studies, all involving australian pilgrims, evaluated the reasons of compliance (or non-compliance) of using facemask during hajj. 18, 29, 41 the most reported reasons for wearing facemask were to avoid transmission of infectious organisms and protection from air pollution. 29 however, discomfort and difficulty in breathing were the most reported reasons for not wearing facemask. 18,41 thirteen studies investigated the effectiveness/efficacy of facemask against respiratory infections, but the endpoints varied very widely. most of these studies (9 out of 13) used a combination of respiratory symptoms (syndromic) as endpoints with varying definitions. for instance, acute respiratory infections (ari) was used as an endpoint in three studies, 20,24,28 ili in two, 18, 37 upper respiratory tract infection (urti) in two, 22, 26 respiratory illness in two 30, 40 and respiratory tract infections in one. 31 however a couple of studies used only one respiratory symptom as an endpoint: fever 39 and cough. 32 only one study established laboratory-proven viral infections 25 as an endpoint. definitions for the endpoints are detailed in table 2 . in regards to the effectiveness of facemask, four out of thirteen studies demonstrated significant effect against respiratory infections, 18, 20, 22, 28 two others showed some effect but did not reach statistical significance. 25, 26 one study assessed its effectiveness against fever but ruled out its protectiveness, 39 and the other six studies did not show effectiveness but results were not statistically significant. 24, [30] [31] [32] 37, 40 the pooled data from all studies revealed significant protectiveness of facemasks against respiratory infections in general at hajj (relative risk [rr] = 0.89, 95% ci: 0.84-0.94, p < 0.01) ( table 2) . according to the ranking system we used, most of the studies were of average quality (c) whereas two studies were ranked above average (b): a pilot rct 18 and a large cross-sectional study, 26 the other seven studies were of below average quality (d) either because of small sample size or poor study quality (table 1) . this systematic review shows that the use of facemask among the attendees of mgs remains essentially unchanged for decades although exceptionally in one study a very high uptake (about 93%) 24 or a very low uptake rate (0.02%) 21 has been reported but such variability can be explained by their unique study designs or population characteristics. the pooled data of this systematic review suggest that facemask is generally effective against respiratory infections at hajj, however the endpoints varied widely. the uptake of facemask among hcws deployed at hajj was generally higher than that among ordinary hajj pilgrims with average compliance among hcws being 72% compared to 46% among pilgrims. this finding is similar to what have been found in other studies that examined the uptake of facemask in other settings such as health care and community settings. for instance, the uptake of facemask among hcws in several studies ranged from 56.6% to 84.3% (average 70.7%). [42] [43] [44] [45] on the other hand, the uptake of facemask among ordinary population in diverse household and community settings ranged from 38% to 80.7% (average 55%). [46] [47] [48] [49] [50] [51] [52] this could be explained by several individual or organisational factors. for example, hcws have firsthand knowledge about the risk of respiratory infections and the role of preventive measurements in hajj. 25 similarly, studies in non-mgs settings showed a positive relationship between hcws' knowledge about the risk of infectious diseases and their compliance to preventive measures including the use of facemask. [53] [54] [55] organisational factors such as ready availability of facemask in health care settings, proper training programs and supportive policy of health care system could have played an important role in improving the compliance of hcws to facemask use. [54] [55] [56] [57] on the other hand, limited studies explored these individual and organisational factors among hajj pilgrims. a few studies showed that providing educational session on protective measures against respiratory infections (including facemask) before hajj was associated with significantly higher uptake of facemasks among pilgrims. 18, 20, 27, 36, 38 moreover, adequate accessibility and availability of facemask during hajj may enhance the compliance of pilgrims. abdin et al and barasheed et al revealed a higher uptake of facemask among groups who were provided with sufficient quantity of free facemask (81.3% versus 33.6%, p < 0.01, and 76% versus 12%, p < 0.01, respectively). 18, 20 however, reasons for not using facemask during hajj have not been explored adequately. while use of facemask at hajj has been officially recommended by saudi ministry of health since 2014, it is too early to have a significant impact on pilgrims' practice of facemask use. 58 although hajj took place in different seasons (spring, winter and autumn), the uptake of facemask among hajj pilgrims during the last decade remained generally stable (figure 2) . findings also showed that there was no significant change in facemask uptake among hajj pilgrims during the course of influenza a (h1n1) pandemic outburst in 2009, and the middle east respiratory syndrome corona virus (mers-cov) outbreak since 2012. this does not concur with what has been reported in published studies involving the members of general public over the several outbreaks of respiratory infections in non-mg settings. [59] [60] [61] [62] [63] [64] those studies showed an increase in facemask use during the outbreaks due to participants' perceived threat of infection. poor awareness among many pilgrims of contemporary outbreaks might explain why their uptake of facemask did not increase even during an ongoing outbreak. [65] [66] [67] interestingly, pilgrims of asian origin (e.g. malaysians) had higher facemask uptake compared to pilgrims from other regions. 37, 40 a polling study that evaluated the uptake of non-pharmaceutical measures during the pandemic influenza a (h1n1) of 2009 found that participants of asian origin (e.g. japan) had the higher facemask uptake (71%) compared to the uptake of participants of western or latin american origin. 68 presence of several peaks of influenza seasons in some asian countries, overcrowding, dense smog and air pollution in many cities may explain the higher uptake of facemask among people from asian countries; 69,70 additionally, cultural acceptance practice of the population around facemask while in public may make a difference. 68 focused studies are required to investigate factors influencing facemask compliance among attendees of hajj and other mgs. in this systematic review, pooled data of facemask effectiveness showed that participants who used facemask during hajj are about 20% less likely to suffer from respiratory infections compared to those who do not use it. this effectiveness of facemask is inconclusive due to great heterogeneity in study questions, assessment methods, study designs and qualities, and endpoints. in regards to the research questions, three out of 13 studies investigated facemask effectiveness as the primary research objective: all three studies yielded significant results; whereas only one out of the other 10 studies that assessed facemask as a secondary or indirect outcome, yielded significant results. further, there was great heterogeneity in how the frequency and duration of facemask use were assessed. although, most of the studies used a self-reported questionnaire to quantify facemask uptake among participants, the qualitative descriptive terms that the studies used (e.g. ''always'', ''mostly'', ''sometimes'' or ''never'') may have introduced subjective bias, since qualitative description varies depending on participants' perception about the frequency and duration of use. however, only one study used measurable criteria in their questionnaires to quantify the number of facemasks used including the duration (in hours) and frequency of use, finding that using facemask more than eight hours per day was associated with significant decrease in ili symptoms among hajj pilgrims. 18 using surveys with more objective options may decrease bias, 71 and provide more accurate estimate of compliance to facemask use in mgs. study designs also may have contributed to variability in results. for instance, two trials, a pilot rct and a non-randomised trial, reported facemask to be significantly effective against respiratory infections at hajj, whereas only two out of six cohort studies reported significant results. in contrast, none of the crosssectional studies yielded significant results. this may indicate that a higher quality study is more likely to produce convincing results. finally, facemask effectiveness also differed depending on the study endpoints. for example, studies that examined effectiveness of facemask against a single respiratory symptom (such as cough, sore throat or fever) either ruled out or did not fully support its effectiveness. 32, 37, 39 this is most likely because singular endpoints are often prone to subjective biases due to their non-specificity. in addition, solitary respiratory symptoms may result from causes other than infections; for instance, cough may result from exposure to dust or smoke during hajj or may be a manifestation of a chronic respiratory condition of non-infectious aetiology, e.g., bronchial asthma. 29 on the other hand, most of the studies that used syndromic criteria (constellation of symptoms) as an endpoint reported facemasks to be effective against respiratory infections during hajj. 18, 20, 22, 26, 28 this is most likely due to the fact that syndromic endpoints are more specific for an illness than a singular symptom. only one study used laboratory-confirmed infection as an endpoint, but its sample size was relatively small (n = 104) and it failed to demonstrate statistically significant protectiveness of facemasks against respiratory viral infections among hajj hcws. 25 similarly, in non-mg settings, effectiveness of facemask varied depending on the study endpoint. [42] [43] [44] [45] [46] [47] [48] [49] [50] [51] [52] [72] [73] [74] metaanalysis of rcts involving facemask in non-mgs showed efficacy against ili but not against laboratory-confirmed influenza. 14, 15, 75 this study is the first focussed systematic review that describes both the uptake and effectiveness of facemasks against respiratory infections in mgs, and it compiles a data pool of 12710 participants originating from more than 50 countries. however, the main limitation is that most of the studies were of 'average' or 'below average' quality. there was only one rct but that was a pilot trial of small sample size, and there was another 'trial' published in a nonindexed journal that did not report methodological details including whether and how randomisation was done. as all included studies were conducted only in the context of hajj, it is not possible to generalise the results to other mgs. a large scale clustered rct is currently in its final phase that will measure the efficacy of facemasks against both 'syndromic' and laboratoryconfirmed viral infections. 75 the full results of the trial, once available, are likely to provide firmer evidence on the usefulness of facemask against respiratory infections among attendees of mgs. in summary, the use of facemask among attendees of a particular mg (hajj) remains almost steady with negligible increase throughout the last decade with an average uptake of 50%. facemasks seem to be beneficial against certain respiratory infections during hajj but not definitively proven. professor robert booy has received funding from baxter, csl, gsk, merck, novartis, pfizer, roche, romark and sanofi pasteur for the conduct of sponsored research, travel to present at conferences or consultancy work; all funding received is directed to research accounts at the children's hospital at westmead. dr harunor rashid received fees from pfizer and novartis for consulting or serving on an advisory board. the other authors have declared no conflict of interest in relation to this work. circulation of respiratory viruses among pilgrims during the 2012 hajj pilgrimage respiratory viruses and bacteria among pilgrims during the 2013 hajj causes of hospitalization of pilgrims in the hajj season of the islamic year influenza outbreaks during world youth day 2008 mass gathering inside the outbreak of the 2009 influenza a (h1n1)v virus in mexico communicable disease alert and response for mass gatherings: key considerations health conditions for travellers to saudi arabia for the pilgrimage to mecca (hajj) vaccinations against respiratory tract infections at hajj mismatching between circulating strains and vaccine strains of influenza: effect on hajj pilgrims from both hemispheres emergence of oseltamivir resistance: control and management of influenza before, during and after the pandemic non-pharmaceutical interventions for the prevention of respiratory tract infections during hajj pilgrimage prevention of influenza at hajj: applications for mass gatherings the open-air treatment of pandemic influenza the use of masks and respirators to prevent transmission of influenza: a systematic review of the scientific evidence unmasking masks in makkah: preventing influenza at hajj the use of facemasks to prevent respiratory infection: a literature review in the context of the health belief model physical interventions to interrupt or reduce the spread of respiratory viruses: systematic review pilot randomised controlled trial to test effectiveness of facemasks in preventing influenza-like illness transmission among australian hajj pilgrims in 2011 preferred reporting items for systematic reviews and meta-analyses: the prisma statement effect of use of face mask on hajj-related respiratory infection among hajjis from riyadh -a health promotion intervention study mass gathering-related mask use during 2009 pandemic influenza a (h1n1) and middle east respiratory syndrome coronavirus the role of using masks to reduce acute upper respiratory tract infection in pilgrims. abstract no. 7. 4th asia pacific travel health conference acceptance and adverse effects of h1n1 vaccinations among a cohort of national guard health care workers during the 2009 hajj season acute respiratory tract infections among hajj medical mission personnel, saudi arabia the prevalance of respiratory viruses among healthcare workers serving pilgrims in makkah during the 2009 influenza a (h1n1) pandemic patterns of diseases and preventive measures among domestic hajjis from central, saudi arabia effect of health education advice on saudi hajjis hajj-associated acute respiratory infection among hajjis from riyadh pilot use of a novel smartphone application to track traveller health behaviour and collect infectious disease data during a mass gathering: hajj pilgrimage protective practices and respiratory illness among us travelers to the 2009 hajj respiratory tract infections and its preventive measures among hajj pilgrims, 2010: a nested case control study the inevitable hajj cough: surveillance data in french pilgrims protective measures against acute respiratory symptoms in french pilgrims participating in the hajj of behavioral risk factors for disease during hajj 1422 h pre-hajj health related advice sources of health education for international arab pilgrims and the effect of this education on their practices towards health hazards in hajj the prevalence of acute respiratory symptoms and role of protective measures among malaysian hajj pilgrims epidemiological pattern of diseases and risk behaviors of pilgrims attending mina hospitals, hajj 1427 h (2007 g) health related experiences among international pilgrims departing through king abdul aziz international airport the prevalence and preventive measures of the respiratory illness among malaysian pilgrims in 2013 hajj season australian hajj pilgrims' infection control beliefs and practices: insight with implications for public health approaches use of surgical face masks to reduce the incidence of the common cold among health care workers in japan: a randomized controlled trial a cluster randomised trial of cloth masks compared with medical masks in healthcare workers a cluster randomized clinical trial comparing fit-tested and non-fit-tested n95 respirators to medical masks to prevent respiratory virus infection in health care workers a randomized clinical trial of three options for n95 respirators and medical masks in health workers surgical mask to prevent influenza transmission in households: a cluster randomized trial facemasks and hand hygiene to prevent influenza transmission in households: a cluster randomized trial preliminary findings of a randomized trial of non-pharmaceutical interventions to prevent influenza transmission in households impact of non-pharmaceutical interventions on uris and influenza in crowded, urban households face mask use and control of respiratory virus transmission in households the role of facemasks and hand hygiene in the prevention of influenza transmission in households: results from a cluster randomised trial findings from a household randomized controlled trial of hand washing and face masks to reduce influenza transmission in attitudes and behaviour of hospital health-care workers regarding influenza a/h1n1: a cross sectional survey facemasks for the prevention of infection in healthcare and community settings behind the mask: determinants of nurse's adherence to facial protective equipment compliance with universal precautions among health care workers at three regional hospitals sars transmission among hospital workers in hong kong health conditions for travellers to saudi arabia for the umra and pilgrimage to mecca (hajj) -2014 who is that masked person: the use of face masks on mexico city public transportation during the influenza a (h1n1) outbreak avian influenza risk perception and preventive behavior among traditional market workers and shoppers in taiwan: practical implications for prevention prevalence of preventive behaviors and associated factors during early phase of the h1n1 influenza epidemic widespread public misconception in the early phase of the h1n1 influenza epidemic anticipated and current preventive behaviors in response to an anticipated human-to-human h5n1 epidemic in the hong kong chinese general population factors influencing the wearing of facemasks to prevent the severe acute respiratory syndrome among adult chinese in hong kong hajj pilgrims knowledge about middle east respiratory syndrome coronavirus attitudes and practices concerning middle east respiratory syndrome among umrah and hajj pilgrims in samsun australian hajj pilgrims' knowledge about mers-cov and other respiratory infections public response to the 2009 influenza a h1n1 pandemic: a polling study in five countries influenza seasonality and vaccination timing in tropical and subtropical areas of southern and south-eastern asia air pollution and health -counselling options for physicians validity and reliability of measurement instruments used in research mask use, hand hygiene, and seasonal influenza-like illness among young adults: a randomized intervention trial surgical mask vs n95 respirator for preventing influenza among health care workers: a randomized trial efficacy of face masks and respirators in preventing upper respiratory tract bacterial colonization and co-infection in hospital healthcare workers a clusterrandomised controlled trial to test the efficacy of facemasks in preventing respiratory viral infection among hajj pilgrims the authors would acknowledge the support of ms. trish bennett, manager, medical library, the children's hospital at westmead, nsw, australia, for help with literature search. key: cord-262673-j2ot35lt authors: ahmed-hassan, hanaa; sisson, brianna; shukla, rajni kant; wijewantha, yasasvi; funderburg, nicholas t.; li, zihai; hayes, don; demberg, thorsten; liyanage, namal p. m. title: innate immune responses to highly pathogenic coronaviruses and other significant respiratory viral infections date: 2020-08-18 journal: front immunol doi: 10.3389/fimmu.2020.01979 sha: doc_id: 262673 cord_uid: j2ot35lt the new pandemic virus sars-cov-2 emerged in china and spread around the world in <3 months, infecting millions of people, and causing countries to shut down public life and businesses. nearly all nations were unprepared for this pandemic with healthcare systems stretched to their limits due to the lack of an effective vaccine and treatment. infection with sars-cov-2 can lead to coronavirus disease 2019 (covid-19). covid-19 is respiratory disease that can result in a cytokine storm with stark differences in morbidity and mortality between younger and older patient populations. details regarding mechanisms of viral entry via the respiratory system and immune system correlates of protection or pathogenesis have not been fully elucidated. here, we provide an overview of the innate immune responses in the lung to the coronaviruses mers-cov, sars-cov, and sars-cov-2. this review provides insight into key innate immune mechanisms that will aid in the development of therapeutics and preventive vaccines for sars-cov-2 infection. severe acute respiratory syndrome coronavirus 2 (sars-cov-2) reportedly emerged at a live animal market in the chinese city of wuhan is currently causing a pandemic and negatively affecting global health (1) (2) (3) . there are more than 11 million confirmed sars-cov-2 infections with an mortality rate that widely varies by country and region (4) . even in industrialized countries, sars-cov-2 led healthcare systems approach the brink of collapse by overwhelming their capacity and straining resources. governments and local leaders ordered the shutdown of cities, regions, countries leading to massive disruptions in the local and global economy. unlike previous coronavirus (cov) infections, the rapid global spread, high transmission rate, longer incubation time, and disease severity of sars-cov-2 requires a better understanding of the epidemiology and immunopathogenesis of this viral outbreak in order to learn from this experience and to manage future pandemics. sars-cov-2 is a highly pathogenic cov (5) (case-fatality rate of 3.6-3.8%) (4, 6) that is related to severe acute respiratory syndrome cov (sars-cov) (case-fatality rate of 14-15%) and the middle east respiratory syndrome cov (mers-cov) (case-fatality rate of 34.4%), see also table 1 (138, 139) . sars-cov, sars-cov-2 and mers-cov target the lower respiratory system, causing respiratory illnesses, including severe pneumonia, acute lung injury (ali) and acute respiratory distress syndrome (ards) (140, 141) . sars-cov-2 infection results in higher hospitalization rates in the elderly (>65) and persons with pre-existing conditions including hypertension, diabetes and obesity compared to rates among younger populations without pre-existing conditions ( table 1 ) (142, 143) . in addition to an age disparity, males with covid-19 appear to have higher risk for worse outcomes and death (143, 144) . epidemiological research of the sars and mers infections also showed that males had a higher mortality rate than females (144) (145) (146) . while sars-cov-2 is a novel coronavirus, several important insights have already been made about its basic mode of transmission. virus particles are inhaled in respiratory droplets expelled from the airways of infected individuals. angiotensinconverting enzyme 2 (ace2), expressed on the ciliated bronchial cells, endothelial cells, and type i and ii alveolar cells, is the host receptor for cell entry into the respiratory tract by both sars-cov-2 and sars-cov ( table 1 ) (147) (148) (149) (150) . the spike protein (s) of cov is responsible for the entry of the virus into the target cell (figure 1 ) (147, 151) . ace2 is a type i transmembrane metallocarboxypeptidase that plays a vital role in the renin-angiotensin system (ras) (152, 153) , which in turn is critical in maintaining blood pressure homeostasis as well as fluid and salt balance in mammals. ace2 is found in vascular endothelial cells, in the renal tubular epithelium, and in leydig cells of the testes (154) . studies have shown that ace2 is expressed in gastrointestinal (gi) tissues, making it a potential site for harboring sars-cov (155) . this may be one of the reasons for gi pathology reported in some patients with covid-19 and viral shedding in stool. in contrast, mers-cov uses dipeptidyl-peptidase 4 (dpp4) as an entry receptor, which is expressed on endothelial cells and some epithelial tissues ( table 1 ) (19, 156) . accumulating data suggest that the innate anti-viral response and adaptive immunity may contribute to a cytokine storm leading to systemic hyper inflammation and exacerbation of the disease in patients with (a) comorbidities (b) older than 65 years of age (c) of the male sex. the exact role of the innate immune system in disease pathogenesis and prevention between the sexes and the impact of age is not fully elucidated. in addition, the potential dysregulation of the innate immune response by sars-cov and sars-cov-2 is not completely understood which warrants further research. the cells of the airway epithelium are the first line of defense, providing a mechanical barrier (mucociliary escalator) that expels particles and pathogens via cilia, mucus, and induced coughing (157, 158) . this barrier includes cells of the pulmonary epithelium and tissue-resident macrophages and dendritic cells (dcs). the macrophages and dcs express pattern recognition receptors (prrs) that can detect molecules from pathogens (pathogen-associated molecular patterns-pamps) or molecules released from damaged cells (damage or danger-associated molecular patterns-damps) (158) (159) (160) . in the lung, there are two populations of macrophages, alveolar and interstitial macrophages (161) . in addition to these macrophages, dcs play a vital role in facilitating the host defenses against respiratory diseases (162) (163) (164) . dcs can be divided into plasmacytoid (pdc) and myeloid types (mdc) (165) (166) (167) . macrophages and the two dc subtypes trigger antiviral responses by generating a substantial amount of type i interferon and these cells play important roles in immune surveillance in the airways and the distal lung (74, (168) (169) (170) (171) (172) . during steady state, the dc density in lung associated tissue declines from the trachea to the alveoli (173) while the representation of cells in macrophage compartment seems more complex (174) . if a virus infects airway epithelial cells, the viral rna would be sensed via intrinsic innate receptors, including rig-1, mda5, nlrp3 inflammasome, and the rna sensing tlrs 7 and 8. in the case of influenza a infection, triggering the prrs causes a strong induction of type 1 interferon (ifn) in epithelial cells (175, 176) . in other viral infections, such as respiratory syncytial virus (rsv), alveolar macrophages are the predominant source of type 1 ifns (161). furthermore, respiratory epithelial cells and lung macrophages are capable of secreting a broad range of chemokines like il-8, macrophage inflammatory protein-1 (mip-1), rantes and cytokines including tnf-α, il-6, il-1β that influence the types of immune cells being recruited to the area in response to acute viral infections (177, 178) . macrophages, depending on their polarization status of either m1 or m2, and in a similar way as airway epithelial cells, can further elicit a th1 or th2 response (158, 161, 178) . in the case of influenza virus infection, the magnitude of epithelial cell response can be proportional to the amount of virus which result in paracrine induction of ifn λ (175) . not only can airway epithelial cells produce a large array of cytokines/chemokines in response to an acute viral infection, but, depending on the magnitude of prr engagement and the combination of pamps and damps triggered, these epithelial cells can modulate the type of chemokines/cytokines produced and influence the influx of innate and adaptive immune cells (158, 160) . the response to different viral infections is generally similar, however, the response can be tailored in timing, magnitude and the induced gene signatures in response to each virus (179) . unlike rsv and mers-cov, which both productively infect alveolar multiple cell types in the lower respiratory tract were found to be infected, including type i alveolar epithelium, macrophages, and putative cd34 + oct-4 + stem/progenitor cells in human lungs (15) (16) (17) ciliated bronchial epithelial cells and type ii pneumocytes (7, 18) un-ciliated bronchial epithelial cells and type ii pneumocytes (19) (20) (21) infect mostly human type i and type ii pneumocytes and alveolar macrophages (22) respiratory, nasal, corneal and intestinal epithelial cells (23) club cells, ciliated cells, type i and type ii alveolar cells (24) ciliated epithelial cells of the upper and lower respiratory tract (25) the ciliated cells of the human airway epithelium are the main target, it also infects basal cells (26) and immune cells, such as macrophages, b cells cd4 + and cd8 + t cells (27) upper and lower airways epithelial cell ( no specific antiviral drugs (134) antiviral drugs may be a treatment option (135) no antiviral agents symptomatic treatment supportive care (136) there are no approved antiviral medications (137) ace2 macrophages (73, 180) , seasonal influenza and sars-cov usually lead to non-productive infections in these cells (181) . in addition, sars-cov infection of primary monocytes yielded little virus, likely due to the suppressive effects of ifn-α (182) . thus, the initial cell type(s) involved in propagating a viral infection intensifies the complexity of the immune response. another key factor that determines the magnitude of the immune response is the induction and rate of cell death. although related, mers-cov induces widespread cell death when compared to sars-cov in human bronchial epithelial cell cultures (183) . however, the sars-cov open reading frame 3a (orf3a) protein can induced necrotic cell death in a variety of cell lines (184) . the same orf3a protein can activate the nlrp3 inflammasome, leading to activation of nf-κb and elevated secretion of active il-1β in cell culture (185) . cytokine (186), with slightly different cytokine/chemokine profiles. this delay in cytokine induction was confirmed in another study using the same epithelial cell lines (187) as well as in human alveolar type ii cells (18) . in both cell lines and primary alveolar type ii cells, sars-cov induced ifn-β, ifn-λ, cxcl10, cxcl11, il-6, ip-10, and tnf-α (18, 187) . mers-cov did not induce ifn-β but induced higher level of il-8 transcript in cell culture. however, no difference in il-8 production was observed between sars-cov and mers-cov at 48 h post-infection (186) . this was confirmed in-vivo using a non-human primate model comparing the immune responses to sars-cov infection between young adult cynomolgus macaques (3-5 yrs) and older macaques (10-19 yrs) (188) . interestingly, the high induction of il-8 was observed on a transcript level in the older animals, while the younger once showed higher levels of ifn-β transcript (188) . in all animals, the expression of ifn-β was inversely correlated with the pathology score, supporting the role of ifn-β in controlling disease severity (188) and introducing a potential area of research to define age disparity observed in patients infected with sars-cov-2. both older age and male sex are important factors associated with high mortality of sars-cov and sars-cov-2 infection (189, 190) . many viruses have evolved to disrupt and subvert the immune responses. a common virus that is well-known to affect the lower airway and counteract the immune response is rsv (178, 191) . the rsv genome encodes non-structural proteins ns1 and ns2 that can block type 1 ifn production and signaling in cell cultures (191) . similar to rsv, the measle virus v protein blocks ifn-α and β signaling by inhibiting stat1 and stat2 signaling in cell lines (192) , whereas mers-cov m protein also suppresses type 1 ifn by inactivating irf3 (193) , leading to the low expression of ifn-β. in contrast to reports in epithelial cell lines or primary alveolar type ii cell culture and observations in non-human primates, sars-cov nucleocapsid (n) protein and membrane (m) protein, as well as nsp1, can suppress ifn response via various mechanisms in cell lines (194) (195) (196) . to bridge the dichotomy of inhibition of ifn signaling in cell lines, and the ifn expression in vivo, cells recruited by the infection need to be considered as a potential source. as previously discussed, infected epithelial cells via paracrine signaling to neighboring cells and resident macrophages, secrete chemokines and cytokines to attract other immune cells. in general, monocytes/macrophages are recruited by ccl3 (mip-1a), ccl2 (mcp-1), and neutrophils are recruited by il-8 (cxcl8), cxcl2, and cxcl5 chemokines, all of which can be secreted by airway epithelial cells (178, 197, 198) . both monocytes and neutrophils are also recruited by complement fragment (anaphylatoxin) c5a (figure 1) . both influenza and sars virus can induce acute lung injury (ali) which is accompanied by high levels of c5a, leading to the influx and activation of innate immune cells (199) (figure 1) . serum samples and lung tissue of sars patients showed high-level expression of cxcl10 (ip-10), which is also found to be induced by sars-cov in the epithelial cell line calu-3 (200) . significant neutrophil, macrophage and cd8 t-cell infiltration can be found in the lung of sars patients by immunohistochemistry (76, 77, 201) . in addition to post-mortem lung histology analysis in patients with sars-cov, experiments using rhesus macaques infected with sars-cov found monocyte and macrophage recruitment. the accumulation of pathogenic inflammatory monocytemacrophages (imms) was also observed in a sars-cov mouse model. the accumulation of imms resulted in heightened lung inflammatory cytokine/chemokine levels, extensive vascular leakage, and impaired virus-specific t cell responses (202) . a strong infiltration of cd68 and mac387 positive monocytes/macrophages were found in the human and animals lung samples (203, 204) . macrophages further stimulate fibroblasts to deposit extracellular matrix leading to pulmonary fibrosis (205) , which was also observed in patients who recovered from sars (206, 207) . autopsy samples acquired from patients with sars-cov-2 patients contained viral nucleocapsid protein (np) positive cd68 + macrophages in the capillaries of the spleen and in lymph nodes, indicating that sars-cov-2 might migrate into the spleen and lymph nodes through macrophages. this study also found that cd169 + macrophages appear to mediate sars-cov-2 into these tissue sites, contributing to virus dissemination (208) . similar to sars-cov-2, sars viral particles and genomic sequences were detected in monocytes, macrophages as well as within different organs of sars patients (15) . sars-cov was shown to infect both immature and mature human monocyte-derived dcs by electron microscopy and immunofluorescence. the detection of negative strands of sars-cov rna in dcs suggests viral replication, but no increase in viral rna was observed (209) . as mentioned above, there was no perceivable increase to sars-cov replication in primary monocytes (182) . another study looked at sars-cov and mers-cov replication in human macrophages, human monocyte-derived macrophages, and dendritic cells (mddcs) and found that both viruses replicated poorly. mers-cov induced ifn-λ1, cxcl10, and mxa mrnas in both macrophages and mddcs, however, sars-cov was unable to induce such responses (69) . interestingly, depletion of inflammatory monocyte-macrophages in the mouse model partially protected from lethal sars infection (210) . these data suggest that monocytes, macrophages and dendritic cells have essential roles in cov infection. the severity of disease and the response to these viruses seems to be dependent on the induced cytokine/chemokine profiles and the amplification of the immune response by the recruited cells. growing evidence of dysregulation of an innate anti-viral response originates from studies using clinical samples (211) and murine models (202, 212, 213) . in addition to dysregulated cellular responses, the complement system may play an important role in sars-cov-2 infection (figure 1) . evidence comes from sars infected patients who had lower levels of mannan binding lectin (mbl) in serum compared to healthy controls (214) . the sars patients with a higher frequency of mbl gene polymorphisms were associated with lower serum levels or deficiency of mbl (214) . it is still unknown if this is also true for covid-19 patients, which requires further investigation. in cell culture experiments sars-cov was able to bind and activate the complement cascade and block viral infection (214) . preliminary findings in a limited number covid-19 patients found significant deposits of the membrane attack complex (mac), c4d and mbl-associated serine protease (masp)2 in the microvasculature indicating sustained, systemic activation (215) . the sars-cov-2 spike protein was co-localized with c4d and mac (215) . in a non-peer reviewed publication by gao et al., mers-cov, sars-cov and sars-cov-2 n protein are able to bind to masp-2 leading to enhanced complement activation (216) (figure 1) . in a later phase of the infection, the complement system might be also triggered via antibodies bound to the virus (classic activation pathway, figure 1 ). this excessive complement activation might play a role in multi organ damage in severe covid-19 cases (217) . in a mers-cov mouse model the blockade of the c5a-c5ar axis alleviated not only lung damage but also spleen damage (218) . mice treated with a monoclonal antibody to c5a showed reduced lung infiltration of cd68 + cells and significantly lower cytokine levels of il-1 β, tnf-α, inf-γ and il-12 (218) . complement blockade might be an important way to curb part of the immune dysregulation associated with covid-19. overall, we need to look closer at the role of the complement system, the recruited innate immune cells and their combined role in pathogenesis, viral clearance and the eventual resolution of the infection. the most abundant leukocytes, neutrophils, play a critical role in clearing viral infections. neutrophils, attracted by chemokines/cytokines released by tissue-resident macrophages and dcs, swarm to the site of infection. they recognize and release bioactive compounds, including cytokines, chemokines and ros, as well as nos in the very early phase of the infection (219, 220) . neutrophils modulate other innate and adaptive immune responses via cytokine/chemokine release and cell death and, therefore, can ameliorate or exacerbate disease progression. neutrophils infiltrate tissues infected by cov, including sars-cov, rat coronavirus (rcov), and mouse hepatitis virus (mhv). a high neutrophil count in the blood of sars patients at the time of hospital admission is associated with a poor prognosis (221, 222) . gao et al. suggested that patients with sars-cov-2 pneumonia can be stratified by neutrophil to lymphocyte ratio (nlr) and age (216) . patients older than 50 years of age and having an nlr ≥ 3.13 had more severe illness, so rapid access to the intensive care unit is required (79, 223) . experiments in mice showed that sars-cov disease severity in older mice correlated with increased pulmonary inflammation and influx of neutrophils (224, 225) . infection of rats with rcov could lead to neutrophil infiltrating into the respiratory tract early after inoculation, followed by the recruitment of macrophages and lymphocytes (226) . infection of mice with a neurotropic murine cov (mhv-jhm) showed infiltration of neutrophils into the brain as early as the first day after inoculation, which then promoted the recruitment of other types of inflammatory cells into the brain, likely through the loss of the blood-brain barrier integrity (227) . gene expression analysis in experimentally infected rhesus macaques with mers-cov revealed the recruitment of neutrophils into infected lung tissue (228, 229) . angiotensin-converting enzyme inhibitors (ace-is) could serve as a potential risk for fatal covid-19 through the upregulation of ace2 (230) and may provide a direct linkage to neutrophils and disease progression. investigators found that ace2 modulates il-17-mediated neutrophil influx by impacting stat3 activity (231) . animal models used to study the pathogenesis of sars-cov-2 have revealed important roles of neutrophils in infection and confirmed findings observed in patients. a new aspect in sars-cov-2 infection is the potential role of neutrophil extracellular traps (nets). the process of net formation is a specific type of cell death that can be triggered under inflammatory conditions (232, 233) , such as gm-csf+c5a, il-8, ifn-α+c5a or other tlr response mediators; all conditions present in severe sars-cov-2 infection (232, 233) . the net formation has been observed in covid-19 patients and may contribute to thrombotic complications in covid-19 patients (234, 235) . microvascular injury and thrombosis have been reported in covid-19 patients, increasing the likelihood that neutrophil net formation might play a role (215, 236, 237) . net formation was reported to be involved in clot formation and thrombosis and can further increase inflammation (232, 233) . therefore, neutrophils can attract a second wave of immune cells to the site of infection by cytokine/chemokine secretion as well as via netosis (238, 239) , which included monocytes and natural killer cells. on the other hand aggregated nets were reported to limit inflammation by degrading cytokines and chemokines and disrupting neutrophil recruitment and activation (240) . despite the presence of neutrophils in sars-cov-2-infected tissues, their role in the clearance and/or immunopathology of the viral infection remains unclear. future studies on the responses of neutrophils to sars-cov-2-infection or infected cells in vitro may elucidate the role of neutrophils in the pathogenesis of respiratory cov infections. natural killer (nk) cells are a heterogenic immune cell subset that acts promptly to combat viral infections. they produce significant amounts of ifn-γ, kill virus-infected cells, provide direct support to other innate immune cells, and aid in the adaptive immune response to counter viruses. nk cells express activating receptors that detect viral antigens, enabling the destruction of infected cells (241) (242) (243) (244) . lectin-like receptor cd94 and killer immunoglobulin-like receptors, such as cd158b, regulate the function of nk cells. a study of 221 patients with sars explored the relationship of the number of nk cells and the expression level of their immunoglobulin-like receptor cd158b in the peripheral blood to the severity of sars (245) . the overall count of nk cells and cd158 + nk cells and the percentage of cd158 + nk cells in patients with sars were significantly lower than counts in healthy subjects (245) . a separate study that analyzed lymphocytes and lymphocyte subsets in a cohort of 38 patients with sars observed reduced nk cell counts in 21 patients (55%) (246) . clinical reports reveal that children appear to have a milder form of sars-cov-2, with peripheral blood lymphocyte levels remaining in the normal range, suggesting less immune dysfunction following the disease (247) . this could be attributed to healthy children expressing lymphocytes, especially nk cells, in a greater quantity compared to healthy adults (248) . interestingly, previous studies found rapid and significant restoration of lymphocyte subsets including, nk cells, in peripheral blood in patients recovering from the initial stages of sars infection (249) . although the primary mechanism for the decrease in nk cells and other subsets during disease onset remains unknown, their contribution to sars-cov-2 needs further study especially from a treatment perspective. innate lymphoid cells (ilcs) are a family of innate immune cells that include ilc1, ilc2 and ilc3. although ilc2 facilitates lung repair after injury, the role of ilcs during respiratory viral infection is not clearly defined (250) . evidence for the potential involvement of ilc2 cells in the lung during viral infection was reported in a murine model (251) . this study found a rapid accumulation of ilc2 cells in the lung after an influenza virus infection, however their initial contribution to exacerbation of the disease was limited (251) . a recent study identified an interaction between ace2-expressing sars-cov-2 target cells and ilcs in the colon (252) . thus, elucidating the role of ilc subsets will be important in understanding the pathogenesis of sars, sars-cov-2 and mers infections. there is distinct evidence indicating an important role of ifns in sars and other cov infections (201, 253) . the sera of patients with sars revealed the presence of high levels of il-1, il-6, infγ, ccl2, cxcl10, and il-8 and products of interferon stimulated genes (254, 255) . high expression levels of isgs such as cd58, ifnar1, and ifngr1 and ifn-stimulated chemokines cxcl10 and ccl2 were observed in another cohort of sars patients and were correlated with the severity of pathogenesis (256) . significant upregulation of cxcl10 gene expression was observed in the severe phase of patients who died from sars. this data is corroborated by studies in patients with mers that found upregulation of cxcl10 in the serum of patients who developed pneumonia (254) . cxcl10 and infα were also correlated with severity of disease (255) . the importance of ifn signaling in response to cov infection has been well-demonstrated in several knockout mouse models. type i, ii, and iii ifn signaling deficient mice have increased susceptibility to mouse-adapted sars-cov strains (257, 258) . studies using mice lacking the ifnar1 and ifnlr1 or stat1 identified higher sars-cov replication in the lungs and delayed virus clearance (259, 260) . another study with mers-cov in mice expressing the human dpp4 receptor showed a role for the ifnar1 in viral clearance and lung inflammation (112) . these mouse models suggest an important role of ifn response for cov clearance. this quick expanding medical literature is very suggestive of an important role of ifn responses for cov control and clearance. many viruses have evolved to disrupt and subvert the immune response. rsv counteracts the immune response (178, 191) ; as discussed earlier, the rsv genome encodes non-structural proteins (ns1 and ns2) that are able to block type 1 ifn production and signaling in cell cultures (191) . similar to rsv, the measle virus v protein blocks ifn-α and β signaling by inhibiting stat1 and stat2 signaling in cell culture lines (192) . covs have developed several ways to escape from innate immune pressure. mers-cov m protein suppresses type 1 ifn by blocking the irf3 activation (193) , explaining the low expression of ifn-β. in various cell lines, sars-cov nucleocapsid (n) protein, membrane (m) protein, as well as nsp1, were reported to suppress ifn response (194, 196, 261) . the nucleocapsid protein (n) of sars-cov interferes with the function of irf3. although it does not form a complex with rig-i or mda5, rna binding activity at the initial recognition stage of viral rna potentially contributes to immune evasion (261, 262) . aside from the hcov, structural proteins, accessory, and non-structural proteins (nsp) are involved in innate immunity modulation. in both sars-cov and mers-cov, host mrna endonucleolytic cleavage is promoted by nsp1 protein, which modifies capped non-viral rnas (263, 264) . nsp1 in sars-cov prevents host mrna translation by interacting with the 40s subunit of the ribosome; in turn, transcription and translation of viral rna is given preference over the host mrna (263) . another study found that additional sars-cov nsp1 residues interfered with ifn-dependent signaling (265) . ifn production is affected by nsp3 proteins in sars-cov and mers-cov. these proteins have both papain-like protease (plpro) and a plp2 domain, and the plpro domains in both sars-cov and mers-cov downregulate mrna levels of ccl5, infβ, cxcl10, and other pro-inflammatory cytokines (266) . the suppression of ifn responses by sars-cov plpro is due to the inhibition of phosphorylation of ifn-regulatory factor 3 (irf3) and its subsequent translocation to the nucleus where it enhances ifn gene transcription (267) . mers-cov plpro also suppresses rig-i and mda5 and antagonizes ifn induction (266, 268) . in hcov-229e and sars-cov suppression of ifn responses, the key molecule is a adp-ribose-1-monophosphatase macrodomain encoded within nsp3 (269) . accessory proteins are not key in viral replication; however, in human cov, this group of proteins are involved in diverse cellular signaling, including cell proliferation, apoptosis, and ifn signaling (270) . by downregulating phosphorylation and nuclear translocation of irf3, open reading frame orf3b and -6 interfere with ifnβ synthesis and prevent ifnβ-induced activation of ifnstimulated response element (isre) in the promoter of isg in sars-cov (262) . in cells transfected with orf4a, -4b, and -5 of mers-cov, ifnβ promoter-driven luciferase activity is significantly reduced, and it may follow a similar pattern of suppression of irf3 nuclear translocation (141) . therefore, the suppression of signaling events in infected immune and airway epithelial cells, as well as the magnitude of suppression due to elevated expression levels of these accessory proteins, needs to be further elucidated to understand delayed or hyperimmune responses and cytokine storm that occurs in cov infection. in addition to revealing our unpreparedness of handling a worldwide pandemic by a viral infection, covid-19 exposed our lack of understanding of the pathogenesis of diseases as well as the host immunity. the interaction of the host innate immune system and other factors including age, sex, and pre-existing conditions need further investigation regarding disease severity and morbidity of sars/mers and covid-19. disease severity and its related progression are further associated with dysregulation of multiple components of both innate and adaptive immune responses leading to a cytokine storm and severe pathology. for the development of a therapeutic intervention, it is vital to elucidate the interplay among the different layers of the innate immune response and their relation to the clinical factors associated with increased morbidity and mortality in cov infection. investments in basic science research are needed to help elucidate the roles of different immune cells, and their contribution to disease severity; it will pave the way to prevent or abrogate cov outbreaks and potentially new viruses. nl, td, dh, zl, and nf performed the literature search, analyzed the literature, and wrote the manuscript. ha-h, bs, yw, and rs performed the literature search and wrote the manuscript. all authors contributed to the article and approved the submitted version. a novel coronavirus outbreak of global health concern a novel coronavirus from patients with pneumonia in china korean society of epidemiology -nco vtft. an interim review of the epidemiological characteristics of 2019 novel coronavirus cross-country comparison of case fatality rates of covid-19/sars-cov-2. osong public health res perspect understanding of covid-19 based on current evidence current status of epidemiology, diagnosis, therapeutics, and vaccines for novel coronavirus disease 2019. (covid-19) angiotensinconverting enzyme 2 is a functional receptor for the sars coronavirus structure of mers-cov spike receptor-binding domain complexed with human receptor dpp4 structural basis for the recognition of sars-cov-2 by full-length human ace2 the mers-cov receptor dpp4 as a candidate binding target of the sars-cov-2 spike. iscience the sialic acid binding activity of human parainfluenza virus 3 and mumps virus glycoproteins enhances the adherence of group b streptococci to hep-2 cells cx3cr1 as a respiratory syncytial virus receptor in pediatric human lung rhinoviruses and their receptors multiple organ infection and the pathogenesis of sars a novel subset of putative stem/progenitor cd34+oct-4+ cells is the major target for sars coronavirus in human lung time course and cellular localization of sars-cov nucleoprotein and rna in lungs from fatal cases of sars innate immune response of human alveolar type ii cells infected with severe acute respiratory syndrome-coronavirus dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-emc anaesthetic and medico-legal problems in patients intoxicated by alcohol reverse genetics with a full-length infectious cdna of the middle east respiratory syndrome coronavirus comparative replication and immune activation profiles of sars-cov-2 and sars-cov in human lungs: an ex vivo study with implications for the pathogenesis of covid-19 sars-cov-2 entry factors are highly expressed in nasal epithelial cells together with innate immune genes investigating influenza a virus infection: tools to track infection and limit tropism parainfluenza virus infection respiratory syncytial virus can infect basal cells and alter human airway epithelial differentiation respiratory syncytial virus (rsv) infects cd4+ t cells: frequency of circulating cd4+ rsv+ t cells as a marker of disease severity in young children the emerging role of rhinoviruses in lower respiratory tract infections in children -clinical and molecular epidemiological study from croatia summary table of sars cases by country q&a: influenza and covid-19 -similarities and differences epidemiology of acute respiratory infections in children of developing countries 2019 case definition coronavirus as a possible cause of severe acute respiratory syndrome epidemiological, demographic, and clinical characteristics of 47 cases of middle east respiratory syndrome coronavirus disease from saudi arabia: a descriptive study clinical aspects and outcomes of 70 patients with middle east respiratory syndrome coronavirus infection: a single-center experience in saudi arabia association between age and clinical characteristics and outcomes of covid-19 distribution of influenza virus types by age using case-based global surveillance data from twenty-nine countries human parainfluenza virus 4 outbreak and the role of diagnostic tests age-specific incidence rates and risk factors for respiratory syncytial virusassociated lower respiratory tract illness in cohort children under 5 years old in the philippines. influenza other respir viruses respiratory syncytial virus infection in adults human rhinovirus c: age, season, and lower respiratory illness over the past 3 decades who. consensus document on the epidemiology of severe acute respiratory syndrome (sars) who. contract mers transmission and risk factors: a systematic review estimates of the reproduction number for seasonal, pandemic, and zoonotic influenza: a systematic review of the literature transmission of respiratory syncytial virus among children under 5 years in households of rural communities, the philippines lethal respiratory disease associated with human rhinovirus c in wild chimpanzees investigation of a nosocomial outbreak of severe acute respiratory syndrome (sars) in toronto comparison of incubation period distribution of human infections with mers-cov in south korea and saudi arabia incubation period and other epidemiological characteristics of 2019. novel coronavirus infections with right truncation: a statistical analysis of publicly available case data incubation periods of acute respiratory viral infections: a systematic review human parainfluenza viruses (hpivs) available online at transmission dynamics and control of severe acute respiratory syndrome q&as on covid-19 and related health topics clinical features and short-term outcomes of 144 patients with sars in the greater toronto area acute renal impairment in coronavirus-associated severe acute respiratory syndrome prevalence of comorbidities in cases of middle east respiratory syndrome coronavirus: a retrospective study respiratory tract cell-mediated immunity clinical course and risk factors for mortality of adult inpatients with covid-19 in wuhan, china: a retrospective cohort study prevalence of comorbidities and its effects in patients infected with sars-cov-2: a systematic review and meta-analysis hospitalized patients with 2009. h1n1 influenza in the united states severe morbidity and mortality associated with respiratory syncytial virus versus influenza infection in hospitalized older adults respiratory syncytial virus infection in elderly and high-risk adults clinical characteristics and outcomes of human rhinovirus positivity in hospitalized children detection of human rhinoviruses in the lower respiratory tract of lung transplant recipients upper and lower respiratory tract infections by human enterovirus and rhinovirus in adult patients with hematological malignancies middle east respiratory syndrome coronavirus shows poor replication but significant induction of antiviral responses in human monocytederived macrophages and dendritic cells macrophages: a trojan horse in covid-19? depletion of alveolar macrophages during influenza infection facilitates bacterial superinfections activation of human macrophages by bacterial components relieves the restriction on replication of an interferon-inducing parainfluenza virus 5 (piv5) p/v mutant productive infection of isolated human alveolar macrophages by respiratory syncytial virus alveolar macrophage-derived type i interferons orchestrate innate immunity to rsv through recruitment of antiviral monocytes rhinovirus replication in human macrophages induces nf-kappabdependent tumor necrosis factor alpha production sars-cov regulates immune function-related gene expression in human monocytic cells modeling the early events of severe acute respiratory syndrome coronavirus infection in vitro mers-cov pathogenesis and antiviral efficacy of licensed drugs in human monocyte-derived antigen-presenting cells dysregulation of immune response in patients with covid-19 in wuhan, china depressed human monocyte function after influenza infection in vitro infection and maturation of monocyte-derived human dendritic cells by human respiratory syncytial virus, human metapneumovirus, and human parainfluenza virus type 3 human airway epithelial cells direct significant rhinovirus replication in monocytic cells by enhancing icam1 expression productive replication of middle east respiratory syndrome coronavirus in monocytederived dendritic cells modulates innate immune response heightened innate immune responses in the respiratory tract of covid-19 patients influenza a virus infection of human primary dendritic cells impairs their ability to cross-present antigen to cd8 t cells human parainfluenza virus type 2 vector induces dendritic cell maturation without viral rna replication/transcription innate immune components that regulate the pathogenesis and resolution of hrsv and hmpv infections human rhinoviruses inhibit the accessory function of dendritic cells by inducing sialoadhesin and b7-h1 expression complement activation contributes to severe acute respiratory syndrome coronavirus pathogenesis mers-cov infection is associated with downregulation of genes encoding th1 and th2 cytokines/chemokines and elevated inflammatory innate immune response in the lower respiratory tract innate immunity to influenza virus infection respiratory syncytial virus (rsv) and its propensity for causing bronchiolitis chemokines and inflammation in the nasal passages of infants with respiratory syncytial virus bronchiolitis elevated cytokine concentrations in the nasopharyngeal and tracheal secretions of children with respiratory syncytial virus disease haematological manifestations in patients with severe acute respiratory syndrome: retrospective analysis middle east respiratory syndrome coronavirus efficiently infects human primary t lymphocytes and activates the extrinsic and intrinsic apoptosis pathways high levels of virus-specific cd4+ t cells predict severe pandemic influenza a virus infection infection and immunoregulation of t lymphocytes by parainfluenza virus type 3 protective and dysregulated t cell immunity in rsv infection rhinovirus has the unique ability to directly activate human t cells in vitro lack of peripheral memory b cell responses in recovered patients with severe acute respiratory syndrome: a six-year follow-up study middle east respiratory syndrome coronavirus (mers-cov): infection, immunological response, and vaccine development breadth of concomitant immune responses prior to patient recovery: a case report of non-severe covid-19 a b cell population that dominates the primary response to influenza virus hemagglutinin does not participate in the memory response b-cell fate decisions following influenza virus infection respiratory syncytial virus infection in infants is associated with predominant th-2-like response innate immune signals modulate antiviral and polyreactive antibody responses during severe respiratory syncytial virus infection peripheral blood t and b lymphocyte subpopulations in infants with acute respiratory syncytial virus brochiolitis human rhinoviruses enter and induce proliferation of b lymphocytes neutralizing antibodies in patients with severe acute respiratory syndrome-associated coronavirus infection longitudinally profiling neutralizing antibody response to sars coronavirus with pseudotypes rapid generation of a mouse model for middle east respiratory syndrome persistence of antibodies against middle east respiratory syndrome coronavirus neutralizing antibodies against sars-cov-2 and other human coronaviruses protective antibodies against influenza proteins protective effect of antibody to parainfluenza type 1 virus respiratory syncytial virus neutralizing antibodies in cord blood, respiratory syncytial virus hospitalization, and recurrent wheeze the time course of the humoral immune response to rhinovirus infection dynamic changes in blood cytokine levels as clinical indicators in severe acute respiratory syndrome plasma inflammatory cytokines and chemokines in severe acute respiratory syndrome the relationship between serum interleukins and t-lymphocyte subsets in patients with severe acute respiratory syndrome active replication of middle east respiratory syndrome coronavirus and aberrant induction of inflammatory cytokines and chemokines in human macrophages: implications for pathogenesis cytokine responses in severe acute respiratory syndrome coronavirus-infected macrophages in vitro: possible relevance to pathogenesis contribution of cytokines to tissue damage during human respiratory syncytial virus infection induction of pro-inflammatory cytokines (il-1 and il-6) and lung inflammation by coronavirus-19 (covi-19 or sars-cov-2): anti-inflammatory strategies cytokines induced during influenza virus infection human parainfluenza virus serotypes differ in their kinetics of replication and cytokine secretion in human tracheobronchial airway epithelium human rhinovirus induced cytokine/chemokine responses in human airway epithelial and immune cells the deadly coronaviruses: the 2003. sars pandemic and the 2020 novel coronavirus epidemic in china evaluation and treatment coronavirus (covid-19) cdc. influenza vaccines -united states rhinovirus vaccination: the case against review of the clinical characteristics of coronavirus disease 2019. (covid-19) available online at respiratory syncytial virus: diagnosis, treatment and prevention update on human rhinovirus and coronavirus infections coronavirus-associated pneumonia in previously healthy children clinical disease in children associated with newly described coronavirus subtypes identification of a novel coronavirus in patients with severe acute respiratory syndrome isolation of a novel coronavirus from a man with pneumonia in saudi arabia hospitalization rates and characteristics of patients hospitalized with laboratory-confirmed coronavirus disease presenting characteristics, comorbidities, and outcomes among 5700 patients hospitalized with covid-19 in the new york city area gender differences in patients with covid-19: focus on severity and mortality do men have a higher case fatality rate of severe acute respiratory syndrome than women do? the pattern of middle east respiratory syndrome coronavirus in saudi arabia: a descriptive epidemiological analysis of data from the saudi ministry of health sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor severe acute respiratory syndrome coronavirus infection of mice transgenic for the human angiotensin-converting enzyme 2 virus receptor human coronavirus nl63 employs the severe acute respiratory syndrome coronavirus receptor for cellular entry tissue distribution of ace2 protein, the functional receptor for sars coronavirus. a first step in understanding sars pathogenesis structure, function, and evolution of coronavirus spike proteins angiotensin-i-converting enzyme and its relatives receptor recognition by the novel coronavirus from wuhan: an analysis based on decade-long structural studies of sars coronavirus trilogy of ace2: a peptidase in the renin-angiotensin system, a sars receptor, and a partner for amino acid transporters a novel coronavirus associated with severe acute respiratory syndrome dipeptidyl-peptidase iv from bench to bedside: an update on structural properties, functions, and clinical aspects of the enzyme dpp iv airway epithelial differentiation and mucociliary clearance respiratory epithelial cells orchestrate pulmonary innate immunity the role of toll-like receptors in the production of cytokines by human lung macrophages lung epithelial cells: therapeutically inducible effectors of antimicrobial defense pulmonary macrophages: key players in the innate defence of the airways the development and function of lungresident macrophages and dendritic cells contribution of dendritic cells in protective immunity against respiratory syncytial virus infection the contributions of lung macrophage and monocyte heterogeneity to influenza pathogenesis plasmacytoid dendritic cells: development, regulation, and function human dendritic cell subsets: an update the multifaceted biology of plasmacytoid dendritic cells role of dendritic cells in sars coronavirus infection plasmacytoid dendritic cells and type i ifn: 50 years of convergent history the dendritic cell lineage: ontogeny and function of dendritic cells and their subsets in the steady state and the inflamed setting macrophage polarization in virus-host interactions macrophages: sentinels and regulators of the immune system lung dendritic cells at the innate-adaptive immune interface lung interstitial macrophages: past, present, and future innate immune response to influenza virus at singlecell resolution in human epithelial cells revealed paracrine induction of interferon lambda 1 the nlrp3 inflammasome mediates in vivo innate immunity to influenza a virus through recognition of viral rna innate immunity in the respiratory epithelium the airway epithelium: soldier in the fight against respiratory viruses lung epithelial cells have virus-specific and shared gene expression responses to infection by diverse respiratory viruses helix 1 tryptophan variants in galleria mellonella apolipophorin iii synergy of il-27 and tnfalpha in regulating cxcl10 expression in lung fibroblasts sars-coronavirus replication in human peripheral monocytes/macrophages middle east respiratory syndrome coronavirus infection: virus-host cell interactions and implications on pathogenesis molecular mechanisms of cell death: central implication of atp synthase in mitochondrial permeability transition severe acute respiratory syndrome coronavirus orf3a protein activates the nlrp3 inflammasome by promoting traf3-dependent ubiquitination of asc delayed induction of proinflammatory cytokines and suppression of innate antiviral response by the novel middle east respiratory syndrome coronavirus: implications for pathogenesis and treatment dynamic innate immune responses of human bronchial epithelial cells to severe acute respiratory syndrome-associated coronavirus infection exacerbated innate host response to sars-cov in aged non-human primates analysis on 54 mortality cases of coronavirus disease 2019 in the republic of korea from prevalence of sars-cov antibody in all hong kong patient contacts respiratory syncytial virus measles virus v protein blocks interferon (ifn)-alpha/beta but not ifn-gamma signaling by inhibiting stat1 and stat2 phosphorylation middle east respiratory syndrome coronavirus m protein suppresses type i interferon expression through the inhibition of tbk1-dependent phosphorylation of irf3 severe acute respiratory syndrome coronavirus nsp1 suppresses host gene expression, including that of type i interferon, in infected cells severe acute respiratory syndrome coronavirus m protein inhibits type i interferon production by impeding the formation of traf3.tank.tbk1/ikkepsilon complex the severe acute respiratory syndrome coronavirus nucleocapsid inhibits type i interferon production by interfering with trim25-mediated rig-i ubiquitination neutrophil recruitment and function in health and inflammation host-pathogen interactions during coronavirus infection of primary alveolar epithelial cells the role of c5a in acute lung injury induced by highly pathogenic viral infections characterization of cytokine/chemokine profiles of severe acute respiratory syndrome lung pathology of fatal severe acute respiratory syndrome dysregulated type i interferon and inflammatory monocyte-macrophage responses cause lethal pneumonia in sars-cov-infected mice pulmonary pathological features in coronavirus associated severe acute respiratory syndrome (sars) spatiotemporal interplay of severe acute respiratory syndrome coronavirus and respiratory mucosal cells drives viral dissemination in rhesus macaques location or origin? what is critical for macrophage propagation of lung fibrosis? thinsection ct in patients with severe acute respiratory syndrome following hospital discharge: preliminary experience pathogenesis of severe acute respiratory syndrome the novel severe acute respiratory syndrome coronavirus 2 (sars-cov-2) directly decimates human spleens and lymph nodes. medrxiv chemokine up-regulation in sars-coronavirus-infected, monocyte-derived human dendritic cells sex-based differences in susceptibility to severe acute respiratory syndrome coronavirus infection interferon-mediated immunopathological events are associated with atypical innate and adaptive immune responses in patients with severe acute respiratory syndrome the pathogenicity of sars-cov-2 in hace2 transgenic mice pathogenesis of sars-cov-2 in transgenic mice expressing human angiotensin-converting enzyme 2 mannosebinding lectin in severe acute respiratory syndrome coronavirus infection complement associated microvascular injury and thrombosis in the pathogenesis of severe covid-19 infection: a report of five cases highly pathogenic coronavirus n protein aggravates lung injury by masp-2-mediated complement over-activation. medrxiv the case of complement activation in covid-19 multiorgan impact blockade of the c5a-c5ar axis alleviates lung damage in hdpp4-transgenic mice infected with mers-cov neutrophils in viral infections: current concepts and caveats a role for neutrophils in viral respiratory disease. front immunol severe acute respiratory distress syndrome (sars): a critical care perspective a major outbreak of severe acute respiratory syndrome in hong kong neutrophil-to-lymphocyte ratio predicts severe illness patients with 2019. novel coronavirus in the early stage mouse-passaged severe acute respiratory syndrome-associated coronavirus leads to lethal pulmonary edema and diffuse alveolar damage in adult but not young mice effect of heterophil depletion by 5-fluorouracil on infectious bronchitis virus infection in chickens neutrophils are needed for an effective immune response against pulmonary rat coronavirus infection, but also contribute to pathology neutrophils promote mononuclear cell infiltration during viral-induced encephalitis middle east respiratory syndrome coronavirus (mers-cov) causes transient lower respiratory tract infection in rhesus macaques clinical features and viral diagnosis of two cases of infection with middle east respiratory syndrome coronavirus: a report of nosocomial transmission sars-cov2: should inhibitors of the renin-angiotensin system be withdrawn in patients with covid-19? a dynamic variation of pulmonary ace2 is required to modulate neutrophilic inflammation in response to pseudomonas aeruginosa lung infection in mice neutrophil extracellular traps in immunity and disease neutrophil extracellular traps (nets) -formation and implications targeting potential drivers of covid-19: neutrophil extracellular traps the role of neutrophil extracellular traps in covid-19: only an hypothesis or a potential new field of research? autopsy findings and venous thromboembolism in patients with covid-19 coagulation abnormalities and thrombosis in patients with covid-19 mechanisms underlying neutrophil-mediated monocyte recruitment partners in crime: neutrophils and monocytes/macrophages in inflammation and disease aggregated neutrophil extracellular traps limit inflammation by degrading cytokines and chemokines selective reduction of natural killer cells and t cells expressing inhibitory receptors for mhc class i in the livers of patients with hepatic malignancy expression of killer cell inhibitory receptors is restricted to true nk cell lymphomas and a subset of intestinal enteropathy-type t cell lymphomas with a cytotoxic phenotype modulation of expression of the mhc class i-binding natural killer cell receptors, and nk activity in relation to viral load in hiv-infected/aids patients hla-cw7 zygosity affects the size of a subset of cd158b+ natural killer cells the involvement of natural killer cells in the pathogenesis of severe acute respiratory syndrome expression of lymphocytes and lymphocyte subsets in patients with severe acute respiratory syndrome sars-cov-2 infection in children: transmission dynamics and clinical characteristics will children reveal their secret? the coronavirus dilemma significant changes of peripheral t lymphocyte subsets in patients with severe acute respiratory syndrome innate lymphoid cells promote lung-tissue homeostasis after infection with influenza virus t cells and ilc2s are major effector cells in influenza-induced exacerbation of allergic airway inflammation in mice single cell rna sequencing of 13 human tissues identify cell types and receptors of human coronaviruses how the sars coronavirus causes disease: host or organism? comparative and kinetic analysis of viral shedding and immunological responses in mers patients representing a broad spectrum of disease severity clinical progression and cytokine profiles of middle east respiratory syndrome coronavirus infection mobile group ii introns of yeast mitochondrial dna are novel site-specific retroelements resolution of primary severe acute respiratory syndrome-associated coronavirus infection requires stat1 sars-cov pathogenesis is regulated by a stat1 dependent but a type i, ii and iii interferon receptor independent mechanism lambda interferon renders epithelial cells of the respiratory and gastrointestinal tracts resistant to viral infections combined action of type i and type iii interferon restricts initial replication of severe acute respiratory syndrome coronavirus in the lung but fails to inhibit systemic virus spread sars-cov nucleocapsid protein antagonizes ifn-beta response by targeting initial step of ifn-beta induction pathway, and its c-terminal region is critical for the antagonism severe acute respiratory syndrome coronavirus open reading frame (orf) 3b, orf 6, and nucleocapsid proteins function as interferon antagonists sars coronavirus nsp1 protein induces templatedependent endonucleolytic cleavage of mrnas: viral mrnas are resistant to nsp1-induced rna cleavage middle east respiratory syndrome coronavirus nsp1 inhibits host gene expression by selectively targeting mrnas transcribed in the nucleus while sparing mrnas of cytoplasmic origin identification of residues of sars-cov nsp1 that differentially affect inhibition of gene expression and antiviral signaling mers-cov papain-like protease has deisgylating and deubiquitinating activities regulation of irf-3-dependent innate immunity by the papain-like protease domain of the severe acute respiratory syndrome coronavirus proteolytic processing, deubiquitinase and interferon antagonist activities of middle east respiratory syndrome coronavirus papain-like protease the adp-ribose-1''-monophosphatase domains of severe acute respiratory syndrome coronavirus and human coronavirus 229e mediate resistance to antiviral interferon responses accessory proteins of sars-cov and other coronaviruses the remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © 2020 ahmed-hassan, sisson, shukla, wijewantha, funderburg, li, hayes, demberg and liyanage. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord-264079-u0nkjexi authors: murphy, dianne; todd, james k.; chao, ru kwa; orr, inara; mcintosh, kenneth title: the use of gowns and masks to control respiratory illness in pediatric hospital personnel date: 1981-11-30 journal: the journal of pediatrics doi: 10.1016/s0022-3476(81)80401-5 sha: doc_id: 264079 cord_uid: u0nkjexi nan the appearance of the skin lesion in this case was typical. we recovered e. rhusipathiae from an aspirate of the center of the lesion, but it is usually recommended to obtain the specimen from the margin? ~' we found only one other case report in the pediatric literature? the fever and elevated blood count in our patient suggest a more severe involvement in this age group. the prognosis is good in adults with or without treatment, but penicillin therapy is advised3; we believe that this recommendation is especially warranted in infants. t h e s p r e a d of viral respiratory illness in hospitals poses a problem of considerable magnitude in wards for infants and small children. nosocomial disease often lengthens the hospital stay and, in some instances, may be severe or even fatal> ~ for certain viruses, particularly respiratory syncytial virus, there is a high frequency of infection in nursing, medical, and other stafp ..... which probably also contributes to secondary spread to other infants. children with respiratory disease are usually placed in isolation and hospital personnel may be cohorted or required to wear gowns when administering patient care. the efficacy of this approach in the control of rsv therefore designed a prospective study to examine the effect of various control methods on the acquisition of symptomatic respiratory infections by medical personnel caring for infants with respiratory disease. the study took place in february and march of 1979, when rsv infections were widespread and influenza a/ussr virus was also present in the community. study design. the infants' ward at the children's hospital of denver consists of seven large rooms, two or more of which are designated as respiratory isolation rooms in the winter season. each room contains four to six beds and one or two sinks. before the study, routine precautions involved the use of gown and mask, which were put on before entering the room and discarded on exit. personnel were required to wash hands with an iodine-based soap after entry, between patients, and before leaving the room. all nursing, medical, and respiratory therapy personnel were solicited to participate in the study; 58 of the 70 available individuals participated and completed the study. individuals were randomly assigned to either a handwashing alone or handwashing, gowning and masking group. all personnel signed consent forms before participating in the study and were paid $20 when the study was completed. nursing supervisors on all three shifts were provided with lists of staff assigned to each group. to assure adherence to the procedures, these lists were posted throughout the ward so that individuals entering a room were easily checked. although compliance was not quantitated, daily surveillance by several of us (d. m. and i. o.) showed excellent co-operation throughout. patient and personnel sampling. nasopharyngeal secretions were obtained by gentle manual suction s from all infants admitted to respiratory isolation rooms during the study. personnel were cultured whenever symptomatic (rhinorrhea, fever, cough, wheezing) from five days before the onset of the study until the end of the eight-week period. throat swabs, broken off in transport medium, or nasopharyngeal secretions were placed on ice and cultured within 30 minutes to three hours of collection. sera were obtained from all study personnel at the onset of the study and two to three weeks after the eight-week period was over. any individual in the h group who developed minor respiratory symptoms was asked to wear a mask until the symptoms abated. in both groups, those with more severe symptoms stayed away from work until they had recovered. respiratory symptoms requiring mask in the h group were present for a total of 14.5% of the working time. collection of clinical data. to document length of illness and hours of exposure, each participant completed a card at the end of the daily shift which recorded his or her code number, hours worked in respiratory isolation rooms, and any symptoms (rhinorrhea, fever, cough~ or wheezing) for that day. these cards were collected daily by the nursing supervisor; anyone not completing a card was notified, and this deficiency was rectified. any symptomatic or absent individual was called and arrangements were made to obtain a viral culture. virologic techniques. nps and throat swabs were cultured on hep-2 cells monitored for sensitivity to rsv. in addition, madin-darby canine kidney cells for detection of influenza viruses were inoculated with specimens during the first five weeks of the study. technical problems precluded continuing this practice throughout. infants' and some adults' nps were also processed for rapid detection of rsv by immunofluorescence? paired sera from adults were tested by complement fixation for antibody to rsv, influenza a and b, parainfluenza 1, 2, and 3, adenovirus, and coronaviruses oc43 and 229e. neutralization antibody to rsv was measured by plaque reduction. 7 hemagglutination inhibition tests for influenza a/ussr/90 and 92 and influenza b/hk were performed by standard methods? a fourfold rise in antibody measured by any of these methods was considered to indicate infection. statistical analysis. data were analyzed by the student t test, chi square, or the fischer exact test, depending on the data base. all data collected were entered in a computer program which performed a regression of dependent to independent variables looking for high "t" statistics in the regression coefficient and the correlations among the variables. personnel. of the 70 individuals available for study, nine nurses elected not to participate, and three withdrew from the study. two of these individuals were in the hg&m group and one in the h group. only one withdrew because of distaste for the procedures. two left for other employment. thus, 58 personnel completed the study and form the base for this analysis. there was no significant difference in age, experience on the pediatric ward, number with children under 10 years of age at home, or degree of exposure in the respiratory isolation rooms within the two groups. during the study, 177 patients were admitted to the respiratory isolation rooms; 87 (49%) yielded rsv by culture or fluorescence. sixty-eight of the 87 were positive by both techniques. ten additional samples were positive by culture only and four by fluorescence alone. rsv was isolated throughout the course of the study. by contrast, influenza virus was recovered only twice from children and appears, in comparison to rsv, to have had a lesser impact on the study group (figure) . on the day before the study began, three symptomatic personnel were cultured and rsv was recovered from two of them. in addition, one symptomatic individual, who had only this one illness during the study period and from whom rsv was not recovered, developed a fourfold rise in antibody to rsv. these three infections were not included in our analysis, and the three individuals were omitted from analysis of rsv infections since they would presumably have at least partial immunity to rsv reinfection during the study period. they were, however, introduced into the study after recovery and included in analysis of subsequent illnesses. during the first week of the study, three additional individuals, who had been ill but unavailable during the prior week, were sampled. none was infected with rsv but influenza a was grown from one throat culture and the other two both had evidence of influenza a by serologic testing. these three illnesses were not included brief clinical and laboratory observations 74 9 number 5 in the analysis below since they predated the onset of the study. influenza a virus was not recovered from any other staff members during the study period, although four infections were detected by serologic means. there were four proven rsv infections in the h group and five in the hg&m groups ('fable). six other viral infections-four with influenza a virus and two with parainfluenza virus type 3-occurred among the staff, all detected by antibody titer rise. the number of such infections, like those with rsv, did not differ between the two study groups. as with specific viral infections, there was no difference between the two groups with respect to number of illnesses, number of days ill, number of individuals with more than one illness, and absenteeism (table) . since gowning and masking did not appear to influence either illness or specific virus infection, we attempted to correlate other possible factors. both age and number of hours of exposure were correlated with illness. in a multiple regression analysis (coefficient of determination = 0.25) younger personnel (p = 0.02), and those who spent more than 30 hours in the study rooms (p = 0.04) tended to be ill more often. in a similar analysis, hours of exposure were also correlated (correlation coefficient--0.25) specifically with rsv infection (p = 0.03). in addition, prestudy serum rsv neutralizing antibody correlated with protection (correlation coefficient = 0.31, p = 0.02). the best method for control of respiratory infections among hospital staff remains to be determined. we were unable to demonstrate any effect of adding the use of both gown and mask to the usual handwashing routine on the development of illness in personnel caring for infants with respiratory disease. specific viral infections were identified in only 15 of 49 illnesses (31%). rsv was the most frequent agent found, being present during nine illnesses (18%) during the study. although rsv infections were fewer than expected, the trend followed the more general one, that gown and mask had no discernible effect on risk of acquisition. it was unfortunate that the study began at a time when rsv infections were already occurring among the staff. rsv infections in infants were frequent, and had we begun a week earlier the number of acquisitions by staff might have been larger, and the data for rsv therefore somewhat more convincing. however, it was the practice before the onset of the study to use handwashing, mask and gown in the isolation areas, and infections acquired before the study reflected that policy. culturing asymptomatic personnel might also have increased the number of rsv infections. we chose not to do so, however, since prior studies had shown rsv infections among staff to be largely symptomatic.:' it might be argued that the reason for the lack of effect was the heavy exposure all adults have to respiratory viral illness in the community at large. against this hypothesis, however, is our finding that both illness and rsv infection were significantly associated with longer hours of exposure in the isolation rooms. other possible reasons for lack of effect are poor compliance with the study protocol, and modes of virus spread which would not be blocked by the use of mask and gown. although we did not measure compliance, we did monitor it carefully during two of the three daily shifts, and the same enforcement measures were used at night as during the day. finally, it may be that the viruses involved elude the barriers set up by means of gown and mask. recent data from hall and douglas "~ indicate that rsv can be successfully transmitted through fomites on the hands, which are then put in contact with either the conjunctivae or the nasal mucosa. another possible mode of spread demonstrated in that study was by means of large infected droplets. rhinoviruses, which might have been present but would not have been detected in this study, have been clearly shown to spread more efficiently by hand-to-nose or hand-to-eye contact than by aerosols. 1~ influenza, on the other hand, is thought to spread through droplet nuclei, against which masks might be an inefficient barrier. thus any one of a number of candidate respiratory viruses could bypass gown or mask and render their use ineffective. we can draw no conclusions regarding intrahospital spread to other infants. indeed, the major function of masks may be to prevent the spread of respiratory viruses from personnel to infants, rather than the other way around. nevertheless, we believe that we have shown that the cumbersome and expensive use of gowns and masks serves tittle if any function in protecting personnel. we recommend that, whenever possible, older, more experienced staff should care for children at high risk of severe respiratory viral illness, and that gown and mask should not be used in an attempt to prevent personnel illness in wards with a high density of acute respiratory infections. the utility of gown and mask in preventing spread to other infants, of other barriers such as gloves or goggles, and of cohorting of personnel remains to be proven. we are grateful to harold dickson, ph.d., for programming and statistical analysis, and to kaye swepston, r.n., the nurses, and the respiratory therapists of the denver children's hospital for their cooperation in this study. a 4-month-old female infant was admitted to hartford hospital because of bloody diarrhea. she had been born after an uneventful term pregnancy and delivery. feedings had consisted solely of breast milk for the first three months. the mother had been taking no medication. at three months of age a cow milk formula (enfamil, mead johnson & company) was introduced, and the patient developed watery diarrhea within several days. treatment with an oral glucose-electrolyte solution (pedialyte, ross laboratories) and then a soy formula (prosobee, mead johnson & company) was followed quickly by return of the stools to normal. re-introduction of the cow milk formula at age 31/2 months was attempted, and the patient rapidly developed diarrhea which prompted admission to a local hospital. from the departments of pediatrics and pathology, hartjbrd hospital *reprint address: section of pediatric gastroenterology, hartford hospital, hartford, ct 06115. on admission she was mildly dehydrated and having approximately six to eight watery stools per day which were guaiac positive. the hematocrit was 37%, wbc count 10,000 cells/mm ~ with 30% polys, 13% bands, 48% lymphocytes, and 9% monocytes. three stools were negative for enteric pathogens as well as for ova and parasites. treatment with intravenous fluid therapy was instituted. when attempts to introduce an elemental formula (pregestimil, mead johnson & company) were unsuccessful, she was transferred to hartford hospital. on admission her general physical examination was normal with the exception of a heart rate of 140 beats/minute. laboratory evaluation included: electrolytes (meq/l) na 132, k 4.4, c1 99, hco 3 19; bun 8 mg/dl, albumin 4.5 gm/dl; hematocrit 38%, wbc count 9,600 cells/mm ~ with 32% polys, 5% bands, 56% lymphocytes, 7% monocytes; platelet count 905,000, reticulocyte count 2%, sedimentation rate 66 mm/hour. careful separation of stool and urine revealed watery, bloody stools weighing approximately 400 to 600 gm/day. stool gram stain revealed abundant polymorphonuclear leukocytes and mixed flora. stool electrolyte concentrations (meq/l) were na 106, k 19, c1 98. three stools were negative for enteric pathogens (salmonella, shigella, campylobacter, and yersinia) as well as for ova and parasites. sigmoidoscopy revealed granular and friable mucosa without any pseudomembranes. rectal biopsy showed an intact surface epithelium with minor loss of nuclear polarity. the epithelium showed regenerative changes characterized by increased mitotic figures and hyperchromatic nuclei and loss of goblet cells. a moderate increase in mononuclear cells within the lamina propria, consisting mainly of mature lymphocytes, was present with focal involvement of the muscularis mucosa. brown and brenn stain for bacteria were negative. bastin r: maladies infectieuses acquisition of parainfluenza 3 virus infection by hospitalized children nosocomial respiratory syncytial virus infections control of nosocomial respiratory syncytial viral infections respiratory syncytial virus infection in adults the immunologic response to respiratory syncytial virus infection in infants rapid virus diagnosis: application of immunoftuorescence clinical and immunological response of infants and children to administration of low temperature adapted respiratory syncytial virus diagnostic procedures for viral & rickettsial infections modes of spread of respiratory syncytial virus (rsv) handto-hand transmission of rhinovirus colds key: cord-264059-jf4j00bp authors: lee, chien-chang; chang, julia chia-yu; mao, xiao-wei; hsu, wan-ting; chen, shey-ying; chen, yee-chun; how, chorng-kuang title: combining procalcitonin and rapid multiplex respiratory virus testing for antibiotic stewardship in older adult patients with severe acute respiratory infection date: 2019-11-30 journal: j am med dir assoc doi: 10.1016/j.jamda.2019.09.020 sha: doc_id: 264059 cord_uid: jf4j00bp objectives: virus infection is underevaluated in older adults with severe acute respiratory infections (saris). we aimed to evaluate the clinical impact of combining point-of-care molecular viral test and serum procalcitonin (pct) level for antibiotic stewardship in the emergency department (ed). design: a prospective twin-center cohort study was conducted between january 2017 and march 2018. setting and participants: older adult patients who presented to the ed with saris received a rapid molecular test for 17 respiratory viruses and a pct test. measures: to evaluate the clinical impact, we compared the outcomes of sari patients between the experimental cohort and a propensity score–matched historical cohort. the primary outcome was the proportion of antibiotics discontinuation or de-escalation in the ed. the secondary outcomes included duration of intravenous antibiotics, length of hospital stay, and mortality. results: a total of 676 patients were included, of which 169 patients were in the experimental group and 507 patients were in the control group. more than one-fourth (27.9%) of the patients in the experimental group tested positive for virus. compared with controls, the experimental group had a significantly higher proportion of antibiotics discontinuation or de-escalation in the ed (26.0% vs 16.1%, p = .007), neuraminidase inhibitor uses (8.9% vs 0.6%, p < .001), and shorter duration of intravenous antibiotics (10.0 vs 14.5 days, p < .001). conclusions and implications: combining rapid viral surveillance and pct test is a useful strategy for early detection of potential viral epidemics and antibiotic stewardship. clustered viral respiratory infections in a nursing home is common. patients transferred from nursing homes to ed may benefit from this approach. objectives: virus infection is underevaluated in older adults with severe acute respiratory infections (saris). we aimed to evaluate the clinical impact of combining point-of-care molecular viral test and serum procalcitonin (pct) level for antibiotic stewardship in the emergency department (ed). design: a prospective twin-center cohort study was conducted between january 2017 and march 2018. setting and participants: older adult patients who presented to the ed with saris received a rapid molecular test for 17 respiratory viruses and a pct test. measures: to evaluate the clinical impact, we compared the outcomes of sari patients between the experimental cohort and a propensity scoreematched historical cohort. the primary outcome was the proportion of antibiotics discontinuation or de-escalation in the ed. the secondary outcomes included duration of intravenous antibiotics, length of hospital stay, and mortality. results: a total of 676 patients were included, of which 169 patients were in the experimental group and 507 patients were in the control group. more than one-fourth (27.9%) of the patients in the experimental group tested positive for virus. compared with controls, the experimental group had a significantly higher proportion of antibiotics discontinuation or de-escalation in the ed (26.0% vs 16.1%, p ¼ .007), neuraminidase inhibitor uses (8.9% vs 0.6%, p < .001), and shorter duration of intravenous antibiotics (10.0 vs 14.5 days, p < .001). conclusions and implications: combining rapid viral surveillance and pct test is a useful strategy for early detection of potential viral epidemics and antibiotic stewardship. clustered viral respiratory infections in a nursing home is common. patients transferred from nursing homes to ed may benefit from this approach. ó 2019 amda e the society for post-acute and long-term care medicine. community-acquired respiratory tract infections are among the most common reasons for emergency department (ed) visits and can be caused by both viral and bacterial pathogens. identification of the pathogen causing symptoms is critical for rapid institution of adequate antiviral or antibiotic therapy. because of the challenges in differentiating between viral and bacterial pathogens, patients with viral respiratory infections are usually underdetected, and unnecessary antibacterial agents are more likely to be administered. therefore, laboratory tests providing accurate and timely determination of the infectious agents associated with viral respiratory diseases are important. a broad array of tests is available to detect viral respiratory agents. rapid antigen tests are available for influenza a and b and respiratory syncytial virus (rsv), but these tests have low sensitivity and specificity. 1, 2 the authors declare no conflicts of interest. molecular diagnostic tests using the polymerase chain reaction (pcr) method to detect the rna or dna of the infectious agents show high sensitivity and specificity, but they are technically challenging and time consuming. the advent of sensitive point-of-care (poc) molecular detection methods has made rapid diagnosis of respiratory virus infections possible. the filmarray system (biofire diagnostics, inc, salt lake city, ut) is a desktop automated real-time pcr system that integrates sample preparation, amplification, detection, and analysis into 1 complete process that delivers results in 1 hour. the respiratory panel can detect 17 respiratory viruses and 3 bacterial targets in a single reaction. 3 initial studies demonstrated that such poc multiplex pcr systems identified previously under-evaluated viral or atypical infections in ed dyspneic patients, and the additional information on rapid respiratory infection testing may also change the physician's antibiotic-prescribing behavior, enabling more timely and appropriate treatment. 4e6 the hospital length of stay and direct medical cost for patients with the identified respiratory pathogens decreased. 7e16 despite the availability of highly accurate viral testing results, the discontinuation or the de-escalation of antibiotics still raises concerns because mixed virus-bacteria coinfection, especially influenza with pneumococcus, is common in older adults. 17 in this study, we proposed a diagnostic approach that combines the multiplex pcr respiratory panel with procalcitonin (pct) tests to better guide the antibiotic treatment. pct is a precursor of calcitonin that is constitutively secreted by c cells of the thyroid gland and k cells of the lungs. in healthy individuals, pct is normally undetectable (<0.01 ng/ml). when stimulated by endotoxin, pct is rapidly produced by parenchymal tissue throughout the body. unlike c-reactive protein, pct does not respond to sterile inflammation or viral infection. 18 this distinctive characteristic makes pct a valuable diagnostic marker. multiple randomized controlled trials have demonstrated that pct levels of <0.25 mg/l can guide the decision to withhold antibiotics or stop therapy early. 19, 20 since the approval of filmarray respiratory panel tests, only a few studies have evaluated the clinical impact after implementation of the multiplex pcr respiratory panel on patients with less severe acute reparatory illness. 8,13e15 to date, no study has focused on older adults with severe acute respiratory illness. older adults are more vulnerable to respiratory virus infection. because of undifferentiated clinical manifestation between bacterial and viral infection, antibiotic overuse in this population is common. in this study, we aimed to assess the impact of implementing a diagnostic algorithm that combines rapid respiratory viral surveillance and pct tests on older patients presenting to the ed with severe acute respiratory illness. we conducted a prospective cohort study in the ed of 2 urban medical centers. clinical impact was evaluated through a comparison of the experimental cohort with a propensity score (ps)ematched historical cohort. we conducted a prospective, multicenter, observational study of a sample of ed patients presenting with acute severe respiratory illness. the eds of 2 urban medical centers participated in this project. the annual ed census is around 100,000 for one medical center and 80,000 for the other. the study period included january 2017 through march 2018. we defined the preerespiratory panel system implementation period as january 2016 through december 2016 (12 months) and the posterespiratory panel system implementation period as january 2017 through march 2018. we had 200 multiplex pcr kits, of which 22 were used for rapid pcr respiratory panel system calibration; the remaining 178 kits were aimed for use among the study patients. however, at the planned end date of the study, we could not reach the target sample size. therefore, a 3-month extension in the experimental group was made to collect sufficient samples. the trial was approved by the institutional review board for human research at each participating center. patients aged 65 years or older presenting to the ed with acute severe respiratory illness were eligible for inclusion. we defined a case of severe acute respiratory illness according to the world health organization's case definition. we defined severe acute respiratory illness in adults as physician-diagnosed lower respiratory tract infection with a pulse oxygen saturation (spo 2 ) on presentation of less than 90% or a respiratory rate >20 breaths/min or the requirement of intubation and mechanical ventilation. basic demographic and clinical information and specimens were collected on the day of admission. an episode of lower respiratory tract infection was defined as acute pulmonary disease with or without acute respiratory failure, including pneumonia, influenza-like illness, or an acute exacerbation of a chronic respiratory illness (including an exacerbation of chronic obstructive pulmonary disease, asthma, or bronchiectasis). the exclusion criteria included if the patient was receiving palliative care or declined nasopharyngeal swabbing. all participants provided written informed consent. the filmarray respiratory panel (biofire diagnostics, inc) detects 17 viruses (rsv, influenza a h1, h1-2009, h3, influenza b, adenovirus, parainfluenza virus 1e4, rhinovirus/enterovirus, human metapneumovirus, human coronavirus oc43, 229e, nl63, and hku1), and 3 atypical bacteria (bordetella pertussis, mycoplasma pneumoniae, and chlamydia pneumoniae). we collected a nasopharyngeal swab using a nylon flocked swab that was immediately placed in universal transport media (utm). the study nurse collected all samples and specimens in utm, and they were tested according to the manufacturer's instructions. blood samples were collected within 24 hours of admission. pct concentrations were measured using an immunoluminometric assay with a detection limit of 0.06 ng/ml (brahms pctsensitive kryptor, thermo fisher scientific, brahms gmbh). respiratory swab and blood samples were tested as soon as they were received in the laboratory. during the study period, the study nurse identified eligible patients and explained the study protocol to the treating physicians and patients. eligible patients received a rapid molecular test with 17 respiratory viruses and a pct test. the results of the respiratory panel or pct tests were communicated to the treating physicians directly by the study nurse as soon as they were available and were kept in the medical records. the study nurse reminded the treating physician of the recommendation of antibiotic treatment based on different viral and pct testing results. the detection of influenza initiates isolation or neuraminidase inhibitor use. the detection of a virus with an elevated serum pct level (!0.25 ng/ml) may indicate the possibility of a superimposed bacterial infection and justify the continual use of antibacterial treatment in patients with noneinfluenza virus infection and combined antiviral and antibacterial treatment in patients with influenza infection. a positive result for respiratory virus with a low serum pct level and stable clinical manifestation may allow early discontinuation or de-escalation of empiric antibiotics. de-escalation was defined as changing to a narrower-spectrum antibiotic or shifting the intravenous antibiotics to oral form. a negative respiratory virus test result with a low serum level of pct (<0.25 ng/ml) would prompt clinicians to consider noninfectious causes of respiratory distress, such as acute exacerbation of obstructive airway disease, acute decompensated heart failure, or fluid overload. information regarding laboratory tests, antibiotic or antiviral therapy administration, duration of intravenous antibiotic treatment, length of intensive care unit stays, length of hospital stay, and 30-day mortality was obtained from electronic health records. we compared the outcome and duration of intravenous antibiotic use with a historical cohort with similar baseline characteristics and clinical presentations. the clinical impact was measured via the proportion of stopping or de-escalating antibiotics, neuraminidase inhibitor uses in the ed, duration of intravenous antibiotics treatment, length of hospital stay, length of intensive care unit stays, 30-day mortality, and overall all-cause mortality. to evaluate the clinical impact of combining the respiratory panel and pct testing on the outcomes of patients with severe acute respiratory illness, we established a historical cohort, including all patients presenting to the ed with severe acute respiratory illness from january 1, 2016 to december 31, 2016. the database included the following: demographics, clinical presentations, presenting viral signs, laboratory data, image results, ed and admission, medications used in the ed and hospitalization course, and discharge status. we then used a ps-matching technique to select a group of patients with similar demographics, comorbidities, diagnoses, vital signs, and laboratory results to the experimental cohort that received the respiratory panel and pct test. to increase the statistical power for analysis, we performed a 1-to-3 matching. the final cohort includes 169 older adult severe acute respiratory illness patients who received the respiratory panel and pct test and 507 ps-matched control patients. this composite cohort was used to assess the clinical impact of the rapid respiratory viral surveillance and pct tests. baseline characteristics were summarized using appropriate descriptive statistics. the categorical variables were presented as frequency and percentage and compared using the chi-squared test. the continuous variables were presented by median with interquartile range and compared by nonparametric mann-whitney u tests. the numbers of different respiratory viral isolates and mean serum level of pct for different viral infections were shown by bar graph. to select control patients, we built a ps for matching. ps was defined as the conditional probability of being tested with respiratory panel and pct, which was derived from the logistic regression model that included the following potential predictors: demographics, comorbidity, presenting vital signs, laboratory results, and admission diagnoses. to verify the balancing of baseline covariates after ps matching, we made a standardized difference plot to ensure minimum differences in the baseline covariates between 2 groups of patients (supplementary figure 1 ). in the ps-matched cohort, we compared the outcome between the current cohort and the ps-matched historical cohort using the logistic regression model, adjusting for the residual difference in the baseline covariates. all statistical analyses were performed by sas 9.4 (sas inc, cary, nc), and a p value of < .05 was deemed significant. a total of 178 patients enrolled in the study, of which 9 were excluded because of missing data or loss of follow-up. finally, 169 older adult patients with severe acute respiratory illness were included in the study analysis, of which 36 (21.3%) patients tested positive for respiratory virus. these patients were sick, so they were all hospitalized. the demographics, presenting vital signs, laboratory test results, and underlying comorbidity of the experimental and control cohorts are shown in table 1 . in the experimental cohort, the mean age was 81.2 years and 69.8% were males. diabetes, cancer, and chronic pulmonary disease were the leading 3 comorbidities, and pneumonia, chronic obstructive pulmonary disease with acute exacerbation, and acute respiratory failure were the most prevalent diagnoses. the control cohort had a comparable distribution on the aforementioned characteristics, except for including fewer patients with dementia or chronic liver disease. in the experimental group, 36 patients tested positive for respiratory virus, including 13 influenza a or b virus (7.7%), 9 rsv (5.3%), 9 human rhinovirus/enterovirus (5.3%), 2 coronavirus (1.2%), 2 parainfluenza virus type 3 (1.2%), and 1 human metapneumovirus (0.6%). in the control group, 20 patients (3.3%) were diagnosed with influenza, which was significantly lower than in the experimental group (p ¼ .049) ( table 2) . of the 36 patients who tested positive for virus, 14 (38.9%) had a pct level lower than 0.25 ng/ml. coronavirus, influenza a, and human rhinovirus/enterovirus infections had higher serum levels of pct (figure 1 ). compared with the control group, the patients in the experimental group had more antibiotics de-escalation (21.9% vs 13.2%, p ¼ .007), received more neuraminidase inhibitor in the ed (8.9% vs 0.6%, p < .001), had a shorter duration of intravenous antibiotics use (mealthough the patients in the experimental group had a trend of more antibiotic discontinuation, the small number of patients prevents meaningful comparison. neither 30-day nor in-hospital mortality was significantly different between the 2 groups. to further account for the residual covariate difference after ps matching, we performed logistic or linear regression adjusting for age, temperature, chronic liver disease, and dementia. the results revealed that rapid pcr respiratory panel and pct testing were associated with increased odds of discontinuing or de-escalating antibiotics [odds ratio (or) 1.97, 95% confidence interval (ci) 1.28, 3.02], increased odds of neuraminidase inhibitor prescription (or 17.9, 95% ci 5.02, 63.98), and shorter duration of intravenous antibiotics (-e4.44, 95% ci e2.08, à2.79). there was no difference in length of hospital stay and 30-day mortality (table 3) . this prospective cohort study reports the clinical impact of rapid molecular diagnosis of respiratory pathogens in conjunction with pct testing on older adult patients presenting to the ed with severe acute respiratory illness. the results showed 21.3% of older adult severe acute respiratory illness patients to be having respiratory virus infection, with influenza, rsv, and human rhinovirus/enterovirus being the 3 leading pathogens. we demonstrated that the new diagnostic approach was associated with increased discontinuation or deescalation of antibiotics, reduced length of intravenous antibiotics treatment, and improved influenza detection and antiviral use. these findings are consistent with those of previous studies. brendish et al 8 showed that patients receiving respiratory panel testing were more likely to undergo single doses or brief courses of antibiotics treatment. respiratory panel testing was also associated with a reduced length of stay and improved influenza detection and antiviral use. however, they did not find that routine use of respiratory panel testing could reduce the proportion of patients treated with antibiotics, which they ascribed to the initiation of antibiotics before the results of pct in many patients. a pre-post study showed that the use of the respiratory panel decreased the time to diagnosis of respiratory viruses, hospital admission rate, length of stay, number of chest radiographs, and duration of antimicrobial use. 13 gelfer et al 21 combined the respiratory panel and pct tests, but found no significant differences in overall antibiotic exposure between the experimental and standard-of-care groups. nevertheless, they found significantly fewer patients discharged on antibiotics and a shorter duration of therapy in a subgroup of patients with positive viral and negative pct testing results. they stressed the importance of proactive communication between the antibiotics stewardship team and physicians. our results showed that the proposed diagnostic approach could reduce intravenous antibiotics treatment duration by 4.44 days without compromising patient outcomes. historically, it has been advised to complete the course of intravenous antibiotics treatment despite the resolution of clinical symptoms. however, there is little evidence to support this practice. 22, 23 overuse of antibiotics was associated with increased risk of clostridioides difficile infection, and a prolonged course of intravenous antibiotics may increase the risk of adverse drug events, organ dysfunction, or mortality. 24 it is noteworthy that the identification of respiratory virus alone may not be sufficient to reduce antibiotic use because of the concerns regarding mixed virus-bacteria coinfection, especially influenza with pneumococcus infection. 25 low serum level of pct may help alleviate the concerns of mixed infection. in addition, communicating the results to the treating physicians is important. 13, 16 although we did not have a formal antibiotic stewardship team, the study nurse communicated the results to the treating physicians and promoted antibiotics stewardship. another finding is the underdiagnosis of influenza in older adult patients. older adult patients were less likely to undergo a provider-ordered influenza test. they usually lack the typical presentation of influenza-like illness and may present with respiratory distress or confusion. 26 a recent study showed that the diagnosis of influenza based on clinical grounds alone was associated with a suboptimal sensitivity of 36% and a specificity of 78%. 27 effect estimates for dichotomous outcomes were calculated by logistic regression whereas those for continuous outcomes were calculated using quantile regression. both models were adjusted for covariates not balanced after ps matching, including age, temperature, chronic liver disease, dementia, and chronic obstructive pulmonary disease with acute exacerbation. the proposed algorithm for respiratory virus infection diagnosis and antibiotic stewardship may also have implications for nursing home (nh) residents. acute respiratory virus infection outbreaks are a common problem in nhs. 28, 29 a recent systematic review reported a 1.21% to 85.2% annual incidence of influenza or rsv infection in long-term care facilities. 28 other than influenza and rsv, human metapneumovirus is the third most common causative pathogen for nh respiratory infection outbreaks. 30 nhs often do not have on-site equipment to evaluate suspected infection; therefore, a lower threshold for antibiotic prescription is common. it is estimated that approximately two-thirds of nh residents received antibiotics each year, and up to 75% of the treatment is inappropriate. nhs become the reservoirs for resistant bacteria within a community. 31 although the present protocol cannot be implemented in nhs, it can be used among severe nh patients who are transferred to the ed. in a less severe outbreak, the nasopharyngeal samples of nh residents can be collected and sent to contracted laboratories for respiratory panel testing. the early detection of acute respiratory infection enables early isolation of infected patients and early antiviral drug administration, which can prevent or contain a respiratory virus infection outbreak. cost is an important consideration for the large-scale clinical implementation of rapid multiplex pcr testing. previous analyses showed that rapid multiplex pcr testing was the most cost-effective testing strategy for the detection of influenza in children. 12, 32 the cost-effectiveness of respiratory panel testing is highly influenced by the prevalence of influenza and the proportion of patients treated with antivirals. the significant improvement in influenza diagnosis and antiviral treatment in our study suggests that a combination of respiratory panel and pct testing may be cost-effective in our study setting. such speculation, however, requires future validation. our results have to be interpreted in light of several limitations. first, pct tests were not used in the comparison cohort. we cannot determine the impact of the viral panel and pct tests separately. second, the study nurse only enrolled patients during working hours of weekdays. selection or spectrum bias is less likely because we did not find significant difference in the outcomes of patients presenting to the ed on different time shifts. third, the postdischarge follow-up data of the historical comparison cohort cannot be retrieved. we therefore could not compare the duration of oral antibiotics between the 2 cohorts. the reduction of intravenous antibiotic duration alone is important because it has been shown to be a strong risk factor for the development of resistant bacteria strains. fourth, the generalization of the results to other settings should be taken into consideration. the long hospitalization duration in our study was due to old age, severe illness, and low hospitalization cost. 33, 34 lastly, the incidence of various respiratory viruses may have varied across the 2 seasons of the study period. the strengths of our study include the older adult population, the twin-center prospective cohort design, the simple antibiotic stewardship algorithm, and the comparison to a ps-matched cohort. the findings of our study support the use of rapid multiplex pcr respiratory panels in conjunction with the pct test for early diagnosis of respiratory viral infection and to inform optimizing antibiotic use in older adult patients presenting to the ed with severe acute respiratory illness. respiratory viral infection outbreak is common in nursing homes. performing the proposed diagnostic approach on patients transferred from nhs may enable early detection of the causative pathogens and early isolation of infected patients. as the cost per test is still high, institutions should develop a protocol to prevent indiscriminate testing with multiplex pcr and provide proactive real-time feedback to treating physicians for antimicrobial stewardship. further studies are needed to assess the incremental value of multiplex pcr viral testing compared with pct testing alone in the management of patients with severe acute respiratory infection in the ed. diagnostic accuracy of rapid antigen detection tests for respiratory syncytial virus infection: systematic review and meta-analysis diagnostic accuracy of novel and traditional rapid tests for influenza infection compared with reverse transcriptase polymerase chain reaction: a systematic review and meta-analysis the filmarray(r) respiratory panel: an automated, broadly multiplexed molecular test for the rapid and accurate detection of respiratory pathogens the clinical significance of filmarray respiratory panel in diagnosing community-acquired pneumonia multiplex pcr system for the rapid diagnosis of respiratory virus infection: systematic review and meta-analysis multicenter evaluation of biofire filmarray respiratory panel 2 for detection of viruses and bacteria in nasopharyngeal swab samples the influence of rapid influenza diagnostic testing on antibiotic prescribing patterns in rural thailand routine molecular point-of-care testing for respiratory viruses in adults presenting to hospital with acute respiratory illness (respoc): a pragmatic, open-label, randomised controlled trial effect of the influenza virus rapid antigen test on a physician's decision to prescribe antibiotics and on patient length of stay in the emergency department the rapid diagnosis of viral respiratory tract infections and its impact on antimicrobial stewardship programs the use of a multiplex real-time pcr assay for diagnosing acute respiratory viral infections in children attending an emergency unit randomised controlled trial and health economic evaluation of the impact of diagnostic testing for influenza, respiratory syncytial virus and streptococcus pneumoniae infection on the management of acute admissions in the elderly and high-risk 18-to 64-yearolds impact of early detection of respiratory viruses by multiplex pcr assay on clinical outcomes in adult patients impact of rapid molecular respiratory virus testing on real-time decision making in a pediatric emergency department impact of a rapid respiratory panel test on patient outcomes multiplex respiratory virus testing for antimicrobial stewardship: a prospective assessment of antimicrobial use and clinical outcomes among hospitalized adults viral infection in community-acquired pneumonia: a systematic review and meta-analysis diagnostic accuracy of c-reactive protein and procalcitonin in suspected community-acquired pneumonia adults visiting emergency department and having a systematic thoracic ct scan using procalcitonin to guide antibiotic therapy overview of procalcitonin assays and procalcitonin-guided protocols for the management of patients with infections and sepsis the clinical impact of the detection of potential etiologic pathogens of community-acquired pneumonia should we abandon "finishing the course" of antimicrobials? the antibiotic course has had its day hospital ward antibiotic prescribing and the risks of clostridium difficile infection viral-bacterial coinfection affects the presentation and alters the prognosis of severe community-acquired pneumonia underdiagnosis of influenza virus infection in hospitalized older adults clinical diagnosis of influenza in the ed the burden of respiratory infections among older adults in long-term care: a systematic review influenza in long-term care facilities outbreak of human metapneumovirus in a nursing home: a clinical perspective comparing appropriateness of antibiotics for nursing home residents by setting of prescription initiation: a cross-sectional analysis economic analysis of rapid and sensitive polymerase chain reaction testing in the emergency department for influenza infections in children effect of a modified hospital elder life program on delirium and length of hospital stay in patients undergoing abdominal surgery: a cluster randomized clinical trial predicting the length of hospital stay of postacute care patients in taiwan using the chinese version of the continuity assessment record and evaluation item set biofire diagnostics, llc, salt lake city, ut sponsored the filmarray rp kits for this study. biofire has no role in the interpretation of the results and writing of the manuscript. key: cord-262366-cmjnb0al authors: gerna, giuseppe; campanini, giulia; rovida, francesca; percivalle, elena; sarasini, antonella; marchi, antonietta; baldanti, fausto title: genetic variability of human coronavirus oc43‐, 229e‐, and nl63‐like strains and their association with lower respiratory tract infections of hospitalized infants and immunocompromised patients date: 2006-05-23 journal: j med virol doi: 10.1002/jmv.20645 sha: doc_id: 262366 cord_uid: cmjnb0al in the winter–spring seasons 2003–2004 and 2004–2005, 47 (5.7%) patients with acute respiratory infection associated with human coronavirus (hcov) 229e‐, nl63‐, and oc43‐like strains were identified among 823 (597 immunocompetent and 226 immunocompromised) patients admitted to hospital with acute respiratory syndromes. viral infections were diagnosed by either immunological (monoclonal antibodies) or molecular (rt‐pcr) methods. each of two sets of primer pairs developed for detection of all covs (pancov) failed to detect 15 of the 53 (28.3%) hcov strains identified. on the other hand, all hcov strains could be detected by using type‐specific primers targeting genes 1ab and n. the huh‐7 cell line was found to be susceptible to isolation and identification of oc43‐ and 229e‐like strains. overall, hcov infection was caused by oc43‐like, 229e‐like, and nl63‐like strains in 25 (53.2%), 10 (21.3%), and 9 (19.1%) patients, respectively. in addition, three patients (6.4%) were infected by untypeable hcov strains. nl63‐like strains were not found to circulate in 2003–2004, and 229e‐like strains did not circulate in 2004–2005, while oc43‐like strains were detected in both seasons. the monthly distribution reached a peak during january through march. lower predominated over upper respiratory tract infections in each age group. in addition, hcov infections interested only immunocompetent infants and young children during the first year of life, while all adults were immunocompromised patients. coinfections of hcovs and other respiratory viruses (mostly interesting the first year of life) were observed in 14 of the 47 (29.8%) patients and were associated with severe respiratory syndromes more frequently than hcov single infections (p = 0.002). in conclusion, the use of multiple primer sets targeting different genes is recommended for diagnosis of all types of hcov infection. in addition, the detection of still untypeable hcov strains suggests that the number of hcovs involved in human pathology might further increase. finally, hcovs should be screened routinely for in both infants and immunocompromised patients with acute respiratory infection. j. med. virol. 78:938–949, 2006. © 2006 wiley‐liss, inc. in the winter-spring seasons 2003-2004 and 2004-2005, 47 (5.7%) patients with acute respiratory infection associated with human coronavirus (hcov) 229e-, nl63-, and oc43-like strains were identified among 823 (597 immunocompetent and 226 immunocompromised) patients admitted to hospital with acute respiratory syndromes. viral infections were diagnosed by either immunological (monoclonal antibodies) or molecular (rt-pcr) methods. each of two sets of primer pairs developed for detection of all covs (pancov) failed to detect 15 of the 53 (28.3%) hcov strains identified. on the other hand, all hcov strains could be detected by using typespecific primers targeting genes 1ab and n. the huh-7 cell line was found to be susceptible to isolation and identification of oc43-and 229e-like strains. overall, hcov infection was caused by oc43-like, 229e-like, and nl63-like strains in 25 (53.2%), 10 (21.3%), and 9 (19.1%) patients, respectively. in addition, three patients (6.4%) were infected by untypeable hcov strains. nl63-like strains were not found to circulate in [2003] [2004] , and 229e-like strains did not circulate in [2004] [2005] , while oc43-like strains were detected in both seasons. the monthly distribution reached a peak during january through march. lower predominated over upper respiratory tract infections in each age group. in addition, hcov infections interested only immunocompetent infants and young children during the first year of life, while all adults were immunocompromised patients. coinfections of hcovs and other respiratory viruses (mostly interesting the first year of life) were observed in 14 of the 47 (29.8%) patients and were associated with severe respiratory syndromes more frequently than hcov single infections (p ¼ 0.002). in conclusion, the use of multiple primer sets targeting different genes is recommended for diagnosis of all types of hcov infection. in addition, the detection of still untypeable hcov strains suggests that the number of hcovs involved in human pathology might further increase. finally, hcovs should be screened routinely for in both infants and immunocompromised patients with acute respiratory infection. j. med. virol. 78:938-949, 2006 . ß 2006 wiley-liss, inc. following the first reports in the 1960s [tyrrell and bynoe, 1965; hamre and procknow, 1966; mcintosh et al., 1967] , human coronaviruses (hcovs) were shown to be pathogenic in volunteer studies [bradburne and somerset, 1972] and widespread in the community in a number of seroepidemiological surveys of the two known strains, hcov-229e and hcov-oc43 [mcintosh et al., 1970; kaye et al., 1971 kaye et al., , 1972 hamre and beem, 1972; monto and lim, 1974; gerna et al., 1978 gerna et al., , 1980 macnaughton, 1982] . initially, several other hcovs were described, such as b814 [tyrrell and bynoe, 1965] , hcov-oc16, hcov-oc37, and hcov-oc48 [mcintosh et al., 1967] . however, these viruses could not be grown in tissue culture or animal models. attempts to identify hcovs in clinical specimens or cell cultures by using either polyclonal or monoclonal antibodies were not satisfactory [mcintosh et al., 1978; sizun et al., 1998 ]. animal covs were then investigated extensively in a variety of animal species, while the study of the antigenic relationship of human and animal covs was addressed [kaye et al., 1975; gerna et al., 1981] , and the unique strategy of replication of covs was described [lai and holmes, 2001] . this allowed the rapid identification of the etiologic agent of the severe acute respiratory syndrome (hcov-sars) in 2003 [peiris et al., 2003] . in parallel, the potential pathogenic role of hcovs, and, in particular, hcov-oc43 in diseases of the gastrointestinal tract was investigated by the study of the antigenic relationship of hcov-oc43 and neonatal calf diarrhea coronavirus or bovine cov (bcov), and hcov-oc43 and human enteric covs [chany et al., 1982; gerna et al., 1985] . in addition, molecular techniques permitted the identification of known group i (229e-like) and group ii (oc43-like) hcovs in respiratory secretions [sizun et al., 1998; el-sahly et al., 2000; vabret et al., 2001 vabret et al., , 2003 falsey et al., 2002; pene et al., 2003; vallet et al., 2004] and the discovery of new hcovs. recently, two groups of researchers from the netherlands [fouchier et al., 2004; van der hoek et al., 2004] and one group from usa [esper et al., 2005] reported a new hcov, referred to as nl63 and new haven, respectively. this virus was related distantly to hcov-229e, and described as one viral agent associated with acute respiratory disease in hospitalized children. furthermore, another hcov (referred to as hku1), distantly related to hcov-oc43, was detected in two hong-kong patients with pneumonia [woo et al., 2005a] , and then reported elsewhere [woo et al., 2005b; sloots et al., 2006; vabret et al., 2006] . a similar virus was also reported in sweden by molecular screening of respiratory tract samples [allander et al., 2005] . finally, a few studies have addressed the circulation of hcovs with respect to other respiratory viruses in different countries, as well as their involvement in respiratory syndromes other than the common cold, and their association with asthma and chronic obstructive bronchopneumopathies, including hcov pneumonia in transplanted patients [lina et al., 1996; folz and elkordy, 1999; el-sahly et al., 2000; falsey et al., 2002] . at present, several issues remain to be investigated or defined: (i) the reliability of currently available immunological and molecular methodologies for diagnosing infections caused by different groups of hcovs; (ii) the epidemiology of respiratory infections caused by hcovs; (iii) the role of known hcovs in causing admission to the hospital of patients with respiratory infections as well as their relationship to the severity of the relevant respiratory syndromes; (iv) the comparative pathogenicity of different hcovs in different age groups. in the present study, these issues were investigated in a hospitalized patient population affected by 229e-, nl63-, and oc43-like infections during two consecutive winter-spring seasons. to date, hku1 has not been detected in italy. from december 2003 through may 2005, nasopharyngeal aspirates were collected prospectively from 823 patients admitted to hospital with an episode of acute respiratory infection. of these, 333 were less than 1 year old, 168 2-5 years old, 87 6-20 years old, and 233 >21 years old. study patients were further subdivided based on immunocompetence. in the four age classes, immunocompetents included 333, 143, 44, and 77 patients, while immunosuppressed patients included 2, 25, 43, and 156 subjects, respectively. of the 226 immunosuppressed patients, 70 included in the first three age groups were hematopoietic stem cell transplant recipients. in the >21-year group 108 were solid organ transplant recipients, 46 stem cell transplant recipients, and two aids patients. specimens were handled and aliquoted as reported previously , and used for immunological (monoclonal antibodies) and molecular (rt-pcr) assays, as well as for short-term and long-term virus isolation in cell culture. respiratory samples were examined routinely for influenzaviruses a and b, parainfluenzaviruses 1-4, human respiratory syncytial virus, and human adenoviruses by both direct fluorescent staining and culture. in addition, human metapneumoviruses were examined by both monoclonal antibodies and rt-pcr as recently reported gerna et al., 2006] . finally, hcovs were tested by monoclonal antibodies and rt-pcr, as detailed below, and human rhinoviruses were searched for by rt-pcr [steininger et al., 2001] . in this study, the term coinfection indicates the simultaneous detection of two or more respiratory viruses in the same sample taken from a patient with respiratory infection, while the term sequential infection refers to the sequential identification of two different respiratory viruses in two nasopharyngeal aspirates taken from the same patient within 30 days from each other. direct fluorescent antibody staining was applied to both slides containing smears of respiratory epithelial columnar cells from nasopharyngeal aspirates and cell cultures 48 hr after inoculation of respiratory secretions, as reported [rovida et al., 2005] . in either case, as a first step, cells were stained with a pool (simulfluor respiratory screen reagent, chemicon international, inc., temecula, ca) of fluorescein-labeled monoclonal antibodies to conventional respiratory viruses (influenzavirus types a and b, human parainfluenzavirus types 1-4, human respiratory syncytial virus, and human adenovirus). then, as a second step, positive samples were stained with individual monoclonal antibodies obtained from the same source (chemicon). group-and type-specific monoclonals to human metapneumovirus were developed in the laboratory as reported gerna et al., 2006] . as for hcovs, commercially available monoclonal antibody to oc43 (chemicon) was found to perform satisfactorily, while monoclonal antibodies to 229e were developed in the laboratory and found to be highly specific. finally, monoclonal antibodies to nl63 were not available. inoculated cell cultures included mixtures (mix) of a549 and mv1lu (ratio 1:1) cells [huang and turchek, 2000] , as well as llc-mk2 and mdck cell lines. cell cultures were processed for virus isolation and identification as reported previously [rovida et al., 2005] . in addition, in the last part of the study, huh-7 [ [nakabayashi et al., 1982] provided by maura pizzuti, institute for biomedical research, rome, italy], vero and llc-mk2 cell lines, as well as secondary african green monkey kidney cell cultures, were inoculated with six npas rt-pcr-positive for hcovs (one positive for 229e, one positive for oc43, and four positive for nl63) to investigate the susceptibility of these cells to isolation and propagation of hcovs. rt-pcr assays for respiratory viruses were optimized to detect at least 10 input plasmid copies, as described previously ]. in addition, as reported in table i , npas were tested for hcovs by rt-pcr by using: (i) a primer set (referred to as pancov-03) developed for detection of all coronaviruses and relevant to gene 1ab [poutanen et al., 2003] ; (ii) a second set of primers (referred to as pancov-05) also developed for detection of both human and animal coronaviruses and relevant to gene 1ab ; (iii) three sets of primers specific for oc43, 229e, and nl63 hcovs, respectively, and relevant to gene n; and, finally, three sets of primers specific for oc43, 229e, and nl63, respectively, derived from pancov-05, thus, relevant to gene 1ab. the following fragments of the 1ab gene were amplified (table i) : nt 14321-14536 of 229e (amplicon size, 215 nt) with pan cov-03, nt 14098-14348 of 229e (size, 250 nt) with pan cov-05, nt 14922-15172 (size, 250 nt) with oc43-specific primers, nt 14098-14348 (size, 250 nt) with 229e-specific primers, and nt 14017-14267 (size, 250 nt) with nl-63-specific primers. in addition, the following fragments of the n gene were amplified: nt 715-1073 (size, 378 nt) with oc43-specific primers, nt 578-921 (size, 343 nt) with 229e-specific primers, and nt 563-876 (size, 313 nt) with nl63specific primers. the indicated fragments of genes 1ab and n of hcovs oc43, 229e, and nl63, following amplification with specific primers, were sequenced with the abi prism 3100 automatic sequencer (applied biosystems, foster city, ca). viral sequences of the amplified fragments of genes 1ab and n of different hcov strains as well as reference strains were aligned with the clustal w program version 1.7. distances between pairs of nucleotide sequences were calculated by using the dnadist modules (with kimura's twoparameter method) in the phylip package, version 3.572 (felsenstein, department of genetics, university of washington, seattle, wa). the phylip (njplot) program was used to construct phylogenetic trees with nucleotide sequences by means of the neighbor-joining method from the same distance matrices. bootstrap support was determined by 100 resamplings of the sequences. comparison of the distribution frequencies was performed with the pearson's chi square test. on the whole, 53 hcov strains were identified from 47 patients admitted to hospital as follows: 27 (50.9%) oc43-like strains, 14 (26.4%) 229e-like strains, 9 (17%) nl63-like strains, and 3 (5.7%) untypeable hcov strains (fig. 1a) . six strains (four 229e-like and two oc43-like strains) were recovered twice from the same patients within 2 weeks after initial virus recovery. all hcov-positive samples were tested by two sets of primers aimed at detecting all hcov strains (table i) : (i) pancov-03 [poutanen et al., 2003] ; and (ii) pancov-05 . of the 53 hcov-positive samples, 23 (43.4%) were found positive by both assays, while 15 (28.3%) were detected by pancov-03 only, and 15 (28.3%) by pancov-05 only (fig. 1b,c) . all of the nine nl63-like strains were detected by the pancov-05 primer pair only. in conclusion, 38/53 (71.7%) samples tested by pancov-03 were positive for hcov: 21 (39.6%) for oc43-, 14 (26.4%) for 229e-like strains, and three (5.7%) for untypeable strains, while 15 strains (28.3%) were negative. similarly, of the 53 samples tested by pancov-05, 38 (71.7%) were positive: 22 (41.5%) for oc43-like, 7 (13.2%) for 229e-like, and 9 (17%) for nl63-like strains, while 15 strains (28.3%) were negative (no untypeable strains with this primer set). all 53 hcov strains were typed by sequencing and phylogenetic analysis using the amplification products provided by both or either pancov. subsequently, to confirm results, each of the 53 hcov strains were amplified by using two primer pairs specific for genes 1ab and n of oc43, 229e, and nl63 prototypes, respectively (table i) . pancov typing results were consistently confirmed by type-specific primer sets, with no cross-reactivity among primers. primer-specific amplification products of gene 1ab (fig. 2) and n fragments were used for phylogenetic analysis. oc43-like strains clustered in a group distinct from reference strain oc43 and much more distant from the new hcov hku1, except for a single strain (i-pv 02/ 04-2645) distinct from both oc43 and hku1. similarly, 229e-strains clustered into a single group distinct from the reference strain 229e and distant from nl63. finally, nl63-like strains were grouped together substantially, and distinct from the reference strain nl63 and very distant from 229e. a summary of the genetic variability (nt and aa changes) of different hcov strains (within the limits of the amplified fragment of genes 1ab and n) with respect to reference strains reported in gene bank is given in table ii . weakness of pancov-03 amplification of the three untypeable strains did not allow its sequencing. however, negative results following amplification of n gene with 229e-, oc43-, and nl63-specific primers did exclude their similarity with the reference strains. following identification of hcov-positive nasopharyngeal aspirates by rt-pcr, retrospective attempts were made to grow short-term oc43-, 229e-, and nl63like strains in cell cultures from a small number of specimens. one oc43-and one 229e-like strain were inoculated and found to grow in the huh-7 cell line. viruses were identified (fig. 3a-d) and 229e-like strains (10 6 to 10 7 tcid 50 /ml). no crossreactivity of these strains with other human respiratory viruses was detected either by using rt-pcr or monoclonal antibodies. on the other hand, four nl63like-positive samples showed initial growth in secondary african green monkey kidney cell cultures as well as vero and llc-mk2 cell lines, as indicated by rt-pcr using type-specific primers (fig. 3e) . however, further propagation was unsuccessful. these preliminary results suggest the possible recovery and long-term propagation of 229e-like and oc43-like hcov strains in cell culture, whereas a cell culture system suitable for long-term propagation of nl63-like strains remains to be identified. among 823 patients admitted to hospital with an episode of acute respiratory tract infection in the winter-spring seasons 2003-04 and 2004-05, 47 (5.7%) patients were found to be affected by hcov infections. the list of respiratory viruses associated with acute respiratory infections in 447/823 (54.3%) patients is reported in table iii . the most frequently detected virus was hrsv (26.7%), followed by rhinoviruses (24.2%) and influenza viruses (13.9%), while hmpv and hcovs were circulating at a comparable rate (8.1 and 7.4%, respectively). a great variation in the circulation of different hcovs was observed between the two seasons examined. in fact, 229e-like strains circulated only in 2003-04 (10 infected patients), and nl63-like strains only in 2004-05 (9 infected patients), while oc43-like strains were detected in both seasons, thereby infecting 25 patients (fig. 4a,b) . the peak circulation of hcovs was reached in january through march, while a few strains were observed from november to may. stratification by age showed that 20/333 (6.0%) patients were affected by hcov infection in the first year of life, while 8 (4.8%) were affected in the 2-5 years old group, 13 (14.9%) in the 6-20 years old group, and only 6 (2.6%) were > 20 years old (fig. 5a ). in 33/47 patients (70.2%), respiratory infections were attributed to a single hcov strain, while in 14/47 patients (29.8%) hcovs were associated with one or more other respiratory viruses. analysis of the distribution of single infections and coinfections by age showed that they affected an equal number (n ¼ 10) of patients in the first year of life, while the incidence of coinfections (table iv) greatly decreased in the other age groups (only four patients in the 2-20 years old groups). among the 47 patients affected by respiratory viral infections associated with hcovs, 35 (74.5%) showed lower (bronchitis, bronchiolitis, pneumonia) and 12 (25.5%) upper respiratory tract (rhinitis, pharyngitis, laryngitis) infections. the predominance of lower versus upper infections was observed in patients of each age group with no significant difference in the incidence among different age groups (fig. 5b) . as shown in figure 5c ,d, the mean percentage of immunocompetent patients with hcov infections was 4.5% (27/597), whereas the mean percentage of immunocompromised patients infected by hcovs was 8.8% (20/226) with a statistically significant difference (w 2 test, p ¼ 0.02). the relative risk of contracting hcov infection in immunocompromised patients was twice that of immunocompetent patients. the total number of hcov infections observed during the first year of life included 20/333 (6.0%) immunocompetent infants and young children, while the proportion of immunocompetent children with hcov infections in the older age groups included 5/143 (3.5%) and 2/44 (4.5%) patients in the 2-5 and the 6-20 years old groups, respectively. no hcov infection (0/77) was observed in immunocompetent patients older than 20 years (fig. 5c ). conversely, no immunocompromised patients with hcov infections, were observed in the first year of life (0/2 patients). in older age groups, hcov infections of immunocompromised patients increased progressively up to 12.5% (3/25 patients) in the 2-5 years old group, and to 25.6% (11/43 patients) in the 6-20 years old group, including all infected patients older than 20 years (3.8%, 6/156 patients) (fig. 5d) . in the same group of 47 patients examined with hcov respiratory infections, 25 (53.2%) were infected by oc43-like strains (six in association with other viruses), 10 (21.3%) by 229e-like strains (three coinfections), and 9 (19.2%) by nl63-strains (two coinfections). in addition, three patients (6.3%) were infected by untypeable hcovs (three coinfections). no significant difference in the circulation of different hcovs among different age groups was observed (fig. 4c) . the most common symptoms were rhinorrhea (52.2%), fever (47.8%), and cough (47.8%) in oc43-infected patients. similarly, predominant symptoms were rhinorrhea (70%), fever (50.0%), and cough (50.0%) in 229e-infected patients, while rhinorrhea (57.1%) and cough (42.8%) were most commonly observed in nl63-infected individuals, in association with infrequent episodes of fever (14.2%). severe lower respiratory tract syndromes (bronchiolitis, pneumonia) were observed at presentation in 10/15 lower respiratory tract infections associated with hcov in the first year of life: six (three single infections and three coinfections) were associated with oc43-like strains, two with hcov untypeable strains (both coinfections), one to 229e-like strain (coinfection), and one to an nl63-like strain (coinfection). thus, severe hcov infections in the first year of life appeared to be mostly associated with oc43-like strains. conversely, none of the five adult immunocompromised adult patients with lower respiratory tract infections showed association with bronchiolitis or pneumonia. severe respiratory syndromes were observed in 9/14 (64.3%) coinfected patients (table iv) , and only in 3/33 patients (9.1%) infected by hcov alone (p ¼ 0.002). thus, hcov coinfections appeared to be associated with a significantly higher number of severe syndromes involving the lower respiratory tract. after the 1970s, clinical studies on hcovs were mostly abandoned due to either the presumed low clinical impact of these viruses, or to the great difficulties encountered in their isolation and identification. renewed interest was triggered recently by the identification of the coronavirus responsible for sars in 2003. in addition, the development of viral genome amplification procedures has provided a major contribution to the identification of hcov infections. a major variant of group i hcovs, with the 229e virus as a prototype, was identified in the nl63 strain detected in the netherlands [fouchier et al., 2004; van der hoek et al., 2004] , and in the usa [esper et al., 2005] . more recently, a major variant of group ii hcovs, with the oc43 virus as a prototype, was first reported in hong-kong as hku1 [woo et al., 2005a] and, then, in france, australia, and other regions of china [woo et al., 2005b; sloots et al., 2006; vabret et al., 2006] . a wide range of antigenic variability has been reported by researchers recovering a series of viruses in organ cultures of human embryonic trachea [mcintosh et al., 1967; mcintosh, 2005] . however, a great deal of controversy has been raised about the genetic stability [st-jean et al., 2004] or variability of hcov oc43-like strains. the genetic variability of hcovs is supported by the present study, showing that both sets of pancov primers actually did not detect about 30% of circulating strains. this failure of both pancov primer sets was confirmed by the use of group-specific primer pairs relevant to two different genes. a similar conclusion was recently reported by another study [chiu et al., 2005] . as a result, in order to detect the maximal number of circulating hcovs, specific primers should be used, and results confirmed by a second set of primers, preferably targeting a different gene. in the present study, no hku1 strain was detected by the two pancov primer sets used, while, in the absence of a positive control, hku1-specific primers were not used. a complement to the molecular techniques available for detection of hcovs is represented by the use of the huh-7 cell line, which has been recently employed for respiratory virus isolation [vabret et al., 2001; pene et al., 2003; freymuth et al., 2005] . this cell line, which was shown to be susceptible to infection by a murine coronavirus, the mouse hepatitis virus [koetters et al., 1999] , has been found to be helpful in increasing the rate of respiratory virus recovery from dfa-negative samples . in addition, huh-7 was reported to be permissive for growth of hcovs, rhinoviruses, and enteroviruses. however, in that study, hcovs were cultured in huh7 for 4 days only, and then identified by rt-pcr. in the present study, it was possible to isolate and extensively propagate two strains of 229e-like and oc43-like strains from respiratory secretions, thus obtaining high virus yields after a few passages. a major morphological characteristic of both 229e-like and oc43-like strains was the presence of syncytial formations in inoculated cell cultures, appearing early (24-48 hr) after inoculation and progressively enlarging until detaching from the cell surface. in this respect, it seems important to recall that sars-hcov was reported to produce syncytia in vivo [ksiazek et al., 2003] . as for nl63-like strains, initial growth was confirmed by the positive rt-pcr results in the supernatant of all three types of cell cultures inoculated with four nl63-positive respiratory samples, that is, two cell lines (llc-mk2 and vero) and one secondary culture of african green monkey kidney. however, at subsequent passages, the presence of the viral rna signal in the medium weakened progressively and finally disappeared. since inoculated nl63-like-positive samples were freezethawed more than once prior to inoculation, new isolation attempts will be made as soon as fresh positive specimens become available. the phylogenetic analysis based on sequencing of small fragments from genes 1ab and n showed that each of the three groups of hcovs detected in our study belong to a single cluster, which is clearly distinct from the relevant reference strain. the only exception regarded an oc43-like strain (i-pv 02/04-2645), which was distinct from both pavia strains and the oc43 reference strain and was recovered from a patient who had recently arrived from albany. these data confirm the genetic similarity of the strains circulating in the same geographical area without excluding the simultaneous presence of other unrelated or poorly related strains. the pathologic impact of hcov infections was investigated by analyzing the role of these infections in the context of the patient population admitted to hospital due to an acute respiratory infection during two consecutive winter-spring seasons. on the whole, the overall rate of hcov infections (including both single infections and coinfections) followed that of human respiratory syncytial virus, rhinovirus, and influenza virus infections, and preceded that of human metapneumoviruses, adenoviruses, and human parainfluenza viruses, with comparable rates of coinfections and sequential infections. although different studies have reported variations, these data correlate grossly with previous surveys [lina et al., 1996; freymuth et al., 2005] . in addition, coinfections by different respiratory viruses have been repeatedly reported [el-sahly et al., 2000; rovida et al., 2005] . from an epidemiological standpoint, in the great majority of studies, 229e, oc43, and nl63 van der hoek et al., 2004 outbreaks have been found to occur in the winter-spring season with recurrences every 2-4 years [mcintosh et al., 1970; kaye et al., 1971; monto and lim, 1974] . in our study, while the peak of hcov circulation was reached in january through march, the three hcovs were found to circulate differently during the two consecutive seasons studied. while oc43-like strains were found to circulate in both seasons, 229e-and nl63-like strains were detected in either one of the two seasons, as observed previously by others. on the other hand, all three hcovs were represented in the four age groups in which the hospitalized patient population was divided, as reported previously [el-sahly et al., 2000; vabret et al., 2003] . the study of the age distribution showed a large predominance of hcov infections in the first year of life, when 15/20 (75.0%) patients were affected by lower respiratory tract infections and 10 (66.6%) suffered from severe lower respiratory tract syndromes, such as bronchiolitis or pneumonia. it is important to emphasize that all infected infants and young children were immunocompetent subjects. these results strengthen previous observations on the great pathologic potential of hcovs in very young patients [mcintosh et al., 1974; sizun et al., 1995; el-sahly et al., 2000; vabret et al., 2003; chiu et al., 2005] . in addition to 229e-and oc43like strains, also the newly identified nl63-like strains were shown to be associated with lower respiratory tract infections, including bronchiolitis and pneumonia [arden et al., 2005; bastien et al., 2005; ebihara et al., 2005; esper et al., 2005; vabret et al., 2005] . therefore, hcovs, and, as a result of this study, particularly oc43like strains, appear to behave like other respiratory viruses, such as human respiratory syncytial virus, human metapneumovirus, and human parainfluenza viruses, displaying a greater virulence in infancy and early childhood. on the other hand, adult patients included in this study were all immunocompromised transplanted patients, who had never suffered from severe respiratory syndromes. however, case reports of hcov-related pneumonia have been described [pene et al., 2003] , and the increasing number of transplanted patients undergoing immunosuppressive therapy render the investigation of these viruses mandatory in lower respiratory tract secretions from transplant recipients with bronchiolitis and pneumonia. finally, the significantly greater association of hcov coinfections with severe clinical syndromes, that emerged in the present study, shows a rate of severe lower respiratory tract infections greater than 60% in patients with coinfections compared to less than 10% in patients with a single infection. the major pathologic effect of hcov coinfections was observed again in infants and young children. however, whether the coinfection by hcov was a factor increasing the severity of the associated viral infection remains hypothetical. in conclusion, the genetic variability of hcovs might make the detection of all circulating strains difficult. cultures might be important for new hcov detection. the recently renewed interest in hcovs has allowed the identification of these viruses as major pathogens of infancy and early childhood, while their pathologic role in immunocompromised patients remains to be defined. coinfection by hcovs and other respiratory viruses is often detected in severe lower respiratory tract infections of infants and young children. cloning of a human parvovirus by molecular screening of respiratory tract samples new human coronavirus, hcov-nl63, associated with severe lower respiratory tract disease in australia human coronavirus nl63 infection in canada coronavirus antibody titres in sera of healthy adults and experimentally infected volunteers association of coronavirus with neonatal necrotizing enterocolitis human coronavirus nl63 infection and other coronavirus infections in children hospitalised with acute respiratory disease in hong kong detection of human coronavirus nl63 in young children with bronchiolitis spectrum of clinical illness in hospitalised patients with ''common cold'' virus infection evidence of a novel human coronavirus that is associated with respiratory tract disease in infants and young children rhinovirus and coronavirus infection-associated hospitalizations among older adults coronavirus pneumonia following autologous bone marrow transplantation for breast cancer a previously undescribed coronavirus associated with respiratory disease in humans replication of respiratory viruses, particularly influenza virus, rhinovirus, and coronavirus in huh7 hepatocarcinoma cell line determination of coronavirus 229e antibody by an immune-adherence hemagglutination method human coronavirus oc43 serum inhibitor and neutralizing antibody by a new plaque-reduction assay antigenic and biological relationships between human coronavirus oc43 and neonatal calf diarrhoea coronavirus human enteric coronaviruses: antigenic relatedness to human coronavirus oc43 and possible etiologic role in viral gastroenteritis simultaneous detection and typing of human metapneumovirus strains in nasopharyngeal secretions and cell cultures by monoclonal antibodies virologic studies of acute respiratory disease in young adults. v. coronavirus 229e infections during six years of surveillance a new virus isolated from the human respiratory tract mink lung cells and mixed mink lung and a549 cells for rapid detection of influenza virus and other respiratory viruses seroepidemiologic survey of coronavirus (strain oc43) related infections in a children's population detection of coronavirus 229e antibody by indirect hemagglutination calf diarrhea coronavirus mouse hepatitis virus strain jmh infects a human hepatocellular carcinoma cell line newly discovered coronavirus as the primary cause of severe acute respiratory syndrome coronaviridae: the viruses and their replication surveillance of community-acquired viral infections due to respiratory viruses in rhone-alpes (france) during winter 1994 to 1995 occurrence and frequency of coronavirus infections in humans as determined by enzyme-linked imunosorbent assay coronaviruses in the limelight recovery in tracheal organ cultures of novel viruses from patients with respiratory disease seroepidemiologic studies of coronavirus infection in adults and children coronavirus infection in acute lower respiratory tract disease of infants diagnosis of human coronavirus infections by immunofluorescence: method and application to respiratory disease in hospitalized children a novel pancoronavirus rt-pcr assay: frequent detection of human coronavirus nl63 in children hospitalized with respiratory tract infections in belgium the tecumseh study of respiratory illness. vi. frequency of and relationship between outbreaks of coronavirus infection growth of human hepatoma cell lines with differentiated functions in chemically defined medium coronavirus as a possible cause of severe acute respiratory syndrome coronavirus 229e-related pneumonia in immunocompromised patients rapid detection of human metapneumovirus strains in nasopharyngeal aspirates and shell vial cultures by monoclonal antibodies identification of severe acute respiratory syndrome in canada monoclonal antibodies versus reverse transcription-pcr for detection of respiratory viruses in a patient population with respiratory tract infections admitted to hospital detection and pathogenicity of human metapneumovirus respiratory infections in pediatric italian patients during a winter-spring season neonatal nosocomial respiratory infection with coronavirus: a prospective study in a neonatal intensive care unit comparison of immunofluorescence with monoclonal antibodies and rt-pcr for the detection of human coronaviruses 229e and oc43 in cell culture evidence of human coronavirus hku1 and human bocavirus in australian children human respiratory coronavirus oc43: genetic stability and neuroinvasion early detection of acute rhinovirus infections by a rapid reverse transcription-pcr assay cultivation of a novel type of commoncold virus in organ cultures direct diagnosis of human respiratory coronaviruses 229e and oc43 by the polymerase chain reaction an outbreak of coronavirus oc43 respiratory infection in normandy human coronavirus nl63 detection of the new human coronavirus hku1: a report of 6 cases detection of human coronavirus 229e in nasal specimens in large scale studies using an rt-pcr hybridisation assay identification of a new human coronavirus circulation of genetically distinct contemporary human coronavirus oc43 strains characterization and complete genome sequence of a novel coronavirus, coronavirus hku1, from patients with pneumonia clinical and molecular epidemiological features of coronavirus hku1-associated community-acquired pneumonia we thank linda d'arrigo for revision of the english. we are indebted to the technical staff of the servizio di virologia. this research was partially supported by ministero della salute, ricerca finalizzata 2004 (grants 89282 and 89288). key: cord-265751-q1ecpfyg authors: shahani, lokesh; ariza-heredia, ella j.; chemaly, roy f. title: antiviral therapy for respiratory viral infections in immunocompromised patients date: 2017-01-16 journal: expert rev anti infect ther doi: 10.1080/14787210.2017.1279970 sha: doc_id: 265751 cord_uid: q1ecpfyg introduction: respiratory viruses (influenza, parainfluenza, respiratory syncytial virus, coronavirus, human metapneumovirus, and rhinovirus) represent the most common causes of respiratory viral infections in immunocompromised patients. also, these infections may be more severe in immunocompromised patients than in the general population. early diagnosis and treatment of viral infections continue to be of paramount importance in immunocompromised patients; because once viral replication and invasive infections are evident, prognosis can be grave. areas covered: the purpose of this review is to provide an overview of the main antiviral agents used for the treatment of respiratory viral infections in immunocompromised patients and review of the new agents in the pipeline. expert commentary: over the past decade, important diagnostic advances, specifically, the use of rapid molecular testing has helped close the gap between clinical scenarios and pathogen identification and enhanced early diagnosis of viral infections and understanding of the role of prolonged shedding and viral loads. advancements in novel antiviral therapeutics with high resistance thresholds and effective immunization for preventable infections in immunocompromised patients are needed. the spectrum of immunocompromised hosts has expanded over the past decade owing to prolonged survival of patients with various malignancies and advances in both solid-organ and hematopoietic stem cell transplantation. novel immunosuppressive therapies create diverse immune deficits that generate a substrate for opportunistic infections [1] . these patients are defined by higher susceptibility to infections by organisms with lower native virulence than in immunologically normal hosts. influenza, parainfluenza, respiratory syncytial virus, coronavirus, human metapneumovirus, and rhinovirus represent the most common cause of respiratory viral infections in immunocompromised patients [1] . most of these infections are seasonal, and the viruses cause a wide range of upper respiratory tract infections (urtis) and lower respiratory tract infections (lrtis). however, adverse outcomes are far more likely in immunocompromised patients than in nonimmunocompromised individuals and include progression to pneumonia, respiratory failure, and increased mortality rates (1) (2) (3) (4) . in fact, the lrti rates and mortality rates for hematopoietic stem cell transplant (hsct) recipients and patients with hematological malignancies reportedly range from 10% to 50% [2] [3] [4] [5] . longterm complications associated with respiratory viral infections, such as airflow obstruction and bronchiolitis obliterans, have developed in hsct and lung transplant recipients [6, 7] . figure 1 highlights the high rates of progression to lrti and death among immunocompromised patients with common respiratory viral infections. the management of viral infections is challenging because viruses are intracellular parasites that use many of the host's own pathways to replicate and propagate. therefore, antiviral agents need to target specific viral components to avoid potential damage to host cells. figure 2 highlights the life cycle of viral replication and site of action of various antiviral agents. advances in the treatment of respiratory infections have been made over the past decades. table 1 highlights the current available agents for treatment of respiratory viral infections and table 2 lists the agents currently in the pipeline for these different viruses. the purpose of this review is to provide an overview of the main antiviral agents that are used in the management of respiratory infections in immunocompromised patients focusing on its clinical relevance and our experience, as well as to provide an update on the current investigational agents in the pipeline. the influenza virus is among the most common human respiratory viruses and belongs to the orthomyxoviridae family. four types of influenza viruses are a, b, c, and d. human influenza a and b viruses cause seasonal epidemics of disease almost every winter in the united states. influenza type c infections cause a mild respiratory illness and are not thought to cause epidemics. influenza d viruses primarily affect cattle and are not known to infect humans [17] . influenza a viruses are grouped into subtypes based on antigenic characteristics of 2 proteins on their surfaces, hemagglutinin (ha) and neuraminidase (na) with 18 different ha subtypes and 11 na subtypes. influenza a viruses can be further broken down into different subtypes. the most common subtypes of influenza a virus affecting humans are h1n1 and h3n2. influenza b viruses are not grouped into subtypes but can be further broken down into lineages. currently, circulating influenza b viruses belong to 1 of 2 lineages: b/yamagata and b/victoria [18] . the seasonal prevalence of influenza infections in immunocompromised patients, including solid-organ transplant and hsct recipients, closely parallels the community-wide prevalence, with peaks from december to february, with influenza b activity sometimes seen in april and may [19] . however, the illness has the potential to be more severe in this population than in healthy host [20] . without treatment reported, mortality rate range from 25% to 40% in immunocompromised patients, and is related to complications including pneumonia, and bacterial and fungal superinfections [21] . in a retrospective study, we identified profound lymphocytopenia (absolute lymphocyte count <200 cells/ ml), age greater than 65 years, and neutropenia (absolute neutrophil count <500 cells/ml) as potential risk factors associated with progression from urti to lrti [22] . early antiviral therapy within the first 48 h after presentation has been associated with improved prognosis in several studies [23] [24] [25] . the two main groups of antivirals used to treat influenza are m2 inhibitors (amantadine and rimantadine), which only act against influenza a, and na inhibitors active against influenza a and b: oseltamivir, zanamivir, and peramivir. m2 inhibitors inhibit the ion channel of the m2 protein in the influenza a virus, leading to defects in uncoating and assembly of the virus ( figure 2 ). the influenza virus enters its host cell via receptor-mediated endocytosis; thereafter, it is localized on endocytotic vacuoles. the m2 proton channel transports the ions needed for acidification of the influenza virus inside the vacuoles. this acidification is required for dissociation of the m1 protein from the ribonucleoprotein complexes and the onset of viral replication [26, 27] . the recommended dose of amantadine is 200 mg given once daily or 100 mg given twice daily (duration of therapy is generally 5 days) [8] . the most common side effects of these agents are gastrointestinal (nausea and vomiting) and effects on the central nervous system, including anxiety, insomnia, impaired thinking, confusion, lightheadedness, and hallucinations [8] . resistance of influenza a infection to m2 inhibitors results from mutations of the pore-lining residues in the ion channel, keeping adamantine and rimantadine from entering the channel [28] . according to data from the world health organization collaborating center for surveillance, epidemiology, and control of influenza at the us center for disease control and prevention (cdc), the rates of m2 inhibitors resistance have increased from 0.4% in 1994-1995 season to 12.3% in 2003-2004 [29] . however, during the 2005-2006 season, rates as high as 92% were reported for the influenza a (h3n2) virus [30] . recent cdc data demonstrate high prevalence of m2 inhibitors resistance in all influenza a (h3n2) and influenza a (h1n1) pdm09 virus isolates tested [31] . currently, the current advisory committee on immunization practices (acip) guidelines for treatment of influenza infections, do not recommend the routine use of amantadine and rimantadine in the usa for therapy or chemoprophylaxis for currently circulating influenza a virus strains [8] . nais block the active site of neuraminidase, resulting in uncleaved sialic acid residues on the host cell surface and viral envelopes ( figure 2 ). uncleaved sialic acid bound to viral ha causes viral aggregation on the host cell surface, which reduces the amount of virus released [8] . nais are virustatic, not virucidal, and early administration of them is a key factor in the development of resistance to the virus and their effectiveness. treatment with these inhibitors should neither be delayed while awaiting the results of diagnostic testing nor withheld from infected patients with indications for therapy who present more than 48 h after the onset of symptoms, particularly patients needing hospitalization [8] . in particular, the nais zanamivir and oseltamivir are first-line agents for treatment of and prophylaxis for influenza. oseltamivir is an oral nai usually prescribed as 75 mg orally twice daily (renally adjusted). the recommended duration of antiviral therapy is 5 days [8] . however, a longer duration (10 days) may be considered for severely ill patients or influenza a(h1n1) virus strains h275y substitution leads to resistance [10, 11] zanamavir nai intravenous zanamivir available through compassionate use program single dose of 600 mg administered intravenously [8] influenza a (h1n1) with both an h275y and an e119d or e119g na substitution lead to resistance to zanamivir [12, 13] . peramivir nai longer duration of 5 days in high-risk patients [9] . [8] . this antiviral therapy is most likely to provide the most benefits when initiated within the first 48 h after an infection occurs, so treatment should be initiated as soon as possible [8, 32] . some experts recommend higher dose of orally administered oseltamivir (e.g. 150 mg twice daily in adults with normal renal function) for the treatment of influenza infection in immunocompromised patients and those who are hospitalized and severely ill [8] . however, no clear evidence indicates that doubling the dose of oseltamivir is a more effective treatment than administering the normally prescribed dose in hospitalized patient, with or without severe illness [33, 34] . in a randomized trial of hospitalized patients with severe influenza, mortality rates were similar for patients who received oseltamivir at the double and standard doses [33] . however, 4 patients on the standard dose arm who were infected with influenza a (h1n1) virus without the h275y substitution at baseline acquired this substitution while on treatment. although no inferences can be made so far due to the small number of patients, using higher dose to prevent resistance and clinical failure in severely ill patients or immunocompromised patients still need to be determined in future studies [33] . in addition, a prospective study of adults hospitalized with influenza a and b infections treated with a single or double dose of oseltamivir twice daily demonstrated no differences between the groups in viral clearance, fever duration, oxygen supplementation, or hospitalization length [34] . patients receiving antiviral medications whose infections do not respond to treatment may have infections with antiviral-resistant influenza viruses. authors reported oseltamivir resistance, sometimes occurring within 1 week after treatment initiation, in immunocompromised patients with influenza a (h1n1) viral infections in the 2009 pandemic (pdm09) [35] . genotypic and phenotypic antiviral susceptibility testing are currently available to check the presence of mutations conferring resistance [36] . the more common emergence of resistance to oseltamivir in immunocompromised patients probably partly owes to prolonged viral shedding despite the use of antiviral therapy [37] . use of infection control measures is vital to reduce the risk of oseltamivir-resistant virus transmission in immunocompromised patients [32] . the pooled incidence rate of oseltamivir resistance for seasonal influenza a(h1n1) infections was estimated to be about 2.6% by a systematic review in 2009 [38] . however, in europe during the 2007-2008-winter season, rates of influenza a(h1n1) resistance were higher (up to 68%) [39] . authors reported that a specific substitution of the seasonal influenza a (h1n1) virus strains h275y (histidine-to-tyrosine substitution in neuraminidase), caused resistance in most of these cases [10, 11] . most circulating influenza a (h3n2) and influenza a (h1n1) pdm09 are still susceptible to oseltamivir and zanamivir. of the influenza a (h1n1) pdm09 infections tested during the 2013-2014 influenza season, 98.2% were susceptible to oseltamivir, and 100% were susceptible to zanamivir [40] . usually mild and limited to the first 2 days of treatment, nausea and vomiting are the most common reported toxic effects of oseltamivir, occurring in about 15% of recipients [41, 42] . zanamivir is administered via an inhaler in a dose of 2 inhalations (each inhalation of 5-mg) twice a day [27] . the chemoprophylaxis dosage of zanamivir is 10 mg (2 inhalations) administered once a day [8] . in randomized trials, this treatment shortened the duration of influenza symptoms by 1-3 days [43] [44] [45] [46] . use of intravenous (iv) zanamivir was evaluated in a recent phase iii clinical trial where the efficacy and safety of 300 mg or 600 mg of intravenous zanamivir twice daily were compared to 75 mg of oral oseltamivir twice daily for the treatment of hospitalized patients with influenza infections. the preliminary analysis showed no statistical difference in the time to clinical response (primary outcome variable) between iv zanamivir at 600 mg and oseltamivir, or between iv zanamivir at 600 mg or 300 mg [47] . it is currently available for compassionate use from its manufacturer via a us fda emergent investigational new drug application, and in a compassionate-use program in europe [48, 49] . zanamivir is currently the therapy of choice for oseltamivir-resistant influenza infections. however, the literature contains few cases of influenza virus with zanamivir resistance. infections with the h1n1 influenza strain possessing both an h275y na substitution (oseltamivir resistance) and an e119d (with aspartic acid replacing glutamic acid at position 119) or e119g (with glycine replacing glutamic acid at position 119) na substitution are resistant to zanamivir [12, 13] . the main adverse reactions to zanamivir are related to bronchospasm. use of inhalation powder to treat influenza infection is not recommended for patients with underlying airway issues (i.e. chronic obstructive pulmonary disease, asthma [8] . also, as it contains a lactose carrier, it can clog ventilator tubing nebulizers and mechanical ventilators [50] . peramivir is active against influenza a and b and was approved by the fda in 2014 for treating uncomplicated influenza infections in adults [51] . it is the first nai approved for iv use and is administered as a single iv dose of 600 mg because of its strong and prolonged affinity for the na in influenza virus. peramivir use should be considered for patients who are unable to tolerate oral or enteric drugs [51, 52] . use of a single dose of 600 mg of peramivir administered intravenously, alleviated influenza symptoms an average of 21 h sooner and fever approximately 12 h sooner than in patients given a placebo in a published report [52] . a study of patients at high risk for complications (including patients with diabetes, with chronic respiratory disease, or receiving immunosuppressive therapy), given peramivir for up to 5 days demonstrated shorter durations of illness than in patients given a single dose, and hence a longer duration of treatment for immunocompromised patients is suggested [9] . also, an open-label, randomized study of high-risk patients during the 2009 influenza a (h1n1) pdm09 demonstrated that use of peramivir (300 mg twice daily or 600 mg once daily) for 5-10 days reduced viral shedding and produced clinical improvement [53] . authors have reported cross-resistance of oseltamivir and peramivir in immunocompromised patients infected with influenza a (h1n1) virus containing the h275y variant [54] [55] [56] . therefore, patients infected with influenza a virus with a suspected or documented h275y substitution should not receive peramivir [14] . diarrhea is the most common reported adverse effect of peramivir [52] . more serious reactions associated with the central nervous system have included delirium and abnormal behavior leading to injury in patients with influenza who received oseltamivir or peramivir. primarily reported among children, these neurological events often began abruptly and resolved rapidly [57, 58] . the contribution of treatment with neuraminidase inhibitors to these events has yet to be established [59] , as some of these adverse events may have been related to the influenza infection rather than its treatment. authors have frequently reported neuropsychiatric symptoms in children with influenza infections; these symptoms were not always associated with the treatment with neuraminidase inhibitors [60-64]. das181 (ansun biopharma, san diego, ca, usa) is a recombinant fusion protein with a sialidase derived from actinomycoses viscosus that cleaves sialic acid receptors in host cells ( figure 2 ) [65] . this protein binds to cells and efficiently removes cell-surface sialic acid residues from respiratory epithelium, inhibiting viral infection. considering that das181 targets the host cells rather than the virus, it is less likely than virus-targeted drugs to induce treatment resistance. das 181 is administered via inhalation and has exhibited preclinical activity against numerous strains of influenza (a and b) and parainfluenza viruses (pivs) [65, 66] . in a phase ii double-blind, placebo-controlled clinical trial assessing influenza viral load and patient safety in otherwise healthy influenza-infected participants, an inhaled das181 dosage of 20 mg per day reduced viral loads and viral shedding in the multiple-dose group more than in patients taking a placebo as measured using quantitative polymerase chain reaction (p < 0.05); however, there was no difference in alleviation of flu-like symptoms between the placebo and the treatment arms. overall, das181 was well tolerated for up to 7 days when administered via daily inhalation for 5-7 days except for thrombocytopenia and liver test abnormalities in some instances. favipiravir (t705; toyama chemical, tokyo, japan) is an investigational antiviral drug that functions as a nucleotide analog and inhibitor of the viral rna polymerase of influenza. favipiravir is active against a broad range of influenza a, b, and c viruses, including highly pathogenic avian a (h5n1) and novel avian a (h7n9) viruses [67] , as well as influenza viruses resistant to treatment with nais or m2 inhibitors [68] . studies of preclinical cellular and mice models have demonstrated synergy of favipiravir with oseltamivir [69, 70] . this drug is currently being tested in phase iii clinical trials in the usa, europe, and latin america [69, 70] . laninamivir (cs-8958; biota pharmaceuticals, alpharetta, ga, usa) is a long-acting nai administered via a dry-powder inhaler. a phase iii randomized controlled trial demonstrated the superiority of a single inhalation dose of laninamivir octanoate to a 5-day course of oral oseltamivir in adults with seasonal influenza [71] . the drug is potentially effective against oseltamivir-resistant viruses and is currently available in japan. laninamivir has been demonstrated to be effective in reducing transmission of influenza infection from patients to household contacts. in a randomized trial, household contact of patient with influenza infection were randomly assigned to receive a single dose of laninamivir, 2 doses of laninamivir given daily for 2 days, or a placebo. family members in the laninamivir groups were less likely to develop clinical influenza as compared to the placebo group [72] . daiichi sankyo company, ltd. in japan plans to study the drug for the prevention of influenza, in single inhalation dose, in both adult and children [73] . jnj-63623872 (vx-787; janssen pharma, titusville, usa) is a nonnucleoside inhibitor targeting pb2, an influenza rna polymerase protein, inhibiting production of viral mrna, and preventing cell death [74] . it demonstrated activity against all influenza a strains tested in vitro. human studies have demonstrated significant decrease in virus shedding, when administered at a loading dose of 900 or 1200 mg on the first day followed by 600 mg once daily for 4 days [75] . a phase iib trial evaluating the dosing and frequency of the drug in healthy patients with uncomplicated influenza infection is currently under way [76] . nitazoxanide (nt-300; romark laboratories, florida, usa), an antiparasitic agent, appears to inhibit the maturation of influenza virus ha [77] . in a phase iib/iii trial, the treatment with nitazoxanide 600 mg twice daily for 5 days was associated with reduction in symptoms duration and viral titers among patients with acute uncomplicated influenza infection [15] . nitazoxanide has also shown synergistic effects in vitro with nais [78] and a current phase iii trial to investigate the efficacy of this synergism has been completed and results are awaited [79] . medi8852 (astrazeneca, gaithersburg, maryland, usa) is a monoclonal antibody targeting the highly conserved epitope in the ha stalk of influenza a virus [80] . it is currently being evaluated in a phase ib/iia clinical trial for safety and efficacy of a single intravenous dose in combination with oseltamivir, and as a monotherapy in adult patients with confirmed acute, uncomplicated influenza a infections [81] . vis410 (visterra, inc., cambridge, ma, usa) is a neutralizing human igg1 anti-ha antibody, which binds to a conserved region of the ha stalk of the influenza virus [82] . in mice, it resulted in 100% protection from influenza infection when administered prophylactically [83] . coinfections with bacterial pathogens and influenza infection may lead to significant morbidity and mortality. bacterial coinfection is associated with an increase in disease severity, hospital admission and even mortality, with streptococcus pneumoniae and staphylococcus aureus been the most common pathogens in such setting followed by, haemophilus influenzae, and group a streptococci [84] . a recent meta-analysis by klein et al. (2016) noted that older age, a higher apache ii (acute physiology and chronic health evaluation ii) score, diabetes mellitus, and sepsis were risk factors predisposing to coinfections [84] . the american college on immunization practices (acip) recommends simultaneous antiviral and antibiotic treatment for severely ill patients with influenza infections [8] . consistent with the acip guidelines, the infectious disease society of america (idsa) guidelines recommend appropriate use of diagnostic tests as guidance for targeted antibacterial therapy for hospitalized patients. recommended antibacterial therapy includes cefotaxime, ceftriaxone, and respiratory fluoroquinolones. treatment with vancomycin, linezolid, or other agents directed against methicillin-resistant staphylococcus aureus (mrsa) is recommended for patients with confirmed or a compatible clinical presentation of mrsa infection (i.e. shock and necrotizing pneumonia) [85] . respiratory syncytial virus (rsv), an enveloped, singlestranded rna virus of the family paramyxoviridae, frequently causes seasonal upper respiratory viral infections in infants and young children. symptomatic rsv reinfections in immunocompetent adults often consist of urtis lasting 2-5 days. in immunocompromised patients such as hsct and solid-organ transplant recipients, rsv infections may progress to severe and life-threatening lrtis [86] . investigators at the university of texas md anderson cancer center developed an immunodeficiency scoring index for rsv that accounts for major risk factors that identify hsct recipients who are at high risk for progression of rsv infection to an lrti and rsv-associated mortality [87] . age, neutropenia, lymphocytopenia, graft-versus-host disease, use of myeloablative conditioning regimens, use of corticosteroids, a recent hsct, and pre-engraftment are the main risk factors that are weighed in this index to categorize patients into prognostic risk groups [67] : low (0-2), moderate (3) (4) (5) (6) , and high (7) (8) (9) (10) (11) (12) risk. the authors reported a statistically significant trend of higher incidence of lrti-and rsv infection-associated mortality as the risk increased from low to moderate to high (p < 0.001). patients in the high-risk group demonstrated greatest benefit of ribavirin-based therapy at the urti stage and were at the highest risk for progression to lrti and death in the absence of antiviral therapy. we suggest using the immunodeficiency scoring index for rsv to identify high-risk patients who would benefit from treatment with aerosolized ribavirin. as seen in hsct recipients, researchers noted an association between a low lymphocyte count (mean, 580 cells/mm 3 ) and rsv infection progression to an lrti in solid-organ transplant recipients with lung transplant recipients having the highest risk of adverse outcomes [88] . ribavirin is a nucleoside analog that resembles guanosine. as a monophosphate, ribavirin inhibits the dehydrogenase enzyme, which is essential for the synthesis of guanosine triphosphate, and reduces the cellular deposits of guanidine necessary for viral growth. it inhibits the initiation and elongation of rna fragments resulting in inhibition of viral protein synthesis (figure 2 ) [89] . aerosolized ribavirin is the only fda-approved treatment of severe rsv-lrtis in hospitalized infants and young children with underlying compromising conditions (prematurity, cardiopulmonary disease, or immunosuppression) [90] . rsv infections markedly increase morbidity and mortality rates in hsct recipients. ribavirin-based antiviral therapy is recommended by european guidelines for leukemia patients and hsct recipients at high risk of complications [16, 91] . in a systematic review of the literature by shah et al. [92] and based mainly on retrospective studies, any form of ribavirinbased therapy (alone or in combination with immunomodulators) prevented urtis from progressing to lrtis (from 45% to 16%) and improved mortality rates (from 70% to 35%) when compared to no therapy in adult hsct recipients [92] . whether the benefits of aerosolized ribavirin versus the oral form justify its use in immunocompromised patients remain subject of controversy, especially given the recent drastic increase in the cost of the aerosolized form [93] . researchers have systematically reviewed the use of oral ribavirin to treat various respiratory viral infections, including rsv infections [94] . the authors concluded that mortality rates were highly variable and often dependent on the underlying severity of illness rather than the effects of oral ribavirin; however, there were not randomized or control studies available for evaluation [94] . in 2004, khanna et al. [95] reported that oral ribavirin had a good safety profile in 34 rsv-infected patients with upper or lower respiratory tract infection but could not draw a strong conclusion regarding its efficacy. the doses recommended in the european conference on infections in leukaemia (ecil-4) guidelines included a loading dose of 600 mg followed by 200 mg every 8 h the first day, 400 mg every 8 h the second day, and then escalation daily to a maximum of 30 mg/kg/day [16] . the iv formulation of ribavirin has been beneficial in some cases of rsv infection, but further trials are needed [96, 97] . various other therapies such as, intravenous immunoglobulin (ivig), rsv hyperimmunoglobulin, and palivizumab (a monoclonal rsv igg), have been used for treatment and prevention of rsv infections in immunocompromised patients with mixed results. early studies demonstrated that ribavirin in combination with rsv ivig (respigam; medimmune, gaithersburg, md, usa), an hyperimmune globulin preparation with high concentrations of rsv-neutralizing antibodies, offered a mortality advantage over ribavirin alone in rsv-infected pediatric hsct recipients with lrtis [98] . however, production of rsv ivig has since then been discontinued because of the introduction of alternatives such as palivizumab, an engineered anti-rsv monoclonal antibody. palivizumab is currently approved for prophylaxis for rsv infection in a select group of high-risk infants with bronchopulmonary dysplasia, infants with a history of premature birth (â�¤35-week gestational age), and children younger than 24 months with hemodynamically significant congenital heart disease during the rsv infection season [99] . the american academy of pediatrics recommends a palivizumab dose of 15-mg/kg body weight administered monthly throughout the rsv infection season (first dose administered prior to commencement of the season and a maximum of 5 doses per season) [99] . kassis et al. demonstrated the utility of palivizumab for prophylaxis in a hsct unit following an rsv infection outbreak. palivizumab was useful in preventing rsv infection in 16 rsv-negative patients considered to be at high risk for complications from rsv infection when combined with strict infection-control measures [100] . in contrast, palivizumab failed to demonstrate any impact on progression to lrti or mortality in a case series of 40 allogeneic hsct recipients infected with rsv [101] . given the questionable efficacy and high cost of palivizumab, mainly for adult patients, routine use of it is not encouraged in the adult immunocompromised population [102] . in adult hsct recipients with rsv pneumonia, uncontrolled studies suggested that use of combination therapy with ribavirin and ivig improved survival [103, 104] . additional studies of rsv-infected lung transplant recipients demonstrated that combined treatment with ribavirin (nebulized or iv) with ivig and/or corticosteroids reduced mortality rates, length of mechanical ventilation, and incidence of bronchiolitis obliterans [105] . although combined use of ribavirin and ivig has not been supported by a randomized trial, this expensive treatment is reserved for select patients with rsv-related lrtis and severe immune deficiency [103, 104] . aln-rsv01 (alnylam pharmaceuticals, cambridge, ma, usa) is small-interfering rna (sirna) that inhibits rsv replication by interrupting synthesis of the viral nucleocapsid protein, and treatment with this compound has demonstrated promising results in phase ii clinical trials [106] . rna interference is a natural process and sirnas induce sequence-specific degradation of mrna and thus reduce expression of the corresponding protein [106] . in a randomized, double-blind, placebocontrolled trial, researchers administered prophylactic aln-rsv01 as a nasal spray before experimental inoculation in healthy adults wild-type for rsv and observed a 38% reduction in the number of infections [106] . in a phase iia randomized, double-blind, placebo-controlled trial of adult lung transplant recipients with confirmed rsv urtis, use of aerosolized aln-rsv01 (0.6 mg/kg) daily for 3 days significantly reduced mean cumulative daily symptom scores (p = 0.035) and the incidence of progressive bronchiolitis obliterans syndrome by day 90 more so than in patients given a placebo (6% vs. 50 %; p = 0.027) [107] . also, a recent phase iib trial with lung transplant recipients demonstrated a trend of decreasing new or progressive bronchiolitis obliterans (bos) incidence (14% vs. 30%; p = 0.058) at 180 days. the treatment effect was enhanced with initiation of aln-rsv01 use fewer than 5 days after symptom onset [108] . whether further development of this compound would be pursued remains unknown at the present time. mdt-637 (microdose therapeutx, monmouth junction, nj, usa and gilead sciences, foster city, ca, usa) and the gs-5806 (gilead sciences, foster city, ca, usa) are both antiviral fusion inhibitors. oral gs-5806 has shown safety and tolerability in healthy adults [109] . currently, two phase iib trials are underway to evaluate the antiviral effects, pharmacokinetics, safety, and tolerability of gs-5806 in hsct recipients with either rsv uri or lrti [110, 111] . mdt-637 is delivered as a dry-inhalation powder and has been evaluated in a phase ii trial to assess safety and tolerability in healthy adults [112] . al-8176 (alios, south san francisco, ca, usa) is a nucleoside inhibitor of the l-protein [113] and has demonstrated efficacy in human challenge studies [114, 115] . l-protein is an rna-dependent rna polymerase of rsv, and its inhibition impact future viral replication [113] . in a randomized, double-blind, placebo-controlled phase ii challenge study conducted in healthy adult volunteers who were infected intranasally with rsv, al-8176 was well tolerated and demonstrated significant reduction in rsv viral loads (p < 0.0002) and improvement in symptom scores (p < 0.02) when compared to placebo [114, 115] . polyclonal high-titers rsv immunoglobulin (ri-001; adma biologics, inc., hackensack, nj, usa) is being tested in patients who are immunocompromised to prevent progression of urtis to lrtis. preliminary results are pending [116] . motavizumab is a newly developed monoclonal antibody targeting a highly conserved antigenic site on the fusion glycoprotein of rsv. it had antiviral effects in hospitalized children but was not superior to palivizumab in seasonal rsv prophylaxis in preterm infants with chronic lung disease of prematurity at-risk for rsv related lrti, hospitalization or death [117] . in 2010, fda antiviral drugs advisory committee declined the request for licensure of motavizumab. the concerns raised included the lack of additional benefits of motavizumab over palivizumab and the additional risk of cutaneous hypersensitivity reactions [118] . parainfluenza virus (piv) is a single-stranded, enveloped rna paramyxovirus comprising 4 antigens that share serotypes, although most clinical piv infections are caused by types 1, 2, and 3. in the general population, most clinical piv infections are caused by piv-3 followed by piv-1 and piv-2 [119] . although piv infections often occur year round, peak seasonal activity reportedly occurs from late september to december for piv-1 and during the spring and summer months for piv-3 [119] . piv most commonly affects the upper respiratory tract after an incubation period of 1-4 days and is commonly associated with urtis in children. in immunocompromised patients, authors described progression to lrti in about 37% of hsct recipients and piv-infected patients with hematological malignancies [120] . the risk factors for progression from piv-urti to piv-lrti include lymphocytopenia, neutropenia at the onset of infection, use of corticosteroids during piv-urti, and respiratory coinfections [120] . risk factors for piv-related mortality include lymphocytopenia, young age (<2 years), refractory or relapsed underlying hematological malignancies, an acute physiology and chronic health evaluation ii score greater than 15, respiratory coinfections, and steroid use at infection onset [120] . no antiviral agents are licensed to treat piv, so its management is limited to supportive care. in some instances, physicians have used oral or aerosolized ribavirin with or without ivigs for the treatment of piv lrti in immunocompromised patients with various outcomes [121] . new antiviral agents and vaccines in the pipeline may change the paradigm of piv infection management, particularly in immunocompromised patients. although, as described above, clinical providers have used oral and aerosolized ribavirin to treat piv [122] , the available data on their use for this infection remain controversial. two recent systematic reviews on hsct recipients and hematological malignancy patients demonstrated that ribavirin was not significantly more effective at preventing the progression of urti to lrti or piv-associated mortality than was supportive care alone [120, 123] . also, in lung transplant recipients with piv infections, use of oral ribavirin for 14 days at 15-20 mg/kg/day in 2 divided doses (dose length) was associated with some benefits, including a lower rate of bronchiolitis obliterans syndrome within 6 months after development of the infection than that in a non-ribavirin group (5% vs. 24%; p = 0.02) [124] . given, the lack of clear evidence of a positive outcome in piv-infected patients as well as the absence of control studies, justified recommendation for the use of ribavirin for the treatment of piv in immunocompromised patients cannot be made. as described above, das181 is a novel sialidase fusion protein with activity against piv in vivo and in vitro because it effectively cleaves sialic acid from respiratory epithelial cells, preventing piv entry into the cells (figure 2 ) [125] . das 181 have been administered on a compassionate-use basis for severe piv infections in immunocompromised patients, with apparent clinical benefits and antiviral effects [126] . in a case series, 4 pediatric hsct recipients with piv detected in respiratory specimens (2 from the upper respiratory tract and 2 from the lower respiratory tract) received inhaled das181 for 5-10 days. oxygen requirements and respiratory rates improved in all 4 patients, and their viral loads decreased within 1 week after therapy initiation [127] . in a similar case series, 16 hsct recipients received das181 daily to treat piv infections (14 lrtis and 2 urtis). of the 16 patient, 9 had complete clinical response, and 4 patients had a partial response to das181 therapy. of 7 patients with virological and spirometric data, 5 had reduction in piv viral load in nasopharyngeal secretions and 4 had improved forced expiratory volumes by the end of treatment [128] . in an ongoing phase ii double-blind, placebocontrolled trial, investigators are examining the effects of das181 in immunocompromised patients with piv-related pneumonia [129] . a recent report described the use of das181 in 13 hsct recipients: 56% of them had responses to therapy, and 24% had partial responses. they also had greater than a 1-log reduction in piv viral load [130] . bcx2798 and bcx2855 (biocryst pharmaceuticals, inc., birmingham, al, usa) are new antiviral hemagglutinin neuraminidase inhibitors and have been evaluated in mouse models of infection with a virus similar to piv, recombinant sendai virus [131] . bcx2798 and bcx2855 have demonstrated antiviral activity against piv-3 by markedly reducing pulmonary viral titers and mortality rates in rats when given intranasal within 24 h after development of infection [132] . human studies of these two inhibitors have yet to be undertaken. human rhinoviruses (hrvs) are positive-sense, single-stranded rna viruses with icosahedral symmetry. they are characterized into three genetically distinct groups designated a, b, and c within the genus enterovirus and family picornaviridae. the viral capsid that encases the rna genome is made up of four proteins: vp1, vp2, vp3, and vp4. the remaining nonstructural proteins are involved in viral genome replication and assembly [133] . hrv infections are responsible for more than one half of cold-like illnesses and cost billions of dollars annually in medical visits and missed days of work in the usa [134] . peak incidence occurs in the early fall, with a smaller peak in the spring [135] . both peak incidences are associated with urti, otitis media, and sinusitis [133] . a recent study of patients going to the emergency room with influenza-like illnesses who also had hematological malignancies demonstrated that 40% of the patients (110/272) presented with hrv infections. researchers found that the severity of hrv infection in these patients was similar to that of h1n1 influenza in the 2009 pandemic. nearly 40% of patients with hrv-associated respiratory symptoms were admitted to the hospital, 29% had lrtis, and 11% needed intensive care unit admission [136] . other studies, including those with hsct recipients, have replicated these results [137, 138] . markers for increased immunosuppression and illness severity in patients with hrv infections, including neutropenia (absolute neutrophil count â�¤500 cells/âµl), hypoalbuminemia (serum albumin level â�¤3.2 mg/dl), and infections with a respiratory co-pathogen(s) were associated with progression to hrv-related pneumonia [138] . in contrast, parody et al. [139] described a much lower rate of progression to lrti (13%) in a similar patient population. use of a different case definition for hrv infection may explain the disparity in the prevalence of lrtis in these two reports. chronic hrv infection has occurred in lung transplant patients [140] . furthermore, in a study of 36 adult lung transplant recipients, 13% of all bronchoalveolar fluid specimens obtained from 15 (42%) symptomatic patients over a 2-year period were positive for hrv [141] . currently, treatment of hrv infection consists of supportive care. antiviral medications for hrv are under investigation. the viral capsid was the initial viral protein targeted in the development of drugs to inhibit viral replication. these drugs work by binding to the hydrophobic pocket of the viral capsid, resulting in a conformational change, increasing the stability of the virion and interfering with its ability to interact with the cellular receptor [142] . vapendavir (aviragen therapeutics, alpharetta, ga, usa) is an oral agent that binds to the hrv vp1 capsid protein and prevents the release of viral rna into the target cells. vapendavir exhibits antiviral activity against hrv-a and hrv-b serotypes; however, activity against hrv-c is not yet known. a phase ii randomized, double-blind, placebo-controlled study of asthmatic adults with hrv urtis showed lower severity scores for cold symptoms, greater mean reductions in asthma scores, and higher evening peak expiratory flow in those given vapendavir than placebo [143] . pleconaril (viropharma, exton, pa, usa) was the first developed capsid-binding anti-hrv agent. two phase iii multicenter studies in the usa and canada randomized 2,096 healthy subjects with self-diagnosed colds into groups receiving pleconaril at 400 mg orally twice daily or placebo for 5 days. in the primary-efficacy population, which consisted of 1,363 subjects with hrv rna detected in nasal secretions, pleconariltreated subjects experienced a 1-day reduction in the mean duration of illness compared to the placebo group (7.3 days versus 6.3 days; p = 0.001) [144] . in another study, researchers found an association between hrv susceptibility to pleconaril and clinical outcomes [145] . the fda declined licensing of pleconaril owing to concerns of development of resistant virus strains. additionally, interactions among cytochrome p-450 3a, hormonal contraception, and antiretroviral therapy for human immunodeficiency viral infection may reduce the effectiveness of pleconaril [146] . rupintrivir (agouron pharmaceuticals, inc., san diego, ca, usa) is an in vitro 3c protease inhibitor that acts against many hrvs and enteroviruses. rupintrivir reduced viral loads and respiratory symptoms in healthy volunteers with experimentally induced rhinovirus colds and was well tolerated by the participants [147] . however, in trials of patients with natural infections, rupintrivir failed to reduce viral loads or symptom severity [148] . inhaled interferon-beta (sng001, synairgen plc, southampton, england) was tested in a phase ii, placebo controlled randomized trial of adult asthmatics receiving inhaled corticosteroids and with a history of deterioration with colds, and was associated with significant improvement in asthma symptoms, 65% fewer moderate exacerbations, improved morning peak expiratory flow rates, and reduced use of relief bronchodilators [149] . human metapneumovirus (hmpv) is an enveloped, negativesense, single-stranded rna virus. it is the first human member of the metapneumovirus genus in the pneumovirinae subfamily within the paramyxoviridae family. first identified in the netherlands in 2001, serological studies of antibodies against hmpv indicated that the virus has circulated in humans for at least 50 years [150] . phylogenetic analysis has identified two genotypes of hmpv: hmpv a and hmpv b [151] . hmpv uses a fusion mechanism to penetrate target cells. the fusion process consists of insertion of the hydrophobic fusion peptide into the target cell membrane and refolding of the f protein. this step requires the interaction of two specific domains: heptad repeats a and b [152] . investigators have studied this process for development of it as a potential antiviral treatment. hmpv causes respiratory infections and has a seasonal distribution comparable with those of influenza and rsv infections [153] . although immunocompromised patients acquire hmpv infections at the same frequency as immunocompetent individuals, they are at higher risk for severe infections. this higher risk likely can be attributed to poor viral clearance [153, 154] . a recent systematic review in hsct recipients and hematologic malignancy patients estimated the incidence of progression of hmpv-urti to lrti at 34% and an associated mortality rate of 6% [154] . factors associated with this progression in hsct recipients include early onset of infection after transplantation, steroid use, and a low lymphocyte count [155] . to date, treatment of hmpv infections has been mainly supportive. researchers have investigated several treatment regimens. standard immunoglobulin preparations have inhibited replication of hmpv in vitro [156] , and approaches such as use of selective immunoglobulins and fusion inhibitors have demonstrated antiviral activity in vitro and in animal studies. administration of oral or aerosolized ribavirin with or without polyclonal ivigs has been advocated for the treatment of severe hmpv infections and is currently used in some centers for highrisk patients [156] [157] [158] [159] [160] , although most data are still anecdotal. fusion inhibitors target the initial steps of viral fusion and penetration into the human cell. fusion inhibitors with sequence similarity with the hra and hrb domains of the viral fusion protein have demonstrated important role in viral inhibition. balb/c mice inoculated with lethal intranasal hmpv challenge were completely protected from clinical symptoms and mortality if they simultaneously received the hra2 peptide [152] . hr-1 peptides also have demonstrated effectiveness as viral inhibitors [161] . researchers developed mab 338 (medimmune, gaithersburg, md, usa) to target hmpv fusion proteins. it appeared to effectively neutralize hmpv in golden syrian hamster models and reduce the pulmonary viral titers, thereby limiting severe acute manifestations and bronchial hyper-reactivity [162] . a human monoclonal antibody fragment (human fab ds7) with biological activity against the fusion protein of hmpv demonstrated prophylactic and therapeutic potential against severe hmpv infections when tested in cotton rats [163] . human coronavirus (hcov) is a single-stranded, enveloped rna virus belonging to the family coronaviridae. in temperate climates, hcov infection is transmitted primarily during the winter and is a well-recognized cause of urtis during the respiratory viral season [164] . usually mild in immunocompetent hosts, hcov infection in immunocompromised populations may progress to lrti [16] . emerging hcovs, such as severe acute respiratory syndrome-associated hcov in 2002-2003 and the more recently identified middle east respiratory syndrome in 2012-2013, have prompted a further impetus to develop therapeutics against this infection because current antiviral agents are lacking and treatment of it remains palliative. discovery and in vitro evaluation of hcov therapy is ongoing, including investigation of entry inhibitors, human monoclonal antibodies, and proteosome inhibitors [165] [166] [167] [168] . respiratory viral infections continue to be major clinical problems in immunocompromised patients. high clinical suspicion and the use of rapid diagnostic tests remain crucial, as early treatment is associated with improved outcomes and reduced transmission. several advances in the prevention and treatments of influenza infection have occurred in recent decades. inadequate efficacy of the influenza vaccine as well as the emergence of antiviral resistance, which appears to occur more commonly in immunocompromised patients than in healthy host, underline the difficulties in management of respiratory infections in immunocompromised individuals. rsv and piv infections continue to be associated with high morbidity and mortality, and further advancements in prevention of and therapy for respiratory viral infections are needed. the impact of rhinovirus, coronavirus, and metapneumovirus infection in patients with compromised immune systems is becoming evident as new, widely available molecular testing improves the recognition of these viral infections. over the past decade, important diagnostic advances, specifically, the use of rapid molecular testing has helped close the gap between clinical scenarios and pathogen identification and enhanced early diagnosis of viral infections and understanding of the role of prolonged shedding and viral loads. respiratory viral infections can be complicated for both clinicians and immunocompromised patients. future studies that identify and validate scoring systems to ascertaining patients at highest risk for complications of respiratory viral infections including lrti, are of utmost importance. also, identification of long-term complications after respiratory viral infections in immunocompromised patients and devising interventions for prevention will be of the utmost value. last, advancement in novel antiviral therapeutics with high-resistance thresholds and effective immunization for preventable infections in immunocompromised patients are needed. to curtail the impact of respiratory viruses on our immunocompromised patients, we should focus on prevention of exposure and progression to worse outcomes. multiple interventional modalities should be studied from stimulation of the innate immune system, response to immunizations, to new antiviral therapies, to avert infection and progression to lower tract respiratory infections. one of the main challenges for immunocompromised patients is the ability to clear infections with subsequent complications associated with worsening infections, prolonged shedding, risk of resistance and coinfections. treatment targeting not only viral replication, but also the immune response to these infections may offer better outcomes. last, understanding the role of the microbiome and virome, and its implications on transmission as well as development of infection will be key for development of new strategies. â�¢ respiratory viruses are the most frequent cause of respiratory infections in immunocompromised patients, and are associated with higher rate of progression to pneumonia, respiratory failure and death. â�¢ high prevalence of m2 inhibitors resistance detected in influenza a (h3n2) and 2009 h1n1 virus strains preclude their use for prophylaxis or empiric treatment of seasonal influenza â�¢ neuraminidase inhibitors are the first line agents for treatment of influenza and treatment is most likely to provide the most benefit when initiated within the first 48 h of illness â�¢ zanamivir is currently the therapy of choice for the treatment of oseltamivir-resistant influenza infection â�¢ an immunodeficiency scoring index for rsv, that accounts for the number of risk factors, can be used to identify hsct recipients who are at high risk for progression to rsv lrti and rsv associated mortality â�¢ ribavirin-based therapy (alone or in combination with immunomodulators) can be effective in preventing progression from urti to lrti and may improve mortality in highly immunosuppressed adult hsct recipients â�¢ the safety and efficacy of das181 in immunocompromised patients with piv pneumonia, is currently being studied in an ongoing phase 2 double-blind, placebo-controlled trial. â�¢ vapendavir binds to the hrv capsid protein, preventing the release of viral rna into the target cells and has demonstrated favorable results in asthmatic adults with hrv urtis. â�¢ antiviral agents for hmpv and hcov are still under study in vitro or in animal models this paper was not funded. ej ariza-heredia has received research grants from oxford immunotec. rf chemaly has received research grants from gilead, gsk, and ansun pharmaceuticals and honoraria from adma biologics and gilead. the authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. papers of special note have been highlighted as either of interest (â�¢) or of considerable interest (â�¢â�¢) to readers respiratory infections in immunocompromised patients â�¢ the paper describes the immune defect in immunocompromised patients and their increased susceptibility to respiratory infections viral infections in immunocompromised patients the clinical features and outcome of 2009 h1n1 influenza infection in allo-sct patients: a british society of blood and marrow transplantation study increased mortality of solid organ transplant recipients with h1n1 infection: a single center experience changing epidemiology of respiratory viral infections in hematopoietic cell transplant recipients and solid organ transplant recipients the challenge of respiratory virus infections in hematopoietic cell transplant recipients airflow decline after myeloablative allogeneic hematopoietic cell transplantation: the role of community respiratory viruses antiviral agents for the treatment and chemoprophylaxis of influenza -recommendations of the advisory committee on immunization practices (acip) this article is a comprehensive review of treatment of influenza infection intravenous peramivir for treatment of influenza a and b virus infection in high-risk patients emergence of resistance to oseltamivir among influenza a(h1n1) viruses in europe global update on the susceptibility of human influenza viruses to neuraminidase inhibitors e119d neuraminidase mutation conferring pan-resistance to neuraminidase inhibitors in an a(h1n1)pdm09 isolate from a stem-cell transplant recipient emergence of multidrug-resistant influenza a(h1n1)pdm09 virus variants in an immunocompromised child treated with oseltamivir and zanamivir characterization of neuraminidase inhibitor-resistant influenza a (h1n1) pdm09 viruses isolated in four seasons during pandemic and post-pandemic periods in japan. influenza other respir viruses effect of nitazoxanide in adults and adolescents with acute uncomplicated influenza: a double-blind, randomised, placebo-controlled, phase 2b/3 trial ecil-4): guidelines for diagnosis and treatment of human respiratory syncytial virus, parainfluenza virus, metapneumovirus, rhinovirus, and coronavirus this article provides a comprehensive summary of the treatment options for respiratory viral infection in immunocompromised patients characterization of a novel influenza virus in cattle and swine: proposal for a new genus in the orthomyxoviridae family a revision of the system of nomenclature for influenza viruses: a who memorandum influenza activity -united states, 2015-16 season and composition of the 2016-17 influenza vaccine respiratory viral infections in hematopoietic stem cell and solid organ transplant recipients influenza, including the novel h1n1, in organ transplant patients respiratory viral infections in adults with hematologic malignancies and human stem cell transplantation recipients: a retrospective study at a major cancer center influenza infections after hematopoietic stem cell transplantation: risk factors, mortality, and the effect of antiviral therapy respiratory virus infections in stem cell transplant patients: the european experience differences in clinical outcomes after 2009 influenza a/h1n1 and seasonal influenza among hematopoietic cell transplant recipients antivirals and resistance: influenza virus effect of m1 protein and low ph on nuclear transport of influenza virus ribonucleoproteins adamantane resistance in influenza a incidence of adamantane resistance among influenza a (h3n2) viruses isolated worldwide from 1994 to 2005: a cause for concern adamantane resistance among influenza a viruses isolated early during the 2005-2006 influenza season in the united states update: influenza activity -united states, 2009-10 season h1n1 influenza in healthcare settings, including protection of healthcare personnel effect of double dose oseltamivir on clinical and virological outcomes in children and adults admitted to hospital with severe influenza: double blind randomised controlled trial a prospective intervention study on higher-dose oseltamivir treatment in adults hospitalized with influenza a and b infections oseltamivir-resistant novel influenza a (h1n1) virus infection in two immunosuppressed patients-seattle antiviral resistance in influenza viruses: laboratory testing recovery of drug-resistant influenza virus from immunocompromised patients: a case series systematic review of influenza resistance to the neuraminidase inhibitors oseltamivir-resistant influenza virus a (h1n1), europe, 2007-08 season efficacy and safety of the oral neuraminidase inhibitor oseltamivir in treating acute influenza: a randomized controlled trial. us oral neuraminidase study group efficacy and safety of oseltamivir in treatment of acute influenza: a randomised controlled trial effectiveness of neuraminidase inhibitors in treatment and prevention of influenza a and b: systematic review and meta-analyses of randomised controlled trials efficacy and safety of the neuraminidase inhibitor zanamivirin the treatment of influenza a and b virus infections efficacy and safety of the neuraminidase inhibitor zanamivir in the treatment of influenzavirus infections. gg167 influenza study group randomised trial of efficacy and safety of inhaled zanamivir in treatment of influenza a and b virus infections. the mist (management of influenza in the southern hemisphere trialists) study group a phase iii international, randomized, double-blind, double-dummy study to evaluate the efficacy and safety of 300mg or 600mg of intravenous zanamivir twice daily compared to 75mg of oral oseltamivir twice daily in the treatment of hospitalized adults and adolescents with influenza use conditions of use, conditions for distribution and patients targeted and conditions for safety monitoring addressed to member states for iv zanamivir available for compassionate use clinical experience with intravenous zanamivir under an emergency ind program in the united states relenza (zanamivir) inhalation powder. united states food and drug administration fda approves rapivab to treat flu infection. united states food and drug administration efficacy and safety of intravenous peramivir for treatment of seasonal influenza virus infection intravenous peramivir for treatment of influenza in hospitalized patients early emergence of an h275y mutation in a hematopoietic cell transplant recipient treated with intravenous peramivir oseltamivir-resistant 2009 h1n1 influenza pneumonia during therapy in a renal transplant recipient rapid selection of oseltamivir-and peramivir-resistant pandemic h1n1 virus during therapy in 2 immunocompromised hosts neuropsychiatric adverse effects of oseltamivir in the fda adverse event reporting system efficacy, safety, and pharmacokinetics of intravenous peramivir in children with 2009 pandemic h1n1 influenza a virus infection the association between oseltamivir use and adverse neuropsychiatric outcomes among tricare beneficiaries, ages 1 through 21 years diagnosed with influenza adolescent jump case in japan associated with influenza but not oseltamivir clinical characteristics of children with abnormal behaviors associated with influenza infection clinical characteristics of children with influenza a virus infection requiring hospitalization influenza-associated central nervous system dysfunction in taiwanese children: clinical characteristics and outcomes with and without administration of oseltamivir oseltamivir treatment for children showing abnormal behavior during influenza virus infection a phase ii study of das181, a novel host directed antiviral for the treatment of influenza infection phase 1 clinical trials of das181, an inhaled sialidase, in healthy adults t-705 (favipiravir) activity against lethal h5n1 influenza a viruses in vitro antiviral activity of favipiravir (t-705) against drug-resistant influenza and 2009 a(h1n1) viruses synergistic combinations of favipiravir and oseltamivir against wild-type pandemic and oseltamivir-resistant influenza a virus infections in mice in vitro activity of favipiravir and neuraminidase inhibitor combinations against oseltamivir-sensitive and oseltamivir-resistant pandemic influenza a (h1n1) virus long-acting neuraminidase inhibitor laninamivir octanoate versus oseltamivir for treatment of influenza: a double-blind, randomized, noninferiority clinical trial long-acting neuraminidase inhibitor laninamivir octanoate as post-exposure prophylaxis for influenza daiichi sankyo receives additional dosage regimen approval in japan for long-acting neuraminidase inhibitor inavir dry powder inhaler 20mg discovery of a novel, first-inclass, orally bioavailable azaindole inhibitor (vx-787) of influenza pb2 preclinical activity of vx-787, a first in-class, orally bioavailable inhibitor of the influenza virus polymerase pb2 subunit study of acute uncomplicated seasonal influenza a in adult subjects nitazoxanide: a first-in-class broad-spectrum antiviral agent synergistic effect of nitazoxanide with neuraminidase inhibitors against influenza a viruses in vitro trial to evaluate the efficacy and safety of nitazoxanide in the treatment of acute uncomplicated influenza meeting report: 4th isirv antiviral group conference: novel antiviral therapies for influenza and other respiratory viruses a phase 2a to evaluate the safety of medi8852 in adults with uncomplicated influenza a broadly neutralizing human monoclonal antibody is effective against h7n9 safety and upper respiratory pharmacokinetics of the hemagglutinin stalk-binding antibody vis410 support treatment and prophylaxis based on population modeling of seasonal influenza a outbreaks the frequency of influenza and bacterial coinfection: a systematic review and meta-analysis. influenza other respir viruses infectious diseases society of america/american thoracic society consensus guidelines on the management of community-acquired pneumonia in adults respiratory syncytial virus infection in elderly and high-risk adults an immunodeficiency scoring index to predict poor outcomes in hematopoietic cell transplant recipients with respiratory syncytial virus infections this article describes the immunodeficiency scoring index that can identify high-risk hematopoietic cell transplant recipients with respiratory syncytial virus infection clinical and radiological features of respiratory syncytial virus in solid organ transplant recipients: a single-center experience mechanisms of action of ribavirin against distinct viruses report of the committee on infectious diseases impact of aerosolized ribavirin on mortality in 280 allogeneic haematopoietic stem cell transplant recipients with respiratory syncytial virus infections management of rsv infections in adult recipients of hematopoietic stem cell transplantation this systematic review highlights the importance of ribavirin among high-risk hct recipients with rsv infection aerosolized ribavirin: the most expensive drug for pneumonia oral ribavirin for the treatment of noninfluenza respiratory viral infections: a systematic review respiratory syncytial virus infection in patients with hematological diseases: single-center study and review of the literature intravenous ribavirin for respiratory syncytial viral infections in pediatric hematopoietic sct recipients intravenous ribavirin is a safe and cost-effective treatment for respiratory syncytial virus infection after lung transplantation respiratory syncytial virus immune globulin treatment of lower respiratory tract infection in pediatric patients undergoing bone marrow transplantation -a compassionate use experience committee on infectious diseases and bronchiolitis guidelines committee. updated guidance for palivizumab prophylaxis among infants and young children at increased risk of hospitalization for respiratory syncytial virus infection detection and control of a nosocomial respiratory syncytial virus outbreak in a stem cell transplantation unit: the role of palivizumab palivizumab treatment of respiratory syncytial virus infection after allogeneic hematopoietic stem cell transplantation the medicaid cost of palivizumab combination therapy with aerosolized ribavirin and intravenous immunoglobulin for respiratory syncytial virus disease in adult bone marrow transplant recipients respiratory syncytial virus upper respiratory tract illnesses in adult blood and marrow transplant recipients: combination therapy with aerosolized ribavirin and intravenous immunoglobulin treatment of respiratory syncytial virus pneumonia in a lung transplant recipient: case report and review of the literature a randomized, double-blind, placebo-controlled study of an rnai-based therapy directed against respiratory syncytial virus rna interference therapy in lung transplant patients infected with respiratory syncytial virus aln-rsv01 for prevention of bronchiolitis obliterans syndrome after respiratory syncytial virus infection in lung transplant recipients discovery of an oral respiratory syncytial virus (rsv) fusion inhibitor (gs-5806) and clinical proof of concept in a human rsv challenge study presatovir in hematopoietic cell transplant recipients with respiratory syncytial virus infection of the upper respiratory tract presatovir in hematopoietic cell transplant recipients with respiratory syncytial virus (rsv) infection of the lower respiratory tract a trial to assess the safety, tolerability, and pharmacokinetics of mdt-637 in healthy volunteers discovery of 4ê¹-chloromethyl-2ê¹-deoxy-3ê¹,5ê¹-di-o-isobutyryl-2ê¹-fluorocytidine (als-8176), a first-inclass rsv polymerase inhibitor for treatment of human respiratory syncytial virus infection oral gs-5806 activity in a respiratory syncytial virus challenge study activity of oral als-008176 in a respiratory syncytial virus challenge study ri-001 in immunosuppressed respiratory syncytial virus (rsv) infected patients at risk of lower tract rsv illness motavizumab for prophylaxis of respiratory syncytial virus in high-risk children: a noninferiority trial center for drug evaluation and research division of anti-viral products (davp) advisory committee briefing document. u.s. food and drug administration anderson lj seasonal trends of human parainfluenza viral infections: united states parainfluenza virus infections in hematopoietic cell transplant recipients and hematologic malignancy patients: a systematic review this paper provides a comprehensive review of parainfluenza virus infections in immunocompromised patients antiviral therapy of respiratory viruses in haematopoietic stem cell transplant recipients the characteristics and outcomes of parainfluenza virus infections in 200 patients with leukemia or recipients of hematopoietic stem cell transplantation efficacy of oral ribavirin in hematologic disease patients with paramyxovirus infection: analytic strategy using propensity scores single-centre experience with oral ribavirin in lung transplant recipients with paramyxovirus infections clinical potential of das181 for treatment of parainfluenza-3 infections in transplant recipients das 181 treatment of hematopoietic stem cell transplant patients with parainfluenza virus lung disease requiring mechanical ventilation successful treatment of parainfluenza virus respiratory tract infection with das181 in 4 immunocompromised children das181 for treatment of parainfluenza virus infections in hematopoietic stem cell transplant recipients at a single center an open label study to examine the effects of das181 administered by dry powder inhaler (dpi) or nebulized formulation in immunocompromised subjects with parainfluenza (piv) infection. ansun biopharma i das181 for treatment of parainfluenza virus infections in hematopoietic stem cell transplant recipients at a single center efficacy of the novel parainfluenza virus haemagglutinin-neuraminidase inhibitor bcx 2798 in mice -further evaluation efficacy of novel hemagglutinin-neuraminidase inhibitors bcx 2798 and bcx 2855 against human parainfluenza viruses in vitro and in vivo human rhinoviruses the economic burden of non-influenza-related viral respiratory tract infection in the united states picornavirus infections in children diagnosed by rt-pcr during longitudinal surveillance with weekly sampling: association with symptomatic illness and effect of season severity of human rhinovirus infection in immunocompromised adults is similar to that of 2009 h1n1 influenza clinical and molecular epidemiology of human rhinovirus infections in patients with hematologic malignancy human rhinovirus infections of the lower respiratory tract in hematopoietic stem cell transplant recipients upper and lower respiratory tract infections by human enterovirus and rhinovirus in adult patients with hematological malignancies chronic rhinoviral infection in lung transplant recipients detection of human rhinoviruses in the lower respiratory tract of lung transplant recipients combating enterovirus replication: state-of-the-art on antiviral research vapendavir significantly improves upper respiratory symptoms of naturally acquired rhinovirus infection in asthmatic adults: results of a phase 2 clinical trial efficacy and safety of oral pleconaril for treatment of colds due to picornaviruses in adults: results of 2 double-blind, randomized, placebo-controlled trials relationship of pleconaril susceptibility and clinical outcomes in treatment of common colds caused by rhinoviruses fda panel rejects common cold treatment phase ii, randomized, double-blind, placebo-controlled studies of ruprintrivir nasal spray 2-percent suspension for prevention and treatment of experimentally induced rhinovirus colds in healthy volunteers in vitro antiviral activity and single-dose pharmacokinetics in humans of a novel, orally bioavailable inhibitor of human rhinovirus 3c protease the effect of inhaled ifn-î² on worsening of asthma symptoms caused by viral infections a newly discovered human pneumovirus isolated from young children with respiratory tract disease genetic variability of human metapneumovirus infection: evidence of a shift in viral genotype without a change in illness identification and evaluation of a highly effective fusion inhibitor for human metapneumovirus a prospective study comparing human metapneumovirus with other respiratory viruses in adults with hematologic malignancies and respiratory tract infections human metapneumovirus infections in hematopoietic cell transplant recipients and hematologic malignancy patients: a systematic review human metapneumovirus infections following hematopoietic cell transplantation: factors associated with disease progression comparison of the inhibition of human metapneumovirus and respiratory syncytial virus by ribavirin and immune serum globulin in vitro effect of ribavirin and glucocorticoid treatment in a mouse model of human metapneumovirus infection treatment of severe human metapneumovirus (hmpv) pneumonia in an immunocompromised child with oral ribavirin and ivig treatment with oral ribavirin and ivig of severe human metapneumovirus pneumonia (hmpv) in immune compromised child the human metapneumovirus: a case series and review of the literature examination of a fusogenic hexameric core from human metapneumovirus and identification of a potent synthetic peptide inhibitor from the heptad repeat 1 region isolation and characterization of monoclonal antibodies which neutralize human metapneumovirus in vitro and in vivo prophylactic and therapeutic benefits of a monoclonal antibody against the fusion protein of human metapneumovirus in a mouse model update on rhinovirus and coronavirus infections human monoclonal antibodies against highly conserved hr1 and hr2 domains of the sars-cov spike protein are more broadly neutralizing severe acute respiratory syndrome coronavirus replication is severely impaired by mg132 due to proteasome-independent inhibition of m-calpain virucidal activity of a scorpion venom peptide variant mucroporin-m1 against measles, sars-cov and influenza h5n1 viruses key: cord-255711-8lojw5cz authors: palmu, arto a.; ware, robert s.; lambert, stephen b.; sarna, mohinder; bialasiewicz, seweryn; seib, kate l.; atack, john m.; nissen, michael d.; grimwood, keith title: nasal swab bacteriology by pcr during the first 24‐months of life: a prospective birth cohort study date: 2019-01-04 journal: pediatr pulmonol doi: 10.1002/ppul.24231 sha: doc_id: 255711 cord_uid: 8lojw5cz background: most respiratory bacterial carriage studies in children are based on cross‐sectional samples or longitudinal studies with infrequent sampling points. the prospective observational research in childhood infectious diseases birth cohort study intensively evaluated the community‐based epidemiology of respiratory viruses and bacteria during the first 2‐years of life. here we report the bacteriologic findings. methods: pregnant women in brisbane, australia were recruited between september 2010 and october 2012, and their healthy newborn children were followed for the first 2‐years of life. parents kept a daily symptom diary for the study child, collected a weekly anterior nose swab and completed an illness burden diary when their child met pre‐defined illness criteria. specimens were tested for respiratory bacteria by real‐time polymerase chain reaction (pcr) assays and those containing human genomic dna, deemed as high‐quality, were analyzed. results: altogether 8100 high‐quality nasal swab specimens from 158 enrolled children were analyzed. streptococcus pneumoniae, moraxella catarrhalis, and haemophilus influenzae were detected in 42.4%, 38.9%, and 14.8% of these samples, respectively. concomitant detection of bacteria was common. in contrast, bordetella pertussis, b. parapertussis, mycoplasma pneumoniae, chlamydia pneumoniae, and simkania negevensis were rarely identified. the prevalence of the three major bacteria was higher with increasing age and in the winter and spring months. siblings and childcare attendance were the other risk factors identified. conclusions: we confirmed the feasibility of frequent nasal swabbing by parents for studying bacterial colonization. pcr detected the major respiratory tract bacteria with expected high frequencies, but atypical bacteria were found rarely in this cohort. acute respiratory infections (ari) are responsible for substantial global morbidity and are important causes of mortality in children from lowincome countries. 1 carriage of respiratory bacterial pathogens, such as streptococcus pneumoniae, moraxella catarrhalis, and haemophilus influenzae, is a prerequisite for two important childhood aris; acute otitis media, which is the most frequently reported pediatric bacterial infection, 2 and bacterial pneumonia, a leading cause of childhood deaths. 1, 3 these bacteria are also carried in the upper airways of healthy young children who are considered the principal reservoirs for maintaining these organisms within populations. 4 (clinicaltrials.gov: nct01304914). 6 in this prospective, communitybased birth cohort study, children were enrolled at their mothers' antenatal visits and followed from birth until their second birthday. recruitment was progressive over 2-years at two metropolitan hospitals (one private and one government-funded). to be eligible to participate, children needed to be healthy, without congenital abnormalities or underlying chronic disorders, and to be born at term (36-42 weeks gestation). participants were able to leave the study temporarily (such as during family holidays) and re-join at a later date. the primary aim of the orchid study was to describe the nature and timing of viral respiratory and gastrointestinal infections during the first 2-years of life. 6 after enrolment, data on demographic, social, and health history, pregnancy, and birth details were collected by parental interview. subsequently, telephone interviews were conducted every 3-months to update feeding, vaccination, and childcare attendance details. childcare was categorized as formal (regulated care outside the child's home) or informal (non-regulated care provided by family or friends). parents received training to recognize respiratory symptoms. parents used diary cards to record daily, pre-defined respiratory symptoms, or diagnoses in a designed tick-box format. parents also recorded in a separate illness impact diary any health visits and antibiotic prescriptions for their child's ari episodes. 9 both diaries were returned by mail each month to the research team. research personnel collected anterior nasal swabs soon after birth and, following training and receiving written instructions, parents obtained swabs weekly thereafter. all swabs were collected using a plastic-shaft, rayon-budded swab in a transport tube with a foam pad reservoir soaked with viral transport medium (virocult mw950, medical wire & equipment, wiltshire, england) as one of the primary aims of the orchid study was to detect respiratory viruses. swabs were sent by standard surface mail to the laboratory where they were stored at −80°c, at a median of 3 (interquartile range 2-4) days after swabbing. since the swab's viral transport medium contained chloramphenicol and amphotericin b, polymerase chain reaction (pcr) assays rather than culture were used to detect the bacterial pathogens of interest. for nucleic acid extraction, each swab was resuspended in 2 ml of phosphate buffered saline from which 200 μl was used for extraction. samples were extracted on the qiaxtractor automated highthroughput extraction platform using dx reagents following the manufacturer's instructions (qiagen, australia). total dna and rna were eluted into 150 μl of elution buffer. each sample was spiked with whole equine herpesvirus (ehv) to assess nucleic acid extraction quality and presence of potential pcr inhibitors. any extract having a >3 cycle threshold (ct) difference to that of the expected value by ehv real-time pcr assay was considered to have failed quality control and the sample was re-extracted. nasal swab specimen quality was determined by screening for human dna using an endogenous retrovirus-3 (erv3) assay as described previously. 10 swabs with erv3 ct values >38 were deemed lower-quality and they, and associated person-time, were excluded from analyses. table) . batch testing methods and results for 17 respiratory viruses from the orchid cohort were available and have been described previously. 6, 7 ct values <40 for individual bacteria were considered positive. 11 the characteristics of the 158 enrolled cohort children, of whom 154 provided daily symptom data, are shown in table 1 overall, the prevalence of the major respiratory bacterial pathogens in the 8100 high-quality nasal swabs were: s. pneumoniae table) . bacterial prevalence was higher in symptomatic children (table 2) . higher prevalence of bacteria was detected in swabs taken during respiratory symptoms, especially when acute otitis media was diagnosed. this was observed also in swabs negative for any virus (supplemental digital content 4, table) . conversely, the prevalence of respiratory symptoms was more common in bacteria-positive figure) . a similar pattern with even higher differences between the aors was seen for the other bacterial species (data not shown). in concordance with earlier studies, we also observed the association of bacterial detection with respiratory symptoms. 25 furthermore, although symptoms were more prevalent in virusthis study confirms previous findings that the main risk factors for carrying respiratory bacterial pathogens are age, having the strengths of the orchid study include its prospective design, the comprehensive surveillance provided by daily symptom diaries, and weekly sample collections from birth until age 24-months. enrolment over two calendar years and follow-up of 4-years allowed for different seasonal and year-to-year epidemiologic patterns. considering its intensive nature, cohort retention was good with >70% followed for at least 18-months, and 68% of the maximum expected swabs were collected with a median interval of 1-week as stated in the protocol. 6 routine laboratory quality assurance included assaying samples for the human dna marker, erv3, to ensure human cells were present. while important for viral detection, 10 it seems also relevant for bacteria as we observed considerably higher bacterial detection rates in samples containing erv-3. consequently, only highquality nasal swabs were used to avoid compromising sensitivity in the analysis. there are, however, important limitations to consider. we did not validate upper airway bacterial detection by nasal swab and pcr against the gold standard of nps culture. however, our current analyses using previously validated pcr targets resulted in expected bacterial prevalence rates. the specificity of the pcr assays are high, yet the ply-pcr may cross-react with oral streptococci. 36 we in conclusion, we have used a novel method to evaluate bacterial carriage in children using nasal swabs optimized initially for viral detection. this method, however, is suitable also for bacterial detection and is especially feasible when frequent sampling is required. future analyses will explore interactions between nasal viruses, bacteria, and ari symptoms, while subtyping will allow carriage dynamics to be explored in greater detail. estimates of the global, regional, and national morbidity, mortality, and aetiologies of lower respiratory tract infections in 195 countries: a systematic analysis for the global burden of disease study otitis media epidemiology of community-acquired pneumonia and implications for vaccination of children living in developing and newly industrialized countries: a systematic literature review the fundamental link between pneumococcal carriage and disease carriage of mycoplasma pneumoniae in the upper respiratory tract of symptomatic and asymptomatic children: an observational study observational research in childhood infectious diseases (orchid): a dynamic birth cohort study viruses causing lower respiratory symptoms in young children: findings from the orchid birth cohort detection of viruses in weekly stool specimens collected during the first 2 years of life: a pilot study of five healthy australian infants in the rotavirus vaccine era the burden of community-managed acute respiratory infections in the first 2-years of life nasal swab samples and real-time polymerase chain reaction assays in community-based, longitudinal studies of respiratory viruses: the importance of sample integrity and quality control mailed versus frozen transport of nasal swabs for surveillance of respiratory bacteria in remote indigenous communities in australia standard method for detecting upper respiratory carriage of streptococcus pneumoniae: updated recommendations from the world health organization pneumococcal carriage working group comparison of four different sampling methods for detecting pharyngeal carriage of streptococcus pneumoniae and haemophilus influenzae in children alternative sampling methods for detecting bacterial pathogens in children with upper respiratory tract infections evaluation of swabbing methods for estimating the prevalence of bacterial carriage in the upper respiratory tract: a cross sectional study the association between anterior nares and nasopharyngeal microbiota in infants hospitalized for bronchiolitis. microbiome culture and pcr detection of haemophilus influenzae and haemophilus haemolyticus in australian indigenous children with bronchiectasis density interactions among streptococcus pneumoniae, haemophilus influenzae and staphylococcus aureus in the nasopharynx of young peruvian children molecular surveillance of nasopharyngeal carriage of streptococcus pneumoniae in children vaccinated with conjugated polysaccharide pneumococcal vaccines nasopharyngeal carriage of streptococcus pneumoniae in finnish children younger than 2 years old otitis-prone children have immunologic deficiencies in naturally acquired nasopharyngeal mucosal antibody response after streptococcus pneumoniae colonization a longitudinal study of streptococcus pneumoniae carriage in a cohort of infants and their mothers on the thailand-myanmar border epidemiologic studies of streptococcus pneumoniae in infants: acquisition, carriage, and infection during the first 24 months of life epidemiologic studies of streptococcus pneumoniae in infants. the effects of season and age on pneumococcal acquisition and carriage in the first 24 months of life relationship between rhinitis symptoms, respiratory viral infections and nasopharyngeal colonization with streptococcus pneumoniae, haemophilus influenzae and staphylococcus aureus in children attending daycare modelling the co-occurrence of streptococcus pneumoniae with other bacterial and viral pathogens in the upper respiratory tract epidemiological markers for interactions among streptococcus pneumoniae, haemophilus influenzae, and staphylococcus aureus in upper respiratory tract carriage density, serotype diversity, and fitness of streptococcus pneumoniae in upper respiratory tract cocolonization with nontypeable haemophilus influenzae prevalence of bordetella pertussis and bordetella parapertussis in infants presenting to the emergency department with bronchiolitis a controlled trial of two acellular vaccines and one whole-cell vaccine against pertussis. progetto pertosse working group bordetella parapertussis infection in children: epidemiology, clinical symptoms, and molecular characteristics of isolates ten years' experience with yearround active surveillance of up to 19 respiratory pathogens in children simkania negevensis may be a true cause of community acquired pneumonia in children crowding and other strong predictors of upper respiratory tract carriage of otitis media-related bacteria in australian aboriginal and non-aboriginal children evaluation and improvement of real-time pcr assays targeting lyta, ply, and psaa genes for detection of pneumococcal dna molecular surveillance of true nontypeable haemophilus influenzae: an evaluation of pcr screening assays nasal swab bacteriology by pcr during the first 24-months of life: a prospective birth cohort study key: cord-257171-kp2huia0 authors: brand, kim h.; ahout, inge m.l.; de groot, ronald; warris, adilia; ferwerda, gerben; hermans, peter w.m. title: use of mmp‐8 and mmp‐9 to assess disease severity in children with viral lower respiratory tract infections date: 2012-07-18 journal: j med virol doi: 10.1002/jmv.23301 sha: doc_id: 257171 cord_uid: kp2huia0 matrix metalloproteinases (mmps) play an important role in respiratory inflammatory diseases, such as asthma and chronic obstructive pulmonary disease. it was hypothesized that mmp‐8 and mmp‐9 may function as biological markers to assess disease severity in viral lower respiratory tract infections in children. mmp‐8 and mmp‐9 mrna expression levels in peripheral blood mononuclear cells (pbmcs) and granulocytes obtained in both the acute and recovery phase from 153 children with mild, moderate, and severe viral lower respiratory tract infections were determined using real‐time pcr. in addition, mmp‐8 and mmp‐9 concentrations in blood and nasopharyngeal specimens were determined during acute mild, moderate, and severe infection, and after recovery using elisa. furthermore, pbmcs and neutrophils obtained from healthy volunteers were stimulated with rsv, lps (tlr4 agonist), and pam3cys (tlr2 agonist) in vitro. disease severity of viral lower respiratory tract infections in children is associated with increased expression levels of the mmp‐8 and mmp‐9 genes in both pbmcs and granulocytes. on the contrary, in vitro experiments showed that mmp‐8 and mmp‐9 mrna and protein expression in pbmcs and granulocytes is not induced by stimulation with rsv, the most frequent detected virus in young children with viral lower respiratory tract infections. these data indicate that expression levels of the mmp‐8 and mmp‐9 genes in both pbmcs and neutrophils are associated with viral lower respiratory tract infections disease severity. these observations justify future validation in independent prospective study cohorts of the usefulness of mmp‐8 and mmp‐9 as potential markers for disease severity in viral respiratory infections. j. med. virol. 84:1471–1480, 2012. © 2012 wiley periodicals, inc. respiratory viral infections are an important cause of hospitalization among children younger than 5 years of age with estimated population-based hospitalization rates of 1-2% [shay et al., 1999; henrickson et al., 2004; iwane et al., 2004] . human respiratory syncytial virus (rsv) is the most commonly identified virus with detection rates up to 40-85% in infants hospitalized for respiratory infections during winter epidemics [wang et al., 1995; boyce et al., 2000; black, 2003] . the clinical manifestations range from a simple common cold to severe lower respiratory tract symptoms requiring mechanical ventilation. about 6-11% of the children admitted to hospital with rsv infection require intensive care admission [purcell and fergie, 2004; berger et al., 2009] . up to 35% of the children hospitalized with bronchiolitis did not receive any supportive intervention [mansbach et al., 2008] . on the other hand, it is crucial to avoid discharge of those children who may experience clinical deterioration. among children sent home with the diagnosis bronchiolitis, 4.6-6.8% required hospitalization later on during infection [roback and baskin, 1997; norwood et al., 2010] . biomarkers to assess severity of viral lower respiratory tract infections, in particular rsv infection, may be helpful to clinicians in the decision whether a child needs to be hospitalized. lung injury during severe rsv infection is thought to be mediated by both direct cytotoxic effects of the virus and the result of the induced inflammation. pathologic features of severe rsv infection include extensive bronchiolar epithelial destruction, peribronchial lymphocyte infiltration, necrosis of bronchial epithelium, and mucus plugs in the small bronchioles [johnson et al., 2007; welliver et al., 2008] . matrix metalloproteinases (mmps) are family of zinc endopeptidases capable of degrading components of the cellular matrix, and consequently, are suggested to be important in several diseases associated with tissue remodeling. pronounced increase in their expression is thought to be associated with a variety of inflammatory disease, including respiratory diseases [greenlee et al., 2007] . mmps play a role in cellular migration of neutrophils, lymphocytes, and other immune cells to the lungs by degrading extracellular matrix, but also have pro-and anti-inflammatory properties. the activity of mmps is regulated through binding to tissue inhibitor of metalloproteinases (timps) leading to inactivation. an imbalance in production and activation, or inactivation by timps might augment airway inflammation through direct or indirect effects upon signaling pathways that influence migration of leukocytes through the tissues [greenlee et al., 2007; schuurhof et al., 2012] . increased concentrations and activity of mmp-8 and mmp-9 have been observed in respiratory samples obtained from adults and children with acute lung injury and pneumonia [hartog et al., 2003; fligiel et al., 2006; schaaf et al., 2008; kong et al., 2009] as well as in chronic lung diseases such as asthma [prikk et al., 2002; obase et al., 2010] . in addition, a relation between mmp-9 concentrations and disease severity of pneumonia [hartog et al., 2003; schaaf et al., 2008] and asthma [belleguic et al., 2002; mattos et al., 2002] has been described. yeo et al. [2002] have reported that mmp-9 protein expression is increased in human airway epithelial cell lines infected with rsv. in addition, mmp-9 gene expression is increased in the lungs of rsv-infected mice [li and shen, 2007] . another study demonstrated that nasopharyngeal samples from infants infected with rsv and parainfluenza virus (piv) contain increased mmp-9 and timp-1 concentrations [elliott et al., 2007] . in the current study, it was hypothesized that mmp-8 and mmp-9 gene expression levels, and consequently, mmp-8 and mmp-9 plasma concentrations may function as biomarkers for disease severity in viral lower respiratory tract infections. children younger than 5 years of age with laboratory confirmed viral lower respiratory tract infections were prospectively included during three consecutive winter seasons (november-april in the years 2006-2009). patients with congenital heart or lung disease, known immunodeficiency's or glucocorticoid use were excluded. viral lower respiratory tract infections was defined as an acute infection of the lower airways, characterized by increased respiratory effort (tachypnea and/or use of accessory respiratory muscles and/or expiratory wheezing and/or crackles and/or apnea) in combination with a confirmed viral etiology by multiplex real-time polymerase chain reaction (rt-pcr) on nasopharyngeal washes as described previously [templeton et al., 2004] . the multiplex rt-pcr assay detect 15 different viral pathogens; influenza virus types a and b, coronavirus 229e and oc43, human bocavirus, enterovirus, adenovirus, parechovirus, piv types 1-4, human metapneumovirus, rhinovirus (rv), and rsv. written informed consent was obtained from all parents and the study was approved by the committee on research involving human subjects of the university nijmegen medical centre. within 24 hr after admission a blood sample and nasopharyngeal aspirate was collected and parents from hospitalized children were asked permission to draw a second blood sample and nasopharyngeal aspirate 4-6 weeks after admission. medical history, demographics, and clinical parameters were collected from questionnaires and medical records. patients were classified into three different groups based on severity of disease. children without hypoxia or severe feeding problems were allocated in the mild group, those requiring hospitalization for supplemental oxygen (oxygen saturations <93%) and/or nasogastric feeding in the moderate group and children requiring mechanical ventilation in the severe group. a nasopharyngeal aspirate was collected by introducing a catheter, connected to a collection tube and an aspiration system, into the nasopharyngeal cavity. then, 1.5 ml of saline was instilled into the catheter and, while slowly retracting the catheter, the nasopharyngeal fluid was aspirated in a collection tube. afterwards the catheter was flushed with 1 ml of saline and added to the collection fluid. the samples were kept cold and immediately transferred to the laboratory. the nasopharyngeal aspirate was centrifuged at 500g for 10 min at 48c to spin down the mucus and cells, after which the supernatant was frozen at à808c. five milliliters of blood was collected into sodium heparin tubes and directly transferred to the laboratory. a thin blood smear was prepared and stained with (may-grunwald-)giemsa to determine the percentages of granulocytes and pbmcs. pbmcs were obtained by density gradient centrifugation (lymphoprep 1 ; axis shield, oslo, norway) and stored in trizol at à808c for rna isolation. plasma samples were stored at à808c for elisas. quantitative mrna expression of mmp-8 and mmp-9 in pbmcs and granulocytes rna from pbmc and granulocytes was extracted using trizol (invitrogen life technologies, bleiswijk, the netherlands) according to the manufacturers' protocol. subsequently, a clean-up was performed on total rna with the rneasy minikit (qiagen, venlo, the netherlands) according to the manufacturers' instructions. total rna (2 mg, measured with spectrophotometry, nanodrop, wilmington) was reverse transcribed using a high-capacity cdna reverse transcription kit according to the manufacturers' instructions (applied biosystems, foster city, ca) and cdna was stored at à208. the relative gene expression was measured with sybr green pcr mastermix (applied biosystems; p/n 4367659) on the abi 7500 fast real time pcr system using standard program and software. after 40 repetitions a dissociation curve was performed as control for the specificity of the pcr reaction. the following primers were used: hactin f: cgtcacacttcatgatggagttg, hactin r: ctt-ccttcctgggcatgga; hmmp-9 f: gcccccc-ttgcataagga, hmmp-9 r: cagggcgaggacca-tagag; and hmmp-8 f: ccagtttgacatttga-tgctatcac, hmmp-8 r: ctgaggatgccttctcc-agaa. all reactions were performed in duplo. actin was used as reference gene. after a quality check (melting temp, curve of reaction, and standard deviation c t ) the dc t of the mmp-8 and mmp-9 to actin was calculated and expressed as relative expression. concentrations of total mmp-8 and mmp-9 in plasma, nasopharyngeal aspirate, and supernatants of cell stimulation assay were measured by elisa according to the manufacturers' protocol (duoset, r&d systems, abingdon, uk). in addition, timp-1 concentrations in plasma were determined as described above. after informed consent, blood was drawn from healthy volunteers and collected in edta tubes. blood was diluted 1:1 with pyrogene-free pbs (lonza, basel, switzerland). pbmcs and granulocytes were obtained by density gradient centrifugation (lymphoprep 1 ; axis shield). after washing, pbmcs were brought at a concentration of 5 â 10 6 cells/ml in serum-free rpmi (gibco, invitrogen, paisley, uk) with 100 u/ml of penicilin/streptavidin (gibco, invitrogen). granulocytes were purified by lysing the red blood cells (0.155 m nh 4 cl, 0.0001 m na 2 edta and 0.01 m khco 3 ), and, after washing, granulocytes were suspended at a concentration of 5 â 10 6 cells/ml in rpmi supplemented with 0.5% human serum albumin (sanquin, amsterdam, the netherlands). mononuclear cells (5 â 10 5 in 100 ml) were added to round-bottom 96-well plates and stimulated with either 100 ml culture medium (negative control), 1 ng/ ml lps (escherichia coli serotype 055:b5, sigma-aldrich, purified as described previously [hirschfeld et al., 2000] or moi 1 of rsv a2 (kindly provided by dr. r. de swart, erasmus mc, rotterdam, the netherlands). rsv a2 was cultured in hela cells and purified by ultracentrifuge over a sucrose 30% gradient. after incubation for 24 hr at 378c and 5% co 2. supernatant was collected and stored at à808c. neutrophils (5 â 10 5 in 100 ml) were stimulated and incubated in the same way for 4 hr and supernatant was stored at à808c. apoptosis was determined on the facscalibur by annexin v apoptosis detection kit (bd) according to the manufacturers' instructions and no differences between stimuli were found after 4 hr. values are expressed as percentages for categorical variables and as mean and standard error (se) or median and interquartile range (iqr) for continuous variables. for variables that were not normally distributed, kruskal-wallis test was performed to compare continuous variables followed by mann-whitney u-tests for individual comparisons. chi-squared tests were performed to compare categorical data. a twosided value of p < 0.05 was considered statistically significant. in total, 153 patients were included. in 109 patients (71%) rsv was detected. rsv positive children were significantly younger than rsv negative children. no other significant differences were observed between these groups (table i) . a total of 54, 60, and 39 children were classified as having mild, moderate, and severe disease, respectively. patients with severe disease were significantly younger compared to those with mild disease (105 days vs. 278 days; p < 0.05). more prematurely born children were observed in the severe group compared to the mild and moderate group. no other significant differences in clinical parameters were found between the different severity groups (table ii ). in addition, total leukocytes and neutrophil counts were comparable between all groups. disease severity is associated with increased gene expression levels of in both granulocytes and pbmcs during acute viral infection we observed increased expression of the mmp-8 and mmp-9 genes in both pbmcs and granulocytes compared to recovery. no differences in gene expression of mmp-8 and mmp-9 in both pbmcs and granulocytes were found between rsv positive and rsv negative children during acute infection. in general, gene expression of the mmp-9 gene was higher in granulocytes than in pbmcs. for mmp-8, the same trend was noticed (figs. 1a, b and 2a, b) . increased disease severity was associated with higher expression levels of the mmp-8 and mmp-9 genes in both pbmcs and granulocytes. to determine whether this association was dependent on the type of virus, rsv positive and rsv negative children were analyzed separately. for rsv positive patients, the same association was found between disease severity and gene expression levels (figs. 1c, d and 2c, d) . for rsv negative patients, mmp-8 and mmp-9 gene expression was higher in children with severe disease compared to those with mild. in addition, rsv negative children with severe disease had higher expression levels of the mmp-8 gene in both pbmcs and granulocytes compared to those with moderate disease (data not shown). the plasma concentration of mmp-8 was increased during acute rsv infection compared to recovery. in rsv negative patients this difference was not significant (fig. 3a) . higher mmp-8 plasma concentrations were found in children with severe and moderate disease compared to those with mild disease (fig. 4a) . in nasopharyngeal washes, the concentration of mmp-8 was increased during acute rsv infection compared to recovery washes (fig. 3d) . no significant differences of mmp-8 concentrations in the nasopharyngeal washes were observed between the different severity groups (fig. 4d) . in both rsv positive as rsv negative patients mmp-9 concentrations in the nasopharyngeal washes were increased during infection (fig. 3e ). children with moderate disease had increased mmp-9 concentrations in nasopharyngeal washes compared to those with mild disease. however, no significant differences in mmp-9 concentrations were observed in children with severe disease compared to those with mild and moderate disease (fig. 4e) . timp-1 concentrations in plasma or nasopharyngeal washes were not increased during acute infection and there was no correlation with disease severity (figs. 3c and 4c). the ratio between mmp-9 and timp-1 plasma concentrations, an indicator for enzyme activity, was not increased during acute rsv infection. this ratio was significant higher in the recovery plasma of rsv negative patients compared to the acute samples (fig. 3f) . no relation between disease severity and the plasma mmp-9 and timp-1 ratio was found (fig. 4f ). no differences in plasma or nasopharyngeal levels of mmp-8, mmp-9, and timp-1 plasma concentrations were observed between rsv positive and rsv negative children during acute viral respiratory infection. in general, mmp-8 and mmp-9 concentrations were higher in nasopharyngeal samples compared to plasma. mmp-9 plasma concentrations correlated with the number of granulocytes measured during acute rsv infection (pearson's correlation coefficient 0.33; p ¼ 0.019). no correlation was found between the number of granulocytes and mmp-8 plasma and nasopharyngeal concentrations and mmp-9 nasopharyngeal concentrations (data not shown). furthermore, there was no correlation between symptomatic days before presentation and levels of mmp-8 and mmp-9 gene expression or concentration of the protein in plasma and nasopharyngeal washes (data not shown). to investigate whether the source of plasma mmp-8 and mmp-9 during rsv infection was the result of direct interaction of pbmcs or neutrophils with rsv, pbmcs and neutrophils were stimulated with rsv in vitro. stimulation of pbmcs with lps (tlr4 agonist) induced mmp-9 secretion, whereas stimulation with rsv had no effect. none of the stimuli induced mmp-8 secretion by pbmc (fig. 5a) . stimulation of pbmcs with rsv did not result in increased gene expression of mmp-8 and only a moderate increase of mmp-9 expression was observed (fig. 5b) . unstimulated neutrophils secreted high levels of mmp-8 and mmp-9. stimulation with lps and rsv had no effect on the release of mmp-8 and mmp-9 by neutrophils (fig. 5c) . this study demonstrates that disease severity of viral lower respiratory tract infections in children is associated with increased gene expression levels of the mmp-8 and mmp-9 genes in both pbmcs and granulocytes. these associations were observed in children with lower respiratory tract infections caused by either rsv or other respiratory viruses. the in vitro experiments in this study show that mmp-8 and mmp-9 mrna and protein expression in pbmcs and granulocytes is not induced by stimulation with rsv. consequently, other factors than direct viral interaction induce gene expression in pbmcs and granulocytes. this is the first study that describes an association between mmp-8 and mmp-9 gene expression and disease severity of viral lower respiratory infections in children. this association was significant for children with a rsv infection and there was a trend for children with a viral lower respiratory infection caused by other viruses, indicating to a more general marker for disease severity during respiratory viral infections. several studies have shown that transcriptional analysis of peripheral blood cells can be used to discriminate the etiology and disease outcome [alizadeh et al., 2000; allantaz et al., 2007; ramilo et al., 2007; chaussabel et al., 2008] . ramilo et al. [2007] compared the transcriptional profiles of pbmcs of children with infectious diseases, and identified a set of genes that could separate influenza a infections from bacterial infections (staphylococcus aureus, escherichia coli, and streptococcus pneumoniae). both mmp-8 and mmp-9 were not represented in the selected set of classifier genes. retrospective analysis of the microarray data set was performed by us, and showed that mrna expression of mmp-8 and mmp-9 were elevated in all groups compared to controls, indicating a more general marker for inflammatory mmp-9 (e) in mild, moderate, and severe disease are given. ratio's between plasma mmp-9 and timp-1 (f). concentrations (ng/ml) are given in mean ae standard error. mann-whitney u-tests were performed to compare mild, moderate, and severe disease. a two-sided value of p < 0.05 was considered statistically significant. disease (data not shown). no data were available on disease severity, so it is not possible to exclude an association with disease severity. in an experimental model of viral infection of the upper respiratory tract in adults with rsv, influenza, and rv, no upregulation of mmp-8 and mmp-9 was detected in whole blood transcriptional profiles [zaas et al., 2009 ]. however, these infections were all mild with consequently low levels of inflammatory markers. this study shows that in mmp-8 plasma concentrations were increased during acute infection with rsv. although mmp-8 plasma concentrations were higher in moderate and severe disease compared to mild disease, there was no step-wise relation with disease severity. this is in contrast with the gene expression data and indicates a different source of plasma proteins than the circulating cell population. this is in line with the study of hartog et al. [2003] in which they found elevated mmp-8 concentrations in plasma and lung fluid in adults with hospital-acquired bacterial pneumonia compared to healthy controls. they found an association between clinical severity scores and mmp-8 concentrations in bal fluid, but not in plasma. although mmp-9 plasma concentrations were increased during acute viral respiratory infections in children, no association between mmp-9 plasma concentrations and disease severity was found in this study. previous studies have described such an association for several inflammatory diseases, such as pneumonia [hartog et al., 2003] , tuberculosis infections [hrabec et al., 2002] , septic shock [nakamura et al., 1998] , and asthma [belleguic et al., 2002] . this may be related to the fact that, in consistent with other studies [ricou et al., 1996; vignola et al., 1998; yang et al., 2005] , mmp-9 concentrations were correlated to neutrophil counts, although no significant differences in neutrophil counts between the different severity groups were observed in this study. timp-1 is an inhibitor of the protease activity of all known mmps [gomez et al., 1997 ]. previous studies have described an association between an imbalance between mmp-9 and timp-1 and tissue degradation and airflow obstruction in asthma and chronic bronchitis [vignola et al., 1998; mautino et al., 1999] . in addition, elevated mmp-9/timp-1 ratios have been observed in plasma from patients with status asthmatics [belleguic et al., 2002] . furthermore, it has been shown that increased timp-1 concentrations, but not mmp-9, in nasopharyngeal washes of rsvinfected children correlated with disease severity and this suggests that a disturbed mmp-9/timp-1 homeostasis contributes to disease severity [elliott et al., 2007] . the ratio of mmp-9 and timp-1 concentration in plasma did not show a correlation with disease severity in this study and indicates that mmp-9 is differentially regulated at the mucosal level during infection. although both mmp-8 and mmp-9 concentrations in nasopharyngeal samples were increased during acute infection compared to recovery samples no association with disease severity was observed. the wide range of nasopharyngeal concentrations between individuals is partly due to the variation induced by aspiration volumes from the nasopharyngeal cavity. currently, more standardized methods have been developed, such as flocked swabs, which can be used for viral diagnostics as well as protein analysis [dezzutti et al., 2011; munywoki et al., 2011] . normalization of protein levels to stable metabolites present in the mucus might further improve the use of nasopharyngeal samples for diagnostics, although these methods are not available yet. further it should be taken in account that upper respiratory samples do not necessarily represent the situation in the lower airways and the systemic inflammatory response. the in vitro experiments in this study show that mmp-8 and mmp-9 mrna and protein expression in pbmcs and granulocytes were not induced by stimulation with rsv. other factors than direct interaction between rsv and host cells could explain the increased gene expression levels of mmp-8 and mmp-9 in children with viral lower respiratory tract infections. influx of bone marrow-derived neutrophil precursors in blood from children with severe rsv infections can result in higher mmp-9 expression due to granule protein production, such as mmp-8 and mmp-9, during immature stages of neutrophil development [lukens et al., 2010] . also inflammatory mediators, such as growth factors, pro-inflammatory cytokines, oxidative stress upon viral infection can induce elevated gene expression levels of mmps [greenlee et al., 2007] . it has also been shown that the lung injury caused by mechanical ventilation has resulted in increased mmp-8 and mmp-9 expression [albaiceta et al., 2010] . however, in this study, the last mentioned cannot completely explain the differences in gene expression since also differences in gene expression between patients with mild and moderate disease were observed, all non-ventilated patients. the results of this study indicate that neutrophils are the major source of mmp-9 production. the higher mmp-8 and mmp-9 concentrations in nasopharyngeal samples compared to plasma may therefore reflect the influx and degranulation of neutrophils in the airways during infection. this is in contrast to observations made by others that suggest that airway epithelial cells are the primary source of mmps. it has been shown that mmp-9 gene expression is increased in human airway epithelial cell lines infected with rsv [yeo et al., 2002] . however, another study indicated that infected human airway epithelial cells are not the primary source of mmps and timp-1 and that infiltrating leukocytes are responsible for mmp-9 in airway samples [elliott et al., 2007] . also in a rsv infection model in mice, of which it is known that the epithelial cells are not infected, it was demonstrated that gene expression of mmp-9 is elevated in the lungs most likely by infiltrating cells [li and shen, 2007] . for mmp-8, no correlation with neutrophil counts was observed and gene expression levels in granulocytes and pbmcs were comparable indicating that mmp-8 transcription and secretion was different regulated than mmp-9. this is supported by differences in the degranulation of subcellular neutrophilic granules, in which mmp-8 and mmp-9 are stored [faurschou and borregaard, 2003] and differences in transcriptional events that induce mmp-8 and mmp-9 mrna expression. for example, it has been shown that pro-inflammatory cytokines, particularly il-1b, play a central role in the modulation of mmp-8 expression [knauper et al., 1993; abe et al., 2001] . future studies may reveal the role for mmp-8 plasma concentrations as a potential biomarker to assess disease severity in viral lower respiratory tract infections in children. the relation of mmp-8 and mmp-9 with viral load in the nasopharyngeal cavity have not been investigated in this study. in experimental respiratory viral infection models, inflammatory markers such as cytokines correlated with viral load and the symptom scores, indicating that the amount of virus is the driving force for inflammation [devincenzo et al., 2010] . also in children, disease severity has been associated with high viral titers [houben et al., 2010; el saleeby et al., 2011] . further research might reveal the role of inflammatory mediators in the pathogeneses of severe respiratory viral infections [openshaw, 2005] . in this light, it is interesting to consider inflammatory mediators, such as mmps, as potential targets for therapy. some limitations of this study need to be considered. first, the younger age of the children with the most severe lower respiratory tract infections may have caused a bias in the results. however, age was not correlated with mmp-8 and mmp-9 plasma concentrations, which suggests that age alone cannot explain the observed differences. according to these results, thrailkill et al. [2005] did not found significant differences in mmp-8 and mmp-9 serum concentrations in children, 2-18 years of age. second, multiple viruses were detected in 40% children with lower respiratory tract infections and it cannot be entirely exclude that the presence of multiple viruses have induced a different inflammatory response. the results of this study suggest that the expression of mmp-8 and mmp-9 genes are potential marker candidates for diagnostic use to assess disease severity in children with viral lower respiratory tract infections. markers for disease severity do not have clinical implication at present, because currently antiviral treatment is not available. however, new antiviral treatment of patients will be based most likely on diagnostics predicting disease severity and susceptibility [quinn, 2010; hoggatt, 2011] . furthermore, markers for disease severity are also important for research purposes to study effects of interventions, such as treatment. although currently no biomarkers at transcription level are available in the clinic, because the processing is time consuming, innovative techniques may enable rapid analysis of the expression of multiple genes at transcriptional level in the near future [brand et al., 2010] . in conclusion, increased expression of the mmp-8 and mmp-9 genes was observed in pbmcs and granulocytes obtained from children with severe viral lower respiratory tract infections. mmp-8 and mmp-9 gene expression levels in circulating cells may be useful markers to support clinical evaluation of disease severity in viral respiratory infections. these results justify future follow-up, that is, the validation in independent prospective study cohorts of the usefulness of mmp-8 and mmp-9 as a potential markers for disease severity in viral respiratory infections. we would like to thank all parents and children for their participation in the study. we are grateful to the medical staff of the department of pediatrics and intensive care unit at the umcn st. radboud and canisius wilhelmina hospital for assistance in obtaining clinical samples. in addition, we would like to thank e. voorbrood and p. ruijs from the laboratory of hematology at the umcn st. radboud for counting the blood smears. induction of collagenase-2 (matrix metalloproteinase-8) gene expression by interleukin-1beta in human gingival fibroblasts absence or inhibition of matrix metalloproteinase-8 decreases ventilator-induced lung injury distinct types of diffuse large bcell lymphoma identified by gene expression profiling blood leukocyte microarrays to diagnose systemic onset juvenile idiopathic arthritis and follow the response to il-1 blockade increased release of matrix metalloproteinase-9 in the plasma of acute severe asthmatic patients prospective population-based study of rsv-related intermediate care and intensive care unit admissions in switzerland over a 4-year period systematic review of the biology and medical management of respiratory syncytial virus infection rates of hospitalization for respiratory syncytial virus infection among children in medicaid host biomarkers and paediatric infectious diseases: from molecular profiles to clinical application a modular analysis framework for blood genomics studies: application to systemic lupus erythematosus viral load drives disease in humans experimentally infected with respiratory syncytial virus performance of swabs, lavage, and diluents to quantify biomarkers of female genital tract soluble mucosal mediators respiratory syncytial virus load, viral dynamics, and disease severity in previously healthy naturally infected children matrix metalloproteinase-9 and tissue inhibitor of matrix metalloproteinase-1 in the respiratory tracts of human infants following paramyxovirus infection neutrophil granules and secretory vesicles in inflammation matrix metalloproteinases and matrix metalloproteinase inhibitors in acute lung injury tissue inhibitors of metalloproteinases: structure, regulation and biological functions matrix metalloproteinases in lung: multiple, multifarious, and multifaceted pulmonary matrix metalloproteinase excess in hospital-acquired pneumonia national disease burden of respiratory viruses detected in children by polymerase chain reaction cutting edge: repurification of lipopolysaccharide eliminates signaling through both human and murine toll-like receptor 2 personalized medicine-trends in molecular diagnostics: exponential growth expected in the next ten years disease severity and viral load are correlated in infants with primary respiratory syncytial virus infection in the community circulation level of matrix metalloproteinase-9 is correlated with disease severity in tuberculosis patients population-based surveillance for hospitalizations associated with respiratory syncytial virus, influenza virus, and parainfluenza viruses among young children the histopathology of fatal untreated human respiratory syncytial virus infection fragmentation of human polymorphonuclearleucocyte collagenase matrix metalloproteinase activity in pediatric acute lung injury effect of respiratory syncytial virus on the activity of matrix metalloproteinase in mice a systemic neutrophil response precedes robust cd8(þ) t-cell activation during natural respiratory syncytial virus infection in infants prospective multicenter study of bronchiolitis: predicting safe discharges from the emergency department matrix metalloproteinase-9 expression in asthma: effect of asthma severity, allergen challenge, and inhaled corticosteroids balance in asthma between matrix metalloproteinases and their inhibitors improved detection of respiratory viruses in pediatric outpatients with acute respiratory illness by real-time pcr using nasopharyngeal flocked swabs modulation of plasma metalloproteinase-9 concentrations and peripheral blood monocyte mrna levels in patients with septic shock: effect of fiber-immobilized polymyxin b treatment prospective multicenter study of bronchiolitis: predictors of an unscheduled visit after discharge from the emergency department effects of inhaled corticosteroids on metalloproteinase-8 and tissue inhibitor of metalloproteinase-1 in the airways of asthmatic children antiviral immune responses and lung inflammation after respiratory syncytial virus infection airway obstruction correlates with collagenase-2 (mmp-8) expression and activation in bronchial asthma driscoll children's hospital respiratory syncytial virus database: risk factors, treatment and hospital course in 3308 infants and young children payers and the assessment of clinical utility for companion diagnostics gene expression patterns in blood leukocytes discriminate patients with acute infections matrix metalloproteinases and timp in acute respiratory distress syndrome failure of oxygen saturation and clinical assessment to predict which patients with bronchiolitis discharged from the emergency department will return requiring admission hospital acquired pneumonia with high-risk bacteria is associated with increased pulmonary matrix metalloproteinase activity proteins involved in extracellular matrix dynamics are associated with rsv disease severity bronchiolitis-associated hospitalizations among us children rapid and sensitive method using multiplex real-time pcr for diagnosis of infections by influenza a and influenza b viruses, respiratory syncytial virus, and parainfluenza viruses 1, 2 3, and 4 physiological matrix metalloproteinase concentrations in serum during childhood and adolescence, using luminex multiplex technology sputum metalloproteinase-9/tissue inhibitor of metalloproteinase-1 ratio correlates with airflow obstruction in asthma and chronic bronchitis pediatric investigators collaborative network on infections in canada (picnic) prospective study of risk factors and outcomes in patients hospitalized with respiratory syncytial viral lower respiratory tract infection respiratory syncytial virus and influenza virus infections: observations from tissues of fatal infant cases excessive matrix metalloproteinase-9 in the plasma of community-acquired pneumonia respiratory syncytial virus infection induces matrix metalloproteinase-9 expression in epithelial cells gene expression signatures diagnose influenza and other symptomatic respiratory viral infections in humans key: cord-007222-31o180fz authors: mufson, maurice a.; levine, harry d.; wasil, raymond e.; mocega-gonzalez, hilda e.; krause, helen e. title: epidemiology of respiratory syncytial virus infection among infants and children in chicago date: 1973-08-17 journal: am j epidemiol doi: 10.1093/oxfordjournals.aje.a121542 sha: doc_id: 7222 cord_uid: 31o180fz from january 1, 1967 to december 1971, the temporal pattern of respiratory syncytial virus infection was investigated in infants and children younger than 18 months hospitalized for acute lower respiratory tract disease. of 4696 infants and children with acute lower respiratory tract disease admitted to the cook county hospital, 2530 were tested for virus infection by virus isolation or serologic procedures or both. overall, respiratory syncytial virus infections were detected in 12% and parainfluenza 3 virus in 10.8% of individuals tested. other respiratory viruses were less commonly identified. respiratory syncytial virus epidemics occurred annually and were temporally synchronous with the peak periods of respiratory disease admissions. only during epidemics of respiratory syncytial virus did admission for respiratory tract disease usually reach 40 patients or more weekly. the peak months of respiratory syncytial virus epidemics were december 1966, january 1968, february-march 1969, april 1970 and january 1971. epidemics lasted about 17 weeks. no similar annual epidemic pattern was seen with the other myxoviruses. bronchiolitis; epidemiology; infants; myxovirus; parainfluenza virus; pneumonia; respiratory syncytial virus; respiratory tract diseases long term surveillance programs of lower ratory syncytial virus (rsv) is a major respiratory tract disease in infants and cause of pneumonia and bronchiolitis in inchildren have provided evidence that respi-fants; during epidemic periods, rsv has abbreviations: hek, human embryonic kid-"division of pediatrics, cook county hospital ney; rmk, rhesus monkey kidney; rsv, respira-and hektoen institute for medical research. chitory syncytial virus. cago^ illinois. 1 departments of medicine and preventive • west side veterans administration hospital, medicine and community health, abraham lin-ohieano illinois coin school of medicine, and the epidemiology " , , • t u t <• *• tv -a u " ,. " , , , _ ... " ... tt . ., , supported in part bv infectious disease branch section, school of public health, university of _, " "" " """ tlmltr , " . , x . tir • i ,>. ,, j-, o , , ., ", , ". , been associated with approximately onehalf of such cases (1) (2) (3) (4) (5) (6) (7) (8) (9) . respiratory syncytial virus epidemics occur annually, usually during the winter months in temperate zones, but the pattern of their epidemic occurrence, which has been systematically investigated in only a few metropolitan areas, has been reported to vary somewhat among differing population groups and cities (2, 3, 6, 9, 10) . in washington, d.c., rsv epidemics exhibited alternating periodicity or a two-year biniodal cycle, appearing early in winter one year and later in the next year (6) . a similar pattern has been reported for the occurrence of this infection among infants and children in chapel hill, north carolina (10) . by comparison, in chicago, illinois, and seattle, washington, during the last six years rsv infection apparently followed a four-year cycle, with successively later epidemics annually for three years, and then an early winter occurrence after a short fourth cycle (9, 11) . this report describes the periodic pattern of rsv infection in infants and children with lower respiratory tract disease admitted to the cook county hospital in chicago, between january 1, 1967 and december 31, 1971. four epidemiologic years of rsv infection are included in this 60-month study period. in a previous communication, a mathematical model was described which simulated the occurrence of rsv epidemics during the first three years (11) . the occurrence during this time of coronavirus infections is reported in detail elsewhere (12) . population. the study group comprised infants and children younger than 18 months of age admitted to the cook county hospital because of acute lower respiratory tract disease. the population has been described previously (13) . categories of acute respiratory 7 tract disease were pneumonias, bronchiolitis, laryngo-tracheo-bronchitis. most infants and children ill with respi-ratory tract disease admitted between sunday and friday mornings were tested for virus infection by virus isolation or serologic procedures or both. on some days the number of infants and children admitted with acute lower respiratory tract disease exceeded the capacity of our laboratory, and in these instances individuals whose illnesses were of most recent onset were tested. vinis isolation procedures. oropharyngeal swab specimens for virus isolation were obtained within -24 hours after admission from study children (13) . the oropharynx was vigorously rubbed with a sterile swab and the swab was then extracted in 4 ml of veal infusion broth containing 0.5 per cent bovine serum albumin. for virus isolation, 0.2 ml of broth was inoculated into each of two roller tube cultures of primary rhesus monkey kidney (rmk), hep-2 and fetal human diploid (strain wi-38) cells. infrequently, during the first two years of the study, primary human embryonic kidney (hek) cells were also inoculated. each oropharyngeal swab specimen was pre-treated with penicillin, streptomycin and amphotericin at 4 c ior one hour prior to inoculation, as previously described (13) . cell cultures were incubated on a rotating drum at 33 c for at least 18 days, and often for as long as 28 days. subpassages of negative cultures were not made. hep-2, wi-38 and hek cell cultures were examined for cytopathic effects at about five-day intervals, and the culture media were changed at these times. viral isolates were identified by serologic procedures (13) . rmk cultures were tested for hemadsorption at five-to seven-day intervals, and the culture media changed at these times. for hemadsorption, 0.25 ml of a 0.4 per cent suspension of guinea pig erythrocytes was added to each culture tube and the tubes refrigerated at 4 c for 30 minutes. hemadsorbing isolates were identified by hemadsorption-inhibition procedures using antisera for influenza a and b, parainfluenza 1, 2 and 3, mumps and sv-5 viruses. antibody determinations. acute and convalescent phase sera collected usually about 18 to 21 days apart, but at least 10 days apart, were tested for complement-fixing antibodies to parainfluenza 1, 2 and 3, rsv, influenza a and b viruses, adenoviruses, coronavirus 229e and m. •pneumoniae. microtiter complement-fixation tests were employed with overnight fixation at 4 c and 1.7 to 1.8 units of complement. eight units of rsv and the parainfluenza virus antigens and four units of the other antigens were used. evidence of infection was a fourfold or greater rise in antibody titer between the acute and convalescent phase sera. data analyses. the occurrence of virus infections was analyzed by epidemiologic year, extending from july 1 to june 30. cases were plotted by day of admission. demographic, virus isolation and antibody assay data were coded for computer processing. computer assisted analyses were done using the ibm 360/155 computer in the research resources laboratory of the university of illinois college of medicine. during the 60-month study period, 4696 infants and children younger than 18 months of age with acute lower respiratory tract diseases were admitted to the hospital (table 1) . usually between 900 and 1000 infants and children were admitted each epidemiologic year. most infants suffered from pneumonia or bronchiolitis and these comprised nearly 85 per cent of the total population. of the total population of infants admitted for lower respiratory tract disease, 2530 (53.8 per cent) were tested for virus infection by virus isolation or serologic procedures or both. frequency of virus infection. overall, rsv infections were detected in 304 (12.0 per cent) of lower respiratory tract illnesses tested (table 2) . it was the predominant infection in respiratory disease in children younger than 18 months of age. as a group, however, myxovirus infections were even more frequently associated with such cases. parainfluenza 3 virus, the second most common infection, was associated with 274 (10.8 per cent) of these illnesses. in contrast, parainfluenza 1 and 2 viruses, influenza a virus and adenovirus infection occurred much less often. influenza b virus infections were rare (table 2) . during the five-year observation period, rsv epidemics occurred annually. proportionately more respiratory syncytial infections were detected in alternate epidemiologic years ( periodicity oj respiratory syncytial virus epidemics. epidemics of rsv temporally paralleled the peak periods of respiratory disease admission (figure 1). the start of each epidemic was signalled by a marked increase in admission of infants and young children for serious lower respiratory tract disease. during these epidemics, admissions to the hospital exceeded 20 patients weekly and usually reached 40 patients weekly at the peak of the epidemic. other virus infection outbreaks did not have as evident an impact on the number of admissions for lower respiratory tract diseases. the pattern of rsv disease epidemics can be clarified and simplified by plotting a three-week moving average of the raw admissions data (figure 1). using this curve, the peak months of rsv epidemics were from the three-week moving average plot of admissions, the time intervals between peaks were calculated (table 4). the first three epidemics after january 1967 occurred on a nearly regular cycle. these epidemics occurred at 55-to 58-week intervals from the peak of one epidemic to the peak of the next, the peak of each appearing successively later each year than the previous one. the following epidemic, however, occurred between december 1970 and march 1971, after only a 39-week interval after the previous epidemic peak. the peak of the 1972 epidemic in march was about 63 weeks after this early peak. prevalence of other myxoviruses. in addition to the five major admission peaks associated with epidemics of rsv infection, other smaller peaks of admission of infants and children with respirator)' tract disease can be delineated (figure 1). although parainfluenza 3 virus occurred endemically in this population, clusters of infection ocseasonal occurrence of respiratory syncytial virus infeclian a7iwng children younger than 18 months of age requiring hospitalization for acute lower respiratory tract disease, january 1, 1967 to december si, 1971 year 1966-1967 1967-196s 1969 1970 1970-1971 1972 sions of infants and children with lower respiratory tract disease, these were not as large as those observed during respiratory syncytial virus epidemics. a brief, sharp peak of admission follower respiratory tract diseases occurred "unseasonally" in august 1968, and was not associated with infections with viruses included in this study. although we have no specific etiologic explanation for this peak, the data indicate that other infectious agents are etiologically related to these diseases. discussion this continuing surveillance study of virus infections in infants and young children with acute lower respiratory tract disease describes the cyclical pattern of occurrence of rsv infections. from the winter of 1966-1967 until april 1970 in chicago, rsv epidemics occurred on a regular cycle with a periodicity of about 58 weeks. this cycle interval placed succeeding epidemics slightly later each year. following the spring epidemic in april 1970, the interval between this peak and the next was only 39 weeks, reorientating the epidemics to the colder months. thus it appears that a four-year cycle of respiratory syncytial virus epidemics occurred with about three equal long cycles and one short one. whether this four-year pattern was coinci-dental or will repeat during the next four years remains to be investigated. the most recent epidemic of rsv, however, occurred in march 1972 or at a relatively later time compared to the 1968 epidemic. data on the timing of rsv epidemics from other populations located in different large cities agree with our findings (6, 9, 10) . in the largescale study of respiratory tract infections conducted in seattle, washington, rsv epidemics occurred in nearly the same months as in chicago during the same period of observation. peak months of these epidemics in seattle were january 1967 , february 1968 , march 1969 , april-may 1970 , january 1971 and march-april 1972 . from these data it appears that the cyclical occurrence in seattle was the same as it is in chicago. similarly, for the years 1967 to 1970 in washington, d.c., peak months of rsv epidemics in children's hospital were january 1967 , february 1968 , february 1969 and april 1970 . in chapel hill, north carolina, the peak months were january 1967, march 1968 and january-february 1969, slightly different than in chicago in these three years (10) . it is of interest that from 1963 to 1967 berglund (3) described the occurrence of rsv epidemics in children in turku, finland, which followed a pattern similar to the latter half of the four-year cycle in chicago. for chicago, seattle and washington, d.c., three widely separated cities with differing but characteristic climatic conditions, the temporal occurrence of rsv epidemics was very similar. respiratory syncytial virus infection does not appear to move wavelike over the country but rather starts nearly simultaneously in these three areas, and perhaps throughout the country as a whole. weather conditions do not appear to influence the occurrence of these epidemics, except that outbreaks do not develop in epidemic fashion during the summer months in the northern temperate zone. in chicago, temperature and humidity as predictors (or independent variables) of rsv occurrence showed a minor, but statistically significant, effect. other variables which might contribute to the initiation of respiratory syncytial virus epidemics are undefined, as are those that terminate the outbreaks. infants and children younger than one year of age comprise the major group of individuals with significant clinical illness consequent to rsv infection. in any one year, this group of susceptibles to a large extent were born subsequent to the previous respiratory syncytial virus epidemic. as the susceptible pool of infants enlarges it is probable that a critical size is reached which encourages effective spread of the virus, and an epidemic is initiated. however, the short cycle epidemic of 1970-1971 is evidence against the hypothesis that the accumulation of a sufficient pool of susceptibles requires more than one year for an rsv epidemic to develop, unless the previous epidemic failed to exhaust the susceptible pool or new susceptibles entered the community at a higher rate. in either instance, no data exist to explain the short cycle epidemic. lacking confirmation on the variables which might contribute to rsv epidemics precludes describing a mathematical model of the epidemics. previously we reported on a model to describe the regular cycle of outbreaks (11), viz: . ?, [* 8i n (t) the dependent (predicted) variable was the number of admissions, possibly smoothed by moving average, of infants and children with lower respiratory tract disease for a given week. the a's and b's are the coefficients determined by least squares, t is time measured from the start of the study, k the number of observed points in a single cycle and i is the harmonic number. auto-correlations computed for the 1966-1967, 1967-1968 and 1968-1969 epidemics were found to peak at 58 weeks, and suggested that this interval was the fundamental frequency. the intervals between peaks computed from the seven-week moving average plot agree fairly well with this value. using three harmonics, the model generated values for admissions which fit the three-week moving average data closely; the unbiased multiple correlation was 0.93. because of the shortened interval between the march 1970 and april 1971 epidemics, the model did not predict the april 1971 epidemic. although the model cannot be modified to simulate the four-year cycle observed, it does suggest that the variables which influence rsv outbreaks are cyclical, and studies to delineate such variables in rsv epidemics are required. the definition of these factors could contribute to a means for modifying the impact of rsv epidemics on the population of susceptible infants and young children. acute respiratory disease in infancy and childhood: present understanding and prospects for prevention influenza respiratory syncytial virus and pneumonia in glasgow studies on respiratory syncytial vims infection studies on the role of viruses, bacteria, and m. pneumoniae as causes of lower respiratory tract infections in children influence of immunological factors in respiratory syncytial disease of the lower respiratory tract recovery of respiratorj' syncytial virus during the years 1960-1970 respiratorj' syncytinl and other viruses associated with respiratory disease in infants respiratorj' disease in group daj' care incidence and etiologj' of pneumonia, croup and bronchiolitis in preschool children belonging to a prepaid medical care group over a four-j'ear period epidemiology of acute lower respiratorj' disease in children periodicity, predictability and respiratory syncytial (rs) virus infection epidemiologj' of coronavirus infections in infants with acute lower respiratory tract disease viruses, mycoplasma pneumoniae and bacteria associated with lower respiratory tract disease among infants key: cord-018421-wy3mtafh authors: waghmare, alpana; boeckh, michael title: rhinovirus, coronavirus, enterovirus, and bocavirus after hematopoietic cell transplantation or solid organ transplantation date: 2016-02-15 journal: transplant infections doi: 10.1007/978-3-319-28797-3_32 sha: doc_id: 18421 cord_uid: wy3mtafh respiratory viral infections represent a significant cause of morbidity and mortality in immunocompromised hosts. newer molecular detection assays have allowed for the characterization of several respiratory viruses not previously recognized as having significant clinical impact in the immunocompromised population. human rhinoviruses are the most common respiratory viruses detected in the upper respiratory tract of hematopoietic cell transplant and lung transplant recipients, and evidence on the impact on clinical outcomes is mounting. other respiratory viruses including enteroviruses (evs), coronaviruses (covs), and bocavirus may also contribute to pulmonary disease; however, data is limited in the immunocompromised population. further studies are needed to define the epidemiology, risk factors, and clinical outcomes of these infections; this data will help inform decisions regarding development of antiviral therapy and infection prevention strategies. human rhinoviruses (hrvs), the viruses predominantly associated with the common cold, are highly prevalent in both immunocompetent and immunocompromised individuals. prior to the development of sensitive molecular viral detection assays, infl uenza, respiratory syncytial virus, and parainfl uenza virus were the most common and most concerning respiratory viral pathogens detected in hematopoietic cell transplant (hct) recipients [ 1 ] . due to the development of polymerase chain reaction (pcr) assays for viral detection, hrvs are now known to be the most common viruses detected from respiratory specimens in hct recipients and can account for 25-40% of cases of viral respiratory infections in these patients [ 2 -4 ] (figure 32-1 ). due to their high prevalence and their ability to cause progressive infection, hrvs are also a signifi cant cause of lower respiratory tract infection (lrti) in hct recipients (table 32-1 ) . hrv infection is also common in solid organ transplant (sot) recipients , although the incidence is not known among sot recipients as a whole. in lung transplant recipients , data from older retrospective and prospective studies suggests an incidence of 35-55% among patients with positive respiratory samples [ 5 -7 ] (figure 32-2 ) . in a recent prospective surveillance study of 112 lung transplant recipients, hrvs represented 62% of all positive samples [ 8 ] . among symptomatic lung transplant recipients, hrv represented 34% of all respiratory viruses detected [ 9 ] . hrvs are members of the picornaviridae family and are classifi ed into three species, hrv-a, hrv-b, and hrv-c, based on similarity in genome organization, capsid features, and conserved sequences [ 10 ] . the total number of genotypes continues to grow as new genotypes are characterized; currently at least 160 unique genotypes are described. due to poor growth in traditional viral culture models, hrv-c was only recognized after the development of molecular diagnostic techniques. thus, hrv-c is not a novel species, but rather one that has been circulating unnoticed due to lack of an appropriate diagnostic assay. there are several biologic characteristics of hrv-c that differentiate the species from hrv-a and hrv-b. hrv-a and hrv-b both use icam-1 or ldlr for cell attachment and entry, whereas it appears that hrv-c may utilize a distinct receptor, cadherin-related family member 3, that is associated with asthma susceptibility [ 11 , 12 ] . additionally, hrv-c species are stable at higher temperatures and readily infect upper and lower airways, whereas hrv-a and hrv-b species tend to be more limited to the sinuses and upper airways [ 13 , 14 ] . these biologic characteristics are thought to play a role in variations in clinical outcomes observed among the different species. most immunocompetent patients with hrv present with an afebrile, self-limited syndrome characterized by rhinorrhea, nasal congestion, and malaise, and less frequently sore throat, mild cough, and hoarseness [ 15 -19 ] . hrv may also be associated with exacerbations of sinusitis, chronic bronchitis, and asthma, and with lower respiratory tract syndromes and atypical pneumonias in otherwise healthy people, including the young and the elderly [ 20 , 21 ] . the specifi c mechanisms by which hrvs produce lung diseases are not well understood. hrvs are also implicated in asthma and chronic obstructive pulmonary disease (copd) exacerbations, but again the mechanisms are poorly defi ned. with the widespread availability of pcr diagnostics, data are emerging on the incidence and clinical relevance of hrv infections in immunocompromised patients. early studies relied on culture to detect hrv, a specifi c but insensitive method because the standard viral culture systems are not optimized for hrv detection, especially hrv-c [ 22 ] . for example, a fred hutchinson cancer center surveillance study from 1987 to 1992 detected hrvs in 29 specimens, and only one was from a lower respiratory tract specimen [ 2 ] . a prospective 5-year study at md anderson cancer center cultured specimens specifi cally for hrvs at lower temperatures with roller culture methods, and reported that hrv infections were associated with substantial morbidity and mortality in 7 of 22 (32%) myelosuppressed patients [ 23 ] . in that study, approximately one third of the adult hct recipients who developed symptomatic hrv infections prior to engraftment had progression of upper respiratory tract symptoms to lrti, and all cases with pneumonias were fatal. lung biopsies and autopsies revealed fi ndings consistent with interstitial pneumonitis and/or ards, but no in situ evaluation was performed to defi nitively assess hrv infection. similar reports with evidence of lrti based on radiographic and bal fi ndings continue to be noted [ 24 -26 ] , but it remains unknown if pneumonia is a direct cause of viral invasion of the lung tissue or by host responses in the lung. evidence for in vitro and in vivo replication in lower respiratory tract has been shown in experimental infection, where hrv was isolated from human volunteers after intranasal hrv challenge by in situ hybridization [ 27 ] . the use of rt-pcr continues to provide new information about the frequency of hrv infection. in a study of bal samples from 77 hct recipients that were tested using rt-pcr , hrv was detected in six patients (8%), mortality rate was very high (83%) and two of the six patients showed persistent hrv infection. however, all of the hrv-infected patients had signifi cant coinfections and it was not certain whether hrv infection was the direct cause of poor prognosis [ 25 ] . in a small cohort of patients with hematologic malignancy, lrti was associated with hypoalbuminemia and bacterial copathogens were seen in 25% of patients [ 28 ] . recent studies have shown that immunocompromised adults with hrv demonstrated similar hospital admission rates, intensive care unit admissions, and mortality rates as patients with pandemic h1n1 infl uenza [ 29 ] . several reports have linked hrv infection to severe respiratory failure and even death [ 23 -25 ] . furthermore, recently presented data suggest that lrti associated with hrv leads to a mortality rate comparable to that of rsv, infl uenza virus, and piv [ 30 ] , independent of the presence of co-pathogens. risk factors for mortality following hrv lrti included bone marrow stem cell source, oxygen requirement at time of diagnosis, and steroid use ≥1 mg/kg prior to diagnosis [ 30 ] . other factors that may infl uence clinical severity include the presence of hrv rna in blood, viral load, and hrv species type; however, no data exist in immunocompromised patients to date. hrv viral rna was detected in the sera of 30 (12%) of 243 pediatric patients with severe hrv respiratory infection, with hrv-c being the predominant species [ 31 ] . in healthy pediatric patients, increased respiratory viral load has been associated with hrv lrti and hrvc has been implicated as a more virulent pathogen [ 32 -34 ] . others, however, have shown lack of correlation between hrv-c and oxygen requirement, length of hospitalizations, and coinfections [ 35 ] . the predominance of hrv-c in hct recipients has also been described in small studies, with higher rates of pneumonia in patients with hrv-c detected from the upper respiratory tract [ 36 ] . in a small cohort of patients with hematologic malignancies, the rate of lrti was not different between patients infected with hrv-a, hrv-b, or hrv-c [ 28 ] . the relative risk of hrv-c infection in the immunocompromised population remains unknown, and more research is needed to defi ne the role of strain differences on outcomes. detection and diagnosis of respiratory viral infections prior to transplant is a common clinical concern that has until recently only been evaluated in small cohorts for certain viruses [ 37 -40 ] . in a large, prospective surveillance cohort of allogeneic hct recipients, detection of hrv pretransplant was associated with signifi cantly fewer days alive and out of the hospital, and signifi cantly higher mortality at 100 days posttransplant [ 41 ] . further, larger prospective studies are needed to determine risk factors for posttransplant complications, the role of viral load and symptom burden at the time of transplantation, and the need to potentially delay transplantation for patients with hrv present prior to transplantation. ultimately, the issue of viral causality of disease and evaluation of prophylactic and treatment modalities will need to be addressed. the impact of hrv infection prior to sot is not known. like hct recipients, sot recipients are exposed to highly immunosuppressive regimens that leave them susceptible to respiratory viral infections. lung transplant recipients have the added disadvantage of altered lung immunity due to factors such as impaired ciliary clearance, poor cough refl ex, and abnormal lymphatic drainage. these factors can predispose to lower respiratory tract infections. the impact of hrv on outcomes in lung transplant recipients can range from asymptomatic infection to severe disease. in a pooled analysis of all respiratory viruses detected in lung transplant recipients, viruses were detected fi ve times more frequently when respiratory symptoms were present [ 42 ] . a correlation between higher symptom scores and higher rhinovirus load in the upper respiratory tract has been demonstrated, although even asymptomatic patients can have relatively high viral loads [ 43 ] . the relative rate of progression from upper to lower tract disease for hrv specifi cally is not known, although the effect on lung function has been evaluated in aggregate for all respiratory viruses and suggests a decline in forced expiratory volume (fev1) of −5% to −30% [ 42 ] . for hrv specifi cally, the fev1 loss was similar to that seen in other respiratory viruses [ 8 ] . the correlation between respiratory viral infections and acute rejection, chronic rejection, and bronchiolitis obliterans syndrome (bos) remains somewhat unclear, with several confl icting fi ndings when respiratory viruses were evaluated in aggregate [ 6 -8 ] . a recent large cohort of 250 lung transplant recipients, however, showed an independent association between respiratory viral infections (34% hrv) and chronic lung allograft dysfunction in multivariate models [ 9 ] . this association was infl uenced by time, with more of an effect within a shorter period following respiratory infection. larger, prospective studies investing individual viruses are needed to clearly assess the impact on these outcomes. unlike paramyxoviruses, hrv infection cannot be diagnosed based on characteristic histopathologic changes or changes in cell morphology. in the past, cell culture was used to diagnose hrv infection using multiple cell lines at low temperatures of 33-34 °c, often in rolling tubes. the cell lines utilized for the detection of hrvs may detect enteroviruses; hrv isolates are distinguished from enteroviruses by their lability in acid (loss in viral titer following exposure to a ph of 5). there are no commercially available antigen-detection assays or simple kits for the detection of hrv. rt-pcr has dramatically improved the ability to both detect and characterize hrvs, with current assays at least two to three times more sensitive than conventional culture methods [ 44 ] . some pcr assays are able to distinguish between enteroviruses and hrvs instead of the acid lability assays [ 45 ] . typing of hrvs based on pcr amplifi cation sequence variations in 5′-noncoding region also has been described [ 46 ] . new standardized methods to detect more of the over 100 strains of hrv have now been described [ 47 ] ; however, commercially available multiplex respiratory viral pcr panels contain primer/probe sets that can cross-react between enterovirus and hrv strains. new strains and types of hrv are being detected frequently and more diseases associated with hrv are being described using new and diverse molecular methods. there are no approved antivirals for the treatment of hrv infections. several agents have been evaluated in preclinical and clinical trials for the treatment of hrv infection in immunocompetent hosts, including capsid binding inhibitors, protease inhibitors, and rna synthesis inhibitors [ 48 ] . none of these agents have been evaluated in immunocompromised hosts. given the high prevalence and potential severity of hrv infection in this population, there is a great need for drug development and clinical trials for the prevention and treatment of lrti. outside of transplant recipients, there is a potential need for intervention in other populations such as patients with asthma or copd to prevent disease exacerbation [ 49 , 50 ] . covs are a frequent cause of the common cold, but little is known about the role of covs in immunocompromised patients [ 51 ] (table 32 -1 ). human group 1 (subtypes 229e and nl63) and human group 2 (oc43 and hku1) covs were originally reported as causes of human respiratory illnesses. the availability of more sophisticated diagnostic tools, such as rt-pcr , has facilitated the detection of covs in normal and immunocompromised persons. these improved molecular methods of viral discovery facilitated the recent identifi cation of the novel group 1 and 2 human cov subtypes-nl63 in 2004 [ 52 ] and hku1 in 2005 [ 53 ] . a more accurate clinical epidemiology of cov infection is beginning to emerge. it is now known that all four known subtypes of cov circulate simultaneously [ 54 ] , and that in addition to the common cold, cov is associated with upper respiratory tract infection and lrti in persons with and without underlying conditions [ 55 , 56 ] . in lung transplant recipients, covs appear to be the second most common respiratory viruses after picornaviruses with a detection rate of 13-27% of positive samples [ 5 -7 ] (figure 32-2 ) . in a prospective surveillance cohort of lung transplant recipients, coronaviruses were detected in 13% of all positive samples, again only second to picornaviruses [ 8 ] . two additional covs associated with outbreaks are the severe acute respiratory syndrome-associated cov (sars-cov) and the recently described middle east respiratory syndrome-cov (mers-cov) . the sars outbreak originated in guangdong province in china in 2002 and was characterized by a life-threatening, atypical pneumonia and was spread by close contact with infected humans, mostly to household contacts and health care workers [ 57 ] . sars-cov is not currently circulating in the world with the most recent human cases of infection reported in china in 2004 [ 58 ] . mers-cov fi rst emerged in the arabian peninsula in 2012, and since then travel-associated cases have been found in a number of countries outside the region [ 59 ] . in adults, the fatality rate is estimated to be 40%; in children asymptomatic infection is common but patients with underlying medical conditions are at increased risk [ 60 , 61 ] . there is little data on the incidence of sars-cov and mers-cov in immunocompromised hosts, although immune suppression is considered a risk factor. sars-cov has been described in liver transplant recipients and in patients with myelodysplastic syndrome [ 62 , 63 ] . mers-cov has been described in patients on chronic immunosuppression and in renal transplant recipients with a broad range of clinical presentations [ 64 , 65 ] . other covs have been reported to cause pneumonia in children and immunocompromised patients treated for hematologic malignancies [ 66 -68 ] . the role of coronavirus virus infection prior to transplantation is not known. although most cov infections result in relatively mild upper respiratory tract infection, these viruses have been associated with more severe lrti (e.g., bronchiolitis and pneumonia) in patients who are immunosuppressed, have asthma, or are premature. in one retrospective study carried out over 1 year, cov was detected in six immunocompromised children-fi ve with acute lymphocytic leukemia and one renal transplant recipient [ 54 ] . five patients were febrile at the time coronavirus was present, with fevers lasting 1-7 days. all patients initially presented with rhinorrhea and nasal discharge; two children had cough as a presenting symptom. chest radiographs of only one of the three children were abnormal; lrti based on decreased oxygen saturation, tachypnea, and abnormal chest radiograph was present in only one child with leukemia, who was signifi cantly neutropenic and lymphopenic at the time cov was detected. covs have been associated with lrti in hct recipients with sometimes fatal outcomes [ 66 , 67 , 69 -71 ] . the clinical characteristics of sars-cov and mers-cov infection in hct and sot patients are not well described, and presentation can range from mild symptoms to respiratory failure and death [ 62 -65 , 72 ]. until the advent of rt-pcr , techniques for the detection of cov were limited and the reliable identifi cation of cov was problematic. early detection techniques isolated two subtypes-oc43 and 229e, originally using organ cultures of human embryonic trachea, with morphology determined using negative staining with electron microscopy [ 73 ] . with the advent of molecular detection methods and increased interest in cov detection during the sars outbreak, new strains of covs have been discovered and new rt-pcr assays developed that facilitate further studies of these viruses. based on rt-pcr assays, four strains of non-sars covs (oc43, 229e, nl63, and hku1) appear to cocirculate during the non-summer months in temperate climates, and are associated with symptomatic disease in immunocompromised hosts [ 54 ] . guidance on rt-pcr and serologic assays for the confi rmation on mers-cov can be found on the world health organization website [ 74 ] . there are no approved antivirals for prophylaxis or treatment of cov infections and supportive care remains paramount in managing patients infected with coronaviruses. though several antivirals were used during the sars-cov epidemic, no clear benefi t could be established on systematic review [ 75 ] . oral ribavirin was evaluated in retrospective studies for the treatment of mers-cov in immunocompetent individuals; decreased survival was noted in one study when compared to matched controls [ 76 , 77 ] , however, larger prospective studies are needed to show true effi cacy. shedding of all coronaviruses may persist for up to months, and routine infection control practices are encouraged. evs are part of the picornaviridae family of viruses and can be associated with severe illness in immunocompromised hosts. evs include polioviruses, coxsackieviruses, and echoviruses; these are now all classified into four species: enterovirus a (ev-a), ev-b, ev-c, and ev-d. risk for infection and subsequent poor outcomes appears to be heavily influenced by age, although factors such as sex and socioeconomic status play a role in the general population. ev activity can be either sporadic or epidemic, and several outbreaks have been described. evs are typically found during the summer and early autumn in temperate climates. enterovirus-d68 (ev-d68) was fi rst identifi ed in california in 1962 [ 78 ] and has since been associated with several small outbreaks, both in the us and internationally, from 2009 to 2013 [ 79 -84 ] . in the summer of 2014, several hundred cases of severe respiratory illnesses in children in the united states were found to be associated with ev-d68 infection [ 85 ] , and several additional clusters have been described worldwide [ 86 -96 ] . evs can cause a wide spectrum of illnesses in immunocompetent individuals including asymptomatic infection, poliomyelitis, meningitis, encephalitis, cardiac disease, muscle disease, eye infections, respiratory infections, exanthems, and neonatal disease. the most frequently described manifestation in immunocompromised patients is respiratory disease, although the incidence and spectrum of disease is not known. according to one study, evs can be associated with lower respiratory tract infection and mortality; however, larger studies are needed to establish specifi c risk factors for worse outcomes [ 97 ] . most confi rmed cases of ev-d68 infection have been in children, occurring primarily in patients with underlying lung disease such as asthma or a history of wheezing. ev-d68 was also associated with several cases of acute fl accid paralysis in children during the 2014 outbreak in the united states, although defi nitive causation has not yet been established [ 98 , 99 ] . the impact of ev-d68 infection in immunocompromised hosts is not known; however, the association between ev-d68 and severe illness was described in eight adult immunocompromised patients with presumptive ev-d68 infection including hct recipients [ 100 ] . additionally, one recent report of adults with confi rmed ev-d68 infection included solid organ transplant recipients [ 87 ] . depending on the clinical scenario, evs can be detected from a number of clinical specimens including cerebral spinal fl uid, serum, respiratory specimens, cardiac tissue, and stool. evs may be identifi ed in throat samples as well as fecal specimens and cerebrospinal fl uid. commercial multiplex pcr assays contain primer/probe sets that may cross react between rhinoviruses and enteroviruses. a specifi c ev-d68 rt-pcr has been developed by the cdc and has been made publically available [ 101 ] . there are no approved antivirals approved for the treatment of evs. intravenous immunoglobulin (ivig) has been used in the treatment of neonatal enteroviral sepsis, but the effect on clinical outcomes is highly dependent on the presence of specifi c neutralizing antibodies and timing of administration [ 102 , 103 ] . pleconaril , an oral capsid inhibitor with activity against picornaviruses, has been evaluated in treatment of enteroviral infections including meningitis, neonatal sepsis, and respiratory infections [ 104 -108 ] but is not available for treatment. other capsid binders, protease inhibitors, and polymerase inhibitors are in various stages of development, but none are currently available for treatment of enteroviral infections [ 109 ] . no studies have shown effi cacy in immunocompromised hosts. human bocavirus (hbov) is a newly identifi ed human parvovirus that was originally identifi ed by random pcr amplifi cation/cloning technique on pooled respiratory secretions from hospitalized children with respiratory tract symptoms [ 110 ] . this virus was named "human bocavirus," due to its relatedness to the genome organization of two other parvoviruses, bovine parvovirus and minute virus of canines, in the family parvoviridae. this virus continues to be detected in young children with a winter seasonality [ 111 -113 ] . the relationship of hbov and respiratory disease in immunocompromised patients is not yet clear. preliminary evidence to date demonstrates case reports of disseminated hbov infection with involvement of the respiratory tract, blood, and stool in several patients, sometimes associated with gvhd and prolonged viral shedding in the feces [ 114 , 115 ] . other studies have reported little evidence linking this virus with pulmonary pathology or severe respiratory disease in hct or lung transplant recipients [ 116 -118 ] . further research is necessary to link this virus with the disease in the transplant recipient. no specifi c antiviral therapy is available. respiratory viruses are a signifi cant concern following hct and sot and can be associated with substantial morbidity and mortality, even among viruses traditionally not concerned pathogenic. new, sensitive diagnostic assays allow for routine detection of rhinoviruses, enteroviruses, coronaviruses, and bocavirus, and additional data on the epidemiology, risk factors, outcomes of infection, and the impact of different viral strains are desperately needed. preliminary studies suggest that detection of these viruses prior to transplant may affect outcomes, but additional studies are needed to explore this important clinical area. furthermore, as new antivirals are being developed, it will be important to identify high-risk patients that may benefi t from treatment. finally, a better understanding of these viruses will be able to inform better infection prevention strategies that will remain the mainstay of viral control. respiratory virus infections after stem cell transplantation: a prospective study from the infectious diseases working party of the european group for blood and marrow transplantation respiratory virus infections after marrow transplant: the fred hutchinson cancer research center experience respiratory viral infections after bone marrow/peripheral stem-cell transplantation: the christie hospital experience human rhinovirus and coronavirus detection among allogeneic hematopoietic stem cell transplantation recipients clinical impact of community-acquired respiratory viruses on bronchiolitis obliterans after lung transplant upper and lower respiratory tract viral infections and acute graft rejection in lung transplant recipients a prospective molecular surveillance study evaluating the clinical impact of community-acquired respiratory viruses in lung transplant recipients incidence and outcomes of respiratory viral infections in lung transplant recipients: a prospective study symptomatic respiratory virus infection and chronic lung allograft dysfunction analysis of the complete genome sequences of human rhinovirus the minor receptor group of human rhinovirus (hrv) includes hrv23 and hrv25, but the presence of a lysine in the vp1 hi loop is not suffi cient for receptor binding cadherin-related family member 3, a childhood asthma susceptibility gene product, mediates rhinovirus c binding and replication clinical and molecular features of human rhinovirus c. microbes infect biological characteristics and propagation of human rhinovirus-c in differentiated sinus epithelial cells frequency and natural history of rhinovirus infections in adults during autumn natural and experimental rhinovirus infections of the lower respiratory tract fields virology respiratory viral infections in immunocompetent and immunocompromised persons clinical virology detection of rhinovirus rna in lower airway cells during experimentally induced infection community-acquired pneumonia requiring hospitalization among u.s. adults manual of clinical microbiology rhinovirus infections in myelosuppressed adult blood and marrow transplant recipients rhinovirus as a cause of fatal lower respiratory tract infection in adult stem cell transplantation patients: a report of two cases rhinovirus infections in hematopoietic stem cell transplant recipients with pneumonia human rhinovirus infections of the lower respiratory tract in hematopoietic stem cell transplant recipients rhinoviruses infect the lower airways clinical and molecular epidemiology of human rhinovirus infections in patients with hematologic malignancy severity of human rhinovirus infection in immunocompromised adults is similar to that of 2009 h1n1 infl uenza human rhinovirus rna detection in the lower respiratory tract of hematopoietic cell transplant recipients: association with mortality. abstract presented at: asbmt tandem meeting detection of human rhinovirus c viral genome in blood among children with severe respiratory infections in the philippines molecular epidemiology of human rhinovirus infections in the pediatric emergency department association between human rhinovirus c and severity of acute asthma in children rhinovirus load and disease severity in children with lower respiratory tract infections molecular epidemiology of severe respiratory disease by human rhinoviruses and enteroviruses at a tertiary paediatric hospital in human rhinovirus c in adult haematopoietic after hematopoietic cell transplantation… stem cell transplant recipients with respiratory illness pretransplantation respiratory syncytial virus infection: impact of a strategy to delay transplantation respiratory virus infection in immunocompromised patients prospective study of respiratory viral infections in pediatric hemopoietic stem cell transplantation patients parainfl uenza virus 3 infection pre-and posthaematopoietic stem cell transplantation: re-infection or persistence? clinical outcomes associated with respiratory virus detection before allogeneic hematopoietic stem cell transplant respiratory viruses in lung transplant recipients: a critical review and pooled analysis of clinical studies role of rhinovirus load in the upper respiratory tract and severity of symptoms in lung transplant recipients use of polymerase chain reaction for diagnosis of picornavirus infection in subjects with and without respiratory symptoms classifi cation of respiratory tract picornavirus isolates as enteroviruses or rhinoviruses by using reverse transcription-polymerase chain reaction typing of human rhinoviruses based on sequence variations in the 5′ non-coding region real-time reverse transcription-pcr assay for comprehensive detection of human rhinoviruses the human rhinovirus: humanpathological impact, mechanisms of antirhinoviral agents, and strategies for their discovery toward antiviral therapy/prophylaxis for rhinovirus-induced exacerbations of chronic obstructive pulmonary disease: challenges, opportunities, and strategies rhinovirus and asthma: a storied history of incompatibility identifi cation of a new human coronavirus characterization and complete genome sequence of a novel coronavirus, coronavirus hku1, from patients with pneumonia clinical disease in children associated with newly described coronavirus subtypes coronavirusassociated pneumonia in previously healthy children characterization of human coronavirus oc43 and human coronavirus nl63 infections among hospitalized children <5 years of age a review of studies on animal reservoirs of the sars coronavirus severe acute respiratory syndrome-retrospect and lessons of 2004 outbreak in china update on the epidemiology of middle east respiratory syndrome coronavirus (mers-cov) infection, and guidance for the public, clinicians, and public health authorities estimation of mers-coronavirus reproductive number and case fatality rate for the spring 2014 saudi arabia outbreak: insights from publicly available data middle east respiratory syndrome coronavirus disease in children severe acute respiratory syndrome (sars) in a liver transplant recipient and guidelines for donor sars screening a major outbreak of severe acute respiratory syndrome in hong kong clinical features and viral diagnosis of two cases of infection with middle east respiratory syndrome coronavirus: a report of nosocomial transmission mers cov infection in two renal transplant recipients: case report fatal lower respiratory tract disease with human corona virus nl63 in an adult haematopoietic cell transplant recipient coronavirus 229e-related pneumonia in immunocompromised patients human coronavirus oc43 pneumonia in a pediatric cancer patient with down syndrome and acute lymphoblastic leukemia genetic variability of human coronavirus oc43-, 229e-, and nl63-like strains and their association with lower respiratory tract infections of hospitalized infants and immunocompromised patients coronavirus pneumonia following autologous bone marrow transplantation for breast cancer use of a novel virus detection assay to identify coronavirus hku1 in the lungs of a hematopoietic stem cell transplant recipient with fatal pneumonia short-term outcome of critically ill patients with severe acute respiratory syndrome viral infections of humans world health organization. laboratory testing for middle east respiratory syndrome coronavirus sars: systematic review of treatment effects ribavirin and interferon alfa-2a for severe middle east respiratory syndrome coronavirus infection: a retrospective cohort study ribavirin and interferon therapy in patients infected with the middle east respiratory syndrome coronavirus: an observational study a probable new human picornavirus associated with respiratory diseases clusters of acute respiratory illness associated with human enterovirus 68-asia enterovirus 68 among children with severe acute respiratory infection, the philippines detection of enterovirus 68 in serum from pediatric patients with pneumonia and their clinical outcomes. infl uenza other respi viruses enterovirus 68 in children with acute respiratory tract infections circulating viruses associated with severe complicated enterovirus infection in taiwan: a multi-year analysis a fatal central nervous system enterovirus 68 infection severe respiratory illness associated with enterovirus d68-missouri and illinois high frequency of enterovirus d68 in children hospitalised with respiratory illness in norway, autumn 2014. infl uenza other respi viruses the emergence of enterovirus d68 in a dutch university medical center and the necessity for routinely screening for respiratory viruses molecular epidemiology of enterovirus d68 from 2013 to 2014 in philippines characterization of enterovirus activity, including that of enterovirus d68, in pediatric patients in alberta, canada, in 2014 genome sequence of enterovirus d68 and clinical disease two cases of acute severe fl accid myelitis associated with enterovirus d68 infection in children enterovirus d68 infection low-level circulation of enterovirus d68-associated acute respiratory infections emergence of enterovirus d68 in denmark clinical characteristics and molecular epidemiology of enterovirus infection in infants <3 months in a referral paediatric hospital of barcelona enterovirus d68 nosocomial outbreak in elderly people upper and lower respiratory tract infections by human enterovirus and rhinovirus in adult patients with hematological malignancies a novel outbreak enterovirus d68 strain associated with acute fl accid myelitis cases in the usa (2012-14): a retrospective cohort study a cluster of acute fl accid paralysis and cranial nerve dysfunction temporally associated with an outbreak of enterovirus d68 in children in colorado clinical disease due to enterovirus d68 in adult hematologic malignancy patients and hematopoietic cell transplant recipients ev-d68) 2014 outbreak strain-specifi c real-time reverse transcription/polymerase chain reaction (rrt-pcr) assay instructions neonatal enterovirus infection: virology, serology, and effects of intravenous immune globulin effect of intravenous immunoglobulin for neonates with severe enteroviral infections with emphasis on the timing of administration enteroviral meningitis: natural history and outcome of pleconaril therapy oral pleconaril treatment of picornavirus-associated viral respiratory illness in adults: effi cacy and tolerability in phase ii clinical trials effi cacy and safety of oral pleconaril for treatment of colds due to picornaviruses in adults: results of 2 double-blind, randomized, placebo-controlled trials relationship of pleconaril susceptibility and clinical outcomes in treatment of common colds caused by rhinoviruses a randomized, double-blind, placebo-controlled trial of pleconaril for the treatment of neonates with enterovirus sepsis replication and inhibitors of enteroviruses and parechoviruses from the cover: cloning of a human parvovirus by molecular screening of respiratory tract samples human bocavirus infection frequent and prolonged shedding of bocavirus in young children attending daycare human bocavirus 1 primary infection and shedding in infants disseminated bocavirus infection after stem cell transplant persistence of human bocavirus dna in immunocompromised children rare and emerging viral infection in the transplant population human bocavirus: passenger or pathogen in acute respiratory tract infections? absence of human bocavirus in bronchoalveolar lavage fl uid of lung transplant patients key: cord-261598-w2tyongu authors: babady, n esther title: the filmarray® respiratory panel: an automated, broadly multiplexed molecular test for the rapid and accurate detection of respiratory pathogens date: 2014-01-09 journal: expert rev mol diagn doi: 10.1586/14737159.2013.848794 sha: doc_id: 261598 cord_uid: w2tyongu the filmarray respiratory panel (rp) (biofire(™) diagnostics, inc., salt lake city, ut, usa) is the first multiplex molecular panel cleared by the us fda for the detection of both bacterial and viral respiratory pathogens in nasopharygeal swabs. the filmarray rp targets 20 pathogens including 17 viruses and subtypes and three bacteria, and is performed with minimal sample manipulation. the filmarray rp has a fully automated sample-to-answer workflow with a turn-around-time of approximately 1 h. the reported sensitivity and specificity of the assay ranges from 80 to 100 and 100%, respectively, with the sensitivity for the adenovirus as low as 46%. a new version of the filmarray rp assay (version 1.7) with improved sensitivity for the adenovirus was released in 2013. the performance characteristics and simplified workflow have allowed its implementation in a wide range of laboratories. the filmarray rp has changed the diagnostic landscape and will have a significant impact on the care of patients with respiratory tract infection. the filmarray respiratory panel (rp) (biofire diagnostics, inc., salt lake city, ut, usa) is the first multiplex molecular panel cleared by the us fda for the detection of both bacterial and viral respiratory pathogens in nasopharygeal swabs. the filmarray rp targets 20 pathogens including 17 viruses and subtypes and three bacteria, and is performed with minimal sample manipulation. the filmarray rp has a fully automated sample-to-answer workflow with a turn-around-time of approximately 1 h. the reported sensitivity and specificity of the assay ranges from 80 to 100 and 100%, respectively, with the sensitivity for the adenovirus as low as 46%. a new version of the filmarray rp assay (version 1.7) with improved sensitivity for the adenovirus was released in 2013. the performance characteristics and simplified workflow have allowed its implementation in a wide range of laboratories. the filmarray rp has changed the diagnostic landscape and will have a significant impact on the care of patients with respiratory tract infection. community-acquired respiratory tract infections occur with varied frequency throughout the year. these infections are among the most common reasons for healthcare visits and can be caused by both viral and bacterial pathogens [1] . symptoms caused by viruses are non-specific and include sore throat, runny nose, watery eyes, cough, wheezing, shortness of breath, sputum production and nasal congestion. additionally, these symptoms are indistinguishable from those caused by bacterial pathogens [2] . mixed infections with two or more respiratory pathogens are not uncommon and might be underreported due to the limited sensitivity of methods currently used for diagnosis [3] [4] [5] . the identity of the pathogen causing symptoms is critical for rapid institution of adequate antiviral or antibiotic therapy as well as proper isolation of infected patients to prevent health-care associated infection, which can have devastating effects in an immunocompromised population [6, 7] . empirical, broad-spectrum therapy is often administered due to limitations of conventional diagnostic methods to provide timely results to guide clinician's decision. a great illustration of the importance of specific diagnosis came with the emergence of the novel h1n1 influenza virus in 2009. unlike the seasonal h1n1 influenza virus, the novel virus was sensitive to the antiviral drug oseltamivir (tamiflu ò ), but resistant to amantadine and rimantadine (symadine ò and flumadine ò ). since respiratory symptoms of these two influenza viruses are similar, accurate and timely identification of the correct virus was critical for the administration of appropriate therapy [8] . in normal hosts, most respiratory viral infections are selflimited, although complications can occurs. in the last two decades, respiratory viruses have become increasingly recognized as a serious cause of morbidity and mortality in hematopoietic stem cell transplant (hsct) and solid organ transplant (sot) recipients [9] [10] [11] [12] [13] . as part of their treatment, patients undergoing hsct and sot become severely immunosuppressed, rendering them highly susceptible to infectious pathogens. serious complications, which can be fatal, include viral pneumonia, lateral airflow obstruction syndrome, graft rejection and superinfection with bacterial and fungal pathogens [9, 12] . similarly, respiratory viruses can have a significant impact in young children and elderly patients resulting in frequent hospitalization and significant health costs [14, 15] . the most common viruses causing respiratory illness include influenza virus type a, influenza virus type b, respiratory syncytial virus (rsv), parainfluenza viruses (piv) 1, 2 and 3, human metapneumovirus (hmpv) and picornaviruses (rhinoviruses and enteroviruses) [7] . in children undergoing treatment for hematologic malignancies, infections due to influenza, parainfluenza parainfluenza viruses, and rsv are particularly common and have significant impact on oncologic care [16, 17] . the clinical significance of other viruses such as bocavirus, human coronaviruses nl63, human coronaviruses hku1, parainfluenza virus 4 and rhinovirus is less clear [18] . respiratory tract infections caused by bacterial pathogens includes streptococcus pneumoniae, haemophilus influenzae, as well as atypical bacterial that are often difficult to culture in the laboratory including chlamydophila pneumoniae and mycoplasma pneumoniae [19] . influenza viruses are enveloped, negative sense, single-stranded rna (ssrna) viruses belonging to the orthomyxoviridae family. there are three types of influenza viruses: influenza virus type a, influenza virus type b and influenza virus type c. types a and b are the most commonly isolated while type c is rare and often not included in routine diagnostic test. influenza viruses type a are further sub-typed based on the sequence of two glycoproteins: hemagglutinin (h) and neuraminidase (n) [20] . influenza viruses cause annual, seasonal epidemic characterized by an increase in the frequency of upper respiratory symptoms seen in the community between december and may in the northern hemisphere [21] . although the infection usually remains limited to the upper respiratory tract in most patients, complications due to influenza viruses have been reported in hsct and sot patients, especially lung transplant recipients [18, 22, 23] . in one study, the frequency of pneumonia caused by influenza viruses was 30% with a fatality rate of 15% in hsct recipients [12] . however, reports on frequency of complications has varied (7-44%) with fatality ranging from 15 to 28% in hsct recipients [18] . in sot, the complication rate varies based on the organ transplanted with lung transplants recipients being particularly at increased risk [22, 23] . rsv is an enveloped, non-segmented, ssrna virus belonging to the paramyxoviridae family. the seasonality of rsv follows closely that of influenza viruses with increased incidence in the winter months in the northern hemisphere [24] . in transplant patients and young children, infection with rsv can result in serious complications [25, 26] . as such, in most centers, detection of rsv pre-transplant results in a delay of sct until patient clears the infection. in one study of hsct recipients, 80-90% of patients with upper respiratory rsv infection developed pneumonia with 30-40% exhibiting symptoms within 7 days following upper respiratory tract symptoms [26] . in a large study of children under 5 years of age, the incidence of rsv infection was 18%. rsv infections occurred in previously healthy children with no pre-existing conditions. risk factors were identified only for those under 2 year of age [25] . piv are non-segmented, negative sense rna viruses, structurally related to rsv and belong to the paraxmyxoviridae family. there are four predominant piv serotypes including: piv-1, piv-2, piv-3 and piv-4 [27] . until recently, piv-4 was rarely isolated since most diagnostic assays available did not include reagents for the detection of piv-4 and so less information is available on this piv subtype. in the northern hemisphere, piv occurs year-round with piv-1 occurring most frequently in the fall, piv-2 occurring in the fall to early winter and piv-3 occuring in the spring and summer months [24, 27] . infections caused by piv are usually mild, ranging from asymptomatic shedding to the common cold, croup and bronchiolitis. similar to rsv, piv infection occurs frequently in children, with most children exposed to piv-3 by the age of two and piv-1 by the age of 5 [24] . the reported incidence of piv in hstc populations ranges from 0.2 to 18% with 13 to 43% progressing to lower respiratory tract infections (lrtis) and 12 to 50% of these lrti resulting in death with most infections caused by piv-3 [18, 28] . in lung transplant recipients, piv lrti have been associated with high rate of allograft rejection [29] . hmpv hmpv is another member of the paraxmyxoviridae family and is structurally related to rsv and piv. it is a relatively newly described virus and has become increasingly recognized as a cause of significant respiratory illness in young children, older adults and immunocompromised patients [30, 31] . the incidence of hmpv in hsct patients ranges from 3 to 7% with complications and fatality rate similar to those of rsv [18, 32] . similar to other paramyxoviridae viruses, hmpv can result in graft dysfunction and/or rejection in lung transplant recipients [33, 34] . adenoviruses are non-enveloped, double stranded dna viruses belonging to the adenoviridae family. adenoviruses are divided into seven types (a-g) and 52 serotypes, with type b/c most commonly associated with respiratory illnesses [35] . adenoviruses can remain dormant in lymphoid cells and may reactivate following immunosuppression [36] . adenoviruses are present year-round and common in pediatric populations where they generally cause self-limited, mild infections [37] . the incidence of adenovirus in hsct and sot patients ranges from 3 to 29% and 5 to 10%, respectively and varies with patient age, type of transplant, the degree of immunosuppression, and the organ being transplanted [13, 18, 38] . rhinoviruses are small, ssrna viruses of the picornaviridae family. rhinoviruses are a diverse group of viruses with more than 100 serotypes identified to date [39] . rhinoviruses are the most common cause of the 'common cold' and can be isolated throughout the year with a peak incidence in september [40] . in healthy individuals, rhinovirus infections are usually self-limited with symptoms lasting between 7-14 days [41] . several reports have linked rhinoviruses with more severe presentations including otitis media, bronchiolitis and exacerbation of asthma in children [41] . in immunocompromised hosts, severe complications including pneumonia, can occur. in one recent study, the severity of rhinovirus infection in immunocompromised patients was similar to that of the 2009 h1n1 pandemic influenza a [42] . with the use of current diagnostic molecular methods, information of the frequency and severity of infections caused by rhinoviruses will become available. coronaviruses (cov) are the largest rna viruses and belong to the coronaviridae family along with toroviruses [43] . cov are divided into three groups based on sequence homology with group 1 including cov-229e and cov-nl63, group 2 including cov-oc43 and cov-hku1 and group 3 which does not include any human covs. cov are responsible for a wide range of upper respiratory tract infections (urti) but are associated mainly with the 'common cold' [43] . only a limited number of reports on cov infections in immunocompromised hosts are available. in one study, asymptomatic shedding of cov was detected in the first 100 days following allogeneic hsct [44] . in other reports, lirts, with fatal outcomes have been reported in patients following sct [45, 46] . bacterial pathogens included in the respiratory panel (rp) include bordetella pertussis, mycoplasma pneumoniae and chlamydophila pneumoniae. all three organisms can cause both upper and lower respiratory tract infections including acute sinusitis, bronchitis and pneumonia [47] . b. pertussis causes pertussis or whooping cough primarily in non-vaccinated children and in adults, the infection is associated with a persistent cough of up to 2 weeks [48] . atypical organisms, including m. pneumoniae and c. pneumoniae, account for about one third of cases of bacterial pneumonia [19, 49] . unlike other common bacteria associated with these symptoms, b. pertussis, m. pneumoniae and c. pneumoniae do not grow readily on media used for routine bacterial culture and therefore require a high level of suspicion from the clinician to inform the laboratory and request specific diagnostic tests. in one study, persistent cough of at least 2 weeks was observed in children infected with one or more of these three organisms underscoring the need for sensitive and specific diagnosis to guide therapy [50] . diagnostic assays for respiratory pathogens conventional methods used to diagnose respiratory viral infections include rapid antigen tests, direct fluorescent antibody assays, shell vials and viral culture [51] . each of these methods has advantages and disadvantages with sensitivities and specificities ranging from 44 to 99% and 74 to 100%, respectively requiring additional testing of negative specimens. in addition, these assays can be subjective, require expertise for interpretation of cytopathic effect, have a limited range of detection and a turn-around-time as long as 14 days [51] . molecular assays, based on pcr, are rapid and sensitive compared to conventional methods. until recently, individual laboratories with the appropriate expertise, developed and implemented their own real-time pcr assays for each of the most common respiratory viruses [31, [52] [53] [54] . several commercial assays, for both research-use only (ruo) and those cleared by the fda (table 1) are now widely available, facilitating the implementation of these technologies in diagnostic microbiology laboratory. until recently, most fda-approved molecular assays targeted only the most commonly isolated viruses, including influenza a and b viruses and rsv, either as singleplex or duplex assay. although this method has the benefit of targeted approach to diagnosis of respiratory infections, it is limited to only the most common viruses, requires clinicians to order multiple tests and does not always permit the detection of co-infecting pathogens. an alternative approach was the development of broadly multiplexed assay that allow simultaneous detection of a wider range of possible pathogens (5 or greater) in one single tube [4, [55] [56] [57] [58] [59] . in 2008, the fda cleared the first broadly multiplexed molecular assay, the xtag ò respiratory viral panel ( two studies have evaluated the performance of the esensor rvp compared to other multiplex respiratory panels including the filmarray rp, the xtag rvp and the xtag rvp fast and to laboratory developed tests [58, 59] . the sensitivity and specificity of the esensor rvp varied from 90 to 100% and 99 to 100% depending on the viral target. the filmarray rp assay is the first and only fda-cleared assay for the qualitative detection of nucleic acid targets from both viruses and bacteria in nasopharyngeal swab specimens. the filmarray rp was initially fda-cleared for the detection of 15 viruses including the same viruses as the xtag rvp plus cov nl63 and hku1 and piv-4. in 2012, five more targets were cleared including cov 229e and oc43, and three bacterial targets b. pertussis, c. pneumoniae and m. pneumoniae (version 1.6). to increase the detection rate for adenovirus, a second assay for the virus was added to the rp and this was fda cleared in 2013 (version 1.7). the assay is performed on the filmarray instrument and can be completed in approximately 1 h. the filmarray is an integrated platform that combines automated sample preparation, total nucleic acid extraction with nested, multiplex pcr and reverse transcriptase pcr and automated detection of amplified targets [62] . the instrument has a small footprint (39.1 â 25. 4 â 16.3 cm) and contains pneumatic and electrical components to move reagents through the pouch and perform the functions described. nested pcr is effected using two peltier devices, and melt curves are detected using a blue led light source and a charge couple device (ccd) camera (figure 1) [62, 63] . nucleic acids are released from cells by the combined action of denaturing buffers, ceramic beads and a bead beater. once lysed, cells are transferred to a blister containing magnetic beads which binds the released nucleic acids. following application of a magnet and several washes, the nucleic acids are eluted and moved to pcr sites for amplification. once purified, the dna/rna extract is mixed with the first stage multiplexed pcr reagents, including a set of outer primers, and temperature cycling occurs on the first peltier device. a reverse transcription pcr step is performed prior to the first pcr cycle. amplification of target nucleic acids is then accomplished using the principle of nested pcr which uses a two stages approach to increase the sensitivity and specificity of the pcr reaction [64] . the resulting mixture is diluted and moved to the second stage pcr where the amplified products from the first stage pcr are used as templates and further amplified using a second set of inner primers with cycling occurring on the second peltier device. during the second stage pcr, lcgreen plus, a fluorescent, dna intercalating dye, is incorporated into the dna as it is amplified. positive reactions are determined based on dna melting curve analysis of amplified product. each target is run in triplicate, in three separate wells of the microarray. the melt curve of each individual replicate for each target is measured following amplification and a positive reaction is determined if the melt curve shape and peak falls within pre-established ranges. control of the instrument and interpretation of the results are done automatically by the filmarray software [62, 63] . reproduced with permission from [63] ó biofire diagnostics (2010). the filmarray ò rp diagnostic profile www.expert-reviews.com the pouch all reagents necessary to perform the assay are contained in the vacuum sealed filmarray pouch. the lyophilized reagents, which are distributed in 12 separate reservoirs, are rehydrated with a buffer solution just prior to adding the specimen. additional reagents are contained in three of the pouch's six blisters, including ceramic beads for lysis, magnetic beads for dna/ rna purification and the oligonucleotides for the first stage pcr. the blisters are connected to channels which are used to move the liquid reaction from one blister to another. the last section of the pouch is the 'solid-phase array', a set of 102 wells with each well containing primers for the second stage pcr (figure 1) [62, 63] . the filmarray rp targets 20 viruses and bacteria including influenza virus type a (including subtypes h1n1, h3n2 and the 2009-h1n1), influenza virus type b, rsv, adenovirus, hmpv, rhinoviruses/enteroviruses, cov hku1, nl63, 229e, oc43, piv-1, piv-2, piv-3, piv-4, b. pertussis, c. pneumoniae and m. pneumoniae. in addition, the assay contains two target controls, a rna target from schizosaccharomyces pombe, which controls the entire process from extraction to dna melt analysis, and a dna target control, which is included in the array well and controls for the second stage pcr. each target has at least three replicate assays and the final interpretation is determined from the number of replicates that are positive. the filmarray rp has multiple targets for some of the pathogens detected (e.g., influenza virus type a has two pan-influenza targets and one ha specific target). published reports on the performance of the filmarray rp are summarized in table 2. the filmarray rp has been evaluated against other fda cleared molecular assays [59, [65] [66] [67] . most of the published reports evaluated the premarket version of the filmarray rp on samples from pediatric patients where the prevalence of respiratory viruses is expected to be higher. overall, the filmarray rp has a sensitivity and specificity >80%. however, the reported sensitivity of the first version of filmarray rp for adenovirus is around 50%. as noted above, the manufacturer has released a new version of the filmarray rp with improved adenovirus detection. rand et al. compared the performance of the filmarray rp and xtag rvp on 200 specimens from both adult and pediatric patients, previously tested by viral culture and antigen testing [67] . the complete agreement between the two tests was excellent at 91.5% with more viruses detected by the filmarray rp (160 viruses by filmarray rp versus 149 viruses by xtag rvp). notably, the filmarray rp had greater sensitivity for rsv than the xtag rvp (100 vs 82.2%). loeffelholz and colleagues compared the filmarray rp to the series of fda cleared prodesse (genprobe) real-time pcr assays including the proflu+, pro-fast+, proparaflu+, prohmpv+ and proadeno+ [66] . the fil-marray rp and prodesse assays showed good overall agreement (94.3%) when tested on 192 specimens from pediatric patients, with the filmarray rp being more sensitive for piv-1 and -3, detecting an additional two piv-1 and three piv-3 not detected by the proparaflu + assay. the proadeno+ was more sensitive for adenoviruses than the filmarray rp, detecting a total of 11 adenoviruses compared to five adenovirus detected by the filmarray rp. additionally, the authors compared the filmarray rp to ldt for targets not included in the prodesse assays with the sensitivity ranging from 50 to 100% although the number of positive specimens tested was small (n = 1 to 8), except for rhinovirus/enterovirus (n = 118) targets. we compared the performance of the filmarray rp to the pre-market version of the xtag rvp fast on 358 respiratory specimens from pediatric patients [65] . in our report, the filmarray rp was overall more sensitive than the xtag rvp fast for most viruses common to both assays, especially for rsv (100 vs 60%) and flub (100 vs 50%). however, the xtag rvp fast was more sensitive than the filmarray rp for piv-4 (100 vs 33.3%) and rhinoviruses (97.6 vs 91.6%). in a recent study, the filmarray rp was evaluated along other multiplexed fda-cleared molecular assays including the xtag rvp, the xtag rvp fast and the genmark esensor [59] . the filmarray rp had an overall sensitivity of 84.5% with sensitivity varying from 92-100% for influenza virus a/h3, mpv, piv-1, piv-2, piv-3 and rsv b and sensitivity varying from 73-83% for influenza virus a h1/2009, influenza virus, rsv a and rhinoviruses/enteroviruses. of note, the sensitivity of the filmarray rp and the xtag rvp for rsv was similar [59] . the sensitivity of the filmarray rp for adenoviruses was especially low at 57% as reported in other studies [66, 68] . other studies have evaluated the filmarray rp against conventional methods, laboratory-developed assays and ruo assays [57, [68] [69] [70] . in one study done using mock specimens prepared by combining previously positive nasal washes, nasal swabs, bronchoalveolar lavage fluids, sputum and tracheal aspirates, the filmarray rp detected 90% of viruses identified by the ldt assays with discordant results mainly occurring at low viral loads [69] . the fil-marray rp was also evaluated against the resplex ii panel v2.0 (qiagen) and the two tests had an overall agreement of 83.8% with the filmarray rp detecting about 10% more positive specimens and 21% more viruses than the resplex assays [57] . pierce et al. reported an overall agreement between the filmarray rp and their ldt real-time pcrs of 98.6% with the filmarray rp adenovirus assay being less sensitive than the ldt adenovirus real-time pcr, missing 13/24 adenovirus positive specimens. the filmarray rp was unable to specifically detect adenovirus serotypes 6 and 41 at any viral concentrations and serotypes 2, 20, 35 and 37 at low viral concentrations [68] . according to the manufacturer's product insert, the low sensitivity of the filmarray rp for adenovirus specifically relates to adenovirus c, serotypes 2 and 6 as confirmed in pierce et al. study, and recommended testing of negative specimens using alternative methods to detect adenoviruses when suspected (filmarray rp pi v1.6). in april 2013, a new version of the filmarray rp assay (version 1.7) was released. the filmarray rp v1.7 was designed to enhance the detection of adenoviruses by adding a second adenovirus assay to the panel. no peer-reviewed published reports are yet available on the performance of the improved assay for adenovirus. abstracts presented at the 29th clinical virology symposium showed increased sensitivity of the filmarray rp v1.7 when compared to filmarray rp v1.6 (81.6 vs 23.8%) but still missed adenoviruses detected by the xtag rvp or prodesse proadeno+ assay and ldt adenovirus singleplex [71, 72] . respiratory tract infections can be caused by a wide range of pathogens including viruses and bacteria. in addition, novel viruses, including bocavirus and novel covs, are increasingly being recognized as causes of respiratory tract illness. given the non-specific nature of the respiratory symptoms, the ability to detect these novel viruses as well as other significant pathogens is paramount for optimal patient care. the filmarray rp is the most extensive panel that is currently fda cleared for rapid detection of respiratory tract pathogens. although its adenovirus assays still require improvement, the overall clinical sensitivity and specificity of the filmarray rp assay is better than conventional methods and comparable to other high complexity multiplexed molecular assays on the market. multiplex molecular diagnostic assays are ideal for infections with a wide differential diagnosis. however, the design of broadly multiplexed assays is challenging as multiplex pcr assays are subject to decreased sensitivity due in part to, targets' competition and primer dimers formation [64] . the overall sensitivity and specificity of the filmarray rp in published reports varied between 85-100% and 100%, respectively with the sensitivity of the adenovirus assays remaining a challenge even in the new version of the assay. however, even with the lower sensitivity of the adenovirus, the filmarray rp has drastically changed the diagnostic landscape and allowed implementation of a sensitive and rapid molecular assay in a wide range of diagnostic settings [73, 74] . the filmarray rp was the second highly multiplexed molecular assay to be cleared by the fda and the first one to include bacterial pathogens in the panel. it is important to note however that as extensive as the filmarray rp panel is, other important bacterial causes of pneumonia including s. pneumoniae and h. influenzae, are not included on the panel. when compared to other fda cleared multiplexed assay (table 1) , the filmarray rp has the greatest number of targets in its panel, the simplest workflow, the shortest turn-around-time and the highest reagent cost [59, 65] . the true value of the filmarray rp assay lies in its ability to provide actionable results to clinicians and to facilitate the flow of patients in the hospital by providing infection control staff with real-time information. the only specimen type that is currently cleared by the fda for testing is naso-pharyngeal swabs. however, other respiratory tract specimens are routinely submitted to the laboratory for testing including nasal swabs, nasal washes, throat swabs, bronchoalveolar lavage fluids and sputum. all studies included in this article evaluated the off-label use of filmarray rp on other specimen types with sensitivity and specificity similar to that of np swabs suggesting that rp would be useful for diagnosing both upper and lower respiratory tract infections. five-year view five years ago, the fda cleared the xtag rvp as the first broadly multiplexed molecular assay for the detection of respiratory viruses in nasopharyngeal swabs. today there are four fda-cleared broadly multiplexed assays, the xtag rvp and the xtag rvp fast (luminex corp.), the esensor rvp (genmark inc.) and the filmarray respiratory panel (rp) (biofire diagnostics). the filmarray rp tests for a wide range of pathogens with high sensitivity and specificity and in a timely manner. as such, the filmarray rp has raised the expectations of both the clinical laboratories and clinicians as to what is possible for the rapid and sensitive diagnosis of infectious diseases in general. the next 5 years should see the continued increase in the options for rapid, sensitive and simple to perform molecular assays for infectious disease diagnosis. one of the limitations of the filmarray rp is still its cost compared to traditional methods and other commercially available molecular diagnostic tools, although when considering workflow and labor costs, the overall cost of the filmarray rp is comparable to other highly multiplexed assays (i.e., xtag rvp and the esensor rvp) [59] . in this era of health care reform, clinical laboratories and hospitals are constantly faced with the challenge of delivering the best patient care possible in the most cost-effective way. for the filmarray rp to fully realize its potential, the cost of the assay will have to decrease so to be available to a wider range of patients. the filmarray rp is currently classified as a moderate complexity test and as such is subject to the requirements associated with performing moderate/high complexity tests including quality control and assessment and performance of the test in accredited laboratory settings [75] . it is possible that the filmarray rp might eventually obtain a waived complexity category, which will allow its use in point of care settings like an emergency room or doctor's office. as antiviral drugs become available to treat viruses other than influenza and rsv, the ability of any molecular platform to rapidly diagnose the specific virus responsible for a respiratory syndrome will become even more important. furthermore, quantitative or semi-quantitative assays that provide information on viral loads will be useful for monitoring of patient response to treatment or to gain a better understanding of the infectious doses for each of the viruses included in multiplexed panels. the next five years will see an emergence of outcome studies on the real impact and benefit of these assays on patient care and public health in general. finally, the impact of new and emerging infections cannot be denied. in the last year, the avian h7n9 influenza a virus and the middle eastern syndrome coronaviruses have reminded us of how quickly new and emerging viruses can appear and spread. the challenge for manufacturers of these molecular assays will be to design the assays to be sensitive and specific and yet broad enough to accommodate potential emerging pathogens. the author has no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. this includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties. no writing assistance was utilized in the production of this manuscript. • respiratory tract infections occur all year long with different pathogens peaking at different times but overlapping in their seasonal distributions. • clinical symptoms caused by respiratory pathogens, including bacteria and viruses are non-specific. • there are currently four highly multiplexed respiratory panels that are us fda approved for diagnosis of respiratory infections. • the filmarray respiratory panel is sensitive and specific, has the simplest workflow and fastest turn-around-time of all assays. • the filmarray respiratory panel is the only panel that includes both viral and bacterial causes of upper respiratory tract infections/lower respiratory tract infections . national ambulatory medical care survey: 2002 summary etiology of community-acquired pneumonia in 254 hospitalized children respiratory viral infections in children with leukemia evaluation of multiple test methods for the detection of the novel 2009 influenza a (h1n1) during the new york city outbreak early pulmonary complications after hematopoietic stem cell transplantation in pediatric patients: association with cytomegalovirus infection nosocomial infections in patients with cancer detection of respiratory viruses by molecular methods h1n1 influenza the challenge of respiratory virus infections in hematopoietic cell transplant recipients respiratory virus infections after marrow transplant: the fred hutchinson cancer research center experience community respiratory virus infections in bone marrow transplant recipients: the m.d. anderson cancer center experience respiratory viral infections in adults with hematologic malignancies and human stem cell transplantation recipients: a retrospective study at a major cancer center viral infections in immunocompromised patients: what's new with respiratory viruses? infectious disease hospitalizations among infants in the united states management of respiratory infections in the elderly parainfluenza virus infections in children with hematologic malignancies symptomatic parainfluenza virus infections in children undergoing hematopoietic stem cell transplantation changing epidemiology of respiratory viral infections in hematopoietic cell transplant recipients and solid organ transplant recipients approach to common bacterial infections: community-acquired pneumonia a revision of the systerm of nomenclature for influenza viruses: a who memorandum influenza viruses. in: manual of clinical microbiology community respiratory viral infection in adult lung transplant recipients influenza virus infection in adult solid organ transplant recipients respiratory syncytial virus and parainfluenza virus the burden of respiratory syncytial virus infection in young children community-acquired respiratory syncytial virus and parainfluenza virus infections after hematopoietic stem cell transplantation: the fred hutchinson cancer research center experience parainfluenza viruses the characteristics and outcomes of parainfluenza virus infections in 200 patients with leukemia or recipients of hematopoietic stem cell transplantation the epidemiology of parainfluenza virus infection in lung transplant recipients human metapneumovirus infections in young and elderly adults clinical characterization of human metapneumovirus infection among patients with cancer respiratory viral infections in transplant and oncology patients human metapneumovirus infection in lung transplant recipients: clinical presentation and epidemiology incidence and morbidity of human metapneumovirus and other community-acquired respiratory viruses in lung transplant recipients adenovirus infections in transplant recipients adenovirus infection in hematopoietic stem cell transplantation: effect of ganciclovir and impact on survival treatment of adenovirus infections in the immunocompromised host adenoviruses in immunocompromised hosts sequencing and analyses of all known human rhinovirus genomes reveal structure and evolution new developments in the epidemiology and clinical spectrum of rhinovirus infections human rhinoviruses severity of human rhinovirus infection in immunocompromised adults is similar to that of 2009 h1n1 influenza in: manual of clinical microbiology human rhinovirus and coronavirus detection among allogeneic hematopoietic stem cell transplantation recipients fatal lower respiratory tract disease with human corona virus nl63 in an adult haematopoietic cell transplant recipient coronavirus 229e-related pneumonia in immunocompromised patients the atypical pneumonias: clinical diagnosis and importance pertussis of adults and infants role of chlamydia pneumoniae and mycoplasma pneumoniae as causative agents of community-acquired pneumonia in hospitalised children and adolescents bordetella pertussis, bordetella parapertussis, mycoplasma pneumoniae, chlamydia pneumoniae and persistent cough in children. scand detection of respiratory viruses using non-molecular based methods comparison of conventional and molecular detection of respiratory viruses in hematopoietic cell transplant recipients h1n1 influenza infection in cancer patients and hematopoietic stem cell transplant recipients evaluation of the cepheid respiratory syncytial virus and influenza virus a/b real-time pcr analyte specific reagent development of a rapid automated influenza a, influenza b, and respiratory syncytial virus a/b multiplex real-time rt-pcr assay and its use during the 2009 h1n1 swine-origin influenza virus epidemic in milwaukee detection of respiratory viruses with a multiplex polymerase chain reaction assay (multicode-plx respiratory virus panel) in patients with hematologic malignancies comparison of two broadly multiplexed pcr systems for viral detection in clinical respiratory tract specimens from immunocompromised children comparison of the genmark diagnostics esensor respiratory viral panel to real-time pcr for detection of respiratory viruses in children comparison of the biofire film array rp, genmark esensor rvp, luminex xtag rvpv1, and luminex xtag rvp fast multiplex assays for detection of respiratory viruses principles of the xtag respiratory viral panel assay (rvp assay) comparison of the luminex xtag respiratory viral panel with xtag respiratory viral panel fast for diagnosis of respiratory virus infections an automated nested multiplex pcr system for multi-pathogen detection: development and application to respiratory tract infection biofire diagnostics inc. filmarray ò operator's manual ruo in vitro nucleic acid amplification techniques comparison of the luminex xtag rvp fast assay and the idaho technology filmarray rp assay for detection of respiratory viruses in pediatric patients at a cancer hospital comparison of the filmarray respiratory panel and prodesse real-time pcr assays for detection of respiratory pathogens comparison of two multiplex methods for detection of respiratory viruses: filmarray rp and xtag rvp comparison of the idaho technology filmarray system to real-time pcr for detection of respiratory pathogens in children comparison of filmarray respiratory panel and laboratory-developed real-time reverse transcription-polymerase chain reaction assays for respiratory virus detection respiratory virus detection in immunocompromised patients with filmarray respiratory panel compared to conventional methods evaluation of the biofire filmarray respiratory panel v1.6 and v1.7 for the detection of respiratory viruses. presented at: 29th clinical virology symposium evaluation of a modified version of the filmarray respiratory panel (ruo v1.7) for detection of adenovirus from nasopharyngeal specimens. presented at: 29th clinical virology symposium implementation of filmarray respiratory viral panel in a core laboratory improves testing turnaround time and patient care evaluation of the filmarray(r) respiratory panel for clinical use in a large children's hospital code of federal regulations. title 42, cfr 493.17. test categorization key: cord-263927-hnsyas9q authors: peci, adriana; winter, anne‐luise; gubbay, jonathan b.; skowronski, danuta m.; balogun, elizabeth i.; de lima, cedric; crowcroft, natasha s.; rebbapragada, anu title: community‐acquired respiratory viruses and co‐infection among patients of ontario sentinel practices, april 2009 to february 2010 date: 2012-08-09 journal: influenza other respir viruses doi: 10.1111/j.1750-2659.2012.00418.x sha: doc_id: 263927 cord_uid: hnsyas9q please cite this paper as: peci et al. (2012) community‐acquired respiratory viruses and co‐infection among patients of ontario sentinel practices, april 2009 to february 2010. influenza and other respiratory viruses 7(4), 559–566. background respiratory viruses are known to cocirculate but this has not been described in detail during an influenza pandemic. objectives to describe respiratory viruses, including co‐infection and associated attributes such as age, sex or comorbidity, in patients presenting with influenza‐like illness to a community sentinel network, during the pandemic a(h1n1)pdm09 in ontario, canada. methods respiratory samples and epidemiologic details were collected from 1018 patients with influenza‐like illness as part of respiratory virus surveillance and a multiprovincial case–control study of influenza vaccine effectiveness. results at least one virus was detected in 668 (65·6%) of 1018 samples; 512 (50·3%) had single infections and 156 (15·3%) co‐infections. of single infections, the most common viruses were influenza a in 304 (59·4%) samples of which 275 (90·5%) were influenza a(h1n1)pdm09, and enterovirus/rhinovirus in 149 (29·1%) samples. the most common co‐infections were influenza a and respiratory syncytial virus b, and influenza a and enterovirus/rhinovirus. in multinomial logistic regression analyses adjusted for age, sex, comorbidity, and timeliness of sample collection, single infection was less often detected in the elderly and co‐infection more often in patients <30 years of age. co‐infection, but not single infection, was more likely detected in patients who had a sample collected within 2 days of symptom onset as compared to 3–7 days. conclusions respiratory viral co‐infections are commonly detected when using molecular techniques. early sample collection increases likelihood of detection of co‐infection. further studies are needed to better understand the clinical significance of viral co‐infection. a novel influenza virus, a(h1n1)pdm09 emerged in april 2009 and spread rapidly, primarily through human-tohuman transmission. several million people were infected globally. 1 an important feature of this virus was that it mostly affected younger people with 60% of patients under 18 years of age, suggesting possible pre-existing immunity in the elderly due to previous exposure to antigenically related influenza strains. 2, 3 the assumption made in most pandemic plans before 2009 was that the pandemic virus would be the dominant circulating respiratory virus. 4 few studies performed extensive respiratory testing beyond influenza during the pandemic, and fewer still focused on community cases. casalegno et al. documented cocirculation and co-infection of a(h1n1)pdm09 with rhinovirus during the pandemic. 5 watanabe et al. found a wide range of etiologic agents were identified among respiratory samples that were influenza negative, highlighting the need to diagnose other viral organisms that can co-circulate with influenza. 6 louie et al. investigated samples from laboratory-confirmed fatal a(h1n1)pdm09 cases during the pandemic and bacterial pathogens were identified in 22 of 77 samples. 7 prior to the 2009 pandemic, respiratory viral co-infection was reported in 7-27% of respiratory samples submitted for viral diagnosis. [8] [9] [10] [11] [12] [13] higher proportions of influenza a, respiratory syncytial virus (rsv), parainfluenza viruses, and rhinovirus, compared with other circulating viruses have been detected in patients with co-infections. [14] [15] [16] [17] [18] co-infection has not been fully explored due to limitations of several studies. some studies focused on younger age groups, hospitalized patients or deceased individuals, which does not represent the general population. [8] [9] [10] 12, 15, 19, 20 others have utilized a small sample size or limited their focus to certain viral pathogens, underestimating the role of other viruses in co-infection. 9, 15, 16, 20, 21 this study enrolled community patients presenting with (ili) to a community sentinel network, during the influenza pandemic a(h1n1)pdm09 in ontario, canada and documented the profile of respiratory viruses causing ili symptoms. this study aimed to describe respiratory viruses including co-infections and host-associated attributes such as age, sex, and comorbidity. data were collected as part of a multiprovincial case-control sentinel network study that has been described elsewhere. 21 the sentinel network included 117 sentinels across the province of ontario (with a population of 13ae4 million) who volunteered to participate in the study. it was anticipated that each sentinel would submit an average of 1-2 samples per week from their clinical practice during the study period, april 21, 2009 to february 25, 2010 . this period was chosen to span the full pandemic in ontario. eligible patients were ontario residents, who presented to a sentinel's office with influenza-like illness (ili) within seven days of symptom onset; number and selection of eligible patients was at the sentinel's discretion. ili was defined as acute onset of fever and cough and one or more of the following: sore throat, myalgia, arthralgia, headache or prostration. standard information was collected including date of birth, sex, chronic conditions, symptom onset, and sample collection date. the main outcome was the number of respiratory viruses detected per sample. samples were categorized as negative, single infection or co-infection when no virus, one virus, or at least two viruses were detected, respectively. age was determined as age at symptom onset and categorized as 0-4, 5-14, 15-29, 30-54, 55-64, and 65+years. time to sample collection was calculated as the difference between sample collection and symptom onset dates and categorized as less than or equal to twodays or 3-7 days. chronic condition was defined as heart ⁄ lung ⁄ renal ⁄ metabolic ⁄ blood ⁄ immune conditions or conditions that compromise the management of respiratory secretions and increase risk of aspiration and categorized as yes ⁄ no. this study was approved by the university of toronto's ethics board and all patients gave verbal consent to participate. a nasal or nasopharyngeal sample was collected from each patient using starswabò multitrans collection swab and transported at 4°c for testing at public health ontario laboratory (phol)-toronto; in this study, each sample represents one patient. viral rna was extracted directly from samples using nuclisens ò easymag ò (biomérieux, inc., marcy l'etoile, france). samples were tested for influenza a and influenza b by influenza real-time reverse transcriptase (rrt)-pcr and also for influenza a, influenza b, enterovirus ⁄ rhinovirus, rsv, parainfluenza, adenovirus, coronaviruses, and metapneumovirus by a commercial multiplex pcr method [luminex respiratory viral panel (luminex molecular diagnostics, toronto, on, canada) or seeplex rv (seegene usa, rockville, md, usa)]. in the event of discrepant results between the two methods, positive results for influenza a by either method were considered positive. rrt-pcr was used for subtyping of influenza a samples; all influenza a specimens were subtyped, but not all attempts were successful. statistical analyses were performed using stata software version 10.0 (statacorp, college station, tx, usa). descriptive analyses were conducted to derive the proportion of single, co-infection and no infection as well as describe patient characteristics using chi-square. crude and adjusted multinomial logistic regression were employed to evaluate any association of single, co-infection and noinfection with patient characteristics including age, sex, chronic condition, and time to sample collection. odds ratios (or) with 95% confidence intervals (ci) were calculated. a total of 1018 respiratory samples from 1018 patients with influenza-like illness were included in this study after excluding 102 (9ae1%) samples that did not meet study inclusion criteria (figure 1 ). at least one respiratory virus was detected in 668 (65ae6%) of the samples. of the 831 detected viruses, influenza a was the most frequent accounting for 452 (54ae4%) followed by enterovirus ⁄ rhinovirus 194 (23ae3%) and rsv 120 (14ae4%) ( table 1) . of 452 influenza a viruses, 408 (90ae3%) were a(h1n1)pdm09, two (0ae4%) were h3, and 42 (9ae3%) could not be subtyped presumably due to low viral load. peaks in detection for influenza a occurred in june and october 2010, for enterovirus ⁄ rhinovirus in september 2010, and for rsv in october 2010 ( figure 2 ). a single virus was detected in 512 (50ae3%) samples. of these, 304 (59ae4%) were influenza a and 208 (40ae6%) were other respiratory viruses, the most common being enterovirus ⁄ rhinovirus, detected in 149 (29ae1%) samples (table 2) . peaks for single infection occurred in june and september 2009, which were mainly due to the increase in influenza a and enterovirus ⁄ rhinovirus, respectively ( figure 3 ). viral co-infection was detected in 156 (15ae3%) of the samples of which 149 (95ae5%) were dual infections and seven (4ae5%) triple infections. one hundred and fortyeight (94ae9%) of the co-infections were combination of a(h1n1)pdm09 and another respiratory virus and eight (5ae1%) were non-influenza combinations. the most common co-infections were influenza a ⁄ rsv b and influenza a ⁄ enterovirus ⁄ rhinovirus, responsible for 64ae1% and 21ae8% of co-infections, respectively ( table 3 ).the highest proportion of co-infection was detected in october, corresponding with peak activity of influenza a and rsv ( figure 3 ). the median age of patients in the study was 23 years with a range of 3 months-96 years of age ( table 4 ). the highest proportion of single and co-infections was observed in children 5-14 and 0-4 years of age, respectively. the proportion of those with no infection detected steadily increased with age, peaking at the elderly, aged 65 years and over ( figure 4 ). females were overrepresented, comprising 599 (58ae8%) of the patients included in this study. patients with no-infection, single infection, and co-infection did not differ with regards to sex. two hundred and nineteen (21ae6%) patients had a chronic condition. of these, 37ae4% had no virus detected, 44ae8% had single infections, and 17ae8% had co-infections, whereas among the 795 participants without comorbidities, the distribution was 33ae6%, 51ae8%, and 14ae6%, respectively; however, that was not statistically significant ( table 4 ). the median number of days from symptom onset to sample collection was two with a range of 0-7 days. five hundred and eighty-two (57ae2%) and 436 (42ae8%) samples were collected within 2 days and 3-7 days, respectively. of the 582 samples collected within 2 days of onset, 32ae9% had no virus detected, 48ae5% had single infections, and 18ae6% had co-infections, whereas among those collected within 3-7 days, the distribution was 36ae3%, 52ae8%, and 11%, which was statistically significant. in crude and adjusted multinomial logistic regression, patients with single and co-infections were compared to those with no infection. compared to the elderly, patients under 65 years of age were more likely to have a single infection; the highest likelihood was observed in children 5-14 years of age (table 5) . patients under 30 years of age were more likely to have co-infections compared with patients 65 and over; this was most evident in the 0-4 age group. presence of a chronic condition did not increase the likelihood of single infection but increased the likelihood of co-infection; this did not achieve significance. co-infection was more likely detected in patients who had samples collected within 2 days as compared to 3-7 days; this did not apply for those with single infections. there was no sex difference. in this study, 66% of samples tested during the 2009 pandemic in ontario had at least one virus detected and 15% had co-infections. these findings are consistent with reports from other studies with the range of co-infection reported from 7-27%. [8] [9] [10] [11] [12] [13] however, positivity and co-infection rates vary widely between studies. there are various reasons for this finding: firstly, detection methods differ notably between studies, which impacts sensitivity, specificity and other technical parameters; secondly, viruses targeted differ from one study to another as does the study population. 21 this study was conducted during the influenza pandemic a(h1n1)pdm09 which was associated with an increased number of samples submitted and high detection of figure 2 ). this demonstrates the importance of monitoring circulating respiratory viruses when advising clinicians to prescribe antivirals empirically during a pandemic. 2 (0-7) 2 (0-7) 2 (0-7) 2 (0-7) *p-value <0ae05 is considered significant. **time to specimen collection = collection date-symptom onset date. despite the higher prevalence of enterovirus ⁄ rhinovirus (23ae4%) than rsv (14ae5%), co-infection with a(h1n1) pdm09/rsv was more common than a(h1n1)pdm09 ⁄ enterovirus ⁄ rhinovirus, accounting for 64ae1% and 22ae8% of the co-infections, respectively. this may reflect the younger age of patients infected by a(h1n1)pdm09, who were therefore also at greater risk of rsv. in addition, rsv cocirculated with a(h1n1)pdm09 more than enterovirus ⁄ rhinovirus, which peaked before the second wave (figure 2 ). there may also be preferential interactions among certain pathogens; viral interactions were not assessed in this study. 23 when other respiratory samples positive for enterovirus ⁄ rhinovirus were further evaluated at our laboratory, they all were confirmed as rhinovirus, not enterovirus. 24 single infection was more commonly detected in those less than 65 years of age. it is known that respiratory infections are more common in children for several reasons, including an immature immune system, lack of preexisting immunity particularly to new emerging viruses, and greater viral exposure opportunities. 11, 18, 25 younger patients shed higher levels of virus when infected and also may be brought for medical care earlier than older patients, facilitating detection in these groups. 26 in addition, lower detection of single and co-infection in elderly may be explained by pre-existing immunity to a(h1n1)pdm09 and other respiratory viruses. 2 co-infection was more common in persons less than 30 years of age as compared to older adults. these data are congruent with findings from a previously published study where co-infection was more likely in younger than older adults. 18 the combined effect of predominance of a(h1n1)pdm09 and the greater likelihood of infection with other respiratory viruses among younger ages likely explains our age-related findings of co-infection during the pandemic, which may not be generalizable to a typical influenza season. the presence of comorbidities did not increase the likelihood of having a single infection but increased the likelihood of co-infections; this did not achieve statistical significance. patients with chronic conditions are at higher risk of severe disease and consequently may be more likely to seek medical care. 26 selection bias is unlikely to influence these results as the proportion of patients with comorbidities was similar (21ae5%) to that in ontario's population (20ae3%). 27 sample collection within 2 days of symptom onset was found to independently increase the likelihood of detecting a viral co-infection but not single infection. long et al. reported an inverse relationship between duration of symptoms and viral detection rate due to greater viral shedding earlier in the disease process. 11 this study was designed to examine circulating viruses and co-infection. the presence of more than two viruses in the same sample may not always indicate clinical infection. as viruses may be detected in asymptomatic patients, it is impossible to determine which viruses caused symptoms. previous studies suggest co-infection may manifest higher disease severity, which may shorten the time to medical care and viral detection; disease severity was not assessed in the current study. 19, 28 as viral-bacterial co-infections also occurred during the pandemic, it will be interesting for further studies to investigate their characteristics and impact on disease severity. 28, 29 in summary, a(h1n1)pdm09 was frequently detected among community patients with ili. however, other respiratory viruses cocirculated with a(h1n1)pdm09 during the pandemic, reinforcing the need to test for other viral agents even during a pandemic to appropriately guide clinical treatment decisions. viral diagnosis, primarily a(h1n1)pdm09, was made more often in patients less than 65 years of age. viral co-infection was commonly detected in this study and was most likely detected in individuals less than 30 years of age. earlier sample collection improves the detection of viral co-infections. understanding the contribution of other circulating respiratory pathogens during a pandemic may lead to improved individual diagnosis and recommendations for community-based clinicians, and more effective prevention and treatment of respiratory infections, including use of influenza antivirals. novel swine-origin influenza a (h1n1) virus in humans a (h1n1) influenza virus pandemic: a review complications of seasonal and pandemic influenza who checklist for influenza pandemic preparedness planning 2005. department of communicable disease surveillance and response global influenza programme. available online: http: ⁄ ⁄ a (h1n1)v, rvs: the race for hivernal pandemics respiratory virus infections among hospitalized patients with suspected influenza a h1n1 2009 virus during the first pandemic wave in brazil bacterial coinfections in lung tissue specimens from fatal cases of the association of newly identified respiratory viruses with lowers respiratory tract infections in korean children association of rhinovirus infection with increased disease severity in acute bronchiolitis single versus dual respiratory virus infections in hospitalized infants: impact on clinical course of disease and interferon-y response prospective evaluation of a novel multiplex real-time pcr assay for detection of fifteen respiratory pathogens. duration of symptoms significantly affects detection rate frequent detection of viral coinfection in children hospitalized with acute respiratory tract infection using a real-time polymerase chain reaction multipathogen infections in hospitalized children with acute respiratory infections development of a respiratory virus panel test for detection of twenty human respiratory viruses by use of multiplex pcr and a fluid microbead-based assay low mortality rates related to respiratory virus infections after bone marrow transplant incidence of common respiratory viral infections related to climate factors in hospitalized children in hong kong respiratory viral infections in infants: causes, clinical symptoms, virology, and immunology prevalence of human respiratory viruses in adults with acute respiratory tract infections in beijing correlation of viral load of respiratory pathogens and co-infections with disease severity in children hospitalized for lower respiratory tract infection impact of respiratory virus infections on persons with chronic underlying conditions human metapneumovirus and respiratory syncytial virus in hospitalized danish children with acute respiratory tract infection association between the 2008-09 seasonal influenza vaccine and pandemic h1n1 illness during spring-summer 2009: four observational studies from canada evidence from multiplex molecular assays for complex multipathogen interaction in acute respiratory infections rhinovirus outbreaks in long-term care facilities viruses in communityacquired pneumonia in children aged less than 3 years old; high rate of viral confection correlation of pandemic (h1n1) 2009 viral load with disease severity and prolonged viral shedding in children self-reported ph1n1 influenza vaccination coverage for ontario streptococcus pneumonia coinfection is correlated with the severity of h1n1 pandemic influenza invasive group a streptococcal infection concurrent with 2009 h1n1 influenza authors wish to acknowledge the staff of molecular diagnostics and virus detection departments at public health ontario laboratory-toronto, the ontario and national sentinel influenza vaccine effectiveness study and the college of family physicians of canada funded by the canadian institute of health research. dms was principal investigator on a clinical trial of pediatric influenza vaccine dose response for which influenza vaccine was provided free by sanofi pasteur. jbg has received research grants from glaxosmithkline inc. and hoffman-la roche ltd to study antiviral resistance in influenza. key: cord-006325-3no74e74 authors: jeannoël, m.; lina, g.; rasigade, j. p.; lina, b.; morfin, f.; casalegno, jean sebastien title: microorganisms associated with respiratory syncytial virus pneumonia in the adult population date: 2018-10-23 journal: eur j clin microbiol infect dis doi: 10.1007/s10096-018-3407-3 sha: doc_id: 6325 cord_uid: 3no74e74 respiratory syncytial virus (rsv) has been recognized as responsible for severe respiratory illness in adults, especially in the elderly. while pneumonia is commonly observed during rsv infection, the burden and epidemiology of bacterial superinfection is poorly understood. the aim of this study was to identify microorganisms associated with rsv-positive pneumonia in adults. a retrospective study was conducted during three consecutive winters (october to april 2013–2016) in the university hospital of lyon, france. during rsv circulation periods, a systematic rsv screening was performed by reverse-transcription pcr on all respiratory samples collected from adults. records of rsv-positive patients were subsequently analyzed to identify radiologically confirmed pneumonia cases. bacteria were identified by standard bacteriology cultures or urinary antigen screening and classified as potentially causative of pneumonia if quantification was above the specific threshold as defined by the european manual of clinical microbiology. overall, 14,792 adult respiratory samples were screened for rsv detection by pcr. in total, 292 had a positive rsv detection (2.0%) among which 89 presented with pneumonia including 27 bacterial superinfections (9.3%) with streptococcus pneumonia, haemophilus influenza, staphylococcus aureus, pseudomonas aeruginosa, and moraxella catarrhalis. most patients were elderly (55.6%) and patients with comorbidities (77.8%). a more severe outcome was observed for rsv-bacteria-associated pneumonia compared with rsv pneumonia: length of stay was significantly longer (16 days vs 10 days) and icu hospitalization more frequent (66.7% vs 21.0%) (p < 0.05). in conclusion, we did not observe major differences in the epidemiology of bacterial superinfections in rsv-positive pneumonia compared to reports on post-influenza pneumonia. respiratory syncytial virus (rsv) is now recognized as an important cause of respiratory illness in adults, especially in the elderly [1] . secondary bacterial pneumonia after or during an rsv infection is among the more severe complications reported in this population [2] . few studies have described superinfection or coinfection with bacteria among adults with rsv-positive pneumonia in inpatient setting [3, 4] . diagnostic testing for rsv is usually not routinely performed in this population. however burden and epidemiology of rsv-positive pneumonia in adults is important to assess in regards of the current antibiotic therapy recommendations [5] . the aim of our study was to gain insights in the epidemiology of bacterial superinfections associated with rsv pneumonia in adults. we conducted a retrospective, monocenter, and observational study on three successive winter periods (october to april, 2013 april, -2016 . in the university hospital of lyon (hospices civils de lyon, france), upper and lower respiratory tract samples from patients with respiratory symptoms are systematically screened for rsv and metapneumovirus (hmpv) using a reverse transcriptase polymerase chain reaction (rt-pcr). we included all adult patients (≥ 18 years old) positive for rsv with radiologically confirmed pneumonia as diagnosed in the medical chart. hospital-acquired pneumonia (hap) was defined as radiologically confirmed pneumonia that manifests ≥ 48 h after the patient's admission to the hospital, and not present at the time of intubation. data collected included laboratory results, demographics (age, sex), underlying conditions (cardiovascular disease, pulmonary disease, diabetes mellitus, and immunodeficiency), as well as clinical outcomes (length of stay, acute respiratory distress syndrome (ards), intensive care unit (icu) admission, in-hospital death). for virus detection, clinical specimens comprised nasopharyngeal swabs, endotracheal aspirates, and broncho-alveolar lavages. respiratory specimens were extracted using easymag (biomerieux, marcy l'étoile, france). rsv detection was performed using a rt-pcr (abi 7500; thermofisher, illkirch, france), or duplex rt-pcr detecting rsv and hmpv (r-gene detection kit, biomérieux-argene, france) as previously described [6] . for bacteria detection, pathogens were classified as potentially causative of pneumonia if identified in pleural fluid or blood, or if identified within a sputum specimen, endotracheal aspirate, and broncho-alveolar lavage with good-quality criteria and if quantification was above the appropriate emcm decision threshold. semi-quantitative bacterial culture process and interpretation was performed following the european manual of clinical microbiology (emcm) guidelines [7] . maldi-tof (vitek-ms) was used for species identification. statistical analyses were performed using graphpad prism software (version 5.0, graphpad software, la jolla, ca, usa). univariate analysis was used to compare rsvbacteria-associated pneumonia and rsv-positive pneumonia. quantitative variables (age and length of stay) were expressed as median and interquartile range (iqr). difference between the age groups was assessed by student's t test. difference between lengths of stay was assessed by non-parametric mann-whitney test. qualitative variables were expressed as number and percentage. a fisher's exact test was used for univariate comparisons. a p value < 0.05 was considered significant. during the study period, viral rt-pcr was performed on 14,792 respiratory samples collected from adult patients. influenza was detected in 1441 patients (9.7% of all respiratory samples), rhinovirus in 438 patients (3.0% of all respiratory samples), rsv in 292 patients (2.0% of all respiratory samples), and hmpv in 114 patients (0.8%). the median age of patients tested positive for rsv was 68 years (interquartile range 54 to 82), with a sex ratio of 1 (51.9% were men). among rsv-positive patients, 95.5% of case records were retrieved (n = 279/292). primary discharge diagnoses in patients without respiratory symptoms were as follows: 10.0% (n = 28/279) of systematic sampling without acute respiratory infection in immunosuppressed patients, 6.5% (n = 18/279) of acute heart failure, 2.5% (n = 7/279) of sepsis, and 1.4% (n = 4/279) of neurological disorders. 79.6% (n = 222/279) patients presented with respiratory disorders, including 19.4% (n = 54/279) of respiratory failure, 15.8% (n = 44/279) of acute respiratory infection, 12.5% (n = 35/279) of acute respiratory distress syndrome, and 31.9% (n = 89/279) radiologically confirmed pneumonia. among these 89 pneumonia, 27 cases were associated with bacteria classified as potentially causative (rsv-bacteria-associated pneumonia) and 62 cases had no bacterial documentation (rsv-positive pneumonia). among these 62 rsvpositive pneumonia, 5 cases were associated with another virus (2 rsv and influenza coinfections, 1 rsv and picornavirus coinfection, 1 rsv and bocavirus coinfection, and 1 rsv and cmv coinfection). among the 27 rsv-bacteria associated pneumonia, hospital-acquired pneumonia (hap) was observed mostly in adults ≥ 65 years old (n = 8/10) whereas community-acquired pneumonia (cap) occurred mostly in 18 to 64-year-old adults (n = 10/17). most of the patients presenting a pneumonia were elderly (median age 74 years (interquartile range 58 to 85) and had underlying conditions (77.8%) ( table 1 ). only 11.1% were resident of nursing homes. demographic characteristics did not differ between patients with an rsv-bacteria-associated pneumonia and patients with an rsv-positive pneumonia. among these 89 pneumonia, 31 patients received an empiric antibiotic treatment and 3 immunosuppressed patients received an antiviral treatment: 1 patient received ribavirin, 1 patient received polyvalent immunoglobulins, and 1 patient received ribavirin and polyvalent immunoglobulins. in total, 22 patients presented an ards, 31 patients were admitted to icus and in-hospital mortality occurred in 18 adults of which all were ≥ 65 years old (n = 14/18) or immunosuppressed (n = 4/18). rsv and bacteria coinfection was statistically associated with a more severe outcome than rsv-positive pneumonia as length of stay was significantly longer (16 days vs 10 days) and icu hospitalization more frequent (66.7% vs 21.0%) (p < 0.05). however no significant difference was observed regarding in-hospital mortality ( table 1) . among the 27 cases of rsv-positive pneumonia with superinfection, the most frequent bacteria were streptococcus pneumoniae, enterobacteriaceae ( kleb siella p neu monia , en teroba cte r cloa ca e , citrobacter koseri), pseudomonas aeruginosa, haemophilus influenza, staphylococcus aureus, and moraxella catarrhalis. in two patients, more than one bacterial pathogen was detected. in patients hospitalized f o r c a p, t he m o s t f r e qu en t ba ct er i a w e re s . pneumoniae, h. influenzae, and s. aureus whereas in hap the most frequent bacteria were p. aeruginosa and enterobacteriacae (table 2 ). in this study, detection rates of the screened viruses were consistent with previous reports describing the epidemiology of respiratory viruses in hospitalized adults [8] [9] [10] . rsv detection in our study was low (2.0%) compare to rates of rsv detection reported in previous studies ranging from 7% of hospitalized adult patients to 10% of hospitalization for acute respiratory illness in an elderly population [11] [12] [13] . it is probably due to the systematic testing strategy associated to a species distribution of pathogenic bacteria involved in rsv-positive pneumonia (cap) and hospital-acquired pneumonia (hap) sampling bias toward influenza-like illness. those symptoms are indeed poorly predictive of an rsv infection [14] . among rsv infected patients, 31.9% presented a radiologically confirmed pneumonia and 9.7% had an rsv-bacteriadocumented pneumonia. despite the large number of rsv testing among the adult population, we identified only a few rsv radiologically confirmed pneumonia. although this is a low prevalence, it is in agreement with the current literature in which 9 to 39% of rsv-bacteria co-infections were reported [3] . in our study, a higher prevalence was noted in the elderly (15/27), immunosuppressed, and patients with comorbidities (21/27). as it has been previously described, we observed that rsv-bacteria-associated pneumonia have a more severe outcome than rsv-positive pneumonia [15] . enterobacteriaceae and pseudomonas aeruginosa were the most frequently detected pathogens in hap and did not differ from the pathogens usually responsible for hap in icu [16] . s. pneumonia and h. influenza were the most frequently detected pathogens in cap. in a previous study in adults with cap, s. pneumonia and h. influenza were also mainly reported in rsv-bacteria coinfections [4] . we did not observed major differences to influenza-bacterial coinfections [17] . those results suggest that there is no main difference between the microbiological epidemiology of rsv-bacterial coinfections and influenza-bacterial coinfections. this observation needs to be confirmed by other studies. further research should focus on estimating the rsv cap burden in adult as well as understanding the underlying mechanisms of copathogenesis. conflict of interest the authors declare that they have no conflict of interest. ethical approval this article does not contain any studies with human participants performed by any of the authors. informed consent for this type of study formal consent is not required. risk of mortality associated with respiratory syncytial virus and influenza infection in adults viral-bacterial coinfection affects the presentation and alters the prognosis of severe community-acquired pneumonia respiratory syncytial virus infection in elderly and high-risk adults incidence and characteristics of viral community-acquired pneumonia in adults community-acquired pneumonia genetic characterization of respiratory syncytial virus highlights a new ba genotype and emergence of the on1 genotype in lyon european manual of clinical microbiology. basel: european society for clinical microbiology and infections diseases : société française de microbiologie can analysis of routine viral testing provide accurate estimates of respiratory syncytial virus disease burden in adults? prevalence of respiratory viruses among adults, by season, age, respiratory tract region and type of medical impact of viral multiplex real-time pcr on management of respiratory tract infection: a retrospective cohort study respiratory syncytial virus hospitalization in middle-aged and older adults rates of hospitalizations for respiratory syncytial virus, human metapneumovirus, and influenza virus in older adults respiratory syncytial virus infection in adults clinical characteristics and outcome of respiratory syncytial virus infection among adults hospitalized with influenza-like illness in france elucidation of bacterial pneumonia-causing pathogens in patients with respiratory viral infection hospital-acquired pneumonia in icu the co-pathogenesis of influenza viruses with bacteria in the lung key: cord-256930-bz80uxnx authors: xiao, ni‐guang; zhang, bing; xie, zhi‐ping; zhou, qiong‐hua; zhang, rong‐fang; zhong, li‐li; ding, xiao‐fang; li, jia; song, jing‐rong; gao, han‐chun; hou, yun‐de; duan, zhao‐jun title: prevalence of human metapneumovirus in children with acute lower respiratory infection in changsha, china date: 2013-01-07 journal: j med virol doi: 10.1002/jmv.23501 sha: doc_id: 256930 cord_uid: bz80uxnx human metapneumovirus (hmpv) causes acute respiratory infections in children. the prevalence and clinical characteristics of hmpv were determined in nasopharyngeal aspirates of children in changsha, china. reverse transcription‐polymerase chain reaction (rt‐pcr) or pcr was employed to screen for both hmpv and other common respiratory viruses in 1,165 nasopharyngeal aspirate specimens collected from children with lower respiratory tract infections from september 2007 to august 2008. all pcr products were sequenced, and demographic and clinical data were collected from all patients. seventy‐six of 1,165 (6.5%) specimens were positive for hmpv, of which 85.5% (65/76) occurred in the winter and spring seasons. the hmpv coinfection rate was 57.9% (44/76), and human bocavirus was the most common virus detected in conjunction with hmpv. phylogenetic analysis revealed that 94.7% of the hmpv detected were of subgroup a2, 5.3% were subgroup b2, and none belonged to either the a1 or b1 subgroups. no significant differences were found in terms of the frequency of diagnosis and clinical signs between either the co‐ and mono‐infection groups, or between patients with and without underlying diseases. it was concluded that hmpv is an important viral pathogen in pediatric patients with lower respiratory tract infections in changsha. only hmpv genotypes a2 and b2 were co‐circulating in this locality; human bocavirus was the most common coinfecting virus, and coinfection did not affect disease severity. j. med. virol. 85:546–553, 2013. © 2013 wiley periodicals, inc. human metapneumovirus (hmpv) was first identified in 2001 in nasopharyngeal specimens from children with acute respiratory tract illness in the netherlands [van den hoogen et al., 2001] . this virus has been classified as a member of the genus metapneumovirus of the subfamily pneumovirinae of the family paramyxoviridae [van den hoogen et al., 2001; boivin et al., 2002; bastien et al., 2003] . subsequently, hmpv was described as a cause of acute respiratory disease in many countries, including canada, the united states, australia, japan, france, hong kong, and korea [ordá s et al., 2006] . hmpv is recognized as a common cause of respiratory infections, ranging from upper respiratory tract infection to severe lower respiratory tract infection, in individuals of all ages, particularly in infants and children freymouth et al., 2003; peiris et al., 2003; van den hoogen et al., 2003; mcadam et al., 2004; loo et al., 2007] . however, limited data exist for hmpv infection, especially concerning its prevalence, and molecular and clinical characterizations of hmpv in children with lower respiratory tract infections in china, with the exception of a previous study in lanzhou city [xiao et al., 2010] . hmpv strains are divided into two main groups, a and b, based on their nucleotide sequences. each group is subdivided into two sublineages, a1 and a2, and b1 and b2 [loo et al., 2007] . in addition, parti-tioning further the sublineage a2 into two genetic clusters designated a2a and a2b has been suggested [huck et al., 2006] , and the relationship of strain differences to clinical features has not been elucidated fully [schildgen et al., 2005; agapov et al., 2006; manoha et al., 2007; pitoiset et al., 2010] . in this study, 1,165 children with lower respiratory tract infections in changsha city were screened for hmpv and several other common respiratory viruses, and the epidemiological and clinical features of infection with the various hmpv genotypes were characterized. the objective of this study was to investigate the prevalence and clinical characteristics of hmpv in chinese children with lower respiratory tract infections. nasopharyngeal aspirate samples were collected from 1,165 children with lower respiratory tract infection in the hunan province people's hospital, china, on 2 days each week from september 2007 to august 2008. all patients were 14 years of age or younger, and informed consent was obtained from their parents/guardians. all patients had symptoms of lower respiratory tract infection on admission. all nasopharyngeal aspirate samples were collected 1-3 days after the onset of lower respiratory tract infection. demographic data and details of the clinical findings and severity of disease were recorded. the study protocol was approved by the hospital ethics committee. all nasopharyngeal aspirate specimens were collected and transported immediately to the laboratory at the national institute for viral disease control and prevention, china cdc, and stored at à808c until required for further testing. viral dna and rna were extracted from 140 ml of each nasopharyngeal aspirate specimen using the qiaamp viral dna and the qiaamp viral rna mini kits (qiagen, shanghai, china) according to the manufacturer's instructions. cdna was synthesized using random hexamer primers with the superscript ii rh à reverse transcriptase (invitrogen, carlsbad, ca). screening for hmpv was conducted using conventional polymerase chain reaction (pcr) methods. hmpv forward (5 0 -ccc ttt gtt tca ggc caa-3 0 ) and reverse (5 0 -gca gct tca aca gta gct g-3 0 ) primers, which target the m gene and generate a 416bp product, were used as described previously [pujol et al., 2005] . all pcr products were purified using the qiaquick pcr purification kit (qiagen) and sequenced by sinogenomax (beijing, china). the reaction mix contained 10 pmol of each primer and 1.25 units of extaq dna polymerase (takara bio, tokyo, japan). reactions were incubated at 948c for 8 min, followed by 35 cycles at 948c for 30 sec, 558c for 30 sec, and 728c for 45 sec, followed by a final extension at 728c for 10 min. a standard reverse transcription (rt)-pcr was used to screen for respiratory syncytial virus (rsv), human rhinovirus, influenza a virus, influenza b virus, parainfluenza virus, human coronaviruses hku1 and nl63, and pcr for adenovirus and human bocavirus [hierholzer et al., 1993; pujol et al., 2005] . all positive sequences were determined and analyzed using the dnastar software package. a neighbor-joining tree was constructed using the mega software package (version 3.1). based on variables reported in previous studies [caracciolo et al., 2008] a severity index was defined a priori by assigning one point to each of the following: use of supplemental oxygen, duration of hospital stay of more than 7 days, and admission to an intensive care unit (icu). the significance of differences in rates among various groups was evaluated using the chi-square test, fisher's exact test, or student's t-test. all analyses were performed using spss version 13.0 software (spss, inc., chicago, il). p < 0.05 was considered statistically significant. in total, 1,165 patients were included; the study sample represented 36.7% of the total of 3,174 admissions for acute lower respiratory disease to the hunan province people's hospital from september 2007 to august 2008. patient ages ranged from 3 hr to 156 months with a median of 15.4 months. the majority of patients (97.5%) were 5 years old or younger. the male:female ratio was 1.9:1 (763:402). all subjects were inpatients. at least one respiratory virus was detected in 871 of the 1,165 samples and 76 (6.5%) were positive for hmpv by rt-pcr. hmpv accounted for 8.7% of the total viral agents detected. forty-four of 76 (57.9%) children who were hmpv-positive were found to be coinfected with other respiratory viruses, including 16 with human bocavirus, 13 with rsv, 10 with human rhinovirus, 7 with parainfluenza 3 virus, 4 with adenovirus, 3 with influenza b virus, 2 with hcov-hku1, and 1 with hcov-nl63. human bocavirus was the most common coinfecting virus, accounting for 16/56 (28.6%) coinfections. no differences in coinfection rates were observed between hmpv a and hmpv b (p ¼ 0.106). hmpv was detected in every month except for september and october 2007 and august 2008. the number of positive specimens peaked in march (n ¼ 20; 26.3%) and april (n ¼ 19; 25%; fig. 1 ). the age of patients infected with hmpv varied from 20 days to 12 years of age (median, 15.9 months) and 93.4% (71/ 76) were 5 years of age. of a subset of 42 children >60 months of age, five (11.9%) acquired hmpv infection (fig. 2 ). the male:female ratio of the patients infected with hmpv was 3.5:1 (x 2 ¼ 5.300; p ¼ 0.021). information on clinical symptoms was available for all patients infected with hmpv. the main clinical features of patients infected with hmpv included bronchitis (one, 1.3%), bronchopneumonia (30, 39.5%), bronchiolitis (33, 43.4%), and pneumonia (12, 15.8%). of the patients infected with hmpv, all (100%) presented with cough, 40 (52.6%) had a fever, 37 (48.7%) had respiratory crepitations, 33 (43.4%) exhibited wheezing, 2 (2.6%) suffered vomiting, and 7 (9.2%) had diarrhea. five patients with hmpv infection had a fever exceeding 408c. duration of stay in hospital ranged from 1 to 26 days (mean, 8.3 days). none of these patients died; however, five required supplemental oxygen and intensive care. erythrocyte sedimentation rates and c-reactive protein analysis were not performed in all patients infected with hmpv in the study. the majority of patients infected with hmpv had normal erythrocyte sedimentation rates, c-reactive protein and leukocyte counts. fourteen (20.6%) patients infected with hmpv also had elevated glutamic-pyruvic transaminase, 31 (45.6%) had elevated glutamic-oxaloacetic transaminase, and 46 (73%) had elevated creatine kinase-mb (table i) . chest radiographs were taken in 57 patients; abnormal infiltrates were noted in 42 (73.7%) subjects (interstitial lung disease, 13 cases; scattered consolidation of the lung, 33 cases; consolidation in lobar distribution, 1 case; pleural effusion, 1 case; single hila of pulmonary swelling, 1 case; and emphysema, 10 cases). no significant differences were observed in any of the epidemiological characteristics, clinical presentations between the hmpv mono-(group 1) and coinfection groups (group 2), or between the hmpv monoand human bocavirus coinfection groups (group 3; table ii ). note that a significant difference was observed in the rate of coinfection with rsv between those 12 months and those >12 months (p ¼ 0.045). we also found that wheezing was more prevalent in subjects coinfected with rsv than in those with hmpv mono-infection (69.2% vs. 37.5%), although this difference was not significant (p ¼ 0.053 a ; table ii) . a total of 163 of 1,165 (13.99%) patients with lower respiratory tract infection and 14 of 76 (18.4%) who were hmpv-positive had an underlying illness. no significant difference was observed in the detection rate between these two groups (p ¼ 0.250). a significant difference was detected in the prevalence of diarrhea between the two groups (p ¼ 0.024). however, no significant difference was found for the duration of hospital stay, age, gender, the majority of clinical diagnoses, and all clinical symptoms (table iii) . the sequences of positive products shared high homology with standard sequences from genbank (97-100%). single nucleotide mutations and nucleotide insertions were found, indicating a slow genetic variation rate. phylogenetic analyses indicated that the 76 hmpv specimens were classified into the two main genetic lineages, a and b. seventy-two (94.7%) hmpv strains were group a2, four (5.3%) strains were subgroup b2, and none were either subgroup a1 or b1 (fig. 3) . during the epidemic season, sublineages a2 and b2 co-circulated, with 94.7% (72/76) of the circulating viruses belonging to sublineage a2. between the a2 and b2 genotype strains, the sequence identities of m gene fragments were 86.06-87.15% and 81.14-82.46%, at the nucleotide and amino acid levels, respectively. the identities within subgroup a2 were 98.91-99.56% and 98.58-99.13%, and within subgroup b2 were 95.91-97.98% and 96.37-97.38%, respectively. of the 1,165 children with lower respiratory tract infection included in this study, 6.5% were hmpvpositive by rt-pcr. a similar incidence was reported in singapore [loo et al., 2007] , the united states [mcadam et al., 2004] , hong kong [peiris et al., 2003] , and in lanzhou city [xiao et al., 2010] , although the findings were different from those of other studies [mcadam et al., 2004; chung et al., 2006; bosis et al., 2008; heikkinen et al., 2008] . [esper et al., 2003; dollner et al., 2004; mcadam et al., 2004] . by the age of 5 years, >90% of individuals screened have evidence of hmpv infection. in this study, the majority of patients who were hmpv-positive (93.4% 71/76) were less than 5 years old. no significant differences in the age distribution rate were detected, but a study from chongqing, a city in southwestern china, reported that younger children (less than 6 months old) had the highest rate of hmpv infection [chen et al., 2010] . van den hoogen et al. [2001] and bastien et al. [2003] reported that no significant difference in the prevalence of hmpv existed between male and female patients. in this study, the majority of patients infected with hmpv (59/76) were male, and statistical analysis found differences in prevalence between males and females, which indicated that male patients are at a higher risk of hmpv infection. hmpv was detected in each month with the exception of september and october 2007 and august 2008. positive specimens peaked in march and april, in agreement with findings from the united states [mcadam et al., 2004] , canada [bastien et al., 2003] , and a previous study [xiao et al., 2010] . however, hmpv was detected year-round in singapore [loo et al., 2007] and peaked during the summer months in hong kong [peiris et al., 2003] . although these data suggest that hmpv may follow varying epidemiologic patterns in different regions, elucidation of the exact epidemiologic characteristics of hmpv infection requires further investigation. approximately 58% of patients who were hmpvpositive were coinfected with other respiratory viruses, in agreement with previous reports [caracciolo et al., 2008; cilla et al., 2008; xiao et al., 2010] . human bocavirus and rsv were the most common coinfecting viruses (36.4% and 29.5%, respectively). the data regarding the impact of coinfections of human bocavirus and rsv with hmpv on disease severity are conflicting [greensill et al., 2003; semple et al., 2005; caracciolo et al., 2008] . in this study, no significant difference in clinical symptoms, age, sex, or duration of hospital stay between the mono-and coinfection groups was detected (table ii) . considerable evidence suggests that hmpv is responsible for both upper respiratory tract infection and lower respiratory tract infection in infants and young children freymouth et al., 2003; van den hoogen et al., 2003] . indeed, arabpour et al. [2008] reported a higher prevalence of lower respiratory tract infection in children infected with hmpv. however, the present study encompasses only hospitalized children with lower respiratory tract infection; therefore, the prevalence of upper respiratory tract infection and lower respiratory tract infection could not be elucidated. additionally, williams et al. [2004] reported that one-third of children with hmpvassociated lower respiratory tract infection were diagnosed with concomitant acute otitis media. however, in this study, no acute otitis media occurred in subjects who were infected with hmpv. bronchopneumonia and bronchiolitis were the most frequent clinical diagnoses in this study, as has been reported previously [peiris et al., 2003; loo et al., 2007; xiao et al., 2010] . fever, cough, respiratory crepitations, and wheezing were the most common symptoms of these patients. these symptoms are identical to those reported in children in canada and korea [chung et al., 2006; caracciolo et al., 2008] . no significant difference was observed in the frequencies of cough, fever, respiratory crepitations, wheezing, vomiting, diarrhea, or duration of stay in hospital between the hmpv mono-and coinfection groups. more than one-half of patients with hmpv had normal erythrocyte sedimentation rates, c-reactive protein and leukocyte counts, and a majority had hepatic and renal injury. data on whether hmpv infection can induce hepatic and renal injury are limited; therefore, this aspect requires further study. chest radiographs of the majority of patients who were hmpv-positive showed sporadic consolidation of the lung, 21.1% (12/57) showed interstitial lung disease and emphysema, and only one patient showed consolidation in lobar distribution, pleural effusion, and single hila of pulmonary swelling, which is consistent with a report from korea [chung et al., 2006] . a majority of patients who were hmpv-positive reportedly had underlying diseases kaida et al., 2007] , and 14 of 76 such patients had at least one underlying disease (table iii) . a significant difference in the incidence of diarrhea was observed between subjects who were hmpv-positive with and without underlying illnesses (p ¼ 0.024), which suggests that hmpv infection may cause different clinical presentations in patients depending on the underlying conditions. however, no significant difference was detected in the detection rate, age, sex, duration of stay in hospital, the majority of clinical diagnoses, or clinical symptoms between the two groups (table iii) . these data suggest that hmpv infection did not aggravate clinical symptoms or contribute to duration of hospital stay in subjects with underlying illnesses. three subjects had congenital heart disease, which ranked first in terms of underlying diseases. two each had gastroesophageal reflux and measles. further study is needed to investigate whether congenital heart disease, gastroesophageal reflux, or measles poses a major risk factor for hmpv infection. previous data have suggested that the two hmpv genotypes co-circulate and that different subgroups may predominate from year to year mackay et al., 2004] . in this study, phylogenetic analysis demonstrated the simultaneous existence of two groups (a and b) and two of the four subgroups (a2 and b2). the majority of these strains (94.7%, 72/76) clustered predominantly with group a hmpv, and all belonged to subgroup a2 (100%, 72/72), which is consistent with the work of boivin et al. [2004] . moreover, a previous study in lanzhou city showed that sublineages a1, a2 (a2a and a2b), and b1 cocirculated during the 2006-2007 epidemic, but only a2 circulated during the 2007-2008 epidemic [xiao et al., 2010] . these data suggest that the circulation pattern of hmpv in china is complex, which poses a challenge for future vaccine development that relies on more molecular epidemiologic studies. to summarize, the prevalence and clinical characteristics of hmpv in children with lower respiratory tract infection in changsha, china were described. hmpv was detected in 76 of 1,135 (6.5%) nasopharyngeal aspirate specimens collected. approximately 58% of subjects infected with hmpv were coinfected with other respiratory viruses, most commonly human bocavirus. the most common symptoms and clinical diagnosis in those infected with hmpv were cough and bronchopneumonia, and the predominant circulating genogroup was subgroup a2. statistical analysis indicated that male subjects and those less than 5 years of age were at a higher risk of hmpv infection, and coinfection with other respiratory viruses did not affect disease severity. fig. 3 . phylogenetic analysis of the partial m gene sequences of 76 human metapneumovirus strains from nasopharyngeal aspirate specimens. phylogenetic trees were constructed by the neighbor-joining method using mega ver. 3.1. viral sequences in marks were generated from the present study; other reference sequences were obtained from genbank. bootstrap values are shown at each branching point. genetic variability of human metapneumovirus infection: evidence of a shift in viral genotype without a change in illness the highest prevalence of human metapneumovirus in ahwaz children accompanied by acute respiratory infections human metapneumovirus infection in the canadian population virological features and clinical manifestations associated with human metapneumovirus: a new paramyxovirus responsible for acute respiratory-tract infections in all age groups global genetic diversity of human metapneumovirus fusion gene association between high nasopharyngeal viral load and disease severity in children with human metapneumovirus infection human metapneumovirus infection in young children hospitalized with acute respiratory tract disease: virologic and clinical features acute lower respiratory tract infections by human metapneumovirus in children in southwest china: a 2-year study human metapneumovirus infection in hospitalized children with acute respiratory disease in korea hospitalization rates for human metapneumovirus infection among 0-to-3 year-olds in gipuzkoa outbreak of human metapneumovirus infection in norwegian children human metapneumovirus infection in the united states: clinical manifestations associated with a newly emerging respiratory infection in children presence of the new human metapneumovirus in french children with bronchiolitis human metapneumovirus in severe respiratory syncytial virus bronchiolitis human metapneumovirus infections in children detection of adenovirus in clinical specimens by polymerase chain reaction and liquid-phase hybridization quantitated by time-resolved fluorometry novel human metapneumovirus sublineage co-infection of human metapneumovirus with adenovirus or respiratory syncytial virus among children in japan human metapneumovirus in children use of the p gene to genotype human metapneumovirus identifies four viral subtypes epidemiologic and clinical features of hmpv, rsv and rv infections in young children human metapneumovirus in children tested at a tertiary-care hospital role of metapneumovirus in viral respiratory infections in young children children with respiratory disease associated with metapneumovirus in hong kong characterization of human metapneumoviruses isolated from patients in north america human metapneumovirus genotypes and severity of disease in young children (n ¼ 100) during a 7-year study in dijon hospital, france development of three multiplex rt-pcr assays for the detection of 12 respiratory rna viruses human metapneumovirus rna in encephalitis patient dual infection of infants by human metapneumovirus and human respiratory syncytial virus is strongly associated with severe bronchiolitis a newly discovered human pneumovirus isolated from young children with respiratory tract disease prevalence and clinical symptoms of human metapneumovirus infection in hospitalized patients human metapneumovirus and lower respiratory tract disease in otherwise healthy infants and children prevalence and clinical and molecular characterization of human metapneumovirus in children with acute respiratory infection in china key: cord-312691-ynh84b98 authors: mohd, hamzah a.; memish, ziad a.; alfaraj, sarah h.; mcclish, donna; altuwaijri, talal; alanazi, marzouqah s.; aloqiel, saleh a.; alenzi, ahmed m.; bafaqeeh, fahad; mohamed, amal m.; aldosari, kamel; ghazal, sameeh title: predictors of mers-cov infection: a large case control study of patients presenting with ili at a mers-cov referral hospital in saudi arabia date: 2016-09-24 journal: travel med infect dis doi: 10.1016/j.tmaid.2016.09.008 sha: doc_id: 312691 cord_uid: ynh84b98 background: a case control study to better characterize the clinical features, laboratory, and radiological abnormalities associated with mers-cov infection in order to help with early identification of this syndrome from other respiratory infections. methods: eighty patients admitted to a hospital in riyadh, diagnosed with mers-cov infection based on rt-pcr were matched on age, sex, and the presence of a co-morbid condition on a basis of 1:2 to other patients admitted with respiratory symptoms and tested negative for mers-cov on rt-pcr. results: none of the reported mers-cov presenting symptoms was significantly associated with being infected with mers-cov. on the other hand, wbc count was significantly lower in patients with confirmed mers-cov infection (median 5.7 vs 9.3, p: 0.0004). neutrophil count was as well significantly lower in mers-cov patients (median 3.7 vs 6.7, p: 0.0001). both ast, and alt values were significantly higher in mers-cov infected group (ast median 42 vs 36, p: 0.03, and alt median 33 vs 28, p: 0.003). overall our mers-cov mortality rate was (10%) below the national figure of (40%). conclusions: none of the presenting symptoms are specific for mers-cov infection. and out of all the investigations wbc, neutrophil counts, ast and alt values have some predictive utility. mers-cov is a novel betacoronavirus that was discovered in 2012 after it was isolated from the respiratory secretions of a patient in saudi arabia who died with acute respiratory syndrome [1] . so far, all reported cases were diagnosed in the arabian peninsula (mainly saudi arabia) or epidemiologically linked to it [2e7] . up to april 2016 more than 1360 cases have been reported in saudi arabia alone with a mortality rate that exceeded 40% [8] . though the disease is believed to be zoonotic with strong evidence towards a camel reservoir [9e14], major outbreaks are believed to be nosocomial involving transmission within health care facilities [2,15e18] . early after its discovery, mers-cov infection screening was limited to those critically ill with severe acute respiratory illness [17,19e21] . later on the saudi arabian ministry of health issued guidelines for mers-cov screening in which they limited suspect cases to patients with clinical or radiological evidence of pneumonia, patients with respiratory illness along with a history of possible exposures to a mers-cov patient, and patients with unexplained acute febrile illness with hematological laboratory abnormalities (leukopenia or thrombocytopenia) and gastrointestinal or respiratory symptoms [22] . many studies were conducted in saudi arabia to characterize the illness associated with mers-cov [17,19e21] , but they were limited by the small sample size. studies done elsewhere were limited as well by the extremely small sample size [3e7], except in south korea where a large outbreak occurred between may and july of 2015 due to an imported case from the arabian peninsula [2] . travel associated cases have been observed in europe, notably in uk, france, germany, austria and italy with secondary cases in close contacts of index cases without a travel history suggesting person-to-person transmission [22e25] . in this study we tried to overcome the previous limitation by studying a larger cohort of mers-cov patients at our facility and comparing it to a control group in an attempt to look for predictors of mers-cov infection. in this study we followed a cohort of patients tested for mers-cov infection at prince mohammed bin abdulaziz hospital (pmah) emergency department, a governmental hospital in riyadh, saudi arabia, between april 1st 2014 and september 30th 2015. patients who were felt to have influenza like illness (ili) were screened for mers-cov and all patients who required admission and had any respiratory symptom (cough, shortness of breath, sore throat) were screened for mers-cov infection and followed up until discharged. we also included patients who were called for admission after a positive mers-cov test that was done at an earlier visit to our emergency department and individuals who were hospital quarantined due to a significant exposure history to a mers-cov patient. as pmah is the mers-cov referral center for governmental (public) and private hospitals in riyadh, saudi arabia, we excluded patients who were already diagnosed with mers-cov at other facilities and referred to our hospital for isolation and further management. due to the great heterogeneity of this cohort, and in an attempt to look for predictors of mers-cov infection, we matched the patients who tested positive for mers-cov (cases) to a control group from the rest of the study population who were admitted and repeatedly tested negative for mers-cov. we extracted a matched control for the positive cases based on age (within 5 years of age), gender, and presence of any comorbidity. the attempted matching was 1 case to 2 controls. respiratory samples (nasopharyngeal swapping or tracheal aspirates) were obtained from all patients and tested for mers-cov infection using real-time reverse-transcription polymerase chain reaction (rt-pcr). the specimens were submitted to and testing was carried out at the saudi ministry of health mers-cov regional laboratory. the test amplified both the upstream e protein (upe gene) and orf1a for mers-cov. a positive case was determined if both assays were positive. each patient was tested at least twice, each on a different day. reporting suspected and confirmed cases was the original purpose of data collection which was initially limited to date of admission, sex, age, nationality, designated hospital ward, results/dates of mers-cov testing, and patients' outcomes. data concerning presenting symptoms: fever, sore throat, cough, shortness of breath, and gastrointestinal symptoms (nausea, vomiting or diarrhea), as well as the initial laboratory work up: white blood cell count (wbc) 10 9 per liter, hemoglobin (hgb) grams per deciliter, platelets 10 9 per liter, creatinine micromoles per liter, albumin gram per liter, aspartate aminotransferase (ast) units per liter, alanine aminotransferase (alt) units per liter, and initial chest-x-ray (cxr) results were later extracted from the electronic medical records. we summarized the data by descriptive statistics. frequencies and percentages were calculated for categorical variables. continuous variables (mainly laboratory values) tended to have skewed distributions; thus we used medians rather than means. a conditional logistic regression analysis was conducted to identify the variables that are independently associated with mers-cov infection. the magnitude of association, presented as the odds ratio and 95% ci was also determined this way. all reported p values in this article are also based on the logistic regression. the laboratory measures were found to not be linearly related to the logit of mers-cov infection, so the values were broken into 4 approximately equally sized groups (via quartiles, referred to as quarters) and odds ratios estimated for each as compared to the first quartile as reference. note that for analysis of laboratory measures, a few people had to be excluded due to missing values. we used sas version 9.4 (sas institute, cary nc) to perform all of the analysis. the significance level for all of the statistical tests was set at 0.05. from 24,606 patients who presented to our emergency department between april 1st 2014 and september 30th 2015 with respiratory complaints, 3148 were tested for mers-cov. from those who were screened, only 757 patients were admitted. eighty one patients tested positive for mers-cov and the rest, 676 patients, repeatedly tested negative and were used to find matches to mers-cov cases. see fig. 1 . we were able to match all but two of the confirmed mers patients. for one of them we were only able to find one control, and for the other no match was found. this resulted in a total of 239 patients for analysis. table 1 shows the characteristics of both the mers-cov infected group and the control group. the median age for mers-cov patients was 40 years; 60% were males, 66.2% were saudi, and 16.2% were health care workers. 46% had at least one co morbidity; hypertension and diabetes were the most common. 80% of mers-cov patients were symptomatic; fever was the most common symptom among mers-cov infected group 58.7% (73% of the symptomatic patients), followed by cough 52.5% (73% of symptomatic patients). shortness of breath was the third common symptom that was reported by 37.5% of mers-cov infected patients (47% of the symptomatic patients). around 17.5% of mers-cov infected patients reported gi symptoms (22% of the symptomatic patients). sore throat was reported in 13.7% (17% of the symptomatic patients). in mers-cov infected group the median value for wbcs was 5.7 (iqr: 4.1e8.6), neutrophils 3.6 (iqr: 2.2e5.3), hgb 145 (iqr: 128e157), platelets 205 (iqr: 148e270), albumin 37 (iqr: 34e42), ast 42 (iqr: 26e105), alt 33 (iqr: 21e93), and creatinine 68.1 (iqr: 60.8e81.9). all of the above mentioned medians were within the normal ranges. almost 51% of mers-cov patients had chest x-ray findings upon admission. around 19% were sick enough to be admitted directly to the icu, and around 10% of mers-cov infected patients expired. as discussed above, matching was based on age (within 5 years of age), gender, and presence of any comorbidity. 64.8% of the control group were saudi and11.3% were health care workers. hypertension and diabetes were the most common comorbidities. 95% of the control group patients were symptomatic; fever 64.1% and cough 59.1% were the most commonly reported symptoms in the control group. around 35% complained of shortness of breath, and 14.5% had a sore throat. 13.8% of the control group reported gi symptoms. laboratory analysis showed a median wbcs value of 9.3 (iqr: 6.0e14.2), neutrophils 6.7 (iqr: 3.7e11.1), hgb 138 (iqr: 114e153), platelets 238 (iqr 170e338), albumin 36 (iqr: 32e41), ast 36 (iqr: 23e62), alt 28 (iqr: 14.5e48.5), and creatinine 70.1 (iqr: 61.4e86.9). almost 58% of the control group patients had chest x-ray findings upon admission. around 16.3% of the control group patients were sick enough to be admitted directly to the icu on presentation, and 4.4% of the control group patients expired during their hospital stay. there was no statistical difference in the proportion of saudi nationals (66.2% vs 64.8% or, 1.07; p z 0.82) as well as health care workers (16.2% vs11.3% or, 2.20; p z 0.16) between the confirmed and matched groups. though confirmed mers-cov patients were statistically less likely to be symptomatic (80% vs 95% or: 0.21; p: 0.001), no statistically significant differences between the two groups were found in regards to frequency of a specific symptom (fever, cough, shortness of breath, gastrointestinal symptoms or sore throat with p values of 0.43, 0.35, 0.74, 0.42, and 0.89 respectively). this was as well the case in regards to the presence of chest-x-ray findings upon admission (50.7% vs 57.8%, or: 0.72; p: 0.27). no significant statistical difference was observed between the mers-cov confirmed group and the control group in regards to intensive care unit need upon admission (18.7% vs 16.3%, or: 2.29; p: 0.67). though mortality rate seemed to be higher among mers-cov infected group, this was not statistically significant (10% vs 4.4%, or: 2.29; p: 0.11). the median wbcs counts (5.7 vs 9.3), as well as the median neutrophil counts (3.6 vs 6.7) were both lower in the infected group, and that was statistically significant (p values 0.0004 and 0.0001 respectively). the median alanine aminotransferase (alt) value was higher among mers-cov infected group compared to the control group (33 vs 28, p: 0.003). that was as well the case with aspartate aminotransferase (ast) (42 vs 36, p: 0.031). there was no statistical difference in the median values for hgb, platelets, albumin, and creatinine between the two groups, although the relationship for hgb was marginally significant (p values of 0.07, 0.22, 0.84, and 0.58 respectively). table 2 displays comparative analysis of significant laboratory values between confirmed and suspected cases. the odds of being a confirmed case was significantly lower for those with wbc in the 3rd and 4th quarters (wbcs 8.2) as compared to the reference category of patients with wbc less than the 25th percentile (wbcs < 4.8). the odds of being a confirmed case was significantly lower for those with neutrophil values in the 3rd and 4th quarters as compared to the reference category of patients with neutrophil less than the 25th percentile, with odds ratios 0.22 and 0.18 respectively (quartiles 2.93, 5.06, 9,38) for neutrophil. the median alt values were higher for those with confirmed mers-cov infection as compared to those without (median 33 vs 28 respectively). the odds ratios were significantly increased for patients with confirmed mers when alt values were in the 2nd and 4th quarters (alt 17e29, and alt > 60) as compared to patients with alt values in the lowest quarter (alt < 17); or z 3.72 and 5.94 respectively. patients with values between 29 and 60 did not have statistically significantly increased odds of confirmed mers. patients with confirmed mers had higher median values of ast (42 vs 36). patients with ast greater than 76.5 had statistically significant higher odds of having confirmed mers than those in the lowest quarter (values less than 24) with an odds ratio of 3.31, 95% ci: 1.24, 8.86. as observed in previous studies [17,19e21] , we found that having wbcs and neutrophil counts within the normal range is more likely to be associated with mers-cov. by comparing our cohort of mers-cov patients to another cohort of 47 patients diagnosed with mers-cov between september 2012 and june of 2013 described by assiri et al. [21] we can notice that both cohorts were predominated by male sex (60% vs 77%), though ours had a lower male proportion. male predominance, which was observed in almost every surveillance study [17,19e21] could be related to the culture in saudi arabia, where women wear veils that cover both the nose and mouth and may help protect from exposure, along with decreased outdoor activities compared to men. our patients' median age was 40 years compared to a median age in the range of 50e59 in the previous cohort. fever (59% vs 98%), cough (53% vs 83%), and shortness of breath (38% vs 72%) were the main symptoms, though our patients were less likely to be symptomatic. we also noticed that our patients were less likely to have co morbid conditions (46% vs 96%), less likely to have chest-x-ray abnormalities (51% vs. 100%) and had a significantly lower mortality (10% vs 60%). all this implies that, earlier in the outbreak, screening and diagnoses were limited to the very sick population who subsequently had a high mortality. in our patient population we were liberal in screening any potential admission who complained of respiratory symptoms and due to very strict infection control program we included individuals who were quarantined due to a significant exposure history to a mers-cov patient. by doing so we were aiming at preventing a possible mers-cov outbreak related to inadequate infection control measures. this helped uncover many asymptomatic or mildly symptomatic cases. this might also imply that the true burden of the disease in the kingdom is still uncovered and that we might be just seeing the tip of the iceberg. this theory was first brought up after a nationwide, crosssectional, serological study done between december 1st 2012 and december 1st 2013 in which serum samples from just over ten thousand individuals, whom age and sex distribution largely matched the general population [26] . this report has far reaching implications. in this study we found that none of the presenting symptoms helped distinguish those with mers-cov infection from the matched control group presenting with ili symptoms. almost half of mers-cov patients had no cxr abnormalities on presentation. in addition to raising significant questions on the validity of the current moh suspect case definition, this will challenge the practicing physicians in the emergency room in endemic and non-endemic countries on how to deal with patients presenting with ili symptoms [27, 28] . even with access to full viral panel on all ili patients and with evidence of influenza virus as the etiology, mers-cov can't be ruled out. this is based on data from iran where 3/5 mers cases had concomitant influenza infection [29, 30] . our study has a few limitations, the main being the lack of comprehensive testing for viral respiratory panels for patients admitted with ili symptoms (cases and matched controls). recently this has been added to the testing of all patients admitted with ili. a larger, prospective, multicenter study in the endemic areas is needed to better characterize the illness associated with mers-cov infection and specify its predictors. none. isolation of a novel coronavirus from a man with pneumonia in saudi arabia middle east respiratory syndrome coronavirus outbreak in the republic of korea middle east respiratory syndrome coronavirus (mers-cov) the investigation team. first cases of middle east respiratory syndrome coronavirus (mers-cov) infections in france, investigations and implications for the prevention of human-to-human transmission on behalf of the mers-cov outbreak investigation team of the netherlands. middle east respiratory syndrome coronavirus (mers-cov) infections in two returning travellers in the netherlands laboratory-confirmed case of middle east respiratory syndrome coronavirus (mers-cov) infection in malaysia: preparedness and response first confirmed cases of middle east respiratory syndrome coronavirus (mers-cov) infection in the united states, updated information on the epidemiology of mers-cov infection, and guidance for the public, clinicians, and public health authorities -may ccc) statistics. kingdom of saudi arabia: ministry of health (moh) evidence for camel-to-human transmission of mers coronavirus replication and shedding of mers-cov in upper respiratory tract of inoculated dromedary camels middle east respiratory syndrome coronavirus neutralizing serum antibodies in dromedary camels: a comparative serological study antibodies against mers coronavirus in dromedary camels middle east respiratory syndrome coronavirus infection in dromedary camels in saudi arabia middle east respiratory syndrome (mers) coronavirus seroprevalence in domestic livestock in saudi arabia an observational, laboratory-based study of outbreaks of middle east respiratory syndrome coronavirus in jeddah and riyadh, kingdom of saudi arabia hospital-associated outbreak of middle east respiratory syndrome coronavirus: a serologic, epidemiologic, and clinical description hospital outbreak of middle east respiratory syndrome coronavirus synthesizing data and models for the spread of mers-cov, 2013: key role of index cases and hospital transmission middle east respiratory syndrome coronavirus: a case-control study of hospitalized patients clinical aspects and outcomes of 70 patients with middle east respiratory syndrome coronavirus infection: a singlecenter experience in saudi arabia epidemiological, demographic, and clinical characteristics of 47 cases of middle east respiratory syndrome coronavirus disease from saudi arabia: a descriptive study infection prevention and control guidelines for middle east respiratory syndrome coronavirus (mers-cov) infection imported cases of middle east respiratory syndrome: an update travel implications of emerging coronaviruses: sars and mers-cov middle east respiratory syndrome coronavirus: current situation and travelassociated concerns presence of middle east respiratory syndrome coronavirus antibodies in saudi arabia: a nationwide, crosssectional, serological study an adult returned traveler from dubai hospitalized with an influenzalike illness (ili): middle east respiratory syndrome (mers) or influenza? infection control implications from a near mers case middle east respiratory syndrome (mers): a zoonotic viral pneumonia serial intervals of respiratory infectious diseases: a systematic review and analysis cluster of middle east respiratory syndrome coronavirus infections in iran none of the authors declared coi. key: cord-019100-rce6kyu4 authors: heymann, peter w.; zambrano, juan c.; rakes, gary p. title: virus-induced wheezing in children: respiratory syncytial virus (rsv) and rhinovirus date: 1998-02-01 journal: immunol allergy clin north am doi: 10.1016/s0889-8561(05)70345-3 sha: doc_id: 19100 cord_uid: rce6kyu4 the strong association between infantile wheezing and respiratory tract infections caused by the respiratory syncytial virus (rsv) has been well established. in studies of older children, rhinovirus becomes the major virus associated with asthma. these relationships are outlined in the box on page 36. in the past, this relationship was more difficult to appreciate, because rhinovirus does not always grow well in culture. in addition, the linkage between asthma and atopy during childhood has raised the question whether viral infections alone can precipitate exacerbations of asthma. use of the polymerase chain reaction (pcr) to measure viral nucleic acid material has provided the opportunity to study virus-induced wheezing among children in greater detail, and investigations of experimental rhinovirus infections in adults have demonstrated how this virus can augment both the early and late phase manifestations of airway hyperreactivity. this article reviews recent advances that have enhanced our understanding of virus-induced wheezing, along with new information indicating that interactions between viral infections and allergic inflammation may be critical to the pathogenesis of acute symptoms. a. early childhood wheezing (1st 2 years of life): 1. predisposing risk factors: prematurity; lower lung function at birth; maternal smoking during and following pregnancy; frequent exposure to viral infections at day care. 2. major risk factors for acute attacks: viral infections, especially rsv; tobacco smoke exposure. 1. predisposing risk factors: genetic predisposition (maternal asthma and atopy is a stronger influence than asthma and atopy in the father); atopic dermatitis and/or food hypersensitivity: increased exposure to aeroallergens early in life. 2. major risk factors for acute attacks: viral infections, especially rhinovirus; exposure to aeroallergens (dust, pollen, weed, mold allergens); tobacco smoke exposure. the interaction between rhinovirus infections and allergic inflammation becomes significantly associated with wheezing after the age of 2 years. b. development of asthma: *datafrom information and literature cited in references 11, 17, 23, 35 , and 48. a vast majority of children become infected with rsv during their first 2 years of life, and approximately 90% of children have a serologic response to this virus by the age of z. 31 in keeping with this, rsv is the most common viral respiratory tract pathogen isolated from infants who wheeze. many of the attacks that lead to emergency room visits and hospitalizations occur within the first 6 months of life, when infants are more likely to experience their initial infection.", **, 31 current information indicates that rsv infections during infancy are not, by themselves, responsible for the development of a~thma.3~ it has become clear, for example, that most infants who wheeze (approximately 60% to 70%) become symptom-free as they grow older.", 4x nevertheless, up to one third of the children who wheeze with rsv are at risk for recurrent and persistent wheezing, and there is considerable interest in research that will improve our ability to identify those children early in life. it has become apparent, for example, that infants whose parents have allergic symptoms or asthma, especially the mother, have a greater risk for developing asthma and that predisposing events that may influence this process may even begin in utero.zu, 35 infants with atopic dermatitis or those who become sensitized to food allergens early in life also have an increased risk for developing allergic respiratory tract symptoms, including asthma, as they grow ~l d e r .~, '~, 3u among infants who die from serious rsv disease, a vigorous perivascular lymphocyte response has been observed in the lungs at however, a different pathogenic process also has been observed during clinical trials of a formalin-inactivated rsv vaccine; this process led to serious pulmonary disease as well as peripheral blood eosinophilia in over half of the vaccine recipients when they acquired a subsequent rsv infection? pulmonary eosinophilia, characteristic of lung inflammation in asthma, also was noted in autopsy specimens from some of the recipients who died.33 mechanisms for the development of this eosinophilic response in infants are not clear. in previous studies, an ige mechanism for wheezing was proposed for some infants based on the detection of ige antibody specific for rsv antigen in their nasopharyngeal secretion^.^^ increased levels of histamine in secretions from the same patients also were reported. in addition, elevations in nasal eosinophil cationic protein (ecp) were observed by the same investigators in a separate study of rsv infected infants.'* at present, however, it is not clear that ige to rsv has a positive predictive value for the development of asthma;* and high levels of nasal ecp and ige to rsv have not yet been measured in the same patients. thus, the functionality of ige to rsv in the pathogenesis of infantile wheezing and its relevance to the development of asthma is uncertain. under certain circumstances, there is evidence that rsv infections may be capable of inducing a th, lymphocyte response leading to the development of eosinophil inflammation. in mice, th, responses to the capsular rsv-attachment antigen (g protein) have been demonstrated, leading to the production of il-5 when lymphocytes from sensitized mice are stimulated in vitro with rsv-g. these mice also developed pulmonary eosinophilia following subsequent intranasal infection with rsv.4v,4y the quantities of il-4 or ige antibody produced by these animals is not significant, and the eosinophil response in lungs also has been produced in b-cell-deficient mice, suggesting a mechanism independent of ige antibody. this eosinophil process, however, requires prior immunization (sensitization) and may not explain the pathogenesis of rsv-induced wheezing in humans when they experience initial infections during early infancy. other studies have examined the elaboration of cytokines (le., il-11) that may be important in the pathogenesis of airway inflammation caused by rsv, as well as parainfluenza virus.13 taken together, our understanding of infantile wheezing induced by rsv has increased substantially. however, research leading to new treatments (vaccines and pharmacologic agents) to counter the adverse effects of rsv and the identification of wheezing infants who are predisposed to developing asthma remain important problems. in studies of school-age children with asthma, common cold viruses were associated with 80% to 85% of wheezing exacerbations in a community-based study of asthmatic children aged 9 to 11 yearsz6 using pcr analysis, rhinovirus accounted for two thirds of the viruses isolated. inspection of symptom score charts revealed sudden falls in peak flow values within 2 days of the onset of cold symptoms, followed by recovery to baseline during the subsequent 2 to 3 weeks. in another study, a strong relationship between rhinovirus infection and asthma was observed in children treated in a pediatric emergency department for acute wheezing." rhinovirus was the dominant virus cultured in nasal aspirates from wheezing children after the age of 2 years. from this age on, rsv was not significantly associated with wheezing, whereas the combination of allergen-specific ige antibody and a positive culture for rhinovirus was particularly striking (odds ratio for wheezing = 10.8). when nasal washes from another study of children seen in the emergency room were tested using pcr techniques, rhinovirus was again the most common virus associated with wheezing after the age of 2.43 once again, the combination of allergen-specific ige antibody and viral infection increased the risk for wheezing (odds ratio = 16.4). even more striking among the wheezing children was a strong relationship between a positive pcr test for rhinovirus and the presence of eosinophils or ecp in nasal secretions. although school-age children experience approximately six to eight colds per year as compared with two to three colds per year for adults, recent pcr data also indicate that rhinovirus infections were associated with 38% of asthma exacerbations in adult individuals 19 to 46 years old.3y other respiratory tract pathogens have been isolated from children and adults during asthma exacerbations. coronavirus, which has been identified as a causative agent in 10% to 20% of common colds, also has been implicated in attacks of asthma.41 less frequently, influenza and adenovirus have been detected. although mycoplasma pneumoniae has been observed in up to 20% of community-acquired episodes of pneumonia, this organism probably is an infrequent cause of asthma exacerbations.36 thus, evidence from several studies indicate that rhinovirus is the major viral pathogen linked to attacks of asthma in children and adults. this information has stimulated additional studies, primarily experimental infections in adults, to explore how rhinovirus alters lower airways function. because of the importance of rhinovirus as a cause of common cold symptoms, there is considerable information characterizing the structure of rhinovirus and its interactions with the intercellular adhesion molecule-1 (icam-1) receptor. the human rhinoviruses represent a large genus within the class of picornaviruses (small rna viruses). the genus contains up to 100 distinct serotypes showing antigenic diversity. 44 the molecular structure of rhinovirus (type 14) has been characterized in great detail. this virus is a small (30 mm) nonenveloped particle within an icosahedral protein capsule composed of 60 protomeric units with four capsid proteins (vp 1, vp 2, vp 3, and vp 4). x-ray diffraction studies have shown canyons on the capsid surface that contain recognition binding sites for icam-1.44 the receptor icam-1 belongs to the immunoglobulin super-group of proteins and is expressed on the luminal surface of epithelial and endothelial cells. its natural ligand is lfa-1 (lymphocyte function associated antigen), which is widely expressed on leukocytes including eosinophils. its upregulation can be induced by a number of cytokines (i.e., ifn-?) and is associated with the recruitment of leukocytes to the sites of inflammation. icam-1 is also the host receptor used by the vast majority (goo/,) of rhinovirus serotypes for cell attachment and entry.sn it has been suggested that its up-regulation in allergic inflammation may predispose allergic individuals to higher rates of rhinovirus infection, leading to further increases in icam-1 expression and inflammatory cell infiltration^.^ its proposed up-regulation in asthma also may increase the probability of a rhinovirus infection and the capacity of this virus to provoke an asthma attack. standard methods for detecting rhinovirus have included viral culture, serology, and immunofluorescent techniques. many epidemiologic studies of the common cold and initial studies of experimental rhinovirus infections relied on cultures for detection. recovery rates of rhinovirus in nasal secretions from children experiencing natural colds have varied from 10% to 3oy0.~' however, rhinovirus does not grow optimally in culture, and positive results in population based studies may underestimate the prevalence of rhinovirus infections. more recently, pcr methods that detect rhinovirus rna have been compared with cultures in children with common cold symptoms. the results demonstrated a detection rate up to threefold greater using pcr analysis.z8 however, during experimental colds, rhinovirus can be recovered in culture in up to 90% of nasal washes obtained from patients at the height of symptoms (2 to 3 days following inoculation).ir this raises the possibility that the lower detection rates for rhinovirus using cultures from individuals during natural colds may result from the delay in obtaining samples and abatement of viral shedding once the infection is in progress. several investigations have examined rhinovirus infections and the pathogenesis of common cold symptoms in nonallergic individuals as outlined in the box below. these studies have explored the effects of rhinovirus on the upper and lower respiratory tract and have established a framework for more recent studies of rhinovirus infections in allergic individuals. the timing of viral shedding in relation to cold symptoms has been carefully examined following experimental rhinovirus inoculation of adult volunteers.ir viral replication in the nasopharynx was noted to increase 1 day after inoculation and peaked at 48 hours. cold symptoms including rhinitis, pharyngitis, and bronchitis began within 24 to 48 hours and then peaked by day 3 or 4. in a study of ct scans from adults, sinus involvement with membrane thickening and mucous hypersecretion was evident in 87% of patients within 2 to 4 days after experimental rhinovirus inoc~lation.'~ this indicates that rhinovirus colds are likely to encompass a viral rhinosinusitis, not just rhinitis. during experimental infections, no significant cytopathic changes have been found in either ciliated or nonciliated nasal epithelial in addition, no destruction of the nasal mucosa or the inferior turbinate was observed by scanning electron microscopy. implications of these findings are that the production of cold symptoms with rhinovirus are caused by mechanisms other than tissue destruction. these findings are in marked contrast to epithelial cell damage caused by adenovirus and influenza a and b virus in cell cultures. 57 in situ hybridization has been used to localize sites and determine the extent of rhinovirus replication. in experimentally infected volunteers, rhinovirus replicated in a small proportion (patchy involvement) of epithelial cells distributed in the nasal epithelium and in nonciliated cells in the nasopharynx." following the onset of cold symptoms, the number of neutrophils in the epithelium and subepithelial cell layer were noted to increase by day 2 during an experimental infection.5h this was followed by an influx of neutrophils into nasal secretions leading to a purulent discharge in experimentally infected volunteers 4 to 5 days after the onset of symptoms. this change was not accompanied by associated changes in bacterial pathogens, suggesting that rhinovirus itself may be involved in initiating this neutrophil response. although the number of ivmdhocvtes observed in the nasal mucosa did not change significantly, a mild lymphocyte infiltration was noted after a period of 2 to 4 i~ even though the local immunopathologic and pathophysiologic processes of colds induced by rhinovirus are not fully understood, recent information about mediators and cytokines is likely to contribute to knowledge of the pathogenesis of events leading to symptoms. generally, levels of histamine in nasal secretions do not change significantly during acute experimental rhinovirus infections in nonallergic individuals.'*, 38 however, increased concentrations of kinins, which act as potent vasoactive substances, have been detected in nasal secretions of adults given rhinovirus experimentally; and such symptoms as sore throat, nasal congestion, and rhinorrhea have been observed in healthy volunteers after administering bradykinin intranasally.3r, 42 increased levels of il-8, a potent leukocyte chemoattractant, have been measured in nasal secretions from subjects with wild-type rhinovirus infection^.^^ increases in the production of other proinflammatory cytokines (i.e., il-11, gm-csf, ifn-y, and tnf-a) also have been reported?, i5fz9 the capacity of rhinovirus to cause infection and replicate in the lower respiratory tract has been difficult to confirm. in part, this derives from difficulty in obtaining specimens that are free of contamination. in studies of adults with allergic rhinitis who were infected experimentally with rhinovirus (type 16), virus was not isolated either from fluid or cells obtained by bronchoalveolar lavage (bal)." by comparison, virus always was found in high titers in nasal washes. explanations for the preferential replication of rhinovirus in the upper airways has been attributed to its temperature sensitivity in culture, which may tend to favor its replication in the upper a i r~a y s . 4~ more specifically, reducing the temperature of incubation of rhinovirus in culture from 37â°c to 33â°c greatly enhances the efficacy of virus isolation. while studies continue to document rhinovirus replication and infection in the lower airways, investigations focused on inflammatory pathways involving mediators and cytokines will continue to be important in defining the immune response stimulated by this virus. in contrast, there is less doubt that other respiratory viruses (adenovirus, influenza virus, rsv, and parainfluenza virus) can infect the lower airways. several studies have compared the severity of upper respiratory tract symptoms and lower airway responsiveness in allergic and nonallergic adults following experimental rhinovirus inoculation. in studies of experimental viral rhinitis by bardin and colleagues, no difference between atopic patients and controls was observed with respect to shedding of rhinovirus or symptom scores.' a particularly interesting finding was that the severity of colds experienced by nonallergic individuals was increased in those who lacked preinoculation neutralizing antibodies in their sera to the rhinovirus serotype used for inoculation. this was not observed in atopic individuals, who developed severe colds and had a significant increase in nasal wash albumin in spite of the presence of neutralizing antibody. doyle and colleagues found little evidence to support an increased susceptibility of atopic subjects to rhinovirus.lu however, atopic patients did experience an earlier onset of sneezing and nasal congestion. these investigators examined peripheral blood lymphocyte responses during experimental infection. a consistent feature was the persistence of t-cell activation to rhinovirus in atopic subjects up to 3 weeks after i n f e~t i o n .~~ skoner and others from this group noted that experimental rhinovirus (type 39) infection induced significant increases in total serum ige, leukocyte histamine release, and platelet aggregation during the acute phase of infection in adults with allergic r h i n i t i~.~~ however, the acute phase rise in total serum ige could not be correlated to a rise in specific ige antibody to allergens tested, and there was no correlation between the rise in serum ige and enhanced release of histamine from leukocytes stimulated with anti-ige antibody. similarly, total ige levels in atopics given a rhinovirus infection did not correlate with the severity of rhinitis in studies by others.' thus, the functional significance of the rise in total ige during infection is unclear. no changes in plasma histamine or in histamine in nasal secretions have been observed in nonatopic individuals during rhinovirus infections.'2, 38 however, increased histamine secretion was seen more frequently during the initial days of rhinitis in nasal washes from allergic adults following rhinovirus inoculati~n.~~ in studies of bronchial reactivity, increased airway hyperresponsiveness demonstrated by spirometry and methacholine challenges were observed in some but not all asthmatic subjects during experimental infection with rhinovirus (type 39).*' these changes were not observed in nonallergic subjects. in a more recent study, ragweed-allergic patients with rhinitis developed increased lower airway responsiveness to inhaled histamine and ragweed allergen during an acute infection with rhinovirus (type 16).32 of interest in this study by lemanske and colleagues was that their patients also had an increased likelihood of developing late phase bronchoconstrictor responses following allergen challenge. these changes remained persistent up to 4 weeks after the experimental infection. overall, these observations suggest an important relationship between rhinovirus infection and the potentiation of allergic inflammation in the airways. the effects of bronchoprovocation with ragweed allergen during rhinovirus infection were examined in individuals with allergic rhinitis by calhoun and colleague^.^ using a segmental allergen bronchoprovocation method, the investigators detected increased levels of histamine in bal samples, both immediately and 48 hours after antigen challenge. during the infection, tryptase from mast cells was initially detected in bal samples; however, a significant increase in histamine, but not tryptase, during the late phase response suggested that basophils rather than mast cells may be the cell source of histamine at 48 hours. the most striking observation during the acute infection was an augmented recruitment of eosinophils into the airways 48 hours after allergen challenge. moreover, these effects persisted and were apparent up to 1 month after rhinovirus inoculation. these data indicate that rhinovirus infection in the upper respiratory tract can significantly augment both immediate and late phase cell and mediator responses in the lower airways of allergic individuals. in another study of adult volunteers, including six allergic asthmatics, an increase in lower airways responsiveness to histamine was noted during the acute phase of rhinovirus infection.= this was accompanied by increases in submucosal lymphocytes followed by a fall in lymphocyte numbers in the epithelium and submucosa detected in bronchial biopsy specimens during the convalescent period. an increase in epithelial eosinophils also was noted during the cold, and the rise in eosinophil numbers persisted into convalescence in the asthmatic, but not in the normal subjects. increased sensitivity to histamine in the asthmatics also persisted in the convalescent period. taken together, these studies have demonstrated a relationship between rhinovirus upper respiratory tract infections and augmented late phase bronchial responsiveness associated with eosinophil recruitment into the lungs. more detailed studies directly linking infections to the development of this eosinophil response and studies to define the mechanisms leading to this reaction are of great interest. recent studies provide evidence for rhinovirus activation of t cells through a monocyte-dependent mechanism that also promoted eosinophil survival in vitro.lh a link between virus infections and allergic eosinophil inflammation has also been implicated in a mouse model, demonstrating that a bystander cd4 + th, cellular response to ovalbumin can switch virus peptidespecific cd8 + t cells in the lung to produce il-5, leading to eosinophil infiltration following virus peptide challenge.$ epidemiologic studies have shown a correlation between the seasonality of viral upper respiratory tract infections-particularly rsv and rhinovirus-and asthma exacerbations. in the northern hemisphere, rsv infections are most common during the midwinter months (december through february). these are months when infants are more likely to be seen in clinics, emergency rooms, and the hospital for acute attacks of wheezing (fig. 1).11,22 in a time/trend analysis, the seasonal patterns of respiratory tract infections and hospital admissions were evaluated for older children and a particularly strong correlation was found between the seasonal pattern of upper respiratory tract infections, particularly rhinovirus, and hospital admissions for asthma among children. upper respiratory tract infections and admissions for asthma also are more frequent during periods of school attendance and less frequent during school holidays.*7, 51 consistent with this, peak admissions to the pediatric emergency room for wheezing at the university of virginia are observed annually in the fall and spring months when rhinovirus infections are most common combined with increased exposure to tree and grass pollens in the spring and ragweed, alternaria, and higher levels of household dust mite allergen during the fall (fig. 2) . this visiting pattern already becomes apparent for wheezing exacerbations in young children, aged 2 and 3, when the diagnosis of asthma often is considered (figure 2 ). an awareness of the combined seasonal influences of allergen exposures and viral infection can be very helpful in designing treatment plans for individual patients. these treatment plans should include allergen avoidance and emphasize seasonal requirements for daily antiinflammatory medications. physicians also should be aware that these seasonal influences are likely to vary geographically. evidence that infections with rhinovirus may augment the late phase eosino-phi1 response in asthmatic individuals provides a rationale for using steroids to minimize the severity and persistence of airway inflammation and decrease the reliance of patients on inhaled bronchodilators during colds. when children with asthma develop a cold, peak flow tests can be useful to monitor changes in lower airway function. morning peak flow tests should be checked with the onset of cold symptoms and for the duration of the infection, with particular attention focused on changes that may occur during the first 2 to 4 days. recent studies indicate that inhaled steroids may benefit asthmatic children during 45, 55 those who are already using inhaled steroids daily may benefit from an increased dose for 2 to 3 weeks. should symptoms worsen, the need for systemic steroids should be considered with the patient's physician. important questions remain about the capacity of viral respiratory tract pathogens to augment inflammatory pathways in the lungs of asthmatic children. more research is needed to learn whether the examples of pulmonary eosinophilia stimulated by rsv antigens (i.e., the attachment g protein) in animals and humans represent an immune response similar to parasite induced eosinophilia or whether this is a process characteristic of allergic inflammation in an individual who is genetically predisposed to develop allergic respiratory symptoms and asthma. the absence of cytopathic changes in the respiratory tract epithelium during rhinovirus colds, the patchy involvement of infected epithelial cells, and the lack of direct evidence for virus replication and infection in the lower airways suggest mechanisms involving the enhanced production of proinflammatory cytokines in the pathogenesis of inflammation induced by this virus. candidate cytokines such as tnf-a, ifn-y, il-5, il-8, il-11, and gm-csf currently are being investigated with respect to their cell sources and production during rhinovirus infections in allergic and nonallergic patients. in addition, efforts to develop reagents to block the binding of rhinovirus to icam-1, the major receptor used by rhinovirus to infect cells, also represent a therapeutic approach for preventing episodes of asthma induced by this overall, our understanding for the capacity of viral infections to precipitate wheezing attacks in infants and children has improved significantly during the last decade. the need for new information and strategies to treat virus-induced asthma exacerbations is now becoming more important as the prevalence of asthma continues to increase. acknowledgment manuscript. the authors are grateful to richard leahy for his assistance in the preparation of this amplified rhinovirus colds in atopic subjects lower airways inflammatory response during rhinovirus colds. int arch allergy immunol prognosis of asthma in children a common cold virus, rhinovirus 16, potentiates airway inflammation after segmental antigen bronchoprovocation in allergic subjects icam-1 on epithelial cells in allergic subjects: a hallmark of allergic inflammation field evaluation of a respiratory syncytial virus vaccine and trivalent parainfluenza virus vaccine in a pediatric population prevention of viral induced attacks using inhaled budesonide virus-specific cd8 + cells can switch to interleukin 5 production and induce airway eosinophilia localization of human rhinovirus replication in the upper respiratory tract by in situ hybridization rhinovirus 39 infection in allergic and nonallergic subjects risk factors for acute wheezing in infants and children: viruses, passive smoke, and ige antibodies to inhalant allergens mediators of immediate hypersensitivity in nasal secretions during natural colds and rhinovirus infection interleukin-11: stimulation in vivo and in vitro by respiratory syncytial virus and induction of airway hyperresponsiveness eosinophil degranulation in the respiratory tract during naturally acquired respiratory syncytial virus infection the effects of rhinovirus infections on allergic airway responses rhinovirus produces non-specific activation of lymphocytes through a monocyte-dependent mechanism natural history of sensitization in atopic dermatitis rhinovirus infection of the normal human airway computed tomographic study of the common cold influence of maternal allergic disease during pregnancy on ige, alternaria skin tests and asthma in children exacerbations of asthma in adults during experimental rhinovirus infection the etiologic and epidemiologic spectrum of bronchiolitis in pediatric practice allergy: early childhood asthma: what are the questions? analysis of nasal secretions during experimen-92~535-540 tal rhinovirus upper respiratory tract infections eosinophil cationic protein in serum and nasal washes from wheezing infants and children community study of role of viral infections in exacerbations of asthma in 9-11 year old children the relationship between upper respiratory infections and hospital admissions for asthma: a time-trend analysis use of polymerase chain reaction for diagnosis of picornavirus infection in subjects with and without respiratory symptoms natural and experimental rhinovirus infections of the lower respiratory tract a clinical and immunologic study of allergy to hen's egg white stuntman hr respiratory syncytial virus puzzle: clinical features, pathophysiology, treatment and prevention rhinovirus upper respiratory tract infection increases airway hyperreactivity and late asthmatic reactions respiratory syncytial virus disease in infants despite prior administration of antigenic inactivated vaccine a soluble form of intercellular adhesion molecule-1 inhibits rhinovirus infection asthma and wheezing in the first six years of life mycoplasmal infections. epidemiology, immunology, diagnostic techniques, and therapeutic strategies risk factors for developing wheezing and asthma in childhood et a1 kinins are generated during experimental rhinovirus colds respiratory viruses and exacerbations of asthma in adults pulmonary eosinophilic response to respiratory syncytial virus infections in mice sensitized to the major surface glycoprotein g viruses as precipitants of asthma symptoms, i: epidemiology la capra s, et a1 nasal provocation with bradykinin induces symptoms of rhinitis and a sore throat human rhinovirus in wheezing children: relationship to serum ige and nasal eosinophil cationic protein inhaled corticosteroids provide better protection against worsening asthma following respiratory infection effect of rhinovirus 39 infection on cellular immune parameters in allergic and non-allergic subjects effect of rhinovirus 39 (rv-39) infection on 13 immune and inflammatory parameters in allergic and non-allergic subjects natural history of asthma in childhood: a birth cohort study virus-specific memory and effector t lymphocytes exhibit different cytokine responses to antigens during experimental murine respiratory syncytial virus infection a cell adhesion molecule, icam-1, is the major surface receptor for rhinoviruses school holidays and admissions with asthma increased levels of il-8 in the nasal aspirates of children with virus-associated asthma the development of respiratory syncytial virusspecific ige and the release of histamine in nasopharyngeal secretions after infection the relationships of rsv-specific immunoglobulin e antibody responses in infancy, recurrent wheezing, and pulmonary function at age 7-8 years treatment of acute, episodic asthma in preschool children using intermittent high dose inhaled steroids at home et a1 histopathologic examination and enumeration of polymorphonuclear leukocytes in the nasal mucosa during experimental rhinovirus colds respiratory virus infection of monolayer cultures of human nasal epithelial cells bronchiolitis-state of the art key: cord-262623-lmf2h6oc authors: light, r. bruce title: plagues in the icu: a brief history of community-acquired epidemic and endemic transmissible infections leading to intensive care admission date: 2009-01-31 journal: critical care clinics doi: 10.1016/j.ccc.2008.11.002 sha: doc_id: 262623 cord_uid: lmf2h6oc the ability to diagnose and treat infectious diseases and handle infectious disease outbreaks continues to improve. for the most part, the major plagues of antiquity remain historical footnotes, yet, despite many advances, there is clear evidence that major pandemic illness is always just one outbreak away. in addition to the hiv pandemic, the smaller epidemic outbreaks of legionnaire's disease, hantavirus pulmonary syndrome, and severe acute respiratory syndrome, among many others, points out the potential risk associated with a lack of preplanning and preparedness. although pandemic influenza is at the top of the list when discussing possible future major infectious disease outbreaks, the truth is that the identity of the next major pandemic pathogen cannot be predicted with any accuracy. we can only hope that general preparedness and the lessons learned from previous outbreaks suffice. intensive care, the epitome of the application of modern technology to medicine, arguably began as a response to the increasing numbers of older children and young adults developing paralytic polio during the early 1950s. severe paralytic polio itself turns out to have been among the products of increasing modernity in the western world. clinical recognition of the syndromes associated with the polio virus date back about 200 years. the fact that the disease was caused by a transmissible viral particle was demonstrated by 1908. until early in the twentieth century the virus was readily transmitted throughout the population almost continuously by personal contact and by the fecal-oral route via water. the result was that most people's first exposure to the virus occurred in infancy. at this age most infections resulted in a clinically unapparent infection, in part due to partial protection by maternal antibody, after which lifelong immunity was established. only a few suffered a paralytic episode with the infection, at the time termed ''infantile paralysis.'' 1 as hygienic standards of the advanced economy nations rose throughout the first half of the century, early childhood exposure to the virus declined. an increasing fraction of the population had their first exposure in late childhood or during young adulthood. in these age groups the likelihood that the infection will cause a paralytic syndrome is greatly increased, so both the incidence of clinically recognized infection and that of paralysis rose. by the early 1950s clinically recognized cases had reached 15-25 annually for every 100,000 people in the united states, making it a major public health concern and a source of a great deal of fear in the general populace. 2 more than 90% of polio virus infections are asymptomatic. however, at the more severe end of the clinical spectrum are the paralytic syndromes which range from paralysis of one or more limbs (''spinal polio'') to syndromes with respiratory muscle or bulbar paralysis (''respiratory polio'' or ''bulbospinal polio'') with loss of respiratory or upper airway muscle function or both. these more severe outcomes rise in incidence from about 0.1% in infants to more than 1% in older children and adults. in the early part of the century development of polio with bulbar involvement was associated with a death rate of greater than 90%, generally from respiratory failure. use of a mechanical respirator to try to avert death was first attempted at the children's hospital in boston in 1928, using an ''iron lung.'' 3 the machine was basically a sealed box with a hole at one end for the patient's head to protrude, attached to two vacuum cleaners. the motors were then cycled to alternately create vacuum inside the box, expanding the patient's chest and causing the patient to inhale through the mouth (outside the box), then allowing air back into the box to permit exhalation. the design was further improved in 1931, and the machines came into increasingly broad use throughout north america and europe during the severe outbreaks of the 1930s. adoption of this therapy resulted in a significant reduction in mortality during these years. iron lungs, however, were cumbersome, difficult to use when trying to provide nursing care, and expensive. a more cost-effective and user-friendly approach to providing respiratory support was clearly needed. this finally came by bringing the positive pressure ventilation (ppv) concept out of the operating room. ppv was first used for respiratory support for polio victims at blegdam hospital in copenhagen, denmark, an innovation attributable to danish anesthetist bjorn ibsen. 4 during this large outbreak in 1952, some 200 medical students were put to work hand-ventilating dozens of patients through tracheostomies until the worst of the paralytic phase of the illness had passed, often several weeks. the concept quickly spread elsewhere and was widely adopted, yielding substantial reductions in mortality. for reasons of efficiency and convenience, patients needing respiratory support were often grouped in a single location where the necessary expertise and equipment were available. the introduction of ppv into a defined area of the hospital used to support respiratory failure was the genesis of the modern icu and represents a signal event in the development of the field of critical care medicine. the introduction of effective vaccines, the salk (inactivated) vaccine in 1952 and later the sabin (live attenuated oral) vaccine in 1962, immediately and dramatically reduced the incidence of polio to less than one per 100,000 population by the early 1960s and the incidence continued to fall thereafter. the last case of wild strain polio in north america was reported in 1979, and since that time the only cases of paralytic polio have been rare instances of disease due to variants of the live oral vaccine strain. polio does, however, still contribute to illnesses that may require intensive care in the form of the ''post-polio syndrome.'' this occurs in patients who survived paralytic polio decades ago and who, over the years, develop a gradual decline in function in the originally affected nerves and muscles which can years later once again threaten them with disability and, in some cases, respiratory failure requiring intensive care. 5 in july 1976, american legion members attending a convention at a hotel in philadelphia suddenly began falling ill with an acute febrile illness with pneumonia, often associated with extrapulmonary symptoms such as myalgia or diarrhea. 6 many developed acute respiratory failure requiring mechanical ventilatory support in icu. over 200 were affected and 34 died, an alarming mortality rate, especially since physicians caring for the patients had no idea what was causing the illness. conventional microbiologic investigations yielded no convincing pathogens despite intensive investigation for the usual bacteria and viruses and other potential pathogens. epidemiologic and various biologic investigations were quickly implemented by local health authorities and by the centers for disease control and prevention (cdc). these showed that the disease was likely airborne and that it occurred more frequently in older individuals who had underlying lung disease, smoked, or were relatively immunocompromised. analysis of the likelihood of death revealed that those who received tetracyclines or macrolide antibiotics were more likely to survive than those who received only betalactams. however, no causal agent was uncovered, though many potential causes were excluded-most known bacteria and viruses, many biologic toxins and many environmental agents such as toxic chemicals and metals. in 1977, joseph mcdade and charles shepard of the cdc reported the isolation of a novel fastidious gram-negative bacillus from the available clinical specimens. 7 they named it legionella pneumophila. this discovery was quickly followed by an explosion of knowledge about the organism and its ecology, antimicrobial susceptibility, and of other bacteria within the genus. over time, demonstration that it was a widely occurring colonizer of brackish water, particularly in air conditioners, cooling towers, and water heaters and pipes, led to the implication of these reservoirs in several hundred outbreaks of the disease worldwide, especially in hospitals and other public health institutions, and hotels. thus began widespread development of regulations and guidelines for limiting the degree of colonization of these water sources by legionella, resulting in a reduction in the size, number, and scope of subsequent outbreaks. since the initial description of the disease, legionella has, of course, been shown to be one of the major causes of community-acquired pneumonia (cap), particularly in the more severe subset requiring icu care; this fact underlies the major lesson from plagues in the icu the outbreak-the nearly universal recommendation for inclusion of antimicrobial therapy for legionell spp in any treatment regimen for severe cap without another obvious cause. although we now know that no amount of continuing effort can completely eliminate this organism from our environment, and that we will continue to see endemic cases, we also know that clusters of cases should trigger an investigation into finding the point source of the outbreak, a situation that continues to occur several times a year somewhere in the world. in addition, the 1976 philadelphia outbreak that defined ''legionnaire's disease'' was the first in the modern icu era to demonstrate that major unknown infectious disease syndromes of severe consequence still exist, presaging the new infectious disease syndromes to be discovered in the years that followed. in the late 1970s, emergency rooms and icus throughout north america began to see an increasing number of young menstruating women presenting with a previously little-known syndrome characterized by sudden onset of a high fever, often associated with vomiting and diarrhea, quickly followed by severe hypotension. early in the course most patients developed a diffuse macular rash, often with mucous membrane inflammation, with subsequent desquamation during convalescence. patients frequently required massive fluid resuscitation because of systemic capillary leak, as well as vasopressor support, mechanical ventilation for adult respiratory distress syndrome, and even renal replacement therapy for acute renal failure, complicating the shock episode. one of the early clusters of observed cases was reported in 1978, and the term ''toxic shock syndrome'' was coined based on the isolation of an exotoxin-producing staphylococcus aureus from mucosal surfaces or the site of a minor infection in the absence of bacteremia. 8 as the case numbers rapidly increased case definitions for the syndrome were formulated and epidemiologic studies mounted. by 1980, over 1000 cases had been formally reported to the cdc. 9 the case fatality rate was reported to be as high as 15% in the more severe cases included in the earliest reports, falling to about 3%-5% as recognition of the syndrome improved. by this time there were clear epidemiologic links between the syndrome and menstrual use of high-absorbency tampons which were often left in place longer than less absorbent products. colonization of the tampon with staphylococcus aureus was also implicated, consistent with the postulated toxin-mediated disease mechanism. 10 within months of these revelations the main manufacturer of the implicated tampons withdrew them from the market and women began changing tampons with greater frequency or stopped using them at the urging of public health authorities. the incidence of the syndrome immediately began to fall and within a few years, with the changing of use patterns of tampons and changes in their manufacture, toxic shock syndrome disappeared, for the most part, but not entirely, from the icu. even at the height of tss incidence in the united states, about 6% of the cases reported were nonmenstrual and 4% were in males. 11 subsequent development of the knowledge that the clinical syndrome was due to strains of staphylococcus aureus that secrete a particular toxin (toxic shock syndrome toxin 1, or tsst-1), which is both absorbable from mucosal surfaces and capable of producing a profound shock syndrome even in the absence of significant invasive infection, soon led to the more widespread recognition of the nonmenstrual toxic shock syndrome. this syndrome, which was almost certainly extant before but little-recognized, was perhaps the main lesson from the outbreak: even trivial staphylococcal skin or wound infections light or mucosal surface colonization in the presence of a foreign body such as a nasal pack for nosebleed can lead to a severe shock syndrome if the organism is present and produces this toxin or one of several related ones. the recognition of the staphylococcal toxic shock syndrome also led to increasing understanding of the role of ''superantigens'' as a mechanism of disease-bacterial toxins capable of activating a large fraction (up to 20%) of the total t-lymphocyte population. such superantigens have since been implicated in a number of other disease syndromes, among them the streptococcal toxic shock syndrome (see below). over the past two decades, the incidence of menstrual and nonmenstrual staphylococcal tss has been about one per 100,000 population in most areas. busy icus will, therefore, continue to see occasional cases. however, there is some recent evidence that case numbers may be on the rise again in at least some areas, possibly because of a resurgence in the prevalence of toxin-producing strains in the community. 12 in 1980, physicians working in infectious diseases and critical care medicine thought they knew all about pneumocystosis. the organism, then thought to be a protozoon, had been first described in 1909 by carlos chagas in brazil, and since then had been clearly implicated as a cause of interstitial pneumonia in debilitated and malnourished children (in the aftermath of world war ii) and, later, a cause of severe opportunistic pneumonia in immunocompromised patients, usually those being treated with highdose corticosteroids for connective tissue diseases or lymphoreticular neoplasms. 13 in these patients it caused an impressively aggressive bilateral pneumonia leading to acute respiratory failure. this pneumonia was notoriously difficult to definitively diagnose, requiring bronchoscopy or open lung biopsy to demonstrate the small numbers of characteristic pneumocystis organisms on special silver stains of clinical specimens. the mainstay of treatment at that time was pentamidine, generally given intramuscularly, giving way to trimethoprim/sulfamethoxazole after the publication in 1978 of a randomized clinical trial showing that it was at least as effective and generally better tolerated. 14 in the early 1980s, a new form of the infection began to be seen with regularity. young men began to present to hospital with a rather more indolent diffuse bilateral pneumonia that nevertheless went on to cause respiratory failure and which, when investigated, proved to be due to pneumocystis. 15 the course of the disease was quite different from what physicians had been used to up to then. it began more gradually, progressed at a slower pace and was associated with a much lesser systemic inflammatory response. microscopy of respiratory specimens revealed exponentially more organisms than previously seen, such that many patients could be diagnosed from sputum specimens rather than bronchoscopy, and biopsy was virtually never needed. nobody had any idea why this was happening, although it quickly became apparent that the underlying cause of the infection was a new form of severe deficiency of cell-mediated immunity. epidemiologic investigations were soon underway. patterns began to emerge. many of the young men were haitian or had been to haiti. many were homosexual, bisexual, or had worked in the sex trade; others had abused intravenous drugs. the many fewer women with the disease had similar exposures. theories proliferated. was it immunologic exhaustion from exposure to too many microbial stresses? toxins? drugs used in the sex trade? multiple and synergistic viral infections? through the early 1980s, the case load grew. icus throughout north america and, later, in europe and elsewhere, saw increasing numbers of young people, mainly men, plagues in the icu with severe respiratory failure due to pneumocystis pneumonia. by 1981 they were being called patients with acquired immunodeficiency syndrome (aids) on the basis of demonstration of low numbers of cd4 lymphocytes in the blood, but the cause remained unclear. then, in 1984, montagnier and barre-sinoussi at the pasteur institute in france isolated a viral pathogen that they named lymphadenopathy-associated virus (lav). at the national institutes of health in the united states, gallo demonstrated that the virus (which he referred to as human t-cell leukemia virus iii, based on an unproven relationship to other viruses he had previously discovered) definitively caused aids. the virus, now called human immunodeficiency virus (hiv)-1 was isolated and described and the first diagnostic kits devised, resolving the mystery of causation. montagnier and barre-sinoussi would eventually win the nobel prize in medicine in 2008 for their contribution. during the 1980s, patients continued to present with severe pneumonia requiring respiratory support and intensive antimicrobial therapy, often with less than satisfactory results. as knowledge progressed, hiv-associated pneumocystis infection in the icu changed its face several times over the years. at the beginning of the epidemic, most patients presenting for care with hiv/aids and pneumocystosis were severely ill with diffuse pneumonia and hypoxemic respiratory failure and many died, 80%-90% in most centers, prompting widespread debate about whether such patients should even be admitted to icu for mechanical ventilatory support. however, as experience with the disease developed it became clear that an early and aggressive approach could improve prognosis. it was found that in the aids population even minor respiratory symptoms with few or no abnormalities on chest radiograph could be due to pneumocystis infection in the earlier stages, and that even modest degrees of arterial oxygen desaturation signaled impending respiratory failure. earlier bronchoscopy for diagnosis followed by prompt antimicrobial therapy, with pentamidine predominantly in the early 80s and later primarily with trimethoprim/sulfamethoxazole, led to overall mortality rates falling to the 50%-60% range by the middle of the decade. the advent of systemic corticosteroids therapy for early respiratory failure in aidsassociated pneumocystosis was then shown to further reduce the numbers of patients progressing to advanced respiratory failure, leading to reductions in the numbers of cases needing icu admission and further reducing overall mortality rates to the 15%-20% range. 16 but for patients requiring icu care mortality rates were as high as before the use of steroids and often higher, likely related to the fact that most patients developing respiratory failure had already failed to improve or had progressed despite intensive antimicrobial and corticosteroid therapy. 17 along with these developments in management of the disease, progress was being made on hiv itself. following identification of the virus in 1984, there soon followed increasingly reliable diagnostic tests for the infection, leading to earlier identification of cases and monitoring of cd4 lymphocyte counts. by the early 1990s, studies supporting widespread use of chemoprophylaxis against pneumocystis in all patients with cd4 counts <200/mm 3 were available and became standard public health agency recommendations. pneumocystosis, which in the 1980s and 1990s was one of the principal causes of hypoxemic respiratory failure in many icus in north america and western europe, began to decline rapidly in incidence, becoming relatively uncommon even before the widespread adoption of highly active antiretroviral therapy, which has, since the mid-1990s, caused the disease to all but disappear from our icus. although many lessons can be drawn from the battle against aids-related pneumoncystis pneumonia during the 1980s and 1990s, for icu and infectious diseases practitioners one of the main ones comes from the sad fact that once patients had developed full-blown hypoxemic respiratory failure even the best intensive care could only deliver 20% survival rates. the really large gains in survival came not from better icu technology but from pre-empting the disease on multiple fronts, including earlier diagnosis of hiv infection, early diagnosis and antimicrobial treatment of pneumonia, steroid treatment of early respiratory failure, antimicrobial prophylactic regimens and, later, effective antiretroviral therapy. until 1993, the only members of the bunyaviridae family of viruses known to cause disease in north america were members of the genus bunyavirus, all causing mosquito-borne viral encephalitis, mainly in children (california encephalitis). other members of the family were known to cause serious febrile illnesses, encephalitides and hemorrhagic-fevers in africa and asia (rift valley fever, crimean-congo hemorrhagic fever, hemorrhagic fever with renal syndrome). however, in the spring of 1993 wetter-than-usual conditions in the american southwest led to increased availability of food for deer mice, leading to a population explosion and increasing movement of rodents into human-occupied spaces, increasing the chance that humans might be exposed to the rodents and their excreta. in rapid succession, several previously healthy young people, mainly navajos, presented to health care institutions in the four corners area of the southwestern united states, all with fulminant illnesses leading to shock and acute respiratory failure requiring icu care. by early june that year, 24 cases had been identified and 12 had died. 18 in most cases the illness had started with fever and widespread myalgia, soon followed by cough, then by cardiovascular collapse due to a severe systemic capillary leak syndrome and by acute respiratory failure due to low-pressure pulmonary edema. in some cases the time from onset to icu or death was as little as 24 hours, in others a few days. 19 remarkably, although no pathogen was initially identified from blood or tissues, in less than a month after the first report of a possible outbreak, serologic testing had demonstrated antibody cross-reactivity with a range of known pathogens of the hantavirus group, suggesting that the disease was due to a previously unknown member of this group. shortly thereafter exposure to deer mice and their excreta was implicated as the likely source of the infection. the mortality rate for the early cases of hantavirus pulmonary syndrome (hps) was extremely high-80% in the initially reported group of patients-mostly due to intractable shock and unsupportable hypoxemic respiratory failure due to acute respiratory distress syndrome (ards). however, this improved with clinical experience as it became evident that administration of large amounts of intravenous fluids in the face of profound capillary leak only resulted in much worse generalized and pulmonary edema, with little improvement in the shock state and only worsening of the respiratory failure. management changed to an approach limiting the amount of fluid administered early in the course together with earlier institution of inotropic support, resulting in a much improved survival rate of about 60%, generally with minimal or no long-term sequelae in survivors. 20 in subsequent years development of increasingly specific serologic and virologic testing has demonstrated that this disease had been present but unrecognized throughout north and south america long before this outbreak, and that there are several related viruses, each associated with a particular rodent, causing endemic disease and the occasional outbreak. by the mid-1990s, over 150 cases were reported in 25 states, mainly in the southwest, and cases have since been reported plagues in the icu in small numbers in most other states, canada, mexico, and south america, where several outbreaks have occurred. whereas occasional cases continue to be seen in icus in all these areas, no further major outbreaks have yet occurred in the united states or canada, though clearly remaining a threat under the right conditions; the only currently available preventive measure is avoiding rodent contact. 21 steven simpson, md, one of the intensivists at the health sciences center in albuquerque, new mexico, who was closely involved in the initial four corners outbreak, points out that the event highlights several trends in subsequent disease outbreaks in north america. one is the extreme rapidity with which novel pathogens and potential pharmacotherapeutic agents can now be identified. whereas the pathogen in the legionnaire's outbreak took almost a year to identify, researchers identified the hps pathogen and its source in just months. computerized access to data and data analysis along with virtually instantaneous electronic transmission of information plays a central role in this development. the initial hps outbreak has several icu-related lessons to teach us. while the aforementioned treatment strategies effective in a systemic capillary leak syndrome have been absorbed by the critical care community, it appears that one lesson taken to heart by the local icu teams failed to disseminate to the broader icu community. the initial outbreak was accompanied by a marked element of fear and concern among health care workers, including those in the icu, and a significant level of panic in the local community; a combination of this fear, the requirement for rigorous quarantine precautions, and a marked increase in transfers to the icu of any severely ill patients with symptoms remotely compatible with hps resulted in some compromise of icu operations, due to being completely overwhelmed. this might potentially have been avoided by an awareness that for an effective epidemic response, it is essential to include both hospital and icu operations in each locale. the outbreak also reinforces the principle that nearly all old and most new epidemic infectious diseases have their origin in close contact between humans and other species of animal, both wild and domestic, and new kinds and quantities of such contact are likely to cause new, or newly recognized, disease syndromes. streptococcus pyogenes was one of the first bacteria ever to be conclusively linked to human disease (puerperal infection associated with childbirth). however, over the past 125 years the nature of the diseases stemming from it has changed dramatically on several occasions. at the turn of the last century, it was well known as a cause of streptococcal pharyngitis, erysipelas, and wound infections. it also caused severe septicemic illnesses that frequently led to death. osler 22 knew streptococcus pyogenes as a principal cause of thoracic empyema following pneumonia or severe cases of scarlet fever, and also as a major cause of primary bacteremia with sepsis. these more severe manifestations of streptococcal infection became increasingly uncommon as the twentieth century progressed, particularly after the arrival of antibiotics mid-century. notably, osler did not mention streptococcus as a cause of necrotizing fasciitis or being associated with soft tissue necrosis in wound infections. this syndrome was first described by meleney in 1924; 23 at that time, it was characteristically a slowly evolving gangrenous infection, usually of surgical wounds, which often responded well to debridement and was associated with a mortality rate of only 20%. 24 for over a generation after the advent of the modern antibiotic era, streptococcus pyogenes was seldom a problem that led to critical illness-soft tissue infections and light the occasional bacteremia were generally very amenable to treatment; extensive surgery or drainage was seldom required, and cases requiring icu support for shock or respiratory failure were rare. beginning in the mid-1980s, medical practitioners in centers across north america and europe began seeing previously unknown forms of severe streptococcal disease, soon labeled streptococcal necrotizing fasciitis and streptococcal toxic shock syndrome. 25 streptococcal toxic shock syndrome (strep tss) is any infection with streptococcus pyogenes that is associated with a rapidly progressing systemic toxic response characterized by early onset of high fever and myalgia, often with prominent gastrointestinal symptoms, and by rapid progression to hypotension and multiple organ system failure. the illness usually requires icu support for massive fluid resuscitation, vasopressor and inotropic support and mechanical ventilation. although some cases have primary bacteremia, many others have a localized focus of infection, most often in soft tissues, that only becomes clinically apparent after the onset of shock. streptococcal necrotizing fasciitis is often associated with strep tss and, as mentioned, is often only correctly diagnosed after the onset of shock. the most characteristic story is presentation to a physician or an emergency room with abrupt onset of severe pain, often in an extremity with minimal or no evidence of cutaneous injury. at this stage severe systemic toxicity is usually not present and, since examination of the painful site is also at this stage quite unremarkable, patients are frequently sent home with analgesics and reassurance. over the next 4-48 hours pain at the site of infection continues to increase, soft tissue swelling and redness appear above the deeper tissues that are undergoing ongoing necrotizing infection, eventually resulting in full-thickness necrosis evidenced by ecchymosis, cutaneous necrosis, and bullae formation. 26 early or later in this course strep tss frequently occurs. when these cases first began to appear, clinicians' approach to both the sepsis and the tissue necrosis was essentially the same as that used for apparently similar syndromes caused by other bacteria. a broad spectrum antimicrobial was started, fluid resuscitation begun and imaging studies ordered to better define the source of infection causing pain or localized swelling. imaging frequently demonstrated only soft tissue swelling consistent with cellulitis, so surgery was often deferred until superficial signs of tissue necrosis became obvious, and then when surgery was done it was often performed using the conventional approach of trying to conserve as much tissue as possible. the result was that treatment was often too little and too late, with mortality rates exceeding 70% in many reported series. with the realization that treatment, to be successful, must be swift and aggressive, approaches to therapy changed. emergency physicians were increasingly alerted to the fact that severe pain at any body site, even with relatively minimal localized physical findings and particularly if accompanied by signs of systemic inflammation, could represent necrotizing fasciitis. surgeons began to be consulted much earlier, and any localized pain with swelling more often led to diagnostic surgical exploration rather than imaging and waiting. antimicrobial strategies changed. addition of clindamycin to the usual penicillin or other beta-lactam therapy was advocated and widely adopted, based on results from animal models of the syndrome and on pharmacologic and physiologic considerations, including its ability to inhibit bacterial protein (ie, toxin) synthesis, penetrate necrotic tissues, and inhibit inflammatory cytokine synthesis. 27 toxin neutralization using pooled intravenous gamma globulin was also advocated with the support of primarily historical case-control studies. 28 in most centers, implementation of these approaches has led to dramatic reductions in mortality rates to about 20%-30% although, in the absence of any adequate controlled trials, it remains unclear what the relative contribution of each of these measures has been to the improved outcome. unlike several of the other ''plagues'' discussed above, this is one that is still very much with us. the streptococcus pyogenes strains most strongly associated with severe invasive disease (m-protein types 1 and 3) have increasingly been supplanting those associated with less severe disease resulting in an endemic sporadic case-rate for severe disease of one to 20 cases/100,000 population yearly, with intermittent larger-scale community outbreaks, both of which will continue to require vigilance and an aggressive therapeutic stance from the critical care community. 29 the first case of this apparently novel severe viral respiratory infection occurred in guangdong province in southern china in november 2002. the victim, a farmer, died of an undiagnosed ''atypical pneumonia.'' over the ensuing weeks several more cases of severe respiratory syndromes began to appear in the region, also undiagnosed. by the end of november there had been enough such cases to generate considerable alarm among the medical community in china, generating internet communications between institutions which were picked up by international monitoring agencies. this led to a request from the world health organization (who) for information about the outbreak, but no information was forthcoming from chinese authorities. the first official report about the outbreak was made to public health authorities in guangdong in early january 2003, with a later report to the who in february that, in retrospect, did not fully make clear either the nature or the scale of the problem. 30 transmission of the disease within china continued to occur, leading to rapidly increasing numbers of cases in south china, then throughout the country and to the capitol beijing (where one of the largest outbreaks occurred). exposure of chinese travelers and visitors to the country was inevitable, given the scale of the outbreak. one exposed individual was a physician from mainland china who, incubating the disease during his travel, stayed at the metropole hotel in hong kong in early march. later investigations showed that he transmitted the virus to at least 16 other guests at the hotel, who then carried it by international air travel to taiwan, singapore, vietnam, and canada. one of these contact cases was an american businessman headed for singapore. becoming ill while in transit, he stopped in hanoi where he was admitted to hospital with a severe pneumonia, to which he eventually succumbed. soon after, a number of health care workers who had been in contact with him also became acutely ill. fortunately for the course of the outbreak, one of the consultants on the case was an italian physician working with the who in vietnam, dr. carlo urbani. he immediately recognized that this was a previously unknown severe atypical pneumonia that was relatively easily transmissible and reported it to the who; this led to immediate mobilization of investigative efforts and worldwide alerts about the threat. 31 unfortunately, in the course of caring for the victims of the disease in hanoi, dr. urbani himself contracted the infection and died of it later. as information from china became more available, it became clear that by this time there had already been hundreds of cases and numerous deaths. the majority of the initial wave of cases were noted to have occurred primarily in farmers and food handlers, particularly those working in food markets where live wild animals were kept and sold for food. the second large wave of those affected were health care workers exposed in hospital to patients with the disease. the illness was characterized by fever and myalgia with gastrointestinal symptoms in the initial phase, occurring an average of 5 days after exposure (range 2-10 days). many cases got no worse than this, but others went on to develop dyspnea associated with radiographic evidence of a diffuse, patchy pneumonitis which, in some, progressed to ards. an average of light 15% required mechanical ventilatory support, and when the data were all in from later phases of the outbreak, mortality rates averaged about 10% overall, worse in the aged and debilitated, lower in the young and healthy. the largest outbreak outside asia occurred in toronto, canada. the index case, a visitor to china, returned to canada and died of pneumonia at home, undiagnosed, in early march 2003. shortly thereafter, one of his sons was admitted to hospital with a severe respiratory illness and died a few days later. by this time, four other family members had become ill and had been admitted to hospital; the first cases of affected health care workers appeared soon after among those who had cared for the dying son of the index case. within days, other instances of transmission from undiagnosed contacts of the initial cases in hospitals, doctors' offices, emergency rooms, and at social events were leading to admission of cases to several hospitals throughout toronto. the response of the public health authorities, beginning soon after the who global alert and coincident with the recognition of the first local cases, was quick and vigorous, including closure of the main affected hospital, intensive follow-up of probable contacts, quarantine of suspected cases based on a fairly inclusive case definition and strict institution of barrier contact protection for health care workers. 32 by mid-april the number of new cases was rapidly declining, although there was one cluster of late cases related to exposure of a large number of health care workers during the resuscitation and difficult intubation of a critically ill patient. a later cluster of cases also occurred in a rehabilitation hospital, where it appeared that unrecognized contacts from the first phase of the outbreak had been transferred and transmitted the disease to other patients and staff. the worldwide outbreak was essentially over by july 2003. there were a total of 8098 reported cases from 26 countries, with 774 deaths. intensive epidemiologic and laboratory study of the disease by investigators and laboratories worldwide led to unprecedented rapid growth in knowledge about the causative agent. the virus, more or less simultaneously characterized at a number of laboratories around the world, proved to be a previously unknown coronavirus (severe acute respiratory syndrome [sars]-cov) with capacity to infect and spread from a variety of wild animals to humans. epidemiologic, serologic, and virologic evidence was developed linking human cases to exposure to infected wild animals, including masked palm civets, raccoon dogs, ferrets and ferret badgers, all being sold for human consumption in markets in china. 33 control of their transport and sale and exposures to humans by chinese health authorities was probably one of the major factors in bringing the first outbreak under control, the partial failure of which later led to a second, much smaller outbreak late in 2004. although the initial speculation was that one or more of these wild animals were the reservoir in nature for the infection, it now appears more likely that the viral reservoir is actually bats, with crosstransmission of the virus between bats, food animals, and humans in crowded markets leading to development of strains with the capacity to transmit between humans. 34 public health authorities worldwide learned much from sars about the importance of effective international communication in developing a rapid and effective response to outbreaks of novel viruses, and more about how to go about containing such infections within communities and hospitals. several intensivists involved in the outbreak credit e-mail communications from other international outbreak sites for effective advice on critical elements of disease protection (eg, powered air purifying respirators and full contact rather than droplet precautions) and therapy. for the critical care community, perhaps one main lesson was the importance of ''super-spreading incidents'' in propagating the disease in hospitals. many of these occurred in critically ill patients undergoing resuscitation with difficult or traumatic intubation, generating aerosols in closed spaces which contained many superfluous and inadequately protected health care workers. handling these situations safely depends crucially on identifying the potential risk and undertaking the resuscitation and intubation using the most experienced operators available, adequately protected with basic barrier precautions (eye protection, gloves and surgical face-masks), using sedation or paralysis as necessary to minimize trauma and aerosol generation, and with only essential and adequately protected staff in the room. 35 this likely applies to many other situations with potential for disease transmission to health care workers. unfortunately, this epidemic again points out the primary lesson that was not absorbed from the earlier hps outbreak, namely, the need for detailed preplanning and preparation for a major infectious disease epidemic that is inclusive of hospital and icu operations in each locale. according to participants, the sars outbreak demonstrated many of the same early icu operational problems that plagued the hps outbreak albeit on a larger scale. in the icu era, there has yet to occur a true influenza pandemic with a high attack rate in all age groups and associated high hospitalization and mortality rates, as was seen in the great 1918 pandemic. in that worldwide disaster, it is estimated that 30% of all people became ill with the virus and an estimated 50-100 million died. 36 minor recent pandemics in 1968 and 1977 had less than one twentieth of the impact of the 1918 influenza, not greatly different from the yearly interpandemic influenza the world has been experiencing in the 30 years since. interpandemic influenza epidemics since 1977 have been caused primarily by h3n2 and h1n1 influenza viruses, to which most of the population has developed some degree of immunity from prior infection or vaccination. the result is what public health authorities have become used to seeing: each year a slightly different influenza a appears in asia with minor antigenic changes in the ha or na surface proteins (termed drift), making it infectious once again for humans whose immune systems have yet to be exposed to the new variant, and a new epidemic is launched. when the ''flu'' arrives in an area, cases begin to appear suddenly and there is rapid spread in the population, usually with 20%-30% becoming infected over a 6-week period with a peak in case numbers at week two or three. about half of those infected will seek medical attention, many more than once, and one to about 25 per thousand infected will be admitted to hospital with a respiratory syndrome such as pneumonia, chronic obstructive pulmonary disease exacerbation, asthmatic attack, or cardiac failure, the rate depending on age and underlying comorbidities. overall, about 0.1% of those infected die, with mortality rates among those with major comorbidities up to 5%. these latter cases constitute most of the increase in the icu case load which most units experience every winter. the load is sometimes taxing but usually not overwhelming. a true pandemic is unlikely to play out this way. how different it would be depends on a number of factors: the antigenic difference in the new influenza virus compared with the old (ie, the antigenic ''shift'' to a different one of 15 ha or 9 na protein subtypes due to introduction of a variant from another influenza-susceptible species), how transmissible the new virus is, how virulent it is, how susceptible it is to antiviral drugs, and whether the world is prepared for it with drug availability and vaccines. the prototype severe pandemic was the spanish influenza of 1918, an h1n1 virus. the most recent circulating influenza virus just before that time was an h3n8 that had arrived in 1901. current evidence suggests that an avian influenza virus underwent a period of evolutionary adaptation, possibly in another susceptible species such as swine, fitting it for transmission to humans, which it then did. 36, 37 this h1n1 virus had not been previously experienced by any segment of the population except the very old, so nearly everyone, particularly non-elderly adults and children, was without immunity and was at risk of severe infection. attack rates, as noted earlier, were extremely high everywhere as were rates of primary influenza pneumonia, complicating bacterial pneumonia, and death. in the united states, death rates were more than 20-fold higher than in any influenza pandemic since. an outbreak of influenza on this scale, if unchecked by effective antiviral therapy or vaccines, would render icu care such as mechanical ventilatory support for respiratory failure irrelevant. even today, with maximal respiratory support, most patients with diffuse primary viral pneumonia complicated by respiratory failure cannot be saved, and the numbers presenting for such care in a short space of time, if comparable to the 1918 pandemic, would overwhelm our current icu capacity within days. currently the main apparent threat of a new pandemic comes in the form of the h5n1 influenza virus. this virus is now present nearly worldwide in migratory and, intermittently, domestic bird populations. from time to time, transmission of the virus from birds to humans occurs, generally from close contact situations. who data indicate that there have been 387 laboratory-confirmed cases of such transmission from 2003 to mid-2008. 38 the mortality rate has exceeded 60%, although it is likely that many less severe cases do not come to medical attention and are therefore not counted as confirmed case survivors. to date no instances of transmission to humans by humans or other mammals has been documented. however, the threat remains that if this virus were to become capable of human-to-human transmission by adaptation in another susceptible mammalian host such as swine, a pandemic on the order of the 1918 event could occur. with no true pandemic for over 30 years, including all of the icu era, health authorities worldwide are deeply engaged in trying to learn the lesson of this new ''plague'' before it actually occurs. it is clear that we will need excellent international communication, rapidly enactable containment and quarantine plans and, if possible, effective antivirals and vaccines to deal with the h5n1 virus. if it evolves as feared and becomes easily transmissible while retaining its current virulence; modern life-sustaining technology alone will be no shield at all. the last 60 years have seen remarkable advances in the ability to diagnose and treat infectious diseases and handle infectious disease outbreaks. for the most part, the major plagues of antiquity remain historical footnotes. however, despite these advances, there is clear evidence that major pandemic illness is always just one outbreak away. in addition to the hiv pandemic, the smaller epidemic outbreaks of legionnaire's disease, hantavirus pulmonary syndrome, and sars, among many others, points out the potential risk associated with a lack of preplanning and preparedness. although pandemic influenza is at the top of the list when discussing possible future major infectious disease outbreaks, the truth is that the identity of the next major pandemic pathogen cannot be predicted with any accuracy. we can only hope that general preparedness and the lessons learned from previous outbreaks suffice. a history of poliomyelitis polio: an american story an apparatus for the prolonged administration of artificial respiration the physiologic challenges of the 1952 copenhagen poliomyelitis epidemic and a renaissance in clinical respiratory physiology post-poliomyelitis syndrome legionnaire's disease: description of an epidemic of pneumonia legionnaire's disease: isolation of a bacterium and demonstration of its role in other respiratory diseases toxic-shock syndrome associated with phagegroup-1 staphylococci toxic-shock syndrome -united states toxic-shock syndrome in menstruating women: association with tampon use and staphylococcus aureus and clinical features in 52 cases epidemiologic notes and reports, toxic-shock syndrome, united states reemergence of staphylococcal toxicshock syndrome in intensity of immunosuppression and the incidence of pneumocystis carinii pneumonia comparison of pentamadine isothionate and trimethoprim/sulfamethoxazole in the treatment of pneumocystis carinii pneumonia pneumocystis pneumonia -los angeles a controlled trial of early adjunctive treatment with corticosteroids for pcp in aids aids-related pneumonia in the era of adjunctive steroids outbreak of acute illness -southwestern united states hantavirus pulmonary syndrome: a clinical description of 17 patients with a newly recognized disease cardiopulmonary manifestations of hantavirus pulmonary syndrome hantavirus pulmonary syndrome -united states: updated recommendations for risk reduction the principles and practice of medicine hemolytic streptococcus gangrene hemolytic streptococcal gangrene: the importance of early diagnosis and operation clinical and bacteriological observations of a toxic-shock-like syndrome due to streptococcus pyogenes streptococcal necrotizing fasciitis: comparison between histological and clinical features antibiotic effects on bacterial viability, toxin production and host response intravenous immunoglobulin therapy for streptococcal toxic-shock syndrome -a comparative observational study. the canadian streptococcal study group epidemiologic analysis of group a streptococcal serotypes associated with severe systemic infections, rheumatic fever, or uncomplicated pharyngitis who -epidemic and pandemic alert and response (epr) acute respiratory syndrome in hong kong special administrative region of china/vietnam public health measures to control the spread of the severe acute respiratory syndrome during the outbreak in toronto isolation and characterization of viruses related to the sars coronavirus from animals in southern china bats are natural reservoirs of sars-like coronaviruses critically ill patients with severe acute respiratory syndrome influenza: the mother of all pandemics molecular basis for the generation in pigs of influenza a viruses with pandemic potential who-epidemic and pandemic alert and response (epr) key: cord-253502-v2hh3w3r authors: leung, c.w.; chiu, w.k. title: clinical picture, diagnosis, treatment and outcome of severe acute respiratory syndrome (sars) in children date: 2004-11-05 journal: paediatr respir rev doi: 10.1016/j.prrv.2004.07.010 sha: doc_id: 253502 cord_uid: v2hh3w3r children are susceptible to infection by sars-associated coronavirus (sars-cov) but the clinical picture of sars is milder than in adults. teenagers resemble adults in presentation and disease progression and may develop severe illness requiring intensive care and assisted ventilation. fever, malaise, cough, coryza, chills or rigor, sputum production, headache, myalgia, leucopaenia, lymphopaenia, thrombocytopaenia, mildly prolonged activated partial thromboplastin times and elevated lactate dehydrogenase levels are common presenting features. radiographic findings are non-specific but high-resolution computed tomography of the thorax in clinically suspected cases may be an early diagnostic aid when initial chest radiographs appear normal. the improved reverse transcription-polymerase chain reaction (rt-pcr) assays are critical in the early diagnosis of sars, with sensitivity approaching 80% in the first 3 days of illness when performed on nasopharyngeal aspirates, the preferred specimens. absence of seroconversion to sars-cov beyond 28 days from disease onset generally excludes the diagnosis. the best treatment strategy for sars among children remains to be determined. no case fatality has been reported in children and the shortto medium-term outcome appears to be good. the importance of continued monitoring for any long-term complications due to the disease or its empiric treatment, cannot be overemphasised. severe acute respiratory syndrome (sars), a newly described infectious disease caused by the novel sarsassociated coronavirus (sars-cov), has become a major threat to public health globally. 1-4 sars is highly contagious and has been aptly coined 'the first plague of the twenty-first century'. the disease is characterised by transmission in healthcare and household settings and through intriguing superspreading events which were pivotal in its global spread. [5] [6] [7] [8] [9] [10] [11] superspreading events including a major hospital outbreak, in-flight transmission on board commercial paediatric respiratory reviews (2004) summary children are susceptible to infection by sars-associated coronavirus (sars-cov) but the clinical picture of sars is milder than in adults. teenagers resemble adults in presentation and disease progression and may develop severe illness requiring intensive care and assisted ventilation. fever, malaise, cough, coryza, chills or rigor, sputum production, headache, myalgia, leucopaenia, lymphopaenia, thrombocytopaenia, mildly prolonged activated partial thromboplastin times and elevated lactate dehydrogenase levels are common presenting features. radiographic findings are non-specific but highresolution computed tomography of the thorax in clinically suspected cases may be an early diagnostic aid when initial chest radiographs appear normal. the improved reverse transcription-polymerase chain reaction (rt-pcr) assays are critical in the early diagnosis of sars, with sensitivity approaching 80% in the first 3 days of illness when performed on nasopharyngeal aspirates, the preferred specimens. absence of seroconversion to sars-cov beyond 28 days from disease onset generally excludes the diagnosis. the best treatment strategy for sars among children remains to be determined. no case fatality has been reported in children and the short-to medium-term outcome appears to be good. the importance of continued monitoring for any long-term complications due to the disease or its empiric treatment, cannot be overemphasised. ß 2004 elsevier ltd. all rights reserved. abbreviations: sars, severe acute respiratory syndrome; sars-cov, sars-associated coronavirus; rsv, respiratory syncytial virus; ards, acute respiratory distress syndrome; cxr, chest radiograph; hrct, high-resolution computed tomography; boop, bronchiolitis obliterans-organising pneumonia; npa, nasopharyngeal aspirate; rt-pcr, reverse transcription-polymerase chain reaction; ifa, immunofluorescence assay; elisa, enzyme-linked immunosorbant assay. *correspondence to: c.w. leung; e-mail: leungcw@ha.org.hk. airliners, transmission in a hotel and a large-scale community outbreak in a densely populated residential complex, primarily resulting from environmental contamination by a 'superspreader' with diarrhoea, were well described. 5, 6, [12] [13] [14] [15] the disease first started as a mysterious outbreak of atypical pneumonia in the guangdong province of southern china in november 2002. by july 31, 2003, up to 29 countries and regions of the world had been affected by sars. a worldwide total of 8098 cases of probable sars, 1707 (21%) of these being healthcare workers and 774 deaths (9.6%) were recorded. 16 in hong kong, the toll was 1755 affected individuals, including 386 (22%) healthcare workers and 299 deaths (17%). 16 the subsequent reemergence of the first six sporadic cases of sars, two of which were probably laboratory-acquired, did not result in local transmission in singapore, taiwan and china. [17] [18] [19] [20] children appeared to be less affected by the disease, with smaller case numbers and less severe illness reported. [21] [22] [23] [24] all age groups are susceptible to sars-cov, which is new to humans. however, rapid isolation of diseased adults, whose infectivity is lower in the first few days of illness, has contributed to reduced frequency of household exposure for children. the exact number of children affected by sars worldwide is unknown as the age breakdown of reported cases was not available or incomplete for some of the affected countries (who sars surveillance team, personal communication). it is estimated that children <18 years of age only accounted for about 5% of the total affected. there was no reported mortality in children (who sars surveillance team, personal communication). a total of 121 children aged <18 years were registered in the e-sars database of the hospital authority of hong kong, accounting for 7% of all patients notified. the crude age-specific attack rate for children in hong kong was 8.9 per 100 000 persons <18 years of age. serologic confirmation of sars was documented in 89 children (6.6 per 100 000 persons <18 years of age). sixty-four children with clinical disease and seroconversion to sars-cov were managed in the authors' hospitals. the experience with this cohort of laboratory-confirmed patients forms the basis of the clinical information presented in this review. 22, 23 most children reported worldwide were previously healthy and there was no sex predominance. thirty-five (55%) of the 64 children managed by the authors were girls. the male to female ratio was 1:1.2. their mean and median ages were 11.7 and 12 years, respectively. the youngest patient was a 56-day-old premature infant, which is the youngest case reported to date. 25 comorbidity was only present in 5 children (8%) but none of them were immunocompromised. an epidemiologic link was available in the vast majority of children with sars, which appeared to be the most important clue leading to diagnosis in an epidemic situation. worldwide, children were usually secondary household contacts of affected adults, some of whom were healthcare workers or international travellers returning from areas with local transmission of sars. transmission among children or from children to adult contacts was uncommon. about 60% of serologically confirmed children in hong kong were victims of a point source community outbreak due to environmental contamination. 14 the actual proportion of children being secondary household contacts in the particular outbreak could not be determined given the short incubation period between exposure, either to a common environmental source or an index household member, and presentation. there is no published report on the differences in susceptibility and communicability between children and adults. any apparent difference might be related to different risks of exposure for the two age groups. sars is largely an atypical pneumonia with minimal or no extrapulmonary manifestation, apart from diarrhoea. 26 cellular tropism of the sars-cov has been demonstrated primarily in pneumocytes and surface enterocytes of the small bowel. 27 the clinical presentation of sars is nonspecific, with features overlapping those of atypical pneumonia caused by other respiratory pathogens such as influenza virus (including highly pathogenic avian influenza viruses), parainfluenza virus, adenovirus, respiratory syncytial virus (rsv), mycoplasma pneumoniae, chlamydia pneumoniae, chlamydia psittaci and legionella pneumophila. the clinical course of sars in adult patients is well described and appears to follow a triphasic pattern. 6, [28] [29] [30] [31] [32] following an incubation period of 2-10 days (mean 6.4 days, 95% ci 5.2 to 7.7), adults present with a prodrome characterised by high fever (temperature >38 8c), chills or rigor, malaise, headache, dizziness and myalgia. upper respiratory symptoms such as coryza and sore throat are mild and uncommon. diarrhoea is a presenting feature in 6-20% of adult patients. 6, 26, 30 after 2-7 days the disease progresses to involve the lower respiratory tract and a dry, non-productive cough or dyspnoea becomes prominent. in 10-20% of cases, progression to acute respiratory distress syndrome (ards) necessitating intubation and assisted ventilation is observed. mortality results primarily from respiratory failure and a significant proportion of patients recover from pulmonary destruction over an extended period. sars appears to run a less aggressive clinical course in children compared with adults. the severity of illness varies and the extent of asymptomatic infection is unknown, although it is believed to be uncommon. children are usually hospitalised 3-4 days after the onset of symptoms. in one paediatric case series, the mean duration of fever before admission was 3.7 ae 0.6 days (median 3, range 0-12). 23 the most common presenting clinical features in children include fever, malaise, cough, coryza, chills or rigor, sputum production, headache and myalgia (table 1) . 22, 23 lethargy, poor feeding or anorexia, nausea, vomiting, diarrhoea, abdominal pain, sore throat, dyspnoea and dizziness are less commonly encountered. less than 20% of children may pass loose to watery stools, but profuse diarrhoea is rare throughout the course of illness. blood and mucus in the stool, features suggestive of inflammatory enterocolitis, have not been reported. cough, predominantly unproductive in nature, is only found in just over half of the children at presentation. definite physical signs of consolidation are hardly evident and crepitations (crackles) on chest auscultation are unusual despite prominent radiographic evidence of pulmonary infiltrates, even in patients who develop respiratory distress, hence the description of 'atypical' pneumonia. lymphadenopathy, hepatosplenomegaly or clinical bleeding is absent. skin rash is an exceedingly rare manifestation. 22 hypoxaemia is seldom noted at presentation and generally develops towards the end of the first week or the beginning of the second week of illness in severe cases. 33 the youngest patient, however, presented with a cyanotic attack, dyspnoea, cough and hypothermia with subsequent development of fever. 25 teenagers (aged >12 years) may resemble adults in presentation and disease progression. they tend to have more constitutional upsets and systemic symptoms of malaise, chills or rigor, headache, myalgia and dizziness are significantly more common ( table 2) . they appear sicker, have a greater need for oxygen therapy and other respiratory support and may require intensive care. 33 children 12 years of age generally have milder symptoms and coryza is significantly more common ( table 2) . they appear to run a milder and shorter clinical course. the clinical picture is sometimes indistinguishable from other viral infections of the upper respiratory tract, thus posing a diagnostic challenge. the clinical course of sars in the majority of children follows a biphasic pattern. the phase of viral replication, which lasts for a few days, is characterised by an abrupt onset of fever and constitutional symptoms in association with an increase in body viral load. 34 the phase of immunopathologic damage is marked by the progression of pneumonia and hypoxaemia, when the body viral load declines and an exaggerated host immune response supervenes. 35 the prodromal and pneumonic phases of the disease, however, may be less distinct in comparison with adult patients. progression to ards, or the third phase as in adults, is only seen in a very small number of children, predominantly adolescents. the natural history of untreated sars in both adults and children remains unclear. as most patients worldwide had received some form of empiric treatment in the form of antiviral agents with or without corticosteroids, the probability of spontaneous recovery could not be ascertained. nevertheless, three children with mild disease had recovered on supportive therapy alone in the authors' cohort. 22, 23 anecdotal reports of extrapulmonary manifestations of sars, in the form of central nervous system dysfunction and probable viral hepatitis, have been described in adults. [36] [37] [38] atypical presentation of sars, in the form of non-specific febrile illness or febrile non-pneumonic respiratory illness, have been observed in both children and adults. 23, 39, 40 such cases are likely to evade clinical detection in the absence of a definite contact history with patients with suspected or confirmed sars. the full spectrum of clinical as well as subclinical illnesses caused by infection with sars-cov will unfold with further epidemiological studies and case reports. as sars is basically a pneumonic infection, chest radiograph (cxr) is therefore an essential diagnostic tool. the principal radiographic abnormality of sars in children is illdefined airspace shadowing, which presents as ground-glass opacities and/or unifocal, lobar or multifocal areas of consolidation. [21] [22] [23] [24] 41, 42 unilateral focal opacity was reported as the most common finding in one paediatric case series and was evident in 86% of children at presentation (fig. 1 ). 22 in adults, regions of airspace disease predominate in the lower lobes but are also noted elsewhere. 6 there appears to be no predominant distribution pattern of consolidation in children. [21] [22] [23] cxr opacities are most often peripheral or mixed central and peripheral in location. the lung opacities show a tendency to progress, with increase in size or involvement of multiple areas either unilaterally or bilaterally in moderate to severe cases. rapid progression to unilateral multifocal or bilateral involvement, with reduction in lung volumes in the second week of illness, is typical in children who develop severe hypoxaemia (fig. 2) . 23 in the advanced stage of the disease, which only occurs in a very small number of children, widespread ground-glass opacities and diffuse patchy consolidations are seen, likely representing progression to ards. pneumonic changes may not be apparent at presentation as mildly symptomatic individuals may be identified early in the prodromal period through contact tracing of patients diagnosed with sars. repeat cxr examination, as guided by failure of resolution of symptoms or change in clinical condition, will clarify the picture by revealing new pulmonary infiltrates as the disease progresses. frequent monitoring of cxr changes has the additional benefit of detecting early radiographic deterioration in many patients, heralding clinical deterioration. radiographic resolution, on the other hand, generally lags behind clinical improvement. complete resolution of the airspace opacities can take more than a month in the most severely affected children. 23 no preliminary evidence of pulmonary fibrosis, bronchial wall thickening, bronchiectasis or lung volume loss was observed on follow-up in one paediatric case series. 23 viral pneumonias tend to show reticulo-nodularity as well as a symmetrical perihilar peribronchial pattern of infiltration which is sometimes marked by hilar adenopathy. 43 in contrast to pneumonias caused by other respiratory pathogens, the cxr of children with sars shows no evidence of interstitial disease, hilar adenopathy, mediastinal widening, significant pleural effusion, cavitation, abscess formation, pneumatocele, pneumothorax or pneumomediastinum. [21] [22] [23] [24] 41, 42 nevertheless, the radiographic features of sars in children are non-specific. radiological differentiation of sars from other commonly encountered childhood respiratory illnesses causing airspace disease can be difficult. 41 high-resolution computed tomography (hrct) of the chest has been used as an early diagnostic tool in clinically suspected children with initial negative or equivocal chest radiographs. [21] [22] [23] [24] 41 hrct findings may include groundglass opacification, unifocal or multifocal consolidation in subpleural, peripheral or central regions and interlobular septal and intralobular interstitial thickening (fig. 3) . the characteristic peripheral alveolar opacities are reminiscent of bronchiolitis obliterans-organising pneumonia (boop). 6, 21, 44 in general, hrct is sensitive in detecting more extensive airspace consolidation and ground-glass attenuation than cxr. the investigation is particularly useful when lung parenchymal abnormalities are minimal early in the course of illness, or being obscured by the diaphragm and the cardiac silhouette. the utility of chest hrct lies in the early confirmation of airspace disease in radiographically inapparent cases with a strong contact history and clinical features highly suspicious of sars, thus allowing prompt isolation and monitoring for clinical and radiological deterioration. the haematological and biochemical abnormalities of sars in children are neither diagnostic nor specific. like adults, the most consistent haematological finding is lymphopaenia, which is present in about 70% of children at presentation and about 90% during the course of illness. 22, 23 depletion of lymphocytes may be secondary to the direct cytopathic effect of the virus, cytokine-mediated apoptosis, lymphocyte margination due to increased cortisol secretion from activation of the hypothalamic-pituitary-adrenal axis or the administration of high-dose glucocorticoids, which have a profound lympholytic effect, especially on t lymphocytes. [45] [46] [47] other haematological abnormalities such as leucopaenia, thrombocytopaenia and mildly prolonged activated partial thromboplastin times are observed in about 30% of children. anaemia is rarely found at presentation and is only detected in <5% of children. 22, 23 unlike adults, a significant drop in the haemoglobin level during the course of illness that necessitates discontinuation of empiric antiviral therapy, namely ribavirin, has not been observed. [22] [23] [24] reactive thrombocytosis on recovery from sars is significantly more common in children 12 years of age. 23 this phenomenon is sometimes observed in children recovering from systemic viral infections and is probably not related to the use of corticosteroids. despite an abnormal clotting profile with elevated d-dimer levels and the detection of lupus anticoagulants in a small number of children, bleeding events or thrombotic complications have not been reported. 22, 48 the most common biochemical abnormality in children with sars is an elevated lactate dehydrogenase level, which is present in about 50% at presentation and about 70% during the course of illness. elevated alanine aminotransferase levels are seen in <20% of children at presentation and <50% during the course of illness. elevation of creatine kinase levels vary from 10% to 40% between case series. 22, 23 teenage patients tend to have more derangement of laboratory parameters and they may take longer to resolve. 22 similar to human infection with avian influenza a h5n1 virus, cytokine dysregulation is believed to be pivotal in the immunopathogenesis of sars among adults and children. serial monitoring of the plasma inflammatory cytokine profile using flow cytometry in a cohort of eight paediatric patients suggests that the caspase-1-dependent pathway in infected macrophages is selectively activated, as reflected by substantial elevation of circulating interleukin-1b levels. 49 conversely, interleukin-6 and tumour necrosis factor-a levels, which are markedly increased in human infection with avian influenza a h5n1 virus, are not overtly elevated throughout the course of illness. 50, 51 the predominant activation of the th1 immune response facilitates viral clearance and may explain the rapid recovery of children. as sars is a newly emerging infectious disease with unknown aetiology initially, the initial case definitions of suspected and probable sars promulgated by the world health organization were meant for surveillance and were necessarily broadly inclusive and non-specific. patients were categorised based on clinical, radiologic and epidemiologic features and after exclusion of alternative diagnoses. the original who surveillance case definitions for sars required that lower respiratory symptoms of cough, short-ness of breath or difficulty breathing were present. applying this would have missed many children who do not present with the above symptoms. the lack of sensitivity and specificity of the initial who case definitions have generated uncertainty in individual case management at the point of care. 24, 52 with more understanding of the disease and identification of a novel coronavirus as the causative agent, the case definitions of sars were revised on may 1, 2003. 53 as the clinical and radiologic features were non-specific, much emphasis was placed on the identification of an epidemiologic link to suggest the diagnosis. the vast majority of patients in the last epidemic had a clear history of exposure, either to patients suspected of or diagnosed with sars, or to a setting where recent local transmission was occurring. when the epidemic was over, an epidemiologic clue became more difficult to ascertain in sporadic cases that re-emerged. the latest who case definitions in the post-outbreak period now incorporate both clinical and laboratory elements, with further emphasis on clearly defined microbiologic criteria besides exclusion of alternative diagnoses (table 3) . 54 nevertheless, careful epidemiologic history taking remains essential in the diagnostic work-up and early implementation of appropriate infection control measures in suspected patients. important questions to ask in the 'peace time' include: (1) history of recent travel to pre-viously sars-affected areas or areas with an increased likelihood of animal to human transmission of sars-cov infection; (2) close contact with a suspected sars patient; (3) history of recent hospitalisation or contact with a healthcare facility; (4) individuals who are either healthcare workers or laboratory workers with potential exposure to sars patients or live sars-cov; and (5) link to a cluster of cases of unexplained respiratory illness in the community. microbiological investigations are the cornerstones for the confirmation of sars. the diagnostic work-up should include tests for pathogens which cause communityacquired pneumonia in children. 23 a blood culture is also needed. for children with productive cough who are old enough to produce a reliable specimen, sputum for bacterial culture should be performed. nasopharyngeal aspirate (npa) should be saved for rapid antigen detection of influenza a and b, rsv, adenovirus and parainfluenza types 1, 2 and 3, using direct immunofluorescence assays. urine samples may be tested for legionella pneumophila and streptococcus pneumoniae antigens. npa specimens should also be inoculated into different cell lines for isolation of respiratory viruses. serologic studies should include mycoplasma pneumoniae igm and paired acute and convalescent sera for igg against mycoplasma pneumoniae, chlamydia pneumoniae, chlamydia psittaci, legionella pneumophila, influenza a and b, rsv, adenovirus and parainfluenza types 1, 2 and 3. specific tests for the detection of sars-cov include: (1) molecular or nucleic acid amplification test using reverse transcription-polymer-ase chain reaction (rt-pcr); (2) antibody tests; and (3) cell culture. 55 in view of the high transmissibility of sars in hospitals, laboratory confirmation of the diagnosis early in the course of illness is vital to allow for the best utilisation of the limited isolation and cohorting facilities in most hospitals. rapid diagnosis with rt-pcr tests targeting specific segments of the sars-cov genome, primarily the polymerase gene, were used extensively during the last epidemic. [1] [2] [3] [56] [57] [58] [59] the method can be applied to nasopharyngeal aspirates, nose and throat swabs, saliva, sputum, endotracheal aspirates, bronchoalveolar lavage, stool, urine, plasma and serum. nasopharyngeal aspirates, combined nose and throat swabs and stool are the most commonly used. experience in hong kong and toronto suggests that the first generation conventional rt-pcr assays in use at the time of the initial outbreak lacked sufficient sensitivity to clinically rule out sars. 1, 60 despite initial optimism, the test has a sensitivity of 30% in npa, 28% in combined nose and throat swabs and 20% in stool in the first 5 days of illness. 61 it only reaches a maximum sensitivity of 60% when performed on upper respiratory specimens collected between days 9 to 11 from onset of fever (government virus unit, public health laboratory centre, hong kong special administrative region. data on file), where day 10 coincides with the maximum viral load in npa specimens as clinical picture, diagnosis, treatment and outcome of sars in children 281 a person with a history of: fever (!38 8c) and one or more symptoms of lower respiratory tract illness (cough, difficulty breathing, shortness of breath) and radiographic evidence of lung infiltrates consistent with pneumonia or rds or autopsy findings consistent with the pathology of pneumonia or rds without an identifiable cause and no alternative diagnosis can fully explain the illness laboratory definition of sars a person with symptoms and signs that are clinically suggestive of sars and with positive laboratory findings for sars-cov based on one or more of the following diagnostic criteria: (a) pcr positive for sars-cov using a validated method from: at least two different clinical specimens (e.g. nasopharyngeal and stool) or the same clinical specimen collected on two or more occasions during the course of the illness (e.g. sequential nasopharyngeal aspirates) or two different assays or repeat pcr using a new rna extract from the original clinical sample on each occasion of testing (b) seroconversion by elisa or ifa negative antibody test on acute serum followed by positive antibody test on convalescent phase serum tested in parallel or four-fold or greater rise in antibody titre between acute and convalescent phase sera tested in parallel (c) virus isolation isolation in cell culture of sars-cov from any specimen and pcr confirmation using a validated method measured in adult patients. 35 the low viral load in the upper respiratory tract in the initial few days of illness poses a diagnostic challenge. the lower respiratory tract as the primary target of sars-cov infection is the probable explanation. sputum specimens appear to have a higher diagnostic yield but productive cough is uncommon in sars patients in the early phase of illness and sputum is difficult to obtain in children. the overall diagnostic yield in the second week of illness increases to >80% when stool specimens are also examined, with stool yielding better results than respiratory specimens. 61 improving rna extraction from the specimen can markedly improve the sensitivity of conventional rt-pcr assays. when a modified rna extraction protocol is combined with an optimised real-time rt-pcr assay, a sensitivity of 80% and specificity of 100% can now be achieved in the first 3 days of illness, using npa as the preferred specimen. 57 a recently described real-time nested pcr assay performed on throat swabs is capable of detecting <10 copies of viral genome per reaction and achieves a much shorter turn-around time than conventional nested rt-pcr. 62 the technique of real-time rt-pcr has also been applied to plasma and serum samples. it has been shown that 50% of plasma and 78% of serum samples are positive for sars-cov rna during the first week of illness in adult sars patients. 63 a detection rate of 87.5-100% obtained in the plasma of eight paediatric patients within the first week of illness similarly suggests that plasma sars-cov rna quantification is a very sensitive and potentially useful early diagnostic tool. 34 the potential advantages of realtime rt-pcr include an increase in sensitivity, reduction in analytical time, reduction of risk of carry over contamination and availability of quantitative result for disease monitoring and prognostic purposes. 63 interestingly, despite a milder clinical course in paediatric patients, no significant differences in plasma viral loads are observed in plasma samples taken from paediatric and adult sars patients within the first week of admission and at day 7 after fever onset. 34 obtaining an npa specimen has been regarded by some as a hazardous procedure posing significant risk to the operator, although it is the best specimen for the rapid diagnosis of sars and the exclusion of other pathogens in the early phase of illness. to obviate the need for the protection of healthcare workers, an ingenious method for self-obtaining nasopharyngeal specimens through conjunctiva-upper respiratory tract irrigation (curti) has been described as an alternative. 64 the lack of serologic evidence of prior sars-cov infection in humans suggests that the virus has only recently entered the human population, presumably from an animal reservoir in southern china. 65, 66 specific igm and igg antibodies appear in response to sars-cov infection, with their levels changing during the course of the infection. serum antibody testing by immunofluorescence assay (ifa) or enzymelinked immunosorbant assay (elisa) have been developed to diagnose sars. 1, 3, 35, 67 the ifa test detects igm and igg antibodies and yields positive results in 16% and 55% of cases, respectively, after 10 days of illness. both are detectable in 91% of ifa tests by 25 days. 68 an indirect immunofluorescence test for igg antibody provides a sensitivity and specificity of 100%. 67 the elisa test detects a mixture of serum igm and igg antibodies, 80% and 85% respectively being positive by the second week. detection rate for both is 100% by week 3. the decay curves suggest that igm seropositivity is lost by about 12 weeks, while igg titres peak at 4 weeks and remain elevated until 12 weeks. 69 the antibody response is usually negative until 10 days from onset of symptoms. by day 28, seroconversion is demonstrated in 93% of sars patients despite corticosteroid therapy. 35 seroconversion from negative to positive or a !four-fold rise in igg antibody titres indicates recent infection. no detection of antibody in serum obtained >28 days from onset of illness indicates an absence of sars-cov infection and is the only laboratory method for excluding the diagnosis. 70, 71 serologic testing appears to be the best method for confirming sars, with positive rates ranging from 93% to 99%. 35, 52, 61 igm or other antibody assays have not been successful in closing the diagnostic window within the first week of illness. 65 even if some patients seroconvert early, the utility of serology is confined to retrospective diagnosis given the generally long lag time to seroconversion. igg usually remains detectable after resolution of the illness but the duration of persisting protective neutralising antibodies and their boosting response remain unknown. sars-cov can be isolated from respiratory secretions, blood or stool by inoculating cell cultures and growing the virus. vero e6 cells and fetal rhesus monkey kidney cells are suitable to support the viral growth, with the cytopathic effect demonstrable by 2-6 and 2-4 days respectively after inoculation. [1] [2] [3] the cultured virus must be identified as sars-cov with further tests, primarily rt-pcr assays. 61 the major limitation of viral culture in sars is its very low sensitivity. in one paediatric series, the virus was only successfully isolated from npa cultures in 16% of children. 23 negative cell culture results, like negative rt-pcr results, do not exclude sars infection. cell culture is also a very demanding test and primary virus isolation takes too long to be meaningful for early diagnosis. furthermore, amplification of the viable virus is associated with a potential biohazard, necessitating biosafety level three containment. culture-based diagnostic techniques are unlikely to be widely available but with the exception of animal inoculation, it is the only way to show the existence of viable sars-cov. 4, 72 the usual 'gold standard' of microbiological diagnosis, namely the isolation of the pathogen, has limited application in sars. during the global outbreak of sars, it was understandable that treatment was empiric, given the explosive epidemic of a life-threatening infection in multiple countries before the viral agent was even identified. time for planning, let alone conducting, a well-designed prospective clinical trial to assess the efficacy of any treatment regimen was simply not there. a proposed regimen consisting of antibiotics, ribavirin and corticosteroids was based on initial anecdotal successes in 2 outbreak studies in adult patients. 6, 28 subsequently, a standard treatment protocol was developed by a group of physicians in hong kong, which included (1) antibiotics for treatment of community-acquired pneumonia caused by usual and by atypical pathogens, (2) ribavirin as a broad-spectrum antiviral agent targeting the presumed viral etiology of sars, and (3) immunomodulating agents in the form of glucocorticoids. 73 a similar regimen in children consisting of antibiotics and ribavirin, with or without corticosteroids, was used. 21, 74, 75 in adult patients, the high incidence of deranged liver function, leucopaenia, severe lymphopaenia, thrombocytopaenia and progression to ards suggests severe systemic inflammatory damage induced by sars-cov. 1 the pathogenesis of the infection is postulated as an over-exuberant immunopathological reaction or a ''cytokine storm'' resulting from unrestricted viral replication during the early stages of the disease. findings consistent with cytokine dysregulation are the radiological changes of multifocal, flitting, boop-like features with progression to ards, the histological changes of macrophage infiltration and diffuse alveolar damage and the dramatic clinical and radiologic improvement with high-dose corticosteroid therapy. 1, 76 the viral load in sars followed an inverted v pattern, with progressive fall in viral shedding after day 10-15, correlating with seroconversion. 35 the logical approach to preventing severe disease is to restrict viral replication and to modulate inappropriate immunological responses. in principle, antiviral agent should be prescribed first during the phase of active viral replication, followed by an immunomodulator if the former fails and the patient is affected by immune hyperactivation. the use of ribavirin in adults and children has been reported by groups of investigators worldwide. 1, 6, [21] [22] [23] [24] [28] [29] [30] 35, [77] [78] [79] [80] [81] [82] ribavirin was empirically chosen in sars because of its broad-spectrum of activities against dna and rna viruses. ribavirin was also known to be effective in the treatment of fulminant murine hepatitis, which is caused by an animal coronavirus. in the murine hepatitis model, ribavirin exerted an immunomodulatory effect by decreasing the release of proinflammatory cytokines from the macrophages and switching the immune response from a th 2 to a th 1 response. 83, 84 however, it was later learnt that ribavirin demonstrated no or minimal activity against sars-cov isolates in vitro. 85, 86 in vitro testing indicated that ribavirin failed to inhibit replication or cell to cell spread at low drug concentrations. 87 although inhibitory activity was demonstrated at high drug concentrations, the resultant cytotoxic effects were undesirable. 88 it appeared that due to the low activity of ribavirin in vitro, inhibitory concentrations might not be achieved clinically without causing significant toxicity. investigators in canada have generally used ribavirin at a higher dosage similar to that recommended for treatment of several viral haemorrhagic fever syndromes and have observed severe adverse events in adult patients. booth et al. reported that 40% of patients had elevated hepatic transaminase levels, 14% had sinus bradycardia, 76% had haemolysis with haemoglobin levels declining by at least 2g/ dl in 49% and that 18% had to discontinue treatment. 30 knowles et al. reported that 61%, 58% and 46% of 110 patients had haemolytic anemia, hypocalcemia and hypomagnesaemia, respectively. 89 children appear to tolerate ribavirin much better than their adult counterparts. [21] [22] [23] [24] solid clinical data to demonstrate the efficacy of ribavirin is lacking. the limited data suggest that, at least in adults, dosages of about 2g/d might be effective while not causing severe adverse reactions. such doses should be considered for further studies. doses lower than 1g/d appear ineffective. 85 the only randomised controlled trial involving the use of ribavirin in the treatment of sars was conducted in china by zhao et al. the open-label study failed to demonstrate any efficacy and led the investigators to conclude that ribavirin, given at 400-600 mg/d, was less effective than early and aggressive use of corticosteroids combined with non-invasive ventilatory support. 77 non-randomised studies of corticosteroids have been reported in both adults and children with seemingly favourable outcomes in terms of clinical and radiologic improvements, suggesting that the combined use of ribavirin and corticosteroids might be effective. 1, 6, [21] [22] [23] 28, 33, 35, 73, 82, 90, 91 other reports on the combined regimen were inconclusive or failed to demonstrate obvious benefit. 30, 78, 79 in the paediatric series reported by leung et al., 95% and 84% of the 44 children with laboratory-confirmed sars were treated with ribavirin and corticosteroids respectively, without significant adverse events and all patients recovered. 23 in the series reported by chiu et al., 95% and 62% of the 21 children received ribavirin and corticosteroids, respectively and achieved similar outcomes. 22 all were subsequently confirmed by seroconversion to sars-cov after the report was published. bitnun et al. reported the use of ribavirin without corticosteroids in 10 children with probable sars but virologic confirmation was lacking. 24 in contrast, zeng et al. treated 33 children with chinese traditional medicine and antibiotics with good results. only 10 of the children had an epidemiologic link to sars, however, and virologic data were not available. 92 the use of corticosteroids in viral infections is controversial and is potentially hazardous. as an immunosuppressive agent, corticosteroids might promote viral replication, enhance infectivity and possibly cause a rebound of infection. it is known that in acute viral respiratory infections, early-response cytokines such as tumour necrosis factor, interleukin-1 and interleukin-6 mediate lung injury. the rationale for using corticosteroids is to suppress the ''cytokine storm'' which is thought to be the main factor accounting for the progression of disease. but using corticosteroids with possibly ineffective antiviral therapy in patients with viral pneumonitis can be hazardous. 93 despite the initial success of corticosteroids in the treatment of sars, the report of an adult patient whose clinical course was complicated by fatal aspergillosis was disturbing and had even led others to recommend close laboratory monitoring for aspergillosis in sars patients receiving corticosteroids. 94, 95 in retrospect, we do not think that ribavirin alone has any significant effect in halting disease progression and corticosteroids are probably unnecessary for children who do not develop moderate to severe hypoxaemia. in our experience, as with others, corticosteroids may be life saving in patients who are threatened by impending acute respiratory failure. we cannot categorically recommend this treatment strategy in view of the small number of children treated and the lack of objective evidence from a controlled trial. the place of corticosteroids in the rescue therapy of patients who have clearly experienced failure of supportive care remains to be determined. no evidence-based therapeutic approach for sars exists although more than 30 papers have been published internationally that mention antiviral treatment. various other antiviral and immunomodulating agents have been used in adult patients with preliminary success. these include the use of lopinavir / ritonavir in combination with ribavirin and corticosteroids, interferon a plus corticosteroids and convalescent plasma from patients. [96] [97] [98] [99] their true role in the treatment of children is unknown. knowledge generated by detailed bioinformatic analysis of the sars-cov genome can be harnessed to identify possible targets for antiviral therapy, such as enzymatic proteins of the viral replicase-transcriptase complex. this approach has been reviewed by davidson and siddell who concluded that the most economical and effective way to contain the virus would be the therapeutic use of antiviral agents to block viral entry to target cells or to inhibit intracellular viral replication. 100 in vitro studies have highlighted the antiviral potential of several compounds, including recombinant human interferon b-1a, interferon b-1b, glycyrrhizin, human monoclonal antibody against the spike protein of sars-cov and small interfering rna. [101] [102] [103] [104] [105] with more understanding of the pathogenesis as well as the clinical course of the disease, treatment will evolve. the best treatment for sars in adults and children remains unknown. time is now on our side to plan for clinical trials should the disease re-emerge. with increased vigilance, rapid detection and effective infection control measures, outbreaks of sars seem less likely. it might never be possible, therefore, to recruit a sufficient number of patients to complete the trials and give us an early answer. in adults, the risk factors for severe illness are advanced age, high initial absolute neutrophil counts, low platelet counts, high initial or peak lactate dehydrogenase levels and positive rt-pcr results for npa specimens. 6, [106] [107] [108] [109] only one paediatric series has identified risk factors for severe illness in terms of requirements for oxygen and intensive care. these include a sore throat, a high neutrophil count at presentation, and peak neutrophilia. the finding of sore throat as an independent risk factor is intriguing but may be incidental, given the small number of patients. no association between the presence of sore throat and the detection of sars-cov by rt-pcr or culture in npa specimens, which might correlate with higher viral load, could be demonstrated. 23 the short-term outcome of sars among children is good in comparison to adults. no case fatality has been reported. the need for intensive care and mechanical ventilation was up to 23.2% and 13.8% respectively in adults. 6 chiu et al. reported that 9.5% of children required oxygen supplementation and none required assisted ventilation. 22 leung et al. reported an oxygen requirement in 20.5% and assisted ventilatory support in 6.8% of children. 23 the figures for oxygen requirement and assisted ventilation in the two paediatric series combined are 17% and 5%, respectively. diffuse thinning and shedding of hair was observed in 41.5% of children in one series, generally at 2-3 months after disease onset. the condition was self-limiting and spontaneous recovery occurred within 1-3 months. this is consistent with acute telogen effluvium secondary to febrile systemic illness, critical care or severe psychologic stress in life-threatening situations. 23 li et al. examined the radiologic and pulmonary function outcomes of 47 children, 6 months after diagnosis and detected mild radiologic abnormalities with hrct and in pulmonary function testing in 34% and 10.5% respectively. 110 however, all children were asymptomatic and had normal clinical examination, premorbid hrct and pulmonary function test results were not available for comparison. in contrast, some adult patients have devel-oped pulmonary fibrosis despite recovery from the primary illness. 111 the psychological impact of separation, isolation in an intimidating hospital environment, bereavement and family disintegration following the death of close adult family members in children who recovered from sars are immense. however, children appear to be more resilient than adults in psychological adjustment to sars and serious psychological sequelae were not evident 3 months after discharge. 23 continued monitoring for delayed onset of psychological problems in children is essential. children who have recovered from the acute illness should be monitored for the possibility of continued viral shedding and the development of pulmonary sequelae and postviral complications (e.g. chronic fatigue), as well as for any long-term complications of high-dose corticosteroid therapy. children are susceptible to infection by sars-cov. despite the milder clinical picture, the good short-to medium-term outcome and the availability of reliable early diagnostic techniques, treatment remains controversial. the long-term outcome of sars in children remains unknown. there are still enormous gaps in our knowledge about sars. much work needs to be done, urgently. sars is largely an atypical pneumonia with minimal or no extrapulmonary manifestation apart from diarrhoea. the clinical picture of sars is milder in children but teenagers may develop severe illness resembling adults. the clinical, radiologic and laboratory features of sars are non-specific. an epidemiologic link is the most important clue to diagnosis in an outbreak situation. refined rt-pcr assays can achieve a sensitivity of 80% in the early diagnosis of sars in the first 3 days of illness. npa specimens are the preferred specimens for rt-pcr assays in the first week of illness. both npa and stool specimens should be tested in the second week. a negative rt-pcr result cannot exclude the diagnosis. absence of seroconversion beyond 28 days from disease onset generally excludes the diagnosis. apart from supportive treatment, including oxygen therapy and assisted ventilation, other treatment modalities remain unproven. molecular biology of sars-cov and mechanisms of its genome expression. pathogenesis of sars-cov infection. natural history and full spectrum of sars-cov infection. improved early diagnostic techniques. 'gold standard' of diagnosis. novel therapy and vaccine. longitudinal follow-up for long-term outcome and persistence of protective immunity to reinfection. coronavirus as a possible cause of severe acute respiratory syndrome identification of a novel coronavirus in patients with severe acute respiratory syndrome a novel coronavirus associated with severe acute respiratory syndrome koch's postulates fulfilled for sars virus update: outbreak of severe acute respiratory syndrome -worldwide a major outbreak of severe acute respiratory syndrome in hong kong sars transmission among hospital workers in hong kong sars infection among health care workers in beijing, china cluster of severe acute respiratory syndrome cases among protected health-care workers -toronto, canada probable secondary infections in households of sars patients in hong kong secondary household transmission of sars in-flight transmission of severe acute respiratory syndrome (sars): a case report transmission of the severe acute respiratory syndrome on aircraft outbreak of severe acute respiratory syndrome (sars) at amoy gardens, kowloon bay, hong kong: main findings of the investigation world health organization. summary of probable sars cases with onset of illness from 1 severe acute respiratory syndrome (sars) in singapore -update 2 -sars case in singapore linked to accidental laboratory contamination severe acute respiratory syndrome (sars) in taiwan, china update 4 -review of probable and laboratory-confirmed sars cases in southern china new case of laboratory-confirmed sars in guangdong, china -update 5 clinical presentations and outcome of severe acute respiratory syndrome in children severe acute respiratory syndrome in children: experience in a regional hospital in hong kong severe acute respiratory syndrome among children children hospitalized with severe acute respiratory syndrome-related illness in toronto a young infant with severe acute respiratory syndrome enteric involvement of severe acute respiratory syndrome-associated coronavirus infection tissue and cellular tropism of the coronavirus associated with severe acute respiratory syndrome: an in-situ hybridization study of fatal cases a cluster of cases of severe acute respiratory syndrome in hong kong identification of severe acute respiratory syndrome in canada clinical features and short-term outcomes of 144 patients with sars in the greater toronto area world health organization. preliminary clinical description of severe acute respiratory syndrome severe acute respiratory syndrome adolescent twin sisters with severe acute respiratory syndrome (sars) serial analysis of the plasma concentration of sars coronavirus rna in pediatric patients with severe acute respiratory syndrome clinical progression and viral load in a community outbreak of coronavirus-associated sars pneumonia: a prospective study possible central nervous system infection by sars coronavirus detection of sars coronavirus rna in the cerebrospinal fluid of a patient with severe acute respiratory syndrome sars-associated viral hepatitis caused by a novel coronavirus: report of three cases relative rates of non-pneumonic sars coronavirus infection and sars coronavirus pneumonia severe acute respiratory syndrome without respiratory symptoms or abnormal chest radiograph findings severe acute respiratory syndrome (sars): chest radiographic features in children severe acute respiratory syndrome (sars) in a paediatric cluster in singapore roentgenographic features of common pediatric viral respiratory tract infections thin-section ct of severe acute respiratory syndrome: evaluation of 73 patients exposed to or with the disease haematological manifestations in patients with severe acute respiratory syndrome: retrospective analysis lymphopenia in sars (letter) apoptosis may explain lymphopenia of sars (letter) severe acute respiratory syndrome and lupus anticoagulants in children inflammatory cytokine profile in children with severe acute respiratory syndrome pathology of fatal human infection associated with avian influenza a h5n1 virus induction of proinflammatory cytokines in human macrophages by influenza a (h5n1) viruses: a mechanism for the unusual severity of human disease evaluation of who criteria for identifying patients with severe acute respiratory syndrome out of hospital: prospective observational study world health organization. case definitions for surveillance of severe acute respiratory syndrome (sars) world health organization. alert, verification and public health management of sars in the post-outbreak period severe acute respiratory syndrome (sars): laboratory diagnostic tests rapid diagnosis of a coronavirus associated with severe acute respiratory syndrome (sars) early diagnosis of sars coronavirus infection detection of sars coronavirus in patients with severe acute respiratory syndrome by conventional and real-time quantitative reverse transcription-pcr assays evaluation of reverse transcription-pcr assays for rapid diagnosis of severe acute respiratory syndrome associated with a novel coronavirus interpretation of diagnostic laboratory tests for severe acute respiratory syndrome: the toronto experience detection of sars coronavirus in patients with suspected sars sensitive and quantitative detection of severe acute respiratory syndrome coronavirus infection by real-time nested polymerase chain reaction quantitative analysis and prognostic implication of sars coronavirus rna in the plasma and serum of patients with severe acute respiratory syndrome conjunctival-upper respiratory tract irrigation for early diagnosis of severe acute respiratory syndrome crouching tiger, hidden dragon: the laboratory diagnosis of severe acute respiratory syndrome isolation and characterization of viruses related to the sars coronavirus from animals in southern china immunofluorescence assay for serologic diagnosis of sars the application of indirect immunofluorescence assay in the diagnosis of severe acute respiratory syndrome profile of specific antibodies to the sarsassociated coronavirus combining clinical and epidemiologic features for early recognition of sars public health guidance for community-level preparedness and response to severe acute respiratory syndrome (sars) version 2. supplement f: laboratory guidance. appendix f7 -fact sheet for clinicians: interpreting sars-cov test results from cdc and other public health laboratories newly discovered coronavirus as the primary cause of severe acute respiratory syndrome development of a standard treatment protocol for severe acute respiratory syndrome sars reference pmh/pwh interim guideline on the management of children with sars lung pathology of fatal severe acute respiratory syndrome description and clinical treatment of an early outbreak of severe acute respiratory syndrome in guangzhou, pr china severe acute respiratory syndrome (sars) in singapore: clinical features of index patient and initial contacts clinical description of a completed outbreak of sars in vietnam icu management of severe acute respiratory syndrome identification and containment of an outbreak of sars in a community hospital clinical course and management of sars in health care workers in toronto: a case series inhibition of murine hepatitis virus infections by the immunomodulator 2,3,5,6,7,8-hexahydro-2-phenyl-8,8-dimethoxy-imidazo[1,2a]pyridine (pr-879-317a) ribavirin inhibits viral-induced macrophage production of tnf, il-1, the procoagulant fgl 2 prothrombinase and preserves th 1 cytokine production but inhibits th 2 cytokine response antiviral treatment of sars: can we draw any conclusions? ribavirin in the treatment of severe acute respiratory syndrome (sars) severe acute respiratory syndrome (sars) and coronavirus testing -united states inhibition of sars coronavirus infection in vitro with clinically approved antiviral drugs common adverse events associated with the use of ribavirin for severe acute respiratory syndrome high-dose pulse versus nonpulse corticosteroid regimens in severe acute respiratory syndrome treatment of severe acute respiratory syndrome in health-care workers clinical characteristics and prognosis of 33 children with severe acute respiratory syndrome in guangzhou area sung jj (authors' reply). the use of corticosteroids in sars fatal aspergillosis in a patient with sars who was treated with corticosteroids real time assay of aspergillus should be used in sars patients receiving corticosteroids treatment of severe acute respiratory syndrome with lopinavir/ritonavir: a multicentre retrospective matched cohort study role of lopinavir/ritonavir in the treatment of sars: initial virological and clinical findings interferon alfacon-1 plus corticosteroids in severe acute respiratory syndrome: a preliminary study treatment of severe acute respiratory syndrome with convalescent plasma potential for antiviral treatment of severe acute respiratory syndrome interferon-b 1a and sars coronavirus replication treatment of sars with human interferons glycyrrhizin, an active component of liquorice roots, and replication of sars-associated coronavirus potent neutralization of severe acute respiratory syndrome (sars) coronavirus by a human mab to s1 protein that blocks receptor association inhibiting sars coronavirus by small interfering rna severe acute respiratory syndrome: clinical outcome and prognostic correlates outcomes and prognostic factors in 267 patients with severe acute respiratory syndrome in hong kong coronavirus-positive nasopharyngeal aspirate as predictor for severe acute respiratory syndrome mortality prognostic factors for severe acute respiratory syndrome: a clinical analysis of 165 cases radiological and pulmonary function outcome of children with sars thin-section ct in patients with severe acute respiratory syndrome following hospital discharge: preliminary experience key: cord-006261-yw5k8qkz authors: heath, gregory w.; macera, caroline a.; nieman, david c. title: exercise and upper respiratory tract infections: is there a relationship? date: 2012-10-23 journal: sports med doi: 10.2165/00007256-199214060-00003 sha: doc_id: 6261 cord_uid: yw5k8qkz nan runners and other exercise enthusiasts are traditionally motivated to participate in sports for competitive or recreational reasons. however, some exerciseparticipants are convinced that exercisewill provide health benefits (nash 1986 ). indeed, regular exercise and physical activity have been established as a means of reducing cardiovascular disease risk and contributing to longevity (paffenbarger et al. 1986; powell et al. 1987 ). in addition, habitual exercisers often report other health-related rewards from endurance exercise such as weight control (heath et a1. 1981) , improved energy levels (hughes 1984) and stress reduction (blumenthal et al. 1980; sinyor et al. 1983 ). many exercisers also believe that exercise improves their resistance to infection and anecdotally report that they have fewer colds and other upper respiratory tract infections (urti) [simon 1987 ]. despite these beliefs, few studies have examined the relationships that exist among exercise and clinical expressions of upper respiratory tract infections. this article seeks to examine the existing studies that have investigated the relationship between exercise and upper respiratory tract infections and to determine ifany such relationship exists. it begins by defining and discussing the specific health outcome of infections and the classification of these diseases. secondly, the epidemiology and natural history are discussed with the clinical and experimental evi-dence of the relationship between upper respiratory tract infections and exercise explored. the suggested immunological and physiological mechanisms of this possible relationship are examined, followed by a review of the epidemiological data linking an association of exercise to upper respiratory tract infections. finally, guidelines for the exercising public are presented as an application of our attempt to answer the question : exercise and upper respiratory tract infections -is there a relationship? upper respiratory tract infections consist of a number of acute illnesses that occur in the upper portion of the respiratory tract. the most common infections include pharyngitis, croup, bacterial tracheitis, epiglottitis and the common cold. since the common cold is the leading acute illness and cause of visits to a physician in the developed world, the common cold is a focus of the article. the term 'cold' carries different meanings for many people; however, it is usually defined as an acute illness that involves nasopharyngitis and catarrh , little or no fever and insignificant systemic symptoms. ruses it is not known which of these modes of spread are most important. for rhinovirus and respiratory syncytial virus close contact with an infected person or infected secretions is necessary, with a number of studies demonstrating that people infected with rhinovirus colds have recoverable infectious virus on their hands (gwaltney 1983; gwaltney & hendley 1978; hendley et al 1973) . furthermore, these studies have shown that rhinovirus may be readily transferred from the contaminated hands of one person to the hands of another who, if susceptible, may acquire infection by touching his nasal or conjunctival mucosa. the aetiology of this group of upper respiratory tract infections is viral in nature. the predominant group of viruses which cause colds are the rhinoviruses followed by the coronaviruses (table i) . the parainfluenza viruses, respiratory syncytial virus and influenza viruses are all epidemic viruses that are associated with colds. however, these viruses cause more serious upper respiratory tract infections which tend to mask the milder colds (hall & mcbride 1989 ). transmission of upper respiratory tract infection-causing viruses may occur through several modes. suspension of viral particles in large droplets produced by a cough or sneeze with direct contact into the eyes or on to the upper respiratory passages is a common mode, usually produced by close contact with the infected individual. virus suspended in the small particle aerosol of a sneeze or cough is capable of travelling greater distances and is another common mode of infection. finally, the virus can be spread by contact with contaminated secretions by hand from contaminated surface to mucous membranes (self-inoculation) [hall & mcbride 1989] . for most of the respiratory vi-when examining the numbers and types of viruses causing upper respiratory tract infections it is clear that this is a rather ubiquitous group of infections. in general, the number of infections acquired per year decreases with age (bader et al 1953; brimblecombe et al. 1958; dingle et al. 1964; fox et al. 1972; gwaltney et al. 1966) . infants and children have the highest incidence with 4 to 8 infections per year. this rate may even double when children are in day care or nursery school. in schoolaged children the incidence is 2 to 6 colds per year (badger et al. 1953; brimblecombe et al. 1958; dingle et al. 1964; fox et al. 1972) . adults acquire 2 to 5 upper respiratory tract infections per year, with women and adults who live in households with children tending to suffer even more colds per year (gwaltney et al 1966) . smoking has been shown to aggravate the signs and symptoms of infection but not to increase the attack rate (gwaltney et al. 1966 (gwaltney et al. , 1967 . exposure to cold or to chilling does not increase the chance of acquiring or aggravating a cold (douglas et al. 1968 ). recent studies have demonstrated that psychological stress is a risk factor for the development of an upper respiratory tract infection (cohen et al. 1991; graham 1986) . finally, in studies exploring the prophylactic use of vitamin c in prevention, the attack rate of upper respiratory tract infections was not diminished alfig. 2 . effect of marathon running on lymphocyte responsiveness to concanavalin a (con a) at pre-exercise andat 30 min and 3h recovery from the marathon. though symptoms were ameliorated (coulehan et al. 1976; karlowski et al. 1975; miller et al. 1977 ). human and animal experimental evidence has demonstrated a relationship between exercise and upper respiratory tract infections (nieman & nehlsen-cannarella 1991 a, 1992 . furthermore, these studies provide insight into potential mechanisms that might explain the relationship between physical activity and infection. (nk) cell activity at 3 effector: target (e: t) ratiosat 1.5, 6 and 21h following a 3h run at 70%~02max. several researchers have reported that various aspects of immune function are depressed following intense, prolonged endurance exercise (nieman & nehlsen-cannarella 1991a) . heavy exertion is a form of physiological stress that causes large increases in epinephrine (adrenaline) and cortisol levels, hormones which have been consistently associated with a suppression of immune function, and rapid perturbations in circulating levels ofleucocyte and lymphocyte subsets. nieman et al. (1989a) and berk et al. (1990) ran 10 seasoned marathoners at their fastest marathon pace on treadmills for 3 hours. cortisol rose 59% abo ve baseline levels after the 3-hour run , remain-ing elevated for 1.5 hours of recovery before falling to normal daytime levels. this increase in cortisol correlated inversely with a 25 to 46% decrease in natural killer (nk) cell activity at 1.5 hours after recovery, which persisted for nearly 6 hours ( fig. 1 ). pedersen et al. (1988 pedersen et al. ( , 1989 pedersen et al. ( , 1990 and kappel et al. (1991) have carefully demonstrated that the post exercise suppression of natural killer cell activity is also related to increased levels of prostaglandins released from monocytes. eskola et al. (1978) and gmunder et al. (1988) have reported a significant decrease in lymphocyte proliferative response for several hours after a marathon (42.2km) [ fig. 2 ]. macneil et al. (1991) have demonstrated that the lymphocyte proliferative response is decreased for at least 2 hours following cycle ergometer exercise, especially following high intensity exercise by athletes. following 1 hour of cycling at 80% v02max by untrained individuals, tvede et al. (1989) found suppression of b lymphocyte function for at least 2 hours because of an inhibitory effect of activated monocytes. have determined that neutrophil killing capacity is decreased in elite athletes engaging in prolonged periods of intensive tra ining in comparison to untrained controls. nieman et al. (1989b) and have reported significantly lower serum complement in long distance runners relative to sedentary controls. a significant decrease in salivary immunoglobulin concentrations following 2 hours of intense cycling or 50km of cross-country ski racing z 20 10 has been described by 2 groups of investigators (mackinnon et al. 1987; tomasi et al. 1982) . israel et al. (1982) have reported that serum immunoglobulins fall 10 to 28% for at least 1 day after athletes run 45 or 75km at high intensity. russian investigators have related that exhaustion of immune reserves can be observed during periods of important competitions, manifested by lowered immunoglobulin levels and suppression of phagocytic activity ofneutrophils (pershin et al. 1985; petrova et al. 1983 petrova et al. , 1985 . results from animal studies have rather consistently supported the view that heavy acute and chronic exertion are related to negative changes in immune function. several researchers have reported that exhaustive single bouts of exercise by both trained and untrained animals, or 6 days to 4.5 months of heavy exercise training, are linked to increased splenic epinephrine and cortisol levels and decreased splenic natural killer and t cell lymphocyte function (ferry et al. 1990; hoffman-goetz et al. 1986; mahan & young 1989; simpson & hoffman-goetz 1990) . thus, both circulating immune cells and those found in secondary lymphoid tissues may have their function suppressed because of the increase in cortisol and catecholamine levels that occur following heavy exertion. further research is needed to better elucidate the clinical significance of exercise induced changes in immune status and function (many of which are transient in nature), and which variables best predict potential changes in host protection. the data at present are not consistent enough between studies to even suggest thresholds for various immune system markers that may indicate increased risk of upper respiratory tract infection. psychological factors may also play an important role in the relationship between exercise and upper respiratory tract infection. exercise is a form of physiological and psychological stress, varying according to the intensity and duration of the training programme. interestingly, the acute re-sports medicine 14 (6) 1992 sponse of the immune system to psychological stressors alone is in many ways similar to those that occur in response to acute exercise (naliboff et al. 1991) . if the exercise training programme is deemed stressful by the athlete, the combined psychological and physiological impact may overwhelm the ability of the immune system to protect the host (nieman & nehlsen-cannarella 1992) . mental stress alone has been related to a wide variety of negative changes in immunity. bereavement, major depression, loneliness, schizophrenia, marital discord and other forms of mental stress have all been associated with suppression of immune function (jemmott & locke 1984; khansari et al. 1990) . a biochemical basis for bidirectional communication between the immune system and neuroendocrine system has been established (blalock 1989) .these systems produce and use many of the same signal molecules in the form of hormones, lymphokines and monokines for inter-and intrasystem communication and regulation. lymphoid organs are innervated by the autonomic nervous system, and lymphocytes have receptors for the various stress hormones. in the other direction, for example, products of leucocytes have been shown to alter neuronal activity in certain areas of the brain. thus, stress of any form may decrease host protection from infection through both autonomic nervous system and hormonal mechanisms. research by graham and associates (1986) , for example, has demonstrated that during a given 6month period, highly stressed individuals have twice as many days with respiratory infection symptoms compared with low-stressed people. cohen et al. (1991) gave nasal drops containing respiratory viruses to 394 subjects and reported that psychological stress was associated in a dose-response manner with an increased risk of acute infectious respiratory illness. although specific research in this area has not yet been conducted, it would seem logical to assume that athletes around the time of competition, when both physiological and psychological stress are high, would be most vulnerable to respiratory infections. another factor that may be important for the risk of respiratory infection in athletes is the involvement of the immune system in the tissue repair process that occurs following strenuous exercise (for a review see neiman & nehlsen-cannarella 1991 b). it has been well established that both heavy acute and chronic exertion are associated with muscle cell damage, local inflammation and the stereotyped sequence of host defence reactions known as the acute phase response (evans & cannon 1991; nieman & nehlsen-cannarella 1991a) . the acute phase response following endurance exercise involves the complement system, neutrophils, macro phages, various cytokines and acute phase proteins, and can last for several days, promoting clearance of damaged tissue and setting the stage for repair and growth. lymphocytes, neutrophils and macrophages are attracted to the injured muscle cells, and invade the area to aid in the process. neutrophils phagocytise tissue debris and release a wide variety of factors that aid in the digestion of adjacent dead tissue cells (smith 1991) . macrophages have surface receptors which allow them to react nonspecifically to a variety of substances, a process enhanced by the presence of opsonins (primarily complement and antibody). macrophages also are a prime source of cytokines that mediate most of the physiological and inflammatory reactions accompanying muscle cell injury. dufaux and order (1989b) have shown that plasma elastase-a j-antitrypsin, neopterin, tumour necrosis factor and soluble interleukin-2 receptor increase during recovery from a 2.5-hour running test, supporting the concept of a functional involvement of polymorphonuclear neutrophils and an activation of macrophages .and t-lymphocytes. dufaux and order (1989a) have also provided evidence for complement activation after 2.5 hours of running. could the active enmeshment of the immune system in the muscle tissue repair and inflammation process mean that protection from respiratory infection is compromised? research to answer this question is certainly warranted, and may greatly increase our understanding as to how and why ath-357 letes appear to be more susceptible to respiratory tract infections during periods of heavy training. few studies have investigated the common belief that moderate physical activity is beneficial in decreasing risk of respiratory tract infections and improving immune function. more research is certainly warranted to investigate this interesting area. since the turn of the century, the influence of exercise training on resistance to infection has been investigated using animal models. cannon and kluger (1984) have reviewed the animal literature and concluded that moderate exercise prior to infection may increase resistance to infection, but that exhaustive exercise after contracting an infection may be detrimental, in accordance with this viewpoint, slubik et al. (1987) have reported that moderate physical exercise preceding irradiation diminishes radiation injury in animals while intensive exercise and stress may aggravate the damage. have shown that 1 hour of cycling at 60%~02max may increase resistance to infection by improving the 'killing capacity' of neutrophils, an effect which persists for at least 6 hours of recovery. given that neutrophils are the body's best phagocyte, these findings suggest that regular episodes of moderate exercise may increase resistance to infection. in a randomised controlled study by nieman et al. (1990) and nehlsen-cannarella et al. (1991) , the effects of walking on immune response and acute respiratory. infection tract symptomatology were measured on a group of sedentary, mildly obese women. the exercise subjects walked 45 minutes per session, 5 times per week, for 15 continuous weeks on a measured course under supervision. subjects recorded health problems in a daily log book using 10 codes supplied by the centers for disease control. exercise subjects experienced half the number of days with respiratory infection symptoms during the 15 weeks compared with the sedentary control group (5.1 â± 1.2 vs 10.8 â± 2.3 days, respecthus, the response of the immune system to exercise may have much to do with the degree of intensity and total exertion load, and corresponding changes in concentrations of cortisol and epinephrine. both of these hormones have been associated with many negative effects on immune function (cavallo et al. 1986; crary et al. 1983; cupps & fauci 1982) . lymphocytes have~-adren ergic receptors, and the presence of epinephrine during exercise increases receptor number on t suppressor/cytotoxic and natural killer cells, which are the major lymphocyte subsets that increase in response to an exercise challenge (field et al. i991; maisel et at. 1990; van tits et at. 1990 ). the degree to which these lymphocyte subsets increase in the peripheral blood as they are recruited from lymphoid tissue pools is highly dependent on the magnitude of change in epinephrine. additionally, the effect on lymphocyte function is also dependent on the change in both epinephrine and cortisol. moderate exercise such as walking does not increase the concentration of epinephrine and cortisol in the blood, resulting in a small lymphocytosis in contrast to intense exercise, in which levels of both hormones increase (nieman 1991) [ fig. 4 ]. thus , it may be argued that moderate exercise induces a small increase in natural killer cells and t cytotoxic/suppressor cells without the potential suppressive effect of epinephrine or cortisol, creating a milieu which may be favourable for host protection. there is only a finite number of lymphocytes that is specific to any particular antigen. theoretically, by recruiting lymphocytes from the periphery, exercise may increase the rate of lymphocyte circulation through the body, improving the potential for interaction of lymphocyte and antigen without the attending negative effects of stress hormones (nieman & nehlsen-cannarella 1992) . 3 ]. moderate exercise training led to a 20% net increase in each of the 3 serum immunoglobulins, and was significantly correlated with fewer respiratory infection symptoms days (nieman & nehlsen-cannarella 1991 b) . moderate exercise training also led to a significant increase in natural killer cell activity which was correlated with a decrease in the duration of respiratory infection symptoms per episode. these results are similar to those of crist et al. (1989) who moderately exercised elderly women for 16 weeks and measured a 33% higher natural killer cell activity at rest, and a heightened increase following maximal testing. laboratory and clinical studies suggest that changes in immune parameters associated with heavy train ing lead to adverse health effects, particularly an increase in the incidence or severity of upper respiratory tract infections. however, anecdotal reports by marathon runners that running increased (or decreased) their incidence of upper respiratory tract infections were not correlated with immune system changes (green et al. 1981) . if the documented changes in immune parameters are associated with upper respiratory tract infections, there are several methods by which epidemiological investigations can be used to evaluate this effect. to use the epidemiological approach, the following must be present: a defined population from which the exposed group and a comparison group can be selected, and a clear operational definition of the outcome of interest. the exposure (training) can be measured in a number of ways that would indicate relative levels of individual exertion. while this is not as accurate as the measurement that would occur in a laboratory setting, there is confidence that measuring levels of the exposure is possible in a population-based setting. however, defining the appropriate control or comparison group is a more difficult problem. among studies involving runners, there have been a variety of methods used to determine the comparison group: faster runners were compared with slower runners of the same road race (peters et al. 1983 ); low mileage runners were compared to high mileage runners over the same time period (heath et al. 1991) ; marathon runners were compared with those who ran shorter races on the same day nieman et ai. 1989c) ; marathon runners were compared with trained nonrunners (nieman et al. 1990b) , or marathon runners were compared with nonrunning friends (peters et al. 1983 ). each of these comparison groups has its advantages and drawbacks. the true measure of the outcome (upper respiratory tract infections) is impossible to ascertain without a clinical examination and a laboratory workup. in lieu of a clinical assessment, the best surrogate measure for upper respiratory tract infection is self-reported symptoms. however, many other disease processes or allergic reactions may present with symptoms similar to those of upper respiratory tract infections. unless these symptoms are severe or proceed to disability, most affected individuals will not be seen by medical personnel, and the symptoms will soon resolve without a medical diagnosis. in spite of these limitations, selfreport is the best choice, and steps can be taken to operationally define upper respiratory tract infection symptomatology so that definitions are consistent across studies. in spite of these measurement difficulties, several studies have explored the relationship between exercise and upper respiratory tract infection symptomatology among population-based samples, usually runners. among these studies, some have supported the suggestion that athletes engaging in marathon type events or very heavy training are at increased risk of respiratory tract infections (heath et al. 1991; nieman et al. 1990b; peters et al. 1983 ), while others have found no difference among exposed and unexposed groups nieman et al. 1989c) ,and still others have found a decrease in infections among the exposed compared with the unexposed (nieman et al. 1990a; schouten et al. 1988 ). peters and bateman (1983) studied the incidence of respiratory tract infections in 1550 randomly selected runners who took part in a 56km race compared with matched controls who did not run. those who ran the race reported more symptoms of respiratory tract infections during the 2week period following the race than those who did not run, and those who ran the faster race times reported more symptoms, indicating a doseresponse relationship. nieman et al. (l990b) studied the incidence of respiratory tract infection in a large group of marathon runners who varied widely in running ability and training habits. those who ran the marathon reported more symptoms during the week following the race than similarly experienced runners who had applied but did not participate in the race for sports medicine 14 (6) 1992 possible confounders, the researchers concluded that, compared with nonrunners, runners experience increased risk for respiratory tract infection during heavy training or following a marathon race event. heath et al. (1991) followed a cohort of 530 runners who self-reported any symptoms of respiratory infections daily for 1 year. the average runner was about 40 years of age, ran 20 miles (32km) per week, and experienced a rate of 1.2 respiratory infections per year. controlling for various confounding variables, the lowest odds ratio for respiratory infection was found among those running less than 10 miles (16km) per week. the odds ratio more than doubled for those running more than 17 miles (27km) per week. the authors concluded that running mileage was a significant risk factor for upper respiratory tract infection symptoms. in another study, no difference (for either men or women) was found in the incidence of upper respiratory tract infection symptomatology during the month after the race for marathon runners compared with those who ran on the same day, but for shorter distances. in this study, the most important determinant for symptoms of upper respiratory tract infection after the race was the presence of upper respiratory tract infection symptoms before the race. these results persisted after controlling for total training mileage and percentage increase in training mileage during the month before the race. nieman et al. (l989c) studied the incidence of upper respiratory tract infection symptoms among participants in 5km, iokm and half-marathon road races. during the 2 months before the race, those running more than 15 miles (24km) per week reported more events than those running fewer miles. there was no increase in symptoms after the road race compared with the week before the road race, indicating that the race did not appear to be associated with an increased risk of acute upper respiratory tract infection. in a study of young adults, schouten et al. (1988) found no difference in the incidence or duration of upper respiratory tract infection symptoms when level of sports activity or maximum aerobic power. they found a slight inverse association between symptoms and level of sports activity for women only. although this study had the advantage of measuring several types of physical activity as well as aerobic power, the upper respiratory tract infection measurement relied on memory recall for the past 6 months, leading to possible misclassification. in a 15-week study of immune response and exercise, 36 mildly obese women were randomly assigned to walking or nonexercising groups (nieman et a1. 1990a) . during this time, the women in the exercise group experienced fewer upper respiratory tract infection symptom days, improved cardiorespiratory fitness, and increased natural killer cell numbers compared with their sedentary controls. this study indicates that intensity of exercise may playa role, and that moderate activity may actually improve immune function and associated upper respiratory tract infection symptoms. because this is a new field it is not surprising that the results of the few studies done to date have not been consistent. while most, but not all, of the published studies have found a relationship between increased training and upper respiratory tract infections, and considering that the type and intensity of exercise has not been fully explored, no definitive conclusions can be drawn. however, on the basis of the available studies, some general guidelines can be provided for future studies. separate comparison groups, a lower intensity running or exercise comparison group as well as a nonexercising or sedentary comparison group should be considered. during the analysis stage the comparison groups could be used to sort out some of the biases present in the previous studies. because reporting behaviour differs between men and women, the sample size should be large enough to allow separate analyses for men and women. a consistent time frame for reporting upper respiratory tract infection symptoms after racing events should be used. the studies noted here have used follow-up times ranging from i to 4 weeks. when using self-report data, every attempt 361 should be made to differentiate symptoms due to allergy from symptoms because of upper respiratory tract infections. because allergic rhinitis may be present (but undiagnosed) the case definition of upper respiratory tract infections should be defined in such a way as to capture most of the events. data should also be collected on duration of the event, and for longer studies, the number of symptom-free days between events should be defined. at present, insufficient evidence exists to recommend precisely what laboratory tests of immune function should be conducted to ascertain when an athlete is at increased risk of an infectious episode due to overtraining and/or psychosocial stress. although some evidence would suggest that low immunoglobulin and complement levels, decreased lymphocyte proliferative response, diminished neutrophil phagocytic activity, depressed natural killer cell activity, low total lymphocyte count and low helper/suppressor t cell ratio are each important markers of increased risk, the exact level at which one or a combination of some or all of these immune components becomes predictive is unknown (nieman & nehlsen-cannarella 1992) . there is an interesting similarity in the metabolic and immunological responses to intense endurance exercise and to an infectious challenge (lewis et a1. 1986; schaefer et a1. 1987) . in both conditions, the number of circulating leucocytes increases, lymphopenia occurs (especially t cells with cells trafficking to peripheral tissues), the lymphocyte responses to phytohaemagglutinin (pha) and concanavalin (con-a) decrease, body core temperature rises, plasma levels of acute phase proteins increase and degranulation of neutrophils develops. since endurance exercise is associated with muscle cell damage and an increased intake of potential pathogens through heightened ventilation, it is logical that in preparation for such a challenge, the immune system receives a signal from the neuroendocrine network that activates the immune system. why then do clinical experience and epidemiological data point toward an increased risk of respiratory infection in some athletes? the mass of evidence favours the view that psychosocial variables play an important role in effecting immunological competence. the net effect of combined psychological and physiological stress from unusually heavy endurance exercise, especially during times of competition, may lead to suppression or down-regulation of the immune system. for those athletes who must exercise intensely for competitive reasons, several precautions can help decrease the risk of sickness. these include spacing vigorous workouts and race events as far apart as possible, eating a well balanced diet, keeping other life stresses to a minimum, avoiding overtraining and chronic fatigue and obtaining adequate sleep. before and after intense race events, the athlete should try to avoid contact with sick people if at all possible. for the fitness enthusiast, the area of concern is not so much the harm that may come from overexertion, but the benefits that may derive from engaging in regular, moderate forms of exercise. at this time, even though investigative evidence suggests improved host protection and immunosurveillance from moderate physical activity, more research is needed to improve our understanding of the workload threshold below or above which exercise becomes protective rather than detrimental. should athletes exercise when they have an upper respiratory tract infection? most clinical authorities in this area recommend that if the athlete has symptoms of a common cold with no constitutional involvement, then regular training may be safely resumed a few days after the resolution of symptoms (roberts 1986; simon 1987) . mild exercise during sickness with the common cold does not appear to be contraindicated. however, if there are symptoms or signs of systemic involvement (e.g. fever, extreme fatigue, muscle aches, swollen lymph glands), then 2 to 4 weeks should be allowed before resumption of more intense training. these precautions are advised because of the well documented relationship between intense. exercise and sports medicine 14 (6) 1992 the risk of developing a viral cardiomyopathy and other severe form of viral infection (sharp 1989) . physical activity and exercise produce a variety of alterations of the immune system, most of which have not been fully investigated. however, the effects of vigorous exercise appear to depress immune function and may compromise host defense against upper respiratory tract infections. because of the complexity of host defence mechanisms and the physiologyof exercise,further research is needed in this area. clinical studies examining the effects of moderate levels of physical activity have shown possible enhanced immune responses with a concomitant impact on the length and severity of an upper respiratory tract infection. therefore, the relationship between exercise and upper respiratory tract infections appears to be 'j' shaped with the most sedentary at greatest risk of upper respiratory tract infections along with the vigorously active, with those engaged in moderate levels of activity manifesting the apparently better host defence. epidemiological studies have generally demonstrated a greater risk of upper respiratory tract infection with vigorous levels of exercise; however, these studies must be considered to be limited because of the lack of adequate nonexercising control groups. further studies are necessary to demonstrate the clinical and public health significance of these relationships. engaging in regular moderate level physical activity is most beneficial for maintaining health and in preventing an initial upper respiratory tract infection. moderate level physical activity coupled with generally good hygiene and avoidance of close contact with individuals known to have an active upper respiratory tract infection appear to be the most commonsense measures for avoiding contracting an upper respiratory tract infection. however, since avoidance of a high risk situation, such as the presence of children, is often difficult, the hygienic measures of frequent handwashing and the thorough washing of potential fomitic surfaces will aid in reducing exposure to the upper respiratory tract infection agent. for those athletes who are required to engage in high intensity training and competition, further steps should be taken to minimise contact with situations where exposure to upper respiratory tract infection agents is high. in addition, training techniques should take into consideration the need for the organism to restore host resistance by including lower intensity training sandwiched between higher intensity training bouts. a study of illness in a group of cleveland families: ii. incidence ofcommon respiratory diseases the effect of long endurance running on natural killer cells in marathoners a molecular basis for bidirectional communication between the immune and neuroendocrine systems effects of exercise on the type a (coronary prone) behaviour pattern family studies of respiratory infections exercise enhances survival rate in mice infected with salmonella typhimurium cortisol and immune interferon can interact in the modulation of human natural killer cell activity psychological stress and susceptibility to the common cold vitamin c and acute illness in navajo school children epinephrine-induced changes in the distribution of lymphocyte subsets in peripheral blood of humans physical exercise increases natural cellular-mediated tumor cytotoxicity in elderly women corticosteroid-mediated immunoregulation in man illness in the home: study of 25 000 illnesses in a group of cleveland families exposure to cold environment and rhinovirus common cold: failure to demonstrate effect complement activation after prolonged exercise plasma elastase-alpha l-antitrypsin, neopterin, tumor necrosis factor, and soluble interleukin-2 receptor after prolonged exercise effect of sport stress on lymphocyte transformation and antibody formation the metabolic effects of exercise-induced muscle damage immunomodulations induced in rats by exercise on a treadmill circulating mononuclear cell numbers and function during intense exercise and recovery the seattle virus watch: ii. objectives. study population and its observation data processing and summary of illnesses effect of long-term physical exercise on lymphocyte reactivity: similarity to spaceflight reactions stress and acute, respiratory infection immune function in marathon runners rhinovirus transmission, one ifby air, two if by hand rhinovirus colds: epidemiology, clinical characteristics and transmission rhinovirus infections in an industrial population: i. the occurrence of illness rhinovirus infections in an industrial population: ii. characteristics of illness and antibody response upper respiratory tract infections: the common cold, pharyngitis, croup, bacterial tracheitis and epiglottitis. in pennington (ed.) respiratory infections: diagnosis and management exercise and the incidence of upper respiratory tract infections a physiological comparison of young and older endurance athletes transmission of rhinovirus colds by self-inoculation psychological effects of habitual aerobic exercise: a critical review chronic exercise stress in mice depresses splenic t lymphocyte mitogenesis in vitro die konzentration der immunglobuline a, g und m im serum bei trainierten und untrainierten sowie nach verschiedenen sportlichen ausdauerleistungen psychosocial factors, immunologic mediation, and human susceptibility to infectious diseases: how much do we know? evidence that the effect of physical exercise on nk cell activity is mediated by epinephrine ascorbic acid for the common cold: a prophylactic and therapeutic trial effects of stress on the immune system influenza virus infection induces functional alterations in peripheral blood lymphocytes running and upper respiratory tract infections postrace morbidity among runners the effect of exercise on secretory and natural immunity lymphocyte proliferation response after exercise in men: fitness, intensity, and duration effects immune parameters of untrained or exercise-trained rats after exhaustive exercise b-adrenergic receptors in lymphocyte subsets after exercise: alterations in normal individuals and patients with congestive heart failure therapeutic effect of vitamin c: a co-twin control study immunological changes in young and old adults during brief laboratory stress can exercise make use immune to disease? the effects of moderate exercise training on immune response effects of long endurance running on immune system parameters and lymphocyte function in experienced marathoners effects of endurance exercise on immune response the effects of acute and chronic exercise on immunoglobulins exercise and infection the effects of acute moderate exercise on leucocyte and lymphocyte subpopulations the effects of moderate exercise training on natural killer cells and acute upper respiratory tract infections munoglobulin levels in athletes and sedentary controls infectious episodes in runners before and after a roadrace infectious episodes in runners before and after the los angeles marathon a prospective study of respiratory infections in 12-year-old children actively engaged in sports physical activity, all cause mortality, and longevity of college alumni natural killer cell activity in peripheral blood of highly trained and untrained persons modulation of natural killer cell activity in peripheral blood by physical exercise indomethacin in vitro and in vivo abolishes post-exercise suppression of natural killer cell activity in peripheral blood reserve potentials of immunity ultramarathon running and upper respiratory tract infections the phenomenon of the formation of universal rosette-forming cells under superextreme loads neutrophil phagocytic activity and the humoral factors of general and local immunity under intensive physical loading physical activity and the incidence of coronary heart disease viral illnesses and sports performance jogger's leukocytes physical activity and upper respiratory tract infections in a normal population of young men and women: the amsterdam growth and health study viruses and the athlete exercise stress and murine natural killer cell function exercise and infection aerobic fitness level and reactivity to psychosocial stress: physiological, biochemical, and subjective measures the combined effect of ionizing radiation and physical exercises on some indices of nonspecific protection and immunity exercise, training and neutrophil microbicidal activity effect of exercise on complement activity acute inflammation: the underlying mechanism in delayed onset muscle soreness? immune parameters in athletes before and after strenuous exercise mechanisms of b-iymphocyte suppression induced by acute 365 physical exercise catecholamines increase lymphocyte beta 2-adrenergic receptors via a beta 2-adrenergic, spleen-dependent process division of chronic disease control and community intervention, ms-k47, centers for disease control venue: netanya, israel for further information, please contact: hony tenenbaum international congress wingate institute for physical education and sport wingate post office netanya key: cord-024188-d7tnku8z authors: nissen, michael d.; lambert, stephen b.; whiley, david m.; sloots, theo p. title: respiratory infections date: 2010-03-27 journal: pcr for clinical microbiology doi: 10.1007/978-90-481-9039-3_5 sha: doc_id: 24188 cord_uid: d7tnku8z until recently, conventional culture techniques and immunofluorecence assays were considered the gold standard for the detection of respiratory viruses, even though results are mostly available too late or lacked specificity and sensitivity. these methods are now widely replaced with appropriate dnaand rna-based amplification techniques, in particular real time pcr amplification, for the detection of an extended number of agents responsible for acute respiratory infections. real-time pcr offers rapid results, efficiencies in work flow and a reduced risk of false positive results due to contamination. as a result, better patient management or reduction of unnecessary antibiotic administration will be possible leading to enhanced efficiencies in health care. in applying molecular methods to diagnostic use, the laboratory can optimise its diagnostic strategy by applying a combination of real-time amplification tests for respiratory viruses and the non-viral respiratory bacterial pathogens. however this must be done within a context of resource availability, technical expertise available and clinical utility. it seems certain that molecular microbiology will continue to develop, leading to further applications in diagnostic technology, thereby improving our understanding of disease processes and enhancing our knowledge of the pathogens responsible. they account for about two million deaths each year [22] and rank first among causes of disability-adjusted life-years (dalys) lost in developing countries (94.6 million, 6.3% of total [43] ). the populations most at risk for developing a fatal respiratory disease are the very young, the elderly, and the immunocompromised. while upper respiratory infections (uris) are very frequent but seldom lifethreatening, lower respiratory infections (lris) are responsible for more severe illnesses such as influenza, pneumonia, tuberculosis, and bronchiolitis that are the leading contributors to aris' mortality ( fig. 5.1) . pneumonia, with a global burden of 5,000 childhood deaths every day, is a tangible threat that needs to be dealt with accordingly. the incidence of aris in children aged younger than 5 years is estimated to be 0.29 and 0.05 episodes per child-year in developing and industrialized countries, respectively, which translates into 151 million and 5 million new episodes each year, respectively [28] . most cases occur in india (43 million), china (21 million), pakistan (10 million), bangladesh, indonesia and nigeria (56 million each). pneumonia is responsible for about 21% of all deaths in children aged younger than 5 years, leading to estimates that of every 1,000 children born alive, 12-20 die from pneumonia before their fifth birthday [43] . the main aetiological agents responsible for aris in children include streptococcus pneumoniae, haemophilus influenzae type b (hib), staphylococcus a wide variety of well known and newly identified agents cause respiratory illness and disease in humans. it is not possible to differentiate with certainty the aetiological agent in an infection based on clinical symptoms alone. in pre-school aged children, for example, illnesses due to respiratory viruses have seasonal variations and differences in the presence of fever and other symptoms, the likelihood of household transmission, and the impact they have in terms of medical visits and disruption to family life. but none of these features in isolation or combination is sufficiently specific to link illness to a pathogen with certainty. diagnosis in individual mild illnesses may not alter management, but can prevent unnecessary hospitalisation, antibiotic therapy, or further invasive investigation. laboratory confirmation of the cause of infections has been made more sensitive and rapid through the use of pcr technology. pcr has taught us that the constellation and severity of symptoms can cluster with particular infectious agents. for example, recent findings from the new vaccine surveillance network in the united states show that despite respiratory syncytial virus (rsv), parainfluenza viruses (pivs), and human coronaviruses (hcovs) all being common in early childhood; rsv and pivs are more common causes of hospital admission with acute febrile and respiratory illness than hcovs [33, 41] . despite such clustering, in individual illnesses it can be said that even viruses more typically associated with severe childhood illness can cause milder symptoms, modified by immune or possibly genetic factors, and that severe disease, whilst more commonly caused by a small group of well-studied viruses, can result from infection due to any virus. viruses that are typically considered to cause infrequent or mild disease, such as influenza c virus and piv-4, may cause more significant illness in vulnerable populations, including the young and immunocompromised. influenza, the most studied of respiratory viruses, provides broad insights for other common myxovirus and paramyxovirus respiratory agents. a review of healthy adult human volunteer studies showed that viral shedding increased sharply between 0.5 and 1 day after influenza virus challenge, peaking on day two; shedding can be detected 24 to 28 h before clinical onset, and has a mean duration of 4.8 days; two-thirds of subjects had symptomatic infection, and total symptom scores peaked on day three [11] . the natural history of infection may differ in the elderly and children. for example, pre-symptomatic influenza virus shedding has been seen for 6 days before clinical onset and mean duration of virus isolation from hospitalised children not receiving an antiviral was 6.8 days [29] . respiratory viruses can be transmitted through a number of modes: direct contact and fomites, large droplet, and airborne small particles. the importance of each of these modes depends on the virus in question, the site of infection, and the environment. for example, the eyes and nose appear to be much more important routes of infection for rsv than the mouth. modern molecular methods have resulted in the identification of previously unknown viruses from specimens collected from the respiratory tract. testing for new viruses along with known viruses, including rhinoviruses [6] by pcr, is filling the diagnostic void in respiratory illness and infection, and has improved our understanding of the epidemiology of such illnesses. in all but tropical climates there are a group of respiratory viruses that occur more frequently in the non-summer months, often peaking in winter; these viruses include influenza viruses, rsv, human metapneumovirus (hmpv), pivs, and hcovs ( fig. 5.2 ). human rhinoviruses (hrvs) are the most commonly identified group of viruses in both community-managed and more severe respiratory illness in children and older age-groups, having a year round presence but being more common in the spring and autumn months [6] . whilst it is clear rhinoviruses are a major pathogenic group, there is still uncertainty about the predictive value of a positive molecular test for a picornavirus, particularly from children. in tropical settings, influenza and other respiratory viruses can have a high background year-long presence [38] . respiratory viruses circulate freely in all populations, but moderate to severe illness tends to disproportionately affect certain groups. infections due to common viruses that result in disease severe enough to warrant laboratory testing, notification, or hospitalisation occur in the young, the very old, or both, such as with rsv and influenza [9, 14] . in spite of the inclusion of the live attenuated measles vaccine in the expanded program of immunization (epi), measles virus was still responsible in 2002 for some 213,000 deaths worldwide, essentially due to insufficient vaccine coverage [27] . the situation has fortunately been substantially improved lately, but the leading cause of serious respiratory illness in young children is respiratory syncytial virus (rsv), the agent of infantile bronchiolitis, which is associated with substantial morbidity and mortality [21] . parainfluenza viruses (piv-1, piv-2 and piv-3), especially piv-3, are second in incidence immediately after rsv. all children by the age of 2 years have had at least one episode of piv and/or rsv illness. in addition, both viruses can cause severe disease in the elderly, especially in patients with a chronic respiratory or cardiac condition [18] . although the disease burden due to these pathogens has not been accurately quantified in developing countries, extrapolation from known figures in industrialised countries, such as 125,000 reported cases of rsv per year in the usa, leads to the impressive global estimates of 64 million cases and 160,000 deaths per year from rsv infection worldwide. rsv was identified in 15-40% of pneumonia or bronchiolitis cases admitted to hospital in developing countries, followed by influenza viruses, parainfluenza viruses, human metapneumovirus and adenovirus [40] . the elderly also are at risk for severe rsv disease, and 14,000-60,000 rsv-related hospitalisations of the elderly are reported to occur annually in the usa [14] . human metapneumovirus, a member of the paramyxoviridae, is a recognised cause of a large fraction of severe aris in infant, elderly and immunocompromised populations [15] . other viruses that cause respiratory infections are coronaviruses, adenoviruses and rhinoviruses. recently discovered coronaviruses hcov-hku1 and hcov-nl63 are significant pathogens that contribute to the hospitalisation of children for ari [24, 35] . among other members of the coronaviridae are human coronaviruses hcov-229e and hcov-oc43, agents of the common cold. another recently identified coronavirus is that of the severe acute respiratory syndrome (sars), sars-cov, which emerged in southern china in late 2002 and spread in the spring of 2003 to some 30 countries within asia, europe and north america. in the elderly, influenza-related pneumonia remains a leading cause of infectious disease-related deaths. the threat of an avian influenza pandemic has been looming ever since the emergence in 1997 in hong kong of the h5n1 avian influenza virus, the new h5n1 variant is highly pathogenic for poultry and wild birds and can lethally infect cats and humans. at this time, however, it still is not possible to predict which virus is going to eventually cause a pandemic and when it is going to happen, but the preparation of pandemic influenza vaccines is being actively pursued, generating broad new knowledge on how to improve seasonal influenza vaccine immunogenicity. this has proven to be of the utmost importance with the recent emergence of influenza virus a h1n1 (2009) ("human swine influenza") in mexico and the usa in 2009. regarding influenza virus, the average global burden of inter-pandemic influenza may be on the order of 1 billion cases per year, leading to 300,000-500,000 deaths worldwide. however, the substantial reduction in ari mortality observed in developing countries that have implemented simple case management, including provision of antibiotics to children with ari, suggests that bacterial pneumonia contributes to a large proportion of deaths in these populations. available data suggest that dual infections with viral and bacterial pathogens may be quite common, as seen by the fact that, in the industrialized world, epidemics of rsv and/or influenza coincide with epidemics of s.pneumoniae year after year [32] . while influenza virus is the most commonly met pathogen in this context, other respiratory viruses, including rsv, measles virus, parainfluenza viruses, or adenoviruses may also predispose to secondary bacterial infections. several different bacterial species may be implicated, including h. influenzae, staphylococcus aureus, streptococcus pyogenes, mycoplasma pneumoniae, and, most importantly of all, s. pneumoniae [25] . half or more of the flu-associated mortality in the 1918-1919 spanish flu epidemic is believed to have resulted from pneumococcal super-infections. the same is true for developing countries. as an example, the observation was made in south africa that children vaccinated with the 7-valent conjugate pneumococcal vaccine showed 31% reduction in virus-associated pneumonias requiring hospitalisation, strongly emphasising the presumed importance of dual infections involving s. pneumonia [13] . dual infection seems to increase the severity of the disease and to result in higher mortality. this might be due to inhibition of pulmonary antibacterial defenses during recovery from viral infectiions. streptococcus pneumoniae (pneumococcus) was identified in 30-50% of bacterial pneumonia cases in developing countries in the 1990s, followed by haemophilus influenzae type b (hib; 10-30% of cases), then staphylococcus aureus and klebsiella pneumoniae [28] . non-typable h. influenzae (nthi), and non-typhoid salmonella spp. have also been implicated in some but not all studies. other organisms, such as mycoplasma pneumoniae, chlamydia spp., pseudomonas spp. and escherichia coli also can cause pneumonia. the most common syndromes associated with m. pneumoniae infections are acute bronchitis, pharyngitis and otitis, but 10% of infected children develop pneumonia [39] . the introduction of hib conjugate vaccines has resulted in a truly remarkable decline in hib disease where the vaccine has been introduced. however, the vaccine is not yet routinely made available to a majority of children worldwide. as a result, 400,000 deaths are still estimated to occur from hib disease each year [12] . in view of their safety and remarkable efficacy, the who has recommended the global implementation of the hib conjugate vaccines. s. pneumoniae is estimated to cause more than one-third of the 2 million deaths due to aris, especially in developing countries where the bacterium is one of the most important bacterial pathogens of infancy and early childhood [45] . virtually every child in the world is colonised with one or more strains of pneumococcus and becomes a nasopharyngeal carrier during their first few years of life. many children will go on to develop otitis media, and a few will eventually develop invasive pneumococcal disease including bacteraemic pneumonia and/or meningitis. the introduction of the conjugate pneumococcal vaccine in routine infant immunization should have a major impact on pneumonia in children less than 5 years of age worldwide, as already documented in the usa [26] . tuberculosis (tb) continues to be a leading cause of deaths worldwide, with an estimated one third of humanity infected and about 1.7 million deaths each year, a global toll of 4,650 lives daily. the emergence of mycobacterium tuberculosis (mtb) strains carrying drug-resistance mutations against first-line dugs (mdr-tb) and, more recently, against both first-and second-line drugs (xdr-tb), shows that it will most probably be impossible to contain the tb pandemic with drugs alone. more than one hundred new tb vaccine candidates have been tested in animal models and some have moved into clinical trials. testing such a wide variety of vaccine types using different strategies will obviously require time and a lot of coordination, especially as surrogate markers of protection still remain mostly unknown at this time. finally, it should be emphasized that nosocomial or hospital-acquired pneumonia is a major public health problem: pneumonia is the second most common type of all nosocomial infections, with an associated case fatality rate of 20-50%. infectious fungal respiratory diseases can be divided into those that occur opportunistically in immunosuppressed patients and those that occur in generally healthy individuals. fungi which affect immunosuppressed individuals are frequently pneumocystis jiroveci, and species of aspergillus and candida as well as cryptococcus neoformans [5] , while organisms such as histoplasma capsulatum, coccidioides immitis and blastomyces dermatitidis are frequent pathogens in healthy individuals in certain endemic regions. the causative fungi of respiratory infections vary with the population selected and the geographical region. the majority of fungal infections in lungtransplant recipients involve aspergillus spp., followed by candida, pneumocystis, cryptococcus, geographically restricted agents and newly emerging fungal pathogens. aspergillus infection remains the main fungal complication in lung-transplantation recipients. there are various fungal agents responsible for pulmonary fungal infection in patients with haematological malignancies, but aspergillus spp. and other molds such as zygomycetes or fusarium spp. represent the most frequently isolated microorganisms [19] . less commonly, pneumonia could be due to candida spp., cryptococcus spp. or pneumocystis jirovecii [17, 20] . although invasive aspergillosis is an uncommon complication of haematopoietic stem cell transplants (hsct) and solid organ transplants (sot), it continues to be associated with poor outcomes [5] . invasive pulmonary aspergillosis is rare in patients with chronic obstructive lung disease and is commonly associated with high doses of corticosteroids and multiple broad-spectrum antibiotics [19] . candida infection is predominant in patients with non-haematologic malignant tumours and in non-lung sot recipients. candida albicans and candida parapsilosis were the predominant isolates of pulmonary candidiasis in ventilated preterm infants with a birth weight of less than 1,250 g; the incidence rate of pulmonary candidiasis during the first month of life was 8.6% (20/233 cases) [17] . in intensive care units, c. albicans was also the most frequently isolated fungal species in all sites (68.9%). isolation of fungi allowed a diagnosis of fungal infection in 121 patients (7.7%) [47] . cryptococcus infection occurred in both immunosuppressed and immunocompetent individuals. pulmonary cryptococcosis is usually the primary site of a disseminated or central-nervous-system cryptococcal infection that may be fatal [46] . traditionally, capsule-deficient cryptococcus neoformans was considered to have low virulence. however, a recent study showed that the presentations and outcomes did not differ significantly between patients with proven pulmonary cryptococcosis caused by capsule-deficient cr. neoformans and six patients with pulmonary cryptococcosis caused by capsule-intact cr. neoformans [46] . the predisposing factors for fungal respiratory infections are increasing with the emergence of new immunosuppressive treatment. the increase of fungal associated respiratory tract infections may predominantly be attributed to the development of invasive diagnostic tools and the use of new methods for the identification of isolates, such as molecular techniques. recent advances in molecular biology have greatly improved the detection of viral respiratory pathogens. yet, even with the most sensitive molecular techniques, only 40-60% of infections are consistently diagnosed. this suggests that additional respiratory viruses are likely to exist. in fact, since 2001, seven previously undescribed viruses have been identified by analysis of clinical specimens from the human respiratory tract (table 5 .1). these new viral agents were detected by novel molecular methods such as virus discovery based on cdna-aflp (amplified fragment length polymorphism) (vidisca), pan-viral dna microarrays and high throughput sequencing [4] . more random pcr [2, 16] broadly, the advent of these new technologies has greatly stimulated efforts to identify novel viruses in the respiratory tract and in other human disease states. of the viruses discovered over the last 7 years, human metapneumovirus (hmpv) and the newly emerging human coronaviruses (hcov) are considered causative agents of respiratory disease. however, to date, sars coronavirus has been restricted geographically and has only been associated with limited and sporadic outbreaks. recently, human bocavirus (hbov) and the new human polyomaviruses kiv, wuv and merkel cell polyomavirus (mcv) were detected in respiratory secretions, and although an association with the respiratory tract has been postulated, it still remains to be proven [2, 8, 16 ]. hmpv infection is associated with a broad spectrum of clinical signs in patients of all age groups, and is the cause of upper and lower respiratory tract infection in infants and young children [30] . it is second only to rsv as a significant cause of bronchiolitis in early childhood and children are most likely to be hospitalised with severe disease. studies have linked hmpv with acute otitis media and asthma exacerbations in children and with exacerbations of both asthma and chronic obstructive pulmonary disease (copd) in adults. however, severe disease may occur in all patients with underlying medical conditions such as cardiopulmonary disease, the elderly and immunocompromised subjects. in these subjects the virus can cause prolonged and serious infections, particularly severe lung disease including fatality [10] (fig. 5.3) . the first reports of sars coronavirus infection were published in 2003, and the causative agent was subsequently characterised as a novel human coronavirus [23] . the sars epidemic was halted by a highly effective global public health response coordinated by the world health organization, and there is no further evidence that sars cov is currently circulating in humans. however, the sars outbreak focused renewed attention on coronaviruses generally, resulting in the discovery of two more new human coronaviruses, nl63 and hku1. hcov-nl63 was first detected in 2004 in a child from the netherlands with bronchiolitis, shown to be a cause of severe lower respiratory tract infection (lrti) in young children, and an agent of laryngotracheitis (croup) [37] . hcov-hku1 was detected in 2005 in an adult with chronic pulmonary disease in hong kong [44] and was subsequently shown to be globally distributed. hku1 infection presents with common respiratory symptoms as well as a more severe clinical presentation including bronchiolitis and pneumonia [24] . in healthy adults hcov nl63 and hku1 infections are generally not life threatening. this suggests that these coronaviruses, like 229e and oc43, only cause more-severe disease in young children, elderly persons, and the immunocompromised. they may be detected in 1 to 10% of patients with acute respiratory tract infections, and co-detection of these viruses with other respiratory viruses is common [37] . human bocavirus was first described in 2005 with a prevalence of 3.1% in swedish children with lrti [1] and subsequently in australia with 5.2% prevalence in children with arti during winter [31] . although a positive association of hbov with arti was suggested, the results remain inconclusive, because a high prevalence of other respiratory viruses was found in hbov-positive patients. to confirm the role of hbov as a respiratory pathogen, more extensive studies including matched control populations need to be performed. one such study using control subjects [3] proposed hbov as a cause of acute wheezing in children. hbov has been frequently detected in immunosuppressed adults but only rarely in immunocompetent adult subjects. however, it is uncertain if the presence of hbov in these subjects is the result of re-infection, viral persistence or reactivation. recently, three new human polyomaviruses, kiv, wuv and mcv were detected in specimens of patients with arti [2, 8, 16] . allander et al. [2] reported a prevalence of 1% for kiv in nasopharyngeal aspirates (npa) collected from a swedish population and gaynor et al. [16] showed a prevalence of 3% and 0.6% for wuv in respiratory samples from australia and the usa respectively. more recently, although originally found in merkel cell carcinoma, mcv was also found in 5.9% of npa collected from australian subjects with arti [8] . since these first reports, kiv, wuv and mcv have been detected in a number of geographic locations, suggesting a global presence for these viruses. one striking feature of early findings concerning kiv and wuv is their high rate of co-detection with other respiratory viruses. a co-detection rate of 74% has been observed for kiv and rates ranging from 68% to 79% for wuv [7] . so, even though an aetiological role in childhood respiratory disease has been proposed, it is difficult to assess a pathogenic role for these viruses unless observations are compared with those for matched control populations. further studies will need to be completed before the role of kiv and wuv as respiratory pathogens can be confirmed, and it remains possible that these viruses are not involved in respiratory disease, but that their presence in the respiratory tract simply reflects their mode of transmission. laboratory diagnosis of respiratory virus infections requires specimens containing cells from the respiratory tract collected early in the clinical illness. the most appropriate specimens are npas and bronchoalveolar lavage (bal). where the generation of aerosols may pose an infection risk to collection personnel, nose and throat swabs are a viable alternative. recent studies however, have shown that swabs are not as effective as npas for the detection of adenovirus and respiratory syncytial virus. limited data is available suggesting that flocked swabs are superior to traditional swabs for detecting respiratory virus infections. collection swabs should be dry and not contain bacterial transport medium as these often contain substances that are inhibitory to pcr reactions. in the case of influenza virus detection, the cdc recommends that viral transport medium (vtm) is added to swabs to assist in virus preservation. if vtm is to be added to dry swabs, they should be vortexed to release cells in to the medium and transferred to a sterile vial for transportation to the laboratory. vtm cannot be added to swab receptacles as they will invariably leak in transit and pose an infection risk. all respiratory specimen types should be transported to the laboratory at 4 â�¢ c. some npas and sputum samples received in the laboratory are very mucoid. these may either be diluted in vtm or digested with sputasol to facilitate the extraction process prior to molecular analysis. it is preferable to use an extraction method that will extract both rna and dna with equal efficiency as additional testing for agents such as bordetella pertussis and mycoplasma pneumoniae are often requested if a viral cause cannot be found. lung tissues are often collected at autopsy for either culture or molecular viral studies. the specimen requirements for both molecular and culture based respiratory virus detection methods are similar and therefore laboratories can choose the detection method that suits their role. all respiratory specimens must be handled with appropriate personal protection equipment as specified for pc2 laboratories and opened and aliquotted only in a class ii biological safety cabinet. specimens should be stored at 4 â�¢ c until they are processed. c. bletchly (b) molecular diagnostic unit, microbiology division, pathology queensland central laboratory, brisbane, qld 4029, australia e-mail: cheryl_bletchly@health.qld.gov.au traditional laboratory respiratory virus diagnosis involved virus culture with, or without immunofluorescence staining with specific antibodies. culture is highly sensitive if the appropriate cell lines are utilised and once a virus is isolated it can be further characterised and amplified if required. viral culture provides the added advantage that it will only detect infectious virus and not persistent nucleic acid. it has the disadvantages however of being expensive due to the requirement to maintain cells lines and tissue culture media and the requirement for expertise in sterile technique and interpretation of cytopathic effect (cpe) and fluorescence staining. direct immunofluorescence assay (dfa) of respiratory tract cells provides rapid results but does involve a series of manual manipulations and washes along with interpretation by highly skilled personnel (fig. 5.4) . the laboratory's primary role will most often dictate their method of choice for respiratory virus detection. diagnostic laboratories require rapid result turn-around and will most likely opt for molecular detection which can be readily adapted to high throughput batching and multiplexing. most molecular assays can be reported within 24 h of sample receipt whereas culture methods will take from 1 to 14 days and often much longer for confirmation. direct antigen tests are available commercially for some respiratory viruses but lack sensitivity (approximately 70%) although the positive predictive value is high. the expense and false negative rate of direct antigen tests need to be balanced with their rapidity and ease of use. negative direct antigen results should be confirmed by a more sensitive assay such as pcr. public health laboratories will most likely employ viral culture to enable viral isolate characterisation and the ability to detect unsuspected viruses. despite the popularity of molecular detection methods for respiratory viruses very few well validated commercial assays are available for the wide range of respiratory pathogens that are of clinical significance. most laboratories utilise "in-house" methods in combination with a rigorous quality assurance programmes. cell culture and direct immunofluorescent assay (dfa) staining using monoclonal antibodies were previously the most commonly used laboratory techniques for detecting respiratory viruses. although still used widely in the united states, these traditional techniques have gradually been superseded by highly sensitive and rapid reverse transcriptase polymerase chain reaction (rt-pcr) assays, with most laboratories in australia now using rt-pcr methods. additional advantages of rt-pcr detection of respiratory viruses include results that are not significantly affected by a loss of viral viability during specimen transport or storage, and that rt-pcr does not require the presence of intact, infected cells within the specimen. the pcr revolution has also been further stimulated through improvements in the technology, including the advent of real-time pcr and multiplex pcr methods, both of which reduce staff hands-on time, decrease result turnaround-times, increase through-put and are more user friendly compared to conventional pcr techniques. standard quality control practices should be implemented when testing for respiratory viruses by pcr, including the use of a suitable positive control, negative control, extraction control and inhibition control. sequence-related issues (discussed in more detail in chapter 8) are also very relevant to respiratory viruses. consideration needs to be given to the type of probe used when designing real-time pcr methods for respiratory viruses. the main issue is that respiratory viruses, particularly the rna viruses, show considerable genetic variation. for this reason, it is often difficult to identify a sufficiently large and conserved region to accommodate two hybridisation probes and so the single-probe taqman format is more commonly utilised for respiratory virus detection. on the other hand, we have also observed problems using the smaller minor-groove binder (mgb) taqman probes. the issue stems from mgb taqman probes being more susceptible to single nucleotide polymorphisms than standard taqman probes. for example, we have found that some rsv strains provided poor fluorescent signal, barely above the background negative signal, in an rsv mgb assay [42] . cloning of a human parvovirus by molecular screening of respiratory tract samples identification of a third human polyomavirus human bocavirus and acute wheezing in children virus discovery by sequence-independent genome amplification fungal infections in primary immunodeficiencies frequent detection of human rhinoviruses, paramyxoviruses, coronaviruses, and bocavirus during acute respiratory tract infections presence of the newly discovered human polyomaviruses ki and wu in australian patients with acute respiratory tract infection merkel cell polyomavirus in respiratory specimens: a possible route of transmission? vaccine preventable diseases and vaccination coverage in australia human metapneumovirus in a haematopoietic stem cell transplant recipient with fatal lower respiratory tract disease time lines of infection and disease in human influenza: a review of volunteer challenge studies haemophilus influenzae vaccines efficacy of nine-valent pneumococcal conjugate vaccine against pneumonia and invasive pneumococcal disease in the gambia: randomised, double-blind, placebo-controlled trial respiratory syncytial virus infection in elderly and high-risk adults human metapneumovirus infection in young children hospitalized with respiratory tract disease identification of a novel polyomavirus from patients with acute respiratory tract infections congenital candida pneumonia and sepsis: a case report and review of the literature respiratory syncytial virus pneumonia among the elderly: an assessment of disease burden invasion of the alveolar-capillary barrier by aspergillus spp.: therapeutic and diagnostic implications for immunocompromised patients with invasive pulmonary aspergillosis cryptococcosis: an emerging respiratory mycosis respiratory synsytial virus and parainfluenza virus vaccines human vaccine research and development: an over-view a novel coronavirus associated with severe acute respiratory syndrome coronavirus hku1 and other coronavirus infections in hong kong insights into the interaction between influenza virus and pneumococcus impact of pediatric vaccination with pneumococcal conjugate vaccine on the risk of bacteremic pneumococcal pneumonia in adults epidemiology and etiology of childhood pneumonia viral shedding in children with influenza virus infections treated with neuraminidase inhibitors evidence of human coronavirus hku1 and human bocavirus in australian children seasonality of invasive pneumococcal disease: temporal relation to documented influenza and respiratory syncytial viral circulation coronavirus infection and hospitalizations for acute respiratory illness in young children a newly discovered human pneumovirus isolated from young children with respiratory tract disease human coronavirus nl63, a new respiratory virus human coronavirus nl63 infection is associated with croup influenza in tropical regions community outbreak of mycoplasma pneumoniae infection: school-based cluster of neurologic disease associated with household transmission of respiratory illness respiratory syncytial virus infection in tropical and developing countries parainfluenza virus infection of young children: estimates of the population-based burden of hospitalization sequence variation can affect the performance of minor groove binder taqman probes in viral diagnostic assays estimates of world-wide distribution of child deaths from acute respiratory infections characterization and complete genome sequence of a novel coronavirus, coronavirus hku1, from patients with pneumonia make any mother and child count pulmonary cryptococcosis in non-aids patients fungal infections in the icu key: cord-265257-p9f0pl3y authors: masoud, khaldoun; hanna-wakim, rima; zaraket, hassan; kharroubi, samer; araj, george f; matar, ghassan m; dbaibo, ghassan title: viral etiology of acute respiratory infections in pediatric patients in lebanon date: 2019-11-01 journal: mediterr j hematol infect dis doi: 10.4084/mjhid.2019.059 sha: doc_id: 265257 cord_uid: p9f0pl3y background: acute respiratory infections (ari) are the leading cause of death worldwide, especially among children. the majority of these infections in children are of viral etiology. in this study, we evaluated the incidence of viral ari among children in lebanon. patients and methods: children presenting with symptoms of ari were prospectively recruited between september 2009 to february 2012. nasopharyngeal aspirates were obtained from patients and screened for 11 respiratory viruses using a multiplex luminex-based pcr assay. results: two hundred twenty-one patients were recruited with a median age of 1 year (iqr: 0 – 5). out of 221 patients, 116 (52.5%) were positive for at least one virus, the majority (103/116; 88.8%) of which were in children under 6-year of age. overall, 188 viruses were detected. rhinovirus (rhv) was the most common virus detected in 81 (69.8%) patients followed by coxsackie virus and echovirus (cvev) which were detected as one target in the panel in 45 (38.8%), and parainfluenza viruses (piv types: 1, 2, 3, 4) in 24 (20.7%) patients. coinfection with more than one virus was detected in 49 (42.9%) patients. rhv and cvev were the most common viruses associated with co-infections and higher risk of rhinorrhea. conclusions: viral pathogens account for at least half of the aris in lebanon, with a high frequency of co-infections being detected. introduction. acute respiratory tract infections (aris) are among the most common reasons for primary care consultations. 1 the world health organization (who) ranks aris as the fourth major killer after cardiovascular diseases, general infections and parasitic diseases, and cancer. 2 aris cause 4 million death globally. the burden is especially high in children where aris are responsible for 11-22% of deaths. 3 aris can lead to severe complications requiring hospitalizations and can have fatal outcomes. viruses are the most common etiology of aris in children. 6, 7 these include rhinovirus (rhv), respiratory www.mjhid.org mediterr j hematol infect dis 2019; 11; e2019059 pag. 2 / 7 syncytial virus (rsv), influenza (ifn), parainfluenza virus (piv), coronavirus (cov), human metapneumovirus (hmpv), enteroviruses (ev), adenovirus (adv), and human bocavirus (hbov). 6, 8, 9 each of these viruses poses a significant health burden. nair et al. estimated that 111 500 deaths in children <5 years were attributable to influenza-associated lower respiratory tract infections (lri) in 2008, the vast majority of which occurred in developing countries. 10 rsv was estimated to have caused 33.8 million lri episode in children under five, of which 3.4 million were severe causing up to 199 000 deaths. 11 in addition to the health burden of viral respiratory tract infections (rtis), the economic impact is also high if we account for health care costs (direct cost) and loss of productivity (indirect cost). a study by fendrik et al. estimated the total economic impact of non-influenzarelated viral rtis in the united states at $40 billion annually. 12 the advancements that have been achieved in developing antiviral drugs, some of which have already been approved, against respiratory viruses allow for targeted therapy of viral aris. [13] [14] [15] this possibility calls for better and faster diagnosis of the etiologic agents in ari patients to benefit from the full potential of these drugs. 6 furthermore, aris are associated with the greatest amount of excess use of antibiotics that has led to unprecedented increase in antimicrobial drug resistance; 16, 17 therefore, proper and timely diagnosis of viral infections can help reduce unnecessary antibiotic prescriptions. 5, 6 in lebanon, studies investigating the viral etiologies of aris are very scarce. in this study, we determined the viral etiologies among ari patients at a tertiary care hospital that serves an ethnically and socioeconomically diverse patient population. patients and samples collection. infants and children younger than 18 years of age with symptoms of ari disease presenting to the emergency department or the departments of pediatrics of the american university of beirut medical center (aubmc), beirut, lebanon were prospectively recruited between september 2009 to february 2012. an ari was defined as an acute infection of the upper and lower respiratory airways. recruited patients had one or more of the following symptoms: fever, cough, sore throat, rhinorrhea, headache, conjunctivitis, wheezing, dyspnea, and vomiting. medical history and demographic data were obtained from the patients and their medical records. a respiratory sample was collected and stored at -80°c for viral assessment. the study was approved by the institutional review board (irb) of the aubmc, and written informed consent was obtained from all parents. nucleic acid extraction and viral detection. nucleic acid was extracted from clinical specimens by using the qiaamp minelute virus spin kit (qiagen) according to the manufacturer's protocol. a 200 µl aliquot of each specimen was used for nucleic extraction. specimens were then analyzed by the resplex ii panel (qiagen) using the manufacturer's protocol. the resplex ii panel can detect 11 viral targets: rsva, rsvb, infa, infb, piv1, piv2, piv3, piv4, hmpv, cvev (coxsackievirus and echovirus), and rhv. briefly, 10 µl of each specimen were added to 40 µl reverse transcription-pcr (resplex ii) master mix, including the supplied primers. targets were detected by mixing 5 µl portions of amplification products with resplex ii bead in hybridization buffer at 52°c for10 min. streptavidin-phycoerythrin conjugate was added, and mixtures were incubated at 52°c for a further 5 min before the addition of stop buffer. the samples were then analyzed on a luminex bio-rad bioplex 200 system (bio-rad laboratories) using bio-rad bioplex manager software. the cutoff value for each target was determined, as previously described by lia et al. 19 statistical analysis. the data were checked for completeness, and responses were coded and entered into the statistical package for the social sciences (spss) software version 23 for windows, which was later used for statistical analyses. 32 descriptive statistics were presented to summarize the study variables of interest as counts and percentages for the categorical variables and as medians and interquartile range (iqr) for the continuous ones. the chi-square test was used to calculate the association between two categorical variables. pearson's chi-square analysis with bonferroni-holm p-value correction was used for multiple comparisons to assess infectivity enhancing correlations. univariate and multivariate logistic regression analyses were applied to determine which factors are associated with rhinorrhea. in the regression model, rhinorrhea was used as the dependent variable. odds ratios and their respective 95% confidence intervals were calculated. for all analysis done, a pvalue of less than 0.05 was considered statistically significant. patient characteristics. a total of 221 specimens were collected from children presenting with symptoms of ari between september 2009 and february 2012 ( table 1) . the socio-demographic characteristics of the study patients are presented in table 1 . overall, the study consisted of 130 males (58.8%) and 91 females (41.2%) with patients' median age of 1 (iqr: 0 -5) years. seventy-four patients (33.5%) were children under one year of age, 105 (47.5%) were between 1 to 6 years old, 33 (14.9%) were 6 to 12 years old, and 9 (4.1%) were 12 to 18 years old. sixty-seven (30.3%) of the children had an underlying disease (asthma, immune-deficiency, allergic rhinitis, or cystic fibrosis). at the time of diagnosis 13 (5.8%) patients were receiving chemotherapy, and 95 (43%) had received an antibiotic. virological characterization. samples were screened for 11 virus targets included in the resplexii respiratory panel. of the 221 ari episodes, 116 (52.5%) were confirmed to be of viral etiology being positive for at least one of the virus targets tests ( table 2 ). the majority (n=103; 88%) of viral ari episodes were observed in children under 6-year of age (chisquare, p<0.05). figure 1 shows the frequency of each of the viruses detected in the study population. overall 188 viruses were detected. rhinovirus (rhv) was the most common virus detected in 81 (69.8%) patients followed cystic fibrosis 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 2) . a significant majority (n=46; 93.9%; chi-square p-value<0.05) of coinfections occurred in children under six years of age. the most frequent viral co-infections involved two viruses (n=33), 10 cases had a triple infection, 5 had four viruses detected, and one case had five viruses. almost half cases of rhinovirus (50.6%) were positive for at least another virus in the panel. cvev positive cases also had a high rate of coinfection (73%). moreover, hmpv and infb were detected in 9 samples, and all were co-infected. table 3 summarizes the correlation of different viruses among our patients. several correlations enhancing infectivity were evident in our analysis. of note, rhv was the most frequently detected virus in co-infections and was significantly associated with rsvb, infb, piv3, hmpv, and cvev. underlying conditions and clinical presentation. we next analyzed the correlation between each of the viral etiologies or co-infection with the underlying conditions ( table 2) . to simplify the analysis, we treated subtypes or genotypes of a virus as a single group (e.g. rsv for rsva and rsvb, etc.). having an underlying condition or receiving chemotherapy or a course of antibiotics were not found to be a risk factor for having a viral etiology or co-infection ( table 2) . additionally, we investigated the seasonal variation of viruses. rhinovirus infections were detected throughout the year however the peak rate occurred during the main rainy months (november, december), likely for coxsackie/echovirus and rsv. on the other hand,influenza a virus infections had a peak in the fall (september, october); (figure 3 ). in terms of clinical symptoms, fever, cough and rhinorrhea were major symptoms observed in most of the patients infected with one or more respiratory virus ( table 2) . chisquare analysis revealed a significant correlation between rhinorrhea and inf, cvev, and rhv and coinfection ( table 2) . bivariate logistic regression was then performed to determine the risk associated with these infections. our analysis revealed that rhv or a multivariate logistic regression model was used to examine the correlates of rhinorrhea in the study patients. variables were put in the model in order of strength of their correlation with rhinorrhea as per the bivariate analysis. the effect of each variable on the model was assessed, and the variable was kept if it significantly contributed to a better fit of the model. the final model included the following variables: rhv and cvev. the results of the multivariate model showed that cvev was independently associated with rhinorrhea (or: 4.73; ci: 1.59 -14.07). cvev infected patients were 4.73 times more likely to have rhinorrhea compared to none-cvev patients controlling for rhv. unlike the bivariate analysis, rhv was not significantly associated with rhinorrhea (or: 1.78; ci: 0.91 -3.48). rhv infected patients were 1.78 times more likely to have rhinorrhea compared to none-rhv patients controlling for cvev; however, this was not statistically significant. discussion. we demonstrated that viral infections are responsible for at least half of the aris in children in lebanon. rhinovirus infection was the most common etiology of ari consistent with other studies from lebanon and other countries. 18, 20, 21 in neighboring jordan and egypt rhinovirus incidence was second to rsv, but the population captured in these studies was younger than that included in our study. 22, 23 the overall viral ari incidence (52.5%) in our study lower than that recently reported by finianos et al. (70%) in lebanon. 18 both studies targeted children; however, finianos et al. screened their specimens for more viral targets than those included in our analysis. in our study we did not test for hcov, adv, ev, and hbov which collectively accounted for 50% of viral ari in the study by finianos et al. the coinfection rate in our study (42.9%) was higher than that previously reported in lebanon (37%), qatar (21.4%), and egypt (10.8%). 18, 23, 24 this incongruence could be because cvev, which was frequently detected with other viruses in our study, was not screened in the previous studies from the region. 18 cvev infections are not commonly reported in studies investigating respiratory infections. in our study, cvev infection constituted 38.8% of all viral ari cases and was independently associated with rhinorrhea. this incidence is much higher than that reported in other countries. a recent study in latin america reported that cvev was associated with 3% of the ari cases. 25 in central america, cvev was even much lower (0.3%). 26 the very low prevalence of cvev in other regions might have discouraged its testing. given the high prevalence of cvev in lebanon, we recommend testing for enteroviruses, including (cvev). co-infections were found to be more common younger children in lebanon, and that is similar to a previous study done in mexican children showing that the majority of coinfections occur in children <6 months of age. 27 younger children are likely to be more prone to infections due to their lack or still weak immunity to respiratory viruses. the effect of coinfections on disease outcomes is not well understood. 28 patients coinfected with pandemic h1n1 influenza and rhinovirus tended to have milder clinical severity when compared with non-rhinovirus coinfections; 29 while the patients coinfected with hmpv and rsv were prone to a higher risk of severe bronchiolitis. 30 additionally, the prevalence and severity of obstructive airway disease were higher in patients with coinfections. 31 in our study, coinfection was associated with higher risk of rhinorrhea but not with more severe symptoms like dyspnea. in contrast, some studies showed no correlation between coinfection status and clinical severity. 32, 33 the complexity of viral coinfections and the large number of respiratory viruses involved make challenging to study the effect of coinfection on disease outcome in a clinical setting. therefore, there is a need for developing in vitro or in vivo models to allow a better understanding of coinfections. for example, dual infection with inf was shown to suppress rsv growth in vitro. 34 the suppression of rsv by inf was suggested to be due to competition for protein synthesis and budding from the cell surface. further studies are warranted to investigate the interactions among respiratory viruses during coinfection and their effect on the host. our study had a couple of limitations. first, we have not screened for hbov, and hcov which are not included in resplex ii kit and thus the prevalence of viral ari is expected to be higher than 52%. another limitation was our inability to rule out bacterial etiologies which was not tested for in the current study. in conclusion, viral etiologies contribute to a large proportion of aris many of which involve more than one viral agent. the burden of acute respiratory infections in crisis-affected populations: a systematic review who | the global burden of disease estimates of world-wide distribution of child deaths from acute respiratory infections respiratory viruses other than influenza virus: impact and therapeutic advances detection of respiratory viruses by molecular methods epidemiology and prevention of pediatric viral respiratory infections in health-care institutions identification of new respiratory viruses in the new millennium global burden of respiratory infections due to seasonal influenza in young children: a systematic review and meta-analysis global burden of acute lower respiratory infections due to respiratory syncytial virus in young children: a systematic review and meta-analysis. the lancet the economic burden of non-influenza-related viral respiratory tract infection in the united states recent advances in diagnosis, prevention, and treatment of human respiratory syncytial virus developing new antiviral agents for influenza treatment: what does the future hold? advances in antivirals for non-influenza respiratory virus infections. influenza other respir viruses use of antibiotics for adult upper respiratory infections in outpatient settings: a national ambulatory network study excessive antibiotic use for acute respiratory infections in the united states viral infections of the lower respiratory tract: old viruses, new viruses, and the role of diagnosis simultaneous detection and high-throughput identification of a panel of rna viruses causing respiratory tract infections etiology, seasonality and clinical characterization of viral respiratory infections among hospitalized children in beirut, lebanon viral and atypical bacterial aetiologies of infection in hospitalised patients admitted with clinical suspicion of influenza in thailand, vietnam and indonesia. influenza other respir viruses viral aetiology of acute respiratory infections with cough in infancy: a community-based birth cohort study human metapneumovirus in hospitalized children in viral etiologies of lower respiratory tract infections among egyptian children under five years of age the role of human metapneumovirus in pediatric respiratory tract infection in qatar human rhinoviruses and enteroviruses in influenza-like illness in latin america influenza and other respiratory viruses in three central american countries. influenza other respir viruses viral coinfection in acute respiratory infection in mexican children treated by the emergency service: a cross-sectional study respiratory viral infections in infants: causes, clinical symptoms, virology, and immunology rate and influence of respiratory virus co-infection on pandemic (h1n1) influenza disease dual infection of infants by human metapneumovirus and human respiratory syncytial virus is strongly associated with severe bronchiolitis single versus dual respiratory virus infections in hospitalized infants: impact on clinical course of disease and interferon-gamma response human metapneumovirus infections cause similar symptoms and clinical severity as respiratory syncytial virus infections viral and atypical bacterial detection in acute respiratory infection in children under five years in vitro growth profiles of respiratory syncytial virus in the presence of influenza virus key: cord-265354-ajfjrnr9 authors: vu, d.‐l.; bridevaux, p.‐o.; aubert, j.‐d.; soccal, p. m.; kaiser, l. title: respiratory viruses in lung transplant recipients: a critical review and pooled analysis of clinical studies date: 2011-04-26 journal: am j transplant doi: 10.1111/j.1600-6143.2011.03490.x sha: doc_id: 265354 cord_uid: ajfjrnr9 lung transplant recipients present an increased risk for severe complications associated with respiratory infections. we conducted a review of the literature examining the clinical relationship between viral respiratory infection and graft complications. thirty‐four studies describing the clinical impact of influenza, respiratory syncytial virus, parainfluenza, human metapneumovirus, rhinovirus, enterovirus, coronavirus, bocavirus or adenovirus were identified. the detection rate of respiratory viral infection ranged from 1.4% to 60%. viruses were detected five times more frequently when respiratory symptoms were present [odds ratio (or) = 4.97; 95% ci = 2.11–11.68]. based on available observations, we could not observe an association between respiratory viral infection and acute rejection (or = 1.35; 95% ci = 0.41–4.43). we found a pooled incidence of 18% (9/50) of bronchiolitis obliterans syndrome (bos) in virus‐positive cases compared to 11.6% (37/319) in virus‐negative cases; however, limited number of bos events did not allow to confirm the association. our review confirms a causal relationship between respiratory viruses and respiratory symptoms, but cannot confirm a link between respiratory viruses and acute lung rejection. this is related in part to the heterogeneity and limitations of available studies. the link with bos needs also to be reassessed in appropriate prospective studies. respiratory viruses comprise different viruses, such as influenza, respiratory synctial virus (rsv), parainfluenza (piv), human metapneumovirus (hmpv), rhinovirus, enterovirus, bocavirus and adenovirus. although most respiratory viral infections cause self-limited upper respiratory diseases, lung transplant recipients (ltrs) are particularly prone to develop complications (1) (2) (3) (4) (5) (6) . this is related to the immunosuppressive therapy that could promote protracted infection, but also to the direct exposure of the graft to the infectious agent together with an impaired mucociliary function and lymphatic drainage, and the absence of cough reflex. apart from the direct infection-related morbidity, it is commonly accepted that these infections could promote rejection and subsequently lead to bronchiolitis obliterans syndrome (bos), the main limitation to long-term survival. however, this association is based on reports that have focused mainly on paramyxoviruses or on influenza and adenovirus to a lesser extent (7) . these studies are heterogeneous and have several technical limitations in terms of design, case selection and diagnostic procedures (1, 8) . over the years the improvement of molecular tools, including real-time pcr technology, has contributed to increase the sensitivity of our diagnostic procedures and new species (hmpv, coronavirus nl63 and hku1, bocavirus, rhinovirus c) have also emerged. to assess appropriately the evidence supporting a role of respiratory viruses as a cause of symptoms and graft complications in ltrs, we conducted a systematic review of the published literature (9) . we searched the medline database from 1 january 1985 to 31 march 2010 using the following key words: 'lung transplant recipients or immunocompromised hosts' and 'influenza, parainfluenza, rsv, metapneumovirus, coronavirus, bocavirus, adenovirus and respiratory viruses', respectively. in addition, reference lists from review articles and selected papers were hand-searched and matched to our database. only peer-reviewed original articles reporting at least three lung transplant cases with a description of virological methods, design and clinical end-points were included. data were collected in standardized report forms with the following information: year of the screening period; design (cohort, case series, retrospective, prospective); age and size of the population screened; number and type of specimens/viruses tested; number of virus-positive episodes analyzed; type of assays used; clinical conditions; association with acute rejection/chronic rejection/bos and histopathological results; antiviral treatment and survival rate. the potential limitations and any other comments considered as relevant were noted. we calculated confidence intervals (ci) around proportions for studies on viral frequency using the agresti and coull method. odds ratios (or) were calculated for each study to determine the association of respiratory viruses with acute rejection or respiratory symptoms. due to significant heterogeneity between studies, we used random effect models to calculate meta-analytic summaries of the association between respiratory viruses and acute rejection or respiratory symptoms. all analyses were performed with stata 11 (77845; college station, tx, usa). we identified 34 studies; 26 focused on ltrs only and eight analyzed also other immunocompromised populations. viruses considered in our review cover influenza a, b and c, rsv a and b, piv 1-4, hmpv, rhinovirus, enterovirus, coronavirus 229e, oc43, nl63 and hku1, bocavirus and adenovirus, but not herpes viruses. the main characteristics of the 34 studies (1-34) are presented in table 1 and it can be estimated that more than 4000 specimens from ltrs have been screened for the presence of at least one of the above-mentioned respiratory viruses. in approximately one-third of studies (29%), screening was within the frame of prospective cohort studies investigating the cause and/or the clinical impact of acute viral respiratory tract infections; all others were retrospective or case series. in 21 (61.7%) studies, patients were recruited from outpatient clinics; in six (17.6%), patients were hospitalized; one recruited both in-and outpatients and the recruitment setting could not be determined precisely for the remaining six (2, 11, 12, 20, 24, 34) . diagnostic procedures were performed only in symptomatic patients in 12 (35.2%) studies, and in 20 (58.8%) they were also performed as routine posttransplant surveillance or as a control procedure after treatment of an acute rejection. reasons for the procedure were not identifiable in two retrospective studies (2, 27 ). an 8.8% of studies concerned children only, 17.6% enrolled both children and adults and 73.5% were in adults. clinical conditions analyzed ranged from uncomplicated upper respiratory tract infection to severe pneumonia requiring intensive care unit (icu) admission. overall, it was possible to identify that viral investigations were performed in upper respiratory specimens (nasopharyngeal swabs or aspirates) in 8.8% of studies and in bronchoalveolar lavage (bal) specimens in 38.2%. both types of specimens were used in the remaining 52.9% studies, but in 61.1% of these it was not possible to clearly establish the respective proportion of upper versus lower respiratory specimens. we identified only one study that compared systematically upper versus lower respiratory tract viral screening performed simultaneously in a given individual (28) . thus, we were unable to compare the respective sensitivity and role of viral screening in the upper versus the lower respiratory tract. as expected, there was a significant heterogeneity of the different diagnostic procedures used. at least one molecular assay was used in 53% of investigations and only five studies used a large panel to target at least 12 of the above-mentioned 18 viruses. in the older studies, classical methods, such as immunofluorescence or viral culture, were the sole diagnostic tools. the type of technique used (immunofluorescence-based, culture or nucleic acid detection) and the completeness of the screening performed in each of the available studies selected are shown in table 1 . in terms of viral screening, influenza was screened in most studies (91% and 88% for influenza a and b, respectively, but only 3% for influenza c), followed by rsv (85%), piv 1-3 (82-85%, but only 15% for piv 4) and adenovirus (71%). other respiratory viruses that require mainly molecular assays to be detected were less frequently screened; 41% for rhinovirus (rhinovirus c screened in only one study); 35% for hmpv; 11-24% for the different subtypes of coronaviruses; 18% for enterovirus and 12% for bocavirus. overall, only 15% of studies screened at least 75% of the 18 respiratory viruses listed in table 1 . when assessable, the overall detection rate of respiratory viral infection in the screened population varied from 1.4% to 60%. this wide range can be explained in part by the heterogeneity of the population enrolled (asymptomatic cases versus subjects with limited upper respiratory symptoms versus patients hospitalized with complications). table s1 depicts the prevalence of virus positivity for each individual study and as a pooled prevalence according to diagnostic method, number of viruses screened and sample size. as expected, the virus positivity rate was higher for studies with small sample size using pcr technique and screening for numerous viruses. for example, studies using pcr techniques had a higher detection rate (12.0%) compared to those not using pcr (1.4%). this can be explained in part by the greater number of viruses searched for by pcr techniques (8.4% for studies identifying nine or more viruses; 3.0% for those identifying eight or less viruses). the respective contribution of each species in positive cases was available in 82% of studies. when a large panel of molecular tools was used, viruses most frequently detected were rhinovirus and coronavirus. in the three studies continued that screened at least 14 respiratory viruses (22, 28, 34) , rhinovirus represented 35-55% of all positive cases and coronaviruses 13-27%. when including three supplementary studies screening up to 12 or 13 viruses (3, 24, 29) , the most frequent virus detected was still rhinovirus (8.8-55.5%; table s1 ). of note, for some targets, such as coronaviruses, not all species (oc43, e229, nl63, hku1) were included. ten of 34 studies compared the rate of viral infections observed in symptomatic cases versus those without respiratory symptoms. we found that smaller studies tended to include more symptomatic patients and that larger studies were associated with a lower virus detection rate (22.3% for studies with less than 150 specimens versus 0.6% for studies with 150 specimens or more; table s1 ). figure 1a highlights that in all but one study, the association between laboratory-proven respiratory viruses and symptoms was present. we found that viruses were detected five times more frequently when respiratory symptoms were present (or = 4.97; 95% ci = 2.11-11.68). in terms of objective assessment of the graft function during the acute phase, lung function assessment was available in 53% of studies and showed a forced expiratory volume (fev 1 ) decline that ranged from -5% to -30% for the overall enrolled population. the fev 1 decline was usually similar or even more important among symptomatic patients, but very few studies provided a specific comparison of fev 1 variability according to the presence or absence of a viral illness (28) , which prevented further analysis. short-term crude mortality rate was evaluated in 52.9% of studies and ranged from 0% to 25%. antiviral treatment was used in 53% of studies, mostly ribavirin for rsv infection, but also neuraminidase inhibitors and amantadine for influenza infection. of these studies, 72% discussed treatment efficacy, but only 28% considered treatment efficacy as an end-point. based on the clinical outcome of treated subjects, it is reported that early antiviral therapy might be associated with a reduction of complications and mortality. nevertheless, given the small number of cases, the lack of randomization and appropriate control groups, and the absence of analysis reporting a precise rate of reduction in mortality and/or morbidity, these trends could only considered as non evidence-based conclusions. twenty-five of 34 studies representing more than 2900 ltr specimens reported that transbronchial lung biopsies had been performed and a total of 923 pathological examinations were potentially available. however, the presence of acute rejection or obliterative bronchiolitis (ob) was reported only in 68% and 2.6% of cases, respectively. among a total of 282 virus-positive and 553 virus-negative cases, 21 overall (95% ci) 1 biopsy-proven. 2 biopsy-proven or fev decline >/= 20%. * random effect. results and an acute rejection rate. three studies were not suitable for the present analysis, thus leaving 19 studies reporting a total of 267 acute rejection events graded ≥ a2. in these 19 investigations, the frequency of acute rejections ≥ a2 ranged between 5.9% and 47.6% (table s4) . the association with acute rejection can only be estimated by comparing the rate observed in virus-positive cases with the one observed in virus-negative cases; this was available in only four studies (7, 21, 28, 34) . one study suggests a significant positive association (34) , which could not be confirmed in the three others ( figure 1b) . overall, we found no statistically significant association between respiratory viruses and acute rejection (or = 1.35; 95% ci = 0.41-4.43). ob/bos incidence following respiratory viral infections was reported for a period of time ranging from a few months to 1 year. in 11 studies (32.5%), all except one (1) used either biopsy-proven chronic rejection (defined by the presence of ob) or a sustained fev 1 decline of 20% according to the international society for heart and lung transplantation guidelines (35) . bos incidence ( table 2 ) following a respiratory viral infection ranged from 5.4% to 62.5% in virus-positive cases and was reported in only three studies for virus-negative cases (5, 21, 29) with a rate ranging from 9.1% to 52.9%. pooled incidence rates of these three studies revealed a bos incidence of 18% (9/50) in virus-positive cases compared to 11.6% (37/319) in virus-negative cases. the low number of bos events analyzed in these three investigations limited our ability to provide any meta-analytic summary that could be considered as relevant. four of the 11 studies (table 2) provided a statistical analysis testing the potential association with bos, but two without providing clearly the bos rate in virus-negative cases. one (17) failed to show any significant association and three (7,12,29) described a significant higher rate of bos in subjects experiencing a respiratory viral infection. during seasonal peaks, ltrs living in the community are exposed to rna and dna respiratory viruses. given the concomitant presence of a significant immunosuppression and impaired protective mechanisms of the grafted lung, these viral infections will promote complications and graft rejection (7, 21, 22, 34) . in the present review of 34 studies, our goal was to assess the strength and the characteristics of this association in available clinical reports and whether this translates into an observable association in real-life conditions. incomplete microbiological investigations or insensitive diagnostic tools limited the completeness of viral investigations; only the most recent reports have used a large panel of molecular tests and can provide a less biased image of the respective role of each viral agent. in the early 1990s, studies used mainly viral culture or direct immunofluorescence and, if available, pcr was limited to influenza, rsv or parainfluenza viruses. the recent emergence of new viruses such as hmpv, coronavirus nl63, coronavirus, hku1, bocavirus and human rhinovirus c need to be included in any modern molecular panel; these new agents have been systematically studied in two studies only. interestingly, when tested, the so-called 'common cold' viruses like rhinoviruses and coronavirus revealed to be the most frequent compared to others such as influenza or paramyxoviruses, an observation consistent with other hospital-based studies. depending on the type and number of technique used, the size of the study or enrolment criteria, the observed frequency of viral infections can dramatically change-ranging for each individual virus from less than 1% to more than 20% in our pooled analysis (table s1 ). the clinical significance of a positive viral nucleic acid detection result is a critical point that needs to be confronted with the presence or absence of respiratory symptoms. this type of analysis has been done in at least 11 studies (table s3 and figure 1a ) in which ltrs submitted to a routine respiratory screening for graft follow-up were used as controls and compared to symptomatic cases. it was consistently shown that in the presence of a viral infection, the likelihood of respiratory symptoms was five times higher. this observation could guide clinicians in their interpretation of microbiological results in an era where increasingly sensitive molecular diagnostic panels are available. even if a background positivity rate is expected, for example, following unnoticed or asymptomatic infection, or when seasonal outbreaks are ongoing in the community, these viruses likely contributed to symptom production in most cases and cannot be regarded as innocent bystanders (3, 22, 28, 29) . although mainly expected in the upper respiratory tract, viral infections are also present in lower respiratory specimens. this raises several issues such as the respective ability of each respiratory virus to infect the lower respiratory tract and whether all of them should be considered as equally able to cause graft complications in ltrs. despite being expected, our pooled analysis was unable to confirm a positive association between acute rejection and a previous viral infection. however, this conclusion needs to be considered carefully since the three largest studies (7, 28, 34) representing 96% of all cases brought discordant results; two of these failed to observe a positive association (7, 28) , whereas a third (34) reported a 33.3% rejection rate in 48 virus-positive cases compared to 6.7% in virusnegative cases (p value = 0.001). of note, this latter study considered not only biopsy-proven cases as rejection criteria, but also a fev 1 decline of 20% or more. another potential limitation of our pooled analysis is related to the heterogeneity of the design of each study: some reported an acute rejection rate during the acute phase of the viral infection and others during a follow-up period of 3 months. although the present report focuses on respiratory viruses, it must also be kept in mind that these agents could be associated, or promote other bacterial or fungal infections that subsequently could lead to graft complications. with regard to chronic rejection, a relationship between a previous respiratory viral infection and the subsequent development of bos was reported as statistically significant in three studies (14, 24, 41) . in at least seven other studies in which bos incidence was evaluated or discussed, the risk could not be linked to respiratory viruses or was not evaluable. the median number of virus-positive cases in the 10 studies in which bos was analyzed was five (range 1-13). four of these studies compared the rate in virus-positive versus negative cases for a total of only nine bos events in those virus-positive cases ( table 2 ). the low number of events, incompleteness, heterogeneity and the retrospective design of published reports, did not allow us to conduct any appropriate statistical analysis. of note, some studies have suggested that selected viruses, such as rsv, piv, influenza and possibly (7, 21, 22, 34) hmpv (6, 21) , are particularly prone to trigger graft rejection. in most of these studies, control groups were incomplete and thus again the clinical relationship between one specific respiratory viral family and graft rejection needs to be reconsidered carefully. in conclusion, our review confirms a causal relationship between respiratory viral infections and respiratory symptoms, even when these infections are documented by molecular assays. however, the respective role of each respiratory virus, especially with respect to picorna-and coronavirus, needs to be reconsidered. although it is certain that lower respiratory viral infection will promote graft complication, we highlight that the clinical link between respiratory viruses and acute lung rejection or bos needs to be characterized in prospective and appropriately designed cohort studies. additional supporting information may be found in the online version of this article. table s3 : relative proportion of virus-positive cases according to the presence of respiratory symptoms in lung transplants recipients screened for respiratory viruses table s4 : summary of studies analyzing the potential association between acute rejection and respiratory viral infections in lung transplant recipients please note: wiley-blackwell are not responsible for the content of functionality of any supporting materials supplied by the authors. any queries (other than missing material) should be directed to the corresponding for the article. community respiratory viral infection in adult lung transplant recipients clinical features and outcomes of paramyxoviral infection in lung transplant recipients treated with ribavirin lower respiratory viral illnesses: improved diagnosis by molecular methods and clinical impact respiratory viruses and severe lower respiratory tract complications in hospitalized patients a single-season prospective study of respiratory viral infections in lung transplant recipients human metapneumovirus in lung transplant recipients and comparison to respiratory syncytial virus respiratory viral infections are a distinct risk for bronchiolitis obliterans syndrome and death the epidemiology of parainfluenza virus infection in lung transplant recipients efficacy of oral ribavirin in lung transplant patients with respiratory syncytial virus lower respiratory tract infection parainfluenza and influenza virus infections in pediatric organ transplant recipients incidence and significance of noncytomegalovirus viral respiratory infection after adult lung transplantation adenovirus infection in the lung results in graft failure after lung transplantation respiratory syncytial virusassociated infections in adult recipients of solid organ transplants influenza pneumonia in lung transplant recipients: clinical features and association with bronchiolitis obliterans syndrome the value of polymerase chain reaction for the diagnosis of viral respiratory tract infections in lung transplant recipients influenza and parainfluenza respiratory viral infection requiring admission in adult lung transplant recipients respiratory viruses and chronic rejection in lung transplant recipients influenza virus infection in adult solid organ transplant recipients indirect fluorescent antibody testing of nasopharyngeal swabs for influenza diagnosis in lung transplant recipients detection of severe human metapneumovirus infection by real-time polymerase chain reaction and histopathological assessment human metapneumovirus infection in lung transplant recipients: clinical presentation and epidemiology clinical impact of community-acquired respiratory viruses on bronchiolitis obliterans after lung transplant intravenous ribavirin is a safe and cost-effective treatment for respiratory syncytial virus infection after lung transplantation impact of human metapneumovirus and human cytomegalovirus versus other respiratory viruses on the lower respiratory tract infections of lung transplant recipients diagnosis of human metapneumovirus infection in immunosuppressed lung transplant recipients and children evaluated for pertussis absence of human bocavirus in bronchoalveolar lavage fluid of lung transplant patients outcome of influenza infection managed with oseltamivir in lung transplant recipients upper and lower respiratory viral infections and acute graft rejection in lung transplant recipients community-acquired respiratory viral infections in lung transplant recipients: a single season cohort study correlation of rhinovirus load in the respiratory tract and clinical symptoms in hospitalized immunocompetent and immunocompromised patients respiratory viral infections within one year after pediatric lung transplant what role for human rhinovriuses in the lower respiratory tract a multi-drug regimen for respiratory syncytial virus and parainfluenza virus infections in adult lung and heart-lung transplant recipients a prospective molecular surveillance study evaluating the clinical impact of communityacquired respiratory viruses in lung transplant recipients bronchiolitis obliterans syndrome 2001: an update of the diagnostic criteria this work was supported by a grant of the swiss national science foundation attributed to l. kaiser (3200b-101670). the authors of this manuscript have no conflicts of interest to disclose as described by the american journal of transplantation. key: cord-263749-bbhh5xb1 authors: larenas-linnemann, désirée; rodríguez-pérez, noel; arias-cruz, alfredo; blandón-vijil, maría virginia; del-río-navarro, blanca e.; estrada-cardona, alan; gereda, josé e.; luna-pech, jorge a.; navarrete-rodríguez, elsy maureen; onuma-takane, ernesto; pozo-beltrán, césar fireth; rojo-gutiérrez, maría isabel title: enhancing innate immunity against virus in times of covid-19: trying to untangle facts from fictions date: 2020-10-09 journal: world allergy organ j doi: 10.1016/j.waojou.2020.100476 sha: doc_id: 263749 cord_uid: bbhh5xb1 introduction in the light of the current covid-19 pandemic, during which the world is confronted with a new, highly contagious virus that suppresses innate immunity as one of its initial virulence mechanisms, thus escaping from the first-line human defense mechanisms, enhancing innate immunity seems a good preventive strategy. methods without the intention to write an official systematic review, but more to give an overview of possible strategies, in this review article we discuss several interventions that might stimulate innate immunity and thus our defense against (viral) respiratory tract infections. some of these interventions can also stimulate the adaptive tand b-cell responses, but our main focus is on the innate part of immunity. we divide the reviewed interventions in: 1) lifestyle related (exercise, >7 hours sleep, forest walking, meditation/mindfulness, vitamin supplementation); 2) non-specific immune stimulants (letting fever advance, bacterial vaccines, probiotics, dialyzable leukocyte extract, pidotimod) and 3) specific vaccines with heterologous effect (bcg vaccine, mumps-measles-rubeola vaccine, i.e.). results for each of these interventions we briefly comment on their definition, possible mechanisms and evidence of clinical efficacy or lack of it, especially focusing on respiratory tract infections, viral infections and eventually a reduced mortality in severe respiratory infections in the intensive care unit. at the end a summary table demonstrates the best trials supporting (or not) clinical evidence. conclusion several interventions have some degree of evidence for enhancing the innate immune response and thus conveying possible benefit, but specific trials in covid-19 should be conducted to support solid recommendations. context of the covid-19 pandemic, extrapolated evidence from previous studies shows that regular, 128 moderate to vigorous exercise improves immune competency across the lifespan and may help to 129 reduce the frequency of upper-respiratory tract infections (urtis).(9, 10) importantly, this has been 130 observed also in older individuals (>65 year-olds) who regularly exercise, whose urtis incidence per 131 year has been inversely associated with the energy expenditure utilized during physical activity. conversely, some studies report that during prolonged periods of strenuous exercise (like marathons) 155 and after approximately 2 weeks following intense competitions, there is an immunodepression span 156 (the "open window" period), related to a higher rate of urti-like symptoms (mainly self-reported) vs. 157 those who undergo lower physical activity. (21, 22) this theory, attributed to the consequences of 158 psychological stress and excessive training,(23, 24) is however highly debated today in the light of a 159 better understanding of the variability in the intensity-related response to physical activity, which 160 depends largely on the functional capacity of the immune system, so up-to-date perceptions on this 161 topic tend to discard the concept of exercise-induced immunosuppression. in fact, it is widely accepted 162 that, when performed regularly, during and after exercise the immune system is in a heightened state of 163 immune surveillance and regulation.(25) 164 during the covid-19 quarantine it is important to stay active and to exercise regularly (gradually 165 increasing intensity), not only as a way to keep a healthy lifestyle, but also as an immunoprotective and 166 immunoregulatory activity. it is possible that the older adults (who are at higher risk of a severe sars-167 cov-2 infection) may obtain the greatest exercise-induced benefits to their immunological health. 168 169 2.2. forest bathing (shinrin yoku) 170 in a series of several, subsequent small studies, japanese investigators in cooperation with experts from 171 the stanford university have shown that walking in a forest seems to enhance nk cell activity, partly 172 stimulated by volatile substances produced by trees (particularly phytoncides: antimicrobial essential 173 oils from trees). the first experiment showed in vitro that phytoncides activate nk cell activity and 174 augment nk cell granules content.(26) then they confirmed this in peripheral blood lymphocytes of 175 subjects submitted to a prospective trial of a 3-day, 2-night trip to the forest including three 2-hour 176 walks.(27) in a third case-control trial they demonstrated that the effect was not related to the walking 177 j o u r n a l p r e -p r o o f 8 tour, as they compared the rise in nk-cell activity after a 3-day tour to a small city with that after a 3-day 178 tour to the forest. both tours included three 2-hour walks, but nk-cell activity was only enhanced after 179 the forest trip. moreover, nk-cell activity stayed elevated one week and still somewhat 30 days after the 180 tour. ( other studies have shown that sleep deprivation, both complete or only selective to the rapid eye 198 movement (rem) stage, modifies various components of the innate immune system, such as dendritic 199 cells (dcs) and the percentage of some subpopulations of t cells (cd4+, cd8+ and nk) and cytokine 200 levels (ifn-γ, tnf-α and il-1).(32-34) specifically, in a clinical study in women 77 hours of sleep 201 deprivation (total and rem phase) resulted in a proinflammatory state and affected the cellular immune 202 response, presenting changes in the production of ifns and in the phagocytic activity. sleep deprivation 203 also decreased lymphocytic blastogenesis, nk cell activity, and regulation of il-1 and il-2.(35) 204 j o u r n a l p r e -p r o o f 9 moreover, in the elderly, a sleep pattern with poor quality (self-reported) was related to increased levels 205 of il-6 and this association was not explained by other factors, such as depressive symptoms, stress or 206 loneliness, but exclusively by sleep disturbance as an independent factor.(36) 207 as for the clinical evidence, an adequate sleep pattern also appears to be beneficial in modulating the 208 adaptive immune response. spiegel the previously mentioned interventions are potential options that could improve the immune system 303 against sars-cov-2, but more evidence is required.(70) see table 2. 304 zinc is an essential trace element for the proper functioning of multiple biological processes within the 306 human body, and its contribution to immunity is not an exception.(72) 307 the complete mechanism by which zinc could decrease the number or severity of viral infectious 308 processes in general and of covid-19 in particular is not exactly understood yet; however, effects have 309 been observed on the binding of the viral agent to the mucosa and on its replication, as well as on the 310 regulation of the inflammatory process;(73) enhanced benefits have been hypothesized when co-311 administered with other medications such as (hydroxy)chloroquine that could function as a ionophore, 312 facilitating the entrance of zinc into the cells.(74) the human body's ability to store zinc is known to be 313 low; its deficiency compromises the immune system, as has been evidenced occasionally by thymic 314 atrophy, lymphopenia and altered lymphocyte responses. is not rock-solid yet, could be considered options that might help enhance the innate immune system 604 and thus worthwhile, especially, as most lack the risk for serious adverse events. together with the 605 techniques focusing on reduction of the viral load, such as social distancing, equipment for personal 606 protection and adequate hand hygiene, improving our first-line defense seems vital. finally, we add in 607 exercise regular exercise has a probable immunoprotective and immunoregulatory effect. older adults may obtain the greatest exercise-induced benefits for their immunological health. forest walks might be beneficial to boost innate immunity, particularly in times of a pandemic; evidence is limited, but there are no direct health-related safety issues. the potential benefit of adequate sleep patterns overweighs the low level of evidence available up to date mindfulness a constant program of a professionally guided meditation strategy could be beneficial to achieve an effective stress reduction and probably to reduce the burden of urtis, mainly in older adults the published study results so far have not been conclusive. subjects with vitamin deficiency would likely benefit most from supplementation. the intervention could be considered relatively safe a cochrane collaboration systematic review showed daily doses of 0.2mg reduced the duration of common cold. in ards patients very high iv doses might be beneficial. more data are needed. all 4 meta-analyzes have reported beneficial effects of zinc supplementation in children for the prevention of pneumonia. important to restore the redox balance. adverse effects are considered mild a modest efficacy of some probiotics (lactobacillus and bifidobacterium strains) was found in reducing the incidence and duration of viral respiratory infections though it might seem of benefit, evidence of the reduction of infections is very scarce and quality trials are lacking. with the use of pidotimod there is a lower recurrence of respiratory tract infections as compared to conventional treatment and less use of antibiotics we encourage to wait for the results of the ongoing rct before using bcg vaccination for the prevention of covid-19 heterothe immune stimulation produced by heterologous vaccines enhances the response to other pathogens, different from the one in the vaccine and was associated with reduced all-cause infant mortality (e.g. measles vaccine, bcg). influenza: the once and future pandemic estimation of the basic reproduction number, average incubation time, 615 asymptomatic infection rate, and case fatality rate for covid-19: meta-analysis and sensitivity analysis preliminary prediction of the basic 618 reproduction number of the wuhan novel coronavirus 2019-ncov sars coronavirus pathogenesis: host innate immune responses and viral 620 antagonism of interferon covid-19: consider 622 cytokine storm syndromes and immunosuppression is a "cytokine storm" relevant to covid-19? targets of t cell 626 responses to sars-cov-2 coronavirus in humans with covid-19 disease and unexposed individuals therapeutic potential of b-1a cells in covid-19 moderate to vigorous 630 physical activity and risk of upper-respiratory tract infection physical activity, 632 stress, and self-reported upper respiratory tract infection the symptomatology of upper respiratory tract 634 infections and exercise in elderly people exercise and the aging 636 immune system moderate exercise enhances the production of 638 interferon-gamma and interleukin-12 in peripheral blood mononuclear cells acute exercise 641 mobilises cd8+ t lymphocytes exhibiting an effector-memory phenotype exercise-induced redistribution of t 644 lymphocytes is regulated by adrenergic mechanisms t-regulatory cells exhibit 646 a biphasic response to prolonged endurance exercise in humans exercise and psychosocial factors 649 modulate immunity to influenza vaccine in elderly individuals signaling via trif contributes to a protective innate immune response to severe acute respiratory 653 syndrome coronavirus infection influence of 655 exercise training and age on cd14+ cell-surface expression of toll-like receptor 2 and 4 exercise amelioration of depression-like behavior in ovx mice 658 is associated with suppression of nlrp3 inflammasome activation in hippocampus does exercise increase the risk of upper respiratory 664 tract infections exercise, infection, and immunity exercise and airway injury in athletes debunking the myth of exercise-induced immune suppression: 669 redefining the impact of exercise on immunological health across the lifespan phytoncides (wood 672 essential oils) induce human natural killer cell activity forest bathing enhances 675 human natural killer activity and expression of anti-cancer proteins visiting a forest, but not 678 a city, increases human natural killer activity and expression of anti-cancer proteins effect of phytoncide 681 from trees on human natural killer cell function using psychoneuroimmunity against covid-19 galphas-684 coupled receptor signaling and sleep regulate integrin activation of human antigen-specific t cells number and function of circulating human antigen 687 presenting cells regulated by sleep rem sleep deprivation in rats results in 689 inflammation and interleukin-17 elevation effects of acute and chronic sleep loss on 691 immune modulation of rats the bidirectional relationship between sleep and immunity 694 against infections sleep disturbance and 696 older adults' inflammatory responses to acute stress effect of sleep deprivation on response to immunization. 698 sleep habits and susceptibility to the 700 common cold behaviorally assessed sleep and susceptibility 702 to the common cold psychological stress and susceptibility to the common cold relationships between mindfulness practice and levels of mindfulness, 706 medical and psychological symptoms and well-being in a mindfulness-based stress reduction program meditation or exercise for 709 preventing acute respiratory infection: a randomized controlled trial epidemic influenza and 711 vitamin d vitamin d and the intracrinology of innate immunity the interplay between vitamin d and viral 715 infections vitamin d in allergic disease: shedding light on a complex problem unexpected actions of vitamin d: new perspectives on the regulation of 719 innate and adaptive immunity vitamin d supplementation could reduce risk of influenza and covid-19 infections and deaths. 722 nutrients administration in ventilated intensive care unit patients: a pilot double blind randomized controlled 725 trial vitamin d and respiratory tract 727 infections: a systematic review and meta-analysis of randomized controlled trials vitamin d 730 supplementation to prevent acute respiratory tract infections: systematic review and meta-analysis of 731 individual participant data effect of vitamin d3 supplementation on 733 respiratory tract infections in healthy individuals: a systematic review and meta-analysis of 734 randomized controlled trials vitamin d supplementation for the prevention 736 of childhood acute respiratory infections: a systematic review of randomised controlled trials the 2011 report on 739 dietary reference intakes for calcium and vitamin d from the institute of medicine: what clinicians need 740 to know vitamin-d and covid-19: do deficient risk a poorer outcome? lancet diabetes 745 endocrinol vitamin d: deficiency, sufficiency and toxicity intravenous vitamin c for reduction of cytokines storm in acute respiratory 749 distress syndrome vitamin c is an essential factor on the anti-viral 751 immune responses through the production of interferon-alpha/beta at the initial stage of influenza a 752 virus (h3n2) infection vitamin c and common cold-induced asthma: a systematic review and statistical 754 analysis acid potentially regulates immune and inflammatory response associated 757 with coronavirus infections: a perspective from system biology analysis hypovitaminosis c and 759 vitamin c deficiency in critically ill patients despite recommended enteral and parenteral intakes. crit 760 care functional role of 763 dietary intervention to improve the outcome of covid-19: a hypothesis of work vascular endothelialitis, thrombosis, and angiogenesis in covid-19 modulation of host defence 768 against bacterial and viral infections by omega-3 polyunsaturated fatty acids flavonoids from houttuynia cordata attenuate 771 h1n1-induced acute lung injury in mice via inhibition of influenza virus and toll-like receptor signalling individual risk management 774 strategy and potential therapeutic options for the covid-19 pandemic the clinical benefits of chinese patent 776 medicines against covid-19 based on current evidence traditional chinese medicine in the treatment of 778 cov-2): a review and perspective pharmacological perspective: glycyrrhizin may be an efficacious therapeutic 781 agent for covid-19 the role of zinc in antiviral immunity zinc and 785 respiratory tract infections: perspectives for covid19 (review) improving the efficacy of chloroquine and hydroxychloroquine against 787 sars-cov-2 may require zinc additives -a better synergy for future covid-19 clinical trials zinc in human health: effect of zinc on immune cells role of zinc administration in prevention of childhood diarrhea 791 and respiratory illnesses: a meta-analysis low zinc status: a new risk factor for pneumonia in the 793 elderly? prevention of diarrhea and 795 pneumonia by zinc supplementation in children in developing countries: pooled analysis of randomized 796 controlled trials. zinc investigators' collaborative group zinc supplementation for the prevention of acute lower 798 respiratory infection in children in developing countries: meta-analysis and meta-regression of 799 randomized trials zinc lozenges may shorten the duration of colds: a systematic review fever and the thermal regulation of immunity: the immune 803 system feels the heat fever-like thermal conditions 805 regulate the activation of maturing dendritic cells conservation of 807 il-6 trans-signaling mechanisms controlling l-selectin adhesion by fever-range thermal stress from fever to immunity: a new role for igfbp-6? activation of the sympathetic nervous 814 system modulates neutrophil function the regulation of interleukin-6 implicates skeletal muscle as an integrative 816 stress sensor and endocrine organ human monocyte stimulation by 818 experimental whole body hyperthermia functional expression 820 of trpv channels in t cells and their implications in immune regulation the acute phase 822 protein haptoglobin regulates host immunity passive heat therapy protects 826 against endothelial cell hypoxia-reoxygenation via effects of elevations in temperature and circulating 827 factors community-acquired pneumonia in the elderly: association of 829 mortality with lack of fever and leukocytosis the effect of antipyretic 831 therapy upon outcomes in critically ill patients: a randomized, prospective study the value of sanitarium treatment in respiratory diseases washington, d.c.: review and 836 herald publishing assn antipyretic drugs in patients with fever and infection: literature review the effect of antipyretic 841 medications on mortality in critically ill patients with infection: a systematic review and meta-analysis population-level effects of suppressing fever therapeutic targeting of trained 846 immunity bcg-induced trained 848 immunity in nk cells: role for non-specific protection to infection trained immunity: a 852 program of innate immune memory in health and disease human dendritic cells activated with mv130 induce th1, th17 and il-10 responses via ripk2 and myd88 855 signalling pathways mv140, a 857 sublingual polyvalent bacterial preparation to treat recurrent urinary tract infections, licenses human 858 dendritic cells for generating th1, th17, and il-10 responses via syk and myd88 efficacy of whole-cell killed bacterial vaccines in preventing 864 pneumonia and death during the 1918 influenza pandemic sublingual therapeutic 866 immunization with a polyvalent bacterial preparation in patients with recurrent respiratory infections: 867 immunomodulatory effect on antigen-specific memory cd4+ t cells and impact on clinical outcome primary prevention of severe lower 870 respiratory illnesses in at-risk infants using the immunomodulator om-85 trained immunity in newborn 873 infants of hbv-infected mothers probiotics and the gut immune system: indirect regulation beneficial effects of 877 probiotic consumption on the immune system immunobiotic strains modulate toll-like receptor 3 agonist induced innate 879 antiviral immune response in human intestinal epithelial cells by modulating ifn regulatory factor 3 880 and nf-kappab signaling type i/ii 882 interferon in hiv-1-infected patients: expression in gut mucosa and in peripheral blood mononuclear 883 cells and its modification upon probiotic supplementation prospective study of probiotic 885 supplementation results in immune stimulation and improvement of upper respiratory infection rate probiotics and paraprobiotics in viral infection: 888 clinical application and effects on the innate and acquired immune systems probiotics and covid-19: one size does not fit all effectiveness of probiotics on the duration 893 of illness in healthy children and adults who develop common acute respiratory infectious conditions: a 894 systematic review and meta-analysis probiotics for preventing acute upper respiratory tract infections serum and 898 fecal profiles of aromatic microbial metabolites reflect gut microbiota disruption in critically ill patients: 899 a prospective observational pilot study is diet partly 901 responsible for differences in covid-19 death rates between and within countries? the cellular transfer of cutaneous hypersensitivity to tuberculin in man cd8+ t cells produce a 906 dialyzable antigen-specific activator of dendritic cells immune memory by dialyzable leukocyte extract from a cd8+ t cell line early differentiation of human cd11c(+)nk cells with gammadelta t cell activation properties 912 is promoted by dialyzable leukocyte extracts adjuvant treatment with a dialyzable leukocytes extract contributes to maintain 915 hpv-infected women free of low-grade cervical lesions dialyzable leukocyte extracts activate tlr-2 on monocytes the dialyzable leukocyte extract transferon(tm) inhibits tumor growth and brain 921 metastasis in a murine model of prostate cancer dialyzable leukocyte extract (transferon) administration in sepsis: 924 experience from a single referral pediatric intensive care unit innate immune system to improve survival traits in high risk pathogen scenarios adjuvant treatment using transfer factor 929 for bronchogenic carcinoma: long-term follow-up immunostimulants in respiratory diseases: focus on pidotimod effect of pidotimod on phagocytosis and 933 intracellular killing of staphylococcus aureus by human circulating polymorphonuclear neutrophils and 934 alveolar macrophages pidotimod stimulates natural killer cell 936 activity and inhibits thymocyte cell death pidotimod shows a chemokine-like activity through cxc chemokine receptor 3 (cxcr3) pidotimod promotes 941 functional maturation of dendritic cells and displays adjuvant properties at the nasal mucosa level immunomodulatory effects 944 of pidotimod in adults with community-acquired pneumonia undergoing standard antibiotic therapy pidotimod, an immunostimulant in pediatric recurrent respiratory 947 tract infections: a meta-analysis of randomized controlled trials safety and immunogenicity of early 949 bacillus calmette-guerin vaccination in infants who are preterm and/or have low birth weights: a 950 systematic review and meta-analysis treating bcg-induced disease in 952 children neonatal bcg vaccination against hospitalization due to respiratory infection and sepsis. clin infect 955 dis early bcg-denmark 957 and neonatal mortality among infants weighing <2500 g: a randomized controlled trial the efficacy of bacillus calmette-guerin 960 vaccinations for the prevention of acute upper respiratory tract infection in the elderly bcg 963 vaccination enhances the immunogenicity of subsequent influenza vaccination in healthy volunteers: a 964 randomized, placebo-controlled pilot study bcg vaccination protects 966 against experimental viral infection in humans through the induction of cytokines associated with 967 trained immunity bcg vaccine protection from severe coronavirus 969 disease 2019 (covid-19) sars-cov-2 rates in bcg-vaccinated and unvaccinated young 971 adults bcg-induced trained immunity: can it offer protection against covid-19? 973 heterologous immunity: role in natural and vaccine-induced resistance to 975 infections vaccination and heterologous immunity: 977 educating the immune system non-specific effect of vaccines: immediate 979 protection against respiratory syncytial virus infection by a live attenuated influenza vaccine. front 980 microbiol vitamin d supplementation and risk of respiratory tract infections: a meta-982 analysis of randomized controlled trials vitamin d supplementation for preventing infections 984 in children under five years of age effect of vitamin c 986 infusion on organ failure and biomarkers of inflammation and vascular injury in patients with sepsis 987 and severe acute respiratory failure: the citris-ali randomized clinical trial allergic rhinitis and its impact on asthma (aria) guidelines for allergic rhinitis based on grading of 991 recommendations assessment, development and evaluation (grade) and real-world evidence enteral omega-3 994 fatty acid, gamma-linolenic acid, and antioxidant supplementation in acute lung injury association of bcg, dtp, and measles containing vaccines with childhood mortality: systematic review. 998 days (enteral supplementation of n-3 fatty acids, γ-linolenic rice unlike the normal anti-viral response with increased ifn type i and iii and with it the activation of genes 1006 stimulated by ifn in adjacent cells and thereby increasing its anti-viral defense, the coronavirus has 1007 mechanisms that lower this anti-viral innate defense mechanism by interfering with ifn production and 1008 its effects. in addition, chemotactic molecules are released in a viral infection that attract macrophages 1009 (m∅), natural killer (nk) cells, and neutrophils. this reaction is not fully achieved during early infection 1010 with coronavirus, so the initial innate immune response appears incomplete and slow. after this first 1011 innate response, adaptive immunity is triggered via activation of dendritic cells (dc) that stimulate 1012 specific th1 lymphocytes, which in turn activate cytotoxic t cells (tccell) to eliminate infected cells, in 1013 the more advanced stages of the infection, along with plasma cell development and the production of 1014 antiviral igm and igg antibodies (not shown). however, in some patients with covid-19 at this stage a 1015 dysregulated activation of macrophages (i.e. by il6) is seen, causing the feared cytokine storm. 1016 the conversion of 25 ohd 2 to 1,25-(oh) 2 d 3 can be done directly in some cells of the immune system 1018 such as macrophages. the macrophage, like kidney cells, contains the enzyme 1-alpha hydroxylase that 1019 is capable of transforming vitamin d into its active form, calcitriol. 1020exposure of the macrophage to some pathogens induces the production of cp27b (step 1) (which allows 1021 25 ohd to enter the mitochondria for transformation into its active form 1,25-(oh) 2 d 3 ), (step 2) as well 1022 as vitamin d receptor (vdr) (step 3), which by binding to 1,25-(oh) 2 d3 increases the production of 1023 cathelicidin. (step 4) 1024the antimicrobial activity of vitamin d appears to be primarily dependent on the induction of 1025 cathelicidins, which perform numerous functions that enhance both innate and adaptive immunity; help 1026 improve the digestion process within the phagolysosome through a non-oxygen dependent mechanism 1027 key: cord-253148-3t4o27xp authors: chow, brian d.w.; huang, yung t.; esper, frank p. title: evidence of human bocavirus circulating in children and adults, cleveland, ohio date: 2008-09-19 journal: j clin virol doi: 10.1016/j.jcv.2008.07.009 sha: doc_id: 253148 cord_uid: 3t4o27xp background: viral respiratory illness is a major cause of morbidity and mortality. the human bocavirus (hbov) is a recently recognized parvovirus isolated from human respiratory secretions. objectives: to define the clinical and epidemiologic characteristics in adult and pediatric patients with evidence of hbov. study design: from october 2005 through october 2006, we screened respiratory samples from children and adults negative for common respiratory pathogens for hbov by pcr. demographic and clinical characteristics were obtained from medical records of hbov positive individuals. results: of 2075 samples screened, 1826 (88.0%) represented distinct respiratory events: 1539 (84.3%) were pediatric (<18 years), and 273 (15.0%) adult (≥18 years). forty (2.2%) patients had hbov: 36 (2.3%) children and 4 (1.5%) adults. hbov positive children had history of prematurity (31.3%) and cardiac disease (18.8%). adults had underlying pulmonary (100%) and cardiac (50%) disease. twenty-seven children (84.4%) were hospitalized; 9 (28.1%) required intensive care. all adults were hospitalized; none required intensive care. nosocomial acquisition likely occurred in 3 patients. conclusions: hbov circulates in cleveland, oh, in children and adults with similar frequencies, and can warrant hospitalization and intensive care. further study would clarify our understanding of this newly recognized human pathogen. respiratory illness is an important reason for adult and pediatric emergency department visits. 1 in the united states, the proportion of pediatric hospitalizations caused by asthma, pneumonia, and acute bronchitis is nearly 22%. of all hospital discharges, 9.7% are attributed to diseases of the respiratory system. 2 in the united states, the annual burden to society for respiratory infections is estimated to be $112 billion. 3 allander et al. (2005) described a novel parvovirus isolated from human respiratory secretions from patients with pneumonia named the human bocavirus (hbov). 4 since that time, its presence in both the pediatric and adult population has been confirmed throughout the world. the incidence of this pathogen has ranged from 1.8% to 19%, with a variety of clinical presentations. [5] [6] [7] human bocavirus has been found in patients with upper and lower respiratory tract disease, 5, 8, 9 as well as gastrointestinal illness. [10] [11] [12] cases reported in children constitute the majority; adult cases are reported less frequently. 7, 13, 14 common presentations include cough, wheezing, fever, and emesis. 7, 14, 15 thai adults have been reported to have pneumonia associated with hbov severe enough to warrant hospitalization. 13 multiple studies have reported frequent detection of other viral pathogens in hbov positive samples. 16 it has been hypothesized that prolonged shedding or persistence may be a reason. because of this, questions remain about the role of hbov in respiratory infection. we sought to further define the clinical and epidemiologic characteristics of hbov in adult and pediatric patients in cleveland, oh. cleveland. samples were submitted to the core laboratory at the discretion of the primary medical teams. submitted samples originated from the emergency department, inpatient wards, intensive care units and hospital-affiliated primary care outpatient clinics. we obtained all clinical specimens from children and adults that had negative results for rsv, parainfluenza viruses (1-3), influenza a and b, and adenovirus by direct immunofluorescence assay (dfa). for dfa, samples were processed, applied to slides by cytocentrifugation, stained with respiratory panel 1 tm direct immunofluorescence assay (millipore, temecula, ca), and examined. from nucleic acid from each respiratory specimen was extracted with the magmax tm -96 viral rna isolation kit (applied biosystems, foster city, ca) according to the manufacturer's protocol. this product recovers both rna and dna from samples allowing us to screen for multiple respiratory pathogens. samples were then screened by pcr for the presence of hbov with platinum taq polymerase (invitrogen) according to the manufacturer's specification. primers used in the screening of the respiratory specimens were based on sequences in published reports and targeted the hbov np-1 gene. 15 the forward primer, 5 -gacctctgtaagtactattac-3 and reverse primer, 5 -ctctgtgttgactgaatacag-3 produce a 353 base-pair amplicon that corresponds to nucleotides 2351-2704 of the hbov np-1 gene. each set of pcr reactions contained appropriate negative controls. pcr amplification cycles were as follows: 95 • c for 3 min followed by 40 cycles of 94 • c for 1 min, 54 • c for 1 min, 72 • c for 30 s and completed with a 10-min 72 • cycle. sequencing was performed on abi prism 3730 dna analyzer automated sequencer. isolates positive for hbov were screened for common respiratory viruses by rt-pcr with published primer sets. viruses screened include respiratory syncytial virus, human parainfluenza virus 3, human metapneumovirus, adenovirus, rhinovirus, influenza, wu polyomavirus, and the human coronaviruses nl63, hku1, oc43, 229e. 17-21 the phylogenetic analysis included representative samples of cleveland isolates in addition to isolates from published reports. the region of analysis corresponded to a 228-base pair region of the np-1 gene spanning nucleotides 2426-2654 of the hbov genome. clustalw alignment was generated using bioedit 7.0.5.2 alignment software. five-hundred bootstrap data sets were created using the phylip program seqboot. phylogenetic analyses were conducted using the phylip program dnaml, with the default transition to transversion ratio of 2.0 and 1 jumble. available medical records for hbov positive-individuals were reviewed. demographic data and clinical characteristics of each individual were recorded on a standard collection form. pediatric cases were defined as patients whose age at the time of sample collection was less than 18 years; adult cases were defined as patient age at time of collection of 18 years and older. of 2075 samples screened, 1826 (88.0%) were determined to be separate respiratory events. samples collected greater than 28 days apart from a single individual were considered separate respiratory events. of these, 1539 (84.3%) were obtained from pediatric patients, and 273 (15.0%) from adult patients. demographic information was unavailable for 14 (0.8%) patients. samples were collected throughout the year, with more samples obtained during the winter months in cleveland. monthly sample collections ranged from 63 (july) to 271 (march) (fig. 1) . forty samples (2.2%) tested positive for hbov by pcr: 36 (90%) pediatric patients and 4 (10%) adult patients. medical records were unavailable for 1 (2.5%) subject. four (10.0%) samples had other respiratory pathogens identified by rt-pcr. these include rhinovirus (2) and coronaviruses hcov-nl63 (1) and hcov-229e (1). all coinfected samples originated from children and were excluded from clinical analysis. hbov samples were primarily identified in fall and winter, peaking in november where 6.8% of samples screened positive. hbov continued to circulate until may. no hbov was detected in the summer months of june, july, and august (fig. 1) . the median pediatric age was 12 months, with the range of ages from the 24 days to 8 years 5 months. the median adult age was 58 years, ranging from 20 years to 86 years. the 12-18 month age group had the highest prevalence, with hbov detected in 6.0% of respiratory samples screened (fig. 2) . the clinical presentation of the pediatric populations is described in table 1 . of pediatric patients testing positive, 66.7% were male, 53.1% were caucasian, and 31.3% were african-american. underlying conditions included prematurity (31.3%), cardiac disease (18.8%), immunodeficiency (9.3%), and pulmonary disease (9.3%). the most common symptoms reported included cough (75.0%), rhinorrhea (62.5%), and fever (53.1%). gastrointestinal symptoms were reported in 11 (34.3%) patients, and 8 (25.0%) patients had wheezing. of pediatric patients who screened positive for hbov, 27 (84.4%) were admitted to the hospital, including 9 (28.1%) who required intensive care. for patients admitted to an intensive care unit, the median stay in intensive care was 2 days (range 1-5 days). reasons for icu admission included respiratory distress (66.6%), apnea (11.1%), cardiac arrhythmia (11.1%), and shock (11.1%). of the remaining pediatric patients, 18 (56.3%) were admitted to a pediatric ward. disposition for one patient was unavailable. the median duration of hospitalization was 3 days (range 1-8 days). admission diagnoses for patients not requiring intensive care include bronchiolitis (22.2%), dehydration (16.7%), exacerbation of asthma or reactive airway disease (16.7%), pneumonia (11.1%), apparent life threatening event (11.1%), hypoxia (11.1%), and bronchopulmonary dysplasia exacerbation (5.6%). of all pediatric patients admitted, oxygen therapy was initiated or increased over baseline requirements in 10 (31.3%) patients. one patient (3.1%) was discharged on oxygen therapy not previously required. mean duration of oxygen therapy before return room air or baseline oxygen requirements was 4.7 days. in pediatric patients positive for hbov, there were no deaths. three (9.4%) pediatric patients were already hospitalized when respiratory samples were obtained. one patient with hypoplastic left heart syndrome was admitted in respiratory failure with respiratory syncytial virus (rsv). subsequent respiratory samples screened negative for rsv and hbov. thirty-one days into her admission a nasopharyngeal sample tested positive for hbov and negative for other respiratory pathogens by rt-pcr. two other cases involved newborn patients who had never been discharged from the hospital. one patient in the neonatal icu was not previously on respiratory support, and presented with multiple drops in oxygen saturation. she required oxygen therapy for 20 days. two subjects are twin siblings in the same household, both born prematurely with bronchopulmonary dysplasia, and were both admitted to the hospital sequentially with respiratory symptoms. in the adult group, all patients had underlying pulmonary disease; half had underlying cardiac disease ( table 1 ). the most common presenting symptoms were rhinorrhea (50%), cough (50%), and wheeze (50%). all were hospitalized, with median hospitalization of 1.5 days (range 1-4 days). admission diagnoses include change in mental status (50.0%) and asthma exacerbation (50.0%). none required oxygen therapy or intensive care, and there were no deaths. adult chest radiographs, while abnormal, were unchanged from previous studies. of the 24 patients who had a complete blood count with differential drawn, 44.4% were abnormal. abnormalities include immature granulocytes (37.5%), atypical lymphocytes (29.1%), and leukocytosis (20.8%). twenty-six (81.3%) pediatric patients had chest radiographs taken, with 76.9% of chest radiographs reported as abnormal. abnormalities included perihilar/peribronchial thickening or central inflammatory changes (42.3%), infiltrate (19.2%), ground glass disease (7.7%), pleural effusion (7.7%), hyperinflation (7.7%), and atelectasis (7.7%). phylogenetic analysis confirmed that a single strain of hbov circulates in cleveland, oh, and is similar to strains described in various studies reported worldwide. there was a pediatric diagnoses include multifocal atrial tachycardia, ventricular septal defect,atrial septal defect, patent ductus arteriosus, and hypoplastic left heart. in adults, diagnoses include heart failure, atrial fibrillation/flutter, and prosthetic mitral and tricuspid values. b include kippel-feil syndrome with asplenia, neutropenia secondary to chemotherapy, and common variable immune deficiency. c in pediatric patients, excludes lung disease associated with prematurity. includes asthma and hunter's disease. in adults, includes asthma, chronic obstructive pulmonary disease, transudative pleural effusion, and restrictive lung disease. d specific respiratory findings (number, %) -pediatric patients respiratory distress (13, 40.6%), hypoxia (9, 28.1%), ronchi (10, 31.2%), wheeze (8, 25 .0%), tachypnea (8, 25 .0%), rales (7, 21.8%), cyanosis (4, 12.5%), apnea (3, 9.3%). adult patients: tachypnea (3, 75%), wheeze (2, 50%), rales (1, 25%), and hypoxia (1, 25%). no difference in strains circulating among children and adults (fig. 3) . this study demonstrates that hbov circulates in cleveland, oh. in our study, hbov predominates in the winter months, and extends into the late spring and early fall. the predominate age group affected are children under 18 months. in our study, hbov is present in many patients who have cardiac or pulmonary disease. in many institutions, respiratory sample collection and viral screening occur infrequently in adults. however, it is notable that hbov was found at similar rates in both pediatric and adult patients in our population. we found hbov at slightly higher rates in adults than other reports. 6, 7 this underscores that viral respiratory disease leading to hospitalization in the adult population may be underappreciated. like prior studies, we find a substantial number of patients with gastrointestinal symptoms including emesis and diarrhea. gastrointestinal symptoms are reported with other respiratory viruses, but generally with lower frequency. 19, 22 by screening respiratory samples, this study is biased towards finding respiratory tract disease. further investigation of patients with gastrointestinal illness will improve our understanding of this virus in gastrointestinal pathology. our data suggests nosocomial acquisition of hbov in three cases, and close household contact in another. for the two patients who screened hbov positive in the nicu, vertical transmission cannot be ruled out. siblings with sequential hospitalization both screening positive for hbov does not prove that the children infected each other although it seems likely their infections originated from close contact. these cases highlight the importance of universal caregiver and visitor hygiene. further study on modes of transmission are needed to elucidate specific precautions needed for patients with hbov. hbov was the only identified pathogen in 36 (90%) isolates. this differs from other reports which demonstrate a higher rate of viral co-infection. 7 this discrepancy is likely due to our selection of samples that are negative for common respiratory pathogens routinely screened at this institution. as such, the co-infection rate seen in this study cannot be compared to those of other studies. screening of samples positive for other respiratory pathogens will likely increase the number of hbov positive samples. however, this report suggests that clinical disease associated with hbov alone may be severe enough to require admission to the hospital in both adults and children and to the intensive care unit in children. limitations of our study include its retrospective nature and lack of a control group. by screening samples of patients who present to a tertiary care center, there is likely an overrepresentation of patients with severe disease. in our study all 80-90% of samples screened were collected from patients who were hospitalized. other reports show that many patients with hbov can have either mild or severe respiratory tract disease. 23 the sensitivity of the nucleic acid extraction and primer set used has not been established. using different primer sets or alternate dna isolation techniques may identify further hbov positive samples. we find that hbov is circulating in cleveland, oh, in children and adults. disease associated with hbov is severe enough to warrant hospitalization and intensive care support. our study suggests that hbov may be transmitted between close household contacts and within the hospital. further study on the prevalence in adult patients, both in hospital settings and outpatient settings, would clarify our understanding of this virus. influenza other respiratory virus-related emergency department visits among young children hcup fact book no. 6. ahrq publication no. 05-0056 economic burden of respiratory infections in an employed population cloning of a human parvovirus by molecular screening of respiratory tract samples human bocavirus and acute wheezing in children human bocavirus infection human bocavirus infections in hospitalized children and adults human bocavirus in italian patients with respiratory diseases prospective study of human bocavirus (hbov) infection in a pediatric university hospital in germany human bocavirus a respiratory and enteric virus clinical and molecular epidemiology of human bocavirus in respiratory and fecal samples from children in hong kong detection of human bocavirus in children hospitalized because of acute gastroenteritis human bocavirus: a novel parvovirus epidemiologically associated with pneumonia requiring hospitalization in thailand severe pneumonia and human bocavirus in adult human bocavirus infection in young children in the united states: molecular epidemiological profile and clinical characteristics of a newly emerging respiratory virus human bocavirus: passenger or pathogen in acute respiratory tract infections? identification of a novel polyomavirus from patients with acute respiratory tract infections rapid multiplex nested pcr for detection of respiratory viruses a one year experience with human metapneumovirus in children less than 5 years old evidence of a novel coronavirus associated with respiratory tract disease in infants and children evidence of coronavirus hku1 infection in the united states: clinical manifestations associated with hku1 in children < 5 years of age excretion patterns of human metapneumovirus and respiratory syncytial virus among young children human bocavirus in febrile children we are indebted the staff of the core laboratory at university collection of specimens and clinical data were approved by the university hospitals human investigation committee. none. key: cord-258386-aiwzgkq3 authors: mclaren, rodney a.; london, viktoriya; atallah, fouad; mccalla, sandra; haberman, shoshana; fisher, nelli; stein, janet l.; minkoff, howard l. title: delivery for respiratory compromise among pregnant women with covid-19 date: 2020-05-23 journal: am j obstet gynecol doi: 10.1016/j.ajog.2020.05.035 sha: doc_id: 258386 cord_uid: aiwzgkq3 objective while rapid recourse to delivery after failed cpr has been shown to improve outcomes of pregnant patients with cardiac arrest,1,2 it is not known whether delivery improves or compromises the outcome of covid patients with respiratory failure.3,4 our objective was to evaluate the safety and utility of delivery of covid-19 infected pregnant women needing respiratory support. study design this is a retrospective observational study of covid-19 infected pregnant women (pcr diagnosed), with severe disease (defined per prior publications.3). a subset of these cases was previously presented, but without detail on the effect of delivery on disease (london, et al. “the relationship between status at presentation and outcomes among pregnant women with covid-19” am j perinatol., in press). the study was exempted by irb. results of 125 confirmed cases of covid-19, twelve (9.6%) had severe disease (table 1). among the 12, three resolved spontaneously after transient respiratory support in hospital and were discharged home (one subsequently returned in preterm labor and delivered by cesarean two weeks later). of the remaining nine who continued to need respiratory support, seven (77.8%) had iatrogenic preterm deliveries (six by cesarean delivery) for maternal respiratory distress (needing increasing levels of respiratory support without improved oxygen saturation), one had an early term delivery due to prom, and one, now 30 weeks, has required intensive care with high-flow nasal cannula for three weeks. of the eight patients delivering with maternal respiratory distress, seven did not require intubation, and one was intubated for emergent cesarean delivery, and remained on a ventilator for 19 days. among the non-intubated, four had an improvement in oxygenation within two hours postpartum; two required less respiratory support, and two were taken completely off respiratory support. none of the other three required an increased level of respiratory support, and were off of all support between four and seven days postpartum. conclusion delivery did not worsen the respiratory status of women with persistent oxygen desaturation and the need for increasing respiratory support. among women not needing a ventilator, return of normal respiratory status after delivery occurred within hours to days. the one patient intubated intraoperatively took longer to recover. it is possible delivery may be less salutary when damage to the lungs are sufficient to warrant intubation. this series suggests that maternal respiratory distress should not be a contraindication to delivery. with covid-19" am j perinatol., in press). the study was exempted by irb. 34 35 results: of 125 confirmed cases of covid-19, twelve (9.6%) had severe disease (table 1) . 36 among the 12, three resolved spontaneously after transient respiratory support in hospital and 37 were discharged home (one subsequently returned in preterm labor and delivered by cesarean 38 two weeks later). of the remaining nine who continued to need respiratory support, seven 39 (77.8%) had iatrogenic preterm deliveries (six by cesarean delivery) for maternal respiratory 40 distress (needing increasing levels of respiratory support without improved oxygen saturation), 41 one had an early term delivery due to prom, and one, now 30 weeks, has required intensive 42 care with high-flow nasal cannula for three weeks. 43 of the eight patients delivering with maternal respiratory distress, seven did not require 45 intubation, and one was intubated for emergent cesarean delivery, and remained on a ventilator 46 for 19 days. among the non-intubated, four had an improvement in oxygenation within two 47 hours postpartum; two required less respiratory support, and two were taken completely off 48 respiratory support. none of the other three required an increased level of respiratory support, 49 and were off of all support between four and seven days postpartum. 50 51 conclusion: delivery did not worsen the respiratory status of women with persistent oxygen 52 desaturation and the need for increasing respiratory support. among women not needing a 53 ventilator, return of normal respiratory status after delivery occurred within hours to days. the 54 one patient intubated intraoperatively took longer to recover. it is possible delivery may be less 55 salutary when damage to the lungs are sufficient to warrant intubation. this series suggests that 56 maternal respiratory distress should not be a contraindication to delivery. 57 58 as noted in a recent smfm-soap guideline, it's not known whether uterine decompression 59 improves respiratory status; we are unable to shed light on that issue. 4 while we saw no harm, we 60 cannot be certain that delivery per se caused the improvement we saw, or whether a similar 61 outcome could have been achieved with ongoing respiratory support (although one of three 62 patients managed conservatively, remained on respiratory support for three weeks). in sum, 63 while more data on the effects of delivery are needed, we have shown in a small series that 64 women with covid-19 requiring respiratory support fared well when they underwent delivery. society for maternal-fetal medicine (smfm) amniotic fluid embolism: diagnosis and 68 management perioperative and resuscitation, council on cardiovascular diseases in the young society for maternal-fetal medicine. management considerations for pregnant patients 76 with covid-19 covid_pos_preg_patients_4-29-20_final.pdf. accessed labor and delivery covid-19 considerations soap_covid_ld_considerations_-_revision_4-14-20_-_changes_highlighted key: cord-271172-y48dovux authors: potter, christopher william title: chapter 25 respiratory tract viruses date: 1998-12-31 journal: principles of medical biology doi: 10.1016/s1569-2582(97)80009-8 sha: doc_id: 271172 cord_uid: y48dovux summary respiratory tract infections are among the commonest of illnesses, and most individuals will experience two to five infections during each year of their lives. the illnesses vary from relatively mild common colds caused by rhinoviruses and coronaviruses, to severe bronchiolitis and pneumonia caused by adenoviruses and influenza viruses and respiratory syncytial virus (rsv) in infants: the former is associated with little morbidity and no mortality, while influenza is responsible annually for between 1 and 25 thousand deaths per 50 million population. over 140 viruses cause respiratory tract infections, with the added complications of influenza viruses where new antigenic variants are recognized almost annually; and immunity to infection by one virus strain offers little or no protection to infection by others. knowledge of the mechanisms of spread of respiratory viruses is largely understood and has helped in infection control; however, the clinical signs and symptoms of infection tend not to be diagnostic of the causative agent; and although vaccines have been developed for the more serious infections such as influenza and some adenovirus infection, none are available for other important infections. treatment is largely symptomatic, but the compounds ribovirin for rsv infection and amantadine for influenza virus infection have been shown to be effective. much remains to be discovered before more effective measures can be implemented to limit the enormous costs incurred by these infections. the number of viruses involved is large, and the spectrum of illness complex: in the present chapter, the viruses are described, together with the features of the epidemiology, pathogenesis, clinical disease, and treatment. virus infections of the respiratory tract affect all people in all places at all times throughout life. over 140 viruses are involved: the average child under the age of five years or adult in the developed countries will experience four to five and two to four infections each year, respectively; and although many infections are relatively mild, some are life-threatening. douglas and edelstein (1988) have said that, "about three hundred million cases of acute respiratory disease occur in the united states each year, accounting for about one hundred and fifty million visits to physicians. the cost of these illnesses exceeds one billion dollars exclusive of time lost for work": this statistic, adjusted for population, can be directed at any country. the size of the problem demands much of a general physician's time, while the importance has focused the attention of epidemiologists, microbiologists, pharmaceutical companies and many others. the viruses which cause respiratory tract infections in man include both rna and dna viruses in six families: this does not include many viruses which can cause respiratory symptoms, but where the principal tissue or organ of infection is other than the respiratory tract. these families are listed in table 1 , together with data on the finer classification into sub-families, genera, or sub-genera, the number of distinct types of virus in each group and the number of these that cause human respiratory infections. thus, there are six families encapsulating 21 sub-divisions and over 240 viruses, of which over 140 cause respiratory tract infections in man, while the remainder cause other types of infection or infections in other species. the exact numbers are complicated by the rhinoviruses where more than 100 serotypes are known, but many more probably exist, and by influenza a and b virus which exhibit continuing genetic change producing new antigenic variants in most years. the respiratory tract can be considered to be stratified horizontally into levels, beginning with the nasal passages and descending sequentially to the throat, trachea, bronchi, bronchioles, and the alveoli: it is convenient to divide the respiratory tract in this manner, since many viruses have a predilection for infection of a particular level, and these associations are shown in table 2 . thus, the common cold, limited to the nasal passages, is caused commonly by rhinoviruses and virology. rhinoviruses belong to the family picornaviridae and are distinguished from other members of this family by a higher particle density, acid lability which limits intestinal infection, relative heat stability, resistance to a variety of detergents and survival for several hours or even days outside the body. virus particles contain an internal rna which is single-stranded with positive polarity surrounded by a capsid of 60 capsomeres arranged in icosahedral symmetry: particles are approximately 30 nm in diameter. rhinoviruses replicate in human cells and less well in the cells of other primates: the conditions for growth are a relatively low ph of 7.0 to 7.2, a low temperature of 33 °c and aeration of cell cultures; and under these conditions cytopathic effects can be observed. the growth cycle takes some 11 to 17 hours; and each infected cell yields 10-200 virus particles. cross-neutralization tests indicate over 100 distinct serological types, but some sharing of epitopes does exist (hamparian et al., 1987) . epidemiology. the frequency of respiratory infections is 2 to 4 each year for adults, with a higher incidence in children: most are conmion colds of which some 30% are due to the rhinoviruses, and a number of serotypes can circulate concurrently with no evidence of cross-immunity. infections tend to be most common in spring and autumn, and are spread by large and small droplets of nasal secretions from infected persons, by direct spread from nasal secretions via hand to hand contact and auto-inoculation of nasal or conjunctival mucosa or via inanimate objects: infection can be initiated by very little virus, and the viruses can survive for long periods outside the body. peak titers appear in nasal secretions 1 to 3 days after the onset of symptoms, and virus persists for 5 to 7 days; however, virus secretion can continue for 2 to 3 weeks. a serum antibody response is seen in 30 to 90% of affected patients and a local iga response in 70 to 100%: immunity lasts 2 to 4 years, but the multiplicity of circulating serotypes gives litde or no protection against clinical disease (gwaltney, 1982) . pathogenesis. the pathogenesis of rhinovirus infection is not understood: viruses grow to high titer in the nasal epithelium, but biopsies of antigen-positive nasal mucosa cells show litde pathological change. infection causes progressive loss of ciliated epithelial cells, an inflammatory cell infiltrate, edema, hyperemia and a seromucinous exudation (hendley, 1983) . virus replication is predominantly in the nasal mucosa: other cell types such as in the nares and throat are relatively resistant. clinical features. after an incubation period of 1 to 4 days during which virus titers increase, symptoms of nasal obstruction and discharge, sneezing and coughing occur; a sore throat with erythema and headache may be experienced, but fever and systemic symptoms are usually absent. the symptoms usually improve after 2 to 3 days; but symptoms may last for 7 days, and in 25% of cases for 14 days. complications include sinusitis and otitis media; rhinoviruses have been isolated from the middle ear, but more often a bacterium has been found in conjunction with virus. small particle infection can cause tracheobronchitis and bronchiolitis, and exacerbations of asthma and chronic bronchitis are commonly precipitated by rhinovirus infection. diagnosis. rhinoviruses can be recovered from infected persons by inoculating nasal secretions onto monkey or human cells and incubating these in a roller drum at 33 °c to maximize the cytopathic effects; this appears 2 to 6 days after infection (al-nakib and tyrrell, 1988) . isolates are serotyped using a battery of neutralizing antisera in various combinations, but this is seldom carried out except for research purposes; and the lack of any specific treatment means that laboratory diagnosis is seldom attempted. treatment. there is no specific treatment for rhinovirus infections, and the treatments recommended are to relieve symptoms: nasal decongestants can be used to relieve obstruction; gargles will relieve the sore throat; and analgesics can be used if necessary. if the cough is severe, suppressants such as codeine or dextromethorphan can be given. the multiplicity of antigenic types makes vaccine development impractical, and volunteer studies have shown that inactivated vaccines have litde protective effect. locally applied interferon a suppresses virus replication, but long-term use is cytotoxic causing irritation, bleeding, and ulceration in some patients. virology. coronaviruses, within the family coronaviridae are divided into four antigenic groups containing a total of 13 serotypes of which two are infectious agents of man. the singular structure and method of replication distinguish these viruses. virus particles are 80 to 160 nm in diameter, and contain a large singlestranded rna with positive polarity and a nuclear protein coiled internally to form the nucleocapsid. this core is surrounded by a lipid bilayer into which are inserted three distinct glycoproteins which radiate from the surface: these are a receptor binding glycoprotein which initiates cell fusion and induces neutrahzing antibody production; a glycoprotein with combined neuraminidase and hemagglutinating activity; and a glycoprotein which binds to the internal nucleocapsid and effects virus budding. coronaviruses can be cultivated in organ cultures of human embryonic trachea or less successfully in tissue cultures of human cells. the serotypes are distinguished by neutralization tests, or more commonly by immunofluorescence or elisa tests. epidemiology. coronavirus infections usually occur in the winter and early spring months, with epidemics every 2 to 3 years: the two serotypes causing human infection tend to be exclusive, and 5 to 30% of common colds are due to these viruses. infection is spread by small particle aerosols; many infections are asymptomatic, and may not induce an immune response, but clinical infections invariably induce detectable antibody; however, antibody titers may not be sustained, and reinfection with the same serotype can occur within months (isaacs et al., 1983) . almost 100% of adults have detectable antibody to both serotypes. pathogenesis and clinical disease. these features are similar to those described for rhinoviruses; however, the incubation period is usually longer at 2 to 5 days, the illness persists for 2 to 18 days, some 25% of patients exhibit a mild fever but throat infection and cough is less frequent. complications of infection are unusual, but isolated cases of pneumonia are recorded; and these viruses cause exacerbations of asthma and chronic bronchitis. the pathogenesis of infection is little understood, but the evidence suggests a slow and progressive destruction to the ciliated epithelium. diagnosis and treatment. diagnosis of coronavirus infection is both difficult and unsatisfactory: organ cultures are the most sensitive for virus isolation but are rarely available, and tissue culture systems are relatively insensitive. direct demonstration of virus in cells in respiratory secretions by immunofluorescence or elisa are used in some laboratories, but most diagnoses are made retrospectively by demonstrating a significant rise of antibody following infection (schieble and kapikian, 1979) . as for rhinovirus infection, treatment is symptomatic: no antiviral drugs have been developed and no vaccine is available for coronavirus infections. experimentally, a-interferon has been shown to ameliorate symptoms and reduce virus replication. some 50% of common colds, characterized as an afebrile illness localized to the nasal epithelium and presenting with nasal obstruction and discharge, sneezing and coughing are caused by rhinoviruses and coronaviruses; the remainder are caused by a number of other viruses including echoviruses, coxsackie a and b viruses. respiratory syncytial virus, influenza c and parainfluenza viruses ( table 2) . as these infections are relatively mild and a large number of agents are involved, exact laboratory diagnosis is seldom undertaken except for research purposes, and there are no specific treatments. virology. there are 47 serotypes of human adenoviruses in six subgenera in the genus mastadenovirus in the family adenoviridae: eight serotypes in three subgenera cause respiratory infection in man (table 2 ). virus particles contain single-stranded dna surrounded by at least 11 polypeptides. the outer capsid is made up of 252 capsomeres arranged in an isocosahedral of 20 triangles with 12 vortices giving a particle diameter of 60 to 80 nm: 240 capsomeres are hexons with six neighbors and are composed of a single protein with common epitopes for all serotypes; and the remaining 12 capsomeres are pentons with five neighbors, each composed of a base with a fiber-like projection radiating outwards and are serotype specific. human adenoviruses only replicate sequentially in human cells. virus particles attach to cell receptors via the penton fiber and then to other cell receptors by the hexons: following entry, cellular dna and protein synthesis are inhibited; replication takes place in the cell nucleus; newly-formed pentons are toxic to cells; and virus is released after cell death. the division of human adenoviruses into six subgenera is based on the sequence homology of the dna genome, dna fragment analysis following restriction enzyme treatment, oncogenicity for newborn hamsters, the molecular mass of the internal proteins, length of fibers and precent g + c in the viral dna. the division into serotypes is based on neutralization tests which show no cross-reactivity. this classification is important, since it is consistent with the association of the viruses with various human infections (wadell, 1984) . epidemiology. adenoviruses cause both sporadic and epidemic infections: five per cent of acute respiratory infections and ten per cent of febrile infections of children, together with 3 and 7% of these infections in adults, are caused by these viruses; and some 10% of all pneumonias in children are due to adenovirus infection. transmission is by respiratory droplets, whilst in children fecal/oral transmission is important: fecal excretion can continue for weeks and months after acute infection. by the age of 10 years, 50% of children have antibody to serotypes 1,2, and 5; antibody to other serotypes is less common, but in total the results reflect the importance of these viruses in human infection. pathogenesis. adenoviruses, spread by droplet infection, impinge on epithelial cells in the pharynx or in the lower respiratory tract to enter and kill cells by a combination of inhibition of cellular metabolism, virus replication and the toxic effects of the penton: the results are extensive desquamation of affected areas, causing sore throat, necrotizing bronchitis, bronchiolitis and interstitial pneumonia. in lymphoid cells, infection causes hypertrophy: affected cells can harbor latent virus for months, years or throughout life; and although 50% of excised tonsils and adenoids contain latent virus, there is no evidence that this can exacerbate, except possibly in pertussis syndrome. clinical disease. adenoviruses cause some 5% of all acute respiratory disease (ard) seen in children aged five years or less (brandt et al., 1969) . these are predominantly due to serotypes 1,2 and 5 (subgenera b); and the frequency of these infections in children means adult infections are unusual. spread is by droplet infection: after an incubation period of 2 to 4 days, symptoms begin with pharyngitis, cough, nasal congestion and coryza, whilst fever, exudative tonsillitis, malaise, headache and myalgia are often seen. infection tends to progress over a 2 to 3 day period, and resolves in 5 to 8 days. infection may extend to cause laryngotracheobronchitis, bronchitis and pneumonia, the last being particularly important in young children and the cause of some deaths. sporadic infections of both children and adults are also caused by adenovirus types 3 and 7 (subgenera c): these present as above, but laryngotracheobronchitis and pneumonia are more commonly seen, with 40 to 50% showing radiological evidence of pneumonia; and with conjunctivitis, when the infection is known as pharyngo-conjunctival fever, and that occasionally is seen as epidemics among children and in families. the respiratory tract complications are particularly severe in immunocompromised patients. probably due to overcrowding and fatigue, adenovirus types 3,4 and 7 cause epidemic ard in military recruits: some 80% of recruits can become infected, 20 to 40% require hospitalization for lower respiratory tract infection and pneumonia, and deaths are recorded. adenoviruses have been frequendy isolated from patients with whooping cough syndrome in conjunction with bordetella pertussis', this is usually reported as adenovirus type 5. however, there is little evidence that adenoviruses can produce this syndrome alone: the presence of adenovirus may be due to reactivation from tonsillar tissue by bordetella pertussis to complicate the infective process. diagnosis. adenoviruses are present in throat swabs, throat or nasal washings or feces of infected persons. virus growth is best in tissue culture of human embryonic kidney, but hela and kb cells are a more available alternative. growth is recognized by a characteristic cytopathic effect; however, this may take days or weeks to develop, but virus can be demonstrated early in infected cells by immunofluorescence with specific antibody. isolates can be identified as adenoviruses with antisera to the common epitopes of the hexon using complement fixation, immunofluorescence or elisa tests; and specific serotypes identified by neutralization tests. there are no effective antiviral agents for the treatment of adenovirus infection, and treatment is limited to the relief of symptoms. in addition, there is no adenovirus vaccine; however, the problem of ard in military recruits is of such importance that a vaccine has been developed: live adenovirus types 4 and 7, the main viruses responsible for ard, are given orally as a coated enteric preparation; this establishes an intestinal infection, induces an immune reaction and, in bypassing the respiratory tract, does not cause symptoms (tarafuji etal, 1979) . virology. epstein-barr virus (eb v) is a member of the family herpetoviridae, and the only member of the sub-family gammaherpesvirinae to cause infection in humans: the virus is distinguished from other herpetoviridae in replicating or establishing a latent infection in p-lymphocytes, and the potential for promoting tumorgenicity of these cells. the virus is relatively large at 150 to 200 nm diameter: particles are composed sequentially of linear double-stranded dna and an internal core of proteins; a capsid of 162 capsomeres arranged in an icosahedral form; a protein tegument; and a trilaminar envelope derived from the cellular membrane into which are inserted spikes of several virus-specific glycoproteins. infection is primarily of the epithelial cells of the oropharynx, and via the complement receptor cd21: virus infection from these cells spreads to p-lymphocytes where a few cells undergo lytic infection while the majority support a latent infection that leads to cell proliferation. the mechanism for this latency is not fully understood, but is believed to be due to the absence or low-level of host cell transcriptional factors that are essential for virus replication: the result is a persistent, life-long p-lymphocyte infection. epidemiology serological studies from most developed countries have indicated that eb v infection is common, and 80 to 90% of adults have been infected: in developing countries, infection occurs earlier and is more common. once infected, a subject will remain a virus excretor for months, years and probably life, with continuous virus production from a variable number of p-lymphocytes in the oral cavity. infection is accompanied by an intense immune response; and it is the balance between virus production and immune status which determines the extent of virus secretion at any one time. the immune response is not sufficient to resolve the illness, as infected lymphocytes appear to be resistant to cytotoxic t cells and there is a down-regulation of hla antigen expression. the immortalization of p-lymphocytes is polyclonal and leads to cell proliferation; this can be further exaggerated in immunosuppressed patients, such as hiv or malaria infected persons, which in turn can lead to a chromosome translocation where the c-myc oncogene comes adjacent to a strong promoter: the outcome is a malignant cell transformation giving rise to burkitt's lymphoma (lenair and bornkamm, 1987) . pathogenesis. infection is from saliva of previously infected subjects: since virus production is low, transmission requires close contact, and peaks of infection are seen at ages 1-6 and 14-20 years corresponding to the ages of early and adolescent intimacy. the exact site of infection remains unknown, but waldeyer's ring, rich in lymphocytes, epithelial cells or the salivary glands are suggested by various authors. virus-infected (3-lymphocytes are disseminated throughout the body via the blood stream, and antigen-positive cells can be detected in most organs and tissues. infection is accompanied by an intense immune reaction to virus and to the proliferating p cells which includes virus-specific antibody, heterophile antibody, autoantibodies and rheumatoid factor (robinson and stevens, 1984) ; indeed, the clinical disease is due to the nature and the intensity of the immune responses. the changing pattern of the immune state regulates virus secretion, and the infectivity of the patient for others. clinical features. following contact with infected saliva, there is an incubation period of 30-45 days followed by a short prodromal illness of headache, malaise and fatigue: after this, the definitive symptoms of glandular fever occur. most patients complain of a sore throat with hyperemia and hyperplasia of the lymphoid tissue; exhibit an exudate over the pharynx; have a fever which lasts for some 10 days; and have cervical or general lymphadenopathy: fever and malaise can persist for weeks or months; secondary infections are common; blood dyscrasias occur; splenic enlargement is common, but rarely leads to rupture; and a mild hepatomegaly is seen in 5 to 10% of patients associated with a transient jaundice. mild rubilliform skin rashes can occur; however, ampicillin causes a maculopapular rash and is not used in patients with sore throats. oral cavity obstruction due to massive enlargement of tonsils, adenoids and epiglottis may require emergency treatment. fatalities are recorded, but are usually associated with immunocompromised patients. diagnosis. the symptoms of glandular fever usually alert the physician who can confirm his suspicions by a number of laboratory tests. the virus is difficult to cultivate: in vitro growth is only seen in lymphocytes, but only a few cells support virus replication; however, virus protein or dna can be demonstrated in infected cells by western blotdng or dna hybridizafion tests, respectively. serological tests include the demonstration of serum igm antibody to virus capsid proteins using an elisa test, or the detecuon of the heterophile antibody response using the paul bunnell test. atypical monocytes can form up to 20% of the peripheral blood leucocytes. treatment and control. both interferon and acyclovir have been shown to diminish virus secretion during treatment, but relapses occur when treatment is stopped: more importantly, neither treatment significantly ameliorates symptoms, and are therefore not recommended. in the absence of specific therapy, treatment is supportive: the sore throat can be treated with analgesics; and some suggest corticosteroids limit the duration of illness, possibly by the effect on the immune response. the importance of ebv in initiating burkitt's lymphoma in african children where the incidence is 1:5000, has focused attention on developing an ebv vaccine; such vaccines are undergoing clinical trials at the present time. although febrile sore throats are caused by adenovirus and ebv infection, a large number of other viruses can produce the same clinical symptoms; these include influenza a, b, and c, numerous serotypes of coxsackie and echoviruses and parainfluenza viruses. collectively, these latter viruses are responsible for more than half the febrile sore throats which occur; and collectively, viruses are responsible for over 90% of the febrile sore throats caused by infectious agents. virology. influenza viruses are the only viruses of the genus orthomyxovirus in the family orthomyxoviridae, affecting man, birds, horses, pigs, and other species. the viruses are approximately spherical with a diameter of 80 to 120 nm. each virus particle consists of single-stranded rna of negative polarity segmented into eight fragments of varying size. the rna is closely associated with a nuclear protein (np) and the polymerase enzyme complex to form a helical structure: the np takes one of three antigenic forms which allows influenza viruses to be classified into types a, b, and c. surrounding a nuclear protein is the matrix or membrane protein, and this in turn is surrounded by a lipid bilayer. inserted into the bilayer, and radiating from the surface, are two virus glycoproteins. the most numerous glycoprotein is the hemagglutinin (ha) which is the receptor binding component; the classification of influenza types into subtypes is based principally on the different antigenic forms of the ha (hi, h2, h3) molecule. the second glycoprotein is the neuraminidase (na) which is important in both cell infection and in facilitating the release of newly formed virus from the surface of infected cells; the na is antigenically variable (n,,n2) and this variation is used in the subtype classification of the viruses. influenza viruses grow in human and monkey cells and in the amniotic and allantoic cavity of embryonated hens' eggs. absorbed virus is uncoated and the virus rna together with the polymerase enzyme complex pass to the cell nucleus. replication, which is dependent on cell rna synthesis, produces virus components which pass to the cell membrane for assembly, and are then budded from the cell surface: the complete cycle takes 8 to 10 hours. epidemiology. influenza holds a unique position among the viruses causing respiratory tract infection, since it commonly and dramatically causes local outbreaks or widespread epidemics, and these occur in most parts of the world and in some countries in most years. epidemics occur suddenly and without warning, and the number of people infected range from few hundreds to hundreds of thousands: although short-lived, epidemics can infect up to 70% of a population, with clinical disease occurring in 50% of infected subjects, and deaths due direcdy or indirectly to influenza number from 1000 to 25,000 per 50 million persons per year in developed countries. the importance of this infection in causing morbidity and mortality is reflected in the enormous scientific effort made to understand the virus, the nature of the disease and to devise methods for control. when influenza virus isolates are cross-referenced to patients, time and place, several patterns are seen. firstly, most pandemics and widespread epidemics are caused by influenza a viruses; influenza b viruses are associated with self-limiting epidemics which occur in families or small communities; and influenza c virus is associated with sporadic infections, mainly among young children. influenza a exhibits the greatest antigenic diversity; influenza b exhibits some variation; and influenza c is relatively stable. secondly, the recorded patterns of influenza a epidemics during the past century exhibit two phenomena. every 10 to 15 years since records began in 1890, influenza virus has undergone major antigenic changes in the ha molecule, termed antigenic shift: the emergence of these new subtypes has resulted in the pandemics seen in the years 1933 (hi), 1947(h1), 1957(h2) and 1968(h3); and caused the epidemics which followed until the next new subtype emerged. these subtypes are distinct, and immunity to one provides no protection against infection by others. the origin of new subtypes cannot be by simple mutation from previously existing strains, since many genetic changes occur and intermediary strains are not found. two theories for the origin of new subtypes are advanced. firstly, two influenza viruses, one of human origin and the other probably of avian origin, infect the same cell: due to the segmental nature of the virus genomes, reassortant virus is easily produced combining the properties to infect man and the ha glycoprotein of the non-human strain. the new subtypes can now cause pandemic infection in populations with no previous immunity. an alternative theory is that the various subtypes circulate sequentially over a period of 70 to 80 years, since on two occasions antibody to new serotypes has been detected in sera from elderly people years prior to the emergence of that new subtype to cause pandemic infection: where the viruses survive between times is unknown. between the times of antigenic shift, epidemics occur in most years and the strains which cause them exhibit antigenic drift; these strains belong to the same subtype, but do not cross-react completely, and infection by one strain does not induce solid immunity to later emerging strains. this sequential accumulation of mutations arises naturally, and is selected by antigenic pressure in the immune and partially-immune population. in addition to antigenic drift and shift, viruses isolated from different places at the same time, and even viruses from different individuals in the same epidemic, can exhibit antigenic differences; this is known as inter-and intra-epidemic variation, and both underline the difficulty in matching vaccine virus to epidemic strains, and contributes to the disappointing low levels of immunity induced by inactivated influenza vaccines. the degree of crossprotection is directly related to the degree of cross-reaction of the virus ha, and this is shown in table 3 . influenza b viruses exhibit antigenic drift but not antigenic shift. pathogenesis. the pathogenesis of influenza has not been agreed among researchers, and many features are not understood. virological investigations indicate that infection is from virus inhaled as droplets on to the epithelial cells of both the upper and lower respiratory airway. histological studies of nasal exudate and tracheal biopsies indicate that the major site of infection is the ciliated columnar epithelial cells which become progressively rounded and swollen, and exhibit vacuolation with loss of ciliation; the progression usually begins in the tracheal bronchial epithelium and then ascends. the result is the widespread destruction of the ciliated epithelium down to the basement membrane which itself is not affected; the lesions become increasingly permeable with polymorphonuclear infiltration and edema. because of the generalized symptoms of uncomplicated influenza, viremic spread has been suspected; however, there is no conclusive evidence that viremia occurs. in contrast, virus infections have been associated with ecg and eeg changes; some unconfirmed observations of virus antigen in brain and heart tissue have been published; and infection can be associated with viral encephalitis, particularly among children. these findings suggest dissemination of either virus or virus products from the respiratory tract; virus is known to grow in leucocytes and the release of pyrogens or cytokines offers an explanation for some systemic symptoms. clinical disease. the symptoms of influenza tend to be constant regardless of the subtype or strain of virus; however, the clinical features of influenza in young children may vary from that of adults, with croup a more common symptom in children and sore throats more common in adults. droplet infection is followed by an incubation period of approximately 24 to 96 hours: the onset of illness is usually abrupt. symptoms include fever, headache, photophobia, shivering, dry cough, malaise, aching of muscles and a dry, ticking throat which can lead to the voice becoming husky or lost. the eyes are often watery, burning and painful on movement. fever is usually continuous, and typically lasts some 3 days: in a percentage of patients, a second rise in temperature may occur, usually smaller than the first, which gives the infection a biphasic fever curve. the cough may persist for several days; the nose can be blocked or show a purulent discharge; and myalgia is most severe in leg muscles, but also may involve the other extremities. acute illness usually resolves within 7 days, but patients frequently complain of feeling listless for weeks, and depression is a common residual complaint. infections caused by influenza b resemble closely those caused by influenza a; in contrast, influenza c is usually a mild upper respiratory tract infection. the complications of infection include tracheobronchitis and bronchiolitis: these patients exhibit a productive cough and chest tightness, and crepitations are commonly heard but the lungs are usually radiologically clear. these complications are most conmionly seen in patients with obstructive bronchitis and in older people, and death from influenza can result in such patients. pneumonia in patients with influenza virus infection can be primary or secondary. in viral pneumonia, patients developed a persistent fever and leucocytosis, dyspnea, hypoxia, and cyanosis; this follows the acute symptoms described above. sputum specimens show no bacterial cause, and a proportion of these patients die of diffuse hemorrhagic pneumonia. more commonly, pneumonia following influenza is due to secondary bacterial infection, principally with staphylococcus aureus, but also with streptococcus pneumoniae, hemophilus influenzae and other bacterial species: this complication is a major cause of death among elderly people and those with underlying disease such as congestive heart failure and chronic bronchitis. in addition, patients with diabetes, renal disease, alcoholism and those who are pregnant also have an increased susceptibility to secondary bronchopneumonia. influenza is also associated with myalgia, a common feature of acute disease, but clinical myositis and myoglobinuria can occur: the symptoms develop after the onset of respiratory infection, when muscles become painful and tender, but without neurological symptoms. an important complication of influenza infection is the syndrome known as reye's syndrome characterized by encephalopathy and fatty liver degeneration; this is chiefly seen at age 8 to 15 years, and among those hospitalized the mortality can be as high as 50%. the association of reye's syndrome following infection by influenza a or b or other viruses has been fully demonstrated, but the pathogenesis remains obscure (carey et al., 1976) : researchers have highlighted the association of virus infection with treatment of fever with high concentrations of aspirin, and for this reason aspirin should not be given to patients in this age group. more conjectural is the association of influenza infection in pregnancy with congenital abnormality; this is not justified with our current knowledge. further complications reported are ketoacidosis in diabetic patients, acute viral encephalitis in children, guillain-barre syndrome, sudden infant death syndrome and toxic shock syndrome resulting from the dual association of staphylococcus aureus and influenza infection. diagnosis. influenza viruses can be recovered from throat washings or swabs by inoculating tissue cultures of kidney tissue from rhesus monkeys, chicks, and a variety of other species. after incubation, newly produced virus can be detected in supernatant fluids by the ability to agglutinate erythrocytes (hemagglutination), or the adherence of erythrocytes to virus particles assembled on the cell surface (hemadsorption). influenza virus can also be cultured in the amniotic cavity of embyronated eggs: after incubation, high titers of virus are found in the amniotic fluid, and are detected by hemagglutination. the viruses are recognized as influenza a, b, or c by complement fixation tests using extracts of infected cells containing high concentrations of np antigen, and type-specific antisera. further identification of influenza isolates into sub-types and strains is dependent upon antigenic differences in the ha. this is determined by hemagglutination inhibition (hi) tests against antisera raised in experimental animals against a range of virus subtypes and strains: the titer of each antisera against homologous virus is known prior to testing, and the pattern of hi titers found against an unknown influenza virus determines the strain and type. however, this is a highly specialized typing system which is the responsibility of who reference laboratories who constantly type new isolated viruses in a worldwide endeavor to detect new virus variants as they arise. proof of influenza infection can also be obtained by demonstrating a rise in specific complement fixating or hi antibodies in sera collected early after the onset of symptoms and 14 to 21 days later. control and treatment. the constant, almost annual antigenic changes seen in influenza virus a and to a lesser extent influenza b means that vaccines need to be developed for each new epidemic strain. at present, vaccines are produced by inoculating virus into embryonated eggs, purifying and inactivating the resultant virus growth to give a whole virus, disrupted virus or virus subunit (ha and na) vaccines (potter, 1982) . to date, no live attenuated virus vaccines are available. inactivated vaccines produce few reactions, but most are mild and ephemeral; induce serum antibody in the majority of subjects, but immunity in only 60 to 90% of vaccinees. due to the severity and fatalities from influenza, vaccine is offered annually to at-risk patients: these include persons aged 65 yrs of age, patients with a history of chronic chest or heart disease, and patients with asthma, renal dysfunction and metabolic disorders. in some years, such as when a new subtype is recognized, key personnel in industry and social services should be offered vaccine. at present, the treatment of influenza is symptomatic: patients are advised to remain in bed for 2 to 3 days until the acute symptoms subside; symptoms of headache and fever are treated with paracetamol; codeine linctus can relieve the cough; insomnia may be treated by barbiturates or promethazine; and antibiotics are indicated when chest complications are present or suspected. the use of prophylactic antibiotics in patients with chronic chest disease is common, but not recommended. the compound amantadine, and the analog rimantidine, are active against influenza: an oral dose of 100 mg per day given to people in contact with influenza decreases the chance of infection by some 70%, and given to patients with clinical disease can reduce both the length and severity of disease (dolin et al., 1976) . virology. parainfluenza viruses of the genus paramyxovirus in the family paramyxoviridae are distinguished by the size and shape of the nucleocapsid, biochemical similarity, antigenic cross-reactivity and the presence of a surface glycoprotein with combined hemagglutination and neuraminidase activity. virus particles consist of single, non-segmented, negative-strand rna, and three internal proteins surrounded by a lipid bilayer with a fourth protein, into which are inserted the hemagglutinin/neuraminidase molecule and a fusion protein which both radiate from the surface of the virion particle. the complete virion has a diameter of 150-200 nm. the viruses replicate in primary human and monkey cells with assembly of new virus particles taking place in the cytoplasm and release by budding: the effect on the cells is lytic with cytopathic effect and occasionally syncytia formation. viruses can be detected by hemadsorption of guinea-pig erythrocytes to infected cells through virion particles budding through the cell membrane. four serotypes cause respiratory infection in man, and are individually recognized by various tests including hemagglutination inhibition, hemadsorption inhibition and neutralization tests. epidemiology. parainfluenza viruses types 1, 2, and 3 are the major cause of tracheobronchitis and croup in young children; type 3 is frequently associated with pneumonia; and type 4 causes mild upper respiratory tract infections. infection is by droplets and requires only a small dose of virus; virus from infected persons is shed for 3 to 10 days, but in some cases can continue for 3 to 4 weeks. infection by all types is worldwide with peak numbers occurring in the winter months: epidemics are frequently recorded, and reinfection common (chapman et al., 1981) . over 50% have antibodies to one or more of these viruses by age 2 years, and over 75% by age 4 years. pathogenesis. mild infections are mostly of the nose and throat with minimal involvement of the lower respiratory tract; more extensive infection by types 1 and 2 involves the larynx, trachea, and bronchi with pneumonia occurring in some 15 to 20% of patients; and type 3 infection causing a higher incidence of bronchiolitis and pneumonia. virus replication has a lytic effect on the epithelial cells, whilst in the trachea and bronchi infection causes excess mucus production leading to atelectasis and pneumonia. infection induces an ige antibody response in serious cases which in turn initiates histamine release: it is thought that these responses are important in the pathogenesis of infection. clinical features. following an incubation period of 2 to 4 days, primary infection in children is seen as a rhinitis and pharyngitis with erythema: some evidence of bronchitis is commonly seen with hoarseness, cough with croup and bronchitis with rhonchi. fever is recorded and lasts 2-3 days. in more severe cases, infection extends to produce a heightened fever, a laryngotracheobronchitis with a barking cough and croup which lasts for 48 to 72 hours; symptoms may worsen to cause air hunger and cyanosis, sternal and intercostal retractions, airway obstruction and glottic and subglottic narrowing (parrott et al., 1962) : if pneumonia develops, the cough is productive, and radiological examination may show interstitial and perihylar infiltration. diagnosis. viruses present in throat washings can be cultivated on monkey kidney cells: after replication, virus particles can be demonstrated on the surface of infected cells by hemadsorption. alternatively, virus can be detected directly by immunofluorescence tests on respiratory secretions (ray and minnich, 1987) . diagnosis based on serological tests is less satisfactory: primary infection induces a type-specific antibody response detectable by hemagglutination inhibition, complement fixation, or neutralization tests, but subsequent infections induce a heterotypic response. control and treatment. much research has been carried out on the development of a parainfluenza virus vaccine but none is available at the present time: inactivated vaccines induce serum antibodies, but only partial immunity. treatment is symptomatic: children may be nursed in plastic tents supplied with cool moistened oxygen for 2 or 3 days to relieve respiratory symptoms; severe obstruction may require endotracheal intubation or tracheostomy; and accumulative and excessive tracheobronchial secretion may require bronchoscopy aspiration. antibiotics are used where investigations indicate secondary bacterial infection. the use of corticosteroids is contentious, but aerosolized preparations of the antiviral compound ribavirin may be valuable. some 50% of cases of laryngotracheitis and croup in children under age three years are due to parainfluenza virus infection; the remaining cases are due to influenza a and b, respiratory syncytial virus (rsv) and various serotypes of coxsackie and echoviruses. the severity of infection indicates laboratory investigation; thus, the contribution of these latter agents to the syndrome is well-documented. acute bronchitis, or more commonly tracheo-bronchitis since contiguous respiratory compartments are usually involved, has been associated with adenovirus type 3, 4 and 7 infection in both children and adults, but other serotypes have been identified. among the other infections causing bronchitis are rhinoviruses and rsv virus in children, and measles and influenza a virus infections in children and adults: these infections may precede secondary bacterial infection. exacerbation of chronic bronchitis is frequently associated with virus infections; these include a wide range of viruses, but are most commonly caused by rsv, rhinoviruses, and parainfluenza viruses. virology. respiratory syncytial virus (rsv) belongs to the genus pneumovirus within the family paramyxoviridae; and is distinguished by the form of a nucleocapsid, replication entirely in the cell cytoplasm and the absence of hemagglutinin and neuraminidase glycoproteins. the virus particle is structurally similar to that of other members of the family consisting of a single, non-segmented, negative strand of rna and three internal proteins surrounded by a lipid bilayer with two associated proteins: inserted into the outer aspect of the lipid bilayer are spikes of an attachment protein and a fusion protein. the diameter of the virion is 120 to 300 nm. rsv replicates in the cytoplasm of a range of human and animal cells: cell death is principally the result of cell fusion, and the formation of multinucleate syncytia indicates the presence of virus and gives the virus its name. antigenic variants are known, and this has resulted in recognition of two subgroups. epidemiology. rsv causes annual epidemics in the winter months in most countries which are indicated by an abrupt rise in the number of pediatric admissions to hospital (glezen and denny, 1973) . infection is spread by large droplets and therefore require close contact; by hand from nasal and conjunctival secretions; or via inanimate objects and self-inoculation: the virus is highly infectious, and 50% of children are infected by 1 year of life, and all children by age 2 years, with recurrent infection common. the virus causes 75% of all bronchiolitis and 25% of all pneumonia cases seen in children under 1 year of age: the mortality rate is 0.5 to 2.5% with most in children with underlying heart or respiratory disease. infection is essentially an upper respiratory tract infection in children aged less than 6 weeks or over 6 months; however, between these age limits 30% of infections involve the lower respiratory tract. recovery is accompanied by serum antibody and a cell-mediated immune response which protects against subsequent lower respiratory tract infection; the local iga antibody response is ephemeral, allowing further upper respiratory tract infections in later life. pathogenesis. after an incubation period of 3 to 6 days, infection begins in the nasopharynx with virus titers reaching a maximum at 2 to 3 days, declining between 3 to 6 days but can be detected in some patients for 3 weeks. spread to the lower respiratory tract is by cell-to-cell interaction in the respiratory epithelium and via aspirates. cell infection is cytopathic following cell fusion, causing inflammation and necrosis with associated plugging of the airways; but other factors are involved in the disease which are not fully defined: these include immunopathology due to antibody production, the formation of antigen-antibody complexes, delayed hypersensitivity reactions, an exaggerated cytotoxic t-cell response and an ige response as described for parainfluenza virus infection (welliver et al., 1984) . clinical disease. in children aged less than 6 weeks or greater than 6 months infection is usually seen as an upper respiratory tract (urt) infection with rhinitis, a mild fever, sneezing, and wheezing; some 40% of children exhibit a lower respiratory tract involvement with tachypnea, rales, and rhonchi. more severe lower respiratory tract (lrt) infection may occur, but this is more common at age 6 to 24 weeks: following the mainly urt infection, patients develop a bronchiolitis with dyspnea, severe tachypnea, and intercostal and substernal retraction; and in most severe cases an added pneumonia occurs with hypoxia and cyanosis. radiological appearances vary from normal to that of a bacterial pneumonia, but clinical severity is not mirrored by the radiological changes. the infection lasts 6 to 12 days with patients showing improvement after 3 to 4 days, but in severe cases symptoms may persist for several weeks. diagnosis. aspirates or nasal secretions contain virus that can be detected by inoculating tissue cultures which show syncytia formation following virus replication, or by direct tests for virus antigen using immunofluorescence tests. infection induces a rise in serum antibody detected by complement fixation or neutralization tests. treatment. patients with lrt infection commonly require hospitalization for supportive therapy: reduction of fever and hydration is commonly adequate, but in more severe cases oxygen may be required to assist breathing; mechanical removal of respiratory secretions may be necessary, and blood gases should be monitored. the infection responds to treatment with the antiviral compound ribovirin, and administration of this compound as a small particle aerosol has proven effective (hall et al., 1983) . no effective vaccine has been developed despite 20 years of dedicated research. studies have shown that 50 to 90% of bronchiolitis cases are caused by rsv; and characterized by necrosis and sloughing of the bronchiolar epithelial leading to the plugging of small airways, obstruction and atelectasis (hall et al., 1986) . however, other viruses more appropriately associated with other compartments of the respiratory tract, can produce the same pathological changes and clinical symptoms. thus, bronchiolitis has been associated with infections by influenza viruses a and b, and adenoviruses; and in young children with parainfluenza virus infection: these virus infections are described under separate headings. pneumonia, characterized by radiological changes, physical signs and pathology, is uncommonly related to infection by any virus; however, three reservations should be admitted. firstly, cases of acute pneumonia due to adenovirus and influenza viruses, although unusual, are well-documented, and fatalities have been recorded following these infections. secondly, severe infection by viruses in higher compartments of the respiratory tract, can extend to cause pneumonia: these include adenovirus and influenza viruses again, and rsv and parainfluenza viruses in young children. thirdly, primary pneumonia is a rare presentation by measles, chicken pox (varicella/zoster) and cytomegalovirus (cmv) infection: although unusual in normal subjects these are more commonly seen in immunocompromised persons, where the infection can be devastating. pneumonia in the immunocompromised by measles, chicken pox or cmv is usually an extension of typical infection to involve the lungs, but can present without a rash or with an atypical rash in patients with no history of infection. patients develop cough and chest pains, and more seriously dyspnea and cyanosis; x-rays may show evidence of viral pneumonia with atypical, patchy consolidation; and deaths are recorded in 40% or more of immunocompromised patients. pathologically, the alveoli contain edema fluid with macrophages, but few polymorphonuclear cells; and typically and diagnostically, giant multinuclear cells. the viruses can be grown from the bronchial secretions; however, since these are rapidly progressing infections and suggested treatments are available for two of these viruses, quicker methods such as the direct demonstration of virus by immunofluorescence or elis a tests on cells in secretions are needed. there is no treatment for measles virus pneumonia; acyclovir or ganciclovir are reported to be of value in the treatment of cmv; and acyclovir will prevent pneumonia in chicken pox patients, but has no proven value in treating established pneumonia. respiratory tract infections are among the commonest of illnesses, and most individuals will experience two to five infections during each year of their lives. the illnesses vary from relatively mild common colds caused by rhinoviruses and coronaviruses, to severe bronchiolitis and pneumonia caused by adenoviruses and influenza viruses and respiratory syncytial virus (rsv) in infants: the former is associated with little morbidity and no mortality, while influenza is responsible annually for between 1 and 25 thousand deaths per 50 million population. over 140 viruses cause respiratory tract infections, with the added complications of influenza viruses where new antigenic variants are recognized almost annually; and immunity to infection by one virus strain offers little or no protection to infection by others. knowledge of the mechanisms of spread of respiratory viruses is largely understood and has helped in infection control; however, the clinical signs and symptoms of infection tend not to be diagnostic of the causative agent; and although vaccines have been developed for the more serious infections such as influenza and some adenovirus infection, none are available for other important infections. treatment is largely symptomatic, but the compounds ribovirin for rsv infection and amantadine for influenza virus infection have been shown to be effective. much remains to be discovered before more effective measures can be implemented to limit the enormous costs incurred by these infections. the number of viruses involved is large, and the spectrum of illness complex: in the present chapter, the viruses are described, together with the features of the epidemiology, pathogenesis, clinical disease, and treatment. common cold viruses-rhinoviruses infections in 18,000 infants and children in a controlled study of respiratory tract disease. i. adenovirus pathogenicity in relation to serological type and illness syndrome the epidemiology of tracheobronchitis in pediatric practice a nationwide outbreak of reye's syndrome. amer a controlled trial of amantadine and rimantadine in the prophylaxis of influenza a infection epidemiology of acute lower respiratory disease in children rhinoviruses. in: viral infections of humans: epidemiology and control aerosalised ribavirin treatment of infants with respiratory syncytial virus infection respiratory syncytial viral infection in children with compromised immune function a collaborative report: rhinoviruses-extension of the numbering system from 89-l(x) rhinovirus colds: immunology and pathogenesis epidemiology of coronavirus respiratory infections burkitt's lymphoma, a human cancer model for the study of the multistep development of cancer: proposal for a new scenario inactivated influenza virus vaccine efficiency of immunofluorescence for rapid diagnosis of common respiratory viruses production of autoantibodies to cellular antigens by human b cells transformed by epstein-barr virus coronaviruses. in: diagnostic procedures for viral, rickettsial and chlamydial infections simultaneous administration of live, enteric-coated adenovirus 4, 7 and 21 vaccines: safety and immunogenicity molecular epidemiology of human adenoviruses defective regulation of immune responses in respiratory syncytial virus infection rhinoviruses in seattle families 1975-1979 the tecumseh study of respiratory disease vi. frequency of the relationship between outbreaks of coronavirus infection world-wide epidemiology of human adenovirus infections influenza seminars in respiratory infections parainfluenza virus bronchiolitis: epidemiology and pathology respiratory syncytial virus epidemiology of respiratory syncytial virus infection in washington d.c. ill composite analyses of eleven consecutive yearly epidemics respiratory syncytial virus: brief review key: cord-266822-ecq50ye2 authors: rath, barbara; conrad, tim; myles, puja; alchikh, maren; ma, xiaolin; hoppe, christian; tief, franziska; chen, xi; obermeier, patrick; kisler, bron; schweiger, brunhilde title: influenza and other respiratory viruses: standardizing disease severity in surveillance and clinical trials date: 2017-05-12 journal: expert rev anti infect ther doi: 10.1080/14787210.2017.1295847 sha: doc_id: 266822 cord_uid: ecq50ye2 introduction: influenza-like illness is a leading cause of hospitalization in children. disease burden due to influenza and other respiratory viral infections is reported on a population level, but clinical scores measuring individual changes in disease severity are urgently needed. areas covered: we present a composite clinical score allowing individual patient data analyses of disease severity based on systematic literature review and who-criteria for uncomplicated and complicated disease. the 22-item vivi disease severity score showed a normal distribution in a pediatric cohort of 6073 children aged 0–18 years (mean age 3.13; s.d. 3.89; range: 0 to 18.79). expert commentary: the vivi score was correlated with risk of antibiotic use as well as need for hospitalization and intensive care. the vivi score was used to track children with influenza, respiratory syncytial virus, human metapneumovirus, human rhinovirus, and adenovirus infections and is fully compliant with regulatory data standards. the vivi disease severity score mobile application allows physicians to measure disease severity at the point-of care thereby taking clinical trials to the next level. influenza-like illness (ili) and acute respiratory infections (ari) in children are common. the clinical presentation may range from subtle to severe symptoms requiring advanced medical care [1, 2] . the wide spectrum of disease presentations and the role of risk factors (rfs) in terms of disease severity are poorly understood. laboratory diagnostics are not usually ordered in routine care [3] [4] [5] [6] . surveillance programs should rely on laboratory-confirmed cases rather than clinical suspicion to solve the denominator problem. this will allow the timely detection of virus-specific seasonality in a given (sub)population [7] . an even greater challenge will present itself when investigators wish to determine the impact of different respiratory viruses on disease burden [8] . a deeper understanding of disease severity in relation to specific respiratory viruses will help in the monitoring of the real-world impact of 'natural' or untreated disease as well as preventive measures and therapeutic interventions such as vaccines and antivirals. the timely detection of seasonality will help with the targeted and costeffective use of viral diagnostics in hospital-based surveillance settings. ideally, viral diagnostics should be aligned with simultaneous standardized disease severity assessments. standardized measures of disease severity are urgently needed for clinical trials of vaccines and antivirals currently in development for ari caused by influenza (flu), respiratory syncytial virus (rsv), human metapneumovirus (hmpv), adenovirus (adv), or human rhinovirus (hrv) [9] [10] [11] [12] [13] [14] [15] [16] [17] . furthermore, it would be desirable to assess, at the point of care, which patients are suffering from severe disease in relation to their perceived rf profile, and to use such point-of-care assessments to individualize the use of anti-infective therapy. experience during the recent influenza pandemic has shown that influenza disease severity appears rather unpredictable, especially in young patients. whilst the majority of adults with severe disease did have previously identifiable rfs, the majority of children affected by severe disease did not [18, 19] . the expected or perceived risk of severe outcomes may also influence a physician's decision to test a patient for influenza and other respiratory viruses [20] . there is little consensus on which symptoms should trigger a physician's suspicion, and local practices differ significantly from site to site and from season to season [19, [21] [22] [23] [24] . comprehensive reviews of the published literature and disease severity measurements used in clinical trials and surveillance systems are lacking. the numerous observational studies and clinical trials assessing the prevention and treatment influenza and other respiratory viruses have been rather inconsistent. commonly used indicators of disease severity such as 'hospitalization,' a diagnosis of 'pneumonia,' and other adverse outcomes including mortality are known to be highly dependent on the studied population, the medical setting, the choice of data sources, and the availability of resources [25] . head-to-head comparisons and meta-analyses comparing different preventive and treatment interventions will require universally accepted disease severity measurements. sentinel surveillance systems tend to focus on private practices and laboratory testing based on clinical suspicion on behalf of primary care providers working at surveillance sites [26] . with children being the most prominent transmitters of influenza, pediatric emergency rooms and large tertiary care hospitals are ideal sites to monitor seasonality covering the entire spectrum of clinical presentations [27] [28] [29] . to create a model system free of selection bias, a perennial quality management (qm) program was instituted at a large pediatric academic center in collaboration with the national reference centre for influenza and other respiratory viruses [30] [31] [32] [33] [34] . the specific aims of the presented analyses are (1) to develop a standardized approach to measuring ili disease severity based on literature review and who guidelines and (2) to apply new mathematical models to the real-time surveillance of ili in large tertiary care centers. (2) studies were not one of the following: randomized clinical trials, non-randomized clinical trials, observational studies, or epidemiological studies; and (3) studies lacked any clinical criterion for disease severity. animal studies, adult studies, meta-analysis, and review papers were also excluded. based on the systematic literature review, the vivi disease severity score was developed as a 22-item weighed clinical composite score, according to who-criteria of uncomplicated and complicated disease [35] . the vivi disease severity score is comprised of 9 items describing signs and symptoms of uncomplicated disease (disease severity, uncomplicated: dsu, weighed single-fold) reflecting 'regular' ili activity, whereas the 13 items describing parameters consistent with complicated disease (disease severity, complicated: dsc, weighed threefold) indicate high-impact clinical presentations in the target population (textbox 1). the vivi disease severity score was subsequently user tested as a web-user interface as well as a mobile application for tablet computers, to be used at the point of care. based on the 16 most commonly cited rfs for severe disease in the pediatric or adolescent age group, a simple rf score was composed [35] [36] [37] [38] . the vivi risk factor score (textbox 2) was implemented on the same mobile application to allow the reporting of disease severity in relation to previously identifiable rfs in the individual patient. the consultation index is an epidemiological indicator reported weekly by the national reference centre for influenza and other respiratory viruses and the influenza working group, based on the proportion of ari at representative sentinel practices across the country [39] . the consultation index represents a timely indicator of any deviation from a baseline rate of ari cases presenting to the respective sentinel practices. a 'normal ari activity' is assumed if the consultation index remains below 115. increased activities are typically measured during the winter months, when seasonal viruses circulate in the community. fluctuations in ari activity as measured by the consultation index represent a useful indicator of disease burden based on actual case numbers. reporting of the number of cases, however, does not reveal information on disease severity with each individual case. by plotting the consultation index with the corresponding average vivi disease severity score in the same graph, we obtain a comprehensive picture of ari disease burden that is based on both actual case numbers and case severity. figure 4 (a) illustrates that disease severity does not always follow the peaks and troughs of case numbers as measured using the consultation index [39] . the vivi score and the consultation index are therefore measuring opposing end points; one is based on individual disease severity per patient (vivi disease severity score) and the other serves as an epidemiological indicator of ari activity and the overall disease burden within the national surveillance system (consultation index). the vivi disease severity and risk factor score were user tested in the context of a qm program for children with ili at a large pediatric hospital in germany as described previously [30] [31] [32] [33] [34] . according to the standard operating procedures, patients with a physician diagnosis of ili and/or fulfilling predefined case criteria (body temperature ≥38°c and ≥1 respiratory symptom) admitted to the emergency department (ed) or pediatric inpatient wards, participated in the qm program [30] [31] [32] [33] [34] . independent of routine clinical care, a specifically trained qm team obtained nasopharyngeal samples and performed standardized clinical assessments using the vivi disease severity score in line with who criteria for uncomplicated and complicated influenza [30, 35, 40] . the vivi disease severity score was recorded at the first consultation with patients participating in the qm program. physicians in routine care were blinded to the results of the clinical assessments by qm staff, and they were unaware of the vivi disease severity scores assigned by the qm team. qm staff on the other hand assessed patients prior to allocation and treatment decisions on behalf of the clinical team in routine care [30] . nasopharyngeal specimens were delivered to the national reference centre for influenza and other respiratory viruses for individual rt-pcr testing influenza virus a and b, rsv, hmpv, hrv, and adv as described below. from december 2009 until april 2015, a total of 6073 children aged 0-18 years participated in the qm program. the qm program included both in-and outpatients to represent the broadest possible spectrum of disease severity. from 2009 to 2015, all patients presenting the ed were screened for ili criteria once weekly, regardless of whether they were subsequently admitted to the hospital or not. from 2011 onward, daily screenings of all inpatients were added (including weekends and holidays). the qm team performed the disease severity assessments independently and the results remained unknown to the routine staff. hence, the data acquired by the qm team did not have any influence on treatment or hospitalization decisions. also, the treating physician did not know the result of the rt-pcr testing when deciding on neuraminidase inhibitor treatment. patients with laboratory-confirmed influenza infection were invited to participate in follow-up assessments whenever feasible. follow-up visits in the qm program were voluntary and scheduled according to the parent's preferences. during follow-up visits, the vivi disease severity score assessment was repeated and recorded by the qm team using the same procedure as during the initial assessment. nasopharyngeal samples were repeated and sent for analogous rt-pcr testing [32] . the qm program was approved by the institutional review board (ea 24/008/10). informed consent procedures were waived for enhanced quality of care and infection control [30] [31] [32] [33] [34] 40 ]. nasopharyngeal swabs were washed out in a total volume of 3 ml of cell culture medium either individually or pooled per patient. rna was extracted from 300 µl of patient specimen using the magattract viral rna m48 kit (qiagen, hilden, germany) and eluted in 80 µl elution buffer. alternatively, rna was extracted using the magna pure 96 dna and viral na small volume kit (roche deutschland holding gmbh, mannheim, germany) from 200 µl specimen with an elution volume of 50 µl. a volume of 25 µl of extracted rna was subjected to cdna synthesis applying 200 u m-mlv reverse transcriptase (invitrogen, karlsruhe, germany) in a total volume of 40 µl. all cdna samples were analyzed by rt-pcr for the presence of each of the pathogens influenza virus a and b, rsv, hmpv, hrv, and adv as published previously [41] [42] [43] [44] [45] . a descriptive analysis of the study sample was performed by calculating proportions and summarizing continuous variables using mean (standard deviation and range) and median (interquartile range). histograms and box plots were used to illustrate the distribution of vivi disease severity scores. correlations between the vivi disease severity score and the consultation index were assessed using scatter plots and pearson's correlation coefficient. the mean difference in vivi disease severity scores was compared across patient and clinical characteristics. statistical significance was assessed using the t-test or the chisquared test as appropriate. to test whether patients with elevated vivi disease severity scores also had elevated rf scores, we performed correlation analysis using pearson's correlation coefficient. these analyses were conducted using stata version 14 (statacorp lp, texas, usa). we further performed regression analyses to identify a set of influential rfs that could model a linear correlation: vivi disease severity score = w 1 × rf 1, w 2 × rf 2, . . ., w n × rf n. here, w i is the respective weight factor for feature i in the regression model [46] . in a subset of patients with laboratory-confirmed influenza infection during the 2011/12 and 2012/13 winter seasons, the vivi disease severity score was also used to follow patients longitudinally with respect to viral load and disease severity over time [32] . to assess the relationship between vivi disease severity score and virus load, we performed pearson correlation analyses for all records, for which more than two followup time point with virology and vivi disease severity scores was available. decision tree analysis [47] was performed to study the relationship between subgroups with a strong positive and negative correlation between disease severity and virus load. as an objective and data-driven measure to detect seasonality of respiratory viral infections in acute care settings, we introduced time series analysis with change point (cp) detection. the goal of cp detection algorithms is to identify changes in the dynamical behavior within a time series [48] . the main difference to a statistically oriented analysis is that it assumes that an intrinsic dynamics model generates the data. cp detection therefore identifies those time points, when time series trends start differing significantly from previous data. this procedure allows identification of critical time points when weekly average numbers of laboratory-confirmed influenza infections start to increase (or decrease) compared to preceding weeks. for further detail on cp detection, please refer to the supplemental data. in this paper, we used the cp detection approach to analyze the qm dataset, which allowed computing averages of target variables assigned to respective calendar weeks (such as average rates of laboratory-confirmed influenza infections per calendar week, average disease severity per calendar week, etc.). for the detection of seasonal patterns, we used the following three-step algorithm. (1) the data were clustered using k-means clustering [49] into potential seasons. we used k = 3 to model two main seasons (high and low) and a transition between those seasons. (2) we assigned a preliminary cp to a week w t , if the cluster assignment c(w t ) to the respective week differed from the cluster assignment to the preceding week, i.e. if c(w t ) ≠ c(w t−1 ). (3) finally, we computed a list of preliminary cps that would split the dataset into time frames tf 1 . . .tf n , where each time frame t i was defined to lie between two consecutive cps. we then checked for each preliminary cp, whether the values before and after the cp (for the two time frames tf i−1 . . .tf i ) differed significantly (p < 0.05) based on a t-test. this procedure ensures that two regions separated by a cp are indeed different. all preliminary cps fulfilling the above criteria were reported. the systematic literature search yielded 613 potentially relevant articles. among these, 529 articles were excluded based on the criteria mentioned above. an additional 56 studies lacked specific criteria for disease severity. finally, a total number of 84 eligible articles were identified, the characteristics of which are summarized in textbox 3. it became evident that several clinical parameters were shared by multiple studies, as for example hospitalization, oxygen requirement, labored breathing, (p)icu admission, mortality, feeding problems/dehydration/vomiting, fever, wheezing or abnormal breath sounds, etc. all of these commonly used criteria were included in the vivi disease severity score (see also: tief et al. [30] , in textbox 3) except for mortality, which is usually recorded separately in hospital records. the vivi disease severity score was validated in the full qm cohort comprised of 6073 patients aged 0-18 years (mean: 3.13 years; sd: 3.89; range: 0-18.79 years). a percentage of 33.6 of the qm program participants was under the age of 1 year, 51.0% were aged 1-5 years, 13.6% were in the age group 6-15 years, and 1.8% were aged 16-18 years. a total of 3399 (56.0%) of the participants were male. a total of 1685 (27.8%) participants were prescribed antibiotics while only 202 (3.3%) were prescribed antivirals in hospital. at presentation, 3172 (52.2%) were assessed as being in need of hospitalization with 997 (16.4%) being in need of intensive care (including assisted ventilation and extracorporeal membrane oxygenation). with regard to viral etiology, in decreasing order of frequency, we identified rhinovirus (22.9%), rsv (17.2%), adv (9.6%), a(h1n1) influenza virus (4.5%), metapneumovirus (4.4%), a(h3n2) influenza virus (2.8%), influenza b viruses of the victoria-lineage influenza (1.7%), and type b viruses of the yamagata-lineage (2.0%). in 5.8% of the cases, there was more than 1 concurrent viral infection. table 1 summarizes the findings from the rf assessment exercise carried out as part of the quality monitoring and table 2 summarizes the clinical symptoms at presentation. a total of 702 patients (11.6%) had chest-radiography in the ed. chest radiography findings showed that 438 (7.2%) had pneumonia, 84 (1.4%) had bronchitis, 1 (0.02%) had bronchiectasis, 3 (0.05%) had bronchiolitis, and 33(0.5%) had other non-pneumonia abnormalities. one hundred and nineteen (2.0%) had a lumbar puncture done in the ed and 113 (1.9%) had cerebrospinal fluid (csf) chemistry done. sixty-nine (1.1%) had csf cultures done with only four (0.07%) sample positive for bacteria (1 bacillus species, 1 staphylococcus epidermidis, 1 staphylococcus hominis, and 1 unspecified bacteria positive). no cases of streptococcus pneumoniae were identified on culture. during hospitalization, 119 (2.0%) had a lumbar puncture and 97 (1.6%) had csf chemistry and culture done. four (0.08%) samples were positive for bacteria including escherichia coli, micrococcus, staphyococcus epidermidis and streptococcus salivarius, streptococcus mitis/oralis as well as enterovirus in two cases. no cases of streptococcus pneumoniae were identified on culture. a total of 603 (9.9%) had a chest radiograph during their inpatient stay. inpatient chest radiography findings showed that 354 (5.8%) had pneumonia, 48 (0.8%) had bronchitis, 2 (0.03%) had bronchiolitis, 3 (0.05%) had bronchiectasis, and 53 (0.9%) had other non-pneumonia abnormalities. in total, 698 (11.5%) of the study participants had been diagnosed with pneumonia on chest radiography at some point during hospitalization. there were two (0.03%) deaths recorded in the emergency room. one of the deaths was attributed to encephalitis and sepsis following infection. the cause of death in the second patient was related to serious underlying cardiac disease in a young infant. the vivi disease severity scores showed a normal distribution with a mean score of 14.5 (sd: 6.0; range 0-34) at initial assessment ( figure 1 ). the vivi disease severity score was significantly higher in patients with the need for hospitalization (mean difference [95% ci]: −7.51 [−7.76 to −7.26]; p < 0.001), with a need for critical care facilities (mean difference [95% ci]: −6.24 [−6.58 to −5.91]; p < 0.001) as well as in those with signs of primary or secondary bacterial lower respiratory tract infections (mean difference [95% ci]: −6.46 [−6.71 to −6.20]; p < 0.001). the median risk factor score in this cohort was 1 (iqr: 0-1); the median rf score was 0.88 (sd: 0.78) and scores ranged from 0 to 6. the cp analysis was applied to detect seasonal patterns for each virus detected in the qm cohort. we define a virus to be seasonal if it is not present during the whole year. with this definition, we found that influenza viruses (figure 2 ), as well as rsv and hmpv, showed a strong seasonal behavior ( figure 3 ) with predominance during the northern hemisphere winter months. adv and hrv were 'rapid cyclers' with frequent and brief peaks throughout the year ( figure 3 ). the cp method showed that in a hospital-based syndromic surveillance system, seasons can be detected and defined in real time for each of the respiratory viruses. during the post-pandemic 2010/11 season for example, influenza a(h1n1)pdm09 viruses continued to predominate in the qm cohort. influenza a(h3n2) viruses, on the other hand, were absent during the 2009/10 and 2010/11 seasons but replaced pandemic h1n1 strains during the subsequent season (see figure 2 ). also, differentiation of influenza b lineages revealed that influenza b yamagata and victoria viruses did not always circulate annually but instead showed alternating patterns. the cp analysis was also used to identify fluctuations in average disease severity per calendar week in the qm program ( figure 4 ). the initial period until summer of 2011, when the qm program was restricted to once-weekly screening of in-and outpatients in the ed, is visually separated from the full surveillance phase beginning with the 2011/12 winter season, when daily screenings of all inpatients hospitalized with suspected ili fever defined as >=38°c. * includes the following items: supplemental oxygen and clinical parameters, such as: oxygen saturation (o2sat), o2sat-to-fio2 ratio (o2sat/ fio2), or hypoxia. there was a small but significant difference in vivi disease severity scores between those subjects where no viral etiology could be detected (mean vivi disease severity score: 13.85; sd 5.81), those identified with a single viral infection (mean vivi disease severity score: 14.90; sd 6.00) and those with more than 1 concurrent viral infection (mean vivi disease severity score: 15.73; sd 6.10); p (anova) < 0.001. for each patient in the qm cohort, we computed the overall vivi disease severity scores as well as the component of the dsu and dsc symptom category, respectively (table 3) . average disease severity with different respiratory viral infections revealed that rsv induced the highest level of disease severity followed by hmpv, influenza a(h3n2), and hrv infections. disease severity with adv, influenza b, and influenza a (h1n1)pdm09 viruses remained below average ( table 3 ). the vivi disease severity score distributions including viral coinfections are displayed in figure 5 . to illustrate the comparison, we computed the average vivi disease severity score per calendar week and compared to the consultation index during the same week ( figure 6(a) ). as expected, no significant correlation was observed between weekly vivi disease severity scores and the consultation index (pearson's correlation coefficient r = 0.10; p = 0.1309) during corresponding weeks, indicating disease severity and case numbers are not linked. to allow visual interpretation, we also plotted the weekly average vivi disease severity score (in the ed prior to october 2011 and in ed and inpatient units thereafter) against the time course of the respective seasonal viruses. the results are shown in figure 6 (b). the viruses circulating (represented in % of all qm patients tested: y axis) are shown in relationship to the average vivi disease severity score during the respective calendar week. some viruses peaked simultaneously with the average disease severity but a cumulative effect was more common. the effect of viruses prevalent during the summer months was more pronounced when inpatients were included in the qm program, thus including severe cases requiring hospital admission. the pearson's correlation coefficient r was 0.1923 indicating a statistically significant but weak positive correlation between vivi disease severity and risk factor scores (p < 0.001). the distribution of vivi disease severity scores by different rfs is illustrated in table 4 . to evaluate which of the rfs as defined by who [35] (textbox 2) had the highest impact on disease severity (i.e. vivi disease severity score), we performed regression analysis as follows: vivi disease severity score = w 1 × rfs 1, w 2 × rf 2, . . ., w n × rf n. regression analysis revealed that there was no specific set of variables that could be used to model this relationship significantly well. the best subset of rf variables was 'rf 1: infant <2 years of age,' 'rf 3: cardiac condition,' 'rf 2: pulmonary condition,' 'rf 6: obesity,' and 'rf 4: diabetes' together yielded a r 2 goodness-of-fit of 0.06. using all rf variables yielded a r 2 of 0.07. to further explore the relationship between age and rf, we studied median and mean vivi disease severity score in infants in children below 5 years of age, and in children aged 6 years and above (table 5) . we then performed pearson correlation to test for a potential relationship between vivi disease severity score and patient age. the pearson collation revealed r = −0.073, suggesting that there is in fact no significant correlation between the vivi disease severity score and patient age. an increasing vivi disease severity score indicates increasing disease severity. the key aspect of the vivi disease severity score is that it provides data standardization across the full spectrum of severity as well as comparison within a cohort, and between different seasons or sites. in the future, this may allow the comparison of various treatment decisions in clinical trials and observational settings. as described above, physicians in routine care were unaware of the results of vivi disease severity score assessments by qm staff and reversely, qm staff were unaware of treatment decisions when assessing a patient. analysis of vivi disease severity score results revealed that disease severity in patients (with any virus) who were prescribed neuraminidase inhibitors was 19 considering the variability in disease presentations and courses of illness with influenza and other respiratory viral infections in children, the vivi disease severity score is not intended to be validated against future clinical events or outcomes. to assess whether the vivi disease severity score could be used to standardize consecutive follow-up visits in clinical trials, a total number of 216 qm patients with influenza diagnoses were followed longitudinally with virology (pcr) and disease severity assessments over time. the overall pearson correlation between vivi disease severity score and virus load (using cycle threshold = ct values) over time was 0.501. a closer look at the correlation histogram ( figure 7 ) revealed three major subgroups: the largest group of 161 patients can be categorized as having a moderate to strong positive correlation (r ≥ 0.5) between disease severity and viral load over time; a second group of 35 patients showed a strong negative correlation (r ≤ −0.5). a third group of 20 patients showed a weak (positive or negative) correlation (−0.5 < r < 0.5). preliminary decision tree analysis of these groups suggested that a vivi disease severity score below 11 and the rf 'infant below 2 years of age' were connected to a negative correlation between virus load and disease severity. respiratory infections are among the most common reasons for children to be admitted to pediatric hospitals. hospitalbased surveillance of respiratory viral infections is of great value to understand the full disease spectrum, from mild symptoms to serious presentations. children are the most avid transmitters of respiratory viral infections, and the information gained from syndromic surveillance in children's hospitals can complement decentralized sentinel surveillance systems in a meaningful way [133] . with the advent of rapid diagnostics and mobile health applications, it has now become possible to monitor virological and clinical end points in real time [134] [135] [136] [137] [138] [139] [140] . traditionally, disease activity is monitored based on epidemiological parameters such as ili or ari incidence, hospitalization rates, or mortality [141, 142] . the consultation index was developed by the robert koch institute and has proven to be a sophisticated epidemiological tool to assess background ari activity at representative sentinel practices. fluctuations in ari activity in private practices represent a useful indicator of disease burden based on actual case numbers. reporting of the number of cases, however, does not reveal information on disease severity with each individual case [143] . the vivi disease severity score aims to fill this gap. the vivi disease severity score is a 22-item weighed clinical composite score consisting of dsu items reflecting 'regular' ili activity and dsc items indicating 'high-impact' clinical presentations in the target population [144] . the vivi disease severity score opens avenues to new individual patient data (ipd) analyses, for example to identify clinically relevant seasonal patterns of disease severity linked to different viral diagnoses confirmed in the same group of patients. the vivi disease severity score also allows consistent measurements of disease severity when following individual patients over time, as would be the case in clinical trials [40] . follow-up assessments are useful whenever standardized severity data need to be recaptured over time. when frequent 'snap shots' of disease severity are combined with virology data, it may be possible to generate a 'moving image' with interesting new applications in clinical research. the introduction of standardized disease severity scores will facilitate headto-head comparisons and the 'meta-analyzability' of clinical trials and observational studies. full compliance of the vivi disease severity score mobile application with clinical data interchange standards consortium (cdisc) standards further expands data interoperability and compliance with reporting formats to regulatory agencies [145] [146] [147] [148] [149] [150] . it is important to note the scope of the proposed disease severity measure. this expert review does not intend to raise expectations that a disease severity score could or should be used to predict future events or physician behavior. instead, statistically significant mean differences are highlighted in bold (t-test p value < 0.05). *the interpretation of this risk factor was limited or **very limited by a low (*n < 100) or very low (**n < 10) prevalence rate in the qm population (see also table 1 ). we introduce a simple 22-item weighted clinical composite score allowing the assessor to translate the current condition of the patient into a two-digit number, which allows comparison of one patient to another, regardless of the setting. to this end, the paper provides the descriptive account of how a standardized score can be utilized to assess the relationships observed between the score and various (independent) treatment and management decisions for readers to draw their own conclusions about how they may in turn use the vivi disease severity score in clinical practice or research. this expert review also introduces time series analysis with change point detection as a mathematical model applied, for the first time, to determining the timing and seasonality or respiratory viruses circulating in a hospital ad emergency room. while the observation that several viruses may circulate in a seasonal pattern is not new, the authors demonstrate that purely data-inherent definitions of seasonality could be an interesting addition to traditionally used methods. comparisons of average vivi disease severity score in the hospital system with the consultation index in the same calendar week (i.e. simultaneous ari consultations in sentinel practices) revealed that the two parameters are intrinsically different. a 'heavy' season with a high frequency of ari consultations is not the same as a 'light' season with fewer but more severe cases. the individual assessments in the qm cohort detected fluctuations in disease severity at a time when increases in overall ari incidence in the general population were not evident. especially during atypical influenza seasons with unusually few or unusually severe cases, the monitoring of disease severity in addition to incidence rates will provide important complementary information. standardized disease severity assessments also enable the cross-cohort comparison of disease burden between different viral pathogens. it is not surprising that rsv was identified as a key contributor to disease severity in a tertiary children's hospital, followed by hmpv disease. a better understanding of the real-world impact of different respiratory viruses on child health will help in the prioritization of drug and vaccine development. the development of the vivi disease severity score is based on a systematic review of the published literature. the review showed that severity assessments have been inconsistent. four clinical management parameters were used commonly as indicators of disease severity: hospitalization, intensive care treatment, oxygen supplementation, and mechanical ventilation (both invasive or noninvasive). the availability of any such measure, however, is highly dependent on the setting. the vivi disease severity score therefore uses the 'need for hospitalization' or 'need for icu admission' (as determined by the assessor) instead. if the assessor determines that a patient would benefit from any such measures, the item can be scored regardless of the availability of icu or hospital beds at the respective time or location. to ensure inter-rater consistency, the qm team was specifically trained to apply established who definitions and standard criteria for the assessment of each aspect of the vivi disease severity score. for example, fever was defined according to marcy et al. [151] and acute lower respiratory tract infection as per roth et al. [152] . for use in multicenter settings, the vivi disease severity score app will include help menus in the user interface to ensure that assessors are aware of the same criteria and age-appropriate values. acknowledging that the content, structure, and quality of standardized data are of paramount importance, the development team worked closely with the cdisc to ensure full compliance of terminologies and data elements with industry and regulatory guidance. the literature review showed that grading severity is not the same as predicting severity. especially in young children, disease severity will fluctuate over time, until the episode is resolved eventually. the course of illness may or may not be linear. the vivi disease severity score is designed to help the physician measure and monitor the situation ad hoc, or repeatedly over time, but not to predict the future of the patient. several scores have been designed, not to measure severity ad hoc, but to predict the likelihood of fatal outcomes in the future as is the case with the respiratory index of severity in children [153] , the pediatric index of mortality score (pims) [83] , and the pediatric risk of mortality score (prims) [84, 85] . these latter two scores (pims and prims) were specific to rsv infections [83] in infants [84, 85] . the kristjansson clinical respiratory score for rsv infections in children [82] was designed to include children beyond the infant age group. the index of severity was studied in bocavirus infections in infants and children <5 years [130] , as was the symptom score for coronavirus infections in children [132] . very few scores were developed to measure disease severity regardless of the type of respiratory virus causing the disease. the clinical severity score was used to monitor rsv, hrv, and hmpv infections in children <3 years [91, 117, 122] . the systematic literature review, updated in 2016, confirmed that vivi disease severity score was the only score covering all pediatric age groups and any respiratory virus encompassing any of the key parameters outlined in the published literature to date [18, 30] . the vivi disease severity score was also the only composite score that has been validated in a prospective cohort of more than 6000 children and adolescents from 0 to 18 years, yielding a normal distribution. regular severity assessments over time can be combined with cp detection methodology to detect of significant changes in disease severity in cohorts. hospital surveillance will thus become feasible in real time, as rapid-turnaround diagnostic tests are evolving [154] [155] [156] [157] . the use of rapid diagnostic tests can then be targeted according to the local surveillance information. bioinformatics analyses and machine learning algorithms may provide new avenues for the identification of virus-specific seasonality patterns [158] . during past influenza seasons, differences in the composition of subtypes and disease severity have been significant. the linkage of simultaneous virus surveillance with point-of-care disease severity assessments will advance the understanding of local epidemiology. local epidemiology is key to understanding the impact of different strains on different populations. in north america, influenza a h3n2 viruses reappeared 1 year sooner than in europe, i.e. in 2010/11 [159] followed by an unusually 'light' season with few or late cases during the winter of 2011/12 [160] . public health agencies in the uk reported a particularly 'severe' season in 2010/11 [161, 162] , whereas australia reported increased rates of severe influenza disease in 2014 [163] [164] [165] , similar to mexico during 2013/14. classically, seasons have been regarded as 'severe' when coinciding with high overall case numbers, hospitalization rates, or mortality [166, 167] . in the future, it will be important to distinguish the impact of fluctuations in influenza (sub)types on disease severity in specific patient groups, based on ipd. the use of standardized measures of severity may also be helpful in the study of medical decision-making and diagnostic algorithms. physicians in routine care often report that their decision to order virus diagnostics is often dependent on a variety of factors such as levels of training, media attention [168, 169] , specific requests by patients or parents, 'typical' versus 'atypical' disease presentations, availability and cost of diagnostic tests, insurance status of the patient, time constraints, etc. [20] . the same applies to the decision to hospitalize a patient. it is safe to assume that testing and rates of hospitalization are not the same at the beginning, peak, and end of an influenza season. standardized disease severity scores may allow hospitals to set objective thresholds for diagnostic testing or admission decisions, depending on local conditions and epidemiology. when population-based indicators are used instead of individual clinical outcome parameters, considerable bias may be introduced due to differences in patient reporting, access to health care [170] as well as physician awareness and reimbursement [171] creating challenges in global surveillance systems [172, 173] . some surveillance programs use retrospective chart reviews and icd coding. icd codes, however, do not always distinguish between laboratory-confirmed cases and clinical diagnoses [174] . interpersonal variability and the unpredictable nature of respiratory viral infections pose a challenge to surveillance and preparedness programs [175] . influenza seasons in particular vary with respect to case numbers and disease severity attributable to various viral subtypes and population strata [67, 68, 113, 127] . the prospective monitoring of disease severity associated with laboratory-confirmed diagnoses will help to delineate vulnerable subpopulations expressing disease severity differently compared to the population average. real-time surveillance of disease severity may provide public health stakeholders with crucial information to adjust the allocation of hospital beds and resources [52] . the introduction of ipd disease severity assessments in a hospital-based surveillance system facilitates the timely identification of abnormal patterns of disease severity, i.e. though network analysis [176] or during time periods when the overall ili disease severity is different from previous seasons or the rest of the year. importantly, fluctuations in disease severity measured by the vivi disease severity score are independent of incidence-based surveillance indices. traditional disease severity estimates have focused on extreme presentations such as mortality rates [177, 178] or icu admission [179] but were not designed to monitor the full spectrum of mild-to-severe disease presentations. additional granularity will be required for clinical trials. when the vivi disease severity score was used to follow patients longitudinally, disease severity was measured consistently from the time of initial presentation until resolution of symptoms. the vivi disease severity score has also been used to measure of subtle changes in disease severity in icu patients requiring organ replacement therapy [40] . once standardized scores are used consistently, this will open the path to headto-head comparisons of antivirals and vaccines and to prospective ipd meta-analyses. it will be important to investigate the complex relationship between virus load and disease severity and expected outcomes, which would provide important clues for clinical trial design [40] . patients showing atypical patterns of disease severity for example (such as a negative correlation between virus load and disease severity) may represent individuals where antivirals do not exert the desired effect. additional analyses are underway to understand this relationship better. standardized disease severity measures will facilitate biomarkers studies and the identification of viral and host factors associated with severe outcomes [180] . a precision medicine approach would lead to individualized risk communication strategies to improve the acceptance of vaccines and antivirals where they are most effective. the low uptake in influenza vaccines in the qm cohort indicates that significant numbers of symptomatic influenza cases might have been prevented through immunization [181] . the presented work has several limitations: the current experience with the vivi disease severity score is based on a single-center tertiary care setting. additional decentralized studies will be needed to validate the vivi disease severity score in international settings and in private practice networks, where severity may be lower. further studies are planned in adults and the elderly, including the development of a compatible score for patient-reported outcomes. it is possible that different populations yield different results, but standardization is the prerequisite to study any such difference. mobile applications will be particularly useful in low-resource settings, where disease severity may be higher and decisions have to be taken instantly. finally, the effect of antiviral treatment or vaccine prevention on disease severity could not be assessed due to a minimal use of neuraminidase inhibitors and influenza vaccines in the current setting [182] . the only conclusion that can be drawn is that physicians in this setting hardly ever used antivirals but were more likely to prescribe antibiotics if a patient appeared severely ill, as expressed in significantly higher vivi disease severity scores [30] . it will be interesting to study decision-making processes and the impact of different forms of medical interventions on disease severity in a variety of settings in the future. at this point, the majority of sentinel surveillance systems are laboratory based yielding limited clinical information but important data with respect to the evolution of influenza viruses, subtypes, resistance, seasonality, and transmissibility. it would be of great benefit to monitor disease severity individually, along with regional and geographic differences in virus circulation, using standardized disease severity measurements such as the vivi disease severity score. our contributions are the following: (a) the design of a hospital-based surveillance program and a unique qm cohort of more than 6000 children, where an independent qm team monitored patients daily using standardized clinical assessments and virology at the national reference centre for influenza and other respiratory viruses. (b) a novel disease severity score (the vivi disease severity score) and mobile application to detect specific changes in ipd and the individual course of illness in pediatric clinical trials and observational settings. the presented tools are in line with the priorities issued by regulatory agencies with regards to data standardization and the development of clinical outcome measures for the development of new antivirals. with composite disease severity scores, the focus will shift from virological to clinical end points, and the impact of therapeutic interventions on the quality of disease presentations. only the systematic unbiased and prospective assessment of all cases, whether mild or severe, throughout several seasons, will provide objective insight into the actual disease burden with influenza and other respiratory viruses. mobile health technologies enable new precision medicine approaches not only in clinical trials but also in routine patient care. individualized disease severity assessments in children with influenza and other respiratory viruses will allow the physician to communicate better with the parent or patient, providing the current status as a validated measure of disease severity compared to similar age and population strata. in patents receiving antiviral therapy, progress can be measured and communicated accordingly and again, individually. most importantly, with the availability of validated disease severity measures and standardized datasets, the physician will be able to determine which patients may be 'lagging behind' in their response to therapeutic interventions. a better understanding of the complex relationship between virus load and disease severity in children with different respiratory viruses will provide important clues for a personalized approach to antiviral therapy. biomarker analyses linked to standardized disease severity assessments will help to elucidate why some patients improve rapidly as soon as virus loads decline, whereas a smaller group of patients does not improve as expected. this latter subgroup of patients may benefit from different therapeutic approaches, for example immunomodulation. precision medicine tools such as the vivi disease severity score mobile application will provide important tools for the objective evaluation of new antivirals for soon-to-be treatable respiratory viruses. • regulatory agencies and public health stakeholders have repeatedly called for international consensus on disease severity measures in influenza and other respiratory viruses. • this need has become imminent with the rapid development of new anti-infective therapies for respiratory viral infections in children and adults. • the challenge of data standardization is greatest in infants and young children, who may present with subtle and atypical symptoms. • based on a systematic review of the literature we developed a 22-item composite clinical score (the vivi disease severity score) for the immediate measurement of disease severity with acute reparatory infections the point-of-care. • the vivi disease severity score was made available as a web-user interface and mobile application for validation in a quality management program including more than 6000 children 0-18 years of age. • linking standardized diseases severity scores with rapid diagnostics will allow the instantaneous monitoring of incidence rates of acute respiratory viral infections along with the severity of each case. • with this comprehensive manuscript, we are providing insight into the future of observational studies and clinical trials of antivirals for soon-to-be-treatable acute respiratory diseases in children. • the reader is guided through novel analytic approaches that have become possible through rigorously standardized individual patient-data (ipd) analyses of disease severity. br wrote the initial draft of the manuscript. ma, ft, xc and po were in charge of data aggregation, acquisition and qc/qa, and provided important input into the manuscript. xm conducted and interpreted the systematic literature review. bk and ch were in charge of data standardization, ch provided database maintenance and management. bs designed and supervised the laboratory analyses. tc, pm conducted the data analysis. br designed the qm program and the vivi disease severity score and supervised the project. all authors take responsibility for the integrity of the data and the accuracy of the data analysis. all authors have seen and approved the final version of the manuscript. the authors would like to express their gratitude to the team at the robert koch institute for providing virology testing in-kind and to the vienna vaccine safety initiative for providing the vivi disease severity score and mobile application. tc was funded by the german ministry of research and education (bmbf) project grant 3fo18501 (forschungscampus modal). the authors would also like to express their thanks to members of the vivi think tank for their expert feedback and encouragement throughout the course of the project. papers of special note have been highlighted as either of interest (•) or of considerable interest (••) to readers burden of severe 2009 pandemic influenza a (h1n1) infection in children in southeast spain influenza burden for children with asthma during the summer 2009 outbreak of "swine flu" in scotland what respiratory pathogens were diagnosed as h1n1 clinical features of the initial cases of 2009 pandemic influenza a (h1n1) virus infection in china unique data describing clinical features of pandemic influenza infection (see supplementary data) case-control study of clinical features of influenza in hospitalized patients is influenza an influenza-like illness? clinical presentation of influenza in hospitalized patients seasonal patterns of respiratory syncytial virus, influenza a virus, human metapneumovirus, and parainfluenza virus type 3 infections on the basis of virus isolation data between a robust parameter estimation method for estimating disease burden of respiratory viruses current landscape of antiviral drug discovery fight against h1n1 influenza a virus: recent insights towards the development of druggable compounds the paramyxovirus polymerase complex as a target for next-generation anti-paramyxovirus therapeutics advances in antivirals for non-influenza respiratory virus infections. influenza other respir viruses key review paper on antiviral drug development for non-influenza respiratory viruses update on new antivirals under development for the treatment of double-stranded dna virus infections toward antiviral therapy/ prophylaxis for rhinovirus-induced exacerbations of chronic obstructive pulmonary disease: challenges, opportunities, and strategies replication and inhibitors of enteroviruses and parechoviruses combating enterovirus replication: state-of-the-art on antiviral research university of texas medical branch at galveston. the university of texas medical branch at galveston towards a personalised approach to managing influenza infections in infants and children -food for thought and a note on oseltamivir • position paper describing the overall concept of the quality management in children with ili, including systematic disease severity assessments and point-of-care diagnostics modelling estimates of agespecific influenza-related hospitalisation and mortality in the united kingdom the continued rise of respiratory viruses the validity of clinical practice guidelines for empirical use of oseltamivir for influenza in thai children influenza in the emergency department: vaccination, diagnosis, and treatment: clinical practice paper approved by american academy of emergency medicine clinical guidelines committee the role of influenza in the epidemiology of pneumonia clinical outcomes associated with respiratory virus detection before allogeneic hematopoietic stem cell transplant inequities in ambulatory care and the relationship between socioeconomic status and respiratory hospitalizations: a population-based study of a canadian city example of a study illustrating the limitations of using hospitalization as an indicator of disease severity evaluation of a pilot respiratory virus surveillance system linking electronic health record and diagnostic data high costs of influenza: direct medical costs of influenza disease in young children the underrecognized burden of influenza in young children clinical aspects of 2009 pandemic influenza a (h1n1) virus infection in austria an inception cohort study assessing the role of pneumococcal and other bacterial pathogens in children with influenza and ili and a clinical decision model for stringent antibiotic use evaluation of novel secondgeneration rsv and influenza rapid tests at the point of care quantitative influenza follow-up testing (qift)-a novel biomarker for the monitoring of disease activity at the point-of-care early detection of influenza a and b infection in infants and children using conventional and fluorescence-based rapid testing virus load kinetics and resistance development during oseltamivir treatment in infants and children infected with influenza a(h1n1) 2009 and influenza b viruses clinical management of human infection with pandemic (h1n1) 2009: revised guidance identification of children at risk of influenza-related complications in primary and ambulatory care: a systematic review and meta-analysis risk factors associated with severe influenza virus infections in hospitalized children during the 2013 to 2014 season surveillance for severe acute respiratory infections (sari) in hospitals in the who european region -an exploratory analysis of risk factors for a severe outcome in influenza-positive sari cases calculation of the incidence of primary care visits due to acute respiratory infections •• rki publication describing the consultation index pharmacokinetics of oral and intravenous oseltamivir treatment of severe influenza b virus infection requiring organ replacement therapy differentiation of influenza b virus lineages yamagata and victoria by real-time pcr genetic variability of group a human respiratory syncytial virus strains circulating in germany from 1998 to diagnostic approach for the differentiation of the pandemic influenza a(h1n1)v virus from recent human influenza viruses by real-time pcr development of a pcr-based assay for detection, quantification, and genotyping of human adenoviruses human metapneumovirus: insights from a ten-year molecular and epidemiological analysis in germany regularization paths for generalized linear models via coordinate descent c 4.5: programs for machine learning sequential change point detection in molecular dynamics trajectories cluster analysis of multivariate data: efficiency versus interpretability of classifications viral etiology of acute lower respiratory tract infections in hospitalized young children in northern taiwan development of the respiratory index of severity in children (risc) score among young children with respiratory infections in south africa increased healthcare resource utilization for acute respiratory illness among latino infants predictors of severe disease in a hospitalized population of children with acute viral lower respiratory tract infections virus type and genomic load in acute bronchiolitis: severity and treatment response with inhaled adrenaline viruses as sole causative agents of severe acute respiratory tract infections in children epidemiologic characteristics and the relationship with disease severity of respiratory syncytial virus genotypes from children with lower respiratory tract infection in the southern zhejiang province multiple versus single virus respiratory infections: viral load and clinical disease severity in hospitalized children. influenza other respir viruses correlation of viral load of respiratory pathogens and co-infections with disease severity in children hospitalized for lower respiratory tract infection example of a study exploring the complex relationship between virus load and disease severity the first wheezing episode: respiratory virus etiology, atopic characteristics, and illness severity infection with multiple viruses is not associated with increased disease severity in children with bronchiolitis example of a study exploring the complex relationship between viral coinfections and disease severity in children illness severity and work productivity loss among working adults with medically attended acute respiratory illnesses: us influenza vaccine effectiveness network comparing the clinical severity of the first versus second wave of 2009 influenza a (h1n1) in a new york city pediatric healthcare facility the burden and severity of illness due to 2009 pandemic influenza a (h1n1) in a large us city during the late summer and early fall of interleukin and neurotrophin up-regulation correlates with severity of h1n1 infection in children: a case-control study novel influenza a(h1n1) in a pediatric health care facility in new york city during the first wave of the 2009 pandemic hospitalised malaysian children with pandemic (h1n1) 2009 influenza: clinical characteristics, risk factors for severe disease and comparison with the 2002-2007 seasonal influenza characteristics of hospitalized cases with influenza a (h1n1)pdm09 infection during first winter season of post-pandemic in china association between the severity of influenza a(h7n9) virus infections and length of the incubation period example of a study exploring the complex relationship between incubation time and disease severity influenza pneumonia among adolescents and adults: a concurrent comparison between influenza a (h1n1) pdm09 and a (h3n2) in the post-pandemic period children hospitalised with influenza-associated pneumonia during the 2009 pandemic displayed increased disease severity burden of seasonal influenza in children with neurodevelopmental conditions clinical predictors of disease severity during the 2009-2010 a(hin1) influenza virus pandemic in a paediatric population viral load at diagnosis and influenza a h1n1 (2009) disease severity in children. influenza other respir viruses example of a study exploring the complex relationship between virus load and disease severity comparative community burden and severity of seasonal and pandemic influenza: results of the flu watch cohort study example of a study of disease burden based on epidemiological criteria prevalence and clinical aspects of respiratory syncytial virus a and b groups in children seen at hospital de clinicas of uberlandia, mg, brazil what is the clinical relevance of respiratory syncytial virus bronchiolitis?: findings from a multi-center, prospective study human respiratory syncytial virus in children with acute respiratory tract infections in china correlation between inflammatory mediators in the nasopharyngeal secretion and in the serum of children with lower respiratory tract infection caused by respiratory syncytial virus and disease severity whole blood gene expression profiles to assess pathogenesis and disease severity in infants with respiratory syncytial virus infection example of a study exploring the complex relationship between biomarkers and disease severity innate immune dysfunction is associated with enhanced disease severity in infants with severe respiratory syncytial virus bronchiolitis clinical course of communityacquired respiratory syncytial virus pneumonia in newborns hospitalized in neonatal intensive care unit value of a risk scoring tool to predict respiratory syncytial virus disease severity and need for hospitalization in term infants fio2 predicts outcome in infants with respiratory syncytial virus-induced acute respiratory distress syndrome infants with severe respiratory syncytial virus needed less ventilator time with nasal continuous airways pressure then invasive mechanical ventilation pulmonary matrix metalloproteinase-9 activity in mechanically ventilated children with respiratory syncytial virus risk factors for respiratory syncytial virus bronchiolitis hospital admission in new zealand distribution and clinical impact of human respiratory syncytial virus genotypes in hospitalized children over 2 winter seasons epidemiology of respiratory syncytial virus in children in cyprus during three consecutive winter seasons (2010-2013): age distribution, seasonality and association between prevalent genotypes and disease severity molecular epidemiology and disease severity of human respiratory syncytial virus in vietnam disease severity and viral load are correlated in infants with primary respiratory syncytial virus infection in the community nasopharyngeal bacterial burden and antibiotics: influence on inflammatory markers and disease severity in infants with respiratory syncytial virus bronchiolitis respiratory syncytial virus genomic load and disease severity among children hospitalized with bronchiolitis: multicenter cohort studies in the united states and finland five study g. epidemiological and clinical data of hospitalizations associated with respiratory syncytial virus infection in children under 5 years of age in spain: five multicenter study. influenza other respir viruses epidemiologic, socioeconomic, and clinical factors associated with severity of respiratory syncytial virus infection in previously healthy infants respiratory syncytial virus infections in hospitalized infants: association between viral load, virus subgroup, and disease severity surfactant protein a2 polymorphisms and disease severity in a respiratory syncytial virusinfected population respiratory syncytial virus load, viral dynamics, and disease severity in previously healthy naturally infected children effect of dexamethasone on respiratory syncytial virus-induced lung inflammation in children: results of a randomized, placebo controlled clinical trial children hospitalized with respiratory syncytial virus infection in saskatchewan pediatric tertiary care centers relating plaque morphology to respiratory syncytial virus subgroup, viral load, and disease severity in children novel inflammatory markers, clinical risk factors and virus type associated with severe respiratory syncytial virus infection risk factors in children hospitalized with rsv bronchiolitis versus non-rsv bronchiolitis olfactomedin 4 serves as a marker for disease severity in pediatric respiratory syncytial virus (rsv) infection hospitalization for respiratory syncytial virus bronchiolitis and disease severity in twins viral specific factors contribute to clinical respiratory syncytial virus disease severity differences in infants children with down syndrome are high-risk for severe respiratory syncytial virus disease rsv infection among children born moderately preterm in a community-based cohort il1rl1 gene variants and nasopharyngeal il1rl-a levels are associated with severe rsv bronchiolitis: a multicenter cohort study serum palivizumab level is associated with decreased severity of respiratory syncytial virus disease in high-risk infants value of chest radiographic pattern in rsv disease of the newborn: a multicenter retrospective cohort study severe respiratory syncytial virus bronchiolitis in infants is associated with reduced airway interferon gamma and substance p proteins involved in extracellular matrix dynamics are associated with respiratory syncytial virus disease severity high pneumococcal density correlates with more mucosal inflammation and reduced respiratory syncytial virus disease severity in infants right ventricular function in children with severe respiratory syncytial virus (rsv) bronchiolitis molecular epidemiology and disease severity of respiratory syncytial virus in relation to other potential pathogens in children hospitalized with acute respiratory infection in jordan evaluation of igg antibodies against respiratory syncytial virus (rsv), and associated risk factors for severe respiratory tract infections in pre-school children in north-central decreased innate immune cytokine responses correlate with disease severity in children with respiratory syncytial virus and human rhinovirus bronchiolitis comparison of risk factors for human metapneumovirus and respiratory syncytial virus disease severity in young children respiratory syncytial virus, human bocavirus and rhinovirus bronchiolitis in infants clinical disease and viral load in children infected with respiratory syncytial virus or human metapneumovirus human metapneumovirus infections are associated with severe morbidity in hospitalized children of all ages human metapneumovirus viral load is an important risk factor for disease severity in young children incidence, morbidity, and costs of human metapneumovirus infection in hospitalized children human metapneumovirus infection in young children hospitalized with acute respiratory tract disease: virologic and clinical features human metapneumovirus infection in jordanian children: epidemiology and risk factors for severe disease human rhinovirus and disease severity in children impact of human rhinovirus types and viral load on the severity of illness in hospitalized children with lower respiratory tract infections epidemiologic, clinical, and virologic characteristics of human rhinovirus infection among otherwise healthy children and adults: rhinovirus among adults and children clinical severity of rhinovirus/ enterovirus compared to other respiratory viruses in children. influenza other respir viruses high human bocavirus viral load is associated with disease severity in children under five years of age human bocavirus in children with acute respiratory infections in vietnam severity and outcome associated with human coronavirus oc43 infections among children success factors of european syndromic surveillance systems: a worked example of applying qualitative comparative analysis innovative digital tools and surveillance systems for the timely detection of adverse events at the point of care: a proof-of-concept study enabling precision medicine with digital case classification at the point-of-care can mobile technologies improve on-time vaccination? a study piloting maternal use of immunizeca, a pan-canadian immunization app evaluating the impact of a computerized surveillance algorithm and decision support system on sepsis mortality development and usage of wiki-based software for point-of-care emergency medical information an evaluation of the feasibility and usability of a proof of concept mobile app for adverse event reporting post influenza vaccination mpneumonia: development of an innovative mhealth application for diagnosing and treating childhood pneumonia and other childhood illnesses in low-resource settings has estimation of numbers of cases of pandemic influenza h1n1 in england in 2009 provided a useful measure of the occurrence of disease? influenza other respi viruses • study exploring various parameters commonly used for disease burden estimates a sentinel platform to evaluate influenza vaccine effectiveness and new variant circulation influenza surveillance in australia: we need to do more than count an inception cohort study assessing the role of pneumococcal and other bacterial pathogens in children with influenza and ili and a clinical decision model for stringent antibiotic use using semantic web technologies for the generation of domain-specific templates to support clinical study metadata standards electronic healthcare record and clinical research in cardiovascular radiology. hl7 cda and cdisc odm interoperability using electronic health records for clinical research: the case of the ehr4cr project current applications and future directions for the cdisc operational data model standard: a methodological review standardized data sharing in a paediatric oncology research network-a proof-ofconcept study harmonization of detailed clinical models with clinical study data standards fever as an adverse event following immunization: case definition and guidelines of data collection, analysis, and presentation acute lower respiratory infections in childhood: opportunities for reducing the global burden through nutritional interventions optimizing virus identification in critically ill children suspected of having an acute severe viral infection survey of influenza and other respiratory viruses diagnostic testing in us hospitals diagnostic accuracy of rapid antigen detection tests for respiratory syncytial virus infection: systematic review and meta-analysis antibiotic discontinuation rates associated with positive respiratory viral panel and low procalcitonin results in proven or suspected respiratory infections evaluation of the becton dickinson rapid influenza diagnostic tests in outpatients in germany during seven influenza seasons example of a study exploring the use of rapid diagnostics in respiratory virus surveillance in germany global influenza seasonality: reconciling patterns across temperate and tropical regions influenza vaccine effectiveness: maintained protection throughout the duration of influenza seasons update: influenza activity -united states, 2011-12 season and composition of the 2012-13 influenza vaccine a new sentinel surveillance system for severe influenza in england shows a shift in age distribution of hospitalised cases in the post-pandemic period influenza a(h1n1)pdm09 in england influenza outbreak preparedness: lessons from outbreaks in residential care facilities in 2014 review of the 2014 influenza season in the southern hemisphere influenza epidemiology in adults admitted to sentinel australian hospitals in 2014: the influenza complications alert network (flucan) substantial morbidity and mortality associated with pandemic a/h1n1 influenza in mexico, winter 2013-2014: gradual age shift and severity intense seasonal a/h1n1 influenza in mexico forecasting influenza outbreak dynamics in melbourne from internet search query surveillance data. influenza other respir viruses accurate estimation of influenza epidemics using google search data via argo the impact of workplace policies and other social factors on self-reported influenza-like illness incidence during the 2009 h1n1 pandemic australia's notifiable disease status, 2009: annual report of the national notifiable diseases surveillance system early reporting of pandemic flu and the challenge of global surveillance: a lesson for southeast asia improving global influenza surveillance: trends of a(h5n1) virus in africa and asia nationwide surveillance of influenza during the pandemic (2009-10) and post-pandemic (2010-11) periods in taiwan influenza pandemic epidemiologic and virologic diversity: reminding ourselves of the possibilities network analysis of global influenza spread epidemiology of influenza a (h1n1)pdm09-associated deaths in the united states estimating absolute and relative case fatality ratios from infectious disease surveillance data pandemic influenza a(h1) pdm09 in hospitals and intensive care units -results from a new hospital surveillance exploring cell tropism as a possible contributor to influenza infection severity increasing the intent to receive a pandemic influenza vaccination: testing the impact of theory-based messages influenza antiviral prescribing practices during the 2007-08 and 2008-09 influenza seasons in the setting of increased resistance to oseltamivir among circulating influenza viruses key: cord-256065-zz2907h0 authors: barral-arca, ruth; gómez-carballa, alberto; cebey-lópez, miriam; bello, xabier; martinón-torres, federico; salas, antonio title: a meta-analysis of multiple whole blood gene expression data unveils a diagnostic host-response transcript signature for respiratory syncytial virus date: 2020-03-06 journal: int j mol sci doi: 10.3390/ijms21051831 sha: doc_id: 256065 cord_uid: zz2907h0 respiratory syncytial virus (rsv) is one of the major causes of acute lower respiratory tract infection worldwide. the absence of a commercial vaccine and the limited success of current therapeutic strategies against rsv make further research necessary. we used a multi-cohort analysis approach to investigate host transcriptomic biomarkers and shed further light on the molecular mechanism underlying rsv-host interactions. we meta-analyzed seven transcriptome microarray studies from the public gene expression omnibus (geo) repository containing a total of 922 samples, including rsv, healthy controls, coronaviruses, enteroviruses, influenzas, rhinoviruses, and coinfections, from both adult and pediatric patients. we identified > 1500 genes differentially expressed when comparing the transcriptomes of rsv-infected patients against healthy controls. functional enrichment analysis showed several pathways significantly altered, including immunologic response mediated by rsv infection, pattern recognition receptors, cell cycle, and olfactory signaling. in addition, we identified a minimal 17-transcript host signature specific for rsv infection by comparing transcriptomic profiles against other respiratory viruses. these multi-genic signatures might help to investigate future drug targets against rsv infection. respiratory syncytial virus (rsv) is the main cause of lower respiratory tract infections in early life [1] , and one of the major causes of morbidity and mortality, especially in children younger than six months [2] . re-infections are also common in patients older than two years and adults, although usually less severe [3, 4] . patients with premature birth, chronic lung disease, congenital heart disease, and immunodeficiency are more likely to suffer from severe forms of rsv and may require hospitalization [5] ; however, most of the children hospitalized with severe rsv disease lack known identifiable risk factors [6, 7] . it has been shown that host genetic susceptibility to the disease may play a key role in the different pathogenesis produced by rsv in children [8, 9] , but many aspects of the host-pathogen interaction a principal component analysis (pca ) of the combined data (rsv samples and healthy controls) was carried out to detect batch effects in the eight datasets analyzed. this analysis revealed that the transcriptomes of the rsv cases in the gse80179 form a separate cluster when compared to the rest of the samples ( figure s1 ). this anomalous pattern could be explained by different severities, strand or sampling time points with regard to the other samples, or undesirable batch effects. this dataset was therefore eliminated from further analysis. thus, the meta-analysis was finally conducted on a total of seven datasets containing transcriptomic data from 922 samples: 296 rsv, 4 coronaviruses, 4 enteroviruses, 188 influenzas, 71 rhinoviruses, 93 coinfections, and 266 healthy controls; from both adult and pediatric patients (table s1) . a new pca was conducted with the remaining seven datasets and based on the 100 most deg (figure 1 ). the first principal component (pc1; accounting for~46% of the variation), in combination with pc2 (accounting for~19% of the variation), separates all the samples in two clusters according to their disease status. it is remarkable that, while the healthy controls appear more scattered in the plot, rsv patients are more tightly clustered, suggesting that the rsv alters the transcriptome in a more specific manner. it is also noteworthy that the 93 cases of co-infection included in the meta-analysis do not generate a pattern of sub-clustering in the pca plot. by comparing the transcriptomes of rsv-infected patients to healthy controls, we identified 1,562 genes differentially expressed (678 over-expressed and 884 under-expressed; fdr of 5%; table s2 ). this differential gene expression pattern explains the clustering pattern observed in the pca ( figure 1 ). our functional enrichment study using reactome showed that the deg were enriched in the host cell-cycle pathway in rsv infected patients when compared to controls (r-hsa-69620, adjusted p-value = 1.16 × 10 −5 ; r-hsa-69278, adjusted p-value = 3.83 × 10 −5 ; r-hsa-1640170, adjusted p-value = 2.37 × 10 −5 ) (table s3) . among the deg between rsv-infected patients and healthy controls, a number of them affecting immunologic-related pathways are strongly over-represented: (i) immune system (r-hsa-168256; adjusted p-value = 4.06 × 10 −27 ), (ii) cytokine signaling in immune system (r-hsa-1280215: stat1, stat2, mmp-9; adjusted p-value = 7.90 × 10 −16 ), (iii) innate immune system (r-hsa-168249; adjusted p-value = 5.92 × 10 −19 ) and (iv) adaptive immune system pathways (r-hsa-1280218; adjusted p-value = 1.11 × 10 −7 ). we also observed an over-representation of deg belonging to pathways related to (i) interleukins signaling (r-hsa-449147; adjusted p-value = 1.36 × 10 −7 ) such as interleukin-1 family signaling (r-hsa-446652; adjusted p-value = 1.45 × 10 −6 ) and (ii) interleukin-1 signaling (r-hsa-9020702; adjusted p-value = 7.67 × 10 −6 ; table s3 ). rsv infection also causes an overrepresentation deg related to the interferon signaling pathway (r-hsa-913531; table s3 ) and interferon-inducible genes such as eif2ak2, mx1 and ifitm1. over-representation is also observed by comparing the transcriptomes of rsv-infected patients to healthy controls, we identified 1,562 genes differentially expressed (678 over-expressed and 884 under-expressed; fdr of 5%; table s2 ). this differential gene expression pattern explains the clustering pattern observed in the pca ( figure 1 ). our functional enrichment study using reactome showed that the deg were enriched in the host cell-cycle pathway in rsv infected patients when compared to controls (r-hsa-69620, adjusted p-value = 1.16 × 10 −5 ; r-hsa-69278, adjusted p-value = 3.83 × 10 −5 ; r-hsa-1640170, adjusted p-value = 2.37 × 10 −5 ) (table s3) . among the deg between rsv-infected patients and healthy controls, a number of them affecting immunologic-related pathways are strongly over-represented: (i) immune system (r-hsa-168256; adjusted p-value = 4.06 × 10 −27 ), (ii) cytokine signaling in immune system (r-hsa-1280215: stat1, stat2, mmp-9; adjusted p-value = 7.90 × 10 −16 ), (iii) innate immune system (r-hsa-168249; adjusted p-value = 5.92 × 10 −19 ) and (iv) adaptive immune system pathways (r-hsa-1280218; adjusted p-value = 1.11 × 10 −7 ). we also observed an over-representation of deg belonging to pathways related to (i) interleukins signaling (r-hsa-449147; adjusted p-value = 1.36 × 10 −7 ) such as interleukin-1 family signaling (r-hsa-446652; adjusted p-value = 1.45 × 10 −6 ) and (ii) interleukin-1 signaling (r-hsa-9020702; adjusted p-value = 7.67 × 10 −6 ; table s3 ). rsv infection also causes an over-representation deg related to the interferon signaling pathway (r-hsa-913531; table s3) and interferon-inducible genes such as eif2ak2, mx1 and ifitm1. over-representation is also observed in essential proteins of the innate immune response such as oligoadenylate synthase (oas) family proteins and rnase l, which are responsible for rna degradation hereby blocking viral replication [25] . in addition, other pathways related to host response to infectious disease (r-hsa-5663205; adjusted p-value = 2.08 × 10 −21 ) and others involved in the viral infection process, such as r-hsa-168254 (influenza infection; adjusted p-value = 2.56 × 10 −17 ), r-hsa-162906 (hiv infection; adjusted p-value = 8.62 × 10 −7 ), r-hsa-168273 (influenza viral rna transcription and replication; adjusted p-value = 3.91 × 10 −17 ) and r-hsa-192823 (viral mrna translation; adjusted p-value = 5.14 × 10 −18 ), were found to be clearly enriched in rsv patients (table s3 ). rsv infection also provokes an over-representation of deg related to the neutrophil degranulation pathway (r-hsa-6798695; adjusted p-value = 1.59 × 10 −14 ). rsv additionally induced over-representation of differential expressed pattern recognition receptor genes as compared to healthy controls (table s3 ): (i) toll-like receptors (tlr) cascades (r-hsa-168898; adjusted p-value = 1.23 × 10 −3 ), including tlr3 (r-hsa-168164; adjusted p-value = 2.45 × 10 −2 ), which are specialized in the recognition of conserved molecular features of different pathogens such as bacteria, viruses, fungi, and parasites; and (ii) c-type lectin receptors (r-hsa-5621481; adjusted p-value = 5.12 × 10 −6 ), capable of sensing glycans present in viral pathogens to activate antiviral immune responses such as phagocytosis, cytokine production, antigen processing and presentation, and subsequent t cell activation. related to this, aim2-like receptors (interferon inducible protein aim2; adjusted p-value = 7.90 × 10 −24 ; table s2 ) were found to be over-expressed (table s2) . aim2-like receptors can act as enzymatic complexes (inflammasomes) and are known to be activated by the presence of microbial dna within the cytosol [26] . in response to viral and bacterial infection these inflammasomes trigger caspase-1 activation and subsequently induce the release of mature pro-inflammatory interleukins such as il-18, contributing to the immune response to pathogens [27] (table s2) . other enriched pathways (table s3 ) are the tlr4 cascade (r-hsa-166016), which acts as co-receptor with cd14 (adjusted p-value = 2.29 × 10 −8 ) detecting bacterial lipopolysaccharide, and other tlr4-related pathways such as trif(ticam1)-mediated tlr4 signaling (r-hsa-937061) and myd88-independent tlr4 cascade (r-hsa-166166). finally, we found that the rsv infection causes an under-representation of deg related to the olfactory signaling pathway (r-hsa-381753; adjusted p-value = 1.09 × 10 −12 ) (table s3 ). to investigate a specific rsv transcriptome signature, we compared rsv expression patterns against a viral multi-cohort set following a cross-validation strategy that randomly divides the whole dataset into a training and a test set. representative samples of coronaviruses, influenzas, rhinoviruses, enteroviruses, and rsv transcriptomes were present in both the training and the test sets. the volcano plot of figure 2a shows the genes differentially expressed when comparing rsv-infected children to children infected by other pathogens. to investigate a specific rsv transcriptome signature, we compared rsv expression patterns against a viral multi-cohort set following a cross-validation strategy that randomly divides the whole dataset into a training and a test set. representative samples of coronaviruses, influenzas, rhinoviruses, enteroviruses, and rsv transcriptomes were present in both the training and the test sets. the volcano plot of figure 2a shows the genes differentially expressed when comparing rsvinfected children to children infected by other pathogens. among the 100 most significant genes in this analysis (indicated in red in figure 2a ), we searched for the minimum transcriptome signature allowing to discriminate between rsv from other pathogens using the optimal gene model size according to prems algorithm. figure 2b shows the optimal model characterized by 17 genes that conform to an optimal signature to distinguish rsv from other viral conditions. this 17-transcript signature (table 1; figure 3a ) identified in the discovery training set distinguished rsv from other viral conditions with area under de curve (auc) of 90.2% (95%ci: 87-93.0), sensitivity of 81.3% and specificity of 93.0%. in spite of the global burden of rsv, our knowledge of how rsv interacts with the host remains incomplete. moreover, the study of the host gene expression response to rsv infection is usually restricted to specific and very homogeneous cohorts, hampering the interpretation of the results, the impact of the conclusions obtained and their translation into a molecular test that could be used in routine clinical practice. the present meta-analysis shows that rsv infection leads to global changes in the host transcriptome, affecting not only the transcriptomic machinery of the airway infected cells [28] , but also the expression of hundreds of genes in blood cells (table s2 ). in particular, according to our results, rsv alters the expression of >1,500 genes in the host compared to healthy controls, involving e.g., cell cycle and immune system genes. this host response can be differentiated to those from other viruses with only 17-transcripts, which might eventually facilitate its use for clinical diagnosis. when applying this signature to the test set, the auc decreases to 83.6% (95%ci: 76.0-91.3), with a sensitivity of 73.6% and specificity of 87.5%. when we evaluated the performance of the model to differentiate rsv from each pathogen individually, we obtained auc values ranging from 77.0% for coronavirus, to 97.9% for enterovirus in the training cohort, and values ranging from 77.1% for influenza to 97.1% for enterovirus in the test cohort (table 2 ; figure 3b ,c). in spite of the global burden of rsv, our knowledge of how rsv interacts with the host remains incomplete. moreover, the study of the host gene expression response to rsv infection is usually restricted to specific and very homogeneous cohorts, hampering the interpretation of the results, the impact of the conclusions obtained and their translation into a molecular test that could be used in routine clinical practice. the present meta-analysis shows that rsv infection leads to global changes in the host transcriptome, affecting not only the transcriptomic machinery of the airway infected cells [28] , but also the expression of hundreds of genes in blood cells (table s2 ). in particular, according to our results, rsv alters the expression of >1,500 genes in the host compared to healthy controls, involving e.g., cell cycle and immune system genes. this host response can be differentiated to those from other viruses with only 17-transcripts, which might eventually facilitate its use for clinical diagnosis. in mammals, the cell cycle is governed by a complex molecular machinery. the data indicate that patients infected with rsv have an over-representation of differentially expressed cell cycle-related genes, a well-known strategy employed by many other viruses to facilitate their replication [29, 30] . as expected, we observed deg and pathways related to the immunological response that are particularly relevant in the context of rsv infection, e.g., cytokines, which are small proteins released by cells that are known to play a major role in interactions and communications between cells. rsv infection causes a significant alteration of cytokine pathways and related pathways (adjusted p-value = 7.90 × 10 −16 ; table s3 ), a finding that is in agreement with other authors, indicating that host genetic variation in cytokines predisposes to suffering complications during rsv infection [31] . although more studies are needed to confirm this hypothesis, overall, these results indicate that measuring cytokines might help identify children at high risk of suffering rsv complications; this would allow starting treatments in earlier stages of the disease. also, among the genes that are over-represented during rsv infection are the stat family members, which act as transcription activators in response to cytokines. for instance, the absence of stat1 resulted in airway dysfunction in knockout mice infected with rsv [32] , and variation at stat2 has been associated with rsv susceptibility in preterm children [33] . matrix metallopeptidases (mmps) are proteins mostly known for degrading extracellular matrix proteins, but these are also known to be involved in other processes such as cytokine inactivation [34] . experiments in mice carried out by kong et al. [35] indicated that rsv infection raises mmp-9 levels, whereas the reduction of mmp-9 resulted in decreased viral replication, suggesting that mmp-9 may be a potential therapeutic target for rsv disease. our results are in line with these findings as we found this gene up-regulated, supporting the hypothesis that mmp-9 could be a promising drug target. like other authors, we found up-regulated interferon-inducible genes such as eif2ak2 [5] , which according to previous results plays a role in rsv susceptibility and immunological response. it has been described that mrna level of this gene (and expression of tlr4) depends at least partially on the patient ethnicity, suggesting that the transcriptional response of individuals may be affected by the populational background [36] [37] [38] . another interferon-induced gene we found up-regulated was ifitm1, which, according to zhang et al. [39] , inhibits rsv infection interfering with the viral entry and replication processes. remarkably, our infected patients expressed as almost 50% more interferon-induced gtp-binding protein mx1 gene (log 2 fc = 1.48) than the healthy controls. this finding is interesting as it has been argued that polymorphisms in mx1 predispose to severe form of rsv in infants [36] . we also found an up-regulation of genes belonging to the innate immune response. for instance, oas1 and oas2 are responsible for the activation of ribonuclease l, which is part of the innate immune defense, during viral infection [40] . ribonuclease l is an interferon ifn-induced ribonuclease that, when activated, destroys all single-stranded rnas within the cell (cellular and viral) including rrnas [41] . the destruction of all rna within the cell is its last attempt to fight back against a virus before the onset of apoptosis [42] . rsv infection also induces an over-representation of deg related to the neutrophil degranulation pathway; this pathway was found to be related to the immune response to rsv [43] and other viral respiratory diseases [44] . it has been shown that neutrophils degranulate into the airway in response to rsv, pointing to a local innate response to the infection. neutrophils degranulation implies the release of antimicrobial substances that may contribute to the control of commensal bacteria in the upper respiratory tract [28] . according to our meta-analysis, five pattern recognition receptors (cd14, tlr4, tlr5, tlr7 and tlr8) are overexpressed during rsv infection. variants at these genes might be associated with the development of rsv bronchiolitis in different human populations, including israel, finland, argentina, japan, and greece [45] [46] [47] [48] [49] [50] [51] . in 2000, kurt-jones et al. [24] reported that rsv persisted longer in the respiratory organs of infected tlr4-deficient mice in comparison to controls, and suggested that therapies that target the expression of tlrs could be useful to fight rsv infections [24] . moreover, tlr4 polymorphisms asp299gly and thr399ile seem to be associated with an enhanced risk of developing severe rsv bronchiolitis [45] . furthermore, zhou et al. [52] indicated that the tlr4 signaling pathway, in conjunction with myd88 up-regulation, could be responsible for the activation of immune responses to rsv infection in airway epithelial cells [52] . our results are in agreement with their observations, as we detected the up-regulation of both genes in the blood of rsv-infected patients when compared to healthy controls (table s2) . the up-regulation of the tlr3 cascade is particularly interesting since this receptor recognizes viral dsrna, ultimately stimulating the production on type i interferons, a family of cytokines that regulate immune response to viral and other intracellular infections [53] . the over-representation in patients of c-type lectin receptors (clrs) pathway (r-hsa-5621481; table s3 ) is also remarkable as it has been described that the detection of viral glycans by these receptors help to fight viral infections. up-regulation of clrs activates antiviral immune responses such as antigen processing and presentation, t cell activation and phagocytosis, suppressing viral dissemination within the host. nevertheless, clrs can be a double-edged sword as some viruses have evolved to use these receptors for viral entry into host cells, avoiding immune recognition, where kidnapping the host cell machinery they produce hundreds of new copies of themselves spreading their copies further into the host [54] . whole exome sequencing studies have revealed host biomarkers of susceptibility to rsv belonging to the olfactory and taste receptors. single nucleotide polymorphism (snp) variation at these genes has been observed to be associated with rsv infection [8] . rsv has also been described to cause post-viral olfactory loss [55] . according to our meta-analysis, rsv infection also has an impact on the deg related to on the olfactory signaling pathway (r-hsa-381753; adjusted p-value = 1.09 × 10 −12 ). last but not least, we identified a 17-transcript blood signature specific for rsv that differentiates it from other respiratory viruses of similar etiology such as influenza, rhinovirus, coronavirus and enterovirus. this indicates that rsv alters the host transcriptome in a specific manner that can be distinguished from other respiratory viruses. the identified signature could be relevant for rsv diagnosis. even though current automatic pcr-based technologies already show good performance [56] , these 17 transcripts might further our understanding of host molecular processes specifically altered by the rsv, which could eventually lead to the discovery of new drug targets for the treatment of rsv patients. future laboratory validation will be necessary before the discovered signature can be used in clinical settings. a major advantage of the present meta-analysis is that, by gathering data from different studies, we substantially increase sample size and consequently the statistical power to detect deg when compared to controls and other pathogens and reduce the potential bias derived from selecting patients in particular populations, geographic areas and/or seasons. to reduce batch effects the data were carefully normalized and preprocessed (see methods section); a limitation of this procedure is however that it not only reduces artificial sources of variability between datasets, but it might also reduce biological sources. the normalization procedure also reduces the number of probes analyzed to those that are shared between all microarrays. another limitation of our study is that information on the rsv serotype (a/a2 or b), neither the disease status of the patients, are not available for all the datasets. the present study is a stepping-stone towards understanding how rsv affects the host transcriptome, but further studies are needed to better understand how the host transcriptomic response change among serotypes, and during the progression of the disease. these studies would allow to shed further light in the mechanism responsible of rsv pathogenicity. we queried the public gene expression microarray repository gene expression omnibus (geo) for human gene expression datasets using the following terms: "rsv" and/or "syncytial". we retained only those studies containing microarray expression data from whole blood samples of rsv infected patients. more information on the datasets used is provided in table s1 . to merge and integrate the public domain rsv microarray studies, we first normalized and preprocessed each dataset separately using the package lumi [61] for illumina ® microarrays data and the package oligo [62] for affymetrix ® datasets ( figure s2 ). we also conducted a principal component analysis (pca) to check for outliers and evaluate the presence of strong batch effects that could affect the analysis of the data. subsequently, we used the r package coconut (combat co-normalization using controls) to combine all datasets into one and reduce batch effects in the meta-analysis [21] . finally, to determine which genes are significantly up-or down-regulated during the rsv infection (n = 296) when compared to both healthy controls (n = 193) and other viral conditions as a single group (n = 360; table s2 ), we used the r package limma [63] and a moderated t-statistic. a linear model was fitted considering the age as a categorical covariate of the model (children / adult); this allowed us to minimize confounding effects considering that the study gse68310 was carried out on adults whereas the rest of studies were conducted in children. multiple testing correction was performed using the false discovery rate (fdr). a summary of the methodological procedure is shown in figure 4 . more information on the datasets used is provided in table s1 . to merge and integrate the public domain rsv microarray studies, we first normalized and preprocessed each dataset separately using the package lumi [61] for illumina ® microarrays data and the package oligo [62] for affymetrix ® datasets ( figure s2 ). we also conducted a principal component analysis (pca) to check for outliers and evaluate the presence of strong batch effects that could affect the analysis of the data. subsequently, we used the r package coconut (combat co-normalization using controls) to combine all datasets into one and reduce batch effects in the meta-analysis [21] . finally, to determine which genes are significantly up-or down-regulated during the rsv infection (n = 296) when compared to both healthy controls (n = 193) and other viral conditions as a single group (n = 360; table s2 ), we used the r package limma [63] and a moderated t-statistic. a linear model was fitted considering the age as a categorical covariate of the model (children / adult); this allowed us to minimize confounding effects considering that the study gse68310 was carried out on adults whereas the rest of studies were conducted in children. multiple testing correction was performed using the false discovery rate (fdr). a summary of the methodological procedure is shown in figure 4 . we used the reactome pathway database to examine biological pathways associated with the genes differentially expressed in rsv patients. thus, to categorize differentially expressed genes (deg) for overrepresentation of reactome pathways we used panther classification system with the following parameters: reactome pathways overrepresentation test (released on 2019-03-08) with reactome version 65 (released on 2019-03-12), and homo sapiens as a reference list. statistical significance was evaluated using fisher's exact test and fdr as the multiple test correction method. our hypothesis is that rsv affects the transcriptome in a particular way distinguishable from other viral conditions. from the list of deg obtained when compared rsv patients against other viral conditions (table s2) , we investigated a minimum specific transcript signature of rsv infection. we used parallel regularised regression model search (prems) [64] in a randomly split dataset removing the healthy controls: training set (n = 521) and validation set (n = 135). prems explores different logistic regression models constructed from optimal subsets of the candidate genes while increasing the model size iteratively. prems [64] was chosen instead of other methods because it tends to select signatures with fewer genes without sacrificing model accuracy, which would facilitate its future translation into a clinical test [17] . the bio-signature was searched among the top 100 deg between rsv and non-rsv categories. finally, the accuracy of the model estimated by prems was calculated as the area under the receiver operator curve (auc) using the r package proc [65] in both training and test cohorts. all analyses were carried out using r software version 3.5.2 [66] . the present integrated multicohort analysis suggests that rsv alters the expression of >1500 genes in the host. a number of them are related to different pathways, namely cell cycle, immunological response to viral infection (including pattern recognition receptors), and olfactory signaling. in addition, rsv modifies the host transcriptome in a very specific manner, different from other respiratory viruses with similar phenotypes. we found a 17-transcript signature (validated in several independent cohorts), that allows the discrimination of rsv infection from other respiratory viruses. considering the small number of transcripts involved, this signature might be potentially translated into a point of care test. this study is a step forward to a better understanding of the molecular mechanism underlying rsv infection. the biomarkers of rsv infection detected may help discover new drug targets and improve the development of vaccines. using pathway-based approaches such as go term enrichment or ingenuity pathway analysis (ipa) to prioritize the genes whose expression is altered by rsv infection may help discovering new drug targets and improve the development of vaccines. supplementary materials: supplementary materials can be found at http://www.mdpi.com/1422-0067/21/5/1831/ s1. table s1 . description of samples and cohorts included in the present study. table s2 . genes differentially expressed according to r package limma. table s3 . reactome pathway enrichment analysis results. figure s1 . pca plot of the eight datasets initially selected for the meta-analysis. the data points clustering on the right side of the plot (pc1) correspond to the outlier transcriptome profiles of the rsv samples from the dataset gse80179; the control samples in gse80179 cluster with the other controls. this study received support from the instituto de salud carlos iii: project gepem (instituto de salud carlos iii(isciii)/pi16/01478/cofinanciado feder), diavir (instituto de salud carlos iii(isciii)/dts19/00049/cofinanciado feder; proyecto de desarrollo tecnológico en salud) and resvi-omics (instituto de salud carlos iii(isciii)/pi19/0103; 9/cofinanciado feder) given to a.s.; and project resvinext (instituto de salud carlos iii(isciii)/pi16/01569/cofinanciado feder), and enterogen (instituto de salud carlos iii(isciii)/ pi19/01090/cofinanciado feder) given to f.m.-t. biomarkers of respiratory syncytial virus (rsv) infection: specific neutrophil and cytokine levels provide increased accuracy in predicting disease severity global, regional, and national disease burden estimates of acute lower respiratory infections due to respiratory syncytial virus in young children in 2015: a systematic review and modelling study whole blood gene expression in infants with respiratory syncytial virus bronchiolitis risk of primary infection and reinfection with respiratory syncytial virus review of epidemiology and clinical risk factors for severe respiratory syncytial virus (rsv) infection risk factors in children hospitalized with rsv bronchiolitis versus non-rsv bronchiolitis the burden of respiratory syncytial virus infection in young children whole exome sequencing reveals new candidate genes in host genomic susceptibility to respiratory syncytial virus disease increased concordance of severe respiratory syncytial virus infection in identical twins disease severity in respiratory syncytial virus infection: role of host genetic variation respiratory syncytial virus bronchiolitis in children up to 5 years of age in spain: epidemiology and comorbidities: an observational study diagnosis of childhood tuberculosis and host rna expression in africa diagnostic test accuracy of a 2-transcript host rna signature for discriminating bacterial vs viral infection in febrile children whole blood gene expression profiles to assess pathogenesis and disease severity in infants with respiratory syncytial virus infection a 2-transcript host cell signature distinguishes viral from bacterial diarrhea and it is influenced by the severity of symptoms a qpcr expression assay of ifi44l gene differentiates viral from bacterial infections in febrile children diagnosis of kawasaki disease using a minimal whole-blood gene expression signature gene expression and regulation in systemic lupus erythematosus how to read a systematic review and meta-analysis and apply the results to patient care: users' guides to the medical literature power failure: why small sample size undermines the reliability of neuroscience genome-wide expression for diagnosis of pulmonary tuberculosis: a multicohort analysis host-response-based gene signatures for tuberculosis diagnosis: a systematic comparison of 16 signatures a 20-gene set predictive of progression to severe dengue a community approach to mortality prediction in sepsis via gene expression analysis 2 -5 -oligoadenylate synthetase-like protein inhibits respiratory syncytial virus replication and is targeted by the viral nonstructural protein 1 aim2 recognizes cytosolic dsdna and forms a caspase-1-activating inflammasome with asc mechanisms of nod-like receptor-associated inflammasome activation airway response to respiratory syncytial virus has incidental antibacterial effects the cell cycle: a review martinon-torres, f. strong down-regulation of glycophorin genes: a host defense mechanism against rotavirus infection respiratory syncytial virus (rsv) evades the human adaptive immune system by skewing the th1/th2 cytokine balance toward increased levels of th2 cytokines and ige, markers of allergy-a review respiratory syncytial virus infection in the absence of stat 1 results in airway dysfunction, airway mucus, and augmented il-17 levels genetic susceptibility to respiratory syncytial virus bronchiolitis in preterm children is associated with airway remodeling genes and innate immune genes chemokine and cytokine processing by matrix metalloproteinases and its effect on leukocyte migration and inflammation matrix metalloproteinase-9 mediates rsv infection in vitro and in vivo a genetic model of differential susceptibility to human respiratory syncytial virus (rsv) infection ancestry patterns inferred from massive rnaseq data gene-expression variation within and among human populations human respiratory syncytial virus infection is inhibited by ifn-induced transmembrane proteins activation of rnase l is dependent on oas3 expression during infection with diverse human viruses cell-type-specific effects of rnase l on viral induction of beta interferon the role of 2 -5 oligoadenylate-activated ribonuclease l in apoptosis respiratory syncytial virus stimulates neutrophil degranulation and chemokine release a role for neutrophils in viral respiratory disease. front. immunol. 2017, 8, 550 association between common toll-like receptor 4 mutations and severe respiratory syncytial virus disease tlr4 genotype and environmental lps mediate rsv bronchiolitis through th2 polarization association of tlr4 polymorphisms with symptomatic respiratory syncytial virus infection in high-risk infants and young children toll-like receptor 4 asp299gly polymorphism in respiratory syncytial virus epidemics cd14 -550 c/t, which is related to the serum level of soluble cd14, is associated with the development of respiratory syncytial virus bronchiolitis in the japanese population the role of tlr4 and cd14 polymorphisms in the pathogenesis of respiratory syncytial virus bronchiolitis in greek infants he, q. tlr5 rs5744174 gene polymorphism is associated with the virus etiology of infant bronchiolitis but not with post-bronchiolitis asthma transcriptomic profiling in childhood h1n1/09 influenza reveals reduced expression of protein synthesis genes type i interferons in host defense a simple screening approach to prioritize genes for functional analysis identifies a role for interferon regulatory factor 7 in the control of respiratory syncytial virus disease postviral olfactory loss comparison of cepheid xpert flu/rsv xc and biofire filmarray for detection of influenza a, influenza b, and respiratory syncytial virus plasticity and virus specificity of the airway epithelial cell immune response during respiratory virus infection nasopharyngeal microbiota, host transcriptome, and disease severity in children with respiratory syncytial virus infection host gene expression in nose and blood for the diagnosis of viral respiratory infection host transcriptional response to influenza and other acute respiratory viral infections-a prospective cohort study a pipeline for processing illumina microarray a framework for oligonucleotide microarray preprocessing limma powers differential expression analyses for rna-sequencing and microarray studies parallel regularised regression model search for sparse bio-signature discovery proc: an open-source package for r and s+ to analyze and compare roc curves r: a language and environment for statistical computing this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license we gratefully acknowledge cesga (supercomputing centre of galicia, santiago de compostela, spain) for computing availability, web hosting, and support. we would also like to thank timothy e sweeney (inflammatix, inc) for assistance with the coconut package. the authors declare no conflict of interest. key: cord-272143-6ej3eibd authors: benavides‐nieto, marta; méndez‐echevarría, ana; del rosal, teresa; garcía‐garcía, maría luz; casas, inmaculada; pozo, francisco; de la serna, olga; lopez‐granados, eduardo; rodriguez‐pena, rebeca; calvo, cristina title: the role of respiratory viruses in children with humoral immunodeficiency on immunoglobulin replacement therapy date: 2018-12-21 journal: pediatr pulmonol doi: 10.1002/ppul.24214 sha: doc_id: 272143 cord_uid: 6ej3eibd background: the role of viruses in children with respiratory tract infections and humoral immunodeficiencies has hardly been studied. we have evaluated these infections in children with humoral immunodeficiencies who required immunoglobulin replacement therapy, considering their relationship with symptoms, lung function, bacterial co‐infection, and outcomes. methods: we conducted a prospective case‐control study during a 1‐year period, including children with humoral immunodeficiencies receiving immunoglobulin replacement therapy. for each patient, at least one healthy family member was included. respiratory samples for viral detection were taken every 1‐3 months, and in case of respiratory tract infections. symptoms questionnaires were filled biweekly. spirometry and sputum culture were performed in every episode. results: sixty‐six episodes were analyzed in 14 patients (median age 12 years; iqr 7‐17), identifying 18 respiratory viruses (27.3%), being rhinovirus the most frequently isolated one (12/18; 66%). positive viral episodes were associated with clinical symptoms (89% vs 43%), more frequent antibiotic treatment (44% vs 15%) or hospital admission (22% vs 0%) than negative ones. patients with positive viral detection showed impaired lung function, with lower fev1 and fvc values. conclusions: in our experience, viral respiratory tract infections can cause significant respiratory symptoms and impaired lung function, in children with hid, despite immunoglobulin replacement therapy. these patients could benefit from the monitoring of viral infections, as these may be a gateway for ongoing lung damage. children with severe t-cell immunodeficiencies present impaired clearance of respiratory viruses, and pulmonary complications of viral infections are leading causes of morbidity and mortality in this group of patients. 1 however, the role of respiratory viruses in children with other types of primary immunodeficiency (pid), mainly those with humoral immunodeficiencies (hid) or diseases of immune dysregulation, has hardly been studied. children with hid usually suffer from recurrent bacterial respiratory infections, resulting in progressive bronchiectasis and chronic lung disease. [2] [3] [4] [5] immunoglobulin replacement therapy (irt) reduces the frequency of these infections. however, despite adequate irt, recurrent respiratory infections are still one of the leading causes of morbidity and mortality in these patients. [2] [3] [4] little data are currently available regarding the susceptibility to respiratory viruses of hypogammaglobulinemic patients receiving irt. 4, 6, 7 however, some other viruses have been described which play significant roles in these patients. [8] [9] [10] [11] human herpesvirus 8 (hhv-8) has been associated with granulomatous/lymphocytic interstitial lung disease (glild) in patients with common variable immunodeficiency (cvid), 8 and enterovirus is a known cause of fatal meningoencephalitis in patients with x-linked agammaglobulinemia (xla). 9, 10 in addition, there are several recent reports showing an increased susceptibility to viral respiratory infections in adults with cvid receiving irt, 4-7 that can contribute to chronic and persistent pulmonary inflammation. 2, 7 we report, to the best of our knowledge, the first study that analyses respiratory viruses in pediatric patients with predominantly antibody deficiency who required irt, considering their relationship with clinical symptoms and pulmonary function, bacterial co-infection, treatment and outcomes. we conducted a prospective case-control single-center study during 1 year (november 2016 to october 2017) in a national reference unit for pid in madrid, spain. we included patients less than 18 years of age diagnosed with hid who required irt. patients with combined immunodeficiency and/or reduced proliferative responses to mitogens (pha, pwm, cona) were excluded. patients with hid who did not require irt and/or patients receiving immunosuppressive treatments other than rituximab were excluded. committee. informed consents were obtained from patients and parents. for each patient, a healthy control was included, usually one of their parents. respiratory samples (nasopharyngeal aspirate [npa] for patients and nasopharyngeal swab for controls) were obtained every one to three months, coinciding with intravenous immunoglobulin administration or outpatient clinic evaluations, both in the patient and in the control group. other investigations performed during the same visit in the patient group included: spirometry (in patients older than 5 years), spontaneous sputum culture (in patients older than 10 years), igg levels, full blood count (fbc), c-reactive protein, and erythrocyte sedimentation rate (esr). if patients developed respiratory symptoms between visits, additional sputum culture and nasopharyngeal aspirates were performed in the first 3 days after symptom onset, as well as an additional spirometry. in children older than 10 years of age who needed antibiotic for treating respiratory infections, the sputum samples were obtained before starting the therapy. every moment in which a respiratory sample was obtained from a patient was defined as an episode. symptoms questionnaires elaborated ad hoc were filled out systematically biweekly by patients and controls, recording fever, increased respiratory secretions, cough, respiratory distress, sputum three independent rt-pcr assays were performed to detect sixteen respiratory viruses as previously published by our group. [12] [13] [14] influenza a, b, and c viruses were detected by using previously described primer sets only to amplify influenza viruses in a multiplex pcr assay. 12 a second multiplex pcr was used to detect parainfluenza viruses 1-4, human coronaviruses 229e and oc43, enteroviruses, and rhinoviruses (rv). 13 presence of respiratory syncityal virus (rsv) a and b types, human metapneumovirus, human bocavirus, and adenoviruses were investigated by a third multiplex rt-nested pcr-brq method. 14 spontaneous sputum samples were collected using sterile specimen containers and immediately processed or stored at 4°c until benavides-nieto et al. | 195 processing was feasible. samples were cultured on standard media, and potential respiratory tract pathogens were identified and tested for antimicrobial susceptibility. pulmonary function tests were performed on all patients at study entry and repeated with every sample collection. spirometry was performed according to established guidelines 15 16 during the study period, 14 patients with predominantly antibody deficiency were included (5 girls and 9 boys). their main immunological diagnoses are reported in table 1 . the median age of patients was 12 years (iqr 7-17), and only two of them were younger than 5 years. eleven patients were receiving intravenous irt and three patients, subcutaneous irt. immunological status at inclusion is described in table 2 . eighteen healthy adult family members (mother and/or father) of the patients were included as healthy control subjects (at least one control per case included). on the other hand, other authors have hypothesized that persistent and recurrent viral respiratory infections could adversely affect the microbiome, leading to an increased bacterial density in children's nasopharyngeal tract, predisposing to sinopulmonary bacterial infections. 17, 18 as other authors, 4 we recommend screening for viral infection in children with hid and acute respiratory symptoms to avoid unnecessary antibiotic treatment. however, viral testing is routinely performed only in children with severe combined immunodeficiency (scid). 1, 4 regarding hid, protocols present a great deal of variation across europe in the management of lung complications. 19 there is an urgent need for consensus guidelines on how to monitor lung complications and how to treat respiratory infections in hid patients. 19 not all our patients were typical hid such as xla or cvid. three of our cases were scid patients who had received a hematopoietic cell transplant, but presented persistent b-cell lymphopenia and hypogammaglobulinemia. although most children with scid recover cellular immunity 1 year after transplantation, many patients are likely to be on irt for a considerable period until full reconstitution of b-cell immunity has been achieved. 20,21 b-cell immune reconstitution and its consequences regarding viral respiratory infections have not been deeply investigated in these patients. our data support that these patients are also prone to viral respiratory infections, which impact lung function and lead to antibiotic consumption. another patient was an ad-hies. regarding humoral immunity, many patients with ad-hies require immunoglobulin infusions due to memory b-cell lymphopenia and decline in specific antibody titers. 22 recurrent respiratory tract infections have been described in these children, and more than 90% of them develop pneumonia. 22 although bacteria are detected in 44% of respiratory exacerbations, viral pneumonia has also been reported. 22 lung sequelae in these patients are frequent, mainly as bronchiectasis. 22 thus, monitoring viral infections in these children may be important. one patient in our series suffered from apds2. while the disease is heterogeneous, respiratory infections and their complications are frequent and often severe. 23 the spectrum of pathogens is highly reminiscent of other primary antibody deficiency syndromes such as cvid. 23 coulter et al 24 seem to play a role in children with hid, as has also been described in adults. 7, 17 further studies are needed to improve our knowledge about the impact of respiratory viral infections, their prevention and management in children with irt, with the aim of avoiding short-and longterm consequences. clinical features before hematopoietic stem cell transplantation or enzyme replacement therapy of children with combined immunodeficiency lung disease in primary antibody deficiency subclinical infection and dosing in primary immunodeficiencies immune deficiency: changing spectrum of pathogens respiratory infections and antibiotic usage in common variable immunodeficiency virus shedding after human rhinovirus infection in children, adults and patients with hypogammaglobulinaemia recurrent and persistent respiratory tract viral infections in patients with primary hypogammaglobulinemia granulomatous-lymphocytic interstitial lung disease (glild) in common variable immunodeficiency (cvid) enteroviruses in x-linked agammaglobulinemia: update on epidemiology and therapy x-linked agammaglobulinemia: report on a united states registry of 201 patients primary immunodeficiency diseases associated with increased susceptibility to viral infections and malignancies simultaneous detection of influenza a, b, and c viruses, respiratory syncytial virus, and adenoviruses in clinical samples by multiplex reverse transcription nested-pcr assay simultaneous detection of fourteen respiratory viruses in clinical specimens by two multiplex reverse transcription nested-pcr assays detection of new respiratory viruses in hospitalized infants with bronchiolitis: a three-year prospective study standardisation of spirometry grading the severity of airways obstruction: new wine in new bottles the lung in primary immunodeficiencies: new concepts in infection and inflammation outgrowth of the bacterial airway microbiome after rhinovirus exacerbation of chronic obstructive pulmonary disease screening protocols to monitor respiratory status in primary immunodeficiency disease: findings from a european survey and subclinical infection working group immune reconstitution and survival of 100 scid patients post-hematopoietic cell transplant: a pidtc natural history study the kinetics of early t and b cell immune recovery after bone marrow transplantation in rag-2-deficient scid patients autosomal dominant stat3 deficiency and hyper-ige syndrome: molecular, cellular, and clinical features from a french national survey respiratory manifestations of the activated phosphoinositide 3-kinase delta syndrome clinical spectrum and features of activated phosphoinositide 3-kinase δ syndrome: a large patient cohort study escmid study group for infections in compromised hosts (esgich) consensus document on the safety of targeted and biological therapies: an infectious diseases perspective (agents targeting lymphoid cells surface antigens [i]: cd19, cd20 and cd52) the role of respiratory viruses in children with humoral immunodeficiency on immunoglobulin replacement therapy to claire marsden for the revision of the english language. this study has been partially supported by fis (fondo de investigaciones sanitarias-spanish health research fund) grant no. pi15ciii/00028. the authors declare no conflict of interest. key: cord-001070-zlzag6a8 authors: camargo, c.n.; carraro, e.; granato, c.f.; bellei, n. title: human rhinovirus infections in symptomatic and asymptomatic subjects date: 2012-06-01 journal: braz j microbiol doi: 10.1590/s1517-838220120004000049 sha: doc_id: 1070 cord_uid: zlzag6a8 the role of rhinovirus asymptomatic infections in the transmission among close contacts subjects is unknown. we tested health care workers, a pair of one child and a family member and immunocompromised patients (n =191). hrv were detected on 22.9% symptomatic and 3.6% asymptomatic cases suggesting lower transmission among contacts. describing adults was realized by bellei and colleagues (2007) detecting 37.7%. the dynamic of rhinovirus transmission is relevant to address for the epidemiology characteristics of infection within families, schools and nosocomial outbreaks. rhinovirus can be easily transmitted from person to person mainly through hand contact with infected respiratory secretions (15) . studies of asymptomatic infected individuals pointed to 15 -30% rates (9, 19, 14, 20, 7, 19) of hrv infection but the role of infected subjects as reservoirs for secondary cases infections is unknown. many studies have investigated the occurrence of rhinovirus among community cases but there is a lack of information about the frequency of rhinovirus asymptomatic cases. we investigated hrv infections rates on selected populations of a pair of one child and one family member, health care workers (hcw), and immunocompromised patients with and without respiratory symptoms from june to september. in this study, a total of 191 nasal swab (ns) specimens were collected from three groups. one hundred and eleven health care workers (hcw) from são paulo hospital, 36 pairs of one child and one family member and 8 blood marrow transplanted hospitalized patients (bmt). they were considered eligible symptomatic patients if possible viral etiology within 7 days of symptoms onset. the clinical criterion was presentation of at least one respiratory symptom (cough, sore throat, or nasal congestion) and one constitutional symptom (headache, malaise, myalgia, chills). for shown in table 1 . influenza and rhinovirus infections among health-care workers acute respiratory infection and influenza-like illness viral etiologies in brazilian adults respiratory consequences of rhinovirus infection rhinovirus infections in myelosuppressed adult blood and marrow transplanted recipients two outbreaks of severe respiratory disease in nursing homes associated with rhinovirus rhinovirus infections in hematopoietic stem cell transplant recipients with pneumonia persistence of rhinovirus and enterovirus rna after acute respiratory illness in children identification of respiratory viruses in asymptomatic subjects: asymptomatic respiratory viral infections use of polymerase chain reaction for diagnosis of picornavirus infection in subjects with and without respiratory symptoms comparison of conventional and molecular detection of respiratory viruses in hematopoietic cell transplant recipients new vaccine surveillance network rhinovirus-associated hospitalizations in young children respiratory illness caused by picornavirus infection: a review of clinical outcomes risk factors for lower respiratory complications of rhinovirus infections in elderly people living in the community: prospective cohort study human picornavirus and coronavirus rna in nasopharynx of children without concurrent respiratory symptoms rhinovirus transmission within families with children: insidence of symptomatic and assymptomatic infections rhinovirus and respiratory syncytial virus in wheezing children requiring emergency care. ige and eosinophil analyses genetic clustering of all 102 human rhinovirus prototype strains: serotype 87 is close to human enterovirus 70 phylogenetic analysis of rhinovirus isolates collected during successive epidemic seasons viral respiratory infections in hospitalized and community control children in alaska frequent detection of respiratory viruses in adult recipients of stem cell transplants with the use of real-time polymerase chain reaction, compared with viral culture respiratory pathogens in children with and without respiratory symptoms a rhinovirus outbreak among residents of a long-term care facility all the content of the journal, except where otherwise noted, is licensed under a creative commons license we acknowledge dr. eurico arruda (fmrp-usp) for providing hrv 39 isolate.the authors acknowledge the financial support of key: cord-023942-vrs3je1x authors: powers, karen s. title: acute pulmonary infections date: 2011-12-16 journal: pediatric critical care study guide doi: 10.1007/978-0-85729-923-9_25 sha: doc_id: 23942 cord_uid: vrs3je1x acute lower respiratory infection is a common cause of morbidity in infants and children, and at times, requires intensive care and mechanical ventilation. viral bronchiolitis and bacterial pneumonia account for the majority of lower respiratory tract infections that lead to respiratory insufficiency and pediatric intensive care admission. twenty-seven percent of children who require mechanical ventilation for at least 24 h in pediatric intensive care units are diagnosed with bronchiolitis and 16% have the diagnosis of pneumonia. the median length of time intubated for an acute pulmonary infection leading to respiratory failure is approximately 7 days. acute lower respiratory infection is a common cause of morbidity in infants and children, and at times, requires intensive care and mechanical ventilation. viral bronchiolitis and bacterial pneumonia account for the majority of lower respiratory tract infections that lead to respiratory insuffi ciency and pediatric intensive care admission. twenty-seven percent of children who require mechanical ventilation for at least 24 h in pediatric intensive care units are diagnosed with bronchiolitis and 16% have the diagnosis of pneumonia. the median length of time intubated for an acute pulmonary infection leading to respiratory failure is approximately 7 days. viral bronchiolitis remains the leading cause for hospital admission in infancy and the most frequent cause of acute respiratory failure in children admitted to pediatric intensive care units in north america. pneumonia in children younger than 5 years of age has an annual incidence karen s. powers of 34-40 cases per 1,000. community acquired pneumonia can also lead to severe respiratory compromise especially in children with pre-existing disease. a detailed understanding of the diverse etiologies and distinct clinical courses of acute pulmonary infections is essential for the pediatric critical care practioner. this chapter will focus on bronchiolitis and pneumonia as the two leading causes of pulmonary infections leading to picu admission. approximately one third of children develop bronchiolitis during the fi rst 2 years of life. of these, only 1 in 10 (3% of all infants in the united states) will require hospitalization. although hospitalization rates have increased over the last three decades, mortality remains low. overall mortality rate is 1-2%, but as high as 5% in high risk infants. most deaths occur in infants younger than 6 months of age with co-morbidities such as prematurity, congenital heart disease, congenital or acquired lung disease or immunodefi ciency. respiratory syncytial virus (rsv) was fi rst isolated in 1957 and still represents the major cause of bronchiolitis. other causative viruses include parainfl uenza, adenovirus, enterovirus, infl uenza and most recently human metapneumovirus and human bocavirus (hbov). in the northern hemisphere, rsv outbreaks occur from october to june. human metapneumovirus (hmpv) recently has been identifi ed as the causative agent in 3-19% of bronchiolitis cases, possibly surpassing parainfl uenza as the second most common etiology. its prevalence is slightly higher in the late winter and spring. parainfl uenza infections peak at 10 months of age, representing approximately 7-10% of cases of bronchiolitis. parainfl uenza (piv-3) is endemic throughout the year, but especially common in the late spring. males are 1.5-2 times more likely to require hospitalization for bronchiolitis and are likely to have more severe disease. an x-linked genetic trait that results in a reduced tolerance to hypoxia has been postulated and would be consistent with the observation of increased mortality in newborn males with infant respiratory distress syndrome. virtually all children by the age of two will have been infected with rsv, all children by the age of fi ve will have been infected with hmpv, and all children by the age of nine will have been infected with hbov. the remainder of the discussion on bronchiolitis will be divided into rsv and non-rsv bronchiolitis. although etiologic agents may differ, clinical courses are often similar. respiratory syncytial virus (rsv) accounts for 50-80% of bronchiolitis, infecting one-half of all infants within the fi rst year of life and hospitalizing approximately 120,000 infants yearly (about 3% of affected infants). approximately 10% of these infants require mechanical ventilation. co-infection with either hmpv or rhinovirus occurs in 10-30% of young children. two types of rsv exist -types a and b. type a is more common and is believed to cause more severe disease, although data is not conclusive. both types may exist simultaneously in the community. infants less than 1 year will typically shed the virus for about 9 days. children with immunodefi ciencies may shed the virus for months. the immune response varies with age and contributes to both termination of the disease and its pathologic features. the virus is transmitted from respiratory secretions by close contact with infected persons or by contact with contaminated objects or surfaces. there is a 45% rsv transmission rate within families and about one-half of hospital workers will acquire rsv. therefore, hand washing and the wearing of gowns and gloves is of primary importance to attenuate transmission. mortality from rsv bronchiolitis continues to decline with better intensive care and the use of preventive therapies. male infants are more likely to require hospitalization and usually manifest more severe disease. about ½ of all infants will be infected with rsv bronchiolitis in their 1st year of life; 3% will be hospitalized; 10% of hospitalized infants will require mechanical ventilation. antibody-mediated immunity rsv introduced onto the nasal or conjunctival mucosal surface causes profuse rhinorrhea within a few days. during the fi rst 2 months of life, passively acquired maternal antibodies are protective. however, as maternal antibody titers gradually decrease, infants become susceptible to severe disease. cell-bound iga may develop to help clear the virus. circulating igg directed against the glycoprotein (g) and fusion (f) proteins (operative in syncytia formation) on the viral surface will develop several days later. infants less than 3 months of age appear to induce a weaker antibody response likely due to the presence of maternal antibodies. virus-specifi c ige in the respiratory tract is associated with disease severity. often, complete and effective immune responses are not induced, thus re-infections are possible even during the same season. epithelial cells and alveolar macrophages are key activators of cellular immunity. although these cells enhance viral clearing, they also contribute to airway infl ammation through the release of cytokines and chemokines. these include interleukin (il)-1, tumor necrosis factor-alpha, il-6, il-8, macrophage-infl ammatory protein (mip)-1-alpha and rantes (regulated upon activation, normal t cell expressed and secreted). release of these cytokines and chemokines are believed to be partially responsible for airway infl ammation and hyperreactivity. the effects of these mediators persist beyond the acute infection and contribute to prolonged pulmonary dysfunction. children who require mechanical ventilation have lower peripheral t cell counts compared to hospitalized infants not requiring mechanical ventilation. these infants demonstrate low t cell proliferative responses and interferon (ifn-g ) production. il-12 is required for the initiation of cellular immunity. the length of time requiring mechanical ventilation has been found to be inversely related to il-12 production. the role of th1/th2-like cytokine profi les, expressed as ifn-g /il-4 ratios, is controversial. in some studies, these ratios decreased after polyclonal stimulation in hospitalized infants with rsv. however, more recent studies have shown normal ratios following polyclonal stimulation. neutrophils are the predominant cell found in the airways of infants with rsv bronchiolitis. elevated levels of il-8 are found in high concentrations in the nasal secretions of infected children and act as a neutrophil chemoattractant. further evidence of cellular induced injury is seen in post-mortem examination where peribronchial lymphocyte infi ltration with bronchial epithelial necrosis is typically present. infants typically present with tachypnea, rhinorrhea, cough, low-grade fever, irritability, poor feeding and vomiting. respiratory rates greater than 60 breaths per minute are often associated with room air saturations of less than 96%. infants may also have tachycardia, mild conjunctivitis, otitis media, or pharyngitis. low-grade fever usually persists for 1-3 days. in addition, infants may develop a metabolic acidosis from poor caloric and fl uid intake. apnea often is the fi rst presenting symptom of rsv bronchiolitis in small infants. the etiology of apnea remains unknown; however, is likely related to the immaturity of the respiratory control center in the brainstem. the incidence of apnea in infants with bronchiolitis is approximately 16-20%. the heterogeneous nature of rsv induced lung disease can cause atelectasis in some areas and overdistension in others. chest roentgenograms often show hyperinfl ation with fl attening of the diaphragms and patchy or peribronchial infi ltrates. atelectasis, especially of the right upper lobe, is often seen. infants may have high lung volumes with the functional residual capacity often being twice normal. the decrease in dynamic compliance and increase up regulation of the infl ammatory cascade with release of chemokines and cytokines are contributory to the airway infl ammation and hyperreactivity. c hapter 25 • ac ute pu lmonary i n fections in airway resistance leads to marked increase in work of breathing, often worse during expiration from lower airway obstruction. alterations in gas exchange and hypoxemia are secondary to a ventilation-perfusion mismatch. the anatomical differences between young infants and older children contribute to the severity of the disease in the young. due to the highly compliant cartilaginous chest wall and poor thoracic musculature, the infant's chest wall has diffi culty countering the lung's inherent tendency towards collapse. this leads to a greater propensity of small infants towards atelectasis compared with older children. the absence of effective collateral ventilation in infants also contributes to the development of atelectasis and impaired gas exchange. cellular debris in small airways and peribronchial edema increase airways resistance leading to wheezing as the predominant symptom in some infants. despite the potential for severe impairment in lung function, most hospitalized infants improve within 3-5 days. typically, by 2 weeks, they have normal respiratory rates, oxygenation, and ventilation. chest radiographs usually normalize by day 9. however, about 20% of infants will have a protracted course, with some mild respiratory symptoms persisting for months. viral respiratory infections have been linked to the development of asthma later in childhood. the tucson children's respiratory study group prospectively followed for 13 years, 880 infants who had bronchiolitis and found an increased risk for subsequent wheezing episodes. some infants are at an increased risk for severe rsv disease such as those with chronic lung disease due to prematurity (bronchopulmonary dysplasia), cystic fi brosis, congenital heart disease, and immunodefi ciencies. in children with cystic fi brosis, rsv accounted for 18% of symptomatic infections, 33% of hospitalizations for infants less than 1 year, and 43% of infants requiring mechanical ventilation. in a study of hospitalized infants with congenital heart disease infected with rsv, 33% required intensive care, 19% received mechanical ventilation, and 3.4% died. children having undergone hematopoietic stem cell transplants who develop rsv infections have an extremely high mortality of 60-80% despite mechanical ventilation and antiviral therapy. environmental factors such as crowding, passive exposure to tobacco smoke, and lack of breast-feeding are associated with the development of severe disease. compared to national averages, native american and alaskan children younger than 1 year of age have higher rates of infections. there are three subtypes of human parainfl uenza viruses. hpiv-3 is most frequently isolated from children with bronchiolitis, while piv-1 and piv-2 most commonly cause croup. similar to rsv, both cell-mediated hyper-responsiveness to viral antigen and virus-specifi c ige responses are observed in children with parainfl uenza bronchiolitis. upper airway edema with concomitant obstructive symptoms may be present. children that are infected with parainfl uenza have a signifi cant likelihood of developing asthma later in life. the human metapneumoviruses (hmpv) are a group of rna viruses of the paramyxoviridae family identifi ed in humans in 2001. hmpv appears to be the second most common cause of bronchiolitis in children throughout the world. the majority of children are born with maternal hmpv specifi c igg which wanes to around 25% by 6-12 months of age. by age fi ve, essentially 100% of children have been exposed to hmpv and will have neutralizing antibody to hmpv. there are two subgroups, a and b, with group a having more severe clinical symptoms. clinical presentation of children with this virus is similar to rsv. the pulmonary infl ammation generally peaks on day 5 which includes interstitial edema and infl ammatory cell infi ltrates of the bronchioles and alveoli. these infl ammatory changes can persist for up to 21 days. about half of infected children are 0-12 months of age, and infection is primarily in the winter months. human bocavirus (hbov) was recently discovered in 2003. with amino acid sequencing, this new member of the parvoviridae family was found to be closely related to the bovine parvovirus and the canine minute virus, hence the name bocavirus (bo for bovine and ca for canine). detection of the hbov from the respiratory tract in symptomatic children and its absence of detection in non-symptomatic controls strongly suggest the virus to have a role in respiratory infections in children. co-infection is commonly described in up to 60% of samples. it remains unclear if hbov is a primary pathogen or acts to exacerbate other viral illnesses. the pathogenesis of hbov has not been well described, but with the high occurrence of wheezing and lower respiratory tract symptoms in children infected with the virus, it is speculated that this virus may be a signifi cant contributor to asthma exacerbations. the majority of infected children have rhinorrhea, cough, and wheezing, however, diarrhea has been reported in up to 25% of these children. in children with high viral loads, hbov has been detected in the serum suggesting the potential for disease beyond the respiratory tract. both infl uenza a, including novel infl uenza strains such as h1n1, and infl uenza b can cause a clinical picture consistent with bronchiolitis in the small infant. these viruses may cause severe multisystem disease and are discussed in greater detail in the viral pneumonia section. rapid diagnostic assays are available for early detection of many viruses. the older assays are antigen-based and include indirect immunofl uorescence/direct immunofl uorescence (ifa/ dfa), enzyme immunoassay (eia), optical immunoassay (oia), and neuraminidase activity assays. although still widely used because they are inexpensive and technically simple, they have a low specifi city and sensitivity. molecular assays are becoming the new "gold standard" for respiratory virus detection -replacing tissue culture that may take days. the published sensitivities and specifi cities approach 100% when compared to tissue culture or antigen assay. these assays generally use polymerase chain reaction (pcr) amplifi cation. signifi cant advancements in these assays are being made to simplify the performance of the assay and decrease the required time. the most important cause of false negative test results remains poor specimen handling or inadequate sample collection. other than aiding with cohorting of hospitalized patients, serologic detection of respiratory viruses is rarely clinically useful. regardless of the viral etiology of bronchiolitis, supportive care remains the mainstay of treatment. supplemental humidifi ed oxygen is frequently needed. due to many infants being obligate nasal breathers, frequent nasal suctioning may be benefi cial to maintain an unobstructed upper airway. the affected infant or child is often unable to take adequate fl uids complicated by increased insensible losses from the respiratory tract; hence, intravenous fl uids may be required. infants and children with severe respiratory distress should be kept npo in the event respiratory failure ensues and endotracheal intubation is required. antibiotics are not routinely indicated in previously healthy children infected with rsv. progressive disease, leukocytosis, persistent fever, consolidation on radiograph or systemic toxicity should prompt an evaluation of bacterial co-infection and the use of empiric antibiotics. high risk patients often require close monitoring and care in an intensive care unit. these include infants less than 6 weeks of age or infants with a history of prematurity, congenital heart disease, bronchopulmonary dysplasia, immunodefi ciency or neurologic disease. infants with rsv bronchiolitis typically have a combination of hyperinfl ation, pulmonary infi ltrates, supportive therapy is the mainstay of treatment for bronchiolitis. ribavirin, bronchodilators, and corticosteroids have not shown to be of benefi t. secondary bacterial infections are rare. and atelectasis. therefore, no one mode of ventilation can be recommended for all infants. non-invasive positive pressure (niipp) modes (cpap or bipap) may be attempted in infants where their primary respiratory embarrassment is secondary to atelectasis. however, this may not be suitable if the disease process appears severe or protracted as prolonged use of nipp may make feeding diffi cult, cause breakdown of facial tissue, or be diffi cult to maintain without signifi cant sedation that further compromises ventilation. if an infant requires endotracheal intubation, the mode of mechanical ventilation should be tailored to the predominant lung pathology present (i.e. atelectasis versus hyperinfl ation). children with signifi cant air trapping may need mechanical ventilation similar to a child with asthma, providing low respiratory rates and longer inspiration and exhalation times. the more typical infant will lose functional residual capacity (frc) because of atelectasis and alveolar infi ltrates. therefore, despite having some air trapping, these infants often need peep to be adjusted to recruit alveoli and return frc to normal. in the setting of elevated pulmonary vascular resistance (pvr) which may occur in infants with congenital heart disease or bronchopulmonary dysplasia, lowering pvr by traditional methods such as maintaining oxygenation, deep sedation, muscle relaxation and even nitric oxide may be indicated. ribavirin is the only fda-approved antiviral drug for rsv. ribavirin inhibits viral replication and is active against rsv, infl uenza a and b, adenoviruses, and hepatitis viruses. for lower respiratory tract diseases, ribavirin is typically administered via aerosolization. in 1996, a meta-analysis of studies involving ribavirin was discouraging and was consistent with the common clinical experience that ribavirin did not improve clinical outcomes. therapy targeted at attenuating the virus-induced infl ammatory cascade has also been disappointing. corticosteroid administration was not associated with reduction in clinical scores, the need for hospitalization, or the length of hospitalization. routine use of any corticosteroid given via any route (intravenous, enteral or aerosolized) is not indicated, except in patients with pre-existing chronic lung disease. bronchodilators have not shown a clear benefi t in patients with acute rsv bronchiolitis. in 12 randomized control trials, involving 843 infants, evaluating the effect of salbutamol or albuterol on bronchiolitis, 9 (75%) showed no effect. the remaining three studies demonstrated only a small transient improvement in the acute clinical score. although the routine use of bronchodilator therapy cannot be recommended, it has become acceptable practice to attempt to see if individual infants are beta agonist responsive or not. if no clinical response is seen after a trial of a beta agonist, its use should be discontinued. in the 1990s, fi ve randomized trials involving 225 infants, evaluating the effect of nebulized adrenaline (epinephrine) on bronchiolitis showed clinical improvement, with reductions in oxygen requirement, respiratory rate, wheezing, and decrease in pulmonary vascular resistance. two of these studies showed lower hospital admission rates and earlier discharge. a 2004 cochrane systematic review suggested a potential benefi t with epinephrine administration. however, subsequent studies have not supported its routine use. as with albuterol, a clinical trial in selected infants seems reasonable. nebulized hypertonic saline has been used for treating hospitalized, as well as ambulatory, children with viral bronchiolitis with variable success. a recent cochrane meta-analysis of nebulized hypertonic saline has shown an improvement in clinical scores and decrease in hospital duration. several studies have evaluated the benefi t of surfactant and nitric oxide for severe respiratory distress. the results have been inconclusive and do not currently support their routine use. heliox, a mixture of oxygen (20-30%) and helium (70-80%) with lower viscosity than air has been used successfully in cases of airway obstruction, croup, airway surgery, and asthma to reduce respiratory effort during the period of airway compromise. several studies have shown improved respiratory distress scores in patients on heliox with continuous positive airway pressure obviating the need for intubation and mechanical ventilation. palivizumab is a neutralizing humanized mouse monoclonal antibody directed against the rsv-f glycoprotein. it was licensed by the food and drug administration (fda) in 1998 for premature infants and infants with bronchopulmonary dysplasia. the randomized, double blind, placebo controlled impact-rsv trial involving 1,502 high risk infants found a signifi cant reduction of 55% in hospitalizations. with the exception of very rare anaphylaxis, no signifi cant adverse effects have been observed. palivizumab has been approved for use in infants with congenital heart disease. the cardiac synagis study group included 1,287 children with congenital heart disease in a randomized, double blind, placebo controlled trial; it found a 45% relative reduction in rsv associated hospitalizations with no deaths attributable to the palivizumab. since cardiopulmonary bypass can decrease serum drug concentrations by about 58%, it is recommended that an additional dose be given following surgery, if continued protection is desired. palivizumab should be administered intramuscularly as 15 mg/kg every 30 days for a total of fi ve doses during rsv season, which is generally from november through march, to high risk infants. infants or children that develop an rsv infection should continue to receive prophylaxis following recovery because the naturally acquired antibodies are not fully protective. motavizumab, a new, enhanced potency, humanized rsv monoclonal antibody has demonstrated 50-100 times greater neutralizing activity against rsv. in completion of a phase iii trial, motavizumab was found equal to palivizumab for the prevention of rsv hospitalization and superior to palivizumab for reduction of rsv-specifi c outpatient medically attended lower respiratory tract infections (malris). pneumonia describes any infl ammatory condition of the lung in which the alveoli are compromised by aspirated foreign matter, infl ammatory fl uid, or cellular debris. infection is the primary cause of parenchymal injury to the lung. pathogens include viruses, bacteria and fungi. signs and symptoms of pneumonia are non-specifi c and may be occult in the young infant. children often have fever, chills, headache, malaise, restlessness, and irritability. gastrointestinal complaints such as abdominal pain, distention, or emesis may also be present in young children. the symptoms are often preceded by minor upper respiratory tract infections characterized by low-grade fever and rhinorrhea. with more signifi cant involvement of the lower respiratory tract, tachypnea, dyspnea, cough, nasal fl aring, grunting, or retractions may be seen. the older child may demonstrate productive sputum and complain of pleuritic chest pain. on auscultation of the chest, rales and/or decreased breath sounds might be heard over areas of consolidation or pleural effusions. however, due the short path for transmission of breath sounds and the small chest size in infants, breath sounds may not be decreased, even in the presence of effusions. children with pleural irritation might prefer to lie on the affected side with legs fl exed and may complain of radiating pain to the neck and shoulder or into the abdomen. community acquired pneumonia (cap) is a common, and at times, a serious infection in children. the incidence of cap is 35-40 cases per 1,000 children less than 5 years of age and 11-16 cases per 1,000 children 5-14 years of age. the exact prevalence of the etiologic agents causing pediatric pneumonia is diffi cult to ascertain. it is often diffi cult to differentiate viral from bacterial pneumonia based solely on clinical examination. specifi c pathogens causing cap can be determined in only approximately one-third of children using commonly available cultures, antigen detection, or serologic techniques. blood cultures yield pathogens in only about 10-15% of infants and children with bacterial cap and many children do not undergo viral testing as it is often unnecessary. with these inherent limitations, it is generally thought that viruses account for approximately 80% of cap in children under the age of 2 years and approximately 50% of cap in preschool children ages 2-5 years. palivizumab should be used as preventive therapy in infants with chronic lung disease and congenital heart disease. cardiopulmonary bypass signifi cantly lowers the serum level of palivizumab, so it should be redosed following surgery if continued protection desired. viral causes decline in the school age and adolescent child and bacterial causes such as streptococcus pneumoniae and mycoplasma become important pathogens ( fig. 25-1 ) . overall, bacteria account for 20-30% of community-acquired pneumonias. the likelihood of infection with different bacteria varies by age. in the newborn period, organisms from the maternal genital tract are likely causes and include group b streptococcus , escherichia coli , enteric gram-negative bacilli, listeria , and chlamydia . in older infants, streptococcus pneumoniae becomes a signifi cant cause and remains so until 6 years of age. group a streptococcus and staphylococcus aureus are uncommon causes. moraxella catarralis is a common cause of upper respiratory tract disease, but rarely causes pneumonia. about 20% of infants with pertussis will have bacterial co-infection. in children older than 6 years of age, streptococcus pneumoniae remains the most common cause. hemophilus infl uenzae type b (hib), and most recently streptococcus pneumoniae , have decreased signifi cantly as causes of cap due to the widespread use of effective vaccines. in the older child and young adolescent, the atypical pneumonias, mycoplasma and chlamydia , become more prevalent and viral causes less common. rare bacterial pneumonias can occur with animal contact and include: francisella tularensis (rabbits); chlamydia psittaci (parrots and birds); coxiella burnetii (sheep); and salmonella choleraesuis (pigs). children with congenital anatomical defects, immunodefi ciencies, and genetic disorders are at increased risk for bacterial, viral and fungal pneumonia. the airways are normally sterile below the sublaryngeal area to the lung parenchyma. there are several protective mechanisms that include anatomic and mechanical factors, local immune defenses, and the systemic immune response. microbes are fi ltered by nasal hairs or are expelled from the airways by the epiglottic refl ex, cough refl ex, and mucociliary apparatus. immunoglobulin a (iga) is the predominant immunoglobulin present in the upper respiratory tract. iga is able to bind two antigens simultaneously, forming large antigen-antibody complexes. in this manner, the microbes are neutralized and removed by ciliary clearance, thus preventing microbial binding to the epithelium. in the lower tract, immunoglobulin g (igg) provides humoral protection by opsonizing microbes for phagocytosis by neutrophils and macrophages, activating the complement cascade, and by neutralizing bacterial it is diffi cult to determine the etiologic agent causing pneumonia, but when microbial agents are identifi ed, bacteria are isolated in 20-30%. etiology of community acquired pneumonia based on age endotoxin. alveolar macrophages produce superoxide anions, hydrogen peroxide, and hydroxyl radicals that serve an important role in the host defense; however, uncontrolled production can lead to lung injury. in addition to oxygen radicals, a number of cytokines are produced by the alveolar macrophages. these include il-1, il-6, tnf, transforming growth factor-β (tgf-b ), chemotactic factors, platelet derived growth factor, and m-csf. these cytokines play a central role in phagocytic recruitment and activation. infection occurs when one or more of the defense mechanisms is altered or if the inoculum is too large. pathogens typically gain entry through inhalation of aerosolized material or through aspiration of resistant organisms inhabiting the upper airways. less frequently, pneumonia can occur via hematogenous spread. in children with bacterial pneumonia, a signifi cant portion will have a concurrent or preceding viral infection. viral infection may predispose to bacterial superinfection by reducing clearance mechanisms and by weakening the host immune response. pathogens entering the lower airways evoke an exudative consolidation of pulmonary tissues. initially, there is hyperemia of lung parenchyma due to vascular engorgement and capillary leak causing exudation and intra-alveolar fl uid accumulation. fibrin is then deposited and the airways are infi ltrated with neutrophils. consolidation causes a decrease in lung compliance and vital capacity and a total reduction in the surface area available for gas exchange. a physiologic shunt (v/q mismatch) occurs as there is increased blood fl ow through poorly ventilated segments of lung, resulting in hypoxia. compensatory hypoxic vasoconstriction may occur in an attempt to reduce v/q mismatch and hypoxia, especially in localized areas of consolidation. with treatment, resolution of consolidation will occur in 8-10 days. the exudate undergoes enzymatic digestion and is either reabsorbed or removed by coughing. if the bacterial infection extends into the pleural cavity, an empyema may result. streptococcus pneumoniae is a gram-positive diplococcus that is frequently found in the upper respiratory tract. there are over 80 capsular serotypes with 80% of infections caused by 14 serotypes. it is the most common bacterial cause for pneumonia occurring at a peak age of 13-18 months. typically, it causes a lobar or segmental consolidation, but it may manifest as patchy infi ltrates in infants. pleural effusions occur in up to 20% of children that require hospitalization (fig. 25-2 ) . pneumatocoele formation is rare. hemolytic uremic syndrome is associated with neuraminidase-producing strains. treatment is typically with a penicillin or cephalosporin. emerging resistance may require initial therapy with vancomycin. in hospitalized patients, parenteral therapy is generally needed for 48-72 h after fever resolves, followed by completion of 7-10 days of enteral therapy. pneumococcal conjugate vaccines (pcv) have been developed that confer immunity against 7 and 13 serotypes. the 7-valent pcv (prevnar) was licensed for use in the united states in 2000. a 13-valent pcv has been recently introduced and will replace the 7-valent pcv. the pcvs have been highly effective at reducing hospitalizations among children younger than 2 years for pneumococcal pneumonia. pcv is now recommended universally for children younger than 24 months of age and older children at high risk due to underlying diseases. high risk children include those with sickle cell disease and other types of functional asplenia, human immunodefi ciency syndrome, primary immunodefi ciency, children receiving immunosuppressive therapy, and children with chronic pulmonary or cardiac disease. a 23-valent pcv is available for pneumonia occurs when one or more of the host defense mechanisms are altered. viruses enhance the host susceptibility to bacterial pathogens by affecting clearing mechanisms and by weakening the host immune response. streptococcus pneumoniae is the most common bacterial cause for pneumonia. c hapter 25 • ac ute pu lmonary i n fections high risk children who need expanded serotype coverage. children with sickle cell disease or functional asplenia should continue to receive antibiotic prophylaxis regardless of whether or not they have received pneumococcal vaccines. approximately 50-75% of infants born to chlamydia trachomatis -infected mothers will become infected at one or more anatomical site, including conjunctiva, nasopharynx, rectum, and vagina. about 30% of infants with nasopharyngeal infections will develop pneumonia. the infants usually present at about 4-12 weeks of age with cough and congestion, but an absence of fever. the cough often interferes with the ability to feed. infants generally have tachypnea and rales on examination and chest x-ray frequently shows hyperinfl ation. a peripheral eosinophilia may be present. c . trachomatis is susceptible to macrolides, tetracyclines, quinolones, and sulfonamides. erythromycin for 2-3 weeks is the treatment of choice for neonatal pneumonia. mycoplasma pneumoniae and chlamydia pneumoniae play a greater role in causing respiratory tract disease in children then previously thought. an indolent course that develops over 5-7 days manifested by low-grade fever, scratchy sore throat, aches, and headaches characterizes both pathogens. after a few days, rales may be heard, particularly in the bases where the infi ltrates tend to occur. these organisms have been associated with the initiation, promotion, and exacerbation of asthma in children. in addition, a pertussis-like illness with acute bronchitis has been described. a recent study has shown that nearly half of the cases of community-acquired pneumonia in children aged 2-14 years were associated with m . pneumoniae or c . pneumoniae . classic atypical pneumonias caused by these organisms are usually mild and self-limited. however, a number of studies have suggested that severe pulmonary infection may occur in otherwise healthy children. pleural effusions, pneumatocoeles, lung abscesses, pneumothoraces, bronchiectasis, chronic interstitial fi brosis, and acute respiratory distress syndrome although rare complications, have all been reported. serological testing is the most common means of diagnosis, but this is often retrospective. cultures obtained from swabbing the nasopharynx may take several days to grow. pcr techniques are currently being refi ned and standardized. treatment with antibiotics reduces the rate of recurrent wheezing episodes, decreases morbidity, and shortens the duration of symptoms. the organisms are susceptible to tetracyclines, macrolides, and quinolones. the optimal doses and duration of treatment is unclear; however, some data suggest that prolonged treatment for greater than 2 weeks may be more desirable to decrease symptoms and eradicate the organism from the nasopharynx. chlamydia pneumoniae have an increased prevalence in older children. chest radiograph of 3 year old female with streptococcus pneumoniae pneumonia. note the combination of consolidation and effusion affecting the right lung. (image provided courtesy of fa maffei) staphylococcus aureus is a gram-positive organism that can be found on the skin, nasal mucosa, and other mucus membranes. about 20-30% of children are carriers. it is generally spread by direct contact or by respiratory particles. s . aureus is an unusual cause of lower airway disease in otherwise healthy children. it is more typically isolated from infants and young children with debilitating conditions. primary s . aureus pneumonia presents in the winter or early spring with a short febrile prodrome and a rapid onset of pulmonary symptoms. blood cultures are positive in 20-30% of patients. secondary staphylococcal pneumonia will have a more prolonged prodrome with no seasonal predilection, but is often seen after infl uenza infections. as this secondary pneumonia is usually a result of hematogenous spread, blood cultures are positive in about 90% of patients. unilateral lobar disease is more typical with primary disease, while diffuse bilateral infi ltrates are more frequent with secondary pneumonia. effusions can be diagnosed in about 15% of children at presentation, but ultimately will develop in about 75% of cases. pneumatocoeles occur in up to 45-65% of children. treatment is with nafcillin or oxacillin, but more organisms are becoming resistant and require therapy for serious or invasive disease with vancomycin, linezolid, daptomycin, or quinupristin-dalfopristin. methicillin resistant staphylococcus aureus (mrsa) was once considered to be restricted to hospitals and long-term care facilities. however, community acquired mrsa (ca-mrsa) is now a signifi cant cause of a variety of infections (including pneumonia) in children without prior health care facility exposure. the majority of community acquired mrsa infections involve minor skin and soft tissue infections, but invasive and sometimes fatal infections can occur in otherwise healthy individuals. ca-mrsa and healthcare-associated mrsa (ha-mrsa) can be distinguished by several important features. patients with ca-mrsa by defi nition have not had recent hospitalization (acute or chronic care), prolonged antibiotic use or chronic underlying disease. toxin production also distinguishes ca-mrsa from ha-mrsa. panton valentine leukocidin (pvl) is a toxin which is present in most ca-mrsa isolates, but rarely in ha-mrsa isolates. pvl toxin lyses white blood cells leading to leukopenia and a decreased ability to kill s . aureus . its production has been implicated as a contributor to the development of ca-mrsa necrotizing pneumonia. ca-mrsa isolates, unlike ha-mrsa, lack multi-drug resistance. ca-mrsa is generally more susceptible to clindamycin, trimethoprim-sulfamethoxazole and doxycycline than ha-mrsa, probably because ha-mrsa has developed resistance to survive in the healthcare setting. group a betahemolytic streptococcus (gabhs) is a gram-positive organism responsible for about 15% of pharyngitis and tonsillitis in children. it is rare as a primary cause of pneumonia. when it does occur, the children generally have high fever and appear toxic. the pneumonia is typically lobar. associated empyemas are common and pneumatocoeles may develop. there are several virulent toxin-producing gabhs m-serotypes that are associated with toxic shock syndrome. pre-existing varicella disease with disruption of skin and soft tissue as the port of entry is reported approximately 40-50% of the time. an associated pneumonia occurs in 10-20% of children with toxic shock syndrome. gabhs are highly susceptible to penicillins and cephalosporins. in cases of toxic shock, clindamycin is often added to inhibit the production of streptococcal pyrogenic exotoxins a (spe-a) and b (spe-b). about 30-40% of infants with perinatally acquired group b streptococcus (gbs) infections will have pneumonia. the infant usually has systemic disease and blood cultures are frequently positive. late-onset gbs is predominantly caused by the type iii serotype. in these infants, the infection is usually manifest as bacteremia without a focus or with meningitis. pneumonia is rare in late-onset disease. gbs is uniformly sensitive to penicillin. while staphylococcus aureus pneumonia is uncommon, effusions ultimately develop in about 75% of cases and pneumatocoeles occur in 45-60%. pertussis, or "whooping cough" is a highly contagious respiratory tract infection caused by the gram-negative pleomorphic bacillus bordetella pertussis and less commonly bordetella parapertussis . with the development and widespread use of a vaccine in the 1940s, a significant and sustained decrease in incidence has occurred. however, despite immunization rates greater than 80%, cyclical recurrences of the disease have occurred every 3-4 years since the 1980s. this is likely secondary to the waning of immunity in adolescents and young adults. under-immunized or unimmunized infants are the most vulnerable. nearly all deaths reported from pertussis occur in infants younger than 3 months of age. pertussis is often divided into catarrahal (fever, rhinnorhea and initiation of cough), paroxysmal (severe coughing episodes, lymphocytosis, potential for complications) and convalescent stages (slow waning of cough over weeks to months). complications include secondary bacterial or viral pneumonia, apnea, malnutrition, pulmonary hypertension and neurologic involvement including seizures and encephalopathy. infants less than 6 months of age are at highest risk for complications and mortality. characteristic paroxysms of cough with an end inspiratory whoop occur in children. infants may present with a nonspecifi c cough with associated apnea and cyanosis, without a whoop. adolescents may be asymptomatic or have only a mild prolonged cough. an increased white blood count up to 100,000 with a lymphocytosis is characteristic early in the course of the disease. the preferred test for laboratory confi rmation is the detection of b. pertussis dna by pcr assay. bacteriologic culture provides a defi nitive diagnosis. if administered during the early stages of the disease (fi rst 7-10 days of illness), erythromycin for 14 days may decrease symptoms and reduce the risk of spread. a 5 day course of azithromycin or a 7-10 day course of clarithromycin has been found to be as effective with less gastrointestinal symptoms. corticosteroids, bronchodilators, or intravenous immunoglobulins have not demonstrated effi cacy. supportive care with supplemental oxygen, mechanical ventilation, intravenous fl uids, maintenance of adequate caloric intake, and treatment of secondary bacterial infections are the mainstay of therapy. the use of extracorporeal membrane oxygenation in infants with hypoxemia, pulmonary hypertension and right heart failure refractory to conventional mechanical ventilation has resulted in poorer outcomes than expected. vaccination in infancy with booster doses in adolescence is preventative. about 80-85% of pneumonias in children are caused by viruses. there is considerable evidence that viral infections often precede bacterial pneumonias and cause weakening of the host defenses. viral pneumonias with rsv and parainfl uenza are discussed in more detail in the bronchiolitis section. infl uenza is the main viral cause of pneumonia in school-aged children requiring hospitalization. there are three serotypes, a, b, and c which are further divided into subtypes based on the hemagglutinin and neuraminidase genes. hemagglutinin 1, 2, and 3 and neuraminidase 1 and 2 typically infect humans. the gene segments for the surface glycoproteins are unstable, so mutations, called antigenic shift, occur regularly. epidemics occur annually during the winter months with a short, 1-3 day incubation period. the virus causes destruction of the ciliated respiratory epithelium within 1 day of symptoms. airway edema and infi ltration with infl ammatory cells into the airway mucosa and epithelium follows. slow repair occurs over 2-4 weeks. a severe fulminating pneumonia may result in hemorrhagic exudates that contain many polymorphonuclear and mononuclear cells. destruction of the respiratory epithelium often leads to secondary bacterial infections. during the 2003-2004 infl uenza season, 143 infl uenza-related deaths occurred in children; of these, 41% were less than 2 years of age. forty-fi ve percent of the older children (2-17 years of age) did not have an underlying medical condition. rare complications of although death from infl uenza pneumonia is uncommon, a signifi cant number of the children that died were previously healthy. infl uenza include acute myositis, rhabdomyolysis, myocarditis, pericarditis, reye syndrome, encephalitis, transverse myelitis, and guillain-barré syndrome. children may present with an abrupt clinical course manifested by high fever, myalgias, headaches, scratchy sore throats, and dry cough. peripheral white blood counts are usually less than 5,000. pulmonary infi ltrates often involve multiple lobes. bacterial co-infection, especially with mrsa, increases morbidity and mortality signifi cantly. rimantidine and amantadine can shorten the course for infl uenza type a disease by limiting viral replication, but only if given within the fi rst 48 h of the disease. prophylactic dosing is 70-90% effective and does not interfere with antibody production from the vaccine. both drugs have central nervous system and gastrointestinal side effects, including an increase in the incidence of seizures. oseltamivir and zanamivir have recently been approved for the treatment of infl uenza infections in children. they inhibit neuraminidase, an enzyme produced by infl uenza a and b. the course of disease in healthy adults can be reduced by 1-2 days, if started within 48 h of the onset of symptoms. zanamivir is a dry powder aerosol that must be delivered by a special breath-activated device. bronchospasm in patients with asthma has been reported. aspirin or aspirin-containing products should be avoided due to the risk of reye syndrome. immunoprophylaxis is the most effective strategy for the prevention of infl uenza infection. inactivated vaccines have effi cacy rates from 70% to 90%. currently, the inactivated vaccine is recommended for all children older than 6 months of age with high risk conditions including chronic pulmonary or cardiac disease, immunosuppressive disorders, sickle cell disease and other hemoglobinopathies, diseases requiring long-term aspirin therapy, chronic metabolic and renal diseases; healthy children aged 6-23 months; and household contacts over the age of 6 months of high risk persons. a live, attenuated infl uenza vaccine was licensed in 2003. it is administered by the intranasal route and is approved for healthy children aged 5-17 years. avian infl uenza viruses do not normally infect species other than birds and pigs. however, in 1997, the fi rst human death from avian infl uenza occurred in hong kong in a 3 year old with reye syndrome. subsequently, an epidemic occurred among humans in hong kong with close contact to live, infected poultry. the subtype h5n1 appears to be the most ominous due to its ability to rapidly mutate and infect new species. the overall mortality rate is greater than 70%. the avian viruses are not believed to be transmissible from person-to-person, but some recent cases are being investigated for this possibility. children uniformly present with fever and cough. symptoms range from typical infl uenza-like symptoms to conjunctivitis to respiratory disease and failure. signifi cant laboratory data include leukopenia and thrombocytopenia. all children who developed pneumonia and progressed to ards died. diagnosis remains diffi cult, as no tests are widely available. of the antiviral drugs available for infl uenza a, the most recent h5n1 strains in southeast asia are resistant to rimantadine and amantadine. therefore, treatment is mainly supportive. a prototype h5n1 vaccine was made available to manufacturers in april 2004, but production is diffi cult because the standard means of producing infl uenza vaccines from specially grown chicken eggs is not feasible. h5n1 kills the embryo before enough viruses can be harvested for vaccine production. in april, 2009, the centers for disease control confi rmed the emergence of a novel infl uenza a (h1n1) virus with genes from swine viruses of the eurasian lineage and genes from avian infl uenza viruses. by june, 2009, the fi rst infl uenza pandemic since 1968 was declared, affecting over 191 countries and territories. in comparison to illnesses with seasonal infl uenza, the majority of cases occurred in individuals younger than 65 years of age, with nearly half of the cases occurring in children under 18 years of age . the clinical symptoms can be typical for infl uenza; fever, sore throat, cough, and muscle aches with the addition of vomiting and diarrhea in children. a wide range of complications although antiviral medications may attenuate the course of infl uenza when given early, immunoprophylaxis with vaccines is the most effective strategy for the control of infl uenza infections. avian infl uenza has occurred in epidemics among persons with close contact to live, infected poultry. all children with pneumonia that progressed to ards succumbed to the disease. have been reported that include mild-to-moderate (otitis media, sinusitis, myositis, and febrile seizures) to more severe complications such as myocarditis, rhabdomyolysis or encephalitis. severe complications may frequently involve invasive bacterial co-infection (i.e. mrsa) and/or exacerbation of underlying medical conditions in particular asthma. children who present initially with uncomplicated infl uenza may have rapidly progressive hypoxemic respiratory failure and multiorgan system dysfunction that is refractory to all therapies ( fig. 25-3 ) . of reported h1n1 deaths, approximately 20% were in children. the majority of these children had comorbid asthma, neuro-developmental conditions, or obesity. an american academy of pediatrics work group identifi ed children at greatest risk for life-threatening h1n1 infl uenza disease (table 25-1 ) . the centers for disease control has recommended prompt empiric antiviral therapy for infants, children, and adolescents of any age presenting with suspected or confi rmed h1n1 infl uenza and any of the following conditions: illness requiring hospitalization ■ progressive, severe, or complicated illness, regardless of previous health ■ presence of signifi cant risk factors (see table ■ 25-1 ) the h1n1 strain has been found to be resistant to amantadine and rimantadine, but is usually sensitive to neuraminidase inhibitors, specifi cally oseltamivir or zanamir. in 2009, oseltamivir was emergently approved for treatment in children less than 12 months of age. resistance to oseltamivir has been reported and is thought due to the h275y mutation. interestingly, the mutation confers resistance to oseltamivir, but not to zanamivir. peramivir, a neuraminidase inhibitor, an unapproved (investigational) antiviral available in an intravenous formulation received an emergency use authorization permit from the fda for use in children with confi rmed severe refractory h1n1 infl uenza. its use should be restricted to children that are not responding to either oral or inhaled antiviral drugs or if the parenteral route is the only dependable method of drug delivery. a vaccine was manufactured and licensed using the same standards as seasonal infl uenza by late 2009. a single dose was found to provide adequate protection in children older than 10 years of age, younger children requiring two doses separated by at least 21 days. adenoviruses have been implicated in 4-10% of pneumonias in children. adenoviruses are classifi ed into 49 serotypes with types 3, 7, 7a, 11, and 21 being the most common etiologic agents of lower respiratory disease and causing a severe necrotizing pneumonitis. these serotypes are associated with serious pulmonary sequelae, such as bronchiectasis, bronchiolitis obliterans, unilateral hyperlucent lung, and persistently abnormal pulmonary function tests. adenovirus infections peak between 6 months and 5 years of age. mortality from severe respiratory infections can be high, because the disease often involves multiple organ systems. survivors may have permanent lung injury often in the form of bronchiolitis obliterans. in the immunocompromised host, mortality rates are as high as 50-80%. cidofovir has in vitro activity against adenovirus, but proof of effi cacy is limited. therapy is supportive. severe acute respiratory syndrome is a newly described pulmonary infection caused by a novel sars-associated coronavirus. sars-cov is highly contagious and was coined "the fi rst plague of the twenty-fi rst century". the disease rapidly spreads among household contacts and healthcare personnel. children less than 18 years of age account for only approximately 5% of those affected, with a mean age of 12 years. no deaths were reported among children in the 2003 outbreak. children and adults present with fever, malaise, cough, coryza, chills or rigor, sputum production, headache, myalgia, leukopenia, lymphopenia, thrombocytopenia, mildly elevated activated partial thromboplastin times, and elevated levels of lactate dehydrogenase. radiographs of the chest show non-specifi c infi ltrates. apart from diarrhea, patients have minimal extrapulmonary symptoms. early diagnosis by reverse transcription-polymerase chain reaction (rt-pcr) can be made with 80% sensitivity on nasopharyngeal aspirates within the fi rst 3 days of the illness. the clinical course follows a triphasic pattern. there is an incubation period of 2-10 days with a prodrome of high fever, chills, malaise, headache, and myalgias. diarrhea occurs in up to 20% of adults. after 2-7 days, the disease progresses to involve the lower airways with a dry non-productive cough and dyspnea. in 10-20% of cases, acute respiratory distress syndrome (ards) follows and often patients require mechanical ventilation. deaths occur from respiratory failure. young children run a milder and shorter biphasic clinical course. cough is found in approximately half the children, and crackles are rarely heard despite radiographic evidence of infi ltrates. a regimen of antibiotics, ribavirin, and corticosteroids was proposed based on initial anecdotal success. however, ribavirin has demonstrated minimal activity against sars-cov isolates in vitro . non-randomized studies of corticosteroids have reported favorable outcomes. a pediatric series of 44 children with confi rmed sars treated with ribavirin and corticosteroids showed no adverse effects and all survived. mortality from adenovirus infections remains high because of multiple organ system involvement. sars rarely affects children, and when it does, morbidity is less, with no reported mortalities. 1. neurological disorders, such as epilepsy, cerebral palsy, developmental delay and neuromuscular disorders 2. chronic respiratory diseases associated with impaired pulmonary function and/or diffi culty handling lung secretions, moderate and especially severe persistent asthma, technology-dependent children (e.g., those requiring oxygen, tracheostomy, or a ventilator) 3. primary immunodefi ciencies or conditions that require medications or treatments that result in secondary immunodefi ciencies 5. congenital heart disease 6. metabolic (e.g., mitochondrial) or endocrine disorders, especially if cardiopulmonary function is impaired adapted from http://www.aap.org/new/swinefl u.htm hantavirus cardiopulmonary syndrome is a viral zoonotic disease that affects healthy children and adolescents who are exposed to aerosols of rodent excreta. the deer mouse is the main rodent reservoir. most cases occur in the southwestern united states, but cases have been confi rmed in 30 states. hcps presents with a prodrome of fever, chills, myalgia, headache, and gastrointestinal symptoms. respiratory compromise requiring supplemental oxygen generally occurs within 72 h. the disease can progress to respiratory distress and ards. the majority of deaths result from hypoxemia and cardiac dysfunction with marked hypotension and ventricular arrhythmias. in adults, the case fatality rate is approximately 38%. a recent case series of 13 children aged 10-16 years, revealed that 92% of infected children developed hcps, 33% died, and 67% were critically ill and required mechanical ventilation. treatment is supportive as ribavirin has not been proven to reduce mortality. extracorporeal membrane oxygenation was used on two patients, one of which survived. laboratory evaluation reveals thrombocytopenia, leukocytosis, and circulating immunoblasts. an elevated prothrombin time of ³ 14 s is predictive of severe disease. no deaths were reported in children younger than 14 years of age. diagnosis can be made by detection of hantavirus-specifi c immunoglobulin m, hantavirus-specifi c rna by polymerase chain reaction, or hantavirus antigen by immunohistochemistry. respiratory infections in children with primary or acquired immunodefi ciencies requiring intensive care are not uncommon. these infants and children are susceptible to many organisms that are rarely pathogenic in a normal host. primary immunodefciencies include abnormalities or defi ciencies in immunoglobulins and antibodies, t and b cells, phagocytes, natural killer cells, and complement. acquired immunodefi ciencies include asplenia, human immunodefi ciency virus (hiv), corticosteroid therapy, and immunosuppresion used for marrow or solid organ transplants. immunocompromised children can present with attenuated signs and symptoms of respiratory infections. in addition to physical examination and chest roentgenograms, these children often require chest computed tomography to better delineate the extent of disease. bronchoalveolar lavage, needle aspiration, or lung biopsies might be required to make a defi nitive diagnosis. pulmonary specimens should be tested for common bacteria as well as for pneumocystis carinii, acid-fast bacilli, nocardia, legionella, crytococcus, aspergillus, candida, histoplasma, coccidioides, and blastomyces. viruses such as cytomegalovirus, varicella, herpes virus, and measles should be considered. pneumocystis carinii (now known pneumocystis jiroveci ) is an opportunistic pulmonary pathogen in infants and children with human immunodefi ciency virus (hiv) and other primary immunodefi ciencies, malnutrition, hematological malignancies, solid organ and bone marrow transplant recipients, and patients on high dose corticosteroid therapy for infl ammatory and collagen-vascular diseases. it is a unicellular organism that exists as a cyst (the diagnostic form). the organism attaches to the type i alveolar cells resulting in an alveolitis characterized by ventilation-perfusion mismatch and decreased pulmonary compliance. if untreated, pcp carries a mortality rate of 25-50%, and nearly 100% in the hiv-seropositive child. fortunately, the incidence has markedly decreased with the administration of chemoprophylactic agents to high risk patients. children typically present with fever, tachypnea, non-productive cough, and hypoxia with an absence of rales on auscultation of the chest. initially, they may have an elevated ph and low carbon dioxide levels. lactate dehydrogenase levels are generally elevated. bilateral diffuse alveolar infi ltrates are seen with initial hilar involvement subsequently spreading to the periphery (fig. 25-4 ) . diagnosis is made by demonstrating the organism with the methenamine silver nitrate stain on pulmonary tissue, respiratory secretions, or lung fl uid. bronchoalveolar lavage is the most widely used technique to obtain lung fl uid for diagnosis. treatment consists of supportive therapy hantavirus is rare in infants and school-aged children. no deaths have been reported in children less than 14 years of age. with supplemental oxygen; ultimately continuous positive airway pressure or mechanical ventilation may be necessary if respiratory failure occurs. trimethoprim-sulfamethoxazole (tmp-smx) is the recommended initial treatment. in patients that cannot tolerate tmp-smx, then pentamidine isoethionate should be used. corticosteroids in anti-infl ammatory doses as an adjunct to antimicrobial therapy have improved clinical outcomes. concurrent pulmonary infections were found in 35% of patients, most frequently bacterial or cytomegalovirus pneumonia. determination of the etiologic agent in pneumonia is diffi cult. fortunately, in most community-acquired pneumonias, identifi cation of the specifi c causative organism is not critical. however, in children with a complicated course that fails to respond to standard therapies, defi nitive diagnosis of the etiologic agent is essential. complete blood counts, infl ammatory markers, and chest radiographs do not differentiate the causative agents for pneumonia. blood cultures are rarely positive outside of the neonatal period. rapid antigen tests are available for rsv, parainfl uenza, infl uenza, and adenovirus. nasopharyngeal swabs for viral cultures generally take 7-8 days to become positive, and in one study, 86% of the patients had been discharged prior to the positive results. older children and adolescents might be able to produce sputum for gram stain and culture. an adequate specimen should contain more the 25 leukocytes and fewer than 25 squamous epithelial cells per low-power fi eld. in the intubated patient, sputum can be more easily acquired. however, interpretation of the results of gram stains and cultures is at times diffi cult in differentiating colonizing from pathologic organisms. colonization of the endotracheal tube may occur as early as 12 h, but most frequently between 60 and 96 h. the oropharynx becomes colonized within 36 h, the stomach at 36-60 h, and the lower respiratory tract between 60 and 84 h. in addition, a comparison of infectious agents isolated by both tracheal aspirates and bronchoalveolar lavage found only 36% concordance. bronchoalveolar lavage (bal) can be safely used to obtain secretions from the lower airways for gram stain and culture. it is especially useful in the diagnosis of pneumonia in the immunocompromised child. however, bal performed directly through the bronchoscope carries a risk of contamination. the smallest bronchoscope that can accommodate a protected specimen brush is 4.8 mm and requires a 6.5 mm endotracheal tube for passage. the smallest fl exible fi beroptic bronchoscope with a suction channel has an external chest radiograph of severe pneumocystis carinii pneumonia in a 13 month old male with combined immunodefi ciency. note the diffuse alveolar involvement and air bronchograms. (image provided courtesy of fa maffei) diameter of 2.8 mm and is too small to admit a double-sheathed brush. non-bronchoscopic double-lumen plugged catheters can be inserted blindly through the endotracheal tube to obtain a non-contaminated specimen. the sensitivity and specifi city of these samples are similar to those obtained by a bronchoscopic guided protected specimen. transthoracic needle aspirations are performed in some centers with good results. one study reported a diagnostic success rate in 59% of patients. the incidence of pneumothorax was approximately 20%, but none required subsequent placement of a pleural drainage catheter. a lung biopsy is rarely needed to make a defi nitive diagnosis. supportive treatment with oxygen and intravenous fl uids are often standard therapies. as both pneumonia and mechanical ventilation can cause an elevation in anti-diuretic hormone levels, careful fl uid monitoring is essential to avoid overhydration, excessive lung water and hyponatremia. initial antibiotic choices should be empiric and based upon the likely organisms for each age group, because of the diffi culty in identifying the causative agent. the child's respiratory status including respiratory rate, work of breathing, pulse oximetry, and central nervous system response should be closely monitored. non-invasive bi-level positive airway pressure (bipap) has been effective for use in children with mild to moderate respiratory insuffi ciency, defi ned as an a-a gradient >100 and <250 or pao 2 /fio 2 ratio <200 but >100 mm hg. serial evaluation of mask-face contact areas is essential to avoid skin breakdown. children with moderate or severe respiratory insuffi ciency often require intubation and mechanical ventilation. children with respiratory failure secondary to pneumonia often require increased positive end expiratory pressure (peep), increased inspiratory time, and aggressive pulmonary toilet to recruit alveoli. for patients requiring high levels of peep, adequate sedation is often required to prevent patient/ventilator asynchrony and barotrauma. spontaneous respirations should be encouraged while on mechanical ventilation. rarely, the use of neuromuscular blockade is required to allow mechanical ventilation. prone positioning may improve ventilation/perfusion (v/q) mismatching in dependent lung regions. lung protective strategies allowing permissive hypercapnea with small lung volumes to ventilate and appropriate peep to maintain alveolar recruitment is recommended for children with pneumonia. high frequency oscillatory ventilation can also be utilized to maintain mean airway pressure and alveolar recruitment. airway pressure release ventilation (aprv) provides recruitment of alveoli while allowing spontaneous respirations. in children with severe respiratory distress syndrome, treatment with bovine surfactant may improve oxygenation. extracorporeal life support continues to have a role in children with reversible severe acute hypoxemic respiratory failure refractory to mechanical ventilation. pneumonias can often be complicated by the development of pleural effusions and empyemas. these occur when the fl uid production by the interstitial lung tissue exceeds the maximum pleural lymphatic fl ow. parapneumonic effusions often occur from pneumonia as white blood cells and other debris of infection block the lymphatics resulting in elevation of protein in the pleural space, increase in colloid osmotic pressure, and consequent failure of fl uid reabsorption. on physical exam, the child will have decreased breath sounds over the effusion. in older children, auscultatory percussion changes might be appreciated. plain chest radiographs can reveal most clinically signifi cant effusions. ultrasound and chest computed tomograms are useful in determining the volume and quality of the fl uid and the presence of loculations. simple parapneumonic effusions or transudates can also be differentiated from exudates by using the criteria of light et al. (table 25 -2 ). a pleural fl uid ph less than 7.2 indicates a complicated effusion that is likely exudative and requires drainage whereas a pleural fl uid ph more than 7.3 suggests that the effusion may be managed with systemic antibiotics alone. complicated parapneumonic effusions or empyemas occur when the fl uid becomes purulent. during this stage, the effusions undergo a fi brinopurulent stage with many polymorphonuclear leukocytes, bacteria, and cellular debris entering the fl uid. fibrin is deposited over the pleural surfaces and loculations begin to form. the ph and glucose levels fall as the ldh levels rise. if untreated, they often progress to a third organizing stage in which the exudate non-invasive bipap ventilation can be effective for children with moderate respiratory insuffi ciency. develops into an inelastic, fi brotic peel that restricts the lung. simple parapneumonic effusions usually resolve with thoracentesis or tube thoracostomy and antibiotic treatment of the pneumonia. more complicated parapneumonic effusions have been successfully treated with thoracotomy tubes and fi brinolytics. however, although risks for bleeding are reportedly low, this therapy requires close monitoring of chest tube drainage and instillation of expensive medications with intermittent clamping of the chest tube. no single recommendation for the choice of fi brinolytic agent or dosage has been established. also, if tried late in the organizing phase, this is often unsuccessful due to loculations and the high viscosity of the purulent fl uid. surgical debridement either by open procedure or by video-assisted thorascopic surgery (vats) is often needed for organizing, complicated parapneumonic effusions. multiple studies have reported that early vats or thoracotomy for empyema leads to a shorter hospital stay. the treatment modality is best determined by the temporal stage and nature of the effusion. acute pulmonary infections are common diagnoses that require admission to the pediatric intensive care units. understanding the pathophysiology of lower respiratory infections enables the intensivist to tailor therapy to the individual child and pathogen. early establishment of a specifi c etiology and the selection of the correct treatment plan directly impacts clinical outcome. video-assisted thorascopic surgery (vats) for the treatment of empyemas has been associated with shorter hospital stay. which of the following therapies have been proven to be a consistent benefi t for rsv bronchiolitis? a. aminophylline b. bronchodilators c. corticosteroids d. ribavirin e. supportive care 2. palivizumab is indicated for which of the following children? a. a 5 month old, former 27 week premature infant who just underwent surgical repair of a large ventricular septal defect who received palivizumab 2 weeks ago b. a 9 month old, former 28 week premature infant with mild bronchopulmonary dysplasia who received palivizumab 2 weeks ago c. a 1 month old, former 36 week premature infant with peripheral pulmonic stenosis who has never received palivizumab d. a 2 month old full term infant with a urea cycle defect who has never received palivizumab e. an 8 month old, former 25 week premature infant with bronchopulmonary dysplasia who received his fi fth dose of palivizumab a month ago pleural fl uid may be classifi ed as exudative, if one or more of the following criteria are met: ■ pleural fl uid protein divided by serum protein >0.5 (sensitivity 98%, specifi city 83%) ■ pleural fl uid lactate dehydrogenase (ldh) divided by serum ldh > 0.6 (sensitivity 86%, specifi city 84%) ■ pleural fl uid ldh is more than two-thirds of the upper limit of normal for serum ldh (sensitivity 82%, specifi city 89%) adapted from light (2002) human bocavirus and acute wheezing in children american academy of pediatrics subcommittee on diagnosis and management of bronchiolitis. diagnosis and management of bronchiolitis the yield of fl exible fi beroptic bronchoscopy in pediatric intensive care patients immunological mechanisms of severe respiratory syncytial virus bronchiolitis human metapneumovirus infection in young children hospitalized with acute respiratory tract disease: virologic and clinical features a multicenter, randomized, controlled trial of dexamethasone for bronchiolitis natural infection of infants with respiratory syncytial virus subgroups a and b: a study of frequency, disease severity, and viral load the use of albuterol in hospitalized infants with bronchiolitis advances in the treatment and prevention of severe viral bronchiolitis the presence and sequence of endotracheal tube colonization in patients undergoing mechanical ventilation palivizumab prophylaxis reduces hospitalization due to respiratory syncytial virus in young children with hemodynamically signifi cant congenital heart disease systemic corticosteroids in infant bronchiolitis: a meta-analysis drainage, fi brinolytics, or surgery: a comparison of treatment options in pediatric empyema does vats provide optimal treatment of empyema in children? a systematic review intravenous ribavirin treatment for severe adenovirus disease in immunocompromised children clinical picture, diagnosis, treatment, and outcome of severe acute respiratory syndrome (sars) in children noninvasive therapy with helium-oxygen for severe bronchiolitis pleural effusion pleural effusions: the diagnostic separation of transudates and exudates nebulized 3% hypertonic saline solution treatment in hospitalized infants with viral bronchiolitis development of wheezing disorders and asthma in preschool children heliox therapy in infants with acute bronchiolitis nasal continuous positive airway pressure with heliox versus air oxygen in infants with acute bronchiolitis: a crossover study diagnosis and management of pneumonia in children community-acquired pneumonia in children selected populations at increased risk from respiratory syncytial virus infection human metapneumovirus and human bocavirus in children mixed respiratory virus infections mycoplasma pneumoniae and chlamydia pneumoniae cause lower respiratory tract disease in paediatric patients children's hospital respiratory syncytial virus database: risk factors, treatment and hospital course in 3308 infants and young children infection with sin nombre hantavirus: clinical presentation and outcome in children and adolescents ribavirin for respiratory syncytial virus lower respiratory tract infection: a systematic overview risk of bacterial infection in previously healthy respiratory syncytial virus-infected young children admitted to the intensive care unit infl uenza in pediatric intensive cure unit do bronchodilators have an effect on bronchiolitis? comparison of conventional viral cultures with direct fl uorescent antibody stains for diagnosis of community-acquired respiratory virus infections in hospitalized children respiratory syncytial virus immune globulin for prophylaxis against respiratory syncytial virus disease in infants and children with congenital heart disease respiratory syncytial virus in early life and risk of wheeze and allergy by age 13 years h1n1 infl uenza the impact-rsv study group. palivizumab, a humanized respiratory syncytial virus monoclonal antibody, reduces hospitalization from respiratory syncytial virus infection in high-risk infants high incidence of pulmonary bacterial co-infection in children with severe respiratory syncytial virus (rsv) bronchiolitis bench-to-bedside review: ventilator strategies to reduce lung injury -lessons from pediatric and neonatal intensive care etiological diagnosis of childhood pneumonia by use of transthoracic needle aspiration and modern microbiological methods a multicenter, randomized, double-blind, controlled trial of nebulized epinephrine in infants with acute bronchiolitis respiratory syncytial virus and other respiratory viruses effect of exogenous surfactant (calfactant) in pediatric acute lung injury. a randomized controlled trial current concepts on pulmonary host defense mechanisms in children nebulized hypertonic saline solution for acute bronchiolitis in infants bronchiolitis: recent evidence on diagnosis and management key: cord-023721-e0zp2gux authors: meissner, h. cody title: bronchiolitis date: 2013-02-10 journal: principles and practice of pediatric infectious diseases doi: 10.1016/b978-1-4377-2702-9.00033-7 sha: doc_id: 23721 cord_uid: e0zp2gux nan all references are available online at www.expertconsult.com bronchiolitis, a disease primarily of the first 2 years of life charac terized by signs and symptoms of obstructive airway disease, is caused most commonly by viruses. 1 approximately 2% to 3% of infants in the first 12 months of life are hospitalized with bron chiolitis, accounting for approximately 125,000 hospitalizations and 200 to 500 deaths annually in the united states. 2 data from the centers for disease control and prevention (cdc) indicate that the number of yearly hospital admissions attributable to bronchiolitis increased more than twofold between 1980 and 1996. 3 increasing survival rates for premature infants as well as infants with compromised cardiac, pulmonary, and immune status increase the number of children at risk for severe bronchiolitis. many viruses can cause bronchiolitis, although respiratory syn cytial virus (rsv), human metapneumovirus, and parainfluenza virus type 3 are the most common etiologic agents. [1] [2] [3] [4] [5] [6] [7] other viruses are implicated less frequently (table 331) . [8] [9] [10] during the winter months, rsv is identified as the etiologic agent by cell culture or antigen detection assays in up to 80% of children hos pitalized with bronchiolitis or pneumonia. epidemics of bronchi olitis in early spring and fall often are caused by parainfluenza virus type 3. [11] [12] [13] [14] the yearly cycles of these respiratory viruses are depicted in figure 331 . other viral causes of bronchiolitis include rhinoviruses and coronaviruses. bordetella pertussis, mycoplasma pneumoniae, measles, influenza, and adenovirus have been associated with a severe form of bron chiolitis, bronchiolitis obliterans. [15] [16] [17] [18] this uncommon obstructive pulmonary disease is characterized histologically by the progres sion of acute airway inflammation to necrosis of the cells lining the lumen with severe obliterative fibrosis in the final stages. the pathogenesis of bronchiolitis obliterans probably differs from that of simple viral bronchiolitis. bronchiolitis may be defined as an episode of obstructive lower airway disease precipitated by a viral infection in infants younger than 24 months of age. the peak incidence of severe disease occurs between 2 and 6 months of age. 1, 19, 20 rates of hospitalization are higher in boys and among infants living in industrialized urban settings rather than in rural settings. 21 hospitalization rates are about 5 times higher among infants and children in highrisk groups than among nonhighrisk infants. highrisk groups include premature infants (<35 weeks' gestation), infants born with hemodynamically significant congenital heart disease, as well as infants with chronic lung disease of prematurity (previously called bronchopulmonary dysplasia). [22] [23] [24] [25] [26] [27] although mortality has been reduced in recent years, morbidity among highrisk patients can be high, with average hospital length of stay and intensity of care several times that of previously healthy infants. 2, 28 occurrence of the respiratory virus season is predictable, even though the severity of the season, the date of onset, the peak of activity, and the end of the season cannot be predicted with preci sion. there can be variation in timing of community outbreaks of disease due to rsv from year to year in the same community and among neighboring communities, even in the same season. in the u.s., communities in the south tend to experience the earliest limited numbers of cases of bronchiolitis occur during summer and early fall, and they are likely to be caused by viruses other than rsv, such as rhinovirus and parainfluenza viruses. these cases are generally milder than rsvrelated cases. in tropical coun tries, the annual epidemic of rsv coincides with the rainy season or "winter," although sporadic cases can occur throughout the year. 10 rsv can be divided into a and b strains, each with numerous subtypes or genotypes. type a strains may be associated more commonly with epidemics, severe disease, and a higher hospitali zation rate than type b strains, although not all studies are consist ent with regard to differences in severity. [30] [31] [32] [33] both strains may circulate during the same season, and infants may be reinfected within the same year. a progressive increase in hospitalization rates for bronchiolitis in the u.s. has occurred since the late 1980s. 3, 22 this increase may be related to a greater ability to identify hypoxic infants through the use of pulse oximetry. alternatively, the increase in hospitaliza tion may reflect increased use of daycare centers or changes in criteria for admission. 3 household crowding is an important risk factor for severe viral lower respiratory tract illness due to rsv as well as other respiratory viruses. 34, 35 generally it is recognized that as the number of household members increases, the risk of expo sure to infectious respiratory secretions also increases. childcare attendance has been correlated with an increased risk of bronchi olitis in some studies. unlike other respiratory virus infections, exposure to passive household tobacco smoke has not been asso ciated with an increased risk of rsv hospitalization on a consist ent basis. in contrast to the welldocumented beneficial effect of breastfeeding against some viral illnesses, existing data are con flicting regarding the specific protective effect of breastfeeding against rsv infection. [35] [36] [37] [38] [39] parental history of bronchiolitis or week no. onset of rsv activity and the midwest tends to experience the latest onset. 29 the duration of the season for the west and the northeast is typically between that in the south and the midwest. nevertheless, these variations occur within the overall pattern of rsv outbreaks, usually beginning in november or december, peaking in january or february, and ending by the end of march or april. all references are available online at www.expertconsult.com asthma is associated with a higher risk for the development of lower respiratory illness in offspring. 40, 41 young chronologic age at the beginning of the rsv season is a consistent risk factor for rsv hospitalization. several reasons may account for this increase in risk. most severe rsv disease occurs in the first 6 months of life so that birth shortly before or early after the onset of the rsv season will result in a longer period of exposure to rsv earlier in life. second, maternal antibody concen trations to rsv show seasonal variation and infants born early in the rsv season are more likely to be born to mothers with low serum antibody concentrations to the f (fusion) protein of rsv. 42,43 low concentrations of rsv antibody correlate with susceptibility to severe rsv disease in infants. acute bronchiolitis generally implies disease of infectious etiology, usually due to viruses with specific tropism for bronchiolar epi thelium. because most healthy infants recover from bronchiolitis without incident, information regarding the pathologic changes caused by infection is inferred from animal studies and from biopsy or autopsy materials in severe cases. viral infection causes profound alterations in the epithelial cell and mucosal surfaces of the human respiratory tract. the characteristic histopathology in bronchiolitis is a lymphocytic infiltration of the bronchiolar walls and edema of the surrounding tissue. disease progression is asso ciated with proliferation and necrosis of the bronchiolar epithe lium. the sloughed necrotic epithelium and the increased mucus production lead to obstruction of the lumen of the infant's small airways. air movement is restricted during inspiration and expira tion but is more restricted during expiration when the lumen is further compromised by positive expiratory pressure, resulting in expiratory wheezing. the obstruction results in air trapping and the characteristic appearance of hyperinflation on chest radio graphs. as this air is absorbed, the radiographic pattern evolves to show atelectasis. [44] [45] [46] [47] [48] [49] the presence of high serum concentrations of immunoglobulin igg antibodies to rsv (whether transplacentally acquired or administered intramuscularly) ameliorates rsv illness. 50-54 severe obstructive illness may be related to stimulation of virusspecific igemediated hypersensitivity responses or altered cellmediated immune responses. 55-60 bronchiolitis represents the late stage of a respiratory disease that progresses over several days. upper respiratory tract symptoms consisting of nasal discharge and mild cough begin about 3 to 5 days after onset of infection. approximately 30% to 40% of rsv infected infants have progression of disease to involve the lower respiratory tract. spread to the lower airway occurs either by aspi ration of rsvinfected epithelial cells or by celltocell spread of the virus. lowerairway involvement is marked by a sudden increase in the work of breathing, cough, tachypnea, wheezing, crackles, use of accessory muscles, and nasal flaring. 61,62 the respi ratory rate often exceeds 60 to 70 breaths/minute in young infants, and expiration is prolonged. intercostal and subcostal retractions with wheezing are evident. initially, wheezing occurs during the expiratory phase only and is only audible through a stethoscope. as wheezing progresses, it can be heard without a stethoscope. the chest becomes hyperexpanded and hyperresonant, respirations more labored, and retractions more severe. hypoxemia out of proportion to clinical distress is typical of rsv infection. mild hypoxemia occurs even in otherwise wellappearing infants, the socalled happy wheezers. respiratory failure can be due to hypox emia (an early and sometimes sudden occurrence) or progressive hypercapnia due to fatigue. the small airways of young infants can become so narrowed that wheezing is inaudible. in this setting disease severity is recognized by the absence of audible air exchange, flaring of the alae nasae, expiratory grunting, severe subcostal, supraclavicular, and intercostal retractions, and hypox emia. progressive illness often is accompanied by a rapid fall in oxygen saturation after minimal manipulation. a child with these findings usually requires intubation and ventilatory support. apnea can be an early manifestation of rsv infection, at times resulting in respiratory failure. 63 rsvrelated apnea is mediated by the central nervous system, occurring in young, often prematurely born infants. 64 because the severity of bronchiolitis often waxes and wanes prior to consistent improvement, assessment of respira tory status can vary markedly over a short period. the ability of the young infant to breast or bottlefeed without distress over time often provides a practical guide to disease severity and man agement. an infant who has substantial difficulty feeding as a result of respiratory distress has moderate or severe illness and usually requires hospitalization. otherwise healthy infants younger than 2 months of age, infants born prematurely (less than 35 weeks' gestation), and infants with chronic lung disease of prematurity (previously called broncho pulmonary dysplasia) or infants born with congenital heart disease have the highest morbidity and mortality rates due to bronchiolitis. 65, 66 infants born with congenital heart disease at greatest risk of hospitalization due to bronchiolitis include those with moderate to severe pulmonary hypertension and infants with cyanotic heart disease. rsvinfected infants and children with the following hemodynamically insignificant heart disease are gener ally not considered to be at increased risk of hospitalization: secundum atrial septal defect, small ventricular septal defect, pulmonic stenosis, uncomplicated aortic stenosis, mild coarcta tion of the aorta, and patent ductus arteriosus, as well as infants with lesions adequately corrected by surgery (unless they continue to require medication for management of congestive heart failure). 67, 68 severe respiratory distress with bronchiolitis can be the presenting manifestation of previously unrecognized con genital heart disease. once hospitalized, the rsvinfected infant may have a highly variable course of illness. [69] [70] [71] [72] [73] among otherwise healthy infants, intensive care unit admission because of respiratory deterioration is uncommon. 74 a decision to admit to the intensive care unit is based on the possible need for intubation because of progressive hypercapnia, increasing hypoxemia despite supplemental oxygen, or apnea. the typical course for a previously healthy infant older than 6 months is one of improvement over 2 to 5 days, as evi denced by decreases in respiratory rate, retractions, duration of expiration, and oxygen requirement. the median duration of symptoms in 95 infants with firsttime bronchiolitis who came to medical attention at an emergency department in wisconsin was 15 days and onequarter of the infants remained symptomatic after 3 weeks. 75 pulmonary function abnormalities and evidence of mild desaturation (oxygen saturations in the range of 93% to 95%) can persist for several weeks. 76 the differential diagnosis of bronchiolitis includes airway hypersensitivity to environmental irritants, anatomic abnormality of the airway, cardiac disease with pulmonary edema, cystic fibrosis, foreignbody aspiration, and gastroesophageal reflux. the diagnosis of bronchiolitis is based on clinical criteria with supporting radiographic findings. typical chest radiographic find ings are hyperinflation, with flattening of the diaphragms and hyperlucency of the lungs, and patchy atelectasis, especially involving the right upper lobe (figures 332 and 333) . 74, 76 atel ectasis is due to airway narrowing or mucous plugging and is associated with volume loss; it may be confused with lobar con solidation or aspiration pneumonia, both of which are generally volumeexpanding lesions. bacterial pneumonia infrequently occurs as a complication of bronchiolitis but should be suspected in the infant with fever persisting for more than 2 to 3 days and lack of response to supportive management. establishing a specific etiologic diagnosis is helpful in predict ing the clinical course, in cohorting in the hospital, and may become increasingly useful as more antiviral agents effective against respiratory viruses become available. although viral culture of respiratory secretions has been the "gold standard" for reverse transcription-polymerase chain reaction-enzyme hybridi zation assay (hexaplex) is available to test a single nasopharyngeal sample for rsv, influenza a and b viruses, parainfluenza virus, and adenoviruses. 84 antigen detection tests are useful in diagnosing certain viral infections, but, as with all tests, the positive predictive value decreases as disease incidence goes down. specificity of antigen detection assays are lowest during the off season and at the onset and end of the respiratory virus season. most infants with bronchiolitis can be managed at home with supportive care, but hypoxia or inability to feed adequately neces sitate hospitalization. once hospitalized, most infants respond to administration of supplemental oxygen and replacement of fluid deficits. 1 the value of mist inhalation by vaporizer or tent is not proven; its use can provoke reflex bronchoconstriction. the spe cific treatment strategies used differ widely across children's medical centers. 71 fewer than 10% of previously healthy infants hospitalized for bronchiolitis require intubation and mechanical ventilation because of respiratory failure or apnea; the percentage is higher for prematurely born infants and infants with chronic lung disease or congenital heart malformations. the therapeutic role of bronchodilator agents in bronchiolitis is controversial. 1,85 bronchodilator therapy is not recommended for routine management of first time wheezing associated with rsv bronchiolitis. occasionally, a single administration of an aero solized bronchodilator elicits a response, but this improvement is not seen in most infants with bronchiolitis and is not generally reproducible with subsequent doses. [86] [87] [88] [89] [90] [91] [92] modest improvement in clinical scores and in tests of pulmonary function have been reported with use of inhaled racemic epinephrine [91] [92] [93] and î²adrenergic agents, principally salbutamol and albuterol. [92] [93] [94] however, clinical improvement following repeated doses of epine phrine is not sustained and favorable response to î²adrenergic agents, as measured by clinical score and oxygenation, is inconsistent. 95-100 flores and horwitz 101 performed a metaanalysis of eight studies with similar designs. overall, their analysis sup ported a beneficial effect in certain infants, but identifying those infants could not be consistently accomplished at the time of initial presentation. on balance, an initial trial of bronchodilator therapy for the hospitalized infant with bronchiolitis is reasona ble, although brief episodes of hypoxia can be precipitated by adrenergic agents. bronchodilator therapy should only be contin ued if consistent improvement in respiratory distress or oxygen saturation is observed. racemic epinephrine should not be con tinued beyond one or two doses. although corticosteroids reduce the inflammatory changes observed with bronchiolitis, they may increase viral replication and prolong shedding. most studies examining the role of corti costeroids alone in the treatment of bronchiolitis have not demonstrated a consistent clinical benefit. [102] [103] [104] [105] [106] [107] [108] [109] [110] although one metaanalysis of previously published reports of corticosteroid use in bronchiolitis concluded that there may be slight improvements in duration of symptoms, length of hospital stay, and clinical scores, these benefits appear to be limited. 111 the routine use of corticosteroids in bronchiolitis is not recommended. however, a national collaborative, blinded, placebocontrolled trial con ducted in canada demonstrated that the combination of neb ulized epinephrine and oral dexamethasone treatment for children with bronchiolits evaluated in emergency departments reduced the subsequent rate of hospitalization by 9% compared with placebo or either treatment alone (p=0.07) and was less costly. 112, 113 diagnosis of rsv infection, it often is too slow a method to be clinically useful. enzyme immunoassays and direct fluorescent antibody (dfa) techniques for the identification of rsv, influenza virus, parainfluenza viruses, and adenoviruses permit rapid and accurate diagnoses. [77] [78] [79] [80] [81] nasal wash is the preferred method of specimen collection. the dfa test permits evaluation of adequacy of the specimen's number of epithelial cells for antigen detection. a respiratory screening of nasal secretions using pooled mono clonal antibodies to the common agents of bronchiolitis, followed by specific identification for a positive reaction, is a cost and timesaving procedure compared with standard tissue culture iso lation. amplification of virus using the shell vial method, fol lowed by use of specific monoclonal agents, and amplification of viral genome by the polymerase chain reaction offer the promise of improved sensitivity for rapid detection but are not as widely available nor as rapid as enzyme immunoassay and fluorescent antibody techniques. 82,83 a multitest system for quantitative in infancy increases the likelihood of childhood asthma. numer ous studies have defined a higher risk of recurrent wheezing throughout childhood after bronchiolitis in infancy, and abnor malities of smallairway function have been identified in school aged children with a history of bronchiolitis in infancy. however, each of these findings may simply be a reflection of hereditary tendencies that are expressed both at the time of bronchiolitis and upon allergen exposure in later childhood. [130] [131] [132] [133] [134] moreover, by adolescence, the rate of recurrent wheezing in subjects who had bronchiolitis in infancy appears to fall to the rate observed in subjects without a history of bronchiolitis. 134 thus, it is uncertain whether bronchiolitis is causally associated with longterm respi ratory morbidity. strategies that reduce contact of vulnerable infants with individu als with respiratory tract infections, minimizing passive exposure to cigarette smoke, and limiting nosocomial transmission of caus ative agents offer immediate opportunities to reduce bronchiolitis morbidity. monthly administration of monoclonal antif anti body (palivizumab) throughout the rsv season reduces the inci dence of hospitalization due to rsv infection in infants with bronchopulmonary dysplasia, congenital heart disease, and pre maturity by about 50% (see chapter 225, respiratory syncytial virus). the high cost and modest effect of palivizumab limit its use for passive immunoprophylaxis to the most medically fragile infants. no vaccine to prevent infection with rsv or parainfluenza viruses, the most common causes of bronchiolitis, is licensed or near licensure. trivalent influenza vaccine is recommended for all infants older than 6 months of age during the influenza season. because this is not approved for use in infants younger than 6 months, routine influenza vaccination is important for family members and caregivers of these young patients. potential rsv vaccine candidates currently being evaluated include inactivated preparations of the purified fusion protein of rsv, dna vaccines coding for the major immunogenic proteins of the virus, and rep licating mutants of the virus that replicate in the upper respiratory tract but are inactivated at the higher temperatures of the lung. 135 ribavirin is a nucleoside analogue with in vitro activity against rsv, adenovirus, influenza a and b viruses, and parainfluenza viruses. early trials indicated that ribavirin therapy was associated with modest improvement in clinical scores, oxygenation, and duration of mechanical ventilation for infants with severe bron chiolitis due to rsv infection. these studies were challenged on the basis that control groups received water aerosols, which may produce bronchospasm in individuals with hyperreactive airways. clinical trials with ribavirin have not demonstrated a consistent decrease in need for mechanical ventilation, decrease in length of stay in the intensive care unit, or reduction in days of hospitaliza tion. conflicting results from efficacy trials, concern about poten tial toxic effects among exposed healthcare professionals, aerosol route of administration, and high cost have all resulted in limited use of ribavirin. [114] [115] [116] guidelines for the use of ribavirin in rsv disease are presented in chapter 225, respiratory syncytial virus. potential options for the treatment of bronchiolitis, if caused by influenza a or b viruses, are discussed in chapter 229, influ enza viruses. [117] [118] [119] [120] [121] immune globulins and other therapies antibody preparations containing high titers of neutralizing anti body against rsv as well as a preparation of monoclonal antibod ies directed against one of the two major rsv surface glycoproteins (fusion glycoprotein) reduce the risk of hospitalization due to rsv infection. 50,51 used therapeutically, they result in more rapid clear ing of virus from the respiratory tract but do not alter the course of illness and should not be used for the treatment of rsv infection. [122] [123] [124] [125] [126] although vitamin a levels have been demon strated to be low in infants with rsv bronchiolitis, a therapeutic benefit of vitamin a therapy has not been demonstrated. [127] [128] [129] most otherwise healthy infants recover completely from acute bronchiolitis, although subtle pulmonary abnormalities can persist for weeks. 38 diagnosis and management of bronchiolitis the burden of rsv infection in young children bronchiolitis associated hospitalizations among us children, 1980-1966 the epidemiology of acute respiratory tract infection in young children: comparison of findings from developing countries epidemiologic patterns of acute lower respiratory disease of children in a pediatric group practice a newly discovered human pneumovirus isolated from young children with respiratory tract disease incidence and etiology of pneumonia, croup and bronchiolitis in preschool children belonging to a prepaid medical care group over a fouryear period epidemiology of respiratory syncytial virus infection in washington, dc. i: importance of the virus in different respiratory tract disease syndromes and temporal distribution of infection differential detection of rhinovirus and enterovirus rna sequences associated with classical immunofluorescence assay detection of respiratory virus antigens in nasopharyngeal swabs from infants with bronchiolitis etiologic and clinical evaluation of acute lower respiratory tract infections in young argentinean children: an overview reducing the impact of viral respiratory infections in children parainfluenza viruses seasonal trends in human parainfluenza viral infections: united states studies on the role of viruses, bacteria and m. pneumoniae as causes of lower respiratory tract infections in children pathologic manifestations of bronchiolitis, constrictive bronchiolitis, cryptogenic organizing pneumonia, and diffuse panbronchiolitis review bronchiolitis obliterans, bronchiectasis, and other sequelae of adenovirus type 21 infection in young children an outbreak of adenovirus type 7 infection in children in montreal persistent adenoviral infection and chronic airway obstruction in children the etiologic and epidemiologic spectrum of bronchiolitis in pediatric practice risk of primary infection and reinfection with respiratory syncytial virus national surveillance for respiratory syncytial virus, united states, 1985-1990 rehospitalization for respiratory syncytial virus among premature infants fatal respiratory syncytial virus infection in severe combined immunodeficiency syndrome selected populations at increased risk from rsv infections respiratory syncytial virus infection in children with bronchopulmonary dysplasia preterm twins and triplets: a highrisk group for severe respiratory syncytial virus infection annual variation in rsv season and decisions regarding immunoprophylaxis with palivizumab bronchiolitis associated mortality and estimates of rsv associated deaths among united states children 1979-1997 substantial variability in community rsv season timing distribution and clinical impact of human respiratory syncytial virus genotypes in hospitalized children over two winter seasons multicenter study of strains of respiratory syncytial virus occurrence of groups a and b of respiratory syncytial virus over 15 years: associated epidemiologic and clinical characteristics in hospitalized and ambulatory children correlation between rsv genotype and severity of illness the unresolved issue of risk factors for hospitalization of infants with rsv infection born after 33-35 weeks gestation environmental and demographic risk factors for rsv lower respiratory tract disease daycare center attendance and hospitalization for lower respiratory tract illness longterm prospective study in children after respiratory syncytial virus infection relationship between urinary cotinine level and diagnosis in children admitted to hospital risk factors for respiratory syncytial virusassociated lower respiratory illnesses in the first year of life parental childhood respiratory illness and respiratory illness in their infants. group health medical associates airway response to a bronchodilator in healthy parents of infants with bronchiolitis respiratory syncytial virus related apnea in infants central apneas in infants with bronchiolitis admitted to the paediatric intensive care unit revised indications for the use of palivizumab and rsv immune globulin intravenous for the prevention of rsv infections revised indications for the use of palivizumab and rsv immune globulin intravenous for the prevention of rsv infections rsv immune globulin for prophylaxis against rsv disease in infants and children with congenital heart disease palivizumab prophylaxis reduces hospitalization due to rsv in young children with hemodynamically significant congenital heart disease prediction of the duration of hospitalization in patients with respiratory syncytial virus infection: use of clinical parameters duration of hospitalization in previously well infants with respiratory syncytial virus infection the effect of practice variation on resource utilization in infants hospitalized for viral lower respiratory illness predicting deterioration in previously healthy infants hospitalized with rsv infection clinical and physiological manifestations of bronchiolitis and pneumonia: outcome of respiratory syncytial virus radiologic appearance of viral disease of the lower respiratory tract in infants and children duration of illness in infants with bronchiolitis evaluated in the emergency department roentgenographic features of common pediatric viral respiratory tract infections respiratory syncytial virus: a comparison of diagnostic modalities efficiency of immunofluorescence for rapid detection of common respiratory viruses application of directigen flua for the detection of influenza a virus in human and nonhuman specimens simulfluor respiratory screen for rapid detection of multiple respiratory viruses in clinical specimens by immunofluorescence staining rapid diagnosis of respiratory viral infections by using a shell vial assay and monoclonal antibody pool use of pcrenzyme immunoassay for identification of influenza a virus matrix rna in clinical samples negative for cultivable virus detection of respiratory syncytial virus a and b parainfluenzavirus 3 sequences in respiratory tracts of infants by a single pcr with primers targeted to the lpolymerase gene and differential hybridization rapid simultaneous diagnosis of infections with respiratory syncytial viruses a and b, influenza viruses a and b, and human parainfluenza viruses types 1, 2, and 3 by multiplex quantitative reverse transcription polymerase chain reactionenzyme hybridation assay (hexaplex) bronchodilators for bronchiolitis decreased neutrophil betaadrenergic receptors in the neonate influence of the pattern of structural growth of lung on susceptibility to specific infectious diseases in infants and children effect of salbutamol on histamineinduced bronchoconstriction in healthy infants lung function and bronchial challenges in infants the effect of age on bronchodilator responsiveness effect of racemic epinephrine and salbutamol on clinical score and pulmonary mechanics in infants with bronchiolitis a randomized trial comparing the efficacy of epinephrine to salbutamol in acute bronchiolitis inhaled nebulized adrenaline improves lung function in infants with acute bronchiolitis assessment of bronchodilator responsiveness in infants with bronchiolitis: a comparison of the tidal and the raised volume rapid thoracoabdominal compression technique recent advances in the treatment of bronchiolitis and laryngitis bronchodilator responsiveness in infants with bronchiolitis bronchodilators for treatment of mild bronchiolitis: a factorial randomised trial oral versus nebulized albuterol in the management of bronchiolitis in egypt the use of albuterol in hospitalized infants with bronchiolitis a multicenter, randomized, double blind, controlled trial of nebulized epinephrine in infants with acute bronchiolitis efficacy of beta 2agonists in bronchiolitis: a reappraisal and meta analysis dexamethasone in salbutamoltreated inpatients with acute bronchiolitis: a randomized, controlled trial the effect of high dose inhaled corticosteroid on wheeze in infant after rsv infection: randomized double blind placebo controlled trial a multicenter randomized controlled trial of dexamethasone for bronchiolitis corticosteroids do not affect the clinical or physiological status of infants with bronchiolitis longterm effects of prednisolone in the acute phase of bronchiolitis caused by respiratory syncytial virus early nebulized budesonide in the treatment of bronchiolitis and the prevention of postbronchiolitic wheezing efficacy of corticosteroids in acute bronchiolitis: shortterm and longterm followup efficacy of oral dexamethasone in outpatients with acute bronchiolitis oral prednisolone in the acute management of children age 6 to 35 months with viral respiratory infectioninduced lower airway disease: a randomized, placebocontrolled trial systemic corticosteroids in infant bronchiolitis: a metaanalysis epinephrine and dexamethasone in children with bronchiolitis costeffectiveness of epinephrine and dexamethasone in children with bronchiolitis committee on infectious diseases. reassessment of the indications for ribavirin therapy in respiratory syncytial virus infections aerosolized ribavirin treatment of infants with respiratory syncytial viral infection: a randomized doubleblind study ribavirin aerosol treatment of bronchiolitis associated with respiratory syncytial virus infection in infants prevention and control of influenza with vaccines neuraminidase inhibitors for treatment of influenza a and b infections use of the oral neuraminidase inhibitor oseltamivir in experimental human influenza infection: randomized controlled trials for prevention and treatment clinical efficacy and safety of the orally inhaled neuraminidase inhibitor zanamivir in the treatment of influenza: a randomized, doubleblind, placebocontrolled european study zanamivir for treatment of symptomatic influenza a and b infection in children five to twelve years of age: a randomized controlled trial intravenous immunoglobulin treatment of respiratory syncytial virus infections in infants and young children controlled trial to evaluate protection of high risk infants against rsv by using standard intravenous immune globulin respiratory syncytial virus (rsv) immunoglobulin therapy for rsv lower respiratory tract infection in infants and young children at high risk for severe rsv infection respiratory syncytial virus immune globulin treatment of rsv lower respiratory tract infection in previously healthy children reduction of respiratory syncytial virus (rsv) in tracheal aspirates in intubated infants by the use of humanized monoclonal antibody to rsv f protein a randomized controlled trial of vitamin a in children with severe measles serum vitamin a levels in respiratory syncytial virus infection vitamin a and respiratory syncytial virus infection the relationship of childhood respiratory illness to adult obstructive airway disease pulmonary function changes in children after respiratory syncytial virus infection in infancy the relationship of rsvspecific immunoglobulin e antibody responses in infancy, recurrent wheezing, and pulmonary function at age 7-8 years asthma and wheezing in the first six years of life respiratory syncytial virus in early life and risk of wheeze and allergy by age 13 years evaluation of combined live, attenuated rsv and parainfluenza 3 virus vaccines in infants and young children key: cord-021277-smw6owql authors: nan title: respiratory distress syndrome: recent research date: 2013-02-17 journal: nan doi: 10.2165/00128413-199107860-00060 sha: doc_id: 21277 cord_uid: smw6owql nan in infants effects in adults use of corticosteroids in the adult respiratory distress syndrome -a clinical review adult respiratory distress syndrome associated with parainfluenza virus type i in children effects of prostaglandin ei on oxygen delivery and consumption in patients with the adult respiratory distress syndrome prophylactic treatment with an aerosolized corticosteroid liposome in a porcine model of early ards induced by endotoxaemia physiologic effects and side effects of prostaglandin e i in the adult respiratory distress syndrome respiratory distress and acute renal failure due to synergic bleomycin-cisplatin toxicity key: cord-266260-t02jngq0 authors: ramshaw, rebecca e.; letourneau, ian d.; hong, amy y.; hon, julia; morgan, julia d.; osborne, joshua c. p.; shirude, shreya; van kerkhove, maria d.; hay, simon i.; pigott, david m. title: a database of geopositioned middle east respiratory syndrome coronavirus occurrences date: 2019-12-13 journal: sci data doi: 10.1038/s41597-019-0330-0 sha: doc_id: 266260 cord_uid: t02jngq0 as a world health organization research and development blueprint priority pathogen, there is a need to better understand the geographic distribution of middle east respiratory syndrome coronavirus (mers-cov) and its potential to infect mammals and humans. this database documents cases of mers-cov globally, with specific attention paid to zoonotic transmission. an initial literature search was conducted in pubmed, web of science, and scopus; after screening articles according to the inclusion/exclusion criteria, a total of 208 sources were selected for extraction and geo-positioning. each mers-cov occurrence was assigned one of the following classifications based upon published contextual information: index, unspecified, secondary, mammal, environmental, or imported. in total, this database is comprised of 861 unique geo-positioned mers-cov occurrences. the purpose of this article is to share a collated mers-cov database and extraction protocol that can be utilized in future mapping efforts for both mers-cov and other infectious diseases. more broadly, it may also provide useful data for the development of targeted mers-cov surveillance, which would prove invaluable in preventing future zoonotic spillover. middle east respiratory syndrome coronavirus (mers-cov) emerged as a global health concern in 2012 when the first human case was documented in saudi arabia 1 . now listed as one of the who research and development blueprint priority pathogens, cases have been reported in 27 countries across four continents 2 . imported cases into non-endemic countries such as france, great britain, the united states, and south korea have caused secondary cases [3] [4] [5] , thus highlighting the potential for mers-cov to spread far beyond the countries where index cases originate. reports in animals suggest that viral circulation could be far more widespread than suggested by human cases alone [6] [7] [8] . to help prevent future incidence of mers-cov, public health officials can focus on mitigating zoonotic transfer; however, in order to do this effectively, additional research is needed to determine where spillover could occur between mammals and humans. previous literature reviews have looked at healthcare-associated outbreaks 9 , importation events resulting in secondary cases 10, 11 , occurrences among dromedary camels 12, 13 , or to summarize current knowledge and knowledge gaps of mers-cov 14, 15 . this database seeks fill gaps in literature and build upon existing notification data by enhancing the geographic resolution of mers-cov data and providing occurrences of both mammal and environmental detections in addition to human cases. this information can help inform epidemiological models and targeted disease surveillance, both of which play important roles in strengthening global health security. knowledge of the geographic extent of disease transmission allows stakeholders to develop appropriate emergency response and preparedness activities (https://www.jeealliance.org/ global-health-security-and-ihr-implementation/joint-external-evaluation-jee/), inform policy for livestock trade and quarantine, determine appropriate demand for future vaccines (http://cepi.net/mission) and decide where to deliver them. additionally, targeted disease surveillance will provide healthcare workers with updated lists of the methods and protocols summarized below have been adapted from previously published literature extraction processes [18] [19] [20] [21] [22] , and provide additional context surrounding our systematic data collection from published reports of mers-cov. data collection. we identified published reports of mers-cov by searching pubmed, web of science, and scopus with the following terms: "middle eastern respiratory syndrome", "middle east respiratory syndrome", "merscov", and "mers". the initial search was for all articles published about mers-cov prior to april 30, 2017 , and was subsequently updated to february 22, 2018. these searches were conducted through the university of washington libraries' institutional database subscriptions. we searched the web of science web of science core collection (the subscribed edition includes science citation index expanded, 1900-present; social sciences citation index, 1975-present; arts & humanities citation index, 1975-present; emerging sources citation index, 2015-present). we searched the standard scopus database and the standard, freely available pubmed database; these products have a single version that is consistent across institutional subscriptions or access points. in total, this search returned 7,301 related abstracts, which were collated into a database before a title-abstract screening was manually conducted (fig. 1. flowchart) . articles were removed if they did not contain an occurrence of mers-cov; for example, vaccine development research or coronavirus proteomic analyses. non-english articles were flagged for further review and brought into the full text screening stage. the accompanying supplementary file highlight the title and abstract screening process and the inclusion and exclusion criteria. full text review was conducted on 1,083 sources. to meet the inclusion criteria, articles must have contained both of the following items: 1) a detection of mers-cov from humans, animals, or environmental sources, and 2) mers-cov occurrences tagged with spatial information. additionally, extractors attempted to prospectively manually remove articles containing duplicate occurrences that were already extracted in the dataset. extractors only prospectively manually removed articles if it was clear the articles contained data we were confident had already been extracted and had high-quality data. we excluded 885 sources based on full text review. in addition, we reviewed citations and retroactively added relevant articles to our database if they were not already included. we retroactively added and subsequently marked ten articles for extraction using this process. in total, we extracted 208 peer-reviewed sources reporting detection of mers-cov that included geographic and relevant epidemiological metadata. geo-positioning of data. google maps or arcgis 23 was used to manually extract location information at the highest resolution available from individual articles. we evaluated spatial information as either points or polygons. the geography was defined as a point if the location of transmission was reported to have occurred within a 5 × 5 km area. point data are represented by a specific latitude and longitude. a point references an area smaller than 5 × 5 km in order to be compatible with the typical 5 × 5 km resolution of satellite imagery used for global analyses. the geography was defined as a polygon if the location of transmission was less clear, but known to have occurred in a general area (e.g. a province), or the location of transmission occurred within an area greater than 5 × 5 km (e.g. a large city). we used contextual information to determine location in instances where the author's spelling of a location differed from google maps or arcgis. maps provided by authors were digitized using arcgis. we used three different types of polygons: known administrative boundaries, buffers, and custom polygons. relevant administrative units were sourced from the global administrative unit layers curated by the food and agricultural organization of the un 24 for known administrative boundaries of governorates, districts, or regions, and paired with the occurrence record. buffers were created to encompass areas in cities and regions without corresponding administrative units. to ensure that buffers encompassed the entirety of the area of interest, google maps was used to determine the required radius. in areas with unspecified boundaries (e.g. table mountain national park and the border region between saudi arabia and uae) arcgis was used to generate custom polygons, which were assigned a unique code within a defined shapefile for ease of re-identification. this database is publicly available online 16, 17 . each of the 861 rows represents a unique occurrence of mers-cov. rows containing an index, unspecified, or imported case represent a single case of mers-cov. rows containing mammal and secondary cases may represent more than one case but are still unique geospatial occurrences. table 1 shows an overview of the content available in the publicly available dataset. in addition, online-only table 1 lists occurrences by geography, origin, 405 shape type, and publication and online-only table 2 provides citations of the data. index 34 99 1 0 93 7 234 unspecified 86 50 1 4 35 27 203 mammal 53 56 7 30 43 19 208 import 11 2 0 2 10 9 34 secondary 82 30 1 1 26 8 148 absent 3 8 0 0 7 3 21 environmental www.nature.com/scientificdata www.nature.com/scientificdata/ 15. pathogen: name the pathogen identified (e.g. mers-cov, bat coronaviruses, and other mers-cov-like pathogens). 16. pathogen_note: miscellaneous notes regarding pathogen. 17. patient_type: index, unspecified, na, secondary, import, or absent. • index: any human infection of mers-cov resulting after direct contact with an animal and no reported contact with a confirmed mers-cov case or healthcare setting. • unspecified: cases that lacked sufficient epidemiological evidence to classify them as any other status (e.g. serosurvey studies). • na: non-applicable field; case was not a patient (e.g. mammal) • secondary: defined as any cases resulting from contact with known human infections. cases reported after the index case can be assumed to be secondary cases unless accompanied by specific details of likely independent exposure to an animal reservoir. • import: cases that were brought into a non-endemic country after transmission occurred elsewhere. • absent: suspected case(s) ultimately confirmed negative for mers-cov. 18 . transmission_route: zoonotic, direct, unspecified, or animal-to-animal. • zoonotic: transmission occurred from an animal to a human. • direct: only relevant for human-to-human transmission. • unspecified: lacked sufficient epidemiological evidence to classify a human case as zoonotic or direct. • animal-to-animal: transmission occurred from an animal to another animal. 19 . clinical: describes whether the mers-cov occurrence demonstrated clinical signs of infection. denoted by yes, no, or unknown. • yes: clinical signs of infection were present/reported. clinical signs among humans may range from mild (e.g. fever, cough) to severe (e.g. pneumonia, kidney failure). clinical signs among camels include nasal discharge. • no: clinical signs of infection were not present/reported. • unknown: subject(s) may or may not have been demonstrating clinical signs of infection. for example, some authors did not explicitly mention symptoms, but individuals reportedly sought medical care. another example being when a diagnostic serosurvey was conducted during an ongoing outbreak. the term "unknown" was used when articles lacked sufficient evidence for extractors to definitively label as "yes" or "no". 20. diagnostic: describes the class of diagnostic method that was used. pcr, serology, or reported. 21 . diagnostic_note: more detailed information related to the specific test used (e.g. rk39, igg, or igm serology). 22. serosurvey: describes the context if serological testing was used. • diagnostic: testing of symptomatic patients. • exploratory: historic exposure determined among healthy asymptomatic individuals. 23. country: iso3 code for country in which the case occurred. 24 . origin: open-ended field to provide more details on the specific in-country location of mers-cov case. 25. problem_geography: this field was utilized if the mers-cov case was reported in a location that could cause uncertainty when determining exact geographic occurrence (e.g. hospital, abattoir). 26. lat: latitude measured in decimal degrees. 27. long: longitude measured in decimal degrees. 28. latlong_source: the source from which latitude and longitude were derived. 29. loc_confidence: states the level of confidence that researchers had when assigning a geographic location to the mers-cov case (good or bad). an answer of 'good' meant the article stated clearly that the case occurred in a specific geographic location and no assumptions were required on part of the researcher. an answer of 'bad' meant the article did not clearly state the specific geographic location of the mers-cov case, but the researcher was able to infer the location of occurrence. the field site_notes was utilized to detail the logic behind researchers' decisions when inference was required. 30. shape_type: the geographic shape type assigned to the mers-cov occurrence (point or polygon). 31. poly_type: if the mers-cov occurrence was assigned a shape_type of polygon, was it admin (gaul), custom, or buffer? 32. buffer_radius: if a mers-cov occurrence was assigned a buffer, what is the radius in km? 33. gaul_year_or_custom_shapefile: file path used to reach the necessary shape file in arcgis. users of this dataset can find custom shapefiles created for this dataset at: https://cloud.ihme.washington.edu/index. php/s/dgoykyqnbjg54f2/download 34. poly_id: a standardized and unique identifier assigned to each gaul shapefile. 35 . poly_field: which type of polygon was used to geo-position the occurrence? (e.g. if admin1 polygon was used, enter adm1_code) 36. site_notes: miscellaneous notes regarding the site of occurrence. 37. month_start: month that the occurrence(s) began. if the article provided a specific month of illness onset, the month was assigned a number from 1-12 (1 = january, 2 = february, etc.). if the article did not provide a specific month of illness onset, then researchers assigned a value of 'na' . month that the occurrence(s) ended, defined as the date a patient tested negative for mers-cov. if the article provided a specific month for recovery, the month was assigned a number from 1-12 (1 = january, 2 = february, etc.). if the article did not provide a specific month of symptom onset, then researchers assigned a value of 'na' . 39. year_start: year that the occurrence(s) began. if the year of illness onset was not provided in the article, the ihme standard was used: (year_start = publication year -3). year that the occurrence(s) ended. if the article did not provide a specific year for recovery, the ihme standard was used: (year_end = publication year -1). 41. year_accuracy: if years were reported, this field was assigned a value of '0' . if assumptions were required, this field was assigned a value of '1' . all data extracted from the original search (october 2012 to april 30, 2017) was reviewed independently by a second individual to check for accuracy. challenging extractions from the updated search (may 1, 2017 to february 22, 2018) were selected for group review during bi-weekly team meetings. upon extraction completion, all data were checked to ensure they fell on land and within the correct country. while the protocol implemented above was designed to reduce the amount of subjective decisions made by extractors, total elimination was not possible. wherever a subjective decision had to be made, the extractor utilized the various notes fields in order to document the logic behind decisions. these decisions were subsequently reviewed by other extractors. the techniques described here can be applied to collect and curate datasets for other infectious diseases, as has been previously demonstrated with dengue 20 and leishmaniasis 18 . additionally, since these data were collected independently through published reports of mers-cov occurrence, they may be used to build upon existing notification data 26, 27 . our ability to capture occurrences in this dataset is contingent on the data contained within published literature. therefore, this dataset does not represent a total count of all cases. instead, this dataset's value lies within its geo-precision. data were extracted with a focus on obtaining the highest resolution possible. these data may be merged with other datasets, such as who 26 or oie 27 surveillance records, and are intended to complement, not replace, these resources. together, published reports and notification data can provide a more comprehensive snapshot of current disease extent and at-risk locations. an important consideration, whether using the literature data alone, or in combination with other databases, is the potential for duplication. various pieces of metadata can be used to evaluate where potential duplicates could lie, such as common date fields (month_start, month_end, year_start, year_end) or consistent geographic details (lat, long, poly_id, shape_type) or shared epidemiological tags (patient_type). researchers may wish to consider further steps, such as fuzzy matching of geographic data (e.g. matching a point with an overlapping buffer) or temporal data (e.g. matching a precise month with an overlapping month interval). we acknowledge this duplicate-removal process will not catch all matching records, but it will likely catch several. we recommend occurrences are layered from top to bottom in the following order: index (green), unspecified (orange), mammal (yellow), import (blue), secondary (purple). points were plotted using their assigned latitudes and longitudes, and shape files were created for polygons. buffers were also plotted using assigned latitudes and longitudes, after which each buffer's custom radius was drawn. higher resolution geographies (points, buffers, governorates) were plotted on top of lower resolution geographies (countries, regions). www.nature.com/scientificdata www.nature.com/scientificdata/ this approach because it will allow researchers to remove several duplicates without erroneously deleting any two occurrences that are truly unique (i.e. not duplicates). essentially, we recommend a sensitive approach above a more specific approach, as the latter simply risks culling too many records that aren't actually duplicates. when merging with other databases, consistency in metadata tagging is essential. for the who disease outbreak news data feed 26, 27 for instance, nomenclature for case definitions is slightly different, with who definitions of "community acquired" and "not reported" comparable to "index" and "unspecified" respectively. in addition, it is important to recognize what information is beyond the scope of these additional databases. again, when comparing to the who dataset, it is important to recognize that serologically positive cases do not meet the case definition used in the who database. these adjustments need to be identified on a dataset-to-dataset basis. among cases tagged as index or unspecified. occurrences tagged as index are coloured green, those tagged as unspecified are coloured orange. points were plotted using their assigned latitudes and longitudes, and shape files were created for polygons. buffers were also plotted using assigned latitudes and longitudes, after which each buffer's custom radius was drawn. higher resolution geographies (points, buffers, governorates) were plotted on top of lower resolution geographies (countries, regions). points were plotted using their assigned latitudes and longitudes, and shape files were created for polygons. buffers were also plotted using assigned latitudes and longitudes, after which each buffer's custom radius was drawn. higher resolution geographies (points, buffers, governorates) were plotted on top of lower resolution geographies (countries, regions). www.nature.com/scientificdata www.nature.com/scientificdata/ this database can be combined with other covariates (e.g. satellite imagery) to produce environmental suitability models of mers-cov infection risk and potential spillover on both global and regional scales as achieved with other exemplar datasets [28] [29] [30] [31] . this information can be useful in resource allocation aimed at improving disease surveillance and contribute towards a better understanding of the factors facilitating continued emergence of index cases. the addition of sampling techniques and prevalence data may improve this dataset. researchers were ultimately unable to add these data due to inconsistencies in the way literature reported sampling techniques and prevalence date by geography. an attempt to extract these data using the current approach would have led to sporadic inclusion of this information and would not have been comprehensive for the entire dataset. moving forward, we recommend authors report sampling technique and prevalence data at the highest resolution geography possible, as seen in miguel et al. 32 . we encourage continued presentation of paired epidemiological and geographic metadata that would allow for more detailed analysis in the future. this database may also be utilized in clinical settings to provide an evidence-base for diagnoses when used in conjunction with patient travel histories. additionally, it can be used to identify geographies for surveillance, particularly areas where mers-cov has been documented in animals but not humans (e.g. ethiopia and nigeria). identifying locations for surveillance will, in turn, inform global health security. while models will increase the resolution at which these questions can be addressed, datasets such as this provide an initial baseline. a major limitation of this database is the potential for sampling bias, which stems from higher frequency of disease reporting within countries where there exists strong healthcare infrastructure and reporting systems. this database does not attempt to account for such biases, which must be addressed in subsequent modelling activities where such biases are of consequence. similarly, another limitation is potential duplicate documentation of singular occurrences. this can happen when the same occurrence is assigned different geographies (e.g. point, polygon) in multiple publications. even though extractors made efforts to prospectively manually identify duplicate occurrences, this was challenging because the process relied upon papers providing sufficient details for extractors to determine a duplicate occurrence (e.g. geography, patient demographics, dates of occurrence, diagnostic methods, etc.). however, the majority of papers did not report such details for each occurrence. in those instances, it was impossible for extractors to discern whether occurrences may have been duplicates from a previous artic le. even case studies inconsistently reported patient details and demographic information. these are some examples of challenges faced by extractors when we attempted to identify duplicates. without sufficient contextual clues, extractors lacked evidence to determine duplicity and thus likely extracted some unique occurrences more than once. despite efforts to remove duplicate occurrences from the database, it is possible that some remain. geographic uncertainty is similarly problematic for analyses such as this. in some cases, polygons, as opposed to points, are utilised as a geographic frame of reference, reflecting the uncertainty in geotagging in the articles themselves. for some occurrences, there is a strong assumption that the geography listed corresponds to the site of infection. while the use of 5 km × 5 km as the minimum geographical unit allows for some leeway in this precision, it is possible that even with the point data (often corresponding to household clusters) these may not map directly with true infection sites. this must be considered in any subsequent geospatial analysis. finally, this database represents a time-bounded survey of the literature. while all efforts were made to be comprehensive within this period, articles, and therefore data, will continue to be published. efforts to streamline ongoing collection processes are still to be fully realized 33 . regardless, we hope that this dataset provides a solid baseline for further iteration. isolation of a novel coronavirus from a man with pneumonia in saudi arabia middle east respiratory syndrome coronavirus (mers-cov) clinical features and viral diagnosis of two cases of infection with middle east respiratory syndrome coronavirus: a report of nosocomial transmission enhanced mers coronavirus surveillance of travelers from the middle east to england control of an outbreak of middle east respiratory syndrome in a tertiary hospital in korea molecular evolution of mers coronavirus: dromedaries as a recent intermediate host or long-time animal reservoir? middle east respiratory syndrome coronavirus in dromedary camels: an outbreak investigation presence of antibodies but no evidence for circulation of mers-cov in dromedaries on the canary islands comparative analysis of eleven healthcare-associated outbreaks of middle east respiratory syndrome coronavirus (mers-cov) from 2015 to 2017 assessing the risk of observing multiple generations of middle east respiratory syndrome (mers) cases given an imported case risk of mers importation and onward transmission: a systematic review and analysis of cases reported to who a systematic review of mers-cov seroprevalence and rna prevalence in dromedary camels: implications for animal vaccination a rapid scoping review of middle east respiratory syndrome coronavirus in animal hosts what have we learned about middle east respiratory syndrome coronavirus emergence in humans? a systematic literature review. vector borne zoonotic dis middle east respiratory syndrome coronavirus (mers-cov) infection: epidemiology, pathogenesis and clinical characteristics geopositioned middle east respiratory syndrome coronavirus occurrences. database 1983-2017. institute for health metrics and evaluation (ihme) database of geopostioned middle east respiratory syndrome coronavirus occurrences global database of leishmaniasis occurrence locations the contemporary distribution of trypanosoma cruzi infection in humans, alternative hosts and vectors a global compendium of human dengue virus occurrence a global compendium of human crimean-congo haemorrhagic fever virus occurrence a comprehensive database of the geographic spread of past human ebola outbreaks food and agricultural organization of the united nations. the global administrative unit layers (gaul): technical aspects global distribution maps of the leishmaniases updates to the zoonotic niche map of ebola virus disease in africa the global distribution of crimean-congo hemorrhagic fever the global distribution and burden of dengue risk factors for mers coronavirus infection in dromedary camels in a supervised learning process to validate online disease reports for use in predictive models critical contribution of laboratories to outbreak response support for middle east respiratory syndrome coronavirus international health regulations (2005) facilitate communication for in-flight contacts of a middle east respiratory syndrome case first confirmed case of middle east respiratory syndrome coronavirus infection in the kingdom of bahrain: in a saudi gentleman after cardiac bypass surgery. case rep event based surveillance of middle east respiratory syndrome coronavirus (mers-cov) in bangladesh among pilgrims and travelers from the middle east: an update for the period middle east respiratory syndrome coronavirus antibodies in dromedary camels middle east respiratory syndrome coronavirus neutralising serum antibodies in dromedary camels: a comparative serological study definitive diagnosis in suspected middle east respiratory syndrome coronavirus cases characteristics of traveler with middle east respiratory syndrome complete genome sequence of middle east respiratory syndrome coronavirus (mers-cov) from the first imported mers-cov case in china imported case of mers-cov infection identified in china mers-related betacoronavirus in vespertilio superans bats prevalence and genetic diversity analysis of human coronaviruses among cross-border children no mers-cov but positive influenza viruses in returning hajj pilgrims, china two deletion variants of middle east respiratory syndrome coronavirus found in a patient with characteristic symptoms human neutralizing monoclonal antibody inhibition of middle east respiratory syndrome coronavirus replication in the common marmoset complete genome sequence of middle east respiratory syndrome coronavirus isolated from a dromedary camel in egypt cross-sectional surveillance of middle east respiratory syndrome coronavirus (mers-cov) in dromedary camels and other mammals in egypt mers coronavirus neutralizing antibodies in camels mers coronaviruses in dromedary camels seroepidemiology for mers coronavirus using microneutralisation and pseudoparticle virus neutralisation assays reveal a high prevalence of antibody in dromedary camels in egypt systematic, active surveillance for middle east respiratory syndrome coronavirus in camels in egypt middle east respiratory syndrome coronavirus: epidemiology and disease control measures geographic distribution of mers coronavirus among dromedary camels first cases of middle east respiratory syndrome coronavirus (mers-cov) infections in france, investigations and implications for the prevention of human-to-human transmission clinical features and virological analysis of a case of middle east respiratory syndrome coronavirus infection a case of imported middle east respiratory syndrome coronavirus infection and public health response laboratory investigation and phylogenetic analysis of an imported middle east respiratory syndrome coronavirus case in greece cluster of middle east respiratory syndrome coronavirus infections in iran epidemiological and clinical characteristics of patients with middle east respiratory syndrome coronavirus in iran in 2014 health care associated middle east respiratory syndrome (mers): a case from iran influenza virus but not mers coronavirus circulation in iran phylogenetic analysis of merscov in human and camels in middle east respiratory syndrome coronavirus specific antibodies in naturally exposed israeli llamas, alpacas and camels the prevalence of middle east respiratory syndrome coronavirus (mers-cov) antibodies in dromedary camels in israel detection of coronaviruses in bats of various species in italy investigation of an imported case of middle east respiratory syndrome coronavirus (mers-cov don't forget the migrants": exploring preparedness and response strategies to combat the potential spread of mers-cov virus through migrant workers in sri lanka high prevalence of middle east respiratory coronavirus in young dromedary camels in jordan. vector borne zoonotic dis arabia: an index case investigation middle east respiratory syndrome coronavirus (mers-cov) serology in major livestock species in an affected region in jordan stillbirth during infection with middle east respiratory syndrome coronavirus antibodies against mers coronavirus in dromedary camels mers-cov antibodies in humans, africa no serologic evidence of middle east respiratory syndrome coronavirus infection among camel farmers exposed to highly seropositive camel herds: a household linked study serological evidence of mers-cov antibodies in dromedary camels (camelus dromedaries) in laikipia county occurrence of the middle east respiratory syndrome coronavirus (mers-cov) across the gulf corporation council countries: four years update emergence of mers-cov in the middle east: origins, transmission, treatment, and perspectives laboratory-confirmed case of middle east respiratory syndrome coronavirus (mers-cov) infection in malaysia: preparedness and response dromedary camels in northern mali have high seropositivity to mers-cov middle east respiratory syndrome coronavirus (mers-cov) infections in two returning travellers in the netherlands middle east respiratory syndrome coronavirus (mers-cov) in dromedary camels in nigeria lack of serological evidence of middle east respiratory syndrome coronavirus infection in virus exposed camel abattoir workers in nigeria asymptomatic mers-cov infection in humans possibly linked to infected dromedaries imported from oman to united arab emirates middle east respiratory syndrome coronavirus (mers-cov) in dromedary camels the middle east respiratory syndrome coronavirus (mers-cov) zoonotic origin and transmission of middle east respiratory syndrome coronavirus in the uae imported case of middle east respiratory syndrome coronavirus (mers-cov) infection from oman to thailand serologic evidence for mers-cov infection in dromedary camels contact tracing the first middle east respiratory syndrome case in the philippines effectiveness of the middle east respiratory syndrome-coronavirus protocol in enhancing the function of an emergency department in qatar high proportion of mers-cov shedding dromedaries at slaughterhouse with a potential epidemiological link to human cases isolation of mers coronavirus from a dromedary camel mers-cov infection of alpaca in a region where mers-cov is endemic middle east respiratory syndrome coronavirus (mers-cov) rna and neutralising antibodies in milk collected according to local customs from dromedary camels occupational exposure to dromedaries and risk for mers-cov infection risk factors for primary middle east respiratory syndrome coronavirus infection in camel workers in qatar during 2013-2014: a case-control study mers-cov outbreak in jeddah-a link to health care facilities a case of long-term excretion and subclinical infection with middle east respiratory syndrome coronavirus in a healthcare worker a comparative study of clinical presentation and risk factors for adverse outcome in patients hospitalised with acute respiratory disease due to mers coronavirus or other causes a family cluster of middle east respiratory syndrome coronavirus infections related to a likely unrecognized asymptomatic or mild case acute management and long-term survival among subjects with severe middle east respiratory syndrome coronavirus pneumonia and ards acute middle east respiratory syndrome coronavirus: temporal lung changes observed on the chest radiographs of 55 patients acute myocarditis associated with novel middle east respiratory syndrome coronavirus an outbreak of middle east respiratory syndrome (mers) due to coronavirus in al-ahssa region, saudi arabia antibody response and disease severity in healthcare worker mers survivors brief report: family cluster of middle east respiratory syndrome coronavirus infections characteristics and outcomes of middle east respiratory syndrome coronavirus patients admitted to an intensive care unit in jeddah, saudi arabia clinical and laboratory findings of the first imported case of middle east respiratory syndrome coronavirus to the united states clinical aspects and outcomes of 70 patients with middle east respiratory syndrome coronavirus infection: a singlecenter experience in saudi arabia clinical course and outcomes of critically ill patients with middle east respiratory syndrome coronavirus infection co-circulation of three camel coronavirus species and recombination of mers-covs in saudi arabia community case clusters of middle east respiratory syndrome coronavirus in hafr al-batin, kingdom of saudi arabia: a descriptive genomic study description of a hospital outbreak of middle east respiratory syndrome in a large tertiary care hospital in saudi arabia descriptive epidemiology and characteristics of confirmed cases of middle east respiratory syndrome coronavirus infection in the makkah region of saudi arabia detection of the middle east respiratory syndrome coronavirus genome in an air sample originating from a camel barn owned by an infected patient epidemiological, demographic, and clinical characteristics of 47 cases of middle east respiratory syndrome coronavirus disease from saudi arabia: a descriptive study evidence for camel-to-human transmission of mers coronavirus exposures among mers case-patients first confirmed cases of middle east respiratory syndrome coronavirus (mers-cov) infection in the united states, updated information on the epidemiology of mers-cov infection, and guidance for the public, clinicians, and public health authorities hospital outbreak of middle east respiratory syndrome coronavirus human infection with mers coronavirus after exposure to infected camels, saudi arabia identified transmission dynamics of middle east respiratory syndrome coronavirus infection during an outbreak: implications of an overcrowded emergency department impact of middle east respiratory syndrome coronavirus (mers-cov) on pregnancy and perinatal outcome lack of middle east respiratory syndrome coronavirus transmission from infected camels longitudinal study of middle east respiratory syndrome coronavirus infection in dromedary camel herds in saudi arabia transmission and evolution of the middle east respiratory syndrome coronavirus in saudi arabia: a descriptive genomic study spread, circulation, and evolution of the middle east respiratory syndrome coronavirus an observational, laboratory-based study of outbreaks of middle east respiratory syndrome coronavirus in jeddah and riyadh, kingdom of saudi arabia multifacility outbreak of middle east respiratory syndrome in taif, saudi arabia epidemiology of a novel recombinant middle east respiratory syndrome coronavirus in humans in saudi arabia mers coronavirus in dromedary camel herd, saudi arabia mers cov infection in two renal transplant recipients: case report mers-cov in upper respiratory tract and lungs of dromedary camels middle east respiratory syndrome (mers) coronavirus seroprevalence in domestic livestock in saudi arabia middle east respiratory syndrome coronavirus (mers-cov): a cluster analysis with implications for global management of suspected cases middle east respiratory syndrome coronavirus in al-madinah city, saudi arabia: demographic, clinical and survival data middle east respiratory syndrome coronavirus in children middle east respiratory syndrome coronavirus infection during pregnancy: a report of 5 cases from saudi arabia middle east respiratory syndrome coronavirus infection in dromedary camels in saudi arabia middle east respiratory syndrome coronavirus transmission in extended family middle eastern respiratory syndrome corona virus (mers cov): case reports from a tertiary care hospital in saudi arabia molecular epidemiology of hospital outbreak of middle east respiratory syndrome notes from the field: nosocomial outbreak of middle east respiratory syndrome in a large tertiary care hospital-riyadh, saudi arabia outbreak of middle east respiratory syndrome at tertiary care hospital patient characteristics infected with middle east respiratory syndrome coronavirus infection in a tertiary hospital predictors of mers-cov infection: a large case control study of patients presenting with ili at a mers-cov referral hospital in saudi arabia presence of middle east respiratory syndrome coronavirus antibodies in saudi arabia: a nationwide, crosssectional, serological study presentation and outcome of middle east respiratory syndrome in saudi intensive care unit patients report of middle east respiratory syndrome coronavirus (mers-cov) infection in four patients with hematological malignancies treated at king fahad medical city risk factors for middle east respiratory syndrome coronavirus infection among healthcare personnel spontaneous intracranial hemorrhage in a patient with middle east respiratory syndrome corona virus successful recovery of mers cov pneumonia in a patient with acquired immunodeficiency syndrome: a case report surveillance and testing for middle east respiratory syndrome coronavirus, saudi arabia the critical care response to a hospital outbreak of middle east respiratory syndrome coronavirus (mers-cov) infection: an observational study the first case of the 2015 korean middle east respiratory syndrome outbreak travel-related mers-cov cases: an assessment of exposures and risk factors in a group of dutch travellers returning from the kingdom of saudi arabia treatment outcomes for patients with middle eastern respiratory syndrome coronavirus (mers cov) infection at a coronavirus referral center in the kingdom of saudi arabia middle east respiratory syndrome coronavirus transmission among health care workers: implication for infection control histopathology of middle east respiratory syndrome coronovirus (mers-cov) infection -clinicopathological and ultrastructural study the calm before the storm: clinical observations of middle east respiratory syndrome (mers) patients the prevalence of middle east respiratory syndrome coronavirus (mers-cov) infection in livestock and temporal relation to locations and seasons assessing the detection of middle east respiratory syndrome coronavirus igg in suspected and proven cases of middle east respiratory syndrome coronavirus infection cross-sectional study of mers-cov-specific rna and antibodies in animals that have had contact with mers patients in saudi arabia a cohort-study of patients suspected for mers-cov in a referral hospital in saudi arabia conveyance contact investigation for imported middle east respiratory syndrome cases recovery from the middle east respiratory syndrome is associated with antibody and t-cell responses outbreak of middle east respiratory syndrome-coronavirus causes high fatality after cardiac operations prevalence of antibodies against the middle east respiratory syndrome coronavirus, influenza a and b viruses among blood donors, saudi arabia serological evidence of coronavirus infections in native hamadryas baboons (papio hamadryas hamadryas) of the kingdom of saudi arabia hematologic, hepatic, and renal function changes in hospitalized patients with middle east respiratory syndrome coronavirus middle east respiratory syndrome coronavirus and pulmonary tuberculosis coinfection: implications for infection control outcome of strict implementation of infection prevention control measures during an outbreak of middle east respiratory syndrome outbreak of middle east respiratory syndrome coronavirus in saudi arabia: a retrospective study sero-prevalence of middle east respiratory syndrome coronavirus (mers-cov) specific antibodies in dromedary camels in tabuk, saudi arabia seroepidemiology of middle east respiratory syndrome (mers) coronavirus in saudi arabia (1993) and australia (2014) and characterisation of assay specificity risk factors for primary middle east respiratory syndrome coronavirus illness in humans middle east respiratory syndrome coronavirus disease in children close relative of human middle east respiratory syndrome coronavirus in bat rooting the phylogenetic tree of middle east respiratory syndrome coronavirus by characterization of a conspecific virus from an african bat mers outbreak in korea: hospital-to-hospital transmission a case report of a middle east respiratory syndrome survivor with kidney biopsy results an unexpected outbreak of middle east respiratory syndrome coronavirus infection in the republic of korea atypical presentations of mers-cov infection in immunocompromised hosts clinical implications of 5 cases of middle east respiratory syndrome coronavirus infection in a south korean outbreak emergency cesarean section in an epidemic of the middle east respiratory syndrome: a case report epidemiologic features of the first mers outbreak in korea: focus on pyeongtaek st. mary's hospital epidemiological investigation of the 119th confirmed middle east respiratory syndrome coronavirus case with an indefinite mode of transmission during the pyeongtaek outbreak in korea extensive viable middle east respiratory syndrome (mers) coronavirus contamination in air and surrounding environment in mers isolation wards detection of severe acute respiratory syndrome-like, middle east respiratory syndrome-like bat coronaviruses and group h rotavirus in faeces of korean bats high fatality rates and associated factors in two hospital outbreaks of mers in daejeon, the republic of korea infectivity of an asymptomatic patient with middle east respiratory syndrome coronavirus mers epidemiological investigation to detect potential mode of transmission in the 178th mers confirmed case in pyeongtaek mers-cov outbreak following a single patient exposure in an emergency room in south korea: an epidemiological outbreak study middle east respiratory syndrome in 3 persons, south korea middle east respiratory syndrome-coronavirus infection: a case report of serial computed tomographic findings in a young male patient outbreaks of middle east respiratory syndrome in two hospitals initiated by a single patient in daejeon successful treatment of suspected organizing pneumonia in a patient with middle east respiratory syndrome coronavirus infection: a case report surveillance operation for the 141st confirmed case of middle east respiratory syndrome coronavirus in response to the patient's prior travel to jeju island transmission among healthcare worker contacts with a middle east respiratory syndrome patient in a single korean centre host susceptibility to mers-cov infection, a retrospective cohort study of the 2015 korean mers outbreak a study of the probable transmission routes of mers-cov during the first hospital outbreak in the republic of korea a middle east respiratory syndrome screening clinic for health care personnel during the 2015 middle east respiratory syndrome outbreak in south korea: a single-center experience evaluation and clinical validation of two field-deployable reverse transcription-insulated isothermal pcr assays for the detection of the middle east respiratory syndrome-coronavirus serologic responses of 42 mers-coronavirus-infected patients according to the disease severity the clinical and virological features of the first imported case causing mers-cov outbreak in south korea mers-cov antibody responses 1 year after symptom onset neurological complications during treatment of middle east respiratory syndrome impact of middle east respiratory syndrome outbreak on the use of emergency medical resources in febrile patients hospital outbreaks of middle east respiratory syndrome zero transmission of middle east respiratory syndrome: lessons learned from thailand family cluster of middle east respiratory syndrome coronavirus infections further evidence for bats as the evolutionary source of middle east respiratory syndrome coronavirus a phylogenetically distinct middle east respiratory syndrome coronavirus detected in a dromedary calf from a closed dairy herd in dubai with rising seroprevalence with age acute middle east respiratory syndrome coronavirus infection in livestock dromedaries antibodies against mers coronavirus in dromedary camels clinicopathologic, immunohistochemical, and ultrastructural findings of a fatal case of middle east respiratory syndrome coronavirus infection in the united arab emirates epidemiological investigation of middle east respiratory syndrome coronavirus in dromedary camel farms linked with human infection in abu dhabi emirate middle east respiratory syndrome coronavirus antibody reactors among camels in dubai middle east respiratory syndrome coronavirus during pregnancy polyphyletic origin of mers coronaviruses and isolation of a novel clade a strain from dromedary camels in the united arab emirates prevalence of middle east respiratory syndrome coronavirus (mers-cov) in dromedary camels in abu dhabi emirate time course of mers-cov infection and immunity in dromedary camels transmission of middle east respiratory syndrome coronavirus infections in healthcare settings response to emergence of middle east respiratory syndrome coronavirus diversity of middle east respiratory syndrome coronaviruses in 109 dromedary camels based on full-genome sequencing identification of diverse viruses in upper respiratory samples in dromedary camels from united arab emirates mers-cov in pregnancy some epidemiological studies on mers coronavirus in dromedaries in the united arab emirates -a short communication emerging and reemerging diseases in the world health organization (who) eastern mediterranean region-progress, challenges, and who initiatives melinda gates foundation opp#1181128 and s.i.h. was supported by opp1132415 curated and catalogued the database. s.s. provided managerial support. all authors participated in interpreting and summarizing the results. r.e.r. wrote the first draft of the manuscript. all other authors critically reviewed the manuscript had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis s.i.h. and d.m.p. are members of the editorial board of scientific data. supplementary information is available for this paper at https://doi.org/10.1038/s41597-019-0330-0.correspondence and requests for materials should be addressed to d.m.p.reprints and permissions information is available at www.nature.com/reprints. open access this article is licensed under a creative commons attribution 4.0 international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.the creative commons public domain dedication waiver http://creativecommons.org/publicdomain/zero/1.0/ applies to the metadata files associated with this article. key: cord-262347-ejhz9rra authors: kappes, matthew a.; faaberg, kay s. title: prrsv structure, replication and recombination: origin of phenotype and genotype diversity date: 2015-03-07 journal: virology doi: 10.1016/j.virol.2015.02.012 sha: doc_id: 262347 cord_uid: ejhz9rra porcine reproductive and respiratory disease virus (prrsv) has the intrinsic ability to adapt and evolve. after 25 years of study, this persistent pathogen has continued to frustrate efforts to eliminate infection of herds through vaccination or other elimination strategies. the purpose of this review is to summarize the research on the virion structure, replication and recombination properties of prrsv that have led to the extraordinary phenotype and genotype diversity that exists worldwide. porcine reproductive and respiratory syndrome virus (prrsv) is the etiological agent of a worldwide epidemic designated porcine reproductive and respiratory syndrome (prrs). prrsv is highly host and tissue restricted to swine cells of the monocyte lineage, preferentially infecting the porcine alveolar macrophage (amφ) and is a persistent virus (duan et al., 1997; villarreal et al., 2000) . prrsv first emerged in the late 1980s as a "mystery" disease progressing through swine populations in both europe and north america collins et al., 1992; hopper et al., 1992; morin et al., 1991; wensvoort et al., 1991) . prevailing clinical symptoms were noted to be respiratory distress in young swine and widespread reproductive failure in pregnant sows including mummified, stillborn and aborted fetuses (goyal, 1993) . initial characterization of circulating european (type 1) and north american (type 2) genotype isolates was found to be surprisingly genetically divergent. although overall disease phenotype, gross clinical symptoms, genomic organization and temporal emergence were all similar, these strains differed by $ 40% at the nucleotide level collins et al., 1992; meulenberg et al., 1994; nelsen et al., 1999; wensvoort et al., 1991) . the degree of genetic heterogeneity suggests a protracted period of independent evolution on the two continents . molecular clock analysis predicts the divergence of the two genotypes from a common ancestor between a decade to over a century prior to clinical recognition (forsberg et al., 2001; yoon et al., 2013) , presumably from another host species (hanada et al., 2005; plagemann, 2003) . the origin of prrsv remains unknown and no secondary animal, human, or arthropod vectors have been identified to date (otake et al., 2003a (otake et al., , 2003b schurrer et al., 2005 schurrer et al., , 2004 zimmerman et al., 1997) . in the $ 25 years since the first emergence of prrsv, a global near worldwide epidemic has been sustained by a set of emerging and re-emerging strains supported by high frequency mutation and recombination (goldberg et al., 2003; meng, 2012; murtaugh et al., 2010) . prrsv remains the most economically devastating disease of swine and contributes to the deterioration of animal health through disease and the continual emergence of increasingly divergent and often virulent strains (gauger et al., 2012; han et al., 2006; holtkamp, 2011; tian et al., 2007) . other reviews have recently assessed the nidovirus family in broad terms, concentrating on what is similar between the family members and choosing to concentrate on cellular pathogenesis, but often leaving critical prrsv-specific details out of their discussions. therefore, the intent of this review is to examine what is known about prrsv virion structure and replication mechanisms that contribute to the highfrequency mutation and recombination observed, resulting in a vast array of phenotypically and genotypically divergrent strains. prrsv is a member of the arteriviridae family within the order nidovirales, which also includes the coronaviridae and roniviridae families. the nidovirus order constitutes a group of singlestranded positive-sense rna viruses which share a hallmark replication/transcription strategy, similar genomic organization, and a defining set of genetic elements, but differ in host species and range, disease phenotype, virion morphology, cellular tropism, genomic size and encoded content . the arteriviridae family is composed of five viruses which share similar genetic and biological characteristics such as genomic organization and content, morphology and a cellular tropism for the macrophage lineage . viral species include prrsv, simian hemorrhagic fever virus (shfv), lactate-dehydrogenase elevating virus (ldv), newly recognized wobbly possum disease virus (wpdv) in free-ranging australian brushtail possums (trichosurus vulpecula) and equine arteritis virus (eav; arterivirus prototype species) dunowska et al., 2012; plagemann and moennig, 1992) . the similar genomic organization, characteristic genetic elements and common functionality of orthologous proteins however have led to the acceptance of many putative functions for prrsv proteins, often derived from studies of the arterivirus prototype species eav and the more distantly related nidoviruses. each arterivirus infects only one animal species, in contrast to other nidoviruses such as some coronaviruses, which have been shown to transmit between species (hilgenfeld and peiris, 2013) . the prrsv genome varies from 14.9 kb to 15.5 kb in length and expresses a range of accessory and structural proteins through two distinct transcription mechanisms. the genomic organization and associated expression profiles are depicted in fig. 1 . the prrsv genome encodes a 5 0 proximal noncoding element (5 0 -untranslated region; 5 0 utr) of 217-222 nucleotides (nt; type 1) and 188-191 nt (type 2) in length yun and lee, 2013) . the 5 0 utr functions will be discussed below. directly downstream of the 5 0 utr has the large overlapping replicase open reading frames (orf). the orf1a/b share a single translational start site but are augmented by two ribosomal frameshift (rfs) sites at genomic positions 3889 nt (rfs1; nonstructural protein (nsp) 2) and 7695 nt (rfs2; nsp8/9) [vr-2332 (u87392) reference sequence] meulenberg et al., 1993b; nelsen et al., 1999; li et al., 2015) . two products are generated from the rfs1 site; a -1 rfs occurs approximately 7% of the time and results in an immediate termination of translation (nsp2n) , and a -2 rfs event occurs at $ 20% efficiency and yields a translational extinction in the -2 coding frame through the putative transmembrane domain of nonstructural protein (nsp2) . the large replicase polyproteins pp1a, pp1a-nsp2n, pp1a-nsp2tf, and pp1ab are generated from full-length genomic rna. using the type 2 prototype strain vr-2332 (u87392) for a reference, orf1a/b is encoded by a 5 0 proximal segment of approximately 12 kb [7512 nt orf1a, 4374 nt orf1b] yielding four distinct polyproteins including pp1a-nsp2n (1234 amino acids (aa); -1 rfs at rfs1), pp1a-nsp2tf (1,403aa; -2 rfs at rfs1), pp1a (2,503aa), and pp1ab (3,960aa; -1 rfs at rfs2) (fig. 1) . the replicase polyproteins are co-translationally and posttranslationally processed into at least 16 distinct nonstructural proteins (nsp) via the rfss and four virally encoded proteinases including papain-like cysteine proteinases 1α (plp1α; nsp1α), plp1β (nsp1β) and plp2 (nsp2), and the main serine proteinase (sp; nsp4) snijder et al., 2013) . plp1α and plpβ function to cleave the nsp1α ↓ nsp1β and the nsp1β ↓ nsp2 junction, respectively; plp2 is responsible for the cleavage of the nsp2 ↓ nsp3 junction and the main sp processes all remaining nsp products (nsp3-12) (han et al., 2009; snijder et al., 2013) . orf1a encodes pp1a encompassing nsps 1-8 and orf1ab encodes pp1ab composed of all known nsps (nsp1α/β, nsp2-6, nsp7α/β, nsp8-12) whereas nsp9 is a translational extension of nsp8 via a programmed -1 rfs at position vr-2332 7695 nt (rfs2) ( fig. 1 ) (meulenberg et al., 1993b; nelsen et al., 1999) . in contrast, the previously recognized structural proteins are encoded and individually expressed by a set of subgenomic rnas (sgrna) generated through a negative-strand intermediate (sgrna2-7; fig. 1 ) (van marle et al., 1999a) . sgrnas are genetically polycistronic (except rna7) but are assumed to be functionally monocistronic/bicistronic, where only the 5 0 terminal orf(s) is expressed (fig. 1) . sgrna 2 encodes orf2a/b which is translated to yield glycoprotein 2 (gp2) and a small unglycosylated envelope protein (e); orf3 is expressed from sgrna3 to yield gp3; and sgrna4 encodes orf4 yielding gp4. together gp2, gp3, and gp4 form a trimeric complex resulting in the minor glycoprotein complex which functions in viral entry and is heavily nglycosylated (das et al., 2010; wissink et al., 2005) . sgrna5 encodes orf5 and orf5a. orf5a codes for the orf5a protein, a small unglycosylated protein that is required for virus viability and orf5 codes for gp5, the major glycoprotein with a variable number of n-glycan residues surrounding the cell attachment domain (johnson et al., 2011; mardassi et al., 1996; robinson et al., 2013) . orf6 is expressed from sgrna6, resulting in the generation of the membrane protein (m). gp5 and m form a disulfide-linked heterodimer and together constitute the major glycoprotein complex on the virion, as was first shown for ldv (faaberg et al., 1995; mardassi et al., 1996) . finally, the nucleocapsid protein (n) is encoded by orf7 and is expressed from sgrna7. n is the major structural element within the prrsv virion which forms disulfide-linked homodimers, functions to package the viral genomic rna (grna), and is the only known structural protein which does not encode a transmembrane domain or to not have an ectodomain upon the prrsv virion (bautista et al., 1996; dea et al., 2000; doan and dokland, 2003; loemba et al., 1996; spilman et al., 2009; wissink et al., 2005; wootton and yoo, 2003) . recently, the nsp2 protein, coded for by the most variable region of the genome with insertions and deletions, was also shown to be incorporated into or onto ultrapurified virions of several prrsv strains as a set of differently sized protein isomers, presumably through its four to five membrane spanning regions near the c-terminal end (kappes et al., 2015) (han et al., 2010; kappes et al., 2013) . this surprising result increases the number of viral proteins, 10 or more (full-length nsp2 and its isomers, nsp2tf, gps 2-5, e, m, n, orf5a), that are exposed to the porcine immune system on entry of prrsv into swine alveolar macrophages veit et al., 2014) . the original work suggests that only 3 of these (gp5, m, and n) make up the majority of the protein content of prrsv (drew et al., 1995; mardassi et al., 1994a mardassi et al., , 1995 meulenberg et al., 1995; nelson et al., 1994) . however, the immense genetic and protein variation of all of these structural proteins, from the least conserved nsp2 region (han et al., 2006; tian et al., 2007) to the most conserved m protein (murtaugh et al., 1995; veit et al., 2014) , shows the complexity and the plasticity of the prrsv genome and virion structure. the 5 0 and 3 0 utrs flank the core protein coding regions of the prrsv genome (fig. 1 ). both the 5 0 and 3 0 utrs are implicated as essential components contributing to the viral strategies imparting replicative and translational functionality; however, the exact functions of the 5 0 and 3 0 utrs, and the associated mechanisms of interaction, are poorly understood. both encode conserved putative rna secondary structures important to replicative function. the 5 0 utr is encoded first within the prrsv genome and possesses a putative type i 5 0 cap structure (sagripanti et al., 1986) . the 5 0 utr is genetically variable, type 1 and 2 strains share approximately 50% genetic homology, and within each genotype, the pairwise identity is about 96% (lin et al., 2002; meulenberg et al., 1993a; nelsen et al., 1999; oleksiewicz et al., 1999; tan et al., 2001) . detailed studies of the distantly related coronavirus species, as well as arteriviruses, have shown the 5 0 utrs are regulators of genomic replication, transcription, and mrna translation, and are considered a necessary docking site for a variety of viral and host factors to complete these functions (choi et al., 2006; gao et al., 2012; liao and lai, 1994; lu et al., 2011; tahara et al., 1994; zhang et al., 1994) . the 3 0 utr is located directly downstream of orf7 and is encoded by approximately 150 nt excluding the polyadenylation site. the 3 0 utr [114 nt (type 1), 148 nt (type 2)] is also genetically diverse, sharing approximately 70% nucleotide identity between type 1 and type 2 sequenced isolates but about 96% pairwise nucleotide identity within each genotype choi et al., 2006; verheije et al., 2002; yin et al., 2013) . recent reviews extend upon the brief description presented here wang et al., 2014; yun and lee, 2013) . due to the unique attributes of nidovirus transcription and replication, including uncharacteristically large polycistronic rna genomes and the transcription of a nested set of 5 0 , 3 0 co-terminal sgrnas through a discontinuous transcription strategy, which is in itself a mechanism of recombination, nidoviral rna synthesis mechanisms have been suggested to be of unparalleled complexity among positive strand rna viruses van hemert et al., 2008) . prrsv replication closely ties three key features: rearrangement of host membranes to establish viral replication complexes (rc), synthesis and expression of grna, transcription of sgrna for the efficient expression of structural proteins, at the same time as the unique ability to produce aberrant prrsv sgrnas known as heteroclites (yuan et al., 2000 (yuan et al., , 2004 . genesis of grna (replication) and sgrna is inherently tied through the shared negative strand synthesis mechanism. modulation of negative-sense transcription through a non-stochastic mechanism yields either grna or one of six standard sgrnas (rna2-7) through an abortive disjoining/rejoining discontinuous transcription strategy ( fig. 1) (meng et al., 1996b; nelsen et al., 1999; pasternak et al., 2001; van marle et al., 1999a) . the characterization of the cellular entry mechanism that prrsv utilizes has been studied in detail, and will not be covered in this review (van breedam et al., 2010) . little is known about the establishment of prrsv infection, however, from the point postentry to the development of rc, including the formation of characteristic perinuclear double-membrane vesicles (dmvs) (knoops et al., 2012) . dmvs are believed to be derived from the endoplasmic reticulum (er) which are apparent sites of viral replication (pedersen et al., 1999) . it has been shown that the eav replicase proteins (orf1a/b) are sufficient to support viral replication (molenkamp et al., 2000b) , but that infectivity is dependent on the presence of the structural genes encoded at the 3 0 -end of the arterivirus genome molenkamp et al., 2000b; verheije et al., 2002) . upon entry, the grna serves as the mrna for immediate translation of the large replicase polyproteins. within orf1a, three proteins are recognized putative transmembrane proteins (nsp2, nsp3, and nsp5). the eav nsp2 and nsp3 were shown to be sufficient to modulate host cellular membranes into structures similar to those observed during viral infection . it is believed that membrane integration and possibly protein-protein interactions of these transmembrane proteins function to torque the existing membrane structures to form the dmvs; tethering the genesis and processing of the polyprotein (s) at the site of replication. additional viral or cellular interacting partners are not well defined. the mechanism of dmv formation is unknown but may include the modulation of autophagy and/or apoptosis pathways (breckenridge et al., 2003; chen et al., 2012; costers et al., 2008; cottam et al., 2014; huo et al., 2013; labarque et al., 2003; razi et al., 2009; sun et al., 2012; wang et al., 2014; yin et al., 2012; yu et al., 1999) . the core replicative machinery of prrsvthe rna dependent rna polymerase (rdrp; nsp9), the zinc-binding domain (zbd; or z; nsp10), the rna helicase (nsp10), and the conserved nidovirus uridylate-specific endoribonuclease, (nendou or u; nsp11)is encoded within orf1b (ulferts and ziebuhr, 2011) . the calculated rfs efficiency of the rfs1 (6-7% -1 rfs; 16-20% -2 rfs) and the rfs2 ( $ 20%) (den boon et al., 1991) demonstrates that orf1b (nsp9-12) is generated approximately once out of every six translational events (15%), suggesting the stoichiometric requirements for the core replicative machinery is low compared to the 5 0 encoded replicase proteins. rdrps form a characteristic right hand configuration (thumb, palm, finger(s)) with the thumb and fingers in contact to create a pocket for substrates (ferrer-orta et al., 2006) . comparison of single-and double-stranded rna virus rdrps show structural similarity even though there is low sequence homology between classes (ferrer-orta et al., 2006) . all polymerases share a core set of conserved motifs, suggesting a common ancestor (sabanadzovic et al., 2009) . the structure of the rdrp possesses an additional conserved motif (o'reilly and kao, 1998) . the rdrp of nidoviruses is phylogenetically clustered with the picorna-like virus superfamily koonin, 1991) but possesses a sdd (ser-asp-asp) signature, located within the active site on the palm side of the rdrp. the nidoviral sdd motif is a hallmark of the viral family that discriminates it from all other positive-sense rna virus groups that contain a gdd (gly-asp-asp) motif (den boon et al., 1991) . the sdd motif at this position was shown to be critical for eav replication (van dinten et al., 1999) ; surprisingly a s-g mutation within the prrsv rdrp was replication competent (grna) but displayed deficiencies in sgrna synthesis . another salient fact is that the arteriviral rdrp does not possess the 3 0 proofreading abilities that other nidoviruses display (lauber et al., 2013) . the rate of random mutation introduction is therefore elevated (forsberg, 2005; forsberg et al., 2001 forsberg et al., , 2002 , contributing to an abnormally high evolution rate estimated at between 4.71 â 10 2 and 9.8 â 10 2 /synonymous site/year (hanada et al., 2005) . nsp10 encodes the prrsv helicase protein (bautista et al., 2002) . the prrsv multi-domain helicase (hel) is composed of the core 1a and 2a canonical domains found in super-family 1 type helicases, a flexible accessory domain (1b), and a unique zinc-binding domain (zbd) (deng et al., 2014) . the prrsv hel functions to unwind dsrna in a 5 0 to 3 0 polarity (bautista et al., 2002) . both the flexible accessory domain and the zbd are critical to replicative function of eav including generation of grna and sgrna (van dinten et al., 2000; van marle et al., 1999b) . the helicase is predicted to function in concert with the rdrp to facilitate replication and transcription; however, it is not understood how the 5 0 to 3 0 directionality of the helicase and the 3 0 to 5 0 rdrp synthesis coordinate these activities (fang and snijder, 2010) . nsp11 harbors the nidoviral uridylate-specific endoribonuclease (nendou) domain (ulferts and ziebuhr, 2011) . originally described in coronaviruses, the nendou of eav was shown to be required for genome replication, and mutation of critical residues had varying deleterious effects on replication, and particularly on sgrna synthesis (posthuma et al., 2006) . in examining the core nuclease region of eav and prrsv, single-stranded rna was the preferred substrate, as was shown for the coronavirus sudden acute respiratory virus (sars). however, no dependence on the divalent cation mn 2 þ was seen. sars nsp15, the coronavirus orthologue, was previously shown to mn 2 þ dependent. eav nendou protein was also shown cleave 3 0 of pyrimidines, preferring uridine over cytidine, and releasing products with 2 0 ,3 0 -cyclic phosphate and 5 0 -oh ends. in addition, cleavage after unpaired over paired pyrimidines was preferred (nedialkova et al., 2009; ulferts and ziebuhr, 2011) . the function of nendou in nidovirus replication has not been established. how the prrsv rdrp initiates replication, either by a primer dependent mechanism or by de novo synthesis is also unknown. to assess the activity of the eav rdrp, a recombinant version of the polymerase was generated and assessed. eav rdrp activity was found in the absence of a primer with poly(u) or poly (c) templates but not with poly(a) templates, indicating a de novo initiation method in a template specific manner . introduction of primers to either the poly(u) or poly (c) templates reduced rdrp activity . incubation with non-complementary bases (i.e. for poly (u) template¼gtp and utp) did not result in isotopic labeling, which shows the rdrp did not function as a terminal transferase, and radioactively labeled primers were not incorporated into the synthetic non-viral templates . de novo polymerase activity could not be detected on viral templates however. this finding suggests the arterivirus rdrp is catalytically active without other viral factors and capable of de novo synthesis, but may require other viral or cellular co-factors to efficiently perform replication or transcription processes. similar research on prrsv has not been accomplished to date. it is generally believed that positive-sense rna viruses use conformational switches in their terminal noncoding regions in the form of higher order rna secondary structure to regulate translation, transcription of sgrnas, and genomic replication . to define the minimal cis-acting 3 0 genomic element required for efficient prrsv replication, progressive 3 0 deletions were introduced into self-limiting prrsv replicons encoding an internal ribosome entry site (ires)-driven luciferase (luc) reporter within the deleted region (choi et al., 2006) . only the smallest deletion, encoding the full m and n proteins, replicated to similar levels as the positive control. the next smallest deletion removing the m protein coding region but maintaining the complete n orf resulted in significant loss of genome replication (choi et al., 2006) . taken together, including the rdrp, hel, 5 0 utr, 3 0 utr, orf6, orf7, and other unknown viral proteins, these data show that key genetic elements or protein interacting partners are required for efficient prrsv replication and transcription, and are interspersed within multiple coding regions of the genome. viruses require the ability to selectively regulate transcription and translation processes both temporally and quantitatively in a highly ordered and balanced process (pasternak et al., 2000) . the expression mechanisms between the nonstructural replicase proteins and the structural proteins are fundamentally separated within the prrsv genome facilitating rapid expression of nsps from the grna and subsequent amplification of sgrna transcripts through a differential transcription cascade (pasternak et al., 2004) . sgrnas are synthesized by the viral rdrp through a highly ordered process encoded within the prrsv genome. the set of nested (nido latin: nested) sgrnas encode noncontiguous grna sequence including both the 5 0 utr and the polyadenylated 3 0 utr as well as one or more orfs from the 3 0 region of the genome (orf2-7) but lack the entire large $ 12 kb replicase coding region (orf1a/b) (fig. 1) pasternak et al., 2006; van berlo et al., 1982) . it was originally hypothesized that nidoviral sgrnas could be generated through a free 5 0 utr priming stage (baker and lai, 1990 ; baric et al., 1983 ) but was ultimately shown the 189 bp 5 0 untranslated of strain vr-2332 is shown as a bar encompassing two discrete regions, the 5 0 terminal sequence of 183 bases that differs approximately 5% between strains of the same genotype (gray bar) and the 100% conserved u/guaacc hexanucleotide on the distal end that serves as the transcription regulatory sequence (trs; black bar). the prrsv replicase complex is represented by a multi-point star. (a) production of conanical sgmrna. (step 1) sgrna synthesis initiates as ( à ) strand replication (blue bar) from the full-length ( þ ) sense (green bar) genome. rdrp interaction with a trs either results with a read-through and a continuation of ( à ) strand replication or, in the case of sgrna synthesis, ( step 2) disassociation of the replicating strain (body aauugg; white body) and ( step 3) re-joining at the 5 0 leader trs (leader aauugg; leader-body junction) through sequence complementarity annealing followed by completion of ( à ) strand sgrna synthesis. all sgrnas possess identical 3 0 and 5 0 termini (see fig. 1 ). (step 4) ( à ) sense sgrnas serve as template for generation of ( þ ) sense sgrna synthesis, required for structural protein translation. (b) production of heteroclite sgrna at unconventional leader-body junction sites to express aberrant proteins. that nidoviruses utilize a discontinuous sgrna transcription strategy (sawicki and sawicki, 1995; van marle et al., 1999a) . discontinuous replication proceeds through a replicative fusion of the viral genome 5 0 utr to one of many downstream 3 0 sites through base pairing interactions between sense and antisense stem-loop (sl) structures via long-range rna-rna interactions during negative strand synthesis (van marle et al., 1999a) . specifically, an antisense transcription-regulating sequence (trs) at or near the 5 0 end of each structural protein coding region (orf2-7) can each individually form a kissing-loop interaction with a conserved trs sequence (uuaacc), located at the 3 0 terminus of the 5 0 utr. sgrnas synthesis and grna replication utilize a similar initial synthesis mechanism, whereby negative strand transcriptional extension from the 3 0 termini is completed until a trs signal sequence within the body of the genome is encountered ( fig. 2a ) (den boon et al., 1996; pasternak et al., 2004) . the body trs is ordered into a sl structure encoding a conserved heptanucleotide primary sequence (body trs signal) (den boon et al., 1996) within the loop structure. this signal halts transcription of the negative strand and a "decision" is made between transcriptional readthrough and continuation of synthesis, or a disjoining of the transcriptional machinery and rejoining to the common leader trs (antisense to body-trs) by complementary base-pairing with a second sl structure within 5 0 utr (pasternak et al., 2003; van marle et al., 1999a) . the leader trs is located within the 5 0 utr directly upstream of the aug start codon of nsp1α and is part of a highly ordered and well conserved rna secondary structural motif between the 5 0 utr and the 5 0 nsp1α coding region. transcriptional read-through of all body trs sites will result in grna synthesis. decoupling from the genomic strand at the body trs and rejoining at the leader trs results in noncontiguous transcription that lacks a large central region of the genome, yielding one of at least six sgrna products (dependent on which body trs is utilized) ( fig. 2a) (pasternak et al., 2001) . the structural integrity of the eav nsp1 region, composed of two papain-like protease domains and a predicted n-terminal zinc finger, was also indispensible for transcription, and has been shown to interact with the cellular cofactor p100 (tijms et al., 2007 (tijms et al., , 2001 tijms and snijder, 2003) . again, all research completed on the arterivirus mechanism of subgenomic synthesis was through the use of the model eav strain adapted for growth in tissue culture. similar research on prrsv, with strain differences in replication rates and its immense depth of variation, may yield novel findings in this field. while the mechanism of sgrna generation is conserved, the discontinuous transcription process has been shown to be able to utilize both canonical and non-canonical body trs sites and can have strain specific derivations (den boon et al., 1996; meng et al., 1996b; nelsen et al., 1999) . alternative canonical and noncanonical body trs sites often precede the coding region of prrsv structural proteins, which function to drive sgrna synthesis to various degrees of efficiency, yielding major and minor sgrna species encoding the same structural protein. for instance, the na prrsv prototype strain (vr-2332) utilizes at least two different leader-body junction sites for the generation of sgrna4 and sgrna7 subspecies, and produces a separate species, sgrna5-1, for the expression of a truncated gp5 utilizing a downstream aug . other nuances for individual prrsv strains have been noted (lin et al., 2002; meng et al., 1996b) . examining mutational studies within the eav orf7 body trs (nucleocapsid) to abolish the generation of sgrna7, the most abundantly produced sgrna (van marle et al., 1999a) found that elimination of sgrna7 synthesis resulted in obvious increase in sgrna6, 5, 4, and 2 but production of sgrna3 remained unchanged. additionally, it was noted that a mutation within the leader trs, altering the fifth nucleotide of the conserved sequence (5 0 -ucaag-3 0 ) eliminated synthesis of all sgrnas except sgrna3 ( van marle et al., 1999a) . this effect is due to a unique non-canonical trs semi-independent generation of sgrna3.1, produced by both eav and prrsv (meng et al., 1996b; van marle et al., 1999a) . sgrna3.1 uses a non-trs body sequence of 5 0 -ucaauaccc-3 0 which lacks the 3 0 terminal c residue of the canonical trs sequence but possesses an additional five nucleotides (uaccc) that match the adjoining sequence downstream of the leader trs, allowing for sense/antisense base pairing. (van marle et al., 1999a) . data from single and double knockout mutagenesis studies of alternative (non-canonical) trs body sequences for eav showed that the expression of solely the minor sgrna species from alterative joining sites of the gp3, gp4, and gp5 structural proteins was sufficient for production of infectious progeny virus (pasternak et al., 2000) . it is not clear if the alternative trs body sites serve as a secondary mechanism to rescue deleterious mutations from the error-prone rdrp, or if they serve a dedicated purpose within the viral life cycle. knockout of the canonical eav trs body sequence of orf3, 4, or 5 resulted in infection rates at perceived wild-type levels (defined by ifa cellto-cell spread; 1-3 log pfu/ml reduction in titer) by utilizing secondary trs sequences within these coding regions (pasternak et al., 2000) . double knockout mutants of both the canonical and accessory body trs sequences surprisingly still resulted in generation of progeny virus, but at reduced levels (orf3 ¼2 log reduction, orf4¼ 3 log reduction, orf5¼5 log reduction; pfu/ ml). it is presumed that even if the use of alternative trs sequences results in inefficient sgrna synthesis, the two amplification cycles (genomic rna-( à ) sense sgrna synthesis-(þ ) sense sgrna) may result in sufficient sgrna copy numbers to allow a productive infection cycle to proceed. the authors noted that the reduction in pfu titers of the single or double trs mutants corresponded "very well" to the reduction in molar ratios of each respective rna subspecies (pasternak et al., 2000) . prrsv eu prototype strain lelystad virus (lv) sgrnas possess a conserved six nucleotide junction sequence of ucaacc (or similar sequence), but show heterogeneity at the junction site, suggesting the joining mechanism may be "imprecise" (meulenberg et al., 1993a) . studies on whole rna rt-pcr (cdna) of virally infected cells with na prrsv strains further identified a similar common leader-body junction sequence u(g)ua(g/c)acc (meng et al., 1996b; nelsen et al., 1999; oleksiewicz et al., 1999) . genetic heterogeneity was also noted at these junction sites, differing by a single base to a couple of nucleotides, showing there is slight flexibility within the disjoining and reattachment of the viral rdrp during this step (meng et al., 1996b; nelsen et al., 1999) . furthermore, the body trs motifs and the distance upstream from the starting aug for the expression of sgrnas differ between type 1 and type 2 prrsv, except for the predominant sgrna7 transcript (meulenberg et al., 1993a; nelsen et al., 1999) . on top of this, surveying northern analyses of different prrsv strains shows that each strain has different quantities of each sgrna transcript size, and often differ in their trs motifs (gauger et al., 2012; guo et al., 2013b; wang et al., 2008) . thus, the mechanism(s) regulating prrsv sgrna synthesis when comparing different viral strains appear to be more complex than appears when examining one viral strain in depth. defective interfering (di) rnas are a normally observed byproduct of positive-sense rna virus replication, particularly under high multiplicity of infection (m.o.i.) culturing conditions (masters, 2006; molenkamp et al., 2000a; pattnaik et al., 1992; xiao et al., 2011) . di rnas are generated through nonhomologous recombination between viral genomes resulting in random internal deletions but still encode the replication elements essential for generation of defective progeny virus including the genes encoding for the polymerase, essential replicase proteins, and capsid protein(s) (yuan et al., 2000) . unlike dis that have been described in other nidoviruses, a group of "heteroclite" sgrnas (heteroclite¼deviating from common forms or rules) were identified within prrsv replicative products of unusual structure but containing large internal deletions (yuan et al., 2000) . heteroclite sgrnas species were identified within infected cells, purified virions, porcine alveolar macrophages infected with field isolates, under natural infection conditions, within plaque-purified viral infections, and are assumed to result from homologous recombination at atypical nucleotide stretches (fig. 2b) (yuan et al., 2000 (yuan et al., , 2004 . the essential replicative products such as the viral rdrp were found to be absent within the heteroclite rnas, discriminating them from prototypical di genomes. in addition, heteroclites do not appear to interfere with ongoing genomic rna and sgrna transcription (yuan et al., 2000 (yuan et al., , 2004 . sequencing analysis showed a short site of two to seven conserved nucleotides between the 5 0 and 3 0 joining regions, but these aberrant trs motifs varied in sequence (yuan et al., 2004) . when rt-pcr products of culture supernatants of strain vr-23332 using a 5 0 and 3 0 primer pair were analyzed, at least 9 bands were discriminated (s1-s9). the result of this analysis and subsequent nucleotide sequencing showed that many similar-sized heteroclites were present in each band, but each band represented several individual heteroclites with different trs motifs. the 5 0 region of the heteroclites encoded terminal orf1a proteins including one or more of the papain-like proteases and joined within the downstream coding region either in-frame or within alternative reading frames, perhaps producing aberrant proteins (yuan et al., 2000 (yuan et al., , 2004 . these rna species persist in a range of experimental culturing conditions including low m.o.i. passage and plaque purification (yuan et al., 2000) , and were found to be packaged within the prrsv virion (yuan et al., 2004) . all identified heteroclites included at least the first 476 nt of the type 2 prototype, strain vr-2332, and later studies pinpointed 35 nucleotides in nsp1 that bound to the n protein and therefore was an important element in viral packaging (baig and zakhartchouk, 2011) . additional di rna that possesses many similar genetic features has been identified, but each contains a smaller deletion (nsp2-9) and encodes all structural proteins (xiao et al., 2011) . currently there is no known function for heteroclite sgrnas, but they have been proposed as a packaging vector to study the effect of viral factors, or the effect of exogenous elements on viral replication, translation, progeny phenotype, or immune response/modulation (yuan et al., 2000) . they may also allow for recombination events to occur in the background of an ongoing prrsv coinfection of two or more dissimilar strains. in this way, new viral sequences may be allowed to coexist with and interact with nascent viruses via recombination in the background, occasionally leading to new viral species with enhanced properties. hypothetical at this juncture, this concept should be evaluated by defined recombination studies. the mechanism of prrsv recombination is ill described (fig. 3) . homologous recombination was first described for nidoviruses using mouse hepatitis virus (mhv), a coronavirus, and the authors posited that less than full-length rna intermediates might be generated during viral rna replication. these early studies led to the proposal that mhv replication proceeded in a discontinuous and nonprocessive manner, perhaps at sites of secondary and tertiary structure thus generating free rna intermediates, which could be used in rna recombination via a copy-choice mechanism (lai, 1992; lai et al., 1985; makino et al., 1986) . in the absence of selection pressure, mhv rna recombination was found to be random, but that only certain recombinants are selected over passage in tissue culture, leading to the conclusion that there were "hotspots" for recombination (banner and lai, 1991) . recombination was detected during both negative and positive strand rna transcription, and took place not only between two different viral strains but also between one replicating viral rna and transfected non-replicating mhv rna fragments. furthermore, the recombinants could be detected after viral growth in cell culture and in animals, and successful recombination occurred more frequently within a hypervariable region which was also subject to deletion (liao and lai, 1992) . these results were shown to be representative of other coronaviruses, most notably infectious bronchitis virus (ibv), where numerous reports detail the ability of the virus to recombine in the field (kottier et al., 1995; toro et al., 2012; wang et al., 1993) . in the case of ibv, a pathogen of poultry, recombination has been shown to be robust, perhaps due to housing poultry in large flocks (cavanagh and davis, 1993) . most of these early conclusions mirror what has been shown to occur in prrsv. kapur et al. (1996) provided strong statistical evidence for intragenic recombination or gene conversion in orfs 2, 3, 4, 5 and 7, but not in orf 6. the first laboratory examination of prrsv recombination was done using two different prrsv strains to infect ma-104 cells. differential primer pairs were used in rt-pcr studies to examine cloned cell culture progeny for recombination events over an 1182 nucleotide span encompassing part of orf3 to orf5 of type 2 prrsv. five clones were selected for sequence analysis, which revealed that four clones each represented a single unique crossover, and one clone appeared to be a triple crossover recombinant. rnase treatment of the cell supernatant before rt-pcr analysis proved that the recombinant rnas were protected from degradation and therefore represented packaged viral rnas. finally, the investigators showed that the recombinants could be detected for up to three passages, but eventually were overtaken by one parental strain that had increased replication kinetics in the cultured cell line. recombination frequencies of up to 10% were estimated and recombinants could also be found in animals (murtaugh et al., 2001; yuan et al., 1999) . a similar experiment was completed with type 1 prrsv, reporting frequencies of only 0.1 and 2.5% rna recombination occurring within a 621 bp fragment, but also noting that recombination events are correlated with the size of the fragment analyzed (van vugt et al., 2001) . there are many reported algorithmically detected instances of recombination occurring between prrsv field strains of the same genotype, as well as defined coinfection studies in swine (li et al., 2009; liu et al., 2011; martin-valls et al., 2014; shi et al., 2013a) . recombination hotspots have been observed to take place within the 3 0 end structural genes, as well as in nsp2, and nsp9 (li et al., 2009; liu et al., 2011) . a more thorough analysis has shown that multiple breakpoints of recombination were detected by genetic algorithm recombination detection (gard) software all along the genome of both type 1 and type 2 isolates. gard analysis of 25 type 1 genomes produced 11 statistical breakpoints. similarly, 55 type 2 genomes led to the identification of 9 breakpoints (martin-valls et al., 2014) . rare evidence exists for nonhomologous recombination between the prrsv genome and other rna segments. a survey of 3188 field viruses (18 type 1) sequenced in the gp5 region (597-618 bp) by the university of minnesota veterinary diagnostic laboratory showed that a key segment coding for gp5 hypervariable region 1 was subject to insertions and deletions (faaberg, 2007) . this region contains 219 different patterns for an 8 amino acid stretch in hypervariable region 1, inducing non-neutralizing antibodies (ostrowski et al., 2002; plagemann et al., 2002) , but was also seen to have additions (up to 6 amino acids) or deletions (1 amino acid). two particular field isolates, encoding an extra 6 amino acids in the hypervariable region (nggmrtaansnsss), were found to be identical in nucleotide sequence (ggggggau-gaggaccgcc) in that 6 amino acid stretch to prrsv orf1 sequence, as well as many other swine host transcripts and other pathogens (unpublished data). although breakpoints and recombinants are valuable tools to understand viral evolution, there is a paucity of research directed toward understanding the viral and host machinery that prrsv utilizes to successfully carry out recombination. except for the illustration that mutations in the sdd motif of nsp9 of prrsv results in viral replication without sgrna transcription, and the hel and nendou domains being critical for both genomic and sgrna replication in eav and prrsv, no detailed mechanism for arterivirus homologous and nonhomologous recombination has been put forward. the molecular evolution of prrsv has been examined by many investigators in detail and will not be addressed in this review (frossard et al., 2013; shi et al., 2013a shi et al., , 2010a shi et al., , 2010b shi et al., , 2013b stadejek et al., 2013) . the cause of such rapid evolution may be primarily due to the lack of prrsv rdrp proofreading and tremendous viral recombination, resulting in an extraordinary diverse composition of isolates with varying pathogenicity. from the emergence of prrs in the united states in 1987 (keffaber, 1989) , it was apparent that there were several circulating viruses besides the usa prototype virus, vr-2332 . the disease was first recognized as mostly a reproductive disease, causing anorexia, late term abortions, and delayed return to estrus in sows. the infection of sows also led to increased preweaning mortality in young pigs that survived. histologically, interstitial pneumonitis, lymphomononuclear encephalitis, and lymphomononuclear myocarditis in piglets and focal vasculitis in the brain of the sow were seen. in nursing, growing, and finishing pigs, mild flu-like symptoms are evident, with pronounced hyperpnea, fever, and interstitial pneumonitis . in europe, similar disease phenotypes were observed, with the additional finding that sows sometimes had blue ears, but antigenic differences were seen between european virus isolates from different countries and these were radically dissimilar from united states and canadian isolates drew et al., 1995; hopper et al., 1992; paton et al., 1991; plana et al., 1992; wensvoort et al., 1992 wensvoort et al., , 1991 . similar findings based on herd clinical symptoms as well as seroconversion were also reported in the usa and canada (dea et al., 1992; morrison et al., 1992) . the first nucleotide sequencing efforts directed at the prrsv 3 0 -end of the genome confirmed these antigenic findings, revealing approximately 10% nucleotide differences between type 2 isolates and 40% between type 1 and type 2 isolates (drew et al., 1997; kapur et al., 1996; mardassi et al., 1994b; meng et al., 1994; meulenberg et al., 1994; pesch et al., 2005) . further evolution now places the divergence within both genotypes at 415% when comparing whole genomes (han et al., 2006; van doorsselaere et al., 2012) . investigators soon found that these differences were also reflected in the degree of pathogenicity caused by different viral isolates (halbur et al., 1995 (halbur et al., , 1996 . key events in prrsv diversity were the emergence of an "atypical" or "acute" variant that appeared in iowa, usa in the mid 1990s (meng et al., 1996a; mengeling et al., 1998) , the sudden appearance in 2001 of a novel class of prrsv named mn184 in minnesota, usa (han et al., 2006) , the notable type 2 highlypathogenic prrsv (hp-prrsv) in 2006 in china and subsequently most of asia (tian et al., 2007) , and the recent demonstration of enhanced pathogenicity of the type 1 lena strain (karniychuk et al., 2010) . some prrsv isolates have been shown to be neurovirulent (rossow et al., 1999; tian et al., 2007) , and most recently the ability of particular strains to depress the swine immune response has been shown to vary (brockmeier et al., 2012; guo et al., 2013a guo et al., , 2013b wang et al., 2013) . since the beginning, instances of increased virulence have emerged episodically in different regions of the world. in each case, however, the appearance of the novel isolates is sudden and the result of a significantly divergent prrsv genome sometimes accompanied by insertions or deletions in most often nsp2 (brockmeier et al., 2012) , and often containing detectable recombination breakpoints (shi et al., 2013a) . the remarkable phenotypic and genetic diversity is most likely the result of the innate attribute of prrsv persistence, replication, and recombination to yield an extraordinarily flexible viral genome, attempting to circumvent attempts to eradicate the pathogen from swine by the host response, vaccination or other means. mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the u.s. department of agriculture. an enzyme linked immunosorbent assay (elisa) for the detection of antibodies to the porcine reproductive and respiratory syndrome (prrs) virus new insights into rna packaging in porcine reproductive and respiratory syndrome virus nidovirales, virus taxonomy: ninth report of the international committee on taxonomy of viruses an in vitro system for the leader-primed transcription of coronavirus mrnas random nature of coronavirus rna recombination in the absence of selection pressure characterization of replicative intermediate rna of mouse hepatitis virus: presence of leader rna sequences on nascent chains report on the first outbreaks of the porcine reproductive and respiratory syndrome (prrs) in france. diagnosis and viral isolation functional properties of the predicted helicase of porcine reproductive and respiratory syndrome virus structural polypeptides of the american (vr-2332) strain of porcine reproductive and respiratory syndrome virus de novo initiation of rna synthesis by the arterivirus rnadependent rna polymerase an rna pseudoknot in the 3 0 end of the arterivirus genome has a critical role in regulating viral rna synthesis characterization of swine infertility and respiratory syndrome (sirs) virus (isolate atcc vr-2332) regulation of apoptosis by endoplasmic reticulum pathways genomic sequence and virulence comparison of four type 2 porcine reproductive and respiratory syndrome virus strains sequence analysis of strains of avian infectious bronchitis coronavirus isolated during the 1960s in the u.k induction of autophagy enhances porcine reproductive and respiratory syndrome virus replication identification of 5 0 and 3 0 cis-acting elements of the porcine reproductive and respiratory syndrome virus: acquisition of novel 5 0 au-rich sequences restored replication of a 5 0 -proximal 7-nucleotide deletion mutant isolation of swine infertility and respiratory syndrome virus (isolate atcc vr-2332) in north america and experimental reproduction of the disease in gnotobiotic pigs porcine reproductive and respiratory syndrome virus modulates apoptosis during replication in alveolar macrophages coronavirus nsp6 restricts autophagosome expansion the minor envelope glycoproteins gp2a and gp4 of porcine reproductive and respiratory syndrome virus interact with the receptor cd163 prrs syndrome in quebec: isolation of a virus serologically related to lelystad virus current knowledge on the structural proteins of porcine reproductive and respiratory syndrome (prrs) virus: comparison of the north american and european isolates equine arteritis virus subgenomic mrna synthesis: analysis of leader-body junctions and replicative-form rnas equine arteritis virus is not a togavirus but belongs to the coronaviruslike superfamily structural basis for the regulatory function of a complex zincbinding domain in a replicative arterivirus helicase resembling a nonsensemediated mrna decay helicase structure of the nucleocapsid protein of porcine reproductive and respiratory syndrome virus variation in open reading frames 3, 4 and 7 among porcine reproductive and respiratory syndrome virus isolates in the uk production, characterization and reactivity of monoclonal antibodies to porcine reproductive and respiratory syndrome virus effects of origin and state of differentiation and activation of monocytes/macrophages on their susceptibility to porcine reproductive and respiratory syndrome virus (prrsv) identification of a novel nidovirus associated with a neurological disease of the australian brushtail possum (trichosurus vulpecula) arterivirus structural proteins and assembly family arteriviridae disulfide bonds between two envelope proteins of lactate dehydrogenase-elevating virus are essential for viral infectivity the prrsv replicase: exploring the multifunctionality of an intriguing set of nonstructural proteins efficient-2 frameshifting by mammalian ribosomes to synthesize an additional arterivirus protein a comparison of viral rna-dependent rna polymerases divergence time of porcine reproductive and respiratory syndrome virus subtypes a molecular clock dates the common ancestor of european-type porcine reproductive and respiratory syndrome virus at more than 10 years before the emergence of disease the genetic diversity of european type prrsv is similar to that of the north american type but is geographically skewed within europe porcine reproductive and respiratory syndrome virus: genetic diversity of recent british isolates cis-acting structural element in 5 0 utr is essential for infectivity of porcine reproductive and respiratory syndrome virus genetic and phenotypic characterization of a 2006 united states porcine reproductive and respiratory virus isolate associated with high morbidity and mortality in the field quasispecies variation of porcine reproductive and respiratory syndrome virus during natural infection nidovirales: evolving the largest rna virus genome porcine reproductive and respiratory syndrome experimental infection of united states swine with a chinese highly pathogenic strain of porcine reproductive and respiratory syndrome virus chinese and vietnamese strains of hp-prrsv cause different pathogenic outcomes in united states high health swine comparison of the pathogenicity of two us porcine reproductive and respiratory syndrome virus isolates with that of the lelystad virus comparative pathogenicity of nine us porcine reproductive and respiratory syndrome virus (prrsv) isolates in a five-week-old cesarean-derived, colostrum-deprived pig model the porcine reproductive and respiratory syndrome virus nsp2 cysteine protease domain possesses both trans-and cis-cleavage activities proteolytic products of the porcine reproductive and respiratory syndrome virus nsp2 replicase protein complete genome analysis of rflp 184 isolates of porcine reproductive and respiratory syndrome virus the origin and evolution of porcine reproductive and respiratory syndrome viruses from sars to mers: 10 years of research on highly pathogenic human coronaviruses industry study: assessment of the economic impact of porcine reproductive and respiratory syndrome virus on an outbreak of blue-eared pig disease (porcine reproductive and respiratory syndrome) in four pig herds in great britain involvement of unfolded protein response, p53 and akt in modulation of porcine reproductive and respiratory syndrome virus-mediated jnk activation novel structural protein in porcine reproductive and respiratory syndrome virus encoded by an alternative orf5 present in all arteriviruses highly divergent strains of porcine reproductive and respiratory syndrome virus incorporate multiple isoforms of nonstructural protein 2 into virions porcine reproductive and respiratory syndrome virus nonstructural protein2 (nsp2) topology and selective isoform integration in artificial membranes genetic variation in porcine reproductive and respiratory syndrome virus isolates in the midwestern united states pathogenesis and antigenic characterization of a new east european subtype 3 porcine reproductive and respiratory syndrome virus isolate reproductive failure of unknown etiology ultrastructural characterization of arterivirus replication structures: reshaping the endoplasmic reticulum to accommodate viral rna synthesis the phylogeny of rna-dependent rna polymerases of positivestrand rna viruses experimental evidence of recombination in coronavirus infectious bronchitis virus apoptosis in the lungs of pigs infected with porcine reproductive and respiratory syndrome virus and associations with the production of apoptogenic cytokines genetic recombination in rna viruses recombination between nonsegmented rna genomes of murine coronaviruses the footprint of genome architecture in the largest genome expansion in rna viruses recombination in vaccine and circulating strains of porcine reproductive and respiratory syndrome viruses proteolytic processing of the porcine reproductive and respiratory syndrome virus replicase transactivation of programmed ribosomal frameshifting by a viral protein rna recombination in a coronavirus: recombination between viral genomic rna and transfected rna fragments requirement of the 5 0 -end genomic sequence as an upstream cis-acting element for coronavirus subgenomic mrna transcription leader-body junction sequence of the viral subgenomic mrnas of porcine reproductive and respiratory syndrome virus isolated in taiwan recombination analyses between two strains of porcine reproductive and respiratory syndrome virus in vivo kinetics of humoral immune response to the major structural proteins of the porcine reproductive and respiratory syndrome virus a 5 0 -proximal stem-loop structure of 5 0 untranslated region of porcine reproductive and respiratory syndrome virus genome is key for virus replication high-frequency rna recombination of murine coronaviruses porcine reproductive and respiratory syndrome virus: morphological, biochemical and serological characteristics of quebec isolates associated with acute and chronic outbreaks of porcine reproductive and respiratory syndrome intracellular synthesis, processing, and transport of proteins encoded by orfs 5 to 7 of porcine reproductive and respiratory syndrome virus identification of major differences in the nucleocapsid protein genes of a quebec strain and european strains of porcine reproductive and respiratory syndrome virus molecular analysis of the orfs 3 to 7 of porcine reproductive and respiratory syndrome virus, quebec reference strain analysis of orf5 and full-length genome sequences of porcine reproductive and respiratory syndrome virus isolates of genotypes 1 and 2 retrieved worldwide provides evidence that recombination is a common phenomenon and may produce mosaic isolates the molecular biology of coronaviruses spread like a wildfire-the omnipresence of porcine circovirus type 2 (pcv2) and its ever-expanding association with diseases in pigs molecular cloning and nucleotide sequencing of the 3 0 -terminal genomic rna of the porcine reproductive and respiratory syndrome virus characterization of a highvirulence us isolate of porcine reproductive and respiratory syndrome virus in a continuous cell line, atcc crl11171 a nested set of six or seven subgenomic mrnas is formed in cells infected with different isolates of porcine reproductive and respiratory syndrome virus clinical consequences of exposing pregnant gilts to strains of porcine reproductive and respiratory syndrome (prrs) virus isolated from field cases of "atypical subgenomic rnas of lelystad virus contain a conserved leader-body junction sequence lelystad virus, the causative agent of porcine epidemic abortion and respiratory syndrome (pears), is related to ldv and eav lelystad virus belongs to a new virus family, comprising lactate dehydrogenase-elevating virus, equine arteritis virus, and simian hemorrhagic fever virus characterization of proteins encoded by orfs 2 to 7 of lelystad virus isolation and characterization of an arterivirus defective interfering rna genome the arterivirus replicase is the only viral protein required for genome replication and subgenomic mrna transcription porcine reproductive and respiratory syndrome in quebec serologic evidence incriminating a recently isolated virus (atcc vr-2332) as the cause of swine infertility and respiratory syndrome (sirs) comparison of the structural protein coding sequences of the vr-2332 and lelystad virus strains of the prrs virus the everexpanding diversity of porcine reproductive and respiratory syndrome virus genetic interaction between porcine reproductive and respiratory syndrome virus (prrsv) strains in cell culture and in animals biochemical characterization of arterivirus nonstructural protein 11 reveals the nidovirus-wide conservation of a replicative endoribonuclease porcine reproductive and respiratory syndrome virus comparison: divergent evolution on two continents serum immune responses to the proteins of porcine reproductive and respiratory syndrome (prrs) virus analysis of rna-dependent rna polymerase structure and function as guided by known polymerase structures and computer predictions of secondary structure determination of 5 0 -leader sequences from radically disparate strains of porcine reproductive and respiratory syndrome virus reveals the presence of highly conserved sequence motifs identification of neutralizing and nonneutralizing epitopes in the porcine reproductive and respiratory syndrome virus gp5 ectodomain survival of porcine reproductive and respiratory syndrome virus in houseflies evaluation of mosquitoes, aedes vexans, as biological vectors of porcine reproductive and respiratory syndrome virus genetic manipulation of arterivirus alternative mrna leader-body junction sites reveals tight regulation of structural protein expression regulation of relative abundance of arterivirus subgenomic mrnas nidovirus transcription: how to make sense…? sequence requirements for rna strand transfer during nidovirus discontinuous subgenomic rna synthesis the stability of the duplex between sense and antisense transcription-regulating sequences is a crucial factor in arterivirus subgenomic mrna synthesis blue ear' disease of pigs infectious defective interfering particles of vsv from transcripts of a cdna clone open reading frame 1a-encoded subunits of the arterivirus replicase induce endoplasmic reticulumderived double-membrane vesicles which carry the viral replication complex new insights into the genetic diversity of european porcine reproductive and respiratory syndrome virus (prrsv) porcine reproductive and respiratory syndrome virus: origin hypothesis lactate dehydrogenase-elevating virus, equine arteritis virus, and simian hemorrhagic fever virus: a new group of positivestrand rna viruses the primary neutralization epitope of porcine respiratory and reproductive syndrome virus strain vr-2332 is located in the middle of the gp5 ectodomain porcine epidemic abortion and respiratory syndrome (mystery swine disease). isolation in spain of the causative agent and experimental reproduction of the disease site-directed mutagenesis of the nidovirus replicative endoribonuclease nendou exerts pleiotropic effects on the arterivirus life cycle early endosomes and endosomal coatomer are required for autophagy purifying selection in porcine reproductive and respiratory syndrome virus orf5a protein influences variation in envelope glycoprotein 5 glycosylation porcine reproductive and respiratory syndrome virus infection in neonatal pigs characterised by marked neurovirulence permutation of the active site of putative rna-dependent rna polymerase in a newly identified species of plant alpha-like virus the cap structure of simian hemorrhagic fever virion rna coronaviruses use discontinuous extension for synthesis of subgenome-length negative strands retention of ingested porcine reproductive and respiratory syndrome virus in houseflies spatial dispersal of porcine reproductive and respiratory syndrome virus-contaminated flies after contact with experimentally infected pigs recombination is associated with an outbreak of novel highly pathogenic porcine reproductive and respiratory syndrome viruses in china molecular epidemiology of prrsv: a phylogenetic perspective phylogeny-based evolutionary, demographical, and geographical dissection of north american type 2 porcine reproductive and respiratory syndrome viruses the spread of type 2 porcine reproductive and respiratory syndrome virus (prrsv) in north america: a phylogeographic approach arterivirus molecular biology and pathogenesis non-structural proteins 2 and 3 interact to modify host cell membranes during the formation of the arterivirus replication complex cryo-electron tomography of porcine reproductive and respiratory syndrome virus: organization of the nucleocapsid molecular evolution of prrsv in europe: current state of play porcine reproductive and respiratory syndrome virus induces autophagy to promote virus replication coronavirus translational regulation: leader affects mrna efficiency comparison of the 5 0 leader sequences of north american isolates of reference and field strains of porcine reproductive and respiratory syndrome virus (prrsv) emergence of fatal prrsv variants: unparalleled outbreaks of atypical prrs in china and molecular dissection of the unique hallmark arterivirus subgenomic mrna synthesis and virion biogenesis depend on the multifunctional nsp1 autoprotease equine arteritis virus non-structural protein 1, an essential factor for viral subgenomic mrna synthesis, interacts with the cellular transcription co-factor p100 a zinc fingercontaining papain-like protease couples subgenomic mrna synthesis to genome translation in a positive-stranded rna virus genetic diversity and selection regulates evolution of infectious bronchitis virus nidovirus ribonucleases: structures and functions in viral replication equine arteritis virusinfected cells contain six polyadenylated virus-specific rnas porcine reproductive and respiratory syndrome virus entry into the porcine macrophage proteolytic processing of the open reading frame 1b-encoded part of arterivirus replicase is mediated by nsp4 serine protease and is essential for virus replication the predicted metal-binding region of the arterivirus helicase protein is involved in subgenomic mrna synthesis, genome replication, and virion biogenesis the in vitro rna synthesizing activity of the isolated arterivirus replication/transcription complex is dependent on a host factor arterivirus discontinuous mrna transcription is guided by base pairing between sense and antisense transcription-regulating sequences characterization of an equine arteritis virus replicase mutant defective in subgenomic mrna synthesis high frequency rna recombination in porcine reproductive and respiratory syndrome virus occurs preferentially between parental sequences with high similarity membrane proteins of arterivirus particles: structure, topology, processing and function kissing interaction between 3 0 noncoding and coding sequences is essential for porcine arterivirus rna replication acute and persistent viral life strategies and their relationship to emerging diseases comparative analysis of apoptotic changes in peripheral immune organs and lungs following experimental infection of piglets with highly pathogenic and classical porcine reproductive and respiratory syndrome virus evidence of natural recombination within the s1 gene of infectious bronchitis virus enhancing neutralizing antibody production by an interferoninducing porcine reproductive and respiratory syndrome virus strain attenuation of porcine reproductive and respiratory syndrome virus strain mn184 using chimeric construction with vaccine sequence antigenic comparison of lelystad virus and swine infertility and respiratory syndrome (sirs) virus mystery swine disease in the netherlands: the isolation of lelystad virus envelope protein requirements for the assembly of infectious virions of porcine reproductive and respiratory syndrome virus homo-oligomerization of the porcine reproductive and respiratory syndrome virus nucleocapsid protein and the role of disulfide linkages identification of new defective interfering rna species associated with porcine reproductive and respiratory syndrome virus infection activation of c-jun nh(2)-terminal kinase is required for porcine reproductive and respiratory syndrome virus-induced apoptosis but not for virus replication conserved nucleotides in the terminus of the 3 0 utr region are important for the replication and infectivity of porcine reproductive and respiratory syndrome virus complete genome sequences of porcine reproductive and respiratory syndrome viruses: perspectives on their temporal and spatial dynamics the endoplasmic reticulum stressresponsive protein grp78 protects neurons against excitotoxicity and apoptosis: suppression of oxidative stress and stabilization of calcium homeostasis heteroclite subgenomic rnas are produced in porcine reproductive and respiratory syndrome virus infection characterization of heteroclite subgenomic rnas associated with prrsv infection recombination between north american strains of porcine reproductive and respiratory syndrome virus overview: replication of porcine reproductive and respiratory syndrome virus coronavirus leader rna regulates and initiates subgenomic mrna transcription both in trans and in cis mutational analysis of the sdd sequence motif of a prrsv rna-dependent rna polymerase studies of porcine reproductive and respiratory syndrome (prrs) virus infection in avian species this work builds upon the doctoral thesis of matthew kappes, department of veterinary microbiology and preventive medicine, iowa state university, ames, ia, 2014, entitled "identification and characterization of a novel structural protein of porcine reproductive and respiratory syndrome virus, the replicase nonstructural protein 2". project 3625-32000-108-00d of the usda agricultural research service provided support for dr. faaberg. dr. kappes was supported by bi vetmedica, inc., and by the usda agricultural research service. usda is an equal opportunity provider and employer. key: cord-255011-7oqfod62 authors: erles, kerstin; brownlie, joe title: canine respiratory coronavirus: an emerging pathogen in the canine infectious respiratory disease complex date: 2008-05-22 journal: vet clin north am small anim pract doi: 10.1016/j.cvsm.2008.02.008 sha: doc_id: 255011 cord_uid: 7oqfod62 infectious respiratory disease in dogs is a constant challenge because of the involvement of several pathogens and environmental factors. canine respiratory coronavirus (crcov) is a new coronavirus of dogs, which is widespread in north america, japan, and several european countries. crcov has been associated with respiratory disease, particularly in kenneled dog populations. the virus is genetically and antigenically distinct from enteric canine coronavirus; therefore, specific tests are required for diagnosis. r espiratory disease in dogs is generally of greatest importance in establishments in which dogs are housed in groups, such as shelters, boarding kennels, and veterinary hospitals. disease outbreaks involving only one species of infectious agent are possible, as seen in distemper outbreaks in susceptible populations [1] . most commonly, however, infectious respiratory disease in dogs has a multifactorial etiology and is best described as canine infectious respiratory disease (cird) complex (also known as ''kennel cough''). viruses detected in dogs with cird include canine parainfluenza virus (cpiv) [2] , canine adenovirus (cav) type 2 [3] and canine herpesvirus [4] . canine influenza virus, which recently has been detected in some parts of the united states, is likely to become part of the disease complex because it often causes mild respiratory disease characterized by nasal discharge and persistent cough [5] . bacteria are important in cird as primary pathogens and as a cause of secondary infections. bordetella bronchiseptica is the bacterium most frequently associated with cird [6] , but mycoplasmas, particularly mycoplasma cynos, have also been linked to the disease [6, 7] . streptococcus equi subsp. zooepidemicus has been isolated from severe cases of respiratory disease that were frequently fatal [8] . vaccines have been developed for protection against several canine respiratory pathogens. combination vaccines routinely contain canine distemper virus and cav-1 or cav-2. cav vaccines confer cross-protection against type 1 (the cause of canine infectious hepatitis) and type 2 (associated with respiratory disease). cpiv is also included in several multivalent vaccines, or it is available in combination with b bronchiseptica as a ''kennel cough vaccine.'' this work was supported by battersea dogs and cats home and the guide dogs for the blind association. canine respiratory coronavirus (crcov) was first detected in 2003 in dogs housed at a uk rehoming center [9] . the center had a high turnover of dogs and was reporting problems with enzootic respiratory disease despite regular vaccination. an investigation into pathogens associated with cird in this population led to the detection of a coronavirus in tracheal and lung samples by reverse transcriptase polymerase chain reaction (rt-pcr). coronaviruses are large enveloped viruses containing a positive-sense singlestranded rna genome. the structural proteins located in the viral envelope include the spike protein (s), the membrane protein (m), and the small membrane protein (e). initial sequence analysis of crcov showed a high similarity to bovine coronavirus (bcov) and human coronavirus oc43 (96% amino acid identity with bcov in the variable spike protein). coronaviruses had been described before in dogs with gastroenteritis [10] ; however, it was shown that crcov was distinct from the previously known canine coronavirus (ccov). the virus showed only 69% nucleotide identity in the highly conserved polymerase region and only 21% amino acid sequence identity in the spike protein, indicating that crcov was a novel coronavirus of dogs. members of the family coronaviridae are separated into groups according to their genetic similarities [11] . most members of group 2 of coronaviruses contain an additional gene coding for a surface hemagglutinin-esterase protein. this gene was found to be present in crcov, confirming its place in group 2 together with its closest relative, bcov (fig. 1) . ccov, in contrast is a member of group 1, which includes feline coronavirus and porcine transmissible gastroenteritis among others. currently, the oldest samples that tested positive for crcov are canine lung samples collected in canada in 1996 [12] . one of the reasons precluding earlier discovery of crcov may be its poor growth in cell culture and the requirement for specific host cells. the close genetic relation to bcov throughout the crcov genome indicates that the virus was probably transmitted to dogs from cattle [13] . interestingly, it has recently been shown that human coronavirus oc43 also may have emerged after viral transmission from cattle to people [14] . coronaviruses are a cause of respiratory disease in many species, including human beings, poultry, and cattle [15] [16] [17] [18] [19] [20] . the presence of crcov in dogs was first described in a large study of dogs with cird [9] . in this investigation, performed at a shelter, clinical signs were graded by veterinary clinicians into (1) no signs of respiratory disease; (2) mild cough; (3) mild cough and nasal discharge; (4) cough, nasal discharge, and inappetence; and (5) severe respiratory disease with evidence of bronchopneumonia. because of a small number of samples in grade 4, grades 3 and 4 were merged and referred to as ''moderate respiratory disease.'' crcov was most frequently detected in the trachea of dogs with mild clinical signs (grade 2). it was less frequently recovered from dogs with moderate or severe clinical signs or from dogs without clinical signs at the time of sampling. crcov was also detected in the lung, albeit less frequently. table 1 summarizes the detection of crcov in clinical samples from dogs. after 3 weeks of stay at a shelter, almost 100% of dogs tested positive for antibodies to crcov compared with 30% on the day of entry, indicating that the virus was highly prevalent in the population and was easily transmitted. it was also found that the presence of antibodies to crcov on the day of entry led to a significantly reduced risk for contracting cird, supporting the hypothesis that crcov played a role in the etiology of the disease [9] . after this initial investigation, crcov was also detected in two uk training kennels for working dogs [21] . serum samples had been collected during two outbreaks of respiratory disease at one of the kennels and 4 weeks after. almost all dogs housed at the kennel showed seroconversion to crcov after the outbreaks. moreover, crcov was detected by pcr in two oropharyngeal swabs taken from dogs with clinical respiratory disease. not all dogs that developed antibodies to crcov also showed signs of cird; nevertheless, this was the second study associating crcov with respiratory disease in dogs. two further studies identified crcov in nasal and oral swabs from dogs in japan that had respiratory disease [22, 23] . an analysis of 126 archival tissue blocks of cases of respiratory disease identified two crcov-positive samples by immunohistochemistry. both were from dogs with bronchitis and bronchiolitis, and crcov antigen was found to be present in respiratory columnar epithelial cells. one of those dogs was also positive for canine distemper virus [12] . the detection rate of crcov in the archival study may seem quite low; however, the tissue samples were mostly derived from dogs with severe respiratory disease with a fatal outcome, whereas crcov has mostly been associated with mild respiratory disease so far. serologic studies to determine the prevalence of antibodies to crcov have been performed to date for the united kingdom, republic of ireland, italy, united states, canada, and japan. the highest seroprevalence was detected in canada (59.1% of sera tested were found to be positive) and the united states (54.7% of sera tested were found to be positive). samples from the united states had been collected from 33 states, and positive samples were identified in 29 [24] . in those states that allowed a meaningful interpretation of seroprevalence (more than 10 samples), the prevalence ranged from 31.3% (maine) to 87.5% (kentucky). the prevalence in the united kingdom and the republic of ireland was lower, with 36% and 30.3%, respectively [24] . two studies performed in italy showed seroprevalence ranging from 20% to 32.5% [25, 26] . the lowest seroprevalence was detected in japan, with 17.8% [23] . further data are not yet available, but it is likely that crcov is present throughout the united states and in other european countries. although crcov was detected throughout the year in a kennel with enzootic cird, another study reported a seasonal occurrence of the virus in the winter months. no seroconversions to crcov and few cases of respiratory disease were recorded in the summer months [21] . a similar seasonality has been reported for human coronaviruses involved in the common cold [27] . crcov infections can occur in dogs of all ages. dogs younger than 1 year of age were significantly more likely to be seronegative than older dogs, however [24, 25] . this is in contrast to the prevalence of enteric ccov, which is frequently found in dogs younger than 1 year of age [10] . this may reflect different patterns of transmission of the two viruses. the seroprevalence of crcov was increasing after the age of 1 year for all studies and then reached a plateau between the ages of 2 and approximately 8 years. this is probably a consequence of the greater probability of exposure to the virus with increasing contact with other dogs. it is not certain how long crcov antibody levels in dogs remain stable after infection. one study showed a twofold decrease in antibody titers in 6 of 14 dogs tested and a fourfold decrease in 4 dogs in less than 1 year [21] . because these were naturally occurring infections, it is not known if the antibody response measured reflected primary or repeated infections. the viral dose encountered by dogs would also influence the level and duration of the antibody response. the rapid spread of crcov through kenneled populations indicates that the virus is highly contagious. this, in conjunction with the predominant detection of crcov in respiratory samples, suggests that it is mostly spread by means of respiratory secretions. crcov probably enters the respiratory tract by inhalation of droplets or contact with secretions and contaminated surfaces. it is not possible to discuss the pathogenesis and clinical signs associated with crcov without considering the cird complex as a whole. crcov has been detected in several studies in dogs with respiratory disease. in most of these cases, however, other respiratory pathogens were also present. in two detailed studies into the causes of cird in which evidence of crcov was reported, the dogs presented with the typical signs of a dry cough and nasal discharge [21, 28] . concurrent infections were most frequently caused by cpiv and b bronchiseptica. crcov has also been detected in dogs that have nonrespiratory disease. it was detected in the lung, spleen, mesenteric lymph nodes, and intestines of a dog that had died from hemorrhagic gastroenteritis [26] . the dog also tested positive for canine parvovirus type 2 and ccov. similarly, crcov was detected in a rectal swab from a dog with vomiting and diarrhea, which was also positive for ccov and cpiv [22] . in both cases, the concurrent infections with canine parvovirus or ccov are likely to have been the cause of the clinical signs. studies of the tissue distribution of crcov in 10 naturally infected dogs showed that crcov was most frequently detected in the nasal cavity, nasal tonsil, and trachea and less frequently in the lung, bronchial lymph nodes, and palatine tonsil. it was also detected in samples from the spleen, mesenteric lymph nodes, and colon but not in the enteric content (k. erles, unpublished data, 2004) . the tissue tropism of crcov therefore seems not to be exclusively respiratory, and fecal-oral transmission of crcov may be possible. crcov may show a dual tropism, similar to bcov, but the ability of the virus to replicate in the epithelium of the gastrointestinal tract and the clinical consequences need further investigation. experimental studies using crcov have not been reported to date. a study using bcov showed that dogs became infected and transmitted the virus to contact dogs. bcov was detected in rectal and oral swabs, and the dogs developed neutralizing antibodies to the virus [29] . the dogs did not develop fever or any clinical signs of respiratory or gastrointestinal disease. despite their high similarity, bcov may be less pathogenic in dogs compared with crcov. furthermore, the etiology of cird has been shown to involve multiple pathogens. viral infections can aid the entry of other pathogens by facilitating their attachment or by inhibition of the mucociliary clearance. many pathogens known to be involved in the cird complex have also been found in dogs without clinical signs, including cpiv and b bronchiseptica [28, 30] . when assessing the pathogenesis of complex diseases, it is important to consider the possible interaction of pathogens during coinfections and contributing factors, such as stress. because of the involvement of multiple pathogens in the etiology of cird, it is not possible to diagnose crcov solely by clinical signs. the most suitable test to diagnose crcov in respiratory samples is nested rt-pcr based on the spike glycoprotein gene [9] or the hemagglutinin-esterase gene [22] . this test has a high sensitivity, which is particularly useful when analyzing samples with a potentially low number of cells, such as oropharyngeal or nasal swabs. both pcr methods are specific for crcov and do not detect enteric ccov. because the virus was found most frequently in the nasal cavity, nasal swabs are suitable diagnostic samples. crcov has also been detected in oral swabs, and, furthermore, nasal or tracheal washes are likely to yield crcov during an active infection. if postmortem samples are available, nasal cavity, nasal tonsil, trachea, and lung samples should be collected for analysis. isolation of crcov in cell culture has been achieved; however, it is not recommended to use virus isolation alone to diagnose crcov. to date, isolation of crcov has only succeeded on the human rectal tumor cell line hrt-18 and its clone hrt-18g [13] . even on hrt-18 cells, the isolation of crcov from rt-pcr-positive samples is often unsuccessful [22, 23, 26] . the only isolate of crcov that has so far been studied in detail did not produce a cytopathic effect on hrt-18 cells, and infection had to be confirmed by using immunofluorescence or pcr. supernatants from infected cell cultures were found to agglutinate chicken erythrocytes at 4 c [13] . hemagglutination assays may aid in detection of crcov-infected cell cultures if isolation is attempted. crcov has also been detected by immunohistochemistry on formalin-fixed tissues using an antibody directed against bcov [12] . the sensitivity of immunohistochemistry in comparison to pcr has not been evaluated; however, this method is useful for testing archival respiratory samples. serology is a valuable tool for the detection of crcov infections if paired serum samples are collected during an outbreak of respiratory disease and at least 2 to 3 weeks afterward. the high similarity of crcov and bcov allows the use of bcov antigens to test canine sera by elisa [9, 26] . similarly, bcov has been used instead of crcov in serum neutralization tests [23] . a hemagglutination inhibition test based on bcov has also been evaluated but was assessed as having poor sensitivity and specificity compared with an elisa based on bcov [26] . an elisa assay using crcov antigen was found to have slightly higher sensitivity and specificity compared with an assay based on bcov; however, overall, the agreement between the two elisa tests was high [24] . antibodies to crcov have also been detected by using an immunofluorescence assay on crcov-infected hrt-18 cells [24] . specific tests for crcov are becoming increasingly available; however, most assays offered for the detection of coronaviruses in dogs are specific for enteric ccov. antibodies to crcov do not cross-react with enteric ccov. it is important to use an assay capable of detecting antibodies to crcov or related group 2 coronaviruses, such as bcov. consequently, the requirement for crcov detection should be discussed with the diagnostic laboratory before submitting samples for rt-pcr, virus isolation, or serology. there is no specific treatment for infections caused by crcov. as for other causes of cird, patient care should focus on the prevention and treatment of bacterial infections. although many pathogens involved in cird are reported to be associated with mild clinical disease, it is important to bear in mind that mixed infections can potentially be much more severe. patients should be monitored, because the condition may rapidly worsen. severe cases of cird with sudden death have been reported after infections with streptococcus equi subsp. zooepidemicus [8] . to date, no vaccines against crcov are available. vaccines against ccov are unlikely to protect against infection with crcov because of a low similarity in the spike proteins that are the major immunogenic proteins of coronaviruses. vaccines against other respiratory pathogens may not prevent cird, particularly in large populations because of the presence of other infectious agents. nevertheless, they have the potential to reduce the number of circulating pathogens if given to all dogs on entry. vaccines against canine distemper virus and cav are widely used, and this may account for the inability to identify either virus in a population with enzootic cird [28] . although no specific tests have been performed to determine the stability of crcov in the environment, other coronaviruses have been reported to remain infectious in respiratory secretions for more than 7 days [31] . thorough cleaning and disinfection of kennels after outbreaks of respiratory disease are therefore required. coronaviruses are inactivated by disinfectants commonly used for surface disinfection in kennels and veterinary practices. the role of fecal shedding and the potential transmission of crcov among dogs sharing common facilities, such as outdoor runs, have yet to be resolved. other generally recommended measures, such as washing one's hands after handling animals with respiratory disease should also help to reduce the spread of the virus. crcov has been detected in dogs up to 4 weeks after entry into a kennel. because the time of infection in those naturally occurring cases is not known, it is unclear how long crcov is being shed. after experimental infection of dogs with bcov, the virus was detected in a rectal swab after 11 days [29] . quarantine of newly arriving dogs, if feasible in training kennels or shelters, should therefore last for at least 2 weeks. group 1 ccovs, referred to in this article as ccovs, have previously been associated with mild gastroenteritis. according to their similarity to feline coronaviruses, they are divided into type i (related to feline coronavirus type i) or type ii (related to feline coronavirus type ii) [32] . although ccov has been isolated from the lung after experimental infections [33] , it was generally considered to be restricted to the gastrointestinal tract during naturally occurring infection. recently, an outbreak of a systemic fatal disease was described from which a type ii ccov was isolated [34] . although the dogs presented with vomiting, diarrhea, and neurologic signs, postmortem examination also revealed bronchopneumonia. ccov was detected in internal organs, including the lung, kidney, and brain. sequence analysis of the ccov isolate identified a mutation in open reading frame 3b, leading to a truncated nonstructural protein. it is not clear if this mutation is responsible for the extended tropism of this isolate [35] . further studies are required to determine the presence of type ii ccovs in cases of severe systemic disease and in cases of respiratory disease. crcov is a novel coronavirus of dogs distinct from ccov. it is present in north america, europe, and japan. crcov is frequently detected in dogs with clinical respiratory signs and may contribute to the cird complex. increased awareness of the existence of crcov and the development of routinely available diagnostic tests should enhance our knowledge of the presence of crcov in canine populations with and without respiratory disease. it is recommended to use pcr methods or serology on paired serum samples to diagnose crcov infections, because the sensitivity of virus isolation is low. identification of causative agents during outbreaks of respiratory disease in canine populations ought to be performed more frequently. this would help to determine the importance of individual viruses and bacteria, not only in the investigated population but in the cird complex as a whole. the etiology of cird is multifactorial and is likely to change continuously, because some pathogens are controlled by vaccination, although other infectious agents emerge to take their place. canine distemper: re-emergence of an old enemy sv-5-like parainfluenza virus in dogs association of canine adenovirus (toronto a 26/ 61) with an outbreak of laryngotracheitis (''kennel cough''): a preliminary report canine tracheobronchitis: isolation and characterization of the agent with experimental reproduction of the disease transmission of equine influenza virus to dogs bordetella and mycoplasma respiratory infections in dogs and cats mycoplasmas associated with canine infectious respiratory disease the association of streptococcus equi subsp. zooepidemicus with canine infectious respiratory disease detection of a group 2 coronavirus in dogs with canine infectious respiratory disease studies on the epizootiology of canine coronavirus coronavirus pathogenesis and the emerging pathogen severe acute respiratory syndrome coronavirus detection of coronavirus in cases of tracheobronchitis in dogs: a retrospective study from 1971 to 2003 isolation and sequence analysis of canine respiratory coronavirus complete genomic sequence of human coronavirus oc43: molecular clock analysis suggests a relatively recent zoonotic coronavirus transmission event coronavirus as a possible cause of severe acute respiratory syndrome viruses and bacteria in the etiology of the common cold identification of a new human coronavirus characterization and complete genome sequence of a novel coronavirus, coronavirus hku1, from patients with pneumonia coronavirus avian infectious bronchitis virus coronavirus and pasteurella infections in bovine shipping fever pneumonia and evans' criteria for causation investigation into the causes of canine infectious respiratory disease: antibody responses to canine respiratory coronavirus and canine herpesvirus in two kennelled dog populations survey of dogs in japan for group 2 canine coronavirus infection the prevalence of a group 2 coronavirus in dogs in japan serological prevalence of canine respiratory coronavirus serological prevalence of canine respiratory coronavirus in southern italy and epidemiological relationship with canine enteric coronavirus serological and molecular evidence that canine respiratory coronavirus is circulating in italy epidemiology of coronavirus respiratory infections longitudinal study of viruses associated with canine infectious respiratory disease the infectivity and pathogenicity of a group 2 bovine coronavirus in pups respiratory disease in kennelled dogs: serological responses to bordetella bronchiseptica lipopolysaccharide do not correlate with bacterial isolation or clinical respiratory symptoms survival of severe acute respiratory syndrome coronavirus genetic diversity of a canine coronavirus detected in pups with diarrhoea in italy canine coronavirus infection in the dog following oronasal inoculation canine coronavirus highly pathogenic for dogs molecular characterisation of the virulent canine coronavirus cb/05 strain key: cord-017897-mbwm0ytg authors: chiumello, davide; marino, antonella; cammaroto, antonio title: the acute respiratory distress syndrome: diagnosis and management date: 2018-10-01 journal: practical trends in anesthesia and intensive care 2018 doi: 10.1007/978-3-319-94189-9_11 sha: doc_id: 17897 cord_uid: mbwm0ytg acute respiratory distress syndrome (ards) is characterized by a new acute onset of hypoxemia secondary to a pulmonary edema of non-cardiogenic origin, bilateral lung opacities and reduction in respiratory system compliance after an insult direct or indirect to lungs. its first description was in 1970s, and then several shared definitions tried to describe this clinical entity; the last one, known as berlin definition, brought an improvement in predictive ability for mortality. in the present chapter, the diagnostic workup of the syndrome will be presented with particular attention to microbiological investigations which represent a milestone in the diagnostic process and to imaging techniques such as ct scan and lung ultrasound. despite the treatment is mainly based on supportive strategies, attention should be applied to assure adequate respiratory gas exchange while minimizing the risk of ventilator-induced lung injury (vili) onset. therefore will be described several therapeutic approaches to ards, including noninvasive mechanical ventilation (nimv), high-flow nasal cannulas (hfnc) and invasive ventilation with particular emphasis to risks and benefits of mechanical ventilation, peep optimization and lung protective ventilation strategies. rescue techniques, such as permissive hypercapnia, prone positioning, neuromuscular blockade, inhaled vasodilators, corticosteroids, recruitment maneuvers and extracorporeal life support, will also be reviewed. finally, the chapter will deal with the mechanical ventilation weaning process with particular emphasis on extrapulmonary factors such as neurologic, diaphragmatic or cardiovascular alterations which can lead to weaning failure. the first of the acute respiratory distress syndrome (ards) description was in 1821 by laennec. since that many and more accurate definitions followed. nowadays almost 5% of hospitalized and mechanically ventilated patients present ards diagnostic criteria [1] . ards can be generally defined as a new acute onset of hypoxemia and bilateral opacities after an insult direct or indirect to the lungs [2] [3] [4] . in 1994 there was the first shared definition, and then, in 2001, an update known as "berlin definition" was made by an expert panel of the european society of intensive care medicine [4] . according to this new definition, ards is an acute form of diffuse lung injury that happens in patients with predisposing factors, with: -symptoms onset within 1 week of a known clinical insult or new or worsening respiratory symptoms -bilateral opacities not fully explained by effusions, lobar/lung collapse, or nodules -respiratory failure not fully explained by cardiac failure or fluid overload -hypoxia, classified by pao 2 /fio 2 ratio measured with at least peep of 5 cmh 2 o into: mild (200 mmhg ≤ pao 2 /fio 2 ≤ 300 mmhg), moderate (100 mmhg ≤ pao 2 / fio 2 ≤ 200 mmhg), severe (pao 2 /fio 2 ≤ 100 mmhg) [2] [3] [4] . this new definition brings a small, but very important, improvement in predictive ability for mortality (area under the curve [auc] 0.577) [5] . evaluate lung morphology, which in ards is characterized by consolidated regions (homogeneous areas with increased density without identifiable vessels or bronchi), ground glass regions (areas with increased density but with still visible vessels), and normally aerated regions. since lungs are characterized by diffuse edema, with the superimposed pressure causing atelectasis and collapse of dependent lung zones, consolidated areas are typically located in dependent lung regions [18] . lung ct scan is also helpful in lung potential of recruitment evaluation, i.e., the proportion of consolidated lung that regain aeration after an increase in alveolar pressures. in ards patients, potential of recruitment could range between 0% and 70%. moreover, lung ct scan could help clinician in identifying ards etiology; in fact, in pulmonary ards consolidated and ground glass areas are similar, while in extrapulmonary ards ground glass areas are predominant [19] . next to ct scan, ultrasound of lung parenchyma, pleura, and air may be helpful in diagnostic evaluation, clinical management, and monitoring of ards patients [20] [21] [22] [23] . in respiratory failure patients, lung ultrasound is characterized mainly by b-line (hyperechogenic vertical artifact line that starts from pleura) [21] , while the b-pattern composed of three or more b-lines appears to be correlated with an interstitial pathological process [24] . a bilateral homogeneous b-pattern is not decisive between ards and cardiogenic edema and deserves further analysis [25] , while bilateral, nothomogeneous b-pattern plus c-pattern composed of consolidated areas and pleura abnormalities are suggestive of ards etiology [26] . figure 11 .2 shows possible lung ultrasound patterns. the acute respiratory failure management includes early recognition of the triggering cause and timely targeted treatment. besides that, supportive treatments must be started to assure adequate respiratory gas exchange while minimizing the risk of ventilator-induced lung injury (vili) onset. actual knowledge suggest that in most severe ards patients, spontaneous respiratory triggering could be dangerous; thus the spontaneous breathing approach should be used only in mild and moderate ards patients. different therapeutic targets should be met using different pharmacological and non-pharmacological approaches and different mechanical ventilation modalities. noninvasive mechanical ventilation (nimv) is able to reduce patient's work of breathing and intrapulmonary shunt, improving gas exchange, avoiding patient's deep sedation, and reducing the ventilator-associated pneumonia risk. however, nimv use is widely debated due to the high risk of failure (i.e. an intubation rate between 30% and 86% and a mortality rate between 15% and 71%) [27] , and the consequent risk of delaying intubation and mechanical ventilation in patients who fail this kind of support. high-flow nasal cannula (hfnc) represents an additional noninvasive ventilatory support that ensures patient's administration of a heated and humidified high flow of oxygen through the patient's nose and has shown to be able to reduce respiratory work while improving oxygenation and co 2 elimination, providing the patient with a positive end-expiratory pressure (peep) that varies between 4 and 6 cmh 2 o. a recent study carried out on patients diagnosed with ards, as in the case of niv [28] , showed however a high rate of hfnc failure, equal to 40% [29] . invasive mechanical ventilation is a supportive therapy able to guarantee adequate gas exchange (increase pao 2 and clear co 2 adequately) and reduce the respiratory muscle activity [30] . mechanical ventilation presents a double effect on patient oxygenation: it allows a continuous and precise fio 2 titration and during the inspiratory phase applies a positive airway pressure that reopens collapsed alveolar units. this second effect is likely to be limited in time, unless an adequate positive endexpiratory pressure is applied during the expiratory phase to avoid the alveolar recollapse [31] . the ventilatory setting in ards patient remains a daily challenge, and the choice should be adapted to each patient considering his/her hemodynamic parameters, respiratory mechanics, and gas exchange. during the last 30 years, literature has already widely demonstrated that high-volume/high-frequency ventilation can damage the lungs [32] mainly through the cycling collapse-reopening and alveolar overdistention phenomena that contribute to the so-called atelect-trauma [33] . the application of high peep allows collapsed alveoli reopening and intrapulmonary shunt reduction, moreover it reduces the repetitive alveolar opening and closing which occurs during the respiratory cycle [34] . however, it's not always useful to set high peep levels, as it could appear at first; indeed, two randomized and controlled trials comparing ards patients treated with low vs. high peep [35, 36] have not shown any benefit from the use of the high peep strategy. these contradictory results can be explained by the pulmonary recruitment concept, i.e., the volume of collapsed pulmonary parenchyma in which is possible to re-establish a normal aeration by an increase in the airway pressure [37] . to recruit collapsed lung regions and keep them open, it is necessary to apply an airway pressure higher enough to counterbalance the superimposed pressure, i.e., the pressure generated by the weight of the lung and the rib cage that acts on the lung below [38] . several maneuvers can be used to recruit the lung: the sigh (i.e., one high-volume breath intermittently provided by the ventilator), the extended sigh (i.e., a progressive increase in peep or a progressive increase in both peep and plateau pressure), and the sustained inflation (i.e., a static sustained increase in the airway pressure [35] [36] [37] [38] [39] [40] protracted for 20-40 seconds) [39] . the target of these maneuvers is to increase the transpulmonary pressure for a period of time sufficiently long to reinflate the alveolar units previously closed. while these maneuvers are able to improve the oxygenation for a variable period of time, their systematic use did not result in a mortality reduction [40] . while the lung ct scan is the gold standard for the potential of recruitment evaluation, the lung ultrasound seems to be a promising alternative available at the patient's bedside with several advantages such as safety and repeatability; however further studies are necessary to confirm this data [41] . as known, the choice of a too low end-expiratory positive pressure could cause the collapse of otherwise recruited parenchymal areas, while the choice of a too high end-expiratory positive pressure could increase dead space and tissue stretch thus raising the risk of lung damage. the peep optimization is therefore crucial in the individual patient to avoid the continuous opening and closing and the overdistention phenomena in some parenchymal areas. different approaches have been proposed to choose the best peep, but the most commonly used is the one based on the peep/fio 2 table, which use the patient's saturation/oxygenation as target [36] . another method is based on the respiratory mechanics: peep is progressively increased while keeping the tidal volume constant and the airway pressure within a safety range (26-28 cmh 2 o) [42] . conversely, our group uses the esophageal pressure variation during the breath, to evaluate the transpulmonary pressure. it is measured as: (plateau pressure − total peep) − (esophageal pressure at plateau − esophageal pressure at zeep the main determinants of the ventilator-induced lung injury are strain (defined as the lung deformation induced by the application of the tidal volume) and stress (i.e., the transpulmonary pressure determined by the strain) [44] . therefore, to maintain low stress and strain, it is necessary to apply a low tidal volume or have a high residual functional capacity [42, 45] . a recent meta-analysis has shown how the use of "protective ventilation," with a tidal volume of 6 ml/kg (calculated on kg of ideal body weight), guarantees a reduction in mortality [46] . since the actual body weight isn't an accurate index of lung size, it is recommended the use of the ideal weight (calculated based on gender and height) to calculate the best tidal volume; however, the ideal weight is not correlated with the functional residual capacity of the lung, highlighting that the same tidal volume can generate very different stress and strain values [47] in people with the same gender and height but different functional residual capacity. amato, in a recent study performed over a group of 3500 patients with ards ventilated with different combinations of peep and tidal volume, showed that the variable most closely associated with the outcome of patients is represented by the driving pressure of the airways, calculated as (plateau pressure − total peep). furthermore in that study was demonstrated that high levels of peep appeared protective only when associated with reduced driving pressure, with a pressure cutoff of 15 cmh 2 o [48] . however, the use of driving pressure has several limitations: the main one is the fact that the pressure that extends the lung is the transpulmonary pressure and not the airway pressure. current recommendations encourage the use, in mechanically ventilated patients, of a conservative oxygen strategy with an o 2 arterial saturation target ranging from 88% to 95%. the associated use of a "protective ventilation," with the aim of reducing the damage induced by ventilation, can however cause the development of hypercapnia; however a paco 2 around 70 mmhg and a ph of about 7.20 have been proved to be safe [49, 50] , except in special cases such as patients with intracranial hypertension or severe heart failure. the rationale for this permissive strategy lies in the known effect that hypercapnic acidosis exerts on arterial and tissue oxygenation [51] . in order to guarantee a better patient adaptation to the ventilator, to reduce the oxygen consumption related to the respiratory muscle activity and to guarantee a protective transpulmonary pressure, the use of neuromuscular blockers is accepted in clinical practice [49] . moreover, neuromuscular blockers have the ability to reduce stress and strain applied to the parenchyma. neto demonstrated that, in patients with severe ards, a short course of treatment with neuromuscular blockers was associated with a mortality decrease [52] . the indications for the prone positioning have changed over time: once it was used to improve arterial oxygenation in the most severe forms of respiratory failure [53, 54] ; while nowadays it aims to achieve a more homogeneous distribution of stress and strain within the lung parenchyma, acting in synergy with the remaining therapies and protecting against the ventilator induced lung injury [55] . the prone positioning improves ventilation/perfusion coupling thus improving the co 2 elimination and improves the ventilation distribution across the dorsal regions of the pulmonary parenchyma [55, 56] . the association of prone positioning and the use of neuromuscular blockers, in patients with severe ards, seems to have a synergistic effect on oxygenation and overall duration of mechanical ventilation and seems to be associated with a better outcome. however, these data needs further studies to confirm. in any case, the prone positioning presents few absolute contraindications, namely, pregnancy, open abdominal treatment, unstable fractures, and hemodynamic instability [55] . as shown above, the central role in the pathogenesis of ards is played by the inflammatory response that develops in the lung. several trials have been performed over time to evaluate the use of corticosteroids in the treatment of respiratory distress syndrome, but the results appeared controversial [57, 58] . meduri in his study carried out in the early phases of the ards, showed that the use of a decremental infusion scheme of corticosteroids leads to a mortality reduction in intensive care [57] . however, in other studies, this result has not been confirmed [58, 59] . although nitric oxide (no) has a known vasodilatory effect on the pulmonary vessels thus ensuring an improvement of the ventilation/perfusion coupling, its use in patients with ards is not universally accepted [60] . in fact, it has not been clearly demonstrated its benefit in terms of mortality, while its use is burdened by possible serious complications, such as renal failure [61] . the use of extracorporeal membrane oxygenation (ecmo) in the treatment of severe respiratory failure was born around the 70s with the aim of properly oxygenating the patient ensuring a protective ventilation, reducing the chances of lung damage. several observational studies have demonstrated various ecmo's benefits in patients with respiratory failure. however, the cesar study, a recent randomized trial, showed an increase in survival at 6 months (63% vs. 47%) but no difference in quality of life and spirometric parameters between patients undergoing conventional mechanical ventilation and extracorporeal support in reference ecmo centers [62] . therefore, considering the non-univocal interpretation of the data coming from this trial, nowadays it is not possible to conclude for a superiority of ecmo support compared with the association of the supportive therapy listed above [63] . it is of crucial importance the choice of the right moment to start the weaning from mechanical ventilation and to extubate the patient: any delay in extubation increases the risk to develop ventilator-associated pneumonia [64] ; while a premature extubation can lead to a prolonged stay in icu [65] and/or to a new need of invasive respiratory support. the weaning from mechanical ventilation is considered difficult in the 20-30% of mechanical-ventilated patients: the failure of the weaning process is defined as the inability to overcome a spontaneous breathing test or as the need for re-intubation within the first 48 hours from the endotracheal tube removal [66] . the causes of weaning failure are complex and determined by different factors; the main ones are listed below. in the patient with a difficult weaning, an increase in the airways resistance should be considered. moreover, a secondary tracheal obstruction caused by tracheal stenosis, tracheomalacia or the development of granulation tissue, can contribute to a complicated weaning from mechanical ventilation [67] . in ards patients an increase in airway resistances is typically due to bronchial walls edema of the small airways. the delirium seems to be the more frequent neurological alteration associated with a difficult weaning, with a four-time extubation failure rate than a patient without neurological complications [68] . delirium diagnosis is simple thanks to the use of validates scales, such as cam-icu. psychiatrists and psychologists could be helpful in other cognitive disturbances diagnosis different from delirium. a well-known risk factor for delirium is represented by sedation, in particular when midazolam is used [69] . the implementation of a daily sedative wash-out protocol, possibly together with a spontaneous breathing trial, can be associated to a reduction in ventilatory support length [70] . the depression development, common in patients staying in icu for long periods, seems to be associated to an increased risk of weaning failure [71] . antidepressant drugs seem to foster weaning from mechanical ventilation, even if only few data are available at the moment [72] . in the patient affected by an alteration of the myocardial contractility, the shift from mechanical ventilation to spontaneous breathing causes an increase in the cardiovascular work, mainly due to two factors: an intrathoracic pressure variation that causes changes in preload and afterload and an increase of the oxygen consumption by respiratory muscles [73] . an accurate cardiovascular evaluation in mechanically ventilated patients makes the introduction or the optimization of the appropriate therapy possible: this allows a reduction of the weaning failure risk. the beginning of weaning causes an increase in respiratory muscle workload that frequently appears to be already weakened. in assessing the cause of muscle weakness, it is important to bear in mind that the respiratory muscle dysfunction can result from a damage located anywhere on the axis from the afferent chemoreceptors, to the respiratory center, to the single muscle fiber [73] . the cause of the failure is frequently represented by a diaphragm alteration that can be secondary to two conditions that often coexist in the same patient: the critical illness polyneuropathy (cip) involving the phrenic nerve and, more often, the critical illness myopathy (cim). several works have demonstrated that in mechanicalventilated patients, there is often an alteration of the respiratory muscle contractility [74] . before implementing weaning-from-mechanical-ventilation protocols, it is necessary to carefully assess the diaphragmatic function so as to exclude the presence of alterations. to do so, some tests used in clinical practice are here below displayed. • p 0.1 : it is the most frequently used test for the respiratory drive evaluation in mechanically ventilated patients. in order to carry out this test, the ventilator's inspiratory valve is closed, and the pressure fall within the first 100 msec after the patient's inspiratory attempt is recorded. usually, the p 0.1 value varies between 0.5 and 1.5 cmh 2 o. it is important to note that this parameter depends both on the inspiratory muscular strength and on the respiratory drive. • maximal inspiratory pressure (mip): represents the maximum pressure that the patient can generate by inhaling against a completely occluded airway, starting from functional residual capacity (frc). the minimum thresholds are -75 cmh 2 o for men and -50 cmh 2 o for women [75] . theoretically, the most negative values exclude the presence of a significant muscular weakness. • rapid shallow breathing index (rsbi): introduced by tobin [76] , it is one of the most common indexes used to evaluate patients in weaning process. it is defined as the ratio between the respiratory rate and the tidal volume expressed in liter. patients that tend to breathe with a higher respiratory rate and with a smaller tidal volume have a high rsbi and more probably a higher risk of weaning failure. the majority part of centers considers a rsbi < 105 adequate to start weaning the patient from mechanical ventilation [77] . mechanical ventilation is a life-saving intervention in patients affected by acute respiratory distress, but it is also associated with complications. therefore it is desirable to wean patients from mechanical ventilation as soon as the underlying cause that led to the need for ventilatory support is resolved or the patient has sufficiently improved and is able to sustain spontaneous breathing with adequate respiratory mechanics and gas exchange. recently, there were published some guidelines aimed at giving indications on which weaning/extubation techniques it is recommended to use in patients under mechanical ventilation [78] : -for acutely hospitalized patients ventilated more than 24 h who are able to make a weaning attempt, it is recommended to carry out an initial spontaneous breathing trial with inspiratory pressure support (5-8 cmh 2 o). -for acutely hospitalized patients ventilated for more than 24 h, it is suggested to use protocols to minimize sedation or guarantee sedative suspension periods, during which carry out a spontaneous breathing trial. -for acutely hospitalized patients ventilated more than 24 h at high risk for extubation failure and who have passed a spontaneous breathing trial, it is recommended the application of noninvasive ventilation (niv) following extubation. still today, ards represents a syndrome with a globally high incidence and a high mortality rate that varies between 40% and 60%. the use of a systematic diagnostic approach can help physicians to rapidly identify the triggering cause of the syndrome, making it possible to quickly start with the right therapy. chest imaging, mainly represented by ct scan, is of primary relevance both in the diagnostic pathway and in the evaluation of lung parenchyma recruitability. the use of lung ultrasound is gaining a pivotal role in the daily bedside evaluation of the patient, thanks to its role in the differential diagnosis and to the possibility to evaluate right and left ventricular function. the supportive treatment guaranteed to patients with respiratory distress needs to be oriented to the maintenance of vital functions, to the improvement of gas exchange and to the reduction of lung injury risk. in order to avoid ventilator-induced lung injury and to set a lung protective ventilation, it is useful to monitor functional residual capacity (frc) and transpulmonary pressure. in the most severe cases, it can be useful to use neuromuscular-blocking drugs and prone position so as to improve ventilation/perfusion ratio. another challenge for physicians seems to be the weaning from mechanical ventilation: the aim is to exclude all the alterations that may delay or make fail the respiratory weaning. the latest guidelines written by the american thoracic society and the american college of chest physicians are useful to treat the patient in this crucial phase. evolution of mechanical ventilation in response to clinical research acute respiratory distress in adults the american-european consensus conference on ards. definitions, mechanisms, relevant outcomes, and clinical trial coordination acute respiratory distress syndrome: the berlin definition current concepts of ards: a narrative review lung recruitability is better estimated according to the berlin definition of acute respiratory distress syndrome at standard 5 cm h2o rather than higher positive end-expiratory pressure: a retrospective cohort study the berlin definition of ards: an expanded rationale, justification, and supplementary material the acute respiratory distress syndrome viral community-acquired pneumonia in non immunocompromised adults incidence and characteristics of viral community-acquired pneumonia in adults virus-induced acute respiratory distress syndrome: epidemiology, management and outcome viral infection in patients with severe pneumonia requiring intensive care unit admission clinical aspects of pandemic 2009 influenza a (h1n1) virus infection diagnostic workup for ards patients molecular diagnosis of legionella infections--clinical utility of front-line screening as part of a pneumonia diagnostic algorithm acute respiratory distress syndrome mimickers lacking common risk factors of the berlin definition vertical gradient of regional lung inflation in adult respiratory distress syndrome adult respiratory distress syndrome due to pulmonary and extrapulmonary causes: ct, clinical, and functional correlations ultrasound in the management of thoracic disease international evidence-based recommendations for point-of-care lung ultrasound ultrasound assessment of antibiotic-induced pulmonary reaeration in ventilator-associated pneumonia the value of lung ultrasound monitoring in h1n1 acute respiratory distress syndrome relevance of lung ultrasound in the diagnosis of acute respiratory failure: the blue protocol early detection of acute lung injury uncoupled to hypoxemia in pigs using ultrasound lung comets chest sonography: a useful tool to differentiate acute cardiogenic pulmonary edema from acute respiratory distress syndrome role of noninvasive ventilation in acute lung injury/acute respiratory distress syndrome: a proportion meta-analysis predictors of failure of noninvasive positive pressure ventilation in patients with acute hypoxemic respiratory failure: a multi-center study use of high-flow nasal cannula oxygen therapy in subjects with ards: a 1-year observational study ultra-protective ventilation and hypoxemia recruitment and derecruitment during acute respiratory failure: an experimental study experimental pulmonary edema due to intermittent positive pressure ventilation with high inflation pressures. protection by positive end-expiratory pressure multiple system organ failure. is mechanical ventilation a contributing factor? lung opening and closing during ventilation of acute respiratory distress syndrome higher versus lower positive end-expiratory pressures in patients with the acute respiratory distress syndrome ventilation strategy using low tidal volumes, recruitment maneuvers, and high positive end-expiratory pressure for acute lung injury and acute respiratory distress syndrome: a randomized controlled trial lung recruitment assessed by respiratory mechanics and computed tomography in patients with acute respiratory distress syndrome. what is the relationship? compressive forces and computed tomography-derived positive end-expiratory pressure in acute respiratory distress syndrome recruitment maneuvers in acute respiratory distress syndrome and during general anesthesia effects of alveolar recruitment maneuvers on clinical outcomes in patients with acute respiratory distress syndrome: a systematic review and meta-analysis bedside ultrasound assessment of positive end-expiratory pressure-induced lung recruitment lung stress and strain during mechanical ventilation: any safe threshold? low-dose chest computed tomography for quantitative and visual anatomical analysis in patients with acute respiratory distress syndrome stress and strain within the lung the assessment of transpulmonary pressure in mechanically ventilated ards patients lung protective ventilation strategy for the acute respiratory distress syndrome lung stress and strain during mechanical ventilation for acute respiratory distress syndrome driving pressure and survival in the acute respiratory distress syndrome balancing neuromuscular blockade versus preserved muscle activity low mortality rate in adult respiratory distress syndrome using low-volume, pressure-limited ventilation with permissive hypercapnia: a prospective study driving pressure and intraoperative protective ventilation neuromuscular blocking agents in patients with acute respiratory distress syndrome: a summary of the current evidence from three randomized controlled trials the prone position in ards patients use of extreme position changes in acute respiratory failure prone position in acute respiratory distress syndrome. rationale, indications, and limits prone positioning and neuromuscular blocking agents are part of standard care in severe ards patients: yes effect of prolonged methylprednisolone therapy in unresolving acute respiratory distress syndrome: a randomized controlled trial efficacy and safety of corticosteroids for persistent acute respiratory distress syndrome high-dose corticosteroids in patients with the adult respiratory distress syndrome pulmonary vasodilators. respir care inhaled nitric oxide does not reduce mortality in patients with acute respiratory distress syndrome regardless of severity: systematic review and meta-analysis efficacy and economic assessment of conventional ventilatory support versus extracorporeal membrane oxygenation for severe adult respiratory failure (cesar): a multicentre randomised controlled trial extracorporeal life support in critically ill adults respiratory failure in the elderly effect of failed extubation on the outcome of mechanical ventilation weaning from mechanical ventilation significant tracheal obstruction causing failure to wean in patients requiring prolonged mechanical ventilation: a forgotten complication of long-term mechanical ventilation neurologic status, cough, secretions and extubation outcomes a clinical prediction rule for delirium after elective noncardiac surgery efficacy and safety of a paired sedation and ventilator weaning protocol for mechanically ventilated patients in intensive care (a wakening and breathing controlled trial): a randomised controlled trial depressive disorders during weaning from prolonged mechanical ventilation treatment of depression with methylphenidate in patients difficult to wean from mechanical ventilation in the intensive care unit clinical review: the abc of weaning failure--a structured approach is weaning failure caused by low-frequency fatigue of the diaphragm? maximal respiratory pressures: normal values and relationship to age and sex a prospective study of indexes predicting the outcome of trials of weaning from mechanical ventilation effect on the duration of mechanical ventilation of identifying patients capable of breathing spontaneously liberation from mechanical ventilation in critically ill adults: an official american college of chest physicians/ american thoracic society clinical practice guideline: inspiratory pressure augmentation during spontaneous breathing trials, protocols minimizing sedation, and noninvasive ventilation immediately after extubation key: cord-258139-x4js9vqe authors: callan, robert j; garry, franklyn b title: biosecurity and bovine respiratory disease date: 2005-03-04 journal: vet clin north am food anim pract doi: 10.1016/s0749-0720(02)00004-x sha: doc_id: 258139 cord_uid: x4js9vqe although biosecurity practices play a role in minimizing respiratory disease in cattle, they must be used in combination with other management strategies that address the many other risk factors. because the pathogens involved in bovine respiratory disease are enzootic in the general cattle population, biosecurity practices aimed at the complete elimination of exposure are currently impractical. several animal husbandry and production management practices can be used to minimize pathogen shedding, exposure, and transmission within a given population, however. various combinations of these control measures can be applied to individual farms to help decrease the morbidity and mortality attributed to respiratory disease. and treatment, it seems that respiratory disease remains one of the foremost cattle health concerns. the challenge that the authors were presented with in writing this article was to consider the role that biosecurity could play in reducing the occurrence or effect of respiratory disease. it seems that little research has specifically evaluated the effects of biosecurity management practices on the occurrence of the problem in livestock operations. indeed, recognizing the multifactorial etiology of infectious respiratory disease and the ubiquitous presence of the pathogens involved leads to the conclusion that attempts to decrease disease prevalence must incorporate multiple management steps, of which biosecurity practices are only a single component. although biosecurity practices have equal potential to decrease respiratory disease losses in all food animal species, the authors focus this article primarily on bovine respiratory disease complex. this article addresses major areas of respiratory pathogen control and provides some suggestions for practical intervention. bovine respiratory disease is not a single entity, nor is it attributable to a single cause [2] . one useful scheme for characterizing respiratory tract diseases in a practical manner distinguishes three different categories of problems [40] . these include the bovine respiratory disease complex (brdc), epitomized by shipping fever pneumonia and enzootic calf pneumonia; acute interstitial pneumonias; and metastatic pneumonia. this scheme excludes many problems that involve only the upper respiratory tract, although these problems may predispose to lower tract infections. the interstitial pneumonias are most commonly attributed to toxicoses, and metastatic pneumonias are secondary complications of disease in other organ systems that spread hematogenously to the lung. although these disease problems are frequently fatal for affected cattle, they occur sporadically and are generally not considered to be contagious. the authors focus their attention for this discussion on brdc. this problem has an infectious origin, and it is by far the most frequently occurring form of cattle respiratory disease. cattle of all ages and in a variety of circumstances can be affected by brdc, but the disease most commonly manifests in young dairy calves (enzootic calf pneumonia) and in beef calves recently arrived at feedlots (shipping fever pneumonia). research over the past several decades has provided an increasingly clear picture of how brdc occurs and why it is so common. unfortunately, this knowledge has not led to a commensurate decrease in the morbidity and mortality associated with this problem, primarily because animals are commonly managed in ways that predispose to disease development. bovine respiratory disease complex refers to bacterial bronchopneumonia that may or may not be complicated by previous or concurrent viral or mycoplasma infection [2] . numerous bacterial species can be isolated from the lungs of affected animals. in feedlot cattle and adult cattle, mannheimia (pasteurella) haemolytica is considered the most important pathogen, with lesser roles attributed to pasteurella multocida and hemophilus somnus. in younger calves, these same pathogens play a role, but mycoplasma spp. are also considered to be important. arcanobacterium pyogenes, fusobacterium spp., and bacteroides spp. are frequently isolated from animals with chronic, abscessing lung lesions but do not play a major role in acute bronchopneumonia. less common bacterial isolates, including streptococcus spp., staphylococcus spp., pseudomonas aeruginosa, and chlamydia spp., are also occasionally identified in young calves. all of the bacterial pathogens considered important in brdc can be isolated from the upper respiratory tract of healthy cattle and calves. these pathogens are considered ubiquitous in cattle populations, not because they can be found in each animal, but because they are readily identified in the nasopharynx of some animals in most populations. in the absence of other predisposing causes of disease, it seems that the simple presence of these bacterial agents is not of major significance. the disease complex is best characterized as being multifactorial, only occurring when a combination of factors involving the animal, environment, and infectious agents are present. viral pathogens are implicated in the development of brdc, although the final pulmonary pathology is primarily caused by bacterial pathogens [2] . the principal viruses involved in brdc include bovine herpesvirus 1 (infectious bovine rhinotracheitis), bovine parainfluenza virus type 3, bovine respiratory syncytial virus, and bovine viral diarrhea virus. lesser roles are attributed to bovine coronavirus, adenovirus, rhinovirus, reovirus, and enterovirus. these viral pathogens primarily infect the upper respiratory tract, resulting in rhinitis, tracheitis, and bronchitis. their ability to cause direct pulmonary disease is generally limited except for bovine respiratory syncytial virus, which can also cause severe lung damage as the primary agent. all of these viral pathogens predispose the lung to bacterial infection and bronchopneumonia. the primary role of these agents in brdc is to promote bacterial challenge to the lungs by compromising respiratory tract defense mechanisms. the predisposing causes of brdc act synergistically and are most commonly identified in combination rather than as single causative problems. the list of predisposing animal factors is long and includes animal age, decreased immune responsiveness due to animal stress, lack of previous viral exposure or vaccination, inadequate passive immunoglobulin transfer in young calves, nutritional deficiencies, and dehydration. environmental risk factors include high air humidity or dust content, rapidly changing environmental temperatures, extreme heat or cold, and high concentrations of noxious gases such as ammonia. several risk factors may increase pathogen density or pathogen exposure, although these risk factors probably act by other means as well. for example, commingling cattle from multiple sources may increase exposure to antigenically heterogeneous viral pathogens, while also increasing animal stress. poor ventilation and high humidity can increase pathogen density and survival time but also can increase noxious gas concentrations and adversely affect pulmonary function. animal crowding increases airborne pathogen exposure but also induces animal stress and reduces immune responsiveness. when evaluating the rate of brdc occurrence in cattle populations, it is clear that efforts to prevent this disease have not been effective on an industry-wide basis, although some individual producers have successfully used prevention strategies. the two biggest areas of brdc effect are in the form of enzootic calf pneumonia of dairy calves and shipping fever pneumonia of feedlot cattle. given our current understanding of this disease problem, it is clear that the animal management systems employed for these groups of animals (i.e., dairy calf-rearing systems and feedlot cattle-receiving systems) have failed to rigorously apply knowledge of disease pathogenesis and prevention into their processes. this situation may be changing currently, as the beef production industry increasingly uses quality-assurance principles in production systems and develops marketing procedures and animal-purchasing practices that reward improvements in animal health [12, 35, 46] . similarly, the dairy industry has begun to recognize the economic benefit of improved calf health and increasingly uses specialized calf-rearing systems [1, 44] . because the purpose of this article is to examine the role of biosecurity management in respiratory disease prevention, the authors do not attempt to provide a complete review of brdc preventive practices. many of the important means of preventing brdc do not employ biosecurity but are targeted toward enhancing animal immune preparedness and enhancing animal response to infectious challenge. effective respiratory disease preventive practices are those targeted at reducing identified risk factors for disease development [1, 2, 35, 46] . these practices include management to improve animal nutrition with special emphasis on micronutrient nutrition, practices that reduce animal stress, reduced commingling of animals, improved animal transportation and feedlot receiving practices, improved preconditioning and vaccination programs that emphasize vaccination before shipment and during times of low calf stress, and improved ventilation with reduced crowding. it is important to consider the factors that drive the development and implementation of disease prevention and biosecurity programs. the most apparent of these factors is the effect on animal production and growth; however, all interventions have their cost, and these costs must always be considered relative to the potential economic returns. unfortunately, information regarding the financial impact of herd biosecurity programs is limited, and estimates based on clinical experience must often be applied. other issues, including herd pathogen status and its effect on livestock marketing, food product quality assurance, drug residues, injection site lesions, antimicrobial resistance, and animal welfare also contribute to the forces that drive the development of biosecurity programs. ultimately, a biosecurity program must be integrated into the overall herd management. it must be developed using a team approach that addresses the concerns of the producer, the economic effect on the production unit, the influence on product quality, and public health concerns. the veterinarian is best suited to effectively develop and implement such programs. the multifactorial nature of brdc and the ubiquitous presence of respiratory pathogens are important concepts when considering the role that biosecurity can play in decreasing the prevalence of disease. for infectious diseases in which point source pathogen exposure, high susceptibility, and high virulence are prominent features of disease transmission (e.g., anthrax, footand-mouth disease, rabies, and so forth), limiting animal contact with the pathogen is a key feature of disease prevention and may even provide the means of disease eradication. alternatively, when the causative pathogens are endemic in a population and individual susceptibility is dependent on numerous interrelated factors, the management of animal resistance and risk factors may be proportionally more important for disease prevention than biosecurity practices. it appears that brdc prevention requires a combination of management to enhance animal resistance plus management to reduce exposure to the pathogens. the important point is not to de-emphasize the value of reducing pathogen introduction, exposure, and transmission (i.e., biosecurity) but to also stress the importance of other management features that promote animal resistance. it is particularly important that preventive management practices be coordinated and used in combination, because no single management procedure will be successful without the complement of other practices. it is likely that our inability to reduce the prevalence of respiratory disease in cattle is, in part, attributable to our failure to integrate multiple aspects of respiratory disease prevention practices, including biosecurity. the fundamental concept of biosecurity is to decrease pathogen transmission between animals. transmission of respiratory pathogens occurs by close nose-to-nose contact, environmental or fomite exposure, and airborne exposure. increased contact between shedding and susceptible individuals increases pathogen spread. environmental exposure through common areas and equipment that involve oral or nasal contact such as feed bunks, water troughs, and salt blocks may be an even greater risk, however. total environmental pathogen load is extremely important in considering respiratory pathogen transmission. environmental contamination from animals in contact is the primary source of most respiratory pathogens. individual animal shedding is quite variable and depends on the etiologic agent, the time course of the disease, the clinical severity, and the immune response of the host. in general, clinically ill animals shed greater numbers of pathogens than normal or asymptomatic animals; however, it must be recognized that individuals periodically shed both viral and bacterial respiratory pathogens without evidence of disease. well-vaccinated animals may also periodically shed pathogens and should not necessarily be considered completely safe from disease transmission. the persistence of the pathogen in the environment also contributes to pathogen exposure. environmental pathogen survival times depend on many factors, including organic material, moisture, direct sunlight, and exposure to disinfectants. environmental survival times for most viral respiratory pathogens are probably on the order of minutes to several hours [18, 47] . survival times for bacterial pathogens may be longer depending on the environmental conditions and the organism. airborne transmission is dependent on numerous factors, including ambient temperature, relative humidity, airborne particle (dust) density, ventilation, prevailing wind, and structural or geographic obstructions [47] . airborne transmission of typical viral respiratory pathogens can occur over distances as far as 4 meters and possibly further [29, 30] . airborne transmission of other viruses such as footand-mouth disease virus or pseudorabies virus has been shown to occur over many miles, however [10, 11, 15, 45, 47] . adding to the complexity of pathogen transmission, it seems that the efficiency of transmission is different between different strains of a given pathogen [30] . understanding how management practices can reduce either pathogen shedding or exposure is the key to creating effective biosecurity programs. the term biosecurity is used for those management and hygiene practices that reduce introduction, exposure, and transmission of infectious agents. although biosecurity may not provide the single most important component of respiratory disease prevention, reducing pathogen exposure is a valuable part of any infectious disease management system. little information is available to specifically evaluate the effect of individual biosecurity practices in prevention of brdc, but there are some important respiratory disease prevention practices that limit pathogen exposure and good reason to more closely evaluate the role that biosecurity could play in the future. the authors emphasize five areas of biosecurity management that should be more rigorously applied for the reduction of respiratory disease prevalence in cattle, including (1) strategic vaccination, (2) calf biosecurity, (3) housing ventilation, (4) commingling and animal contact, and (5) bovine viral diarrhea virus control. many improvements in vaccine technology have occurred over the past few decades, and practitioners have an array of improved bovine respiratory pathogen vaccines at their disposal [35] . unfortunately, the current respiratory pathogen vaccines have not all been scrutinized for efficacy to the most desirable degree, and many do not protect against respiratory disease nearly as effectively as some veterinarians and producers would like to believe. although vaccines directed at specific conserved proteins, such as toxoid vaccines, may completely prevent a particular disease, vaccines against complex disease agents that have multiple antigenic strains are unlikely to be capable of such levels of protection. respiratory vaccines are better viewed as disease modifiers than absolute preventive agents. vaccines are usually used as a means to decrease the likelihood or severity of disease occurrence in the individual animal receiving the vaccination. indeed, vaccine efficacy may be evaluated in many ways, but the more rigorous evaluations involve the ability of a vaccinated animal to withstand a challenge of disease or pathogen exposure [42] . practitioners tend to view vaccination as one of the management factors that enhance animal resistance to infection and thus augment the value of biosecurity management by working to reduce susceptibility to infectious disease rather than decrease exposure and transmission. for respiratory disease prevention, however, effective vaccination can also serve as part of a biosecurity management system. in addition to preventing disease, a vaccine's efficacy might also be considered for its ability to limit pathogen shedding when infection does occur. vaccineinduced immunity often results in decreased magnitude and duration of pathogen shedding [6, 16, 59] . because exposure is directly related to pathogen concentration in the environment, it follows that vaccine-induced reductions in shedding should decrease transmission within a susceptible population. proper vaccine use and a well-managed vaccination program can be viewed as part of a complete biosecurity program. at a minimum, a good vaccination program should include the following: â�¢ proper storage and administration of the vaccine as indicated by the manufacturer's labeled recommendations. â�¢ vaccination of all susceptible animals, including both resident and incoming animals. â�¢ application of the vaccine to systemically healthy, well-nourished, minimally stressed, and immunocompetent cattle. â�¢ strategic timing of vaccination so that it precedes contact with new animals long enough to allow an appropriate immune response. â�¢ revaccination as recommended for the particular vaccine product. biosecurity management of calves is extremely important for development of healthy animals. many of the biosecurity recommendations for newborn calves focus on decreasing the transmission of enteric pathogens; however, these same principles can be important for minimizing respiratory disease problems. several details of calf biosecurity management deserve emphasis. environmental and housing factors significantly affect calf health and viability. differences in calf management for cow-calf herds versus dairies are related to the relative risk of respiratory disease between these two production groups. beef calves are generally raised in open-range situations that effectively dilute the exposure to respiratory pathogens. although beef calves are continually exposed to pathogens shed from adult cattle and other calves, the magnitude of pathogen exposure before weaning is generally low, resulting in relatively little respiratory disease. in contrast to many enteric pathogens, the environmental survival time of the respiratory pathogens is limited [18] , and accumulation of pathogens in the environment is not considered a primary concern. dairy calf housing has a significant effect on the incidence of respiratory disease in neonatal calves. although the common viral respiratory pathogens can be transmitted over distances up to 4 meters [29, 30] , properly spaced calf hutches seem to effectively limit aerosol transmission of respiratory pathogens. the short survival of these pathogens in the environment limits the transmission between successive occupants of an individual hutch. disinfection procedures that are used for enteric diseases should be more than sufficient to decrease respiratory pathogen transmission (see article by barrington et al. in this issue). in contrast, there is a high risk of respiratory disease transmission in group-raised neonatal calves. factors including the number of animals, relative animal density, housing facilities, and ventilation conditions significantly contribute to transmission in grouped calves and are discussed in subsequent sections of this article. numerous management practices can decrease exposure and transmission of respiratory pathogens to calves in dairy operations. feeding pasteurized milk or milk replacer is a useful biosecurity practice for minimizing the spread of enteric agents such as salmonella spp. or mycobacterium avium subsp. paratuberculosis. these practices are also effective at limiting ingestion of potential respiratory pathogens. mycoplasma spp. bacteria are commonly implicated in newborn calf disease, including enzootic calf pneumonia [2, 37, 48, 49] . although mycoplasma spp. may spread by the airborne route, it is also a common mastitis pathogen and can be shed from clinically or subclinically infected cows [24, 37, 49] . nasopharyngeal colonization occurs after oral ingestion of contaminated milk, potentially resulting in clinical respiratory disease in calves [37] . mycoplasma spp. and other pathogens can also spread hematogenously after ingestion by a susceptible calf [24, 37] . similarly, other potential respiratory pathogens such as streptococcus spp., staphylococcus spp., salmonella spp., and escherichia coli can be recovered from milk and spread hematogenously to the lungs after oral ingestion. bovine viral diarrhea virus is shed in the milk of persistently infected cattle. ingestion of bovine viral diarrhea virus-contaminated milk can result in respiratory and systemic infections, possible immune suppression, and respiratory disease. proper cleaning and disinfection of calf feeding equipment, including nursing bottles, buckets, and mixing utensils, should be performed. equipment should be cleaned with a detergent and disinfected between uses. a common and economical disinfectant is standard household bleach used at a 1:10 dilution. bottles and equipment that are potentially shared between multiple animals should be soaked for 15 to 20 minutes in this solution. although bleach will not completely kill all potential pathogens, it is effective at significantly decreasing viable numbers and thus contributing to decreased exposure and transmission between feedings. prompt removal of dairy calves from the maternity pen environment, where they are exposed to numerous adult cow pathogens, can also decrease transmission of potential respiratory pathogens. newborn calves should not have direct contact with older calves and adults. calf hutch spacing should be evaluated, with a minimum of 4 feet of separation between calves. worker hygiene can minimize contamination of calf feed and the calf environment. appropriate vaccination of dams before colostral production can increase passive transfer of effective antibodies, reducing the risk of exposure and potential shedding after infection. it has been demonstrated that good colostral transfer to beef calves was associated with decreased occurrence of disease episodes and improved calf performance all the way through the growing and finishing period in feedlot animals [36] . it is unlikely that the passive transfer of immunoglobulins per se is specifically responsible for beneficial effects on the long-term health of animals, but profound effects may result from management that improves newborn health and disease resistance. this in turn provides for improved nutrition, growth, physiologic well-being, and decreased total pathogen load. numerous calfhood husbandry procedures should be considered as standard biosecurity protocols for all infectious diseases, including respiratory disease. sick animals should be identified and separated from healthy animals. a specific calf-isolation area should be established, with consideration to animal comfort and ease of cleaning and disinfection. where practical, individual equipment should be used for each separate calf. specific care and treatment personnel should be identified, and animals with suspected infectious diseases should be treated after handling healthy animals. additional personnel hygiene protocols include dedicated coveralls to be used in the sick pens, the use of rubber overboots, and disinfectant footbaths. personnel should be encouraged to wash their hands before and after entering the sick pens and between caring for animals with dissimilar disease conditions. in many cases, equipment and facilities need to be made available to help establish such procedures. similar biosecurity management practices can be used in cow-calf herds. although feeding pasteurized milk or milk replacer is obviously not a practical management practice, milk-borne exposure to pathogens can be minimized by proper attention to the adult cows. adult cattle must be appropriately vaccinated to provide optimal colostral immunity to the calves and to decrease adult cow infections and shedding. adult cow nutrition should be optimized to improve colostrum quality. adult cow nutrition can also have a dramatic effect on calving ease and decrease the incidence of dystocia. special attention should be placed on high-risk calves, including calves delivered with manual assistance, cesarean section, born in inclement weather, weak or premature calves, and multiple births. such calves often do not nurse colostrum in a timely fashion or have impaired absorption of immunoglobulin. cows should be evaluated for evidence of clinical mastitis and treated or culled as appropriate. decreased morbidity can be observed by minimizing the time that beef cow-calf pairs spend in a designated calving area, where pathogen loads tend to increase throughout the calving season. bovine viral diarrhea virus surveillance and eradication in cows and calves should also be used (see discussion in a following section). as can be seen from the preceding discussion, many of the management practices that contribute to biosecurity of respiratory disease are standard quality-assurance practices that are recommended for basic calf health. good ventilation is a critical aspect of animal management and can profoundly affect respiratory health. several discussions of ventilation and its effect on animal health are present in the literature [2] [3] [4] [5] 13, 14, 19, 27, 31, 34, 38, 39, 43, 51, 58] . proper ventilation serves eight primary functions: 1. it decreases the airborne pathogen concentration 2. it eliminates noxious gases (ammonia, hydrogen sulfide, carbon dioxide, carbon monoxide, and methane) 3. it decreases airborne dust contamination 4. it decreases airborne endotoxin levels 5. it maintains optimum ambient temperature 6. it maintains optimum environmental humidity levels 7. it eliminates drafts 8. it eliminates areas of stagnant air with respect to biosecurity, one of the most important aspects of proper ventilation is the reduction in the concentration of airborne pathogens. all of the important viral and bacterial respiratory pathogens can spread aerogenously and can attain high concentrations in poorly ventilated housing areas. airborne pathogen concentration is a function of many factors, including animal type, housing system, stocking rate, bedding, humidity, dust particle density and size, and finally, elimination through ventilation. improved ventilation is one important means whereby airborne pathogen concentration can be readily decreased within the given constraints of an operation; however, pathogen removal is not a linear function, and practical and theoretical limits are often observed [33] . studies of building ventilation for humans demonstrate potential reductions in airborne exposure of pathogens and disease incidence, although improved ventilation beyond that which provides comfort may not be practical or provide significant additional benefit [33] . as the airborne pathogen load rises, ventilation provides progressively less protection against respiratory infections. it is important to realize that stocking rate has a more dramatic effect on airborne pathogen density than ventilation [33, 58] . for example, a two-fold increase in stocking rate requires nearly a 10-fold increase in ventilation to maintain the same airborne pathogen density [58] . ventilation cannot overcome grossly inadequate housing, management, or hygiene within a production unit. along with stocking density, there are other practical concerns that contribute to airborne pathogen density and transmission. one of these is related to animal handling and excitement. it is extremely important to handle grouped animals in a calm environment with minimal animal activity and stress. increased animal activity not only increases dust exposure (which contains airborne pathogens) but also increases ventilatory rate, ventilatory effort, and tidal volume, which in turn increases the amount of aerosolized pathogen shed by infected animals and the amount of pathogen inhaled by susceptible animals. the increased dust exposure will also adversely affect mucociliary clearance and respiratory defense mechanisms. part of the effect of ventilation is to minimize airborne contaminants that can impair respiratory function and defense mechanisms [34, 38, 39, 58] . significant airborne contaminants include ammonia, hydrogen sulfide, carbon dioxide, carbon monoxide, methane, dust particles, and endotoxin. ammonia and hydrogen sulfide are toxic gases and can contribute to respiratory damage, decreased mucociliary clearance, decreased alveolar macrophage activity, and overall compromise to respiratory defense mechanisms. carbon dioxide, carbon monoxide, and methane contribute primarily as asphyxiative gases and generally do not contribute to significant impairment of the respiratory tract. dust particles also contribute to the impairment of respiratory defense mechanisms. dust particles can arise from both organic and inorganic sources. in general, particles greater than 5 lm are filtered out by the nasal passages; most particles from 2 to 5 lm are removed by the mucociliary clearance of the trachea and bronchi, and particles less than 2 lm can penetrate to the alveolar spaces [47, 58] . organic and inorganic dust particles can impair mucociliary clearance and overload alveolar macrophage phagocytic clearance [58] . organic dust particles are generally of more concern in confinement and intensive housing situations. in animal housing environments, most of the organic dust arises from fecal material, skin, and hair. organic dust is significant in that it often contains high endotoxin and pathogen levels [38, 39] . inhaled endotoxin can contribute to pulmonary compromise by initiating inflammatory reactions within the alveoli and alveolar vascular endothelium. appropriate ventilation is also important in maintaining acceptable humidity and ambient temperature levels within confinement or semi-open housing. observed thermoneutral ranges (the range of air temperature that sustains optimal performance) for a variety of domestic livestock are available (table 1 ) [58] . in general, livestock can perform adequately within a fairly wide thermoneutral range. higher temperatures, especially when combined with high humidity, tend to be more problematic than low temperatures [34] . depending on the given climate and temperature ranges of a geographic region, housing ventilation will need to be designed to provide either heating or cooling or both. cold temperatures and perhaps temperature fluctuations can decrease mucociliary clearance and predispose animals to respiratory disease [17] . often, wide temperature fluctuations are more detrimental to animal health because they do not allow suitable adaptation over time. there is minimal information on how ambient temperature directly relates to airborne pathogen biosecurity. increased ambient temperature results in increased respiration and may increase pathogen shedding from infected animals. the direct effects of ambient temperature on pathogen survival are relatively unknown. some studies suggest that the concentration of airborne particles is increased at low temperatures, and airborne bacterial concentrations were higher in winter than in summer [47] . there is slightly more information concerning the effects of relative humidity on pathogen survival and thus, airborne biosecurity [47, 58] . in general, viruses with a hydrophobic lipid outer shell (i.e., enveloped viruses) survive better in lower humidity, and lipid-free viruses (i.e., foot-and-mouth disease virus) are more stable in moist air [18, 47] . the four primary viral respiratory pathogens in cattle (bovine herpsevirus 1, bovine parainfluenza virus type 3, bovine respiratory syncytial virus, and bovine viral diarrhea virus) are all enveloped viruses and would be considered more stable in dry air, although the authors are unaware of specific studies documenting this conclusion. gram-negative bacteria have outer phospholipid membranes and are also expected to be more stable in dry air [47] . mycoplasma are reported to be sensitive to relative humidity between 40% and 70% [58] . extrapolation of these limited data suggests that typical airborne pathogens associated with respiratory disease in domestic animals survive better in cool, dry air such as is observed in the late fall, winter, and early spring months. although this correlates with clinical observations concerning the relative seasonal incidence of respiratory disease, a direct association has not been established. in beef cattle, seasonal increases in respiratory disease also correlate with seasonal management practices associated with movement of cattle to feedlots and increased animal density. it is likely that climate and management factors act together to dramatically increase pathogen exposure and transmission in feedlots. alternatively, the high humidity that can be observed with dairy confinement housing in cold weather probably contributes to increased respiratory disease because of the higher pathogen density associated with increased aerosolized particle concentrations. ventilation systems should be constructed to provide even airflow throughout the structure without areas of air stagnation or drafts. pockets of air stagnation have higher levels of airborne contaminants and contribute to the exposure and transmission of respiratory pathogens. air stagnation can often be remedied by appropriate use of inexpensive fans. correcting draft conditions can be more problematic and often requires complete evaluation of the housing structure for air leaks and evaluation of the ventilation system, especially air intake vents. guidelines for housing of livestock have been reported, including recommendations for ventilation (table 2) [2, 5, 13, 27, 34, 51, 58] . appropriate ventilation should flow from younger to older animals to minimize spread of pathogens to the more susceptible animals. the total air volume should be completely changed 4 times per hour in winter, and it should be changed up to 30 times per hour in summer [5, 51 ]. the ventilation system should confinement housing â�¢ minimum of four air changes per hour (winter) â�¢ total exhaust capacity for up to 30 air changes per hour (summer) â�¢ continuous (not intermittent) ventilation â�¢ single-speed fans, not variable-speed fans, should be used â�¢ fans must be able to sustain 1/8-inch static pressure â�¢ one must allow for two to four different ventilation rates using multiple fans â�¢ enough inlet slot area should be provided to allow minimal inlet velocity of 100 fpm (winter) and 800 fpm (summer) â�¢ thermostats should be used to control ventilation fans â�¢ thermostats should be located at eye level near the center of the barn â�¢ the ventilation rate should be altered by stepping up the number of fans used for each level â�¢ wall fans should be mounted near the ceiling but collect air using ducts from within 38 cm (15 in) of the floor â�¢ the fresh air intake should be located near the ceiling but at least 4 feet from any exhaust fan â�¢ adjustable eave slot inlets should be used to distribute incoming air uniformly provide constant rather than intermittent airflow. in the winter, the goal of ventilation is to minimize airborne pathogen density, remove excess moisture from animal respiration, and maintain adequate ambient temperature (10-13â°c, 50-55â°f). although higher ventilation rates improve air quality, they are inefficient because they require excessive heating costs. supplemental heating may be necessary as the outside temperature falls or stocking density decreases. at optimal stocking densities, livestock generally produce enough animal heat to maintain adequate ambient temperature in confined housing when outside temperatures remain above ã¿8â°c [51] . winter ventilation is a compromise between the removal of airborne contaminants and the maintenance of ambient temperature. the primary goal of summer ventilation is to minimize ambient temperature and relative humidity. this requires high ventilation flow rates, which also enhance air quality. the goal is to maintain an ambient housing temperature to no more than 2â°c above the outside temperature [5, 51] . relative humidity levels should be maintained between 50% and 80%, and ammonia levels should not exceed 10 ppm [2, 34, 47, 51, 58] . maximum recommended stocking densities should not be exceeded (see table 2 ) [26] . separate age groups of cattle should be maintained in separate barns or be separated by barrier walls. calf hutches for individual dairy calves provide an ideal means of managing relative calf isolation and limiting airborne transmission if they are properly positioned and spaced. recommendations for calf hutches include one calf per hutch with a minimum separation of 4 feet between hutches. hutches should be placed at least 10 feet from oldercattle enclosures and 50 feet from livestock building exhaust fans. many cattle management systems provide numerous opportunities for exchange of respiratory pathogens from animal to animal. assembling groups of beef calves for a feedlot often involves mixing calves from different origins, congregation of animals at sale barns or other holding pens, and movement in congested cattle transports. these activities are well known to increase the rate of respiratory disease occurrence by stressing the animals and providing circumstances that decrease disease resistance. these same animal contact and crowding circumstances can dramatically increase exposure to pathogens, often including pathogens to which the animal has not developed prior immunity. in a recent national survey, more than 50% of dairy producers housed sick animals in a manner that allowed direct nose-to-nose contact with healthy herdmates [54] . many dairy producers expand their herds by purchasing animals from other sources, but less than 25% of them provide any quarantine time for the incoming animals. for producers who introduced 15% or more of their total animal inventory during an expansion, 16.6% reported an increase in occurrence of respiratory disease during the year [55] . during the early phases of respiratory disease, the shedding rates of pathogens via respiratory secretions increases dramatically. commingling, crowding, and the animal stresses that are involved in animal movement can precipitate respiratory problems. these same factors can increase spread of pathogens to other animals with close contact. although quarantine may not be effective against diseases with chronic carrier states such as johne's disease, it can substantially decrease the risk of spreading respiratory pathogens. furthermore, the duration of respiratory pathogen shedding has also been well characterized. in general, nasal shedding of viral respiratory pathogens is significantly reduced by 14 days after infection but may persist longer in individual animals, which suggests that quarantine for approximately 14 to 21 days should significantly reduce the exposure and transmission of these pathogens within an operation. practical suggestions for limiting pathogen spread by contact include quarantine of incoming livestock, maintenance of hospital areas that do not allow contact with healthy animals, prevention of animal contact between different age groups of cattle, minimizing the time animals spend in market channels, and limiting the introduction of new animals to assembled herds or pens of cattle. the concepts of pathogen transmission within grouped housing can be effectively applied to weaned dairy calves. calves receive relatively low pathogen exposure while in calf hutches. on weaning and grouping in calf pens, the risk of exposure increases dramatically. it is important to appreciate that the risk of exposure rises with the number of calves housed together. for example, if one estimates that 5% of calves born in a herd with bovine viral diarrhea virus are persistently infected, then the probability of bovine viral diarrhea virus exposure in a group of 10 calves is approximately 0.4 (1-0.95 10 ). if the stocking rate increases to 30 calves, the probability nearly doubles to 0.78 (1-0.95 30 ). limiting the number of calves per pen to less than seven is associated with decreased respiratory disease mortality [28] (also see article by smith in this issue). it must be emphasized that one animal can expose an entire pen of animals by simple close contact, airborne transmission, or environmental transmission at common housing areas such as feed bunks and water troughs. by dividing animals into smaller groups, the number of animals exposed is lowered significantly. using the same example of bovine viral diarrhea virus exposure, if one splits the 30 calves into three separate pens, the probability of having all 30 calves exposed to bovine viral diarrhea virus falls from 0.78 to the comparatively negligible level of 0.064 [(1-0.95 10 ) 3 ]. simple segregation of animals is not sufficient unless physical barriers for fence line contact, separation of food and water troughs, segregation of likely fomites, and blocking airborne spread are used. these same principles can be applied to any group-housing situation. such management and housing decisions must be made based on a balance between the risk and cost of disease versus the availability and cost of facilities and labor. it was noted previously that the common bovine respiratory pathogens are considered to be ubiquitous in cattle populations in the united states and most other countries. although this does not suggest that every animal harbors each pathogen, these agents can be found routinely in the nasopharynx of healthy and diseased animals within most herds. in contrast, some european countries have successfully eradicated some viral respiratory pathogens such as bovine herpesvirus 1 and bovine viral diarrhea virus (bvdv) from cattle populations. under the currently prevailing practices within the united states and many other countries, the authors do not suggest testing or identification of most respiratory pathogens as a viable means to identify carriers or to exclude the animals from introduction into a herd. the exception to this is bvdv. although bvdv is not considered a primary pneumopathogen, it is considered to have an important role in respiratory disease of cattle [2, 41] . the immunosuppressive effects of the virus and the close association of bvdv infection and respiratory disease occurrence in some epidemiologic studies suggest that the virus plays a role by promoting secondary bacterial lung infection. although bvdv vaccines have been improved over the past several years, vaccination alone rarely eliminates bvdv from an infected herd. an effective bvdv biosecurity program must include the identification and removal of persistently infected animals, bvdv screening of incoming animals and their calves, and a comprehensive vaccination program [7, 8, 25, 57] . persistently infected cattle do not mount an effective immune response against the virus and are capable of shedding large amounts of the virus into the environment through multiple routes. persistently infected animals have been implicated as the primary means by which bvdv infection is maintained in assembled dairy herds, and they are also considered a significant threat for transmission in cow-calf and feedlot operations [20] [21] [22] [23] 32, 50, 60] . with the development of new tests over the past several years, our ability to accurately and expediently identify persistently infected animals has dramatically improved [9, 25] . the serum immunoperoxidase monolayer assay (ipma) and antigen capture elisa tests and the immunohistochemistry test of skin biopsy material have appropriate sensitivity and specificity for detecting persistently infected cattle. the authors do not know of significant published research that evaluates the effect of test-and-cull strategies for bvdv on the occurrence of brdc; however, elimination of persistently infected animals from herds can have significant positive effects in decreasing other bvdv manifestations such as reproductive failure. implementing test-and-cull procedures for persistently infected animals may prove to be a powerful means of decreasing brdc prevalence. in general, all cattle introduced into a herd should be tested for bvdv before purchase or entry. acute bvdv infections of pregnant cattle can result in animals that are bvdv negative at the time of testing while the fetus is persistently infected. it is critical that all calves from newly introduced pregnant animals also be tested immediately after birth. to establish a bvdv-negative herd, it is generally more effective and economical to test calves as they are born rather than screen adult populations. a negative result for a calf indicates that not only the calf but also all of the calf's maternal ancestors are not persistently infected. a single positive test on a calf does not differentiate between acute and persistent infection. a confirmatory test may be performed in 4 weeks, or the animal may be assumed to be persistently infected and euthanized or sold for slaughter. the dams of all persistently infected calves should be traced and tested as well to determine their status. in most cases, these animals will test negative, indicating fetal exposure due to acute infection during gestation. bulls should also be tested because they can contribute to animal exposure within a herd. although biosecurity practices play a role in minimizing respiratory disease in cattle, they must be used in combination with other management strategies that address the many other risk factors. because the pathogens involved in bovine respiratory disease are enzootic in the general cattle population, biosecurity practices aimed at the complete elimination of exposure are currently impractical. several animal husbandry and production management practices can be used to minimize pathogen shedding, exposure, and transmission within a given population, however. various combinations of these control measures can be applied to individual farms to help decrease the morbidity and mortality attributed to respiratory disease. dairy calf pneumonia: the disease and its impact the bronchopneumonias (respiratory disease complex of cattle, sheep, and goats) appraising the adequacy of environment for confined animals influence of improved ventilation on health of confined cattle calculation of ventilation needs for confined cattle a new inactivated bvdv genotype i and ii vaccine: an immunisation and challenge study with bvdv genotype i control of bovine viral diarrhea virus infection without vaccines control of bovine viral diarrhea infection by use of vaccination diagnosis of bovine viral diarrhea virus infections further evidence of long distance airborne transmission of aujeszky's disease (pseudorabies) virus evidence of long distance airborne transmission of aujeszky's disease (pseudorabies) virus preconditioning calves for the feedlot ventilation of sheep and goat barns air environment and animal performance airborne spread of foot-and-mouth disease in saskatchewan. canada, 1951-1952 cross-protective efficacy of a bovine viral diarrhea virus (bvdv) type 1 vaccine against bvdv type 2 challenge pulmonary particle deposition and airway mucociliary clearance in cold-exposed calves factors influencing the dispersal, survival and deposition of airborne pathogens of farm animals ventilation system to minimize airborne bacteria, dust, humidity, and ammonia in calf nurseries age distribution of animals persistently infected with bovine virus diarrhea virus in twenty-two danish dairy herds epidemiological features and economical importance of bovine virus diarrhoea virus (bvdv) infections epidemiology of bovine viral diarrhea virus prevalence of cattle persistently infected with bovine viral diarrhea virus in 20 dairy herds in two counties in central michigan and comparison of prevalence of antibody-positive cattle among herds with different infection and vaccination status mastitis, polyarthritis and abortion caused by mycoplasma species bovine group 7 in dairy cattle testing and management strategies for effective beef and dairy herd bvdv biosecurity programs preventing calf pneumonia sheep housing and health management variables associated with high mortality rates attributable to respiratory tract problems in female calves prior to weaning airborne transmission of bhv1. brsv, and bvdv among cattle is possible under experimental conditions airborne transmission of bovine herpesvirus 1 infections in calves under field conditions ventilation systems for swine housing a long term epidemiological study of bovine viral diarrhoea infections in a large herd of dairy cattle airborne infection: theoretical limits of protection achievable by building ventilation housing of ruminants: ii. requirements of good housing and effects of bad housing advances in pulmonary immunology failure of passive transfer: risk factors and effects on lifetime performance mycoplasma bovis as an agent of mastitis, pneumonia, arthritis and genital disorders in cattle hazards in confinement housing-gases and dusts in confined animal houses for swine, poultry, horses and humans characterization of particles, ammonia and endotoxin in swine confinement operations clinical classification of pneumonias in cattle bovine respiratory tract disease caused by bovine viral diarrhea virus assessing vaccine efficacy housing of livestock: ii. practical considerations of ventilation and construction descriptive epidemiology of morbidity and mortality in minnesota dairy heifer calves an integrated model to predict the atmospheric spread of foot-and-mouth disease virus the importance of preventing bovine respiratory disease: a beef industry review the role of infectious aerosols in disease transmission in pigs mycoplasma infection in cattle: i pneumonia-arthritis syndrome clinical study of the disease of calves associated with mycoplasma bovis infection the prevalence of bovine viral diarrhea virus infection in a population of feedlot calves in western canada housing and environment for dairy calves aphis:vs centers for epidemiology and animal health nahms. beef '97, part ii: reference of 1997 beef cow-calf health & health management practices aphis:vs centers for epidemiology and animal health nahms. cattle and calves death loss aphis:vs centers for epidemiology and animal health nahms. dairy '96, part i: reference of 1996 dairy management practices aphis:vs centers for epidemiology and animal health nahms. dairy '96, part iii: reference of 1996 dairy health and health management aphis:vs centers for epidemiology and animal health nahms. feedlot '99, part iii: health management and biosecurity in vaccination of cattle against bovine viral diarrhoea ventilation, air hygiene and animal health the efficacy of modified-live bovine respiratory syncytial virus vaccines in experimentally infected calves persistent bovine viral diarrhoea virus infection in us beef herds key: cord-263764-2ewz8ok4 authors: kutter, jasmin s; spronken, monique i; fraaij, pieter l; fouchier, ron am; herfst, sander title: transmission routes of respiratory viruses among humans date: 2018-01-17 journal: curr opin virol doi: 10.1016/j.coviro.2018.01.001 sha: doc_id: 263764 cord_uid: 2ewz8ok4 respiratory tract infections can be caused by a wide variety of viruses. airborne transmission via droplets and aerosols enables some of these viruses to spread efficiently among humans, causing outbreaks that are difficult to control. many outbreaks have been investigated retrospectively to study the possible routes of inter-human virus transmission. the results of these studies are often inconclusive and at the same time data from controlled experiments is sparse. therefore, fundamental knowledge on transmission routes that could be used to improve intervention strategies is still missing. we here present an overview of the available data from experimental and observational studies on the transmission routes of respiratory viruses between humans, identify knowledge gaps, and discuss how the available knowledge is currently implemented in isolation guidelines in health care settings. viral respiratory tract infections are a leading cause of morbidity and mortality worldwide, representing an enormous economic and disease burden [1] . respiratory viruses replicate in the respiratory tract from where they are subsequently shed and transmitted via respiratory secretions. they are classified in different virus families and differ in virulence and target groups. respiratory tract infections may range from asymptomatic to acute live threating disease thereby posing a major health threat to young children, elderly, and immunocompromised people. respiratory viruses spread via three different transmission routes: contact (direct or indirect), droplet and aerosol transmission (table 1) [2, 3] . contact transmission refers to direct virus transfer from an infected person to a susceptible individual (e.g. via contaminated hands) or indirect virus transfer via intermediate objects (fomites). transmission of virus through the air can occur via droplets or aerosols. the commonly accepted cut-off size between the large droplets and small aerosols is 5 mm, although this varies considerably between studies, ranging up to 12 mm [4] [5] [6] [7] [8] . droplets generated during coughing, sneezing or talking do not remain suspended in air and travel less than 1 m before settling on the mucosa of close contacts or environmental surfaces. aerosols have a slow settling velocity, thus they remain suspended in the air longer and can travel further [5, 9, 10] . transmission via each of these three routes is complex and depends on many variables such as environmental factors (e.g. humidity and temperature), crowding of people, but also on host factors such as receptor distribution throughout the respiratory tract. the fact that all these variables affect the different transmission routes of the different respiratory viruses in a dissimilar way, makes it very difficult to investigate them experimentally [9, 11] . here, we summarize the evidence from experimental and observational studies on inter-human transmission routes of important respiratory viruses (summarized in table 2 ). a literature search was conducted for each respiratory virus using 'human transmission experiments' and 'transmission (routes)' of the virus of interest as search criteria in pubmed and google scholar. subsequently, the backward snowball method was applied in which additional papers were identified based on the reference list of a paper of interest. as this review focuses on the evidence on inter-human transmission routes, data from animal studies were excluded. in addition, intervention studies, (aircraft) outbreak reports and household studies were excluded if the transmission route was not specifically investigated. the strengths and weaknesses of the different methods employed in transmission studies are summarized in table 3 . finally, we discuss our findings in the light of several available (inter)national guidelines on infection control. our observations underscore the urgent need for new knowledge on respiratory virus transmission routes and the implementation of this knowledge in infection control guidelines to advance intervention strategies for currently circulating and newly emerging viruses and to improve public health. measles is one of the most contagious viral diseases in humans that has been associated with aerosol transmission for a long time [12,13,14 ,15-17,18 ]. however, it should be noted that mv also replicates systemically, and that there is a role for dead cell debris-associated virus spread via fomites. in the late 1970s and early 1980s, data from retrospective observational studies obtained during outbreaks in pediatric practices, a school, and a sporting event suggested transmission through aerosols [14 ,15-17,18 ] . indeed, those studies showed that most secondary cases never came in direct contact with the index patient and some were never even simultaneously present in the same area as the index case [14 ,18 ] . examination of airflow in the pediatricians' offices showed that aerosols were not only dispersed over the entire examination room but also accumulated in the hallway and other areas [14 ,18 ] . furthermore, based on the investigation of air circulation in a sport stadium, in which a mv outbreak occurred, authors suggested that mv had been dispersed through the ventilation system [16] . thus it was concluded that mv can be transmitted via aerosols. although coughing is a common symptom associated with measles disease, index patients were described to cough inter-human transmission of respiratory viruses kutter et al. 143 table 1 commonly accepted respiratory routes of transmission [18 ] . superspreaders are individuals who are able to infect a disproportionally large number of susceptible contacts when compared to a typical individual [19] [20] [21] [22] , which may contribute to the efficient transmission of mv. table 3 overview of the methods to study human-to-human transmission and their respective pro's and con's usually not conclusive on transmission route or relative importance of transmission routes. [120] [121] [122] [123] outbreak reportaircraft relatively easy to perform outbreak in closed setting retrospective which can result in recall-bias and hard to trace back passenger movements. inconclusive. only reported in case of secondary infections and in these cases infections may also occur before or after the flight. [118, [124] [125] [126] [127] non-pharmaceutical intervention can help to discriminate between transmission routes if performed properly. usually no controlled environment. difficult to determine ideal time-point of the intervention. risk of drop-out or perseverance. [35, [128] [129] [130] [131] pharmaceutical intervention characterization of droplet/ aerosol size. can be used in parallel with human studies or outbreaks. can gain information on possible aerosol spread. in a nosocomial setting aerosol-generating procedures can play a major role. frequently only detection by pcr. direct human-to-human transmission is not studied (circumstantial). technical issues (procedure may affect virus viability) or false interpretation. visualize airstream usually performed retrospectively and not during outbreaks [134, 135] computational modeling/simulation describes transmission in a greater context. can account for heterogeneity of transmission within a population. human mannequins can be used as replacement for humans theoretical (for mathematical modeling). artificial setting. [82, [136] [137] [138] [139] [140] [141] there is a substantial lack of (experimental) evidence on the transmission routes of piv (types 1-4) and hmpv. for both viruses, contact and droplet transmission are commonly accepted transmission routes [23] [24] [25] . however, only virus stability on various surfaces has been investigated so far and it has been shown that piv and hmpv are stable on non-absorptive surfaces and can barely be recovered from absorptive surfaces [26-30]. transmission of rsv among humans is thought to occur via droplets and fomites [1, 7] . in the 1980s three potential transmission routes of rsv were studied in humans by dividing infected infants and healthy volunteers into three groups, representing: firstly, all transmission routes, secondly, transmission via fomites and finally, airborne transmission by allowing the volunteers to have either, firstly, direct contact with infants (cuddlers), secondly, touching potential fomites (touchers) or finally, sitting next to the infant (sitters). volunteers in the group of the cuddlers and touchers but not the sitters became infected, suggesting that direct contact and droplet transmission were the probable routes for efficient infection of the volunteers and that transmission via aerosols was less likely [31] . another study on the transmission via fomites showed that rsv could be recovered from countertops for several hours, but only for several minutes from absorptive surfaces such as paper tissue and skin [32 ] . later on, in the late 1990s, aintablian et al. detected rsv rna in the air up to 7 m away from a patient's head [33] . in spite of that, since virus infectivity could not be demonstrated, potential airborne transmission of rsv has been considered negligible and transmission of rsv was thought to occur mainly through contact and droplet transmission. however, in a recent study authors were able to collect aerosols that contained viable virus from the air around rsv infected children [34 ] . although the detection of viable virus in the air is by itself not enough to confirm aerosol transmission, the general presumption that rsv exclusively transmits via droplets should be reconsidered and explored further. . in a three-day rhinovirus experiment with healthy volunteers different exposure modes were used to investigate the rhinovirus transmission route: firsrtly, smallparticle exposure (separating donor and recipients by wire mesh), secondly, large particle exposure (encouraging contact, coughing and sneezing while wearing gloves) and finally, direct contact exposure (hand contact followed by self-inoculation). from the results it was concluded that direct contact was the main transmission route [36] . furthermore, rhinovirus rna was detected in offices by air sampling studies and subsequent sequencing resulted in a matched air-mucus pair [37] . in a miniature field trail, experimentally infected donors with severe colds participated in a card game with susceptible recipients for 12 hours [38 ,39 ,40]. a restraining device, preventing touching of the head and face, was used in the aerosol condition and heavily contaminated cards and exaggerated hand-to-face movements in the fomite condition. in these experiments aerosol transmission was suggested [40] . in general, transmission rates and exposure time varied between studies, which may contribute to the different routes of transmission that were observed. therefore, the donor-hours of exposure was determined using donors with severe rhinovirus infections. at 200 hours of exposure to donors, transmission had occurred to 50% of the susceptible recipients, though the transmission route itself was not investigated [38 ]. due to the severity of the yearly influenza epidemics and the potential of zoonotic influenza a viruses to cause severe outbreaks, there have been many studies on influenza a virus transmission among humans. different kinds of studies, such as air sampling and intervention studies, as well as human challenge studies have been conducted. in addition, transmission events have been described extensively after outbreaks in aircrafts, households and hospital settings. however, until today, results on the relative importance of droplet and aerosol transmission of influenza viruses stay inconclusive and hence, there are many reviews intensively discussing this issue [10, [45] [46] [47] [48] [49] [50] . already in the mid-1900s human challenge models were used to assess the transmission route of influenza virus [51 ,52-54]. it was shown that illness outcome is dependent on the inoculation route and tends to be milder in intranasally infected volunteers in comparison to inoculation through inhalation [52, 53] . furthermore, illness seemed to be milder in experimentally infected volunteers than in naturally infected individuals [51 ] . increasing numbers of studies focused on the detection and quantification of influenza viruses contained in droplets and aerosols expelled into the air through breathing, sneezing and coughing of infected individuals the sars outbreak was primarily linked to healthcare settings, with 49% of the cases linked to hospitals [71] , most probably caused by aerosol-generating procedures on severely ill patients [72, 73] . aerosol-generating procedures like intubation, the use of continuous positivepressure ventilation and drug delivery via nebulizers are likely to produce 'fine infectious droplets', which travel further than droplets from coughs [74] . additionally, superspreading events contributed to the dispersion of the sars outbreak [73, [75] [76] [77] , particularly in the hotel metropole and the prince of wales hospital in hong kong [76] . moreover, a link with transmission to healthcare workers was observed when they were in close proximity (<1 m) to an index patient, suggesting direct contact or droplet transmission [73,78 ,79 ] . air samples and swabs from frequently touched surfaces in a room occupied by a sars patient tested positive by pcr, although no virus could be cultured from these samples [80] . in the amoy gardens outbreak fecal droplet transmission was suggested [81, 82] . to date, there is little data on the human-to-human mers-cov transmission route [83] . mers-cov remained stable on non-absorptive for 8 up to 48 hours and for 10 min at 20 c and 40% relative humidity in aerosols [84] . mers-cov outbreaks in humans are, like those with sars-cov, primarily linked to healthcare settings, with a link to hospitals in 31% of the cases [71, 85, 86] and healthcare associated human-to-human transmission was observed [87, 88] . superspreader events were shown to play an important role in nosocomial outbreaks [71, 89] . virus was isolated from environmental samples in hospital rooms, suggesting direct contact or fomite transmission. moreover, the airborne potential of mers was investigated by air sample analysis [90, 91 ] . viral rna was detected on the inlet of air ventilation equipment [90] and virus was isolated from air samples and surfaces from inaccessible areas like the ventilator exit, implicating potential aerosol transmission [91 ] . human adenoviruses can cause respiratory disease (mainly type 1-5, 7, 14 and 21) [92, 93] , conjunctivitis or infantile gastroenteritis (type 40 and 41) [94] . they are a common cause of respiratory illness and pneumonia in children [95, 96] , whereas infections are generally asymptomatic in adults [92] . adenoviruses cause nosocomial outbreaks, especially in pediatric care facilities, where they spread rapidly [95, 97, 98] . moreover, adenovirus type 4 and 7 are responsible for large outbreaks of acute respiratory disease, especially in crowded conditions. this is illustrated by, for example, outbreaks among military recruits for which airborne spread was suggested [92, 94, 99] . it is difficult to eliminate adenovirus from skin, fomites and environmental surfaces [100] . an outbreak in a mental care facility was probably enhanced by spending the day mainly in a crowded room while sharing cigarettes and soda cans, suggesting indirect fomite spread [101] . in a study published in 1966, experimental infections with adenovirus administered as aerosols (0.3-2.5 mm) or droplets (15 mm) to healthy, male inmates, resulted in infection of all volunteers, although the resulting illness resembled a natural infection only in the aerosol group [102] . during a military training period, increased numbers of adenovirus infections occurred over time, which correlated with an increased detection of pcr-positive air filters. additionally, a correlation between disease and the extent of ventilation was observed, with more ventilation resulting in fewer disease cases [103 ] . in a more recent study in military recruits, positive viral dna samples were mainly obtained from pillows, lockers and rifles, although adenovirus dna was also detected in air samples. no consistent correlation between increased positive environmental samples and disease was observed [104] . studies on the transmission routes of respiratory viruses have been performed since the beginning of the 20th century [105] . despite this, the relative importance of transmission routes of respiratory viruses is still unclear, depending on the heterogeneity of many factors like the environment (e.g. temperature and humidity), pathogen and host [5, 19] . differences in virus shedding between individuals can contribute to the transmissibility rate, especially in the case of superspreaders [75, 106] . in addition, the sars-cov outbreak highlighted the impact of aerosol-generating procedures on the increased risk of human-to-human transmission [74, 107] , demonstrating that for these procedures additional containment measures are necessary. inter-human transmission has been studied under many different (experimental) conditions. a summary of the advantages and disadvantages of the different study designs (table 3 ) highlights the difficulty of human transmission experiments. as a consequence, contrasting results have been obtained for many viruses. this is also reflected in table 2 , summarizing the experimental data on inter-human transmission. besides the difficulty of performing studies under well-controlled conditions, another key issue is that often (attenuated) laboratory strains are studied in healthy adults, which does not reflect the natural circumstances and target group and hence influence the outcome of the studies. respiratory viruses are an important cause of nosocomial infections, especially in children. therefore, we consulted the guidelines on infection prevention from national [108] , european [109] , american [3, 110] and international [111]) organizations for their information on transmission routes (table 2 ) and associated isolation guidelines (figure 1) . unfortunately, terms and definitions of respiratory transmission routes and isolation guidelines are not always used in a uniform way, leaving room for personal interpretation. but more importantly, information on the transmission route does not always reflect the isolation guidelines (e.g. for piv and rhinovirus, figure 1) . as a proxy for transmission route, virus stability is often referred to in the guidelines, however, this can only imply a role for indirect contact transmission but is by no means conclusive on the transmission route. inter-human transmission of respiratory viruses kutter et al. 147 precautions such as strict hand hygiene and cough etiquette. it is important to note differences in isolation guidelines between different organizations and the lack of correlation to scientific data. the variation in described transmission routes and associated isolation guidelines among the different organizations underscores the lack of convincing data. well-designed human infection studies could be employed to investigate the role of transmission routes of respiratory viruses among humans [112 ] . however, since human transmission experiments are very challenging, animal transmission models can provide an attractive alternative and should be explored and developed for all respiratory viruses. in such experiments, the influence of environmental factors on transmission routes can also be investigated [113] . however, before extrapolating experimentally generated data to humans, it is important to understand the limitations of these models, and appreciate the heterogeneity of experimental setups employed in laboratories [114] . furthermore, quantitative data such as viral load in the air can be obtained by air sampling methods in various environments, such as hospital settings. air sampling of viruses is an increasingly used technology in animal and human experiments. however, whereas most studies rely on the detection of viral genome copies, viability assays such as plaque assays or virus titration should be included to gain information on virus infectivity. ultimately, the knowledge gap on inter-human transmission should be filled by developing and performing stateof-the art experiments in a natural setting. combined with animal transmission models and air sampling in different (health care and experimental) settings, these data should result in a thorough scientific understanding of the inter-human transmission routes of respiratory viruses. eventually, this knowledge will help with an evidence-based risk assessment of the different transmission routes to improve existing infection prevention strategies. papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest infection prevention and control of epidemic and pandemic-prone acute respiratory diseases in health care: who interim guidelines guideline for isolation precautions: preventing transmission of infectious agents in health care settings the role of particle size in aerosolised pathogen transmission: a review inactivation of influenza a viruses in the environment and modes of transmission: a critical review influenza virus aerosols in human exhaled breath: particle size, culturability, and effect of surgical masks aerosol transmission of influenza a virus: a review of new studies environmental factors affecting the transmission of respiratory viruses aerobiology and its role in the transmission of infectious diseases drivers of airborne human-to-human pathogen transmission measles outbreak in a pediatric practice: airborne transmission in an office setting detailed retrospective analysis including airflow studies of a measles outbreak in a pediatric practice an explosive point-source measles outbreak in a highly vaccinated population, modes of transmission and risk factors for disease an outbreak of measles at an international sporting event with airborne transmission in a domed stadium airborne spread of measles in a suburban elementary school airborne transmission of measles in a physician's office detailed retrospective analysis including airflow studies and a mathematical airborne transmission model of a measles outbreak in a physician's office super-spreaders in infectious diseases spatial and temporal dynamics of superspreading events in the 2014-2015 west africa ebola epidemic superspreading and the effect of individual variation on disease emergence an epidemic of measles in southern greenland, 1951; measles in virgin soil. ii. the epidemic proper cdc: human parainfluenza viruses (hpivs) parainfluenza viruses transmission of sars and mers coronaviruses and influenza virus in healthcare settings: the possible role of dry surface contamination stability of sars coronavirus in human specimens and environment and its sensitivity to heating and uv irradiation transmission characteristics of mers and sars in the healthcare setting: a comparative study a major outbreak of severe acute respiratory syndrome in hong kong cluster of severe acute respiratory syndrome cases among protected health-care workers -toronto protecting health care workers from sars and other respiratory pathogens: a review of the infection control literature mers, sars, and ebola: the role of superspreaders in infectious disease progress in global surveillance and response capacity 10 years after severe acute respiratory syndrome superspreading sars events cluster of sars among medical students exposed to single patient authors demonstrate sars-cov transmission in healthcare workers and a link with proximity to the index case investigation of a nosocomial outbreak of severe acute respiratory syndrome (sars) in toronto, canada authors describe the risk of sars-cov transmission regarding distance and aerosol-generating procedures during a nosocomial outbreak detection of airborne severe acute respiratory syndrome (sars) coronavirus and environmental contamination in sars outbreak units transmission and control of sars evidence of airborne transmission of the severe acute respiratory syndrome virus middle east respiratory syndrome coronavirus: review of the current situation in the world stability of middle east respiratory syndrome coronavirus (mers-cov) under different environmental conditions transmission of middle east respiratory syndrome coronavirus infections in healthcare settings mers-cov outbreak in jeddah -a link to health care facilities hospital outbreak of middle east respiratory syndrome coronavirus clinical features and viral diagnosis of two cases of infection with middle east respiratory syndrome coronavirus: a report of nosocomial transmission middle east respiratory syndrome coronavirus superspreading event involving 81 persons environmental contamination and viral shedding in mers patients during mers-cov outbreak in south korea extensive viable middle east respiratory syndrome (mers) coronavirus contamination in air and surrounding environment in mers isolation wards first time that mers-cov was demonstrated in air samples from outbreak units. virus was also detected on fomites and environmental surfaces how contagious are common respiratory tract infections? human adenovirus associated with severe respiratory infection fields virology evolution of an adenovirus outbreak in a multidisciplinary children's hospital hospital-acquired adenovirus 7h infantile respiratory infection in chile outbreak of human adenovirus type 3 infection in a pediatric long-term care facility -illinois outbreak of adenoviral infections in a longterm paediatric facility epidemiology of adenovirus respiratory infections in military recruit populations healthcare-associated atypical pneumonia multiple cases of life-threatening adenovirus pneumonia in a mental health care center aerosol-induced adenoviral illness resembling the naturally occurring illness in military recruits detection of adenoviruses (adv) in culturenegative environmental samples by pcr during an advassociated respiratory disease outbreak authors show a link between adenovirus dna in air filters and adenovirusrelated hospitalizations transmission dynamics and prospective environmental sampling of adenovirus in a military recruit setting the aerial conveyance of infection infectious disease: the tolerance of superspreaders possible sars coronavirus transmission during cardiopulmonary resuscitation wip: w.i.p. ziekenhuizen: indicatie voor isolatie manual of childhood infections: the blue book use of a human influenza challenge model to assess person-to-person transmission: proof-of-concept study a human proof-of-concept study, in which authors assessed infectivity rate of experimentally inoculated volunteers and secondary attack rate among susceptible individuals influenza virus transmission is dependent on relative humidity and temperature complexities in ferret influenza virus pathogenesis and transmission models cdc: interim infection prevention and control recommendations for hospitalized patients with middle east respiratory syndrome coronavirus (mers-cov) viruses as agents of airborne contagion effectiveness of precautions against droplets and contact in prevention of nosocomial transmission of severe acute respiratory syndrome (sars) transmission of the severe acute respiratory syndrome on aircraft transmission of experimental rhinovirus colds in volunteer married couples avian influenza h5n1 transmission in households, indonesia respiratory syncytial virus infections within families nosocomial transmission of respiratory syncytial virus in an outpatient cancer center probable secondary infections in households of sars patients in hong kong measles transmission in immunized and partially immunized air travellers low risk of measles transmission after exposure on an international airline flight influenza a(h1n1)pdm09 during air travel contact tracing for influenza a(h1n1)pdm09 virus-infected passenger on international flight facemasks and hand hygiene to prevent influenza transmission in households: a cluster randomized trial findings from a household randomized controlled trial of hand washing and face masks to reduce influenza transmission in bangkok, thailand. influenza other respir viruses feasibility of elementary school children's use of hand gel and facemasks during influenza season. influenza other respir viruses the effect of placebo and virucidal paper handkerchiefs on viral contamination of the hand and transmission of experimental rhinoviral infection short-term treatment with zanamivir to prevent influenza: results of a placebo-controlled study detection of measles virus rna in air and surface specimens in a hospital setting protection from microbial contamination in a room ventilated by a uni-directional air flow the metropole, superspreaders, and other mysteries potential for airborne transmission of infection in the waiting areas of healthcare premises: stochastic analysis using a monte carlo model relative contributions of four exposure pathways to influenza infection risk calculating the potential for within-flight transmission of influenza a (h1n1) a study quantifying the hand-to-face contact rate and its potential application to predicting respiratory tract infection factors involved in the aerosol transmission of infection and control of ventilation in healthcare premises we thank dr. rik de swart, dr. bart haagmans, dr. arno andeweg, and dr. sabrina schreiner for helpful discussions. jk and sh are supported by an nwo vidi grant (contract number 91715372), and ms, rf and sh by niaid/nih contract hhsn272201400008c. pf receives funding from the eu fp7 project prepare (grant number 602525). the sponsors had no role in the collection, analysis and interpretation of data, in the writing of the report, and in the decision to submit the article for publication. key: cord-002801-6myqgme3 authors: yoon, byung woo; lee, seung hyeun title: possible therapeutic effect of orally administered ribavirin for respiratory syncytial virus-induced acute respiratory distress syndrome in an immunocompetent patient: a case report date: 2017-12-20 journal: j med case rep doi: 10.1186/s13256-017-1514-x sha: doc_id: 2801 cord_uid: 6myqgme3 background: human respiratory syncytial virus usually causes self-limiting upper respiratory infection and occasionally causes pneumonia in immunocompromised hosts. respiratory syncytial virus-induced severe pneumonia or acute respiratory distress syndrome in immunocompetent adults has been rarely described. unfortunately, optimal treatment has not been established for this potentially fatal condition. we report a case of respiratory syncytial virus-induced acute respiratory distress syndrome occurring in a previously healthy man successfully treated with orally administered ribavirin. case presentation: an 81-year-old previously healthy korean man presented with cough, dyspnea, and febrile sensation. he had hypoxemia with diffuse ground glass opacity evident on chest radiography, which progressed and required mechanical ventilation. all microbiological tests were negative except multiplex real-time reverse transcriptase polymerase chain reaction using respiratory specimen, which was positive for human adenovirus. under the diagnosis of respiratory syncytial virus-induced acute respiratory distress syndrome, orally administered ribavirin was administered and he recuperated completely without complications. conclusion: this case demonstrates the potential usefulness of orally administered ribavirin as a therapeutic option for severe respiratory syncytial virus infection, at least in an immunocompetent host. human respiratory syncytial virus (rsv) is an enveloped ribonucleic acid (rna) virus belonging to the family paramyxoviridae. rsv is a major pathogen causing lower respiratory tract infection in babies and young children, leading to hospitalization and death [1] . in adults, it usually causes upper respiratory infection that is self-limiting. however, with recent increases in hematopoietic stem cell and solid organ transplantation, rsv infection is attracting clinical attention as a key pathogen of opportunistic infections which is associated with high mortality and morbidity [2] . rsv-induced severe pneumonia or acute respiratory distress syndrome (ards) in immunocompromised patients is not uncommon. however, it has been rarely described in immunocompetent adults. here we report a case of ards due to rsv occurring in a previously healthy adult successfully treated with orally administered ribavirin. he denied any previous medical histories. he stopped smoking tobacco 30 years ago, and never drank alcohol in recent years. his vital signs were: blood pressure 140/ 80 mmhg, heart rate 96 beats/minute, respiratory rate 22 breaths/minute, and body temperature 38.2°c. on physical examination, crackle was noted in both lungs. laboratory tests revealed a white cell count of 7800/ mm 3 with slight left shift (neutrophils 88.6%), c-reactive protein (crp) level of 223.6 mg/dl (normal < 5.0 mg/ dl), total bilirubin level of 1.5 mg/dl, and alanine transaminase and aspartate transaminase levels of 59 and 61 iu/l, respectively. his sodium level was 125 meq/ml. in arterial blood gas analysis, which was checked in ambient conditions, ph, partial pressure of carbon dioxide in arterial blood (paco 2 ), partial pressure of oxygen in arterial blood (pao 2 ), bicarbonate, and oxygen saturation levels were 7.50, 30 mmhg, 48 mmhg, 23.4 mmol/l, and 87%, respectively. the result of a test for human immunodeficiency virus was negative. serologic tests for mycoplasma and chlamydia were negative. streptococcal and legionella urinary antigens were negative. antinuclear and anti-neutrophilic cytoplasmic antibodies were negative. a chest x-ray revealed diffuse haziness dominant in his right lung field (fig. 1a) . chest computed tomography revealed ground glass opacity in both lungs with small amounts of pleural effusion dominant in the right hemithorax (fig. 1b) . with an initial assessment of community-acquired pneumonia, we administered nasal oxygen at 4l/minute and empirical antibiotics with a respiratory quinolone. at hospital day 2, thoracentesis was conducted in the right hemithorax and a turbid yellowish fluid was obtained. pleural fluid analysis revealed lymphocyte-dominant exudate with white cell count of 560/mm 3 and adenosine deaminase level of 4.4 iu/l. on the same day, opacities were found on chest x-ray and hypoxemia rapidly progressed to require high flow oxygen supply with fraction of inspired oxygen (fio 2 ) 0.8 at a flow rate of 40 l/minute (fig. 2a) . at hospital day 3, he had to be intubated and mechanically ventilated due to worsening hypoxemia. the initial pao 2 /fio 2 after application of mechanical ventilator was 65, which was compatible with the definition of "severe" ards [3] . potential cardiac dysfunction was ruled out using transthoracic echocardiography. antibiotics were escalated to carbapenem. multiplex real-time reverse transcriptase polymerase chain reaction (rt-pcr) was conducted using advansure tm respiratory virus real-time rt-pcr kit (lg life sciences, seoul, korea) to detect respiratory viruses using tracheal aspirate. results revealed positive for human rsv type b. under the diagnosis of rsv-induced ards based on the berlin definition [4] , we started antiviral therapy of orally administered ribavirin 400 mg every 12 hours with concomitant intravenously administered methylprednisolone 30 mg every 24 hours. after treatment, hypoxemia and lung lesions gradually improved. at hospital day 17, he was extubated and we tapered methylprednisolone to orally administered prednisolone 15 mg. finally, his chest x-ray cleared and he was discharged on hospital day 27 without any complications or drug-related adverse events (fig. 2b ). orally administered ribavirin was maintained until his discharge. we summarized the whole clinical course with the drugs administered in fig. 3 . rsv pneumonia in adults occurs mostly in immunocompromised patients and is characterized by rapid clinical deterioration often leading to death. however, ards due to rsv in previously healthy adults is extremely rare and only two cases have been reported to date [5, 6] . of note, patients in those cases were treated without ribavirin or with inhaled ribavirin. to the best of our knowledge, this is the first case that reports successful treatment of rsvinduced ards using orally administered ribavirin in an immunocompetent patient. rsv has been considered a less significant pathogen in adults as it usually causes mild and self-limiting upper respiratory tract infection. the clinical significance of rsv infection in adults has been acknowledged in recent years. a study has estimated that rsv infects 3 to 10% of adults annually and it may be associated with 5 to 15% of community-acquired pneumonia [7] . in a retrospective cohort study, rsv infection was complicated with pneumonia in two thirds of infected patients from which one tenth required mechanical ventilation, and the mortality rate was as high as 15 to 20% comparable to that of seasonal influenza [8] . in that study, severe rsv-related lower respiratory tract infections occurred mostly in elderly patients and those with major medical comorbidities [8] . in this case, the patient denied previous medical history and had no relevant comorbidities. therefore, the severe rsv infection may be attributable to his advanced age. similar to other viral diseases, rsv pneumonia is difficult to diagnose. its respiratory symptoms are nonspecific, and laboratory and radiologic findings are usually indistinguishable from other respiratory viral infections. definitive diagnosis of rsv can be confirmed by identification of typical plaque morphology with syncytium formation using immunofluorescent staining. however this is time consuming and costly. nucleic acid detection using multiplex real-time rt-pcr test is used in clinical practice as it enables rapid detection with increased sensitivity [9] . in this case, atypically rapid deterioration of clinical manifestations led us to suspect infections caused by atypical pathogen including viral pneumonia. therefore, we conducted the multiplex real-time rt-pcr test. although diagnostic performances of different multiplex real-time rt-pcr assays for respiratory viral infections can vary depending on devices used [10, 11] , the assay used for diagnosis of our case had revealed relatively high sensitivity and specificity for respiratory viral pathogens with performance that is comparable to other commercial assays despite shorter turnaround time [12] . in a previous study, rogers et al. reported the clinical impact of multiplex rtpcr test for respiratory viruses on clinical outcome using more than 1000 children patients including 344 rsv pneumonias [13] . they demonstrated that the use of a multiplex rt-pcr test was associated with decreased duration of antibiotic use, length of in-patient stay, and duration of isolation of patients admitted for acute respiratory infections. in addition, rappo et al. using a retrospective cohort (n = 337) had compared clinical outcomes for adult patients diagnosed by multiplex rt-pcr for respiratory viruses with those diagnosed by conventional methods at a tertiary care center [14] . in that study, influenza (63% from all isolates) and rsv (15%) were the predominant viruses identified. they found a significantly lower rate of admission, length of hospital stay, duration of antimicrobial use, and number of chest radiographs, after adjusting potential confounders in the multiplex rt-pcr group [14] . taken together, multiple rt-pcr tests are clinically useful methods for early detection of viral pathogens for respiratory tract infection and for cost effectiveness during treatment. our case was compatible with the berlin definition of ards, defined by the timing (within 1 week of clinical insult or onset of respiratory symptoms), radiographic changes (bilateral opacities not fully explained by effusions, consolidation, or atelectasis), and origin of edema (not fully explained by cardiac failure or fluid overload). the severity of our case corresponded to "severe" [4] . there is debate about the use of corticosteroids for patients with ards, and current data do not support routine use of corticosteroids in those patients [3] . however, several studies suggested that low-dose corticosteroids (1 to 2 mg/kg per day of intravenously administered methylprednisolone) may be beneficial in terms of short-term mortality for patients with ards which is less than 14 days after onset [15, 16] . our case was compatible with the definition of ards and duration of onset was less than 3 days, thus we started 30 mg of methylprednisolone intravenously. a current guideline states that a low dose of systemic steroid used in the early stage may improve hypoxemia and reduce the period of mechanical ventilation, length of intensive care unit (icu) stay, and mortality [3] . optimal treatment duration of corticosteroids has not been established, as corticosteroids were used for different durations at different trials. we maintained the initial dose of methylprednisolone by the time of his extubation and slowly tapered it until his discharge. the clinical benefits of corticosteroids for respiratory virus-associated ards as well as their optimal dose or treatment duration need to be elucidated. of note, pathogens other than rsv could have contributed to the development of his ards and a spontaneous resolution could have contributed to the outcome. however, his atypical clinical presentation and no evidence of other disease or infectious pathogen except rsv after vigorous microbiologic examination led us to suspect rsv-related ards. optimal treatment for rsv pneumonia has not been established. oral neuraminidase inhibitors have been widely used in severe influenza infection, however, they failed to show efficacy against paramyxoviridae family viruses including rsv. aerosolized ribavirin, a nucleoside analogue with broad antiviral activity, has been reported to be effective in preventing severe pneumonia in non-influenza respiratory viral infections in babies and children [17] . however, there is concern about its use due to its high cost, teratogenicity, and potentially administered risk of lung function decline. in addition, intravenously administered or aerosolized ribavirin unfortunately is not readily available in our country because we can acquire this agent only through the korea orphan & essential drug center (koedc), which may cause delay in the start of treatment. meanwhile, orally administered ribavirin is relatively safe and economic, and several reports have suggested that it is associated with favorable clinical outcomes in rsv infection [18, 19] . thus, we selected orally administered ribavirin with systemic corticosteroids for our case. our treatment is supported by a previous study reporting that orally administered ribavirin and corticosteroid were a well-tolerated and cost-effective regimen for lung and heart/lung transplant recipients with paramyxovirus infection [20] . this case suggests that orally administered ribavirin may be an option, although not optimal treatment, even for cases of severe rsv, especially in a situation where other forms of ribavirin are not readily available. rsv-induced ards is very uncommon but can be lethal in immunocompetent patients. the present case highlights the significance of early clinical suspicion and active use of multiplex real-time rt-pcr test. in addition, this case suggests that orally administered ribavirin could be a therapeutic option even for severe pneumonia or ards due to rsv, at least in immunocompetent hosts, especially if other antiviral agents are unavailable. future studies are needed to determine its efficacy for selected patients. global burden of acute lower respiratory infections due to respiratory syncytial virus in young children: a systematic review and meta-analysis respiratory syncytial virus infection in recipients of allogeneic stem-cell transplantation: a retrospective study of the incidence, clinical features, and outcome clinical practice guideline of acute respiratory distress syndrome acute respiratory distress syndrome: the berlin definition respiratory syncytial virus infection: a cause of respiratory distress syndrome and pneumonia in adults inhaled ribavirin therapy in adult respiratory syncytial virus-induced acute respiratory distress syndrome respiratory syncytial virus infection in adults high morbidity and mortality in adults hospitalized for respiratory syncytial virus infections rapid diagnosis of respiratory syncytial virus infections in immunocompromised adults multiplex pcr point of care testing versus routine, laboratory-based testing in the treatment of adults with respiratory tract infections: a quasi-randomised study assessing impact on length of stay and antimicrobial use respiratory syncytial virus infection control challenges with a novel polymerase chain reaction assay in a comparison of the advansure real-time rt-pcr and seeplex(®) rv12 ace assay for the detection of respiratory viruses impact of a rapid respiratory panel test on patient outcomes impact of early detection of respiratory viruses by multiplex pcr assay on clinical outcomes in adult patients use of corticosteroids in acute lung injury and acute respiratory distress syndrome: a systematic review and meta-analysis steroid treatment in ards: a critical appraisal of the ards network trial and the recent literature respiratory viruses other than influenza virus: impact and therapeutic advances oral ribavirin therapy for lower respiratory tract infection of respiratory syncytial virus complicating bronchiolitis obliterans after allogeneic hematopoietic stem cell transplantation successful treatment of parainfluenza virus 3 pneumonia with oral ribavirin and methylprednisolone in a bone marrow transplant recipient single-centre experience with oral ribavirin in lung transplant recipients with paramyxovirus infections an 81-year-old korean man visited our out-patient clinic complaining of cough, dyspnea, and febrile sensation. the present study was not funded. the dataset supporting the conclusions of this article are included within the article.authors' contributions bwy contributed to diagnoses and management, collected data, and wrote and reviewed the manuscript. shl contributed to the study design and reviewed the manuscript. both authors read and approved the final manuscript.ethics approval and consent to participate this case report was performed in accordance with international ethical rules. written informed consent was obtained from the patient for publication of this case report and any accompanying images. a copy of the written consent is available for review by the editor-in-chief of this journal. the authors declare that they have no competing interests.• we accept pre-submission inquiries • our selector tool helps you to find the most relevant journal submit your next manuscript to biomed central and we will help you at every step: key: cord-252037-rj61mzqj authors: gerna, g.; campanini, g.; rovida, f.; sarasini, a.; lilleri, d.; paolucci, s.; marchi, a.; baldanti, f.; revello, m. g. title: changing circulation rate of human metapneumovirus strains and types among hospitalized pediatric patients during three consecutive winter-spring seasons date: 2005-06-28 journal: arch virol doi: 10.1007/s00705-005-0581-2 sha: doc_id: 252037 cord_uid: rj61mzqj from 2001 through 2004, 808 pediatric patients admitted to hospital because of acute respiratory infections were examined for presence of respiratory viruses by either direct fluorescent staining using monoclonal antibodies or rt-pcr during three consecutive winter-spring seasons. on the whole, 336 (42%) patients were detected as positive for one or more respiratory viruses. the most widely circulating virus was human respiratory syncytial virus (hrsv) infecting 50% of positive patients, followed by human metapneumovirus (hmpv) found in 13% of patients, and then by influenza virus type a, human parainfluenzaviruses and coinfections. significant variations in the circulation rate of hrsv, hmpv and influenzavirus type a were observed during the individual seasons. in addition, the circulation rates of the different types of hmpv changed yearly. in 2001–2002 and 2002–2003 hmpv circulated at a significant lower proportion than hrsv, while in 2003–2004 the circulation rates of the two viruses were closer. in conclusion, the 4 hmpv subtypes circulated yearly in northern italy flanking hrsv as major respiratory pathogens in the infantile patient population. young children are among patients most severely affected by hmpv infections [2, 12, 22] . however, several epidemiological features of hmpv infections remain to be investigated. in particular, differences in the circulation rate of hmpv strains as well as different hmpv types and subtypes during different seasons, and the relative pathogenicity of hmpv with respect to human respiratory synctial virus (hrsv) remain to be defined. in this study, we examined: i) the circulation rate of hmpv among the other respiratory viruses during 3 consecutive winter-spring seasons; ii) the relative circulation of the 2 types and the 4 subtypes of hmpv during the same 3-year period; iii) the relative impact of hmpv as compared to hrsv in determining admission to the hospital of infants with acute respiratory infections. from december 2001 through november 2004, 808 infants and young children were admitted to hospital because of an acute respiratory virus infection. all patients had fever, cough, and lower respiratory tract involvement. nasopharyngeal aspirates (npa) were collected upon admission and then divided into 4 aliquots: one was used for molecular assays (rt-pcr), the second for direct fluorescent antibody (dfa) staining of respiratory cells, the third for short-term and long-term virus isolation in cell cultures, and the fourth was frozen as a back-up sample [13] . specimens were examined for influenzaviruses a and b, parainfluenzaviruses (hpiv) 1 to 3, hrsv, and human adenoviruses (hadv) by dfa and virus isolation in cell cultures. in addition, hcovs, group i (229e-like) and ii (oc43-like), and hmpv types a and b were sought by rt-pcr. typing of hmpv strains was achieved by sequencing and phylogenetic analysis. similarly, grouping of hcovs was achieved by sequencing. the study was approved by the irccs policlinico san matteo ethics committee. the simultaneous detection of 2 respiratory viruses in the same npa was referred to as coinfection. rapid virus recovery was achieved in cell cultures by inoculating npa samples onto 2 shell vials of a mixture of a549 and mv1lu (1:1) cells [9] , and 2 shell vials of both llc-mk2 and mdck cells, which were stained with mabs at 2 and, if required, 7 days p.i. conventional virus recovery was obtained following inoculation of 2 tubes of llc-mk2 and mdck cells, which were stained with mabs 7 days p.i. or, if required, during the following 2 weeks. a pool of fluorescein-labeled mabs to conventional respiratory viruses (influenzavirus a and b, hpiv 1-3, hrsv, and hadv), as well as the single fluorescein-conjugated mabs included in the pool (purchased from chemicon international, inc., temecula, ca) were used for both dfa and virus identification in cell cultures. following rt reaction, pcr assays for the identification of hcovs and hmpv strains were optimised to detect at least 10 input plasmid copies. to this purpose, primers for hcov (groups i and ii) were selected from a published protocol [12a] , whereas primers for hmpv types a and b (genes n and f) were originally designed from genbank published virus sequences (table 1) . amplification products were cloned in pcr2.1 plasmid vector (ta cloning kit, invitrogen, carlsbad, ca) and quantitative standards were obtained following titration of insert-containing plasmids [13] . thus, defined amounts of target sequences could be amplified in parallel with clinical samples. nucleic acids were extracted using nuclisens ® iso kit (biomerieux, lyon, france). rt and pcr reactions were performed as reported [13] . pcr products were examined on 3% agarose gel. classification of hmpv strains of this study into types a and b, and subtypes a1-a2, and b1-b2, was achieved by phylogenetic analysis of 2 fragments of both genes f and n of hmpv strains, respectively. viral sequences of the amplified gene f (nt 661 to1094), and gene n (nt 121 to 562) fragments of hmpv isolates as well as reference strains were aligned with the clustal w program version 1.7, whereas sequence similarity comparisons were carried out with the megalign program (dnastar inc., madison, wi). the philips (njplot) program was used to construct phylogenetic trees with nucleotide sequences by means of the neighbour-joining method from the same distance matrices. bootstrap support was determined by 100 resamplings of the sequences. statistical differences in the circulation rate of respiratory virus infections among the 3 consecutive seasons were determined by the chi square test. the relative distribution of different respiratory viruses causing severe infections requiring admission to the hospital of infants and young children in three consecutive winter-spring seasons from 2001 through 2004 is reported in table 2 within the aliquot of patients found positive for some respiratory virus, no difference in the circulation rate was observed for respiratory virus infections caused by influenzavirus b, hpivs, hadvs, hcovs, and coinfections along the three years studied. on the contrary, significant differences in the circulation rate table 2 were found for influenzavirus a, hrsv, and hmpv (table 2) . however, while hrsv and hmpv infections predominated during the first 3 months of age, the other infections were evenly distributed during the first 5 years of age. (table 2) . surprisingly, in the group of 33 coinfections, simultaneous infection of the same patient by hmpv and hrsv was observed in a single case in 2003-2004 ( table 3) . the circulation rate of hmpv reached its peak (fig. 4) . due to the high similarity of the pathology caused in newborns and infants, the circulation rates of hmpv and hrsv were analysed by comparing the relevant number of infected patients admitted to hospital in the winter-spring (december through may) and summer-fall (june through november) seasons of each year. both viruses were nearly absent during the summer-fall seasons, whereas they were highly circulating, at a different proportion, during the winter-spring seasons. although the incidence of hrsv infections in pediatric patients admitted to the an epidemiological survey on the circulation rate of the most important respiratory viruses responsible for admission to the hospital of infants and young children in a defined geographical area during the 3-year period from 2001 through 2004 was conducted using npa samples as clinical specimens. in order to contain costs, on the basis of recently acquired results showing a comparable sensitivity of the molecular (rt-pcr) and the immunological (monoclonals) diagnostic approaches [13] , influenzaviruses a and b, hpiv 1-3, hrsv, and hadvs were detected by monoclonal antibodies, while hcovs and hmpvs were detected by rt-pcr. while no significant difference was observed in the circulation rate of the majority of respiratory viruses, a significant difference in the circulation of influenzavirus a, hrsv and hmpv, was found during the 3 consecutive winterspring seasons. apart from influenzaviruses a, which are known to circulate differently in different years, hmpv and hrsv circulated at a stable rate in the first 2 seasons examined. on the contrary, the relative proportion of patients infected by hrsv decreased sharply in the most recent season, while that of patients infected by hmpv increased significantly. as a consequence, the number of young patients admitted to the hospital because of lower respiratory tract infections caused by hmpv increased accordingly. yearly variation in the circulation rate of hmpv has been recently reported [7, 8, 11, 19] . besides a striking variation in the overall circulation rate of hmpv, a significant variation in the circulation of the 4 hmpv subtypes was observed during the study period. whether different hmpv types and subtypes may be differentiated only on the basis of nucleotide sequence (phylogenetic analysis) or may represent true serotypes or subserotypes remains to be determined. in fact, while ferret immune sera have been reported to identify two distinct serotypes (a and b) showing a ≥16fold dilution difference between homologous and heterologous neutralizing antibody titer [18] , several primate as well as hamster immune sera did not show any discriminating capacity [15] . similarly, in our experience, as already reported by others [16] , guinea pig immune sera were not able to identify two distinct serotypes. thus, at the moment, the terms types and subtypes indicate genotypes and subgenotypes rather than serotypes and subserotypes. in a recent study by our group, using a fragment of gene n (nt 80 to 596), it was possible to identify 8 amino acid substitutions at positions 14, 31, 52, 56, 63, 66, 70, and 96, that might be used to differentiate type a from type b strains [14] . monoclonal antibodies reactive with epitopes containing these differential amino acids might discriminate between the two hmpv types. the simultaneous circulation of hrsv and hmpv in the same period of the year (winter-spring season) and in the same patient population (infants and young children), along with a comparable degree of pathogenicity, could hypothetically facilitate coinfections by these two members of the paramyxovirinae subfamily. in a recent population-based prospective multicenter study of the children requiring intensive care conducted in germany over 2 years, 18% had hmpv infections, and 60% of these children were infected with hmpv in combination with hrsv, whereas hmpv was not detected in a large series of hrsv-positive children not requiring intensive care support. this data support the hypothesis that coinfections with hrsv and hmpv are more severe than infections with either hrsv or hmpv alone, at least in children less than 3 years of age [10] . in our study, outside the intensive care unit, surprisingly a single case of coinfection by hmpv and hrsv was found, thus suggesting that this coinfection is very infrequent in the immunocompetent host. with reference to the italian pediatric population admitted to hospital during the winter-spring season, the following conclusions can be drawn from our epidemiological study: i) hmpv strains circulate at a different rate in different years; ii) changes in the prevalence of hmpv types and subtypes occur yearly; iii) the prevalence of hmpv versus hrsv infections may also change significantly in different years. virological features and clinical manifestations associated with human pneumovirus: a new paramyxovirus responsible for acute respiratory-tract infections in all age groups human metapneumovirus infections in hospitalized children global genetic diversity of human metapneumovirus fusion gene human metapneumovirus infection in the canadian population human metapneumovirus detection in patients with severe acute respiratory syndrome evidence of a novel human coronavirus that is associated with respiratory tract disease in infants and young children human metapneumovirus infections in young and elderly adults evidence of human metapneumovirus in children in argentina mink lung cells and mixed mink lung and a549 cells for rapid detection of influenza virus and other respiratory viruses prospective study of human metapneumovirus infection in children less than 3 years of age human metapneumovirus associated with respiratory tract infections in a 3-year study of nasal swabs from infants in italy children with respiratory disease associated with metapneumovirus in hong kong identification of severe acute respiratory syndrome in canada monoclonal antibodies versus reverse transcription-pcr for detection of respiratory viruses in a patient population with respiratory tract infections admitted to hospital detection and pathogenicity of human metapneumovirus respiratory infections in pediatric italian patients during a winter-spring season the two major human metapneumovirus genetic lineages are highly related antigenically, and the fusion (f) protein is a major contributor to this antigenic relatedness analysis of the genomic sequence of a human pneumovirus a newly discovered human pneumovirus isolated from young children with respiratory tract disease antigenic and genetic variability of human metapneumoviruses prevalence and clinical symptoms of human metapneumovirus infection in hospitalised patients identification of a new human coronavirus human metapneumovirus and lower respiratory tract disease in otherwise healthy infants and children author's address: prof. giuseppe gerna, servizio di virologia, irccs policlinico san matteo this work was partially supported by grants from ministero della salute, ricerca finalizzata irccs policlinico san matteo (convenzione n. 118), and ricerca corrente (research code 80557). we thank daniela sartori for preparing the manuscript and linda d'arrigo for revision of the english. we are also indebted to dr. massimo fabbi for preparing guinea pig hyperimmune sera. key: cord-259927-xh9cw9ao authors: papadopoulos, nikolaos g.; megremis, spyridon; kitsioulis, nikolaos a.; vangelatou, olympia; west, peter; xepapadaki, paraskevi title: promising approaches for the treatment and prevention of viral respiratory illnesses date: 2017-07-21 journal: j allergy clin immunol doi: 10.1016/j.jaci.2017.07.001 sha: doc_id: 259927 cord_uid: xh9cw9ao viral respiratory tract infections are the most common human ailments, leading to enormous health and economic burden. hundreds of viral species and subtypes have been associated with these conditions, with influenza viruses, respiratory syncytial virus, and rhinoviruses being the most frequent and with the highest burden. when considering prevention or treatment of viral respiratory tract infections, potential targets include the causative pathogens themselves but also the immune response, disease transmission, or even just the symptoms. strategies targeting all these aspects are developing concurrently, and several novel and promising approaches are emerging. in this perspective we overview the entire range of options and highlight some of the most promising approaches, including new antiviral agents, symptomatic or immunomodulatory drugs, the re-emergence of natural remedies, and vaccines and public health policies toward prevention. wide-scale prevention through immunization appears to be within reach for respiratory syncytial virus and promising for influenza virus, whereas additional effort is needed in regard to rhinovirus, as well as other respiratory tract viruses. discuss this article on the jaci journal club blog: www.jacionline.blogspot.com. the respiratory system is one of the main portals of entry for human pathogens. although precise calculations are challenging because of methodology and inherent variability, the number of potentially infectious viruses we breathe every day can be in the range of many thousands. 1 thus it is not surprising that viral respiratory tract infections (vrti) are the most common human diseases, leading to enormous health and economic burden. 2 a wide variety of conditions fall within the spectrum of vrtis. many of these are by themselves major public health concerns: influenza, acute bronchiolitis, viral pneumonia, and common colds. together with their downstream effects (ie, acute exacerbations of asthma and chronic obstructive pulmonary disease [copd] ), all result in vast amounts of morbidity, mortality, and health care costs, including primary care visits, hospitalizations, and deaths but also inappropriate use of antibiotics, loss of productivity, and effects on quality of life. [3] [4] [5] respiratory tract viruses have been isolated and characterized during the last century, starting from influenza virus (ifv) in the 1930s and followed by respiratory syncytial virus (rsv), coronaviruses, adenoviruses, and rhinoviruses in the 1950 to 1960s; nevertheless, ''new'' viruses or subtypes, such as human metapneumovirus or rhinovirus c, are still being identified. 6, 7 even though several of these viruses are typically associated with a clinicopathologic entity (eg, ifv with influenza, rsv with bronchiolitis, and rhinovirus with the common cold), there is also extensive overlap, and it is often difficult to identify the etiologic agent based on clinical grounds alone. 8 consequently, when considering prevention and treatment of vrti, potential targets include specific pathogens, the immune response, disease transmission, or just symptoms. here we provide an overview of the options and highlight some of the most promising approaches in vrti treatment, including symptomatic medication, immunomodulatory drugs, antiviral agents, and natural products, as well as in vrti prevention, ranging from vaccines to immunostimulators and public health policies. this is a vast field, and thus we emphasize advances that might be relevant in tackling the virus-induced aspects of allergic disease, such as asthma exacerbations. most mild viral respiratory illnesses are managed symptomatically with over-the-counter medications, such as nasal decongestants, antipyretics/analgesics, antitussives, or expectorants, on which no major improvements are foreseen. although generally well tolerated for short-term relief, some agents can have adverse effects, especially in young children. therefore the us food and drug administration has issued a warning against the use of over-the-counter cough and cold products in children younger than 4 years of age. 9 furthermore, the use of decongestants should be minimized, especially in children, 10 whereas codeine has been restricted in children by the european medical agency since 2015. 11 selective cox inhibitors, such as celecoxib and mesalazine, have been widely used in clinics for their antipyretic, analgesic, and antiinflammatory properties in patients with airway diseases, 12 whereas their combination with neuraminidase inhibitors (nais) has significantly improved the survival of ifv-infected mice. 13 recent studies have revealed a new genus of specialized proresolving lipid mediators (spms), including lipoxins, resolvins, protectins, and maresins, enhancing anti-inflammatory, antiviral, and proresolving mechanisms. 14 medications interfering with prostanoid and lipoxygenase biosynthesis and signaling, thus affecting resolution and spm switching, such as aspirin and nonsteroidal anti-inflammatory drugs, have been suggested as potential agents modulating antiviral immunity, 15, 16 whereas several spm resolution agonists are in clinical development programs. symptomatic relief can also be sought in severe cases. noninvasive ventilation can reduce respiratory distress in patients with acute viral bronchiolitis. 17 very recently, new devices delivering totally conditioned gas (378c at 100% relative humidity) through a very high-flow nasal cannula (up to 60 l/min) have been indicated for bronchiolitis mainly as rescue therapy to reduce the need for admission to the intensive care unit. 18 immune and antiviral pathway modulators although vrtis are most often short-lived events, impaired antiviral clearance and/or activation of inflammatory pathways lead to important downstream complications, such as exacerbations of asthma or copd. 19 the immune and antiviral mechanisms leading from infection to exacerbation have been scrutinized, 20 and medications targeting these pathways are being evaluated as promising candidates to reduce disease burden. impaired interferon production has been observed in patients with various obstructive respiratory diseases, potentially contributing to enhanced susceptibility to and/or severity of virus-induced acute airway exacerbations. 21 although inhaled ifn-b supplementation has not shown a clear effect in preventing virus-induced symptom worsening in patients with mild asthma, subanalysis in patients with severe asthma showed a protective effect. 22 interestingly, in an experimental model exogenous administration of ifn-l1 induced a strong and more prolonged antiviral state than ifn-b. 23 moreover, experimental studies in an allergic asthma model showed that ifn-l supplementation enhanced t h 1 immunity by inducing ifn-g and suppressing t h 2 and t h 17 responses through modulation of lung cd11c 1 dendritic cell function. 24, 25 novel antibody-based drugs with antirhinovirus and immunomodulatory effects act through ifn-b induction and suppression of t h 2 responses in experimental models. 26 the prototype synthetic toll-like receptor (tlr) 4 antagonist eritoran (e5564) and anti-tlr4 igg therapy have been shown to block ifv lethality in mice by suppressing lung pathology, clinical symptoms, and viral titers. 27, 28 other innate immune receptors, such as tlr2, also have potential for host-targeted therapeutic approaches. 21 interestingly, omalizumab, an anti-ige mab, prevents asthma exacerbations either by decreasing the duration and shedding of rhinovirus infection or by blocking the synergistic effect of rhinovirus infection on allergy. 29, 30 because high-affinity ige receptor (fcεri) cross-linking on plasmacytoid dendritic cells reduces ifn-a responses after viral infections, it is plausible that omalizumab enhances virus-induced ifn-a production in asthmatic patients, thus limiting virus spreading and infection severity. 31 ''severe cytokine storm,'' an entity associated with markedly higher levels of proinflammatory cytokines, has been associated with severe influenza infections; immunomodulatory agents have been proposed as potential therapeutic strategies. 32 peroxisome proliferator-activated receptor g agonists (eg, rosiglitazone and pioglitazone) are critical regulators of inflammation and have been promising in improving the clinical outcome of severe influenza infections. 33 their development slowed down from 2000 to 2005 because of possible cardiovascular side effects; however, in 2015, the us food and drug administration lifted restrictions based on new safety data. 34 moreover, sphingosine-1-phosphate receptor 1 agonists 1, which are located mainly on pulmonary endothelial cells, exhibit cytokine storm-blunting activity by suppressing both innate cellular and cytokine/chemokine responses, particularly when combined with antiviral agents. 35 there is increasing interest in the use of macrolides to treat or prevent virus-induced asthma exacerbations, although microbial resistance remains a major hurdle, and therefore they are not currently indicated. early in vivo evidence suggested that azithromycin has anti-inflammatory and antiviral effects through induction of interferon-stimulated gene mrna expression and reduced viral replication and release in patients with asthma and chronic obstructive lung disease. 36, 37 in a randomized clinical trial including wheezing preschool-aged children, early azithromycin administration significantly reduced the likelihood of a severe lower respiratory tract infection. 38 novel macrolides (mycobacterium avium complex 5) with anti-inflammatory, antibacterial, and, more importantly, interferon-augmenting activity in airway epithelium have been identified. 39 finally, in vitro models have demonstrated that a 1 -antitrypsin exerts anti-inflammatory effects in airway epithelial cells from rhinovirus-infected patients with copd, potentially through inhibition on caspase-1 activity, suggesting a 1 -antitrypsin as a potential anti-inflammatory agent. 40 antivirals vrtis are usually characterized by an acute and self-limiting course, which means that the peak of viral replication usually precedes or parallels the appearance of clinical symptoms. as a result, the time window from verification and/or typing of the pathogen, allowing a specific therapeutic intervention, is extremely narrow. additional challenges need to be overcome, such as the structural variation of viral proteins, multiple genotypes, and high mutation rates. accordingly, only a very limited number of specific antiviral drugs are currently licensed, and promising approaches mostly aim to control severe complications, reduce disease burden, or transmission. antiviral strategies aim to block particular stages of the viral lytic cycle, including attachment and entry to the host cell, replication, transcription, and translation (fig 1) . 41 in principle, preventing a viral pathogen from entering the host cell represents the ideal antiviral strategy because the virus is not allowed to ''hack'' the host: ifv nais have been successfully used to competitively bind the sialic acid-binding pocket of neuroaminidase and are good examples of this approach. oseltamivir and zanamivir have been used as anti-flu therapies, 42 whereas laninamivir and peramivir show antiviral activity against wild-type but also against oseltamivir-resistant and nai-resistant strains, respectively. 43, 44 the nonenveloped rhinoviruses use viral capsid structures to bind their receptors (intercellular adhesion molecule 1 [icam-1], low-density lipoprotein receptor, and cadherin-related family member 3). 45 even though more than 50% of rhinovirus strains use icam-1 for cell entry, an icam-1 competitor, tremacamra, did not make it into the clinic despite initially promising results, 46 and no anti-icam-1 drugs are currently available. another strategy is to prevent capsid uncoating and further assembly of new virions. this strategy has been successfully used against ifv and severe acute respiratory syndrome (sars)coronavirus, which use a class i fusion mechanism. 47 das181 (fludase, nexbio, inc, san diego, calif) is a fusion construct that cleaves the sialic acid receptors on host cells, and its antiviral spectrum includes ifv and parainfluenza viruses (pivs). 48 nonenveloped viruses, such as rhinovirus, release their genomes through a conformational shift of the capsid proteins accompanied by an expansion of the viral shell along with the opening of symmetry-related channels (pores) from which the genome is released (virus uncoating). 49, 50 various capsid-binding compounds against rhinoviruses have been tested (r and win series) without ultimate success. 51 pleconaril, bta798 (vapendavir), and pocapavir (v-073) are still under clinical evaluation. 52 of note, a major drawback of capsid binders is the rapid emergence of resistance. 52 several fusion inhibitors are being developed for the treatment of rsv and have been reviewed elsewhere. 3, 53 because of their limited coding capacity, viruses rely on the production of polyproteins that need to be cleaved into functional subunits by viral proteases. the enterovirus polyprotein is cleaved by a family of cysteine proteases, which are highly conserved among different subtypes but lack homology with human proteases. unfortunately, after failed attempts with ruprintrivir (ag7088) and ag7404, which showed antiviral activity in vitro but not in vivo, no similar agents are being pursued currently. 52 the use of hiv protease inhibitors, such as lopinavir and ritonavir, in patients with sars has not been associated with any proved benefit, although retrospective studies reported that severe outcomes (acute respiratory distress syndrome or death) occurred less often in those receiving a combination of lopinavir/ritonavir and ribavirin with corticosteroids. 54 polymerase inhibitors (nucleoside/nucleotide analogs) act by leading to termination of the polynucleotide chain elongation. ribavirin has been used for the treatment of severe rsv-related disease in high-risk infants and in combination with protease inhibitors in patients with sars, but its use has been limited because of cost and unconfirmed efficacy toward severe outcomes. als-008176 is a promising orally bioavailable prodrug of the novel rsv replication inhibitor als-008112 (a cytidine nucleoside analogue), which inhibits rsv replication. 55 other promising polymerase inhibitors include amiloride (competitive inhibitor of coxsackie virus b3 rna polymerase) and gpc-n114 (multiple genera in picornaviridae) but are still in the early stages. 52 favipiravir (t-705) is an antiviral drug that selectively inhibits the rna-dependent rna polymerase of ifv, as well as several other viruses. 56 umifenovir has been shown to inhibit various human respiratory rna viruses, including several strains of ifv-a and ifv-b, rsv, piv3, and rhinovirus b14. it also demonstrates inhibitory activity against other viruses, enveloped or not, responsible for emerging or globally prevalent infectious diseases. 57 finally, a most promising but also challenging antiviral approach is through use of antisense oligonucleotides. antisense oligonucleotides are single-stranded deoxyribonucleotide oligomers with a sequence complementary to a target mrna transcript. thus viral genomic rna or viral mrna can be targeted directly. antisense technology and rna interference have been experimentally explored in targeting measles virus, sars-coronavirus, coxsackievirus, enteroviruses and rhinoviruses, piv, human metapneumovirus, ifv, and rsv genomes. 58, 59 the rna inhibition-based therapeutic that is furthest advanced in clinical development at this time is against rsv. 58 aln-rsv01 is an unmodified, naked, small interfering rna designed to inhibit the replication of rsv by interrupting the synthesis of the viral n protein. the sequence of the target is well conserved throughout naturally occurring rsv a and b genotypes. 58 in all, new antivirals are being explored continuously, particularly for life-threatening viruses, such as ifv (influenza) and rsv. rhinoviruses, even though simple in terms of genome organization and protein coding, have proved extremely difficult to target, mostly because of their high diversity and immuneevading strategies but also to some extent to the underestimation of rhinovirus infection clinical consequences. within the past few years, scientific communities all over the world have shown renewed interest in the search for novel immune-stimulating or antiviral agents of plant origin for either treatment or prevention, often using ethnopharmacologic approaches. 60 natural compounds are widely recognized as privileged structures trimmed by evolutionary processes to interact with macromolecular targets. plants use a diverse set of biochemical pathways to generate several secondary metabolites representing ecosystem adaptations to help plants to survive various environmental stresses and protect them from infections and infestations. 61 the antiviral potential of plant extracts or compounds varies among viruses. 62 natural compounds occupy an equally large and complex chemical space as synthetic compounds. in the case of antiviral agents, 80% of 46 entities registered in the last approximately 30 years (1981-2010) can be classified as natural product botanicals, synthetic but natural product mimics, natural product pharmacophores, or a combination of the latter 2. oseltamivir, a success story in ifv drug synthesis, has its roots in nature: the abundant plant constituents quinic acid and shikimic acid are used as its starting materials. 63 a screening strategy was applied to investigate crude extracts from 260 plant species on their inhibiting potential toward nais of clostridium perfringens. 64 moreover, 14 bioactive compounds from cleistocalyx operculatus buds were discovered by using an anti-ifv screening approach. 65 the chinese academy of medical sciences tested more than 10,000 plants. among them, a pronounced neuroaminidase-inhibiting effect was observed for the herb extract of elsholtzia rugulos. some extracts from agrimonia pilosa, 66 echinacea purpurea, 67 and prunus mume 68 or the multicomponent mixtures polyphenol fractions from punica granatum 69 and secoiridoid glucosides from ligustrum lucidum 70 have shown a significant reduction of virus-induced cytopathic effects and in general antiviral or anti-influenza activity. a 10% to 20% risk reduction of common cold incidence with the use of echinacea species supplements has been shown. 71 moreover, a recent meta-analysis demonstrated benefit on long-term (2-4 months) prevention with echinacea species on recurrent respiratory tract infections (rtis). 72 another promising compound is bno 1016, a fixed combination of 5 herbal substances that significantly reduced symptoms and led to faster recovery in patients with acute viral rhinosinusitis. 73 reported antiviral effects from natural products, regardless of whether obtained from clinical trials or empiric knowledge, can only give clues for further research. however, it appears that we are entering a new golden age of natural product drug discovery. prevention of viral respiratory illness is attempted by either avoiding exposure or strengthening immune defenses, either nonspecifically with immunostimulators or specifically with vaccines. often, but not always, interventions are targeted toward high-risk groups for a particular infection (eg, rsv in infants and the elderly and ifv in patients with asthma). a variety of compounds (of microbial, herbal, or synthetic origin) have been used and are still being developed as nonspecific immunostimulatory agents to enhance or modulate the immune response against respiratory pathogens in a preventive or sometimes also therapeutic context. the effectiveness of these agents is usually moderate, and therefore they are only used as secondary supportive measures. as such, however, their potential should not be underestimated. among several agents based on bacterial components (om-85 bv, lw 50020, pmbl, d53, and ru 41740), om-85 bv, a lyophilisate of water-soluble fractions of bacteria commonly detected in patients with rtis, has been extensively studied, and a role in the prevention of both acute and recurrent rtis has been shown. 74, 75 mechanistic studies have confirmed pleiotropic immunomodulating effects on both innate and adaptive immunity. 76, 77 pidotimod, a synthetic dipeptide molecule, induces a variety of immunomodulatory effects 78, 79 and has shown some efficacy in preventing rtis, although this was not always confirmed. 80, 81 probiotic supplementation has been shown to reduce the incidence, duration, and severity of upper respiratory tract infections through immune modulation 82 and in particular rhinovirus infection through altering nasal innate inflammatory responses. 83 vitamin d (25-hydroxyvitamin d) has a modulatory role in host defense, inflammation, immunity, and epithelial repair after respiratory tract infections. 84 a recent meta-analysis has confirmed that vitamin d supplementation reduces the overall risk of acute respiratory tract infections. 85 data from in vitro rhinovirus-infected human primary bronchial epithelial cells showed that exogenous vitamin d can reduce rhinovirus replication through increasing interferon and cathelicidin gene expression. 86 a significant amount of research is still dedicated to the efficacy of vitamin d supplementation, although not without controversy. hopefully, specific indications will be consolidated soon. despite widespread use and a multitude of studies, the role of vitamins c or zinc supplements is still inconclusive in relation to their action against the common cold. 87 interestingly, meditation and exercise might significantly contribute to the reduction of rti burden, 88 suggesting that the immunostimulatory capacity of nonpharmacologic measures should also be considered. the high transmission rate and epidemic nature of respiratory tract viruses indicate that effective public health measures to reduce transmission can have a substantial role in the overall prevention of these infections. a plethora of studies and metaanalyses delineated the important contribution of health policies in reducing transmission of epidemic respiratory tract viruses. in an elegant randomized control trial, an automated web-based intervention that maximized handwashing intention was associated with fewer episodes of influenza-like illness, shorter duration of symptoms, and fewer antibiotic prescriptions in the intervention group. 89 although similar results regarding handwashing have been confirmed in a cochrane meta-analysis, 90 hand hygiene interventions in educational settings were not as unequivocally effective. 91, 92 low adherence to hand hygiene recommendations was correlated with higher incidence of ifv infection among health care workers during the 2009 pandemic. 93 the use of face masks has been shown to be highly effective in the interruption of respiratory viral spread. 90 this has been demonstrated further in a cluster randomized trial in which a reduced odds ratio of influenza infection secondary attack was observed in the intervention group. 94 face masks are now regularly worn in some communities, especially in asia, but much less so in western societies. taken together, it seems that public health measures might provide a valuable ally in decreasing the burden of respiratory tract infections in the community. both vaccines and mabs (passive immunization) are relevant interventions. vaccines for ifv, rhinovirus, and rsv were initially developed as long ago as the 1940s to 1960s, although with mixed success, mostly because of rapid virus evolution. improved understanding of vaccine immunology and technologic developments place us now closer than ever to developing highly effective vaccines against the major respiratory tract viruses. mab therapies to viral infections, such as ebv (rituximab) or rsv (palivizumab), provide passive immunization and are licensed, whereas similar agents targeting influenza and other viruses are in preclinical development. 95 neutralizing antibodies can bind and inactivate viruses, inhibit viral cell entry (blocking receptor binding or conformational changes), prevent the release of virions from the cell, or modulate immune effector functions. 96, 97 engineering and production strategies to produce antibody fragments, higher-affinity binding, and longer half-life are contributing to a lower overall cost for therapy, 95 although vaccines are still considered preferable in most cases. it is notable that effective neutralizing mab epitopes can also inform the rational design of vaccines. 98 different types of vaccines to respiratory viruses exist, and these are shown in fig 2. traditionally, either live attenuated or inactivated viruses are used. more recently, subunit vaccines made of detergent-disrupted whole viruses or purified viral proteins are also common. furthermore, promising approaches use microparticle/nanoparticle material and recombinant technologies to produce broadly immunogenic, often self-adjuvanting, reproducible, and safe vaccine responses. 99 these delivery systems include synthetic polymers, virosomes, virus-like particles (vlps), liposomes, lipid nanoparticles, proteins, emulsions, and immune-stimulating complexes. 22 currently, naturally occurring particles are favored because of safety concerns, 100 even though synthetic polymers, such as poly lactic-co-glycolic acid, are in use, 101 and gold nanoparticles have shown promising results. 102 self-assembling protein nanoparticles, such as ferritin cages and vaults, have also shown promising preclinical data. 103, 104 layer-by-layer peptide-fabricated vaccine containing alternately charged poly-l-glutamic acid and poly-l-lysine layers with rsv peptides added have been efficacious in animals. 105 a virosomal adjuvanted vaccine composed of reconstituted ifv envelope, effectively removing the core proteins and rna, has been available for years with excellent tolerability and efficacy. 106 several vlp vaccines based on hepatitis b virus surface antigen have been approved for viral infections, such as human papilloma virus 107 and other microbes (eg, malaria 108 ), although an ifv candidate has not progressed. 109 nevertheless these and other vlps offer promise because of their valency, similar immune presentation to pathogens, and antigenic preservation. 110 adjuvants form a vital part of many vaccines; however, only aluminum hydroxide and oil in water emulsions are currently approved. a number of novel adjuvants, such as microcrystalline tyrosine, matrix m, pathogen-associated molecular patterns, and chitosan, are in development. [111] [112] [113] [114] dna and rna vaccines induce an immune response to the nucleic acid-encoded antigen. 115 impressive results have been reported in animals for a single low-dose intradermal, nonreplicating dna vaccine for rsv; however, whether this will translate effectively to human subjects is not yet known. 116 to enhance immunogenicity, rna vaccines have been encapsulated in nanoparticles, achieving sterilizing immunity for zika virus in mice, 117 as well as being incorporated into virus-based self-replicating constructs known as replicons. 118, 119 active ifv vaccination already forms the core of the global strategy against severe seasonal and pandemic influenza. trivalent and more recent quadrivalent vaccines are largely efficacious in healthy adults provided an adequate match between circulating and vaccine strains. 120 higher-dose (60 mg) and mf59-adjuvanted vaccines are available for elderly patients. 121, 122 similarly, pandemic vaccines can offer greater cross-clade protection because of the presence of improved (as03 or mf59) adjuvants. 123 the current frontier of ifv vaccine development is ''universal'' vaccines (table i) . ideally, these would protect not only from circulating and pandemic strains but also from novel epitopes that might evolve in the future. many such vaccines are currently in preclinical and early clinical stages. heterosubtypic cross-reactive antibodies to ifv-a against the hemagglutinin (ha) stalk 147 have been isolated from immune subjects, 148 leading to mabs now in phase 2. 98,124 similar multilineage ha-stalk antibodies to ifv-b have also been reported. 149 other conserved proteins have also been targeted, and an anti-m2e antibody is in development. 150 therefore passive immunization or postinfection treatment might soon become another tool to combat ifv. 125, 126, 151 ha-stalk and chimeric head/stalk-based vaccines have also shown encouraging preclinical results. 103, [152] [153] [154] a further vaccine strategy based on conserved epitopes in proteins, such as m1, np, and pb1, involves induction of cd4 1 and cd8 1 t-cell immunity, 155 leading to development of a promising mva viral vector vaccine. other vaccines use multiepitope peptides to induce ifv-specific t-cell responses, reducing viral shedding in human subjects. 127, 133 self-replicating rna nanoparticles also encoding multiple proteins and hepatitis b virus-based vlps expressing m2e and ha epitopes also appear promising. 156 there are currently no licensed vaccines and only 1 mab (palivizumab) approved for the prevention of rsv infection. however, there are numerous candidates in clinical trials, as recently reviewed. 3 suptavumab, an anti-f mab, 157 has reached phase iii trials in preterm infants. medi8897 offers 9-fold greater potency than palivizumab and has extended half-life in primates, suggesting a once per season dosing. 128 candidate vaccines are based on live attenuated strains, subunit, vector, and nanoparticle technologies with a range of adjuvants. chimeric and combination vaccines using expression vectors in vlps show much promise. 158 recent preclinical results exhibit effective neutralization of rsv. [159] [160] [161] [162] [163] the most advanced of these is the novavax f-protein vlp nanoparticle vaccine with aluminum hydroxide adjuvant, which is in phase iii for maternal vaccination. 129, 130 transplacental transmission of neutralizing antibodies has been demonstrated in preclinical studies, although this has not conferred significant protection from rsv. 164 recombinant dna vaccines are also promising because of their apparent ability to induce a balanced t h 1/t h 2 response, with a broad igg/iga profile mimicking live rsv challenge. 165 intranasal and oral vaccine formulations are now in the early stages of clinical studies. 166 fig 2. vaccine types. a, live attenuated vaccines are grown in culture to make them less virulent but can have the problem of reversion. b, inactivated vaccines are treated with uv or formaldehyde to crosslink proteins and make them nonviable. c, proteins can be purified, extracted, or dissolved by using detergents. d, naked nucleic acids are also used as vaccines. e, nanoparticle vaccines encompass natural and synthetic materials. membranes can be used to make liposomes to contain and deliver an antigen to a target cell. f, viruses can have nucleic acid and core protein removed to form virosomes. g, viral proteins, such as ha stalks or antigens, can be engineered onto immunogenic core proteins (eg, ferritin or vaults). this example is ha on ferritin adapted from pbd codes 3bve and 5c0s. h, viruses, such as the vaccinia virus ankara, with coat proteins and genetic material removed can be engineered to express other antigens, such as influenza m2 ion channel protein. i, vlps can be engineered to express antigens and naturally glycosylated proteins and have adjuvants incorporated into the coat. pamp, pathogen-associated molecular pattern. j, synthetic nanoparticles made from polymers (polystyrene or poly lactic-co-glycolic acid), gold, or carbon nanotubes can have peptides adsorbed, admixed, or encapsulated. ag, antigen. initial vaccination attempts 167 and more recent preclinical experiments show that inactivated rhinovirus vaccines are type specific and not cross-neutralizing. 8 however, although in animals 168 rhinovirus antibody responses might be weakly cross-neutralizing, data from human subjects suggest that responses are mainly misdirected to internal epitopes. 169 understanding the full extent of rhinovirus diversity would probably be required to develop a panspecies vaccine. multiple strategies are being developed to reduce the burden of viral respiratory illnesses. it is likely that many of these strategies will find a relevant indication: antiviral strategies will most probably make sense in severe life-threatening situations or when a window of opportunity is clearly present, such as in specific virus seasons and susceptible populations. ideally, prevention at a wide scale through immunization will be able to reduce the overall burden of respiratory infections with a huge effect. this appears to be within reach for rsv and ifv, whereas additional effort is needed toward rhinovirus. in the meantime, symptomatic and immunostimulatory measures provide relief, and they hold promise in relation to postviral reactive airway disease. public health measures should be expanded because they can be critical in reducing the effect and contain potential epidemics. drivers of airborne human-to-human pathogen transmission global epidemiology of non-influenza rna respiratory viruses: data gaps and a growing need for surveillance lower respiratory tract infection caused by respiratory syncytial virus: current management and new therapeutics estimates of hospitalization attributable to influenza and rsv in the us during 1997-2009, by age and risk status asthma research in europe: a transformative agenda for innovation and competitiveness influenza: exposing the true killer identification of new respiratory viruses in the new millennium a polyvalent inactivated rhinovirus vaccine is broadly immunogenic in rhesus macaques over-the-counter medications: update on cough and cold preparations limits and danger of ephedrine and pseudoephedrine as nasal decongestants comprehensive evidence-based review on european antitussives cyclooxygenase 2: its regulation, role and impact in airway inflammation delayed antiviral plus immunomodulator treatment still reduces mortality in mice infected by high inoculum of influenza a/h5n1 virus the resolution code of acute inflammation: novel pro-resolving lipid mediators in resolution targeted prostaglandin e2 inhibition enhances antiviral immunity through induction of type i interferon and apoptosis in macrophages role of the lipoxygenase pathway in rsv-induced alternatively activated macrophages leading to resolution of lung pathology non-invasive ventilation improves respiratory distress in children with acute viral bronchiolitis: a systematic review high-flow warm humidified oxygen versus standard low-flow nasal cannula oxygen for moderate bronchiolitis (hfwho rct): an open, phase 4, randomised controlled trial pathogenesis of viral infection in exacerbations of airway disease viruses and bacteria in acute asthma exacerbations-a ga(2) len-dare systematic review novel strategies for targeting innate immune responses to influenza the effect of inhaled ifn-beta on worsening of asthma symptoms caused by viral infections. a randomized trial efficacy of ifn-lambda1 to protect human airway epithelial cells against human rhinovirus 1b infection il-28a (ifn-lambda2) modulates lung dc function to promote th1 immune skewing and suppress allergic airway disease host dna released by netosis promotes rhinovirus-induced type-2 allergic asthma exacerbation efficacy of novel antibody-based drugs against rhinovirus infection: in vitro and in vivo results the tlr4 antagonist eritoran protects mice from lethal influenza infection the toll-like receptor 4 antagonist eritoran protects mice from lethal filovirus challenge preseasonal treatment with either omalizumab or an inhaled corticosteroid boost to prevent fall asthma exacerbations omalizumab is associated with reduced acute severity of rhinovirus-triggered asthma exacerbation interferon at the crossroads of allergy and viral infections the cytokine storm of severe influenza and development of immunomodulatory therapy immunomodulatory therapy for severe influenza fda drug safety communication: fda eliminates the risk evaluation and mitigation strategy (rems) for rosiglitazone-containing diabetes medicines dissecting influenza virus pathogenesis uncovers a novel chemical approach to combat the infection azithromycin induces anti-viral responses in bronchial epithelial cells azithromycin induces anti-viral effects in cultured bronchial epithelial cells from copd patients early administration of azithromycin and prevention of severe lower respiratory tract illnesses in preschool children with a history of such illnesses: a randomized clinical trial identification of novel macrolides with antibacterial, anti-inflammatory and type i and iii ifn-augmenting activity in airway epithelium the anti-inflammatory effect of alpha-1 antitrypsin in rhinovirus-infected human airway epithelial cells current progress in antiviral strategies influenza neuraminidase inhibitors: synthetic approaches, derivatives and biological activity japanese surveillance systems and treatment for influenza detection and management of antiviral resistance for influenza viruses viral entry pathways: the example of common cold viruses efficacy of tremacamra, a soluble intercellular adhesion molecule 1, for experimental rhinovirus infection: a randomized clinical trial viral membrane fusion drugs to cure avian influenza infection-multiple ways to prevent cell death viral uncoating is directional: exit of the genomic rna in a common cold virus starts with the poly-(a) tail at the 3'-end picornavirus rna is protected from cleavage by ribonuclease during virion uncoating and transfer across cellular and model membranes selective inhibitors of picornavirus replication replication and inhibitors of enteroviruses and parechoviruses challenges and opportunities in developing respiratory syncytial virus therapeutics advances in antivirals for non-influenza respiratory virus infections. influenza other respir viruses als-008176 for respiratory syncytial virus infection favipiravir (t-705), a novel viral rna polymerase inhibitor arbidol as a broad-spectrum antiviral: an update the promise, pitfalls and progress of rna-interference-based antiviral therapy for respiratory viruses gene knockdown in human rhinovirus 1b using 2'-ome-modified sirnas results in the reactivation of the interferon response traditional use of medicinal agents longevity extension by phytochemicals a review on antiviral activity of the himalayan medicinal plants traditionally used to treat bronchitis and related symptoms influenza neuraminidase: a druggable target for natural products neuraminidase inhibitors from reynoutria elliptica structure-activity relationship of flavonoids as influenza virus neuraminidase inhibitors and their in vitro anti-viral activities c-methylated flavonoids from cleistocalyx operculatus and their inhibitory effects on novel influenza a (h1n1) neuraminidase anti-viral properties and mode of action of standardized echinacea purpurea extract against highly pathogenic avian influenza virus (h5n1, h7n7) and swine-origin h1n1 (s-oiv) in vitro inhibition of human influenza a virus infection by fruit-juice concentrate of japanese plum (prunus mume sieb. et zucc) pomegranate (punica granatum) purified polyphenol extract inhibits influenza virus and has a synergistic effect with oseltamivir in vitro evaluation of secoiridoid glucosides from the fruits of ligustrum lucidum as antiviral agents echinacea for preventing and treating the common cold echinacea reduces the risk of recurrent respiratory tract infections and complications: a meta-analysis of randomized controlled trials herbal drug bno 1016 is safe and effective in the treatment of acute viral rhinosinusitis impact of a mixed bacterial lysate (om-85 bv) on the immunogenicity, safety and tolerability of inactivated influenza vaccine in children with recurrent respiratory tract infection clinical and immunological benefits of om-85 bacterial lysate in patients with allergic rhinitis, asthma, and copd and recurrent respiratory infections om-85 is an immunomodulator of interferon-beta production and inflammasome activity bacterial lysate increases the percentage of natural killer t cells in peripheral blood and alleviates asthma in children pidotimod: the state of art metabolomic profile of children with recurrent respiratory infections pidotimod for the prevention of acute respiratory infections in healthy children entering into daycare: a double blind randomized placebo-controlled study efficacy and safety of pidotimod in the prevention of recurrent respiratory infections in children: a multicentre study probiotics for preventing acute upper respiratory tract infections effect of probiotic on innate inflammatory response and viral shedding in experimental rhinovirus infection-a randomised controlled trial vitamin d modulation of innate immune responses to respiratory viral infections vitamin d supplementation to prevent acute respiratory tract infections: systematic review and meta-analysis of individual participant data vitamin d increases the antiviral activity of bronchial epithelial cells in vitro prevention and treatment of the common cold: making sense of the evidence meditation or exercise for preventing acute respiratory infection: a randomized controlled trial an internet-delivered handwashing intervention to modify influenza-like illness and respiratory infection transmission (primit): a primary care randomised trial physical interventions to interrupt or reduce the spread of respiratory viruses effectiveness of hand hygiene interventions in reducing illness absence among children in educational settings: a systematic review and meta-analysis can a school-based hand hygiene program reduce asthma exacerbations among elementary school children? risk factors for influenza among health care workers during 2009 pandemic the role of facemasks and hand hygiene in the prevention of influenza transmission in households: results from a cluster randomised trial monoclonal antibodies for prophylactic and therapeutic use against viral infections the growth and potential of human antiviral monoclonal antibody therapeutics converting monoclonal antibody-based immunotherapies from passive to active: bringing immune complexes into play antibody recognition of a highly conserved influenza virus epitope harnessing nanoparticles for immunomodulation and vaccines assessing toxicity of fine and nanoparticles: comparing in vitro measurements to in vivo pulmonary toxicity profiles current advances in research and clinical applications of plga-based nanotechnology consensus m2e peptide conjugated to gold nanoparticles confers protection against h1n1, h3n2 and h5n1 influenza a viruses hemagglutinin-stem nanoparticles generate heterosubtypic influenza protection self-assembling influenza nanoparticle vaccines elicit broadly neutralizing h1n1 antibodies layer-by-layer nanoparticle vaccines carrying the g protein cx3c motif protect against rsv infection and disease eleven years of inflexal v-a virosomal adjuvanted influenza vaccine human papillomavirus and hpv vaccines: a review as01 malaria vaccine with or without a booster dose in infants and children in africa: final results of a phase 3, individually randomised, controlled trial m2e-based universal influenza a vaccine virus-like particles as a highly efficient vaccine platform: diversity of targets and production systems and advances in clinical development comparison of a novel microcrystalline tyrosine adjuvant with aluminium hydroxide for enhancing vaccination against seasonal influenza matrix m h5n1 vaccine induces cross-h5 clade humoral immune responses in a randomized clinical trial and provides protection from highly pathogenic influenza challenge in ferrets safety and immunogenicity of a recombinant m2e-flagellin influenza vaccine (stf2.4xm2e) in healthy adults chitosan-a versatile semi-synthetic polymer in biomedical applications genetic immunization is a simple method for eliciting an immune response development of an intradermal dna vaccine delivery strategy to achieve single-dose immunity against respiratory syncytial virus modified mrna vaccines protect against zika virus infection dna and rna-based vaccines: principles, progress and prospects replicon rna viral vectors as vaccines transforming growth factor-beta promotes rhinovirus replication in bronchial epithelial cells by suppressing the innate immune response efficacy and safety of high-dose influenza vaccine in elderly adults: a systematic review and meta-analysis effectiveness of mf59-adjuvanted seasonal influenza vaccine in the elderly: a systematic review and meta-analysis a systematic review and meta-analysis of cross-reactivity of antibodies induced by oil-in-water emulsion adjuvanted influenza h5n1 virus monovalent vaccines preclinical pharmacokinetics of mhaa4549a, a human monoclonal antibody to influenza a virus, and the prediction of its efficacious clinical dose for the treatment of patients hospitalized with influenza a efficacy and safety of treatment with an anti-m2e monoclonal antibody in experimental human influenza safety and upper respiratory pharmacokinetics of the hemagglutinin stalk-binding antibody vis410 support treatment and prophylaxis based on population modeling of seasonal influenza a outbreaks priming by a novel universal influenza vaccine (multimeric-001)-a gateway for improving immune response in the elderly population a highly potent extended half-life antibody as a potential rsv vaccine surrogate for all infants safety and immunogenicity of a sf9 insect cell-derived respiratory syncytial virus fusion protein nanoparticle vaccine immunogenicity and safety of a respiratory syncytial virus fusion protein (rsv f) nanoparticle vaccine in older adults evaluation of the immunogenicity and safety of different doses and formulations of a broad spectrum influenza vaccine (flu-v) developed by seek: study protocol for a single-center, randomized, double-blind and placebo-controlled clinical phase iib trial caparr os-wanderley w. synthetic influenza vaccine (flu-v) stimulates cell mediated immunity in a double-blind, randomised, placebo-controlled phase i trial a synthetic influenza virus vaccine induces a cellular immune response that correlates with reduction in symptomatology and virus shedding in a randomized phase ib live-virus challenge in humans a t cell-inducing influenza vaccine for the elderly: safety and immunogenicity of mva-np1m1 in adults aged over 50 years a phase iia study to assess the safety and efficacy of a new influenza candidate vaccine mva-np1m1 in healthy adults-flu002 clinical study report evaluating the immunogenicity and safety of a biondvax-developed universal influenza vaccine (multimeric-001) either as a standalone vaccine or as a primer to h5n1 influenza vaccine back to the future: immunization with m-001 prior to trivalent influenza vaccine in 2011/12 enhanced protective immune responses against 2014/15 epidemic strain safety and immunogenicity of multimeric-001-a novel universal influenza vaccine implication of respiratory syncytial virus (rsv) f transgene sequence heterogeneity observed in phase 1 evaluation of medi-534, a live attenuated parainfluenza type 3 vectored rsv vaccine phase-i study medi-534, of a live, attenuated intranasal vaccine against respiratory syncytial virus and parainfluenza-3 virus in seropositive children a randomized, blinded, controlled, dose-ranging study of a respiratory syncytial virus recombinant fusion (f) nanoparticle vaccine in healthy women of childbearing age efficacy of motavizumab for the prevention of respiratory syncytial virus disease in healthy native american infants: a phase 3 randomised double-blind placebo-controlled trial motavizumab for prophylaxis of respiratory syncytial virus in high-risk children: a noninferiority trial trivalency of a nanobody specific for the human respiratory syncytial virus fusion glycoprotein drastically enhances virus neutralization and impacts escape mutant selection efficacy, safety, and pharmacokinetics of a new 10% liquid intravenous immunoglobulin containing high titer neutralizing antibody to rsv and other respiratory viruses in subjects with primary immunodeficiency disease treatment with novel rsv ig ri-002 controls viral replication and reduces pulmonary damage in immunocompromised sigmodon hispidus structural and functional bases for broad-spectrum neutralization of avian and human influenza a viruses broadly cross-reactive antibodies dominate the human b cell response against 2009 pandemic h1n1 influenza virus infection highly conserved protective epitopes on influenza b viruses human antibodies reveal a protective epitope that is highly conserved among human and nonhuman influenza a viruses tackling influenza with broadly neutralizing antibodies influenza virus vaccine based on the conserved hemagglutinin stalk domain chimeric hemagglutinin influenza virus vaccine constructs elicit broadly protective stalk-specific antibodies chimeric hemagglutinin constructs induce broad protection against influenza b virus challenge in the mouse model epitope specificity plays a critical role in regulating antibody-dependent cell-mediated cytotoxicity against influenza a virus influenza vaccine research funded by the european commission fp7-health-2013-innovation-1 project antibodies to watch in 2017 protective efficacy and immunogenicity of an adenoviral vector vaccine encoding the codon-optimized f protein of respiratory syncytial virus immunogenicity of rsv f dna vaccine in balb/c mice rsv fusion (f) protein dna vaccine provides partial protection against viral infection chimeric virus-like particles containing a conserved region of the g protein in combination with a single peptide of the m2 protein confer protection against respiratory syncytial virus infection baculovirus-expressed virus-like particle vaccine in combination with dna encoding the fusion protein confers protection against respiratory syncytial virus co-immunization with virus-like particle and dna vaccines induces protection against respiratory syncytial virus infection and bronchiolitis respiratory syncytial virus: infection, detection, and new options for prevention and treatment immunogenicity and efficacy of codon optimized dna vaccines encoding the f-protein of respiratory syncytial virus mucosal vaccines against respiratory syncytial virus prevention of colds by vaccination against a rhinovirus: a report by the scientific committee on common cold vaccines challenges in developing a cross-serotype rhinovirus vaccine misdirected antibody responses against an n-terminal epitope on human rhinovirus vp1 as explanation for recurrent rv infections key: cord-048447-chz8luni authors: duffett, mark; choong, karen; ng, vivian; randolph, adrienne; cook, deborah j title: surfactant therapy for acute respiratory failure in children: a systematic review and meta-analysis date: 2007-06-15 journal: crit care doi: 10.1186/cc5944 sha: doc_id: 48447 cord_uid: chz8luni introduction: exogenous surfactant is used to treat acute respiratory failure in children, although the benefits and harms in this setting are not clear. the objective of the present systematic review is to assess the effect of exogenous pulmonary surfactant on all-cause mortality in children mechanically ventilated for acute respiratory failure. methods: we searched the medline, embase, cinahl and ovid healthstar databases, the bibliographies of included trials and review articles, conference proceedings and trial registries. we included prospective, randomized, controlled trials of pulmonary surfactant that enrolled intubated and mechanically ventilated children with acute respiratory failure. we excluded trials that exclusively enrolled neonates or patients with asthma. two reviewers independently rated trials for inclusion, extracted data and assessed the methodologic quality. we quantitatively pooled the results of trials, where suitable, using a random effects model. results: six trials randomizing 314 patients were included. surfactant use reduced mortality (relative risk = 0.7, 95% confidence interval = 0.4 to 0.97, p = 0.04), was associated with increased ventilator-free days (weighted mean difference = 2.5 days, 95% confidence interval = 0.3 to 4.6 days, p = 0.02) and reduced the duration of ventilation (weighted mean difference = 2.3 days, 95% confidence interval = 0.1 to 4.4 days, p = 0.04). conclusion: surfactant use decreased mortality, was associated with more ventilator-free days and reduced the duration of ventilation. no serious adverse events were reported. acute respiratory failure remains the primary indication for admission to north american paediatric intensive care units (picus) and accounts for significant mortality, morbidity and resource utilization [1] . respiratory infections, in particular pneumonia and severe bronchiolitis, are the most common causes of respiratory failure requiring mechanical ventilation in children [1] . alterations in endogenous surfactant play a role in the pathogenesis of many causes of acute lung injury (ali) and acute respiratory distress syndrome (ards) [2] . surfactant dysfunction, destruction and inactivation have also been demonstrated in children with acute respiratory insufficiency due to bronchiolitis [3, 4] . the administration of exogenous surfactant may reduce the need for mechanical ventilation and its associated sequelae by restoring surfactant levels and function. inspired by the success of surfactants in reducing mortality and the need for mechanical ventilation in neonatal respiratory distress syndrome [5] , investigators have studied exogenous surfactant in other populations with various causes of respiratory failure. trials of surfactant in adults with ali and ards have not demonstrated a mortality benefit [6] [7] [8] [9] , perhaps due to inherent differences in the aetiology of lung injury in adults, the design features of the trials, the mode and timing of surfactant administration or the type and dose of surfactant used. in children with respiratory failure, the efficacy of exogenous surfactant has been suggested in uncontrolled studies ali = acute lung injury; ards = acute respiratory distress syndrome; fio 2 = fractional inspired oxygen; pao 2 = arterial oxygen tension; picu = paediatric intensive care unit; rsv = respiratory syncytial virus. (page number not for citation purposes) [10, 11] . the relatively low mortality rate, the diversity of the study populations and the shorter duration of mechanical ventilation are factors that make large-scale randomized controlled trials in this population challenging to conduct. two of the largest trials were stopped early due to slower than expected enrolment [12, 13] . while the use of surfactant in ards/ali has not been previously systematically reviewed, its use in children with bronchiolitis has been [14] . we anticipated that including trials enrolling children with acute respiratory failure from a variety of causes would result in a heterogeneous population and would increase the generalizability of the results. our confidence in the results of the present review would also be increased if a consistent effect is shown in subgroups and across a spectrum of disease severity. the primary objective of the systematic review is to assess the effect of the administration of pulmonary surfactant compared with no therapy or with placebo on all-cause mortality (at or before hospital discharge) in mechanically ventilated children with acute respiratory failure. we included trials that were prospective, that were randomized, that enrolled children intubated and mechanically ventilated for acute respiratory failure and that compared the intratracheal administration or nebulization of at least one dose of natural or artificial pulmonary surfactant with a placebo or no intervention. we excluded trials exclusively enrolling neonates or patients with asthma. we used the trial authors' definitions of paediatric. the primary outcome measure was all-cause mortality at or before hospital discharge. secondary outcomes were ventilator-free days to day 28 (a composite of mortality and duration of ventilation, defined as days alive and free from mechanical ventilation) [15] , the duration of mechanical ventilation (from intubation to extubation, death or trial withdrawal), the duration of picu stay, the use of rescue therapy (such as extracorporeal membrane oxygenation, high-frequency oscillatory ventilation, open label surfactant and nitric oxide), and complications and adverse effects as reported by the trial authors. one of us searched for published and unpublished trials, examining trial registries, conference proceedings and the bibliographies of any identified trials and relevant reviews (the search strategy is available upon request). we polled paediatric intensivists and pharmacists at our institution for additional trials. we selected search terms from the keywords and mesh terms of previous surfactant trials and from the generic and brand names of commercially available surfactants. we imposed no language restrictions. one of us screened the title (and abstract if required) of all citations retrieved. we selected citations for further evaluation if they reported the administration of at least one dose of surfactant to at least one child or if the title or abstract did not give enough information to make an assessment. two reviewers independently reviewed all citations meeting criteria for further review and applied the inclusion criteria. disagreements between reviewers were resolved by consensus in consultation with a third reviewer. we considered agreement between reviewers to be acceptable if the kappa value was greater than 0.8. we used the following characteristics to assess the methodologic quality: allocation concealment (sealed envelopes or central randomization were considered adequate), blinding (which of the trial personnel and caregivers were blinded, and the methods used to ensure blinding), completeness of followup (assessed by the number of patients randomized for whom there were no outcomes), similarity of the groups at baseline (with respect to known prognostic factors: age, aetiology, severity of illness as measured by the pediatric risk of mortality score, and immunosuppression), whether a standard or recommended strategy for mechanical ventilation was used, and whether a priori criteria for the use of co-interventions were used. effective blinding of surfactant is challenging because of the large volumes of milky fluid administered, which can often be seen by caregivers in the patients' ventilator tubing or endotracheal tube, particularly during suctioning. we pretested and refined the developed forms on two trials of surfactant therapy for adults, and clarified definitions based on feedback from the reviewers. two reviewers then independently used these forms to abstract trial quality, blinded to the authors, the journal, the country of origin and the results. we resolved any disagreements by consensus in consultation with a third reviewer if needed. after pretesting and refining the forms on two trials of surfactant therapy in adults and clarifying definitions based on feedback from the reviewers, two reviewers then independently abstracted the data. reviewers were only provided with a full-text version of the trials from which the introduction, conclusions and discussion were omitted and from which the author, journal and country of origin were deleted. we thereafter examined these sections of the reports for any missing data. we resolved any disagreements between reviewers by consensus in consultation with a third reviewer if needed. we asked the authors to supply data not included in the published reports. two reviewers performed data entry in duplicate. we quantitatively pooled the results of individual trials when possible. we expressed the treatment effect as a relative risk for dichotomous outcomes and as a weighted mean difference for continuous outcomes with 95% confidence intervals. we considered effects statistically significant if p < 0.05. a z test was used to statistically test the estimates of treatment effect between groups [16] . we assessed heterogeneity among trials using the i 2 statistic, and considered an i 2 value greater than 50% to indicate substantial heterogeneity [17] . revman 4.2 software and a random effects model were used to perform the analyses [18] . we chose the random effects model because it gives a more conservative estimate of the precision of the treatment effects and because the true effect of the intervention probably varies given the different populations enrolled in these trials [19] . a subgroup analysis was planned based on the aetiology of respiratory failure (trials enrolling exclusively patients with respiratory syncytial virus (rsv)/ severe bronchiolitis compared with all other trials) if sufficient data were available, because these trials were likely to enrol a younger, more homogeneous, population with a lower predicted risk of mortality. we also planned sensitivity analysis based on methodological features of the included trials (trials reporting adequate allocation concealment compared with all other trials). we identified 742 unique citations, six of which met our inclusion criteria ( figure 1 outlines the reasons for exclusion). most reports excluded enrolled neonates or were retrospective or uncontrolled in design. chance corrected agreement was excellent (kappa = 0.91, 95% confidence interval = 0.73-1.1). table 1 presents a complete description of our quality assessment. only one trial did not report allocation concealment [20] . although effective blinding of surfactant is challenging, two trials reported blinding of the picu team [12, 20] . the two flow diagram of included trials flow diagram of included trials. rcts, randomized controlled trials. groups were generally well matched in terms of baseline characteristics in most trials. the most significant imbalance was the numerically higher number of immunosuppressed patients in the placebo group. these patients had higher mortality (56%) than the immunocompetent group (13%). the authors attempted to adjust for this imbalance with logistic regression, which suggested that the treatment effect seemed to be relatively consistent between the two groups [12] . only one trial reported a priori criteria for rescue therapy [13] . table 2 describes the included trials. three trials enrolled exclusively infants with rsv-induced respiratory failure [20, 21] or with severe bronchiolitis [22] . the remaining three trials enrolled a heterogeneous group of patients with ards or ali [12, 23, 24] . while the individual treatment protocols varied, all trials used comparable doses (50-100 mg/kg phospholipids) of natural or modified natural surfactants and each patient typically received one or two doses. a variety of interventions were used in the control groups: no intervention, air placebo or similar sedation and ventilation manoeuvres without a placebo. although one study [20] used a modified natural surfactant, all the products used contained surfactant proteins b and c. all studies administered surfactant early in the course of respiratory failure; most patients were treated within 12-48 hours of requiring mechanical ventilation. the baseline characteristics of the patients are presented in table 3 . while there was significant heterogeneity among and within trials with respect to age and cause of respiratory failure, we considered the initial pediatric risk of mortality scores and the initial pao 2 /fio 2 ratios to be clinically comparable. mortality data were available for all six trials, randomizing 311 patients and reporting data for 305 patients. there were no deaths reported in the three rsv/severe bronchiolitis trials; thus our estimate is based on three trials randomizing 232 patients, 64 of whom died. in the pooled analysis, surfactant was associated with significantly lower mortality (relative risk = 0.7, 95% confidence interval = 0.4-0.97, p = 0.04). there was no evidence of heterogeneity (i 2 = 0%) ( figure 2 ). ventilator-free days to day 28 the number of ventilator-free days to day 28 was available for six trials randomizing 311 patients and reporting data for 305 patients. in the pooled analysis, surfactant was associated with significantly more ventilator-free days (weighted mean dif(figure 3 ). the duration of mechanical ventilation was available for six trials randomizing 311 patients and reporting data for 305 patients. in the pooled analysis, surfactant was associated with a significantly shorter duration of mechanical ventilation (weighted mean difference = 2.3 days, 95% confidence interval = 0.1-4.4 days, p = 0.04) (figure 4) . the duration of picu stay was available for five trials randomizing 273 patients and reporting data for 272 patients. in the pooled analysis, surfactant was associated with a shortened duration of picu stay (weighted mean difference = 2.6 days, 95% confidence interval = 0.02-5.2 days, p = 0.05), but this difference was not statistically significant ( figure 5 ). data on the use of rescue therapy were available for six trials randomizing 311 patients and reporting data for 305 patients. in the pooled analysis, the surfactant was associated with a significantly lower use of rescue therapy (relative risk = 0.4, 95% confidence interval = 0.3-0.7, p < 0.0001). there was no evidence of heterogeneity (i 2 = 0%). this summary estimate should be interpreted with caution as only one trial reported a protocol for initiating rescue therapy. the decision to use a rescue therapy, particularly an open-label surfactant, may be influenced by knowledge of the patient's allocation; furthermore, only two trials reported blinded caregivers and the methods used to ensure blinding may not be adequate. surfactant therapy was well tolerated (see table 4 ), but only three of the trials reported any definitions or a priori criteria or of collecting adverse events [12, 21, 23] . transient hypotension and transient hypoxia were the most commonly reported adverse events in the largest trial. these responded to a brief adjustment in ventilation, to a slowing of the rate of surfactant administration or to fluid administration. there was no difference in the incidence of air leaks in the two trials that reported this outcome. no patient was withdrawn from any of the trials because of adverse events. we did not pool the data on adverse events associated with the trial interventions from the six trials because of the inconsistent manner in which the events were documented and reported. the effect of surfactant on ventilator-free days, the duration of mechanical ventilation and the duration of picu stay was not significantly different when we compared the three trials that enrolled exclusively patients with rsv/severe bronchiolitis with the three other trials (table 5) . a 100% survival in the bronchiolitis trials subgroup precludes formal subgroup analysis for the primary outcome of mortality. all but one of the included trials reported adequate allocation concealment (defined as sealed envelopes or central telephone randomization). since there were no deaths in this trial we could not assess the effect of inadequate allocation concealment on mortality. pooling the five remaining trials did not change the direction of the effect and did not significantly meta-analysis of trials of surfactant in children with acute respiratory failure: mortality meta-analysis of trials of surfactant in children with acute respiratory failure: mortality. ali, acute lung injury; ards, acute respiratory distress syndrome; 95% ci, 95% confidence interval; rr, relative risk; rsv, respiratory syncytial virus. meta-analysis of trials of surfactant in children with acute respiratory failure: ventilator-free days meta-analysis of trials of surfactant in children with acute respiratory failure: ventilator-free days. ali, acute lung injury; ards, acute respiratory distress syndrome; 95% ci, 95% confidence interval; rsv, respiratory syncytial virus; sd, standard deviation; wmd, weighted mean difference. change the point estimates for the secondary outcomes of ventilator-free days, duration of ventilation or duration of picu stay (table 6 ). in the present systematic review and meta-analysis of the effect of surfactant for critically ill children with acute respiratory failure we found that surfactant therapy significantly reduced our primary outcome of mortality. surfactant was associated with more ventilator-free days, with decreased duration of ventilation and with less use of rescue therapy as compared with standard therapy. there was no significant difference in the duration of picu stay. surfactant therapy was well tolerated; while transient hypoxia and hypotension were reported during surfactant administration, no study reported any serious adverse events. the patients enrolled in these trials are representative of the heterogeneous group of children with early, severe acute respiratory failure that is seen in clinical practice. these patients had similar severity of illness scores and a similar degree of respiratory failure (as measured by pediatric risk of mortality scores and pao 2 :fio 2 ratios). the heterogeneity of results for our primary outcome of mortality was low. the presence of significant heterogeneity reduces the strength of inferences we can make regarding the effect of surfactant on the secondary outcomes of ventilator-free days, meta-analysis of trials of surfactant in children with acute respiratory failure: duration of mechanical ventilation meta-analysis of trials of surfactant in children with acute respiratory failure: duration of mechanical ventilation. ali, acute lung injury; ards, acute respiratory distress syndrome; 95% ci, 95% confidence interval; rsv, respiratory syncytial virus; sd, standard deviation; wmd, weighted mean difference. meta-analysis of trials of surfactant in children with acute respiratory failure: duration of picu stay meta-analysis of trials of surfactant in children with acute respiratory failure: duration of picu stay. ali, acute lung injury; ards, acute respiratory distress syndrome; 95% ci, 95% confidence interval; picu, paediatric intensive care unit; rsv, respiratory syncytial virus; sd, standard deviation; wmd, weighted mean difference. duration of ventilation and duration of picu stay. separately pooling the trials that exclusively enrolled patients with rsv/ severe bronchiolitis and those enrolling patients with ards/ ali from a variety of causes did not significantly reduce the heterogeneity. changing ventilation strategies and the use of a variety of natural and modified natural surfactants may have increased the heterogeneity of our results. ventilation strategies, such as the use of lower tidal volumes and earlier use of high-frequency oscillatory ventilation, have evolved significantly in the 10-year span over which the included trials were conducted [25] [26] [27] . the surfactants used in the included trials were all natural or modified natural surfactants; however, these surfactants may have slightly different effects on oxygenation and compliance due to the differences in phospholipid and surfactant protein composition, which may have influenced individual study results. the strengths of the present review include a comprehensive search strategy, broad inclusion criteria (resulting in a representative, heterogeneous population) and abstraction of clinically important outcomes in duplicate, independently blinded to information that may bias evaluation. the strength of the inference we can make from our subgroup analysis is limited because we were unable to extract all subgroup data from these trials. access to individual patient data would allow better examination of the treatment effect in subgroups of patients and would facilitate further exploration of possible causes of heterogeneity. we found that mortality was very different between the trials that exclusively enrolled patients with rsv/severe bronchiolitis and those that enrolled patients with ards/ali from a variety of causes. we pooled the results because both conditions result in abnormal surfactant function and because of the substantial overlap between the two groups; up to 17% of children in the ards/ali trials had rsv and up to 50% of the children in some bronchiolitis studies also had pneumonia. the reduction in mortality and the increased ventilator-free days have important implications as very few trials in paediatric critical care suggest a favourable impact on mortality [28] . the present review suggests that surfactant could be an important adjunct in the management of paediatric respiratory failure. uncertainty exists, however, about the reproducibility of treatment effects generated from relatively small unblinded trials; questions remain about adverse affects, which may be undetected or under-reported in this literature. also, a large proportion of patients and events are reported in one trial [12] . furthermore, issues of the optimal dose and the timing of administration, and which patients are most likely to derive benefit, should be studied in further adequately powered multicentre trials. the pediatric acute lung injury and sepsis investigators network is planning a large rigorous randomized trial enrolling children with acute hypoxemic respiratory failure to address these issues. surfactant use decreased mortality, was associated with more ventilator-free days and reduced the duration of ventilation. no serious adverse events were reported. most trials enrolled small numbers of children, and further well-designed and adequately powered multicentre trials are therefore required. â�¢ surfactant decreased mortality in a heterogeneous population of children with acute respiratory failure. â�¢ surfactant was associated with more ventilator-free days and a reduced duration of ventilation. â�¢ no serious adverse events were reported. â�¢ further well-designed and adequately powered multicentre trials are required. the feasibility of conducting clinical trials in infants and children with acute respiratory failure adult respiratory distress syndrome in pediatric patients. i. clinical aspects, pathophysiology, pathology, and mechanisms of lung injury harwood jl: abnormal surfactant composition and activity in severe bronchiolitis surfactant abnormalities in infants with severe viral bronchiolitis a controlled trial of synthetic surfactant in infants weighing 1250 g or more with respiratory distress syndrome. the american exosurf neonatal study group i, and the canadian exosurf neonatal study group aerosolized surfactant in adults with sepsis-induced acute respiratory distress syndrome. exosurf acute respiratory distress syndrome sepsis study group bovine surfactant therapy for patients with acute respiratory distress syndrome treatment of acute respiratory distress syndrome with recombinant surfactant protein c surfactant safety and potential efficacy of an aerosolized surfactant in human sepsis-induced adult respiratory distress syndrome exogenous surfactant therapy for pediatric patients with the acute respiratory distress syndrome calf's lung surfactant extract in acute hypoxemic respiratory failure in children effect of exogenous surfactant (calfactant) in pediatric acute lung injury: a randomized controlled trial treatment with bovine surfactant in severe acute respiratory distress syndrome in children: a randomized multicenter study efficacy of interventions for bronchiolitis in critically ill infants: a systematic review and meta-analysis statistical evaluation of ventilator-free days as an efficacy measure in clinical trials of treatments for acute respiratory distress syndrome the statistical basis of meta-analysis quantifying heterogeneity in a meta-analysis 2 copenhagen: the nordic cochrane centre, the cochrane collaboration meta-analysis in clinical trials exogenous surfactant supplementation in infants with respiratory syncytial virus bronchiolitis marraro g: multicenter, randomized, controlled study of porcine surfactant in severe respiratory syncytial virus-induced respiratory failure porcine-derived surfactant treatment of severe bronchiolitis instillation of calf lung surfactant extract (calfactant) is beneficial in pediatric acute hypoxemic respiratory failure treatment with bovine surfactant in severe acute respiratory distress syndrome in children: a randomized multicenter study the acute respiratory distress syndrome network: ventilation with lower tidal volumes as compared with traditional tidal volumes for acute lung injury and the acute respiratory distress syndrome high-frequency oscillatory ventilation in pediatric respiratory failure: a multicenter experience prospective, randomized comparison of high-frequency oscillatory ventilation and conventional mechanical ventilation in pediatric respiratory failure alternative outcome measures for pediatric clinical sepsis trials the authors would like to acknowledge the authors of the primary trials (dr marco luchetti, dr jens mã¶ller, dr shane tibby and dr douglas willson) for providing additional information or clarification. thanks to john duffett for reviewing the citations and for data entry. the authors declare that they have no competing interests. md conceived of this review. md, kc, vn and djc participated in the design. md and vn extracted data and assessed the quality of the included studies. md, kc, djc and ar helped to draft the manuscript. all authors read and approved the final manuscript. the following additional files are available online: a word file containing a table listing individual trial inclusion criteria and exclusion criteria. see http://www.biomedcentral.com/content/ supplementary/cc5944-s1.doc key: cord-020700-iko8gy1e authors: calvo, cristina; aguado, isabel; garcía-garcía, maría luz; ruiz-chercoles, esther; díaz-martinez, eloisa; albañil, rosa maría; campelo, olga; olivas, antonio; muñóz-gonzalez, luisa; pozo, francisco; fernandez-arroyo, rosa; fernandez-rincón, adelaida; calderon, ana; casas, inmaculada title: respiratory viral infections in a cohort of children during the first year of life and their role in the development of wheezing() date: 2017-07-06 journal: an pediatr (engl ed) doi: 10.1016/j.anpede.2016.08.008 sha: doc_id: 20700 cord_uid: iko8gy1e introduction: it is known that infants with viral respiratory infections severe enough to require hospital admission have a high risk of developing recurrent wheezing. few data have been published on unselected populations. the main aim of this study was to analyse symptomatic and asymptomatic respiratory viral infections during the first year of life in a cohort of infants, recruited at birth, and the development of recurrent wheezing. patients and methods: a total of 302 newborns were recruited. a nasopharyngeal aspirate was taken when the patients had a respiratory infection, as well as in the visits for vaccination at 2, 4, 6, and 12 months. rt-nested pcr assays were performed to detect 16 viruses. results: a total of 1293 samples were analysed (1005 healthy controls and 288 respiratory infections). samples taken during routine check-ups were positive in 30.8% of cases, while those with respiratory infection were positive in 77.8%, p < .001 (or: 3, 95% ci: 2.4–3.8). a total of 239 (79%) infants had at least 1 positive respiratory viral infection detected. the most frequent virus (71%) was rhinovirus (rv). recurrent wheezing was found in 27 (11%) children during their first year of life (1.2 episodes, sd 2.9). recurrent wheezing was present in 58.3% of patients admitted to hospital during their first viral infection, vs. 8.6% of infants when the first infection was mild or who had asymptomatic viral detection, p < .001 (or: 2.18; 95% ci: 1.05–4.5). conclusions: in our series, severe respiratory infections leading to hospitalisation in the first months of life are risk factors for developing wheezing, but not in the case of mild rv infections. results: a total of 1293 samples were analysed (1005 healthy controls and 288 respiratory infections). samples taken during routine check-ups were positive in 30.8% of cases, while those with respiratory infection were positive in 77.8%, p < .001 (or: 3, 95% ci: 2.4---3.8). a total of 239 (79%) infants had at least 1 positive respiratory viral infection detected. the most frequent virus (71%) was rhinovirus (rv). recurrent wheezing was found in 27 (11%) children during their first year of life (1.2 episodes, sd 2.9). recurrent wheezing was present in 58.3% of patients admitted to hospital during their first viral infection, vs. 8.6% of infants when the first infection was mild or who had asymptomatic viral detection, p < .001 (or: 2.18; 95% ci: 1.05---4.5). conclusions: in our series, severe respiratory infections leading to hospitalisation in the first months of life are risk factors for developing wheezing, but not in the case of mild rv infections. © 2016 asociación española de pediatría. published by elsevier españa, s.l.u. all rights reserved. infecciones respiratorias; infecciones virales; infecciones asintomáticas; rinovirus; sibilancias; lactantes infecciones virales respiratorias en una cohorte de niños durante el primer año de vida y su papel en el desarrollo de sibilancias resumen introducción: las infecciones respiratorias virales que requieren hospitalización parecen conferir riesgo de desarrollar sibilancias recurrentes, pero existen pocos datos publicados en poblaciones no seleccionadas por tener factores de riesgo. nuestro objetivo principal fue analizar si las infecciones respiratorias virales sintomáticas y asintomáticas, de diferente gravedad, durante el primer año de vida en una cohorte de recién nacidos, suponen un mayor riesgo de sibilancias recurrentes. pacientes y métodos: se incluyeron 302 recién nacidos. se recogió aspirado nasofaríngeo a los niños cuando presentaron una infección respiratoria y de forma periódica en los controles de salud (2, 4, 6 y 12 meses). se estudiaron 16 virus respiratorios mediante reacción en cadena de polimerasa (pcr). resultados: se analizaron 1.293 muestras (1.005 controles de salud y 288 infecciones respiratorias). el 30,8% de las muestras tomadas en los controles de salud fueron positivas, frente a un 77,8% en las infecciones respiratorias, p < 0,001 (or: 3, ic 95%: 2,4-3,8). un total de 239 (79%) lactantes tuvieron al menos una detección viral positiva durante el primer año de vida. el virus más frecuentemente identificado (71%) fue el rinovirus (rv). en 27 lactantes (11%) se detectaron sibilancias recurrentes durante su primer año de vida (2,9 de: 1,2 episodios). el 58,3% de los lactantes cuya primera infección respiratoria requirió hospitalización desarrollaron sibilancias de repetición, frente al 8,6% de los niños cuya primera infección fue leve o asintomática, acute respiratory infections (aris) are a major reason for health care use in infants and constitute a substantial economic burden. 1,2 although infections in this age group are most frequently of a viral aetiology, 3,4 inappropriate antibiotic use continues to be widespread. techniques based on the polymerase chain reaction (pcr) have made the aetiological diagnosis of aris possible in children, and made us aware of the high prevalence of asymptomatic viral infections. 5, 6 respiratory syncytial virus (rsv), human metapneumovirus (hmpv) and influenza virus (flu) have been clearly identified as respiratory pathogens, and more recent studies have been analysing the role of other viruses such rhinovirus (rv) and human bocavirus (hbov), although the high prevalence of coinfection with other respiratory viruses or their detection in asymptomatic patients pose challenges to the interpretation of these positive results. 7, 8 it is known that children with severe rsv infection that require hospitalisation are at high risk of developing asthma in the long term, even as late as age 18 years, as sigurs et al. demonstrated. 9 there is also evidence of an increased risk of recurrent wheezing up to age 5 years in infants hospitalised due to bronchiolitis associated to hmpv. 10 there are fewer studies in children that have not been admitted to hospital, among which we would like to mention the classic studies by stein et al., who analysed an unselected cohort and found that milder infections by rsv also increased the risk of developing wheezing. 11 in recent years, the role of rv as a long-term predictor of future asthma has also been investigated. a study of particular interest on the subject is that of lemanske et al., 12 whose cohort of infants that had a rv infection with wheezing in the first year of life were at increased risk of having developed wheezing by age 3 years, with an odds ratio of 10. most of the studies that assess the risk of developing asthma or wheezing have been conducted in selected high-risk populations. 13, 14 furthermore, little is known about mild viral respiratory infections managed in outpatient settings in relation to the future development of wheezing, not to mention asymptomatic viral infections, although it is generally assumed that risk increases with increasing severity. the objective of our prospective study was to analyse asymptomatic and symptomatic infections of varying severity in a cohort of newborns during the first year of life and assess their role in the development of recurrent wheezing. our secondary objective was to analyse the aetiology and clinical characteristics of these infections. we conducted a prospective cohort study in newborns to analyse the association between past viral infections of varying severity and the development of recurrent wheezing. we requested the participation of all newborns delivered in four health care centres in the área sur health zone of madrid during their first neonatal visit (7---14 days of life). the parents or guardians signed an informed consent form and completed a questionnaire of epidemiological data (history of asthma and atopy in the family, exposure to tobacco smoke, pregnancy and delivery data, number of siblings). nasopharyngeal aspirate (npa) specimens were collected during this visit and routine checkups performed at ages 2, 4, 6 and 12 months. furthermore, if the infants developed respiratory symptoms, they were evaluated by a paediatrician, with collection of an additional npa sample. we considered children with specimens collected during routine well-child checkups healthy controls, even if they presented with isolated rhinorrhoea. we defined asymptomatic infection as detection of a virus in the npa of one of these patients. we defined clinically significant respiratory infection as a case that met the clinical criteria for respiratory infection presented in table 1 . we defined ''first detected viral infection'' as pcr detecting a respiratory virus in a sample for the first time in the life of a child, whether the infection was asymptomatic, symptomatic and managed at the outpatient level, or symptomatic and requiring hospital admission. when a child met the criteria for respiratory infection described in table 1 , the paediatrician conducted an evaluation, a clinical data form was completed, and a npa table 1 in the absence of respiratory distress or wheezing. we defined bronchiolitis as the first episode of respiratory infection that manifested with respiratory distress and wheezing. subsequent episodes of similar characteristics were classified as recurrent wheezing. episodes manifesting with inspiratory dyspnoea and wheezing were classified as laryngotracheobronchitis. laryngitis was defined as inspiratory dyspnoea without wheezing. we defined pneumonia as evidence of focal infiltrates or consolidation in chest radiography in the absence of wheezing. we classified all infections requiring hospital admission as severe, and the rest as mild. when the requisite criteria were met, a npa sample was collected and kept at 4 • c to await transportation to the influenza and respiratory viruses laboratory of the centro nacional de microbiología (national centre of microbiology). in hospitalised patients, the npa sample was collected between 7 and 8 am of the morning following admission and kept refrigerated until it was transported. three rt-nested pcr assays were used to test for 16 respiratory viruses. reverse transcription (rt) and the first amplification reaction were performed with onestep rt-pcr kits (qiagen). influenza a, b and c were detected by a previously described pcr assay. 15 a second multiplex pcr assay was used to detect parainfluenza virus (piv) 1 through 4, coronavirus 229e and oc43, enterovirus and rv. 16 the presence of rsv-a and b, hmpv, hbov and adenovirus (adv) was assessed with a third rt-nested pcr assay using the brq method. 3 we have expressed discrete data as percentages and continuous data as mean and standard deviation. we compared clinical and laboratory characteristics using the student's t, mann---whitney u, chi-square and fisher exact tests. we defined statistical significance as a p-value of less than .05 in any of the tests. the statistical analysis was performed with the statistical package for the social sciences (spss) version 13.0. we recruited a total of 302 infants at birth, of which 55.3% were male (167), 80% born to spanish parents, and 14% of latin-american ancestry. thirty-five percent of infants were exposed to tobacco smoke; 36% of mothers and 37% of fathers had asthma and/or atopy. only 2 patients were born preterm at a gestational age of 33 weeks. all infants but 7 completed the yearlong followup. those 7 patients did not attend the 12-month checkup, but we contacted their paediatricians to obtain information about their clinical outcomes, so they were ultimately included in the analysis. a total of 1,239 samples were collected, 1,005 during routine visits and 288 when a respiratory infection was suspected. of the samples collected during the well-child checkups, 30.8% (310) tested positive, while viruses were detected in 77.8% of respiratory infection cases (224/288), p < .001 (odds ratio, 3; 95% confidence interval, 2.4---3.8). the frequency of viral detection in asymptomatic children (healthy controls) increased proportionally with age from 20.8% in newborns aged 15 days to up to 42% in infants aged 12 months. the virus identified most frequently was rv, found in 82% (254) of cases. other viruses were found in a small proportion of cases: adv (9.7%), piv (4.2%), hbov (3.2%), hmpv (2.9%), flu (2.2%) and rsv (1.6%) (fig. 1 ). of the 302 infants included in the study, 239 (79%) had at least one positive viral detection in the first year of life (fig. 2) . the virus detected most frequently was rv (181 samples, 71%), followed by piv (6.3%), adv (5%) and rsv (6%). coinfections were only detected in 21 cases (8.8%). the mean age at detection of the first viral infection was 3.4 months (sd, 2.8). the first detected viral infection was associated with upper respiratory tract infections in 45 patients (20%), bronchiolitis in 19 (8.5%) while remaining 159 samples (70%) corresponded to asymptomatic patients and had been collected during routine well-child checkups. we analysed the outcomes of infants that had at least one positive specimen, and found that 27 (11%) developed recurrent wheezing in the first year of life (mean ± sd, 2.9 ± 1.2 episodes; range, 2---5) (fig. 3) . rhinovirus was the first virus detected in 18 patients (66%), of who 14 (52%) were asymptomatic at the time of the specimen collection (routine checkup). the mean age of infants that developed recurrent wheezing with their first positive viral detection was 2 months (sd, 1.3; all but three were at least 3 months old). ten of the 27 infants with episodes of bronchiolitis required admission (37%). the most frequently involved virus was rv, both in mild bronchiolitis cases managed at the outpatient level (11/17, 69%) and in severe cases that required hospitalisation (7/10, 70%). recurrent wheezing episodes (at least one following the initial bronchiolitis) occurred in 58.3% of infants in who the first viral detection corresponded to a severe infection that required admission, compared to 8.6% of infants whose first detection corresponded to a mild infection managed at the outpatient level or an asymptomatic infection (p < .001; odds ratio, 2.18; 95% ci, 1.05---4.5). a total of 5 infants received montelukast or budesonide during the followup (18%, 3 admitted to hospital and 2 managed at outpatient level at first positive viral detection). there were no differences in the family history of asthma or atopy between children with and without recurrent wheezing. there were 288 episodes of infection managed in outpatient settings that corresponded to 160 infants (53% of the total sample) who had between 1 and 6 episodes each (mean ± sd, 1.6 ± 1). the most frequent symptoms were: fever in 90 cases (38%), rhinorrhoea in 224 (96%), cough in 223 (96%), respiratory distress in 51 (21%) and nonspecific symptoms in 35 (15%). the most frequent diagnoses were upper respiratory tract infection (147, 51%) and bronchiolitis (48, 16%), with a lesser incidence of recurrent wheezing, laryngitis and pneumonia. only 2 patients had infiltrates on radiologic examination, and both required hospital admission during the followup. the viruses detected in these infections were rv in 127 cases (55.8%), followed by piv (13%), rsv (12.5%), hmpv (8.9%) and flu (8%) (fig. 2) . the causative agent of upper respiratory tract infection was rv in 61% of cases, followed by piv (13%) and adv (11%). when it came to bronchiolitis, rv was detected in 53%, rsv in 25% and hmpv in 8% of cases. we analysed a total of 19 hospital admissions corresponding to 14 infants (14/302; 4.6% of the cohort; 5.8% of infants with a positive viral detection). five patients were admitted more than twice in the first year of life. fifty-eight percent of admitted patients (11) were female. the mean age of admission was 3.6 months (sd, 2.5 months; range, 6 days---8 months). forty-two percent presented with fever (mean, 38.6 • c; sd, 0.9 • c) lasting 3.5 days (sd, 2). sixty-eight percent (13/19 episodes) required oxygen supplementation for a mean of 5 days (sd, 3). twenty-six percent (5/19) had infiltrates detected by radiology. the diagnoses were bronchiolitis in 12 cases (63%), wheezing episode in 3 (15%), upper respiratory tract infection in 3 and pneumonia in 1. only 3 infants received antibiotics. respiratory syncytial virus was detected in 42% of the cases and rv in 52%. viral coinfection was detected in 4 cases. a considerable number of infants test positive for at least one respiratory virus in the first year of life, and we observed a percentage of 79% in our cohort. more than half of the cohort experienced at least one symptomatic infection treated at the outpatient level, with a mean of 1.6 symptomatic infections a year (range, 1---6), while approximately 5% required hospital admission. rhinovirus was the virus detected most frequently in symptomatic (43%) as well as asymptomatic (25%) infants. eleven percent of the infants with positive virology results went on to develop recurrent wheezing in the first year of life, but the risk was much higher in patients that required admission due to bronchiolitis (58%; odds ratio, 2.18) compared to patients whose first infection was mild or asymptomatic. our data are consistent with the findings of the study conducted by rhedin et al., who analysed a group of paediatric outpatients with respiratory infections and a group of asymptomatic controls. rhedin et al. detected respiratory viruses in 72.3% of cases (n = 151) and 35.4% of controls (n = 74) (p = .001). rhinovirus was the virus identified most frequently in both cases and controls (47.9 and 21.5%, respectively). 6 although infections by rv are common, a recent study that used nucleotide sequencing demonstrated that the prolonged presence of rhinovirus rna in the respiratory tract following an upper respiratory tract infection is rare in healthy infants (<5%). in these infants, the detection of rv rna in repeat samples at least 30 days apart most likely corresponded to new infections. 17 another study found that the detection of the same virus strain more than 2 weeks apart was unusual. 18 in our study, we found that rv was detected in a significant proportion of infants that were classified as healthy (routine vaccination visit), probably because we did not take isolated rhinorrhoea into account, and this is a frequent symptom in children that does not contraindicate vaccination. however, it is possible that rhinorrhoea is actually a presenting symptom in mild rv infections. unfortunately, we have no data on the percentage of infants considered healthy controls that had rhinorrhoea as the sole symptom. this is consistent with the hypothesis of jartti et al. according to which rv always causes an actual infection, which may or may not be clinically relevant. 5 although there are authors like jansen et al., 19 who found a higher rv load in children with acute symptomatic infections compared to asymptomatic children, or utokaparch et al., 20 who detected significantly higher rv loads in infections of the lower respiratory tract, there are others, like takeyama et al., 21 who have not found a correlation between viral load and disease severity in rv infections. at any rate, the clinical significance of viral loads in respiratory samples is not well established, and we did not measure viral loads in our patients. we also ought to highlight the small percentage of coinfections found in our cohort (8.8%), as other case series, including previous studies by our research group, found a percentages of coinfection of up to 46% in hospitalised children with rhinovirus infections. 22 we believe that the age of our patients (a substantial proportion of the specimens were collected from infants aged less than 6 months) and our decision to include mild and asymptomatic infections with positive virology may account for the low proportion of coinfection. in a cohort study of 140 newborns with a design similar to our own, van der gugten et al. 23 found that rv infections manifesting with wheezing in the first year of life were a risk factor for developing recurrent wheezing in the future. although they did not assess the severity of acute episodes, mild infections without wheezing were not associated with an increased risk of recurrent wheezing. these authors reached the conclusions we have reached based on our cohort, in which infections by rv or rsv that were sufficiently severe to cause bronchiolitis requiring hospital admission were a risk factor for developing wheezing as early as the first year of life. the followup of our cohort in the immediate future will allow us to learn whether the risk of wheezing continues to be higher in early childhood. as lemanske et al. suggested, 12 the severity of infection is a risk factor for the development of asthma and recurrent wheezing. various studies have demonstrated that severe bronchiolitis associated to rv infection increase the risk of asthma, but as we commented above, these studies were conducted in selected populations. 24---26 our case series, which analysed symptomatic and asymptomatic infections and severity at an early age (less than 3 months in most first episodes) in an unselected population contributes a broader picture on the subject. although our study has strengths, such as its unselected sample and testing for all respiratory viruses (as opposed to only the most prevalent) and for the presence of asymptomatic infection, we ought to comment on certain methodological aspects. we did not collect weekly samples to study viral shedding and its duration, and we also did not perform genotyping of rv. the duration of followup was short, and the number of patients with recurrent wheezing small. nevertheless, we believe that the association found in the study is worth considering, and that we will be able to reach broader conclusions in upcoming years. until then, we can state that respiratory infections in the first months of life that are severe enough to require hospitalisation are a risk factor for the development of recurrent wheezing. this study was partially funded by the the economic burden of noninfluenza-related viral respiratory tract infection in the united states viruses associated with acute respiratory infections and influenza-like illness among outpatients from the influenza incidence surveillance project detection of new respiratory viruses in infants hospitalized with bronchiolitis. a three year prospective study respiratory syncytial virus-associated hospitalizations among children less than 24 months of age indentification of respiratory viruses in asymptomatic subjects: aymptomatic respiratory viral infections clinical utility of pcr for common viruses in acute respiratory illness human bocavirus detection in nasopharyngeal aspirates of children without clinical symptoms of respiratory infection high incidence of human bocavirus infection in children in spain asthma and allergy patterns over 18 years after severe rsv bronchiolitis in the first year of life human metapneumovirus bronchiolitis in infancy is an important risk factor for asthma at age 5. pediatr pulmonol respiratory syncytial virus in early life and risk of wheeze and allergy by age 13 years rhinovirus illnesses during infancy predict subsequent childhood wheezing wheezing rhinovirus illnesses in early life predict asthma development in high-risk children rhinovirus induced wheezing in infancy ----the first sign of childhood asthma? simultaneous detection of influenza a, b, and c viruses, respiratory syncytial virus, and adenoviruses in clinical samples by multiplex reverse transcription nested-pcr assay simultaneous detection of fourteen respiratory viruses in clinical specimens by two multiplex reverse transcription nested-pcr assays duration of rhinovirus shedding in the upper respiratory tract in the first year of life serial viral infections in infants with recurrent respiratory illnesses frequent detection of respiratory viruses without symptoms: toward defining clinically relevant cutoff values the relationship between respiratory viral loads and diagnosis in children presenting to a pediatric hospital emergency department rhinovirus load and disease severity in children with lower respiratory tract infections role of rhinovirus c respiratory infections in sick and healthy children in spain human rhinovirus and wheezing: short and long-term associations in children response to infections in patients with asthma and atopic disease: an epiphenomenon or reflection of host susceptibility? rhinovirus-induced bronchiolitis and asthma development rhinovirus bronchiolitis and recurrent wheezing: 1-year follow-up key: cord-001162-z8cbbit3 authors: yun, heather c.; fugate, william h.; murray, clinton k.; cropper, thomas l.; lott, lisa; mcdonald, j. matthew title: pandemic influenza virus 2009 h1n1 and adenovirus in a high risk population of young adults: epidemiology, comparison of clinical presentations, and coinfection date: 2014-01-08 journal: plos one doi: 10.1371/journal.pone.0085094 sha: doc_id: 1162 cord_uid: z8cbbit3 background: in 2009, pandemic h1n1 influenza virus (2009 h1n1) emerged worldwide, causing morbidity and mortality that disproportionately affected young adults. upper respiratory infection (uri), largely due to adenovirus, is an endemic cause of morbidity in military training. whether clinical presentations differ or excess morbidity results from coinfection is unclear. methods: the center for advanced molecular detection evaluates epidemiology and rapid diagnostics of respiratory pathogens in trainees with uri. from may 1, 2009, to november 30, 2009, demographic, clinical, and pcr data from throat and nasal specimens for adenovirus and 2009 h1n1 were prospectively collected. results: 375 trainees with uri enrolled and were tested for both adenovirus and 2009 h1n1 by pcr (median age 20; 89% male). adenovirus pcr was positive in 72% (96% serotype e-4) and 2009 h1n1 in 20%. males were more likely to have adenovirus and females more likely to have 2009 h1n1 (p = 0.047). subjects with 2009 h1n1 presented an average of 1 week earlier in training, had shorter illness duration before enrollment, less sore throat, diarrhea, and fewer abnormal findings on throat exam. coryza and cough were more common with 2009 h1n1 compared to adenovirus. subjects with 2009 h1n1 were less likely to have adenovirus than those without, despite persistently high frequencies of adenovirus detections during peak 2009 h1n1 weeks (15% vs. 83%, p < 0.01). coinfection with adenovirus and 2009 h1n1 was rare (4%). rates of hospitalization and pneumonia did not differ between the adenovirus, 2009 h1n1, or coinfected groups. conclusion: military trainees with 2009 h1n1 vs. adenovirus have differing clinical presentations, and males are more likely to have adenovirus. despite high frequencies of adenovirus infection, coinfection with adenovirus and 2009 h1n1 is rare and apparently does not result in increased morbidity. non-influenza related upper respiratory infections (uri) are universally experienced illnesses that, despite their typically selflimited nature, lead to billions of dollars of lost income, and predispose to serious illnesses including pneumonia. [1] when influenza is responsible, pandemics can result and cause millions of deaths. in 2009, a novel h1n1 influenza virus (2009 h1n1) emerged and rapidly spread worldwide, causing excess mortality in children and young adults. although the global estimate of deaths has been lower than seen in several previous pandemics, the number of life years lost is estimated to be five times higher than those lost to seasonal h1n1 viruses and comparable to the number lost during the 1968 pandemic. [2, 3] military trainees, along with other groups of crowded, stressed individuals, are disproportion-ately affected by respiratory illnesses due to a variety of pathogens. with the exception of the prior adenovirus vaccine era from 1980-1996, adenoviruses have historically been the most common causes of febrile uri in this population, and have also led to serious illness and fatalities. [4] [5] [6] [7] in one large study of transmission dynamics of adenovirus in a military training setting, approximately one-third of incoming trainees were already immune, one-third developed a febrile uri due to adenovirus, and the remainder seroconverted with subclinical or asymptomatic infection. [8] large influenza outbreaks are less common, given the universal immunization of basic trainees and routine use of ring antiviral chemoprophylaxis in training units with known influenza cases, if cases occur within the first two weeks after immunization. [9, 10] however, in 2009, type-specific influenza vaccine was not widely available until well into the full wave of illness. [11] with large numbers of concurrently circulating respiratory pathogens occurring year round in this diverse group of individuals, coming from a variety of geographic locations and backgrounds, and living in close contact for months, coinfection with multiple organisms would be expected to be a regular occurrence. however, whether coinfection contributes to differing clinical presentations or outcomes in this young, healthy adult population is unknown. while coinfections with viral pathogens including 2009 h1n1 have been described in patients with respiratory infections, few prospective studies have related these to clinical presentation and outcomes in adults since molecular diagnostics became available, and none in the setting of high background rates of adenovirus. [12] [13] [14] [15] [16] [17] we sought to describe the epidemiology of 2009 h1n1 and adenovirus in a basic training population, and to correlate differences in clinical presentations and outcomes with each respective pathogen and in coinfections. joint base san antonio-lackland is the only air force location for basic military training with approximately 43,000 recruits per year, 6,000-7,000 recruits training at any given time, and a training period lasting 8.5 weeks. basic military trainees (bmts) are assigned to training units called ''flights'' of 50-60 individuals, with whom they train and reside in bay dormitories; tobacco product use is not allowed. ill trainees present for care at an outpatient clinic; if they are febrile with a respiratory illness they are then cohorted to a ''fever flight'' where they recover until they are afebrile and able to return to training. trainees who require hospitalization are admitted to the tertiary care hospital on base. trainees routinely receive chemoprophylaxis against streptococcus pyogenes during their first week of training; this consists of benzathine penicillin or azithromycin for penicillin allergic recruits. immunizations against meningococcus, hepatitis a and b, and measles, mumps and rubella are also administered during the first week of training. trivalent seasonal influenza vaccine was administered during the first week of training throughout the study period, but 2009 h1n1 vaccine was not available until december 1, 2009 . during the study period, oseltamivir was routinely used for treatment of ill trainees with confirmed 2009 h1n1 infection. oseltamivir was also routinely used for chemoprophylaxis of well trainees in close contact with a confirmed case. the center for advanced molecular detection (59 th medical wing/science and technology, air education and training command) was established in 2003 for prospective evaluation of epidemiology and novel technologies to rapidly detect respiratory pathogens in trainees with uri. subjects were approached for enrollment at the point of care for their uri, and met inclusion criteria if they were bmts 17 years of age or older and had any symptom of upper respiratory tract infection or pneumonia. demographic data, including age, race, gender, week of training, city/state of previous residence, and smoking history, were recorded. additionally, a symptom questionnaire (including respiratory and gastrointestinal symptoms), perceived stress level on a 10-point likert scale, and clinical signs, including vital signs, height and weight, physical exam findings, and physician diagnosis were recorded, as was the ward of hospital admission (intensive care unit vs. ward) where applicable. for this substudy, cases were included if they enrolled in the study and were tested for both adenovirus (using study methodology) and 2009 h1n1 (as part of clinical care). duplicate cases (for numerous presentations for uri nucleic acid extraction primers and probes used have been reported for adenovirus by heim et al. [19] all qpcr was conducted using applied biosystems 7900 and 7500 real-time pcr instruments (applied biosystems, ca). for adenovirus testing, cycling was conducted with 500 nm concentrations of both forward and reverse primers and 300 nm concentration of probe. reaction conditions included an initial 10 min denaturation at 95uc, followed by 45 cycles of 95uc for 15 sec and 60uc for 1 min. a specimen was considered positive if its cycle of threshold (ct value) was equal to or less than 40, as described previously. [19, 20] . all subjects provided written, voluntary informed consent in the presence of an ombudsman. the study was approved by wilford hall medical center/brooke army medical center institutional review board (irb). gender and ethnicity were self-reported. per department of defense (dod) directive 3216.02, for purposes of legal capacity to participate in dod-conducted or -supported research involving human subjects, all active duty service members in a federal duty status are considered to be adults. the participation of such members is not subject to requirements regarding research involving children or minors. when service members are under 18 years of age, students at service academies, or trainees, the irb shall carefully consider the recruitment process and the necessity to include such members as human subjects. the wilford hall medical center/brooke army medical center irb carefully considered the bmt recruitment process for the study and ruled that consenting bmts 17 years of age or older who presented to the health clinics could be enrolled. data were entered in duplicate for quality control. analysis was performed using existing software (spss, version 19.0; spss). continuous variables were analyzed by student's t-test or mann-whitney u test for parametric and nonparametric data, respectively. categorical variables were evaluated by chi-squared, fisher's exact test, or spearman correlation. multiple nonparametric groups of continuous variables were analyzed by kruskal-wallis testing. all p-values are two-tailed and statistical significance at p ,0.05. table 2 , and seasonal variation presented in figure 1 . during the peak of 2009 h1n1 activity among trainees, weeks 33-42, out of 130 subjects, adenovirus was found alone in 58, 2009 h1n1 alone in 51, and 9 were coinfected; neither virus was recovered from 12 subjects. during that time frame, there was a negative association between 2009 h1n1 and adenovirus (p,0.01). subjects with 2009 h1n1 were much less likely to be adenovirus positive than those without 2009 h1n1 (15% vs. 83%). subjects with adenovirus were much less likely to have 2009 h1n1 than those without adenovirus (13% vs. 81%). a comparison of demographic information in subjects infected with 2009 h1n1 vs. adenovirus is presented in table 3 . significant findings include gender disparities of 2009 h1n1 and adenovirus infection, with a larger proportion of males presenting with adenovirus (p = 0.047). including subjects with coinfections, adenovirus was detected in 60% of female subjects vs. 74% of male subjects, and 2009 h1n1 in 30% of female subjects vs. 19% of male subjects. 2009 h1n1 infected subjects also presented one week earlier in training. however, after exclusion of cases diagnosed in the first week of training, the distributions of the epidemic curves were similar and both peaked in the fifth week (data not shown). adenovirus detections peaked earlier in the year compared to 2009 h1n1 infections. the most commonly reported symptoms were fever (100%), cough (90%), headache (88%), and sore throat (87%), and the median duration of symptoms prior to enrollment was 3 days (table 4 ). six patients were diagnosed with pneumonia, and three were admitted to the hospital, all to regular internal medicine wards. clinical differences, presented in table 4 , included a shorter duration of illness prior to presentation for clinical care in the 2009 h1n1 infected group compared to the adenovirus group (2 days vs. 3 days, p , 0.01), increased proportion of subjects complaining of coryza and cough in the 2009 h1n1 group, and increased predominance of sore throat and diarrhea in the adenovirus group. physical exam revealed increased abnormal findings on throat exam in the adenovirus group. there were no differences in pneumonia or hospitalization rates between the two groups, but both of these events were rare. clinical data in subjects infected with adenovirus, with or without concomitant 2009 h1n1, are also presented in table 4 . sore throat was less common in the coinfection group compared to adenovirus alone (71% vs. 94%, p = 0.01), and abnormalities on throat exam were less common in the coinfection group compared to adenovirus alone. oral temperature on presentation was higher for the coinfected group than for those infected with adenovirus alone (101.9 0 f vs. 101.5 0 f, p = 0.03). there were no hospitalizations or diagnoses of pneumonia in the coinfection group; this was not significantly different than the rates seen in the adenovirus group. [16, 21] . a number of studies have also evaluated whether 2009 h1n1was associated with either negative or positive effects (predominantly in terms of acquisition rather than severity) on other respiratory viruses, and, taken together with reference to adenovirus, the results of these are inconclusive. one study performed in the united kingdom in 2009-2010 suggested negative associations between 2009 h1n1 and human metapneumovirus as well as rhinovirus, though not adenovirus. however, few adenovirus detections were found compared to our population (,5%), and most of these were in young children. [22] a south african study of respiratory viruses in hospitalized patients found only six patients with adenovirus and 2009 h1n1 coinfection out of over 8000 subjects enrolled. [23] to our knowledge, negative associations between adenovirus and influenza a virus (either seasonal or 2009 h1n1) detection have not been demonstrated. it is also unclear, if there is a negative association between the two viruses, whether adenovirus is protective against influenza infection or vice versa. in this data set, influenza patients presented earlier, although not after excluding those presenting during the first week of training, who likely would have arrived with their infection. the policy of cohorting together all bmts with fever and uri, regardless of the causative pathogen, would seem to increase the likelihood of coinfection, rather than skewing the data towards the appearance of a negative association. it will also be worthwhile to see whether influenza epidemiology will change since the late 2011 reintroduction of adenovirus serotypes 4 and 7 vaccines in military trainees, or whether issues arise with concurrent administration of both live attenuated influenza and adenovirus vaccines, which could affect current trainee vaccine policies. in the meantime, concerns about cohorting patients that may have either adenovirus or 2009 h1n1 on the basis of syndromic presentation can be alleviated on the relative scarcity of coinfection and on the absence of any evidence of increased illness severity among coinfected subjects. the differing demographics and clinical presentations of adenovirus vs. 2009 h1n1 infection in this study are also of interest. first, the gender differences seen for each virus are intriguing. males represented 89% of the study population, enrolled after presenting with respiratory illness. however, males generally represent only 80% of the air force basic training population, so the study population was already disproportionately male. adenovirus has long been suggested to be predominantly an illness of men in this population, and this study is no exception to this trend. [24, 25] 2009 h1n1, like all influenza a viruses, has similar mechanisms of transmission, yet in this study population disproportionately affected females. the reasons for this gender difference are unclear. both of these infections as captured in this study population would meet the cdc definition for influenzalike-illness (fever plus cough or sore throat), used for surveillance and cohorting purposes, but had differences in presentation of statistical and arguably clinical significance. [26] adenovirus was consistently more likely to produce signs and symptoms referable to the throat, while 2009 h1n1 produced a predominance of cough, as well as shorter illness duration prior to presentation for care, potentially reflecting more rapid development of uncomfortable symptoms. these findings may have importance for both infection control and empiric therapy. the strengths of this study include the molecular characterization of respiratory pathogens together with capture of detailed clinical data in a large cohort of otherwise healthy adult patients with few complicating comorbidities, as well as the closed nature of the population, which lends itself to capture of events such as hospital admissions and pneumonia diagnoses. limitations include the absence of serologic data, or the ability to serially characterize pathogens from the same cohort of individuals, which would provide more granular detail about the time course of developing each infection. subject enrollment was variable throughout the study period, depending on rates of clinical illness within the training population, as well as availability of study personnel to enroll trainees, and given that 2009 h1n1 influenza virus pcr was done as part of clinical care, there could have been some differences in those who enrolled vs. did not enroll, or those who received 2009 h1n1 testing (and thus were included in this study) and those who did not. due to this variability in total enrollment, and because 2009 h1n1 testing became infrequent clinically after november 2009, inferences about the impact of h1n1 towards the end of 2009 are limited. however, data from the naval health research center's febrile respiratory illness surveillance update show a rate of febrile respiratory infection that increased in december of 2009, 82% of which was associated with adenovirus, with no influenza virus detected. [27] additionally, the use of oseltamivir, both for prophylaxis and for treatment, would have impacted both epidemiology and severity of illness. finally, without inclusion of asymptomatic or minimally symptomatic individuals, conclusions can only be drawn about the scarcity of coinfections with individuals at the point of presentation to care. in summary, this epidemiologic survey of young adults in military training presenting with fever and uri demonstrated significant differences in 2009 h1n1 vs. adenovirus in terms of gender predilection and presenting symptoms. in addition, coinfections with 2009 h1n1 and adenovirus were rare despite high endemicity of adenovirus before and during the 2009 h1n1 epidemic, and, beyond a higher temperature on presentation, coinfections were not associated with increased clinical severity compared with adenovirus alone. the economic burden of non-influenza-related viral respiratory tract infection in the united states portrait of a year-old pandemic preliminary estimates of mortality and years of life lost associated with the 2009 a/h1n1 pandemic in the us and comparison with past influenza seasons adult adenovirus infections: loss of orphaned vaccines precipitates military respiratory disease epidemics. for the adenovirus surveillance group acute respiratory disease in military trainees: the adenovirus surveillance program genotype prevalence and risk factors for severe clinical adenovirus infection, united states adenovirus-associated deaths in us military during postvaccination period transmission dynamics and prospective environmental sampling of adenovirus in a military recruit setting respiratory diseases among u.s. military personnel: countering emerging threats influenza a in a basic training population: implications for directly observed therapy cdc: 38 million doses of h1n1 vaccine available do rhinoviruses reduce the probability of viral co-detection during acute respiratory tract infections? simultaneous detection of influenza a, b, and c viruses, respiratory syncytial virus, and adenoviruses in clinical samples by multiplex reverse transcription nested-pcr assay mixed respiratory virus infections pan-viral screening of respiratory tract infections in adults with and without asthma reveals unexpected human coronavirus and human rhinovirus diversity rate and influence of respiratory virus co-infection on pandemic (h1n1) influenza disease high burden of non-influenza viruses in influenza-like illness in the early weeks of h1n1v epidemic in france cdc protocol of realtime rtpcr for influenza a (h1n1) (world health organization collaboration center for influenza at the rapid and quantitative detection of human adenovirus dna by real-time pcr evaluation and validation of a real-time pcr assay for detection and quantitation of human adenovirus 14 from clinical samples respiratory viral coinfection among hospitalized patients with h1n1 2009 during the first pandemic wave in brazil respiratory viral infections during the 2009-2010 winter season in central england, uk: incidence and patterns of multiple virus co-infections respiratory viral coinfections identified by a 10-plex real-time reversetranscription polymerase chain reaction assay in patients hospitalized with severe acute respiratory illness-south africa outbreak of severe respiratory disease associated with emergent human adenovirus serotype 14 at a us air force training facility in 2007 epidemic of adenovirus-induced respiratory illness among us military recruits: epidemiologic and immunologic risk factors in healthy, young adults self-reported influenza-like illness during the 2009 h1n1 influenza pandemic-united states febrile respiratory illness (fri) surveillance update, department of respiratory diseases research, naval health research center we appreciate the administrative assistance of francine stotler with the execution of this study.disclaimer: the opinions or assertions contained herein are the private views of the authors and are not to be construed as official or reflecting the views of the department of the army, department of the air force, department of defense or the us government. this work was prepared as part of their official duties and, as such, there is no copyright to be transferred. key: cord-005583-hmv8jjfl authors: peters, m. j.; tasker, r. c.; kiff, k. m.; yates, r.; hatch, d. j. title: acute hypoxemic respiratory failure in children: case mix and the utility of respiratory severity indices date: 2013-12-27 journal: intensive care med doi: 10.1007/s001340050647 sha: doc_id: 5583 cord_uid: hmv8jjfl objective: acute hypoxemic respiratory failure (ahrf) is a common reason for emergency pediatric intensive care. an objective assessment of disease severity from acute physiological parameters would be of value in clinical practice and in the design of clinical trials. we hypothesised that there was a difference in the best early respiratory indices in those who died compared with those who survived. design: a prospective observational study of 118 consecutive ahrf admissions with data analysis incorporating all blood gases. setting: a pediatric intensive care unit in a national children’s hospital. interventions: none. results: mortality was 26/118,22% (95% confidence interval 18–26%). there were no significant differences in the best alveolar-arterial oxygen tension gradient (a-ado(2), torr), oxygenation index (oi), ventilation index (vi), or pao(2)/fio(2) during the first 2 days of intensive care between the survivors and non-survivors. only the mean airway pressure (map, cm h(2)o) used for supportive care was significantly different on days 0 and 1 (p≤0.05) with higher pressure being used in non-survivors. multiple logistic regression analysis did not identify any gas exchange or ventilator parameter independently associated with mortality. rather, all deaths were associated with coincident pathology or multi-organ system failure, or perceived treatment futility due to pre-existing diagnoses instead of unsupportable respiratory failure. when using previously published predictors of outcome (vi>40 and oi>40; a-ado(2)>450 for 24 h; a-ado(2)>470 or map>23; or a-ado(2)>420) the risk of mortality was overestimated significantly in the current population. conclusion: the original hypothesis was refuted. it appears that the outcome of ahrf in present day pediatric critical care is principally related to the severity of associated pathology and now no longer solely to the severity of respiratory failure. further studies in larger series are needed to confirm these findings. (95 % confidence interval 18-26 % ). there were no significant differences in the best alveolar-arterial oxygen tension gradient (a-ado> torr), oxygenation index (oi), ventilation index (vi), or pao2/fio2 during the first 2 days of intensive care between the survivors and nonsurvivors. only the mean airway pressure (mar cm h;o) used for supportive care was significantly different on days 0 and i (p _< 0.05) with higher pressure being used in non-survivors. multiple logistic regression analysis did not identify any gas exchange or ventilator parameter independently associated with mortality. rather, all deaths were associated with coincident pathology or multi-organ system failure, or perceived treatment futility due to pre-existing diagnoses instead of unsupportable respiratory failure. when using previously published predictors of outcome (vi > 40 and oi > 40; a-ado2 > 450 for 24 h; a-ado 2 > 470 or map > 23; or a-ado2 > 420) the risk of mortality was overestimated significantly in the current population. conclusion: the original hypothesis was refuted. it appears that the outcome of ahrf in present day pediatric critical care is principally related to the severity of associated pathology and now no longer solely to the severity of respiratory failure. further studies in larger series are needed to confirm these findings. acute hypoxemic respiratory failure (ahrf) remains a common reason for emergency pediatric intensive care. even with currently available ventilatory support it re-tains a significant morbidity and mortality with one subgroup, those with acute respiratory distress syndrome (ards), having a mortality rate of 40-75 % [1] [2] [3] [4] [5] [6] . however, it has also been suggested that much lower mortalities (< 10%) may occur in specific conditions such as respiratory syncytial virus-related ards [7] . in this setting, promising new interventions, including permissive hypercarbia, exogenous surfactant, inhaled nitric oxide, high frequency oscillatory ventilation, extracorporeal membrane oxygenation and perfluorocarbon-assisted gas exchange have been described with the purpose of 'rescuing' potential survivors from severe pathology. in order to evaluate effectively such therapies, clinical trials have adopted assessments of disease severity for patient recruitment. such an approach is based on the hypothesis that the initial magnitude of acute severe physiological derangement equates with subsequent mortality. thereby warranting the experimental intervention as well as providing a measure for verifying the similarity between treatment patients and control patients. our experience from a retrospective study of children with severe ahrf [8] questioned the usefulness of the previously reported respiratory predictors of outcome [2] [3] [4] [5] . rather, in common with sarnaik and colleagues [9] , who found in their pediatric study that response to an intervention better predicted intensive care outcome, we reported that greater improvement in oxygenation to an intervention (a standard dose of inhaled nitric oxide) was associated with improved outcome [8] . this suggests that with current clinical expertise, a test of potential ventilation-perfusion mismatch, shunt and lung injury reversibility is a more appropriate predictor of outcome than the status prior to any intervention. in an individual patient, such a response to intervention should be indicated by their best, rather than worst, measure of gas exchange, therefore, the purpose of the present prospective, single institution study of ahrf in children was to assess whether the best, early respiratory indices in non-survivors were significantly different from those who survived. approval for this observational study was obtained from our institution's ethics committee and patient data was stored according to the requirements of the data protection act. between august 1 1995 and march 31 1997, all children older than 1 month and less than 16 years of age admitted to our pediatric intensive care unit (picu) were eligible for inclusion in this prospective study. inclusion criteria were modified from the american-european consensus conference diagnostic criteria for ards [10] : a) acute onset of respiratory failure over less than 48 h, b) evidence of a severe defect in oxygenation (arterial oxygen tension to fraction of inspired oxygen ratio (pao2/fio2) of less than 200 mmhg) for at least six consecutive hours on the day of picu admission, c) no evidence of left atrial hypertension and d) four quadrant interstitial shadowing on chest x-ray. children without the characteristic chest x-ray appearances of ards, but meeting the other criteria for ards, were described as cases of ahre details of the patients' acute diagnoses as well as any underlying conditions were recorded. an electronic patient charting sys-tem (carevue, hewlett packard) was reviewed daily and ventilator and physiological parameters recorded and stored on a separate data base. every blood gas analysis performed throughout the patients' admissions was reviewed and the oxygenation index (oi = mean airway pressure (map) x fio2/pao2) , alveolar-arterial oxygen tension gradient (a-ado2), pao2/fio 2 ratio and ventilation index (vi = respiratory rate x paco 2 x peak inspiratory pressure / 1000) were calculated for each blood gas measurement. analyses carried out used the best value obtained over the period under assessment. for the comparison with previously reported studies [2] [3] [4] [5] every blood gas was reviewed and the respective study criteria applied for patient selection. the ventilatory strategy employed in these patients was one of permissive hypercarbia (target pao 2_< 8kpa, provided ph > 7.25) with limitation of peak inspiratory pressure (< 35 cmh20 ) while employing high maps to ensure maximum lung volume recruitment via the use of peak end expiratory pressure and inverse inspiratory : expiratory ratios. high frequency oscillatory ventilation was employed if oxygenation was inadequate with a map of 20 cmh20 or greater. the use of inhaled nitric oxide therapy throughout the last 12 months of the study was controlled by an institution approved multi-center randomisation protocol. extracorporeal membrane oxygenation was employed when no stability could be achieved with the above techniques. death or survival to discharge from the picu were the end points of the study. in children who died, the mode of death was recorded: failed resuscitation, limitation or withdrawal of support or brain death. the clinical course of patients with ahrf was categorised according to the severity of disrupted gas exchange and whether or not improvement occurred within 3 days. the data were stored in a microsoft access 2.0 data base and analysed with microsoft excel 7.0 and statistical software (statistical package for social sciences 6.13, spss inc.). comparisons between non-survivor and survivor data were performed with an independent sample t-test after transformation to normality if required. multiple logistic regression analysis was performed against survival for a range of respiratory parameters from days 0, 1 and 2 in those patients with available data for those days. parameters found to be significant in the univariate analysis were tested as well as those indices previously suggested to be associated with outcome from ards or ahrf [2] [3] [4] [5] . in addition, age, weight, multi-organ system failure (mosf) score [11] and the presence of underlying immunodeficiency were also tested in the model. beta coefficients from significant independent predictors were converted to adjusted odds ratios with 95 % confidence intervals. comparison with published series [2] [3] [4] [5] included a meta-analysis of reported results, calculation of the likelihood ratio for a positive test resuit, and the two-sample test for proportions. out of 850 admissions to the picu, 118 patients were admitted with ahrf over the 20 months of the study. the median age was 9 months (range 1-167 months), and weight 4.3 kg (1-53 kg). the median length of picu stay was 8 days (range 0-80 days). the picu mortality was 39/732, 5 % (95 % confidence interval 4-7 % ) in the non-ahrf cases, and significantly greater in the ahrf cases, 26/118, 22% (14-30%, p< 0.001). one patient with ahrf died within 6 h of admission, having been inappropriately intubated and resuscitated: since treatment was limited from the time of admission, his respiratory indices data were excluded from analysis. fifty-two children who fulfilled the full criteria for ards in addition to ahrf had a significantly higher mortality in comparison to the non-ards, ahrf patients: 36.5% (19/52) and 10.6% (7/66), respectively (p < 0.001). the ventilatory strategy in these patients is reflected in the group median paco2 and map for survivors and non-survivors for each day of the study (fig. 1 ). nonconventional or specialised intensive care treatments included extra-corporeal membrane oxygenation (4 cases, 1 death), high frequency oscillatory ventilation (25cases, 9deaths), artificial surfactant (15cases, 4 deaths) and intention to use nitric oxide (38 cases, 12 deaths). outcome and acute physiological disturbance the range of physiological parameters most widely suggested to be associated with outcome are shown as a univariate analysis (table 1) for the day of admission (day 0), and the subsequent two complete days on the picu. on days 0 and 1 the eventual survivors do not differ significantly from non-survivors in terms of early a-ado 2, paojfio 2, oi or vi (when using the best values from all blood gases over the particular time period). by day 2, however, the a-ado2 just reached significance (p = 0.05). although it should be noted that up to this time there had been significant attrition in patient numbers with nine of the patients present on day 0 not surviving and seven others improving to extubation, and therefore beyond the need for blood gas monitoring. in relation to the ventilatory parameters, there were significant differences between survivors and non-survivors in the maximum map employed on day 0 and day 1, and the highest peak inspiratory pressures employed on day 0. in stepwise multiple logistic regression analysis, best and worst gas exchange parameters (a-ado2, pao2/ fio> oi, vi, paco2, peak inspiratory pressure, positive end expiratory pressure and map) were not independently associated with poor outcome on days 0, 1 or 2 of admission. the findings were not altered by correction for age, weight, mosf score or the presence of immunodeficiency. the association between ards and increased mortality noted on univariate analysis was not significant when corrected for the presence of mosf score _> 2. respiratory infection (44) non-infective respiratory disease (21)* head injury (2) • non-survivors all cases (118) immunodefieicney (29)* ex-premature (22) normal ( the acute diagnosis associated with admission and the mortality in each category is shown in fig. 2 as well as the underlying or associated diagnosis. the presence of mosf (score _> 2) was the only pattern of acute diagnosis associated significantly with death: 45 % mortality rate (18/40) with an odds ratio (95 % confidence interval) of 4.4 (1.5-13.5). of special note is the favorable outcome for previously healthy children with only 3 deaths from 34 cases (9.6 %) compared with the 23/83 (27.7%) in cases with pre-existing disease (p < 0.001). as with previous reports [1, [4] [5] [6] , the outcome for immunodeficient children who develop ahrf was significantly worse than for the rest of the study population: 13 deaths from 29 cases (45%) versus 13 deaths from 89 cases (15%), p <0.001; odds ratio 3.1 (95% confidence interval 1.1-8.0). in the stepwise multiple logistic regression analysis, adjustment for gas exchange parameters (a-ado> pao2/fio2, oi, vi, paco 2, peak inspiratory pressure, positive end expiratory pressure and map) did not alter the association of mosf and immunodeficiency with non-survival. the clinical course of patients with ahrf was categorised according to the severity of the disruption of gas exchange. using the best daily a-ado2 (but the classification is identical if paojfio 2 or oi are used), five distinct patterns of ahrf were identified (fig. 3) . survivors follow one of three clinical patterns: a course of mild disease only (pattern a: n--45), early improvement within 3 days (pattern b: n = 23) or later improvement longer than 3 days (pattern c: n --24). non-survivors die with persistent, severe, hypoxemic, respiratory failure (pattern d: n --17) or during or following resolution of ahrf (pattern e: n --9). the majority of deaths occur in children with persistently severe abnormal gas exchange (pattern d). of these children, none were previously healthy (ten were immunodeficient, three were ex-premature infants and the others had inherited metabolic disorders or major chromosomal abnormalities). further, only six of these patients died whilst receiving full supportive treatment including cardiopulmonary resuscitation, the remainder had either support withdrawn (4/17) or a limitation of intensive care therapy (7/17) because of the severity of the associated conditions or co-incident organ failure. therefore only six cases with ahrf reached a point of unsupportable respiratory failure, and none of these were previously normal children. the other children who died (pattern e) most frequently did so from severe cerebral injury (5/9 from brain death including the three previously normal children who died) with the other four cases having supportive treatment withdrawn or limited because of the severity of associated diseases. the principal observation in this preliminary report of ahrf in children is that associated or underlying diagnoses -case mix -have significant bearing on population outcomes. not surprisingly, children meeting criteria for ards had a poorer outcome. further, when using a defined ventilatory strategy, which in our practice emphasises permissive hypercarbia and lung volume recruitment, severity of ventilatory parameters (i. e., high map) rather than indices of gas exchange, reflected better the likelihood of poor outcome. most importantly, where the acute physiological parameters fail to differ between good and poor outcome patients, we propose that the presence of severe pre-existing disease or associated pathology, rather than severity of respiratory failure alone is associated with outcome in modern pediatric practice. this hypothesis should be tested in a larger series since, although we recruited 118 patients, there were only 26 deaths on which many of our conclusions are based. comparisons with previously published respiratory predictors of outcome from studies of ahrf and ards in childhood are shown in table 2 . none of the proposed physiological correlates of outcome were applicable to our series. reviewing every blood gas and applying the published criteria we found in each case, the predictor overestimated our patients' risk of mortality, excepting the very severe criteria from the melbourne study in 1991, which used a peak inspiratory pressure greater than 40cmh20 and a-ado2 more than 580mmhg [4] . these were rarely achieved in our population (7 cases) and hence the confidence intervals remain so wide (18-90%) that no useful conclusion could be drawn. the largest study of pediatric ahrf [6] , a multi-center retrospective study including 470 cases from 1991, identified an association between acute respiratory physiological disturbance and outcome. however, chil-dren who became brain dead or had treatment withdrawn because of perceived treatment futility -in the setting of severe neurological insult -were excluded from the subsequent analysis. such an approach (included because the study was principally designed to identify extra-corporeal membrane oxygenation candidates) would have excluded from our analysis all the normal children who died. since brain injury is a possible complication of severe hypoxemia or the disease processes that initiated hypoxemia, our view was that these patients should be included in our attempt to identify factors associated with outcome. the difference between our current findings and those of scores or predictors identified in the late 1980s and early 1990s may, in fact, relate to a fundamental change in ventilatory strategy. as shown in fig. 1 , our median paco2 was 6-8 kpa instead of the 5.3-6 kpa reported in the pediatric critical care study group multi-center retrospective study of children managed in 1991 [6] . table 2 comparison of previously published [2] [3] [4] [5] respiratory severity parameters with the present series (ppv positive predictive value for mortality, vi ventilation index, oi oxygenation index, pip peak inspiratory pressure (cmh20), a-ado 2 alveolar arterial oxygen gradient (mmhg), map mean airway pressure (cmh20), lr + the likelihood ratio for a positive test result, i.e. the ratio of finding the predictor in non-survivors to finding it in survivors) * indicates intermediate to high diagnostic impact, ns not significant proposed ppv lr + ppv p predictors (95 % confidence interval) in present study melbourne 1991 [2] memphis 1991 [3] salt lake city 1991 [4] philadelphia 1993 [5] vi pattern of disease and outcome the patients in our series exhibited one of five patterns in their clinical course. deaths amongst children admitted in ahrf can occur with active and progressive lung disease (pattern d) or in spite of resolving lung disease (pattern e). in children who survive, recovery may be slow or fairly rapid. on inspecting the data, it is apparent that there are similarities in the initial respiratory indices in children who survive despite severe, prolonged gas exchange disruption (pattern c) and those who die despite improving or improved gas exchange parameters (pattern e). the relative proportion of the patients with these patterns in a population being studied will clearly determine the utility of gas exchange parameters in predicting survival: conversely, as is our experience when including patients with underlying immunodeficiency or other associated diseases, these proportions may confound their use. of further note is the mode of death in children with persistently, severely abnormal gas exchange (pattern d): cases rarely reached a level of respiratory failure which was unsupportable by current techniques. instead, in the majority of cases (11/17) treatment was discontinued or limited as a result of other aspects of the clinical situation. worth re-emphasising in this context is the observation that no previously normal child died of unsupportable respiratory failure. observations from adult intensive care studies of lung injury have indicated that outcome is not necessarily related to the level of arterial oxygenation [12, 13] . in contrast, many previous pediatric studies in defined populations have supported a contrary notion [1, [2] [3] [4] [5] [6] . in the present pediatric study, we have observed that mortality from respiratory failure appears to be related to associated disease rather than the severity of initial gas exchange per se. the implications of such a hypothesis are wide. firstly, is there much to be gained by refining further the techniques of respiratory support when mortality is frequently determined by non-pulmonary factors? indeed, it has been suggested that mechanical ventilation should now be considered less a form of treatment than a form of organ support during disease resolution [14] . secondly, can severity systems that solely employ acute pulmonary physiological parameters and do not incorporate underlying etiology be used to good effect, specifically in pediatric ahrf? perhaps the reported value of such pulmonary physiological predictors, with their institution specificity, are more a reflection of physician behavior, i. e. patient selection and local ventilatory strategy employed, than patient pulmonary pathophysiology. in this context, it is of interest that we found the presence or absence of ahrf in all picu patients to be a discriminator. more recently developed non-linear, multiple logistic regression models that predict the risk of death for children less than 16 years of age (e. g., the 'pediatric risk of mortality iii'-prism ifi [15] and the 'paediatric index of mortality' -pim [16] ) may improve the outcome prediction since they incorporate both diagnostic and disease categories as well as acute physiological respiratory parameters. however, these two severity scoring systems do differ: not least in their ability, possibly, to be influenced by the ventilatory strategy employed. prism iii utilizes ph, pco2, poz and fio2 and respiratory rate, whereas pim utilizes po2 and fio2, and whether or not mechanical ventilation is being used. thirdly, how can clinical trials be designed to assess the impact of new respiratory therapies? it is possible that a comparison between heterogeneous treated and control groups using only early respiratory parameters to confirm similarity of disease severity is invalid. in keeping with other reports [17] , the present series indicates that case mix should not be ignored, e.g., ahrf in an ex-premature infant with respiratory syncytial virus is not the same as ahrf in an infant with aspiration pneumonia -even if the respiratory indices suggest they are similar. therefore, is it not time to reconsider disease-specific stratification criteria in any future treatment evaluation, even though this will inevitably mean that studies will take much longer to recruit sufficient patients. finally, since death in a previously normal child is now an infrequent end point in ahrf, our data reiterates a previously discussed idea [18] , that other markers sought. of ventilation-related outcome should be adult respiratory distress syndrome in a pediatric intensive care unit: predisposing conditions, clinical course and outcome predictors of mortality in children with respiratory failure: possible indications for ecmo alveolar-arterial oxygen gradient as a predictor of outcome in patients with non-neonatal respiratory failure mortality rates and prognostic variables in children with adult respiratory distress syndrome adult respiratory distress syndrome in children: associated diseases, clinical course and predictors of death fackler jc and the pediatric critical care study group and the extracorporeal life support organisation (1995) predicting death in pediatric patients with acute respiratory failure acute respiratory distress syndrome caused by respiratory syncytial virus early response to inhaled nitric oxide and its relationship to outcome in children with severe hypoxemic respiratory failure predicting outcome in children with severe acute respiratory failure treated with high-frequency ventilation the american-european consensus conference on ards: definitions, mechanisms, relevant outcomes and clinicai trial coordination outcome of pediatric patients with multiple organ system failure arterial oxygenation does not predict the outcome of patients with acute respiratory failure needing mechanical ventilation is outcome from ards related to the severity of respiratory failure? outcome from mechanical ventilation prism iii: an updated pediatric risk of mortality score wilkinson k (1997) paediatric index of mortality (pim): a mortality prediction model for children in intensive care intensive care society's apache ii study in britain and ireland -i: variations in case mix of adult admissions to general intensive care units and impact on outcome should morbidity replace mortality as an end point for clinical trials in intensive care? key: cord-257656-z7zx46gd authors: ljubin-sternak, sunčanica; meštrović, tomislav; ivković-jureković, irena; kolarić, branko; slović, anamarija; forčić, dubravko; tot, tatjana; mijač, maja; vraneš, jasmina title: the emerging role of rhinoviruses in lower respiratory tract infections in children – clinical and molecular epidemiological study from croatia, 2017–2019 date: 2019-12-03 journal: front microbiol doi: 10.3389/fmicb.2019.02737 sha: doc_id: 257656 cord_uid: z7zx46gd rhinoviruses (rvs) are increasingly implicated not only in mild upper respiratory tract infections, but also in more severe lower respiratory tract infections; however, little is known about species diversity and viral epidemiology of rvs among the infected children. therefore, we investigated the rhinovirus (rv) infection prevalence over a 2-year period, compared it with prevalence patterns of other common respiratory viruses, and explored clinical and molecular epidemiology of rv infections among 590 children hospitalized with acute respiratory infection in north-western and central parts of croatia. for respiratory virus detection, nasopharyngeal and pharyngeal flocked swabs were taken from each patient and subsequently analyzed with multiplex rt-pcr. to determine the rv species in a subset of positive children, 5′utr in rv-positive samples has been sequenced. nucleotide sequences of referent rv strains were retrieved by searching the database with basic local alignment tool, and used to construct alignments and phylogenetic trees using mafft multiple sequence alignment tool and the maximum likelihood method, respectively. in our study population rv was the most frequently detected virus, diagnosed in 197 patients (33.4%), of which 60.4% was detected as a monoinfection. median age of rv-infected children was 2.25 years, and more than half of children infected with rv (55.8%) presented with lower respiratory tract infections. most rv cases were detected from september to december, and all three species co-circulated during the analyzed period (2017–2019). sequence analysis based on 5′utr region yielded 69 distinct strains; the most prevalent was rv-c (47.4%) followed by rv-a (44.7%) and rv-b (7.9%). most of rv-a sequences formed a distinct phylogenetic group; only strains ri/hr409-18 (along with a reference strain mf978777) clustered with rv-c strains. strains belonging to the group c were the most diverse (41.6% identity among strains), while group b was the most conserved (71.5% identity among strains). despite such differences in strain groups (hitherto undescribed in croatia), clinical presentation of infected children was rather similar. our results are consistent with newer studies that investigated the etiology of acute respiratory infections, especially those focused on children with lower respiratory tract infections, where rvs should always be considered as potentially serious pathogens. rhinoviruses (rvs) are increasingly implicated not only in mild upper respiratory tract infections, but also in more severe lower respiratory tract infections; however, little is known about species diversity and viral epidemiology of rvs among the infected children. therefore, we investigated the rhinovirus (rv) infection prevalence over a 2-year period, compared it with prevalence patterns of other common respiratory viruses, and explored clinical and molecular epidemiology of rv infections among 590 children hospitalized with acute respiratory infection in north-western and central parts of croatia. for respiratory virus detection, nasopharyngeal and pharyngeal flocked swabs were taken from each patient and subsequently analyzed with multiplex rt-pcr. to determine the rv species in a subset of positive children, 5 utr in rv-positive samples has been sequenced. nucleotide sequences of referent rv strains were retrieved by searching the database with basic local alignment tool, and used to construct alignments and phylogenetic trees using mafft multiple sequence alignment tool and the maximum likelihood method, respectively. in our study population rv was the most frequently detected virus, diagnosed in 197 patients (33.4%), of which 60.4% was detected as a monoinfection. median age of rv-infected children was 2.25 years, and more than half of children infected with rv (55.8%) presented with lower respiratory tract infections. most rv cases were detected from september to december, and all three species co-circulated during the analyzed period (2017) (2018) (2019) . sequence analysis based on 5 utr region yielded 69 distinct strains; the most prevalent was rv-c (47.4%) followed by rv-a (44.7%) and rv-b (7.9%). most of rv-a sequences formed introduction rhinoviruses (rvs) are small, non-enveloped viruses that belong to the family picornaviridae, genus enterovirus. to date, there are 171 rhinovirus (rv) genotypes recognized and classified into the three species as rv-a (83 types), rv-b (32 types), and rv-c (56 types) (royston and tapparel, 2016; pan et al., 2018) . rv-a and rv-b were discovered by isolation on monkey kidney cells in 1950s (price, 1956) while rv-c genotypes, which are not cultivable using ordinary culture methods, have been identified decades after following the rise of molecular techniques (lamson et al., 2006; lau et al., 2007) . there are also differences between rv species in utilization of cell entry receptor: a majority of rv-a and rv-b attach to the intercellular adhesion molecule (icam)-1 (classified as the major receptor group) and the others alternatively bind low density lipoprotein receptor (ldl-r) (minor receptor group), whereas rv-c utilizes human cadherin-related family member 3 (cdhr3) (bochkov et al., 2015; royston and tapparel, 2016) . rhinovirus genome is a 7.2-kb single-stranded, positive-sense rna with a single open reading frame (orf) joined to a 5 untranslated region (5 utr) and a short viral priming protein (vpg) (jacobs et al., 2013) . orf encodes a poly-protein which is cleaved by virally encoded proteases in 11 proteins. four proteins -vp1, vp2, vp3, and vp4 -make up the viral capsid and account for the virus' antigenic diversity, while the remaining non-structural proteins are involved in viral genome replication and assembly. a rather conserved 5 utr region that harbors internal ribosomal entry site (ires) is usually utilized for rv detection from clinical samples, while more precise genotyping is based on vp4/vp2 or vp1 sequence analysis. rhinovirus have been neglected for decades, primarily because they were considered less virulent and only capable of causing mild common cold, and were not recognized, until recently, as important causative agents of lower respiratory tract infections (lrtis) and severe respiratory disease . introduction of sensitive and technically simple molecular detection assays, especially multiplex pcr, enabled affordable rv detection in line with other common respiratory viruses in clinical samples. together with coronaviruses, rvs are indeed responsible for majority of upper respiratory tract infection (urti), but also for substantial rate of lrtis in all age groups (lauinger et al., 2013; zhao et al., 2018 zhao et al., ,čivljak et al., 2019 . many studies showed that rv is one of the leading causes of pneumonia, bronchiolitis and other form of severe respiratory disease (zhao et al., 2018) , standing side by side with respiratory syncytial virus (rsv) in children and influenza in elderly (esposito et al., 2013; ning et al., 2017; čivljak et al., 2019) . there are several studies that report increased rates of asthma exacerbations and lrtis among children with rv-c when compared to those infected with rv-a and rv-b (bizzintino et al., 2011; lauinger et al., 2013) ; however, recent studies did not find any relationship between a specific rv species and the severity of clinical presentation (jacobs et al., 2015; van der linden et al., 2016; zhao et al., 2018) . data on rv prevalence in croatia are scarce, and molecular epidemiology was thus far not described. this study aims to determine the rv prevalence, compare it with prevalence patterns of other common respiratory viruses, as well as to explore clinical and molecular epidemiological features of rv infections among hospitalized children with acute respiratory infection. the research was performed in accordance with relevant guidelines/regulations and in line with the declaration of helsinki, as revised in 2013. written informed consent was obtained from all participants (children's parents or their legal guardians). the study was approved by the ethics committee of the dr. andrija štampar teaching institute of public health and conducted as part of the croatian science foundation project entitled "new and neglected respiratory viruses in vulnerable groups of patients" (no. ip-2016-06-7556). all standard biosecurity and institutional safety procedures have been adhered to. from may 2017 to april 2019, a total of 590 patients were included from hospitals located in north-western and central part of croatia: clinical hospital zagreb and general hospital karlovac, respectively. inclusion criteria were: age lower than 18 years, a clinical diagnosis of acute respiratory tract infection (ari), and need for hospitalization [on ward (≥1 day) or day hospital (for more than 6 but less than 24 h)]. exclusion criteria were presumed bacterial respiratory infection -including otitis, sinusitis and bacterial pneumonia, healthcare-associated infection, and ambulatory treated patients. patients were categorized into the four groups according to their respective age (i.e., <1, 1-2.99, 3-4.99 and ≥5 years of age), and two groups according to the localization of infection in those with upper respiratory tract infection (urti) and lower respiratory tract infection (lrti). urti was defined by symptoms of the common cold, coryza, cough, and hoarseness with or without fever, so clinical syndromes of respiratory catarrh, rhinitis and/or pharyngitis represented urti category. lrti was defined by symptoms of tachypnea, wheezing, severe cough, breathlessness, and by specific clinical signs such as nasal flaring, jugular and intercostal retractions, cyanosis (in rare instances), as well as wheezing, crackles and inspiratory rhonchi or generally reduced breath sounds during auscultation. clinical syndromes of bronchitis, bronchiolitis and pneumonia were included in lrti category (tregoning and schwarze, 2010; ljubin-sternak et al., 2016) . to avoid unnecessary x-ray exposure, chest radiographs were taken only for some of the patients in order to exclude or confirm bacterial pneumonia. for respiratory virus detection, nasopharyngeal and pharyngeal flocked swabs from each patient were collected, combined, and placed in viral transport medium (utm tm , copan, italy). specimens were immediately transported to the molecular microbiology laboratory at the public health institute where they were stored at −80 • c until tested. the results of virology testing were released to the physicians periodically, approximately once per week. as a part of routine care, nasopharyngeal, pharyngeal swabs, and blood cultures were taken from hospitalized patients and submitted for bacterial diagnostics using standard cultivation methods. demographic and clinical data, antimicrobial use records, and the results of routine bacterial studies were collected by a retrospective review of patient charts. to isolate viral dna and rna from viral transport medium, 300 µl has been extracted according to the manufacturer's protocol using ribospin tm vrd kit (geneall biotechnology, seoul, korea). multiplex rt-pcr for 15 respiratory viruses using seeplex r rv15 detection kit (seegene inc., seoul, korea) was performed. briefly, multiplex pcr and cdna synthesis as onestep reaction was performed and set up in three different tubes with three sets of primers. more specifically, "a set" contained primers for simultaneous amplification of target sequences of adenovirus (adv), human coronavirus (hcov) 229e/nl63, parainfluenza virus (piv) types 1-3, and pcr internal control to check for the presence of substances that may interfere with amplification; "b set" contained primers for hcov oc43, rv groups a/b/c, rsv type a and b, influenza (flu) type a, and pcr internal control; and "c set" contained primers for human bocavirus (hbov), flu type b, human metapneumovirus (hmpv), piv type 4, human enterovirus (hev), and the overall process control (human rnase p was included throughout the entire process as a control from nucleic acid extraction to amplification). amplification was performed on thermal cycler geneamp r 9700 pcr system (applied biosystems, foster city, united states). detection of pcr products was done by microchip electrophoresis on the mce r -202 multina device (shimadzu, kyoto, japan) -including software analysis showing results in the form of electropherograms and virtual image gels. to determine rv species, 5 utr of the genome in rv-positive samples was sequenced. total rna was extracted from 500 µl of rv-positive samples by the method reported by chomczynski and mackey (1998) . reverse transcription was performed at 42 • c for 60 min, in a reaction mix containing 10 µl of isolated rna, 1 × pcr buffer (ge healthcare, united kingdom), 0.1 mm of each dntp, 20 u of rnase inhibitor (thermo fisher scientific, united states), 1.25 mm mgcl2, 2.5 mm of random hexanucleotide primers and 50 u of mulv reverse transcriptase (thermo fisher scientific, united states) in a final volume of 20 µl. pcr reaction was performed by amplifying 5 utr, using ntr + (5 caa gya ctt ctg tyt ccc 3 ) and ntr-(5 cac gga cac cca aag tag t 3 ) primer pair, modified from wisdom et al. (2009) , corresponding to positions 161-178 and 531-549 of strain a2 (acc. no. x02316.1), respectively. final pcr mixtures contained 1 × onetaq pcr buffer (new england biolabs, united states), 10 mm dntp, 0.25 mm mgcl2, 0.25 mm of each primer, and 1.25 u of onetaq dna polymerase (new england biolabs, united states). the amplified products were separated on a 1.5% agarose gel, excised and purified by centrifugation through glass wool (sun et al., 2012) . sequencing reactions were set up with purified dna, one of the specific primers used for amplification, and a bigdye terminator v3.1 cycle sequencing kit (thermo fisher scientific, united states) according to the manufacturer's protocol. sequencing and sequence analysis were performed on a 3130 genetic analyzer (thermo fisher scientific, united states). nucleotide sequences of referent rv strains were retrieved by searching the database with blast (basic local alignment tool) 1 and used to construct alignments and phylogenetic trees. alignments were performed using mafft multiple sequence alignment tool available at the embl-ebi website 2 , and edited in aliview v1.23 (larsson, 2014) . phylogenetic trees were generated using the maximum likelihood method with molecular evolutionary genetics analyses (mega) software v6.06 (tamura et al., 2013) , under the most appropriate model of nt substitution determined with jmodeltest v2.1.4 (darriba et al., 2012) . bootstrap probabilities for 1,000 iterations were calculated to evaluate confidence estimates. sequence conservation (defined as percentage of genomic positions identical in all strains; gaps were ignored during computing) and evolutionary distances (p-distances) within and between groups have been calculated using mega 6.06. the sequences of hrsv strains obtained in this study were deposited in the genbank under acc. nos. mn369460 -mn369528. data analysis was performed using stata/mp (ver.15.1; statacorp llc, college station, united states). age was presented using medians with stated interquartile range (iqr). demographic and clinical parameters were compared by χ 2 -test or fisher's exact test for categorical variables and by kruskal-wallis test for continuous variables. to assess the strength of association between dependent variable (rv positive patients) and age we used univariate logistic regression. p-value was set to <0.05. in total, 590 patients were screened for respiratory viruses. the exact prevalence pattern of detected viruses can be seen in table 1 . rv was the most frequently detected virus, diagnosed in 197 patients (33.4%); 60.4% as monoinfection, and 39.6% as coinfection with other respiratory viruses. this was followed by rsv (19.3%), adv (15.6%), pivs (9.5%), flu types a and b (7.6%), hcov 229/nl63 and oc43 (7.1%), hbov (5.3), hev (4.6%), and hmpv (3.1%). there was a significant difference in age according to the specific virus (p < 0.001) (figure 1) . median age in years of rv infected children (2.25, iqr 6.50) was higher than in children infected with rsv (0.41, iqr 1.29), pivs (1.04, iqr 3.00), hcov (1.33, iqr 9.16), hmpv (0.92, iqr 4.09), and hbov (1.21, iqr 1.71), but lower than in those infected with flu (3.58, iqr 5.59), adv (2.88, iqr 4.44), and hev (3.66, iqr 5.29) (figure 1) . there was no statistically significant difference in the prevalence of rv infection between age groups (p = 0.781). additionally, age (1 year increase) was not significant predictor for rv positivity (or = 1.01, 95% ci = 0.97-1.06; p = 0.534). according to the clinical presentation, there was a significant difference in the proportion of lrtis between the type of the respiratory viruses (p = 0.002) (figure 2) . more than half of children infected with rv (110; 55.8%) presented with lrti; nonetheless, this was not significantly different from the proportion of rv positive children with urti (87; 44.2%) (p = 0.336), while children with rsv infection significantly more often presented with lrti (p < 0.001). out of the 197 rv-positive samples by multiplex pcr, we were able to sequence 76 samples (39%). sequence analysis based on 395 bp of 5' utr region of 76 samples yielded 69 viral types (seven strains had identical sequence) (figure 3) . the most prevalent was rv-c (36/76; 47.4%) followed by rv-a (34/76; 44.7%) and rv-b (6/76; 7.9%). most of rv-a sequences formed a distinct phylogenetic group; only strain ri/hr409-18 (along with a reference strain mf978777) clustered with rv-c strains (figure 3) . of the three respective groups, strains belonging to the group c were the most diverse, with identity of 41.6% (142 of 341 identical positions), while group b was the most conserved with 71.5% identity among strains (241/337). group a comprised strains which shared an overall 54.3% identical positions (183/337). calculated p-distances between groups showed group a and c are more closely related (p-distance 0.234) than to group b. similar p-value was calculated between group b and group a (p-distance 0.31) or group c strains (p-distance 0.33), respectively. no significant difference has been demonstrated in clinical symptoms based on the rv species, with the exception of increased frequency of antibiotic treatment in those infected with rv-a species (p = 0.012) ( table 2) . most rv cases were detected from september to december, and all three species co-circulated during the analyzed period (figure 4) . in this study we initially evaluated the prevalence of rv and other common respiratory viruses, as well as rv species distribution in hospitalized children with symptoms of ari over a period of 2 years. our results are consistent with previously published studies that investigated the etiology of ari, especially those focused on children with lrtis (chen et al., 2015; ning et al., 2017) . indeed, rv is right next to rsv when addressing the most common causes of bronchiolitis in hospitalized children, as demonstrated in very low-birth-weight infants from argentina and in one multicentre prospective study from the united states, respectively (mansbach et al., 2012; miller et al., 2012) . although rv can be found both in upper and lower respiratory tract, the potential for spread and the pathophysiology of infection in those two regions differs (as evidenced by studies conducted on rsv) (kim et al., 2016; gonzález-parra and dobrovolny, 2019) . possible causes of such disparity are fundamental differences in the immune responses and virus-cell interactions between these two anatomical regions, resulting in altered disease manifestation and spread (gonzález-parra and dobrovolny, 2019). there is also evidence that mucus velocity is decreased in small children (akin to elderly individuals) (grubb et al., 2016) , making them more susceptible to lrti and creating in turn a potential niche for more detrimental effect of rv infection. when age is concerned, our study has showed that rv holds a middle ground, not affecting very young children as is the case with rsv. this is comparable with the results found in the study by cebey-lópez et al. (2015) , where rsv infection was also seen in the youngest age groups (less than 1 year of age), rv was present in somewhat older children (mean between 14.4 and 40.9 months), while adv predominated in patients older than 50 months. conversely, some other author groups pointed out how rv can predominate in practically all age groups (tsagarakis et al., 2018) . in order to determine the rv species, we decided to sequence and analyze 5 utr, which has been established as a relatively simple and rapid technique for identifying the rv serotypes in clinical samples. moreover, it has been shown that 5 utr rt-pcr demonstrated greater sensitivity than vp4-vp2 pcr, as reflected by the higher positivity rate in amplification of clinical isolates, and further, no need for a nested pcr or multiple primer pairs -reducing in turn the contamination rate, cost and turnaround time (kiang et al., 2008) . despite using primers which target the region widely used for typing purposes, more than half of the samples produced no amplicons suitable for sequencing. such low rate of successful rv sequencing from clinical samples may be a consequence of mutations in primer regions or low viral load in original sample, but most probably arises due to rna degradation during freezing/thawing cycles. phylogenetic groups were readily distinguished and all three rv groups were detected, with group c and a predominating. the proportion of the three rv species revealed in this study (rv-a 44.7%, rv-b 7.9%, and rv-c, 47.4%) is consistent with prior studies worldwide (rv-a, 35.9-67.7%; rv-b, 1.5-13%; rv-c, 23-59.3%) (lauinger et al., 2013; rahamat-langendoen et al., 2013; jacobs et al., 2015; tsatsral et al., 2015; ratnamohan et al., 2016; van der linden et al., 2016; zhao et al., 2018) . intermixing of groups was not observed, except for a single group a sequence (ri/hr409-18) which clustered with group c sequences, indicating a recombination event or co-infection as was proposed by richter et al. (2015) . furthermore, group b strains were detected sporadically during the analyzed period, which is in accordance with other studies (lauinger et al., 2013; launes et al., 2015; richter et al., 2015) . these results represent the first report on rv diversity in croatia. in previous reports rv-c (and in lesser extent rv-a) have been associated with more severe illness (bizzintino et al., 2011; lauinger et al., 2013; linder et al., 2013; chen et al., 2015) ; however, more recent studies failed to report the connections between species and disease severity (rahamat-langendoen et al., 2013; jacobs et al., 2015; van der linden et al., 2016; zhao et al., 2018) . in this study there were no significant differences observed in clinical symptoms among three species, except more frequent utilization of antibiotic therapy in patients with rv-a species which can be result of subjective clinical assessment of more severe disease and empirical introduction of therapy. rv circulated throughout the 2-year period covered by this study with peaks in autumn and winter months. previous studies reported that rv infections occur all year round, with peaks of infection usually in spring and autumn months (čivljak et al., 2019) . however, recent research endeavors also report peaks in autumn and winter months (zhao et al., 2018) , and some of them specifically note that rv-c demonstrate peak in winter months (linder et al., 2013) . our study also observed no rv-c detection in spring and summer season. there are several limitations of the study. due to crosssectional study approach, a control group of asymptomatic patients could not have been included (which would facilitate assessment of rv infection severity). furthermore, we performed only 5 utr targeted rt-pcr assay and did not confirm our results with vp4/vp2 or vp1 sequences analysis. some authors report discordance between proposed phylogeny groups when sequences from the 5'utr and vp4/vp2 coding regions were analyzed, which can result in imprecise classification (ratnamohan et al., 2016) . more specifically, samples that clustered as rv a using 5 utr analysis can be revealed as rv-c when vp4/vp2 region is analyzed (ratnamohan et al., 2016) . also, complicated rhinovirus infections were excluded from the study. notwithstanding the aforementioned limitations, this is the first study endeavor cataloging a circulation of rv species in croatia over 2 years. in conclusion, in our study more than half of children infected with rv presented with lrti, which (together with other newer studies in the field) underlines the need for paradigm shift where rvs will not be merely associated with urti, but also considered in cases of lrti. regardless of the diversity of rv found in this study and the purported heterogeneity of the rv strains infecting the children, the similarity of clinical presentation negates the notion that certain rv species might be more virulent, at least in our case. more clinical and epidemiological studies are warranted to further elucidate this issue, with inevitable use of animal models to study pathogenic specificities of rv infection. the datasets generated for this study can be found in the genbank-accession numbers from mn369460 to mn369528. the studies involving human participants were reviewed and approved by the ethics committee of the dr. andrija štampar teaching institute of public health and conducted as part of the croatian science foundation project entitled "new and neglected respiratory viruses in vulnerable groups of patients" (no. ip-2016-06-7556) . written informed consent to participate in this study was provided by the participants' legal guardian/ next of kin. slj-s, ii-j, and jv designed the research. slj-s, as, and df performed the experiments. mm and tt collected the data. bk, as, and df analyzed the data. slj-s, tm, mm, tt, ii-j, as, and df interpreted the data and prepared the draft of the manuscript. jv and tm critically reviewed the draft. slj-s and tm wrote the final version of the manuscript. all authors reviewed and approved the final version of the manuscript. this work has been fully supported by the croatian science foundation under the project no. ip-2016-06-7556 titled "new and neglected respiratory viruses in vulnerable groups of patients" (principal investigator slj-s). the funders had no role in the study design, data collection and analysis, decision to publish or preparation of the manuscript. association between human rhinovirus c and severity of asthma in children cadherin-related family member 3, a childhood asthma susceptibility gene product, mediates rhinovirus c binding and replication viral co-infections in pediatric patients hospitalized with lower tract acute respiratory infections epidemiologic, clinical, and virologic characteristics of human rhinovirus infection among otherwise healthy children and adults: rhinovirus among adults and children single-step method of total rna isolated by acid guanidine phenol extraction viral pathogens associated with acute respiratory illness in hospitalized adults and elderly from jmodeltest 2: more models, new heuristics and parallel computing impact of viral infections in children with community-acquired pneumoniae: results of a study of 17 respiratory viruses prophylactic administration of respiratory syncytial virus immune globulin to high-risk infants and young children human rhinoviruses clinical and molecular epidemiology of human rhinovirus infections in patients with hematologic malignancy assay for 5 noncoding region analysis of all human rhinovirus prototype strains ct findings in viral lower respiratory tract infections caused by parainfluenza virus, influenza virus and respiratory syncytial virus masstag polymerase-chain-reaction detection of respiratory pathogens, including a new rhinovirus genotype, that caused influenza-like illness in new york state during aliview: a fast and lightweight alignment viewer and editor for large datasets clinical features and complete genome characterization of a distinct human rhinovirus (rv) genetic cluster, probably representing a previously undetected rv species rv-c, associated with acute respiratory illness in children patient characteristics and severity of human rhinovirus infections in children molecular epidemiology of severe respiratory disease by human rhinoviruses and enteroviruses at a tertiary paediatric hospital in human rhinovirus c: age, season, and lower respiratory illness over the past 3 decades etiology and clinical characteristics of single and multiple respiratory virus infections diagnosed in croatian children in two respiratory seasons prospective multicenter study of viral etiology and hospital length of stay in children with severe bronchiolitis human rhinoviruses in severe respiratory disease in very low birth weight infants the etiology of community-acquired pneumonia among children under 5 years of age in mainland china genome sequences of rhinovirus genotype c56 detected in three patients with acute respiratory illness the isolation of a new virus associated with respiratory clinical disease in humans the significance of rhinovirus detection in hospitalized children: clinical, epidemiological and virological features phylogenetic analysis of human rhinoviruses collected over four successive years in sydney molecular epidemiology of rhinoviruses in cyprus over three consecutive seasons rhinoviruses and respiratory enteroviruses: not as simple as abc a quick, cost-free method of purification of dna fragments from agarose gel mega6: molecular evolutionary genetics analysis version 6.0 rhinovirus-from bench to bedside respiratory viral infections in infants: causes, clinical symptoms, virology, and immunology age-related prevalence of common upper respiratory pathogens, based on the application of the filmarray respiratory panel in a tertiary hospital in greece molecular epidemiology of the human rhinovirus infection in mongolia during 2008-2013 a molecular epidemiological perspective of rhinovirus types circulating in amsterdam from screening respiratory samples for detection of human rhinoviruses (rvs) and enteroviruses: comprehensive vp4-vp2 typing reveals high incidence and genetic diversity of rv species c genotypic diversity and epidemiology of human rhinovirus among children with severe acute respiratory tract infection in shanghai the authors wish to thank matea kvaternik celjak for her technical assistance. key: cord-251986-ajlpb9li authors: li, yan‐chao; bai, wan‐zhu; hashikawa, tsutomu title: the neuroinvasive potential of sars‐cov2 may play a role in the respiratory failure of covid‐19 patients date: 2020-03-11 journal: j med virol doi: 10.1002/jmv.25728 sha: doc_id: 251986 cord_uid: ajlpb9li following the severe acute respiratory syndrome coronavirus (sars‐cov) and middle east respiratory syndrome coronavirus (mers‐cov), another highly pathogenic coronavirus named sars‐cov‐2 (previously known as 2019‐ncov) emerged in december 2019 in wuhan, china, and rapidly spreads around the world. this virus shares highly homological sequence with sars‐cov, and causes acute, highly lethal pneumonia coronavirus disease 2019 (covid‐19) with clinical symptoms similar to those reported for sars‐cov and mers‐cov. the most characteristic symptom of patients with covid‐19 is respiratory distress, and most of the patients admitted to the intensive care could not breathe spontaneously. additionally, some patients with covid‐19 also showed neurologic signs, such as headache, nausea, and vomiting. increasing evidence shows that coronaviruses are not always confined to the respiratory tract and that they may also invade the central nervous system inducing neurological diseases. the infection of sars‐cov has been reported in the brains from both patients and experimental animals, where the brainstem was heavily infected. furthermore, some coronaviruses have been demonstrated able to spread via a synapse‐connected route to the medullary cardiorespiratory center from the mechanoreceptors and chemoreceptors in the lung and lower respiratory airways. considering the high similarity between sars‐cov and sars‐cov2, it remains to make clear whether the potential invasion of sars‐cov2 is partially responsible for the acute respiratory failure of patients with covid‐19. awareness of this may have a guiding significance for the prevention and treatment of the sars‐cov‐2‐induced respiratory failure. increasing evidence shows that coronaviruses are not always confined to the respiratory tract and that they may also invade the central nervous system inducing neurological diseases. the infection of sars-cov has been reported in the brains from both patients and experimental animals, where the brainstem was heavily infected. furthermore, some coronaviruses have been demonstrated able to spread via a synapse-connected route to the medullary cardiorespiratory center from the mechanoreceptors and chemoreceptors in the lung and lower respiratory airways. considering the high similarity between sars-cov and sars-cov2, it remains to make clear whether the potential invasion of sars-cov2 is partially responsible for the acute respiratory failure of patients with covid-19. awareness of this may have a guiding significance for the prevention and treatment of the sars-cov-2-induced respiratory failure. genomic analysis shows that sars-cov-2 is in the same betacoronavirus (βcov) clade as mers-cov and sars-cov, and shares highly homological sequence with sars-cov. 3 the public evidence shows that covid-19 shares similar pathogenesis with the pneumonia induced by sars-cov or mers-cov. 4 moreover, the entry of sars-cov-2 into human host cells has been identified to use the same receptor as sars-cov. 5, 6 most covs share a similar viral structure and infection pathway, 7, 8 and therefore the infection mechanisms previously found for other covs may also be applicable for sars-cov-2. a growing body of evidence shows that neurotropism is one common feature of covs. 1, [9] [10] [11] [12] therefore, it is urgent to make clear whether sars-cov-2 can gain access to the central nervous system (cns) and induce neuronal injury leading to the acute respiratory distress. sars-cov-2 causes acute, highly lethal pneumonia with clinical symptoms similar to those reported for sars-cov and mers-cov. 2, 11 imaging examination revealed that most patients with fever, dry cough, and dyspnea showed bilateral ground-glass opacities on chest computerized tomography scans. 12 however, different from sars-cov, sars-cov-2-infected patients rarely showed prominent upper respiratory tract signs and symptoms, indicating that the target cells of sars-cov-2 may be located in the lower airway. 2 based upon the first-hand evidence from wuhan local hospitals, 2,10,12 the common symptoms of covid-19 were fever (83%-99%) and dry cough (59.4%-82%) at the onset of illness. however, the most characteristic symptom of patients is respiratory distress (~55%). among the patients with dyspnea, more than half needed intensive care. about 46% to 65% of the patients in the intensive care worsened in a short period of time and died due to respiratory failure. among the 36 cases in the intensive care reported by wang et al, 10 11.1% received highflow oxygen therapy, 41.7% received noninvasive ventilation, and 47.2% received invasive ventilation. these data suggest that most (about 89%) of the patients in need of intensive care could not breathe spontaneously. it is now known that covs are not always confined to the respiratory tract and that they may also invade the cns inducing neurological diseases. such neuroinvasive propensity of covs has been documented almost for all the βcovs, including sars-cov, 1 mers-cov, 13 hcov-229e, 14 hcov-oc43, 15 mouse hepatitis virus, 16 and porcine hemagglutinating encephalomyelitis coronavirus (hev). 9, [17] [18] [19] with respect to the high similarity between sars-cov and sars-cov2, it remains to know whether the potential neuroinvasion of sars-cov-2 plays a role in the acute respiratory failure of patients with covid-19. it is believed that the tissue distributions of host receptors are generally consistent with the tropisms of viruses. [20] [21] [22] the entry of sars-cov into human host cells is mediated mainly by a cellular receptor angiotensin-converting enzyme 2 (ace2), which is expressed in human airway epithelia, lung parenchyma, vascular endothelia, kidney cells, and small intestine cells. [23] [24] [25] different from sars-cov, mers-cov enters human host cells mainly via dipeptidyl peptidase 4 (dpp4), which is present in the lower respiratory tract, kidney, small intestine, liver, and the cells of the immune system. 26, 27 however, the presence of ace2 or dpp4 solely is not sufficient in the brain, where they were located almost exclusively in the neurons. [31] [32] [33] experimental studies using transgenic mice further revealed that either sars-cov 34 or mers-cov, 13 when given intranasally, could enter the brain, possibly via the olfactory nerves, and thereafter rapidly spread to some specific brain areas including thalamus and brainstem. it is noteworthy that in the mice infected with low inoculum doses of mers-cov virus particles were detected only in the brain, but not in the lung, which indicates that the infection in the cns was more important for the high mortality observed in the infected mice. 13 among the involved brain areas, the brainstem has been demonstrated to be heavily infected by sars-cov 34, 35 or mers-cov. 13 the exact route by which sars-cov or mers-cov enters the cns is still not reported. however, hematogenous or lymphatic route seems impossible, especially in the early stage of infection, since almost no virus particle was detected in the nonneuronal cells in the infected brain areas. [31] [32] [33] on the other hand, increasing evidence shows that covs may first invade peripheral nerve terminals, and then gain access to the cns via a synapse-connected route. 9, 17, 19, 36 the trans-synaptic transfer has been well documented for other covs, such as hev67 9-10,18-19 and avian bronchitis virus. 36, 37 hev 67n is the first cov found to invade the porcine brain, and it shares more than 91% homology with hcov-oc43. 38, 39 hev first oronasally infects the nasal mucosa, tonsil, lung, and small intestine in suckling piglets, and then is delivered retrogradely via peripheral nerves to the medullary neurons in charge of peristaltic function of the digestive tract, resulting in the so-called vomiting diseases. 18, 19 the transfer of hev 67n between neurons has been demonstrated by our previous ultrastructural studies to use the clathrin-coatingmediated endocytotic/exocytotic pathway. 17 similarly, the trans-synaptic transfer has been reported for avian bronchitis virus. 36, 37 intranasal inoculation in mice with avian influenza virus was reported to cause neural infection besides bronchitis or pneumonia. 36 of interest, viral antigens have been detected in the brainstem, where the infected regions included the nucleus of the solitary tract and nucleus ambiguus. the nucleus of the solitary tract receives sensory information from the mechanoreceptors and chemoreceptors in the lung and respiratory tracts, [40] [41] [42] while the efferent fibers from the nucleus ambiguus and the nucleus of the solitary tract provide innervation to airway smooth muscle, glands, and blood vessels. such neuroanatomic interconnections indicate that the death of infected animals or patients may be due to the dysfunction of the cardiorespiratory center in the brainstem. 11, 30, 36 taken together, the neuroinvasive propensity has been demonstrated as a common feature of covs. in light of the high similarity between sars-cov and sars-cov2, it is quite likely that sars-cov-2 also possesses a similar potential. based on an epidemiological survey on covid-19, the median time from the first symptom to dyspnea was 5.0 days, to hospital admission was 7.0 days, and to the intensive care was 8.0 days. 10 therefore, the latency period may be enough for the virus to enter and destroy the medullary neurons. as a matter of fact, the previous studies 2,14-15 mentioned above has reported that some patients infected with sars-cov-2 did show neurologic signs such as headache (about 8%), nausea and vomiting (1%). more recently, one study on 214 covid-19 patients by mao et al. 43 further found that about 88% (78/88) among the severe patients displayed neurologic manifestations including acute cerebrovascular diseases and impaired consciousness. therefore, awareness of the possible neuroinvasion may have a guiding significance for the prevention and treatment of the sars-cov-2-induced respiratory failure. http://orcid.org/0000-0002-2884-9829 mechanisms of host defense following severe acute respiratory syndrome-coronavirus (sars-cov) pulmonary infection of mice clinical features of patients infected with 2019 novel coronavirus in wuhan measures for diagnosing and treating infections by a novel coronavirus responsible for a pneumonia outbreak originating in wuhan, china. microbes infect from sars to mers, thrusting coronaviruses into the spotlight genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding receptor recognition by novel coronavirus from wuhan: an analysis based on decade-long structural studies of sars cryo-em structures of mers-cov and sars-cov spike glycoproteins reveal the dynamic receptor binding domains coronavirus spike protein and tropism changes coronavirus infection of rat dorsal root ganglia: ultrastructural characterization of viral replication, transfer, and the early response of satellite cells clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus-infected pneumonia in wuhan, china novel coronavirus is putting the whole world on alert epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a descriptive study middle east respiratory syndrome coronavirus causes multiple organ damage and lethal disease in mice transgenic for human dipeptidyl peptidase 4 neurotropism of human coronavirus 229e axonal transport enables neuron-to-neuron propagation of human coronavirus oc43 hepatitis e virus infects neurons and brains neurotropic virus tracing suggests a membranous-coating-mediated mechanism for transsynaptic communication characteristics of a coronavirus (strain 67n) of pigs immunofluorescence studies on the pathogenesis of hemagglutinating encephalomyelitis virus infection in pigs after oronasal inoculation exploring the pathogenesis of severe acute respiratory syndrome (sars): the tissue distribution of the coronavirus (sars-cov) and its putative receptor, angiotensin-converting enzyme 2 (ace2) quantitative temporal-spatial distribution of severe acute respiratory syndromeassociated coronavirus (sars-cov) in post-mortem tissues cleavage of the sars coronavirus spike glycoprotein by airway proteases enhances virus entry into human bronchial epithelial cells in vitro a novel angiotensinconverting enzyme-related carboxypeptidase (ace2) converts angiotensin i to angiotensin 1-9 quantitative mrna expression profiling of ace 2, a novel homologue of angiotensin converting enzyme tissue distribution of ace2 protein, the functional receptor for sars coronavirus. a first step in understanding sars pathogenesis expression of cd26 (dipeptidyl peptidase iv) on resting and activated human t-lymphocytes the multifunctional or moonlighting protein cd26/dppiv persistent infection of sars coronavirus in colonic cells in vitro the clinical pathology of severe acute respiratory syndrome (sars): a report from dipeptidyl peptidase iv, which probably plays important roles in alzheimer disease (ad) pathology, is upregulated in ad brain neurons and associates with amyloid plaques organ distribution of severe acute respiratory syndrome (sars) associated coronavirus (sars-cov) in sars patients: implications for pathogenesis and virus transmission pathways multiple organ infection and the pathogenesis of sars detection of severe acute respiratory syndrome coronavirus in the brain: potential role of the chemokine mig in pathogenesis severe acute respiratory syndrome coronavirus infection causes neuronal death in the absence of encephalitis in mice transgenic for human ace2 lethal infection of k18-hace2 mice infected with severe acute respiratory syndrome coronavirus the vagus nerve is one route of transneural invasion for intranasally inoculated influenza a virus in mice morphogenesis of avian infectious bronchitis virus in primary chick kidney cells a comparative sequence analysis to revise the current taxonomy of the family coronaviridae the evidence of porcine hemagglutinating encephalomyelitis virus induced nonsuppurative encephalitis as the cause of death in piglets brain stem projections of sensory and motor components of the vagus complex in the cat: ii. laryngeal, tracheobronchial, pulmonary, cardiac, and gastrointestinal branches cns innervation of vagal preganglionic neurons controlling peripheral airways: a transneuronal labeling study using pseudorabies virus interaction of the rabies virus p protein with the lc8 dynein light chain neurological manifestations of hospitalized patients with covid-19 in wuhan, china: a retrospective case series study the neuroinvasive potential of sars-cov2 may play a role in the respiratory failure of covid-19 patients key: cord-007030-mewo9w43 authors: hashim, suhana; ayub, zeti n.; mohamed, zeehaida; hasan, habsah; harun, azian; ismail, nabilah; rahman, zaidah a.; suraiya, siti; naing, nyi nyi; aziz, aniza a. title: the prevalence and preventive measures of the respiratory illness among malaysian pilgrims in 2013 hajj season date: 2016-02-08 journal: j travel med doi: 10.1093/jtm/tav019 sha: doc_id: 7030 cord_uid: mewo9w43 background. respiratory illness continues to exert a burden on hajj pilgrims in makkah. the purpose of this study is to determine the prevalence of respiratory illness and its associated factors among malaysian hajj pilgrims in 2013 and to describe its preventive measures. methods. a cross-sectional study was conducted in makkah and malaysia during the 2013 hajj season. a self-administered proforma on social demographics, previous experience of hajj or umrah, smoking habits, co-morbid illness and practices of preventive measures against respiratory illness were obtained. results. a total of 468 proforma were analysed. the prevalence of the respiratory illness was 93.4% with a subset of 78.2% fulfilled the criteria for influenza-like illness (ili). most of them (77.8%) had a respiratory illness of <2 weeks duration. approximately 61.8% were administered antibiotics but only 2.1% of them had been hospitalized. most of them acquired the infection after a brief stay at arafat (81.2%). vaccination coverages for influenza virus and pneumococcal disease were quite high, 65.2% and 59.4%, respectively. for other preventive measures practices, only 31.8% of them practiced good hand hygiene, ∼82.9% of pilgrims used surgical face masks, n95 face masks, dry towels, wet towels or veils as their face masks. nearly one-half of the respondents (44.4%) took vitamins as their food supplement. malaysian hajj pilgrims with previous experience of hajj (or 0.24; 95% ci 0.10–0.56) or umrah (or 0.19; 95% ci 0.07–0.52) and those who have practiced good hand hygiene (or 0.35; 95% ci 0.16–0.79) were found to be significantly associated with lower risk of having respiratory illness. otherwise, pilgrims who had contact with those with respiratory illness (or 2.61; 95% ci 1.12–6.09) was associated with higher risk. conclusions. the prevalence of respiratory illness remains high among malaysian hajj pilgrims despite having some practices of preventive measures. all preventive measures which include hand hygiene, wearing face masks and influenza vaccination must be practiced together as bundle of care to reduce respiratory illness effectively. the hajj is an islamic pilgrimage to makkah. it draws in 3 million muslims surging from all over the world which accounts for the largest gathering of people globally on an annual basis. the 2013 hajj season began from 13 october to 17 october 2013 (8) (9) (10) (11) (12) dhu al-hijjah). they perform specific rituals and follow a detailed route. the pilgrims perform their first circumambulations by walking seven times anticlockwise around the kaaba. then, they are required to walk for a total distance of 2.1 km between the hills of safa and marwah seven times. on the 13 october, they travel to mina and spend one night there for prayers and additional rituals. the next day, the pilgrims around the world gather at arafat. when the sun sets, they leave arafat and move to muzdalifah, located between arafat and mina. they stay at mina in crowded tents for at least two nights. for the completion of the umrah pilgrimage, which is called the lesser pilgrimage; it is different as it can be performed throughout the year, the pilgrims are not required to perform the brief stay at arafat. for 1400 years, the mass gathering during hajj has been associated with the risk of communicable diseases, particularly respiratory infection. 1 extended stays at hajj sites, physical exhaustion, extreme heat and crowded accommodation encourage disease transmission, especially those deriving from airborne agents. 2 crowd densities during hajj are about up to seven people per square metre. 3 respiratory tract infection during hajj continues to exert a burden on pilgrims. the respiratory problems account for 74% of all medical illnesses reported during hajj seasons. 4 pneumonia being the leading reason for hospital admission in 39% of all patients. 5 a recent study involving malaysian hajj pilgrims found that 90% of them had at least one respiratory symptom. 6 respiratory illness is a disease affecting the respiratory system and can be due to infection or non-infection. it is complex to define the syndromes of respiratory illness due to variation in the severity, duration and types of symptoms. 7 the purpose of this study is to determine the prevalence of the respiratory illness among malaysian hajj pilgrims in 2013, to describe its preventive measures and to determine the association between sociodemographic, previous experience of hajj/umrah, co-morbidity, smoking habits, vaccination and preventive measures with respiratory illness. apart from those preventive measures, we also explored the association between good hand hygiene practice and respiratory illness/ili. this is a cross-sectional study involving all consented malaysian hajj pilgrims in november, 2013. an expanded definition of respiratory illness was used for this study. respiratory illness was defined as when the person is having at least one of the respiratory symptoms (non-ili) or ili. with references to other studies and some limitations (mainly logistic problems), ili is defined as the triad of cough, subjective fever and sore throat, those who did not fulfil the criteria of ili were classified into a non-ili group. [8] [9] [10] [11] sample size was calculated based on two proportion formula. the sample size according to potential associated factors, the largest sample size was given 780. after adding a possible non-respondent rate of 10%, the sample size for this study is 858. the sampling frame was consented to the malaysian hujjaj in 2013 who attended the hajj course at universiti sains malaysia (usm) kelantan on 23 august and the 24 august 2013, those who transitted at hajj building complex, malaysia from the 15th of september until the 19 september 2013 and those at the kingdom of saudi arabia (ksa). the sample selection was based on convenient sampling due to logistic problems. the researchers did not have access to the name of all pilgrims and they did not know about the approval of hajj visa status until a few weeks before their departure. the inclusion criteria were hajj pilgrims above 18 years old and able to comprehend and fill up proforma. pilgrims who were very ill and unable to independently respond to the proforma were excluded. data based on the social demographic, co-morbid illness, smoking habits, symptoms of respiratory illness, history of contact with respiratory ill patients, previous experience of hajj or umrah, the practice of preventive measures, influenza and pneumococcal vaccination and supplement intake against respiratory illness were obtained by a self-administered proforma. good hand hygiene or optimal handwashing practices is defined as handwashing for 20 s at least five times per day by using water with soap or a hand sanitizer. 12, 13 in this study, we define good hand hygiene practice as those who frequently wash their hands using hand sanitizers indicated by centres for disease control (cdc). 14 those using water only, handkerchiefs or disposable tissues were considered as poor hand hygiene practices. history of contact with respiratory illness sufferers is defined as pilgrims who have direct contact or close contact (being within 6 feet (2 m) or within the room or care area for a prolonged period of time while not wearing recommended personal protective equipment. 15 brief instructions were given to the hajj pilgrims before receiving proforma. the proforma were given to participants in malaysia before their departure and also at makkah before departing for a brief stay at arafat. all the completed proforma were collected after completion of hajj at makkah and at the local airport upon arrival in malaysia or via postage. the pilgrims were required to complete the proforma at least 2 weeks after their stay at arafat. all respiratory symptoms that occurred 2 weeks after arrival in ksa were considered significant. the statistical package for the social sciences (spss version 22.0) was exercised for data entry and statistical analysis. descriptive statistics were applied to describe the prevalence, practice of deterrent measures and associated factors. the analysis of association between sociodemographic, previous experience of hajj or umrah, co-morbidity, smoking habits, vaccination and the practice of preventive measures with the respiratory illness were done using simple logistic regression and the variables with p value of <0.25 or variables with clinically significant values will be included further with multiple logistic regression analysis. the risk estimation was carried out using the odds ratio (or) and 95% confidence intervals (ci) and the p value of <0.05 were significant. variable interaction and multi-collinearity followed by testing on a model assumption was performed before decisions made on the final model. for the final model, the p value of <0.05 were considered as significant. ethical approval was obtained from the usm research and ethics committee before this study (reference number: a total of 1200 proforma were distributed to the pilgrims, however, only 480 responded, only 40%. out of 480 pilgrims who returned the proforma, 12 were excluded from the analysis as grossly incomplete. altogether 468 proforma were analysed. the age for malaysian hajj pilgrims in this study ranged from 17 to 84 years old with a mean age of 52.52 (sd 10.15). the males (56.2%) dominated the female pilgrims with an obvious male to female ratio of 1.3. more than one-half of the pilgrims had at least one medical illness (60.0%). many of them had hypertension (26.5%), followed by diabetes mellitus (dm) (15.4%), allergic rhinitis (9.0%), bronchial asthma (5.6%) and others (3.6%). some of the pilgrims still had smoked (12.2%) and 17.9% of them were obese ( table 1) . the prevalence of respiratory illness symptoms was 93.4% with a subset of 78.2% fulfil the criteria for ili. most of them had a respiratory illness of <2 weeks (77.8%). approximately 61.8% were administered antibiotics, only 2.1% of them were hospitalized. one-half of them had a history of contact with respiratory illness sufferers (52.2%). they acquired the infection intensely at arafat (81.2%) ( table 2) . for the practice of preventive measures, the total number of pilgrims that received influenza vaccinations was 305 (65.2%). from those, 130 (27.8%) pilgrims had been immunized with influenza and pneumococcal vaccinations. a total of 82.9% of pilgrims wore face mask, i.e. surgical face masks, n95 face masks, dry towels, wet towels or veils as their face mask. only 31.8% of them practiced good hand hygiene. nearly one-half of the respondents (44.4%) took vitamins as their food supplement (table 3) . factors associated with respiratory illness among malaysian pilgrims using simple logistic regression analysis was shown in table 4 . all the supplements were seemed able to protect them from the illness, however they were not statistically significant (table 4 ). malaysian hajj pilgrims with previous experience of hajj (or 0.24; 95% ci 0.10-0.56) or umrah (or 0.19; 95% ci 0.07-0.52) and those with good hand hygiene (or 0.35; 95% ci 0.16-0.79) were significantly associated with lower risk of respiratory illness. otherwise, pilgrims having contact with those with respiratory illness (or 3.01; 95% ci 1.35-6.68) were associated with higher risk ( table 5 ). the percentage of ili was lower (38.9%) in those vaccinated with the influenza vaccine than those unvaccinated (61.1%). however, it was statistically not significant with a p value of 0.15 (table 6 ). the prevalence of the respiratory illness varied by country of origin and by year based on the studies conducted over the past few years. in our study, the prevalence of respiratory illness symptoms among malaysian hajj pilgrims for the 2013 season was 93.4%, with a subset of 78.2% fulfilling the criteria for ili. the respiratory illness prevalence was consistent with french pilgrims during the same year (90.7%). 9 the prevalence for the 2013 african hajj pilgrims was slightly lower (77.6%). 16 the results of our study are in parallel with previous studies, 97.0% and 90.0% for the 2009 saudi arabia hajj pilgrims and the 2007 malaysian hajj pilgrims, respectively. 6, 17 in contradiction, the percentage was lower for the us pilgrims in 2009 who suffered from respiratory illness symptoms (41.3%). 18 crowdedness is a major risk factor for the transmission of respiratory illness. all compulsory rituals of hajj involve crowded places, jam-packed with pilgrims. these conditions contributed to almost one-half of the pilgrims (52.2%) having contact with people suffering from respiratory illness during hajj. they were at a significantly high risk of developing the respiratory illness by three times higher than those who do not have the contact (p value 0.01). practising contact avoidance during hajj is imperative as it can shorten the duration of respiratory illness. 18 practicing social distancing and contact avoidance is effective in reducing the transmission of the respiratory symptoms during the 2009 us pilgrims and ili symptoms in 2014 with the indian pilgrims. 18, 19 the majority of the respondents was inflicted with respiratory illness after a brief stay at arafat (81.2%). it may be correlated with the incubation period of the illness and the peak exposure of pilgrims whilst performing the hajj rituals. other studies estimated approximately one in three pilgrims will experience respiratory symptoms which usually occurred at the end or shortly after performing hajj rituals. 20 influenza vaccination is one of the recommended vaccines for high risk pilgrims to reduce mortality and morbidity. the vaccine uptake for influenza in our study is lower (65.2%) in contrast to malaysian pilgrims in 2007 (72.8%) and saudi arabian pilgrims in 2009 (94.4%). 6, 17 the percentage of vaccine uptake among the 2013 hajj pilgrims from other countries were as low as 31.8% for french pilgrims and 31.0% for pilgrims from saudi arabia, qatar and australia. 21, 22 effective influenza vaccine remains debatable and cannot be proven if the study was conducted without virology confirmation. multiple factors such as mismatch between vaccine strains and circulating strains, inappropriate storage and the handling of the vaccine can reduce its effectiveness or waning of immunity in the population. one study systematically reviewed the available studies assessing the uptake and effectiveness of the influenza vaccine among pilgrims. the effectiveness of the influenza vaccine varied across studies, but was effective against laboratory-confirmed influenza with a p value of <0.001. 23 in our study, the prevalence of ili was not significantly associated with the status of the vaccination which was similar with pilgrims in malaysia (2007), france (2006) and iran (2006). 6, 24, 25 the vaccine was not associated with the reduction and number of acute respiratory symptoms nor any relation to the length of stay for malaysian hajj pilgrims in 2007. 26 the findings were not in accordance with the french pilgrims in 2013 in which the ili symptoms were less frequently reported in the vaccinated group (34.1%) than unvaccinated group (61.5%) and was statistically significant (p value 0.009). 21 in another study, they found that the influenza vaccine appeared to provide some protection in 'at risk' hajj pilgrims but not in the 'not at risk' group. 10 the influenza vaccines prevented clinic visits for ili among malaysian pilgrims in 2000 and pakistani pilgrims in 1999. 11, 27 the influenza vaccination is a protective factor for ili more than for non-ili, giving 70-77% protection for ili and just 20% protection against the non-ili group. 11 one hundred and forty-nine of our respondents adopted good hand hygiene during hajj (31.8%). the results were similar to the french pilgrims in 2012 (46.3%) and the us hujjaj in 2009 (45.5%). 18, 28 however, the adherence of french pilgrims towards hand sanitizer practices in 2009 was higher (77.4%) than in the 2012 hajj season. 29 effective hand hygiene practice in this study could significantly decrease the risk of respiratory illness by 60% than pilgrims who practiced poor hand hygiene. it was parallel with the us pilgrims in 2009 and the french pilgrims in 2012, as regularly washing hands and the use of hand sanitizers significantly causes less of the ili symptoms. 18, 28 a systematic review on hand-hygiene interventions, including education and the use of alcohol-based hand sanitizers towards respiratory illness indicated that some of the interventions were not efficacious against respiratory illnesses. 30 the consistent application of hand hygiene during critical points in the chain of transmission is likely to play a major role in shaping the relative effectiveness of hand-hygiene interventions in terms of disease outcome. face masks are able to limit the spread of microorganisms, mainly from the respiratory droplets. however, the cdc in usa found that the intermittent use of surgical-type masks was associated with more than a 2.5-fold greater risk of infection. disposable face masks should be used once and replaced when they become moist and are to be disposed of properly. most of the malaysian pilgrims admitted to wearing face masks during hajj (82.9%). this finding was in accordance with other studies, 72.9% for malaysian pilgrims in 2007 and 79.6% for french pilgrims in 2009. 8, 29 the percentage was higher if compared with other countries in the 2009 hajj season; saudi arabia (56%) and usa (42%). 17, 18 it has been revealed that face masks either offered no significant protection or were associated with a longer duration of sore throat and fever symptoms among hajj pilgrims. 25, 29, 31 a recent study illustrated that many pilgrims at the 2009 hajj may not have worn masks correctly (e.g. mistakenly positioning the top of the mask below the nose). 32 a recent review by benkouiten et al. 33 in 2014 also mentioned that the effectiveness of the face mask in the prevention of the respiratory symptoms among hajj pilgrims revealed variable results. regular use of a face mask was the most essential practical protective factor in respiratory illness, using it for more than 8 h led to a substantial decrease in the incidence of ili among australian pilgrims in 2011. 34, 35 these masks were potentially effective at preventing respiratory virus acquisition by household contacts of infected people when worn by healthy people. however, the effectiveness depended largely on adherence to mask use. 36 honey is one of the most promising natural substances that can combat or prevent respiratory illness. 37 approximately 22.9% of pilgrims consumed honey during hajj. however, there was no other study to compare the prevalence of honey intake among hajj pilgrims except for one study to determine the effectiveness of honey in reducing respiratory symptoms. 38 during the hajj, pilgrims undergo great physical and emotional strain. the experienced hajj veterans or even umrah is an advantage as it can help them to be more physically prepared as well as mentally and spiritually ready. however, every year is different and every person's experience is individually specific but they are able to prepare themselves as much as possible for the hajj challenges. the association of previous experiences during hajj or umrah with respiratory illness has not been studied before. malaysian hajj pilgrims with previous experience of hajj or umrah were found to be significantly associated with lower risk of developing respiratory illness (p value of 0.001 for both). further study needs to be conducted to explore the factors that contribute to the association of respiratory illness in those who have experienced performing hajj or umrah. the limitations of this study include inadequate sample size and a poor response rate as it could affect the power of the study. the small response rate could be due to a lack of commitment from the respondents following a very packed hajj ritual schedule. shortage of staff to handle and follow-up the respondents in makkah, further added to the problem. in conclusion, the prevalence of respiratory illness remains high among malaysian hajj pilgrims despite having some practicing preventive measures. practicing only certain preventive measures are inadequate. all preventive measures including hand hygiene, wearing face masks and influenza vaccination must be practiced together to reduce the respiratory illness effectively. in our study, good hand hygiene practice was lower compared with other preventive measures; therefore, health authorities should find a way to overcome this problem. further studies are required to develop a health education module to promote a comprehensive preventive measure for hajj pilgrims. prevention of influenza at hajj: applications for mass gatherings the hajj: communicable and non-communicable health hazards and current guidance for pilgrims the quest for public health security at hajj: the who guidelines on communicable disease alert and response during mass gatherings health risks at the hajj pattern of admission to hospitals during muslim pilgrimage (hajj) the prevalence of acute respiratory symptoms and role of protective measures among malaysian hajj pilgrims understanding the symptoms of the common cold and influenza the association between pre-morbid conditions and respiratory tract manifestations amongst malaysian hajj pilgrims lack of mers coronavirus but prevalence of influenza virus in french pilgrims after influenza vaccine in hajj pilgrims: policy issues from field studies a case-control study of influenza vaccine effectiveness among malaysian pilgrims attending the hajj in saudi arabia the who guidelines on hand hygiene in healthcare (advanced draft) mask use, hand hygiene, and seasonal influenza-like illness among young adults: a randomized intervention trial clean hands save lives middle east respiratory syndrome coronavirus (mers-cov) high prevalence of common respiratory viruses and no evidence of middle east respiratory syndrome coronavirus in hajj pilgrims returning to ghana patterns of diseases and preventive measures among domestic hajjis from central, saudi arabia protective practices and respiratory illness among us travelers to the 2009 hajj influenza-like illness (ili): prevalence and preventive practices among indian hajj pilgrims of karnataka influenza a common viral infection among hajj pilgrims: time for routine surveillance and vaccination respiratory viruses and bacteria among pilgrims during the 2013 hajj viral respiratory infections among hajj pilgrims in 2013 vaccinations against respiratory tract infections at hajj viral etiology of acute respiratory infections among iranian hajj pilgrims incidence of hajj-associated febrile cough episodes among french pilgrims: a prospective cohort study on the influence of statin use and risk factors effect of influenza vaccination on acute respiratory symptoms in malaysian hajj pilgrims the incidence of vaccine preventable influenza-like illness and medication use among pakistani pilgrims to the haj in saudi arabia circulation of respiratory viruses among pilgrims during the 2012 hajj pilgrimage protective measures against acute respiratory symptoms in french pilgrims participating in the hajj of 2009 effect of hand hygiene on infectious disease risk in the community setting: a meta-analysis acute respiratory tract infections among hajj medical mission personnel, saudi arabia findings from a household randomized controlled trial of hand washing and face masks to reduce influenza transmission in non-pharmaceutical interventions for the prevention of respiratory tract infections during hajj pilgrimage pilot randomised controlled trial to test effectiveness of facemasks in preventing influenzalike illness transmission among australian hajj pilgrims in 2011 hajj-associated acute respiratory infection among hajjis from riyadh a quantitative assessment of the efficacy of surgical and n95 masks to filter influenza virus in patients with acute influenza infection conventional and alternative medical advice for cold and flu prevention: what should be recommended and what should be avoided? the benefit of tualang honey in reducing acute respiratory symptoms among malaysian hajj pilgrims: a preliminary study the authors acknowledge tabung haji malaysia, especially kelantan branch and sultan ismail petra airport for their continuous support and recommendations. conflict of interest: none declared. key: cord-005818-3mzwliiy authors: speer, c. p. title: surfactantsubstitutionstherapie: ein entscheidender durchbruch in der behandlung des atemnotsyndroms frühgeborener date: 2014-04-24 journal: monatsschr kinderheilkd doi: 10.1007/s00112-002-0434-y sha: doc_id: 5818 cord_uid: 3mzwliiy surfactant replacement therapy is a major breakthrough in neonatal medicine. prophylaxis or treatment of neonatal respiratory distress syndrome (rds) with various surfactant preparations is now a clinical reality. with either strategy the incidence of air leaks and mortality is reduced; more babies with rds survive without signs of chronic lung disease. the earlier the treatment, the better the outcome. an initial dose of 100 mg/kg can be recommended for all natural preparations, and some babies may benefit from multiple dose treatment. meta-analyses show a consistent advantage of natural surfactant preparations over the currently available synthetic ones. there is growing evidence that surfactant may also be beneficial in pulmonary diseases of the newborn which are characterized by inactivation of the surfactant system such as meconium aspiration syndrome or neonatal pneumonia. die surfactantsubstitution stellt einen entscheidenden durchbruch in der behandlung des atemnotsyndroms dar.durch eine prophylaktische surfactantgabe oder die therapie des manifesten atemnotsyndroms konnten die akuten pulmonalen komplikationen beatmeter frühgeborener um 2/3 reduziert und die sterblichkeit nahezu halbiert werden.eine frühe surfactantapplikation innerhalb der ersten 15 lebensminuten ist besonders bei sehr unreifen frühgeborenen einer späteren behandlung bei manifestem atemnotsyndrom überlegen.als initiale dosis werden 100 mg phospholipide/kg empfohlen; einige frühgeborene profitieren von einer mehrfachbehandlung.natürliche surfactantpräparate haben eine bessere klinische wirksamkeit als die zurzeit verfügbaren synthetischen präparate.es gibt einige hinweise, dass eine surfactanttherapie auch bei pulmonalen erkrankungen neugeborener einen therapeutischen effekt haben kann; diese erkrankungen wie mekoniumaspirationssyndrom und neonatale pneumonie führen u.a.über eine inaktivierung von surfactant zu einer sekundären defizienz dieses oberflächenaktiven systems. frühgeborene · atemnotsyndrom · surfactant · therapie die surfactantsubstitutionsbehandlung des atemnotsyndroms frühgeborener stellt einen meilenstein in der neonatalmedizin dar. 1959 konnten avery und mead (boston) bei verstorbenen frühgeborenen mit atemnotsyndrom erstmals belegen, dass das hyaline membranensyndrom mit dem mangel eines oberflächenaktiven materials assoziiert war [2] . 1967 gelang es rüfer (göttingen), die mechanischen eigenschaften surfactantdepletierter isolierter tierlungen durch intrabronchiale instillation einer oberflächenaktiven substanz zu verbessern; ein jahr später "die surfactantsubstitution ist die am besten untersuchte therapie der neonatalmedizin." konnte er diesen positiven effekt einer surfactantwirkung an isolierten lungen frühgeborener, die an einem atemnotsyndrom verstorben waren, nachweisen [49] .enhorning und robertson (toronto, stockholm) publizierten 1972 die viel beachteten ergebnisse einer surfactantsubstitutionstherapie bei beatmeten frühgeborenen kaninchen, die nach dieser behandlung eine nahezu normale lungenfunktion zeigten [14] . fujiwara und mitarbeiter (morioka) berichteten 1980 erstmals von frühgeborenen mit manifestem atemnotsyndrom (engl: respiratory distress syndrom: rds), die nach einer intratrachealen applikation eines rindersurfactants mit einer deutlichen verbesserung des pulmonalen gasaustausches reagierten [18] . in der folgenden dekade wurde die klinische wirksamkeit der surfactantbehandlung in sorgfältig geplanten, multizentrischen kontrollierten und/oder randomisierten studien eindrucksvoll belegt; weltweit wurden mehr als 10.000 frühgeborene mit verschiedensten surfactantpräparationen behandelt [56] .die in den 90iger jahren durchgeführten randomisierten studien hatten im wesentlichen das ziel, eine optimale behandlungsstrategie zu definieren. die surfactantsubstitution ist die am besten untersuchte therapie der neonatalmedizin. in der folgenden darstellung sollen die wesentlichen ergebnisse der klinischen studien -auch mit hilfe von metaanalysen -kurz zusammengefasst und die aktuellen therapieoptionen dargestellt werden. surfactant replacement therapy is a major breakthrough in neonatal medicine.prophylaxis or treatment of neonatal respiratory distress syndrome (rds) with various surfactant preparations is now a clinical reality. with either strategy the incidence of air leaks and mortality is reduced; more babies with rds survive without signs of chronic lung disease.the earlier the treatment, the better the outcome.an initial dose of 100 mg/kg can be recommended for all natural preparations, and some babies may benefit from multiple dose treatment.metaanalyses show a consistent advantage of natural surfactant preparations over the currently available synthetic ones.there is growing evidence that surfactant may also be beneficial in pulmonary diseases of the newborn which are characterized by inactivation of the surfactant system such as meconium aspiration syndrome or neonatal pneumonia. preterm infants · respiratory distress syndrome · pulmonary surfactant · prophylaxis · rescue wonnen [62] . die in diesen präparaten enthaltenen hydrophoben niedermolekularen surfactantproteine b (sp-b) und c (sp-c) verbessern die adsorption und ausbreitung der phospholipide in das bronchoalveoläre system. eine angeborene defizienz der surfactantproteine sp-b und sp-c führt zu einer gravierenden störung der surfactanthomöostase mit der ausbildung einer diffusen lungenerkrankung, die durch die anhäufung großer mengen phospholipid-und proteinhaltiger materialien in den alveolen charakterisiert ist [21] . die hydrophilen hochmolekularen surfactantproteine a (sp-a) und d (sp-d), die vermutlich die sekretion und wiederaufnahme der phospholipide auf zellulärer ebene regulieren und darüber hinaus wichtige abwehrfunktionen gegen verschiedene bakterien, viren und toxine übernehmen, werden während des extraktionsprozesses entfernt. die bedeutung dieser proteine für das unspezifische abwehrsystem wurde in tierexperimentellen untersuchungen an "knockout"-mäusen belegt [71] . neuere molekulargenetische untersuchungen von sp-a-allelen in einer homogenen finnischen population weisen darauf hin, dass der homozygote genotyp sp-a (6a 2 /6a 2 ) zur entwicklung eines rds prädisponiert, während der genotyp (6a 3 /6a 3 ) vor der ausbildung dieser erkrankung schützt [28] . die surfactantpräparate unterscheiden sich deutlich in der zusammensetzung der phospholipidfraktionen, der konzentration und im applikationsvolumen. [56] . die inzidenz der bronchopulmonalen dysplasie wurde durch die surfactanttherapie jedoch nicht beeinflusst.auf der basis dieser zahlen kann man vermuten, dass jährlich in den westlichen nationen zwischen 10.000 und 20.000 frühgeborene das atemnotsyndrom durch eine surfactantbehandlung überleben [27] . unmittelbar nach intratrachealer applikation natürlicher surfactantpräparate konnte bei frühgeborenen mit manifestem rds in allen studien eine -wenn auch recht unterschiedliche -verbesserung der oxygenierung und des gasaustausches beobachtet werden. eine besonders rasche reduktion der inspiratorischen sauerstoffkonzentration und des maschinellen beatmungsdruckes wurde nach gabe des porcinen surfactantpräparats curosurf beobachtet [59] .neuere tierexperimentelle untersuchungen mit diesem präparat belegen, dass radioaktiv markiertes surfactant nach intratrachealer applikation innerhalb von 60 s homogen in allen lungenarealen verteilt ist [34] . synthetische präparate zeigen im vergleich zu natürlichen surfactantpräparationen eine wesentlich langsamere verbesserung des pulmonalen gasaustausches und des beatmungsverlaufs. in verschiedenen studien wurden surfactantinitialdosierungen von 25-200 mg phospholipide/kg körpergewicht verwendet.von tierexperimentellen untersuchungen abgesehen, wurde eine optimale klinische dosisfindungsstudie der ersten surfactantdosis mit keinem der verfügbaren surfactantpräparate durchgeführt. allerdings gibt es hinweise, dass höhere initialdosen eine bessere klinische effektivität aufweisen als niedrigere, so z. b. eine initialdosis von 120 mg/kg surfactant-ta im vergleich zu 60 mg/kg [39] . eine initialdosis von 100 mg/kg alveofact war ebenfalls einer dosis von 50 mg/kg überlegen; neben einer verbesserten oxygenierung wurde in der mit 100 mg/kg behandelten gruppe seltener ein pulmonales interstitielles emphysem beobachtet [19] . eine pilotstudie mit dem porcinen präparat curosurf zeigte dagegen,dass eine initialdosierung von 100 mg/kg im vergleich zu 200 mg/kg einen nahezu identischen akuten effekt auf den gasaustausch hatte [31] . dieser befund wurde in einer großen multizentrischen europäischen studie (n=2168) im wesentlichen bestätigt. nach einer initialdosierung von 100 bzw. 200 mg/kg curosurf fand sich kein unterschied in der mortalität und morbidität zwischen den gruppen von frühgeborenen, die mit einem niedrigdosisregime (maximale kumulative dosis 300 mg/kg) oder einem hochdosisregime (maximale kumulative dosis von 600 mg/kg) behandelt worden waren [25] . frühgeborene mit rds, die nach einem initial guten ansprechen auf die surfactanttherapie innerhalb von 48 h einen erneuten ansteigenden sauerstoff-und beatmungsbedarf zeigten,profitieren von einer wiederholungsbehandlung. durch eine mehrfachtherapie mit surfactant konnten pneumothoraxinzidenz und sterblichkeit von frühgeborenen mit schweren rds weiter gesenkt werden [58] . man sollte sich immer vor augen halten, dass bereits im frühen stadium des atemnotsyndroms durch eine zunehmende alveolare-kapillare leckage serumproteine in die alveolen übertreten und das surfactantsystem inaktivieren (abb.1).diese inaktivierung kann nur durch eine ausreichende surfactantsubstitution mitigiert werden. es gibt hinweise, dass höhere initialdosen eine bessere klinische effektivität haben als niedrigere. neuere untersuchungen zur pathogenese des rds weisen darüber hinaus auf ein bisher wenig beachtetes phänomen hin. eine nicht unerhebliche anzahl von frühgeborenen, die bereits in utero einer chorioamnionitis ausgesetzt waren, weisen entzündliche veränderungen des lungengewebes und zeichen einer systemischen inflammationsreaktion auf [55] .diese frühgeborenen leiden nicht nur an einer primären surfactantdefizienz,sondern auch an einer ausgeprägten sekundären inaktivierung des surfactantsystems. zur behandlung des atemnotsyndroms empfehlen die meisten untersucher daher eine initialdosierung von 100 mg/kg. für eine prophylaktische surfactantsubstitution können möglicherweise etwas geringere dosen verwendet werden, ein beleg für diese annahme liegt jedoch nicht vor. in der klinik sollte ein individualisiertes therapieregime der wiederbehandlung gewählt werden.bei anhaltendem sauerstoffbedarf von >30% und maschineller beatmung sollte eine wiederholungsbehandlung durchgeführt werden. eine prophylaktische surfactantapplikation vor dem ersten atemzug hat theoretische vorteile.aufgrund einer verbesserten surfactantverteilung und gleichmäßigeren lungenbelüftung sollte das verminderte baro-und volumentrauma zu einer geringeren schädigung der alveolaren-kapillaren membran führen und somit den einstrom von inhibitorischen serumproteinen vermindern. durch eine prophylaktische surfactantsubstitution werden aber in unkenntnis des reifungsgrads des pulmonalen surfactantsystems etwa 40% aller frühgeborenen unnötigerweise mit exogenem surfactant behandelt. die konsequenzen dieser strategie sind neben einer unnötigen intubation und einer exposition frühgeborener mit den möglichen nebenwirkungen der therapie die nicht unerheblichen kosten [11] . mehrere randomisierte studien, welche die prophylaktische oder früh vergleichsstudien zwischen natürlichen und synthetischen surfactantpräparaten wurden überwiegend zwischen dem bovinen präparat survanta und dem synthetischen surfactant exosurf durchgeführt. elf vergleichsstudien wurden systematischen begutachtungen und metaanalysen unterzogen [24, 53] surfactantpräparate den zurzeit verfügbaren synthetischen präparationen überlegen sind [35] . möglicherweise ihrer klinischen wirksamkeit. die therapie mit curosurf [59] und infasurf [6] zeigte im vergleich zu survanta eine raschere reduktion des sauerstoffbedarfs und der beatmungssituation. keine der studien hatte jedoch ausreichende patientenzahlen, um mögliche unterschiede in der morbidität und mortalität zu erfassen. aus verschiedenen gründen ist es mehr als fraglich, ob in der zukunft adäquat geplante vergleichsstudien natürlicher surfactantpräparate durchgeführt werden können. eine 1-malige applikation von curosurf mit einer anschließenden cpap-behandlung frühgeborener mit moderatem rds führte im vergleich zu unbehandelten kontrollpatienten, die nur eine cpap-atemhilfe hatten, zu einer deutlichen verbesserung der oxygenierung und reduzierte die notwendigkeit einer nachfolgenden maschinellen beatmung [66] . in einer weiteren studie, in der frühgeborene <30 gestationswochen mit moderatem rds unter cpap-atemhilfe untersucht wurden, konnte gezeigt werden, dass eine frühe surfactantapplikation im verlauf des atemnotsyndroms einer späten behandlung überlegen war; die frühe behandlung verringerte die notwendigkeit einer maschinellen beatmung und/oder sterblichkeit der risikopatienten [67] . wie diese beiden studien zeigen, muss eine surfactantbehandlung nicht zwangsläufig von einer maschinellen beatmung gefolgt sein; ausgewählte patienten können durchaus von einer konsekutiven cpap-therapie nach surfactantapplikation profitieren. dieses neue behandlungsprinzip wird inzwischen unter dem namen "insure" ("intubate-surfactant-extubate") in randomisierten studien weiterverfolgt [5] . natürliche surfactantpräparate werden auf unterschiedliche weise appliziert. folgende techniken wurden in studien verwendet: ◗ bolusgaben eines oder zweier aliquots mit kurzzeitiger unterbrechung der beatmung, ◗ injektion über einen im tubus integrierten applikationskanal unter maschineller beatmung, ◗ gabe von 4 fraktionierten dosen mit unterschiedlicher körperpositionierung des patienten und ◗ diskonnektion vom beatmungsgerät [56] . beim vergleich der verschiedenen techniken konnte kein unterschied zwischen den einzelnen applikationsformen festgestellt werden [72] . wie tierexperimentelle untersuchungen eindrucksvoll belegen, führte eine langsame infusion von curosurf über 44 min zu einer gravierenden surfactantfehlverteilung in der lunge, ohne einen positi-ven effekt auf den gasaustausch zu zeigen; ähnliche befunde wurden nach 4minütiger infusion erhoben [51] . um eine unterbrechung der maschinellen beatmung zu vermeiden, wurde in einer randomisierten multizentrischen curosurf-studie eine 1-minütige surfactantinstillation über einen applikationskanal mit einer bolusgabe verglichen.während der 1-min-instillation hatten die frühgeborenen weniger episoden von transienten abfällen der sauerstoffsättigung sowie der herzfrequenz; die klinische effektivität und die inzidenz pulmonaler und extrapulmonaler komplikationen waren bei beiden applikationsformen identisch [65] . eine nebulisierung von surfactant war bisher nicht erfolgreich [4, 17, 36] . es gibt inzwischen klare belege, dass eine pränatale kortikosteroidbehandlung und die postnatale surfactanttherapie synergistische effekte haben und die morbidität sowie mortalität frühgeborener mit rds reduzieren [37] . die surfactantbehandlung wird von den meisten frühgeborenen problemlos toleriert. während der surfactantapplikation können, jedoch besonders bei präparaten, die in einem relativ großen volumen verabreicht werden, kurzzeitige vorübergehende beeinträchtigungen der patienten beobachtet werden [73] : akute verlegung der atemwege, reflux von surfactant,abnahme der sauerstoffsättigung, zyanose und bradykardien. hämodynamische effekte mit kurzzeitigen verminderungen des pulmonalen und systemischen blutdrucks, die vermutlich durch eine abnahme des peripheren gefäßwiderstandes zu erklären sind [42] , treten nach applikation von natürlichen präparaten auf. während dieser phase wurde auch eine vorübergehende suppression der eeg-aktivität und des zerebralen blutflusses beschrieben [29] . glücklicherweise sind diese veränderungen nicht mit einer erhöhten inzidenz zerebraler komplikationen assoziiert; eine analyse aller verfügbaren kontrollierten studien zeigt eher eine tendenzielle reduktion der inzidenz schwerer hirnblutungen, nach prophylaktischer surfactantgabe ist dieser effekt besonders deutlich [68] . sorgfältige nach◗ unabhängig von der art der surfactantpräparation muss der behandelnde kinderarzt mit allen aspekten der surfactantapplikation, der maschinellen beatmung sowie allen anderen maßnahmen der neonatologischen intensivmedizin vertraut sein untersuchungen von surfactantbehandelten frühgeborenen fanden keine erhöhte rate neurologischer komplikationen und entwicklungsauffälligkeiten [15, 23] . die scheinbar erhöhte inzidenz eines persistierenden ductus arteriosus (pda) ist über den akuten abfall des pulmonalen gefäßwiderstandes zu erklären; sie ist nicht als echte komplikation zu werten, sondern reflektiert vielmehr die wirksamkeit der applizierten surfactantpräparation. ein hämodynamisch wirksamer pda sollte durch adäquate, zeitgerechte maßnahmen behandelt werden. ebenso ist eine hyperventilation der frühgeborenen strikt zu vermeiden; eine hyperventilation ist eindeutig mit einer erhöhten inzidenz der periventrikulären leukomalazie sowie akuter und chronischer lungenschädigungen assoziiert [38] .akute lungenblutungen wurden häufiger nach prophylaktischer applikation synthetischer präparate beobachtet [45, 52] . anfängliche theoretische befürchtungen, dass die surfactantbehandlung das pulmonale abwehrsystem kompromittieren und somit eine erhöhte rate pulmonaler infektionen nach sich ziehen könnte, haben sich nicht bestätigt. in keiner der kontrollierten studien wurde eine erhöhte rate an pneumonien beschrieben. eine reihe von in-vitro-experimenten und tierexperimentellen untersuchungen haben im gegenteil belegt, dass klinisch verwendete surfactantpräparate zum teil potente antimikrobielle fähigkeiten besitzen und die vermehrung von relevanten erregern der neonatalperiode wie streptokokken der gruppe b direkt verhindern; besonders wirksam war das porcine präparat curosurf [46] . vor kurzem wurde in diesem präparat das antibakterielle peptid prophenin identifiziert [69] . die in natürlichen und synthetischen surfactantpräparaten ent-haltenen phospholipide haben darüber hinaus eine vielzahl von antiinflammatorischen eigenschaften, die sich möglicherweise positiv auf die bereits während der frühen stadien des atemnotsyndroms auftretenden intraalveolaren entzündungsreaktion auswirken können [28, 60] . es gibt bisher keine hinweise, dass sich frühgeborene nach behandlung mit natürlichen präparaten gegen bestandteile des surfactant sensibilisiert hätten. vermutlich als ausdruck einer geringeren alveolarschädigung und eines verminderten übertritts von surfactantproteinen in die systemische zirkulation wurden seltener zirkulierende antikörper bei surfactantbehandelten frühgeborenen beobachtet [7] . ebenso wurden bis heute keine hinweise für eine übertragung von atypischen infektionserregern, "slow-virus"-infektionen oder prionen auf frühgeborene nach behandlung mit natürlichen surfactantpräparaten gefunden. eine potenzielle gefährdung ist jedoch bei der gruppe der natürlichen präparate nicht mit sicherheit auszuschließen. die surfactantbehandlung ist, wie ökonomische analysen von hospitalisierungskosten belegen, eine kosteneffektive maßnahme; besonders durch eine prophylaktische oder frühe surfactantsubstitution lassen sich kosten für das gesundheitssystem einsparen [12, 50] . [33] . in einer kleinen, kontrollierten und randomisierten studie erhielten neugeborene mit mekoniumaspiration hohe und repetitive dosen des bovinen surfactantpräparats survanta. die behandelten neugeborenen zeigten im vergleich zu den unbehandelten kontrollpatienten eine deutlich verbesserte oxygenierung und eine geringere inzidenz akuter pulmonaler komplikationen [16] . positive effekte auf den gasaustausch wurden auch in einer großen beobachtungsstudie beschrieben [26] . ebenso profitierten neugeborene mit verschiedenen formen eines akuten lungenversagens von einer exogenen surfactanttherapie; die therapiedauer einer extrakorporalen membranoxygenierung konnte durch surfactant reduziert werden [41] . zur zeit laufende multizentrische studien, u.a. in china, werden den stellenwert der surfactant-behandlung beim mekoniumaspirationssyndrom definieren. ob eine initiale lavage mit verdünntem surfactant die prognose und komplikationsrate des mekoniumaspirationssyndroms verbessern kann, muss trotz der positiven tierexperimentellen ergebnisse ebenfalls in randomisierten, klinischen studien geklärt werden [40, 70] . sorgfältig geplante kontrollierte und randomisierte studien, die mit den zurzeit verfügbaren surfactantpräparationen durchgeführt werden, müssen den stellenwert der surfactanttherapie für die oben genannten neuen indikationsfelder definieren. in der nahen zukunft werden darüber hinaus physiologisch und klinisch aktive neue surfactantpräparate zur verfügung stehen, die aus surfactantlipiden und synthetischen proteinanalogen von sp-b und sp-c bestehen werden [48] . diese präparate werden eine standardisierte komposition aktiver surfactantbestandteile enthalten und reproduzierbare biophysikalische eigenschaften haben. diese neue generation von surfactant kann theoretisch für die verschie-denen formen von lungenerkrankungen früh-und neugeborener hergestellt werden und sich durch eine hohe resistenz gegen inaktivierende substanzen auszeichnen [9, 33] . erste klinische untersuchungen mit einem ein leucin-/lysinpeptid enthaltendem synthetischem surfactant (kl 4 , surfaxin) zeigten eine verbesserung des gasaustausches bei frühgeborenen mit rds und neugeborenen mit mekoniumaspirationssyndrom [8, 70] . in-vitro-untersuchungen und tierexperimentelle studien belegen ebenso die wirksamkeit eines synthetischen präparats (venticute), das rekombinantes sp-c (rsp-c) enthält [22] . im vergleich zu natürlichen surfactantpräparaten weisen diese beiden synthetischen präparationen einen geringeren inaktivierungsgrad auf. sp-a und sp-d enthaltendes natürliches surfactant zeigte allerdings das ausgeprägteste resistenzverhalten gegen eine mekoniuminaktivierung [33, 61] (abb. 2b). ein neuer ansatz zur minimierung der surfactantinaktivierung wurde vor kurzem berichtet. durch zugabe von zuckerpolymeren und anderen nicht ionischen polymeren wie dextran und polyäthylenglykol zu natürlichen surfactantpräparaten konnte die surfactantinaktivierung aufgehoben werden [63, 64] . so ist es denkbar, dass z. b. zur behandlung der neonatalen pneumonie diesen surfactantpräparaten die proteine sp-a und sp-d, polymere, spezifische antikörper oder antibakterielle substanzen beigefügt werden. die großindustrielle herstellung wird zu einer reduktion der kosten führen und das risiko von viralen infektionen und prionenerkrankungen eliminieren. die klinische wirksamkeit dieser neuen surfactantpräparate wird in vergleichsstudien gegen die momentan verfügbaren natürlichen surfactantpräparate zu belegen sein. die surfactanttherapie ist heute ein fester bestandteil der behandlung frühgeborener mit atemnotsyndrom. die faszinierende geschichte der klinischen surfactantforschung lässt sich in folgenden aussagen zusammenfassen: pumactant and poractant alfa for treatment of respiratory distress syndrome in neonates born at 25-29 weeks' gestation: a randomised trial surface properties in relation to atelectasis and hyaline membrane disease randomized multicenter trial of treatment with porcine natural surfactant for moderately severe neonatal respiratory distress syndrome pilot study of nebulized surfactant therapy for neonatal respiratory distress syndrome lung function in premature infants can be improved.surfactant therapy and cpap reduce the need of respiratory support comparison of infasurf (calf lung surfactant extract) to survanta (beractant) in the treatment and prevention of respiratory distress syndrome surfactant proteins and anti-surfactant antibodies in sera from infants with respiratory distress syndrome with and without surfactant treatment the efficacy and safety of kl 4 -surfactant in preterm infants with respiratory distress syndrome future surfactant preparations surfactant replacement therapy: an update on applications surfactant replacement therapy: prophylaxis or treatment? theoretical changes in neonatal hospitalisation costs after the introduction of porcine-derived lung surfactant (curosurf®) mortality, severe respiratory distress syndrome, and chronic lung disease of the newborn are reduced more after prophylactic than after therapeutic administration of the surfactant curosurf lung expansion in the premature rabbit fetus after tracheal deposition of surfactant survival and follow-up of infants born at 23 to 26 weeks of gestational age: effects of surfactant therapy surfactant replacement therapy for meconium aspiration syndrome nebulisation of surfactants in an animal model of neonatal respiratory distress artificial surfactant therapy in hyaline-membrane disease high-dose versus low-dose bovine surfactant treatment in very premature infants early versus late surfactant treatment in preterm infants of 27 to 32 weeks of gestational age: a multicenter controlled clinical trial pulmonale alveolarproteinosen -molekulare grundlagen und konsequenzen für diagnostik und therapie effects of rsp-c surfactant on oxygenation and histology in a rat-lung-lavage model of acute lung injury follow up data from babies treated with surfactant phospholipidkonzentration: 2,5 mg/ml) in abhängigkeit von der zugesetzten mekoniumkonzentration. humanes gepooltes, lyophilisiertes mekonium von reifen neugeborenen wurde mit den natürlichen modifizierten surfactants alveofact, curosurf und survanta (a) bzw. den neuen synthetischen präparationen rsp-c-und kl 4 -surfactant (b) inkubiert, die minimale oberflächenspannung wurde in einem pulsating-bubble-surfactometer bestimmt curosurf und survanta sich durch geringste mekoniumzusätze inaktivieren lassen (a), zeigten sowohl rsp-c-als auch kl 4 -surfactant eine deutlich höhere resistenz gegenüber einer inhibition. natürlicher lavagesurfactant, der das gesamte proteinspektrum, einschließlich sp-a und sp-d enthält, lässt sich erst durch mekoniumkonzentrationen >10 mg/ml hemmen natural versus synthetic surfactants in neonatal respiratory distress syndrome multicenter randomised trial comparing high and low dose surfactant regimens for the treatment of respiratory distress syndrome (the curosurf 4 trial) treatment of severe meconium aspiration syndrome with porcine surfactant infants born very premature -more prospective studies needed surfactant in respiratory distress syndrome and lung injury cerebroelectrical depression following surfactant treatment in preterm neonates sekundärer surfactant-mangel bei respiratorischer insuffizienz im neugeborenenalter -experimentelle und klinische untersuchungen zur pathogenese des mekonium-aspirations-syndroms und der konnatalen pneumonie einfluß von 2 unterschiedlichen dosierungen eines porcinen surfactants auf den pulmonalen gasaustausch frühgeborener mit schwerem atemnotsyndrom surfactant treatment of neonates with respiratory failure and group b streptococcal infection resistance of different surfactant preparations to inactivation by meconium a radio-isotope technique for visualisation of exogenous surfactant spreading in immature lamb lungs at birth which surfactant for treatment of respiratory-distress syndrome surfactant aerosol treatment of respiratory distress syndrome in spontaneously breathing premature infants prenatal dexamethasone treatment in conjunction with rescue therapy of human surfactant: a randomized placebo-controlled multicenter study history and current understanding of surfactant surfactant replacement therapy in neonatal respiratory distress syndrome.a multicenter, randomized clinical trial: comparison of high versus low-dose of surfactant ta surfactant lavage for the management of severe meconium aspiration syndrome multicenter study of surfactant (beractant) use in the treatment of term infants with severe respiratory failure acute effects on systemic circulation after intratracheal instillation of curosurf or survanta in surfactantdepleted newborn piglets prospective randomized multicenter comparison of high-frequency oscillatory ventilation and conventional ventilation in preterm infants of less than 30 weeks with respiratory distress syndrome outcome following pulmonary haemorrhage in very low birthweight neonates treated with surfactant pulmonary hemorrhage and exogenous surfactant therapy: a metaanalysis influence of modified natural or synthetic surfactant preparations on growth of bacteria causing infections in the neonatal period new targets for surfactant replacement therapy: experimental and clinical aspects synthetic surfactants to treat neonatal lung disease der einfluss oberflächenaktiver substanzen auf entfaltung und retraktion isolierter lungen effect of surfactant on morbidity, mortality, and ressource use in newborn infants weighing 500 to 1500 g pulmonary distribution and efficacy of exogenous surfactant in lung lavaged rabbits are influenced by the instillation technique prophylactic synthetic surfactant for preventing morbidity and mortality in preterm infants natural surfactant extract versus synthetic surfactant for neonatal respiratory distress syndrome prophylactic versus selective use of surfactant for peventing morbidity and mortality in preterm infants new insights into the pathogenesis of pulmonary inflammation in preterm infants surfactant therapy in the newborn early versus late surfactant replacement therapy in severe respiratory distress syndrome randomized european multicenter trial of surfactant replacement therapy for severe neonatal respiratory distress syndrome: single versus multiple doses of curosurf randomized clinical trial of two treatment regimens of natural surfactant preparations in neonatal respiratory distress syndrome randomized trial.comparing natural and synthetic surfactant: increased infection rate after natural surfactant? biophysical and physiological properties of a modified porcine surfactant enriched with surfactant protein a current perspectives on the drug treatment of neonatal respiratory distress syndrome nonionic polymers reverse inactivation of surfactant by meconium and other substances dextran reduces surfactant inhibition by meconium a randomized comparison of surfactant dosing via a dual-lumen endotracheal tube in respiratory distress syndrome surfactant therapy and nasal continuous positive airway pressure for newborns with respiratory distress syndrome nasal continuous positive airway pressure and early surfactant therapy for respiratory distress syndrome in newborns of less than 30 weeks of gestation prophylactic administration of porcine-derived lung surfactant is a significant factor in reducing the odds for peri-intraventricular hemorrhage in premature infants porcine pulmonary surfactant preparations contain the antibacterial peptide prophenin and a c-terminal 18-residue fragment thereof advances in the treatment of the meconium aspiration syndrome immunomodulatory functions of surfactant comparison of three dosing procedures for administration of bovine surfactant to neonates with respiratory distress syndrome treatment investigational new drug experience with survanta (beractant) key: cord-023712-nptuuixw authors: bower, john; mcbride, john t. title: bronchiolitis date: 2014-10-31 journal: mandell, douglas, and bennett's principles and practice of infectious diseases doi: 10.1016/b978-1-4557-4801-3.00068-0 sha: doc_id: 23712 cord_uid: nptuuixw nan with bronchiolitis we have to contend with illness that's now and disease that comes then; for many such infants a mold has been cast, perhaps by their unborn and unknown past, which destines that they shall in time wheeze again. for them this disease is the far, boding knell of vulnerable lungs to a microbe's dark spell. -caroline breese hall (1939-2013, the original author of much of this chapter and to whose memory it is dedicated) bronchiolitis is the most common acute viral lower respiratory tract illness occurring during the first 2 years of life. much interest and effort have been aimed at determining the pathogenesis and management of this illness among hospitalized and outpatient children. despite this, concerns and controversies continue. bronchiolitis has acquired during its long lineage a notable number of sobriquets, including "acute catarrhal bronchitis, " "interstitial bronchopneumonia, " "spastic bronchopneumonia, " "capillary or obstructive bronchitis, " and, more commonly, "wheezy bronchitis" and "asthmatic bronchiolitis. " the diversity of these terms is indicative of the past and ongoing confusion and difficulty in clinical differentiation of bronchiolitis from asthma and infectious asthma. these entities usually refer to repeated episodes of wheezing that may be triggered by infectious agents and tend to occur in children beyond infancy. the definition of bronchiolitis varies but usually applies to children younger than 2 years of age with a first episode of wheezing commonly associated with fever, cough, rhinorrhea, and tachypnea. 1,2 consensus does exist, however, that bronchiolitis continues to impose a major and increasing health care burden. bronchiolitis has been estimated to be the leading cause of all hospitalizations among infants in the united states. 3, 4, 5 etiology bronchiolitis was not recognized as a distinct entity until the 1940s and was initially thought to be caused by bacteria. 6, 7 viruses are now known to be the prime cause of the syndrome and the associated characteristic pathology of the lower respiratory tract. respiratory syncytial virus (rsv) is the major pathogen identified. the roles played by other viral agents are controversial and depend partly on the population being studied and the laboratory methods used for detection. correlation with disease is particularly problematic because viruses that commonly infect this young age group can cause high rates of asymptomatic infection or prolonged shedding, including adenoviruses and human bocavirus (hbov). in addition, some agents, such as rhinoviruses, may trigger asthmatic airway inflammation and bronchospasm without causing the small airway pathology characteristic of infection of the lower respiratory tract with bronchiolitis. rsv has been identified as the principal agent in two thirds of the cases of bronchiolitis, and in hospitalized patients the proportion is likely higher. 1, 8, 9 other viruses that commonly have been identified as single or coinfecting agents among children with bronchiolitis include human metapneumovirus (hmpv), the parainfluenza viruses, influenza viruses, rhinoviruses, human coronaviruses (hcov), and hbov (table 68-1) . among the parainfluenza viruses, parainfluenza virus types 1 and 3 are more commonly associated with bronchiolitis in hospitalized children than type 2. 10 illnesses with hmpv and rsv are generally indistinguishable, although lower respiratory tract illness with hmpv is usually less severe. [11] [12] [13] both rsv and hmpv occur from fall to spring, but activity of hmpv in the community is generally less intense. [11] [12] [13] hospitalized children with hmpv infection tend to be slightly older than children with rsv, and almost all children are infected with hmpv by 5 to 10 years of age. 12 influenza a and b viruses frequently cause lower respiratory tract disease among children younger than 2 years of age, but the proportion manifesting as bronchiolitis is less than that observed with rsv. [13] [14] [15] with the use of sensitive molecular techniques, additional viruses have been identified in young children with bronchiolitis including hcovs (see chapter 157) and hbov (see chapter 149). hcovs are composed of four different strains, including the novel strains hcov-nl63 and hcov-hku1, and have been identified in 7% of young children hospitalized with respiratory illness. 16 however, an equal percentage of asymptomatic children younger than 5 years of age have been observed to shed hcov. 16 the parvovirus hbov is increasingly being detected by reverse-transcriptase polymerase chain reaction (rt-pcr) in respiratory and fecal specimens from adults and children • diagnosis is clinically based on presence of rsv in the community, initial episode of wheezing, and evidence of upper respiratory infection. • other causes of wheezing in early childhood should be excluded, such as congenital heart disease with failure, foreign body aspiration, dysphagia, and asthma. • apnea may occur early in the course of viral bronchiolitis, usually in infants younger than 44 weeks' postconceptional age. • therapy is supportive and includes hydration, oxygen, and respiratory support as needed. • corticosteroids and bronchodilators are not generally beneficial. • hypertonic saline aerosols delivered three times daily may hasten recovery but have not been widely adopted. • respiratory support by high-flow nasal cannula may prevent or delay intubation in patients with apnea or respiratory failure. 26 despite the steady rates of hospitalization for bronchiolitis, the mortality rates associated with bronchiolitis have declined in the united states to fewer than 400 deaths per year. 3, 27 most deaths (79%) occur in infants younger than 1 year, primarily during the first several months of life. children with chronic conditions, especially conditions affecting cardiopulmonary function, are most likely to develop severe or fatal bronchiolitis. children with prematurity and the associated chronic lung disease have a fivefold increased risk of developing disease requiring hospitalization than children with no comorbid conditions. 3, 28, 29 multiple demographic, environmental, and biologic factors have been associated with increased rates of hospitalization among otherwise normal children. bronchiolitis is more common in boys, especially among children with more severe illness, with a male-to-female ratio of about 1.5 : 1. 30 other factors that have been associated with a greater likelihood of severe illness include young maternal age, lower cord blood antibody titers to rsv, lower socioeconomic status, tobacco smoke exposure, living in crowded surroundings, having older siblings, daycare attendance, lack of breast-feeding, a predisposition to atopy or hyperreactivity of the airway, and illness caused by rsv. 11, [31] [32] [33] infants with specific genotypes predicted to modify innate mucosal immunity are at greater risk of severe rsv infections. 34 a similar mechanism might explain the fact that certain ethnic groups of infants have higher rates of hospitalization for bronchiolitis. native american and native alaskan children have hospitalization rates two to three times higher than those of the general population of u.s. children of the same age. 35 nevertheless, the major independent risk factor for bronchiolitis requiring hospitalization is young age, within the first 6 months of life. 11, 33 pathophysiology in 1940, engle and newns 6 carefully described the pathology of a severe and often fatal lower respiratory tract disease they observed in young infants. they called this "proliferate mural bronchiolitis. " their findings of the generalized involvement of the respiratory epithelium of the small airways have been confirmed as being characteristic of infection-induced bronchiolitis among young children. the virus initially replicates in the epithelium of the upper respiratory tract, with subsequent spread within a few days to the lower tract airways ( fig. 68-1 ). early inflammation of the bronchial and bronchiolar epithelium occurs along with peribronchiolar infiltration, mostly with mononuclear cells, and edema of the submucosa and adventitia. the respiratory epithelium becomes necrotic and is sloughed into the lumina of the airways. subsequently, the epithelium proliferates and shows cuboidal cells without cilia ( fig. 68-2) . with a spectrum of upper and lower respiratory illnesses, including bronchiolitis. hbov has been reported as a sole pathogen in 1% to 6% of young children with bronchiolitis and as a coinfection in 6% to 20% of bronchiolitis cases. however, hbov is shed for prolonged periods and may be detected long after the clinical manifestations associated with acute infection have resolved. 8, 17, 18, 19, 20 viruses that are primarily agents of upper respiratory tract infections are also commonly identified in specimens obtained from children with bronchiolitis. notable among these are the picornaviruses (rhinoviruses and enteroviruses) and adenoviruses. the direct role of these viruses in causing bronchiolitis is uncertain because their high prevalence in this age group makes them common agents of dual infection. 8, 9 rhinoviruses, with more than 100 serotypes, are identified in 3% to 30% of children with bronchiolitis, and more than half the time they are present as coinfecting agents, most commonly with rsv. 8, 21 interpreting the role of rhinoviruses in children with bronchiolitis is complicated by the association of rhinovirus with episodes of wheezing from reactive airway or asthma exacerbations. 21 one group that appears to be at particular risk for severe lower respiratory tract infection due to human rhinoviruses are very-low-birth-weight infants. 22 enteroviruses have been identified in up to 7% of children hospitalized with bronchiolitis and are usually present as coinfecting viruses. 9 it remains uncertain whether dual viral infections increase the risk of developing more severe illness with bronchiolitis. 8, 9 studies examining whether hmpv coinfection is associated with more severe rsv lower respiratory tract disease suggest an increase in disease severity among hospitalized children, but this has not been consistently demonstrated. 9,23-25 bronchiolitis shows a yearly seasonal pattern that varies according to geography and climate. in temperate climates, the peak occurrence of cases is during the winter to early spring and usually correlates with the prevalence of rsv in the community. outbreaks of bronchiolitis are less distinctive in warmer and tropical climates where rsv occurs over longer periods. bronchiolitis cases in these areas may be seen throughout the year, and the prevalence of cases depends on the seasonal patterns of the known and yet unknown agents associated with bronchiolitis (see table 68 -1). bronchiolitis is most common during the first year of life, with the peak attack rate occurring between 1 and 10 months of age and among hospitalized cases between 2 and 5 months of age. each year, 1% to 3% of infants younger than 12 months of age are hospitalized with bronchiolitis; 80% are younger than 6 months of age. for the period 1997 to 2006, age-specific rates of hospitalization for bronchiolitis remained steady with an overall rate of 26 per 1000 children younger than 1 year of age and 48.9 per 1000 for infants younger than 3 months of age. 4,5 for children older than 1 year of age the rate was 1.8 per 1000. 4 overall, rsv disease accounts for up to 24% of hospitalizations among lethargy and poor feeding. retractions of the chest wall, flaring of the nasal alae, and grunting are evidence of increased work of breathing. the hallmark of bronchiolitis is the rapid variability of the child's respiratory signs. auscultatory findings may vary from only wheezing or crackles, to both, or to neither. decreasing lung sounds on auscultation associated with increasing dyspnea and diminished movement of air may indicate progressive obstruction and impending respiratory failure. dehydration commonly accompanies bronchiolitis, resulting from paroxysms of coughing, which may trigger vomiting, and from poor oral intake related to the child's respiratory distress and lethargy. tachypnea increases the fluid requirement further. of children hospitalized with bronchiolitis in the united kingdom, 82% on admission had feeding difficulties that lasted an average of 27 hours. 36 supplemental oxygen was administered at the time of admission to 22% of the children whose mean pulse oxygen saturation was 94%. within 6 hours of admission, 70% were given supplemental oxygen, although the mean pulse oxygen saturation level decreased an average of 2%. no correlation was observed between the pulse oxygen saturation level obtained at 6 hours and the administration of supplemental oxygen or the length of hospital stay. infants whose feeding difficulties resolved and who continued to be hospitalized for supplemental oxygen administration only had no evidence of clinical deterioration. considering that bronchiolitis is one of the most frequent causes of pediatric ambulatory visits and hospitalization, children at low risk for developing complicated illness have been evaluated to determine which children may be safely discharged home. among children younger than 2 years of age presenting with bronchiolitis at 30 u.s. emergency departments during 2004 to 2006, 57% were discharged to home. 37 characteristics of the children whose home discharge was safe included age 2 months or older, a history of eczema, respiratory rates inflammatory changes of variable severity are observed in most small bronchi and bronchioles. because resistance to airflow is related inversely to the cube of the radius of the airway, the inflammation and edema make the lumina of small airways in infants particularly vulnerable to obstruction (see fig. 68-1) . plugs of necrotic material and fibrin may completely or partially obstruct the small airways. smooth muscle constriction does not seem to be a major factor in the obstruction. in areas peripheral to sites of partial obstruction, air becomes trapped by a process similar to a "ball-valve" mechanism. negative intrapleural pressure exerted during inspiration allows air to flow beyond the point of partial obstruction. on expiration, however, the size of the lumen decreases, resulting in obstruction and gas trapping. in areas peripheral to obstruction, trapped air is eventually absorbed, which results in multiple areas of atelectasis. this absorptive atelectasis is accelerated when a child breathes high concentrations of oxygen, which is absorbed into the blood much faster than nitrogen. the degree of atelectasis or hyperinflation that develops is greater in infants than it would be in older children or adults because collateral channels that maintain alveolar expansion in the presence of airway obstruction are not well developed early in life. the physiologic correlates of airway obstruction are dyspnea, tachypnea, a diminished tidal volume, and a diminished ratio of ventilation to perfusion resulting first in arterial hypoxemia. when an infant is no longer able to compensate for the disordered gas exchange by increasing ventilation, hypercarbia may ensue. the pathologic process may progress to involve the alveolar walls and spaces, producing an interstitial pneumonitis. recovery tends to be slow, requiring several weeks. bronchiolitis commonly has a prodrome of several days that is marked by upper respiratory tract signs, especially coryza, cough, and fever, which is usually mild. lower respiratory tract involvement may be signaled by the development of a prominent cough, followed by an increased respiratory rate, and nonspecific systemic symptoms such as a b management of bronchiolitis. 2 complete blood cell count values vary in children with bronchiolitis and have not been shown to be helpful in determining the diagnosis or therapy of bronchiolitis. 2 additional diagnostic procedures should be reserved for children whose history, findings, or clinical course are not as expected. although rapid diagnostic testing is generally unnecessary, it may be useful at times for implementing appropriate infection control, monitoring seasonal patterns of respiratory pathogens, restricting antimicrobial use, or providing confirmation of the diagnosis in children with unusual clinical presentations or severe disease. timely diagnosis of specific viral respiratory pathogens may occasionally be necessary to guide specific antiviral therapy in children with high-risk conditions or severe illness with influenza or rsv. rapid diagnostic approaches to identifying the common viral agents of bronchiolitis include tissue culture, antigen detection, and pcr. nasopharyngeal washes provide the most appropriate specimen. when available, tissue culture by shell vial technique can provide positive culture results within several days. rapid antigen detection includes direct and indirect immunofluorescent assays, optical immunoassays, and enzyme immunoassays. these rapid viral antigen techniques are most commonly used because of their ease, cost, and availability of results within hours. 52 rapid real-time pcr testing is becoming increasingly available for the simultaneous diagnosis of multiple respiratory viruses and is capable of high sensitivity and specificity, as well as short turnaround times. 53 the positive predictive value of all these viral assays significantly diminishes when the prevalence of the agent, such as rsv or influenza, is low in the community. serologic tests to determine the etiologic agent are rarely helpful in clinical management and may be difficult to interpret because a young infant would have maternally acquired antibody to many of the viral agents of bronchiolitis. the differentiation of wheezing caused by rsv infection from wheezing caused by many other mechanisms in infants is challenging because rsv occurs in epidemics. during the height of the epidemic, it is tempting to assume that rsv is the culprit in any wheezing infant. the differential diagnosis of wheezing in an infant is broad and requires a careful history and examination. 54 congestive heart failure is most important to consider because infants with left-to-right shunt are likely to become symptomatic and present with tachypnea and wheezing at around 8 to 10 weeks of age. gastric reflux and aspiration may produce a picture that is indistinguishable clinically from acute bronchiolitis. an asthma exacerbation precipitated by a viral infection is possible, particularly in infants with a strong family history of asthma. other considerations include foreign body aspiration, vascular ring, cystic fibrosis, and immunodeficiency. supportive care is the mainstay of therapy for outpatient and inpatient children. guidelines for care have been published and updated. 2 at home, care is aimed primarily at comfort, maintaining adequate hydration, and treating fever if necessary. 2,55 young children, especially infants, are particularly compromised by a respiratory rate of 60 or greater per minute and by the increased nasal congestion and mucus production in the lower respiratory tract. these may result in diminished fluid intake, inability to sleep, increased work of breathing, and the risk of requiring assisted ventilation. clearance of secretions by administering chest percussion or deep pharyngeal and tracheal suctioning has been ineffective in the management of bronchiolitis and is not advised. 2 among more severely ill children with hypoxemia, supplemental oxygen administration may be of prime importance. the spo 2 level at which supplemental oxygen should be administered is not well defined, however, and is controversial. although spo 2 levels of 90% to 95% on room air have been commonly used, the american academy of pediatrics has advised for previously healthy infants that supplemental oxygen should be initiated when persistent measurements of spo 2 levels less than 90% are obtained. 2 in fact, the use of pulse oximetry in previously healthy children without signs of respiratory distress has not been associated with a better clinical outcome but has led to increased use of medical services and cost and is not routinely recommended. 2 other factors than just the spo 2 level should be considered in the that were below normal for age, oxygen saturation levels 94% or greater, no or mild chest wall retractions, fewer treatments with bronchodilators during the first hour, and adequate oral intake. the acute course of bronchiolitis typically lasts 3 to 7 days. a minority of children with rsv lower respiratory infection presents with critical hypoxemia, apnea, or respiratory failure and immediately requires intensive care. most children admitted to the hospital are less severely affected, and relatively few of them deteriorate dramatically after admission. in one study, less than 2% of previously healthy children admitted to the regular floor for rsv infection subsequently required intensive care. 38 most infants improve within 3 to 4 days, with a gradual recovery period of 1 to 2 weeks, but cough may persist longer. the median duration of illness in one study of ambulatory children with bronchiolitis was 12 days. after 3 weeks, 18% remained symptomatic, and after 4 weeks, 9% were still ill. 39 gender, weight, or respiratory rate was not predictive of longer illness. complications associated with bronchiolitis occur most frequently in infants within the first several months of life, in premature infants, and in children with chronic cardiac, pulmonary, and immunodeficiency diseases. 3, [27] [28] [29] the most serious complication is progression to respiratory failure. although the risk of respiratory failure is relatively low for most children with rsv bronchiolitis, a small number of severely affected infants will require assisted ventilation in most intensive care units each year. intubation and ventilation are usually indicated by recurrent severe apnea or hypercapnic/hypoxemic respiratory failure. apnea, one of the most frequent acute complications, occurs in 3% to 21% of infants. [40] [41] [42] apnea typically is the presenting manifestation, occurring after several days of respiratory symptoms that may be so mild as to go unnoticed. infants who present with apnea are at risk of developing severe lower respiratory disease even as the apnea typically resolves within a day or two. apnea is most likely to occur in premature infants and in infants within the first 2 months of life (e.g., infants who are younger than 44 weeks' postconceptional age). the apnea does not seem to be obstructive, generally has a good prognosis, and is not associated with an increased risk of sudden infant death syndrome subsequently. aspiration has been shown to be a frequent complication in infants hospitalized with rsv bronchiolitis. 43, 44 it is possible that infants with preexisting dysphagia are at increased risk of severe bronchiolitis with rsv infection, so this may represent association rather than causation. secondary bacterial infections complicating bronchiolitis are uncommon, and concurrent bacterial infections occur in 0% to 7% of bronchiolitis cases. 28, [45] [46] [47] [48] concurrent bacterial infections most frequently are urinary tract infections, unrelated to the bronchiolitis. bacterial coinfections have been less common in children with bronchiolitis than in control children without bronchiolitis. the most frequent clinical association observed in infants hospitalized with bronchiolitis is subsequent episodes of recurrent wheezing, estimated to occur in 30% to 50% of infants hospitalized with bronchiolitis. the pathogenesis of this link is unclear (see chapter 160). controversy continues over the extent to which this association is explained by a genetic predisposition to both severe rsv disease and subsequent wheezing or by an effect of rsv infection itself. 49 nevertheless, the prognosis for most children with recurrent episodes of wheezing during early childhood is good. among most children, the episodes diminish or disappear before reaching the teenage years. 50,51 the diagnosis of bronchiolitis may be made for most children on the basis of the characteristic clinical and epidemiologic findings. these include the acute onset of the typical constellation of respiratory tract findings of cough, wheezing, and increasing respiratory effort after an upper respiratory tract prodrome, particularly during the winter respiratory season, in a child younger than 2 years of age. 2 laboratory and radiologic studies are unnecessary for diagnosis; they do not change the outcome for most children and are not routinely recommended. the assessment of the severity of the bronchiolitis should also be based on the child's history and physical examination according to the american academy of pediatrics' guidelines on the diagnosis and times a day) has been shown to result in more rapid clinical improvement and shortened length of stay in infants with rsv bronchiolitis without evidence of side effects. 62 nevertheless, this therapy has not been universally adopted because several studies have been unable to show benefit and because of the theoretical but unsubstantiated concern that the hypertonic saline aerosol might induce bronchospasm in infants with asthma. ribavirin (1-β-d-ribofuranosyl-1,2,4-triazole-3-carboxamide), a synthetic nucleoside, is available for aerosol treatment for rsv bronchiolitis among hospitalized infants. the drug is not recommended routinely, however, and should be considered only for infants with severe disease at high risk of severe illness (see chapters 44 and 160). 2 several approaches are used to provide direct respiratory support for the small number of young infants or those with underlying abnormalities who develop life-threatening apnea or respiratory failure during an episode of bronchiolitis. noninvasive approaches that have been used to avoid intubation include continuous positive airway pressure, heliox, and high-flow nasal cannula therapy (hfnc). [63] [64] [65] the first two have not been consistently shown to be adequately beneficial to justify the challenge of administration. hfnc, however, may be effective in preventing or delaying the need for intubation. 66, 67 prevention prevention of the clinical entity of bronchiolitis is a goal unlikely to be reached in the near future because of its multiple etiologies and varying pathogenesis. for prevention of bronchiolitis associated with primary rsv infection, prophylactic administration of humanized monoclonal antibodies directed against the rsv f protein has been effective in reducing the rate of rsv hospitalization among high-risk infants who are premature and have comorbid conditions affecting cardiopulmonary function (see chapter 160). 29, 68 the mainstay of preventing bronchiolitis remains the interruption of the spread of the infectious agent to infants and to the young age group of children who develop bronchiolitis. preventing contact of the child with individuals who have signs of illness may be helpful, but many individuals may have infection that is asymptomatic or mild enough that it is unrecognized. multiple infection-control procedures are recommended for rsv and other agents of bronchiolitis, but among these the most effective, whether in the hospital or home, are good hand hygiene and education of personnel and families (see chapter 160). decision to administer supplemental oxygen. additional risk factors to consider include underlying chronic conditions, poor feeding, clinical respiratory distress, fever, and acidosis, which may shift the oxyhemoglobin association curve such that appreciably lower levels of pao 2 may occur at spo 2 levels greater than 90%. therapeutic agents most frequently used for rsv bronchiolitis include bronchodilators, corticosteroids, and antibiotics. of infants hospitalized with rsv infection in north america, europe, and australia, 75% to 80% were treated with bronchodilating agents, 10% to 40% were treated with corticosteroids, and 15% to 40% were treated with intravenous antibiotics. 56 multiple studies have shown these therapies as inconsistently effective, and none is routinely recommended. 2 a cochrane review of the use of bronchodilators for bronchiolitis concluded that the limited transient improvement using various clinical scoring systems observed was associated with questionable clinical benefit. 57 a subsequent review of the evidence by the american academy of pediatrics reached a similar conclusion and recommendation against the routine use of bronchodilators for infants with initial episodes of wheezing. 2 the addition of anticholinergic medications to the therapeutic regimen has not been shown to improve the course of viral bronchiolitis. these recommendations may not apply to children who have had recurrent wheezing before the episode of viral bronchiolitis. multiple trials have examined the use of nebulized, oral, and parenteral corticosteroid medications among children with bronchiolitis. most of these trials have not included specific viral identification and are heterogeneous in design and in the populations included. reviews that analyzed the randomized and controlled trials concluded that the evidence was insufficient to recommend routine use of these medications for bronchiolitis. [58] [59] [60] a subsequent large, placebo-controlled trial of oral dexamethasone therapy was conducted in 20 emergency departments over three rsv seasons among 608 children 2 to 12 months old with their first episode of wheezing. administration of a single oral dose of 1 mg/kg of dexamethasone had no effect on the subsequent rate of hospitalization or the clinical assessment score, even among children with a family history of asthma. 61 review of this and the previous studies resulted in the current recommendation that corticosteroid medications should not be used routinely in the management of bronchiolitis. 2 nebulized hypertonic saline (most commonly 3 ml of 3% saline combined with a bronchodilator delivered by jet nebulization three the complete reference list is available online at expert consult. acute bronchiolitis diagnosis and management of bronchiolitis recent trends in severe respiratory syncytial virus (rsv) among us infants respiratory syncytial virus−associated hospitalizations among infants and young children in the united states hospitalizations associated with influenza and respiratory syncytial virus in the united states proliferative mural bronchiolitis acute bronchiolitis in children sole pathogen in acute bronchiolitis: is there a role for other organisms apart from respiratory syncytial virus? infection with multiple viruses is not associated with increased disease severity in children with bronchiolitis parainfluenza virus infections of young children: estimates of the populationbased burden of hospitalization comparison of risk factors for human metapneumovirus and respiratory syncytial virus disease severity in young children prevalence and clinical symptoms of human metapneumovirus infection in hospitalized patients comparison of human metapneumovirus, respiratory syncytial virus and influenza a virus lower respiratory tract infections in hospitalized young children the underrecognized burden of influenza in young children prospective multicenter study of the viral etiology of bronchiolitis in the emergency department human coronavirus in young children hospitalized for acute respiratory illness and asymptomatic controls human bocavirus commonly involved in multiple viral airway infections broad respiratory virus detection in infants hospitalized for bronchiolitis by use of a multiplex rt-pcr dna microarray system clinical and epidemiologic characteristics of human bocavirus in danish infants: results from a prospective birth cohort study human bocavirus infection in children with respiratory tract disease human rhinoviruses: the cold wars resume human rhinoviruses in severe respiratory disease in very low birth weight infants dual infection of infants by human metapneumovirus and human respiratory syncytial virus is strongly associated with severe bronchiolitis human metapneumovirus and severity of respiratory syncytial virus disease the impact of dual viral infection in infants admitted to a pediatric intensive care unit associated with severe bronchiolitis bronchiolitis: clinical characteristics associated with hospitalization and length of stay bronchiolitisassociated mortality and estimates of respiratory syncytial virus-associated deaths among us children, 1979-1997 selected populations at increased risk from respiratory syncytial virus infection respiratory syncytial virus bronchiolitis in us emergency departments, 1992 to 2000: epidemiology and practice variation environmental and demographic risk factors for respiratory syncytial virus lower respiratory tract disease the burden of respiratory syncytial virus infection among healthy children risk factors in children hospitalized with rsv bronchiolitis versus non-rsv bronchiolitis innate immune dysfunction is associated with enhanced disease severity in infants with severe respiratory syncytial virus bronchiolitis lower respiratory tract infections among american indian and alaska native children and the general population of u.s. children effect of oxygen supplementation on length of stay for infants hospitalized with acute viral bronchiolitis prospective multicenter study of bronchiolitis: predicting safe discharges from the emergency department predicting deterioration in previously healthy infants hospitalized with respiratory syncytial virus infection duration of illness in ambulatory children diagnosed with bronchiolitis respiratory syncytial virus-related apnea in infants: demographics and outcome risk factors for respiratory syncytial virus associated apnoea identifying hospitalized infants who have bronchiolitis and are at high risk for apnea benefits of thickened feeds in previously healthy infants with respiratory syncytial virus bronchiolitis aspiration: a factor in rapidly deteriorating bronchiolitis in previously healthy infants? risks for bacteremia and urinary tract infections in young febrile children with bronchiolitis risk of serious bacterial infection in young febrile infants with respiratory syncytial virus infections a prospective study of the risk for serious bacterial infections in hospitalized febrile infants with or without bronchiolitis office-based treatment and outcomes for febrile infants with clinically diagnosed bronchiolitis causal links between rsv infection and asthma: no clear answers to an old question respiratory syncytial virus bronchiolitis and the pathogenesis of childhood asthma respiratory syncytial virus in early life and risk of wheeze and allergy by age 13 years diagnostic assays for respiratory syncytial virus disease comparison of fast-track diagnostics respiratory pathogens multiplex real-time rt-pcr assay with in-house singleplex assays for comprehensive detection of human respiratory viruses when an infant wheezes: clues to the differential the treatment of bronchiolitis international variation in the management of infants hospitalized with respiratory syncytial virus bronchodilators for bronchiolitis agency for healthcare research and quality. management of bronchiolitis in infants and children. agency for healthcare research and quality systemic corticosteroids in infant bronchiolitis: a meta-analysis glucocorticoids for acute viral bronchiolitis in infants and young children a multicenter, randomized, controlled trial of dexamethasone for bronchiolitis nebulized hypertonic saline solution for acute bronchiolitis in infants use of continuous positive airway pressure (cpap) in acute viral bronchiolitis: a systematic review randomised controlled trial of nasal continuous positive airways pressure (cpap) in bronchiolitis nasal continuous positive airway pressure with heliox versus air oxygen in infants with acute bronchiolitis: a crossover study high flow nasal cannulae therapy in infants with bronchiolitis humidified high-flow nasal cannula: is it the new and improved cpap? phase 3 trial of motavizumab (medi-524), an enhanced potency respiratory syncytial virus (rsv) specific monoclonal antibody (mab) for the prevention of serious rsv disease in high risk infants key: cord-253761-wjm8ju3v authors: haidopoulou, katerina; goutaki, myrofora; damianidou, lambrini; eboriadou, maria; antoniadis, antonis; papa, anna title: human bocavirus infections in hospitalized greek children date: 2010-03-09 journal: arch med sci doi: 10.5114/aoms.2010.13515 sha: doc_id: 253761 cord_uid: wjm8ju3v introduction: the epidemiology of human bocavirus (hbov) infections has not been described in greece, a south-eastern european country. to define the epidemiological profile and the clinical characteristics associated with hbov infection in a population of children hospitalized with respiratory tract infection. material and methods: during a one-year period throat swab samples were collected from 370 previously healthy children, aged 14 days to 13 years, admitted to two different paediatric wards because of respiratory tract infection. samples were tested for hbov by pcr amplifying a part of the ns1 gene. results: human bocavirus was detected in 12 children (3.2%). four of the 12 cases were co-infections, 3 of them with influenza a and 1 with coronavirus oc43. cases were observed only during the cold months. the mean age of children was 1.8 years (range 2 months to 4 years). the most common symptoms were fever, cough and various degrees of respiratory distress. all children were clinically diagnosed as having lower respiratory tract infections, mainly pneumonia and acute laryngotracheobronchitis, and recovered uneventfully. conclusions: hbov infections occur in greece mostly among very young children. they accounted for 3.2% of children hospitalized with acute respiratory disease. cases were observed only in late autumn to early spring. human bocavirus (hbov) (genus bocavirus, family parvoviridae) has been recently identified in children with respiratory tract infection (rti), first in sweden [1] , and subsequently in different parts of the world [2] [3] [4] [5] [6] [7] [8] [9] [10] . however, most studies so far have only retrospectively studied virus prevalence and only a few have addressed whether hbov infection is associated with respiratory disease symptoms. the aim of the present study was to define the epidemiological profile and the clinical characteristics associated with hbov in hospitalized children with respiratory tract infection (rti) in greece. during a one-year period (october 2006 to september 2007) throat swab samples were collected from 370 previously healthy children, aged 14 days to 13 years (mean age ± sd 17 ±13 months) admitted to two dif-ferent large hospitals of thessaloniki, northern greece. samples were taken on the first day of admission and kept at -70°c until use. all children were admitted because of acute infection of the respiratory tract. demographic data and clinical diagnosis including acute infection of the upper respiratory tract, croup, bronchitis, bronchiolitis and pneumonia were obtained from a computer-generated discharge diagnosis based database. the case notes of children were reviewed using a standardised clinical data extraction form. the following data were recorded: sex, age, initial presenting symptoms, signs, discharge diagnosis and routine laboratory examinations upon admission and during hospitalization (complete blood count, c-reactive protein, chest x-ray). extracted dna was subjected to polymerase chain reaction (pcr) which targets the ns1 gene of the hbov genome [8] . all pcr products were sequenced and nucleotide sequences were compared with respective hbov sequences retrieved from genbank. in addition, all samples were tested by molecular methods for mycoplasma pneumoniae, respiratory syncytial virus, coronaviruses, influenza viruses, human metapneumovirus and adenoviruses. hbov dna was detected in samples of 12 children (3.3%), 6 of them males. sequencing and phylogenetic analysis revealed that hbov sequences of 11 cases were identical to each other and to the swedish strain st2 (nc007455), differing by 1 nucleotide from the 12 th case (gr186), which was identical to strain chsd4 (dq471814) from usa. all cases had clinical evidence of lower rti (fever, tachypnoea, hypoxia, retractions, and abnormal auscultation findings). four of the 12 cases were coinfections, 3 of them with influenza a virus and 1 with coronavirus oc43. concerning the 8 patients in whom only hbov was detected, they were 3 males and 5 girls, aged 2-33 months (mean age ± sd: 17 ±9 months), hospitalized with the clinical diagnosis of 2 each: laryngotracheobronchitis, bronchiolitis, pneumonia, and asthma exacerbation. common symptoms of viral respiratory tract infection such as fever, cough, rhinorrhoea and pharyngitis were found in the majority of our patients (87.5-100%). six patients (75%) presented with various degrees of respiratory distress. those patients had tachypnoea (a respiratory rate of 45-90 breaths/min) and low haemoglobin oxygen saturation levels (sao 2 ) in the range 90-94%. these children received oxygen supplementation until 1 day before discharge. wheezing was the most common clinical finding (5 patients) while another 1 child presented with stridor. hoarseness was noticed in 2 children. difficulty in feeding was also a common complaint, reported by 5 patients. other clinical findings included diarrhoea (2 patients, 25%), a symptom previously described in hbov infections [11, 12] , and otitis media (1 patient, 12.5%). the major clinical and laboratory findings in the 8 patients in whom hbov was the sole pathogen detected are presented in table i . chest x-rays were available for all 8 children. the most common finding, present in 6 patients, was bilateral interstitial infiltrates. consolidation was detected in the other 2 children. clinical expression was not different in children with hbov co-infection. the child with coronavirus oc43 co-infection also presented with conjunctivitis, while respiratory distress was the main concern in a child with influenza-a virus co-infection. all children recovered uneventfully. the median hospitalization time was 5 days (range 4-9 days). for 1 patient a second throat swab sample, taken 20 days after the first one, was found negative for hbov dna. prevalence of hbov in our study (3.3%) was found to be relatively low compared to published data from other parts of the world [2, 4, 6, 9, 10, [13] [14] [15] [16] . however, prevalence rates between 1.5 and 19% have been observed and it is possible that differences of study populations and sampling techniques account for the encountered discrepancies [17] . although the number of studies since the first description of hbov is increasing, many of these studies are retrospective on stored samples, or only on samples negative for other pathogens, or on samples collected during a few months and thus suffering from the bias that sampling may be guided from the occurrence of rsv or influenza epidemics. in the present prospective study samples were collected during 1 year and were tested regardless of positivity for another pathogen. in previous whole year studies it was found that the vast majority of hbov positive samples were collected in cold months [14, 18, 19] . we also found that positive cases were observed only during the cold months. the first hbov case was observed in mid december 2006, and the last at the end of april 2007. greece is a mediterranean country in the balkan peninsula, with hot, dry summers and cold, damp winters. nevertheless, the mean temperature of winter 2006-2007 in greece (and worldwide) was above average (warmest winter on record), and the precipitation drier than normal (national observatory of athens), conditions which might have affected the prevalence of hbov. previous studies have implicated hbov as a cause of rti, most commonly in children < 2 years old, a finding similar to our study, where 10/12 children were younger than 2 years, the youngest one being a 2-month boy with acute bronchiolitis [13, 14, [20] [21] [22] . however, frequency of hbov infection was found to be relatively low in children younger than 6 months, and the possibility of passive protection due to maternal antibodies has been questioned. these findings together with the high prevalence rates in young children led to the hypothesis that hbov may be an endemic virus with high attack rates in those susceptible; one would then expect that the majority of the population would be infected during childhood. in support of this hypothesis are the high seroprevalence rates against hbov reported in the adult population [23] . the mere presence of hbov in the airway may not be able to define its role as a pathogen; therefore it is anticipated that serology would be a useful diagnostic addition to the study of hbov infection. four of the 12 cases were co-infections, 3 of them with influenza a virus and 1 with coronavirus oc43. similar findings were reported in other studies, where co-infection was found in one third of patients [5, 24] , while other investigators have reported even higher rates [8, 15, 25] . it has been hypothesized that detection of hbov asymptomatic presence may be enhanced by the symptoms caused by another virus or that hbov may aggravate the clinical course of symptoms due to other viral infections, so that it is frequently detected in hospitalized children [26] . co-infection is frequently described with many respiratory viruses and the question whether this may result in more serious clinical outcomes has not been clarified [27] . the association of hbov with acute wheezing, a symptom found in 62.5% of our patients, has been well described in recently published studies [5, 14, 18, 28] . another 2 of our patients presented with laryngotracheobronchitis, a finding which is in accordance with the study of rihkanen et al. [29] . only 1 of our patients returned for follow-up and a second throat swab sample, taken 20 days after the first one, was found negative for hbov dna. our findings suggest that hbov may have similar clinical features to those of other respiratory viruses such as rsv. one may argue that the fact that hbov is prevalent in samples from patients with respiratory tract infection does not guarantee a causative role for the symptoms, especially when -as in this case -it is frequently detected in combination with other respiratory viruses of known pathogenic potential. on the other hand, hbov was rarely detected in asymptomatic children [16, 22] . in conclusion, in the present study we have provided a first insight into the epidemiology and clinical aspects of hbov infections in hospitalized children in greece. our findings suggest a possible association of hbov with lower rti, manifested mainly by fever, cough and wheezing, most often in infants, and a seasonal pattern with hbov cases occurring in cold months. cloning of a human parvovirus by molecular screening of respiratory tract samples detection of human bocavirus in canadian children in a 1-year study bocavirus infection in hospitalized children detection of human bocavirus in hospitalised children human bocavirus in french children detection of human bocavirus in ill and healthy spanish children: a 2-year study the first detection of human bocavirus 2 infections in china evidence of human coronavirus hku1 and human bocavirus in australian children human bocavirus in very young infants hospitalized with acute respiratory infection in northeast brazil the incidence of human bocavirus infection among children admitted to hospital in singapore human bocavirus: prevalence and clinical spectrum at a children's hospital human bocavirus, a respiratory and enteric virus viruses in community-acquired pneumonia in children aged less than 3 years old: high rate of viral coinfection human bocavirus infection in young children in the united states: molecular epidemiological profile and clinical characteristics of a newly emerging respiratory virus isolation of human bocavirus from swiss infants with respiratory infections frequent detection of bocavirus dna in german children with respiratory tract infections human bocavirus human bocavirus and acute wheezing in children high incidence of human bocavirus infection in children in spain human bocavirus infection in a neonatal intensive care unit human bocavirus: a novel parvovirus epidemiologically associated with pneumonia requiring hospitalization in thailand human bocavirus in italian patients with respiratory diseases seroepidemiology of human bocavirus in hokkaido prefecture the association of newly identified respiratory viruses with lower respiratory tract infections in korean children frequent detection of viral coinfection in children hospitalized with acute respiratory tract infection using a real-time polymerase chain reaction human bocavirus: passenger or pathogen in acute respiratory tract infections? coronaviruses oc43 and 229e lower respiratory tract co-infections: a clinical report of two cases human bocavirus: a cause of severe asthma exacerbation in children respiratory viruses in laryngeal croup of young children we would like to thank sofia komi for technical assistance. key: cord-010233-772e35kx authors: monto, arnold s.; fendrick, a.mark; sarnes, matthew w. title: respiratory illness caused by picornavirus infection: a review of clinical outcomes date: 2002-01-03 journal: clin ther doi: 10.1016/s0149-2918(01)80133-8 sha: doc_id: 10233 cord_uid: 772e35kx background: respiratory infections result from invasion of the respiratory tract, mainly by viruses, and are the leading cause of acute morbidity in individuals of all ages worldwide. during peak season, picornaviruses cause 82% of all episodes of acute nasopharyngitis (the common cold), the most frequent manifestation of acute respiratory infection, and produce more restriction of activity and physician consultations annually than any other viral or bacterial source of respiratory illness. objective: this article reviews the clinical impact and outcomes of picornavirus-induced respiratory infections in specific populations at risk for complications. it also discusses the potential economic impact of the morbidity associated with picornavirus-induced respiratory infection. methods: relevant literature was identified through searches of medline, ovid, international pharmaceutical abstracts, and lexis-nexis. the search terms used were picornavirus, rhinovirus, enterovirus, viral respiratory infection, upper respiratory infection, disease burden, economic, cost, complications, asthma, copd, immunocompromised, elderly, otitis media, and sinusitis. additional publications were identified from the reference lists of the retrieved articles. conclusions: based on the clinical literature, picornavirus infections are associated with severe morbidity as well as considerable economic and societal costs. future research should focus on identifying patterns of illness and the costs associated with management of these infections. new treatments should be assessed not only in terms of their ability to produce the desired clinical outcome, but also in terms of their ability to reduce the burden of disease, decrease health care costs, and improve productivity. respiratory infections are recognized as the leading cause of acute morbidity in individuals of all ages worldwide. in developing countries, the morbidity associated with respiratory infections may be at least as severe as that in industrialized countries, and these infections are the leading cause of death in children under 5 years of age.',* the health interview survey quantifies illnesses that result in disability and/or physician consultation in the us population. based on this survey, the annual incidence of acute respiratory infections in the united states in 1996 was 79%, exceeding the rates of digestive conditions, injuries, and infective/parasitic conditions combined. these infections may result from invasion of the respiratory tract by bacteria, viruses, or, in rare cases, other infectious agents; however, viruses are the most frequently identified pathogens. the viral pathogens primarily associated with acute respiratory infections include picomaviruses, coronaviruses, adenoviruses, paraintluenza viruses, influenza viruses, and respiratory syncytial viruses' (figure 1 3) . picornaviruses are a large group of rna viruses. in terms of causing acute respiratory infection in humans, the most important picornaviruses are the rhinoviruses and enteroviruses. picornaviruses contribute significantly to the incidence of acute respiratory infections. in a study conducted during the autumn,5 picornaviruses were found to cause 82% of all episodes of acute nasopharyngitis, which is the most common manifestation of acute respiratory infection. individually, the incidence of acute nasopharyngitis in adults ranges from 2 to 4 cases per year and in children from 6 to 8 cases per year.6,7 although picornavirus-induced nasopharyngitis is often referred to as the common cold, the morbidity associated with these infections should not be trivialized. in fact, because rhinovirus-induced illnesses are so common, they produce more restriction of activity and physician consultations annually than respiratory illnesses caused by other viruses or bacteria.s this article reviews the clinical impact and outcomes associated with picomavirusinduced respiratory infection in specific populations at risk for complications secondary to these infections. it also discusses the potential economic impact of the morbidity associated with picornavirusinduced respiratory infections. relevant literature was identified through searches of medline, ovid, international pharmaceutical abstracts, and lexis-nexis. the search terms used were picnrnuvirus, rhinovirus, enterovirus, vim1 respiruto~ infection, upper respiratory infection, disease burden, economic, cost, complicutions, asthma, copd, immunocompromised, elderly, otitis media, and sinusitis. additional publications were identified from the reference lists of the retrieved articles. rhinovirus infections are associated with more pronounced clinical manifestations than respiratory infections of other viral etiologies. as many as 70% to 88% of human rhinovirus infections result in symptomatic respiratory episodes characterized by rhinorrhea, nasal obstruction, cough, and hoarseness." the median duration of illness in young adults is 7 days; however, symptoms may last up to 2 weeks in one quarter of cases, and the duration of illness may be even longer in children and the elderly.9 there is a peak in the incidence of rhinovirus infection in early fall and again in mid-to late springjo ( figure 24 ). younger children appear to be more susceptible to viral respiratory infections, as demonstrated by the fact that preschool children can have 5 to 9 respiratory infections per year. i ' in fact, during an outbreak of a new virus strain, over three fourths of the children in a nursery school were infected.12 also, secondary attack rates in family members have been observed to range from 25% to 70%, depending on the immune status of the exposed individual.r2 the high incidence of viral respiratory infections is most likely due to the large number of rhinovirus types and the fact that immunity is type specific and of short duration. for these reasons among others, the role of rhinoviruses has overshadowed that of enteroviruses, which also contribute to the morbidity of viral respiratory infections. it is estimated that enteroviruses infect between 10 and 15 million people annually. episodes of enterovirusinduced respiratory infection are seen throughout the year; however, the most prominent respiratory syndrome caused by these pathogens occurs in children during the summer months.'" these enteroviral infections typically result in a nonspecific febrile illness complicated by respiratory symptoms. i4 although viral respiratory infections due to picornaviruses appear to be selflimiting in most healthy adults, they are increasingly implicated in acute infectious exacerbations of illnesses in high-risk populations. technological advances such as polymerase chain-reaction testing have made virus identification more sensitive and readily available. this, in turn, has made it possible to identify specific patient populations (eg, the elderly, infants, and immunocompromised persons), who are particularly susceptible to picornavirus infection and in whom infectious episodes typically significantly increase the use of health care resources. 15 in addition, these advances in technology have confirmed the results of earlier studies, further demonstrating the significance of rhinoviruses in causing or predisposing patients to otitis media and sinusitis and exacerbating other chronic respiratory diseases such as asthma, cystic fibrosis, and chronic obstructive pulmonary disease (copd). asthma is estimated to affect >17 million people (6% of the population) in the united states, resulting in over i00 million days of restricted activity and 470,000 hospitalizations annually. 16.17 in 1990, the estimated cost of treating asthma was $6.2 billion; 43% of those costs were associated with emergency department visits, hospitalizations, and death. is respiratory infections caused by picornaviruses are believed to be a major contributor to asthma exacerbations, particularly in children. asthmatic children appear to experience a significantly greater number of viral respiratory infections compared with nonasthmatic children. 19 up to 45% of acute asthma exacerbations in children are thought to be related to viral respiratory infections20 the most common cause of these infections are the rhinoviruses, which have been recovered from the respiratory tract at the onset of an asthma attack, suggesting that the virus may have been an initiating factor in the attack.*' several studies have identified rhinovirus as the pathogen most commonly associated with asthma in those >2 years of age.r5 rakes et a122 conducted a crosssectional study examining the prevalence of respiratory viruses in children presenting to the emergency department because of wheezing episodes. they found that respiratory viruses were present in 82% of children <2 years of age and in 83% of children between 2 and 16 years of age. rhinovirus was the predominant pathogen isolated in children >2 years of age (7 i%), indicating that the majority of wheezing episodes in this age group may be associated with rhinoviruses. the prevalence of rhinoviruses in asthmatic children was further investigated by johnston et a1.2" in a 13-month trial, these authors evaluated 108 children between the ages of 9 and 11 years with the respiratory symptoms of wheezing and/or cough and a decrease in peak expiratory flow rate (pefr). respiratory viruses, two thirds of which were rhinoviruses, were detected in 77% of patients. viral respiratory infections were implicated in 85% of episodes with upper respiratory symptoms and 80% of episodes with reductions in pefr. the median reduction in pefr was 81 l/min, and the median duration of decline in pefr was 14 days. based on their results, the authors reported that 80% to 85% of asthma exacerbations in schoolaged children appear to be associated with viral respiratory infections, primarily with rhinoviruses. in only 10% to 21% of adult asthmatic patients.*(' however, beasley et a124 found the prevalence of viral respiratory infections to be 36% in adults with severe asthma and 10% in those with mild exacerbation of asthma. nicholson et a125 conducted a longitudinal trial in asthmatic adults between the ages of 19 and 46 years, evaluating 3 15 episodes of respiratory illness over 2 years. the average duration of asthma in the patient population was -20 years. thirty-eight percent of the patients had previously been hospitalized for asthma. the investigators found that 80% of subjective asthma exacerbations occurred with symptomatic colds and 89% of symptomatic colds were associated with symptoms of asthma. these findings suggest that acute respiratory infections may be as commonly linked to exacerbations of asthma in adults as they are in children. in this study, 84 clinical episodes of respiratory illness were associated with objective evidence of an asthma exacerbation (decrease in pefr 250 l/min). sixty-three percent of laboratory-confirmed acute respiratory infections were found to be caused by rhinoviruses. of the 84 clinical episodes of respiratory illness associated with an objective asthma exacerbation, patients consulted a general practitioner in 49% of episodes, required oral steroids in 38%, required a nebulizer in 2 1 %, and were prescribed antibiotics in 33%. in addition to the clinical impact of rhinoviruses in asthma, their impact on medical resource use has also been demonstrated. teichtahl et al*" compared the incidence of respiratory infection in 79 patients admitted to the respiratory medical unit for acute asthma with that in 54 control subjects admitted to the surgical unit for elective surgery. twenty-nine (37%) of the patients who were admitted for asthma exacerbation had evidence of a respiratory infection, compared with 5 (9%) of the control group (p < 0.001). of the 23 asthma patients with confirmed evidence of a viral infection, 9 (39%) had rhinovirus infections. significant mortality resulting from viral respiratory infections is routinely observed in the elderly. although mortality resulting specifically from rhinoviral respiratory infections has not been demonstrated, considerable morbidity is associated with these infections. in a study by nicholson et a1, 26 533 adults aged between 60 and 90 years were monitored for episodes of viral upper respiratory tract infections. the annual incidence of viral respiratory infections was found to be 1.2 per patient. a total of 23 1 pathogens were identified in 2 11 (42%) of the 497 episodes for which specimens were available. of the pathogens identified, 121 (52%) were rhinoviruses. of the 96 (45%) patients in whom rhinovirus was the sole pathogen isolated, 60 (63%) patients had lower respiratory tract involvement. the median duration of illness in these patients was 16 days. of the 60 patients with lower respiratory tract involvement, 25 (42%) had to restrict their normal activities (eg, cleaning, grocery shopping), and 16 (27%) were bedridden. a total of 4 i (68%) patients sought medical attention, and antibiotics were prescribed for 76%.27 altogether, 3 patients were hospitalized, 1 of whom died from exacerbation of copd by rhinovirus infection. the authors reported that the overall burden of rhinovirus infection was greater than that of the viruses typically associated with significant morbidity and mortality, such as influenza. 26 a second study assessing the impact of rhinovirus infection on the elderly focused on an outbreak of respiratory illness among patients in a long-term care facility.'x specimens from the throat and nasopharynx of 67 patients were obtained and cultured. of the 67 cultures, 33 (49%) were positive for rhinovirus. each of the patients with rhinovirus-positive cultures had upper respiratory tract symptoms, 23 (70%) had systemic symptoms, 22 (67%) had gastrointestinal symptoms, and 1 i (33%) had lower respiratory tract symptoms. more severe disease progression was found in 17 (52%) rhinovirus-infected patients with concomitant copd. five of 17 (29%) patients with copd required pharmacologic bronchodilation, 2 (12%) had to be transferred out of the facility because of declining respiratory function. and 1 (6%) died of respiratory failure. these findings indicate that outbreaks of rhinovirus infections in nursing homes should be considered potentially serious. a study from the 1960s indicated that 2.0% of community respiratory diseases were complicated by otitis media and 0.5% by sinusitis.7 although such percentages may seem insignificant, the high frequency of respiratory infections means that large numbers of patients are affected. a conservative estimate places the inci-dence of viral respiratory infections in the united states at 0.7 infection per person per year." based on these data, an estimated 196 million people in the united states experience a viral respiratory infection each year, with -4 million of these infections complicated by otitis media and almost 1 million complicated by sinusitis. new technologies that permit more accurate identification of viral pathogens will probably reveal that the frequency of virally induced infections is even higher and that viral pathogens are a precursor in more cases of otitis media and sinusitis than originally estimated; however, further research is necessary. it has been estimated that >90% of patients with otitis media also have symptoms of upper respiratory tract infections that are probably the result of a primary viral infection.29 this correlation is supported by the fact that 40% to 50% of patients with otitis media have detectable virus in the nasopharynx, and 17% have detectable virus in the middle ear.29 viruses can also complicate the course of recovery for patients with otitis media. in 2 recent studies,"o,"' viruses in the middle ear have been implicated in the apparent failure of antibiotic treatment by complicating the response to treatment. in a study in 71 patients with combined viral and bacterial otitis media,"' the presence of rhinovirus was associated with a higher failure rate of antibiotic therapy than was respiratory syncytial virus, parainfluenza virus, or influenza virus. rhinovirus has been identified as a potential pathogen in -40% of cases of community-acquired sinusitis.32 although there is evidence that these viruses contribute to the development of sinusitis, their exact role remains undefined. in a study by turner et al,"" 34 healthy adults were experimentally infected with rhinovirus. on magnetic resonance imaging, changes were observed in the paranasal sinuses of 33% of subjects. the relationship between viral infections and sinusitis was further investigated in a study evaluating naturally acquired acute nasopharyngitis.'4 in this study, sinus abnormalities were detected by computed tomography in over 85% (29) of adult patients. after 2 weeks of observation, spontaneous resolution of these abnormalities occurred in almost 80% (27) in some patient populations. is although the role of picornaviruses in these more serious diseases of the lower respiratory tract cannot be determined definitively without more extensive sampling of the lower respiratory tract, recent studies have pointed to the importance of picornavirus infection in these disease processes in particular patient populations.'" infants with bronchopulmonary dysplasia are at a significantly increased risk for acute episodes of severe viral respiratory infection. in a study by daily, 35 27 of 41 (66%) preterm infants with bronchopulmonary dysplasia required rehospitalization, the most common cause being upper respiratory tract infection. whereas some studiesi0,i5 have indicated that respiratory syncytial virus is the pathogen most commonly isolated in these patients, oth-er@.s7 have demonstrated that rhinovirus makes a substantial contribution. in fact, because none of these studies used assays suitable for identifying rhinoviruses, the role of these agents may have been underestimated. chidekel et alx7 studied 40 patients (44 cases) with a history of bronchopulmonary dysplasia for an average of 16 months and identified 8 (18%) cases of severe lower respiratory tract infection that were associated with rhinoviruses. of the 7 patients in these 8 cases, 5 (7 1%) required hospitalization for a mean duration of 11 days, and 4 (57%) were admitted to the intensive care unit. patients with rhinoviral respiratory infections required additional long-term medical therapy, suggesting that rhinoviruses can have lasting clinical implications. patients with cystic fibrosis are also at increased risk for complications of viral respiratory infections. in fact, 18% to 38% of pulmonary exacerbations are preceded by viral respiratory infections. collinson et ais recorded 147 episodes of acute nasopharyngitis over 17 months in 37 children with cystic fibrosis aged between 2 months and 18 years, translating into an annual incidence of 2.7%. cultures were available for 1 19 of the 147 episodes, and the causative pathogen was identified as rhinovirus in 2 1 (18%) episodes. these viral respiratory infections were associated with acute decreases in pulmonary function, increased rates of disease progression, and a predisposition to bacterial pulmonary infection. lower respiratory tract infections are the most common complication of viral respiratory infection in immunocompromised patients.s9 in one study,"o the com-monly identified causes of viral respiratory infection in immunocompromised patients included cytomegalovirus, herpes simplex virus, and varicella-zoster virus; however, viruses such as the picornaviruses were also implicated. in this and other studies, it is not certain that the techniques used were capable of detecting rhinovirus and whether these viruses were accurately represented in the study findings. immunocompromised patients are at risk for developing serious or life-threatening disease as a result of a viral infection of the respiratory tract. in the study by rabella et a14" both rhinoviruses and enteroviruses were identified as viral pathogens in 785 immunosuppressed patients (ie, patients infected with hiv, bone marrow transplant recipients, organ transplant recipients, patients with hematologic malignancies) with a suspected respiratory infection between january 1991 and december 1995. picornavirus infections were associated with respiratory failure in 2 patients, 1 of them requiring mechanical ventilation and the other having a clinical course resulting in pneumonia. in a study conducted at the university of texas m.d. anderson cancer center (mdacc), " 130 cases of picornavirus infection were identified in adult patients with leukemia and bone marrow transplant recipients. more than 90% of the picornavirus infections tested were found to be caused by rhinoviruses. in several patients, the infection progressed to pneumonia that appeared to be bacterial or fungal in origin. however, some patients had a clinical course consistent with viral pneumonia and died of unexplained interstitial pneumonia. in a 5-year retrospective follow-up study of 23 blood and bone marrow transplant recipients who had symptoms of up-per respiratory tract infection,42 rhinovirus was identified as the causative pathogen in all patients. eight (35%) patients developed progressive pneumonia that resulted in death. autopsies on 5 of 6 patients demonstrated interstitial pneumonitis and/or changes of acute respiratory distress syndrome. the deaths were attributed to progressive viral pneumonia on the basis of histologic features and ante mortem isolation of rhinovirus from lower respiratory tract sites. in a prospective study conducted at mdacc4" an evaluation of adult bone marrow transplant recipients hospitalized for an acute viral respiratory infection demonstrated that 18% of the 2 17 identified infections were due to picornaviruses. of 12 patients in whom rhinovirus had been implicated as the causative pathogen, 7 (58%) developed complicating pneumonia, and 3 (25%) died as a result. findings from autopsies performed on 2 of the patients were consistent with progressive viral pneumonia. a third trial at mdacc investigating acute respiratory illnesses in hospitalized patients with leukemia found that 18% of viral infections were caused by picornaviruses. 41 together, the results of these studies demonstrate that immunocompromised hosts are at increased risk from serious picornavirus infections, which have the potential to result in death. although the economic impact of picornavirus infections is most pronounced in the at-risk populations described, these infections have a significant economic impact on the general population as well. picornavirus infection is manifested primarily as acute nasopharyngitis. according to estimates from the national center for health statistics3 >62 million cases of the common cold required medical attention and resulted in 148 million days of restricted activity in 1996. even more significant from the economic and productivity perspectives, acute nasopharyngitis caused -20 million lost workdays in adults aged 218 years and 21 million lost school days in children aged <18 years. also important from an employer's perspective is the fact that employees miss days of work not only when they themselves are ill but when they stay home to care for sick children. productivity is also diminished when employees come to work with a picornavirus-induced respiratory infection. the compromised quality of life associated with these infections in turn has a negative effect on productivity. furthermore, many of the medications currently used for the palliative treatment of respiratory symptoms cause drowsiness, which may further diminish efficiency and productivity in the workplace. finally, because respiratory picornavirus infections are easily transmitted to persons in proximity to infected individuals, other employees are likely to become infected, resulting in additional losses in productivity. because factors affecting productivity and quality of life are increasingly important in today's health care environment, further analyses are needed to better quantify the impact of respiratory picornavirus infections on the overall health care population. to date, the direct use of health care resources associated with viral respiratory illness in the general population has not been investigated comprehensively. the current literature focuses primarily on atrisk populations. these data indicate that use of emergency department and hospital services for exacerbations or compli-cations caused by picomavirus infections is the major factor in the economic impact of respiratory infections within such atrisk subpopulations as asthma patients, the elderly, high-risk patients with diseases of the lower respiratory tract, cystic fibrosis patients, and immunocompromised patients. patients with sinusitis or otitis media are an exception, with physician office visits and antibiotic use accounting for the majority of the economic impact. the economic impact of picornavirus infections is illustrated by findings that asthma exacerbations are linked to rhinovirus infections in 57% of children and 15% of adults hospitalized for asthma exacerbations.2",2" if these statistics are applied to the average 463,500 asthmarelated hospitalizations per year (34.6% of which involve children),ls rhinovirus infections are responsible for an estimated 136,800 hospitalizations annually. based on these figures and an average cost of $3000 per hospitalization for an asthma exacerbation, the cost of hospitalizations due to rhinovirus-induced asthma exacerbations would be ~$410 million annually. a prospective economic analysis of hospitalizations due to respiratory infections in the asthmatic population remains to be conducted. other markers of the economic impact of rhinoviral respiratory infections in the asthmatic population are not as well defined as the costs associated with hospitalization. factors such as the use of other health care resources (medications and physician office visits), productivity, quality of life, and work and school absenteeism have not been measured to a significant extent, and further evaluation of these variables will be crucial to establishing the true economic burden of viral respiratory infections in the asthmatic population. patients with picornavirus infections who are at high risk for complications because of age (infants, the elderly), lower respiratory tract involvement (patients with copd), or immune status (cystic fibrosis patients, immunocompromised patients) constitute additional populations in which hospital and emergency department use are the greatest contributors to overall costs. again, the literature described in this review illustrates a definite link between picornavirus infections and increased health care use in these populations; however, the overall patterns of resource use and costs of illness have not been defined. in addition to the significant morbidity associated with picornavirus infections in the clinical literature, these infections also have substantial economic and societal implications. because the magnitude of these implications is not yet well defined, future research should focus on identifying the patterns of illness and costs associated with the management of these infections. the ability to determine the costs associated with viral respiratory infections will be particularly important when evaluating new treatments, which should be viewed not only in terms of their ability to produce the desired clinical outcome, but also in terms of their ability to reduce the burden of disease, decrease health care costs, and improve productivity. acknowledgment support for this article was provided by viropharma inc, exton, pennsylvania. the clinical impact of human respiratory virus infections acute respiratory infection in children of developing countries: challenge of the 1990s current estimates from the national health interview survey, 1996. national center for health statistics rhinovirus infections in tecumseh, michigan: frequency of illness and number of serotypes frequency and natural history of rhinovirus infections in adults during autumn rhinovirus infections in an industrial population. i. the occurrence of illness illness in the home: study of 25,000 illnesses in a group of cleveland families viral respiratory infections in the community: epidemiology, agents, and interventions principles and practice oj' infectious diseases the common cold respiratory disease in group day care acute respiratory illness in nursery school children: a longitudinal study of the occurrence of illness and respiratory viruses viral isolation rates during summer from children with acute upper respiratory tract disease and healthy children temporal and geographic patterns of isolates of nonpolio enterovirus in the united states rhinoviruses: important respiratory pathogens surveillance for asthma-united states 1960-1995 centers for disease control and prevention. forecasted state-specific estimates of self-reported asthma prevalence-united states an economic evaluation of asthma in the u.s greater frequency of viral respiratory infections in asthmatic children as compared with their nonasthmatic siblings the incidence of respiratory tract infection in adults requiring hospitalization for asthma viruses as precipitants of asthmatic attacks in children rhinovirus and respiratory syncytial virus in wheezing children requiring emergency care. ige and eosinophil analyses community study of role of viral infections in exacerbations of asthma in l i year old children viral respiratory tract infection and exacerbations of asthma in adult patients respiratory viruses and exacerbations of asthma in adults acute viral infections of upper respiratory tract in elderly people living in the community: comparative, prospective, population based study of disease burden risk factors for lower respiratory complications of rhinovirus infections in elderly people living in the community: prospective cohort study a rhinovirus outbreak among residents of a long-term care facility report of a workshop on respiratory viral infections: epidemiology, diagnosis, treatment, and prevention bacteriologic failure of amoxicillinclavulanate in treatment of acute otitis media caused by nontypeable haemophilus influenzae association of rhinovirus infection with poor bacteriologic outcome of bacterial-viral otitis media. c/in infecr dis hayden fg. detection of rhinovirus in sinus brushings of patients with acute community-acquired sinusitis by reverse transcription-pcr physiologic abnormalities in the paranasal sinuses during experimental rhinovirus colds computed tomographic study of the common cold home oxygen therapy for infants with bronchodysplasia: growth and development rhinovirus infection associated with serious respiratory illness in patients with bronchopulmonary dysplasia effects of upper respiratory tract infections in patients with cystic fibrosis community respiratory virus infections in immunocompromised patients with cancer respiratory viral infections in immunocompetent and immunocompromised persons rhinovirus infections in myelosuppressed adult blood and marrow transplant recipients conventional respiratory viruses recov community respiratory virus infections among hospitalized adult bone marrow transplant recipients key: cord-256788-h4iv8crq authors: sumino, kaharu; tucker, jennifer; shahab, muhammad; jaffee, katy f.; visness, cynthia m.; gern, james e.; bloomberg, gordon r.; holtzman, michael j. title: antiviral ifn-γ responses of monocytes at birth predict respiratory tract illness in the first year of life date: 2012-03-27 journal: j allergy clin immunol doi: 10.1016/j.jaci.2012.02.033 sha: doc_id: 256788 cord_uid: h4iv8crq background: viral respiratory tract infections are the leading cause of acute illness during infancy and are closely linked to chronic inflammatory airway diseases later in life. however, the determinants of susceptibility to acute respiratory tract infections still need to be defined. objective: we investigated whether the individual variation in antiviral response at birth determines the risk for acute respiratory tract illness in the first year of life. methods: we studied 82 children who were enrolled in a birth cohort study of inner-city children with at least 1 parent with allergy or asthma. we cultured cord blood monocytes and assessed ifng and ccl5 mrna production at 24 hours after inoculation with respiratory syncytial virus. we also monitored the frequency of acute respiratory tract illness at 3-month intervals and analyzed nasal lavage samples for respiratory tract viruses at the time of illness during the first year. results: respiratory tract infection was reported for 88% of subjects, and respiratory tract viruses were recovered in 74% of symptomatic children. we observed a wide range of antiviral responses in cord blood monocytes across the population. furthermore, a decrease in production of ifng (but not ccl5) mrna in response to respiratory syncytial virus infection of monocytes was associated with a significant increase in the frequency of upper respiratory tract infections (r = −0.42, p < .001) and the prevalence of ear and sinus infections, pneumonias, and respiratory-related hospitalizations. conclusion: individual variations in the innate immune response to respiratory tract viruses are detectable even at birth, and these differences predict the susceptibility to acute respiratory tract illness during the first year of life. viral respiratory tract infections are a common cause of early childhood illness. most of these infections are short-lived and selflimited, but some can be severe enough to require hospitalization. indeed, viral respiratory tract infections are associated with 20% of all mortality in children less than 5 years of age. 1 in addition to the morbidity of the acute infection, viral respiratory tract infections with wheezing are strong indicators of subsequent asthma. 2, 3 therefore predicting those infants at risk for respiratory tract infections is an important first step in preventing acute and chronic respiratory disease. previous studies have identified a variety of potential risk factors for viral lower respiratory tract infections during the first year of life. these factors include day care attendance, number of siblings, small lung size, exposure to tobacco smoke, low birth weight, and premature birth. [3] [4] [5] [6] infections caused by respiratory syncytial virus (rsv) are particularly implicated in acute illness and chronic lung disease in the first 2 years of life. 7 however, the majority of rsv infections in infants occur without any known risk factors. 8 thus we still do not understand the precise mechanism for the wide variation in susceptibility to severe respiratory tract infections among children in these settings. one possible explanation for the range of susceptibility to viral respiratory tract infection in early childhood is that there are definable variations in the antiviral response, such as a congenital deficiency in the innate immune response that can be detected even at the time of birth. a central ingredient of the innate immune response to respiratory viruses is the system for interferon production and signaling. 9 in that regard a decrease in ifn-g production from cord blood mononuclear cells (cbmcs) stimulated by pha or allergens has been associated with increased risk for acute respiratory tract illness during infancy. 10, 11 perhaps more relevant to viral infection, the lack of a detectable ifn-g response to rsv in cbmcs was associated with decreased wheezing in the first year of life, but a detectable response was only found in a third of subjects, and therefore predictive power was limited. 12 therefore in the present study we developed alternative methods to determine whether the innate immune response of virus-infected cbmcs could predict the later development of respiratory tract illness. we used rsv to activate cbmcs based on the well-established association of rsv infection with subsequent childhood asthma. 2, 13, 14 however, to monitor the innate immune response to rsv, we determined the induction of the genes encoding ifn-g and the remarkably virus-responsive chemokine ccl5 based on sensitive and quantitative methods for mrna from 15 we also monitored interferon signal transduction by tracking the level of signal transducer and activator of transcription (stat) 1 activation in response to ifn-b stimulation. in both cases we used cultured/adherent cbmcs to select for monocytes versus the mixed cell population that included t cells in previous studies. we assessed whether each of these immune end points could predict the development of respiratory tract illness during the first year of life in a prospective birth cohort of children at high risk for asthma and allergic disease. the experimental matrix led to the unexpected finding of rsv-induced ifng gene expression in monocytes as a predictor of subsequent viral respiratory tract illness. we analyzed cord blood samples from 82 newborns enrolled in the urban environment and childhood asthma (ureca) study. this group represents a subset of the 178 children enrolled at the st louis site, which in turn was a subset of the total number of children enrolled at the baltimore, boston, and new york city sites between february 2005 and march 2007, as described previously. [16] [17] [18] subjects were required to have at least 1 parent with allergic rhinitis, eczema, and/or asthma and to reside in an area with greater than 20% of the residents below the poverty level, as well as being born at 34 weeks' gestation or later. at the st louis site, a small number of children without an allergic parent (n 5 5) were also recruited for comparison. after enrollment, all subjects were monitored for any episodes of acute respiratory tract illness over the next year along with quarterly assessments of respiratory (and nonrespiratory) tract illness and wheezing by questionnaire. nasal lavage samples were obtained when a caregiver reported an acute respiratory tract illness and at the time of a 1-year follow-up visit. the washington university human research protection office approved the study protocol. cord blood samples were collected in the delivery room, and cbmcs were isolated by means of density gradient centrifugation with accuspin tubes (sigma, st louis, mo) within 16 hours of collection, as described previously. 17, 19 when sufficient amounts of sample were available (ie, in 82/ 178 subjects), the cells were resuspended in rpmi medium with 10% fbs, 2 mmol/l l-glutamine, and 1 mmol/l nonessential amino acids to a final concentration of 8 3 10 5 per milliliter and plated in 4-well lab-tek chambers (500 ml per well; nunc a/s, roskilde, denmark) for the real-time pcr assay and in 2-well lab-tek chambers (1 ml per well) for the stat1 activation assay, as described below. nonadherent cells were removed after 24 hours, and adherent cells were cultured for 5 days with media changes on days 1, 2, and 4 and removal of additional nonadherent cells. at the end of the cell-culture period, the adherent cells were greater than 95% positive for cd68 immunostaining as a marker of monocytic lineage and therefore designated as cord blood monocyte cultures. the approach avoided purification methods (eg, magnetic bead selection or fluorescence-activated cell sorting) that modify the cell membrane or cell-culture methods (eg, growth factor supplementation) that promote full differentiation and polarization and thereby aimed to obtain cells of the monocyte lineage that were representative of naive lung tissue monocytes and macrophages (the target for viral respiratory tract infection in vivo). on culture day 5, cord blood monocytes were infected with rsv (a2 strain) at a multiplicity of infection of 7.5 or an equivalent amount of uvinactivated respiratory syncytial virus (rsv-uv). cellular rna was isolated immediately and 24 hours after inoculation with the rnaeasy mini kit (qiagen, valencia, calif) and transcribed to cdna by using the high-capacity cdna reverse transcription kit (applied biosystems, foster city, calif). single-target quantitative real-time pcr was used to monitor ifng and ccl5 mrna and rsv rna levels. for ifng and ccl5 mrna, primers were obtained from applied biosystems (hs00174575_a1 and hs00174143_a1). for rsv rna, primers 5312f (59-tccctacggttgtgatcgataga-39) and 5396r (59-tgatgggaagtagtagtgtaaagttggt-39) and probe 5349t (59-aggtaatacagccaaatc-39) targeting the viral l gene were based on the sequence of rsv strain crd2 (genbank accession no. dq340570). for glyceraldehyde-3-phosphate dehydrogenase (gapdh) mrna, primers 50f (59-cagccgagccacatccctcagacaccat-39) and 125r (59-ctttaccagagttaaaagcagccctggtgacca-39) and probe 88t (59-aggtcggagtcaaccgatttggtcgtattg-39) were used. plasmids encoding ccl5 and ifng (origene, rockville, md) and a portion of the rsv l gene (nt 5400-7016) and gapdh gene sequence (genbank accession no. nm_002046) were used to generate rna standards. rt-pcr was performed by using taqman real-time pcr master mix with 5 ml of sample cdna in accordance with the manufacturer's protocol (applied biosystems). all data for gene copy number was normalized to gapdh level. in a subset of cord blood samples (n 5 63) with an adequate number of cells, we also assessed interferon signal transduction by monitoring the level of stat1 phosphorylation in response to interferon stimulation. the corresponding cbmcs were processed as described above and serum starved on day 4 of culture. on culture day 5, the cells were incubated with ifn-b (100 u/ml) for 30 minutes. cell lysate was harvested after treatment with cell lysis buffer (cell signaling, danvers, mass). the level of total stat1 was determined by using elisa (invitrogen, carlsbad, calif), and phosphorylated stat1 (tyr701) levels were determined by using a sandwich elisa (pathscan phospho-stat1, cell signaling). nasal lavage samples were obtained during acute respiratory tract illnesses during the first year of life and at the 1-year follow-up visit. for illness samples, a respiratory symptom scorecard was completed as described previously. 10, 18 when the score indicated a moderate-to-severe respiratory tract illness, the site staff obtained a nasal lavage sample within 48 hours. all nasal lavage samples were processed for identification of 9 respiratory tract viruses by using a pcr-based assay, as described previously. 18 descriptive data were expressed as percentages, means 6 sds, or medians with interquartile ranges for nonnormally distributed data. to test differences between specific groups, x 2 or fisher exact tests were used to compare categorical variables, whereas unpaired t tests were used to compare continuous variables. appropriate log transformations were made to the data to yield an approximately normal distribution. for nonnormally distributed data, the wilcoxon rank sum tests (mann-whitney u tests) were used to compare groups. each measurement was standardized as the ratio over control to minimize variability in day-to-day experiments as follows: d ifng mrna response to rsv 5 ifng mrna copies with rsv/ ifng mrna copies without rsv, d ccl5 mrna response to rsv 5 ccl5 mrna copies with rsv infection/ccl5 mrna copies with no rsv infection, and rsv level was expressed as the rsv rna copy number with infection minus the value for no infection. spearman correlations were calculated to test for associations between variables. we examined the possibility of confounding by the following variables: sex, breast-feeding, maternal smoking during pregnancy and the first year of the child's life, an overall sum of bedroom allergen exposure (mus musculus, blattella germanica, and felis domesticus), and exposure to endotoxin (recombinant factor c assay) and ergosterol. we evaluated whether any of these variables were associated with the outcomes under analysis, and we found only that maternal smoking during the first year of life was related to the pneumonia outcome and the sum of bedroom allergen exposures was related to sinus infections. however, in logistic models controlling for these variables, the odds ratios changed by less than 5% when adjusted, and therefore we present unadjusted relationships here. all statistical tests were 2-tailed, and p values of less than .05 were considered statistically significant. statistical procedures were conducted with both sas 9.2 (sas institute, inc, cary, nc) and r 2.12.2 software. we processed all cord blood samples that contained an adequate number of cells, representing 82 of the total of 178 children who were enrolled at the st louis site of the ureca cohort. among the 82 newborns, 85% of the babies were african american, the mean age of the mother at the time of delivery was 23.7 years, and 63% of the infants had at least 1 parent with asthma (table i) . subjects were reported to have an average of 4.2 upper respiratory tract infections (colds), 1.3 wheezing illnesses, and an all-cause hospitalization rate of 15% during the first year of life. the basic demographics and first-year outcomes for this group of 82 children were not significantly different from those of the remaining group of 96 children who were not part of the present analysis (see table e1 in this article's online repository at www.jacionline.org). we collected nasal lavage samples during symptomatic respiratory episodes (n 5 38) and at routine 1-year follow-up visits (n 5 64). respiratory tract viruses were detected in 74% of the symptomatic episodes and in 48% of the routine visits (table ii) . human rhinovirus was found with the highest frequency and rsv with the next highest frequency in subjects with symptomatic episodes, but both could also be detected in asymptomatic subjects. together, the findings indicate that the majority of respiratory tract illnesses during the first year of life are associated with detectable levels of respiratory tract viral pathogens, but asymptomatic infants also have a high rate of apparent viral carriage. the distribution of values for rsv-induced ifng and ccl5 mrna, rsv rna, and stat1 activation showed a broad range among the subject population, suggesting a degree of individual variation among subjects for each of these responses (table iii) . this finding further suggests that the differences in responses might translate to variable degrees of protection against viral infection. we next investigated whether individual variation in antiviral response was associated with the development of respiratory tract fig 1) . there was no correlation between ifng response and available measures that might be associated with a subject's atopy (ie, number of parents with asthma, number of parents with hayfever, and presence of parental asthma at the time of initial screening). the relationship to respiratory tract infections was selective for the ifng response because we found no association between ccl5 response, rsv titer, or stat1 activation with the frequency of upper respiratory tract infections (r 5 20.17, p 5 .17; r 5 20.01, p 5 .94; and r 5 20.02, p 5 .91, respectively). we found no association of ifng response, ccl5 response, rsv titer, or stat1 activation with the frequency of wheezing episodes in the first year of life (r 5 20.05, p 5 .71; r 5 20.12, p 5 .32; r 5 20.04, p 5 .75; and r 5 0.12, p 5 .36, respectively). we also investigated whether there was an association of antiviral responses with the occurrence of infections at other respiratory and nonrespiratory sites (ie, sinus, ear, croup, and stomach), as well as any association with the number of reported hospitalizations for respiratory tract illness. we found that ifn-g responses to rsv in cord blood monocyte cultures were inversely related to the frequency of ear infections (p 5 .01), sinus infections (p 5 .04), pneumonias (p 5 .02), and respiratory-related hospitalizations (p 5 .05, fig 2) . we found no differences in ifn-g responses between those who did and did not have croup, ''stomach flu,'' or unexplained fevers (fig 2 and data not shown) . together, these findings reinforce the association of a decreased ifn-g response to rsv with the development of increased viral respiratory tract infections in the first year of life. in this study we provide evidence that a decreased antiviral interferon response at the time of birth is selectively associated with an increase in acute respiratory tract infections in the first year of life among infants at high risk for asthma and allergic disease. in support of this relationship between antiviral response and respiratory tract infection, we show that (1) rsv-driven induction of ifng mrna production in cord blood monocytes is variable among infants at birth; (2) decreased levels of rsv-induced ifng mrna in cord blood monocytes are associated with a significant increase in the frequency of upper respiratory tract infections, as well as the prevalence of ear infections, sinus infections, pneumonias, and respiratory tract illnesses requiring hospitalization; (3) levels of rsv-induced ccl5 mrna expression (another highly inducible antiviral system) and interferon-driven activation of stat1 (the downstream target of the interferon receptors) are not associated with this phenotype for subsequent illness; (4) symptomatic respiratory tract illnesses were frequently associated with detectable levels of respiratory viral pathogens; and (5) levels of rsv-induced ifng mrna were not linked to other types of infections, such as croup or stomach flu, which are caused by other types of pathogens. together, these findings provide for a close relationship between rsv-driven ifng mrna production and the development of viral respiratory tract illness and in turn suggest the possibility that a decrease in this type of response might lead to an increase in this type of illness. the present findings offer distinct insights from those obtained previously. for example, others found that pha-and allergenstimulated ifn-g production in cbmcs was inversely correlated with the frequency of viral respiratory tract infection in the first year of life. 10, 11 however, these studies likely measured the responsiveness of t cells because nonadherent cells were not eliminated and t-cell mitogen and antigen were used for stimulation. moreover, the effect of viral infection itself was not assessed in these studies. other studies examined the capacity of pbmcs to produce ifn-g during rsv-induced illness in children, but in this case cells were activated with phorbol 12-myristate 13-acetate-ionomycin and cross-linking antibodies to t-cell costimulatory receptors, and the response was localized to cd3 1 t cells. 20 similarly, others again made no attempt to purify cells and then stimulated cells with t-cell mitogen and did not separately quantify ifng mrna levels. 21 the same approach was taken in previous reports of an association between decreased ifn-g production from cbmcs and an increased risk of allergic sensitization and recurrent wheezing. 12, 22, 23 here again, this might reflect the focus on t-cell production of ifn-g and the proposed role of t h 1 versus t h 2 cytokines in the development of atopy and asthma. after these studies, we have come to better recognize the critical role of the innate immune system in controlling viral infection and postviral asthma. 9 in particular, the interferon and monocyte-macrophage systems are required for protective immunity against respiratory tract viruses, such as rsv, [24] [25] [26] and these same systems are capable of driving postviral asthma independent of the adaptive immune system, at least in experimental models. [27] [28] [29] therefore the present approach was designed to directly assess the innate immune response to viral infection and was done so by using the relevant cell type (purified monocytes) and stimulus (rsv infection), as well as more specific and sensitive methods (real-time quantitative pcr) than applied previously. the upshot is the first evidence that the monocyte ifng gene also serves as a marker and might even participate in the susceptibility to infection during infancy. this unexpected finding provides a new lead for control of the antiviral response because the previous view was that ifng gene expression was silenced in the monocyte lineage and was only active in lymphoid cells. 30 in that regard we note that ifn-g production is generally attributed to natural killer (nk) cells, nkt cells, and t cells, whereas monocytes and macrophages are solely a target of ifn-g action. indeed, studies of atopic disease in infancy often focus exclusively on t-cell production of ifn-g. 31 this circumstance is also likely due to the lower levels of ifn-g produced by monocytes and macrophages under conditions used in previous studies. here we are able to measure ifn-g production by using a sensitive assay for the corresponding mrna. whether this level of interferon production has functional consequences is uncertain, but its utility as a biomarker for susceptibility to viral infection proved quite useful. in contrast, it appears that the monocytemacrophage lineage is critical for host defense against respiratory tract viruses (including rsv). in particular, lung macrophages are charged with clearance of infected cells without dying themselves, and this protection derives from an antiapoptotic survival function of the chemokine ccl5. 25 if this function is lost (eg, in mice that are ccl5 deficient), the host is more susceptible to viral respiratory tract infection. other work suggests that ccl5 is also needed to direct dendritic cell traffic in the face of viral infection. 32 each of these observations are consistent with those linking ccl5 promoter gene polymorphisms to susceptibility to severe rsv-induced bronchiolitis. 33 nonetheless, we did not find that rsv induction of ccl5 gene expression was significantly associated with viral respiratory tract infection rates in the first year of life. it is still possible, however, that ccl5 production at the level of the lung macrophage would be predictive of susceptibility to infection, especially given the heterogeneity of monocyte-macrophage populations in the circulation and the lung. our study was conducted in a population selected for high prevalence of atopic disease. however, we do not expect that ifng response or viral susceptibility is attributable to atopy. indeed, we found no association of the ifng response with the available measures associated with atopy in our subject group because full evaluation of atopic status in our subjects was not yet performed at 1 year of age. similarly, others found no association between lower respiratory tract illness in the first year of life and the occurrence of parental atopy or subject eczema. 11 these findings suggest that viral susceptibility can be independent of atopy, and certainly there is evidence that this can be the case in experimental models of viral respiratory tract infection. 25 however, it will require a nonatopic cohort, follow-up of the present cohort, or both to formally test the relationship between virus-induced ifn-g production in monocytes, viral susceptibility, and atopic status in human subjects. the molecular mechanism for rsv induction of ifng gene expression still needs to be defined. thus the pathogen recognition receptor system is responsible for mediating viral induction of the various forms of ifn-a, ifn-b, and ifn-l, and rsv is remarkably effective in blocking the induction (and signaling) of these interferon species. 34, 35 by contrast, the pathogen recognition receptor system of toll-like receptors and retinoic acid inducible gene i (rig-i)-like receptors (rlrs) does not appear to regulate ifng gene expression or signaling. instead, ifng gene expression is subject to a distinct type of positive and negative regulation at pretranscriptional, transcriptional, and posttranscriptional levels, at least in the case of lymphoid (nk, nkt, and t) cells. 30 however, these regulatory mechanisms have not been studied in monocytes or in response to rsv in any cell type. we could not define this regulatory mechanism with such limited human samples, but our work should open this new field of research. rsv is the most common cause of serious respiratory tract illness during infancy, and severe rsv-induced bronchiolitis is linked to subsequent wheezing illness/asthma. furthermore, paramyxoviral infection is established as a high-fidelity experimental model of asthma. moreover, rsv and related paramyxoviruses are easily detected in monocytes and macrophages in the lung. hence we chose rsv for our study of newborns and their monocytes, unaware of course that the subsequent results would predict respiratory tract illness associated with rhinovirus. with such a severe limitation in cell sample size, we were unable to test multiple viruses, but an analysis of the response to other types of viruses (including rhinovirus) and viral strains (including other rsv types) would be a valuable goal in the future. in summary, we report that congenital variations in the innate immune response might predict the susceptibility to acute respiratory tract illness during the first year of life. our effort uncovered evidence that the ifng mrna monocyte response to virus rather than t-cell response to mitogen/allergen might be linked to the development of viral infections and eventually postviral asthma. we were able to define this relationship despite a relatively small sample size. sample availability for complex immunologic analysis served to limit the number of ureca participants that could be studied. however, the demographic characteristics of our cohort were no different from those of the overall group, suggesting that we studied a representative sample of subjects. in addition, our study included mostly african american children, and therefore the result might not be generalized to children with other racial backgrounds. interestingly, others have recently found that interferon-stimulated genes (eg, pyhin1) are also linked to the development of asthma, particularly in subjects of african descent. 36 our findings are also consistent with observations of decreased ifn-g production in response to rsv in pbmcs from older children and adults with allergic asthma. 37, 38 thus these early events in infancy might carry over to a similar deficit in antiviral defense in later life. together, the findings suggest that a full analysis of interferon production pathways might provide key insights into the susceptibility to viral respiratory tract infection and subsequent chronic obstructive lung diseases, such as asthma. world health organization. the world health report 2005: make every mother and child count. geneva: world health organization wheezing rhinovirus illnesses in early life predict asthma development in high-risk children tucson children's respiratory study: 1980 to present day care attendance in the first year of life and illnesses of the upper and lower respiratory tract in children with a familial history of atopy prospective study of healthcare utilisation and respiratory morbidity due to rsv infection in prematurely born infants influence of attendance at day care on the common cold from birth through 13 years of age respiratory syncytial virus infection: immune response, immunopathogenesis, and treatment global burden of acute lower respiratory infections due to respiratory syncytial virus in young children: a systematic review and meta-analysis host epithelial-viral interactions as cause and cure for asthma cytokine response patterns, exposure to viruses, and respiratory infections in the first year of life cord blood cytokines and acute lower respiratory illnesses in the first year of life bidirectional interactions between viral respiratory illnesses and cytokine responses in the first year of life respiratory syncytial virus in early life and risk of wheeze and allergy by age 13 years asthma and allergy patterns over 18 years after severe rsv bronchiolitis in the first year of life virusinducible expression of a host chemokine gene relies on replication-linked mrna stabilization prenatal maternal stress and cord blood innate and adaptive cytokine responses in an inner-city cohort parental characteristics, somatic fetal growth, and season of birth influence innate and adaptive cord blood cytokine responses the urban environment and childhood asthma (ureca) birth cohort study: design, methods, and study population standardization and performance evaluation of mononuclear cell cytokine secretion assays in a multicenter study decreased interferon-gamma response in respiratory syncytial virus compared to other respiratory viral infections in infants type 1 and type 2 cytokine imbalance in acute respiratory syncytial virus bronchiolitis association between antenatal cytokine production and the development of atopy and asthma at age 6 years reduced interferon gamma production and soluble cd14 levels in early life predict recurrent wheezing by 1 year of age the role of ifn in respiratory syncytial virus pathogenesis ccl5-ccr5 interaction provides antiapoptotic signals for macrophage survival during viral infection airway epithelial versus immune cell stat1 function for innate defense against respiratory viral infection induction of high-affinity ige receptor on lung dendritic cells during viral infection leads to mucous cell metaplasia persistent activation of an innate immune response translates respiratory viral infection into chronic inflammatory lung disease cutting edge: cd49d1 neutrophils induce fcεri expression on lung dendritic cells in a mouse model of postviral asthma regulation of interferon-g during innate and adaptive immune responses high ifn-g production by cd81 t cells and early sensitization among infants at high risk of atopy controls for lung dendritic cell maturation and migration during respiratory viral infection rantes promoter gene polymorphisms and suceptibility to severe respiratory syncytial virus-induced bronchiolitis suppression of the induction of alpha, beta, and gamma interferons by the ns1 and ns2 proteins of human respiratory syncytial virus in human epithelial cells and macrophages respiratory syncytial virus nonstructural proteins ns1 and ns2 mediate inhibition of stat2 expression and type i interferon responsiveness meta-analysis of genome-wide association studies of asthma in ethnically diverse north american populations atopic phenotype in children is associated with decreased virus-induced interferon-alpha release impaired virus-induced interferon-alpha2 release in adult asthmatic patients disclosure of potential conflict of interest: k. sumino has received research support from the national institutes of health (nih). j. e. gern is on the scientific advisory board for and owns stock options in 3v biosciences; has consulted for centocor, boeheringer ingelheim, glaxosmithkline, biota, medimmune, and theraclone; and has received research support from astrazeneca. g. r. bloomberg has received research support from the nih/national institute of allergy and infectious diseases. m. j. holtzman has consulted for and received research support from hoffman-la roche and forest laboratories. the rest of the authors declare that they have no relevant conflicts of interest. clinical implications: individual variations in the immune response to respiratory tract viruses are detectable at birth, and these differences predict the susceptibility to acute respiratory tract illness during first year of life. key: cord-023728-fgcldn4e authors: bower, john; mcbride, john t. title: croup in children (acute laryngotracheobronchitis) date: 2014-10-31 journal: mandell, douglas, and bennett's principles and practice of infectious diseases doi: 10.1016/b978-1-4557-4801-3.00061-8 sha: doc_id: 23728 cord_uid: fgcldn4e nan the term croup now generally refers to an acute respiratory tract illness characterized by a distinctive barking cough, hoarseness, and inspiratory stridor in a young child, usually between 6 months and 3 years old. this syndrome results from inflammation of varying levels of the upper respiratory tract, which sometimes spreads to the lower respiratory tract, producing concomitant lower respiratory tract findings. croup is primarily laryngotracheitis and encompasses a spectrum of infections from laryngitis to laryngotracheobronchitis and sometimes laryngotracheobronchopneumonitis. most common among the clinical argot of croup are recurrent, allergic, and spasmodic croup. most children develop croup only once or twice despite multiple infections with the viruses that are prime etiologic agents. some children have recurrent episodes of croup, however, which is often referred to as "spasmodic croup. " spasmodic croup and "allergic croup" also have been applied to cases that tend to be sudden in onset, often at night, with minimal coryza and fever, and that occur among children with a family history of croup or atopy. 4 spasmodic croup generally cannot be differentiated from a single episode of the usual type of croup, however, in its clinical manifestations or in its etiology, which is usually viral. croup is a common illness among outpatients, but few cases require hospitalization. [4] [5] [6] [7] croup occurs in 2% to 6% of young children each year. about 10% to 16% of all children experience at least one attack of croup, and 5% have recurrent croup, consisting of three or more episodes. the peak occurrence is in the second year of life, with most cases occurring between 3 months and 3 years of age. in a seattle prepaid group practice, the annual incidence of croup was 7 per 1000 for all children younger than 6 years, and the peak incidence in the second year of life was 14.9 per 1000 children. 7 among children younger than 2 years of age presenting to emergency departments in alberta, canada, for the period 1999 to 2005, the rates of croup ranged from 30.9 to 49.6 per 1000 emergency department visits. 8 hospital admissions have significantly declined in recent years in correlation with the use of effective outpatient therapy for croup. from 1979 to 1997, croup cases associated with parainfluenza viruses, estimated from the national hospital discharge survey, showed that the number of admissions among children younger than 5 years decreased by approximately one third. 5 outbreaks of measles in the united states and elsewhere serve as a reminder that rubeola in the prevaccine era often resulted in severe and complicated croup. during the 1989 to 1991 upsurge of measles cases in the united states, laryngotracheobronchitis complicated approximately 20% of the cases of measles among hospitalized patients in los angeles and houston. 18, 19 children with croup as a complication of measles tended to be younger, they had a more severe course, and 17% to 22% required intubation. in some children, the outcome was fatal. the epidemiologic patterns of croup reflect mainly the seasonal predilection of the major agents (see fig. 61-1) . parainfluenza virus type 1 predominantly occurs every other year in the fall, resulting in the major outbreaks of croup recognized biennially in odd-numbered years since 1993. 5, 11 other parainfluenza viruses have less distinctive seasonal patterns. parainfluenza type 2 virus also contributes to the cases occurring in the fall and winter, but irregularly and at lower levels. 20 parainfluenza type 3 virus appears yearly, and although it may be present throughout much of the year, parainfluenza type 3 virus predominantly occurs in the spring to fall and is the major cause of the swell of croup cases observed each spring. influenza a and b viruses and rsv also contribute to the cases in the winter and spring. rhinoviruses, enteroviruses, bocavirus, and coronaviruses are present through most of the year. in some areas, enteroviruses have an increased prevalence during the summer and fall and bocavirus is prevalent during the fall to spring (see fig. 61 -1). the shrill sonorous inspiration so characteristic of this complaint, marks very unequivocally its seat.… from some cause there is an unusual approximation of the sides of the glottis … the influence being very analogous to that produced by too strong compression of the reed against the mouthpiece of the clarinet by the lips of one who has made no great proficiency in that instrument, when a harsh, squeaking sound is produced abundantly discordant and grating to the ear. hugh ley, 1836 21 the virus initially infects the upper respiratory tract and usually produces congestion of the nasal passages and nasopharynx. subsequently, especially during primary infection, the larynx, the trachea, and sometimes the bronchi become involved. the classic signs of croup-stridor, hoarseness, and cough-arise mostly from the inflammation of the larynx and trachea. the resulting obstruction is greatest at the subglottic level because this is the least distensible part of the airway because it is encircled by the cricoid cartilage, with the narrow anterior ring and the larger posterior quadrangular lamina forming a "signet ring. " the impeded flow of air through this narrowed area produces the classic high-pitched vibratory sounds, or stridor. this is most apparent on inspiration because high linear velocity in the already narrowed airway creates a negative intraluminal pressure, narrowing the extrathoracic airway further, much as sucking on a partially occluded paper straw causes it to collapse inwardly. airway collapse is enhanced in young children because of the increased compliance of their airway walls. 22 even minimal inflammation of the membranes lining the narrow passages of the larynx and glottis in a young child results in an appreciable degree of obstruction because resistance to airflow is inversely related to the fourth power of the radius of the airway. the mucous membrane is also looser and more vascular, and the cricoid cartilage is less rigid. nasal obstruction and crying can aggravate the dynamic narrowing of the child's airway further. with the subglottic obstruction, the child's tidal volume initially declines. this is compensated by an increase in the respiratory rate to maintain adequate alveolar ventilation ( fig. 61-2 ). if the degree of obstruction worsens, the work of breathing may increase such that the child tires and can no longer maintain an adequate respiratory effort. the tidal volume may decrease further, and, as the respiratory rate declines, hypercarbia and secondary hypoxemia ensue. in addition to airway narrowing related to mucosal swelling and dynamic collapse, it is possible that upper airway inflammation leads rates decreased by 33% from 1.8 to 1.2. in ontario, the estimated annual rates of hospitalization from 1988 to 2002 also showed a decline among children younger than 5 years, and the rates were lower among children 1 to 4 years old than among infants. 9 the decline did not begin until after the winter of 1993 to 1994, however, when the annual rate per 1000 children younger than 5 years was 2.67. in 2001 to 2002, the rate had declined by 86% to 0.37. among children evaluated for croup in an emergency department, one or more viral agents were identified in 80% of specimens by reversetranscriptase polymerase chain reaction (rt-pcr) assay; the parainfluenza viruses were detected most frequently. 10 no matter what means of detection were used, studies over decades have consistently shown that the parainfluenza viruses, especially type 1, are the most frequent cause of croup. [4] [5] [6] [7] [10] [11] [12] only the parainfluenza viruses are associated with the major peaks of occurrence of croup cases (fig. 61-1 ). parainfluenza type 1 has been identified in approximately one fourth to one third of cases. parainfluenza type 3 generally is the second most commonly associated virus, accounting for 6% to 10% of cases. a small proportion of all influenza illnesses among children is associated with croup, but among croup cases, influenza accounts for 1% to 10% of cases depending on the year and circulating strain. similarly, although respiratory syncytial virus (rsv) infections are particularly prevalent among this age group, relatively few (about 5% of rsv infections) manifest as croup. more recent studies using rt-pcr methods have suggested an etiologic role for viruses other than the parainfluenza viruses. rhinoviruses, enteroviruses, adenoviruses, and bocavirus have been detected in 9% to 13% of specimens from children with croup. 10,12, 13 croup has also been observed in a small percentage of children younger than 5 years of age infected with metapneumovirus. 14 the human coronaviruses (hcov) have been identified in up to 7% of young children with acute respiratory tract infections, with the nl63 strain most often associated with croup. 15 in seoul, south korea, hcov nl63 was the second most commonly isolated virus from children presenting with croup. 16 the significance of these associations, however, is unclear because respiratory viruses often appear as coinfections and studies have been limited by small sample sizes. larger studies are needed to confirm these findings. 17 the diagnosis of croup can almost always be made on the basis of the characteristic epidemiologic features, the clinical manifestations, and the history, especially in children 6 months through 3 years of age. diagnostic procedures that upset the child may worsen the respiratory distress and should be avoided. 7 laboratory analysis generally should be limited to tests necessary for management of a more severely ill child, such as tests used to assess dehydration and oxygenation. white blood cell counts and differentials are rarely helpful or distinctive in diagnosing croup. identification of the specific viral agent also is usually unnecessary, and obtaining respiratory tract swabs and secretions is likely to augment the child's respiratory distress. viral identification may be warranted when specific antiviral therapy is being considered, such as for severely ill or high-risk children with influenza. in most instances, a rapid antigen assay, such as immunofluorescent and enzyme immunoassays, is used. 24 rapid multiplex pcr assays for respiratory viruses provide an increasingly available alternative with improved sensitivity and relatively short turnaround times. radiographic evaluation is usually unnecessary for the diagnosis of croup and, as noted earlier, should be undertaken with caution and careful monitoring of the child. among atypical cases, however, the radiologic picture may be helpful in the differential diagnosis. the characteristic manifestation of viral croup noted on an anteroposterior neck film is a 5-to 10-mm narrowed shadow of the trachea in the subglottic area. this is often described as the "hourglass" or "steeple" sign ( fig. 61-3) . the lateral view of the neck may show an increased width of the airspace in the hypopharyngeal area. dilation of the pharyngeal airway often is seen and is indicative of the child's increased respiratory effort. the diagnostic value of these radiographic findings is nevertheless questionable. they are not consistently observed in all cases of viral croup, and some studies have shown them to be of low specificity and sensitivity for confirming or ruling out viral croup. for children presenting with atypical features or history, a broad range of diagnoses should be considered. 25 a case should be considered to active constriction of the muscles of the upper trachea and larynx that might contribute to airway narrowing in some children with croup, particularly those with spasmodic croup. this might explain the association between recurrent croup and asthma or airway hyperreactivity. the disease generally comes on in the evening after the little patient has been exposed to the weather during the day and often after a slight catarrh of some days' standing. at first his voice is observed to be hoarse and pulling … he awakens with a most unusual cough, rough, and stridulous. and now his breathing is laborious, each inspiration being accompanied by a harsh, shrill noise. john cheyne, 1814 2 although abrupt onset of stridor at night may be the initial indication of illness, most children have a prodrome of mild upper respiratory tract signs of rhinorrhea, cough, and sometimes fever 12 to 48 hours before the onset of the distinctive "rough and stridulous" cough of croup. the deepening cough and hoarseness herald the onset of the respiratory stridor. the cough is not productive but has the striking deep brassy tone of a "seal's bark. "* the respiratory stridor may be accompanied by retractions of the chest wall, which are usually most marked in the supraclavicular and suprasternal areas. some children may progress to have inspiratory and expiratory stridor. the respiratory rate may be slightly elevated, but rates greater than 50 per minute are unusual in children with croup, in contrast to the marked tachypnea that is often evident with bronchiolitis. the onset of stridor commonly occurs at night; in milder cases it may improve in the morning, only to worsen again at night. children whose croup is characterized by abrupt nighttime onset with little prodrome of a respiratory tract infection, followed by daytime improvement, are often designated as having "spasmodic croup. " these children tend to have repeated episodes over several days or separated by months. generally, an episode of recurrent croup cannot be differentiated from the usual case of viral croup clinically or by viral etiology. 12 a viral etiology was identified by rt-pcr in 68% of the children, and the proportion with an identified viral infection was not significantly different between children with single and recurrent episodes of croup. a few children with recurrent croup have an underlying condition such as subglottic stenosis or gastroesophageal reflux. for most children, the course of croup is less than 3 to 4 days. although the cough may persist longer, the characteristic barking quality resolves within 2 days in most children. 23 *the characteristic cough and stridor have also been described by ley in 1836 21 as "the crowing of a cock, the yelping of a fox, the barking of a dog, the braying of an ass, or a ringing sound, as if the voice came from a brazen tube. " occasionally, recurrent episodes of stridor may be related to gastrointestinal reflux. 33 appropriate therapy for croup is determined by the severity of the child's illness. accurate assessment of the child's clinical status is essential. the natural fluctuations in the course of croup often confound this evaluation, however, as well as complicate assessment of the success of therapy. most children with mild croup may be cared for at home. keeping a child with croup comfortable and avoiding disturbing procedures are particularly important, because anxiety and crying may enhance the respiratory distress. the child should be given adequate liquids and antipyretics if necessary. despite a plethora of home therapies for croup, none has proved consistently effective. taking a child with croup outside to breathe cold air or into a shower to breathe warm mist are commonly recommended. vaporizers and other means of producing mist in the home have long been advised. in the past century, steaming tea kettles were an integral and often primary mode of therapy. nevertheless, the beneficial effects of mist have not been proved. 7, [34] [35] [36] [37] multiple scoring systems have been used to assess the severity of croup. the scoring system most frequently used is the westley clinical score. 38 the major findings on physical examination used for this score are the degree of stridor, chest wall retractions, air entry, level of consciousness or fatigue, and presence of cyanosis. guidelines for the management of croup generally have classified croup as mild, moderate, and severe, with patients with mild cases having corresponding westley scores of 0 to 2, those with moderately severe cases having scores of 3 to 7, those with severe cases having scores of 8 to 11, and patients at risk for imminent respiratory failure having scores of 12 to 17 (table 61-1) . 4, 7 the therapy recommended varies according to the assessed level of severity, but the mainstay of therapy beyond supportive care is dexamethasone. one dose of dexamethasone orally or, if necessary, intramuscularly, administered to outpatients and in emergency departments has been shown to be effective in reducing the need for hospitalization. 7, 39, 40, 41 repeated doses are seldom necessary. nebulized epinephrine, racemic epinephrine, or l-epinephrine may be added to the dexamethasone for children with severe croup. 38, 42 because improvement after nebulized epinephrine is transient, treatment may be repeated. a child treated with one of these aerosols should be observed for at least 2 hours (see table 61 -1) prior to discharge. administration of a mixture of helium and oxygen has long been used to improve gas exchange in various obstructive disorders of the upper and lower respiratory tract. little evidence exists, however, that administering heliox to children with croup is beneficial. [43] [44] [45] atypical if the child does not have the most characteristic features of croup, especially the seal's bark cough and hoarseness. the physician's most important clinical responsibility in evaluating a child with inspiratory upper airway obstruction is differentiating children with the common and usually benign viral croup from the few children who have life-threatening obstruction from bacterial epiglottitis or tracheitis. the history of a rapidly progressive course, high fever, a toxic appearance, and drooling are most characteristic of these bacterial processes, and the brassy cough of viral croup is characteristically absent. children with these symptoms demand careful evaluation and management. acute bacterial epiglottitis is usually due to infection with h. influenzae type b and has become rare since the widespread use of vaccination. 25, 26 the differentiating features of epiglottitis include the strikingly rapid onset and progression of the illness, high fever, and toxic appearance. the child is often sitting, leaning forward, and anxious and may have a muffled voice, marked dysphagia, and drooling. the history of an upper respiratory tract infection with rhinorrhea and laryngitis usually is not present. epiglottitis is almost always an indication for prompt antibiotic therapy and securing the airway by intubation in a controlled environment. bacterial tracheitis has an acute onset and presentation similar to that of epiglottitis. 7,26-29 its rapid and dramatic onset is characterized by high fever, stridor, and dyspnea with copious amounts of purulent sputum. the child may progress rapidly to complete airway obstruction. the course is unresponsive to therapy with nebulized epinephrine, and suspected cases should be managed as a medical emergency. bacterial cellulitis and abscesses of the deep neck spaces, including peritonsillar and retropharyngeal abscesses, may also manifest with similar findings of high fever, dysphagia, and drooling. 30, 31 the characteristic upper respiratory tract signs, hoarseness and barking cough, are usually not present. c. diphtheriae, although a major cause of stridor in the past, is now rarely seen in the united states and other developed countries but should still be considered in countries with low rates of immunization. 32 all of these diagnoses represent pediatric emergencies, and, as with epiglottitis, usually justify careful intubation. noninfectious causes of obstruction that mimic croup include aspiration of a foreign body, which is common in the same age group as that of viral croup; trauma to the upper airway, such as from toxic ingestions; and angioneurotic edema. 25 anatomic abnormalities, such as vocal cord paralysis and anomalies that impinge on the laryngotracheal area, may cause stridor, especially when a respiratory tract infection augments the obstruction to airflow. these include tracheal stenosis, laryngeal webs, and papillomas. in most cases, the history and lack of acute signs of respiratory tract infection allow differentiation. in the older child, pulmonary function testing may be helpful. croup remains a common illness among young children. with the currently available modalities for management, most children may be cared for at home, and the illness usually resolves within 3 to 4 days. 23 most have mild symptoms, and only about 5% of children discharged from the emergency department after corticosteroid therapy need to return because of worsening of symptoms. 46 if the child's symptoms are minimal at discharge, return within 24 hours is unlikely. in canada, of all children with croup, about 4% have been estimated to require hospitalization and intubation was required for only 1 of the 170 hospitalized children or 1 in 4500 of all children with croup. 7, 23 croup presentations to emergency departments in alberta, canada: a large population-based study parainfluenza virus infection of young children: estimates of the populationbased burden of hospitalization human rhinovirus species associated with hospitalizations for acute respiratory illness in young us children human coronavirus in young children hospitalized for acute respiratory illness and asymptomatic controls burden of human metapneumovirus infection in young children role of human coronavirus nl63 in hospitalized children with croup proving etiologic relationships to disease: the particular problem of human coronaviruses glucocorticoids for croup nebulized epinephrine for croup in children evidence-based child health heliox for croup in children an inquiry into the nature, causes and cure of the croup cambridge history and geography of human disease project clinical practice: croup human parainfluenza virus-associated hospitalizations among children less than five years of age in the united states incidence and etiology of pneumonia, croup, and bronchiolitis in preschool children belonging to a prepaid medical care group over a fouryear period guideline for the diagnosis and management of croup croup presentations to emergency departments in alberta, canada: a large population-based study croup hospitalizations in ontario: a 14-year time-series analysis respiratory viruses in laryngeal croup of young children parainfluenza virus infection of young children: estimates of the populationbased burden of hospitalization the viral aetiology of croup and recurrent croup human rhinovirus species associated with hospitalizations for acute respiratory illness in young us children human coronavirus in young children hospitalized for acute respiratory illness and asymptomatic controls burden of human metapneumovirus infection in young children role of human coronavirus nl63 in hospitalized children with croup proving etiologic relationships to disease: the particular problem of human coronaviruses severe laryngotracheobronchitis as a complication of measles during an urban epidemic severe laryngotracheobronchitis complicating measles respiratory syncytial virus and parainfluenza virus an essay on the laryngismus stridulus or croup-like inspiration of infants stridor in childhood croup: duration of symptoms and impact on family functioning diagnostic assays for respiratory syncytial virus disease pediatric respiratory infections bacterial tracheitis: report of eight new cases and review changing epidemiology of life-threatening upper airway infections: the reemergence of bacterial tracheitis bacterial tracheitis clinical features and treatment of retropharyngeal abscess in children the contemporary approach to diagnosis and management of peritonsillar abscess resurgence of diphtheria recurrent croup presentation, diagnosis, and management a randomized controlled trial of mist in the acute treatment of moderate croup controlled delivery of high vs low humidity vs mist therapy for croup in emergency departments: a randomized controlled trial humidified air inhalation for treating croup lack of efficacy of humidification in the treatment of croup: why do physicians persist in using an unproven modality? can nebulized racemic epinephrine by ippb for the treatment of croup: a double-blind study a randomized trial of a single dose of oral dexamethasone for mild croup glucocorticoids for croup nebulized epinephrine for croup in children evidence-based child health use of heliox in children use of helium-oxygen mixtures in the treatment of croup: a systematic review heliox for croup in children the management of croup key: cord-292587-hp4zd8lr authors: rubino, ilaria; choi, hyo-jick title: respiratory protection against pandemic and epidemic diseases date: 2017-10-31 journal: trends in biotechnology doi: 10.1016/j.tibtech.2017.06.005 sha: doc_id: 292587 cord_uid: hp4zd8lr respiratory protection against airborne pathogens is crucial for pandemic/epidemic preparedness in the context of personal protection, healthcare systems, and governance. we expect that the development of technologies that overcome the existing challenges in current respiratory protective devices will lead to a timely and effective response to the next outbreak. ilaria rubino 1 and hyo-jick choi 1, * respiratory protection against airborne pathogens is crucial for pandemic/epidemic preparedness in the context of personal protection, healthcare systems, and governance. we expect that the development of technologies that overcome the existing challenges in current respiratory protective devices will lead to a timely and effective response to the next outbreak. influenza, a major respiratory disease, poses great risks to global health. influenza epidemics and pandemics are responsible for 250 000-500 000 deaths each year (www.who.int/mediacentre/ factsheets/fs211/en/) and >50 million fatalities worldwide in the past century (www.cdc.gov/flu/pandemic-resources/ basics/past-pandemics.html), respectively. the next influenza pandemic is estimated to cause $60 million deaths [1] . ideally, vaccination within 2 months of the outbreak can provide effective protection [2] . however, because several months are necessary for vaccine development and administration, the infection risk is heightened during the non-vaccine period. this is further supported by the outcomes of 2002-2003 severe acute respiratory syndrome (sars) outbreak that originated in china, in which the disease was transmitted globally within few weeks, but the first vaccine phase i clinical study began a year after the outbreak [3] . logistically, an effective pandemic preparedness plan should include both vaccination and alternative mitigation methods (pharmaceuticalantiviral; non-pharmaceuticalisolation, administrative control, personal protective measures). therefore, respiratory protection devices are a key non-pharmaceutical intervention that is essential to the global strategy for pandemic readiness. the parameters behind respiratory protection and airborne transmission intertwine in a complex system that can be broken down into four bidirectional components: (i) release, (ii) infection, (iii) filtration, and (iv) protection (figure 1 ). once a subject is infected, nanometer-to-millimeter-sized pathogenic particles can be released while breathing, speaking, sneezing, or coughing, and infect a host respiratory tract via different mechanisms that depend on the aerodynamic size of the particles (d a <5 mm, lower respiratory tract; 5 < d a < 100 mm, upper respiratory tract). similarly to infection, current respiratory protection devices filter infectious particles in a size-dependent manner. filtration efficiency, comfort (e.g., breathability), and fit at the face-mask interface govern technical performance. while effective management and availability of control measures are crucial to an outbreak response, the pathogens [ 1 1 4 _ t d $ d i f f ] (virus/bacteria/ fungi) captured on filters are an intrinsic concern because of fear of cross-infection, new aerosol release, and contaminated waste. recurrent recommendations regarding respiratory protective measures by the centers for disease control and prevention (cdc) and the world health organization (who) emphasize their prominent role in emergency preparedness. nonetheless, fewer scientific efforts have been focused on respiratory protection technologies compared to vaccine development technologies. we present here an overview of currently available respiratory intervention technologies and their implications for future research directions in response to pandemic/epidemic outbreaks. surgical masks have been in use for over 100 years as barriers against the development of infection via large droplets produced during surgery. n95 filtering facepiece respirators (n95 respirators) were introduced in 1995 as part of the national institute for occupational safety and health (niosh) 42 code of federal regulations (cfr) part 84 on non-powered air-purifying respirators (www.cdc. gov/niosh/npptl/topics/respirators/ pt84abs2.html). currently, surgical masks and n95 respirators are the two main intervention measures for personal respiratory protection. nonetheless, technical challenges exist, some of which are shared by both devices: (i) filtration efficiency, (ii) cross[ 1 0 9 _ t d $ d i f f ] -infection, (iii) recyclability, and (iv) faceseal. a primary issue concerning the efficacy of surgical masks against airborne pathogens is low filtration efficiency. although performance can vary drastically among models, inconsistent reports on surgical mask efficacy are probably associated with improper application, resulting in performance mismatch. another crucial issue is cross[ 1 0 9 _ t d $ d i f f ] -infection/transmission. because viruses and microorganisms can survive for at least a few hours to several days [4] , masks and respirators become a source of infection for the wearer and others, thus limiting them to single use. infectious aerosols on filters can also be re-released into the environment (i.e., reaerosolization), for example through accidents. with a particle diameter of 1.15 mm, re-aerosolization from n95 respirators as a result of a fall (drop height, 0.76 m) was between 0.002% and 0.012% [5] . various sterilization methods (e.g., ethylene oxide, formalin, uv, bleach, hydrogen peroxide) have been tested to recycle respirators. however, the drawbacks of each method, such as performance deterioration and generation of toxic residues, have restricted their application. as an example, decontamination of n95 respirators by autoclave, 160 c dry heat, 70% isopropanol, and soap and water lowered the filtration efficiency [6] . ethylene oxide treatment of respirators caused deposition of hazardous residues of 2-hydroxyethyl acetate on the straps, and bleach, oxidants, or dimethyldioxirane raised issues of sharp odor and incompatibility with staples/nosepiece [7] . despite the need for further research, with safety as a preponderant concern, mask recyclability would be beneficial because it would reduce the amount of biohazardous waste and derived risks. in addition, reusability would naturally address a shortage of respirators during pandemics. furthermore, aerosols penetrate through loose-fitting masks/respirators, based on wearer facial features, movement, proper and timely fit testing/check, aerosol size, and mask shape. particles <10 mm enter through faceleaks 5-6-fold and up to 10fold more than through the filter of surgical masks and n95 respirators, respectively [8] . thus, although improving filtration efficiency is necessary, better fitting should be a primary objective to fully address aerosol penetration. interestingly, the general public tends to disregard infection control guidelines. as such, although respirators are recommended when airborne transmission is possible, surgical masks have experienced greater acceptance because of advantages such as comfort, availability, and cost. however, inappropriate application of devices may not provide consistent protection. this in turn stimulates research and development of new technologies to close the gap between guideline and practice. safer and more effective respiratory protection for a timely emergency response? diverse methods have been investigated to improve the performance of respiratory protection devices (box 1), such as higher subject releases pathogenic aerosols by breathing, talking, sneezing, and coughing. depending on the size, the particles deposit in different levels of the respiratory tract à upper respiratory tract (green), tracheobronchial region (blue), and alveolar region (red) à as a result of different mechanisms (i.e., interception, impaction, sedimentation, and diffusion). based on the same mechanisms, with additional electrostatic interactions of charged fibers, masks offer respiratory protection by filtration. the degree of respiratory protection is affected by the technical performance of the mask (filtration efficiency, comfort, faceseal, proper donning/doffing, and pathogen infectivity), as well as the infrastructure (available supplies, policies, and cost). in addition, because virus/bacteria infectivity is maintained on the fibers, the filter becomes a source of cross-infection, re-aerosolization, and environmental contamination. neutralization of the pathogens on respiratory protective devices is an approach that can bridge this gap towards pandemic and epidemic preparedness. filtration efficiency without sacrificing breathability. representative examples include fabrication using nanofibers and incorporation of electric charge by plasma treatment and charge-carrying agents. however, major technical challenges remain to be addressed for effective preparedness from the standpoint of contamination and infrastructure. hence, production of a filter that inactivates the collected pathogens would bring key improvements to current surgical masks and respirators, resulting in increased protection, reduced risk of cross[ 1 0 9 _ t d $ d i f f ] -infection, and recyclability without decontamination ( figure 1 ). to inactivate viruses, antimicrobial treatments have been investigated for filters utilizing halogens, metals, quaternary ammonium compounds, antibody-antigen reaction, and salt recrystallization. chlorine compounds such as n-halamines and iodine-treated filters have been assessed against bacteria (micrococcus luteus and escherichia coli) [9] . in addition, surgical masks functionalized with silver nitrate nanoparticles or quaternary ammonium inactivated bacteria (escherichia coli and staphylococcus aureus, and acinetobacter baumannii, enterococcus faecalis, and staphylococcus aureus, respectively) by interaction with thiol groups (100% reduction in $48 h) and membrane permeability damage ($92% reduction in 1 h), respectively [10, 11] . however, antimicrobial technologies based on silver/copper, reactive oxygen molecules, iodine, and titanium dioxide did not exhibit inactivation properties against ms2 virus [12] . another approach to inhibit virus transmission involves modification of the filter surface with the antigen-specific antibodies [13] . despite the merits of each approach, effective protection against virus aerosols is still limited by slow action (rapid inactivation should occur in the order of minutes, not hours) or binding specificity. recently, salt recrystallization was found to physically destroy viruses on surgical mask filters within few minutes in a strainindependent manner, potentially enabling reuse without separate processing steps [14] . notably, most studies have focused on the functionalization of the outermost and middle layers of the mask. the final design of the protection device layers should consider the spatial deposition of aerosols within masks and their contact surface. based on the above observations, we identify three central parameters in developing pathogen-inactivating filters. first, an inactivation mechanism should act rapidly to avoid cross[ 1 0 9 _ t d $ d i f f ] [ 1 0 8 _ t d $ d i f f ] -infection. although additional aspects are involved (e.g., fraction of transferred pathogens, surface area), unsafe handling and people's tendency to touch their face every $4 minutes lead to a risk of contact transmission from a pathogen-laden mask/respirator [15] . second, pathogens should be neutralized in a strainnonspecific way. the pathogen/strain responsible for the next pandemic cannot be exactly predicted because of continuous mutation. as such, antibodyfunctionalized protective devices that target a strain-specific virus would delay the emergency response. thus, the pathogen-killing mechanism should guarantee broad-spectrum protection. third, the ideal technology should be the key technical components of the performance of current respiratory protection devices are filtration efficiency, fit, and comfort. each has a significant role in protective efficacy, and specific parameters can be tuned to improve them. fit: non-filtered air entering through a poor seal between mask and face is a prominent concern. efforts towards reducing faceleaks can be grouped as follows. (i) material selection that allows customizable mask shape/tightness. (ii) investigation of wearers' facial features, and simulation of the spatial distribution of leaks to guide mask design. (iii) fit testing/training optimization to increase efficacy/compliance. filtration efficiency: whereas n95 respirators have a certified filtration efficiency of 95%, surgical masks have low performance. several major parameters can be controlled to decrease particle penetration, (i) decreasing the diameter of fibers. (ii) decreasing the size of filter pores, (iii) controlling fiber electrical charge through manufacturing process/material selection. (iv) increasing the thickness of the filters. user comfort: the wearer's perception of comfort is crucial to correct practices and effective protection. tolerability during mask use is often limited by the following factors. (iv) discomfort from prolonged contact between skin and rough materials. (iv) difficulty in communicating. device-independent. in the case of a pandemic outbreak, time-consuming production and cost would be major limitations to respirator use. although a certified respirator is recommended, considering the heavy use of surgical masks and scarcity of respirators, the technology should be easily extendable to masks and other existing infection control measures. therefore, the aforementioned factors outline the considerations that can enhance respiratory protection for a timely emergency response. the unpredictable nature of airborne pandemic diseases and their impact on our economy and society present a big challenge at the national and global level. only a prompt and coordinated response among different sectors of society can maintain security from this threat, which can be implemented through the help of technological innovations and comprehensive planning. unfortunately, despite being recognized as a key technical element in pandemic/epidemic preparedness, innovation in the design of respiratory protection devices has been sparse. in alignment with the strategic plan for pandemic/epidemic preparedness, we anticipate that incorporation of efficient pathogen-neutralization mechanisms can overcome the existing technical (contact transmission, source control, waste) and non-technical (supply shortage, policies, cost) challenges in respiratory protection. thus, we expect this engaging field to expand further, with the promise to offer enhanced protection to the global population. bacterial biohybrids use the energy of bacteria to manipulate synthetic materials with the goal of solving biomedical problems at the micro-and nanoscale. we explore current in vitro studies of bacterial biohybrids, the first attempts at in vivo biohybrid research, and problems to be addressed for the future. the aim of biohybrids is to harness cell motility and energy for user-desired tasks, including the transport of artificial cargo, drug delivery, or to power a tool for micromanipulation of other objects [1, 2] . bacteria-powered biohybrids (box 1) present new micromachines to perform complex tasks at the micro-and nanoscale. in vitro, biohybrids have demonstrated the ability to selectively sort particles [3] and even build microarchitectures [4] , but real-world applications for bacterial biohybrids have yet to be achieved. however, the biomedical field offers many opportunities to utilize the micromaneuverability and natural sensing capabilities of biohybrids for non-invasive medical applications that are not possible with current technologies, and recent research has been pushing biohybrids towards this goal. current bacterial biohybrids have the potential to be used for cancer or disease detection, targeted drug release, and even disruption of infectious biofilm sites. however, many challenges with external guidance, cargo loading and unloading, and efficient swimming remain, hindering their use in clinical and therapeutic applications. we present here recent attempts and developments to improve bacterial biohybrid performance and move the field closer to in vivo medical applications. bacteria attached to micro-or nanoparticles are some of the best-studied bacterial biohybrid systems. bacteria adhere to the particle and carry it while swimming, creating an effective cargo delivery system. guided cell adhesion of the bacterial body to localized regions of the estimation of potential global pandemic influenza mortality on the basis of vital registry data from the 1918-20 pandemic: a quantitative analysis strategies for mitigating an influenza pandemic severe acute respiratory syndrome persistence of the 2009 pandemic influenza a (h1n1) virus on n95 respirators particle release from respirators. part i: determination of the effect of particle size, drop height, and load effect of decontamination on the filtration efficiency of two filtering facepiece respirator models analysis of residual chemicals on filtering facepiece respirators after decontamination performance of an n95 filtering facepiece particulate respirator and a surgical mask during human breathing: two pathways for particle penetration evaluation of physical capture efficiency and disinfection capability of an iodinated biocidal filter medium antimicrobial effect of surgical masks coated with nanoparticles application of a quaternary ammonium agent on surgical face masks before use for pre-decontamination of nosocomial infection-related bioaerosols evaluation of the survivability of ms2 viral aerosols deposited on filtering face piece respirator samples incorporating antimicrobial technologies protection from avian influenza h5n1 virus infection with antibody-impregnated filters universal and reusable virus deactivation system for respiratory protection a study quantifying the hand-to-face contact rate and its potential application to predicting respiratory tract infection this research was financially supported by startup funds from university of alberta (h-j.c.). key: cord-022084-hap7flng authors: arruda, eurico; cintra, otavio a.l.; hayden, frederick g. title: respiratory tract viral infections date: 2009-05-15 journal: tropical infectious diseases doi: 10.1016/b978-0-443-06668-9.50064-8 sha: doc_id: 22084 cord_uid: hap7flng nan acute respiratory infections (aris) are prevalent worldwide 1 and rival diarrhea as the leading cause of death in developing countries. 1, 2 in some impoverished urban populations in south america, ari symptoms may be present on an almost continuous basis, making it difficult to determine symptom-free days and estimate attack rates. 3, 4 children from these areas may spend 40% to 75% of their time with respiratory symptoms, 1,5 mostly caused by upper respiratory infections (uris). the most striking disparity between developing and developed countries with regard to ari epidemiology is the case-fatality rate of lower respiratory infection (lri), mainly pneumonia, bronchiolitis, and influenza, 6, 7 in children under 5 years of age, which may reach 16% in some areas. 1, 8 several community-based studies have established the importance of common respiratory viral infections in tropical countries 5 (table 59-1) . in impoverished populations, these common viral infections may occur simultaneously with measles, diarrhea, and malnutrition, resulting in complex interactions of pathologic conditions that carry the potential to become life-threatening diseases. 1, 9 unlike certain pathogens restricted to tropical areas, the respiratory viruses have worldwide distribution, efficient person-to-person transmission, and an impact on all age groups. except for a few agents (e.g., adenoviruses, severe acute respiratory syndrome [sars] coronavirus) and rare cases of extrapulmonary dissemination with other respiratory viruses, replication is generally restricted to the respiratory mucosa of humans. in most health-care facility-based studies of acute lri (alri) conducted in tropical countries (table 59 -2), respiratory syncytial virus (rsv) is the virus most frequently detected (11% to 33%), followed by parainfluenza viruses (1% to 13%), adenoviruses (2% to 34%), and influenza viruses (1% to 4%). with few exceptions, human rhinovirus (hrv) and human coronaviruses (hcov) have not been reported frequently in studies in tropical countries, 5 probably because of difficulties in their detection. although previous studies have shown that attending daycare centers can be a risk for ari, 10 providing day care for children has become an important economic issue in developing countries, where mothers must join the workforce to contribute to the family income. consistent with studies in the united states and elsewhere, a study found a high burden of ari in young, low-income children attending day care in salvador, brazil. 11 few specific interventions are available to reduce the impact of respiratory viruses, 2 and the application of these interventions may be further hampered by epidemiologic patterns in ari and socioeconomic differences in temperate, developed countries compared with equatorial regions. for example, housing conditions and crowding pose challenges for optimizing health-care strategies in the tropics. while the incidences of some respiratory viruses, particularly rsv and influenza, show seasonal trends in some tropical areas, the association of seasonal peaks of respiratory viruses in general may be less apparent where fluctuations in temperature are smaller. 12 nutritional and educational interventions, such as reinforcing breast-feeding, 13 vitamin a supplementation for measles, 14 and facilitation of access to oral rehydration therapy, 15 may have significant effect on the morbidity and mortality due to lri alone or in association with diarrhea. in this chapter we focus attention on the most common viral respiratory infections, whose main features are summarized in table 59 -3, and try to highlight features unique to the developing world. in tropical countries influenza activity may occur yearround as well as in outbreaks more typical of temperate regions. these infections cause serious disease in populations weakened by malnutrition, with limited access to medical care. 16 of note, the predisposition induced by influenza to superimposed bacterial infections, mainly streptococcus pneumoniae, may greatly affect morbidity and mortality, mainly among impoverished populations. 17 in addition, influenza viruses can reassort or sometimes cross species barriers to generate emergent strains that may cause localized outbreaks or potentially pandemics with enormous impact for health on a global scale. 18 influenza viruses are pleomorphic, enveloped, with segmented negative-strand rna genomes and belong to the family orthomyxoviridae. influenza viruses are distributed in three genera-a, b, and c-based on the antigenicity of the nucleoprotein (np) and matrix protein. influenza a virus is further classified in subtypes based on its two surface glycoproteins: hemagglutinin (ha) and neuraminidase (na). 19 among the 15 ha and 9 na subtypes recognized in nature, 6 ha (h1, h2, h3, h5, h7, and h9) and three na (n1, n2, and n7) subtypes have now been identified in human isolates of influenza a viruses. 18 however, only three subtypes of ha (h1, h2, and h3) and two of na (n1 and n2) have caused pandemics and sustained circulation in human populations in recent years. 20 the genomes of influenza viruses contain eight rna segments in influenza a and b viruses, and seven rna segments in influenza c. 19 the glycoprotein ha is responsible for the attachment of the virus to sialic acid-containing cell receptors, and it mediates fusion and penetration. proteolytic cleavage of ha by cellular serine proteases exposes hydrophobic fusion domains that mediate membrane fusion. the na cleaves terminal sialic acid from glycoconjugates present on respiratory mucins, cells, and progeny virions. this action destroys receptors recognized by ha and allows budding virus to be released from infected cells and to spread within the respiratory tract. influenza c virus contains a single surface glycoprotein that binds to receptor, promotes fusion of membranes, and also cleaves sialic acid. 19 virus binding to receptor is followed by internalization into endosomes, fusion of viral and endosomal membranes, and release of the genome to the cytoplasm, from where it is transported to the nucleus. in influenza a viruses, the m2 protein serves an ion channel function that facilitates dissociation of the rna segments from the virion interior. transcription of the negative-strand genomic rna into positive-strand messenger rna (mrna) and complementary rna (crna) is mediated by a viral rna polymerase complex in the nucleus. crna serves as a template for the synthesis of negative-strand virion rna genome segments, and mrna directs viral protein synthesis. newly assembled nucleocapsids acquire an envelope as they bud through the cell surface. only viruses with a full complement of genome segments are infectious. 19 influenza a viruses are primarily viruses of aquatic birds, particularly ducks and shore birds, which harbor all subtypes recognized to date. selected subtypes naturally infect a range of terrestrial (swine, horses, humans) and aquatic (seals) mammals; influenza b virus infects humans and uncommonly seals, dogs, cats, and swine, and influenza c virus is primarily a virus of humans. depending on the virus type and subtype, experimental infection can be induced in mice, ferrets, chickens, swine, and primates, and the viruses can be propagated in primary cultures of kidney cells, continuous cell lines (mdck, vero, per.c6, and llc-mk2), and also in embryonated hen' s eggs. 20 the biologic property of influenza viruses to bind erythrocytes is exploited for early detection of the virus in cell culture and for the development of serologic assays by hemagglutination inhibition. 20 influenza viruses are inactivated by temperatures above 50°c and by lipid solvents, acid, formaldehyde, ionizing radiation, and ultraviolet (uv) light. 20 influenza viruses occur throughout the world, causing highly contagious respiratory infections with high morbidity and excess mortality (in seasonal peaks), particularly in infants and the elderly. in developing tropical countries, influenza has been associated with an average of 5% of aris leading to physician contact. 6, 21 this apparently low proportion probably represents only the most severe cases, since 30% to 50% of children under 5 years of age in tropical africa have been found to seroconvert in one outbreak. 21 previously healthy children younger than 1 year of age are hospitalized for influenza at rates similar to those for adults at high risk for influenza, and influenza accounts for a great number of outpatient visits and courses of antibiotics in children of all ages. 22 when human influenza virus is introduced into a malnourished population with limited access to care, high morbidity and mortality rates can occur, as was observed in madagascar in 2001 where a conventional influenza a/h3n2 subtype virus was associated with case-fatality rates of approximately 3%. contrary to the remarkably sharp seasonality of influenza a outbreaks in temperate countries, seasonal patterns in tropical countries have varied between studies. in southern india 23 and thailand 24 influenza has occurred throughout the year with sporadic outbreaks, whereas there have been consistent outbreaks in june-july and november-january, coinciding with the winter seasons in the southern and northern hemispheres, but with no apparent association with meteorologic factors. 12 in the philippines influenza has been more frequent between november and january, 25 while in senegal, nigeria, and taiwan there has been clear association with increased rainfall. 12 in southeastern brazil, 26 argentina, 27 as well as in south africa, 28 seasonal outbreaks of influenza a have occurred annually from may through august (mid autumn through winter) in association with cooler temperatures but not with rainfall. influenza b outbreaks occur periodically, yet less frequently than influenza a, in both temperate and tropical regions, 12, 20 whereas influenza c is generally nonseasonal. 20 minor changes in antigenicity, called antigenic drift, are caused by accumulation of point mutations in the genes coding for influenza ha and na, generating new strains that spread in annual epidemics. influenza viruses b and c are less prone to antigenic drift. major antigenic changes in influenza a are called antigenic shift and result in the emergence of a novel ha subtype with or without new na to which humans lack significant immunity. 18 this may be caused by the acquisition of new gene segments through genetic reassortment in a host infected simultaneously by a human and an animal (typically avian) virus, or by reappearance of a subtype from reservoir. swine are susceptible to both avian and human influenza viruses and may be hosts for reassortment or serve as the mammalian species in which avian viruses can adapt. novel influenza virus subtypes generated by shift have caused catastrophic pandemics, including three in the last century. the h1n1 "spanish flu" pandemic of 1918 is estimated to have caused up to 100 million deaths worldwide, 18 while the h2n2 "asian flu" in 1957 and the "hong kong flu" in 1958 caused an estimated 1 to 3 million deaths. recent clusters of human infections due to avian influenza, particularly h5n1 subtype viruses in asia, have raised concerns about new pandemic threats. in 1997 a highly pathogenic avian influenza h5n1 virus resulting from reassortment among several avian viruses caused lethal outbreaks in domestic poultry and severe illness with 6 deaths among 18 human cases in hong kong. the outbreak was due to exposure to infected poultry in live poultry markets and was later contained by their slaughter. this virus was transmitted inefficiently from person to person. 18, 20 in february 2003 an h5n1 virus caused deaths in a family visiting fujian province in china. since late 2003 wide-scale poultry outbreaks due to h5n1 virus have occurred in at least 10 asian countries. interspecies transmission to humans occurred in at least two countries, causing over 45 cases and 32 deaths in vietnam and thailand. 29 these h5n1 viruses have continued to reassort, evolve antigenically, and extend their host range with documented infections in swine and felids. prolonged nonsymptomatic excretion in ducks and detection in migratory birds indicate that this virus has become endemic in southeast asia. 18, 30 an avian h9n2 virus with human receptor specificity has also spread throughout asia in domestic poultry and pigs and caused mild disease in humans in hong kong and china. 18 an avian h7n7 virus caused conjunctivitis in at least 82 people and one fatal pneumonia in the netherlands in 2003; the outbreak was contained by culling and quarantine of poultry. in february 2004 a highly pathogenic h7n3 virus emerged in domestic poultry in british columbia with two documented human cases of conjunctivitis and mild "flulike" illness. 18, 29 influenza virus is transmitted from person to person by large droplets and small-particle aerosols, as well as possibly by fomites with hand contamination and subsequent selfinoculation. the relative importance of these routes is uncertain for natural influenza. ingestion of infected birds has led to infection by avian viruses in cats. secondary attack rates may reach over 70% in semiclosed populations, especially among schoolchildren and patients debilitated by underlying conditions who live in relative confinement, such as nursing home residents. 20 children play a major role in influenza outbreaks with respect to propagation of the epidemic virus in families and communities. 20 classic influenza starts abruptly after an incubation period of 1 to 2 days, with fever, chills, malaise, headache, myalgia, and prostration, often accompanied by nonproductive cough, sore throat, and mild rhinorrhea. systemic complaints last 3 to 5 days, whereas sore throat, hoarseness, and cough, with substernal discomfort, may increase in severity as the systemic symptoms subside. cough and asthenia often persist for 2 weeks or longer. respiratory symptoms may be minimal or absent initially, especially in the elderly or infants. in frail elderly persons, lassitude, lethargy, confusion, lowgrade fever, and sometimes gastrointestinal complaints may be the primary findings. influenza b tends to be milder than influenza a, and influenza c typically causes colds or bronchitis. 20 influenza may also present as unexplained fever, croup (laryngotracheobronchitis), vomiting, diarrhea, and neurologic manifestations in young children. 31 up to 50% of influenza virus infections in adults are subclinical. 20 influenza causes a variety of viral respiratory complications, including otitis media, sinusitis, tracheobronchitis, pneumonia, and, in young children, bronchiolitis and croup. secondary bacterial infections, especially pneumonia caused by staphylococcus aureus, streptococcus pneumoniae, and haemophilus influenzae, are common complications and should be suspected in relapses of fever, chest pain, and cough. 20 influenza is also associated with invasive meningococcal infections. other complications include exacerbations of asthma, chronic bronchitis, and congestive heart failure. myositis, myoglobinuric renal failure, meningoencephalitis, transverse myelitis, polyneuritis, parotitis, myocarditis, arthritis, and disseminated intravascular coagulation rarely occur after influenza. reye' s syndrome occurs in fewer than 1 per 100,000 cases of influenza in patients under 18 years of age following the use of salicylates. pregnant women, human immunodeficiency virus (hiv)-infected patients, and other immunocompromised hosts are at higher risk for severe disease and complications. 20 the virus infects the respiratory mucosa, where it causes lytic infection of cells and desquamation of the respiratory epithelium, mononuclear cell infiltrates in the lamina propria, and altered mucociliary clearance. tracheobronchitis is a typical feature and often associated with prolonged abnormalities in small airway pulmonary function and airway hyperreactivity. primary influenza viral pneumonia results in diffuse alveolar damage, alveolar hemorrhage and exudate, hyaline membranes, and later reactive fibrosis. fatal cases show pathologic changes in nonrespiratory organs, such as brain congestion and swelling, myocardial inflammation, and fibrinoid changes in arterioles. 20 viral replication in the upper respiratory tract generally peaks within 1 or 2 days of symptom onset and, depending on age and prior immunologic experience, continues for about 3 to 8 days. the severity of illness broadly correlates with upper respiratory tract viral levels. constitutional symptoms with influenza are due in part to the release of proinflammatory cytokines and chemokines. levels of interferon (ifn-α and ifn-γ), tumor necrosis factor (tnf-α), interleukins and chemokines (il-1β, il-6, il-8, il-10, mcp-10, mip-1α and mip-1β) are increased in nasal secretions, and ifn, il-6, and tnf-α are increased in blood in human influenza. 20 the tissue tropism of a strain of influenza virus depends, among other factors, on a combination of susceptibility of its ha to be cleaved by, and tissue availability of proteases with specificity to cleave it, thus rendering the virus infectious. 32 extrapulmonary dissemination of virus has been uncommonly documented in humans, but systemic spread is a regular feature of highly pathogenic avian viruses in chickens and sometimes in rodents or other mammalian hosts. serum and secretory antibodies directed to ha and na appear about 10 days after infection. protection against reinfection by the homologous strain is durable following natural infection and is correlated with serum and nasal neutralizing antibody levels, principally directed against ha. vaccine-induced protection may last for up to 2 to 3 years against homotypic virus. infection also induces cell-mediated immunity, which is detectable 3 to 6 days after infection and seems to be important for recovery. 30 cytotoxic t-lymphocyte responses against internal proteins may provide some degree of heterosubtypic immunity. the diagnosis of influenza is frequently made on the basis of clinical and epidemiologic information. a higher index of suspicion and laboratory diagnostics is needed outside the season, particularly in sporadic individual cases or unexplained outbreaks of febrile respiratory illness. viral isolation from respiratory specimens can be done in several types of cells (e.g., prmk, mdck, llc-mk2) and remains the current standard. 20 the presence of virus may be detected in cell cultures by hemadsorption with guinea pig erythrocytes before or after cytopathic effect (cpe) is visible. blind hemadsorption is positive 3 days after inoculation in almost all positive samples. 20 confirmation of isolates can be done by hemagglutination inhibition or immunofluorescence with typespecific antisera. diagnosis can also be made in 1 to 2 days by immunofluorescence of monolayers of mdck cells inoculated by centrifugation (shell-vial). 33 conserved influenza antigens (m or np) directly in clinical samples can be detected by one of several techniques (e.g., immunofluorescence [if], enzyme immunoassay [eia]), and multiple point-of-care kits are commercially available with turnaround time of 15 to 30 minutes. one commercial assay is based on detection of influenzaspecific na activity. the sensitivities of these assays are higher in children (up to 90%) than in adults (generally 50% to 70%) and depend on duration of illness and sample type. 20 several formats of reverse transcription-polymerase chain reaction (rt-pcr) assays have been used for the detection of influenza a and b rnas in clinical samples, with the advantage of detecting genomes of noninfectious virus. 20 the time to perform rt-pcr is longer but the cost may be lower than for commercial rapid antigen detection kits, especially in developing countries. real-time rt-pcr has enabled the development of assays that provide rapid quantitative detection of influenza a and b with high sensitivity. 34, 35 these assays have great potential to replace other methods, because they are simultaneously rapid, highly sensitive, quantitative, and amenable to being used in multiplex format, which might include probes for several different respiratory pathogens. 35 however, the costs are still prohibitive for most laboratories in developing nations. serologic diagnosis of influenza using paired acute and convalescent serum can be done retrospectively by a variety of techniques but mainly for serologic survey purposes. 20 amantadine and rimantadine are m2 ion channel blockers that inhibit influenza a virus replication at the uncoating step. 20 in uncomplicated influenza a in adults without underlying diseases, treatment with either drug can reduce the duration of influenza illness by approximately 1 to 2 days if started early, within 48 hours from the onset of symptoms. amantadine is excreted in an unchanged state in the urine, while rimantadine is extensively metabolized after absorption and less than 10% of the dose is excreted unchanged in the urine. elderly persons need only half the dose to achieve similar plasma levels. amantadine or rimantadine may cause gastrointestinal upset and central nervous system side effects. central nervous system (cns) intolerance is more common with amantadine and, when severe, can be manifested as agitation, psychosis, seizures, and coma. mild complaints including insomnia, dizziness, anxiety, dry mouth, anorexia, and nausea are reversible upon discontinuation. amantadine and rimantadine are marketed as 100-mg tablets and 10-mg/ml syrup. the recommended dose is 100 mg twice daily for adults older than 65 years of age (100 mg/day for patients ≥65 years of age). for children under age 10 years, a rimantadine dose of 5 mg/kg/day (maximum, 150 mg/day) has been suggested. 20 dose reductions proportional to the creatinine clearance (clcr) are suggested for patients with renal insufficiency (amantadine for clcr <60 to 80 ml/min/1.73 m 2 ; rimantadine for clcr <10 to 20 ml/min/1.73 m 2 ). influenza virus resistant to amantadine-rimantadine emerges in approximately one third of treated patients; such viruses are transmissible to close contacts and cause typical influenza illness. resistance to these drugs renders them ineffective and is sometimes present naturally, including in recent human isolates of h5n1 virus. 20 the neuraminidase inhibitors zanamivir and oseltamivir inhibit both influenza a and b viruses by blocking the active site of the enzyme for cleavage of sialic acid, thus inhibiting virus release from infected cells and spread within the respiratory tract. 36 in adults and children older than 5 years, inhaled zanamivir (10 mg twice daily for 5 days) provides 1to 2.5-day reduction in illness 37 and reduces antibiotic use for lower respiratory complications by 40%. zanamivir is generally well tolerated but may uncommonly induce bronchospasm, particularly in those with influenza and pre-existing airway disease. 20 oseltamivir (75 mg orally twice daily for 5 days) reduces illness severity, time to resumption of daily activities by 1 to 3 days, and rates of complications leading to antibiotic prescription and hospitalization by about 50% in adults. in children 1 to 12 years of age, oseltamivir reduces the frequency of otitis media and, consequently, antibiotic prescriptions. side effects include mild-to-moderate nausea or emesis. dosage of neuraminidase inhibitors does not need to be adjusted for the elderly. 20 resistance emergence is uncommon with both drugs, 20 although a recent study of children treated with oseltamivir detected drug-resistant viruses in 18%, often in association with prolonged viral excretion, and showed that children can be a source of viral transmission, even after 5 days of treatment. 38 antipyretic-analgesic drugs may be used for influenzainduced fever and aches. aspirin should be avoided because of its association with reye' s syndrome. immunization with formalin-inactivated or live-attenuated multivalent influenza virus vaccines and chemoprophylaxis for influenza virus a are the methods available for preventing influenza. influenza vaccine is used prior to the influenza season and currently includes one strain of influenza b and two strains of subtypes h3n2 and h1n1 of influenza a virus, chosen by the world health organization (who) surveillance network among the viruses most likely to circulate in the next influenza season. 20, 39 the inactivated vaccine has an approximate 70% to 90% efficacy in preventing illness in healthy children and adults. 39 it also reduces influenza-related hospitalizations and mortality in elderly and high-risk patients. the centers for disease control and prevention (cdc) recommends the immunization of persons aged 50 years and older; residents of nursing homes; children and adults with chronic cardiovascular or pulmonary disease, including asthma; persons chronically ill with diabetes mellitus, renal dysfunction, or hemoglobinopathies; immunosuppressed patients including those with hiv infection; children and adolescents on chronic aspirin therapy who may develop postinfluenza reye' s syndrome; women who will be pregnant during the influenza season; children aged 6 to 23 months; those who can transmit influenza to persons at high risk, such as health-care workers and household contacts of those at high risk including children 0 to 23 months of age; crew members of cruise ships; providers of essential services; and unimmunized travelers to areas where influenza may be circulating, including the tropics, the southern hemisphere between april and september, and those traveling in large organized tourist groups. in addition, vaccine is made available to anyone interested in reducing the likelihood of becoming ill with influenza. 20, 39 the inactivated vaccine, administered as a single intramuscular (im) dose shortly before influenza season (two doses in previously unimmunized children <9 years of age), is safe during pregnancy but should be avoided in persons with history of anaphylactic reactions to eggs. 39 vaccine safety and efficacy in children has been extensively evaluated and has shown a favorable safety profile with efficacy in 1-to 15-year-old children of 77% to 91%. inactivated vaccine is not currently recommended for children younger than 6 months, but vaccination of household contacts and caregivers should reduce the risk of these high-risk children contracting influenza. healthy people aged 5 to 49 years who are not contacts of immunosuppressed patients can receive either inactivated or intranasal live-attenuated vaccines. 39 influenza inactivated vaccine has been recently introduced in many tropical areas of the world, with a composition based on influenza viruses circulating in the southern hemisphere. the vaccine is given prior to the influenza season, which for most countries in the southern hemisphere is between may and july. 40 in south america, annual vaccination of the elderly has reduced hospitalizations and mortality for respiratory diseases. 41 continuous surveillance has already shown that regional variations of circulating influenza virus strains should be taken in consideration in the formulation of influenza vaccines with compositions more appropriate for south america. 42 live-attenuated, cold-adapted vaccines administered intranasally are well tolerated, genetically stable, and rarely transmissible and have the advantage of inducing local secretory immunoglobulin a (iga) responses. because of potential interference between components, two doses may be required in young children. 20 this vaccine was licensed in the united states in 2003, where it has become an option for healthy persons aged 5 to 49 years, including those in close contact with groups at high risk and those wanting to avoid influenza. 39 this vaccine is not recommended for persons with asthma and other chronic disorders of the pulmonary or cardiovascular systems; persons with underlying medical conditions, including diabetes, renal dysfunction, and hemoglobinopathies; persons with known or suspected immunodeficiency diseases or who are receiving immunosuppressive therapies; children or adolescents receiving aspirin or other salicylates; persons with a history of guillain-barré syndrome; pregnant women; and persons with a history of hypersensitivity to eggs. 39 cold-adapted trivalent influenza vaccine is highly effective (92% in phase 3 studies) in preventing cultureconfirmed influenza in healthy children and has provided protection against drift variant strains in some studies. in young and middle-aged adults, efficacy is generally comparable to that of inactivated vaccine. 39 other investigational approaches have been explored in influenza vaccine development, including recombinant ha produced in insect cells, virosomes incorporating surface glycoproteins, m2 protein conjugated with hepatitis b virus core, and naked dna encoding influenza virus nucleoprotein or ha. 20 cell culture-based vaccines (mdck, vero) have been approved in europe and may offer an alternative to the limitations of the current egg-grown vaccines. the technique of reverse genetics has been used to rapidly produce candidate vaccines against potential pandemic threat viruses. amantadine and rimantadine are approved for use, and are 70% to 90% effective in the prophylaxis of influenza a during outbreaks. unvaccinated elderly persons, immunodeficient patients, patients in chronic care institutions experiencing outbreaks, persons who could not be vaccinated, and those who received a vaccine strain different from the outbreak strain may receive prophylaxis with amantadine or rimantadine. prophylaxis should be started as early as possible at doses equivalent to those used for therapy, and continued until 1 week after the end of the outbreak for a total of at least 2 weeks. 39 amantadine-and rimantadine-resistant mutants of influenza a virus occur in up to 30% of treated patients and may be associated with failure of drug prophylaxis. 43 both oseltamivir (75 mg twice daily) and inhaled zanamivir (10 mg/dose twice daily) are more than 80% effective in the prophylaxis of influenza during outbreaks, but only oseltamivir has been approved for this indication in the united states. 20, 39 antiviral agents, especially the neuraminidase inhibitors, could significantly help in the control of a future influenza pandemic by reducing lower respiratory complications and hospitalizations as well as potentially person-to-person transmission. however, supply limitations 44 pose a real difficulty. therefore, policies to ensure a reasonable supply of these drugs, as well as directions to optimize the use of limited supplies, are important issues to be considered. 18 respiratory syncytial virus (rsv) is the single most important viral cause of lower respiratory disease and a major cause of morbidity and mortality in children worldwide. rsv is the leading cause of hospitalization in young children in developed and developing countries. 45 in tropical areas, rsv has been the most frequently isolated virus in hospital-based ari studies of children. 5 agent rsv, the only known human pathogen of the genus pneumovirus in the family paramyxoviridae, is a pleomorphic rna virus with helical nucleocapsid and lipid-containing envelope. antigenic differences in the surface glycoprotein g permit the classification of rsv into groups a and b, each with antigenic subgroups. 46 the interaction of rsv envelope glycoprotein g with glycosaminoglycans enables adherence to the cell surface. however, g protein-independent mechanisms of attachment must exist, since mutants devoid of g protein can also enter host cells. rsv enters the cell by fusion of viral envelope with cell membranes, a process mediated by binding of the viral f protein to the cell gtpase rhoa. 46 the syncytia resulting from fusion of the infected cells to adjacent ones are the major feature of the cytopathic effect of paramyxoviruses. once in the cytoplasm, the negative-strand rna is transcribed by viral transcriptase into mrnas, which then direct viral protein synthesis. an intermediate positivestrand full-length crna serves as a template for the synthesis of progeny negative-strand rna. as they bud through the cell membrane, the virions acquire a glycoprotein-containing envelope. 46 rsv causes asymptomatic infection in a variety of experimental animals, but natural infection occurs only in humans and chimpanzees. 47 rsv grows well in several human heteroploid cell lines, such as hep-2, hela, and a549, and is sensitive to ether, chloroform, detergents, and a ph less than 5. rsv is inactivated at 55°c, survives poorly on porous surfaces, and loses infectivity significantly by slow freezing and storage at temperatures above 4°c. 46 rsv occurs worldwide and causes annual outbreaks in temperate climates in the winter and early spring, with sporadic cases throughout the year. 47 in tropical regions, where temperature fluctuations are smaller and the only significant seasonal variable is often rainfall, rsv outbreaks tend to occur in the rainy seasons. such has been the case in malaysia, hong kong, india, papua new guinea, colombia, kenya, and the gambia. 12 interestingly, in singapore rsv peak activity occurs from march to august, a period of higher temperature, higher day-to-day temperature variation, and lower relative humidity. 48 in southeast brazil, rsv occurs seasonally, within a broader range of months from february through july, after the rainy season and when temperatures tend to be cooler, with slight variations from year to year. 49 in regions where average winter temperatures are colder, such as in são paulo city and the southernmost parts of brazil, as well as in argentina, rsv peak activity tends to occur in july and august. [50] [51] [52] most children have specific serum rsv antibody by age 2 years, but reinfections occur throughout life. more than one subtype of either rsv group may cocirculate in one season, with group predominance changing from year to year, without apparent correlation with clinical or epidemiologic characteristics of the illness they cause. 49,53 rsv transmission requires close contact and occurs either by large-particle aerosols or by contamination of hands and inoculation into the eye or nose. secondary infections in family contacts of an index case are common, after an average incubation period ranging from 2 to 8 days. 45 it is estimated that 30% of all infants will have rsv infection that requires medical attention and that 2% of them will be hospitalized. 45 an estimated 10% of children will have bronchiolitis in their first year of life, with 60% to 90% of those infections caused by rsv. 54 in southeast brazil, rsv is the leading cause of lower respiratory tract infections in children younger than 1 year of age and is responsible for up to 85% of hospitalizations in this age group during peak months. 52 the spectrum of illnesses caused by rsv ranges from mild uri to severe lri, including pneumonia, bronchiolitis, tracheobronchitis, and croup. 45 in infants and young children, uri with fever and otitis media is common. during outbreaks, rsv rna has been detected in up to 75% of middle ear effusions in children with rsv infection and acute otitis media. 55 the most frequent lri caused by rsv in infants is bronchiolitis, usually preceded by 2 to 3 days of uri symptoms, and progressing to lower respiratory tract involvement characterized by tachypnea, dyspnea, cough, expiratory wheezing, air trapping, and in more severe cases, intercostal muscle retractions and cyanosis. fever is present in only 50% of infants. chest radiographs may show hyperaeration of the lungs and sometimes segmented atelectasis. 45 blood counts usually show lymphocytosis, and an increase in neutrophils with a left shift could be associated with bacterial superinfection. the most frequent bacterial superinfection in children with rsv infections is acute otitis media, which may be found in up to 60% of children with brochiolitis. 56 however, more serious bacterial infections that may require sepsis work-up is uncommon in previously healthy infants with rsv infections. 57 this may be different, however, in developing tropical areas, where rsv frequently causes infections in children previously debilitated by other diseases and malnutrition. infants with congenital heart disease, premature infants, or infants with underlying pulmonary conditions, such as cystic fibrosis and bronchopulmonary dysplasia, as well as immunocompromised hosts of any age, are at risk for severe and fatal rsv infections. hiv-infected children with rsv infections have a higher rate of pneumonia and prolonged illness and virus shedding, but the general severity of the rsv disease is not increased. 45 differential diagnosis of acute bronchiolitis includes asthma, pneumonia, congenital heart and lung diseases, and cystic fibrosis. particular clinical signs are generally not accurate predictors of specific viral causes, but in a study conducted in the philippines, wheezing was a significant predictor of viral lri, while manifestations of higher severity, such as chest indrawing and cyanosis, were more often associated with bacterial lri. 58 the most frequent rsv illness in children over 3 years of age and adults is uri with coryza and cough, sore throat, and hoarseness, often accompanied by low-grade fever. exacerbations of chronic pulmonary diseases and wheezing can also be seen in adults with rsv infection. 45 the role of rsv infections in causing wheezing and asthma exacerbations in infants is well established in studies conducted in temperate areas. 59 similar observations have been made in an emergency room study conducted in southeast brazil, which found that infection with respiratory viruses, especially rsv, and a family history of allergy were independently associated with wheezing. 60 similar findings have been observed in urban nigerian preschool children. 61 rsv has been increasingly recognized as a cause of lri in the elderly, mainly characterized by interstitial pneumonia, prolonged cough, and dyspnea in persons with chronic pulmonary conditions, and it should be considered in the differential diagnosis of flulike illnesses. 62 rsv replicates in respiratory epithelium to reach titers as high as 10 6 tcid 50 /ml in nasal secretions of infected babies, and virus shedding may be as prolonged as 3 weeks after the symptoms disappear. 46 rsv spreads from cell to cell and may involve the entire respiratory tree, reaching bronchioles in 1 to 3 days after the onset of rhinorrhea. replication in the bronchiolar epithelium causes necrosis of ciliated cells, syncytia formation, peribronchiolar inflammation with abundant lymphocytes and macrophages, and impairment of secretion clearance, resulting in small airway obstruction and the hyperinflation characteristic of bronchiolitis. pneumonia frequently coexists, evidenced by interstitial mononuclear infiltrate, eosinophilic cytoplasmic inclusions in epithelial cells, and multinucleated giant cells. the most severe rsv disease occurs in young babies, whose immature airways may be unable to compensate for the pathologic changes. 46 naturally acquired immunity to rsv is incomplete and short-lived, but the severity of illness tends to decrease with reinfections. local secretory iga correlates better with protection than does serum antibody level and age, and pre-existing virus-specific maternal antibodies influence the development of neutralizing antibodies. cell-mediated immune response is central to recovery from rsv infection, and patients with suppressed cell-mediated immune response are at risk of severe rsv pulmonary disease and fatal outcome. 45, 46 the type of immune response to the virus is probably a major factor in the development of wheezing and asthma exacerbations. a bias toward a th2 cytokine response seems to be associated with more severe disease, whereas a th1 response leads to effective viral clearance and milder illness. the virus itself generally triggers a th1 response, but a preexisting th1 deficiency may be associated with disease severity in some children. it has been suggested that rsv bronchiolitis may be a marker of predisposition to wheezing or asthma later in life. 46, 63 children vaccinated with a formalin-inactivated rsv vaccine developed in the 1960s had severe disease when exposed to natural infection, apparently as a consequence of an imbalance between protective and immunopathologic t-cell responses elicited by previous parenteral immunization with inactivated rsv. this would favor a cd4+ th2 cytokine pattern in response to subsequent rsv infections, whereas a previous natural infection would favor a cd4+ th1 pattern in response to reinfection. 64 nasopharyngeal aspirates or swabs, nasal washings, and lower respiratory samples are all appropriate specimens for rsv isolation. this is usually accomplished in cultures of hep-2 cell line, in which rsv induces syncytia in 3 to 5 days. rsv antigen detection by eia, including membrane-based eia, is sensitive and specific and requires virtually no equipment, making it ideal for field studies. rapid rsv detection by if of exfoliated respiratory cells may be even more sensitive than eia-based methods. 45, 46 the increasing use of rapid tests has facilitated the assessment of rsv in tropical areas. 1 ideally, a combination of a rapid method with viral isolation should be used for maximal rsv detection, but the cost may still be prohibitive for the meager resources available in some tropical areas. detection of rsv rna by conventional rt-pcr has shown suboptimal sensitivity, especially when compared with easy-to-perform, more sensitive rapid methods. 45 however, more recently developed assays based on real-time rt-pcr are proving to be more sensitive than conventional rt-pcr assays, with the added conveniences of being rapid, quantitative, and amenable to simultaneous detection and subtyping of rsv directly from clinical specimens. 65 rsv serology has limited value for case management but may be useful for epidemiologic surveys. 45 treatment uri caused by rsv requires no specific treatment, and antibiotics are needed only when bacterial otitis media or sinusitis are present. 45 the supportive treatment of infants with rsv bronchiolitis consists basically in preventing hypoxemia and electrolyte imbalance, in addition to aerosolized bronchodilators. the lack of obvious correlation between radiologic findings and disease severity suggests that a chest film should be recommended only for severely ill or deteriorating infants. 54 to prevent hypoxemia, requirements may vary from simple removal of respiratory secretions and proper positioning of the infant to mechanical respiratory assistance and even extracorporeal membrane oxygenation (ecmo). pulse oximetry has been advocated to assess oxygen needs, but in tropical developing areas, where oximeters may not be available, serial clinical assessment is essential to monitor disease progression. for this purpose, crackles and cyanosis seem to correlate better with hypoxemia than tachypnea and intercostal retraction. 54 correction of hypoxemia can be accomplished with 40% or lower oxygen concentrations. 45 oxygen should be humidified with saline and delivered by mask if head boxes or tents are unavailable. the role of corticosteroids remains unclear with some evidence that they are not beneficial. 54 the only antiviral drug currently approved for the treatment of infants with rsv is the synthetic nucleoside ribavirin, delivered by small-particle aerosol via a mist tent, mask, oxygen hood, or ventilator. it is recommended only for infants and young children with an underlying condition, such as congenital heart disease, cystic fibrosis, or immunosuppression. premature infants, infants younger than 6 weeks of age, and severely ill infants may also be considered for therapy. 66 aerosolized ribavirin is well tolerated, but it is expensive and its prolonged administration requires facilities that may not be available in impoverished tropical areas. passive immunotherapy with rsv immunoglobulin, in combination with aerosolized ribavirin, improved the outcome of rsv pneumonia in bone marrow transplant patients. 67 the use of rsvintravenous immunoglobulin (ivig) or humanized monoclonal antibody against rsv has shown no benefit for the treatment of rsv infections in infants. 45 no vaccine is currently available for rsv prophylaxis. the disease enhancement caused by formalin-inactivated vaccine in the 1960s plus results of more recent unsuccessful trials of live-attenuated vaccines, have significantly slowed progress toward an rsv vaccine. 2 purified fusion protein vaccine has been tested for safety and immunogenicity in seropositive children older than 18 months, and was associated with reduction of lower respiratory tract illness, but not of rsv infection rates, in children with cystic fibrosis. 45 these and other candidate subunit vaccines, as well as intranasal liveattenuated vaccines, should be tested in high-risk children with underlying bronchopulmonary diseases. passive immunization of high-risk infants with monthly infusions of rsv immunoglobulin during the rsv season reduced the incidence and severity of rsv infections in highrisk children. 68 this costly intervention is the only available means of protecting high-risk children against serious rsv lri. monthly intramuscular injections of humanized monoclonal antibody should be considered for passive immunoprophylaxis during rsv season for high-risk infants such as preterm infants less than 6 months old, children with congenital heart disease, and children less than 2 years of age with bronchopulmonary dysplasia. 45 hospitalized infants with rsv infection should be isolated or grouped to prevent cross-infection. hand washing; use of eye-nose goggles, gowns, and gloves; and decontamination of surfaces and fomites are additional nosocomial infection control measures. 45 human parainfluenza viruses (hpivs) are the single most frequent cause of croup in infants and children worldwide and are second only to rsv as cause of lri in infants. 45, 69 little is known about the epidemiology of hpivs in tropical countries, but these viruses have been detected in up to 13% of children in hospital-based ari studies in developing countries. 5,21 hpivs are distributed in two genera of the family paramyxoviridae, sharing the structural and biological characteristics already mentioned in the rsv section. hpivs are classified antigenically into types 1 to 4, and hpiv-4 has subtypes a and b. hpiv types 1 and 3 are classified in the genus respirovirus, while hpiv types 2 and 4 are in the genus rubulavirus. hpiv-1 and -3 are the types most frequently associated with lri in children, the immunocompromised, the chronically ill, and the elderly, whereas piv-4 causes mostly uri in both children and adults. 69 binding of hpiv to sialic acid in the cell membrane is mediated by the glycoprotein hn, which contains hemagglutinin and neuraminidase activities. fusion of viral and cell membranes is mediated by the viral f protein, which is cleaved by cellular proteolytic enzymes. 45 once inside the cell, the cycle is similar to other paramyxoviridae, as summarized in the rsv section. hpivs can be propagated in primary simian or human kidney cells and in several cell lines, such as hep-2, vero, mdck, llc-mk2, bhk, and hela. 69 a variety of experimental animals undergo asymptomatic infection with piv, but only higher primates develop symptoms. 45, 69 epidemiology primary hpiv infection occurs early in childhood, and by age 5 virtually all children are seropositive. 19 an estimated one third of all viral lris in children in the united states are caused by hpiv-1 and -3. 69, 70 in most temperate regions, hpiv-1 and -2 cause epidemics in the fall of alternate years, either in co-circulation or alternating with one another. the biennial pattern of hpiv-1 is found in both hemispheres. 69 hpiv-1 causes most croup epidemics, whereas hpiv-2 more frequently causes illness with milder manifestations, although it can also cause croup. 69 hpiv-3 occurs endemically throughout the year, with sporadic spring outbreaks mainly among infants, and hpiv-4 occurs sporadically throughout the year in children and adults. 45, 69, 70 in tropical areas hpivs may account for up to 15% of child hospital admissions due to lri. 21 community-based ari studies in children under age 5 years show higher hpiv activity during rainy seasons in tropical countries. 3, 24 hpivs were the most frequent viruses detected in school-aged children with bronchial asthma exacerbations in urban nigeria. 61 hpivs spread mainly within families and closed communities, such as nurseries, day-care centers, and pediatric wards, with high secondary attack rates. in a longitudinal study conducted with children less than 2 years of age with ari in a day-care center for low-income families in northeast brazil, hpivs represented 11% of the viruses detected. 11 the virus does not persist long in the environment and is transmitted mainly by large droplets and fomites. 45 viral shedding usually lasts 3 to 10 days, but shedding of hpiv for months has been reported in very young children and immunosuppressed hosts. 71 primary hpiv infection may cause rhinitis, pharyngitis, laryngotracheobronchitis (croup), bronchiolitis, or pneumonia. approximately two thirds of all piv infections in children result in febrile uri with associated otitis media in 10% to 34%. the remaining one third of piv infections are cases of croup, bronchiolitis, or pneumonia. 61, 63 hpivs, mainly of types 1 and 2, cause up to 74% of all cases of croup. 69 croup is the most striking clinical presentation of hpiv infection and is most common between the ages of 6 and 36 months. 72 croup is manifested by inspiratory stridor, barking cough, and hoarseness caused by subglottic edema, preceded by rhinorrhea, mild cough, and low-grade fever. 45, 72 most children recover in 2 to 5 days, but some may develop bronchiolitis and pneumonia and present with a bronchopneumonia-croup syndrome. 45, 69, 72 since immunity to hpivs is incomplete, infections tend to occur throughout life, but little is known about hpiv infections in adults. in general, adults have only nonspecific uri, commonly with hoarseness. 45 hpivs can cause particularly severe diseases in immunocompromised hosts, especially children with severe combined immunodeficiency and bone marrow transplant patients. mortality in bone marrow transplant patients with hpiv infection varies from 10% to 20% in most series. 45, 69 hpivs replicate in ciliated epithelial cells, causing cytolysis of the respiratory mucosa. the infection begins in the upper respiratory tract and tends to disseminate down the respiratory tree. the larynx and trachea are mostly involved in the croup syndrome, and extensive involvement of the lower respiratory tree may be present in tracheobronchitis, bronchopneumonia, and bronchiolitis. 69, 71, 72 similar to influenza, factors determining the extent of hpiv infection include the susceptibility of the viral f protein to be cleaved and tissue-specific differences in the production of proteases to cleave it. 45 host immunity is largely mediated by humoral immunity to the two surface proteins hn and f. virtually all children by the age of 3 years will have seroconverted to hpivs, generally first to hpiv-3 but later also to hpiv-1 and -2. at school age, a significant proportion of children will have seroconverted also to hpiv-4. secretory antibody targeted to the hn glycoprotein is the best marker of protection against piv, 71 but the protection conferred by antibodies is limited, and repeated infections will develop. t-cell immune response seems to be involved in the clearance of virus and additionally in the development of inflammatory infiltrate, edema, and excess mucus secretion, 69 and immunocompromised hosts may develop progressive and even lethal disease. 73 like rsv, pivs cause mononuclear interstitial infiltrate, epithelial necrosis, inflammatory exudate into the alveoli, and hyaline membrane formation in the lungs. 45 piv is present in respiratory secretions until about 8 days from the onset of symptoms and can be isolated in monkey kidney primary cells and several continuous cell lines. virus can be detected in the monolayers by hemadsorption with guinea pig erythrocytes in around 3 days after inoculation and confirmed by if. 69, 71 shell-vial assays have been developed for hpiv detection but with mixed results. 69 if of exfoliated respiratory epithelial cells has produced conflicting and sometimes disappointing results, with most studies reporting sensitivities between 50% and 75% at best. 69 detection of viral rna by rt-pcr, including commercially available multiplex assays for several respiratory viruses, has enhanced the sensitivity of detection of hpiv from clinical samples. 69, 74 real-time pcr for respiratory viruses in multiplex format is sensitive and specific for hpiv. 35 at present, only supportive and symptomatic treatment is available for piv infections. management of croup includes supplemental oxygen and racemic epinephrine nebulization in hospitalized patients. mist therapy, although traditional, has no proven value. 72 short-term, high-dose systemic corticosteroids may reduce the need for intubation, and nebulized budesonide has a rapid effect and is as safe and efficacious as nebulized epinephrine in moderately severe croup. 72 several antiviral agents have in vitro activity against hpivs, but none has reached clinical testing. 69 there have been anecdotal reports of reduced hpiv shedding in immunocompromised patients treated with ribavirin, but this finding has not resisted scrutiny. 69 future possibilities include the bcx 2798 and bcx 2855 compounds, whose design is based on the threedimensional structure of the hn protein, which inhibit the hemagglutinin and neuraminidase activities of the protein and were effective in vitro and in an animal model against hpiv-1, -2 and -3. 75 no interventions are available for the prevention of hpiv infections. early trials with inactivated hpiv vaccine in the 1960s were unsuccessful. 69 recently, a live-attenuated, coldadapted hpiv-3 vaccine was found to be immunogenic for children as young as 1 month of age and holds promise for further development. 69 the same vaccine was tested in combination with a live-attenuated rsv vaccine candidate, showing that this approach is feasible and deserves further study. 76 characterization of hpiv proteins hn and f has led to development of subunit immunogens that showed efficacy in animal models. 69 human rhinoviruses (hrvs) are the most frequent respiratory pathogens of humans. 77 they were the most frequently isolated viruses in children under 5 years of age with ari in an urban slum in northeast brazil. 3 human rhinoviruses are small, nonenveloped, positivestrand rna viruses in the family picornaviridae, with over 100 identified serotypes. 77 hrv serotypes have been classified according to receptorspecificity into three groups. the major group includes 91 serotypes whose receptor is intercellular adhesion molecule-1 (icam-1); the minor group contains 10 serotypes whose receptor is the low-density lipoprotein receptor (ldlr); and the remaining serotype, hrv-87, utilizes a sialoprotein as cell receptor. unlike other picornaviruses, hrvs are acid-labile, a property that distinguishes them from enteroviruses. 77 the hrv genome is a monocistronic single-stranded rna, packed in an icosahedral capsid composed of 12 pentamers. surrounding the fivefold vertex, each pentamer contains a 1.2-to 3.0-nm-wide canyon that contains the receptor binding site. following receptor binding, the viral positivestrand rna is released into the cytoplasm and directs the synthesis of a polyprotein, whose cleavage products include an rna polymerase. this enzyme will produce an expanding pool of positive-strand rna using as a template an intermediate negative-strand rna. the positive-strand rna can be either translated into virion proteins or packaged as a genome into newly assembled virions. the hrv replication cycle takes place in the cytoplasm, and mature virions are released when the host cell is lysed. 77 hrvs are resistant to ethanol, ether, chloroform, and nonionic detergent but are sensitive to uv light; to ph lower than 5 and higher than 9; and to halogens such as chlorine, bromine, iodine, and phenolic disinfectants. they are stable for days on environmental surfaces and for years at minus 70°c. 77 hrv infects only higher primates and causes illness only in humans. several cell lines of primate origin support hrv propagation, but certain strains of hela cells and human embryonic fibroblasts provide higher sensitivity for hrv isolation from clinical specimens. 78 the optimal growth temperature for hrv is 33°c to 35°c. 77 hrv infections occur in people from all continents, including remotely located population groups, such as bushmen from the kalahari desert, native alaskans, and an isolated amazon indian tribe. 79 hrv has been estimated to cause up to 80% of all autumn colds in temperate climates. 80 in tropical countries, very few community-based studies of viral ari have used adequate hrv detection methods, 5 and this has limited the assessment of the actual impact of hrv in those areas. however, available evidence indicates that hrv is frequently associated with ari in children in brazil. in fortaleza, a city in northeast brazil, hrv detected by isolation in cell culture represented 46% of the viruses in children under 5 years of age with ari. 3 in salvador, another city in the same region, hrv represented 52% of the viruses detected by rt-pcr in association with ari in children younger than 2 years of age attending a day-care center for the underprivileged. 11 data on the frequency of hrv among adults in tropical countries are even more scarce. in singapore, hrv was detected in 20% of the samples obtained from adults with ari symptoms attending primary care centers. 81 hrv transmission requires close exposure and occurs mainly by hand-to-hand contact, followed by self-inoculation into the eye or nose, but also by airborne spread. once hrv reaches the nasal cavity, infection occurs in virtually 100% of susceptible subjects, and approximately 75% of those infected develop illness after a 1-to 2-day incubation. 77 children play a central role in spreading the virus in the household. evidence suggests that indoor hrv transmission is favored by high relative humidity and crowding of young children, as occurs in the united states at the beginning of the school term, which may explain the autumn seasonal peak of hrv. 77 in tropical northeastern brazil, however, where relative humidity remains above 70% reaching 90% during the rainy season, longitudinal studies have found no obvious hrv seasonality. 3, 11 hrv colds are indistinguishable from colds of other viral causes and consist of nasal discharge, nasal obstruction, sneezing, sore or scratchy throat, hoarseness, cough, and headache. facial and ear pressure may be present. fever and malaise are uncommon. these symptoms last approximately 7 days but may persist for up to 2 weeks in 25% of cases. infants and toddlers may display only nasal discharge and be otherwise asymptomatic. 77 the majority of patients have obstruction and mucosal abnormalities of the sinus cavities, eustachian tubes, and middle ear, which predispose to secondary bacterial sinusitis and otitis media, each complication found in approximately 2% of all colds. 82 hrv rna may be detected by rt-pcr in maxillary sinus brushings in 40% of adults presenting with acute sinusitis, 83 and in 24% of the samples of middle ear fluid from children less than 7 years of age with diagnosis of acute otitis media. 84 hrv is frequently associated with exacerbations of chronic obstructive pulmonary disease and asthma attacks in children over 2 years of age and in adults. 59, 77, 85 hrv replication is restricted to the respiratory epithelium, taking place in scattered ciliated cells of the nose and in nonciliated cells of the nasopharynx. 86 this tropism seems to be a consequence of receptor availability. infection of a limited number of cells triggers the release of cytokines, chemokines, and inflammatory mediators, which together with stimulation of the local parasympathetic nerve endings, results in the cold symptoms. kinins, prostaglandins, and proinflammatory cytokines and chemokines may contribute to vasodilation, increased vascular permeability, influx of polymorphonuclear leukocytes, exocrine gland secretion, and nerve ending stimulation, resulting in nasal obstruction, rhinorrhea, sneezing, cough, and sore throat. 77 serotype-specific neutralizing igm, igg, and iga antibodies develop in most infected persons in 7 to 21 days and persist for years. protection from infection is partially attributed to the presence of iga antibody in nasal secretions, and recovery from illness is more dependent on cell-mediated immunity. hrv-induced blastogenesis, natural killer cell activity, mitogen-stimulated cell production of il-2 and ifn-γ have been documented during hrv infection. 77, 87 hrv induces the expression of human β-defensin 2 (hbd-2) in the respiratory epithelium, which supports a role for hbd-2 in host defense to hrv infection. 88 hrv can be detected in respiratory secretions by isolation in cultures of susceptible cell lines. 78 hrv shedding peaks around 48 hours after infection and declines rapidly, but may remain at low levels for up to 3 weeks. 77 cultures should be kept at 33°c to 35°c in a roller drum and examined for 10 to 14 days. the presence of hrv, indicated by the typical cpe, is confirmed by the acid sensitivity of the isolate. rapid immunocytochemical methods are not available because of the large number of serotypes. rt-pcr in clinical samples is more sensitive and less tedious than hrv isolation, 83 and the recently introduced real-time pcr-based assay is more sensitive than conventional rt-pcr. 89 pcr-based assays have been useful in studies to assess the impact of hrv in different settings. the homotypic nature of hrv antibodies restricts serology to experimental settings. 77 trials of antiviral agents for hrv have been conducted, but no specific treatment suitable for routine use has yet been identified, mainly because of lack of potency, untoward side effects, and drug delivery problems. 90 ruprintrivir, a selective inhibitor of hrv 3c protease, has potent, broad-spectrum anti-hrv activity in vitro. a double-blind, placebo-controlled clinical trial of intranasal ruprintrivir in experimental hrv infection reduced symptoms by 33% and also decreased viral titers and nasal discharge. 91 symptomatic relief from cold symptoms can be obtained with a broad variety of nonprescription medications. systemic sympathomimetic decongestants, such as pseudoephedrine, may reduce nasal obstruction, first-generation antihistamines may reduce sneezing and rhinorrhea, and nonsteroidal antiinflammatory drugs such as naproxen or ibuprofen may reduce headache, cough, and systemic symptoms. 77 the large number of hrv serotypes with minimal crossantigenicity has hampered the development of an hrv vaccine. it may be possible to reduce exposure to hrv by hand washing after contact with a cold sufferer or after handling objects that may have been contaminated with respiratory secretions. 77 studies in experimentally infected volunteers show that application of the virucidal agents salicylic acid or pyroglutamic acid to the hands reduced recovery of rhinovirus from the hand skin of treated persons as compared with controls. 92 this result suggests that rhinovirus transmission can be prevented by virucidal hand treatments. short-term, postexposure prophylaxis by intranasal ifn-α significantly reduced the incidence of hrv colds in household contacts of an index case. 93 however, the cost and difficulty of making the drug available to homes in a timely fashion reduce the utility of this approach for extended use by populations, especially in tropical countries. ruprintrivir has also been evaluated for prophylaxis of hrv colds starting 6 hours prior to inoculation of human volunteers. this approach reduced the proportion of subjects with positive viral cultures and viral titers but did not affect the frequency of colds. 91 respiratory infections caused by adenoviruses are among the most frequent illnesses that these viruses cause, particularly in children under age 5 years. 94 adenoviruses have been frequently isolated in ari studies in tropical countries. 5 in the south cone of south america, adenoviruses were the second most frequent virus recovered from children hospitalized for ari. 95 adenoviruses are nonenveloped, icosahedral dna viruses of the genus mastadenovirus in the family adenoviridae. 94 adenoviruses are distinguished antigenically by group-specific (a through f) and type-specific (1 through 49) antigens and by genomic subtypes identified by restriction site mapping. 93, 94 the adenovirus capsid consists of three morphologically, antigenically, and functionally distinct types of capsomers: hexons, penton bases, and fibers that project from the penton bases. the hexon and penton bases contain complementfixing, group-specific antigens common to all human adenoviruses, whereas the fibers have primarily neutralizing and hemagglutination-inhibiting, type-specific antigens. adenoviruses are commonly accompanied by small, singlestranded dna parvoviruses known as adenoassociated viruses, which do not seem to cause any specific disease. most people have antibodies to at least one of the four serotypes of adenoassociated virus by age 10 years. 96 the fiber protein binds to the host cell, through the protein coxsackie and adenovirus receptor (car) of the immunoglobulin superfamily, which serves as a high-affinity receptor for adenoviruses. the class i major histocompatibility complex (mhc) also may serve as receptor for adenovirus 5. 94 ligand-receptor interaction facilitates interaction of the penton base with cell surface integrins, which triggers entry. after endocytosis, the double-stranded linear genomic dna is transported to the nucleus, where "early" and "late" sets of viral genes are transcribed, resulting in mrnas coding for structural and nonstructural proteins. virus assembly takes place in the nucleus, and the infectious cycle is completed by the release of up to 1 million virions upon cell lysis. 94 adenoviruses replicate well in continuous cell lines of epithelial origin, such as hep-2, hela, and a549, and can be adapted to grow in human embryonic lung fibroblasts. they are stable over a wide ph range (5 to 9), resistant to isopropyl alcohol, ether, and chloroform, stable for weeks at room temperature and for years at approximately 20°c or colder, and can be lyophilized. they are inactivated by sodium hypochlorite and a temperature of 60°c for 2 minutes. 97 respiratory transmission of adenoviruses occurs at all ages but is of prime importance during epidemics among military recruits. ocular transmission has been associated with swimming pools and physician offices where sterilization or hand washing has been inadequate. asymptomatic infection and a prolonged carrier state are common. 94 low-number adenovirus serotypes (1, 2, 3, and 5) are more frequent before age 5 years and account for 5% to 20% of cases of uri and approximately 5% of cases of lri in children. 98 in adults, adenoviruses occur sporadically and cause mostly uri. infections by adenoviruses types 4 and 7 are usually epidemic, with attack rates of 6% to 16% per week in newly assembled military recruits, whose adenovirus carriage rate may be as high as 18%. 94 in this group, the adenoviral syndromes vary from mild colds to severe lri, but overall attack rates may reach 80%, with 20% to 40% of the individuals needing hospitalization. 94 in temperate climates, adenoviral infections are more frequent in late winter, spring, and early summer, whereas in northeast brazil they seem to occur year-round. 5 in salvador, also in northeast brazil, adenoviruses were detected in 11% of children younger than age 2 years with ari in a day-care center. 11 in tropical areas the incidence of adenovirus infections in military recruits is lower, and different serotypes may be involved. 98 pharyngoconjunctival fever, commonly caused by adenoviruses types 3 and 7, may be epidemic or endemic among children during the summer in temperate climates. inadequate chlorination or filtration of swimming pools and lakes has been associated with epidemics. 99 the incubation period of adenovirus infections averages 10 days. 98 adenovirus respiratory diseases may involve all parts of the respiratory tract, and up to 50% of nonepidemic infections are asymptomatic. in fact, adenoviruses were discovered because of their propensity for latency in adenoidal tissue. 94, 98 in southeast brazil, adenoviruses were detected with equal frequency in wheezing young children and asymptomatic controls. 60 most adenoviral illnesses consist of febrile colds, and in children the fever may be high and long-lasting. pharyngitis is common and may be associated with fever, pharyngeal exudate, granular appearance of the mucosa, and anterior cervical adenopathy, similarly to streptococcal pharyngitis. 94 adenoviruses can be recovered from up to 20% of cases of pharyngitis in small children. pharyngitis may be concurrent with pharyngoconjunctival fever, a syndrome caused by adenovirus types 3 and 7 and characterized by conjunctivitis, frequently unilateral, which may last for 1 to 2 weeks, preauricular adenopathy, cough, rhinitis, malaise, and fever. 99 the most frequent complication of adenoviral colds is acute otitis media, which occurs in up to 30% of cases. 100 adenovirus lris consist mainly of bronchitis and pneumonia, and may make up over 10% of childhood lris in temperate areas. 101 adenoviruses may cause permanent lung parenchymal damage, especially when concurrent with measles. 101 epidemic adenoviral infections in military recruits have a spectrum of clinical manifestation ranging from colds to severe pneumonia. typically, however, the manifestations are fever, pharyngeal symptoms, cough, chest pain, headache, and malaise. 98 overwhelming pneumonitis may be part of disseminated adenoviral infections in newborn infants and patients with immunodeficiencies, including acquired immunodeficiency syndrome (aids). however, the frequent concomitance of other respiratory pathogens in aids patients and the high prevalence of asymptomatic adenovirus infection shed doubt on the causal role of the adenovirus in these patients. 102 adenoviruses are also an important cause of epidemic keratoconjunctivitis. 94, 98 while most adenoviral aris are self-limited and uncommonly associated with death or permanent sequelae, 94 adenoviruses alone or associated with other pathogens have been recovered from 20% of fatal cases of lri in argentina. 103 adenoviral respiratory disease results from necrosis of cells of airway epithelia, and viremia may result in disseminated infection in immunocompromised persons. bronchiolitis, interstitial pneumonitis, and mononuclear cell infiltrates are part of the inflammatory process in the lungs. it remains unclear why certain strains are more virulent than others. for example, the genomic variant b7h was associated with the majority of fatal lower respiratory disease in south america. 95 in addition to lytic infection, adenoviruses may become latent in epithelial and lymphoid cells, which is probably important to maintaining the virus in populations. 94 a possible role of latent adenovirus in the pathogenesis of chronic airway inflammation has been suggested. 94, 96, 104 protection from adenovirus infection and disease is mainly due to type-specific neutralizing antibody, but reinfections, mostly asymptomatic, may occur. a long-lived t-cell immune response develops in most infected immunocompetent persons and is not only responsible for recovery but also is involved in tissue pathologic changes. 94 adenoviruses can be detected in respiratory, ocular, or ear secretions, but clinical correlation is required, because asymptomatic virus shedding is common. isolation of adenoviruses in cell culture with identification by if constitutes the standard diagnostic method, but direct detection of viral antigens or viral dna by pcr in clinical samples is an attractive rapid alternative. 94 rapid antigen detection by immunochromatography is around 95% sensitive in comparison with cell culture, and easily can be used in point-of-care diagnosis of adenovirus. however, both conventional and real-time pcr are more sensitive than cell culture is. 105 positive results by pcr should be interpreted with caution, given the propensity of adenoviruses to cause latency. adenoviruses cause a characteristic cpe in a variety of cell lines of human origin, and maintenance of cultures for 2 weeks combined with blind passage (i.e., passage of cells even without obvious cpe to see if it develops after passage) may increase adenovirus recovery. 3, 94 inoculation of cells by centrifugation followed by immunostaining may shorten the detection time. 106 several serologic tests can detect antibodies to the common hexon antigen. 94 however, their clinical utility is restricted. at present, there is no routine effective antiviral treatment for adenovirus infections. successful therapy of severe adenoviral infections in immunocompromised patients with iv ribavirin has been reported. 107 cidofovir has shown some efficacy in the rabbit ocular model of adenoviral infection. iododeoxyuridine and adenine arabinoside were unsuccessful in the treatment of adenoviral keratoconjunctivitis. 94 a live vaccine consisting of wild-type adenovirus packaged in enteric-coated capsules induces immunity by ensuring enteric infection without infection of the respiratory tree. this approach has been used successfully to vaccinate military recruits against adenoviruses types 4 and 7. 98 proper sterilization, hand washing, and chlorination can prevent adenovirus spread via tonometers, hands, and swimming pools. coronaviruses are enveloped viruses with distinct virion morphology, displaying widely spaced, long petal-shaped spikes at the surface, that confer a crownlike appearance, the origin of the name corona. the envelope contains a long helical nucleocapsid with single, positive-stranded rna, 27 to 32 kb in size, which is the largest known viral rna genome. 108 until very recently, only three human coronaviruses (hcovs) were known to exist: hcov-229e, hcov-oc43, and the cov associated with severe acute respiratory syndrome (sars-cov). recently, two groups in the netherlands almost simultaneously published studies that resulted in the identification of two new strains of hcov: hcov-nl63 109 and hcov-nl. 110 in addition, pcr primers directed to conserved replicase 1a sequences of animal covs led to the identification of yet another hcov detected in 8.8% of children from new haven, connecticut with symptoms of ari. this agent was designated hcov-nh and is likely to represent the same species of hcov-nl and -nl63. 111 on the basis of antigenic and genetic studies, the known human coronaviruses are distributed in three of the four coronavirus groups so far identified. hcov-229e, -nh, -nl, and -nl63 belong to group i, hcov-oc43 belongs to group ii, and sars-cov is the only known constituent of group iv, while group iii contains no known human viruses and consists only of the avian infectious bronchitis virus. coronavirus rna synthesis occurs in the cytoplasm via a negative-strand rna intermediate. the viral rna possesses a 5′ cap followed by a leader sequence and an untranslated region, with another 3′ terminal untranslated region followed by a poly(a) tail. the genome is polycistronic and the synthesis of subgenomic negative-sense rnas is done by discontinuous transcription to originate a nested set of subgenomic mrnas that share the 5′ leader sequence and overlap at the 3′ end. the envelope contains the structural proteins s (spike), m (membrane), e (envelope), and only in the case of some group ii coronaviruses, ha (hemagglutinin). the s glycoprotein contains neutralizing and t-cell epitopes and functions as the cell receptor ligand, thereby determining tissue tropism. the m protein is embedded in the envelope and interacts with the n (nucleocapsid) protein during maturation. in addition to the nucleocapsid and envelope proteins, a replicase is present in cells infected by all coronaviruses. new virions assemble by budding through intracellular membranes and are released through vesicles of the secretory pathway. 108 hcov-229e and -oc43 are considered to be second only to rhinoviruses as agents of common colds, causing infections with variable frequency, depending mainly on the detection method and season of the study. up to 35% of mild upper respiratory tract infections in adults have been attributed to hcov-229e. 112 while hcov-229e and -oc43 are documented causes of colds in temperate regions, their impact as causes of respiratory infections in tropical regions has not been defined. human coronaviruses were first isolated in england, almost 40 years ago, in human organ cultures of tracheal and nasal tissues. there have been relatively few field studies based on hcov isolation in cell culture, likely because these viruses are too fastidious to be propagated, but most respiratory isolates obtained so far have been antigenically similar to either hcov-229e or -oc43. 112 these agents have the same structural features as the other members of the family. the s protein of hcov-229e binds to the metalloprotease human aminopeptidase n at the cell surface, and entry is independent of enzymatic activity of the receptor. the hemagglutinin of hcov-oc43 binds to sialic acid present in glycoproteins on the cell surface and this interaction facilitates infection, but to the best of our knowledge, a specific receptor has not been identified for this agent. 108 hcovs have been found throughout the world and are considered to be the second most frequent cause of common cold, accounting for an average rate of 15% of respiratory illnesses in the general population in the united states. however, the rates may be quite variable from year to year, ranging from 1% to 35% in years of peak activity. hcov infections occur mainly in the winter and spring months, but summer activity has also been documented. during the autumn peak of rhinovirus activity, 8% of the adults with a cold negative for rhinovirus were positive for hcov by rt-pcr in charlottesville, va. 80 hcov-229e has caused well-documented winter outbreaks at 2-to 4-year intervals in temperate regions. 112, 113 similarly, winter outbreak of hcov-oc43 has also been detected in europe. 114 in contrast, little is known about the prevalence of hcov-229e and -oc43 in tropical countries. in brazil, the activity of hcov-229e as cause of respiratory infections in nonhospitalized children was first documented by serology in the early 1970s, with a seropositivity rate of 26% in adults by complement fixation assay. 115 the usual manifestations of hcov infection are typical common colds. the incubation period tends to be 1 day longer that that for rhinovirus colds, with illness duration of 6 to 7 days. low-grade fever may occur in up to 20% of the patients, and in addition to nasal symptoms, cough and sore throat occur frequently. more serious infections of the lower respiratory tract caused by hcov have also been documented, either sporadically in infants with pneumonia and immunocompromised patients, or in up to 33% of previously healthy marine corps recruits with pneumonia. 112, 113 in addition, hcov-229e and -oc43 have been recognized in association with influenza-like illnesses in frail elderly patients. eight of 100 (8%) nasopharyngeal swabs from older patients hospitalized for cardiopulmonary illnesses during the influenza seasonal outbreak in rochester, n.y., were positive for hcov (five for hcov-229e). 116 respiratory hcov infections have been associated with exacerbations of asthma, chronic bronchitis, and recurrent wheezing in children. 112, 113 hcov was detected by rt-pcr in 38 of 292 (13%) episodes of asthma in children 9 to 11 years old in england. 117 in brazil, hcov was detected in respiratory samples from 3 (2 oc43 and 1 229e) of 73 (4%) children younger than 2 years of age who came to the er with wheezing. 60 similarly to hrv, hcov infections have been frequently recognized in association with otitis media and maxillary sinusitis in children and adults. hcov was detected by rt-pcr in the middle ear effusion or nasopharyngeal aspirate from 16 of 92 (17%) children with acute otitis media in finland 84 and in nasal swabs from 3 of 20 adults with acute maxillary sinusitis. 83 there is no convenient small animal model to study the pathogenesis of hcov, and humans naturally or experimentally infected are the only source of information obtained in vivo. hcovs are transmitted by the respiratory route, and experimentally infected volunteers shed virus for approximately 5 days, beginning 48 hours after infection, which is approximately the time of onset of symptoms. 112, 113 the peak of symptoms occurs 2 to 4 days postinoculation. 112 hcov-229e is known to infect airway epithelial cells from the apical surface, where the receptor is constitutively expressed, and to exit productively infected cells through the same route. 118 ultrastructural studies of nasal epithelium of volunteers experimentally infected with hcov-229e revealed significantly greater epithelial cell damage, ciliary loss, and cytolysis in virus-inoculated subjects than in sham-inoculated ones on day 3 postinfection. 119 in the united states, seropositivity to hcov-oc43 and -229e rises during the first 5 years of life, and around 40% of adults are seropositive. symptomatic reinfections are possible, despite the presence of antibodies, suggesting rapidly waning immune response or circulation of closely related but antigenically different viruses. 113, 120 several studies indicate that respiratory hcovs are able to reach the central nervous system. 112, 113, 120 the recently reported temporal association between hcov-nh infection and kawaski disease 121 awaits confirmation. laboratory diagnosis in clinical samples by isolation is tedious, because the two best characterized strains of hcov are difficult to grow in routine cell cultures. since primers can be developed for relatively constant parts of the genome, rt-pcr-based assays for hcov-229e and -oc43 have recently become the best alternative to other methods of detection. 80 more recently, a quantitative real-time pcr-based assay for hcovs has been developed, providing a faster means for detection and determination of viral load with potential applications in clinical studies. 122 serologic diagnosis of hcov by eia is sensitive and specific and has been useful in epidemiologic surveys. 113 intranasal interferon protects against experimental infection with hcov-229e, 123 but no specific antiviral therapy is available, and treatment of hcov-induced colds remains largely symptomatic. no vaccines are currently available for hcov. the hcov that fulfills koch`s postulates as the causative agent of sars 127 shares structural features and genome organization of the family coronaviridae ( fig. 59-1) . the prompt recognition of the peculiar morphology of a coronavirus in the electron microscopic studies of vero e6 cells inoculated with oropharyngeal material from a patient was the initial finding that resulted in the identification of sars-cov. 128 the viral genome is 29,727 nucleotides in length, with more than 11 open reading frames coding for 23 putative proteins, some of which have unknown functions. sars-cov is phylogenetically different and equidistant from all previously known coronaviruses, but isolates from different origins are relatively homogeneous genetically. genome analysis reveals that sars-cov is neither a host-range mutant nor a recombinant of respiratory tract viral infections ■ 651 previously known coronaviruses but rather an independently emerged virus. sars-cov seems to have evolved from an animal sars-like virus, acquiring greater fitness in humans during the course of the outbreaks, probably through the appearance of nucleotide deletions in open reading frame 8. 129 it is also noteworthy that genetic signatures present in the genomes allow for differentiation of isolates obtained from different clusters. 129 the replicative cycle of sars-cov is thought to follow the same main steps as other coronaviruses. sars coronavirus (sars-cov) probably emerged around november 2002 in the province of guangdong, china, where there was no serologic evidence of infection caused by this virus in sera of healthy humans sampled prior to that time. 130 at the beginning of the outbreak, many affected individuals in guangdong were directly or indirectly involved with game trade, and indeed, palm civets and raccoon dogs from wildgame markets in the area were later found to harbor a cov 99% homologous to sars-cov at the nucleotide level. this suggests that animal-to-human interspecies transmission was involved in the outbreak, providing the source of an agent that later adapted to efficient human-to-human transmission. 131, 132 interestingly, shortly after the lifting of a wildlife trade ban that originally had been imposed to control the sars outbreak, new cases were again detected in guangdong, all of them caused by viruses newly introduced from animals. since the ban was reinstalled, there have been no further naturally acquired human cases of sars in guangdong. 131 remarkably, 1.8% of 938 serum samples from adults recruited in 2001 in hong kong tested positive for sars-cov antibodies, suggesting that a small proportion of healthy people from hong kong, as opposed to guangdong, china, had been exposed to sars-related viruses at least 2 years before the outbreak. 133 it is probable that sars-cov precursors previously crossed the species barrier and may even have caused subclinical human infection, but perhaps only occasionally this event generated strains adapted to successful human-to-human transmission. 131 sars-cov is mainly transmitted between humans by the deposition of infected droplets or aerosols on the respiratory epithelium. the number of confirmed secondary cases generated by one index case of sars is relatively low, ranging from 2.2 to 3.7, suggesting relatively inefficient transmission. in addition, transmission is infrequent during the first 5 days of illness, partly because of the low viral load in respiratory secretions during that phase. for reasons not completely understood, some sars patients, identified as superspreaders, disproportionately contribute to the generation of a high number of secondary cases. 131 excretion of sars-cov in sputa and stools may average 21 and 27 days, respectively, after symptom onset, but an excretion period as prolonged as 126 days has been documented in stools. 134 such prolonged shedding of virus in feces raises the possibility of oral-fecal transmission and, in fact, one outbreak of sars was attributed to a faulty sewage system. 131 case-fatality rates estimated based on cases admitted to hospital have been around 13% for patients younger than age 60 and 43% for those older than age 60 years. however, it is likely that case-fatality rates based on all infections occurring in the community would be lower. 135 transmission of sars-cov among health-care workers and between patients in the hospital setting played a pivotal role in outbreak propagation. analysis of data from initial outbreaks indicates that close contact is the most important factor leading to nosocomial transmission of this agent. despite the lack of complete studies on the sensitivity of sars-cov to different environmental conditions, there have been reports of sars-cov persisting for up to 2 days on environmental surfaces and 4 days in diarrheal stools. 136 the median incubation period of sars is 4 to 6 days. clinical symptoms and signs of sars appear 2 to 10 days after exposure, and systemic symptoms, such as fever, chills, myalgia, and malaise, usually appear first. respiratory symptoms appear 2 to 7 days later, represented most frequently by nonproductive cough, dyspnea, chest pain, headache, and sore throat. diarrhea and vomiting may occur. chest radiograms frequently reveal infiltrates consistent with viral pneumonitis, consisting mostly of consolidations and ground-glass opacifications. computed tomography (ct) scans in patients with normal or equivocal chest radiograms may show unilobar or multilobar abnormalities. fever generally subsides in 48 hours, but one or two relapses within 8 to 15 days are frequently observed. lymphopenia with reduction of both cd4+ and cd8+ cells, slight decrease in platelet counts, prolonged coagulation profile, and elevated serum enzymes (lactic dehydrogenase [ldh], creatinine kinase [ck], and c-reactive protein [crp]) are often observed. around one third of patients may have cd4+ lymphocyte counts below 200 cells/mm 3 and higher susceptibility to secondary infections. watery diarrhea with an average of six evacuations per day is common. 130, 131, 137 radiologic worsening of the pulmonary lesions seen at admission, with or without appearance of new lesions, is a frequent observation, with development of diffuse groundglass changes frequently heralding the development of acute respiratory distress syndrome (ards). hypoxemia is noted in approximately half of the patients at around 9 days after the onset of symptoms, and a high proportion of those admitted to the intensive care unit (icu), especially older males, require mechanical ventilation around day 13. development of spontaneous pneumomediastinum during follow-up is not uncommon, probably as a consequence of ruptured peripheral lung lesions into the pleural space. 130 prognosis is related to the level of viral replication in tissues, and patients with high viral loads in serum, nasopharyngeal aspirates, or feces, as well as those in whom virus can be detected from multiple sites, tend to have poor clinical outcome. 131 in addition to old age and severe underlying diseases, ck and crp levels have been identified as predictors of poor outcome. 138 the n-terminal portion of the spike glycoprotein is needed for virus attachment to the virus receptor, identified as the metallopeptidase angiotensin-converting enzyme homolog (ace-2), 139 but it is unclear whether the mechanism of entry is contingent on ph-dependent endocytosis. 131 some inconsistencies between ace-2 and sars-cov tissue distribution suggest that ace-2 may not be the only receptor, or that a coreceptor molecule may be needed for cell infection. sars-cov spike protein can also bind the dendritic cell-specific c-type lectin intercellular adhesion molecule 3-grabbing nonintegrin (dc-sign), which does not result in dendritic cell infection by the agent but allows for sars-cov to be transported to susceptible target cells elsewhere. 140 sars-cov has been detected in studies using different combinations of immunohistochemistry, in situ hybridization, and electron microscopy in pneumocytes and on the apical surface of enterocytes. marked inflammatory infiltrates and mucosal atrophy have not been observed in the intestine, and the pathogenesis of the sars-cov-related diarrhea remains largely unknown. 131 sars-cov viral load in the upper airways is low in the 4 initial days, with the peak at day 10 of illness. quantitative rt-pcr for sars-cov in nasopharyngeal aspirates from patients who tested positive at admission revealed viral loads around 10 5 copies/ml on days 5 and 15 after clinical onset, and peak 10 7 copies/ml on day 10. 130 higher viral loads can be detected in the lower respiratory tract than in the upper airways. pulmonary tissue shows diffuse alveolar damage, mixed infiltrate, lung edema, hyaline membrane, abundant macrophages in alveoli and interstitium, and syncytia formation. besides respiratory secretions and stools, sars-cov can be detected in urine in up to 30% of patients, with titers averaging 10 4.4 copies/ml, in association with abnormal urinalysis results. 141 the effect of sars-cov infection on the immune system is highlighted by pronounced t-cell lymphopenia and elevation of several inflammatory cytokines (il-1β, il-6, and il-12) and chemokines (mcp-1 and ip-10) observed in sars patients. while mcp-1 is likely to be involved in the lung monocytic/macrophagic infiltrate, its role is not firmly established, since other viral diseases that are associated with elevated mcp-1, such as influenza, do not include such prominent histologic features. in addition, since immunologic markers in the peripheral blood may not reflect what happens in the microenvironment of the lung, the pathogenic importance of these findings is not clear. co-inheritance of hla-b*0703 and -b60 is higher among sars patients than in the general population, favoring a role for the genetic background in susceptibility to sars-cov. 131 the pathogenesis of the t-cell lymphopenia remains unknown. seroconversion has been documented in 93% of the patients at around 20 days and the rise in igg titers correlates with decrease in viral load. 130 paradoxically, clinical worsening also occurs during this phase, suggesting that, rather than unchecked viral replication, immunopathologic factors may be responsible for the lung lesions. 130 while infection in experimental animals, such as cynomolgus macaques, ferrets, cats, golden syrian hamsters, mice, and african green monkeys, does not induce disease that mimics that in humans, these models are important for studies of pathogenesis and development of vaccines and therapy. 129 in addition, the development of an infectious cdna clone of sars-cov should permit reverse genetics experiments and may help elucidate determinants of viral pathogenesis. 142 low viral loads in the upper respiratory tract in the first few days of illness account for the relatively poor sensitivity (35% to 65%) of first-generation rt-pcr for diagnosis in that period. sars-cov is detectable by rt-pcr in nasopharyngeal aspirates in only one third of patients at presentation and in two thirds at day 14. rt-pcr may be positive for sars-cov in stools from as much as 97% of patients at day 14, and in urine in 42% of samples at day 15. 130 testing multiple nasopharyngeal, serum, and fecal samples increases the sensitivity of the diagnosis by rt-pcr. 143, 144 to overcome the low sensitivity of conventional rt-pcr, quantitative real-time pcr-based assays for sars-cov have been developed that improve sensitivity and turnaround time, allow for amplification and analysis to be done in a closed system, and thus reduce cross-contamination. in addition, the capability of the assay to quantitate viral load has contributed not only to understanding viral pathogenesis but also to predicting outcome, since high viral loads are associated with poor prognosis. 143 the ability to grow sars-cov in vero e6 cell cultures was critical to identifying the agent. sars-cov can be recovered by isolation from respiratory secretions, feces, and urine in the first 3 weeks of illness, but the overall sensitivity is relatively low and recovery is more likely to be successful from respiratory secretions than from stools and urine. 143 recent small outbreaks of sars-cov originating in laboratories 143 have heightened concern about laboratory safety issues regarding sars specimens. the who guidelines for biosafety in the diagnosis of sars (updates available at the who web site) recommend that propagation of sars-cov in cell culture for isolation or for preparation of viral stocks and cell slides be performed in biosafety level 3 (bsl3) laboratories, whereas handling serum and blood specimens for routine tests and serology can be performed in bsl2 laboratories. nucleic acid extraction procedures, inoculation of bacterial or mycologic cultures, and preparation of sample smears can be done in bsl2 laboratories, observing bsl3 work practices (use of safety cabinets, sealed centrifuges, protective equipment, 5% bleach spillage decontamination, and proper waste disposal). although not useful for early diagnosis, seroconversion determined by ifa or eia remains the gold standard for confirming sars diagnosis. igg seroconversion is detectable in over 90% of patients at around day 28. 130 antibody crossreaction with other human coronaviruses, however rare, remains a possibility; therefore, confirmation of positive serology by an independent neutralization assay should be performed if available. 143 the main component of treatment of sars patients is supportive therapy, chiefly the management of hypoxemia and ards. during the 2003 outbreak, treatment included a broadspectrum antiviral agent (ribavirin) and immunosuppressive doses of corticosteroids, aimed at reducing the immunopathologic damage to the lungs. the use of high-dose steroid therapy is controversial and for the most part supported by anecdotal evidence, whereas the use of ribavirin is based on the broad antiviral spectrum of the drug. however, sars-cov is only modestly susceptible to ribavirin in vitro, and therapeutic doses are difficult to achieve clinically. since it became possible to grow sars-cov in culture, many potential antiviral compounds have been evaluated in vitro, but just a few have been tested in animal models and even fewer are in clinical testing. 131 interferons (ifn-αn1/n3, leukocytic ifn-α, ifn-β) and hiv protease inhibitors were consistently active in vitro and may be considered for animal testing and clinical trials. 131 the resolution of the structure of sars-cov principal protease has prompted studies of the inhibitory capacity of known anti-hiv protease inhibitors for treatment of sars. in one open-label study, a combination of hiv protease inhibitor (lopinavir plus pharmacokinetic booster ritonavir) and ribavirin was used to treat sars patients and the outcomes were compared with historical controls treated with ribavirin alone. at day 21 after onset of symptoms, development of ards or death was significantly less frequent in the group treated with the combination (2.3%) than in historical controls (28.8%). in addition, peak viral loads in respiratory samples and stools were reduced in the group treated with the combination as compared with controls. 145 however, since there were differences in outcome predictors, such as sex, platelet counts, and ldh levels, between the two groups, these results should be interpreted with caution. a preliminary open-label study found that a restricted number of patients treated with subcutaneous interferon alfacon-1 in association with corticosteroids showed reduced oxygen-saturation impairment and faster resolution of radiographic chest findings than those treated with corticosteroids alone. 146 convalescent plasma has also been tested in the treatment of sars patients. in one preliminary uncontrolled study, convalescent plasma may have reduced the frequency of poor outcome when given before 14 days of illness. 147 it is impossible to predict whether naturally reemerging sars-cov would be likely to cause a global outbreak. nevertheless, a vaccine for this agent would be relevant for high-risk individuals, such as workers in laboratories, hospitals, and game-animal farming. therefore, considerable effort has been directed at developing such a vaccine. it has been shown that sars-cov spike protein produced in bacteria and expressed on chimeric parainfluenza virus, as well as spike protein-encoding dna, induced neutralizing antibodies and protected experimental animals from challenge with live virus. at present, no sars-cov vaccine is available for human use. therefore, in the absence of person-to-person transmission of sars-cov worldwide, prevention of future outbreaks of sars requires careful surveillance. the goal is to maximize early detection of new cases of sars to implement control measures, thereby minimizing social disruption. 131 to reach this goal, the cdc recommends testing for sars-cov in patients who require hospitalization for radiographically confirmed pneumonia or ards without identifiable etiology and who have one of the following risk factors in the 10 days before the onset of illness: (1) travel to mainland china, hong kong, or taiwan, or close contact with an ill person with a history of recent travel to one of these areas, or (2) employment in an occupation associated with a risk for sars-cov exposure (e.g., health-care worker with direct patient contact; worker in a laboratory that contains live sars-cov), or (3) belonging to a cluster of cases of atypical pneumonia without an alternative diagnosis (updates on these recommendations are made available at the cdc web site http://www.cdc.gov/ncidod/sars). during times of overt sars activity, prevention of humanto-human transmission is pivotal to curtailing outbreaks. although sars infectiousness relative to the onset and termination of clinical symptoms has not been accurately determined, it is clear that shortening the time from onset to hospital admission and isolation reduces the risk of transmission, thus contributing substantially to curtailing of outbreaks. identification of new cases through contact tracing played an important role in the control of the outbreaks registered so far. stringent isolation procedures must be adopted for confirmed and suspected cases, which require a high level of alertness among health-care workers for early identification of sars cases. the scenario may be further complicated in situations in which other diseases such as influenza and hantavirus pulmonary infections may occur simultaneously. 135 rates of transmission of sars-cov among health-care workers vary, depending on stringency of control measures adopted, presence of so-called superspreaders in the hospital, and kind of activities carried out by personnel, especially as related to proximity to the index case. assisting during intubation, suctioning, and manipulating ventilatory apparatuses seem to be high-risk activities. while studies conducted in different settings have produced conflicting results, one study in toronto found that up to 25% of the nurses who cared for sars patients in critical care units became infected. 148 the presence of severe watery diarrhea may add to the challenge for the infection control team. 130 an updated set of recommendations for health-care and laboratory personnel is available at the cdc web site (http://www.cdc.gov/ncidod/sars). a new paramyxovirus was described in the netherlands in 2001, in association with respiratory illness in children. the agent was first detected by analysis of previously unidentifiable viral isolates that induced cytopathic effect in llc-mk2 cell cultures. the isolates were recovered over a 10-year period in respiratory secretions from 28 children with ari occurring in the winter time. electron microscopy of cell culture isolates revealed paramyxovirus-like particles, and rna sequencing revealed genome sequences and organization consistent with a paramyxovirus of the subfamily pneumovirinae, most closely related to avian pneumovirus of the genus metapneumovirus. rather than an avian virus that can also infect humans, this agent is now recognized as a primarily human pathogen, and thus has been named human metapneumovirus (hmpv). 156,157 hmpv antibodies detected in sera collected in 1958 in the netherlands indicate that this agent has been in circulation for at least 4 to 5 decades. 156 agent hmpv particles are enveloped, pleomorphic, spherical, and filamentous particles, with a mean diameter of about 209 nm. 156,157 complete genome sequences of hmpv are available and, in contrast to the genomic organization of pneumoviruses, metapneumoviruses have different positioning of the genes between m and l and lack ns1 and ns2 genes. 156,158 similar to hrsv, genetic and antigenic studies indicate that hmpv isolates cluster into two main serotype named a and b, with n gene sequences 83% to 85% similar at the nucleotide level, each subgroup including two genetic lineages (a1, a2, b1, and b2). 156, 159, 160 both are globally distributed. there have been no detailed studies of the hmpv replication cycle, but it is likely to be similar to that of other human paramyxoviruses. hmpv is a frequent cause of community-acquired ari in children and adults in all continents, although with variable incidence in different settings. 157, 159, [161] [162] [163] [164] [165] [166] [167] [168] [169] in the united states, hmpv has been reported in up to 20% of lower respiratory tract illnesses whose etiology would have been unidentifiable prior to the development of assays for the detection of hmpv. 170 in canada, during the 2001-2002 winter season, hmpv was detected in 15% of patients of all age groups from four different provinces. 167 similar to hrsv, hmpv infections are more frequent in the colder months in temperate regions, and different strains of both subgroups a and b cocirculate during the same year. 157, 162 however, only limited knowledge is available about hmpv seasonality in more tropical climates. peaks of hmpv activity have been documented in the spring/summer in hong kong, 171 while in south africa hmpv has been detected in 6% to 9% of children with ari admitted to hospitals in the winter season. 161 ,165 hmpv was detected alone or simultaneously with rsv in 24% of children younger than 3 years of age admitted to health-care facilities in aracaju, northeast brazil, in the months of april and may, 2002. 164 interestingly, hmpv was not detected by the same methods in that same city, in the following year. 166 this apparent variability in hmpv incidence from year to year has also been observed in studies conducted in argentina 168 and italy, where hmpv frequencies varied from 7% to 43% in three consecutive annual respiratory virus seasons. 172 long-term prospective studies will be needed to establish whether there is a seasonal pattern in hmpv circulation in tropical regions of the world. clinically, hmpv infections resemble closely those caused by hrsv, ranging from mild upper ari to severe bronchiolitis and pneumonia. the median age of children hospitalized with hmpv infection is older than those with hrsv. hrsv in hospitalized infants and young children may require intensive care and mechanical ventilation, 156,173,174 and dual infection with hmpv and hrsv appears to increase the likelihood of severe illness. 175, 176 the most frequent symptoms in all age groups are fever, dyspnea, cough, wheezing/stridor, rhinitis, and sore throat. 162, 173 all infected children in one study had either pneumonia or bronchiolitis, frequently accompanied by otitis media. 162 hmpv may cause more serious infections in patients with comorbid or immunosuppressive conditions, as well as in the very young and the elderly. 162, 167 in one study, all individuals older than 65 with lower respiratory infection caused by hmpv had at least one underlying chronic or debilitating condition, including lymphoma, leukemia, or neurologic or cardiovascular diseases. 162 hmpv infection in adults may present as influenza-like illness, acute bronchitis, or common cold. 162 in england, in the winter of 2000-2001, hmpv was detected by rt-pcr in association with 2.2% of samples taken from patients in all age groups with influenza-like illnesses negative for hrsv and influenza viruses. 159 hmpv has been increasingly recognized as cause of acute wheezing in children. one study conducted in finland found hmpv in 8% of wheezing children, who presented significantly higher levels of il-8 in nasal secretions as compared to children with hrsv-associated wheezing. 174 a study conducted in brazil found that 47% of the children with hmpv had wheezing and 31% had chest indrawing. 164 previous history of asthma has been more frequently associated with hmpv than with hrsv infection and hmpv-infected patients are more often treated with bronchodilators and corticosteroids than hrvs-infected patients. 173 little is known about specific mechanisms of pathogenesis and host immune response in hmpv infections. hmpv is a pathogen of both the upper and lower respiratory tracts. 162 hmpv replicates efficiently in the respiratory tract of monkeys, with virus shedding peaking between days 2 and 8 following infection. 156 serologic data indicates that hmpv infects young individuals, and by the age of 5 virtually all children have become seropositive for the agent; reinfections at later ages are common. 156, 157, 159 interestingly, coinfection with hmpv has been reported to correlate with increased severity of hrsv infections. a study conducted in the united kingdom found that this coinfection caused a tenfold increase in the relative risk of admission to the icu for mechanical ventilation in children under 2 with hrsv bronchiolitis. 173 a similar finding was also reported in germany. 176 hmpv can be isolated in llc-mk2 cells from nasal aspirates or nasopharyngeal swabs. the cytopathic effect, characteristically negative on hemadsorption testing, develops usually late after inoculation (up to 23 days). 160 sensitive rt-pcr assays for this agent have been developed in many different laboratories and have rapidly become standard for hmpv diagnosis. 159,169 a real-time pcr assay for hmpv showed to be more sensitive than conventional rt-pcr, even when hybridization was used to increase sensitivity of the detection of amplicons generated by the conventional method. 169 using real-time pcr, hmpv was detected in 10% of 329 samples collected from patients with ari in australia from march to october 2001 that were negative for other pathogens. 169 other than supportive measures, oxygen therapy, bronchodilators, corticosteroids and mechanical ventilation, there is no specific antiviral treatment for this agent. 173 ribavirin is inhibitory for hmpv in vitro. 176 although a hmpv vaccine is not available at this time, the demonstration that hamsters, ferrets, and african green monkeys are susceptible to infection by hmpv, and that hamsters vaccinated with serotype a the epidemiology of acute respiratory tract infection in young children: comparison of findings from several developing countries report of a workshop on respiratory viral infections: epidemiology, diagnosis, treatment and prevention acute respiratory viral infections in ambulatory children of urban northeast brazil longitudinal studies of infectious diseases and physical growth of infants in huascar, an underprivileged peri-urban community in at the edge of development: health crises in a transitional society epidemiology of acute respiratory infections in children of developing countries pan american health organization: acute respiratory infections in the americas the magnitude of mortality from acute respiratory infections in children under 5 years in developing countries acute lower respiratory tract infections in hospitalized patients with diarrhea in dhaka day-care center attendance and hospitalization for lower respiratory tract illness viral respiratory infections in young children attending day care in urban northeast brazil epidemiology and seasonality of respiratory tract virus infections in the tropics the cultural context of breastfeeding: perspectives on the recent decline in breast-feeding in northeast and northcentral brazil reduced mortality among children in southern india receiving a small weekly dose of vitamin a search for a solution: blending oral rehydration therapy (ort) and popular medicine pathogenesis of respiratory infections due to influenza virus: implications for developing countries respiratory viruses predisposing to bacterial infections: role of neuraminidase influenza: emergence and control orthomyxoviridae: the viruses and their replication influenza virus viral vaccines for the prevention of childhood pneumonia in developing nations: priorities and prospects the effect of influenza on hospitalizations, outpatient visits, and courses of antibiotics in children etiology of acute respiratory infections in children in tropical southern india a community-based study of acute respiratory tract infection in thai children etiology of acute lower respiratory tract infection in children from alabang, metro manilla outbreak of influenza type a (h1n1) in iporanga antigenic and genomic relation between human influenza viruses that circulated in argentina in the period 1995-1999 and the corresponding vaccine components regional perspectives on influenza surveillance in africa h5n1 influenza: a protean pandemic threat influenza type a and b infections in hospitalized pediatric patients influenza viruses, cell enzymes, and pathogenicity detection of influenza virus by centrifugal inoculation of mdck cells and staining with monoclonal antibodies rapid detection and simultaneous subtype differentiation of influenza a viruses by real time pcr rapid and sensitive method using multiplex real-time pcr for diagnosis of infections by influenza a and influenza b viruses, respiratory syncytial virus, and parainfluenza viruses 1, 2, 3, and 4 rational design of potent sialidase-based inhibitors of influenza virus replication efficacy and safety of the neuraminidase inhibitor zanamivir in the treatment of influenzavirus infections resistant influenza a viruses in children treated with oseltamivir: descriptive study centers for disease control and prevention: prevention and control of influenza: recommendations of the advisory committee on immunization practices (acip) influenza vaccination in 2000: recommendations and vaccine use in 50 developed and rapidly developing countries immunization against influenza in the elderly: the argentinian experience regional perspectives on influenza surveillance in south america clinical and epidemiologic importance of influenza a viruses resistant to amantadine and rimantadine pandemic influenza: is an antiviral response realistic? respiratory syncytial virus and parainfluenza viruses respiratory syncytial virus brief report: respiratory syncytial virus activity-united states seasonal trends of viral respiratory tract infections in the tropics occurrence and severity of infections caused by subgroup a and b respiratory syncytial virus in children in southeast brazil viral etiology of acute respiratory infections among children in porto alegre, rs, brazil respiratory syncytial virus: changes in prevalence of subgroups a and b among argentinian children clinical patterns and seasonal trends in respiratory syncytial virus hospitalizations in sao paulo antigenic characterization of respiratory syncytial virus group a and b isolates in rio de janeiro, brazil management of acute bronchiolitis detection of genomic sequences of respiratory syncytial virus in otitis media with effusion in children acute otitis media in children with bronchiolitis prevalence of serious bacterial infections in febrile infants with respiratory syncytial virus infection etiology of acute lower respiratory tract infection in children from alabang, metro manilla rhinovirus and respiratory syncytial virus in wheezing children requiring emergency care. ige and eosinophil analyses risk factors for wheezing in a subtropical environment: role of respiratory viruses and allergen sensitization microbial inciters of acute asthma in urban nigerian children winter viruses: influenza-and respiratory syncytial virus-related morbidity in chronic lung disease pathogenesis of respiratory syncytial virus bronchiolitis-related wheezing pathogenesis of respiratory syncytial virus vaccine-augmented pathology evaluation of quantitative and typespecific real-time rt-pcr assays for detection of respiratory syncytial virus in respiratory specimens from children american academy of pediatrics committee on infectious diseases: reassessment of the indications for ribavirin therapy in respiratory virus infections combination therapy with aerosolized ribavirin and intravenous immunoglobulin for respiratory syncytial virus disease in adult bone marrow transplant recipients role of antibody and use of respiratory syncytial virus (rsv) immune globulin to prevent severe rsv disease in high-risk children parainfluenza viruses parainfluenza viral infections in pediatric outpatients: seasonal patterns and clinical characteristics biology of parainfluenza viruses viral croup: a current perspective parainfluenza and influenza virus infections in pediatric organ transplant recipients simultaneous detection of fourteen respiratory viruses in clinical specimens by two multiplex reverse transcription nested-pcr assays efficacy of novel hemagglutinin-neuraminidase inhibitors bcx 2798 and bcx 2855 against human parainfluenza viruses in vitro and in vivo evaluation of combined live, attenuated respiratory syncytial virus and parainfluenza 3 virus vaccines in infants and young children clinical virology comparative susceptibilities of human embryonic fibroblasts and hela cells for isolation of human rhinoviruses rhinovirus antibodies in an isolated amazon indian tribe frequency and natural history of rhinovirus infections in adults during autumn acute respiratory symptoms in adults in general practice computed tomographic study of the common cold detection of rhinovirus in sinus brushings of patients with acute community-acquired sinusitis by reverse transcription-pcr detection of rhinovirus, respiratory syncytial virus, and coronavirus infections in acute otitis media by reverse transcriptase polymerase chain reaction viral infections in relation to age, atopy, and season of admission among children hospitalized for wheezing localization of human rhinovirus replication in the upper respiratory tract by in situ hybridization peripheral blood mononuclear cells interleukin-2 and interferon-g production, cytotoxicity, and antigen-stimulated blastogenesis during experimental rhinovirus infection human rhinovirus infection induces airway epithelial cell production of human beta-defensin 2 both in vitro and in vivo rhinovirus detection: comparison of real-time and conventional pcr clinical studies of antiviral agents for picornaviral infections phase ii, randomized, double-blind, placebo-controlled studies of ruprintrivir nasal spray 2-percent suspension for prevention and treatment of experimentally induced rhinovirus colds in healthy volunteers efficacy of organic acids in hand cleansers for prevention of rhinovirus infections prevention of natural colds by contact prophylaxis with intranasal alpha2-interferon fields' virology molecular epidemiology of adenovirus acute lower respiratory infections of children in the south cone of south america (1991-1994) latent viral infections in airway epithelium diagnostic procedures for viral, rickettsial and chlamydial infections viral infections of humans prevention: outbreak of pharyngoconjunctival fever at a summer camp-north carolina acute otitis media and respiratory virus infection pneumonias in childhood: etiology and response to antimicrobial therapy adenovirus infections in human immunodeficiency virus-positive patients: clinical features and molecular epidemiology etiologic, clinical and pathologic analysis of 31 fatal cases of acute respiratory tract infections in argentinian children under 5 years of age latent adenoviral infection in the pathogenesis of chronic airway obstruction evaluation of a bedside immunochromatographic test for detection of adenovirus in respiratory samples, by comparison to virus isolation, pcr and real-time pcr rapid diagnosis of respiratory viral infections by using a shell vial assay and monoclonal antibody pool intravenous ribavirin therapy for disseminated adenovirus infection coronaviridae: the viruses and their replication identification of a new human coronavirus a previously undescribed coronavirus associated with respiratory disease in humans evidence of a novel human coronavirus that is associated with respiratory tract disease in infants and young children clinical virology viral infection of humans an outbreak of coronavirus oc43 respiratory infection in normandy, france seroepidemiologic study of coronavirus infection in brazilian children and civilian adults rhinovirus and coronavirus infection-associated hospitalizations among older adults community study of role of viral infections in exacerbations of asthma in 9-11 year old children human coronavirus 229e infects polarized airway epithelia from the apical surface the effects of coronavirus on human nasal ciliated respiratory epithelium fields' virology. philadelphia association between a novel human coronavirus and kawasaki disease frequent detection of human coronaviruses in clinical specimens from patients with respiratory tract infections by use of a novel real-time reversetranscriptase polymerase chain reaction intranasal interferon as protection against experimental respiratory coronavirus infection in volunteers severe acute respiratory syndrome: identification of the etiological agent update: outbreak of severe acute respiratory syndrome worldwide a novel coronavirus associated with severe acute respiratory syndrome comparative full-length genome sequence analysis of 14 sars coronavirus isolates and commom mutations associated with putative origins of infection clinical progression and viral load in a community outbreak of coronavirus-associated sars pneumonia: a prospective study severe acute respiratory syndrome isolation and characterisation of viruses related to sars coronavirus from animals in southern china sars-related virus predating sars outbreak long-term sars coronavirus excretion from patient cohort epidemiological determinants of spread of causal agent of severe acute respiratory syndrome in hong kong sars: responding to an unknown virus clinical manifestations, laboratory findings, and treatment outcomes of sars patients angiotensin-converting enzyme 2 is a functional receptor for the sars coronavirus ph-dependent entry of severe acute respiratory syndrome coronavirus is mediated by the spike glycoprotein and enhanced by dentritic cell transfer through dc sign viral loads in clinical specimens and sars manifestations reverse genetics with a full-length infectious cdna clone of severe acute respiratory syndrome coronavirus the aetiology, origins, and diagnosis of severe acute respiratory syndrome evaluation of reverse transcription-pcr assays for repid diagnosis of severe respiratory syndrome associated with a novel coronavirus role of lopinavir/ritonavir in the treatment of sars: initial virological and clinical finings interferon alfacon-1 plus corticosteroids in severe acute respiratory syndrome use of convalescent plasma therapy in sars patients in hong kong sars among critical care nurses acute respiratory tract infections among a birth cohort of children from cali, colombia, who were studied through 17 months of age etiology of acute lower respiratory tract infections in children in a rural community in the gambia viral agents of acute respiratory infections in young children in kuala lampur viral etiology and epidemiology of acute respiratory infections in children in nairobi, kenya diagnoses of acute lower respiratory tract infections in children in rawalpindi and islamabad a study of nonbacterial agents of acute lower respiratory tract infection in thai children patterns of acute respiratory tract infection in children: a longitudinal study in a depressed community in metro manila characterization of human metapneumoviruses isolated from patients in north america analysis of the genome sequence of a human metapneumovirus human metapneumovirus as a cause of community-acquired respiratory illness antigenic and genetic variability of human metapneumoviruses human metapneumovirusassociated lower respiratory tract infections among hospitalized human immunodeficiency virus type 1 (hiv-1)-infected and hiv-1-uninfected african infants virological features and clinical manifestations associated with human metapneumovirus: a new paramyxovirus responsible for acute respiratory infections in all age groups human metapneumovirus as a causative agent of acute bronchiolitis in infants human metapneumovirus infection in hospital referred south african children respiratory syncytial virus and metapneumovirus in children over two seasons with a high incidence of respiratory infections in brazil human metapneumovirus infections in the canadian population evidence of human metapneumovirus in children in argentina molecular assays for detection of human metapneumovirus human metapneumovirus in lower respiratory tract disease in otherwise healthy infants and children children with respiratory disease associated with metapneumovirus in hong kong human metapneumovirus associated with respiratory tract infections in a 3-year study of nasal swabs from infants in italy metapneumovirus and acute wheezing in children dual infection of infants by human metapneumovirus and human respiratory syncytial virus is strongly associated with severe bronchiolitis prospective study of human metapneumovirus infection in children less than 3 years of age identification of small animal and primate models for evaluation of vaccine candidates for human metapneumovirus (hmpv) and implications for hmpv design were protected from challenge with either a or b hmpv serotypes opens possibilities for hmpv vaccine design. 176 humanized neutralizing monoclonal antibody to f protein is active in experimentally infected animals. 177 key: cord-275828-c6d6nk7x authors: mikasa, keiichi; aoki, nobuki; aoki, yosuke; abe, shuichi; iwata, satoshi; ouchi, kazunobu; kasahara, kei; kadota, junichi; kishida, naoki; kobayashi, osamu; sakata, hiroshi; seki, masahumi; tsukada, hiroki; tokue, yutaka; nakamura-uchiyama, fukumi; higa, futoshi; maeda, koichi; yanagihara, katsunori; yoshida, koichiro title: jaid/jsc guidelines for the treatment of respiratory infectious diseases: the japanese association for infectious diseases/japanese society of chemotherapy – the jaid/jsc guide to clinical management of infectious disease/guideline-preparing committee respiratory infectious disease wg date: 2016-07-31 journal: journal of infection and chemotherapy doi: 10.1016/j.jiac.2015.12.019 sha: doc_id: 275828 cord_uid: c6d6nk7x nan the japanese association for infectious diseases (jaid) and japanese society of chemotherapy (jsc) announced the "guide for the use of antimicrobial drugs" in 2001 and the "guidelines for the use of antimicrobial drugs" in 2005. subsequently, the "the jaid/ jsc guide to clinical management of infectious diseases 2011" was published. with its revision, guidelines were newly prepared. concerning respiratory infectious diseases, in japan, the japanese respiratory society published guidelines for the management of community-acquired pneumonia, hospital-acquired pneumonia, respiratory tract infection, and -/nursing and healthcare-associated pneumonia. furthermore, the japanese society of pediatric pulmonology and japanese society for pediatric infectious diseases announced the "guidelines for the management of respiratory infectious diseases in children in japan". internationally, many guidelines, including those established by the american thoracic society and infectious diseases society of america, have been published from various countries. thereafter, clinical research on respiratory infectious diseases has advanced, leading to the accumulation of many outcomes regarding epidemiology, clinical diagnosis, and treatment. however, the types of microorganisms that cause respiratory infectious diseases have increased with the number of resistant bacteria. in addition, conditions have also varied with causative microorganisms through the recent compromised host's severe status. the place of treatment varies: from the outpatient clinic to the icu. physicians responsible for treatment also vary: practitioners, hospital doctors, pulmonologists, emergency physicians, board certified member of jaid, japanese antimicrobial chemotherapy physician. there are a large number of options of antimicrobial drugs that are available, including new drugs; therapeutic strategies are confused. on the other hand, recently, the entity of pk-pd has been commonly recognized, and the importance of scientifically using antimicrobial drugs has been emphasized. in addition, the japanese society of chemotherapy established a system for antimicrobial chemotherapy-certified physicians, and promoted the widespread, adequate use of antimicrobial drugs. based on these, the two societies prepared the jaid/jsc guidelines for the treatment of respiratory infectious diseases. if specific treatment guidelines can be presented, this may contribute to an improvement in the treatment responses of respiratory infectious diseases, a reduction in health expenditure, and the prevention of resistant bacteria. the guidelines were prepared based on the ebm so that they reflected the management of respiratory infectious diseases in japan and covered all such diseases in adults and children. to prepare the guidelines, a committee was established in 2012, and a draft was published on homepage based on an approval from the boards of directors at the two societies through a review-based consensus. opinions were collected from the two societies' members. in japan, there have been no such guidelines covering respiratory infectious diseases. in the future, with further advances in research, the contents of the guidelines must be revised. however, we successfully provided treatment guidelines that are the most advanced at present. the guidelines were prepared for all clinicians to understand the treatment of respiratory infectious diseases and manage them with antimicrobial drugs adequately. they do not limit treatment by individual physicians or affect their rights to select it. the guidelines may be commonly applied for respiratory infectious disease management/research/education in japan, improving the quality of respiratory infectious disease management, preventing an increase in the number of resistant bacteria, and contributing to national health. we hope that the guidelines will be utilized by a large number of clinicians in respiratory infectious disease management. lastly, we thank the committee members and secretariat staff for their cooperation. 1. descriptions on the recommendation grade and evidence level 2. definition of firstand second-choice drugs 3. precautions -in this article, with respect to the administration method (especially doses) of antimicrobial drugs, they are recommended based on sufficient doses. considering the products adopted at each medical institution, antibiograms, severity, underlying disease, age, and presence or absence of organ disorder, the dose should be increased or decreased if necessary. -the spectra of third-generation cephems for intravenous injection, ctx and ctrx, are similar, but ctx, which is excreted in the kidney, should be primarily used when liver dysfunction is present, and ctrx, which is excreted in bile, should be primarily used when renal dysfunction is present. -as quinolones exhibit antitubercular actions, patients with pulmonary tuberculosis should be excluded for use. 4 . a list of antimicrobial drug abbreviations and doses for neonates are presented at the end of this volume. 2.1. community-acquired pneumonia 2.1.1. empiric therapy ---executive summary---patient with bacterial pneumonia should be treated primarily with high-dose penicillin (aii). in elderly patients and those with underlying lung diseases, the use of respiratory quinolones may be considered positively (bii). in case of atypical pneumonia, a macrolide or tetracycline is the first choice. respiratory quinolones should be reserved as alternative drugs (bii), but may be used depending on local circumstances about drug resistance (ciii). in case of whether pneumonia or atypical pneumonia dose not diagnose, comobination with high-dose penicillin and a macrolide or tetracycline should be attempted first (bii). respiratory quinolones should be reserved as alternative drugs (bii). in severer cases requiring treatment in the icu, a macrolide or new quinolone should be used aggressively in combination with a broad spectrum b-lactam such as high-dose penicillin from the beginning of treatment (aii). ---explanation---community-acquired pneumonia refers to hospital-acquired pneumonia that develops 48 h or more after admission or pneumonia that develops in healthy adults on social activities other than medical practice-/nursing-associated pneumonia [1e3] . as signs and symptoms, cough, sputum, thoracic pain, and dyspnea appear, and this disease acutely occurs with systemic symptoms such as fever and general malaise [1e3] . however, these symptoms are not marked in some elderly patients. furthermore, atypical pneumonia including mycoplasma is characterized by a small amount of sputum, and can be differentiated (tables 1 and 2) [4, 5] . concerning examination, gram staining and culture of sputum are used to identify causative microorganisms and select subsequent treatment strategies [6, 7] (aii). kits for rapid diagnosis with urine or nasal swab are also used for auxiliary diagnosis [8, 9] (aii). a blood test shows inflammatory findings such as leukocytosis and an increase in the crp level, facilitating a certain assessment of the disease [5, 10] . on thoracic imaging, consolidation or a ground glasslike shadow is observed [1e5] (ii). when patients are in an immunosuppressive state related to an underlying disease, a causative microorganism test should be performed, considering the possibility of opportunistic infection [1e3, 11, 12] (a). in elderly patients, aspiration pneumonia is frequently observed, and the management of this disorder is necessary (refer to the section "2.4 aspiration pneumonia" on page. 19 ). in the presence of renal dysfunction, the type and dose of an antimicrobial drug must be carefully selected [11, 12] (aii). bacterial pneumonia should be differentiated from atypical pneumonia in accordance with "the jrs guidelines for the management of community-acquired pneumonia in adults in 2007" (edited by the committee to prepare guidelines regarding respiratory infectious diseases, japanese respiratory society) (tables 1 and 2) [3] . although legionella pneumonia is routinely classified as atypical pneumonia, various types of atypical pneumonia do not include legionella pneumonia in this differentiation method. a. bacterial pneumonia (1) outpatient treatment bacterial pneumonia is primarily caused by sterptococcus pneumoniae, haemophilus influenzae, and moraxella catarrhalis [1e5, 13, 14] (ii). basically, these types of pneumonia should be treated by orally administering high-dose penicillin [1e4] (aii). in japan, macrolide-resistant s. pneumoniae is detected in most cases; therefore, macrolides are not recommended as the first choice, differing form those in europe and the united states [4, 5, 10, 13, 14] (aii). for outpatient treatment, b-lactamase inhibitorcontaining penicillin is commonly used. therapy with cva/ampc or sbtpc (2 tablets/3e4 times a day) is recommended with respect to the efficacy and suppression of resistant bacteria [1, 4, 11] (aii). however, such high-dose prescriptions are not always accepted by health insurance system in japan, and the following prescriptions (examples) should also be considered. in elderly patients or those with underlying lung diseases such as copd/old pulmonary tuberculosis, the use of respiratory quinolones should be considered positively from the perspective of the effects on penicillin-resistant pneumococcus and tissue transfer [11, 14, 15] (bii). however, many new quinolones also have antimicrobial activities against mycobacterium tuberculosis; therefore, the presence or absence of active tuberculosis must be strictly checked before administration [16] (aii). (2) hospital treatment for hospital treatment, injection is primarily used. however, basic concepts for drug selection are similar to those at the outpatient clinic. considering s. pneumoniae, h. influenzae, and m. catarrhalis, high-dose penicillin or cephems, which are effective for these microorganisms, should be selected [1e4] (aii). if more potent treatment is required, respiratory quinolone injection should be used [15, 17] (bii). ---drugs to be recommended---(1) outpatient treatment a first choices -cva/ampc, oral (125/250 mg), 2 tablets/3e4 times a day -sbtpc, oral (375 mg), 2 tablets/3e4 times a day * concerning cva/ampc and sbtpc, up to 1000 mg of ampc or up to 750 mg of abpc are approved dosage in japan. combination therapy with ampc (oral preparation) should also be considered. [example] cva/ampc, oral (125/250 mg), 1 tablet/3 times a day þ ampc, oral (250 mg), 1 tablet/3 times a day <> second choices -lvfx, oral, 500 mg/once a day -grnx, oral, 400 mg/once a day -stfx, oral, 100 mg/1e2 times a day -mflx, oral, 400 mg/once a day -tflx, oral, 300 mg/twice a day (2) hospital treatment a first choices -sbt/abpc, intravenous drip, 3 g/3e4 times a day -ctx, intravenous drip, 1e2 g/2e3 times a day -ctrx, intravenous drip, 2 g/once a day or 1 g/twice a day <> second choice -lvfx, intravenous drip, 500 mg/once a day b. atypical pneumonia (1) outpatient treatment atypical pneumonia is primarily caused by mycoplasma pneumoniae, chlamydophila pneumoniae, and legionella pneumophila [1e5, 10, 11, 13, 14] (ii). the oral administration of a macrolide or tetracycline is the first choice [1, 4, 5, 7] (aii). to suppress resistant bacteria, respiratory quinolones should be reserved as alternative drugs [1, 4, 11, 12, 18] (bii). however, recently, the appearance of macrolideresistant m. pneumoniae in adults has raised an issue in japan. respiratory quinolones must be used as the first choice depending on local circumstances about drug resistance [18] (ciii). (2) hospital treatment for hospital treatment, injection is primarily used. however, basic concepts for drug selection are similar to those at the outpatient clinic. if more potent treatment is required, new quinolone injection should be used [1e4,11,15,17] (bii). ---drugs to be recommended---(1) outpatient treatment a first choices -azm sustained-release preparation, oral, 2 g/single dose -cam, oral, 200 mg/twice a day -mino, oral, 100 mg twice a day <> second choices -lvfx, oral, 500 mg/once a day -grnx, oral, 400 mg/once a day -stfx, oral, 100 mg/1e2 times a day -mflx, oral, 400 mg/once a day table 1 items used to differentiate between bacterial and atypical pneumonia [3] . 1. under 60 years of age 2. no or minor underlying diseases 3. stubborn cough 4. poor chest auscultatory findings 5. no sputum, or no identified aetiological agent by rapid diagnosis 6. a peripheral white blood cell count below 10,000/ml table 2 criteria for differentiation [3] . in cases using the 6 items in -tflx, oral, 300 mg/twice a day (2) hospital treatment -azm, intravenous drip, 500 mg/once a day -mino, intravenous drip, 100 mg/twice a day -lvfx, intravenous drip, 500 mg/once a day -cpfx, intravenous drip, 300 mg/twice a day. -pzfx, intravenous drip, 500 to 1000 mg/twice a day c. cases in which whether the disease is bacterial pneumonia or atypical pneumonia is unclear (1) outpatient treatment in this case, combination therapy with high-dose penicillin and a macrolide or tetracycline should be selected as the first choice to cover both bacterial and atypical pneumonia [1e4, 11, 13, 14, 17, 18] (bii). as respiratory quinolones cover both bacterial and atypical pneumonia, they are convenient, but should be reserved as alternative drugs from the perspective of suppression of resistant bacteria [1e4, 11, 15, 17, 18] (bii). however, in elderly patients or those with underlying lung diseases such as copd/old pulmonary tuberculosis, the use of respiratory quinolones should be considered positively from the perspective of the effects on penicillin-resistant pneumococcus and tissue transfer [11, 14, 15] (bii). recently, the appearance of macrolideresistant m. pneumoniae in adults has raised an issue. respiratory quinolones may be used as the first choice depending on local circumstances about drug resistance [18] (ciii). (2) hospital treatment for hospital treatment, injection is primarily used. however, basic concepts for drug selection are similar to those at the outpatient clinic. if more potent treatment is required, new quinolone injection should be used [1e4, 11, 15] (bii). (3) severer cases requiring treatment in the icu in severer cases requiring treatment in the icu, s. pneumoniae should be initially considered, and a macrolide or new quinolone should be used aggressively in combination with a broad spectrum b-lactam such as high-dose penicillin from the beginning of treatment primarily to cover latent atypical bacteria (in particular, when l. pneumophila is not covered, the condition may become fatal) [1e4, 11, 17, 18] (aii). in particular, combination therapy with a macrolide is recommended from immunological aspects to suppress excessive inflammation related to cytokines [19] (cii). as the possibility that causative microorganisms may be enteric bacteria including esbl-producing bacteria cannot be ruled out, carbapenem injection should be used as a first-choice drug in patients with a background factor for which esbl-producing bacteria are frequently detected [11, 20] (bii). the sensitivity of a urinary antigen kit to s. pneumoniae and legionella spp. is approximately 60%. therefore, even when the patient is negative for these bacteria in the initial phase, the possibility of pneumonia related to these bacteria should not be ruled out [1e4, 8, 9] (ii). ---drugs to be recommended---(1) outpatient treatment a first choices -cva/ampc, oral (125/250 mg), 2 tablets/3e4 times a day -sbtpc, oral (375 mg), 2 tablets/3e4 times a day * concerning cva/ampc and sbtpc, up to 1000 mg of ampc or up to 750 mg of abpc are approved dosage in japan. combination therapy with ampc (oral preparation) should also be considered. [example] cva/ampc, oral (125/250 mg), 1 tablet/3 times a day þ ampc, oral (250 mg),1 tablet/3 times a day þ one of the followings: -azm sustained-release preparation, oral, 2 g/single dose -cam, oral, 200 mg/twice a day -mino, oral, 100 mg/twice a day <> second choices -lvfx, oral, 500 mg/once a day -grnx, oral, 400 mg/once a day -stfx, oral, 100 mg/1e2 times a day -mflx, oral, 400 mg/once a day -tflx, oral, 300 mg/twice a day (2) hospital treatment a first choices -sbt/abpc, intravenous drip, 3 g/3e4 times a day -ctx, intravenous drip, 1e2 g/2e3 times a day -ctrx, intravenous drip, 2 g/once a day or 1 g/twice a day þ one of the followings: -azm, intravenous drip, 500 mg/once a day -mino, intravenous drip, 100 mg/twice a day -cam, oral, 200 mg/twice a day <> second choices -lvfx, intravenous drip, 500 mg/once a day -pzfx, intravenous drip, 500 to 1000 mg/twice a day (3) severer cases requiring treatment in the icu -taz/pipc, intravenous drip, 4.5 g/3e4 times a day -ipm/cs, intravenous drip, 0.5e1 g/2e4 times a day -mepm, intravenous drip, 1 g/2e3 times a day -bipm, intravenous drip, 0.3e0.6 g/3e4 times a day -drpm, intravenous drip, 0.5e1 g/3 times a day þ one of the followings: -azm, intravenous drip, 500 mg/once a day -lvfx, intravenous drip, 500 mg/once a day -cpfx, intravenous drip, 300 mg/twice a day -pzfx, intravenous drip, 500 to 1000 mg/twice a day -mino, intravenous drip, 100 mg/twice a day ---executive summary----when causative microorganisms are identified based on the results of microbial examination of good-quality sputum, blood culture, and urinary antigen (s. pneumoniae, l. pneumophila) tests and drug susceptibility testing of the causative agents, definitive therapy should be performed if possible [2, 3] (biii). -the place of treatment and drugs should be selected in accordance with the severity of the disease [2, 3] (aii). -antimicrobial drugs should be selected in reference to the susceptibility of isolated bacteria to antimicrobial drugs or a drugsusceptibility tendency in the area [2, 3, 13, 22] when these data are available (aii). -the administration period of an antimicrobial drug is determined in accordance with the improvement of symptoms and laboratory data, with a target of 5e7 days [2, 21] (biii). -when the patient is infected with l. pneumophila or c. pneumoniae, the optimal administration period is about 14 days [21] (biv). a. streptococcus pneumoniae -the clinical and laboratory standards institute (clsi) has established higher criteria for breakpoints for penicillin susceptibility on the administration of parenteral antimicrobial drugs for s. pneumoniae infections other than meningitis [23] , based on the following findings: patients with severe pneumonia due to s. pneumoniae with a low pcg susceptibility (mic: 0.12e4 mg/ml) showed no difference in responses to pcg and outcome [24, 25] (ii). for the treatment of pneumococcal pneumonia, the dose of penicillin should be increased [23, 26] (a). -in japan, most s. pneumoniae isolates are macrolide resistant [13, 22] . -respiratory quinolones have potent anti-pneumococcal activities (iii). the clinical effects of such quinolones are similar to those of high-dose ampc [27] (ii). -in japan, quinolone-resistant s. pneumoniae is detected in 2e3% of the isolates [13] . as quinolone resistance may be readily induced by point mutations of dna gyrase or topoisomerase genes [28] , quinolones must be used adequately (aiii). b. haemophilus influenzae -the abpc-resistant mechanism of h. influenzae involves blactamase production and/or pbp mutation. previously, blactamase production was primarily involved, but, recently, pbp mutation-mediated b-lactamase-negative abpc-resistant (blnar) strains have been increasingly detected. abpcresistant strains with both b-lactamase production and pbp mutation are classified as b-lactamase-positive cva/abpcresistant (blpacr) strains. -according to a national survey in japan, 49 (39.8%) and 7 (5.7%) of 123 h. influenzae strains were blnar and b-lactamase-producing strains, respectively [13] . -blnar strains are also resistant to firstand secondgeneration cephems. -pipc exhibits an antimicrobial activity against blnar strains. however, it is ineffective for blpacr strains. c. klebsiella spp., escherichia coli, proteus spp. -the proportion of extended spectrum b-lactamase (esbl)producing bacteria has slightly increased among isolates from respiratory samples. -according to a national survey in japan, esbl-producing bacteria account for 1.8e3.4% of respiratory sample-derived klebsiella spp. strains [13, 29] . -most esbl-producing strains are simultaneously resistant to quinolones [30] . antimicrobials should be selected according to the drug susceptibility of isolated bacteria. -in japan, carbapenemase-producing strains are extremely rare. d. mycoplasma pneumoniae -in the field of pediatrics, the detection rate of macrolideresistant m. pneumoniae has markedly increased. in adults, that of macrolide-resistant m. pneumoniae also increase [31, 32] . -tetracyclines exhibit potent clinical effects on macrolideresistant m. pneumoniae [33] . -respiratory quinolones have good activities against m. pneumoniae [34, 35] . e. legionella spp. -it should be noted that pneumonia related to legionella spp. other than l. pneumophila sg1 cannot be diagnosed using legionella urinary antigen testing. -as neither b-lactams nor aminoglycosides have antimicrobial activities against legionella spp., which proliferates within host cells, they are clinically ineffective. -quinolones, macrolides, and tetracyclines have been confirmed to exhibit clinical effects on legionella spp. previously, em was the first choice for this infection, but many recent studies have showed the clinical efficacies of lvfx and azm [36, 37] . -rfp is effective when combined with em. the combination of em and rfp is useful. a study suggested the effects of combination therapy with lvfx and a macrolide [38] (ciii). -although there are no marked differences in antimicrobial drug susceptibility among legionella spp., clinical reviews to verify this are limited [39] . f. chlamydophila pneumoniae -only a few studies have supported the clinical effects of antimicrobial drugs against c. pneumoniae pulmonary infections. -tetracyclines, macrolides, and quinolones may be effective. these drugs are recommended primarily based on the results of basic studies [34, 40] . g. staphylococcus aureus -with respect to staphylococcus aureus in japan, there has been an increase in the number of methicillin-resistant strains even in patients with community-acquired pneumonia. in particular, recently, municipal-onset-type mrsa (ca-mrsa) with panton-valentine-leucocidine (pvl) has been detected in japan, raising an issue [41] . -in cases of mssa infection (bacteremia), the clinical effects of cez are superior to those of vcm [42] . -as the susceptibility of mrsa to oral antimicrobial drugs differs among isolates, drugs should be selected according to its drug susceptibility results. h. streptococcus spp. -among various types of streptococcus, the streptococcus anginosus group is frequently detected, and characterized by strong abscess-forming features [43] . streptococcus pyogenes and streptococcus agalactiae may also cause pneumonia. the former may lead to serious pulmonary infection [44] (v). -there is no penicillin resistance, but macrolide resistance is observed at a low frequency [45] . -the anti-streptococcus activities of quinolones vary. among quinolones, grnx, mflx, and stfx have relatively potent antimicrobial activities [13, 46] . i. moraxella catarrhalis -the number of b-lactamaseproducing strains has increased since the 1990's. currently, most strains produce b-lactamase [13, 47] . b-lactamase produced by m. catarrhalis decomposes penicillin. -in japan, macrolide-or quinolone-resistance have not been reported [13] . j. anaerobes -most anaerobes that cause pneumonia exist in the oral cavity. peptostreptococcus spp., prevotella spp., and fusobacterium spp. are involved. mixed infection with microaerophilic streptococci is often observed. -in many cases, infection with anaerobes may be associated with aspiration. -most oral anaerobes (prevotella spp., fusobacterium spp., and porphyromonas spp.) are susceptible to combination drugs consisting of penicillin and a b-lactamase inhibitor, cldm and mnz [48] . k. pseudomonas aeruginosa -in patients with chronic respiratory tract infection, pseudomonas aeruginosa colonizes in the airway, and may cause community-acquired pneumonia [49] . -as the susceptibility of p. aeruginosa to antimicrobial drugs differs among clinical isolates, drugs should be selected according to its drug susceptibility results. ---drugs to be recommended----the drug susceptibility of each clinical isolate should be classified in accordance with the clsi criteria [23] . -establishment of prescriptions recommended in this article * antimicrobials have been approved for specific diseases and specific causative agents by japanese ministry of health and welfare. the approvals are based on the results of clinical studies with good clinical practice. as a general rule, the recommendations in this section refer to this (aii). however, recent trends in drug susceptibility are also considered. * the recent drug susceptibility results of the nationwide studies in japan were referred [13, 22] . * the recommendations without the approvals by japanese ministry are graded by evidence levels. [ -when the susceptibility of identified causative microorganisms is clarified, or after the treatment responsiveness is evaluated, whether or not de-escalation is possible should be reviewed [50e53] (aii). ---explanation---definition: hospital-acquired pneumonia is defined as "pneumonia that newly develops 48 h or more after admission". in many cases, treatment is difficult due to unfavorable patient conditions such as the presence of an underlying disease, immune capacity, and general condition [50e52]. laboratory findings: patients meeting 2 of 3 items, fever, an abnormal leukocyte count, and purulent secretes, in addition to the appearance of an abnormal shadow of the chest should be diagnosed with hospital-acquired pneumonia [50e52]. 1) ventilator-associated pneumonia (vap): vap refers to pneumonia that newly develops 48 h or more after endotracheal intubation/ventilator initiation. its onset within 4e5 days after endotracheal intubation is classified as early-type, and its subsequent onset as late-type [50, 51, 54, 55] . 2) hospital-acquired pneumonia other than vap: several types of hospital-acquired pneumonia other than vap include (1) immunodeficiency (for example, neutropenia during anticancer therapy, cell-mediated immunodeficiency related to the administration of steroids or immunosuppressive drugs) and (2) aspiration pneumonia including latent aspiration (refer to the section "2.4 aspiration pneumonia" on page 19). appropriate management and selection of antimicrobial drugs in accordance with individual conditions are necessary [50] . with respect to microorganisms that are expected, refer to the section "2.2 hospital-acquired pneumonia---2.2.2 empiric therapy: cases in which gram staining is available" (p. 10). ---drugs to be recommended--a. cases in which there is no risk of resistant bacteria antimicrobial drugs should be selected, targeting streptococcus pneumoniae, h. influenzae, and klebsiella spp. as causative microorganisms [50e52] (biii). although it is difficult to estimate/identify causative microorganisms using sputum samples, bacteria that are not isolated/cultured from goodquality sputum may not be causative microorganisms. if resistant bacteria such as mrsa and p. aeruginosa are not detected on sputum culture and there is no deterioration of clinical symptoms, an initial drug should be continued [50] (biii). in patients in whom aspiration episodes are clear, those in whom oral hygiene is not maintained, or those with consciousness disorder, drugs with anti-anaerobe activities should be selected, considering the involvement of anaerobes [50] (biii). if an adequate antimicrobial drug is administered, the treatment period may be 7e10 days, excluding mrsa and p. aeruginosa [50, 53] (bii). a first choices -sbt/abpc, intravenous drip, 3 g/3e4 times a day -ctx, intravenous drip, 1e2 g/3 times a day -ctrx, intravenous drip, 2 g/once a day or 1 g/twice a day * if the involvement of anaerobes is suspected, sbt/abpc should be selected. <> second choice -lvfx, intravenous drip, 500 mg/once a day (as its antimicrobial activity against anaerobes is weak, monotherapy with this drug should be avoided in patients with aspiration pneumonia.). b. cases in which there is a risk of multi-drug-resistant bacteria (table 3 ) [51] to cover multi-drug-resistant bacteria including p. aeruginosa, broad-spectrum antimicrobial drugs with anti-p. aeruginosa activities should be selected [50e52] (aiii). considering the frequency of esbl in each institution, carbapenems should be considered even when enteric bacteria, including klebsiella spp. and escherichia coli, are suspected (biv). if p. aeruginosa is not isolated on good-quality sputum culture, a treatment option should be de-escalated to drugs for cases in which there is no risk of resistant bacteria [50e52] (aii). if aspiration is suspected, or if the involvement of grampositive bacteria is suggested, combination therapy with cldm must be considered (biv). if there is a risk of mrsa carrier (table 4) , combination therapy with anti-mrsa drugs should also be considered. the mean administration period of antimicrobial drugs with respect to causative bacteria in patients with an improvement was approximately 10 days. however, that for resistant bacteria such as p. aeruginosa and mrsa was approximately 12 days [53] (bii). if appropriate antimicrobial drugs can be administered after clarifying causative bacteria, a treatment period of approximately 10 days is recommended [53, 56, 57] (bii). a first choices -taz/pipc, intravenous drip, 4.5 g/3e4 times a day -ipm/cs, intravenous drip, 0.5 g/4 times a day or 1 g/3 times a day -mepm, intravenous drip, 1 g/3 times a day -drpm, intravenous drip, 0.5e1 g/3 times a day -bipm, intravenous drip, 0.3e0.6 g/3e4 times a day <> second choices -cfpm, intravenous drip, 1e2 g/2e4 times a day -cpfx, intravenous drip, 300 mg/twice a day -pzfx, intravenous drip, 500 to 1000 mg/twice a day if the involvement of anaerobes is suspected, one of the following options should be combined with one of the above regimens: -cldm, intravenous drip, 600 mg/2e4 times a day -sbt/abpc, intravenous drip, 3 g/3e4 times a day c. severe cases one of the following options must be combined with one of the regimens for cases in which there is a risk of multi-drugresistant bacteria. when comparing the results between patients undergoing appropriate and inappropriate treatments, the prognosis of the latter was significantly poorer [58, 59] (bii). however, a study reported that the prognosis in a group with compliance with recommended drug selection was significantly poorer than in a non-compliance group in patients in whom infection with drug-resistant bacteria in the icu was suspected even among those in whom the etiology was bacteriologically investigated [60] (bii). therefore, it must be considered that, even when resistant bacteria are etiologically involved, the administration of an appropriate antimicrobial drug that covers them does not always improve the prognosis. -taz/pipc, intravenous drip, 4.5 g/3e4 times a day -ipm/cs, intravenous drip, 0.5 g/4 times a day or 1 g/3 times a day -mepm, intravenous drip, 1 g/3 times a day -drpm, intravenous drip, 0.5e1 g/3 times a day -bipm, intravenous drip, 0.3e0.6 g/3e4 times a day one of the following options should be combined with one of the above regimens: should be estimated based on the results of the clinical microbiological culture (cmc: gram staining and culture) of a lower airway sample immediately before the start of treatment, and not based on bacteria isolated on active surveillance culture (asc), which was conducted as a strategy to prevent/ control infection prior to onset [66] . -microorganisms that cause pneumonia or (colonization of) the lower airway should be estimated based on the presence or absence of neutrophils or phagocytosis (excluding those patients with neutropenia or functional impairment of neutrophil) [50] (bii). ---explanation---[gram staining] diagnostic accuracy of hospital-acquired pneumonia is improved by confirming neutrophils and bacterial cells using gram staining of airway samples. this observation has also been confirmed through an increase in the likelihood ratio of hospital-acquired pneumonia in patients with a clinical pulmonary infection score (cpis) of 6 points or higher [61] . as bacteria isolated from the lower airways of inpatients are common colonizers in many cases, gram staining is also useful for discerning colonization from infection by evaluating the presence or absence of neutrophil and phagocytosis. therefore, it is desirable to combine bacterial culture with gram staining [51,61e65] . antimicrobial-drug selection based on gram staining findings leads to appropriate empiric therapy in two-thirds of patients with hospital-acquired pneumonia, and it can be continued as definitive therapy in many cases [62] . if there are no bacterial cells on gram staining of lower airway sample in whom an antimicrobial regimen was not changed within the past 72 h, it is unlikely that the focus of infection/inflammation is within the lungs (lower airway) [51] . in this case, the possibility of pneumonia mimic, such as pleural effusion, atelectasis, and pulmonary edema, is suggested if the lung field opacity still remains in chest x-ray. if there is no other infectious focus, the discontinuation of an antimicrobial drug may be warranted [50, 66, 67] . a study has reported that the culture results of asc performed as a strategy of routine infection control measure prior to the development of nosocomial pneumonia accurately predicted the causative pathogen in only 35% of cases [66] . therefore, it is necessary to submit airway samples for clinical microbiological culture (cmc) immediately before the start of presumptive treatment. [causative microorganisms and their origin] microorganisms that cause hospital-acquired pneumonia are derived from the oropharynx, airway (including the nasal cavity and nasal sinus), digestive tract, and environment. gastrointestinal tract-derived causative microorganisms are enteric bacteria (primarily, klebsiella spp., e. coli and others such as proteus spp., enterobacter spp., serratia spp., morganella spp., and citrobacter spp.). those derived from the upper airway include s. pneumoniae, h. influenzae, moraxella catarrhalis, s. aureus (namely methicillin sensitive strain), and oral anaerobes. those derived from the environment include methicillin-sensitive s. aureus, pseudomonas spp., acinetobacter spp., and stenotrophomonas spp. [50, 51, 65, 68] . as the above bacteria derived from the airway and gastrointestinal tract basically exert strong virulence to the airway, they can be considered a core pathogen group of hospital-acquired pneumonia. their potential for developing airway inflammatory response is generally believed stronger than those caused by environmental pathogen [67, 68] . table 3 risk factors for multi-drug-resistant bacteria. 1. previous use of antimicrobial drugs within 90 days 2. interval of 5 days or more from admission 3. admission from an area/hospital in which resistant bacteria are frequent 4. immunosuppressive state or treatment table 4 risk factors for carrying mrsa [50] . conditions under which anti-mrsa drug therapy should be considered (including gram stain) mrsa---" (p. 13). (2) diplococcus consisting of a pair of two cocci (gpdc: grampositive diplococci) s. pneumoniae should initially be suspected. enterococcus is also a gpdc in microscopic appearance, but is basically considered a non-pulmonary pathogen [67] . <> cases in which there have been no previous treatment with antimicrobial drugs or risks of penicillin-resistant pneumococcus -pcg, intravenous drip, 2,000,000 to 3,000,000 units/4e6 times a day -abpc, intravenous drip, 2 g/4e6 times a day <> cases in which previous treatment with antimicrobial drugs or a risk of prsp is present -ctrx, intravenous drip, 1 g/twice a day or 2 g/once a day -ctx, intravenous drip, 1e2 g/2e3 times a day -lvfx, intravenous drip, 500 mg/once a day -vcm, intravenous drip, 1 g/twice a day (tdm should be conducted so that a trough is 15e20 mg/ml [69] .) (3) gram-positive coccus in either short or long chain (gpc in chain) aor b-hemolytic streptococci is indicated. -pcg, intravenous drip, 2,000,000 to 3,000,000 units/ 4e6 times a day -abpc, intravenous drip, 2 g/4e6 times a day (4) gram-positive bacillus with a rod-like morphology (gpr: gram-positive rod) corynebacterium spp. may be indicated. -vcm, intravenous drip, 1 g/twice a day (tdm should be conducted so that a trough is 15e20 mg/ml [69] .) c. gram-negative bacteria ---executive summary----when gram-negative bacteria are observed, h. influenzae, m. catarrhalis, enterobacteriaceae, p. aeruginosa, acinetobacter spp., and stenotrophomonas spp. may be indicated [50e52, 65, 68] (bii). -it is difficult to estimate the type of bacteria based on the morphology on gram staining in comparison with grampositive bacteria. -gram-negative bacteria frequently detected as causative microorganisms include enteric bacteria and p. aeruginosa. -it is encouraged important to recognize the basic antimicrobial drug susceptibility pattern of each type (group) of bacteria to make sure that the empiric antimicrobial therapy is appropriate (table 5 ). ---drugs to be recommended--(1) cases of early-onset hospital-acquired pneumonia in which there have been no previous administration of antimicrobial drugs or risk of resistant bacteria aero-respiratory pathogen, such as h. influenzae and m. catarrhalis, and enteric bacteria, such as klebsiella spp., are indicated. -sbt/abpc, intravenous drip, 3 g/3e4 times a day -ctrx, intravenous drip, 1 g/twice a day or 2 g/once a day -ctx, intravenous drip, 1e2 g/2e3 times a day -lvfx, intravenous drip, 500 mg/once a day (2) cases of late-onset hospital-acquired pneumonia or ventilator-associated pneumonia in which the risk of resistant bacteria is high an antimicrobial drug with anti-pseudomonal activity that targets non-glucose-fermentative gram-negative rod should be administered [50, 51, 68] -to treat polymicrobial infection, the administration of an antimicrobial drug with an activity against obligate anaerobe is not always necessary [67, 70] . -in non-severe cases, the administration of antimicrobial agents with anti-mrsa activity may be withheld in the initial phase even when staphylococcus-like bacterial cells are observed [70] . ---explanation---when several types of bacteria differing in gram staining and morphology are observed, the condition is commonly interpreted as aspiration pneumonia, suggesting the involvement of anaerobes. however, the number of hospital-acquired pneumonia (including vap) caused by anaerobes have been reported relatively smaller than generally anticipated. [71] , polymicrobial infection does not always require the prompt antimicrobial therapy that covers anaerobes. even though when aspiration pneumonia is suspected, sbt/abpc is frequently prescribed assuming anaerobic infection, which actually works good on many occasions, it has to be acknowledged that sbt/abpc exert good antimicrobial activity not solely against anaerobes, but also aero-enteric pathogen of pneumonia such as streptococcus pneumonia, oral streptococci, h. influenzae, m. catarrhalis, and klebsiella pneumonia. inpatients are often exposed to gram-negative bacteria residing in the hospital environment. furthermore, there are many opportunities to undergo antimicrobial drug therapy that affects the indigenous microflora. for such reasons, gram-negative bacillus (enteric bacteria or p. aeruginosa) frequently colonize within the oropharyngeal region of the elderly patients or long-term bedbound patients, many of whom need airway suctioning or have tracheostomy that may serve as portal of entry of environmental pathogen. oropharyngeal microflora primarily consisting of these gram-negative bacteria can be aspirated into the airway after surgery requiring sedation or anesthesia, or during or after endoscopic examination [57, 72, 73] . briefly, anaerobes may be an occasional pathoen in polymicrobial infection as seen on gram staining of patients with suspected aspiration pneumonia, but s. pneumoniae, h. influenzae, s. aureus, klebsiella spp., p. aeruginosa, and acinetobacter spp. are more commonly involved in many cases, being similar to the microorganisms that are thought to be the major pathogen of hospital-acquired pneumonia. this is in contrast with the community-onset aspiration pneumonia, represented by lung abscess, in that anaerobes are primarily involved [67, 71] . anaerobes involved in hospital-acquired pneumonia include facultative anaerobic a-hemolytic streptococci in the oral cavity and obligate anaerobes. oral obligate anaerobes include grampositive coccus (peptostreptococcus sp.), gram-negative coccus (veillonella sp.), and gram-negative bacillus "oral pigmented" bacteroides (bacteroides melaninogenicus), prevotella sp., porphyromonas sp., and fusobacterium sp.). many of these types of bacteria are susceptible to b-lactams that do not contain a b-lactamase inhibitor, new quinolones, macrolides, and tetracyclines. therefore, patients with hospital-acquired pneumonia may be basically treated by standard empiric therapy for hospital-acquired pneumonia even when aspiration pneumonia related to several types of bacteria is suspected [67] . ---drugs to be recommended---(1) cases in which it is not necessary to consider the involvement of multi-drug-resistant bacteria, or early hospitalacquired pneumonia the involvement of oral streptococcus, oral anaerobes, s. pneumoniae, h. influenzae, and enteric bacteria should be considered. -sbt/abpc, intravenous drip, 3 g/3e4 times a day -ctrx, intravenous drip, 2 g/once a day or 1 g/twice a day -ctx, intravenous drip, 1e2 g/2e3 times a day -lvfx, intravenous drip, 500 mg/once a day (2) late-onset hospital-acquired pneumonia or cases in which there is a risk of multi-drug-resistant bacteria in addition to the above pathogens, the involvement of non-glucose-fermentative gram negative bacteria or esblproducing enteric bacteria must be considered. -cfpm, intravenous drip, 1e2 g/2e4 times a day -czop, intravenous drip, 1e2 g/2e4 times a day -taz/pipc, intravenous drip, 4.5 g/3e4 times a day table 5 basic susceptibility of various pathogen groups to antimicrobial drugs. ---explanation---if drug susceptibility test is not conducted for some reasons after the identification of causative microorganisms, an antimicrobial drug should be selected with reference to the susceptibility pattern (local sensitivity) of the identified bacteria at each institution. if the local sensitivity is not obtained, a drug should be selected based on the basic susceptibility of various pathogens to antimicrobial drugs (table 5 ) [74] . in the treatment of hospital-acquired pneumonia, the duration of antimicrobial therapy generally tends to be longer than required for the following reasons: opacity on chest x-ray often remains for reasons other than pneumonia even after the start of antimicrobial drug treatment; and there may be a large number of latent nonpneumonia (or non-infectious-disease) factors that may cause increase in body temperature or crp level in inpatients [75] . however, if appropriate antimicrobial drug treatment is performed, it is possible to complete treatment in 1 week [57] . in strains such as enterobacter spp., serratia spp., citrobacter spp., and morganella spp. (table 5 gnr b ), the expression of intrinsic antimicrobial-drugresistance genes encoded in chromosome genes is induced during antimicrobial drug treatment, a phenomenon which is basically rarely seen in e. coli, klebsiella spp., h. influenzae, and m. catarrhalis (table 5 gnr a ) ( table 5) [74, 76, 77] . therefore, if adequately chosen treatment parameters are improved, antimicrobial treatment could be completed with careful follow up of patients' condition. although it is useful to recognize these pathogens, abbreviated as space (serratia, pseudomonas, acinetobacter, citrobacter, and enterobacter), as a representative microorganism group that causes hospital-acquired pneumonia, the space group is essentially a common colonizer. therefore it is important to bear in mind that antimicrobial drug is not always indicated upon the isolation of space to avoid selection of antimicrobial resistant bacteria related to unnecessary or long-term antimicrobial therapy [65, 67] . lzd should be selected [78, 79] (ai). the therapeutic efficacy of lzd is similar to those of glycopeptides [50, 80] . the penetration of lzd into the alveolar epitheliumlining fluid and intra-alveolar sputum is more favorable. therefore, use of lzd should be encouraged in cases of restricted sputum expectoration, such as vap [51] (bii). exclusive use of a single drug may accelerate the acquisition of resistance to the agent [81e83] (ci). as dap is inactivated by pulmonary surfactants, its use should be avoided for mrsa pneumonia. -glycopeptides should be selected as first-line drug for pneumonia caused by corynebacterium sp [84] (aii). ---explanation---there is no significant difference in the therapeutic efficacy for mrsa pneumonia between glycopeptides and lzd. several studies reported that the overall clinical efficacy of lzd, including the incidence of side effects, was superior to vcm in patients with hospital-acquired pneumonia caused by mrsa [85, 86] . however, since the dosing of vcm in these studies have been considered suboptimal, further study is needed [51, 87] . some investigators have recommended that, when mrsa is susceptible to cldm or mino on a susceptibility test, lzd, a protein synthesis inhibitor should be administered given the possible involvement of the panton-valentine leukocidin [78, 88] . if a prompt improvement is achieved by the intravenous drip of lzd 600 mg q12h, or if the patient's condition is not critical, switch from the intravenous administration to an oral preparation of lzd, which shows high bioavailability [89] , is encouraged. as dap is inactivated by pulmonary surfactants, it should not be used to treat mrsa pneumonia. this may not apply to the treatment of septic pulmonary embolism [90] . ---drugs to be recommended---(1) mrsa a first choices -vcm, intravenous drip, 1 g/twice a day -teic, intravenous drip, 400 mg for the first 2 days/ twice a day for loading, 400 mg/once a day from day 3 * tdm should be conducted so that the trough levels of vcm and teic range from 15 to 20 mg/ml [11] . -lzd, intravenous drip or oral administration, 600 mg/ twice a day <> second choices -abk, intravenous drip, 300 mg/once a day (a trough level was established as 2 mg/ml using tdm.) -st combination drug (smx at 400 mg/tmp at 80 mg), oral administration, 2 tablets/twice a day or intravenous drip, 960 mg/twice a day -cldm, intravenous drip, 600 mg/2e4 times a day (the results of drug susceptibility testing must be confirmed). [non-extended-spectrum b-lactamase (esbl)-producing bacteria] (2) hospital treatment " (p.7). (2) e. coli, klebsiella spp., proteus mirabilis (esbl-producing bacteria) refer to the section "2.1 community-acquired pneumonia---2.1.2 definitive therapy--[8] klebsiella spp. [esbl-producing bacteria] (2) hospital treatment" (p. 7). (3) enterobacter spp., serratia spp., citrobacter spp., morganella spp., proteus vulgaris a third-generation cephems or quinolones should be administered [50, 51, 68] (aii). -ctrx, intravenous drip, 2 g/once a day or 1 g/twice a day -ctx, intravenous drip, 1e2 g/2e3 times a day -lvfx, intravenous drip, 500 mg/once a day -cpfx, intravenous drip, 300 mg/twice a day -pzfx, intravenous drip, 1000 mg/twice a day <> if a strain is estimated to constantly express cephalosporinase (highly resistant to b-lactamase inhibitorcontaining b-lactams, oxyimino [¼3rd generation] cephalosporin and cephamycin, through plasmid genes) on an antimicrobial drug susceptibility test, fourthgeneration cephems or carbapenems should be administered. -cfpm, intravenous drip, 1e2 g/4 times a day -czop, intravenous drip, 1e2 g/4 times a day -mepm, intravenous drip, 1 g/3 times a day -drpm, intravenous drip, 0.5e1 g/3 times a day (4) p. aeruginosa anti-pseudomonal penicillins, third-generation or later cephems, carbapenems, or new quinolones should be administered [50, 51] refer to the section "2.1 community-acquired pneumonia---2.1.2 definitive therapy--[3] h. influenza (abpcsusceptible), [4] h. influenza (b-lactamase-producing), [5] h. influenza (b-lactamase-negative ampicillin-resistant (blnar), and [6] h. influenza (b-lactamase-positive amoxicillin clavulanate-resistant (blpacr)" (p. 6e7). ---explanation---according to some investigators, enteric bacteria are classified into 2 types: sensitive, gram-negative rods, such as e. coli, klebsiella pneumoniae, and p. mirabilis, which are susceptible to first-generation cephalosporin, and resistant, gram-negative rods, such as enterobacter spp., serratia spp., and citrobacter spp., which show an intrinsic or inducible resistance to third-generation cephalosporin through chromosomal ampc genes [74, 76, 77] . in addition, the number of extended spectrum of b-lactamase (esbl)-producing strains of e. coli, klebsiella, and proteus sp. that are resistant to all cephalosporin has increased. among resistant gnrs, such as enterobacter spp., strains that constantly produce ampctype b-lactamase (cephalosporinase) (plasmid type) must also be considered [76, 77] . concerning non-fermentative bacteria, their intrinsic susceptibility to antimicrobial agents differs among p. aeruginosa, stenotrophomonas spp., and acinetobacter spp. a study indicated that, in patients with p. aeruginosa pneumonia, monotherapy with a new quinolone might show unfavorable bacteria-eradicating effects or lead to recrudescence [90] . in some patients, combination therapy with a b-lactam (pipc, caz, cfpm, or carbapenems), which has an anti-pseudomonal activity, and aminoglycoside or new quinolone may be considered [51, 90, 94] . most strains of stenotrophomonas spp. are susceptible to mino or an st combination drug. m. catarrhalis and acinetobacter spp. are the frequent types of gram-negative coccus detected in patients with early and late hospital-acquired pneumonia, respectively. many strains of the former produce b-lactamase. the latter is a gnr existing in the hospital environment, and may be resistant to many antimicrobial drugs. however, in japan, the multi-drug resistance of this type of bacteria has not widely distributed. carbapenems and new quinolones should be selected. however, the vast majority of acinetobaccter strains are susceptible to sbt/abpc. in particular, sbt has an antimicrobial activity against this type of bacteria, and their susceptibility to sbt/abpc should routinely be confirmed. primary test drugs for an antimicrobial susceptibility of this type of bacteria (drugs appropriate for a routine examination panel) are sbt/abpc, caz, ipm/cs, mepm, gm, tob, lvfx, and cpfx [95] . pan-sensitive strains of h. influenzae are b-lactamase (bl)-negative, abpc-sensitive (blnas) strains. however, there are various resistance patterns: bl-producing, abpcresistant (blpar), bl-negative, abpc-resistant (blnar), and bl-producing, ampc/cva-resistant (blpacr) strains. blnas strains can be treated with abpc, but sbt/abpc therapy is required to control blpar strains. the administration of ctrx or new quinolones is necessary for blnar or blpacr. a randomized-controlled trial with multivariate analysis has shown that factors for favorable bacteriological effects included the absence of p. aeruginosa-related pneumonia (<0.01), a higher body weight (<0.01), a low apache ii score (severity) (0.03), and cpfx therapy (0.04) [90] . conditions suggesting the use of new quinolones include allergy to b-lactams, the presence of or concern for nephropathy (an aminoglycoside cannot be combined with a b-lactam), necessity of covering obligate intracellular pathogen, or situations in which switching to an oral preparation is indicated [94] . an in vitro study indicated that the alveolar epithelial lining fluid (elf) concentration of lvfx reached as high as its serum concentration. furthermore, a prospective open-label study reported that switching of intravenous drip to oral administration decreased the elf concentration, but it was within the range at which many pathogens are deemed sensitive based on the cumulative data of minimum inhibitory concentrations for causative microorganisms [94] . ---executive summary----nursing and healthcare-associated pneumonia (nhcap) is a category independently defined in japan based on medical circumstances. -the attending physician proposes a treatment category (groups a to d) by evaluating what treatment is necessary as the most important item based on the patient's and his/her family's will ( fig. 1 ) [96] . -risk factors for resistant bacteria are categorized into two items, and initial treatment options are recommended, assuming target causative microorganisms (civ). -in patients in whom the general condition is unfavorable due to complications or in terminal-stage patients, initial treatment options are recommended considering side effects from the perspective of innocent properties (civ). -in group d, in which intensive care is required, combination therapy with broad-spectrum (involving resistant bacteria and legionella) and potent antimicrobial drugs is recommended (bi). ---explanation--in 2011, the japanese respiratory society issued the "guidelines for the management of nursing and healthcare-associated pneumonia (nhcap)" [96] , considering medical circumstances in japan with reference to the entity of healthcare-associated pneumonia (hcap) proposed in the untied states [51] . the definition of nhcap is shown in table 6 . as this committee has no objection to the entity itself, the selection of drugs will be explained based on evidence to avoid duplications with the above guidelines. the mortality rate and frequency of resistant bacteria in patients with nhcap are intermediate between community-acquired pneumonia (cap) and hospital-acquired pneumonia (hap). however, nhcap may be primarily regarded as being similar to geriatric pneumonia [97, 98] . there is no fact that the rate at which resistant bacteria are isolated increases with severity [97] . even when pneumonia is not severe, the host's activities of daily living (adl) and an underlying disease/immunodeficiency reduce the prognosis in many cases [98] . as this type of pneumonia develops in a variety of uneven populations, it is difficult to simply determine severity classification. therefore, considering various conditions, the entity of "treatment category", involving the ethical aspects of geriatric care, was introduced based on evaluation by the attending physician who knows the patient well ( fig. 1 ). frequent basic conditions or concomitant diseases for nhcap in japan include an advanced age, central nervous diseases, aspiration, a reduction in adl, and tubal feeding. their factors are aspiration pneumonia itself or risk factors, and hcap in japan may overlap with aspiration pneumonia [99] . on the other hand, in nhcap patients, mrsa, p. aeruginosa, and anaerobes are more frequently isolated in comparison with cap patients. it is necessary to switch therapeutic strategies, considering these causative microorganisms. refer to the next section "2.4 aspiration pneumonia" (p. 19 ). [type and frequency of causative microorganisms] concerning causative microorganisms in nhcap patients, resistant bacteria are frequently detected, differing from cap patients. however, with respect to microorganisms that cause hcap, the distribution and frequency of streptococcus pneumoniae and h. influenzae, which are frequently isolated in cap patients, as well as mrsa, p. aeruginosa, and gram-negative bacillus, which are frequently detected in hap patients, differ among countries, areas, and institutions due to their variety (iii). concerning causative microorganisms, a study reported that there was no marked difference between nhcap and cap [100] . on the other hand, a study in the united states indicated that s. aureus was frequently detected [101] , and another study in italy, where the rapid aging of society is advanced, as described for japan, reported that aspiration pneumonia, h. influenzae, s. aureus, and gram-negative bacillus were more frequent than in patients with cap [59] . as a result, the rate of resistant bacteria increased, and inappropriate antimicrobial drugs were selected in a high proportion of patients. in addition, the mortality rate was higher than in cap patients, suggesting the association between the two factors. representative causative microorganisms with respect to the presence or absence of risk factors for resistant bacteria are presented in table 7 [96] . of these, resistant bacteria, which are not targeted in cap patients, were isolated in approximately 20%. however, the value was lower than in hap patients. this is a current status of japan (iii). however, we must consider that patients in whom isolated bacteria are unclear account for approximately 50%, with the involvement of aspiration as a background factor [99] . in addition to bacteria commonly isolated in cap patients, the frequency of enteric bacteria and anaerobes has increased [102] . [rules of antimicrobial drug therapy] risk factors for resistant bacteria in nhcap patients include "the previous use of antimicrobial drugs for 2 days or more within 90 days" and "tubal feeding" ( [96] . risk factors for involvement by drug-resistant pathogens. -if no antibiotic therapy in the preceding 90 days or current tube feeding, the patient can be assumed to have no risk of involvement by drug-resistant pathogens. -however, if past medical history indicates isolation of mrsa, the patient should be assumed to have risk of involvement by mrsa. *1) inappropriate when aspiration pneumonia is suspected, because it has insufficient activity against anaerobic bacteria. *2) because of insufficient activity against anaerobic bacteria, when used to treat suspected aspiration pneumonia, it should be used in combination with an antibiotic that has activity against anaerobic bacteria (e.g., mnz, cldm, sbt/abpc). definition of nhcap [96] . 1. pneumonia diagnosed in a resident of an extended care facility or nursing home 2. pneumonia diagnosed in a person who has been discharged from a hospital within the preceding 90 days 3. pneumonia diagnosed in an elderly or disable d person who is receiving nursing care 4. pneumonia diagnosed in a person who is receiving regular endovascular treatment as an outpatient (dialysis, antibiotic therapy, chemotherapy, immunosuppressant therapy) standards for nursing care patients whose performance statues is ps 3 (capable of only limited self-care, confined to bed or a chair more than 50% of their waking hours) or more. item 1 incudes patients on psychiatric wards. not isolated in those who had made favorable daily life activities without a history of antimicrobial drug therapy [103] . another study indicated that tubal feeding was an independent risk factor for infection with p. aeruginosa (odds ratio: 13.9) [104] (ii). this is the reason why treatment category c was established in the guidelines. briefly, patients who do not meet the above two items are regarded as having no risk factor for resistant bacteria, and assigned to group b. patients meeting 1 or 2 items or those in whom mrsa was previously isolated are assigned to group c. respective drugs to be recommended were separately established. patients in whom outpatient treatment is considered to be appropriate are assigned to group a, and those in whom the attending physician considers ventilator or icu management necessary to group d. drugs to be recommended were added, and a treatment category algorithm ( fig. 1 ) [96] was prepared. concerning hcap treatment in europe and the united states, there is a gap between drugs used in clinical practice and those recommended in guidelines [105] (ii). therefore, treatment category-based empiric therapy in japan may be acceptable in clinical practice; future investigation is necessary. there is no evidence regarding the administration period of antimicrobial drugs. an administration period of 7e10 days, which is routinely adopted in the highest percentage of patients, is appropriate (biv). when administering antimicrobial drugs for a longer period, equivalent-spectrum antimicrobial drugs should be selected, or de-escalation of antimicrobial drugs should be performed. in this case, fever, crp, and leukocyte counts are often used as indices of the treatment response. in cases of aspiration pneumonia in which aspiration recurs during treatment despite the efficacy of antimicrobial drugs, it is necessary to evaluate whether the effects of antimicrobial drugs are not obtained or recurrence occurs. ---drugs to be recommended--a. empiric therapy ( fig. 1 ) [96] (1) cases in which there is no risk of resistant bacteria and outpatient treatment is performed (group a) according to a study, chlamydophila spp. and m. pneumoniae accounted for 34.7 and 9.3% of patients in whom the type of microorganisms that cause nhcap was clarified in japan, respectively [98] . the results suggested that chlamydophila spp. is a target of treatment, as described for cap. therefore, in group a, combination therapy with a b-lactam and macrolide or monotherapy with a respiratory quinolone should be performed (bii). in group d, an anti-p. aeruginosa drug should be combined with cpfx, pzfx, or azm for injection, considering legionella or chlamydophila spp. pneumonia. however, concerning combination therapy with a macrolide (cii) in patients without "severe pneumonia requiring intensive care", as described below, the evidence level is not always high from the perspectives of medical economics, side effects, and resistant bacteria [106] . some studies examined the mortality rate with respect to the presence or absence of treatment covering atypical pathogens, and reported that the mortality rate was significantly lower in the presence of such treatment [17] . a recent meta-analysis also showed a difference [107] . respiratory quinolones were established as an option (bii) based on many references describing that their effects are similar to or more potent than those of combination therapy with a b-lactam and macrolide. however, this must be further examined, considering factors such as severity and the presence or absence of concomitant sepsis [108] . furthermore, the prevalence of penicillinresistant pneumococcus, which has been internationally emphasized as an issue, and macrolide-resistant pneumococcus, which has been markedly observed in japan, was also a background factor for establishing respiratory quinolones as an option [109] . the previous use of antimicrobial drugs, which is often observed in patients with nhcap, is considered to be a risk factor for resistant pneumococcus [110] . a study reported that penicillin or em resistance in hcap patients was more advanced than in cap patients [100] . a study indicated that the efficacy of oral therapy with lvfx was similar to that of ctrx injection therapy in 619 patients with cap [111] . another study reported that, among 680 patients with non-severe hcap, oral lvfx was useful in those with no description of causative microorganisms [112] . however, when aspiration pneumonia is suspected, grnx or mflx should be selected, because the effects of lvfx on anaerobes are weak. furthermore, several studies suggested the usefulness of mflx, which is not influenced by the kidney function and does not require dose regulation, in elderly patients with nhcap [113, 114] . as treatment is completed with a single, high dose, compliance is favorable. azm sustained-release preparations [115e117], which simultaneously cover bacteria and atypical pathogens, and stfx, which shows a favorable mic for anaerobes, may also be recommended [118] . a first choices -cva/ampc, oral (125/250 mg), 2 tablets/3e4 times a day -sbtpc, oral (375 mg), 2 tablets/3e4 times a day table 8 risk factors for involvement by drug-resistant pathogens a in nhcap [96] . history of antibiotic therapy for 2 or more days in the preceding 90 days current tube feeding the risk of mrsa should be taken into account whenever there is past history of mrsa isolation. when attempting to predict the isolation of drug-resistant pathogens based on the presence of these risk factors, it should be borne in mind that their sensitivity and negative predictive value are high, but their specificity and positive predictive value are low. a drug-resistant pathogens include pseudomonas aeruginosa, mrsa, acinetobacter, esbl-producing enteric bacteria, and stenotrophomonas maltophilia. table 7 possible pathogens isolated from nhcap patients [96] . when an nhcap patient has no risk factors for involvement by drug-resistant pathogens pneumococcus mssa gram-negative enteric bacteria (including klebsiella and e. coli) haemophilus influenzae oral streptococci atypical pathogens (particularly chlamydophila) when an nhcap patient has a risk factor for involvement by drug-resistant pathogens (the following will be considered in addition to the above-mentioned pathogens) pseudomonas aeruginosa mrsa acinetobacter esbl-producing enteric bacteria þ one of the followings: -azm sustained-release preparation, oral, 2 g/single dose -cam, oral, 200 mg/twice a day <> second choices -mflx, oral, 400 mg/once a day -grnx, oral, 400 mg/once a day -stfx, oral, 100 mg/l to 2 times a day or -ctrx *1 , intravenous drip, 2 g/once a day or 1 g/twice a day -ctx *1 , intravenous drip, 1e2 g/2e3 times a day þ one of the followings: -azm sustained-release preparation, oral, 2 g/single dose -cam, oral, 200 mg/twice a day *1) as the antimicrobial activity of the drug against anaerobes is insufficient, it is inappropriate under a tentative diagnosis of aspiration pneumonia. (2) cases in which there is no risk of resistant bacteria and hospital treatment is performed (group b) in this category, we recommend monotherapy from the perspectives of causative microorganisms resembling those for cap and side effect-based "innocent properties". a study reported that initial treatment with narrow-spectrum antimicrobial drugs did not always lead to a poor prognosis [119] . in particular, according to two articles [120, 121] , it is not necessary to consider resistant bacteria in hcap patients in whom causative microorganisms are unclear; treatment in accordance with cap treatment is sufficient (bii). however, an advanced age, central nervous diseases, aspiration, and a reduction in adl are the clinical characteristics of aspiration pneumonia. the condition of nhcap in japan overlaps with aspiration pneumonia [122] . therefore, in group b, to which patients admitted for the first time, with no recent use of antimicrobial drugs, correspond, antimicrobial therapy with b-lactamase inhibitor-containing penicillins is appropriate, as described for cap. however, when aspiration pneumonia is suspected, ctrx and lvfx should be avoided (biv). for the management of enteric bacteria, candidate drugs for group b, papm/bp, may be selected. actually, it is not necessary to consider p. aeruginosa in patients with cap or non-icu hap. a study indicated that ertapenem, which is not effective for p. aeruginosa, was useful [123] , as demonstrated for papm/bp. however, another study reported that the widespread use of ertapenem induced the cross resistance of p. aeruginosa to other carbapenems; the use of papm/bp alone should be avoided [124] (biv). in elderly persons, with a high risk of aspiration, who are repeatedly admitted and discharged, klebsiella is often involved. taz/pipc is more useful according to a study [125] (bii). in patients in whom gram-negative bacillus is detected on gram staining of sputum or those in whom the involvement of enteric bacteria is suspected, papm/bp or taz/pipc should be selected (biv). -sbt/abpc, intravenous drip, 3 g/3e4 times a day -ctrx *1 , intravenous drip, 2 g/once a day or 1 g/twice a day -ctx *1 , intravenous drip, 1e2 g/2e3 times a day -lvfx *1 , intravenous drip, 500 mg/once a day -papm/bp, intravenous drip, 0.5e1 g/2e4 times a day *1) as the antimicrobial activity of the drug against anaerobes is insufficient, it is inappropriate under a tentative diagnosis of aspiration pneumonia. (3) cases in which there is a risk of resistant bacteria and hospital treatment is performed (group c) the target microorganisms include p. aeruginosa, mrsa, and acinetobacter spp. in addition to frequent microorganisms that cause respiratory infection [97, 99, 103, 126] . as antimicrobial drugs, taz/pipc, with an antimicrobial activity against p. aeruginosa, fourth-generation cephems, carbapenems, and quinolones (cpfx, pzfx) are recommended. taz/ pipc exhibits effects similar to those of ipm/cs and mepm in patients with nursing and healthcare-associated pneumonia [127] (bii). pzfx also has an antimicrobial activity against s. pneumoniae when used at a high dose (2 g/day). when pneumonia related to atypical pathogens such as chlamydophila spp. is suggested, quinolones should be selected. as the antimicrobial activities of fourth-generation cephems and quinolones against anaerobes are weak, these drugs should be combined with mnz, cldm, or sbt/abpc. recently, the resistance of the bacteroides fragilis group to cldm has advanced [128] . therefore, in europe and the united states, mnz is selected as a first-choice antimicrobial drug against anaerobes. however, the rate at which the b. fragilis group is involved in oral anaerobes is low, and combination therapy with cldm may be selected [129, 130] . therefore, for combination therapy with fourth-generation cephems, we recommend the two drugs. when there is a risk of mrsa, such as previous admission, they should be combined with vcm, teic, or lzd. if there is no abscess formation, abk is also effective. a first choices -taz/pipc, intravenous drip, 4.5 g/3e4 times a day -ipm/cs, intravenous drip, 0.5e1 g/2e4 times a day -mepm, intravenous drip, 1 g/2e3 times a day -drpm, intravenous drip, 0.5e1 g/3 times a day <> second choices -cfpm* 2 , intravenous drip, 1e2 g/2e4 times a day -cpr *2 , intravenous drip, 1e2 g/2e4 times a day þ one of the followings: -cldm, intravenous drip, 600 mg/2e4 times a day -mnz, intravenous drip, 500 mg/4 times a day or -cpfx *2 , intravenous drip, 300 mg/twice a day -pzfx *2 , intravenous drip, 1000 mg/twice a day þ one of the followings: -cldm, intravenous drip, 600 mg/2e4 times a day -sbt/abpc, intravenous drip, 3 g/3e4 times a day *in addition to the above drugs, if mrsa infection is suspected, antimicrobial drugs should be added in accordance with the section "mrsa pneumonia". *2) as the antimicrobial activity of the drug against anaerobes is insufficient, it should be combined with a drug with an antimicrobial activity against anaerobes (mnz, cldm, or sbt/abpc) under a tentative diagnosis of aspiration pneumonia. (4) severe cases requiring intensive care (group d) to cover l. pneumophila and atypical pathogens, which are rare as causative microorganisms but may cause severe conditions, group-c antimicrobial drugs should be combined with cpfx, pzfx, or azm injection (bi). concerning the usefulness of combination therapy with a b-lactam and macrolide injection for severe pneumonia, evidence has been accumulated [131] . a study indicated that, in severe community-acquired pneumonia patients with sepsis or requiring icu management, combination therapy with a blactam and macrolide led to a more favorable prognosis compared to that with a quinolone (i), suggesting that antiinflammatory actions are involved in the mechanism [132] . in addition, another study reported that, among pneumonia patients with acute pulmonary disorder, both the ventilator withdrawal and survival rates in a macrolide-treated group were higher than in a non-macrolide-treated group [19] (i). several meta-analyses also support them [107, 133] . a first choices -taz/pipc, intravenous drip, 4.5 g/3e4 times a day -ipm/cs, intravenous drip, 0.5e1 g/2e4 times a day -mepm, intravenous drip, 1 g/2e3 times a day -drpm, intravenous drip, 0.5e1 g/3 times a day þ one of the followings: -cpfx *2 , intravenous drip, 300 mg/twice a day -pzfx *2 , intravenous drip, 1000 mg/twice a day -azm, intravenous drip, 500 mg/once a day <> second choices -cfpm *2 , intravenous drip, 1e2 g/2e4 times a day -cpr *2 , intravenous drip, 1e2 g/2e4 times a day þ one of the followings: -cldm, intravenous drip, 600 mg/2e4 times a day -mnz, intravenous drip, 500 mg/4 times a day þ one of the followings: -cpfx *2 , intravenous drip, 300 mg/twice a day -pzfx *2 , intravenous drip, 1000 mg/twice a day -azm, intravenous drip, 500 mg/once a day in addition to the above drugs, *if mrsa infection is suspected, antimicrobial drugs should be added in accordance with the section "mrsa pneumonia". *2) as the antimicrobial activity of the drug against anaerobes is insufficient, it should be combined with a drug with an antimicrobial activity against anaerobes (mnz, cldm, or sbt/abpc) under a tentative diagnosis of aspiration pneumonia. antimicrobial drugs against identified causative microorganisms should be selected in accordance with the section "2.2 hospital-acquired pneumonia" (p. 8). ---executive summary----as oral indigenous bacteria, including anaerobes, cause aspiration pneumonia, b-lactamase inhibitor-containing penicillins are appropriate (bii). -in cases of nosocomial onset, it is necessary to cover gramnegative bacillus, including p. aeruginosa. -in cases of severe ventilator-associated pneumonia (vap), the selection of broad-spectrum antimicrobial drugs or combination therapy with them should not be hesitated (ai). -the detection rate of esbl-producing gram-negative bacillus has increased, and antimicrobial drugs should be carefully selected. -it is important to prevent subclinical aspiration through oral care and the prevention of gastroesophageal reflux, such as head lifting (bii). -to prevent aspiration pneumonia, it is also important to improve the nutritional status and avoid the overuse of sleeping pills/ sedatives (bii). ---explanation---[characteristic and classification of diseases] aspiration pneumonia occurs with a background factor, dysphagia, which is frequently observed in the presence of a reduction in adl or systemic functions, especially cerebrovascular disorder. its onset is associated with dietary ingestion in elderly persons [134] . currently, aspiration pneumonia is accurately defined only in the guidelines for the management of hospitalacquired pneumonia (hap) in adults, which were prepared by the japanese respiratory society [135] . the guidelines present conditions that may cause dysphagia, which were proposed by the japanese study group on aspiration pulmonary disease (table 9 , modified) [136] . in our guidelines, we primarily explain antimicrobial drugs to be selected for patients with such conditions. the above definition is also adopted in the guidelines for the management of nursing and healthcare-associated pneumonia (nhcap) [96] . in elderly persons admitted to long-term care beds or nursing homes, risk factors include dysphagia and tubal feeding according to international data on pneumonia that develops in nursing homes [137e139]. in japan, frequent underlying diseases in patients with nhcap also include central nervous diseases and dementia, which are closely associated with aspiration. the proportion of patients after percutaneous endoscopic gastrostomy (peg) is high [140] . however, among various types of community-acquired pneumonia, a diagnosis of aspiration pneumonia is made based on onset factor-based classification, and is not equal to nhcap diagnosed primarily based on the place of onset or grade of nursing. according to data in spain, aspiration pneumonia accounts for 20.6% of patients with healthcareassociated pneumonia (hcap) requiring admission. this percentage was markedly higher than in those with communityacquired pneumonia (cap) requiring admission (3.6%), but corresponded to no more than 1/5 [100] . on the other hand, a multicenter cooperative study involving inpatients with pneumonia in japan, where the rapid aging of society is advanced, reported that 60.1% of patients who were admitted with cap had aspiration pneumonia. even in patients with cap, which is not classified as nhcap, the involvement of aspiration cannot be ignored [141] . furthermore, the study indicated the involvement of aspiration in 86.7% of patients, aged over 70 years, with cap/ hap [141] . in the future, the significance of distinguishing aspiration pneumonia among patients with nhcap or hap and changing therapeutic strategies should be examined. however, nhcap more markedly affects adl compared to cap, and the aspect of elderly pneumonia is emphasized; it may be significant to positively diagnose aspiration pneumonia and establish therapeutic strategies different from those for cap [142] . concerning hap, a reduction in the immune function is a background factor. hap has two aspects: pneumonia with a high risk of resistant bacteria and that in which central nervous diseaserelated aspiration is involved. in the guidelines for the management of hospital-acquired pneumonia in adults, which were prepared by the japanese respiratory society, mendelson syndrome and vap are categorized as a group, and 3 classifications, involving diffuse deglutition-related bronchiolitis, in which there are no findings of pneumonia, are proposed. in addition, a flow chart for diagnosis is presented [135] (fig. 2) . with respect to the condition and treatment of vap, refer to a review described by chastre et al. [55] management other than antimicrobial drug therapy should also be considered, and bundle (table 10 )-based prevention should be performed [143] (aii). [type and frequency of causative microorganisms] streptococcus pneumoniae, s. aureus, and enterobacteriacae have been reported. a study indicated that k. pneumoniae was frequent [144] . the involvement of oral indigenous bacteria, such as streptococcus anginosus group. and anaerobes, has been suggested [145, 146] . in cases of nosocomial onset, gram-negative bacillus, including p. aeruginosa, must also be considered. concerning e. coli, klebsiella spp., and proteus spp., the number of esbl-producing strains may increase in the future. [rules of antimicrobial drug therapy] if appropriate antimicrobial drug therapy is not selected under a diagnosis of aspiration-related pneumonia, insufficient treatment may lead to a fatal condition, or excessive treatment may increase the number of resistant bacteria, showing negative effects. there may be differences in options for empiric therapy between patients with vap (most patients show severe conditions) and those with diffuse deglutition-related bronchiolitis, in whom the start of treatment is not accelerated. on the other hand, approaches to prevent pneumonia after aspiration or avoid aspiration are important. oral care, head lifting, and improvement in the nutritional status must be considered, and the overuse of sleeping pills/ sedatives should be avoided (bii). the best option for standard-type aspiration pneumonia is an antimicrobial drug that exists an antimicrobial activity against both aerobes and anaerobes. sbt/abpc and taz/pipc are effective for anaerobes frequently isolated in the respiratory system, such as fusobacterium spp., prevotella spp., and peptostreptococcus spp. [147, 148] . as the resistance rates of these types of bacteria to the two regimens are low, these regimens are also recommended as first choices in the guidelines established by the japanese association for anaerobic infection research [126] . however, a study reported that the previous administration of antimicrobial drugs and adl were correlated with the frequency of enterobacteriacae-or p. aeruginosa-related pneumonia [149] . a retrospective study involving 90 patients with aspiration pneumonia showed that the frequency of k. pneumoniae-related pneumonia was 25% [150] . based on these studies, drugs to be selected should be changed in accordance with the previous administration of antimicrobial chemotherapeutic drugs in patients admitted to general or medical wards. among patients with hospital-acquired pneumonia, broad-spectrum drugs should be selected in those with severe aspiration pneumonia or vap (bii). when causative microorganisms are identified and an improvement in the condition is achieved, de-escalation should be performed. ---drugs to be recommended--a. empiric therapy ➀ no risk of resistant bacteria drugs with potent antimicrobial activities against oral anaerobes are presented. however, no article has provided high-level evidence regarding aspiration pneumonia. as the following drugs affect the intestinal flora, antimicrobial drugassociated diarrhea may occur. if symptom improvement is delayed, patients must be promptly admitted, and drip table 9 conditions that may cause dysphagia [136] [96] . bundles for the prevention of ventilator-associated pneumonia. (1) upper body lifting the head should be lifted at 30e45 . (2) discontinuation of sedatives a sedative should be discontinued once a day to evaluate whether or not extubation is possible. ( in addition to the above items, methods to prevent aspiration pneumonia include oral care, the administration of drugs that improve the deglutition function, such as ace inhibitors and cilostazol, improvement in the nutritional status, eating/swallowing rehabilitation, and anti-pneumococcus vaccination. infusion therapy should be performed (outpatient treatment should not be prolonged). (1) outpatient treatment a first choices -cva/ampc, oral (125/250 mg), 2 tablets/3e4 times a day -sbtpc, oral (375 mg), 2 tablets/3e4 times a day <> second choices -mflx, oral, 400 mg/once a day -stfx, oral, 100 mg/1e2 times a day -grnx, oral, 400 mg/once a day (2) hospital treatment when a diagnosis of aspiration pneumonia is made, sbt/abpc is most frequently used in japan [151] . kaneko et al. reported that the sensitivity of oral anaerobes that may cause aspiration pneumonia, such as peptostreptococcus spp., prevotella spp., and fusobacterium spp., to sbt/abpc was 100%, similar to that to taz/pipc [152] . the effects of cldm on aspiration pneumonia or a pulmonary abscess are similar to those of sbt/abpc (bi) [145] . sbt/abpc and cldm showed similar effects and tolerance on aspiration pneumonia (67.5 and 63.5%, respectively) [147] . oral anaerobes, excluding bacteroides spp., are still susceptible to cldm. a randomized clinical trial (rct) indicated that cldm was more potent than cephems [153] . a first choice -sbt/abpc, intravenous drip, 3 g/3e4 times a day <> second choice -cldm, intravenous drip, 600 mg/2e4 times a day ➁ cases in which there is a risk of resistant bacteria or severe cases in cases in which there is a risk of resistant bacteria or severe cases, drugs should be selected in accordance with options for group c for nhcap. tubal feeding is a risk factor for aspiration, and is also a risk factor for resistant bacteria [97] . when the involvement of enterobacteriacae, such as k. pneumoniae and e. coli, is suggested, empiric therapy should be selected in accordance with cases in which there is a risk of resistant bacteria [55] . in japan, the proportion of esbl-producing bacteria on sputum culture in patients with respiratory infectious diseases is 5% or less [13] , but the number of esbl-producing bacteria has slightly increased; this must be considered in the future [154, 155] . a study reported that the clinical effects of taz/pipc on non-esblproducing k. pneumoniae were more potent than those of sbt/abpc; caution is needed [125] . it must be considered that, in cases of aspiration pneumonia classified as hcap, enterobacteriacae is isolated at a frequency that cannot be ignored. concerning aspiration pneumonia that occurs in hospitals, some reviews proposed that antimicrobial drugs should be selected, regarding the condition as hospitalacquired pneumonia; empiric therapy may be selected in accordance with the guidelines for the management of hospital-acquired pneumonia, which were published by the japanese respiratory society [156, 157] . according to a study, bipm, which does not cause kidney dysfunction in elderly patients, is also effective (civ); therefore, it is presented as an option for cases in which there is a risk of resistant bacteria [158] . the mortality rate in patients with vap is high. if causative microorganisms cannot be initially covered, the mortality rate may increase [51] . therefore, drugs should be selected, regarding the condition as severe aspiration pneumonia. three studies reported that, in a group in which taz/pipc was selected as a drug to be combined with aminoglycosides for vap treatment, the mortality rate was lower than in a group in which caz was selected [159e161]. in particular, when pneumonia was caused by p. aeruginosa, the clinical effects of taz/pipc were more potent than those of ipm/cs (bii). for monotherapy, information on the culture of protected specimen brush (psb) samples or broncho-alveolar lavage (bal) fluid is strongly recommended [70, 162] . on the other hand, an observational study indicated that threedrug therapy (two anti-p. aeruginosa drugs þ an anti-mrsa drug) deteriorated the prognosis; an rct should be conducted in the future [60] . therefore, if there is a risk of resistant bacteria, at least broad-spectrum antimicrobial drugs must be used for empiric therapy. however, assuming causative microorganisms with reference to gram staining reactions, minimum necessary antimicrobial drugs should be selected based on local factors (antimicrobial drug susceptibility pattern of each type of bacteria in each hospital). recently, the entity of ventilator-associated tracheobronchitis (vat) was proposed, and the disadvantages of aggressive treatment have been discussed [163] . in a multicenter cooperative study, patients with vat, which occurred in the icu, were divided into two groups with and without antimicrobial drug therapy, and the results were compared. in the former, the incidence of vap was significantly lower, and the mechanical ventilation-free period was significantly longer. in addition, the icu mortality rate was significantly lower. on the other hand, there was no significant difference in the appearance of resistant bacteria between the two groups [164] . a first choices -taz/pipc, intravenous drip, 4.5 g/3e4 times a day -ipm/cs, intravenous drip, 0.5e1 g/2e4 times a day -mepm, intravenous drip, 1 g/2e3 times a day -drpm, intravenous drip, 0.5e1 g/3 times a day -bipm, intravenous drip, 0.3e0.6 g/3e4 times a day <> second choices -cfpm, intravenous drip, 1e2 g/2e4 times a day -cpr, intravenous drip, 1e2 g/2e4 times a day þ one of the followings: -cldm, intravenous drip, 600 mg/2e4 times a day -mnz, intravenous drip, 500 mg/4 times a day or -lvfx, intravenous drip, 500 mg/once a day -cpfx, intravenous drip, 300 mg/twice a day -pzfx, intravenous drip, 500 to 1000 mg/twice a day þ one of the followings: -cldm, intravenous drip, 600 mg/2e4 times a day -mnz, intravenous drip, 500 mg/4 times a day -sbt/abpc, intravenous drip, 1.5e3 g/3e4 times a day *if mrsa infection is suspected, antimicrobial drugs should be administered in accordance with the section "mrsa pneumonia" in addition to the above drugs. [administration period of antimicrobial drugs] it is recommended that the treatment period of hospitalacquired pneumonia should be 7e10 days. however, the treatment period should be 14 days in patients with pneumonia related to non-glucose-fermenting bacteria such as p. aeruginosa [2] (bii). concerning vap, a study reported that there was no difference in the clinical effects between 8 and 15 days [57] . to control identified causative microorganisms, antimicrobial drugs should be selected in accordance with the section "hospital-acquired pneumonia". if mrsa infection is suspected, antimicrobial drugs should be selected in accordance with the section "mrsa pneumonia". (bii). -in severe or refractory cases in which the efficacy of initial treatment is not sufficient, combination therapy with an antifungal drug should also be considered [171e173] (bii). -as the effects of combination therapy with an azole and amph-b preparation antagonize in some strains, a combination of these drugs should be avoided [174] (aiii). -for the target treatment of this disease, an antifungal drug of which the class is different from that of a drug used for preventive administration should be used (biii). ---explanation---[characteristics of the disease] -symptoms: this disease develops in severe immunocompromised hosts such as patients received chemotherapy for leukemia or hematopoietic stem cell/organ transplantation. symptoms such as fever that does not respond to broadspectrum antimicrobial drugs, cough, dyspnea, sputum, and bloody sputum/hemoptysis are observed. -laboratory findings: chest x-ray shows an infiltrative shadow (typically, a wedge shadow involving the pleura as the base). on thoracic ct, infiltrative and nodular shadows (with a halo sign in some cases) are observed. in the recovery phase of neutrophils, an air crescent sign is noted. an increase in the inflammatory marker such as the crp level, aspergillus galactomannan antigen-positive reactions, and an increase in the (1 / 3)-b-d-glucan level are useful for diagnosis. however, neither the sensitivity nor specificity is sufficient. the results should be carefully evaluated. -causative microorganisms: aspergillus fumigatus is frequently detected, but, recently, an increase in the number of patients with non-fumigatus aspergillus-related ipa has been indicated. -specific condition: lesions are sometimes formed in the nasal sinus and brain; caution is needed. -early diagnosis: early treatment is important for successful treatment for this disease. ---drugs to be recommended---a study reported that, in a group in which vrcz was used for the initial treatment of ipa, the results of treatment were more favorable than in a group in which d-amph was used [166] . furthermore, another study indicated that therapy with l-amb at 3 mg/kg/day was safer than that at 10 mg/kg/ day, although there was no significant difference in the clinical efficacy [167] . cpfg, mcfg, and itcz also have anti-aspergillus activities, and can be used. it is important to consider different strategies in accordance with the appearance of the host's allergy or adverse events and interactions with drugs used to treat an underlying disease. b. chronic progressive pulmonary aspergillosis (cppa) ---executive summary----in japan, various disease types such as aspergilloma with infiltration and enlargement of an existing cavity are included. cppa includes various diseases such as chronic necrotizing pulmonary aspergillosis (cnpa), chronic cavitary pulmonary aspergillosis (ccpa), and chronic fibrosing pulmonary aspergillosis (cfpa). it refers to a series of syndrome for which the administration of antifungal drugs is essential. -treatment should be started with injection. if symptoms and findings are stabilized, injection should be switched to oral drugs. -initial treatment with mcfg or cpfg should be performed [175, 176] (ai). -initial treatment with itcz, vrcz, or l-amb can also be selected in accordance with the host's underlying disease or drugs used to treat the underlying disease. -for maintenance therapy, itcz and vrcz oral preparations are recommended (aiii). ---explanation---[characteristics of the disease] -symptoms: this disease develops in hosts with organic diseases such as a cavity or cystic disease of the lung or bronchus. symptoms such as fever, sputum, bloody sputum/ hemoptysis, and dyspnea are observed. -laboratory findings: chest x-ray and ct show an infiltrative shadow, enlargement of a cavity, thickening of the cavity wall/pleura, and a niveau in the cavity. there is an increase in the crp level in many patients. most patients are positive for anti-aspergillus precipitating antibody. neither aspergillus galactomannan antigen nor b-d-glucan is a clue to diagnosis. -causative microorganisms: a. fumigatus is frequently detected. non-fumigatus aspergillus-related cppa is also often observed. ---drugs to be recommended---a clinical study in japan indicated that there was no marked difference in the efficacy of treatment between mcfg-and vrcz-treated groups, whereas mcfg was safer [175] . another study reported that there was no difference in treatment results between mcfg and cpfg [176] . in the phase of severe symptoms such as fever and bloody sputum, treatment should be started using these injections. -pulmonary aspergilloma is classified into two types: simple and complex aspergilloma based on the grade of difficulty in resection. the former refers to aspergilloma formation in a focus with a thin wall, such as a cyst, without accessory lesions at the periphery. the latter refers to aspergilloma formation in a cavity derived from a strongly destructed existing structure of the lung, such as old pulmonary tuberculosis and bronchiectasis, with marked destructive lesions or pleural adhesion at the periphery of the cavity. ---drugs to be recommended---resection should be selected as a first choice. when resection is impossible, medical treatment can be considered. in preparation, such as l-amb, and 5-fc should be performed for 2 weeks or more. subsequently, treatment should be continued using flcz or f-flcz. -in japan, cryptococcus gattii infection has also been reported. if possible, causative fungus must be isolated/identified. ---explanation---[characteristics of the disease] -symptoms: this disease is often asymptomatic, and is detected on a health checkup in many cases. -laboratory findings: chest x-ray and ct show solitary or multiple nodular and infiltrative shadows. -some cavities are observed in a lot of cases. there is no enhancement of the inflammatory marker in many cases, but glucuronoxylomannan antigen-positive reactions are detected. -causative microorganisms: this disease is caused by cryptococcus neoformans. recently, infection with c. gattii has been reported in vancouver, canada and the north area of the west coast of the united states of america; caution is needed. -c. gattii is primarily distributed in the tropical and subtropical zones. infection in humans has been considered to be rare. however, since 1999, patients infected with c. gattii have been reported in the pacific coast of north america. even healthy adults are infected with c. gattii, and the mortality rate is high. ---drugs to be recommended---although there is no evidence regarding pulmonary cryptococcosis, flcz tablets, which have a potent activity against cryptococcus, are frequently selected when the patient's condition is stable in the absence of an underlying disease. azoles other than this drug can also be selected. [180] (aii). -if the lesion is localized, resection should be considered. -combination therapy with an iron chelating agent and l-amb should be avoided [181] (ai). ---explanation--recently, an increase in the incidence of infection with cunninghamella has also been indicated. -specific condition: nasal/brain-type, dermal, and disseminated zygomycosis is observed. ---drugs to be recommended---currently, only amph preparations may be clinically useful for treating zygomycosis among antifungal drugs that are available in clinical practice in japan. as high-dose therapy must be started as early as possible, not d-amph but l-amb should be selected. -l-amb, intravenous drip, 5 mg/kg/once a day precautions for the use of antifungal drugs (confirm the package inserts.) ➀ vrcz vision disorder, liver dysfunction, and neurological/ mental adverse events may occur. combination therapy with rfp, rbt, efavirenz, ritonavir, carbamazepine, long-acting barbiturate, pimozide, quinidine sulfate, ergot alkaloid, or triazolam is contraindicated. this drug is also contraindicated for pregnant women. as a rule, it is contraindicated for patients with a ccr of <30 ml/ min (injection only). as the blood concentration of vrcz may vary, tdm should be conducted. in patients with mild to moderate liver dysfunction, the dose should be regulated. ➁ itcz hepatopathy and congestive heart failure may occur. combination therapy with pimozide, quinidine, bepridil, simvastatin, triazolam, azelnidipine, ergotamine, nisoldipine, dihydroergotamine, vardenafil, eplerenone, blonanserin, sildenafil, tadalafil, aliskiren, dabigatran(itcz oral only), rivaroxaban, ergometrine, or methylergometrine is contraindicated. this drug is contraindicated for patients with severe liver diseases, pregnant women. patients with a ccr of <30 ml/min are also contraindicated (injection only). ➂ flcz hepatopathy and a prolongation of qt may occur. combination therapy with triazolam, ergotamine, dihydroergotamine, quinidine, or pimozide is contraindicated. this drug is also contraindicated for pregnant women. ➃ f-flcz combination therapy with triazolam, ergotamine, dihydroergotamine, quinidine, or pimozide is contraindicated. this drug is also contraindicated for pregnant women. ➄ l-amb adverse events such as nephropathy, hypopotassiumemia, and fever may occur. this drug is contraindicated during leukocyte transfusion. ➅ cpfg this drug is safe, but hepatopathy may occur. caution is needed for combination therapy with cyclosporin, tacrolimus, rfp, efavirenz, nevirapine, phenytoin, dexamethasone, or carbamazepine. ➆ mcfg this drug is safe, but hepatopathy may occur. ➇ 5-fc anorexia and myelopathy may occur. combination therapy with tegafur-/gimeracil-/oteracil potassium-containing drugs is contraindicated. even after the discontinuation of tegafur-/gimeracil-/oteracil potassium-containing drugs, combination therapy should be avoided within 7 days. this drug is also contraindicated for pregnant women. ---drugs to be recommended---st combination drugs are used as a gold standard of pcp treatment. however, there have been a large number of patients in whom treatment was discontinued due to their side effects. recently, atovaquone also became available in japan as a second-choice drug. in patients with mild pcp, atovaquone tablets were as effective as st combination drugs. in those with moderate pcp, st combination drugs were more effective, but there was no significant difference due to a small number of subjects. in atovaquone-treated patients, the incidence of adverse events for which administration was discontinued was lower than in st combination drug-treated patients, suggesting that the tolerance is high [184] . the administration period should be 21 days in hivinfected patients and 14 days in non-hiv-infected patients. a target daily dose of trimethoprim should be 15e20 mg/kg. -st combination drug, oral, 3 to 4 tablets/3 times a day -st combination drug, intravenous drip, 240e320 mg as trimethoprim/3 times a day (infused over 1e2 h) <> second choices -pentamidine, intravenous drip, 4 mg/kg/once a day (infused over 1e2 h) -atovaquone oral suspension, 5 ml (750 mg as atovaquone)/twice a day for 21 days (orally administered after meals) * adjuvant therapy in patients with a pao 2 of <70 mmhg or a-ado 2 of >35 mmhg in room air, one of the above drugs should be combined with a corticosteroid from the start of treatment. however, the dose may be reduced or administration may be discontinued in the early phase in accordance with symptoms. when the respiratory state is extremely unfavorable, pulse therapy should also be considered. prednisolone days 1e5: oral, 30e40 mg/twice a day days 6e10: oral, 15e20 mg/twice a day days 11e21: oral, 7.5e10 mg/twice a day ---precautions for each drug---(confirm the package inserts.) ➀ st combination drug (baktar tablets) fever, exanthema, digestive symptoms, hepatopathy, nephropathy, and blood disorder may occur. this drug may interact with methotrexate, sulfadoxine, pyrimethamine, diaphenylsulfone, sulfonyl amide/sulfonylurea oral drugs for diabetes, warfarin, phenytoin, cyclosporin, zidovudine, digoxin, tricyclic antidepressants, and lamivudine. this drug is contraindicated for neonates, low-birthweight infants, pregnant women, and patients with g-6-pd deficiency. in patients with renal dysfunction, dose reduction must be considered. side effects such as hypoglycemia, hypotension, nephropathy, taste disorder, numbness of the tongue/lips, ventricular arrhythmia, exanthema, and fever may occur. combination therapy with zalcitabine, pfa, or amiodarone is contraindicated. this drug is contraindicated for patients with severe ventilatory disturbance. ➂ atovaquone nausea/vomiting, exanthema, and diarrhea may occur. this drug should be carefully administered to patients with severe kidney or liver dysfunction. this drug may interact with rfp, rbt, tetracycline, metoclopramide, zidovudine, acetaminophen, benzodiazepines, aciclovir, opioid analgesic drugs, cephalosporin antibiotics, antidiarrheal drugs/laxatives, and indinavir. h. cytomegalovirus (cmv) pneumonia ---executive summary----in the field of transplantation, preemptive treatment with gcv should be conducted through cmv antigenemia test monitoring. -the efficacy of preemptive treatment with vgcv or pfa is similar to that of gcv. -if a diagnosis of cmv pneumonia is made, treatment with gcv should be promptly started [185] (aii). -vgcv and pfa are recognized as alternative drugs for gcv [186, 187] (bii). -combination therapy with an antiviral drug and high-dose immunoglobulin should be performed [188] (aiii). ---explanation-----antimicrobial drugs to be recommended---a first-choice drug for cmv pneumonia treatment is gcv, which has been frequently used. pfa has been used to treat cmv infection in aids patients, but experience on its use is limited in patients after hematopoietic stem cell transplantation. (1) initial administration a first choice -gcv, intravenous drip, 5 mg/kg (over 1 h or more)/ every 12 h for 2e3 weeks þ anti-cmv high-titer gamma globulin, intravenous drip, 2.5e5 g/once a day for the first 3 days <> second choices -pfa, intravenous drip, 60 mg/kg (over 1 h or more)/3 times a day, every 8 h for 2e3 weeks or more þ anti-cmv high-titer gamma globulin, intravenous drip, 2.5e5 g/once a day for the first 3 days or -pfa, intravenous drip, 90 mg/kg (over 2 h or more)/ twice a day, every 12 h for 2e3 weeks or more þ anti-cmv high-titer gamma globulin, intravenous drip, 2.5e5 g/once a day for the first 3 days (2) maintenance administration a first choices -gcv, intravenous drip, 5 mg/kg (over 1 h or more)/once a day, 7 days a week or -gcv, intravenous drip, 6 mg/kg (over 1 h or more)/once a day, 5 days a week * this regimen should be completed after confirming the disappearance of clinical symptoms and negative reactions on two consecutive cmv antigenemia tests. <> second choice -pfa, intravenous drip, 90e120 mg/kg (over 2 h or more)/once a day (in clinical practice in japan, there have been few case reports on once-a-day administration at 120 mg/ kg as maintenance therapy. a dose exceeding 120 mg/ kg should be avoided. for administration at 120 mg/kg, twice-a-day administration at 60 mg/kg is commonly selected.) * this regimen should be completed after confirming the disappearance of clinical symptoms and negative reactions on two consecutive cmv antigenemia tests. ---precautions for each drug---(confirm the package inserts.) ➀ gcv severe leukopenia, neutropenia, anemia, thrombopenia, pancytopenia, aplastic anemia, and bone marrow suppression may occur. an animal experiment showed that this drug induced transient or irreversible spermatogenic dysfunction and reduced fertility. in humans, this drug may cause spermatogenic dysfunction. an animal experiment demonstrated the teratogenicity, mutagenicity, and carcinogenicity of this drug. in the presence of renal hypofunction, it is necessary to regulate the dose. this drug is contraindicated for patients with marked bone marrow suppression (neutrophil count: <500/mm 3 or platelet count: <25,000/mm 3 ) and pregnant women. it may interact with didanosine, zidovudine, ipm/cs, bone marrow-suppressing and kidney function-affecting drugs, zalcitabine, st combination drugs, cyclosporin, probenecid, and mycophenolate mofetil. ➁ vgcv this is a prodrug of gcv. ➂ pfa acute renal failure, shock, heart failure, thrombophlebitis, and convulsion may occur. it is necessary to regulate the dose in accordance with the kidney function. combination therapy with pentamidine is contraindicated. this drug is contraindicated for patients with a ccr of <0.4 ml/min/kg. ---executive summary---for the treatment of community-acquired pneumonia in children, antimicrobial drugs should be selected, considering age and severity. ---explanation--patients with acute respiratory infectious disease symptoms, such as fever, nasal discharge, pharyngeal pain, and cough, and the appearance of a new infiltrative shadow in the lung on imaging examinations such as chest x-ray and ct are regarded as having pneumonia [189] . in patients with pneumonia, thoracic auscultation findings often include accessory murmurs and the attenuation of respiratory sounds. most patients with respiratory infectious diseases consult hospitals with fever and cough. the lesion site of the airway is estimated based on symptoms and physical findings (fig. 3 ) [189] . in addition to thoracic findings, it is necessary to check the presence or absence of dyspnea signs, such as tachypnea, nasal alar breathing, retractive breathing, shoulder breathing, orthopnea, groaning, and cyanosis. to consider the need of antimicrobial-drug administration and options of antimicrobial drugs, pneumonia is classified into three types: bacterial, viral, and atypical pneumonia based on causative microorganisms [189] . [type and frequency of causative microorganisms] microorganisms that cause childhood community-acquired pneumonia differ among ages. according to the data on investigation of causative microorganisms based on lavage sputum culture in japan, bacterial and viral pneumonia is frequent in infants/children aged 1 year or younger. in those aged 2e6 years, the incidences of bacterial, viral, and atypical pneumonia are similar. in those aged over 6 years, the incidence of atypical pneumonia is the highest [189] (fig. 4) insufficient. however, a review reported similar findings [191] ( table 11 ) [189] . in children, it is not easy to investigate causative microorganisms. in institutions in which it is impossible to investigate causative microorganisms, treatment must be performed based on the statistical frequency of causative microorganisms described below. however, in those in which investigation is possible, the etiology should be investigated if possible. [rules of antimicrobial drug therapy] it is important to improve the efficacy of treatment by accurately predicting causative microorganisms and selecting an appropriate antimicrobial drug and its administration method. whether or not an antimicrobial drug should be indicated must be comprehensively evaluated by differentiating bacterial, viral, and atypical pneumonia with reference to age, severity, clinical symptoms, physical findings, laboratory data, and x-ray findings [189] . as a rule, a single antimicrobial drug should be selected. when the type of microorganisms that caused pneumonia is identified, an antimicrobial drug should be selected through de-escalation, considering drug susceptibility and pharmacokinetics. [clinical symptoms, physical findings] wet cough and tachypnea are frequently observed in children with bacterial pneumonia. the proportion of labored breathingfree patients is high in children with mycoplasma pneumonia [191, 192] . auscultation findings include intermittent accessory murmurs (rales) regardless of the type of pneumonia. in children with mycoplasma pneumonia, the proportion of those in whom auscultation findings are not marked is significantly higher than in other groups. in children with chlamydia pneumonia, fever is mild, and cough is protracted. thus, clinical symptoms and physical findings show characteristics related to causative microorganisms, but it is difficult to identify causative microorganisms based on clinical symptoms and physical findings alone in individual patients [193e195] . concerning laboratory findings on admission in children with bacterial and viral pneumonia, there are significant differences in the leukocyte count, crp level, and erythrocyte sedimentation rate between the two groups (p < 0.01). however, measurements overlapping in about one-third of patients are presented [195] (fig. 5) . briefly, it is impossible to accurately differentiate bacterial from viral pneumonia based on inflammatory responses reflected by the leukocyte count, crp level, and erythrocyte sedimentation rate. mycoplasma pneumonia is characterized by increases in the crp level and erythrocyte sedimentation rate, but many patients show normal leukocyte counts or a slight decrease in this parameter. furthermore, it is difficult to differentiate mycoplasma from viral pneumonia based on laboratory data [196] . [chest x-ray] chest x-ray findings show characteristics related to causative microorganisms to some degree, but it is difficult to identify causative microorganisms in individual patients [197] . it is important to evaluate the severity of pneumonia, for selecting outpatient or hospital treatment and reviewing the necessity of antimicrobial drugs and route of administration (oral or intravenous). the classification of severity in the guidelines for the management of respiratory infectious diseases in children in japan 2011 is presented [189] (table 12) . causative microorganisms with respect to age in children with community-acquired pneumonia [190] . the severity classification of childhood pneumonia has not been reached in japan or internationally. this should be examined in the future. [standards for outpatient/hospital treatment] as a rule, outpatient treatment should be performed in mildstatus patients evaluated according to the severity classification, and hospital treatment in mild-status patients with dehydration. in addition, it is necessary to determine admission when outpatient treatment does not lead to an improvement in symptoms or considering social adaptation [189] (table 13) . [initial antimicrobial drug therapy] when examining children with pneumonia, treatment must be started without any precise information about causative microorganism in many cases. basically, empiric therapy should be performed, considering the severity of pneumonia and causative microorganisms. -the type of causative microorganisms depends on age and severity. therefore, the necessity of antimicrobial drugs should be examined, and selected, considering age and severity. in addition, bacterial, viral, or atypical pneumonia should be differentiated, and comprehensively evaluated in reference to clinical symptoms, physical findings, laboratory findings, and xray findings [189] . in particular, acute bronchitis/pneumonia related to intermediately susceptible h. influenzae (mic: 2 mg/ml) can be managed with oral ampc or abpc intravenous injection therapies [204] . recently, the number of abpc-susceptible strains has annually decreased [198, 200] . the proportion of blnar strains (mic: 4 mg/ table 11 age-related distribution of microorganisms that cause pneumonia in children [189] . immediately after birth to 20 5 . inflammatory response on admission in children with pneumonia [196] . ml or more) has increased, raising an issue with respect to drug selection. when the involvement of blnar strains is suspected, high-dose ampc or new oral cephems may be necessary at outpatient clinics [208] . the efficacy of outpatient antimicrobial drugs for blnar strains, which will increase in the future, should be carefully monitored. concerning hospital treatment, the clinical effects of abpc intravenous injection for 3e4 days until the results of a susceptibility test were clarified were investigated, and approximately 80% of patients responded to this therapy. there was no exacerbation in any patient [189] . in non-responders or patients in whom clinical effects are insufficient, it is necessary to switch the antimicrobial drug. pipc, ctx, and ctrx have stable antimicrobial activities. when reviewing the clinical effects of pipc on childhood bronchopulmonary infection, the response rate was 95%; the results were satisfactory [205] . -concerning treatment for m. catarrhalis pneumonia, m. catarrhalis produces b-lactamase. however, when examining the clinical course, ampc is effective [205, 206] . this is because the enzymatic activity of b-lactamase produced by m. catarrhalis is low [207] . (table 14) . [evaluation of the treatment response and administration period] the administration period of antimicrobial drugs is shown in table 15 [211] . classification of the severity of community-acquired pneumonia in children [189] . moderate severe 6 . increases in the number of resistant strains of s. pneumoniae and h. influenzae derived from respiratory infectious diseases [199] . to treat community-acquired pneumonia, antimicrobial drugs should be administered for 3e7 days. the treatment response should be evaluated after 2e3 days. in children, disease progression is often prompt, and the first evaluation should be performed after 2 days in younger and severe-status children, and not after 3 days [189] . if an improvement in clinical symptoms or laboratory data is achieved, the same antimicrobial drug should be continued until an appropriate antimicrobial drug and drug susceptibility are clarified. with respect to the administration period of antimicrobial drugs, factors such as the type of causative microorganisms and patient background differ among individual patients; therefore, it is difficult to establish standardized criteria. as m. pneumoniae and c. pneumoniae slowly proliferate, the treatment period is prolonged (table 15 ). in patients infected with general bacteria, the administration of antimicrobial drugs can be discontinued 3 days after pyretolysis [189] . however, further antimicrobial drug therapy is necessary to treat s. aureus pneumonia. [management for non-responders to antimicrobial drugs] when there are no therapeutic effects of antimicrobial drugs on pneumonia, whether or not a diagnosis of pneumonia is correct should be initially investigated [189] . the possibility of diseases other than pneumonia, with a pneumonia-like shadow, must be reviewed (table 16 ). if it can be ruled out, whether or not the expected type of pathogenic microorganisms is correct should be examined. if the type of causative microorganisms is the same as expected, the possibility of resistant microorganisms should be considered. new therapeutic strategies should be devised carefully and promptly. when the condition further exacerbates despite treatment switching, additional examination should be conducted. [203] . concepts regarding the diagnosis and treatment of childhood mycoplasma pneumonia [211] . 1. as it is often difficult to make a diagnosis of mycoplasma pneumoniae infection based on serum antibody titer-positive findings in the acute stage alone, the mycoplasma pneumoniae nucleic acid identification test (lamp method) should be conducted to make a definitive diagnosis in the acute stage. 2. as first-choice drugs for mycoplasma pneumonia, macrolides should be used. 3 . the effects of macrolides can be evaluated based on pyretolysis within 2e3 days after administration. 4. to treat pneumonia that does not respond to macrolides, the administration of tosufloxacin or tetracyclines should be considered if necessary. however, as a rule, tetracyclines are contraindicated for children aged 7 years or younger. 5. these antimicrobial drugs should be administered in accordance with administration periods recommended for individual drugs. 6. in patients with severe pneumonia, systemic steroid therapy must be considered. however, easy steroid administration should be avoided. however, mycoplasma pneumoniae is not included in bacterial types for which this preparation may be indicated. ---drugs to be recommended--a first choices 1) cases in which there is no risk of resistant bacteria -ampc, oral, 10e15 mg/kg/3 times a day -sbtpc, oral, 10 mg/kg/3 times a day -cdtr-pi, oral, 3 mg/kg/3 times a day -cfpn-pi, oral, 3 mg/kg/3 times a day -cftm-pi, oral, 3 mg/kg/3 times a day 2) cases in which infection with resistant bacteria is suspected i) two years or younger, ii) pretreatment with an antimicrobial drug (within 2 weeks), iii) concomitant development of otitis media, iv) history of pneumonia/repeated otitis media -ampc, oral, 20e30 mg/kg/3 times a day -cva/ampc (1:14 preparation), oral, 48.2 mg/ kg/twice a day -cdtr-pi, oral, 6 mg/kg/3 times a day -cfpn-pi, oral, 6 mg/kg/3 times a day -cftm-pi, oral, 6 mg/kg/3 times a day 3) cases in which onset/recurrence/recrudescence is observed despite previous treatment 2) -tbpm-pi, oral, 4e6 mg/kg/twice a day -tflx, oral, 6 mg/kg/twice a day <> second choices -azm, oral, 10 mg/kg/once a day for 3 days -cam, oral, 7.5 mg/kg/twice a day (2) admission (moderate, general ward) a first choices -abpc, intravenous injection or drip, 50 mg/kg/3 times a day -pipc, intravenous injection or drip, 50 mg/kg/3 times a day -sbt/abpc, intravenous injection or drip, 75 mg/kg/ 3 times a day * when m. pneumoniae, chlamydia trachomatis, or c. pneumoniae infection is strongly suspected, one of the above regimens should be combined with a macrolide [with respect to the administration method/ dose, refer to the section "➁ six years or older---(1) outpatient clinic (mild)".]. <> second choices -ctx, intravenous injection or drip, 40 mg/kg/3 times a day -ctrx, intravenous injection or drip, 25e60 mg/kg/ once to twice a day (50e60 mg/kg/day) (3) admission (severe, icu) -papm/bp, intravenous drip, 20 mg/kg/3 times a day -mepm, intravenous drip, 20 mg/kg/3 times a day -taz/pipc, intravenous drip, 112.5 mg/kg/3 times a day * when legionellosis cannot be ruled out, one of the above regimens should be combined with a macrolide [with respect to the administration method/dose, refer to the section "➁ six years or older---(2) admission (moderate, general ward)".]. ➁ six years or older (1) outpatient clinic (mild) a first choices -azm, oral, 10 mg/kg/once a day for 3 days -cam, oral, 7.5 mg/kg/twice a day <> second choices -ampc, oral, 10e15 mg/kg/3 times a day -sbtpc, oral, 10 mg/kg/3 times a day -cdtr-pi, oral, 3 mg/kg/3 times a day -cfpn-pi, oral, 3 mg/kg/3 times a day -cftm-pi, oral, 3 mg/kg/3 times a day -mino, oral, 1e2 mg/kg/twice a day (in children aged 7 years or younger, the use of this drug is limited to those in whom other drugs cannot be used or nonresponders to other drugs.) ( a first choices 1) cases in which bacterial pneumonia is suspected -abpc, intravenous injection or drip, 50 mg/kg/3 times a day -pipc, intravenous injection or drip, 50 mg/kg/3 times a day -sbt/abpc, intravenous injection or drip, 75 mg/ kg/3 times a day -ctx, intravenous injection or drip, 40 mg/kg/3 times a day -ctrx, intravenous injection or drip, 25e60 mg/ kg/once to twice a day (50e60 mg/kg/day) 2) cases in which atypical pneumonia is suspected -azm, oral, 10 mg/kg/once a day for 3 days -cam, oral, 7.5 mg/kg/twice a day -em, intravenous drip, 10 mg/kg/3e4 times a day -mino, oral or intravenous drip, 1e2 mg/kg/twice a day (in children aged 7 years or younger, the use of this drug is limited to those in whom other drugs cannot be used or non-responders to other drugs.) 3) cases in which it is impossible to differentiate bacterial from atypical pneumonia one drug each should be selected from choices 1) and 2), and combined. -em, oral, 10e15 mg/kg/3 times a day -azm, oral, 10 mg/kg/once a day for 3 days -cam, oral, 7.5 mg/kg/twice a day -em, intravenous drip, 10 mg/kg/3e4 times a day -mino, oral or intravenous drip, 1e2 mg/kg/twice a day (in children aged 7 years or younger, the use of this drug is limited to those in whom other drugs cannot be used or non-responders to other drugs.) ---executive summary---for the treatment of hospital-acquired pneumonia in children, antimicrobial drugs should be selected, considering severity and the involvement of resistant bacteria. empiric therapy should be started by combining two drugs if necessary, considering various resistant microorganisms, differing from that for communityacquired pneumonia (biii). hospital-acquired pneumonia is defined as pneumonia that newly develops 48 h or more after admission. ventilator-associated pneumonia is defined as pneumonia that develops 48 h or more after endotracheal intubation [211] . these conditions may become severe due to an underlying disease, reduced immune capacity, or the deterioration of the general condition, and are caused by drugresistant microorganisms in many cases; treatment is difficult in most cases. not only microorganisms acquired in the community but also those existing in the hospital environment cause hospital-acquired pneumonia in children, as reported in the adult field. bacteria that cause community-acquired pneumonia (s. pneumoniae, h. influenzae), enteric bacteria (e. coli, k. pneumoniae), s. aureus, non-glucose-fermenting bacteria, such as p. aeruginosa, and acinetobacter spp., and anaerobes cause hospital-acquired pneumonia [212] . in addition, not only general bacteria but also fungus and viruses sometimes cause hospital-acquired pneumonia in patients with immunodeficiency. among patients with nosocomial infection, causative microorganisms differ, and drug-resistant microorganisms are involved in many cases. in children, it is not easy to investigate causative microorganisms, but, if drug susceptibility is clarified, it contributes to successful treatment. therefore, lavage or aspiration sputum culture should be conducted to investigate the etiology, if possible [189, 211] . [rules of antimicrobial drug therapy] basically, empiric therapy should be performed, considering the severity of pneumonia, an underlying disease, and causative microorganisms. in particular, the involvement of drug-resistant microorganisms, such as mrsa, extended-spectrum b-lactamase (esbl)-producing bacteria, and multi-drug-resistant p. aeruginosa (mdrp), must always be considered for treatment. empiric therapy should be started by combining two drugs if necessary, considering various resistant microorganisms, differing from that for community-acquired pneumonia [211] . as the state of resistant bacteria differs among institutions, antimicrobial drug options should be customized based on records on antimicrobial drug susceptibility (antibiograms) at each institution. a consensus regarding the administration period of antimicrobial drugs has not been reached. with respect to the administration period of antimicrobial drugs, factors such as the type of causative microorganisms and patient background differ among individual patients with nosocomial infection; therefore, it is difficult to establish standardized criteria. however, when complications such as severe immunodeficiency, pulmonary suppuration, lung abscess, and pleuritis are absent, antimicrobial drugs should be administered for 5 days after pyretolysis (7e10 days) [50] . considering an underlying disease or the immune state, flexible management must be performed. in children, disease progression is often prompt, and the first evaluation should be performed after 2 days in younger and severe-status children, and not after 3 days [189] . if an improvement in clinical symptoms or laboratory data is achieved, the same antimicrobial drug should be continued until an appropriate antimicrobial drug and drug susceptibility are clarified. when the type of microorganisms that caused pneumonia is identified, a target-focused antimicrobial drug should be selected through de-escalation, considering drug susceptibility and pharmacokinetics [211] . concerning multi-drug-resistant microorganisms, it is important to promote standard preventive strategies and those to control nosocomial infection, such as the prevention of droplet/contact infection, thoroughly. furthermore, oral care and devised postures (if there is no medical contraindication, the head should be lifted at 30e45 .) are necessary to prevent vap [211] . in this article, the classification of severity (table 12 , p. 30) in the guidelines for the management of respiratory infectious diseases in children in japan 2011 was used [189] (refer to the section "3. pneumonia (children), 3.1 community-acquired pneumonia".). ---drugs to be recommended--a first choices -caz, intravenous injection or drip, 50 mg/kg/3 times a day -czop, intravenous injection or drip, 40 mg/kg/3e4 times a day -cpr, intravenous injection or drip, 40 mg/kg/3e4 times a day if necessary, one of the above regimens should be combined with one of the following drugs: 1) cases in which anaerobe infection is suspected (such as aspiration pneumonia) -cldm, intravenous drip, 10 in children with immunodeficiency-/blood disease-related pneumonia, antimicrobial drugs should be selected, considering an underlying disease, the grade of immunodeficiency, and involvement of various causative microorganisms. initial antimicrobial drug therapy should be started by combining two drugs if necessary, considering various causative microorganisms, differing from that for community-acquired pneumonia (biii). [characteristics and classification of the disease] as immunodeficiency-/blood disease-related pneumonia in children often develops in hospitals, it has the characteristics of hospital-acquired pneumonia in many cases. it may become severe due to the patient's unfavorable conditions, such as an underlying disease, reduced immune capacity, and the deterioration of the general condition. even non-pathogenic microorganisms may cause pneumonia in many cases. furthermore, drug-resistant microorganisms often cause pneumonia; treatment is difficult in many cases [213, 214] . to achieve multidisciplinary, comprehensive treatment to save children, it is necessary to cooperate with other special fields. [type and frequency of causative microorganisms] not only microorganisms acquired in the community in the presence of various immunodeficiency states but also nonpathogenic microorganisms existing in the hospital environment cause immunodeficiency-/blood disease-related pneumonia in children. bacteria that cause community-acquired pneumonia (s. pneumoniae, h. influenzae), enteric bacteria (e. coli, k. pneumoniae), s. aureus, non-glucose-fermenting bacteria, such as p. aeruginosa, and acinetobacter spp., and anaerobes cause this type of pneumonia. in addition, not only general bacteria but also fungus and viruses often cause this type of pneumonia. furthermore, drugresistant microorganisms are involved in many cases, as indicated for hospital-acquired pneumonia [213, 214] . [type of immunodeficiency, causative microorganisms to be monitored, and precautions for diagnosis] ➀ humoral immunodeficiency: as bacterial opsonization and complement activation are affected, patients with humoral immunodeficiency are prone to be infected with general bacteria. among immunodeficiency patients with hyper-igm-emia, pneumocystis pneumonia should be considered in those with conditions related to cd40 ligand abnormalities. ➁ cellular immunodeficiency: in addition to infection with general bacteria, infection with intracellular parasitic bacteria, fungus, or protozoa may become severe, and be protracted. as the differentiation and induction of b and killer t cells by cd4-positive lymphocytes are affected, the eradication of virus-infected cells is inhibited (table 17 ) [215] . ➂ neutrophil abnormalities: neutrophil abnormalities are classified into two types: neutropenia and neutrophil functional disorders. patients with a peripheral blood absolute neutrophil count (anc) of <500/ml or those in whom the anc is estimated to reach <500/ml within 48 h are regarded as having neutropenia [215] . of these, the risk is higher in patients with an anc of 100/ml or less in whom the period is estimated to exceed 7 days. many patients with neutropenia do not show purulent sputum or abnormal findings on chest x-ray even in the presence of pneumonia. therefore, when fever persists, thoracic ct should be performed in the early stage. all microorganisms including general bacteria (gram-positive bacteria, gramnegative bacteria), fungus, and viruses may cause pneumonia. in particular, in addition to neutropenia early after homologous hematopoietic stem cell transplantation, infection-prone features associated with humoral/cellular immunodeficiency related to the administration of immunosuppressives persist over a long period. furthermore, the concomitant development of acute/chronic graft-versus-host disease (gvhd) is a risk factor for the onset of pneumonia. in addition, non-infectious pulmonary disorder related to drugs/radiation for pretreatment may occur, and it is important to differentiate it from infectious diseases (fig. 9 ) [214, 216] . a representative neutrophil functional disorder, chronic granulomatosis, induces active oxygen production disorder of neutrophils, affecting bactericidal actions. therefore, patients with this disorder are prone to be infected with non-h 2 o 2 -producing catalase-positive bacteria (s. aureus, k. pneumoniae, e. coli, candida spp., aspergillus spp.). ➃ complement deficiency: patients with complement deficiency are prone to be infected with bacteria with capsules, such as s. pneumoniae, h. influenzae (capsule strains), and neisseria meningitidis [214] . in children, it is not easy to investigate causative microorganisms, but, if drug susceptibility is clarified, it contributes to successful treatment. therefore, various cultures should be conducted to investigate the etiology, if possible. in addition, testing of various antigens, such as urinary s. pneumoniae/legionella antigens, b-dglucan, aspergillus antigen, cryptococcus antigen, candida antigen, and cytomegalovirus antigen, and tests using nucleic acid amplification methods, such as the pcr method, should be utilized, if possible. [rules of antimicrobial drug therapy] initial antimicrobial drug therapy should be performed, considering the severity of pneumonia and an underlying disease. for the treatment of immunodeficiency-/blood disease-related pneumonia in children, antimicrobial drug therapy should also be basically selected, considering causative microorganisms. as described for nosocomial infection, the involvement of drugresistant microorganisms, such as mrsa, extended-spectrum blactamase (esbl)-producing bacteria, and multi-drug-resistant p. aeruginosa (mdrp), must always be considered for treatment. initial antimicrobial drug therapy should be started by combining two drugs, if necessary, considering various resistant microorganisms, differing from that for community-acquired pneumonia [213, 214] . as the state of resistant bacteria differs among institutions, antimicrobial drug options should be customized based on records on antimicrobial drug susceptibility (antibiograms) at each institution. a consensus regarding the administration period of antimicrobial drugs has not been reached. with respect to the administration period of antimicrobial drugs, factors such as the type of causative microorganisms and patient background differ among individual patients with nosocomial infection; therefore, it is difficult to establish standardized criteria. in children, disease progression is often prompt, and the first evaluation should be performed after 2 days in younger and severe-status children, and not after 3 days. if an improvement in clinical symptoms or laboratory data is achieved, the same antimicrobial drug should be continued until an appropriate antimicrobial drug and drug susceptibility are clarified. when the type of microorganisms that caused pneumonia is identified, a target-focused antimicrobial drug should be selected through de-escalation, considering drug susceptibility and pharmacokinetics [189] . monitoring culture of the airway is useful for treating immunodeficiency-/blood disease-related pneumonia in children [214] . general preventive methods are presented in table 18 [213] . in addition, long-term low-dose macrolide therapy or the intermittent administration of b-lactams to prevent p. aeruginosa fixation is effective in some patients with chronic bronchitis [217, 218] . in those with chronic granulomatosis, the oral administration of itcz (4e6 mg/kg/day, maximum: 100 mg/day) or subcutaneous injection of ifn-g (250,000 domestic standard units/m 2 , 1e3 times a week) is useful for preventing infection [219] . in this article, the classification of severity (table 12 , p. 30) in the guidelines for the management of respiratory infectious diseases in children in japan 2011 was used (refer to the section "3. pneumonia (children), 3.1 community-acquired pneumonia".). ---drugs to be recommended--(1) pneumonia related to mild immunodeficiency in the initial phase after admission -sbt/abpc, intravenous injection or drip, 75 mg/kg/3e4 times a day -ctx, intravenous injection or drip, 40 mg/kg/3e4 times a day -ctrx, intravenous injection or drip, 50 mg/kg/twice a day (2) pneumonia related to moderate/severe immunodeficiency -caz, intravenous injection or drip, 50 mg/kg/3e4 times a day -czop, intravenous injection or drip, 50 mg/kg/3e4 times a day -cpr, intravenous injection or drip, 50 mg/kg/3e4 times a day in severe cases, -papm/bp, intravenous drip, 20 mg/kg/3 times a day -mepm, intravenous drip, 20 mg/kg/3 times a day -drpm, intravenous drip, 20 mg/kg/3 times a day -taz/pipc, intravenous drip, 112.5 mg/kg/3 times a day -vcm, intravenous drip, 15 mg/kg/3 times a day -abk, intravenous drip, 4e6 mg/kg/once a day -teic, intravenous drip, 10 mg/kg/every 12 h, 3 times, subsequently: 6e10 mg/kg/once a day when there is no response, -amk, intravenous drip, 5e7.5 mg/kg/twice a day -tob, intravenous drip, 3.3 mg/kg/3 times a day if necessary, an antifungal drug (mcfg) and st combination drug should be additionally administered. when aspergillus infection is suspected, treatment should be started with vrcz or l-amb instead of mcfg. in patients with cellular immunodeficiency, one of the above drugs should be combined with mcfg and an st combination drug in the early stage. in those with neutrophil abnormalities, one of the above drugs should be combined with mcfg in the early stage. when legionellosis cannot be ruled out in the severest cases, one of the above regimens should be combined with a macrolide (with respect to the administration method/dose, refer to the section "3.1 community-acquired pneumonia---➁ six years or older---(2) admission (moderate, general ward)" (p. 32).). in the presence of gcv resistance, the intravenous drip of pfa (foscarnet), 60 mg/kg/3 times a day should be performed. ---executive summary---in the treatment of neonatal pneumonia, antimicrobial drugs should be selected after differentiating congenital from acquired pneumonia. initial antimicrobial drug therapy should be started by combining two drugs, regarding the condition as severe systemic infection and considering various causative microorganisms. the administration method and dose should be selected based on the age (days) and birth weight (biii). [characteristics and classification of the disease] the incidence of neonatal pneumonia is not high. according to sakata, pneumonia occurred in only 40 (0.25%) of approximately 16,000 neonates who were admitted during a 10-year period [220] . neonatal pneumonia is frequently observed as a portion of systemic infection represented by sepsis, and not as a single condition. it is classified into two types: congenital and acquired pneumonia [221] . in most cases, neonatal pneumonia does not cause any typical symptoms such as fever or cough in comparison with infantile/ childhood pneumonia. the neonatal protective capacity against infection is physiologically immature, often leading to a severe condition. in the neonatal phase, artificial respiration management is performed in many cases, and ventilator-associated pneumonia (vap) is frequently observed [222e224]. in addition, many neonates are admitted to the neonatal intensive care unit (nicu) for a long period; therefore, the incidence of nosocomial infection is high. [type and frequency of causative microorganisms] several types of congenital pneumonia include transplacental infection, intrauterine infection related to aspiration of infected amniotic fluid on premature rupture, and birth canal infection with vaginal discharge on delivery. these types of congenital pneumonia are primarily caused by various viruses (cytomegalovirus, herpes simplex virus), bacteria (streptococcus agalactiae (gbs), e. coli, listeria monocytogenes), chlamydia, and fungus. the clinical onset of perinatal pneumonia is frequently observed immediately to 7 days after birth [221] . postnatal pneumonia usually develops 2 weeks or more after birth. it is caused by viruses and bacteria. viral infection may occur when rs virus, parainfluenza virus, or adenovirus prevails in the ward. however, it rarely leads to the onset of pneumonia. frequent causative bacteria include s. aureus, e. coli, p. aeruginosa, acinetobacter spp., enterobacter spp., and legionella spp., which exist in the environment. these often cause nosocomial infection [221] . in premature babies, gram staining of tracheal aspirate samples is useful for selecting antimicrobial drugs to be administered in the initial phase [225] . in children, it is not easy to investigate causative microorganisms, but, if drug susceptibility is clarified, it contributes to successful treatment. therefore, blood or bronchial lavage fluid culture should be conducted to investigate the etiology, if possible. [rules of antimicrobial drug therapy] as many neonates have systemic infection in an immunodeficiency state, the same administration method and dose as selected for the treatment of sepsis should be used [221] . therefore, the severity of pneumonia is not considered. when bacterial pneumonia is suspected, combination therapy with abpc and ctx is commonly selected as initial treatment. considering l. monocytogenes infection, combination therapy with abpc is recommended. after causative microorganisms are identified, antimicrobial drugs should be selected with reference to their drug susceptibility. in cases of vap, antimicrobial drug options should be customized based on records on the drug susceptibility (antibiograms) of microorganisms that cause nosocomial infection at each institution. in neonates, there are marked age-related differences in the pharmacokinetics of antimicrobial drugs; therefore, these drugs must be carefully administered in the neonatal phase. concretely, the same dose as established in the field of pediatrics should be used, and the administration interval should be prolonged in accordance with age [221] . in cases of severe asphyxia or acute renal failure, the administration interval must be further prolonged. it should be shortened with an improvement in the kidney function [226e229] . a consensus regarding the administration period of antimicrobial drugs has not been reached. however, the standard administration period is 14 days. with respect to the administration method/dose, refer to the section "xi. doses for neonates". in neonates, disease progression is often prompt, and the treatment response should be evaluated after 2 days, and not after 3 days. if an improvement in clinical symptoms or laboratory data is achieved, the same antimicrobial drug should be continued until an appropriate antimicrobial drug and drug susceptibility are clarified. when the type of microorganisms that caused pneumonia is identified, a target-focused antimicrobial drug should be selected through de-escalation, considering drug susceptibility and pharmacokinetics [189] . ---drugs to be recommended-- ---executive summary----pyothorax refers to a condition in which pus accumulates in the thoracic cavity. usually, pleural effusion puncture is performed, and a diagnosis of pyothorax is made based on the results of various examinations, such as (macroscopic) purulent pleural effusion, microorganisms detected on gram staining or culture of pleural effusion, or pleural effusion ph: <7.2 (biii). -in patients with acute pyothorax related to community-acquired pneumonia, treatment should be performed in accordance with that for community-acquired pneumonia, considering microorganisms that cause community-acquired pneumonia, such as streptococcus pneumoniae (biii). -in patients with slowly progressing pyothorax, mixed infection with oral aerobes/anaerobes is frequently observed. combination therapy with pcg or abpc and cldm or mnz, which show anti-anaerobe activities, or therapy with a single antimicrobial drug with an anti-anaerobe activity, such as sbt/abpc, should be selected (biii). -when there is a risk of multi-drug-resistant bacteria, monotherapy with a carbapenem, combination therapy with a fourthgeneration cephalosporin and cldm or mnz, and combination therapy with a quinolone and cldm or mnz should be considered, assuming esbl-producing enteric bacteria, resistant p. aeruginosa, anaerobes, and acinetobacter (biii). -if the results of culture/susceptibility tests are clarified, antimicrobial drugs should be changed in accordance with them (aiii). -the penetration of aminoglycosides to the thoracic cavity is poor, and their activities reduce when the ph is low. therefore, the use of aminoglycosides should be avoided as a rule (biii). -if a diagnosis of pyothorax is made, the administration of an appropriate antimicrobial drug should be started, and drainage must be performed (aii). if possible, the attending physician should consult a surgeon in the early stage (aiii). -in some patients with marked pleural thickening or multilocular pleural effusion, thoracoscopic debridement is necessary (biii). in addition, a fibrinolytic drug, such as streptokinase, is administered through a thoracic drain, or surgical interventions such as thoracotomy or decortication is performed in some cases (biii). ---explanation--[diagnosis] -pyothorax is defined as a condition in which pus accumulates in the thoracic cavity, but this definition has no diagnostic objectivity. for this reason, usually, pleural effusion puncture is performed, and a diagnosis of pyothorax is made based on the results of various examinations, such as (macroscopic) purulent pleural effusion, microorganisms detected on gram staining or culture of pleural effusion, or pleural effusion ph: <7.2 [230, 231] . [causative microorganisms] -pleural effusion appears in 30e40% of patients with bacterial pneumonia, but leads to pyothorax in 0.5e2%. other etiological factors for pyothorax include surgery, trauma, and esophageal perforation. -microorganisms that cause pyothorax depend on its etiology and course. in the presence of bacterial pneumonia, pyothorax is caused by the same microorganisms as caused bacterial pneumonia. furthermore, acute pyothorax is frequently caused by s. pneumoniae and s. aureus. however, in many patients with chronic pyothorax, mixed infection primarily with anaerobes is involved. among anaerobes, fusobacterium spp. (especially fusobacterium nucleatum), prevotella spp., peptostreptococcus spp., and bacteroides spp. are frequently detected [232e234]. according to recent studies, the detection rate of the streptococcus anginosus group is high [235, 236] . -in many cases, pyothorax with a relatively slow course is associated with m. tuberculosis. it must be considered that pulmonary lesions do not always concurrently exist with tuberculous pleuritis. [treatment] -no randomized comparative study regarding antimicrobial drug treatment for pyothorax has been conducted. an antimicrobial drug with an activity against microorganisms expected or obtained on culture should be selected and administered. in acuteonset patients in whom there is no risk of resistant bacteria, for example, those with pyothorax accompanying communityacquired pneumonia, antimicrobial drugs such as pcg and abpc should be initially selected, considering s. pneumoniae. these drugs simultaneously cover fusobacterium, peptostreptococcus, and the viridans streptococcus group. however, prevotella and bacteroides produce b-lactamase; therefore, when the results of culture are not obtained, combination therapy with antimicrobial drugs with activities against anaerobes, such as cldm and mnz, or therapy with such a single drug, such as sbt/abpc, should be selected. -when there is a risk of multi-drug-resistant bacteria, monotherapy with a carbapenem should be selected as a first choice, assuming esbl-producing enteric bacteria, resistant p. aeruginosa, and acinetobacter. a fourth-generation cephalosporin should be combined with cldm, or a quinolone should be combined with cldm or sbt/abpc. -if the results of culture/susceptibility tests are clarified, antimicrobial drugs should be changed in accordance with them. however, the culture of anaerobes is difficult, or is not conducted in some cases. therefore, this should be confirmed to the laboratory. when only one type of bacteria are detected on a culture test despite several types of bacteria detected on gram staining of pleural effusion, anaerobes must also be considered. -the penetration of aminoglycosides to the thoracic cavity is poor, and their activities reduce when the ph is low. therefore, for pyothorax treatment, the use of aminoglycosides should be generally avoided [237e240]. [treatment period] -the pyothorax treatment period has not been established. however, when pneumonia promptly responds to treatment and thoracic drainage is successfully achieved in patients with pneumonia-related pyothorax, a treatment period of 10e14 days is required. in patients in whom drainage is unsuccessful, those with marked pleural thickening, or those with encapsulated/septum-like pyothorax, a treatment period of about 4 weeks is often required. [treatments other than antimicrobial drug therapy] -after a diagnosis of pyothorax is made, the administration of an appropriate antimicrobial drug should be promptly started, and drainage is necessary. when the pleural fluid is purulent and viscous in the absence of a multilocular pattern, chest-tube insertion is routinely performed. the position of insertion should be confirmed using thoracic ct within 24 h after insertion. wozniak et al. performed multivariate analysis involving 104 patients with pyothorax and indicated that failure in the first drainage was strongly correlated with mortality, suggesting the necessity of early consultation with surgeons [241] . -in some patients with marked pleural thickening or multilocular pleural effusion, thoracoscopic debridement is necessary. in addition, a fibrinolytic drug, such as streptokinase, is administered through a thoracic drain, or surgerical interventions such as thoracotomy or decortication is performed in some cases [242] . ---drugs to be recommended--a first choice -sbt/abpc, intravenous drip, 3 g/3e4 times a day <> second choices -pcg, intravenous drip, 2,000,000 to 3,000,000 units/4 times a day -abpc, intravenous drip, 2 g/3e4 times a day þ one of the followings: -cldm, intravenous drip, 600 mg/2e4 times a day -mnz, intravenous drip, 500 mg/4 times a day ➁ cases in which there is a risk of multi-drug-resistant bacteria a first choices -taz/pipc, intravenous drip, 4.5 g/3e4 times a day -ipm/cs, intravenous drip, 0.5e1 g/2e4 times a day -mepm, intravenous drip, 1 g/2e3 times a day -drpm, intravenous drip, 0.5e1 g/3 times a day <> second choices (i) -cfpm, intravenous drip, 1e2 g/2e4 times a day -czop, intravenous drip, 1e2 g/2e4 times a day -cpr, intravenous drip, 1e2 g/2e4 times a day þ one of the followings: -cldm, intravenous drip, 600 mg/2e4 times a day -mnz, intravenous drip, 500 mg/4 times a day <> second choices (ii) -lvfx, intravenous drip, 500 mg/once a day -cpfx, intravenous drip, 300 mg/twice a day -pzfx, intravenous drip, 500 to 1000 mg/twice a day þ one of the followings: -cldm, intravenous drip, 600 mg/2e4 times a day -sbt/abpc, intravenous drip, 3 g/3e4 times a day -mnz, intravenous drip, 500 mg/4 times a day * with respect to the risk of multi-drug-resistant bacteria, refer to the section "2.2 hospital-acquired pneumonia" table 3 (p. 10). * in particular, mrsa infection must be considered in patients with nosocomial onset or the previous administration of antimicrobial drugs. when staphylococcus infection is suspected on gram staining of pleural effusion, an anti-mrsa drug should be used as an empiric therapy. if mssa is identified as causative bacteria, deescalation should be performed. 2. definitive therapy -antimicrobial drugs against causative microorganisms identified should be selected in accordance with the section "2. ---executive summary---for the treatment of pyothorax in children, antimicrobial drugs should be administered after investigating the etiology using thoracentesis or blood culture, if possible. in addition to antimicrobial drug therapy, treatment for retention fluid (pleural effusion drainage, continuous drainage) must also be considered (biii). [characteristics and classification of the disease] pleuritis refers to inflammation of the pleura. fluid (pleural effusion) is retained in the pleural cavity. pleuritis is classified into 3 types: fibrinous (dry) pleuritis, exudative (wet) pleuritis, and purulent pleuritis (pyothorax) based on conditions [243] . on auscultation, the attenuation of respiratory sounds, as well as pleural friction rubs, are heard. percussion dullness is noted. in the chronic stage, localized pleural thickening in various shapes is observed. when pus is obtained on thoracentesis, a definitive diagnosis of pyothorax is made. when the protein level in nonpurulent pleural effusion is high, tuberculous pleuritis should be differentiated [244] . [type and frequency of causative microorganisms] previously, pyothorax was associated with s. aureus in many cases. however, recently, such cases have been rare. according to data form a national survey in the former half of the 1990's, there were only a few patients with s. pneumoniae-or anaeroberelated pyothorax [243] . in many cases, pleuritis follows m. pneumoniae-or virus-related pneumonia. decubitus-view imaging shows the retention of pleural effusion in approximately 20% of patients with mycoplasma pneumonia [245] . if the drug susceptibility of causative microorganisms is clarified, it contributes to successful treatment. therefore, pleural effusion obtained by thoracentesis should be cultured to investigate the etiology, if possible. when bacterial infection-related pyothorax is suspected, the intravenous injection or drip of sbt/abpc, ctx, or ctrx should be selected in community-onset patients without an underlying disease. on the other hand, combination therapy with sbt/abpc (intravenous injection or drip) and cldm (intravenous drip) or carbapenem therapy (intravenous drip) should be started in those with an underlying disease or nosocomial onset, considering s. pneumoniae, anaerobes, h. influenzae, and s. aureus [243] . based on the gram staining reactions of pleural effusion, causative microorganisms should be estimated, and antimicrobial drugs must be reviewed. if necessary, tuberculosis should also be investigated. the administration period of antimicrobial drugs must be longer than that for pneumonia. as the type of causative microorganisms, patient background, and state of retention-fluid drainage differ among individual patients, it is difficult to establish standardized criteria. however, target administration periods for s. pyogenes (gas)-, s. pneumoniae-, and s. aureus-related pyothorax are 10, 14, and 21 days or more, respectively. the treatment response should be evaluated 3e4 days after the start of administration. if an improvement in clinical symptoms or laboratory data is achieved, the same antimicrobial drug should be continued until causative microorganisms and their drug susceptibility are clarified. when the type of microorganisms that caused pneumonia is identified, a targetfocused antimicrobial drug should be selected through deescalation, considering drug susceptibility and pharmacokinetics [189] . for the treatment of pyothorax, treatment for retention fluid (pleural effusion drainage) is also a basic procedure in addition to antimicrobial drug therapy. if necessary, continuous drainage should be performed (table 19 ) [243] . if pleural thickening leads to underexpanded lung, decortication should be indicated. the widespread application of thoracoscopic surgery has facilitated minimally invasive surgery [246] . ---drugs to be recommended--(1) community onset (without an underlying disease) -sbt/abpc, intravenous injection or drip, 75 mg/kg/3 times a day -ctx, intravenous injection or drip, 40 mg/kg/3e4 times a day -ctrx, intravenous injection or drip, 50 mg/kg/twice a day (2) community-acquired infection (with an underlying disease), nosocomial onset -cldm, intravenous drip, 10 mg/kg/3 times a day þ -sbt/abpc, intravenous injection or drip, 75 mg/kg/3 times a day or one of the following drugs alone should be administered: -taz/pipc, intravenous drip, 112.5 mg/kg/3 times a day -papm/bp, intravenous drip, 20 mg/kg/3 times a day -mepm, intravenous drip, 20 mg/kg/3 times a day -drpm, intravenous drip, 20 mg/kg/3 times a day 2. definitive therapy refer to the section "3.1 community-acquired pneumonia---3.1.2 definitive therapy" (p. 33) or "3.2 hospital-acquired pneumonia---3.2.2 definitive therapy" (p. 34). table 19 indications for continuous drainage [243] . ➀ cases in which pleural effusion obtained on thoracentesis is purulent ➁ cases in which clinical effects are not achieved by antimicrobial drug therapy alone (within 72 h) ➂ cases in which retention fluid affects the respiratory function ---executive summary----as initial treatment, four drugs (inh, rfp, and pza þ eb or sm) should be administered for 2 months. subsequently, as a rule, standard treatment (a), in which two drugs, inh and rfp, are administered for 4 months, should be performed as maintenance treatment (ai). -when pza cannot be used for initial treatment for some reason, inh, rfp, and eb or sm, should be administered for 2 months as initial treatment. subsequently, standard treatment (b), in which two drugs, inh and rfp, are administered for 7 months, should be performed as maintenance treatment (aii). -in patients in whom chest x-ray shows a cavity on initial consultation to during initial treatment and the septum culture is still positive at the completion of initial treatment, maintenance treatment should be prolonged over 3 months. in addition, the prolongation of maintenance treatment should also be considered in patients with severe tuberculosis, such as military tuberculosis and tuberculosis of the central nervous system, those with immune depression, and those with relapse of tuberculosis (aii). -for the treatment of latent tuberculosis infection, inh should be administered for 6 or 9 months (ai). when m. tuberculosis, as the source of infection, is resistant to inh, or when the oral administration of inh is difficult due to side effects, rfp should be used as a second-choice drug for 4 or 6 months (ai). ---explanation--[254, 255] . in particular, this method is named "standard treatment (a)" in the guidelines for the management of tuberculosis in 2012 in japan. in this article, this name is also used. -the secondary assessment of a study examining the efficacy of rifapentine and inh showed that factors for unsuccessful treatment/recurrence included a cavity on chest x-ray at the start of treatment and positive findings on culture at the completion of initial treatment for 2 months [256] . similarly, when the treatment period was extended from 6 to 8 months in patients with silicotuberculosis, in whom the unsuccessful treatment/recurrence rates are high, the recurrence rate decreased from 22 to 7%. therefore, various guidelines recommend that maintenance treatment should be prolonged over 3 months in patients with a cavity and those showing positive findings on septum culture at the completion of initial treatment [254, 255] . -in 1990, combs et al. compared the results of treatment between a group treated with inh, rfp, and pza for 2 months and, then, with inh and rfp for 4 months and that treated with inh and rfp for 9 months, and reported that the efficacy and incidence of side effects were similar [257] . based on such a study, the following regimen is recommended as standard treatment (b) in the guidelines for the management of tuberculosis in 2012 in japan: when pza cannot be used for some reason, inh, rfp, and eb or sm, are administered for the first 2 months, and, subsequently, two drugs, inh and rfp, are administered for 7 months. -patients who are infected with m. tuberculosis, but do not develop tuberculosis are regarded as having latent tuberculosis infection (ltbi). inh administration for ltbi decreases the incidence of tuberculosis by 25e92% [258] . previously, this was called preventive therapy, but is currently termed ltbi treatment. the decrease in the incidence of tuberculosis is correlated with compliance with inh, and more marked preventive effects may be achieved when compliance is higher [258e260]. in a study involving inh administration to ltbi patients with an old shadow on chest x-ray, this therapy inhibited the onset of tuberculosis in 65 and 75% of patients treated for 6 and 12 months, respectively (there was no significant difference between the two groups) [261] . based on the data, some studies recommended that the period of standard inh administration for ltbi should be 9 months [262e264]. however, a consensus regarding an effective administration period (6 or 9 months) has not been reached from various aspects including the efficacy, compliance, expenses, and incidence of side effects. actually, the administration period should be determined based on compliance and the incidence of side effects. -in ltbi treatment, rfp should be used for 4 or 6 months as an alternative drug when the oral administration of inh is impossible for some reason. the preventive effects of rfp on the onset of tuberculosis in ltbi patients may be similar to those of inh. furthermore, the incidence of liver dysfunction is lower than that related to inh [265e267]. however, for the use of rfp, drug interactions must be considered. ---drugs to be recommended---a first choice -inh, oral, 5 mg/kg/once a day (maximum: 300 mg/ day) þ rfp, oral, 10 mg/kg/once a day (maximum: 600 mg/ day, orally administered before meals as a rule) þ pza, oral, 25 mg/kg/once a day (maximum: 1500 mg/day) þ eb, oral, 15 mg/kg/once a day (maximum: 750 mg/day) or sm, intramuscular injection, 15 mg/kg/once a day (maximum: 750 mg/day)/2e3 times a week. * the above 4 drugs should be administered for 2 months, and, subsequently, two drugs, inh and rfp, should be administered for 4 months. <> second choice -inh, oral, 5 mg/kg/once a day (maximum: 300 mg/ day) þ rfp, oral, 10 mg/kg/once a day (maximum: 600 mg/ day, orally administered before meals as a rule) þ eb, oral, 15 mg/kg/once a day (maximum: 750 mg/day) or sm, intramuscular injection, 15 mg/kg/once a day (maximum: 750 mg/day)/2e3 times a week. * the above 3 drugs should be administered for 2 months, and, subsequently, two drugs, inh and rfp, should be administered for 7 months. [269] . in japan, tanaka et al. conducted combination therapy with cam (10 mg/kg) and eb/rfp/km in 39 patients, and indicated that 89.5% of those who underwent initial treatment became negative for mac [270] . concerning cam, many studies have reported a correlation between the in vitro drug susceptibility and treatment response [271e273]. furthermore, a study indicated that azm was as effective as cam [274] . -rbt, which was published in the drug price in nhi scheme in japan in 2008, was also approved for tuberculosis and non-tuberculous mycobacterium infection, including pulmonary mac infection. the drug interactions of rbt are less marked than those of rfp, and rbt is used as a first-choice drug for disseminated mac infection in hiv-infected patients [275] . on the other hand, rbt induces side effects such as uveitis. in elderly patients, in whom pulmonary mac infection frequently develops, various side effects, such as gastrointestinal disorder, make long-term therapy difficult [276] . in addition, no study has indicated that rbt is more effective than rfp for pulmonary mac infection in non-hiv-infected patients. therefore, rfp should be selected as a first-choice drug for pulmonary mac infection in non-hiv-infected patients. -kobashi et al. divided 146 patients with pulmonary mac infection into two groups: a group treated with cam, rfp, eb, and sm (intramuscularly injected at 15 mg/kg 3 times a week for 3 months) and a group treated with saline, and conducted a randomized, double-blind, comparative study [277] . in the sm-treated group, the rate at which the culture of sputum became negative was higher than in the salinetreated group (71.2 vs. 50.7%, respectively). there has been no high-quality study demonstrating the usefulness of combination therapy with aminoglycosides other than their study. however, various guidelines recommend that combination therapy with sm, amk, or km should be performed for 2e3 months in the initial phase of treatment in patients with a cavity or severe nodular/bronchodilatation type infection based on experience [276, 278, 279] . guidelines in the united states recommend sm or amk, and comment that no study has showed which of two drugs, sm and amk, is more effective, although sm has been more frequently used [276] . in japan, sm or km is recommended [279] . -based on the results of these studies, the non-tuberculous mycobacterium infection control committee, japanese society for tuberculosis recommends the following doses and administration methods for chemotherapy for pulmonary mac infection in the "opinions regarding chemotherapy for pulmonary non-tuberculous mycobacterium infection---revision in 2012": rfp: 10 mg/kg (up to 600 mg)/day, once a day, eb: 15 mg/kg (up to 750 mg)/day, once a day, cam: 600e800 mg/day (15e20 mg/kg), once a day or two divided doses (800 mg: two divided doses), and sm or km: 15 mg/kg or less (up to 1000 mg), intramuscularly injected 2 or 3 times a week [279] . -a cooperative statement on non-tuberculous mycobacterium infection by the american thoracic society/infectious diseases society of america recommends therapy with cam at 500 to 1000 mg/day or azm at 250 mg/day, eb at 15 mg/kg/day, and rfp at 10 mg/kg/day (up to 600 mg) for patients with a cavity or severe nodular/bronchodilatation type infection. in addition, it is recommended that combination therapy with sm or amk (8e10 mg/kg, 2e3 times a week, patients aged over 50 years: 500 mg or less) for 2e3 months in the initial phase should be considered [276, 280] . -the treatment period is established as about 1 year after the culture becomes negative in the above guidelines, but this is not based on evidence. in the guidelines for the management of non-tuberculous mycobacterium infection, which were published by the british thoracic society, the treatment period of pulmonary mac infection is established as 2 years. in the future, an optimal treatment period should be investigated. ---drugs to be recommended----cam, oral, 200 mg/3 times a day or 400 mg/twice a day þ rfp, oral, 10 mg/kg/once a day (maximum: 600 mg/ day, orally administered before meals as a rule) þ eb, oral, 15 mg/kg/once a day (maximum: 750 mg/day) * severe-status patients and those with cavity-type lesions in addition to the above regimen, the intramuscular injection of sm or km should be added. abscessus is sometimes susceptible to lzd, tgc (tigecycline), and ketolides, but it is unclear whether there is a correlation between the drug susceptibility and clinical effects [286, 287] . -combination therapy with cam and several intravenous antimicrobial drugs (amk, cfx, and ipm/cs) may control the symptoms and progression of pulmonary infection with m. abscessus [288, 289] . however, actually, hospitalization is required to administer these intravenous antimicrobials, and the administration period is limited to 2e3 months. subsequently, treatment with oral drugs is performed, but cam is the only reliable oral drug, as described above. on the other hand, monotherapy with cam should be avoided from the perspective of resistance induction. although some studies reported combination therapy with lzd or quinolones, its efficacy has not been established. -based on such a background, a combination of surgical resection of the lesion and combination chemotherapy is the only treatment that is expected to achieve the complete cure of pulmonary infection with m. abscessus in which the lesion is localized [284, 288, 289] . ---drugs to be recommended--a first choice based on the results of a drug susceptibility test, the following antimicrobial drugs should be combined: -cam, oral, 200 mg/3 times a day or 400 mg/twice a day þ amk, intravenous drip, 15 mg/kg/once a day þ ipm/cs, intravenous drip, 0.5 g/4 times a day or 1 g/3 times a day * surgery must be considered. the treatment period should be at least 12 months after culture becomes negative. ---executive summary----for the treatment of childhood tuberculosis, several drugs should be combined, and administered for a specific period (aii). -for the treatment of non-tuberculous mycobacterium infection, several drugs should be combined, and administered for a specific period. however, mycobacterium often resists treatment. if there is no treatment response, surgery must be considered (ciii). ---explanation--[characteristics and classification of the disease] in japan, tuberculosis is still an important infectious disease. when encountering patients with chronic infection who do not respond to general antimicrobial drugs, tuberculosis should be considered for differential diagnosis. mycobacteria that can be cultured are classified into two types: m. tuberculosis complex and non-tuberculous mycobacteria (ntm) [247] . m. tuberculosis is a major type of m. tuberculosis complex, and has a strong infectivity from humans to humans. childhood tuberculosis is classified into two types based on age [247] . briefly, primary tuberculosis represented by hilar lymph node tuberculosis and meningitis, which develop following primary infection, is characteristic of infants and children. the interval from infection until onset is short, and the morbidity rate is high. in addition, this disease may lead to a severe condition. pulmonary/hilar lymph node tuberculosis in infants and children is asymptomatic, or the general condition is favorable even in the presence of fever or cough in many cases. when primary tuberculosis is detected based on dyspnea or an unfavorable general condition in addition to fever or cough, many infants/children have military tuberculosis or meningitis. on the other hand, secondary tuberculosis with a cavity lesion or nodular shadow in the lung field is frequent in junior high school students or older. symptoms such as cough, sputum, fever, and thoracic pain are often observed. in most children, the source of infection can be clarified through detailed peripheral contact screening at the time of onset. usually, the source of infection is clarified in 2/3 to 3/4 of children. it is often their fathers/mothers or grandfathers/grandmothers [290e292]. as childhood tuberculosis does not form a cavity in the lung field, the m. tuberculosis level in the focus is lower than in adults. in many cases, it is difficult to bacteriologically or histologically make a definitive diagnosis in comparison with adult tuberculosis. usually, it is possible to make a definitive diagnosis by comprehensively evaluating epidemiological/clinical information such as opportunities for the source of infection to contact with tuberculosis patients, tuberculin reaction-or quantiferon tb (qft)-based verification of infection, imaging findings suggestive of tuberculosis, such as chest x-ray findings, verification of m. tuberculosis from sputum or gastric juice, and individuals' resistance including the grade of bcg vaccination-acquired immunity and age, as well as by considering treatment responses in some cases. qft is a very useful testing method to quantitatively measure ifn-g and diagnose tuberculosis infection without being influenced by bcg. however, assessment in infants/children should be further examined in the future [293] . when a definitive diagnosis of pulmonary tuberculosis cannot be made based on chest x-ray findings alone, thoracic ct, which facilitates the detailed evaluation of the presence or absence and extent of tuberculous lesions, is useful for diagnosis. furthermore, imaging findings of tuberculosis do not change in a short period in many cases. non-tuberculous mycobacterium belongs to mycobacterium, the same category as reported for m. tuberculosis. therefore, it is often detected as a mycobacterium-positive smear of sputum, that is, gaffky's positive reaction. initially, some patients are regarded as having infectious tuberculosis, and admitted to a tuberculosis ward. symptoms and imaging findings are also similar between nontuberculous mycobacterium-and m. tuberculosis-infected patients. unless detected bacteria are identified, or unless either gene is detected using the nucleic acid amplification method, it is difficult to differentiate the two types of bacteria. however, it is important to recognize that tuberculosis and non-tuberculous mycobacterium infection are different diseases [276, 294] . the most important point is that non-tuberculous mycobacterium infection does not transmit from humans to humans, differing from tuberculosis, an infectious disease in humans. therefore, it is not necessary to isolate the patient, and, as a rule, patients requiring admission should be managed in a general ward. as there are no public hygieneassociated problems, it is not necessary to submit a report to a health center. [type and frequency of causative microorganisms] in japan, the number of patients with childhood tuberculosis has markedly decreased. the number of newly registered patients with tuberculosis decreased from 53,229 (1963) to 95 (2008) in children aged 0e14 years [290e292]. however, a decrease in the incidence of smear-positive pulmonary tuberculosis, which is important as the source of infection, is not marked in great urban areas. we cannot conclude that the opportunity of infection in children is favorably decreasing; caution is needed. the number of patients who newly develop non-tuberculous mycobacterium infection in japan is estimated to be approximately 8000. in the adult field, it accounts for about 1/3 of that of patients who newly develop tuberculosis. however, it is relatively low in children. approximately 80% of patients with non-tuberculous mycobacterium infection are infected with m. avium complex (m. avium and mycoboterium intracellulare, pulmonary mac infection), and approximately 15% are infected with m. cansasii. [rules of antimicrobial drug therapy] -the characteristics of antitubercular chemotherapy in children are that children are tolerable to a relatively high dose per body weight in comparison with adults with respect to pharmacokinetics, and that the incidence of side effects is low [247] . in the pediatric field, 6-month treatment with inh, rfp, and pza for childhood pulmonary tuberculosis is internationally selected as standard chemotherapy: three drugs, inh, rfp, and pza, are administered every day for the first 2 months, and inh and rfp every day for the subsequent 4 months. when drug resistance is suspected, these drugs should be combined with sm or eb in the initial phase until the results of a resistance test are clarified. in patients with secondary tuberculosis, 4-drug combination therapy with inh, rfp, pza, and sm (or eb) should be initially performed. in addition, as a rule, follow-up must be continued for 2 years after the completion of treatment [290e292]. on the other hand, drug resistance, referral to another hospital, and discontinued treatment are present among patients who drop out of treatment, although the number of such patients is small. it is necessary to support the resistance and continuation of treatment. in particular, recently, the number of patients in whom it is difficult to continue treatment has increased. potent compliance support must be considered in connection with direct observed therapy (dot) by health centers and welfare activities [247] . side effects during treatment include liver dysfunction. however, if the maximum ast or alt levels are approximately 100, administration should be carefully continued without discontinuing treatment. if these levels exceed 100, treatment should be transiently discontinued, and additional administration at a low dose should be conducted after confirming the normalization of the liver function. the dose should be gradually increased. liver dysfunction requiring a change of treatment is not frequent. furthermore, there is an increase in the serum uric acid level, but continuous treatment leads to normalization. there have been few patients with arthralgia. -prevention of tuberculosis: to prevent the onset of tuberculosis in uninfected persons, bcg vaccination should be performed. concerning its efficacy, a consensus regarding its potent preventive effects on severe disseminated tuberculosis, such as tuberculous meningitis and military tuberculosis, has been reached. considering the importance of tuberculous meningitis prevention, bcg vaccination in the early phase of infancy (5e8 months after birth, or earlier in accordance with the state of peripheral tuberculosis prevalence) is still necessary in japan [290] . -treatment for latent tuberculosis: to prevent the onset of tuberculosis in persons with a history of tuberculosis, treatment for latent tuberculosis (conventional chemoprevention) should be conducted. a large-scale controlled study reported that inh therapy decreased the incidence of tuberculosis by approximately 50e60%. for drug administration, the risk of tuberculosis onset should be concretely and flexibly evaluated based on the tuberculin reaction, opportunity of infection, age, and state of bcg vaccination in individual patients [247] . -treatment for non-tuberculous mycobacterium infection: nontuberculous mycobacterium infection is refractory despite combination therapy with antitubercular drugs. in particular, there is no evidence regarding treatment in children [276] . the effects of monotherapy are weak, and monotherapy with cam may lead to the appearance of cam-resistant bacteria within a few months [279] ; therefore, this therapy should be avoided. the responses of m. kansasii to antitubercular drugs are relatively favorable, and cure may be achieved. however, pulmonary mac infection is often resistant to treatment. if there is no response, surgery must be considered. recurrence after the completion of treatment is also often observed. ---drugs to be recommended--* as the administration period is longer than that for tuberculosis patients, the development of vision disorder should be considered even at these doses. * if there is no response, surgery must be considered. ---explanation---acute bronchitis is characterized by cough that persists for 5 days or more. in most cases, cough persists for 1e3 weeks, but spontaneously subsides [295, 296] . sputum is present in some cases, but is absent in others. sputum may be purulent even when viral infection is etiologically involved. neither chest x-ray nor ct shows the appearance of a new abnormal shadow, differing from pneumonia. viruses . there is no evidence that infection with other bacteria directly causes acute bronchitis in adults without an underlying disease [296] . however, in a study using the transtracheal aspiration method in japan, h. influenzae, s. pneumoniae, and m. catarrhalis were primarily isolated in patients diagnosed with bacterial acute bronchitis in the absence of a chronic lower respiratory infectious disease as an underlying disease [299] . in cases of pertussis, cough persists particularly over a long period, and paroxysmal coughing, inspiratory whooping, and vomiting after coughing may occur [300] . acute bronchitis caused by m. pneumoniae also induces severe, persistent cough. in cases of influenza, fever, headache, general malaise, and arthralgia are observed. furthermore, acute viral bronchitis may lead to acute bacterial exacerbation in patients with chronic respiratory lesions as underlying diseases; fever and an increase in the amount of purulent sputum are observed. as a rule, when an underlying disease or complication is absent, the routine administration of antimicrobial drugs for acute bronchitis is not recommended [297, 298] . to control symptoms such as cough, symptomatic therapy should be performed if necessary. on the other hand, antimicrobial drug treatment with macrolides is indicated for patients with pertussis. treatment after the catarrhal period does not reduce the degree or duration of cough, but antimicrobial drugs are necessary to prevent infection to peripheral persons [301, 302] . when performing antimicrobial drug treatment for acute bronchitis caused by m. pneumoniae or c. pneumoniae, macrolides should be selected as first-choice drugs. however, an increase in macrolide-resistant m. pneumoniae must be considered [303] . in cases of influenza, anti-influenza therapy should be conducted within 48 h after onset [304] . in patients with underlying diseases or elderly persons with complications, bacterial (e.g., s. pneumoniae) infection may occur following viral infection, although this is not frequent in healthy adults. when acute bronchitis related to bacterial infection, including secondary infection, is strongly suspected based on cough/sputum, fever, leukocytosis, or findings suggestive of the presence of causative microorganisms on gram staining of sputum despite the absence of a new infiltrative shadow on chest x-ray, antimicrobial drug treatment is considered in accordance with treatment for community-acquired bacterial pneumonia [305, 306] . ---drugs to be recommended--when there are no complications such as chronic respiratory diseases, the administration of antimicrobial drugs for acute bronchitis are not recommended as a rule (with respect to the selection of antimicrobial drugs for acute bronchitis complicated by chronic respiratory diseases with secondary bacterial infection, refer to the section "6. miller & jones classification of purulent sputum. m1: saliva, complete mucous sputum m2: mucous sputum containing a small volume of purulent sputum p1: sputum in which the purulent area comprises 1/3 or smaller p2: sputum in which the purulent area comprises 1/3 to 2/3 p3: sputum in which the purulent area comprises 2/3 or greater fever and shortness of breath, in addition to bacterial infection-related respiratory symptoms, such as increases in the frequency of cough, volume of purulent sputum, and degree of purulence, from the chronic, stable conditions of underlying diseases, such as copd, bronchiectasis, and old pulmonary tuberculosis. concerning laboratory data, inflammatory responses involving the leukocyte count and crp level are enhanced, and pao 2 is often reduced on blood gas analysis. [imaging findings] imaging findings are necessary to differentiate chronic respiratory disease-related airway infection from pneumonia. the absence of a shadow must be confirmed. ct should also be performed to evaluate underlying diseases such as pulmonary emphysema and bronchiectasis. [estimation of causative microorganisms and gram staining] it is possible to collect sputum in many patients. gram staining is useful for predicting causative microorganisms or differentiating respiratory tract infection in the presence of chronic respiratory disease from persistent infection. according to a study, the tone of sputum suggests the presence of pathogenic microorganisms rather than the degree of purulence; macroscopic examination is also necessary [319] . sputum involves much information, and is the most important sample. samples should be collected before the administration of antimicrobial drugs. those collected on waking-up early in the morning are ideal. to evaluate the degree of sputum purulence, the miller & jones classification [305] (table 21) is used, but, if samples are evaluated as p2 or higher, causative microorganisms may be predicted using gram staining. on gram staining, an area where the number of inflammatory cells is large should be initially searched at a low magnification, and detailed observation should be conducted at a high magnification. before the administration of antimicrobial drugs, sputum should always be submitted for a susceptibility test. as causative microorganisms, h. influenzae, p. aeruginosa, m. catarrhalis, and s. pneumoniae are frequently detected. persistent infection with p. aeruginosa is often observed, but it must be differentiated from acute exacerbation based on clinical symptoms and laboratory data. in addition, s. aureus and k. pneumoniae should be considered [310] . the involvement of atypical pathogens such as c. pneumoniae or mixed infection with viruses and bacteria must also be considered. [treatment] the purpose of treatment is to reduce clinical symptoms, prevent recurrence, prolong the interval until subsequent exacerbation, and inhibit lung tissue damage. the administration of appropriate antimicrobial drugs relieves clinical symptoms, and maintains the respiratory function [320] . on the other hand, inappropriate antimicrobial drugs may deteriorate the prognosis, inducing recurrence. in japan, the resistance of s. pneumoniae and h. influenzae to macrolides and b-lactams is advanced [13, 29] . several studies have reported that new quinolones are more useful than b-lactams [15,312e315] . respiratory quinolones have potent antimicrobial activities against all types of causative microorganisms, and against resistant bacteria [321e323]. concerning the administration period, a study indicated that the efficacy of administration for 5 days was similar to that for 7 days, and that the former was safer than the latter. the administration period should be shortened [324] . ---drugs to be recommended--a. empiric therapy internationally, some studies have supported the usefulness of b-lactams [325, 326] . however, in japan, the resistance of s. pneumoniae and h. influenzae to macrolides and b-lactams is advanced, and p. aeruginosa is also sometimes isolated. therefore, the use of b-lactams and macrolides is limited to patients without risk factors. an international comparative study reported that the efficacy of azm sustained-release preparations was similar to that of new quinolones [327] . however, in japan, the long-term administration of macrolides is performed in many patients; therefore, circumstances differ. beta-lactamase-producing strains are detected in approximately 10e20% of h. influenzae strains. beta-lactamasenegative, ampicillin-resistant (blnar) strains account for approximately 20%. therefore, when drug susceptibility is unclear, new quinolones should be selected as first-choice oral antimicrobial drugs. if drug susceptibility is clarified, they should be switched to effective and narrow-spectrum drugs. as injection, penicillins should be initially selected, followed by b-lactamase inhibitor-containing penicillins, carbapenems, and new quinolones. ➁ m. catarrhalis beta-lactamase-producing strains account for 100% of m. catarrhalis strains. as oral antimicrobial drugs, macrolides should be initially selected, followed by b-lactamase inhibitor-containing penicillins, second-/third-generation cephems, and new quinolones. as injection, b-lactamase inhibitor-containing penicillins, second-/third-generation cephems, new quinolones, or carbapenems should be selected. ➂ p. aeruginosa as oral drugs, new quinolones should be selected. as injection, anti-p. aeruginosa penicillins, cephems, monobactums, carbapenems, or new quinolones should be selected. as the drug susceptibility of this type of bacteria markedly differs among strains, drugs should be selected based on the results of culture tests. ➃ s. pneumoniae as oral drugs, penicillins should be initially selected, followed by new quinolones. in patients with a risk of resistant bacteria, respiratory quinolones such as lvfx and grnx should be selected. as injection, penicillins or ctrx should be selected, but carbapenems must be considered in severestatus patients. ---explanation---[characteristics/classification of the disease] dpb is a chronic inflammatory disease of the respiratory tract, which is frequently observed in east asians including japanese. there is no gender difference, and this disease frequently develops in persons aged 40e50 years. it is often detected in patients with a history of chronic sinusitis or in those with the concomitant development of chronic sinusitis. this disease is classified as the category of sinobronchial syndrome. [symptoms] the most typical symptoms of dpb are persistent cough and purulent sputum. symptoms such as exertional shortness of breath and dyspnea appear in accordance with disease progression. in patients with a complication of chronic sinusitis, purulent nasal discharge and nasal obstruction are observed. chest x-ray shows pulmonary overexpansion or a diffuse scattered nodular shadow. thoracic hrct reveals a diffuse centrilobular nodular shadow. furthermore, obstructive respiratory dysfunction, hypoxemia, and an increase in the cold agglutinin value (64-fold or more on the hemagglutination method) are observed. [type and frequency of causative microorganisms] in patients with dpb, persistent respiratory tract infection with h. influenzae, s. pneumoniae, or m. catarrhalis is often observed. however, the incidence of persistent infection with p. aeruginosa increases with progression. [long-term macrolide therapy] previously, the prognosis of dpb was unfavorable; respiratory failure gradually progressed through repeated acute exacerbation related to respiratory tract infection, leading to a fatal outcome; however, the prognosis of dpb has been markedly improved since long-term macrolide therapy with low-dose administration of em or other 14-membered ring macrolides was established [328e340]. early diagnosis/treatment have facilitated the complete cure of dpb. therefore, if once a diagnosis of dpb is made, long-term macrolide therapy should be started promptly. ---drugs to be recommended---➀ persistent infection -the oral administration of em at 400e600 mg/day should be continued for 6 months to be evaluated its clinical effects [328] . -in many cases, an improvement in symptoms (such as a decrease in the volume of sputum) will be achieved within 1e3 months after the start of administration. -in addition, an improvement in imaging findings or the respiratory function will be achieved after 3e6 months of treatment. croup syndrome is characterized by acute laryngeal stenosisassociated respiratory disturbance such as barking cough, hoarseness, and inspiratory stridor. most lesions involve not only the larynx but also the trachea/bronchus. this disease is sometimes called laryngotracheobronchitis [352] . etiological factors are classified into two types: infectious and non-infectious (allergy-/ foreign body-related) factors [352, 353] . the incidence of infectious croup syndrome is high in infants/children aged 7 monthse3 years [354, 355] . [type and frequency of causative microorganisms] croup syndrome is primarily caused by viruses. parainfluenza virus type 1 is the most common virus [356] . in addition, parainfluenza virus type 2/3, influenza a/b virus, rs virus, human metapneumovirus, coronavirus, adenovirus, and measles virus are relatively frequently isolated [353, 357] . [rules of antimicrobial drug therapy] in most cases, croup syndrome is caused by viruses, and antimicrobial drugs are not necessary. therefore, no study has evaluated the efficacy of antimicrobial drugs in patients with croup syndrome. there are no treatment guidelines regarding croup syndrome in which antimicrobial drugs are recommended [358, 359] . ---executive summary---bronchiolitis is caused by viruses, and the administration of antimicrobial drugs is not necessary (ai). ---explanation--bronchiolitis is an acute, inflammatory, obstructive disease involving the bronchiole. narrowing of the bronchiolar lumen related to mucosal epithelial injury, inflammatory-cell infiltration, interstitial edema, or an increase in mucus secretion causes air trapping in the peripheral respiratory tract, leading to obstructive respiratory disorder. this disease frequently develops in children aged 2 years or younger. however, infants aged 11 months or younger account for 80% or more [360] . [type and frequency of causative microorganisms] bronchiolitis is primarily caused by viruses. rs virus accounts for 60e80%. in addition, parainfluenza virus, human metapneumovirus, adenovirus, and influenza virus are relatively frequently isolated [361e363]. [rules of antimicrobial drug therapy] in most cases, bronchiolitis is caused by viruses, and antimicrobial drugs are not necessary. basic treatment is symptomatic therapy. in double-blind comparative studies involving abpc and non-treated groups [364] , azm and non-treated groups [365] , and abpc intravenous injection/oral em and non-treated groups [366] , respectively, there were no significant differences in the admission period or symptom improvement. however, a small-scale doubleblind comparative study reported that the interval until recovery in the cam-treated group was shorter than in the non-treated group [367] . according to another study, the incidence of secondary bacterial infection during the course of rs virus-related bronchiolitis was 1.2%, and there was no difference between antimicrobial drug-treated and non-treated groups [368] . therefore, it is not necessary to administer antimicrobial drugs to children with bronchiolitis for routine treatment or the prevention of secondary bacterial infection. however, follow-up must be carefully continued during the course of bronchiolitis. when a diagnosis of secondary bacterial infection-related pneumonia or otitis media is made, antimicrobial drug therapy should be started. ---executive summary---bacterial tracheitis is a bacterial disease with the rapid progression of dyspnea. if symptoms are progressive, antimicrobial drugs should be used even when a definitive diagnosis is not made (aiii). ---explanation--fever and croup syndrome-like cough/stridor initially appear, and respiratory disorder rapidly progresses, but there is no specific posture, salivation, or dysphagia, which are characteristic of acute epiglottitis. a definitive diagnosis can be made based on characteristic clinical features and purulent secretion in the respiratory tract. in some cases, a lateral view of the larynx on x-ray shows stenosis below the larynx [352] . this disease frequently develops in children aged 3e8 years [369] . [type and frequency of causative microorganisms] s. aureus-related tracheitis accounts for approximately 60%, followed by that related to m. catarrhalis, h. influenzae, streptococcus pneumoniae, and streptococcus pyogenes [370e373]. mixed infection with viruses and bacteria is frequent, and parainfluenza virus type i [373] and influenza a virus [374] are often detected. [rules of antimicrobial drug therapy] antimicrobial drugs should be intravenously administered for the following reasons: this disease rapidly progresses, and oral administration is difficult in many cases. as empiric therapy, combination therapy with vcm, which may be effective for infection with s. aureus (including mrsa), and third-generation cephems (ctrx, ctx), which have potent antimicrobial activities against m. catarrhalis, h. influenzae, s. pneumoniae, and s. pyogenes, should be performed. the administration period is 10e14 days [369] . ---drugs to be recommended---refer to the section "3. pneumonia (children)---drugs to be recommended 2. definitive therapy---" (p. 33). ---executive summary---acute bronchitis is primarily caused by viruses, and the necessity of antimicrobial drug administration is low (ai). when acute bronchitis is caused by m. pneumoniae, c. pneumoniae, or b. pertussis, antimicrobial drugs should be administered if necessary (aiii). secondary infection with s. pneumoniae or h. influenzae may occur, although its incidence is unclear. therefore, when there is no improvement, the administration of antimicrobial drugs should be considered (aiii). ---explanation---[characteristics and classification of the disease] bronchitis causes symptoms such as cough, fever, and general malaise. various causative microorganisms induce inflammation of the epithelial tracheobronchial tissue, leading to the onset of bronchitis. clinically, there are no special findings on auscultation, or only rough respiratory sounds (intermittent accessory murmurs) are heard. chest x-ray does not also show any marked infiltrative shadow. usually, patients in whom the interval after onset is less than 3 weeks are regarded as having acute bronchitis [360] . however, bronchitis diagnosed in japan slightly differs from that in europe and the united states. the latter primarily causes persistent cough. in japan, patients in whom there are no findings on chest x-ray despite clinical signs of pneumonia or those in whom chest x-ray is not performed are often diagnosed with bronchitis; the disease entity must be arranged. [type and frequency of causative microorganisms] viruses, such as rhinovirus, influenza virus, rs virus, adenovirus, parainfluenza virus, human metapneumovirus, and human bocavirus, account for 90% of causative microorganisms. m. pneumoniae, c. pneumoniae, and b. pertussis also cause bronchitis, although such cases are relatively rare [360, 375] . [rules of antimicrobial drug therapy] acute bronchitis is primarily caused by viruses, and the administration of antimicrobial drugs is not necessary. a metaanalysis compared adults to whom antimicrobial drugs were administered for bronchitis treatment with non-treated adults, and indicated that there was no difference in the efficacy [376] . few reports on clinical studies involving children have been published, and the scale is small; objective data are insufficient, but no study has reported that antimicrobial drugs are effective [377e379]. however, if secondary bacterial infection following viral infection causes fever, purulent sputum, leukocytosis, or an increase in the crp level, antimicrobial drugs should be administered, considering s. pneumoniae and h. influenzae. other indications for antimicrobial drug administration include m. pneumoniae-, c. pneumoniae-, or b. pertussis-related bronchitis with protracted cough. as m. pneumoniae-or c. pneumoniae-related bronchitis tends to show spontaneous cure, the administration of antimicrobial drugs is not always necessary, but the necessity of administration should be evaluated, considering the severity of symptoms and course (with respect to indications and administration methods, refer to the section "3. pneumonia (children)".). first-choice drugs for m. pneumoniae-, c. pneumoniae-, or b. pertussis-related bronchitis are macrolides. in patients with b. pertussis-related bronchitis, antimicrobial drugs relieve symptoms only during the catarrhal period, but, if b. pertussis-related bronchitis is suspected based on clinical symptoms, previous vaccination, lymphocyte-predominant leukocytosis, an anti-pt antibody titer, and lamp findings, antimicrobial drugs should be used. however, 16-membered ring macrolides are ineffective for b. pertussis-related bronchitis. symptoms are similar to those of pneumonia, but, when chest x-ray does not show any abnormalities, or, when it is impossible to strictly differentiate bronchitis from pneumonia due to difficulty in chest x-ray, treatment should be performed in accordance with pneumonia. ---drugs to be recommended--secondary bacterial infection after viral infection (cases in which fever, purulent sputum, leukocytosis, or an increase in the crp level is observed) a first choices -ampc, oral, 10e15 mg/kg/3 times a day -sbtpc, oral, 10 mg/kg/3 times a day -cdtr-pi, oral, 3 mg/kg/3 times a day -cfpn-pi, oral, 3 mg/kg/3 times a day -cftm-pi, oral, 3 mg/kg/3 times a day <> second choices -azm, oral, 10 mg/kg/once a day, 3 days -cam, oral, 7.5 mg/kg/twice a day 2. definitive therapy ➀ b. pertussis -em, oral, 10e15 mg/kg/3 times a day -cam, oral, 7.5 mg/kg/twice a day -azm, oral, 10 mg/kg/once a day, 3 days ➁ m. pneumoniae ▪ macrolide-sensitive strains -em, oral, 10e15 mg/kg/3 times a day -azm, oral, 10 mg/kg/once a day, 3 days -cam, oral, 7.5 mg/kg/twice a day ▪ macrolide-resistant strains -mino, oral or intravenous drip, 1e2 mg/kg/twice a day (in children aged 7 years or younger, the use of this drug is limited to those in whom other drugs cannot be used or non-responders.) -tflx, oral, 6 mg/kg/twice a day (administration is limited to children aged 7 years or younger in whom mino cannot be used.) ➂ chlamydia (c. pneumoniae, c. psittaci, c. trachomatis) -em, oral, 10e15 mg/kg/3 times a day -azm, oral, 10 mg/kg/once a day, 3 days -cam, oral, 7.5 mg/kg/twice a day 8. influenza ---executive summary----both m2 protein inhibitors and neuraminidase inhibitors (nais) are commercially available as anti-influenza drugs as of july 2013. -influenza viruses a (h3n2) and a (h1n1) pdm09 (seasonal influenza) have been reported to be resistant to amantadine, an m2 protein inhibitor which can be used in japan. the use of this drug as an anti-influenza drug should be avoided for a while [380, 381] . -during the influenza outbreak period, anti-influenza therapy should be promptly started based on a clinical diagnosis even when patients with influenza-like symptoms show negative results on a rapid diagnosis kits (because influenza cannot be completely ruled out) [382] (ai). -nais significantly improve influenza survival, and nai administration within 2 days after onset significantly reduces the rate at which the condition becomes severe [383, 384] (ai). -currently, the following nais can be selected in japan. during the outbreak period, an appropriate drug should be selected based on the patient background and latest information on a prevalent influenza strain: -oseltamivir (oral), efficacy: a (h1n1) pdm09, a (h3n2), b, resistance: h275y mutant -zanamivir (inhalation), efficacy: type a/b -laninamivir (inhalation), efficacy: type a/b -peramivir (intravenous drip), efficacy: type a/b ---drugs to be recommended---there is no meta-analysis of anti-influenza drugs other than oseltamivir and zanamivir as of july 2013. however, it has been shown that the early introduction of anti-influenza therapy for influenza significantly inhibits not only the mortality and admission rates but also the incidences of influenza-associated pneumonia, otitis media, and ischemic heart disease in comparison with symptomatic therapy. nais such as laninamivir and peramivir have also been confirmed to be as effective as oseltamivir at the time of development [385] (ai). <>outpatient treatment -oseltamivir, oral, 75 mg/twice a day, 5 days (as a rule, administration to children/adolescents aged 10e19 years should be avoided.) -zanamivir, inhalation, 10 mg/twice a day, 5 days -laninamivir, inhalation, 40 mg/single dose -peramivir, intravenous drip, 300 mg/single dose <>hospital treatment ➀ patients with severe, life-threatening influenza in patients with severe, life-threatening influenza requiring admission, respiratory failure or encephalopathy is present. in either case, the complication must be treated, but, as a rule, nais should be introduced within 48 h after the onset of influenza to obtain their effects. -oseltamivir, oral, 75 mg/twice a day, 5 days (as a rule, administration to children/adolescents aged 10e19 years should be avoided.) -peramivir, intravenous drip, 600 mg/single dose (this drug can be repeatedly administered every day in accordance with symptoms.) ➁ non-life-threatening influenza patients with pneumonia -oseltamivir, oral, 75 mg/twice a day, 5 days (as a rule, administration to children/adolescents aged 10e19 years should be avoided.) -peramivir, intravenous drip, 300 mg (600 mg for patients in whom the condition may become severe)/single dose (this drug can be repeatedly administered every day in accordance with symptoms.) ➂ non-life-threatening influenza patients without pneumonia -oseltamivir, oral, 75 mg/twice a day, 5 days (as a rule, administration to children/adolescents aged 10e19 years should be avoided.) -zanamivir, inhalation, 10 mg/twice a day, 5 days -laninamivir, inhalation, 40 mg/single dose -peramivir, intravenous drip, 300 mg/single dose (this drug can be repeatedly administered every day in accordance with symptoms.) * patients with a (h7n9) -basic treatment is the early administration of anti-influenza drugs [386] . -according to an article, there were no significant differences in the viral level or mortality rate 5 days after the start of administration between double-and standard-dose oseltamivir therapies [387] . however, treatment was not started within 48 h after onset in all patients in the article. ---executive summary---influenza is caused by influenza virus. antimicrobial drugs are not necessary. it is recommended that neuraminidase inhibitors should be administered within 48 h after onset (ai). if pneumonia or otitis media occurs through secondary bacterial infection, the administration of antimicrobial drugs must be considered (biii). ---explanation---[characteristics and classification of the disease] influenza often appears with sudden fever and shivering/ headache/general malaise/muscular pain/dry cough, followed by marked respiratory or digestive symptoms. in underlying diseasefree children, recovery is achieved after 3e7 days [388, 389] . however, during the outbreak period, even underlying disease-free children are often admitted with serious symptoms such as encephalopathy, myocarditis, and pneumonia requiring artificial respiration. although the outbreak period is from december until march every year, outbreaks are observed in the summer in some areas [388, 389] . [type and frequency of causative microorganisms] since influenza a (h1n1) pdm09 prevailed in 2009, the outbreaks of 3 types of influenza, a (h1n1) pdm09, a (h3n2), and b, have been repeated. their incidences differ among years. [rules of antimicrobial drug therapy] no antimicrobial drug is indicated for influenza. it is recommended that neuraminidase inhibitors should be administered within 48 h after onset [390] . the doses of neuraminidase inhibitors approved in japan are presented in table 22 . during the course of influenza, bacterial infection such as pneumonia and otitis media may occur. previously, the incidence of secondary bacterial infection in children exceeded 10% [391] , but it has been 3% or less since neuraminidase inhibitors were developed [392, 393] . however, bacterial infection is observed in 25e33% of severe-status patients requiring intensive care [394, 395] . streptococcus pneumoniae, s. aureus, and h. influenzae are frequently detected as causative microorganisms [388, 389, 394, 395] . no study has demonstrated that the prophylactic administration of antimicrobial drugs at the onset of influenza prevents secondary bacterial infection. among children with influenza, antimicrobial drug therapy should be considered in those in whom signs of pneumonia or otitis media do not subside despite the administration of a neuraminidase inhibitor. 9. parasitic diseases of the respiratory system ---executive summary----parasitic diseases are widely distributed throughout the world. in addition to domestic infection, japanese travelers may be infected in overseas endemic areas. furthermore, parasitic diseases always considered in foreign patients from endemic areas [396] (biv). -when peripheral blood eosinophilia is observed in addition to respiratory symptoms and abnormal findings of the chest imaging, examinations should be performed to differentiate parasitic diseases [396] (biv). -parasitic diseases of the respiratory system are caused by paragonimus spp., ascaris lumbricoides, ancylostoma duodenale, necator americanus, strongyloides stercoralis, toxocara canis, toxocara cati, wuchereria bancrofti, brugia malayi, or dirofilaria immitis. -there are two major diagnostic tests for parasitic diseases: (1) detection of parasite eggs or larvae in sputum or stools, and (2) detection of parasite-specific antibodies using serum or pleural effusion samples (immunodiagnosis). -an anti-parasite antibody screening test against 12 species of parasite is commercially available; paragonimus spp., strongyloides sp., t. canis, d. immitis, ascaris suum, anisakis simplex, gnathostoma spp., fasciola hepatica, clonorchis sinensis, spirometra erinaceieuropaei, and cysticercus cellulosae. -diagnosis/treatment consultations regarding parasitic diseases by the japanese society of parasitology are available (as of 2013). refer to the homepage (http://jsp.tm.nagasaki-u.ac.jp). ---explanation----paragonimus spp. several paragonimus species are known to cause human infection. paragonimus westermani and p. miyazakii are distributed in japan. infection occurs through the consumption of freshwater crabs (intermediate host: eriocheir japonica, e. sinensis, geothelphusa dehaani) or wild boars (paratenic host:sus scrofa) contaminated with metacercaria (infective larvae) as a raw or insufficiently cooked food [397] . typical symptoms are cough, sputum, thoracic pain, and exertional dyspnea. in patients with such symptoms, the presence of peripheral blood eosinophilia suggests this disease. in many cases, a diagnosis is made based on the peripheral blood eosinophilia, a history taking of food and table 22 standard doses of neuraminidase inhibitors in children. oseltamivir a oral 2 mg/kg, twice a day, 5 days (the use of this drug should be avoided in children/adolescents aged 10e19 years.) zanamivir b inhalation 10 mg, twice a day, 5 days laninamivir inhalation 10 years or older: 40 mg 9 years or younger: 20 mg, single dose peramivir intravenous drip 10 mg/kg, once a day a the preventive administration of this drug at 2 mg/kg (once a day) for 10 days was approved, but is not covered by health insurance. b the preventive administration of this drug at 10 mg (once a day) for 10 days was approved, but is not covered by health insurance. immunodiagnosis. some of paragonimiasis patients are asymptomatic, and the presence of lung lesions is detected on a health checkup. extrapulmonary paragonimiasis such as cutaneous and cerebral paragonimiasis are classically known form of the ectopic infection with paragonimus spp. in cases of cutaneous paragonimiasis, a slowly moving nodular lesion in the subcutaneous tissue is a characteristic symptom. the worms may migrate through mediastinal soft tissues to the brain, causing eosinophilic meningitis or cerebral paragonimiasis in some cases [397] . since paragonimus spp. is widely distributed around the world, japanese travel to endemic area such as china, korea, thailand, and philippines possible to infect with paragonimus spp. as well as foreign patients from endemic areas, this disease always is considered when a patient has respiratory symptoms and/or lung lesions with eosinophilia [398] . chest x-ray findings vary: not only pulmonary parenchymal lesions, such as nodular (±cavity formation) and infiltrative shadows, but also pleural lesions, such as the retention of pleural effusion and pneumothorax, are sometimes observed [399] . many patients show peripheral blood eosinophilia and/or elevated serum total ige. immunodiagnosis to detect parasite-specific antibody has been proven as the most useful and reliable tool. not only patient's serum but also pleural effusion could examine by immunological test. commercially available anti-parasite antibody screening test is including paragomimus spp. in japan, egg-detection rate among paragonimiasis patients nowadays is low; for example 51.2% in sputum and 53.8% in balf [66] , 66.7% in bronchoscopic aspirate [397, 399] . after a definitive diagnosis is made, oral treatment should be started. the type of treatment to be selected, outpatient or hospital treatment, depends on the patient's general condition. however, usually, outpatient treatment is possible. in patients having pleural effusion, pleural fluid must be extensively drained off before starting chemotherapy [400] (biii). in patients with chronically encapsulated pleural effusion, surgery is required [401] (ciii). ---drugs to be recommended---a first choice (same dose for adults and children) -praziquantel, oral, 25 mg/kg/3 times a day, 2e3 days [402] (aiii) -a. lumbricoides, a. duodenale, n. americanus the larvae of these parasites pass through the lung in the human body, causing asthma-or pneumonia-like symptoms such as transient fever, cough, and dyspnea. on chest x-ray, transient nodular/infiltrative shadows are observed. many patients show peripheral blood eosinophilia and/or elevated serum total ige. previously, the condition was called loffler syndrome. currently, it is classified as simple pulmonary eosinophilia in the category of pie syndrome [402] . symptoms appear 1e2 weeks after the oral ingestion of a. lumbricoides embryonated eggs. the latency period of percutaneous infection with n. americanus larvae or percutaneous/oral infection with a. duodenale larvae is approximately 10 days. larvae appeared in the small intestine penetrate the intestinal wall, enter the portal vein, migrate through liver to heart/lung. larvae penetrate the human skin transfer to the lung with blood flow. then these larvae migrate up the bronchi and trachea, over the epiglottis, down the esophagus/stomach, and reach the small intestine. in the intestine the larvae develop into mature adults [403] . simple pulmonary eosinophilia related to parasites spontaneously resolved in a few weeks. in many cases, a definitive diagnosis is made based on parasite eggs detected on a stool examination, although larvae are sometimes detected in sputum [403] . ---drugs to be recommended---a first choice (same dose for adults and children) -pyrantel pamoate, oral, 10 mg/kg/single-dose administration * dry syrup for children is available. * this drug is effective for adult worms in the intestinal tract, but not for larvae migrating in the human body. therefore, a stool examination should be performed 2 weeks after oral administration. if parasite eggs are detected, additional administration should be conducted. <> second choices (same dose for adults and children) -albendazol, oral, 400 mg/single-dose administration -mebendazole, oral, 100 mg/twice a day, 3 days, or 500 mg/single-dose administration -ivermectin, oral, 150e200 mg/kg/single-dose administration (after fasting) -s. stercoralis in japan, s. stercoralis is distributed in nansei islands, chain of islands extending from southwestern kyushu to northern taiwan. there are few young persons newly infected with s. stercoralis, but the incidence of infection is high in elderly persons [404] . internationally, s. stercoralis is widely distributed in tropical/subtropical areas. not only domestic infection but also japanese travel to endemic area possible to infect with this parasite. foreign patients from endemic areas, strongyloidiasis always considered when a patient has respiratory symptoms and/or lung lesions with eosinophilia. s. stercoralis percutaneously infects humans. larvae penetrate the human skin transfer to the lung with blood flow. then these larvae migrate up the bronchi and trachea, over the epiglottis, down the esophagus/stomach, and reach the small intestine. in the intestine the larvae develop into mature adults. parasite eggs delivered by adults are hatched while descending the intestinal tract, and larvae excreted in stools. some larvae developed to infective form again invade/transfer through the mucosa around the anus, maintaining a life cycle in the human body. such a mode of infection is termed autoinfection. with the transfer of larvae to the lung, asthma-or pneumonia-like symptoms, such as transient fever, cough, and dyspnea, appear, as indicated for simple pulmonary eosinophilia related to a. lumbricoides, a. duodenale, or n. americanus. on chest x-ray, transient nodular/infiltrative shadows are observed. many patients show peripheral blood eosinophilia and/or an increase in the total ige level. in immunocompromised patients, suppressing the cellmediated immunity such as atl patients, hiv/aids patients, those receiving immunosuppressive drugs, the number of s. stercoralis increases through acceleration of autoinfection, and larvae are disseminated in various organs, leading to a severe condition (disseminated strongyloidiasis). in such cases, stridor, bloody sputum, tachypnea, protein-losing gastroenteritis, ileus, and mobile exanthema are observed. furthermore, severe pneumonia related to enteric bacteria disseminated with larvae, lung abscess, or bacterial meningitis concomitantly occurs [403] . a diagnosis is made based on s. stercoralis larvae detected in stools/sputum. immunological diagnosis is also useful [403] . ---drugs to be recommended---a first choice (same dose for adults and children) -ivermectin, oral, 200 mg/kg/single-dose administration (after fasting), additional administration at the same dose after 2 weeks (ai) * repeated administration must be considered in cases of disseminated strongyloidiasis [405] . * a study reported that transrectal or percutaneous administration was useful in patients in whom oral administration was difficult due to digestive symptoms [406] . <> second choice (same dose for adults and children) -albendazol, oral, 400 mg/twice a day, 7 days (bi) -t. canis, t. cati infection to humans occurs through the oral ingestion of the embryonated eggs of t. canis or t. cati [407] . it also occurs through the consumption of beef/chicken liver or meat contaminated with larvae as a raw or insufficiently heated food. this is considered to be a dominant route of infection in japan [407] . as humans are not definitive host for t. canis and t. cati, they do not become adults in the human body [408] . larvae invading the human body transfer to various organs with blood flow through the intestinal mucosa. target organs are the lung, liver, eyes, and central nervous system including spinal cord. toxocariasis is a typical larva migrans. lesions are often observed in the lung/liver (visceral larva migrans). there are few symptoms, or nonspecific symptoms are present. in many cases, a diagnosis is made based on peripheral blood eosinophilia and multiple nodular shadows of the lung or liver on ct. ocular or central nervous symptoms appear in some patients. it is impossible to make a diagnosis on a stool examination due to larva migrans. immunological diagnosis is useful. after a definitive diagnosis is made, treatment should be started. when visceral larva migrans is asymptomatic, follow-up may be continued. ---drugs to be recommended---a first choice (same dose for adults and children) -albendazol, oral, 5 mg/kg/2 or 3 times a day, 4e8 weeks [409] (biii) * this drug should be taken with meals for 28 days. a 14-day period of discontinuation should be established and then restarted drug if required. <> second choices (same dose for adults and children) -albendazol, oral, 400 mg/twice a day, 5 days [410] (biii) -mebendazole, oral, 100e200 mg/twice a day, 5 days [411] (ciii) -w. bancrofti, b. malayi w. bancrofti and b. malayi are called lymphatic filaria. infection occurs when these parasites are transmitted to humans through mosquitoes. the pathogenesis of lymphatic filariasis is relate to structural and functional abnormality of lymphatic channels induced by parasitized adult worms. infection with w. bancrofti is characterized by febrile attacks, lymphedema/elephantiasis, hydrocele, and chyluria. in patients infected with b. malayi, neither hydrocele nor chyluria is observed, and lymphedema of the lower limbs/ elephantiasis are localized in the lower thighs. microfilaria produced by adult worms is not pathogenic, but rarely induces allergic reactions in the lung, contributing to tropical pulmonary eosinophilia (tpe) [403] . this condition shows a chronic course, differing from simple pulmonary eosinophilia. cough, dyspnea, and stridor with exacerbation at night are observed. fever, malaise, and weight loss are noted in some patients. on chest x-ray, bilateral reticular nodular lesions are detected. peripheral blood eosinophilia and an increase in the anti-filaria antibody titer are observed. in case of tpe, no microfilaria is detected [403] . if treatment is not performed, the condition may gradually exacerbate. it is important to adequately make a diagnosis for the differentiation of this disease. in the world, 130,000,000 persons are infected with lymphatic filaria, but tpe occurs in less than 0.5% of these. the risk of this disease in travelers is unclear, and most patients consist of foreign persons from endemic areas [412] . ---drugs to be recommended---a first choice (same dose for adults and children) -diethylcarbamazine, 2 mg/kg/3 times a day, 12 days * in cases of malayan filariasis, marked side effects, such as digestive symptoms, fever, lymphangitis/ lymphadenitis, and orchitis/epididymitis, may appear. therefore, a half dose (1 mg/kg) should be administered 3 times a day for 12 days [413] . -d. immitis d. immitis parasitize in the dog right ventricle and pulmonary artery, where mature female worms produce microfilaria that circulate in peripheral blood. infection to humans is mediated by microfilaria-ingesting mosquitoes. d. immitis larvae in human subcutaneous tissue inserted by mosquito bite, some larvae migrate to the heart and die. dead worms produce infarcts then they lodge in pulmonary vessels. there are few symptoms [402] . in patients with symptoms, thoracic pain, cough, bloody sputum, stridor, and fever have been reported. typical chest x-ray findings include solitary coin lesions. in many cases, an abnormal shadow of the chest x-ray is indicated on a health checkup, and d. immitis infection is pathologically diagnosed from a tissue specimen extirpated under a tentative diagnosis of lung cancer. at this point, peripheral blood eosinophilia is rarely observed [403] . ---drugs to be recommended (same dose for adults and children)----as a diagnosis is made using pathological specimens in many patients, oral treatment is not necessary. -if necessary, diethylcarbamazine at 2 mg/kg should be orally administered 3 times a day for 12 days. ---precautions for each drug--in the japanese package inserts, it is described that this drug at 40 mg/kg/day should be orally administered twice a day for 2 days for the treatment of paragominiasis. however, we recommend administration at 75 mg/kg/day (3 times a day) for 2e3 days based on reference no. 400. the incidence of side effects is low, but fever, abdominal discomfort, nausea, diarrhea, and headache are sometimes observed. masahumi seki: speaker's honorarium from shionogi & co., ltd., and taisho toyama pharmaceutical co., ltd dolin r, editors. mandell, douglas, and bennett's principles and practice of infectious diseases infectious diseases society of america/american thoracic society consensus guidelines on the management of community-acquired pneumonia in adults the committee for the japanese respiratory society guidelines for the management of respiratory infections. the japanese respiratory society guidelines for the management of community-acquired pneumonia in adults. tokyo: the japanese respiratory society nationwide survey on the 2005 guide-lines for the management of community-acquired adult pneumonia : validation of differentiation between bacterial pneumonia and a typical pneumonia evaluation of an assessment system for the jrs 2005: a-drop for the management of cap in adults sputum gram stain assessment in community-acquired bacteremic pneumonia assessment of the usefulness of sputum culture for diagnosis of communityacquired pneumonia using the port predictive scoring system detection of legionella pneumophila antigen in urine samples by the binaxnow immunochromatographic assay and comparison with both binax legionella urinary enzyme immunoassay (eia) and biotest legionella urin antigen eia rapid urinary antigen test for diagnosis of pneumococcal community-acquired pneumonia in adults revision of the severity rating and classification of hospital-acquired pneumonia in the japanese respiratory society guidelines the committee for the japanese respiratory society guidelines for the management of respiratory tract infection. the guidelines for the management of nursing and healthcare-associated pneumonia. the japanese respiratory society the committee for the japanese respiratory society guidelines for the management of respiratory tract infection. the guidelines for the management of hospital-acquired pneumonia in adults. the japanese respiratory society nationwide surveillance of bacterial respiratory pathogens conducted by the surveillance committee of japanese society of chemotherapy, japanese association for infectious diseases, and japanese society for clinical microbiology in 2009: general view of the pathogens' antibacterial susceptibility prospective multi center study of the causative organisms of communityacquired pneumonia in adults in japan clinical evaluation of levofloxacin versus oral b-lactams for acute exacerbation of copd fluoroquinolone exposure prior to tuberculosis diagnosis is associated with an increased risk of death a worldwide perspective of atypical pathogens in community-acquired pneumonia infectious diseases to be focused on mycoplasma pneumoniae combination anti-biotic therapy with macrorides improves survival in intubated patients with community-acquired pneumonia antibiotic therapy for klebsiella pneumoniae bacteremia: implications of production of extended-spectrum b-lactamases dolin r, editors. mandell, douglas, and bennett's principles and practice of infectious diseases the study group on antimicrobial susceptibility of pathogens isolated from respiratory infections. antimicrobial susceptibility of pathogens isolated from more than 10000 patients with infectious respiratory diseases: a 25-year longitudinal study performance standards for antimicrobial susceptibility testing. twenty-third informational supplement. clsi resistance to penicillin and cephalosporin and mortality from severe pneumococcal pneumonia in rationale for revised penicillin susceptibility breakpoints versus streptococcus pneumoniae: coping with anti microbial susceptibility in an era of resistance bts guidelines for the management of community acquired pneumonia in adults: 2009 update cap 5 moxifloxacin study group. oral moxifloxacin vs high-dosage amoxicillin in the treatment of mild-to-moderate, community-acquired, suspected pneumococcal pneumonia in adults spafloxacin resistance in clinical isolates of streptococcus pneumoniae: involvement of multiple mutations in gyra and parc genes nationwide surveillance of bacterial respiratory pathogens condected by japanese society of chemotherapy in 2008: general view of the pathogens' antibacterial susceptibility molecular characteristics of extended-spectrum b-lactamase-producing escherichia coli in japan: emergence of ctx-m-15-producing e. coli st131 macrolide-resistant mycoplasma pneumoniae: characteristics of isolates and clinical aspects of communityacquired pneumonia macrolide-resistant mycoplasma pneumoniae in adolescents with communityacquired pneumonia rapid effectiveness of minocycline or doxycycline against macrolide-resistant mycoplasma pneumoniae infection in a 2011 outbreak among japanese children in vitro and in vivo antibacterial activities of dc-159a, a new fluoroquinolone in vitro activities of 11 antimicrobial agents against macrolide-resistant mycoplasma pneumoniae isolates from pediatric patients: results from a multicenter surveillance study antimicrobial chemotherapy for legionnaires disease: levofloxacin versus macrolides macrolides versus quinolones in legionella pneumonia: results from the community-acquired pneumonia organization international study community-acquired legionella pneumonia in the intensive care unit: impact on survival of combined anti biotic therapy infection due to legionella species other than l. pneumophila in vitro activities of azithromycin, clarithromycin, and other antibiotics against chlamydia pneumoniae dissemination of panton-valentine leukocidine positive metchicillinresistant staphylococcus aureus in okinawa comparative effectiveness of nafcillin or cefazolin versus vancomycin in metchicillin susceptible staphylococcus aureus bacteremia the streptococcus milleri group as a cause of pulmonary infections review of 17 cases of pneumonia caused by streptococcus pyogenes distribution of emm type and antibiotic susceptibility of group a streptococci causing invasive and non invasive diseases in vitro activities of sitafloxacin compared with several fluoroquinolones against streptococcus anginosus and streptococcus constellatus moraxella catarrhalis: from emerging to established pathogen antimicrobial susceptibility of 800 anaerobic isolates from patients with dentoalveolar infection to 13 oral antibiotics community-acquired pneumonia due to gram-negative bacteria and pseudomonas aeruginosa: incidence, risk, and prognosis the committee for the japanese respiratory society guidelines for the management of respiratory tract infection. the guidelines for the management of hospital-acquired pneumonia in adults. tokyo: the japanese respiratory society infectious diseases society of america. guidelines for the management of adults with hospital-acquired, ventilator-associated, and healthcare-associated pneumonia guidelines for the management of hospital-acquired pneumonia in the uk: report of the working party on hospital-acquired pneumonia of the british society for antimicrobial chemotherapy hap study group: multicenter survey on hospital-acquired pneumonia and the clinical efficacy of first-line antibiotics in japan international conference for the development of consensus on the diagnosis and treatment of ventilator-associated pneumonia ventilator-associated pneumonia short-course empiric antibiotic therapy for patients with pulmonary infiltrates in the intensive care unit. a proposed solution for indiscriminate antibiotic prescription comparison of 8 vs 15 days of antibiotic therapy for ventilator-associated pneumonia in adults: a randomized trial nosocomial pneumonia: a multivariate analysis of risk and prognosis study group of the italian society of internal medicine. outcomes of patients hospitalized with community-acquired, health care-associated and hospital-acquired pneumonia implementation of guidelines for management of possible multidrugresistant pneumonia in intensive care: an observational, multicentre cohort study diagnosing pneumonia during mechanical ventilation: the clinical pulmonary infection score revisited value of gram stain examination of lower respiratory tract secretions for early diagnosis of nosocomial pneumonia evaluation of bronchoscopic techniques for the diagnosis of nosocomial pneumonia invasive and noninvasive strategies for management of suspected ventilatorassociated pneumonia: a randomized trial hospital-acquired pneumonia in the 21st century: a review of existing treatment options and their impact on patient care role of serial routine microbio-logic culture results in the initial management of ventilator-associated pneumonia pneumonia essentials clinical practice guidelines for hospital-acquired pneumonia and ventilatorassociated pneumonia in adults the committee for the japanese society of chemotherapy guidelines for tdm of antimicrobial agents. guidelines for tdm of antimicrobial agents the role of an aerobes in patients with ventilatorassociated pneumonia and aspiration pneumonia: a prospective study mandell, douglas, and bennett's principles and practice of infectious diseases physiologic factors contributing to a transition in oral immunity among mechanically ventilated adults indigenous microflora and innate immunity of the head and neck antimicrobial treatment of hospital-acquired pneumonia nosocomial fever of unknown origin. fever of unknown origin. ny: informa health-care b-lactam resistance in the 21st century updated functional classification of b-lactamases. antimicrob mrsa working party of he british society for antimicrobial chemotherapy. guidelines (2008) for the prophylaxis and treatment of methicillin-resistant staphylococcus aureus (mrsa) infections in the united kingdom clinical practice guidelines by the infectious diseases society of america for the treatment of methicillin-resistant staphylococcus aureus infections in adults and children linezolid versus vancomycin for the treatment of infections caused by methicillin-resistant staphylococcus aureus in japan response to emerging infection leading to outbreak of linezolid-resistant enterococci nosocomial spread of enterococcus faecium resistant to vancomycin and linezolid in a tertiary medical center emergence of linezolid resistance in the vancomycin-resistant enterococcus faecium multilocus sequence typing c1 epidemic lineage clinical microbiology of coryneform bacteria linezolid vs vancomycin: analysis of two double-blind studies of patients with methicillin-resistant staphylococcus aureus nosocomial pneumonia the linezolid nosocomial pneumonia study group. linezolid (pnu-100766) versus vancomycin in the treatment of hospitalized patients with nosocomial pneumonia: a randomized, double-blind, multi center study guidelines for uk practice for the diagnosis and management of methicillin-resistant staphylococcus aureus (mrsa) infections presenting in the community oral antibiotic therapy of serious systemic infections daptomycin versus standard therapy for bacteremia and endocarditis caused by staphylococcus aureus treatment of severe pneumonia in hospitalized patients: results of a multicenter, randomized, double-blind trial comparing intra-venous ciprofloxacin with imipenem-cilastatin ventilator-associated pneumonia caused by potentially drug-resistant bacteria acinetobacter infection acinetobacter baumannii: emergence of a successful pathogen alvarez-beltr an m. levofloxacin in the treatment of ventilator-associated pneumonia performance standards for antimicrobial susceptibility testing; twentieth informational supplement. m100es20. wayne pa: clinical and laboratory standards institute the committee for the japanese respiratory society guidelines for the management of nursing and healthcare associated pneumonia. the japanese respiratory society guidelines for the management of nursing and healthcare associated pneumonia. tokyo: the japanese respiratory society health-care-associated pneumonia among hospitalizes patients in a japanese community hospital a prospective comparison of nursing homeacquired pneumonia with health care associated pneumonia in nonintubated elderly characteristics and disease severity of healthcare-associated pneumonia among patients in a hospital in kitakyushu health care-associated pneumonia requiring hospital admission: epidemiology, antibiotic therapy, and clinical outcomes epidemiology and outcomes of healthcare-associated pneumonia: results from a large us database of culture-positive pneumonia what is health-care associated pneumonia, and how should it be treated? indicators of potentially drug-resistant bacteria in severe nursing home acquired pneumonia community-acquired pneumonia through enterobacteriaceae and pseudomonas aeruginosa: diagnosis, incidence and predictors the hcap gap: differences between self-reported practice patterns and published guidelines for health care-associated pneumonia how good is the evidence for the recommended empirical antimicrobial treatment of patients hospitalized because of community-acquired pneumonia? a systematic review macroride-based regimens and mortality in hospitalized patients with communityacquired pneumonia: a systematic review and meta-analysis respiratory fluoroquinolones for the treatment of community-acquired pneumonia: a meta-analysis of randomized controlled trials combination antibiotic therapy lowers mortality among severely ill patients with pneumococcal bacteremia risk factors and response to antibiotic therapy in adults with bacteremic pneumonia caused by penicillin-resistant pneumococci a comparative study of levofloxacin and ceftriaxone in the treatment of hospitalized patients with pneumonia effect of a clinical pathway to reduce hospitalizations in nursing home residents with pneumonia: a randomized controlled trial randomized controlled trial of sequential intravenous (i.v.) and oral moxifloxacin compared with sequential i.v. and oral co-amoxiclav with or without clarithromycin in patients with community-acquired pneumonia requiring initial parenteral treatment moxifloxacin monotherapy is effective in hospitalized patients with community-acquired pneumonia: the motiv studyda randomized clinical trial azithromycin vs cefuroxime plus erythromycin for empirical treatment of communityacquired pneumonia in hospitalized patients: a prospective, randomized, multicenter trial meta-analysis of randomized controlled trials on the comparative efficacy and safety of azithromycin against other antibiotics for lower respiratory tract infections efficacy of azithromycin in the treatment of community-acquired pneumonia, including patients with macrolide-resistant streptococcus pneumoniae infection in vitro antibacterial activity of dx-619, a novel des-fluoro (6) quinolone management of patients with community-acquired pneumonia treated in hospital in sweden a comparison of culture-positive and culture-negative health-care-associated pneumonia effect of antibiotic guidelines on outcomes of hospitalized patients with nursing home-acquired pneumonia health-care-associated pneumonia is primarily due to aspiration pneumonia ertapenem versus cefepime for initial empirical treatment of pneumonia acquired in skilled-care facilities or in hospitals outside the intensive care unit selectivity of ertapnem for psudomonas aeruginosa mutants cross-resistant to other carbapenems retrospective investigation of the clinical effects of tazobactam/piperacillin and sulbactam/ampicillin on aspiration pneumonia caused by klebsiella pneumoniae japanese society of chemotherapy and journal of japanese association for anaerobic infection research. guidelines for treatment of anaerobic infections prospective randomized comparison study of piperacillin/tazobactam and meropenem for health care-associated pneumonia in japan clinical evaluation of bedridden patients with pneumonia receiving home health care national survey on the susceptibility of bacteroides fragilis group: report and analysis of trends for 1997d2000 examination of anaerobes in patients with respiratory infectious diseases using the transtracheal aspiration method impact of initial antibiotic choice on clinical outcomes in community-acquired pneumonia: analysis of a hospital claims-made database management of community-acquired pneumonia macrolide antibiotics and survival in patients with acute lung injury aspiration pneumonitis and aspiration pneumonia the committee for the japanese respiratory society guidelines for the management of respiratory tract infection. the guidelines for the management of hospital-acquired pneumonia in adults. tokyo: the japanese respiratory society japanese study group on aspiration pulmonary disease. diagnosis and treatment for aspiration pulmonary disease epidemiology of community-acquired pneumonia in the elderly risk factors for pneumonia and other lower respiratory tract infection in elderly residents of long term care facilities pneumonia in residents of long-term care facilities: epidemiology, management, and prevention hospitalized nursing home-acquired pneumonia: comparison with community-acquired pneumonia in older adults high incidence of aspiration pneumonia in community-and hospital-acquired pneumonia in hospitalized patients: a multi-center, prospective study in japan rethinking the concepts of communityacquired and health-care-associated pneumonia effectiveness of a multi dimensional approach for prevention of ventilator-associated pneumonia in adult intensive care units from 14 developing countries of four continents: findings of the international nosocomial infection control consortium the sanford guide to antimicrobial therapy 2011 pathogenic bacteria in aspiration pneumonia microbiology of severe aspiration pneumonia in institutionalized elderly ampicillin þ sulbactam vs clindamysin ± cephalosporin for the treatment of aspiration pneumonia and primary lung abscess tazobactam/piperacillin for moderate-to-severe pneumonia in patients with risk for aspiration: comparison with imipenem/cilastatin indicators of potentially drug-resistant bacteria in severe nursing home-acquired pneumonia changing bacteriology of adult community-acquired lung abscess in taiwan: klebsiella pneumoniae versus anaerobes survey regarding antimicrobial drug therapy for aspiration pneumonia treatment of aspiration pneumonia based on the antimicrobial susceptibility pattern of oral bacteria pathogens reappraisal of clindamycin iv monotherapy for treatment of mild-tomoderate aspiration pneumonia in elderly patients regional variation in the prevalence of extended-spectrum b-lactamase-producing clinical isolates in the asia-pacific region (sentry 1998d2002) special issue, current topics on urological infectious diseases---refractory factors and management---, 2 resistance of bacteria isolated from patients with urinary tract infection to antimicrobial drugs and methods to prevent drug resistance aspiration pneumonia in adults uptodate, topic 7024 version 9.0. this topic last updated anaerobic bacterial infection of the lung evaluation of the efficacy and safety of biapenem against pneumonia in the elderly and a study on its pharmacokinetics treatment of ventilator-associated pneumonia with piperacillin-tazobactam/amikacin versus ceftazidime/amikacin: a multicenter, randomized controlled trial prospective randomized comparison of imipenem-cilastatin and piperacillin-tazobactam in nosocomial pneumonia or peritonitis ventilatorassociated pneumonia due to pseudomonas aeruginosa preemptive antibiotic treatment based on gram staining reduced the incidence of ards in mechanically ventilated patients ventilator-associated tracheobronchitis: the impact of targeted antibiotic therapy on patient outcomes antimicrobial treatment for ventilator-associated tracheobronchitis: a randomized, controlled, multicenter study imaging findings in acute invasive pulmonary aspergillosis: clinical significance of the halo sign voriconazole versus amphotericin b for primary therapy of invasive aspergillosis liposomal amphotericin b as initial therapy for invasive mold infection: a randomized trial comparing a high-loading dose regimen with standard dosing (ambiload trial) caspofungin use in daily clinical practice for treatment of invasive aspergillosis: results of a prospective observational registry micafungin (fk463), alone or in combination with other systemic antifungal agents, for the treatment of acute invasive aspergillosis intravenous itraconazole followed by oral itraconazole in the treatment of invasive pulmonary aspergillosis in patients with hematologic malignancies, chronic granulomatous disease, or aids combination of voriconazole and caspofungin as primary therapy for invasive aspergillosis in solid organ transplant recipients: a prospective, multicenter, observational study combination antifungal therapy for invasive aspergillosis liposomal amphotericin b in combination with caspofungin for invasive aspergillosis in patients with hematologic malignancies. a randomized pilot study triazole-polyene antagonism in experimental invasive pulmonary aspergillosis: in vitro and in vivo correlation intravenous micafungin versus voriconazole for chronic pulmonary aspergillosis: a multicenter trial in japan a doubleblind comparative study of the safety and efficacy of caspofungin versus micafungin in the treatment of candidiasis and aspergillosis aspergilloma: a series of 89 surgical cases fluconazole monotherapy for cryptococcosis in non-aids patients cryptococcosis in human immunodeficiency virus-negative patients in the era of effective azole therapy primary treatment of zygomycosis with liposomal amphotericin b: analysis of 28 cases the deferasirox-ambisome therapy for mucormycosis (defeat mucor) study: a randomized, double-blinded, placebo-controlled trial pneumocystis pneumonia the national institute of health-university of california expert panel for corticosteroids as adjunctive therapy for pneumocystis pneumonia. consensus statement on the use of corticosteroids as adjunctive therapy for pneumocystis pneumonia in the acquired immunodeficiency syndrome comparison of atovaquone (566c80) with trimethoprim-sulfamethoxazole to treat pneumocystis carinii pneumonia in patients with aids international consensus guidelines on the management of cytomegalovirus in solid organ transplantation oral valganciclovir is noninferior to intravenous ganciclovir for the treatment of cytomegalovirus disease in solid organ transplant recipients forscarnet vs ganciclovir for cytomegalovirus (cmv) antigenemia after allogeneic hemopoietic stem cell transplantation (hsct): a randomised study treatment of interstitial pneumonitis due to cytomegalovirus with ganciclovir and intravenous immune globulin: experience of european bone marrow transplant group committee to prepare the guidelines for the management of respiratory infectious diseases in children. pneumonia. the guidelines for the management of respiratory infectious diseases in children in japan limitations of the etiological diagnosis of bronchopulmonary infectious diseases---appearance of emb community-acquired pneumonia in children fifth pediatric respiration seminar. review of chest x-ray findings of pneumonia, childhood pneumonia with respect to 3 causative pathogens world health organization: programme for the control of acute respiratory infections: technical bases for the who recommendations on the management of pneumonia in children at first-level health facilities pneumonia in pediatric outpatients: cause and clinical manifestations etiology of pediatric inpatients with pneumoniaeanalysis of clinical symptoms, physical examination and simple laboratory findings basic examination regarding "the guidelines for the management of pneumonia in children", selection of treatment differential diagnosis of viral, mycoplasmal and bacteraemic pneumococcal pneumonias on admission to hospital current status of pneumonia in children, review of chest x-ray findings problems associated with high prevalence of multidrug-resistant bacteria in patients with community-acquired infections nationwide survey of the development of drug-resistant pathogens in the pediatric field: drug sensitivity of streptococcus pneumonia in japan nationwide survey of the development of drug-resistance in the pediatric field: drug sensitivity of haemophilus influenzae in japan macrolide-resistant mycoplasma pneumoniae: characteristics of isolates and clinical aspects of communityacquired pneumonia 18th informational supplement. performance standards for antimicrobial susceptibility testing annual change of ampicillin susceptibility of h. influenzae and the clinical efficacy of penicillins in bronchopulmonary infections caused by b-lactamase negative h. influenzae with an ampicillin-minimum inhibitory concentration (mic) of 2 mg/ml clinical effects of piperacillin and tazobactam/piperacillin on haemophilus influenzae lower respiratory tract infection in pediatric patients clinical studies on lower respiratory tract infections caused by moraxella catarrhalis in childhood (1st report) clinical studies on lower respiratory tract infections caused by moraxella catarrhalis in childhood (2nd report)-indirect pathogenicity of moraxella catarrhalis increased prevalence of penicillin-resistant viridans group streptococci in japanese children with upper respiratory infection treated by beta-lactam agents and in those with oncohematologic diseases strong macrolide resistance in children/ pandemic of mycoplasma pneumoniae infection macrolide resistance of mycoplasma pneumoniae in primary hospitals committee to prepare the guidelines for the management of respiratory infectious diseases in children, vaccination/infectious disease control committee of the japan pediatric society. supplement revision (2013) of the guidelines for the management of respiratory infectious diseases in children in 2011 regarding the diagnosis and treatment of pneumonia with mycoplasma pneumoniae in children ventilator-associated pneumonia in neonatal and pediatric intensive care unit patients committee to prepare the guidelines for the management of respiratory infectious diseases in children. pneumonia in the presence of an underlying disease 1. blood disease. guidelines for the management of respiratory infectious diseases in children committee to prepare the guidelines for the management of respiratory infectious diseases in children. pneumonia in the presence of an underlying disease 2. immunodeficiency. guidelines for the management of respiratory infectious diseases in children clinical practice guideline for the use of antimicrobial agents in neutropenic patients with cancer: 2010 update by the infectious diseases society of america indicative therapy during blood disease treatment experience on long-term therapy with macrolides in the field of pediatrics refractory respiratory infectious diseases. tokyo: van medical co itraconazole to prevent fungal infections in chronic granulomatous disease special issue---respiratory infectious disease---neonatal pneumonia committee to prepare the guidelines for the management of respiratory infectious diseases in children. pneumonia in the presence of an underlying disease 2. neonates. guidelines for the management of respiratory infectious diseases in children nosocomial infection in a newborn intensive care unit (nicu), south korea a 10-year prospective surveillance of nosocomial infections in neonatal intensive care units risk factors and outcomes for ventilatorassociated pneumonia in neonatal intensive care unit patients usefulness of gram staining of tracheal aspirates in initial therapy for ventilatorassociated pneumonia in extremely preterm neonates childhood infectious diseases-i. review of childhood infectious diseases with respect to the type of infection, 13. neonatal infectious diseases chapter 2. various infectious diseases and how to select/use drugs, neonatal period, neonatal infectious diseases neonatal care/basic knowledge v. management method, use of antimicrobial drugs antimicrobial/antiviral drugs for severe infectious diseases iii. clues to diagnosis and selection of drugs in patients with various infectious diseases/conditions, infectious diseases in neonates medical and surgical treatment of parapneumonic effusions: an evidence-based guideline parapneumonic effusions and empyema aerobic and anaerobic microbiology of empyema. a retrospective review in two military hospitals a retrospective review of cases of anaerobic empyema and update of bacteriology anaerobic microbiology in 198 cases of pleural empyema: a bulgarian study thoracic empyema in patients with community-acquired pneumonia controlled trial of intrapleural streptokinase for pleural infection antibiotic therapy of pleural empyema pharmacokinetics and therapeutic efficacy of gentamicin in an experimental pleural empyema rabbit model amikacin concentrations in uninfected post thoracotomy pleural fluid and in serum after intravenous and intrapleural injection penetration of aminoglycosides in uninfected pleural exudates and in pleural empyemas choice of first intervention is related to outcomes in the management of empyema; discussion 30d1 management of pleural infection in adults: british thoracic society pleural disease guideline 2010 committee to prepare the guidelines for the management of respiratory infectious diseases in children. pleuritis/pyothorax. guidelines for the management of respiratory infectious diseases in children pleural tuberculosis radiological study of mycoplasma pneumonia in children principle and practice of pediatric surgery committee to prepare the guidelines for the management of respiratory infectious diseases in children. primary diseases for which vaccination should be performed 5. tuberculosis. guidelines for the management of respiratory infectious diseases in children controlled clinical trial of four short-course (6-month) regimens of chemotherapy for treatment of pulmonary tuberculosis controlled clinical trial of four short-course (6-month) regimens of chemotherapy for treatment of pulmonary tuberculosis controlled trial of 2, 4, and 6 months of pyrazinamide in 6-month, three-times-weekly regimens for smear-positive pulmonary tuberculosis, including an assessment of a combined preparation of isoniazid, rifampin, and pyrazinamide. results at 30 months a controlled trial of 6 months' chemotherapy in pulmonary tuberculosis. final report: results during the 36 months after the end of chemotherapy and beyond short-course antituberculous chemotherapy for pulmonary and pleural disease: 5 years' experience in clinical practice joint tuberculosis committee of the british thoracic society. chemotherapy and management of tuberculosis in the united kingdom: recommendations 1998 infectious diseases society of america: treatment of tuberculosis the japanese society for tuberculosis. guidelines for the management of tuberculosis rifapentine and isoniazid once a week versus rifampicin and isoniazid twice a week for treatment of drug-susceptible pulmonary tuberculosis in hiv-negative patients: a randomised clinical trial usphs tuberculosis short-course chemotherapy trial 21:effectiveness, toxicity, and acceptability. the report of final results controlled chemoprophylaxis trials in tuberculosis. a general review prophylactic effect of isoniazid in young tuberculin reactors isoniazid chemoprophylaxis of tuberculosis international union against tuberculosis committee on prophylaxis. efficacy of various durations of isoniazid preventive therapy for tuberculosis: five years of follow-up in the iuat trial clinical practice. latent tuberculosis infection how much isoniazid is needed for prevention of tuberculosis among immune competent adults? this statement was endorsed by the council of the infectious diseases society of america. (idsa) madras/british medical research council. a double-blind placebo-controlled clinical trial of three antituberculosis chemoprophylaxis regimens in patients with silicosis in hong kong rifampin preventive therapy for tuberculosis infection: experience with 157 adolescents adverse events with 4 months of rifampin therapy or 9 months of isoniazid therapy for latent tuberculosis infection: a randomized trial the research committee of the british thoracic society. pulmonary disease caused by mycobacterium avium-intracellulare in hiv-negative patients: five-year follow-up of patients receiving standardized treatment clarithromycin regimens for pulmonary mycobacterium avium complex. the first 50 patients effect of clarithromycin regimen for mycobacterium avium complex pulmonary disease clarithromycin in the treatment of mycobacterium avium lung infections in patients without aids initial clarithromycin monotherapy for mycobacterium avium-intracellulare complex lung disease clinical and molecular analysis of macrolide resistance in mycobacterium avium complex lung disease azithromycin activity against mycobacterium avium complex lung disease in patients who were not infected with human immunodeficiency virus two controlled trials of rifabutin prophylaxis against mycobacterium avium complex infection in aids infectious disease society of america. an official ats/idsa statement: diagnosis, treatment, and prevention of nontuberculous mycobacterial diseases a double-blind randomized study of aminoglycoside infusion with combined therapy for pulmonary mycobacterium avium complex disease sub committee of the joint tuberculosis committee of the british thoracic society. management of opportunist mycobacterial infections: joint tuberculosis committee guidelines 1999 the scientific assembly for infection and tuberculosis of the japanese respiratory society. guidelines for chemotherapy of pulmonary nontuberculous mycobacterial disease e 2012 revised version this official statement of the american thoracic society was approved by the board of directors treatment of pulmonary disease due to mycobacterium kansasii: recent experience with rifampin detection of rpob mutations in rifampicin-resistant mycobacterium kansasii treatment of pulmonary disease caused by mycobacterium kansasii: results of 18 vs 12 months' chemotherapy clinical features of pulmonary disease caused by rapidly growing mycobacteria. an analysis of 154 patients clinical and taxonomic status of pathogenic nonpigmented or late-pigmenting rapidly growing mycobacteria pulmonary disease caused by rapidly growing mycobacteria mycobacterium abscessus from respiratory isolates: activities of drug combinations antibiotic treatment of mycobacterium abscessus lung disease: a retrospective analysis of 65 patients clinical and microbiologic outcomes in patients receiving treatment for mycobacterium abscessus pulmonary disease supervised by the tuberculosis and infectious diseases control division, health service bureau, ministry of health, labour and welfare. statistics of tuberculosis in 2009 special issue---new vaccination system of bcg-for the prevention of tuberculosis based on scientific grounds usefulness of interferon-gamma release assays for diagnosing tb infection and problems with these assays subsidy for scientific research by the ministry of health, labour and welfare in 2009, research business for new/old infectious diseases such as new influenza "evaluation of tuberculosis control strategies and study regarding the development/application of new diagnostic/treatment techniques japanese new guidelines for nontuberculous mycobacterial pulmonary disease clinical practice. acute bronchitis uncomplicated acute bronchitis principles of appropriate antibiotic use for treatment of uncomplicated acute bronchitis: background chronic cough due to acute bronchitis: accp evidence-based clinical practice guidelines clinical study of acute bacterial bronchitis health burden of pertussis in adolescents and adults recommended antimicrobial agents for the treatment and post-exposure prophylaxis of pertussis: 2005 cdc guidelines antibiotics for whooping cough (pertussis). cochrane database syst rev increased macrolide resistance of mycoplasma pneumoniae in pediatric patients with community-acquired pneumonia antivirals for treatment of influenza: a systematic review and meta-analysis of observational studies the committee for the japanese respiratory society guidelines for the management of respiratory tract infection. the guidelines for the management of respiratory tract infection. basic concept of respiratory tract infection in adults. tokyo: the japanese respiratory society antibiotic prescribing for self limiting respiratory tract infections in primary care: summary of nice guidance antibiotic therapy in exacerbation of chronic obstructive pulmonary disease characterisation of exacerbations of chronic bronchitis and copd in europe: the giant study indicators of bacterial infection in patients with acute exacerbation of chronic bronchitis for application in clinical trials of anti bacterial drugs infection in the pathogenesis and course of chronic obstructive pulmonary disease a multicentre surveillance study on the characteristics, bacterial aetiologies and in vitro antibiotic susceptibilities in patients with acute exacerbations of chronic bronchitis clinical response of levofloxacine 500 mg qd to respiratory tract infection open study of sitafloxacin in patients with respiratory tract infections e pk/pd study efficacy and safety of moxifloxacin in acute exacerbations of chronic bronchitis: a prospective, multicenter, observational study (avanti) five-day moxifloxacin therapy compared with 7-day co-amoxiclav therapy for the treatment of acute exacerbation of chronic bronchitis levofloxacin 750 mg qd for five days versus amoxicillin/clavulanate 875 mg/125 mg bid for ten days for treatment of acute bacterial exacerbation of chronic bronchitis: a post hoc analysis of data from severely ill patients shortcourse antibiotic treatment in acute exacerbations of chronic bronchitis and copd: a meta-analysis of double-blind studies shortversus long-duration antimicrobial treatment for exacerbations of chronic bronchitis: a meta-analysis sputum colour and bacteria in chronic bronchitis exacerbations: a pooled analysis antibiotics are associated with lower relapse rates in outpatients with acute exacerbations of copd in vitro antibacterial activity of garenoxacin molecular characterization of quinolone-insensitive streptococcus pneumonia isolates from japanese patients comparative mutant prevention concentration and mutant selection window of sitafloxacin versus other quinolones using strains of haemophilus influenzae with decreasing susceptibility to levofloxacin randomized, double-blind study comparing 5-and 7-day regimens of oral levofloxacin in patients with acute exacerbation of chronic bronchitis the efficacy of cefditoren pivoxil in the treatment of lower respiratory tract infections, with a focus on the per-pathogen bacteriologic response in infections caused by streptococcus pneumoniae and haemophilus influenzae: a pooled analysis of seven clinical trials role of oral extended-spectrum cephems in the treatment of acute exacerbation of chronic bronchitis efficacy and safety of 3-day azithromycin versus 5-day moxifloxacin for the treatment of acute bacterial exacerbations of chronic bronchitis final draft report of the treatment guideline of dpb. diffuse lung disease study group, scientific research specific disease study business by the ministry of health, labour and welfare. study report in 1999 macrolides for diffuse panbronchiolitis study regarding the clinical efficacy of long-term therapy with low-dose erythromycin for diffuse panbronchiolitis long-term therapeutic effects of erythromycin and newquinolone antibacterial agents on diffuse panbronchiolitis longterm low-dose administration of erythromycin to patients with diffuse panbronchiolitis report by the diffuse lung disease survey/study group of the ministry of health and welfare in 1990 diffuse panbronchiolitis: followup ct examination effects of long-term erythromycin therapy on diffuse panbronchiolitis leading to chronic respiratory failure long term effect of erythromycin therapy in patients with chronic pseudomonas aeruginosa infection improvement of survival in patients with diffuse panbronchiolitis treated with low-dose erythromycin diffuse panbronchiolitis: role of macrolides in therapy efficacy of lomg-term macrolide antibiotic therapy in patients with diffuse panbronchiolitis: comparison between hla-b54-positive and negative cases long-term macrolide antibiotic therapy in the treatment of chronic small airway disease clinically mimicking diffuse panbronchiolitis analysis of cases allowed to cease erythromycin therapy for diffuse panbronchiolitisecomparative study between patients with cessation of the therapy and patients continuing the therapy long-term efficacy and safety of clarithromycin treatment in patients with diffuse panbronchiolitis roxithromycin treatment in patients with chronic lower respiratory tract diseaseeits clinical efficacy and effect on cytokine clinical and immune regulatory effects of roxithromycin therapy for chronic respiratory tract infection study on azithromycin in treatment of diffuse panbronchiolitis effects of long-term low-dose azithromycin in patients with non-cf bronchiectasis effect of long-term, low-dose erythromycin on pulmonary exacerbations among patients with non-cystic fibrosis bronchiectasis: the bless randomized controlled trial effect of azithromycin maintenance treatment on infectious exacerbations among patients with non-cystic fibrosis bronchiectasis: the bat randomized controlled trial inhibitory effects of macrolide antibiotics on exacerbations and hospitalization in chronic obstructive pulmonary disease in japan: a retrospective multicenter analysis azithromycin for prevention of exacerbations of copd the guidelines for the management of respiratory tract infection" basic concept of respiratory tract infection management in adults acute inflammatory upper airway obstruction (croup, epiglottitis, laryngitis, and bacterial tracheitis) croup (laryngitis, laryngotracheitis, spasmodic croup, laryngotracheobronchitis, bacterial tracheitis) croup: an 11-year study in a pediatric practice croup hospitalizations in ontario: a 14-year time-series analysis a current perspective respiratory viruses in laryngeal croup of young children croup: an overview croup-assessment and management wheezing, bronchiolitis, and bronchitis feigin and cherry's textbook of pediatric infectious diseases bronchiolitis-associated hospitalizations among us children prospective multicenter study of the viral etiology of bronchiolitis in the emergency department antibiotic treatment of epidemic bronchiolitisea double-blind trial azithromycin does not improve disease course in hospitalized infants with respiratory syncytial virus (rsv) lower respiratory tract disease: a randomized equivalence trial management of bronchiolitis without antibiotics: a multicentre randomized control trial in bangladesh clarithromycin in the treatment of rsv bronchiolitis: a double-blind, randomised, placebo-controlled trial risk of secondary bacterial infection in infants hospitalized with respiratory syncytial viral infection bacterial tracheitis in children: approach to diagnosis and treatment branhamella catarrhalis as a cause of bacterial tracheitis bacterial tracheitis: report of eight new cases and review is bacterial tracheitis changing? a 14-month experience in a pediatric intensive care unit feigin and cherry's textbook of pediatric infectious diseases antibiotics for acute bronchitis the value of antibiotics in minor respiratory illness in children. a controlled trial amoxycillin and cotrimoxazole in presumed viral respiratory infections of childhood: placebo-controlled trial cough illness/bronchitisdprinciples of judicious use of antimicrobial agents high levels of adamantane resistance among a (h3n2) viruses and interim guidelines for use of anti-viral agentseunited states, 2005d06 influenza season update: drug susceptibility of swineorigin influenza a (h1n1) viruses accuracy of rapid influenza diagnostic tests. a meta-analysis critically ill patients with 2009 influenza a (h1n1) in mexico enteric absorption and pharmaco-kinetics of oseltamivir in critically ill patients with pandemic (h1n1) influenza antivials for treatment of influenza. a systemic review and meta-analysis of observation studies influenza committee of the japanese association for infectious diseases, proposal by the japanese association for infectious diseases effect of double dose oseltamivir on clinical and virological outcomes in children and adults admitted to hospital with severe influenza: double -blind randomised controlled trial influenza viruses feigin and cherry's textbook of pediatric infectious diseases ameican academy of peiatrics. haemophilus influenza infections a study of illness in a group of clevel and families. xvii. the occurrence of asian influenza clinical characteristics of pediatric hospitalizations associated with 2009 pandemic influenza a (h1n1) in northern bavaria otolaryngological complications in patients infected with the influenza a (h1n1) virus severe influenza cases in paediatric intensive care units in germany during the pre-pandemic seasons critically ill children during the 2009d2010 influenza pandemic in the united states the current status of parasitic diseases in japan tropical lung disease paragonimiasis cases recently found among immigrants in japan clinicoradiologic features of pleuropulmonary paragonimus westermani on kyusyu island efficacy of chemotherapy of paragonimiasis in southern kyushu, japan. in: the 5th asian-pacific congress for parasitic zoonosis a case of chronic pleural empyema by paragonimus westermani infection resistant to chemotherapy and cured by surgical decortication paragonimiasis: experimental and clinical experience with praziquantel in korea parasitic infections of the lung:a guide for the respiratory physician current status of strongyloides infection in okinawa pulmonary strongyloidiasis: the varied clinical presentations non-oral treatment with ivermectin for disseminated strongyloidiasis how common is human toxocariasis? towards standardizing our knowledge toxocariasis in japan lessons from eight cases of adult pulmonary toxocariasis :abridged republication thiabendazole vs. albendazole in treatment of toxocariasis: a clinical trial a case of adult hepatic toxocariasis tropical pulmonary eosinophilia: a case series in a setting of nonendemicity study group regarding the establishment of medical management by appropriate treatment with rare-disease drugs for imported tropical diseases/parasitosis. guidelines for drug therapy for parasitosis. study group regarding drugs for tropical diseases drug to be contraindicated for combination therapy: rfp precautions for combination therapy: decrease in the blood concentration: dexamethasone, phenytoin, carbamazepine, chloroquine increase in the blood concentration: cimetidine ➁ pyrantel pamoate patients can take this drug regardless of meals. there are few side effects. ➂ albendazol this drug should be taken with meals for 28 days. a 14day period of discontinuation should be established and then restarted drug if required.as liver dysfunction is frequently observed, caution is needed during the administration period. bone marrow suppression and stevens-johnson syndrome must also be considered.precautions for combination therapy: decrease in the blood concentration: ritonavir, phenytoin, carbamazepine, phenobarbital increase in the blood concentration: praziquantel ➃ mebendazolealthough this drug has been used in few children, the same dose as established for adults is used. in children weighing 20 kg or less, a half dose should be used. this drug is contraindicated for pregnant women or those who may be pregnant. combination therapy with cimetidine may increase the blood concentration of mebendazole. combination therapy with metronidazole may cause toxic epidermal necrolysis (ten) and stevens-johnson syndrome. after fasting, this drug should be taken with water. the incidence of side effects is low, but nausea/vomiting and mild hepatic disorder are sometimes observed. the safety of this drug in pregnant women or children weighing less than 15 kg has not been established. to these patients, this drug should be administered only when its therapeutic advantage is considered to exceed its risk. ➅ diethylcarbamazine as side effects, fever, lymphangitis/lymphadenitis, and orchitis/epididymitis, which result from anti-parasitic actions, are observed in addition to abdominal discomfort, nausea, and abdominal pain. the safety of this drug in pregnant women has not been established. upper row: dose (mg/kg), lower row: frequency of administration per day key: cord-291961-usl8z6ep authors: zheng, wen-zhi; wei, tian-li; ma, fen-lian; yuan, wu-mei; zhang, qian; zhang, ya-xin; cui, hong; zheng, li-shu title: human polyomavirus type six in respiratory samples from hospitalized children with respiratory tract infections in beijing, china date: 2015-10-13 journal: virol j doi: 10.1186/s12985-015-0390-5 sha: doc_id: 291961 cord_uid: usl8z6ep background: hpyv6 is a novel human polyomavirus (hpyv), and neither its natural history nor its prevalence in human disease is well known. therefore, the epidemiology and phylogenetic status of hpyv6 must be systematically characterized. methods: the vp1 gene of hpyv6 was detected with an established taqman real-time pcr from nasopharyngeal aspirate specimens collected from hospitalized children with respiratory tract infections. the hpyv6-positive specimens were screened for other common respiratory viruses with real-time pcr assays. results: the prevalence of hpyv6 was 1.7 % (15/887), and children ≤ 5 years of age accounted for 80 % (12/15) of cases. all 15 hpyv6-positive patients were coinfected with other respiratory viruses, of which influenza virus a (ifva) (8/15, 53.3 %) and respiratory syncytial virus (7/15, 46.7 %) were most common. all 15 hpyv6-positive patients were diagnosed with lower respiratory tract infections, and their viral loads ranged from 1.38 to 182.42 copies/μl nasopharyngeal aspirate specimen. the most common symptoms were cough (100 %) and fever (86.7 %). the complete 4926-bp genome (bj376 strain, genbank accession number km387421) was amplified and showed 100 % identity to hpyv6 strain 607a. conclusions: the prevalence of hpyv6 was 1.7 % in nasopharyngeal aspirate specimens from hospitalized children with respiratory tract infections, as analyzed by real-time pcr. because the coinfection rate was high and the viral load low, it was not possible to establish a correlation between hpyv6 and respiratory diseases. electronic supplementary material: the online version of this article (doi:10.1186/s12985-015-0390-5) contains supplementary material, which is available to authorized users. the family polyomaviridae contains viruses that are among the smallest known to infect humans, in terms of both their particle sizes and genome lengths. the 40-45 nm nonenveloped icosahedral particles carry a circular doublestranded dna genome of approximately 5000 bp, which is divided into three functional regions: the noncoding control region, the early gene region encoding the large t antigen and the small t antigen, and the late coding region, encoding capsid proteins vp1, vp2, and vp3 [1] . human polyomaviruses (hpyvs) have not been associated with any severe acute disease in healthy humans. however, the viral proteins expressed by polyomaviruses (pyvs) can initiate the transformation and immortalization of cultured cells and cause cancer in experimental animals [2] . the hpyv family currently consists of 13 members, including jcpyv [3] , bkpyv [4] , wupyv [5] , kipyv [6] , mcpyv [7] , hpyv6 [8] , hpyv7 [8] , tspyv [9] , hpyv9 [10] , hpyv10 [11] [12] [13] , stlpyv [14] , hpyv12 [15] and njpyv-2013 [16] . previous studies have indicated that a number of hpyvs are associated with human diseases, such as progressive multifocal leukoencephalopathy (jcpyv), hemorrhagic cystitis (bkpyv), merkel cell carcinoma (mcpyv), and trichodysplasia spinulosa (tspyv) [3, 7, 9, [17] [18] [19] . a serological study demonstrated that the seroprevalence of these viruses in 1501 plasma samples from healthy adult blood donors was bkpyv (82 %), jcpyv (39 %), kipyv (55 %), wupyv (69 %), mcpyv isolate 350 (25 %); and mcpyv isolate 339 (42 %). the seroprevalence of all polyomaviruses in children under 21 years of age (n = 721) was similar to that in the adult population, suggesting that primary exposure to these viruses occurs in early childhood and seems to result in lifelong persistence [20] . nevertheless, the natural histories of most hpyvs and their prevalence in human diseases are not yet well known. in 2008, mcpyv was discovered by feng et al. [7] . hpyv6 and hpyv7 were discovered with mcpyv in skin swabs from the foreheads of healthy volunteers [8] . however, later research could not demonstrate a relationship between hpyv6 and merkel cell carcinoma or other skin diseases. a phylogenetic analysis of the complete hpyv6 genome indicated that hpyv6 shared a branch with kipyv and wupyv. previous reports have shown that genomic fragments of kipyv and wupyv have been regularly detected in nasopharyngeal aspirates of children with respiratory tract infections (rtis) and are suspected of a causal relationship with respiratory disease. however, the link between these pyvs and respiratory diseases remains speculative [5, 6, 21, 22] . a real-time pcr assay was established here to determine the prevalence of hpyv6 throughout a time period of 12 months (from october 2011 to september 2012). the prevalence of hpyv6 was 1.7 % (15/887). a complete hpyv6 genome was amplified, sequenced and found to be identical with a hpyv6 isolate from usa. with the standard curve derived from serial dna dilutions, the dynamic range of the real-time pcr assay was 10 0 -10 10 copies/μl and the limit of detection was one copy. the coefficient of determination (r 2 = 0.99667) showed a good linear correlation. the taqman-based real-time pcr assay to detect hpyv6 did not amplify any other viral pathogen, showing the excellent specificity of this assay. four different dna concentrations (10 5 -10 8 copies per reaction) were repeated five times in each run. the maximum coefficient of variation was 0.66 %, which indicates good precision (data not shown). a total of 887 npa samples were obtained from 887 children with rti. the sex ratio (male:female) was 524:363 (1.44:1) and the median age was 24 months (ranging from 3 days to 14 years). the prevalence of hpyv6 was 1.7 % (15/887) in the 887 npa specimens tested. of the 15 hpyv6-positive patients, eight were male (8/524, 1.53 %) and seven were female (7/363, 1.93 %), so the prevalence was similar in both sexes (p > 0.05). the ages of the infected patients ranged from 4 days to 13 years, and children ≤ 5 years of age accounted for 80 % (12/15) of the total hpyv6-positive children. the age distribution of the hpyv6-infected children indicated that those aged 37-48 months had the highest infection rate table 1) . all 15 hpyv6-positive patients were diagnosed with lower rti, including bronchopneumonia in nine (60 %), acute bronchitis in three (20 %), bronchitis in two (13.3 %), and pneumonia in one (6.7 %). the most common symptom was cough, which occurred in all 15 patients (100 %). other clinical presentations included fever (n = 13, 86.7 %), gasping (n = 3, 20 %), vomiting (n = 3, 20 %), and diarrhea (n = 1, 6.7 %). among the hpyv6-positive specimens, the hpyv6 genome copies ranged from 1.38 to 182.42 copies/μl npa on a realtime pcr assay ( table 2 ). the hpyv6 genome copy number was 52.07 copies/μl npa in children suffering lower rtis only, and was slightly higher than 29.75 copies/μl npa in those infected with lower rtis and other diseases, but these did not differ significantly (p > 0.05, mann-whitney u test). to determine the complete hpyv6 genomic sequence, overlapping genomic fragments were amplified with nested pcr using 15 pairs of virus-specific primers (listed in additional file 1: table s2 ), and the complete 4926-bp genome was compiled with bioedit 9.0 software. the genome sequence of hpyv6 was deposited in genbank under accession number km387421. blast analysis with the complete hpyv6 genome showed a high level of nucleic acid identity to the six full hpyv6 genomes available in genbank. relationsship to the hpyv6 strain 607a was 100 %. hpyv6, thought to be a skin-tropic polyomavirus, was initially described in 2010. since then, subgenomic fragments of hpyv6 dna have been detected in a variety of specimen types, including skin, respiratory secretion samples, and various tumor samples (for instance, the answers to these questions will require more information on the biology and epidemiology of the hpyvs. the genomes and proteins of hpyv6, one of the novel hpyvs, show little sequence homology with previously reported hpyvs (bkpyv and jcpyv). although hpyv6 encodes a conserved, potentially carcinogenic ltag, previous studies have shown no association between hpyv6 and tumors [1] . furthermore, a phylogenetic analysis indicated that ltag of hpyv6 (km387421) is only distantly related to its homologues in other cancerassociated hpyvs. the hpyv6, wupyv, and kipyv strains formed a clade in the complete genome and vp1 amino acid phylogenies, whether hpyv6 also associate with respiratory infection which need more clinical and experimental evidences to support. hpyv6 has been detected in specimens from the human respiratory tract, but there are as yet insufficient epidemiological data to demonstrate a correlation between hpyv6 and respiratory disease. because initial infections with most hpyvs occur in infancy, the prevalence of hpyv6 in npas from children was detected with real-time pcr. hpyv6 displayed an overall prevalence of 1.7 % in npa samples collected from children in a hospital in china, which is similar to its prevalence reported previously (0.5-2 %) [34, 35] . it has not been confirmed that hpyv6 infects humans via the respiratory tract, but the respiratory tract may be a possible route of transmission. in this study, hpyv6 was mainly detected in children less than 5 years of age, and the peak incidence occurred in spring. all 15 hpyv6-positive patients were coinfected with other respiratory viruses, of which ifva and rsv were the most common. the hpyv6-positive patients were diagnosed with lower rtis, 60 % had bronchopneumonia, and the most common symptoms were cough and fever. although known hpyvs cause disease in patients with immune-system imbalances, they do not seem to cause obvious illnesses in the great majority of infected individuals. however, bkpyv can induce nephropathy in kidney transplantation recipients and jcpyv causes progressive multifocal leukoencephalopathy. progressively increasing or high viral loads are also associated with high-level viral replication and disease. for instance, progressive multifocal leukoencephalopathy and hemorrhagic cystitis are related to high viral loads of jcpyv and bkpyv, respectively. the present study cannot confirm that hpyv6 is the cause of rtis in hospitalized children, because the viral loads of hpyv6 were low (1.38-182.42 copies/μl) and the coinfection rate with other respiratory viruses was high. in previous reports, hpyvs were detected in the respiratory tract and skin but, to date, there has been insufficient evidence that hpyv6 is associated with any respiratory tract disease or skin disease [36] . whether hpyv6 induce any disease requires the analysis of further data. the detection rate for hpyv6 by real-time pcr assay was 1.7 % in 887 npa samples collected from hospitalized children with rti. an association between hpyv6 and respiratory diseases could not been revealed due to the high coinfection rate and the low hpyv6 viral load. from 1 october, 2011, to 30 september, 2012, 887 nasopharyngeal aspirate (npa) specimens were collected continuously from 887 hospitalized children with rti, who ranged in age from 3 days to 14 years, at beijing friendship hospital, beijing, china. the patients' parents or guardians gave their written informed consent for specimen collection and testing, and the project was approved by the ethical committee of the beijing friendship hospital. the npa specimens were collected and transported immediately to the national institute for viral disease control and prevention, china cdc, and stored at −80°c until further processing. all demographic data and clinical findings were recorded on a standard form. total nucleic acids were extracted from each npa specimen using the qiaamp minelute virus spin kit (qiagen, beijing, china). a taqman-based real-time pcr assay for the detection of vp1 gene of hpyv6 was designed with primer express 3.0 software. the primer sequences used were hpyv6-f 5'-ttaacacccttctttgtgctgcta-3' and hpyv6-r 5'-gcccaattattcaaagcagctaa-3' , and the probe sequence was hpyv6-p fam-ctgtcacag gcctgctgagcaatagatttc-tamra. the specificities of the primers and probe were evaluated in genbank with blast. the primers and probe were synthesized by invitrogen (beijing, china). a common reaction mix was prepared for the real-time pcr assays. briefly, the final 20 μl reaction mix contained 10 μl of taqman gene expression master mix, 1.8 μl of each primer (10 pmol/μl), 0.2 μl of probe (10 pmol/μl), 2 μl of pmd18-t/vp1 plasmid template (plasmid pmd18-t linked to the vp1 gene of hpyv6), and 4.2 μl of h 2 o. the amplification conditions included an initial incubation at 50°c for 2 min and 95°c for 15 min, followed by 40 cycles of 95°c for 15 s and 60°c for 1 min, using the mx3005p qpcr system (agilent stratagene). ten-fold serial dilutions of the pmd18-t/vp1 plasmid (from 10 0 to 10 10 copies/μl) were added to the real-time pcr reactions in duplicate. the results were used to generate a standard curve for hpyv6. specificity was assessed by testing mixed samples of other common hpyvs, including wupyv, kipyv, jcpyv, bkpyv, and hpyv7. to test the reproducibility of the assay, we added 10 5 -10 8 copies/μl of pmd18-t/vp1 plasmid to each reaction and each concentration of dna was repeated five times. in addition, housekeeping gene glyceraldehyd-3-phosphate dehydrogenase (gapdh) was used as internal control. 2 μl of nucleic acid of each npa specimen was added to each reaction. only samples that were positive according to both pcr and dna sequencing were considered "positive". fifteen overlapping fragments of the complete genome of hpyv6 strain bj376 were pcr amplified (table 2) with the takara ex taq kit. the 15 overlapping fragments were then cloned into pmd18-t and sequenced (invitrogen). the nucleotide sequence of the full-length hpyv6 genome was then compiled using bioedit 9.0 software. the full-length hpyv6 sequence was aligned with the sequences of other hpyvs and other hpyv6 strains available in genbank with dnastar software. a neighbor-joining tree was constructed with mega 6.0. the significance of differences between the prevalence rates and viral loads of various groups was tested with fisher's exact test and the mann-whitney u test. all analyses were performed with spss 19.0 software. genome analysis of the new human polyomaviruses human polyomaviruses in disease and cancer cultivation of papova-like virus from human brain with progressive multifocal leucoencephalopathy new human papovavirus (b.k.) isolated from urine after renal transplantation identification of a novel polyomavirus from patients with acute respiratory tract infections identification of a third human polyomavirus clonal integration of a polyomavirus in human merkel cell carcinoma merkel cell polyomavirus and two previously unknown polyomaviruses are chronically shed from human skin discovery of a new human polyomavirus associated with trichodysplasia spinulosa in an immunocompromized patient a novel human polyomavirus closely related to the african green monkeyderived lymphotropic polyomavirus complete genome sequence of a tenth human polyomavirus discovery of a novel polyomavirus in acute diarrheal samples from children identification of mw polyomavirus, a novel polyomavirus in human stool discovery of stl polyomavirus, a polyomavirus of ancestral recombinant origin that encodes a unique t antigen by alternative splicing identification of a novel human polyomavirus in organs of the gastrointestinal tract identification of a novel polyomavirus in a pancreatic transplant recipient with retinal blindness and vasculitic myopathy association between a high bk virus load in urine samples of patients with graftversus-host disease and development of hemorrhagic cystitis after hematopoietic stem cell transplantation progressive multifocal leukoencephalopathy and promyelocytic leukemia nuclear bodies: a review of clinical, neuropathological, and virological aspects of jc virus-induced demyelinating disease sv40 and human cancer: a review of recent data seroepidemiology of human polyomaviruses presence of the newly discovered human polyomaviruses ki and wu in australian patients with acute respiratory tract infection molecular epidemiology of ki and wu polyomaviruses in infants with acute respiratory disease and in adult hematopoietic stem cell transplant recipients rapid and sensitive method using multiplex real-time pcr for diagnosis of infections by influenza a and influenza b viruses, respiratory syncytial virus, and parainfluenza viruses 1, 2, 3, and 4 development and assay of rna transcripts of enterovirus species a to d, rhinovirus species a to c, and human parechovirus:assessment of assay sensitivity and specificity of real-time screening and typing methods wu and ki polyomaviruses in respiratory samples from allogeneic hematopoietic cell transplant recipients development and evaluation of real-time pcr assays for the detection of the newly identified ki and wu polyomaviruses human bocavirus in patients with respiratory tract infection pring-akerblom p. rapid and quantitative detection of human adenovirus dna by real-time pcr real-time reverse transcriptase pcr assay for detection of human metapneumoviruses from all known genetic lineages design and performance testing of quantitative real time pcr assays for influenza a and b viral load measurement impact of human coronavirus infections in otherwise healthy children who attended an emergency department exploring the prevalence of ten polyomaviruses and two herpes viruses in breast cancer prevalence of human polyomaviruses in common and rare types of non-merkel cell carcinoma skin cancer detection of novel polyomaviruses, tspyv, hpyv6, hpyv7, hpyv9 and mwpyv in feces, urine, blood, respiratory swabs and cerebrospinal fluid human polyomaviruses in children undergoing transplantation no evidence for association of hpyv6 or hpyv7 with different skin cancers submit your next manuscript to biomed central and take full advantage of: • convenient online submission • thorough peer review • no space constraints or color figure charges • immediate publication on acceptance • inclusion in pubmed, cas, scopus and google scholar • research which is freely available for redistribution submit your manuscript at www we thank the beijing friendship hospital, capital medical university, for providing the samples, and the laboratory staff who contributed to this study. no compensation was given to subjects for participation in this study. additional file 1: table s1 . primers and probes used to detect respiratory viruses. table s2 . primers used to amplify hpyv6 with nested pcr (docx 28 kb) the authors declare that they have no competing interests. key: cord-257220-fe2sacjj authors: butler, j. e.; lager, k. m.; golde, william; faaberg, kay s.; sinkora, marek; loving, crystal; zhang, y. i. title: porcine reproductive and respiratory syndrome (prrs): an immune dysregulatory pandemic date: 2014-07-01 journal: immunol res doi: 10.1007/s12026-014-8549-5 sha: doc_id: 257220 cord_uid: fe2sacjj porcine reproductive and respiratory disease syndrome (prrs) is a viral pandemic that especially affects neonates within the “critical window” of immunological development. prrs was recognized in 1987 and within a few years became pandemic causing an estimated yearly $600,000 economic loss in the usa with comparative losses in most other countries. the causative agent is a single-stranded, positive-sense enveloped arterivirus (prrsv) that infects macrophages and plasmacytoid dendritic cells. despite the discovery of prrsv in 1991 and the publication of >2,000 articles, the control of prrs is problematic. despite the large volume of literature on this disease, the cellular and molecular mechanisms describing how prrsv dysregulates the host immune system are poorly understood. we know that prrsv suppresses innate immunity and causes abnormal b cell proliferation and repertoire development, often lymphopenia and thymic atrophy. the prrsv genome is highly diverse, rapidly evolving but amenable to the generation of many mutants and chimeric viruses for experimental studies. prrsv only replicates in swine which adds to the experimental difficulty since no inbred well-defined animal models are available. in this article, we summarize current knowledge and apply it toward developing a series of provocative and testable hypotheses to explain how prrsv immunomodulates the porcine immune system with the goal of adding new perspectives on this disease. north american-like (prototype vr-2332). the virus is a member of the family arteriviridae in the order nidovirales, which includes lactate dehydrogenase-elevating virus of mice (ldv), simian hemorrhagic fever virus (shfv), equine arterivirus (eav) and the recently described wobbly possum disease virus (wpdv) [5, 6] . as the name implies, except for wpdv that appears to be only neurologic, they are associated with some form of vasculitis. the virus can be transmitted across the placenta to infect the fetus [7, 8] despite the fact that the porcine placenta is impermeable to maternal antibodies [9] . prrs is the number one disease problem in major swine producing areas around the world. it is estimated to cost the industry 660 million dollars a year just in the usa with proportional losses recognized in other countries. this is attributed to the remarkable ability of prrsv to: (1) infect swine at all stages of production, (2) be shed in the semen of boars for extended periods of time, (3) be easily transmitted between farms, (4) tolerate a high mutation rate, and (5) negatively modulate the host's immune response. prrs has been a troubling disease because of its persistence and because [20 years of research has failed to produce an efficacious vaccine. this has been somewhat surprising since eav infections are resolved in 7-14 days and a number of efficacious vaccines are available [10] . the rapid resolution of eav is reminiscent of the pattern of sterilizing immunity seen with porcine influenza even in germfree (gf) piglets, so it is not simply a case of neonatal incompetence. rather, prrsv is more similar to ldv in which both the virus and the antibody response persist in mice [11] . as implied by its name, prrs causes two separate pathologies: fetal abortion and respiratory disease in young and older pigs. there is some evidence that prrsv replicates predominately in the thymus, which results in thymic atrophy [8, 12, 13] . this feature separates prrsv from both eav and ldv. while this is especially pronounced with highly pathogenic strains (hp-prrsv) [14, 15] , it is not necessarily the case for all isolates. more than 2,000 papers have been published on prrs, nearly all of which describe studies using conventional animals [1, 2, [16] [17] [18] . most initial studies focused on adaptive immunity, although it is well recognized that viral infection also affects the innate immune system [19] . few studies have focused on immune dysregulation by prrsv, but recent work describes how prrsv can suppress innate immunity (''the innate immune response to prrsv'' section). murtaugh and genzow propose that ''identification of the viral structures that elicit the protective immunity in pigs and factors that modulate the efficacy of protection in vivo is essential to rational development of immunological tools to prevent and control prrs.'' this focus is very important but as general guderian advised hitler in 1942 ''if what you are doing is not working, try something different'' [20] . what is lacking in prrsv research is a greater effort to determine the mechanisms, whereby the virus modulates the porcine immune response. in this review, we describe testable hypotheses to explain how this virus modulates the host immune system. both prrsv and ldv are immune modulatory and although not retroviruses, may have more in common with hiv than eav. ldv elevates igg levels in mice with little production of virus-specific antibodies [11, 21] , which is almost identical to what is seen in isolator piglets infected with prrsv [22] (''the effect of age, rearing, complement and the role of mucosal immunity'' section). polyclonal b cell activation is often associated with autoimmunity and is common to a number of viral infections that are genetically unrelated to the arteriviridae [23] . many viral infections such as bovine viral diarrhea virus [24] interfere with ''normal'' immune processes, which prolong the replication window for the viruses and thus increase the opportunity for contagious spread. thus, virus classification may be a poor predictor of the effect of a virus on the immune system. with rare exception, interference with the immune response is not the cause of death; good parasites rarely kill their host. rather, secondary bacterial infections are more likely to cause death in prrsv-infected conventional animals [8, 16, 25, 26] . renukaradhydad et al. [27] showed that coinfection with prcv (porcine respiratory coronavirus) reduced nk cell function more than prrsv alone and dual infection caused more pathology [28] . likewise, prrs decreased the efficacy of siv vaccination and increased clinical disease [29] , and mycoplasma hyopneumoniae infection significantly prolonged and increased the severity of prrs [30] . as implied in the name of the disease, the clinical manifestations of prrs involve reproductive failure in sows and respiratory disease in young and growing pigs. historically, field reports described ''uncomplicated'' prrsv infections in young pigs as a mild-to-moderate pneumonia recognized clinically as an increased respiration rate at rest that would become labored with exertion. these observations were readily demonstrated experimentally. reproductive failure, which became the hallmark sign of prrs, included abortion ''storms'' and a sudden increase in dead fetuses and weak-born pigs that would affect most of the sows in the herd. in experimental sow infections during late gestation, fetal death and weak-born pigs are a predictable outcome, but prrsv-induced abortions are uncommon. the course of clinical disease following prrsv infection has been well chronicled. in the hundreds of animal experiments that have been reported since 1991, it has become clear that there is considerable variation in clinical responses. most of this is attributed to the use of different prrsv isolates, and collectively, it appears that the isolates from the early 1990s are less pathogenic than isolates from the late 1990s and certainly much less pathogenic when compared to asian hp-prrsv. although differences in viruses may be a major factor in clinical variability, differences do occur when using the same virus under similar conditions suggesting that the host is also an important variable. fortunately, there is considerable knowledge and expertise in prrsv genetics to allow this to be further tested (''prrs the virus'' section). at this time, variation in clinical response is attributed to genetics, age, and coinfections [31] . based on early field reports and experimental data, swine become more resistant to clinical disease with age, and boars and sows exhibit fewer clinical signs. this is not completely accurate since there is growing evidence that as prrsv mutates overtime, it may gain in virulence. why adults are more resistant to clinical disease and more likely to resolve the disease with vn antibodies [32] is unclear, but it may reflect the less well-developed immune system of neonates (fig. 1) . likewise, how the virus develops a chronic infection in the boar and is shed in the semen for extended periods of time is not known. current swine husbandry practices are almost completely dependent on the use of artificial insemination resulting in a population of boar studs that may supply semen to tens of thousands of sows. this practice dramatically magnifies the danger of using prrsv-contaminated semen. similarly, the concentration of sows in large buildings certainly contributes to possible horizontal transmission of virus and subsequent clinical and economic affects. at a cellular level, prrsv antigens and nucleic acids have been demonstrated in cells of the monocyte and dendritic cell lineage in a variety of organs. prrsv in the lung is often associated with lesions; however, the presence of virus and lesions is less frequent in other organs. the observations support a tropism of the virus for the lung, which could lead to pneumonia. however, when compared to other swine pathogens, the presence of prrsv in the lung and other organs seems minimal in relationship to clinical disease. one explanation for this may be that the pathogenic mechanism(s) of prrsv is(are) not necessarily a simple cytolytic effect on a tissue with influenza a that infects airway epithelia. instead, prrsv may just affect a smaller group of cells that have important regulatory controls, which could lead to a variety of diseases most likely those of hematopoietic/lymphoid tissues. the behavior of good parasites like viruses is to cause a delay in their eviction to allow for reproduction and transfer of their offspring to another host. others may revert to a low virulence state and continue to survive in the host. viruses such as those in the herpes family that are persistent for life have all evolved mechanisms that dysregulate the immune system. few investigative groups have seriously focused on immune dysregulation during prrsv infections. a great many viruses foil antigen presentation by interfering with mhc expression. rapid reduction of mhc adaptive immunity fig. 1 the critical window of immunological development. neonates are vulnerable during this period since their adaptive immune system is undeveloped, and they depend on innate and passive immunity. within this period, healthy gut colonization takes place which drives the development of adaptive immunity and both oral tolerance and immune homeostasis develop. in some mammals, passive maternal antibodies are provided in utero as well as post-natally through suckling. the colors are a result of blending overlapping events. modified from butler and sinkora [237] class i surface expression is a common feature of viral infections and is seen with foot-and-mouth disease virus [33] . in epstein barr virus (ebv) infection, degraded peptides from the ebna-1 nuclear antigen are not degraded, and so, these peptides are not presented [34] . something similar happens with presentation of peptides derived from a 72-kda transcription factor in human cytomegalo virus (hcmv) [35] . while the complex mechanism in these two examples is incompletely understood, there is better data for several other herpes viruses that inhibit the tap complex. tap is required for the transport of cytosolic peptides (including those derived from a virus) across the er. this step is required in their eventual presentation to cd8 t cells. tap inhibition is found in herpes infection of swine, dogs, and cattle but not in rodents or lagomorphs [36] . an adenovirus protein (e19) retains degraded peptides in the er and thus also prevents their presentation to t cells [37] . in hcmv, several gene products target mhc i for proteasome degradation [38] . in hiv, the nef and vpu proteins downregulate expression of surface mhc i [39] . in both human and bovine papilloma viruses, the gene product e6 is believed to interfere with the processing of cellular proteins and could thus affect presentation of peptides [40] . viruses may also interfere with mhc ii expression that is induced by ifn [41] . viral infection also disrupts cell cycling and interferes with cytokine and chemokine production and also cytokine action. the list of examples is long but in general, il-1, il-12, both type i and ii interferons are affected. as reviewed above, interference with innate cytokine synthesis may be especially important. these effects have been reported for a wide variety of viruses including pox viruses, herpes viruses, adenoviruses, and others. this further indicates that immune dysregulation is widespread among viral infection and that many families are involved indicating that it is a feature of the type of particular pathogens and but not their place in phylogeny. viral gene products also interfere with effector functions of the immune system. for example, they can interfere with apoptosis, and in swine, fmdv has been shown to inhibit the natural killer (nk) cell response to infection [42] . it is known that adenoviruses can cause lysosomal degradation of fas that is part of the complex used by cytotoxic t cells and nk cells to induce apoptosis of virus-infected cells [43, 44] . more than 30 viral genes affect this part of the anti-viral defense [45] . infecting viruses may also interfere with virus neutralization. the mechanism of viral neutralization has been a matter of conjecture for[40 years. do neutralizing antibodies bind those viral epitopes that prevent their recognition by the receptors on potentially permissive cells or do they inhibit the fusion of the viral membrane with the endocytic membrane? if it is simple blocking, multiple antibodies appear to be needed since as many as 25 % of such viral epitopes must be antibody bound to prevent infection [45, 47] . is simple blocking by antibodies enough or is help needed from an immune complex? in the case of eav, adding fresh serum as a source of complement, greatly increased the effectiveness of vn. covalent binding of c3 and c4 can facilitate clearance by cells that express complement receptors. in addition to merely facilitating clearance, complement-containing immune complexes can augment b cell activation [46] , whereas igg complexes without complement can downregulate b cell responses through crosslinking to fccriib [47] . non-neutralizing antibodies may also act as a trojan horse in facilitating virus uptake through fccrs, a process dubbed as antibody-dependent enhancement that can increase infectivity 10-100 fold [48] . recently, attention is being given to another immunosuppressive player in cancer and persistent viral infection. myeloid-derived suppressor cells (mdsc) were first described from a mouse model of lung cancer in which these cells inhibited t cell proliferation [49] . these cells function through reactive oxygen species (ros), inos and arginase-1 [50] . acting through ros, tcr can become nitrated preventing peptide binding [51] . ros-dependent suppression of cd4 ? and cd8 ? t cells by mdsc in hcv infections [52] . current understanding suggests that mdsc also inhibit nk cell function. mdsc suppression is also known for hiv, vsv, and vaccinia [50] . since prrsv can be persistent, a role for mdsc should not be ignored. if viral neutralization is complement dependent, viruses that interfere with this mechanism can prolong their replication time in the host. there is evidence that vaccinia, cowpox, and variola secrete proteins that block c3 convertase action [53, 54] . while the mechanism involved is unclear, herpes viruses can also inhibit complement activation [55, 56] . it has been known for some time that many viruses that cause persistent infection including ldv and prrsv are strong polyclonal b cell activators and often lead to the appearance of autoantibodies, a symptom that the preimmune repertoire has been expanded [21] [22] [23] [57] [58] [59] [60] [61] [62] . tumorigenic viruses like ebv that target b cells give rise to elevated levels of monoclonal antibodies not directed to ebv [63] . in these cases, immunoglobulin (igg) levels are a poor indicator of the anti-viral response. the innate immune response to prrsv host innate immune responses play a key role against early viral infection. host pattern recognition receptors for rna viruses include rig (retinoic-acid-inducible gene)-i-like receptors (rlrs) and toll-like receptors (tlrs) [64, 65] . activation of rlr and tlr signaling pathways leads to activation of interferon regulatory factor 3 (irf-3), irf7, and nf-jb, followed by induction of type i ifns (i.e., ifna and b) and expression of inflammatory cytokines. type i ifns are critical to innate immunity against viral infections and play an important role in the stimulation of adaptive immune response [66, 67] . prrsv is sensitive to type i ifns, and the sensitivity is confirmed in vivo. pigs that were inoculated with recombinant adenovirus for ifn-a expression and challenged with prrsv 1 day later had reduced lung lesion and delayed viremia and antibody response [68] . the presence of ifn-a at the time of infection alters innate and adaptive immune responses to prrsv [69] . prrsv appears to inhibit synthesis of type i ifns in pigs, while swine transmissible gastroenteritis virus (tgev) and porcine respiratory coronavirus (prcv) induced high level of ifna [70, 71] . ifn-a could not be detected in the lungs of pigs in which prrsv actively replicated. it was estimated that the ifn-inducing capacity of prrsv is at least 159-fold lower than that of prcv [71] . prrsv infection of pulmonary alveolar macrophages (pams) does not lead to ifn-a production [70] . plasmacytoid dendritic cells (pdcs) are thought to be the major source of ifn-a in vivo. prrsv also fails to induce porcine pdcs to produce ifn-a, while pseudorabies virus (prv), swine influenza virus (siv), and tgev stimulated the pdcs to synthesize ifn-a [72, 73] . however, nf-jb activation occurred in the presence of prrsv. loving et al. [74] showed that prrsv replicated in monocyte-derived dcs but not lung dcs and that dc response to prrsv was merely limited to ifn-b transcription but no ifn-alpha transcription. prrsv replication in marc-145 cells significantly inhibits the doublestranded rna-induced type i ifn transcription [75] . the prrsv proteins that are found to be antagonists of ifn induction include nsp1, nsp2, nsp11, and n (see review [76] ). nsp1 has been studied in more detail than the others. nsp1 is self-cleaved into nsp1a and nsp1b subunits, both of which mainly localize in the cell nucleus and dramatically inhibit ifn-b expression [77] . beura et al. [78] showed that nsp1b inhibited double-stranded rna (dsrna)-induced irf3 phosphorylation and nuclear translocation. however, kim et al. [79] showed that nsp1 inhibited irf3 association with creb-binding protein (cbp) in the nucleus but had no effect on irf3 phosphorylation and nuclear translocation. the discrepancy is possibly because an nsp1b that is 14-residue longer than its authentic form was used in the beura's study. another possible reason is that different prrsv strains were used. nsp2 inhibits ifn induction by blocking irf3 activation, and the ovarian tumor (otu) protease domain interferes with the nf-jb signaling [80] . nsp2 also inhibits the antiviral function of isg15 by the deubiquitinase activity of the out domain [81] . nsp11, an endonuclease, is also an ifn antagonist [78] . the ifn antagonizing activity is not restricted to nonstructural proteins. nucleocapsid (n) protein inhibits ifn-b induction by interfering with dsrna-induced irf3 activation [82] . the multiple components of nsps interfere with ifn induction. the nsps are early proteins, and n is a late one, which may play roles at different stages of viral replication. prrsv interferes not only with ifn induction, but also with ifn-activated signaling. ifns bind to their receptors on cell surface and activate jak/stat signaling, resulting in the expression of ifn-stimulated genes (isgs) [83] . prrsv inhibits the ifn-activated jak/stat signal transduction and isg expression in both marc-145 and pam cells [84] [85] [86] . prrsv replication in marc-145 cells suppresses jak/stat signaling stimulated by addition of ifn-a [84] . prrsv infection of pam cells also blocks jak/stat signaling, while a vaccine strain ingel-vac prrs mlv has little effect, possibly due to its less efficient replication in the primary cells [84] . nsp1b inhibits the jak/stat signaling via inducing the degradation of karyopherin-alpha1 (kpna1, also called importin-alpha5), which is known to mediate the nuclear import of stat1 [81] . prrsv infection of marc-145 cells also reduces kpna1 expression. besides nsp1b, other prrsv proteins including nsp7, nsp12, gp3, and n were also found to be able to inhibit ifn signaling [85] . prrsv field isolates have variable suppressive effect on ifn-a induction in pam cultures, and the suppression was found at post-transcriptional stage [87] . this is not unexpected as prrsv strains are divergent in genomic sequences (''prrs the virus'' section). prrsv infection of monocyte-derived dendritic cells (mo-dc) induces the transcription of ifn-a/b but no detectable ifn-a in culture supernatant, suggesting a blockage at post-transcriptional stage [88] . prrsv infection of marc-145 cells inhibits ifn expression by interfering with the rlr signaling pathway [89] . a variety of type 1 and 2 prrsv were found to stimulate ifn-a secretion by pdc via tlr-7 pathway, and the effect did not require live virus [90] . the suppressive effect on pdc was thought to be strain dependent. a novel isolate, a2mc2, induced ifns in both marc-145 and pam cells, and virus replication was needed for ifn induction [91] . type 1 ifns and isgs were detected in a2mc2-infected cells. a2mc2 infection of pigs resulted in higher level neutralizing antibody than a mlv vaccine strain that is highly homologous in sequence [92] . variable effect on ifn signaling among prrsv strains was also found [85] . among six prrsv strains (vr-2385, ingelvac prrs mlv, vr-2332, nvsl97-7895, mn184, and lelystad) tested, all but mn184 inhibited ifn signaling in marc-145 cells, and all but mlv and nvsl blocked the ifn activation in pams. nsp1b from the six strains were cloned, and all but mlv nsp1b inhibited ifn signaling when overexpressed [92] . there is good agreement that prrsv infections are not resolved rapidly in piglets, e.g., not in 7-14 days, in contrast to infections with swine influenza, fmdv, or eav in horses [10, 93, 94] . further, the carrier state may exist for up to 150 days [95] , and viral rna can be detected out to 251 dpi [95, 96] . antibodies to prrsv can be detected as early as 1 week after infection [97] (fig. 2 ), yet viral neutralizing (vn) antibodies are not usually detected prior to 4 weeks [98, 99] (fig. 3) . maximum titers may not be reached until 10-18 weeks dpi, and the peak titers are usually modest [98, 100] . igg antibody levels appear to peak at 21-35 dpi in piglets but persist at lower levels thereafter [97] . some reports indicate that viremia and viral replication can persist even in the presence of vn antibodies [1, 101] , and viremia can be resolved before vn antibodies are detected [100, 102, 103] . in the case of prrsv, ldv, and eav, gp5 is considered the most important neutralizing epitope in vn [10, [104] [105] [106] . focus has been on the hydrophilic ectodomain of gp5 [107] . however, gp5 has numerous glycosylation sites that might influence the avidity and specificity of antibodies to gp5. in general and because of the high frequency of mutation in rna viruses, there is considerable variation in gp5 among various strains of prrsv (''prrs the virus'' section). thus, the concept of the dependence of antibodies to gp5 for vn is complicated. using recombinant polypeptides, li and murtaugh [107] showed that the titer of antibody to the gp5 ectodomain did not correlate with the vn antibody titer. vane et al. [108] used peptide-specific antisera to show that the largest number of antigenic sites was associated with gp3 and no neutralizing targets were associated with either gp5 or m. using chimeric viruses, lu et al. [109] showed that gp5 and m were not responsible for tissue tropism. furthermore, other studies have shown that viremia is resolved before vn antibodies appear [100] (fig. 3) and animals are protected from the european variant without them [110] . evidence suggests that recognition may depend on strain variants/types. mabs to gp4 recognize the european variant but not the north american variant [111] . in spite of these often contradictory reports, the bulk of the evidence supports the view that vn neutralizing antibodies are important for protection [32, 101, 112, 113] . unfortunately, the mechanism of vn for prrs has not been researched. as regards vn antibodies to prrsv, there are some concerns about work already published. one concern is the amount of data available and from what experimental animal group they was obtained. if vn depends on labor intensive culture studies, it is likely that data currently available are from a few time points and a few animals. whatever viral epitopes or whole virus variants are used, a high throughput microtiter system should be adapted. it would be a shame if the current belief in poor vn activity is a consequence of selected and limited sampling. one can also question the methods used. in most studies, vn is tested using a lab strain virus and marc 145 cells to which the virus has become adapted in vitro. this is a valid assay for the cell line and the prrsv strain used but does it test whether neutralization has occurred in vivo in infected animals in which different target cells and virus variants are interacting? the failure of swine to develop a sterilizing immune response has raised the issue of whether this virus produces fig. 3 in piglets, the appearance of neutralizing antibodies is delayed, but other antibodies appear shortly after infection. from lopez et al. [101] suppression or tolerance [114] . some have reported the presence of cd4 ? cells with a suppressor phenotype (cd4 ? cd25 ? foxp3 ? ) after infections with prrsv [115, 116] . silva-campa et al. [117] showed that porcine cells with the treg phenotype make il10 and tgfb, confirming their analogous function to those in mice. it is known that pulmonary dendritic cells can induce tolerance through il-10 [118] . however, in a three virus study using isolator piglets, an increase in cd4 cells with a suppressor phenotype was not associated with prrs [119] . few studies have experimentally tested whether prrsv is functionally immunosuppressive while many show inhibition of type i interferons by prrsv (''the innate immune response to prrsv'' section). if tregs in conventional animals are functional, they appear not to interfere with the antibody response to klh in prrsv-infected pigs [97] . the thymic atrophy caused by prrsv can result in subnormal levels of double-positive thymocytes drives t cell development and loss of peripheral cd4 cells [70, 120] . some coinfection studies suggest that prrsv can interfere with protective responses to other viruses (''history and discovery of the causative virus'' section), which is supported by extensive field reports of synergy between prrsv infections and endemic infections within herd. infections with asian hp-prrsv elevate a large number of cytokines associated with both innate and adaptive immunity, both pro-inflammatory and otherwise [16] . this ''cytokine storm'' suggests that prrsv affects many pathways leading to innate and adaptive responses or their suppression. an element in the kinetics of prrsv infection is the age of the host. klinge et al. [121] showed that prrsv antibodies are detected at the same time in infected piglets and adults, yet viremia is immediate and resolved in sows, but develops late and remains persistent in piglets (fig. 2 ). the delayed increase in viremia in piglets is correlated with a delay in the infection-induced increase in il-10; the increase in this suppressive cytokine seems correlated with viral replication, but not the time of infection. the muchcited viral persistence seems to be a feature of piglets since, except for boars, the virus does not persist in swine infected later in life [121] (fig. 2) . furthermore, the presence of vn antibodies in older pigs is correlated with elimination of the virus [32] . by contrast isolator piglets appear much more susceptible to b cell immune dysregulation (''response to prrsv infection in germfree piglets'' section) and prrsv is most immune dysregulatory during the critical window of immunological development before immune homeostasis has been established ( fig. 1) . figure 2 shows that viremia persists in piglets but not in adults. figure 3 shows that antibodies detected by elisa appear early but the appearance of those with vn activity is delayed. this could reflect a difference in sensitivity between elisa-based assays and vn assays. early protection to all infections depend on innate immunity which then raises the question of whether persistence of viremia in piglets (fig. 2 ) reflects suppression of innate responses in piglets (''the innate immune response to prrsv'' section). while this may initially be critical, there is still too little information to conclude that the adaptive immune response is not impaired. there are reports that the amnestic antibody response to prrsv is poor or absent [97] , yet little is known about t helper and memory cells in response to prrsv infection. t cell recognition of viral epitopes has been described [122, 123] , but a tetramer assay system for these epitopes has not been developed for prrsv. despite the fact that so many viruses interfere with class i presentation, little attention has been given to prrs. overall, there is insufficient information as to whether the b cell or the t cell systems are most affected by prrsv and about the extent to which one or the other is impaired. the genetic variability of prrsv (''prrs the virus'' section) could also be a major player in the puzzle that has confounded investigators for [20 years. hard evidence for escape mutants during infection is lacking but heterologous challenge studies indicate immunity to one strain does not confer immunity to all [124] . in conventional herds, persistence might be due to re-infection with extrinsic variants for which crossprotection is absent. a particularly useful observation comes from so-called herd closure [125, 126] . this essentially involves immunizing adult animals in a virus-free herd and then isolating them from exposure to outside animals. that these animals remain prrsv-free suggests that: (1) vaccinated adult swine can develop sterilizing immunity if isolated from other animals and (2) escape mutants are unable to establish a re-infection in such herds. however, these experiments have not been performed with asian hp-prrsv or with very young piglets whose immune system is just developing (fig. 1 ). more than 20 vaccines have been developed for prrs, although no single product has been totally successful [17] . these vaccines and their efficacy are the subject of another review (k.m. lager, submitted). the functional, cellular response in adaptive immunity is characterized by the activation and expansion of antigenspecific, mhc-restricted cytotoxic t lymphocytes (ctl). in general, this is the primary effector function and most efficient immunity against viruses in mammalian species as university of iowa immunology 2014 (2014) 59:81108 87 because ctl kill virus-infected cells and arrest the generation of new viral particles. the role of this aspect of the immune response in prrsv infection is poorly understood. costers et al. [127] published that induction of virusspecific ctl in prrsv-infected swine is very weak and slow to develop. they analyzed this by using prrsvinfected autologous cells as targets of ctl killing. by comparison, these authors show a strong response of similar pigs infected with pseudo rabies virus (prv) in ctl assays using prv-infected target cells. in chronic viral infections, the regulatory element ppp2r2d plays a significant role in ctl dysfunction [128] . other in vivo studies have not tested for the predicted prrsv epitopes that would induce ctl responses [129] and have used nonswine animal models. this complicates interpretation of the small literature available on this subject. furthermore, analysis of ctl induction is complicated by the nature of this effector function. experimentally, ctl killing is measured by analysis of these cells killing virus-infected cells in vitro in an antigen-specific, mhc-restricted manner. in most cases, the virus also kills the virus-infected cells. provided it is allowed by the in vitro system, killing takes days to occur. thus, new approaches are needed. the role of c/d t cells in prrs is unclear. several reports describe that c/d cells are affected by prrsv and other viral infections [44, 130, 131] . the latter shows that c/d t cells behave similarly to cytotoxic and nk cells. in isolator piglets, only the subset of cd2 ? cd8 ? c/d t cells was increased, which is the only subset is known to be cytotoxic [119] . the paucity of information at this point is insufficient to construct a meaningful hypotheses regarding the role of c/d t cells in prrs. however, depleting them in vivo using mabs could determine whether they play a role in either disease resolution or pathology. prrsv affects lymphocyte development in thymus prrsv infection can cause an acute lymphopenia, thymic atrophy, and lymphadenopathy associated with the presence of prrsv antigen in the thymus. thus, development of a protective, adaptive immune response to prrsv may be impaired because prrsv infection negatively impacts circulating and developing lymphocyte populations, and reconstitution of the peripheral lymphocyte pool can be impaired. lymphopenia appears soon after infection [7, 120, 132, 133] and follows an influx of macrophage-like cells in the thymus and secondary lymphoid organs that contain prrsv [134, 135] . there is also a loss of immature t cells in the thymus [15, 136, 137] accompanied by significant lymphadenopathy [13, 22, 134-136, 138, 139] . it seems important to connect these observations to understand how prrsv affects the development of prrsv-specific immunity. the two mechanisms on which the animal relies to return balance to the circulating t cell pool are thymopoiesis and homeostatic proliferation of peripheral cells [140] . homeostatic proliferation, or expansion of the existing peripheral t cell pool, is the primary means for reconstitution following peripheral depletion. in mice, both peripheral memory t cells and naïve t cells undergo homeostatic proliferation, though at different rates (fast vs. slow, respectively) and with differing signal requirements (mhc, il-7, etc.). naïve t cells undergo slow homeostatic proliferation in secondary lymphoid organs (such as lymph nodes) that is dependent on il-7 and self-peptide:mhc presentation by an apc [141] . this type of proliferative recovery has been implicated in autoimmunity because of preferential expansion of t cells with greater specificity and stronger avidity for self, which has been observed following administration of lymphodepleting drugs [142] . prrsv infection has been shown to result in production of autoantibodies [22, 59, 139] , which may be related to the expansion of autoreactive t cells and/or the failure of the pre-immune repertoire to diversify (''response to prrsv infection in germfree piglets'' section). memory t cells can proliferate outside secondary lymphoid organs, and the signal does not require mhc contact. collectively, the noted lymphadenopathy associated with prrsv infection may be the result of homeostatic proliferation of peripheral t cells, and possibly b cells, to repopulate the peripheral pool. if lymphoid hyperplasia is the result of homeostatic proliferation, it requires determining why the cells do not egress from the lymph node. in addition to proliferation of existing t cells, newly developed thymic emigrants can contribute to restoring the peripheral pool to a normal level following a lymphopenicinducing event. however, reports indicate a loss of t cells in the thymus following prrsv infection [8, 15] . development of t cells in thymus is well described in textbooks, and at a certain stage, cd4 ? cd8 ? cells (double-positive,dp) interact with cortical thymic epithelial cells (ctec) to scan for positively selecting antigens. positive selection occurs when the t cell receptor has an intermediate affinity/avidity interaction with self-peptide presented by mhc on the ctec. positively selected cells then commit to the cd4 or cd8 lineage (single-positive, sp) and rapidly relocate to the medulla where they sample antigen presented by medullary tecs (mtec) and/or dendritic cells. these dp cells should not be confused with those dpc cells in the periphery of normal pigs [143] . medullary tecs are unique in the expression of autoimmune regulator (aire) gene, which controls the expression of tissue-restricted antigens. tissue-restricted antigens (i.e., self-proteins) are picked up by neighboring thymic medullary dendritic cells for presentation to developing sp t cells, which drives t cell selection. if a high affinity/avidity signal through the t cell receptor at this stage is received, cells die by negative selection to prevent release of autoreactive cells into the periphery, which is referred to as central tolerance [144] . mature naïve t cells, presumably those that only recognize foreign antigen, are then released into the periphery. various groups have shown a population of macrophage-like cells in the thymus stains for prrsv antigen by immunohistochemistry [12, 136, 138] . in addition, reports have highlighted the negative impact of prrsv infection on thymic cellularity [15, 120] , primarily as a loss of cd4/ cd8 dp cells in the thymus of prrsv-infected pigs [8] . the loss of developing t cells in the thymus likely affects the number and nature of newly developed t cells exiting the thymus during prrsv infection. the presentation of prrsv antigens in the thymus may also induce tolerance (loss of naïve cells that would recognize prrsv antigen) and provide a mechanism for the reported increase in regulatory t cells after prrsv infection [117] . these data together give support to the notion that infection of apcs in the thymus has a detrimental effect on the development of naïve t cells, and this likely has a negative impact on the development of a protective immune response to clear the virus from the pig. some of the lymphopenia that occurs shortly after birth may reflect the rapidly expanding blood volume but whatever the cause, it is not due to a selective depletion of t cells [119] . in young pigs, prrsv induces a reduction in circulating lymphocytes early after infection, but not in age-matched controls (c. loving, pers com). since the decrease in circulating lymphocytes occurs before obvious phenotypic changes in the thymus, the lymphopenia is: (1) not due to thymus infection by prrsv, (2) an effect by prrrv on the peripheral t cell compartment, or (3) a red herring in the quest to understand how prrsv dysregulates the piglets immune system. it is unclear if the drop in circulating lymphocytes is related to the lymphadenopathy observed later in the infection, but could be a compensatory attempt to repopulate the peripheral lymphocyte pool. response to prrsv infection in germfree piglets ''isolator piglets'' are recovered by caesarian surgery and reared in germfree isolators [145, 146] . these animals have not encountered gut flora, which drives development of adaptive immunity through stimulation of toll-like receptors [147, 148] (fig. 1) . furthermore, they obtain no passive maternal antibody in utero and receive no colostrum that could protect them from pathogens or interfere with immune responsiveness [9] . finally, isolator piglets have no exposure to other pathogens or to other strains of prrsv. the response of isolator piglets is intrinsic and not modulated by other pathogens, subclinical infections, maternal antibodies, or exposure to other environmental factors. these piglets provide the best in vivo opportunity to identify the direct in vivo effects of prrsv on the neonatal immune system. isolator piglets can also be considered as ex vivo fetal piglets and, therefore, a good model to study prrsv-infected fetuses. since the adaptive immune system is not developed in fetuses, their intrinsic response is either innate or driven by fetal infections that promote development of adaptive immunity (fig. 1) . rna viruses are often sensed by intracellular by toll-like receptors which sense either positive or negative single-stranded rna or double-stranded rna (a recognized adjuvant) generated as part of viral replication. these molecules can drive development of adaptive immunity as shown with swine influenza [149] . fetal piglets are immunocompetent as early as 79 days of gestation (dg) [150] and have lymph nodes, an active bone marrow, ig gene class-switch recombination has occurred, and the ileal peyer's patches are especially well developed. while some changes are likely to occur between dg 80 and birth (dg 114), these have not been identified. when fetuses are confronted with prrsv, they respond in the same manner as isolator piglets [151] (see below). studies using prrsv-infected isolator piglets [22, 119, 152, 153] have revealed a number of features about the immune response to prrsv that may provide clues as to how this virus modulates the host immune system. immediately obvious is hypergammaglobulinemia, lymphoid adenopathy, and the appearance of autoantibodies [22] (fig. 4) . polyclonal b cell activation, hypergammaglobulinemia, and the appearance of autoantibodies are also seen in infections by unrelated viruses [23] . polyclonal b cell activation is also a feature on ldv infection in mice, a related arterivirus that is also persistent [154] . autoantibodies in prrsv-infected isolator piglets to golgi proteins [22] are also a feature of ldv infections [57, 61] and may be in part due to the site of morphogenesis of arteriviruses [60] . in addition to hypergammaglobulinemia and autoimmunity, prrsv-infected isolator piglets exhibit abnormal antibody repertoire and b cell development. measured as a repertoire diversification index, the values are in the range of 0.5, not significantly greater than for fetal piglets or sham control isolator piglets but 40-100 fold less than sivinfected isolator piglets and conventionally reared piglets (pic; fig. 5a ). sequence analyses revealed that the cdr3 binding sites of the ig from prrsv-infected piglets are even more hydrophobic than in newborns and sham controls while those for siv and pic are shifted to the hydrophilic region (fig. 5b) [153] . hydrophobic binding sites are incompatible with antibodies that recognize glycoproteins and are a feature of the pre-immune antibody repertoire [155] . in these animals, b cell differentiation is extremely rapid and cells representing the activated b cell stage are nearly undetectable indicating that b cells rapidly become plasma cells [119] . comparative cellular studies of isolator piglets infected with prrsv and siv failed to reveal any evidence of immune suppression, i.e., lack of evidence for elevation of fox3p cd4 ? , cd25 ? t cells. however, cells with a suppressor phenotype were observed in parallel studies using pcv2-infected piglets [119] in which functional immune suppression has been reported [156] . accepting the fact that the effect of a viral, bacterial, or fungal infection in germfree reflects a direct effect of the pathogen, our data suggest that dysregulation of b cell differentiation is one of the principal feature of neonatal infections with prrsv during the critical window (fig. 1) . (figs. 2, 3) . by contrast, adult animals make good vn antibodies and eliminate the infection [32] . some additional support comes from studies using homologous variants [124] . osorio et al. [112] demonstrated that passively administered ig-containing vn antibodies obtained from convalescent sows could provide sterilizing immunity in piglets although a follow-up study showed that while viremia was ablated, viral replication persisted in some tissues [101] . in the same studies, passive administration of non-neutralizing anti-prrsv serum had little effect although the mechanism of vn was not described. it would be wise to know whether active complement was also transferred. since prrsv is a respiratory infection, it would also seem important to know whether passive antibodies would have reached the respiratory tract. it is known that parenteral and oral vaccination of the sow generates passive antibodies that are protective against tgev [157, 158] . these and other studies support the view that effective antibodies were made by adults [32, 112, 121, 125] . tgev is a gastrointestinal infection, so ingestion of passive maternal antibodies, via milk and colostrum, has access to the site of infection. by analogy to ww ii: ''you need to stop them on the beaches.'' the respiratory tract, especially the upper portion, is the domain of the mucosal immune system. thus, parenterally administered passive antibodies to prrsv are unlikely to reach mucosal sites. this may explain why follow-up studies by lopez et al. [101] showed that virus still replicated in some tissues. the differences among result obtained using isolator versus conventional piglets might provide clues as to the nature of the apparent neonatal immune dysregulation. while lymph node adenopathy and some thymic atrophy are common to both groups, the extraordinary hypergammaglobulinemia of all isotypes and b cell expansion has only been consistently reported for gf isolator piglets (fig. 4) . this may in part be due to the fact that investigators who studied conventional piglets rarely measure ig levels in serum or bal. such measurements in conventional piglets would be difficult to interpret since conventional piglets would have ingested maternal ig through suckling. conventional piglets used in these studies would be from prrsv-free herds, so very little of the ingested and absorbed ig would be prrsv specific and therefore not protective. this may explain why the extent of the disease is similar. both groups of animals make virusspecific antibodies but because of the extraordinary hypergammaglobulinemia seen in isolator piglets, and because absorbed ig are from prrs-free sows, only a tiny proportion would be virus specific [22] . however, knowing how many cells are virus-specific relative to other viral infections would be a much more useful parameter for comparing both groups. the b cell clonal analysis done with isolator piglets showing selected expansion of the pre-immune repertoire has not been performed in studies of conventional piglets. the opposite is true for cytokine studies. however, cytokine studies in conventional piglets might be misleading because of undetected secondary infection or the effect of regulatory elements in colostrum or the impact of gut colonization [159] . while the impact of normal gut flora can impact cytokine levels in conventional animals, investigators typically compare their data to control littermates raised in the same environment, so this should play little role. however, the lack of gut colonization of isolator piglets might be in part responsible for the differences in the degree of hypergammaglobulinemia, since elements received via colostrum could establish immune homeostasis which might dampen polyclonal b cell activation and proliferation [159] . in limited studies, no differences were found between isolator piglets colonized with benign escherichia coli and their colonization-free littermates [22] . however, studies in mice and rabbits indicate that all colonizers are ''not created equal'' [160] , so results obtained using only e. coli could be misleading. difference in the innate immune response in isolator versus conventional piglets has not been reported. in summary, prrsv infections that result in fetal abortion, b cell dysregulation in isolator piglets suggests that piglets are more susceptible during the critical window of immunological development (fig. 1 ). since siv infections are rapidly resolved even in gf piglets, it suggests that age-related neonatal immune incompetence cannot alone explain the persistence of prrsv. this would appear to shift blame to active immune dysregulation. while siv is quickly evicted, one must remember it infects primarily epithelial cells, not cells of the hematopoietic/ immune system. thus, siv infections would theoretically provide less opportunity for immune dysregulation of the developing neonatal immune system. in any case, investigators need to be careful about assuming that what happens in piglets, also happens in adults. the prrsv genome as indicated previously, prrsv is a member of the family arteriviridae, in the order nidovirales, which also includes university of iowa immunology 2014 (2014) 59:81108 91 the viral families of coronaviridae, inclusive of coronavirinae and torovirinae, and roniviridae [161] . the nidovirales order (latin: nested set) contains viruses with similar genomic organization and replication strategy. the arterivirion contains a polyadenylated molecule of singlestrand, positive-sense rna (which is itself infectious) that varies in length for prrsv (14,876-15,520 bp) and eav (12,704-12,731 bp), but not as yet in complete published genomes for shfv (15,717 bp) and wpdv (12,093 bp). the particles are roughly spherical with an average virion diameter of 54 nm and consist of a helical nucleocapsid surrounded by a lipid bilayer containing several proteins [6, 162] . all arteriviruses replicate in alveolar macrophages of their respective host, apart from wpdv, for which the host cell type is not known. except for wpdv, which was only recently genetically characterized [5] , each individual arterivirus species consists of many diverse genomes. prrsv has been most studied in terms of host pathogenesis. there are two recognized prrsv genotypes: type 1 or european-like (prototype lelystad) and type 2 or north american-like (prototype vr-2332) [6] . the two main genotypes share approximately 60 % nucleotide identity, but each may vary more than 20 % in nucleotide sequence. the genome length of type 1 (14,876-15,098 bp) not only differs from type 2 (14,968-15,520 bp), but discrete sections of the genomes are different as well. prrsv rna includes a 5 0 untranslated region (utr) of 220-221 (type 1) or 188-191 (type 2) followed a large replicase gene of variable length processed into at least 16 recognized nonstructural proteins (nsp1a, 1b, 2(2tf, 2n), 3-7a, 7b-12) by self-encoded proteases. the proteases include papain-like protease (plp) 1a and plp1b in nsp1, plp2 in nsp2, and a serine protease (sp) in nsp4 [6, 163, 164] . presently, most of the cleavages have been defined using eav. plp1a and 1b, and plp2 cleave once cotranslationally, directly downstream of the respective enzyme. sp completes the remaining cleavages. nsp9 harbors the core rna-dependent rna polymerase (rdrp), nsp10 is a helicase, and nsp11 contains a mn 2?dependent rnase that cleaves at u stretches (nendou) and is involved in rna replication [165] . downstream of the replicase gene is overlapping open reading frames (orfs) enumerated as orf2 encoding for glycoprotein (gp) 2, orf2b encoding non-glycosylated envelope protein e, orf3 encoding gp3, orf4 encoding gp4, orf5a encoding non-glycosylated protein 5a, orf5 encoding gp5, orf6 encoding the non-glycosylated membrane protein m, and orf7 encoding the nucleocapsid protein n. since these orfs overlap, mutations to one coding sequence may affect adjacent orfs. they are transcribed as a nested set of at least six subgenomic rnas (sgrnas) in infected cells. all of the downstream orfs encode structural proteins [6, 162] . as mentioned above, type 1 prrsv differs in the length of most structural orfs when compared to type 2 viruses. a remarkable feature of the prrsv genome has been the rate of mutational diversification. it has been estimated that prrsv rna may have evolved at a higher rate (10 -2 /site/ year) than other rna viruses (10 -3 -10 -5 /site/year) [165] although another investigator estimates the rate is similar to other rna viruses [166] . the frequency of mutation includes not only simple mutation, but also is accounted for by a high rate of recombination [167] [168] [169] [170] . it is estimated that there now exist as many as four major subtypes of type 1 prrsv, based on orf5 and orf7 phylogeny [171, 172] . even more subtypes, as many as nine, have been identified for type 2 prrsv when based on orf5. the husbandry of commercial swine, with large numbers of hogs from different source herds and artificial insemination with boar stud semen, is believed to have accelerated the evolution of prrsv [173] . there is also ample evidence that two or more prrsv strains may infect an individual pig [174, 175] . the combination of husbandry with genetic mutation and recombination between different viral strains has made the study of prrsv evolution challenging. the major envelope proteins of prrsv consist of gp5 and m [100, 176] . gp5 forms a heterodimeric complex with m linked by a disulfide bond [177] . both gp5 and m are thought to traverse the viral envelope three times and have only a small extravirion domain and a longer intravirion domain, much as was shown for ldv and eav [178, 179] . gp5 is the most variable structural protein, and the predicted ectodomain after signal sequence cleavage is approximately 32 residues [180, 181] . within these 32 amino acids, two hypervariable regions surround a quite conserved region, which contains the completely conserved cysteine disulfide-linked to m and two potential n-glycosylation sites [104, 180] . the conserved domain has been shown to harbor a neutralization domain, and the n-terminal sequence has been termed a decoy epitope that is not neutralizing [101, 103, 104, [182] [183] [184] [185] [186] . however, since the conserved domain is surrounded by complex oligosaccharides, it is shielded from neutralizing antibodies [184, 186] . the m protein, which is believed to act as glue to bring all virion components together, has also been implicated in neutralization [187] [188] [189] . in addition, two of the minor glycoproteins (gp3 and gp4) have also been shown to harbor neutralizing epitopes [108, 176, [190] [191] [192] [193] [194] . as shown for eav, gp2:gp3:gp4 are thought to be disulfidelinked heterotrimers on the extravirion of prrsv and are thought to be in very low amounts compared to gp5 [195, 196] . although the minor glycoproteins may play a role in neutralization of some or all prrsv strains, there is little else known about the viral functions these proteins perform in prrsv [197] [198] [199] . the phosphorylated n protein encapsidates the rna genome, probably in a helical conformation [200, 201] , and is most likely involved in capsulation and budding from the endoplasmic reticulum as was shown for eav [202] . the swine host synthesizes the most antibodies to the abundant n protein, which are non-neutralizing [203] . replicase proteins that have been shown to induce high levels of antibody are nsp1, nsp2, and nsp7 [204] . nsp2 has also been shown to harbor many b cell epitopes from different prrsv strains [80, [205] [206] [207] and has recently been shown to be incorporated into the virion [208] . several infectious clones of prrsv have been produced [16, [209] [210] [211] [212] [213] [214] [215] [216] [217] [218] [219] [220] . most of the clones were developed using type 2 viruses. these infectious clones represent only a fraction of the variability seen in the field, but are extremely useful in probing the genome for dispensable regions [211, 217, 220, 221] , insertion of foreign genes to develop diva viruses [210, 211, 221] , investigation of structurefunction relationships [87, 105, 213, [222] [223] [224] [225] , examination of host virulence [218, 220, [226] [227] [228] , and/or the probing of host response [222, 229, 230] . there are also several studies using chimeric viruses, either within or between certain arteriviruses. some chimeric studies have led to the conclusion that the minor glycoproteins, not gp5, are important for tropism in cell culture [109, [231] [232] [233] and that the m protein is also not involved [234] . other investigators have explored combining different regions of type 1 prrsv with type 2 to examine viability [232, 235] or to explore the effect of nglycosylation differences between strains [192] . in an attempt to develop broader crossneutralizing antibody, researchers have mixed regions of the prrsv genome from different strains, creating a panel of chimeric viruses to explore changes in the virus as well as the swine host antibody response [193] . the same investigators used this technique to attenuate a strain of prrsv [137] . lastly, researchers have attempted to define regions of the prrsv genome responsible for attenuation/virulence [219, 227] or to act as vaccines [236] . these studies have led to the knowledge that it appears that attenuation, as well as virulence, is multifactorial, involving two or more regions that can differ based upon the lineage of virus used for study. the main lesson learned from these studies is that each strain of prrsv, derived from field isolates or those with defined mutations, harbors individual characteristics that influence the specific pathogenesis seen. these characteristics include viral replication rate, the amount of specific subgenomic messages, the relative ability to process viral replicase proteins, the amount of n-glycans displayed on the virion, the amount of each individual viral protein, the relative interaction rate between viral proteins, and the relative ability of each strain to inhibit type i interferon and to induce humoral and cellular immunity. added to these viral causes of pathogenic differences under defined clinical conditions are the host response to each individual viral strain, host genetics, climate effects, and herd immunity, among other factors. the need for new experimental tools and approaches advances in science have mostly succeeded because the experiments employed were focused on testing a specific hypothesis and because they were designed so that the number of variables was minimized. naturally, this is much more difficult in biology because of the complexity of living systems and because many variables are unknown when the study begins. the image that emerges from the cumulative literature on prrs is that many: (a) represent a category that is often derogatorily referred to as fishing expeditions, i.e., exploratory research, (b) are repetitious of other work already done or represents near re-publication of the same work in another journal, and (c) are noncomparative studies. the work appears to be driven by the pressure to produce a vaccine, not to understand how prrsv modulates the immune system. the combination of swine and prrs offers a particular challenge to immunologists. prrsv does not replicate in mice, there are no practical inbred strains of swine, immunological reagents are limited, and producing stable cells lines has proven to be difficult. most studies have been done using conventionally reared piglets, which represents a complex model as illustrated in the following hypothetical example. consider 100 pigs infected with prrsv and 100 noninfected controls. since pigs are outbred, difference in responses can be genetic. if they are conventional, each animal in each group has not had the same experience since it may have a different mother, and its passive immune experience could differ in terms of colostral regulatory factors obtained and their dosage. suckling patterns differ within a litter giving rise to the often used ''hind teat'' syndrome. if you split the litter, you must then move some piglets to surrogate mothers, which introduces another set of variables. gut colonization plays university of iowa immunology 2014 (2014) 59:81108 93 an important role in development of adaptive immunity [147, 148] , and colonizers do not have an equal effect [160] . colonization typically occurs by contamination at the birth canal and thereafter by contact with the mother through suckling or contact with her feces. assuming that each newborn piglet in each experimental group encounters the same environmental experience is extremely difficult to prove. all of these assumes they have the same living conditions and have no contact with other animals that not part of the study. the ''closed herd'' studies cited earlier is an example of how this latter aspect can be properly controlled. conventional animals almost invariably contact other microorganism, some that are pathogens and some that are merely commensals. while experimenters may control for serious pathogens, they typically do not control for subclinical infection or for differences in the make-up and effect of benign colonizers. all of these may affect how a young pig responds to an experimental infection with prrs or a prrs vaccine. the literature shows that animals studied differ in age and there appears to be an age factor in their immune responsiveness and in the persistence of the virus (''the effect of age, rearing, complement and the role of mucosal immunity'' section). if the purpose of a study is to understand how a virus affects the immune system, conventional piglets are probably a poor choice. if on the other hand, the goal is only to test a vaccine under farm conditions, then the approach is fine. after all, the sabin and sauk vaccines and many successful bacterial vaccine before them prevented the spread of many horrible diseases but it would take decades to understand the etiology of the disease and just why these vaccines worked. the story of prrs is more like the story of hiv; the old time vaccine recipes do not work, and so, it is now time to understand the etiology of the viral infection and how it interferes with its immune-based eviction. while there is no mouse model for prrs, there is a mouse model for ldv. the superficial similarities in outcome are such that one wonders why the ldv model has not been used more for prrsv given the vast number of immunological reagents that are available for mouse immunology. assuming that for other reasons, ldv is not a good model, then perhaps the next approach would be to compare how siv, prrsv, and fmdv affect the porcine response in a controlled in vivo setting such as the isolator piglet. one glance at the literature reveals that compared to their counterparts in mainstream immunology/virology, those in the veterinary field are at a disadvantage. one obvious problem is the lack of reagents for work on the swine immune system. however, the literature also suggests an apparent reluctance to employ some of the 30-year-old technologies already available. notably, simple assays like quantification of igs are rarely used, as are immunohistochemical assays that measure ig-containing cells and elispots that measure isotypic distributions, antigenspecific b cells, and cytokine secretions. while elispot and pcr assays have been used in prrs research, neither of these methods provide data on where the cells responsible are located within the geography of the organs studied. refining these to single cells in situ assays as used in other species would provide more useful information. single cell sorting and recovery of rna by micromanipulation are also available. given the many studies done in conventional piglets that refer to the lack of vn early in development of prrsv infection, why there are no assays to determine the mechanism of vn to test if complement is required or if antibody affinity is important is puzzling. likewise for a disease that affects the respiratory tract, the lack of studies on the mucosal/local immune response to prrsv is conspicuous. while using more controlled in vivo studies can help to understand prrs, they cannot address questions about what prrsv does at the cell and molecular level. without in vitro studies, it will be difficult to understand how prrsv affects the host immune system. as mentioned above, the lack of stable cell lines presents a real problem. this can partially explain why there are no mixed culture studies to determine whether mhc i is downregulated by prrsv and how infected macrophages or the virus itself affects t and b cells and their interactions. even a question still exists as to the exact cell population that can be infected. for example, does prrsv infect lymphocytes or only macrophages/dendritic cells? if this should occur, lymphocytes are present at all different stages of development, and if a particular viral receptor is needed, it may not be present at all times during lymphocyte differentiation. since porcine cell lines immortalized at each stage of lymphocyte development are not available, the question is more difficult to answer. it may also be dangerous to use only laboratory strain for infection studies and only established cell lines to which the strain has been adapted. for example, marc 145 cells used to propagate prrsv do not show downregulation of type 1 ifn, while this is not true for pdcinfected in vivo. to address whether the remarkable polyclonal b cell proliferation seen in gf isolator piglets is the direct effect of the virus, studies involving t-b cell interactions or contact between b cells and infected macrophages are needed. the same applies to cytokines: what cells are making which cytokines and where are these cells histologically located since cytokines typically act at short distances? especially useful for these studies would be engineered prrsv mutants lacking the ability to make certain gene products. the wealth of information on the prrsv genome, the many variants, and engineered mutants, provide a rich resource of research material (''prrs the virus'' section). in the last two decades, which covers the same period in which prrs has been studied, tetramer assays to quantify t cell specificity and involvement have become well established and can now be used with some limitation for cattle and swine. studies that concern innate immunity are already being conducted in vitro (''the innate immune response to prrsv'' section). perhaps the best way to determine how prrsv modulates or dysregulates the immune system is to start with fetal and neonatal animals since the pandemic nature of prrs appears developmentally linked. that the effectiveness of neonatal vaccines is age-dependent is no surprise to any immunologist and forms the basis for the timing of childhood vaccination schemes. while for prrsv and other viruses that cross the placenta, studying the fetal immune response would be wise, but quite impractical. fortunately, in swine and other artiodactyls, newborns are essentially ex vivo fetuses since they can be reared in gf isolators in which maternal regulatory factors and the effects of gut colonization are absent [9, 237] . given the experimental ''cleanliness'' of using isolator piglets (''response to prrsv infection in germfree piglets'' section), why they are so seldom used is surprising. first, there is a matter of expense which is not trivial. second is the rather subjective view that isolator piglets are artifacts because they do not reflect the farm experience and environment. so what is the purpose of prrs research: to simulate the farm experience and produce a vaccine ''in the blind'' or to first understand how the virus affects the host? if the former is successful, the latter usually becomes mute. unfortunately, the latter does not seem to be the case for prrs since the virus was identified [20 years ago and the disease has not been controlled. one argument favoring isolator piglets is their use as a model for fetal piglets that are aborted after in utero infection. the most compelling argument for the use of isolator piglets to understand how the virus dysregulates the immune system is that it minimizes the number of variables, always a feature of good experimental design. finally, if prrs is primarily a persistence problem in neonates, the use of isolator piglets automatically confines studies to the critical window of immunological development (fig. 1) . all studies in biology must grapple with what is ''normal.'' eviction of the virus shortly after infection might be considered ''normal'', while those that are not might be ''abnormal.'' this reasoning is certainly open to discussion. from a practical position, this is a good starting point if the goal is to understand how certain infectious agents affect the immune system. good experiments cannot be done in a vacuum. a glance of the literature shows that many experimental studies compare virus-infected piglets only with noninfected controls. this overlooks the possibility that the changes observed are common to all viral infections including suppression of nk function, interference with class i presentation, and polyclonal b cell activation. rather, experiments need to be designed in a manner to identify ''prrs-specific'' immune dysregulatory factors. a number of those done in studies on innate immunity have been done comparatively (''the innate immune response to prrsv'' section). coinfection studies are really relevant. for example, renukaradhyad et al. [27] showed that while prcv reduced nk activity by 30 %, dual infection with prrsv reduced this 80-100 %. in nearly all coinfection studies, there was an increase in disease [29, 238, 239] as might be expected resulting in increased morbidity and mortality. it would be surprising if coinfection did not result in more pathology and perhaps a delayed/depressed immune response. thus, such studies would seem unreliable in the identification of virulence factors of prrsv. there are also parallel studies using siv, pcv2, fmdv, and tgev to distinguish ''normal'' versus ''abnormal.'' however, these viruses have different cell tropism. are there any other porcine virus that infect macrophages and are eliminated in 7-14 days? there is also the issue of virulence. in the case of prrsv, one expects the degree of immune dysregulation to parallel the degree of virulence. hp-prrsv is more virulent because it kills the host in a shorter time or produces more severe clinical symptoms. does it also cause more severe immune dysregulation? if not, then assuming all events seen with vaccine strains of prrsv are due to immune dysregulation could lead in the wrong direction. the purpose of this review was to allow individual specialists to review their area of expertise and then to ask each to contribute a subhypothesis. we then assembled these separate views into global hypothesis. our goal was to especially provide new investigators with a number of testable hypotheses that could explain how prrsv dysregulates the neonatal porcine immune system. prrsv suppresses innate immunity, which delays adaptive immune responses prrsv infection in pigs leads to delayed production and low titer of neutralizing antibodies [113] as well as weak cell-mediated immune response [240] . we hypothesize that the suppression of innate immunity can be an important contributing factor to the modulation of host immune responses because type i ifns promote antigen presentation and natural killer cell functions, enhance antibody production of b cells, and play an important role in the differentiation of both cd4 ? and cd8 ? t cells. the prrsv interference with the innate immunity is at multiple levels, from ifn induction, ifn-activated signaling to activity of isgs. therefore, viral-mediated suppression of innate immunity not only inhibits early host defense against the infection, but also interrupts the development of adaptive immunity, especially in the young pigs. this may explain why young pigs develop more severe disease and poorer protective immune response during the critical window of development (fig. 1) . therefore, we would suggest comparative studies using siv and tgev to determine at the cytokine/cellular level, if prrsv-infected pams or pdcs alter the signal to t and b cells or even developing thymocytes. using the ifn-inducing prrsv strain a2mc2 could add to the value of the model. we further hypothesize that given the divergence of prrsv strains in sequences and clinical features that experiments utilize various strains and engineered mutants. since type i ifns are proinflammatory, the proper amount at the right site and time may be protective, whereas extreme elevation could result in damaging inflammation. a typical example is that hp-prrsv induces high-level ifn-a, but causes high mortality in pigs [16] . polyclonal b cell activation resulting in hyperplastic lymph nodes packed with ig-containing cells (igcc) is a hallmark of prrsv-infected isolator piglets. this is paralleled by hypergammaglobulinemia in which de novo-synthesized ig levels can increase as much as 1,000-fold in 3 weeks postinfection although \1 % of these are virus specific [22, 152] (fig. 4) . we assume that the same type of immune dysregulation occurs in conventional piglets, although it may be masked by the high concentration of absorbed passive ig that increase serum ig levels to [20 mg/ml. the extraordinary hypergammaglobulinemia simultaneously occurs as b cells rapidly differentiate to plasma cells in a manner in which the intermediate stage of activated b cells (cd2 ? cd21 -) is virtually absent [119] . future studies in both conventional and isolator piglets need to confirm or reject the observation that a very small proportion of specific antibodies characterizes the response to prrsv. if confirmed, it would lend support to the view that rapid b cells differentiation allows little time for diversification of the antibody repertoire. this can be tested after pcr recovery and cloning of the rearranged vdj from various tissues. using labeled probes specific for the nonmutated cdr1 and cdr2 regions of the seven porcine vh genes, a repertoire diversification index (rdi) can be calculated as described previously and shown in fig. 5 [241, 242] . since the rdi is largely a measure of the degree of somatic hypermutation, it indirectly tests whether gc formation and function have been normal. it would be nice to confirm this in conventional piglets and adult swine, but the data would be uninterpretable since conventional piglets and adult swine have been antigenized through contact with other microorganisms, and changes could not be ascribed to prrsv. suspicion about abnormal gc activity might also explain the findings of mulupuri et al. [97] . they used in vitro restimulation assays to suggest that there is a poor memory b cell response to prrsv. work by raymond and rowland [12] identified gc in newborn prrsv-infected piglets using a mab to cdw75 that has not been validated in swine. the gc and memory cell questions need to be pursued using better reagents and better experimental designs. the delay in development of vn antibodies in prrsvinfected piglets while the anti-viral response continue to rise (fig. 2 ) might be because early antibodies are: (1) complement dependent for vn, (2) of low affinity, (3) specific for non-neutralizing epitopes, or (4) of the wrong antibody isotype. alternatively, the differences between iddex elisa titers and vn merely reflect differences in assay sensitivity. in a single study, the addition of fresh serum did not improve vn to ldv, but it did improve the efficiency of vn to eav in horses suggesting that vn is complement dependent in horses but not in mice [10] . this is a simple assay and should be done with sera from prrsv-infected swine. a most likely possibility is that antibody affinity is too low in neonates to perform as effective vn antibodies. in the case of denge virus, at least 25 % of the neutralizing epitopes must be bound by antibodies for vn to occur [47] . immunochemists over the last 50 years have developed a plethora of methods to determine antibody affinity. most of these were developed to study antibody interactions with defined haptens. these studies established a number of very important principles including the observation that avidity, i.e., the staying power of an antibody, was determined by the ratio of the on-rate to the off-rate. thus, some ''quick and dirty'' methods have surfaced based on the principle that antibodies that remain bound in the presence of denaturants like urea or guanidine hcl are used [243] , which are of high affinity. using this procedure, the relative affinity of a non-vn serum could be compared to that from adult swine that has vn capacity. should the experiments designed to test the role of complement or antibody affinity give negative results, another approach would be to test the specificity of early antibodies for certain viral epitopes. as reviewed in ''humoral responses of conventional animals'' section, vn antibodies to the lelystad virus preferentially recognize gp3. assuming gp3 is the critical epitope, and affinity has been ruled out; it might suggest that antibodies to gp3 appear late during infection or that gp3 is poorly expressed on the virions used in the assay. once bound, the fate of the virus-antibody complex can also depend on the isotype of the antibody, which brings us to the fourth possibility. multivalency such as with pentameric igm can compensate for intrinsic binding site affinity and, therefore, perform much better than nonpolymeric igg so that early igm should provide good vn activity. the subclass of the igg antibody can also play a functional role in the effectiveness of complement-mediated vn. in swine, igg3 is the most totipotent igg based on its motifs for complement and fccr binding [244] . however, actual functional comparisons have not been carried out. igg3 is expressed very early in fetal and newborn piglets but after antigen exposure, other igg subclasses, especially igg1 replace igg3 [245, 246] . during the period in which vn has been typically measured (fig. 2) , there is at least tenfold more igg than igm present, and thus, igg is most likely the antibody in serum that is being measured in current vn tests. to determine which subclass of igg is involved would be extremely difficult. first, all commercially available mabs to swine igg are more or less pan specific [247] . even if such reagents were available, those which bind the virus would almost certainly be a mixture, so most probably antibodies of all subclasses involved, albeit probably dominated by igg1. perhaps the only way to truly test the effector function of the different igg subclass antibodies seems at this point unjustifiable. this would require construction of chimeric antibodies for each subclass each with a binding site that recognizes a neutralizing epitope of prrsv akin to the method we have described for expression and recovery of individual porcine igg subclass proteins [247] . confirmation of this subhypotheses might explain the initial ineffectiveness of the humoral response to prrsv during the critical window, but it does not explain why the extraordinary b cell expansion occurs and what force is driving this event. these require other subhypotheses and experiments to test them. we hypothesize that prrsv infects a population of antigen-presenting cells that migrate to or are constituent in the thymus of fetal or newborn animals, e.g., tecs, macrophages, and pdc that are engaged in thymocytes development and compromises proper t cell development. the interaction of thymocytes with these infected apcs might result in cytokine production/transcription and other protein transcription, which is abnormal compared with agematched controls. furthermore, the emerging t cell populations could be tested for their ability to recognize peptides derived from prrsv or a control antigens like ovalbumin. contrived in vitro systems should be developed to determine whether t cells developed in prrsv-infected thymi can provide t cell help for antibody responses, activation of macrophages, or can behave as ctls. we propose that the role of ctls in prrsv infection is fundamentally different in the infection of neonatal pigs compared to adults. we propose that the ability of pigs infected in utero or shortly after birth to mount any ctl response against prrsv is compromised by the impaired development of ctl precursors due to reduction of thymic selection. further, t cell selection that does occur could suffer from prrsv antigens being seen as self-antigen, as a result of infection of thymic cells involved in t cell selection. contrarily, in animals infected with prrsv as adults, ctl precursors have developed normally, and even though the infection impairs innate immunity, the presence of virus-infected cells eventually could lead to a protracted development of a moderate ctl response. further, we propose that the dysregulation of b cell function favors expansion of cd4 helper t cells not those required for induction of ctls. this could also contribute to or be the sole cause of the protracted development of antiviral ctl responses in adult animals. we describe below techniques to test theses hypotheses. first, we can use live, virulent virus in the short (hours long) assays to detect ctl killing. alternatively, avirulent strains of the virus can be used as surrogates, allowing the cell death to be solely a result of ctl killing of the target cell. in other circumstances, viral proteins can be delivered to target cells artificially, by vectors for instance [248] . since the ctl are from an infected animal and the autologous cells (or mhc matched target cell line) are given the vector expressing viral proteins, the measure of killing is now attributable to the ctl, as there is no live virus. a dominating concept of the immunopathogenesis of prrsv infection is the immunosuppression or dysregulation of the adaptive immune response. as with many livestock studies, there is a body of work describing the antibody response but little analysis of ctls. the single report of ctl function describes a basic analysis of a single strain of virus and concludes there is a low-level ctl response that is protracted in the kinetics of development [127] . a better understanding of ctl biology in prrsv infection will require a more sensitive assay for ctl function. using tools available today, class i mhc tetramers can be designed and tested to track ctl development and function. for instance, cd107a (lamp1a) is an integral membrane protein that lines the vesicles that contain the granules that mediate killing by nk cells and ctls. these granules are released by the vesicle membrane fusing with the cell membrane and releasing the contents. as a consequence, cd107a is now detected on the cell surface. so, a tetramer-positive, cd107a expressing cell is a prrsv-specific ctl that has just killed a virus-infected cell. so, not only is the cell phenotype determines, i.e., prrsv-specific cd8 t cells but also whether these cells function as ctls. another possibility to explain the decrease in ctls might be the action of mdsc [50, 52] . these macrophages accumulate at the site of chronic viral infections and tumors and suppress ctls. therefore, highly infected sites such as thymus, lung, and certain lymph nodes [8, 13, 136, 138] may harbor these cells. since prrsv targets macrophages, could their infection result in differentiation of myeloid cells to mdsc? with these tools, hypothesis testing can determine whether ctls are efficiently induced, induced but not functional, develop early but are rapidly downregulated, develop late, etc. elevation of p3 expressing, cd4 ? , cd25 ? treg populations reported in prrsv-infected isolator pigs is controversial (''humoral responses of conventional animals'' section). however, if class ii sla tetramers could be used to focus on the prrsv reactive cells in that population exclusively, this antigen-specific population may be highly induced, but masked by the present methods of analysis. however, given the evidence available, a more likely hypothesis is that the normal, t cell differentiation is dysregulated as reflected in the apparent dysregulation of helper t cells that promote excessive b cell proliferation while preventing prrsvspecific ctls from expanding that become activated to kill virus-infected cells. the opportunity to manipulate the prrsv genome provides the opportunity to test whether certain viral genes/proteins are responsible for immune dysregulation. nsp2 is the most variable protein in the virus, subject to insertion/deletion(s) compared to the prototype type 2 strain, vr-2332. the fact that the nsp2 protein is an early protein and also a structural component of virions [208] suggests that it may be in contact with host macrophages and dcs, and stimulators derived from those and other host cells. it also possesses that a key protease, plp2, whose ability to downregulate ifn-a and can act to deubiquinate proteins is well established, has a key role in the viral replication cycle by cleaving the nsp2/ 3 junction. lastly, this protein is the largest protein of the virus. a prior in vivo study has shown that a specific deletion of 87 aa in nsp2 of strain vr-2332 resulted in virus (vr-2332d87) with replication kinetics in 4-week-old swine about 1 log lower than the parent strain, while other deletions elsewhere in nsp2 had a more dramatic effect on viral replication (''prrs the virus'' section). it was also shown that swine inoculated with vr-2332d87 had no delay in onset of antibodies to the nucleocapsid protein. what was intriguing was that these same animals showed a delay in serum ifn-c and a significant decrease in lymph node enlargement over that seen with vr-2332. unfortunately, no comparison was completed on the thymic tissue or any other immune response measurement. these prior studies must now be examined using more virulent prrsv strains, and we must delineate the amino acids responsible for immune evasion. two strains that we will develop deletion mutants for and test our hypothesis are type 2 strains mn-184 and asian hp-prrsv. one can begin by deleting the nucleotides of these more virulent viruses that represent the same region as vr2332d87. however, other regions of nsp2 may serve to evade immune responses. only the hypervariable regions (aa 12-24; aa 323-817 of vr-2332) of the respective viruses have been shown to be mutable, so work should concentrate on those areas and make successive deletions based on nsp2 secondary structural predictions in the infectious clones of the parent viruses. once developed, these mutants will be used in in vivo studies with conventional and isolator piglets and in in vitro studies. since infected mq and pdcs fail to secrete ifna [73, 74] , they would also poorly stimulate the antiviral state, so the first event is to compromise the first line of defense (innate immunity), which would allow spread of the virus. second, the ifna-deficient infected mq may then present to peripheral t cells in lymph nodes and without normal levels of il-12 from dcs and pdcs, would not favor a th1 profile and differentiation to ctls. thus, a major element in adaptive antiviral immunity is impaired. rather these events favor a th2 profile that might cause proliferation of cd4 helper cells at the expenses of tregs and cd8 ctls. the suggestion that infected mq and pdcs could induce apoptosis of thymocytes might indicate they could have the same effect on the peripheral t cell compartment. this could create a lymphopenic state. the increase in il-10 suggests suppression that could account for the increase in tregs [117] and may be derived from mdsc [50] . the elevation of tregs might be a delayed event, which would have been overlooked by sinkora et al. [119] who worked only with isolator piglets. it is still difficult to accept that if adaptive immunity is forced to a th2 profile, it explains the polyclonal b cell activation and runaway b cell proliferation. the third event is that these infected mq, cdcs, and pdcs move to the developing thymus as apcs where they interact with dp thymocytes in the medulla that for reasons unknown, resulting in atrophy of dp thymocytes. together with help from thymic epithelial cells (nurse cells), prrsv may be therefore recognized as a self-antigen so surviving thymocytes could enter the periphery and recognize prrsv as self, as reported by the wieland for anti-golgi antibodies. in fact, the vasculitis that is a feature or arterivirus infections may be due to self-antibodies that coat the vascular as shown by lemke et al. [22] . while the loss of dp thymocytes might lead to the loss of emerging t cells and in t cell lymphopenia, there is little evidence to support this. however, the quality and quantity of emerging cd4, cd8, and tregs might be altered as described above for the peripheral t cell compartment. without functional tregs, activated b cells may initially proliferate out of control as suggested from sinkora et al. [119] . could an abundance of selfreactive th2 cells, some of which may crossreact with prrsv, be sufficient to drive rapid differentiation to plasma cells or perhaps il-6 from infected mq? alternatively, gc may not form or are abnormal, so there is little selection and the resultant plasma cells show little repertoire diversification (fig. 6 ) and therefore poor affinity to viral epitopes so that few which are strongly virus specific. while prrsv-specific vn antibodies can control the peripheral spread of the virus, ctls are needed to eliminate virus-infected cells. in most viral infections, pdcs secrete il-12 that promotes th1 cells that can also activate mq to kill their intracellular parasites/viruses. if chronically infected tissues are infiltrated by mdsc, such t cells may be inhibited [50] . in any case, since events in the thymus might reduce the number of peripheral th1 helpers, the infection would persist. perhaps of greatest effect is that if the number of virus-specific peripheral cd8 cells is low, there would be fever potential ctls to attack the infected mq. not trivial is that most scenarios described for ctl involve killing of epithelial cells like in siv. in the case of prrsv, it would involve the killing of infected mq. how easy is that? r d i fig. 6 a antibody repertoire diversification measured as a repertoire diversification index (rdi). prrs = isolator piglets infected with prrsv; gf = germfree controls; c/ v = isolator piglets colonized with benign e. coli or infected with siv; pic = young, helminth-infected conventionally reared pigs (pic). b hydropathicity profiles calculated from sequence analysis of the hcdr3 region of ig from prrsv-infected piglets compared to pic animals (top) and compared to newborns (bottom). the numbers in parentheses indicate the number of sequences examined. hydrophobic hcdr3 regions i and ii are a feature of an undiversified pre-immune repertoire whereas region iii is characteristic of a diversified repertoire. from butler et al. [153] adult model while what we have written above might explain the impact of prrsv on neonates, the literature we have reviewed suggests that a separate model is required for the situation in adult swine. while we may be dealing with one disease at the cellular/molecular level, we may be dealing with two disease models at the organismal level as regards the immunological perspective: one for adults and one for neonates. for all sorts of reasons, we believe that immune homeostasis is developing during the critical window of immune development (fig. 1 ) when most piglets are prrsv infected. when an adult pig is considered, they have already properly developed their t cell repertoire and compartment. that means they have normal levels of cd8 cells that are potential ctls. likewise, they have th2 cells to form gc and tregs to prevent uncontrolled b cell expansion. as a result, adult animals mount effective immune responses with vn antibodies and ctls that resolve the disease, regardless of whether the innate response continues to be compromised since host protection is now heavily dependent on de novo adaptive immunity (fig. 1) . in fetal and newborn piglets, innate immunity probably plays the major role in immune defense but after development of adaptive immunity, it become compensatory, not primary. this most likely explains why studies like those of robinson et al. [32] show that prrs is resolved in adults, presumably by both vn antibodies and ctls. thus, the host adaptive response override the negative effect of prrsv on innate immunity in adult animals. this suggests that the principal impact of prrsv is on the fetus and the neonate during the critical window and is thereafter not a serious threat to adults. from the position of vaccinologists, it would seem wise to supply neonatal vaccinates with the ingredients that would promote immunocompetence as summarized in fig. 1 . all of the events described for fetal/neonatal and adult animals are relevant to the common vaccine version of prrsv. however, is the effect of hp-prrsv merely a quantitative difference or does it have a qualitative effect? namely, does hp-prrsv primarily target the thymus so its greatest impact is on t cell cells development? since hp-prrsv has a greater effect than vaccine strain, prrsv on post-natal lymphopenia suggests that hp-prrsv also acts in the periphery. as previously described, failure to produce vn antibodies could be epitope dependent, so that differences between animals with and without vn antibodies could be epitope specificity, not a difference in affinity regardless of the mechanism of vn. the beauty of prrrv genetics is that a large number of variant are available and others can be engineered (''prrs the virus'' section). the availability and expertise of the investigators in this area provide an unusual opportunity for the experimental design of studies to determine how certain viral genes affect immune dysregulation and how epitopes differs in their ability to stimulate protective immune responses. testing the global hypothesis the working hypothesis offers numerous opportunities for testing. exactly, how each step in the scheme is tested is left to the ingenuity of the investigators. suffice to say there is a great need to know the cytokine, co-stimulatory molecule expression and signaling features of prrsvinfected macrophages when acting as apc versus noninfected macrophages both in thymus and in the periphery. do these ifna-impaired macrophages preferentially or inappropriately stimulate certain t cell subsets or do they promote differentiation of mdsc? using engineered mutants, one might determine what genetic features of the virus are responsible for any aberrant signaling. the core protein of hcv promotes mdsc differentiation [51] . such ''defective mutants'' might also be the basis for future vaccines. likewise, it would be wise to know what signaling events are aberrant in thymocytes from prrsvinfected animals. as the runaway b cell proliferation still lacks an explanation, it would seem important to know whether infected macrophages can explain that part of the puzzle. testing for germinal center formation, antibody affinity and the complement dependence of vn are relatively straightforward. the issue of tregs should be resolved. are those with a suppressor phenotype functionally suppressive? could it be the lack of functional tregs that permits runaway b cell proliferation and differentiation? while tetramer assays could be valuable, they will remain in the distant future given the state of swine genetics. in the meantime, it is reasonable to assume that the same co-stimulatory molecules and signaling pathways that operate in mice also operate in swine so the information gained from studies in pigs can take advantage of the vast resource of information assembled from mouse research. using simplified systems such as in vitro assays and isolator piglets has the best chance of determining how prrsv dysregulates the neonatal porcine immune system. if pandemic prrs is a neonatal phenomenon, developing vaccines that stimulate development and maturation of the adaptive immune system, e.g., probiotic cultures, should also be considered. porcine reproductive and respiratory syndrome epidemiology of porcine reproductive and respiratory syndrome (prrs): an overview isolation of swine infertility and respiratory syndrome virus (isolate atcc vr-2332) in north america and experimental reproduction of the disease in gnotobiotic pigs mystery swine disease in the netherlands: the isolation of lelystad virus identification of a novel nidovirus associated with a neurological disease of the australian brushtail possum (trichosurus vulpecula) virus taxonomy: classification and nomenclature of viruses: ninth report of the international committee on taxonomy of viruses pathogenesis of porcine reproductive and respiratory syndrome virus infection in mid-gestation sows and fetuses in vitro infection by procine reproductive and respiratory syndrome virus is sufficient to increase susceptibility of piglets to challenge by streptococcus suis type ii immunoglobulins of the mammary secretions the immune responses to equine arteritis virus: potential lessons for other arteriviruses polyclonal activation of b cells by lactate dehydrogenase-elevating virus is mediated by n-glycans on short ectodomains of the primary envelope glycoprotein the interaction between prrsv and the late gestation pig fetus pathogenesis of porcine reproductive and respiratory syndrome virus infection in gnotobiotic pigs characterization of thymus atrophy in piglets infected with highly pathogenic porcine reproductive and respiratory syndrome virus experimental infection of united states swine with a chinese highly pathogenic strain of porcine reproductive and respiratory syndrome virus immunological responses of swine to porcine reproductive and respiratory syndrome virus infections immunological solutions for treatment and prevention of porcine reproductive and respiratory syndrome (prrs) assessment of the economic impact of porcine reproductive and respiratory syndrome on swine production in the united states natural killer cell frequency and function in pigs selectively bred for high and low antibody and cell-mediated immune response: response to vaccination with modified live transmissible gastroenteritis virus in vivo polyclonal b-lymphocyte activation elicited by murine viruses lymphoid hyperplasia resulting in immune dysregulation is caused by porcine reproductive and respiratory syndrome virus infection in neonatal pigs hypergammaglobulinemia and autoantibody induction mechanisms in viral infection the evolution of bovine viral diarrhea: a review effect of intranasal inoculation with bordetella bronchiseptica, porcine reproductive and respiratory syndrome virus, or a combination of both organisms on subsequent infection with pasturella multocida in pigs secondary infection with streptococcus suis serotype 7 increase the virulence of highly pathogenic porcine reproductive and respiratory syndrome virus in pigs porcine reproductive and respiratory syndrome virus-induced suppression exacerbates the inflammatory response to porcine respiratory coronavirus in pigs proinflammatory cytokines and viral respiratory disease in pigs vaccine efficacy and immune response to swine influenza virus challenge in pigs infected with porcine reproductive and respiratory syndrome virus at the time of siv vaccination effect of vaccination on the potentiation of porcine reproductive and respiratory syndrome virus (prrsv)-induced pneumonia by mycoplasma hyopneumoniae genome-wide associated and genomic prediction for host response to porcine reproductive and respiratory syndrome virus infection humoral response to porcine reproductive and respiratory syndrome virus infection with foot-and-mouth disease virus results in a rapid reduction of mhc class i surface expression isolation of cytotoxic lymphocytes from healthy seropositive individuals specific for peptide epitopes from epstein-barr virus nuclear antigen 1: implications for viral persistence and tumor surveillance selective interference with class i major histocompatibility complex presentation of the major immediate-early proteins following infection with human cytomegalovirus molecular mechanism and species specificity of tap inhibition by herpes simplex virus icp47 herpes simplex virus inhibitor icp47 destabilizes the transporter associated with antigen processing (ytap) heterodimer retention of adenovirus e19 glycoprotein in the endoplasmic reticulum is essential to its ability to block antigen presentation dissociation of type i membrane proteins from the er to the cytosol is sensitive to changes in redox potential the downregulation of cd4 and mhc-i by primate lentiviruses: a paradigm for the modulation of cell surface receptors interaction of the bovine papillomavirus e6 qirh rhw clathrin adapter complex. ap-1 modulation of the major histocompatibility complex antigen expression by viral infection apoptosis regulators from dna viruses rapid and transient activation of cdt cells to interferon gamma production, nk cell-like killing and antigen processing during acute virus infection the adenovirus e3/10.4k-14.5 proteins down modulate the apoptosis receptor fas/apo-1 by inducing its internalization subverison of the immune system structural insights into the mechanisms of antibody-mediated neutralization of flavivirus infection: implications for vaccine development c3d of complement as a molecular adjuvant: bridging innate and acquired immunity fc-dependent inhibition of mouse b cell activation by whole anti-mu antibodies measles virus nucleocapsid protein binds to fcgammarii and inhibits human b cell antibody production monoclonal antibody-mediated enhancement of dengue virus infection in vitro and in vivo and strategies for prevention dengue viremia titer, antibody response pattern and virus serotype correlate with disease severity inhibition of the complement cascade by the major secretory protein of vaccine virus regulation of complement activity by vaccinia virus complement-control protein herpes simplex virus glycoproteins gc-1 and gc-2 bind to the third component of complement and provide protection against complement-mediated neutralization of viral infectivity immune evasion properties of herpes simplex virus type 1 glycoprotein gc lactate dehydrogenaseelevating virus induces anti-golgi apparatus antibodies infection of mice with lactate dehydrogenase-elevating virus leads to stimulation of autoantibodies autoantibodies against golgi apparatus induced by arteriviruses autoimmunity induced by lactate dehydrogenase-elevating virus: monoclonal autoantibodies against golgi antigens and other subcellular elements polyclonal activation of b cells occurs in lymphoid organs from porcine reproductive and respiratory syndrome virus (prrsv)-induced pigs neonatal infection of mice with lactate dehydrogenaseelevating virus results in suppression of humoral anti-viral immune response but does not alter the course of viremia or the polyclonal activation of b cells and immune complex formation hodgkins disease and the epstein-barr virus the toll-like receptor 7 (tlr7)-specific stimulus loxoribine uncovers a strong relationship within the tlr7, 8 and 9 subfamily innate immune recognition of viral infection immunomodulatory functions of type i interferons interferon signalling network in innate defence adenovirus-mediated expression of interferon-alpha delays viral replication and reduces disease signs in swine challenged with porcine reproductive and respiratory syndrome virus the presence of alpha interferon at the time of infection alters the innate and adaptive immune responses to porcine reproductive and respiratory syndrome virus interferon-alpha response to swine arterivirus (poav), the porcine reproductive and respiratory syndrome virus in vivo and in vitro interferon (ifn) studies with the porcine reproductive and respiratory syndrome virus (prrsv) characterization of the cytokine and maturation responses of pure populations of porcine plasmacytoid dendritic cells to porcine viruses and toll-like receptor agonists north american porcine reproductive and respiratory syndrome viruses inhibit type i interferon production by plasmacytoid dendritic cells differential type i interferon activation and susceptibility of dendritic cell populations to porcine arterivirus interferon type i response in porcine reproductive and respiratory syndrome virus-infected marc-145 cells interplay between interferon-mediated innate immunity and porcine reproductive and respiratory syndrome virus identification of two autocleavage products of nonstructural protein 1 (nsp1) in porcine reproductive and respiratory syndrome virus infected cells: nsp1 function as interferon antagonist porcine reproductive and respiratory syndrome virus nonstructural protein 1beta modulates host innate immune response by antagonizing irf3 activation modulation of type i interferon induction by porcine reproductive and respiratory syndrome virus and degradation of creb-binding protein by non-structural protein 1 in marc-145 and hela cells the cysteine domain of porcine reproductive and respiratory syndrome virus nonstructural protein 2 possesses deubiquitinating and interferon antagonism functions nonstructural protein 2 of porcine reproductive and respiratory syndrome virus inhibits the antiviral function of interferon-stimulated gene 15 porcine reproductive and respiratory syndrome virus nucleocapsid protein modulates interferon-beta production by inhibiting irf3 activation in immortalized porcine alveolar macrophages jak-stat pathways and transcriptional activation in response to ifns and other extracellular signaling proteins porcine reproductive and respiratory syndrome virus inhibits type i interferon signaling by blocking stat1/stat2 nuclear translocation variable interference with interferon signal transduction by different strains of porcine reproductive and respiratory syndrome virus porcine reproductive and respiratory syndrome virus nsp1beta inhibits interferon-activated jak/stat signal transduction by inducing karyopherin-alpha1 degradation mutations within the nuclear localization signal of the porcine reproductive and respiratory syndrome virus nucleocapsid protein attenuate virus replication porcine reproductive and respiratory syndrome virus activates the transcription of interferon alpha/beta (ifn-alpha/beta) in monocyte-derived dendritic cells (mo-dc) porcine reproductive and respiratory syndrome virus (prrsv) suppresses interferon-beta production by interfering with the rig-i signaling pathway impact of genotype 1 and 2 of porcine reproductive and respiratory syndrome viruses on interferon-alpha responses by plasmacytoid dendritic cells induction of type i interferons by a novel porcine reproductive and respiratory syndrome virus isolate enhancing neutralizing antibody production by an interferon-inducing porcine reproductive and respiratory syndrome virus strain antibody response to porcine reproductive and respiratory syndrome virus (prrsv) nonstructural proteins and implications for diagnostic detection and differentiation of prrsv types i and ii studies of quantitative parameters of virus excretion and transmission in pigs and cattle experimentally infected with foot-and-mouth disease virus porcine reproductive and respiratory syndrome virus: description of persistence in individual pigs upon experimental infection duration of infection and proportion of pigs persistently infected with porcine reproductive and respiratory syndrome virus antigen-specific b cell responses to porcine reproductive and respiratory syndrome virus infection modulation of host cell responses and evasion strategies for porcine reproductive and respiratory syndrome virus characterization of swine infertility and respiratory syndrome (sirs) virus (isolate atcc vr-2332) serum immune response to the proteins of porcine reproductive and respiratory syndrome (prrs) virus protection against porcine reproductive and respiratory syndrome virus (prrsv) infection through passive transfer of prrsv-neutralizing antibodies is dose dependent antibody production and blastogenesis response in pigs experimentally infected with porcine reproductive and respiratory syndrome virus the challenge of prrs immunology the primary neutralization epitope of porcine respiratory and reproductive syndrome virus strain vr-2332 is located in the middle of the gp5 ectodomain the major envelope proteins gp5 of a european reproductive and respiratory syndrome virus contains a neutralizing epitope in it n-terminal ectodomain monoclonal antibodies to the gp5 of porcine reproductive and respiratory syndrome virus are more effective in virus neutralization than monoclonal antibodies to the gp4 dissociation of porcine reproductive and respiratory syndrome virus neutralization from antibodies specific to major envelope protein surface epitopes characterization of antigenic regions in the porcine reproductive and respiratory syndrome virus by the use of peptide-specific serum antibodies chimeric viruses containing the nterminal ectodomains of gp5 and m proteins of porcine reproductive and respiratory syndrome virus do not change the cellular tropism of equine arteritis virus different european-type vaccines against porcine reproductive and respiratory syndrome virus have different immunological properties and confer different protection to pigs posttranslational processing and identification of a neutralization domain of the gp4 protein encoded in orf4 of the lelystadt virus passive transfer of virusspecific antibodies confer protection against reproductive failure induced by a virulent strain of porcine reproductive and respiratory syndrome virus and establishes sterilizing immunity effect of cellular changes and onset of humoral immunity on the replication of porcine reproductive and respiratory syndrome virus in the lungs of pigs a review of evidence for immunosuppression due to porcine reproductive and respiratory syndrome virus role of porcine reproductive and respiratory syndrome virus nucleocapsid protein in induction of interleukin-10 and regulatory t-lymphocytes (treg) induction of inducible cd4 ? cd25 ? porcine reproductive and respiratory syndrome virus induces cd4 ? pulmonary dendritic cells producing il-10 mediate tolerance induced by respiratory exposure to antigen comparative lymphocyte profile and the t and b cell spectratype of germfree piglets infected with three important viruses thymocyte and peripheral blood t lymphocyte subpopulation changes in piglets following in utero infection with porcine reproductive and respiratory syndrome virus age-dependent resistance to porcine reproductive and respiratory syndrome virus replication in swine identification of immunodominant t cell epitopes present in glycoprotein 5 of the north american genotype of porcine reproductive and respiratory syndrome virus cell-mediated immunity to porcine reproductive and respiratory syndrome virus in swine evaluation of protective immunity in gilts inoculated with the nadc-8 isolate of porcine reproductive and respiratory syndrome virus (prrsv) and challenged-exposed with an antigenically distinct isolate control and elimination of porcine reproductive and respiratory syndrome virus effect on total pigs weaned of herd closure for elimination of porcine reproductive and respiratory syndrome virus functional impairment of prrsv-specific peripheral cd3 ? cd8 high cell in vivo discovery of immunotherapy targets in the tumour microenvironment identification of cytotoxic t lymphocyte epitopes on swine viruses: multi-epitope design for universal t cell vaccine gamma delta lymphocyte response to porcine reproductive and respiratory syndrome virus efficacy of a modified live porcine reproductive and respiratory syndrome virus (prrsv) vaccine in pigs naturally exposed to heterologous european (italian cluster) field strain: clinical protection and cell-mediated immunity hematological and immunological parameters of 4 1/2-month old pigs infected with prrs virus effects of different us isolates of porcine reproductive and respiratory syndrome virus (prrsv) on blood and bone marrow parameters of experimentally infected pigs experimental porcine reproductive and respiratory syndrome virus infection in one-, four-, and 10-week-old pigs immunohistochemical identification of porcine reproductive and respiratory syndrome virus (prrsv) antigen in the heart and lymphoid system of three-week-old colostrum-deprived pigs comparison of the pathogenicity of two us porcine reproductive 138 and respiratory syndrome virus isolates with that of the lelystad virus attenuation of porcine reproductive and respiratory syndrome virus by molecular breeding of the virus envelope genes from genetically divergent strains comparative pathogenicity of nine us porcine reproductive and respiratory syndrome virus (prrsv) isolates in a five-week-old cesarean-derived, colostrum-deprived pig model temporal and morphologic characterization of the distribution of porcine reproductive and respiratory syndrome virus (prrsv) by in situ hybridization in pigs infected with isolates of prrsv that differ in virulence lymphodepletion and homeostatic proliferation: implications for transplantation autologous regulation of naive t cell homeostasis within the t cell compartment a causal link between lymphopenia and autoimmunity characterization of a porcine cd1-specific mab that distinguishes cd4/cd8 double-positive thymic from peripheral t lymphocytes antigen presentation in the thymus for positive selection and central tolerance induction the piglet as a model for b cell and immune system gnotobiotic pigs derivation and rearing antibody repertoire development in fetal and neonatal piglets. viii. colonization is required for newborn piglets to make serum antibodies to t-dependent and type 2 tindependent antigens antibody repertoire development in fetal and neonatal piglets. ix. three pamps act synergistically to allow germfree piglets to respond to ti-2 and td antigens antibody repertoire development in fetal and neonatal piglets. xvi. influenza stimulates adaptive immunity, class switch and diversification of the igg repertoire encoded by downstream cc genes development of the humoral immune response of the pig antibody repertoire development in fetal and neonatal piglets. xxiii. fetal piglets infected with a vaccine strain of prrs virus display the same immune dysregulation seen in isolator piglets antibody repertoire development in fetal and neonatal piglets. xix. undiversified b cells with hydrophobic hcdr3s preferentially proliferate in prrs porcine reproductive and respiratory syndrome virus (prrsv) subverts development of adaptive immunity by proliferation of germline-encoded b cells with hydrophobic hcdr3s lactate dehydrogenase-elevating virus and related viruses forced enrichment of hydrophobic amino acids in immunoglobulin cdr3-h3 impairs splenic b cell development but not antibody production porcine circoviruses: a minuscule yet mammoth paradox contribution of passive immunity to porcine respiratory coronavirus to protection against transmissible gastroenteritis virus challenge exposure in suckling pigs immunization of pregnant gilts with prcv induces lactogenic immunity for protection of nursing piglets from challenge with tgev the mammary gland in mucosal and regional immunity role of commensal bacteria in development of gut-associated lymphoid tissues and preimmune antibody repertoire order nidovirales novel structural protein in porcine reproductive and respiratory syndrome virus encoded by an alternative orf5 present in all arteriviruses efficient -2 frameshifting by mammalian ribosomes to synthesize an additional 8. arterivirus protein the origin and evolution of porcine reproductive and respiratory syndrome viruses arterivirus molecular biology and pathogenesis divergence time of porcine reproductive and respiratory syndrome virus subtypes genetic variation in porcine reproductive and respiratory syndrome virus isolates in the midwestern united states recombination is associated with an outbreak of novel highly pathogenic porcine reproductive and respiratory syndrome viruses in china recombination between north american strains of porcine reproductive and respiratory syndrome virus high frequency rna recombination in porcine reproductive and respiratory syndrome virus occurs preferentially between parental sequences with high similarity a bayesian phylogeographical analysis of type 1 porcine reproductive and respiratory syndrome virus (prrsv) molecular evolution of prrsv in europe: current state of play recombination analyses between two strains of porcine reproductive and respiratory syndrome virus in vivo genetic and phenotypic characterization of a 2006 united states porcine reproductive and respiratory virus isolate associated with high morbidity and mortality in the field complete genome analysis of rflp 184 isolates of porcine reproductive and respiratory syndrome virus characterization of proteins encoded by orfs 2 to 7 of lelystad virus intracellular synthesis, processing, and transport of proteins encoded by orfs 5 to 7 of porcine reproductive and respiratory syndrome virus structural proteins of equine arteritis virus the envelope proteins of lactate dehydrogenase-elevating virus and their membrane topography arterivirus structural proteins and assembly signal peptide cleavage from gp5 of prrsv: a minor fraction of molecules retains the decoy epitope, a presumed molecular cause for viral persistence neutralizing antibody responses of pigs infected with natural gp5 n-glycan mutants of porcine reproductive and respiratory syndrome virus role of neutralizing antibodies in prrsv protective immunity identification of neutralizing and nonneutralizing epitopes in the porcine reproductive and respiratory syndrome virus gp5 ectodomain apoptosis induced in vivo during acute infection by porcine reproductive and respiratory syndrome virus gp5 ectodomain epitope of porcine reproductive and respiratory syndrome virus, strain lelystad virus monoclonal antibody analysis of porcine reproductive and respiratory syndrome virus epitopes associated with antibody-dependent enhancement and neutralization of virus infection effect of virusspecific antibodies on attachment, internalization and infection of porcine reproductive and respiratory syndrome virus in primary macrophages involvement of the matrix protein in attachment of porcine reproductive and respiratory syndrome virus to a heparinlike receptor on porcine alveolar macrophages gp4 of porcine reproductive and respiratory syndrome virus contains a neutralizing epitope that is susceptible to immunoselection in vitro molecular assessment of the role of envelope-associated structural proteins in cross neutralization among different prrs viruses immune evasion of porcine reproductive and respiratory syndrome virus through glycan shielding involves both glycoprotein 5 as well as glycoprotein 3 broadening the heterologous cross-neutralizing antibody inducing ability of porcine reproductive and respiratory syndrome virus by breeding the gp4 or m genes dna shuffling of the gp3 genes of porcine reproductive and respiratory syndrome virus (prrsv) produces a chimeric virus with an improved cross-neutralizing ability against a heterologous prrsv strain proteins encoded by open reading frames 3 and 4 of the genome of lelystad virus (arteriviridae) are structural proteins of the virion glycosylation of minor envelope glycoproteins of porcine reproductive and respiratory syndrome virus in infectious virus recovery, receptor interaction, and immune response formation of disulfidelinked complexes between the three minor envelope glycoproteins (gp2b, gp3, and gp4) of equine arteritis virus glycosyl-phosphatidylinositol (gpi)-anchored membrane association of the porcine reproductive and respiratory syndrome virus gp4 glycoprotein and its co-localization with cd163 in lipid rafts potential role of porcine reproductive and respiratory syndrome virus structural protein gp2 in apoptosis inhibition the structural biology of prrsv cryo-electron tomography of porcine reproductive and respiratory syndrome virus: organization of the nucleocapsid nuclear localization of non-structural protein 1 and nucleocapsid protein of equine arteritis virus competitive elisa for detection of antibodies to porcine reproductive and respiratory syndrome virus using recombinant e. coli-expressed nucleocapsid protein as antigen pathogenicity of swine influenza h1n1 virus antigenically distinguishable from classical and european strains serologic marker candidates identified among b-cell linear epitopes of nsp2 and structural proteins of a north american strain of porcine reproductive and respiratory syndrome virus epitope mapping porcine reproductive and respiratory syndrome virus by phage display: the nsp2 fragment of the replicase polyprotein contains a cluster of b-cell epitopes monoclonal antibody and porcine antisera recognized b-cell epitopes of nsp2 protein of a chinese strain of porcine reproductive and respiratory syndrome virus highly divergent strains of porcine reproductive and respiratory syndrome virus incorporate multiple isoforms of nonstructural protein 2 into virions identification of 5 0 and 3 0 cis-acting elements of the porcine reproductive and respiratory syndrome virus: acquisition of novel 5 0 au-rich sequences restored replication of a 5 0 -proximal 7-nucleotide deletion mutant a full-length cdna infectious clone of north american type 1 porcine reproductive and respiratory syndrome virus: expression of green fluorescent protein in the nsp2 region identification of nonessential regions of the nsp2 replicase protein of porcine reproductive and respiratory syndrome virus strain vr-2332 for replication in cell culture infectious clonederived viruses from virulent and vaccine strains of porcine reproductive and respiratory syndrome virus mimic biological properties of their parental viruses in a pregnant sow model a dna-launched reverse genetics system for porcine reproductive and respiratory syndrome virus reveals that homodimerization of the nucleocapsid protein is essential for virus infectivity an infectious cdna clone of a highly pathogenic porcine reproductive and respiratory syndrome virus variant associated with porcine high fever syndrome infectious transcripts from cloned genomelength cdna of porcine reproductive and respiratory syndrome virus generation of an infectious clone of vr-2332, a highly virulent north american-type isolate of porcine reproductive and respiratory syndrome virus recovery of viable porcine reproductive and respiratory syndrome virus from an infectious clone containing a partial deletion within the nsp2-encoding region a highly pathogenic porcine reproductive and respiratory syndrome virus generated from an infectious cdna clone retains the in vivo virulence and transmissibility properties of the parental virus attenuation of porcine reproductive and respiratory syndrome virus strain mn184 using chimeric construction with vaccine sequence the 30-amino-acid deletion in the nsp2 of highly pathogenic porcine reproductive and respiratory syndrome virus emerging in china is not related to its virulence expression of a foreign epitope by porcine reproductive and respiratory syndrome virus influence of nlinked glycosylation of porcine reproductive and respiratory syndrome virus gp5 on virus infectivity, antigenicity, and ability to induce neutralizing antibodies the nsp1alpha and nsp1 papain-like autoproteinases are essential for porcine reproductive and respiratory syndrome virus rna synthesis cysteine residues of the porcine reproductive and respiratory syndrome virus small envelope protein are nonessential for virus infectivity functional mapping of the porcine reproductive and respiratory syndrome virus capsid protein nuclear localization signal and its pathogenic association chinese and vietnamese strains of hp-prrsv cause different pathogenic outcomes in united states high health swine identification of virulence determinants of porcine reproductive and respiratory syndrome virus through construction of chimeric clones viable porcine arteriviruses with deletions proximal to the 3 0 end of the genome identification of amino acid residues important for anti-ifn activity of porcine reproductive and respiratory syndrome virus non-structural protein 1 a virulent strain of porcine reproductive and respiratory syndrome virus does not up-regulate interleukin-10 in vitro and in vivo construction of chimeric arteriviruses reveals that the ectodomain of the major glycoprotein is not the main determinant of equine arteritis virus tropism in cell culture replacement of the heterologous 5( 0 ) untranslated region allows preservation of the fully functional activities of type 2 porcine reproductive and respiratory syndrome virus arterivirus minor envelope proteins are a major determinant of viral tropism in cell culture chimeric arteriviruses generated by swapping of the m protein ectodomain rule out a role of this domain in viral targeting chimeric porcine reproductive and respiratory syndrome viruses reveal full function of genotype 1 envelope proteins in the backbone of genotype 2 vaccine efficacy of porcine reproductive and respiratory syndrome virus chimeras the isolator piglet: a model for studying the development of adaptive immunity dual infections of feeder pigs with porcine reproductive and respiratory syndrome virus followed by porcine respiratory coronavirus or swine influenza virus: a clinical and virological study negative impact of porcine reproductive and respiratory syndrome virus infection on the efficacy of classical swine fever vaccine the level of virus-specific t-cell and macrophage recruitment in porcine reproductive and respiratory syndrome virus infection in pigs is independent of virus load antibody repertoire development in fetal and neonatal pigs. xiii. ''hybrid vh genes'' and the preimmune repertoire re-visited antibody repertoire development in fetal and neonatal piglets. xxi. vh usage remains constant during development in fetal piglets and postnatally in pigs exposed to environmental antigen adaptation o a commercial elisa to determine the igg avidity in sweep experimentally and naturally infected with neospora caninum porcine igg: structure, genetics and evolution linkage haplotype for igg and iga subclass genes antibody repertoire development in fetal and neonatal piglets. xvii. igg subclass transcription revisited with emphasis on new igg3 resolution of an immunodiagnostic dilemma: heavy chain chimeric antibodies for species in which plasmacytomas are unknown induction of foot and mouth disease virus (fmdv) specific cytotoxic t cells killing by vaccination acknowledgments the authors acknowledge and thank nancy wertz for her help in assembly of the manuscript and to dr. eric nelson for scientific review of the manuscript. key: cord-017784-4r3fpmlb authors: foccillo, giampiero title: the infections causing acute respiratory failure in elderly patients date: 2019-08-06 journal: ventilatory support and oxygen therapy in elder, palliative and end-of-life care patients doi: 10.1007/978-3-030-26664-6_5 sha: doc_id: 17784 cord_uid: 4r3fpmlb the immune system of older individuals declines with advancing age (“immunosenescence”) increasing susceptibility to infection, as well as to an increased risk of a worse outcome. severe community-acquired pneumonia and acute exacerbations of chronic obstructive pulmonary disease (aecopd) are causes of acute respiratory failure (arf) in elderly patients. non-invasive mechanical ventilation (niv) is effective in the treatment of patients with arf, above all in case of aecopd. aging is accompanied by profound morphological and physiological alterations. in particular, the immune system undergoes a complex series of remodeling/restructuring events, involving almost all compartments, both cell-mediated immunity and humoral immune responses. this process termed immunosenescence or immune dysregulation, together changes in lung function who occur with advancing age, play a critical role in the manifestation of age-related pulmonary diseases such as infections (i.e., pneumonia), chronic obstructive pulmonary disease (copd), and increased the risk for develop sepsis [1] . respiratory failure is not a disease per se but a consequence of the problems that interfere with the ability to breathe. the term refers to the inability to perform adequately the fundamental functions of respiration: to deliver oxygen to the blood and to eliminate carbon dioxide from it. respiratory failure has many causes and can come on abruptly (acute respiratory failure), when the underlying cause progresses rapidly, or slowly (chronic respiratory failure), when it is associated over months or even years with a progressive underlying process. triggering causes of arf in advanced aged patients are especially acute heart decompensation, severe community-acquired pneumonia (cap), acute exacerbations of copd (aecopd), and pulmonary embolism. pneumothorax, lung cancer, severe sepsis, and acute asthma were less frequent (<5%) [2] . acute respiratory failure is a condition in which the respiratory system fails in one or both of its gas functions, i.e., oxygenation (pao 2 <60 mmhg) of and/or elimination of carbon dioxide (arterial carbon dioxide tension (paco 2 ) >45 mmhg). in practice, it may be classified as either hypoxemic or hypercapnic. hypoxemic respiratory failure (type i) is characterized by an arterial oxygen tension (pao 2 ) lower than 60 mm hg with a normal or low arterial carbon dioxide tension (paco 2 ). this is the most common form of respiratory failure, and it can be associated with virtually all acute diseases of the lung, which generally involve fluid filling or collapse of alveolar units. the four pathophysiological mechanisms related to hypoxemic arf are [3, 4] : 1. ventilation/perfusion inequality which is the main mechanisms in an emergency setting (congestive heart failure or pneumonia) 2. increased shunt (acute respiratory distress syndrome) 3. alveolar hypoventilation (chronic obstructive pulmonary disease) 4. diffusion impairment (pulmonary fibrosis) hypercapnic respiratory failure (type ii) is characterized by a paco 2 higher than 45 mmhg. hypoxemia is common in patients with hypercapnic respiratory failure who are breathing room air. common etiologies include drug overdose, neuromuscular disease, chest wall abnormalities, and severe airway disorders (e.g., chronic obstructive pulmonary disease). in case of a copd exacerbation, an acute inflammation in the airways increases resistance to air flow with consequent air trapping. increased resistance and elastic load due to air trapping place respiratory muscles at a mechanical disadvantage and increases the work of breathing and leads to arf. infectious factor such as pneumonia with/without sepsis caused by a variety of pathogens, including bacteria, viruses, malaria, and fungal is the medical condition that is most commonly associated with acute respiratory distress syndrome (ards). sepsis due to nonpulmonary infections, aspiration of gastric contents, and major trauma with shock also commonly precipitate the injury [5] . since the same kinds of immune cells and immune proteins, including immunoglobulins and complements, are observed in the pathologic lesions of pneumonia, ards, and other organ-specific pathologic lesions, it may be a reasonable assumption that sepsis and ards have similar underlying mechanisms, characterized by inflammation and endothelial dysfunction [6] . ards is a heterogeneous syndrome characterized by increased permeability of pulmonary capillary endothelial cells and alveolar epithelial cells, leading to hypoxemia that is refractory to usual oxygen therapy [7] . the severity of ards is associated with poor prognosis and higher mortality, and, by the berlin definition, diagnostic hypoxemia is defined as decreased arterial pao 2 /fio 2 ratio with parameters of 201-300 mmhg for mild ards, 101-200 mmhg for moderate ards, and <100 mmhg for severe ards [8] . considering the immunopathogenesis of pneumonia, sepsis, and ards, early and appropriate antimicrobial therapy is critical to reduce the number of pathogens and pathogen-originated substances, thereby inducing early recovery from the disease and better outcome. similarly, although the knowledge about the effectiveness of antibiotics in the management of acute exacerbations of copd remains limited by the lack of strong evidence, studies show that early antibiotic administration is associated with improved outcomes among patients hospitalized for acute exacerbations of copd [9] . furthermore inadequate antibiotic therapy, which through incomplete resolution of the initial exacerbation and persistent bacterial infection, is likely to influence the risk of relapse. exacerbations of chronic obstructive pulmonary disease (copd) are defined as sustained worsening of a patient's condition beyond normal day-to-day variations that is acute in onset, and that may also require a change in medication and/or hospitalization [10] . an acute copd exacerbation can be viewed as an acute inflammatory event superimposed on chronic inflammation associated with copd. most aecopd may be due to viral infection or bacterial infection (50%), but irritants such as smoke or environmental factors such as low temperature and air pollution account for 15-20% of exacerbations [11] . in patients with mild disease, streptococcus pneumoniae, haemophilus influenzae, and moraxella catarrhalis are the predominant microorganisms, whereas in patients with severe copd, requiring mechanical ventilation, levels of these bacteria are reduced and other microorganism such as haemophilus parainfluenzae and pseudomonas aeruginosa predominate. it has also been observed that the severity of lung function, measured by fev1, has an impact on the microbiology of exacerbation [12] . patients with increased airway obstruction and frequent exacerbations, the microbiology of the exacerbations is often more complex, with a predominance of enterobacteriaceae and pseudomonas aeruginosa. the role of atypical microorganisms is the subject of debate. according to some authors, there is no association between the presence of organisms such as chlamydia pneumoniae, legionella spp., mycoplasma pneumoniae, and aecopd [13] . however, in aecb, several older studies and a few recent studies have implicated atypical bacteria in only 5-10% of episodes [14] . although it is difficult to define precisely the proportion of exacerbations caused by viruses, they are often implicated in aecopd (up to 30% of cases) [15] . the most common viruses associated with exacerbations of copd are rhinoviruses, but in more severe exacerbations requiring hospitalization, influenza virus is more common. viral infection may, also, facilitate subsequent bacterial infection or increase the number of bacteria already present in the lower airways. the use of antibiotics in exacerbations copd remains controversial; therefore, in the era of antibiotic resistance, the identification of clinical characteristics that identify patients with aecopd that can be safely treated without antibiotics is extremely important. there is evidence supporting the use antibiotics in exacerbations when patients have clinical signs of a bacterial infection, e.g., increased sputum purulence, especially when there are changes in the color of sputum [16] . antibiotics are also recommended for patients with exacerbations requiring mechanical ventilation, as this has been shown significantly to reduce mortality and the risk of secondary pneumonia. in summary, antibiotics should be given to patients with exacerbations of copd who have three cardinal symptoms: increase in dyspnea, sputum volume, and sputum purulence; have two of the cardinal symptoms if increased purulence of sputum is one of the two symptoms; or require mechanical ventilation (invasive or non-invasive) [10] . (table 5. after the decision to initiate empirical antibiotic therapy, the choice of empirical antibiotic regimen most appropriate for treating an episode of aecopd should be based on the following: 1. the severity of copd, established by fev1 and the history of more than three exacerbations in the previous 12 months. 2. the patients age (≥ or <65 years). 3. significant comorbidity (diabetes mellitus, liver cirrhosis, chronic renal failure, or heart disease). 4. the risk of infection due to p. aeruginosa, which must be considered in the presence of two of the following risk factors: recent hospitalization, frequent (>4 times/year) or recent (within the previous 3 months) administration of antibiotics, severe disease (fev1 <30%), or the use of oral corticosteroids (>10 mg of prednisone daily in the previous 2 weeks). between 10% and 20% of patients with moderate to severe exacerbations do not respond to initial empirical therapy and require a change of antibiotic. in these cases, the infection can be caused by staphylococcus aureus, p. aeruginosa, or some atypical microorganism not covered by the initial regimen or because of resistant organisms (such as s. pneumoniae); hence, a microbiological assessment would help to adjust the antibiotic treatment of second choice [17] . there are not enough data concerning the role of antiviral therapy in respiratory failure due to copd. there are no standard procedures that determine the dose and duration of antibiotic treatment in patients with aecopd. therefore, several studies demonstrate that short-term antibiotic use is associated with very important advantages such as reduction of exposure that will result in decreased bacterial resistance, enhanced compliance, and decreased side effects. the recommended length of antibiotics therapy is 5-7 days [10]. the incidence of community-acquired pneumonia increases with age, reaching 25-44 cases per 1000 inhabitants/year in the population over the age of 65 years, up to four times that of younger patients [18] . elderly patients are more predisposed to pneumonia because of their impaired gag reflex, decreased mucociliary function, waning immunity, impaired febrile response, and chronic disease (diabetes mellitus, chronic obstructive pulmonary disease, chronic heart failure, cancer and chronic renal insufficiency). furthermore, central nervous system disorders and/or an impaired gag reflex predispose elderly patients to aspiration pneumonia. with respect to the youngest population, pneumonia in the elderly subjects is more severe, requires often hospitalization, and is characterized by a longer length of stay and by greater mortality, particularly in patients with comorbidities [19] . it is important to remember that clinical presentation of pneumonia in the elderly may be subtle and may be afebrile. altered mental status, a sudden decline in functional capacity, worsening of underlying diseases, and falls may be the only findings. as stated by sir william osler [20] "in old age, pneumonia may be latent, coming on without chill, the cough and expectoration are slight, the physical signs ill defined and changeable, and the constitutional symptoms out of all proportion." as with any infectious disease, identifying the causative agent in cap can be extremely useful in guiding antimicrobial therapy. unfortunately, despite the use of more sensitive and specific diagnostic methods to define an etiologic pathogen in community-acquired pneumonia requiring hospitalization, in the majority of patients, a microbiologic diagnosis cannot be made [21] . in most cases, the microbiologic patterns observed in the elderly do not differ significantly from those observed in younger populations although with a different age-related distribution. streptococcus pneumoniae is still the most important pathogen in younger as well as older adults and the incidence of pneumococcal pneumonia generally increases with age. haemophilus influenzae is relatively more common in elderly patients than in non-elderly adults and are the second most frequently identified microorganisms, while moraxella catarrhalis is of particular importance as a cause of community-acquired pneumonia in patients with chronic bronchitis. the most discernible differences between the two groups were gram-negatives, especially enterobacteriaceae were found more frequently in the elderly, whereas certain atypical pathogens (legionella pneumophila, mycoplasma pneumoniae, and coxiella burnetii) were more frequent in younger patient [22] . among the atypical bacteria, chlamydophila pneumoniae is the most common agent, while mycoplasma pneumoniae is less frequently associated with cap in this age group. although uncommon, legionella pneumophila should be considered in elderly adults presenting with atypical symptoms (e.g., headache, weakness, altered mental status, gastrointestinal disturbances, or bradycardia in the setting of a paucity of respiratory symptoms) or those with severe pneumonia. gram-negative organisms are an uncommon cause of cap. however, in severely debilitated or chronically ill elderly patients from the community, a high index of suspicion for gram-negative bacilli may be warranted, especially in those who fail to improve on standard therapy. the probability of mrsa in patients hospitalized in conventional wards is low being more frequent in severe cap understood as the need for admission in an intensive care unit (icu). thus, in the presence of less than two factors of multiresistance (severe pneumonia, hospitalization in the previous 90 days, living in a residence, severe basal dependence for basic daily life activities, immunodepression, or the taking of antibiotics in the previous 6 months) coverage against mrsa should be included if the patient presents severe disease. pseudomonas aeruginosa is not a frequent pathogen in cap; factors increasing the likelihood for pseudomonas infection are structural lung disease such as bronchiectasis or severe copd with fev1 <35%, severe pneumonia requiring icu admission, frequent or recent use of antibiotics, recent hospital admission and steroid use. aspiration pneumonia refers to an infection that develops after the entrance of pathologic oropharyngeal microbes into the lung. major risk factors for aspiration include depressed consciousness, compromised airway defenses, dysphagia, gastroesophageal reflux disease, and recurrent vomiting. most patients with communityacquired aspiration pneumonia have a mixed infection with anaerobic and aerobic bacteria, whereas those with hospital-acquired pneumonia will more likely have gram-negative infections, including pseudomonas aeruginosa [23] . in regard to viral etiology, the influenza virus and respiratory syncytial virus are most important cause of pneumonia in the elderly, often within the context of epidemic outbreaks and may cause both viral primary pneumonias such as bacterial superinfection by s. pneumoniae, s. aureus, and haemophilus influenzae. (table 5. antimicrobials are the cornerstone of therapy for cap in any population, but limited data are available regarding the specific treatments for elderly patients. the only guidelines for the management of community-acquired pneumonia in the elderly patient are published in the year 2014 in spanish [24] . initial antibiotic selection for cap is often empirical, as the causative pathogen cannot be predicted from clinical laboratory or radiological findings and therapy must be started swiftly as rapid antibiotic delivery is associated with a better outcome [25] . empirical antimicrobial therapy must include coverage for the most prevalent pathogens and should be based on several aspects such as the severity of diseases, local patterns of antimicrobial resistances, multi-drug-resistant risk factors, and drug allergies. international guidelines agree that patients hospitalized with non-severe cap should be started on empirical combination therapy using a β-lactam plus a macrolide or a respiratory fluoroquinolone alone (levofloxacin, moxifloxacin, and gatifloxacin). the respiratory quinolones are quinolones that are highly active against both the typical and atypical respiratory pathogens causing cap. because ciprofloxacin is relatively inactive against streptococcus pneumoniae, even though it is active against the atypical pathogens, it is not termed a "respiratory quinolone." in patients with severe community-acquired pneumonia (admission in icu or intermediate care), the guidelines recommend a minimum of a β-lactam plus either a macrolide or a quinolone. the combination treatment offers an advantage over monotherapy by expanding the antimicrobial coverage and probably by immunomodulation (macrolides, quinolones). in severe patients with risk factors for mrsa vancomycin or linezolid should be added. cap patients with risk factors for p. aeruginosa should receive empirical combination therapy as an antipneumococcal anti-pseudomonal β-lactam plus either ciprofloxacin or high-dose levofloxacin or the above β-lactam plus an aminoglycoside and azithromycin is an appropriate regimen. in patients with risk factors of aspiration, an antibiotic should be used which should also cover s. pneumoniae and be effective against anaerobes and enterobacteriaceae since these may be the causal microorganisms involved. in most guidelines, β-lactam/β-lactamase inhibitors (amoxicillin-clavulanate, ampicillin/sulbactam/piperacillin/tazobactam) are considered to be the antibiotics of choice. ceftriaxone plus clindamycin or metronidazole and respiratory fluoroquinolone are the alternative. for patients at the increasing rise of resistances of enterobacteriaceae ertapenem this therapeutic option is valid mainly for its sensitivity versus anaerobes, s. pneumoniae, and all the enterobacteriaceae, including extended-spectrum β-lactamase-producing (esbl). antibiotic agents with specific anaerobic activity (metronidazole or clindamycin) are not routinely warranted and may be indicated only in patients with severe periodontal disease, putrid sputum, or evidence of necrotizing pneumonia or lung abscess on radiographs of the chest. antiviral treatment is recommended as early as possible for any patient with confirmed or suspected influenza pneumonia and should not be delayed by confirmatory laboratory testing results. neuraminidase inhibitors, oseltamivir and zanamivir, are the agents of choice. the usual dosing of oseltamivir for the treatment of influenza is 75 mg orally twice daily and of zanamivir is 10 mg (2 inhalations) twice daily. the recommended duration for antiviral treatment is 5 days. the optimal duration of antibiotic therapy is not yet known. bts guidelines [26] propose 7-10 days treatment for most patients with severe cap, whereas european guidelines [27] suggest that the duration of treatment should generally not exceed 8 days in a patient who responds to initial therapy. the use of biomarkers such as procalcitonin or the c-reactive protein may be useful to shorten the duration of antibiotic treatment [28] . the duration of antibiotic treatment may be prolonged in patients receiving initial inadequate antibiotic therapy, patients with extrapulmonary infection (e.g., endocarditis, meningitis), patients infected with multi-drugresistant organism (e.g., s. aureus) and patients with necrotizing pneumonia. regarding the dosing (table 5. 3), there are many considerations to be made when caring for elderly patients: multiple comorbidities, polypharmacy potentially resulting in drug interactions, pharmacokinetic and pharmacodynamic changes that can result in altered drug metabolism and subsequent toxic effects of drug accumulation [29] . despite the complexity of all of these changes, however, the singular impairment that most significantly influences the medical management of infection in older patients is the decline in renal function commonly observed in this group. in patients with impaired renal function, antibiotics should always be started at full initial doses and then adjusted based on renal function. lower respiratory tract infections, including pneumonia and exacerbation of chronic obstructive pulmonary disease, are among the most common causes of arf in elderly people and the most important cause of hospitalization. moreover pulmonary infection is also the most frequent single cause of ards. in the management of respiratory failure, it is mandatory reversing and/or preventing tissue hypoxia through conventional oxygen therapy or invasive or non-invasive mechanical ventilation. besides, early administration of appropriate antimicrobials has been postulated as a key strategy in the survival of patients with very severe infections requiring intensive care unit admission taking into account the most risk of adverse events of an antimicrobial in a frail elderly patient. 1. acute respiratory failure is a frequent complication in elderly patients. 2. infections (cap and aecopd) are among the most frequent triggering causes of arf in advanced aged patients. 3. antimicrobials are the cornerstone of therapy for cap and in copd exacerbations when patients have clinical signs of a bacterial infection. 4. antibiotic duration should be as shorter as possible, based on the pathogen, clinical response, and presence of complications. 5. a judicious use of antibiotic therapy in the respiratory tract infections is critical in the era of antibiotic resistance. the impact of immunosenescence on pulmonary disease acute respiratory failure in the elderly: etiology, emergency diagnosis and prognosis respiratory failure acute respiratory failure in the elderly: diagnosis and prognosis acute respiratory distress syndrome, and early immune-modulator therapy the acute respiratory distress syndrome acute respiratory distress syndrome acute respiratory distress syndrome: the berlin definition antibiotic therapy and treatment failure in patients hospitalized for acute exacerbations of chronic obstructive pulmonary disease global initiative for chronic obstructive lung diseases. global strategy for the diagnosis, management and prevention of chronic obstructive pulmonary disease infection in the pathogenesis and course of chronic obstructive pulmonary disease infective exacerbations of chronic bronchitis: relation between bacteriologic etiology and lung function the role of atypical pathogens in exacerbations of chronic obstructive pulmonary disease infectious etiology of acute exacerbations of chronic bronchitis prevalence of viral infection detected by pcr and rt-pcr in patients with acute exacerbation of copd: a systematic review sputum colour and bacteria in chronic bronchitis exacerbations: a pooled analysis pathogen-directed therapy in acute exacerbations of copd pneumonia in the very old community-acquired pneumonia in very elderly patients: causative organisms, clinical characteristics, and outcomes bacterial pneumonia in the elderly: the observations of sir william osler in retrospect community-acquired pneumonia requiring hospitalization among u.s. adults pneumonia in the elderly: a review of the epidemiology, pathogenesis, microbiology, and clinical features aspiration-related pulmonary syndromes guidelines for the management of community-acquired pneumonia in the elderly patient infectious diseases society of america/ american thoracic society consensus guidelines on the management of community-acquired pneumonia in adults guidelines for the management of communityacquired pneumonia in adults: update guidelines for the management of adult lower respiratory tract infections blood biomarkers for personalized treatment and patient management decisions in community-acquired pneumonia antibacterial agents in the elderly key: cord-291217-fpte1q9m authors: adams, madeleine; doull, iolo title: management of bronchiolitis date: 2009-05-13 journal: paediatr child health (oxford) doi: 10.1016/j.paed.2009.03.001 sha: doc_id: 291217 cord_uid: fpte1q9m bronchiolitis is the commonest cause of hospital admission in infancy. severity varies from mild and self-limiting through to respiratory failure requiring intensive care and ventilation. many viruses cause bronchiolitis, the commonest being respiratory syncytial virus (rsv). supportive care is the mainstay of treatment, with emphasis on fluid replacement and oxygen therapy. agents with evidence of no benefit in acute bronchiolitis include β(2) agonists, ipratropium, montelukast, corticosteroids, antiviral agents such as ribavirin or rsv immunoglobulin, physiotherapy, nebulized deoxyribonuclease or antibiotics. it is possible that nebulized epinephrine has a small short-term effect, and that nebulized 3% hypertonic saline administered with a bronchodilator may decrease length of stay in hospital. preventative strategies such as rsv immunoglobulin or the anti-rsv monoclonal antibody palivizumab can decrease disease severity. in 1963 eor reynolds concluded that 'oxygen therapy is vitally important in bronchiolitis and there is little convincing evidence that any other therapy is consistently or even occasionally useful'. it is arguable that there has been little progress in the subsequent 46 years. treatments that might be effective include nebulized 3% hypertonic saline mixed with a bronchodilator, and ventilatory support for respiratory failure. for the majority of patients, however, supportive management is the mainstay, with emphasis on treating insufficient fluid intake and hypoxia. bronchiolitis is the commonest cause of hospitalization in infancy. it usually affects infants aged 1-6 months, although it can occur up to 2 years of age, and is usually a mild self-limiting illness that does not require medical intervention. it is a clinical syndrome characterized initially by coryzal symptoms followed madeleine adams mrcpch by onset of harsh cough, tachypnoea and wheezing. on examination there may be chest hyperinflation with costal recession, and fine inspiratory crackles and polyphonic expiratory wheeze on auscultation. a significant contributor to confusion over the management of bronchiolitis is the absence of an internationally agreed common definition. in the united kingdom, australasia, and parts of europe, bronchiolitis is interpreted as the presence of tachypnoea, hyperinflation of the chest, and characteristically widespread fine end inspiratory crackles on auscultation. wheeze is commonly but not invariably present. the pattern of illness is virtually always seen in the first year of life, most commonly in the first 6 months of life. in contrast, in north america and other parts of europe, bronchiolitis is a term for any viral infection of the lower respiratory tract in the first 2 years of life, and may include children with recurrent wheeze. the confusion over definition is compounded by different types of study; many studies in inpatients will be closer to the uk and australasian definition, while many studies in outpatients will include mostly children corresponding to the north american and european definition. furthermore there is no widely accepted validated scoring system to measure illness severity, making comparisons between studies difficult. commonly, outcome measures in interventional studies will include summation scores of symptoms and signs, oxygen saturations, respiratory rate, and length of stay in hospital. approximately 1-3% of infants will be hospitalized with bronchiolitis, of whom 1-2% will require ventilation for either respiratory failure or apnoeas. hospitalization for bronchiolitis is commoner in boys and in lower socioeconomic groups. preterm infants (less than 32 weeks) and infants with chronic lung disease, congenital cardiac abnormalities, or immune deficiencies are at higher risk of severe disease. mortality rates in hospitalized infants are 0.5-1.7%. respiratory syncytial virus (rsv) accounts for 50-90% of cases of bronchiolitis resulting in approximately 20000 uk admissions per year. annual epidemics of rsv occur between late autumn and spring (october to march in the northern hemisphere). the virus rapidly infects the respiratory tract epithelium, causing epithelial necrosis and destruction of the cilia. there is a florid inflammatory response with neutrophil and lymphocytic cellular infiltration and oedema of the submucosa, and an imbalance in cytokines with an excess of th type ii over type i cytokines demonstrated by high interleukin 4 (il4)/interferon γ ratio. there is increased mucus production from goblet cells which, in combination with the decimated epithelial cells, results in mucus plugging. mucus plugging causes obstruction of bronchioles with resultant air trapping leading to areas of hyperinflation and airway collapse. as the respiratory epithelium regenerates, the new non-ciliated cells are poorly equipped to clear the inflammatory debris. hypoxaemia is primarily due to ventilation/perfusion mismatch, and hypercapnoea is a relatively late phenomenon. after rsv, the next most frequent cause is probably human metapneumovirus (hmpv) which causes a similar clinical picture as rsv, although dual infection with both rsv and hmpv may cause more severe disease. rhinovirus may be a commoner cause in older infants, and less frequent causes include adenovirus, parainfluenza, influenza, coronavirus, and the more recently identified bocavirus. it is possible that bacterial superinfection is associated with increased severity of disease. interventions can be classed as supportive, therapeutic or preventative. the current management of bronchiolitis is primarily supportive, concentrating on the major effects of the condition, namely inadequate feeding, respiratory distress and apnoeas. inadequate feeding is usually secondary to respiratory distress and the consequent increased work of breathing. the infant will be tachypnoeic, may have bouts of coughing with increased upper airway secretions, and thus may struggle to feed adequately. tachypnoea increases fluid loss, and so the infant can easily become dehydrated. infants with bronchiolitis are generally intolerant of interventions, and so minimal handling is recommended. for milder cases giving small volumes of feed at regular intervals may be sufficient. administration of oxygen may be enough to decrease work of breathing, so allowing regular fluid intake. if this proves inadequate, a nasogastric tube can be passed to administer enteral fluids -initially bolus feeds, but if this worsens respiratory distress, by continuous feeding. ultimately if the infant is unable to tolerate enteral feeds, fluids can be administered intravenously. rsv infection can result in inappropriate syndrome of inappropriate antidiuretic hormone secretion (siadh), and so after determining serum electrolytes, intravenous fluids are usually restricted to 75% of maintenance requirements. the ventilation/perfusion mismatch characteristic of bronchiolitis results in hypoxia and increased work of breathing. oxygen is the mainstay of treatment for respiratory distress, and is usually administered via head-box to minimize handling. for higher concentrations, a combination of head box and facemask or rebreathing bag may be used, although this usually implies significant respiratory failure and often precedes respiratory collapse. increasing impairment of gas exchange will result in hypoxia unresponsive to head-box oxygen and hypercarbia, eventually leading to physical exhaustion and complete respiratory failure. between 2% and 5% of bronchiolitis admissions will have respiratory failure, and increasingly non-invasive ventilation is used to offer continuous positive airway pressure (cpap) support, usually via nasal prongs to decrease the need for intubation and sedation. it is thought that the action of cpap is through prevention of airway collapse during the respiratory cycle, so preserving ventilation and decreasing ventilation/perfusion mismatch. observational studies suggest that in bronchiolitis cpap decreases the respiratory rate, pulse rate, and partial pressure of carbon dioxide, and so decreases the intubation rate and consequently the rate of ventilator-associated pneumonia. cpap does not appear to affect length of hospital stay or duration of ventilation. a controlled study demonstrated that if initiated early, nasal cpap resulted in a significant decrease in pco 2 and was well tolerated without any significant complications, suggesting that earlier intervention would improve outcome. if respiratory failure continues despite nasal cpap, full intubation and ventilation is warranted. in those that are mechanically ventilated, administration of exogenous surfactant may decrease duration of mechanical ventilation. if mechanical ventilation is ineffective, there may be a role for extracorporeal membrane oxygenation (ecmo). interpretation of the evidence for the efficacy of bronchodilators in bronchiolitis is hampered by the heterogeneity of the inclusion criteria in different studies. many studies, particularly if outpatient-based, will include infants up to 2 years old with recurrent wheeze, and it is arguable that many of these infants may have an asthma phenotype. three classes of bronchodilators have been trialled in bronchiolitis: β 2 agonists, ipratropium, and adrenergic agents, all of which have shown benefit in asthmatics. bronchodilators are the treatment of choice for acute asthma in older children and adults, and there is some evidence that bronchodilators may produce short-term improvement in clinical scores in outpatient-based studies of bronchiolitis. an earlier meta-analysis by flores and horwitz concluded that β 2 agonists had a statistically significant but clinically insignificant effect on oxygen saturations and heart rate when used in milder cases in an outpatient setting, but had no significant effect on hospitalizations. a later systematic review of all pharmacological treatments concluded that β 2 agonists had no significant beneficial effects. the most recent cochrane review of 22 trials involving 1428 infants with bronchiolitis who received inhaled bronchodilators (including β 2 agonists, ipratropium and adrenergic agents) reported a significant improvement in overall average clinical score, but had no effect on either pulse oximetry measurements or on risk of hospitalization. however, the inclusion criteria for many of the studies allowed children with recurrent wheezing up to 24 months of age, and subgroup analysis suggested that benefit was primarily in outpatient studies of shorter duration, suggesting that many of those who showed benefit may have had recurrent wheeze rather than isolated bronchiolitis. an earlier systematic review identified four studies assessing the use of nebulized ipratropium bromide in bronchiolitis and concluded that there was no significant benefit. a subsequent cochrane analysis reached similar conclusions. it is likely that ipratropium has a role in young infants with recurrent wheeze. adrenergic agents such as epinephrine appear attractive as they combine the β-adrenergic effects of bronchodilation with the α-adrenergic effects of vasoconstriction of the bronchiolar vasculature. the vasoconstrictive action in particular could potentially decrease the characteristic mucosal oedema and mucus hyper-secretion. a meta-analysis of 14 studies concluded that nebulized epinephrine had beneficial effects compared to either placebo or salbutamol, but the effects were short-term. in studies of inpatients, when compared to placebo epinephrine resulted in a significantly better clinical score at 60 minutes, and when compared to salbutamol resulted in a significantly lower respiratory rate after 30 minutes. short-term benefits were more pronounced in outpatient studies, but the benefits lasted less than 60 minutes and had no effect on hospitalization rates. thus in summary there is very little evidence of benefit for any bronchodilators in bronchiolitis. what evidence there is of very short-term benefit from epinephrine, mostly in the outpatient setting, which may reflect benefit in infants with recurrent wheeze phenotypes. the leukotriene receptor antagonist montelukast may be beneficial in young children and infants when started early in acute (mostly virus-induced) asthma/wheezing. however, a recent placebo-controlled study of montelukast in 53 infants with a first episode of bronchiolitis demonstrated absence of benefit, with no significant differences in length of hospitalization, clinical severity score, or inflammatory mediators between the two groups. corticosteroids would appear to be an attractive therapy for bronchiolitis; bronchiolitis is characterized by a florid inflammatory response, and the anti-inflammatory actions of corticosteroids are beneficial in older children and adults with asthma. unfortunately there is a large body of evidence for their lack of benefit in bronchiolitis. the most recent cochrane review of 13 trials included nearly 1200 children aged 0-30 months who had received the equivalent of 0.5-10 mg/kg of systemic prednisone for 2-7 days. although the length of hospitalization was 0.38 days shorter in the steroid-treated group, the difference was not significant, and there was a similar lack of significant differences in respiratory rate, oxygen saturations, need for supplemental oxygen, need for supportive fluids, and need for bronchodilators. furthermore corticosteroids showed no significant benefits even in subgroup analyses of infants who were less than 12 months of age, had confirmed respiratory syncytial virus (rsv) infection, or had no previous history of wheezing. it is interesting to note that a recent large trial of oral corticosteroids in older preschool children with acute wheeze also demonstrated no benefit. the mode of action of hypertonic saline (hs) in bronchiolitis is unclear. in vitro, hs increases the airway surface liquid (asl) height in an epithelial cell line model, and in vivo hs increases the mucociliary clearance of radiolabelled aerosol in both normal controls and asthmatics. hypertonic saline is increasingly utilized in cystic fibrosis (cf) where it increases mucociliary clearance for at least 8 hours in a dose-dependent manner, and increases sputum expectoration. in a randomized placebo-controlled study 4 ml of 7% hs administered twice daily via a nebulizer resulted in significant decreases in the number of both mild and severe respiratory exacerbations, in the days antibiotics received per year; and in the number of days absent from work or school. hs can induce bronchospasm even in normal adults, and so in cf it is recommended that hs is only administered after a bronchodilator. there is increasing evidence that disease pathogenesis in cf is due to increased sodium absorption from the airway surface, leading to airway dehydration and subsequent loss of the normal asl volume and thus decreased ciliary clearance. administration of hs is thought to attract water into the airway, so increasing asl volume and ciliary clearance. other agents that attract water into the airway, such as the sugar alcohol mannitol, have similar effects. it is unclear whether a similar mode of action is applicable to bronchiolitis, and other postulated modes of action include breakdown of ionic bonds within airway mucus, resulting in decreased mucus viscosity and so easing clearance of airway secretions, and increased prostaglandin e 2 release which stimulates ciliary beat frequency; or possibly hs simply induces coughing, even in normal subjects, and so some of its action could be simply through its tussive effect. a cochrane review of four trials of hs in 254 infants with bronchiolitis demonstrated significantly decreased duration of hospitalization. three trials were from the same group of investigators, and used a similar treatment protocol of administering 4 ml of 3% hs every 8 hours, mixed with either 1.5 mg of epinephrine or 5 mg of terbutaline. although administration of a bronchodilator was not essential in the other trial, the majority of subjects concomitantly received a bronchodilator. the pooled data suggested that 3% hs decreased the median duration of stay by 0.94 days (95% confidence interval: −1.48 to −0.4, p = 0.0006) compared to nebulized 0.9% saline, although there was no significant effect on hospitalization rate. a controlled trial of the antiviral agent ribavirin via aerosol in infants receiving mechanical ventilation for severe rsv infection initially showed significant benefit, although there were concerns over the choice of aerosolized water as the control. a repeat study using aerosolized saline as control demonstrated no benefit, and a subsequent meta-analysis of all controlled trials concluded that there was no significant benefit from aerosolized ribavirin, although there may be a slight reduction in the duration of mechanical ventilation. there may however be a role for intravenous ribavirin in severe disease in immunocompromised patients. intravenous rsv immune globulin is ineffective in the acute treatment of rsv lower respiratory tract infection. other treatments that have shown no benefit in acute bronchiolitis include nebulized deoxyribonuclease, chest physiotherapy, and antibiotics. infants known to be at risk of severe bronchiolitis can receive passive immunization against rsv, either with rsv immunoglobulin (rsvig) by monthly intravenous injections or with monthly intramuscular injections of the humanized monoclonal antibody palivizumab. neither has any risk of rsv transmission, and neither interferes with the routine childhood vaccination schedule. the prevent study was a randomized double-blind comparison of rsv immunoglobulin prophylaxis every 30 days versus placebo in over 500 premature infants and infants with bronchopulmonary dysplasia; it demonstrated a significant reduction of hospitalization for rsv and duration of hospitalization, but demonstrated no significant effect on the need for mechanical ventilation, days on intensive care, or mortality. rsvig is no longer used because of the need for monthly intravenous infusions and the small theoretical risk of viral transmission. there is probably no agent that divides neonatologists and respiratory paediatricians as much as palivizumab. palivizumab is a humanized immunoglobulin g1 that binds to the rsv fusion protein, and in the cotton rat model it is 50-100 more potent than rsv immunoglobulin. it prevents entry of rsv into the cells lining the respiratory tract, and when administered intramuscularly has a half life of 18-20 days. it is administered by monthly intramuscular injections during the rsv season, and is currently licensed for use in preterm infants (less than 35 weeks' gestation) who are less than 6 months old at the start of the bronchiolitis season, any child under 2 years of age who has received treatment for bronchopulmonary dysplasia in the previous 6 months, or in children with haemodynamically significant heart disease. the impact study was a placebo-controlled trial of intramuscular palivizumab every 30 days in over 1500 high-risk infants; the infants were either born at or earlier than 35 weeks' gestation and were less than 6 months of age, or were less than 2 years of age and had had a clinical diagnosis of bronchopulmonary dysplasia which required treatment (oxygen, corticosteroids, bronchodilators or diuretics) in the preceding 6 months. palivizumab significantly decreased the risk of hospitalization, duration of hospitalization, and the need for intensive care, although it had no significant effect on either the need for ventilation or death. although the relative effect was striking, the absolute effect was much less so, and the calculated number needed to treat (nnt) was 17. due to its high cost, its economic value has been questioned, with an estimated cost of preventing one hospital admission of £43,000. in 2005 the joint committee for vaccinations and immunizations (jcvi) recommended that palivuzimab should be given to all children less than 2 years of age who had previously been treated with home oxygen for chronic lung disease, or those with haemodynamically significant congenital heart disease, pulmonary hypertension, or severe congenital immune deficiency. despite these recommendations, the use of palivuzimab varies greatly in different areas of the uk. at present there is no effective vaccine for bronchiolitis. attempts have been made to develop a vaccine against rsv; however, when this vaccine was entered into clinical trials patients given the vaccine went on to develop more severe symptoms than those not vaccinated. current efforts to develop a vaccine suitable for human trials focus on using non-animal-derived materials to obtain viral plasmid dna; at present the efficiency of viral recovery is 30-100%. fluids and oxygen are the mainstays of management. there is increasing evidence for the role of nebulized 3% hypertonic saline administered with a bronchodilator. cpap and if necessary mechanical ventilation are effective for respiratory failure. palivizumab decreases the risk of hospitalizations in high-risk infants, although there is controversy over its benefits. ◆ wheezing with chest hyperinflation with costal recession on examination, and fine inspiratory crackles and polyphonic expiratory wheeze on auscultation • rsv accounts for 50-90% of cases of bronchiolitis -other causative agents include human metapneumovirus (hmpv), rhinovirus and less frequently adenovirus, parainfluenza, influenza, coronavirus and the more recently identified bocavirus • management of bronchiolitis is primarily supportive, concentrating on the major complications of inadequate feeding, respiratory distress and apnoeas. fluids (either enteral or parenteral) and oxygen are the mainstays, although cpap or mechanical ventilation are effective for respiratory failure • there is no role for bronchodilators, corticosteroids, antiviral agents, physiotherapy, nebulized dnase or antibiotics, but nebulized 3% hypertonic saline administered with a bronchodilator may decrease length of stay in hospital • preventative strategies such as rsv immunoglobulin or the anti-rsv monoclonal antibody palivuzimab can decrease disease severity and need for hospitalization, although there is controversy over their benefits a double-blind, placebo-controlled, randomized trial of montelukast for acute bronchiolitis deshpande sa, northern v. the clinical and health economic burden of respiratory syncytial virus disease among children under 2 years of age in a defined geographical area a controlled trial of long-term inhaled hypertonic saline in patients with cystic fibrosis palivizumab for preterm infants. is it worth it? bronchodilators for bronchiolitis basic epidemiology and immunopathology of rsv in children a meta-analysis of randomized controlled trials evaluating the efficacy of epinephrine for the treatment of acute viral bronchiolitis non-invasive ventilation as primary ventilatory support for infants with severe bronchiolitis pharmacologic treatment of bronchiolitis in infants and children: a systematic review humanized respiratory syncytial virus monoclonal antibody, reduces hospitalization from respiratory syncytial virus infection in high-risk infants. the impact-rsv study group glucocorticoids for acute viral bronchiolitis in infants and young children high incidence of pulmonary bacterial co-infection in children with severe respiratory syncytial virus (rsv) bronchiolitis human metapneumovirus and lower respiratory tract disease in otherwise healthy infants and children nebulized hypertonic saline solution for acute bronchiolitis in infants • although for most infants bronchiolitis is a mild, self limiting illness, it is nevertheless the commonest cause of hospital admission in infancy -1-3% of infants will be hospitalized • it is a clinical syndrome characterized by coryzal symptoms followed by onset of harsh cough, tachypnoea and key: cord-265054-52eqdlef authors: schaller, matthew; hogaboam, cory m.; lukacs, nicholas; kunkel, steven l. title: respiratory viral infections drive chemokine expression and exacerbate the asthmatic response date: 2006-08-03 journal: j allergy clin immunol doi: 10.1016/j.jaci.2006.05.025 sha: doc_id: 265054 cord_uid: 52eqdlef a number of investigations have linked respiratory vial infections and the intensity and subsequent exacerbation of asthma through host response mechanisms. for example, it is likely that the immune-inflammatory response to respiratory syncytial virus can cause a predisposition toward an intense inflammatory reaction associated with asthma, and adenovirus might cause exacerbation of the immune response associated with chronic obstructive pulmonary disease. in each of these situations, the host's immune response plays a critical mechanistic role through the production of certain cytokines and chemokines. specific aspects of these augmented immune responses are determined by the biology of the virus, the genetic variability of the host, and the cytokine-chemokine phenotype of the involved tissue. for instance, the type 1/type 2 cytokine ratio in the airways during infection with rhinovirus determines how long the viral infection endures. by this same theory, it has been demonstrated that chemokine levels produced during respiratory syncytial virus infection determine host responses to later immune stimuli in the lung, with the potential to augment the asthmatic response. further research in this area will clarify cytokines, chemokines, or cell targets, which will provide the basis for next-generation therapies. increasing clinical evidence supports the concept that certain respiratory viral infections play an important mechanistic role in the initiation of acute lung pathology, as well as set the foundation for longer-term chronic effects that were initiated during the original virus-host interaction. a number of clinical epidemiology studies have identified that the exposure to respiratory syncytial virus (rsv) during early childhood can provide the underpinnings for the development of chronic asthma in later childhood. furthermore, respiratory viruses, such as rsv and rhinovirus, appear to be intimately linked to exacerbations in the physiologic and immunologic intensity of an asthmatic response in many individuals. these exacerbations are associated with increased airway hyperresponsiveness and a significant influx of leukocytes into the lungs. the intensity of the inflammatory response has been directly correlated to the expression of chemokines by virally infected pulmonary structural cells, resident immune cells, and infiltrating leukocytes. in the clinical arena investigations have incriminated a number of chemokines, including cxcl8 (il-8), ccl3 (macrophage inflammatory protein 1a), and ccl5 (rantes), as major mediators released during respiratory viral infection, and the level of these chemokines correlates with the severity of disease. these studies collectively indicate an intimate connection between chemokine expression and respiratory viral infections. they support the growing notion that exposure to infectious agents in early life profoundly influences subsequent immune events that might facilitate the development of severe chronic obstructive airway disease. it is becoming clear that not all respiratory viruses induce the same lung pathology; instead, they are responsible for disparate immune responses. clinical studies examining patients infected with respiratory viruses have found that rsv, rhinovirus, and adenovirus cause airway obstruction and in some cases cause decreased forced expiratory volume readings. 1 interestingly, this correlation is not present in patients infected with the influenza virus. additionally, studies in mouse models have shown that although rsv infection causes the production of (t h 2) cytokines, as measured on the basis of mrna, this is not the case for influenza. 2 one experimental mouse model examining the role between influenza and allergy has determined that influenza infection can decrease the allergic response, although the mechanism is not clear. 3 these studies are supported by clinical investigations that demonstrate that a relatively low number of influenza cases, versus rsv or rhinovirus infections, actually cause exacerbation of asthma and acute bronchiolitis in adults. [4] [5] [6] some of the differences in the end physiologic response to viral infection likely come from the differences in the profile of inflammatory mediators that are produced in response to each virus. one particular difference in the host response to infection with rsv or rhinovirus is the chemokine profile that each infection elicits. although there are often cellular similarities in the response to infection, there are clear differences in the chemokines that are present, the levels of those chemokines expressed, and the presence of the predominant chemokine-generating cell types. chemokines, or chemotactic cytokines, were first described for their ability to attract leukocytes to sites of inflammation, a phenomenon known as chemotaxis. however, data now support a varied and eclectic role for these cytokines during immune-inflammatory processes. chemokines are produced by a variety of cells, including stromal cells, epithelial cells, and all immune cells. these molecules are known to have functions other than chemotaxis, including regulation of inflammation, cellular proliferation, mucus production, tumorigenesis, and angiogenesis. in addition, various chemokines have been found to be important in the initiation and maintenance of the host's response to pathogens. several studies have examined the differences in the host response to respiratory viral infection in human subjects. although none of these studies have come up with a single cytokine or chemokine that can account for the differences in physiologic and immunologic responses that occur on infection, several interesting findings have been published. although many studies have looked at the production of ccl3 and ccl5 in vitro and in vivo, there is no difference in the level of these ligands found in the airways of patients infected with influenza or rsv. 7, 8 however, one report demonstrated that in patients infected with either rsv or influenza, higher levels of ccl3 protein levels in the bronchoalveolar lavage (bal) fluid correlated with hypoxic bronchiolitis, which was not the case for ccl5. 8 another study found that patients infected with rsv have higher serum levels of ccl5, soluble intercellular adhesion molecule, il-4, il-5, and ige than patients infected with influenza. 9 this latter study suggests the importance of examining additional compartments for cytokine and chemokine levels and not only the lung when studying respiratory viral infections. cxcl8 (il-8) is the most studied chemokine and appears to correlate with the severity of respiratory disease. some differences have been found in cxcl8 production in patients infected with respiratory viruses. a comparative study examining the effects of respiratory viruses on cxcl8 production found that although levels of cxcl8 protein in the nasal lavage fluid (nlf) of influenza virusand rhinovirus-infected patients correlated with a higher symptom score, this was not the case for rsv-infected patients. interestingly, in this same study the rsv-infected group had the greatest incidence of wheezing, 1 an indicative correlate for the development of asthma. another group found that influenza infection was associated with higher cxcl8 levels in nasal washes than rsv. 10 additionally, the peak production of cxcl8 is different when comparing infections. in experimental rsv infection of human subjects, there is an initial spike of cxcl8 protein levels in nlf on day 1, with a more significant peak production occurring between days 6 and 14 of infection. 11 however, during influenza infection, cxcl8 protein in nlf peaks between days 4 and 6 and is back to baseline by day 7. 12 rhinovirus infection mimics the cxcl8 secretion pattern seen in influenza infection, with the peak production occurring between days 2 and 3 and dropping off by day 5. 13 these data clearly demonstrate that the biology between the viruses is different, which might account for some of the differences in inflammation and pathophysiology of the different pathogens. differences have also been found in the levels of some nonchemotactic cytokines when comparing respiratory viruses, specifically influenza and rsv. one study found that il-6 levels were higher in the nlf of rsv-infected patients than in uninfected control subjects, which was not the case for influenza-infected patients. an additional difference between the groups was that although levels of il-11 were increased in asthmatic subjects infected with rsv, this was not the case for influenza-infected asthmatic patients. 10 experimental infection of human subjects with viruses has yielded additional valuable data on the duration of infection and peak of chemokine and cytokine production. although a direct comparative study has not been conducted in experimental infection, it is clear that rsv infection lasts longer than influenza or rhinovirus infection. rsv is detected in nasal washes between days 5 and 14, whereas influenza and rhinovirus titers peak at day 2 and disappear by day 8. 11, 12, 14 in contrast, adenovirus might be able to establish a latent infection that is correlated with chronic obstructive pulmonary disease (copd). 15 thus the phenotypic cytokine and chemokine production profiles and disease severity are likely affected by the ability of the virus to propagate. infecting cells in vitro and measuring chemokine production has been beneficial in understanding which chemokines are relevant during viral infection. although several chemokines, such as ccl3, ccl5, ccl11, and cxcl8, are consistently found to be produced in cell culture systems upon viral infection, microarray data from rsv-infected epithelial cells suggests that other chemokines might also play a role in the response to viral infection. 16 interestingly, there seems to be some disparity in the chemokines produced during a given viral infection. for example, pbmcs infected with influenza do not produce cxcl8, [17] [18] [19] [20] and epithelial cells infected with adenovirus do not produce ccl3 7 (summarized in fig 1) . 7, 13, chemokines and their receptors might also play a more direct role in the outcome of viral infection than cell recruitment or activation. for example, in vitro ccl5 production has been shown to inhibit binding of rsv to cells. 41 additionally, the rsv g protein was shown to mimic the ligand for cx3cr1 42 and might inhibit t cells from migrating to the lung during rsv infection. 43 although the host response to viral infection differs depending on the infectious agent, perhaps the most interesting and clinically relevant differences occur well after the infection has taken place. for example, many have speculated that rsv infection can result in childhood asthma, and research has been published to suggest this hypothesis might be true. other research suggests that adenovirus infection might predispose children to chronic obstructive brochitis. 44 another report implicated that exacerbation of copd might be the result of latent adenoviral infection. 45 it is clear that some viruses can have a lasting effect on the structure and function of the lung as shown in fig 2. interestingly, no research studies to date draw a correlation between influenza infection and the later development of respiratory disorders. one study did find that the percentage of influenza-infected children affected by bronchiolitis, which is thought be a marker for wheezing later in life, was only 5%. 46 the most powerful research suggesting that rsv infection predisposes children to later asthma followed the same cohort of children for 13 years. one group of these children was hospitalized as infants for rsvinduced bronchiolitis, whereas the control group did not have an rsv infection during infancy. the studies show that children infected with rsv have increased wheezing and allergies when compared with control subjects. 47, 48 analyzing the cytokine secretion of t cells from these individuals in response to a panel of aeroallergens revealed that t cells from children hospitalized for rsv in their infancy secreted more il-4 in response to aeroallergens than control subjects. 49 another publication demonstrated that although rsv infection at a young age leads to a significant increase in wheeze up to age 11 years, the incidence of wheeze in rsv-infected individuals decreases by age 13 years. 50 two other studies found that although there was no correlation between rsv infection in infancy and clinically diagnosed asthma later in life, children infected with rsv had impaired lung function when compared with control subjects. 51, 52 these investigators argue that rsv does not cause skewing of the immune system, but rather those children affected by rsv infection have preexisting lung abnormalities. these same abnormalities cause decreased lung function later in life. in accordance with this research, another study followed a cohort of children admitted to a hospital for acute bronchiolitis, the cause of which was not determined. nine years after admission, children in the index group had a higher incidence of asthma when compared with control subjects. 53 further research will clarify whether viral infection is necessary for predisposition to asthma or whether it merely uncovers abnormalities in individuals who are already predisposed to have asthmatic responses. murine studies have also yielded conflicting results in regard to the ability of viral infection to enhance subsequent allergic responses. although some research suggests that rsv infection occurring during the allergen sensitization phase prolongs airway hyperreactivity and increases inflammation and mucus production in the lungs, 54 other reports suggest that rsv given before allergen sensitization reduces airway hyperreactivity, eosinophilia, and il-13 production. 55 another study with repeated rsv infections before and during allergen challenge demonstrated a decrease in mucus-producing cells and alveolitis, although infection did not alter lymphocytic infiltration into the lungs. 56 this research suggests that the timing of rsv infection is critical in determining how the immune system responds to subsequent stimuli. if applied clinically, these data could determine which rsv-infected patients receive treatment. several studies in which mice were infected with rsv and then sensitized to allergen suggest ccl5 might be important in predisposing virally infected mice to more severe allergies. in these studies rsv infection was initiated 21 days before allergen sensitization. in control mice previous rsv infection increased airway hyperreactivity and the level of chemokines in the lungs. blocking il-13 during rsv infection reduced the levels of chemokines, as well as airway hyperreactivity, in the lungs of mice that were subsequently sensitized and challenged with allergen. 57 when ccl5 was blocked during the course of rsv infection, subsequent allergen sensitization and challenge was reduced to the phenotype of allergen-sensitized uninfected mice. this was correlated to a reduced level of leukotriene production. 58 additionally, the absence of ccr1 in this model caused a reduction in airway hyperreactivity and mucus, which was accompanied by a reduced amount of il-13 in the lungs, as well as reduced numbers of t cells and eosinophils. 59 these studies implicate that ccl5 and other ccr1 ligands might be important in setting up the immune system in the lung to respond inappropriately to allergens after rsv infection. fig 3 shows some of the chemokines induced during rsv infection and the cell types on which the receptors for these chemokines are present. many viruses have been implicated in the exacerbation of allergic asthma, including influenza, rhinovirus, rsv, adenovirus, and coronaviruses. experimental infection of human subjects with rhinovirus demonstrated that exacerbation was associated with increased airway hyperreactivity in response to allergen. 60 interestingly, rsv appears to cause the most severe exacerbations, 61 whereas rhinovirus appears to be responsible for the majority of exacerbations. 6, 62 for this reason, the bulk of human studies have examined the role of rhinovirus in exacerbation of allergic responses. the exacerbation of allergic asthma by viruses seems to be correlated with both ccl5 and cxcl8. there is a definite link between rhinovirus infection in exacerbation of asthma and increased cxcl8 levels. one report found that cxcl8 levels correlated with severity of symptoms in asthmatic subjects who had rhinovirus infection, 63 which mimics what is seen in subjects infected with rhinovirus alone. not surprisingly, there was also a correlation between cxcl8 levels and neutrophils found in the airway. this report found no differences in airway hyperreactivity of asthmatic subjects on rhinovirus infection, which is in contrast to other published studies, but did find an increased sensitivity to histamine challenge. another investigation examining the different cell types recruited to the respiratory tract during the common cold found a difference in the number of mast cells present in allergic patients versus those seen in nonallergic patients on viral infection. 64 this result could account for the increased sensitivity to histamine that was observed by van benten et al. 64 this report also found that the number of ccl5-and ccl11-producing cells increased on viral infection in both allergic and nonallergic individuals. the investigators also observed an increase in almost every type of inflammatory cell, including eosinophils, t cells, macrophages, and neutrophils, regardless of allergen sensitization. other research has demonstrated a correlation between ifn-g levels and symptom scores in rhinovirus-infected asthmatic subjects. those patients with a higher ratio of ifn-g/il-5 mrna in the airways had less severe symptom scores. interestingly, these subjects also had no detectable virus 14 days after infection, whereas those patients with a lower ifn-g/il-5 mrna ratio had worse symptoms and detectable virus at 14 days after infection. 65 the implications of this latter study are that the virus itself might not cause the exacerbation of asthma, but rather the individual's host response to the virus could be responsible. in this case perhaps higher levels of il-5 or lower levels of ifn-g are responsible for delayed viral clearance. the mechanism could be due to recruitment of inappropriate cells or unnecessary regulation of cells already recruited. although these results suggest that there are few differences in the types of inflammatory processes that occur in allergic individuals infected with a respiratory virus when compared with nonallergic individuals, it is likely that exacerbation of allergic asthma by viral infection still exists. one criterion that might cause the exacerbation of asthma appears to be the presence of allergen at the time of viral infection. if both viral infection and allergen exposure do not occur at the same time, then the increase in inflammation caused by the virus has a limited ability to exacerbate an allergic response. confirmation of this hypothesis stems from a report demonstrating that there was a higher risk of hospital admission for exacerbation of asthma in patients who were both infected with a respiratory virus and exposed to allergen at the same time. 66 another study in which allergic individuals were infected 1 week after allergen exposure reported that subjects infected with rhinovirus had no differences in airway hyperreactivity when compared with infected nonallergic individuals. this provides further evidence that viral infection and allergen exposure need to be concurrent for exacerbation to occur. 67 however, concomitant exposure to allergen and virus is likely not the only cause of asthma exacerbation because nonallergic asthmatic subjects can also experience periods of increased wheezing. 68 a number of studies have been done in murine models to examine the role of viral infection in allergen-sensitized mice. these models sensitize mice to an allergen and then infect the mice with virus before allergen challenge. although several models have proved that influenza 69 and rsv 70 can increase inflammation in the lungs of allergic mice on infection and augment the allergic response, other studies have actually proved the opposite. for example, one study shows that influenza infection actually inhibits the recruitment of t h 2 cells to the bal fluid of allergic mice. 3 a report comparing the effects of several viruses on animals previously sensitized to allergen showed that although both influenza and rsv induced similar patterns of inflammation, influenza downregulated the expression of type 2 cytokines in the lung. 2 it is possible that some of the changes in inflammation that take place on respiratory viral infection of allergic mice are due to differences in chemokine expression. for instance, rsv infection increased levels of ccl11, 71 ccl3, and ccl5 in the lungs of allergic mice (schaller and lukacs, unpublished data). the increase in chemokines could cause an increase in t cells to the lungs and bal fluid of allergen-sensitized mice. this has been shown in both an influenza 69 and an rsv model of exacerbated asthma (schaller and lukacs, unpublished data). the research using the influenza model demonstrated that allergen-specific cells can be recruited to the lymph node on viral infection and secrete il-4. this suggests a prominent role for t cells during the virally exacerbated response. further evidence supporting the hypothesis that increased t-cell recruitment might be responsible for asthma exacerbation comes from research using an influenza virus expressing the mhc i and mhc ii epitopes of the ovalbumin (ova) peptide. these studies have shown that ova-specific t cells of both the cd4 1 and cd8 1 subsets can be recruited to the lung and bal fluid nonspecifically, even in the absence of an influenza infection. 72, 73 viral infection with wild-type influenza increased recruitment of these t cells to the lung and lymph node; however, only the influenza virus expressing ova peptide was able to induce activation and expansion of the ova-specific t-cell population. these studies provide insight into the mechanism of asthma exacerbation. although respiratory viral infection causes increased recruitment of t cells to the lung and lymph node, only some of these t cells are specific for viral antigen. in addition, some of the recruited t cells might be specific for inhaled allergens. if allergen exposure occurs at the time of viral infection, the increase in allergen-specific t cells to the lung would cause an increased allergic response. in addition to an ongoing viral infection, this could cause increased airway hyperreactivity and increased pathology in the respiratory tract. the recruitment of allergen-responsive t cells to the lung and draining lymph nodes has been linked to ccr1 in a murine model of rsv-induced exacerbation of allergic asthma (schaller and lukacs, unpublished data). studies initiated in our laboratory have demonstrated that rsv infection increases the numbers of both cd4 1 and cd8 1 t cells in the lungs and lymph nodes of allergic mice. this was correlated with increased inflammatory cytokine levels and increases in the chemokines ccl3 and ccl5. these studies also provide evidence that ccr1 might be in part responsible for exacerbation because ccr1 2/2 mice do not have an exacerbated phenotype. ccr1 2/2 mice exhibited a reduction in recruitment of allergen-specific cd8 1 t cells to the lymph node. thus it is possible that ccr1 is part of the chemokine receptor profile of allergen-specific and virus-specific t cells, and the production of ccr1 ligands during viral infection could cause the recruitment of t cells to the site of infection and enhance allergic responses. the role of ccr1 in t cell-mediated allergen exacerbation correlates well with data from multiple laboratories indicating that ccl5 is one of the most highly expressed chemokines during virally induced disease. 7, 8, 74 a subset of memory cd8 1 t cells can also express the cxcr1 receptor and chemotax in response to cxcl8. 75 these t cells are activated, expressing high levels of perforin and granzyme, and are more cytotoxic than the larger population of memory cd8 1 t cells. interestingly, in patients infected with influenza, a high percentage of cxcr1 1 cd8 1 t cells are specific for the virus. 76 conclusions taken together, a variety of investigations suggest an important role for chemokines during respiratory viral infection. in cases of viral infection alone, the differences in production of chemokines, such as ccl5, ccl3, ccl11, and cxcl8, on infection might cause differences in the host response. these chemokines might also be important in setting up later immune responses in the lung. the way the host responds to infection with different viruses might cause the association of various viral infections with chronic diseases. for example, it is possible that the host response to rsv can cause a predisposition toward asthma and that adenovirus causes exacerbation of copd, thereby leading to do the production of certain cytokines and chemokines. although some of this response is determined by the biology of the virus, genetic variability of the host also plays a role. for instance, the t h 1/t h 2 cytokine ratio in the airways during infection with rhinovirus determines how long the viral infection endures. by this same theory, it is possible that the amount of ccl5 produced during rsv infection determines host responses to later immune stimuli in the lung and that the amount of cxcl8 produced during adenoviral infection correlates with the degree of exacerbation of a patient with copd. further research in this area might clarify what cytokines, chemokines, or cell types need to be targeted to prevent viral infection from influencing other immune responses. although certain viruses might set up later immune responses in the respiratory tract, it is likely that all respiratory viruses are able to exacerbate asthmatic responses in the lung. this is because the production of excess chemokines in the lung will not only recruit virus-specific t cells but also allergen-specific t cells. these allergen-specific cells will augment any allergic response that is already ongoing in the lung. although a previous study has shown that ccr1 is responsible for this, there are likely other chemokine receptors that are also shared between allergenspecific and virus-specific cells. with the identification of the chemokine receptors responsible for virally induced respiratory diseases, treatments might become more likely as therapies develop that could target cells that express these receptors. although it is clear that many different cell types are responsible for clearing viral infection, perhaps targeting ones that are responsible for much of the pathology could eliminate many of the side effects of viral infection that result in skewing of later immune responses. for example, by targeting ccr1 1 cd8 1 t cells, but not cxcr1 1 cd8 1 t cells, viral clearance would not be delayed, and the pathology of viral infection could be reduced. a better understanding of how the host responds to different respiratory viral infections will also contribute significantly to our understanding of immunology in general. for example, how virus biology affects cell signaling is key in understanding how the host later responds to an infection. because there are clear differences in which chemokines are produced on viral infection, this might be a good beginning in our understanding of how different viral infections cause disparate responses in the host. relationships among specific viral pathogens, virus-induced interleukin-8, and respiratory symptoms in infancy respiratory syncytial virus, pneumonia virus of mice, and influenza a virus differently affect respiratory allergy in mice influenza a virus infection inhibits the efficient recruitment of th2 cells into the airways and the development of airway eosinophilia respiratory syncytial virus infection in elderly and high-risk adults the relationship between respiratory syncytial virus infections and the development of wheezing and asthma in children respiratory viruses and exacerbations of asthma in adults macrophage inflammatory protein-1alpha and rantes are present in nasal secretions during ongoing upper respiratory tract infection a comparison of epidemiologic and immunologic features of bronchiolitis caused by influenza virus and respiratory syncytial virus a comparison of cytokine responses in respiratory syncytial virus and influenza a infections in infants interleukin-6, interleukin-8, interleukin-11, and interferon-gamma levels in nasopharyngeal aspirates from wheezing children with respiratory syncytial virus or influenza a virus infection chemokines in nasal secretions of normal adults experimentally infected with respiratory syncytial virus local and systemic cytokine responses during experimental human influenza a virus infection. relation to symptom formation and host defense rhinovirus stimulation of interleukin-8 in vivo and in vitro: role of nf-kappab effect of induced interferon in experimental rhinovirus infections in volunteers latent adenoviral infection in the pathogenesis of chronic airways obstruction expression of respiratory syncytial virus-induced chemokine gene networks in lower airway epithelial cells revealed by cdna microarrays selective induction of monocyte and not neutrophil-attracting chemokines after influenza a virus infection differential mononuclear leukocyte attracting chemokine production after stimulation with active and inactivated influenza a virus chemokine receptor expression and chemotactic responsiveness of human monocytes after influenza a virus infection influenza a and sendai viruses induce differential chemokine gene expression and transcription factor activation in human macrophages cell-specific expression of rantes, mcp-1, and mip-1alpha by lower airway epithelial cells and eosinophils infected with respiratory syncytial virus expression of cytokine and chemokine genes by human middle ear epithelial cells induced by influenza a virus and streptococcus pneumoniae opacity variants rhinovirus infection up-regulates eotaxin and eotaxin-2 expression in bronchial epithelial cells cytokines contribute to airway dysfunction in antigen-challenged guinea pigs: inhibition of airway hyperreactivity, pulmonary eosinophil accumulation, and tumor necrosis factor generation by pretreatment with an interleukin-1 receptor antagonist the release of inflammatory cytokines from human peripheral blood mononuclear cells in vitro following exposure to adenovirus variants and capsid rsv infection of human airway epithelial cells causes production of the beta-chemokine rantes expression of rantes by normal airway epithelial cells after influenza virus a infection proinflammatory cytokine responses induced by influenza a (h5n1) viruses in primary human alveolar and bronchial epithelial cells adenovirus vector-induced inflammation: capsid-dependent induction of the c-c chemokine rantes requires nf-kappa b rhinovirus replication causes rantes production in primary bronchial epithelial cells human metapneumovirus elicits weak ifn-g memory responses compared with respiratory syncytial virus defense against influenza a virus infection: essential role of the chemokine system airway epithelial cell-induced activation of monocytes and eosinophils in respiratory syncytial viral infection influenza virus a stimulates expression of eotaxin by nasal epithelial cells respiratory syncytial virus-induced cytokine production by a human bronchial epithelial cell line influenza virus a infection induces interleukin-8 gene expression in human airway epithelial cells type-specific induction of interleukin-8 by adenovirus cytokine (tumor necrosis factor, il-6, and il-8) production by respiratory syncytial virus-infected human alveolar macrophages il-8 release from human neutrophils by the respiratory syncytial virus is independent of viral replication rhinoviruses induce interleukin-8 mrna and protein production in human monocytes inhibition of respiratory syncytial virus infection with the cc chemokine rantes (ccl5) cx3c chemokine mimicry by respiratory syncytial virus g glycoprotein respiratory syncytial virus g protein and g protein cx3c motif adversely affect cx3cr11 t cell responses persistent adenoviral infection and chronic obstructive bronchitis in children: is there a link? persistent adenoviral infection and chronic airway obstruction in children influenza-a infection in children severe respiratory syncytial virus bronchiolitis in infancy and asthma and allergy at age 13 respiratory syncytial virus bronchiolitis in infancy is an important risk factor for asthma and allergy at age 7 enhanced il-4 responses in children with a history of respiratory syncytial virus bronchiolitis in infancy respiratory syncytial virus in early life and risk of wheeze and allergy by age 13 years respiratory morbidity 20 years after rsv infection in infancy wheezing, asthma, and pulmonary dysfunction 10 years after infection with respiratory syncytial virus in infancy respiratory status and allergy nine to 10 years after acute bronchiolitis respiratory syncytial virus infection prolongs methacholine-induced airway hyperresponsiveness in ovalbumin-sensitized mice immune interaction between respiratory syncytial virus infection and allergen sensitization critically depends on timing of challenges timing of infection and prior immunization with respiratory syncytial virus (rsv) in rsv-enhanced allergic inflammation respiratory syncytial virus predisposes mice to augmented allergic airway responses via il-13-mediated mechanisms respiratory syncytial virus-induced ccl5/rantes contributes to exacerbation of allergic airway inflammation respiratory syncytial virus-induced exaggeration of allergic airway disease is dependent upon ccr1-associated immune responses rhinovirus upper respiratory infection increases airway hyperreactivity and late asthmatic reactions altered eosinophil levels as a result of viral infection in asthma exacerbation in childhood community study of role of viral infections in exacerbations of asthma in 9-11 year old children effect of experimental rhinovirus 16 colds on airway hyperresponsiveness to histamine and interleukin-8 in nasal lavage in asthmatic subjects in vivo prolonged nasal eosinophilia in allergic patients after common cold relationship of upper and lower airway cytokines to outcome of experimental rhinovirus infection synergism between allergens and viruses and risk of hospital admission with asthma: case-control study are rhinovirus-induced airway responses in asthma aggravated by chronic allergen exposure? allergic vs nonallergic asthma: what makes the difference? allergic airway inflammation is exacerbated during acute influenza infection and correlates with increased allergen presentation and recruitment of allergen-specific t-helper type 2 cells influence of respiratory syncytial virus infection on cytokine and inflammatory responses in allergic mice recurrent respiratory syncytial virus infections in allergen-sensitized mice lead to persistent airway inflammation and hyperresponsiveness antigen-specific and non-specific cd4(1) t cell recruitment and proliferation during influenza infection the role of antigen in the localization of naive, acutely activated, and memory cd8(1) t cells to the lung during influenza pneumonia chemokine concentrations in nasal washings of infants with rhinovirus illnesses cutting edge: expression of chemokine receptor cxcr1 on human effector cd81 t cells il-8 responsiveness defines a subset of cd8 t cells poised to kill key: cord-315598-qwh72inx authors: mendoza, jose luis accini; estrada, victor hugo nieto; lópez, nelly beltrán; bolaños, elisabeth ramos; franco, daniel molano; castell, carmelo dueñas; moreno, albert alexander valencia; amaya, iván camilo alarcón; flórez, john serna; valencia, bladimir alejandro gil; camilo pizarro, g; polo, yulieth maría zabaleta; meza, carmen lucia chica title: actualizacion de la declaración de consenso en medicina critica para la atención multidisciplinaria del paciente con sospecha o confirmación diagnóstica de covid-19 date: 2020-10-06 journal: nan doi: 10.1016/j.acci.2020.09.004 sha: doc_id: 315598 cord_uid: qwh72inx antecedentes y objetivos: la enfermedad por coronavirus de 2019 (covid-19) es una enfermedad ocasionada por el nuevo coronavirus del síndrome respiratorio agudo grave (sars-cov-2). se identificó por primera vez en diciembre de 2019 en la ciudad de wuhan, en los meses siguientes se expandió rápidamente a todos los continentes y la organización mundial de la salud (oms), la reconoció como una pandemia global el 11 de marzo de 2020. la mayoría de los individuos son asintomáticos pero una baja proporción ingresan a cuidados intensivos con una alta morbilidad y mortalidad. este consenso tiene como objetivo actualizar la declaratoria inicial emitida por la asociación colombiana de medicina crítica (amci) para el manejo del paciente críticamente enfermo con covid-19 dentro de las áreas críticas de las instituciones de salud. métodos: este estudio utilizó dos técnicas de consenso formal para construir las recomendaciones finales: delphi modificada y grupos nominales. se construyeron preguntas por la estrategia pico. 10 grupos nominales desarrollaron recomendaciones para cada unidad temática. el producto del consenso fue evaluado y calificado en una ronda delphi y se discutió de forma virtual por los relatores de cada núcleo y los representantes de sociedades médicas científicas afines al manejo del paciente con coid-19. resultados: 80 expertos nacionales participaron en la actualización del consenso amci, especialistas en medicina critica y cuidados intensivos, nefrología, neurología, neumología, bioeticistas, medicina interna, anestesia, cirugía general, cirugía de cabeza y cuello, cuidados paliativos, enfermeras especialistas en medicina crítica, terapeutas respiratorias especialistas en medicina crítica y fisioterapia, con experiencia clínica en la atención del paciente críticamente enfermo. la declaratoria emite recomendaciones en los ámbitos más relevantes para la atención en salud de los casos de covid-19 al interior de las unidades de cuidados intensivos en el contexto nacional de colombia. conclusiones: un grupo significativo multidisciplinario de profesionales expertos en medicina crítica emiten mediante técnicas de consenso formal recomendaciones sobre la mejor práctica para la atención del paciente críticamente enfermo con covid-19. las recomendaciones deben ser adaptadas a las condiciones específicas, administrativas y estructurales de las distintas unidades de cuidados intensivos del país. background and objectives: the 2019 coronavirus disease (covid-19) is caused by the new severe acute respiratory syndrome coronavirus (sars-cov-2). it was first identified in december 2019 in wuhan, china. in the following months it spread quickly to all continents and was recognised as a global pandemic by the world health organization (who) on march 11th, 2020. most cases of infection remain asymptomatic, while a low proportion require intensive care, experiencing high morbidity and mortality. this consensus aims to update the initial statement issued by the colombian association of critical medicine (amci) for the management of the critically ill patient with covid-19 within the critical areas of health institutions. methods: this study used two formal consensus techniques to construct the final recommendations: modified delphi and nominal groups. questions were constructed using the pico strategy. recommendations for each thematic unit were developed by 10 nominal groups. the consensus product was evaluated and qualified in a delphi round, and was discussed virtually by the speaker of each nucleus, as well as the representatives of scientific medical societies related to the management of the patient with covid-19. results: a total of 80 national experts participated in the update of the amci consensus, all specialists in critical and intensive care medicine, nephrologists, neurologists, chest physician, bioethicists, internal medicine specialists, anaesthetists, general surgeons, head and neck surgery, palliative care, nurses specialised in critical medicine, respiratory therapists specialised in critical medicine and physiotherapy, with clinical experience in the care of critically ill patients. this update issues recommendations in the most relevant areas for health care of covid-19 patients within the intensive care units, contextualised for colombia. conclusions: a significant multidisciplinary group of professionals, who are experts in critical medicine, reviewed and issued recommendations on best practice for the care of critically ill patients with covid-19 through formal consensus techniques. recommendations must be adapted to the specific, administrative, and structural conditions of the different intensive care units in the country. para la actualización de la declaratoria se utilizaron dos técnicas para el desarrollo de consensos de tipo formal, técnica delphi modificada y grupos nominales. un consenso formal permite integrar las opiniones de un colectivo de expertos que están expuestos a un tema específico (experto afectado) con la mejor evidencia científica disponible, utilizando técnicas que permitan reducir los sesgos de subjetividad. la técnica delphi es una metodología que plantea enviar cuestionarios a un grupo de expertos, para que califiquen una serie de recomendaciones en rondas reiteradas con retroalimentación de los resultados y respuestas anónimas, la técnica delphi empleada fue modificada, variante a la versión original propuesta por la corporación rand en 1948, pero se mantuvo las ventajas de la técnica, la iteración y retroalimentación para reflexión de las propias opiniones. los grupos nominales es una técnica que reúne a un grupo de expertos bajo la coordinación de un facilitador para evaluar y calificar información o preguntas (1, 2) . para la actualización se convocó 10 grupos nominales con 50 expertos multidisciplinarios cada uno con un líder o jefe de núcleo. los grupos construyeron las preguntas por metodología pico y desarrollaron progresivamente las recomendaciones hasta las versiones finales. el proyecto se desarrolló en 3 fases, fase 1: formulación del problema y socialización; fase 2: elaboración de las preguntas, fase 3: formulación de las recomendaciones y ronda de calificación. las estrategias de búsqueda se desarrollaron en bases de datos especializadas (medline, embase, lilacs, central), en las circunstancias donde no se encontró evidencia directa, se utilizó y se adaptó evidencia indirecta del tópico relevante en el paciente críticamente enfermo general. 80 expertos con un promedio de 15 años de experiencia en la atención del paciente crítico evaluaron y calificación las recomendaciones en la metodología delphi mediante un cuestionario distribuido por medio de correo electrónico, respetando la política de privacidad de datos vigente. recomendación se recomienda que los prestadores de servicios de acuerdo con su infraestructura física y la disponibilidad de recursos (tecnológico, humano, de interdependencia y apoyo) definan su modelo de atención para pacientes con covid-19 en estado crítico basado en principios de factibilidad, efectividad, seguridad y la relación entre la demanda (momento epidemiológico) y capacidad/capacidades de respuesta:  modelo 1. atención de pacientes con sospecha o confirmación diagnóstica covid19 . este enfoque permite concentrar, optimizar y racionalizar recursos y reducir el riesgo potencial de contagio al equipo de atención, de apoyo y de pacientes.  modelo 2. atención mixta, de pacientes con y sin diagnóstico de covid-19, en escenarios que cuentan con unidades de aislamiento normatizados (presión negativa y >12-renovaciones completas de aire por hora) soportado en el documento institucional de gestión organizacional y operativo del servicio de cuidados intensivos, descrito en los procesos prioritarios. amci ® menos accidentalidad y violencia). la reducción de procedimientos quirúrgicos complejos electivos es una opción razonable condicionado a las posibilidades del paciente. sin embargo, situaciones como la progresión y descompensación de las patologías crónicas asociadas a las medidas de restricción social puede plantear un efecto bumerang con mayor demanda de camas de uci. con base en las predicciones simuladas de las tasas esperadas de ingreso a uci de pacientes con covid-19 contrastado con el déficit de servicios y camas de cuidados críticos en el país a partir de la capacidad instalada se han planteado 4 fases de desarrollo cuyas características en términos de servicios, recursos y cronología se aprecian en la ilustración 1 y 2.  fase 1 (para el gobierno nacional: ampliación de la capacidad instalada*). parte de la liberación de camas de cuidados intensivos destinadas para atención covid-19 bajo el modelo 1 y/o 2. la liberación de camas y servicios con mínima adaptación es la fase más inmediata y resolutiva que debe acogerse a la exigencia normativa (resolución 3100 de 2019), en la que se espera menos mortalidad, morbilidad y tasa de complicaciones asociados con la atención de pacientes con covid-19 en estado crítico.  fase 2 (optimización para el gobierno nacional*) representan el reordenamiento de las camas de cuidados intermedios adultos en camas de cuidados intensivos y de hospitalización en intermedio. de adultos. el gobierno nacional toma en cuenta la ampliación de la capacidad instalada hospitalaria descrita en el plan territorial. para esta fase se necesitan equipos de ventilación mecánica (excluidos por la norma de estos servicios), monitoreo básico y avanzado y un número mayor de talento humano multidisciplinario competente. amci ® acuerdo con las etapas (1-3) y los perfiles requeridos priorizados como primera, segunda y tercera línea de respuesta*. en la ilustración 1 podemos observar que avanzar de las fases 1 a 4 se va a necesitar mayor intervención en términos de organización, planeación operativa, formación por competencias y apoyo por telesalud (teleapoyo o teleexperticia). el personal no especializado o especializado de servicios hospitalarios diferente a urgencia, quirófano o uci pueden constituir grupos de apoyo para la gestión administrativa y de índole humanitaria (líneas de respuesta) la integración de estos requerimientos adaptativos se ha puesto de manifiesto en la experiencia del centro médico new york -presbyterian weill en la ciudad de nueva york, en donde la demanda de camas de unidades de cuidados intensivos (uci) y ventilación mecánica excedió su capacidad.(3) se recurrió a los quirófanos y de recuperación las cuales no estaban en uso porque los procedimientos electivos habían sido pospuestos. se hicieron adecuaciones físicas para garantizar la vigilancia continua de los pacientes y la seguridad del equipo de atención. se capacito a todo el personal de cuidado perioperatorio disponible y fueron distribuidos en las áreas recién configuradas. las enfermeras familiarizadas con las máquinas de anestesia asumieron como terapeutas respiratorios y los intensivistas de anestesia supervisaban estas unidades. recomendación se recomienda la adopción de un modelo simulado de predicción (basado en el cociente de fatalidad, tasa de ingreso a uci y el número de reproducción ro) para proyectar, de manera anticipada las necesidades de recurso físico, tecnológico y humano de cuidados críticos en fase de preparación de la pandemia por covid-19. las tomas de decisiones relacionadas con el fortalecimiento de la capacidad/capacidades de la oferta de cuidados críticos (habilitadas o adaptadas) pueden deducirse mediante la aplicación de modelos matemáticos que intentan, desde la dinámica epidemiológica, establecer el efecto simulado de las medidas de mitigación o supresión adoptadas sobre la tasa de contagio a través del tiempo. con ello se busca planear el uso de recursos e implementar acciones de prevención y de distanciamiento social más eficientes, así como establecer las necesidades en materia de cama de cuidados críticos (intensivos e intermedios) habilitadas o adaptadas en áreas de expansión y con ello los recursos resolutivos como tecnologías, interdependencias y talento humano capacitado. la capacidad predictiva del modelo resulta de establecer: la información en términos de casos proyectados (población susceptible), el modelo de transmisión, el impacto de las amci ® intervenciones no farmacológicas (mitigación y supresión) para lograr disminuir el número de reproducción r (tasa de contagio), la distribución de la gravedad de la enfermedad y con ello el porcentaje esperado de casos críticos el decreto 417 del 17.03.20 (4) , por el cual se declara un estado de emergencia económica, social y ecológica en todo el territorio nacional proyectó para una tasa de contagio de 2.68 y cerca de 4 millones de casos, la ocurrencia de 187 mil casos de pacientes con covid-19 en estado crítico (4.7%) y la necesidad de incrementar las camas de cuidados intensivos en un 10% que con una estancia promedio de 14 días tendría un costo de 200 mil millones de pesos. colombia cuenta con cerca de 8500 camas de cuidados intensivos (1/3 parte son intermedio) de las cuales el 60% podría ser utilizada para la atención de pacientes covid-19 (5100). con base en el modelo matemático ha proyectado la necesidad de 9500 camas de uci indicando por deducción la necesidad de 4400 camas a partir de un plan adaptativo de expansión y extensión. colombia reporta a la fecha actual 113,389 casos confirmados y 3984 nuevos casos de covid-19 confirmados y cerca del 4% ocupan una cama de cuidados intensivos (esta cifra es mayor si se tomaran en cuenta los casos sospechosos). de este modo, los modelos matemáticos permiten predecir el comportamiento epidemiológico de la enfermedad y con esto anticiparse a proyectar el plan de fortalecimiento hospitalario (incluyendo camas, tecnologías, talento humano) y los recursos financieros para respaldar la expansión del cuidado críticos. estos modelos deben ser predictivos y no reactivos al comportamiento epidemiológico de la enfermedad y solo debe des escalarse hasta después de reducirse a menos de 1.0 el índice ro. (5-10) recomendación se sugiere la implementación de una estrategia de telesalud (teleapoyo o teleexperticia) en el marco de la pandemia covid-19, cuando no se cuente con un intensivista presencial, que, mediante una tecnología adecuada complemente la atención en las áreas de cuidados críticos realizado por personal capacitado. aún cuando es considerada ventajosa sus implicaciones en términos de resultados clínicos, económicos y de riesgos legales no se ha demostrado. en situaciones donde se declara una pandemia los sistemas de salud pueden tener dificultades para hacer frente a una demanda exponencial y fuera de control. esto puede ser así en el marco pandemia covid-19 que prevé un 5% de pacientes en condición crítica y que amerita reorganización y/o adaptación de su capacidad de respuesta. incrementar la disponibilidad de camas y servicios de cuidados críticos mediante una estrategia de expansión supone retos asociados a la insuficiencia que se puede presentar en talento humano especializado específicamente de especialistas en medicina crítica y cuidado amci ® intensivo, escenario que se puede complicar en la medida que intensivistas sean separados o aislados en el curso de la epidemia. en este contexto se hace necesaria la implementación de modelos ágiles de telesalud (ts) para el acompañamiento de las unidades de cuidado crítico en expansión y de instituciones prestadoras de servicios de salud de baja y mediana complejidad para la regulación con los equipos de referencia y contrarreferencia de pacientes que pueden necesitar atención en cuidados intensivos. (11) (12) (13) (14) (15) (16) el decreto ley 538 de 2020 (17) plantea la adopción de medidas en el sector salud para garantizar la prestación de los servicios de salud y para facilitar la implementación de modelos de atención que incluyan la telesalud y la prestación de los servicios en la modalidad de telemedicina se determinan algunas medidas temporales para: i) adecuar temporalmente un lugar no destinado a la prestación de servicios de salud, intra o extra mural. ii) prestar servicios en modalidades o complejidades diferentes a las habilitadas dentro de las cuales puede estar la telemedicina iii) prestar servicios de salud no habilitados. en este decreto también se establecen condiciones temporales para la implementación de plataformas tecnológicas para la telesalud. en complemento, la resolución 3100 de 2019(18) (estándares de habilitación) plantea la modalidad de la telemedicina (prestador remisor-prestador de referencia) para las unidades de cuidado intermedio e intensivo. la telesalud se puede prestar de dos maneras: teleapoyo y teleexperticia. tabla 2. teleapoyo (ta) soporte solicitado por un profesional de la salud a otro profesional de la salud a través de tic siendo responsable de la conducta quién solicita el apoyo. no requiere habilitación y por tanto no requiere autorización transitoria relación a distancia con comunicación sincrónica o asincrónica utilizando tic entre dos profesionales de la salud, uno de los cuales atiende presencialmente al usuario y otro atiende a distancia. el primero es responsable de las decisiones/recomendaciones entregadas al paciente y el segundo es responsable de la calidad de la opinión que entrega y debe especificar las condiciones en las que se da dicha opinión, lo cual debe consignarse en la historia clínica. requiere autorización transitoria (decreto 358 2020) (17) la telesalud y la prestación de servicios de salud en esta modalidad son estrategias seguras y efectivas para guiar, a distancia, el diagnóstico y el tratamiento del paciente hospitalizado y en estado crítico. sus ventajas generales se presentan en la tabla 3. 14 asociación colombiana de medicina crítica y cuidados intensivos. amci ® tabla 2. ventajas generales de la telesalud decreto legislativo 538 de 2020.  facilita la viabilidad de aplicación modelos organizativos que favorecen la continuidad y la integridad asistencial y la atención centrada al entorno del paciente, aplicando conceptos de globalidad e interoperabilidad a las organizaciones sanitarias, dando lugar a nuevas formas de organización y trabajo en red.  mejora de la calidad asistencial, ya que facilitan el acceso y la disponibilidad de servicios asistenciales en condiciones de calidad.  mejora calidad de vida del paciente por la disminución de desplazamientos para la atención ya que permite la atención o monitorización remota con tic en su domicilio.  mejora la oportunidad y la resolutividad de la atención.  facilita la equidad en el acceso a los servicios de salud independientemente de la localización geográfica (acerca la atención especializada a toda la población).  mejora la atención integral y seguimiento tanto de los pacientes crónicos, como los de las enfermedades de baja prevalencia.  reduce los tiempos de espera (tanto en la realización del diagnóstico como en el tratamiento), evitando complicaciones por no atención oportuna.  posibilita realizar atención remota de mediana y alta complejidad en la baja complejidad, reduciendo el número de remisiones.  disminuye la posibilidad de infección cruzada entre usuarios de los servicios de salud y el personal de salud.  incide en la formación y competencia del talento humano en salud.  facilita la educación de pacientes en medicina preventiva y salud pública.  descongestiona servicios de urgencias y consulta externa.  contribuye a la reducción de movilidad de personas en la ciudad.  responde a las necesidades inmediatas en salud de la comunidad.  es un medio de racionalización de costos en salud. puede abarcar otros servicios de gestión administrativa como entrega de fórmulas o facturación. amci ® se recomienda la implementación de un modelo de cuidados críticos covid-19 liderado por intensivistas, en áreas habilitadas o adaptadas, con el beneficio preponderante de disminuir la mortalidad, tiempo de estancia y optimización de recursos. se recomienda una cobertura por intensivistas de al menos 12 horas diarias y un cociente intensivista/paciente cercano a 1 intensivista por cada 8-10 pacientes, basado en la alta complejidad de la enfermedad critica covid-19 con un alto porcentaje de pacientes en ventilación mecánica, largos tiempos de estancia y alto riesgo de mortalidad. fuerte a favor fundamento un intensivista es un profesional médico capacitado en medicina crítica y de cuidados intensivos conforme a los estándares establecidos por una institución de educación superior debidamente reconocida ante el ministerio de salud. este especialista debe liderar y tomar todas las decisiones con respecto al cuidado de los pacientes críticos, incluyendo admisiones y egresos, qué médicos consultar, estándares de atención, gestión de la calidad y seguridad, gestión humana y ética, interacción con la familia e implementación de un programa de investigación y de formación continua para mejorar capacidades y competencias del equipo de atención, control de conflictos, entre otras.(20) existe una enorme validez conceptual y una preponderancia de evidencia que sugiere que ser atendido por un especialista en cuidados críticos (intensivista) es "bueno" para los pacientes de la uci. la mayoría de los estudios demuestran el impacto positivo de un uci dirigida por intensivistas los modelos de personal médico de la uci más ampliamente estudiados difieren en el nivel al cual los intensivistas están involucrados en el manejo de los pacientes. las uci de alta intensidad son aquellas donde un intensivista de tiempo completo u obligatorio maneja a la mayoría de los pacientes diariamente. las uci de baja intensidad no tienen participación intensivista u ofrecen consultas intensivistas electivas. un metaanálisis mostró que un modelo de alta intensidad en comparación con uno de baja intensidad estuvo asociado con una menor mortalidad en la uci, menor mortalidad hospitalaria, y una reducción significativa en la duración de la estancia hospitalaria. un modelo de alta intensidad por la noche se asoció con menor mortalidad solo cuando durante el día era de baja intensidad. (21) (22) (23) (24) (25) (26) (27) (28) (29) (30) (31) (32) (33) (34) (35) (36) 17 asociación amci ® forzosa" por condición de riesgo siempre en consonancia con los términos establecidos en el decreto ley 588 de 2020 (39). según la normatividad vigente todo miembro del equipo de atención, especialista no intensivista o no especialista (enfermeras, fisioterapeutas/terapeutas respiratorias) y auxiliares de enfermería deben tener constancia de asistencia a acciones de formación continua y/o capacitación en atención covid-19 en cuidados críticos las cual puede ser parte de un programa institucional de capacitación liderado por el intensivista coordinador o titular del servicio o a partir de cursos respaldados por instituciones académicas acreditadas o los ofrecidos por la asociación colombiana de medicina crítica y cuidados intensivos (amci). las tablas 5 y 6 nos muestra como dentro de este proceso adaptativo por estado de emergencia y atribuido a un desequilibrio entre la oferta y la demanda, otras especialidades, profesionales de la salud y personal en formación pueden hacer parte de los equipos de atención bajo las siguientes premisas: 1. la supervisión, coordinación y liderazgo del intensivista es necesaria y 2. el intensivista establece los roles y competencias del th no intensivista y no normatizado, de acuerdo a sus perfiles, y delegada acciones asistenciales (vía aérea, accesos vasculares, reanimación cardiopulmonar, pronación), administrativas (ordenes médicas, notas clínicas) o de naturaleza humanitaria (comunicación con la familia, apoyo emocional al th, etc.). es necesario considerar los roles de las especialidades que formarán parte de la gestión asistencial y/o administrativa de pacientes con covid-19 en áreas críticas habilitadas o adaptadas de manera transitoria en colombia tabla 7. integridad de interdependencia (norma3100/10) integralidad e interdependencia (adaptiva) gfa integridad de interdependencia (norma3100/10) integralidad e interdependencia (norma 3100/10) integridad de interdependencia (norma3100/10) obligatorio en gestión asistencial integralidad e interdependencia(norma 3100/10) obligatorio gestión asistencial apoyo a tomas de decisiones(gfa) apoyo a tomas de decisiones (gfa) a:formacion continua + covid-19 (curso virtual); b: requiere capacitación covid-19 (curso virtual); c: gfa: grupo focal asistencial, sistema alerta-acción, rcp, pronación, accesos vasculares, ingreso a uci. d: gfad: grupo focal administrativo: consentimiento, notas de evolución. ts: telesalud. se recomienda la aplicación de la escala news-2 por parte de un equipo de respuesta rápida, para establecer el lugar de atención de pacientes con diagnóstico definitivo o sospecha de infección por covid-19 que encuentran en los servicios de urgencias o de hospitalización. se recomiendan escalas como el qsofa y el curb-65 para apoyar la decisión tomada con base en la escala news-2. sin embargo, un qsofa mayor o igual a 2 puntos (mínimo 2/3) y un crb-65 mayor o igual 2 puntos tienen baja sensibilidad (alta incidencia de falsos negativos) para identificar pacientes que puedan requerir ingreso a uci. se recomienda el score de riesgo covid-19-gram para identificar el riesgo de desarrollar un estado de la enfermedad crítico en pacientes con covid-19 y como herramienta complementaria a la escala news-2 en escenarios de alta demanda y escasez de recursos, de manera que la decisión de ingreso a uci se haga sobre aquellos pacientes que realmente se beneficiarán de la misma en términos de vidas salvadas y número de años salvados. la escala news fue construida y validada en paciente con infección por el virus de la influenza a/h7n9 y se recomienda como una herramienta objetiva para decidir nivel de atención, incluyendo ingreso a uci o situaciones terminales que requieren acompañamiento familiar y medidas de cuidado paliativo. esta escala incluye 6 variables fisiológicas que son: frecuencia respiratoria, saturación arterial de oxígeno (spo2), uso de oxígeno suplementario, presión arterial sistólica, frecuencia cardiaca (pulso), temperatura y nivel de conciencia. es de mencionar que esta escala no contempla la edad del paciente. en 2007 un reporte del acute medicine task force of the royal college, london, uk, recomendó la utilización de la escala news en los servicios de urgencias (41) . un estudio que evaluó una base de datos con 198.755 signos vitales obtenidos de 35.585 pacientes demostró que esta escala tiene una buena capacidad para discriminar pacientes en riesgo para un desenlace combinado de paro cardiaco, ingreso no anticipado a uci o muerte dentro de las primeras 24 horas de atención; de esta manera genera una gran oportunidad para el establecimiento temprano de una intervención clínica que cambie el pronóstico del paciente (42) . la escala news-2 tampoco contempla la edad, pero sí incluye la presencia de hipercapnia bajo diferentes niveles de spo2 y oxígeno suplementario. esta escala es la que ha sido estudiada como herramienta para identificar pacientes en riesgo de desarrollar un estado de enfermedad crítica por covid-19 con un valor ≥ 5 puntos (5/20) (43) . con base en la escala news-2 se establece el grado de riesgo, el tipo de alerta, y la intensidad de monitoreo requerido; y de acuerdo con el puntaje arrojado, se define claramente el nivel de atención que requiere el paciente con diagnóstico de infección por covid-19, incluso ingreso a uci (tabla 7). esto se establece a través de una escala de puntuación así: -score 0 puntos: manejo domiciliario bajo aislamiento y signos de alarma. -score 1 punto: manejo domiciliario y seguimiento clínico en casa. -score 2-4 puntos: manejo en salas de hospitalización. -score 5-7 puntos: manejo en uci, área covid-19. -score ≥ 7 puntos sin condición extremadamente grave o irreversible y con alta posibilidad de recuperación: traslado a uci, área covid-19. amci ® -score ≥ 7 puntos con condición extremadamente grave y con datos de irreversibilidad o enfermedad terminal: no ingresa a uci y se traslada a salas de hospitalización con acompañamiento familiar y consulta a experto en bioética y cuidados paliativos. la ilustración 4 establece un flujograma de conductas basado en el puntaje del news-2. tabla 6. news-2 score. el qsofa con un valor ≥ 2 puntos es otra herramienta recomendada para decidir qué pacientes que ingresan a uci. esta herramienta fue recomendada por la tercera definición de consenso (sepsis-3) para identificar pacientes con alto riesgo de muerte o estadía prolongada en uci entre aquellos con sospecha de infección (44) . en este score, un punto es asignado para 3 variables así: frecuencia respiratoria ≥ 22/min, presión arterial sistólica ≤ 100 mmhg y escala de coma de glasgow (ecg) < 15. el score curb-65 y su versión simplificada, el crb-65 se utilizan para evaluar la severidad de enfermedad en personas hospitalizadas con neumonía adquirida en comunidad (nac). ambos scores han sido adoptados por la sociedad británica de tórax para predecir la necesidad de soporte respiratorio o vasopresor intensivo (srvi) en pacientes con covid-19 (40) . el score crb-65 asigna un punto para 4 variables así: confusión de reciente inicio, frecuencia respiratoria ≥ 30/min, presión arterial sistólica < 90 mmhg o presión arterial diastólica ≤ 60 mmhg y edad ≥ 65 años. pacientes con un score ≥ 2 puntos necesitan hospitalización (43) . en un estudio clínico observacional realizado sobre los primeros 42 pacientes ingresados en un hospital de noruega con diagnóstico confirmado de covid-19, se evaluó la utilización de 4 sistemas de score clínicos al momento del ingreso: news-2, qsofa, crb-65 y sirs, con los puntos de corte previamente mencionados (43) . la enfermedad se clasificó como enfermedad severa y enfermedad crítica. solo 9 pacientes (21%) se clasificaron como enfermedad crítica. al evaluar los scores con sus puntos de corte, 2 pacientes presentaron un qsofa ≥ 2 [1 con enfermedad severa (3%) y 1 con enfermedad crítica (11%)], solo 7 pacientes presentaron un crb-65 ≥ 2 [6 con enfermedad severa (18%) y 1 con enfermedad crítica (11%)] y 19 pacientes presentaron un news-2 ≥ 5 [(11 con enfermedad severa (33%) y 8 con enfermedad crítica (89%)]. la mediana del score news-2 para pacientes con enfermedad severa fue de 3.3 [riq: 3-9] vs 7.6 [riq: 8.4 -10] para pacientes con enfermedad crítica. los autores concluyen que el qsofa y el crb-65 se comportan similar y con una baja capacidad para la identificación de enfermedad crónica en pacientes con covid-19; por otro lado, los datos indican que el news-2 podría ser una herramienta más útil para identificar pacientes con riesgo de un curso más agresivo de la enfermedad (33% vs. 89%) (43) . amci ® un estudio de cohorte retrospectivo realizado en un hospital en liverpool (uk), el puntaje qsofa se comportó como el más específico (79%; 95% ic: 77% -81%) pero el menos sensible (37%; 95% ic: 31% -43%) en comparación al puntaje sirs y la escala news como predictor de mortalidad hospitalaria en un grupo de 1818 paciente admitidos por sepsis al servicio de emergencias (45) . otro estudio retrospectivo de un único centro comparó el rendimiento de 3 sistemas de score, qsofa, curb-65 y crb-65 para predecir la necesidad de soporte vasopresor o respiratorio intensivo (svri) en 116 pacientes ingresados con diagnóstico confirmado de covid-19 a un hospital de wuhan (china) (40) . un total de 25 pacientes (21.6%) necesitaron svri durante su estadía en el hospital. la tasa de mortalidad hospitalaria en esta cohorte fue de 9 pacientes (7.8%). se evalúo el rendimiento de las escalas con análisis de curva roc (auc), puntos de corte óptimo, sensibilidad, especificidad y valores predictivos. el punto de corte óptimo del crb-65 para predicción de srvi fue de 2 puntos, con una sensibilidad del 64% y una especificidad del 93.4%. el valor auc del score crb-65 para predecir la necesidad de svri fue significativamente más alto que el del qsofa (0.81 ± 0.05 vs. 0.70 ± 0.06, p= 0.02). los valores de auc fueron similares entre crb-65 y curb-65 para predecir svri (0.81 ± 0.05 vs. 0.85 ± 0.05, p= 0.08). los autores concluyen que el crb-65 podría ser mejor que el qsofa para identificar paciente con covid-19 en riesgo de necesitar svri. su et al consideran que fue la inclusión de la edad ≥ 65 años dentro del score crb-65 lo que le dio un mayor grado de superioridad sobre el qsofa (40) . el crb-65 puede ser una herramienta de puntuación útil para covid-19 debido a su simplicidad en la aplicación, especialmente en emergencias y condiciones de escasez de recursos. finalmente, el score de riesgo covid-19-gram fue descrito por un grupo de investigadores en covid-19 quienes reunieron datos de 1590 pacientes en 575 hospitales en china (46) . esta fue una cohorte retrospectiva multicéntrico en la que se recogieron un total de 72 variables entre demográficas, médicas, clínicas (signos y síntomas), imagenológicas y de resultados de laboratorios. utilizando la metodología de regresión lasso (least absolute shrinkage and selection operator) construyen un modelo de regresión multivariable resultando en un score de riesgo predictivo para desarrollar enfermedad critica en pacientes con covid-19 confirmado al momento de la admisión. de las 72 variables iniciales 10 fueron predictores independientes estadísticamente significativos para el desarrollo de enfermedad crítica. estas variables fueron: anormalidad en los rx tx (or: 3.39; 95%ic: 2. 14 -5.38 recomendación se recomienda la aplicación de un algoritmo basado en una evaluación dinámica del score news 2 que involucre una escala de evaluación funcional, para priorizar el ingreso a uci con transparencia científica y ética con equidad social, y de ser posible respaldado por un comité de priorización clínica (cpc) integrado por expertos de cuidado intensivo y un representante del comité de ética hospitalaria durante la pandemia por covid-19. se recomienda un modelo determinado por 4 prioridades para definir criterios de ingreso a uci, permite establecer rápidamente qué pacientes se benefician de ingreso uci y qué pacientes deben permanecer en servicios de hospitalización, o con acompañamiento familiar y cuales con medidas de cuidado paliativo. fuerte a favor page amci ® fundamento la pandemia covid-19 nos ha enseñado que la disponibilidad de camas de uci puede ser insuficiente y el plan estratégico diseñado para ampliar la capacidad de respuesta debe ir de la mano con la implementación rigurosa de un protocolo de triaje y de priorización de ingreso a cuidados intensivos, como medida extraordinaria para optimizar los recursos, mitigar y controlar los efectos de la pandemia sobre el balance oferta (efectiva y resolutiva) y la demanda. los protocolos de triaje y priorización están diseñados para asignar los limitados recursos de una manera justa y transparente donde, por definición, algunas personas serán excluidas del acceso a la atención orientado a aumentar la disponibilidad de camas de cuidados intensivos. sin embargo, es necesario enfatizar que la disponibilidad de camas no es un fin en sí mismo. la intención implícita y explícita de los protocolos de clasificación debiera ser el «bien público» de maximizar la supervivencia de la población. pero es incorrecto suponer que este bien público se logra al maximizar la supervivencia entre los que reciben cuidados intensivos. si bien muchos protocolos de triaje reconocen esto al tratar de excluir a los pacientes que no lo necesitan absolutamente (el «demasiado sanos») y los que tienen menos probabilidades de beneficiarse (él «demasiado enfermo»), no prestan suficiente atención a las diferencias entre grupos en términos de la duración de los cuidados intensivos necesarios para lograr resultados. si el objetivo del triage es mejorar la supervivencia de la población con un recurso escaso, entonces el recurso escaso no son camas, sino días de cama; no son ventiladores, sino tiempo de ventilación. de ello se deduce que el triaje no será efectivo si en la valoración, no se discrimina adecuadamente y se considera de manera equívoca que la gran mayoría de las personas que requieren cuidados intensivos tienen una probabilidad similar de supervivencia y una duración de estadía anticipada similar. (47) de este modo, el ingreso a uci debe acogerse a los criterios habituales, científicos y éticos, bajo el rigor de "idoneidad clínica" tomando en cuenta parámetros como la gravedad de la enfermedad, la presencia de comorbilidades (severidad, clase funcional), potencial de recuperabilidad, deseo del paciente (o la familia), de equidad distributiva y el uso de las escalas validadas de severidad y de predicción de ingreso a cuidados intensivos. los pacientes con covid-19 tienden a progresar después del inicio de los síntomas dentro de los 7 a 12 días a una forma grave con síndrome de dificultad respiratoria aguda (sdra) o falla multiorgánica órgano. la identificación temprana y simple de pacientes que requieren respiración intensiva o el soporte vasopresor sería de gran valor durante el brote covid-19. (48) (49) (50) (51) la implementación de un algoritmo "dinámico" que vincule uno o más de las escalas fisiológicas (news con o sin qsofa y/o crb-65 ≥2), una escala de predicción de ingreso a uci (covid-19-gram) y un puntaje de fragilidad (vipi) puede informarnos sobre el estado actual y evolutivo de la enfermedad y a priorizar el ingreso de pacientes a uci permitiendo un uso óptimo de los recursos y tomar decisiones éticas, transparentes y centradas en la dignidad de los pacientes y el bien público ilustración 4-5. se recomienda no usar escalas de severidad de enfermedad (criterios objetivos) para definir el traslado de pacientes de uci hacia un nivel de menos complejidad de atención, ya que estas escalas no han sido validadas para este uso. se recomienda en la atención por covid-19 en cuidados intensivos utilizar los mismos criterios de egreso que se emplean para el traslado desde uci hacia una unidad de menor complejidad de pacientes sin infección por covid-19. se recomienda contar con áreas de bajo nivel de complejidad asignadas solo a la atención de paciente con infección por covid-19, las cuales serán las áreas hacia donde se realiza el de-escalamiento gradual de los pacientes basado en su evolución clínica. cuando se habla de criterios objetivos, se hace referencia a escalas de severidad de enfermedad que ayuden a tomar decisiones más racionales y no basadas en consideraciones tradicionales de resolución de cuadros clínicos. no hay una recomendación definida sobre el uso de escalas de severidad de enfermedad para definir el de-escalamiento de la atención para pacientes críticos. los sistemas de evaluación de severidad de enfermedad generales y específicos pueden identificar una población específica de pacientes en alto riesgo de deterioro clínico luego del traslado fuera de la uci(53); sin embargo, su valor para evaluar que tan preparado está un paciente individual para ser trasladado a un nivel inferior de cuidado no ha sido evaluado(53). los criterios que recomienda el colegio americano de cuidado intensivo(53) para definir el traslado desde uci a un nivel de menor complejidad (unidad de cuidados intermedios o sala de hospitalización) se basan en 3 principios:  cuando el estado fisiológico del paciente se ha estabilizado y ya no es necesario monitoreo y tratamiento en uci.  cuando el paciente cumpla con los criterios de admisión del nivel de menor complejidad, teniendo en cuenta la disponibilidad actual del recurso, el pronóstico del paciente y la presencia de intervenciones activas en curso. específicamente para los pacientes con alto riesgo de muerte y reingreso a uci en quienes se decide no hacer intervenciones adicionales (alta severidad de enfermedad, inestabilidad fisiológica, soporte orgánico), el colegio americano de cuidado intensivo(53) siguiere pasarlos a una unidad de menor nivel de atención o a un hospital de cuidado agudo de largo plazo; siempre con un formato escrito donde se deje claro la decisión para reducir la tasa de reingreso a uci(53). específicamente en situaciones de pandemia y escasez de recursos, la sociedad española de medicina intensiva, crítica y unidades coronarias (semicyuc) recomienda la realización de la escala sofa al menos cada 48 horas para identificar pacientes con evolución tórpida y progresiva a un fallo multiorgánico luego de la iniciación de tratamientos de soporte vital. esto permitirá la adecuación y reorientación de medidas terapéuticas a un objetivo más paliativo, incluyendo la transferencia del paciente a un nivel más bajo de complejidad y la consulta al servicio de cuidado paliativo para que brinde la atención respectiva(52). finalmente, se debe mencionar que no se ha establecido una diferencia en los criterios de egreso de pacientes con covid-19 para su traslado desde uci hacia una unidad de menos complejidad, con respecto a los utilizados para pacientes sin infección por covid-19(54). todo paciente hospitalizado en uci sea covid-19 o no, debe ser valorado diariamente para establecer en qué momento su condición clínica permite que sea trasladado fuera de la uci hacia una unidad de menor complejidad. en caso de tratarse de pacientes con sospecha o diagnóstico de covid-19, este traslado puede hacerse a una unidad de cuidados intermedios dispuesta como área covid-19 o una sala de hospitalización con igual asignación(54); esto es lo que se conoce como de-escalamiento gradual de la atención hasta el final egreso del paciente a casa. se recomienda para pacientes con covid-19 que evolucionan hacia la mejoría, utilizar los criterios clásicos de de-escalamiento del nivel de atención (unidad de cuidados intermedios o sala de hospitalización) que propone el colegio americano de cuidado intensivo(53) . se recomienda aplicar la escala sofa al menos cada 48 horas sumado al criterio de fragilidad y años de vida saludables salvados estos criterios podrían ser válidos en pacientes con fallo terapéutico a las medidas óptimas y orientar decisiones de deescalamiento de medidas y de nivel de complejidad de atención cuando la demanda potencial supera la oferta (capacidad y capacidades), estas decisiones deben ser tomadas idealmente en junta médica. amci ® se recomienda generar procesos administrativos más eficientes para el traslado de pacientes fuera de la uci hacia niveles de menor complejidad, estos ayudarán a liberar recursos para otros pacientes. se recomienda no utilizar el resultado de la rt-pcr positiva para decidir el egreso de la unidad de cuidados intensivos. la literatura no ha definido unos criterios de flexibilidad en uci para el traslado de pacientes con sospecha o diagnóstico confirmado de covid-19; y los criterios que definen la posibilidad de egreso de uci y que aplican a todos los pacientes, incluso aquellos ingresados por una condición crítica en relación con infección por covid-19, están claramente definidos por el colegio americano de cuidado intensivo en su documento-guía del 2016.(53). los principios fundamentales han sido revisados en los fundamentos de la pregunta 8. es importante implementar una estrategia de identificación temprana para aquellos pacientes con soporte vital avanzado que evolucionan progresivamente a fallo multiorgánico y pocas probabilidades de recuperación; el de-escalamiento de medidas y su posterior traslado fuera de la uci, liberará espacio para otros pacientes en situaciones de desborde de la demanda. la sociedad española de medicina intensiva, crítica y unidades coronarias (semicyuc) recomienda que en caso de complicaciones o que se prevea una mala evolución tanto clínica como funcional, se plantee el retiro terapéutico por futilidad y se inicie un manejo con medidas de soporte paliativo, tal como ha sido considerado en los fundamentos de la pregunta 8 (55) . para el grupo español es claro que, adecuar procesos administrativos más eficientes para traslado de pacientes fuera de la uci a los usualmente utilizados en condiciones normales de práctica clínica, ayudaría a ser más eficientes en la gestión de la demanda (55) . finalmente, la persistencia de una rt-pcr positiva no es una contraindicación para el traslado del paciente fuera de la uci siempre y cuando se aseguren condiciones de aislamiento por contacto y aerosol en aquellas áreas de menor complejidad de atención; estas incluyen zonas de expansión o área del hospital específicamente acondicionadas para el manejo de pacientes estables o pacientes con limitación de esfuerzo y manejo paliativo de su condición (55). recomendación se recomienda que la disposición final de cadáveres de personas con sospecha o diagnóstico de covid-19 se haga preferiblemente por cremación. en tal caso, las cenizas pueden ser objeto de manipulación sin que suponga ningún riesgo. amci ® se recomienda que la disposición final del cadáver se haga por inhumación en sepultura o bóveda individualizada cuando no se cuente con instalaciones para cremación en el territorio donde ocurrió el deceso o la disponibilidad de esta tecnología desborda la capacidad económica de las personas. se recomienda realizar siempre el aislamiento del cadáver en el lugar del deceso, siguiendo las recomendaciones del ministerio de salud y la protección social (msps). se recomienda que en los casos que se requiera necropsia médico legal y estuviese indicada la cremación, esta deberá contar con la orden del fiscal del caso. se recomienda que la institución establezca en sus procesos prioritarios un protocolo humanizado de despedida bajo estrictos criterios de bioseguridad. no existe una evidencia fuerte que recomiende hacer una disposición de cadáveres de pacientes fallecidos con sospecha o diagnóstico confirmado de covid-19 con un acto de cremación o con inhumación y sepultura en féretro; sin embargo, siempre y cuando la manipulación y manejo del cadáver hasta su disposición final se haga manteniendo todas las medidas de precaución para evitar la diseminación del virus y siguiendo la normatividad legal vigente, ambas formas son aceptadas. la infección por covid-19 es una enfermedad con gran variabilidad en presentación clínica, alta tasa de contagio y para la cual no existe en el momento un tratamiento definido. el riesgo de contagio al personal que ejecuta autopsias o procedimientos de tanatopraxia y la probabilidad de diseminación de la enfermedad por la manipulación de cadáveres no se conoce, pero se considera que puede ser alto, teniendo en cuenta que, en ausencia de la aplicación de un método de diagnóstico masivo, todo caso debe considerarse potencialmente positivo. por tanto, el manejo de cuerpos de personas fallecidas con diagnóstico confirmado, sospechoso o probable de sars-cov-2 (covid-19), debe realizarse con la mínima manipulación posible(56). el cadáver debe ser transferido lo antes posible al depósito y entregado al servicio funerario antes de 24 horas luego del fallecimiento(57). el transporte, la cremación o inhumación, según sea el caso, se efectuará en el menor tiempo posible, con el fin de prevenir la exposición de los trabajadores y comunidad general al virus sars-cov-2 (covid-19). se debe evitar la realización de rituales fúnebres que conlleven reuniones o aglomeraciones de personas(56). el alistamiento del cadáver será realizado en el ámbito hospitalario del mismo sitio del deceso. las personas que accedan a la habitación donde se encuentre el cadáver, deben tomar las precauciones de transmisión por contacto y gotas, y para ello deben contar con todos los elementos de protección personal (epp) y seguir los procedimientos de amci ® bioseguridad de acuerdo con lo establecido en el protocolo del msps.(58) para hacer el alistamiento del cadáver, se debe cubrir todos los orificios naturales con algodón impregnado de solución desinfectante y se deberá envolver en su totalidad sin retirar catéteres, sondas o tubos que puedan contener los fluidos del cadáver, en tela antifluido o sábana; luego se deberá envolver en dos bolsas plásticas biodegradables que cumplan con las características técnico-sanitarias de impermeabilidad y resistencia a la presión de gases en su interior(57). se debe rociar el interior y el exterior de ambas bolsas con solución desinfectante de hipoclorito sódico que contenga 5.000 ppm de cloro activo(57) (exceptuando los casos asociados de covid-19 y muerte violenta). una vez el cadáver esté adecuadamente dispuesto en las bolsas, se podrá movilizar sin riesgo hacia el depósito de cadáveres siguiendo la ruta intrahospitalaria dispuesta para este traslado. luego el cadáver podrá será entregado al personal del servicio funerario para su depósito en ataúd o contenedor de cremación o inhumación y posterior traslado al sitio de destino final (horno crematorio y/o cementerio), luego de completar toda la documentación necesaria. cuando deba practicarse necropsia médico legal, el cuerpo será entregado a los servidores del sistema judicial quienes asumirán la custodia(56). si se han seguido correctamente todas estas indicaciones, se asume que no hay ninguna diferencia entre disponer del cuerpo enviándolo al crematorio o colocarlo en ataúd para llevarlo al tanatorio y realizar el entierro. si se opta por lo primero, las cenizas pueden ser objeto de manipulación sin que supongan ningún riesgo.(57) se recomienda que el trabajador de la salud conozca a través de la institución donde labora, los riesgos éticos, de salud y seguridad a que se expone por la atención en el paciente covid-19, evitando así conflictos e incertidumbres que afecten la atención. se recomienda que las instituciones prestadoras de salud a través de los líderes de atención médica, guíen y orienten a los trabajadores, para ofrecer una mejor atención médica y menor daño emocional durante la pandemia. se recomienda dar a conocer las directrices institucionales sobre el nivel de atención (uci vs. sala médica); inicio del tratamiento de soporte vital (incluyendo rcp y soporte de ventilación); retirada del tratamiento de soporte vital; y derivación a cuidados paliativos (centrados en la comodidad) en la atención médica de los pacientes durante la pandemia, esto genera una atención caracterizada por alivio del sufrimiento, no abandono, respeto a amci ® los derechos y preferencias de los pacientes, igualdad moral de las personas y la equidad en la distribución de riesgos y beneficios en la sociedad. el apoyo de la práctica ética es necesario integrarlo al cuidado de la salud y al bienestar de la fuerza laboral del cuidado en salud. reconociendo los desafíos especiales a que se enfrentan al responder al covid-19. esto forma parte del liderazgo en la atención médica y del servicio del deber cívico. berlinger n. el 29 de abril del 2020. en su artículo "respondiendo a covid-19 como un desafío regional de salud pública pautas preliminares para la colaboración regional que involucra hospitales". refiere que los trabajadores del equipo médico tienen el deber de conocer la gestión asistencial de los "desafíos éticos" previsibles durante la emergencia de salud pública (pandemia covid-19). que los desafíos éticos surgen cuando existe incertidumbre acerca de cómo "hacer lo correcto" es cuando los deberes o valores en los trabajadores entran en conflicto. estos desafíos afectan a la fuerza laboral (carga moral y emocional ante una decisión no prevista) en la atención médica. así como la operatividad en la atención médica (falta de epp y recursos que pueden limitar el buen desempeño por temor a infectarse).(59) los líderes de atención médica tienen el deber de guiar a los trabajadores de atención médica que experimentan condiciones laborales exigentes, mayor riesgo de daños ocupacionales, incertidumbre ética y angustia moral durante una emergencia de salud pública.(60) chih chen a, t. el 6 de abril del 2020. en su editorial ¿cómo deben prepararse los sistemas de salud para la evolución de la pandemia de covid-19? sugiere un apoyo emocional adecuado para el personal y horas razonables de exposición al riesgo para evitar el agotamiento, ya que los profesionales de la salud luchan por cuidar a los pacientes y proteger sus vidas y sus familias. se refiere que a medida que aumenta el número de casos, los médicos y los trabajadores de la salud en la primera línea deben reducir al mínimo su carga de trabajo clínico. las instituciones de atención médica deben reasignar al personal realizar tareas no clínicas, incluidos el papeleo y la recopilación de datos, tanto como sea posible. los hospitales deben tomarse su tiempo para capacitar al personal para implementar eficazmente las precauciones de contacto y los procesos de flujo. (61) jick j.l. el 5 de marzo del 2020. en relación con la obligación de planificar la atención médica, considera que: los líderes de atención médica tienen el deber de planificar la gestión de los desafíos éticos previsibles durante una emergencia de salud pública. la planificación de los desafíos éticos previsibles incluye la identificación de posibles decisiones de triage, herramientas y procesos. en una emergencia de salud pública que presenta una enfermedad respiratoria grave, es posible que se deban tomar decisiones de clasificación sobre el nivel de atención (uci vs. sala médica); inicio del tratamiento de soporte vital (incluyendo rcp y soporte de ventilación); retirada del tratamiento de soporte vital; y derivación a cuidados paliativos (centrados en la comodidad) si el tratamiento de soporte vital no se iniciará o se suspenderá. es posible que también se deban tomar decisiones de clasificación en relación con la escasez de personal, espacio y suministros. el deber de cuidado fundamental requiere fidelidad al paciente (no abandono como una obligación ética y legal), alivio del sufrimiento y respeto a los derechos y preferencias de los amci ® pacientes. el deber de cuidado y sus ramificaciones son el enfoque principal de la ética clínica, a través de los servicios de consulta de ética clínica a pie de cama, el desarrollo de políticas institucionales y la educación y capacitación en ética para los médicos. deberes de promover la igualdad moral de las personas y la equidad (justicia en relación con la necesidad) en la distribución de riesgos y beneficios en la sociedad. estos deberes generan deberes subsidiarios para promover la seguridad pública, proteger la salud de la comunidad y asignar de manera justa recursos limitados, entre otras actividades. estos deberes y sus ramificaciones son el foco principal de la ética de la salud pública. no se puede emitir una recomendación a favor o en contra acerca del uso de los medicamentos y dispositivos "prototipos" utilizados en el manejo del covid-19 denominados de uso compasivo o fuera de etiqueta, se considera sin embargo que no pueden ser utilizados por fuera de ensayos clínicos o protocolos institucionales estandarizados de evaluación del perfil de riesgo/beneficio y bajo la aplicación de consentimiento informado. se entiende como uso compasivo la utilización, en pacientes aislados y al margen de un ensayo clínico. dichos medicamentos experimentales no han sido aprobados aún por la fda, (64) y no se ha demostrado su seguridad y eficacia. es importante recordar que el medicamento médico puede tener efectos secundarios inesperados y graves, y que los pacientes deben considerar los posibles riesgos cuando procuran acceder a un producto médico experimental. hay que tener en cuenta que, para utilizar un medicamento bajo las condiciones de uso compasivo, se requerirá el consentimiento informado por escrito del paciente o de su representante legal, un informe clínico en el que el médico justifique la necesidad de dicho tratamiento. la regulación de la utilización de medicamentos por la vía del uso compasivo se ha realizado dentro de un texto legal sobre la realización de ensayos clínicos. una interpretación común del uso no indicado en la etiqueta y el uso compasivo de medicamentos es que, si el paciente murió, murió de la enfermedad, pero si el paciente sobrevivió, sobrevivió debido al medicamento administrado. recomendación se recomienda en la comunicación inicial con los familiares del paciente adulto con sospecha o diagnóstico de covid-19 críticamente enfermo incluir de forma clara y transparente los aspectos relacionados con el derecho al final de la vida que incluye: proporcionalidad en el tratamiento, adecuación del esfuerzo terapéutico, documento de voluntad anticipada, adecuación del esfuerzo terapéutico y la atención paliativa. situaciones estas que se pueden presentar durante la evolución hospitalaria y que requieren de una decisión conjunta entre el médico y el familiar del paciente. se recomienda dar una información específica, y adecuada a los familiares del paciente con sospecha o diagnóstico de covid-19, para que firmen el consentimiento informado, generando esta información confianza y comprensión en el familiar. la información del consentimiento que recibe el familiar debe constar dentro de la historia clínica. fundamento el ministerio de salud y protección social del 23 de marzo del 2020 a través del documento de "recomendaciones generales para la toma de decisiones éticas en los servicios de salud durante la pandemia covid-19". ante la posible circunstancia de pacientes sin capacidad para la toma de decisiones, por deterioro del estado general o requerimiento de aislamiento, en el cual no se puede contactar a su representante, conduzca la toma de decisiones teniendo en cuenta la prioridad de no hacer daño y la modificación en las condiciones de disponibilidad de los recursos en caso de presentarse deterioro. recuerde que esta situación debe preverse y debe ser informada a los representantes desde la admisión del paciente". durante toda la atención debe darse información sobre la posibilidad de que se presenten limitaciones de acceso a los soportes necesarios incluido al personal de salud , lo anterior puede limitar los derechos individuales o preferencias, esto debe ser informado al paciente y su familia, para que les permita entender que bajo la emergencia, puede presentarse una circunstancia que en condiciones habituales pudiera ser reversible de ser tratada pero en el contexto actual los recursos pueden verse trágicamente limitados, sin que esto implique abandono en el cuidado. el documento se refiere a la información que debe recibir el paciente o su familiar sustituto durante su evolución o fallecimiento. (65)la información durante la evolución también debe incluir: la información sobre el ejercicio de derechos al final de la vida incluyendo la adecuación de los esfuerzos terapéuticos y la suscripción de documentos de voluntad anticipada la consulta y revisión de existencia de este en todos los casos. se recomienda tener un consentimiento informado al ingreso hospitalario del paciente covid-19, se deben tener en cuenta las circunstancias del paciente al ingreso hospitalario, si la capacidad para la toma de decisiones está limitada por su estado clínico o incapacidad mental. de ser estas las circunstancias se dará la información al familiar en primera línea de consanguinidad quien asume por el paciente el consentimiento de la información (consentimiento sustituto). se recomienda tener el consentimiento informado en situaciones de excepción o urgencia ante la pandemia por covid-19, debe ser universal, en el que se informe el ingreso a la uci, o a cualquier otra área hospitalaria, realización de procedimientos, administración de tratamientos, posibles riesgos, beneficios durante su hospitalización. con esto se respeta el derecho a la autonomía personal en el paciente competente. en caso contrario el familiar tomará la información y asume el consentimiento a la información dada. es importante que el familiar esté informado de las decisiones que se vayan tomando durante la evolución hospitalaria (realización de procedimientos, inicios o cambios de tratamientos, movilización dentro del área hospitalaria. etc.) fuerte a favor fundamento el ministerio de salud y protección social, el 25 de marzo del 2020 ha elaborado un formato de "consentimiento informado para acompañante de casos probable/confirmado de covid-19". en que se expresa: "que de manera detallada se me ha suministrado información completa, suficiente, con un lenguaje sencillo y claro. el profesional de la salud me ha explicado la naturaleza de la enfermedad, acerca del significado de caso sospechoso o confirmado del coronavirus covid-19 en cuanto a su presentación clínica, modo de contagio, medidas para contenerla, posibilidad de sufrir la enfermedad, complicaciones o muerte, mientras permanezca como acompañante del paciente". este documento se firma al ingreso por el acompañante o familiar quien asume las decisiones durante su estancia hospitalaria. (67) el consentimiento informado en los pacientes covid-19 será un consentimiento sustituto para su ingreso a la uci y para los procedimientos que en la uci se realicen (colocación de tubo orotraqueal, diálisis, colocación de catéteres, reanimación cardiopulmonar, ecmo, etc.). previa información y autorización del familiar. en circunstancias normales el consentimiento debe ser firmado por el paciente quien en su autonomía acepta la información sobre su manejo y tratamiento. feld ad. recomendación se sugiere ante la pandemia del covid-19, si es posible, que el grupo de expertos en bioética y/o comité de ética institucional sean consultados y estén informados por el médico responsable para la orientación o consejo en la toma de directrices ante decisiones difíciles. se sugiere en lo posible que el médico tratante no asuma solo la responsabilidad moral de la decisión y que la decisión sea institucional y documentada en la historia clínica e informada a los familiares. en caso de no contar con un comité de bioética y/o ética hospitalaria el médico responsable podrá tomar la decisión fundamentada en principios éticos y derechos del paciente o convocar a una junta médica u otro comité relacionado con su dilema o consultar un apoyo externo en bioética. débil a favor fundamento el ministerio de salud y protección social. (65) el 23 de marzo del 2020 en el documento "recomendaciones generales para la toma de decisiones éticas en los servicios de salud durante la pandemia covid-19 establece: "que en caso de que la institución cuente con un comité de bioética y/o ética, con el servicio de bioética o ética clínica, o consultoría clínico-ética, se debe definir una ruta de consulta para los casos que de forma concreta puedan superar las recomendaciones generales. de igual forma establece que en las circunstancias actuales que se viven el actuar ético es parte integral del profesionalismo del cuidado. teniendo en cuenta que los profesionales de la medicina serán los llamados a tomar decisiones de alto estrés moral, al tener que adherirse y promover conductas concretas basados en las circunstancias que les rodean para dar o retirar tratamientos de las personas infectadas, quienes esperan confirmación del diagnóstico y de las personas que acuden a la atención en salud por razones diferentes a la infección por covid-19. partiendo de lo antes referenciado, se recomienda que las acciones emprendidas, busquen siempre poder responder a: a. no hacer daño b. beneficiar c. actuar con justicia sobre la persona en el contexto de la emergencia frente a la justicia sanitaria de la población d. mantener la integridad profesional" el comité de bioética y/o comité de ética institucional en tiempos normales o en tiempos de pandemia deberá mantener actualizadas las directrices de toma de decisiones. que apoyaran al médico responsable en la decisión. de no contar el médico con dicho comité o directrices institucionales y deba tomar una decisión que no permite interconsultar, deberá justificar en la historia clínica fundamentado en los principios éticos y derechos del paciente las razones que lo llevaron a tomar la decisión e informar y dialogar con los familiares del hecho. esto es importante que siempre quede documentado en la historia clínica la acción moral y ética de la decisión y el diálogo con la familia. en caso de que el médico responsable no cuente con un comité de bioética y/o comité de ética institucional, ni con el apoyo externo de expertos en bioética. y no quiera tomar la amci ® decisión a título personal podrá consultar a otro médico de la institución su decisión y entre ambos definir la acción a seguir. esta decisión conjunta debe ser documentada en la historia clínica e informada al grupo de trabajo y a los familiares como junta médica. si los familiares después de recibir la información no quedan satisfechos ellos tienen el derecho a la segunda opinión. los comités de bioética y/o comités de ética hospitalaria son entes administrativos, consultores, orientadores, asesores y consejeros de las situaciones que tienen que ver con el respeto y cumplimiento de los principios éticos, deberes y derechos de los pacientes, sus recomendaciones no son vinculantes, apoyan y orientan la decisión médica. aconsejando la mejor decisión ante una situación que genera un dilema moral o ético en el médico responsable. las decisiones médicas son tomadas por el consultor en bioética, quien es médico. los comités institucionales fuera de un comité de bioética y/o ética que podría dar apoyo al médico responsable y que serían otras instancias consultivas serian el comité de humanización, comité de historias clínicas, comité de bioseguridad, comité de infectología, comité de mortalidad hospitalaria, comité de paliativos o un comité de gerencia. pues todos los mencionados tiene que ver con el bienestar del paciente y la seguridad del médico ante una decisión. las decisiones especiales deben ser tomadas inicialmente a través de la realización de un comité (pueden ser los mencionados), en su defecto una junta médica. una vez se tenga la decisión esta debe ser consultada a la familia como una decisión institucional respaldada por el comité o junta médica realizada. se recomienda que todo ensayo clínico que se realice en la institución debe ser presentado, revisado y aceptado por un comité de investigación local o un comité de investigación externo nacional o internacional. se recomienda que todo paciente que se incluya en un ensayo clínico debe contar con un consentimiento informado el cual garantiza la aceptación voluntaria a participar y la comprensión de los objetivos, riesgos, beneficios, derechos y responsabilidades que tiene dentro de la investigación. se recomienda el consentimiento informado en todo ensayo clínico, el cual debe ser debe ser individual en tiempos de normalidad como en tiempos de pandemia por covid-19. solo el comité de ética en investigación podrá establecer en tiempos de normalidad o de pandemia las condiciones de dispensa o excepción al requisito de obtener el consentimiento informado. refiere que el consentimiento informado tiene sus raíces en el código de núremberg de 1947 y la declaración de helsinki de 1964 y ahora es un principio rector para la conducta en la investigación médica. en el consentimiento informado para investigaciones clínicas es claro que los participantes deben entender ampliamente los componentes del consentimiento. (69) thanh tam, n. et al. el 22 de enero 2015. mediante una revisión sistemática de pubmed, scopus y google scholar y revisando manualmente las listas de referencias para publicaciones hasta octubre de 2013. realizó un metaanálisis de los resultados del estudio utilizando un modelo de efectos aleatorios para tener en cuenta la heterogeneidad. evaluó la proporción de participantes en ensayos clínicos que entienden los diferentes componentes del consentimiento informado. encontrando que los participantes en ensayos clínicos deben comprender los componentes fundamentales del consentimiento informado como: la naturaleza y los beneficios del estudio, la libertad de retirarse en cualquier momento y la naturaleza voluntaria de la participación, así como la comprensión de otros componentes, como la aleatorización y el placebo. la proporción de participantes en ensayos clínicos que comprendieron diferentes componentes del consentimiento informado varió de 52.1% a 75.8%. esto asegura que la toma de decisiones de los participantes es significativa y que sus intereses están protegidos. (70) la red de américa latina y el caribe de cnb-unesco, (26 de marzo de 2020) que agrupa a las comisiones y consejos nacionales de bioética cuya finalidad es la de asesorar sobre los problemas éticos relativos a las ciencias de la vida y la salud humana expresa su preocupación ante la realización de investigaciones biomédicas en relación con la pandemia de enfermedad infecciosa por coronavirus covid-19. reconociendo lo siguiente en relación con el consentimiento informado: que la investigación con seres humanos durante las emergencias debe contar con garantías éticas mayores, no menores, que en las situaciones ordinarias. que en situación de excepción o emergencia los participantes deben seleccionarse en forma justa y proporcionar una justificación adecuada cuando se escogen o excluyen determinadas poblaciones, distribuyendo en forma equitativa las posibles cargas y beneficios de participar en esa investigación. "que se debe obtener el consentimiento informado individual de los participantes incluso en una situación de excepción o alarma, a menos que se cumplan las condiciones para la dispensa del consentimiento informado. las cuales el comité de revisión ética solo puede decidir dar la dispensa al requisito de obtener consentimiento informado: a) si no es factible obtenerlo; y si además los estudios: b) tienen un importante valor social y científico, c) solo suponen riesgos mínimos para los participantes, d) no implican agravio comparativo con otros grupos en situación o no de vulnerabilidad; y si e) se garantiza que no se privará a la población investigada de acceder en forma preferencial al beneficio derivado. de otorgarse un consentimiento informado amplio, éste debería ser única y exclusivamente para los procesos asociados con covid-19".(71) amci ® se recomienda considerar la transición del cuidado intensivo al cuidado paliativo en todo paciente con sospecha o diagnóstico de covid-19 sin mejoría a pesar de las intervenciones óptimas, con empeoramiento progresivo de su pronóstico vital y ante un evidente deterioro; aplicando medidas generales en control de síntomas ( manejo de secreciones -tratamiento del dolor -tratamiento de la disnea -sedación paliativa), así como apoyo espiritual, siempre acompañando al paciente y nunca abandonarlo en el final de la vida. fuerte a favor fundamento la sociedad española de anestesiología, reanimación y terapéutica del dolor en su documento: "marco ético pandemia covid-19" madrid, 20 de marzo de 2020 refiere: la sedación paliativa en pacientes hipóxicos con progresión de la enfermedad no subsidiaria de tratamiento debe considerarse como una expresión de buena práctica clínica y debe seguir las recomendaciones existentes. si se prevé un período agónico no corto, se debe proporcionar una transferencia a un entorno no intensivo.(71) se recomienda la utilización de guías establecidas previamente a la pandemia por el ministerio de salud y sociedades científicas para orientar las decisiones que se tomen al final de la vida en pacientes con sospecha o diagnóstico de covid-19. estas guías deben ser divulgadas al equipo de atención y aplicadas en los pacientes en casos de: adecuación del esfuerzo terapéutico (aet), orden de no reanimar (onr), consentimiento sustituto, voluntades anticipadas, cuidados paliativos. se recomienda fundamentar las decisiones del final de la vida individualizadas a cada paciente y a cada situación sin llegar a tomar decisiones apresuradas sin fundamento científico o ético, solicitando de ser posible una valoración por medicina paliativa para el manejo de síntomas. se recomienda indagar durante la hospitalización de todo paciente con sospecha o diagnóstico de covid-19, si en tiempos de salud hizo válida su autonomía y realizó un documento de voluntad anticipada, teniendo en cuenta que esta será equivalente al consentimiento informado. amci ® terapéuticos y la suscripción de documentos de voluntad anticipada.(72) el inicio de sedación paliativa con reubicación del pacientede ser necesario el des escalonamiento por deterioro clínico. en situación de pandemia covid-19 cuando se refiere a situaciones del final de la vida se relaciona a la adecuación del esfuerzo terapéutico, la sedación paliativa la cual será la maniobra terapéutica que se utilizará en pacientes no recuperables y que no son candidatos a cuidados intensivos por covid-19,(73) que evolucionan desfavorablemente y tienen mal pronóstico a corto plazo, así como la disnea refractaria y la limitación del esfuerzo terapéutico. el delirium o síndrome confusional por fallo cerebral agudo, es un problema habitual en situaciones de alteración orgánica severa, y ha sido descrito como uno de los síntomas neurológicos presente en los pacientes que sufren infección por el covid-19(74). wilson c. 24 de abril del 2020. en su artículo "la crisis golpea al final de la vida" se refiere a que el brote de coronavirus está obligando a las personas a enfrentar dilemas en torno a la cantidad de atención médica que se debe brindar al final de la vida y apresurar decisiones controvertidas sobre rechazar ciertos tratamientos. dicen los expertos que esto ha alentado a más personas a tomar decisiones de tratamiento anticipadas relacionadas con la rcp y la ventilación,(75) haesen s. el 16 de mayo de 2018. en su artículo "dirigir a los ciudadanos a crear directivas anticipadas" las voluntades o directrices anticipadas son para las personas que quieran asumir plenamente su papel de ciudadanos responsables tomando decisiones proactivas. la decisión de redactar directivas anticipadas marca un cambio del enfoque actual de "aceptación" a un escenario de "exclusión voluntaria".(76)al emitir una directiva de tratamiento anticipado, una persona autónoma puede expresar formalmente qué tipo de tratamiento desea y no desea recibir en caso de que se enferme o se lastime y no pueda decidir de manera autónoma sobre su tratamiento. (77) ministerio de salud y protección social en su documento de voluntades anticipadas que es el documento en el que toda persona capaz, sana o en estado de enfermedad, en pleno uso de sus facultades legales y mentales y como previsión de no poder tomar decisiones en el futuro, declara, de forma libre, consciente e informada su voluntad sobre las preferencias al final de la vida que sean relevantes para su marco de valores personales.(72) se recomienda que el paciente crítico con covid-19 que no es candidato para ingresar o continuar recibiendo cuidados intensivos y que presente deterioro rápido con mal pronóstico a corto plazo, se le brinde una adecuación del esfuerzo terapéutico orientada a acompañamiento al final de la vida, alivio del sufrimiento y control de síntomas. dependiendo de la disponibilidad de recursos se sugiere dentro del plan de atención hospitalaria contar con un área destinada a la atención del final de vida con el recurso físico, humano y de procesos necesario. marzo 13 del 2020. refiere: en situación de adecuación terapéutica, retirada de medidas y/o mala evolución es adecuado derivar al paciente a un área de menor complejidad para establecer el plan de cuidados paliativos. consultar al servicio de cuidados paliativos para procurar la continuidad de cuidados de los pacientes en los que se haya acordado la limitación de tratamientos y aliviar su sufrimiento, incluyendo la sedación paliativa en los casos en los que sea precisa.(71) schmidhauser tf. el 8 de abril 2020. considera en su publicación que: los cuidados paliativos durante la pandemia de covid-19 deben adaptarse a un estilo de" cuidados paliativos de emergencia" ya que los pacientes pueden deteriorarse rápidamente y requieren decisiones rápidas y planes de tratamiento claros. estos deben ser seguidos fácilmente por los miembros del personal de salud que atienden a estos pacientes. además, los cuidados paliativos deben estar a la vanguardia para ayudar a tomar las mejores decisiones, atender a las familias y ofrecer apoyo espiritual.(71) se recomienda como estrategia de protección personal en las unidades de cuidado intensivo sin presión negativa y cubículos abiertos utilizar de forma continua el respirador n95 o fpp2, adicional a otros elementos de protección para prevención del contagio por covid-19. la atención segura en áreas crítica para todas las modalidades de atención se fortalece a través de las medidas de precauciones estándar en el manejo de todos los pacientes, establecidos en el "manual de medidas básicas para control de infecciones en prestador servicios de salud" ley 9 de 1979, por la cual se dictan medidas sanitarias. resolución 4445 de 1996, numeral 3 y numeral 4.3 epp. el respirador, n95 o fpp2, puede utilizarse de manera continua por 8 a 12 horas, o desecharlo antes si está visiblemente contaminada o si está húmeda. luego de colocar la n95 se debe verificar prueba de ajuste antes de ingresar a la unidad para atención de pacientes con covid-19 de la siguiente manera: mascarillas sin válvula de exhalación: cubra la totalidad de la mascarilla con ambas manos y exhale con fuerza. si nota fugas de aire por sus bordes, reajuste la posición del respirador. mascarillas con válvula de exhalación: cubra el respirador con ambas manos e inhale con energía. deberá sentir una presión negativa dentro de la mascarilla. si detecta alguna pérdida de presión o entrada de aire, reajuste la posición del respirador. no se puede emitir una recomendación a favor o en contra acerca de la efectividad de bioseguridad del uso extendido, continuo o intermitente de los respiradores n95 ó ffp2. sin embargo, se considera que puede ser una alternativa, bajo la adopción de un protocolo riguroso, cuando se debe optimizar el uso de los epp en el contexto de un acceso limitado. el reúso no está permitido en colombia. la duración máxima del uso continuo de la n95 es de 8 a 12 horas, siguiendo las recomendaciones del manual de medidas básicas para control de infecciones en ips de minsalud. pero en la vida real, ningún trabajador tolera 8 a 12 horas continuas con un respirador. por esto, su uso continuo en el sitio de trabajo dependerá de la necesidad de pausar para comer, para ir al baño, etc. en este caso, se guardará en una bolsa de papel para su nueva colocación, si tiene menos de 12 horas, o desechará si está visiblemente contaminada o se torna húmeda. el reúso de la n95 dependerá de la casa del fabricante, de si contiene o no celulosa en su estructura del respirador. por ejemplo, la recomendación de desinfección para los respiradores sin celulosa es con peróxido de hidrógeno vaporizado al 58% por 28 minutos (tabla 9 y 10). los respiradores n95 de uso industrial tienen mayor contenido de celulosa que los de uso médico por lo tanto para procesos de esterilización, solo los n95 de uso médico podrán ser esterilizados mediante de peróxido de hidrógeno vaporizado (sterrad®) (84-88) 22. ¿existe superioridad en términos de protección personal para el personal sanitario y de apoyo dentro de las unidades de cuidados intensivos con la utilización del overol frente a la bata manga larga con antifluido durante la atención del paciente con covid-19? no se puede emitir una recomendación a favor o en contra si los overoles ofrecen mayor protección por cobertura corporal frente a otros elementos como vestidos largos, batas y delantales. resulta intuitivo que su uso genera una mayor protección en especial en servicios cohortizados. sin embargo, su uso está asociado con una mayor dificultad para su colocación y retiro, lo que puede potencializar el contagio del usuario, la utilización debe hacerse bajo un protocolo supervisado y chequeado. las batas modificadas para ajustarse firmemente en el cuello pueden reducir la contaminación. en estudios ya descritos el uso de un respirador eléctrico purificador de aire con overol puede proteger contra el riesgo de contaminación mejor que una máscara n95 y una bata con un rr: 0.27, intervalo de confianza (ic) del 95%: 0.17 a 0.43 pero fue más difícil su retiro con rr 7.5, ic del 95% 1.81 a 31.1. en un eca (59 participantes), las personas con una bata larga tenían menos contaminación que aquellas con un overol. las batas pueden proteger mejor contra la contaminación que los delantales.(89-92) los epp como batas y overoles deberían estar hechos de un material que cumpla con los requisitos mínimos de la asociación americana de químicos textiles:  tipo a: buena repelencia al agua, resistente a la penetración, pero mala permeabilidad al aire.  tipo b: buena repelencia al agua, buena permeabilidad al aire, pero poca resistencia a la penetración del agua.  tipo c: bata quirúrgica que tiene poca repelencia al agua y resistencia a la penetración de agua.  tipo d: hecho de fibras de polietileno de alta densidad, tela no tejida (tyvek®), tiene buena repelencia y resistencia al agua, mala permeabilidad al aire. no se puede emitir una recomendación a favor o en contra para el uso de respiradores elastoméricos como elementos de protección personal dentro de las unidades de cuidados amci ® intensivos. no hay evidencia que soporte la superioridad de los respiradores elastoméricos frente a los n95, son más costosos, difíciles de utilizar y pueden implicar algún riesgo para el paciente. por tanto, su uso sólo debería considerarse frente a un desabastecimiento de los n95 y bajo la adopción de un protocolo institucional riguroso y bajo chequeo. los respiradores elastoméricos son respiradores ajustados a media cara o cara completa, esta última otorga protección ocular. su filtración está determinada por el filtro que se utilice, estos van desde partículas de nivel n95 a p100. están hechos de material sintético o de goma que les permite desinfectarse, limpiarse y reutilizarse repetidamente. están equipados con cartuchos de filtro reemplazables. al igual que los respiradores n95, los respiradores elastoméricos requiere entrenamiento adecuado para su correcta colocación y retiro. por eso es muy importante revisar el manual del usuario antes de su uso. los respiradores elastoméricos no deberían utilizarse en entornos quirúrgicos, debido al riesgo potencial de contaminación del campo quirúrgico, con el aire que sale de la válvula de exhalación. como recomendación de buena práctica, aprobado por la fda, debe colocarse una máscara quirúrgica encima de la válvula de exhalación para evitar este riesgo. solo se debe permitir el uso del respirador elastoméricos por clínica para evitar infecciones cruzadas, esto permitirá una protección esencial contra agentes infecciosos y la auto contaminación. aunque los cartuchos de filtro son finalmente desechables, están destinados a ser reutilizados hasta que ya no se puede respirar o se vuelven visiblemente sucio. generalmente se recomienda, en la mayoría de los casos, hacer recambios cada 30 días. deben tener procedimientos de limpieza/desinfección actualizados y aprobados por su manufacturador.(78, 93-110) recomendación se recomienda realizar la limpieza y desinfección de equipos biomédicos y de superficies las veces que sean necesarias y en el momento de egreso del paciente siguiendo los protocolos de cada institución. el desinfectante para este proceso debe ser de nivel intermedio o alto para superficies y equipos biomédicos y cumplir con las recomendaciones del fabricante según lo aprobado en el registro sanitario. fuerte a favor fundamento para la desinfección de las superficies ambientales hospitalarias y domiciliarias, la oms recomienda emplear un desinfectante que sea efectivo contra virus cubiertos (el coronavirus pertenece a esta categoría), específicamente, recomienda emplear alcohol etílico para la desinfección de algunos equipos biomédicos reusables (p. ej.: termómetros) y para las superficies, el hipoclorito de sodio o precursores de sodio como el dicloroisocianurato de sodio (nadcc) que tiene la ventaja de la estabilidad, la facilidad en la dilución y que no es corrosivo.(87) (tabla 11). page se recomienda que el ingreso de un paciente a uci debe hacerse bajo un procedimiento estandarizado que incluya la coordinación y comunicación de los servicios vinculados, adecuación de la unidad de atención a las necesidades del paciente y la garantía de la bioseguridad del equipo multidisciplinario. se recomienda que cada institución establezca en sus procesos prioritarios el circuito del traslado que incluye el itinerario del traslado, el uso de ascensor, el número y la organización de los intervinientes sanitarios y no sanitarios (celadores, seguridad, limpieza), las medidas de protección empleadas por los mismos (epp, limpieza) y los recursos materiales necesarios durante el traslado. el traslado de pacientes con casos sospechosos o confirmados de covid-19 se puede presentar entre servicios a nivel hospitalario o entre instituciones con diferentes niveles de atención y deben tenerse las precauciones universales de un traslado seguro. una posición responsable es evitar el traslado de estos pacientes el máximo posible, a menos que sea imprescindible, teniendo en cuenta el riesgo/beneficio. considerar evitar traslados interinstitucionales solo por temas administrativos. el personal sanitario que realice el traslado debe contar con todos los epp, considerando este traslado como de alto riesgo de transmisión vírica. se debe utilizar mascarilla quirúrgica o n-95, de acuerdo con el riesgo amci ® de aerosolización. hasta que la rt-pcr para sars-cov-2 este negativo se podrían retomar las prácticas habituales de traslado de los pacientes . (111) (112) (113) capítulo 2. abordaje diagnóstico y covid-19 se recomienda en pacientes con diagnóstico o sospecha de infección por sars-cov-2 clasificar la enfermedad en leve, severo o paciente crítico, teniendo en cuenta los criterios de la clasificación por las fases y estadios de la enfermedad. se recomienda en pacientes con diagnóstico o sospecha de infección por sars-cov-2, clasificados como críticos y que requieren de intubación orotraqueal realizar la clasificación por fenotipos (1 o 2), con el fin de proyectar una estrategia de ventilación mecánica. el covid-19, es una enfermedad con una presentación clínica diversa, desde formas leves hasta presentaciones graves que incluyen el sdra, la mediana del período de incubación desde la exposición hasta el inicio de los síntomas es de aproximadamente 4 a 5 días, y el 97.5% de los pacientes sintomáticos tendrán síntomas dentro de los 11.5 días después de la infección (114) , que incluye fiebre, tos, disfagia, malestar general, mialgias, anorexia, náuseas, diarrea, anosmia y ageusia; la disnea se presentó entre los 5 y 13 días (115) y puede representar progresión a covid-19 severo, que se manifiesta con hipoxemia, disfunción orgánica múltiple, documentación de arritmias cardíacas, rabdomiólisis, coagulopatía y choque (116) . dentro del espectro de enfermedad, siddiki et al, proponen un enfoque estructurado por fases expresados en tres estadios (historia natural de la enfermedad), siendo el primero donde la patogenicidad viral es dominante, se incluye el periodo de incubación, síntomas leves, con multiplicación del sars-cov-2 centrándose principalmente en el sistema respiratorio gracias a la unión del virus con el receptor de la enzima convertidora de angiotensina 2 (ace2), el hemograma puede revelar linfopenia y neutrofilia sin otras anormalidades significativas. el estadio 2 es la enfermedad pulmonar establecida, neumonía viral, tos, fiebre con progresión en algunos casos a hipoxia con trastorno de los índices de oxigenación (pao2/fio2 menor 300 mmhg), hallazgos en imágenes de tórax (radiografía y/o tomografía) de infiltrados alveolares o vidrio esmerilado, mayor linfopenia y elevación de transaminasas (2a: sin hipoxemia, 2b: con hipoxemia). el estadio 3 o fase de híper inflamación sistémica extrapulmonar se caracteriza por elevación de biomarcadores inflamatorios y estado protrombótico (il -2, il-6, il-7, ftn -α, proteína c reactiva, ferritina y el dímero d), con presencia en las formas más graves de disfunción orgánica múltiple, lesión miocárdica (troponina y péptido natriurético de tipo b elevados), con fenómenos trombóticos, progresión a sdra y choque (117) . amci ® la neumonía por sars-cov-2, se característica por disociación entre la severidad de la hipoxemia y el mantenimiento relativamente bueno de la mecánica respiratoria, con compliance del sistema respiratorio en promedio de 50 ml / cmh2o; gattinoni, marini et al, proponen dos fenotipos de presentación de la insuficiencia respiratoria; el primero (tipo 1) con una mecánica pulmonar adecuada, con baja probabilidad de reclutabilidad y con hipoxemia, relacionado al desbalance entre la perfusión y la ventilación; el segundo (tipo 2) más acorde a las definiciones de sdra (csdra "covid-19 patient with sdra"), con una compliance pulmonar baja y reclutabilidad potencial(118, 119) (120). se propone la siguiente clasificación clínica del covid-19. (121, 122 , y pacientes críticos de 5%, con tasa global de mortalidad de 2,3%, siendo mayor entre los pacientes de 70 a 79 años con 8% y entre los mayores de 80 años con 14.8%, dentro del grupo de pacientes clasificado como crítico la mortalidad descrita fue del 49%(121). recomendación se recomienda en cuidado intensivo, realizar el diagnóstico de covid-19 del paciente sospechoso por medio de rt-pcr conociendo su alta especificidad, su variabilidad en relación con el tiempo y pérdida de rendimiento diagnóstico luego de la primera semana de inicio de los síntomas. se recomienda tomar la primera muestra para rt-pcr de hisopo nasofaríngeo o de cornete medio sobre hisopado oro faríngeo o de saliva, de ser negativo se puede repetir la prueba de 24 a 48 horas preferiblemente de tracto respiratorio inferior, esputo no inducido o en aspirado traqueal en paciente intubado. se recomienda el uso conjunto de rt-pcr e igm por elisa en pacientes con sospecha de covid-19, primera rt-pcr negativa, que se encuentren entre la segunda y tercera semana desde el inicio de los síntomas, con el objetivo de mejorar la sensibilidad en la identificación de infección por sars-cov-2. en cuidado intensivo, el diagnóstico de covid-19 se fundamenta con base en la presentación clínica compatible y factores epidemiológicos asociados con probabilidad de infección; el diagnóstico definitivo se realiza con pruebas de amplificación de ácido nucleico del virus (naat), la detección del genoma viral del sars-cov-2 se realiza por medio de reacción en cadena de la polimerasa por transcriptasa reversa (rt-pcr) dado a su especificidad del 100% (123, 124) ; por lo cual todo paciente que cumple con la definición de caso sospechoso se le debe realizar rt-pcr, sars-cov-2 independientemente de si se encuentra otro patógeno respiratorio (124) . las muestras para el diagnóstico por rt pcr se recolecta de las vías respiratorias superiores, nasofaringe, cornete medio u orofaringe; todos con alta especificidad. sin embargo, se sugiere recolectar los hisopos nasofaríngeos o de cornete medio por tener mayor sensibilidad (125 (17/34) . en pacientes con neumonía severa a quienes se le realizó lavado broncoalveolar (bal) y rt-pcr entre los días 8 y 14 el 100% de las muestras fueron positivos, en pacientes no intubados con esputo no inducido el 83% de las muestras fueron positivas (126, 127). wang et al, en un estudio de 205 pacientes con covid-19, las rt-pcr con tasas positivas más altas fue en muestras extraídas por bal (95%; 14 de 15 muestras) y esputo 72% (72 de 104 muestras) (128) . para la detección del sars-cov-2 por rt-pcr en pacientes en cuidado intensivo, teniendo en cuenta la rigurosidad de aspectos de bioseguridad y aerosolización, se debe tomar la primera muestras en nasofaringe o cornete medio, si esta prueba es negativa se puede repetir en 24 a 48 horas, si este es el caso o existe más de 8 días desde el inicio de los síntomas se prefiere una muestra del tracto respiratorio inferior, por esputo no inducido por personal de salud o por aspirado traqueal en pacientes intubados (126, 129), aunque el rendimiento diagnóstico del bal es alto por lo general, se debe evitar la broncoscopia para minimizar la exposición de los trabajadores de la salud (130) . la probabilidad de detección del arn de sars-cov-2 puede variar según la fase de la enfermedad, si bien una rt-pcr positiva confirma el diagnóstico de covid-19, los reportes falsos negativos y la sensibilidad se ve influenciado por el tiempo desde la exposición e inicio de síntomas. kucirka et al, en un análisis de siete estudios evaluaron el rendimiento diagnóstico de la rt-pcr en relación con el tiempo desde el inicio de los síntomas o la exposición, con resultados expresados en tasa estimada de falsos negativos, siendo del 100% el día de la exposición, del 38% el día 5 (estimado como primer día de síntomas, ic: 18% a 65%), 20% en el día 8 (día 3 desde el inicio de síntomas, ic: 12% a 30%) luego comenzó a aumentar nuevamente de 21% en el día 9 (ic: 13 a 31%) a 66% en el día 21 (ic: 54 a 77%) (131) . la precisión y los valores predictivos de rt-pcr para sars-cov-2 no se han evaluado sistemáticamente, la sensibilidad de las pruebas moleculares está influenciada por múltiples factores como sitio y calidad de la muestra, técnica de procesamiento; probablemente las menores tasas de falsos negativos (sensibilidad entre 65 y 80%) está entre el día 3 y 10 luego de inicio de los síntomas (131) . a partir de aquí el rendimiento diagnóstico disminuye, por lo tanto, es importante que el intensivista valore estas consideraciones en el momento de tomar conductas, en cuanto tipo de aislamiento, tratamiento y pronóstico. amci ® las pruebas serológicas detectan anticuerpos contra el sars-cov-2 y ayudan a identificar pacientes que han tenido la enfermedad y algunos con la enfermedad activa, la seroconversión se ha descrito entre el día 13 y 21, sin embargo, hay incertidumbre en la incidencia de la seroconversión (132) . estas pruebas se usan principalmente en tamizaje poblacional y estudios de seroprevalencia; en cuidado intensivo el análisis de la igm por elisa contribuye a la detección de pacientes con infección reciente, además con el análisis conjunto con la igg se clasifica el estado de infección en agudo o convaleciente. las pruebas serológicas se realizan por diferentes técnicas como la inmunocromatográfica de flujo lateral, la inmunofluorescencia indirecta (ifi) y el ensayo de inmunoadsorción ligado a enzima (elisa) (133, 134) . las pruebas serológicas no deben usarse como la única prueba para diagnosticar o excluir la infección activa por sars-cov-2. la sensibilidad y la especificidad de muchas de estas pruebas serológicas son inciertas, así como su valor predictivo positivo. los anticuerpos detectables generalmente tardan varios días en desarrollarse. guo et al, documenta niveles de anticuerpos por elisa, con una mediana de detección de anticuerpos igm e iga de 5 días (iqr, 3-6) y de igg de 14 días (iqr, 10-18) después del inicio de los síntomas, con una probabilidad de resultados positivos de 85.4%, 92.7% y 77.9 % respectivamente; es probable que el rendimiento diagnóstico de igm por elisa sea mayor que la de rt-pcr después del quinto día luego de inicio de síntomas; cuando se combinan estas técnicas (elisa igm con rt-pcr) la tasa de detección positiva es del 98.6% (135) . zaho et al, en un estudio de 173 pacientes con covid-19, donde el 18,5% estaba en condición crítica, la mediana del tiempo desde el inicio de los síntomas hasta la detección de anticuerpos (técnica elisa) fue de 12 días para igm y 14 días para igg; dentro de los primeros 7 días desde el inicio de los síntomas solo el 38.3% tenía anticuerpos detectables, entre los días 8 a 14 la sensibilidad de igm fue 73.3% e igg de 54.1%, luego de los 15 días la sensibilidad igm e igg fue de 94.3% y 79.8% respectivamente; el uso combinado de rt-pcr y elisa igm presentó una sensibilidad del 78% entre los días 1 a 7 y del 97% entre los días 8 a 14. (136) la rt-pcr tiene especificidad del 100%, con adecuado rendimiento diagnóstico entre los días 3 y 10 luego del inicio de los síntomas con sensibilidad que varía entre el 65 y 80%, con presencia ascendente de falsos negativos luego del día 9, por lo cual el diagnóstico debe tener consistencias epidemiológicas y clínicas (síntomas y hallazgos radiológicos compatibles con covid-19) donde una rt pcr negativa no excluye la enfermedad; la precisión y el tiempo para la detección de anticuerpos varían con la técnica utilizada, su uso es limitado en cuidado intensivo, sin embargo su identificación por técnica elisa en conjunto con rt-pcr mejora la sensibilidad y la probabilidad de falsos negativos, especialmente entre los días 3 y 14 desde el inicio de síntomas. faltan estudios que evalúen el rendimiento diagnóstico de las diferentes pruebas. amci ® se recomienda la medición de marcadores de severidad al ingreso a uci del paciente críticamente enfermo por covid-19 (hemograma, transaminasas, ldh, ferritina, troponina, dímero d y pcr) los cuales se han asociado con peor pronóstico en la enfermedad por covid-19, logrando ofrecer intervenciones más tempranas. se recomienda no utilizar una periodicidad de rutina para la medición de seguimiento de biomarcadores de severidad en el paciente con sospecha o diagnóstico de covid-19. en un estudio cohorte retrospectivo que evaluó 140 pacientes diagnosticados con covid-19 desde el 18 de enero de 2020 hasta el 12 de marzo de 2020, fang liu y colaboradores encontraron correlación en la elevación de il-6 y pcr con la gravedad clínica, lo que sugiere podrían usarse como factores independientes para predecir la severidad del cuadro, los pacientes con il-6> 32.1 pg./ml o pcr> 41.8 mg/l tenían más probabilidades de tener complicaciones graves (137) , así mismo en otro estudio multicéntrico retrospectivo de 150 pacientes infectados se identificó resultados de laboratorio con diferencias significativas con elevación de glóbulos blancos, valores absolutos de linfocitos, plaquetas, albúmina, bilirrubinas, función renal, transaminasas, troponina, proteína c reactiva e interleucina (il 6) en el grupo con desenlace de mortalidad contra los dados de alta (138) . entre otros marcadores la troponina como lesión cardiaca (elevación de troponina por encima del percentil 99 límite de referencia superior) se ha reportado en 7% a 23% de los pacientes con covid-19 en wuhan, china, en dos estudios retrospectivos por xiabo yang y colaboradores (37, 139) . en una revisión sistemática de mayo 2020, kermali m. y colaboradores exponen que existe evidencia a favor de los valores bajos de linfocitos y plaquetas y valores elevados de los biomarcadores il-6, pcr, troponina, ldh, ferritina, proteína amiloidea a y dímero d, pueden relacionarse con la gravedad de la infección por covid-19 y su fuerte asociación con la mortalidad (140) . estos resultados pueden usarse como un complemento en la práctica clínica para guiar a los médicos a identificar pacientes con mal pronóstico y la rápida implementación de medidas de soporte, monitorización y reanimación en la evolución de los pacientes críticos en la unidad de cuidados intensivos. solo en 1 estudio, karmali et al, en 2002, determinan en promedio entre 48 a 72 horas, la periodicidad en el seguimiento de estos, sin embargo, no se discrimina entre pacientes críticos y no críticos. consideramos que el seguimiento de estas pruebas debe estar ajustado al juicio clínico del médico intensivista tratante, según la evolución de los pacientes. se muestran el comportamiento de los biomarcadores mas frecuentes en la tabla 12. tabla 11. biomarcadores en pacientes críticos con sospecha o diagnóstico de covid-19. tendencia de biomarcador en relación con la gravedad covid-19 pcr aumentada recomendación se recomienda la no medición de marcadores de inflamación o de severidad de forma rutinaria solo con el objetivo de iniciar un tratamiento específico o algoritmos terapéuticos en la enfermedad por covid-19 en pacientes críticos. el síndrome de liberación de citocinas o denominado "tormenta de citocinas" parece asociarse en pacientes con afecciones graves por covid-19. la citocina proinflamatoria il-6 es la citocina mejor documentada en covid-19 correlacionada con la gravedad, el estado crítico del paciente, la carga viral y el pronóstico (138, 141, 142) . se han descrito mayores niveles de citoquinas proinflamatorias (il-2, il-6, il-7, factor estimulante de colonias de granulocitos, factor de necrosis tumoral e interferón gamma) asociadas a compromiso pulmonar severo en pacientes con infección por coronavirus, determinado por la rápida replicación del virus, infiltración masiva de células inflamatorias y trastorno severo de la inflamación (143, 144) . igualmente, está asociada la presencia de linfopenia como biomarcador de mal pronóstico para covid-19 (145) . hallazgos similares se encontraron en la pandemia de influenza a (h1n1) de 2009 sin ser especificó su valor (146) . las manifestaciones clínicas de la tormenta de citocinas incluyen síndrome de respuesta inflamatoria sistémica, hipotensión, síndrome de fuga capilar, insuficiencia renal, sdra, miocarditis, entre otras (116) , algunos autores han determinado este cuadro como un síndrome de linfohistiocitosis hemofagocitica secundaria. es razonable pensar que, en pacientes con sospecha de tormenta de citocinas basado en los hallazgos de laboratorio, el manejo con inmunomoduladores puede resultar beneficioso, sin embargo, los resultados del manejo de la hiperinflación basado en pruebas diagnósticas han tenido resultados encontrados en pacientes con covid-19. el uso de esteroides, inmunoglobulina endovenosa, inhibidores del receptor de citoquinas (tocilizumab) o inhibidores de janus kinasa, han disminuido los valores de los biomarcadores, días de hospitalización (147) o necesidad de fracciones elevadas de oxígeno (148) , sin embargo no han demostrado beneficio sobre la mortalidad y en algunos casos, si un aumento en la incidencia de infecciones bacterianas o fúngicas sobreagregadas (116) . se recomienda realizar radiografía simple de tórax para todos los pacientes con sospecha o diagnóstico de covid-19 en uci. se recomienda realizar tac de tórax según disponibilidad de tecnología institucional, ante la incertidumbre diagnostica, teniendo en cuenta las condiciones clínicas, la tolerancia del paciente al traslado y los protocolos administrativos de seguridad. fuerte a favor fundamento se reconoce como el gold estándar diagnóstico de infección pulmonar por covid-19 a los estudios moleculares, sin embargo, estos presentan limitaciones: a) contaminación de las muestras b) errores en la técnica de la toma, c) muestra insuficiente para ampliación genética favoreciendo falsos negativos d) demora de reporte de los resultados. por lo anterior, se recomienda imágenes diagnósticas en la aproximación de paciente con sospechas de infección pulmonar por covid-19 (149, 150) .  radiografía simple de tórax: ventajas: mayor accesibilidad que la tac de tórax y realización de la prueba a la cabecera del enfermo desventaja: baja sensibilidad en estadio temprano, después del día 9 de inicio de los síntomas presenta aumento en el rendimiento diagnóstico  tac de tórax: ventaja: es ampliamente recomendada. alta sensibilidad en estadios tempranos. permite describir extensión, distribución, localización, densidades parenquimatosas, aplicables en clasificaciones y puntajes diagnósticos, pronósticos y de seguimiento en permanente evolución y mejoría. algunas asociaciones referentes como asociación china de radiología, en su recomendación de expertos propone clasificación tomografía en 4 estadios 1 temprana 2 avanzada 3 severo 4 disipación desventaja: traslado del paciente hasta el tomógrafo (no todos los enfermos toleran el transporte) y tecnología no disponible en todos los niveles de atención page no se puede emitir una recomendación a favor o en contra para el uso de ecografía pulmonar a la cabecera del paciente crítico como herramienta diagnóstica o de pronóstico en con covid-19. se puede considerar como una alternativa para la valoración imagenológica pulmonar en el paciente crítico con covid-19 cuando las condiciones del paciente no permitan su traslado. no se sugiere la utilización de la ecografía pulmonar para el seguimiento de lesiones pulmonares agudas en el enfoque del paciente crítico con covid-19. puede utilizarse para determinar complicaciones asociadas a la enfermedad o en la inserción de dispositivos invasivos. en general los estudios de imágenes no representan un papel concluyente para el diagnóstico de covid-19. la ultrasonografía en específico requiere estudios de validación, un programa de entrenamiento es operador dependiente, y se le atribuido limitaciones en la capacidad de discriminación en la cronicidad de las lesiones pulmonares. la ultrasonografía pulmonar puede servir como herramienta a la cabecera del paciente para mejorar la evaluación del compromiso pulmonar y reducir el uso de radiografías de tórax y tomografía computarizada (151) , sin embargo no debe usarse para el diagnóstico inicial, pues éste se compone de criterios clínicos, radiográficos y microbiológicos que actualmente son el estándar de oro; la ecografía no los reemplaza debido a la baja especificidad en relación con el virus, se sugiere su uso como complemento en la valoración diaria del paciente, ojalá realizada por el mismo observador. la ecografía pulmonar es altamente sensible y puede revisar de forma rápida y precisa la condición pulmonar, creando un potencial para evaluar los cambios o la resolución con el tiempo, especialmente en la uci, escenario en el que cada vez se usa más para la detección de múltiples patologías pulmonares que se pueden demostrar junto con covid-19, sin embargo, hasta la fecha no hay hallazgos específicos, ni patognomónicos que se relacionen con covid-19 en el examen ecográfico del paciente (152) . la adopción de ultrasonido pulmonar puede reducir la necesidad de exposición a la radiación ionizante y, a su vez, reducir la cantidad de radiografías necesarias para la evaluación rutinaria del paciente, disminuyendo también la exposición de personal asistencial adicional como el uso de elementos de protección personal (153) . es bien conocido el beneficio de la ecografía durante y después de la colocación de accesos venosos centrales para establecer la presencia o no de complicaciones inmediatas como neumotórax. a la fecha no hay publicaciones acerca de la utilidad del ultrasonido como herramienta para establecer pronóstico. se necesitan más estudios para evaluar la utilidad de la ecografía pulmonar en el diagnóstico y manejo de covid-19 (153) . se recomienda no establecer un punto de corte en el valor de dímero d para el inicio rutinario de anticoagulación plena en el contexto de infección por covid-19. se recomienda la administración de profilaxis antitrombótica según protocolo institucional independiente de niveles de dímero d en el paciente críticamente enfermo por covid-19. los fenómenos inflamatorios inherentes a procesos infecciosos son considerados desde décadas previas, factores protrombóticos, no siendo una excepción la infección por covid-19. en algunas publicaciones se hallan asociaciones con desenlaces cardiovasculares negativos (154) y sugieren asociación entre niveles elevados de dímero d ( marcador de estado de trombosis ) y riesgo de embolismo pulmonar con or crecientes or de 1.7 a los 3 días hasta 2.4 a los 9 días de seguimiento (155) . de igual manera, zhou et al, reportan asociación de dímero d mayor a 1 mcg/ml y mortalidad (37) sin embargo los estudios presentan limitaciones en su diseño, a pesar de ello algunos autores proponen anticoagulación como factor de protección en mortalidad sin precisar precisión en la dosis, tipo de heparina y selección de enfermos (156) . finalmente la european heart journal en su entrega de farmacología cardiovascular desarrolla una propuesta en la cual combina un puntaje previo de riesgo de cid en uci a niveles de fibrinógeno; esta pudiera ser una herramienta para selección de pacientes a recibir anticoagulación sin embargo aún está en proceso de validación (157) . por estos motivos, hasta el momento, no se tiene suficiente cuerpo de evidencia que permita hacer una recomendación basado en los niveles de dímero d como variable aislada para administración de anticoagulación terapéutica. se recomienda no utilizar de rutina la procalcitonina en un algoritmo diagnóstico, para diferenciar entre neumonía viral vs bacteriana o confirmar la presencia de una sobreinfección bacteriana en el paciente con sospecha o diagnóstico de covid-19. se recomienda no medir de forma rutinaria la procalcitonina en pacientes con sospecha o diagnóstico de covid-19 como factor pronóstico. la procalcitonina es un biomarcador que ha sido incluido en algoritmos de diagnóstico y pronóstico durante los últimos años. schuetz et al, en 2017, concluyen en una revisión sistemática de 26 estudios, que la procalcitonina es segura dentro de un algoritmo, para guiar a los médicos tratantes entre iniciar o suspender antibióticos en neumonía adquirida en la comunidad; sin embargo en una revisión sistemática más reciente, kamat et al, reportaron una sensibilidad 55% [ic 95% 37%-71%; i2 = 95.5%], especificidad 76% [95% ic, 62%-86%; i2 = 94,1%]) para el inicio de tratamiento antibiótico para neumonía bacteriana, lo cual nos determina que la prueba es inespecífica para diferenciar entre infecciones virales vs bacterianas. en neumonía por sars-cov-2, se han publicado algunos artículos evaluando el uso de la procalcitonina como prueba asociada al pronóstico de los pacientes. liu et al encontraron que la procalcitonina se asoció a mayor severidad de los cuadros de neumonía. hr, 4.908; 95% ci, 1.797-13.402; p=0.002. en este estudio también se tuvo un resultado similar con la proteína c reactiva y la il-6. plebani et al, publican un metaanálisis, donde sugiere que los niveles elevados de procalcitonina se asocian a mayor severidad de la infección. (or, 4.76; 95% ci, 2.74-8.29). es importante mencionar, que en estos estudios se evaluaron otros biomarcadores de inflamación como interleucina 6, proteína c reactiva y ferritina; presentando todos ellos, aumento en sus valores y asociándose a severidad de la enfermedad; por tanto, se considera que, en particular, la procalcitonina elevada, no representa una diferencia en el pronóstico, comparado con otros biomarcadores de inflamación. hasta el momento no se han publicado estudios en infección por sars-cov-2 donde se evalué el papel de prueba diagnóstica para confirmar sobreinfección bacteriana o diferenciar entre neumonía viral vs bacteriana. (137, (158) (159) (160) se recomienda no usar de forma rutinaria el uso de pruebas clínicas de laboratorios clínicos para determinar la resolución de la enfermedad crítica por covid-19. se recomienda considerar la ausencia de dificultad respiratoria y fiebre por más de 72 horas, requerimiento de oxígeno a baja concentración y bajo flujo, como indicadores clínicos de resolución de la fase crítica de la enfermedad por covid-19. se recomienda no utilizar de forma rutinaria el uso de pruebas microbiológicas de erradicación viral, para determinar la resolución de la enfermedad en pacientes en uci con covid-19. amci ® enfermedad está asociado tanto a la carga viral como a la respuesta hiperinflamatoria del huésped a la infección viral. en cuanto a la carga viral, en pacientes que tienen un curso leve de infección, el pico de la carga viral en muestras nasales y orofaríngeas ocurre durante los primeros 5-6 días tras el inicio de síntomas y prácticamente desaparece al día 10, mientras que en los que cursan con neumonía severa en uci, la carga viral es 60 veces mayor y puede persistir la excreción viral hasta el día 21 a 28 (161) . por esta razón, consideramos que en los pacientes en uci no es necesario confirmar la erradicación del virus o su negativización en muestra respiratorias, orina o heces, para determinar la mejoría clínica, curación o para el egreso del paciente crítico (162) . en el contexto clínico, el pronóstico se ha asociado a la presencia de marcadores bioquímicos elevados, sin embargo, no existe evidencia que el seguimiento con estos marcadores iniciales de inflamación determine el momento exacto de la resolución de la enfermedad. varias organizaciones internacionales como el cdc de usa y el european centre for disease prevention and control (10 mar 2020) (162, 163), national centre for infectious diseases (ncid) singapore (164), world health organization (13 de marzo de 2020)(165), han establecido criterios para resolución clínica y egreso hospitalario de los pacientes. estos criterios incluyen: ausencia de fiebre mayor a 72 horas sin antipiréticos, mejoría de los síntomas respiratorios, ausencia de requerimiento de hospitalización por otras patologías, el resultado de dos (2) rt-pcr para sars-cov-2 negativas, con intervalo de muestra mayor a 24 horas. la utilidad de dichos criterios no ha sido evaluada en pacientes en cuidado intensivo. en uci, huang et al (115) , describieron en 28 pacientes a quienes se dio egreso, la ausencia de fiebre por 10 días, mejoría radiológica y evidencia de erradicación viral, como criterios de alta. sin embargo, consideramos que deben primar los criterios clínicos sobre los paraclínicos, en el momento de definir el egreso de un paciente de cuidado intensivo, teniendo como principal indicador la ausencia de dificultad respiratoria y la mejoría en los índices de oxigenación, con requerimiento de oxígeno suplementario a bajos flujos y concentración (166) . se recomienda la cánula de alto flujo, donde esté disponible, en pacientes con covid-19 a nivel del mar con hipoxemia leve (pao2/fio2<300 y >200 o sao2/fio2<300 y>200). en alturas superiores a los 2000 mts por encima del nivel del mar esta terapia se puede considerar en pacientes que no tengan hipoxemia severa (pafi< 100). amci ® se recomienda en pacientes críticos por covid-19 el uso de la cánula de alto flujo en salas de presión negativa, donde estén disponibles, que garanticen la seguridad del recurso humano. si no se dispone de habitación con presión negativa se puede optar por habitación individual cerrada. se debe contar con todo el equipo de protección personal necesario para el personal sanitario y de apoyo. se recomienda colocar mascarilla quirúrgica por encima de la cánula nasal en el paciente con sospecha o diagnóstico de covid-19 y mantener una distancia mínima de 2 metros con otros pacientes. se recomienda la intubación inmediata en pacientes críticamente enfermos con sospecha o diagnóstico covid-19 con índice de rox ([spo2/fio2] / frecuencia respiratoria) < 3 a las 2 horas de iniciada la oxigenación con cánula de alto flujo teniendo en cuenta que el retraso en la intubación aumenta la mortalidad. se recomienda considerar la cánula de alto flujo en caso de agotamiento de ventiladores mecánicos. la cánula de alto flujo ofrece flujos de hasta 60 litros/minuto, que aportan una fracción inspirada de oxígeno (fio2) constante que reduce el espacio muerto y produce una presión positiva que genera reclutamiento alveolar y puede redistribuir el líquido alveolar (167) (168) (169) . se ha reportado que al generar aerosoles, aumenta el riesgo de contagio para el personal de salud (167, (170) (171) (172) . hasta el punto de que se ha recomendado su uso en salas con presión negativa (167) . recientes publicaciones han establecido que la cánula de alto flujo genera una corta distancia de dispersión de aerosoles con lo cual las medidas de distanciamiento, un adecuado equipo de protección personal y donde estén disponibles, realizarla en salas de presión negativa darían seguridad al uso de la cánula de alto flujo (167, 173) . a pesar de que la experiencia en la actual pandemia ha sido escasa, basada en series de casos, estudios retrospectivos y de metodología limitada, ha resultado ser promisoria en cuanto a la mejoría en la oxigenación y la tolerancia por el paciente (37, 121, 139, 166, 167, (174) (175) (176) (177) (178) . sin embargo, hasta el momento no se ha establecido que la cánula de alto flujo evite la intubación. la caf podría convertirse en un alto riesgo de mortalidad al prolongar la decisión de intubación y al favorecer la lesión pulmonar autoinducida (p-sili) por aumento del esfuerzo inspiratorio. por todo ello es necesario un estricto y estrecho monitoreo del paciente durante una o dos horas para definir si ha habido mejoría o no mientras se realizan estudios que demuestren que evita la intubación y genera desenlaces clínicos importantes como menor estancia en uci, menor estancia hospitalaria y menos días de ventilación mecánica. la terapia de oxígeno de caf podría ser considerada para pacientes atendidos en altitudes mayor a 2000 mts, que no tienen hipoxemia severa (pafi < 100), la respuesta debe evaluar dentro de los 30 a 60 minutos posteriores a su inicio y los pacientes que no mejoran amci ® significativamente y progresa la dificultad respiratoria no deben mantenerse con esta terapia. el monitoreo del paciente con caf para la toma de decisión de éxito o fracaso de esta estrategia y considerar la posibilidad de intubación incluye la evaluación gasométrica, la oximetría de pulso, así como considerar los criterios para intubación: frecuencia respiratoria >30 por minuto, deterioro de conciencia, inestabilidad hemodinámica, pao2/fio2<150 (a nivel del mar), sao2/fio2<150, índice de rox<2,85, spo2<93% (119, 166-169, 175-177, 179,180) . se recomienda crear o ajustar protocolos institucionales de sedoanalgesia basado en objetivos con escalas validadas. se recomienda el uso de analgesia multimodal que incluya analgésicos opioides, no opioides y bloqueos regionales en el paciente crítico por sospecha o diagnóstico de covid-19. se recomienda sedación profunda con agentes como midazolam o propofol para mantener rass entre -4 y -5 en pacientes covid-19 con sdra severo, necesidad de uso de relajantes neuromusculares o posición prona. se puede considerar en planos de sedación superficial agentes como propofol o dexmedetomidina (coadyuvante) para mantener rass entre 0 y -3 en pacientes seleccionados con sospecha o diagnóstico de covid-19 con sdra no severo. en la actualidad no se encuentra evidencia de alta calidad proveniente de ensayos clínicos, sino editoriales, series, reportes de casos y artículos de revisión de expertos (180) (181) (182) . la creación y aplicación de protocolos de sedoanalgesia adaptados a cada institución ha mostrado disminución del tiempo en la uci y menores complicaciones (183, 184) . es importante definir objetivos guiados por escalas, recomendándose sedación profunda o completa en situaciones especiales como ventilación mecánica invasiva por sdra severo, disincronía ventilatoria persistente, posición prona y bloqueo neuromuscular (bnm), como puede observarse en pacientes covid-19 con compromiso pulmonar severo. mientras que se debe procurar sedación ligera, cooperativa o no sedación en pacientes en ventilación mecánica invasiva en pacientes con sdra no severo, ventilación no invasiva y en el retiro de la ventilación (185, 186) . los opioides han sido el pilar de la analgesia en dolor moderado a severo. el fentanilo es actualmente el más usado; el remifentanilo reduce el tiempo en ventilación en pacientes amci ® renales, hepáticos, ancianos y neurológicos; la hidromorfona se prefiere en el retiro de la ventilación y en pacientes extubados; y la metadona ha mostrado disminuir la tolerancia a opioides (187) . se propone el uso de estrategias de analgesia multimodal asociando medicamentos no opioides como el paracetamol, ketamina a dosis analgésicas (<0,5 mg/kg) en dolor somático, lidocaína en dolor visceral, y pregabalina en dolor neuropático (188) . la sedación ligera o cooperativa son mejores con propofol en cuanto a tiempo de despertar y con dexmedetomidina para preservar funciones cognitivas y el impulso respiratorio, con menor desacondicionamiento (189) . en sedación profunda, el propofol ha mostrado más fácil titulación y menos acumulación que el midazolam; sin embargo, su uso se ve limitado hasta 48 horas y a dosis < 3,0 mg/kg/h, ante el riesgo de pris (síndrome relacionado con la infusión de propofol). el midazolam, sin dosis techo ni tiempo límite y de bajo costo, ha sido el más utilizado de los sedantes, disminuyendo su uso por su asociación con delirium y de retraso en los tiempos en ventilación; sin embargo, la pandemia covid-19 ha vuelto a aumentar su uso. la dexmedetomidina ha sido utilizada como adyuvante en sedación profunda, disminuyendo el consumo de sedantes, con menos efectos secundarios (190, 191) . se muestran los medicamentos para sedoanalgesia y relajación neuromuscular que se pueden utilizar en los pacientes con covid-19 en la tabla 13. se recomienda iniciar una estrategia individualizada de ventilación mecánica ajustadas a las condiciones específicas de severidad en el paciente crítico con covid-19. amci ® se recomienda la ventilación protectora en modos controlados por volumen o por presión que garanticen un volumen corriente < 8 cc/kg de peso predicho teniendo como metas una presión meseta < 30 cm h2o y una presión de conducción < 15 cm h2o. se recomienda emplear fracciones inspiradas de oxígeno para lograr metas de saturación de acuerdo con la pao2/fio2 entre 88 y 92% en el paciente con sospecha o diagnóstico de covid-19. en la paciente embarazada entre 92 y 95%. se recomienda en el paciente crítico por covid-19 iniciar con peep individualizado a la severidad del compromiso pulmonar y ajustar el nivel de peep de acuerdo con la tabla de fio2/peep. el estudio arma (192) demostró que la ventilación con bajos volúmenes corrientes (vt) se asocia con reducción de: mortalidad (p=0.007), en días libres de ventilación mecánica (p=0.007) y días libres de falla orgánica (p= 0.006). una revisión sistemática posterior confirmó que el uso de bajos volúmenes se asocia con menor progresión a sdra (193) . un metaanálisis que revisó 159 estudios y 29 meta-análisis en uci confirmó que la ventilación protectora era una de las tres intervenciones que mejora la sobrevida en pacientes con sdra (194) . esto fue confirmado por landoni en un análisis de estudios multicéntricos con impacto en mortalidad en uci (195) . recientes publicaciones han sugerido que en covid-19 puede haber dos fenotipos que se diferencian en la distensibilidad (119, 196) . sin embargo, el mismo estudio arma demostró que "el beneficio de ventilación con vt más bajo fue independiente de la distensibilidad de las vías respiratorias, lo que sugiere que el vt más bajo fue ventajoso independientemente de la distensibilidad pulmonar". más aún, el uso de bajos vt se asoció con una reducción en las concentraciones de interleuquina 6 lo cual explicaría el mayor número de días sin falla orgánica múltiple y sugeriría una reducida respuesta inflamatoria asociada a la ventilación protectora (192) . con el tiempo la evidencia ha demostrado que la ventilación protectora, además de vt y presión meseta bajos, debe incluir presiones de conducción menores de 15 cm h2o (197) (198) (199) . existe suficiente evidencia que demuestra que fio2 y pao altas se asocian con aumento en la morbimortalidad (200) (201) (202) . en sdra el estudio arma y una más reciente publicación demostraron que el tener metas conservadoras de pao2 se asocia con mayor sobrevida (192, 203) . hasta el momento, la literatura en ventilación mecánica ha demostrado que la mejor estrategia para ajustar el nivel de peep en sdra es la tabla de fio2/peep (192, 204) . amci ® se recomienda la ventilación mecánica protectora en sdra por covid-19 independiente del fenotipo de presentación. se recomienda la clasificación de fenotipos en sdra para pacientes con covid-19 para ajustar la toma de decisiones de manera individualizada en ventilación mecánica. el manejo ventilatorio en covid-19 tiene los mismos principios generales de los pacientes con sdra (205) . sin embargo, la identificación de fenotipos podría impactar en la evolución y pronóstico (206) . gattinoni ha postulado el desarrollo de un sdra típico (fenotipo h: con alta elastancia, alto cortocircuito, alto peso pulmonar) o una presentación atípica (fenotipo l: caracterizado por baja elastancia, bajo shunt, bajo peso pulmonar). (207) . pelosi et al sugiere clasificar los pacientes con tres fenotipos similares (208) . con base en tales fenotipos se han propuesto estrategias ventilatorias diferenciales para minimizar el daño inducido por el ventilador (vili) (209): 1. el primer paso, en el fenotipo l, es revertir la hipoxemia aumentando la fio2. 2. en el tipo l, hay varias opciones no invasivas: cánula nasal de flujo alto, presión positiva continua en la vía aérea (cpap) o ventilación no invasiva (niv). se debe evaluar el esfuerzo inspiratorio y, de estar disponible, medir la presión esofágica. la peep alta puede disminuir los cambios de presión pleural y detener el ciclo vicioso que exacerba la lesión pulmonar. sin embargo, la peep alta, en pacientes con distensibilidad normal, puede tener efectos hemodinámicos perjudiciales. en cualquier caso, las opciones no invasivas son cuestionables, ya que pueden asociarse con altas tasas de fracaso y retraso de la intubación. 3. la magnitud de las presiones pleurales inspiratorias puede determinar la transición del tipo l al h. la presión esofágica > 15 cmh2o aumenta el riesgo de vili y, por lo tanto, la intubación debe realizarse lo antes posible. los tipo l, si son hipercápnicos, pueden ser ventilados con volúmenes > 6 ml / kg (hasta 8 ml / kg). la posición prona debe ser usada solo en último caso, ya que las condiciones pulmonares son buenas. la peep debe reducirse a 8-10 cm h2o, dado que la capacidad de reclutamiento es baja y el riesgo de falla hemodinámica aumenta. la intubación puede evitar la transición al fenotipo tipo h. 4. los pacientes tipo h deben ser tratados como sdra grave, incluyendo mayor peep, si es compatible con la hemodinamia, posición en pronación y soporte extracorpóreo. en el fenotipo 3 se deben aplicar las estrategias de ventilación protectora convencional (208, 209) . amci ® se recomienda en paciente con covid-19 considerar hipoxemia refractaria cuando no se obtienen las metas de oxígeno propuestas, a pesar de las maniobras ventilatorias recomendadas y cumple con los siguientes parámetros: pafi < 150, fio2 > 0,6 y peep apropiado, considerando la altitud. se recomienda considerar la utilización de ecmo, en sitios donde esté disponible y con alta experiencia para obtener resultados aceptables, en pacientes con hipoxemia refractaria luego de haber implementado ventilación protectora, relajación neuromuscular y posición prona. se recomienda administrar tromboprofilaxis en todos los pacientes con covid-19 con hipoxemia refractaria que no presenten contraindicaciones. la hipoxemia refractaria no es un concepto estático y absoluto, según la definición de berlín del sdra, se clasifica el sdra en leve, moderado y severo de acuerdo con la relación pao2 y fracción inspirada de oxígeno, con peep mayor de 5 cms de h2o. la hipoxemia severa es aquella que cuenta con una pafi menor de 100 (210) (211) (212) . la hipoxemia refractaria hace referencia a un estado de hipoxemia severa que a pesar de las diferentes estrategias ventilatorias no aumenta la pafi y tiene consecuencias en el estado ácido básico y metabolismo celular permitiendo una anaerobiosis (213, 214) . para definir la hipoxemia refractaria deben coincidir varios escenarios , una pafi menor de 150, una fracción inspirada de oxígeno mayor de 0.6, a pesar de un peep apropiado no se tiene en cuenta en la definición el ph ni la paco2 ni el tiempo transcurrido (122) . algunas de las medidas terapéuticas no ventilatorias que se han empleado en sdra y covid-19 con hipoxemia refractarias son la oxigenación con membrana extracorpórea (ecmo) y la tromboprofilaxis o anticoagulación de rutina. la oxigenación extracorpórea a través de una membrana ha sido una estrategia controvertida en pacientes con hipoxemia refractaria de diferentes orígenes, en la epidemia de la influenza por el virus h1n1 fue usada en pacientes con hipoxemia refractaria teniendo resultados aceptables (215) . en el estudio eolia los resultados no mostraron mejoría en la supervivencia, aunque hay diferentes posiciones y estudios post hoc de este ensayo clínico con beneficios, su uso se limita a casos muy restringidos y en sitios de alta experiencia para obtener resultados aceptables (216) . en sdra por covid-19 el ecmo se ha usado en hipoxemia refractaria entre un 4 a 11.5% en diferentes series con resultados variables (205, 217, 218) . un tipo de pacientes hipoxémicos y con ventilación mecánica han presentado cuadros tromboembólicos pulmonares en estos casos la trombólisis de rescate con activador de plasminógeno tisular rtpa (alteplase) se ha recomendado con resultados alentadores en serie de casos, pero su evidencia es muy débil para ser recomendada(219-223). dada la alta frecuencia de enfermedad tromboembólica reportada en covid-19 se ha reportado la utilidad de la tromboprofilaxis, especialmente en casos de dímero d o índice de sic elevado (156) . amci ® se recomienda monitorizar sistemáticamente la oxigenación con los índices: pao2/fio2 y sao2/fio2, y en donde esté disponible el monitoreo continuo con capnografía. se recomienda monitorizar de forma rutinaria la presión meseta y la presión de conducción como estrategia al pie de la cama para verificar la ventilación protectora. el sdra y covid-19, es una condición dinámica que apenas se está caracterizando, hay varias presentaciones que no cumplen con todos los criterios de berlín (209, 211) . gattinoni ha caracterizado en dos presentaciones el sdra en los pacientes con neumonía por coronavirus sars-cov-2, una con alta compliance, mínima reclutabilidad; la otra con baja compliance, pulmones pesados y reclutabilidad, tal vez esta presentación sea el verdadero sdra (118, 224, 225) . los pacientes que requieren ventilación mecánica por falla ventilatoria en covid-19, son los que mayor mortalidad tienen al parecer por la lesión pulmonar inducida por la intubación tardía y el gran esfuerzo respiratorio con presiones transpulmonares oscilantes y muy negativas (212, 213, 226) . la monitoria de estos pacientes soportados con ventilación mecánica tiene dos objetivos: el primero detectar el deterioro clínico para sugerir estrategias más avanzadas como el ecmo, y el segundo es evitar el daño pulmonar inducido por la ventilación mecánica. se debe tener presente la mayor posibilidad de contagio con el número de manipulaciones en el paciente, por esto nunca olvidar el perfecto uso de los elementos de protección personal y disminuir el número de contacto con el paciente. las metas que se buscan con la ventilación mecánica en el paciente con sdra por covid-19 son mantener una oxigenación adecuada teniendo en cuenta la altura sobre el nivel del mar con pao2 entre 70 y 80 mmh20 y metas de saturaciones reportadas entre 89 y 92% a 90 y 95% , mantener una ventilación adecuada evitando el espacio muerto , disminuir el trabajo respiratorio y protegiendo el pulmón del daño ocasionado por la ventilación mecánica y las repercusiones hemodinámicas (214) .  el confort de los pacientes en ventilación mecánica es la principal señal de un uso adecuado del modo ventilatorio y los parámetros ventilatorios apropiados para la patología y demanda del paciente (214) .  las curvas y bucles son herramientas indispensables para valorar la mecánica respiratoria del paciente soportado con ventilación mecánica, se puede diagnosticar amci ® las asincronías del paciente y el ventilador, el origen, tipo y frecuencia además de la respuesta al manejo. también se evalúa la resistencia de la vía aérea (214) . variables fisiológicas:  es importante valorar la oxigenación del paciente, la literatura actual sugiere el monitoreo de la pafi es el más representativo y sencillo test para valorar la oxigenación y representa el shunt pulmonar, se debe hacer mínimo diariamente, o cuando se haga una intervención en el ventilador o paciente; en sdra por covid-19 la hipoxemia se relaciona directamente con mortalidad, debe mejorar con la ventilación mecánica (214, 227) .  la medición de la paco2 indica la de la ventilación, la hipercapnia tiene relación directa con el espacio muerto en el paciente con sdra, y varios estudios la relacionan con la mortalidad. puede evaluarse directamente en los gases arteriales o relacionarla con el pco2 expirado por medios de la capnografía, gattinoni propone una forma de evaluarla al lado de la cama del paciente relacionando el etco2/paco2, cuando es < de 1 sugiere un shunt elevado y mayor espacio muerto; áreas de pulmón ventiladas y no aireadas. otras tecnologías incluidas en el ventilador moderno como la capnografía volumétrica se está validando para evaluar el espacio muerto, la reclutabilidad y la titulación de peep (214, 228) .  la saturación venosa mixta svo2 , refleja de manera subrogada la función ventricular, no todos los pacientes tienen catéter de arteria pulmonar para su medición por lo que se está reemplazando con el ultrasonido en la cabecera del paciente; recordar que el 30% de los pacientes con sdra cursan con falla ventricular derecha (214) . monitoria de mecánica ventilatoria y protección pulmonar:  para evitar el daño pulmonar debe propender por un volumen corriente bajo (4-8 ml/kg de peso predicho) y presión plateau menor de 30 cms h20, para garantizar la ventilación con protección pulmonar (192, 229) .  driving pressure ( presión cambiante de la vía aérea, presión diferencial o presión de conducción) es la presión plateau (presión pico en ventilación controlada por presión) menos peep, debe ser menor de 15 cm h20, está relacionado con aumento en la mortalidad en pacientes ventilados por que representa una medición indirecta del strain pulmonar porque relaciona el volumen corriente con la compliance del sistema respiratorio y este a su vez se relaciona con el volumen espiratorio pulmonar final (198) .  la medición de la compliance del sistema respiratorio es necesaria y nos clasifica el paciente de acuerdo con su fenotipo para trazar el plan de manejo ventilatorio, cuando la compliance es baja, esto se puede hacer al lado de la cama del paciente con los ventiladores modernos (214, 225) .  la construcción de la curva presión/volumen aún es una herramienta útil para ubicar el área de ventilación segura del paciente, evitando el atelectrauma y la sobredistensión pulmonar(estrés) , el peep se calcula dos puntos por encima del punto de inflexión inferior y el punto de inflexión superior nos indica hasta dónde podemos aumentar el volumen corriente este punto debe estar por debajo de 30 cms h20 para evitar la sobre distensión, con los ventiladores modernos se puede construir esta curva (214, 229) . amci ®  las curvas presión tiempo en pacientes ventilados con modos volumétricos pueden monitorizar la resistencia de la vía aérea, la compliance pulmonar, el trabajo respiratorio, las curvas de flujo puede también indicar si se presenta autopeep, resistencia aumentada de la vía aérea entre otras (214) .  presión transpulmonar, en casos más complicados donde es más difícil obtener la meta de oxigenación a pesar del peep en aumento una opción es el catéter esofágico, para medir la presión transpulmonar en la inspiración y espiración y calcular así el stress pulmonar y evitar las presiones oscilatorias y sobre todo negativas para evitar el daño pulmonar. esta herramienta también ayuda a evaluar el trabajo respiratorio, y el diagnóstico de las asincronías que se presenten en el paciente ventilado (214, 230) . se recomienda no utilizar de forma rutinaria la relajación neuromuscular en el paciente crítico con covid-19 con sdra. se recomienda utilizar la relajación neuromuscular en pacientes en posición supino o prono, que están fuera de parámetros de protección pulmonar (presión de conducción mayor 15 y presión plateau mayor a 30) con pafi menor 150 y cuando ya no es posible limitar el volumen corriente. se debe considerar la utilización de protocolos estandarizados con el fin de disminuir la variabilidad, y según disponibilidad seleccionar el cisatracurio como primera opción, en caso de escasez se pueden utilizar otras opciones teniendo en cuenta su farmacodinamia y farmacocinética. el 13 de marzo de 2020, la organización mundial de la salud emite una serie de orientaciones para el manejo de la infección respiratoria aguda grave (irag) en pacientes con sospecha o diagnóstico de covid-19. en el paciente críticamente enfermo con sdra moderado o grave (pao2/fio2 <150) por covid-19 no está indicado de forma sistemática el bloqueo neuromuscular mediante infusión continua debido a que no se cuenta con evidencia suficiente que sustente mejoría en la supervivencia con respecto a una estrategia de sedación ligera sin bloqueo neuromuscular, se debe considerar su uso cuando se evidencia asincronía paciente-ventilador a pesar de la sedación, hasta el punto de que no se pueda limitar el volumen corriente de forma fiable, hipoxemia o hipercapnia que no mejoran con el tratamiento (231) . recomendación se recomienda no utilizar de forma rutinaria oni en pacientes adultos que presenten sdra e infección por sars-cov-2. fuerte en contra fundamento a la fecha (mayo 18 de 2020) no contamos con estudios sobre el uso de óxido nítrico inhalado (oni) como tratamiento de pacientes con infección covid-19. existe evidencia indirecta sobre el uso de oni en el síndrome de dificultad respiratoria aguda (sars-cov), y la infección por coronavirus en el síndrome respiratorio de oriente medio (mers-cov) (233) . en 2016 cochrane realizó una revisión sistemática que incluyó 14 ensayos de calidad moderada con 1275 pacientes adultos con sdra tratados con óxido nítrico inhalado. los resultados no mostraron ningún efecto estadísticamente significativo sobre la mortalidad (rr 1.04, 95% ci 0.9 -1.19). se mostró mejora transitoria en el índice de oxigenación a las 24 horas (md (diferencia media) -2,31, ic del 95% -2,73 a -1,89) y mejoría en pao2/fio2 a las 24 horas (md 15,91, ic del 95% 8,25 a 23,56). no se identificó diferencia significativa en los días libres de ventilación y finalmente se presentó aumento estadísticamente significativo en la incidencia de insuficiencia renal en pacientes con óxido nítrico inhalado (rr 1,59, ic del 95% 1,17)(234). amci ® en 2014 se realizó un estudio observacional que incluyó 14 pacientes tratados de dos hospitales de beijing con oni como tratamiento para sars (235) . en comparación con ningún tratamiento, oni mejoró la saturación arterial de oxígeno (spo2) de 93% a 99% (p<0.05); se asoció a menor necesidad de oxígeno suplementario (p< 0.05) y menor necesidad y retiro de cpap y bial (p < 0.05). los cambios en radiografía de tórax mejoraron en 5 de los 6 pacientes que recibieron oni. sin embargo, debido a problemas graves de validez por pequeño tamaño de la muestra (n= 16, oni=6, control=8), no aleatorización y no enmascaramiento en la asignación, se considera que este estudio cuenta con baja calidad metodológica, lo cual limita la interpretación de los resultados. en un estudio retrospectivo multicéntrico que incluyó 330 pacientes con mers-cov en condición crítica en arabia saudita, se mostró que el manejo con ventilación no invasiva (niv) tenía mayor probabilidad de requerir óxido nítrico en comparación con los pacientes con ventilación mecánica invasiva (20,0% vs 11,7%, p a 0,05) (236) . en una serie de casos en la que participaron 12 pacientes con infección por mers-cov confirmada o probable, 6 pacientes recibieron oni debido a hipoxemia refractaria. en el seguimiento a 90 días, cinco de los pacientes continuaron vivos (237) . los estudios sobre mers-cov se limitaron a una serie de casos y una cohorte retrospectiva con baja calidad de evidencia. en ambos estudios, los pacientes recibieron otras terapias de rescate (relajación neuromuscular, ventilación oscilatoria de alta frecuencia, ecmo y posición en prono), por lo tanto, se desconoce el efecto terapéutico clínico del oni en el tratamiento de la infección por mers-cov. a la fecha (18 de mayo de 2020) tres ensayos clínicos evalúan el papel del óxido nítrico inhalado en pacientes con covid-19 y sdra leve/moderado, y como profilaxis para los trabajadores de la salud covid-19(tabla 14). tabla 13. comparación de ensayos clínicos que evalúan el papel del óxido nítrico inhalado en pacientes con covid-19 y sdra leve/moderado, y como profilaxis para los trabajadores de la salud covid-19. no se cuenta con evidencia por el momento que respalde el uso de óxido nítrico inhalado en pacientes con covid-19. los resultados de los ensayos en curso, así como ensayos clínicos de alta calidad son necesarios para apoyar su uso. sólo evidencia indirecta metodológicamente limitada de óxido nítrico en pacientes con sras mostró una mejor oxigenación, una menor necesidad de oxígeno suplementario y mejoría en la radiografía de tórax. en pacientes con sdra y mers-cov, no mostró un beneficio claro e incluso mostró un mayor riesgo de insuficiencia renal (238) . otros estudios han evaluado el efecto tóxico asociado a su uso documentando metahemoglobinemia(239), inhibición de la agregación plaquetaria y formación de dióxido de nitrógeno (240) . razones por la cuales guías recientemente publicada no recomienda su uso de forma rutinaria(205). recomendación se recomienda el uso temprano de la ventilación en posición prona, por al menos 16 horas continuas, en pacientes con sdra por covid-19 con pao2/fio2<150 mmhg. la ventilación en posición prono como estrategia ventilatoria propuesta desde los años 70 (241), cuenta con evidencia que demuestra resultados positivos en cuanto a mejoría de mortalidad, mejoría en el trastorno de oxigenación y el reclutamiento alveolar en pacientes con sdra. los mecanismos por los cuales la posición prona conduce a la mejoría en el trastorno de oxigenación y del reclutamiento alveolar en los pacientes con sdra, incluyen (242) (243) (244) (245) : amci ®  mejoría de la relación ventilación/perfusión y mayor homogeneidad en la distribución de aire en los pulmones.  aumento del volumen de fin de espiración.  disminución del efecto compresivo del corazón en los pulmones.  mejoría del drenaje de las secreciones.  optimización del reclutamiento alveolar, con mejoría de la distribución del volumen corriente, a su vez, limita el desarrollo del daño alveolar pulmonar. los estudios coinciden en el efecto benéfico que esta terapia tiene en la mejoría de la oxigenación, el objetivo se centrará en evaluar las recomendaciones con respecto a:  beneficio de la terapia con respecto a la mortalidad al día 28, al día 90 y a los 6 meses.  beneficio de la terapia según el grado de severidad de sdra con respecto la relación pao2/fio2.  tiempo de terapia en posición prono con mayor beneficio.  número de sesiones de la terapia en posición prono.  recomendaciones según balance riesgo/beneficio en lo que respecta a los efectos adversos: retiro o desplazamiento no planeado de catéteres, obstrucción de tubo endotraqueal, neumonía asociada a ventilador, lesiones de presión. se eligieron 8 artículos que con las características metodológicas consistentes (246) (247) (248) (249) (250) (251) (252) (253) . de estos ocho artículos se excluyó uno por corresponder a pacientes pediátricos (253) . al evaluar los escritos de forma cronológica, se puede apreciar en los primeros artículos (246, 247) , la dificultad para lograr enrolar (reclutar) el suficiente número de pacientes, de tal manera que algunos estudios fueron detenidos de forma prematura (246, 247, 251, 252) . era entonces esperable que los resultados no fueran concluyentes, y que no lograran ser robustos, al no alcanzar el tamaño de muestra deseado, comprometiéndose la confiabilidad, el poder y corriendo el riesgo de obtener resultados falsamente negativos. sin embargo, se podía percibir en los diferentes estudios, una notoria mejoría en la oxigenación, sin repercusión en la mortalidad (246) (247) (248) (249) (250) (251) . por otro lado, es necesario tener en cuenta que los estudios iniciales (246) (247) (248) (249) no se realizaron con el uso de ventilación protectora asociada a la pronación. esto es un elemento pertinente, pues la ventilación protectora puede per se, brindar un efecto adicional en la mejoría de la oxigenación. otro efecto importante que debe analizarse es el bloqueo neuromuscular, cuya evidencia hoy sugiere su uso en criterios ya mencionados (254) . con el tiempo y según la experiencia de cada centro, se fue utilizando con menor dificultad (255) . sin embargo, no se tenía aún claro qué tipo de pacientes obtenían el mayor beneficio de la terapia, además que el tiempo de la terapia en posición prona, seguía siendo una incógnita. la evidencia reflejaba hasta el momento que los pacientes con sdra más severos y la terapia aplicada por más tiempo se asociaban con una tendencia a la reducción en mortalidad. para dirimir qué tipo de pacientes se beneficiarían más de la terapia, se tomó como base, la severidad del sdra según la relación pao2/fio2 (210) . a pesar de que los estudios iniciales(246-250) enfrentaban el reducido tamaño de muestra, algunos análisis identifican beneficio de la mortalidad (246) en pacientes con sdra más severo, constituyendo un probable umbral de beneficio cercano y por debajo de 140-150 mmhg (256) . amci ® antes del estudio proseva (252) en totalidad 2 estudios reportan mortalidad al día 28 (247, 251) , un estudio mortalidad al día 30 (246) , uno estudio mortalidad al día 60 (250)y uno mortalidad al día 90 (248) . sin embargo, al tomar todos los estudios, aún no se alcanzaba suficiente significancia estadística para reducir la mortalidad a los 28 días y 6 meses. con el estudio proseva -guerin et als -en el año 2013 (252) , se alcanza más poder estadístico, demostrando un beneficio importante en pacientes con pao2/fio2 menor de 150 mmhg, ventilados con bloqueo neuromuscular y ventilación protectora en su totalidad, y logrando una reducción de la mortalidad al día 28 y mantenida hasta el día 90 con respecto al grupo control (16.0% (38 de 237 participantes) versus 32.8% (75 de 229) (p<0.001). de tal manera que, según la evidencia disponible, en lo que al trastorno de oxigenación se refiere (según la relación pao2/fio2), sugiere que el mejor candidato para esta estrategia ventilatoria es el paciente con sdra severo con una pao2/fio2 menor de 150 mmhg. es de primordial importancia detenerse a considerar por otra parte, el número de horas que se implementaría la terapia. el estudio inicial realizado por gattinoni et al. (246) , llevó a los pacientes a un período corto de 6 horas, sin encontrar resultados positivos en mortalidad. (resultados que no se pueden solamente atribuir a la calidad del estudio, sino también, al reducido número de horas de la ventilación en posición prono). mancebo et als (la abreviación latina para "otros "es et al.), y fernández et al. (249, 250) por su parte, optan por ventilar un mayor número de horas (18-20 horas) obteniendo resultados (que sugieren una reducción en mortalidad) con tendencia a disminuir mortalidad. si bien diferentes metaanálisis sugieren no (la ausencia de beneficio) beneficio de la ventilación en prono (256, 257) , los resultados son diferentes cuando se aborda la terapia con períodos mayores a 12 horas (256) . teniendo en cuenta, que el estudio con más poder estadístico (252) postula 16 horas de terapia en posición prono, la recomendación es la pronación por un tiempo mayor a 16 horas, contemplando hasta las 20 horas por sesión. otra pregunta que surge con frecuencia es el número de veces que se puede implementar la terapia. existen diferentes estudios en los cuales se indica el número de veces en promedio en que se llevó el paciente a posición prono (247, 248) , otro protocolo en donde se estipula un número límite de días en los que se llevó a cabo la terapia(249), y otro estudios en los cuales no se precisó el número de sesiones de la terapia (246, 250) . se podría entonces recomendar con respecto a la mayor evidencia disponible (252) , pronar a los pacientes en varias sesiones (4 en promedio) (depende de los criterios para continuar o suspender el prono que deben ser individualizados para cada paciente, en cada zona geográfica y en cada unidad de cuidados intensivos, es difícil saber si 4 es el promedio para todos), y considerarla más veces si es necesario. en lo que respecta a los efectos adversos, tres estudios reportaron barotrauma y neumonía asociada a ventilador (247, 249, 250) ; dos estudios desplazamiento de catéter central (246, 251) y siete estudios reportaron extubación (246) (247) (248) (249) (250) (251) (252) . sin embargo, los efectos adversos no alcanzaron significancia estadística para proscribir la terapia (256) . recientemente fue publicado el consenso colombiano de sdra(204) en el cual se hace referencia a las recomendaciones para realización de ventilación prono. se presenta la lista de chequeo, y medidas que deben ser realizadas en la maniobra para lo cual se cuenta con la participación de terapia respiratoria, enfermería y médico. se recomienda implementar un protocolo de retiro de ventilación mecánica basado en la prueba de respiración espontánea y articulado con un protocolo de sedación y analgesia en el paciente críticamente enfermo por sospecha o diagnóstico de covid-19. desde diciembre de 2019, un número de casos de neumonía por síndrome respiratorio agudo severo sars-cov2/covid-19 en wuhan china se identificaron, como causa de insuficiencia respiratoria aguda(258). el síndrome de dificultad respiratoria aguda (sdra) ocurre en el 20% de los 138 pacientes hospitalizados y en el 61% de los 36 pacientes admitidos a la unidad de cuidados intensivos (uci) en wuhan (174) . mientras la ventilación mecánica es una intervención que potencialmente salva la vida, esta puede llevar a múltiples complicaciones y contribuir a la lesión pulmonar (259) . es por todo esto que el retraso en el retiro de la ventilación mecánica puede aumentar el riesgo de amci ® infecciones, aumenta la sedación innecesaria, el trauma de la vía aérea y aumento en el costo de la atención de estos pacientes (260) . el retiro de la ventilación mecánica es un proceso de tres pasos, el primero es conocido como preparación la cual depende de variables fisiológicas, criterios clínicos y predictores de weaning (destete), el segundo paso es el propio weaning, el cual consiste en la disminución del soporte ventilatorio entregado al paciente, con el objetivo de llevar al último paso que es la extubación(261), ilustración 7. por todo lo anterior, se recomienda realizar un proceso de retiro de ventilación mecánica invasiva adoptando un protocolo, seleccionando adecuadamente a los pacientes, ya que evidencia sugiere que la adecuada selección de los pacientes disminuye los días de ventilación mecánica, disminuye la estancia hospitalaria y la estancia en uci (262) . se recomienda realizar el retiro de la ventilación mecánica siguiendo los pasos antes mencionados, iniciando con la preparación, la cual consiste en: preparación 1. asegurarse que la lesión pulmonar que llevó a la falla respiratoria esté resuelta. 2. adecuado intercambio de gas, definido como adecuados índices de oxigenación con peep (presión positiva al final de la expiración) 5 a 8 cmh2o, y fio2 (fracción inspirada de oxígeno) < 0.5. al igual que el proceso de intubación, es un proceso que genera aerosoles por lo cual se recomienda la aplicación de lidocaína dosis de 0,5 a 1,5 mg por kilogramo de peso, 1 a 5 minutos antes la extubación con el objetivo de disminuir el reflejo de tos (266) y la exposición del personal de salud. se recomienda en el paciente con sospecha o diagnóstico de covid-19 a quien se considera realizar vmni, utilizarla en salas de presión negativa, donde estén disponibles, que garanticen la seguridad del recurso humano, con todo el equipo de protección personal necesario. se recomienda considerar la vmni en pacientes con covid-19 con hipoxemia leve (pao2/fio2<300 y >200 o sao2/fio2<300 y>200) en salas de presión negativas, donde estén disponibles que garanticen la seguridad del recurso humano, con todo el equipo de protección personal necesario. fuerte a favor se recomienda considerar la vmni en pacientes con covid-19 con hipoxemia leve (pao2/fio2<300 y >200 o sao2/fio2<300 y>200) y con historia de epoc o cuadro de edema pulmonar agudo asociado en salas de presión negativas, donde estén disponibles que garanticen la seguridad del recurso humano, con todo el equipo de protección personal necesario. se recomienda colocar doble filtro en el circuito del ventilador para reducir el riesgo de generación de aerosoles en vmni del paciente crítico con sospecha o diagnóstico de covid-19. se recomienda la intubación inmediata en pacientes críticamente enfermos por covid-19 si se evidencia respiración toraco-abdominal, uso de músculos accesorios, frecuencia respiratoria > 30, hipoxemia (pao2/fio2<200 o sao2/fio2 <200), fracaso ventilatorio (ph<7.35 con paco2>45 mmhg), hacor>5. existe suficiente evidencia que demuestra que la vmni es una estrategia que reduce la mortalidad en pacientes críticos (195, 267, 268) . además, reduce la necesidad de intubación. los números de casos necesarios a tratar (nnt) son: ocho para salvar una vida y 5 para evitar una intubación (268) . estos desenlaces son fundamentalmente en pacientes con epoc y edema pulmonar agudo (268) (269) (270) ; también hay evidencia a favor, aunque menos fuerte, en pacientes inmunosuprimidos (259, 271) . se ha planteado mayores tasas de éxito con interfaces faciales totales o con helmet (195, (267) (268) (269) (270) (271) . por el contrario, en falla respiratoria hipoxémica hay evidencia en contra del uso de vmni (272) . la experiencia previa con h1n1, sars y mers no apoya el uso de la vmni en falla respiratoria hipoxémica de origen viral (273) (274) (275) (276) (277) (278) . además, se ha cuestionado el uso de vmni en covid-19 por el riesgo de contagio al generar aerosoles (279) . recientemente se demostró que la distancia de dispersión de aerosoles era menor de un metro (280) . por otro lado, el fracaso de la vmni se ha asociado con alta morbimortalidad (281) (282) (283) . ello obliga a evaluar estrictamente la posibilidad de éxito o fracaso. así, en falla respiratoria amci ® hipoxémica la escala hacor ha sido validada para este fin y un puntaje>5 contraindicaría la vmni (284) . adicionalmente si se emplea la vmni el prolongar la decisión de intubación puede aumentar la mortalidad y es por ello necesario monitorizar estrictamente al paciente y evaluarlo para establecer, ojalá antes de dos horas, si el paciente responde a la vmni (167, 285, 286) . las indicaciones para intubación en este caso son respiración toracoabdominal, uso de músculos accesorios, frecuencia respiratoria mayor de 30, hipoxemia (pao2/fio2<200 o sao2/fio2 <200), fracaso ventilatorio (ph<7.35 con paco2>45 mmhg), hacor>5 o índice de rox (spo2/fio2)/frecuencia respiratoria) < 3 (119, 167, 179, 180, 285, 286) . recomendación se sugiere el uso de posición prono en pacientes no ventilados críticamente enfermos por covid-19 que no responden a la oxigenoterapia convencional de acuerdo con los protocolos institucionales, las condiciones de cada servicio y la tolerancia individual de cada paciente. débil a favor fundamento los pacientes con enfermedad por coronavirus 2019 (covid-19) están en riesgo de desarrollar un síndrome de dificultad respiratoria aguda (sdra) (287) . en pacientes intubados con síndrome de dificultad respiratoria aguda grave, la posición prona (pp) temprana y prolongada (al menos 16 horas diarias) mejora la oxigenación y disminuye la mortalidad (252, 288) .debido a que las unidades de cuidados intensivos (uci) están sobrecargadas con pacientes con covid-19, la pp en paciente despierto con respiración espontánea puede ser útil para mejorar la oxigenación y prevenir las transferencias hacia uci. un estudio describió la viabilidad del uso de la ventilación no invasiva y la cánula de alto flujo asociado a la pp estableciendo su tolerancia y su seguridad en pacientes con sdra moderado y severo(289); la pronación puede reclutar todas las regiones pulmonares y favorecer el drenaje de secreciones de la vía respiratoria, mejorando el intercambio gaseoso y la supervivencia en el síndrome de dificultad respiratoria (sdra) (252) . en una comunicación corta proveniente de italia en el cual se incluyeron 15 pacientes que son sometidos al pp asociada con el uso de ventilación no invasiva, se concluye que proporcionar niv en la posición prona a los pacientes con covid-19 y sdra en salas generales en un hospital en italia era factible. la frecuencia respiratoria disminuyó durante su implementación y la oxigenación mejoró durante una pronación posterior a su línea de base. si la intubación se evitó o se retrasó, queda por determinar (290) . en otro reporte de caso, publicado recientemente por un grupo francés(291) de pacientes con covid-19 e insuficiencia respiratoria hipoxémica manejados fuera de la uci, el 63% amci ® fue capaz de tolerar pp durante más de 3 horas. sin embargo, la oxigenación aumentó durante el pp solo un 25% y no se mantuvo en la mitad de los pacientes después del regreso a la posición supina. estos resultados son consistentes con los hallazgos de pequeños estudios previos de pp en pacientes no intubados (289) . un ensayo clínico controlado que evalúe el uso del pp en pacientes no intubados puede ser un mecanismo para seleccionar pacientes que bien puedan beneficiarse de esta estrategia terapéutica. dada la evidencia débil que soporta el uso del pp en pacientes no intubados en términos de la disminución de la necesidad de entubación o ingreso a cuidados intensivos y la duda razonable de aumentar desenlaces deletéreos en aquellos pacientes en los cuales se retarde el tiempo de intubación, no se emite recomendación, a favor o en contra, del empleo de esta estrategia de manera rutinaria. en situaciones en las cuales hay limitación de recursos y de disponibilidad de camas en cuidados intensivos el uso de la pp asociada a vni o cánula de alto flujo podría ser una estrategia útil para mejorar la oxigenación en pacientes infectados con covid-19 e hipoxemia. se recomienda considerar la elevación de biomarcadores como la troponina i o t y el nt-pro-bnp en el paciente con covid-19 como indicadores de injuria miocárdica aguda, sin embargo, no reemplazan la ecocardiografía en el enfoque del paciente con sospecha de disfunción miocárdica. fuerte a favor fundamento la injuria miocárdica aguda asociada a covid-19 se reporta con frecuencia teniendo en cuenta los cambios en biomarcadores como la troponina y cambios electrocardiográficos, pero su impacto en la función cardíaca se desconoce y mucho menos su correlación con los cambios ecocardiográficos. los pacientes con covid-19 pueden desarrollar una serie de complicaciones cardiacas desde injuria miocárdica, arritmias, infarto, hasta miocarditis fulminante con falla cardiaca aguda y shock cardiogénico (292) . la troponina i, se ha encontrado más elevada en pacientes con curso fatal por covid-19 (37) . los niveles de nt-pro-bnp han sido reportados con elevación severa en pacientes con miocarditis y disfunción sistólica, con una disminución progresiva en relación a la mejoría de los pacientes, pero no parece tener un correlación significativa con el cambio de la fracción de eyección (fevi) (293) . la evidencia clínica sugiere que la elevación de los biomarcadores es más relacionada al compromiso sistémico que el daño miocárdico directo, q. deng y colaboradores en un análisis retrospectivo de 112 pacientes, reportaron niveles iniciales de troponinas normales casi en la mayoría, en el 37,5% de los casos los niveles incrementaron significativamente, principalmente en los que fallecieron y solo 6 pacientes tenían fevi menor al 50% y ninguno inferior al 40%, lo cual no sugiere una asociación entre las dos amci ® pruebas (294) . en una publicación donde se compara el fenotipo de 18 pacientes con covid-19 con un histórico de 23 pacientes con sdra por influenza (295) , se encontró que los primeros tenían mayor elevación de troponinas 73% vs 53%, pero en los parámetros ecocardiográficos contrario a lo que se esperaría los índices de rendimiento ventricular fueron mayores para el grupo de covid-19: índice cardiaco 3.1 vs 2.5 l/m/m 2 ; fevi 52 vs 44%; tapse 25 vs 18mm, nuevamente aunque no fue uno de los objetivos del estudio, parece no encontrarse correlación entre los biomarcadores que sugieren injuria miocárdica y los parámetros ecocardiográficos, el cual constituye uno de los pilares de la exploración cardiaca. se recomienda considerar como marcadores iniciales de mal pronóstico en el paciente crítico con covid-19 con sospecha de disfunción miocárdica aguda la elevación persistente de troponina i, mioglobina o creatin kinasa; independiente de la fracción de eyección del ventrículo izquierdo evaluada mediante ecocardiografía. fundamento los pacientes con covid-19 admitidos a la unidad de cuidados intensivos presentan con frecuencia disfunción cardiaca primaria, que puede corresponder a cardiomiopatía por estrés o miocarditis viral, pero también pueden ser consecuencia del compromiso sistémico (294, 296) . aunque parece que en los fenotipos cardiovasculares estudiados, el compromiso hemodinámico severo de la función sistólica izquierda y derecha es menor (295) . en el estudio de deng y colaboradores con 112 pacientes con covid-19, la fiebre, la disnea, hipoxemia, la obesidad y niveles elevados de cpk, troponina y nt-pro-bnp se relacionaron significativamente con mayor severidad (294) . en el subgrupo de pacientes con miocarditis frente a controles, el perfil clínico se describe con mayor edad, niveles de temperatura más elevados (38.8 ± 0.8 vs 38.2 ± 1.0; p:0.03), mayor proporción de disnea (92,8 vs 51%), y de dolor torácico (92,8 vs 61,2%). en los 14 pacientes fallecidos el 100% tuvieron picos de elevación de troponina i y de nt-pro-bnp dentro la semana que precedió la muerte, el 78% presentaron alteraciones electrocardiográficas y solo el 28% presentaron fracción de eyección menor o igual al 45% (294) . shi y colaboradores, enrolaron 671 pacientes para describir el significado clínico del compromiso miocardio de pacientes con covid-19 en wuhan, 62 pacientes fallecieron, de los cuales el 75% presentaron injuria miocárdica aguda (297) . el área bajo la curva (auc) de la troponina i inicial para predecir muerte intrahospitalaria fue de 0,92 (ic 95%, de 0,87-0,96) con una sensibilidad y especificidad del 86%, el auc para mioglobina fue de 0.88 y para cpk-mb fue de 0,87(297). un punto de corte para el pico más alto de troponina i de 4.5, tuvo un hazard ratio para mortalidad de 1.25 (ic 95%, 1.07-1.46; p= 0.004). en un análisis multivariado la edad avanzada, la respuesta inflamatoria y las enfermedades cardiovasculares subyacentes se asociaron con mayor riesgo de lesión miocárdica en pacientes con covid-19. con la información disponible parece que los biomarcadores de lesión miocárdica aguda elevados amci ® al ingreso y de forma persistente pueden predecir el riesgo de mortalidad intrahospitalaria en los pacientes con sospecha o diagnóstico de covid-19 con afectación cardiovascular. se recomienda no realizar de forma rutinaria ecocardiografía en pacientes críticos con covid-19. se debe practicar ecocardiografía en pacientes con sospecha o diagnóstico de covid-19 si presenta alguna de las siguientes condiciones: 1. síntomas y signos de insuficiencia cardíaca aguda de novo. 2. shock o deterioro súbito hemodinámico refractario a líquidos y/o vasoactivos con sospecha de origen cardiogénico. 3. sospecha de infarto agudo de miocardio o embolismo pulmonar para determinar intervenciones terapéuticas con un beneficio clínico. 4. cambios en el electrocardiograma, arritmias ventriculares o paro cardiorrespiratorio no explicados por otra causa. para nuestro conocimiento, en el momento no existe estudios clínicos que evalúen los criterios para realización de ecocardiograma en el paciente con covid-19. las sociedades de ecocardiografía han recomendado realizar el ecocardiograma en el contexto clínico en el cual, la información obtenida proporcione un cambio en la conducta o se espere un beneficio clínico al realizar este procedimiento (298) (299) (300) (301) . igualmente, se recomienda realizar el examen a la cabecera del paciente y el escaneo debe ser dirigido a contestar preguntas específicas según el contexto clínico del paciente (298) (299) (300) (301) (302) . ward et al, en su publicación describe cómo el uso del ecocardiograma limitado (dirigido) en la university of chicago medicine (ucm), aumentó significativamente durante la pandemia (15% frente a 34%, p <0.001), posterior a la implementación de recomendaciones sobre el uso apropiado de la ecocardiografía en tiempos de pandemia (303) . los pacientes con infección por sars -cov-2 pueden presentarse con comorbilidades cardiovasculares que potencialmente estén descompensadas y/o compromiso cardiovascular por covid-19. en este último, podemos encontrar alguno de los siguientes fenotipos: falla cardiaca aguda en el marco de compromiso directo viral o secundario al estrés metabólico y liberación de citoquinas, síndrome coronario agudo, cor-pulmonar secundario a tep o por compromiso secundario al sdra (299, 300, 304, 305) . las manifestaciones cardiovascular puede sospecharse en el marco de choque que no esté explicado por causas extracardiacas evidentes que no responde a líquidos, dolor torácico con clínica de síndrome coronario agudo, cambios electrocardiográficos y elevación de biomarcadores de lesión miocárdica, signos de falla cardiaca descompensada, deterioro súbito de la oxigenación, arritmias o paro cardiorrespiratorio (301, 304, 305) . ante estas manifestaciones, el ecocardiograma podría ser útil para entender el origen de la descompensación aguda, al estar enfocado a amci ® responder preguntas acerca de la función ventricular global y segmentaria (en el abordaje de síndrome coronario agudo), compromiso del ventrículo derecho, alteraciones valvulares, derrame pericárdico, si existe una contribución cardiovascular al compromiso pulmonar, si en el marco del choque existe evidencia de componente cardiogénico y cómo podría guiarse/optimizarse el soporte hemodinámico de estos pacientes(300-304). se sugiere en pacientes críticamente enfermos con covid-19 que cursan con shock y sdom, ajustar la monitoria a las condiciones clínicas del paciente y recursos disponibles. se puede considerar el cap para el monitoreo del gasto cardiaco, la valoración de la perfusión y orientar los elementos hemodinámicos del tipo de shock, el cap de gasto cardiaco continuo puede disminuir la exposición del personal de salud frente al catéter de medición convencional. se sugiere la utilización de la tdtp dependiendo de la disponibilidad del recurso para orientar el diagnóstico diferencial del sdra versus edema pulmonar cardiogénico en los pacientes con covid-19. fundamento los pacientes con infección severa por sars-cov-2, cursan con alto riesgo de falla renal y cardiovascular, con necesidad de un manejo restrictivo de líquidos, lo que justifica la monitoria estricta en uci (306, 307) . el catéter venoso central es útil para la monitoria inicial de estos pacientes, sin embargo, su predicción a respuesta a volumen está limitada (306) (307) (308) . la monitoría no invasiva tiene limitaciones en casos severos de inestabilidad hemodinámica, ventilación espontánea y en presencia de peep alto, lo que limita su uso para el cálculo de gasto cardiaco y la predicción de respuesta a líquidos (308, 309) . la monitoria con cap puede ser considerada en pacientes con covid-19 que cursan con choque y doms, con el objetivo de realizar un diagnóstico definitivo de los componentes del choque, valorar la hipoperfusión, la función cardiaca y el estado de volemia (307, 308) . igualmente, los pacientes con sospecha tep o compromiso del ventrículo derecho pueden beneficiarse de esta monitoria(308). richard et al, en su estudio determinaron los desenlaces asociados al uso de cap vs cvc en pacientes con shock, sdra, o ambos, sin evidenciar diferencias en mortalidad (49.9% vs 51.3% p =.70) o estancia hospitalaria. el uso de cap no garantiza la mejoría de desenlaces en pacientes con covid-19, sin embargo, la presencia de una monitoria continua ayudaría a optimizar los recursos y disminuiría la interacción con el paciente, con menor exposición del equipo médico (310) . la monitoria por tdtp puede utilizarse en pacientes con covid-19 que cursan con choque, buscando optimizar el manejo hídrico, valorar el agua extravascular pulmonar (evlw) y el índice de permeabilidad vascular pulmonar (pvpi) con el fin de establecer el diagnóstico amci ® definitivo del edema pulmonar: sdra vs cardiogénico (311) (312) (313) . hu et al, en su estudio evaluaron los desenlaces del uso de evlw y la presión de cuña de la arteria pulmonar (pawp) como estrategias para el manejo de líquidos en pacientes sdra, no encontraron diferencias significativas en las tasas de supervivencia (p = 0,542). no obstante, en el grupo de evlw la duración de la ventilación mecánica y la estancia en la uci fueron significativamente menor (p <0,05), al igual que el balance hídrico (p <0.05), con mejoría significativa en los índices de oxigenación (p = 0.006)(314). no se puede emitir una recomendación a favor o en contra para la utilización de un protocolo de ultrasonido rutinario a la cabecera del paciente (pocus). sin embargo, se podría considerar el uso en pacientes seleccionados, con los adecuados epp y desinfección de los equipos; donde el pocus pueda tener ventajas sobre otras modalidades de monitoria o en pacientes con limitaciones para monitoria invasiva que requieren evaluación del estado hemodinámico o determinación de severidad del compromiso pulmonar. las recomendaciones sobre la utilidad de pocus en pacientes con covid-19 están enfocadas principalmente en la evaluación de la severidad/progresión de la lesión pulmonar, diagnóstico de manifestaciones cardiovasculares, monitoria hemodinámica y en la guía de fluidoterapia (302, (315) (316) (317) . en la valoración del compromiso pulmonar por covid-19, el pocus ofrece una ventaja sobre otras modalidades de monitoreo, debido a la capacidad de enmarcar el compromiso pulmonar en una línea de tiempo según sus hallazgos: desde la aparición de un patrón de "líneas b", consolidaciones subpleurales con evolución a consolidaciones multilobares, irregularidades en el artefacto de la línea pleural y finalmente aparición de patrón de "líneas a" una vez inicie la recuperación, con adecuada correlación tomográfica (318) (319) (320) (321) . en la diferenciación del origen del choque, el pocus ha demostrado superioridad versus el concepto clínico al evaluar: función ventricular (incluyendo ventrículo derecho), vena cava inferior, líquido libre abdominal, lesiones aórticas, compromiso pulmonar y búsqueda de trombosis venosa profunda identificando tep (322, 323) . en cuanto a la monitoria hemodinámica de pacientes con covid-19, adicional a la función y gasto cardíacos, se recomienda variables dinámicas de respuesta a líquidos y ultrasonido pulmonar en el diagnóstico de sobrecarga hídrica (205) . la variabilidad de gasto cardiaco calculado por pocus durante la elevación pasiva de miembros inferiores, identifica los respondedores a líquidos con sensibilidad del 88% y un lr (-): 0.13 [95%ci, 0.07-0.22]) (324) . la variabilidad de la vena cava inferior muestra limitaciones en pacientes con aumento de presiones de cavidades derechas y respiración espontánea (325) . en cuanto a otras modalidades de monitoría no invasiva, es importante conocer las limitaciones en el marco de compromiso hemodinámico severo, ventilación espontánea, alteraciones valvulares aórticas, entre otras (309) . marik (326) . el ultrasonido tiene limitaciones específicas como la necesidad de un operador experimentado y la adecuada calidad de las imágenes. adicionalmente, en los pacientes con covid-19, esta modalidad de monitoreo requiere una mayor interacción con el paciente, mayor uso de epp en comparación con otras modalidades el pac(307). se recomienda perseguir al inicio de la reanimación del paciente críticamente enfermo con covid-19, metas clínicas de fácil medición, como la presión arterial media (entre 60 y 65 mmhg) o el gasto urinario (mayor a 0,5 cc/k/h) y metas de perfusión como el lactato en sangre arterial (menor a 2 mmol), la saturación venosa central de oxígeno (entre 65 y 70%) y la diferencia veno arterial de co2 (menor a 6 mmhg). se han reportado casos de disfunción ventricular como causa de choque asociado a covid-19, sin embargo, no se ha descrito un manejo específico o cambios en las metas de reanimación para estos pacientes, las manifestaciones de hipoperfusión tisular son: alteración de la conciencia, oliguria, piel fría y moteada y pulso débil. a nivel de gases sanguíneos las metas de reanimación pueden ser globales como el lactato (en sangre arterial vn< 2 mmol) y la diferencia veno arterial de co2(pv-aco2 vn < 6 mmhg) o regionales como la saturación venosa central de oxígeno medida en la sangre venosa tomada de un catéter central (vn: 65-70%). se puede optimizar la perfusión, interviniendo los principales determinantes: fluidos para aumentar el volumen intravascular, inotrópicos para aumentar la fuerza de contractilidad, vasopresores para recuperar la presión de perfusión y transfusión de glóbulos rojos para aumentar la hemoglobina como transportador de oxígeno (44) , las recomendaciones dadas en el documento anterior siguen siendo válidas e incluyen entre otras que todo paciente con covid-19 en estado de shock debe ser ingresado de forma inmediata a la unidad de cuidados intensivos, garantizando el aislamiento indicado, procurando recuperar la presión arterial media a valores > 65 mmhg, para ello la utilización de un catéter venoso central en los pacientes que no responden al manejo inicial y el procedimiento debe ser realizado por el médico con mayor entrenamiento, idealmente guiado por ecografía si hay disponibilidad y las competencias, así mismo las estrategia de control estricto de fluidos para no generar efectos deletéreos relacionados a la sobrecarga de volumen, es lo más indicado. se recomienda en la reanimación inicial de pacientes en estado de shock con sospecha o amci ® diagnóstico de covid-19, guiar la fluidoterapia con el uso de índices clínicos como el tiempo de llenado capilar, temperatura de la piel y depuración de lactato; en fases avanzadas donde el monitoreo clínico es insuficiente utilizar medidas dinámicas como la variabilidad de presión de pulso (vpp), la variabilidad de volumen sistólico (vvs), la respuesta a la maniobra de elevación pasiva de piernas o la prueba de oclusión teleespiratoria de acuerdo a los recursos disponibles y la experiencia. fuerte a favor fundamento sobre los objetivos de intervención y el tipo de agente vasoactivo a utilizar en pacientes con covid-19 y shock no existe una evidencia directa y las recomendaciones se basarán en evidencia indirecta de pacientes críticos con diversos tipos de shock en especial el séptico y vasopléjico. la falla circulatoria aguda asociado a sdra en covid-19 se presenta con una frecuencia del 6-30% y en la admisión a urgencias la hipotensión y un lactato ≥2 es infrecuente(38, 327). la baja sensibilidad del qsofa y crb-65 para predecir la severidad del covid-19 y la necesidad de intervenciones de terapia intensiva refleja lo infrecuente del shock en esta condición. factores como la vasoplejia, fuga capilar asociada al estado hiperinflamatorio, altos requerimientos de peep y disfunción cardiaca pueden ser generadores o contribuyentes del shock y deben ser considerados en el abordaje diagnóstico, en su interpretación para lograr tomas de decisiones adecuadas. en pacientes con sdra, una reanimación óptima de líquidos debe tomar en cuenta aspectos como tiempo (oportunidad), tipo (cristaloides balanceados y/o no balanceados o coloides) y volumen (ni mucho, ni poco) con el objetivo de disminuir la mortalidad, el tiempo de vm y de cuidados intensivos, sin que ello afecte los índices de oxigenación, de perfusión tisular y la morbilidad asociada con su uso inadecuado. la administración agresiva de líquidos puede empeorar la oxigenación y la disfunción ventricular, lo que potencializa un mayor tiempo de ventilación mecánica e incluso la mortalidad. la evidencia ha demostrado que una estrategia conservadora de fluidos (balance -136+/amci ® en una revisión sistemática y un metaanálisis de 7 rct (n = 1.301) una terapia dirigida al aclaramiento temprano de lactato frente a una terapia guiada por la saturación venosa central de oxígeno (svo2), se asoció con una reducción significativa de la mortalidad (rr 0,68), menor estadía en la uci (dm 1,64 días), y menor duración de la ventilación mecánica (dm -10,22 horas). pero se debe resaltar que un nivel alto de lactato no siempre indica hipovolemia; también puede ser causada por uso de adrenalina, agonistas beta o por disfunción mitocondrial, insuficiencia hepática e isquemia mesentérica (331) . por otra parte, el llenado capilar (crt), una prueba técnicamente fácil y accesible, realizada cada 30 minutos se asoció con una reducción no significativa de la mortalidad (hr 0,75) en comparación con la medición de lactato sérico cada 2 horas. dado el potencial beneficio sobre mortalidad, duración de estancia en uci y la duración de la ventilación mecánica, así como su accesibilidad, sugerimos utilizar parámetros dinámicos de temperatura de la piel, tiempo de llenado capilar y / o medición de lactato sobre parámetros estáticos para evaluar la capacidad de respuesta a la fluidoterapia en pacientes con covid-19 y shock(332). se recomienda en pacientes adultos con covid-19 y estado de shock, escoger la norepinefrina como el vasopresor de primera línea y a la vasopresina el de segunda. si no se cuenta con norepinefrina el uso de vasopresina o epinefrina serían la primera elección; la dopamina no se recomienda por el mayor riesgo de arritmias. se recomienda iniciar dobutamina frente al aumento de la dosis de norepinefrina en pacientes en estado de shock con evidencia de disfunción cardíaca e hipoperfusión persistente a pesar de la reanimación inicial. fuerte a favor fundamento sobre los objetivos de intervención y el tipo de agente vasoactivo a utilizar en pacientes con covid-19 y shock no existe una evidencia directa y las recomendaciones solo pueden basarse en evidencia indirecta de pacientes críticos con sepsis y sdra. en pacientes en shock séptico los agentes vasoactivos para alcanzar una pam de 60-65 es un objetivo razonable. una presión media más alta puede incrementar 2.5 veces el riesgo de arritmias cardiacas y no está exento de riesgo de isquemia en las extremidades (333) . para aproximarnos a la escogencia de los vasoactivos en shock séptico, basados en su perfil de riesgo/beneficio, la guía scandinavian society of anaesthesiology and intensive care medicine (ssai ) task force for acute circulatory failure(334), la revisión sistemática de cochrane database 2 con 28 rct con un n:3497 (335) y el ensayo clínico controlado de honarmand k et al con un n:3,737 (336) amci ®  la noradrenalina es el agente vasoactivo más ampliamente estudiado con el menor riesgo a priori de efectos no deseados, razón por la cual se sugiere usar como el agente vasoactivo de primera línea en pacientes con covid-19 y shock.  si la noradrenalina no está disponible la vasopresina o epinefrina se muestran como la mejor alternativa. los factores que determinan la elección entre vasopresina y epinefrina pueden incluir disponibilidad y el perfil de seguridad de estos agentes. con la vasopresina, la isquemia digital puede ser una preocupación y con epinefrina, la taquicardia, la isquemia miocárdica y el exceso de producción de lactato.  el uso de dopamina se ha asociado un 2.5 veces mayor de riesgo de aparición de arritmias frente a la norepinefrina y un posible aumento del riesgo de mortalidad. por ello la dopamina no debe utilizarse en pacientes con covid-19 y shock donde haya disponible de norepinefrina o las alternativas señaladas  en shock distributivo la adición de vasopresina a las catecolaminas evidenció baja certeza de reducción de la mortalidad (rr 0,91; ic del 95%: 0,85 a 0,99), alta certeza de una reducción de la fibrilación auricular (rr 0,77; ic del 95%: 0,67 a 0,88) y certeza moderada de un mayor riesgo de isquemia digital (rr 2,56; ic del 95%: 1,24 a 5,25). en vista de estos hallazgos se plantea la vasopresina como un agente vasoactivo de segunda línea a ser utilizado si la pam objetivo no se ha alcanzado con norepinefrina en pacientes con covid-19 y shock. no existe evidencia directa en pacientes con covid-19 y shock, para establecer una recomendación sobre cuál es el agente inotrópico óptimo. en una guía de práctica clínica de 2018 que evalúa el agente inotrópico óptimo en pacientes con insuficiencia circulatoria aguda (shock), no se identificaron rct que comparen dobutamina versus placebo o ningún tratamiento. con base en una justificación fisiopatológica, sugerimos agregar dobutamina, más que no suministrar ningún tratamiento, en pacientes con covid-19 y shock con evidencia de disfunción cardíaca e hipoperfusión persistente a pesar de la reanimación con líquidos y altas dosis de norepinefrina. el uso de dobutamina en estado de shock, incluso en pacientes con covid-19 con shock, debe ser investigado. recomendación se recomienda no suspender la medicación estándar para falla cardiaca en pacientes con sospecha o diagnóstico de covid-19, especialmente los iecas, ara-ii, y b-bloqueadores, si la condición clínica permite continuar el uso de esta medicación, ya que no se ha podido confirmar una asociación nociva. amci ® previos alrededor de la relación independiente de la edad avanzada, enfermedad cardiovascular subyacente (enfermedad coronaria, insuficiencia cardíaca y arritmias), tabaquismo activo y epoc con muerte por covid-19 (337) (338) (339) ; esos mismos reportes sugieren que las mujeres son proporcionalmente más propensas a sobrevivir a la infección por covid-19 que los hombre; existen además consideraciones especiales desde el punto de vista cardiovascular, que se deben tener en cuenta al decidir cualquier terapia en paciente afectados por covid-19 y existe la hipótesis de un efecto nocivo de la terapia estándar para falla cardiaca en estos pacientes (338) . en un estudio observacional que incluye 8910 paciente con infección por covid-19, se evaluó la relación entre la enfermedad cardiovascular subyacente, y la asociación entre la terapia farmacológica cardiovascular y la mortalidad (337); respecto a los factores de riesgo cardiovascular, el 30.5% de los pacientes tenían hiperlipidemia, el 26.3% tenía hipertensión, el 14.3% tenía diabetes mellitus, y en relación a los medicamentos, los sobrevivientes usaron más comúnmente ieca y las estatinas que los no sobrevivientes, mientras que no se encontró asociación entre la supervivencia y el uso de ara ii; respecto a los otros medicamentos incluidos b-bloqueadores, antiplaquetarios e insulina no se encontraron diferencias significativas; sin embargo, y teniendo en cuenta el impacto sobre mortalidad del uso de betabloqueadores en la falla cardiaca, consideremos que la decisión de continuar su uso debe basarse en el análisis clínico de cada paciente y su estabilidad clínica. se recomienda establecer un protocolo de reanimación ajustado al contexto del paciente con covid-19 con una organización administrativa ajustada a la pandemia que incluya las siguientes estrategias:  desarrollar una estrategia de prevención del paro cardiorrespiratorio en el paciente con sospecha o diagnóstico de covid-19 basada en la detección oportuna.  formar un equipo multidisciplinar formado en rcp con líderes médicos, de en enfermería y en terapia respiratoria, los cuales deben educar a todo el equipo de trabajo en la identificación de signos de alerta temprana, cambios abruptos de variables clínicas, técnicas de monitoreo, interpretación de paraclínicos y de alarmas de monitoreo.  entrenamiento del personal sanitario a través del uso de la simulación clínica en manejo de crisis, epp, y procesos de atención fundamentales en la atención del paro cardiaco en el paciente con sospecha o diagnóstico de covid-19  promover la comunicación asertiva, planeación y retroalimentación de las intervenciones realizadas antes y después de un evento de paro cardiaco (briefing y debriefing), con el fin de establecer modificaciones que conllevarán a mejoras en la atención de futuros eventos. amci ® el pronóstico y sobrevida de un paciente con sospecha o diagnóstico de covid-19 que presenta paro cardiaco depende de la prevención a través del reconocimiento oportuno de las causas reversibles de éste, la no presencialidad y un ritmo de paro no desfibrilable lo hacen de mal pronóstico. la prevención va a depender del nivel de entrenamiento del equipo previamente, y en época de pico de pandemia cuando el talento humano especializado disminuya, se hace necesario que en los equipos de trabajo estén liderados por especialistas en la disciplina para que puedan guiar al equipo. los nuevos procesos de atención del paciente, la alta contagiosidad del virus, y el uso de nuevos medicamentos hacen que se requiera un entrenamiento del personal para estandarizar los procesos y disminuir el error médico. la crisis de covid-19 está ejerciendo una presión sin precedentes sobre las personas, los equipos y los sistemas organizacionales, conllevando a errores médicos que van desde la infección cruzada por el personal sanitario con la posibilidad de cometer errores en la atención. cada día trae nuevos desafíos: picos en volumen y gravedad, escasez de equipos y estrés en los médicos sobrecargados, que se manifiesta según la experiencia de wuhan en insomnio y depresión. se propone implementar una estrategia antes, durante y después del trabajo clínico, denominada circle up covid-19 desarrollada por center of medical simulation dirigida a convertir equipos de trabajo muchas veces insustituibles , en eficientes , seguros, fuertes y que se apoyan mutuamente incluyendo en mejora de la salud psicológica . impactando en el rendimiento del equipo , y promoviendo el bienestar y la resiliencia(340-342) se recomienda establecer un protocolo de reanimación ajustado al contexto clínico del paciente con covid-19 que incluya las siguientes modificaciones:  implementar criterios de selección e inicio de maniobras de rcp en la atención del paro cardíaco basados en la bioética y en el pronóstico de supervivencia a corto y largo plazo de los pacientes.  promover la prevención del paro cardiaco mediante la detección oportuna del riesgo y definir intubaciones programadas.  asegurar la correcta protección con los epp necesarios al abordar el paro cardiaco y la intubación que son procedimientos generadores de aerosoles.  priorizar el manejo de la vía aérea antes del inicio de las compresiones torácicas, haciendo énfasis en la reducción de la exposición de aerosoles (código azul protegido).  utilizar filtro de alta eficiencia contra virus para todas las estrategias de ventilación (bolsa mascarilla con cierre hermético y en el circuito del ventilador).  promover la realización de la intubación por el operador de mayor experticia con uso de videolaringoscopio si está disponible y considerar el acceso supraglótico solo si el intento de intubación es fallido. amci ®  en caso de parada cardiaca en ventilación mecánica iniciar el masaje cardiaco evitando las desconexiones del circuito del respirador.  en caso de paro en posición prono si el paciente se encuentra vigil retornar rápidamente a la posición supino y si está en ventilación mecánica es razonable realizar compresiones en la espalda. fuerte a favor fundamento se hace necesario el entrenamiento en el manejo de los procedimientos generadores de aerosoles y se sugiere que el que realiza la intubación orotraqueal debe ser el más experto. el riesgo de aerosolización es de 6.6 en el proceso de intubación orotraqueal, el de compresiones torácicas es de 4,5, ventilación mecánica no invasiva de 3,1, ventilación manual pre-intubación de 2,8, succión después de intubación 1,3. se ha descrito que si no se ha capacitado previamente en el retiro de los epp existe más riesgo de auto contaminación y aumenta ésta sin un líder supervisor al retirarlo. según revisión de cochrane sobre ropa y equipo de protección para los trabajadores sanitarios para evitar que se contagien con el coronavirus y otras enfermedades altamente infecciosas da a conocer que la capacitación presencial, la simulación por ordenador y la capacitación por vídeo dieron lugar a menos errores a la hora de quitarse el epp que la capacitación impartida solo como material escrito o una conferencia tradicional (343) (344) (345) (346) (347) (348) . se recomienda no considerar la existencia de manifestaciones neurológicas específicas o típicas atribuidas a la infección por sars-cov-2. se recomienda realizar la valoración neurológica integral del paciente con diagnóstico o sospecha de sars-cov-2 teniendo en cuenta manifestaciones frecuentes relacionadas a covid-19: disgeusia, anosmia, cefalea, vértigo, confusión, delirium, alteración de estado de consciencia, eventos cerebrovasculares, ataxia, polineuropatías inflamatorias y convulsiones. una revisión sistemática realizada por asadi-pooya et al. entre diciembre y marzo de 2020 mostró que el 25% de los pacientes con covid-19 presentaron sintomatología neurológica. analizaron cinco artículos (n=765) donde 4 eran retrospectivos, y 1 era prospectivo, encontrando cefalea entre el 6 y el 13%, vértigo entre el 9 y el 17%, confusión en el 9%, alteración del estado de consciencia en el 8%, eventos cerebrovasculares en un 3%, ataxia en un 0,5% y convulsiones en un 0,5% (349) . amci ® menor a 1000 participantes donde recolectarán información sobre el compromiso neurológico en pacientes con covid-19 con un seguimiento hasta febrero de 2021 (350) . la hiposmia y la disgeusia de aparición súbita son manifestaciones clínicas muy prevalentes en pacientes con covid-19 evidentes aun en ausencia de sintomatología respiratoria alta. lechien et al publicaron un estudio multicéntrico que incluyeron 12 hospitales europeos reclutando 412 pacientes infectados con covid-19 levemoderado donde 85,6% presentaron alteraciones relacionadas con el olfato y 88,5% presentaron alteraciones relacionadas con el gusto, donde la anosmia se presentaba antes que cualquier otro síntoma en el 11,8% de los casos y el 18,2% de los casos no presentaba rinorrea u obstrucción nasal, la recuperación del olfato fue presente en el 44% de los pacientes y las mujeres fue el grupo poblacional más afectado (p=0,001) (351) . otras manifestaciones clínicas son las alteraciones de la agudeza visual, y dolor tipo neuralgia (352) . respecto a las patologías psiquiátricas; la serie de mao comenta que el 7,5% de los pacientes tiene clínica de alteración del estado de consciencia, concepto que se aproxima a la defunción de delirium. severance et al. encontraron que 106 pacientes con sintomatología psicótica aguda presentaron niveles elevados de inmunoglobulina g para coronavirus del tipo hku1, nl63 y oc43 con diferencias estadísticamente significativas respecto a los individuos controles (n=106) (p<0,001). donde la respuesta inmune para nl63 fue asociado con el espectro-esquizofrenia (or: 3.10, ci 5 1.27-7.58, p= 0.013) pero no se correlaciona con desórdenes afectivos (353) . aún no se ha descrito una correlación directa de este trastorno psicótico con covid-19. los síntomas musculares se han observado en pacientes infectados por covid-19 incluyendo la miopatía del paciente en estado crítico (miopatía difusa no necrotizante con degeneración grasa de fibras musculares), la miopatía necrotizante (ligada a falla orgánica múltiple) y la miopatía de filamentos gruesos(354). se recomienda considerar como predictores clínicos neurológicos de alerta para sospechar covid-19: anosmia, disgeusia, delirium y alteraciones neuromusculares inespecíficas sin otra causa aparente de explicación. se recomienda no establecer de rutina predictores neurológicos específicos de mal pronóstico en el paciente críticamente enfermo con covid-19. se deben tener en cuenta los factores de riesgos generales de mal pronóstico para la población general como la edad avanzada, las comorbilidades cardiovasculares y el tabaquismo. page la proteína spike (s) del covid-19 es reconocida por la enzima convertidora de antígenos 2 (eca2) de la célula huésped cuyo papel es el punto de entrada molecular a tejidos pulmonares, gastrointestinales y neuronales (355) . la forma como el covid-19 ingresa al sistema nervioso central es desconocido, pero se especula que inicialmente invade terminales nerviosas periféricas y después llega al sistema nervioso central a través de una ruta guiada por sinapsis nerviosas con un patrón ascendente (ruta dada por el coronavirus hev67 y el oc-43) (352, 356) . en modelos de roedores el covid-19 ingresaría al cerebro a través del nervio olfatorio, atravesando la lámina cribiforme propagándose por el tálamo y el tallo cerebral; explicándose por la expresión de los receptores de la enzima convertidora de antígenos (eca2) en la superficie de las mucosas nasales, las neuronas y la glía (352, 357, 358) . una segunda forma de ingreso es a través de la vía hematológica mediante arterias cerebrales atravesando la barrera hemato-encefálica utilizando las células inflamatorias como un modelo similar al del caballo de troya (352, 359, 360) logrando una ubicación definitiva en células neuronales y endoteliales del lóbulo frontal como lo demostraron en estudios post-mortem descritos por paniz -mondolfi et al, lo que explicaría los cambios comportamentales de paciente (360) . la tercera forma de acceso al sistema nervioso central es mediante el drenaje del sistema linfático cerebral invadiendo ganglios linfáticos hiliares y mesentéricos con sintomatología gastrointestinal asociada (361) . una vez ha logrado ingresar tiene la capacidad de infectar macrófagos, microglía, y astroglía los cuales secretan factores proinflamatorios como interleuquina 12, interleuquina 15, y factor de necrosis tumoral alfa (362) . esta condición se exacerba con el desencadenamiento de la tormenta de citoquinas liderada por la interleuquina 6, interleuquina 2, interleuquina 7 e interferón gamma (355) . de esta forma los coronavirus siendo neurotrópicos ocasionan múltiples manifestaciones clínicas ya mencionadas, así como encefalitis, parálisis flácida, incluyendo la asociación con guillain-barré (357) . los síntomas que harían sospechar la presencia de neurocovid-19 son la náusea, el vómito y la anorexia, estos síntomas pueden ser el reflejo del compromiso del virus en el área postrema del piso del cuarto ventrículo que hace parte del complejo vagal dorsal de la médula oblonga. sin embargo, estos síntomas pueden enmascararse como una respuesta inespecífica relacionada con un compromiso gastrointestinal (363) . como se mencionó previamente la anosmia y la disgeusia son síntomas significativos para sospechar en covid-19 (358) ; esto es debido a una lesión directa sobre el nervio olfatorio (i par craneal), y la lesión de alguno de los tres nervios encargados de registrar el sentido del gusto como lo son el vii, ix y x pares craneales, así como el compromiso del núcleo solitario y del tálamo como zona de relevo; de hecho el núcleo del tracto solitario es muy cercano al centro respiratorio que podría ocasionar disnea de origen central (361) , otros núcleos como el núcleo dorsal motor del vago y el núcleo ambiguo están relacionados con funciones cardiovasculares a tener en cuenta (364) . respecto a los factores de riesgo destaca el tabaquismo el cual aumenta la posibilidad de neuroinfección debido a interacciones funcionales entre el receptor nicotínico de acetilcolina y el receptor eca2 el cual está sobreexpresado en pacientes fumadores (365) . amci ® en pacientes con infecciones severas vs infecciones no severas (45.5% vs 30.2%, p = 0.02), incluyendo eventos cerebrovasculares (5,7% vs 0,8% p=0,03), alteración del estado de consciencia (14.8% vs 2.4%; p<0.001) y lesiones musculoesqueléticas (19,3% vs 4,8% p< 0001) (366) . la encefalopatía que se manifiesta como una alteración aguda o subaguda del estado de consciencia presentándose en pacientes con comorbilidades, factores de riesgo cardiovasculares, edad avanzada y deterioro cognitivo previo (362, 366, 367) . así como aquellos individuos con hipoxemia la cual induce metabolitos anaerobios en el sistema nervioso central, edema celular, intersticial e isquemia (362) . los eventos cerebrovasculares pueden ser desencadenados por cuadros de hipoxia, inmovilización, un incremento de la respuesta proinflamatoria o por predisposición a la hipercoagulabilidad (359) . respecto a este último rubro tanto la edad (hazard ratio 1,05/por año 95% ic 1,004 -1,01) y la coagulopatía definida como como un tiempo de protrombina mayor a 3 segundos, o tiempo de tromboplastina mayor a 5 segundos (hr 4,1 95% ic 1,9 -9,1) fueron considerados predictores independientes de complicaciones trombóticas (352) ; otros trabajos reportan incremento del conteo plaquetario y niveles elevados de dímero d (352) . de hecho, un scoping review realizado por wilson y jack muestra que la presencia de eventos cerebro vasculares es un factor de riesgo de mal pronóstico para pacientes infectados por covid-19 (368) . mao et al. reportaron 14 eventos cerebro vasculares en 214 pacientes con covid-19, los factores de riesgo más relevantes fueron los clásicos factores de riesgo cardiovasculares (diabetes, hipertensión y edad avanzada), así como una presentación sistémica severa, teniendo como un denominador común el compromiso estructural de grandes vasos (366) . la presencia de convulsiones (clínicas o subclínicas) puede ser una manifestación de eventos cerebro vasculares, meningoencefalitis o hipoxia cerebral. siendo los factores de riesgo más importantes en su exacerbación las alteraciones electrolíticas como hipocalcemia, las reacciones adversas a medicamentos y la epilepsia como comorbilidad de base (359, 368) . otros tipos de coronavirus como lo son el 229e, 293, y oc43 se han aislado de líquido cefalorraquídeo (lcr) de pacientes con esclerosis múltiple sugiriendo posiblemente sean agentes etiológicos en la exacerbación de brotes de esta patología sin embargo aún no se ha documentado la asociación entre covid-19 y ésta condición (356) . otras patologías en las cuales se ha asociado la presencia de esta familia de microorganismos es la enfermedad de parkinson, la esclerosis lateral amiotrofia, la neuritis óptica y la encefalitis aguda diseminada (356, 362) . múltiples trabajos han documentado la asociación entre polineuropatía y coronavirus. la mayoría de ellos consideran que existe una estrecha relación entre una polineuropatía autoinmune exacerbada por la infección por coronavirus o bien un compromiso nervioso periférico inducido por bloqueo neuromuscular, alteraciones hidroelectrolíticas o disvitaminosis(356). amci ® se recomienda que todo paciente con acv isquémico se considere sospechoso de infección por covid-19 si presenta: sintomatología asociada sospechosa de infección por covid-19, contacto cercano con individuos con sintomatología infecciosa, allegados con viajes recientes, si la historia clínica es atípica, si la información suministrada no es clara, si presenta deterioro del estado de alerta inexplicable, y si al examen físico presenta hallazgos compatibles con una infección por covid-19. se recomienda que todo paciente con acv isquémico de quien no se pueda recibir información se considere sospechoso de covid-19 ya que el evento cerebro vascular es una complicación que se ha reportado en pacientes con infección por covid-19. hay una gran evidencia que correlaciona la presencia de infección por covid-19 con factores de riesgo cardiovasculares. esto se demostró en un metaanálisis que incluyó 7 estudios de china (n=1576) donde las comorbilidades más frecuentes fueron hipertensión (21,1%, 95% ic: 13 -27,2%), diabetes (9,7%, 95% ic 7,2 -12,2%), enfermedad cardiovascular (8.4%, 95% ic: 3.8-13.8) y patologías respiratorias (1.5%, 95% ic: 0.9-2.1%). al comparar severidad vs no severidad la presencia de hipertensión tuvo un or de 2.36 (95% ci: 1.46-3.83), y la enfermedad cardiovascular un or de 3.42 (95% ci: 1.88-6.22) respectivamente (369) . existieron pocos casos de hipercoagulabilidad en pacientes sin factores de riesgo cardiovasculares (356) . la cohorte de wuhan (n=138) publicada por wang et al. mostró otros factores de riesgo que podrían eventualmente estar asociados a estado de embolia y trombosis como shock en 8,7%, arritmias 16,7% y miocarditis en un 7,2%. situaciones que conllevarían a hipercoagulabilidad, lesión endotelial y eventualmente la aparición de acv. (174) . en este orden de ideas existe una estrecha correlación entre la presencia de factores de riesgo cardiovasculares que ocasionarían acv y que podrían eventualmente estar relacionados con la patogenia de covid-19. ante esta inquietud khosravani et al. publicaron en stroke un informe especial donde realizan ciertas recomendaciones para el abordaje de los pacientes con acv en el contexto de la pandemia por covid-19 de una forma rápida, eficaz y segura para los diferentes profesionales de la salud. surge así el código stroke protegido el cual consiste en:  usar elementos de protección personal y una mascarilla al paciente  ejecutar protocolo de aislamiento de contacto y gotas  ejecutar protocolo de aerosoles si el paciente está sometido a ventilación mecánica no invasiva, manejo de aspiración de secreciones o maniobras de reanimación cardiopulmonar básica y avanzada  si el paciente presenta deterioro del estado de alerta con necesidad de soporte ventilatorio alto con fracción inspirada de o2 mayor a 50% se recomienda intubar temprano y proceder con el transporte. se debe proceder con el código stroke protegido si el paciente presenta alguna de las siguientes condiciones:  si el paciente presenta sintomatología sospechosa de covid-19 (fiebre, tos, dolor torácico, disnea, cefalea, mialgias, emesis)  si existe algún contacto cercano con sintomatología infecciosa  si el paciente o alguno de sus allegados ha presentado viajes recientes amci ®  si el paciente es covid-19 positivo  si refiere al interrogatorio una historia clínica atípica o poco clara  si el paciente o alguno de sus acudientes es incapaz de suministrar información  si el paciente presenta deterioro del estado de alerta  si al examen físico se encuentran signos compatibles con patologías diferentes a covid19 por último, estas son las recomendaciones en el momento de realizar el traslado a saber:  no apresurarse dentro de la sala de reanimación o dentro de la unidad de cuidados intensivos y mantener la calma  designar un líder para el traslado del paciente y para supervisar el uso adecuado de los elementos de protección personal  limitar el número de personas encargadas del transporte  evitar la contaminación con otras áreas del hospital(370). se recomienda no realizar neuroimagen de rutina en pacientes críticos por covid-19 con cefalea y anosmia, dado que no existe una evidencia concluyente que demuestre una estrecha correlación entre estos síntomas y hallazgos imagenológicos. fuerte en contra fundamento en la mayoría de los casos la cefalea es un síntoma no específico que no es característico de irritación meníngea la ocurrencia de cefaleas aisladas en ausencia de otros síntomas sugiere un mecanismo benigno más que un compromiso de sistema nervioso central (356) . sin embargo, el covid-19 al tener una capacidad neuroinvasiva, se han reportado casos de encefalitis virales con o sin necrosis hemorrágicas de compromiso temporal mesial y talámico que ameritarían estudio de imagen diagnóstica (368) . un estudio retrospectivo publicado por kandenmirli et al. evaluó 749 pacientes en 8 hospitales infectados con covid-19 de los cuales 235 requirieron manejo en uci donde el 21% de ellos (n=50) presentaron sintomatología neurológica. la resonancia magnética cerebral fue realizada en 54% (n=27) de estos pacientes. el 44% tuvieron hallazgos agudos, el 37% tuvieron alteraciones corticales de la captación de señal en el modo flair, 3 pacientes tuvieron anormalidades en la señal flair en la sustancia blanca profunda y subcortical, 4 pacientes con lesiones en el lóbulo frontal, 3 en el lóbulo parietal, 4 en el lóbulo occipital, 1 en el lóbulo temporal, 3 en la corteza de la ínsula, y 3 en el giro cingular (371) . amci ® un paciente presentó trombosis de seno venoso y otro presentó un infarto en el tercio medio del territorio de la arteria cerebral media. en 56% de los pacientes no se encontraron hallazgos que sugieran compromiso intracraneal de covid-19. una correspondencia escrita por helms et al. reportaron 58 pacientes con sdra y covid-19 en dos unidades de cuidados intensivos en francia entre marzo y abril del 2020, el 84% presentaron signos neurológicos como delirium evaluados mediante cam-icu (65%), agitación (69%), signos del tracto cortico espinal (67%), síndrome disejecutivo (36%). de ellos se realiza resonancia magnética en 13 pacientes encontrando alteraciones en la perfusión en un 100%, un realce de leptomeninges en un 62%, y un acv isquémico en un 23%; dos pacientes asintomáticos presentaron áreas isquémicas con hiperintensidad focal (372) . ante estos hallazgos los principales diagnósticos diferenciales son las patologías autoinmunes, encefalitis, convulsiones e hipoglucemia. los pacientes con compromiso frontal bilateral poseen hipoxemia que ocasionan hipoperfusión fronto temporal. las microhemorragias corticales (y no) son consecuentes de la ruptura de la membrana hematoencefálica resultando en este patrón mencionado. el estado postictal muestra un compromiso simétrico de la sustancia blanca. de esta forma hay que considerar otras condiciones como las comorbilidades cardiovasculares, las reacciones adversas a los medicamentos, e hipoxia inducida por sdra que ocasionarían patrones imagenológicos de confusión, que ponen en duda la estrecha relación entre covid-19 y hallazgos de resonancia magnética (371) . en este orden de ideas necesitamos más datos para determinar cuáles son los hallazgos imagenológicos relacionados con neurotropismo y qué patrones pueden encontrarse directamente relacionados con la presencia de convulsiones, hipoxia o el desencadenamiento de una tormenta de citoquinas(362, 371, 372). se recomienda la valoración neurológica completa en los pacientes con sospecha o infección por covid-19. se recomienda la monitorización electroencefalográfica continua por al menos 24h o según la consideración del especialista en neurociencias en el paciente en estado crítico con sospecha o infección por covid-19 en quien se sospeche crisis epilépticas o estatus no convulsivo. las manifestaciones neurológicas en pacientes con infección por covid-19 pueden estar presentes en el 36% de los casos (366, 373, 374) . en el paciente en estado crítico con infección por covid-19 se han observado complicaciones vasculares que puede causar ataque cerebrovascular agudo isquémico o hemorrágico con tazas de entre el 5 al 6% por lo que el examen neurológico debe realizarse para documentar la presencia de déficit amci ® neurológico focal que haga sospechar esta patología (366, 375, 376) . es conocido que los pacientes en unidades de cuidado crítico tienen patologías que aumenta el riesgo de crisis o estado epiléptico, entre las cuales se encuentran (377) (378) (379) (380) :  pacientes sin patologías neurológicas hospitalizados en unidad de cuidado intensivo  hemorragia subaracnoidea  hemorragia intracraneal  trauma cráneo encefálico moderado a severo  infección del sistema nervioso central  tumor cerebral  encefalopatía hipóxico-isquémica algunas de estas patologías se presentan más frecuentemente en pacientes por covid-19 por lo que la monitorización electroencefalográfica continuar es requerida en este grupo de pacientes. el virus puede producir descompensación de paciente con epilepsia conocida, crisis por fiebre, crisis producidas por el estado crítico del paciente o las patologías subsecuentes que se han observado en esta infección (381) . el tiempo de monitorización requerido debe ser entre 12 a 24 horas para lograr una sensibilidad de 82 a 88%(380, 381). se recomienda realizar tomografía cerebral simple ante las manifestaciones neurológicas focales que nos hagan sospechar ataque cerebrovascular isquémico o hemorrágico. la resonancia cerebral puede ser necesaria como estudio complementario para determinar otros diagnósticos diferenciales en los pacientes con sospecha o infección por covid-19. las manifestaciones neurológicas en pacientes con infección por covid-19 pueden estar presentes en el 36% de los casos (366, 373, 374) . en el paciente en estado crítico con infección por covid-19 se han observado complicaciones vasculares que puede causar ataque cerebrovascular agudo isquémico o hemorrágico con tazas de entre el 5 al 6% por lo que el examen neurológico debe realizarse para documentar la presencia de déficit neurológico focal que haga sospechar esta patología (366, 375, 376) . la tomografía cerebral hace parte de la valoración inicial del ataque cerebrovascular isquémico y del estudio para determinar la presencia de otros diagnósticos diferenciales como ataque cerebro vascular hemorrágico en paciente con dicha patología puede ser necesario realizar angiotac con extensión a tórax en caso de sospecha de oclusión proximal o estudios endovasculares en pacientes que sea indicado (382) . la realización de resonancia cerebral usualmente documenta alteraciones en los pacientes con covid-19 en el 37 -62 % de los casos evaluados no relacionada con el acv. se recomienda la realización de punción lumbar en el paciente con sospecha o diagnóstico de covid-19 con base en los reportes de casos disponibles:  paciente con crisis epilépticas de novo.  paciente con alteración del estado de conciencia persistente a pesar de encontrarse metabólicamente compensado, descartado ataque cerebrovascular u otra causa de encefalopatía.  paciente con manifestaciones como mielitis, neuropatía craneal múltiple o sospecha de polineuropatía desmielinizante aguda. las capacidades neurotrópicas de los coronavirus en general han sido expuestas desde la infección por sarscov. en cuanto al sars-cov-2, asadi-pooya y colaboradores(383), han descrito, la posibilidad de ingreso al sistema nervioso central tras el ingreso por la mucosa nasal o por una gran viremia en el torrente sanguíneo. se han descrito procesos inflamatorios asociados (encefalitis) y en previamente con el sars y el mers hasta lesiones desmielinizantes (encefalomielitis aguda diseminada). estas primeras manifestaciones en 2002 con el sars fueron presencia de crisis epilépticas de novo, en quienes excluyendo otras causas tanto por neuroimagen además de pruebas microbiológicas en lcr, les fue descubierto el sars cov mediante pcr rt. en la presente pandemia, takeshi moriguchi y colaboradores (384) describieron el primer caso de encefalitis asociado a sars-cov-2, en un hombre joven en japón, que, tras 7 días de clínica respiratoria, desarrollo crisis epilépticas y alteración del estado de conciencia, sin antecedente conocido de epilepsia. se realizo imagen por resonancia cerebral, demostrando hiperintensidades a nivel temporal y lcr que mostró pleocitosis linfocitaria, se descartaron otros virus ( herpes, herpes zoster) y se le realizó pcr rt para sars-cov-2 siendo positiva. inclusive en este paciente los primeros hisopados faríngeos fueron negativos, pero los hallazgos en tomografía de tórax hicieron sospechar la infección por sars-cov-2. otro caso descrito de encefalitis hemorrágica aguda, fue también descrito en la revisión de ahmad y colaboradores (385) . donde una mujer joven presentó cuadro respiratorio de fiebre de 3 días de evolución y compromiso del estado de conciencia. en esta paciente se aisló el sars-cov-2 en hisopado faríngeo y ante el compromiso severo del estado de conciencia, se realizó resonancia que mostró compromiso hemorrágico bitalámico, en regiones temporales e ínsula, apoyando que se tratara de una diseminación tras neuronal probablemente con puerta de entrada mucosa olfatoria y siguiendo la diseminación por el los tractos del primer nervio hasta la corteza entorrinal, que es la vía propuesta de infección descrita inclusive por grupos como el de montalvan (356) y natoli (386) . amci ® precisamente, en la revisión sistemática de montalvan (356) se describen 1 caso de mielitis en contexto de paciente con infección por sars-cov-2, en quien se documentó la infección en lcr. se recomienda la medición de ácidos nucleicos por rt-pcr para sars-cov-2 en líquido cefalorraquídeo en los pacientes con sospecha o confirmación de infección por sars-cov-2 que realice crisis epilépticas de novo, en quien se descarte otras causas de estructuralidad (acv, tumores) o causas metabólicas (alteraciones hidroelectrolíticas, hipoglicemia, uremia etc.). se recomienda la medición de ácidos nucleicos por rt-pcr para sars-cov-2 en líquido cefalorraquídeo en pacientes con sospecha o confirmación de infección por sars-cov-2 con alteración franca del estado de conciencia, en quien se haya descartado como causante hipoxia, ataque cerebrovascular, alteración hidroelectrolítica o estatus no convulsivo mediante imágenes y electroencefalograma. se recomienda la medición de ácidos nucleicos por rt-pcr para sars-cov-2 en líquido cefalorraquídeo en pacientes con sospecha de encefalitis, mielitis o síndrome de guillain barré. en pacientes críticos, se ha recomendado búsqueda activa de compromiso del snc por sars-cov-2, sobre todo en pacientes con crisis epilépticas que no sean sintomáticas a trastornos metabólicos, además de hallazgos imagenológicos y curso clínico. las descripciones hechas por asadi y takeshi (383, 384) , muestran pacientes con cursos tórpidos, en los que el común denominador es una alteración del estado de conciencia persistente a pesar que otras variables (metabólicas, vasculares e infecciosas diferentes al sars-cov-2). de igual forma pacientes que realicen en contexto de la enfermedad, un cuadro de debilidad generalizada aguda, con arreflexia e incluso compromiso de nervios craneales como los descritos por zahra sedaghat(387). page amci ® se recomienda en los pacientes críticos con compromiso pulmonar por covid-19, un manejo orientado de fluidos frente a una estrategia liberal, ajustando el balance de fluidos de acuerdo con la evaluación clínica y/o a la capacidad de respuesta a volumen para garantizar la perfusión renal. se recomienda ajustar la intensidad de la monitoria en el paciente crítico con covid-19 al grado de severidad de la enfermedad para alcanzar tempranamente metas de reanimación que se reflejen en menor riesgo de lesión renal aguda. se recomienda evitar el uso de medicamentos nefrotóxicos teniendo en cuenta la farmacocinética y farmacodinamia individual, así como las interacciones farmacológicas en el paciente crítico con covid-19. el manejo de la volemia en los pacientes críticos ha cambiado considerablemente en las últimas décadas orientándose a un manejo titulado, evitando la administración empírica de altos volúmenes de líquidos durante la fase de reanimación de los pacientes siendo el flujo sanguíneo el determinante primordial del aporte tisular de oxígeno; los principales componentes de este deben optimizarse y balancearse para evitar la disoxia tisular. en estados de bajo flujo, los mecanismos compensadores neurohumorales producen una redistribución del flujo a lechos no esplácnicos y a nivel renal una redistribución corticomedular convirtiendo el tejido medular renal en una zona vulnerable a la lesión. de igual forma, el flujo sanguíneo renal se ve reducido de forma refleja en presencia de hipoxemia y/o hipercapnia. diversas fuentes de información nos han indicado cómo orientar adecuadamente el manejo del estado de perfusión tisular en los pacientes críticos con o sin una condición de shock, siendo la estimación aproximada del estado de volumen del paciente el pilar fundamental sobre el cual se basará toda la estrategia de restablecimiento o mantenimiento de la volemia del paciente con el fin de mantener la perfusión adecuada. aunque históricamente se prioriza la normalización del volumen en la reanimación de un paciente inestable, hemos comprendido la importancia que tiene el tiempo para lograr las metas en el pronóstico general del paciente y evidencias como el trabajo de ospina y cols. soportan el uso temprano de vasopresores para lograr de forma temprana metas de perfusión mientras buscamos la normovolemia. los pacientes con enfermedad pulmonar asociada a covid-19 deben mantenerse normovolémicos para preservar el flujo sanguíneo renal siendo la evaluación del estado de volumen un verdadero reto clínico. la fiebre, el aumento de pérdidas insensibles, la baja ingesta o las pérdidas por el tracto digestivo, pueden hacer que un paciente con covid-19 tenga hipovolemia, situación que puede desencadenar daño renal de no ser revertida apropiadamente y a tiempo. del otro lado del espectro, la administración liberal de líquidos además del potencial de empeorar la lesión pulmonar en presencia de una membrana alveolo capilar seguramente alterada, puede producir por sí misma un incremento en el riesgo de desarrollar lesión renal como lo demostró el estudio de grissom. desde el punto de vista de las metas amci ® hemodinámicas que se deben tener con un paciente con covid-19, se recomienda seguir los lineamientos de la campaña sobreviviendo a la sepsis, orientadas a mantener un óptimo estado de volumen, unas presiones de perfusión en un rango que permita la regulación de los flujos regionales en los distintos órganos y un gasto cardíaco dentro de unos rangos establecidos para una perfusión sistémica óptima. el examen clínico sigue teniendo vigencia absoluta para una adecuada aproximación al paciente, por ello debemos buscar los indicadores clínicos tradicionales de hidratación (piel, mucosas, enoftalmos, edema, etc.), el llenado capilar como lo describe hernández y cols en el estudio andrómeda-shock, el estado de alerta, las funciones cognitivas y la rata urinaria son entre otros unos marcadores aceptables para hacernos una idea del estado de adecuación de la perfusión periférica. la oliguria como marcador de perfusión renal está presente en 1/4 parte de los pacientes al ingreso a uci sin tener en sí sola una implicación pronóstica, sin embargo, la persistencia de esta en el tiempo es un indicador de alerta y obliga a una evaluación más detallada de las diversas variables que pudieran ocasionarla. debemos recordar que la administración de cargas de líquidos solamente está justificada cuando hay una respuesta cardiovascular a dicha administración, situación que se puede prever con una prueba de elevación pasiva de las piernas o con métodos más invasivos como la variabilidad de la onda de pulso, del volumen sistólico y/o del gasto cardíaco, entre otros. el esfuerzo respiratorio del paciente, los volúmenes utilizados en las estrategias de ventilación protectora y las arritmias frecuentemente presentes en los pacientes con compromiso pulmonar limitan el rendimiento diagnóstico de diversos dispositivos utilizados para la evaluación del estado de volumen y el gasto cardíaco, situación que debemos conocer y manejar(332, 388-394). se recomienda no utilizar de forma rutinaria la administración de tratamientos específicos antivirales para el paciente crítico con covid-19, con lesión renal aguda o crónica se recomienda no utilizar de rutina remdesivir en los pacientes con falla renal crónica y debe suspenderse en los pacientes que desarrollan lesión renal aguda con tfg < 30 ml/min. las intervenciones farmacológicas en los ensayos clínicos deben ajustarse a la farmacocinética y farmacodinamia específicas de cada molécula. la incidencia de falla renal reportada por criterios de kdigo, en los estudios chinos fue de 0.5% de los pacientes críticamente enfermos y de 4.7% en los pacientes con covid-19 y amci ® sdra. sin embargo, otros estudios han demostrado que hasta un 40%de pacientes con covid-19 que ingresan a la uci pueden presentar falla renal aguda. la falla renal en los pacientes con covid-19 es multifactorial como se ha descrito en preguntas anteriores por lo que se recomienda la toma diaria de creatinina sérica y el seguimiento continuo del gasto urinario y otros parámetros de la función renal como hematuria, proteinuria, tasa de filtrado glomerular, nitrógeno ureico en sangre, dímero d. los medicamentos que se emplean en el manejo de la infección covid-19 que actualmente incluyen oseltamivir, lopinavir/ritonavir, ribavirina, y la cloroquina o hidroxicloroquina son metabolizados principalmente en el hígado, aunque en la orina se encuentran metabolitos derivados de oseltamivir, ribavirina y de la hidroxicloroquina. por esto en ninguno de los estudios realizados en torno a la infección por sars-cov-2 se ha realizado ninguna recomendación en cuanto a la modificación de su dosis. la hidroxicloroquina por su parte se metaboliza a cloroquina, que a su vez se metaboliza a monodesetilcloroquina y a bisdesetilcloroquina. este medicamento no es dializable en las diálisis intermitentes y la única recomendación en los pacientes con falla renal, es hacer seguimiento electrocardiográfico estrecho para vigilar la prolongación del qtc. el favipiravir es un inhibidor de la polimerasa dependiente de rna que se encuentra en fase experimental para el tratamiento de la infección por sars-cov-2. la eliminación de este medicamento se realiza por vía renal y en los pacientes con falla renal en estadios leves a moderados se ha encontrado una concentración dos veces mayor en el riñón que sus niveles en sangre; sin embargo, esto no se ha asociado con ningún evento adverso por lo que la recomendación actual es no disminuir la dosis en pacientes con falla renal. el remdesivir se elimina por vía renal por lo cual no se recomienda administrar en pacientes con falla renal y los pacientes que desarrollan falla renal con el medicamento durante los estudios han sido retirados de los ensayos clínicos. no se cuenta con estudios que evalúen el remdesivir en una tfg <30 ml/min. en la tabla 15 se describen algunas intervenciones farmacológicas propuestas en medio de la pandemia para el manejo del covid-19 y su relación con la tasa de filtración según tfg. recientemente , izzedine et al publicaron una carta editorial en el kidney international may 21, 2020 donde alertan sobre el posible efecto nocivo de la hidroxicloroquina en la aparición de falla renal aguda al inhibir la autofagia celular que es un proceso importante en la remodelación de los túbulos renales, siendo estas células de alto recambio, pudiendo todo esto contribuir a la aparición de falla renal aguda(166, 327, 395-398) se recomienda aplicar las indicaciones tradicionales de terapia de soporte renal en pacientes críticamente enfermos con covid-19. se recomienda el inicio de terapia de soporte renal en pacientes críticos con covid-19 con hipercalemia severa o acidosis metabólica severa, refractarias a pesar del manejo médico óptimo o cuando el balance positivo de fluidos es deletéreo, con mayor requerimiento de oxígeno suplementario y que no responde a diuréticos. se recomienda el inicio temprano de terapia de soporte renal dentro de las primeras 24 horas de una indicación absoluta, asegurando previamente una adecuada reanimación de la perfusión tisular. en ausencia de trastornos hidro-electrolíticos y severa sobrecarga de volumen, el tiempo de inicio de diálisis es controversial. amci ® se recomienda en pacientes críticos con covid-19 que requieren soporte renal, las modalidades de terapia continua o extendida si cursa con inestabilidad cardiovascular, de acuerdo con la disponibilidad institucional. se debe considerar en pacientes críticos con covid-19, que requieren inicio de soporte renal preferir la vía femoral para inicio de la terapia para disminuir el riesgo de contaminación por proximidad, la siguiente vía se establecerá de acuerdo con la evolución y condiciones del paciente. en pacientes diagnosticados con covid-19 se puede presentar la insuficiencia renal aguda como parte de su enfermedad. en estudios observacionales de usa y china la ira se reportó entre un 3 y 37% de los pacientes. la enfermedad renal en pacientes con covid-19 se puede manifestar como ira, hematuria o proteinuria, y conllevan un mayor riesgo de mortalidad. la ira se asocia con cambios hemodinámicos y liberación de citocinas, pero no se descarta citotoxicidad directa por el virus. en un estudio realizado en nueva york con 5450 pacientes covid-19 positivos se diagnosticó lesión renal aguda en 37% de ellos, siendo leve con aumento de creatinina dos veces por encima del nivel basal en 47% de los pacientes, moderada en 22% de los pacientes, y severa con más del triple de la creatinina basal en 31%. hubo hematuria en el 46% de los pacientes y proteinuria en el 42%. se requirió terapia dialítica en el 15% de todos los pacientes con diagnóstico de ira, y el 97% de los pacientes que requirieron diálisis estaban en ventilación mecánica. la ira fue notada dentro de las primeras 24 horas de admisión a uci en el 33% de los pacientes y se relaciona con la severidad de la enfermedad. existen además predictores independientes: edad, raza negra, diabetes, hipertensión, enfermedad cardiovascular, ventilación mecánica, y uso de vasopresores. la terapia dialítica debe instaurarse precozmente una vez realizado el diagnóstico, idealmente dentro de las primeras 24 horas, después de asegurado que se ha completado el proceso de reanimación correspondiente. las indicaciones te trr en pacientes críticos con ira en covid-19 no difieren del paciente crítico general y se debe considerar ante: manifestaciones severas de uremia, sobrecarga de volumen, trastornos ácidos básico, refractarios, hipercalemia severa con manifestaciones cardiovasculares. pero no hay datos clínicos que respalden el inicio temprano vs tardío en esta población particular. pero un planteamiento válido es que la sobrecarga de volumen en pacientes que desarrollan sdra es perjudicial, dificultando el soporte ventilatorio óptimo, por lo cual se puede considerar un umbral más bajo para el inicio de trr con esta indicación específica: sdra + sobrecarga de volumen + infección covid-19 (399) . amci ® el paciente debe ser dializado en el cubículo de cuidado intensivo o en la habitación de aislamiento en los casos en que esté disponible y siempre evitar traslado a unidades con otros pacientes. la crrt es la modalidad preferida para este tipo de pacientes, pero debe quedar claro que esto depende de las facilidades de la institución que albergue al paciente y de la experticia de los profesionales. el acceso vascular en el paciente crítico general debe ser en su orden: vena yugular interna derecha, venas femorales comunes, vena yugular interna izquierda, y debe ser colocado por el médico encargado del paciente si está capacitado para ello, para evitar exposiciones innecesarias del personal de la salud. sin embargo, por precaución por el riesgo de contaminación, recomendaciones de expertos basadas en seguridad sugieren la utilización el catéter femoral. el tipo de catéter recomendado es un catéter doble lumen transitorio. sería ideal el monitoreo a través de cámaras del procedimiento para evitar el contacto prolongado del personal de enfermería durante el procedimiento de diálisis. en algunos casos específicos y de acuerdo con la disponibilidad, la diálisis peritoneal puede ser una alternativa. en los casos de crrt el líquido efluente no es contaminante para el personal de la salud. para terminar, es importante hacer énfasis en que en algunos hospitales ha habido escasez de insumos y esto puede llegar a convertirse en un serio problema. se recomienda para casos de fuerza mayor:  un litro de solución salina al 0.9% con cloruro de potasio a necesidad  un litro de dextrosa al 5% en agua con 150 meq de bicarbonato de sodio  un litro de solución salina al 0.9% con 1 gr de cloruro de magnesio  un litro de solución salina al 0.9% con 1 gr de cloruro de calcio esto nos da una solución de cuatro litros que contienen: 153 meq/l de sodio, 37.5 meq/l de bicarbonato, 2.6 mmol/l de magnesio y 2.25 mmol/l de calcio, más una cantidad variable de potasio. esta solución se puede usar como líquido dializante en pacientes en terapias de reemplazo renal continuo. especial cuidado se debe tener en el proceso de anticoagulación, pudiéndose usar heparina no fraccionada, hbpm, y citrato en los centros donde se tenga experiencia (139, 339, (400) (401) (402) (403) (404) (405) (406) (407) (408) (409) (410) (411) . se sugiere no utilizar de rutina la trrc más hemoperfusión en el paciente crítico con covid-19. débil en contra page amci ® se puede considerar en el paciente crítico con covid-19 con lesión renal aguda en quien se considere inicio de trrc, considerar la utilización de filtros de fibra hueca con propiedades adsortivas o asociado con cartuchos para hemoperfusión directa. el síndrome de liberación de citocinas (tormenta de citocinas) es un importante determinante en la transformación de infección por covid-19 de leve a moderado y progresión de la lesión de un órgano como el pulmón con neumonía y sdra a compromiso sistémico con inestabilidad hemodinámica, cid y fom. los pacientes afectados de tormenta de citocinas se encuentran con niveles altos de il-6 especialmente, además de il-10, tnf, que se relacionan con pobres pronósticos y mayor mortalidad. la asociación entre la lesión alveolar y renal (eje pulmón-riñón) es evidenciada en estudio del 2019 por panitchote y cols con 357 pacientes con sdra secundaria a neumonía y sin enfermedad renal preexistente que desarrollaron lesión renal aguda en el 68% con aki 3 en el 50% de ellos. recientemente en estudio en china zhou, con 191 pacientes afectados de covid-19 se encontraron como indicadores de mal pronóstico pacientes con altos niveles de dímero d, il-6, troponina i, dhl, ferritina y choque séptico. las terapias de depuración extracorpórea han sido utilizadas como tratamiento en pacientes con lesión severa por covid-19; dentro de estas se cuentan la crrt, hemoperfusión aislada, intercambio plasmático (tpe), plasmafiltración y adsorción (cpfa) y crrt + hemoperfusión. dentro de los beneficios de la crrt se cuentan la estabilidad hemodinámica, estabilidad del medio interno, depuración de toxinas pequeñas y medianas, incluidos mediadores inflamatorios, cuando se utiliza terapia convectiva, además de permitir soporte nutricional. la asociación de este procedimiento con membranas especiales (an69 + metilsulfonato y polietilamina oxiris) permiten hacer adsorción de citocinas (il-6) y endotoxinas, por periodos de 24h por 2 días consecutivos para manejo de tormenta de citocinas. la hemoperfusión aislada o asociada a crrt, también permite la remoción de il-6, utilizando cartuchos ha330, con procedimientos de 2 a 4 horas de duración por 3 días consecutivos (37, (412) (413) (414) (415) (416) (417) . no se puede emitir una recomendación a favor o en contra sobre el uso rutinario de la plasmaféresis como opción terapéutica en la fase de inflamación del paciente con covid-19. amci ® el coronavirus covid-19 puede inducir el síndrome respiratorio agudo severo (sars), que conduce a la disfunción inmune, la liberación excesiva de citoquinas inflamatorias, y a una serie de reacciones en cascada de activación de citoquinas, que resultan en lesiones alveolares difusas, formación de membrana hialina, exudación de fibrina y otras manifestaciones de lesión del pulmón. en casos severos, la tormenta de citoquinas sistémicas invade el sistema circulatorio, lo que lleva a una inestabilidad hemodinámica, shock y mods (418) . los niveles de il-6, il-10, tnf-a y otras citoquinas inflamatorias en pacientes con covid-19 grave son significativamente más altos, lo que puede estar relacionado con un mal pronóstico (419) . por lo tanto, la plasmaféresis se puede usar con seguridad y efectividad en pacientes con covid-19 grave, para eliminar mediadores inflamatorios de gran peso molecular. la seguridad depende como todas las terapias extracorpóreas de un personal de la uci entrenado, preparado y capacitado para aplicar las intervenciones en forma óptimas (420) . las terapias extracorpóreas de soporte de órganos pueden representar una parte importante de la respuesta y los médicos y otros profesionales de la salud deben estar familiarizados con estas terapias sofisticadas. se debe hacer un llamado a la acción, para crear conciencia sobre las diferentes técnicas extracorpóreas, cada una con criterios específicos y modalidades de prescripción, entrega y monitoreo(421, 422). se recomienda no utilizar de forma rutinaria el uso de un tratamiento específico dirigido a pacientes con infección por sars-cov-2/covid-19 comparado con el manejo estándar para mejorar desenlaces clínicos fuertes. actualmente no existe una terapia dirigida que se a efectiva para el manejo del virus; un número alto de estudios han surgido en los últimos dos meses, la mayoría sin el rigor metodológico suficiente para tomar decisiones adecuadas con respecto al manejo del amci ® paciente con infección por sars-cov-2. el conocimiento en la estructura del virus y el mejor entendimiento en la fisiopatología de la enfermedad genera un sinnúmero de potenciales fármacos que han sido ensayados para el manejo de la enfermedad. en tiempos de pandemia, con una patología catastrófica en términos de vidas humanas y costos hospitalarios; es importante encontrar soluciones a desenlaces importantes como mortalidad, días de estancia en uci y en el hospital, aumento en los días libres del ventilador, disminución de complicaciones mayores debido a la enfermedad entre otros. hasta el momento no se ha documentado ninguna terapia específica que pueda impactar sobre estos desenlaces; pero la calidad de los trabajos, tampoco dejan claro sin él no usar ningún tratamiento específico mejora los desenlaces al menos al disminuir el número de complicaciones. este nuevo beta-coronavirus es similar al coronavirus del síndrome respiratorio agudo severa (sars-cov) y del síndrome respiratorio del medio este (mers-cov); por lo tanto, varias moléculas que habían sido evaluadas en este tipo de enfermedad rápidamente se abrieron paso a ensayos clínicos en paciente con covid-19. estos ensayos principalmente observacionales, aleatorios pero abiertos con un número pequeño de pacientes no han permitido sacar adecuadas conclusiones y es frecuente como ver las diferentes guías de las principales sociedades del mundo cambiar de forma frecuente sus recomendaciones; no existes evidencia de estudios clínicos aleatorios y controlados que midan desenlaces fuertes, la premura de un tratamiento efectivo ha sacrificado el rigor metodológico que una investigación requiere. una estructura viral y replicación conocidas generan posibles dianas para que diferentes fármacos puedan ser investigados, antivirales tipo arbidol el cual inhibe la fusión de la membrana en la envoltura viral a algunos receptores; antimaláricos como la hidroxicloroquina y la cloroquina, las cuales inhiben la entrada viral y endocitosis por múltiples mecanismos, así como los efectos inmunomoduladores demostrados en el huésped; antivirales que impiden la replicación como el lopinavir o darunavir inhibiendo las proteasas o la ribavirina, el remdesivir o el favipiravir que actúan como análogos de nucleótidos o fármacos que actúan modulando la respuesta específica del huésped como el tocilizumab el cual se une al receptor de la il-6 inhibiendo el punto de acción de esta; los corticosteroides con múltiples efectos en la modulación del sistema inmunológico del paciente o los fármacos para evitar la respuesta secundaria a esta cascada inflamatoria como son los anticoagulantes. por último, se han buscado estrategias con el fin de mejorar la inmunización pasiva del huésped en el uso del plasma de pacientes convalecientes o el uso de inmunoglobulinas enriquecidas entre otros tratamientos propuestos para esta enfermedad. como vamos a ver más adelante, actualmente no existe un tratamiento específico con el nivel de evidencia suficiente para recomendar de manera generalizada; tampoco existe suficiente evidencia del manejo del soporte básico sin el uso de fármacos dirigidos, que demuestre que esta estrategia se deba implementar de manera sistemática en todos los pacientes; por lo tanto, a continuación trataremos de resolver las inquietudes con respecto a los diferentes medicamentos que han sido usados en la pandemia del sars-cov-2/covid-19. se recomienda no utilizar antimaláricos tipo hidroxicloroquina (hcq) o cloroquina (cq) para el manejo de pacientes con infección por sars-cov-2/covid-19. para la fecha no hay un adecuado sustento bibliográfico que soporte el uso de antimaláricos en la prevención o manejo de pacientes con infección por sars-cov-2 tanto leve, moderada como severa. los mayores estudios no muestran utilidad clínica y tendencia a mayores eventos cardiovasculares con el uso de antimaláricos en pacientes con infección por covid-19 comparado con no darlo. su utilidad se deriva principalmente de resultados en estudios preclínicos e in vitro; como los presentados por wang y cols donde evaluaron 7 medicamentos de manera in vitro contra el covid-19, siendo el remdesivir y la cq efectivos de manera in vitro contra el nuevo coronavirus(424); liu y cols, donde la hcq fue efectiva en inhibir la infección por sars-cov-2 in vitro que junto con su potencial antiinflamatorio tenía potencial para el uso clínico (425) y yao y cols, donde la hcq fue más potente que la cq para inhibir el sars-cov-2 in vitro y fue recomendado para el uso en humanos en dosis de 400 mg dos veces al día por el primer día, seguido de 200 mg dos veces al día por 4 días más mantendría la concentración efectiva del fármaco en el tejido pulmonar (426) . los pocos estudios clínicos, son de baja calidad y no han mostrado mejoría ni eficacia en el uso de antimaláricos para el manejo de paciente adultos con covid-19, algunos estudios iniciales con pocos pacientes con resultados favorables (427) e incluso con recomendaciones para uso en las primeras versiones de guías internacionales para la amci ® hcq y cq, encontrando superioridad en estudios observacionales, de pocos pacientes, sin comparadores para inhibir la exacerbación de la neumonía, hallazgos de las imágenes pulmonares, promover una conversión negativa al virus y acortar el curso de la enfermedad; la cq tuvo un efecto notable tanto en términos de resultado clínico como de eliminación viral (428) ; considerando la hcq y la cq como un tratamiento costo efectivo (429) . estudios posteriores con un mayor número de pacientes no han logrado reproducir los estudios preclínicos iniciales; mahévas y cols, evaluaron la efectividad de la hcq en 181 pacientes admitidos a cuatro hospitales en francia, con neumonía por covid-19 quienes requieren oxígeno, pero no se encontraban en uci, comparado con una población con manejo estándar; la hcq se usó a dosis de 600 mg día en las primeras 48 horas a la admisión, este estudio no soporte el uso de la hcq en pacientes admitidos al hospital con covid-19 que requieren oxígeno al no reducir de forma significativa la admisión a la uci, el sdra o muerte en el día 7 después del ingreso (430) ; por el contrario, se han reportado efectos secundarios frecuentes (prolongación del intervalo qt, hipoglucemia, cambios en el estado mental, alteraciones gastrointestinales y retinopatía); silvia borda y cols, evaluó la seguridad y eficacia de dos dosis de cq en pacientes con covid-19 severo en un estudio aleatorizado, doble ciego fase iib en 81 pacientes adultos hospitalizados con infección por sars-cov-2, los pacientes fueron expuestos a dosis altas de cq (600 mg dos veces al día por 10 días) o dosis bajas (450 mg dos veces al día en el día 1 y una vez al día por 4 días), los hallazgos preliminares de este estudio sugieren que la dosis más alta de cq no debe recomendarse para pacientes críticos con covid-19 debido a sus posibles riesgos de seguridad, especialmente cuando se toman simultáneamente con azitromicina y oseltamivir; estos hallazgos no pueden extrapolarse a pacientes con covid-19 no severo (431); tang y cols, evaluaron la eficacia y seguridad de la hcq con el manejo estándar en un estudio multicéntrico, abierto, aleatorio y controlado en china, 150 pacientes con covid-19 positivo se incluyeron en el análisis de intención a tratar (75 en el grupo de hcq y 75 en el grupo estándar), la hcq fue administrada a dosis de 1200 mg día por tres días y mantenimiento con dosis de 800 mg día (duración del tratamiento: dos a tres semanas en pacientes con enfermedad leve a moderada o enfermedad severa respectivamente); la administración de hcq no resultó en una significativa mayor probabilidad de conversión negativa comparado con el tratamiento estándar, los efectos adversos fueron mayores en el grupo de hcq (432) . con todo esto la hcq y la cq, si se usan deberá ser bajo estudios experimentales aprobados con una estricta monitorización y vigilancia clínica de la frecuencia cardíaca y el intervalo qt, los niveles de glucosa, la función hepática y renal, y el cribado clínico de trastornos mentales y visuales en pacientes que reciben estos fármacos. debe evitarse hcq/cq en pacientes con enfermedades cardiovasculares subyacentes. nuevas evidencias con un mayor número de pacientes podrían sacar la hcq y la cq inclusive de estudios clínicos; barbosa y cols, evaluaron en un estudio cuasialeatorio comparativo el uso fuera de registro de la hcq en pacientes positivos por el sars-cov-2, el pronóstico primario fue la necesidad de escalar el soporte ventilatorio, cambio en el conteo de linfocitos o cambio en el índice de neutrófilos/linfocitos, un total de 63 pacientes fueron incluidos, 32 en el brazo de hcq. la administración de hcq fue asociada con la necesidad de aumentar el nivel del soporte ventilatorio comparado con aquellos que no recibieron hcq al día 5 del estudio, no hubo beneficios en la mortalidad, reconstitución inmunológica y riesgo de intubación (433) . el estudio con un mayor número de pacientes proviene de la ciudad de new york; geleris y cols, examinaron la asociación entre el uso hcq y la intubación o muerte en un centro médico de ny, se analizaron 1. amci ® estaban más enfermos en términos de oxigenación, en este estudio observacional la administración de hcq no fue asociada con una disminución en el riesgo compuesto de intubación o muerte (434) . se recomienda no utilizar antimaláricos tipo hidroxicloroquina (hcq) o cloroquina (cq) en combinación con azitromicina (az) para el manejo de pacientes con infección por sars -cov-2/covid-19. fundamento para la fecha la evidencia no favorece el uso combinado de los antimaláricos en combinación con la azitromicina; por el contrario, la combinación de estos dos medicamentos puede ser deletérea, inclusive con un aumento reportado en la mortalidad y la aparición de arritmias ventriculares de novo; estudios iniciales fueron promisorios, gautret y cols, evaluaron inicialmente el efecto de la hcq en la carga viral respiratoria en conjunto con el uso de azitromicina, la presencia del virus al día 6 fue el pronóstico primario; 20 pacientes con tratamiento mostraron una significativa reducción en la carga viral al día 6 de la inclusión comparado con los controles; la azitromicina adicionada a la hcq fue significativamente más eficiente en la eliminación viral (435) ; nuevamente gautret y cols, realizaron un estudio observacional, no controlado, no comparativo de 80 pacientes tratados con la combinación de hcq más azitromicina, presentando una mejoría significativa en disminución de la carga nasofaríngea del virus y una menor tiempo de enfermedad (436) ; luego million y cols, evaluaron la combinación de hcq y az en un estudio retrospectivo de 1.061 pacientes con sars-cov-2 tratados con hcq (200 mg tres veces al día por 10 días) + az (500 mg en el día 1, seguido de 250 mg al día por los próximos 4 días), el pronóstico fue mortalidad, empeoramiento clínico (ingreso a uci) o persistencia viral; la administración de hcq+az en combinación antes de que aparecieran las complicaciones del covid-19 es segura y asociada a una baja mortalidad en los pacientes (437) ; soportado además por estudios in vitro que demuestran que la combinación de hcq y az tienen efectos sinérgicos para el sars-cov-2 a concentraciones compatibles con las que se obtienen en pulmones humanos (438) . otros estudios por el contrario no han encontrado resultados positivos, es así como, magagnoli y cols, en un análisis retrospectivo de pacientes confirmados con infección por sars-cov-2 en centros de veteranos de los eeuu, un total de 368 pacientes fueron evaluados (hcq, n=97; hcq+az, n=113; no hcq, n=158), en este estudio no hubo evidencia que el uso de la hcq tanto sola o en combinación con la az, redujo el riesgo de ventilación mecánica en pacientes hospitalizados con covid-19; una asociación con un aumento en la mortalidad fue identificada en pacientes tratados con hcq sola (439) . nuevamente los estudios con un mayor número de pacientes se encuentran en la ciudad de new york; rosenberg y col, describieron la asociación entre hcq, con o sin az en el pronóstico de pacientes hospitalizados con covid-19; un estudio de cohorte multicéntrico retrospectivo en pacientes hospitalizados con covid-19 en 25 hospitales de ny, los pacientes recibieron ; en pacientes hospitalizados en el área metropolitana de ny con covid-19, el tratamiento con hcq, az o ambos, comparado con ningún tratamiento, fue no significativamente asociado con diferencias en la mortalidad hospitalaria (440) . recomendación se recomienda no utilizar de forma rutinaria el uso rutinario del lopinavir/ritonavir para el manejo de pacientes con infección por sars-cov-2/covid-19. en la actualidad no existe evidencia a favor o en contra en el uso del tratamiento con antirretrovirales con lopinavir/ritonavir en el manejo de pacientes adultos hospitalizados con covid-19; no se observó ningún beneficio con lopinavir/ritonavir más allá de la atención estándar. se está en espera de cierre de diferentes ensayos futuros que confirme o excluyan el uso de lopinavir/ritonavir en el paciente covid-19. en diciembre de 2019, un nuevo coronavirus, designado sars-cov-2, ha causado una pandemia (115, 142, 174, 441) ; cuando hablamos de enfermedad producida por covid-19 hablamos de enfermedades que van desde las enfermedades leves autolimitantes del tracto respiratorio hasta neumonía rápidamente progresiva, neumonía grave, falla multiorgánica y muerte. hasta este momento no existen agentes terapéuticos específicos para las infecciones por coronavirus. después de la aparición del síndrome respiratorio agudo grave (sars) en 2003, entre los fármacos aprobados se identificó lopinavir, un inhibidor del aspartato proteasa tipo 1 del virus de inmunodeficiencia humana (vih), que tiene actividad inhibitoria in vitro contra el sras-cov, el virus que causa el sars en los seres humanos y el ritonavir combinado con lopinavir para aumentar su vida media plasmática a través de la inhibición del citocromo p450 (442) . se comenzaron estudios evaluando la respuesta antiviral in vitro de la combinación de lopinavir/ritonavir y ribavirina en 41 pacientes con sars; comparados con 111 pacientes tratados con ribavirina sola, que sirvieron como controles históricos; el pronóstico adverso (sdra o muerte) fue significativamente más bajo en el grupo de tratamiento comparado con los controles históricos (2.4% vs 28.8%, p = 0.001) al día 21 del inicio de los síntomas; una reducción adicional en el uso de esteroides y de infecciones nosocomiales fue vista en el grupo de tratamiento con una disminución en la carga viral y aumento en el conteo de linfocitos (443) ; del mismo modo, el lopinavir tiene actividad, tanto in vitro como en modelo animal, contra el coronavirus del síndrome respiratorio de oriente medio (mers-cov) (444) . estos estudios previos son el soporte inicial para el uso del lopinavir/ritonavir en la epidemia del covid-19; cao y cols, en mayo de 2020 publicaron en china, un estudio controlado, aleatorizado en pacientes hospitalizados con prueba ; los efectos adversos gastrointestinales fueron más comunes con el lopinavir-ritonavir, pero los eventos adversos serios fueron más común con el grupo control; el tratamiento con lopinavir-ritonavir fue suspendido en 13 pacientes (13.8%) secundario a los eventos adversos (445) . otro estudio hung y cols, en hong kong, evaluaron en un trabajo multicéntrico, prospectivo, aleatorizado, fase 2 la eficacia y seguridad de la terapia combinada por 14 días de lopinavir 400 mg y ritonavir 100 mg cada 12 h, ribavirina 400 mg cada 12 h y tres dosis de 8 millones de ui de interferón beta-1b en días alternos en pacientes con covid-19 comparado con lopinavir/ritonavir cada 12 h (grupo control); el resultado primario fue tiempo en la negativización de la pcr viral en el hisopado nasofaríngeo en paciente con covid-19; 127 pacientes fueron ingresados, 86 en el grupo de combinación y 41 en el grupo control; en el grupo de intervención de forma significativa se negativizo la prueba de pcr de forma más rápida (7 días [iqr 5-11]) que el grupo control (12 días [8] [9] [10] [11] [12] [13] [14] [15] ; hr 4.37 [ic 95% 1.86-10.24], p = 0.0010); los eventos adversos fueron similares entre los grupos; ningún paciente murió durante el estudio (446) . por último, un pequeño estudio de zhu y cols en china, con 50 pacientes con sars-cov-2; evaluaron de forma retrospectiva los efectos antivirales y seguridad del lopinavir/ritonavir y el arbidol (antiviral aprobado en china y rusia para el sasr y la influenza), 34 pacientes en el grupo de lopinavir/ritonavir y 16 en el grupo de arbidol; los pacientes lopinavir/ritonavir presentaron un mayor tiempo para la negativización de la prueba de pcr viral (p < 0.01)(447). se recomienda no utilizar de forma rutinaria remdesivir como antiviral para el manejo de pacientes con infección por sars-cov-2/covid-19. se debe considerar su uso en escenarios de estudios de investigación clínica aprobados. actualmente no hay disponibilidad del medicamento en el país (colombia) por lo cual no se incluye en los protocolos de manejo de paciente con covid-19. en estados unidos el primer paciente con covid-19 mostró una mejoría significativa de sus síntomas con 24 horas de tratamiento con remdesivir (448) , lo que abrió la puerta a un nuevo tratamiento para el sars-cov-2; el remdesivir (gs-5734) es un análogo de los nucleótidos que inhibe la rna polimerasa; con un amplio espectro antiviral, puede inhibir la replicación de múltiples coronavirus en las células epiteliales del sistema respiratorio (449) ; estudiado (451) . por último, un estudio publicado por antinori y cols, en milán, italia; de manera prospectiva (compasional) incluyó pacientes con neumonía por sars-cov-2 mayores a 18 años bajo ventilación mecánica o con una saturación de oxígeno ≤ 94% al aire ambiente o un puntaje del national early warning score 2 ≥ 4; el pronóstico primario en cambio en el estado clínico en una escala ordinal de 7 categorías (1 = no hospitalizado de regreso a sus actividades diarias normales; 7 = muerte); de los 35 paciente ingresados, 18 se encontraban en uci y 17 en un piso de hospitalización de enfermedades infecciosas; un curso de 10 días de remdesivir fue completado por 22 pacientes (63%) y suspendido en 13, de os cuales 8 (22.8%) se descontinuo por eventos adversos; a los 28 días, 14 (82.3%) pacientes de piso fueron egresados, 2 permanecían hospitalizados y uno murió (5.9%), en la icu 6 (33.3%) fueron egresados, 8 (44.4%) pacientes murieron, 3 (16.7%) aún se encontraban en ventilación mecánica y 1 (5.6%) estaba con mejoría pero aún hospitalizado; la hipertransaminasemia y la injuria renal aguda fueron los eventos adversos más frecuentes reportados (42.8% y 22.8%, respectivamente); los datos sugieren que el remdesivir puede beneficiar a pacientes con neumonía por sars-cov-2 hospitalizados por fuera de la unidad de cuidado intensivo (452). page se recomienda no utilizar de rutina la ivermectina para el manejo de pacientes con infección por sars-cov-2/covid-19. para la fecha no se cuenta con la suficiente evidencia para emitir una recomendación para el uso de la ivermectina en pacientes con covid-19, en estudios iniciales in vitro, caly y cols, demostró como la ivermectina, una droga autorizada por la fda como antiparasitario tiene un efecto antiviral de amplio espectro de manera in vitro, con una reducción significativa de la replicación viral en modelos experimentales (453) , un estudio aún sin publicar, observacional multicéntrico de casos y controles (n: 704 casos y n: 704 controles), realizado entre el 1 de enero y 31 de marzo de 2020, incluyó pacientes diagnosticados con covid-19 confirmados por laboratorio, la dosis fue de 150 mcg/kg de ivermectina más la terapia médica de soporte en comparación con terapia médica sin ivermectina, el resultado principal fue la medición de supervivencia; en pacientes que requirieron ventilación mecánica, la mortalidad fue menor en el grupo de ivermectina (7,3% y 21,3% respectivamente) y las tasas de mortalidad global fueron más bajas con ivermectina ( no se puede emitir una recomendación a favor o en contra sobre el uso compasivo o rutinario de tocilizumab en pacientes con infección por sars-cov-2/covid-19. en pacientes individualizados se ha reportado desenlaces clínicos favorables. se puede considerar su uso en pacientes que cumplan con todos los siguientes criterios: ver tabla 16. el tocilizumab (tcz) un anticuerpo monoclonal humanizado igg1k, el cual se puede unir de manera específica a los receptores solubles de membrana para la il-6 (sil-6r and mil-6r) y ha sido ampliamente usado en el tratamiento de enfermedades autoinmunes, tales como la artritis reumatoide, la enfermedad de still del adulto o vasculitis de grandes vasos (459) . un primer estudio de xiaoling xu y cols, describió en china, 21 pacientes tratados con tocilizumab, se documentó una mejoría en los síntomas, en los requerimientos de oxígeno y en los hallazgos imagenológicos de la tomografía de tórax; los niveles promedio de il-6 antes de la terapia fueron de 132.38 pg./ml; todos los pacientes recibieron lopinavir y metilprednisolona antes de la terapia. se trata de una serie con una muestra pequeña de pacientes en donde solo 4 (19%) pacientes estaban en condición crítica (148) . posteriormente luo y cols, en china, reportan el uso de tocilizumab en 15 pacientes, con mejoría en el aumento de la pcr en todos los pacientes, excepto uno, y una disminución de la il6. el nivel sérico de il-6 tendió a aumentar inicialmente y luego disminuyó en 10 pacientes. los niveles medios de il-6 antes de la terapia fueron de 111.05 pg./ml; 53% de los pacientes recibieron metilprednisolona; el 20 % fallecieron. es otra serie pequeña, con 7 pacientes (46,6%) en condición crítica (147) . roumier y cols, en francia, estudiaron 30 pacientes que recibieron tcz, observando que se redujo la necesidad de ventilación mecánica en comparación con los controles ( amci ® 0.001); en este estudio, no hubo diferencias en la reducción de la mortalidad y 7 pacientes (23%) se encontraban en uci (460) . klopfenstein y cols, también en francia, en un estudio retrospectivo de casos y controles, encontraron que pacientes que recibieron tcz (n=20), a pesar de tener más requerimiento de oxígeno, con resultados biológicos más pobres (mayor linfopenia y un nivel de pcr superior) al inicio del estudio que los pacientes sin tcz (n = 25), presentaron el objetivo combinado (ingreso a uci y mortalidad) menor que los pacientes sin la terapia (25% vs 72%, p = 0.002); es otra serie, que disminuye la necesidad de ventilación mecánica (0% vs 32%, p = 0.006) de manera significativa (461) . luego rimland ca y cols informan los primeros datos de 11 pacientes con covid-19 tratados con tcz en los estados unidos, 9 de ellos en ventilación mecánica; la pcr y el fibrinógeno mejoraron rápidamente, pero no hubo mejoría en otros marcadores o resultados clínicos. sólo a seis pacientes les tomaron niveles previos de il 6 y de ellos dos tenían niveles bajos (462) . en otro estudio mikulska y cols, próximo a salir en jama, en pacientes con sdra moderado a severo, hay mayor disminución de la mortalidad con el tratamiento combinado de tocilizumab y esteroides, en relación con cada una de estas terapias. por último, no todos los estudios han mostrado resultados positivos; kimmig y cols, de chicago (eeuu), en 28 de los 60 pacientes críticos con covid-19, que recibieron tcz, se asoció con una mayor incidencia de infecciones bacterianas secundarias, incluida la neumonía asociada al ventilador (64.3% vs. 31.3% p = 0.010) (463) . posterior a la recomendación hay nueva evidencia publicada y estudios aún sin publicar que puede soportar el uso de los inhibidores de la il-6 en pacientes con sars-cov-2/covid-19; morena y col, en un estudio de tocilizumab como uso "off-label" en el tratamiento de neumonía por sars-cov-2 en milán, italia, este estudio abierto, prospectivo, describe las características clínicas y el pronóstico de 51 pacientes con covid-19 confirmado y severo tratados con tcz iv, todos los pacientes, presentan niveles plasmáticos elevados de il-6 (> 40 pg/ml) y saturación de oxígeno < 93% al aire ambiente, 45 pacientes (88%) se encontraban con sistema de alto flujo de oxígeno y 6 en ventilación invasiva, a los 7 días luego del tratamiento se observa una caída dramática en la temperatura corporal y la pcr, con un incremento significativo en el conteo de linfocitos (p < 0.001); a los 34 días del tratamiento, 34 pacientes (67%) mostraron una mejoría en la severidad del cuadro; 31 fueron dados de alta; 17 (33%) mostraron empeoramiento de su cuadro clínico y de estos, 14 murieron (27%). la mortalidad fue significativamente asociada con el uso de ventilación mecánica al inicio (83.3% vs 20% de los pacientes en soporte de oxígeno no invasivo, p = 0.0001), el efecto adverso más frecuente reportado fue la elevación de las enzimas hepáticas (29%), trombocitopenia (14%) e infecciones bacterianas serias e infecciones fúngicas en un (27%); los autores concluyen que el tcz ejerce un rápido beneficio sobre los marcadores inflamatorios y la fiebre, aunque no se consiguió un impacto clínico sobre el pronóstico, el riesgo aumentado de infecciones severas no es despreciable (464) . capra y col, describieron 85 pacientes en un hospital de italia con neumonía por covid-19 y falla respiratoria sin soporte ventilatorio y al menos uno de los siguientes: frecuencia respiratoria ≥ 30 respiraciones/min, saturación ≤ 93% o pao2/fio2 <= 300 mmhg, los pacientes recibieron la terapia estándar para el momento (hidroxicloroquina, lopinavir y ritonavir) y fueron considerados el control; 62 pacientes recibieron tzc con 4 días de la admisión más el manejo estándar, los pacientes en el grupo de tratamiento mostraron de manera significativa una mayor sobrevida comparado con los pacientes control (hr para muerte, 0.035; 95% ic], 0.004 a 0.347; p = 0.004), ajustado para las características clínicas de base; 2 de 62 pacientes en el grupo de tcz y 11 de 23 en el grupo control murieron; 92% y 42.1% de los pacientes que se dieron de alta en el grupo de tcz y en el control se recuperaron; la función respiratoria mejoró en el 64.8% de los amci ® pacientes con tcz que aún se mantenía hospitalizados, donde el 100% de los controles empeoro y requirieron ventilación mecánica, dando al tcz un espectro positivo en términos de curso clínico y sobrevida en pacientes con covid (465). guaraldi y col; evaluaron el papel del tcz en reducir el riesgo de ventilación mecánica invasiva en pacientes con neumonía severa por covid-19 quienes recibían tratamiento estándar para el momento (hidroxicloroquina, azitromicina, antirretrovirales y heparinas de bajo peso molecular) en un estudio retrospectivo, observacional en bologna, reggio emilia y módena, italia; el tcz fue dado a dosis de 8 mg/kg de peso corporal de forma iv (con un máximo de 800 mg) en dos infusiones separadas 12 h o 162 mg sc administradas en dos dosis simultáneas, una en cada muslo (324 mg en total), cuando la formulación iv no se encontraba disponible; el pronóstico primario fue la combinación de ventilación mecánica invasiva o muerte; de 1351 pacientes ingresados, 544 (40%) tenían neumonía severa por covid-19 y fueron incluidos, 57 (16%) de 365 pacientes en el grupo estándar requirieron ventilación mecánica, comparados con 33 (18%) de 179 pacientes tratados con tcz (p = 0·41; 16 [18%] de 88 pacientes tratados iv y 17 [19%] de 91 pacientes tratados sc); 73 (20%) pacientes en el grupo estándar murieron, comparado con 13 (7%; p < 0.0001) pacientes con tcz (6 [7%] del grupo iv y 7 [8%] sc); luego de ajustar para sexo, edad, centro de reclutamiento, duración de los síntomas y puntaje de sofa, el tratamiento con tcz fue asociado con una reducción en el riesgo de ventilación mecánica invasiva o muerte (hr 0.61, 95% ic 0.40-0.92; p = 0.020); 24 (13%) de 179 pacientes tratados con tcz fueron diagnosticados con nuevas infecciones en comparación con 14 (4%) de 365 pacientes en el grupo estándar (p < 0.0001) (466) . campochiaro y col, en un solo centro evaluó la eficacia y seguridad del tcz en pacientes con covid-19 severo, se diseñó un estudio retrospectivo en pacientes con características de hiper-inflamación (definida como una elevación tanto en la pcr, ≥ 100 mg/l, normal <6 mg/l o ferritina ≥ 900 ng/ml, normal <400 ng/ml en presencia de un incremento en la dhl > 220 u/l), acompañado de un compromiso respiratorio severo, definido como hallazgos típicos en la radiografía y/o tomografía, la presencia de una saturación de oxígeno ≤ 92% al aire ambiente o una pao2:fio2 ≤ 300 mmhg ingresados a la uci, comparando pacientes con tcz iv al manejo estándar, 65 pacientes fueron incluidos de los cuales 32 fueron tratados con tcz; los pacientes se encontraban con alto flujo o ventilación mecánica no invasiva, a 28 días de seguimiento, 69% de los pacientes con tcz experimentaron mejoría clínica comparado con un 61%del tratamiento estándar (p = 0.61); la mortalidad fue 15% en el grupo de tcz y 33% en el grupo estándar (p = 0.15); la incidencia de infección y trombosis pulmonar fue similar en ambos grupos (467); somers y col, evaluaron un estudio observacional en pacientes con neumonía por covid-19 severo que se encontraban en ventilación mecánica, evaluando como pronóstico la probabilidad de sobrevida posterior a la extubación; 154 pacientes fueron incluidos, 78 recibieron tcz y 76 no; en los modelos ajustados, el tcz, fue asociado con una reducción del 45% en el riesgo de muerte [hr 0.55 95% ic 0.33 a 0.90]; aunque el tcz, fue asociado con un incremento en la proporción de pacientes con superinfecciones (54% vs. 26%; p < 0.001), no hubo diferencias en la mortalidad a 28 días entre los pacientes tratados con tcz con o sin superinfecciones [22% vs. 15%; p=0.42] (468). price y col, también publicaron un estudio observacional de pacientes hospitalizados con covid-19, los pacientes recibieron tcz si cumplían criterios del síndrome de liberación de citoquinas, se evaluaron 239 pacientes; de los cuales 153 (64%) recibieron tcz, estos pacientes que recibieron ventilación mecánica la sobrevida fue del 75% (95% ic, 64-89), luego del tcz pocos eventos adversos fueron reportados y tanto la oxigenación como los biomarcadores de inflamación mejoraron (469) y por último, en un estudio preliminar con datos aún sin publicar perrone y col, evaluaron la eficacia del tcz en pacientes con neumonía por covid-19, en un estudio amci ® multicéntrico fase 2 en italia; se utilizó tcz, a dosis de 8 mg/kg iv, una o dos administraciones con 12 horas de diferencia; 301 y 920 casos fueron disponibles para un análisis de intención a tratar, 67 pacientes murieron; las tasas de letalidad fueron de 18.4% (97.5% ic, 13.6-24.0, p = 0.52) y 22.4% (97.5% ic, 17.2-28.3, p < 0.001) a 14 y 30 días; el tcz redujo la tasa de letalidad a 30 días pero no a 14 días comparado con las esperadas sin presentar una toxicidad significativa; la eficacia fue más evidente en los paciente que no requerían ventilación mecánica (469). recomendación se recomienda no utilizar de rutina bloqueadores de interleuquina-1 (anakinra) en pacientes con infección por sars-cov-2/covid-19. aunque su ventaja está en el perfil de seguridad, su vida media corta (3 horas) y porque las infecciones oportunistas son raras ,no hay suficiente evidencia para emitir una recomendación sobre el uso de este medicamento; cavalli y cols, de milán, italia, realizaron un estudio de cohorte retrospectivo en pacientes con sdra moderado a severo con hiperinflamación (pcr ≥ 10 mg/dl, ferritina ≥ 900 ng/ml o ambos), manejados con ventilación mecánica no invasiva fuera de la uci y que recibieron tratamiento con hidroxicloroquina y lopinavir, los pacientes que recibieron anakinra, 5 mg/kg dos veces al día intravenosa (n= 29, dosis alta) o 100 mg dos veces al día subcutánea (n= 7 pacientes, dosis baja) fueron comparados con una cohorte retrospectiva que no recibió anakinra (tratamiento estándar); la duración del tratamiento se prolongó hasta el beneficio clínico sostenido (reducción del 75% en la pcr, y una pafi > 200, durante al menos 2 días consecutivos) o hasta la muerte, bacteriemia, o efectos secundarios (alt > 3 veces valores de referencia); el tratamiento con anakinra a bajas dosis se interrumpió después de 7 días debido a la escasez de efectos sobre la pcr y el estado clínico. a los 21 días, el tratamiento con dosis altas de anakinra se asoció con una reducción en la pcr y mejoría en la función respiratoria en 21 de 29 pacientes (72%); en el grupo estándar, ocho de 16 pacientes (50%) mostraron mejoría respiratoria a los 21 días. en 21 días de seguimiento, la sobrevida fue del 90% en el grupo de dosis altas de anakinra y del 56% en el grupo estándar (p = 0.009). se trata del primer estudio que demuestra seguridad y mejoría en los pacientes covid-19, pero en el contexto fuera de la uci (470) . huet y cols, de parís, francia, realizaron el estudio llamado ana-covid-19 en el que compararon 52 pacientes tratados con anakinra subcutánea 100 mg dos veces al día durante 72 h, luego 100 mg diarios durante 7 días, con 44 pacientes históricos; su criterio de inclusión fue tener una saturación de oxígeno del 93% o menos con un soporte de mínimo de 6 l / min de oxígeno. la admisión a la uci por ventilación mecánica invasiva o muerte se produjo en 13 (25%) pacientes en el grupo de anakinra y 32 (73%) pacientes en el grupo histórico (hr 0. amci ® pacientes en el grupo histórico tuvieron un aumento en las aminotransferasas hepáticas (471). recomendación se sugiere que la terapia con interferón sólo sea considerada en pacientes con formas graves de infección por covid-19 en el marco de un estudio clínico. estudios preliminares muestran que el virus del covid-19 induce una expresión muy débil de interferones en las células infectadas, lo que obstaculiza la respuesta inmune innata temprana a la infección y sugiere que el uso de interferón (ifn) exógeno para estimular la inmunidad antiviral (473) . zhou q y col, en china, en un estudio observacional de 70 pacientes con covid-19 con gravedad mixta proporcionó evidencia de muy baja calidad que la adición de interferón-α a la terapia con umifenovir no afecta el tiempo de eliminación viral o la duración en la estancia hospitalaria cuando se comparó con el umifenovir solo (474) . hung if y cols, de hong kong, realizaron un ensayo multicéntrico, en adultos con covid-19, en los que 86 pacientes recibieron una combinación de lopinavir/ritonavir y tres dosis de 8 millones de unidades internacionales (0·25 mg) de interferón beta-1b en días alternos (grupo de combinación) y 41 pacientes recibieron lopinavir/ritonavir (grupo control); la terapia de combinación fue segura y superior al control, para aliviar los síntomas y acortar la duración de la eliminación del virus y la estancia hospitalaria; se trata de un estudio fase 2, en pacientes con covid-19 leve a moderado (ningún paciente con ventilación en el grupo de combinación), en el que el ifn se administró en los primeros 7 días de inicio de los síntomas y con el uso de un análogo de nucleósido oral (ribavirina), que no está en nuestras guías (446) . se necesitan estudios de ifn solo o combinado en pacientes críticos con covid-19. los efectos adversos de los ifn tipo i pueden limitar su uso para una intervención generalizada, como se propone en el brazo ifn-β con lopinavir/ritonavir del ensayo solidaridad de la oms. la administración por inhalación de vapor que se realiza actualmente en china ofrece la ventaja de acceso rápido al tracto respiratorio; sin embargo, la farmacodinamia y la farmacocinética de este modo de administración nunca se han evaluado. se sugiere no utilizar de rutina corticoides en el tratamiento de pacientes con sospecha o diagnóstico de covid-19. no se puede considerar su uso profiláctico ni en pacientes con enfermedad leve sin requerimiento de oxígeno. débil en contra page en diciembre de 2019, una serie de casos de neumonía de causa desconocida surgió en wuhan, hubei, china, con presentaciones clínicas muy parecidas a una neumonía viral; los análisis de secuenciación profunda de muestras del tracto respiratorio inferior indicaron un nuevo coronavirus, que se denominó novel coronavirus 2019 (covid-19-2019) (115) . en la actualidad, en ausencia de terapia preventiva para sars-cov-2, la piedra angular de atención para pacientes con covid-19 sigue siendo el manejo de apoyo, que va desde el tratamiento ambulatorio sintomático hasta el tratamiento intensivo completo con medidas de soporte en cuidados intensivos (475) . dentro del manejo farmacológico se ha planteado la opción del uso de corticoides, la justificación estaría basada en la disminución de la respuesta inflamatoria del huésped a nivel pulmonar; es decir, un efecto inmunomodulador, ya que esta infección puede conducir a un síndrome de distrés respiratorio agudo (sdra); sin embargo, el beneficio puede verse superado por los efectos adversos, incluido el retraso en el aclaramiento viral y mayor riesgo de infección secundaria. a pesar de que la evidencia directa de corticoides en covid-19 es limitada, revisiones de los resultados en otras neumonías virales nos podrían orientar en principio en esta actual situación (476) . teniendo en cuenta lo anterior; stockman y cols, en el 2.006, realizaron una revisión sistemática sobre ensayos en pacientes con sars; quince ensayos examinan el uso de corticoides con diez o más pacientes en tratamiento; 13 ensayos también recibían ribavirina; trece de estos estudios no fueron concluyentes; dos estudios describen un daño potencial con el uso de esteroides; en la literatura china estos autores encontraron catorce estudios con uso de esteroides en sars; doce fueron suspendidos por posible daño, la mayoría de estos ensayos se realizaron con muestras pequeñas de pacientes y de manera retrospectiva (477) . arabi y cols, en noviembre 21 de 2.017, realizaron un estudio multicéntrico de cohorte retrospectivo en 14 hospitales de atención terciaria de arabia saudita, donde se incluyeron 309 pacientes; el uso de corticoides en pacientes con mers no se asoció con un cambio significativo a los 90 días en la mortalidad y se documentó un retraso en la eliminación del arn de mers-cov (478) . en cuanto hace referencia a la situación actual de pandemia por sars-cov-2 y compromiso pulmonar; wu y cols, en marzo de 2.020 realizaron un estudio retrospectivo de 201 pacientes con covid-19 en china; para aquellos pacientes que desarrollaron sdra, el tratamiento con metilprednisolona estuvo asociado con una disminución del riesgo de muerte (23/50 [46%] con esteroides vs 21/34 [62%] sin esteroides; hr, 0.38 [ic 95%, 0.20-0.72]), con las limitaciones de los estudios retrospectivo, de un solo centro, con un limitado número de pacientes (400). zha y cols, en marzo de 2.020, describen el uso de corticosteroides en el tratamiento de pacientes con covid-19; no hallaron asociación entre la terapia con esteroides y el pronóstico de los pacientes sin sdra, siendo un estudio con una serie muy pequeña de pacientes (479) . yang y cols, en marzo de 2.020, en una revisión sistemática y meta-análisis que incluyó 5.270 pacientes de 15 estudios, describen como el tratamiento con corticoides estuvo asociado con una mayor mortalidad (rr = 2.11, ic 95% = 1.13-3.94, p = 0,019), mayor estancia (wmd = 6.31, ic 95% 5.26-7.37, p = <0,001) y una mayor tasa de infección bacteriana (rr = 2.08, ic 95% 1.54-2.81, p = <0.001); con algunas limitaciones en este metaanálisis, la mayoría de los estudios incluidos son estudios de cohorte retrospectivos, controles históricos, con un bajo nivel de evidencia y una falta de ensayos controlados aleatorizados con buen diseño, sin un estándar uniforme para el tiempo y la dosis de los corticoides utilizado en los estudios; los efectos de los corticosteroides pueden ser influenciado también por otras opciones terapéuticas, como los medicamentos antivirales (480) . por último, li y cols, en mayo de 2.020, en otra revisión sistemática y metaanálisis, con respecto al uso de corticosteroides en sujetos con amci ® infecciones por sars-cov-2, sars cov y mers-cov, se determinó que hubo retraso en la eliminación del virus sin mejoría en la supervivencia, reducción en la duración de la hospitalización o tasa de admisión en la uci y/o uso de ventilación mecánica; presentándose varios efectos adversos. debido a la preponderancia de los estudios observacionales en el conjunto de datos y los sesgos de selección y publicación, se concluye especialmente con respecto al sars-cov-2, que se necesita mayor investigación con ensayos clínicos aleatorizados. internamente en este meta-análisis sugiere precaución al usar esteroides en pacientes con covid-19 (481). horby y col en una rama del ensayo de evaluación aleatorizada de la terapia covid-19 (recovery), estudio aleatorizado, controlado, abierto que compara una gama de posibles tratamientos con la atención habitual en pacientes hospitalizados con covid-19, compararon el uso de la dexametasona a dosis de 6 mg día (oral o intravenosa) una vez al día por 10 días o el alta según lo que ocurriera primero contra el manejo habitual; en 2104 pacientes aleatorizados que recibieron dexametasona se compararon con 4321 pacientes en manejo estándar; 454 (21.6%) pacientes en el grupo de dexametasona y 1065 (24.6%) pacientes en el grupo control murieron a los 28 días, con un riesgo relativo ajustado para la edad (rr 0.83; 95% ic 0.74 a 0.92; p < 0.001). la mortalidad relativa y absoluta variaron significativamente en relación al soporte ventilatorio al momento de la aleatorización; la dexametasona redujo las muertes en una tercera parte de los pacientes que recibieron ventilación mecánica invasiva (29.0% vs. 40.7%, rr 0.65, 95% ic 0.51 a 0.82; p < 0.001), y una quinta parte en los pacientes que reciben oxígeno sin ventilación mecánica invasiva (21.5% vs. 25.0%, rr 0.80, 95% ic 0.70 a 0.92; p = 0.002), pero sin reducir la mortalidad en paciente que no recibieron soporte respiratorio al momento de la aleatorización (17.0% vs. 13.2%, rr 1.22, 95% ic 0.93 a 1.61]; p = 0.14) (482). se recomienda no utilizar plasma convaleciente como tratamiento de rutina en paciente con sars-cov2 -covid-19. se debe considerar su uso en el marco de un ensayo clínico y con alguno de los dos escenarios siguientes: escenario a (enfermedad severa), definida como uno o más de los siguientes: disnea, frecuencia respiratoria > 30/min, spo2 < 93%, pao2/fio2 < 300 o empeoramiento radiológico con aumento > 50% de los infiltrados pulmonares en 24 -48 horas. escenario b (enfermedad que amenaza la vida), definida como uno o más de los siguientes: falla respiratoria, choque séptico, o disfunción multiorgánica. se recomienda no utilizar plasma convaleciente para profilaxis clínica de rutina contra la infección por sars-cov-2, solo se debe considerar en el marco de un ensayo clínico. (485) . una revisión sistemática y meta-análisis exploratorio realizado en 2014 identificó 32 estudios de infección por coronavirus sars e influenza severa, el estudio reveló una reducción de la mortalidad, especialmente si el plasma convaleciente se emplea en la fase temprana de la enfermedad cuando se comparó con placebo o no tratamiento (or 0.25; ic del 95% 0.14 -0.45); sin embargo, hay que tener presente que los estudios son de baja calidad, carecen de grupos control y puede tener riesgo moderado a alto de sesgo (486) . se ha sugerido que el plasma convaleciente de pacientes que se han recuperado de covid-19 puede ser una terapia potencial, proporcionando inmunidad pasiva de los anticuerpos específicos contra sars-cov-2 y podría servir para prevenir y tratar la enfermedad (487) . las personas que se han recuperado de la infección por sars-cov-2 pueden generar anticuerpos neutralizantes (488, 489) que podrían tener aplicación en la prevención de infección en ciertos escenarios, como las personas con comorbilidades subyacentes que predisponen a enfermedad grave y aquellas con exposición de alto riesgo como los trabajadores de la salud y los expuestos a casos confirmados de covid-19. existen algunos riesgos asociados con el uso de plasma convaleciente, unos conocidos y otros teóricos; los riesgos conocidos son aquellos asociados con la transfusión de hemocomponentes, incluida la transmisión de virus (ej. vih, vhb, vhc, entre otros) (490); riesgo muy bajo, con los estándares de calidad actuales de los bancos de sangre; también se pueden presentar complicaciones no infecciosas, como las reacciones alérgicas, anafilaxia, reacción febril a la transfusión, lesión pulmonar aguda relacionada con la transfusión (trali), sobrecarga cardiaca asociada a transfusión (taco) y hemólisis si se administra plasma abo incompatible (491) . los riesgos teóricos incluyen el empeoramiento de la infección dependiente de anticuerpos (antibody-dependent enhancement of infection -ade); el ade puede ocurrir en varias enfermedades virales e implica una respuesta inflamatoria exagerada ante la presencia de ciertos anticuerpos (492) . otro riesgo teórico es que la administración de anticuerpos a las personas expuestas al sars-cov-2 puede evitar la enfermedad, pero modifica la respuesta inmune de tal manera que esos individuos monten respuestas inmunes atenuadas, lo que los haría vulnerables a la reinfección posterior, si se comprueba que este riesgo es real estos individuos podrían ser vacunados contra covid-19 cuando exista una vacuna disponible (487) . durante el brote actual en china, se utilizó plasma convaleciente en algunos pacientes con covid-19 (493), desde esta publicación se identificaron 9 publicaciones relacionadas con el tema, entre todos los estudios fueron tratados con plasma convaleciente un total de 5063 pacientes(494-502) (tabla 17). shen y cols en marzo 2020 describieron en china el primer reporte en el cual el plasma convaleciente puede ser una opción de tratamiento en pacientes con covid-19; reportaron una serie de casos de 5 pacientes críticamente enfermos con covid-19 y síndrome de dificultad respiratoria aguda (sdra), todos en ventilación mecánica, a quienes se les administró plasma convaleciente con anticuerpos neutralizantes [10 a 22 días después del inicio de la enfermedad (ddie)], todos ellos posteriormente mostraron mejoría clínica, la carga viral de los pacientes disminuyó y fueron negativas en los 12 días posteriores a la intervención (494) . de forma similar, duan y cols en mazo 2020 en china reportaron mejoría clínica en una serie prospectiva de 10 casos de paciente severamente enfermos con covid-19, que recibieron plasma convaleciente con un tiempo medio de 16.5 ddie (11 a 20 días) después del inicio de los síntomas, e hicieron amci ® una comparación con un grupo control histórico comparables en edad, género y severidad de la enfermedad (495) . zhang y cols en mazo 2020, en china reportaron una serie de 4 casos de pacientes con covid-19 críticamente enfermos, 3 en falla respiratoria con ventilación mecánica y dos de ellos con ecmo (membrana de oxigenación extracorpórea), a quienes se les dio tratamiento con plasma convaleciente en un tiempo medio 15.5 ddie, posteriormente todos tuvieron mejoría clínica (496) . posteriormente ahn y cols en abril 2020 en corea, describieron una serie de dos casos de paciente con covid-19 severamente enfermos, en falla respiratoria y con ventilación mecánica, quienes además de recibir hidroxicloroquina, lopinavir/ritonavir y metilprednisolona, fueron tratados con plasma convaleciente entre 6 -10 ddie, ambos pacientes se recuperaron y fueron liberados de la ventilación mecánica, uno fue dado de alta al momento del reporte (497) . ye y cols en abril 2020 en china, describieron una serie de 6 pacientes con covid-19 con anormalidades imagenológicas y deterioro clínico a pesar del tratamiento estándar y con pcr para sars -cov-2 persistentemente positiva, aunque no estuvieron en falla respiratoria o con ventilación mecánica, de hecho, una de las pacientes era portadora asintomática; todos recibieron plasma convaleciente entre 32 a 49 ddie, en todos los pacientes, excepto 1, hubo resolución de los cambios de vidrio esmerilado y consolidación, todos mejoraron y fueron dados de alta (498) . zeng y cols en abril 2020 en china, reportaron una serie de 6 casos con covid-19 en falla respiratoria y se compararon con 21 controles que no recibieron plasma convaleciente por limitación en la disponibilidad y compatibilidad abo; a este grupo de paciente, se les administró plasma convaleciente en promedio 21.5 ddie, en todos los casos se negativizó la pcr para sars-cov-2 a los 3 días después del tratamiento; sin embargo, contrario a los reportes de los 5 estudios previos, en este grupo se murieron 5 pacientes (83% vs 93%, p = 0.184), pero tuvieron mayor porcentaje de aclaramiento del virus (pcr sars-cov-2 negativa 100% vs 21.4%, p = 0.005) antes de la muerte e incluso el tiempo de sobrevida fue mayor en el grupo de tratamiento (p = 0.029) (499) . salazar y cols en mayo 2020, en houston, texas, reportaron una serie de 25 pacientes con covid-19 severa o amenazante para la vida, el desenlace primario fue seguridad y el secundario fue el estado clínico de los pacientes al día 14 luego de la transfusión; al día 7 posttransfusión, 9 pacientes (36%) mejoraron con relación a su estado clínico basal, 13 (52%) no tuvieron cambios y 3 pacientes tuvieron deterioro clínico. siete de los nueve pacientes que mejoraron (28%) habían sido dados de alta; para el día 14 post-transfusión, 19 (76%) de los pacientes presentaron mejoría y 4 pacientes más, habían sido dados de alta; tres pacientes permanecían sin cambios, 3 pacientes se deterioraron y uno murió por una condición no relacionada con el plasma; el promedio de estancia hospitalaria fue de 14.3 días y la estancia hospitalaria luego de la transfusión fue en promedio 11 días; hubo una disminución de los valores promedio de pcr desde 14.66 mg/dl el día 0, a 2,9 mg/dl y 0.45 mg/dl los días 7 y 14 respectivamente; al momento de la publicación del artículo, solo permanecían intubados 2 pacientes; todos los pacientes que requirieron ecmo ya se habían liberado y 20 (80%) fueron dados de alta (500) . también en texas, estados unidos, ramachandruni y col en mayo 2020, reportaron una serie de 5 casos con covid-19 severa, falla respiratoria y en ventilación mecánica o pao2/fio2 < 100, todos con comorbilidades; a los cuales les administraron metilprednisolona y posteriormente plasma convaleciente; compararon los valores basales de pao2/fio2 y posterior a la intervención; encontrando, mejoría de la pao2/fio2 en 48% luego del tratamiento con esteroides y en 26% luego de la administración de plasma convaleciente (501) . finalmente, en respuesta al brote de covid-19 en los eeuu y las tasas de mortalidad reportadas, la fda en colaboración con la mayo clinic y la comunidad nacional de bancos de sangre desarrollaron un programa nacional de acceso ampliado para recolectar y distribuir plasma convaleciente donado por amci ® individuos que se han recuperado de covid-19; entre abril 3 y mayo 11, 2020; fueron incluidos 14.288 pacientes con covid-19 severa o potencialmente mortal o con riesgo alto de progresión a covid-19 severa o potencialmente mortal en el programa nacional de acceso ampliado; en ese tiempo, un total de 8.932 pacientes inscritos recibieron transfusión de plasma convaleciente covid-19. en una publicación reciente, joyner y cols en mayo 2020, en estados unidos, hicieron un análisis de seguridad después de la transfusión de plasma convaleciente covid-19 humano con compatibilidad abo en 5.000 adultos hospitalizados con covid-19 grave o potencialmente mortal, 66% de los cuales se encontraban en uci; la incidencia de eventos adversos serios (eas) durante las 4 horas siguientes a la transfusión de plasma convaleciente fue < 1%, incluyendo mortalidad (0.3%); de los 36 eas reportados, hubo 25 incidentes reportados como eas relacionados, incluyendo 4 muertes, 7 eventos de taco, 11 trali y 3 reacciones alérgicas graves asociadas a la transfusión; sin embargo, solo 2 (de 36) eas fueron considerados definitivamente relacionados con la transfusión de plasma convaleciente por el médico tratante; en este grupo de pacientes, la tasa de mortalidad a los siete días luego de la administración del plasma convaleciente, fue del 14,9% (502) . a pesar que la tasa general de letalidad para la covid-19 parece ser aproximadamente 6.4% (503), la tasa de mortalidad reportada parece no ser excesiva si la comparamos con los informes de wuhan que sugieren tasas de letalidad del 14% para los pacientes hospitalizados (504) y 57% entre los pacientes en unidad de cuidados intensivos (139) . los nueve estudios mostraron mejoría en muchos aspectos, incluyendo el aclaramiento del virus, la disminución del suplemento de oxígeno y la ventilación mecánica, la normalización de los valores de laboratorio, y la recuperación en los hallazgos pulmonares radiológicos. todos los estudios, reportaron que no se presentaron eventos de seguridad o reacciones adversas serias relacionadas con la administración de plasma convaleciente en pacientes con covid-19, excepto en 2 casos relacionados, según criterio de los médicos tratantes, en el estudio publicado por joyner y cols (502) . esta serie de estudios son alentadores; sin embargo, la mayoría de los reportes de casos tienen limitaciones significativas: carecen de los ajustes para factores de confusión críticos, incluidos los co-tratamientos, las características basales, la gravedad de la enfermedad y el momento de administración del plasma y deben ser seguidas de investigaciones adicionales. para establecer mejor el papel del plasma convaleciente es necesario realizar estudios dirigidos a los siguientes escenarios: 1. el uso como profilaxis post-exposición 2. evaluar si el plasma convaleciente es útil en paciente con enfermedad leve 3. el efecto del plasma convaleciente en pacientes con enfermedad moderada 4. el tratamiento de rescate con plasma convaleciente en pacientes que requieren ventilación mecánica debido a covid-19 5. finalmente, trabajos que evalúen la seguridad y farmacocinética del plasma convaleciente en los pacientes pediátricos con alto riesgo. actualmente están en curso varios estudios para evaluar el tratamiento de pacientes infectados con sars-cov-2 con plasma convaleciente. una búsqueda realizada el 23 de mayo de 2020 en clinicaltrials.gov con los términos "plasma convaleciente y covid-19" mostró 82 ensayos en curso sobre el uso de plasma convaleciente en pacientes con amci ® covid-19, que nos ayudarán a resolver las inquietudes relacionadas con esta intervención (505) . page se recomienda no utilizar de rutina las las inmunoglobulinas hiperinmunes en pacientes con infección por sars-cov-2/covid-19. la inmunoglobulina hiperinmune (h-igiv) se deriva de individuos con altos títulos de anticuerpos contra patógenos específicos y se ha utilizado con éxito en el tratamiento de infecciones, como el citomegalovirus y la gripe h1n1 (506) . se propone que la inmunoglobulina hiperinmune combinada con medicamentos antivirales puede ser efectiva en el tratamiento de pacientes con covid-19, estos anticuerpos (ac) recogidos de los pacientes recuperados serán específicos contra covid-19 al aumentar la respuesta inmune en pacientes recién infectados (507) . existe evidencia más sólida para el uso de h-igiv en el tratamiento de enfermedades virales. cheng y cols en enero de 2005, realizaron una revisión retrospectiva en hong kong, que reveló que el plasma convaleciente de los sobrevivientes de sars-cov administrados a pacientes con sars-cov que tenían enfermedad progresiva resultó en tasas de alta significativamente más altas en el día 22 y tasas de mortalidad más bajas, en comparación con los controles históricos (485) . hung y cols en febrero de 2011, realizaron un estudio de cohorte prospectivo sobre la efectividad del plasma convaleciente de los sobrevivientes de h1n1 con un título de ≥ 1:160 ofrecido a pacientes de la uci con infección grave por h1n1; los pacientes que rechazaron las infusiones de plasma convalecientes fueron controles; veinte de los 93 pacientes recibieron sueros convalecientes, el tratamiento con plasma convaleciente condujo a una reducción significativa de la carga viral respiratoria, los niveles séricos de citocinas (il-6, il-10, tnfα) y la mortalidad (484) . posteriormente hung y cols en agosto de 2013, publicaron un estudio multicéntrico prospectivo, doble ciego, aleatorizado y controlado en el que compararon la efectividad de la inmunoglobulina hiperinmune (h-igiv) del plasma convaleciente de los sobrevivientes de h1n1 versus la inmunoglobulina iv (igiv) normal, en pacientes con h1n1 en uci con soporte respiratorio y recibiendo oseltamivir; este estudio mostró, una reducción de la carga viral y una mayor supervivencia en el grupo que recibió h-igiv dentro de los 5 días posteriores al inicio de los síntomas, demostrando la superioridad de la inmunoglobulina hiperinmune sobre la igiv en el tratamiento de la infección grave por h1n1(508). el uso de inmunoglobulina hiperinmune ha demostrado una clara efectividad en el tratamiento de la gripe y el sars-cov; sin embargo, el plasma se debe recolectar y procesar de pacientes convalecientes y verificar que tenga títulos adecuados. según la experiencia con el sars-cov, lo ideal es recolectar plasma de pacientes con un curso de enfermedad más leve (506) . poco se sabe sobre la seguridad de la inmunoglobulina hiperinmune cuando se usa para el tratamiento de infecciones por coronavirus, los riesgos incluyen la exacerbación de la infección dependiente de anticuerpos (antibody-dependent enhancement of infection -ade)(509); el ade puede ocurrir en varias enfermedades virales, e implica una respuesta inflamatoria exagerada ante la presencia de ciertos anticuerpos; sin embargo, los estudios en sars y mers no proveen información suficiente para extrapolarse a la infección por sars-cov-2. no se encontraron estudios con inmunoglobulina hiperinmune en el tratamiento de pacientes con covid-19. no se puede emitir una recomendación a favor o en contra para el uso de la inmunoglobulina intravenosa como tratamiento adyuvante en pacientes con covid-19 severo. se debe considerar la inmunoglobulina intravenosa como tratamiento adyuvante en pacientes con covid-19 severo, en el contexto de estudios clínicos en los siguientes escenarios. escenario a (enfermedad severa), definida como uno o más de los siguientes: disnea, frecuencia respiratoria > 30/min, spo2 < 93%, pao2/fio2 < 300 o empeoramiento radiológico con aumento > 50% de los infiltrados pulmonares en 24 -48 horas. escenario b (enfermedad que amenaza la vida), definida como uno o más de los siguientes: falla respiratoria, choque séptico, o disfunción multiorgánica. en las enfermedades virales, los anticuerpos ejercen su efecto por neutralización viral (bloqueo de la entrada de células virales y, por lo tanto, replicación), activación del complemento, opsonización y mediación de citotoxicidad celular dependiente de anticuerpos. la neutralización viral es específica de antígeno; otras actividades antivirales son antígeno-inespecíficas y se realizan en parte a través de interacciones fc: fc receptor. en la infección por sars-cov-2, el principal antígeno objetivo asociado con la neutralización es la proteína spike, que es responsable de la unión del sars-cov-2 a las células epiteliales, incluidos los neumocitos; los anticuerpos en las inmunoterapias pasivas covid-19 son de naturaleza policlonal, con múltiples epítopos contra los paratopes de sars-cov-2, incluido el dominio de unión al receptor en la proteína spike (509) . la inmunoglobulina intravenosa (igiv) es un producto derivado del plasma de miles de donantes utilizados para el tratamiento de inmunodeficiencias primarias y secundarias, afecciones autoinmunes/inflamatorias, trastornos neuroinmunológicos y secuelas relacionadas con infecciones; la igiv proporciona protección inmune pasiva contra una amplia gama de patógenos; actualmente, la experiencia con el uso de igiv en el tratamiento de la infección por sars-cov-2 es muy limitada; sin embargo, la justificación del uso de ivig en la infección por sars-cov-2 es la modulación de la inflamación (506) . la igiv se ha usado en el tratamiento de otros coronavirus, incluido el sars cov. stockam y cols, en septiembre de 2006, en respuesta a una petición de "the world health organization -who", realizaron una revisión sistemática de los efectos del tratamiento en los pacientes con síndrome respiratorio agudo, incluida la igiv o el plasma convaleciente; se evaluaron cinco estudios sobre el uso de igiv o plasma convaleciente administrado además de corticosteroides y ribavirina, se consideró que estos estudios no fueron concluyentes ya que los efectos de la igiv o el plasma convaleciente no podían distinguirse de otros factores que incluían comorbilidades, estadio de la enfermedad o el efecto de otros tratamientos (477) . wnag y cols, en mayo 2004, hicieron un estudio prospectivo, en un solo centro, de infección por sars en taiwán, se administró igiv si el paciente tenía leucopenia amci ® o trombocitopenia, o si había progresión rápida de la enfermedad en la radiografía; un total de 40 pacientes recibieron igiv, de los cuales 22 tenían citopenias graves, uno de ellos tenía evidencia de síndrome hemofagocítico y 18 paciente tuvieron progresión radiológica de la enfermedad; el estudio sugiere que la igiv condujo a una mejora significativa en el recuento de leucocitos y plaquetas, pero reconoce que no había un grupo de control para evaluar objetivamente las respuestas (510) . lew y cols, en julio 2003, reportaron un estudio retrospectivo de un solo centro en singapur, se encontró que los pacientes adultos con sars tratados con un régimen de pulso de metilprednisolona (400 mg) e igiv (0.4 mg/kg) diariamente durante tres días consecutivos tuvieron una hazard ratio ajustada de 0.41 para mortalidad en comparación con el grupo no tratado, con una tendencia hacia una recuperación más temprana; sin embargo, este hallazgo no fue estadísticamente significativo (ic del 95%: 0.14 a 1.23; p = 0.11); además, este resultado tuvo como factor de confusión el uso concurrente de esteroides (511) . aunque algunas de las preparaciones de igiv comercializadas actualmente (gamunex-c y flebogamma) contienen anticuerpos que reaccionan de forma cruzada contra el sars-cov-2 y otros antígenos de virus, in vitro(512), hasta la fecha, ningún ensayo clínico de alta calidad ha demostrado eficacia y seguridad convincentes de igiv en epidemias de coronavirus. a pesar de que los datos para el uso de igiv en la infección por sars y mers son débiles, la dosis alta de ivig puede ser útil en la infección grave por sars-cov-2 a través de la modulación inmune, saturando fcγr y reduciendo ade (506) . en general, la inmunoglobulina intravenosa es bien tolerada y el perfil de seguridad es bien conocido. las reacciones adversas comunes son leves y autolimitadas, pero se sabe que en pacientes de alto riesgo se producen efectos adversos graves, como trombosis, disfunción renal y muerte. en cuanto la evidencia (tabla 18); xie y cols en abril, en wuhan, china, realizaron un estudio retrospectivo, revisando 58 casos de covid-19 severa (disnea, fr > 30/min, spo2 < 93% en reposo, pao2/fio2 < 300, progresión imagenológica > 50% en 24 -48 horas) o críticamente enfermos (falla respiratoria con ventilación mecánica, choque, disfunción orgánica múltiple) en el cual evaluaron la mortalidad a 28 días como desenlace primario y como desenlace secundario evaluaron la mortalidad a 14 días, días de estancia hospitalaria, de uci y la necesidad de ventilación mecánica; reportaron que el tratamiento con igiv dentro de las 48 horas posteriores al ingreso no sólo redujo el uso de la ventilación mecánica comparado con el tratamiento luego de 48 horas del ingreso (6.67% vs 32.14%, p = 0.016), sino que también redujo la duración de la estancia en el hospital (11.50 ± 1.030 vs 16.96 ± 1.620 días, p = 0.0055) y la uci (9.533 ± 1.089 vs 13.50 ± 1.632 días, p = 0.0453); mejorando en última instancia la mortalidad a los 28 días (p = 0.0215); concluyen, que el estudio demostró que el tratamiento con igiv en pacientes con covid-19 con neumonía grave puede mejorar los indicadores en poco tiempo y mejorar la eficiencia del tratamiento de los pacientes con alta efectividad (513) . el tratamiento con dosis altas de igiv (25 g/día durante 5 días) al inicio del distrés respiratorio, sumado al tratamiento de soporte y en un caso combinados con antivirales (lopinavir/ritonavir) y metilprednisolona en covid-19 grave publicado por cao y cols en marzo, en wuhan, china (514), demostró la elevación de los recuentos de linfocitos, disminución de los marcadores inflamatorios, recuperación de la oxigenación, resolución parcial/completa de las alteraciones radiológicas pulmonares y las pruebas de hisopos nasales y orofaríngeos negativos dentro de unos pocos días después del inicio tratamiento (< 6 días). lanza y cols en mayo 2020, reportaron en nápoles, italia, el caso de una mujer de 42 años que tenía covid-19 severa y que venía con deterioro clínico a la cual se le venía dando tratamiento con hidroxicloroquina más azitromicina, no se le administraron amci ® esteroides por el riesgo de disminuir la depuración de la viremia, a quién se le administró igiv el día 16 después de iniciados los síntomas con mejoría clínica rápida, normalización de los gases arteriales y disminución marcada de los infiltrados pulmonares al día 5 y 8 respectivamente; como evento adverso, reportaron hipotensión durante el inicio de la infusión que se mejoró al disminuir la velocidad de infusión. la paciente finalmente se recuperó, negativizó rt-pcr sars-cov-2 y fue dada de alta (515) . se recomienda la tromboprofilaxis farmacológica en todos los pacientes confirmados o sospechosos de covid-19 severo, a menos que está contraindicada, en cuyo caso es razonable la implementación de profilaxis no farmacológica. en términos generales se reconoce que los pacientes hospitalizados con enfermedad médica aguda, incluidas infecciones como la neumonía, tienen un mayor riesgo de eventos tromboembólicos (516) . tang y cols, en china describieron múltiples trastornos de la coagulación en pacientes con covid-19, aquellos pacientes que no sobrevivieron tenían significativamente mayores niveles de dímero-d y productos de degradación de la fibrina y con tiempos de coagulación convencionales más alargados al ingreso (p < 0.05); 71.4% de los no sobrevivientes y 0.6% de los sobrevivientes cumplían criterios para coagulación intravascular diseminada(517); este mismo grupo realizó un estudio con 449 pacientes con covid-19 severo, pacientes con coagulopatía asociada a covid-19 (cac), definida por un sic score ≥4 la utilización de tromboprofilaxis redujo de manera significativa la mortalidad a 28 días (40.0% vs 64.2%, p = 0.029) así como también en aquellos con un se recomienda no utilizar de rutina antiagregación en pacientes con covid-19 severo con el fin de prevenir desenlaces neurológicos adversos. no se encuentran estudios en la literatura para el uso de antiagregantes para el manejo específico del covid-19; el manejo de los eventos cardiovasculares en pacientes covid-19 no difiere de la población general sin la enfermedad. no se establecen diferencias en los estudios descritos, ni en las publicaciones hasta la fecha de la revisión (518) . no se puede emitir una recomendación a favor o en contra sobre el uso de la anticoagulación terapéutica de rutina con heparinas de bajo peso molecular o heparina no fraccionada en pacientes con bajo riesgo de sangrado y con un curso clínico grave o crítico, que además tengan elevación del dímero d mayor a 1 mcg/ml (1000 ng/ml) y/o fibrinógeno mayor a 900 mg/dl. diferentes publicaciones describen como una estrategia de tratamiento basada en profilaxis con heparina de bajo peso molecular (hbpm) para tratar la coagulopatía grave por covid-19 podría no ser suficiente. especialmente porque estos pacientes tienen dentro de su coagulopatía, una predisposición mayor a la presencia de trombosis que al sangrado. además, los bajos niveles de antitrombina que se han descrito en estos pacientes, los hace más resistentes a la heparina, lo que sugiere que las dosis profilácticas ya sea de heparina no fraccionada o hbpm pueden ser inadecuadas(519-521). 135 asociación colombiana de medicina crítica y cuidados intensivos. amci ® no se puede emitir una recomendación a favor o en contra sobre la anticoagulación terapéutica de rutina con heparinas de bajo peso molecular en pacientes con covid-19 en estado crítico, que presenten elevación del dímero d mayor a 1 mcg/ml (1000 ng/ml). tang y cols, en china con un estudio retrospectivo donde se incluyeron 449 pacientes con covid-19 severo, se evaluó la presencia de trombosis como desenlace asociado. este estudio describe como niveles de dímero d por encima de 1500 ng/ml estaban asociados a una mayor probabilidad de muerte y en estos pacientes el tratamiento anticoagulante reduce de manera significativa la mortalidad (0.44, ic 95% 0.22-0.86) (156, 521) . no se puede emitir una recomendación a favor o en contra para la medición rutinaria de niveles de anti xa en pacientes con covid-19 en los que se decide hacer anticoagulación terapéutica con hbpm. se puede considerar la medición de niveles anti xa si se cuenta con la disponibilidad del recurso. harr y cols, en un estudio en donde se incluyeron pacientes con hiperfibrinogenemia relacionada a trauma, se evidenció como los niveles de fibrinógeno se correlacionaron significativamente con la consistencia del coágulo y adicionalmente como se genera una relación inversa entre los niveles de fibrinógeno y la actividad de las hbpm, lo que sugeriría una potencial resistencia a la heparina. basados en que los pacientes covid-19 presentan niveles de fibrinógeno en muchas ocasiones con niveles > 700 mg/dl e incluso >900 mg/dl lo que hace razonable considerar que aquellos pacientes covid-19 que requieren dosis terapéuticas de hbpm y es posible evaluar los niveles de anti xa, hacer ajustes y monitoreo del nivel de anticoagulación sería una opción razonable (522) . se recomienda el uso de hbpm o hnf para la anticoagulación terapéutica en pacientes con una indicación específica con diagnóstico de covid-19. amci ® como previamente se describió, una revisión sistemática comparó las dosis fijas de hbpm subcutánea con dosis ajustadas de hnf intravenosa o subcutánea en personas con clínica sugestiva de tep, esta revisión demostró que la incidencia de tep recurrente fue menor con hbpm que en los participantes con hnf (or 0.71, ic 95% 0.56 a 0,90), también se asoció con una reducción en el tamaño del trombo (or 0.71, ic 95% 0.61 a 0.82), evidencia de baja calidad. sin embargo, no hubo diferencias en la mortalidad general entre los participantes tratados con hbpm y los tratados con ufh (or 0.84, ic 95% 0.70 a 1.01). por otra parte, los protocolos de manejo en escenarios de coagulación intravascular diseminada (cid), proponen el uso de hnf por encima de la hbpm en pacientes en los que se indica la anticoagulación. más aún, la presencia de falla renal aguda es común en los pacientes con covid-19, por lo que la opción de tratamiento con hnf tiene también escenarios en donde podrían ser de elección (519, 523) . capítulo 8. procedimientos y covid-19 recomendación se recomienda realizar la preoxigenación en pacientes con sospecha o diagnóstico de covid-19, cuando estén disponibles, en áreas de presión negativa con mínimo 12 recambios de aire por hora o en instalaciones con ventilación natural o que tengan un recambio de aire de mínimo 160 l/seg si están disponibles. se recomienda como complemento opcional durante la preoxigenación en el paciente crítico con diagnóstico o sospecha de covid-19 la caja de acrílico para protección durante la intubación, la caja no protege contra la generación de aerosoles fuera de esta y requiere para su uso, entrenamiento previo. si es difícil su uso retírela inmediatamente. se recomienda utilizar en la mascarilla quirúrgica sobre la mascarilla de oxigenación en el paciente crítico con sospecha o diagnóstico de covid-19. se recomienda utilizar filtros hpfa entre la máscara y el dispositivo disponible para la preoxigenación en el paciente crítico con sospecha o diagnóstico de covid-19. se recomienda en el paciente con sospecha o diagnóstico de covid-19 preoxigenación por 3 a 5 min, si el paciente luego de 3 minutos no tiene incremento en la mejoría inicie la administración de medicamentos. se recomienda en caso de compromiso hemodinámico considerar ventilación a dos manos con cierre hermético de la máscara sobre la cara del paciente con frecuencias altas (>25 por minuto y baja presión). amci ® fundamento el manejo de la vía aérea es un procedimiento considerado generador de aerosoles, la enfermedad covid-19 tiene una alta tasa de transmisión y el personal de salud requiere el uso estricto del epp (524, 525) , revise el enunciado para epp recomendado en esta guía. la posibilidad de permanencia del virus en algunos ambientes puede durar hasta 4 horas, por esto una estrategia para proteger al equipo de salud y otros pacientes podrías ser estar en áreas con presión negativa, lo cual no es fácil de encontrar en nuestro contexto o que tenga un adecuado recambio de aire(526). como medidas complementarias se pueden utilizar opciones como las cajas acrílicas para intubación, esta disminuye el riesgo de contaminación por gotas, pero no elimina los aerosoles, es necesario previo entrenamiento. en caso de que la caja sea una limitante retírela inmediatamente (527) . los pacientes covid-19 , clásicamente se presentan con tos, esto es un factor de riesgo para quienes manipulan la vía aérea, la utilización de mascarilla por parte del paciente debajo del dispositivo bolsa mascarilla , la cual también deberá tener un filtro de alta eficiencia para disminuir el número de partículas que pueden estar en el ambiente durante la preoxigenación y posible ventilación, ésta última la cual será evitada al máximo (524, 526, 528) . la preoxigenación es una técnica que pretende barrer el nitrógeno y aumentar la disponibilidad de oxígeno para que cuando el paciente presente apnea por los medicamentos para la intubación o por su condición clínica, se disponga de un tiempo mayor sin desaturación crítica y riesgo de colapso cardio-respiratorio. considere que los pacientes con formas moderadas y severas de covid-19, pueden tener más comprometida esta reserva respiratoria y puede no ser efectiva la preoxigenación y cuando inicia la apnea, la desaturación será más precoz. si después de 3 minutos de preoxigenación no hay mejoría de la saturación arterial, considere fallida esta maniobra y considere mayor riesgo de hipoxemia severa con probabilidad de bradicardia extrema y paro cardiorrespiratorio. en caso de requerir ventilación por disminución rápida de la saturación de oxígeno arterial y considere necesario ventilación manual bolsa mascarilla a dos manos no debe ser vigorosa y debe utilizar filtros hpfa, estos reducirán los aerosoles en el ambiente(526, 529). se recomienda la utilización de cajas de acrílico para intubación del paciente con sospecha o diagnóstico de covid-19 como complemento durante la intubación para disminuir el riesgo de contaminación por gotas, sin embargo, no protege 100 % la generación de aerosoles y si ésta hace más difícil la intubación retírela inmediatamente. amci ® se recomienda minimizar los intentos de intubación orotraqueal en el paciente con sospecha o diagnóstico de covid-19, el primer intento debe procurar ser realizado por el más experimentado en el manejo de la vía aérea. se recomienda la intubación con videolaringoscopio en pacientes con covid-19 teniendo en cuenta disponibilidad y entrenamiento); esta alternativa puede ofrecer ventajas frente a la laringoscopia tradicional. se recomienda administrar medicamentos para asegurar la vía aérea en secuencia de inducción rápida, para obtener en el menor tiempo posible condiciones para la intubación (45 a 60 s). se recomienda en caso de intubación fallida por personal experto, considerar dispositivos supraglóticos como las máscaras laríngeas como medida de rescate con el riesgo de generación de aerosoles. se recomienda contar con disponibilidad de vasopresor y atropina en el sitio donde se realizará la intubación. en caso de contar con lidocaína se recomienda utilizar a dosis de 1 mg/kg sin epinefrina. administrada 3 minutos antes de la intubación. como se describió en el aparte de preoxigenación, se deben garantizar todas las medidas basadas en bioseguridad y protección adecuada para el personal de la salud, ubicación en áreas que cuenten con presión negativa o salas con un óptimo recambio de aire pueden ayudar a disminuir el riesgo de contagio. las medidas complementarias como la caja de taiwán o caja de intubación puede complementar de forma opcional estas medidas durante el proceso de aseguramiento definitivo de la vía aérea pero no garantiza 100 % la eliminación de aerosoles, pudiendo ser un obstáculo para quien realiza la intubación, de ser así, se deberá retirar inmediatamente, por eso sólo es un complemento al epp, que es la verdadera protección en estos escenarios (se debe incluir máscaras n95 o ffp2 fpp3) (524, 527) . el paciente críticamente enfermo puede tener comprometida de forma significativa su reserva respiratoria y hemodinámica por su cuadro clínico y puede empeorar por los medicamentos utilizados durante la intubación orotraqueal. las complicaciones en el manejo de la vía aérea se presentan cuando se realiza más de un intento dentro de los cuales están el traumatismo, desaturación e hipoxemia (530) . es por esto por lo que se debe procurar que el primer intento sea realizado por el más experimentado en el manejo de la vía aérea e idealmente se logre la intubación en el primer intento con la menor hipoxemia secundaria (166, 526, (531) (532) (533) . basado en el planteamiento anterior y considerando la ventaja de intubar pacientes críticamente enfermos en el primer intento dadas sus condiciones clínicas, la revisión de amci ® demandas asociadas a manejo de vía aérea en los estados unidos sigue siendo preocupante al considerar posibles causas la falta de entrenamiento y falta de aplicación de las guías y recomendaciones de manejo así como en la utilización de equipos adecuados para pacientes específicos (534) ; se ha planteado especialmente en los pacientes covid-19 los cuales tienen un importante compromiso pulmonar y alto riesgo de desaturación, la posibilidad de encontrar una vía aérea difícil no predicha y dificultades en su manejo(526, 533). es por esto que se requiere del mejor dispositivo para manejo de vía aérea invasiva disponible, siempre y cuando se cuente con el adecuado entrenamiento previo en su uso, la ventaja de los videolaringoscopio se debe a la superioridad al compararla con la laringoscopia convencional, siendo en algunos grupos la primera opción para intubaciones electivas (535, 536) . como se ha mencionado, el riesgo de una rápida desaturación en pacientes con enfermedad pulmonar, así como en pacientes con covid-19, se debe utilizar la inducción de secuencia rápida la cual se utiliza para pacientes con estómago lleno en los cuales se quiere lograr condiciones de inconciencia y de intubación óptimas en el menor tiempo posible para disminuir el riesgo de broncoaspiración. en estos casos de falla respiratoria con tan mala reserva se quiere aprovechar la ventaja que ofrece esa técnica para tener en poco tiempo al paciente intubado con menor riesgo de desaturación. es así como los medicamentos en una inducción de secuencia rápida incluyen el opioide, hipnótico y relajante, estos dos últimos administrados simultáneamente y lavados con un bolo de 20 cc. la opción del opioide en nuestro contexto suele ser fentanilo a dosis de 2 a 5 mcg / kg iv, dando 2 a 3 minutos de latencia para su efecto, luego el hipnótico que puede ser propofol entre 1 y 1,5 mg / kg si la estabilidad hemodinámica lo permite o considera usar vasopresor simultáneo. en caso de preferir evitar la hipotensión la ketamina a dosis de 1 a 1, 5 mg / kg es una opción más estable hemodinámicamente. con relación al relajante neuromuscular la succinilcolina es la clásicamente utilizada dosis de 1 a 1,5 mg/kg pero debido a sus efectos secundarios como hiperpotasemia, fasciculaciones, mialgias y un importante riesgo de hipertermia maligna algunos grupos no lo consideran, el rocuronio a dosis de 1,2 mg/kg ha demostrado lograr tiempos y condiciones de intubación similar a la succinilcolina sin los efectos secundarios de esta (533, 535) algunos grupos han considerado no utilizarlos si la condición clínica del paciente es crítica pues éste período de latencia puede ser acompañado de una hipoxemia severa y paro cardíaco, por lo tanto sólo usan hipnótico y relajante neuromuscular. ante una vía aérea difícil no predicha en la cual no se logre la intubación, considere los dispositivos supraglóticos como las máscaras laríngeas los cuales son más fáciles de insertar en comparación con la técnica de intubación orotraqueal, y deben estar dentro del planeamiento y organización de elementos para manejo de la vía aérea invasiva. al lograr ventilar con este dispositivo se logrará una recuperación del paciente, pero se pueden generar aerosoles pues este mecanismo de cierre puede permitir escape de aire y macropartículas durante el ciclo respiratorio ya sea manual o mecánico (535, 536) . el cuadro clínico de pacientes críticos y particularmente covid-19, puede asociarse a inestabilidad hemodinámica y requerimiento de soporte vasopresor. la adición de medicamentos como los opioides o hipnóticos pueden asociarse a hipotensión la cual puede no responder a volumen, es necesario evitar episodios de hipotensión en especial en pacientes ancianos, con enfermedades cardiovasculares de base las cuales pueden tolerar menos estos cuadros de hipotensión, así como también pueden favorecer amci ® desbalance en la relación ventilación/perfusión a nivel pulmonar empeorando los cuadros de hipoxemia. la hipoxemia puede acompañarse de bradicardia y si no se corrige la ventilación o la bradicardia es muy probable que el paciente presente paro cardiorrespiratorio, por lo cual se recomienda utilizar una dosis de atropina para corregir la bradicardia, no mejorará la oxigenación, pero tendrá un tiempo adicional para recuperar la oxigenación y ventilación del paciente. en estos casos debe tener disponibles vasopresores y atropina desde el planeamiento de los medicamentos necesarios para el manejo de la vía aérea (166) . se recomienda la intubación orotraqueal oportuna y no retrasar el inicio de la ventilación mecánica invasiva en los pacientes con sdra severo por covid-19 debido a mayor riesgo de desenlaces adversos. fundamento definir el momento de la intubación en esta población es un reto. la mayoría de los autores recomiendan el inicio "temprano" de la ventilación mecánica, sin embargo, la definición de cuando es temprano no es clara. este punto es motivo de análisis dado que a la luz de la evidencia actual la utilización de métodos no invasivos como la ventilación no invasiva y la cánula de alto flujo, para manejo inicial de pacientes con algún grado de hipoxemia es controvertido entre otras por el riesgo que supone al personal sanitario al ser un procedimiento generador de aerosoles. en el 2015 un estudio mostró que la intubación retrasada después de la falla al utilizar cánula de alto flujo o la ventilación no invasiva para pacientes con insuficiencia respiratoria moderada y grave se asoció con una mayor mortalidad. publicaciones recientes muestran que solo la quinta parte de los pacientes que murieron por covid-19 recibieron ventilación mecánica invasiva y soporte respiratorio más agresivo antes de la muerte, lo que indica que en muchos pacientes se habría retrasado la intubación. de los pacientes fallecidos solo el 27% recibieron tratamiento de oxígeno nasal o con mascarilla facial antes de su muerte. esta baja proporción puede tener varias explicaciones. primero, algunos pacientes con hipoxemia severa no tenían otros síntomas, como dificultad para respirar o disnea, es decir, desarrollaron una especie de hipoxemia silenciosa. en segundo lugar, la falta de suficientes ventiladores mecánicos invasivos es una razón importante que evitaría que los pacientes reciban intubación. tercero, el manejo de estos pacientes por un equipo de médicos no intensivistas; por lo tanto, pueden no estar seguros del momento en que un paciente requiere intubación. la serie de casos de la epidemia de covid-19 en wuhan mostró que la intubación tardía era común en la etapa inicial de la epidemia, mostrando que una de esas posibles razones del retraso incluía falta de ventiladores mecánicos invasivos y falta de capacitación clínica específica para el soporte respiratorio. recomendación se recomienda tomar la decisión de intubación orotraqueal en el paciente crítico con sospecha o diagnóstico de covid-19 utilizando una combinación de variables clínicas, gasométricas y hemodinámicas. tabla la intubación orotraqueal (iot) efectiva y segura, programada debe prevenir el colapso respiratorio y hemodinámico. siempre es necesario asegurar la escena del procedimiento de intubación con las consideraciones técnicas y de protección personal adecuadas. conocer los pasos para la realización del procedimiento de intubación orotraqueal (iot) contextualizados al paciente covid-19, reduce los riesgos innecesarios. la iot es un procedimiento generador de aerosoles por lo tanto lo ideal es realizarlo en una habitación con presión negativa, sin embargo, la baja disponibilidad en el país obliga a utilizar otras alternativas de seguridad. una habitación de presión negativa es un cuarto que tiene una presión más baja que las áreas adyacentes, lo que mantiene el flujo de aire fuera de la habitación y hacía habitaciones o áreas contiguas. las puertas de la sala deben mantenerse cerradas, excepto al entrar o salir de la sala, y la entrada y la salida deben minimizarse. la intubación en el paciente crítico con covid-19 es de los procedimientos que mayor riesgo de aerosolización tiene, por lo tanto, se debe adoptar una posición de intervención oportuna, pero también segura, para evitar desenlaces desfavorables en el paciente y disminuir el riesgo de contaminación en el personal de salud, se recomienda individualizar cada caso mediante la combinación y análisis de los criterios clínicos, gasométricos y hemodinámicos de cada paciente. (533, (537) (538) (539) se recomienda no realizar de rutina broncoscopia en los pacientes con sospecha o diagnóstico de covid-19, debido al riesgo de generación de aerosoles. se puede considerar en atelectasias masivas con compromiso significativo de la oxigenación adicional a la lesión pulmonar per sé y la hemorragia alveolar para control local directo. en general la realización de broncoscopia en pacientes con sospecha o confirmación de covid-19 debe ser evitada y realizarse sólo con indicación de emergencia como cuerpo extraño en la vía aérea, hemoptisis masiva, obstrucción grave de la vía aérea central o atelectasia lobar o pulmonar completa(540). esto debido a la alta carga viral en la mucosa nasal y faríngea de los pacientes con infección por sars-cov-2 (102) y la alta producción de aerosoles infecciosos que se generan durante este procedimiento. en caso de ser necesaria su realización, deberá ser llevada a cabo por el operador con mayor experiencia amci ® minimizando el tiempo de exploración y el personal expuesto en la sala. es mandatorio utilizar epp completo que incluya respirador fpp2 o fpp3, bajo protocolo supervisado de donning y doffing (541) . son de elección los broncoscopios desechables de un solo uso, pero de no ser posible se prefiere el uso de un broncoscopio flexible por encima de uno rígido por la más fácil manipulación de este. idealmente el procedimiento se llevará a cabo en el mismo cubículo del paciente que deberá contar con presión negativa y recambio de aire de 12 a 15 veces por hora. recomendación se recomienda no realizar de rutina la broncoscopia para la recolección de muestras para el diagnóstico de covid-19 en el paciente críticamente enfermo. la broncoscopia es una prueba de segunda elección para la toma de muestras respiratorias en los pacientes con sospecha o confirmación de covid-19 (542) . la toma de muestras del tracto respiratorio superior por hisopado nasofaríngeo u orofaríngeo es el método primario y de elección para determinar la infección por sars-cov-2. solo si resultaran dos pruebas negativas y persistiera una alta sospecha diagnóstica estaría indicado tomar muestras del tracto respiratorio inferior por broncoscopia, ya sea aspirado endotraqueal (bas) o lavado bronco alveolar (bal) (543), prefiriendo la realización de minibal para la recolección de muestras (540). las muestras deberán ser recogidas en un recipiente estéril, e introducidas en una bolsa con autocierre. deben manejarse con cuidado extremo evitando manipulaciones innecesarias y bajo protocolos de protección para el personal que las maneja, y trasladarse al laboratorio para su análisis. pueden almacenarse a 2-8ºc las primeras 72 h de su recolección; si se demorara más el análisis, precisa almacenarse a temperatura de -70ºc (124) . en una serie china la sensibilidad del bal fue de 93% frente a 72% en muestra de esputo (no recomendada) y 63% en hisopado nasofaríngeo (128) por lo que el especialista que realiza el procedimiento deberá sopesar el riesgo de este procedimiento en cada caso, valorando que se beneficiarán aquellos pacientes que tengan una indicación adicional para su realización. se recomienda la realización de la traqueostomía cuando está indicada, en los pacientes covid-19 sospechosos y confirmados con pronóstico razonable de vida, después del 5 o 7 día de ventilación , previa valoración y consenso por el equipo quirúrgico y de cuidado intensivo, asegurando que las condiciones clínicas, ventilatorias y hemodinámicas se encuentran controlada. en el contexto de pacientes hospitalizados en cuidado intensivo, la traqueostomía se realiza para facilitar el destete de la ventilación mecánica, mejorar la limpieza de la vía aérea y el manejo de las secreciones, aumentar la comodidad de los pacientes y la movilización y disminuir la probabilidad de complicaciones como la estenosis traqueal; sin embargo no hay una clara disminución en la mortalidad (544) (545) (546) . la infección actual por sars-cov2, tiene diferentes estadios de gravedad, uno de ellos es el compromiso pulmonar el cual se caracteriza por un síndrome de dificultad respiratorio agudo (sdra). de acuerdo con el comportamiento de la covid-19, entre un 10% a un 15% de los pacientes requieren ventilación mecánica (115, 121) , este grupo de pacientes con manifestación grave del compromiso pulmonar requiere estrategias de protección pulmonar en la ventilación mecánica, sedación profunda y posiblemente parálisis muscular y puede tener una mortalidad entre el 30% y el 50% (48, 547) . una de las principales características en éste grupo de pacientes es la mortalidad temprana, definida ésta como aquella que se produce en menos de 14 días; de acuerdo a la experiencia en wuhan, china; leung (548), reporta que la mortalidad se presenta en los primeros cinco días luego de la admisión hospitalaria y de acuerdo a lo referido por graselli et al (287) , en la región de lombardía, italia, la mediana de mortalidad se presenta al día siete después del ingreso. con estas consideraciones, al principio de la pandemia y en las aproximaciones iniciales no se recomendaba realizar la traqueostomía en los primeros 14 días posteriores a la intubación orotraqueal. sin embargo, con el conocimiento de la fisiopatología y las experiencias en otras series, como medida para facilitar la liberación de la ventilación mecánica, se ha podido realizar el procedimiento después de la primera semana de inicio de la ventilación. de acuerdo con el curso natural de la enfermedad, el paciente en promedio se intuba al día 10 a 12 de iniciado los síntomas, una semana posterior a la intubación para la traqueostomía, estaríamos alrededor del día 21 de la enfermedad, donde los pacientes ya tendrán una disminución de la carga viral. esto sin embargo no evita la utilización de los epp necesarios. esta medida en específico fue discutida y consensuada entre la sociedad de medicina critica amci y la asociación colombiana de cirugía. no existe evidencia que permita evaluar el riesgo real de infección del personal asistencial de los pacientes con sospecha o diagnóstico de covid-19 en la realización de traqueostomías. se debe considerar en la traqueostomía y la realización de ésta como un procedimiento generador de aerosoles (organización mundial de la salud). se recomienda que los epp requeridos para la realización del procedimiento, deben incluir máscaras ffp3 o n95, protección ocular, vestido antifluido idealmente desechable y amci ® guantes; este nivel de protección representa el mayor nivel de seguridad para realizar el procedimiento en el paciente con sospecha o diagnóstico de covid-19. no existe actualmente artículos que permitan evaluar cual es el riesgo real de infección del personal asistencial en la realización de traqueostomías en pacientes con covid-19; quizás el ejemplo más cercano, es una serie de casos, reportada durante la epidemia del síndrome respiratorio agudo grave en el 2004 (549), en el cual se realizaron 23 traqueostomías sin ninguna infección del personal, en este reporte se aseguró un adecuado uso del equipo de protección personal (epp), el cual incluía las medidas de barrera, máscaras ffp3 y cuando existía la disponibilidad, respiradores con suministro de aire purificado. la traqueostomía y la realización de ésta es considerada por la organización mundial de la salud (oms) un procedimiento generador de aerosoles, bajo esta perspectiva, el epp requerido para la realización del procedimiento, debe incluir máscaras ffp3 o n95, protección ocular, vestido antifluido idealmente desechable y guantes; este nivel de protección representa el mayor nivel de seguridad para realizar el procedimiento mencionado. es obligatorio, que todo el personal reciba el entrenamiento para la postura, el uso y el retiro de los epp, si estos pasos no se realizan de la forma adecuada, representan una fuente de contaminación(550). se recomiendan no esperar la negativización de la pcr para sars-cov2 para realizar la traqueostomía en el paciente con diagnóstico de covid-19. fuerte en contra fundamento los estudios de zou et al y lescure et al (102, 551) , muestran que la carga viral de los hisopados nasales y faríngeos es elevada en la primera fase de la enfermedad, con una disminución entre el día nueve al quince, pero esta puede permanecer detectable hasta por tres semanas (37) . existen recomendaciones acerca de la necesidad de realizar la traqueostomía una vez la pcr para sars-cov2 sea negativa (552, 553) ;aunque este esquema suena lógico, es importante tener presentes las siguientes consideraciones. la sensibilidad de una sola muestra para ruta-pcr puede ser sólo del 70%(554) y es posible que sea necesario realizar una segunda prueba para minimizar el riesgo para quien realiza el procedimiento, aunque esta aproximación no siempre puede ser viable desde el punto de vista clínico, epidemiológico y administrativo. amci ® recomendamos que la mejor estrategia es diferir la realización de la traqueostomía hasta 5 7 días luego de la intubación cuando esta indicada, con el conocimiento acerca de la evolución natural de la enfermedad, en ese momento en la mayoría de los pacientes, lo más probable es que la condición ya se encuentre en la tercera semana desde el inicio de los síntomas, en cuyo caso lo más probable es que ya exista una disminución de la carga viral; este hecho no evita que se deba utilizar de la forma correcta los epp. recomendación se recomienda escoger la técnica teniendo en cuenta la experticia que tenga en el grupo tratante y la anatomía del paciente para la decisión de la técnica. se recomienda la guía ecográfica para disminuir la probabilidad de complicaciones del procedimiento si la anatomía es desfavorable para la realización de traqueostomía percutánea. se recomienda la traqueostomía quirúrgica en pacientes de riesgo elevado de complicaciones donde se requiere un control más rápido y seguro de la vía aérea. se recomienda no utilizar de forma rutinaria la utilización de broncoscopia para la realización de traqueostomía por vía percutánea. no hay evidencia directa hasta la fecha de publicación del consenso si existe superioridad entre las dos técnicas en el paciente con sospecha o diagnóstico covid-19. en pacientes críticos no existen diferencia en los desenlaces cuando se evalúa el rendimiento de la técnica percutánea y la técnica quirúrgica, la elección de uno u otro método está dado por la anatomía del paciente, el entrenamiento de los profesionales y la disponibilidad de los diferentes insumos y técnicas. tampoco se ha logrado hasta la fecha evaluar durante la pandemia de covid-19 si existe una diferencia entre las dos técnicas y por lo tanto no es posible acercarse a una recomendación basada en la literatura. sin embargo, es importante que para la decisión de la técnica a utilizar se considere la anatomía del paciente y los siguientes aspectos:  no se recomienda la utilización de broncoscopia para la realización de traqueostomía por vía percutánea, ésta aumenta la generación de aerosoles y el número de personas expuestas a estos (555, 556) . amci ®  si la anatomía es desfavorable para la realización de traqueostomía percutánea, la guía ecográfica puede disminuir la probabilidad de complicaciones del procedimiento (557) .  la traqueostomía quirúrgica es una alternativa para la realización del procedimiento en las unidades de cuidado intensivo en momentos de sobrecarga laboral y adicionalmente puede tener un mejor y más rápido control de la vía aérea, especialmente en pacientes con riesgo elevado de complicaciones (549) . recomendación se recomienda el uso de la terapia ecmo en sdra severo refractario por covid-19 (pao2/fio2 < 150, posición prona, requerimiento de relajantes neuromusculares en algunos casos vasodilatadores pulmonares y maniobras de reclutamientos) sin respuesta clínica manifestado por:  pao2/fio2 < 60 mmhg por mas de 6 horas  pao2/fio2 < 50 mmhg por mas de 3 horas  ph < 7.20 + paco2 > 80 mmhg por mas de 6 horas además del criterio anterior, se recomienda tener en cuenta la edad, las comorbilidades y la expectativa de sobrevida del paciente con buena calidad de vida y en circunstancias donde no exista limitación de recursos. no hay estudios clínicos aleatorizados sobre el uso del ecmo en pacientes con covid-19 (166, 558) . existe el estudio eolia (216) , el cual fue detenido durante su realización, y de acuerdo a un análisis bayesiano posterior puede interpretarse como una disminución de la mortalidad en los pacientes en ecmo con sdra severo (211, 216, (559) (560) (561) (562) . de igual manera debe considerarse la racionalización de los recursos y el estado de prevalencia de la pandemia en un lugar determinado. el inicio de la terapia puede evaluarse en función de la cantidad de pacientes en falla respiratoria y la disponibilidad de personal y otros recursos; si el hospital debe comprometer todos los recursos en proveer medidas básicas de cuidado intensivo no debe utilizar el ecmo (563, 564) . los pacientes jóvenes sin comorbilidades son considerados de alta prioridad al igual que los trabajadores de la salud (565, 566) . amci ® se recomienda no desarrollar nuevos centros de ecmo en época de pandemia, sobre todo en situaciones con limitación de recursos. fuerte en contra fundamento actualmente se recomienda el uso del ecmo con las mismas indicaciones para sdra basado en la capacidad de las instituciones de salud para iniciar éste tipo de terapias (567) . en épocas de crisis la capacidad de los hospitales está saturada y obliga a la reubicación y optimización de los recursos (568, 569) . los centros que ofrecen la terapia en ecmo deben ser centros con resultados favorables y tiempos de soporte de pacientes relativamente cortos (570) . cuando estamos en tiempos de capacidad hospitalaria convencional y existe disponibilidad de camas de cuidado intensivo se pueden ofrecer los servicios de ecmo vv, va, e-cpr inclusive a pacientes no covid-19, cuando estamos en contingencia y capacidad nivel 1 se debe hacer un triage respecto a pacientes jóvenes y ofrecer ecmo vv, va, escoger muy bien los casos para pacientes no covid-19 y no ofrecer e-cpr, cuando estamos en contingencia y capacidad nivel 2 es porque ya se están usando sitios de expansión y están casi saturados se restringe el ecmo a todas las indicaciones y se prioriza a pacientes con indicaciones no covid-19 con mayor riesgo de sobrevida, el ecmo vv queda para pacientes jóvenes, con disfunción de 1 órgano y covid-19 positivo, no se ofrecerá ecmo va o e-cpr y cuando estamos en capacidad de crisis es porque la capacidad total hospitalaria está sobresaturada y no es posible realizar ecmo tanto en pacientes covid-19 como en los no covid-19 (564, 571) . se recomienda practicar e implementar medidas de capacitación y vigilancia continua para mejorar la higiene de manos, evaluando la adherencia a protocolos establecidos en los trabajadores de la salud mediante listas de chequeo y supervisión para evitar infecciones cruzadas en el entorno del paciente con sospecha o diagnóstico de covid-19. se recomienda establecer protocolos específicos para reducir el riesgo de infecciones que se deriven de la interacción y el cuidado del paciente crítico con sospecha o diagnóstico de covid-19. amci ® se recomienda implementar prácticas de cuidado para la prevención de contagio de covid-19. se debe utilizar el equipo de protección personal (epp) para la prevención de enfermedades de componente infeccioso asociado a exposición con fluidos corporales derivados del paciente crítico con sospecha o diagnóstico de covid-19. las infecciones relacionadas con la atención sanitaria (iras) son definidas por la organización mundial de la salud como aquellas "infecciones que se presentan en un paciente durante el proceso de atención en un hospital u otro centro sanitario que no estaban presentes o no se estaban incubando en el momento del ingreso; se incluyen las infecciones contraídas en el hospital pero que se manifiestan tras el alta hospitalaria y también las infecciones profesionales entre el personal del centro sanitario". las iras representan una importante carga de enfermedad que se asocia a un impacto negativo en la economía del paciente y del sistema sanitario. la organización mundial de la salud plantea la higiene de manos como la principal medida necesaria para reducir y prevenir las iras; por esta razón establece directrices sobre la higiene de manos en la atención sanitaria y basada en esta propone la estrategia multimodal para la mejora de higiene de manos. las estrategias mencionadas anteriormente han demostrado el incremento en el cumplimiento de higiene de manos y disminución en las infecciones relacionadas con la atención en salud. la estrategia multimodal se articula a través de cinco componentes: cambio del sistema, formación, evaluación/ retroalimentación, recordatorios en el lugar de trabajo clima institucional. a través de estos componentes, se garantiza que el centro sanitario cuenta con la infraestructura necesaria para practicar adecuadamente el lavado de manos incluyendo dentro de este el acceso a un suministro seguro continuo de agua, jabón, preparado alcohólico y toallas; a su vez se proporciona educación, evaluación y retroalimentación con regularidad a todos los profesionales sanitarios (572 se recomienda realizar un plan de cuidados organizado y específico en paciente crítico con sospecha o diagnóstico de covid-19, ofreciendo el uso óptimo de recursos e intervenciones. se recomienda evitar el uso de excesivo de papelería relacionada con los registros usados para gestión de insumos y atención de los pacientes. se recomienda realizar intervenciones educativas enfocadas a mejorar la adherencia y adecuado uso de los epp. se debe procurar el cuidado de los elementos de protección personal bajo un protocolo que conserve las condiciones de integridad de estos. la actual reserva de elementos de protección personal (epp) es insuficiente debido al aumento de la demanda global, por el incremento de casos de covid-19 y por la información errónea que ha conllevado a compras de pánico y almacenamiento. por esta razón la organización mundial de la salud a través de su guía: uso racional del equipo de protección personal para la enfermedad por covid-19 ha implementado las siguientes estrategias para optimizar la disponibilidad de (epp): usar los (epp) adecuadamente, minimizar la necesidad de (epp) y coordinar el suministro adecuado de (epp). (574) la duración máxima del uso continuo de la n95 es de 8 a 12 horas, siguiendo las recomendaciones del manual de medidas básicas para control de infecciones en ips de minsalud. pero en lo cotidiano, ningún trabajador tolera 8 a 12 horas continuas con un respirador. por esto, su uso continuo en el sitio de trabajo dependerá de la necesidad de pausar para comer, para ir al baño, etc. en este caso, se guardará en una bolsa de papel para su nueva colocación, si tiene menos de 12 horas, se desechará si está visiblemente contaminada o se torna húmeda. el reusó de la n95 dependerá de la casa del fabricante, de si contiene o no celulosa en su estructura del respirador. por ejemplo, la recomendación del consenso colombiano acin sobre la desinfección para los respiradores sin celulosa es con peróxido de hidrógeno vaporizado al 58% por 28 minutos. los respiradores n95 de uso industrial tienen mayor contenido de celulosa que los de uso médico por lo tanto para procesos de esterilización, solo los n95 de uso médico podrán ser esterilizados mediante de peróxido de hidrógeno vaporizado (sterrad®)(84-88). amci ® se recomienda elaborar el protocolo de pronación del paciente críticamente enfermo por covid-19, garantizando el entrenamiento al personal de salud, organizando el recurso humano, dispositivos de apoyo y tiempo establecido para cambios de posición para prevenir las lesiones por presión en el paciente. fuerte a favor fundamento la estrategia de pronación es una alternativa eficiente en el manejo del síndrome de dificultad respiratoria en pacientes críticos y es fundamental la gestión del profesional de enfermería en la prevención de complicaciones y eventos adversos, lo cual aportará significativamente a la calidad del cuidado ofrecido favoreciendo las mejoras en la oxigenación. es importante optimizar los cuidados de enfermería en torno de los cuidados de piel en los pacientes en ventilación mecánica en decúbito prono, los estudios han demostrado como principal complicación las lesiones de presión con una incidencia hasta de 25.7%, siendo las más frecuentes las grado 1 y 2(573). una lesión se puede producir si se supera una presión en el tejido capilar arterial de 32 mmhg denominándose interfaz de presión. basándose en lo anteriormente mencionado, el cambio de posición es un componente integral de la prevención y el tratamiento de las upp, con una justificación sólida y de amplia recomendación en la práctica de enfermería (575) . el uso de superficies especiales para el manejo de la presión (semp) a partir del estudio de defloor (576), se determinó un antes y un después en el uso de las semp en conjunción de los cambios posturales. este realizo un importante aporte para reducir la incidencia de lesiones por presión comparado con los colchones de estándar. se recomienda promover actividades para controlar el nivel de estrés en el personal durante las jornadas de trabajo: identificar y reconocer los propios límites, buscar o proponer ayuda psicológica profesional cuando sea requerido, promover estilos de vida saludables, y organizar los turnos de trabajo asegurando periodos de descanso. se recomienda compartir las emociones con pares y superiores, analizar objetivamente las situaciones adversas, manejar fuentes de información objetivas y científicas, realizar pausas activas durante el turno y brindar espacios grupales para expresar emociones, miedos e incentivar al equipo de trabajo reconociendo su labor. amci ® se recomienda utilizar recursos de salud mental ocupacional, apoyo por enfermedad y licencia familiar, además de garantizar una adecuada dotación de personal. los estilos de superación personal y el crecimiento psicológico desempeñan un papel importante en el mantenimiento de la salud mental de las enfermeras. es razonable suponer que los niveles de ansiedad y estrés entre los profesionales de la salud son proporcionalmente más altos que los de la población general debido al contacto directo con pacientes infectados. esto puede explicar por qué las enfermeras de primera línea son excepcionalmente vulnerables a la fatiga y al agotamiento (wang, okoli, et al.2020) , agotamiento mental, falta de moral del personal, control / autonomía de decisión, menor calidad de vida y baja satisfacción laboral (cheung y yip, 2015). (577) . una investigación reciente realizada en china continental menciona el impacto negativo de la pandemia de covid-19 en los trabajadores de atención médica de primera línea, incluidos los mayores niveles de ansiedad (shanafelt, ripp y trockel, 2020 ), depresión (xiang et al. 2020 ), estrés postraumático síntomas, soledad e impotencia (xiang et al.2020) (577, 578) . los aspectos traumáticos y estresantes de la participación en una pandemia también ponen en riesgo el daño psicológico a los médicos (579) . la experiencia psicológica de las enfermeras que atienden a pacientes con covid-19 se puede resumir en 4 temas: primero, las emociones negativas presentes en la etapa inicial consisten en fatiga, incomodidad e impotencia que fue causado por el trabajo de alta intensidad, el miedo y la ansiedad, y la preocupación por los pacientes y sus familiares. segundo, los estilos de auto afrontamiento incluyeron ajustes psicológicos y de vida, actos altruistas, apoyo de equipo y coordinación racional. tercero, encontramos crecimiento bajo presión, que incluía un mayor afecto y agradecimiento, desarrollo de posición de responsabilidad profesional y autorreflexión. finalmente, encontraron que las emociones positivas ocurrieron simultáneamente con emociones negativas(580). se recomienda ofrecer mecanismos de apoyo para amortiguar el estrés relacionado con la pandemia por covid-19. esto incluye intervenciones para pacientes y familias ofreciendo recursos de salud mental y educación al egreso, previo a este reforzar visitas virtuales. se recomienda anticipar las necesidades de salud mental de los pacientes, el personal y las familias para ofrecer una respuesta integral de salud pública. se debe incluir atención psicológica en la hospitalización para pacientes, familiares y personal afectado por covid-19. se recomienda proporcionar atención de salud mental en las comunidades, mientras que se requiere distanciamiento social y los recursos del sistema de salud son limitados. amci ® se recomienda mantener una estrategia de comunicación asertiva con la familia, teniendo en cuenta la formación del personal sobre las estrategias para comunicar malas noticias. ser solidarios con el duelo de las familias y acompañar el proceso de afrontamiento aún en la distancia, identificando factores de riesgo para patología mental o duelo complicado, utilizando los recursos institucionales de salud mental para mejorar las intervenciones. la pandemia tiene el potencial de crear una crisis secundaria de angustia psicológica y desbordamiento del sistema de salud mental. los miembros de la familia pueden experimentar angustia, miedo o ansiedad por la hospitalización de un ser querido, particularmente cuando las medidas de control de infecciones restringen las visitas. la telesalud (incluida la cobertura de seguro para la telesalud), el suministro extendido de medicamentos, el aumento de la capacitación en salud mental del proveedor, el apoyo virtual de pares y los grupos virtuales de apoyo al uso de sustancias pueden ayudar a garantizar que se satisfagan las necesidades de salud mental de la comunidad (581). el sistema de salud y los líderes de enfermería deben asegurarse de que su personal de enfermería clínica esté protegido y respaldado para que puedan proporcionar esta dimensión crucial de la atención de covid-19. se recomienda crear grupos centralizados y definidos para atención de pacientes con sospecha o diagnóstico de covid-19 que se encarguen de elaboración, socialización e implementación de protocolos. estos deben incluir los aspectos de infraestructura, áreas delimitadas, utilización de epp, listas de chequeo, observadores, insumos y recursos que permitan atención integral. se recomienda organizar el plan de atención del paciente con sospecha o diagnóstico de covid-19 de enfermería con la asignación de actividades, número de personas según escalas que midan escalas de carga laboral para definir el número adecuado de los miembros del equipo de trabajo, tiempo de atención, gestión de recursos, gestión de riesgo y un líder por turno que garantice el cumplimiento fuerte a favor fundamento la implementación de estrategias de gestión en contingencias genera un trabajo organizado, enfocado en la prevención y tratamiento centralizado, elaboración y socialización de protocolos claros, áreas específicas, delimitadas y asignadas, con un uso racional del recurso humano que se despliega en fases, desde el inicio de la emergencia considerado como detección temprana hasta la atención directa de pacientes con sospecha amci ® o confirmación de sars-cov-2. dentro de las fases tempranas, se busca la gestión de los recursos necesarios para la atención de estos pacientes, con una asignación de zonas o servicios y unas condiciones particulares, tratamientos específicos y actividades de atención especiales para las cuales se discriminan medicamentos, dispositivos e insumos necesarios para el cuidado de enfermería. los grupos de atención deben contar con capacitación, gestión y supervisión, apoyo logístico, apoyo psicológico y retroalimentación (573, 580) . el plan de atención de enfermería debe tener presente la minimización de exposición, la prevención de infecciones en el personal y cuidados especiales derivados de la condición clínica de los pacientes con esta infección, altamente contagiosa y con síntomas o necesidades que rompe el modo operacional convencional y que requiere implementación basada en la práctica clínica. por lo tanto, el plan debe ser centralizado oportuno, ordenado, seguro y eficiente e incluye: relación enfermería/paciente de acuerdo a criticidad, capacitaciones y entrenamiento al personal de enfermería de línea de frente en el área crítica de aislamiento mediante videos, infografías y procesos prácticos (el contenido de capacitación incluye el uso de elementos de protección personal, higiene de manos, desinfección de áreas, manejo de residuos y esterilización de dispositivos de atención al paciente y manejo de exposición ocupacional), asignación de actividades clínicas (atención directa) y administrativas (supervisión, observador, líderes, gestión de recurso humano y medicamentos), soporte y contratación de personal adicional ante la contingencia con preparación académica o inducción, asignación de turnos razonables con períodos de descanso (alimentación, eliminación), coordinación con otros departamentos y optimización de flujos de trabajo, estrategias de control de infecciones y trabajo en equipo (577, 579) . se recomienda que las muestras clínicas tomadas para el diagnóstico de covid-19 deben conservarse a temperatura entre -2 a 8°c, y luego de las 48 horas deben permanecer congeladas a una temperatura de -70°c. se recomienda que se realice el envío al laboratorio de salud pública de referencia dentro de las 48 horas posteriores a la toma de la muestra del paciente. se recomienda que el transporte de las muestras debe realizarse con geles o pilas congeladas. se recomienda considerar que las muestras del tracto respiratorio bajo presentan la mejor certeza diagnóstica en pacientes con neumonía para adultos intubados y ventilados mecánicamente con sospecha de covid-19. se recomienda contar con elementos de protección personal de acuerdo con las precauciones establecidas para el paciente con sospecha o diagnóstico por covid-19 para evitar la transmisión a profesionales de la salud. se debe evitar perder el circuito cerrado en los pacientes ventilados mecánicamente y valorar el riesgo de las acciones en pacientes con peep alta. se recomienda realizar la toma de muestra post mortem no invasiva por hisopado nasofaríngeo dentro de las primeras seis (6) horas posteriores al fallecimiento, para que esta sea útil para su análisis. las muestras clínicas tomadas para el diagnóstico de coronavirus deben conservarse a temperatura entre -2 a 8°c, y luego de las 48 horas deben permanecer congeladas a una temperatura de -70°c. sin embargo, la muestra puede conservarse en un tiempo máximo de refrigeración por 72 horas. no obstante, se sugiere que se realice el envío al laboratorio de salud pública de referencia dentro de las 48 horas posteriores a la toma. si no se conserva la cadena de frío adecuada, la muestra puede ser inviable. el transporte de las muestras debe realizarse con geles o pilas congeladas (128, 582, 583) .se debe tener en cuenta que no conservar la cadena de frío durante el transporte de la muestra, degradan la partícula viral, obteniéndose falsos negativos (583) . las muestras del tracto respiratorio bajo presentan la mejor certeza diagnóstica en pacientes con neumonía. para adultos intubados y ventilados mecánicamente con sospecha de covid-19 en comparación al tracto respiratorio superior (nasofaríngeo u orofaríngeo). en el caso de aspirado traqueal, es importante considerar que para la obtención de las muestras para el diagnóstico de covid-19 se deben contar con elementos de protección personal de acuerdo a las precauciones estándar para evitar la transmisión a profesionales de la salud, circuito cerrado y valorar su realización en aquellos pacientes con peep alta (128) . la toma de muestra post mortem no invasiva por hisopado nasofaríngeo se debe hacer antes de seis (6) horas post mortem, para que esta sea útil para su análisis(166, 582-585). se recomienda en los pacientes con diagnóstico covid-19, monitorizar continuamente la oxigenación mediante saturación arterial de oxígeno con pulso oxímetro y la aparición temprana de signos clínicos de dificultad respiratoria durante la monitorización (aleteo nasal, cianosis, tirajes intercostales). se recomienda no suministrar de forma rutinaria suministrar oxígeno si la saturación de oxígeno (spo2) está por encima de 94%, y no se evidencian signos clínicos de dificultad respiratoria durante la monitorización continua del patrón respiratorio. se recomienda como parámetro importante para evaluar la oxigenación y guiar el suministro de oxígeno mediante los diferentes dispositivos la transferencia de oxígeno, medida por la pao2/ fio2 o sao2/fio2. se propone iniciar la oxigenoterapia por cánulas de bajo flujo y ajustar el flujo (máximo 5 l) hasta alcanzar la spo2 objetivo ≥ 94%; si el paciente se encuentra en estado crítico iniciar con mascarilla con bolsa de reserva (a 10-15 l / min). una vez que el paciente esté estable, el objetivo de oxigenación es mantener niveles de spo2 entre 88 y 92% en pacientes no embarazadas y entre 92-95% en pacientes embarazadas. se recomienda no utilizar de forma rutinaria el uso de dispositivos que generan aerosoles durante la administración de oxígeno (dispositivos venturi o nebulizador de alto flujo o jet) en pacientes con sospecha o diagnóstico covid-19. en las diferentes guías publicadas para manejo de pacientes positivos para covid-19 las metas de oxigenación durante la terapia de oxígeno en adultos recomiendan iniciar la oxigenoterapia a 5 l / min y ajustar el flujo hasta alcanzar la spo2 objetivo ≥ 94% durante la reanimación; o use mascarilla con bolsa de reserva (a 10-15 l / min) si el paciente está en estado crítico. una vez que el paciente esté estable, el objetivo de oxigenación es > 90% de spo2 en pacientes, no embarazadas y ≥ 92-95% en pacientes embarazadas(165). los dispositivos para la oxigenoterapia se pueden dividir en dos grupos, dependiendo de si cubren la totalidad o una parte de los requerimientos respiratorios del paciente. unos son de bajo flujo o para esfuerzos mínimos del paciente, estos dispositivos completan su ventilación con aire ambiente y los sistemas de alto flujo cubren la totalidad de los requerimientos inspiratorios del paciente. escalones terapéuticos: oxigenoterapia convencional a diferentes concentraciones de bajo flujo (son las cánulas nasales, las mascarillas simples y las mascarillas con reservorio), es el primer escalón terapéutico ante cualquier paciente que presente una situación de hipoxemia (spo2) < 90% respirando aire ambiente. el objetivo debe ser ajustar la fio2 (hasta 0.4) para mantener un nivel de oxigenación adecuado, considerado este como una spo2 > 91%. la administración de oxígeno se considera un procedimiento generador de aerosoles de riesgo bajo y por lo tanto es adecuado para pacientes covid-19 positivos(586). b. en adultos con signos de emergencia (respiración obstruida o ausente, dificultad respiratoria severa, cianosis central, shock, coma y / o convulsiones) deben recibir vía aérea amci ® de emergencia manejo y oxigenoterapia durante la reanimación para apuntar a spo2 ≥ 94%. una vez el paciente está estable, objetivo> 90% de spo2 en adultos no embarazadas y ≥ 92-95% en mujeres embarazadas. c. para el manejo del paciente con covid-19 la máscara de no re inhalación se considera como la opción de preferencia para escalar el paciente antes de la intubación y considerar la transferencia a uci; esto se debe a que puede proporcionar altas fracciones inspiradas de oxígeno (587) . d. los dispositivos que generan aerosoles durante la administración de oxígeno (dispositivos venturi o nebulizador de alto flujo o jet), no están indicados para manejo de covid-19 (588). se recomienda aplicar las estrategias de retiro de la ventilación mecánica habituales para pacientes adultos críticos en general, hasta el momento no se ha construido una evidencia contundente para el destete en covid-19. se recomienda en el paciente críticamente enfermo por covid-19 un descenso de la presión de soporte (psv) según tolerancia clínica, de esta forma el paciente podrá ser sometido a la realización de prueba de respiración espontánea con una presión de soporte de entre 5-8 cm h2o. se recomienda que el destete automatizado puede ser considerado como una herramienta útil según disponibilidad de equipos para realizarlo. se recomienda no utilizar las maniobras que incrementan la aerosolización como la prueba de respiración espontánea en pieza en t o el cuff-leak test en el momento de realizar la medición de los predictores de éxito en el destete. fuerte en contra fundamento la realización de las pruebas de respiración espontánea sigue siendo un factor predictor importante en el éxito en el retiro del soporte ventilatorio mecánico y la indicación de tiempo de duración sigue siendo de 30 a 120 minutos debido a que las intubaciones realizadas en el mismo periodo de tiempo no han tenido diferencias significativas en el éxito del destete (589, 590) . en los pacientes que han sido ventilados por más de 24 horas y que el motivo por el cual fueron llevados a ventilación mecánica ya ha sido superado se debe establecer un protocolo de destete que debe incluir una prueba diaria de respiración espontánea y la minimización o retiro de la sedación (si no existe alguna contraindicación)(165, 591). la movilización temprana como factor coadyuvante en el éxito de la liberación mecánica ya se ha documentado en otros escenarios similares, razón por la cual la implementación amci ® temprana de este tipo de estrategias será un punto de vital importancia para recuperar la funcionalidad de los pacientes con covid-19 (121) . se recomienda que la extubación de los pacientes críticamente enfermos por covid-19 se debe realizar con los elementos de protección personal requeridos para el riesgo de aerosoles. se recomienda no estimular la tos y el esputo inducido en los pacientes con sospecha o diagnóstico de covid-19 posterior a la extubación inmediata. se recomienda no utilizar de forma rutinaria la vmni en la falla respiratoria post extubación en pacientes críticos que no tengan una enfermedad concomitante que sea respondedora a la vmni como el epoc o edema pulmonar de origen cardiogénico en pacientes con sospecha o diagnóstico de covid-19. fuerte en contra se recomienda mantener un umbral bajo para decidir intubación en caso de sospecha de fallo en la extubación en el paciente con sospecha o diagnóstico de covid-19. la estricta monitoria y manejo del paciente posterior a la extubación surgen como un reto insoslayable para el personal de cuidado intensivo, enfocando todos sus esfuerzos en evitar la re-intubación, lo que se traducirá en un descenso significativo de la morbilidad y la mortalidad que supone una re-intubación (592), la cual se puede definir como el no requerimiento de re intubación en las primeras 72 horas post extubación (593, 594) . en los últimos años la cánula nasal de alto flujo (caf) se ha convertido en una herramienta útil en el soporte de oxigenoterapia en los pacientes extubados que presenten riesgo de reintubación (595, 596), y a la vez no presenten hipercapnia (597) . la utilización de ventilación mecánica no invasiva de manera profiláctica en la falla respiratoria post extubación no ha demostrado tener éxito evitando la re-intubación en las primeras 48 horas (598, 599) excepto en las situaciones donde el paciente presente una enfermedad pulmonar o alteración cardiaca concomitante que sea respondedora a el manejo con vmni como lo son la enfermedad pulmonar obstructiva crónica (epoc) y el edema pulmonar de origen cardiogénico (267, 600) . los pacientes extubados en los que se halla documentado epoc, se sugiere posterior a la extubación la implementación de una estrategia de niv de manera protocolaria (600, 601), con una intensidad de 1 hora cada 3 horas durante un período mínimo de 48 horas (602). se recomienda utilizar en los pacientes con extubación reciente que no expresen predictores de riesgo de fracaso, sistemas de oxigenoterapia convencionales de bajo flujo que generen menos riesgo de aerosolización, fuerte a favor se podría considerar cánulas de alto flujo de oxígeno y/o la ventilación mecánica no invasiva (con una máscara facial adecuadamente ajustada y ramas inspiratorias y espiratorias separadas) como terapia de puente después de la extubación, pero se deben brindar las condiciones estructurales necesarias (habitaciones de presión negativa o habitaciones aisladas de puertas cerradas) y con epp estrictos para el personal sanitario. fuerte a favor fundamento las pautas de anzics establecen que la caf y/o la ventilación no invasiva (con una máscara facial bien ajustada y ramas inspiratorias y espiratorias separadas) pueden considerarse como terapia de puente después de la extubación, pero deben proporcionarse epp estricto en el aire. la terapia cpap o bipap (con alta presión espiratoria final) podría ser útil para prevenir la eliminación del reclutamiento en estos pacientes. en el momento de la extubación, los pacientes a menudo han estado enfermos durante más de una semana. es probable que su carga viral disminuya en ese punto, por lo que el riesgo de transmisión del virus puede ser menor (en comparación con la intubación inicial) (603) . de no contar con predictores de que nos indiquen que podría fracasar la extubación se deben utilizar entonces sistemas de oxigenoterapia convencionales de bajo flujo que generen menos riesgo de aerosolización(604). se recomienda limpiar y desinfectar con frecuencia el área de retiro de epp, incluso después de que se haya completado cada procedimiento de eliminación. se debe limpiar esta zona, pasando de las áreas más limpias a las más sucias, antes de ingresar a la habitación del paciente y realizar el manejo y disposición final de residuos. se recomienda realizar la limpieza de superficies con un desinfectante adecuado o con una solución de hipoclorito sódico que contenga 5000 ppm de cloro activo (por ejemplo, un producto con hipoclorito en una concentración de 40-50 gr/litro, se hará una dilución 1:10 en el momento de su utilización). amci ® se recomienda que los recipientes que contengan los residuos deberán quedar en el lugar designado a tal efecto, que permanecerá cerrado hasta que, según el procedimiento de gestión de residuos de la institución sean retirados. los circuitos, filtros, succión cerrada y tot deben ser dispuestos en bolsas de color rojo las cuales deben ser de polietileno de alta densidad de 1.6 milésimas de pulgada y deben contar con un rótulo donde se indiquen: el nombre del generador, las palabras residuos biológicos (covid19 2019) . una vez dispuesto, apretar y asegurar con nudo la bolsa de residuos y remover la bolsa de residuos del recipiente de residuos. posteriormente, desinfectar el exterior de la bolsa con solución desinfectante. luego colocar la bolsa de residuos en otra bolsa adicional de residuos y apretar y asegurar con nudo la bolsa de residuo. finalmente desinfectar la exterior bolsa de residuos con solución desinfectante. una vez terminada la disposición de los residuos de extubación, desinfectar los guantes con que manipuló los residuos con solución desinfectante y ubicar la bolsa de residuos dentro del vehículo de recolección interna de residuos. finalmente desinfectar el exterior de la bolsa de residuos con solución desinfectante. una vez terminada la disposición de los residuos de extubación, desinfectar los guantes con que manipuló los residuos con solución desinfectante y ubicar la bolsa dentro del vehículo de recolección interna. acogerse a la ruta sanitaria que asegure el menor riesgo de contaminación en el traslado interno de los residuos en la habitación del paciente (zona limpia) y zona sucia, se debe garantizar la ubicación de recipiente plástico de color rojo, liviano, resistente a los golpes, en material rígido impermeable, de fácil limpieza, y resistentes a la corrosión. los recipientes deberán ser lavados y desinfectados de acuerdo con los procedimientos establecidos por el prestador de servicios de salud(605-608). se recomienda utilizar un ajuste de peep del paciente crítico por covid-19, basado adicional a la tabla de peep, en las condiciones clínicas del paciente, en los índices de oxigenación, en la mecánica respiratoria del paciente y en los métodos de monitoreo disponibles. se recomienda titular la peep más alta que mantenga o mejore la relación safi y permita una presión plateau ≤ 30 cmh2o. se recomienda utilizar otras estrategias de titulación de peep probadas y con las cuales el equipo de trabajo esté familiarizado, dependiendo de la disponibilidad del recurso: ensayo peep decremental precedido por una maniobra de reclutamiento; titulación mediante la amci ® estimación de la presión transpulmonar con catéter esofágico o tomografía de impedancia eléctrica. fuerte a favor fundamento la titulación de la peep debe hacerse en función de la distensibilidad, oxigenación, espacio muerto y estado hemodinámico. puede titularse la peep mediante la estimación de la presión transpulmonar con catéter esofágico o tomografía de impedancia eléctrica. podría también titularse a partir de la fórmula (dp=plateau-peep) teniendo en cuenta que sea lógico el acoplamiento matemático fisiológico (lo que resultaría en una peep de 15 cmh2o si la presión plateau es de 30 cmh2o). la titulación de la peep requiere consideración de los beneficios (reducción de atelectrauma y mejora del reclutamiento alveolar) frente a los riesgos (sobre distensión inspiratoria final que conduce a lesión pulmonar y mayor resistencia vascular pulmonar)(165, 609, 610). se recomienda aplicar los protocolos de rehabilitación física como estrategia beneficiosa en el tratamiento respiratorio y físico de pacientes críticamente enfermos por covid-19. se recomienda realizar la movilización precoz del paciente críticamente enfermo por covid-19 durante el curso de la enfermedad siempre que sea posible hacerlo de forma segura, asegurando la protección personal del personal sanitario. derivado del tratamiento médico intensivo para algunos pacientes con covid-19, incluida la ventilación pulmonar protectora prolongada, la sedación y el uso de agentes bloqueantes neuromusculares, los pacientes con covid-19 que ingresan en la uci pueden presentar un elevado riesgo de desarrollar debilidad adquirida en la uci empeorando su morbilidad y mortalidad. por lo tanto, es esencial la rehabilitación temprana después de la fase aguda del síndrome de distrés respiratorio agudo (sdra) para limitar la gravedad de la debilidad adquirida en uci y promover la recuperación funcional. según la guía de la oms y la ops, enfatizan extremar el uso de los elementos de protección personal (epp) durante las intervenciones de rehabilitación física. la rehabilitación física proporciona intervenciones a través de movilizaciones, ejercicio terapéutico y programas individualizados a las personas que superan la enfermedad crítica asociada con covid-19 durante la ventilación mecánica y luego de esta, con el fin de permitir un retorno al hogar con funcionalidad. la prescripción de la movilización y ejercicio terapéutico debe de ser considera cuidadosamente en función del estado del paciente teniendo en cuenta, la estabilidad hemodinámica y clínica de la función respiratoria. cuando las movilizaciones, ejercicio terapéutico o programas de rehabilitación están indicados, debe realizarse una correcta planeación teniendo en cuenta amci ® la identificación/uso del personal mínimo necesario para realizar la actividad de manera segura. y el aseguramiento de todo el material que requerido esté a la mano y funcione correctamente y esté perfectamente limpio y desinfectado. si el material/equipo tiene que ser compartido con otros pacientes, límpielo y desinféctelo después de cada uso, entre paciente y paciente. se requiere personal entrenado específicamente para la limpieza y desinfección de los equipos, en una habitación aislada. y siempre que sea posible, evitar el traslado del material entre las áreas infectadas y no infectadas del hospital, manteniendo el equipamiento en las zonas aisladas (611) (612) (613) (614) (615) . ( basados en un estudio preliminar aún sin publicar, se podría sugerir el uso de dexametasona a dosis de 6 mg (oral o venosos) por 10 días o hasta el alta si ocurre primero en pacientes hospitalizados con sospecha o diagnóstico de covid-19 que requieren suplencia de oxígeno, incluyendo aquellos con ventilación mecánica, que sean menores de 70 años y con más de 7 días de síntomas. amci ® actualmente no existe una terapia dirigida que se a efectiva para el manejo del virus; un número alto de estudios han surgido en los últimos dos meses, la mayoría sin el rigor metodológico suficiente para tomar decisiones adecuadas con respecto al manejo del paciente con infección por sars-cov-2. el conocimiento en la estructura del virus y el mejor entendimiento en la fisiopatología de la enfermedad genera un sinnúmero de potenciales fármacos que han sido ensayados para el manejo de la enfermedad. en tiempos de pandemia, con una patología catastrófica en términos de vidas humanas y costos hospitalarios; es importante encontrar soluciones a desenlaces importantes como mortalidad, días de estancia en uci y en el hospital, aumento en los días libres del ventilador, disminución de complicaciones mayores debido a la enfermedad entre otros. hasta el momento no se ha documentado ninguna terapia específica que pueda impactar sobre estos desenlaces; pero la calidad de los trabajos, tampoco dejan claro sin él no usar ningún tratamiento específico mejora los desenlaces al menos al disminuir el número de complicaciones. este nuevo beta-coronavirus es similar al coronavirus del síndrome respiratorio agudo severa (sars-cov) y del síndrome respiratorio del medio este (mers-cov); por lo tanto, varias moléculas que habían sido evaluadas en este tipo de enfermedad rápidamente se abrieron paso a ensayos clínicos en paciente con covid-19. estos ensayos principalmente observacionales, aleatorios pero abiertos con un número pequeño de pacientes no han permitido sacar adecuadas conclusiones y es frecuente como ver las diferentes guías de las principales sociedades del mundo cambiar de forma frecuente sus recomendaciones; no existes evidencia de estudios clínicos aleatorios y controlados que midan desenlaces fuertes, la premura de un tratamiento efectivo ha sacrificado el rigor metodológico que una investigación requiere. una estructura viral y replicación conocidas generan posibles dianas para que diferentes fármacos puedan ser investigados, antivirales tipo arbidol el cual inhibe la fusión de la membrana en la envoltura viral a algunos receptores; antimaláricos como la hidroxicloroquina y la cloroquina, las cuales inhiben la entrada viral y endocitosis por múltiples mecanismos, así como los efectos inmunomoduladores demostrados en el huésped; antivirales que impiden la replicación como el lopinavir o darunavir inhibiendo las proteasas o la ribavirina, el remdesivir o el favipiravir que actúan como análogos de nucleótidos o fármacos que actúan modulando la respuesta específica del huésped como el tocilizumab el cual se une al receptor de la il-6 inhibiendo el punto de acción de esta; los corticosteroides con múltiples efectos en la modulación del sistema inmunológico del paciente o los fármacos para evitar la respuesta secundaria a esta cascada inflamatoria como son los anticoagulantes. por último, se han buscado estrategias con el fin de mejorar la inmunización pasiva del huésped en el uso del plasma de pacientes convalecientes o el uso de inmunoglobulinas enriquecidas entre otros tratamientos propuestos para esta enfermedad. amci ® a los diferentes medicamentos que han sido usados en la pandemia del sars-cov-2/covid-19. de manera reciente en datos preliminares aún sin publicar horby y col en una rama del ensayo de evaluación aleatorizada de la terapia covid-19 (recovery), estudio aleatorizado, controlado, abierto que compara una gama de posibles tratamientos con la atención habitual en pacientes hospitalizados con covid-19, compararon el uso de la dexametasona a dosis de 6 mg día (oral o intravenosa) una vez al día por 10 días o el alta según lo que ocurriera primero contra el manejo habitual; en 2104 pacientes aleatorizados que recibieron dexametasona se compararon con 4321 pacientes en manejo estándar; 454 (21.6%) pacientes en el grupo de dexametasona y 1065 (24.6%) pacientes en el grupo control murieron a los 28 días, con un riesgo relativo ajustado para la edad (rr 0.83; 95% ic 0.74 a 0.92; p < 0.001). la mortalidad relativa y absoluta variaron significativamente en relación al soporte ventilatorio al momento de la aleatorización; la dexametasona redujo las muertes en una tercera parte de los pacientes que recibieron ventilación mecánica invasiva (29.0% vs. 40.7%, rr 0.65, 95% ic 0.51 a 0.82; p < 0.001), y una quinta parte en los pacientes que reciben oxígeno sin ventilación mecánica invasiva (21.5% vs. 25.0%, rr 0.80, 95% ic 0.70 a 0.92; p = 0.002), pero sin reducir la mortalidad en paciente que no recibieron soporte respiratorio al momento de la aleatorización (17.0% vs. 13.2%, rr 1.22, 95% ic 0.93 a 1.61]; p = 0.14) (423) . no se emite recomendación a favor ni en contra para el inhibidor de la janus quinasa (baricitinib) en los pacientes con sospecha clínica o diagnóstico de covid-19 severo. uno de los reguladores conocidos de la endocitosis es la proteína quinasa 1 asociada a ap2 (aak1); la interrupción de aak1 podría, a su vez, interrumpir el paso del virus a las células y también el ensamblaje intracelular de partículas del virus. uno de los seis fármacos de unión a aak1 de alta afinidad es el inhibidor de la janus quinasa (jak1 y jak2), llamado baricitinib, que también se une a la quinasa asociada a la ciclina g, otro regulador de la endocitosis (23) . el baricitinib alcanza concentraciones plasmáticas suficientes para inhibir aak1 con 2 mg o 4 mg una vez al día; por su baja unión a proteínas plasmáticas y a su mínima interacción con las enzimas cyp, permite combinarlo con los antivirales. sin embargo, algunos piensan que el bloqueo de la señal jak-stat por baricitinib puede producir un deterioro de la respuesta antiviral mediada por interferón, con un posible efecto facilitador sobre la evolución de la infección por sars-cov-2; otras limitantes son la linfopenia (no dar si < 500 cel./ mm 3 ) y el aumento de la cpk. (24, 25) . cantini y cols, en abril 2020, en italia, administraron baricitinib a 4 mg/día vía oral por 2 semanas a 12 pacientes con covid-19 moderado y los compararon con un grupo control; la terapia mejoró significativamente los parámetros clínicos, respiratorios y de laboratorio (pcr); ninguno de los pacientes requirió uci vs 33% del grupo control, sin eventos adversos. se amci ® trata de un estudio piloto de seguridad e impacto clínico en pacientes que no estaban en uci (472) . ¿en pacientes hospitalizados con sospecha o diagnóstico de covid-19 el uso de n-acetil cisteína modifica el curso clínico de la enfermedad o genera beneficios en desenlaces clínicos de interés? basados en evidencia indirecta para el manejo del sdra y resultados observaciones en covid-19, se podría utilizar el uso de n-acetil cisteina a dosis de 200mg/kg/día durante los primeros cinco días del sdra, aunque no se ha demostrado impacto en la mortalidad, su utilización parece relacionarse con una disminución significativa en la estancia en la unidad de cuidados intensivos y con disminución de los marcadores inflamatorios en pacientes con covid-19 . la severidad de la infección en covid-19, en gran parte depende de la respuesta inmunológica de cada persona, sin embargo, se encuentran 3 mecanismos fisiopatológicos de relevancia. sobreproducción de moco en vía aérea superior e inferior, que en parte explica la dificultad en la mecánica ventilatoria y los retos de ventilación en estos pacientes, la descarga desmedida de citoquinas proinflamatorias que se asocian a la falla multiorgánica y la coagulopatía asociada a la disfunción endotelial. esto mecanismos fisiopatológicos son comunes en el sdra, incluido los casos asociados a covid-19(616). amci ® enfermo con falla respiratoria aguda, la cual engloba falla respiratoria hipóxica (tipo 1), falla respiratoria hipercápnica aguda (tipo 2), sdra y lesión pulmonar aguda, se revisaron 13 ensayos clínicos, más de 1712 pacientes. el análisis del grupo de n acetilcisteína intravenoso mostró una reducción de estancia en uci, de 4.7 días, con una heterogeneidad muy baja del 25%, con valoración de la evidencia calificada como de alta calidad y baja probabilidad de sesgo (621) . en covid-19, fue utilizada con recuperación completa en un caso severo de un paciente con déficit de glucosa 6 fosfato deshidrogenasa (g6pd), con control de la hemolisis y resolución del compromiso pulmonar. en pacientes sin déficit de g6pd, también ha sido asociada a mejoría clínica y disminución significativa de los niveles de pcr y ferritina (622) . en una revisión de costo efectividad nacional, se identificaron 222 referencias, 3 de ellos era revisiones sistemáticas de la literatura, dos de las cuales incluían metaanálisis (lu 2019 y zhang 2017), y fueron incluidos en la evaluación. estos estudios incluyeron información de ensayos clínicos que comparaban la aplicación de nac intravenosa frente a placebo o cuidado usual en pacientes con sdra. los tres estudios reportan como resultado de mortalidad rr de 0.83 con ic al 95% de 0.62 a 1.11 (lu 2019), rr de 0.64 con ic al 95% de 0.32 a 1.30 (lewis 2019) y rr de 0.73 con ic al 95% de 0.50 a 1.07 (zhang 2017) . para el tiempo de estancia en uci solo las revisiones con metaanálisis reportaron resultados, encontrando una diferencia de promedio de días de estancia de -4.47 días con ic al 95% de -8.79 a -0.14 (lu 2019) y de -4.56 días con ic al 95% de -7.32 a -1.80 (zhang 2017). una de las revisiones reportó que en ninguno de los estudios analizados se presentaron eventos adversos. no se encontraron resultados para los desenlaces de infección, sobreinfección, ni uso y tipo de antibiótico utilizado. en el análisis se encontró una reducción estadísticamente significativa de los días de estancia en uci de los pacientes que recibieron tratamiento con n-acetilcisteína intravenosa con dosis entre 150 y 200 mg/kg/día durante los primeros cinco días del sdra, en comparación con los pacientes que recibieron placebo o manejo usual. no se reportó diferencia estadísticamente significativa en la reducción de la mortalidad de los pacientes que recibieron nac. (623) calidad de vida 1. ¿cómo podemos medir la calidad de vida, en los pacientes con covid-19 que egresan de la uci? se recomienda utilizar los marcadores disponibles de severidad y del riesgo de mortalidad por covid-19 en los pacientes internados en la uci. amci ® las secuelas inmediatas en los pacientes víctimas del devastador ataque sistémico del covid-19 durante su estancia en la uci son valorables, pero no se dispone de herramientas que permita medir el grado de afectación de la calidad de vida de estos pacientes posterior al egreso de la uci o de alta hospitalaria, por lo tanto, se sugiere realizar estudios de creación, validación y utilización de instrumentos de valoración de la calidad de vida en pacientes con covid-19 posteriores al alta hospitalaria. parte importante de los pacientes con diagnóstico de covid-19 que ingresan a la uci, evolucionan tórpidamente presentando deterioro progresivo de los diferentes órganos llegando en pocos días a una falla multiorgánica (327) , estos pacientes presentan características clínicas y de laboratorio que se relacionan de manera significativa con mayor severidad y riesgo de mortalidad (294, 624) . a pesar de conocer con alguna precisión el riesgo de severidad y mortalidad de los pacientes que ingresan a la uci, no disponemos de un score que nos permita evaluar y predecir el grado de afectación en la calidad de vida de los pacientes que logran sobrevivir. aproximadamente un 6,1% del total de pacientes con enfermedad por covid-19 ingresan a uci, y de estos 2,3% sometidos a ventilación mecánica (327); lamentablemente los pacientes con enfermedad severa que logran sobrevivir y recuperarse han sido sometidos a una larga estancia en la uci y a ventilación mecánica invasiva con una intubación prolongada, que puede producir disfunción en la deglución impidiendo a la persona alimentarse de forma correcta y segura. es importante diagnosticar esta disfagia en los pacientes que se están recuperando del covid-19 y tratarla correctamente desde el principio para evitar complicaciones importantes como la malnutrición y la deshidratación, así como el riesgo de neumonía aspirativa. además de la disfagia, la fibrosis pulmonar y el riesgo de trombos son los problemas más frecuentes, pero no los únicos. una de las características de la enfermedad severa por covid-19 es que el virus provoca una enfermedad multiorgánica, con un amplio y heterogéneo abanico de secuelas cuyo alcance todavía se desconoce y aunque el órgano más afectado es el pulmón, puede afectar también otros órganos o sistemas incluido el snc, que en los casos más graves puede presentar encefalitis, delirios, desorientación y confusión, síntomas que pueden persistir tras el alta de la uci. otra secuela frecuente son las polineuropatías, esta afectación suele comenzar con una sensación de hormigueo en las extremidades y en los pacientes con covid-19 se presenta además con un cuadro de miositis que provoca debilidad y cansancio al caminar, a veces incluso en reposo; en algunos pacientes se presenta tal debilidad que dificulta llevar el alimento a la boca e incluso deglutirlo. sin embargo, la primera y más frecuente de las manifestaciones neurológicas del covid-19 es la pérdida del olfato, que a veces perdura como secuela tiempo después del alta. un estudio en 900 pacientes ingresados en el hospital clínico san carlos de madrid revela que el 70% había sufrido anosmia en mayor o menor grado. la importancia de este síntoma radica en que las fosas nasales pueden ser la vía de acceso del virus al sistema nervioso central. amci ® otras posibles secuelas neurológicas asociadas a la infección por covid-19 son la ageusia, la cefalea y amnesia a corto y mediano plazo. también son importantes las secuelas que afectan al sistema cardiovascular. un estudio publicado en la journal o the american medical association advierte que un 20% de los pacientes presenta una elevación de las enzimas que indican daño en el miocardio. la inflamación que provoca el virus puede provocar directamente ese daño y también puede agravar el estado de pacientes que ya tengan una patología cardiovascular de base, muchas miocarditis son reversibles, pero hay una parte importante que deja como secuela una pérdida de la función contráctil. todavía se desconoce el alcance y es difícil medir el impacto de la enfermedad sobre el corazón porque en algunos casos, los síntomas de insuficiencia cardíaca se confunden con los de la neumonía. otra de las complicaciones más frecuentes, y potencialmente más grave, afecta al mecanismo de coagulación de la sangre. durante el ingreso hospitalario se han visto numerosos casos de ictus. la secuela más importante es el riesgo de que se formen trombos, que pueden ir al pulmón o al cerebro, y si se producen en las arterias, pueden dar lugar a un infarto, aunque este efecto es mucho menos frecuente. eso explica que algunos pacientes de covid-19 dados de alta hayan tenido que volver a ingresar por trombosis. finalmente es importante tener presente que a las afectaciones que haya podido producir el virus, hay que sumar las secuelas neurológicas propias de una estancia prolongada en una unidad de cuidados intensivos que también pueden ser graves y a veces no se distinguen bien unas de otras. debilidad muscular, desorientación, depresión y problemas psicológicos son secuelas muy habituales entre los pacientes que salen de la uci por enfermedades diferentes. por la anterior razón es difícil, por ahora, saber qué es efecto directo del virus y que puede derivarse del proceso de hospitalización. aún es difícil decir si los daños a largo plazo dependen del propio virus o de los efectos adversos del proceso tratante. sin embargo, este análisis de las posibles secuelas del covid-19 en el cuerpo, se presenta con más dudas que certezas. como es habitual en medicina, las causas pueden ser múltiples y muchas veces reflejan la participación de varias complicaciones que se han podido dar durante el proceso infeccioso directo o por la hospitalización, la información sobre los mecanismos de invasión del sars-cov-2 en todos los órganos sigue siendo, por ahora, escasa. y también lo es nuestro conocimiento sobre los efectos adversos de los medicamentos, muchos de ellos experimentales, que se han utilizado durante esta crisis. gran parte de lo que conocemos actualmente sobre los efectos de este virus proviene de la experiencia clínica de otros colegas y de las historias de pacientes que han sufrido la enfermedad, quedando todavía mucho por descubrir. dado el actual panorama, diferentes hospitales e instituciones de salud se preparan en torno a la rehabilitación, habilitando ya unidades multidisciplinares poscovid-19 para el seguimiento de estos pacientes y algunos centros están contactando con los pacientes dados de alta para evaluar su estado y hacer un seguimiento a su salud. también están en marcha varios estudios multicéntricos para evaluar el alcance de las secuelas, prácticamente todos los centros sanitarios deberán tener pautas de seguimiento y control para los pacientes ya dados de alta, creándose necesario la utilización simultánea de amci ® instrumentos de medición de calidad de vida, que en nuestro país colombia, ya se han utilizado previamente con este fin (625, 626) . con este instrumento de medición de la calidad de vida se realizaron algunos estudios piloto tanto en pacientes crónicos como en la población general con el fin de determinar la comprensión del instrumento y factibilidad de aplicación del mismo en cuanto a la consistencia interna, la revisión realizada por vilagut y cols demostró que la aplicación de la escala arrojó en diversos estudios un alfa de cronbach igual o superior a 0.7 en todas las escalas excepto en función social (626) . y aunque un número cada vez mayor de estudios mide los resultados físicos, cognitivos, de salud mental y de calidad de vida relacionada con la salud (cvrs) en los sobrevivientes adultos de la uci, los datos sobre las propiedades de medición de tales instrumentos son escasos y, en general, de calidad deficiente a justa. se necesitan análisis empíricos que evalúen el rendimiento de los instrumentos en adultos sobrevivientes de la uci para avanzar en la investigación en este campo (627) . finalmente, el conocimiento de las secuelas y complicaciones dejadas por la infección del covid-19, permitirá identificar importantes variables clínicas que acompañan a esta enfermedad y que afectan de manera importante la calidad de vida de los pacientes que padecen la enfermedad severa en la unidad de cuidados intensivos. en la actualidad no existen estudios para evaluar el riesgo de malnutrición aguda en pacientes hospitalizados por sars-cov-2. experiencias con otras infecciones virales por influenza, se han identificado como factores asociados con mortalidad, la presencia de malnutrición, la adquisición de infección intrahospitalarias, la falla respiratoria y la presencia de infiltrados en la radiografía de tórax (628) . las guías espen 2020 recomiendan utilizar el must o el nrs-2002(2), para la tamización del riesgo nutricional, estos puntajes de tamización previamente han sido validados en múltiples patologías y contextos clínicos; sin embargo, existen otros puntajes útiles desde la perspectiva clínica como la valoración global subjetiva, mini-nutritional asessment(629), puntaje nutric (630) y la global leadership initiative on malnutrition (glim) (631) . el proceso de diagnóstico nutricional debe involucrar, dos componentes: la identificación del riesgo con la utilización de alguno de los puntajes previamente validados en otros contextos y posteriormente el diagnóstico de los pacientes con malnutrición y la valoración de la gravedad de ésta; en este último paso es importante la valoración del índice de masa corporal, los hábitos de consumo calórico y proteico, la presencia de inflamación, los trastornos gastrointestinales, las enfermedades de base y siempre que sea factible el cálculo de la masa muscular. tabla 20. en vista del riesgo de infección al personal de salud, no siempre será necesario la visita nutricional al paciente, ésta podría ayudarse con entrevista al familiar, interrogatorio vía amci ® telefónica y sólo en caso necesario el examen del paciente para lo cual se requiere el uso de equipo de protección personal completo. esta estrategia de interrogatorio al familiar o al paciente por vía remota o telefónica puede ayudar a identificar los patrones de consumo y los hábitos nutricionales de riesgo y en caso de ser necesario la valoración nutricional disminuye el tiempo de exposición a un ambiente contaminado. para la atención presencial de pacientes en el ámbito de cuidado intensivo, es necesario definir cuál es el riesgo que existe de infección para el personal de salud, para aclarar esta pregunta se debe definir si hay un riesgo de generación de aerosoles(88). aunque no existen pautas específicas para la nutrición en pacientes con covid-19, las diferentes sociedades científicas han desarrollado guías de pauta clínica para la nutrición de pacientes con esta enfermedad (632, 633) . idealmente la nutrición debe ser iniciada de forma temprana, esto se refiere al inicio en las primeras 24 a 36 horas del ingreso a cuidado intensivo o en las primeras 12 horas luego de la intubación y el inicio de la ventilación mecánica(633) y se prefiere la vía enteral. aunque no existen estudios para evaluar el momento del inicio de la nutrición en pacientes con infección por sars-cov2, el inicio temprano de la nutrición ha mostrado beneficios en términos de mortalidad y reducción de infecciones con dicha estrategia (634, 635) . adicionalmente es importante, evaluar el riesgo de morbilidad y mortalidad asociado a la malnutrición aguda en el ámbito del paciente crítico, en los pacientes que no se alcance la meta de aporte calórico y proteico por vía enteral o que exista contraindicación para ésta, se debe considerar el inicio de nutrición por vía parenteral, especialmente cuando su riesgo nutricional agudo sea elevado (puntaje nutric ≥5, nrs ≥5) (636) (637) . el choque no es una contraindicación para la utilización de nutrición enteral (638) y no es una indicación para el uso de nutrición parenteral, quizás la mejor estrategia, es vigilar la presencia de disfunción gastrointestinal, en combinación con la presencia de intolerancia a la nutrición enteral, especialmente en pacientes con acidosis láctica en progreso y cuando sea necesario escalar la dosis de vasopresores o exista incapacidad para la reducción de éstos. no es necesario medir el residuo gástrico de rutina, es preferible iniciar procinéticos de forma rutinaria. la sonda debe colocarse con cuidado de evitar riesgo de contaminación, preferiblemente al entubar al paciente. algunos pacientes pueden presentar diarrea, ya que se ha descubierto la presencia de la proteína ace2 (receptor del virus sars-cov-2) en células del esófago, estómago, duodeno y recto. no existe evidencia que indique que la nutrición enteral durante la posición prono aumente el riesgo de complicaciones. sugerimos no suspender nutrición enteral al durante la pronación, se debe iniciar con dosis trófica de 20 ml/h. amci ® se recomienda una estrecha monitorización de la tolerancia a la nutrición enteral para pacientes en posición prono. se recomienda para aumentar la tolerancia de la ne a los pacientes en posición prona, una elevación del tórax entre 10-25º (posición de trendelenburg inversa) no realizar endoscopias digestivas para ubicación de sondas avanzadas recomendaciones de nutrición parenteral los pacientes con covid-19 pueden requerir niveles significativos de sedación y bloqueo neuromuscular, lo que puede aumentar la incidencia de intolerancia gastrointestinal. la nutrición parenteral (np) debe utilizarse donde la alimentación enteral no está disponible o no logra completar el 60% de los requerimientos. si existen limitaciones para la ruta enteral, se podría recomendar nutrición parenteral periférica (npp) en la población que no alcanza el objetivo proteico energético por nutrición oral o enteral. la np temprana debe iniciarse lo antes posible en el paciente de alto riesgo para el cual la ne gástrica o yeyunal temprana no es factible. los pacientes de alto riesgo incluyen aquellos con sepsis o shock que requieren múltiples vasopresores o en aumento, o cuando se requiere soporte respiratorio de alta presión (niv, cpap o peep). metodología de calificación y resumen de las opiniones dentro de consensos formales how to use the nominal group and delphi techniques transforming ors into icus decreto 417 del 17 de marzo 2020 impact of non-pharmaceutical interventions (npis) to reduce covid-19 mortality and healthcare demand how will countrybased mitigation measures influence the course of the covid-19 epidemic? incubation period and other epidemiological characteristics of 2019 novel coronavirus infections with right truncation: a statistical analysis of publicly available case data early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia pattern of early human-to-human transmission of wuhan estimates of the severity of coronavirus disease 2019: a model-based analysis global telemedicine implementation and integration within health systems to fight the covid-19 pandemic: a call to action telemedicina durante la epidemia de covid-19 en chile: guía de buenas prácticas y recomendaciones evaluating tele-icu cost--an imperfect science amci ® 19. colombia pdlr an international survey of training in adult intensive care medicine do intensivist staffing patterns influence hospital mortality following icu admission? a systematic review and meta-analyses nighttime physician staffing improves patient outcomes: no impact of critical care physician staffing on patients with septic shock in a university hospital medical intensive care unit on-site physician staffing in a community hospital intensive care unit. impact on test and procedure use and on patient outcome effect on icu mortality of a full-time critical care specialist intensive care unit physician staffing: seven days a week, 24 hours a day intensivist-to-bed ratio: association with outcomes in the medical icu the effect of multidisciplinary care teams on intensive care unit mortality potential reduction in mortality rates using an intensivist model to manage intensive care units physician staffing patterns and clinical outcomes in critically ill patients: a systematic review association between critical care physician management and patient mortality in the intensive care unit variation in intensive care unit outcomes: a search for the evidence on organizational factors variation in critical care services across north america and western europe benchmark data from more than 240,000 adults that reflect the current practice of critical care in the united states impact of telemedicine intensive care unit coverage on patient outcomes: a systematic review and meta-analysis the use of nonphysician providers in adult intensive care units clinical course and risk factors for mortality of adult inpatients with covid-19 in wuhan, china: a retrospective cohort study presenting characteristics, comorbidities, and outcomes among 5700 patients hospitalized with covid-19 in the new york city area comparison of crb-65 and quick sepsis-related organ failure assessment for predicting the need for intensive respiratory or vasopressor support in patients with covid-19 acute medical care. the right person, in the right setting--first time: how does practice match the report recommendations the ability of the national early warning score (news) to discriminate patients at risk of early cardiac arrest, unanticipated intensive care unit admission, and death covid-19: symptoms, course of illness and use of clinical scoring systems for the first 42 patients admitted to a norwegian local hospital the third international consensus definitions for sepsis and septic shock sirs and news for predicting inhospital mortality and icu admission in emergency admissions treated as sepsis development and validation of a clinical risk score to predict the occurrence of critical illness in hospitalized patients with covid-19 triage: care of the critically ill and injured during pandemics and disasters: chest consensus statement intensive care during the 2019-coronavirus epidemic therapeutic and triage strategies for 2019 novel coronavirus disease in fever clinics critical care utilization for the covid-19 early experience and forecast during an emergency response icu admission, discharge, and triage guidelines: a framework to enhance clinical operations, development of institutional policies, and further research amci ® 54. swiss society of intensive care medicine. recommendations for the admission of patients with covid-19 to intensive care and intermediate care units manual de bioseguridad para prestadores de servicios de salud que brinden atención en salud ante la eventual ethical framework for health care institutions & guidelines for institutional ethics services responding to the coronavirus pandemic how should health systems prepare for the evolving covid-19 pandemic? reflections from the perspective of a tertiary cancer center compassionate use of remdesivir for patients with severe covid-19 recomendaciones generales para la toma de decisiones éticas en los servicios de salud durante la pandemia covid-19 col dsyps. resolución 2665 de 2018 para los suscritos después de junio col dsyps. consentimiento informado para acompañante de casos probable/confirmado de covid-19 informed consent: not just for procedures anymore palliative-end-of-life-and-bereavement-care-22-march-2020.pdf2020 [ 74. asedp. guía práctica de cuidados paliativos en situación de pandemia covid-19 directing citizens to create advance directives who guidelines on hand hygiene in health care: first global patient safety challenge clean care is safer care pdf;jsessionid =bd8875d77b112c71b7b5743aa9428b0c?sequence=12009 [ 80. standardization ecf. protective clothing-performance requirements and tests methods for protective clothing against infective agents cdc. guidance for the selection and use of personal protective equipment (ppe) in healthcare settings filtering facepiece respirators in healthcare settings n95-respirators-surgical-masks-and-face-masks2020 [ 86. anvisa. guidelines for health services: prevention and control measures that should be adopted when assisting suspected or confirmed cases of infection with the new coronavirus personal protective equipment for the ebola virus disease: a comparison of 2 training programs effect of single-versus doublegloving on virus transfer to health care workers' skin and clothing during removal of personal protective equipment assessment of self-contamination during removal of personal protective equipment for ebola patient care equipment-infection-control/n95-respirators-surgical-masks-and-face-masks2020 [ 94. cdc. interim domestic guidance on the use of respirators to prevent transmission of sars infection control measures for operative procedures in severe acute respiratory syndrome-related patients commentary: protecting health workers from airborne mers-cov-learning from sars safety recommendations for evaluation and surgery of the head and neck during the covid-19 pandemic aerosol generating procedures and risk of transmission of acute respiratory infections to healthcare workers: a systematic review minimally invasive surgery and the novel coronavirus outbreak: lessons learned in china and italy sars-cov-2 viral load in upper respiratory specimens of infected patients covid-19 and the otolaryngologist: preliminary evidence-based review what we do when a covid-19 patient needs an operation: operating room preparation and guidance a retrospective study of success, failure, and time needed to perform awake intubation detection of sars-associated coronavirus in throat wash and saliva in early diagnosis the infection evidence of sars-cov-2 in ocular surface： a singlecenter cross-sectional study ophthalmologic evidence against the interpersonal transmission of review article: gastrointestinal features in covid-19 and the possibility of faecal transmission liver injury in covid-19: management and challenges plan de contingencia para los servicios de medicina intensiva frente a la pandemia covid-19 plan de contingencia para los servicios de medicina intensiva frente a la pandemia covid-19 seeiuc. plan de contingencia para los servicios de medicina intensiva frente a la pandemia covid-19 the incubation period of coronavirus disease 2019 (covid-19) from publicly reported confirmed cases: estimation and application clinical features of patients infected with 2019 novel coronavirus in wuhan covid-19: consider cytokine storm syndromes and immunosuppression covid-19 illness in native and immunosuppressed states: a clinical-therapeutic staging proposal covid-19 pneumonia: ards or not? management of covid-19 respiratory distress stages or phenotypes? a critical look at covid-19 pathophysiology characteristics of and important lessons from the coronavirus disease 2019 (covid-19) outbreak in china: summary of a report of 72 314 cases from the chinese center for disease control and prevention sars-cov-2 rna, qualitative real-time rtpcr interim guidelines for collecting, handling, and testing clinical specimens for evaluating the accuracy of different respiratory specimens in the laboratory diagnosis and monitoring the viral shedding of comparative accuracy of oropharyngeal and nasopharyngeal swabs for diagnosis of covid-19 detection of sars-cov-2 in different types of clinical specimens global surveillance for covid-19 caused by human infection with covid-19 virus intensive care management of coronavirus disease 2019 (covid-19): challenges and recommendations variation in false-negative rate of reverse transcriptase polymerase chain reaction-based sars-cov-2 tests by time since exposure severe acute respiratory syndrome coronavirus 2-specific antibody responses in coronavirus disease serology for sars-cov-2: apprehensions, opportunities, and the path forward profile of igg and igm antibodies against severe acute respiratory syndrome coronavirus 2 (sars-cov-2) profiling early humoral response to diagnose novel coronavirus disease (covid-19) antibody responses to sars-cov-2 in patients of novel coronavirus disease 2019 prognostic value of interleukin-6, c-reactive protein correction to: clinical predictors of mortality due to covid-19 based on an analysis of data of 150 patients from wuhan clinical course and outcomes of critically ill patients with sars-cov-2 pneumonia in wuhan, china: a single-centered, retrospective, observational study the role of biomarkers in diagnosis of covid-19 -a systematic review cytokine release syndrome in severe covid-19 epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a descriptive study pathogenic human coronavirus infections: causes and consequences of cytokine storm and immunopathology analysis of serum cytokines in patients with severe acute respiratory syndrome covid-19 infection: the perspectives on immune responses pneumonia and respiratory failure from swine-origin influenza a (h1n1) in mexico tocilizumab treatment in covid-19: a single center experience effective treatment of severe covid-19 patients with tocilizumab diagnosis and treatment of novel coronavirus pneumonia based on the theory of traditional chinese medicine a rapid advice guideline for the diagnosis and treatment of 2019 novel coronavirus (2019-ncov) infected pneumonia (standard version) our italian experience using lung ultrasound for identification, grading and serial follow-up of severity of lung involvement for management of patients with covid-19 point of care and intensive care lung ultrasound: a reference guide for practitioners during covid-19. radiography (lond) point-of-care lung ultrasound findings in patients with covid-19 pneumonia cardiovascular considerations for patients, health care workers, and health systems during the covid-19 pandemic d-dimer in patients infected with covid-19 and suspected pulmonary embolism anticoagulant treatment is associated with decreased mortality in severe coronavirus disease 2019 patients with coagulopathy anticoagulation in covid-19 procalcitonin to initiate or discontinue antibiotics in acute respiratory tract infections procalcitonin to distinguish viral from bacterial pneumonia: a systematic review and meta-analysis procalcitonin in patients with severe coronavirus disease 2019 (covid-19): a meta-analysis viral load of sars-cov-2 in clinical samples discharge criteria for confirmed covid-19 cases -when is it safe to discharge covid-19 cases from the hospital or end home isolation? epidemiologic features and clinical course of patients infected with sars-cov-2 in singapore. jama. 2020. 165. who. clinical management of severe acute respiratory infection when novel coronavirus (2019-ncov) infection is suspected: interim guidance surviving sepsis campaign: guidelines on the management of critically ill adults with coronavirus disease 2019 (covid-19) clinical consensus recommendations regarding non-invasive respiratory support in the adult patient with acute respiratory failure secondary to sars-cov-2 infection current evidence for the effectiveness of heated and humidified high flow nasal cannula supportive therapy in adult patients with respiratory failure high-flow nasal cannula oxygen therapy is superior to conventional oxygen therapy but not to noninvasive mechanical ventilation on intubation rate: a systematic review and meta-analysis comparison of high-flow nasal cannula versus oxygen face mask for environmental bacterial contamination in critically ill pneumonia patients: a randomized controlled crossover trial covid-19): protecting hospitals from the invisible high-flow nasal cannula may be no safer than noninvasive positive pressure ventilation for covid-19 patients high-flow nasal cannula for covid-19 patients: low risk of bio-aerosol dispersion hospitalized patients with 2019 novel coronavirus-infected pneumonia in wuhan, china the use of high-flow nasal oxygen in covid-19 high-flow nasal cannula oxygenation revisited in covid-19 high flow nasal cannula is a good treatment option for covid-19. heart lung patient selfproning with high-flow nasal cannula improves oxygenation in covid-19 pneumonia expert recommendations for tracheal intubation in critically ill patients with noval coronavirus disease intubation and ventilation amid the covid-19 outbreak: wuhan's experience sedation for critically ill patients with covid-19: which specificities? one size does not fit all covid-19: icu delirium management during sars-cov-2 pandemic protocolized intensive care unit management of analgesia, sedation, and delirium improves analgesia and subsyndromal delirium rates changing sedation practices in the intensive care unit--protocol implementation, multifaceted multidisciplinary approach and teamwork effects of neuromuscular blockers on transpulmonary pressures in moderate to severe acute respiratory distress syndrome early deep sedation is associated with decreased in-hospital and two-year follow-up survival pain management protocol implementation and opioid consumption in critical care: an interrupted time series analysis. rev bras ter intensiva ketamine added to morphine or hydromorphone patient-controlled analgesia for acute postoperative pain in adults: a systematic review and meta-analysis of randomized trials dexmedetomidine vs midazolam for sedation of critically ill patients: a randomized trial a randomized, double-blind pilot study of dexmedetomidine versus midazolam for intensive care unit sedation: patient recall of their experiences and short-term psychological outcomes effect of dexmedetomidine added to standard care on ventilator-free time in patients with agitated delirium: a randomized clinical trial ventilation with lower tidal volumes as compared with traditional tidal volumes for acute lung injury and the acute respiratory distress syndrome lower tidal volume at initiation of mechanical ventilation may reduce progression to acute respiratory distress syndrome: a systematic review effects of interventions on survival in acute respiratory distress syndrome: an umbrella review of 159 published randomized trials and 29 meta-analyses mortality in multicenter critical care trials: an analysis of interventions with a significant effect covid-19 pneumonia: different respiratory treatment for different phenotypes? incidence, clinical course, and outcome in 217 patients with acute respiratory distress syndrome driving pressure and survival in the acute respiratory distress syndrome driving pressure: a marker of severity, a safety limit, or a goal for mechanical ventilation? arterial hyperoxia and mortality in critically ill patients: a systematic review and meta-analysis association between administered oxygen, arterial partial oxygen pressure and mortality in mechanically ventilated intensive care unit patients oxygen exposure resulting in arterial oxygen tensions above the protocol goal was associated with worse clinical outcomes in acute respiratory distress syndrome documento de rionegro surviving sepsis campaign: guidelines on the management of critically ill adults with coronavirus disease 2019 (covid-19) covid-19 pneumonia: different respiratory treatments for different phenotypes? intensive care medicine subphenotyping ards in covid-19 patients: consequences for ventilator management distinct phenotypes require distinct respiratory management strategies in severe covid-19 acute respiratory distress syndrome: advances in diagnosis and treatment acute respiratory distress syndrome the berlin definition an official american thoracic society/european society of intensive care medicine/society of critical care medicine clinical practice guideline: mechanical ventilation in adult patients with acute respiratory distress syndrome covid-19 pandemic: overview of protective-ventilation strategy in ards patients respiratory support for adult patients with covid-19 respiratory monitoring in adult intensive care unit extracorporeal membrane oxygenation for ards in adults extracorporeal membrane oxygenation for severe acute respiratory distress syndrome extracorporeal membrane oxygenation as treatment of severe covid-19 infection: a case report optimizing provision of extracorporeal life support during the covid-19 pandemic: practical considerations for canadian jurisdictions rescue therapy for severe covid-19 associated acute respiratory distress syndrome (ards) with tissue plasminogen activator (tpa): a case series early outcomes with utilization of tissue plasminogen activator in covid-19 associated respiratory distress: a series of five cases tissue plasminogen activator (tpa) treatment for covid-19 associated acute respiratory distress syndrome (ards): a case series inhaled nitric oxide for acute respiratory distress syndrome (ards) and acute lung injury in children and adults outpatient inhaled nitric oxide in a patient with vasoreactive ipah and covid-19 infection lung recruitability in covid-19-associated acute respiratory distress syndrome: a single-center observational study acute respiratory failure in covid-19: is it "typical respiratory failure in patients infected with sars-cov-2 association between hypoxemia and mortality in patients with covid-19 ventilatory ratio in hypercapnic mechanically ventilated patients with covid-19-associated acute respiratory distress syndrome ventilator-induced lung injury esophageal pressure monitoring: why, when and how? manejo clínico de la infección respiratoria aguda grave (irag) en caso de sospecha de covid-19 early neuromuscular blockade in the acute respiratory distress syndrome inhaled nitric oxide in acute respiratory distress syndrome: from bench to bedside inhaled nitric oxide for acute respiratory distress syndrome (ards) in children and adults inhalation of nitric oxide in the treatment of severe acute respiratory syndrome: a rescue trial in beijing noninvasive ventilation in critically ill patients with the middle east respiratory syndrome. influenza other respir viruses clinical course and outcomes of critically ill patients with middle east respiratory syndrome coronavirus infection effect of nitric oxide on oxygenation and mortality in acute lung injury: systematic review and metaanalysis the toxicology of inhaled nitric oxide the pathophysiology of nitrogen dioxide during inhaled nitric oxide therapy use of extreme position changes in acute respiratory failure effects of the prone position on respiratory mechanics and gas exchange during acute lung injury the prone position eliminates compression of the lungs by the heart effects of prone positioning on lung protection in patients with acute respiratory distress syndrome body position changes redistribute lung computed-tomographic density in patients with acute respiratory failure: impact and clinical fallout through the following 20 years effect of prone positioning on the survival of patients with acute respiratory failure effects of systematic prone positioning in hypoxemic acute respiratory failure: a randomized controlled trial prone positioning improves oxygenation in post-traumatic lung injury--a prospective randomized trial a multicenter trial of prolonged prone ventilation in severe acute respiratory distress syndrome prone positioning in acute respiratory distress syndrome: a multicenter randomized clinical trial prone positioning in patients with moderate and severe acute respiratory distress syndrome: a randomized controlled trial prone positioning in severe acute respiratory distress syndrome effect of prone positioning on clinical outcomes in children with acute lung injury: a randomized controlled trial neuromuscular blockers in early acute respiratory distress syndrome the efficacy and safety of prone positional ventilation in acute respiratory distress syndrome: updated study-level meta-analysis of 11 randomized controlled trials prone ventilation reduces mortality in patients with acute respiratory failure and severe hypoxemia: systematic review and meta-analysis an updated study-level meta-analysis of randomised controlled trials on proning in ards and acute lung injury noninvasive versus invasive mechanical ventilation for immunocompromised patients with acute respiratory failure: a systematic review and meta-analysis discontinuing mechanical ventilatory support the ventilator discontinuation process: an expanding evidence base delayed weaning in mechanically ventilated patients: a call for implementation of weaning protocols in low-and middle-income countries effect of pressure support vs t-piece ventilation strategies during spontaneous breathing trials on successful extubation among patients receiving mechanical ventilation: a randomized clinical trial mechanical ventilation weaning issues can be reconnection to mechanical ventilation for 1 h after a successful spontaneous breathing trial reduces reintubation in critically ill patients: a multicenter randomized controlled trial lidocaine during intubation and extubation in patients with coronavirus disease (covid-19) official ers/ats clinical practice guidelines: noninvasive ventilation for acute respiratory failure non-invasive positive pressure ventilation to treat respiratory failure resulting from exacerbations of chronic obstructive pulmonary disease: cochrane systematic review and meta-analysis meta-analysis: noninvasive ventilation in acute cardiogenic pulmonary edema noninvasive ventilation in acute heart failure noninvasive ventilation in immunosuppressed patients with pulmonary infiltrates, fever, and acute respiratory failure is there a role for noninvasive ventilation in acute respiratory distress syndrome? a meta-analysis pandemic 2009 influenza a in argentina: a study of 337 patients on mechanical ventilation early noninvasive ventilation treatment for severe influenza pneumonia critically ill patients with 2009 influenza a(h1n1) infection in canada clinical characteristics and outcomes of patients with 2009 influenza a(h1n1) virus infection with respiratory failure requiring mechanical ventilation the sars, mers and novel coronavirus (covid-19) epidemics, the newest and biggest global health threats: what lessons have we learned? more awareness is needed for severe acute respiratory syndrome coronavirus 2019 transmission through exhaled air during noninvasive respiratory support: experience from china covid-19 pandemic and non invasive respiratory management: every goliath needs a david. an evidence based evaluation of problems clinical outcomes of patients requiring ventilatory support in brazilian intensive care units: a multicenter, prospective, cohort study comparative effectiveness of noninvasive and invasive ventilation in critically ill patients with acute exacerbation of chronic obstructive pulmonary disease assessment of heart rate, acidosis, consciousness, oxygenation, and respiratory rate to predict noninvasive ventilation failure in hypoxemic patients an index combining respiratory rate and oxygenation to predict outcome of nasal high-flow therapy british thoracic society/intensive care society guideline for the ventilatory management of acute hypercapnic respiratory failure in adults baseline characteristics and outcomes of 1591 patients infected with sars-cov-2 admitted to icus of the lombardy region prone position for acute respiratory distress syndrome. a systematic review and meta-analysis efficacy and safety of early prone positioning combined with hfnc or niv in moderate to severe ards: a multi-center prospective cohort study respiratory parameters in patients with covid-19 after using noninvasive ventilation in the prone position outside the intensive care unit use of prone positioning in nonintubated patients with covid-19 and hypoxemic acute respiratory failure first case of covid-19 complicated with fulminant myocarditis: a case report and insights. infection cardiac involvement in a patient with coronavirus disease 2019 (covid-19) suspected myocardial injury in patients with covid-19: evidence from front-line clinical observation in wuhan, china cardiovascular phenotypes in ventilated patients with covid-19 acute respiratory distress syndrome covid-19 and acute heart failure: screening the critically ill -a position statement of the cardiac society of australia and new zealand (csanz) characteristics and clinical significance of myocardial injury in patients with severe coronavirus disease 2019 ase statement on protection of patients and echocardiography service providers during the 2019 novel coronavirus outbreak clinical guidance regarding provision of echocardiography during the covid-19 pandemic guidance for the diagnosis and management of cv disease during the covid-19 pandemic echocardiography during the coronavirus disease 2019 (covid-19) pandemic: expert opinion of the working group on echocardiography of the polish cardiac society echocardiography in the time of covid-19 utilization and appropriateness of transthoracic echocardiography in response to the covid-19 pandemic covid-19 illness and heart failure: a missing link? covid-19 clinical guidance for the cardiovascular care team surviving sepsis campaign: international guidelines for management of sepsis and septic shock hemodynamic monitoring options in covid-19 advanced pulmonary and cardiac support of covid-19 patients: emerging recommendations from asaio hemodynamic assessment in the contemporary intensive care unit: a review of circulatory monitoring devices early use of the pulmonary artery catheter and outcomes in patients with shock and acute respiratory distress syndrome: a randomized controlled trial experts' opinion on management of hemodynamics in ards patients: focus on the effects of mechanical ventilation the clinical usefulness of extravascular lung water and pulmonary vascular permeability index to diagnose and characterize pulmonary edema: a prospective multicenter study on the quantitative differential diagnostic definition for acute lung injury/acute respiratory distress syndrome extravascular lung water and pulmonary arterial wedge pressure for fluid management in patients with acute respiratory distress syndrome recommendations for echocardiography laboratories participating in cardiac point of care cardiac ultrasound (pocus) and critical care echocardiography training: report from the american society of echocardiography bedside focused cardiac ultrasound in covid-19 from the wuhan epicenter: the role of cardiac point-of-care ultrasound, limited transthoracic echocardiography, and critical care echocardiography echocardiography in pandemic: front-line perspective, expanding role of ultrasound, and ethics of resource allocation can lung us help critical care clinicians in the early diagnosis of novel coronavirus (covid-19) pneumonia? ccusg) cccusg. findings of lung ultrasonography of novel corona virus pneumonia during the 2019-2020 epidemic ultrasound in covid-19: a timeline of ultrasound findings in relation to ct a preliminary study on the ultrasonic manifestations of peripulmonary lesions of non-critical novel coronavirus pneumonia effect of focused bedside ultrasonography in hypotensive patients on the clinical decision of emergency physicians accuracy of point-of-care multiorgan ultrasonography for the diagnosis of pulmonary embolism will this hemodynamically unstable patient respond to a bolus of intravenous fluids? predict) priedic. does respiratory variation in inferior vena cava diameter predict fluid responsiveness: a systematic review and meta-analysis does central venous pressure predict fluid responsiveness? a systematic review of the literature and the tale of seven mares clinical characteristics of coronavirus disease 2019 in china comparison of two fluid-management strategies in acute lung injury the surviving sepsis campaign bundle: 2018 update incorporating dynamic assessment of fluid responsiveness into goal-directed therapy: a systematic review and meta-analysis relative efficacy and safety of early lactate clearance-guided therapy resuscitation in patients with sepsis: a meta-analysis effect of a resuscitation strategy targeting peripheral perfusion status vs serum lactate levels on 28-day mortality among patients with septic shock: the andromeda-shock randomized clinical trial pooled analysis of higher versus lower blood pressure targets for vasopressor therapy septic and vasodilatory shock scandinavian ssai clinical practice guideline on choice of first-line vasopressor for patients with acute circulatory failure vasopressors for hypotensive shock canadian critical care society clinical practice guideline: the use of vasopressin and vasopressin analogues in critically ill adults with distributive shock cardiovascular disease, drug therapy, and mortality in covid-19 cardiovascular considerations in treating patients with coronavirus disease 2019 (covid-19) clinical characteristics of 113 deceased patients with coronavirus disease 2019: retrospective study inclusion of high-fidelity simulation training in a critical care nursing internship high-fidelity simulation in a clinical care unit is feasible and safe interim guidance for basic and advanced life support in adults, children, and neonates with suspected or confirmed covid-19 ropa y equipo de protección para los trabajadores sanitarios para evitar que se contagien con el coronavirus y otras enfermedades altamente infecciosas adult advanced life support for covid-19 covid-19 reverse cpr: a pilot study of cpr in the prone position central nervous system manifestations of covid-19: a systematic review an italian multicenter retrospective-prospective observational study on neurological manifestations of covid-19 (neurocovid) olfactory and gustatory dysfunctions as a clinical presentation of mild-to-moderate forms of the coronavirus disease (covid-19): a multicenter european study update on the neurology of covid-19 coronavirus immunoreactivity in individuals with a recent onset of psychotic symptoms potential neurological impact of coronaviruses: implications for the novel sars-cov-2 neurological insights of covid-19 pandemic neurological manifestations of covid-19 and other coronavirus infections: a systematic review human coronaviruses and other respiratory viruses: underestimated opportunistic pathogens of the central nervous system? viruses neurological manifestations in covid-19 caused by sars-cov-2 cns penetration of potential anti-covid-19 drugs central nervous system involvement by severe acute respiratory syndrome coronavirus-2 (sars-cov-2) neurological manifestations of patients with covid-19: potential routes of sars-cov-2 neuroinvasion from the periphery to the brain neurological complications of coronavirus and covid-19 autonomic brain centers and pathophysiology of covid-19 neuropathologists play a key role in establishing the extent of covid-19 in human patients does covid19 infect the brain? if so, smokers might be at a higher risk neurologic manifestations of hospitalized patients with coronavirus disease neurological complications of coronavirus disease (covid-19): encephalopathy. cureus coronavirus disease 2019 (covid-19) in neurology and neurosurgery: a scoping review of the early literature prevalence of comorbidities and its effects in patients infected with sars-cov-2: a systematic review and metaanalysis protected code stroke: hyperacute stroke management during the coronavirus disease brain mri findings in patients in the intensive care unit with covid-19 infection neurologic features in severe sars-cov-2 infection evidence of the covid-19 virus targeting the cns: tissue distribution, host-virus interaction, and proposed neurotropic mechanisms epidemiology of covid-19 in a long-term care facility in king county, washington acute cerebrovascular disease following covid-19: a single center neurological manifestations of hospitalized patients with covid-19 in wuhan, china: a retrospective case series study improved epileptic seizure detection combining dynamic feature normalization with eeg novelty detection acute systemic complications of convulsive status epilepticus-a systematic review outcome predictors for status epilepticus--what really counts tratamiento del estado epiléptico, consenso de expertos consensus for prevention and management of coronavirus disease 2019 (covid-19) for neurologists. stroke vasc neurol recomendaciones de diagnóstico y tratamiento del ataque cerebrovascular isquémico agudo durante la contingencia por covid-19 seizures associated with coronavirus infections a first case of meningitis/encephalitis associated with sars-coronavirus-2 neurological manifestations and complications of covid-19: a literature review does sars-cov-2 invade the brain? translational lessons from animal models guillain barre syndrome associated with covid-19 infection: a case report lung-kidney interactions in critically ill patients: consensus report of the acute disease quality initiative (adqi) 21 workgroup intravascular volume assessment in the critically ill patient the dose response multicentre investigation on fluid assessment (doremifa) in critically ill patients fluid management with a simplified conservative protocol for the acute respiratory distress syndrome* surviving sepsis campaign: guidelines on the management of critically ill adults with coronavirus disease 2019 (covid-19) effects of very early start of norepinephrine in patients with septic shock: a propensity score-based analysis the clinical relevance of oliguria in the critically ill patient: analysis of a large observational database clinical characteristics of coronavirus disease 2019 (covid-19) in china: a systematic review and meta-analysis management of acute kidney injury in patients with covid-19 liver and kidney injuries in covid-19 and their effects on drug therapy clinical characteristics of risk factors associated with acute respiratory distress syndrome and death in patients with coronavirus disease mitigating risk of covid-19 in dialysis facilities additional guidance for infection prevention and control recommendations for patients with suspected or confirmed covid-19 in outpatient hemodialysis facilities sars-cov-2 in the peritoneal waste in a patient treated with peritoneal dialysis antibody response and viraemia during the course of severe acute respiratory syndrome (sars)-associated coronavirus infection kidney disease is associated with in-hospital death of patients with covid-19 acute kidney injury in patients hospitalized with covid-19 acute kidney injury in hospitalized patients with coronavirus disease 2019 (covid-19): a meta-analysis collapsing glomerulopathy in a patient with coronavirus disease 2019 (covid-19) renal histopathological analysis of 26 postmortem findings of patients with covid-19 in china collapsing glomerulopathy in a covid-19 patient renal involvement and early prognosis in patients with covid-19 pneumonia expert consensus on the application of special blood purification technology in severe covid-19 pneumonia kidney involvement in covid-19 and rationale for extracorporeal therapies factors associated with acute kidney injury in acute respiratory distress syndrome effect on extrapulmonary sepsis-induced acute lung injury by hemoperfusion with neutral microporous resin column kdigo guidelines:modality of renal replacement therapy for patients with aki potential effect of blood purification therapy in reducing cytokine storm as a late complication of critically ill covid-19 new fronts emerge in the influenza cytokine storm coronavirus epidemic: preparing for extracorporeal organ support in intensive care diagnosis and treatment novel coronavirus pneumonia (covid (covid-19): international expert opinions and coronavirus epidemic and extracorporeal therapies in intensive care: si vis pacem para bellum group rc. effect of dexamethasone in hospitalized patients with covid-19: preliminary report remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus (2019-ncov) in vitro hydroxychloroquine, a less toxic derivative of chloroquine, is effective in inhibiting sars-cov-2 infection in vitro in vitro antiviral activity and projection of optimized dosing design of hydroxychloroquine for the treatment of severe acute respiratory syndrome coronavirus 2 (sars-cov-2) zhejiang da xue xue bao yi xue ban breakthrough: chloroquine phosphate has shown apparent efficacy in treatment of covid-19 associated pneumonia in clinical studies treating covid-19 with chloroquine clinical efficacy of hydroxychloroquine in patients with covid-19 pneumonia who require oxygen: observational comparative study using routine care data effect of high vs low doses of chloroquine diphosphate as adjunctive therapy for patients hospitalized with severe acute respiratory syndrome coronavirus 2 (sars-cov-2) infection: a randomized clinical trial hydroxychloroquine in patients with mainly mild to moderate coronavirus disease 2019: open label, randomised controlled trial clinical outcomes of amci ® patients with covid-19: a quasi-randomized comparative study observational study of hydroxychloroquine in hospitalized patients with covid-19 hydroxychloroquine and azithromycin as a treatment of covid-19: results of an openlabel non-randomized clinical trial clinical and microbiological effect of a combination of hydroxychloroquine and azithromycin in 80 covid-19 patients with at least a six-day follow up: a pilot observational study early treatment of covid-19 patients with hydroxychloroquine and azithromycin: a retrospective analysis of 1061 cases in marseille, france in vitro testing of combined hydroxychloroquine and azithromycin on sars-cov-2 shows synergistic effect outcomes of hydroxychloroquine usage in united states veterans hospitalized with covid-19. medrxiv association of treatment with hydroxychloroquine or azithromycin with in-hospital mortality in patients with covid-19 in new york state treatment of severe acute respiratory syndrome with lopinavir/ritonavir: a multicentre retrospective matched cohort study clinical characteristics of novel coronavirus cases in tertiary hospitals in hubei province role of lopinavir/ritonavir in the treatment of sars: initial virological and clinical findings screening of an fda-approved compound library identifies four small-molecule inhibitors of middle east respiratory syndrome coronavirus replication in cell culture a trial of lopinavir-ritonavir in adults hospitalized with severe covid-19 triple combination of interferon beta-1b, lopinavir-ritonavir, and ribavirin in the treatment of patients admitted to hospital with covid-19: an open-label, randomised, phase 2 trial arbidol monotherapy is superior to lopinavir/ritonavir in treating covid-19 first case of 2019 novel coronavirus in the united states remdesivir for severe acute respiratory syndrome coronavirus 2 causing covid-19: an evaluation of the evidence remdesivir: review of pharmacology, preclinical data and emerging clinical experience for covid-19 remdesivir in adults with severe covid-19: a randomised, double-blind, placebo-controlled, multicentre trial compassionate remdesivir treatment of severe covid-19 pneumonia in intensive care unit (icu) and non-icu patients: clinical outcome and differences in post-treatment hospitalisation status the fda-approved drug ivermectin inhibits the replication of sars-cov-2 in vitro usefulness of ivermectin in covid-19 illness icon (ivermectin in covid nineteen) study: use of ivermectin is associated with lower mortality in hospitalized patients with covid19 adult haemophagocytic syndrome macrophage activation-like syndrome: a distinct entity leading to early death in sepsis development and validation of the hscore, a score for the diagnosis of reactive hemophagocytic syndrome coronavirus disease 2019 (covid-19): a clinical update interleukin-6 blockade for severe tocilizumab therapy reduced intensive care unit admissions and/or mortality in covid-19 patients clinical characteristics and early outcomes in patients with covid-19 treated with tocilizumab at a united states il6 inhibition in critically ill covid-19 patients is associated with increased secondary infections off-label use of tocilizumab for the treatment of sars-cov-2 pneumonia in impact of low dose tocilizumab on mortality rate in patients with covid-19 related pneumonia tocilizumab in patients with severe covid-19: a retrospective cohort study efficacy and safety of tocilizumab in severe covid-19 patients: a single-centre retrospective cohort study tocilizumab for treatment of mechanically ventilated patients with covid-19. medrxiv tocilizumab treatment for cytokine release syndrome in hospitalized covid-19 patients: survival and clinical outcomes interleukin-1 blockade with high-dose anakinra in patients with covid-19, acute respiratory distress syndrome, and hyperinflammation: a retrospective cohort study anakinra for severe forms of covid-19: a cohort study baricitinib therapy in covid-19: a pilot study on safety and clinical impact weak induction of interferon expression by sars-cov-2 supports clinical trials of interferon lambda to treat early covid-19 pharmacologic treatments for coronavirus disease 2019 (covid-19): a review clinical evidence does not support corticosteroid treatment for 2019-ncov lung injury sars: systematic review of treatment effects corticosteroid therapy for critically ill patients with middle east respiratory syndrome corticosteroid treatment of patients with coronavirus disease 2019 (covid-19) the effect of corticosteroid treatment on patients with coronavirus infection: a systematic review and meta-analysis impact of corticosteroid therapy on outcomes of persons with sars-cov-2, sars-cov, or mers-cov infection: a systematic review and meta-analysis group rc. effect of dexamethasone in hospitalized patients with covid-19: preliminary report meta-analysis: convalescent blood products for spanish influenza pneumonia: a future h5n1 treatment? convalescent plasma treatment reduced mortality in patients with severe pandemic influenza a (h1n1) 2009 virus infection use of convalescent plasma therapy in sars patients in hong kong the effectiveness of convalescent plasma and hyperimmune immunoglobulin for the treatment of severe acute respiratory infections of viral etiology: a systematic review and exploratory meta-analysis the convalescent sera option for containing covid-19 characterization of spike glycoprotein of sars-cov-2 on virus entry and its immune cross-reactivity with sars-cov establishment and validation of a pseudovirus neutralization assay for sars-cov-2 prevention of transfusion-transmitted infections noninfectious serious hazards of transfusion molecular mechanism for antibody-dependent enhancement of coronavirus entry treatment of 5 critically ill patients with covid-19 with convalescent plasma effectiveness of convalescent plasma therapy in severe covid-19 patients treatment with convalescent plasma for critically ill patients with severe acute respiratory syndrome coronavirus 2 infection use of convalescent plasma therapy in two covid-19 patients with acute respiratory distress syndrome in korea treatment with convalescent plasma for covid-19 patients in wuhan effect of convalescent plasma therapy on viral shedding and survival in patients with coronavirus disease treatment of covid-19 patients with convalescent plasma in efficacy of convalescent plasma and short course of corticosteroids in patients with covid-19 early safety indicators of covid-19 convalescent plasma in 5 15-day mortality and associated risk factors for hospitalized patients with covid-19 in wuhan, china: an ambispective observational cohort study immunoglobulins in the treatment of covid-19 infection: proceed with caution! could intravenous immunoglobulin collected from recovered coronavirus patients protect against covid-19 and strengthen the immune system of new patients? hyperimmune iv immunoglobulin treatment: a multicenter double-blind randomized controlled trial for patients with severe 2009 influenza a(h1n1) infection active therapy with passive immunotherapy may be effective in the fight against covid-19 clinical manifestations, laboratory findings, and treatment outcomes of sars patients acute respiratory distress syndrome in critically ill patients with severe acute respiratory syndrome currently available intravenous immunoglobulin contains antibodies reacting against severe acute respiratory syndrome coronavirus 2 antigens effect of regular intravenous immunoglobulin therapy on prognosis of severe pneumonia in patients with covid-19 high-dose intravenous immunoglobulin as a therapeutic option for deteriorating patients with coronavirus disease successful intravenous immunoglobulin treatment in severe covid-19 pneumonia esc guidelines for the diagnosis and management of acute pulmonary embolism developed in collaboration with the european respiratory society (ers) abnormal coagulation parameters are associated with poor prognosis in patients with novel coronavirus pneumonia covid-19 and thrombotic or thromboembolic disease: implications for prevention, antithrombotic therapy, and follow-up: jacc state-of-the-art review isth interim guidance on recognition and management of coagulopathy in covid-19 isth interim guidance on recognition and management of coagulopathy in covid-19: a comment thromboembolic risk and anticoagulant therapy in covid-19 patients: emerging evidence and call for action postinjury hyperfibrinogenemia compromises efficacy of heparin-based venous thromboembolism prophylaxis fixed dose subcutaneous low molecular weight heparins versus adjusted dose unfractionated heparin for the initial treatment of venous thromboembolism perioperative management of patients infected with the novel coronavirus: recommendation from the joint task force of the chinese society of anesthesiology and the chinese association of anesthesiologists outbreak of a new coronavirus: what anaesthetists should know barrier enclosure during endotracheal intubation exhaled air dispersion during bag-mask ventilation and sputum suctioning -implications for infection control optimizing preoxygenation in adults a model to describe the rate of oxyhaemoglobin desaturation during apnoea first-attempt success is associated with fewer complications related to intubation in the intensive care unit complications of endotracheal intubation in the critically ill consensus guidelines for managing the airway in patients with covid-19: guidelines from the difficult airway society, the association of anaesthetists the intensive care society, the faculty of intensive care medicine and the royal college of anaesthetists management of difficult tracheal intubation: a closed claims analysis difficult airway society 2015 guidelines for management of unanticipated difficult intubation in adults incidence and predictors of difficult mask ventilation and intubation practical recommendations for critical care and anesthesiology teams caring for novel coronavirus (2019-ncov) patients disease jot. society for advanced bronchoscopy consensus statement and guidelines for bronchoscopy and airway management amid the covid-19 pandemic lack of sars transmission among public hospital workers american association for bronchology and interventional pulmonology (aabip) statement on the use of bronchoscopy and respiratory specimen collection in patients with suspected or confirmed covid-19 infection updated guidance on evaluating and testing persons for coronavirus disease early tracheotomy versus prolonged endotracheal intubation in unselected severely ill icu patients risk factors for posttracheostomy tracheal stenosis systematic review and meta-analysis of studies of the timing of tracheostomy in adult patients undergoing artificial ventilation outcomes and prognostic factors in 267 patients with severe acute respiratory syndrome in hong kong clinical features of deaths in the novel coronavirus epidemic in china surgical considerations for tracheostomy during the covid-19 pandemic: lessons learned from the severe acute respiratory syndrome outbreak 2020) he. covid-19: guidance for infection prevention and control in healthcare settings clinical and virological data of the first cases of covid-19 in europe: a case series aao position statement: tracheotomy recommendations during the covid-19 pandemic recommendations from the cso-hns taskforce on performance of tracheotomy during the covid-19 sensitivity of chest ct for covid-19: comparison to rt-pcr performing tracheostomy during the covid-19 pandemic: guidance and recommendations from the critical care and acute care surgery committees of the american association for the surgery of trauma recommendation of a practical guideline for safe tracheostomy during the covid-19 pandemic tracheotomy in the intensive care unit: guidelines from a french expert panel. ann intensive care venovenous extracorporeal membrane oxygenation for acute respiratory distress syndrome: a systematic review and meta-analysis extracorporeal membrane oxygenation for severe acute respiratory distress syndrome and posterior probability of mortality benefit in a post hoc bayesian analysis of a randomized clinical trial surviving sepsis campaign international guidelines for the management of septic shock and sepsis-associated organ dysfunction in children use of ecmo in ards: does the eolia trial really help? mechanical ventilation management during extracorporeal membrane oxygenation for acute respiratory distress syndrome. an international multicenter prospective cohort treatment limitations in the era of ecmo initial elso guidance document: ecmo for covid-19 patients with severe cardiopulmonary failure extracorporeal life support for adults with respiratory failure and related indications: a review preparing for the most critically ill patients with covid-19: the potential role of extracorporeal membrane oxygenation research in extracorporeal life support: a call to planning and provision of ecmo services for severe ards during the covid-19 pandemic and other outbreaks of emerging infectious diseases association of hospital-level volume of extracorporeal membrane oxygenation cases and mortality. analysis of the extracorporeal life support organization registry position paper for the organization of extracorporeal membrane oxygenation programs for acute respiratory failure in adult patients guía de aplicación de la estrategia multimodal de la oms para la mejora de la higiene de las manos special attention to nurses' protection during the covid-19 epidemic initiation of a new infection control system for the covid-19 outbreak preparing your intensive care unit for the covid-19 pandemic: practical considerations and strategies practical recommendations for the perioperative management of the patient with suspection or serious infection by coronavirus sars-cov covid-19 under the sars cloud: mental health nursing during the pandemic in hong kong kilinc balci fs. ersonal protective equipment for preventing highly infectious diseases due to exposure to contaminated body fluids in healthcare staff what are the effects of multimodal campaigns to improve hand hygiene of healthcare workers? orientaciones para el manejo, traslado y disposición final de cadáveres por covid-19 guía para el cuidado crítico de pacientes adultos graves con coronavirus (covid-19) en las américas guia para toma de muestras respiratorias en pacientes con sospecha de infeccion por coronavirus detection of 2019 novel coronavirus (2019-ncov) by real-time rt-pcr soporte respiratorio en pacientes covid-19 dispersal of respiratory droplets with open vs closed oxygen delivery masks: implications for the transmission of severe acute respiratory syndrome regarding non-invasive respiratory support in the adult patient with acute respiratory failure secondary to sars-cov-2 infection desconexión de la ventilación mecánica . ¿ por qué seguimos buscando métodos alternativos ? a comparison of four methods of weaning patients from mechanical ventilation. spanish lung failure collaborative group use of weaning protocols for reducing duration of mechanical ventilation in critically ill adult patients: cochrane systematic review and meta-analysis epidemiology of weaning outcome according to a new definition. the wind study the decision to extubate in the intensive care unit factores de riesgo asociados con la extubación fallida en pacientes adultos de una unidad de cuidados intensivos de la ciudad de cali respiratory support after extubation: noninvasive ventilation or high-flow nasal cannula, as appropriate. ann intensive care effect of postextubation high-flow nasal cannula vs noninvasive ventilation on reintubation and postextubation respiratory failure in high-risk patients: a randomized clinical trial high-flow nasal cannula to prevent postextubation respiratory failure in high-risk nonhypercapnic patients: a randomized multicenter trial noninvasive positive-pressure ventilation for respiratory failure after extubation noninvasive positive-pressure ventilation for postextubation respiratory distress: a randomized controlled trial the japanese respiratory society noninvasive positive pressure ventilation (nppv) guidelines (second revised edition) weaning from mechanical ventilation intermittent noninvasive ventilation after extubation in patients with chronic respiratory disorders: a multicenter randomized controlled trial (vhyper) anzics). dcidaynz. pautas covid-19 hispaflow) endgemdtdscafea. respiratory support therapy after extubation: who and how? orientaciones para el uso adecuado de los elementos de protección personal por parte de los trabajadores de la salud expuestos a covid-19 en el trabajo y en su domicilio orientaciones para el m anejo de residuos generados en la atención en salud ante la eventual introducción del virus covid-19 a colombia orientaciones para el manejo de residuos generados en la atención en salud ante la eventual introducción del virus covid-19 a colombia ministerio de sanidad. documento técnico manejo en atención primaria del covid-19 gobierno de españa mds. documento técnico manejo clínico del covid-19: unidades de cuidados intensivos ventilacion mecanica en covid-19. una aproximación práctica expert consensus and recommendations on safety criteria for active mobilization of mechanically ventilated critically ill adults melbourne: anzics mobilization of intensive care patients: a multidisciplinary practical guide for clinicians fisioterapia en el manejo del paciente covid19 en fase aguda hospitalaria extrapulmonary manifestations of covid-19 cytokine storms: understanding covid-19 postulated adjuvant therapeutic strategies for covid-19 war to the knife" against thromboinflammation to protect endothelial function of covid-19 patients effects of n-acetylcysteine treatment in acute respiratory distress syndrome: a meta-analysis mucoactive agents for acute respiratory failure in the critically ill: a systematic review and meta-analysis therapeutic blockade of inflammation in severe covid-19 infection with intravenous n-acetylcysteine eficacia y seguridad del uso de n-acetilcisteína parenteral en el manejo de los pacientes con síndrome de dificultad respiratoria del adulto (sdra) analysis of factors associated with disease outcomes in hospitalized patients with 2019 novel coronavirus disease confiabilidad del cuestionario de calidad de vida en salud sf-36 en the spanish version of the short form 36 health survey: a decade of experience and new developments a systematic review finds limited data on measurement properties of instruments measuring outcomes in adult intensive care unit survivors population-based surveillance for 2009 pandemic influenza a (h1n1) virus in guatemala association between malnutrition and clinical outcomes in the intensive care unit: a systematic review identifying critically ill patients who benefit the most from nutrition therapy: the development and initial validation of a novel risk assessment tool glim criteria for the diagnosis of malnutrition -a consensus report from the global clinical nutrition community espen expert statements and practical guidance for nutritional management of individuals with sars-cov-2 infection nutrition therapy in the patient with covid-19 disease requiring icu care guidelines for the provision and assessment of nutrition support therapy in the adult critically ill patient: society of critical care medicine (sccm) and american society for parenteral and enteral nutrition guidelines for the provision and assessment of nutrition support therapy in the adult critically ill patient: society of critical care medicine (sccm) and american society for parenteral and enteral nutrition espen guideline on clinical nutrition in the intensive care unit safety and outcomes of early enteral nutrition in circulatory shock gastrointestinal function in intensive care patients: terminology, definitions and management. recommendations of the esicm working group on abdominal problems key: cord-022082-1dq623oe authors: greaves, peter title: respiratory tract date: 2007-09-28 journal: histopathology of preclinical toxicity studies doi: 10.1016/b978-044452771-4/50007-9 sha: doc_id: 22082 cord_uid: 1dq623oe the chapter describes different aspects of the respiratory tract. in preclinical safety studies, pathologies of the respiratory system can be a result of an intercurrent disease or can be induced by systemically administered drugs. intranasal or inhalation modes of therapy pose particular challenges in terms of the formulations and technologies required to administer a drug. a complex technology is developed to support the assessment of adverse effects of inhaled substances in rodent and nonrodent species, and the extrapolation of experimental findings to humans. the nasal chambers are the structures that are first to be subjected to the effects of inhaled substances, whether microorganisms or chemical substances. in rodents, the relatively small size of the nose and nasal sinuses facilitates a histological examination. findings show that infectious agents cause inflammation in the nose and nasal sinuses, and this may be associated with inflammation in the conjunctiva, the middle ear, and the oral cavity. it has been observed that a particular response of the rodent nasal mucosa to some irritant substances, including pharmaceutical agents, is the formation of rounded eosinophilic inclusions in the cytoplasm of sustentacular cells of the olfactory epithelium, and to a lesser extent in respiratory and glandular epithelial cells. by far and away the most important pulmonary diseases in humans are related to the smoking of tobacco. however, occupational lung diseases caused by inhalation of industrial chemicals, particulate matter and antigens, are also important causes of morbidity and mortality. for this reason, considerable effort has been directed to the examination of airborne pollutants over recent years, including study of their effects in laboratory animals when administered by the inhalation route. extensive study has shown that a complex array of defensive mechanisms protects the lung against the adverse effects of airborne substances and pathogenic organisms. aerodynamic factors prevent access of particles larger than 10|im diameter for these are deposited on the walls of the nasal passages. particles measuring between 2 and 10 (im diameter tend to be trapped by the mucus-covered ciliated epithelium lining the bronchial tree and removed by mucociliary transport aided by the cough reflex. smaller particles may reach the alveoli where they are ingested and transported by pulmonary macrophages.^ considerations of airborne delivery to the lungs are also important in the development of therapies to be administered via the respiratory tract. whilst the inhalation route has been used for many years for volatile anaesthetic gases, the respiratory tract is increasingly being employed for delivery of therapy in not only for asthma and other lung diseases but also as a means of systemic delivery of polypeptides such as insulin. in contrast to the adverse pulmonary effects of cigarette smoke and industrial pollutants, therapeutic agents remain a relatively minor cause of pulmonary toxicity in humans although actual incidence is difficult to ascertain. however, drug-induced pulmonary disease appears to be an increasingly frequent clinical problem and the drugs associated with parenchymal pulmonary injury in humans continue to increase.^ although it is difficult to assess in the context of the underlying disease process, it has been suggested that about 10% of patients receiving wellestablished anticancer drugs develop various forms of pulmonary toxicity some novel antineoplastic therapies may have a similar liability.^ drug-induced toxicity usually occurs after exposure of lung tissue via the circulation to parent drug or metabolites, although increasingly the adverse effects of direct administration of drugs to the lungs needs consideration in preclinical studies. in patients a number of drugs have been associated with pulmonary toxicity which can occur through different mechanisms and take different forms.^"^ through their specific pharmacological action drugs can produce excessive effects on bronchial calibre or pulmonary function. drugs mediate allergic reactions in the bronchi or lungs. they may also produce a variety of obscure, diffuse pulmonary alveolar conditions including a pulmonary syndrome resembling systemic lupus er3^hematosis. as the respiratory tract is a major route by which microorganisms gain entry into the body, opportunistic pulmonary infections with bacteria, viruses, fungi or protozoa are consequences of immunosuppression or broad-spectrum antibacterial therapy. as in other organs, drugs that disturb coagulation may precipitate pulmonary thromboembolism or haemorrhage. localized lung lesions also result from accidental, diagnostic or therapeutic inhalation of xenobiotics. mucociliary clearance is also sensitive to therapeutic agents that affect the secretion of mucus and fluid, ciliary activity and transport.^ treatment with antacids or histamine h2 blockers can also increase the risk of pneumonia developing in patients in intensive care units through increasing gastric ph, which leads to an overgrowth of gram-negative bacteria in the stomach and retrograde pharyngeal colonization.^ in preclinical safety studies, pathology of the respiratory system can be the result of intercurrent disease or be induced by drugs administered systemically. intranasal or inhalation modes of therapy pose particular challenges in terms of the formulations and the technologies required to administer drug. the different anatomical and physiological characteristics of the airways also influence drug toxicity, disposition and metabolism. the development of drugs to be administered by inhalation or intranasal routes is particularly difficult because of the perceived risks of high local drug concentration in respiratory tissues and their use in potentially vulnerable patients with pulmonary disease.^ a complex technology has been developed to support the assessment of the adverse effects of inhaled substances in rodent and non-rodent species and the extrapolation of the experimental findings to humans.^'^ in order to administer drugs by inhalation, it is necessary to generate aerosols (suspensions of particles in a gas) with a well-defined composition, particle size and shape. they must be delivered to the respiratory tract of laboratory animals in a way that parallels the likely human exposure. in case of therapeutic agents, this should avoid non-respiratory pathways through the skin and food. when aerosols are inhaled, various fractions of the particles are deposited at different locations in the respiratory tract. site of deposition depends primarily on particle size, but variability in the sites of deposition occurs among different laboratory animal species and humans by virtue of the differences in the size and shape of the respiratory passages as well as breathing patterns. ^^ in addition, there are different types of inhalers used in human therapy to consider in the assessment, which can deliver different materials to the lungs, for example nebulizers, propellant-driven metered dose inhalers and dry powder inhalers for asthma treatment. ^^ the subsequent fate of inhaled particles depends not only on their size but also their shape, chemical nature, and solubility in body fluids. soluble substances are absorbed into the blood stream and are removed by the pulmonary circulation. they may also undergo metabolism by enzymes present in the cell populations of the respiratory tract and reactive metabolites may cause local pulmonary damage. insoluble, inert particles are removed primarily by the mucociliary transport system of the trachea and bronchi or through phagocytosis by macrophages. overload of the lung by even relatively inert, nonfibrous particles such as titanium dioxide or carbon black may impair alveolar macrophage-mediated particle clearance. ^^ this may lead in turn to accumulation of dusts over time with eventual fibrotic and tumorigenic responses, particularly in rats.^^ measurements of respiration rate, tidal volume, airway resistance, pulmonary gas exchange and the disposition of the inhaled substances have an important place in the evaluation of chemically induced lung damage in laboratory animals.^"^'^^ however, the key component of the evaluation of the adverse effects of inhaled substances is careful morphological assessment of the fixed tissues. even though there are novel and very sensitive physiological methods for the characterization of oedema following lung injury in rodents, light and electron microscopy of lung tissue provides vital qualitative evidence of the nature of injury. ^^ the nasal chambers are the structures which are first to be subjected to the effects of inhaled substances, whether microorganisms or chemical substances. although these chambers are not usually examined in great detail in conventional toxicity studies in which substances are administered orally or by parenteral routes, they are carefully examined histologically when drugs are administered by inhalation. study of nasopharyngeal silicone rubber casts has shown considerable species differences in the anatomy of this part of the airway. ^^"^^ relative to total nasal length, the nasopharynx is longest in rats and shortest in humans with the dog in an intermediate position. maxilloturbinates are relatively simple structures in man and non-human primates but highly complex in dogs and rodents. as a consequence, regional nasal airflow and disposition patterns vary considerably and this influences the distribution of lesions produced by inhaled xenobiotics in the nasal cavity.^^ however, comparison of the nasal cavity of rhesus monkey and humans using magnetic resonance imaging and nasal casts have shown that many similarities in structure exist in these species. ^^ the anterior nares are lined by stratified squamous epithelium. in other zones the sinuses are covered either by respiratory or olfactory epithelium with a zone of transitional epithelium at the junction between the two types. respiratory epithelium is similar to that found elsewhere in the respiratory passages being composed of ciliated cells, serous and mucous cells, brush cells, intermediate cells and progenitor basal cells. it represents a cellular system engaged in mucociliary clearance carrying surface secretions to the nasopharynx to be cleared by swallowing. although this epithelium is similar to that lining the other large airways, key differences are the particularly rich complement of secretory cells and the complex vasculature of the nose which can modulate capillary, arterial and venous blood flow through the mucosa.^^ mucins may be particularly important. it has been postulated that they not only have a physical protective function but also possess antioxidant properties by virtue of the scavenging behaviour of their high proportion of sugar groups. ^^ the proportion of the nose lined by olfactory mucosa is variable between species, being disposed over a much larger area in dogs and rodents than in primates. ^^ however, it is structurally similar in humans and rodents. it is located in more dorsal or posterior regions of the nasal passages out of the direct line of airflow during normal respiration. olfactory mucosa is a pseudostratified columnar epithelium composed of basal cells, sustentacular cells and sensory cells with mucus-secreting bowman's glands situated in the lamina propria. basal cells are composed of two distinct types, light and dark cells. the light type represents the primitive, stem cell population. sustentacular or supporting cells are non-ciliated, columnar cells possessing microvilli that extend into the overlying layer of mucus. cell bodies of olfactory sensory neurons are situated in the middle layer of the epithelium between sustentacular and basal cells. their dendritic processes extend above the epithelial surface to end in a ciliated expansion referred to as the olfactory vesicle that is believed to be the receptor of odour perception. olfactory axons extend from the cell body, penetrate the basement membrane in bundles to become surrounded by schwann cells and eventually join with the olfactory bulb. the olfactory system is of importance in toxicology for it can be selectively damaged by xenobiotics, including therapeutic agents, presumably as a result of its high metabolizing capacity. the superficial location of neural cells in the olfactory epithelium also provides a model system for the study of the effects of xenobiotics on neural cells. submucosal mucous glands have been well characterized in the rat, hamster and dog, where they are divided into lateral nasal glands and maxillary recess glands. these are both situated in the posterior parts of the nasal cavity and composed of mucus-secreting cells.^^"^^ immunocytochemical study using antisera raised against the major isoenzymes of rat hepatic microsomal cytochromes p450 induced by (3-napthoflavone, 3-methylcholanthrene, phenobarbitone and pregnenolone-16-a-carbonitrile as well as nadph-cytochrome p450 reductase, epoxide hydrolase and glutathione s-transferases b, c and e, has shown their presence in rat nasal mucosal cells.^^ cyp2a enzymes appear to be expressed at high levels in the respiratory tract mucosa.^^"^^ this suggests that the nasal mucosa not only has a capacity for metabolizing and activating xenobiotics by oxidation, but also for hydration and inactivation of potentially toxic epoxides and conjugating electrophilic, reactive metabolites with reduced glutathione. it has been shown that the distribution of immune-reactive enzymes is different in olfactory and respiratory mucosa.^^ xenobiotics can be metabolized within both olfactory and respiratory mucosa but the olfactory regions appear to possess greatest capability for oxidative metabolism. consequently, regional differences in nasal toxicity and tumour formation from inhaled materials may not only be a response to different water solubility and deposition patterns but also differences in the formation of reactive metabolites.^'* another feature of this metabolizing activity is that it can be induced by systemically administered xenobiotics and this can alter the distribution of enzyme activity in the nasal mucosa.^^ studies of the mouse olfactory mucosa have shown that whilst typical hepatic inducers of cyp2a5 do not significantly change its expression in the mucosa, olfactory toxicants can alter the pattern of enzyme distribution.^^'^^ like many other tissues exposed to external environmental agents, the nasal mucosa possesses aggregates of lymphoid tissue in the underlying lamina propria. in rats these areas, characterized by follicles containing both t and b cell areas, are located in the ventral aspects of the lateral walls of the nasal airways at the opening of the nasopharyngeal duct.^^'^^ like the gutassociated lymphoid tissue, these nasal follicles have been shown in the rat to be covered by specialized epithelium with islands of cells with microvilli, socalled m or membranous cells. little is known of any toxicity occurring in this tissue despite its strategic position in the respiratory tract.^^ in rodents, the relatively small size of the nose and nasal sinuses facilitates histological examination. usually this area is sectioned transversely into several standardized blocks following decalcification.^^ there have been a number of detailed publications describing the histological preparation and assessment and recording of pathology of the rodent nasal cavity.^^"^^ careful standardized histological sections, careful recording of lesions with the use of diagrams of the rodent nasal cavity are useful in the assessment of lesions in the nasal cavity found in inhalation studies."*^ in larger species, particularly dogs and primates, sectioning and blocking is more complex. although dissection is required, a similar procedure following decalcification can be adopted. careful examination of haematoxylin and eosin stained sections remains paramount in the assessment of the nasal cavity, although special stains may be helpful. examination of cytokeratin expression in the respiratory mucosa has been used as a marker of epithelial differentiation in the respiratory tract>^ a test system that relates to the innervation of the nasal mucosa is that proposed by alarie.^^ the trigeminal nerve endings in the nasal mucosa of mice mediate the response to sensory irritants and this can be measured by a decrease in respiratory rate. it has been shown that a good correlation exists between the decrease in respiration rate in mice exposed to airborne chemicals and the nasal irritancy potential of the chemicals in humans.^^ this enables the detection of airborne sensory irritants and the prediction acceptable levels of exposure to the upper respiratory tract in people. microbial pathogens infectious agents cause inflammation in the nose and nasal sinuses and this may be associated with inflammation in the conjunctiva, middle ear and oral cavity. murine pathogens may cause alterations in the respiratory tract that can confound the assessment of changes induced by xenobiotics.^^ in rats, microbiological agents implicated in the development of rhinitis and sinusitis include corynebacterium kutscheri (pseudotuberculosis), streptococcus pneumonia, pasteurella pneumotropica, klebsiella pneumoniae, mycoplasma pulmonis and the sialodacryoadenitis virus or rat corona virus.^^ rats infected with the sialodacryoadenitis virus show inflammation and necrosis of the upper respiratory epithelium as well as damage to salivary and lachrymal glands. the sendai virus, a paramyxovirus, also has marked tropism for the respiratory tract, including the nasal cavity, and is associated with systemic effects that can compromise studies in laboratory rodents. occasionally, fungal infections of the airways with. aspergillus fumigatus are reported.^^ a variable that has been shown to influence the severity of the rhinitis produced by mycoplasma pulmonis is the strain of rat. following housing of lewis and fischer 344 strains together to eliminate microbial and environmental differences it was shown that the lewis strain developed a more severe rhinitis following inoculation with mycoplasma pulmonis than fischer 344 rats, although the reason for the difference was unclear."^^ rats exposed to ammonia, a common pollutant of the air in laboratory animal cages, have also been shown to develop lesions of the dorsal meatus, dorsal nasal septum and prominence of the turbinates.^^ these lesions are characterized histologically by swelling or mild degeneration of the epithelium. it appeared that ammonia exposure potentiated the acute inflammatory response of the nasal cavity to microbiological pathogens. a microorganism reported in the nasal cavity of rhesus monkeys employed in inhalation studies is the nematode of the genus anatrichosoma.^^ sections of this nematode are found in the squamous epithelium of the nasal vestibule and are associated with acanthosis and hyperkeratosis of the epithehum and a multifocal or diffuse granulomatous inflammation in the submucosa. administration of toxic or irritant substances to laboratory animals by the inhalation route produces degenerative, inflammatory and reactive changes in the nasal mucosa. the range of histological features is similar to those found in other mucosal surfaces damaged by other exogenous agents. whilst therapeutic agents administered by the inhalation route do not usually produce a severe degenerative or inflammatory responses in the nasal mucosa, at least at therapeutic doses, the simple categories proposed by hardisty and colleagues in recording of degenerative and reactive lesions following exposure to volatile chemicals are useful.'^^ categories suggested are: degeneration, regeneration, atrophy (postdegenerative), respiratory metaplasia and basal cell hyperplasia and inflammation. degeneration is usually the earliest morphological change characterized by loss of sensory and sustentacular cells resulting in a thinner mucosa. bowman's glands and nerve bundles, individual cell necrosis may be seen in more severe cases. regeneration is characterized by proliferation of basal cells associated with an epithelium that loses its regular structural features. post-degenerative atrophy usually follows severe damage and is characterized by loss of sensory and sustentacular cells. respiratory metaplasia is a process whereby the normal olfactory mucosa is replaced by pseudostratified epithelium of respiratory type often with cilia. basal cell hyperplasia represents a longer term effect where the proliferating basal cells form a distinct layer of cells below the respiratory epithelium. an example of the type and distribution of the degenerative and inflammatory conditions which can be induced by inhaled irritants is provided by the study in which swiss-webster mice were given various irritants by inhalation for periods of 6 hours per day for 5 days at concentrations that produced a 50% decrease in respiratory rate (alarie test). although the degree of histological changes varied with different agents, the changes were broadly similar in type and distribution.^^ most agents examined produced little or no alteration in the squamous mucosa lining the anterior part of the nose apart from some mild increase in thickness of the squamous layers. principal sites of damage were shown to be the anterior respiratory epithelium adjacent to the vestibule and the olfactory epithelium of the dorsal meatus. there was a distinct decrease in severity in posterior regions. histologically, the lesions in respiratory epithelium ranged from mild loss of cilia and small areas of epithelial exfoliation to frank erosion, ulceration and necrosis of the epithelium and underlying tissues including bone. variable polymorphonuclear cell infiltration was also found. in some cases, early squamous metaplasia developed on the free margins or the naso-maxillo-turbinates. changes to the olfactory epithelium varied from focal to extensive loss of sensory cells associated with damage to sustentacular cells. in severe cases, complete loss of olfactory epithelium occurred. although the degree of histological change was shown to vary with different agents, lesions induced by the more water-soluble chemicals tended to remain localized in the anterior part of the nasal cavity whereas agents with relatively low water solubility produced lung lesions in addition. it was suggested that these findings demonstrated the powerful 'scrubbing' action of the nasal cavity for water soluble, airborne xenobiotics.^^ inflammatory alterations have been induced in the nasal cavity of rodents treated with therapeutic agents at high doses by inhalation. significantly irritant substances do not make viable therapies. however, the precise relevance of such changes for human therapy by the inhalation route are sometimes questionable when the nasal damage is limited to high doses and it is not associated with alterations in other parts of the respiratory tract. in the case of tulobuterol, a 32-adrenergic receptor agonist, it was argued that the nasal inflammation induced in rats in a one month inhalation toxicity study was the result of a particularly high exposure of the nasal epithelium to drug, not representative of the likely human exposure to tulobuterol by inhalation, where little or no nasal exposure would occur.^^ rp73401 [3cyclopentyloxy)-ar-(3,5-dichloro-4-pyridy)-4-methoxybenzamide], a novel type iv phosphodiesterase inhibitor which was being developed for the treatment of asthma and rheumatoid arthritis, was also reported to produce degeneration of the olfactory epithelium in rats but neither dogs nor mice after single and repeated oral doses and by inhalation.^^ histologically, the olfactory epithelium showed necrosis of the superflcial epithelial layers including the sustentacular and sensory cells, with sparing of the basal cell layer. there was also damage to bowman's glands. the development of proliferative lesions and ultimately tumours of principally neuroectodermal origin followed chronic treatment. as rp73401 was highly metabolized and the nasal lesions could be inhibited by treatment of rats with metyrapone, a non-speciflc inhibitor of cytochromes p450, it was postulated that the changes were the result of p450mediated activation in the olfactory tissues, not linked to its pharmacological phosphodiesterase activity.^^ nasal epithelial degeneration and necrosis has also been reported in both rats and dogs treated with another candidate anti-inflammatory drug ci-959 by the intranasal route. this agent affected olfactory epithelium more than respiratory mucosa, suggesting that metabolism was important in the generation of this toxicity^^ although the nasal cavity has not been often examined histologically in great detail in toxicity studies conducted on drugs administered orally or by parenteral routes, damage to the nasal mucosa can be induced by drugs administered by these routes. one example is methimazole, a thioureylene antithyroid drug used in clinical practice where oral doses of 0.2-2mg/kg/day are employed and abnormalities of taste and smell have been described.^^ administration of methimazole at relatively high doses to long-evans rats by single oral (50mg/kg) or intraperitoneal (25mg/kg) routes was shown to produce damage to the sustentacular and sensory cells with sparing of the basal cells and basement membrane.^^ bowman's glands were also involved. methimazole is metabolized by the flavin-containing monooxygenase system and it is employed as a model substrate for this enzyme in vitro. the presence of flavin-containing monooxygenase isoforms in olfactory mucosa of long-evans rats suggested that reactive intermediates may be responsible for the nasal toxicity ^^ similar changes have been reported in mice where depletion of glutathione in the olfactory mucosa has been demonstrated also suggesting formation of local reactive metabolites.^^ histological examination has also shown that intravenous administration of a single dose of vincristine to mice damages the olfactory epithelium.^^'^^ vincristine is a vinca alkaloid derivative used in cancer therapy which has antimitotic activity and binds to tubulin. cell death was noted in olfactory cells 2-5 days after dosing with a peak of cell proliferation at 5 days and repair after about 10 days. these features resemble those that can be seen in other proliferating tissues after single doses of antimitotic drugs. the risk of damage to human olfactory cells from agents with these effects in rat nasal mucosa often remains uncertain because an understanding of relative exposure and metabolism in different species and a better understanding of the metabolic potential of human olfactory mucosa is required. a particular response of the rodent nasal mucosa to some irritant substances, including pharmaceutical agents, is the formation of rounded eosinophilic inclusions in the cytoplasm of sustentacular cells of the olfactory epithelium and to a lesser extent in respiratory and glandular epithelial cells.^^'^^ these inclusions are pas-negative and ultrastructural examination shows that they are membrane-bound, ellipsoid bodies containing homogenous electron dense matrix. their significance remains uncertain. a consensus classification for the variety of proliferative, non-neoplastic changes and atypical epithelial lesions and neoplasms found in the rat nasal cavity has been defined by schwartz and colleagues.^^ the classification of the international agency for research on cancer provides a similar perspective for rats and mice.^^'^^ proliferative lesions may be occasionally seen in untreated rodents in carcinogenicity studies but are much more commonly induced by administration of xenobiotics in inhalation carcinogenicity studies. spontaneous nasal tumours are uncommon but most often squamous in type in rats whereas in mice spontaneous squamous tumours are extremely rare and haemangiomas and respiratory adenomas predominate.^^'^^ the generally agreed categories are described below: mucous (goblet) cell hypertrophy and hyperplasia affects the nasal respiratory epithelium and are characterized by the presence of enlarged mucus-filled goblet cells, some of which form clusters suggestive on intraepithial glands. squamous cell hyperplasia is seen in the stratified squamous epithelium of the nares and is characterized by a focal increase in the number of cell layers. cells may show atypia with irregular enlarged, pleomorphic nuclei and nucleoli. squamous metaplasia occurs to respiratory epithelium under conditions of chronic damage. it is characterized histologically by the presence of three or more layers of epithelial cells with eosinophilic cytoplasm and clear cell boundaries whereas advanced lesions show typical keratinization and formation of intercellular bridges. cellular atypia may also be seen and should be characterized when found. respiratory epithelial metaplasia (of the olfactory epithelium) represents atrophy and degeneration of the olfactory epithelium with loss of sensory cells and in advanced cases loss of sustentacular cells with replacement by ciliated and non-ciliated respiratory epithelium. it may be seen as a spontaneous focal lesion in aged rats. epithelial hyperplasia with cellular atypia (atypical hyperplasia, basal cell hyperplasia, dysplasia) is a term used to embrace proliferative lesions in the respiratory and olfactory mucosa in the nasal cavity in which there is varying degrees of altered differentiation and atypia. there is perturbation of the growth pattern of the epithelium such that the changes are not those found in the normal regenerative response to transient mucosal damage. adenomas (polypoid or villous adenoma, adenomatous or villous polyp) usually develop in the anterior part of the nasal cavity and are usually exophytic lesions that develop from respiratory epithelium or nasal glands. adenomas of respiratory epithelium may be papillary in form but are, by definition, well circumscribed with minimal cellular pleomorphism and atypia. they may very occasionally occur spontaneously in aged rats.^^ adenomas of nasal glands usually show an acinar pattern. squamous cell papillomas develop in the squamous epithelium of the nares or in areas of squamous metaplasia in respiratory or olfactory epithelium. they are exophytic lesions with limited connective tissue stroma. they may develop spontaneously in aged rats.^^ carcinomas of either squamous or glandular differentiation develop in the nasal mucosa. histologically, they have similar characteristics to those in other epithelial tissues. they are rare spontaneous lesions in aged laboratory rodents but may be induced by xenobiotics administered by inhalation, orally or by the parenteral route. squamous carcinomas have been reported to develop in a small number of untreated fischer 344 rats used in carcinogenicity studies in association with point mutations in the c-h-ras and c-k-ras gene.^^ olfactory neuroblastoma (ethesioneuroblastoma, olfactory neuroepithelioma, olfactory neuroepithelial carcinoma) show olfactory differentiation and arise from olfactory epithelium. they do not seem to occur as spontaneous lesions in rats or mice and only rarely induced.^^'^^ cells are arranged in lobules or in solid sheets with scanty stroma. cells are relatively uniform with scanty cytoplasm with round or oval hyperchromatic nuclei. true rosettes with lumens or pseudorosettes are also seen. poorly differentiated tumours of this type may require ultrastructural study for diagnosis. olfactory neuroblastomas typically show the presence of electron-dense neurosecretory granules, neurofilaments or axons. as there is no detailed understanding of the biological behaviour of these neoplasms in laboratory rodents, the generic term olfactory neuroblastoma is usually preferred. they are almost always invasive tumours.^^ olfactory carcinomas forming glands, follicles and rosettes have been occasionally reported in aged syrian hamsters.^^'^^ mesenchymal neoplasms may be seen in the nasal cavity, particularly after exposure to potent carcinogens. their histological features are similar to those in the soft tissues and bone elsewhere in the body (see chapter 2). the mucosa lining the larynx and trachea becomes involved as part of an upper or lower respiratory tract infection. for instance, in rats, an acute laryngitis or tracheitis has been shown to accompany experimental infection with mycoplasma pulmonis and the sialodycroadenitis virus."^^'^^ a spontaneous degenerative condition of tracheal and laryngeal cartilage of uncertain pathogenesis associated with granulomas has been reported in fischer 344 rats.^^ the condition increases in severity and incidence with advancing age although it is seen in rats as young as 6 weeks of age. tracheal cartilage rings may also show alterations in genetically engineered animals, such as the c57bl/6j-tgn(c3-l-tag)cjeg (tag) mice that have generalized defects in cartilage development.^^ the larynx of rodents is also susceptible to the effect of inhaled substances, notably tobacco smoke but also pharmaceutical agents and propellants.^^'^^ in view of the localized nature of induced lesions in the larynx, standardized histological sectioning techniques have been proposed for rats, mice and hamsters using anatomical landmarks.^^"^^ the target site is located on the ventral floor of the larynx near the base of the epiglottis cranial to the ventral laryngeal diverticulum. lesions tend to occur in the ventrolateral region, which is covered by respiratory epithelium and the inner aspect of the arytenoid processes which is lined by squamous mucosa. the larynx responds to inhaled irritants by inflammatory, degenerative and regenerative changes in a manner similar to other regions of the respiratory tract. these include disruption of the epithelial cells, inflammatory cell exudates and infiltration, goblet cell hyperplasia and squamous metaplasia.^^ however, these changes are not specific to inhaled irritants but also occur as a response to natural respiratory tract pathogens in conventionally housed rats.'^^ the pseudostratified ciliated and non-ciliated mucosa of the trachea may also show pathological alterations in inhalations studies, although sites at the bifurcation (carina) are those often first affected. consequently, the carina should be systematically included in examination of the respiratory tract for induced lesions.^^'^^ as in the nasal passages a range of proliferative lesions including squamous hyperplasia, mucous cell hyperplasia, as well as papilloma, carcinoma and mesenchymal tumours are occasionally reported in the airways in laboratory rodents. in humans and laboratory animals, the trachea terminates at the bifurcation giving rise to two main bronchi which serve left and right lungs. depending on species, the main bronchi subdivide into further branches that enter the different lobes. various forms of branching are recognized. bronchi may arise as side-branches from a parent or stem bronchus (monopodial). the parent bronchus can divide into two equal daughter bronchus (dichotomous) or several daughter bronchi ipolychotomous)j^ study of silicone rubber casts of the respiratory tract has shown that the bronchial trees of humans and non-human primates are essentially dichotomous, in contrast to the monopodial pattern of rodents.^^ the comparatively long trachea of the dog gives rise to dichotomous upper airways but monopodial branching develops peripherally within each lobe. the size of the lungs is generally dependent on size and weight of the different species. auometric studies have shown that lung volume, alveolar surface area and diffusing capacity increase proportionally with body weight across a broad range of mammalian species, although cell size and surface area appear to be more determined by cell function rather than species size.^^ dogs have comparatively smaller body mass and higher airway dimensions compared to humans.^^ the number of lobes is species-dependent. the human lung possesses an upper and lower left lobe and an upper, middle and lower right lobe. this contrasts with the upper, middle and lower left lobes and a fourth, azygos right lobe in rhesus monkeys and baboons.^^ the dog has three lobes on both right and left sides. rats, mice and hamsters show cranial, middle, caudal and postcaval right lobes with a single, left lobe in mice and rats and a superior and inferior lobe on the left side in hamsters. cell types lining the bronchi are generally similar between species.^ the majority of cells are the ciliated cells that are accompanied by variable but relatively smaller proportions of basal cells, intermediate cells, mucous or goblet cells, serous cells, neuroendocrine and brush cells. in addition, mucous cells line the adjacent bronchial glands.^^ unlike the tracheal mucosa, which is pseudostratified, the mucosa of intra-pulmonary bronchi is non-stratified. ciliated cells are tall, columnar cells attached to basal and intermediate cells by desmosomal junctions. tight junctions exist between adjacent specialized cells at the apex. each cell possesses 200 or more cilia that are engaged in mucociliary clearance.^^ the superficial cell surface also shows a pronounced glycocalyx. the cytoplasm of ciliated cells contains scattered profiles of rough endoplasmic reticulum, a supranuclear golgi and numerous mitochondria particularly near the apex where a prominent cytoskeleton is also found. mucous or goblet cells are typical mucus-secreting cells representing about 10% of the bronchial mucosa cell population in man but less than 1% in pathogenfree rats.^ the serous cell is a cylindrical or pyramidal cell containing small, round, closely packed serous granules.^^ basal cells are compact, pyramidal cells resting on the basement membrane. they are believed to be progenitor stem cells with the intermediate cells representing an intermediate stage of cell differentiation. the mucus-secreting and ciliated cells form the cellular basis for the mucociliary clearance mechanism of the main conducting airways. the epithelium is covered by a mucous blanket that is fairly complete in humans and rabbits but patchier in rats.^ the mucous layer is segregated into an upper layer or gel phase separated from epithelial cells by a serous layer or sol phase. the complex carbohydrates of the glycocalyx and secreted mucosubstances show species-related differences in their sugar residues, which can be demonstrated histochemically by the use of labelled-lectins.^^ mucociliary clearance mechanisms are sensitive to the effects of many therapeutic agents, particularly those that alter mucins, fluid or electrolyte balance and ciliary activity. anaesthetic gases, barbiturates, narcotics and alcohol depress clearance function. by contrast, topical, oral or parenteral administration of (3-adrenergic agonists, isoprenaline and adrenalin, produce a dose-dependent stimulation of mucociliary transport by an effect on ciliary beat frequency, probably mediated by increasing levels of cyclic adenosine monophosphate in ciuated cells rather than through vascular changes. although basal mucociliary function is dependent on normal vagal tone, parasympathomimetic agents can affect mucociliary transport. acetylcholine and cholinergic agents stimulate ciliary activity whereas anticholinergic drugs, atropine and hyoscine, inhibit ciliary activity and mucociliary transport. these substances may alter deposition of inhaled particles in the lung.^ clara cells or non-ciliated bronchiolar cells located in the bronchiolar epithelium, first described by clara in 1937, are small and cylindrical in shape with highly infolded nuclei, surface microvilli, well developed golgi, abundant endoplasmic reticulum and characteristic oval, homogeneous electron-dense granules in the apical cytoplasm. in rats, rabbits and humans the granules are pas-positive, although they are usually considered pas-negative in hamster and mouse.^^ clara cells have high metabolic activity. they contain cytochrome p450-dependent enzymes and secrete a variety of proteins.^^"^^ clara cell secretory protein is the major component of their cytoplasmic granules and they have been shown to produce mucin following antigen challenge.^^ in most laboratory rodents, the conducting airways terminate abruptly at the non-cartilaginous terminal bronchiole that opens directly into an alveolar type airway, the alveolar duct which in turn communicates with the alveoli.^^ squamous epithelial or type i cells form only about 10% of all lung cells but they line over 90% of the alveolar surface, by virtue of extremely long cytoplasmic extensions. the principal gas exchange takes place across this cell. in the rat, the typical thickness of this barrier is 20 nm for a cytoplasmic extension of a type i pneumocyte, 90 nm for basal lamina and 90 nm for an endothelial cell.^^ the type i cell contains juxtanuclear mitochondria and the long smooth cytoplasmic extensions contain many ribosomes and pinocytotic vesicles. the anatomical configuration and function of type i cells render them highly vulnerable to inhaled gases and particles. the other main alveolar lining cell is the granular pneumocyte or type ii cell which constitutes about 10% of all lung cells, but which covers only about 5% of the alveolar surface.^^ this cell does not possess the long cytoplasmic processes typical of type i cells and it shows many microvilli on its luminal surface. the cell cytoplasm contains rough endoplasmic reticulum, golgi apparatus, some mitochondria and characteristic oval, osmiophilic lamellar inclusions. surfactant, a microaggregate of phospholipid and protein which modifies alveolar surface tension at low inflation volumes, is secreted by type ii alveolar cells. ultrastructural immunocytochemistry has shown the presence of surfactant apoproteins in the synthetic organelles and in the lamellar bodies of these cells, in agreement with the concept that the surfactant apoproteins are synthesized in the rough endoplasmic reticulum, glycosylated in the golgi and are stored in lamellar bodies.^^ type ii cells are more resistant to the damaging effects of xenobiotics and unlike type i cells they retain the ability to undergo mitotic division. following damage to type i cells, increased numbers of mitoses are evident in type ii cells which results in the appearance of large undifferentiated epithelial cells which ultimately differentiate into type i and type ii cells.^^ the lung also contains a dense neural network and a population of endocrinelike cells believed to be important in lung function.^^ these neurosecretory cells (kultschitsky or apud cells) are scattered sparsely in the epithelial surface of the larynx, trachea bronchi, bronchioles and alveoli. these cells are oval or cuboidal with oval nuclei, and argyrophilic cytoplasm which electron microscopic examination shows to contain dense core granules. the role of neuroendocrine cells in the lung is uncertain but immunocytochemical study has shown them to contain a number of neuroendocrine substances including neurone-specific enolase, synaptophysin, chromogranin and a variety of other peptides similar to vasoactive intestinal peptide, bombesin, calcitonin, serotonin, leu-encephalin, p endorphin and acth.^^'^^ cells lining the bronchi, bronchioles and alveolar walls are capable of metabolizing xenobiotics. immunocytochemical study has shown the presence of immune-reactive cytochromes p450, nadph cytochrome p450 reductase, epoxide hydroxylase and glutathione s-transferase in bronchial epithelial cells, ciliated bronchiolar cells, clara cells, type ii and possibly type i pneumocytes in the rat lung.^^ different cell populations contain different amounts of enzymes, clara cells containing the greatest concentrations of the phenobarbitoneinducible isoenzyme of cytochrome p450, nadph-cytochrome p450 reductase and epoxide hydrolase. studies of microsomal enzyme activities suggest that lung tissue contains fewer p450 isoenzymes than liver, principally forms cyplal cyp2b1, cyp3a2 and cyp4bl^^ whereas p450 enzyme activity is highly concentrated in specific cell types, overall microsomal enzyme activity is low compared with liver on the basis of microsomal protein weight.^^ other important cells are the pulmonary alveolar macrophages and lymphocytes. lymphocytes are found in the epithelium of the airways, in the interstitium of alveoli and as part of follicles in bronchial walls. pulmonary macrophages form part of the specific immune defence system of the lung, involving, as elsewhere in the body, antigen presentation. in the rat and mouse, distinctive populations of pulmonary macrophages have been described based on enzyme activities and reactivity to monoclonal antibodies against monocyte and macrophages surface determinates.^^'^^ bronchus associated macrophages in rat and mouse have more acid phosphatase and less non-specific esterase activity than the populations found in the pulmonary alveoli and interstitial tissues. an important aspect of the immune system is the bronchus-associated lymphoid tissue or balt, which forms part of the mucosal lymphoid system found in other epithelia. the morphology of balt is a useful guide to the nature and degree of immune stimulus in the lung. balt is organized in a way that is characteristic of other peripheral lymphoid organs. it is structurally similar in the laboratory rat, mouse, rabbit, guinea pig as well as in man but its size and prominence is species and strain-dependent as well as a function of the degree of antigenic stimulus^"^"^^. in the rat, the balt is composed of lymphoid aggregates or fouicles located mostly between a bronchus and artery with a zone of lymphocytes situated immediately under the bronchial epithelium. as in other peripheral lymphoid tissue, balt is organized into b and t cell zones but in no predetermined manner. immunocytochemical staining has shown that b and t lymphocyte zones differ in location from one aggregate to another. there are about two t lymphoc3^es for every three b cells compared with a ratio of 2:5 in rat peyer's particles.^^ the ratios may be different in other species. quantitative observations of t cell subsets using monoclonal antibodies have also shown that rat balt normally contains twice as many t-helper as t-suppressor/cytotoxic lymphocytes.^^ the t cells are confined to one or two discrete zones with a light scattering of t cells within the b cell zones and immediately under the bronchial epithelium. in common with lymph nodes, interdigitating cells are also found. the epithelium overl3dng balt shows anatomical modifications. it is composed of ciliated and non-ciliated cells covered by microvilli. in conventional, untreated laboratory rats, balt shows little activity and germinal centres are usually absent, although balt may be more prominent in some rat colonies in association with non-specific inflammatory lesions in lungs.^^'^^ in one colony of young wistar rats germinal centres were not seen in balt in untreated animals but they developed following the administration of a single intratracheal dose of lipopolysaccharide, a t cell-independent antigen.^^^ single intratracheal doses of t cell dependent antigens such as horseradish peroxidase, bovine serum albumin and bcg have been shown to produce only minor morphological changes which include expansion of the zone of lymphocytes immediately under the epithelium and infiltration of the bronchial epithelium overlying balt by lymphocytes.^^^ in addition, perivascular, peribronchial or alveolar infiltrates of small and large lymphocytes and macrophages were observed in the lungs of rats given bcg. immunoc3^ochemical study of the rat balt following intratracheal challenge with horseradish peroxidase showed that the majority of cells that infiltrated the bronchial epithelium were t helper (cd4 positive) lymphocytes. ^^^ furthermore, la antigen expression of the epithelial cells overlying the balt was shown to increase, associated with an increase in the number and size of microvilli, a more pronounced glycocalyx and a decrease in number and size of cilia. immunocytochemical study of the balt tissue in c57b1/6 mice using monoclonal antibodies to lymphoid and macrophage populations has demonstrated quite similar arrangements of cells to those in the rat with the majority of t cells belonging to the t helper (cd4 positive) class.^^ the pulmonary lymphatic system drains into mediastinal or cervical lymph nodes. although among rat strains differences in the location of lymph nodes and their drainage occur, tracer studies in the fischer 344 rat using colloidal carbon have shown that the lung lymphatics drain mainly into posterior mediastinal lymph nodes and those in the tracheal wall drains primarily to the internal jugular and posterior cervical nodes.^^^ although a variety of fixation, embedding and staining procedures are available for light and electron microscopic examination of lung tissue, there is no substitute for initial, careful visual inspection of the lungs at autopsy. uneven collapse of lungs on opening the thoracic cavity, discoloration or alteration in texture of the pleural or cut surface, congestion and presence of fluid in the larger airways may indicate structural damage. in rodent lungs, small pulmonary adenomas may be detectable by inspection in good light. fresh lung weight is also a helpful measure in lung assessment, although passive vascular engorgement can significantly affect this value. nevertheless, studies in the normal fischer 344 rat have shown that after exsanguination, wet lung weights show a close relationship to body weight and that dry weight of lungs consistently represents about 20% of the wet weights regardless of age or body weight. ^^^ an increase in wet weight over dry weight appears to be a good index of pulmonary oedema. ^^ various methods of fixation have been employed although simple immersion fixation in formalin for conventional light microscopy has the virtue of simplicity and it avoids the risk of translocation or removal of exudates from airways and alveoli. mixtures of formaldehyde, paraformaldehyde and glutaraldehyde are used in initial fixation for electron microscopy.^^ the best overall appreciation of lung architecture is achieved by instillation of fixative via the trachea under an appropriate constant pressure or by perfusion fixation of the pulmonary arteries that is less liable to dislodge intra-alveolar exudate. in a review of methods employed routinely in rodent toxicity studies, instillation of fixative via the trachea was the preferred method in most laboratories because its advantages were seen to outweigh its disadvantages.^^ the sampling procedure is an important aspect of histological examination of the bronchi and lungs, particularly those of large laboratory animals. the extent of histological sectioning in conventional toxicity studies should be modulated to take account of lesions found by macroscopic examination, the type of study and the nature of the test substance. the bronchi should be carefully sampled to allow assessment of any alterations in bronchial epithelium. morphometric analysis represents a sensitive tool of value in the evaluation of drug-induced lung changes, but it requires particularly rigorous sampling and evaluation procedures.^^^'^^^ a tiered, multiple stage or cascade sampling technique is normally considered the most appropriate for morphometric studies.^^'^ this involves dividing the lung into a series of homogeneous compartments or strata from which randomly selected samples can be examined by appropriate light or electron microscopic techniques. conventional special stains for reticulin and collagen as well as pas and alcian blue for mucins are helpful in the characterisation of lung damage and changes to the respiratory epithelium. immunocytochemistry and enzyme cytochemistry are also useful in the study of the heterogeneous cell population of the lung. xenobiotic metabolizing activity can be studied both by enzyme cytochemical methods as well as by immunocytochemical techniques using antisera specific for pulmonary monooxygenases and related enzymes.^^ important structural components, particularly collagen and laminin can be studied both at light and ultrastructural level with immunocytochemical methods.^^^ cytokeratin immunocytochemistry can be used as a method for the characterization of changes to epithelial cells.^^ clara cells can be localized by the presence of clara cell secretory protein and ciliated cells by the presence of tubulin.^^ endocrine cells are visualized by immunocytochemistry using antibodies to general neuroendocrine markers such as chromogranin and synaptophysin or regulatory peptides.^^ other useful antigens, which can be demonstrated in the lung, include surfactants, lysozyme, immunoglobulins and those of microorganisms that infect the lung.^^^ electron microscopy is particularly useful for the detailed characterization of injury to the cells of the alveolar epithelium and endothelium ( figure 6 .1). pulmonary oedema is a component of many inflammatory conditions of the lung, including those induced by infectious agents. however, the term oedema is reserved for a poorly cellular exudate characterized by the presence of pale, homogenous eosinophilic material in the alveoli, sometimes associated with a similar exudate in the lung septae and perivascular connective tissue. it occurs in a number of spontaneous conditions such as in congestive cardiac failure, metastatic pulmonary neoplasms or as an agonal change in association with pulmonary congestion and haemorrhage. drugs may induce cardiogenic pulmonary oedema as a consequence of pulmonary hypertension or impaired ventricular contractility. cardiogenic oedema is often associated with vascular congestion and red blood cells and haemoglobin may leak into airspaces. this can give rise to the presence of haemoglobin crystals within the oedema fluid in formalin-fixed tissue sections. most importantly, pulmonary oedema may be a manifestation of acute lung injury. inhalation or systemic administration of toxic chemicals may produce acute pulmonary oedema (see figure 6 .1). some substances such as phenylthiourea and a-naphlythiourea produce massive pulmonary oedema in laboratory animals when administered orally, principally as a result of damage to the endothelium of pulmonary capillaries and venules. ^^^ over 30 drugs have been reported to produce non-cardiogenic pulmonary oedema in humans, either directly or through poorly understood immunogenic mechanisms.^ another form of pulmonary oedema involves the main airways. allergic reactions in sensitized airways of asthmatic individuals is believed to result from cross-linking of ige and activation of mast cells that degranulate and release inflammatory mediators. ^^^ this has been reproduced in the main airways of rats sensitized to ovalbumin and then challenged with ovalbumin by the intratracheal route. ^^^ this treatment leads to rapid accumulation of bronchial exudate, degranulation of mast cells and the development of mucosal oedema, most marked immediately below the respiratory epithelium. congestion and haemorrhage is a frequent finding in the lungs of laboratory animals, where it is usually related to certain modes of death. it can be associated with administration of drugs and chemicals that have adverse effects on cardiac function or on the coagulation system. administration of heparin to rats produces a characteristic extravasation of blood into the air spaces.^^^ lower respiratory tract infection is generally not a major health hazard among laboratory animals but it is nevertheless an ever-present threat that can cause overt respiratory disease within a colony or develop following administration of xenobiotics. subclinical pulmonary infections and infestations can also produce histological alterations in the bronchial airways or pulmonary parenchyma which mimic changes induced by inhaled irritants or systemically administered drugs.^^'^^ furthermore, some respiratory pathogens alter immune defences and exacerbate the effects of inhaled substances. ^^^ a range of bacterial and viral pathogens may produce inflammatory lung changes.^^ typically, bacterial pathogens such as steptococcus pneumoniae produce acute bronchitis associated with a variable degree of acute inflammation of the lung parenchyma (bronchopneumonia) or a confluent lobar pneumonia. viral agents are generally associated with histological features of bronchiolitis and interstitial pneumonia, characterized by an increase in mononuclear cells in the respiratory bronchioles and alveolar septa. the histological features are variable for they depend on the particular pathogen, species and strain, immune status, presence or absence of secondary infection and the particular stage at which the infection is examined. respiratory infections are frequently mixed. changes due to secondary bacterial infection are frequently superimposed on those induced by viruses. sequential histopathological examination of the lungs of laboratory animals following inoculation with respiratory tract pathogens has been able to characterize the evolution of pathological changes produced by individual organisms. for instance, following inoculation with mycoplasma pulmonis, one of the more important respiratory pathogens among laboratory rodents both lewis and fischer 344 rats were shown to develop upper and lower respiratory tract inflammation. in the lewis strain this was characterized after 28 days by a variable acute inflammatory exudate in bronchi and bronchioles with focal bronchiectasis, inflammation and hyperplasia of the epithelium with a predominantly macrophage infiltration of the alveoli and alveolar walls.^^'^^^ these changes were associated with marked hyperplasia of the bronchusassociated lymphoid tissue (balt), which extended down the airways and blood vessels towards the periphery of the lungs. although the lymphoid hyperplasia was also found in inoculated fischer 344 rats, it was less marked and accompanied by little or no mucopurulent exudate or active inflammation of the bronchial walls. this disparity in response suggested that differences were related to the degree of lymphocyte activation in the two strains, an imbalance in regulation of lymphocyte proliferation in lewis rats, or both.^^^ other studies have been conducted in both rats and mice infected with another important respiratory pathogen of laboratory rodents, the sendai virus (parainfluenza type 1). sequential studies showed that the initial damage to bronchial and bronchiolar epithelium is associated with polymorphonuclear and lymphocytic inflammation (bronchiolitis). immunocytochemical and ultrastructural studies revealed the presence of viral antigen in the mucosa.^^^ hyperplastic and multinucleated syncytial epithelial cells develop in the hyperplastic terminal bronchiolar epithelium and the inflammatory process extended to involve peribronchial or peribronchiolar parenchyma with infiltration of alveolar walls by mononuclear cells, macrophages and neutrophils. a similar cell population accompanied by cell debris and oedema fluid develops in air spaces. pulmonary arteries show only minor involvement with inflammatory cells and focal reactive hyperplasia of the endothelium. immunocytochemistry and ultrastructural examination suggested that virus replication takes place in alveolar type i and type ii epithelial cells and macrophages but not in endothelial or interstitial cells of the alveolar septae.^^^ it was shown that when repair occurs there may be residual distortion of bronchiolar and alveolar walls by collagen and hyperplastic cuboidal epithelium may line the thickened alveolar septa. air spaces may also contain enlarged macrophages with pale vacuolated cytoplasm.^^^ strain differences in susceptibility have also been demonstrated to this virus. there is differential pulmonary interleukin 12 (il-12) gene expression between virus-susceptible brown norway rats and resistant fischer 344 rats and il-12 treatment provides protection from virus-induced chronic airway inflammation and remodelling. moreover increased tumour necrosis factor a (tnfa) expression has been shown to be an important regulatory factor in the development of sendai virus-induced bronchiolar flbrosis in infected rats.^^^ virus-inoculated brown norway rats had increased tnfa pulmonary mrna levels and increased numbers of bronchiolar macrophages and fibroblasts expressing tnfa protein compared with virus-inoculated f344 rats.^^^ the corona virus, which causes sialodacryoadenitis in many rat colonies, also produces lower respiratory tract inflammation. this is characterized by acute bronchitis and bronchiolitis with focal extension into lung parenchyma. thickened oedematous, hypercellular alveolar walls infiltrated by monocytic cells are found.^^ immunocytochemistry has shown the presence of viral antigen in bronchial and bronchiolar epithelial cells. there is also peribronchial lymphocytic infiltration and increased prominence of balt. ultimately complete resolution occurs. viruses remain a potential source of spontaneous respiratory disease in laboratory dogs. canine adenovirus type 2, parainfiuenza sv5, canine herpes virus, coronavirus and parvovirus have all been isolated from laboratory dogs developing respiratory disease.^^^ the syndrome of visceral larva migrans also incites focal inflammation, granulomas and fibrosis in the lungs of species such as dog and primate in which parasites are prevalent. the syndrome of visceral larva migrans is usually defined as that which results from the migration of nematode larvae into the viscera. it has been well described in the beagle dog lung where it results from the larvae of toxocara species or metastrongyloid nematodes.^^^'^^^ the precise identification of parasites is not always possible in tissue sections. histological appearances of infested lungs are highly variable. nematodes surrounded by granulomas and granulomatous inflammation, mostly in a subpleural location, may be visible in sections. in affected lungs there may be perivasculitis and active arteriolitis, bronchiolitis and peribronchiolitis. pleural involvement by the inflammatory process can be marked, particularly in regions overlying granulomas. scarring develops and pleural and sub-pleural fibrosis is frequently associated with epithelial hyperplasia and squamous metaplasia of the associated airways ( figure 6 .2).^^^ the lesions may sufficiently severe to resemble those induced by high doses of anticancer drugs such as bleomycin (see below). pulmonary acariasis is a common infestation of many species of non-human primates caused by various species of the mite pneumonyssus. reproduction of the mites appears to take place in the terminal bronchioles. pneumonyssus simicola is the recognized form found in rhesus monkeys.^^^ although it is most prevalent in wild caught primates, the disease is not easily eliminated during breeding in captivity. ^^^ even when eliminated by ivemectin the lesions of chronic bronchiolitis, bronchiectasis and pigmentation may persist as an incidental finding. ^^^ as the mite can produce significant destructive pulmonary pathology and render animals susceptible to secondary pulmonary bacterial infections, it can disrupt or confound the interpretation of toxicity studies performed in primates. the lesions are located most frequently in cranial lobes and are characterized by the presence of bullae distending the pleural surface, parenchymal cysts, nodules and scar tissue.^^^'^^^ histologically, there is a wide range of inflammatory activity. fully developed lesions are characterized by granulomatous bronchiolitis and peribronchiolitis with involvement of immediately adjacent alveoli. cystic lesions involving the bronchiolar walls develop around the parasites giving rise to the appearance of walled-off cysts composed of highly cellular granulation tissue, associated with neutrophils, lymphocytes, macrophages, multinucleated giant cells and various pigments (see below). in less active lesions, dilated, cystic airways with walls composed of thick bands of smooth muscle and lined by squamous or cuboidal epithelium are found. pneumocystis carinii is an important cause of pneumonia in patients with the acquired immunodeficiency syndrome (aids) as well as in other immunocompromised patients, including those treated with immunosuppressive drugs.^^"^ the natural habitat of pneumocystis carinii is pulmonary alveoli and it is widely encountered in the human population without being associated with overt disease. both clinical and experimental evidence suggests that impaired cellular immunity is much more important as a predisposing factor than impaired humoral immunity. ^^^ as in humans, laboratory animals may have latent pneumocystis infection that becomes clinically evident following immunosuppression. it has been shown in the rat that chronic administration of various regimens of adenocorticosteroids, low protein diets, cyclophosphamide and other immunosuppressive drugs with concomitant antibiotic administration to prevent other infections gives rise to typical pneumocystis pneumonia. ^^^ rodents with genetically deficient cellular immunity also develop pneumocystis pneumonia. the importance of pneumocystis pneumonia in toxicology is that it can be considered as a sentinel of chronic immune depression. in haematoxylin and eosin stained sections, pneumocystis pneumonia is characterized in both humans and rodents by the presence of alveoli filled with foamy eosinophilic material containing a few macrophages and indistinct nuclei of pneumocystis (figure 6 .2fe). ovoid or crescent-shaped structures of the organisms become clearly visible with gomori methenamine silver or toluidine blue stains. ultrastructural study of rats with pneumocystis pneumonia shows that trophozoites attach themselves most frequently to type i pneumocytes by altering their morphology to the contours of the pneumocytes rather than by a process of invasion. ^^^ systemically administered therapeutic agents may produce histological changes within the lung parenchyma that mimic components of the normal response to respiratory pathogens. however, there is no sharp separation between agents that produce pulmonary oedema with those that are associated with acute inflammatory changes and histological features overlap because an acute inflammatory process is often accompanied by exudate within airspaces. an example of drug-induced pulmonary inflammation in laboratory animals and humans is reported following the administration of interleukin 2 (il-2). il-2 is a glycoprotein lymphokine, molecular weight 15kda, which is normally produced by activated t cells and mediates immunoregulatory responses. it has been produced in large quantities by recombinant dna technology for use in tumour immunotherapy. however, high doses have been associated with a number of adverse effects, notably the 'vascular leak' syndrome, characterized clinically by pulmonary oedema, pleural effusions and ascites. ^^^ the vascular leak syndrome has been reported in laboratory animals given high doses of this agent. histological examination of the lungs of b6d2f mice developing this syndrome following administration of il-2 showed infiltration of the alveolar walls with large lymphocytes and intra-alveolar proteinaceous exudate containing large lymphocytes, macrophages and red blood cells.^^^'^^^ pulmonary venules and arterioles showed the presence of lymphocytes attached to or lying beneath the endothelium, infiltrating vessel walls or in a perivascular location where they were accompanied by oedema fluid or red blood cells. similar, but less severe changes have been demonstrated in rats given il-2.^^^ in addition, treated rats showed an infiltration of pulmonary vasculature with eosinophils probably secondary to an eosinopoietic cytokine produced by il-2 stimulated lymphocytes. immunocytochemical evaluation of the lymphoid infiltrate in mice showed that most of the cells were thy 1.2positive (cd90) lymphocytes. furthermore, co-administration of asialo gml (ganglio-n-tetrosyl-ceremide) with il-2 not only abrogated the clinical signs but also reduced the number of asialo gml-positive lymphocytes in the tissue sections. as lymphoid cells expressing lyt-2 (cds, suppressor/cytotoxic t cells) were unaffected by asialo gml treatment, it was postulated that the vascular leak syndrome (but not antitumour efficacy) in these mice was mediated by an endogeneous subset of il-2 stimulated lymphocytes or lymphokine-activated killer cells. ^^^ corresponding changes were also observed in liver and lymphoid tissue. immunocytochemical and detailed electron microscopic studies in rats have supported the concept that il-2 induces cytotoxic vascular damage that is mediated both directly by lymphokine-activated killer cells and cytotoxic t lymphocytes with secondary release of inflammatory cytokines.^^^ as in humans, severe chronic pulmonary inflammatory disease in laboratory animals may compromise pulmonary function and lead to secondary alterations in other organs. although the mechanisms were not explored in detail, a diffuse interstitial pulmonary inflammatory process with lung haemorrhage was induced in rats treated for two years with prizidilol (skandf 92657-a2), an antihypertensive agent with both vasodilator and (3 adrenoceptor blocking properties.^^^ affected animals developed dyspnoea associated with reduction in lung volume, deformity of the thoracic spinal column and marked cardiac hypertrophy. inflammation with granulomas develops in the lungs of laboratory animals under a variety of different circumstances, which have been alluded to above. a common cause in rodents is granulomatous pulmonary inflammation resulting from aspiration of stomach contents or food particles (aspiration pneumonia). this is sporadically observed in aged rodent where it is associated with general ill health, particularly resulting from pressure effects of large pituitary adenomas and subsequent disturbance of pharyngeal or laryngeal reflex mechanisms. ^^^ histologically, the lungs show peribronchial and peribronchiolar granulomatous inflammation with macrophages and foreign body cells associated with fragments of refractive vegetable matter. the associated bronchial mucosa may also show reactive changes including goblet cell hyperplasia in long-standing cases. as dogs and primates are more liable to be infested by parasites, granulomatous inflammation in response to pulmonary larvae is more common in these species. pulmonary tuberculosis represents a potential problem among non-human primate colonies in view of its insidious onset and its liability for transmission from monkeys to humans. ^^^ pathological findings are similar to those so well known in the human disease. the disease is characterized by the presence of granulomas in lung parenchyma and lymph nodes. in florid cases there may be caseation surrounded by epithelioid and multinucleated giant cells and variable numbers of lymphocytes, plasma cells and flbroblasts. diffuse granulomatous pneumonia as a result of tuberculosis is also reported in non-human primates. granulomatous pneumonitis is also produced in laboratory animals by the intravenous injection of bcg. twenty-eight days following intravenous injection of bacille calmette-guerin (bcg), the lungs of c57b1/6 mice contained numerous granulomas composed of histiocytes and round cells which were surrounded by alveoli with thickened walls and associated with mild interstitial pneumonitis. ^^^ these histological changes were associated with an increase in the number of thy 1.2-positive (cd90) cells, especially lyt-1 (cds) positive lymphocytes. the histological changes were abrogated by treatment with cyclosporin a suggesting an important role for cd5-positive lymphocytes in the development of the granulomas. discrete granulomas occur in the lungs of experimental animals in response to intra-tracheal or intravenous injection of certain relatively insoluble substances (figure 6.3) . intra-tracheal administration of insoluble polymerized dextran and latex micro-particles to mice showed that the morphology and the systemic effects of granulomas depends on the nature of the injected substances. it has been shown that large granulomas develop rapidly in the pulmonary parenchyma around dextran particles that subsequently regress quickly, whereas latex particles produce small, discrete stable granulomas. ^^^ although both forms of granulomas are of foreign body or non-immunological in type, those produced by dextran but not latex beads, are associated with anergy-like immunosuppression, probably caused by release of soluble factors from the granulomas. it has been reported that granuloma formation after instillation of sephadex beads is associated with increases in the interleukin 1-(il-1) like activity in the lung.^^^ studies comparing the effects of inhaled crystalline silica and titanium dioxide have shown a correlation between the release of the macrophage derived cytokine il-1 and granuloma formation, suggesting that il-1 might be a useful biomarker for granuloma formation. ^^^ localized, angiocentric granulomas of foreign body type, clustered around pulmonary arteries and arterioles and occasionally alveolar capillaries and venules also develop following intravenous injection of relatively insoluble polysaccharides or other polymers. ^^^ characteristic epithelioid and large, foreign body type giant cells efface the smaller vessels although overt necrosis is not usually observed (figure 6.3) . haemosiderin-laden macrophages accumulate in the alveou of laboratory animals in association with chronic pulmonary congestion and haemorrhage. similar changes occur in patients in congestive cardiac failure where the haemosiderin-laden macrophages are termed 'heart failure' cells. the lungs of non-human primates are especially liable to contain alveolar, perivascular and peribronchial aggregates of macrophages laden with various brown pigments. iron-containing pigments have been associated with the inflammatory changes produced by simian lung mites (pneumonyssus simicola) which are prevalent in many non-human primates. in addition, lungs from some primate colonies may show perivascular and peribronchial collections of brown-grey macrophages containing highly refractive spicules and plates composed of high concentrations of silica.^^^'^^^ it has been shown that in old world primates including rhesus and cynomolgus monkeys, this pigment contains fossil diatomaceous material, compatible with the concept that the animals inhale dusts containing diatoms and other silicon fragments to which they are exposed in their semi-arid, natural habitats.^^^ chronic lung injury from a variety of different causes is frequently associated with the development of pulmonary fibrosis characterized by the replacement of the normal pulmonary structure by a thickened collagenous matrix with consequent reduction in the capacity for gas exchange. regardless of the inciting agent, the fibrogenic process appears to be generally characterized by disruption of the normal alveolar-capillary structure, leakage of exudate from the vascular compartment into the airspaces, subsequent invasion by infiammatory cells and fibroblasts associated with excess matrix formation. studies in laboratory animals with different fibrogenic agents as well as in humans have suggested that central to pulmonary fibrogenesis is increased production of tnfa by macrophages.^^'^^^'^^^"^^^ this cytokine is a not only a mitogen for fibroblasts but also a potent activator and chemo-attractant for macrophages, capable of stimulating release of other cytokines and inducing expression of adhesion molecule expression on endothelial cells. moreover, it has been shown that tnfa receptor knockout mice appear protected from the fibroproliferative effects of inhaled asbestos. ^^^ pulmonary fibrosis is a common sequel of chronic lower respiratory tract inflammation. it may be associated with, or preceded by interstitial pneumonitis, characterized by infiltration of lymphocytes, plasma cells and macrophages with scattered polymorphonuclear cells. ^^^ focal pulmonary fibrosis occurs spontaneously in laboratory animals, although this is usually most prevalent in dogs and non-human primates as a response to chronic infestation by parasites, which are not easily eliminated during breeding. in humans, conditions leading to pulmonary fibrosis vary widely. they include infections, shock lung syndrome, ionizing radiation, inhalation of irritant particulate matter, exposure to antigens or excessive amounts of oxygen as well as the results of the toxicity of paraquat and a range of both cytotoxic and noncytotoxic therapeutic agents which cause pulmonary parenchymal injury.^'^^^ the principal therapeutic agents that produce pulmonary fibrosis in both humans and laboratory animals are anticancer drugs. bleomycin, a glycopeptide preparation derived from streptomyces verticillus, is the best known example but pulmonary fibrosis is also associated with the clinical use of a number of other anticancer agents, including l,3-bis-(2-chloroethyl)-l-nitrosourea (bcnu or carmustine), cyclophosphamide, busulphan, mitomycin c and methotrexate.2 '108,147-150 the precise mechanisms involved in the induction of pulmonary fibrosis by antineoplastic drugs in humans are poorly understood. the true incidence for a particular drug is difficult to estimate because of confounding factors in cancer patients, such as concomitant administration of several drugs, radiation and oxygen therapy, diffuse pulmonary cancer and opportunistic infections. it is probable that drug-induced fibrosis is accentuated by concomitant administration of several antineoplastic agents, radiation therapy, hyperoxia, preexisting pulmonary damage and age of the patient. severity is often related to total dose of drug received.^^^ novel antineoplastic drugs may also produce lung toxicity.^ bleomycin is associated with the development of interstitial pneumonia and pulmonary fibrosis in clinical use and this can be reproduced in experimental animals. the histopathological appearances of bleomycin-induced pulmonary fibrosis in patients are in many instances different from those seen in laboratory animals because the lungs of patients treated with bleomycin are modified by the primary neoplastic disease, smoking, multiple drugs, radiation therapy and secondary pulmonary infections, interstitial pneumonitis and fibrosis.^^^ it has been postulated that tnfa is an important mediator in the development of bleomycin-induced fibrosis. ^'^ in the preclinical evaluation of bleomycin beagle dogs were given cycles of drug by the intravenous route for periods of up to 26 weeks. ^^^ dogs developed anorexia, weight loss, a variety of epithelial lesions as well as focal interstitial pneumonia and fibrosis. the focal lung lesions were characterized by increased elastic fibres, reticulin, collagen and acid mucosubstances. the lesions were situated predominantly in the pleural and subpleural zones, suggestive of a potentiating effect of friction between the pleural surfaces. histologically the lesions resembled those produced by larvae migrans in the dog (see figure 6 .2a). similar histological changes have also been described in both rats and mice treated with bleomycin by both the intravenous and intratracheal route.^^^'^^^ as fibrosis is such a consistent change, bleomycin-treated rodents have been extensively employed as a model for pulmonary fibrosis. early changes include mild, diffuse increases in interstitial lymphocytes, macrophages, polymorphonuclear cells and perivascular or interstitial oedema. after about a week, interstitial infiltrates also comprise fibroblasts with early collagen deposition, associated with proliferation of macrophages and type ii pneumocytes.^^^'^^^ subsequently, the amount of interstitial collagen increases, with eventual scarring and collapse of lung tissue in proportion to the cumulative dose given. ^^^ immunohistochemical and ultrastructural study of rats and mice treated with bleomycin shows a large accumulation of immune-reactive laminin and reduplication of the basement lamina within the thickened alveolar walls. ^^^ in bleomycin-treated rats three-dimensional scanning electron microscopy shows drug-induced capillary remodelling comprising irregular alveolar and pleural capillaries with increased diameter and decreased branching. ^^^ certain strains of mice have been shown to possess greater sensitivity to bleomycin fibrogenesis. the c57bl/6 strain produces a greater fibroblastic response than dba/2 and swiss mice and the balb/c strain demonstrates a particularly poor fibroblastic response. ^^^ therapeutic use of cyclophosphamide is also occasionally associated with the development of pulmonary interstitial fibrosis.^^^'^^^ it appears to be associated with two forms of pathology: an early-onset pneumonitis and a late onset progressive pulmonary fibrosis. ^^^ similar changes have been less easy to reproduce in laboratory animals. when mice were sequentially examined for periods of up to one year after a single intravenous dose of loomg/kg of cyclophosphamide, only slight pulmonary interstitial thickening and hypercellularity was observed in association with progressive multifocal accumulation of intra-alveolar macrophages. ^^^ however, these changes were also accompanied by a progressive increase in pulmonary hydroxyproline content and a decrease in pulmonary compliance with time in treated animals compared with controls. the changes were amplified by exposure to 70% ambient oxygen. the bronchiolitis, alveolar septal infiammation and fibrosis induced by gold therapy in patients with rheumatoid arthritis is probably immune-mediated. this condition is associated with peripheral eosinophilia and drug-induced alterations to the immune system.^^^ emphysema is characterized by abnormal, permanent enlargement of airspaces distal to terminal bronchioles, accompanied by destruction of their walls without obvious fibrosis. three principle types, centriacinar, panacinar and distal acinar emphysema, are recognized in humans. enlargement of air spaces as a result of congenital factors or fibrous scarring are grouped separately and not regarded as emphysema. ^^^ emphysema has been reported as an age-related spontaneous change in laboratory rats.^^^ however, several experimental rodent emphysema models have been developed, using intratracheal instillation of proteolytic enzymes papain, pancreatic and neutrophil elastase. this gives rise to histological appearances resembling panacinar emphysema in humans.^^^ irritant gases, notably oxides of nitrogen, are also capable of inducing changes in the lungs of laboratory rats and hamsters following long term exposure which resemble mild human, centrilobular emphysema.^^^'^^^ a variety of different names have been applied to membrane-bound, acid phosphatase-positive cytoplasmic inclusions with a lamella or crystalloid ultrastructural matrix. these include myeloid bodies, myelinoid bodies, myelin figures or myelinosomes. these lysosomal inclusions are seen in small numbers in a variety of normal cell types but they accumulate in various organs in laboratory animals following administration of a wide variety of drugs of diverse therapeutic classes.^'^^^"^^^ the generalized accumulation of these lysosomal cytoplasmic bodies is generally called phospholipidosis, a term coined to describe the tissue accumulation of phospholipids. ^^^ at the light microscopic level, phospholipids are characterized by the increase in the number of foam cells in the airspaces. examples of drug-induced phospholipidosis include the anorectic drug chlorphentermine, tricyclic antidepressants, inhibitors of cholesterol biosynthesis such as triparanol, the antihistamine chlorcyclizine and its analogues, the selective oestrogen receptor antagonist tamoxifen, chloroquine and the cardiovascular drugs amiodarone, 4,4'diethylamino-ethoxyhexestrol and perhexiline.^^^'^^^'^^^ many tissues and organs may develop the cytoplasmic inclusions, including lymphoid cells, liver, pancreas, endocrine tissue, nervous system, muscle cells, eyes and particularly lungs. aminoglycoside antibiotics may produce laminated phospholipid inclusions in the renal tubular cell and imidazol antifungals in hepatocytes (see under liver and kidney, chapters 9 and 10). many drugs that induce phospholipidosis usually share structural features, notably a hydrophilic cationic side chain, a primary, secondary or tertiary amine and a hydrophobic region that is usually an aromatic ring or ring system. as this structural pattern renders these molecules amphiphilic, these drugs probably bind with polar lipids by means of electrostatic and hydrophobic forces.^^"^ this leads to formation of drug-lipid complexes which are poorly degraded by lysosomal enzymes and which accumulate in the cell cytoplasm to form the inclusions described above. as the binding is not covalent, its reversibility depends on the dissociation rate constant under the particular intracellular conditions and drug concentration achieved. predictions of this activity based on molecular structure have shown reasonably good correlation with the ability of compounds to produce phospholipidosis in cultured rat peritoneal macrophages. more recently other cell based systems have been proposed for screening for phospholipidosis.^^^'^^^ however these perform less well in the prediction of in vivo potency, presumably because of differences in drug disposition in blood and tissues. it should be underlined that the accumulation of foamy macrophages in the alveolar spaces may also be a spontaneous change in laboratory animals. it has long been recognized as a spontaneous alteration in ageing rats.^^^ it may also be found in lung tissue distal to bronchial lesions that impede clearance mechanisms. in contrast to drug-induced changes, the spontaneous lipidosis characterized by accumulation of alveolar foam cells occurs sporadically in older rats and is observed in both controls and treated animals. drug-induced phospholipidosis occurs within a period of several months during which lungs of control animals remain fairly free of foam cell accumulation. the lungs appear especially vulnerable to drug-induced phospholipidosis, possibly because macrophages are in very close proximity to blood-borne agents. phospholipidosis is also more clearly visible microscopically in alveoli whereas it can be easily overlooked in other organs. the continuous uptake of phospholipid-rich surfactant material from the alveoli by macrophages leads to excessive accumulation of phospholipids when their catabolism is impaired.^^^'^^^ the fact that lungs are commonly affected is a potentially useful diagnostic feature because in many organs phospholipidosis can be extremely difficult to recognize in haematoxylin and eosin stained sections. although the changes in the lungs are not specific for drug-induced phospholipidosis, an increase in the number of lipid-containing lung macrophages in treated animals compared with controls is relatively easy to detect and provides a simple way for the pathologist to screen for this effect. in severe generalized phospholipidosis in rats, the lungs show irregular pale grey or yellowish patches of discoloration of the pleura and parenchyma. this is a result of patchy or confluent aggregates of large, pale, foamy macrophages. they may be free lying or packed in alveoli and accompanied by granular, extracellular material. their abundant cytoplasm shows a vacuolated appearance in which fine eosinophilic granules are sometimes visible. the nuclei are rounded and centrally located structures of variable size (figure 6.4) . multi-laminated cells are also occasionally seen, as are vacuolated cells firmly attached to alveolar walls, probably pneumocytes. these foamy cells stain typically for phospholipids (e.g. acid haematin), although neutral lipids may also be present and stain with oil red o. semi-thin plastic-embedded sections stained with toluidine blue allow better characterization of phospholipidosis in all organs, including the lungs. the macrophages in the air spaces contain unmistakable dense, dark round cytoplasmic inclusions of variable size, some over 5 mm diameter. ^^^ plasticembedded sections also show the inclusions in other pulmonary cells including pneumocytes attached to the alveolar walls, from which they can be seen discharging into the alveolar spaces. as in other organs affected by phospholipidosis, ultrastructural examination reveals dense, multi-lamellar membranes and numerous heterogeneous dense bodies of lysosomal origin (figure 6.4) . these bodies need to be distinguished from membranous bodies that form as a result of fixation for ultrastructural study. lipids tend to leach out and become hydrated to form myelinoid membranes during glutaraldehyde fixation. these structures are subsequently fixed by osmium to give rise to electron-dense membranous figures both outside and inside cells, particularly in mitochondria where they may be mistaken for pathological lesions.^^^ the lamella patterns seen in phospholipidosis may be simple alternating dense and clear lines spaced at 4-5 nm, or more complex arrangements of clear and dense lines. the other typical crystalloid inclusions of hexagonal aggregates of tubular subunits seen in other organs are not usually found in the lungs. the significance of these various forms is uncertain but they probably represent the various phases in which phospholipids exist and are influenced by proportions of lipids present. electron microscopic examination reveals that not only are pulmonary macrophages affected by these changes but that inclusions may be present in pneumocytes types i and ii, pulmonary capillary endothelial cells, smooth muscle cells, bronchiolar epithelium and occasionally neutrophils.^^^"^^^ the changes are typically still visible several weeks after withdrawal from treatment with the offending agent. although the extent of pulmonary phospholipidosis in the lungs varies between dosage regimen and animal species, studies with chlorphentermine, 4,4'diethylaminoethoxyhexestrol and amiodarone indicate that similar cytological and ultrastructural changes occur in most laboratory animal species studied including rats, mice, hamsters, guinea pigs, rabbits and dog.^^^'^^^'^^^'^^^ safety assessment -amphiphilic drugs what are the imphcations for humans of drugs that induce phosphohpidosis in laboratory animals? novel compounds continue to be found that possess the property of producing phosphohpidosis in laboratory animals with varying degrees of severity.^^^'^^^ although not all drugs that produce phosphohpidosis in animals have been studied in humans, only very few drugs that produce phosphohpidosis in animals have been shown capable of inducing significant phosphohpidosis in human clinical practice.^ agents such as chloroquine, 4,4'diethylamindethoyhexestrol and amiodarone, which have been shown to produce phosphohpidosis in patients, can also induce cellular damage in the same organs. however the phenomenon of phosphohpidosis where phospholipids are packaged behind lysosomal membranes may not be causally related to cellular damage in humans. indeed the weight of evidence suggests that druginduced phosphohpidosis per se is an adaptive phenomenon and does not in itself have functional or deleterious consequences unless excessive. ^^^ hence, the finding of phosphohpidosis in animal studies with a novel drug requires careful assessment on a case by case basis with respect to its implications for the safety of humans. an example of this issue is the iodinated benzofuran derivative amiodarone, a potent antiarrhythmic drug effective against ventricular arrhythmia. lung toxicity continues to be a problem in patients treated for cardiac arrhythmias with this drug.^^"^ not only does phosphohpidosis occur in a wide variety of organs in laboratory animals treated with amiodarone,^^^'^^^ but also in liver, peripheral nerve cells, skin, lymphoid cells and lungs in patients at therapeutic doses.^^^'^^^'^^^ although pulmonary interstitial fibrosis occurs in association with phosphohpidosis in patients, amiodarone-induced phosphohpidosis in rodents is not associated with pulmonary fibrosis or significant functional alterations. several theories have been proposed for the pulmonary alveolitis and interstitial fibrosis in humans. the weight of evidence to date suggests that the accumulation of lipid-laden histiocytes is not causally related to the alveolitis or pulmonary fibrosis. ^^^ indeed, overall there is little evidence that the mere presence of phosphohpidosis is deleterious to the organism.^^^ cytotoxicity, possibly through the metabolite desethylamiodarone, has been proposed and an immune-mediated mechanism has been postulated, possibly favoured by the binding of drug to components of pulmonary tissue.^^^ it might also involve free radical formation or indirect influences on inflammatory mechanisms.^^^ it is also possible that pulmonary disease results from an interaction of several mechanisms and metabolic factors unique to particular patients. ^^^ despite undoubted differences in tissue and species sensitivity to development of phosphohpidosis, dose, drug disposition, metabolism and elimination and the degree of tissue exposure to drug are important considerations in safety assessment of drugs that produce phosphohpidosis in laboratory animals. although phosphohpidosis is more likely to occur at high doses employed in toxicity studies than at lower therapeutic doses used in patients, it has been suggested that this may be offset by faster ehmination of the drug, characteristic of small laboratory animals. ^^^ the potential for drugs to accumulate in critical tissues such as eye and heart is especially important when drugs are administered for long periods of time, particularly as tissue/plasma ratios of some amphiphilic drug may exceed 100, following repeated administration.^^^ consequently, although phospholipidosis may not have functional consequences, any implications for humans of drugs that induce phospholipidosis in laboratory animals can only be assessed on a case by case basis, with due consideration of mechanism, drug disposition and clinical risk-benefit analysis. it is important to underline that similar morphological changes due to the increased presence of phospholipids in lysosomes can also result from treatment with compounds that are not cationic amphiphilic structures. mechanisms include direct or indirect inhibition of lysosomal enzyme activity. this reenforces the need to understand the mechanism of any chemically induced increase of phospholipids in the lungs of laboratory animals. for example, it has been shown that when glycosaminoglycans accumulate in inherited human lysosomal disorders they inhibit other lysosomal enzymes, thereby inducing lysosomal phospholipid inclusions. ^^^ this is reflected by administration of high doses of the trypanocidal drug suramin to rats which induces intracellular storage of glycosaminoglycans associated with phospholipid inclusions in diverse organs including lungs. ^^^ although at light microscopy clear vacuoles are typically seen, electron microscopic examination shows the presence of both clear vacuoles containing glycosaminoglycans and lamellar phospholipid inclusions. a similar effect seems to have been produced in rats by elmironâ®, a semi-synthetic heparin-like macromolecular carbohydrate derivative, chemically and structurally similar to glycosaminoglycans used clinically for anticoagulant effects and interstitial nephritis.^^^ another example is the induction of lysosomal inclusions in the lungs of rat and dogs by the macrolide antibiotic erythromycin.^^^ collections of foam cells were described in the lungs and lymphatic tissues of dogs and rats treated with high oral doses. foam cells in the lung showed a pattern of small whorls in vacuoles similar to that seen with other drugs that induce phospholipidosis. in vitro studies have suggested all macrolide antibiotics have the potential to cause phospholipidosis. biochemical studies suggest that drug binds to phosphatidylinositol-containing liposomes and inhibits activity of lysosomal phospholipase in close correlation with the number of cationic groups carried by each of the drugs. ^^^ hook reviewed other agents, such as oxidant gases and insoluble particles including silica, that can also increase phospholipid levels and histological appearances of phospholipidosis in the lungs.^^^ some of these agents inhibit phospholipid catabolism in the lungs giving rise to accumulation of surfactant protein a and surfactant lipoproteins and a clinico-pathological picture similar to pulmonary alveolar proteinosis in humans. studies from humans have shown that three chnically distinct forms of this condition occur: congenital, secondary or acquired. the congenital disease is caused by a diverse range of mutations in the genes encoding surfactant proteins or the (3c chain of the receptor of granulocyte-monocyte colony stimulating factor (gm-csf). the secondary form occurs in association with conditions where there is functional impairment or reduced numbers of alveolar macrophages, such as in haematological cancers, following immune suppression or inhalation of silica or toxic fumes. acquired or idiopathic alveolar proteinosis that accounts for over 90% of all cases (0.37 per 100000 persons) has been an enigma until recently. patients are at risk from infections, particularly nocardia, and the 5 year survival rate appears to be about 75%. studies from transgenic mouse models and in humans have shown that autoantibodies against gm-csf are important in the development of the acquired form of the disease as this antibody causes a defect in macrophages function which impairs the catabolism of surfactant lipids and proteins. ^^^ in this context it is of interest to note that treatment with imatinib, a tyrosine kinase inhibitor of the bcr-abl tyrosine kinase constitutively expressed in philadelphia chromosome positive myeloid leukemia, has been associated with the accumulation of lamellar inclusions in pulmonary macrophages in a leukaemia patient, although this might have been a result of the primary disease. ^^^ studies in mice have suggested that imatinib mesylate actually inhibits the fibrogenic activity of transforming growth factor (3 and prevents fibrosis induced by bleomycin.^^^ various forms of hyperplasia are found in the airways and lungs of laboratory animals. the mucosal surface of the bronchi may show hyperplasia of the goblet cells, squamous hyperplasia or metaplasia. the cells lining the terminal bronchiole and alveolus may also show hyperplasia and squamous metaplasia. standard classifications for the characterization of these changes in histological sections have been developed for use in rodent studies.^^"^^ goblet cell hyperplasia is a well-recognized response of the mucosa of conducting airways to chronic inflammation and inhalation of irritant substances such as cigarette smoke and sulphur dioxide.^^'^^'^^^'^^^ the degree of goblet cell hyperplasia is dictated by the severity and duration of the irritation or inflammatory process. florid cases of goblet cell hyperplasia are characterized by thickening and pseudostratification of the tracheal or bronchial mucosa by a population of tall, mucus secreting cells with abundant pale cytoplasm. in addition, goblet cells extend further down the airways than in normal animals and mucus may fill or distend the airways or impact in the alveoli. in less florid cases, a simple increase in the number of goblet cells may be found without other structural change.^^ goblet cell hyperplasia of the lining epithelium may be accompanied by an increase in size of the underlying submucosal glands. this has clearly been demonstrated in patients with chronic bronchitis and in rats where submucosal glands are normally quite prominent.^^^'^^^ species differences may exist because the airways of laboratory animals are variably endowed with goblet cells and submucosal mucous glands. the normal rat has more goblet cells lining the airways than either mouse or hamster. ^^^ the factors controlling these alterations are uncertain but is has been long suggested that increased mitotic activity as well as cell conversion, probably by metaplasia of serous or clara cells to mucous cells, is involved.^^^ it has more recently been shown in mice sensitized to ovalbumin and subject to a single antigen challenge by aerosol that clara cells in the proximal airways show great plasticity and become mucin-secreting cells.^^ pharmacological agents can induce goblet or mucous cell hyperplasia. rats given six or 12 daily injections of isoprenaline, a non-selective (3 receptor agonist showed a dose-and time-dependent increase in the number and size of alcian blue-positive goblet (mucous) cells as well as serous cells in the tracheal and bronchial mucosa. this was associated with an increase in length, width and depth of submucosal glands.^^^ similar changes were produced by pilocarpine, although both alcian blue-and pas-positive cells were increased in number following this agent, suggesting that pilocarpine induced both acid and neutral glycoprotein secretion. comparison of the distribution of these changes in the rat following isoprenaline, with those of salbutamol, pilocarpine and tobacco smoke, showed that there were regional differences in the distribution of these changes in the airways.^^'* isoprenaline produced a greater increase in secretory cells in peripheral airways than tobacco smoke, which itself produces a greater increase in mitotic activity. isoprenaline and pilocarpine produced a more diffuse change than the more selective (3 agonist, salbutamol. the changes induced by these therapeutic agents are presumably the result of their pharmacological activity.^^'^ sturgess and reid showed that the changes in the rat were accompanied by hypertrophy of the pancreas, submaxillary and parotid salivary glands^^^ (see digestive system, chapter 8). unlike the rat and mouse, the hamster appears predisposed to develop minor multifocal epithelial hyperplasia of the tracheal and bronchial mucosa spontaneously with advancing age. these changes are flat or polypoid in nature and are composed of clear cells and goblet cells.^^'^^ the epithelium of the bronchi shows squamous metaplasia in response to chronic irritation or injury. it is characterized by three or more layers of epithelial cells with abundant eosinophilic cytoplasm with prominent cell boundaries. it may be associated with degenerative alterations to the mucosa or goblet cell hyperplasia. squamous metaplasia can also develop in the alveolar parenchyma as a response to prolonged damage such as produced by large burden of inhaled irritant or insoluble dusts. the metaplasia is also characterized by the presence of several layers of flattened epithelial cells showing squamous differentiation. the term pulmonary keratinizing cyst has been recommended for pulmonary cystic lesions lined by non-neoplastic squamous epithelium without excessive proliferative change.^^^ hyperplasia, bronchiolo-alveolar (type ii cell hyperplasia) hyperplasia may involve the lining epithelium of the alveoli or bronchioli. this form of hyperplasia has been termed alveolar hyperplasia, adenomatosis, alveolar bronchiolization or epithelialization. it occurs spontaneously but can be induced by infections and administration of irritant xenobiotics in rats/^'^^^"^^^ j^j^g64,209 ^^^ hamsters.^^^ histologically, the lesions consist of localized but unencapsulated foci of hyperchromatic regular, cuboidal or columnar cells investing airspaces without appreciable distortion of alveolar walls. neuroendocrine hyperplasia is well described in hamsters. although small aggregates of neuroendocrine cells (neuroepithelial bodies) are found at various levels of the bronchi and bronchioli in normal hamsters, administration of nitrosamines and 4-nitroquinoline 1-oxide produces neuroendocrine hyperplasia.^^^"^^^ hyperplastic lesions are recognizable as groups of non-ciliated cuboidal, oval or columnar cells located in the bronchial or bronchiolar epithelium. they contain argyrophilic granules that show immunoreactivity for corticotrophin (acth) and neurone-specific enolase. ultrastructural examination reveals the presence of dense-core cytoplasmic granules of apud type. proliferative changes have also been reported in other species, including rats and humans in hypoxic conditions, although it has been suggested that these changes might be a result of increased peptide content rather than cell proliferation.^^'^^^ the most frequently diagnosed neoplasm world wide is lung cancer, where it is usually caused by smoking tobacco.^^^ bronchogenic squamous carcinoma is generally the most common subtype but in north america the incidence of adenocarcinoma now exceeds that of squamous cell tumours for reasons not fully understood. some of the most aggressive subtypes are small and large cell neuroendocrine lung cancers, defined as small or large tumour cells with greater than ten mitoses per 2mm^.^^^ these seem to be seen almost exclusively in heavy cigarette smokers. in contrast to findings in people, squamous cell lung tumours are only occasionally seen arising spontaneously in laboratory animals. even laboratory animals -rats, mice, hamster, monkeys and dogs -exposed to tobacco smoke for long periods and at high doses fail to develop an increase in lung tumours.^^^'^^^ moreover, there appears to be no good experimental model for neuroendocrine lung cancer. thus, particular caution is merited if using animal models for prediction of lung tumorigenic potential of inhaled substances. by far the most common primary pulmonary neoplasms found in laboratory rats, mice and hamsters are adenomas and adenocarcinomas. these appear to develop from the bronchiolar or alveolar epithelium, although their precise histogenesis is somewhat disputed. although spontaneous squamous neoplasms are uncommon in rodents cystic keratinizing lesions can be induced in rats by high burdens of particulate material in the lungs.^^^ pleural mesotheliomas and mesenchymal neoplasms also occur in these species but are uncommon. they can be induced in rodents by mineral fibres.^^^ mesenchymal tumours have similar histological features to those in soft tissues and mesotheliomas may show either epithelial or mesenchymal differentiation or both. in most rat strains alveolar or bronchiolar neoplasms occur spontaneously in relatively small numbers, but morphologically identical neoplasia can be induced by administration of chemical carcinogens.^^^ the most common are classified as bronchiolo-alveolar adenoma (pulmonary adenoma) and bronchioloalveolar carcinoma. the national toxicology program database on control fischer 344 rats used in carcinogenicity studies indicates an overall percentage of less than 3% of animals with bronchiolo-alveolar adenomas and less than 1% with bronchiolo-alveolar carcinomas.^^ however, the range of bronchioloalveolar adenomas in different studies was between 0 and 14% in this series. histologically, bronchiolo-alveolar tumours are mostly small, discrete, rounded nodules located in the lung parenchyma and composed of fairly uniform cells with moderately hyperchromatic nuclei arranged in solid (alveolar), tubular, papillary or mixed growth patterns. they usually compress surrounding tissues without infiltration or metastatic spread (adenoma), although loss of differentiation, infiltration and spread to adjacent tissues can occur (adenocarcinoma). ultrastructural study of bronchiolar-alveolar neoplasia in fischer 344 rats has shown the presence of osmiophilic, lamellated inclusion bodies similar to those found in alveolar type ii cells. therefore it has been suggested that the neoplasms are derived from this cell type.^^^ pulmonary squamous carcinoma occurs but is a very uncommon spontaneous neoplasm in the rat.^^ the large proliferative but benign cystic lesions found in the lungs of rats following accumulation of large amounts of particulate matter have been termed pulmonary cystic keratinizing epitheliomas for they have been regarded as benign neoplasms. when these lesions show evidence of tissue invasion they are regarded as pulmonary squamous cell carcinomas. similar lesions are very occasionally reported as spontaneous lesions.^^^ analogous neoplasms are found more commonly in most strains of laboratory mice used in carcinogenicity bioassays although considerable variation in incidence is reported. they are common in strain a mice where they are observed in low frequency at 3-4 months of age and incidences reach nearly 100% by 24 months of age.^^^ fewer, but significant numbers are found in b6c3fi mice, although there is considerable inter-laboratory variation.^^^ the national toxicology program database on control b6c3fi mice used in carcinogenicity studies indicates an overall percentage of about 16% of males and 6% of females with bronchiolo-alveolar adenomas but only about 5% and 2.5% respectively with bronchiolo-alveolar carcinomas.^^ however, the range of bronchioloalveolar tumour varied considerably between studies in this series. even in the same laboratory, mice housed under similar conditions show variation in incidence in these neoplasms with time. the incidence of lung adenomas and adenocarcinomas occurring in cd-i mice used as controls in 18-month carcinogenicity bioassays in the same laboratory under similar conditions for a period of 3 years varied from between 19 and 36% in males and 6 to 16% in females.^^^ by contrast, some strains of mice such as the c5781/10j strain show a very low predisposition to the development of lung adenomas.^^^ although these mouse pulmonary adenomas and adenocarcinomas do not resemble the common lung tumours in humans, strain differences have been exploited to study genetic susceptibility and resistance to pulmonary adenomas and carcinomas.^^^'^^^ histologically, pulmonary tumours of this type in mice are generally small, sharply circumscribed nodules composed of fairly uniform, closely packed columns of cuboidal or columnar cells arranged in tubular or papillary structures with scanty fibrovascular stroma ( figure 6 .5). they may be less well differentiated, with cellular pleomorphism, and show intrabronchial growth, invade lung parenchyma and produce metastatic spread. the histogenesis of mouse pulmonary adenomas and adenocarcinomas is disputed. on the basis of sequential light and electron microscopic study of pulmonary adenomas induced in bagg-webster swiss mice by transplacental exposure to ethylnitrosourea, it has been suggested that they develop from either alveolar type ii cells or clara cells.^^^'^^^ careful, stepwise analysis using light microscopic and electron microscopic examination has suggested that adenomas can be divided into three principal groups. some are composed of solid growths of uniform cuboidal cells with expanding margins limited to alveolar septae (alveolar pattern). these cells contained concentrically arranged cytoplasmic lamellar bodies and abundant, large mitochondria similar to mitochondria found in alveolar type ii cells. tubular or papillary patterns are composed of cuboidal cells showing histological and ultrastructural features of clara cell differentiation.^^^ however, immunocytochemical studies of chemically induced and spontaneous pulmonary neoplasia in b6c3f1, balb/c or a strain mice have shown that the majority of adenocarcinomas, including those showing papillary patterns, contain surfactant apoprotein, typical of type ii antigens, suggesting that most neoplasms show alveolar type ii differentiation.^^^ however, in view of the plasticity of clara cells, this does not exclude a clara cell origin of the tumours. immunocytochemistry of specific clara cell secretory protein expression in a transgenic mouse model of lung carcinomas developing from clara cells has shown that the protein is lost during tumour cell progression.^^^ it has also been shown in strain a mice that the proportion of tumours with papillary and solid/alveolar growth patterns varies with the inducing agent.^^^ this also suggests biological differences exist between histological subtypes. very few squamous carcinomas are reported in most series of mouse studies. a chemically induced mouse model of squamous cell carcinoma has been generated by administration of n-nitroso-tris-chloroethylurea. strain differences in susceptibility to squamous cancer development have been demonstrated in this model, with nih swiss, a/j and swr/j being highly susceptible, akr/j and c57bl/6j being resistant and fvb/j and balb/cj mice showing an intermediate response to carcinogen.^^^ the high incidence and the inherent variabihty of pulmonary adenomas and adenocarcinomas in conventional mouse carcinogenicity bioassays sometimes gives rise to statistically significant differences between control and treatment groups. there is considerable risk in over-interpretation of such group differences in conventional mouse bioassays. in the analysis of group differences, consideration needs to be given to tissue sampling procedure, age-standardization, historical control incidence, effects on food intake as well as the results of mutagenicity studies and carcinogenicity bioassays in other rodent species. indeed, a considerable number of widely employed therapeutic agents of different classes have produced an increase in benign or malignant pulmonary tumours in carcinogenicity studies performed in mice without this proving of any significance to humans. davies and monro counted at least 17 drugs of this type in the 1994 physicians' desk reference of the united states.^^^ for instance, in a carcinogenicity bioassay in which cfl mice were treated for 80 weeks with the synthetic analgesic tilidine fumarate, a statistically significant difference (p < 0.01) was reported in the incidence of lung adenocarcinomas between the top dose female group (24%) and concurrent controls (10%).^^^ it was argued that group differences did not indicate tumorigenic potential of tilidine fumarate on the basis that the incidence in the high dose group was within the historical control range (27%) and that there was no tumorigenic effect in a parallel 104 week rat carcinogenicity study. a more difficult evaluation concerned metronidazole, a nitroimidazole which is an important therapeutic agent active against anaerobic organisms and trichomonas species. administration of this compound led to an increased incidence of pulmonary adenomas and carcinomas in three separate mouse carcinogenicity bioassays.^^^'^^^ the analysis of these findings was somewhat complicated by evidence that metronidazole shows mutagenic activity in bacterial assays using some strains of salmonella typhimurium. it was argued that the risk to human patients was slight because the increase in prevalence in pulmonary tumours was likely to be a result of changes in nutritional status of the mice through the effect of metronidazole on gut fiora, as similar differences could occur between ad libitum fed mice and those fed the same but restricted diet.^^^ it was also postulated that the positive findings in bacterial mutagenesis assays were an inherent part of the antibacterial activity of metronidazole as a result of nitroreduction that does not occur in normal mammalian tissues. this conclusion was supported by negative effects in hamster carcinogenicity bioassays as well as lack of excess cancer risk in women followed up for 10 years or more.^^^ the common occurrence of lung adenomas in strain a mice has been utilized in the development of a quantitative bioassay for carcinogenic activity. this followed the demonstration that administration of carcinogens such as 3-methylcholanthrene to this strain could significantly increase the incidence of pulmonary adenomas within periods of up to six months.^^^ over many years the strain a mouse pulmonary tumour assay has been used to test a large number of chemicals of different classes, including polycyclic hydrocarbons, nitrosamines, food additives, alkyl halides, metals and chemotherapeutic agents.^^^'^^^ however, as with many test systems, correlation of results in the strain a test with 2 year carcinogenicity study data and genotoxicity results have been shown to be poor so prudence is needed in the use of this test.^^^ hamsters develop lung adenomas spontaneously in small numbers with advancing age. they are composed of uniform cylindrical cells similar to those found in bronchial epithelium or goblet cells showing distinct mucus production.^^'^^'^^^ an immunohistochemical study of similar pulmonary neoplasms induced in hamsters by n-nitrosodiethylamine showed the presence of clara cell antigen in early phase of development, but as the tumours developed they became more squamous in type and showed immunoreactivity for cytokeratins.^^^ a clara cell origin was suggested for most of these neoplasms. lung defenses against infection: a clinical correlation drug-induced lung toxicity pulmonary toxicity from novel antineoplastic agents drug-induced diseases. drug-induced respiratory disease mucociliary clearance and mucus secretion in the lung risk factors for pneumonia and fatality in patients receiving continuous mechanical ventilation considerations for toxicology studies of respiratory drug products overview of inhalation toxicology inhalation exposure technology, dosimetry, and regulatory issues pulmonary deposition: determinants and measurement techniques inhalation therapy: technological milestones in asthma treatment the relevance of the rat lung response to particle overload for human risk assessment: a workshop consensus report interpretation of new techniques used in determination of pulmonary function in rodents measurement of respiratory patterns in rodents using whole-body plethysmography and a pneumotachograph evaluation of lung injury in rats and mice comparative pathology of the nasal mucosa in laboratory animals exposed to inhaled irritants comparative aspects of nasal airway anatomy. relevance to inhalation toxicology comparative anatomy, physiology, and function of the upper respiratory tract comparative anatomy of mammalian respiratory tracts: the nasopharyngeal region and the tracheobronchial region airflow, gas deposition, and lesion distribution in the nasal passages a comparative analysis of primate nasal airways using magnetic resonance imaging and nasal casts the upper airways. 1. nasal physiology and defence of the lungs antioxidant protection: a function of tracheobronchial and gastrointestinal mucus the histochemical and microscopical differentiation of the respiratory glands around the maxillary sinus of the rat hamster nasal glands: their structure, sialic acid content, and vulnerability to actinomycin d the lateral nasal gland of the dog, its structure and secretory content sites for xenobiotic activation and detoxication within the respiratory tract: implications for chemically induced toxicity cytochrome-p450 2a of nasal epithelium -regulation and role in carcinogen metabolism purification and characterization of heterologously expressed mouse cyp2a5 and cyp2g1: role in metabolic activation of acetaminophen and 2,6-dichlorobenzonitrile in mouse olfactory mucosal microsomes differential xenobiotic induction of cyp2a5 in mouse liver, kidney, lung, and olfactory mucosa nasal cytochrome p450 2a: identification, regional localization, and metabolic activity toward hexamethylphosphoramide, a known nasal carcinogen cell-specific expression of cyp2a5 in the mouse respiratory tract: effects of olfactory toxicants biotransformation enzymes in the rodent nasal mucosa: the value of a histochemical approach nasal lymphoid tissue in the rat toxicity to nasal associated lymphoid tissue histopathologic examination of the rat nasal cavity structural evaluation of the respiratory system normal histology of the nasal cavity and application of special techniques approaches to the identification and recording of nasal lesions in toxicology studies nasal diagrams -a tool for recording the distribution of nasal lesions in rats and mice histopathology of nasal olfactory mucosa front selected inhalation toxicity studies conducted with volatile chemicals cytokeratin expression patterns in the rat respiratory tract as markers of epithelial differentiation in inhalation toxicology. 1. determination of normal cytokeratin expression patterns in nose, larynx, trachea, and lung irritating properties of airborne materials to the upper respiratory tract bioassay for evaluating the potency of airborne sensory irritants and predicting acceptable levels of exposure in man infectious diseases of the upper respiratory tract: implications for toxicology studies respiratory tract murine respiratory mycoplasmosis in lew and f344 rats: strain differences in lesion severity spontaneous proliferative lesions in the nasopharyngeal meatus of f344 rats spontaneous tumors and common diseases in three types of hamsters spontaneous tumors and common diseases in two colonies of syrian hamsters. ii respiratory tract and digestive system sialodacyoadenitis virus-associated lesions in the lower respiratory tract of rats oropharyngeal granulomas and tracheal cartilage degeneration in fischer-344 rats articular chondromatosis and chrondroid metaplasia in transgenic tag mice cigarette smoke induced pathology of the rat respiratory tract: a comparison of the effects of the particulate and vapour phases histological sectioning of the rodent larynx for inhalation toxicity testing histologic methods and interspecies variations in the laryngeal histology of f344/n rats and b6c3f1 mice interspecies variations in the histology of toxicologically important areas in the larynges of crl:cd rats and syrian golden hamsters revised guides for organ sampling and trimming in rats and mice. part 2: a joint publication of the rita and nacad groups a comparison of the pathology of the larynx from spf, germ-free, conventional, feral and myoplasma-infected rats modelling of biological tree structures allometric relationships of cell numbers and size in the mammalian lung the ultrastructure of various cell types in the lung of the rat length and distribution of cilia in human and canine airways lectin-binding affinities of the respiratory tract. a light microscopical study of ciliated epithelium in rat, guinea pig and hamster clara cell proteins identification, cellular-locahzation, isolation, and characterization of human clara cell-specific 10-kd protein mucin is produced by clara cells in the proximal airways of antigen-challenged mice morphology of the terminal bronchiolar region of common laboratory mammals morphological changes in the lung during the life span of fischer 344 rats immunocytochemical localization of the major surfactant apoproteins in type ii cells, clara cells and alveolar macrophages of rat lung quantitative microscopical methods for the identification and localisation of nerves and neuroendocrine cell markers in mammalian lung what can the biology of small cell cancer of the lung teach us about the endocrine lung? the role of metabolism in chemical-induced pulmonary toxicity characterization of pulmonary macrophages and bronchus-associated lymphoid tissue (balt) macrophages in the rat. an enzyme-cytochemical and immunocytochemical study subpopulations of lymphoid and non-lymphoid cells in bronchus-associated lymphoid tissue (balt) of the mouse bronchial lymphoid tissue. 1. morphologic characteristics the specificity of the high endothelial venule in bronchus-associated lymphoid tissue (balt) t cells and t cell subsets in rat bronchus associated lymphoid tissue (balt) in situ and in suspension have you seen this?' infiammatory lesions in the lungs of rats inflammatory lesions in the lungs of wistar rats histological changes in rat bronchus-associated lymphoid tissue after administration of five different antigens changes occurring in the epithelium covering the bronchus-associated lymphoid tissue of rats after intracheal challenge with horseradish peroxidase patterns of lymphatic drainage to individual thoracic and cervical lymph nodes in the rat age-body weight relationships to lung growth in the f344 rat as indexed by lung weight measurements application of morphometric methods to study diffuse and focal injury in the lung caused by toxic agents morphometric assessment of pulmonary toxicity in the rodent lung the connective tissue of the rat lung: electron immunohistochemical studies immunohistochemical techniques and their applications in the histopathology of the respiratory system systemically applied chemicals that damage lung tissue bronchial mucosal mast cells and their implications in the pathogenesis of asthma morphological-changes in rat tracheal mucosa immediately after antigen challenge comparative studies of heparin and heparin fragments: distribution and toxicity in the rat interactions between sendai virus and bacterial pathogens in the murine lung: a review murine respiratory mycoplasmosis in f344 and lew rats -evolution of lesions and lung lymphoid-cell populations pathogenesis of bronchiolitis and pneumonia induced in neonatal and weanling rats by parainfluenza (sendai) virus respiratory tact lesions in weanling outbred rats infected with sendai virus increased tumor necrosis factor-alpha (tnf-alpha) gene expression in parainfluenza type 1 (sendai) virus-induced bronchiolar fibrosis il-12 reduces the severity of sendai virus-induced bronchiolar inflammation and remodeling studies of respiratory disease in random source laboratory dogs: viral infections in unconditioned dogs visceral larva migrans in the dog lesions produced by a new lung worm in beagle dogs diagnostic exercise: macaque with dyspnoea treatment of pulmonary acariasis in rhesus macaques with ivermectin a primer of primate pathology: lesions and nonlesions attachment of microbes to host cells: relevance o^ pneumocystis carinii animal model: pneumocystis carinii pneumonia in the immunosuppressed rat attachment of pneumocystis carinii to rat pneumocytes a progress report on the treatment of 157 patients with advanced cancer using lymphokine-activated killer cells and interleukin 2 or high-dose interleukin 2 alone toxicity of human recombinant interleukin-2 in rats. pathologic changes are characterized by marked lymphocytic and eosinophilic proliferation and multisystem involvement toxicity of human recombinant interleukin-2 in the mouse is mediated by interleukin-activated lymphoc3^es. separation of efficacy and toxicity by selective lymphocyte subset depletion characterization of the pulmonary lesions induced in rats by human recombinant interleukin-2 dyspnoea and thoracic spinal deformation in rats after oral prizidilol (skandf 92657-a2) diagnostic exercise: pneumonia in a rat multidrug chemotherapy of tuberculosis in rhesus monkeys granulomatous pneumonitis induced by bacille calmette-guerin in the mouse and its treatment with cyclosporin a anergy-like immunosuppression in mice bearing pulmonary foreign body granulomatous inflammation direct evidence for granulomainducing role of interleukin-1 cytokine and growth factor release by alveolar macrophages: potential biomarkers of pulmonary toxicity animal model: pulmonary granulomatous vasculitis induced in rats by treatment with glucan naturally occurring diatomaceous pneumoconiosis in subhuman primates ultrastructural and micropulse analysis of simian lung mite pigments lung c3^okine production in bleomycin-induced pulmonary fibrosis treatment of human recombinant soluble tnf receptor of pulmonary fibrosis induced by bleomycin or silica in mice the physiology of transforming growth factor-a tnf-a receptor knockout mice are protected from the fibroproliferative effects of inhaled asbestos extensive laminin and basement membrane accumulation occurs at the onset of bleomycin-induced rodent pulmonary fibrosis gold-induced pulmonary disease: clinical features, outcome, and differentiation from rheumatoid lung disease methotrexate pneumonitis induced by intrathecal methotrexate therapy. a case report with pharmacokinetic data cytotoxic drug-induced pulmonary disease: update 19s0 lung toxicity associated with cyclophosphamide use. two distinct patterns interstitial pneumonitis associated with bleomycin therapy bleomycin lung toxicity: who are the patients with increased risk? preclinical toxicologic evaluation of bleomycin (nsc 125 066), a new antitumor antibiotic bleomycin-induced pulmonary toxicity in the rat intratracheal versus intravenous administration of bleomycin in mice: acute effects an investigation of possible models for the production of progressive pulmonary fibrosis in the rat. the effects of repeated intraatracheal instillation of bleomycin capillary remodeling in bleomycin-induced pulmonary fibrosis the role of strain variation in murine bleomycin-induced pulmonary fibrosis pulmonary toxicology of cyclophosphamide: a 1-year study animal models of emphysema lung scleroproteins in young and adult rats and in rats with spontaneous emphysema: comparative studies by biochemical and histochemical approach a quantitative study of stenosis in the respiratory bronchiole of the rat in n02-induced emphysema long-term sequelae of bronchiolitis induced by nitrogen dioxide in hamsters cationic amphiphilic drug-induced phospholipidosis drug-induced phospholipidoses drug-induced lipidosis and the alveolar macrophage drug-induced lysosomal disorders in laboratory animals: new substances acting on lysosomes pulmonary and generalized lyosomal storage induced by amphiphilic drugs drug-induced generalized phospholipidosis tamoxifen-induced generalized lipidosis in rats subchronically treated with high doses fine structural alterations in the lungs of iprindole-treated rats a cell-based approach for the early assessment of the phospholipidogenic potential in pharmaceutical research and drug development validation of an in vitro screen for phospholipidosis using a high-content biology platform multifocal histiocytosis in the lungs of rats the induction of pulmonary phospholipidosis and the inhibition of lysosomal phospholipases by amiodarone collecting and processing tissues for diagnostic electron microscopy chlorphentermine-induced lipidosis like ultrastructural alterations in lungs and adrenal glands of several species amiodarone lung toxicity: a human and experimental study generalized phospholipidosis induced by amphiphilic cationic psychotropic drug morphological and biochemical changes in the liver of various species in experimental phospholipidosis after diethylaminoethoxyhexestrol recovery from amiodarone-induced lipidosis in laboratory animals. a toxicological study epididymal and systemic phospholipidosis in rats and dogs treated with the dopamine d3 selective antagonist pnu-177864 myopathy related to administration of a cationic amphiphilic drug and the use of multidose drug distribution analysis to predict its occurrence drug-induced phospholipidosis: are there functional consequences? an 82-year-old man with dyspnea and pulmonary abnormalities amiodarone induced phospholipidosis. biochemical, morphological and functional changes in the lungs of rats chronically treated with amiodarone granular cells as a marker of early amiodarone hepatotoxicity: a pathological and analytical study amiodarone-associated pulmonary fibrosis. evidence of an immunologically mediated mechanism an evaluation of possible mechanisms underlying amiodarone-induced pulmonary toxicity lipidosis induced by amphiphilic cationic drugs pathological discussion. case record of the massachusetts general hospital organomegaly and histopathology in an animal model of mucopolysaccharidosis induced by suramin lysosomal-storage disorder induced by elmiron following 90-days gavage administration in rats and mice foam cell response in the lung and lymphatic tissues during long-term high-level treatment with erythromycin interactions of macrolide antibiotics (erythromycin a, roxithromycin, erythromycylamine [dirithromycin], and azithromycin) with phospholipids: computer-aided conformational analysis and studies on acellular and cell culture models alveolar proteinosis and phospholipidosis of the lungs pulmonary alveolar proteinosis imatinib-associated pulmonary alveolar proteinosis imatinib mesylate inhibits the profibrogenic activity of tgf-(3 and prevents bleomycin-mediated lung fibrosis an experimental study of hypersecretion of mucus in the bronchial tree goblet cell glycoprotein and tracheal gland hypertrophy in rat airways: the effect of tobacco smoke with or without the antiinflammatory agent phenylmethyloxadiazole measurement of the bronchial mucous gland layer: a diagnostic yardstick in chronic bronchitis mitotic activity of airway epithelium after short exposure to tobacco smoke and the effect of the anti-inflammatory agent phenylmethyloxadiazole the effect of isoprenaline and pilocarpine on (a) bronchial mucus-secreting tissue and (b) pancreas, salivary glands, heart, thymus, liver and spleen experimental chronic bronchitis classification of cystic keratinising squamous lesions of the rat lung: report of a workshop pathological changes during aging in barrier-reared fischer 344 male rats neoplastic and nonneoplastic lesions in aging f344 rats histopathological profile of a wistar rat stock including a survey of the literature naturally occurring sendai disease of mice immunohistochemical demonstration of clara cell antigen in lung tumors of bronchiolar origin induced by n-nitrosodiethylamine in syrian golden hamsters sequential morphologic alterations in the bronchial epithelium of syrian golden hamsters during n-nitrosomorpholine-induced pulmonary tumorigenesis lung endocrine-like cells in hamsters treated with diethylnitrosamine: alterations in vivo and in cell culture 4-nitroquinoline 1-oxide-induced pulmonary endocrine cell hyperplasia in s3rrian golden hamsters increased intracellular levels of calcitonin gene-related peptide-like immunoreactivity in pulmonary endocrine-cells of hypoxic rats the 2004 world health organization classification of lung tumors pleura, thymus and heart a review of chronic inhalation studies with mainstream cigarette smoke in rats and mice a review of chronic inhalation studies with mainstream cigarette smoke, in hamsters, dogs, and nonhuman primates animal models of mesothelioma induced by mineral fibers: implications for human risk assessment morphology of spontaneous and induced tumors in the bronchiolo-alveolar region of f344 rats spontaneous cystic keratinising epithelioma in the lung of a sprague-dawley rat strain a mouse lung tumor bioassay variability in the rates of some common naturally occurring tumors in fischer 344 rats and (c57bl/6nxc3h/hen)fl (b6c3f1) mice a carcinogenicity study in mice of a (3-adrenegic antagonist, primidolol; increased total tumour incidence without tissue specificity effect of diet on spontaneous disease in the inbred mouse strain c57b1/10j pol iota is a candidate for the mouse pulmonary adenoma resistance 2 locus, a major modifier of chemically induced lung neoplasia cancer susceptibility in the mouse: genetics, biology and implications for human cancer histogenesis of the papillary clara cell adenoma histologic and ultrastructural features of the clara cell adenoma of the mouse lung immunocytochemical localization of the surfactant apoprotein and clara cell antigen in chemically induced and naturally occurring pulmonary neoplasms of mice immunohistochemical analysis of clara cell secretory protein expression in a transgenic model of mouse lung carcinogenesis strain a/j mouse lung adenoma patterns vary when induced by different carcinogens a chemically induced model for squamous cell carcinoma of the lung in mice: histopathology and strain susceptibility marketed human pharmaceuticals reported to be tumorigenic in rodents evaluation of chronic toxicity and carcinogenesis in rodents with the synthetic analgesic, tilidine fumarate induction of lung tumors and malignant lymphomas in mice by metronidazole toxicologic evaluation of metronidazole with particular reference to carcinogenic, mutagenic, and teratogenic potential induced pulmonary tumors in mice. ii: reaction of lungs of strain a mice to carcinogenic hydrocarbons. arc/iii;es of pathology strain a mouse pulmonary tumor test results for chemicals previously tested in the national cancer institute carcinogenicity tests animal model: spontaneous carcinoma of the lung in hamsters key: cord-023711-xz5ftnat authors: moreno-lópez, j. title: acute respiratory disease in cattle date: 2013-11-17 journal: virus infections of ruminants doi: 10.1016/b978-0-444-87312-5.50075-8 sha: doc_id: 23711 cord_uid: xz5ftnat nan acute respiratory disease in cattle is a serious problem in countries with intensive or semi-intensive systems of feeding and management. the etiology is complex and many reviews list the microorganisms incriminated. some of the viruses which can infect the respiratory tract of cattle are summarized in table 34 . viruses and bacteria, together with physical stress, transport, overcrowding, irregular feeding and poor standard of hygiene as well as environmental factors (ventilation, temperature and humidity) play an important role in pathogenesis. the disease has world-wide distribution. it affects cattle of all races and ages, tends to recur and responds poorly to preventive and therapeutic treatment. even if the disease for the most part occurs during calfhood, adult cattle may also be involved. the symptomatology of acute respiratory disease is similar in different outbreaks and includes depression, inappétence, dullness, increased respiration and pulse rates in the early stages. the respiratory disorder may or may not be accompanied by diarrhea. acute respiratory disease in cattle is not attributable to a single etiological agent. in many instances, mixed infections with two or more agents occur, as shown in table 34 . such mixed infections can contribute to the severity of the disease. moreover, the microorganisms involved appear to act synergetically. examples of synergism have been demonstrated in the usa (shipping fever, caused by parainfluenza-3 (pi3) virus and pasteurellae) and sweden (umeâ disease, caused by pi3 virus and bvdv). it is difficult to estimate the economic losses, but in terms of mortality, loss of milk production and delayed weight gains, the acute respiratory disease is considered as one of the most costly and troublesome problems for the cattle industry. it is generally accepted that viruses are the primary agents and that bacteria like pasteurellae may, as secondary invaders, accentuate the pathology of the respiratory tract, as in "shipping fever". some of the viruses are circulating in the cattle population, as for instance pi3 virus, which is common in the respiratory tract and is easily transmitted. high percentages of seropositive animals were found in various countries. serological surveys have shown that, on the contrary, bvdv was much less frequent on farms separated geographically than on farms with a close contact of animals on common pastures. collectivization of calves with a different immunological status like that occurring on breeder farms or in large feedlots, leads to accumulation of viruses introduced by animals in the incubation or convalescence stages or by latent carriers. under these circumstances, explosive outbreaks of respiratory and enteric disease may occur. it is unknown how stress factors during massive transport and crowding promote pathogenicity of various respiratory viruses, because it is not possible to mimic a natural outbreak by experimental infection. all too frequently only a mild disease is reproduced. some of the viruses per se have an adverse influence on defence mechanisms. it was shown that infection with pi3 virus may initially diminish the cell-mediated immune response; also, bvdv may act on lymphocytes and neutrophils, suppressing antibody production and phagocytosis, respectively. suppression may promote invasion of other agents, particularly through close contact and crowding in stables lacking proper ventilation. as regards diagnosis, a somewhat pathetic conclusion can be drawn: if foot-and-mouth disease virus is isolated from an animal, the disease has been identified. however, if pi3 virus or bvdv has been isolated, you have diagnosed the infection but not an "acute respiratory/enteric disease", because other viruses and also bacteria might be etiologically involved. as diagnostic techniques become more widely applied, an increasing number of viruses has been isolated from cattle with acute respiratory disease. at the end of the 1950s, "shipping fever" was described as a disease following massive transports to or from the enormous feedlots of the usa, and pi3 virus was isolated as the first virus of a condition with a multiple viral etiology. in sweden, at about the same time, there were outbreaks of a severe acute respiratory-enteric syndrome on farms in the umeâ district in the northern part of the country. local veterinarians provisionally called the syndrome "umeâ disease". thus bvdv became the second member of a multiple etiology. at that time, pi3 virus was suspected to be the cause of the infection in the respiratory tract (manifested by tracheitis and pneumonia), and bvdv to be the cause of infection in the intestinal tract (manifested by erosions in the oral and intestinal mucosa). both viruses were indeed identified. however, it has to be kept in mind that the terms "parainfluenza" and "bovine viral diarrhea" are merely taxons by which these viruses are classified. during the following decade, the first two bovine adenoviruses (types 1 and 2) were described in the usa, and subsequently a third type was identified in the uk. these are the isolates that grow in kidney cells with a hexon antigen common to all mastadenoviruses. then hungarian workers reported the isolation of two new bovine adenoviruses, types 4 and 5, which preferentially grow in cultured calf testicle cells. then additional adenoviruses, types 6 through 8 were reported as "testicle-cell types" from various countries and, finally, from bulgaria, a type 9 being a "kidney-cell type". regarding the "testicle-cell type" adenoviruses, the proof for the presence of antigen(s) common to mastadenoviruses is equivocal at present. the types 4, 5 and 7 have been described each as a single causative agent of acute respiratory-enteric disease in hungary and japan, respectively. during the 1970s the first records appeared on bovine respiratory syncytial virus (brsv), closely related to the human respiratory syncytial virus and causing pneumonia in cattle often less than 2 years of age. brsv was found either as a single agent or as a member of a multiple etiology (as recently reported from uk and usa) condition. in some countries the bovid herpesvirus-1, better known as infectious bovine rhinotracheitis (ibr) virus, is a "solo" pathogen. ibrv was the first viral agent definitely shown to cause respiratory infection in cattle. herpesvirus type 3 (malignant catarrhal fever virus), with a low and sporadic incidence, is also incriminated in the bovine respiratory-enteric disease complex. the role of herpesvirus type 4 (prototype strain movar 33/63) has not been elucidated. however, a respiratory illness caused by this virus in association with pasteurella multocida was quite severe. it is easy to extend this listing by adding, for example, bovine enteroviruses, rhino-and parvoviruses, coronaviruses and the reoviruses. the isolation of these viruses from apparently healthy cattle as well as from those with acute respiratory-enteric disease indicates that they are widespread. however, because they appear to be infrequently implicated in disease processes, it seems that they cause infection only under certain conditions. bacteria such as chlamydia, pasteurellae and mycoplasmas may influence the cycles of infection with viruses. young animals require more care than adult animals. calves fare best when confined individually or in very small groups. this facilitates individual observation, control of diseased animals and maintenance of a higher standard of hygiene. a large number of animals maintained in close contact provides ideal conditions for the spread of disease agents via the respiratory tract. the immunological status, especially passive immunity of the animals, is important in connection with respiratory disease. protective titers of serum antibodies have been calculated for some of the respiratory pathogens. pi3 virus infection seems to be prevented by a serum neutralizing antibody titer of 32 and ibrv infection by a titer of 2. the significance of local nasal antibodies and cmi against respiratory disease is of special importance. with the complex etiology as that associated with respiratory infections it is logical that control measures are not easy. control of the disease may be attempted by diminishing the frequency of infections and by implementing a vaccination program against the most frequent agents. some authors have tried to suppress infections by interferon or interferon inducers, i.e. avirulent viruses known to stimulate nonspecific defence mechanisms, including interferon production; however, no efficient control procedures have become available. the development of effective and practical vaccines for the prevention of respiratory disease is notoriously difficult, due to the multiplicity of agents which are involved. as shown in table 34 , at least 15 distinct microorganisms have been identified. certain viruses, however, produce more severe disease than others; vaccines against them may be expected to break the chain of events leading to respiratory disease. the multiplicity of agents necessitates the use of polyvalent vaccines; polyvalence does not appear to affect the immunogenicity of the individual agents. although desirable, the incorporation of all potential pathogens in a single vaccine is difficult, if not impossible. the results of some limited field trials with a tetravalent vaccine have in no way been encouraging. finally, it must be reemphasized that a problem of this complexity is not solved by a single measure but demands that attention is given to both the infectious and environmental causes of the disease. a serosurvey of viruses during outbreaks of acute respiratory and/or enteric disease in swedish cattle key: cord-261941-xf1k5uj1 authors: stackhouse, robin a. title: severe acute respiratory syndrome and tuberculosis date: 2005-03-01 journal: anesthesiol clin north am doi: 10.1016/j.atc.2004.06.002 sha: doc_id: 261941 cord_uid: xf1k5uj1 respiratory infectious diseases such as severe acute respiratory syndrome and tuberculosis create unique risks for anyone who may be exposed. a brief history of each disease is discussed in this article. the pathogenesis, manifestations, and therapy (where applicable) are also addressed. recommendations for limiting secondary transmission are given based on the centers for disease control and prevention guidelines on infection control in health care facilities. agent. three months and many missed opportunities later, the chinese government informed the who on february 11, 2003 , of 305 cases of an atypical pneumonia that had resulted in five deaths. even then, representatives of who and the centers for disease control and prevention (cdc) did not receive governmental access to data or patients until early march. by this time, a worldwide outbreak had begun. a sars-infected physician from guangdong province had traveled to hong kong on february 21, where he checked into room 911 of the metropole hotel. he was later hospitalized and died of sars, but not before transmission of sars to other guests at the hotel who subsequently seeded the infection in hong kong, vietnam, singapore, canada, the united states, and ireland (no local transmission). on march 12, and again on march 15, when it was clear that sars had spread beyond china, the who issued a global health alert [1] [2] [3] [4] . through unprecedented international cooperation involving public health officials, scientists, physicians, and the media, combined with basic infection control measures, the disease was brought under control by july 5, 2003 . there were 8098 probable cases and 774 deaths [5] . there are currently three identified groups of coronaviruses. groups i and ii are known to cause fairly mild upper respiratory diseases in humans. group iii is only known to cause animal disease [6] . based on recent sequencing data, sars-cov does not clearly fit into any of these three groups. although coronaviruses possess an rna-dependent rna polymerase that can switch template strands when a cell is infected with several coronaviruses, making it very prone to recombination, point mutations, and large deletions, sars-cov does not show evidence of being a recombinant of other known viruses. it is an enveloped, single-stranded rna virus containing 29,727 nucleotides and is approximately 100 to 150 nm in size. a similar virus has been identified in a number of wild animal species found in the animal markets of guangdong, china. sequence data from animal isolates show an extra 29-nucleotide sequence not found in humans who have contracted sars. otherwise the sequence data is only different by 43 -57 nucleotides over the entire genome [7] [8] [9] [10] [11] [12] [13] . the outer envelope is embedded with club-shaped spike glycoproteins that give the virus its characteristic crown-like appearance [14] . a rapid sequence evolution of the spike glycoprotein is believed to be responsible for the increased infectivity that developed (the infectivity rate went from 3% initially to 70% by february 2003) [15] . this may explain the occurrence of superspreading events characterized by a large number of transmissions by a relatively few number of source patients. a dramatic pictorial representation of the explosive spread of the disease in singapore can be seen in fig. 1 [16] . there are no laboratory tests that are rapid, sensitive, and specific early in the course of disease, so sars is considered suspect or probable based on clinical and epidemiologic criteria (box 1). it is confirmed through laboratory testing showing an acute rise in sars-cov antibody titers within 4 weeks of developing the disease. current data suggest that after exposure to sars, individuals are not contagious until they become symptomatic. this is generally within 4 to 6 days of contact (range 2 -10 days), though there are rare instances in which the contact may have occurred 14 days prior. with the caveat that respiratory symptoms are improving, infectivity ceases within 10 days of fever resolution. however, it is not clear what effect the use of steroids has on transmissibility, nor the duration of viral shedding. transmission is through close contact, which is defined as living or caring for an individual who has sars or having direct contact with respiratory secretions or bodily fluids of an individual who has sars (eg, direct body contact, sharing eating or drinking utensils, talking to someone [within 3 feet], or exposure to large droplets of bodily fluid [from talking, coughing, sneezing, singing]). airborne and fecal -oral modes of transmission have not been ruled out, necessitating a higher level of precaution when caring for patients who have sars. early symptoms include fever (95% -100%), chills/rigors (73% -90%), headache (20% -70%), and myalgias (20% -83%). this progresses within 2 to 7 days to include a nonproductive cough and shortness of breath. it is important to note that the fever may have resolved before the onset of respiratory symptoms. there is generally radiographic evidence of pneumonia within 7 to 10 days of symptom onset. the incidence of gastrointestinal symptoms varies widely (diarrhea, 10% -67%; nausea/vomiting, 10% -24%). this may be related to the mode of transmission. those who became sick at the amoy garden apartments in hong kong may have been exposed through a fecal -oral route and had a higher incidence of gastrointestinal symptoms than other patient populations. accompanying laboratory findings include: lymphopenia, thrombothe cdc has published an algorithm for the evaluation and management of suspect sars (fig. 2) [19] . work-up should include a review of symptoms consistent with sars as well as questions that would elicit an etiologic link to the source of infection. anyone presenting with respiratory symptoms should be put on droplet precautions (standard face masks on the patient and those in close contact with the patient; avoidance of droplet contact with mucous membranes; hand hygiene). patients who meet the criteria for suspect sars should immediately be placed in a private respiratory isolation room that has been specially engineered to contain negative pressure in relation to the outside hallway and have a minimum of 12 air exchanges per hour. if this is unavailable, the patient should be in a closed room with a high-efficiency particulate air (hepa) filter that generates the appropriate number of air exchanges. during the outbreak in 2003, hospitals cohorted patients because the number of infected patients overwhelmed the capacity of individual isolation rooms. if chest radiograph confirms pneumonia, the health department should be notified and the following tests should be performed: complete blood count with differential, pulse oximetry, blood cultures, sputum gram stain and culture, testing for other respiratory pathogens (influenza a and b, respiratory syncytial virus, legionella, and pneumococcus). if an alternative diagnosis that fully explains the patient's disease has not been identified within 72 hours, testing for sars-cov should alternate diagnosis that fully explains illness sars-cov antibody negative more than 28 days after onset of symptoms case reported based on contact with person who has been ruled out for sars [17] yes to one of three questions. no to three questions. treat as clinically indicated. proceed. patients who do not have radiographic evidence of pneumonia initially should be re-evaluated with a chest radiograph after 72 hours. only if there is still no evidence of pneumonia should discontinuation of sars respiratory precautions be considered. the sars virus may be detected in sputum, bronchi-alveolar lavage, pleural fluid, nasopharyngeal washes and aspirates, naso-and oropharyngeal swabbings, serum, blood, and stool. samples from multiple sites and at different times during the course of the illness should be collected for real-time reverse-transcriptase polymerase chain reaction testing for evidence of viral antigen (table 1) . a negative reverse-transcriptase polymerase chain reaction result does not rule out sars, because there is a window of time during which the virus is present in any given specimen type, and this may be missed [12] . a positive result indicates true infection, assuming no contamination of the sample before or during testing and that there is no cross-reactivity between sars-cov and other coronaviruses (not yet fully delineated). antibody to sars-cov may be identified using one of three tests: immunofluorescent antibody, enzyme-linked immunosorbent antibody (eia or elisa), or a neutralization test. a positive antibody test may indicate prior exposure, acute disease, or cross-reactivity with another virus [20] . antibody crossreactivity is an ongoing area of investigation. many workers in the markets of southern china have tested positive for sars-cov antibody (40% wild animal traders, 20% wild animal butchers, 5% vegetable traders) without ever having evidence of sars infection [21] . sars is confirmed in patients that have an acute illness consistent with sars and convert from negative to positive sars-cov igg, or have a fourfold increase in titers to sars-cov in acute versus convalescent serum [2, 17, 22] . there are no specific recommendations for treatment of sars other than supportive care. many countries that were seriously affected during the initial outbreak treated patients with a combination of steroids and antivirals such as ribavirin. there is no in vitro evidence in support of this regimen, and the cdc does not currently recommend it. many potential antiviral agents are under investigation, as is the use of sars hyperimmune-globulin and sars-cov vaccination. infection control measures in the hospital setting are based on the mechanisms with which the disease is known to be transmitted (close contact, droplet) and those that are potential routes (airborne, fecal -oral). precautions include respiratory isolation, use of personal protective equipment, hand washing, and disinfection of environmental surfaces. respirators disposable, fit-tested n-95 or greater respirators that have been approved by the national institute of occupational safety and health should be used when coming in contact with patients. a surgical mask placed over the nose and mouth of the patient is sufficient to trap the large particles generated through coughing or sneezing. however, these masks are incapable of filtering the virus once the expectorated material dries and the virus becomes airborne as a droplet nucleus. n-95 masks attain a filter efficiency of more than 95% through mechanisms including electrostatic filtration, sedimentation, and diffusion [23 -28] . goggles, disposable gowns, and gloves should be worn when entering a patient's room and during patient contact. when leaving the patient room, gowns and gloves should be removed, avoiding self-contamination [24 -28] . hands should be washed with soap (antimicrobial or plain) and water after patient contact regardless of whether or not gloves have been worn. alcoholbased hand rubs may be used if there has been no visible soiling [24 -28] . when possible, patient care equipment should be left in the patient's room. equipment and environmental surfaces should be disinfected with an agent that has been approved by the environmental protection agency (such as a quaternary ammonium or phenolic compound) that is recommended for the particular item. disinfectants that have proven to be effective include 75% ethanol, 2% phenol, hypochlorite (500 ppm available chlorine), and household detergent [24 -28] . anyone who suspects that they have sars is advised to contact the health care facility before their arrival. patients should put on surgical masks and be isolated from other individuals at the earliest opportunity. sars patients should only be outside their room for required medical procedures. elective procedures should be postponed until the patient is no longer deemed to be infectious. while outside their room, patients should wear a surgical mask. transport personnel should use full barrier protection. when surgery is required, efforts should be made to limit exposure of personnel and other patients. this includes performing the procedure when the least number of people are present in the operating room (or) and limiting personnel in the patient's or to only those who are essential for the procedure. ors generally have positive pressure in relation to the outside hallways to decrease surgical infection risk. if available, ors with antechambers are preferable for cases in which the patient may expose personnel to infection risk. all unnecessary equipment should be removed from the room to prevent contamination. patients should be transferred directly to the or in which the surgery will be performed. everyone in the or should use the full precautions discussed earlier. bacterial/viral filters should be used on both the inspiratory and expiratory limbs of the anesthesia machine. there are no recommendations regarding anesthetic technique. care should be taken to avoid contamination of the anesthesia machine and cart. this may be accomplished by double-gloving and changing the outer pair of gloves after each patient contact. after the procedure, the patient must be recovered in isolation. this may require that recovery occur in the or itself, or the patient's isolation room. all personal protective equipment should be removed before leaving the or, because it may have become contaminated. new personal protective equipment should be put on for transport of the patient when leaving the or. there is controversy as to whether or not n-95 masks should be reused. if adequate supplies are available, it is preferable to dispose of the masks after use, as they are potential fomites for the transfer of infection. if there is a shortage of masks, one recommendation is to wear a surgical mask over (not under-this defeats the purpose of a tight seal that only allows air that is filtered inside the mask) the n-95 mask. this would decrease the gross soiling of the mask. the or should remain vacant for a sufficient period of time to allow for 99.9% air turnover. for a room with five air changes per hour (ach), 83 minutes would be required. at 10 ach, this drops to 41 minutes, and only 28 minutes for 15 ach. all surfaces should be disinfected with an agent that has been approved by the environmental protection agency. the circuit and the gas sampling line should be disposed. all trash should be properly bagged and disposed of as per standard or requirements. the tragically high infection rates of health care workers at the early stages of the epidemic makes it clear that these recommendations must be strictly adhered to in order to protect oneself and other health care workers [17,26,28 -30] . the sars outbreak was controlled by a combination of ancient infection control procedures (isolation, quarantine of contacts) and an unprecedented cooperation among governments, scientists, news agencies, and citizens. the disease has clearly not been eradicated though. there have been cases of sars in laboratory workers where the source of contact was easily identified. however, beginning in december 2003, a growing number of sporadic cases, where the source of contact has yet to be identified, have been occurring. a coronavirus that has 99.8% sequence homology with human sars-cov has been identified in many animal species sold in the markets of southern china for human consumption (eg, palm civets, raccoon, dog, ferret, badger, cyanomolgus macaque, fruit bat, snakes, wild pigs). it is not yet known whether any of these animals can transmit the disease to humans, but the chinese government instituted an eradication campaign for all palm civets in captivity and the us government has placed an embargo on importation of the animals [31] . whether sars can be eliminated will depend on whether there is an animal reservoir or if it has became endemic in the human population. it remains to be seen if it will re-emerge with seasonal outbreaks similar to influenza. will the disease become more or less virulent over time? will it be possible to develop a treatment or neutralizing vaccine that the virus doesn't circumvent through mutation? as new information and answers to the many questions surrounding sars are found, information is available on several public health web sites, including: centers for disease control and prevention (cdc): www.cdc.gov world health organization (who): www.who.int/en/ international society of infectious diseases (isid): www.isid.org tuberculosis tb, a scourge for many centuries, became a treatable disease after the discovery of effective chemoprophylaxis beginning in 1944 (streptomycin). mycobacterium tuberculosis (mtb) develops drug resistance within months when treated with a single therapeutic agent, however. multidrug therapy of adequate duration limits the probability that drug resistance will occur. over the next two decades, many more drugs effective against mtb were identified: p-aminosalicylic acid (1949), isoniazid (1952), pyrazinamide (1954), cycloserine (1955), ethambutol (1962), and rifampin (1963). between the mid-1950s and 1984 there was a steady decrease in the incidence of tb. it was thought to be a disease of the past. resources (staff, funding, clinics, medication) for tb had dwindled. the world health organization (who) had only two fulltime people assigned to work on tb. however, hiv, which increases the risk of developing active tb and subsequently the duration of infectivity, had begun to spread throughout the world. the conditions were ideal for reemergence of tb. between 1985 and 1992, surveillance for tb in the united states showed a 20% increase in the number of cases secondary to the hiv copandemic, poverty, medically underserved populations, congregate living situations (prisons, shelters, long-term care facilities), immigration from countries with endemic tb (haiti, india, mexico, china, korea, vietnam), and a decrease in public health services. in the early 1990s, an estimated 1.7 billion people (one third of the world population) were tb exposed, there were nearly 8 million new cases per year, and 2.9 million deaths were attributed to tb. it became clear that nothing short of a global tb control program would be effective. a program known by the acronym dots (directly observed therapy short-course), which relies on increased governmental commitment, surveillance, diagnosis, availability of drugs, and verification of treatment compliance, was initiated. in the united states, this has led to an annual decline in tb of 5% to 6%. all 22 of the highest burden countries-which account for 80% of the global cases of tb-have adopted dots. the world health assembly has established targets of reaching 85% cure rates and 70% detection rates of infectious cases [32 -37] . mtb is an aerobic rod measuring 2 to 4 mm by 0.3 mm that thrives at a po 2 of 140 mm hg. the bacilli are released into the air as droplets when a person with pulmonary or laryngeal tb coughs, sneezes, speaks, or sings. droplets travel no more than 3 feet from the source. particles larger than 10 mm are trapped and cleared by the cilia in the upper airway without causing disease. however, as the droplets desiccate, they form droplet nuclei that may stay suspended in air currents and spread more widely. droplet nuclei may be inhaled deeper into the bronchioles, thereby evading the mucociliary defenses of the host and leading to infection. in addition to pulmonary tb, other tissues that may become affected include the kidneys, brain, bones, joints, and genitourinary tract [38] . when mtb disseminates, it is known as miliary tb. although rare, tb infection can occur through vertical transmission. congenital tb is usually disseminated and carries a 50% mortality risk [39] . there is an approximately 10% lifetime risk of developing active tb after exposure. the highest risk is in the first 1 to 2 years. however, certain conditions predispose an individual to progression. individuals who have hiv coinfection have a 7% to 10% risk per year of converting to active tb [40] . at-risk populations should be screened for evidence of tb exposure with a mantoux skin test. five units (0.2 ml) of purified protein derivative (ppd) are deposited subcutaneously. in the absence of anergy, induration will develop within 48 to 72 hours of testing if there has been exposure. induration of 5 mm or more is a positive result in anyone who has hiv, chest radiograph consistent with active disease, duration of less than 12 weeks since exposure, or anyone who is immunocompromised. in all others, 10 mm or more is a positive response. there is cross-reactivity between bacille of calmette and guã©rin (bcg) and ppd. the response to the bcg vaccine wanes with time, however, so a positive response to ppd testing is generally indicative of exposure to tb. the response to ppd may be attenuated if it has been a long time since tb exposure or previous testing. periodic screening of at risk populations should use 2-step testing in anyone who has a negative ppd and if it has been more than a year since the previous test. the first test boosts the immune response and increases the positive predictive value of a second test performed 1 to 3 weeks later [24] . the quantiferon-tb test, another screening test for latent tb infection, was approved by the us food and drug administration in 2001. a whole blood sample is tested against antigen to ppd from mtb and mycobacterium avium. it quantitatively measures the release of interferon g in response, and can differentiate between these infections. a newer test is in development that can distinguish between mtb and previous vaccination with bcg. characteristic signs and symptoms of active tb include: cough (74%), weight loss (71%), fever (30%), malaise (30%), and hemoptysis (19%). individuals who present with this constellation of symptoms should have sputum sampling (three samples collected 8-24 hours apart) for acid-fast bacillus staining and culture and a chest radiograph. respiratory isolation precautions should be instituted until the patient is deemed not to be infectious (acid-fast bacillus -negative on three successive sputum samples). chest radiograph evidence of an acute process correlates with infectivity in those who are not immunocompromised or on steroid treatment. culture-positive samples should be tested for drug sensitivity. mtb rapidly develops drug resistance in circumstances of nonadherence to therapy or inadequate treatment regimens. the worldwide prevalence of resistance to at least one drug is 10.7% (range 2% -36%) and multidrug-resistant tb (mdr-tb), defined as resistance to at least isoniazid and rifampicin, is 1% (range 0% -14%). it is currently estimated that 3.2% of the world's new tb cases are mdr-tb. we recently had a patient at our hospital with a strain of tb resistant to nine drugs. treatment should be initiated based on the current cdc recommendations (fig. 3 , table 2 ). because of the implications of coinfection with hiv regarding the choice of therapy and the duration of treatment, hiv counseling and testing is recommended for anyone that has been diagnosed with tb. baseline laboratory values for aspartate aminotransferase, alanine aminotransferase, bilirubin, alkaline phosphatase, serum creatinine, and platelet count should be performed. subsequent laboratory work should be based on known toxicities of the drugs used in treatment. if ethambutol is used, visual acuity and color vision should be assessed [40] . prevention of transmission in medical facilities requires a combination of early identification, isolation, and treatment of infectious individuals with active disease, engineering controls, basic infection control measures, and the use of personal protective equipment. elective surgery should be postponed in patients with tb until they are no longer contagious (three negative sputum smears, improving symptoms and chest radiograph). for surgeries that cannot be delayed, precautions should be used to minimize the risk of transmission. the patient should wear a surgical mask when outside of an isolation room. a surgical mask is adequate for trapping the droplets before they are released into the environment to become droplet nuclei. unlike respiratory isolation rooms, ors are never at negative pressure in relation to the outside hallways because of the risk it would carry for surgical site infections. however, choosing an or with an antechamber or that is physically separated from other areas may reduce environmental exposure. whenever possible, the case should be scheduled when the fewest health care workers risk exposure. the operating room doors should be kept closed, and personnel in the room kept to a minimum. although there have been no reports of tb transmission from a contaminated ventilator, it is recommended that a hepa filter be placed in the circuit between the patient and the ventilator. hepa filters are rated to remove 99.97% of particles larger than 0.3 mm in diameter. when entering any area that could contain respiratory infectious particles, masks conforming to the national institute for occupational safety and health n95 standard should be used. for proper protection, fit testing for the specific brand of mask is required. the mask must make an airtight seal fig. 3. treatment algorithm for tb. patients in whom tb is proved or strongly suspected should have treatment initiated with isoniazid, rifampin, pyrazinamide, and ethambutol for the initial 2 months. a repeat smear and culture should be performed when 2 months of treatment has been completed. if cavities were seen on the initial chest radiograph or the acid-fast smear is positive at completion of 2 months of treatment, the continuation phase of treatment should consist of isoniazid and rifampin daily or twice weekly for 4 months to complete a total of 6 months of treatment. if cavitation was present on the initial chest radiograph and the culture at the time of completion of 2 months of therapy is positive, the continuation phase should be lengthened to 7 months (total of 9 months of treatment). if the patient has hiv infection and the cd4 + cell count is less than 100/ml, the continuation phase should consist of daily or three times weekly isoniazid and rifampin. in hiv-uninfected patients having no cavitation on chest radiograph and negative acid-fast smears at completion of 2 months of treatment, the continuation phase may consist of either once weekly isoniazid and rifapentine, or daily or twice-weekly isoniazid and rifampin, to complete a total of 6 months. patients receiving isoniazid and rifapentine, and whose 2-month cultures are positive, should have treatment extended by an additional 3 months (total of 9 months of treatment there are no data to support intermittent administration there are no data to support intermittent administration children g dose per weight is based on ideal body weight. children weighing more than 40 kg should be dosed as adults. for purposes of this document, adult dosing begins at 15 years of age. a dose may need to be adjusted when there is concomitant use of protease inhibitors or nonnucleoside reverse transcriptase inhibitors. b the drug can likely be used safely in older children but should be used with caution in children less than 5 years of age, in whom visual acuity cannot be monitored. in younger children ethambutol at the dose of 15 mg/kg/d can be used if there is suspected or proven resistance to isoniazid or rifampin. it should be noted that, although this is the dose recommended generally, most clinicians with experience using cycloserine indicate that it is unusual for patients to be able to tolerate this amount. serum concentration measurements are often useful in determining the optimal dose for a given patient. c the single daily dose can be given at bedtime or with the main meal. d dose: 15 mg/kg/d (1 g), and 10 mg/kg/d in persons more than 59 years of age (750 mg). usual dose: 750 -1000 mg administered intramuscularly or intravenously, given as a single dose 5 -7 d/wk and reduced to two or three times per week after the first 2 -4 months or after culture conversion, depending on the efficacy of the other drugs in the regimen. e the long-term (more than several weeks) use of levofloxacin in children and adolescents has not been approved because of concerns about effects on bone and cartilage growth. however, most experts agree that the drug should be considered for children who have tuberculosis caused by organisms resistant to both isoniazid and rifampin. the optimal dose is not known. f the long-term (more than several weeks) use of moxifloxacin in children and adolescents has not been approved because of concerns about effects on bone and cartilage growth. the optimal dose is not known. g the long-term (more than several weeks) use of gatifloxacin in children and adolescents has not been approved because of concerns about effects on bone and cartilage growth. the optimal dose is not known. adapted from centers for disease control and prevention. available at: http://www.cdc.gov/mmwr/pdf/rr/rr5211.pdf. with the face to ensure that all inspired air is filtered through the mask. if the available n95 masks have an exhalation valve, a regular surgical mask must be worn over it to protect the surgical field from infection. those individuals who cannot be successfully fit tested for an n95 mask should use a higher level of respiratory protection, such as a powered air purifying respirator. recovery from anesthesia must take place with the same level of precautions. if there is no isolation room in the recovery room, this will generally mean the patient needs to recover in the or or a respiratory isolation room. a room in which a patient with active tb has been present should not be entered unless necessary until there has been a 99.9% turnover of the air. the duration is dependent on the number of air exchanges per hour, but is generally in the range of 30 to 60 minutes for most ors [24, 30] . bacterial, viral, prion, and possibly as yet unidentified etiologic agents continue to cause human health risks through their ability to circumvent the immune system and our capability to eradicate, treat, or effectively prophylax against them. of current concern to world health are the avian influenza viruses. the h5n1 strain is easily transmitted to humans from its primary host and is highly lethal (>75% mortality). the poultry industry has been severely affected in many countries. its global impact on human health has only been limited by its current lack of transmission from human to human. this could easily change through a recombination event between an avian and a human influenza virus. we will continue to be exposed to new disease-causing organisms through exposure to areas with no prior human habitation and increasing globalization. in addition, we are increasing the selection pressure on these organisms through agricultural pressure (farming conglomerates) and the widespread overuse of antimicrobial agents. our ability to avoid global epidemics in the future depends on our intelligence in responding to what we have learned from our prior failures in controlling infectious diseases. as sars outbreak took shape, health agency took fast action world health organization. consensus document of the epidemiology of severe acute respiratory syndrome (sars) 2003. available at: www.who.int/csr/sars/en/whoconsensus update: outbreak of severe acute respiratory syndrome-worldwide global surveillance, national surveillance, and sars world health organization. available at: www.who.int/en increasing clinician preparedness for severe acute respiratory syndrome (sars) identification of a novel coronavirus in patients with severe acute respiratory syndrome a novel coronavirus associated with severe acute respiratory syndrome novel coronavirus and severe acute respiratory syndrome coronavirus as a possible cause of severe acute respiratory syndrome characterization of a novel coronavirus associated with severe acute respiratory syndrome prospective study of the clinical progression and viral load of sars associated coronavirus pneumonia in a community outbreak. world health organization the genome sequence of the sars-associated coronavirus sars coronavirus: a new challenge for prevention and therapy the chinese sars molecular epidemiology consortium. molecular evolution of the sars coronavirus during the course of the sars epidemic in china severe acute respiratory syndrome-singapore public health guidance for community-level preparedness and response to severe acute respiratory syndrome (sars) severe acute respiratory syndrome (sars) clinical guidance on the identification and evaluation of possible sars-cov disease among persons presenting with community-aquired illness, version 2. available at: www world health organization. summary of the discussion and recommendations of the sars laboratory workshop sars-related virus predating sars outbreak wresting sars from uncertainty now that sars has arrived, will it ever leave world health organization. hospital infection control guidance for severe acute respiratory syndrome (sars) recommendations for isolation precautions in hospitals california department of health services: severe acute respiratory syndrome (sars)-infection control recommendations infection control measures for operative procedures in severe acute respiratory syndrome-related patients guidelines for preventing the transmission of mycobacterium tuberculosis in health-care facilitates guidelines for environmental infection control in health-care facilities the tb epidemic from 1992 to 2002 the global situation of mdr-tb tuberculosis trends in the united states tuberculosis control: past 10 years and future progress global burden of tuberculosis-estimated incidence, prevalence, and mortality by country tuberculosis morbidity-united states harrison's principles of internal medicine tuberculosis: an old disease but a new threat to the mother, fetus, and neonate key: cord-007445-2folsh35 authors: tuffaha, amjad; gern, james e.; lemanske, robert f. title: the role of respiratory viruses in acute and chronic asthma date: 2000-06-01 journal: clin chest med doi: 10.1016/s0272-5231(05)70267-7 sha: doc_id: 7445 cord_uid: 2folsh35 respiratory tract infections caused by viruses, 24, 70 chlamydia, 18, 19, 43, 55, 116 and mycoplasma(61) have been implicated in the pathogenesis of asthma. viruses have been demonstrated to be associated with asthma epidemiologically in at least two ways (fig. 1). first, during infancy, certain viruses have been implicated as potentially being responsible for the inception of the asthmatic phenotype. second, in patients, particularly children, with established asthma, viral upper respiratory tract infections play a significant role in producing acute exacerbations of airway obstruction that may result in frequent outpatient visits or hospitalizations. 24, 55, 56, 57 this article reviews these two areas by focusing first on mechanisms by which virus infections may lead to the development of asthma in infants and children and, second, on mechanisms by which virus infections may produce acute asthmatic symptoms in patients who already have established disease. respiratory tract infections caused by viruses,24, 70 chlamydia,'*, 19, 43* 55* 116 and myco-plasma6i have been implicated in the pathogenesis of asthma. viruses have been demonstrated to be associated with asthma epidemiologically in at least two ways (fig. 1) . first, during infancy, certain viruses have been implicated as potentially being responsible for the inception of the asthmatic phenotype. second, in patients, particularly children, with established asthma, viral upper respiratory tract infections play a significant role in producing acute exacerbations of airway obstruction that may result in frequent outpatient visits or hospitalization^.^^, [55] [56] [57] this article reviews these two areas by focusing first on mechanisms by which virus infections may lead to the development of asthma in infants and children and, second, on mechanisms by which virus infections may produce acute asthmatic symptoms in patients who already have established disease. supported by nih grants a134891, hl56396, and lrolhl61879. infections with respiratory syncytial virus (rsv) or parainfluenza virus (piv) have received much attention because of their predilection to produce a pattern of symptoms termed bronchiozitis that parallels many of the features of childhood and adult asthma.67 respiratory syncytial virus causes about 70% of these episodes and it is estimated that, by age 1 year, 50% to 65% of children will have been infected with this virusn and 40% of these infections involve the lower respiratory tract.92 by age 2, nearly all children will have been infected with rsv at least once. children aged 3 to 6 months are most prone to develop lower respiratory tract symptoms, suggesting that a developmental component (e.g., lung or immunologic maturation) may be involved as 83 the relationship between rsv infections during the first few years of life and the subsequent development of the asthmatic phenotype has been the subject of much interest as well as controversy. variations in reporting longitudinal outcomes (e.g., recurrent wheezing, measurements of airway hyperresponsiveness, diagnosis of asthma) appear to be influenced mostly by the criteria used to define "bronchiolitis." these criteria include the type of virus producing the symptoms (in addition to rsv, viruses that may contribute to the development of bronchiolitis in this age group could be piv, coronavirus, influenzavirus, and rhin~virus~~); the age at the time of infection; the nature and severity of symptoms required for inclusion; and, finally, the characteristics of both the study population (community versus hospital-based) and the study design (retrospective versus prospective). a number of long-term prospective studies of children admitted to a hospital with documented rsv-induced bronchiolitis have shown that about 75% experience wheezing in the first 2 years after the initial illness, more than 50% still wheeze 3 years later, and approximately 40% continue to wheeze after 5 years?,, 49, 733 91, 117, additional insight into these areas recently was provided by the results of an ll-year prospective study involving 880 children who were enrolled at birth, followed for the development of lower respiratory tract illnesses (lris) in the first 3 years of life, and then evaluated for the presence or absence of physician-diagnosed asthma or a history of current wheezing at ages 6 and 11 years.14 most importantly, lung function was evaluated in the first few months of life in a subset of these children prior to the development of a documented lri. during the first 3 years of life, 7.4% had pneumonia documented radio-graphically and 44.7% had a significant lri without pneumonia. respiratory syncytial virus and piv were identified in 36.4% and 7.3%, respectively, in the subjects with pneumonia, and in 35.6% and 15.2%, respectively, of the subjects with a lri. at age 6, physiciandiagnosed asthma was present in 13.6% (or = 3.3), 10.2% (or = 2.4), and 4.6% of the subjects with pneumonia, lri, and no lri, respectively. by age 11, these values increased to 25.9% (or = 2.8), 16.1% (or = 1.6), and 11%, respectively. mean maximum volume at functional residual capacity values before any lri were lower in children with pneumonia and with lris than in children with no lris. these values continued to be lower at age 6 and by age 11, when forced expiratory volume in 1 second (fevl) and fef, , were recorded, similar group relationships persisted. interestingly, despite the persistence of lowered baseline lung function in both the pneumonia and lri groups, many of these deficits were markedly (but not completely) reduced following administration of albuterol. in a second report, further follow-up of this large cohort of children demonstrated that the risk for both frequent (more than three episodes of wheezing per year) and infrequent (three episodes of wheezing per year) wheezing in relation to rsv lower respiratory illnesses decreased markedly with age and became nonsignificant by age 13.1°4 these data suggest that, although rsv infections contribute substantially to the expression of the asthmatic phenotype, other factors (e.g., genetic, environmental, developmental) ap-pear to contribute as well, either in terms of its initial expression or the modification of the phenotype over time. in addition to premorbid lung function, the influence of atopy on the development of the asthmatic phenotype in relationship to viral infections has also been evaluated. interactions between these two factors appear to be bidirectional and dynamic, in that the atopic state can influence the lower airway response to viral infections? 71 viral infections can influence the development of allergen sensitization,28, 29, 99 and interactions can occur when individuals are exposed simultaneously to both allergens and viruses.1z, 68, atopy can be defined as the genetic predisposition to the preferential development of an immunoglobulin (1g)e antibody response to a variety of environmental allergens. as stated previously, atopy has been considered to be a risk factor for the development of childhood asthma and its influence on the pattern of responses following viral infections has been of interest to many investigative groups. it has also been suggested that atopy could be a significant predisposing factor for the development of acute bronchiolitis during rsv epid e m i c~.~~ although some have found that children most likely to have persistent wheezing were those born to atopic parents,64, 91, lz1 others have not.14, 73, 84 some have found that personal atopy is not more prevalent in symptomatic children after br~nchiolitis'~, 73; others have found that documented rsv bronchiolitis significantly increases a child's chances (32% versus 9% in controls) of subsequently developing ige antibody% or lymphocyte proliferative responses75 to both food and aeroallergens. respiratory syncytial virus infections may interact with immunoinflammatory mechanisms involved in immediate hypersensitivity responses in a number of ways.18 first, it has been suggested that viruses capable of infecting lower airway epithelium may lead to enhanced absorption of aeroallergens across the airway wall, predisposing to subsequent sensitizati~n.~~, 94 second, rsv-specific ige antibody formation may lead to mast-cellmediator release within the airway, resulting in the development of bronchospasm and the ingress of eo~inophils.~~, 60, 85, 115 respiratory syncytial virus belongs to the family paramyxoviridae, the genera pneumovirus, and can be differentiated into two serologic subgroups, a and b.m, it has 10 genes, with 12 potential gene products. the g (attachment) and f (fusion) proteins are the major su$ace glycoproteins against which neutralizing antibody is directed. interestingly, in both murine2 and hurnan5l in vitro experiments, it has been noted that the g protein elicits a predominant th2 response, whereas the f protein produces a predominant thl response. in mice, to test the activities of t cells recognizing individual rsv proteins in vivo, virus-specific t-cell lines have been produced using recombinant vaccinia viruses that express either the g or f proteins. following passive transfer of these cell lines to naive recipients and subsequent intranasal inoculation with rsv mice receiving g-specific cells have more severe illnesses, characterized by lung hemorrhage, pulmonary neutrophil recruitment, and intense pulmonary eosinophi1ia.l these experiments are of interest based on the adverse clinical response noted in many infants who received a formalin-inactivated rsv vaccine and subsequently became infected with rsv? these intriguing observations regarding rsv and its influence on thl /th2 responses have recently been expanded. roman et a1 evaluated 15 hospitalized infants (1-15 months) with an acute lower respiratory tract infection caused by rsv. compared with control infants, peripheral blood cells from infected children had suppressed ifn-?/ production ex vivo and, although il-4 production was also decreased, the il-4/ ifn-y ratio was significantly increased. renzi et a p prospectively followed 26 infants hospitalized with bronchiolitis by obtaining blood samples at the time of illness and 5 months later, and found that immune responses during the acute infection correlated with long-term pulmonary outcomes. blood lymphocytes, obtained during the time of bronchiolitis, produced less ifn-?/ ex vivo in response to il-2 and more il-4 in response to d. furinue antigen in children who went on to develop a pattern of recurrent wheezing.8s finally, lower ifn-y production at the time of bronchiolitis has been demonstrated to be an indicator of reduced pulmonary function and increased responsiveness to histamine 5 months after bronchiolitis, and was related to the development of asthma after bronchiolitis in infants. 87 in contrast, other groups have noted increased levels of ifn-y respiratory tract secretions during rsv illnesses in infants and children with bronchiolitis and recurrent wheezing compared with those with upper respiratory tract symptoms unfortunately, in all of the studies reported thus far, the pattern of cytokine response these infants had prior to infection was not evaluated, begging the question as to which of the observed results may be cause and which effect. to more comprehensively evaluate the relationships among virus infection, atopy (cytokine dysregulation of thl / th2 imbalance), and immune system or lung developmental components, a rat model of virus-induced airway dysfunction has been studied extensively.'l' in this model, infection with piv type 1 during a critical developmental time period (when the animals are weaning [ 3 4 weeks of age] as opposed to when they are neonates [4-5 days] or adults) produces chronic (8-12 weeks fol-lowing infection), episodic, reversible airway inflammation and remodeling with associated alterations in airway physiology (increased resistance and rnethacholine responsiveness) that resemble human asthma in high (brown norway strain) but not low (f344 strain) igeantibody producing rats.62 the temporal progression of this asthma-like syndrome is associated with a thl / th2 imbalance within the lung, and its development can be significantly attenuated by the exogenous administration of ifn-8 just prior to and during the viral infection in the brown norway responder strain.lo2 this model further supports the concept of both genetic (atopy; cytokine dysregulation or imbalance) and environmental factors (virus infection) being important in the inception of the asthmatic phenotype, as well as a developmental component contributing. respiratory viruses are common causes of asthma exacerbations in asthmatic subjects of different age 74, 86 serology or culture detection methods of viruses initially indicated an association during asthma exacerba-tionss2 despite the fact that these detection methods are relatively insensitive for viruses such as rhinovirus (rv). the use of reverse transcription polymerase chain reaction (rt-pcr) assays that are more sensitive for detection of rv have confirmed and expanded these initial observation^.^^ indeed, johnston et a157 found that 80% to 85% of school-aged children with acute wheezing episodes tested positive for a virus using rt-pcr and other standard virologic techniques. the virus most often detected was rv. seasonal patterns of upper respiratory virus infections correlate closely with hospital admissions for asthma, particularly in pediatric age these studies indicate that rv infections are the most common cause of asthma exacerbations in children, especially during spring and fall. similar studies, performed in found that about half of asthma exacerbations were associated with rv infection. as discussed previously, in infancy, atopy may define a susceptibility of the host to wheezing with respiratory infections. duff et a1,= for example, studied children who presented to an emergency department with wheezing. children over 2 years of age were more likely to have respiratory allergies or a confirmed respiratory viral infection compared with children with no wheezing. children with the highest risk for wheezing were those who had respiratory allergies and respiratory viral infection, implying that respiratory viral infections and respiratory allergies may have synergistic effects on lower airway physiology and enhance the likelihood of wheezing with respiratory infection. in children less than 2 years of age, wheezing was also noted, but risk factors for wheezing were quite different. these infants were not allergic, had rsv as the major viral isolate, and had passive tobacco smoke exposure as a major risk factor for wheezing. available epidemiologic data in children and adults have shown that episodic drops in peak flow measurements are associated with rv infections. this was found to correlate with an increase in asthma symptoms and nonspecific airway hyperresponsiveness following experimentally infecting asthmatic subjects with rv.15, 41 further studies by griinberg et a140 demonstrated that experimental rv16 infection leads to a transient drop in daily home recordings of fev, in subjects with asthma. this variable airway obstruction correlated significantly with cold symptoms, asthma symptoms, and the increase in airway hyperresponsiveness to histamine. such daily variability in fev, reflects the inflammatory changes within the airway wall, which can be induced by the natural rv infection. following infections with rv8 and influenza a.@, 72 in a study by cheung et all5 14 subjects with mild asthma were inoculated with rv16 or placebo. the maximal contractile response to inhaled methacholine was significantly greater during the rv16 infection and remained elevated for up to 15 days after the acute infection. this study indicates that an upper respiratory viral infection can enhance the reactivity of the lower airway and the magnitude of bronchonstriction changes, which can persist for weeks after the acute infection. respiratory viral infections' effect on lower airway responses are also influenced by host factors. in particular, allergic subjects experience greater changes in airway responsiveness after viral infection than nonallergic control s~b j e c t s .~, 34 furthermore, subjects with lower fev, values tend to have greater changes in airway responsiveness during viral infecti0n.3~ these studies suggest that effects of pre-existing conditions such as allergy and intrinsic lower airway function on caliber are likely to contribute to airway hyperresponsiveness during respiratory viral infection. potential mechanisms through which viral infections could potentially cause bronchoconstriction and increased airway responsiveness include enhancing parasympathetic bronchoconstrictive responses, stimulation of airway sensory nerves, and interference with the bronchodilatory functions of the nonadrenergic, noncholinergic neurons (table 1) . because of difficulties in assessing dysfunction of pulmonary neural regulation in humans, most data that support these proposed mechanisms were derived in animal models of acute respiratory viral infection. further definition of these pathways in humans will depend upon the development of new experimental techniques or inhibition of specific neural pathways. increased bronchial responsiveness has been found in normal and asthmatic subjects changes in small airways structure and function may also contribute significantly to the severity of hyperinflation and gas exchange abnormalities noted in acute asthma exacerbations. the maximal airway contractile response to methacholine in mild asthmatic subjects is increased during a cold, which is probably secondary to excessive airway narrowing attributable to airway wall thickening, airway parenchymal uncoupling, or abnormalities in smooth muscle ~0ntraction.l~ significant changes in airway morphology are noticed in animals with acute viral respiratory illness that leads to marked bronchiolar narrowing and plugging. these changes include bronchiolar airway edema and cell infiltration, epithelial hyperplasia, and folding and sloughing of airway epithelial surfaces. in addition, rats with mild increases in pulmonary resistance and methacholine sensitivity during acute viral respiratory illness have evidence of air trapping and ventilationperfusion mismatches.101 these latter findings indicate that viruses can induce significant changes in the peripheral airways that have significant functional outcomes in the absence of marked changes in measurements of airway obstruction and hyperresponsiveness. respiratory viruses can cause inflammation and injury to healthy airways and can worsen injury in airways that are already inflamed, as demonstrable in asthma. respiratory viruses can induce an inflammatory process by direct cytopathic effects on the airway epithelium (e.g., rsv bronchiolitis) and can induce an immune response to stop viral replication and eradicate the virus. the immune re-sponse to viral infection may be a doubleedged sword, however, as virus-induced inflammation can also contribute to airway obstruction and respiratory symptoms. indeed, although many common cold viruses (e.g., rv) do not produce significant cytopathic effects, possibly because few cells are infected, the immunoinflammatory response to the virus is probably the major cause of respiratory symptoms. in this section, the association between virus-induced immune responses and respiratory symptoms is explored. respiratory viruses replicate primarily in airway epithelial cells. in addition to serving as host cells, it is now well documented that epithelial cells also initiate the immune response to infections through the secretion of cytokines and chemokines. in vitro studies of epithelial cells or cell lines have demonstrated that respiratory viruses such as rv, rsv, and parainfluenzavirus can induce the secretion of many different proinflammatory cytokines (il-1, tnf-a, gm-csf, il-6, il-11) and chemokines (rantes, il-8, mip-la).lo, 20, 23, 96, 98, lo5 epithelial-derived chemokines are likely to be an important signal in initiating antiviral responses through the recruitment of leukocytes to the airway. in support of this concept, il-8, a potent neutrophil chemoattractant, is found in high levels in nasal secretions of children with virus-induced asthma, and levels of il-8 correlate with the number of airway neutrophils and neutrophil myeloperoxidase levels (suggesting neutrophil activation).lo8 there is also evidence, however, that enhanced airway inflammation caused by chemokine secretion may also disturb normal air-way physiology. chemokine levels in nasal secretions correlate closely with cold symptoms,"o for example, and il-8 levels correlate with virus-induced changes in airway respon-~i v e n e s s .~~ levels of epithelial-derived cytokines such as il-6 and il-11 also correlate with respiratory syrnpt0ms,2~ and animal studies indicate that overexpression of il-11 can cause bronchial hyperresponsi~eness.~~~ io7 in addition to stimulating cytokine production, rv can upregulate epithelial cell surface expression of intercellular adhesion molecule-1,79 which, in addition to facilitating cell-cell adhesion, is the receptor for the major group of rv3" lo3 this enhanced expression of adhesion proteins may contribute to the persistence and severity of inflammation in asthmatic subjects and, possibly, the greater susceptibility of asthmatic children to colds compared with nonasthmatic children. mechanisms for the activation of cytokine genes in epithelial cells and adhesion molecules are under investigation. it is known that nuclear factor-lc b activation is important in virus-induced transcriptional regulation of il-650 and, possibly, for the synthesis of a variety of inflammatory cyt~kines.~ in addition, nitric oxide may regulate virus-induced chemokine production through a posttranscriptional mechanism and by inhibiting viral repli~ation;~ although a clinical study did not find a relationship between il-8 and nitrate levels in nasal secretion^.^^ granulocyte recruitment and activation seem to have an important role in the pathogenesis of virus-induced asthma exacerbations. griinberg et a1y1 for example, experimentally inoculated 35 atopic asthma subjects with either rv16 or placebo and found that neutrophil counts in the peripheral blood correlated with the cold and asthma symptom scores and cold-induced changes in airway hyperresponsiveness. in addition, eosinophil granular proteins and leukotriene c, have been detected in the nasal secretions of infants and children with virus-induced wheezing i l l n e~s e s .~~~ 86, 97, increased concentrations of sputum eosinophil cationic protein found during the acute phase of rv infection corre-lated with increases in airway responsiveness in a group of adults with asthma after experimental inoculation with rv16.39 in vitro experiments indicate that rv does not activate eosinophils dire~tly,~; it is more likely that inflammatory mediators and cytokines, secreted by virus-activated cells in the lung, contribute to eosinophil activation. finally, guinea pigs infected with piv develop airway eosinophils and airway hyperresponsive-nessz5 and this outcome is blocked if the guinea pigs are pretreated with il-5neutralizing antibody.l12 most respiratory viruses replicate quickly and, within a few days of inoculation, the quantity of viruses and viral proteins is sufficient to activate mononuclear cells in the airway. in vitro infection of human monocytes with respiratory viruses, for example, leads to a potent proinflammatory cytokine response by release of il-8, il-1, and tnf-cx.~~, 54, 90 interleukin-1 and tnf-a can increase cell recruitment into the airway by enhancing adhesion molecule expression on endothelial cells. in addition, tnf-a has been associated with wheezing illnesses in infancy6 and the development of late-phase allergic reaction and asthma.3, 37 monocytes and macrophages also produce interferon (inf), and its appearance in nasal secretions coincides with the onset of the recovery process. in addition to cytokine production, macrophages incubated with rsv or piv produce lipid mediators such as prostaglandin e, platelet-activating factor, and thromboxane b248, 78, 114 that can augment airway inflammation. lymphocytes, including natural killer cells, cd8+ cytotoxic t cells, and cd4+ t cells, are involved in limiting viral replication and viral clearance. to test the possibility that variations in lymphocyte responses might account for variability in the ability to clear viral infections, parry et apo measured in vitro lymphocyte responses in a group of allergic subjects who were then inoculated with rv 16. vigorous virus-induced responses (lymphocyte proliferation or ifn--y secretion) before inoculation correlated with reduced viral shedding after inoculation. these results sug-gest that factors related to the host cellular response help determine the degree of viral replication during respiratory viral infections. further characterization of these host factors may lead to new therapeutic strategies for respiratory infections, a goal that is particularly important for people with asthma. several studies have shown that viral infections activate a wide range of t cells. evidence for this comes from experiments in mice, in which most of the t cells found in the lung after an acute viral infection are not virus-specific,2l and in vitro studies, in which 25% to 50% of human peripheral blood t cells express the early activation marker cd69 after 24 hours in culture with rv.36 rantes, induced by respiratory viruses, at high concentrations can also induce antigen-independent t-cell activation? these studies suggest that respiratory viruses can induce early, nonspecific t-cell activation and recruitment that could significantly increase the intensity of airway inflammation, resulting in airway dysfunction and respiratory symptoms. this hypothesis is supported by studies of volunteers infected with rhinovirus. respiratory virus infections usually cause peripheral lymphopenia and increased numbers of lymphocytes in the upper and lower airways, for example. the degree of peripheral blood lymphopenia and lymphocytic infiltration of the airway epithelium has been correlated with the increases in airway responsi~eness.~~~ 26 although viral infections cause similar upper respiratory symptoms in allergic and nonallergic individual^,^^, loo there is evidence of interactions between virus-and allergen-induced responses in the lower airway. lemanske and colleag~es,~~ for example, identified 10 patients with allergic rhinitis and experimentally infected them with rv16. the viral infection increased airway reactivity to both inhaled allergen and histamine, and also increased the frequency of a late allergic reaction to inhaled antigens. moreover, calhoun and colleag~es'~ used bronchoscopy to study the inflammatory response to allergen in indi-vidual lung segments before, during, and 1 month after rv16 infection. rv infection enhanced the immediate antigen-induced release of histamine, and also increased eosino-phi1 recruitment of eosinophils to the lung. respiratory infections can have dual effects related to asthma. first, there is increasing evidence that severe infections with rsv and piv in infancy can alter lung development and physiology to increase the risks of subsequent wheezing and asthma. second, infections with common cold viruses and influenza commonly precipitate wheezing symptoms in children and adults who already have established asthma, and rv appears to be the most important virus in producing exacerbations of the disease. the principal mechanisms by which this occurs appears to be viral replication in epithelial cells, triggering a cascade of inflammation involving granulocytes, macrophages, t cells, and secreted cytokines and mediators. the inflammatory process, although essential to clear the infection, augments pre-existing airway inflammation in asthma, leading to increased airway obstruction and lower respiratory tract symptoms. greater understanding of virus-induced changes in inflammation and corresponding changes in airway physiology may lead to new therapeutic approaches to the treatment and prevention of virus-induced airway dysfunction. association of radiologically ascertained pneumonia before age 3 years with asthma-like symptoms and pulmonary function during childhood. a prospective study. openshaw pjm distinct types of lung disease caused by functional subsets of antiviral t cells phenotypic and functional characterization of t-cell lines specific for individual respiratory syncytial virus proteins induction of human airway hyperresponsiveness by tumour necrosis factor-a cytokine (il-8, il-6, tnf-a) and soluble tnf receptor-i release from human peripheral blood mononuclear cells after respiratory syncytial virus infection virusinduced alterations in macrophage production of tumor necrosis factor and prostaglandin-e2 rhinovirus infection induces expression of its own receptor intercellular adhesion molecule 1 (icam-1) via increased nf-kappab-mediated transcription rhinovirusinduced peripheral blood mononuclear cell responses and outcome of experimental infection in allergic subjects interleukin-la mediates the enhanced expression of intercellular adhesion molecule-1 in pulmonary epithelial cells infected with respiratory syncytial virus viruses as precipitants of asthma symptoms. i. epidemiology development of allergen-specific t-cell memory in atopic and normal children wheezing, asthma, and pulmonary dysfunction 10 years after infection with respiratory syncytial virus in infancy increase in cd23+ cells in infants with bronchiolitis is accompanied by appearance of ige and igg4 antibodies specific for respiratory syncytial virus rhinovirus and respiratory syncytial virus in wheezing children requiring emergency ca-ige and eosinophil analyses reduced interferon-y production in infants with bronchiolitis and asthma cellular immunity is activated and a th-2 response is associated with early wheezing in infants after bronchiolitis different effects of influenza virus, respiratory syncytial virus, and sendai virus on human lymphocytes and macrophages interleukin-1 and interleukin-1 inhibitor production by human macrophages exposed to influenza virus or respiratory syncytial virus the relationship between proved viral bronchiolitis and subsequent wheezing ruuskanen 0, ogra pl: respiratory syncytial virus enhancement by parainfluenza 3 infection of contractile responses to substance p and capsaicin in airway smooth muscle from the guinea pig effect of influenza virus infection on allergic sensitization to aerosolized ovalbumin in mice nitric oxide inhibits rhinovirus-induced cytokine production and viral replication in a human respiratory epithelial cell line rhinovirus replication causes rantes production in primary bronchial epithelial cells increased levels of eosinophil major basic protein in nasal secretions in rhinovirus infection [abstract respiratory syncytial virus-induced release of rantes and mip-1 by bronchial epithelial and peripheral mononuclear cells asthma and immunoglobulin-e antibodies after respiratory syncytial virus bronchiolitis: a prospective cohort study with matched controls lower airway responses to influenza a virus in healthy allergic and nonallergic subjects virusinduced airway obstruction and parasympathetic hyperresponsiveness in adult rats prevention of chronic post-bronchiolitis airway sequelae with interferon-y treatment in rats a cell adhesion molecule, icam-i, is the major surface receptor for rhinoviruses respiratory syncytial virus in early life and risk of wheeze and allergy by age 13 years infection of a human respiratory epithelial cell line with rhinovirus. induction of cytokine release and modulation of susceptibility to infection by cytokine exposure respiratory syncytial virus infection of neonatal monocytes stimulates synthesis of interferon regulatory factor-1 and interleukin-lp (il-lp)-converting enzyme and secretion of il-1p targeted expression of il-11 in the murine airway causes lymphocytic inflammation, bronchial remodelling, and airways obstruction role of nasal interleukin-8 in neutrophil recruitment and activation in children with virus-induced asthma respiratory syncytial virus-induced cytokine production by neonatal macrophages association between interleukin-8 concentration in nasal secretions and severity of symptoms of experimental rhinovirus colds parainfluenza virus-induced persistence of airway inflammation, fibrosis, and dysfunction associated with tgf-pi expression in brown norway rats antibody to interleukin-5 inhibits virus-induced airway hyperresponsiveness to histamine in guinea pigs jncreased production of ifn--y and cysteinyl leukotrienes in virus-induced wheezing respiratory syncytial virus infection of human mononuclear phagocytes stimulates synthesis of platelet-activating factor the release of leukotrienes in the respiratory tract during infection with respiratory syncytial virus: role in obstructive airway disease asthma, atopy and chlamydia pneumoniae antibodies in adults continuing respiratory problems three and a half years after acute viral bronchiolitis immunologic mechanisms of virus-induced wheezing and asthma predictive value of respiratory syncytial virus-specific ige responses for recurrent wheezing following bronchiolitis the development of respiratory syncytial virus-specific ige and the release of histamine in nasopharyngeal secretions after infection patterns of allergic respiratory disease in children with a past history of bronchiolitis key: cord-005774-7z6uyn6p authors: hammer, j.; newth, c. j. l. title: infant lung function testing in the intensive care unit date: 1995 journal: intensive care med doi: 10.1007/bf01704742 sha: doc_id: 5774 cord_uid: 7z6uyn6p as a result of the previous shortage of tools to assess objectively the overall physiological status of the respiratory system in infants and young children, it has been difficult to measure the degree of physiological disorder or the response to therapy in respiratory diseases such as bpd, the pediatric version of ards, bronchiolitis, pneumonia, asthma and croup in this patient population. the newborn — four-year old child is particularly difficult to study because of their lack of cooperation and size. the recent progress in computer technology made pulmonary function testing available for this age range and opened up new possibilities for monitoring changes in disease processes affecting the respiratory system. this may improve medical management of infants and children with lung and heart diseases in particular. in 1989, shannon [49] proposed in this journal that the minimum physiological information needed for the intelligent use of mechanical ventilation (particularly if lower airway and/or pulmonary parenchymal disease was apparent) required the measurement of at least 4 variables: i) arterial partial pressure of carbon dioxide; ii) arterial oxygen saturation; iii) the mechanical time constant of the lung and iv) frc. in many circumstances, arterial co(2) is approximated by alveolar (end-tidal) co(2) and the arterial oxygen saturation is obtained from pulse oximetry accurately if perfusion is adequate. the mechanical time constant and frc are easily measured by the techniques described above and together provide important information concerning appropriate ventilator settings for a given disease. the described techniques bring new insights and awareness, but also new responsibilities in the management of infants and children with respiratory compromise. not all of these techniques need to be applied to all infants in the icu. not all the assumptions upon which some of the techniques we have described are based will prove true. any such methods which do not withstand solid scientific testing must be quickly discarded and replaced with better and (hopefully) easier methods. until recently, there has been limited ability to assess objectively the deviations from normal in lung function in infants and children, either within or without the intensive care unit (icu). however, the application of the rapid data acquisition and processing abilities of the personal computer and miniaturization of equipment has led to major changes in this field, particularly for the newborn-4-year-old child age range. the modifications of old and development of new pulmonary function tests have allowed those involved in the care of such patients novel measurements and new perspectives in both the assessment and management of respiratory failure. this review will focus on techniques which are used to measure thoracoabdominal asynchrony, tidal breathing flow-volume loops, small airway function (forced expiratory maneuvers), respiratory mechanics and lung volumes in critically ill infants and children. the major goals of these techniques in an intensive care setting are to: i) understand the underlying pathophysiology, ii) aid with diagnosis, iii) provide assessment of therapeutic response, and iv) provide a guide to changes in a patient's condition which will allow timely interventions to support the patient. ideally, such devices should also provide a measure of the disease and a prediction of outcome, in addition to being inexpensive and noninvasive. however, at this point in the evolution of intensive care, our most useful monitors, diagnosticians and medical prognosticators are almost certainly highly trained and "disease-smart" physicians, nurses and respiratory therapists at the bedside. some applications of pulmonary function testing generally require that the infants are heavily sedated, and under neuromuscular blockade when mechanically assisted with ventilation. the latter situation also requires (usually) that there be no leak around the ett in order to obtain good quality studies. this can be achieved in most cases with either a cuffed ett or with an uncuffed tube with pharyngeal packing. cuffed ett are not recommended for use in children under the age of 8 years [1] , but in a recent prospective study involving 250 infants and children, using cuffed ett one-half size smaller than the calculated uncuffed ett for age, we had no greater incidence of complications either short-term (post-extubation stridor) or long-term (tracheal stenosis) [2] . we now use cuffed ett routinely in our pediatric icu in infants and young children with pulmonary disease. it is not the intent of this report to justify as "useful" or "essential" any of the techniques which are subsequently mentioned. at one end of the spectrum, demonstration of a significant reduction of mortality and morbidity associated with a change in practice provides a dra-matic justification for the use of a diagnostic or monitoring tool. unfortunately, unlike therapeutic interventions, it is rarely possible to provide evidence of such changes with routine monitoring devices in intensive care. in addition, little has been published on the efficacy or cost effectiveness of the various procedures. at the other end of the spectrum, one can argue that any intervention which provides more understanding of disease processes can be justified, providing there is little or no deleterious effect on the patient. intermittent arterial blood gas analysis is regarded as the standard diagnostic tool for respiratory failure and is fundamental for accurate assessment of pulmonary gas exchange and ventilator management [3] . however, technologic advances have made non-invasive devices available for continous monitoring of oxygen (02) and carbon dioxide (co2). these include end-tidal co2, transcutaneous co 2 and 0 2, and pulse oximetry. if the caregiver is aware of their limitations, they allow quick feedback on rapidly changing conditions and are helpful in the continuous supervision of respiratory therapy. non-invasive blood gas monitoring has the potential to reduce significantly the frequency of abg sampling. detailed descriptions of their operation and limitations are beyond the scope of this paper, but are available in recent reviews [4, 51. thoracoabdominal asynchrony (taa) and paradoxical breathing are often observed in infants and children with various forms of respiratory diseases including upper airway obstruction (uao), parenchymal processes (such as hyaline membrane disease, pneumonia and pulmonary edema), obstructive lower airways disease (asthma, bronchiolitis, bpd) and neuromuscular diseases. this phenomenon has generally been descriptive and was referred to as chest wall retractions in clinical scoring systems. however, phase angle analysis of the lissajous figure allows us to easily detect, quantify and monitor taa in a non-invasive manner [6] . in this technique, rib cage (rc) and abdominal (abd) movements are recorded by use of an uncalibrated respiratory inductance plethysmograph, the bands of which are placed at the levels of the nipples and upper abdomen. the analog output of the rc and abd movements is acquired by a computerized data acquisition system that is programmed to calculate continuously phase angles utilizing the method of agostoni and mognoni [7] . phase angle (0) is thus calculated according to the equation: m sin 0 = -s where m is the length of the midpoint of the rc excursion and s is the length depicting the abd excursion (fig. 1 ). in addition, rc and abd movements can be continuously displayed as an x-y plot giving optical information about changes in loop shape and loop direction. except during rem sleep, the rc and abd expand and decrease in synchrony in normal full-term infants and children, producing a closed or very narrow loop with a positive slope on the x-y plot (mean 0 = 8 o, range = 0 to 25 o). however, during taa the loop opens and becomes progressively wider as taa increases. paradoxical breathing also creates a closed or very narrow loop, but with a negative slope. important information can further be obtained from the loop direction. this indicates which compartment (rc or abd) precedes the other. counterclockwise loops indicate that the abd compartment (diaphragm) leads the rc as usually observed in normal quiet breathing and most forms of respiratory distress in children. clockwise loops signify the opposite which is typically associated with diaphragmatic paralysis [8] . continuous phase angle measurement is a promising non-invasive technique for the objective assessment of taa in a variety of respiratory diseases. phase angles are elevated in uao and decrease after (z-agonist therapy (fig. 2) , but demand further research and a better understanding of their predictive value for respiratory failure [6]. we have recently validated phase angle measurements by showing that they correlate with the level of the imposed respiratory load, but do not detect respiratory muscle fatigue, in uao [9]. however, it has been demonstrated that phase angle measurements correlate with transcutaneous co 2 in infants with severe laryngotracheobronchitis and provide a useful, additional way to . phase angle measurements also correlated with improvement after bronchodilator therapy in children with obstructive airway disease such as asthma and bpd [11] . the use of phase angle analysis to monitor taa in infants and children with neuromuscular disease (e.g. infant botulism, guillain-barr6 syndrome, myopathies, neuropathies, spinal cord injuries) needs to be further clarified, but offers a promising tool to monitor sprinting or weaning processes from mechanical ventilation. diaphragmatic paralysis can be easily detected at the bedside by the characteristic generation of clockwise loops, even when this may not be obvious clinically as in the case of unilateral paralysis (e.g. post cardiothoracic surgery) [8] . a problem often encountered with this technique is that at various times phase angle loops are not based on clear sinusoidal rc and abd movements and produce numerous types of non-sinusoidal patterns [12] . we have recently suggested that a sine-wave independent mathematical approach in loop analysis improves the accuracy of phase shift calculations under such circumstances [13, 141. other methods used in the assessment of respiration involve techniques measuring the "work of breathing". the classic method for "work of breathing" has been thoroughly studied in adults by collett and co-workers [15] . however, modified approaches have been used in infants. the "work of breathing" can be measured relatively non-invasively by use of an oesophageal balloon to record pleural pressure changes and subsequent calculation of the pressure-time index. this index is an estimate of the energy cost of the "work of breathing" because 02 consumption by muscle is proportional to the integral of muscle tension (or pressure) with respect to time. klein and reynolds demonstrated that when the unintegrated pleural pressure signal was used in the index, they were able to show a response to therapy with continuous inflating pressure in sleep-related uao [16] . since breathing slows and inspiratory pressure is greater with uao, the "raw" pressure-time index underestimates the true integrated pressure-time index. nonetheless, this seems a simple and effective objective measurement for upper airway obstruction. a further variation was suggested by wolfson et al. [17] in their study on helium breathing in infants with bpd. these techniques await further validation. tidal breathing flow-volume loops tidal breathing flow-volume or pressure-volume loops are increasingly used in continuous or intermittent monitoring of mechanical ventilation in both neonatal and pediatric icus. they can be measured on special "standalone" equipment, or are increasingly incorporated into modern ventilators. these allow the measurement of tidal volume (vt), tidal flows and pressures generated during mechanical and spontaneous breaths. thus, impacts of alteration in ventilator settings or lung physiology on these parameters can be readily detected [18, 19] . spontaneous tidal volume breaths and ventilator breaths can be compared (especially along the expiratory flow limb where flow-limitation may be readily seen) which enables an estimate of the ventilatory reserve provided by mechanical ventilation (fig. 3 ). the generation of inadverunless it is certain that flow is limited (effort independent) at a particular lung volume, changes in flow rates after a therapeutic maneuver (e.g. bronchodilator) may still be attributed to factors other than a simple response to therapy. we have recently shown that the fd technique is capable of producing forced expiratory flows at flow limitation in intubated animals and infants with normal and obstructed airways [22, 23] . for the test procedure, the lungs are inflated by squeezing a breathing bag filled from a continuous compressed o~ supply to +40 cmh20 inflation pressure, defined as total lung capacity (tlc). inflation pressures are held static for at least 3 s, after which a sliding valve is activated to expose the airways to a 100-l capacity, constant negative pressure source of -40 cmh20 deflation pressure. the lungs are deflated until expiratory flow ceases at residual volume (rv) or for at least 3 s. vc and mef at various subdivisions are measured by an interposed pneumotachograph. throughout the procedure the individual is usually under neuromuscular blockade and/or heavy sedation. normal values for vc and mef at the various subdivisions still need to be defined, but in our laboratory lie in the range of 50-70 ml.kg -1 for vc and 24-38 ml.kg -t 9 s -1 for mef25 and 6-15ml.kg-t.s -~ for meft0. since it has become standard of care to use inhaled bronchodilators on intubated and ventilated patients in a variety of diseases, their effectiveness with respect to bronchodilatation can easily be documented by the fd technique [24, 25] . obstructive airway and restrictive lung diseases produce very characteristic patterns (fig. 4) which are helpful in assessing the underlying pathophysiology (e.g. rsv infection causing bronchiolitis, ards or pneumonia). serial vc and mef assessments are helpful in a variety of lung diseases like bpd and ards requiring long term mechanical ventilation and document the resolution or progression of the disease process [26, 27]. compliance and resistance reflect the mechanical properties of the lungs and require the measurement of flow, volume and pressure. compliance (c) is defined as the change in volume per unit change in pressure: it must be emphasized that compliance is a function not only of the elastic properties of the respiratory system, but also of its volume. in other words, the value obtained is different at various lung volumes, dependent on the shape of the pressure-volume curve, which in turn depends on the amount of lung disease and therapeutic maneuvers such as peep or surfactant administration. sudden changes in compliance often reflect the opening and closing of individual lung units rather than changes in lung tissue and surface tension characteristics and represents the resistive properties of the airways, lung tissue and chest wall. several methods have been designed to measure compliance and resistance in ventilated infants which has led to a confusing nomenclature for the practitioner. compliance is referred to as either dynamic compliance (cdyn) when it is measured when ventilation is in motion, or as static (passive) compliance (crs) when respiratory muscles are inactive during the test procedure. the same applies to the resistance of the respiratory system, which is referred to as either dynamic (re) or total respiratory system resistance (rrs). cdyn can be simply calculated by dividing vt by the total change in pressure necessary to deliver that volume. these numbers can be easily extracted from mechanical ventilation. however, it is understood that cdyn is related to both elastic and flow resistive characteristics according to the equation of motion of the single compartment model of the respiratory system: p = vx+r f c where p = transpulmonary pressure, vx = tidal volume and f = tidal flow. thus, cdyn changes with alteration of mechanical ventilation settings including respiratory frequency, inspiratory and end-expiratory pressure [30] . the classic technique of determining cdyn is based on the measurement of oesophageal pressure as a quantification of pleural pressure [31] . this allows differentiation of cdyn into its components of lung compliance (cl) and chest wall compliance (ccw). however, ccw is usually very high in infants and its contribution to total respiratory compliance (cxox) can often be neglected [32, 33], since cl and ccw are related as follows: this technique is invasive by virtue of the need of an oesophageal catheter and the accuracy of such measurements in intubated infants and children is controversial [34, 351. newer methods measure static compliance (crs) and resistance (rrs) and are based on relaxation of both inspiratory and expiratory muscles during brief airway occlusions during exhalation. the most widely used methods are the passive deflation and the multiple occlusion techniques [36] [37] [38] . muscle relaxation is achieved either by invoking the hering-breuer inflation reflex or by use of neuromuscular blockade. we favor the use of shortterm neuromuscular blockade together with sedation for a mechanically ventilated patient in the controlled setting of an icu because it guarantees complete muscle relaxation during the whole expiratory phase. in the following we will concentrate on the discussion of the passive deflation technique (single breath occlusion) and refer the reader to recent literature for the other methods [39] . the passive deflation technique involves measuring pressure during occlusion of the airway at endinspiration and fitting a straight line to the fv-curve obtained during the subsequent passive exhalation [40, 41] . if there is no muscle activity during exhalation, the expiratory time constant (trs) or emptying time of the respiratory system will be entirely dependent on the mechanical properties of the lungs and can be described as follows: thus, both crs and rrs can be obtained from a single breath. the determination of trs gives some idea of how rapidly the lung empties following a mechanical breath. a single time constant is defined as the time required to exhale 63 % of the tidal volume. three time constants are needed to exhale 95~ of the delivered tidal volume. this permits the determination of respiratory rates allowing complete exhalation or the detection of rate settings which lead to inadvertent peep. the passive deflation technique relies on the assumption that the respiratory system can be regarded as a single compartment model. this is valid in most healthy infants especially over the tidal volume range. however, in the presence of lung disease, the respiratory system will not always behave like a single compartment model and a single time constant will not adequately describe all the respiratory mechanics [42, 43]. we have noted multiple time constants in infants with restrictive lung diseases such as acute ards or pulmonary edema, or with severe obstructive airway disease [44] . in all these circumstances crs and rrs are best measured over the longest linear fit of the passive expiratory fv-curve. however, calculation of time constants at different intercepts may give additional information and better describe the respiratory mechanics over the whole expiration phase [451. pattern recognition adds valuable information to the interpretation of results obtained by measuring respiratory mechanics. while obstructive lung disease is characterized by a concave slope of the passive expiratory fv-loop, restrictive lung disease often results in convex loop patterns. it is important to note that in the case of intubated patients, crs and rrs measurements include the physical properties of the ett. unfortunately, there is still a great lack of normal values for crs and rrs in intubated infants and children with normal lungs. according to our studies, such normal data lie in the range of 0.8-1.2ml'cmh:o -1 9 kg -1 for crs and 0.4-0.sml'cmh20-l"s (up to 1.0 with ett < 3.5 mm i.d.) for rrs [25] . pfenninger and aebi [46] have recently used the passive deflation technique for rrs and crs to compare the response to inhaled and intravenous salbutamol in ventilator-dependent infants with chronic lung disease, and concluded there was no difference. such careful objective physiological measurements should influence how weaning such infants from the ventilator is approached. although the most fundamental interest in lung volume measurements in infancy and childhood relates to the assessment of normal and abnormal lung growth [47, 48], the determination of lung volumes is an important part of the respiratory management of infants and children [49] . lung volume measurements can help in diagnosing respiratory disorders, in evaluating responses to therapy, and in finding suitable ventilator settings with respect to rate and ventilating pressures [50-52]. lung volume is also an important variable when lung mechanics are measured [53] because specific compliance and specific resistance are normalized by lung volume, i.e. the functional residual capacity (frc). currently, frc and v t are the only lung volumes that can be accurately, repeatedly, and reliably measured in infants and small children. hence, they are the only lung volumes that can be routinely determined for clinical reasons either in the icu or in the out-patient clinic. other lung volumes such as tlc, vc and rv can also be measured, but the techniques are employed mainly for research, and require an endotracheal tube. frc can be measured by three techniques: plethysmographic (infant body box), helium (he) dilution (a closed-circuit method), and nitrogen (n2) washout (in its modern form, an open-circuit method). except in the smallest of infants, sedation is required for each technique. sulphahexafluoride has recently been used in a promising washin-washout technique [54, 55] , and has now been validated [66] . the body plethysmograph technique is labor-intensive, as is the calibration of the infant body box. edberg and colleagues [56] [57] [58] have recently applied modern computing techniques to the infant body box and obtained useful data in the neonatal icu setting. however, their lung volume data was measured using the nitrogen (n2) washout method. for most workers, the use of plethysmographic methods is impractical in the icu environment, particularly for mechanically ventilated patients. the recent advent of a commercially available computerized infant box (j~tger) may modify this opinion. the closed-circuit he dilution method has been adapted to measure frc on ventilated patients by heldt et al. [59] . the patient is connected via the ett and a sliding valve to both a bag (which is situated inside a transparent plexiglas box) and to the ventilator. in normal pre-test position the patient is ventilated directly by the ventilator through the valve. the bag, which contains a known amount of gas with known he concentration (and thus a known amount of helium) is sealed and is not connected to the patient. at end-exhalation the valve is switched so that the patient is directly connected only to the he-containing bag while the ventilator ventilates the box surrounding the bag and compresses the bag accordingly. the patient is thus ventilated by the bag which is externally compressed by the ventilator cycle. after several breaths, equilibration of he concentration between the lungs and the bag is achieved and frc can be calculated in the same way as in non-ventilated subjects. helium dilution can be used in patients on very high inspired oxygen concentrations (fio2 = 0.97). however, most thermal conductivity based he analyzers are inaccurate when o2 concentrations are high. moreover, calibration depends on the oz/he mixture ratio and should be repeated each time. leak-free connections in intubated infants are more difficult to achieve in this age group, where cuffed tubes are rarely used. if the leak is minimal, it may be eliminated by gentle tracheal pressure during the recording period. although a method for correcting leaks during frci~e measurements has been described by fox et al. [60] and is currently incorporated into some automated systems. this has not been fully evaluated nor validated and may result in significant errors. the technique is based on washing out the n 2 from the lungs by giving the subject 100% o 2 to breathe. if the amount of n 2 washed out is measured and the initial alveolar n 2 concentration is known, then the lung volume from which point the washout started can be derived. in this open circuit method, the patient is switched to breathing 100% 02 and from this point the volume of n 2 exhaled is determined by integration with respect to time of the instantaneous n2 concentration flowing in the exhalation circuit multiplied by the instantaneous flow. in 1985, gerhardt and co-workers [61] devised a new open washout system to which a constant background o2 flow was delivered. the patient inhaled from and exhaled to that circuit with background flow. although the instantaneous flow rate of the washout circuit changes continuously as the subject breathes, the average flow leaving the system over time remains unchanged because the volume of gas subtracted during inspiration is added back to the system during exhalation (this is true as long as the temperature and humidity of the inhaled and exhaled gas are equal -a condition which is easy to meet by using a humidifier). because the method ignored the instantaneous change in flow and used only the average constant flow for calculation, it was essential that sampling of n 2 for concentration measurements would "see" a continuous decrease of n 2 concentration as the washout proceeds, without the effect of the respiratory phase. this was achieved by incorporating a mixing chamber in the exhalation circuit before the sampling port from which mixed expired gas was sampled for n2 analysis. the technique developed by gerhardt et al. for spontaneously breathing infants [62] is not immediately applicable to ventilated children mainly because the gas flow during calibration does not equal the flow during the test. in order to overcome this difficulty, sivan and co-workers [63] used the respiratory mass spectrometer already "in-line" for measuring the instantaneous n 2 concentration, to record the minute ventilation by the argon dilution technique [64] . at frc the patient is switched to a second ventilator delivering 100~ 02 (washout ventilator) and washout starts. this n2 washout technique (which unlike the he dilution method is limited to patients at fio 2 < 0.65) allows accurate determination of frc during mechanical ventilation and correlates well with those values produced using the douglas bag technique [52] . in patients with restrictive lung disease, including a group with ards [44], frc measured at clinically chosen levels of peep (4-10cmh20) was 45~ below predicted frc for nonintubated normal children and 60~ below that of ventilated children with normal lungs at physiological levels of peep (2-4 cmh20). the use of progressively greater levels of peep produced increases in frc towards predicted normal values [63] . however, this suggests that in ards at least, normalization of frc would require sufficient peep to contribute to barotrauma or to compromise cardiac output and systemic oxygen transport. in spontaneously breathing infants and children, frc is the same whether determined by he dilution or n2 washout methods [65] , and is in the range of 16-22 ml 9 kg-1 (mean = 20.4 ml" kg-1). the only published data on ventilated infants and children with normal lungs demonstrated frcs up to 50~ more than the nor-mal values for spontaneously breathing (i.e. not ventilated) children on peeps of 2-4 cm h20 [63] . as a result of the previous shortage of tools to assess objectively the overall physiological status of the respiratory system in infants and young children, it has been difficult to measure the degree of physiological disorder or the response to therapy in respiratory diseases such as bpd, the pediatric version of ards, bronchiolitis, pneumonia, asthma and croup in this patient population. the newborn -four-year old child is particularly difficult to study because of their lack of cooperation and size. the recent progress in computer technology made pulmonary function testing available for this age range and opened up new possibilities for monitoring changes in disease processes affecting the respiratory system. this may improve medical management of infants and children with lung and heart diseases in particular. in 1989, shannon [49] proposed in this journal that the minimum physiological information needed for the intelligent use of mechanical ventilation (particularly if lower airway and/or pulmonary parenchymal disease was apparent) required the measurement of at least 4 variables: i) arterial partial pressure of carbon dioxide; ii) arterial oxygen saturation; iii) the mechanical time constant of the lung and iv) frc. in many circumstances, arterial co2 is approximated by alveolar (end-tidal) co 2 and the arterial oxygen saturation is obtained from pulse oximetry accurately if perfusion is adequate. the mechanical time constant and frc are easily measured by the techniques described above and together provide important information concerning appropriate ventilator settings for a given disease. the described techniques bring new insights and awareness, but also new responsibilities in the management of infants and children with respiratory compromise. not all of these techniques need to be applied to all infants in the icu. not all the assumptions upon which some of the techniques we have described are based will prove true. any such methods which do not withstand solid scientific testing must be quickly discarded and replaced with better and (hopefully) easier methods. ed) textbook of pediatric intensive care. williams & wilkins recognition and management of respiratory failure noninvasive assessment of blood gases effect of positive end-expiratory pressure on respiratory compliance in children with acute respiratory failure functional residual capacity and ventilation homogeneity in mechanically ventilated small neonates measurement of functional residual capacity by sulfur hexafluoride in small-volume lungs during spontaneous breathing and mechanical ventilation immediate effects on lung function of instilled human surfactant in mechanically ventilated newborn infants with irds lung volume, gas mixing, and mechanics of breathing in mechanically ventilated very low birth weight infants with idiopathic respiratory distress syndrom ekstr6m-jodal b, hjalmarson o (1991) a plethysmographic method for assessment of lung function in mechanically ventilated very low birth weight infants a simplified method to determine functional residual capacity during mechanical ventilation effects of endotracheal tube leaks on functional residual capacity determination in intubated neonates a simple method for measuring functional residual capacity by n 2 washout in small animals and newborn infants functional residual capacity in normal neonates and children up to 5 years of age determined by a n 2 washout method functional residual capacity in ventilated infants and children the measurement of metabolic gas exchange and minute volume by mass spectrometry alone comparison of helium dilution and nitrogen washout measurements of functional residual capacity in infants and very young children measurement of functional residual capacity by sulfur hexafluoride in small-volume lungs during spontaneous breathing and mechanical ventilation key: cord-255734-038xu4hq authors: taylor, deborah r. title: obstacles and advances in sars vaccine development date: 2006-02-13 journal: vaccine doi: 10.1016/j.vaccine.2005.08.102 sha: doc_id: 255734 cord_uid: 038xu4hq the emergence of the severe acute respiratory syndrome (sars) that resulted in a pandemic in 2003 spurred a flurry of interest in the development of vaccines to prevent and treat the potentially deadly viral infection. researchers around the world pooled their scientific resources and shared early data in an unprecedented manner in light of the impending public health crisis. there are still large gaps in knowledge about the pathogenesis of this virus. while significant advances have been made in the development of animal models, the practicality of their use may be hampered by a lack of pathological similarity with human disease. described here are issues related to progress in vaccine development and the obstacles that lie ahead for both researchers and regulatory agencies. severe acute respiratory syndrome (sars) was first reported as a disease of unknown etiology in guangdong * tel.: +1 301 827 3660; fax: +1 301 480 7928. sars is characterized by fever, cough and flu-like symptoms. severe cases resulted in alveolar damage, interstitial mononuclear cells and heavy fibrin deposition in the lungs. respiratory distress resulted in atypical pneumonia, requiring ventilation for approximately 20% of patients [2] . the aim of this review is to describe the advances made in the development of animal models for sars and to identify gaps in scientific understanding that need to be filled. by addressing and possibly overcoming these challenges and making use of the advances made, a safe and effective vaccine may be attainable. the information here may provide the scientific basis for facilitating future regulatory decisions related to the licensing of a sars vaccine. the causative agent for sars is a novel member of the coronavirus family, termed sars-cov [3] . coronaviruses are large enveloped rna viruses, so named for the radiating spike proteins found at the surface of the virion ( fig. 1 ; [4] ). there are three groups of related coronaviruses and sars may be a member of a new fourth group [4] . classification of the sars coronavirus has been controversial, although phylogenetic similarities may place the virus in a subgroup of the group 2 coronaviruses [5] . human coronaviruses include three members that cause common respiratory infections [6] . nonhuman coronaviruses include those that cause respiratory infections in birds, and enteric infections in cattle, pigs, dogs and cats [7] . some of these viruses affect multiple organs, for example, both mouse hepatitis virus (mhv) and feline infectious peritonitis (fip) virus are capable of causing respiratory, enteric, neurologic and hepatic infections [7] . coronaviruses are positive-sense rna viruses that replicate by a unique mechanism whereby the structural genes are expressed as a nested set of subgenomic mrnas, characterized by shared common 3 ends and a conserved, capped 5 leader sequence [4] . the nonstructural genes are transcribed from the 5 end as a polyprotein that is processed by viral proteases ( fig. 2; [4] ). proteins are translated from the 5 open reading frame of each mrna [4] . sars cov has eight open reading frames of unknown function, but has structural proteins found in all coronaviruses that include the envelope (e), the matrix or membrane protein (m), spike (s) and nucleocapsid (n) [4] . the s protein is glycosylated and required for viral attachment and possibly entry. the nucleocapsid protein coats the viral genomic rna. viruses that belong to group 2, such as mhv, also contain a hemagglutinin-esterase (he) protein, which is not present in other groups [4] . the flu-like symptoms and atypical pneumonia, characteristic of sars-cov infection, was also frequently accompanied by lymphopenia [8] . alveolar macrophages were prevalent in patients with fatal sars [8] and contributed to the immune-mediated nature of the disease. in situ hybridization showed that viral infection was present in alveolar epithelial cells and viral rna could also be detected the structural genes (spike, envelope, membrane, and nucleocapsid) are located in the 3 end, which includes as many as eight genes of unknown function [4] . in alveolar macrophages and bronchiolar epithelial cells [9] . several studies have suggested that the immune system may be impaired by sars cov. t-cell lymphopenia was observed in 94% of patients observed, with a decline in cd4+ and cd8+ cell types [10] . two weeks after disease onset, th1 cell-mediated immunity and inflammatory response was noted by the marked elevation of cytokines, ifn gamma, the neutrophil chemokine il-8, il-1, -6 and -12, but not tnf, il-2, -4, or -10. accumulation of monocytes/macrophages and neutrophils was also observed [11] . li et al. [12] noted that a rapid decline of t-cell subsets in the periphery was observed in patients during the acute phase of sars infection, but they observed restoration of t cells during recovery. the presence of proinflammatory cytokines may result from activated alveolar macrophages, suggesting that they may play a role in the pathogenesis of sars [8] . antibodies to the sars coronavirus were found retrospectively in 1.8% of samples collected in 2001, indicating that the 2003 outbreak was not the first time that sars had entered the human population [13] . most infected patients developed a humoral response to sars-cov and antiviral antibodies (igg and igm) were detected at 14 days post-onset of symptoms [14] . igm antibodies declined after 30 days and igg antibodies persisted up to day 210 [14] and antiviral neutralizing antibodies were obtained from convalescent patients [14] . morbidity rates were greater for older individuals, while children under 12 years of age did not develop the severe disease that was seen in adults [15] . taken together these data may suggest that the quality of the immune response may play a role in the outcome of virus infection. an additional challenge related to the containment of sars-cov is the lack in identification of its natural host. virus has been detected in wild and domestic animals [16] . in 2003, the first people to be infected were animal handlers in a food market in guangdong province, china, suggesting a role for zoonotic transmission [17] . the sars strain observed in animals varies only slightly from the human virus and may represent a recent jump across species. the development of good animal models will not only be useful for identifying the natural host, but will be invaluable for determining correlates of immunity, for testing therapeutics and vaccine development. a remarkable advance in sars research came with the discovery that mice were susceptible to infection with sars-cov [18] . balb/c mice were infected with 10 3 or 10 5 50% tissue culture infective doses (tcid 50 ) and by day 2 after infection, yielded 10 6 and 10 7 tcid 50 per gram, respectively, from lung tissue. although no clinical disease was observed, mild and focal peribronchiolar mononuclear inflammatory infiltrates were observed upon microscopic examination of the respiratory tract on day 2 [18] after infection. the presence of these infiltrates may suggest some mimicry with human clinical features, although much milder. the respiratory tracts of the mice were cleared of the virus by day 7 after infection. wentworth et al. found sars-cov in the stomach, intestine, and duodenum, in addition to the respiratory tracts of infected mice [19] . subbarao et al. also protected mice from infection by passive administration of sars-cov neutralizing antibody from previously infected mice, suggesting that neutralization in vivo is possible [18] . mice clear the virus by day 7 post-infection, while humans begin to clear the infection by days 9-14 [8] . a small animal model will allow researchers to test therapeutics and vaccines, and because the mice recover from virus infection so efficiently, also identify host factors that contribute to virus resolution. hamsters have also been shown to be a good model for sars-cov infection, reaching similar titers to those seen in mice [20] . surprisingly, immunodeficient mice can clear a sars-cov infection. mice (c57bl/6 background) that lack nk-t cells (cd1 −/− ), nk cells (beige) or those that lack t and b cells (ragl −/− ) cleared the virus by day 9 after infection [21] . the mice displayed high induction of proinflammatory cytokines, suggesting that the adaptive immune response and nk cells were not required for viral clearance in mice. furthermore, it indicates that the involvement of the innate immune response is important in controlling the virus. it is interesting to speculate that interferon pathways may be important in viral clearance. more evidence for the importance of innate immunity was provided through the infection of stat1-deficient mice with sars-cov [22] . stat1 is important to the regulation of interferons and stat1-deficient mice produced a two log increase in viral titer over control mice. additionally, the mutant mice developed interstitial pneumonia, not seen in the control mice [22] but not alveolar damage as seen in the lungs of human patients. it is unclear at this time if the observed pathological differences between human and stat1-deficient mouse lungs were due to time of sampling or differences in host responses [22] . domestic cats and ferrets have also been tested for use as a sars-cov animal model. cats and ferrets were inoculated with 10 6 tcid 50 sars-cov. cats showed no clinical symptoms [23] , while three out of six ferrets became lethargic and one died. virus was recovered from the lungs of infected cats (10 3 tcid 50 /1 gram of tissue) and ferrets (10 6 tcid 50 /1 gram of tissue) [23] . experiments also showed that horizontal transmission of the virus may have occurred between cats that were housed together or ferrets that were housed together, although the kinetics and mode of transmission are still unknown. the non-inoculated infected ferrets became lethargic, developed conjunctivitis and died at 16 and 21 days post-infection. while the ferrets did not show evidence of pneumonia, they did exhibit hepatic lipidosis and emaciation [23] . ter meulen et al. showed that ferrets that were infected with sars-cov showed signs of multifocal pulmonary lesions affecting about 5-10% of the lung [24] . alveolar damage and lymphocyte infiltration was also observed upon histological examination of infected ferret lung tissue. three species of monkeys have been tested for infectivity with sars-cov: cynomolgus, rhesus and african green. african green monkeys supported the highest level of viral replication, yielding a viral titer of approximately 10 4 tcid 50 /ml from nasal swabs [25] . some researchers working with cynomolgus macaques reported signs of dis-ease resembling sars after the monkeys were infected with sars-cov [26, 27] . from day 3 post-infection, macaques became lethargic, had a temporary skin rash, and one animal showed signs of respiratory distress [26] . two of the macaques had interstitial pneumonia with lesions present, similar to autopsy tissue obtained from sars patients [26] . other groups have reported that cynomolgous macaques show limited pathology, mild disease and upper respiratory symptoms [28, 29] . there is no apparent explanation for the discrepancy between these groups, possibly virus strain differences or monkey subspecies differences may account for the differences in outcome, but to conclude that non-human primates most similarly mimic human disease is still controversial. vaccine efficacy is measured by the ability of the antigen to raise a protective immunologic response from b and t cells after exposure to the viral agent. ideally, by creating memory within the immune system, individuals will be protected from infection for decades. several veterinary coronavirus vaccines are currently available, but their efficacy is variable. the vaccine for prevention of infectious bronchitis virus (ibv), which infects chickens, is effective [30] , but the canine and porcine vaccines are only partially effective [31] . the feline infectious peritonitis (fip) vaccine is actually deleterious to the health of the animal and is discussed in further detail below [32] . vaccines can be produced by inactivation of the virus, by using an attenuated or weak form of the virus, or by using recombinant forms of viral components. inactivated virus vaccines are relatively safe because they cannot revert back to the live form. they are also relatively stable and may not even require refrigeration. this is important in developing countries and for ease in mobilization during outbreak or emergency situations. however, there are limitations to their use. inactivated vaccines usually require several doses and some are weakly effective at stimulating an immune response. the vaccine to prevent hepatitis a is an example of an inactivated viral vaccine [33] . live attenuated viral vaccines may require special laboratory development and cannot always be obtained. to reach effective levels, the virus must be capable of robust replication, but must have lost the ability to cause disease. several problems are associated with the use of a live attenuated vaccine. these vaccines must be kept refrigerated or frozen, and have safety issues related to the possibility of reversion to the wild-type form. additionally, they are almost never given to immunocompromised individuals for fears that the attenuated form may cause disease in the absence of an effective immune response [34] . recombinant dna or viral vectors have been constructed in the lab for use as potential vaccines or to study the tissue tropism of the sars virus [25, 35] . the vectors can be used to deliver foreign antigens using attenuated or nonpathogenic organisms. the safety of these types of vaccines [36] centers around persistence of expression in vivo, possible genomic integration of the foreign dna and possible evolutionary changes that may cause instability of the viral vector. the potential transmission of the viral vector, including its introduction into the environment should also be evaluated. an ideal recombinant vaccine might be engineered to include the inherent ability of the foreign substance to be cleared by drug treatments that are proven safe, such as an antibiotic. antibiotic sensitivity introduced into a recombinant vector may allay fears of future adverse events although these designs may raise additional safety concerns. recombinant proteins can also be used to stimulate the immune response. these proteins are purified from yeast or bacteria and currently used in the manufacture of a licensed hepatitis b vaccine [36] . the cell substrate used to manufacture all of these vaccines is also a concern so vaccine production must be performed in a well-characterized cell substrate. vero e6 cells have been used to produce the licensed poliovirus vaccine [37] and may be appropriate for use in the development of a sars vaccine as sars grows well in vero cells. the fda center for biologics evaluation and research (cber) issued a letter to sponsors using vero cells as a cell substrate for investigational vaccines which can be found on the cber website [38] . another consideration is the use of fetal bovine serum and bovine derivatives in the growth of cells. bovine tissues may contain the bovine spongiform encephalitis (bse) agent. guidelines on the use of bse-free blood products appear on the fda website [39] as do guidelines on the use of bse-free bovine derivatives in the production of vaccines [40] . for viruses that have variable strains, a combination vaccine may be effective. a combination vaccine is one that consists of two or more live organisms, inactivated organisms or purified antigens combined [41] . this type of vaccine may be useful to prevent multiple organisms or strains of 1 organism. each component must make a contribution to the whole and compatibility of the components is necessary. rna viruses replicate through rna-dependent rna polymerase encoded by the virus. this type of polymerase has no proof-reading mechanism associated with it, which results in a high rate of uncorrected mutations. these mutations may or may not be lethal to virus replication and may even persist, resulting in rapid evolution of the virus. for this reason, many rna viruses have multiple genomic strains, or quasispecies, present at one time in an individual. quasispecies may arise in response to selective immune pressure, thus allowing for escape mutants. the existence of quasispecies during sars-cov infection is just coming to light [42] and their importance in escape from immune surveillance is still unknown. most coronaviruses are thought to have the ability to recombine due to homologous sequences in the 5 and 3 ends of the mrnas. evidence suggests that the sars-cov originated by recombination between coronaviruses [43, 44] and that there was an additional host-species drift [43] . both large and small animals can be infected with sars-cov, a giant advance for vaccine research. examining infected animal models will provide information that will lead to an understanding of the correlates of immunity. mice have been used to further the understanding of virus neutralization, cytokine upregulation and the minimum requirements for viral clearance, but have yet to show disease that mimics the atypical pneumonia seen in adult humans. while there have been some reports of disease in cynomolgous macaques [26, 27] , many groups have not reproduced these findings [28] . a promising animal model may be the domestic ferret. ferrets show elevated liver enzymes, lymphocytic infiltration and alveolar damage, which has also been observed in humans [8] . despite the usefulness of these animal models, many challenges lie ahead. first, animal models of sars-cov infection do not mimic human disease. in mice, the virus is cleared in less than 1 week and minor pathology in the lung is observed [18] . histopathology performed on necropsy samples suggests that lung epithelial cells are involved, although the absence of pneumonia and infiltrating macrophages [18] is disappointing. stat1-deficient mice may prove promising as an animal model that most similarly mimics human disease [22] . second, in order to test efficacy, large human populations must be tested in areas where the virus is endemic. finally, if sars fails to return, how will vaccine manufacturers test candidate vaccines for efficacy? while the animal rule has been provided for just this type of case, an animal model should mimic human disease in order to be applicable. the final rule was published in the federal register and can be found on the federal register website [45] . additionally, coronaviruses may induce a short-lived immunity. this may be the reason that humans are subject to multiple infections with coronaviruses that cause the common cold. long-term immunity studies for sars-cov are currently underway. antibody-dependent enhancement (ade) has been observed in vaccinated and wild-type infections of fip. ade is thought to potentiate viral infection through the infection of macrophages. viral entry into macrophages occurs when antibodies bind the virus and attach to macrophages via the fc region of the antibody and its interaction with cell surface expressed fc receptors [46] . neutralizing antibodies can also be enhancing antibodies if antibody titer is low or is of the igg class [47, 48] . because macrophages increase with viral disease, this cell type may provide an abundant reservoir for the virus and thus expansion of the virus in the host. some similarities between fip and sars exist. first, in both cases macrophages can be infected with the virus [9, 49] , and in the case of sars, the etiology of disease is contributed by infiltrating alveolar macrophages leading to pneumonitis [8] . second, the treatment with corticosteroids and/or interferon alpha ameliorates sars disease [50] , suggesting an inflammatory, immune-mediated disease. while there has been no observation of ade during sars infection, it is worth noting that one coronavirus, fipv, is capable of eliciting ade and in the evaluation of vaccines, we may want to consider this possible outcome. however, the difficulty in testing animal models for ade bears the caveat that if ade is not observed; it has not proved that vaccines are safe with regard to ade in humans. in contrast, if an animal model for ade is developed, we may learn more about the mechanism of sars-induced ade, which may help form the basis for developing guidelines for safe vaccine development. a potential sars vaccine might target the virus specifically through humoral or cell-mediated immune responses. alternatively, therapeutic vaccines may be useful in the treatment of viral infection. spike-specific monoclonal and polyclonal antibodies that neutralize the virus have been developed [51, 52] and passive transfer of immune serum into naive mice protected them from infection with sars-cov [18] . this suggests that neutralizing antibody alone can prevent viral infection. neutralization may not require host recognition of the fc region of the antibody, but the need to develop humanized forms of these types of antibodies may be critical if they are to be considered for use as a treatment. a human monoclonal antibody, derived from a phage display library, was administered to ferrets and protected the ferrets from lung disease and the shedding of virus in pharyngeal secretions [24] . neutralizing antibodies from convalescent patients have been identified and characterized. usually neutralizing epitopes are located in the spike protein of the virus [20, 52] . recent evidence has determined that virus neutralization is sensitive to deglycosylation of the spike protein, suggesting that conformational epitopes are important in antibody recognition [53] . sars-cov can be efficiently inactivated by ultraviolet (uv) irradiation [54] . mice immunized with uv-inactivated sars-cov develop humoral and cell-mediated immune responses [55] . both t cell proliferation and cytokine upregulation was observed after boosting with the inactivated virus. beta-propiolactone-inactivated virus also elicited neutralizing antibodies when administered to balb/c mice [56] formalin-inactivated sars-cov yielded potent humoral responses in balb/c mice as well [57] . recombinant viruses may be used to elicit responses to introduced sars-cov genes. the first type of recombinant virus is a defective or non-pathogenic vector that expresses sars-cov proteins. the second type is one that is stimulated to assemble virus-like particles (vlp) in vitro. vlps containing the structural envelope proteins including spike (s), envelope (e) and membrane protein (m) have been assembled by coinfecting insect cells with three baculoviruses expressing one of the three structural proteins [58] . structural proteins expressed by the live attenuated bovine parainfluenza virus type 3 (bhpiv3) were evaluated for efficacy in hamsters [20] and african green monkeys [25] . high titer neutralizing antibodies were obtained after only one intranasal immunization with this vector. single immunization with bhpiv3 expressing s alone provided complete protection upon challenge with sars-cov [20, 25] . recombinant live attenuated modified vaccinia virus ankara (mva) was used to deliver the sars spike protein (rmva-s) into balb/c mice [59] . neutralizing antibodies were obtained and a reduction in the viral titer was observed after challenge with live sars-cov [59] . only ferrets that were challenged with sars-cov after vaccination with rmva-s showed enhanced liver disease as demonstrated by increases in alt values and the presence of mononuclear hepatitis upon histological examination [60] . these data suggest enhanced disease due to vaccination with a sars protein. adenoviruses expressing the s, m or n proteins were used in combination to vaccinate rhesus macaques [61] . the immunized animals all had antibody responses to the s protein and t-cell responses to the n protein [61] . highly infectious hiv particles expressing the s protein have been made, primarily to study the host-cell distribution of the putative sars-cov receptor [35] . additionally, investigating the requirements for viral receptor binding and entry will also enhance our understanding of the requirements for viral control. recombinant hiv particles that express the sars spike protein may provide insight into cell tropism and receptor expression profiles [35] . another retrovirus, murine leukemia virus, was used to generate infectious particles containing most of the s protein. convalescent serum was able to neutralize infection of the recombinant virus in vero cells [62] . high cytotoxic t-lymphocyte (ctl) and antibody responses were observed after mice were injected three times with a recombinant plasmid vector expressing the n protein [63] . mice immunized with a plasmid containing the s protein produced anti-sars-cov igg [64] and developed neutralizing antibodies and a t-cell mediated response resulting in a six-fold reduction in viral titer in the lungs [65] . plasmids encoding either the s1 or s2 regions of the spike protein elicited antibody production in mice [66] . neither the s1 or s2 antibodies alone were capable of neutralizing the virus; how-ever, cooperatively they enabled neutralization of the virus, suggesting that both regions of the spike protein are important for host-cell viral entry [66] . the nucleocapsid protein may also stimulate an effective immune response. dna vaccination with calreticulin fused to the n protein generated sars-specific humoral and cellular immunity in c57bl/6 mice [67] . calreticulin was used because it was found to enhance major histocompatibility complex class i presentation of fusion proteins to cd8 (+) t cells [67] . recombinant viruses may be generated from the fulllength infectious cdna clone of sars-cov [68] . this clone may provide a source for genetic manipulation of the genome [68] . once the viral virulence factors are understood, attenuated strains may be obtained by engineered mutation of the virus. vaccine development may proceed through the undertaking of a systematic approach to understanding the correlates of immunity raised by sars-cov. much of the focus has centered towards the humoral response and neutralizing epitopes, but cell-mediated immunity may also be important. ctl epitopes within sars-cov that may be presented by 99% of the human leukocyte antigen supertypes were identified by advanced bioinformatics [69] . further characterization of these epitopes, including their recognition by convalescent serum, should advance the understanding of important immunological features in the control of sars-cov. while there has been intense study of sars, there is still much that is unknown about the pathology of the virus. many research questions will need to be answered, thus providing the resources necessary to develop an effective and safe vaccine. to be sure that a vaccine will provide coverage for a potentially variable virus, we need to know what the occurrences of viral quasispecies are and how variable are the important epitopes. what are the viral virulence factors and can we use this information to develop an attenuated strain? can the virus establish persistent infection and can humans be repeatedly infected? we also need to determine the innate, humoral and cell-mediated immune responses in recovered sars patients in order to understand how viral clearance comes about. to understand viral pathogenesis we must know how the immune system mediates disease. what is the role of lymphocytic infiltrates and do alveolar macrophages enhance disease or control infection? if we define the ability of the virus to replicate in monocytes/macrophages can we be assured that antibody-dependent enhancement will not be a problem for a sars vaccine? most importantly, identification of the natural animal host may launch the further development of large and small animal models for correlates of immunity and drug and vaccine screening. development of an animal model that mimics human disease will be the single most important advance in the development of a sars vaccine. development of a vaccine for sars-cov is imperative and research headed in a for-ward direction will enable the public health community to be ready. toronto sars critical care group. critically ill patients with severe acute respiratory syndrome sars working group. a novel coronavirus associated with severe acute respiratory syndrome sars-beginning to understand a new virus severe acute respiratory syndrome coronavirus phylogeny: toward consensus identification of a new human coronavirus coronaviridae: the viruses and their replication lung pathology of fatal severe acute respiratory syndrome sars coronavirus-infected cells in lung detected by new in situ hybridization technique expression of lymphocytes and lymphocyte subsets in patients with severe acute respiratory syndrome plasma inflammatory cytokines and chemokines in severe acute respiratory syndrome significant changes of peripheral t lymphocyte subsets in patients with severe acute respiratory syndrome sars-related virus predating sars outbreak dissection study on the severe acute respiratory syndrome 3c-like protease reveals the critical role of the extra domain in dimerization of the enzyme: defining the extra domain as a new target for design of highly specific protease inhibitors sars in newborns and children isolation and characterization of viruses related to the sars coronavirus from animals in southern china role of china in the quest to define and control severe acute respiratory syndrome prior infection and passive transfer of neutralizing antibody prevent replication of severe acute respiratory syndrome coronavirus in the respiratory tract of mice mice susceptible to sars coronavirus contributions of the structural proteins of severe acute respiratory syndrome coronavirus to protective immunity mechanisms of host defense following severe acute respiratory syndromecoronavirus (sars-cov) pulmonary infection of mice resolution of primary severe acute respiratory syndrome-associated coronavirus infection requires stat1 virology: sars virus infection of cats and ferrets human monoclonal antibody as prophylaxis for sars coronavirus infection in ferrets mucosal immunisation of african green monkeys (cercopithecus aethiops) with an attenuated parainfluenza virus expressing the sars coronavirus spike protein for the prevention of sars koch's postulates fulfilled for sars virus newly discovered coronavirus as the primary cause of severe acute respiratory syndrome macaque model for severe acute respiratory syndrome replication of sars coronavirus administered into the respiratory tract of african green, rhesus and cynomolgus monkeys protection of chickens after live and inactivated virus vaccination against challenge with nephropathologenic infectious bronchitis virus safety and efficacy of a modified-live canine coronavirus vaccine in dogs antibody-mediated enhancement of disease in feline infectious peritonitis: comparisons with dengue hemorrhagic fever inactivated hepatitis a vaccine: active and passive immunoprophylaxis in chimpanzees live attenuated varicella vaccine highly infectious sars-cov pseudotyped virus reveals the cell tropism and its correlation with receptor expression genetically engineered vaccines: an overview developing new smallpox vaccines current status and future trends in vaccine regulation-usa sars-associated coronavirus quasispecies in individual patients sars associated coronavirus has a recombinant polymerase and coronaviruses have a history of host-shifting mosaic evolution of the severe acute respiratory syndrome coronavirus antibody-dependent enhancement of virus infection and disease the role of igg subclass of mouse monoclonal antibodies in antibody-dependent enhancement of feline infectious peritonitis virus infection of feline macrophages localization of antigenic sites of the s glycoprotein of feline infectious peritonitis virus involved in neutralization and antibody-dependent enhancement antibody-dependent enhancement of feline infectious peritonitis virus infection in feline alveolar macrophages and human monocyte cell line u937 by serum of cats experimentally or naturally infected with feline coronavirus interferon alfacon-1 plus corticosteroids in severe acute respiratory syndrome: a preliminary study development and characterisation of neutralising monoclonal antibody to the sars-coronavirus identification of an antigenie determinant on the s2 domain of the severe acute respiratory syndrome coronavirus spike glycoprotein capable of inducing neutralizing antibodies synthesis and characterization of a native, oligomeric form of recombinant severe acute respiratory syndrome coronavirus spike glycoprotein inactivation of the coronavirus that induces severe acute respiratory syndrome, sars-cov subcutaneously injected uv-inactivated sars coronavirus vaccine elicits systemic humoral immunity in mice inactivated sars-cov vaccine prepared from whole virus induces a high level of neutralizing antibodies in balb/c mice immune responses in balb/c mice induced by a candidate sars-cov inactivated vaccine prepared from f69 strain assembly of human severe acute respiratory syndrome coronavirus-like particles severe acute respiratory syndrome coronavirus spike protein expressed by attenuated vaccinia virus protectively immunizes mice immunization with modified vaccinia virus ankara-based recombinant vaccine against severe acute respiratory syndrome is associated with enhanced hepatitis in ferrets effects of a sars-associated coronavirus vaccine in monkeys retroviral vectors pseudotyped with severe acute respiratory syndrome coronavirus s protein induction of sars-nucleoprotein-specific immune response by use of dna vaccine dna vaccine of sars-cov s gene induces antibody response in mice a dna vaccine induces sars coronavirus neutralization and protective immunity in mice characterization of humoral responses in mice immunized with plasmid dnas encoding sars-cov spike gene fragments generation and characterization of dna vaccines targeting the nucleocapsid protein of severe acute respiratory syndrome coronavirus reverse genetics with a full-length infectious cdna of severe acute respiratory syndrome coronavirus sars ctl vaccine candidates; hla supertype-, genome-wide scanning and biochemical validation kanta subbarao, edward tabor, miriam darnell, robin levis and hira nakhasi are gratefully acknowledged for comments on the manuscript. key: cord-007234-hcpa8ej5 authors: renwick, neil; schweiger, brunhilde; kapoor, vishal; liu, zhiqiang; villari, joseph; bullmann, reinhard; miething, robert; briese, thomas; lipkin, w. ian title: a recently identified rhinovirus genotype is associated with severe respiratory-tract infection in children in germany date: 2007-12-15 journal: j infect dis doi: 10.1086/524312 sha: doc_id: 7234 cord_uid: hcpa8ej5 acute respiratory infection is a significant cause of morbidity and mortality in children worldwide. accurate identification of causative agents is critical to case management and to prioritization in vaccine development. sensitive multiplex diagnostics provide us with an opportunity to investigate the relative contributions of individual agents andmayalso facilitate the discovery of new pathogens. recently, application of masstag polymerase chain reaction (pcr) to undiagnosed infuenza-like illness in new york state led to the discovery of a novel rhinovirus genotype. here we report the investigation, by masstag pcr, of pediatric respiratory-tract infections in germany, studying 97 cases for which no pathogen was identified through routine laboratory evaluation. respiratory viruses were identified in 49 cases (51%); of the 55 identified viruses, 41 (75%) were rhinoviruses. the novel genotype represented 73% of rhinoviruses and 55% of all identified viruses. infections with the novel genotype were associated with upper-respiratory-tract symptoms but, more frequently, with bronchitis, bronchiolitis, and pneumonia. human rhinoviruses (hrvs) are the most frequent cause of acute respiratory illness worldwide. although hrvs are most commonly associated with mild upperrespiratory-tract disease [1] [2] [3] , infection of lower airways does occur [4 -7] . lower-respiratory-tract infections (lrtis) related to hrv are increasingly being reported in infants, elderly persons, and immunocompromised patients [8] . hrvs are also implicated in exacerbations of asthma [9, 10] , chronic bronchitis [11] , and acute bronchiolitis [12] . taxonomically, hrvs are currently grouped into 2 species, human rhinovirus a (hrv-a) and human rhi-novirus b (hrv-b), in the genus rhinovirus of the family picornaviridae ( [13, 14] ). these nonenveloped, positive-sense, single-stranded rna viruses have been classified serologically [15, 16] and on the basis of their antiviral susceptibility profiles [17, 18] , their nucleotidesequence relatedness [19, 20] , and their use of receptors (intercellular adhesion molecule 1, low-density lipoprotein receptor, and decay-accelerating factor) [21] [22] [23] . phylogenetic analyses of the vp4/vp2 and vp1 coding regions have indicated the presence of 76 serotypes in genetic group a and 25 serotypes genetic group b [18 -20, 24 ]. an agent is commonly not implicated in up to 50% of cases of severe respiratory disease, despite the application of polymerase-chain-reaction (pcr) assays as well as classical diagnostic methods, including antigen tests, serology, and culture methods. broad-range molecular assay systems, such as multiplex pcr (hexaplex [25] , genescan [26] , and masstag [27] ), or microarrays (vi-rochip [28] and panmicrobial greenechips [29] ) may therefore allow us to gain new insights into epidemiology and clinical associations [30, 31] . with respect to hrv, recent studies employing sensitive pcr systems for these difficult-to-isolate organisms have shown an increased detection rate, compared with culture methods [1, [32] [33] [34] [35] . applying a multiplex masstag pcr platform, we recently detected numerous agents of respiratory illness in samples that had been submitted for laboratory diagnosis but that had tested negative during routine diagnostic assessment [30] . hrvs were identified at high frequency in this set of samples. detailed genetic analysis indicated that a large fraction of these viruses represent a previously uncharacterized genotype of rhinovirus, one that diverges from either hrv-a or hrv-b. in an attempt to gather additional information on the potential pathogenicity, as well as temporal and geographic distribution, of rhinoviruses, including the recently identified genotype, we evaluated specimens collected, during the 2003-2006 seasons in bad kreuznach, germany, from children hospitalized because of severe lrti. nasopharyngeal aspirates were obtained from children admitted, because of acute respiratory-tract infection, to the kreuznacher diakonie hospital (bad kreuznach, germany) during the interval of 2003-2006. individuals ranged in age from 2 weeks to 5 years (mean age, 5 months; median age, 10 months); 46% were male, 54% female. specimens collected at the time of admission were forwarded undiluted to the robert koch institute (berlin, germany) for laboratory evaluation. rna extraction was performed by use of qiaamp viral rna kits (qiagen). the 97 samples for which no pathogen had been diagnosed after assessment by realtime reverse-transcription (rt)-pcr assay for influenza virus [36] and respiratory syncytial virus infection were stored at assay procedures. the 97 rna samples representing cases of undiagnosed respiratory diseases were employed as a template for cdna synthesis by use of superscript ii kits with random hexamer priming (invitrogen), and they were analyzed by masstag pcr by using a viral primer panel [27] that targeted influenza virus a and b (fluav and flubv, respectively), respiratory syncytial virus a and b (rsv-a and rsv-b, respectively), human parainfluenza virus 1, 2, 3, and 4 (hpiv-1, hpiv-2, hpiv-3, and hpiv-4, respectively), human coronavirus 229e and oc-43 (hcov-229e and hcov-oc43, respectively), human metapneumovirus, entero-and rhinoviruses, and adenoviruses. the fidelity of the masstag pcr signal was verified by reamplification of products and by sequence analysis for all positive specimens. in instances in which masstag pcr indicated the presence of a picornavirus, the vp4/vp2 region was amplified [37] . amplification products were purified from agarose gels (qia gel extraction kit; qiagen), and nucleotidesequencing reactions were performed on both strands by use of the abi prism big dye cycle sequencing kits and the abi prism genetic analyzer systems (applied biosytems). identical results were obtained with duplicate aliquots processed at the new york and berlin laboratories. sequence analyses, alignments, and phylogenetic reconstructions were performed by use of programs from the wisconsin gcg package (accelrys) and by mega 3.1 software [38] . nucleic-acid sequences generated during this work are available at genbank, under the accession numbers eu081778 -eu081816. we used masstag pcr to investigate 97 nasopharyngeal aspirates from children hospitalized because of acute respiratory illness for which no pathogen was identified through routine laboratory testing. masstag pcr identified at least 1 candidate respiratory-viral pathogen in 49 specimens. although there was variability across the 3 seasons clinical associations. hrvs were the viruses most frequently detected in our set of samples, representing 75% (41/55) of the identified viruses; coinfection with another virus was observed in only 12% (5/41) of these cases (table 2). the frequency of fever or cough in infections with hrv (82%) was comparable to that in infections with the other viruses (89%); the frequency of rhinitis or pharyngitis with hrv (79%) was comparable to that with other viruses; and the frequency of lrti symptoms (bronchitis, bronchiolitis, and pneumonia) with hrv (71%) was comparable to that with the other viruses (67%). whereas pneumonia was more common in infections with hrv a/b (56%) than in infections with hrv x (36%), the frequency of bronchiolitis with hrv a/b (11%) was comparable to that with hrv x (12%), and the frequency of bronchitis with hrv a/b (67%) was comparable to that with hrv x (60%). lrti was recorded in 72% of hrv x infections; however, some cases were related to milder disease (table 2) . molecular epidemiology of identified picornaviruses. masstag pcr targets conserved sequences in the 5'untranslated region of entero-and rhinoviruses; thus, to facilitate phylogenetic analysis of hev and hrv, we amplified and sequenced the vp4/vp2 gene region. however, when we used the basic local alignment search tool for analysis at the nucleotide level, we did not find, for 30 of the vp4/vp2 sequences, a significant match with hrv-a, hrv-b, or hev sequences; analysis at the amino-acid level revealed homology to enteroand rhinoviral sequences, with a sequence identity of 60%-65%. high similarity at both the nucleotide-and amino-acid levels was evident when sequences were aligned with an unclassified genetic clade of picornaviruses recently identified in new york state [30] . however, detailed phylogenetic analysis indicated significant sequence diversity among the 30 viruses ( in this study of samples collected, during a 3-year interval, from hospitalized children with severe undiagnosed respiratory infection, masstag pcr allowed us to detect viral pathogens in 49 (51%) of 97 cases. the pathogens most commonly identified were hrvs. these findings are consistent with other studies, which have indicated that rhinoviruses or picornaviruses ac-count for 20%-80% of acute respiratory infections [1, 32, 33, 39 -41]-exceeding, in some instances, even the frequency of rsv infection in pediatric-patient populations [34, [41] [42] [43] . the presence of hrv is not sufficient to prove causation. asymptomatic hrv infection has been described; however, the extent to which infection without disease represents carriage, incubation, or convalescence is unknown [35, 39, 40, 42, 44] . although we did not have samples to test for the presence of hrv in the lower respiratory tract, the high frequency at which hrv was identified as being the sole virus detected suggests a correlation between the agent and the observed lrti symptoms. support for the plausibility of hrv being pathogenic in lrti comes from the facts that (1) in situ hybridization has demonstrated that hrvs exist in lower airways and (2) hrvs have been shown to trigger inflammatory processes in the infected cells and tissues [5] [6] [7] [45] [46] [47] [48] . among the hrvs identified in the present study were representatives of the novel genetic clade recently discovered in new york state [30] ; indeed, these viruses comprised the majority of hrvs detected. hrv-a and hrv-b have been implicated in common colds as well as in severe lrti. in our patients, viruses of the novel genetic clade were also associated with a wide range of diseases, ranging from rhinitis to bronchitis to severe pneumonia, necessitating supplemental oxygen in ϳ50% of cases. a seasonal pattern of hrv infections has been described [2, 3, 43] ; however, data regarding (43) pathogens detected (no.) hpiv-2 (1) rsv-b (1) hpiv-1 (3) hadv (1) hpiv-3 (1) hpiv-2 (1) hev/hrv (12) hpiv-4 (1) hmpv (1) hcov-oc43 (1) hev/hrv (15) hadv (2) hev/hrv (15) specific identification (no. either serotype-or genotype-specific patterns of seasonality or disease symptoms are limited [49 -51] . a temporal trend of sequence diversity or of correlation between genotype (within the novel hrv clade) and clinical diagnosis was not apparent in our data (figure 1). no detailed information is available yet concerning the history of the novel hrv clade; nonetheless, the sequence diversity observed within it (figure 1) is not consistent with a re-cent introduction. this clade may account, in part, for earlier reports of nontypeable rhinoviruses [41, 44] . indeed, its discovery may reflect the implementation of new technologies rather than novelty of the agent itself. we anticipate that future work will define more than 1 serogroup. our findings reinforce other groups' recent work indicating the significance of hrvs in pediatric lrti. the presence of novel hrvs in 2 disparate geographic locations, in association with serious respiratory disease in children as well as in adults, mandates further work in epidemiology and pathogenesis. frequency and natural history of rhinovirus infections in adults during autumn viruses and bacteria in the etiology of the common cold the seasonality of rhinovirus infections and its implications for clinical recognition characterization and classification of echo 28-rhinovirus-coryzavirus agents detection of rhinovirus rna in lower airway cells during experimentally induced infection rhinoviruses infect the lower airways quantitative and qualitative analysis of rhinovirus infection in bronchial tissues rhinovirus and the lower respiratory tract interleukin-10 gene expression in acute virus-induced asthma rhinovirus viremia in children with respiratory infections viral infections of human association of rhinovirus infection with increased disease severity in acute bronchiolitis international committee on taxonomy of viruses. universal database of the international committee on taxonomy of viruses virus taxonomy: eighth report of the international committee on taxonomy of viruses antigenic groupings of 90 rhinovirus serotypes a collaborative report: rhinoviruses-extension of the numbering system from 89 to 100 two groups of rhinoviruses revealed by a panel of antiviral compounds present sequence divergence and differential pathogenicity alignment of capsid protein vp1 sequences of all human rhinovirus prototype strains: conserved motifs and functional domains molecular relationships between 21 human rhinovirus serotypes genetic clustering of all 102 human rhinovirus prototype strains: serotype 87 is close to human enterovirus 70 many rhinovirus serotypes share the same cellular receptor the major and minor group receptor families contain all but one human rhinovirus serotype human rhinovirus 87 and enterovirus 68 represent a unique serotype with rhinovirus and enterovirus features vp1 sequencing of all human rhinovirus serotypes: insights into genus phylogeny and susceptibility to antiviral capsid-binding compounds rapid simultaneous diagnosis of infections with respiratory syncytial viruses a and b, influenza viruses a and b, and human parainfluenza virus types 1, 2, and 3 by multiplex quantitative reverse transcription-polymerase chain reaction-enzyme hybridization assay (hexaplex) genescan reverse transcription-pcr assay for detection of six common respiratory viruses in young children hospitalized with acute respiratory illness diagnostic system for rapid and sensitive differential detection of pathogens microarray-based detection and genotyping of viral pathogens panmicrobial oligonucleotide array for diagnosis of infectious diseases masstag polymerase-chainreaction detection of respiratory pathogens, including a new rhinovirus genotype, that caused influeza-like illness in new york state during microarray detection of human parainfluenzavirus 4 infection associated with respiratory failure in an immunocompetent adult improved detection of rhinoviruses in nasal and throat swabs by seminested rt-pcr molecular diagnosis of human rhinovirus infections: comparison with virus isolation detection of rhinoviruses by tissue culture and two independent amplification techniques, nucleic acid sequence-based amplification and reverse transcription-pcr, in children with acute respiratory infections during a winter season picornavirus infections in children diagnosed by rt-pcr during longitudinal surveillance with weekly sampling: association with symptomatic illness and effect of season application of a fluorogenic pcr assay for typing and subtyping of influenza viruses in respiratory samples simultaneous detection of fourteen respiratory viruses in clinical specimens by two multiplex reverse transcription nested-pcr assays mega3: integrated software for molecular evolutionary genetics analysis and sequence alignment use of polymerase chain reaction for diagnosis of picornavirus infection in subjects with and without respiratory symptoms human picornavirus and coronavirus rna in nasopharynx of children without concurrent respiratory symptoms novel rhinovirus genotype in lrti • jid respiratory picornaviruses and respiratory syncytial virus as causative agents of acute expiratory wheezing in children rhinovirus and respiratory syncytial virus in wheezing children requiring emergency care: ige and eosinophil analyses rhinovirus-associated hospitalizations in young children persistence of rhinovirus and enterovirus rna after acute respiratory illness in children lower airways inflammation during rhinovirus colds in normal and in asthmatic subjects low grade rhinovirus infection induces a prolonged release of il-8 in pulmonary epithelium rhinovirus replication causes rantes production in primary bronchial epithelial cells rhinovirus infection increases 5-lipoxygenase and cyclooxygenase-2 in bronchial biopsy specimens from nonatopic subjects rhinovirus infections in an industrial population. v. change in distribution of serotypes the seattle virus watch. v. epidemiologic observations of rhinovirus infections, 1965-1969, in families with young children rhinoviruses in britain 1963-1973 we thank ingrid zadow, bettina bauer, manuela friedrich, marlies hartwig, and sven tietze for their excellent technical assistance. key: cord-254770-ot9ys10r authors: uçkay, ilker; gasche-soccal, paola m.; kaiser, laurent; stern, richard; mazza-stalder, jesica; aubert, john-david; van delden, christian title: low incidence of severe respiratory syncytial virus infections in lung transplant recipients despite the absence of specific therapy date: 2009-10-17 journal: j heart lung transplant doi: 10.1016/j.healun.2009.08.012 sha: doc_id: 254770 cord_uid: ot9ys10r background: respiratory syncytial virus (rsv) infections in lung transplant recipients (ltrs) have been associated with significant morbidity and mortality. immunoglobulins, ribavirin, and palivizumab are suggested treatments for both pre-emptive and therapeutic purposes. however, in the absence of randomized, placebo-controlled trials, efficacy is controversial and there is toxicity as well as cost concerns. methods: we retrospectively reviewed cases of lower respiratory tract rsv infections in adult ltrs. diagnosis was based on clinical history, combined with a positive polymerase chain reaction (pcr) and/or viral cultures of bronchoalveolar lavage (bal) specimens. results: ten symptomatic patients were identified (7 men and 3 women, age range 28 to 64 years). all were hospitalized for community-acquired respiratory tract infections. two patients had a concomitant acute grade a3 graft rejection, and 1 patient had a concomitant bacterial pneumonia. eight patients did not receive a specific anti-rsv treatment because of clinical stability and/or improvement at the time of rsv diagnosis. only 2 patients (1 with grade a3 allograft rejection and 1 requiring mechanical ventilation) received ribavirin and palivizumab. all patients recovered without complications and with no persistent rsv infection. however, bronchiolitis obliterans (bos) staging worsened in 6 patients during the mean follow-up of 45 months. conclusions: our data suggest that mild rsv infections in ltrs might evolve favorably in the absence of specific anti-viral therapy. however, this observation needs confirmation in a large clinical trial specifically investigating the development of bos in untreated vs treated patients. no placebo-controlled trial has clearly established their indication and efficacy in this population. moreover, their widespread use is limited by concerns of toxicity (mainly nephrotoxicity) and elevated costs. recommendations are few, often controversial, and have been established primarily for bone marrow transplant (bmt) recipients. 4,8 -17 little has been published on the treatment of rsv infection in solid-organ transplant recipients. 5, 6, 18 official recommendations specifically for the management of rsv in transplant patients are available only in a few countries, such as the united states, 4 sweden, 19 and switzerland. 20 in switzerland, pre-emptive therapy in cases of low-grade immunosuppression, prophylaxis in severe immunosuppression, and combined treatment with immunoglobulin (ig), ribavirin, and palivizumab in cases of proven infection have been suggested for both bmt recipients and ltrs. 20 to get a better overview on the clinical evolution of rsv infections in ltrs we retrospectively searched our virology reports and identified 10 adult ltrs with proven lower respiratory tract rsv infection. 11 herein we describe their clinical evolution according to treatment. the study was conducted at the hôpitaux universitaires de genève (hug) and centre hospitalier universitaire vaudois (chuv), switzerland. both hospitals belong to the "centre universitaire romand de transplantation," which performed 194 ltrs since 1993. both hospitals use similar immunosuppressive regimens with initial anti-interleukin-2r induction, and long-term triple associations, including either cyclosporine (trough target levels between 150 and 200 g/liter), tacrolimus (trough target levels between 10 and 15 g/liter), or everolimus (trough target levels between 3 and 15 g/liter), with mycophenolate mofetil (2 ϫ 1 g/day) and low-dose prednisone (5 to 25 mg/day). since 2003, all ltrs have been followed in a prospective cohort study (n ϭ 77). we identified all cases of lower respiratory tract infection due to rsv in ltrs from 2003 to the 77 ltrs in our cohort witnessed a total of 68 viral respiratory tract infections between november 2003 and march 2006, including 10 episodes with respiratory secretions positive for rsv in 10 patients (7 men and 3 women, age range 28 to 64 years). thus, rsv accounted for 14.7% of these viral respiratory infections. diagnosis of rsv was established by viral culture in 4 patients, by pcr in 5 cases, and by both techniques in 1 patient. no other concomitant viral pathogens were found. in addition, in 1 ltr, rsv was detected by pcr during an annual control in the absence of any respiratory symptoms. this patient was excluded from analysis. none of our patients had a secondary respiratory specimen positive for rsv in later time periods. the clinical characteristics of the 10 cases are presented in detail in table 1 . all patients were symptomatic for community-acquired respiratory tract infection at the time of their positive respiratory tract specimen. all episodes occurred during the winter and early spring, without any epidemiologic inter-case link. seven episodes occurred at least 1 year after transplantation, 2 occurred at 6 months, and 1 occurred at 15 days (range 15 to 144 days posttransplantation). three patients had a concomitant biopsyproven allograft rejection 25 : patients 3 and 7 had acute grade a3 rejection and were treated with anti-thymocyte globulins and intravenous methylprednisolone, respectively, and patient 5 had acute grade a1 rejection that did not require specific anti-rejection therapy. two patients had concomitant infection requiring specific therapy: patient 1 had a bacterial pneumonia, and patient 10 had a symptomatic cytomegalovirus (cmv) disease. patient 6 was treated for an asymptomatic concomitant low-grade re-activation of cmv replication. a new infiltrate on the chest x-ray was noted for 3 patients (patients 4, 6, and 8). individual evolution data for forced expiratory volume in 1 second (fev 1 ) are shown in table 2 . compared with 1 month prior to rsv infection, the fev 1 changes ranged from ϩ5% to ϫ42%, with only 2 patients having a ͼ10% fev 1 reduction (patients 4 and 7). four patients never recovered their pre-rsv infection fev 1 value (patient 5: ϫ5%; patient 6: ϫ8.3%; patient 8: ϫ10.9%; and patient 9: ϫ3.7%). all patients recovered from their rsv infection. eight of the 10 patients already had clinical improvement at the time of rsv diagnosis. in 2 of these patients the immunosuppression had been temporarily reduced; however, no patient was given specific rsv treatment. strikingly, this group included 7 patients who concomitantly received methyprednisolone for a rejection episode and had an fev 1 reduction of 41%; patient 4 also had a transient fev 1 reduction of 42%. two patients were treated with ribavirin for 7 days (orally with 1,600 mg/day or intravenously with 10 mg/kg 3 times daily) concomitant with intravenous palivizumab (a single dose of 15 mg/kg) ( table 1) , including patient 3, who required anti-thymocyte globulins for non-responding con the detection of rsv by pcr in respiratory secretions is highly sensitive and specific, 24 and is currently considered the best available test for the diagnosis of respiratory tract infections in adult lung transplant recipients. 15, 20, 21, 23 it has been implemented in many lung transplant centers and may increase the number of patients diagnosed with rsv infections. therefore, guidelines are needed to help clinicians decide whether all ltrs with documented rsv require specific treatment. herein we have reported 10 cases of proven community-acquired lower respiratory tract rsv infections in adult lung transplant recipients. surprisingly, as a result of delayed diagnosis, 8 of them had already improved clinically before the diagnosis of rsv infection was made. these patients therefore recovered without receiving specific anti-rsv therapy. in only 2 of these cases was the immunosuppression temporarily reduced. in 1 patient a new ground-glass opacity on computed tomography scan 18, 26, 27 (for which no other cause than rsv was found) cleared spontaneously. as previously described in bmt recipients, 8, 10 rsv can also be recovered from the lung of asymptomatic lung transplant recipients. 28 indeed, an eleventh, asymptomatic patient not included in this case description had a positive rsv pcr in an annual control bal assessment. he remained clinically stable without any treatment. this case illustrates a possible detection bias in our study. clearly, patients with asymptomatic rsv infections would not seek medical advice, so the true incidence of respiratory tract infections due to rsv in ltrs could be higher. our observation of a high number of rsv-infected ltrs spontaneously evolving favorably contrasts with previous reports supporting early specific anti-rsv therapy (especially aerosolized ribavirin), 5, 6, 18 and raises critical questions regarding the more aggressive therapeutic approaches recommended. 20 according to our review of the 86 previously published cases of rsv infections among adult ltrs, our patients were not less immunosuppressed than those described elsewhere (table 3 ). previous studies suggested that severe rsv infections may occur early after transplantation when the immune response is most compromised. 6 however, a more recent study reported only 24% of infections during the first 3 months post-transplantation. 5 in our cohort, only 1 patient developed an rsv infection during the first 3 months post-transplantation. he evolved favorably without any specific therapy. clearly, further studies are needed to determine whether time after transplantation impacts on the severity of disease and necessity of treatment. both rsv infections and acute rejection episodes have been suggested to be risk factors for the development of bos. 7, 8, 29, 30 it has also been suggested that rsv infections may trigger acute rejection. 3, 18, 29 strikingly, we observed post-rsv infection worsening of bos stage in 60% of patients, during a mean follow-up time of 45 months. interestingly, half of these patients also experienced ն3 post-rsv acute rejection episodes. because of the small number of cases, and confounding rejection episodes, it remains difficult to ascertain the potential responsibility of rsv infections in the development and/or worsening of bos in our cohort. however, this warrants further investigation in large ltr cohorts. a possible causality between mild rsv infections that per se evolve favorably without specific treatment and bos would potentially have major diagnostic and therapeutic implications. indeed, such an association would support screening for rsv in ltrs, even those with mild symptoms. moreover, one would have to establish in controlled trials whether specific anti-rsv treatment could prevent bos worsening in such conditions. one should not forget both the increased costs and potential adverse effects associated with specific rsv therapy. immunoglobulins, 31,32 ribavirin, 33 and pavilizumab 34, 35 may all have significant adverse effects. in some studies the incidence of serious adverse effects of ribavirin was high, with hemolytic anemia occurring in 61% of treated patients. 36 in our study, none of the 2 patients treated with ribavirin developed serious adverse effects. likewise, pelaez et al reported only 1 episode of mild reversible anemia among ltrs treated with oral ribavirin for rsv. 37 they further suggested that oral ribavirin might be as efficient-but 20-fold less expensive-than nebulized ribavirin. cost-effectiveness analyses for pavilizumab for the treatment of rsv infections in adults are missing. such studies are presently available only for prophylaxis in infants, 38,39 who require much smaller doses than adults. 40 in conclusion, our observations support that ltrs without severe disease due to rsv, and without particularly enhanced immunosuppression (eg, anti-thymocyte globulins), do not necessarily require specific anti-viral treatment. this contrasts with previous reports and expert opinions, [41] [42] [43] [44] [45] [46] which favor early specific anti-rsv treatment in ltrs. however, most of these earlier studies were retrospective, included small numbers of patients, assessed several different respiratory viruses, and contained incomplete clinical information. finally, many recommendations were derived from bmt recipients, 4,8 -17 who represent a clinically distinct patient population. we therefore suggest that ltrs positive for rsv at least 3 months after transplantation, with only minor clinical symptoms, may be observed carefully with a transient reduction of immunosuppression, and that a specific anti-rsv treatment be initiated only in cases of clinical deterioration. however, a randomized clinical trial is warranted to determine whether this less aggressive, step-by-step approach is safe, and does not expose ltrs to an increased risk of bos development. we thank the teams from the laboratory of virology and the lung transplantation program at geneva university hospitals and vaud university hospital for their help in the clinical management of the cases. none of the authors has any conflicts of interest to disclose. a single-season prospective study of respiratory viral infections in lung transplant recipients community respiratory viruses: organ transplant recipients community acquired respiratory viral infections after lung transplantation: clinical features and long-term consequences community-acquired respiratory viruses clinical features and outcomes of paramyxoviral infection in lung transplant recipients treated with ribavirin community respiratory viral infection in adult lung transplant recipients intravenous ribavirin is a safe and cost-effective treatment for respiratory syncytial virus infection after lung transplantation respiratory virus infection among hematopoietic cell transplantation recipients: evidence for asymptomatic parainfluenza virus infection randomized controlled multicenter trial of aerosolized ribavirin for respiratory syncytial virus upper respiratory tract infection in hematopoietic cell transplant recipients preemptive treatment of pediatric bone marrow transplant patients with asymptomatic respiratory syncytial virus infection with aerosolized ribavirin respiratory virus infections after stem cell transplantation: a prospective study from the infectious diseases working party of the european group for blood and marrow transplantation community respiratory virus infections among hospitalized adult bone marrow transplant recipients community-acquired respiratory viruses after hemopoietic stem cell or solid organ transplantation respiratory syncytial virus upper respiratory tract illnesses in adult blood and marrow transplant recipients: combination therapy with aerosolized ribavirin and intravenous immunoglobulin frequent detection of respiratory viruses in adult recipients of stem cell transplants with the use of real-time polymerase chain reaction, compared with viral culture immune-globulin prophylaxis of respiratory syncytial virus infection in patients undergoing stem-cell transplantation pre-emptive oral ribavirin therapy of paramyxovirus infections after haematopoietic stem cell transplantation: a pilot study respiratory syncytial virusassociated infections in adult recipients of solid organ transplants management of infections caused by respiratory syncytial virus respiratorisches syncytial-virus (rsv) infektion: massnahmen beim immunsupprimierten patienten lower respiratory viral illnesses: improved diagnosis by molecular methods and clinical impact a prospective hospital-based study of the clinical impact of non-severe acute respiratory syndrome (non-sars)-related human coronavirus infection the value of polymerase chain reaction for the diagnosis of viral respiratory tract infections in lung transplant recipients development of a quantitative taqman rt-pcr for respiratory syncytial virus revision of the 1990 working formulation for the classification of pulmonary allograft rejection ct manifestations of respiratory syncytial virus infection in lung transplant recipients respiratory viral infections in lung transplant recipients: radiologic findings with clinical correlation incidence and significance of noncytomegalovirus viral respiratory infection after adult lung transplantation respiratory viral infections are a distinct risk for bronchiolitis obliterans syndrome and death respiratory viruses and chronic rejection in lung transplant recipients intrainfusion and postinfusion adverse events related to intravenous immunoglobulin therapy in immunodeficiency states adverse reactions of prophylactic intravenous immunoglobulin infusions in iranian patients with primary immunodeficiency ribavirin: adverse drug reactions phase 1 evaluation of the respiratory syncytial virus-specific monoclonal antibody palivizumab in recipients of hematopoietic stem cell transplants results of clinical surveillance during the japanese first palivizumab season in 2002-2003 common adverse events associated with the use of ribavirin for severe acute respiratory syndrome in canada efficacy of oral ribavirin in lung transplant recipients with respiratory syncytial virus lower respiratory tract infection palivizumab for respiratory syncytial virus prophylaxis in high-risk infants: a costeffectiveness analysis cost effectiveness of palivizumab for respiratory syncytial virus prophylaxis in high-risk children: a uk analysis american academy of pediatrics committee on infectious diseases and committee on fetus and newborn. revised indications for the use of palivizumab and respiratory syncytial virus immune globulin intravenous for the prevention of respiratory syncytial virus infections respiratory syncytial virus infections in pediatric liver transplant recipients prevention and treatment of respiratory syncytial virus and parainfluenza viruses in immunocompromised patients steroid-responsive bronchiolitis after human heart-lung transplantation respiratory syncytial virus pneumonia in a lung transplant recipient continuous aerosolised ribavirin for respiratory syncytial virus infection in lung transplant recipients paramyxovirus infection in lung transplant recipients key: cord-253145-1fbj1rdv authors: fox, julie d. title: nucleic acid amplification tests for detection of respiratory viruses date: 2007-10-31 journal: journal of clinical virology doi: 10.1016/s1386-6532(07)70005-7 sha: doc_id: 253145 cord_uid: 1fbj1rdv abstract nucleic acid amplification tests (nats) are increasingly being used for diagnosis of respiratory virus infections. the most familiar formats use dna or rna target amplification methods for enhanced sensitivity above culture and antigen-based procedures. although gel and plate-hybridisation methods are still utilised for analysis of amplified products, detection using “real-time” methods which do not require handling of amplified products are favoured in many laboratories. assays based on nucleic acid amplification and detection can be designed against a broad range of respiratory viruses and have been particularly useful for detection of recently identified viruses such as human metapneumovirus and coronaviruses nl63 and hku1. however, the wide range of potential pathogens which can cause similar respiratory symptomology and disease makes application of individual diagnostic assays based on detection of dna and rna both complex and expensive. one way to resolve this potential problem is to undertake multiplexed nucleic acid amplification reactions with analysis of amplified products by suspension microarray. the respiratory virus panel (rvp) from luminex molecular diagnostics is one example of such an approach which could be made available to diagnostic and public health laboratories for broad spectrum respiratory virus detection. appropriate management of patients with a respiratory virus infection requires rapid identification of the etiologic agent. nats are emerging as the preferred (gold standard) approach for diagnosis of respiratory infections, either as an adjunct to other testing or as a replacement (lee et al., 2006) . nats are not easily compromised by sample quality and timing of collection related to onset of symptoms. there are also benefits in laboratory safety and turn-around time if diagnostic testing can be undertaken without prior culture of the unknown organism. diagnosis of respiratory infections is complex because of the wide range of potential pathogens which can present with the same clinical symptoms, and nats can assist in this process. picornaviruses (rhinoviruses and enteroviruses) and coronaviruses 229e and oc43 have long been recognized as a cause of respiratory symptoms but they are not identified efficiently using standard virological approaches undertaken in the majority of laboratories. detection of these important viruses is increasingly being undertaken using nats allowing full realization of the likely role of these viruses in respiratory infection and disease (garbino et al., 2004; jartti et al., 2004; monto, 2004; vallet et al., 2004; arden et al., 2006; esposito et al., 2006; jacques et al., 2006; kusel et al., 2006; lee et al., 2006; loens et al., 2006; manoha et al., 2007) . in recent years many new respiratory virus pathogens have been identified and it is important that tests for these viruses are included in the respiratory virus testing algorithm. this has proved most efficient by inclusion of at least some nats in the diagnostic testing repertoire although antigen and culture methods are available for some of these viruses (see the article by ginocchio in this supplement). human metapneumovirus (hmpv) is increasingly recognized as an important viral pathogen in the young and elderly (bosis et al., 2005; sivaprakasam et al., 2007; van den hoogen, 2007) . human bocavirus (hbov), although often present with other co-pathogens, has been associated with significant disease, particularly in the young (arden et al., 2006; arnold et al., 2006; bastien et al., 2006) . the recently identified human coronaviruses nl63 and hku1 have also both been evaluated and clinical relevance assessed using nats (chiu et al., 2005; kaiser et al., 2005; woo et al., 2005; esposito et al., 2006; gerna et al., 2006; koetz et al., 2006; lau et al., 2006; han et al., 2007; kupfer et al., 2007) . use of nats allows assessment of the impact of a wider array of potential pathogens on respiratory infections than previously possible and target-specific (multiplex) approaches have proved feasible for enhanced broadspectrum respiratory virus diagnosis. in this review an overview of established methods and new procedures for respiratory virus nucleic acid detection and diagnosis is given. a wide range of both target and signal amplification nucleic acid amplification methods could be applied to respiratory virus detection. any target amplification method has the advantage of sensitivity above signal amplification but the latter may be simpler and less laborious for high specimen throughput. this review focuses on methods where detailed publications using either "in house" or commerciallyavailable assays for respiratory virus nucleic acid detection are available. critical to down-stream nucleic acid detection methods is the quality of extracted nucleic acid which needs to be purified away from any inhibitors. in some cases concentration of the sample can be undertaken during the extraction procedure which enhances clinical sensitivity. it is most convenient if the eluate contains purified nucleic acid suitable for both dna and rna amplification and detection methodologies. although the majority of respiratory virus targets have an rna genome, notable exceptions include advs and hbov both of which are important to include in a viral diagnostic screen. an extract containing total nucleic acid can also be used for analysis of bacteria which may present with similar symptoms to viruses (e.g. mycoplasma pneumoniae, chlamydophila pneumoniae). for many laboratories, automation of the extraction, concentration and nucleic acid purification is critical to utility and application of diagnostic nats and ensures high quality, reproducible results. individual laboratories tend to evaluate extraction procedures as part of their nats and there are very few studies which systematically compare extraction and nucleic acid preparation for respiratory viruses. ideally, a control should be included as a spike into each specimen before extraction to confirm all steps in the process were optimal. this could be a modified/ manufactured nucleic acid (dingle et al., 2004) or could make use of unrelated virus controls (niesters, 2004) . further enhancement of sample preparation will involve higher specimen throughput and, ultimately, a direct link to amplification and detection methods using robotics. primers and probes have been designed and validated for amplification of a range of viral respiratory targets. the main problem is lack of capacity (throughput of specimens) and the laborious and expensive nature of approaches which rely on undertaking many individual nats on a single sample. non-selective amplification procedures have been widely reported as an alternative to target specific amplification. in general, degenerate or conserved primers are useful in amplifying or identifying sequence variants or new members within a virus family but sequence-independent (random) amplification needs to be utilized for unknown targets and for virus discovery (ambrose and clewley, 2006) . one disadvantage of using degenerate or random primers in a broad spectrum amplification method is that the amplification efficiency may be reduced once homologous primers in a mix have been incorporated into products. design of complex multiplex amplification reactions requires a good database of available sequences and suitable software for multiple-sequence alignments. this may be beyond the means of many diagnostic laboratories. kitbased solutions to broad-spectrum respiratory pathogen diagnosis incorporate enhanced multiplex pcr approaches and negate the need for individual laboratories to undertake complex design and validation (e.g., brunstein and thomas, 2006; lee et al., 2007; li et al., 2007; mahony et al., 2007) . pcr (or rt-pcr) is still the most common nucleic acid target amplification method used in the diagnostic laboratory. this is partly because the procedure was widely publicised before the alternative isothermal means of amplifying targets. there have been many complex reactions and diagnostic algorithms reported to try and represent the wide-spectrum of potential pathogens causing respiratory symptoms. despite the well-recognised problems, there are reports of large studies using "in house" procedures where multiplexed primer combinations have proved successful for pcr amplification (coiras et al., 2004; pehler-harrington et al., 2004; syrmis et al., 2004; gunson et al., 2005; weigl et al., 2007) . one interesting adaptation of the pcr amplification is to use a "touchdown" approach which allows more flexibility in design of primer sets. the idea is that assays are run concurrently with simultaneous amplification and subsequent detection of 12 respiratory virus targets (coyle et al., 2004) . this approach avoids the need for complex multiplexing but increases the number of reactions necessary/sample. nucleic acid sequence based amplification (nasba) is an isothermal target amplification procedure which utilizes three enzyme activities (rt, rnaseh and t7 rna polymerase) in order to amplify sense or anti-sense target rna. the method has been applied successfully to respiratory virus detection and diagnosis of associated infection and disease hibbitts and fox, 2002; moore et al., 2004; landry et al., 2005; lee et al., 2006; loens et al., 2006; moore et al., 2006; dare et al., 2007) . loop-mediated isothermal amplification (lamp) was originally developed for rapid amplification of dna targets but can be combined successfully with a reverse transcription step for rna respiratory viruses (e.g., nagamine et al., 2002; hong et al., 2004) . the method utilises 4−6 target specific regions in a strand displacement synthesis resulting in a very rapid isothermal amplification of target. examples of amplified product analysis include gel-based detection [often of semi-nested or nested pcr (coiras et al., 2004; coyle et al., 2004) or lamp (hong et al., 2004) products], automated fluorescent capillary electrophoresis (erdman et al., 2003) or separate hybridisation of products to target specific probes (coiras et al., 2005) . nested pcr was first introduced to enhance sensitivity and specificity of pcr. however, even experienced laboratories have had problems with amplicon contamination using these methods (apfalter et al., 2005) . many diagnostic laboratories prefer a method with separate probe-based hybridization detection for analysis of amplified products or they use "realtime" assays, as described below. separate hybridization methods for analysis of amplified products ensure good control of specificity for analysis of amplified products and have heralded the way for array based methodologies (see below). use of novel labels incorporated into amplified products may facilitate analysis and allow detection of a broader range of viruses in a single assay. one reported methodology targets 20 respiratory viruses using multiplex pcr incorporating masstag labels in the forward and reverse primers (briese et al., 2005) . the principle behind real-time assays is that amplification of the nucleic acid target is combined with detection in a single reaction. there is no need for manipulation of amplified products, which minimises problems with amplicon carry-over and potential false positive reactions. for many real-time assays, detection of amplified products utilises a target-specific probe. various formats and chemistries are available for labelling of these probes in order to discriminate between those free in solution from those bound to target. where multiple target-specific primer sets are used in amplification (ensuring specificity) "in tube" detection of amplified products may utilise intercalating dyes, turbidity measurements or other generic nucleic acid detection procedures. discussion of practical applications for real-time pcr is provided in a recent review (gunson et al., 2006) . choice of real-time amplification and detection format depends on the laboratory throughput, level of expertise available and number of targets to be tested. fluorescentreporting real-time assays are limited by the spectral overlap of labels and, in general, multiplex real-time nats are limited to 5 separate target detections. thus, in tube amplification of target combined with a specificity check by probe hybridization (real-time assays) is extremely useful and convenient if one or a few possible causes of a respiratory illness are to be considered. unfortunately, this approach is not very flexible if there is a need to enhance capacity and broaden the detection. attempts to build diagnostic capacity for real-time assays by introducing multiple primer and probe sets (multiplex primer and probe approaches) have not been entirely successful as there is a tendency for a reduction in amplification efficiency when a complex master mix is utilized. one way to increase the diagnostic capacity is to separate the nucleic acid amplification away from the hybridization and detection reaction. using this approach it is easier to increase the number of targets without compromising the diagnostic efficiency. the move away from a real-time assay could be seen as a step backwards in technology if it were not for the advantage of multiple pathogen detection in a single assay. thus, in some of the latest diagnostic assays, the convenience of individual real-time assays is replaced by the enhanced capacity of separate amplification and detection to allow testing based on clinical presentation rather than a pre-conceived idea about the viral cause. for such an approach to be useful in a diagnostic setting enhancements to both nucleic acid amplification procedures and hybridization methods and formats have been necessary. microarrays have the potential to resolve complex amplified product mixtures. the array (or chip) substrate may be nylon, membrane, glass, silicon or polystyrene microbeads. they may have variable density (numbers of specific probes and thus targets to be queried), and probe design and hybridization conditions can be adjusted to allow some mismatch of sequences. detection of products on a solid-phase microarray can make use of conventional hybridization, flow-through or re-sequencing procedures. all these solid-phase array formats have been utilized for respiratory virus detection and analysis (kessler et al., 2004; coiras et al., 2005; wang et al., 2006; lin et al., 2007) . suspension microarrays employ a liquid phase bead conjugated array technology known as luminex ® xmap tm for detection of amplified products. such suspension microarrays exhibit rapid hybridisation kinetics, flexibility in assay design and format and low cost (dunbar, 2006) . new beads (and probes) can be added or others replaced without having to reformat and print new arrays (a disadvantage for solid-phase arrays). some key respiratory targets are already part of early release commercial assays utilizing multiplex amplification with detection using the luminex ® system. in one format, multiplex pcr products are detected and discriminated using template-specific probes conjugated to different microspheres (brunstein and thomas, 2006; li et al., 2007) . in an alternative strategy, a multiplex pcr is used in a first step followed by primer directed (and target specific) strand extension and labeling. each target-specific primer used in this labeling reaction incorporates a unique capture sequence. it is these capture sequences which are used for detection of amplified products in a universal suspension microarray (lee et al., 2007; mahony et al., 2007) . the rvp assay (luminex molecular diagnostics) is described in further detail in other sections of this review supplement. a summary of main advantages and references to nat enhancements for detection and analysis of respiratory viruses is given in table 1 . the majority of reported studies using nats utilize individual or small multiplex assays targeting important respiratory virus targets such as influenza virus (ifv) a and b, parainfluenza viruses (pivs), adenoviruses (advs) and respiratory syncytial virus (rsv). many feasibility studies have confirmed that nats improve detection of potential pathogens from lower respiratory tract specimens as well as from respiratory swabs even where alternative methods such as dfa and culture are available (hibbitts and fox, 2002; moore et al., 2004; lee et al., 2006) . in the case of respiratory adenoviruses detection by dfa is known to be particularly poor (as illustrated in coyle et al., 2004) . although culture is quite sensitive for detection of advs the shorter turn-around time for diagnosis by nat ensures appropriate early management and employment of infection prevention and control procedures for vulnerable (e.g. immunocompromised) individuals as well as differentiation between designated serotypes for epidemiological study (pehler-harrington et al., 2004; vabret et al., 2004) . many laboratories retain use of dfa methods for rapid analysis of respiratory samples and, in particular, for triage and cohorting of vulnerable hospitalized patients. the yield for dfa can be good during seasonal peaks of rsv and ifv, especially when appropriately taken nasopharyngeal (np) samples are available from children (who tend to shed large amounts of virus compared with adults). diagnostic yield of dfa for non-np sample types and for targets other than ifva, ifvb, piv or rsv is much less. table 2 gives results for analysis of dfa negative np samples using sensitive real-time nats for ifva, ifvb, piv1−4 and rsv (nat methodology as previously published, lee et al., 2006) . the enhanced sensitivity of such nats above dfa for these critical targets is clearly demonstrated, despite the fact that only np samples are included in the analysis. the enhanced pick up of piv by nats is particularly obvious. although this is due partly to the lack of dfa testing reagents for piv4 (which is known to cause some respiratory infections) the vast majority of dfa negative piv nat positives are piv1−3. the dfa test is most useful for ifva and rsv but, even for these targets, a significant number of positives are missed if this procedure alone is used for decisions on patient management and infection prevention and control. while antigen and culture-based procedures have diagnostic utility for detection of ifva, ifvb, piv1−3, rsv and advs, other viruses will be missed if nats are not included in the diagnostic algorithm. as shown in table 1 , nats have well-established advantages for enhanced detection of picornaviruses (enteroviruses and rhinoviruses), coronaviruses 229e and oc43 and piv4. these viruses have long been acknowledged as important causes of respiratory infection and disease but are not identified easily by antigen or culture procedures. practical application of a multiplex pcr amplification with novel masstag labels led to identification of previously unidentified rhinoviruses as a cause of non-specific respiratory illness (lamson et al., 2006) . analysis of recentlyidentified coronaviruses using nats has confirmed that, although nl63 seems to be found most commonly (chiu et al., 2005; kaiser et al., 2005; esposito et al., 2006; gerna et al., 2006; koetz et al., 2006; han et al., 2007) hku1 has also been associated with severe disease in some cases (woo et al., 2005; lau et al., 2006; kupfer et al., 2007) . epidemiological study of hbov requires use of nats, and this virus is now recognized as a cause of acute respiratory virus infection, either alone or with a co-pathogen, in young children (arden et al., 2006; arnold et al., 2006; bastien et al., 2006; ma et al., 2006) . the use of nats has led to identification of more respiratory virus co-infections than were previously recognised using less-sensitive antigen and culture methods. if individual nats are utilized, multiple infections are identified frequently but use of real-time multiplex reactions may lead to competition between amplification and detection table 1 enhanced detection of respiratory viruses using nats virus target nat enhancement (s) compared with antigen or culture procedures example reference(s) to illustrate nat diagnostic enhancement (s) ifv a improved sensitivity. rapid sub-typing and strain analysis available from the same nucleic acid. faster turn-around than culture. infectious virus is not amplified (important safety factor compared with culture for pandemic and avian viruses). erdman et al., 2003; coyle et al., 2004; jennings et al., 2004; moore et al., 2004; syrmis et al., 2004; weinberg et al., 2004; lee et al., 2006; van de pol et al., 2006; wang et al., 2006; li et al., 2007; mahony et al., 2007 ifv b improved sensitivity. faster turn-around than culture. erdman et al., 2003; coyle et al., 2004; jennings et al., 2004; weinberg et al., 2004 rsv improved sensitivity. faster turn-around than culture. van elden et al., 2002; erdman et al., 2003; jennings et al., 2004; garbino et al., 2004; syrmis et al., 2004; weinberg et al., 2004; kusel et al., 2006; lee et al., 2006; moore et al., 2006; van de pol et al., 2006; lee et al., 2007; li et al., 2007 piv 1−3 improved sensitivity. faster turn-around than culture. van elden et al., 2002; erdman et al., 2003; coiras et al., 2004; coyle et al., 2004; jennings et al., 2004; syrmis et al., 2004; weinberg et al., 2004; lee et al., 2006 piv 4 not detectable routinely except by nat. coiras et al., 2004; lee et al., 2007 hmpv improved sensitivity. faster turn-around than culture. jennings et al., 2004; koetz et al., 2006; lee et al., 2006; dare et al., 2007; lee et al., 2007 hbov not detectable routinely except by nat. arden et al., 2006; arnold et al., 2006; bastien et al., 2006 adv improved sensitivity compared with antigen detection. faster turn-around than culture. coyle et al., 2004; jennings et al., 2004; pehler-harrington et al., 2004; syrmis et al., 2004; vabret et al., 2004; arden et al., 2006; lee et al., 2006; lee et al., 2007; li et al., 2007 picornaviruses some picornaviruses are only detectable routinely by nat. faster turn-around and better sensitivity for culturable rhinoviruses and enteroviruses van elden et al., 2002; coiras et al., 2004; garbino et al., 2004; jartti et al., 2004; jennings et al., 2004; landry et al., 2005; arden et al., 2006; jacques et al., 2006; kusel et al., 2006; lamson et al., 2006; lee et al., 2006; van de pol et al., 2006; lee et al., 2007 coronaviruses some coronaviruses are only detectable routinely by nat (nl63, hku1). faster turn-around and better sensitivity for culturable viruses (229e, oc43, sars coronavirus). infectious virus is not amplified (important safety factor compared with culture for sars). jennings et al., 2004; vallet et al., 2004; chiu et al., 2005; kaiser et al., 2005; arden et al., 2006; esposito et al., 2006; gerna et al., 2006; koetz et al., 2006; lau et al., 2006; lee et al., 2006; han et al., 2007; lee et al., 2007 samples were all nasopharyngeal samples (swabs and aspirates) collected and tested 1 targets and a resultant underestimation of co-infection rate. in some cases, co-infections have been linked with more severe illness [e.g. for coronaviruses (gerna et al., 2006) and for the paramyxoviruses rsv and hmpv (semple et al., 2005) ] but the hypothesis that severe symptoms occur because of the additive effect of multiple virus infections is controversial and warrants further study. despite the demonstrated enhanced sensitivity of nats there have been delays in the diagnostic implementation of such assays. the reasons for this include the technical complexity, the cost and the lack of proper validation/ standardization of assays. particular problems have been noted when proficiency studies for use of nats in diagnostic laboratories have been undertaken, showing that false positives and false negatives may be reported (apfalter et al., 2005; templeton et al., 2006) . one study utilizing solid-phase microarray hybridization of randomly amplified pcr products from respiratory cultures and clinical samples demonstrated comparable results to alternative culture or individual pcr methods (palacios et al., 2007) . the potential use of flow-through array procedures for detection and typing of influenza a in a single reaction has also been suggested (kessler et al., 2004) . application of re-sequencing arrays to clinical studies demonstrated correct sequence and strain identification using an array targeting 57 genes for 26 respiratory pathogens (lin et al., 2007) . the particular application of re-sequencing arrays to tracking of influenza genetic variation confirmed utility of this approach to inform vaccine development (wang et al., 2006) . one disadvantage of using re-sequencing arrays for molecular epidemiological studies, however is the need for re-design of components of the array regularly to reflect rna virus sequence variation. evaluation of suspension microarray approaches for retrospective analysis of respiratory specimens confirmed good sensitivity and specificity compared with antigen and culture based procedures (described in more detail in other articles of this review supplement). nats have the advantage of enhanced sensitivity compared with many antigen and culture-based assays and short turnaround times (especially compared with traditional culture). if a sample contains a possible level 3 pathogen there are also safety and logistical advantages to using nats above culture since the sample can be inactivated prior to analysis and then tested in a level 2 environment. however, the broad range of pathogens which can cause similar respiratory symptomology makes it difficult to apply individual (monoplex) or small multiplex nats to comprehensive respiratory diagnosis. such an approach can be cost prohibitive and may not even be possible if specimen quantity is limited. as more clinically-relevant respiratory pathogens are identified, a technological change in how nats are performed is necessary to meet the ever expanding diagnostic need. such technology enhancement and validation will be required before diagnostics based on nats can be considered as practical in an outbreak situation where the causative agent may not be known. as respiratory virus amplification and detection procedures that utilize microarrays are developed and evaluated outside of the research laboratory they will become more accessible to diagnostic laboratories. further development and evaluation of the methods in prospective diagnostic studies is necessary, and sensitivity, specificity and other assay parameters will need to be compared with alternative formats for these types of assays (particularly individual nats for all targets). more targets will also need to be incorporated and validated as novel viruses (and other pathogens) are identified and based on needs for pandemic preparedness. once evaluations of suspension microarray nats have been completed, their utility for high-and low-throughput diagnostic laboratories and the cost implications of applying this technology will be defined. it is likely that technologies based on the currently available commercial assays or other similar methods in development will become widely utilized for respiratory virus diagnosis. approval and regulation of assays by appropriate agencies with concurrent availability of suitable quality control and proficiency panel materials will establish amplification methods combined with array-based detection as the next "gold standard" for respiratory virus diagnosis. nucleic acid amplification tests, in modified format, making use of enhanced amplification and array-based hybridization, have the potential to impact on diagnosis and identification of novel viruses. care must be taken, however, to consider quality control issues and to learn from the problems noted with use of non-standardized individual nats. ideally, the next range of respiratory virus diagnostics should utilize validated (fda, health canada and ce mark) assays provided by commercial companies. availability of suitable proficiency panels for respiratory viruses will be critical to ensure standardization and quality control of new diagnostic procedures. none declared. virus discovery by sequence-independent genome amplification in-house nucleic acid amplification assays in research: how much quality control is needed before one can rely upon the results? frequent detection of human rhinoviruses, paramyxoviruses, coronaviruses, and bocavirus during acute respiratory tract infections human bocavirus: prevalence and clinical spectrum at a children's hospital human bocavirus infection impact of human metapneumovirus in childhood: comparison with respiratory syncytial virus and influenza viruses diagnostic system for rapid and sensitive differential detection of pathogens direct screening of clinical specimens for multiple respiratory pathogens using the genaco respiratory panels 1 and 2 human coronavirus nl63 infection and other coronavirus infections in children hospitalized with acute respiratory disease in hong kong simultaneous detection of fourteen respiratory viruses in clinical specimens by two multiplex reverse transcription nested-pcr assays oligonucleotide array for simultaneous detection of respiratory viruses using a reverse-line blot hybridization assay a touchdown nucleic acid amplification protocol as an alternative to culture backup for immunofluorescence in the routine diagnosis of acute viral respiratory tract infections diagnosis of human metapneumovirus infection in immunosuppressed lung transplant recipients and children evaluated for pertussis stable and noncompetitive rna internal control for routine clinical diagnostic reverse transcription-pcr applications of luminex xmap technology for rapid, highthroughput multiplexed nucleic acid detection genescan reverse transcription-pcr assay for detection of six common respiratory viruses in young children hospitalized with acute respiratory illness impact of human coronavirus infections in otherwise healthy children who attended an emergency department development and evaluation of nucleic acid sequence based amplification (nasba) for diagnosis of enterovirus infections using the nuclisens basic kit lower respiratory viral illnesses: improved diagnosis by molecular methods and clinical impact genetic variability of human coronavirus oc43-, 229e-, and nl63-like strains and their association with lower respiratory tract infections of hospitalized infants and immunocompromised patients real-time rt-pcr detection of 12 respiratory viral infections in four triplex reactions practical experience of high throughput real time pcr in the routine diagnostic virology setting human coronavirus-nl63 infections in korean children the application of molecular techniques to diagnosis of viral respiratory tract infections development and evaluation of a novel loop-mediated isothermal amplification method for rapid detection of severe acute respiratory syndrome coronavirus association of respiratory picornaviruses with acute bronchiolitis in french infants respiratory picornaviruses and respiratory syncytial virus as causative agents of acute expiratory wheezing in children viral etiology of acute respiratory tract infections in children presenting to hospital: role of polymerase chain reaction and demonstration of multiple infections human coronavirus nl63 associated with lower respiratory tract symptoms in early life use of the dna flow-thru chip, a three-dimensional biochip, for typing and subtyping of influenza viruses detection of human coronavirus nl63, human metapneumovirus and respiratory syncytial virus in children with respiratory tract infections in south-west sweden two cases of severe obstructive pneumonia associated with an hku1-like coronavirus role of respiratory viruses in acute upper and lower respiratory tract illness in the first year of life: a birth cohort study masstag polymerase-chain-reaction detection of respiratory pathogens, including a new rhinovirus genotype, that caused influenza-like illness in new york state during real-time nucleic acid sequence-based amplification using molecular beacons for detection of enterovirus rna in clinical specimens coronavirus hku1 and other coronavirus infections in hong kong enhanced identification of viral and atypical bacterial pathogens in lower respiratory tract samples with nucleic acid amplification tests a high-throughput, sensitive and accurate multiplex pcr-microsphere flow cytometry system for large-scale comprehensive detection of respiratory viruses simultaneous detection and high-throughput identification of a panel of rna viruses causing respiratory tract infections using a resequencing microarray as a multiple respiratory pathogen detection assay detection of rhinoviruses by tissue culture and two independent amplification techniques, nucleic acid sequence-based amplification and reverse transcription-pcr, in children with acute respiratory infections during a winter season detection of human bocavirus in japanese children with lower respiratory tract infections development of a respiratory virus panel (rvp) test for the detection of twenty human respiratory viruses using multiplex pcr and a fluid microbead-based array epidemiological and clinical features of hmpv, rsv and rvs infections in young children occurrence of respiratory virus: time, place and person development and evaluation of a real-time nucleic acid sequence based amplification assay for rapid detection of influenza a enhanced clinical utility of the nuclisens easyq rsv a+b assay for rapid detection of respiratory syncytial virus in clinical samples accelerated reaction by loop-mediated isothermal amplification using loop primers molecular and diagnostic clinical virology in real time panmicrobial oligonucleotide array for diagnosis of infectious diseases rapid detection and identification of human adenovirus species by adenoplex, a multiplex pcr-enzyme hybridization assay dual infection of infants by human metapneumovirus and human respiratory syncytial virus is strongly associated with severe bronchiolitis life-threatening human metapneumovirus infections in west of scotland a sensitive, specific, and cost-effective multiplex reverse transcriptase-pcr assay for the detection of seven common respiratory viruses in respiratory samples a multi-centre pilot proficiency programme to assess the quality of molecular detection of respiratory viruses development of a pcr-and hybridization-based assay (pcr adenovirus consensus) for the detection and the species identification of adenoviruses in respiratory specimens detection of human coronavirus 229e in nasal specimens in large scale studies using an rt-pcr hybridization assay respiratory tract infection due to human metapneumovirus among elderly patients polymerase chain reaction is more sensitive than viral culture and antigen testing for the detection of respiratory viruses in adults with hematological cancer and pneumonia identifying influenza viruses with resequencing microarrays ten years' experience with year-round active surveillance of up to 19 respiratory pathogens in children superiority of reverse-transcription polymerase chain reaction to conventional viral culture in the diagnosis of acute respiratory tract infections in children characterization and complete genome sequence of a novel coronavirus, coronavirus hku1, from patients with pneumonia results presented in this review are from routine testing of respiratory samples submitted for investigation at provincial laboratory for public health (provlab, alberta, canada). technologists and laboratory scientists in calgary and edmonton virology and molecular diagnostics areas undertook all the nucleic acid testing. dr bonita lee (provlab and university of alberta) provided invaluable assistance with data analysis. key: cord-004397-ypli7wtu authors: ma, zhan-ying; deng, hua; hua, li-dong; lei, wen; zhang, chang-bin; dai, qi-qiang; tao, wei-jing; zhang, liang title: suspension microarray-based comparison of oropharyngeal swab and bronchoalveolar lavage fluid for pathogen identification in young children hospitalized with respiratory tract infection date: 2020-02-22 journal: bmc infect dis doi: 10.1186/s12879-020-4900-8 sha: doc_id: 4397 cord_uid: ypli7wtu background: respiratory tract infection (rti) in young children is a leading cause of morbidity and hospitalization worldwide. there are few studies assessing the performance for bronchoalveolar lavage fluid (balf) versus oropharyngeal swab (ops) specimens in microbiological findings for children with rti. the primary purpose of this study was to compare the detection rates of ops and paired balf in detecting key respiratory pathogens using suspension microarray. methods: we collected paired ops and balf specimens from 76 hospitalized children with respiratory illness. the samples were tested simultaneously for 8 respiratory viruses and 5 bacteria by suspension microarray. results: of 76 paired specimens, 62 patients (81.6%) had at least one pathogen. balf and ops identified respiratory pathogen infections in 57 (75%) and 49 (64.5%) patients, respectively (p > 0.05). the etiology analysis revealed that viruses were responsible for 53.7% of the patients, whereas bacteria accounted for 32.9% and mycoplasma pneumoniae for 13.4%. the leading 5 pathogens identified were respiratory syncytial virus, streptococcus pneumoniaee, haemophilus influenzae, mycoplasma pneumoniae and adenovirus, and they accounted for 74.2% of etiological fraction. for detection of any pathogen, the overall detection rate of balf (81%) was marginally higher than that (69%) of ops (p = 0.046). the differences in the frequency distribution and sensitivity for most pathogens detected by two sampling methods were not statistically significant. conclusions: in this study, balf and ops had similar microbiological yields. our results indicated the clinical value of ops testing in pediatric patients with respiratory illness. respiratory tract infection (rti) in children is a leading cause of morbidity and hospitalization [1, 2] . especially, severe pneumonia ranks the second most common cause of mortality in children younger than 5 years worldwide according to a recent report [3] . viral and bacterial infections are the primary etiology of rti. accurate and rapid identification of the etiologic agents has an essential role in ensuring the appropriate and effective treatment for patients with respiratory illness, which could avoid unnecessary usage of antibiotics, reduce the overall costs and shorten the period of hospitalization [4] . laboratory diagnosis of respiratory infections is performed traditionally using culture and immunological assays. although microbial culture is regarded as the gold standard, it is time-consuming and labor-intensive. specially, some fastidious pathogens are difficult to cultivate. antigen/antibody detection is fast and simple, however, insufficient sensitivity limits its usage in clinical settings. nowadays, molecular techniques are becoming widely used for identification of respiratory etiologies in clinical practice. multiplex pcr can detect several targets at one time, however, design and optimization of dozens of primers and probes remain challenging. microarray has the benefits of high-throughput, speed and low-cost. suspension microarray enables simultaneously detect a number of pathogens in a single assay, it has faster hybridization kinetics and more flexibility in array construction compared to traditional solid-phase array. a variety of sampling methods have been used in detecting respiratory pathogens in clinical practice. recently, several studies have described the performance of nasal swab, nasal wash, nasopharyngeal aspirate, nasopharyngeal swab (nps), oropharyngeal swab (ops), sputum and bronchoalveolar lavage fluid (balf) samples in microbiological findings using qrt-pcr or multipathogen detection platforms [5] [6] [7] [8] [9] . when testing for respiratory agents, the recommended and commonly collected sample is an upper respiratory swab because nasopharynx and oropharynx are two of the most common portals for the introduction of microbes into the respiratory tract. however, collection of nps is an uncomfortable sampling method for young children, particularly pediatric patients with nasal congestion. compared to nps, ops is less technically challenging and more acceptable to children because it is quick and simple [10] . in addition, some reports have showed that ops can increase the number of viral infections identified by 15%, compared to the nps alone [11] . specifically, it has been found to be significantly more sensitive than nps for the detection of certain viruses, such as adenovirus and 2009 pandemic influenza a (h1n1) virus [12] . so far, few studies have compared balf and paired ops samples from young children hospitalized with rti. in the present study, we used the suspension microarray, a multipathogen detection platform, to simultaneously detect viral and bacterial respiratory pathogens in matched ops and balf specimens from pediatric patients for comparison of the sensitivities between the two sample types. the study protocol was approved by the institutional ethics board of dongguan maternal and child health care hospital, china. written informed consent for the balf procedure and ops sampling was obtained from each parent or guardian. children aged 1 month to 7 years with signs and symptoms of rti admitted to the study hospital between october 2017 and september 2018 were enrolled into this study. ops and balf specimens were collected within 3-5 days of hospital admission. first, we collected the ops samples, a cottontipped swab was used to swab over the posterior pharynx and tonsils. balf specimens were obtained with bronchoscope according to the 2009 and 2018 guidelines of pediatric bronchoscopy in china. samples were stored at 4°c until analysis and all specimens were tested within 24 h of collection. nucleic acid was isolated from balf and ops specimens using a magnetic bead-based blood total nucleic acid kit (magcares™, #m1701, genehar technologies inc., guangzhou, china) according to the manufacturer's protocol. dna and rna concentrations were measured using a quawell q5000 uv spectrophotometer. to detect multiple respiratory pathogens in a single assay, suspension microarray was developed in-house based on the luminex xmap system. the procedure consisted of multiplex pcr, probe design, the attachment of probes to microspheres and hybridization as previously reported [13, 14] . to assess the sensitivity for each sampling method, the presence of a pathogen in either of the specimens was deemed to be a true positive [15, 16] . we compared the differences in sensitivity for each pathogen between two sample types using the chi-squared test or fisher's exact test, as appropriate. statistical analyses were conducted with the software graphpad prism 5.0. a two-tailed p-value < 0.05 was considered statistically significant. a total of 76 young children were subjected to respiratory pathogens detection in this project. clinical characteristics of the patients are summarized in table 1 . the patients included 50 males and 26 females. the median age of children was 16 months (range 1 month to 7 years), and 84.2% of included children were younger than 5 years old. according to the 2013 who definition of severe pneumonia and guideline for children with community acquired pneumonia in china (2013 version), 22 patients were considered as severe respiratory tract infection (srti) and admitted to picu with clinical presentation of cough, difficulty in breathing/tachypnoea, and one or more of the general danger signs such as an inability to drink, persistent vomiting, convulsions, lethargy or unconsciousness, central cyanosis. to compare the ops and paired balf for pathogens detection in young children with rti, we tested the two sample types using suspension microarray. of the 76 cases included in the analysis, 62 patients (81.6%) had at least one respiratory pathogen. balf and ops identified respiratory pathogen infections in 57 (75%) and 49 (64.5%) cases, respectively ( table 2 ). there was not a significant difference in detection rates between the two sample types (p > 0.05). of these, 39 (51.3%) had concordant ops and balf suspension array results: 25 (32.9%) had the same pathogens identified from the ops and balf suspension array, and 14 (18.4%) had concordant negative results. the overall distribution of the respiratory pathogens tested is shown in table 3 . a total of 97 pathogens were found in 76 young patients. the etiology analysis revealed that viruses were responsible for 53.7% of patients, whereas bacteria accounted for 32.9% and mycoplasma pneumoniae for 13.4%. the leading 5 pathogens identified were rsv, sp, hi, mp and adv, and they accounted for 74.2% of etiological fraction. for detection of any pathogen, the overall detection rate of balf (81%) was marginally higher than that (69%) of ops (p = 0.046). also, balf was more sensitive than ops for detecting moraxella catarrhalis (p < 0.05). the differences in frequency distribution and sensitivity for each single pathogen except moraxella catarrhalis of two sampling methods were not statistically significant. virus/virus and virus/bacterium co-infections were found in ops and balf (table 4 ). in ops, a single pathogen was found in 34 cases (69.3%), two pathogens in 13 cases (26.6%), and three pathogens in 2 cases (4.1%). in balf, a single pathogen was identified in 41 cases (71.9%), two pathogens in 13 cases (22.9%), three pathogens in 2 cases (3.5%), and four agents in 1 case (1.7%). the most common co-infections observed was sp plus mp (n = 3) in ops samples, rsv plus hi (n = 3) and followed by rsv plus sp (n = 2) in balf specimens. in the present study, we used suspension array to simultaneously detect multiple viral and bacterial pathogens in paired balf and ops specimens from symptomatic patients hospitalized with respiratory illness. to validate the reliability and accuracy of the multipathogen testing platform, we have compared the yield of suspension array with that of metagenomic next-generation sequencing for microbiological findings and highlighted the high concordance of 13 targets between the two methods (manuscript in preparation). these results showed that our suspension array can reliably identify pathogens in patients with rti. the fast turnaround time (within 4-5 h) makes it possible to be a valuable tool in clinical settings. the reliability of the specimens taken via oropharyngeal/nasopharyngeal swab or wash compared to the deep samples such as balf is a matter of debate and it would be interesting to investigate if they are really useful. here, balf and ops had similar microbiological yields (75% vs. 65%, p > 0.05). the differences in the frequency distribution and sensitivity for most targeted pathogens except moraxella catarrhalis of two sampling methods were not statistically significant. selection of a sampling method for detecting respiratory pathogens must take into account its sensitivity, feasibility and costs. the collection of ops is relatively simple, quick and less invasive compared to other sampling methods. for these reasons, we consider the ops as the preferred method of respiratory tract sampling for pathogen detection. for moraxella catarrhalis, we detected 4 cases in balf and none in paired ops specimens. in general, moraxella catarrhalis causes mainly upper respiratory tract infection (otitis media) in children and lower respiratory tract infection in adults with previously compromised airways such as chronic obstructive pulmonary (3) hi + piv (1) rsv + sp (2) hi + adv (1) sp + mp (1) bp + rsv (1) bp + rsv (1) rsv + piv (1) mp + rsv (1) hbov+sp (1) rsv + adv (1) hbov+rsv ( disease [17, 18] . however, some reports have demonstrated that moraxella catarrhalis may be involved in lower respiratory tract infections in children [19] , which is consistent with our results. more studies are needed to investigate its role in respiratory illness in hospitalized children. among the 76 patients, 25 (32.9%) had the same pathogens identified from the ops and balf suspension array, and 14 (18.4%) had concordant negative results. for the 14 cases, they might be infected by some rare pathogens that are not covered by our suspension array. the top 5 pathogens were rsv, sp, hi, mp and adv, accounting for 74.2% of etiological fraction. our data are in agreement with other recent findings in multi-country case-control studies that found rsv was the most common cause of severe childhood pneumonia [20, 21] . thus, rsv could be a primary target for children hospitalized with respiratory illness. in general, rsv is most commonly found in lower respiratory tract infections particularly in infants [22] . however, the sensitivities of rsv between ops and balf was not significantly different in our testing. rsv infection and replication initiates in the nasopharynx. the virus could be found in both upper and lower airway via high-sensitivity molecular techniques when it spreads from the upper respiratory tract to the lower in individuals with compromised immunity. bordetella pertussis was rarely identified in infants perhaps due to high vaccination rates. in this study, we detected bordetella pertussis in 5 young children. identification of bordetella pertussis in balf or ops specimens may provide predictive value for the outcome of respiratory illness at the individual case level [23] . in terms of some pathogens such as influenza a, adenovirus, mycoplasma pneumoniae and streptococcus pneumoniae, relatively low concordance between balf and ops specimens for them may reflect different cell tropisms for different parts of the respiratory tract. notably, none of the 76 cases was tested positive for hrv in this work, which is somewhat surprising given that this virus is often associated with upper respiratory infection. however, another project in our group showed that the detection rate of hrv was~2% in ops among 2895 pediatric outpatients with respiratory illness. here, the included children were inpatients. although hrv infections are frequent, they are mostly limited to the upper respiratory tract and generally cause relatively mild symptoms [24, 25] . the contribution of hrv may vary by disease severity of included patients and other factors. cultivation is regarded as the gold standard in etiological identification. as shown in table 1 , we have performed sputum culture in 42 of 76 cases. compared with pcr-based methods, the detection rate of it was significantly lower. one of possible explanations is the empirical antibiotic therapy in patients before sampling. here, we focused on comparison of ops and paired balf in detecting respiratory pathogens, rather than the sensitivity of nucleic acid-based array compared to the gold standard, i.e. cultivation. based on the same consideration, a healthy control group was not tested for ruling out false positives in this work because of our specific interest and aim. similarly, a prior study, 86 patients enrolled and no healthy controls included, has also applied this strategy to compare the yields of bronchoalveolar lavage samples with that of nasopharyngeal swabs by using filmarray respiratory panel [8] . this study has several limitations. first, our sample size was relatively small (n = 76) because we focused on paired balf and ops specimens collected from hospitalized children. as a result, there was limited power to compare the sensitivities of balf and ops for specific respiratory pathogens in patients. further studies in a larger cohort may generate a relatively high degree of precision when performing comparative statistical analysis. second, although balf are regarded lower respiratory tract samples, oropharyngeal intubation for balf might result in potential contamination by upper respiratory tract "contaminants", particularly for bacteria/ viruses known to colonize the oropharynx. thus, the balf specimen might be actually both an upper and a lower airway combined sample. however, it does not affect our primary purpose that focuses on the microbiological findings of ops sampling. third, empirical antibiotic use in clinical practice may reduce the sensitivity of assays, particularly for bacteria. collectively, identification of a pathogen does not necessarily equate to the etiological agent, particularly in a multipathogen testing and laboratory results require further interpretation by experienced clinicians. in addition, we here focused on a subset of potential pathogens because the 13 agents (8 viruses and 5 bacteria) are key respiratory pathogens in children based on previous epidemiological investigations in china. in fact, fungi are also important pathogens causing severe infections of the respiratory system. another project in our group by using metagenomic nextgeneration sequencing found that the infection rates of candida albicans, pneumocystis jiroveci and aspergillus fumigatus in young children admitted to picu with respiratory illness were 3.52, 1.68 and 1.23%, respectively. given the high prevalence and importance of the airborne fungal pathogens in respiratory infections, we plan to add fungal species to our upgraded in-house array, which would be a separate study due to many experiments and large undertakings. estimates of worldwide distribution of child deaths from acute respiratory infections acute lower respiratory infections in developing countries global, regional, and national causes of under-5 mortality in 2000-15: an updated systematic analysis with implications for the sustainable development goals impact of multiplex molecular assay turn-around-time on antibiotic utilization and clinical management of hospitalized children with acute respiratory tract infections comparison among nasopharyngeal swab, nasal wash, and oropharyngeal swab for respiratory virus detection in adults with acute pharyngitis concordance between rt-pcr-based detection of respiratory viruses from nasal swabs collected for viral testing and nasopharyngeal swabs collected for bacterial testing comparison of sputum and nasopharyngeal swabs for detection of respiratory viruses filmarray respiratory panel assay: comparison of nasopharyngeal swabs and bronchoalveolar lavage samples improved detection of respiratory pathogens by use of high-quality sputum with taqman array card technology pneumonia methods working g: specimen collection for the diagnosis of pediatric pneumonia added value of an oropharyngeal swab in detection of viruses in children hospitalized with lower respiratory tract infection comparison of nasopharyngeal and oropharyngeal swabs for the diagnosis of eight respiratory viruses by real-time reverse transcription-pcr assays applications of luminex xmap technology for rapid, highthroughput multiplexed nucleic acid detection multiplexed detection and identification of respiratory pathogens using the nxtag(r) respiratory pathogen panel comparison of combined nose-throat swabs with nasopharyngeal aspirates for detection of pandemic influenza a/h1n1 2009 virus by real-time reverse transcriptase pcr comparing nose-throat swabs and nasopharyngeal aspirates collected from children with symptoms for respiratory virus identification using real-time polymerase chain reaction nosocomial transmission clusters and risk factors in moraxella catarrhalis prevalence and resistance pattern of moraxella catarrhalis in community-acquired lower respiratory tract infections moraxella catarrhalis: from emerging to established pathogen causes of severe pneumonia requiring hospital admission in children without hiv infection from africa and asia: the perch multi-country casecontrol study. the lancet microorganisms associated with pneumonia in children <5 years of age in developing and emerging countries: the gabriel pneumonia multicenter, prospective, case-control study viral and host factors in human respiratory syncytial virus pathogenesis pertussisassociated pneumonia in infants and children from low-and middleincome countries participating in the perch study rhinoviruses, allergic inflammation, and asthma role of rhinovirus load in the upper respiratory tract and severity of symptoms in lung transplant recipients publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations not applicable. china. 2 we used suspension-array to compare balf and paired ops specimens for detecting multiple pathogens in children hospitalized with respiratory illness. the similar sensitivities between the two sampling methods indicated the clinical value of ops testing in clinical settings. this research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. the data set used and/or analyzed during the current study is available from the corresponding author on reasonable request. the study protocol was approved by the institutional ethics board of dongguan maternal and child health care hospital. written informed consent was obtained from each parent or guardian. not applicable. the authors declare that they have no competing interests.author details 1 dongguan maternal and child health care hospital, dongguan 523120, key: cord-289697-g24xib4l authors: macdowell, ana l.; bacharier, leonard b. title: infectious triggers of asthma date: 2005-03-01 journal: immunol allergy clin north am doi: 10.1016/j.iac.2004.09.011 sha: doc_id: 289697 cord_uid: g24xib4l there is abundant evidence that asthma is frequently exacerbated by infectious agents. several viruses have been implicated in the inception and exacerbation of asthma. recent attention has been directed at the role of infections with the atypical bacteria mycoplasma pneumoniae and chlamydia pneumoniae as agents capable of triggering asthma exacerbations and potentially as inciting agents for asthma. this article examines the evidence for interaction between specific infectious agents and exacerbations of asthma, including the immunopathology of infection-triggered asthma, and the current therapeutic options for management. the rise in the incidence of atopic disease, including asthma, over the past several decades has not been limited to a particular geographic area and has occurred in developed and developing countries. several factors influence the development and severity of asthma, including atopy, environmental exposures, genetic predisposition, gene-environment interactions, stress, obesity, diet, socioeconomic status, and infection. the ''hygiene hypothesis'' [1, 2] has focused attention on the role of infection in the development of allergic disease. this hypothesis suggests that infections in early life can have a protective effect on the development of asthma and atopy. other researchers have suggested, however, that infection may be a cause for the onset and persistence of asthma. in this ''hit and run hypothesis,'' a pathogen promotes dysregulation of the immune system, leading to prolonged inflammatory responses even after the pathogen has been cleared [3] . thus, the role of infectious agents in the development of asthma is complex: evidence implicates infections as causal and protective with respect to asthma development. in addition to the potential role of infection in the inception of asthma, infection has been implicated as the most common precipitant of asthma exacerbations. several clinical observations have indicated that most asthma episodes are precipitated by factors other than allergen exposure. many asthma episodes are preceded by upper respiratory tract symptoms and may last several days to weeks, in contrast with allergen-induced asthma exacerbations, where exposure often leads to a rapid onset of symptoms with a recovery time of approximately 24 hours [4, 5] . infections have been linked to asthma exacerbations since the 1950s, and over the past several decades there has been extensive investigation 0889 of infectious agents as they relate to asthma development and exacerbations. in this article, we examine infections as triggers of asthma, with a focus on asthma exacerbations. respiratory tract infections (rtis) are the most common cause of acute illness in adults and children, with upper respiratory infections (uris) constituting the majority of such illnesses [6] . adults typically experience two to four uris per year, and children may have up to 12 uris per year [7] . rtis are the major cause of visits to primary care physicians [8] and are associated with significant work and school absenteeism, with an estimated 150 million lost workdays annually. consequently, rtis have great economic impact, with an estimated cost of $40 billion annually in the united states [9] . numerous viruses produce uris (table 1) , and because the symptom patterns are common between many viruses, it is difficult to determine clinically the specific viral etiology of an acute illness ( table 2) . viral infections commonly trigger asthma exacerbations, having been noted in nearly half of asthma exacerbations in adults [10] and in an even greater percentage of exacerbations in children. this was demonstrated in a 13-month study investigating the role of viral infections in asthma exacerbations in 114 children 9 to 11 years of age with asthma [11] . peak expiratory flow (pef) rate was performed twice daily, and upper and lower respiratory tract symptoms were recorded daily. virologic samples were obtained within 48 hours of an increase in upper or lower respiratory symptoms, a fall in pef by more than 50 l/min from the child's baseline, or if the parent subjectively felt that the child was developing a cold. evidence of a viral infection was detected in 80% to 85% of episodes with respiratory tract symptoms, fall in pef, or both. the highest detection rate occurred during reported episodes of wheeze, cough, and upper respiratory tract symptoms, together with a decline in pef. in addition, the severity of a respiratory illness may influence the outcome of a uri because more severe viral infections seem more likely than mild infections to lead to exacerbations of asthma [5] . viral infection has been noted more often during severe exacerbations of asthma than during milder exacerbations [12] . the advent of more sensitive diagnostic tools to detect specific infectious pathogens, such as detection of microbial dna or rna using the polymerase chain reaction (pcr), has strengthened the evidence for viruses as a primary triggering factor in asthma exacerbations [13] . a recent study confirmed a significant increase in the weighted average viral identification in patients of all ages with asthma exacerbation in studies that used pcr when compared with the pre-pcr studies [14] . the same study suggests that viral recovery occurs more often in asthmatic patients who are having an acute exacerbation than in asymptomatic asthmatics or nonasthmatic individuals. almost 100% of children are infected with rsv by 2 y of age. it is the most common cause of bronchiolitis and pneumonia in infants. varied -includes cough, coryza, fever, irritability, anorexia, wheezing, pharyngitis, vomiting, or diarrhea unknown by 5 y of age nearly 100% individuals have been infected most commonly causes respiratory illnesses such as acute bronchitis, including pharyngitis, and occasionally otitis media, which may be bullous. ten percent of infected individuals develop pneumonia within a few days that may last for 3-4 wk. causes disease only in humans; it is highly transmissible by droplets. epidemics occur every 4-7 y because immunity is not long lasting. the long incubation period (ranging from 1-4 wk) along with long asymptomatic carriage (for weeks to months) facilitates familial spread, which may continue for months. responsible for a variety of respiratory diseases including pneumonia, acute bronchitis, and, less commonly, pharyngitis, laryngitis, otitis media, and sinusitis. many infected patients are asymptomatic or mild to moderately ill. a prolonged illness may be present with cough persisting for 2-6 wk, sometimes with a biphasic course. assumed transmission is person-toperson, via infected respiratory secretions. recurrent infection is common, especially in adults. in tropical, less-developed areas, infection seems to occur earlier in life. in the united states, 50% of adults have antibodies by 20 y of age, with initial infection peaking between 5 and 15 y of age. another important observation that links viral infection with asthma exacerbation is the seasonal pattern of distribution of viral infections and asthma exacerbations, especially severe cases requiring hospitalization. in a 2-year study comparing asthma exacerbations due to seasonal allergens, other environmental triggers, and viral infections, a strong relationship was found between the seasonal incidence of asthma and viral infection, although there was no correlation with pollen and spore counts [15] . similarly, viral infections were the major identifiable risk factor for autumnal asthma exacerbations [16] . in addition to viral infections, rtis with atypical organisms, such as mycoplasma pneumoniae and chlamydia pneumoniae, precipitate a significant proportion of acute episodes of wheezing, contribute to the severity and persistence of asthma, and may serve as the initial insult that leads to development of asthma [17] [18] [19] . human rhinovirus (rv) causes nearly half of all upper respiratory illnesses. although rv infection was initially believed to be limited to the upper airways [20] , lower airway epithelial rv infection has been demonstrated [21] . although infection of the lower respiratory tract may occur, the mechanisms through which viral infections, including rv, provoke asthma are unclear but may include direct extension of upper rtis to the lower respiratory tract. the mechanism may be indirect and involve effects on airway responsiveness independent of the direct epithelial damage and inflammation associated with lower rtis (lrtis). rv infection can enhance the immediate and late-phase responses to allergen [22] , potentially augmenting the allergic inflammation within the airway and precipitating asthma exacerbations. rv infection can lead to profound exacerbation of asthma and is responsible for the majority of hospitalizations for childhood asthma, although less so in adults [20] . rv infections are associated with declines in lung function in asthmatics compared with normal subjects within 2 days after development of a rv infection [23] . rv infection augments airways hyper-responsiveness 4 days after experimental rv infection, an effect that was more pronounced in those with a severe cold [24] . the rise in airways hyper-responsiveness was accompanied by an increase in nasal interleukin (il)-8 in the rv-infected group at days 2 and 9; the increase in nasal il-8 at day 2 correlated significantly with the change in airway responsiveness at day 4. coronavirus is the second most common virus associated with asthma episodes in children and adults. infections due to coronavirus may be associated with less severe lower respiratory tract symptoms than infections with other viruses. this is suggested by the finding that coronavirus-associated asthma episodes in asthmatic school-age children were associated with smaller median declines in pef (56 l/min) compared with episodes triggered by other viruses (85.5 l/min) [11] . in a study of elderly adults, coronavirus was associated with lower respiratory illness in more than 40% of patients, and one quarter of patients consulted a medical practitioner and received antibiotics. more impressive was the observation that coronavirus infection produced a greater disease burden value than influenza or respiratory syncytial virus [25] . influenza virus triggers asthma exacerbations in all age groups [11, 26] . in addition, asthmatic individuals seem to be more susceptible to death associated with influenza infections, as observed in the asian pandemic in 1957 [27] . the time course of influenza-induced asthma exacerbations was examined retrospectively in 20 asthmatic children 8 to 12 years of age with acute respiratory symptoms [28] . fifteen of 20 patients had decreases in fev 1 n20% from baseline during the acute stage, beginning from onset of symptoms in all but one subject, whose fev 1 decreased during the incubation period. fev 1 decreased maximally on the second day of illness by an average of 30%. improvement began on the third day, and fev 1 returned to within 10% of normal between the seventh and tenth day. the rate of adenoviral infection declines with age until 9 years and then increases. the exception to this pattern is infection with serotype 7, whose infection rate increases with age [29] . infection is frequently associated with wheezing, as demonstrated in a retrospective chart review study [30] where wheezing was noted in 58.3% of nonasthmatic children under 2 years of age admitted to an intensive care unit with adenoviral acute lrti. in this study, the mortality rate was 16.7%, generally in the setting of infection with adenoviral serotype 7. adenoviral infection has been demonstrated during acute asthma episodes, but the frequency of adenoviral infection is substantially lower that the frequency for rhinovirus and coronavirus [31] . latent adenoviral infection may have a role in the genesis of asthma. furthermore, adenoviral shedding may be prolonged, lasting up to 906 days. when nasopharyngeal swabs from 50 asymptomatic asthmatic children and 20 healthy control subjects were examined by pcr, adenovirus dna was found in 78.4% of asthmatic children, compared with only 5% of healthy control subjects [32] . adenovirus has been recovered from bronchoalveolar lavage (bal) in children with asthma 12 months or more after acute infection [33] . in this study, bal was performed in 34 children (mean age of 5 years) with unfavorable responses to standard corticosteroid and bronchodilator therapy. adenoviral infectious triggers of asthma antigens were detected in bal fluid (balf) from 94% of subjects. repeat studies within 1 year showed that six of eight subjects were positive for adenovirus on two occasions and that three were positive when sampled three times. cultures of the balf were positive for adenovirus in all cultures performed, indicating that the virus was capable of replication. similar studies performed in control patients without persistent asthma failed to detect evidence of adenovirus. respiratory syncytial virus (rsv) infects almost 100% of children by 2 years of age and is the most common cause of bronchiolitis and pneumonia in infants [34] . in addition to causing acute lrti, rsv serves as a trigger for exacerbations of asthma and other chronic lung diseases. infants who experience severe rsv bronchiolitis seem to have increased frequencies of wheeze and asthma later in life. a comparison of several retrospective studies of children admitted for bronchiolitis found that the postbronchiolitis group had a significantly higher frequency of bronchial obstructive symptoms 2 to 10 years later and, when pulmonary function studies were performed, diminished fev 1 or increased bronchial reactivity compared with healthy control subjects [35] . these findings were confirmed in a prospective study when children hospitalized with confirmed rsv bronchiolitis were evaluated at 7.5 years of age and compared with age-and gender-matched control subjects [36] . by 7.5 years of age, the cumulative prevalence of asthma was 30% in the rsv group versus 3% in the control group, and current asthma was present in 23% of the rsv group versus 2% of the control group. however, the duration of the effect of rsv infection on asthma-related symptoms appears to be limited. in a prospective study of 1246 children enrolled at birth, 207 developed an rsv ltri not requiring hospitalization during the first 3 years of life [37] . when compared with a control group of children with no lrti documented during the first 3 years of life, the group with mild rsv lrti had a substantially increased risk of frequent wheeze at 6 years of age (odds ratio [or] 4.3), and the risk for frequent wheeze remained significantly increased at 11 years of age (or 2.4), at which time prebronchodilator fev 1 , but not postbronchodilator fev 1 , was significantly lower in the rsv group. by age 13 years, there were no significant between-group differences in terms of increased risk for frequent or infrequent wheezing. these studies demonstrate that rsv bronchiolitis is a significant independent risk factor for subsequent frequent wheezing, although this effect seems to decrease with age and may be dependent upon the severity of the rsv infection. similar to adenoviral infection, the persistence of rsv may underlie in part the sequelae of severe rsv disease. infection may lead to alteration in the patterns of local interferon, chemokine, and cytokine production [38] , potentially leading to chronic inflammation [39] . furthermore, the age at first viral infection may direct the pattern of disease later in life by generating a th2-biased memory response to rsv, which may direct responses to other antigens in the lung toward an allergic phenotype. this is suggested by a study in which mice infected with rsv at different ages (1, 7, 28, or 56 days) demonstrated stronger th2 responses in the group primed at the youngest age when reinfected with rsv at 12 weeks of age [40] . the parainfluenza viruses (piv) cause a spectrum of respiratory illness similar to that caused by rsv but result in fewer hospitalizations [41, 42] . most illnesses are limited to the upper respiratory tract [41] , although approximately 15% involve the lower respiratory tract, and 2.8 of every 1000 children with such infections required hospitalization [42] . although less common than rv or coronavirus infection, piv was detected in 14% of episodes of increased symptoms or decreased pef in school-aged children [11] . more frequent and severe wheezing has been correlated with elevated levels of ige antibody to rsv and piv in nasal secretions of children with bronchiolitis due to rsv and piv [43] . human metapneumovirus (hmpv) was identified in 2001 in respiratory samples from children with respiratory disease in the netherlands [44] . the clinical symptoms experienced by infected individuals are diverse and may consist of upper or lower respiratory tract symptoms ranging from otitis media to bronchiolitis, croup, pneumonia, and possibly exacerbations of asthma [45] . hmpv is responsible worldwide for community-acquired acute rtis affecting children and other age groups, with a mean age of illness of 11.6 months and a male predominance (male/female ratio 1.8:1). the broad epidemic seasonality and the evidence of genetic variability suggest that there may be more than one serotype of hmpv [44] . wheezing is part of the clinical symptomatology associated with hmpv infection. more than half of otherwise healthy children presenting with acute respiratory illness and evidence of hmpv infection experienced wheezing in one study [45] . in series of 19 children with evidence of hmpv infection, bronchiolitis was the most common diagnosis, and 50% of patients had wheezing [46] . both of these studies evaluated specimens collected from previously healthy children during an acute respiratory illness during which no other pathogen was identified and detected evidence of hmpv in 6.4% [46] and 20% [45] of the previously negative samples. although hmpv infection is often accompanied by wheezing, there have been conflicting reports linking hmpv infections and asthma exacerbations [47, 48] . nevertheless, bronchiolitis is a common cause for hospitalization, and given the increasing hospitalization rates over the past two decades [49] , it is possible that hmpv may be responsible for a portion of hospitalizations in children with infectious triggers of asthma 53 bronchiolitis and wheezing unrelated to rsv infection [46] . furthermore, coinfection with rsv and hmpv may augment the severity of bronchiolitis [47] . m pneumoniae and c pneumoniae initial evidence suggested that infection with m pneumoniae and c pneumoniae was associated with asthma chronicity. several case reports suggest associations between infections with atypical organisms with decreased expiratory flow rates and increased airway hyper-responsiveness in nonasthmatic individuals [50] and the onset of asthma symptoms in previously healthy nonasthmatic adults [51, 52] . most of these individuals present with complaints of malaise, shortness of breath of gradual onset, and wheezing, which typically resolve after treatment with macrolide antibiotics or oral corticosteroids [51] . symptoms may progress and persist, as illustrated by an adult male with fever, severe cough, shortness of breath, consolidation on chest radiograph, and evidence of m pneumoniae infection based on a rise in serum antibody titers who subsequently developed wheezing episodes with reversible airway obstruction and airway reactivity to methacholine [52] . infections with these organisms can persist for months, and animal studies show that m pneumoniae can be detected by pcr for up to 200 days after infection, even though the animals become antibody and culture negative by 70 days [53] . these reports suggest that m pneumoniae may serve as a cause of acute wheezing and a triggering factor for the onset of asthma. the most comprehensive evaluation of the role of m pneumoniae and c pneumoniae infections in patients with chronic asthma evaluated 55 adult patients with chronic asthma and 11 control subjects by using pcr, culture, and serology to detect m pneumoniae species, c pneumoniae species, and viruses from the nasopharynx, lung, and blood [54] . fifty-six percent of the asthmatic patients had positive pcr studies for m pneumoniae (n = 25) or c pneumoniae (n = 7), which were mainly found in balf or biopsy samples. only 1 of 11 control subjects had a positive pcr finding for m pneumoniae. cultures for these organisms were negative in all patients. a distinguishing feature between pcr-positive and pcr-negative patients was a significantly greater number of tissue mast cells in the group of patients who were pcr positive. of additional significance is the link of atypical infectious organisms with asthma exacerbations. in a serologically based prospective study, 100 adult patients hospitalized with exacerbations of asthma were compared with hospitalized surgical patients with no history of lung disease at any time or uri in the month before admission [55] . in this series, m pneumoniae was identified more often than any other pathogen in the asthmatic group (18 m pneumoniae, eight c pneumoniae, 11 influenza a, five influenza b, three piv-1, two piv-2, one piv-3, six adenovirus, two rsv, three s. pneumoniae, and five legionella spp.) and in the control group (three m pneumoniae). however, only 8 of the 18 patients had m pneumoniae identified as the sole infectious agent, making it difficult to ascertain the culpability of m pneumoniae as the cause of hospitalization. a study of 71 children with acute wheezing and 80 age-matched healthy children detected m pneumoniae in 22.5% and c pneumoniae in 15.5% of children with wheezing compared with 7.5% and 2.5%, respectively, in healthy control subjects [56] . when the children who were infected with either organism were treated with clarithromycin, improvement in the course of the disease was observed, further supporting the role of these atypical organisms in the exacerbation of asthma. these findings were recently confirmed in a french series, where m pneumoniae infection was found in 20% and c pneumoniae infection was found in 3.4% of children during an acute asthma exacerbation [19] . acute m pneumoniae infection was confirmed in 50% and c pneumoniae in 8.3% of patients experiencing their first wheezing episode. further studies are needed to confirm the association between infection and asthma exacerbation, to determine the prevalence of such infections in patients with acute exacerbations of asthma, and to examine if infection with these organisms modifies the severity of the exacerbation or the response to therapy. viral-induced wheeze (viw) is characterized by brief episodes of lower respiratory symptoms and decreased pulmonary function in the setting of an acute viral uri, interspersed with longer asymptomatic periods with normal pulmonary function [11, 57] . this differs from classic childhood asthma, which is characterized by chronic symptoms, with atopy being a major risk factor [58] . classic asthma and viw were considered two different entities until 1969, when a report suggested that the two groups have similar characteristics [59] and benefited similarly from the same prophylactic treatment [60] . in the 1990s, there was a division of the wheezing phenotypes, especially in children [58] . patients with viw alone seem to outgrow the symptoms by age 6; however, in some patients, the pattern of viw may continue into adulthood with less severe symptoms, negative methacholine challenges, and pulmonary functions that remain normal [61] . the inability to reliably differentiate between viw and asthma, especially in young children, complicates the evaluation of the influence of viral infections on exacerbations of wheezing. furthermore, this heterogeneity in wheezing phenotypes has implications in terms of the efficacy of therapies used to treat such episodes. viruses typically enter the body through contact with mucosal surfaces. the cell-specific distribution of viral receptors determines the viral tropism. once the viral particles are internalized, nucleic acids are released, and transcription and production of viral proteins starts. the viral genome is replicated, and virions are one of the earliest responses to viral infection is the production of ifns by different cell types; ifn-a is produced by leukocytes, ifn-b is produced by fibroblasts, and inf-g is produced by th1 cells and natural killer (nk) cells. ifns induce transcription of many genes, including two with direct antiviral activity, and lead to increased expression of mhc class i and ii genes. interferons are potent activators of antiviral effector cells such as nk cells, cd8 t lymphocytes, and macrophages. although the inflammatory process generated by virus infection is generally viewed as a th1 pattern with a predominance of interferons, especially inf-g, in atopy there is a predominance of the th2 cytokine profile. however, viral infections promote increased cytokine-mediated inflammation through direct induction of specific cytokines produced by different viral agents [62] . the ability of certain pathogens to stimulate the production of th2 cytokines [63] may explain why certain pathogens are more strongly associated with asthma exacerbation than others. viruses have been implicated in the inception of asthma because viral infections with a propensity for lower airway involvement during infancy have been associated with chronic lower respiratory tract symptoms and asthma [64] . this seems to be particularly relevant to rsv bronchiolitis, which has been demonstrated to be a significant independent risk factor for subsequent frequent wheezing [37] . the sequelae of severe rsv disease could be explained in part by viral persistence [39] . this has been supported by a recent study demonstrating the persistence of viral genomic and messenger rna in lung homogenates of balb/c mice up to 100 days post rsv infection, whereas virus could no longer be detected in balf after day 14 post-infection [65] . another possible mechanism by which a virus could promote asthma is by generating changes in patterns of pro-inflammatory cytokine production, which could facilitate virus persistence, as demonstrated with rsv [38] . viral infection may exert direct effects on airway cells. an increase in the production of il-10 by nonspecifically stimulated peripheral blood mononuclear cells during acute and convalescent phases of rsv infection requiring hospitalization has been demonstrated [66] . in animal studies, it was suggested that il-10 may have a direct effect in airway smooth muscle and in the regulation of airway tone [67] . although there is evidence supporting the role of viral infections in the development of asthma, further investigation is necessary to confirm this hypothesis because the mechanisms that could allow persistency or latency of viral infection are poorly understood. it has been hypothesized that asthmatic individuals have increased susceptibility to viral infections. some researchers have found an increased incidence of viral infections in asthmatic children when compared with nonasthmatics [14, 26] , a pattern that could be explained by the increased expression of icam-1, the receptor for rv, in asthmatics subjects [68] . however, this finding was not confirmed in a study that followed cohabitating couples consisting of an atopic asthmatic and a healthy nonatopic, nonasthmatic individual [23] . in this study, subjects completed daily diary cards of upper and lower respiratory tract symptoms and measured pef twice daily. nasal aspirates were taken and examined for rhinovirus every 2 weeks. rhinovirus was detected in 10.1% of samples from the asthmatics and 8.5% of samples from the nonasthmatic participants. after adjustment for confounding factors, asthma did not significantly increase the risk of infection with rhinovirus in asthmatic individuals (or 1.15). the effect of atopic status on the rate of viral infection is unclear; evidence exists suggesting no difference between the rate of viral infection between atopic and nonatopic individuals [69] or an even lower rate of viral infections among atopic individuals [15, 70] , although these studies did not have adequate statistical power to confirm this trend. there is an increased risk of acute wheezing when atopy is combined with viral infection when compared with atopy or virus infection alone [70] , and infants with a family history of atopy seem more likely to develop bronchiolitis with a higher rate of hospitalization [71] . even if asthmatics do not experience more frequent infections than nonasthmatics, it is possible that asthmatics have a higher incidence of symptoms when experiencing viral infections. during rhinoviral infection, there is a greater incidence of symptoms in asthmatics compared with nonasthmatics [72] . this is further suggested by a report that asthmatics experienced seroconversion to influenza a virus at the time of asthma exacerbation even in the absence of signs of respiratory infection [5] . although there is evidence supporting the role of infection in the genesis of asthma and allergy, a protective effect of infections in the development of atopy has also been postulated. an inverse relationship between infection and allergy was first noted when a study comparing white families with native americans reported that ige levels and the prevalences of asthma and eczema were higher in the white population, whereas helminthic, viral, and bacterial infections were more prevalent in the native americans [1] . it was observed that increased family size, often associated with more frequent infections in early childhood, had an inverse relationship with the prevalence of allergic rhinitis [2] and asthma [73] . this was further supported by studies reporting an inverse relationship between the age of day care entry and the diagnosis of asthma [74, 75] . one potential explanation for this pattern is that at birth there is a predominant th2 response, and, as exposure to infections occurs, there is a gradual shift toward a th1dominant response. however, if the skewing of the immune response to th1, which regulates response to viral infection, is impaired, a th2 response would infectious triggers of asthma predominate, favoring the development of allergy. ex vivo studies have shown that asthmatics exposed to viral infections lack the capacity to mount a strong th1 response [76, 77] . there is no clinically effective treatment for the common cold. as the mechanisms of viral-induced wheezing and asthma are elucidated, new forms of treatment may emerge. the involvement of many inflammatory pathways suggests that antiviral and anti-inflammatory therapies have potential roles for intervention after onset of symptoms; however, a combination of both therapeutic approaches may have the greatest impact. prophylaxis for the acquisition of viral infections, in the form of vaccination or pharmacologic therapy, offers the best hope of disease control. the major obstacle for treatment is the wide variety of organisms associated with uris, including viral and bacterial agents ( table 2 ). in addition, accurate and timely diagnosis is essential for the appropriate targeting of specific antiinfective therapies. the rapid rate of mutation of viruses leads to the emergence of resistant strains. in addition, there are difficulties with the delivery, expense, and efficacy of drugs [78] . treatment for viral rtis remains symptomatic, although future approaches will likely be directed toward reducing the inflammatory response elicited by the virus. vaccination remains the mainstay of prophylaxis against infections. however, with the exception of influenza, vaccine development for respiratory viruses has been slow and disappointing. influenza vaccine contains three strains (two a and one b) of inactivated virus, one or two of which are modified yearly based upon predictions of the upcoming viral strains. they are produced in embryonated hen eggs and are highly immunogenic, conferring protection in 70% to 80% of the vaccine recipients with minimal adverse effects. whole-cell influenza vaccine is no longer available, and the current vaccines consist of subvirion (prepared by disrupting the lipid membrane) or purified surface antigen. recently, a liveattenuated, cold-adapted, trivalent, intranasal influenza vaccine (flumist) has been introduced, but it is contraindicated in asthmatics [79] . a long-standing concern that influenza vaccination may trigger exacerbations of asthma was addressed in a multicenter, randomized, double-blind, placebocontrolled, crossover trial in 2032 patients with asthma (age range 3-64 years). this study confirmed the safety of the influenza vaccine in asthmatics by demonstrating that the frequency of exacerbations of asthma was similar in the 2 weeks after vaccination with the active influenza vaccine or placebo (28.8% and 27.7%, respectively) [80] . although yearly influenza vaccination is recommended as a routine element of asthma management [81] , a recent study generated concern about the usefulness of influenza vaccine in preventing influenza-related asthma exacerbations. this randomized, double-blind, placebo-controlled trial showed that the number, se-verity, and duration of influenza-related asthma exacerbation was similar between the group receiving influenza vaccination and the group receiving placebo over the course of one influenza virus season [82] . vaccinated children tended to have shorter exacerbations (by approximately 3 days) than nonvaccinated children. antiviral therapy targets the source of infection directly, decreasing the number of infectious agents and therefore reducing inflammatory process. the only licensed antiviral therapies are directed against influenza a (amantadine and rimantadine), influenza a and b (zanamivir and oseltamivir), and rsv (ribavirin). the neuraminidase inhibitors, zanamivir and oseltamivir, have an advantage over adamantanes, amantadine, and rimantadine because they have a broader spectrum and are effective against the a and b strains of influenza virus. the inhibition of neuraminidase, whose active site consists of 11 amino acids conserved in all naturally occurring influenza virus [83] , prevent cleavage of sialic acid from newly acquired membrane, leaving emerging virus inactive and thereby decreasing infectivity [84] . both neuraminidase inhibitors improve respiratory outcomes in patients with asthma and acute influenza infections [78] and have the added benefit of being effective in the prophylaxis against influenza infections [85] . although it is generally well tolerated, there are case reports of bronchospasm after treatment with inhaled zanamivir [86] ; however, it is difficult to separate these symptoms from the effects of the influenza infection. the disadvantage of current antiviral therapy is the specificity for influenza and the need for initiation of treatment within 48 hours of onset of infection. the toxicity profile of ribavirin, approved for use in severe rsv infections, limits its clinical use except in settings of severe illness in immunocompromised hosts. antibiotic use is appropriate if there is evidence of bacterial infection contributing to asthma exacerbations, although pyogenic lung infections rarely exacerbate asthma and are rarely associated with wheezing. although some macrolide antibiotics have been reported to have antiviral effects in vitro against rhinoviruses [87] , these effects have not been confirmed in vivo, and a recent cochrane review does not support the use of antibiotics for the treatment of the common cold [88] . the anti-inflammatory effects of macrolide antibiotics are not limited to their ability to interfere with corticosteroid metabolism [89] , as evidenced by inhibition of the neutrophil oxidative burst [90] , reduction of cytokine formation [91] , and reduction of icam-1 production [92] . asthmatic patients infected with m pneumoniae or c pneumoniae may benefit from prolonged treatment with clarithromycin, as evidenced by significant improvement in fev 1 [18, 93] . furthermore, in a double-blind, randomized, crossover study, 17 patients with stable mild or moderate asthma not evaluated for m pneumoniae or c pneumoniae received 200 mg of clarithromycin or placebo twice daily for 8 weeks. methacholine responsiveness improved in all the patients after 8 weeks of clarithromycin treatment [94] . improvement in airway hyperresponsiveness after 8 weeks of clarithromycin treatment was confirmed in a group of patients with asthma receiving concomitant therapy with inhaled corticosteroids who were not selected on the basis of infection with m pneumoniae or c pneumoniae [95] . it remains unclear as to the mechanism by which macro-infectious triggers of asthma lide antibiotics improve airway hyper-responsiveness in patients with asthma, but possibilities may include treatment of occult or chronic infection, interference with steroid metabolism, or the anti-inflammatory properties of this class of antimicrobials. although there are international guidelines for the management of asthma [81, 96] , there is a relative paucity of evidence regarding therapeutic strategies specifically for viw in asthmatics or healthy subjects. because most acute exacerbations of asthma are induced by viral infections and because many forms of asthma therapy, especially inhaled corticosteroids, reduce the frequency and severity of exacerbations, one would presume that the current treatment for chronic asthma would be efficacious in preventing viw. however, the varying phenotypes of wheezing, especially in childhood, seem to respond differently to such management approaches. this is particularly true for rsv-associated wheezing, which does not consistently respond to medications often used to treat asthma exacerbations, including bronchodilators and corticosteroids [97] . thus, despite the efficacy of inhaled corticosteroids in the control of asthma and reduction of exacerbations, patients continue to experience exacerbations, particularly in the setting of viral rtis. several treatment approaches have been investigated in an attempt to reduce the morbidity associated with wheezing associated with rtis. brunette et al [98] examined the effect of a short course of oral corticosteroid administered in an unblinded manner at onset of uri symptoms in a group of children with histories of recurrent wheezing in the setting of viral infections. over a 1-year period, the group receiving oral corticosteroids at the early signs of rtis experienced reductions in the frequencies of wheezing, emergency room visits, and hospitalizations. however, a recent double-blind, placebo-controlled trial evaluating the use of parent-initiated oral corticosteroids at the early signs of an episode of presumed viral-induced wheezing did not detect a difference between oral corticosteroid therapy and placebo in terms of symptom scores and rate of hospitalization [99] . thus, the role for the use of oral corticosteroids at the early signs of illness in children with recurrent viral wheezing is unclear, and additional investigation is required to determine the efficacy of this approach in the management and attenuation of wheezing episodes. the repeated use of systemic corticosteroids for such episodes remains a clinical concern. given the efficacy of inhaled corticosteroids (ics) in the daily management of asthma and their favorable safety profile when compared with systemic corticosteroids, the use of ics in the management of viw has been explored. although ics are effective in the management of persistent asthma, current evidence suggests a lack of efficacy in the regular use of ics in patients with mild viw [100, 101] . a recent meta-analysis concluded that the use of ics episodically for viral-triggered wheezing in children not using them as maintenance may decrease the rate of oral corticosteroid requirement [101] . in patients receiving daily ics therapy, the common clinical practice of doubling the dose of ics at the onset of an asthma exacerbation has been shown to be ineffective in preventing symptom progression [102] . however, a recent study in adults demonstrated the valuable effects of quadrupling the ics dose with acute asthma exacerbations [103] . these data suggest that corticosteroids, taken orally or inhaled, may be used as treatment and preventive therapy for asthma exacerbations in the setting of rtis. the cysteinyl leukotrienes (cyslts) have been identified as important mediators in the complex pathophysiology of asthma. cyslts are detectable in the blood, urine, nasal secretions, sputum, and balf of patients with chronic asthma. elevated cyslts have been detected in respiratory secretion of children with viral induced wheezing [104] . similar to elevated levels in asthmatics, 20 infants with prolonged or persistent wheeze (mean 14.9 months) and a history of viral illness at wheeze onset had significant elevations of leukotrienes in bal despite the fact that 12 of 20 infants were receiving daily ics therapy ( 450 mg/d) [105] . these findings suggest that, similar to asthma pathophysiology, cyslts play a role in the pathophysiology of viral-induced wheeze. additionally, based on the above study, the cyslts are not fully suppressed by the preferred standard antiinflammatory therapy, inhaled corticosteroids. thus, antagonism of the effects of cyslt using the leukotriene receptor antagonists may provide clinical benefit to patients with viw. the relative efficacies of these intervention strategies aimed at reduction of wheezing and asthma in the setting of rtis depend upon the wheezing phenotype and probably the timing of the initiation of therapy. investigation of other therapeutic approaches to viw is ongoing and may provide insight as to the optimal treatment approach for this challenging condition. infections have been implicated in asthma exacerbations and in the inception of asthma. several studies support the concept that viruses and atypical infectious agents may induce asthma exacerbations and contribute to the chronicity of asthma. the further elucidation of the mechanisms that underlie the interaction between infectious agents and asthma will lead to improvements in treatment and prevention of such exacerbations. studies are needed to explore the vast domain of infectious triggered asthma exacerbations. serum ige levels in white and metis communities in saskatchewan hay fever, hygiene, and household size viral induction of a chronic asthma phenotype and genetic segregation from the acute response recurrent wheezy bronchitis and viral respiratory infections viral respiratory infections in asthamtic children staying in a mountain resort microbiology and epidemiology of upper respiratory tract infections acute respiratory illness in the community: frequency of illness and the agents involved repeat consultations after antibiotic prescribing for respiratory infection: a study in one general practice the economic burden of non-influenza-related viral respiratory tract infection in the united states risk factors for lower respiratory complications of rhinovirus infections in elderly people living in the community: prospective cohort study community study of role of viral infections in exacerbations of asthma in 9-11 year old children viral respiratory tract infections and exacerbations of asthma in adult patients viruses as precipitants of asthma symptoms. i: epidemiology epidemiology and diagnosis of virus-induced asthma respiratory virus infections and aeroallergens in acute bronchial asthma respiratory infections and the autumn increase in asthma morbidity chronic chlamydia pneumoniae infection and asthma exacerbations in children detection of mycoplasma pneumoniae in the airways of adults with chronic asthma mycoplasma pneumoniae and asthma in children viral and other infections of the human respiratory tract. london7 chapman & hall rhinoviruses infect the lower airways rhinovirus upper respiratory infection increases airway hyperreactivity and late asthmatic reactions frequency, severity, and duration of rhinovirus infections in asthmatic and non-asthmatic individuals: a longitudinal cohort study effect of experimental rhinovirus 16 colds on airway hyperresponsiveness to histamine and interleukin-8 in nasal lavage in asthmatic subjects in vivo acute viral infections of upper respiratory tract in the elderly people living in the community: comparative, prospective, population based study of disease burden viruses as precipitants of asthmatic attacks in children asiatic influenza in allergic patients with bronchial asthma the effects of influenza virus infection on fev1 in asthmatic children: the time-course study the seattle virus watch. viii: observations of adenovirus infections adenovirus type 7 associated with severe and fatal acute lower respiratory infections in argentine children epidemiology of respiratory viruses in patients hospitalized with near-fatal asthma, acute exacerbations of asthma, or chronic obstructive pulmonary disease persistence of viruses in upper respiratory tract of children with asthma persistent adenoviral infection and chronic airway obstruction in children epidemiology of respiratory syncytial virus infection among infants and children in chicago epidemiologic and clinical evidence of a respiratory syncytial virus-reactive airway disease link respiratory syncytial virus bronchiolitis in infancy is an important risk factor for asthma and allergy at age 7 respiratory syncytial virus in early life and risk of wheeze and allergy by age 13 years persistence of respiratory syncytial virus in macrophages alters phagocytosis and pro-inflammatory cytokine production medical consequences of persistent viral infection age at first viral infection determines the pattern of t cell-mediated disease during reinfection in adulthood parainfluenza viral infections in pediatric outpatients: seasonal patterns and clinical characteristics epidemiology and clinical impact of parainfluenza virus infections in otherwise healthy infants and young children b 5 years old immunologic mechanisms of virus-induced wheezing and asthma a newly discovered human pneumovirus isolated from young children with respiratory tract disease human metapneumovirus and lower respiratory tract disease in otherwise healthy infants and children human metapneumovirus infection in the united states: clinical manifestations associated with a newly emerging respiratory infection in children human metapneumovirus in severe respiratory syncytial virus bronchiolitis astma exacerbations in children associated with rhinovirus but not with human metapneumovirus infections bronchiolitis-associated hospitalizations among us children, 1980-1996 mycoplasma pneumoniae: acute illness, antibiotics, and subsequent pulmonary function association of mycoplasma pneumoniae antigen with initial onset of bronchial asthma experience with newer techniques for the laboratory detection of mycoplasma pneumoniae infection: adelaide, 1978-1992 a link between chronic asthma and chronic infection atypical pathogen infection in adults with acute exacerbation of bronchial asthma importance of acute mycoplasma pneumoniae and chlamydia pneumoniae infections in children with wheezing the effect of inhaled corticosteroid on episodes of viral associated wheezing in school age children asthma and wheezing in the first six years of life prevalence, natural history and relationship of wheezy bronchitis and asthma in children: an epidemiologic study underdiagnosis and treatment of asthma in childhood outcome of wheeze in childhood: symptoms and pulmonary function 25 years later respiratory infections in allergy and asthma. new york7 marcel dekker predominant type-2 response in infants with respiratory syncytial virus (rsv) infection demonstrated by cytokine flow cytometry association of radiologically ascertained pneumonia before age 3 yr with asthma-like symptoms and pulmonary function during childhood: a prospective study latency and persistence of respiratory syncytial virus despite t cell immunity monocyte il-10 production during respiratory syncytial virus bronchiolitis is associated with recurrent wheezing in a one-year follow-up study the failure of interleukin-10-deficient mice to develop airway hyperresponsiveness is overcome by respiratory syncytial virus infection in allergen-sensitized/challenged mice association of rhinovirus infection with asthma association of viral and mycoplasma infections with exacerbations of asthma rhinovirus and respiratory syncytial virus in wheezing children requiring emergency care. ige and eosinophil analyses family history of atopy and clinical course of rsv infection in ambulatory and hospitalized infants role of viral infection and host factors in acute episodes of asthma and chronic bronchiolitis family size, atopic disorders in parents, asthma in children, and ethnicity siblings, day care attendance and the risk of asthma and wheezing during childhood age of entry to day nursery and allergy in later childhood a defective type 1 response to rhinovirus in atopic asthma relationship of upper and lower airway cytokines to outcome of experimental rhinovirus infection treatment of common colds recommendations for influenza immunization of children american lung associations asthma clinical research centers. the safety of inactivated influenza vaccine in adults and children with asthma expert panel report ii: guidelines for the diagnosis and management of asthma. bethesda (md)7 us department of health and human services influenza vaccination in children with asthma: randomized double-blind placebo-controlled trial comparison of efficacies of rwj-270201, zanamivir, and oseltamivir against h5n1, h9n2, and other avian influenza viruses the molecular biology of influenza virus pathogenicity inhaled zanamivir for the prevention of influenza in families. zanamivir family study group respiratory distress associated with zanamavir erythromycin inhibits rhinovirus infection in cultured human tracheal epithelial cells antibiotics for the common cold roxithromycin reduces the degree of bronchial hyperresponsiveness in children with asthma erythromycin and roxithromycin potentiate human neutrophil locomotion in vitro by inhibition of leukoattractant activated superoxide generation and autooxidation erythromycin supresses interleukin 6 expression by human bronchial epithelial cells: a potential mechanism of its anti-inflammatory action effect of erythromycin on hemophilus influenzae endotoxin-induced release of il-6, il-8, and sicam-1 by cultured human bronchial epithelial cells mycoplasma pneumoniae and chlamydia pneumoniae in asthma: effect of clarithromycin clarithromycin suppresses bronchial hyperresponsiveness associated with eosinophilic inflammation in patients with asthma clarithromycin reduces the severity of bronchial hyperresponsiveness in patients with asthma global initiative for asthma: global strategy for asthma management and prevention. bethesda (md)7 national institutes of health the role of corticosteroids in bronchiolitis and croup childhood asthma: prevention of attacks with short-term corticosteroid treatment of upper respiratory tract infection efficacy of a short course of parent-initiated oral prednisolone for viral wheeze in children aged 1-5 years: randomised controlled trial effect of continuous treatment with topical corticosteroid on episodic viral wheeze in preschool children inhaled steroid for episodic viral wheeze of childhood treatment of acute asthmatic exacerbations with an increased dose of inhaled steroid low-dose budesonide with the addition of an increased dose during exacerbations is effective in long-term asthma control increased production of ifn-gamma and cysteinyl leukotrienes in virus-induced wheezing persistent wheezing in very young children is associated with lower respiratory inflammation key: cord-000602-z5p3a64x authors: bhat, niranjan; o'brien, katherine l.; karron, ruth a.; driscoll, amanda j.; murdoch, david r. title: use and evaluation of molecular diagnostics for pneumonia etiology studies date: 2012-03-08 journal: clinical infectious diseases doi: 10.1093/cid/cir1060 sha: doc_id: 602 cord_uid: z5p3a64x comprehensive microbiological testing will be a core function of the pneumonia etiology research for child health (perch) project. the development stage of perch provided the time and resources necessary for us to conduct a comprehensive review of the current state of respiratory diagnostics. these efforts allowed us to articulate the unique requirements of perch, establish that molecular methods would be central to our testing strategy, and focus on a short list of candidate platforms. this process also highlighted critical challenges in the general design and interpretation of diagnostic evaluation studies, particularly in the field of respiratory infections. although our final molecular diagnostic platform was ultimately selected on the basis of operational and strategic considerations determined by the specific context of perch, our review highlighted several conceptual and practical challenges in respiratory diagnostics that have broader relevance for the performance and interpretation of pneumonia research studies. comprehensive microbiological testing will be a core function of the pneumonia etiology research for child health (perch) project. the development stage of perch provided the time and resources necessary for us to conduct a comprehensive review of the current state of respiratory diagnostics. these efforts allowed us to articulate the unique requirements of perch, establish that molecular methods would be central to our testing strategy, and focus on a short list of candidate platforms. this process also highlighted critical challenges in the general design and interpretation of diagnostic evaluation studies, particularly in the field of respiratory infections. although our final molecular diagnostic platform was ultimately selected on the basis of operational and strategic considerations determined by the specific context of perch, our review highlighted several conceptual and practical challenges in respiratory diagnostics that have broader relevance for the performance and interpretation of pneumonia research studies. the development of a comprehensive microbiological testing strategy has been a core principle in the conception and design of the pneumonia etiology research for child health (perch) project [1] . in formulating the most effective approach for respiratory diagnosis, we determined that a multiplex molecular diagnostic platform would be an essential component in our approach. many of the technical and operational considerations encountered through this process proved relevant to the overall design of the project. we describe here the theoretical and practical challenges encountered in the evaluation and selection of a molecular platform for the diagnosis of pneumonia. as described elsewhere in this issue [2, 3] , microbiological evidence of infection must be considered in the context of several fundamental difficulties found in respiratory diagnostics, including the frequent lack of access to the site of infection, the insensitivity of available tests, insufficient assay validation, and complexities in determining whether a detected pathogen has a causal role in the illness. the specific research-related demands of perch added to these constraints, requiring that our diagnostic strategy must exclude any prior assumptions regarding the likely importance of specific pathogens; must include a full range of respiratory tract specimens, including upper respiratory swab or aspirate, induced sputum, lung aspirate, bronchoalveolar lavage, and pleural fluid; must be comprehensive, yet realistic; must appropriately balance the demands of accuracy and efficiency; must account for both clinical and research ethical issues; and must be feasible for use and support at all participating field sites. to begin the selection process, the perch investigators conducted an extensive review of the microbiologic diagnosis of respiratory infections. using published and unpublished data, as well as user and developer experiences, our team prepared a strategic summary of the available technologies that could detect pathogens from respiratory tract specimens. we evaluated each major assay category, including traditional bacteriology and viral culture, direct antigen and immunofluorescent antibody detection, and nucleic detection acid tests. it was evident that molecular diagnostics should be among the mix of diagnostic tools required to meet the needs of perch. nucleic acid detection tests (nadts) have a number of advantages over other diagnostic platforms for the evaluation of respiratory specimens [4] . they demonstrate superior sensitivity in detecting organisms that are fastidious, less viable, or present in only small amounts [5] . molecular diagnostics can also be quickly adapted to detect evolving or emerging pathogens and are amenable to efficiencies of scale such as automation. they also allow the simultaneous detection of multiple targets (multiplexing), which in turn allows for testing by clinical syndrome and the detection of co-infections. nadt methods present less of a safety hazard for laboratory personnel compared with culture, typically require less time compared with bacterial culture, and require less technical capacity compared with viral culture. given these advantages, nadts have been extensively evaluated in the detection of several viruses and bacteria of the respiratory tract and have become the diagnostic tool of choice for many agents that are difficult to isolate [4] . molecular diagnostic platforms are not without their disadvantages. cost and complexity remain significant barriers to adoption in many laboratories, and nadts often risk problems of laboratory contamination with amplified products, particularly if the assay procedure requires opening of the reaction tube prior to the target detection step [6] . measures to limit contamination often require additional laboratory space that may not be available in resource-constrained settings. nevertheless, nadt methods represent one of the more productive areas of diagnostics research, promising future improvements in automation and speed, smaller devices, improved cost efficiencies, and better detection of emerging pathogens [7] . the focus on molecular methods for respiratory pathogen detection yielded a large variety of potential technologies for consideration in perch (table 1) . polymerase chain reaction (pcr) technology is more common at research sites worldwide, can be adapted to various platforms, and easily allows for multiplex amplification. multiplexing, in which several targets are assayed for simultaneously, is commonly employed in pcr-based assays and offers significant advantages over single-pathogen assays in terms of efficiency and pathogen coverage. still, developers must overcome considerable complexities in harmonizing the reaction requirements of each individual target and limiting potential competition among the analytes. these factors may result in a measurable decrease in sensitivity compared with single-plex assays. several techniques have been developed to address such factors, such as alterations in cycling protocols [8] , nested primer combinations [9, 10] , complex primer structures and concentrations [11, 12] , and the use of nontraditional nucleotides [13] [14] [15] . other nadt technologies, such as nucleic acid sequence-based amplification and loop-mediated isothermal amplification, have been used for the detection of respiratory pathogens, but experience with multiplexing is limited [16] [17] [18] [19] . technologies for target detection take on an even larger variety of formats. older methods include agarose gel electrophoresis, reverse-transcription pcr enzyme hybridization assay [20] [21] [22] and enzyme-linked oligonucleotide capture [18] . more recently, solid-and liquid-phase array platforms have become more useful for the detection of multiple targets. solid-phase arrays use a variety of formats, typically embedding target-specific oligonucleotides onto a glass or silicon microchip [23] [24] [25] [26] [27] [28] [29] [30] [31] to detect anywhere between dozens to hundreds of thousands of amplified sequences. several respiratory diagnostic systems have been based on a liquid-phase technology using polystyrene microbeads (luminex) [9, 10, [13] [14] [15] 32] or mass spectroscopy [33, 34] for amplicon discrimination. although these approaches have greatly expanded the versatility and sensitivity of multiplex pcr, their complexity, specialized equipment, and high start-up costs have limited their widespread adoption to date. moreover, these platforms typically require separate steps for amplification and detection, increasing both the workload and the risk of operator error or amplicon contamination. real-time pcr assays address these issues by combining amplification and detection in one reaction tube, thus facilitating automation and reducing contamination. in addition, this technique allows for the quantification of pathogens and the assessment of replication efficiency. as with conventional pcr, multiplex real-time assays are subject to competition and inhibition among primers [5, 35] . real-time assays are also restricted in the number of reaction products that can be detected in parallel [35] , although this problem can be partially circumvented using arrays of uniplex real-time reactions at very small volumes [36] . successful in-house realtime assays directed against respiratory pathogens have been developed using uniplex [37] and multiplex [38, 39] approaches, but data on the performance of commercialized versions are not readily available. much of the effort in nadt development for respiratory diagnostics has focused on the detection of viruses, given the advantages of these techniques over conventional methods in terms of speed, sensitivity, and versatility for detecting this class of pathogens. multiplex approaches for viral detection have become more common as technologies have improved (reviewed by [5, 7, 40, 41] ). in addition, nadts have now become the gold standard for the detection of mycoplasma pneumoniae [42] and chlamydophila pneumoniae [43] and a useful addition to antigen testing for legionella species [44] . multiplex assays for the detection of more traditional bacterial pathogens have not been studied as frequently in respiratory specimens, primarily because culture techniques are usually adequate for clinical practice. moreover, molecular methods provide no additional advantage over culture in differentiating infection from colonization of the upper respiratory tract. nevertheless, multiplex nadts for bacteria such as s. pneumoniae, haemophilus influenzae, and streptococcus pyogenes have been evaluated in respiratory specimens [45] and will likely be incorporated into larger multiplexing assays. having conducted our survey of the field, we narrowed our list of candidate molecular diagnostic platforms even further on the basis of the unique needs of our research study. as with clinical laboratories, we closely examined factors such as cost, feasibility, quality assurance, capital investment, platform versatility, and future utility. in contrast with clinical laboratories, we considered issues such as the rapid return of results or regulatory approval for use in patient care to be less crucial to our objectives. moreover, our approach emphasized comprehensive pathogen detection, rather than focusing primarily on pathogens relevant for clinical management or infection control. finally, our selected platform would be deployed in low-resource settings, where requirements for a reliable or continuous power supply, adequate access to reagents, sensitivity to extreme environmental conditions, and access to technical support would be highly relevant. as our appraisals progressed, we encountered many challenges in interpretation that are common to the field of diagnostics evaluation. most basic among these was confusion regarding the usage of the terms ''sensitivity'' and ''specificity,'' and the evaluations needed to measure these parameters [46] . the distinction between ''analytic'' performance characteristics, as opposed to ''diagnostic'' or ''clinical'' performance characteristics, is essential for properly assessing the validation of any assay, but it is particularly true in the field of molecular diagnostics. for nadts, analytic sensitivity refers to the lowest concentration of target that can be detected, whereas analytic specificity measures the ability of the test to exclude undesired targets despite similar genetic sequences. in contrast, diagnostic or clinical sensitivity of a nucleic acid detection test refers to the appropriate identification of all patients carrying the agent, and diagnostic or clinical specificity describes the assay's ability to exclude uninfected patients. clinical performance characteristics are subject to a number of factors, including the patient's disease status, variations in the concentration of the target throughout the course of illness, inhibition by other substances present in the specimen, sample quality, sampling variability, and specimen degradation. generally, assays should be tested against a reference or gold standard. for tests of microbial detection, the reference standard typically is culture, but molecular diagnostics are often much more sensitive in detecting nucleic acid than is culture for viable organisms, leading to difficulties in interpreting the clinical relevance of false-positive results. the challenges of assessing diagnostic tests have been increasingly recognized in recent years. for instance, the standards for reporting of diagnostic accuracy initiative [47, 48] offers guidelines on the reporting of diagnostic studies, whereas the quality assessment tool for diagnostic accuracy studies provides corresponding guidance on their evaluation [49] . nevertheless, respiratory diagnostics are particularly limited by the inability to determine whether the detection of a particular pathogen in a symptomatic patient indicates that it is causative of the illness or results from contamination, colonization, or prolonged shedding from a prior unrelated infection, particularly when testing specimens from the upper respiratory tract. this issue is not typically addressed in diagnostic evaluation studies, but it has become more relevant as molecular diagnostics have expanded the lower limits of pathogen detection by several orders of magnitude. attempts to answer this question have suggested an additional category of test performance, the ''epidemiological'' specificity of a test, to describe the ability of an assay to assign true etiologic status to a pathogen for a specific illness. ultimately, determination of the epidemiologic specificity of a respiratory diagnostic would require the interpretation of microbiologic results in conjunction with all other clinical and laboratory data, perhaps in the form of a predictive model. such analyses are uncommon but will be a main focus of the perch study. respiratory diagnostics are further complicated by the absence of a perfect gold standard. culture is difficult or insensitive for some pathogens and unavailable for others (eg, human metapneumovirus, parainfluenzavirus type 4, rhinovirus group c, or pneumocystis jiroveci). serologic tests are often not available and usually require paired serum specimens for accurate results. statistical methods to adjust for such alloyed gold standards, such as discrepant analysis, have been frequently employed, but they can be susceptible to significant bias [50] . comparative evaluations of respiratory diagnostic assays must also take into account variations in which panel of pathogens is selected, which genetic sequences are targeted, what specimen sources are used [3, 51] , and even what methods are used for nucleic acid extraction [52] . the us food and drug administration has recently published industry guidance that may encourage additional work in this area [53] . as the perch evaluation progressed, the concepts derived from our deliberations were distilled into a list of desirable and essential attributes summarizing our strategy for evaluation ( table 2 ). this list addressed issues such as assay performance (range of targets, acceptable specimen sources, sensitivity, and specificity), operational concerns (space requirements, assay throughput, quality assurance programs, maintenance requirements, and reagent availability), and strategic issues (capacity for automation, versatility and future utility, start-up and maintenance costs, and developer engagement). for additional input, we presented our summary to the pneumonia methods working group, an expert committee formed to advise perch. ultimately, this outline of key qualities and data allowed us to articulate our thoughts and communicate our strategy more effectively to collaborators, advisors, and assay developers. we applied our list of attributes to more than a dozen candidate diagnostic systems that met our initial criteria, and developed a short list of candidate platforms. we then tested these final assays in our perch-affiliated laboratories, using a standardized set of mock specimens. this process allowed us to engage with the assay manufacturers and their academic partners, directly compare the performance characteristics of nucleic acid extraction procedure included in overall process (and automated) ability to process a variety of respiratory tract specimens small specimen volume requirements specimen collection requirements well-characterized and suitable for field studies readily available reagents with long expiry dates and room-temperature storage requirements the platforms, and gain essential information that could only be acquired through hands-on experience, such as capabilities for technology transfer, ease of use, and workflow. details of this evaluation will be the subject of a separate article. by including a phase for protocol development, the perch investigators were able to perform an extensive literature review of respiratory diagnostics, clearly outline the major theoretical and practical concerns, and engage a group of experts for critical input. through this process, we confirmed the suitability of molecular diagnostics for our needs and identified critical information gaps. our evaluation highlighted numerous advantages of this technology, including excellent sensitivity and adaptability for a full range of respiratory pathogens and specimen sources, as well as clear capabilities for multiplexing and automation. we nevertheless realized that our conclusions represent but a snapshot in time, and the field of molecular diagnostics is rapidly evolving, with constant improvements in accuracy, speed, automation, and cost. yet it can also be expected that methods for evaluating respiratory diagnostics will continue to evolve in parallel, providing new answers to the practical and conceptual challenges that shaped the development of a diagnostic testing strategy for perch. perch: a 21st century childhood pneumonia etiology study laboratory methods for determining pneumonia etiology in children specimen collection for the diagnosis of pediatric pneumonia emerging advances in rapid diagnostics of respiratory infections nucleic acid amplification tests for detection of respiratory viruses in-house nucleic acid amplification assays in research: how much quality control is needed before one can rely upon the results? molecular diagnosis of respiratory viruses a touchdown nucleic acid amplification protocol as an alternative to culture backup for immunofluorescence in the routine diagnosis of acute viral respiratory tract infections evidence from multiplex molecular assays for complex multipathogen interactions in acute respiratory infections simultaneous detection and high-throughput identification of a panel of rna viruses causing respiratory tract infections rapid detection and identification of 12 respiratory viruses using a dual priming oligonucleotide system-based multiplex pcr assay comparison of the seeplex reverse transcription pcr assay with the r-mix viral culture and immunofluorescence techniques for detection of eight respiratory viruses high-throughput, sensitive, and accurate multiplex pcr-microsphere flow cytometry system for large-scale comprehensive detection of respiratory viruses evaluation of a multiplexed pcr assay for detection of respiratory viral pathogens in a public health laboratory setting multicode-plx system for multiplexed detection of seventeen respiratory viruses loop-mediated isothermal amplification (lamp): a rapid, accurate, and cost-effective diagnostic method for infectious diseases rapid real-time nucleic acid sequence-based amplification-molecular beacon platform to detect fungal and bacterial bloodstream infections development of multiplex nucleic acid sequence-based amplification for detection of human respiratory tract viruses predicting viruses accurately by a multiplex microfluidic loop-mediated isothermal amplification chip rapid diagnosis of human parainfluenza virus type 1 infection by quantitative reverse transcription-pcr-enzyme hybridization assay evaluation of the hexaplex assay for detection of respiratory viruses in children detection of 11 common viral and bacterial pathogens causing community-acquired pneumonia or sepsis in asymptomatic patients by using a multiplex reverse transcription-pcr assay with manual (enzyme hybridization) or automated (electronic microarray) detection use of the dna flow-thru chip, a three-dimensional biochip, for typing and subtyping of influenza viruses oligonucleotide array for simultaneous detection molecular diagnostics for pneumonia studies d cid 2012:54 (suppl 2) d s157 of respiratory viruses using a reverse-line blot hybridization assay identifying influenza viruses with resequencing microarrays panmicrobial oligonucleotide array for diagnosis of infectious diseases pan-viral screening of respiratory tract infections in adults with and without asthma reveals unexpected human coronavirus and human rhinovirus diversity detection of respiratory viruses and subtype identification of influenza a viruses by green-echipresp oligonucleotide microarray using a resequencing microarray as a multiple respiratory pathogen detection assay utility of dna microarrays for detection of viruses in acute respiratory tract infections in children comparison of automated microarray detection with real-time pcr assays for detection of respiratory viruses in specimens obtained from children development of a respiratory virus panel test for detection of twenty human respiratory viruses by use of multiplex pcr and a fluid microbead-based assay diagnostic system for rapid and sensitive differential detection of pathogens ibis t5000: a universal biosensor approach for microbiology using multiplex real time pcr in order to streamline a routine diagnostic service application of taqman low-density arrays for simultaneous detection of multiple respiratory pathogens comparison of real-time pcr assays with fluorescent-antibody assays for diagnosis of respiratory virus infections in children viral etiology of severe pneumonia among kenyan infants and children prospective evaluation of a novel multiplex real-time pcr assay for detection of fifteen respiratory pathogens-duration of symptoms significantly affects detection rate detection of respiratory viruses by molecular methods utilization of nucleic acid amplification assays for the detection of respiratory viruses acute respiratory infection due to mycoplasma pneumoniae: current status of diagnostic methods acute respiratory infection due to chlamydia pneumoniae: current status of diagnostic methods diagnosis of legionella infection development and evaluation of a novel multiplex pcr technology for molecular differential detection of bacterial respiratory disease pathogens sensitivity'' and ''specificity'' reconsidered: the meaning of these terms in analytical and diagnostic settings towards complete and accurate reporting of studies of diagnostic accuracy: the stard initiative the stard statement for reporting studies of diagnostic accuracy: explanation and elaboration the development of quadas: a tool for the quality assessment of studies of diagnostic accuracy included in systematic reviews evaluation of nucleic acid amplification tests in the absence of a perfect gold-standard test: a review of the statistical and epidemiologic issues optimal sampling sites and methods for detection of pathogens possibly causing community-acquired lower respiratory tract infections comparison of commercial systems for extraction of nucleic acids from dna/rna respiratory pathogens center for devices and radiological health. guidance for industry and fda staff: class ii special controls guidance document: respiratory viral panel multiplex nucleic acid assay evaluation of commercial resplex ii v2.0, multicode-plx, and xtag respiratory viral panels for the diagnosis of respiratory viral infections in adults acknowledgments. we thank bhagvanji thumar and trevor anderson for their technical support and advice.disclaimer. the contents of this article are solely the responsibility of the authors and do not necessarily represent the official view of ncrr or the national institutes of health.financial support. key: cord-016070-e9ix35x3 authors: perret pérez, cecilia; ferrés garrido, marcela title: pneumonia caused by emerging viral agents date: 2020-02-01 journal: pediatric respiratory diseases doi: 10.1007/978-3-030-26961-6_34 sha: doc_id: 16070 cord_uid: e9ix35x3 emerging viruses that cause pneumonia in humans are agents which normally circulate in the animal population but can move to human hosts under certain circumstances, which determines the occurrence of a new type of disease. the middle east respiratory syndrome (mers) is caused by a coronavirus. the disease has a wide symptomatic spectrum that can range from asymptomatic infections to fulminant respiratory failure. diagnostic confirmation is achieved through viral isolation. severe acute respiratory syndrome (sars), also produced by a coronavirus, is capable of producing a serious pulmonary disease outbreak with no reappearance. the clinical presentation includes fever, malaise, cough, and headache followed by diarrhea. other coronaviruses (hcov-nl63 and hcov-hku1) can cause serious lower respiratory infections in small children, the elderly, and immunosuppressed patients. influenza virus is widespread in nature, and avian virus may spread to humans, as has been reported with h7n9, h5n1, h10n8, and h6n1. cardiopulmonary hantavirus syndrome, a feverish disease characterized by respiratory insufficiency and shock, is produced by andes virus. other emerging viruses are enterovirus d68 and polyomavirus. emerging viruses that cause pneumonia in humans have a common trait: they are all zoonoses. normally, these agents circulate in the animal population, often without causing morbidity, but under certain circumstances they can move to human hosts, which determines the occurrence of a new type of disease. nature holds many examples of such diseases, including avian influenza, ebola, and severe acute respiratory syndrome (sars) . in this chapter we review agents that have provoked major concern outside their country of origin, but also hanta virus, because of the endemic nature it has acquired in chile. the middle east respiratory syndrome (mers) is caused by mers-cov, a recently identified coronavirus. several coronaviruses can cause upper respiratory tract infections, but in some cases they can also produce lower respiratory tract infections and flu-like states. the sars coronavirus and mers-cov are two pathogens from the coronavirus family that predominantly cause serious lower tract respiratory infections with a high mortality rate, but they are genetically different viruses. mers-cov was first identified in 2012 following the death of a patient in saudi arabia with a serious respiratory infection. this finding led to the retrospective diagnosis of the first cases of an inhospital outbreak during 2012 in jordan. most cases have been recorded in the middle east, with more than 75% of the cases in saudi arabia and some cases outside this region affecting travelers. the mortality rate of mers is approximately 30%. information about the mers mechanism of transmission is still limited, but it is likely to occur through droplets and by direct and indirect contact with infected respiratory secretions. aerosol transmission has not been ruled out. currently, transmission between humans is limited and occasional, with a low secondary attack rate. isolated cases have been recorded, consisting of nosocomial and household outbreaks, but transmission is not sustained over time. this was the situation in the major south korean outbreak, which originated through a case in saudi arabia and affected more than 180 people. adequate infection control measures in healthcare rapidly limit in-hospital transmission. genetic analysis shows that the human mers-cov is quite similar to the virus found in bats and essentially identical to that observed in camels. the virus appears to have originated in bats, transitioning through camels, probably in africa, and afterward being transmitted to humans. serological studies do not show its presence in humans before 2012, but it has been observed in camels since the 1990s. this observation suggests that camels are the reservoirs of the virus, which can be transmitted to humans through direct contact with these animals or through consumption of their milk: 1599 cases had been diagnosed by july 2015, with 574 deaths [world health organization (who)]. of these patients, 63% are male, with an average age of 48 years ( fig. 34.1 ). in children, the disease tends to be milder or asymptomatic, with severe cases resulting from a comorbidity. incubation, defined as the period between the primary and the secondary case, is estimated to last an average of 5 days (2-14 days). clinical presentation is characterized by fever, cough, myalgia, and diarrhea. the disease has a wide symptomatic spectrum, which can range from asymptomatic infections to fulminant respiratory failure, which is related to high mortality. patients evolve rapidly (5-day period on average) to respiratory and kidney failure, requiring ventilation support and intensive care management in more than 60% of cases. diagnostic confirmation is achieved through viral isolation in laboratories with biosecurity clearance level 3; alternatively, the virus is detected in respiratory samples through molecular biology techniques (polymerase chain reaction, pcr), which are only available in reference laboratories. in patients who have displayed symptoms for more than 14 days, the determination of specific antibodies is recommended. there is no specific treatment, because no antiviral therapy is available, only support measures in intensive care for the most serious cases. there is no specific preventive vaccine. this infection should be suspected in every traveler who reports having traveled to the arabian peninsula during the past 14 days and who also presents with respiratory symptoms and fever. sars is the first identified coronavirus capable of producing a serious pulmonary disease, unlike those previously known (hcov-229e and hcov-oc 43), which cause the common cold. sars emerged in late 2002 in china and quickly disseminated through southeast asia, europe, africa, and america. bats were its presumed reservoir, and it was transferred to humans through civets. this virus caused great international alarm because of its rapid progression and its seriousness, resulting from extensive and rapidly progressing pneumonia with a mortality rate around 50%. health workers were particularly affected. the most constant symptoms during admissions were high fever, malaise, cough, and headache, followed by diarrhea and prolonged fever. more than 8000 cases were noted, causing more than 700 deaths. thanks to international coordination under who leadership, the epidemic was controlled in july 2003. there were five clear objectives, which were achieved in record time: identification of the etiological agent, development of diagnostic tests for virus detection, creation and evaluation of epidemiological treatment protocols to reduce morbidity and mortality, definition of key epidemiological parameters to control transmission, and formulation of appropriate public health measures. sars demonstrated that coronaviruses can cause serious lower respiratory infections, which would later be observed for hcov-nl63 and hcov-hku 1. to date, no new circulation of this agent has been proven. coronavirus nl 63, which belongs to coronavirus group i, was discovered for the first time in a child with bronchiolitis in the netherlands in 2004, and then ratified in small children hospitalized for serious respiratory infections. hku1, which belongs to the coronavirus group ii identified in 2005, was also shown to be capable of causing a lower respiratory tract infection. both have been described throughout the world, proving their ubiquity. this virus is detected in up to 10% of acute respiratory disease cases, and its symptoms range from upper respiratory disease, including flu-like disease, fever, rhinitis, odynophagia, and cough, to serious conditions with a rapidly progressing respiratory disease. infection by hcov-n63 in children manifests as an obstructive laryngotracheitis in up to 45% of cases in comparison with children not infected by this virus. the first hcov-hku1 viruses were described in elderly patients with preexisting conditions that caused their death. some later studies, which considered children as well as adults, have confirmed that infection caused by this coronavirus may worsen the health status of individuals with underlying diseases, so that they need to be hospitalized more often. as with hcov-n63, infection causes upper respiratory tract symptoms, such as fever, coryza, odynophagia, and coughing. wheezing, pneumonia, and bronchiolitis may also be present. in children a greater fre-quency of feverish convulsions has been observed in comparison with children who were not infected by hcov-hku1. hcov-nl63 and hcov-hku1 are viruses that tend to manifest as a common cold, just as the usual coronaviruses hcov-229e and hcov-oc43; nevertheless, in small children, elderly patients, and immunosuppressed patients, they can cause serious respiratory disease with a high mortality rate. the influenza a virus is widespread in nature: its main reservoir is domestic and feral birds. several types exist, according to their hemagglutinin (h) and neuraminidase (n) makeup, only two of which have recently circulated among humans and are causing seasonal outbreaks: h1n1 and h3n2. the animal reservoir of the virus is large and varied. several subtypes have been described, which include 17 hemagglutinin and 9 neuraminidase types. these zoonotic viruses may cause diseases and infections in several animals, generally pigs and poultry. these zoonotic viruses in particular adapt very poorly to humans, so they seldom cause human diseases, which are limited to outbreaks circumscribed in terms of time and population. nevertheless, human cases of a h5n1 were observed in hong kong for the first time in 1997 and have continued being reported to date. more than 700 cases have been notified, all in asia and northern africa, with 413 deaths. most cases are isolated, with some intrafamilial clusters being described. human-to-human transmission is rare, which reflects the poor ability of the virus to adapt to human respiratory mucosa. the persistence of the circulation of these viruses and the great genetic variability of the influenza a virus make us fear that the avian virus may adapt to humans, which would ease its transmission among the human population, potentially causing a pandemic: this is what happened with the h1n1 influenza a, which had a porcine origin and adapted to humans, creating a pandemic in 2009. the first cases of human infection caused by h7n9 influenza a were reported in china during 2013, and most of these were related in one way or another to contact with poultry, whether direct or environmental in markets where live birds are commercialized. by 2015 there had been 488 confirmed h7n9 influenza cases in china since the beginning of the outbreak, with 185 deaths. this virus does not seem to spread easily among humans, and there has been no proof of sustained human transmission, although its transmission potential seems to be more effective than that of the h5n1 influenza a virus. a couple of cases have been reported outside china: a traveler in malaysia who stayed in china and a man and his wife who were diagnosed in canada in january 2015 after traveling to china (who). there are no recorded secondary cases in these countries, which confirms its low probability of human-tohuman transmission. cases have been observed during the coldest seasons in china. from december 2014 to february 2015, 83 new cases were diagnosed, with an average age of 56 years and 19 deaths. of these newly diagnosed patients, 72% are male, and 93% of patients have had direct contact with poultry markets. the incubation period is 4 days (2-8 days). even though mild cases have been described, most diagnoses have been serious, with a mortality rate close to 35%. hospitalized patients have a febrile disease, with temperatures above 102.2 °f and cough. the disease progresses swiftly from moderate to severe. in contrast to human seasonal influenza, most patients do not report rhinorrhea or odynophagia. in a set of hospitalized patients, the disease progressed swiftly for an average of 3 days after its onset, counting from the beginning of the symptoms until hospitalization. almost 70% required invasive mechanical ventilation, with a death rate of 30%. deaths from respiratory failure reached 38%, and 62% were caused by septic shock. most deaths corresponded to elderly patients and with a underlying disease, as well as to the use of systemic steroids. diagnosis is performed by identifying viral rna through rt-pcr in respiratory tract samples, obtained through swabbing or nasal suctioning. as well as other avian influenza a virus, this virus is sensitive to oseltamivir and is resistant to adamantines. oseltamivir is indicated for hospitalized or ambulatory patients, whether they have been confirmed or are under suspicion of being infected by h7n9 influenza, even if more than 48 h have passed since the onset of symptoms. the dosage and timeframe of therapy are not clearly established for serious patients, but a longer timeframe is suggested, around 10 days and in higher doses. in patients with mild and noncomplicated infections, therapy must continue for 5 days. patients with mild infection, who require ambulatory treatment and whose only exposure factor is travel to an area where there are recorded cases, in humans or birds, have no empirical indication for oseltamivir. isolated cases of avian origin in humans caused by the influenza h10n8 virus and h6n1 have been observed in china. these facts prove that in these zoonotic influenza cases vigilance is extremely important, given its pandemic potential, so it is crucial to pay close attention to travelers and enforce local vigilance. in 1993, a new virus from the the bunyaviridae family was identified in the united states of america. it was named "virus with no name" and was deemed responsible for what is now known as cardiopulmonary hantavirus syndrome (síndrome cardiopulmonar por hantavirus, scph), a feverish disease characterized by respiratory insufficiency and shock. this discovery, which was a new zoonosis, in practice extended over the next years across the whole american continent. in this process, new clinical manifestations were recognized and new agents identified, including the andes, laguna negra, araquara, and choclo viruses, which are prevalent in chile, argentina, paraguay, brazil, and panama, respectively. their natural reservoir are sigmondontinae rodents, which belong to the muridae family. these animals develop a chronic infection with an intermittent viral and asymptomatic excretion. stress situations, such as lack of food during birthing periods, cold, or habitat interventions such as logging, have been associated with greater virus excretion in these rodents. mice excrete the virus through their feces, urine, and saliva, contaminating the environment. the most representative virus-carrying rodents are peromyscus maniculatus or "deer mouse" (which carries the no-name virus), oligoryzomys longicaudatus or "long-tailed mouse," and calomys laucha, among others. humans acquire the infection through the inhalation of secretions (stools, urine, saliva) of infected rodents. the andes virus, predominant in southern argentina and the single causal agent in chile, is only transmitted through close human-to-human contact. hantaviruses are spherical viruses with a trisegmented rna genome with a lipid envelope through which two glycoproteins (gn and gc) protrude. these three segments code through proteins such as rna polymerase (l segment); glycoproteins gn and gc (m segment), which are important in the recognition of β3 integrins that the virus use as receptors; and nucleoprotein (s segment), a highly conserved protein used for the laboratory diagnosis of these agents. the lipid envelope is sensitive and is destroyed by detergents, chloride, desiccation, and sun exposure. all these actions form the basis of the prevention and control recommendations for hantavirus infections. the agent enters the respiratory tract through inhalation of the aerosolized virus in the environment or through contaminated human secretions. after an incubation period that ranges from 1 to 6 weeks (18 days on average), nonspecific symptoms begin, including fever, myalgias, and headache, plus digestive symptoms (more common in children). this stage is followed by progressive respiratory disease and finally by respiratory failure, which is the most serious manifestation of this infection. in 100% of the cases of acute infection, the virus is present in all the white cells and, in variable proportions, in plasma, respiratory secretions, saliva, and urine. also, viral rna has been detected in white cells up to 15 days before and 90 days after the first symptoms. interaction with β3 integrins and the replication of viral endothelial cells of various tissues appear to alter the modulation functions of permeability in these cells, especially increasing the permeability in small lung vessels, which favors arterial hypotension, thrombocytopenia, and hypoxia from plasma flooding into alveolar spaces. protein n and superficial glycoproteins stimulate the production of specific and neutralizing antibodies, which have been associated with better survival outcomes when they increase prematurely. patients who progress to the cardiopulmonary phase, where respiratory failure sets in, require supplementary oxygen; in addition, more than two thirds need mechanical ventilation, and 50% of patients develop cardiogenic shock, which constitutes a poor prognosis factor. the increased vascularity rate explains the pulmonary edema observed during this stage. this functional alteration is transitory, lasting from 48 to 72 h; afterward, pulmonary function is quickly recovered following a brief period of noticeable diuresis. cardiogenic shock is difficult to manage and is the main cause of death. the lethality of hanta cardiopulmonary syndrome is about 35%, and most deaths occur during the first 48 h of evolution. the hemogram is the most useful general laboratory test for hypothesizing a diagnosis, because it can, from an early stage, reveal manifest reductions in the number of platelets as well as the presence of lymphocytes, which take the shape of immunoblasts. a late onset of hematocrit increase has been observed, which is concomitant with the beginning of the cardiopulmonary phase of the disease. it is also helpful to test for ldh and transaminases, which increase nonspecifically. a chest x-ray may change in a matter of hours from a nonspecific interstitial pattern to diffuse pulmonary edema. the virological diagnosis is confirmed through rt-pcr in white cells or through the elisa detection of specific igm/igg for each regional virus. currently, there is no specific treatment for hantavirus infection other than cardiopulmonary support. patient should be monitored closely, preferably in an intensive care unit, to provide measures such as mechanical ventilation for breathing support, restriction of liquids, and vasoactive drugs. the use of an antiviral such as ribavirin has not been shown to be an effective treatment. extracorporeal membrane oxygenation (ecmo) has been used as a rescue therapy in some cases of serious cardiopulmonary failure that do not respond to conventional ventilation and vasoactive drugs. in chile, two therapeutic options have been investigated for hantavirus: methylprednisolone in high doses and immune plasma with high neutralizing antibody titers. only the latter strategy has shown promising results relative to mortality rate reduction when used immediately after symptom onset. as a control measure in hospitalized patient management, and considering that hantavirus has been described as capable of causing nosocomial transmission, standard precautions must be taken: ideally, interning the patient in an individual room and wearing protection equipment [apron, gloves, face mask (no. 95) with a high efficiency filter, and security glasses], especially in procedures during which there is close contact with the patient's fluids, such as intubation, secretion suctioning, and retrieval of samples for laboratory tests. this viral agent, enterovirus d68, has been known since 1962, when it was isolated in children suffering from bronchiolitis and pneumonia. because no widely available trials have been conducted, not much is known about its epidemiology and clinical manifestations. during the fall of 2014, missouri and illinois hospitals reported an unusual rise in the number of serious cases of children, with or without a background of obstructive disease, who presented with acute respiratory infection. enterovirus d65 was detected in these patients by applying molecular biology techniques to respiratory samples. the infection is more frequent in school-age children, whose most relevant clinical antecedent was the presence of previous persistent coughing, asthma, or wheezing episodes that may have required intensive care unit (icu) management. the virulence of this agent is more impressive than that of other enteroviruses, considering that it was also identified as a causal agent of obstructive episodes in previously healthy children who, when treated with antiasthma therapy, did not respond adequately and had to be hospitalized in intensive care because of hypoxemia and, with some exceptions, had to receive mechanical ventilation. the most common laboratory findings were high total neutrophils and chest x-ray showing peribronchial interstitial infiltrations, hyperinsufflation, and atelectasis. as diagnostic tests improve in sensitivity and specificity, and their use becomes more widespread in pediatric centers, our understanding of the epidemiology and pathogeny of this viral agent will increase. two polyomaviruses with respiratory tropism were discovered in 2007 through deep sequencing of samples taken from respiratory secretions of symptomatic patients: polyomaviruses ki (kipyv) and wu (wupyv). these viruses can be found in the lower and upper respiratory tract of immunocompetent as well as immunocompromised patients. their pathogenic role is not completely clear, because they usually occur at low frequency, have a low viral load, and are related to pathogens whose morbidity is better characterized. diagnosis requires molecular techniques (real-time pcr), whose inclusion in future research will allow us to better characterize the epidemiology and clinical spectrum of the infections caused by these agents. clinical course and outcomes of critically ill patients with middle east respiratory syndrome coronavirus infection confirmed human cases of avian influenza a (h7n9) reported to who prospective evaluation of household contacts of persons with hantavirus cardiopulmonary syndrome in chile hantaviruses and cardiopulmonary syndrome in south america prospective population-based study of viral lower respiratory tract infections in children under 3 years of age (the pride study) human infection with a novel avian-origin influenza a (h7n9) virus human hantavirus infections: epidemiology, clinical features, pathogenesis and immunology person-to-person household and nosocomial transmission of andes hantavirus middle east respiratory syndrome coronavirus disease in children clínica de prevención, diagnóstico y trata-miento del sindrome cardiopulmonar por hantavirus web site minsal the novel human coronaviruses nl63 and hku1 update on the epidemiology of middle east respiratory syndrome coronavirus (mers-cov) infection, and guidance for the public, clinicians, and public health authorities hantavirus immunology of rodent reservoirs: current status and future directions summary of current situation, literature update and risk assessment novel avian-origin influenza a (h7n9) in critically ill patients in china key: cord-292261-gh9cifjr authors: feng, fan; tuchman, sylvie; denninger, john w.; fricchione, gregory l.; yeung, albert title: qigong for the prevention, treatment, and rehabilitation of covid-19 infection in older adults date: 2020-05-15 journal: am j geriatr psychiatry doi: 10.1016/j.jagp.2020.05.012 sha: doc_id: 292261 cord_uid: gh9cifjr the elderly are at high risk of contracting respiratory infectious diseases, including covid-19 infection. the recent pandemic has the potential to cause significant physical and mental damage in older adults. similarly to other mind-body exercises in traditional chinese medicine, qigong features regulation of breath rhythm and pattern, body movement and posture, and meditation. given these traits, qigong has the potential to play a role in the prevention, treatment, and rehabilitation of respiratory infections, such as covid-19. potential mechanisms of action include stress reduction, emotion regulation, strengthening of respiratory muscles, reduction of inflammation, and enhanced immune function. three forms of qigong; abdominal breathing, ba duan jin and liu zi jue, all of which are gentle, smooth, and simple for the elderly to practice, are recommended in this context. the outbreak of coronavirus disease 2019 , that was first reported by local health facilities in wuhan studies for inclusion were identified by querying pubmed, the china national knowledge infrastructure, the china science and technology journal database, and wanfang data. the main mechanisms for the occurrence and development of covid-19 are immunosuppression and cytokine storm. 4 covid-19 patients with severe symptoms may develop respiratory impairment and need rehabilitation, including respiratory muscle training, whole-body movement and psychological rehabilitation. 5 we searched in the above databases using terms to address the potential mechanism of qigong in the prevention, treatment and rehabilitation of covid-19, including "immune function", "inflammation", "cytokine", "respiratory muscle", "stress" , "mood" and "emotion", combined with terms which address different types of qigong: "qigong", "qi gong", "tai-chi", "tai chi", "taichi", "taiji", "yi jin jing", "yijinjing", "wu qin xi", "wuqinxi", "liu zi jue", "liuzijue", "ba duan jin", "baduanjin", as well as "abdominal breathing" and "abdominal respiration". given that clinical studies on the intervention of qigong for covid-19 are limited, we used the terms "respiratory infection" and "respiratory rehabilitation", combined with terms mentioned above to search for clinical evidence about the application of qigong in the treatment and rehabilitation of respiratory infection. the abstract and the full text of each article were reviewed and included if it identified as clinical research or as a clinical systematic review published in english or chinese. "qigong" is composed of two chinese characters "qi" and "gong". "qi" refers to the energy that motivates human life activities, and "gong" refers to the regulation of qi through practice. the concept of qi in tcm is very broad, and it is involved in nearly all physiological and pathological processes. 6 according to its different functions, qi can be divided into different types, for example defensive wei qi, and the organ qi that regulates the function of each organ. the channels through which qi moves in the body are called meridians, which are distributed on the surface of the limbs and trunk and extend to the inside organs. qigong is a mind-body training skill that can regulate body, breath and mind under the guidance of theory of tcm to guide qi operation in the meridian, to regulate physical function, and to prevent and treat diseases. 7 qigong regulates the body through an adjustment of body movement and posture. qigong's body regulation is aimed at relaxation, so the movements are typically gentle and smooth. regulation of breath involves changes in respiratory movement, rhythm, and pattern. breath in qigong needs to be slow, long, and deep. sometimes changes in breath pattern are also required, such as abdominal breathing, and breathing with phonation, both of which are typical patterns of qigong respiration. abdominal breathing refers to a breathing pattern with obvious abdomen movement, and breathing with phonation is a combination of breath and the production of speech sounds. regulation of mind includes focusing attention and visualization. most operations of mind regulation are similar to meditation, therefore qigong is also considered a meditative movement. 8 qigong originated in the primeval time of china as a means of self-care. according to the first historical record in china "shang shu", 4000 years ago, ancient chinese people found that stretching and dancing could release pain. this is the rudiment of qigong. almost all religions and philosophical schools, such as taoism, buddhism, traditional chinese medicine, and martial arts, have elements of qigong practice methods, with different appellations. in the 1950s, experts and scholars reached a consensus and coined this methodology "qigong", and the first qigong institute was established in china in 1954. many studies on qigong have been carried out through modern research methods, including the observation of physiological and psychological changes during or after qigong practice, along with clinical trials of treating various diseases with qigong. qigong is particularly appropriate for older people due to its gentle and smooth movements, and there are wide applications of qigong in geriatric medicine, 9 including in the treatment of musculoskeletal disorders, pain relief, and muscle strengthening. as a mind-body skill, qigong has been found to impact internal and psychosomatic diseases, such as asthma, hypertension, peptic ulcers and diabetes. qigong is also used as a meditative movement for treating geriatric mental conditions including mood disorders and cognitive impairment. 10, 11 according to different operations, qigong techniques can be divided into two groups: dynamic qigong is more successful than passive qigong with regards to physical regulation, therefore it can be more effective in treating musculoskeletal and psychosomatic disease. practitioners who have difficulty focusing their attention can concentrate on movements and actions in dynamic qigong, which is an easier skill to master. passive qigong pays more attention to mind regulation. attention training is an important and common technique of mind regulation that asks practitioners to focus attention on an object or on the present, which is similar to mindfulness meditation. according to theory of tcm, through extensive practice of focusing attention, practitioners can enter a state of tranquility. passive qigong has few requirements for physical strength, as it can be practiced in any posture without movement. in addition, for those with impaired body movement ability, passive qigong is a better choice than dynamic qigong. a study on mindfulness, conducted by lacaille et al. 12 , indicated that prolonged mindfulness practice was associated with an increase in mindful responding, which was in turn associated with increased positive affect and with less perceived stress and negative affect. thus, those who engage in extensive practice of passive qigong may be likely to experience better psychological outcomes. respiratory infectious diseases belong to the category of external pathogens diseases in tcm. its pathogenesis is that external pathogens invade the human body and produce tension in the balance between "good and evil". the "evil" refers to exogenous pathogens, which can be considered similar to the pathogen of infection. "good" refers to the defensive function of the human body. when exogenous pathogens invade the human body, defensive wei qi fights against them. it can be considered that wei qi represents immune function from the perspective of modern medicine. the relationship between wei qi and exogenous pathogens determines whether the disease will develop and the prognosis of the disease. if wei qi is strong enough to defend against the exogenous pathogen, the disease would not occur, or would be easier to heal, and the prognosis would be good. because of a decline in organ function and an increase in chronic medical conditions, older people are considered to be in a state of weakness or insufficient energy, conceptualized as qi and blood deficiency in tcm. wei qi is thought of as being scarce in the elderly. therefore, according to theories of tcm, when encountering infectious diseases such as covid-19, the elderly are more likely to be affected, and infections are more likely develop into severe diseases with poor prognoses. given that qigong regulates the function of qi in the human body, in which wei qi is included, it may prevent respiratory infection or promote recovery from respiratory infection in the elderly. outbreaks and illness are a source of stress, and stress reactions or emotional problems can occur in hospital inpatients, people in isolation, and those in the general population. benson et al. 13 observed physiological changes during meditation, and found that meditation can counteract stress response. benson coined the physiological change elicited by meditation a "relaxation response". as a meditative movement, qigong has been studied as a tool for stress management. ryu et al. 14 observed changes in stress hormones during qigong practice, and found that beta-endorphins increased in the middle of training while levels of adreno-cortico-tropic-hormone declined mid and post-practice suggesting decreased stress levels. it has been suggested that qigong regulates emotion through enhancing nonreactivity to aversive thoughts and impulses by focusing attention, regulating the hypothalamus-pituitary-adrenal axis reactivity and the balance of the autonomic nervous system, and through changing the function of the brain, limbic system, and expression of genes linked to inflammatory responses and stress-related pathways. 15 in a meta-analysis on treating chronic obstructive pulmonary disease (copd) with qigong, wu et al. 16 qigong can reduce both inflammatory factors and inflammatory response. irwin et al. 22 examined the cytokines in older adults who had participated in a 6-month tai-chi program, and found reductions in levels of il-6 in subjects in the intervention group who previously showed high levels of this inflammatory marker. in another study, irwin et al. 23 found that in older adults with insomnia, tai-chi reduced proinflammatory gene expression and marginally reduced c-reactive protein by the end of the 4-month practice, and reduced monocyte production of proinflammatory cytokines at the end of the program and at the follow-up after 7 and 16 months, when compared to the control group. additionally, a 12-week program of tai-chi has been found to increase levels of the anti-inflammatory cytokine il-10 in middle-aged adults. 24 chen et al. 25 found that in copd patients, 60-day liu zi jue practice lowered the level of il-4、il-13 and il-17, and increased the level of il-10 when compared to a regular treatment control group. qigong's enhancement of immune function has manifested in both non-specific immune response and specific immune response. regarding non-specific immune response, qigong can increase the amount or activity of immune cells in the body. yeh et al. 24 found that in middle-aged healthy people, after a 12-week program of tai some studies have demonstrated the effectiveness of qigong in preventing respiratory infectious diseases. hu et al. 34 selected elderly men as experimental subjects and randomly divided participants into either a qigong intervention group or a control group who performed jogging. compared with the control group, the experimental group experienced significantly fewer respiratory tract infections after qigong exercise for two years, and the difference between the two groups increased with exercise time. wright et al. 35 found that in swimmers who practiced qigong at least once per week, cold and flu symptoms showed a significant non-linear association with frequency of qigong practice, with a strong, inverse relationship between practice frequency and symptom scores. there are few studies on the intervention of qigong in the acute phase of respiratory infection, but according to limited research results qigong can be found to shorten the course of infection. in ties' study, 36 90 female healthy students were separated into three groups; a control group, a three times a week movement group, and a five times a week movement group, after a six-month tai-chi training. in the two tai-chi groups, the levels of iga and igg became higher, as compared with the control group. there was no difference in the frequency of respiratory tract infection in the three groups, however the duration of each onset became shorter in the tai-chi group. improved when compared with the control group who received regular treatment. some simple qigong can be learned independently through watching qigong videos. before learning qigong, consulting with doctors is necessary for safety reasons. learners can begin with physical movements of the forms. after practitioners acquire the sequences of both isometric and isotonic segmental movements in upper and lower extremities, they can try to combine breathing techniques and focus their attention on movement, breath and qi. considering the physiological characteristics of the elderly, the pathological features of respiratory diseases, and the psychosocial factors in the face of the covid-19 epidemic, we recommend ba duan jin, liu zi jue, and abdominal breathing. according to the research results mentioned above, these three kinds of qigong are often used in the prevention and treatment of respiratory infections, for the movement is smooth with low intensity, and easy to learn. in addition, the range of these three qigong movements is small, and the space requirements are not significant. thus, they are suitable for home practice during the current epidemic. the type of respiratory pattern of pursed lip breathing performed by expiration to produce six different sounds (xu, he, hu, si, chui, and xi) is similar to the pursed-lips breathing in rehabilitation training for copd patients 43 . it can modify rapid shallow breathing patterns and retard the expiratory flow rate. additionally, the different sounds can produce vibrations with different frequencies, which is commonly used in neurorehabilitation 44 and tension relaxation 45 . research indicates that liu zi jue might help tissue and organs in respiratory recovery through these vibrations. liu zi jue is a good choice for people seeking to recuperate from respiratory dysfunction and sequela of covid-19 infection. the available biological and psychological evidence suggest qigong may be potentially useful for the prevention, treatment, and rehabilitation of respiratory infections, including covid-19. the elderly, in particular, could benefit from qigong during the ongoing pandemic, for it is easy to practice. future studies are needed to confirm the effectiveness of qigong in this context and to provide more evidence on this topic. clinical characteristics of refractory covid-19 pneumonia in wuhan, china state administration of traditional medicine of china: diagnosis and treatment protocol for covid-19 (trial version 7) [state administration of traditional medicine of china web site dysregulation of immune response in patients with covid-19 in pulmonary rehabilitation guidelines in the principle of 4s for patients infected with 2019 novel coronavirus (2019•ncov) foundation theory of traditional chinese medicine china press of traditional chinese medicine effect of meditative movement on affect and flow in qigong practitioners physical and psychological health outcomes of qigong exercise in older adults: a systematic review and meta-analysis the beneficial effects of qigong on elderly depression the effect of continuous fitness qigong exercise on mild cognitive impairment in the elderly daily mindful responding mediates the effect of meditation practice on stress and mood the role of practice duration and adherence relaxation response acute effect of qigong training on stress hormonal levels in man qigong and tai-chi for mood regulation effect of qigong on self-rating depression and anxiety scale scores of copd patients: a meta-analysis qigong practice among chronically ill patients during the sars outbreak coping with future epidemics: taichi practice as an overcoming strategy used by survivors of severe acute respiratory syndrome (sars) in post-sars hong kong the effect of qigong on muscle function of older adults effect of different breathing training methods on the fatigue and diaphragm muscle function of stroke patients effectiveness of water-based liuzijue exercise on respiratory muscle strength and peripheral skeletal muscle function in patients with copd mitigating cellular inflammation in older adults: a randomized controlled trial of taichi chih cognitive behavioral therapy and taichi reverse cellular and genomic markers of inflammation in late-life insomnia: a randomized controlled trial regular taichi chuan exercise enhances functional mobility and cd4cd25 regulatory t cells effect of six-word breath exercise on the inflammatory immune factors and pathogen distribution in asthma patients study on anti-aging effect of qigong baduanjin effects of exercising building-up qigong-wuqinxi on middle-aged and old people' s nk cell activity upper respiratory tract infection, and the immune system taichi chuan increases circulating myeloid dendritic cells acute effects on the counts of innate and adaptive immune response cells after 1 month of taoist qigong practice effect of "taichi" exercise on antioxidant enzymes activities and immunity function in middle-aged participants augmenting immune responses to varicella zoster virus in older adults: a randomized, controlled trial of taichi effects of a taiji and qigong intervention on the antibody response to influenza vaccine in older adults effect of qigong on the prevention and treatment of respiratory tract infection in the elderly a pilot study of qigong practice and upper respiratory illness in elite swimmers research on the impact of the taichi chuan or the upper respiratory tract infection for female students the therapeutic effects of qigong in patients with chronic obstructive pulmonary disease in the stable stage: a meta-analysis effects of long-term home-based liuzijue exercise combined with clinical guidance in elderly patients with chronic obstructive pulmonary disease effect of baduanjin on respiratory symptoms in chronic bronchitis abdominal breathing increases tear secretion in healthy women ba duan jin --an ancient chinese exercise effects of qigong exercise on non-motor symptoms and inflammatory status in parkinson's disease: a protocol for a randomized controlled trial pursed-lips breathing reduces dynamic hyperinflation induced by activities of daily living test in patients with chronic obstructive pulmonary disease: a randomized cross-over study focal vibration in neurorehabilitation vibrational therapies for vocal fatigue the other authors report no conflicts with any product mentioned or concept discussed in this article. the authors alone are responsible for the content and writing of this article. key: cord-017715-99ri6x0y authors: zhou, bo-ping; lu, pu-xuan; chen, qiu; xiao-ping, tang; yu-juan, guan; jin-xin, liu; xing, lu; zhen-wei, lang; xin-chun, chen title: sars date: 2015-07-25 journal: diagnostic imaging of emerging infectious diseases doi: 10.1007/978-94-017-7363-8_2 sha: doc_id: 17715 cord_uid: 99ri6x0y severe acute respiratory syndrome (sars) is an acute respiratory tract infectious disease induced by sars-cov and mainly transmitted through the short-distance air droplets and close contact. its main clinical characteristics is abrupt onset of the disease and the initial symptom is fever accompanied with systematic symptoms of headache, soreness and fatigue, and respiratory tract symptoms such as cough, chest dullness, and dyspnea. a few cases may progress to acute respiratory distress syndrome (ards). due to its self-limiting feature, the prognosis is predominantly good but may be poor in severe cases, with mortality about 9.3 %. some patients may develop such complications such as lung fibrosis and necrosis of the head of femur. on april 8, 2003, sars was defined as a legal infectious disease by the ministry of heath of china. 1. the fi rst sars case was reported around the world on january 2, 2003; a hospital in heyuan city of guangdong province hospitalized two patients' with severe pulmonary infection of unknown cause, which was the fi rst traceable report of "sars" around the global. 2. on february 28, 2003 , sars was fi rst diagnosed by the who expert carlor urbani in hanoi of vietnam, who also named this new unexplained disease " s evere a cute r espiratory s yndrome" (sars). following the emergence of sars, who and governments around the world attached great importance and took consecutively effective control and prevention measures, which reined in the infection rapidly controlled in a short time. on april 28, 2003 , who eliminated vietnam from the name list of the sars-infected area, which made vietnam the fi rst to get rid of sars outbreaks. since then, numerous countries were also ticked off the list. on july 5, 2003 , who announced to remove taiwan of china from sars-affected area list, which is also the last. at present, there is no sars-affected area around the world, signaling the victory of global battle against sars. by the end of september 26, 2003 , there were in total 8,098 people infected and 774 deceased, with the mortality at 9.5 %. on april 16, 2003, a new coronavirus never seen was announced as the cause of sars by who. it is the joint efforts of global scientists and biological scientifi c and technological advances that enabled establishment of international sars research network, separation and identifi cation of sars coronavirus, and confi rmation of sars pathogenesis. some canadian laboratories excluded infl uenza virus a and b; para-infl uenza virus type 1, 2, and 3; adenovirus; and respiratory syncytial virus by scanning electron microscopy and direct fl uorescent antibody test. they also conducted immunohistochemistry on the corpse tissues of dead patients about viruses including infl uenza virus a and b, respiratory syncytial virus, adenovirus, circovirus, hantaan virus, measles virus, intestinal tract virus, and pneumonia mycoplasma and chlamydia, which showed negative. scholars from german, france, america, hong kong, and taiwan applied specifi c pcr tests targeted at corresponding pathogens to examine pneumonia mycoplasma, pneumonia chlamydia, human cytomegalovirus, circovirus, herpes virus, human coronavirus oc43 and 229e and arenaviruses, bunyavirus, hantaan virus, crimean-congo hemorrhagic fever virus, which also indicated negative. on march 21, 2003 , hong kong university fi rstly separated and cultured coronavirus from the nasopharyngeal specimen of sars patients by vero cells, and then several laboratories of canada and america disease center and sars international coordination group also cultured coronavirus. on april 16, 2003 , based on the aforementioned research fi ndings, who declared a new coronavirus as the pathogen of sars and named it as sars coronavirus (sars-cov). sars-cov is a single-stranded and positive rna virus belonging to the genus coronavirus of coronaviridae family of nidovirales order. it bears great morphological resemblance with known human coronaviruses. under the electric microscopes, sars coronavirus presents with pleomorphic spherical particles with envelopes in vero e6 cell in vitro and with a diameter of about 80-140 mm. it has distinct coronavirus morphological characteristics that rodlike surface projections about 20-40 nm long protrude outside viral envelope to form a corona. for separation and culture of viruses in vero e6 cells, viral particles are mostly found in vacuoles inside cells under electric microscope, mainly hollow particle, and viruses outside cells observed with spikes. the coronavirus is mainly composed of nucleic acid, protein, carbonate, and lipid. sars-cov are relatively stable in human specimens and environment but relatively sensitive to chemical and physical factors. the viruses could be killed by exposure to ultraviolet rays for 60 min. as the envelope of sars-cov contains lipoids, they are sensitive to lipid solvents and could be inactivated by ether, chloroform, tween, 70 % ethyl alcohol, methanol, pancreatic enzymes, and ultraviolet rays. ether could sterilize the virus completely under 4°cafter 24 h, 75 % ethyl alcohol could deactivate virus after 5 min, and disinfectants containing chlorine may kill the virus aft. sars is the fi rst newly emerging infectious disease in the twenty-fi rst century. since the fi rst case was detected in guangdong on january 2, 2003, sars spread to 29 countries at fi ve states at an unprecedented speed with several months. according to the report of who, sars epidemics was found in total 29 the main source of infection is sars patients, and other infectious sources such as animals need to be further consolidated. it is regarded that only symptomatic patients can effectively spread sars-cov. at present there still lacks clinical proof that asymptomatic sars patients could spread sars coronavirus. few cases are highly contagious and able to directly infect over 10 patients, who are called super-spreader. the fi ve super-spreaders in singapore have been found infecting 103 people. such powerful infectiousness is attributable to the high viral load inside patients who could discharge abundant viruses via respiratory tract in a short time. specialists from the department of microbiology of hong kong university and guangdong provincial disease control and prevention center tested abundant sars coronaviruses from the samples collected from civet cats sold in guangzhou and shenzhen, suggesting that the human sars coronavirus might originate from civet cats. sars virus is spread mainly through the respiratory tract and also spread by aerosol and droplet. high level of sars coronavirus could be detected in the throat swabs and sputum specimens of the patients in acute phase. viruses discharged by patients via cough would exist in the air within certain radius, thus forming short-distance respiratory tract droplet spread. it is a main route of sars dissemination. indirect or direct contact spread could also spread sars coronavirus. it could be spread through contact with the respiratory tract secretion or through the mouth and nose via contaminated hands and toys. as viruses proliferate abundantly in the respiratory tract, exfoliated cells containing infectious viruses could be expelled outside the body through patients' respiration, cough, and sneeze and suspend in the air, thus forming the aerosol of sars virus. cough and sneeze could greatly contribute to production of sars virus aerosol. sars is a new type of infectious diseases, and people effectively exposed to sars pathogens are vulnerable. present data reveal that sars is an acute self-limiting infectious disease, and neutralizing antibodies to sars could be detected 3 weeks after onset. however, the change patterns of neutralizing antibody titer have not been fully elucidated. the epidemics of sars mainly take place in the winter and spring. sars outbreaks are mainly concentrated at coastal tourism cities with prosperous economy, dense population, and convenient transport, particularly air transport. from the perspective of international geography, sars primarily occurs in southeastern countries or regions, including china mainland, hong kong, taiwan, singapore, and vietnam. analysis of the statistics of various countries shows that sars onset is mainly recorded in young populations aged 20-50 years old, occupying around 80 % of the total. sars coronavirus (sars-cov), a mutant of coronaviridae, is pathogenic to hosts in the following two aspects: fi rstly, viral infections could directly damage the structure and functions of infected cells and trigger cell apoptosis; secondly, immune reactions and release of various cytokines induced by viral infections could not only eliminate infectious pathogens but also may deal severe damages on histiocytes due to excessive immune reactions. the member of coronaviridae is a single-chain positive rna virus, which taxonomically belongs to nidovirales with arteriviridae. the homological analysis of sars-cov structural protein amino-acid sequences shows that the main structural protein s (spike protein), m (membrane protein), n (nucleocapsid protein), and e (envelope protein) bear very low homology with other known coronaviruses, between 20 and 30 %. besides, bioinformation analysis infers that sars-cov may also encode 5-9 nonstructural proteins of unknown functions. all these sars-cov specifi c proteins may play key roles in determining the viral virulence. s-protein, existent on the viral surface in form of trimer, is the main component in constructing the coronal structure and major structural protein for the integration of virus and host cell receptors and fusion of viral envelope and cell membrane to enable invasion of viruses. after infection with sensitive cells, sars-cov could replicate enormously and release virions, which may disturb cell metabolism and directly result in injury of body tissues and cells. in the early phase of the disease and in the continual progression phase, the active immune cells such as cd4+ and cd8+ t lymphocytes, nk cells, and dc cells are decreased prominently, to an extent positively correlated with severity of conditions and speed of progression. besides, all dead patients are detected with irreversible signifi cant decline. however, along with remission of conditions and entry into recovery phase, the number of aforementioned immune cells could be recovered to some extent, whose change presents distinct par-allel correlation with disease evolution. dynamic observations suggest that serum pre-infl ammatory cytokine il-6, il-8, il-16, and inf-α level of sars patients could reach the peak at about second week of disease course, which relatively corresponds to the acute aggravation phase. all these consolidate the immune damage of sars-cov infections. autopsies of the over 20 patients who died after infected with sars revealed that sars is a disease detrimental to organs of the whole body, particularly lung tissue and immunity system. primary pathologic changes can be classifi ed into severe lung lesions, immune organ impairment, toxic changes of other organs, and secondary infections. the causes of death were as follows: (1) diffusive lung alveolar damages lead to progressive respiratory failure; (2) impairment of liver, kidney, heart, and several other organs may induce multiple organ failures and exacerbate conditions; (3) due to injury of immune organs particularly lymph nodes and spleen, the lymphocytes are decreased and immunity compromised, therefore resulting in secondary mycotic infections. lung tissues become swollen and heavier. according to domestic report, the left lung may reach 500-1480 g (average 811.4 g) and right lung 700-1125 g (average 869.3 g), with the total lung up to 1170-2605 g (average 1460.7 g). the lung tissues appear in dark red. texture is hard, indicative of consolidation. the surface is relatively smooth and free of pleural adhesions. lung tissues are observed with blood vessel dilation and congestion, spotted and patchy necrosis, and hemorrhagic infarct foci and focal compensatory emphysema. sections of various lung lobes are visualized with outfl ows of light red or/and a few foamy blood-stained fl uids. the trachea and bronchi are found with exudations of slight mucoid or blood-stained secretions. pulmonary hilar lymph nodes are revealed with slight enlargement and pleural cavity without or with a few effusions. microscopy reveals bilateral diffusive severe injuries of alveoli, characterized by acute exudative, hemorrhagic, and fi brinous infl ammation. however, the injury may be inhomogeneous and diverse at different lung lobes and different parts of the same lobe. the characteristic change in early phase is pulmonary edema, with homogeneous pink serous or fi brinous effusions fi lling alveolar cavities as well as erythrocytes leakages in some cavities. the effusions from some alveolar cavities may condense to form thin-layer membranelike substance which adheres to alveolar walls, namely, formation of hyaline membrane ( fig. 2.1 ). alveolar epithelia present with such diffusive injuries as degeneration and necrosis, with exfoliated or/and apoptotic type ii alveolar epithelial cells observable in alveolar cavities, which are similar to changes of desquamative pneumonia ( fig. 2.2 ). among them, some appear like apoptotic bodies. partial regional type ii alveolar epithelial cells and macrophages proliferate actively, with obvious increase of cell volume and distinct nucleolus. in some case, cells are detected fusing with each other to form syncytial monocytes and multinucleated giant cells (fig. 2.3 ) . the alveolar epithelia of some cases are examined with structures similar to viral inclusion bodies, mostly spherical and with eosinophilic staining, around which a transparent halo is visible (fig. 2.4 ) . pulmonary interstitium and alveolar spaces are detected with high dilation and congestion of capillaries, swelling and exfoliation of epithelial cells, as well as widening of the latter, which present slight lymphocyte and monocyte infi ltration, small vessel proliferation, and enlargement characteristic of vasculitis changes. some regions are observed with pulmonary vascular embolism or fi brinous microthrombi in alveolar capillaries, while some alveolar capillary cavities manifest dilation but free of blood components. in addition, some patients are detected with lung tissue focal hemorrhage, compensatory emphysema, and small airway necrotizing infl ammation and alveolar collapse or shrinkage. patients with a disease course of over three weeks manifest fi broblast proliferation in alveolar septa, and individual cases demonstrate organization of fi brinous exudates from alveoli and fi broblast proliferation, which gives rise to glomeruloid structures, named "glomeruloid organizing pneumonia" (fig. 2 .5 ). the bronchial epithelial cells are revealed with exfoliation, scarce and exfoliated cilia, as well as structures similar to viral inclusions and squamous cell metaplasia at the inside. the bronchioli are detected with submucosal edema, infl ammatory cell infi ltration, and serous grandular hyperplasia with hyperactive secretion. pulmonary hilar lymph nodes demonstrate light swelling and present different degrees of congestion, hemorrhagic necrosis, and decrease of innate lymphocytes under the microscope. blood sinus in the lymph nodes presents with grave congestion and dilatation, lymphoid nodule atrophy or disappearance, prominently the cortex, and lymphatic tissue focal necrosis, accompanied by apoptosis of some lymphocytes ( fig. 2.6 ). the spleen mostly shrinks with soft texture, and has patchy and focal hemorrhage at the surface and sections. splenic white pulp is detected obviously atrophic or absent, with central arterial wall thickened, endothelial cells swollen and partially shed, peripheral lymphatic sheath lymphocyte depleted greatly, and germinal center disappeared. splenic corpuscle is observed with plasma protein sedimentation in central arterial walls, white pulp and marginal sinus lymph tissues with large patchy necrosis, and partial remaining lymphocytes with apoptosis. the splenic sinus inside red pulp is revealed with obvious congestion and focal hemorrhagic necrosis as well as proliferation of histiocytes ( fig. 2.7 ). the number of hematopoietic cells in bone marrow declines, and granulocyte system and megakaryocytic system are relatively inhibited. polychromatic erythroblasts present with small focal proliferation. tissues of the brain, liver, kidney, and heart demonstrate varying degrees of congestion, edema, degeneration, and necrosis as well as infi ltration of a few lymphocytes. secondary bacterial pneumonia and fungal infection may occur ( fig. 2.8 ). the initial symptom of sars is frequently fever, accompanied by headache and general muscular and joint souring soreness, and often without nasal discharge, sore throat, and other upper respiratory tract catarrhal symptoms. pulmonary signs are not obvious and signs and distinct systematic symptoms are inconsistent, which is typical to sars. the clinical manifestations of sars may vary, which can be divided into light type, common type, severe type, and atypical type, with the common type most commonly seen. based on the clinical development, it could be divided into early phase, progression phase, and recovery phase. generally, the incubation phase lasts 2-14 days, with an average of 4-7 days and a median of 6 days, predominantly 2-10 days. the initial symptom primarily is fever, mostly high fever, accounting for 94.4-100 %. patient body temperature may reach 38-40 °c and 42 °c at the highest. the fever type varies, including remittent fever, irregular fever and continuous fever. the duration of fever is mostly 5-9 days. it presents as headache, joint or/and systematic soreness, and chest pain. it is reported that the incidence of headache in sars patients is 17-90 %. most sars patients have cough, which is dry with scanty sputum. generally sars does not incur upper respiratory tract catarrhal symptoms. severe cases may suffer from accelerated respiration, shortness of breath, or even acute respiratory distress syndrome, characterized by progressive dyspnea or even distress with respiratory frequency up to >20 times. patients complain of heart beat or palpitation accompanied often by precordial discomfort. the incidence is 35-90 %. the incidence of diarrhea is reported mostly at 7-44 %, with single case up to 70 %. one characteristic of sars is severe symptoms and light signs. the signs are mostly not obvious or even absent. dyspnea presents irregular respiratory frequency, depth, and rhythm. the incidence of dyspnea of sars patients is 21-57 %. severe patients may manifest nose fanning, orthopnea, and cyanosis, involvement of accessory respiratory muscles in respiration, as well as "three depression signs" upon inhalation. according to report, 11 % of typical patients and 14 % of severe patients have sinus tachycardia, and 2.3 % of sars patients suffer from paroxysmal supraventricular tachycardia. the pulmonary signs of sars patients are frequently unobvious or absent in the early phase. light moist rales may be audible (17-62 %) . patients may also demonstrate pulmonary consolidation sign and attenuated breath sounds. there may also be a few pleural effusions. in light of conditions, it could be classifi ed in clinics into light type, common (typical) type, severe type, and special (atypical) type. the follow-up visit of 267 sars patients via x-ray and hrct by choi et al. found that about 3-4 % patients developed fi brotic changes of lung interstitium and shrinkage of lung volume. application of glucocorticoids could to some extent alleviate lung injury of patients with severe infectious atypi-cal pneumonia. however, long-term and abundant use of glucocorticoids may generate numerous side effects, and it is an undisputable fact that hormones may induce femoral head ischemic necrosis. literature reports suggest that the incidence of femoral head ischemic necrosis is 5-25 %. the white blood cell count remains within normal range in majority patients and may decrease in some patients (24.2 %). 95 % of severe patients without secondary infections are revealed with decreased total white blood cell, and normal neutrocyte and monocyte differential white blood cell count. however, 80 % initially diagnosed patients manifest a decrease of absolute lymphocyte count, suggestive of a tendency of gradual decline, and morphocytology change, with abnormal lymphocytes observable. 13.2-41 % patients are examined with thrombocytopenia. many evidences prove that sars-cov could directly invade the immune system and act mainly on lymphocytic system, especially the t lymphocytes, which could lower peripheral blood lymphocytes. the peripheral blood white cell count is normal or decreased in most sars patients, but cd3+, cd4+, and cd8+ t lymphocyte counts are distinctly lower compared with those of healthy population. the more severe the condition, the more drastically declined the t lymphocyte count. blood gas analysis reveals hypoxemia of different degrees and commonly no carbon dioxide retention. generally under 3-5 l/min oxygen uptake, patients with severe hypoxemia are measured of arterial partial pressure from oxygen (pao 2 ) <70 mmhg or blood oxygen saturation (spo 2 ) <93 %, or develop acute lung injury (ali) or acute respiratory distress syndrome (ards). one of the main immunopathogenesis changes in sars patients is insuffi ciency of t-lymphocyte-mediated specifi c cellular immunity, characterized by distinct damages of t lymphocytes and its subgroups, particularly cd3 + , cd4 + , and cd8 + . there exists distinct correlation between the extent of t lymphocyte damages and severity of the disease. the method recommended by who for test of serum sars-cov antibodies is enzyme-linked immunosorbent assay (elisa). upon test of the sars-cov-specifi c antibodies in the infected patients, including igg, igm, or total antibody, seroconversion or increase by 4 times and above of any serum antibody in progress phase and recovery phase could serve as evidence for defi nite diagnosis. polymerase chain reaction (pcr), a type of molecular biological test, could identify sars virus genes from various specimens (blood, feces, respiratory tract secretions, tissue sections, etc.), that is, sars virus rna. presence of any of the following three circumstances could be sars virus positive : 1. clinical specimens from at least two different sites are tested positive (nasopharyngeal secretions and feces). 2. the same clinical specimens collected by at least 2 days apart are tested positive (two or more specimens of nasopharyngeal excretions). 3. the same clinical specimen is tested positive by two different methods or in different laboratories. 4. repeated pcr examination indicated positive. clinical diagnosis 1. the patient has a history of intimate contact with patients or is one of the infected populations or consolidated having infected others by defi nite evidences. 2. the patient had been to or lived in the areas with sars patients and secondary infection epidemics. the onset is acute and initial symptom is fever, with body temperature >38 °c, and occasionally accompanied by concurrent chillness. patients may also suffer from headache, joint and muscular soreness, fatigue, and diarrhea, and generally there is no upper respiratory tract catarrhal symptom. patients may have cough, mostly dry with scanty sputum, and chest dullness. severe patients demonstrate accelerated respiration, shortness of breath, or obvious respiratory distress. pulmonary signs are not obvious, and light moist rales may be audible or pulmonary consolidation observable. there is generally no increase of peripheral white blood cell count or decrease. besides, the lymphocyte count is found generally declined. lungs present different degrees of patchy and plaque-shaped infi ltration shadows or reticulate changes, which may progress rapidly into large patchy shadows. these commonly occur at multiple lobes or both sides, and absorption and dissipation of shadows are relatively slow. pulmonary shadows and symptoms and signs may be inconsistent. if the test indicates negative, reexaminations shall be conducted after 1-2 days. there are no remarkable responses to antibiotics. patients in compliance with any of the following three circumstances are diagnosed as suspected cases. 1. the patient has a history of intimate contact with patients, or is one of the infected population, or is confi rmed having infecting other people by concrete evidences, who also manifest aforementioned clinical symptoms but not high white blood cell count by test of peripheral blood. 2. the patient has been to or lived in areas reported with infectious sars patients and patients suffering from secondary infections 2 weeks before the disease onset, who also have abovementioned clinical symptoms and consistent pulmonary shadows on chest x-ray fi lms with the abovementioned characteristics. 3. despite the lack of epidemic data, the patient has abovementioned clinical manifestations and not high peripheral white blood cell count, as well as pulmonary shadows on chest x-ray that accord with abovementioned characteristics. in other words, the patient conform to the aforementioned 1 (1) + 2 + 3 or 1 (2) +2 + 4 or 2 + 3 + 4. in case of any of the three following circumstances, the patient could be clinically diagnosed with sars. 1. the patient has a history of intimate contact with patients, or is one of the infected population, or is confi rmed having infecting other people by concrete evidences, who also manifest aforementioned clinical symptoms and pulmonary shadows on chest x-ray image. 2. the patient has been to or lived in areas reported with infectious sars patients and patients suffering from secondary infections 2 weeks before the disease onset, who also have abovementioned clinical symptoms, not high peripheral blood white cell count and pulmonary shadows on chest x-ray fi lms. 3. the patient has been to or lived in areas reported with infectious sars patients and patients suffering from secondary infections 2 weeks before the disease onset, who also have abovementioned clinical symptoms, pulmonary shadows on chest x-ray fi lms, and no obvious response to anti-infectious treatment. in other words, the patient conform to aforementioned 1 (1) + 2 + 4 or 1 (2) +2 + 3 + 4 or 1 (2) + 2 + 4 + 5. the conditions are mild without pulmonary fi brosis and other sequela. the prognosis is good. patients have typical clinical manifestations of the early phase, progression phase, and recovery phase. the onset is acute, and the fi rst symptom is fever, with body temperature generally >38 °c. besides, the patients have no obvious respiratory distress or hypoxemia. some patients are auscultated with slight moist rales or observed with pulmonary consolidation. the prognosis is good without sequelae. severe type accounts for about 30 %. it is same with the common type in clinical process but presents severe conditions and rapid progression. some cases may progress to acute pulmonary injury or ards, rapidly incurring respiratory failure or even death. the diagnosis could be reached in case of any of the following three circumstances: 1. dyspnea adult respiratory frequency is ≥30 times /min, accompanied by one of the following conditions: the chest imaging shows that the total surface of multilobar lesions or foci occupies more than 1/3 of the two lungs on orthotopic imaging. conditions progress, with the focal area increased by over 50 % within 48 h or accounting for over 1/4 of the two lungs on orthotopic imaging. 2. obvious hypoxemia is detected, with the oxygenation index lower than 300 mmhg. 3. shock or multi-organic dysfunction syndrome occurs. there are still some atypical cases of this disease. this generally refers to the fi rst 1-7 days. the onset is acute and initial symptom is fever. half of the patients are accompanied by the nonspecifi c systemic symptoms such as chillness, headache, and joint and muscular soreness. this phase spans the day 8-14 of the disease course. infectious toxic symptoms such as fever, fatigue, muscular soreness continue to exist or even aggravate. patient's body temperature rises, possibly to 39 °c or higher in a short time, and the high fever is persistent. lung lesions demonstrate progressive exacerbation. a few patients (10-15 %) may develop ards, which is life threatening. it mostly takes place at day 15-21 of disease course. most patients have good prognosis after recovery for about 2 weeks. some severe patients could gradually recover within 2-3 months after discharge. these mainly encompass standing chest x-ray and bedside chest x-ray, which are economic and effective examination methods and fi rst optional examination for preliminary diagnosis and reexamination. mobile x-ray bedside imaging should be applied for patients under observation and inpatients. as lung disease of sars patients progress rapidly, chest x-ray reexamination within a short period could help observe changes of conditions, commonly once every 1-2 days at the early phase and progression phase. regular chest x-ray also need to be conducted during recovery phase so as to ascertain absorption and dissipation of lesions, residual foci, and pulmonary interstitial fi brosis. for sars patients, ct scan generally is not a preferred method and, if necessary, could be conducted under strict disinfected quarantine. if the foci could not be absorbed for a long term in sars recovery phase or patients still have symptoms but normal chest presentations, ct scan need to be carried out for further observation as it can better visualize the subtle pulmonary interstitial changes, such as lung interlobular septum thickening, intralobular septum thickening, subpleural linear shadow, and small ground-glassdensity lesion and regional and segmental bronchiectasis, and therefore is helpful for clinical diagnosis of pulmonary interstitial fi brosis. thin-layer ct scan or hrct scan can better visualize intrapulmonary low-density small lesions, thus contributory to early diagnosis of pulmonary interstitial fi brosis. ct examination could also aid identifi cation of other concurrent lung lesions. medical staff in the room of imaging examination and department of radiology shall prevent infections in strict compliance with sars disinfection and prevention requirements and meanwhile conscientiously implement x-ray protection. 1. ward shall be equipped with bedside x-ray machine; imaging for all sars inpatients shall be conducted in wards and patients shall wear masks. 2. the personal protection for imaging technician should be subject to the level-two protection requirements and, when necessary (imaging for critically ill patients), to level-three protection requirements. 3. x-ray fi lm magazine should be put into the isolation bag before imaging (one isolation bag for each person). 4. the imaging technicians should sterilize their hands before return to the radiological department and then remove protective clothing, gloves, and protective glasses at the designated place. they should wash hands thoroughly, gargle, take a shower, and change clean working clothes before work. 1. the department must strengthen interior ventilation and air disinfection . ultraviolet irradiation may be assumed at night, 2-3 times per day, 30 minutes per time, and peracetic acid spray conducted 2 times per day. 2. rigorous isolation measures: all subjects must wear masks. the operation staff must wear protective masks, gloves, and gowns, with masks changed every 4 h and gowns replaced in time, and should use disposal bed sheet and shoe covers, and dispose of and replace them timely after the examination. 3. sars examination zone should be separated from conventional patient examination zone and examination staff shall be fi xed. if feasible, special chest x-ray examination room and x-ray machines may be set up in the isolation zone to avoid cross-infections. 1. the protective measures for subjects and operation staff are similar to those required about isolation protection in radiological department. 2. all the subjects must wear masks and, for examination in ct room, shall minimize the stay time as much as possible. 3. after the patient leaves, the environment and articles which may be polluted by patients shall be given terminal disinfection. 4. the department must enhance interior ventilation and air disinfection and, in addition to use of chemical disinfectant spray, may also initiate long-term ultraviolet irradiation at night for disinfection. 5. a pathway specifi c for sars patients should be established. chest x-ray diagnosis the imaging manifestations of sars are closely related to its pathological changes. besides edema, infl ammatory cell infi ltration and other nonspecifi c infl ammatory changes, the more prominent pulmonary pathological feature of sars is abundant exfoliation of epithelial cells, inducing obvious thickening and damage of alveolar septum, as well as significant organization of effusions inside alveolar cavities. all the three changes of infl ammation in early phase (degeneration, exudation, hyperplasia) are observable. the mechanism of acute lung injury induced by sars may be ascribed to sars coronavirus direct attack of alveolar epithelia and alveolar capillary epithelia or (and) indirect damage via lymphocyte, macrophage and effector cells, and lymphokine, cytokine, and infl ammatory mediator released by them. this suggests that besides viral direct infection, immune reactions are also involved in lung injury. in severe lung injury, pulmonary interstitial and pulmonary parenchymal air cavity changes detected by autopsy pathology, especially pulmonary air cavity not completely fi lled by lesion or fi lled but coexistent with unfi lled alveoli, are visualized as ground-glass-density shadows on x-ray and ct. varying degrees of residual lung cavity present with different densities of ground-glass shadows on thin-layer ct or high-resolution ct (hrct). complete fi lling of the alveolar space leads to pulmonary consolidation shadows. once pulmonary edema and alveolar hyaline membrane are formed, patients clinically manifest decreased lung compliance, progressive dyspnea, and intractable hypoxemia. it is such rapid pathological changes of sars that give rise to characteristic presentations by chest x-ray examination, including rapid progression of lesions, diverse morphological states, and wide range, often spanning several segments and lobes. therefore, dynamic observation of chest image holds great signifi cance for diagnosis of sars. ground-glass-density shadows are pathologically the result of lesions mainly at pulmonary interstitium and alveoli, with pulmonary interstitial lesions frequently complicated by alveolitis. ground-glass-density shadows incurred by alveolar consolidation are attributable to partial fi lling of alveoli or coexistence of fi lling and unfi lled alveoli. such shadows on x-ray and ct can be determined if the density of lesions is lower than that of blood vessels, and blood vessel presentations are observable inside. the density of lesions lower than that of pulmonary hila on x-ray could also help identify ground-glass-density shadows. pulmonary consolidation shadows are pathologically due to fi lling of alveoli by pathological tissues, which is frequently complicated with lung interstitial lesions. pulmonary consolidation could be confi rmed if the lesions are found with higher density than vascular shadows and without vascular shadows inside but with air bronchogram on x-ray and ct. it could also be determined if the lesions present higher density than pulmonary hila or similar density to mediastinum. the lesions are solitary or multiple in small patches, and some lesions are distributed along lung lobes and segments. 96 % of sars patients may develop subsequently solitary and multiple small patchy shadows within seven days of onset, with relatively low density, blurry borders, and irregular shapes, predominantly solitary. occasionally, the pulmonary textures around lesions are revealed increased and thickened and mainly distributed at peripheral area, more commonly at bilateral inferior lungs. since chest x-ray can poorly visualize relatively small lesions and at sometimes posteroanterior x-ray can hardly display lesions overlapping with heart shadows, orthotopic chest imaging should be conducted at the same time ( fig. 2.9 ). the conditions of most patients become worse within 14 days after onset. the small patchy shadows in the early phase may progress into large patchy, multiple, or diffusive lesions. the lesions may spread from unilateral lung to both lungs and from one lung fi eld to several fi elds. severe patients may demonstrate obvious changes 1-2 days after onset. lesions present mainly as ground-glass-density shadows or coexist with lung consolidation shadows. the center of some shadows with high density indicates consolidation, and relatively low-density peripheral area suggests ground-glass fig. 2.9 the 18-year-old male patient diagnosed with sars. the specifi c segment rt-pcr of srs virus is positive. on day 5 of onset, the right inferior lung fi eld was observed with patchy ill-demarcated shadows of increased density density which may be complicated with thickening and increase of pulmonary textures. severe patients may develop "white lung." some cases may have pneumothorax, pneumomediastinum, and subcutaneous emphysema after the use of ventilator. a small part of lesions adjacent to the pleura may be complicated with local pleural thickening or revealed with mild tentiform adhesion. pleural changes may reside after absorption of intrapulmonary lesions. complication of slight pleural effusions may also be observable (fig. 2.10 ). the lesions of sars patients begin to be absorbed in 15-30 days, which can be completely absorbed for majority patients. a few patients may show pulmonary fi brosis or pulmonary interstitial hyperplasia, with apparent pulmonary interstitial hyperplasia occurring 30-40 days after the onset. the intrapulmonary patchy shadows present with shrinkage and increase of density to gradually form high-density cordlike and honeycomb-shaped shadows in lungs. severe pulmonary interstitial proliferation could dwindle the lung volume and make the mediastinum shift to the affected side. the imaging may reveal local irregular high-density plaque and cord-shaped shadows. occurrence of intrapulmonary honeycomb-shaped shadows and tractional bronchiectasis is the characteristic of pulmonary interstitial fi brosis (fig. 2.11 ). as rapid change is an important feature of sars chest x-ray manifestations, dynamic x-ray studies and observations could provide evidences for sars defi nite diagnosis. therefore, observation of the disease dynamic change is critical to sars chest x-ray, which is a major difference from x-ray examinations of other pneumonia. in the early phase and progression phase of sars, lung foci may undergo remarkable changes within a short time (24 h at the shortest), such as expansion and dissemination, which are characterized by changes in shape, extent, and site of the foci. this suggests that clinical physicians shall conduct chest orthotopic imaging once every 24-48 h after the sars patients are hospitalized so as to ascertain the disease changes. imaging dynamic changes are associated with multiple factors, such as age, original underlying disease, treatment method, and effi cacy. the lesions are generally absorbed 14 days after onset but possibly on day 7 in some mild patients. the focal shadows may become smaller with the density gradually decreased. one characteristic dynamic change of sars is migrating change of lesions in some patients, suggesting that the foci fi rstly occur at the unilateral inferior lung fi eld and then spread to contralateral and/or superior and middle lung fi eld. patients with worse lesions may manifest wider imaging extent and new foci, and severe patients may develop "white lung." ards is a key cause leading to death of patients (fig. 2.12 ) . lung imaging examination is an important basis for sars diagnosis, and continual imaging examination could visualize the dynamic change characteristics of the disease. after sars infection, chest abnormal changes frequently occur on day 1-7. in initial phase, focal shadows are frequently seen, which might be unilateral but dominantly bilateral. ct reveals small patchy ground-glass-density shadows, some of which are quasi-circular. lesions present as solitary small patchy lung consolidation and multiple small patchy or relatively large patchy shadows. large foci may appear as ground-glass-density shadows and concurrent lung consolidation shadows, with relatively higher-density vascular shadows observable in the former. besides, some foci are found with increased vascular shadows at the periphery. in addition, lesions are unveiled frequently at the bilateral lung inferior fi elds and lung margins (fig. 2.13 ). lesions in majority patients may progress and worsen within 14 days after onset. the small patchy shadows in early phase could expand into large patchy, multiple, or diffusive lesions within 3-7 days. besides, lesions extend from unilateral lung to bilateral lungs, from one lung fi eld to multiple lung fi elds. most patients could develop the severest infi ltration of lungs on day 8-14, namely, peak phase or "critical" phase. ct presentations are still predominated by ground-glass-density shadows, which may be complicated by pulmonary consolidation. lesions are commonly multiple and in diffusive distribution at both lungs, and lesions of varying shapes may coexist, with quasi-circular foci relatively common. some cases appear with ground-glass-density shadows all the time from onset to absorption of lesions. when ground-glass-density shadows are complicated by pulmonary consolidation, the large patchy, small patchy, or quasi-circular ground-glass-density shadows could be observed with pulmonary consolidation shadows of relatively high density. ground-glass density and pulmonary consolidation may also occur at different parts of the lung and could be visualized on the same layer or different layers of ct. for lesions typical of pulmonary consolidation, pulmonary consolidation appears as plaque-shaped high-density shadows or consolidation signs at lung lobes and segments. in addition, lesions are frequently recorded at lung lobe segment of the inferior lobe and outer band of lung fi eld (fig. 2.14 ) . lesions usually begin to be absorbed 2-3 weeks after onset, with the shadow shrunk and density gradually reduced and absorbed. although the clinical symptoms of some patients improve and disappear, light ground-glass-density shadows could still be seen in lungs on ct. it may prolong a relatively long time. during the absorption of intrapulmonary lesions, there is also concurrent pulmonary interstitial proliferation, which begins to be absorbed under dynamic observation. some lesions may develop into pulmonary interstitial fi brosis, characterized by lobular septum, intralobular septum, interstitial thickening, and subpleural curvature imaging signs as well as regional irregular high-density plaque and cord-like presentations. occurrence of intrapulmonary honeycomb manifestations and tractional bronchiectasis signifi es pulmonary interstitial fi brosis ( fig. 2.15 ). sars complication is an important factor affecting prognosis, which should be given enormous attentions. during sars treatment, antiviral drugs are administered as well as glucocorticoids and broad-spectrum antibiotics. some patients may still need airway positive pressure ventilation, tracheotomy, and tracheal intubation. reasonable use of such measures is of great signifi cance for improving the treatment rate and lowering mortality of sars patients. however, to be noted, such treatment drugs and methods may incur adverse reactions and cause complications. sars complications already detected encompass pulmonary secondary infection, mediastinal and subcutaneous emphysema, pneumothorax, bone aseptic necrosis, empyema, brain secondary infection, and pulmonary interstitial interstitial fibrosis in the early phase, sars manifests bronchiolar and peripheral interstitial pneumonia, which could further progress into patchy ground-glass blurry shadows at bilateral superior, middle and inferior lung fi elds, and consolidation and air bronchogram at different layers alveolar consolidation, with interstitial lesions present persistently and mostly reversible. in the recovery phase, only some cases develop into pulmonary interstitial proliferation and then cause fi brotic changes. pulmonary interstitial proliferation and fi brosis degree and outcome are associated with the extent of pulmonary involvement in peak phase, occurrence of complications, and treatment. generally, if lesions on chest image are light in peak phase, there will be fewer pulmonary changes left in recovery phase. contrarily, patients with diffusive lung interstitial thickening in recovery phase are often those suffering from relatively severe pulmonary injury in peak phase and involvement of the great mass of lungs, particularly patients with recurrent conditions, protracted course, and concurrent infections. researches demonstrate that 7-8 % of sars patients have pulmonary fi brosis of varying degrees and male patients display relatively more obvious pulmonary impairment than female patients. primary manifestations are lung dysfunction, such as decrease of pulmonary diffusion capacity, lung capacity impairment, and change of total lung capacity particularly residual volume. generally obvious pulmonary interstitial proliferation occurs at about 30-50 days after onset, which presents fi rstly lobular septum and intralobular thickening and subpleural curvature shadows. then there is the shrinkage of intrapulmonary patchy shadows with increase of density and gradual formation of high-density cord and honeycomb shadows inside lungs. severe pulmonary interstitial proliferation could diminish lung volume and compel the mediastinum to affected side. besides, pulmonary interstitial proliferation could occur extensively inside lungs, predominated by lobular septum, intralobular interstitial thickening, and subpleural curvature shadows, as well as irregular high-density plaque and cord-like shadows at some regions. honeycombshaped shadows and tractional bronchiectasis are signs of pulmonary interstitial fi brosis. ct reveals cord, reticulate, and honeycomb-shaped shadows, and hrct could better visualize the subtle abnormal changes of pulmonary interstitium, such as lobular septum thickening, intralobular interstitial thickening, subpleural curvature shadows, and honeycomb-shaped shadows. whether the cord-like and reticulate changes visualized by ct are completely absorbed or induce persistent fi brosis still entails long-term follow-up observation ( fig. 2.16 ). numerous sars patients develop osteonecrosis after recovery, which mostly appears as femoral head necrosis and also possibly necrosis of the ankle joint, knee joint, and shoulder joint. scans of bilateral hips and knees of 82 sars patients in rehabilitation phase disclosed that 51 patients (62 %) suffered from osseous abnormality, of which bone ischemia accounts for 17 % and implication of three sites for 20 %. its pathogenesis still remains not completely ascertained, and it may be ascribed to improper use of hormones and injury of sclerotin from sars viruses, bacterial embolism, lipid embolism, or multiple mixed factors. the primary pathological change of aseptic necrosis of femoral head is degenerative alteration due to femoral head blood circulation disturbance, mainly characterized by limping and pain. the lower limb of the affected side is relatively shorter and presents with light fl exion and adduction, as well as with slightly limited abduction and intorsion. initial x-ray examinations suggest that the involved femoral head has normal appearance but inhomogeneous density, with spotted osteoporosis. gradually ensue the increase of density as a result of osteonecrosis, which meanwhile is interspersed with some transparent shadows. femoral head may become fl at, fragmented, and irregular in contour from pressures. typical femoral head necrosis appears in the shape of wedge, with the base toward articular surface and the top toward metaphysis. since lesions are dominantly confi ned at the anterosuperior area of femoral head, lateral imaging of hip joints could clearly reproduce the extent of lesions. the necrotic area is encircled by a ring of transparent band. hip articular cavity may become wider and irregular due to fl attening of the femur. sometimes, there may be complication of dislocation. the fragments of femoral head may be shed off into the joint, giving rise to joint mouse. normal femoral head is located at the acetabular center in the transverse ct image and may appear in the shape of sphere or hook on different layers, with delicate high-density bone cortex at the periphery. the high-density trabeculae inside femoral head are arranged in stelliform formation from the center continually to bone cortex, named as "stelliform sign." ct manifestations of aseptic necrosis of femoral head can be divided into early and advanced phase. in the early phase, the stelliform sign formed by the trabeculae of femoral head is deformed or absent, presenting spotted and patchy shadows of increased density inside femoral head and fasciculate changes or mutual fusion at its periphery. in the advanced phase, femoral head become ruptured and out of shape, and absorption of sclerotin between bone fragments appears as irregular low-density area, with stelliform sign disappeared. mri manifestations of aseptic necrosis of femoral head include: necrotic areas of femoral head of early cases have no repair reactions or mechanic collapse and still maintain normal lipid signals of medullary bones. however, lowsignal margins incurred by sclerosis reactions could be seen at the periphery of foci. the t1 high signals of normal femoral head on t1-weighted image are detected with black linear low signals (representing demarcation between normal and ischemic bone tissues). on t2-weighted image, a high-signal line is visible at the medial low-signal sclerotic reaction line, thus forming the typical double-line sign. the double-line sign could refl ect congestion and infl ammation of granula-tion tissues and proves specifi c to aseptic necrosis of femoral head. severe infl ammation, congestion, fi brosis, and sclerosis could greatly deplete the amount of lipids of femoral head, which presents liquid-like signals on mri, low signals on t1-weighted image, and high signals on t2-weighted images. advanced cases are typical of fi brosis and sclerosis, and the affected area present with fi brotic mri features, that is, low signals on t1-and t2-weighted and proton densityweighted imaging. complication with effusions in hip joints appears as t1 low signals and t2 high signals. based on mri presentations, aseptic necrosis of femoral head could be classifi ed into four phases: phase i is characterized by homogenous or inhomogeneous low-signal areas adjacent to the joint above the femoral head on t1-weighted imaging; phase ii is visualized with wedge-shaped lowsignal bands; phase iii presents with sequestrum crescent sign and cortex collapse; phase iv manifests joint degeneration and joint space narrowing besides presentations of phase iii. in light of the aforementioned mr staging criteria, sars cases complicated by aseptic necrosis of femoral head already reported are mostly in phase ii. additionally, aseptic necrosis after use of hormones, besides most frequently involvement of the femoral head, could also implicate other joints, such as the knee joint, elbow joint, ankle joint, and acetabulum. therefore, if necessary, mr examinations of other joints shall be conducted to ascertain the extent and degree of lesions. if sars patients complain of pain at both hip joints and limited movement, ct scan or mri examination of both hip joints is recommended to conclude defi nite diagnosis. x-ray plain scan is not sensitive to early bone ischemic necrosis, which therefore is not preferred (fig. 2.17 and 2.18 ). the fi rst case of severe acute respiratory syndrome in heyuan city, guangdong province analyses on one case of severe acute respiratory syndrome "super transmitter" and chain of transmission a novel coronavirus associated with severe acute respiratory syndrome coronavirus as a possible cause of severe acute respiratory syndrome identifi cation of a novel coronavirus in patients with severe acute respiratory syndrome identifi cation of severe acute respiratory syndrome in canada a cluster of cases of severe acute respiratory syndrome in hong kong characterization of a novel coronavirus associated with severe acute respiratory syndrome the genome sequence of the sars-associated coronavirus identifi cation and molecular cloning and sequence analysis of a novel coronavirus from patients with sars by rt pcr koch's postulates fulfi lled for sars virus comparative analysis of the sars coronavirus genome: a good start to a long journey the quantitative detection of anti-coronavirus antibody titer in medical personnel closely contacted with severe acute respiratory syndrome patients measurement of subsets of blood t lymphocyte in 93 patients with severe acute respiratory syndrome and its clinical signifi cance isolation and characterization of viruses related to the sars coronavirus from animals in southern china mass spectrometric characterization of proteins from the sars virus: a preliminary report research advance in origin of severe acute respiratory syndrome virus severe acute respiratory syndrome in 78 patients:a retrospective study clinical features and short-term outcomes of 144 patients with sars in the greater toronto area epidemiological determinants of spread of causal agent of severe acute respiratory syndrome in hong kong preliminary analysis of the infectious atypical pneumonia and its prevention and control measures clinical analysis of 18 pediatric cases with severe respiratory syndrome association of disease progression with dynamic alterations of clinical immunological and virology factors in patients with severe acute respiratory syndrome detection and analysis of antibody of severe acute respiratory syndrome associated coronavirus early diagnosis of sars coronavirus infection by real time rt-pcr sars. people health publishing house clinical pathology and pathogenesis of severe acute respiratory syndrome study on etiology and pathology of severe acute respiratory syndrome a clinicopathological study on 3 cases of severe acute respiratory syndrome pathological and ultramicrostructural changes of tissues in a patient with severe acute respiratory syndrome lung pathology of fatal severe acute respiratory syndrome angiotensin-converting enzyme 2 is a functional receptor for the sars coronavirus clinical progression and viral load in a community outbreak of coronavirus-associated sars pneumonia: a prospective study in vitro detection of apoptosis in monocytes/macrophages infected with human coronavirus haematological manifestations in patients with severe acute respiratory syndrome sars coronavirus replicase proteins in pathogenesis development of a standard treatment protocol for severe acute respiratory syndrome sars coronavirus pathogenesis: host innate immune responses and viral antagonism of interferon management of critical severe acute respiratory syndrome and risk factors for death clinical and chest x-ray characteristics of 5 cases with severe acute respiratory syndrome in children in shenzhen area severe acute respiratory syndrome in taiwan: analysis of epidemiological characteristics in 29 cases mri in the following up of the osteonecrosis in sars patients measurement of subgroups of peripheral blood t lymphocytes in patients with severe acute respiratory syndrome and its clinical signifi cance establishment of fl uorescent pcr method for detection of sars coronavirus and the clinical trial morphology and morphogenesis of severe acute respiratory syndrome (sars) associated virus albert osterhaus profi le. the virus collector diagnosis of severe acute respiratory syndrome (sars) by detection of sars coronavirus nucleocapsid antibodies in an antigen-capturing enzyme-linked immunosorbent assay proliferative growth of sars coronavirus in vero e6 cells chest x-ray imaging of patients with sars investigation of chest x-ray manifestations of severe acute respiratory syndrome clinical characteristics and chest x-ray manifestations in sars ct appearances and dynamic changes in severe acute respiratory syndrome early x-ray and ct manifestations of sars severe acute respiratory syndrome: radiographic review of 40 probable cases in toronto canada thin-section ct in patients with severe acute respiratory syndrome following hospital discharge: preliminary experience ct and radiography of bacterial respiratory infections in aids patients thin-section ct of severe acute respiratory syndrome: evaluation of 73 patients exposed to or with the disease severe acute respiratory syndrome: radiographic and ct fi ndings imaging of severe acute respiratory syndrome in hong kong chest ct manifestations of severe acute respiratory syndrome chest roentgenographic features of severe acute respiratory syndrome imaging follow-up of sars patients complicated with pulmonary fi brosis x-ray contrast study of severe and ordinary sars patients complicated with pulmonary fi brosis and femoral head necrosis overview and outlook of treatment of sars patients clinical presentations and outcome of severe acute respiratory syndrome in children transmission dynamics and control of severe acute respiratory syndrome key: cord-292871-vgposxom authors: falsey, ann r.; mccann, robert m.; hall, william j.; criddle, mary m.; formica, maria a.; wycoff, dennis; kolassa, john e. title: the “common cold” in frail older persons: impact of rhinovirus and coronavirus in a senior daycare center date: 2015-04-27 journal: j am geriatr soc doi: 10.1111/j.1532-5415.1997.tb01474.x sha: doc_id: 292871 cord_uid: vgposxom objective: to evaluate the incidence and impact of rhino‐virus and coronavirus infections in older persons attending daycare. design: prospective descriptive study. setting: three senior daycare centers in rochester, new york. patients: frail older persons and staff members of the daycare centers who developed signs or symptoms of an acute respiratory illness measurements: demographic, medical, and physical findings were recorded on subjects at baseline and during respiratory illness. nasopharyngeal specimens for viral culture as well as acute and convalescent sera for coronavirus 229e enzyme immunoassay (eia) were obtained for all illnesses. results: during the 44 months of study, 352 older persons experienced 522 illnesses. thirty‐five (7%) of 522 cultures were positive for rhinovirus and 37 (8%) of 451 serologies were positive for coronavirus 229e infection. the clinical syndromes associated with rhinovirus and coronavirus infection were similar and characterized by nasal congestion, cough, and constitutional symptoms. no patient died or was hospitalized, but approximately 50% had evidence of lower respiratory tract involvement. the average illness lasted 14 days. during the same period, 113 staff developed 338 respiratory illnesses. eight percent were identified as coronavirus and 9% as rhinovirus. cough, sputum production, and constitutional symptoms were significantly more common among older persons. conclusions: rhinovirus and coronavirus 229e are common causes of moderately debilitating acute respiratory illnesses among older persons attending daycare. objective: to evaluate the incidence and impact of rhinovirus and coronavirus infections in older persons attending daycare. design: prospective descriptive study. settlng: three senior daycare centers in rochester, new york. patients: frail older persons and staff members of the daycare centers who developed signs or symptoms of an acute respiratory illness measurements: demographic, medical, and physical findings were recorded on subjects at baseline and during respiratory illness. nasopharyngeal specimens for viral culture as well as acute and convalescent sera for coronavirus 229e enzyme immunoassay (eia) were obtained for all illnesses. results: during the 44 months of study, 352 older persons experienced 522 illnesses. thirty-five (7%) of 522 cultures were positive for rhinovirus and 37 (8%) of 451 serologies were positive for coronavirus 229e infection. the clinical syndromes associated with rhinovirus and coronavirus infection were similar and characterized by nasal congestion, cough, and constitutional symptoms. n o patient died or was hospitalized, but approximately 50% had evidence of lower respiratory tract involvement. the average illness lasted 14 days. during the same period, 113 staff developed 338 respiratory illnesses. eight percent were identified as coronavirus and 9% as rhinovirus. cough, sputum production, and constitutional symptoms were significantly more common among older persons. conclusions: rhinovirus and coronavirus 229e are common causes of moderately debilitating acute respiratory illnesses among older persons attending daycare. j am geriatr soc 45:706-711,1997. r morbidity and mortality in older persons. viruses, such as influenza and respiratory syncytial virus (rsv), have been shown to be the cause of serious disease in this age group. ' less is known about the impact of rhinoviruses and coronaviruses, the causative agents in the majority of "common colds.'" approximately 20 to 40% of upper respiratory tract infections (uris) in adults are caused by rhinoviruses, and 5 to 15% are caused by coronaviruses. more than 100 serotypes of rhinoviruses and two major serotypes of coronavirus (229e and oc43) have been ider~tified.~-~ reinfections with both viruses occur throughout life, in part because of multiple serotypes and incomplete immunity.'-' while these infections lead to significant time lost from work and school, they generally do not result in serious disease in children or young adults."i2 rates of acute respiratory tract infections diminish with advancing age, most likely as a result of less frequent exposure^.'^ however, frail older persons attending adult daycare centers may represent a special population at increased risk of infection and at risk for complications from these common infection^.'^ the purpose of this study was to evaluate prospectively the prevalence and clinical features of rhinovirus and coronavirus 229e infections in frail older persons attending senior daycare. volunteers were recruited from three sites of a senior daycare program in rochester, new york. these facilities allow frail older persons who are nursing home-eligible by new york state medicaid standards to be maintained at home by providing comprehensive medical and social services. all attendees of the daycare program were recruited to take part in the study. daycare participants were scheduled to attend the center, on average, 3.5 days per week, with a minimum of once a week. ill subjects were encouraged to attend the program for evaluation by center physicians. in addition, all staff members with direct contact with older participants were also recruited for the surveillance study. informed consent was obtained from volunteers upon entrance into the daycare program. if volunteers were unable to provide informed consent, consent was obtained from their legal guardians. baseline information, including medical history and demographics, were obtained from daycare participant's medical records. subjects were examined at base-line, and arterial oxygenation saturation (sao,) was measured percutaneously by pulse oximetry (ohmeda-biox iv-boulder, co). baseline serum samples were collected from daycare participants and staff members. surveillance for acute respiratory illnesses took place between january 30, 1992, and october 2, 1995. possible respiratory illnesses among daycare workers or attendees were reported by the daycare staff to the project nurse for evaluation. an acute respiratory illness was defined as nasal congestion, sore throat, new or increased cough, wheezing, sputum production, or respiratory difficulty with or without fever. illness evaluations consisted of a directed history and physical exam, mcasurement of sao,, and nasopharyngeal swab for viral culture. subjects were evaluated each day they attended the center until symptoms resolved. end of illness was defined as resolution of symptoms and physical findings. if participants were well upon return to the center after an absence of several days, they were questioned as to when symptoms resolved. staff members who became ill answered brief questionnaires and had nasopharyngeal cultures taken. four-week convalescent sera were obtained from as many staff and participants as possible. viral cultures and sera were not collected from asymptomatic individuals. nasopharyngeal swabs were performed by gently rubbing the posterior nasal turbinates and posterior pharynx with cotton tip swabs. swabs were placed in veal infusion broth, transported to the laboratory on ice, and inoculated onto wi-38 cell cultures (biowhittaker, walkersville, md) within 6 hours of collection. tubes were incubated at 33°c on roller drums and observed for 10 days for cytopathic effect (cpe). rhinovirus infection was identified by typical cpe and confirmed by acid lability testing. serologic evidence of coronavirus infection was defined as a greater than 4 rise in coronavirus-specific igc as measured by enzyme immunoassay (eia). coronavirus antigens were prepared by expanding coronavirus 229e virus in wi-38 cells. at the point of visible cpe, monolayers were scraped, and cellular material was pelleted in a sorvall at 500 g for 15 minutes. pellets were resuspended in 0.5% np40, and suspensions were sonicated every 15 minutes for 1 hour. eia plates were prepared by coating 229e antigen to immulon round bottom plates in bicarbonate buffer and stored at 4°c overnight. control plates were prepared by using uninfected wi-38 cell lysates prepared by the above procedure. acute and convalescent sera was added in serial 2-fold dilutions from 1:400 to 1:102,400 in duplicate to control and antigen plates. serum igc was detected with alkaline phosphatase conjugated goat a human igg followed by substrate. coronavirus titer was defined as the highest titer with an optical density (o.d.) 2 0.100 and at least twice the o.d. of the control plate. as part of an ongoing surveillance project for respiratory infections in the daycare centers, all nasal specimens were cultured for other viruses (influenza, rsv, parainfluenza, enteroviruses), and all sera were tested by eia for influenza a and b, parainfluenza, and rsv infection. details of these methodologies and the results of this project will be presented in a future publication. during the 44 months of study, 352 older daycare attendees were enrolled and participated in the surveillance project for a mean of 43.6 2 33.7 months. all illnesses were evaluated with viral cultures, and 451 of 522 specimens (86%) had acute and convalescent sera available for coronavirus eia. both coronavirus 229b and rhinovirus infections were identified in approximately 7% of all illnesses. viral cultures were positive for rhinovirus in 35/522 (6.8%) specimens and coronavirus serology was positive in 37/451 (8.0%) paired sera tested. sixty-one subjects experienced 72 separate infections. fifty persons had a single infection, and 11 subjects had multiple infections. three subjects had two different rhinovirus infections, two subjects had two episodes of coronavirus infection, and six people had one rhinovirus and one coronavirus infection each during the study period. six individuals had evidence of concurrent mixed viral infcctions. four persons had 4-fold rises in both rsv and coronavirus titers. two subjects, one with coronavirus infection and one with rhinovirus, had serologic evidence of parainfluenza infection. thus, in 32 illnesses, coronavirus 229e was the sole pathogen identified, and in 34 illnesses, rhinovirus was the only organism found. the clinical features associated with illnesses were analyzed only in cases where either coronavirus or rhinovirus was the only agent identified. coronavirus 229e infections were identified most commonly during the winter and early spring whereas rhinovirus activity was sporadic but tended to be more frequent in the summer and fall ( figure 1 ). interestingly, when coronavirus was circulating, rhinovirus activity nearly ceased. while no clear outbreaks of infection occurred at any daycare center, periods of viral activity typically involved small clusters of three to six older persons and several staff members. clusters of rhinovirus or coronavirus infections were not infrequently preceded by an ill staff member. in the spring of 1995, a 4-week period of increased coronavirus activity at one center, involving seven people, was preceded by 2 days with a coronavirus-infected staff member. the mean age of the 61 subjects who experienced illnesses was 78.5 ? 7.1 years old. the demographics and clinical characteristics of the group that became infected were reflective of the group as a whole except that diabetes was less common in the infected group (1 1 vs 27%, p = .01) ( table 1 ). approximately two-thirds of subjects had underlying cardiac disease, and 21 yo had chronic pulmonary disease. in addition, there were no significant differences between the group infected with rhinovirus compared with the group infected with coronavirus. the clinical syndromes produced by coronavirus 229b and rhinovirus were nearly identical ( table 2 ). most illnesses were characterized by nasal congestion, cough, and constitutional symptoms. low-grade fever was not uncommon, but temperature of 101°f or greater occurred in only three subjects, two with coronavirus and one with rhinovirus. although subjects generally recovered without significant sequelae, illnesses lasted, on average, 14 days. approximately 50% of illnesses were associated with evidence of lower respiratory tract involvement as defined by the presence of sputum production, shortness of breath, new wheezing and/or new rales on exam. thirty-six percent complained of feeling short of breath. twenty-two percent had wheezing, and 46% had rales found on auscultation of the chest. notably, few subjects had wheezing (3%) or rales (13%) on baseline examinations. although mean sao, measurements for the group dropped only a modest amount from 95.3 ? 1.9 at baseline to 94.2 ? 2.0, p = .003 when ill, seven individuals had a greater than 4-point drop in sao, during illness. one individual with coronavirus had a fall in sao, from a baseline of 95% to 89% while ill. four illnesses were evaluated with chest roentgenograms, of which three were normal and one showed congestive heart failure. nine percent of subjects with rhinovirus and 16% with coronavirus received bronchodilators. antibiotics were frequently prescribed in both groups ( table 3) . one individual with rhinovirus infection was hospitalized and treated for congestive heart failure with complete recovery. no deaths occurred. of note, the six illnesses associated with rsv or parainfluenza were not significantly different from those with rhinovirus or coronavirus alone. when individuals with underlying cardiac or pulmonary disease were compared with those without, no significant difference in the severity of rhinovirus or coronavirus infections was noted. wheezing was found to be equally prevalent in those with chronic lung disease as in those without pulmonary problems (23 vs 22%). subjects who developed wheezing during their illnesses were symptomatic slightly longer (15.3 5 6.6 days vs 12.4 2 5.9, p = .095) and received antibiotics much more frequently (86% vs 33%, p < .001) than those who had no evidence of bronchospasm. during the same time period, 113 staff members developed 338 respiratory illnesses ( figure 1b) cough, sputum production, and constitutional symptoms were significantly more common among older persons. twenty percent of daycarc staff missed work secondary to rhinovirus or coronavirus illnesses. this study represents the largest series to date of prospectively identified cases of coronavirus 229e and rhinovirus infections in older persons. our data show that both viruses are common in the daycare setting, and although infections did not generally result in serious complications, many were clinically significant with prolonged symptoms and evidence of lower respiratory tract involvement. relatively little information has been reported on the impact of these common viruses in frail older populations. in a study of acute respiratory infections in nursing home patients by nicholson et al., 12 persons were identified as having coronavirus infection by eia, and three had evidence of lower respiratory in~olvement.'~ in the same study, 11 individuals were found to have rhinovirus infection, one of whom had lower respiratory tract disease. in our previous study of 14 nursing home residents with rhinovirus infections, all illnesses were mild, with only 21 % complaining of sputum production and 14% noting shortness of breath. no patient was hospitalized or died.16 in contrast, in a recent report by wald et al. describing an outbreak of rhinovirus infection that affected 35 institutionalized older persons, a high percentage (66%) of subjects had lower respiratory tract symptoms, and 52% had new abnormalities on lung exam.i7 persons with underlying lung disease had more severe illnesses, with two individuals requiring hospitalization, one radiographically documented pneumonia, and one death secondary to respiratory failure. although all participants of the present study recovered without serious sequelae in contrast to our previous study in the nursing home, these subjects were more seriously ill. similar to the study by wald and colleagues, our current subjects frequently had evidence of lower respiratory tract involvement with new auscultatory findings, symptoms of dyspnea, and a drop in arterial oxygen saturation. additionally, subjects were ill for approximately 2 wecks compared with the usual 2 to 4 days of illness in the young healthy adult. it is also note worthy that, during this era of increasing antimicrobial resistance, antibiotics were prescribed during 50% of illnesses caused by rhinoviruses and coronaviruses. rhinovirus and coronavirus infections have only rarely been found to be the cause of pneumonia in adults, even in severely immunocompromised patient^.^.^.'".'^ however, both viruses have been implicated as a precipitating factor in exacerbations of asthma and copd.''-2z consistent with the published literature, no older subjects in our study had evidence of invasive disease or pneumonia. this contrasts sharply with infection with influenza, rsv, or parainfluenza in older persons where rates of pneumonia can be high and excess mortality rates have been n~t e d . ' * '~-~~ the relatively milder illnesses associated with rhinoviruses and coronaviruses likely reflect the biological characteristics of these viruses. rhinovirus replicates poorly at core body temperature of 37°c and appears to produce symptoms via chemical mediators rather than direct viral invasion.26 although less well studied because of fastidious growth requirements, coronaviruses also do not appear to cause significant damage to respiratory epithelium. the lower respiratory signs of wheezing and rales without evidence of pneumonia in our patients suggest that these viruses cause disease in older persons by aggravating preexisting congestive heart failure or inducing bronchospasm. the incidence of rhinovirus and coronavirus 229e infections in the daycare centers was found to be nearly identical. since we tested only for one of the two most common serotypes of coronavirus infection and the incidence of 229e and oc43 are roughly equivalent, it is possible that the number of illnesses attributable to coronavirus may actually have been double what was reported. the small intermittent clusters of infections in staff and participants at each center suggest that these viruses were introduced into the centers from outside sources. however, once introduced, some element of nosocomial spread is also likely because of close contact between staff and older persons. the daycare policy that encourages participants to attend daycare so they can receive medical attention from the on-site physicians may have influenced the overall incidence of infection in the daycare. however, this concern must be balanced with the need to provide medical care to this very debilitated group of older persons. the daycare center requires that employees with febrile illnesses and/or uncontrolled respiratory symptoms stay out of work until symptoms resolve. however, many of the common respiratory viruses do not cause fever or severe symptoms in young healthy persons, and, therefore, most healthcare workers suffering from upper respiratory illnesses do not miss work. because many individuals, both staff and participants, will be experiencing 'colds' and be in close contact throughout the winter months, good infection control practices in daycare centers are critical. most respiratory viruses, with the exception of influenza viruses, require relatively close contact for transmission.'' rhinoviruses can be transmitted either by fomites and autoinoculation or by aerosol ~p r e a d . '~.~~ although less information is available about the transmission of coronavirus, it is likely they are also spread by fomites and close contact.' many authorities in the field of pediatrics feel that control of respiratory infections in children's daycare centers is nearly impossible because of the nature of young children's activities.30 however, in senior daycare centers, the outlook for infection control may be more hopeful. since transmission of these agents is caused, in part, by fomites, careful handwashing may interrupt ~pread.'~ in addition, architectural design of centers with attention to square feet per resident and adequate ventilation may be important for future control of respiratory infection^.^' in summary, coronavirus 229 and rhinoviruses were found to be common causes of acute respiratory illnesses among the staff and participants of a senior daycare program. although illnesses were not as severe as those associated with other viral pathogens such as influenza and rsv, older subjects were moderately debilitated by these infections. attention should be paid to basic infection control principles to limit spread of these common viruses. respiratory syncytial virus or influenza? lancet isolation of rhinoviruses and coronaviruses rhinovirus infections in tecumseh, michi-1993 gan: frequency of illness and number of serotypes vitro and in volunteers: evidence of heterogeneity among 229e-related strains the time course of the immune response to experimental coronavirus infection of man characteristics of illness and antibody response the behavior of recent isolates of human respiratory coronavirus in rhinovirus infections in rhinovirus infections in an industrial population. 1. the occurrence of illness virologic studies of acute respiratory disease in young adults coronavirus infection in working adults seroepidemiologic survey of coronavirus 2298 infections in a population of children rhinoviruses in seattle families, 1975-1979 acute respiratory illness in an american community: the tecumseh study acute respiratory tract infections in daycare centers for older persons viral respiratory infections in the institutionalized elderly: clinical and epidemiologic findings a rhinovirus outbreak among residents of a long-term care facility principles and practices of infectious diseases impact of respiratory virus infection in patients with chronic chest disease association of viral and mycoplasma pneumoniae infections with acute respiratory illness in patients with chronic obstructive pulmonary diseases viruses as precipitants of asthmatic attacks in children respiratory syncytial virus and influenza a infections in the hospitalized elderly parainfluenza outbreaks in extended-care facilities -united states excess pneumonia and influenza hospitalization in the us due 26 epidemiology and control of nosocomial virus infec hand-to-hand transmission of to infmenza epidcmics, 1970-78 726. home architecture and influenza-a attack rates the authors thank the staff and attendees of ils for their participation, christine brower for data management, and joanne prives for transcription assistance. key: cord-017252-88b3preq authors: morgan, carrie i.; shah, samir s. title: pneumonia date: 2014-02-20 journal: pediatric critical care medicine doi: 10.1007/978-1-4471-6356-5_6 sha: doc_id: 17252 cord_uid: 88b3preq respiratory diagnoses continue to make up a large number of admissions to the pediatric intensive care unit (picu), most notably lower respiratory infections including pneumonia. this chapter will focus on pediatric community-acquired pneumonia (cap), immunocompromised pneumonia, and aspiration pneumonia. the pathogenesis for developing pneumonia varies; it can occur by direct inhalation of infectious particles in the air or aspiration, direct extension from the upper airways, and hematogenous spread. there are multiple levels of defense against pathogen invasion including anatomic barriers, as well as innate and adaptive immunity, which may be compromised in picu patients. the etiologies of pediatric pneumonia vary depending on age, host condition, and environmental factors like time of year and location. viruses remain the most common form of lower respiratory tract infection in children, especially in neonates. community-acquired bacterial pneumonia continues to be most prevalent in younger children as well, most often affecting children less than 5 years of age who are otherwise healthy. despite immunizations and public health initiatives, the most common bacterial causes of cap have remained largely unchanged over the last several decades and include: streptococcus pneumoniae, staphylococcus aureus, haemophilus influenzae (including non-typable strains) and moraxella catarrhalis. pulmonary infection in an immunocompromised host provides a much broader differential and must be aggressively treated without delay. this chapter will also address various imaging modalities and typical findings with pediatric pneumonia. methods for pathogen identification are broad and range from non-specific markers of illness to invasive techniques for culture. the mainstay of therapy continues to be antibiotics tailored to the patient and presumed etiology; more novel therapies may include corticosteroids or macrolide antibiotics for immune modulation. in those patients with pneumonia with effusion or empyema, drainage therapies with thoracostomy tubes or a vats procedure may be indicated. respiratory diagnoses continue to make up a large number of admissions to the pediatric intensive care unit (picu) [ 1 ] . lower respiratory tract infections are considered to be any infection beneath the anatomic level of the vocal cords, including bronchitis, bronchiolitis, tracheitis, and pneumonia [ 2 ] . pneumonia remains an important cause of pediatric morbidity and mortality. there are nearly two million pneumoniarelated deaths worldwide each year among children 5 years of age and younger [ 3 , 4 ] . in the u.s., pneumonia causes over three million outpatient visits and more than 150,000 hospitalizations each year [ 5 , 6 ] . in the developed world, early recognition and availability of antimicrobial therapies and respiratory support have lessened the mortality of pneumonia, but its morbidities remain. while widespread use of the heptavalent pneumococcal conjugate vaccine in 2000 was associated with fewer pneumonia-associated complications in infants <1 year of age, complications remained unchanged or increased in school-age children and adolescents [ 5 ] . thus, despite our best efforts at prevention through vaccination, morbidities continue to plague our patients and pneumonia remains a common cause of pediatric hospital admission. this chapter will focus on pediatric community-acquired pneumonia (cap), immunocompromised pneumonia, and aspiration pneumonia. hospital acquired pneumonia is an important type of lower respiratory infection found in the picu, but it is discussed extensively in the chapter on hospital-acquired infections elsewhere in this textbook. the defi nition of pneumonia is generally accepted to be a lower respiratory illness with fever, respiratory symptoms including tachypnea, and often, radiologic evidence of parenchymal infi ltrates [ 7 ] . the world health organization (who) has defi ned pneumonia solely based on clinical fi ndings due to the lack of radiologic studies in many parts of the world [ 8 ] . determining the type of pneumonia can help guide clinical management. previously healthy children presenting with the signs and symptoms of a lower respiratory tract infection are generally considered to have cap. aspiration involves inhaling foreign material beyond the vocal cords, often causing aspiration pneumonitis (chemical pneumonitis) or pneumonia (an infectious process secondary to the aspiration) [ 9 , 10 ] . commonly aspirated materials in children include oropharyngeal secretions, gastric contents, water, hydrocarbon, lipid, and foreign bodies [ 11 ] . guidelines for admission to the icu are available for both young children and adults, and are summarized in table 6 .1 [ 12 , 13 ] . pneumonia can occur by direct inhalation of infectious particles in the air or aspiration, direct extension from the upper airways, and hematogenous spread. anatomic and cellular protection serves as the fi rst line of defense against potential pathogens. airway mucus traps inhaled toxins and microbes and helps to transport them up and out of the respiratory tract via ciliary beating and cough, a mechanism referred to clinically as mucociliary clearance [ 14 ] . when the microbe burden or virulence of the organism surpasses the abilities of these simple mechanical protections, the innate immune response is activated. the innate immunity is responsible for immediate recognition and control of microbial invasion. in mammals, conserved receptors enable rapid recognition of pathogens to begin elimination of the infection as well as initiate the adaptive immune response. activating the innate immune receptors in the airway epithelium leads to mobilization and activation of dendritic cells, t cells, and b cells that amplify antigen recognition, antibody production, and further cellular recruitment and infl ammation [ 15 ] . the specifi cs of these interactions and signaling cascades are beyond the scope of this chapter, but are further discussed in other chapters within this text. the lower respiratory tract remains generally clear of pathogens [ 2 ] . the mechanisms by which microbes are able adapted from refs. [ 12 , 13 ] to overwhelm defensive measures and result in pneumonia vary and depend on host conditions. the most common mechanism of pathogen entry is via inhalation of infectious particles, particularly in the case of specifi c organisms that spread via respiratory droplets such as mycobacterium tuberculosis. many viruses that cause lower respiratory tract infections are also spread utilizing aerosolized modes of transmission, including respiratory syncytial virus (rsv), infl uenza, and rhinoviruses. due to their smaller size compared with bacteria, viruses consolidate more effi ciently on smaller particles [ 16 , 17 ] . hematogenous spread results in pneumonia when bacteria in the bloodstream directly deposit in lung tissue. pulmonary aspiration can occur as a result of swallowing dysfunction, gastroesophageal refl ux, anatomic anomalies such as tracheoesophageal fi stulas, or an inability to protect the airway from oropharyngeal secretions. in the picu, many patients have neurologic diseases that coexist with one, if not several, of these aforementioned mechanisms. furthermore, impaired consciousness, as may occur with head injury, intoxication, sedation, and tracheal intubation, can also impair the ability to protect the airway, diminish the cough refl ex, and exploit the patency of the anatomical connection between the larynx and trachea [ 9 , 10 , 18 ] . direct aspiration of a large inoculum of infectious organisms can result when there is impairment of the host's anatomic defense, usually the gag and cough refl ex. this most commonly occurs in children with profound neurologic impairment or during tracheal intubation [ 19 , 20 ] . viruses still remain the most common cause of lower respiratory tract infection, especially in infants [ 21 ] . the occurrence of primary viral infections and co-infections with bacterial pneumonia are receiving more attention in recent years due to advances in detection methods to improve the reliability and sensitivity in diagnosis [ 22 ] . viruses have been found in approximately 50 % of sampled patients with a range of 43-67 %, although this prevalence is diffi cult to compare across studies that utilize different identifi cation techniques [ 22 -28 ] . the most commonly noted infectious viruses were rhinovirus, human bocavirus, human metapneumovirus (hmpv), and respiratory syncytial virus (rsv). human metapneumovirus causes signifi cant respiratory infection, accounting for 5-8 % of viral pneumonia cases [ 29 , 30 ] . human bocavirus, fi rst described in 2005, is detected in up to 10 % of children with respiratory infections [ 31 ] . however, co-infection with another virus occurs in more than half of human bocavirus infected children, making its role as a predominant respiratory pathogen unclear. one possible explanation for the high prevalence of viral coinfection with human bocavirus is that this virus is shed in respiratory tract secretions for a longer period of time than other viruses [ 32 -34 ] . other important respiratory tract pathogens include adenovirus, parainfl uenza viruses, and infl uenza a or b, all of which vary in prevalence based on season and epidemic periods. the most common complication of viral pneumonia is a secondary bacterial infection. bacterial co-infection occurs in about 15-33 % of pediatric patients hospitalized with a lower respiratory tract infection [ 23 ] . the most often occurring combination was rhinovirus and streptococcus pneumoniae , though it remains diffi cult to interpret the causal role of rhinovirus in lower respiratory tract infections [ 23 , 25 ] . rsv remains an important cause of bronchiolitis in infants and can often progress to pneumonia. a recent study noted that 40 % of children admitted to the picu with rsv bronchiolitis had bacterial co-infection [ 35 ] . community-acquired bacterial pneumonia continues to be most prevalent in younger children as well, most often affecting children less than 5 years of age who are otherwise healthy. despite immunizations and public health initiatives, the most common bacterial causes of cap have remained largely unchanged over the last several decades and include: streptococcus pneumoniae , staphylococcus aureus , haemophilus infl uenzae (including non-typable strains) and moraxella catarrhalis [ 7 , 8 , 21 , 23 ] . in developing countries, other bacterial and viral etiologies must be considered, including mycobacterium tuberculosis, h. infl uenzae type b (in unvaccinated areas of the world), and the measles virus [ 8 ] . in infants under 3-4 weeks of life, the most common etiologic agents include group b streptococcus, listeria monocytogenes, and gram-negative enteric bacteria. mycoplasma pneumoniae and chlamydophila pneumoniae (formerly chlamydia pneumoniae ), once considered to occur primarily among adolescents and young adults, are increasing being recognized as a cause of cap in younger children, including those less than 5 years of age [ 21 ] . there are many causes of immunodefi ciency in pediatrics including congenital, acquired (hiv/aids), or iatrogenic (during chemotherapy or after solid organ or stem cell transplant). these states can result in defi ciencies in humoral immunity, cellular immunity, and neutrophil availability or function, making the host susceptible to not only typical pneumonia etiologies, but many opportunistic agents. thus, the approach to an immunocompromised patient must be altered to consider the type and severity of immunodeficiency, as well as the temporal pattern after chemotherapy or transplant. other considerations that are important in immunocompromised patients include neutropenia, where a low white blood cell count can hinder the patient's ability to exhibit cxr fi ndings and the lack of infl ammation can alter the clinical presentation, and environmental factors and exposures that can cause geographic and temporal clustering of pathogens [ 11 ] . the causes of pneumonia following solid organ and stem cell transplant may follow a predictable temporal relationship. in the early post-transplant period (<1 month), infections from nosocomial or iatrogenic sources are most common. in the middle post-transplant period (1-6 months), donor-associated and opportunistic infections, including reactivation of latent infections, predominate; specifi c causes include cytomegalovirus (cmv), epstein-barr virus (ebv) or human herpes virus 6 (hhv6). late post-transplant period (>6 months) etiologies include community-acquired infections as well as infections associated with profound immunosuppression [ 36 , 37 ] . in an effort to diminish the risk associated with post-transplant immunosuppression, immunosuppressive agents (e.g., calcineurin inhibitors, high-dose corticosteroids) are used sparingly when possible and most protocols include anti-viral (especially cmv), anti-fungal, and pneumocystis jiroveci (pcp) prophylaxis [ 36 ] . still, many common infections continue to pose a great risk. for example, viral infections (e.g., rsv, infl uenza, adenovirus) cause greater virulence following solid organ or stem cell transplantation immediately after transplant when cellular immunity is profoundly low. later in the course of transplantation, fungi such as aspergillus spp. and candida spp. become more prevalent causes of pneumonia with long-term steroid therapy [ 11 , 37 ] . thus, when a pulmonary process is suspected, aggressive treatment with broad-spectrum antibiotics, antifungals, and antivirals must be employed. immunocompromised patients with pulmonary infi ltrates may rapidly progress to respiratory failure and, thus, often require icu care. infection must be aggressively treated without delay, but other conditions must also be sought including pulmonary hemorrhage, malignancy, idiopathic pneumonitis, or cardiac disease [ 11 , 38 ] . the clinical presentation of aspiration pneumonitis or pneumonia can vary and like other pneumonia etiologies, aspiration can result in acute lung injury (ali) or acute respiratory distress syndrome (ards) manifested by severe pulmonary infl ammation and alveolar-capillary permeability injury. it is estimated that approximately one-third of patients with aspiration pneumonitis develop ali/ards [ 39 ] . etiologies of aspiration pneumonia depend if the aspiration is community acquired or hospital acquired. bacteriologic studies in aspiration patients have shown that community acquired aspiration pneumonias are generally the same bacterium as cap, including h. infl uenzae , s. pneumoniae , s. aureus , and enterobacteriaceae species. in those patients who aspirated in a hospital setting, the most common organisms cultured were gram-negative enteric bacteria including pseudomonas aeruginosa . these recent studies failed to grow any anaerobic organisms, refuting the prior studies that endorsed anaerobes as common etiologies [ 10 ] . the role for imaging in pediatric pneumonia is to detect the presence of pneumonia, determine the location and extent, and identify complications such as effusion or empyema. modalities include chest radiographs (cxr), ultrasound (us), and computed tomography (ct) [ 11 ] . the presence of an infi ltrate on cxr, combined with clinical and other laboratory fi ndings can aid in the diagnosis of pneumonia. however, these modalities are not suffi ciently sensitive or specifi c to reliably differentiate between viral, bacterial, and atypical bacterial causes [ 40 ] . the main use for us is to identify and characterize a parapneumonic effusion or empyema and provide image guidance for chest tube placement. this modality is limited by availability of equipment and operators. chest ct is helpful to further evaluate diffi cult cases, particularly immunocompromised children with ill-defi ned infi ltrates on cxr, complex empyema or effusion, or recurrent or chronic pneumonia [ 11 ] . imaging fi ndings in pneumonia can be non-specifi c, but when combined with other factors such as patient age, immune status, and historical information, they may help to narrow the differential diagnosis. in viral pneumonia, the most common fi ndings are bilateral symmetrical parahilar and bronchial opacities with or without atelectasis and air trapping; pleural effusions are rare ( fig. 6.1 ). this is in contrast to bronchopneumonia, a form of bacterial pneumonia that begins as peribronchiolar infl ammation and spreads to the lung parenchyma. bacterial pneumonia is characterized by consolidation and fi lling of the alveolar air spaces with exudate, infl ammation, and fi brin. bronchopneumonia is typical of many bacteria including s. pneumoniae , h. infl uenzae , s. aureus , and gram-negative enteric bacteria. the cxr often reveals fl uffy lobar consolidation or diffuse bilateral opacities extending peripherally, with or without associated pleural effusion. in aspiration pneumonia, the cxr may reveal ground-glass or consolidative opacities predominantly involving the middle and lower (dependent) lobes [ 41 ] . finally, atypical pneumonia etiologies include mycoplasma pneumoniae , chlamydophila pneumoniae and, less commonly, legionella species. the cxr fi ndings for these atypical causes are varied. diffuse interstitial infi ltrates are characteristic though other fi ndings include lobar consolidation, small bilateral pleural effusions, perihilar and peribronchial opacities that resemble butterfl y wings, or a bi-lobar reticular pattern ( fig. 6. 2 ) [ 42 , 43 ] . the etiology of pneumonia in the immunocompromised patient can be diffi cult to determine though further imaging can help elucidate the cause. respiratory failure in an immunocompromised child frequently necessitates a chest ct to better visualize the pattern and extent of disease, aid in diagnosis of the etiology, determine the need for more invasive procedures, and to increase the sensitivity of assessing treatment response [ 11 ] . fungal infections are more diffi cult to diagnose; classic fi ndings include pulmonary nodules on chest ct (fig. 6.3 ). the "gold standard" diagnosis of pneumonia is microbiological identifi cation of a pathogen from the lower respiratory tract [ 2 ] . obtaining a lrt specimen can be diffi cult, especially in children, as it may require an invasive procedure and can be contaminated with oropharyngeal bacteria. most children younger than 8 years of age cannot produce a suffi cient sputum sample, defi ned as <10 squamous or epithelial cells and >25 polymorphonuclear white blood cells per low power fi eld. therefore, most samples are obtained through either an endotracheal tube via aspiration or bronchoalveolar lavage [ 44 ] . other laboratory tests helpful in identifying the causative agent in cap can include blood cultures, viral polymerase chain reaction (pcr) tests, and bacterial serologies. commonly used diagnostic methods available for an individual microorganism may be found in table 6 .2 [ 8 ] . the clinician may also be limited by the capabilities of the laboratory in their institution for performing these tests. because of the diffi culties in determining the etiology of pneumonia, non-microbiologic approaches have been sought to differentiate serious bacterial infections from nonbacterial pneumonia [ 21 ] . many studies have evaluated markers including serum c-reactive protein (crp), blood white cell count (wbc), serum procalcitonin (pct), and erythrocyte sedimentation rate (esr), attempting to fi nd a test, or combination of tests, that would differentiate viral pneumonia from serious bacterial pneumonia necessitating antibiotic therapy [ 8 , 45 -49 ] . all of the aforementioned tests have limited utility in reliably differentiating viral from bacterial pneumonia, but when one or more of the markers are signifi cantly elevated, a bacterial etiology is more likely. thus, taken together with the clinical examination and radiologic fi ndings, these tests can aid the clinician in deciding which patients require antibiotic therapy. pct levels appear to be more sensitive than wbc, esr, and crp in identifying children with bacterial pneumonia and have been used to identify children who may benefi t from a longer duration of antibiotic therapy [ 50 ] . when non-invasive identifi cation techniques are inadequate, or when identifying the cause is especially important, such as when treating an immunocompromised host, invasive diagnostic procedures may be necessary. fiberoptic bronchoscopy with bronchoalveolar lavage (bal) is the preferred diagnostic procedure in an immunocompromised host with an unknown pathogen [ 51 ] . the sensitivity for diagnosis varies and depends on the host, pathogen, and the post-collection microbiologic detection methods employed. while many atypical organisms may be diffi cult to culture, p. jiroveci and mycobacterium infections are more easily detected in bal because of high organism burden in the lungs. the diagnosis of aspiration pneumonia is mainly clinical, often based on historical or witnessed events or conditions, and thus can be diffi cult to ascertain. if a bal is performed in suspected aspiration, the presence of lipid-laden macrophages can help diagnose the aspiration of lipophilic foods such as formula [ 52 ] . a lipid-laden macrophage index can be obtained using the oil red o stain and when high, can be very sensitive and specifi c for aspiration [ 53 ] . other invasive procedures include transbronchial biopsy if diffuse infi ltrates are present but the bal is negative, or ct-guided needle biopsy of a focal lesion. the improved diagnosis with these invasive procedures must be balanced against the risks to critically ill patients [ 54 ] . important noninfectious etiologies to rule out with these invasive procedures include lung rejection (if transplanted), post-engraftment syndrome, idiopathic pneumonitis, graft versus host disease, and bronchiolitis obliterans. children with severe pneumonia requiring admission to the picu are likely to receive intravenous antimicrobial therapy even if only until the possibility of bacterial infection can be excluded. in critically ill children with respiratory failure from pneumonia, prompt initiation of broad-spectrum antimicrobials is crucial. one study in pediatric patients with cap showed that longer delays in receipt of antibiotics were independently associated with adverse outcomes [ 55 ] . however, antibiotic resistance is increasing and the principles of appropriate antibiotic utilization must be adhered to: use of drug with narrowest spectrum, aiming for high tissue penetration, short half-life, and abiding to a short, intense duration of therapy [ 7 ] . the duration of therapy is typically 7-14 days, with 10 days being the best studied. a 7-day course may be reasonable in non-severe cases of pneumonia [ 12 ] . the choice of antimicrobial agent is based on many things including the patient's age, the type of pneumonia, and clinical and epidemiologic factors. recent guidelines published by the pediatric infectious diseases society and the infectious diseases society of america offer guidance for empiric antibiotic selection in children hospitalized with cap (table 6. 3 ) [ 12 ] . pneumonia causes a profound infl ammatory response in the lungs and it has long been postulated that regulating this infl ammation with steroid therapy may help to modulate local tissue damage and accelerate recovery for the patient. in addition, steroids are frequently utilized in other pulmonary infl ammatory conditions such as reactive airway a b the fi nding of at least a quadrupling of serum antibody levels between the acute phase and convalescence the fi nding of igm antibody in serum late in the acute phase or early in convalescence is helpful, as is a positive pcr assay of secretions from a throat or a nasopharyngeal swab rapid igm assays can provide results within 10 min. in younger children, an elevated igm titer is often diagnostic; in older children, the fi nding of at least a quadrupling of serum antibody levels between the acute phase and convalescence is diagnostic. cold agglutinin titers lack sensitivity and specifi city and thus are no longer recommended separate serum specimen because some agents (e.g., piperacillintazobactam) may cross-react with the assay. if invasive aspergillosis is suspected in high-risk patients, serial sampling is recommended. the false positive rate is higher in children than adults [ 91 , 92 ] adapted from mcintosh [ 6 ] . with permission from massachusetts medical society disease (rad) and acute respiratory distress syndrome (ards) [ 56 ] . the infl ammatory responses in pneumonia and ards are similar with increases in pro-infl ammatory cytokines concurrent with illness severity; severe pneumonia can often progress to acute lung injury (ali) or ards [ 57 -59 ] . while preclinical data support the use of steroids, current studies have not demonstrated a reduction in mortality among corticosteroid recipients compared with non-recipients. several trials, however, have shown some secondary benefi ts of steroids, including reduced length of hospital stay and reduced infl ammatory markers [ 60 , 61 ] . in contrast, a multi-center, retrospective cohort study using administrative data found that among patients not receiving concomitant beta-agonist therapy (used as a proxy for wheezing), corticosteroid recipients had a longer los and higher readmission rate compared with non-recipients [ 62 ] . at present, the lack of high quality data supporting the effi cacy of corticosteroids prevents the recommendation for the use of steroids in most patients with severe pneumonia. however, corticosteroids may provide benefi t to certain subgroups of patients such as those with acute onset of wheezing and those who meet the criteria for ali/ards [ 59 ] . macrolide antibiotics have important anti-microbial as well as anti-infl ammatory properties, though the relative importance of these two mechanisms in children with pneumonia is unknown. in adult studies, macrolides have recently been touted for their immunomodulatory effects and clinical benefi t in multiple chronic pulmonary conditions such as asthma, chronic obstructive pulmonary disease (copd), and cystic fi brosis (cf). the specifi c immunomodulatory effects are vast and include inhibition of intracellular signaling to suppress the production of transcription factors such as nf-îºb and decrease production of infl ammatory cytokines that recruit neutrophils [ 63 , 64 ] . several recent studies in adult patients with severe cap and sepsis have shown a benefi t in survival in patients treated with macrolide antibiotics in addition to the recommended antibiotics based on pathogen [ 63 , 65 -68 ] . the role of macrolides in children with pneumonia is unclear. in pediatrics, several small retrospective studies have shown that among children with atypical cap, those treated with macrolides were less likely to have persistence of signs and symptoms after 3 days of therapy [ 69 , 70 ] . among children with m. pneumoniae infection, lu et al. found a shorter duration of fever among macrolide recipients compared with non-recipients [ 71 ] . finally, a large multi-center study of 690 patients with m. pneumoniae infection defi ned by discharge diagnosis codes, the median length of hospital stay was 3 days (interquartile range, 2-6 days); macrolide recipients had a 32 % shorter length of stay compared with non-recipients [ 72 ] . pneumonia-associated complications such as empyema affect 7.5-15 % of children hospitalized with pneumonia [ 5 , 73 -76 ] . the progression from simple parapneumonic effusion to empyema occurs in stages that represent a continuous spectrum (table 6 .4 ) [ 77 ] . in the fi rst stage, there is a rapid infl ux of exudative fl uid into the pleural space as a result of increased pulmonary interstitial fl uid traversing the pleura and an increase in vascular permeability due to pro-infl ammatory cytokines. the pleural fl uid is marked by the absence of bacteria, fl uid ph >7.20, normal glucose, and ldh <3 times the upper limit of normal. at this stage, drainage is not generally required for resolution but if the effusion becomes large and piperacillin-tazobactam if concern for gram negative enteric bacteria iv cefotaxime if >20 days of age adapted from refs. [ 8 , 12 ] impairs respiratory mechanics, drainage might become necessary. the fl uid in the pleural space can fl ow freely and often layers along the lateral chest wall in decubitus fi lms or along the posterior chest wall in supine fi lms [ 37 , 78 ] ( fig. 6.4a , b) . if left untreated, exudative effusions can progress to fi bropurulent effusions characterized by the new presence of bacteria or positive microbial cultures. cellular lysis and phagocytosis in the fl uid can result in ph < 7.20, higher ldh, and low glucose. loculations begin to develop, causing these effusions to now be referred to as "complicated." a chest radiograph may be diffi cult to interpret with respect to evidence of complicated effusions. thoracic us is more accurate than chest radiographs in distinguishing simple from complicated pleural effusions. complicated effusions are associated with fl oating debris and echogenic material or septations. ultrasound is also useful in guiding pleural aspiration and drainage. chest computed tomography (ct) may be indicated to better defi ne pulmonary and pleural anatomy. thickening of the parietal pleura on a contrasted ct scan is suggestive of empyema, even if the effusions are small in size ( fig. 6.4c ) . finally, stage three is the organizing phase where fi broblasts grow into the pleural space and eventually results in a pleural peel, restricting chest mechanics. this stage often necessitates surgical decortication, especially if there is restrictive impairment [ 78 ] . the typical organisms responsible for the development of an empyema include s. pneumoniae and s. aureus . pleural fl uid cultures identify an organism in only 20-30 % of children with empyema. blood cultures are positive in 13-30 % of children with empyema [ 79 -82 ] . s. aureus is most often identifi ed in pleural fl uid culture. however, molecular identifi cation techniques reveal that most culture-negative cases are attributable to s. pneumoniae [ 83 , 84 ] . regardless of the type of effusion present, antibiotic coverage based on treatment guidelines for pneumonia are essential. a recent study on the impact of early antibiotic therapy on the laboratory analysis of pleural fl uid found that pre-treatment signifi cantly hindered a bacterial diagnosis but did not alter the biochemical parameters of the fl uid [ 85 ] . however, delaying antibiotic treatment for a thoracentesis would not be recommended in a critically ill child with respiratory failure secondary to pneumonia. the treatment of complicated effusions and empyema remains controversial but recent studies have better defi ned protocols. a complete list of the available treatments for effusions and empyema is found in table 6 .5 . small, uncomplicated pleural effusions do not routinely require drainage. moderate or large pleural effusions as well as those with evidence of septations or loculations usually require drainage. the medical options include appropriate antimicrobials and chest tube insertion with or without fi brinolytic therapy. surgical options include video-assisted thoracoscopic surgery (vats) or open thoracotomy and decortication. recent guidelines concluded that chest tube drainage with the addition of fi brinolytic agents and vats are equivalent methods of treatment and emphasize the importance of local expertise in determining the optimal approach for individual patients [ 12 , 86 ] . vats has gained popularity over conservative medical therapy as a way to directly visualize the pleural space, mechanically disrupt the adhesions, and strategically place the chest tube for optimal drainage [ 73 , 87 ] . the higher cost and risk of anesthesia with vats must be balanced against the more frequent requirement for additional drainage procedures for those undergoing primary chest tube placement. thoracotomy and decortication are rarely needed. the argument of medical management versus surgical management remains controversial. to date, at least two prospective trials in pediatrics have been completed directly comparing these methods. both trials failed to show any outcome superiority with surgical management [ 80 , 88 ] . certainly children who have a very high white blood cell count in their pleural fl uid (> 15,000), poor output drainage by chest tube, low pleural ph, the presence of bacteria in the pleural fl uid and/or bloodstream, or failure of medical therapy alone may benefi t from early vats [ 86 ] . patients who underwent vats required fewer adapted from refs. [ 12 , 78 , 93 ] additional drainage procedures, but had no difference in hospital length of stay [ 74 ] . however, one study of adults with empyema found that patients treated with a combination of tpa and recombinant human dnase required fewer surgical interventions and had a shorter length of hospital stay [ 89 ] . cost-effectiveness, balance of risks, and availability of resources also plays a role in considerations for surgical management. a comparison of multiple strategies for pediatric empyema noted that the most cost effective method was insertion of a chest tube with fi brinolytic therapy [ 90 ] . abscesses develop in localized areas of parenchymal infection that becomes necrotic and cavitates ( fig. 6.5a , b ) . primary lung abscesses can develop either in previously healthy children or in children with underlying lung disease such as congenital cystic lesions, cystic fi brosis, or immunodefi ciency. mechanisms for abscess development can include direct aspiration of infectious material, embolic phenomena, hematogenous spread from septicemia, or local extension from abdominal or oropharyngeal processes. the most common organisms include gram-positive bacteria such as streptococci, staphylococcus aureus or anaerobes. most abscesses resolve with intravenous antibiotics alone, but aspiration or drainage with a pigtail catheter may be necessary [ 37 ] . vaccines against specifi c bacteria that predominantly cause pneumonia in children, specifi cally pneumococcal conjugate vaccine (pcv-7) and h. infl uenzae vaccine (hib) have drastically lowered the prevalence of infections causes by these strains. since the introduction of pcv-7, several studies have documented its effi cacy, and the decrease in cases of h. infl uenzae are equally striking [ 7 , 21 ] . however, while pcv-7 has decreased the prevalence of invasive pneumococcal disease, the incidence of empyema is rising, the reason for which is unclear [ 76 ] . the licensure of pneumococcal conjugate vaccines that include even more serotypes (e.g., 13-valent) may further change the epidemiology of childhood pneumonia. other vaccines, such as for measles (mmr) and infl uenza, can also aid to reduce these viral infections that so commonly lead to secondary bacterial pneumonia. while vaccines appear to be our greatest effort toward preventing pneumonia in children, more work needs to be done to increase their microbial coverage and availability throughout the world. operative technique in which a small camera and instruments are inserted into the pleural space through 2-3 small (1-2 cm) incisions of the skin and muscle on the lateral chest wall to mechanically remove purulent material and pleural adhesions. a thoracostomy tube is placed through one of the existing incisions following completion of the procedure open thoracotomy operative technique where instruments are inserted into the pleural space through a single 5-8 cm incision of the skin and muscle on the posterolateral chest wall to mechanically remove purulent material and pleural adhesions. a thoracostomy tube is placed through a second smaller 1-2 cm incision following completion of the procedure general anesthesia reprinted from swami and shah [ 43 ] . with permission from mcgraw-hill three decades of pediatric intensive care: who was admitted, what happened in intensive care, and what happened afterward defi ning pneumonia in critically ill infants and children world health organization (who) childhood pneumonia mortality-a permanent global emergency national hospitalization trends for pediatric pneumonia and associated complications ambulatory visit rates and antibiotic prescribing for children with pneumonia pneumonia and other respiratory infections community-acquired pneumonia in children pulmonary complications of pediatric neurological diseases aspiration pneumonitis and aspiration pneumonia pneumonia in normal and immunocompromised children: an overview and update the management of community-acquired pneumonia in infants and children older than 3 months of age: clinical practice guidelines by the pediatric infectious diseases society and the infectious diseases society of america infectious diseases society of america/ american thoracic society consensus guidelines on the management of community-acquired pneumonia in adults airway mucus function and dysfunction beyond infl ammation: airway epithelial cells are at the interface of innate and adaptive immunity the role of particle size in aerosolised pathogen transmission: a review distribution of airborne infl uenza virus and respiratory syncytial virus in an urgent care medical clinic aspiration lung disease the impact of tracheal intubation on host defenses and risks for nosocomial pneumonia feeding the disabled child community-acquired pneumonia: a review and recent advances viruses in community-acquired pneumonia in children aged less than 3 years old: high rate of viral coinfection viruses and bacteria in sputum samples of children with community-acquired pneumonia viral pneumonia induced sputum in the diagnosis of childhood community-acquired pneumonia etiology of community-acquired pneumonia in 254 hospitalized children etiology of community-acquired pneumonia in hospitalized school-age children: evidence for high prevalence of viral infections etiology of community-acquired pneumonia in hospitalized children based on who clinical guidelines the changing face of pediatric respiratory tract infections: how human metapneumovirus and human bocavirus fi t into the overall etiology of respiratory tract infections in young children population-based incidence of human metapneumovirus infection among hospitalized children human bocavirus human bocavirus in children with acute lymphoblastic leukemia frequent and prolonged shedding of bocavirus in young children attending daycare human bocavirus: passenger or pathogen in acute respiratory tract infections? high incidence of pulmonary bacterial coinfection in children with severe respiratory syncytial virus (rsv) bronchiolitis infection in solid-organ transplant recipients respiratory infections: pneumonia, lung abscess, and empyema approach to the immunocompromised host with infection in the intensive care unit aspiration-induced lung injury community-acquired pneumonia in children: what's old? what's new? lipoid pneumonia: spectrum of clinical and radiologic manifestations pulmonary infections pediatric practice: infectious diseases murray and nadel's textbook of respiratory medicine value of the c-reactive protein test in the differentiation of bacterial and viral pneumonia serum procalcitonin, c-reactive protein and interleukin-6 for distinguishing bacterial and viral pneumonia in children differentiation of bacterial and viral communityacquired pneumonia in children white blood cells, c-reactive protein and erythrocyte sedimentation rate in pneumococcal pneumonia in children non-specifi c host response markers in the differentiation between pneumococcal and viral pneumonia: what is the most accurate combination? procalcitonin measurements for guiding antibiotic treatment in pediatric pneumonia role of fl exible bronchoscopy in immunocompromised patients with lung infi ltrates chronic pulmonary aspiration in children lipid-laden macrophages in induced sputum are a marker of oropharyngeal refl ux and possible gastric aspiration open lung biopsy in pediatric bone marrow transplant patients timing of correct parenteral antibiotic initiation and outcomes from severe bacterial community-acquired pneumonia in children activation and regulation of systemic infl ammation in ards: rationale for prolonged glucocorticoid therapy understanding the infl ammatory cytokine response in pneumonia and sepsis: results of the genetic and infl ammatory markers of sepsis (genims) study infl ammatory markers at hospital discharge predict subsequent mortality after pneumonia and sepsis steroids in severe pneumonia: a literature review dexamethasone and length of hospital stay in patients with community-acquired pneumonia: a randomised, double-blind, placebo-controlled trial hydrocortisone infusion for severe community-acquired pneumonia: a preliminary randomized study adjunct corticosteroids in children hospitalized with community-acquired pneumonia immunomodulatory agents in the treatment of community-acquired pneumonia: a systematic review mechanisms of action and clinical application of macrolides as immunomodulatory medications combination antibiotic therapy improves survival in patients with community-acquired pneumonia and shock monotherapy may be suboptimal for severe bacteremic pneumococcal pneumonia impact of macrolide therapy on mortality for patients with severe sepsis due to pneumonia combination antibiotic therapy with macrolides improves survival in intubated patients with communityacquired pneumonia role of mycoplasma pneumoniae and chlamydia pneumoniae in children with community-acquired lower respiratory tract infections characteristics of streptococcus pneumoniae and atypical bacterial infections in children 2-5 years of age with community-acquired pneumonia macrolide use shortens fever duration in mycoplasma pneumoniae infection in children: a 2-year experience macrolide therapy and outcomes in a multicenter cohort of children hospitalized with mycoplasma pneumoniae pneumonia primary operative versus nonoperative therapy for pediatric empyema: a meta-analysis comparative effectiveness of pleural drainage procedures for the treatment of complicated pneumonia in childhood primary early thoracoscopy and reduction in length of hospital stay and additional procedures among children with complicated pneumonia: results of a multicenter retrospective cohort study empyema hospitalizations increased in us children despite pneumococcal conjugate vaccine parapneumonic effusions and empyema parapneumonic pleural effusion and empyema blood cultures in the emergency department evaluation of childhood pneumonia thoracoscopic decortication vs tube thoracostomy with fi brinolysis for empyema in children: a prospective, randomized trial impact of the pneumococcal conjugate vaccine on pneumococcal parapneumonic empyema an epidemiological investigation of a sustained high rate of pediatric parapneumonic empyema: risk factors and microbiological associations the changing face of pleural empyemas in children: epidemiology and management molecular analysis improves pathogen identification and epidemiologic study of pediatric parapneumonic empyema impact of antibiotic therapy on laboratory analysis of parapneumonic pleural fl uid in children management of parapneumonic empyema pediatric respiratory diseases: 2011 update for the rogers' textbook of pediatric intensive care comparison of urokinase and video-assisted thoracoscopic surgery for treatment of childhood empyema intrapleural use of tissue plasminogen activator and dnase in pleural infection cost-effectiveness of competing strategies for the treatment of pediatric empyema diagnostic aspects of invasive aspergillus infections in allogeneic bmt recipients diagnostic potential of nested pcr, galactomannan eia, and beta-d-glucan for invasive aspergillosis in pediatric patients medical and surgical treatment of parapneumonic effusions : an evidence-based guideline key: cord-291639-hioh2s35 authors: alfredo, potena; gaetano, caramori; paolo, casolari; marco, contoli; johnston, sebastian l; alberto, papi title: pathophysiology of viral-induced exacerbations of copd date: 2007-12-17 journal: int j chron obstruct pulmon dis doi: nan sha: doc_id: 291639 cord_uid: hioh2s35 inflammation of the lower airways is a central feature of chronic obstructive pulmonary disease (copd). inflammatory responses are associated with an increased expression of a cascade of proteins including cytokines, chemokines, growth factors, enzymes, adhesion molecules and receptors. in most cases the increased expression of these proteins is the result of enhanced gene transcription: many of these genes are not expressed in normal cells under resting conditions but they are induced in the inflammatory process in a cell-specific manner. transcription factors regulate the expression of many pro-inflammatory genes and play a key role in the pathogenesis of airway inflammation. many studies have suggested a role for viral infections as a causative agent of copd exacerbations. in this review we will focus our attention on the relationship between common respiratory viral infections and the molecular and inflammatory mechanisms that lead to copd exacerbation. many epidemiological and clinical studies have suggested a role for respiratory viral infections in the natural history of chronic obstructive pulmonary disease (copd), particularly during their exacerbations highlighting the need for development of effective vaccines and/or treatment for these viruses. the precise cellular and molecular mechanisms underlying these events are still largely unknown (caramori et al 2006; mallia et al 2007) . lower airways infl ammation is a central feature of many lung diseases, including copd. although the specifi c characteristics of the infl ammatory responses and the site of infl ammation differ between one disease to another, they always involve recruitment and activation of infl ammatory cells and changes in structural cells of the lung. infl ammatory responses are associated with an increased expression of a cascade of proteins that includes cytokines, chemokines, growth factors, enzymes, adhesion molecules and receptors. in most cases the increased expression of these proteins is the result of enhanced gene transcription: many of the genes are not expressed in normal cells under resting conditions but they are induced in the infl ammatory process in a cell-specifi c manner. transcription factors regulate the expression of many genes, including infl ammatory genes and play a key role in the pathogenesis of respiratory infl ammatory diseases, including copd (caramori et al 2004) . in this review we will provide an overview of the relationship between respiratory virus infection and the molecular mechanisms involved in the activation of airway infl ammation in copd exacerbations. copd is a disease state characterized by airfl ow obstruction that is not fully reversible. the airfl ow obstruction is usually both progressive and associated with an abnormal infl ammatory response of the lung to noxious particles or gases. the main cause of copd is cigarette smoking (gold 2006) . chronic obstructive pulmonary disease (copd) is the fourth leading cause of mortality worldwide (gold 2006) . costs of copd are mainly related to exacerbations requiring hospitalization (sullivan et al 2000) . in addition to their enormous acute morbidity, mortality and cost, exacerbations are also associated with major reductions in long term quality of life and lung function (seemungal et al 1998; donaldson et al 2002) . despite the clinical and economic importance of severe copd exacerbations, their etiology and mechanisms are poorly understood. both bacterial (sethi and murphy 2001; white et al 2003) and viral infections (seemungal et al 2000 (seemungal et al , 2001 aaron et al 2001; rohde et al 2003; wedzicha 2004; beckham et al 2005; papi et al 2006) have been detected at increased frequencies during copd exacerbations. the most frequently respiratory viruses involved in the etiology of copd exacerbations are represented by rhinoviruses, infl uenza viruses, coronaviruses, and respiratory syncytial virus (rsv). more rarely parainfl uenza viruses and human metapneumoviruses (hmpv) have also been identifi ed (rohde et al 2003; hamelin et al 2005; falsey et al 2006; papi et al 2006) . a recent study has recently addressed the relative importance of viral versus bacterial infections in the etiology of severe (hospitalized) copd exacerbations (papi et al 2006) . viral and/or bacterial infection was detected in 78% of copd exacerbations (29.7% bacterial, 23.3% viral, 25% viral/bacterial co-infection). infectious exacerbations had longer hospitalizations and greater impairment of several measures of lung function than non-infectious exacerbations. importantly, exacerbations with co-infection had more marked lung function impairment and longer hospitalizations. similarly, when copd exacerbations not requiring hospitalization were evaluated, a greater lung function fall was documented in those patients where both bacterial infection and cold symptoms were present simultaneously (wilkinson et al 2006) . this data suggests that bacterial and viral infections could synergistically cooperate to increase severity of the exacerbations. whether viral infections can lead to bacteriological exacerbation (of previously colonizing bacteria) is still an open question that deserves properly designed longitudinal studies. determining the etiology of exacerbations will inform appropriate antibiotic and antiviral therapy. antibiotic therapy is already widely used, but not always appropriately, in the treatment of copd exacerbations (gold 2006) . indeed, a recent meta-analysis supports antibiotics only for those patients with copd exacerbations with increased cough and sputum purulence who are moderately or severely ill. analysis restricted to community-based studies did not fi nd any differences between antibiotic and placebo (ram et al 2006) . in asthma, recent study showed a signifi cation reduction of symptoms at exacerbations in those patients receiving telithromycin as compared to placebo. given that no relationship between bacteriologic status and the response to study treatment was found, the mechanisms of such a benefi t remain unclear (johnston et al 2006) . therefore, (i) there is an urgent need to develop simple clinical or biological markers to identify those patients at highest risk of bacterial infection and who will benefi t most from antibiotic therapy, (ii) further studies are needed to evaluate whether novel class of antibiotics can be more effective in the treatment of copd exacerbations. antiviral therapy, and in particular anti-rhinovirus therapy, is a "still theoretically" relevant therapeutic option since two thirds of exacerbations are associated with viral infections (seemungal 2001) . indeed, so far no study has investigated whether currently available antiviral treatment can reduce viral induced copd exacerbations. the use of anti-infl uenza neuraminidase inhibitors in copd exacerbations during infl uenza epidemic appears appropriate in non vaccinated patients and may reduce the number of hospitalizations (lalezari et al 2001; cooper et al 2003; kaiser et al 2003) , though further controlled clinical trials are needed to confi rm therapeutic benefi t. viral infections are the most frequent cause of copd exacerbation. whether copd patients are more susceptible to virus infection as compared to normal subjects is still debated. a recent study documented that patients with frequent copd exacerbation have more frequent episodes of naturally occurring colds as compared to patients with infrequent exacerbations (hurst et al 2005) . these results suggest that copd subjects with frequent exacerbations may represent a subgroup particularly susceptible to viral infections. thus, while there is solid evidence of impaired innate (wark et al 2005; contoli et al 2006) and possibly acquired (gern et al 2000; papadopoulos et al 2002) immune responses to viral infection in asthmatic patients, it is not yet clear whether copd patients have increased susceptibility to viral infections. intriguingly patients experiencing frequent colds had a signifi cantly higher exposure to cigarette smoke (hurst 2005) . recently, using a mouse model of cigarette smoke exposure, it has been demonstrated that cigarette smoke increases susceptibility to viral infections possibly via alteration/inhibition of immune response (robbins et al 2004) . another possible mechanism leading to increased susceptibility is related to up regulation of icam-1, the receptor for the major group of human rhinoviruses. latent expression of adenoviral e1a protein in alveolar epithelial cells of patients with emphysema increases icam-1 expression and this could be a potential mechanism for greater susceptibility to rhinovirus infection in copd (retamales et al 2001) . patients with copd are chronically colonized with airway bacteria, and the bacterial load is related to airway infl ammation and disease progression (patel et al 2002) . it has been postulated that bacterial colonization could contribute to increased susceptibility to viral infection in copd patients for example by increasing icam-1 expression in bronchial epithelial cells either directly or through induced infl ammation (sajjan et al 2006) . further studies are required to investigate the interaction between chronic bacterial colonization and respiratory viral infection and in particular whether chronic bacterial colonization can increase susceptibility to viral infection or vice versa. as the contribution of viruses to copd exacerbations has only recently been appreciated little research has been carried out into the mechanisms of virus-induced infl ammation. performing airway sampling at exacerbation in copd patients is even more diffi cult than in asthmatics due to their older age and high prevalence of co-morbidities. one way to overcome these obstacles is the development of a human experimental model that would allow studies to take place under controlled conditions. the fi rst step towards development of such a model has been recently realized with the reporting of the fi rst study evaluating the effects of an experimental viral infection in copd patients (mallia et al 2006) . copd exacerbations are associated with an increased numbers of inflammatory cells in the lower airways and increased/decreased release of pro-inflammatory/ anti-infl ammatory mediators (wedzicha and donaldson 2003) . through these mechanisms respiratory viral infections may enhance the pathological processes associated with cigarette smoking and contribute to the lung pathology and loss of lung function associated with copd. the type and the degree of activation of the different infl ammatory cells recruited to the lung during copd exacerbations has not been well studied (zhu et al 2001; qiu et al 2003) . despite growing clinical evidence for a role of respiratory viral infections in the pathogenesis of copd exacerbations, the precise mechanisms of respiratory virus-induced airway infl ammation and of host defenses against respiratory viruses are poorly understood (johnston 2005) . most of the data available relate to rhinovirus (rv) and respiratory syncytial virus (rsv), ie, the respiratory viruses that appear to be more frequently involved in copd exacerbations. human rhinoviruses (rv) are the largest genus in the picornaviridae family. they are single-stranded rna viruses. rhinovirus is the main cause of the common cold and several studies supported the role of this pathogen in both asthma and copd exacerbations. recent data documents that rhinovirus can reach and replicate in the lower airways. there is striking genetic diversity of the rv strains circulating in a given community during a short time (oliveira et al 1993) . rhinovirus serotypes are divided into two groups on the basis of receptor specifi city. the "major" receptor group (including rv14 and rv16) utilizes intercellular adhesion molecule-1 [(icam-1); cd54] as a receptor for infecting the target cells (greve et al 1989; terajima et al 1997) . using icam-1, rv can infect airway epithelial cells, but there is little evidence of productive replication in other cells such as monocytes, macrophages and eosinophils granulocytes. the "minor" receptor group, do not bind icam-1 and instead bind the ldl receptor and related proteins (hofer et al 1994) . rhinovirus is transmitted by aerosol through infectious droplet nuclei emanating from infected subjects (myatt et al 2003; samet 2004 ). respiratory syncytial virus (rsv) is a member of the family paramyxoviridae, subfamily pneumovirus; it is an enveloped rna virus with a negative-sense, nonsegmented, singlestranded rna genome. rsv is a major respiratory pathogen, it is most common in infants where it causes a range of illnesses from asymptomatic infection through upper respiratory tract infection, bronchiolitis, and pneumonia (falsey and walsh 2000; hall 2001; falsey et al 2005) . in addition, during the past two decades, a growing number of studies have clearly established rsv as a severe pathogen in certain adult populations. the elderly, those with underlying cardiopulmonary disease (chronic heart failure, chronic obstructive pulmonary disease, bronchial asthma) and the immunocompromised patients appear to be at greatest risk of developing severe, even life-threatening lung disease following rsv infection (hall 2000; walsh and falsey 2004; falsey 2005; sethi and murphy 2005) . the primary site of rsv replication are the airway epithelial cells, whereas other cell types (monocytes/macrophages, airway smooth muscle cells eosinophils, neutrophils, mast cells, dendritic cells and endothelial cells) are targets of abortive replication or viral attachment only (arnold and konig 2005; de graaff et al 2005; guerrero-plata et al 2006) . in human bronchial epithelial cells rsv colocalize with icam-1, and this binding can be inhibited by an antibody to the fusion f protein. these data suggests that rsv interaction with icam-1 involves the f protein and facilitates rsv entry and infection of human bronchial epithelial cells (behera et al 2001) . interestingly in vitro rsv infection of human lung endothelial cells increase their expression of icam-1 (arnold and konig 2005) . since rsv replication is largely restricted to airway epithelial cells, an hypothesis is that infl ammatory cell recruitment by the infected cells will start the later immunopathology (becker et al 1992; becker and soukup 1999) . the effects of rsv on airway infl ammation may be therefore partly mediated by sequential production of pro-infl ammatory cytokines/chemokines in the infected airway epithelium. the activation of several nuclear factors has recently been studied in copd (caramori 2004) . several studies have shown that both the promoter genomic regions and the related transcription factors that regulate infl ammatory mediator production are deeply affected by respiratory virus infection. most of the data published until now on this issue are mainly related to rhinovirus and respiratory syncytial virus infections. more research is required to clarify the molecular events that determine the changes in gene transcription determined by virus infection in target cells. to date the specifi c molecular mechanisms involved in the production of pro-infl ammatory mediators by bronchial/lung cells after rhinovirus infection are still not fully characterized. however, some studies have examined at the molecular level the transcription factors involved in rv-induced production of proinfl ammatory mediators from structural cells of the lungs. rhinovirus infection of primary bronchial epithelial cells induces a rapid increase of intracellular oxidants (superoxide anion) production that is maximal at the time of nuclear factor κb (nf-κb) activation. intriguingly, it has been shown, in the same model, that reducing agents inhibit rv-induced icam-1 up-regulation, icam-1 promoter activation and nf-κb activation . this data suggests that modulation of rv induced intracellular oxidant burst can be a novel pharmacological approach to treat/prevent rv induced copd exacerbations. several transcription factors appear to be activated after rhinovirus infection on a variety of respiratory cells (caramori 2006) . these proteins include nf-κb, ap-1 and gata families. activation of intracellular signaling pathways is induced by rhinovirus in epithelial cells, which may be dependent on surface receptor binding (icam-1), or by intracellular products during viral replication such as doublestranded rna (dsrna). it has been shown that dsrna can activate components of several signaling pathways including protein kinase r, nuclear factor-κb (nf-κb) and p38 mitogenactivated protein kinase (mapk) (alexopoulou et al 2001) . however, to date, no studies have investigated the role of these molecules in rhinovirus infection. rhinovirus-induced activation of nf-κb leads to an increased expression of proinfl ammatory cytokines (il-1, il-6, granulocyte colony-stimulating factor (g-csf) and gm-csf), cxc chemokines (il-8) and icam-1 (papi and johnston 1999b) , whereas expression of vcam-1 seems to be mediated by the activation of both nf-κb and gata proteins (papi and johnston 1999a) . it has been recently suggested that early activation of p38 mapk pathway by rhinovirus infection, which induces the activation of many transcription factors, could be a key event in the regulation of rhinovirusinduced cytokine transcription, and may provide a new target for inhibition of rhinovirusinduced asthma exacerbations. rsv-induced cytokine production in airway epithelial cells seems to play an important role in the pathogenesis of rsv respiratory tract infections. recent studies on rsv-epithelial cell interactions have demonstrated that rsv can act at the molecular level by altering pathophysiology of viral-induced exacerbations of copd cytokine gene transcription (jamaluddin et al 1998; mastronarde et al 1998) or mrna stability (koga et al 1999) . as for rv it has been shown that early activation of mitogen-activated protein kinases (mapks) by rsv infection is a key event in the regulation of rsv-induced pro-inflammatory transcription factors activation and cytokine/chemokines transcription in bronchial epithelial cells, however both p38 map kinase, mitogen-activated protein kinase kinase kinase 14/ nf-κb-inducing kinase (map3k14/nik) and extracellular signal-regulated kinases (erks) pathways seem to be important (chen et al 2000; kong et al 2004; chouldhary et al 2005; rixon et al 2005) . several transcription factors appear to be activated after rsv infection on a variety of respiratory cells. these proteins include mainly the nf-κb and ap-1 families. nf-κb is activated by the m2-1 protein of rsv (fiedler et al 1996; mastronarde 1998; casola et al 2000; chouldhary 2005; reimers et al 2005; spann et al 2005) . the mechanisms by which virus-enhanced infl ammation may pave the way to copd exacerbations are so far unknown. the recent availability of a human model of rv-induced copd exacerbations (mallia et al 2007) will foster the research and the progress in this important area. in copd this is so far the only model where a specifi c etiology has been experimentally proven to induce exacerbation. in this pilot study mild to moderate copd patients were safely experimentally infected with rhinovirus providing for the fi st time that, in in vivo experimental condition, rhinovirus infection in copd patients is per se suffi cient to trigger exacerbations. lower respiratory tract symptom scores were signifi cantly increased compared to baseline on days 7 to 14, with peak lower respiratory tract symptoms on days 10 and 11. when the individual symptoms were analyzed separately, all fi ve lower respiratory symptom domains (wheeze, cough, sputum production and dyspnea) increased from baseline however the increases were only statistically signifi cant for wheeze, cough and sputum production, but not for dyspnea. in terms of recovery, all symptom domains other than sputum production had recovered to baseline by day 20, however full recovery of sputum production took almost 4 weeks (mallia et al 2006) . this important data suggests that the link between virus-induced copd exacerbation and increased dyspnea and sputum production is probably very complex and needs dedicated studies for a better comprehension. intriguingly, it has been recently shown that viral induced severe copd exacerbations with or without concomitant bacterial infection are characteristically associated with sputum eosinophilia whereas sputum neutrophilia is present during exacerbations in all subgroups independently from etiology (papi et al 2006) . this fi nding is in line with previous studies showing that in vivo experimental rhinovirus infection in normal subjects leads to lower airway eosinophilia (fraenkel et al 1995) . thus, enhanced airway eosinophilic infl ammation of the airway may be a hallmark of viral infection. further studies are required to confi rm and extend this pivotal observation and to evaluate whether modulation of rhinovirus-induced activation of pro-infl ammatory mediators (ie, nf-κb) might be of any relevance in the treatment/prevention of virus-induced copd exacerbations. many studies conducted in the last decade have produced convincing data on the molecular mechanisms involved in the pathogenesis of natural and experimental respiratory virus infections in humans, particularly rhinovirus (rv) and respiratory syncytial virus (rsv). most of the studies have been performed on human respiratory epithelial cells, which can be directly infected by rv and rsv. their infection by rv and rsv determines the production of several pro-infl ammatory molecules (such as cytokines, chemokines and adhesion molecules). conversely, very few studies have been conducted in vivo on the molecular mechanisms underlying the pro-infl ammatory derangement induced by respiratory viruses in the airways during copd exacerbations. collectively these studies suggest a critical role for several transcription factor families, including the nf-κb, ap-1 and gata families, in the production of pro-infl ammatory mediators after rv and rsv infection. the relative importance of each cell, mediator, signalling pathway and transcription factor will hopefully be clarifi ed in the next few years. the recent development of the fi rst human model of virus induced copd exacerbation, that has been demonstrated to be feasible and so far, safe, will facilitate identifi cation of novel pharmacological targets that will provide opportunities to develop new treatments for exacerbations of copd. granulocyte infl ammatory markers and airway infection during acute exacerbation of chronic obstructive pulmonary disease recognition of doublestranded rna and activation of nf-kappab by toll-like receptor 3 respiratory syncytial virus infection of human lung endothelial cells enhances selectively intercellular adhesion molecule-1 expression respiratory syncytial virus infection of human primary nasal and bronchial epithelial cell cultures and bronchoalveolar macrophages airway epithelial cell-induced activation of monocytes and eosinophils in respiratory syncytial viral infection respiratory viral infections in patients with chronic, obstructive pulmonary disease blocking intercellular adhesion molecule-1 on human epithelial cells decreases respiratory syncytial virus infection transcription factors in asthma and copd molecular mechanisms of respiratory virus-induced asthma and copd exacerbations and pneumonia requirement of a novel upstream response element in respiratory syncytial virus-induced il-8 gene expression activation of erk2 by respiratory syncytial virus in a549 cells is linked to the production of interleukin 8 respiratory syncytial virus infl uences nf-kappab-dependent gene expression through a novel pathway involving map3k14/nik expression and nuclear complex formation with nf-kappab2 role of defi cient type iii interferon-lambda production in asthma exacerbations effectiveness of neuraminidase inhibitors in treatment and prevention of infl uenza a and b: systematic review and meta-analyses of randomised controlled trials respiratory syncytial virus infection of monocyte-derived dendritic cells decreases their capacity to activate cd4 t cells relationship between exacerbation frequency and lung function decline in chronic obstructive pulmonary disease detection of respiratory syncytial virus and human metapneumovirus by reverse transcription polymerase chain reaction in adults with and without respiratory illness respiratory syncytial virus infection in elderly and high-risk adults respiratory syncytial virus infection in adults inhibition of viral replication reverses respiratory syncytial virus-induced nf-kappab activation and interleukin-8 gene expression in a549 cells lower airways infl ammation during rhinovirus colds in normal and in asthmatic subjects relationship of upper and lower airway cytokines to outcome of experimental rhinovirus infection global initiative for chronic obstructive lung disease, national institute of health, national heart lung, and blood institute. global strategy for the diagnosis, management and prevention of chronic obstructive pulmonary disease the major human rhinovirus receptor is icam-1 differential response of dendritic cells to human metapneumovirus and respiratory syncytial virus nosocomial respiratory syncytial virus infections: the "cold war" has not ended respiratory syncytial virus and parainfl uenza virus human metapneumovirus infection in adults with community-acquired pneumonia and exacerbation of chronic obstructive pulmonary disease members of the low density lipoprotein receptor family mediate cell entry of a minor-group common cold virus epidemiological relationships between the common cold and exacerbation frequency in copd the major component of ikb proteolysis occurs independently of the proteasome pathway in respiratory syncytial virus-infected pulmonary epithelial cells overview of virus-induced airway disease the effect of telithromycin in acute exacerbations of asthma impact of oseltamivir treatment on infl uenza-related lower respiratory tract complications and hospitalizations virus-inducible expression of a host chemokine gene relies on replication-linked mrna stabilization erk-1/2 activity is required for effi cient rsv infection zanamivir for the treatment of infl uenza a and b infection in high-risk patients: a pooled analysis of randomized controlled trials exacerbations of asthma and chronic obstructive pulmonary disease (copd) an experimental model of rhinovirus induced chronic obstructive pulmonary disease exacerbations: a pilot study activator protein-1 is the preferred transcription factor for cooperative interaction with nuclear factor-κb in respiratory syncytial virus-induced interleukin-8 gene expression in airway epithelium airborne rhinovirus detection and effect of ultraviolet irradiation on detection by a semi-nested rt-pcr assay the structure of human rhinovirus 16 a defective type 1 response to rhinovirus in atopic asthma infections and airway infl ammation in chronic obstructive pulmonary disease severe exacerbations respiratory epithelial cell expression of vascular cell adhesion molecule-1 and its up-regulation by rhinovirus infection via nf-kappab and gata transcription factors rhinovirus infection induces expression of its own receptor intercellular adhesion molecule 1 (icam-1) via increased nf-kappab-mediated transcription reducing agents inhibit rhinovirus-induced up-regulation of the rhinovirus receptor intercellular adhesion molecule-1 (icam-1) in respiratory epithelial cells pathophysiology of viral-induced exacerbations of copd relationship between bacterial colonisation and the frequency, character, and severity of copd exacerbations biopsy neutrophilia, neutrophil chemokine and receptor gene expression in severe exacerbations of chronic obstructive pulmonary disease antibiotics for exacerbations of chronic obstructive pulmonary disease respiratory syncytial virus m2-1 protein induces the activation of nuclear factor kappa b amplifi cation of infl ammation in emphysema and its association with latent adenoviral infection the respiratory syncytial virus small hydrophobic protein is phosphorylated via a mitogen-activated protein kinase p38-dependent tyrosine kinase activity during virus infection cigarette smoke decreases pulmonary dendritic cells and impacts antiviral immune responsiveness respiratory viruses in exacerbations of chronic obstructive pulmonary disease requiring hospitalisation: a case-control study h. infl uenzae potentiates airway epithelial cell responses to rhinovirus by increasing icam-1 and tlr3 expression how do we catch colds? respiratory viruses, symptoms, and infl ammatory markers in acute exacerbations and stable chronic obstructive pulmonary disease time course and recovery of exacerbations in patients with chronic obstructive pulmonary disease effect of exacerbation on quality of life in patients with chronic obstructive pulmonary disease bacterial infection in chronic obstructive pulmonary disease in 2000: a state-of-the-art review rsv infection -not for kids only effects of nonstructural proteins ns1 and ns2 of human respiratory syncytial virus on interferon regulatory factor 3, nf-kappab, and proinfl ammatory cytokines the economic burden of copd rhinovirus infection of primary cultures of human tracheal epithelium: role of icam-1 and il-1beta age related differences in humoral immune response to respiratory syncytial virus infection in adults asthmatic bronchial epithelial cells have a defi cient innate immune response to infection with rhinovirus role of viruses in exacerbations of chronic obstructive pulmonary disease exacerbations of chronic obstructive pulmonary disease chronic obstructive pulmonary disease. 6: the aetiology of exacerbations of chronic obstructive pulmonary disease effect of interactions between lower airway bacterial and rhinoviral infection in exacerbations of copd exacerbations of bronchitis: bronchial eosinophilia and gene expression for interleukin-4, interleukin-5 and eosinophil chemoattractants key: cord-016451-k8m2xz0e authors: chertow, daniel s.; kindrachuk, jason title: influenza, measles, sars, mers, and smallpox date: 2020-01-03 journal: highly infectious diseases in critical care doi: 10.1007/978-3-030-33803-9_5 sha: doc_id: 16451 cord_uid: k8m2xz0e influenza, measles, sars, mers, and smallpox illnesses are caused by highly infectious viral pathogens that induce critical illness. these biologically diverse viruses enter and replicate within host cells triggering viraland host-mediated damage that results in pneumonia and multiorgan failure in severe cases. early case identification and strict infection control limit healthcare transmission. vaccination allowed smallpox eradication and limits global measles and seasonal influenza mortality. while sars-coronavirus (cov) is no longer circulating, mers-cov and zoonotic influenza viruses, with pandemic potential, remain persistent threats. supportive critical care is the mainstay of treatment for severe disease due to these viral infections. measles virus is a pleomorphic, enveloped, negative-sense, single-stranded rna virus of family paramyxoviridae of approximately 100 nm to 300 nm in diameter [2] . measles virus causes mild to severe illness during seasonal outbreaks in endemic areas and intermittent outbreaks in nonendemic area [10] . measles virus codes for six structural and two nonstructural proteins (fig. 5 .1b) [11] . hemagglutinin (h) and fusion (f) glycoproteins project from the viral surface and facilitate viral binding to cellular receptors and fusion with the host cell membrane, respectively. matrix (m) protein underlies the envelope providing structure. the inner nucleocapsid is composed of rna coated by nucleoprotein (n), bound by the polymerase complex which includes the large (l) polymerase protein, and phosphoprotein (p), a polymerase cofactor. the remaining nonstructural proteins include c and v. coronaviruses are spherical, enveloped, positive-sense, single-stranded rna viruses of family coronaviridae of approximately 120 nm in diameter [12] . coronaviruses are the causative agents of an estimated 30% of upper and lower respiratory tract infections in humans resulting in rhinitis, pharyngitis, sinusitis, bronchiolitis, and pneumonia [13] . while coronaviruses are often associated with mild disease (e.g., hcov-229e, hcov-oc43, hcov-nl63, hcov-hku1), severe acute respiratory syndrome coronavirus (sars-cov), a lineage b betacoronavirus, and middle east respiratory syndrome coronavirus (mers-cov), a lineage c betacoronavirus, are associated with severe and potentially fatal respiratory infection [14, 15] . sars-and mers-cov transcribe 12 and 9 subgenomic rnas, respectively, which encode for the spike (s), envelope (e), membrane (m), and nucleocapsid (n) structural proteins (fig. 5 .1c) [14] . s, e, and m are all integrated into the hostderived lipid envelope, and s facilitates host cell attachment to angiotensinconverting enzyme (ace)-2 receptors for sars-cov and dipeptidyl peptidase (dpp)-4 receptors for mers-cov [16, 17] . the n protein encapsidates the viral genome to form the helical nucleocapsid. the viral replicase-transcriptase complex is made up of 16 nonstructural proteins (nsp1-16) including a unique proofreading exoribonuclease that reduces the accumulation of genome mutations [12] . poxviruses are oval-to-brick-shaped double-stranded dna viruses of family poxviridae that range in size from 200 to 400 nm [2] . viruses within genus orthopoxvirus that cause human disease include cowpox virus (cpxv), monkeypox virus (mpxv), vaccinia virus (vacv), and variola virus (varv), the etiologic agent of smallpox [18] . poxviruses contain a biconcave viral core where the dna genome, dnadependent rna polymerase, and enzymes necessary for particle uncoating reside ( fig. 5.1d ) [19] . this nucleosome is surrounded by a core membrane that is flanked by two proteinaceous lateral bodies. a single lipid membrane surrounds the cellassociated form of the mature virion (mv). a second host-derived lipid envelope covers the extracellular virion (ev) [2, 19] . poxvirus genomes are comprised of a large, linear double-stranded viral dna genome that encodes ~200 genes. highly conserved structural genes are predominantly found in the middle of the genome, whereas variable virulence factor genes that function in immune evasion, virulence, and viral pathogenesis are found at the termini of the genome [20] . wild aquatic birds are natural reservoirs for nearly all influenza a virus subtypes, which spread to domestic avian species and mammals, including humans [5] . h17n10 and h18n11 subtypes are exceptions in that they have only been isolated from bats [6, 7] . certain h5 and h7 subtypes are highly pathogenic to domestic poultry when transmitted from wild birds, known as highly pathogenic avian influenza (hpai) viruses [21] . hpai viruses cause spillover infections in humans that may be severe or fatal. examples include outbreaks of h5n1 and h7n9 hpai viruses in asia with high case fatality among humans, although limited human-tohuman transmission [22, 23] has been reported. hpai virus adaptations might lead to sustained human-to-human transmission, and so poultry outbreaks are managed by flock depopulation [24] . influenza a subtypes isolated in swine include h1 to h5, h9, and n1 and n2. subtypes that spillover into humans cause mild to severe illness and are known as swine "variant" viruses [25] . currently circulating seasonal influenza a subtypes h1n1 and h3n2 and influenza b viruses, yamagata or victoria lineage, cause annual epidemics during fall through spring in temperate regions and infections throughout the year in the tropics [26] . antigenic drift of h and n surface glycoproteins drives annual epidemics. from 2017 to 2018, seasonal influenza caused approximately 49 million illnesses, 1 million hospitalizations, and 79,000 deaths in the united states alone [27] . when two or more influenza a viruses infect a common host, such as a bird or pig, individual gene segments may recombine to form a novel virus, known as antigenic shift. influenza pandemics occur when novel viruses emerge into an immunologically naïve population and become adapted for sustained human-to-human spread. the 1918 "spanish" influenza pandemic was the most severe on record, resulting in an estimated 50 million deaths [28] . less severe pandemics occurred in 1957, 1968, and 2009 . in an effort to improve preparedness and response to seasonal, pandemic, and zoonotic influenza, the world health organization (who) conducts global surveillance of influenza a and b isolates (fig. 5 .2a) [29] . measles is pathogenic for humans and nonhuman primates, although sustained transmission occurs only among humans raising potential for global elimination [30] . historically, measles infected an estimated 90% of children by age 5 years, resulting in approximately 2 million global deaths each year [10] . with the introduction of the measles vaccine in 1963 and advances in global vaccination programs, measles cases and mortality have drastically declined (fig. 5.2b ). by 2017, 85% of children worldwide had received at least one dose of the measles vaccine by age 1 year, and during 2000-2017, global measles mortality decreased by 80%, preventing an estimated 21 million deaths [31] . of the 24 known measles genotypes, only five were detected in circulation during 2016-2017. despite these gains, measles remains endemic in many regions of the world including africa, western pacific, south east asia, and europe, and measles has resurged in previously low-incidence areas (e.g., regions within europe and the americas) with epidemics attributable to importation of cases and suboptimal immunization coverage [32] [33] [34] . an estimated 93% population immunity is required to prevent measles transmission within communities, a prerequisite for global elimination [35] . chinese horseshoe bats are the putative reservoir for sars-cov, and dromedary camels are thought to be the reservoir for mers-cov [36] [37] [38] [39] [40] [41] [42] [43] . animal-to-human transmission likely occurs following direct contact with intermediate hosts [38, 44] . during the 2003-2004 sars epidemic, 8096 cases and 774 deaths were reported from 26 countries with no cases reported since ( fig. 5 .2c) [45] . human-to-human transmission of sars-cov occurred primarily in healthcare settings with healthcare workers comprising 22% and >40% of reported cases in china and canada, respectively [45] . mers was first reported in saudi arabia in 2012 with >2000 cases and >800 deaths reported from 27 countries through 2018 [46] . while most cases have been reported from the arabian peninsula, an imported case to south korea in 2015 resulted in a large outbreak in multiple healthcare facilities [47] . mers transmission occurs primarily in healthcare facilities and to a lesser degree within households [48, 49] . while the only known reservoir for varv is humans, it has been postulated that the virus emerged from an ancestral rodent-borne poxvirus more than 10,000 years ago [18, 50] . numerous smallpox epidemics have occurred throughout recorded history including more than 300 million fatalities during the twentieth century alone [51] [52] [53] . smallpox was eventually eradicated following the implementation of the smallpox eradication program by the who from 1966 to 1980 ( fig. 5 .2d) which was facilitated by the absence of a zoonotic reservoir for varv [51] . influenza viruses are transmitted by large respiratory droplets by coughing, sneezing, or talking or through contact with infected surfaces [54] . influenza viruses bind to sugar moieties on the surface of airway epithelial cells where early viral replication, propagation, and shedding occur during an average 1-2 days of incubation period [55] [56] [57] . peak viral replication typically occurs within 4 days of symptom onset and resolves within 7-10 days, lasting longer in children and immunocompromised hosts [58] [59] [60] . on average one person infects -one to two additional people; however, this reproductive number (r 0 ) varies by viral strain and social and environmental factors [61] . viral infection impairs the airway mucosal barrier and disrupts the alveolar-capillary membrane contributing to leakage of fluid and inflammatory cells into the alveolar space which impairs gas exchange resulting in hypoxemia [62, 63] . bacterial coinfection often complicates severe cases contributing to respiratory failure and death, with staphylococcus aureus and streptococcus species as predominant copathogens [64] . seasonal influenza virus infection is largely limited to the respiratory tract; however, h5 and h7 hpai viruses have a polybasic cleave site within the hemagglutinin allowing for replication outside of the respiratory tract [65, 66] . infection with one strain of influenza does not confer complete immunity to other strains or subtypes [67] . measles is among the most highly contagious respiratory infections, spread by exposure to large respiratory droplets through coughing, sneezing, or talking; by indirect contact with infected surfaces; or by small infectious droplets that can remain suspended in air for up to 2 hours [10, 68] . respiratory tract dendritic cells, lymphocytes, and alveolar macrophages are early targets of infection where during an average 8-to 12-day incubation period measles replicates and spreads to local lymphatics and respiratory epithelium and then disseminates in blood via infected lymphocytes to epithelial and endothelial cells in most organs [69] [70] [71] . the infectious period begins with fever onset and extends for several days after rash appears [72] . the estimated r 0 of measles is 9-18 dependent upon host susceptibility and social and environmental factors [73] . measles infects and disrupts tissues throughout the body; however, severe disease is primarily due to lower respiratory tract and neurological complications [72] . natural measles infection confers lifelong immunity, and passive transfer of maternal antibodies protects newborns during the early postnatal period [74] . individuals who recover from measles infection are at increased risk of secondary infection [75, 76] . sars-cov is transmitted by large respiratory droplets and by contact with infected surfaces. epidemiologic data also support small droplet airborne transmission of sars-cov although the estimated r 0 of 0.86-1.83 argues against this being a predominate route of spread [77, 78] . sars-cov binds to angiotensin-converting enzyme (ace)-2 receptors on respiratory epithelial cells, pneumocytes, and alveolar macrophages resulting in diffuse alveolar damage and respiratory failure [79, 80] . sars is a systemic infection with viremia detected in most cases affecting multiple cell types and organs [81, 82] . acute kidney injury is multifactorial with evidence of acute tubule necrosis, vasculitis, and glomerular fibrosis, and central nervous system manifestations are at least in part attributable to direct infection of neurons resulting in edema and degeneration [83] . mers-cov is transmitted by large respiratory droplets and by contact with infected surfaces with an estimated r0 of <1 to >1 outside of versus within healthcare settings, respectively [84] . mers-cov binds dipeptidyl peptidase 4 (dpp4) on respiratory epithelial cells and pneumocytes where it undergoes productive replication during a 2-14 days incubation period [16] . viral shedding from the lower respiratory tract may persist for weeks [85, 86] . viremia, while not documented in all cases, is associated with severe disease and productive infection of dcs, and macrophages is thought to facilitate immune dysregulation [87, 88] . dpp4 is broadly expressed on cells outside of the lung; however, few autopsy data are available to define viral distribution [16, 89] . varv is transmitted primarily by large respiratory droplets and to a lesser degree through contact with contaminated objects such as scabs, bedding, or clothing or by airborne small respiratory droplets [90, 91] . varv is thought to replicate in airway epithelium and spread to regional lymph nodes [92, 93] . varv replicates within lymph nodes and disseminates via the bloodstream seeding distant sights including skin, spleen, bone marrow, liver, kidney, and other organs [94] . fever manifests following an average 12 days incubation, and rash follows fever by 3-4 days, concurrent with high-level viral shedding from oropharyngeal secretions [95, 96] . the estimated r 0 of smallpox is between 3.5 and 6 [97] . high-level viremia is detected more often with hemorrhagic compared with ordinary type smallpox, although exact mechanisms of organ failure observed in fatal case are not well defined [98] [99] [100] [101] . influenza infection manifests as acute onset of fever, chills, malaise, headache, and myalgias following an average 1-2 days asymptomatic incubation period [9] . most infections are self-limited resolving within 1-2 weeks. upper or lower airway complications include otitis media, sinusitis, bronchitis, and pneumonia with or without bacterial coinfection [63, 64, 102] . risk factors for severe infection include age >65 years or <5 years; pregnancy; preexisting respiratory, cardiac, neurologic, or metabolic conditions; immunosuppression; and obesity. progressive lethargy and shortness of breath, typically within 5 days of symptom onset, suggest development of lower respiratory tract complications which may rapidly progress to respiratory failure and death in severe cases [64] . pneumonia due to influenza infection alone versus influenza and bacterial coinfection cannot be reliably distinguished by clinical or radiological grounds, and so a high index of suspicion is needed. influenza complications outside of the respiratory tract include exacerbation of underlying heart disease including ischemic heart disease and heart failure, myocarditis, encephalopathy, and encephalitis [103] . measles infection manifests by acute onset fever, coryza, conjunctivitis, and cough [10] . small white papules, koplik spots, appear on the buccal mucosa within 3 days of fever onset, followed by development of diffuse maculopapular rash 1 or 2 days later. diarrhea commonly begins shortly following rash onset and may result in dehydration. symptoms typically resolve within 7 days of fever onset in self-limited illness. groups at increased risk for measles complications include malnourished infants and those with vitamin a deficiency, adults >20 years old, and immunocompromised individuals [72] . respiratory complications include otitis media, laryngotracheobronchitis (croup), and pneumonia. pneumonia, often complicated by bacterial coinfection, is the most common severe complication of measles contributing to respiratory failure and death [72, 104] . predominant bacterial copathogens include streptococcus pneumonia, staphylococcus aureus, and haemophilus influenzae. three rare but severe neurologic complications occur [105] . acute disseminated encephalomyelitis (adem) is a demyelinating autoimmune process that occurs within weeks of acute illness in approximately 1 in 1000 cases. adem is characterized by fevers, seizures, and neurologic deficits. measles inclusion body encephalitis is a progressive lethal brain infection occurring within months of acute illness primarily among individuals with impaired cellular immunity. subacute sclerosing panencephalitis (sspe) occurs 5-10 years following initial infection resulting in seizures and cognitive and motor decline resulting in death. sspe affects an estimated 1 in 10,000 infants under 1 year of age and is attributed to host responses to defective viral particle production in the brain. following an average 5-day incubation period, sars-cov infection presents with fevers, chills, dry cough, headache, malaise, and dyspnea commonly followed by watery diarrhea [106] [107] [108] . age >60 years and pregnancy are associated with severe disease manifested by progressive respiratory failure within 2 weeks of illness onset [108, 109] . common laboratory features of sars included lymphopenia, thrombocytopenia, abnormal coagulation parameters, and elevated lactate dehydrogenase, alanine aminotransferase, and creatine kinase levels [110] [111] [112] . acute kidney injury and proteinuria were observed in 7% and 84% of patients, respectively [113] . initial symptoms of mers-cov infection include fever, chills, cough, shortness of breath, myalgia, and malaise following a mean incubation period of 5 days [114] . gastrointestinal symptoms, including vomiting and diarrhea, occur in onethird of patients [115] [116] [117] [118] . the median times from symptom onset to hospitalization, icu admission, and death are 4, 5, and 12 days, respectively [118] . mers patients present with a rapidly progressing pneumonia requiring mechanical ventilation and additional organ support with the first week of illness [109] . severe disease has been linked to comorbidities including diabetes mellitus (68%), chronic renal disease (49%), hypertension (34%), chronic cardiac disease (28%), chronic pulmonary disease (26%), and obesity (17%) [114] . the median age of those with confirmed mers is 50 years with a male-to-female ratio of 3.3:1 [114] . laboratory abnormalities include lymphopenia, leukopenia, thrombocytopenia, elevated serum creatinine levels consistent with acute kidney injury, and elevated liver enzymes [114, 115, 117, 119, 120] . high lactate levels and consumptive coagulopathy have also been reported [119, 121] . chest radiographic abnormalities are due to viral pneumonitis with or without secondary bacterial pneumonia, and acute kidney injury occurs in up to 43% of patients [114, 119, 120, [122] [123] [124] . as the smallpox disease course was related to the clinical presentation of disease, rao proposed a clinical classification system [125] that was later adopted by the who in 1972 [51] . ordinary type smallpox was the most common clinical type of smallpox. the incubation period was 7-19 days and was followed by fever onset (38.5-40.5 °c), headaches, backaches, vomiting, and diarrhea [51] . lesions first appeared on mucous membranes (including the tongue, palate, and pharynx) ~1 day prior to macular rash development, where lesions began on the face followed by proximal regions of the extremities, the trunk, and the distal extremities. lesion development followed a centrifugal dispersion pattern, typically most dense on the face, with papules appearing within 2 days of macular rash development. papules became vesicular ~2-4 days later followed by a pustular stage (5-7 days postrash) that peaked ~10 days postrash. pustule resolution quickly followed and was accompanied by lesion flattening, fluid reabsorption, hardening, and scab formation (14-21 days postrash). rao proposed for ordinary type smallpox to be further subdivided based on the macular rash pattern [125] . these included discrete ordinarytype smallpox, characterized by discrete skin lesions; confluent ordinary-type smallpox, where pustular skin lesions were confluent on the face and extremities; and semiconfluent ordinary-type smallpox, where skin lesions were confluent on the face but disparate over the rest of the body. modified-type smallpox, where lesions were less numerous than in ordinary-type smallpox, was primarily associated with vaccinated individuals and had an accelerated nonfatal disease course [125] . flattype and hemorrhagic-type smallpox were the most lethal forms of the disease but were also very rare (~7% and 3% of patients, respectively) [51] . flat-type smallpox had high cfrs in both unvaccinated and vaccinated patients (97% and 67%, respectively). hemorrhagic-type smallpox was nearly 100% fatal in both vaccinated and unvaccinated individuals, and death normally came prior to macular rash development. the clinical symptoms of flat-type smallpox were more severe during the prodromal period and did not subside. skin lesions were flat and often black or dark purple. respiratory complications were common and patients were febrile throughout disease. death typically occurred 8-12 days post-fever onset. hemorrhagic-type smallpox could be divided into early and late hemorrhagic-type smallpox. the early form was characterized by hemorrhage (primarily subconjunctival) early in the disease course. generalized erythema, petechiae, and ecchymosis within 2 days of fever and flat matter lesions formed across the entire body surface. lesions turned purple by day 4 with death by day 6 as a result of cardiac and pulmonary complications. in the late form, hemorrhages occurred following rash development and death followed between 8 and 10 days post-fever onset. in healthcare settings, patients under evaluation for influenza should be isolated, and standard, droplet, and contact precautions should be implemented [126] . traditional antigen-based rapid diagnostic assays (rdas) for influenza lack sensitivity and cannot be relied upon to rule out infection [26] . newer antigen-based rdas that employ a digital scan of the test strip, and molecular assays that employ isothermal amplification technology have improved sensitivity and specificity that more closely approximates highly sensitive and specific reverse transcriptase polymerase chain reaction (rt-pcr)-based assays [127] . acceptable sample types for influenza testing include nasopharyngeal swab or wash and bronchoalveolar lavage specimens. individuals suspected of zoonotic influenza infection should have case evaluation and specimen testing coordinated through local or state public health authorities. measles should be considered in patients without preexisting immunity and a compatible febrile rash illness. travel to a region with ongoing measles transmission or exposure to other individuals with a febrile rash illness should raise suspicion. patients under evaluation for measles require isolation and implementation of standard, airborne, and contact precautions. local or state health authorities should be contacted within 24 hours to assist with confirmatory testing, case finding, and infection control. measles is typically confirmed by measles-specific igm serology or detection of measles rna in a nasopharyngeal, throat, or urine specimen by rt-pcr [10] . a fourfold or greater rise in measles igg titers between acute and convalescent samples tested 2 or more weeks apart can assist with diagnostic uncertainty. virus can also be cultured from respiratory, blood, and urine specimens in appropriate public health laboratories. while sars is no longer circulating, mers should be suspected in individuals with a compatible febrile illness and an epidemiological risk factor [128] . risk factors include travel to the arabian peninsula or contact with a confirmed or suspected case within 14 days of symptom onset. patients under evaluation for mers require isolation and implementation of standard, airborne, and contact precautions. confirmatory testing and infection control should be coordinated through local or state health authorities. mers may be confirmed in designated public health laboratories by rt-pcr testing of lower respiratory tract specimens [129] . multiple other specimen types including upper respiratory tract samples, serum, and stool should also be collected for testing. serologic testing can be used to evaluate for suspected infection among individuals no longer shedding virus [129, 130] . smallpox has not been observed in over 40 years; however, concerns remain for use as a bioweapon. major and minor criteria have been established to assist clinicians in recognition of smallpox [131] . individuals under evaluation should be isolated, and standard, airborne, and contact precautions should be implemented. local or state health authorities should be contacted to assist with confirmatory testing and public health interventions. pcr identification of variola dna or isolation of the virus from a clinical specimen is required to confirm a diagnosis in specialized highcontainment laboratories. annual seasonal influenza vaccination is recommended in the united states for all individuals aged 6 months or older and has been associated with decreased risk of pneumonia and death, particularly among high-risk groups [132] [133] [134] . seasonal influenza vaccination does not provide protection against novel strains. consequently, efforts are underway to develop a vaccine that would protect against most or all influenza strains [135] . three classes of drugs are licensed for the treatment of influenza in the united states [136] . adamantanes, including amantadine and rimantadine, are not currently recommended given resistance of circulating seasonal strains. baloxavir morboxil, a cap-dependent endonuclease inhibitor, was recently approved for the treatment of uncomplicated influenza [137] . neuraminidase inhibitors (nai) include oral oseltamivir, inhaled zanamivir, and intravenous peramivir. prophylactic use of nais is recommended in unvaccinated individuals with risk factors for severe disease and during institutional outbreaks to limit spread. therapeutic use is recommended for individuals with suspected or confirmed influenza that have developed or are at high risk for influenza complications [26] . influenza complications, including respiratory and multiorgan failure, are managed with supportive care. bacterial coinfection should be considered and empirically treated early pending results of microbiologic testing among severe cases. measles can be effectively prevented through vaccination, typically given in combination with vaccines for rubella (mr), mumps (mmr), or varicella (mmr-v). who recommends the first dose of measles vaccine be administered at 9 or 12 months of age in high and low prevalence settings, respectively [138] . a second dose should be administered after a minimum of 4-week interval. nonimmune individuals that have been exposed to measles should receive post-exposure prophylaxis with mmr or immunoglobulin within 72 hours or 6 days, respectively, although not concurrently [139] . clinical management of patients with measles consists of fluid, electrolyte, and nutritional support and early recognition and treatment of bacterial coinfection [10] . two doses of vitamin a in children under 2 years have been associated with reduced risk of pneumonia and death [140] . who recommends administering 200,000 iu of vitamin a daily for 2 days in children aged 1 year and older, with reduced dosing in younger infants [141] . there are currently no licensed therapeutics or vaccines for sars or mers. consequently, supportive care is the mainstay of treatment [142] . renal replacement therapy is frequently required in severe illness [119, 143, 144] . empiric antibiotics are often administered given potential for secondary bacterial infection. ribavirin and pegylated interferon alpha 2b have been administered to mers patients, although effectiveness data is lacking [144] . aerosol-generating procedures including endotracheal intubation are associated with increased risk of healthcare worker infection necessitating strict adherence to infection control measures, including use of eye protection in addition to standard, airborne, and contact precautions [145] . while routine smallpox vaccination ceased at the end of the smallpox eradication program, it is still employed for those at increased risk for exposure. first-generation vaccines comprise a significant proportion of both the us national and global vaccine stockpiles [146] . however, first-generation vaccines carry high risk of adverse events due to use of replication-competent vacv and potential manufacturing contaminants. second-generation smallpox vaccines have reduced concerns for contaminants and are expected to have similar protective efficacy as first-generation vaccines. acam2000® has garnered us food and drug administration licensure for vaccination of those at high risk for orthopoxvirus exposure and is part of the us strategic national stockpile [147] . acam2000® and the lister-derived vaccines rivm and elstree-bn also contribute to the global stockpile. imvamune (mva), a third-generation vaccine, is licensed in europe and canada and is part of the us national stockpile. passive immunization with vig has been employed to treat complications of vaccinations [148, 149] . there has also been increasing interest in the development and licensure of small molecule antivirals for treatment of orthopoxvirus infections. cmx001 (brincidofovir), a dna synthesis inhibitor, has demonstrated protection against lethal varv in nonhuman primates [150] and has been granted ophan drug designation while also being included in the us strategic national stockpile. st-246 (tecovirimat), which inhibits viral egress, has potent (ic50 < 0.010 μm) and selective (cc50 > 40 mm) inhibitory activities against multiple orthopoxvirues [151] , is the only antipoxvirus therapeutic that has been granted approval in the us and has been added to the strategic national stockpile. types of influenza viruses serologic evidence of exposure to influenza d virus among persons with occupational contact with cattle characterization of a novel influenza virus in cattle and swine: proposal for a new genus in the orthomyxoviridae family avian influenza virus surveillance and wild birds: past and present a distinct lineage of influenza a virus from bats new world bats harbor diverse influenza a viruses a revision of the system of nomenclature for influenza viruses: a who memorandum structure, transcription, and replication of measles virus coronaviruses: an overview of their replication and pathogenesis coronavirus infections in hospitalized pediatric patients with acute respiratory tract disease sars and mers: recent insights into emerging coronaviruses a decade after sars: strategies for controlling emerging coronaviruses dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-emc angiotensin-converting enzyme 2 is a functional receptor for the sars coronavirus an overview of poxviruses discovery of antivirals against smallpox avian influenza: assessing the pandemic threat global epidemiology of avian influenza a h5n1 virus infection in humans, 1997-2015: a systematic review of individual case data epidemiology of human infections with avian influenza a(h7n9) virus in china outbreaks of avian influenza a (h5n2), (h5n8), and (h5n1) among birds--united states influenza a (h3n2) variant virus-related hospitalizations: ohio estimated influenza illnesses, medical visits, hospitalizations, and death in the united states -2017-2018 influenza season influenza: the mother of all pandemics world health organization. global influenza surveillance and response system (gisrs biological feasibility of measles eradication progress toward regional measles elimination -worldwide measles cases in europe a measles outbreak in an underimmunized amish community in ohio progress toward regional measles eliminationworldwide a measles epidemic threshold in a highly vaccinated population middle east respiratory syndrome coronavirus in dromedary camels: an outbreak investigation middle east respiratory syndrome coronavirus neutralising serum antibodies in dromedary camels: a comparative serological study evidence for camel-to-human transmission of mers coronavirus mers coronavirus in dromedary camel herd, saudi arabia isolation of mers coronavirus from a dromedary camel isolation and characterization of a bat sars-like coronavirus that uses the ace2 receptor bats are natural reservoirs of sars-like coronaviruses characterization of a novel coronavirus associated with severe acute respiratory syndrome high prevalence of mers-cov infection in camel workers in saudi arabia severe acute respiratory syndrome (sars): a year in review middle east respiratory syndrome coronavirus (mers-cov) mers-cov outbreak following a single patient exposure in an emergency room in south korea: an epidemiological outbreak study transmission characteristics of mers and sars in the healthcare setting: a comparative study transmission of middle east respiratory syndrome coronavirus infections in healthcare settings on the origin of smallpox: correlating variola phylogenics with historical smallpox records smallpox and its eradication. geneva: world health organization smallpox: the fight to eradicate a global scourge jenner and the history of smallpox and vaccination routes of influenza transmission. influenza other respir viruses transmission of influenza: implications for control in health care settings human and avian influenza viruses target different cells in the lower respiratory tract of humans and other mammals influenza virus receptors in the human airway kinetics of influenza a virus infection in humans the timeline of influenza virus shedding in children and adults in a household transmission study of influenza in managua long-term shedding of influenza virus, parainfluenza virus, respiratory syncytial virus and nosocomial epidemiology in patients with hematological disorders estimates of the reproduction number for seasonal, pandemic, and zoonotic influenza: a systematic review of the literature influenza virus-induced lung injury: pathogenesis and implications for treatment the pathology of influenza virus infections bacterial coinfection in influenza: a grand rounds review human infection with highly pathogenic avian influenza a(h7n9) virus, china molecular pathogenesis of h5 highly pathogenic avian influenza: the role of the haemagglutinin cleavage site motif immunobiology of influenza vaccines airborne transmission of measles in a physician's office measles virus host invasion and pathogenesis pathological consequences of systemic measles virus infection predominant infection of cd150+ lymphocytes and dendritic cells during measles virus infection of macaques the clinical significance of measles: a review the basic reproduction number (r0) of measles: a systematic review early waning of maternal measles antibodies in era of measles elimination: longitudinal study measles virus-induced suppression of immune responses long-term measles-induced immunomodulation increases overall childhood infectious disease mortality evidence of airborne transmission of the severe acute respiratory syndrome virus model parameters and outbreak control for sars epithelial cells lining salivary gland ducts are early target cells of severe acute respiratory syndrome coronavirus infection in the upper respiratory tracts of rhesus macaques a crucial role of angiotensin converting enzyme 2 (ace2) in sars coronavirus-induced lung injury multiple organ infection and the pathogenesis of sars tissue distribution of ace2 protein, the functional receptor for sars coronavirus. a first step in understanding sars pathogenesis pathology and pathogenesis of severe acute respiratory syndrome middle east respiratory syndrome coronavirus (mers-cov). who mers-cov global summary and risk assessment viral shedding and antibody response in 37 patients with middle east respiratory syndrome coronavirus infection viral load kinetics of mers coronavirus infection from sars to mers, thrusting coronaviruses into the spotlight viral rna in blood as indicator of severe outcome in middle east respiratory syndrome coronavirus infection differential expression of the middle east respiratory syndrome coronavirus receptor in the upper respiratory tracts of humans and dromedary camels an airborne outbreak of smallpox in a german hospital and its significance with respect to other recent outbreaks in europe transmission potential of smallpox: estimates based on detailed data from an outbreak diagnosis and management of smallpox immunopathogenesis of poxvirus infections: forecasting the impending storm the cause of death in smallpox: an examination of the pathology record virus excretion in smallpox. 1. excretion in the throat, urine, and conjunctiva of patients tropical infectious diseases: principles, pathogens and practice transmission potential of smallpox in contemporary populations viraemia in smallpox virus and virus antigen in the blood of smallpox patients; their significance in early diagnosis and prognosis viraemia in haemorrhagic and other forms of smallpox influenza a virus--induced acute otitis media the hidden burden of influenza: a review of the extra-pulmonary complications of influenza infection. influenza other respir viruses coinfection is common in measles-associated pneumonia measles virus and the nervous system coronavirus as a possible cause of severe acute respiratory syndrome enteric involvement of severe acute respiratory syndrome-associated coronavirus infection the epidemiology of severe acute respiratory syndrome in the 2003 hong kong epidemic: an analysis of all 1755 patients severe acute respiratory syndrome vs. the middle east respiratory syndrome severe acute respiratory syndrome a major outbreak of severe acute respiratory syndrome in hong kong severe acute respiratory syndrome (sars): epidemiology, diagnosis and management acute renal impairment in coronavirus-associated severe acute respiratory syndrome epidemiological, demographic, and clinical characteristics of 47 cases of middle east respiratory syndrome coronavirus disease from saudi arabia: a descriptive study clinical features and viral diagnosis of two cases of infection with middle east respiratory syndrome coronavirus: a report of nosocomial transmission epidemiological findings from a retrospective investigation family cluster of middle east respiratory syndrome coronavirus infections state of knowledge and data gaps of middle east respiratory syndrome coronavirus (mers-cov) in humans clinical course and outcomes of critically ill patients with middle east respiratory syndrome coronavirus infection isolation of a novel coronavirus from a man with pneumonia in saudi arabia middle east respiratory syndrome coronavirus: a case-control study of hospitalized patients middle east respiratory syndrome coronavirus (mers-cov) infection: chest ct findings middle east respiratory syndrome clinical aspects and outcomes of 70 patients with middle east respiratory syndrome coronavirus infection: a single-center experience in saudi arabia bombay: the kothari book depot prevention strategies for seasonal influenza in healthcare settings diagnostic accuracy of novel and traditional rapid tests for influenza infection compared with reverse transcriptase polymerase chain reaction: a systematic review and meta-analysis interim patient under investigation (pui) guidance and case definitions respiratory tract samples, viral load, and genome fraction yield in patients with middle east respiratory syndrome epidemiology of coronavirus-associated respiratory tract infections and the role of rapid diagnostic tests: a prospective study evaluating patients for smallpox: acute, generalized vesicular or pustular rash illness protocol effectiveness of influenza vaccination in institutionalized older adults: a systematic review university of nottingham influenza and the immunocompromised (uniic) study group, nguyen-van-tam js. influenza vaccination for immunocompromised patients: systematic review and meta-analysis by etiology prevention and control of seasonal influenza with vaccines: recommendations of the advisory committee on immunization practices-united states the pathway to a universal influenza vaccine influenza antiviral medications: summary for cli baloxavir marboxil investigators g. baloxavir marboxil for uncomplicated influenza in adults and adolescents world health organization. measles vaccines: who position paper measles (rubeola): for healthcare professionals vitamin a for treating measles in children middle east respiratory syndrome coronavirus: another zoonotic betacoronavirus causing sars-like disease recovery from severe novel coronavirus infection ribavirin and interferon alfa-2a for severe middle east respiratory syndrome coronavirus infection: a retrospective cohort study aerosol generating procedures and risk of transmission of acute respiratory infections to healthcare workers: a systematic review smallpox vaccine and its stockpile in 2005 clinical guidance for smallpox vaccine use in a postevent vaccination program the efficacy of vaccinial immune globulin. a 15-year study experience of anti-vaccinia immunoglobulin in the united kingdom efficacy of tecovirimat (st-246) in nonhuman primates infected with variola virus (smallpox) an orally bioavailable antipoxvirus compound (st-246) inhibits extracellular virus formation and protects mice from lethal orthopoxvirus challenge key: cord-007796-zggk0x2q authors: lindemans, caroline a.; kimpen, jan l. l. title: the immune response to viral lower respiratory tract infection date: 2005 journal: hot topics in infection and immunity in children ii doi: 10.1007/0-387-25342-4_4 sha: doc_id: 7796 cord_uid: zggk0x2q viruses are responsible for the majority of respiratory infections in childhood,causing considerable morbidity and mortality. it is estimated that in the united states approximately $ 652 million per year is spent on medical costs for respiratory syncytial virus (rsv) related disease alone (paramore et al., 2004). viruses cause a variety of respiratory diseases in children from the common cold to life-threatening pneumonia and bronchiolitis. the host reacts to a viral infection with a combination of innate and adaptive immune mechanisms, usually resulting in the clearance of the virus and clinical recovery. however, there is an accumulating evidence for a number of viral infections that the host immune response actually enhances disease in the course of clearing virus from the infected organs. interestingly, the effectiveness of the immune response seems to be dependent on the age and probably genetic background of the child. this has important implications for treatment as well as vaccine development. viruses are responsible for the majority of respiratory infections in childhood, causing considerable morbidity and mortality. it is estimated that in the united states approximately $ 652 million per year is spent on medical costs for respiratory syncytial virus (rsv) related disease alone (paramore et al., 2004) . viruses cause a variety of respiratory diseases in children from the common cold to life-threatening pneumonia and bronchiolitis. the host reacts to a viral infection with a combination of innate and adaptive immune mechanisms, usually resulting in the clearance of the virus and clinical recovery. however, there is an accumulating evidence for a number of viral infections that the host immune response actually enhances disease in the course of clearing virus from the infected organs. interestingly, the effectiveness of the immune response seems to be dependent on the age and probably genetic background of the child. this has important implications for treatment as well as vaccine development. viral infections play an important role in both childhood and adult asthma. they might be instrumental in the inception of asthma and are associated with the majority of exacerbations in asthmatic individuals (johnston et al., 1995; bont et al., 2000) . in respect to the role of viruses in the pathogenesis of acute and chronic airway disease in children, it is of utmost importance that we gain a proper understanding of the underlying mechanisms involved in order to design effective therapeutic and preventive strategies. although viral respiratory tract infections are considered to be mainly pediatric diseases, there is an increasing acknowledgement of their pathogenic potential in the immunocompromised host of all age groups and in the elderly. causative agent is not identified. when an upper respiratory tract infection in an infant progresses to lower respiratory tract disease, bronchiolitis and pneumonia are most common. both disease entities are hard to differentiate and no clinically relevant differences with regard to outcome have been identified (van woensel et al., 2003) . rsv belongs to the paramyxoviridae family and the genus of pneumovirus. it is an enveloped unsegmented single-stranded rna-virus of which two subtypes are known (a and b) . a clear relationship between subtype and disease severity has not been established (kneyber et al., 1996) . since rsv infection does not lead to complete immunity, reinfection is common. immaturity of the immune system during initial infection seems to be the main cause of incomplete memory-response although an as yet undefined mechanism of partial immune evasion by rsv cannot be ruled out . recently, it was suggested that rsv could cause persistent infection or latency (dakhama et al., 1997; schwarze et al., 2004) , although the significance of this is not clear. rsv affects 70% of infants in the first year of life, and by the age of two nearly all children have been infected. (figure 4 .1) it is likely that a specific balance and timeframe of changes in air temperature and humidity are responsible for the well-defined yearly winter outbreaks of rsv (stensballe et al., 2003) . in most infants as well as in older children and adults, rsv is the cause of upper respiratory tract infection with mild symptoms. however, in the very young, the infection spreads to the lower respiratory tract in approximately 40% of cases. one to three percent of infants develop bronchiolitis or pneumonia requiring hospitalization, with a considerable number requiring mechanical ventilatory support. apart from the obvious respiratory symptoms, very young children frequently present with atypical symptoms such as impaired feeding, vomiting, lethargy, and apnea (kneyber et al., 1998) . several risk factors for more severe disease have been identified, including age less than 6 weeks, prematurity, pre-existent cardiorespiratory disease and immunological impairment (bont and kimpen, 2002) . respiratory symptoms are directly related to airway pathology. necrosis of the airway epithelium is a key phenomenon resulting in sloughing of the epithelial cells. together with a dramatic influx of inflammatory cells into the airways and increased mucus production, this leads to the formation of copious secretions that block the small airways. mucosal edema and bronchospasm through irritation of subepithelial nerve endings further compromise airway diameter. although antibiotics are prescribed for up to 50% of children with lower respiratory tract infections, proof of bacterial superinfection is only found in a minority of patients and the role of this event in the pathogenesis of severe disease remains controversial (purcell and fergie, 2002; bloomfield et al., 2004) . on the other hand, it has been demonstrated that rsv infection of airway epithelial cells in vitro enhances adherence of s. pneumoniae (hament et al., 2004) . the influenza virus belongs to the orthomyxoviridae family and is an enveloped segmented single-stranded rna (ssrna) virus (table 4 .1). the structural proteins of the virion are encoded by separate gene segments and include three viral rna polymerases, nucleoprotein, matrix, and the hemagglutinin (ha) and neuraminidase (na) surface glycoproteins. on the basis of their nucleocapsid and matrix protein antigens, the influenza viruses are divided into three distinct immunological types (a, b, and c). although all three influenza viruses cause respiratory disease in humans, only a and b are known to cause epidemics. induction of a memory-response results in long-lasting immunity and it is the antigenic variation that is responsible for frequent reinfection with the virus. the most antigenic variation is seen in the virus that infects both animals and humans, influenza a. fourteen subtypes of hemagglutinin (h1-h14) and nine types of neuraminidase (n1-n9) are circulating in nature. the segmentation of the genome makes exchange of genetic material between subtypes possible, resulting in the structural changes observed in influenza, which has caused pandemics in the past. when two different subtypes of influenza a virus infect the same cell, major changes (antigenic shifts) can occur through rearrangement of genetic segments from both infecting viruses. minor changes (antigenic drifts) in na and/or ha proteins occur through accumulation of point mutations, and provide a mechanism for the virus to escape protective antibodies and cause respiratory symptoms every year. influenza virus epidemics are difficult to separate in time from rsv epidemics, and the diseases caused by both viruses can also be difficult to differentiate (zambon et al., 2001) . (figure 4 .1) compared to other viruses, morbidity caused by influenza is high in all age groups. children with influenza infection of the respiratory tract are more likely to present with fever. infants aged less than 6 months and older children with an impaired immune system, or other serious health problems, have a higher risk of hospitalization and mortality. subclinical infections with influenza in children are common, suggesting children can be an important reservoir and source of transmission. yearly updated vaccines are available and effective, and recently it has been proposed to extend the current recommendations to children less than 2 years of age, children with recurrent acute otitis media or respiratory tract infections, and healthy children attending day-care centers or elementary schools (principi and esposito, 2004 ). adenoviruses, belonging to the adenoviridae family and the genus mastadenovirus, are a group of dna-viruses of which at least 47 serotypes are known. for lower respiratory disease, subtypes 1, 2, 3, 4, 5, 7, and 21 are most important. it is an icosahedral capsid virus with extruding fiber proteins, which are required for viral entry to epithelial cells (howitt et al., 2003) . although human adenoviruses are ubiquitous, and cause primary infection in the first year of life, there is geographical variation in the distribution of serotypes and in the association of serotypes with different age groups. in europe, adenovirus is the cause of infection in approximately 5% of hospitalized patients with viral lower respiratory tract disease. however, in some south american and asian countries, adenovirus is the second most prevalent pathogen for acute lower respiratory tract infection in children after rsv (carballal et al., 2002) . although adenovirus infections in general occur the whole year round, respiratory adenovirus infections are most common during late winter, spring, and early summer. adenovirus type 7, acquired by inhalation, has been associated with more severe lower respiratory tract disease (larranaga et al., 2000) . subtype-specific immunity occurs. however, some types are capable of establishing persistent asymptomatic infections in tonsils, adenoids, and intestines of infected hosts, and shedding can occur for months or years. children under 6-years old. though it is the causative agent of similar disease entities, hospitalizations occur four times less frequently than for rsv infections (hall, 2001) . two subtypes are clinically important respiratory pathogens in children, piv-1 and piv-3. piv-1 is the main cause of croup in 2-6-year olds, while piv-3 is responsible for parainfluenza bronchiolitis in children under 6-months old. piv-4 only causes mild upper respiratory tract infections. because of acute narrowing of the subglottic region of the larynx, moderate to severe croup may require emergency management with systemic or inhaled corticosteroids, which are effective in improving stridor in a few hours (cetinkaya et al., 2004) . the hallmark cytopathic effect of acute infection with piv-3 is comparable to that of rsv with extensive cell fusion resulting in syncytium formation. for fusion to occur, two piv glycoproteins are required, including the hemagglutinin-neuraminidase (hn) glycoprotein interacting with host cell sialic acid receptor and the viral fusion (f) glycoprotein. rhinovirus infections account for the largest number of respiratory tract infections in children. however, rhinovirus infections produce mild symptoms compared to rsv, piv, and influenzavirus. most of the symptoms caused by rhinovirus are confined to the upper respiratory tract. although present in the community the whole year round, rhinovirus infections peak at the onset of fall, which is probably related to schools starting after summer break. rhinoviruses can cause severe lower respiratory tract infection (guittet et al., 2003; papadopoulos, 2004) and in immunocompromised patients, life-threatening pneumonia. rhinovirus is a positive-stranded rna-virus belonging to the picornavirus family and over 100 serotypes exist, making it difficult to develop an effective vaccine. rhinovirus subtypes have been divided into a major and minor group with respect to the receptor used for cell entry (table 4 .1). major group rhinoviruses use epithelial intracellular adhesion molecule l (icam-1) for cell entry, while minor group viruses bind to the low-density lipoprotein (ldl) receptor. during rhinovirus infection, a predominant granulocyte and monocyte recruitments are observed. while specific antibody production occurs, it is probably not required for viral clearance, although neutralizing antibodies can provide some temporary protection against rhinovirus reinfection (van kempen et al., 1999) . icam-1 blocking antibodies have also been utilized and have been shown to decrease inflammation in vitro. there are, however, indications that rhinovirus can adapt to this with changes in receptor usage (reischl et al., 2001 ). in 2001, a new respiratory virus was identified in the netherlands causing infections similar to rsv in children (van den hoogen et al., 2001) . the reported incidence rate of human metapneumovirus (hmpv) infection in children with acute respiratory symptoms varies between 4% and 16%, of which three-quarters occur in children less than 1-year old (williams et al., 2004) . by the age of 5 years, approximately 70% of children have developed antibodies to hmpv. hmpv very much resembles rsv in its clinical spectrum, varying from coryza to bronchiolitis and pneumonia. however, hmpv is less likely to cause pneumonia than rsv and influenza virus. children with hmpv infection present less frequently with atypical symptoms such as vomiting, and on physical examination, rales and wheezing are found less often. co-infection with rsv and hmpv does occur and has been suggested to result in more severe disease (greensill et al., 2003) . there is some evidence that secondary hmpv infection occurs frequently in childhood, probably accompanied only by mild symptoms (ebihara et al., 2004) . several investigators have found chemokine profiles during acute infection to be different in children with hmpv infections compared to those with rsv infections, with higher interleukin-8 (il-8) and lower rantes concentrations in hmpv patients. however in another study, inflammatory cytokine (il-8, tnf-␣, il-1␤) levels in respiratory secretions were 6-fold lower than in children infected with rsv (jartti et al., 2002; laham et al., 2004) . tthe physiological relevance of these observations remains unclear. the outbreak of severe acute respiratory distress syndrome (sars), which started in late 2002 in east asia, and spread throughout the world during that winter, was found to affect mainly health-care workers and close contacts of diseased individuals. sars, which was proved to be caused by a new coronavirus, induces an atypical pneumonia with fever, dry cough, and shortness of breath. many adults also suffer from myalgia, dizziness, chills, and rigors. it was concluded from postmortem examinations that sars-pathology is primarily caused by immunological damage to the lungs. the interstitial space of the lungs was mainly filled with mononuclear infiltrates and there was diffuse hemorrhage on the lung surface. sars coronavirus (sars-cov) spreads mainly via the respiratory route, the epithelial cell being its primary target cell. as for other coronaviruses, the spike proteins, s1 for cell entry and s2 for fusion, also seem to be important for sars entry of host cells, despite the fact that sars is only 20-30% homologous to other coronaviruses. very recently, angiotensine converting enzyme (ace2) was identified as the host cell receptor for sars-cov (li et al., 2003) and surface expression of ace2 on alveolar epithelial cells was demonstrated (hamming et al., 2004) . transmission of sars-cov occurs by droplets and most cases have occurred through close contact exposure. however, recently evidence of airborne transmission has emerged (yu et al., 2004) . although many individuals were infected in the initial 2 weeks of the epidemic and the disease spread rapidly over several countries, the numbers of infected children stayed relatively low in all regions (ͻ5%). furthermore, children tend to develop less severe disease. after an incubation period of 5-10 days, similar to adults, infected children developed symptoms of a mild upper respiratory tract infection, clinically indistinguishable from other common colds. none of the 100 pediatric sars cases in hong kong turned out to be fatal and only one adolescent required mechanical ventilation (leung et al., 2003) . adolescents are more likely to develop severe disease, as observed in adult sars patients (leung et al., 2004) . a sore throat and a high initial and peak peripheral blood neutrophil count were found to be independent risk factors for severe disease in children with a laboratory-confirmed sars infection. furthermore, children seemed to spread the disease less easily to others and there have appeared no reports in the literature demonstrating transmission from children to other individuals. many children with laboratory-confirmed sars do not meet the who criteria for diagnosis of sars. as children seem to have a much milder clinical course, the term "sars" may not represent the disease in children very well. the role of the innate immune system in viral lower respiratory tract infection has not been studied intensively until recently. studies have focused on the adaptive immunity with the goal of developing a vaccine, for example, for rsv. understanding the mechanisms underlying primary and recurrent viral infection has attracted increased attention. the immunological response against viral invasion of the lower respiratory tract comprises both adaptive and innate immune response mechanisms with both beneficial as well as detrimental characteristics. the innate response occurs in the early phase of the infection and increasing evidence suggests that these early events determine disease course and possibly even long-term outcome (garofalo and haeberle, 2000; tasker et al., 2000) . for the rest of the discussion on immunological phenomena, focus will be on rsv as a prototype. epithelial cells are key regulators of the innate immune response against viral infections (garofalo and haeberle, 2000) , producing a number of inflammatory mediators in response to rsv infection. these include cytokines (interleukin-6, -1, tumor necrosis factor (tnf)-␣), several chemokines (il-8, macrophage inflammatory protein (mip)-1␣, monocyte chemotactic protein (mcp-1), rantes), type-l interferon (ifn-␣/␤), and growth factors (gm-csf, g-csf). epithelial-derived levels of chemokines correlate with disease severity (bont et al., 1999; smyth et al., 2002) . surfactant proteins produced by epithelial cells (sp-a and sp-d) may also play a role as opsonins for viruses and bacteria. thus, epithelial cells provide a potential mechanism for serum-independent phagocytosis. many of these mediators are induced both at the level of secretion and transcription. interestingly, some mediators (e.g., il-8 and rantes) are also upregulated by inactive forms of the virus (harrison et al., 1999) . rsv uptake by immune and non-immune cells is a receptor-mediated process. experiments with blocking antibodies against g-protein revealed inhibition of binding of rsv to epithelial cells. the fractalkine receptor, also known as the cx3cr1 chemokine receptor, is involved in g-protein-mediated uptake by epithelial cells . other receptors may very well be involved in uptake by dendritic cells, macrophages, and other cells of the innate immune system (harris and werling, 2003) , and toll-like receptors (tlrs), especially tlr-4, are being investigated as possible candidates for mediating viral uptake (haeberle et al., 2002a, b; monick et al., 2003) . several groups have demonstrated activation of the transcription factor nf-kb in rsv-infected epithelial cells (tian et al., 2002) . many of the exhibited effects observed in epithelial cells can be explained by activation of nf-kb. several cytokines associated with rsv infection have nf-kb binding sites in their promoter or enhancer regions (bitko et al., 1997) . epithelial nf-kb activation has also been observed in other viral infections, including parainfluenza, influenza a, and rhinovirus (pahl and baeuerle, 1995; kim et al., 2000; bose et al., 2003) . nf-kb could be an exciting target for therapy development and experiments in which balb/c mice were treated with perflubron have confirmed this concept. perflubron has already been shown to be effective in clinical trials of patients with respiratory distress syndrome because of its physical characteristics. besides the beneficial physical effect in improvement of gas exchange and of lung compliance, this agent was found to have anti-inflammatory effects. rsv-infected balb/c mice treated with perflubron intranasally showed a reduction in cellular inflammatory infiltrates and decreased chemokine expression in the lung tissue. both the anti-inflammatory effects were directly linked to interference of perflubron with nf-kb-mediated transcription (haeberle et al., 2002a, b) . the chemokines produced by epithelial cells attract t-cells, neutrophils, monocytes, and possibly eosinophils to the respiratory tract. besides induction of secreted products, epithelial cells upregulate expression of adhesion molecules for neutrophils on their surface, allowing neutrophils to adhere firmly to infected cells (wang and forsyth, 2000) . furthermore, neutrophils are the dominant cell type found in bronchoalveolar lavage (bal) fluid of rsv patients (everard et al., 1994 . however, their role in fighting viral infection is not as well established as in bacterial infections. pathological studies of lungs of rsv-infected calves have shown a major influx of neutrophils in the infected airway mucosa, observed earlier than any other cell type involved. furthermore, neutrophils are the dominant cell type found in bronchoalveolar lavage (bal) fluid of rsv patients (everard et al., 1994 " several chemokines and cytokines involved in neutrophil activation have been associated with rsv lower respiratory tract infections. recently, local neutrophil il-9 production has been linked to rsv bronchiolitis (mcnamara et al., 2004) . as shown by wang et al., a major increase in epithelial damage occurs, when rsvinfected epithelial cells are co-cultured with neutrophils (wang and forsyth, 2000) . this is suggestive of a detrimental role for neutrophil-induced immunopathology in lower respiratory tract infections. rantes and mip-1␣ are produced by the epithelium in response to rsv infection and these chemoattractants recruit eosinophils to the inflammatory site. the analogy between clinical features of virus-induced wheezing illnesses and asthma has made eosinophils an attractive subject for studies aimed at improving understanding of rsv pathogenesis. however, mainly because of their absence in bal of rsv patients, their involvement remains controversial. however, eosinophil-derived cationic protein (ecp) has been linked to bronchiolitis and postbronchiolitic wheezing pifferi et al., 2001; dimova-yaneva et al., 2004) . in vitro, eosinophils have also been shown to be susceptible to rsv. eosinophil priming, superoxide production, and degranulation were induced by incubation with rsv kimpen et al., 1992; olszewska-pazdrak et al., 1998; tachibana et al., 2002) . rosenberg and domachowske (2001) have suggested a beneficial role for eosinophils in rsv bronchiolitis. they identified antiviral properties for the eosinophil based on ribonuclease activity of eosinophil-derived neurotoxin (edn) and ecp. this enzymatic activity leads to destruction of extracellular ssrna virions and delayed replication both in vitro and in vivo. recently, there has also been great interest in the involvement of macrophages and dendritic cells in rsv pathogenesis. these cells were already appreciated for their role in antigen presentation, at which dendritic cells are by far superior. macrophages express phagocyte activity, which may be of importance in clearance of infected epithelial cellular debris. fascinating new players in host defense against viruses are pattern recognition receptors. toll-like receptor-4 (tlr-4) and cd14, both present in a complex on these cells, have been found to interact with rsv and receptor-binding results in triggering of the innate immune system. tlr-4 has been shown to activate nf-kb in macrophages of rsv-infected mice (haeberle et al., 2002a, b) and tlr-4-deficient mice have impaired nk-cell and cd14ϩ cell trafficking and delayed viral clearance . furthermore, intracellular pattern recognition receptors tlr-3 and -7, may be involved in recognizing doubleand single-stranded rna (dsrna/ssrna), respectively (akira and hemmi, 2003; lund et al., 2004) . dsrna is produced during replication of rna-viruses and is a potent inducer of ifn-␣/␤. all human cells can produce ifn-␣/␤ in response to viral infection, while only t-cells and nk-cells produce ifn-␥. dsrna also activates dsrna-dependent protein kinase r (pkr) and nf-kb via distinct pathways. transcription of pkr is under control of ifn-␣/␤. pkr controls enzymes directly involved in protein synthesis, thereby inhibiting cellular and viral protein translation. ifn-␣/␤-deficient mice as well as pkrϫ/ϫ mice are extremely sensitive to influenza infection (balachandran et al., 2000) . several viruses, including rsv, have evolved mechanisms to escape the interferon system, which will be discussed below. respiratory epithelial cells are the principal host cells for viral pathogens in lower respiratory tract disease. the degree of replication and the mechanism of spread along the epithelial layer depend on the virus family characteristics. through the fusion (f) protein, rsv is capable of syncytium formation, which allows it to replicate and spread relatively undetected by the immune system for a relatively long period. the virus itself is directly responsible for cytopathology and viral envelope proteins are expressed on the surface of infected epithelial cells. dendritic cells, lining the basal membrane of the respiratory epithelium encounter rsv, pick up viral antigens and migrate to mediastinal lymph nodes where viral antigen is presented to naïve cd4ϩ t-cells. antigen presentation and co-stimulatory molecule expression lead to maturation to the t-helper phenotype. this then induces b-cell proliferation with the production of specific antibodies as well as proliferation of virus-specific cytotoxic cd8-cells. cellular responses are responsible for controlling and terminating acute infection with rsv. in primary infections, the adaptive cellular immune response develops within 10 days. these cd8ϩ cells can recognize and eliminate virus-infected epithelial cells resulting in perforin-mediated cytotoxity. epithelial cells are nonprofessional antigen presenting cells (apc) expressing mhc class l on the surface (garofalo et al., 1996) . when infected, epithelial cells present viral antigen in association with mhc class l molecules. mhc class l restricted antigen presentation to cd8ϩ cells, among other factors, may determine the strength of the cytotoxic response. in cd8-deficient mice, there is delayed viral clearance; however, these mice also exhibit decreased disease severity (graham et al., 1991) . therefore, it is conceivable that cd8ϩ t-cells are crucial in viral clearance while a surplus of cytotoxicity may result in pulmonary injury. in humans, a cytotoxic t-cell response is elicited against all viral proteins, except the g-(attachment)-protein, which is required for cell entry (bangham et al., 1986; hacking and hull, 2002) . it is suggested that a defective response against g-protein is directly associated with enhanced disease. however, g-protein can induce a cd4ϩ response in mice, which is associated with th2-cytokine production and eosinophilia both during primary and secondary infection (openshaw, 1995) . the immune response to the f-protein is dominated by ifn-␥ production and subsequent polarization toward a th1-type cellular response, and therefore it has been postulated that responses to the other viral proteins can modulate the strong th2response to g-protein (graham et al., 2000) . a stronger th1-response seems to induce a more rapid viral clearance and milder disease (bont et al., 1999; legg et al., 2003) . besides activated t-cells, nkcells also produce considerable amounts of ifn-␥ (hussell and openshaw, 1998) . ifn-␥ has important antiviral effects and provides a link between adaptive and innate immune system. it can induce expression of tnf-related apoptosis inducing ligand (trail) on immune cells, which has the potential to trigger apoptosis of virus-infected cells (sedger et al., 1999) . in vitro findings suggest that rsv-infected cells in vivo are susceptible to killing by immune cells through the trail pathway (kotelkin et al., 2003) . nk-cells are also thought to play a role in activating cd8ϩ cells, further modulating the degree of cytotoxicity (hussell and openshaw, 1998) . in summary, in rsv lower respiratory tract infections, cytotoxic cd8ϩ t-cells are involved in viral clearance while the humoral response is required for the protection against reinfection. however, as has been discussed before, memory is incomplete and repeated infections with rsv are common. both igm and igg as well as secretory iga against rsv are formed in infants, and a more vigorous antibody response seems to be protective against rsv infections (meurman et al., 1984; welliver et al., 1989 ). rsv infections are most severe in the youngest age group, which is the least mature in terms of immunity to infections. relative deficiencies in both innate and antigen-specific immunity in infancy have been characterized. these include delayed trafficking of immune cells, less-efficient antigen presentation by dendritic cells, and impaired production of ifn-␥ by t-cells in response to antigen presentation (bont and kimpen, 2002) . the fetus derives maternal igg-antibodies via the placenta fairly late in gestation. this partly explains why prematurity is an important risk factor for severe disease caused by rsv, as well as the physiological characteristics of the small airways. antibody titers produced by infants are relatively low compared to older children. trials with humanized monoclonal antibodies against rsv-f-protein have shown a 50% reduction in rsv lower respiratory tract-related hospitalizations in this highrisk group for severe disease (impact study group, 1998). the cytokine milieu at the time of infection is another factor possibly contributing to the occurrence of severe rsv bronchiolitis especially in the youngest age group. at birth, there is skewing toward a th2-phenotype and rsv bronchiolitis was long thought to be a th2-type disease. this role of th2-skewing is an attractive concept, because it provides some explanation for the association between rsv bronchiolitis and the development of asthma. asthma and allergy have long been acknowledged to beth2-mediated conditions. however, convincing evidence that primary rsv infections are mediated by th2 cytokines is lacking. dendritic cells are thought to have an important function in skewing the th1/th2-ratio. viruses may be important in maturation of dendritic cells, which can then drive differentiation of naive t-cells into either a th2-or a th1-phenotype. the role of regulatory t-cells that suppress both th1 and th2 differentiation has not been studied in rsv bronchiolitis so far. gene polymorphism studies have been undertaken to identify a genetic background to explain the individual susceptibility to rsv lower respiratory tract infection. several polymorphisms situated in genes relevant for the adaptive and innate immunity have been found to correlate with occurrence of rsv infection. polymorphisms of interleukin-4, il-4r, and its receptor, have been associated with rsv bronchiolitis, which is consistent with the th2-hypothesis (choi et al., 2002; hoebee et al., 2003) . very recently, a polymorphism of the gene coding for interleukin-10 (il-10) was found as well (hoebee et al., 2004) . this is particularly interesting since il-10 is a cytokine produced by t-regulatory cells and monocytes, thought to be primarily involved in development of allergy. gene polymorphisms involved in innate immunity include surfactant proteins spa and d (lahti et al., 2002) , the chemokine il-8 (hull et al., 2001) , tlr-4 (tal et al., 2004) , and the chemokine receptor for rantes and mip-1␣, ccr5 (hull et al., 2003) . immunocompromised patients have a higher risk of developing severe disease from viral respiratory tract infections. in particular, the presence of defects in cellular immunity result in an increased duration of viral shedding and enhanced risk of developing severe disease. most cellular immunodeficiencies are iatrogenic in nature. an important cause is intensive immunosuppressive treatment. the number of pediatric patients undergoing organ or stem-cell transplantation is increasing and high doses of chemotherapeutic and immunosuppressive agents are often used in the pre-and posttransplant regimens. immunosuppressive drugs are used in cancer treatment regimens and for a number of inflammatory conditions. community acquired respiratory viruses such as rsv, rhinovirus, adenovirus, influenza a, influenza b, and the parainfluenza group are frequent causes of respiratory disease in these patients (soldatou and davies, 2003) . adenovirus infections have a particularly high risk of adverse outcome, mortality rates are high, and no effective treatment exists. the presence of lower respiratory tract infection and infection in the pre-engraftment phase of hsct is believed to have a particularly poor prognosis (khushalani et al., 2001) . the risk of severe disease is higher during allogenic hsct than autologous hsct. besides causing increased morbidity and mortality, respiratory tract infections are associated with a greater risk of delayed engraftment (abdallah et al., 2003) . in solid organ transplant patients, respiratory virus infections are also associated with a higher incidence of rejection (wendt, 1997) . prolonged shedding of respiratory viruses for weeks or months has been documented in hiv-infected adults and children. this has important implications for infection control in medical facilities. in addition, respiratory viral infection may result in increased hiv replication and, theoretically, hiv disease progression (king, 1997) . in hiv-infected children, rsv infections are less limited by season (madhi et al., 2000) . however, generally, the course of rsv infections in hiv patients is not more severe, unless there is profound lymphopenia or pre-existing lung disease (soldatou and davies, 2003) . other viruses may also cause respiratory complications in the immunocompromised patients. in particular herpesviruses, such as cytomegalovirus (cmv) and varicella zoster virus, can cause severe pneumonia. with a cmv-negative donor and a cmv-positive recipient, there is an especially high risk of reactivation which may lead to severe disease. this reactivation also occurs with epstein barr virus (ebv), human herpes virus (hhv)-6, -7, and -8, although these are much less frequent causative agents of pneumonia. the innate immune defense to viral respiratory tract infections consists of the mucosal layer, type 1 interferons, activated phagocytes, and nk-cells. the impact of primary defects in the innate immune defense has not been well documented. phagocyte defects are primarily related to a higher incidence of bacterial infections. one indication that impaired phagocyte function also leads to increased severity of respiratory viral infection can be derived from a report of severe abnormalities on lung-ct-scans of rsv patients with phagocyte defects (uzel et al., 2000) . interferon-gamma receptor deficiency, which may have implications for both the adaptive and innate immune system, has also been associated with increased susceptibility to viral respiratory pathogens (dorman et al., 1999) . chronic lung disease also increases susceptibility to respiratory viruses (meert et al., 1989; griffin et al., 2002) . premature patients with bronchopulmonary dysplasia are candidates for rsv-immunoprophylaxis because of their increased risk of developing severe lower respiratory tract infections. in children with cystic fibrosis (cf), 39% are already hospitalized with respiratory virus infection in their first year of life. furthermore, there is a correlation between viral infections in infancy and disease progression. infants with cf suffering from a respiratory virus infection are at significant risk for lower respiratory tract disease, hospitalization, and deterioration in lung function that persists months after the acute illness (hiatt et al., 1999) . cf infants were found to be four times more likely to develop an lrti compared with controls. it has been shown that cfderived airway epithelial cells allow a higher degree of piv replication and have an increased production of pro-inflammatory cytokines (zheng et al., 2003) . cfderived epithelial cells are also unable to express no-synthase 2, which results in a decrease production of nitric oxide (no), which has antiviral capacity, reducing effects on replication. furthermore, in cf-cells there is no viral induction of 2ј5јoligoadenylate synthetase (oas), an enzyme that is normally induced by dsrna and ifn-␥. oas is involved in inhibition of cellular protein synthesis, thereby inhibiting viral replication. respiratory viruses can be isolated from the secretions of approximately 75% of children and of more than half of adults during asthma exacerbations (johnston et al., 1995; lemanske, 2003) . recently, copd exacerbations have also been attributed to viral infection by rhinovirus, rsv, and piv (seemungal and wedzicha, 2003) . the underlying mechanisms for this are, however, still a matter of debate. from experimental rhinovirus (rv) infections in humans, it has been shown that rv infection causes increased bronchoconstriction in atopic non-asthmatic and asthmatic individuals, while symptoms in normal individuals are relatively mild. this implies that induction of a wheezing episode requires both rv infection and a preexisting tendency to develop allergic or asthmatic disease (message and johnston, 2001) . rv-specific t-cell responses can be activated by either serotype-specific or shared viral epitopes. cross-reactivity between rv-subtypes could result in vigorous t-cell responses and may amplify allergic inflammation. other proposed mechanisms linking viral infections to asthma exacerbation are epithelial dysregulation, airway remodeling, the immune response to virus, and alterations of neural responses (message and johnston, 2001; gern, 2002) . upregulation of icam-1-expression, which is the entry receptor for major group rhinoviruses, has been found in susceptible individuals. this may be one mechanism predisposing atopic individuals to rv-induced exacerbations. rhinovirus can induce a number of inflammatory mediators (kinins, arachidonic acid) and cytokines (e.g., il-1, il-6, ifn-␣/␤, gm-csf, tnf-␣) that can further enhance inflammation. th1 cytokines seem to have a general antiviral effect while a predominant th2-cytokine response leads to enhanced disease, failure to clear the virus, and amplification of allergic inflammation (message and johnston, 2001) . eosinophil numbers were found to be increased in bronchial biopsies from both healthy and asthmatic human volunteers after experimental rhinovirus infection. this cell type is associated with allergic inflammation in the lung. in allergic rhinitis patients, the increased level of eosinophils in bal even persisted for 6 weeks. these data suggest a potential role for eosinophils in virus-induced asthma, which can be either pathogenic or protective. virus-induced exacerbations of asthma tend to be resistant to treatment with corticosteroids and may require a different therapeutic approach. in vitro, blocking icam-1 has been tried with positive results, which may be of particular relevance to rhinovirus infections. the possibility of other immunomodulating drugs is being investigated and may be of significant benefit to future asthma treatment. a causal relationship between viral respiratory tract infections and asthma exacerbations is generally acknowledged. however, the suggestion that respiratory virus infection is a causal determinant in the development of asthma is highly controversial. according to the hygiene hypothesis, viral infection would be expected to have an inhibitory effect on the development of asthma (an allergy), and this is supported by a study from matricardi et al. (1997) , showing an inverse relation between hepatitis a seropositivity and atopy among soldiers. the hygiene hypothesis is based on the theory that the immune system is directed toward a more th1-skewed immune response with each viral infection. however, this hypothesis is not supported by the observation that rsv infections, severe enough to cause bronchiolitis, are significantly associated with a higher incidence of asthma up to the age of 7-11 years (stein et al., 1999; sigurs et al., 2000) . these data convincingly show a link between rsv bronchiolitis and recurrent wheezing in childhood. in a recent study, wheezing following rsv lower respiratory tract infection was found to develop independent of atopy . it may, however, be true that the transmission route, the organs involved, and exposure to microbial products may be important in determining the final effect of a virus infection on the development of asthma and allergy (gern and busse, 2002) . the link between rsv infection and atopy is even less clear than the one with recurrent wheezing and asthma, at least in humans. animal studies have yielded conflicting results. one group found that rsv infection in mice enhances subsequent allergic inflammation (schwarze et al., 1997) , while others reported a decrease in allergic sensitization and bhr after rsv infection (peebles et al., 2001) . no proof exists that severe rsv infections are associated with atopy that persists into adulthood (peebles, 2004) . a key question is whether the association with the development of asthma is merely an expression of increased susceptibility to both asthma and rsv-induced lower respiratory tract infections or whether true causality is involved. the prevailing theory on this subject involves maturation effects of the th1/th2-balance. the system shifts from a th2-polarization in fetal life, which is an optimal environment for the placenta, to a more balanced th1/th2-phenotype in adulthood. most viruses are known to induce a th1 cytokine response (ifn-␥). this theory states that when infections occur early in infancy, there is a reduced ability to react with an appropriate antiviral th1-response. low ifn-␥ production may result in spread of the virus to the lower respiratory tract. this is in agreement with findings that in children with severe rsv lower respiratory tract infections, lower amounts of ifn-␥ are produced . the dynamics of this shift toward a more balanced th1/th2 immune response may differ between individuals. both environmental factors and genetic make-up may contribute to a slower maturation of th1 competence in some individuals. respiratory virus infections in infancy and an atopic sensitization to aero-allergens, both of which are related to th2-skewed responses and intermittent wheeze, may than synergistically result in persistent wheeze (holt and sly, 2002) . it is likely that more links between atopic sensitization and respiratory infections exist, while preventive rsv-ivig treatment of children results in a decreased sensitization to aeroallergens as well. rsv-prophylaxis may therefore have a long-term benefit in the development of persistent wheeze (piedimonte and simoes, 2002; wenzel et al., 2002) . another theory linking viral infections in childhood to the development of asthma involves the pathologic effects of viral lower respiratory tract infections on airway physiology. wall thickening with consequent increased resistance may predispose the airway to more infections and thus influence bronchial hyperreactivity (bardin et al., 1992) . however, it may also be true that small airways predispose to both asthma and airway symptoms from viral infections. remodeling of the submucosal neural networks by rsv, as observed by piedimonte et al., is also proposed to result in increased responsiveness to airway irritants (piedimonte, 2002) . walter et al. (2002) have proposed that paramyxoviral infection has the ability not only to induce acute hyperresponsiveness, but also to result in long-lasting changes in airway behavior. from mouse-studies with a piv (sev), it has been concluded that viruses cause long-term effects in epithelial cells, associated with airway reactivity and goblet cell hyperplasia. long-term effects are induced by the virus in the acute phase, and later on, the presence of virus is no longer required for the persistence of symptoms. it is speculated that primary paramyxoviral infection within the proper genetic background may result in chronic dysfunction of epithelial cell behavior. their results have indicated that different mechanisms are responsible for the induction of the acute and the chronic response (walter et al., 2002) . several theories on the induction of asthma have thus been proposed. the current view is that virus infection modulates the development of an asthmatic phenotype in a susceptible host. the relationship is, therefore, not purely causal but certainly requires an intrinsic vulnerability. the effectiveness of respiratory virus infections in the host depends partly on the ability to evade the immune system (figure 4 .2). while several viral evasion mechanisms have evolved, not all have been intensively studied in respiratory viruses. viral entry into host cells is one of the first obstacles viruses have to overcome. since the cell membrane is in principle impermeable to macromolecules, viruses 56 caroline a. lindemans and jan l. l. kimpen must first have an effective method to attach to the cell membrane. some viruses bind putative cell surface receptors that do not simply play a role in viral attachment, but also allow viral entry by inducing endocytosis. for some viral pathogens, such as rhinovirus, these receptors have been identified (icam-1 and ldl-r), while for others, such as rsv, the receptor that is used for cellular entry has not been unequivocally defined. the cx3cr1-chemokine receptor, also known as fractalkine-receptor, may be involved and tlrs have also been proposed to play a role. many enveloped viruses have glycosylated proteins, which not only bind to cellular receptors, but also have additional functions as membrane fusion factors, or receptor-destroying enzymatic activity. membrane fusion factors such as the rsv fprotein also allow cell-to-cell transmission of virus, which keeps it relatively hidden from the cellular immune system (smith and helenius, 2004) . escaping the "interferon signaling system" is one of the common mechanisms most viruses have acquired. as mentioned earlier in this chapter, both ifn-␣/␤ and ifn-␥ have potent antiviral properties. both types of interferon regulate transcription of a variety of target genes through activation of interferon inducible transcription factors. ifn-stimulated genes encode a variety of cellular enzymes, including pkr and 2ј5 ј-oligoadenylate synthetase, both involved in inhibition of viral protein synthesis. furthermore, interferons induce cellular apoptosis and upregulate mhc1 expression, targeting cells for cd8ϩ t-cell-mediated cytotoxicity. additionally, ifn-␥ activates the adaptive cellular immune system. rsv infection leads to an increase in ifn-␣/␤, but does not induce ifn-␥ from mononuclear and nk-cells as efficiently. despite the fact that interferons are known for their antiviral properties, intranasal administration of either lfn-␣/␤ or ifn-␥ in the airway does not lead to reduction of symptoms of viral respiratory infections (ramaswamy et al., 2004) . this is suggestive of a viral mechanism to evade the host's interferon response. the paramyxoviridae family (figure 4 .3), responsible for a large part of children's respiratory infections, consists of two subfamilies based on the structural differences in the gene encoding for the polymerase complex (p-protein). the paramyxovirinae subfamily members are able to block interferon-mediated promoter activity. these paramyxovirinae, to which parainfluenza-, measles-, and mumps virus belong to, have a p-gene that encodes for additional proteins besides the p-protein, the v-proteins. it is these v-proteins that have been found to be responsible for evading the interferon signaling pathways in this group of viruses. ifnmediated transcription is predominantly mediated by signal transducers and activators of transcription (stat). v-proteins have the ability to block interferon-mediated signaling by targeting stats for proteosomal degradation (horvath, 2004) . in contrast, rsv, belonging to the pneumovirinae subfamily, fails to inhibit ifn-induced promoter activity. the pneumovirinae consist of only one genus, the pneumovirus, which also includes hmpv. in this subfamily, p-genes only encode for the p-protein and therefore rsv cannot block interferon-mediated signaling (young et al., 2000) . however, recently it was demonstrated that, although rsv does not inhibit interferon-induced promoter activity, rsv replication is still resistant to ifn treatment of infected cells. apparently, an alternative mechanism to circumvent the interferon antiviral response exists. this has been attributed to additional proteins, characteristic of these pneumoviruses (spann et al., 2004) . these are nonstructural proteins (ns1 and ns2) that have no homologs in the paramyxovirinae. however, the underlying molecular pathway has not yet been elucidated. rsv infection of epithelial has been shown to lead to an upregulation of trail-receptor expression on these cells (kotelkin et al., 2003) . apoptosis of infected cells is an effective way to eliminate intracellular pathogens without damage to the surrounding tissue (figure 4.4) . however, several respiratory viruses have developed mechanisms to inhibit apoptosis. it has been demonstrated that rsv is able to effectively inhibit apoptosis of epithelial cells in vitro, in accordance with the limited pathology induced by rsv in epithelial cells during the first few days of the infection (thomas et al., 2002) . eventually, necrosis is observed when mature viral particles are released from the cells, after 2-3 days. furthermore, experiments with both adult and cord blood monocytes have shown a prolonged longevity of cells, when cultured in the presence of rsv (krilov et al., 2000) . of all respiratory viruses, viral evasion techniques of adenoviruses have been studied most intensively. it appears that approximately a third of the adenovirus genome is devoted to counteracting innate and adaptive immune defenses (burgert et al., 2002) . adenoviruses encode the protein e1a that blocks interferon-induced gene transcription. through the va-rna protein that blocks activation of pkr, they also interfere with the antiviral enzymes that are synthesized under interferon control. additionally, adenoviruses have developed several mechanisms to inhibit both constitutive and death receptor induced apoptosis (figure 4.4) . the e1b/55k protein inhibits p53-mediated apoptosis, e3/19k interacts with pro-apoptotic proteins bax and bak, whereas several e3 proteins are involved in removing fas and trail (death) receptors from the cell surface by promoting their degradation in lysosomes (wold et al., 1999) . finally, adenoviruses interfere with recognition of infected cells by cytotoxic lymphocytes. the adenovirus e3/19k protein inhibits transport of mhc1 molecules to the cell surface, resulting in decreased viral antigen presentation to cd8ϩ cells. future studies may unravel further mechanisms of immune evasion that may also be important in viruses involved in lower respiratory tract disease. this knowledge extrinsic and intrinsic signaling pathways of apoptosis. some viruses such as adenovirus interfere with cellular apoptosis. cellular apoptosis can normally occur via activation of the extrinsic (death receptor) pathway or the intrinsic pathway. both lead to activation of effector caspases 3 and 7, which will inevitably result in apoptosis. via production of external factors, such as tnf-␣, fas-ligand, and trail, death-receptors are cross-linked, which leads to apoptosis. adenovirus e3 proteins can remove these death receptors from the surface, thereby inhibiting extrinsic apoptosis. the intrinsic pathway is regulated by pro-and anti-apoptotic proteins of the bcl2-family. the transcription factor p53 has anticarcinogenic activity, induces the intrinsic pathway and promotes the transcription of pro-apoptotic proteins such as bax and bak. these proteins induce mitochondrial leakage of cytochrome c, which activates caspase 9, finally leading to effector caspase activation and apoptosis. adenovirus proteins are involved in both inhibition of p53 and the functioning of bax and bak. an outbreak of respiratory syncytial virus infection in a bone marrow transplant unit: effect on engraftment and outcome of pneumonia without specific antiviral treatment recognition of pathogen-associated molecular patterns by tlr family essential role for the dsrna-dependent protein kinase pkr in innate immunity to viral infection human and murine cytotoxic t cells specific to respiratory syncytial virus recognize the viral nucleoprotein (n), but not the major glycoprotein (g), expressed by vaccinia virus recombinants viruses as precipitants of asthma symptoms. ii. physiology and mechanisms transcriptional induction of multiple cytokines by human respiratory syncytial virus requires activation of nf-kappa b and is inhibited by sodium salicylate and aspirin bacteraemia and antibiotic use in respiratory syncytial virus infections long-term consequences of respiratory syncytial virus (rsv) bronchiolitis peripheral blood cytokine responses and disease severity in respiratory syncytial virus bronchiolitis immunological mechanisms of severe respiratory syncytial virus bronchiolitis seasonality of long term wheezing following respiratory syncytial virus lower respiratory tract infection natural reinfection with respiratory syncytial virus does not boost virus-specific t-cell immunity temporal activation of nf-kappab regulates an interferon-independent innate antiviral response against cytoplasmic rna viruses subversion of host defense mechanisms by adenoviruses adenovirus type 7 associated with severe and fatal acute lower respiratory infections in argentine children a comparison of nebulized budesonide, and intramuscular, and oral dexamethasone for treatment of croup a common haplotype of interleukin-4 gene il4 is associated with severe respiratory syncytial virus disease in korean children persistence of respiratory syncytial virus (rsv) infection and development of rsv-specific igg1 response in a guinea-pig model of acute bronchiolitis eosinophil activation and cysteinyl leukotriene production in infants with respiratory syncytial virus bronchiolitis viral infections in interferon-gamma receptor deficiency human metapneumovirus infection in japanese children analysis of cells obtained by bronchial lavage of infants with respiratory syncytial virus infection eosinophil degranulation in the respiratory tract during naturally acquired respiratory syncytial virus infection respiratory syncytial virus infection of human respiratory epithelial cells up-regulates class i mhc expression through the induction of ifn-beta and il-1 alpha epithelial regulation of innate immunity to respiratory syncytial virus rhinovirus respiratory infections and asthma relationship of viral infections to wheezing illnesses and asthma role of t lymphocyte subsets in the pathogenesis of primary infection and rechallenge with respiratory syncytial virus in mice immune-mediated disease pathogenesis in respiratory syncytial virus infection human metapneumovirus in severe respiratory syncytial virus bronchiolitis winter viruses: influenza-and respiratory syncytial virus-related morbidity in chronic lung disease rhinovirus and acute respiratory infections in hospitalized children. retrospective study respiratory syncytial virus--viral biology and the host response perflubron reduces lung inflammation in respiratory syncytial virus infection by inhibiting chemokine expression and nuclear factor-kappa b activation respiratory syncytial virus-induced activation of nuclear factor-kappab in the lung involves alveolar macrophages and toll-like receptor 4-dependent pathways respiratory syncytial virus and parainfluenza virus enhanced adherence of streptococcus pneumoniae to human epithelial cells infected with respiratory syncytial virus tissue distribution of ace2 protein, the functional receptor for sars coronavirus. a first step in understanding sars pathogenesis binding and entry of respiratory syncytial virus into host cells and initiation of the innate immune response respiratory syncytical virus-induced chemokine expression in the lower airways: eosinophil recruitment and degranulation involvement of toll-like receptor 4 in innate immunity to respiratory syncytial virus effects of viral lower respiratory tract infection on lung function in infants with cystic fibrosis influence of promoter variants of interleukin-10, interleukin-9, and tumor necrosis factor-alpha genes on respiratory syncytial virus bronchiolitis association of severe respiratory syncytial virus bronchiolitis with interleukin-4 and interleukin-4 receptor alpha polymorphisms interactions between rsv infection, asthma, and atopy: unraveling the complexities silencing stats: lessons from paramyxovirus interferon evasion adenovirus interaction with its cellular receptor car unusual haplotypic structure of il8, a susceptibility locus for a common respiratory virus variants of the chemokine receptor ccr5 are associated with severe bronchiolitis caused by respiratory syncytial virus intracellular ifn-gamma expression in natural killer cells precedes lung cd8ϩ t cell recruitment during respiratory syncytial virus infection metapneumovirus and acute wheezing in children community study of role of viral infections in exacerbations of asthma in 9-11 year old children respiratory syncytial virus infection in the late bone marrow transplant period: report of three cases and review role of nf-kappa b in cytokine production induced from human airway epithelial cells by rhinovirus infection activation of human eosinophils in vitro by respiratory syncytial virus community respiratory viruses in individuals with human immunodeficiency virus infection risk factors for respiratory syncytial virus associated apnoea relationship between clinical severity of respiratory syncytial virus infection and subtype respiratory syncytial virus infection sensitizes cells to apoptosis mediated by tumor necrosis factor-related apoptosisinducing ligand alterations in apoptosis of cord and adult peripheral blood mononuclear cells induced by in vitro infection with respiratory syncytial virus differential production of inflammatory cytokines in primary infection with human metapneumovirus and with other common respiratory viruses of infancy surfactant protein d gene polymorphism associated with severe respiratory syncytial virus infection adenovirus surveillance on children hospitalized for acute lower respiratory infections in chile (1988-1996) type 1 and type 2 cytokine imbalance in acute respiratory syncytial virus bronchiolitis viruses and asthma: inception, exacerbation, and possible prevention severe acute respiratory syndrome among children severe acute respiratory syndrome (sars) in children: epidemiology, presentation and management angiotensin-converting enzyme 2 is a functional receptor for the sars coronavirus recognition of single-stranded rna viruses by toll-like receptor 7 increased burden of respiratory viral associated severe lower respiratory tract infections in children infected with human immunodeficiency virus type-1 cross sectional retrospective study of prevalence of atopy among italian military students with antibodies against hepatitis a virus human metapneumovirus-an important new respiratory virus interleukin 9 production in the lungs of infants with severe respiratory syncytial virus bronchiolitis bronchoalveolar lavage cellularity in infants with severe respiratory syncytial virus bronchiolitis clinical characteristics of respiratory syncytial virus infections in healthy versus previously compromised host the immunology of virus infection in asthma immunoglobulin class-specific antibody response in respiratory syncytial virus infection measured by enzyme immunoassay respiratory syncytial virus up-regulates tlr4 and sensitizes airway epithelial cells to endotoxin respiratory syncytial virusinfected pulmonary epithelial cells induce eosinophil degranulation by a cd18-mediated mechanism immunity and immunopathology to respiratory syncytial virus. the mouse model expression of influenza virus hemagglutinin activates transcription factor nf-kappa b do rhinoviruses cause pneumonia in children? economic impact of respiratory syncytial virus-related illness in the us: an analysis of national databases viral infections, atopy, and asthma: is there a causal relationship? immune interaction between respiratory syncytial virus infection and allergen sensitization critically depends on timing of challenges pathophysiological mechanisms for the respiratory syncytial virus-reactive airway disease link respiratory syncytial virus and subsequent asthma: one step closer to unravelling the gordian knot? eosinophil cationic protein in infants with respiratory syncytial virus bronchiolitis: predictive value for subsequent development of persistent wheezing are we ready for universal influenza vaccination in paediatrics? concurrent serious bacterial infections in 2396 infants and children hospitalized with respiratory syncytial virus lower respiratory tract infections specific inhibition of type i interferon signal transduction by respiratory syncytial virus viral evolution toward change in receptor usage: adaptation of a major group human rhinovirus to grow in icam-1-negative cells eosinophils, eosinophil ribonucleases, and their role in host defense against respiratory virus pathogens respiratory syncytial virus infection results in airway hyperresponsiveness and enhanced airway sensitization to allergen latency and persistence of respiratory syncytial virus despite t cell immunity ifngamma mediates a novel antiviral activity through dynamic modulation of trail and trail receptor expression viral infections in obstructive airway diseases respiratory syncytial virus bronchiolitis in infancy is an important risk factor for asthma and allergy at age 7 how viruses enter animal cells respiratory syncytial virus bronchiolitis: disease severity, interleukin-8, and virus genotype respiratory virus infections in the immunocompromised host suppression of the induction of alpha, beta, and gamma interferons by the ns1 and ns2 proteins of human respiratory syncytial virus in human epithelial cells and macrophages respiratory syncytial virus in early life and risk of wheeze and allergy by age 13 years respiratory syncytial virus epidemics: the ups and downs of a seasonal virus respiratory syncytial virus enhances the expression of cd11b molecules and the generation of superoxide anion by human eosinophils primed with platelet-activating factor association between common toll-like receptor 4 mutations and severe respiratory syncytial virus disease time course of severe respiratory syncytial virus infection in mechanically ventilated infants palivizumab, a humanized respiratory syncytial virus monoclonal antibody, reduces hospitalization from respiratory syncytial virus infection in high-risk infants respiratory syncytial virus inhibits apoptosis and induces nf-kappa b activity through a phosphatidylinositol 3-kinase-dependent pathway identification of nf-kappab-dependent gene networks in respiratory syncytial virus-infected cells cx3c chemokine mimicry by respiratory syncytial virus g glycoprotein respiratory syncytial virus infection in patients with phagocyte defects a newly discovered human pneumovirus isolated from young children with respiratory tract disease an update on the pathophysiology of rhinovirus upper respiratory tract infections viral lower respiratory tract infection in infants and young children replication and clearance of respiratory syncytial virus: apoptosis is an important pathway of virus clearance after experimental infection with bovine respiratory syncytial virus viral induction of a chronic asthma phenotype and genetic segregation from the acute response the interaction of neutrophils with respiratory epithelial cells in viral infection respiratory syncytial virus-specific antibody responses in immunoglobulin a and e isotypes to the f and g proteins and to intact virus after natural infection community respiratory viruses: organ transplant recipients respiratory outcomes in high-risk children 7 to 10 years after prophylaxis with respiratory syncytial virus immune globulin human metapneumovirus and lower respiratory tract disease in otherwise healthy infants and children immune responses to adenoviruses: viral evasion mechanisms and their implications for the clinic paramyxoviridae use distinct virusspecific mechanisms to circumvent the interferon response evidence of airborne transmission of the severe acute respiratory syndrome virus contribution of influenza and respiratory syncytial virus to community cases of influenza-like illness: an observational study impaired innate host defense causes susceptibility to respiratory virus infections in cystic fibrosis is likely to be crucial to the improvement of immunotherapies for prevention and treatment of viral respiratory tract infections. key: cord-018408-ttae193b authors: haddad, imad y.; cornfield, david n. title: pneumonia and empyema date: 2008-11-15 journal: the respiratory tract in pediatric critical illness and injury doi: 10.1007/978-1-84800-925-7_17 sha: doc_id: 18408 cord_uid: ttae193b nan pneumonia is defi ned as infection and infl ammation of the lower respiratory tract in association with parenchymal radiographic opacity. this defi nition excludes bronchiolitis, tracheitis, neonatal pneumonia, and noninfectious causes of pneumonia and pneumonitis, and these are not discussed in this chapter. in the pediatric intensive care unit (picu), several pneumonia types may be encountered. first, a previously healthy child may be admitted to the picu because of severe community-acquired pneumonia (cap). the pneumonia is usually caused by organisms that are prevalent in the out-of-hospital environment. second, patients with genetic or acquired immune defi ciency commonly develop severe pneumonia with opportunistic infections that usually do not infect healthy children. these immunocompromised patients commonly have been given chemo-radiotherapy for cancer or are receiving immune-suppressive agents to prevent rejection episodes following solid organ and hematopoietic stem cell transplantation. third, both previously healthy and immunocompromised patients may acquire nosocomial pneumonia during their hospital stay. mechanically ventilated patients are at especially high risk to develop nosocomial ventilator-associated pneumonia (vap). finally, aspiration pneumonia caused by chronic inoculation of the lower respiratory tract with large amounts of less virulent bacteria in a susceptible host prone to aspiration is also observed in the picu. this classifi cation of pneumonia types in the picu is important because it has major implications on the causative microbial agent and, thus, the choice of initial empiric treatment that may be life saving. this chapter reviews respiratory host defenses that maintain sterility of the lower respiratory tract. in addition, the pathogenesis, classifi cation, and treatment options for pneumonia and empyema in the picu patient are briefl y discussed. potent expiratory maneuver is of fundamental importance in preventing material from being aspirated into the lungs. the conducting airways also contain several antimicrobial substances, including immunoglobulins (igg and secretory iga), and complement that bind and enhance the elimination of microbial agents. in addition, airway epithelial and alveolar type (at) ii cells secrete several antimicrobial peptides. one of the best characterized families of antimicrobial peptides are the defensins, which are cysteine-rich peptides possessing broad antimicrobial activity [4] . an important recent discovery is the expanding role of respiratory airway epithelium in innate immune defenses by mechanisms that mimic those noted in phagocytic cells. respiratory epithelial cells, including atii cells, express tlr and are capable of expressing a variety of cytokines that amplify infl ammation. the importance of innate immunity in epithelial cells was confi rmed in mice with specifi c inhibition of nuclear factor (nf) κb activation that was restricted to distal airway epithelial cells. mice lacking the ability to activate nfκb in epithelial cells exhibited impaired infl ammatory response to inhaled lps [5] . these data provide evidence that distal airway epithelial cells and the signals they transduce play a key physiologic role in lung infl ammation in vivo. alveolar type ii cells also secrete surfactant proteins (sp)-a and d. both sp-a and sp-d are collagen-like lectins (collectins) that agglutinate and/or opsonize pathogens and enhance their phagocytosis by innate immune cells such as alveolar macrophages and neutrophils [6] . surfactant proteins a and d may have additional immunoregulatory functions [7] and also may exhibit direct bactericidal effects by inducing damage to the bacterial cell membrane [8] . the functions of sp-a and sp-d in host defense are listed in table 17 .1. in the distal airspaces, alveolar macrophages are the fi rst phagocytic cell type encountered by pathogens entering the lung. macrophages have the capacity to induce the generation of large amounts of cytokines, chemokines, matrix metalloproteinases (mmp), nitric oxide, and potent oxidants that participate in antimicrobial defenses. in contrast, interstitial macrophages are located in the lung connective tissue and serve as both phagocytic cells and antigen-processing cells. tumor necrosis factor (tnf)-α, a macrophage-derived multifunctional cytokine, is expressed early in both patients with and animal models of pneumonia [9] . microbes also induce macrophages to generate potent chemokines that attract circulating neutrophils and monocytes into the lungs. cytokines/ chemokines amplify infl ammatory responses and orchestrate the polarization and transition of innate to adaptive immunity that function to eliminate invading microorganisms [10] . figure 17 .2 summarizes the cellular and secretory peptides that are components of host defense against microbes in the lower respiratory tract. disorders associated with impaired mechanical, innate, and adaptive host responses that may lead to the development of pneumonia in a susceptible host are listed in table 17 .2. the upper respiratory tract is normally colonized with nonpathogenic bacterial fl ora, but physical and immunologic host defenses generally ensure that bacteria that gain access to the lower respiratory tract are cleared. pneumonia occurs because of an impairment of host defenses (as discussed earlier), invasion by a virulent organism, or invasion by an overwhelming inoculum of less virulent organisms. there are fi ve main modes of pathogen entry into the lower respiratory tract. inhalation of infectious particles is probably the most important pathogenic mechanism in the development of cap, with particular importance in pneumonia of those caused by legionella species and mycobacterium tuberculosis. contact with contaminated fomites also may be important in the acquisition of viral agents, especially respiratory syncytial virus. the viral agents that cause pneumonia proliferate and spread by contiguity to involve lower and more distal portions of the respiratory tract. inhalation is also a common cause of pneumonia caused by contaminated ventilator tubes. endosome tlr5 tlr7/ trl8 tlr9 tlr10 tlr11 tlr12 tlr13 cd14 figure 17.1. toll-like receptors (tlr) and their ligands. lps, lipopolysaccharide; hsps, heat shock proteins. in addition to inhalation, pneumonia arises following the aspiration of microorganisms from the oral cavity or nasopharynx. invasive disease most commonly occurs upon acquisition of a new serotype of the organism with which the patient has not had previous experience. most episodes of vap are thought to develop from the aspiration of oropharyngeal secretions containing potentially pathogenic organisms. aspiration of gastric secretions may also contribute, although likely to a lesser degree. tracheal intubation interrupts the body's anatomic and physiologic defenses against aspiration, making mechanical ventilation a major risk factor for vap. the term aspiration pneumonia should be reserved for pneumonia or pneumonitis resulting from the aspiration of large amounts of gastric or oropharyngeal contents that may contain a large inoculum of relatively nonvirulent bacteria. the pathogens that commonly produce cap or vap, such as streptococcus pneumoniae, gram-negative bacilli, and staphylococcus aureus, are relatively virulent bacteria so that only a small inoculum is required and the aspiration is usually subtle. in immunocompromised individuals, an additional mode of pneumonia acquisition is bacteremia and sepsis. hematogenous deposition of bacteria is responsible for some cases of pneumonia caused by staph. aureus, pseudomonas aeruginosa, and escherichia coli. reactivation of pathogens can take place in the setting of defi cits of cell-mediated immunity. pathogens such as pneumocystis carinii/jiroveci, m. tuberculosis, and cytomegalovirus (cmv) may remain latent for many years after exposure, with fl ares of active disease in the face of immune compromise. reactivation tuberculosis occasionally occurs in immunocompetent hosts. direct inoculation rarely occurs as a result of surgery or bronchoscopy but may play a role in the development of pneumonia in patients supported with mechanical ventilation. the direct extension of infection to the lung from contiguous areas such as the pleural or subdiaphragmatic spaces is rare. community-acquired pneumonia community-acquired pneumonia refers to pneumonia in a previously healthy person who acquired the infection outside a hospital. it is one of the most common serious infections in children, with an incidence of 34 to 40 cases per 1,000 children in the industrialized world [11] . a subset of these patients will require picu admission. admission to the intensive care unit should be considered for patients with persistent hypoxemia despite oxygen therapy, recurrent apnea, signs of respiratory fatigue with or without mental status changes, or evidence of compensated or decompensated shock. infants less than 6 months of age and children with comorbid conditions such as bronchopulmonary dysplasia, cystic fi brosis, neuromuscular disorders, congenital heart disease, and immunodefi ciency disorders have limited respiratory reserves and, therefore, are at increased risk for respiratory failure during a pneumonia episode. for the adult population, the american and british thoracic societies have developed guidelines for hospital and icu admissions for patients with severe cap [12] . according to the american thoracic society guidelines, admission to the icu is needed for patients with severe cap, defi ned as the presence of either one of two major criteria, or the presence of two of three minor criteria. the major criteria include need for mechanical ventilation and septic shock; the minor criteria include systolic blood pressure ≤90 mm hg, multilobar disease, and a pao 2 /fio 2 ratio <250. in addition, a pneumonia severity index (psi) score identifi es adults at increased risk of medical complications and death [13] . however, similar guidelines or scores to grade the severity of pneumonia in children have not been developed. children admitted to the picu because of cap are more commonly infected with bacterial than viral pathogens. streptococcus pneumoniae is the most commonly identifi ed bacterial cause of cap in infants and children older than 1 month. pneumonias caused by group a streptococcus and staph. aureus are less frequent. haemophilus infl uenzae pneumonia has become uncommon following the widespread use of haemophilus infl uenza type b immunization. viruses are identifi ed most often in children <5 years of age. respiratory syncytial virus is the most common viral etiology during infancy, with adenovirus, infl uenza virus, parainfl uenza virus, and the recently described human metapneumonovirus (14) also not infrequently detected. mycoplasma pneumoniae and chlamydia pneumoniae are more common in older children and adolescents [11] . in may 1993 an outbreak of an acute febrile illness associated with respiratory failure, shock, and high mortality was identifi ed by investigators from the centers for disease control and prevention (cdc) as being caused by a hantavirus. in the united states, 95% of the cases occurred west of the mississippi after environmental exposure to infected deer mouse saliva, urine, or feces. in addition, a novel coronavirus was identifi ed as the causative agent of severe acute respiratory syndrome (sars), a new respiratory illness that affects adults and children, although the severity of the disease is less in children than in adults [15] . another cause of severe pneumonia that should be considered is tuberculosis. a history of contact with a person with pulmonary tuberculosis is usually elicited. finally, uncommon causes of cap in otherwise healthy children are fungal infections including coccidiodes immitis, histoplasma capsulatum, and blastomyces dermatitidis. these organisms should be included in the differential diagnosis as a cause of pneumonia only if there is a history of residence or travel to an area of endemic infection. occasionally, infection with strep. pneumoniae [16] and mycoplasma pneumoniae [17] can cause necrotic pneumonia secondary to an invasive organism or exaggerated host immune response. compared to patients with pneumonia and parapneumonic effusions, children who developed necrotizing pneumonia exhibited a more protracted hospital course associated with higher rates of complications, including bronchopleural fi stulas and need for thoracotomy for fi stula repair or lobectomy. none of the necrotizing pneumonia patients were immune defi cient [18] . the diagnosis of cap is usually made based on the presence of respiratory symptoms (cough, retractions) in a febrile and tachypneic child. the presence of infi ltrates on chest radiographs confi rms the diagnosis of pneumonia. infi ltrates are generally either interstitial or alveolar. although alveolar infi ltrates are more commonly observed during bacterial pneumonia [19] , in most studies, the pattern of infi ltrates has not been shown to correctly differentiate viral from bacterial pneumonia [20] . chest radiographs will also detect the presence of pleural effusions, pneumatoceles which are observed during staphylococcal pneumonia, or presence of air-fl uid levels indicative of abscess formation. after initial stabilization, diagnostic testing should be performed rapidly, avoiding delays in the administration of initial empiric therapy. in addition to a chest radiograph, an admitted patient should have a complete blood count and differential and routine blood chemistry testing (including glucose, serum sodium, liver and renal function tests, and electrolytes). all admitted patients should have oxygen saturation assessed by pulse oximetry and supplemental oxygen administered as needed. arterial blood gas should be measured in any patient with severe illness to assess both the level oxygenation and the degree of carbon dioxide retention. for critically ill patients with pneumonia, an aggressive approach to determine the causative microbial agent is warranted. microbiologic confi rmation is ultimately obtained for approximately 30%-50% of children with cap [21]. if a pleural effusion is present, aspiration of pleural fl uid for gram stain and culture prior to starting antibiotics is valuable. blood culture may reveal organisms in up to 30% of patients with bacterial pneumonia [22] . sputum collection is usually not practical for infants and children, and bacterial organisms recovered from the nasopharynx do not accurately predict the etiology of pneumonia. however, recovery of viruses and other atypical pathogens from the nasopharynx is more predictive. bacterial organisms recovered from tracheal secretions obtained through an endotracheal tube may or may not refl ect the causative agent(s) responsible for lower respiratory tract infection. specimens are considered appropriate for examination if they contain ≤10 epithelial cells and ≥25 polymorphonuclear leukocytes under low power [23] . the primary purpose of tracheal aspirate samples is to visualize a bacterial morphology of an organism that was not anticipated so that appropriate drugs can be added to the initial antibiotic regimen (e.g., staph. aureus or an enteric gram-negative antibiotic). bronchoalveolar lavage (bal) has been shown to be a rapid, relatively safe, and relatively noninvasive diagnostic procedure to obtain lower respiratory tract samples for microbial identifi cation and analysis. other techniques that can be used to identify pathogens include antigen detection of bacteria and viruses using immunofl uorescence, polymerase chain reaction, and serology such as cold agglutination test for m. pneumonia. the specifi city of the cold agglutination test for m. pneumonia is almost absolute, although the sensitivity is only about 50%. detection of mycoplasma igm by enzyme-linked immunoabsorbant assay (elisa) is a sensitive technique and should be considered for children [24] . immunocompromised patients are those whose immune mechanisms are defi cient because of congenital immune defi ciency syndromes, acquired immunologic disorders, or exposure to cytotoxic chemotherapy and steroids. in addition, recipients of solid organ and hematopoietic stem cell transplantation (hsct) are frequently given life-long treatment with immunosuppressive agents designed to prevent graft rejection or graft-versus-host disease. patients who develop severe neutropenia (i.e., an absolute neutrophil count ≤500 cells/ml) or lymphopenia for prolonged periods of time are at greatest risk to develop a variety of infectious complications, including life-threatening pneumonia. the lung is the predominant site of opportunistic infection in the immunocompromised patient [25] . immunosuppressed patients are predisposed to develop infections by ubiquitous microorganisms that do not normally cause disease in healthy people. they are also more susceptible to the usual causes of pneumonia, which can affect anyone. the sequence in which different organisms appear in the immunosuppressed and post-transplant recipients is fairly characteristic. nosocomial bacterial infections remain the most common cause of pneumonia during the early posttransplant, neutropenic phase. staphylococcus aureus and gramnegative pathogens predominate. in addition, fungal infections with candida and aspergillus species are not uncommonly seen during a severe neutropenic phase. the second period, from 1 to 6 months after solid organ transplant, is the time when opportunistic infections more commonly associated with transplantation, including nocardia, p. carinii/jiroveci, and cmv are observed [26] . during the third period, after 6 months, patients are categorized into different risk groups depending on the level of function of their allograft and the degree of immunosuppression they have received. those who are on minimal immunosuppression therapy are subject mainly to the same pathogens as the rest of the community. those with allograft dysfunction and ongoing heavy immunosuppressive therapy remain subject to all of the opportunistic infections seen during the second period. lung transplant recipients who develop bronchiolitis obliterans and hsct recipients who develop graft-versus-host disease remain especially at risk for infections [26] . pulmonary infi ltrates in the immunocompromised host may be caused by a variety of organisms, and may have noninfectious causes. because progression to respiratory failure may be rapid, an aggressive approach to diagnosis and treatment is necessary to limit morbidity and mortality. initial broad-spectrum therapy is important, with alterations of the empiric regimen once the clinical situation has stabilized and more diagnostic information has been obtained. in the immunocompromised host, bal procedure should be performed promptly to rule out infectious etiologies. lists suggested bal fl uids analysis studies and cultures. bronchoalveolar lavage is very helpful in the diagnosis of p. carinii/jiroveci, cmv, tuberculosis, and some fungal infections. however, the ability of bal fl uids analysis and culture to detect invasive aspergillosis, one of the most lethal infectious complication after transplantation, is limited [27] . the diagnostic yield for aspergillus species infection has been enhanced by the recently developed elisa that detects galactomannan, a fungal cell wall component released during invasive disease [28] . histopathologic analysis and culture of open lung biopsy specimens may provide accurate determination for the cause of pulmonary infi ltrates in pediatric patients [29] . however, open lung biopsy is associated with a signifi cant surgical risk in critically ill patients. open lung biopsy is most effective and least risky when performed early in the course of patients who develop nodular infi ltrates that require rapid differentiation between fungal infections and more benign lesions [30] . chemoprophylaxis against opportunistic infections is an important component of management of the post-transplant immunosuppressed patients. before the widespread introduction of chemoprophylaxis, p. carinii pneumonia (pcp) was observed to be a common opportunistic infection among transplant recipients. with the administration of low-dose trimethoprim-sulfamethoxazole or an alternative prophylactic agent such as pentamidine, pcp can be effectively prevented [31] . prophylaxis is also recommended for cmv in high-risk cmv seronegative recipients. such prophylaxis includes intravenous ganciclovir for 14 days, followed by oral ganciclovir capsules for three months [32] . aspiration pneumonia refers to the pulmonary consequences resulting from the abnormal entry of fl uid, formula, or endogenous secretions into the lower airways. there is usually compromise in host defenses that protect the lower airways, including glottic closure, cough refl ex, and other clearing mechanisms. histories of seizure, anesthesia, or other episode of reduced level of consciousness, neurologic disease, dysphagia, or gastroesophageal refl ux are all risk factors for aspiration. the risk of aspiration is especially high after removal of an endotracheal tube because of the residual effects of sedative drugs, the presence of a nasogastric tube, and swallowing dysfunction related to alterations of upper-airway sensitivity, glottic injury, and laryngeal muscular dysfunction [33] . aspiration pneumonia may be classifi ed into three clinical syndromes: chemical pneumonitis, bacterial infection, and airway obstruction. in animal models, development of chemical pneumonitis requires a 1 to 4 ml/kg inoculum of fl uid with a ph of 2.5 to initiate an infl ammatory reaction that may lead to pulmonary fi brosis [34] . bacteria, present in the aspirated oropharyngeal and gastric secretions, may also lead to pneumonia. aspiration pneumonia may involve particulate matter or foreign body, which, in addition to causing airway obstruction or refl ux airway closure, may synergistically contribute to acid-induced lung injury [34] . true aspiration pneumonia, by convention, usually refers to an infection caused by less virulent bacteria, primarily anaerobes, which are common constituents of the normal fl ora in a susceptible host prone to aspiration. pneumonia is commonly caused by oropharyngeal fl ora, including anaerobic gram-negative bacilli (bacteroides fragilis, fusobacterium nucleatum, peptostreptococcus, and prevotella) and anaerobic gram-positive bacilli (clostridium, eubacterium, actinomyces, lactobacillus, and propionibacterium). aspiration usually occurs when the patient is supine during or immediately after feeding. in the supine position the right upper lobe is the most dependent part of the lung and is most frequently affected. commonly, impaired airway protective responses are observed. the presence of tracheoesophageal malformations should be investigated if recurrent aspiration is noted in an otherwise healthy infant. the clinical presentation and course of chemical pneumonitis after inhalation of gastric contents ranges from mild and selflimited to severe and life threatening, depending on the nature of the aspirate and the underlying condition of the host. in the absence of witnessed inhalation of vomit, diagnosis is diffi cult and requires a high index of suspicion in a patient who has risk factors for aspiration. in the absence of an obvious predisposition, the abrupt onset of a self-limited illness characterized by dyspnea, cyanosis, and low-grade fever associated with diffuse rales, hypoxemia, and alveolar infi ltrates in dependent lobes should suggest aspiration [35] . if bal is performed, assessment of lipid-laden macrophage index using oil-red-o stain is helpful in confi rming the diagnosis [36] . the presence of foul-smelling putrid discharge in sputum or pleural fl uid is regarded as diagnostic of anaerobic infection. patients often have prolonged fever and productive cough, frequently showing blood in the sputum, which indicates necrosis (tissue death) in the lung. if aspiration is persistent, fi brosis and bronchiectasis may result. a number of interventions (e.g., positioning, dietary changes, drugs, oral hygiene, tube feeding) have been proposed to prevent aspiration patients with an observed aspiration should have immediate tracheal suction or bronchoscopy to clear fl uids and particulate matter that may cause obstruction. the use of corticosteroids in the treatment of chemical pneumonitis is controversial [37] , and antibiotics should not be used early in the course unless a superimposed bacterial infection is suspected. the national nosocomial infection surveillance (nnis) program sponsored by the cdc defi nes vap as pneumonia in patients who have been on mechanical ventilation for >48 hr and have developed new and persistent radiographic evidence of focal infi ltrates. in addition, patients had to have two of the following: temperature >38′c, leukocytosis (white blood cell >12,000/mm 3 ), and purulent sputum (>25 white blood cells/high-powered fi eld on tracheal aspirate gram stain). after blood stream infections, vap is the second most common cause of nosocomial infections in picus. the mean vap rate in children ranges from 6 to 12/1,000 ventilator days, accounting for 20%-50% of hospital-acquired infections [38, 39] . infections acquired in the picu are associated with a signifi cantly increased risk of death [40] . nosocomial pneumonia and vap are typically categorized as either early onset (occurring in the fi rst 3-4 days of mechanical ventilation) or late onset. this distinction is important microbiologically. early-onset nosocomial pneumonia and vap are commonly caused by antibiotic-sensitive, community-acquired organisms (e.g., strep. pneumoniae, and staph. aureus). late-onset nosocomial pneumonia and vap are commonly caused by anti-biotic-resistant nosocomial organisms (e.g., p. aeruginosa, methicillin-resistant staph. aureus, acinetobacter species, and enterobacter species). during the winter respiratory viral season, all patients in a medical care environment are at risk for disease due to respiratory syncytial virus, parainfl uenza, and infl uenza viruses. legionnaire's disease is a multisystem illness with pneumonia caused by legionella species usually present in contaminated water. legionnaire's disease is less common in children than adults. compared with postmortem lung biopsies and culture results, the use of clinical criteria to diagnose vap (lung infi ltrates, leukocytosis, purulent secretions, fever) had a sensitivity of 69% and a specifi city of 75% [41] . clearly, a number of noninfectious causes of fever and pulmonary infi ltrates can also occur in these patients, making the above clinical criteria nonspecifi c for the diagnosis of vap. lung infi ltrates may be caused by pulmonary hemorrhage, chemical aspiration, or atelectasis. fever may be caused by a drug reaction, extrapulmonary infection, or blood transfusion. autopsy results in a series of patients with acute lung injury demonstrated that clinical criteria alone led to an incorrect diagnosis of vap in 29% of clinically suspected cases [42] . these limitations have encouraged the use of invasive approaches to sample and culture material from the lower respiratory tract for accurate diagnosis of vap. ventilator-associated pneumonia is most accurately diagnosed by quantitative culture and microscopic examination of lower respiratory tract secretions, which are best obtained by bronchoscopy and bal [43] . cultures of tracheal aspirates are not very useful in establishing the cause of vap [44] . although such cultures are highly sensitive, their specifi city is low even when they are cultured quantitatively [45] . combining clinical and bacteriologic evaluation is probably the best way to achieve the objectives of correctly diagnosing vap and appropriately using antimicrobial agents. the main aims of this diagnostic approach are to rapidly identify patients with true lung bacterial infection, to select appropriate initial antimicrobial therapy, to adjust therapy based on antibiotic sensitivities, and to withhold antibiotics from patients without vap. guidelines for the prevention of vap in children are lacking, but data extrapolated from adult studies support routine elevation of head of bed 30°, appropriate use of sedatives and muscle relaxants, and adequate oral and circuit hygiene [46] . empyema is the presence of purulent material containing polymorphonuclear leukocytes and fi brin in the pleural cavity. empyema is usually a complication of inadequately treated bacterial cap, although it may occur after trauma, thoracic surgery, or intrathoracic esophageal perforation. although parapneumonic pleural effusions are noted in up to 34&-40% of children with pneumonia, empyema is rare, present in 1%-2% of cases [47] . the formation of an empyema can be divided into three stages: exudative, fi brinopurulent, and organizing. during the exudative stage, pus accumu-lates. this is followed by fi brin deposition and loculation of pleural fl uid known as the fi brinopurulent stage. the organizing stage is characterized by fi broblast proliferation; at this time there is the potential for lung entrapment by scarring [48] . typically, the pleural fl uid in empyema is exudative, caused by protein leakage from the capillaries because of increased permeability and increased hydrostatic pressure during the infl ammatory process. although the distinction between transudates and exudates is sometimes diffi cult to make, several features favor an exudative process. if at least one of the following three criteria is present, the fl uid is virtually always an exudate: (1) pleural fl uid protein >2.9 g/dl or protein/serum protein ratio greater than 0.5; (2) pleural fl uid lactate dehydrogenase (ldh)/serum ldh ratio greater than 0.6; and/or (3) pleural fl uid ldh greater than two thirds the serum ldh [49, 50] . the most common organisms that cause empyema in children are strep. pneumoniae, staph. aureus, and group a streptococci. haemophilus infl uenzae is rarely encountered since the advent of the h. infl uenzae b vaccine. mycoplasma pneumoniae and viruses can rarely result in exudative pleural effusions. in a series of 72 pediatric patients with empyema, 24% were secondary to anaerobic infection [51] . these data highlight the importance of anaerobic bacteria in selected cases of empyema in children and adolescents. in addition, tuberculosis should always be considered in the differential diagnosis, and a purifi ed protein derivative test should be performed. the differential diagnosis of patients with pleural effusions is shown in table 17 .4. the presence of fever associated with clinical signs of bacterial pneumonia is a clue to an underlying pneumonia as the cause of the effusion. a lateral decubitus radiograph, ultrasonography, or computed tomography may differentiate whether the fl uid is loculated. a sample of the fl uid should be obtained by thoracentesis in order to determine if the effusion is a transudate versus exudate. pleural cultures are positive in approximately one half of pediatric patients with empyema. blood culture and urine latex agglutination may help to identify a bacterial pathogen. a pneumatocele or pneumothorax seen on chest fi lm suggests staph. aureus as the cause of the empyema. until a specifi c organism is identifi ed, empiric antibiotic therapy should be instituted. this might include a third-generation cephalosporin and antistaphylococcal β-lactamase-resistant penicillin. antibiotics can be adjusted once an organism is identifi ed. antibiotic therapy should be intravenous until the patient becomes afebrile and then should be continued orally for an additional 2-3 weeks. there is major debate as to the proper adjuvant treatment of children with empyema. prospective, randomized and controlled studies of children with empyema are lacking. with the exception of starting appropriate or empiric antibiotics, there is no consensus on when and in whom to place a chest tube, instill fi brinolytic agents, or take to the operating room [52] . in 1992, light suggested that chest tubes should be inserted if the pleural fl uid is gross pus, if the gram stain of the pleural fl uid is positive, if the pleural fl uid glucose level is below 40 mg/dl, or if the pleural fl uid ph level is less than 7.00 [53] . if drainage with a chest tube is unsatisfactory, either urokinase or tissue plasminogen activator (tpa) should be injected intrapleurally [54, 55] . if drainage is still unsatisfactory, a decortication should be considered [56] . a stage-related approach to the management of empyema is perhaps most effi cacious and cost-effective [57] . in the exudative stage, conservative treatment using tube drainage may suffi ce. fibrinolytic treatment may be useful during the fi brinopurulent stage. in contrast, aggressive treatment using surgical decortication may be necessary during the organizing stage. with the advent of video-assisted thoracoscopy (vats), these traditional approaches to management of empyema in children are being challenged. video-assisted techniques offer distinct advantages in the accurate staging of the disease process, effectiveness of management of organizing pleural disease, and post-operative patient comfort [58] . in a retrospective study, the performance of early vats (<48 hr after admission) in children with empyema was associated with signifi cantly decreased length of hospital stay compared with performance of late vats (>48 hr after admission) [59] . children treated for empyema generally recover and have no residual sequelae. radiographs at the time of discharge usually show pleural thickening that later resolves. follow-up pulmonary function tests and physical examination are also usually normal or consistent with mild restrictive disease [60] . most epidemiologic investigations have clearly demonstrated that the indiscriminate administration of antibiotic agents to patients in the picu has contributed to the emergence of multiresistant pathogens with potentially increased morbidity and mortality. the prevalence of penicillin-resistant strains of strep. pneumoniae, methicillin-resistant staph. aureus, vancomycin-resistant enterococcus, and gram-negative bacteria producing extended-spectrum β-lactamase is increasing. despite these concerns, it is clear that patient survival may improve if pneumonia is correctly and rapidly treated. in adults, inappropriate initial antibiotic therapy is strongly associated with fatality [61] . therefore, it may be concluded that empiric antibiotics for the treatment of severe pneumonia are indicated. the choice of antibiotics is based on several factors, including the age of the patient, the type of pneumonia, and the local resistant patterns of predominant bacterial pathogens. suggested choices for initial empiric antibiotic coverage for pneumonia in the picu are listed in table 17 .5. aspiration pneumonia occurring in the community can be treated with ampicillin-sulbactam. empiric treatment for pneumonia in immunocompromised hosts requires broad-spectrum gram-positive and gram-negative coverage. immunocompromised patients are especially susceptible to a variety of life-threatening opportunistic viral and fungal pneumonias that require prompt diagnosis and aggressive treatment. for example, trimethoprim-sulfamethoxazole or pentamidine should be given for p. carinii/jiroveci, amphotericin b or caspofungin for candida and aspergillus species, acyclovir for herpes, amantadine for infl uenza, ganciclovir or foscarnet for cmv, and ribavirin for severe respiratory syncytial virus. empiric regimens may need to be modifi ed once results of cultures and antibiotic susceptibility testing are available. the infl ammatory response to infection is necessary for host defense but can contribute to the systemic toxicity and lung injury that may result from pneumonia. in some settings, adjunctive treatment of lower respiratory infections with antiinfl ammatory agents can reduce morbidity. corticosteroids have a well-documented role in the management of p. carinii/jiroveci pneumonia. in a multicenter trial, infusion of hydrocortisone signifi cantly decreased length of hospital stay and prevented mortality in adult patients with cap [62] . corticosteroids also may be effective under some circumstances in the treatment of infl ammatory sequelae of respiratory tract infection, such as tuberculous pleurisy and bronchiolitis obliterans organizing pneumonia (boop). strategies targeting specifi c cytokines have not been effective to date but remain active areas of investigation. enhanced understanding of the interactions of pathogen components with tlrs may be helpful one day in controlling and containing infectious diseases. immunization has reduced the incidence of several serious childhood diseases. immunization against infl uenza and increasingly resistant pneumococci can play a critical role in the prevention of pneumonia, particularly in immunocompromised patients. pleiotropic function of toll-like receptors myeloid differentiation factor 88 is essential for pulmonary host defense against pseudomonas aeruginosa but not staphylococcus aureus toll-like receptors: linking innate and adaptive immunity epithelial antibiotics induced at sites of infl ammation respiratory epithelial cells regulate lung infl ammation in response to inhaled endotoxin host defense functions of pulmonary surfactant human surfactant protein a suppresses t cell-dependent infl ammation and attenuates the manifestations of idiopathic pneumonia syndrome in mice interactions of pulmonary collectins with bordetella bronchiseptica and bordetella pertussis lipopolysaccharide elucidate the structural basis of their antimicrobial activities role of tnf-alpha in pulmonary host defense in murine invasive aspergillosis host innate defenses in the lung: the role of cytokines community-acquired pneumonia in children guidelines for the initial management of adults with community-acquired pneumonia: diagnosis, assessment of severity, and initial antimicrobial therapy a prediction rule to identify lowrisk patients with community-acquired pneumonia human metapneumovirus and lower respiratory tract disease in otherwise healthy infants and children clinical presentations and outcome of severe acute respiratory syndrome in children clinical manifestations and molecular epidemiology of necrotizing pneumonia and empyema caused by streptococcus pneumoniae in children in taiwan necrotizing pneumonitis caused by mycoplasma pneumoniae in pediatric patients: report of fi ve cases and review of literature necrotizing pneumonia in children comparison of radiological fi ndings and microbial aetiology of childhood pneumonia etiology of childhood pneumonia: serologic results of a prospective, population-based study acute pneumonia microscopic and bacteriologic analysis of expectorated sputum sensitivity and specifi city of fi ve different mycoplasma detection assays infection in organ-transplant recipients pulmonary complications of solid organ and hematopoietic stem cell transplantation diagnostic aspects of invasive aspergillus infections in allogeneic bmt recipients evaluation of serum sandwich enzymelinked immunosorbent assay for circulating galactomannan during caspofungin therapy: results from the caspofungin invasive aspergillosis study open lung biopsy in pediatric bone marrow transplant patients pulmonary nodular lesions in bone marrow transplant recipients: impact of histologic diagnosis on patient management and prognosis should prophylaxis for pneumocystis carinii pneumonia in solid organ transplant recipients ever be discontinued? oral ganciclovir in pediatric transplant recipients: a pharmacokinetic study swallowing dysfunction in patients receiving prolonged mechanical ventilation pathogenesis of gastric particulate lung injury: a comparison and interaction with acidic pneumonitis aspiration pneumonia the lipid-laden alveolar macrophage as a marker of aspiration in parenchymal lung disease effects of corticosteroids in the treatment of patients with gastric aspiration guidelines for preventing health-care-associated pneumonia, 2003: recommendations of cdc and the healthcare infection control practices advisory committee nosocomial infections in pediatric intensive care units in the united states. national nosocomial infections surveillance system prevalence of nosocomial infections in pediatric intensive care unit patients at us children's hospitals fourth decennial international conference on nosocomial and healthcare-associated infections clinical diagnosis of ventilator associated pneumonia revisited: comparative validation using immediate post-mortem lung biopsies diagnosis of nosocomial bacterial pneumonia in acute, diffuse lung injury management of bacterial pneumonia in ventilated patients. protected bronchoalveolar lavage as a diagnostic tool diagnosis and differential diagnosis of ventilatorassociated pneumonia diagnostic effi ciency of endotracheal aspirates with quantitative bacterial cultures in intubated patients with suspected pneumonia. comparison with the protected specimen brush the prevention of ventilator-associated pneumonia pleural empyema pathophysiology of pleural space infections pleural effusions: the diagnostic separation of transudates and exudates diagnostic value of tests that discriminate between exudative and transudative pleural effusions microbiology of empyema in children and adolescents thoracic empyema in children: early surgical intervention hastens recovery pleural diseases medical management of parapneumonic pleural disease tissue plasminogen activator as an adjuvant therapy for pleural empyema in pediatric patients management of postpneumonic empyemas in children rational treatment of empyema in children favorable outcome of parapneumonic empyema in children managed by primary video-assisted thoracoscopic debridement the changing face of pleural empyemas in children: epidemiology and management empyema in children: clinical course and long-term follow-up clinical importance of delays in the initiation of appropriate antibiotic treatment for ventilator-associated pneumonia hydrocortisone infusion for severe community-acquired pneumonia: a preliminary randomized study key: cord-000285-7p3b6tyf authors: hartert, tina v.; carroll, kecia; gebretsadik, tebeb; woodward, kimberly; minton, patricia title: the tennessee children's respiratory initiative: objectives, design and recruitment results of a prospective cohort study investigating infant viral respiratory illness and the development of asthma and allergic diseases date: 2010-04-08 journal: respirology doi: 10.1111/j.1440-1843.2010.01743.x sha: doc_id: 285 cord_uid: 7p3b6tyf background and objective: the ‘attack rate’ of asthma following viral lower respiratory tract infections (lrti) is about 3–4 fold higher than that of the general population; however, the majority of children who develop viral lrti during infancy do not develop asthma, and asthma incidence has been observed to continuously decrease with age. thus, we do not understand how viral lrti either predispose or serve as a marker of children to develop asthma. the tennessee children's respiratory initiative has been established as a longitudinal prospective investigation of infants and their biological mothers. the primary goals are to investigate both the acute and the long‐term health consequences of varying severity and aetiology of clinically significant viral respiratory tract infections on early childhood outcomes. methods: over four respiratory viral seasons, 2004–2008, term, non‐low birth weight previously healthy infants and their biological mothers were enrolled during an infant's acute viral respiratory illness. longitudinal follow up to age 6 years is ongoing. results: this report describes the study objectives, design and recruitment results of the over 650 families enrolled in this longitudinal investigation. the tennessee children's respiratory initiative is additionally unique because it is designed in parallel with a large retrospective birth cohort of over 95 000 mother–infant dyads with similar objectives to investigate the role of respiratory viral infection severity and aetiology in the development of asthma. conclusions: future reports from this cohort will help to clarify the complex relationship between infant respiratory viral infection severity, aetiology, atopic predisposition and the subsequent development of early childhood asthma and atopic diseases. the tennessee children's respiratory initiative (tcri) has been established as a longitudinal prospective investigation of term, non-low birth weight otherwise healthy infants and their biological mothers. the primary goals of the study are: (i) to investigate both the acute and the long-term health consequences of varying severity and aetiology of clinically significant viral respiratory tract infections on the outcomes of allergic rhinitis (ar) and early childhood asthma; and (ii) to identify the potentially modifiable factors that define children who are at greatest risk of developing asthma following infant respiratory viral infection. this study is unique, in that it was designed in parallel with our tennessee asthma bronchiolitis study (tabs), which is a retrospective birth cohort study of over 95 000 infants and their biological mothers similarly designed to elucidate the factors predisposing to childhood asthma and allergic this report describes the study objectives, design and recruitment results of the tennessee children's respiratory initiative, designed to investigate the role of respiratory viral infection severity and etiology in asthma development. future reports will address the complex relationship between infant respiratory viral infection and asthma and atopic diseases. diseases, but lacking biospecimens. thus, we designed the prospective tcri to establish a base for the evaluation of both the risks and benefits of documented significant infant viral respiratory infection of varying severity and aetiology and other environmental exposures on childhood atopy outcomes and to establish a biospecimen repository for analyses including biomarker testing and genotyping. the prospective cohort has the longitudinal design properties that may overcome potential limitations intrinsic to retrospective studies, such as our tabs cohort. [1] [2] [3] [4] [5] it is our eventual goal that the findings from these investigations, in conjunction with other investigations that have helped to elucidate genetic and environmental factors associated with asthma development, will help in the identification of primary and secondary prevention strategies for asthma. this report describes the study objectives, design and recruitment results of this study cohort. the tcri is a prospective cohort of mother-infant dyads enrolled in a longitudinal investigation of the relationship of infant viral respiratory infection severity and aetiology and the interaction of other risk factors on the development of childhood asthma and allergic diseases. the study was approved by the vanderbilt institutional review board, and parents provided written informed consent for both their and their child's study participation. term, non-low birth weight, otherwise healthy infants were enrolled along with their biological mothers, at a single academic institution, vanderbilt children's hospital, at the time of an acute visit (hospitalization, emergency department or unscheduled outpatient visit) for presumed viral bronchiolitis or upper respiratory tract infection (uri) during respiratory viral seasons september through may 2004-2008. inclusion and exclusion criteria are outlined in table 1 . because of the grant funding start date, the first study season did not begin until november 2004. recruitment was solely hospital-and clinic-based, and was performed 7 days/week during the first 2 years of cohort accrual, and 5 days per week for the two subsequent years. screening and recruitment were done by experienced congenital or acquired chronic heart or lung disease, prior requirement for mechanical ventilation for cardiac or pulmonary disease, immunodeficiency, neurologic disease with possible aspiration, significant gastroesophageal reflux disease felt to contribute to pulmonary disease, tracheomalacia ever received one or more doses of rsv-ivig or palivizumab prior study inclusion fever and neutropenia children whose parents or guardians were not able to understand the consent process, or a language barrier † research nurses using computerized medical charts to screen infants with presumed respiratory viral illness. the components and time line of the subject visits are outlined in table 2 mothers underwent prick skin testing to eight common aeroallergens and a blood specimen was obtained by venepuncture for serum immunoglobulin e (ige) and dna. a structured abstraction form was used to obtain information from the medical record regarding the index enrolment visit: current infant weight, confirmation of birth weight, room air pulse oximetry, requirement for supplemental oxygen, medication administration, prior wheezing episodes and detailed information on the current illness and hospital course. following discharge from the hospital or outpatient setting, the final discharge diagnosis and results of culture data were obtained through chart review. there are three phases of cohort follow up. first, mothers and children undergo an in-person wellchild follow-up visit during the child's second year of life conducted in the vanderbilt clinical research center, or during a home visit. second, families are re-contacted every 12-18 months by phone and/or mailings for purposes of cohort retention, and to provide reminders about the remaining study components. third, mothers, or the current guardians, undergo a phone interview during the fourth and sixth years of life to identify children with asthma, transient wheezing, ar and eczema. the 2-year in-person well-child visit is conducted in the vanderbilt clinical research center, or during a home visit offered to those unable to return to the study institution. during the visit, the isaac questionnaire is administered, blood samples are obtained from children and mothers (if not previously collected) and a buccal swab is collected for dna if blood cannot be obtained. a structured telephone questionnaire is administered to the mother/parent when their child is 4 and again at 6 years. trained interviewers employed in the vanderbilt survey research shared resource use a web-based computer system to conduct structured telephone interviews, which capture detailed information on asthma and atopy diagnoses and symptoms, extensive environmental exposure history, physical activity, and comorbidities. asthma, ar and atopic dermatitis outcomes are determined using the isaac questionnaire. for children with report of asthma and/or asthma symptoms in the previous 12 months, the asthma therapy assessment questionnaire is administered, and information on asthma medications, and asthma-related health-care visits are sought. 12, 13 biospecimen collection and laboratory analyses table 3 outlines the details of cohort biospecimen collection, repository and testing, which includes infant nasopharyngeal, urine, blood and nasal epithelial cell sample collection, and maternal prick skin testing and blood samples. the discharge diagnosis and supporting clinical parameters of the infant acute respiratory illness visit were reviewed to confirm whether each child had lower respiratory tract infections (lrti) or uri (n = 628) or another diagnosis (n = 46). lrti and uri were defined using both the physician discharge diagnosis, as well as post-discharge chart review, and those cases that were not clearly identified as either lrti or uri were reviewed by a panel of paediatricians who determined whether the illness represented an lrti, uri, croup or other, which included those that could not be categorized with the available clinical information. acute respiratory illness severity was determined using the ordinal bronchiolitis score that incorporates admission information on respiratory rate, flaring or retractions, room air oxygen saturation and wheezing, into a score ranging from 0 to 12 (12 being most severe). 27, 28 highly sensitive and specific qrt-pcr assays for many common respiratory viruses, including hmpv, hcov, rsv, influenza a and b, parainfluenzaviruses 1-3 and rhinovirus. real-time rt-pcr is performed using the cepheid smart cycler ii. all specimens are first tested for gapdh to confirm integrity of rna and monitor for potential pcr inhibitors. negative and positive controls are included with each run, including rna runoff transcripts to generate a quantitation curve. [14] [15] [16] [17] [18] [19] [20] [21] if rapid antigen and/or culture for rsv or influenza were performed at the discretion of the admitting physicians during the index visit, these results are also captured and entered in the database. the cells are collected using mid turbinate peds nylon flocked swabs (microrheologics, brescia, italy), and placed into collection and digestion media, followed by processing and plating onto collagen iv coated tissue culture and grown at 37°c in a 5% co2 incubator. to develop techniques for isolation, short-term culture, and in vivo modelling of epithelial and stromal cells, a xenograph model has been developed. following short-term growth in culture, cells are transferred to denuded rat tracheas and implanted subcutaneously in nude mice. follow-up well-child visit mother and child dna is collected from both the mother and child during the blood collection, or using a buccal swab if blood can not be obtained. dna is extracted by the vanderbilt center for human genetics research core laboratory and stored following extraction for future studies. enrolment infant urine is collected from hospitalized infants during the acute infant illness at study enrolment, and from a convenience sample of outpatient subjects. urinary measurements including, leukotrienes c4/d4/e4 (ltc4/d4/e4), and urinary metabolite of the isoprostane, 15-f2t-isop (8-iso-pgf2a) will be measured by a gas chromatographic, and other biomarkers and the remainder of the urinary biospecimens will be maintained in the repository. 25, 26 ige, immunoglobulin e; lrti, lower respiratory tract infections; uri, upper respiratory tract infection. the family history of atopy was obtained using a family tree. maternal atopy will be categorized as evidence of atopy by skin testing or specific ige, and/or clinical symptoms of an atopic disease as assessed by the isaac questionnaire. atopic status of the child will be determined by laboratory evidence of specific ige during the second year of life, and by clinical evidence based on the above definitions. the diagnosis of asthma will be determined at age 6 years based on responses to the isaac questionnaire. [6] [7] [8] the following criteria will define asthma during the sixth year of life: (i) 12-month prevalence of symptoms of asthma (current wheeze) or the presence of exercise-induced wheeze or dry cough at night not due to a cold or chest infection; and (ii) physician diagnosis as determined by the isaac questionnaire using either parental reported physician diagnosis of asthma or chronic use/ prescription of asthma-specific medications. probable asthma will be defined as physician diagnosis only and analysed separately. transient early wheezing will be defined as wheezing episodes present in the first 4 years of life, but not meeting the definition for childhood asthma at age 4 and 6 years. 29 allergic rhinitis will be determined through the isaac core questions on ar. 7, 8 children will be considered to have definite ar if each of three conditions is present between age 5 and 6 years: (i) a history of nasal congestion, runny nose, itchy watery eyes, sinus pain or pressure or headaches, sneezing, blocked nose, loss of sense of smell; (ii) substantial variability in symptoms over time or seasonality; and (iii) diagnosed as having allergic rhinitis by a physician or on medications for ar. probable allergic rhinitis will be defined as meeting two of the three criteria, or only criteria 3. atopic dermatitis will be determined through the isaac core questions on atopic dermatitis, which are based on a list of major and minor criteria widely applied in clinical studies. 8, 30, 31 as eczema is probably more readily confirmed by objective tests than either asthma or rhinitis, patients will be considered to have definite atopic dermatitis if between age 5 and 6 years they report ever having an itchy rash that comes and goes for at least 6 months, and being diagnosed with eczema by a physician. 30, 31 probable atopic dermatitis will be defined as one of the two above criteria. in order to standardize and monitor the quality of data collection and processing, all study personnel received training and were certified for all the study procedures. information is recorded on paper case report forms, data are entered and then checked by a second reviewer. logical data checks are programmed and additionally performed by our systems analyst, investigators and again by our biostatisticians. for laboratory analyses, blind qualitycontrol samples are included in each biospecimen run. telephone interviewers complete classroom training, orientation to the study population, computer modules, role play interviewing and training on study-specific protocols, and are formally evaluated at the end of training. a verbatim-recording of the interviewer and participant responses, and 10% participant re-contact allows quality-control staff to verify responses. the outcome variables of interest are the incidence of asthma and allergic rhinitis. the primary exposure variables of interest are the severity and aetiology of the infant viral illness and maternal and familial atopic status and other environmental exposures. cumulative asthma incidence over time, taking into account loss to follow up, will be used for illustrating incidence data. incidence of asthma and allergic diseases among the enrolled infant population with viral respiratory illness will be calculated by dividing the number of incident asthma cases by the person time of follow up. a kaplan-meier plot of cumulative incidence over time, taking into account loss to follow up, will be used for illustrating incidence data. incidence rates will be calculated by dividing the patient population into quartiles/quintiles of bronchiolitis severity scores. the adjusted risk of asthma and allergic rhinitis with bronchiolitis severity will be evaluated using the cox proportional hazard regression model. to assess the relationship between biomarker concentrations and increased risk of infant bronchiolitis, and early childhood asthma outcomes, we will conduct a nested case-control study. geometric means of urinary biomarker concentrations for those who develop and do not develop asthma will be calculated separately and compared using the paired t-test. the association between these biomarkers and the risk of asthma and allergic rhinitis will be assessed using odds ratios and their corresponding 95% confidence intervals from adjusted logistic regression models. the potential confounders that will be considered in multivariable analyses will include demographic and exposure characteristics. subject recruitment for the tcri study occurred over 4 years, and was completed in may 2008. overall, 9329 visits were screened, representing 7632 unique infants, and 2986 of these infants met study eligibility requirements. from the 2986 eligible infants, 674 infants and their biological mothers were enrolled (fig. 1) . among the 2312 subjects who were available during the recruitment periods, the major reasons for non-response were refusal (22%), insufficient time/ unwilling to stay for the visit (outpatients) (39%), conflict with or already enrolled in another study (20%), and other (18%) (includes language barrier, mother/ guardian not present, previously enrolled and other miscellaneous). in 99.9% of the cohort, the nasal/ throat swab was obtained, and in 79% of the hospitalized infants one spot urine sample was obtained at hospital admission. weekly enrolment into the cohort is depicted in figure 2 . the tcri is a large and comprehensive prospective epidemiologic study of mothers and their biologic children enrolled during infancy with a clinically significant lrti or uri who are being followed through early childhood. this study will provide important information about the role of infant respiratory viral infection severity, aetiology, biomarkers and predictors important in the development of early childhood asthma and allergic rhinitis. the tcri is additionally unique because it is designed in parallel with a large retrospective birth cohort of over 95 000 mother-infant dyads with similar objectives to investigate the role of respiratory viral infection severity and aetiology in the development of asthma. as evidence suggests that the development of asthma may result in part from respiratory viral infection during infancy, which has a predilection for infecting, destroying and/or in some way biologically altering lower airway epithelium, this study will help to delineate whether the severity of that infection and other early-life events impact the risk of asthma and allergic disease development in later childhood. 3 despite a high attack rate of developing asthma following viral bronchiolitis, the majority of children who have infant bronchiolitis do not develop childhood asthma. thus while viral respiratory infections may alter lung physiology and target the inflammatory response to the lower airway, this may only occur during a vulnerable time period during development of the immune system or lung, or in the presence of other risk factors. this developmental component may further reflect important gene-environment interactions that regulate both short-and long-term airway physiological alterations that manifest themselves clinically as childhood asthma. efforts to determine and define the role of these factors, including disease severity, maternal atopy and other environmental exposures, such as second-hand smoke, to asthma pathogenesis are the focus and goal of the tcri. several limitations of this study should be noted. first, the study sample was not randomly selected from the general population, but instead was recruited from a single hospital and clinic-based setting, the vanderbilt children's hospital. while vanderbilt hospitalizations represent greater than 90% of davidson county/nashville infant hospitalizations, it represents a smaller proportion of emergency department visits (51%), and likely even fewer paediatric acute care visits. 2 the relatively low participation rate among eligible subjects is multifactorial and the result of the long-term nature of the study, the lack of study personnel to enrol all eligible subjects, as well as lower willingness among outpatients to extend their visit in order to participate in the study. while this impacts the demographics and exposures of the study population, and thus generalizibility of our study results, it should not impact the findings of the role of infant viral infection on the outcomes of interest. next, while airway hyperreactivity is not assessed in making the diagnosis of childhood asthma, the identification of incident asthma cases will take into consideration the positive response to a validated written questionnaire that has been compared with bronchial provocation testing. 6, 8 while such an ascertainment strategy might result in the underdiagnosis of asthma, it is unlikely to result in false positive diagnoses during the sixth year of life. finally, as with many studies, where all eligible participants were approached for participation, difficulty was encountered in follow up of those currently age-eligible for follow up. strategies to address this include study personnel doing a significant number of follow-up visits at the subject's home, and shipping follow-up materials and requests to paediatricians of subjects who have moved from the region. future reports from this cohort will help to clarify the complex relationship between infant respiratory viral infection severity, aetiology, atopic predisposition, and the development of early childhood asthma and other atopic diseases. ultimately, this study, along with the companion tabs cohort, has the potential to provide new approaches to identify infants at high risk of developing early childhood asthma and allergic diseases, as well as provide important information that may contribute to the development of prevention strategies. increasing burden and risk factors for bronchiolitis-related medical visits in infants enrolled in a state health care insurance plan the underrecognized burden of influenza in young children evidence of a causal role of winter virus infection during infancy on early childhood asthma the severity-dependent relationship of infant bronchiolitis on the risk and morbidity of early childhood asthma season of infant bronchiolitis and estimates of subsequent risk and burden of early childhood asthma international study of asthma and allergies in childhood (isaac) written questionnaire: validation of the asthma component among brazilian children validation of a rhinitis symptom questionnaire (isaac core questions) in a population of swiss school children visiting the school health services. scarpol-team. swiss study on childhood allergy and respiratory symptom with respect to air pollution and climate. international study of asthma and allergies in childhood international study of asthma and allergies in childhood (isaac): rationale and methods collection of dietary-supplement data and implications for analysis a data-based approach to diet questionnaire design and testing nutrition quest, assessment tools for health researchers asthma control test: reliability, validity, and responsiveness in patients not previously followed by asthma specialists development of the asthma control test: a survey for assessing asthma control human metapneumovirus plays an etiologic role in acute asthma hospitalization in adults high-throughput, sensitive, and accurate multiplex pcr-microsphere flow cytometry system for large-scale comprehensive detection of respiratory viruses human metapneumovirus infection in children hospitalized for wheezing a prospective study comparing human metapneumovirus with other respiratory viruses in adults with hematologic malignancies and respiratory tract infections association of human metapneumovirus with acute otitis media the role of human metapneumovirus in upper respiratory tract infections in children: a 20-year experience comparison of the mchip to viral culture, reverse transcription-pcr, and the quickvue influenza a+b test for rapid diagnosis of influenza a diverse group of previously unrecognized human rhinoviruses are common causes of respiratory illnesses in infants in vitro assays for the diagnosis of ige-mediated disorders clinical laboratory assessment of ige-dependent hypersensitivity elevations of local leukotriene c4 levels during viral upper respiratory tract infections quantification of the major urinary metabolite of 15-f2t-isoprostane (8-iso-pgf2alpha) by a stable isotope dilution mass spectrometric assay prednisolone plus albuterol versus albuterol alone in mild to moderate bronchiolitis dexamethasone and salbutamol in the treatment of acute wheezing in infants asthma and wheezing in the first six years of life. the group health medical associates diagnostic features of atopic dermatitis evaluation and relevance of atopic basic and minor features in patients with atopic dermatitis and in the general population we would like to thank the many families, paediatricians and their office staff, who have participated in this study, as without them, this work would not have been possible. key: cord-324786-8k81jetq authors: chang, anne b; grimwood, keith; robertson, colin f; wilson, andrew c; van asperen, peter p; o’grady, kerry-ann f; sloots, theo p; torzillo, paul j; bailey, emily j; mccallum, gabrielle b; masters, ian b; byrnes, catherine a; chatfield, mark d; buntain, helen m; mackay, ian m; morris, peter s title: antibiotics for bronchiectasis exacerbations in children: rationale and study protocol for a randomised placebo-controlled trial date: 2012-08-31 journal: trials doi: 10.1186/1745-6215-13-156 sha: doc_id: 324786 cord_uid: 8k81jetq background: despite bronchiectasis being increasingly recognised as an important cause of chronic respiratory morbidity in both indigenous and non-indigenous settings globally, high quality evidence to inform management is scarce. it is assumed that antibiotics are efficacious for all bronchiectasis exacerbations, but not all practitioners agree. inadequately treated exacerbations may risk lung function deterioration. our study tests the hypothesis that both oral azithromycin and amoxicillin-clavulanic acid are superior to placebo at improving resolution rates of respiratory exacerbations by day 14 in children with bronchiectasis unrelated to cystic fibrosis. methods: we are conducting a bronchiectasis exacerbation study (best), which is a multicentre, randomised, double-blind, double-dummy, placebo-controlled, parallel group trial, in five centres (brisbane, perth, darwin, melbourne, auckland). in the component of best presented here, 189 children fulfilling inclusion criteria are randomised (allocation-concealed) to receive amoxicillin-clavulanic acid (22.5 mg/kg twice daily) with placebo-azithromycin; azithromycin (5 mg/kg daily) with placebo-amoxicillin-clavulanic acid; or placebo-azithromycin with placebo-amoxicillin-clavulanic acid for 14 days. clinical data and a paediatric cough-specific quality of life score are obtained at baseline, at the start and resolution of exacerbations, and at day 14. in most children, blood and deep nasal swabs are also collected at the same time points. the primary outcome is the proportion of children whose exacerbations have resolved at day 14. the main secondary outcome is the paediatric cough-specific quality of life score. other outcomes are time to next exacerbation; requirement for hospitalisation; duration of exacerbation; and spirometry data. descriptive viral and bacteriological data from nasal samples and blood markers will also be reported. discussion: effective, evidence-based management of exacerbations in people with bronchiectasis is clinically important. yet, there are few randomised controlled trials (rcts) in the neglected area of non-cystic fibrosis bronchiectasis. indeed, no published rcts addressing the treatment of bronchiectasis exacerbations in children exist. our multicentre, double-blind rct is designed to determine if azithromycin and amoxicillin-clavulanic acid, compared with placebo, improve symptom resolution on day 14 in children with acute respiratory exacerbations. our planned assessment of the predictors of antibiotic response, the role of antibiotic-resistant respiratory pathogens, and whether early treatment with antibiotics affects duration and time to the next exacerbation, are also all novel. trial registration: australia and new zealand clinical trials register (anzctr) number actrn12612000011886. compared to the early 20th century, the prevalence of bronchiectasis has fallen substantially. although regarded as an 'orphan disease' in affluent countries, reports of prevalence of bronchiectasis are increasing [1, 2] . bronchiectasis remains a major contributor to chronic respiratory morbidity [2, 3] and mortality [1, 4] in both indigenous [5] and non-indigenous populations [6] . in our recently completed multicentre study of children newly referred for chronic cough and managed in accordance to a standardised protocol [7] , 31 (9%) of the 346 children had bronchiectasis proven on radiology [8] . in the northern territory in australia, the incidence of bronchiectasis in the first year of life is 118 in 100,000 [9] . the estimated prevalence of bronchiectasis is 1,470 per 100,000 in central australian indigenous children aged below 15 years [10] and 1,600 per 100,000 in alaskan native children [11] . in the united states, reported prevalence in adults range from 4.2 to 271.8 per 100,000 [6] . however, any reported prevalence is likely to be an underestimate as many cases are misdiagnosed or coexist with other diseases like asthma [12] [13] [14] and chronic obstructive pulmonary disease (copd) [15] . even without accounting for these unrecognised cases, globally there are far more patients with bronchiectasis than cystic fibrosis (cf), which has a prevalence of 7.4 to 7.9 per 100,000 in the european union and the united states [14] . effective clinical management reduces both short-and long-term morbidity (and likely mortality) associated with bronchiectasis [16] [17] [18] . there is increasing evidence that intensive treatment of children who either have bronchiectasis or are at risk of developing severe bronchiectasis prevents poor lung function in adulthood [17] [18] [19] [20] . cohort data have shown that approximately 80% of newly diagnosed adults (non-smokers) with bronchiectasis reported symptoms dating back to childhood and that the duration of chronic cough (the most common symptom of bronchiectasis [21] ) was related (r = −0.51, p < 0.001) to lung function at diagnosis [22] . arguably, appropriate overall management and treatment of exacerbations (leading to reduction of persistent symptoms) potentially prevents or reduces deterioration of chronic respiratory disease [23] . determinants of accelerated lung function decline in adults with bronchiectasis are the frequency of hospitalised exacerbations, increased systemic inflammatory markers and pseudomonas aeruginosa infection [24] . amongst other factors, increased mortality risk is associated with the degree of lung function impairment [25] . no prospective data exist in children. our study and a london-based retrospective study found that, with appropriate treatment in specialised centres, lung function improves and can be maintained [18, 20] . however, those with poor lung function at diagnosis, although substantially improved, were likely to still have poor lung function five years later [20] . we also found that the only significant predictor of a decline in forced expiratory volume in one second (fev 1 ) was frequency of hospitalised exacerbations [20] . forced expiratory volume in one second (fev 1 )% predicted decreased by 1.95% with each previous hospitalised exacerbation [20] . as airway injury in children is superimposed upon the physiological changes involving lung growth and development [26, 27] , improvement in childhood bronchiectasis may impact favourably upon future adult lung function. early and effective management of bronchiectasis exacerbations in children may lead to reduced hospitalisations, better quality of life (qol) and improved future adult lung function. antibiotics are one of the key interventions used to treat acute respiratory exacerbations of bronchiectasis [21, 28] . however, it is biologically plausible that antibiotics are not useful for treating some respiratory exacerbations triggered by viral infections. our retrospective study found that 34% of exacerbations were preceded by a viral-like illness [29] . while most respiratory physicians will treat exacerbations intensively (with antibiotics and airway clearance), other doctors do not. those choosing not to use antibiotics routinely argue that most episodes of exacerbations and cough are caused by viral infections and hence do not require antibiotic therapy. this may be appropriate but viral-bacterial interactions in the airways risk prolonged endobronchial bacterial infection that, with the associated inflammatory cascade, may cause further lung injury [23, 30] . better evidence to guide the management of exacerbations in people with bronchiectasis is needed. there are two components of best; here we present the study protocol of the first phase (best-1). the second phase of best (best-2) will address the question "is daily azithromycin non-inferior (within 20% margin) to amoxicillin-clavulanic acid in achieving resolution of exacerbations on day 21?" the protocol for best-2 will be the subject of a later paper. this first phase of our proposed national multicentre double-blind double-dummy randomised controlled trial (rct) is designed to answer our primary questions, as follows. amongst children with non-cf bronchiectasis, does azithromycin improve the resolution of respiratory exacerbations by day 14 compared with placebo, and does amoxicillin-clavulanic acid improve the resolution of respiratory exacerbations by day 14 compared with placebo? our secondary aims are to: 1. determine the effect of azithromycin or amoxicillinclavulanic acid on the qol, systemic inflammation, time to next respiratory exacerbation, and duration of exacerbations; 2. explore factors that predict response to antibiotics, including respiratory pathogens (viruses, bacteria, macrolide-resistant bacteria) present in respiratory secretions and blood markers; and 3. describe the point prevalence and diversity of respiratory viruses, mycoplasma pneumoniae and chlamydia species during exacerbations using sensitive molecular detection techniques. our study tests the primary hypothesis that both oral azithromycin and amoxicillin-clavulanic acid are superior to placebo in improving the resolution rate of respiratory exacerbations by day 14 in children with non-cf bronchiectasis. we are conducting a multicentre, parallel group, double-blind placebo rct (with concealed allocation) to assess the impact of treatment with antibiotics (azithromycin or amoxicillinclavulanic acid) in children with an exacerbation of bronchiectasis. our study plan is summarised in figure 1 . the inclusion criteria are age below 18 years at time of study enrolment; diagnosed with bronchiectasis by a respiratory physician following high resolution computed tomography in the five years immediately prior to study entry or, if diagnosed earlier, have been followed regularly by a respiratory physician for treatment of bronchiectasis; and has experienced two or more respiratory exacerbations in the 18 months prior to study entry. exclusion criteria are current severe exacerbation of bronchiectasis (dyspnoea, hypoxia or hospitalisation), recent (in last 8 weeks) in the entry; cf; liver dysfunction; allergy or sensitivity to penicillin or macrolides; current or recent lower airway infection by a member of the pseudomonas genus group of gram-negative bacteria (in the four months prior to study enrolment); has received antibiotics belonging to the macrolide or penicillin class of antibiotics within three weeks immediately prior to study entry; or is currently receiving oncological treatment. eligible children will be identified from clinics in our centres (brisbane, perth, darwin and melbourne in australia and auckland in new zealand). parents will be approached and informed consent obtained. baseline pre-exacerbation data will be collected ( figure 1 ), parents contacted monthly and children reviewed every three months. parents will be educated specifically on symptoms of exacerbations and asked to contact the research nurse at the onset of an exacerbation. a double-dummy design is planned. if eligibility is fulfilled and after informed consent has been obtained, the child is randomised to one of three arms. at the start of the exacerbation, the child will receive amoxicillinclavulanic acid with placebo-azithromycin, azithromycin with placebo-amoxicillin-clavulanic acid or placeboazithromycin with placebo-amoxicillin-clavulanic acid. amoxicillin-clavulanic acid dose is 22.5 mg/kg/dose (up to 40 kg) twice a day (max 900 mg/dose). azithromycin dose is 5 mg/kg/day, max of 200 mg daily. equivalent volumes in placebo will be given in all arms. all treatments will continue for 14 days. an exacerbation is defined as an increase in sputum volume or purulence, or three or more days of change in cough (> 20% increase in cough score [31] or type (dry to wet) [32] ). we validated this definition in our prospective study and found that the kappa values (between clinicians) of these symptoms and signs were excellent (> 0.75) [33] . daily diaries will also be collected during exacerbations until the scores for two or more days reflect the child's 'baseline' state, which for each child will be established at enrolment, prior to any exacerbations. this assessment consists of a combination of symptoms (daily cough (yes/no), cough quality (wet/dry/none) and cough score [31] averaged over two consecutive days) and signs (sputum colour (if any present) using a colour chart card (bronkotest ltd, london, uk), crackles on chest auscultation). children will be reviewed on days 7 and 14 and at resolution of the exacerbation. the exacerbation is considered 'resolved' when symptoms and signs are the same as 'baseline' state. post exacerbation, the children will be followed-up and clinically evaluated every three months for 18 months or until their next figure 1 overall schematic study design. amox-clav: amoxicillinclavulanic acid; azithro: azithromycin. exacerbation. 'time to next exacerbation' will be determined by the number of days from 'resolution of current exacerbation' to beginning of the next exacerbation. upon enrolment, the child is assigned to the next unique number on the appropriate stratified list. the allocation will be performed by the trial pharmacist at the royal children's hospital in brisbane. randomisation is stratified by site (brisbane, perth, darwin, melbourne, auckland), age (≤ 5 or > 5 years) and underlying aetiology (idiopathic/post-pneumonia or all other causes). the randomisation sequence was computer generated and used permuted blocks. the allocation sequence is concealed at all times throughout the study. the computer generated allocation sequence was prepared by a statistician external to the study team. the placebo medications, specifically manufactured by the institute of drug technology australia limited (melbourne, victoria), have a similar taste and colour to their respective antibiotics. both active medications (amoxicillin-clavulanic acid and azithromycin) are repackaged by the institute of drug technology. thus both the amoxicillin-clavulanic acid and azithromycin and their respective placebos are in identical opaque bottles. for both types of trial medications, equal volumes of water are added using a syringe and needle by punching the seal. adherence will be assessed by parent report and return of empty bottles. all data will be recorded on standardised forms. on enrolment, demographic information (age, gender, ethnicity, household size, and so on), birth history, breast feeding history, prior illness and in utero and household smoke exposure will be recorded, and a physical examination will be performed by a study physician. the primary and secondary outcome measures (see below) are collected at the time points specified above. serious and non-serious adverse effects (nausea, vomiting, diarrhoea, rash) will also be documented and monitored. safety exit points are discussed in end points below. at enrolment (baseline), all children will have a deep nasal swab (ns) specimen collected. in a subset, additional specimens will be collected at baseline and during exacerbations depending on feasibility (some children are unable to attend the study centre at the onset of the exacerbation) and willingness of parents to allow additional venipuncture. these specimens are: a deep ns specimen for respiratory viruses, respiratory bacterial pathogens (with antibiotic susceptibility testing) and other potentially important respiratory pathogens (m. pneumoniae, chlamydia spp) at baseline and at the beginning and resolution of an exacerbation. the technique used is identical to previous studies [34] [35] [36] where the specimens were described as nasopharyngeal swabs. the nss are handled as per our research laboratory protocol (see below). bloods at baseline and at the beginning and end of each exacerbation for c-reactive protein (crp), neutrophilic marker of inflammation (il-6) [37] , serum amyloid a (saa) [33, 38] and markers of viral infection (interferon gamma-inducible protein 10 (ip-10) and il-10). sputum at baseline and at the beginning and end of each exacerbation (when possible) for lower airway microbiology and antibiotic sensitivity. the verbal categorical descriptive score is a validated daily diary score of cough rated on a six-point scale (0 = no cough to 5 = severe cough and cannot perform activities) with increasing scores reflecting greater interference with usual activities. this rating was validated against an objective cough meter measure [31] and changes in cough scores have been shown to reflect changes in objective cough counts [39] . the parent chronic cough quality of life (pc-qol) is a 27-item questionnaire designed to assess the level of frequency of feelings (15 items) and worry (12 items) related to their child's cough. it uses a seven-point likert-type scale with higher scores reflecting less frequency and fewer worry concerns (that is, greater qol) [40, 41] . the minimal important difference is 0.62 determined by the distribution method and 0.9 determined by the anchor method [42] . oropharyngeal sampling under estimates streptococcus pneumoniae carriage by approximately 50% when compared with ns [43] . thus, ns are the preferred method when evaluating the presence of antibiotic-resistant bacteria. culturing, identifying and, when appropriate, serotyping common respiratory bacteria are established techniques at our research laboratory [36, 44] . swabs are stored in skim milk tryptone glucose glycerol broth medium at −80°c before being batch processed for typical respiratory bacterial pathogens, notably haemophilus influenzae (including strains of non-typeable h. influenzae), moraxella catarrhalis and s. pneumoniae. batches of swabs are thawed and 10-μl aliquots cultured overnight on selective media at 37°c in 5% carbon dioxide. growth of s. pneumoniae, h. influenzae and m. catarrhalis is recorded and confirmed by standard techniques [36, 45] . four isolates each of s. pneumoniae and h. influenzae and two isolates of m. catarrhalis per positive swab are tested for antimicrobial resistance and stored [36, 45] . s. pneumoniae isolates are serotyped using the quellung method (antisera from statens serum institute, copenhagen,denmark). in addition to routine susceptibility testing using the calibrated dichotomous susceptibility disc diffusion method, azithromycin minimum inhibitory concentration (mic) will be determined by etests (ab biodisk, solna, sweden) if the azithromycin disc annulus is <6 mm. for s. pneumoniae, the penicillin mic is determined for penicillin non-susceptible isolates (oxacillin and/or penicillin disc annulus < 6 mm) and for h. influenzae, the ampicillin mic is determined for isolates if the ampicillin disc annulus is < 6 mm. interpretive criteria (clinical and laboratory standards institute breakpoints) used for s. pneumoniae are penicillin non-susceptible mic > 0.12 μg/ml, azithromycin resistance mic ≥ 2 μg/ml; and for h. influenzae, ampicillin resistance mic ≥ 4 μg/ml, azithromycin resistance mic > 4 μg/ml. a nitrocephin-based test will identify beta-lactamase activity in h. influenzae and m. catarrhalis isolates. we will use our previous methods [46, 47] . nucleic acids will be extracted from the media using the high pure viral nucleic acid kit (roche diagnostics, sydney, new south wales, australia), according to the manufacturer's instructions. real-time polymerase chain reaction assays will be used to detect respiratory syncytial viruses (a and b), adenoviruses, influenza viruses (a and b), parainfluenza, human metapneumovirus, human coronaviruses (oc43, hk1, 229e, nl63), enteroviruses, rhinoviruses (and subtypes [48] ) and the more recently described human viruses (human bocavirus 1, parechoviruses, human polyomaviruses k1 and wu) and m. pneumoniae and chlamydia species [49] . serum crp (threshold 5 mg/l) are standard tests that will be analysed locally (diagnostic laboratory of each participating centre). saa, il-6 (threshold < 3 pg/ml), il-10 (threshold < 0.5 pg/ml) and ip-10 (threshold 2.8 pg/ml) will be performed using elisa commercial kits (r&d systems, minneapolis, mn, usa) at our research laboratory. spirometry (in children aged ≥ 5 years) will be performed using american thoracic society criteria and the fev 1 % predicted recorded. we elected not to use oscillatory measures as we found no difference in airway resistance between steady and exacerbation states [33] . thus we will use conventional spirometry although we do not expect to detect significant differences. participation is complete when the child's clinical state returns to baseline and the 'time to next exacerbation' has been obtained. other exit points are if the child is clinically worse prior to day 14 or intolerance to the trial medications requiring withdrawal from the study (as determined by the treating clinician). the primary outcome is the proportion of children whose exacerbations have resolved by day 14. exacerbations will be considered resolved when symptoms and signs are the same as the baseline state. children who are withdrawn from the study or receive additional antibiotic treatment will be categorised as non-resolved. the main secondary outcome is the pc-qol score. other outcomes are the time to next exacerbation; requirement for hospitalisation; duration of exacerbation (persistence of symptoms till 'return to baseline state') and fev 1 % predicted. serum markers (crp, saa, il-6, il-10, ip-10) and data on viruses and respiratory bacterial pathogens, including antibiotic susceptibility patterns to penicillin and azithromycin, will be the secondary laboratory outcomes. we plan to enrol 189 children (63 per arm), providing 84% power (α = 0.0245, two-sided) to detect a halving of the number of children in the active arm achieving resolution by day 14 (that is, azithromycin or amoxicillinclavulanic acid: 60% resolved by day 14, compared with placebo: 30% resolved). this is a conservative estimate when compared with our prospective data of persistent symptoms in 24% of children based on the same diary card [50] . as the primary outcome will be obtained in all enrolled children, a drop-out has not been factored in for the intention-to-treat analysis. with 20% drop-out rate, data from 153 children (51 per arm) for 'per protocol' analysis provides a study power of 75%. both treatment arms are compared with the same placebo arm. while the maximum efficiency is attained by allocating more children to the placebo arm (that is, using an allocation ratio of 1:1:√2), we chose to use a 1:1:1 allocation due to ethical concerns of deviating from standard care in respiratory centres. in the main secondary outcome (pc-qol), based on a between-group difference of 0.9 (minimum important difference [42] ) (sd 0.9), our sample size provides power of 100% (α = 0.05) for data from at least 147 children (that is, assuming at least 80% retention of the 189 children enrolled). for secondary aim 2 (exploring factors that predict response to antibiotics), we will be examining eight main factors and thus a sample size of 147 exceeds the recommended minimum (n = 10 per factor) [51] . the eight factors are smoking, age, underlying aetiology, detection of virus (any versus none, then single versus multiple viruses), presence of azithromycin resistance and blood markers (il-6, il-10, ip-10 levels). data will be reported and presented in accordance with the updated consort criteria [52] . children will be analysed according to allocation status (regardless of subsequent management). an interim analysis is planned and a data safety and monitoring committee will determine if the study should be ceased should superiority of any antibiotic be identified after 50% of sample size is achieved. for our primary aim, the main effects of the interventions will be determined by comparing the primary outcome (resolution of exacerbation) between groups ((azithromycin versus placebo) and (amoxicillin-clavulanic acid versus placebo)). children who exit the study as clinically worse or drop-outs prior to the end point will be considered non-resolved. those who exit the study as 'returned to baseline' will be considered resolved. odds ratios will be calculated and, if appropriate, number needed to treat (for benefit) will be expressed. tests of a treatment arm versus placebo at the end of the study for the primary outcome will be performed at the 2.45% significance level to account for spending some alpha at the interim analysis. per protocol analysis will be an a priori secondary analysis. for clinical secondary outcomes (secondary aim 1), t-tests or mann-whitney will be used for continuous variables (according to normality of data distribution). a kaplan-meier curve will be constructed for each group for 'time to resolution' and 'time to next exacerbation' as done previously [53] . for secondary aim 2 (factors that predict response to antibiotics), univariate analysis will be used to examine the biological factors listed above. factors that have a p-value < 0.2 will be included in a logistic regression model. potential interactions (for example, virus with bacteria) will be examined in the model. descriptive data will be used for secondary aim 3 (point prevalence of respiratory pathogens). a data safety monitoring committee has been established and has met prior to commencement of this study. it was determined that, when 50% of the sample size has been achieved, the stopping rules are as detailed below. if superiority between each antibiotic arm and the placebo arm is shown at significance level of 0.001, the study will cease. if superiority of only one antibiotic is shown, we will continue recruiting children to the other antibiotic arm and to the placebo arm but not to the superior antibiotic arm. if the serious adverse events (related to the medications) in each antibiotic arm outnumber the adverse events in the placebo arm at significance level of 0.01 or less, the study will cease. if increased adverse events of only one antibiotic is shown, we will continue recruiting children to the other antibiotic arm and to the placebo arm but not to the antibiotic arm related with increased adverse events. however, the study is not powered to detect between-group differences in total adverse events. the protocol has received ethical approval from the re despite the considerable global burden, bronchiectasis services receive disproportionately fewer allocated resources (clinical and research) when compared with other chronic respiratory diseases [3, 54, 55] . the marked paucity of rcts [21, 55] is reflected in the existence of only a single (small) published placebo-controlled rct in children with bronchiectasis [21, 56] . that study described a reduction in sputum purulence and airway hyper-responsiveness in children receiving roxithromycin (n = 13) [57] . there are no rcts on the management of bronchiectasis exacerbations in children [58] . almost all current recommendations are based on cf management [21, 28] . such extrapolation can, on occasions, be detrimental for those with non-cf bronchiectasis. for example, a large rct found that deoxyribonuclease (efficacious for cf) increased exacerbations and decline in fev 1 in adults with bronchiectasis [59] despite prior case reports advocating its use [60] . the importance of exacerbations in most chronic respiratory diseases is generally accepted. unfortunately, data on triggers, definitions and effective treatment of bronchiectasis exacerbations in both children and adults are scarce [56, 61, 62] . although viral triggers of acute exacerbations are well described in asthma [46] and copd [63, 64] , no such data exist for bronchiectasis. whether other potential respiratory pathogens (m. pneumoniae and chlamydia species) trigger exacerbations has never been examined. our retrospective study found that 34% of exacerbations were preceded by a viral-like illness [29] . thus, for the first time in this population, we will determine the nature and diversity of respiratory viruses m. pneumoniae and chlamydia species associated with bronchiectasis exacerbations. our study addresses a large knowledge gap in an under-researched area [55] . if the intervention is successful, it would lead to improved short-term (and possibly long-term) health benefits. conclusive results would produce changes to evidence-based standard treatment guidelines. in our retrospective data of 115 respiratory exacerbations [29] , we found that 35% of exacerbations failed to respond to oral antibiotic therapy (duration could not be determined) and required hospital admission. in our prospective cohort of 69 children followed for 900 childmonths (156 exacerbations), 36 exacerbations (23%) were treated with intravenous antibiotics following persistence of symptoms, that is, non-resolution of the exacerbation episode. generally, hospitalisation began three to five weeks following the initiation of oral antibiotics and 'return to baseline' occurred within two weeks of hospitalisation. based on our data that 24% of otherwise well children in the community still have a cough associated with a viral infection at day 14 [50] , we chose day 14 as the time point for this rct. we also asked parents and clinicians about their willingness to use placebo for a period of time; 14 days was the limit with a safety exit point at day 7. adult bronchiectasis studies show that qol measures, particularly cough-specific qol, are valid and important outcome measures [62, 65] . likewise, we have shown the utility of a paediatric chronic cough qol (the pc-qol) score in children with bronchiectasis [66] . in summary, our double-blind, double-dummy rct that examines the superiority of azithromycin and amoxicillin-clavulanic acid (compared with placebo) for exacerbations of bronchiectasis in children has the potential to have both short-term gains and a long-term benefit for reducing the morbidity of bronchiectasis. we trends in bronchiectasis mortality in england and wales trends and burden of bronchiectasis-associated hospitalizations: usa a descriptive study of non-cystic fibrosis bronchiectasis in a pediatric population from central and southern italy bronchiectasis in central australia: a young face to an old disease management of bronchiectasis and chronic suppurative lung disease (csld) in indigenous children and adults from rural and remote australian communities prevalence and economic burden of bronchiectasis can a management pathway for chronic cough in children improve clinical outcomes: protocol for a multicentre evaluation a multi-centre study on chronic cough in children hospitalisation of indigenous children in the northern territory for lower respiratory illness in the first year of life non-cf bronchiectasis-clinical and hrct evaluation bronchiectasis in alaska native children: causes and clinical courses outcomes in children treated for persistent bacterial bronchitis qualitative analysis of high resolution computed tomography scans in severe asthma the prevalence of cystic fibrosis in the european union physiological and radiological characterisation of patients diagnosed with chronic obstructive pulmonary disease in primary care bronchiectasis: the consequence of late diagnosis in chronic respiratory symptoms bronchiectasis secondary to primary immunodeficiency in children: longitudinal changes in structure and function non-cystic fibrosis bronchiectasis in childhood: longitudinal growth and lung function longitudinal study of lung function in a cohort of primary ciliary dyskinesia longitudinal growth and lung function in pediatric non-cf bronchiectasis -what influences lung function stability? chest bronchiectasis and chronic suppurative lung disease (csld) in children and adults in australian and new zealand: thoracic society of australia and new zealand and australian lung foundation position statement phenotypes of adult bronchiectasis: onset of productive cough in childhood and adulthood diagnosing and preventing chronic suppurative lung disease (csld) and bronchiectasis factors associated with lung function decline in adult patients with stable non-cystic fibrosis bronchiectasis mortality in bronchiectasis: a long-term study assessing the factors influencing survival pediatric cough: children are not miniature adults state of the art -chronic wet cough: protracted bronchitis, chronic suppurative lung disease and bronchiectasis british thoracic society guideline for non-cf bronchiectasis exacerbations in non cystic fibrosis bronchiectasis: clinical features and investigations inflammation: a two edged sword. the model of bronchiectasis subjective scoring of cough in children: parent-completed vs child-completed diary cards vs an objective method cough quality in children: a comparison of subjective vs. bronchoscopic findings defining pulmonary exacerbation in children with non-cystic fibrosis bronchiectasis bacterial colonization of the nasopharynx predicts very early onset and persistence of otitis media in australian aboriginal infants randomized placebo-controlled trial on azithromycin to reduce the morbidity of bronchiolitis in indigenous australian infants: rationale and protocol respiratory bacterial pathogens in the nasopharynx and lower airways of australian indigenous children with bronchiectasis innate immune mechanisms linking non-esterified fatty acids and respiratory disease quality-of-life determinants in patients with clinically stable bronchiectasis relationship between measurements of cough severity development of a parent-proxy quality-of-life chronic cough-specific questionnaire: clinical impact vs psychometric evaluations validation of a parent-proxy quality-of-life questionnaire (pc-qol) for paediatric chronic cough minimally important change in a parent-proxy quality of life questionnaire for pediatric chronic cough (pc-qol) nasopharyngeal versus oropharyngeal sampling for detection of pneumococcal carriage in adults streptococcus pneumoniae and noncapsular haemophilus influenzae nasal carriage and hand contamination in children: a comparison of two populations at risk of otitis media random colony selection versus colony morphology for detection of multiple pneumococcal serotypes in nasopharyngeal swabs newly identified respiratory viruses in children with asthma exacerbation not requiring admission into hospital a sensitive, specific, and cost-effective multiplex reverse transcriptase-pcr assay for the detection of seven common respiratory viruses in respiratory samples distinguishing molecular features and clinical characteristics of a putative new rhinovirus species, human rhinovirus c (hrv c) molecular diagnosis of medical viruses asthma and protracted bronchitis: who fares better during an acute respiratory infection? relation between several variables consort 2010 statement: updated guidelines for reporting parallel group randomised trials zinc and vitamin-a supplementation in indigenous children hospitalised with episodes of lower respiratory tract infection: a randomised controlled trial bronchiectasis: a neglected cause of respiratory morbidity and mortality clinical challenges in managing bronchiectasis non-cystic fibrosis bronchiectasis exacerbations effect of roxithromycin on airway responsiveness in children with bronchiectasis: a double-blind, placebo-controlled study short courses of antibiotics for children and adults with bronchiectasis rhdnase study group: treatment of idiopathic bronchiectasis with aerosolized recombinant human dnase i clinical benefit from nebulized human recombinant dnase in kartagener's syndrome procalcitonin in stable and unstable patients with bronchiectasis quality of life and inflammation in exacerbations of bronchiectasis role of viral infections in asthma and chronic obstructive pulmonary disease identifying viral infections in vaccinated chronic obstructive pulmonary disease (copd) patients using clinical features and inflammatory markers assessing response to treatment of exacerbations of bronchiectasis in adults the burden of disease in pediatric non-cystic fibrosis bronchiectasis submit your next manuscript to biomed central and take full advantage of: • convenient online submission • thorough peer review • no space constraints or color figure charges • immediate publication on acceptance • inclusion in pubmed, cas, scopus and google scholar • research which is freely available for redistribution we thank the research staff (lesley versteegh, clare wilson, sophie anderson-james, joanne tuppin, stacey spencer, carol willis) for facilitating the study, robert ware for generating the randomisation sequences and anita champion for allocating the children and dispensing the medications. we are also grateful to members of the indigenous reference group of the child health division at menzies for supporting this study and for overseeing the cultural aspects. we also thank professor alan isles, professor craig mellis and associate professor chris blyth for voluntarily providing their time in their participation as members of the data safety monitoring committee. the authors declare that they have no competing interests.authors' contributions ac conceived the study, and participated in its design and coordination and drafted the manuscript. pm, cr, kg, pva, aw, ko, pt and ts participated in its design and submission to the national health and medical research council. eb and gm participated in initiating the project and imm in the viral analysis plan. ibm, cb and hb will assist in recruitment and assessment of the children. mdc advised on statistical issues. all authors read and approved the final manuscript.authors' information abc, cfr, acw, ppva, cab, ibm and hmb are paediatric respiratory physicians, kg is a paediatric infectious disease physician, pjt is an adult respiratory physician, kfo is an epidemiologist, tps and imm are virologists, mdc is a statistician, ejb and gbm are research nurses and pm is a general paediatrician. key: cord-289050-9w7ks01n authors: donoso, alejandro f.; león, josé a.; camacho, jorge f.; cruces, pablo i.; ferrés, marcela title: fatal hemorrhagic pneumonia caused by human metapneumovirus in an immunocompetent child date: 2008-08-25 journal: pediatr int doi: 10.1111/j.1442-200x.2008.02673.x sha: doc_id: 289050 cord_uid: 9w7ks01n nan human metapneumovirus (hmpv) was recently discovered in 2001 in holland. this agent causes acute respiratory tract infections in children and adults. 1 the range of clinical manifestations varies from asymptomatic infection to severe pneumonia and acute respiratory failure. 1 -3 when this agent coexists with the respiratory syncytial virus (rsv) in bronchiolitis, symptoms are more severe. 4 williams et al. found, in more than 25 years of investigation, that 12% of lower respiratory tract infections in children was caused by hmpv. 2 fatal cases of hmpv in both healthy and immunocompromised children have been rarely described in the literature so far. 5, 6 the aim of the present study was to report the case of a pediatric, immunocompetent patient who presented with severe acute respiratory distress syndrome (ards), for whom there was a fatal outcome, and in whom hmpv was the only etiologic agent found even on post-mortem necropsy. the institutional review board approved the presentation of this report. a previously healthy, 2-year-old girl suffered an episode of diarrhea, vomiting and fever 1 month before admission. she was treated with antipyretics and oral fl uids. one week later she developed mild respiratory distress and coughing. bronchopneumonia was diagnosed and amoxicillin and inhaled salbutamol were administered. she developed transient improvement but coughing and vomiting continued, and the subject was febrile. no other family member had respiratory symptoms. she did not travel abroad. at the emergency room her vital signs were temperature 37.5°c, pulse saturation 97% in room air, respiratory rate 52 breaths/min and heart rate 170 beats/min. prior to hospital admission chest radiograph showed a right-sided bronchopneumonia ( fig. 1 ) . thus, the patient received 2 l/min of oxygen through a nasal cannula (saturating 96%), parenteral fl uids, i.v. ampicillin and inhaled salbutamol. laboratory tests indicated hematocrit 31%, hemoglobin 9.7 g/dl, white blood cell (wbc) count 14.800 cells/mm 3 , platelet count 121 000/mm 3 , and c-reactive protein 8 mg/l (normal value <10 mg/l). three hours after hospital admission the patient suffered sudden respiratory and cardiovascular collapse. the clinical parameters at that point were temperature 38°c, tachycardia 176 beats/min, tachypnea 90 breaths/min, and pulse saturation 79% in fraction of inspired oxygen (fio 2 ) of 0.5. the patient was transferred to the pediatric intensive care unit (picu) where she was pale, tachypneic, had capillary refi ll >2 s, and pediatric glasgow coma scale of 10/15. the patient was intubated immediately while invasive monitoring was initiated. she received fl uid boluses of normal saline and 10% albumin solution (60 ml/kg in total), blood products (20 ml/kg of packed red blood cells and 10 ml/kg of fresh frozen plasma), vasoactive drugs (dopamine at 20 g/kg/min and epinephrine 1 g/kg/min) and empiric antibiotherapy (cefotaxime). laboratory tests indicated metabolic acidosis (ph 7.31, bicarbonate 11.4 mmol/l, base excess -11), hyperglycemia (283 mg/ dl) and hypoalbuminemia (1.3 g/dl). full blood count indicated hematocrit 21.7%, hemoglobin 6.7 g/dl, wbc count 23 500 cells/ mm 3 with 11% lymphocytes, and platelet count 90 000/mm 3 . severe pulmonary edema and hemorrhage from the endotracheal tube were observed in picu ( fig. 2 ) . she was switched to high-frequency oscillatory ventilation (mean airway pressure 30 cmh 2 o, amplitude 60 cmh 2 o, frequency 10 hz) but the patient never responded adequately to any therapy. within the next 60 min she rapidly developed systemic hypotension, anuric renal failure, progressive metabolic acidosis (ph 6.91) and fi nally cardiac arrest. although cardiopulmonary resuscitation was performed for 50 min, the patient died 3 h and 30 min after picu admission. an autopsy was conducted indicating vascular congestion of the alveolar septa, macrophage desquamation and recent hemorrhage in the alveoli, and alveolar collapse on the whole right lung and two-thirds of the left lung. these fi ndings were compatible with bilateral hemorrhagic bronchopneumonia. no other abnormalities were found in the other organs. on post-mortem the laboratory tests were as follows: blood and sputum cultures for bacteria and fungi on chocolate agar with media supplements, trypticase soy blood agar, and macconkey's agar were negative; blood polymerase chain reaction (pcr) for mycoplasma pneumoniae was negative. post-mortem lung tissue was inoculated onto shell vial culture of mdck and h292 cells (atcc ccl-34 and crl-1848, respect ively), followed by immunofl uorescence (if) with monoclonal antibodies against rsv, parainfl uenza virus types 1, 2 and 3, infl uenza virus types a and b, and adenovirus (vrk, viral respiratory kit, cat. b1029-86, bartels, issaquah, usa). none of them were detected. in addition, the same lung tissue taken from the autopsy was also inoculated onto conventional cell monolayer culture, including fi broblasts foreskin (fs), human epithelioma (hep-2), human rhabdomyosarcoma, and african green monkey kidney (bsc-1) cells. although a non-typical cytopathic effect was observed in fs cells, the cells were negative for varicella zoster virus (vzv) antigens and the supernatant was negative for vzv, enterovirus and hmpv on pcr (or reverse transcriptase -pcr [rt-pcr]). furthermore, passage to ll-mk2 cells yielded nothing. in order to detect hmpv, rna was extracted from the postmortem lung tissue, using a high pure nucleic acid isolation kit (roche diagnostics, mannheim, germany) according to the instructions, and one-step rt-pcr for two regions from the hmpv genome was performed. the positive control of hmpv was kindly donated by dr guy boivin (centre rechearches infectologuie, quebec, canada). pcr products were analyzed on electrophoresis in a 2% agarose gel with ethidium bromide, and confi rmed to be 347 bp fusion protein (f) gene fragment and the 213 bp nucleoprotein (n) gene fragment of hmpv. 7 this qualitative in-house rt-pcr was positive. additionally, a real-time quantitative rt-pcr assay for andes hantavirus using light cycler tm instrument (roche molecular biochemicals) in peripheral blood mononuclear cells was negative. pcr primers amplifi ed a segment of s segment equivalent to 234 bp (gen bank nc 003466). in addition, igm and igg for hantavirus were also negative. human metapneumovirus has been recently classifi ed as a new member of the paramyxoviridae family, increasing the list of the well-known viral pathogens of the respiratory tract. 1 since its initial report studies about its epidemiologic behavior and clinical manifestations have risen dramatically. in infants and young children the lower respiratory tract infections caused by hmpv are very similar to the ones caused by rsv, infl uenza and parainfl uenza viruses. although the spectrum of clinical manifestations of hmpv infection is not completely defi ned as yet, the most frequent manifestations include cough, coryza and pharyngitis. fever, wheezing, stridor and respiratory failure are observed in more severe cases. 2, 8, 9 non-respiratory symptoms such as conjunctivitis, otitis media, vomiting, diarrhea, or rash are occasionally reported. human metapneumovirus could be more severe in patients with underlying medical conditions such as chronic lung disease caused by prematurity, cardiac disease, and immunodefi ciency. there is currently controversy about its role in increasing the symptoms in asthmatic patients compared with rsv, adenovirus, infl uenza and parainfl uenza virus, but the biologic signifi cance of this association is unclear. 2 co-infection with hmpv and other virus, such as rsv or severe acute respiratory syndrome-coronavirus, has been reported and may contribute to more severe clinical manifestations. 4 -10 because shell vial cultures with if, one of the most reliable laboratory tests for common respiratory viruses, could not detect any other respiratory virus; and rt-pcr, which is considered to be the best laboratory test for diagnosis of hmpv infection, clearly detected hmpv-rna in the present patient, it is reasonable to assume that hmpv was the only agent involved in the fatal pneumonia. the accumulation of cases, however, and comprehensive microbiological analysis including (rt-) pcr for other respiratory viruses are needed to confi rm that infection with hmpv alone can result in such severe lung disease. few studies on fatal cases have been reported in children. pelletier et al. published a report on an infant who died due to severe and refractory ards. that patient had an underlying immunodefi ciency and the exact cause of death could not be established because the autopsy was not performed. 6 ulloa-gutiérrez et al. reported another severely ill, 3-month-old preterm boy with fig. 2 chest radiograph after intubation, indicating bilateral extensive lung consolidations, predominantly on the right side. fatal human metapneumovirus pneumonia 591 © 2008 japan pediatric society hmpv pneumonia who underwent extracorporeal membrane oxygenation for 10 days. 3 an immunocompetent, 1-year-old girl was reported recently. that patient, who presented with fever, cough and diarrhea developed rapidly an extensive pneumonia and then pulmonary edema and hemorrhage, and fi nally refractory shock. that patient died a few hours after admission. 5 that case resembles that of the present patient very well in terms of immunocompetency, severity, rapid onset of symptoms, and outcome. based on genetic analysis of f gene, hmpv can be divided into two major genotypes that are further classifi ed into 22 subgroups. although correlation between genotypes and phenotypes (pathogenicity) is not precisely known, it will be important to determine genotype in such severe cases. 2,6 unfortunately we could not perform genotype analysis of hmpv detected in the present patient. it is very important to understand the real impact of hmpv infections in different populations, the complete spectrum of manifestations, and the risk factors that can predict a poor outcome including age, underlying medical conditions, co-infection with other respiratory virus, and certain genotypes. we believe that the present case should warn us about the occasional role of hmpv as an agent of severe lung infection in children without a predisposing condition. we recommend including this agent within the causes of severe and potentially fatal pneumonia in children. a newly discovered human pneumovirus isolated from young children with respiratory tract disease human metapneumovirus and lower respiratory tract disease in otherwise healthy infants and children life-threatening human metapneumovirus pneumonia requiring extracorporeal membrane oxygenation in a preterm infant human metapneumovirus in severe respiratory syncytial virus bronchiolitis detection of human metapneumovirus in a fatal pediatric pneumonia respiratory tract reinfections by the new human metapneumovirus in an immunocompromised child molecular assays for detection of human metapneumovirus human metapneumovirus infections in hospitalized children metapneumovirus and acute wheezing in children human metapneumovirus detection in patients with severe acute respiratory syndrome key: cord-000877-usz7pnvu authors: abdel-moneim, ahmed s.; kamel, mahmoud m.; al-ghamdi, abdullhamid s.; al-malky, mater i. r. title: detection of bocavirus in children suffering from acute respiratory tract infections in saudi arabia date: 2013-01-30 journal: plos one doi: 10.1371/journal.pone.0055500 sha: doc_id: 877 cord_uid: usz7pnvu human bocavirus (hbov) was recently discovered in children with respiratory distress and/or diarrhea. to our knowledge, no previous study has reported the existence of bocavirus in saudi arabia. swabs samples from 80 children with respiratory tract infections were examined for the presence of hbov. real-time polymerase chain reaction was used as a sensitive method to detect the hbov. direct gene sequencing was used to determine the genotype of the detected virus isolates. hbov was detected in 22.5% of the examined patients. the np1 partial gene sequence from all patients showed that the circulated strains were related to hbov-1 genotype. most of hbov infected patients showed evidence of mixed coinfection with other viral pathogens. the current study clearly demonstrated that genetically conserved hbov1 circulates in saudi arabia. interestingly, most of the hbov1 infected cases were associated with high rates of co-infections with other viruses. bocavirus is a single-stranded dna virus belonging to the family parvoviridae, subfamily parvovirinae, genus bocavirus. bocaviruses are unique among parvoviruses because they contain a third orf between the non-structural and structural coding regions [1] [2] . the genus bocavirus includes viruses that infect bovine, canine, feline, porcine and some simian species as well as sea lions [3] [4] [5] [6] [7] [8] . human bocavirus (hbov) was first found in children with acute respiratory tract infections in 2005 [1] . it was then detected in children with respiratory tract infections in addition to gastroenteritis worldwide [9] [10] [11] [12] . the virus exists in four different serotypes hbov1-4 [1] [2] [13] [14] . although hbov 1 and 2 were reported in respiratory samples, all the 4 genotypes of hbov have been identified in children with acute gastroenteritis. hbov has been reported in various countries, indicating its worldwide endemic nature. the virus has been identified in europe [15] [16] [17] , america [18] [19] , asia [9, 20] , australia [21] [22] , africa [23] , and the middle east [24] . the prevalence of hbov ranges between 1.5 to 19.3% [18, 25] . primary infection with hbov seems to occur early in life and children between the ages of 6-24 months seem to be mostly affected [9] [10] , but older children can also be infected. newborn children may become protected by maternally derived antibodies [9] . hbov infections are rarely found in adults [26] [27] . lindner et al. detected anti-hbov antibodies in 94% of healthy blood donors .19 years of age [28] . hbov detection has been mostly performed on nasopharyngeal aspirates and swabs and relies mostly on classical [10, 18, 19, 21, 22, 26] and real-time pcr [10, 23, 29] . real-time pcr possesses many advantages over conventional pcr, as it offers greater sensitivity, specificity, and reduced expenditure of time. the current study aims to screen the epidemiological status and molecular phylogeny of hbov isolates prevailing in pediatric patients with respiratory infection in saudi arabia. the current study investigated the prevalence of hbov in patients suffering from respiratory tract infections in saudi arabia. the presence of the major viral causes of the respiratory distress in hbov positive cases was also screened. hbov was detected in 18/ 80 of the examined patients (22.5%) with ages ranging from 2 months to 10 years, (table 1-2). clinical findings for hbovpositive patients were indistinguishable from those for patients with other respiratory viruses. previously, hbov has been detected in samples from patients aged between 5 months and 2 years [1, 29] . ma et al, speculated that the antibody against hbov derived from the mother might protect children under 5 months of age from hbov infection [9] , however, we detected hbov in two cases below 5 months: in a 2-month-old and 4-month-old child ( table 1 -2) that may indicate the possibility of hbov infection in very young children. the rate of hbov in respiratory tract infections has been reported to be 1.5 to 19.3% [18, 25] . real-time pcr was used in the current study to screen hbov due to its high diagnostic sensitivity that could be responsible for the higher rate of hbov infection in saudi arabia than the widely accepted upper limit of infection rates worldwide. meanwhile, a recent study showed 21.5% prevalence among children [30] . the evidence of hbov as the main initiator of the disease in the infected cases is still uncertain because of its high co-infection rate with other pathogens, and it remains unclear whether hbov is the sole etiologic agent or just a concomitant virus bystander. in previous studies, none of the nasal swabs obtained from healthy children yielded a positive hbov test. this suggests that hbov is not a frequent commensal virus inhabiting the respiratory tract [16, 19] . hbov infections are frequently present in concomitant with other viruses and often occur in more than 50% of the tested samples [31] . in the current study, only one case was found to be infected only by hbov as a single virus entity while most of isolates (17/18) showed coinfection with other viral pathogens. the most frequently detected co-pathogens were rsv (13/18; 72.2%), iav (12/18 cases, 66.66%), respiratory adenovirus (6/18 cases, 33.33%) while only 1/18 (5.5%) case was coinfected with piv-3 and none was coninfected with piv-1 ( table 2 ). it is assumed that the rate and frequency of coinfections may be higher if more viruses were screened. consistent with other studies [1, 16] , the prevalence rate of bocavirus was higher in children under 2 years of age (table 1) . partial np-1 gene sequence of the eighteen detected hbov strains were obtained in our study. multisequence analysis showed complete identity (100%) between each other, and phylogenetic analysis demonstrated that they belonged to hbov1 (data not shown). blast analysis revealed complete homology to the published sequence of hbov1. furthermore, the phylogenetic analysis results of three selected sequences showed that the saudi hbov1 strains obtained from respiratory samples belonged to group i human bocaviruses (fig. 1) . to the best of our knowledge, this is the first report of hbov1 in saudi arabia. continuous surveillance and genome sequence analysis are needed to obtain more information on the genotypic variation and molecular evolution of hbov in the country. the study protocol was approved by the medical ethics review board of the college of medicine, taif university and by the pediatric hospital ethics committee in accordance with the guidelines for the protection of human subjects. informed written consents from the next of kin of the participants involved in the study were taken. nasopharyngeal swabs from 80 children suffered from moderate to severe lower respiratory tract infections were collected from january to may 2012 from the governmental pediatric hospital-al-taif, saudi arabia. the children's age ranged from two months to ten years of age. clinical manifestations and case histories were recorded. individual swabs were kept in 1 ml sterile saline containing gentamycin sulphate. swabs were routinely processed and kept at 280uc until further analysis. the real-time pcr assay was performed using commercial, taqman hydrolysis probe based, real time pcr bocavirus detection kit (liferiver, shanghai, china) in eppendorf master-cyclerh ep realplex 2 . the detection of the amplified amplicon was performed in fluorimeter channel fam with the fluorescent quencher bhq1. amplification reactions were performed in a volume of 25 ml containing 2.5 ml of dna template, 21.5 ml reaction mix, 0.4 ml enzyme mix, 1 ml internal control according to the manufacturer's instructions. the thermal cycling conditions were as follows: 2 min at 37uc, an initial denaturation of 2 min at 94uc and 40-cycles of 15 sec at 93uc and annealing/elongation step of 1 min at 60uc. hbov positive samples were screened for the presence of respiratory syncytial virus (rsv), influenza a virus (iav), parinfluenza virus 1(piv-1) and parainfluenza virus 3(piv-3), as well as respiratory enteric virus (adenv) using real-time pcr kits (shanghai zj bio-tech co., ltd). pcr amplification of a 354-base pair fragment of the np1 was performed as described previously [1] . the reaction mix contained 20 pmol of each primer and dna master mix (koma bioteck, inc., seoul, korea). the thermal cycling conditions were as follows: an initial denaturation of 5 min at 94uc, 35 cycles of 1 min at 94uc, 1 min at 54uc and 2 min at 72uc, final extension of 10 min at 72uc.positive pcr products were purified using a qiaquick pcr purification kit (qiagen) and were sequenced commercially (macrogen inc., seoul, korea). the nucleotide sequences of the ns1 gene were compared with those of hbov strains available at the genbank site. phylogenetic analyses were conducted with mega, version 4.1. the 3/18 partial sequences of the ns1 gene were submitted to genbank (accession numbers jx982976-jx982978). cloning of a human parvovirus by molecular screening of respiratory tract samples human bocaviruses are highly diverse, dispersed, recombination prone, and prevalent in enteric infections virus taxonomy: the eighth report of the international committee on taxonomy of viruses identification and nearly full-length genome characterization of novel porcine bocaviruses identification and characterization of a new bocavirus species in gorillas widespread infection with homologues of human parvoviruses b19, parv4, and human bocavirus of chimpanzees and gorillas in the wild the fecal viral flora of california sea lions identification and characterization of bocaviruses in cats and dogs reveals a novel feline bocavirus and a novel genetic group of canine bocavirus detection of human bocavirus in japanese children with lower respiratory tract infections epidemiological profile and clinical associations of human bocavirus and other human parvoviruses human bocavirus, a respiratory and enteric virus human bocavirus in children hospitalized for acute gastroenteritis: a case-control study a novel bocavirus associated with acute gastroenteritis in australian children a newly identified bocavirus species in human stool human bocavirus in french children human bocavirus in italian patients with respiratory diseases isolation of human bocavirus from swiss infants with respiratory infections human bocavirus infection human bocavirus infection in young children in the united states: molecular epidemiological profile and clinical characteristics of a newly emerging respiratory virus quantification of human bocavirus in lower respiratory tract infections in china frequent detection of human rhinoviruses, paramyxoviruses, coronaviruses, and bocavirus during acute respiratory tract infections evidence of human coronavirus hku1 and human bocavirus in australian children human bocavirus in hospitalized children human bocavirus infection among children frequent detection of viral coinfection in children hospitalized with acute respiratory tract infection using a real-time polymerase chain reaction severe pneumonia and human bocavirus in adult surveillance and genome analysis of human bocavirus in patients with respiratory infection in guangzhou, china humoral immune response against human bocavirus vp2 virus-like particles human bocavirus and acute wheezing in children high prevalence of human bocavirus 1 in infants with lower acute respiratory tract disease in argentina human bocavirus commonly involved in multiple viral airway infections key: cord-291618-166wvdbt authors: mikulska, małgorzata; del bono, valerio; gandolfo, nemo; dini, simone; dominietto, alida; di grazia, carmen; bregante, stefania; varaldo, riccardo; orsi, andrea; ansaldi, filippo; bacigalupo, andrea; viscoli, claudio title: epidemiology of viral respiratory tract infections in an outpatient haematology facility date: 2013-10-06 journal: ann hematol doi: 10.1007/s00277-013-1912-0 sha: doc_id: 291618 cord_uid: 166wvdbt viral respiratory tract infections (vrti) are an important cause of morbidity and mortality in haematology patients, particularly after haematopoietic stem cell transplantation (hsct). the incidence, clinical presentation and outcome of symptomatic and asymptomatic vrti in hsct outpatient unit were prospectively evaluated during a single influenza season (january–march 2011). pharyngeal swabs were performed at the first visit and if new symptoms were present. molecular multiplex assay for 12 respiratory viruses was performed by the regional reference laboratory. among 264 swabs from 193 outpatients, 58 (22 %) resulted positive for 61 viruses (influenza, n = 20; respiratory syncytial virus [rsv], n = 21; rhinovirus, n = 12; coronavirus, n = 4; adenovirus, n = 3; parainfluenza, n = 1). vrti were detected more frequently in the presence of symptoms than in asymptomatic patients: 49 out of 162 (30 %) vs. 9 out of 102 (9 %), p < 0.001. influenza-like illness syndrome (ili) was significantly associated with a vrti if compared to other presentations (42 %), while the european centre for disease prevention and control definition was not (30 %). positive predictive value (ppv) of ili for influenza was 17 %. influenza and rsv peak periods were contemporary. influenza prophylaxis was given to 25 patients following exposure. low rate of progression from upper to lower respiratory tract infection (approximately 5 % for influenza and rsv), no nosocomial epidemics and no vrti-related deaths were observed. vrti are very frequent in high-risk haematology outpatients, but symptoms are aspecific and ppv of ili is low. symptoms of influenza and rsv overlap. thus, microbiological diagnosis and contact preventive measures are crucial. rather than universal influenza prophylaxis, prompt diagnosis and treatment of only documented infections could be pursued. viral respiratory tract infections (vrti) are an important cause of morbidity and mortality in patients with hematologic malignancies, particularly in allogeneic haematopoietic stem cell transplantation (hsct) recipients [1] [2] [3] . in immunocompromised patients, vrti are usually more severe, with an increased risk of progression from upper to lower respiratory tract infections and more frequent presence of bacterial or fungal co-pathogens than in immunocompetent patients [4] [5] [6] [7] . moreover, long-term sequelae of vrti, such as restrictive or obstructive airway disease, have been reported [8, 9] . during the last decade, rapid and highly sensitive molecular tests have been developed and made available, with the most recent multiplex polymerase chain reaction (pcr) platform that can detect multiple viral pathogens [10] [11] [12] . based on data from observational studies, it is believed that most vrti in patients with haematological malignancies present with fever and/or upper respiratory tract symptoms, while less is known about the asymptomatic circulation of respiratory viruses, even though prolonged viral shedding has been observed [4, [12] [13] [14] [15] [16] [17] [18] [19] [20] . for influenza infection, the management strategy based on the presence of influenza-like illness syndrome (ili) has been successfully used in epidemiological and treatment studies in immunocompetent patients. for hsct recipients who are at an increased risk of developing severe influenza complications, post-exposure and general chemoprophylaxis during an influenza season have been recommended [21] [22] [23] . nevertheless, the efficacy and impact of these strategies, particularly when compared to early diagnosis and treatment, remain unknown. the aims of this study were to prospectively evaluate the incidence, morbidity and severity of vrti in outpatients of an hsct unit during the influenza season of 2011. additionally, the clinical usefulness of the presence of an ili for diagnosing and treating vrti and the possible occurrence of nosocomial transmission or microepidemics were evaluated. patients and study protocol a 3-month prospective study during the seasonal influenza outbreak, i.e. from 1 january 2011 to 31 march 2011, included all adult outpatients seen at least once a month in the hsct unit. most of the subjects were allogeneic hsct recipients, while the other patients either received an autologous hsct or chemotherapy for haematological diseases. the following data were recorded: demographics, type of the underlying disease and the date and type of hsct, including conditioning regimen. informed consent was obtained in accordance with the declaration of helsinki. both the asymptomatic and symptomatic for vrti patients underwent clinical evaluation at every visit to the outpatient unit and were invited to contact their physician if any new symptoms developed. additionally, influenza vaccination status of the patient and household contacts was noted. pharyngeal swabs, which are specimens considered suitable and easily collected by recent who recommendations, were collected for viral testing at the first visit and then only in case of new vrti symptoms [24] . additional swabs were obtained in patients with vrti in order to document the resolution of the infection of prolonged viral shedding but were not included in the analysis. bronchoalveolar lavage (bal) fluid and other clinical respiratory specimens were collected when clinically indicated. the distribution of influenza cases, i.e. the beginning, the peak and the end of epidemic, in this cohort was compared to data available for italy reported weekly by the italian network for influenza surveillance of superior health institute. definitions ili syndrome was defined as the presence of fever plus at least one of the following symptoms: cough, sore throat, rhinorrhoea and/or systemic symptoms as headache, asthenia, malaise and arthromyalgia, in the absence of other documented causes. vrti was considered as detection of a respiratory virus from the upper or lower respiratory tract [25] . similar to the european centre for disease prevention and control (ecdc) definition, in haematology patients, a confirmed case of viral respiratory tract infectious disease was defined as new onset of symptoms, and at least one of the following: cough, coryza, sore throat and shortness of breath, and the clinician's judgment that the illness is due to an infection [25] . a repeated detection of the same virus in a patient was considered as the same infection episode, and only the first positive swab was considered. co-infection was defined as simultaneous detection of more than one virus, while separate infection episodes were diagnosed if a new virus was detected at the onset of new respiratory symptoms. asymptomatic infection was defined as detectable virus in the patient's upper respiratory tract sample without symptoms of respiratory illness. upper vrti was defined as a respiratory viral isolation from a pharyngeal swab and included both symptomatic and asymptomatic infections. upper vrti symptoms included sore throat, cough, rhinorrhoea or nasal congestion. lower vrti was defined as a clinically and radiologically confirmed pneumonia with detection of virus from the lower respiratory tract (bal fluid). a direct contact for a period of at least 1 h with a patient with an upper or lower vrti was regarded as an exposition to vrti case. molecular assay was performed by the regional reference laboratory operating from monday to friday. all specimens were transported in universal transport medium (copan italia s.p.a., brescia, italy), collected and subjected to rna extraction. according to the manufacturer's instructions, viral rna was extracted by spin columns (qiaamp viral rna mini kit, qiagen, valencia, ca, usa), converted to random hexamer-primed cdna by the revertaid system (fermentas, york, uk), and then stored at −20°c until use. each cdna preparation was subjected to the seeplex rv12 ace detection pcr kit procedure (seegene, seoul, south korea). the multiplex pcr assay allowed the simultaneous detection of 12 respiratory viruses: influenza a/b, human adenovirus, respiratory syncytial virus (rsv) a/b, human metapneumovirus virus, human parainfluenza virus 1/2/3, human rhinovirus a/ b, human coronavirus 229e/nl63-oc43/hku1. samples positive only for the m gene of flu a by the centers for disease control and prevention (cdc) real-time reverse transcription polymerase chain reaction (rt-pcr) were typed using the real-time rt-pcr-based kit, established by the cdc, specific for detecting and characterizing a/ h1n1p. given the higher sensitivity of the cdc rt-pcr system than the seeplex kit for detecting a/h1n1p and influenza a viruses in general, throughout this paper, all results pertaining to influenza a virus should be considered to have been obtained by the former method. a small number of influenza a virus positive samples were untypeable because of low viral loads. the performance of this multiplex assay has been determined elsewhere [26] . at the outpatient unit, all patients, visitors and healthcare workers are required to use a surgical mask and to wash their hands before and after all patient contact. routinely, the outpatients received intravenous treatment in three-patient or five-patient rooms. in order to reduce the possibility of nosocomial transmission and to facilitate the evaluation of potential microepidemics, the patients were always placed in the same room, unless vrti was detected. patients with vrti were placed in individual rooms until they were asymptomatic and, if feasible, negative for respiratory viruses. yearly influenza vaccination of patients, hospital staff and family members was recommended [21, 27] . the differences between the groups were assessed by means of the chi-square test for heterogeneity or fisher's exact test when appropriate. all p values are two-sided; a p value of ≤0.05 was considered to be statistically significant. the analyses were performed using the spss version 13.0 statistical package (spss, inc., chicago, il, usa). a total of 193 patients were screened and 264 pharyngeal swabs were performed. among them, 136 patients had 1 swab performed, 45 had 2, 11 had 3 and 1 had 5. fifty-six percent of patients were male, and the majority (68 %, 132 out of 193) received hsct (127 allogeneic and 7 autologous), at a median of 306 days before entry in the study (range, 30-7, 712 days). in particular, 26 % (35 out of 132) received transplant within 90 days, 14 % (18 out of 132) within 90-180 days and 60 % (79 out of 132) within 180 days or more. among 264 pharyngeal swabs, 162 (61 %) came from symptomatic patients and 102 (39 %) from asymptomatic patients. a total of 58 (22 %) swabs positive for 61 viruses were recovered. in case of symptoms, the virological documentation of infection was significantly more frequent: 30 % (49 out of 162) of swabs performed during symptoms were positive, compared to 9 % (9 out of 102) of swabs in asymptomatic subjects (p <0.0001). the main viruses detected were influenza (20 out of 58 samples, 34 %), rsv (21 out of 58, 36 %) and rhinovirus (12 out of 58, 21 %), and influenza and rsv infections were significantly more frequent in symptomatic subjects ( table 1 ). the symptoms of vrti were divided into three main categories, and the rate of positive virological results was as follows: ili 38 % (25 out of 65), fever only 30 % (9 out of 30) and upper vrti symptoms 22 % (15 out of 67) (fig. 1) six patients had pneumonia; thus, bal was performed and four viruses were detected (co-pathogens are reported in table 2 ): one influenza a, two rsv and one coronavirus. in two cases (influenza and rsv), progression from upper to . these three patients with influenza and rsv pneumonia were successfully treated with oseltamivir and ribavirin, respectively. the other two patients were diagnosed with pneumonia due to other pathogens. weekly incidence and distribution of vrti due to different viruses weekly incidence of all vrti was 3.11 per 100 patient-weeks, of influenza was 1.13 per 100 patient-weeks and of rsv was 1.07 per 100 patient-weeks. the analysis of weekly distribution of diagnosed vrti documented that the peak activity for the main two viruses (influenza and rsv) was detected in the same period. the circulation of influenza virus in this cohort was detected later (detection from weeks 2 to 10, with peak activity in weeks 6 and 7), as compared to the general population (detection from week 50 of 2010 to week 9 of 2011, with peak activity from weeks 2 to 6). a similar delayed circulation was detected for rsv: in the study population in table 2 . in particular, approximately half of the patients with either of the following infections: influenza, rsv and rhinovirus, presented with ili syndrome (11 out of 20, 55 %; 9 out of 19, 47 %; and 5 out of 12, 42 %, respectively). the standard prevention measures that included facial masks and hand hygiene were applied to all the patients with vrti. for influenza infection, the possibility of giving pharmacological prophylaxis with oseltamivir was evaluated for all the contacts of patients with a documented influenza infection. among 20 influenza infections, 19 were treated with the full dose of oseltamivir and 1 patient was not treated because the positive result was available after 72 h and the patient was asymptomatic, vaccinated, with no previous allogeneic hsct and well-controlled underlying disease. overall, 56 contacts of 20 cases were identified among outpatients: 6 were symptomatic and received oseltamivir treatment pending the results of viral testing, whereas 50 were asymptomatic. among them, 19 received oseltamivir prophylaxis within 72 h of exposure to a contact patient, while the remaining 31 patients were closely monitored for the development of signs and symptoms of influenza infection, but no prophylaxis was given. the decision to withhold prophylaxis despite the high number of patients exposed to influenza, no nosocomial outbreak was detected. a cluster of influenza infection was observed in week 7 (five patients in 4 days), but the identified types of viruses were different (three type a h3n2, one type a h1n1 and one type b) and there was no evidence of any nosocomial contact with a patient infected with type a h3n2 4 days before. transmission from healthcare workers or visitors could not be assessed as no swabs were performed among them, but no healthcare worker attending the outpatients unit had respiratory symptoms or fever during the study period and visitors do not have access to outpatient therapy rooms. rsv peak activity was observed from weeks 6 to 8 (seven infections). the equal circulation of the a (four) and b (three) subtypes and no contact between the patients involved make unlikely patient-to-patient transmission and large epidemic outbreak. overall, 45 (23 %) patients were vaccinated with the seasonal strains, while among 148 non-vaccinated patients, 52 (35 %) reported that all or some household contacts had been vaccinated. there was no statistically significant difference in the in the group of hsct recipients, vaccination rate was 0 % (0 out of 53) within 6 months after transplant and 34 % (27 out of 79) among those over 6 months from transplant. among seven autologous transplant recipients, one was vaccinated and none developed influenza. the incidence of influenza was 11 % (6 out of 53) among patient with <6 months from transplant, 7 % (2 out of 28) among those >6 months from transplant and vaccinated and 8 % (4 out of 52) among those >6 months from transplant and not vaccinated. this prospective study confirms that vrti are a very common cause of morbidity, affecting during the observation period of 3 months almost one third of outpatients in an hsct unit. asymptomatic vrti was not infrequent (9 %), but none of them developed a clinically significant disease. symptoms of influenza, rsv and rhinovirus infection were similar and classic symptoms have low ppv for influenza (17 %) . thus, rapid and reliable diagnosis is crucial. fortunately, no large outbreak was detected, and low rate of progression from upper to lower respiratory tract infection and no vrti-related mortality were observed. the presence of any symptom was significantly associated with a confirmed vrti, but in this population, classic ili had low ppv for any vrti (38 %), including influenza (17 %). similar results were reported in another prospective observational study, where 25 % of patients had a vrti, and respiratory viruses were detected more frequently in the presence of symptoms, but symptoms in case of influenza and other vrti were similar [20] . ecdc definition adapted for the haematology population was even less specific than ili. in immunocompetent individuals, ili presentation is considered rather specific for influenza during the epidemic season. in fact, in generally healthy older adolescents and adults living in areas with confirmed influenza virus circulation, estimates of ppv, a simple clinical definition of influenza (acute onset of cough and fever), have been reported to be approximately 80-90 % and empirical anti-influenza treatment was recommended in such conditions [28] [29] [30] [31] . however, even in an immunocompetent population, the accuracy of the clinical diagnosis of influenza on the basis of symptoms alone is being questioned (reported incidence of ili in patients with influenza, 44-51 %) and is now considered limited because symptoms from illness caused by other pathogens can overlap considerably with influenza [29] . in immunocompromised patients, the association between ili and influenza is even less pronounced, as systemic symptoms, such as fever or myalgia, are frequently lacking [32] . therefore, considering the co-circulation of influenza, rsv and rhinoviruses in the same period and the similar clinical presentation of these infections, early use of rapid molecular testing to identify a specific virus, instead of universal empirical treatment with fig. 2 the distribution of different vrti anti-influenza drugs in patients with ili, should be pursued. initiating antiviral treatment at the onset of symptoms and then discontinuing it after diagnostic tests result negative for influenza is another feasible management strategy. for influenza, vaccination from 6 months after transplant is recommended for hsct recipients [21, 27] . in this study, the incidence of influenza was similar in vaccinated and nonvaccinated patients. however, the only two patients with asymptomatic influenza infection had been vaccinated, suggesting that active immunization might significantly attenuate the course of infection, even if it is not prevented. the role of chemoprophylaxis of influenza during the peak season remains controversial, even though it is universally recommended in case of nosocomial outbreaks and for the first 2 weeks after vaccination in case of community or nosocomial outbreak [21, 27] . however, in this population of immunocompromised patients, of whom <25 % was vaccinated, the incidence of influenza during the epidemic season was only 10 %. such a low incidence might be a result of a strict isolation policy with daily use of surgical masks and particular attention to hand hygiene, together with a virusspecific attack rate that may vary annually. additionally, a low progression rate to lower respiratory tract infection and low mortality in all vrti was observed (approximately 5 %), possibly due to a rapid diagnosis and an early start of an antiviral therapy-usually 24-48 h after the onset of symptoms. therefore, in a population with low incidence and mortality and in consideration of the possibility of acquiring resistance to antivirals, as reported during the 2009 h1n1 epidemics [33, 34] , an early diagnosis and post-exposure prophylaxis for close contacts seem preferable to a general chemoprophylactic strategy. molecular assays are particularly convenient for the diagnosis of vrti in immunocompromised patients because they are rapid, sensitive and may detect multiple pathogens, although there are always some viruses not included in the assay [26] . however, they may also detect viral genetic material from a resolved or subclinical infection, and the optimal management of these cases remains unclear. in this cohort, nine asymptomatic subjects had vrti and isolation measures were applied in all cases, but only one patient was treated for influenza. it has been reported that, in asymptomatic patients, lower quantities of virus might be present [20, 35] and transmission might be improbable, as demonstrated by on observation that no transmission to household contacts occurred in asymptomatic immunocompetent subjects with rhinovirus [36] . also, in this study, asymptomatic patients did not subsequently develop any clinical symptoms. the strengths of this study include a prospective standardized diagnostic approach for all the outpatients, regardless of respiratory symptoms and the use of a very sensitive diagnostic method. the limitations are a single-centre experience and a short observation period, which did not include the last weeks of 2010 when some infections could occur. thus, data on the incidence cannot be extrapolated to other populations or seasons. additionally, the sensitivity of detection of respiratory viruses may vary for oropharyngeal and nasopharyngeal specimens [37] . although, nasopharyngeal aspirates may be the most sensitive, especially for conventional diagnostic methods, less invasive specimen collection techniques are also acceptable given the very high sensitivity of new molecular diagnostic methods [37] , while recent guidelines recommend combined intranasal sampling using flocked swabs and the pharyngeal swabs in one virus transport medium [25] . finally, other centres with different management strategies for outpatients, including different contact prevention measures, might experience different rates of nosocomial transmission or morbidity. in conclusion, vrti are frequent in patients with haematological malignancies and may occur both in the presence of classic symptoms and in those with fever only. there was a significant overlapping in both symptoms and circulation period for influenza, rsv and rhinovirus. thus, empirical treatment of influenza in the presence of ili might be reserved for severely ill patients. rapid virological diagnosis and treatment only in case of a documented infection have been found feasible and successful in this setting of high-risk haematology outpatients. emerging viral infections after hematopoietic cell transplantation community respiratory virus infections in immunocompromised patients: hematopoietic stem cell and solid organ transplant recipients, and individuals with human immunodeficiency virus infection respiratory virus infections in stem cell transplant patients: the european experience respiratory virus infections after marrow transplant: the fred hutchinson cancer research center experience prospective study of the incidence, clinical features, and outcome of symptomatic upper and lower respiratory tract infections by respiratory viruses in adult recipients of hematopoietic stem cell transplants for hematologic malignancies the challenge of respiratory virus infections in hematopoietic cell transplant recipients respiratory syncytial virus pneumonitis in immunocompromised adults: clinical features and outcome airflow decline after myeloablative allogeneic hematopoietic cell transplantation: the role of community respiratory viruses strong association between respiratory viral infection early after hematopoietic stem cell transplantation and the development of life-threatening acute and chronic alloimmune lung syndromes nucleic acid amplification-based diagnosis of respiratory virus infections rapid virological surveillance of community influenza infection in general practice changing epidemiology of respiratory viral infections in hematopoietic cell transplant recipients and solid organ transplant recipients respiratory viral infections in adults with hematologic malignancies and human stem cell transplantation recipients: a retrospective study at a major cancer center persistent symptomless human metapneumovirus infection in hematopoietic stem cell transplant recipients respiratory syncytial virus infection in patients with hematological diseases: single-center study and review of the literature outcome of influenza infections in outpatients after allogeneic hematopoietic stem cell transplantation respiratory virus infection in immunocompromised patients community-acquired respiratory syncytial virus and parainfluenza virus infections after hematopoietic stem cell transplantation: the fred hutchinson cancer research center experience management of rsv infections in adult recipients of hematopoietic stem cell transplantation respiratory virus infection among hematopoietic cell transplant recipients: evidence for asymptomatic parainfluenza virus infection guidelines for preventing infectious complications among hematopoietic cell transplantation recipients: a global perspective safety and tolerability of oseltamivir prophylaxis in hematopoietic stem cell transplant recipients: a retrospective casecontrol study viral disease prevention after hematopoietic cell transplantation manual for the laboratory diagnosis and virological surveillance of influenza ecil-4): guidelines for diagnosis and treatment of human respiratory syncytial virus, parainfluenza virus, metapneumovirus, rhinovirus, and coronavirus rapid detection and identification of 12 respiratory viruses using a dual priming oligonucleotide systembased multiplex pcr assay vaccination of hematopoietic cell transplant recipients predicting influenza infections during epidemics with use of a clinical case definition prevention and control of influenza: recommendations of the advisory committee on immunization practices (acip) diagnosis of influenza in the community: relationship of clinical diagnosis to confirmed virological, serologic, or molecular detection of influenza clinical signs and symptoms predicting influenza infection how i treat influenza in patients with hematologic malignancies h275y mutant pandemic (h1n1) 2009 virus in immunocompromised patients emergence of oseltamivir-resistant pandemic (h1n1) 2009 virus within 48 hours human rhinovirus and coronavirus detection among allogeneic hematopoietic stem cell transplantation recipients rhinovirus transmission within families with children: incidence of symptomatic and asymptomatic infections comparison of nasopharyngeal and oropharyngeal swabs for the diagnosis of eight respiratory viruses by real-time reverse transcription-pcr assays the authors declare that they have no conflict of interest. key: cord-017364-d9zmdm23 authors: crowe, james e.; williams, john v. title: paramyxoviruses: respiratory syncytial virus and human metapneumovirus date: 2014-02-27 journal: viral infections of humans doi: 10.1007/978-1-4899-7448-8_26 sha: doc_id: 17364 cord_uid: d9zmdm23 human respiratory syncytial virus (rsv) and human metapneumovirus (mpv) are members of the family paramyxoviridae of the mononegavirales order, comprising the nonsegmented negative-strand rna viruses. paramyxoviridae has two subfamilies: paramyxovirinae, which includes the parainfluenza viruses 1–4 and measles and mumps viruses, and pneumovirinae, which includes rsv and mpv. pneumovirinae has two genera: pneumovirus, which includes human rsv, bovine respiratory syncytial virus, and pneumonia virus of mice, and metapneumovirus, which includes human mpv and avian metapneumovirus, sometimes called avian pneumovirus. human respiratory syncytial virus human respiratory syncytial virus (rsv) and human metapneumovirus (mpv) are members of the family paramyxoviridae of the mononegavirales order, comprising the nonsegmented negative-strand rna viruses. paramyxoviridae has two subfamilies: paramyxovirinae , which includes the parainfl uenza viruses 1-4 and measles and mumps viruses, and pneumovirinae , which includes rsv and mpv. pneumovirinae has two genera: pneumovirus , which includes human rsv, bovine respiratory syncytial virus, and pneumonia virus of mice, and metapneumovirus , which includes human mpv and avian metapneumovirus, sometimes called avian pneumovirus. rsv was isolated fi rst in 1956 from an ill chimpanzee in a laboratory setting that had an illness similar to the common cold. a virus causing a similar cytopathic effect in cultured cells was recovered from infants with respiratory illness shortly after, and studies of human antibodies in the serum of infants and children indicated that infection was common early in life [ 1 , 2 ] . now it is known that rsv is the most common viral agent of serious pediatric respiratory tract disease worldwide. in most areas of the world, rsv is the most common cause of pneumonia and bronchiolitis in infants less than 1 year of age. rsv causes severe disease in young infants, but disease is not restricted to the early life period. the virus can cause severe lower respiratory tract illness in large numbers of elderly subjects and also in subjects who are severely immunocompromised such as hematopoietic stem cell transplant recipients [ 3 -7 ] . mortality in infants and children caused by rsv is uncommon in developed countries with modern critical care units. estimates of the mortality rate are about 0.3 % of hospitalized children or less. mortality has been dropping over the last several decades, and by the late 1990s the estimated number of deaths in the usa was several hundred children a year or less [ 8 , 9 ] . large epidemiologic studies report that the us mortality in children may be only about 100 cases a year. interestingly, while many providers think of rsv infection as principally a pediatric illness, there are over 17,000 deaths per year in the elderly, making them the highest risk population for death [ 9 ] . in less developed countries, however, infant deaths due to rsv infection may be more common. infants with underlying illness are at highest risk among young children for morbidity and mortality from rsv infection. infants with chronic lung disease requiring supplemental oxygen following treatment for prematurity, due to bronchopulmonary dysplasia, are perhaps at the highest risk for prolonged, severe, or fatal illness due to rsv [ 10 ] . infants with severe congenital pulmonary or cardiac disease have been reported to be at risk of death in 3-4 % of cases when hospitalized, although this rate is likely decreasing in the usa [ 11 ] . both children and adults with primary immunodefi ciency or medically induced immunosuppression are at high risk of mortality due to rsv infection. the most severely immunocompromised, and thus those at highest risk of mortality, are hematopoietic stem cell transplant patients of any age [ 12 ] . in some settings, the mortality rate of rsv infection in hematopoietic stem cell transplant patients with severe immunosuppression verges on 100 % [ 12 ] . hospitalization rates of infants for rsv disease vary with the setting, probably due to variations in exposure, genetics, socioeconomic, and other risk factors and due to the local practice style of medical providers. many developed countries report hospitalization rates of about 1 in 100-200 infected infants during the fi rst year of life [ 13 , 14 ] . studies of rsv disease in developed countries suggest that of those infants hospitalized, about 9 % require mechanical ventilation [ 15 , 16 ] . there are certain populations at extraordinarily high risk of hospitalization with rsv, for example, alaskan native infants younger than 1 year have been reported to have a hospitalization rate of 53-249 per 1,000 infants [ 16 ] . low socioeconomic status is a risk factor for higher rate of hospitalization in most areas. rsv also is one of the most common viral causes of serious lower respiratory tract illness in the elderly, especially in institutionalized subjects [ 17 ] . exacerbations of chronic obstructive pulmonary disease (copd) are frequently associated with rsv infection [ 18 , 19 ] . the elderly do not exhibit a remarkably diminished level of antibodies to rsv [ 20 ] . decreased levels of t cell memory in the elderly and specifi cally in patients with (copd) may contribute to the increased susceptibility to rsv infection in these populations [ 21 ] . many think of infl uenza virus as the principal viral respiratory pathogen in this population, but in a hospitalized cohort, infl uenza a virus and rsv infection resulted in similar mortality, lengths of stay, and rates of use of intensive care [ 17 ] . rsv infection accounted for over 10 % of hospitalizations for pneumonia. seroepidemiology studies suggest that virtually all children are infected in the fi rst 2 years of life, and early infection is especially common in infants attending group day care. serological methods are helpful in epidemiology and vaccine studies, but serologies are not often used for diagnosis in clinical settings. because of the transfer of maternal rsvspecifi c antibodies across the placenta, and the high prevalence of early infection, it is unusual to fi nd infants who are rsv seronegative. in older children and adults, a fourfold rise in serum antibodies is often used as evidence of rsv infection, but asymptomatic infections in which viral shedding is low in titer often are not accompanied by serum antibody rises. serological tests in infants are even less sensitive, because young infants may not exhibit a large or durable rise in antibodies. neutralizing antibody tests are considered the best functional marker of infection, but sensitivity is much higher in antigen binding assays using individually purifi ed rsv f or g proteins [ 22 ] . isolation of the virus in cell culture provides a defi nitive test for diagnosis of active infection. various methods of obtaining respiratory virus secretions for testing have been compared. most studies suggested that aspiration or gentle fl ushing of nasal secretions using a solution like saline is best, though some types of nasal swab have given reasonable results. the virus is more thermolabile than most, and thus samples should be transported on wet ice to the diagnostic laboratory and processed rapidly. prolonged times of transport to remote reference laboratories reduce the effectiveness of isolation. after inoculation onto susceptible cell culture substrates, highly trained staff can recognize cytopathology in the cell monolayers by visual inspection and conventional bright-fi eld microscopy after about 3-7 days of incubation. detection may be more effi cient when using shell vial cultures and immunofl uorescence [ 23 ] . various cell lines have been used for rsv detection, such as hep-2 epithelial cells, mrc-5 fi broblasts, and rhesus monkey kidney cells, but the r-mix commercial mix of human and mink lung cells may perform better for detection of rsv [ 24 ] . culture is expensive and requires highly trained staff and therefore is not usually available at the point of care, which is often an outpatient clinic or emergency department. therefore, rapid diagnostic methods were developed for the detection of viral proteins or rna in respiratory secretions. rsv antigen tests mostly rely on direct immunofl uorescent assays (dfa) on exfoliated cells in secretions or enzyme immunoassay (eia). nucleic acid detection assays based on rt-pcr are now widely available for rsv, often in a multiplexed panel for detection of multiple respiratory virus pathogens. these tests are typically more sensitive than any of the virus isolation or protein-based detection assays discussed above. enhanced sensitivity is especially helpful when testing adults, who often shed virus in very low titers. positive rt-pcr tests need to be interpreted in the context of the clinical scenario, since the tests can remain positive for prolonged periods of time after infection, well beyond the period during which infectious virus can be isolated. since children may experience symptomatic respiratory infections every few weeks during the winter, caution must be used in interpretation of positive pcr tests, especially when multiple viruses are detected simultaneously in a sample. some instances of multiple pcr test positivity likely represent residual rna from a previous virus infection and a second rna type representing live virus from the active current infection. rsv typically is propagated in monolayer cell cultures of continuous cell lines of human epithelial cell origin, such as hep-2 cells. monkey kidney cells of various types are also used commonly for propagation in the laboratory. in fact, the virus replicates to some extent in most cell lines of mammalian origin. in non-polarized epithelial cells, the virus often causes a typical cytopathic effect in which multinucleated giant cells form due to cell-cell fusion, termed cell syncytia. this in vitro effect is the origin of the virus name, although it is not clear that rsv causes syncytia in vivo. in polarized epithelial cells in culture, the virus assembles and buds from the apical surface of cells, mimicking to some extent the budding of virus into the lumen of the airway. the virus has a negative-sense single-stranded rna genome with 10 genes encoding 11 proteins. figure 26 .1 compares the genomes of rsv and mpv, which are similar in many respects. the replication proteins are common to both of the viruses, as are the matrix (m) protein and the surface fusion (f) and glycosylated attachment (g) glycoproteins. the gene order differs slightly, and rsv possesses two nonstructural (ns) genes ns1 and ns2 that are not present in mpv. the functions of these genes are not fully defi ned, but involve interactions with the host response machinery, especially interferons. the presence of these host response-modifying genes may explain in part why rsv appears to cause severe disease more commonly than mpv. many of the gene sequences exhibit some clear global sequence relatedness; however, the extent of the relatedness of many of the sequences of corresponding proteins is relatively low. based on sequence homology, it is not expected that there is a signifi cant amount of immunologic cross-reactivity in responses to the two viruses. the rsv virion buds from airway epithelial cells, incorporating host cell membrane as the lipid bilayer that forms the envelope of the particle. since the virus is enveloped, chemicals that disrupt lipid bilayers (detergents) inactivate the virus, leading to the strong recommendations for healthcare provider hand washing following patient contact. the genome is a single strand of rna, which forms a helical complex with the nucleoprotein (n). the fi nal nucleocapsid structure likely is formed by the complete set of replication proteins, which also include the phosphoprotein (p) and the large rna-dependent rna polymerase (l). it is suspected that the m protein helps the particle to form by bridging the nucleocapsid and the lipid envelope with its incorporated surface proteins. the surface of the particle incorporates three integral membrane surface glycoproteins, the highly glycosylated rsv g protein suspected to be the attachment protein, the fusion protein f, and the small hydrophobic protein (sh). rsv f (a type i integral membrane protein) and rsv g (a type ii integral membrane protein) form oligomers on the surface of the particles, which appear like small spikes with globular heads when seen in electron microscopic (em) images by negative stain. the morphology of particles in em images or in fl uorescent microscopy images labeled by conjugated antibodies suggests that the virion particles are fi lamentous. however, spherical particles, fi laments, and more pleomorphic forms have been observed; therefore, it is uncertain what the morphology of infectious particles in vivo during natural infection is. the f protein is critical for entry into cells, by breaching the barrier of the cell lipid bilayer. it is thought that binding of virion particles to susceptible cells at physiologic ph triggers a conformational change of the f protein from a metastable pre-fusion state [ 25 ] to an altered post-fusion conformation [ 26 , 27 ] in which the hydrophobic fusion peptide in the protein is exposed and extended to insert into the host cell membrane. this membrane insertion event accomplishes a fusion of the viral and host membranes, allowing delivery of the nucleocapsid to the cytoplasm where viral replication occurs. this event is termed "fusion from without" when the particle enters a cell. an alternative fusion event ("fusion from within") occurs when newly expressed f protein on the surface of infected cells causes fusion of an infected cell to an adjacent cell in culture causing "syncytia." it is not certain whether this latter form of fusion (cell-cell fusion) occurs during natural infection and contributes to pathogenesis or if the formation of syncytia is a tissue culture phenomenon only. although there are many animal forms of rsv, there is no known animal reservoir of human rsv; close contact with infected humans is the only source of rsv infection. early prospective studies showed that approximately half of infants in the usa are infected during their fi rst year of life; most were infected after two winter epidemics [ 28 ] . about a quarter of infants exhibit signs and symptoms of lower respiratory tract disease (wheezing and/or pneumonia) during a primary rsv infection [ 14 , 28 -31 ] . rsv is the most common virus associated with hospitalization for respiratory illness and in fact is one of the most common of all causes of infant hospitalization. for example, rsv caused 13 hospitalizations per 1,000 us children younger than 1 year in one large study [ 32 ] . during winter rsv epidemics, over 75 % of the children hospitalized for acute lower respiratory tract infection are infected with rsv [ 33 , 34 ] . the rate of very severe disease in hospitalized infants is high, with about 2-5 % of hospitalized infants requiring mechanical ventilation for respiratory failure [ 35 ] . although primary infection of infants is probably most efficient, rsv can infect subjects of any age [ 28 , 36 , 37 ] . some adult infections are asymptomatic, and most are limited symptoms related to infection of the upper respiratory tract, such as the common cold [ 28 , 36 , 38 ] . since otherwise healthy adults all possess measurable rsv serum antibodies and rsv-specifi c t cells, it is clear that prior exposure and induction of immune responses may not prevent infection. however, disease severity is usually reduced after one or several infections early in life, thus immune effectors such as serum neutralizing antibodies do prevent severe disease during reinfections. unlike infl uenza virus, rsv does not exhibit a progressive antigenic drift. although rsv antigenic diversity is observed in fi eld strains, diversity of the antigenic proteins is not required for rsv to cause reinfection [ 39 -41 ] . most experts believe that serum neutralizing antibodies protect against severe lower respiratory tract disease but do not result in sterilizing immunity against infection at the respiratory mucosa. thus, healthy adults show signs and symptoms of the common cold in about half of cases of natural or experimental infections, even though they have experienced numerous previous rsv infections [ 39 ] . there is a single serotype of rsv, but two antigenic subgroups of rsv, with about 25 % antigenic relatedness, have been defi ned using immune sera; the subgroups are designated a and b. antigenic dimorphism for rsv had been noted in an early study [ 42 ] and subsequently was delineated using mabs, which identifi ed extensive differences in the g protein and less extensive differences in f and other proteins [ 43 ] . the two subgroups exhibit a three-to fourfold reciprocal difference in neutralization by polyclonal convalescent serum. analysis of glycoprotein-specifi c responses in experimental rodent models or human infants by enzymelinked immunosorbent assay (elisa) with purifi ed f and g glycoproteins showed that the f proteins are 50 % related antigenically and the g proteins are 1-7 % related [ 44 ] . consistent with this level of antigenic relatedness, f protein expressed by a vector immunization was equally protective in small animals against challenge with the homologous or heterologous subgroup virus, whereas the g protein was 13-fold less effective against the heterologous subgroup virus [ 45 ] . thus, the f protein is responsible for most of the observed cross-subgroup neutralization and protection. in some communities a pattern of infection with viruses of alternating subgroups has been described, but this is not a universal phenomenon. rsv subgroup b virus is more diffi cult to isolate and propagate in culture, so subgroup b viruses are less commonly associated with severe disease in some studies. however, clearly viruses of both subgroups can cause severe disease leading to hospitalization [ 46 -50 ] . infants exhibit the highest risk of severe lower respiratory tract disease. this elevated risk is explained by a myriad of physiologic, immunologic, and other factors. first, the highest point of resistance in the airways is the bronchioles, and the resistance of airways is inversely proportional to the airway radius to the fourth power (resistance ~ 1/radius 4 , from poiseuille's law). the bronchioles of infants are narrow, and infl ammation and secretion in the bronchioles leads to turbulent airfl ow, and further reduction of the airway lumen size. these physical factors lead to the clinical signs of wheezing (a sign of outfl ow obstruction) and air retention. increased respiratory rate can compensate to some extent for respiratory compromise, but prolonged tachypnea can lead to fatigue and eventual respiratory failure. also, during primary infection infants do not possess active immunity to infection, which allow this effi cient virus to replicate in the airway to titers as high as 10 7 infectious particles per ml of secretion. mothers pass rsv antibodies to infants across the placenta during the third trimester, but premature infants do not obtain maternal antibodies prior to 32 weeks' gestation. also, the airways of premature infants are smaller than those of term infants. another factor leading to respiratory diffi culty is dehydration. obstruction of the nasal passages with thick secretions can impede the feeding of infants, who are obligate nose breathers. rsv also is an important cause of serious infection in elderly adults; in fact rsv appears to cause substantially more fatalities in elderly adults than in children [ 17 , 51 ] . as above, a large study of the us population showed that rsv was associated with approximately 17,000 all-cause deaths per year among persons of all ages in the usa [ 9 ] , with most of those deaths in the elderly. virtually any serious medical or genetic condition is associated with some increased risk of severe diseases [ 32 ] . certain particular categories of subjects are at highest risk for severe rsv disease, including infants with congenital heart or chronic lung disease, immunodefi cient subjects of any age and the elderly [ 9 , 10 , 17 , 33 , 52 -57 ] . these latter subjects are thought to have reduced competency of rsv-specifi c t cells. prematurity increases risk to a small extent, but more importantly chronic lung diseases are important factors [ 10 , 33 , 56 , 58 -62 ] . infants who are born prematurely and then suffer persistent chronic lung disease, especially those needing oxygen supplementation, are at very high risk of hospitalization with rsv. children with cystic fi brosis are at high risk of severe disease [ 63 , 64 ] . in children younger than 2 years with cystic fi brosis, the consequence of rsv infection may be prolonged respiratory morbidity [ 65 ] . children with congenital heart disease also are at increased risk [ 33 , 56 , 58 , 62 , 66 , 67 ] . rsv is a common cause of severe respiratory disease in immunocompromised patients, including lower respiratory disease [ 68 ] . mortality rates in some populations of immunocompromised patients verges on 50 % [ 7 , 69 ] . infants with congenital severe combined immunodefi ciency are at special risk [ 57 , 70 ] , but any acquired immunosuppressive condition such as cancer or transplantation puts patients at risk, especially when t cell function is compromised [ 68 , 71 , 72 ] . rsv infection can cause severe lung disease in recipients of lung transplants, sometimes with a long-term outcome of obliterative bronchiolitis [ 73 -75 ] . children with hiv infection shed rsv for extended periods, but disease is not especially severe in hiv-infected children prior to onset of aids [ 76 -78 ] . interestingly, although most immunocompromised subjects appear to be at risk because of t cell problems, infants with phagocytic cell defects including those with interferon-γ receptor defi ciency or chronic granulomatous disease also are at risk of severe rsv disease. transmission of rsv in the hospital setting can lead to serious disease, especially in critical care units with neonates or other high-risk infants [ 79 -86 ] . nosocomial outbreaks in inpatient transplantation facilities are sometimes severe and unit outbreaks can be diffi cult to terminate because transplant patients can shed rsv for many months [ 57 , 58 , 69 , 84 , 87 , 88 ] . theoretically, transmission in inpatient healthcare settings should be preventable through strict compliance with infection control practices, especially hand washing and contact precautions, which are universally recommended for rsv patients. a high level of compliance with precautions is diffi cult to achieve in busy care settings but is needed to prevent transmission by healthcare providers. the use of the prophylactic monoclonal antibody palivizumab has been studied to interrupt an outbreak in a neonatal intensive care unit setting [ 89 ] , but currently palivizumab use is not recommended for this purpose. bacterial otitis media is a common complication of rsv upper respiratory tract infection. in fact, rsv infection is probably the most common precipitating factor associated with otitis media. rsv antigens and nucleic acids have been reported in middle ear fl uids [ 90 , 91 ] . the disease is predominantly due, however, to eustachian tube dysfunction, resulting in bacterial stasis in the middle ear and subsequent otitis media. rsv regularly occurs in annual epidemics. the us national respiratory and enteric virus surveillance system (nrevss) considers that the rsv season starts when the fi rst of two consecutive weeks during which the mean percentage of specimens testing positive for rsv antigen is ≥10 %. the rsv season is considered to have ended in a community when the mean percentage of positive specimens is ≤10 % in reference laboratories for two consecutive weeks. rsv infection occurs in infants and adults worldwide, in yearly epidemics. the virus has been isolated in every area of the world in which surveillance studies have been conducted. the principal season varies depending on the climate and region, but infection is ubiquitous. virtually all children in the world are infected within the fi rst few years of life. epidemics occur in the winter and early spring in the usa. onset of the annual epidemic varies by region of the country and by year but typically begins in the usa in october or november and lasts through late spring. within a region, the timing of rsv season changes slightly each year. florida often has the earliest onset of rsv epidemic and the longest lasting season in the usa. near the equator, infections may be more common during rainy season. during community outbreaks of rsv, the venues with the highest level of young infants and children exhibit the highest rates of transmission of virus, especially large families and day-care settings with large numbers of children per room. hospital infection control practices must be used to prevent rsv spread, using measures including careful hand hygiene, contact precautions for patient isolation, gowns and gloves [ 92 , 93 ] , and, when direct coughing occurs, facemasks and goggles [ 94 , 95 ] . rsv rapid diagnostic testing can be used in hospital infection control practice to identify rsv-infected patients during the admission process [ 96 , 97 ] . rsv is shed for prolonged periods. it has been reported that 92-100 % of hospitalized children are still shedding infectious virus after 7 days [ 98 , 99 ] . exposure to tobacco smoke and poor nutrition increase the incidence of disease [ 100 -102 ] . low socioeconomic status increases the risk of severe disease for uncertain reasons. lower income populations exhibit a fi ve-to tenfold increased risk of hospitalization in many studies [ 14 , 32 , 103 ]. breast-feeding may confer some protective benefi t against rsv disease, but the extent of the benefi t of breast-feeding has been controversial. the results of epidemiologic studies on benefi t have been confl icting, although recent studies suggest that breast-feeding may have a strong protective effect only in girls [ 104 ] . the sex of the infant modulates the severity of rsv infection for additional reasons that are not fully understood. even though the same high proportion of both male and female infants become infected, males have a higher incidence of rsv lower respiratory tract disease than girls [ 30 , 32 , 105 -108 ] . ethnic and genetic factors appear to play a role. alaska and other native american children are at increased risk of severe respiratory disease during rsv infection [ 109 ] . direct contact with secretions from an infected human, usually by fomites (contaminated objects, including hands), allows transmission of virus. in some cases, infected subjects may inoculate contacts at short distance by coughing via large-particle droplets. however, the virus is not spread efficiently by small-particle aerosols [ 110 , 111 ] . the nasal and conjunctival mucus membranes are probably the most common portals of entry [ 112 ] . rsv is one of the most infectious viruses spread by contact, and transmission is very effi cient even among subjects who possess partial immunity due to prior infection. spread among family members and day-care contacts is especially common. the infectious doses for humans are probably only a few infectious particles per ml of respiratory secretions, but infants routinely shed at least a millionfold higher concentration of virus during the peak of illness. the incubation period is not known defi nitely but is about 3-6 days and likely varies according to the intensity of exposure and the amount of virus in the inoculum. the period of viral shedding is many days; infants can shed infectious virus for weeks [ 98 , 99 ] . rsv can survive on hard surfaces for greater than 24 h. infection occurs by inoculation of the nasal or conjunctival mucosa, often by self-inoculation with infected secretions from a close contact. adult volunteers were infected experimentally if virus was inoculated onto the conjunctival sac or into the nose, but not following introduction into the mouth [ 112 ] . the incubation period from time of inoculation to onset of illness for rsv is likely about 4-5 days [ 113 ] . virus replication initiates in the nasopharynx and rapidly can reach concentrations of over a million particles per ml in the upper airway in children. adults, who are partially immune, typically shed much lower amounts of virus. virus spreads quickly to the lower respiratory tract, often causing symptoms within days of onset of upper respiratory symptoms. virus may spread from cell to cell, but also it is likely that small aspirations of upper respiratory secretions with virus inoculate the lower tract. higher titers of virus in respiratory secretions usually are associated with increased severity of disease, in prospective studies of natural infection [ 114 ] or of clinical vaccine trials [ 115 ] . rsv infection is an acute infection and virus shedding usually resolves within days to weeks. rt-pcr tests for virus nucleic acid may remain positive for prolonged periods. some animal studies suggest that a negligible amount of infectious virus may persist in airways far after apparent resolution of shedding, as evidenced by the recovery of low amounts of infectious virus during immunosuppression several months after infection [ 116 ] . the virus infects respiratory epithelial cells in the lung and airway. it is not clear whether rsv also replicates productively in macrophages and dendritic cells in the airway or not. rsv protein antigens have been detected in circulating mononuclear cells [ 117 ] , and viral genomic rna and mrna have been detected by rt-pcr in blood cells [ 118 ] , but this probably represents cells from the airway recirculating, as infectious virus in the blood (viremia) is not detected. rsv replicates in the cells at the luminal surface of the respiratory epithelium and virus both enters and is shed from the apical surface of infected epithelial cells [ 119 , 120 ] . studies of polarized, differentiated respiratory epithelial cells in vitro show that rsv infection preferentially infects ciliated cells at the luminal face [ 121 ] , but it is not clear if infection is restricted to such cells in humans. histopathology studies of infected humans are very limited, but show rsv antigens in the superfi cial cells of the airway in a patchy distribution, with antigen-positive cells and debris in the airway lumen. pathology caused by rsv infection during infant bronchiolitis includes necrosis and proliferation of the bronchiolar epithelium and destruction of ciliated epithelial cells [ 120 , 122 ] . there is an infl ux of a large variety of immune cell types including neutrophils, lymphocytes, and macrophages. the respiratory tissues become edematous. mucous secretion, sloughing of dead cell debris, and the infl ux of apparent infl ammatory cells obstruct the lumen of the narrow bronchioles and alveoli. the small diameter of infant bronchioles is easily obstructed in the presence of dead cells and edema of the airway tissues [ 123 ] . virus-infected cells can be identifi ed in the epithelium of the bronchi, bronchioles, and alveoli [ 120 ] . it is surprising, given the extent of disease, that rsv antigen staining is usually patchy or focal, and even in some cases of fatal rsv bronchiolitis, antigen is present only in small amounts [ 119 , 120 ] . the number of cases that have been collected is limited, however, and there is virtually no histopathology from milder cases. rsv upper respiratory infection is complicated frequently by otitis media caused by bacteria. it is unusual to observe frank bacterial pneumonia or sepsis as a complication of rsv infection, in contrast to some other respiratory viruses like infl uenza. although some clinicians may empirically use antibiotics during infant pneumonia, there is no evidence that antibiotic therapy alters the course of rsv bronchiolitis or pneumonia. antimicrobial therapy should not be used in most cases of rsv bronchiolitis or pneumonia because of the lack of benefi t and risk of selection of antibiotic-resistant colonizing organisms. nevertheless, there is some suggestion that bacterial-viral interactions may affect the overall rate of disease in the population. it is interesting that annual rsv and infl uenza virus epidemics correlate directly with the time of peak incidence of invasive pneumococcal disease in many population studies [ 124 ] . a double-blind, randomized, placebo-controlled trial of pneumococcal conjugate vaccine showed a vaccine-attributable reduction in rates of childhood viral pneumonia requiring hospitalization, caused by any of seven respiratory viruses, including rsv [ 125 ] . the immune mechanisms responsible for resolution of infection and protection against reinfection by rsv are not fully defi ned. antibodies . most experts agree that high levels of serum neutralizing antibodies are associated with relative protection against severe lower respiratory tract disease in otherwise healthy subjects. this idea is supported strongly by the observation that prophylaxis of high-risk infants with a neutralizing monoclonal antibody prevents about half of hospitalizations in that group of patients. many population studies suggest that infants born with high levels of transplacental rsv-neutralizing maternal antibodies develop milder illness or illness at an older age than infants with low maternal antibody levels [ 15 ] . most infants and children in whom maternal antibodies have declined to a low level make their own serum and secretory antibodies to both the f and g surface glycoproteins in response to rsv infection [ 126 ] . antibody responses in neonates are particularly low in quality and magnitude due to immunologic immaturity and the suppressive effect of passively acquired maternal antibodies [ 126 , 127 ] . antibody-mediated immune suppression by passive antibodies primarily affects humoral rather than cellmediated immunity [ 128 , 129 ] . high levels of serum antibodies do not appear to provide solid immunity against disease in the upper respiratory tract. mucosal secretory iga appears to contribute to local protection against reinfection in the airway, although potent protective iga responses are likely relatively short lived. in human infants, the decrease in virus shedding in nasal secretions was associated with the appearance of rsv-specifi c iga antibodies [ 130 ] . t cells . t cells clearly play a major role in resolution of active infection. rsv-specifi c t cells with cytolytic activity, thought to be cd8+ t cells, have been detected in peripheral blood mononuclear cells from infants with rsv disease [ 131 ] . immunodefi cient children, especially those with t cell defects, often fail to clear rsv infection and can shed virus for many months [ 57 ] . adults with leukemia or hematopoietic stem cell transplant also have a very high incidence of prolonged rsv infection leading to severe disease and sometimes death. patterns of host response rsv infection usually causes upper respiratory tract symptoms during primary infection in otherwise healthy term infants; asymptomatic primary infection is not common. there is often profuse rhinorrhea, and the upper tract disease is very often complicated by otitis media. symptoms of lower respiratory tract involvement occur in about a third of primary cases [ 28 ] . the principal diagnoses are bronchiolitis (manifested by tachypnea and wheezing) and pneumonia. these entities are probably not discrete processes but more likely represent a continuum of disease involving increasing tissue distribution. the typical illness starts with nasal congestion, followed in a few days by cough. the infection is sometimes associated with fever, which is usually low grade. after several days of upper tract symptoms, infants may wheeze. many infants suffer mild wheezing that resolves, but some cases progress with tachypnea, diffuse inspiratory crackles, and expiratory wheezes. most children recover in 1-2 weeks with supportive care and observation. if expiratory obstruction becomes severe, however, hyper-expansion of the chest occurs due to air retention, and the compliant nature of the infant chest wall leads to intercostal and subcostal retractions during tachypnea. with prolonged tachypnea, fatigue may occur with poor oxygenation and co 2 retention (measured by pulse oximeter or arterial blood gas measurement), markers of respiratory failure. intubation and mechanical ventilation is used in this setting to manage the respiratory failure. infection during the fi rst day or weeks of life may just be characterized by temperature instability or fever, irritability, and lethargy even in the absence of overt respiratory signs or symptoms. in very young infants, especially those born prematurely, apneic spells may occur in response to rsv infection. apnea may be the fi rst reported evidence of infection in some cases, and apneic spells may recur during the acute infection. these events thankfully are usually self-limited and rarely cause neurologic damage. apneic events are an indication for hospitalization and careful medical supervision with respiratory monitoring. because of the association with apnea, some have considered whether rsv is associated with sudden infant death syndrome (sids). although rsv has been detected in the lung tissues of some cases of sids, there is no statistically signifi cant association between rsv and sids. the reported cases likely refl ect a temporal association caused by the high prevalence of rsv in this age group, the prolonged pattern of shedding, and the simultaneous peak incidence of both rsv infection and sids in winter months [ 132 ] . it is not clear whether infection with rsv causes prolonged abnormal pulmonary function during childhood or whether children with underlying predisposition to lower respiratory tract disease of all causes manifest their susceptibility fi rst to rsv because of the young age of rsv infection. certainly measureable pulmonary function abnormalities are common after rsv lower respiratory tract disease, and these fi ndings may persist for a decade or more [ 133 ] . recurrent wheezing is common during subsequent viral infections after severe rsv bronchiolitis or pneumonia, with an incidence of 10-50 % [ 134 ] . a large case-control study of 200 children hospitalized for bronchiolitis or pneumonia in which rsv was the most common cause found that 7 years later there was a strong predisposition in these subjects toward decreased pulmonary function, recurrent cough, wheezing, and bronchitis [ 135 ] . seminal prospective studies by martinez et al. involved measurement of pulmonary function in infants at birth and then found a strong correlation between prior lower pulmonary function and the development of wheezing during rsv infection [ 136 ] . this correlation persisted in children during the fi rst 3 years of life [ 136 ] . even some individuals who do not typically exhibit recurrent wheezing have postexercise or pharmacologically induced bronchial reactivity [ 137 ] , which may be responsive in part to bronchodilators [ 134 ] . symptomatic upper respiratory tract rsv infections manifested by common cold symptoms are common in otherwise healthy adults, especially in those with frequent exposure to small children [ 80 ] . in the elderly, particularly those with underlying medical diseases, severe pneumonia may occur, leading to hospitalization or even death. astute clinicians can often make a presumptive diagnosis of infection based on the clinical signs of wheezing or pneumonia in an infant during a local epidemic. laboratory testing of nasal or lower airway secretions by antigen test (elisa) or nucleic acid detection (by rt-pcr) provides rapid diagnosis of the presence of virus in many cases. the gold standard for diagnosis is isolation of the virus in cell culture, but this test is typically only available in referral laboratories because of the need for extensive equipment and a high level of technical expertise. control and prevention primary treatment is supportive care, which includes oral or intravenous hydration; monitoring of respiratory status, especially of oxygen saturation during tachypnea; use of supplemental oxygen; removal of secretions from the upper airway; and, in the case of respiratory failure, intubation and mechanical ventilation. advances in support care in pediatric critical care units have caused a major decrease in morbidity and mortality from rsv in the developed world. infants hospitalized for rsv disease should be monitored for apnea. investigators have studied nitric oxide [ 138 , 139 ] mixtures of helium and oxygen [ 140 , 141 ] and surfactant treatment [ 142 ] in clinical experimental studies in the support of infants with severe rsv disease. nitric oxide treatment does not appear to mediate a bronchodilator effect during rsv infection [ 139 ] . therapy of rsv disease by any antiviral agent is challenging because it is a rapid acute infection, and by the time the onset of disease is recognized, it may be too late to alter the course of disease by reducing viral load. the guanosine (ribonucleic acid) analog ribavirin is a nucleoside inhibitor that inhibits viral rna synthesis and viral mrna capping. the drug has in vitro antiviral activity against rsv, and aerosolized ribavirin therapy has been associated with a small but statistically signifi cant increase in oxygen saturation during the acute infection in several small studies. decreases in mechanical ventilation and duration of rsv-associated hospitalization have not been proven (reviewed in [ 143 ] ). ribavirin was approved in 1986 in the usa for treatment of rsv infection [ 144 ] . clinically, the drug usually is administered as a small-particle aerosol using a tent, mask, or mechanical ventilator, delivered for 6-18 h daily for a period of 3-7 days. the drug now is not recommended for routine use because follow-up studies have not shown a major benefi t. the drug may be considered for use in select patients with documented, potentially life-threatening rsv infection. over a dozen other experimental small molecule inhibitors of rsv fusion to cells have been described and tested in preclinical studies for inhibition of rsv, but none have progressed in development to date. a third approach employs short interfering rnas (sirnas), taking advantage of an ancient host cell regulatory system. single-stranded and double-stranded rna molecules that exhibit rsv-specifi c small interfering rnas have been developed for treatment, which cause rna interference activity against rsv, destroying the corresponding rsv rna. these novel compounds have shown promising results in preclinical studies [ 145 ] and have been tested in small clinical trials. human immune globulin with a high titer of rsv antibodies and the rsv monoclonal antibody palivizumab have been tested as therapy of acute rsv disease, but they were not effective for treatment of established disease. anti-infl ammatory strategies have been investigated. no benefi t of corticosteroid therapy on disease severity or length of hospital stay has been demonstrated, despite studies in over a dozen randomized clinical trials of outpatients or hospitalized infants with rsv bronchiolitis. since the drug is of no benefi t on its own [ 146 ] and may prolong virus shedding, it is not recommended. in the future, a possibility might be to combine an effective antiviral treatment with an antiinfl ammatory agent [ 147 ] . intravenous antimicrobial therapy is not appropriate in hospitalized infants with rsv bronchiolitis or pneumonia unless there is clear evidence of secondary bacterial infection. otitis media occurs very often in infants with rsv bronchiolitis; oral antimicrobial agents can be used for therapy of otitis media if necessary. it is intuitive to think of using beta-adrenergic agents, commonly used for the treatment of asthma, to treat the wheezing associated with rsv infection. these agents are not usually recommended for routine care of fi rst-time wheezing associated with rsv bronchiolitis. short-term improvements in oxygenation and clinical scores can be achieved by these therapies, but it has not been established that their use results in improvements in duration or severity of illness or disease outcomes. studies in this area have been confl icting, but systematic reviews of randomized clinical trials of nebulized beta-agonist therapy for treatment of bronchiolitis suggest that they offer little benefi t [ 148 , 149 ] . alpha-adrenergic receptor stimulation results may decrease interstitial and mucosal edema [ 150 ] , and use of nebulized epinephrine (with combined alpha-and beta-adrenergic activity) has been studied with confl icting results [ 151 , 152 ] . alphaagonist stimulation of the sympathetic nervous system is expected to reduce capillary leakage by constricting precapillary arterioles, reducing hydrostatic pressure and consequently bronchial mucosal edema [ 150 ] . racemic epinephrine treatment relieves some respiratory distress but does not affect length of stay [ 153 ] . the usefulness of such agents in the management of rsv bronchiolitis is not clear. the most effective mode of prevention is to avoid contact with infected subjects. in the hospital setting, careful adherence to infection control practices is important for the protection of high-risk patients from rsv infection. careful hand washing may reduce transmission in family and daycare settings. pharmacologic intervention is indicated to prevent hospitalization for the highest risk infants, however. passive rsv immunoprophylaxis with antibodies has proven a costly but relatively effective intervention. parenteral infusion of rsv-neutralizing antibodies into experimental animals was shown early on to confer substantial resistance in the respiratory tract to a subsequent rsv virus challenge [ 154 ] . signifi cant reductions in rsv-associated hospitalizations and disease severity in high-risk human infants were fi rst accomplished with prophylactic administration of human immunoglobulin with high rsv-neutralizing activity given by the intravenous route (rsv-ivig; fda licensed in 1996) [ 155 , 156 ] . monthly intravenous infusions during the rsv season reduced the frequency of pediatric hospitalization and duration of stay by approximately 55 % and decreased the number of days spent in intensive care by 97 %. the use of rsv-ivig was superseded by the use of a monoclonal antibody (mab) that was developed subsequently that could be given by intramuscular route, and production of the former has been discontinued. several mabs were developed for immunoprophylaxis against rsv. the most successful of these was based on murine mab 1129 [ 157 ] , which is specifi c to the f protein and effi ciently neutralizes viruses of both rsv subgroups a and b. this mab was humanized by recombinant methods by transferring its variable regions onto a human igg1 backbone, resulting in a recombinant antibody now named palivizumab [ 158 ] . this mab is 50-100-fold more effective for in vitro neutralization on a per weight basis than was rsv-ivig, and thus the total amount of immunoglobulin administered could be reduced to an amount that could be given im. palivizumab (trade name synagis) was licensed in 1998 for rsv prophylaxis of high-risk infants, following studies demonstrating its safety and effi cacy [ 159 -162 ] . palivizumab is administered monthly through the rsv season and has been widely used in high-risk patients with prematurity, chronic lung disease, and hemodynamically signifi cant heart disease [ 163 ] . more potent derivatives of this recombinant antibody have now been developed [ 164 ] ; however, the lead candidate from these affi nity maturation efforts exhibited increased side effects in a large effi cacy study. prevention of severe disease probably will best be accomplished by development and use of an effective vaccine. vaccine development for rsv has proven exceptionally diffi cult, however. first, young infants are a diffi cult population to immunize. obstacles to immunization at this early age include immunologic immaturity and immunosuppression by maternal antibodies, as already noted [ 165 ] . also, severe adverse events occurred in early rsv vaccine trials. a formalin-inactivated rsv vaccine candidate (fi-rsv) was developed and evaluated in infants and children in the 1960s [ 166 , 167 ] . this vaccine suspension was made by mixing concentrated, inactivated virus with alum adjuvant and was delivered by the intramuscular (im) route. this inoculation did not protect against infection or disease, but rather during subsequent natural infection vaccinees experienced more frequent and severe disease. most fi-rsv vaccinees (80 %) required hospitalization during subsequent natural infection, compared to 5 % in the control group. autopsies of two fatalities showed evidence of rsv replication and pulmonary infl ammation [ 167 ] . this event put a chilling effect on rsv vaccine development efforts. therefore, rsv protein vaccines have been problematic for use in infants given their possible potential for disease enhancement, together with the poor immunogenicity in this population. however, an rsv protein vaccine might be useful in boosting immunity in rsv-experienced older children and adults who are at increased risk of severe rsv disease due to underlying disease or advanced age. protein vaccines for rsv have been evaluated clinically for use in such rsvexperienced individuals, in whom they appear to be safe. one experimental subunit vaccine consisted of purifi ed f protein (pfp) isolated from rsv-infected cell culture. this purifi ed protein vaccine candidate was evaluated in adults, in older children with and without underlying medical diseases, and in the elderly [ 168 ] . the pfp vaccine candidate was well tolerated and moderately immunogenic in these settings. a large multicenter study in children 1-12 years of age with cystic fi brosis did not provide evidence of signifi cant protection against rsv infection [ 169 ] . pfp also has been evaluated for maternal immunization in the third trimester of pregnancy. in the single study to date, the increase in antibody titers was only minimal [ 170 ] . maternal immunization studies are being pursued currently with newer non-replicating vaccine candidates such as emulsion vaccines [ 171 ] and nanoparticle protein preparations [ 172 , 173 ] . live-attenuated vaccines represent an attractive strategy for preventing rsv, since live infection induces a balanced immune response that is not associated with enhanced disease on subsequent natural infection. many live-attenuated rsv vaccine candidates have been developed over several decades. it has proven diffi cult to identify a candidate that is satisfactorily attenuated while remaining satisfactorily immunogenic in the youngest infants. clinical trials of a safe, live-attenuated rsv vaccine for intranasal administration have shown restriction of viral replication in infants following administration of a second dose and have been encouraging [ 174 ] , and additional attenuated vaccine candidates are being developed [ 175 ] . despite over 50 years of research on rsv, many challenges and questions remain. there are many unanswered fundamental questions about the biology of the organism and the pathogenesis of disease. why does reinfection occur throughout life? what are the defi nitive mechanisms of immunity in humans? is there a genetic basis for susceptibility to severe disease? does severe rsv disease cause asthma? what drives the seasonality of rsv? the mortality in infants has been greatly reduced in the usa through advances in critical care, but little rsv-specifi c intervention is available. currently, there is little to offer for therapy except for supportive care. prophylaxis of high-risk infant with a mab prevents some hospitalizations but is expensive and is not always effective. there are no licensed vaccines. given the disaster of early fi-rsv trials, it is not clear that a non-replicating vaccine can be proven safe enough in preclinical models to absolutely assure that enhanced disease will not occur. on the other hand, the explosion of new technologies for generation of recombinant rsv strains, the determination of pre-and post-fusion antigen structures, and new tools for the detailed study of the molecular and genetic basis of human immune responses suggest that much progress will be made in the rsv research fi eld in the coming years. mpv was discovered by investigators in the netherlands who cultivated specimens from children with respiratory infection on a variety of cell types [ 176 ] . a hitherto unknown virus produced cytopathic effects (cpe) in tertiary monkey kidney cells, but could not be identifi ed by antibody staining or rt-pcr for common viruses. electron microscopy of infected cells revealed pleomorphic enveloped particles with surface projections suggesting protein spikes, while biochemical experiments showed that the virus contained a lipid envelope and did not hemagglutinate avian or mammalian red blood cells. elegant randomly primed rt-pcr experiments yielded multiple fragments of genome, which sequence analysis identifi ed as related to avian metapneumovirus (ampv). ampv (formerly turkey rhinotracheitis virus), identifi ed in 1979, is an important global pathogen of poultry including turkeys and chickens [ 177 ] . there are four serotypes of ampv (a-d), and mpv is most closely related genetically to ampv-c [ 178 ] . phylogenetic analysis shows that mpv likely diverged from ampv-c ~200 years ago and thus mpv is of zoonotic origin [ 179 , 180 ] . however, mpv exhibits extremely restricted or no replication during experimental infection of chickens or turkeys and thus is a true human pathogen [ 176 ] . human poultry workers exhibit serological evidence of asymptomatic infection with ampv, providing evidence for the feasibility of an original trans-species transmission event [ 181 ] . while recently identifi ed, mpv is not truly a new virus. studies using archived sera collected in the 1950s revealed a 100 % seroprevalence in humans greater than 5 years old [ 176 ] . nasal washes collected prospectively during the 1970s from children with ari had detectable mpv rna upon retrospective testing by rt-pcr 25 years later [ 182 ] . the specialized cell culture requirements, slow growth, and limited cpe of mpv likely prevented the earlier discovery of this common respiratory pathogen. limited mortality data are available and consist of sporadic case reports, case series, or the identifi cation of fatal cases of mpv infection in research studies. mpv is not a reportable infection and there is no specifi c icd-9 diagnostic code. thus, an accurate estimate of the mortality associated with mpv is not feasible. however, lower respiratory infections are a leading cause of death in children worldwide, primarily in developing nations. mpv is a common cause of severe lower respiratory infection and thus likely accounts for a substantial number of deaths globally. a substantial body of literature has accumulated in the last decade describing the epidemiology, disease burden, and clinical features of mpv. many groups have used standard techniques to provide some estimate of the burden of mpv in diverse populations. most reports have been crosssectional studies of selected populations, usually based on convenience samples of patients with acute respiratory illness. these studies are thus limited by potential selection bias, narrow time periods, and incomplete demographic or clinical data and often lack controls. nonetheless, the broad application of these studies to sizable global populations has illuminated the frequency of mpv infection. many of these studies have focused on special populations, such as patients with asthma, immune compromise, or chronic obstructive pulmonary disease (copd), and thus offer valuable information about mpv among these persons. a number of prospective, well-designed studies in adults and children have been published and offer the best estimates of the population-based incidence of mpv infection. some of these used preexisting prospective data and samples collected prior to the discovery of mpv for retrospective analysis. classical methods including active day care and clinic surveillance, as well as newer approaches such as home surveillance with parent-collected swabs, have been used. these studies have been built upon the foundations of seminal longitudinal studies conducted to investigate other viruses including infl uenza, parainfl uenza viruses, and rsv. taken together, these reports provide a broad survey of mpv epidemiology across diverse geographic environments, socioeconomic populations, and high-risk groups. seroprevalence studies using diverse methods have been performed in different populations. most have used enzymelinked immunosorbent assay (elisa) techniques against whole virus or purifi ed proteins to detect igm or igg. a few have used immunofl uorescent detection of mpv-specifi c antibodies or measured serum virus-neutralizing antibodies. these data have mainly been useful in determining the ubiquity of infection with mpv. some studies have measured acute and convalescent sera to diagnose mpv infection, while others have attempted to establish a serum neutralizing titer that correlates with susceptibility to infection. inherent limitations of serological surveys include the potential for cross-reactive antibodies and the lack of standardized reagents for mpv. most studies have used rt-pcr to detect mpv due to the diffi culty in cultivating the virus. the original isolation of mpv in tertiary monkey kidney cells was possible because the investigator had access to a monkey kidney cell source that was free of the endogenous simian foamy virus (a.d.m.e. osterhaus, personal communication). primary monkey kidney cells commercially available in the usa all contain sfv, and even the addition of anti-sfv antisera cannot prevent the growth of this endogenous virus prior to the slow emergence of mpv. further, the fusion protein of mpv requires cleavage by exogenous trypsin for robust in vitro growth. trypsin is added by most clinical virology laboratories only to cultures of madin-darby canine kidney (mdck) cells for the isolation of infl uenza virus; however, mdck cells are very poorly permissive for mpv even in the presence of trypsin. finally, primary isolation of mpv often requires one or more passages prior to visible cpe and few laboratories routinely follow this procedure. unlike rsv, mpv is not particularly labile to freeze-thaw cycles [ 183 ] and thus can be retrospectively isolated from pcr-positive specimens. fluorescent antibody staining of patient specimens or shell vial cultures can facilitate more rapid identification [ 184 -187 ] . thus, molecular diagnostic techniques have been used for virtually all studies of mpv epidemiology. a number of sensitive and specifi c real-time rt-pcr assays have been described [ 188 -195 ] . many early assays were based on limited sequence data and subsequently were found to be suboptimal for detecting multiple diverse strains [ 195 ] . both individual and multiplexed rt-pcr assays offer more sensitive detection of mpv than culture; however, multiplex assays sometimes balance decreased sensitivity for a single agent with the convenience of detecting multiple viruses simultaneously [ 196 ] . another limitation of molecular detection for all viruses is the ability to detect low levels of viral nucleic acid in the absence of infectious virus. it has become common to detect more than one virus in a single specimen, and the interpretation of these data is far from clear. community respiratory viral infections are frequent in childhood, and the likelihood of detecting viral genome prior to the onset of illness or for prolonged periods after illness resolution complicates the assignment of causation to one of several co-detected viruses. mpv is an enveloped pleomorphic virus ranging in size from 150 to 600 nm, containing a single-stranded negative-sense rna genome [ 178 ] . complete genomic sequences of numerous mpv strains have been published [ 178 , 180 , 197 ] . the genome comprises eight separate open reading frames encoding nine distinct proteins (fig. 26.1 ) . mpv genes are analogous to those of rsv (though ns1 and ns2 are absent in mpv), but the organization of genes differs. ampv and mpv have been taxonomically classifi ed in the separate metapneumovirus genus based on the gene order. phylogenetic analysis of mpv genes consistently identifi es four genetic clades, two major groups designated a and b, each with two minor groups designated a1, a2, b1, and b2 [ 180 , 198 -203 ] . one group has suggested further sublineages based on partial f sequence diversity [ 204 ] , but there is no evidence that this further genetic distinction is of any antigenic or immunologic importance. the two major surface proteins are the fusion (f) and attachment (g), with a third integral membrane short hydrophobic (sh) protein. f is the target of neutralizing antibodies in animal models, f-only vaccines induce protection in animals, and f-specifi c monoclonal antibodies provide passive protection [ 205 -213 ] . in contrast, g-specifi c antibodies do not neutralize virus and g-only vaccines induce neither neutralizing antibodies nor protection [ 206 , 209 , 214 ] . thus, it appears that mpv is unique among human paramyxoviruses in that the attachment protein does not contribute to protective antibodies. further, the g protein exhibits a high degree of genetic variability between subgroups, with as low as 29 % amino acid identity between the major a and b subgroups and a minimum 60 % identity within subgroups [ 202 , 215 -218 ] . the selective pressure for this diversity is unclear. in contrast to g, the f protein is conserved, with a minimum 94 % amino acid identity between a and b subgroups and a minimum 98 % identity within subgroups [ 179 , 202 , 203 ] . presumably there are functional constraints on the diversity of f, since the mutation rate of mpv is high, similar to other rna viruses. the major question regarding the diversity between major or minor subgroups is whether it contributes to antigenic variation or escape in human populations. cross-neutralization against heterologous virus from the a and b lineages was tested using experimental infection of ferrets [ 202 ] . this study found relative neutralization of homologous to heterologous virus ranging from 12 to 96-fold difference, thus providing some evidence for antigenic serotypes. however, subsequent experiments using hamsters, african green monkeys, chimpanzees, and rhesus macaques found that the a and b groups were 64-99 % related antigenically [ 219 ] . infected animals developed neutralizing antibodies that were highly effective against heterologous virus, and previously infected primates were protected against challenge with heterologous virus. cynomolgus macaques infected with a or b subgroup viruses or with candidate vaccines exhibited only a 6-16-fold difference in neutralizing titer against homologous and heterologous viruses [ 220 , 221 ] . taken together, these data show that while mpv f exhibits some antigenic diversity, the virus does not have truly distinct serotypes. the potential implications for human epidemiology are discussed further below. descriptive epidemiology numerous studies document the fact that mpv infection is ubiquitous and that reinfection is common. serosurveys testing large sample collections in canada, china, croatia, germany, israel, japan, the netherlands, taiwan, thailand, the usa, and uruguay show that 95-100 % of children have antibodies against mpv by the age of 5 years [ 222 -231 ] . in many of these studies, 50-75 % of children are seropositive by age 2 years, suggesting that most acquire primary mpv infection early. most identify a decrease in serum mpv antibody titer from birth to 6-12 months, presumably due to the expected decline of maternally derived antibodies. studies in japan and india that compared mpv and rsv titers in the same cohort found that after the expected nadir during early infancy, rsv titers began increasing at an earlier age than mpv [ 232 , 233 ] . this fi nding is interesting in light of epidemiologic data suggesting that primary mpv infection peaks between 6 and 12 months of life compared with the peak of rsv at 2-3 months (discussed below). longitudinal studies in adults and children have documented reinfection by a fourfold rise in serum antibody titer [ 222 , 230 , 231 , 234 -237 ] . the serological data show that mpv infection is nearly ubiquitous during the fi rst years of life. further, reinfection occurs throughout life. in children, primary mpv infection is associated commonly with lower respiratory illness, while reinfection is associated with upper tract disease [ 182 , 238 ] . most epidemiologic studies of mpv in children show that the virus is the second leading cause of lower respiratory infection after rsv. the prevalence of mpv in studies of children with lri is 5-25 %. a 25-year prospective study of otherwise healthy children <5 years old detected mpv in 12 % of children with lri; several children experienced recurrent infection [ 182 ] . a 2-years multicenter study of inpatient and outpatient japanese children with ari identifi ed mpv in 57/637 (8.9 %) [ 239 ] . a 5-years observational study of otherwise healthy korean children <5 years old found mpv in 24/515 (4.7 %), similar to the rates for infl uenza and parainfl uenza virus type 3 (piv-3) [ 240 ] . a very large observational study in queensland, australia, tested specimens obtained from patients of all ages with lri from 2001 to 2004; mpv was detected in 707/10,025 (7.1 %). ninety-two percent of patients with mpv were <5 years old, and mpv was the second most common virus after rsv in these children [ 241 ] . a south african group tested specimens from children hospitalized with ari who were subjects in a prospective pneumococcal vaccine trial; mpv was present in 126/1,409 (8.9 %) and was the most common virus after rsv. the estimated incidence of mpv-associated hospitalization in hiv-negative children was 29/1,000; a number of children had repeat infections [ 242 ] . a prospective study of hospitalized children in hong kong over a 13-month period found mpv in 32/587 (5.5 %); the estimated incidence of mpv-associated hospitalization was 4.4 per 1,000 children <6 years old [ 243 ] . a chinese group conducted a prospective 2-years study of children hospitalized with ari and identifi ed mpv in 227/878 (25.9 %), with most <6 years old [ 244 ] . a large, population-based, prospective surveillance study conducted in three us cities over 6 years found that the incidence of hospitalization for mpv in children <5 years old was 1 per 1,000, lower than the rate of rsv-associated hospitalization in the same cohort (3/1,000) but similar to the rates for infl uenza (0.9/1,000) and piv-3 (0.5/1,000) [ 245 ] . respiratory disease is among the leading causes for hospitalization of adults in the usa, and "infl uenza and pneumonia" ranks among the top 10 causes of deaths annually. although the data are limited, mpv appears to be associated with a substantial burden of ari in adults, primarily those with comorbidities. a prospective study in rochester, ny, enrolled four cohorts during four winters: healthy adults >65, high-risk adults >65 with comorbidities, healthy adults 19-40 years old, and adults hospitalized for acute cardiopulmonary illness [ 246 , 247 ] . overall, mpv infection was detected in 8.5 % of ari in the cohort. the rate of mpv infection was highest in young adults at 13 %, though many of these were asymptomatic and detected only serologically. of note, this group had a mean age of 33, was predominantly female, and had daily exposure to children. of the hospitalized patients, the incidence of mpv annually ranged from 4.4 to 13.2 %. more than 85 % of the hospitalized mpvinfected subjects had underlying conditions, chiefl y cardiopulmonary disease or diabetes mellitus. there were six deaths in this study, all with comorbidities; one had concomitant bacteremia with streptococcus pneumoniae . interestingly, the incidence of mpv infection was similar to the annual average infection rate for rsv (5.5 %) and infl uenza a (2.4 %) in these cohorts during the same study period. a prospective, population-based study in nashville, tn, recruited adults hospitalized for ari at several county hospitals over three winters [ 248 ] . of 508 subjects, 23 (4.5 %) had mpv, 33 (6.5 %) infl uenza, and 31 (6.1 %) rsv. notably, mpv-infected subjects were signifi cantly older than infl uenza-infected subjects (mean 76 vs. 60 years) and had higher rates of chronic cardiopulmonary disease (78 % vs. 52 %). the overall population-based rates of hospitalization for the three viruses were similar, at 1/1,000 for mpv, 1.5/1,000 rsv, and 1.2/1,000 for infl uenza. however, for subjects ≥65 years, hospitalization rates were much higher for mpv and rsv at 2.2/1,000 for mpv and 2.5/1,000 for rsv compared to 1.2/1,000 for infl uenza, likely refl ecting the use of infl uenza vaccine for older adults. a prospective study of community-acquired pneumonia in canada found mpv in 4 % of hospitalized cases during an 18-month period, all with underlying conditions [ 249 ] . a dutch group detected mpv in 2 % of bronchoalveolar lavage specimens from intensive care unit patients. all were >50 years old with comorbid conditions and 83 % died [ 250 ] . similarly, a retrospective study in north ireland found mpv in only 0.8 % of residual respiratory specimens from adults, but 33 % of these died [ 251 ] . together, these data show that mpv is an important cause of acute respiratory disease in adults, primarily older adults or those with underlying comorbid conditions. mpv is associated with acute asthma exacerbations [ 252 -254 ] . mpv was detected in 10 of 132 hospitalized finnish children with acute wheezing [ 255 ] . similarly, mpv was isolated from 7 % of adults hospitalized for acute asthma exacerbation, only one of whom tested positive 3 months later [ 256 ] . premature infants who developed mpv bronchiolitis within the fi rst year of life had decreased lung function at 1 year of age [ 257 ] . a prospective, case-control study of children with mpv bronchiolitis during infancy compared to infants with acute gastroenteritis found that mpv infection early in life was signifi cantly associated with a later diagnosis of asthma and recurrent wheezing at 5 years of age [ 258 ] . mpv causes severe disease in children with comorbid conditions such as cardiac and pulmonary disease or prematurity [ 259 -263 ] . a prospective 1-year study of hospitalized children found that 34 % of patients with mpv had a history of prematurity, chronic lung disease, complex congenital heart disease, or immunodefi ciency [ 264 ] . vicente et al. detected mpv by rt-pcr in 6 % of adults >64 years old with acute exacerbations of copd; none had other pathogens identifi ed by culture or pcr [ 265 ] . a canadian study found that 4 % of hospitalized adults with communityacquired pneumonia or copd exacerbations tested positive for mpv, all with comorbid conditions; one also had infl uenza a and s. pneumoniae [ 266 ] . rsv was present in 9 % and infl uenza a in 6 % of the cohort. mpv was detected in 12 % of adults hospitalized for copd exacerbation during one winter in connecticut, none with other viruses codetected; rsv was present in 8 % and infl uenza a in 4 % of the entire cohort [ 267 ] . severe and fatal mpv disease can occur among immunocompromised individuals, including solid organ and stem cell transplant recipients, hiv-infected persons, and chemotherapy patients [ 268 -275 ] . mpv is associated with morbidity and mortality in adults with hematologic malignancies and stem cell transplant; [ 270 , 276 ] mpv was detected in bronchoalveolar lavage specimens from 5/163 (3 %) episodes of acute respiratory infection in stem cell transplant recipients, and four died [ 270 ] . hiv-positive south african children with mpv were signifi cantly more likely to receive a diagnosis of pneumonia and experience longer hospitalization, lower mean oxygen saturation, and bacteremia; further, hiv-positive children were fi vefold more likely to be infected with mpv than hiv-negative children [ 242 ] . mpv has been implicated in several hospital and institutional outbreaks leading to mortality [ 277 , 278 ] . kim and colleagues [ 279 ] report the transmission of mpv to pediatric hematology-oncology patients during a nosocomial outbreak. the incubation period was between 7 and 9 days. standard, but not droplet, precautions were used. in laboratory studies, infectious virus persists on metal and nonporous surfaces for up to 8 h [ 183 ] . due to the signifi cant morbidity and mortality of mpv in high-risk children, isolation precautions are important. mpv is associated with both upper and lower respiratory tract disease [ 182 , 280 -282 ] . rhinorrhea and cough are the most frequent symptoms, while hoarseness, laryngitis, sore throat, and croup are less common [ 238 -241 , 243 , 282 ] . a large prospective study of children with uri detected mpv in 5 % of patients, similar to rsv, infl uenza, and piv but less frequent than adenovirus and rhinovirus. in these children with uri associated with mpv, fever was present in 54 %, coryza in 82 %, cough in 66 %, pharyngitis in 44 %, hoarseness in 8 %, and conjunctivitis in 3 % [ 280 ] . mpv is associated with acute otitis media and has been detected in nasal secretions and middle ear fl uid [ 182 , 280 , 283 -285 ] . signs and symptoms of lri with mpv include cough, wheezing, and rhonchi. a large chinese study of children with acute respiratory infections found that wheezing was more common in children with mpv than with rsv [ 282 ] . in that study, children with mpv were diagnosed with pneumonia signifi cantly more than children with rsv, 47 % versus 31 % ( p = 0.002). conversely, a larger percentage of children with rsv were diagnosed with bronchiolitis compared to mpv, 62 % versus 42 %, respectively ( p < 0.001). other studies also note the trend toward a higher percentage of children with mpv and pneumonia compared to rsv [ 182 , 238 , 245 ] although this is not always statistically signifi cant [ 243 ] . mpv has been associated rarely with neurologic complications, including febrile seizures and altered mental status, but there is no conclusive evidence for direct neural infection. one case report describes a patient who died and had mpv isolated by rt-pcr from brain and lung tissues [ 286 ] . mpv was detected by rt-pcr in nasal specimens from 4 of 1,570 persons with encephalitis of unknown etiology [ 287 ] . several other reports describe the detection of mpv in a respiratory specimen from patients with encephalitis [ 286 , 288 -290 ] . only one case reports detection of mpv in cerebrospinal fl uid [ 291 ] . the incidence of viral infection varies between countries and years, but mpv circulates in every year [ 176 , 182 , 239 -241 , 243 , 282 , 292 -296 ] . epidemiologic studies have verifi ed the presence of mpv worldwide. mpv is present during all months in temperate regions, although predominant in late winter-early spring, often following the peak of rsv (fig. 26.2 ) [ 176 , 182 , 191 , 239 , 241 , 246 , 264 , 280 , 282 , 294 , 297 ] . in subtropical climates such as hong kong, a springsummer season similar to rsv occurs [ 243 ] . biannual peaks of seasonality have been described in some european studies [ 298 , 299 ] . annual rates of mpv-associated ari are lower than rsv and comparable to parainfl uenza virus types 1-3 combined and infl uenza [ 240 , 241 , 243 , 282 , 294 , 300 ] although mpv does on occasion surpass rsv in incidence [ 293 ] . multiple outbreaks have been reported in nursing homes and other long-term care facilities (ltcf). mpv was the only pathogen identifi ed in an outbreak of ari that occurred over a 6-weeks period at a quebec ltcf, with 6 pcr-confi rmed and 96 epidemiologically linked probable cases. there were three deaths among the confi rmed and nine deaths among the probable cases [ 278 ] . a california study described an outbreak of ari in 26/148 (18 %) residents and, importantly, 13 staff of a ltcf; mpv was confi rmed in 5 residents, 2 of whom were hospitalized, and no other viruses were detected in any case [ 301 ] . attendance in out-of-home day care, breast-feeding, and passive smoke exposure are not signifi cantly associated with mpv infection [ 245 ] . mpv infection is not associated with lower socioeconomic status. viral coinfection has been suggested with mpv; dual infection with rsv and mpv increased the risk of picu admission compared to rsv alone in one small study [ 302 ] . however, this fi nding was refuted by subsequent larger reports [ 303 -305 ] . other studies demonstrate no signifi cant difference in children with coinfections, including adenovirus, bocavirus, coronavirus, infl uenza virus, parainfl uenza viruses, or rsv [ 240 , 241 , 282 , 293 , 306 , 307 ] . mpv has four distinct genetic lineages or subgroups: a1, a2, b2, and b2 [ 202 , 203 , 218 ] . the predominant subtype varies by year and location [ 241 , 280 , 293 , 297 , 308 ] . in italy, over a 3-years period, all four subtypes were identifi ed each season but the predominant subtype changed. in 2001-2002, a1 accounted for 59 % of all strains, the following year b1 and b2 were present equally, and in 2003-2004, 72 % of strains were a2 [ 308 ] . similar variation was observed over 20 years in a us study, with multiple subgroups present in most seasons (fig. 26. 3 ) [ 280 ] . it is unclear whether viruses from different subgroups differ in virulence. a study in spain reported that children with group a infection more frequently had pneumonia and higher disease severity [ 309 ] , while in canada, group b was associated with more severe disease in hospitalized patients [ 261 ] . patients with group b strains in a french study were more likely to have abnormal chest radiographs but did not have signifi cant differences in oxygen saturation, hospitalizations, or clinical severity scores [ 310 ] . other studies have found no major distinctions in disease severity [ 239 , 241 , 2 0 0 1 2 0 0 0 1 9 9 9 1 9 9 8 1 9 9 7 1 9 9 6 1 9 9 5 1 9 9 4 1 9 9 3 1 9 9 2 1 9 9 1 1 9 9 0 1 9 8 9 1 9 8 8 1 9 8 7 1 9 8 6 1 9 8 5 1 9 8 4 1 9 8 3 1 9 8 2 311 ], laboratory abnormalities [ 228 ] , or symptoms [ 240 ] between subgroups. group a viruses replicate more efficiently in animal models, suggesting some meaningful biological differences between groups [ 219 , 220 ]. mpv is an enveloped virus and thus inactivated by soap, disinfectants, or alcohols. spread is thought to occur by direct or close contact with contaminated secretions. infectious virus can persist at room temperature, especially nonporous surfaces, for up to 8 h [ 183 ] . contact precautions are recommended as for rsv with meticulous hand hygiene. cohorting of patients and caregivers should be considered during outbreaks in care facilities. human data are limited; studies in rodents and nonhuman primates reveal mild erosive and infl ammatory changes in the mucosa and submucosa of the airways, with viral replication observed in ciliated epithelial cells in the respiratory tract [ 312 -314 ] . infl ammatory infi ltrates with a lymphocytic and monocytic predominance are present in perivascular and peribronchial areas. sumino and colleagues [ 315 ] reviewed the lung pathology of fi ve adults with mpv infection. histopathology in three patients demonstrated acute, organizing lung injury with diffuse alveolar membrane formation and the presence of smudge cells. the fourth patient had no evidence of lower respiratory tract infection, and the fi fth patient had nonspecifi c acute and chronic infl ammation. a similar study in children revealed chronic infl ammatory changes of the airways with intra-alveolar macrophages [ 316 ] . a major limitation of reports of human pathology is that patients had been mechanically ventilated prior to death, making it diffi cult to distinguish virus-induced pathology from barotrauma and nonspecifi c infl ammation. mpv lacks genes present in other paramyxoviruses that inhibit interferon responses; nevertheless, mpv is capable of blocking type i interferon responses by an unknown mechanism [ 317 , 318 ] . mpv and other respiratory viruses induce pulmonary cd8+ t cells that fail to secrete ifnγ or exhibit cytotoxic degranulation in response to viral peptides; these impaired cd8 t cells resemble exhausted cd8 t cells induced by chronic infections such as hiv and hepatitis c virus [ 319 ] . humans develop neutralizing antibodies to mpv, and passive antibodies alone can protect in animal models. however, immunity wanes over time and likely provides limited cross-protection between subgroups, since reinfections occur in children and adults [ 246 , 248 ] with genetically different strains [ 182 , 239 , 240 , 268 , 320 ] as well as strains from the same subgroup [ 280 ] . early protection against reinfection following primary infection was confi rmed in macaques; however, when challenged 12 weeks later, virus replication was detectable despite the presence of serum antibodies [ 220 ] . eleven months later, antibody levels had waned still further, and all macaques challenged with heterologous virus and two of three animals reinfected with homologous virus had no evidence of protection [ 220 ] . a prospective study in humans noted that baseline mpv antibodies were lower in patients who subsequently became infected versus those who did not become infected [ 321 ] . thus, cross-protection and duration of antibody responses are important issues for vaccine development. patterns of host response mpv causes both upper and lower respiratory tract signs and symptoms clinically indistinguishable from disease associated with rsv and other respiratory viruses [ 182 , 239 , 300 , 322 , 323 ] . fever is present in most cases, especially children [ 239 -241 , 243 , 264 , 280 , 293 ] . transient maculopapular rash has been described in a minority of patients [ 241 , 243 , 293 ] , and vomiting or diarrhea are described with low frequency [ 182 , 239 , 243 ] . laboratory abnormalities are uncommon, although one study identifi ed 27 % of patients with elevated alt and ast values [ 293 ] . white blood cell count and c-reactive protein were not signifi cantly different between rsv and mpv [ 238 , 282 ] . mpv is rarely detected in asymptomatic persons [ 182 , 191 , 324 -326 ] , though in otherwise healthy young adults, mpv infection can be subclinical [ 246 ] . the duration of shedding in healthy individuals is approximately 7-14 days [ 239 , 327 ] . detection in cell culture requires prolonged incubation and is both insensitive and often impractical. shell vial culture offers increased sensitivity over traditional culture [ 184 , 328 ] . ifa has demonstrated a sensitivity of 73 % and specifi city of 97 % with rt-pcr as the gold standard [ 329 ] . dfa has shown similar results [ 330 ] . commercial antibody kits for immunofl uorescent detection of mpv are available. rt-pcr is most commonly used for detection of the virus in epidemiologic studies and is becoming more common in clinical laboratories [ 176 , 320 , 331 , 332 ] . real-time rt-pcr targeting the conserved n gene has high sensitivity for detection of all four subgroups [ 189 , 195 ] . control and prevention the primary therapy for mpv infections is supportive care, including oral or intravenous hydration, monitoring of respiratory status and oxygen saturation, supplemental oxygen, and mechanical ventilation for frank respiratory failure. there are no licensed antivirals for mpv. reports of pharmacologic treatment of mpv are limited to severely ill or immunocompromised patients. ribavirin, an antiviral agent used in severe rsv infection, reduced infl ammation and viral replication in mice with mpv infection [ 333 ] . commercial intravenous immunoglobulin (ivig), ribavirin, and nmso3 (a sulfated sialyl lipid) effectively inhibited mpv in vitro [ 334 , 335 ] . ribavirin, with and without ivig, has been used in immunocompromised adults [ 336 ] . bonney and colleagues [ 337 ] reported successful treatment of an immunocompromised child with mpv using iv ribavirin and ivig. subsequently, oral ribavirin and inhaled ribavirin with ivig have been used [ 275 , 338 ] . however, no randomized, controlled trials have been conducted, and these data should be regarded as purely anecdotal. human and murine monoclonal antibodies exhibit therapeutic effi cacy in rodent models and thus offer potential for immunoprophylaxis [ 210 , 211 , 213 ]. a number of vaccine approaches for mpv have been investigated. the f protein is conserved between subgroups, immunogenic, and the only target of neutralizing antibodies; in contrast to rsv, the mpv g protein does not induce neutralizing antibodies and is not a protective antigen [ 206 , 209 , 214 ] . a recombinant parainfl uenza virus encoding the mpv f protein demonstrated protection against mpv [ 339 ] , and soluble f protein vaccines reduced viral titers in cotton rats and hamsters [ 207 , 208 ] . reverse genetics technology has been developed for mpv and has been used to produce recombinant strains for vaccine development [ 197 , 340 ] . viruses lacking the g, m2-1, m2-2, or sh proteins or with point mutations are attenuated and immunogenic in rodent and primate models [ 197 , 221 , 341 -344 ]. the major unresolved problems in mpv epidemiology and research involve understanding mechanisms of disease and developing therapeutic or preventive strategies. abundant evidence shows that mpv is a signifi cant cause of acute respiratory disease, especially in the very young, older adults, and persons with underlying conditions. as with all mucosal viruses, the short incubation period of mpv com-bined with the transient nature of mucosal iga means that reinfection is possible throughout life. however, serum igg appears to offer protection against severe lower respiratory involvement, and thus a vaccine would likely ameliorate the most severe cases. the best candidate vaccine is not yet clear. further, mpv has as yet unidentifi ed mechanisms to subvert host immunity. elucidation of these pathways might help guide vaccine development and uncover novel host targets for immunomodulation. recovery from infants with respiratory illness of a virus related to chimpanzee coryza agent (cca). ii. epidemiologic aspects of infection in infants and young children recovery from infants with respiratory illness of a virus related to chimpanzee coryza agent (cca). i. isolation, properties and characterization respiratory syncytial virus is an important cause of community-acquired lower respiratory infection among hospitalized adults respiratory syncytial virus infections in adult bone marrow transplant recipients respiratory syncytial virus-induced acute lung injury in adult patients with bone marrow transplants: a clinical approach and review of the literature severe respiratory syncytial virus pneumonia after autologous bone marrow transplantation: a report of three cases and review an outbreak of respiratory syncytial virus in a bone marrow transplant center bronchiolitis-associated mortality and estimates of respiratory syncytial virus-associated deaths among us children, 1979-1997 mortality associated with infl uenza and respiratory syncytial virus in the united states respiratory syncytial virus infection in children with bronchopulmonary dysplasia respiratory syncytial virus infection in children with congenital heart disease: a review community respiratory virus infections among hospitalized adult bone marrow transplant recipients twenty years of outpatient respiratory syncytial virus infection: a framework for vaccine effi cacy trials epidemiology of respiratory syncytial virus infection in washington, d.c. i. importance of the virus in different respiratory tract disease syndromes and temporal distribution of infection risk of respiratory syncytial virus infection for infants from low-income families in relationship to age, sex, ethnic group, and maternal antibody level severe respiratory syncytial virus disease in alaska native children. rsv alaska study group respiratory syncytial virus infection in elderly and high-risk adults detection of respiratory syncytial virus in adults with chronic obstructive pulmonary disease respiratory viruses in exacerbations of chronic obstructive pulmonary disease requiring hospitalisation: a case-control study comparison of respiratory syncytial virus humoral immunity and response to infection in young and elderly adults respiratory syncytial virus-specifi c cd8+ memory t cell responses in elderly persons purifi cation and characterization of gp90, one of the envelope glycoproteins of respiratory syncytial virus evaluation of direct immunofl uorescence, enzyme immunoassay, centrifugation culture, and conventional culture for the detection of respiratory syncytial virus r-mix cells are faster, at least as sensitive and marginally more costly than conventional cell lines for the detection of respiratory viruses structure of rsv fusion glycoprotein trimer bound to a prefusion-specifi c neutralizing antibody structural basis for immunization with postfusion respiratory syncytial virus fusion f glycoprotein (rsv f) to elicit high neutralizing antibody titers structure of respiratory syncytial virus fusion glycoprotein in the postfusion conformation reveals preservation of neutralizing epitopes risk of primary infection and reinfection with respiratory syncytial virus composite analysis of eleven consecutive yearly epidemics infection and disease with respect to age, immunologic status, race and sex respiratory syncytial virus and parainfl uenza virus population-based surveillance for hospitalizations associated with respiratory syncytial virus, infl uenza virus, and parainfl uenza viruses among young children griffi n mr. rates of hospitalization for respiratory syncytial virus infection among children in medicaid bronchiolitis-associated hospitalizations among us children respiratory syncytial virus-coded pediatric hospitalizations respiratorysyncytial-virus infections, reinfections and immunity. a prospective, longitudinal study in young children respiratory syncytial virus infections within families respiratory syncytial virus infections in previously healthy working adults antigenic and genetic diversity among the attachment proteins of group a respiratory syncytial viruses that have caused repeat infections in children pattern of respiratory syncytial virus epidemics in finland: two-year cycles with alternating prevalence of groups a and b subgroup characteristics of respiratory syncytial virus strains recovered from children with two consecutive infections an antigenic analysis of respiratory syncytial virus isolates by a plaque reduction neutralization test antigenic characterization of respiratory syncytial virus strains with monoclonal antibodies strain-specifi c serum antibody responses in infants undergoing primary infection with respiratory syncytial virus the g glycoprotein of human respiratory syncytial viruses of subgroups a and b: extensive sequence divergence between antigenically related proteins clinical severity of respiratory syncytial virus group a and b infection in sydney, australia occurrence of groups a and b of respiratory syncytial virus over 15 years: associated epidemiologic and clinical characteristics in hospitalized and ambulatory children severity of respiratory syncytial virus infection is related to virus strain variation in severity of respiratory syncytial virus infections with subtype distribution and clinical impact of human respiratory syncytial virus genotypes in hospitalized children over 2 winter seasons rsv infection-not for kids only impact of respiratory syncytial virus infection on surgery for congenital heart disease: postoperative course and outcome variable morbidity of respiratory syncytial virus infection in patients with underlying lung disease: a review of the picnic rsv database. pediatric investigators collaborative network on infections in canada rehospitalization for respiratory syncytial virus among premature infants fatal respiratory syncytial virus infection in severe combined immunodefi ciency syndrome respiratory syncytial viral infection in infants with congenital heart disease respiratory syncytial viral infection in children with compromised immune function improved outcome of respiratory syncytial virus infection in a high-risk hospitalized population of canadian children. pediatric investigators collaborative network on infections in canada breast-feeding and health in the 1980s: a global epidemiologic review rehospitalization with respiratory syncytial virus after neonatal intensive care unit discharge: a 3-year follow-up picnic (pediatric investigators collaborative network on infections in canada) study of the role of age and respiratory syncytial virus neutralizing antibody on respiratory syncytial virus illness in patients with underlying heart or lung disease rehospitalisations for respiratory disease and respiratory syncytial virus infection in preterm infants of 29-36 weeks gestational age effects of acute viral respiratory tract infections in patients with cystic fi brosis role of respiratory syncytial virus in early hospitalizations for respiratory distress of young infants with cystic fi brosis effects of viral lower respiratory tract infection on lung function in infants with cystic fi brosis respiratory syncytial virus morbidity and mortality estimates in congenital heart disease patients: a recent experience physiologic risk factors for respiratory viral infections and immunoprophylaxis for respiratory syncytial virus in young children with congenital heart disease respiratory viral infections in immunocompetent and immunocompromised persons community respiratory virus infections in bone marrow transplant recipients: the m.d. anderson cancer center experience ribavirin treatment of viral pneumonitis in severe combined immunodefi ciency disease respiratory virus infections after marrow transplant: the fred hutchinson cancer research center experience community respiratory viruses: organ transplant recipients community respiratory viral infection in adult lung transplant recipients respiratory syncytial virusassociated infections in adult recipients of solid organ transplants infectious complications in heart-lung transplantation. analysis of 200 episodes respiratory syncytial virus infection in human immunodeficiency virus-infected children differing manifestations of respiratory syncytial virus-associated severe lower respiratory tract infections in human immunodeficiency virus type 1-infected and uninfected children increased burden of respiratory viral associated severe lower respiratory tract infections in children infected with human immunodefi ciency virus type-1 nosocomial respiratory syncytial virus infections: the "cold war" has not ended nosocomial respiratory syncytial virus infections control of nosocomial respiratory syncytial viral infections neonatal respiratory syncytial virus infection nosocomial respiratory syncytial virus infection in canadian pediatric hospitals: a pediatric investigators collaborative network on infections in canada study nosocomial transmission of respiratory syncytial virus in immunocompromised adults rsv outbreak in a paediatric intensive care unit medical and economic impact of a respiratory syncytial virus outbreak in a neonatal intensive care unit maternal immunization against viral disease respiratory syncytial virus infections in pediatric liver transplant recipients use of palivizumab to control an outbreak of syncytial respiratory virus in a neonatal intensive care unit presence of respiratory syncytial virus genomic sequences in middle ear fl uid and its relationship to expression of cytokines and cell adhesion molecules identifi cation of respiratory virus antigens in middle ear fl uids of children with acute otitis media prospective controlled study of four infection-control procedures to prevent nosocomial infection with respiratory syncytial virus prevention of nosocomial respiratory syncytial virus infections through compliance with glove and gown isolation precautions respiratory syncytial virus (rsv) infection rate in personnel caring for children with rsv infections. routine isolation procedure vs routine procedure supplemented by use of masks and goggles the use of eye-nose goggles to control nosocomial respiratory syncytial virus infection rapid identifi cation of respiratory viruses: impact on isolation practices and transmission among immunocompromised pediatric patients nosocomial respiratory syncytial virus infections: prevention and control in bone marrow transplant patients respiratory syncytial virus infections in infants: quantitation and duration of shedding quantitative shedding patterns of respiratory syncytial virus in infants environmental and demographic risk factors for respiratory syncytial virus lower respiratory tract disease casecontrol study of the risk factors linked to respiratory syncytial virus infection requiring hospitalization in premature infants born at a gestational age of 33-35 weeks in spain the pediatric investigators collaborative network on infections in canada study of predictors of hospitalization for respiratory syncytial virus infection for infants born at 33 through 35 completed weeks of gestation respiratory syncytial virus in infants and children differential gender response to respiratory infections and to the protective effect of breast milk in preterm infants risk factors for respiratory syncytial virusassociated lower respiratory illnesses in the fi rst year of life epidemiologic patterns of acute lower respiratory disease of children in a pediatric group practice epidemiology of acute lower respiratory disease in children clinically useful method for the isolation of respiratory syncytial virus bronchiolitis-associated hospitalizations among american indian and alaska native children modes of transmission of respiratory syncytial virus possible transmission by fomites of respiratory syncytial virus infectivity of respiratory syncytial virus by various routes of inoculation an outbreak of febrile illness and pneumonia associated with respiratory syncytial virus infection respiratory syncytial virus load predicts disease severity in previously healthy infants illness severity, viral shedding, and antibody responses in infants hospitalized with bronchiolitis caused by respiratory syncytial virus latency and persistence of respiratory syncytial virus despite t cell immunity respiratory syncytial virus infection of human mononuclear leukocytes in vitro and in vivo respiratory syncytial virus rna in cells from the peripheral blood during acute infection speculation on pathogenesis in death from respiratory syncytial virus infection demonstration of respiratory syncytial virus in an autopsy series respiratory syncytial virus infection of human airway epithelial cells is polarized, specifi c to ciliated cells, and without obvious cytopathology pathological changes in virus infections of the lower respiratory tract in children age as a factor in the distribution of lower-airway conductance and in the pathologic anatomy of obstructive lung disease seasonality of invasive pneumococcal disease: temporal relation to documented infl uenza and respiratory syncytial viral circulation vaccine trialist g. a role for streptococcus pneumoniae in virus-associated pneumonia effect of age and preexisting antibody on serum antibody response of infants and children to the f and g glycoproteins during respiratory syncytial virus infection passive transfer of respiratory syncytial virus (rsv) antiserum suppresses the immune response to the rsv fusion (f) and large (g) glycoproteins expressed by recombinant vaccinia viruses microplaque immunoperoxidase detection of infectious respiratory syncytial virus in the lungs of infected mice infl uence of maternal antibodies on vaccine responses: inhibition of antibody but not t cell responses allows successful early prime-boost strategies in mice the immunologic response to infection with respiratory syncytial virus in infants type 1-like immune response is found in children with respiratory syncytial virus infection regardless of clinical severity detection of respiratory syncytial virus nucleic acid in archival postmortem tissue from infants effect of pneumonia in childhood on adult lung function association of radiologically ascertained pneumonia before age 3 yr with asthmalike symptoms and pulmonary function during childhood: a prospective study outcome of acute lower respiratory tract infection in infants: preliminary report of seven-year follow-up study initial airway function is a risk factor for recurrent wheezing respiratory illnesses during the fi rst three years of life. group health medical associates study of 8-year-old children with a history of respiratory syncytial virus bronchiolitis in infancy treatment of respiratory failure with inhaled nitric oxide and high-frequency ventilation in an infant with respiratory syncytial virus pneumonia and bronchopulmonary dysplasia effect of inhaled nitric oxide on respiratory mechanics in ventilated infants with rsv bronchiolitis helium-oxygen improves clinical asthma scores in children with acute bronchiolitis heliox therapy in infants with acute bronchiolitis exogenous surfactant supplementation in infants with respiratory syncytial virus bronchiolitis ribavirin for respiratory syncytial virus infection of the lower respiratory tract in infants and young children ribavirin for respiratory syncytial virus infection of the lower respiratory tract in infants and young children inhibition of respiratory viruses by nasally administered sirna glucocorticoids for acute viral bronchiolitis in infants and young children prospects of antiviral and anti-infl ammatory therapy for respiratory syncytial virus infection effi cacy of bronchodilator therapy in bronchiolitis. a meta-analysis bronchodilators for bronchiolitis the pharmacologic mechanism by which inhaled epinephrine reduces airway obstruction in respiratory syncytial virus-associated bronchiolitis short term effects of adrenaline in bronchiolitis: a randomised controlled trial effect of racemic epinephrine and salbutamol on clinical score and pulmonary mechanics in infants with bronchiolitis racemic epinephrine compared to salbutamol in hospitalized young children with bronchiolitis; a randomized controlled clinical trial quantitative aspects of passive immunity to respiratory syncytial virus infection in infant cotton rats prophylactic administration of respiratory syncytial virus immune globulin to high-risk infants and young children. the respiratory syncytial virus immune globulin study group respiratory syncytial virus immune globulin for prophylaxis against respiratory syncytial virus disease in infants and children with congenital heart disease. the cardiac study group neutralization epitopes of the f glycoprotein of respiratory syncytial virus: effect of mutation upon fusion function development of a humanized monoclonal antibody (medi-493) with potent in vitro and in vivo activity against respiratory syncytial virus safety and pharmacokinetics of an intramuscular humanized monoclonal antibody to respiratory syncytial virus in premature infants and infants with bronchopulmonary dysplasia. the medi-493 study group the impact-rsv study group. palivizumab, a humanized respiratory syncytial virus monoclonal antibody, reduces hospitalization from respiratory syncytial virus infection in high-risk infants safety, tolerance and pharmacokinetics of a humanized monoclonal antibody to respiratory syncytial virus in premature infants and infants with bronchopulmonary dysplasia. medi-493 study group palivizumab prophylaxis reduces hospitalization due to respiratory syncytial virus in young children with hemodynamically signifi cant congenital heart disease committee on f, newborn. revised indications for the use of palivizumab and respiratory syncytial virus immune globulin intravenous for the prevention of respiratory syncytial virus infections ultra-potent antibodies against respiratory syncytial virus: effects of binding kinetics and binding valence on viral neutralization immunology of viral respiratory tract infection in infancy an epidemiologic study of altered clinical reactivity to respiratory syncytial (rs) virus infection in children previously vaccinated with an inactivated rs virus vaccine vaccine-enhanced respiratory syncytial virus disease in cotton rats following immunization with lot 100 or a newly prepared reference vaccine safety and immunogenicity of a respiratory syncytial virus subunit vaccine (pfp-2) in the institutionalized elderly immunogenicity of a new purifi ed fusion protein vaccine to respiratory syncytial virus: a multi-center trial in children with cystic fi brosis safety and immunogenicity of respiratory syncytial virus purifi ed fusion protein-2 vaccine in pregnant women a novel inactivated intranasal respiratory syncytial virus vaccine promotes viral clearance without th2 associated vaccine-enhanced disease safety and immunogenicity of a sf9 insect cell-derived respiratory syncytial virus fusion protein nanoparticle vaccine respiratory syncytial virus fusion glycoprotein expressed in insect cells form protein nanoparticles that induce protective immunity in cotton rats identifi cation of a recombinant live attenuated respiratory syncytial virus vaccine candidate that is highly attenuated in infants the interferon antagonist ns2 protein of respiratory syncytial virus is an important virulence determinant for humans a newly discovered human pneumovirus isolated from young children with respiratory tract disease metapneumoviruses in birds and humans analysis of the genomic sequence of a human metapneumovirus evolutionary dynamics of human and avian metapneumoviruses genomic analysis of four human metapneumovirus prototypes serologic evidence of avian metapneumovirus infection among adults occupationally exposed to turkeys human metapneumovirus and lower respiratory tract disease in otherwise healthy infants and children studies of culture conditions and environmental stability of human metapneumovirus detection of human metapneumovirus in clinical samples by immunofl uorescence staining of shell vial centrifugation cultures prepared from three different cell lines simultaneous detection and typing of human metapneumovirus strains in nasopharyngeal secretions and cell cultures by monoclonal antibodies detection of human metapneumovirus by direct antigen test and shell vial cultures using immunofl uorescent antibody staining evaluation of a commercial direct fl uorescent-antibody assay for human metapneumovirus in respiratory specimens molecular assays for detection of human metapneumovirus real-time reverse transcriptase pcr assay for detection of human metapneumoviruses from all known genetic lineages detection and quantification of human metapneumovirus in pediatric specimens by realtime rt-pcr detection of respiratory syncytial virus and human metapneumovirus by reverse transcription polymerase chain reaction in adults with and without respiratory illness molecular epidemiology of human metapneumovirus in ireland clinical evaluation of nuclisens magnetic extraction and nuclisens analytespecifi c reagents for real-time detection of human metapneumovirus in pediatric respiratory specimens detection and characterisation of human metapneumovirus from children with acute respiratory symptoms in north-west england real-time reverse transcriptase pcr assay for improved detection of human metapneumovirus real-world comparison of two molecular methods for detection of respiratory viruses recovery of human metapneumovirus from cdna: optimization of growth in vitro and expression of additional genes sequence analysis of the n, p, m and f genes of canadian human metapneumovirus strains genetic diversity between human metapneumovirus subgroups global genetic diversity of human metapneumovirus fusion gene use of the p gene to genotype human metapneumovirus identifi es 4 viral subtypes antigenic and genetic variability of human metapneumoviruses genetic diversity and evolution of human metapneumovirus fusion protein over twenty years novel human metapneumovirus sublineage effects of human metapneumovirus and respiratory syncytial virus antigen insertion in two 3' proximal genome positions of bovine/human parainfl uenza virus type 3 on virus replication and immunogenicity individual contributions of the human metapneumovirus f, g, and sh surface glycoproteins to the induction of neutralizing antibodies and protective immunity human metapneumovirus fusion protein vaccines that are immunogenic and protective in cotton rats immunization of syrian golden hamsters with f subunit vaccine of human metapneumovirus induces protection against challenge with homologous or heterologous strains an alphavirus repliconbased human metapneumovirus vaccine is immunogenic and protective in mice and cotton rats isolation and characterization of monoclonal antibodies which neutralize human metapneumovirus in vitro and in vivo a recombinant human monoclonal antibody to human metapneumovirus fusion protein that neutralizes virus in vitro and is effective therapeutically in vivo the prophylactic administration of a monoclonal antibody against human metapneumovirus attenuates viral disease and airways hyperresponsiveness in mice prophylactic and therapeutic benefi ts of a monoclonal antibody against the fusion protein of human metapneumovirus in a mouse model soluble recombinant human metapneumovirus g protein is immunogenic but not protective genetic variability of the g glycoprotein gene of human metapneumovirus sequence polymorphism of the predicted human metapneumovirus g glycoprotein genetic heterogeneity of g and f protein genes from argentinean human metapneumovirus strains human metapneumovirus g protein is highly conserved within but not between genetic lineages the two major human metapneumovirus genetic lineages are highly related antigenically, and the fusion (f) protein is a major contributor to this antigenic relatedness experimental infection of macaques with human metapneumovirus induces transient protective immunity immunogenicity and effi cacy of two candidate human metapneumovirus vaccines in cynomolgus macaques estimates of individuals exposed to human metapneumovirus in a community-based taiwanese population in 1999 seroepidemiology of human metapneumovirus (hmpv) on the basis of a novel enzyme-linked immunosorbent assay utilizing hmpv fusion protein expressed in recombinant vesicular stomatitis virus seroprevalence of human metapneumovirus (hmpv) in the canadian province of saskatchewan analyzed by a recombinant nucleocapsid proteinbased enzyme-linked immunosorbent assay mlinaric galinovic g. serosurvey of human metapneumovirus infection in croatia large-scale seroprevalence analysis of human metapneumovirus and human respiratory syncytial virus infections in beijing, china high seroprevalence of neutralizing capacity against human metapneumovirus in all age groups studied in bonn clinical impact of human metapneumovirus genotypes and genotype-specifi c seroprevalence in yamagata serologic evidence of human metapneumovirus circulation in uruguay human metapneumovirus reinfection among children in thailand determined by elisa using purifi ed soluble fusion protein high seroprevalence of human metapneumovirus among young children in israel seroepidemiological study of human metapneumovirus comparison of the seroprevalence of human metapneumovirus and human respiratory syncytial virus development and validation of an enzyme-linked immunosorbent assay for human metapneumovirus serology based on a recombinant viral protein human metapneumovirus infection among children association between seroprevalence of human metapneumovirus and c-reactive protein level and apolipoprotein e-epsilon4 allele in elderly inpatients in japan development and evaluation of a whole virus-based enzyme-linked immunosorbent assay for the detection of human metapneumovirus antibodies in human sera population-based incidence of human metapneumovirus infection among hospitalized children human metapneumovirus infection in japanese children the association of newly identifi ed respiratory viruses with lower respiratory tract infections in korean children human metapneumovirus seasonality, incidence, and repeat human metapneumovirus lower respiratory tract infections in an area with a high prevalence of human immunodefi ciency virus type-1 infection children with respiratory disease associated with metapneumovirus in hong kong acute lower respiratory tract infections by human metapneumovirus in children in southwest china: a 2-year study burden of human metapneumovirus infection in young children human metapneumovirus infections in young and elderly adults human metapneumovirus infections in adults: another piece of the puzzle rates of hospitalizations for respiratory syncytial virus, human metapneumovirus, and infl uenza virus in older adults human metapneumovirus pneumonia in adults: results of a prospective study human metapneumovirus in bronchoalveolar lavage fl uid of critically ill patients with suspected pneumonia human metapneumovirus in adults: a short case series respiratory picornaviruses and respiratory syncytial virus as causative agents of acute expiratory wheezing in children asthma exacerbations in children associated with rhinovirus but not human metapneumovirus infection prevalence of viral respiratory tract infections in children with asthma metapneumovirus and acute wheezing in children human metapneumovirus infection plays an etiologic role in acute asthma exacerbations requiring hospitalization in adults lung function in prematurely born infants after viral lower respiratory tract infections human metapneumovirus bronchiolitis in infancy is an important risk factor for asthma at age 5 the impact of infection with human metapneumovirus and other respiratory viruses in young infants and children at high risk for severe pulmonary disease severe human metapneumovirus pneumonia in a child with chronic illness comparison of risk factors for human metapneumovirus and respiratory syncytial virus disease severity in young children clinical course of hospitalised children infected with human metapneumovirus and respiratory syncytial virus comparison of human metapneumovirus infection with respiratory syncytial virus infection in children a 1-year experience with human metapneumovirus in children aged <5 years human metapneumovirus and chronic obstructive pulmonary disease human metapneumovirus infection in adults with community-acquired pneumonia and exacerbation of chronic obstructive pulmonary disease human metapneumovirus and exacerbations of chronic obstructive pulmonary disease respiratory tract reinfections by the new human metapneumovirus in an immunocompromised child human metapneumovirus-associated lower respiratory tract infections among hospitalized human immunodefi ciency virus type 1 (hiv-1)-infected and hiv-1-uninfected african infants brief communication: fatal human metapneumovirus infection in stem-cell transplant recipients fatal pneumonia associated with human metapneumovirus (hmpv) in a patient with myeloid leukemia and adenocarcinoma in the lung human metapneumovirus infection in immunocompromised child respiratory failure associated with human metapneumovirus infection in an infant posthepatic transplant human metapneumovirus infection in hematopoietic stem cell transplant recipients the human metapneumovirus: a case series and review of the literature a prospective study comparing human metapneumovirus with other respiratory viruses in adults with hematologic malignancies and respiratory tract infections an outbreak of human metapneumovirus infection in hospitalized psychiatric adult patients in taiwan an outbreak of severe respiratory tract infection due to human metapneumovirus in a long-term care facility molecular epidemiological investigation of a nosocomial outbreak of human metapneumovirus infection in a pediatric hemato-oncology patient population the role of human metapneumovirus in upper respiratory tract infections in children: a 20-year experience human metapneumovirus infection in the united states: clinical manifestations associated with a newly emerging respiratory infection in children human metapneumovirus in children with acute respiratory tract infections in suzhou viral load and acute otitis media development after human metapneumovirus upper respiratory tract infection frequency of human metapneumovirus in the upper respiratory tract of children with symptoms of an acute otitis media detection of human metapneumovirus from children with acute otitis media human metapneumovirus rna in encephalitis patient beyond viruses: clinical profi les and etiologies associated with encephalitis a fatal case of encephalopathy possibly associated with human metapneumovirus infection seasonal distribution and phylogenetic analysis of human metapneumovirus among children in osaka city human metapneumovirus associated with central nervous system infection in children human metapneumovirus in the cerebrospinal fl uid of a patient with acute encephalitis characterization of human metapneumovirus infections in israel human metapneumovirus-associated lower respiratory tract infections in korean infants and young children community epidemiology of human metapneumovirus, human coronavirus nl63, and other respiratory viruses in healthy preschool-aged children using parent-collected specimens evidence of human metapneumovirus in children in argentina human metapneumovirus in hospitalized children in amman, jordan epidemiology and genetic variability of human metapneumovirus during a 4-yearlong study in southeastern brazil biennial spring activity of human metapneumovirus in austria human metapneumovirus infections-biannual epidemics and clinical fi ndings in children in the region of basel, switzerland human metapneumovirus infection among children hospitalized with acute respiratory illness a summer outbreak of human metapneumovirus infection in a long-term-care facility dual infection of infants by human metapneumovirus and human respiratory syncytial virus is strongly associated with severe bronchiolitis absence of human metapneumovirus co-infection in cases of severe respiratory syncytial virus infection human metapneumovirus and severity of respiratory syncytial virus disease viral respiratory infections in hospitalized and community control children in alaska co-infection of human metapneumovirus with adenovirus or respiratory syncytial virus among children in japan co-circulation of human metapneumovirus and sars-associated coronavirus during a major nosocomial sars outbreak in hong kong changing circulation rate of human metapneumovirus strains and types among hospitalized pediatric patients during three consecutive winter-spring seasons differences in clinical severity between genotype a and genotype b human metapneumovirus infection in children human metapneumovirus genotypes and severity of disease in young children (n = 100) during a 7-year study in dijon hospital, france genetic variability of human metapneumovirus infection: evidence of a shift in viral genotype without a change in illness experimental human metapneumovirus infection of cynomolgus macaques (macaca fascicularis) results in virus replication in ciliated epithelial cells and pneumocytes with associated lesions throughout the respiratory tract t lymphocytes contribute to antiviral immunity and pathogenesis in experimental human metapneumovirus infection the cotton rat (sigmodon hispidus) is a permissive small animal model of human metapneumovirus infection, pathogenesis, and protective immunity detection of severe human metapneumovirus infection by real-time polymerase chain reaction and histopathological assessment pathology of human metapneumovirus infection: insights into the pathogenesis of a newly identifi ed respiratory virus human metapneumovirus glycoprotein g inhibits innate immune responses human metapneumovirus inhibits ifn-alpha signaling through inhibition of stat1 phosphorylation viral acute lower respiratory infections impair cd8+ t cells through pd-1 characterization of human metapneumoviruses isolated from patients in north america humoral immunity to human metapneumovirus infection in adults human metapneumovirus infections in hospitalized children respiratory viruses in hospitalized children with acute lower respiratory tract infections in harbin, china prevalence and clinical symptoms of human metapneumovirus infection in hospitalized patients human metapneumovirus in the community detection and genetic diversity of human metapneumovirus in hospitalized children with acute respiratory infections in india detection of multiple respiratory viruses by real-time polymerase chain reaction in infants attending an outpatient clinic comparison of different cell lines and incubation times in the isolation by the shell vial culture of human metapneumovirus from pediatric respiratory samples detection of human metapneumovirus antigens in nasopharyngeal secretions by an immunofl uorescent-antibody test rapid detection of human metapneumovirus strains in nasopharyngeal aspirates and shell vial cultures by monoclonal antibodies virological features and clinical manifestations associated with human metapneumovirus: a new paramyxovirus responsible for acute respiratory-tract infections in all age groups comparative evaluation of real-time pcr assays for detection of the human metapneumovirus effect of ribavirin and glucocorticoid treatment in a mouse model of human metapneumovirus infection comparison of the inhibition of human metapneumovirus and respiratory syncytial virus by ribavirin and immune serum globulin in vitro comparison of the inhibition of human metapneumovirus and respiratory syncytial virus by nmso3 in tissue culture assays human metapneumovirus infection in a hematopoietic transplant recipient successful treatment of human metapneumovirus pneumonia using combination therapy with intravenous ribavirin and immune globulin treatment with oral ribavirin and ivig of severe human metapneumovirus pneumonia (hmpv) in immune compromised child a host-range restricted parainfl uenza virus type 3 (piv3) expressing the human metapneumovirus (hmpv) fusion protein elicits protective immunity in african green monkeys recovery of human metapneumovirus genetic lineages a and b from cloned cdna infection of nonhuman primates with recombinant human metapneumovirus lacking the sh, g, or m2-2 protein categorizes each as a nonessential accessory protein and identifi es vaccine candidates deletion of m2 gene open reading frames 1 and 2 of human metapneumovirus: effects on rna synthesis, attenuation, and immunogenicity generation of temperature-sensitive human metapneumovirus strains that provide protective immunity in hamsters deletion of human metapneumovirus m2-2 increases mutation frequency and attenuates growth in hamsters key: cord-017107-sg8n12hs authors: suri, h. s.; li, g.; gajic, o. title: epidemiology of acute respiratory failure and mechanical ventilation date: 2008 journal: intensive care medicine doi: 10.1007/978-0-387-77383-4_18 sha: doc_id: 17107 cord_uid: sg8n12hs acute respiratory failure, and the need for mechanical ventilation, remains one of the most common reasons for admission to the intensive care unit (icu). the burden of acute respiratory failure is high in terms of mortality and morbidity as well as the cost of its principal treatment, mechanical ventilation. very few epidemiologic studies have evaluated the prevalence and outcome of acute respiratory failure and mechanical ventilation in general. most of the published literature has focused on specific forms of acute respiratory failure, particularly acute lung injury (ali) and acute respiratory distress syndrome (ards). in this chapter, we provide a brief review of the pathophysiology of acute respiratory failure, its definition and classification, and then present the incidence and outcomes of specific forms of acute respiratory failure from epidemiologic studies. acute respiratory failure, and the need for mechanical ventilation, remains one of the most common reasons for admission to the intensive care unit (leu). the burden of acute respiratory failure is high in terms of mortality and morbidity as well as the cost of its principal treatment, mechanical ventilation. very few epidemiologic studies have evaluated the prevalence and outcome of acute respiratory failure and mechanical ventilation in general. most of the published literature has focused on specific forms of acute respiratory failure, particularly acute lung injury (ali) and acute respiratory distress syndrome (ards) . in this chapter, we provide a brief review of the pathophysiology of acute respiratory failure, its definition and classification, and then present the incidence and outcomes of specific forms of acute respiratory failure from epidemiologic studies. normal respiration requires the integrated function of several components of the respiratory system (fig. 1) . dysfunction of any component results in the impairment of normal gas exchange and may lead to acute respiratory failure and the need for mechanical ventilation. according to the underlying pathophysiologic mechanism, acute respiratory failure is usually divided into four patterns: types i-iv ( ta ble 1). type i and type ii respiratory failure are also referred to as hypoxemic and hypercapnic respiratory failure, based on a predominant gas exchange abnormality. in many disease states however, more than one pathophysiologic mechanism is operational and clinical criteria that incorporate setting, acuity, and severity are used more often ( table 1) . acute episodes (exacerbations) of respiratory failure in patients with chronic compensated respiratory insufficiency are usually referred to as 'acute on chronic' respiratory failure. a consensus definition of acute respiratory failure is not available and most studies have used the combination of mechanical ventilation (of variable duration) with or without evidence of severe hypoxemia on arterial blood gas analysis. while some studies utilized a more strict definition than others, the essential component in all has been the need for mechanical ventilation. the indications for mechanical ventilation , however, are mostly based on clinical observations (increased respiratory rate, use of accessory muscles, paradoxical chest wall movements, changes in mental state), none of which has sufficient accuracy or precision. therefore, the epidemiology of acute respiratory failure has so far been restricted to 'treated' acute respiratory failure, possibly explaining the wide variations in the reported incidence and outcomes of acute respiratory failure and associated clinical syndromes. since the availability of intensive care and mechanical ventilation vary greatly in different parts of the world, the burden of acute respiratory failure may be severely underestimated depending on the access to leu services. the incidence of acute respiratory failure varies according to the definition used and the population studied ( table 2) . two european studies , one conducted in germany [i] and the other in sweden, denmark, and iceland [2] , estimated very similar incidences, 88.6 and 77.6 cases per 100,000 person-years. both studies used an identical definition (intubation and mechanical ventilation for > 24 h regardless of arterial blood gas findings) and employed a multicenter approach with large patient cohorts over a short period (8 weeks). on the other hand, behrendt reported a much higher incidence of acute respiratory failure in the usa, 137.1 per 100,000 patient-years. this incidence was estimated based on the icd-9-cm disease codes for diagnoses and treatment in patients > 5 yrs old observed over a l-year period [3] . the significant variation between the us and european incidences may in part be explained by the differences in study design ( table 2 ) and in part by incons istent indications and access to mechanical ventilation in different countries. acute respiratory failure is often accompanied or followed by a failure of other vital organs, and death most often occurs becau se of multiorgan failure (mof) and the withdrawal of mechan ical ventilati on when the chances for a meaningful recovery of the patient's qual ity of life are deemed to be exceedingly low. imprecision of clinical prognostic criteria, variations in available resources, and patient and provider preferences limit the interpretation of mortality data from different epidemiologic studies. reported mortality rates for acute respir ator y failure from the 1990s are remarkably similar, approximately 40 % in spite of differences in study designs and the definit ions applied ( table 2 ). lewandowski and coworkers [1] studied 476 patients from 72 icus in berlin, germany and reported mortality rates of 36-58 % depending on the lung injury score (lis). in a large prospective study from scandinavia, luhr and coworkers reported an all-cause 90-day mortality of 41 % [2] . in a large, prospective international cohort involving 361 icus, esteban and coworkers reported an icu mortality of 30.7 %. mortality increased significantly in patients with sepsis, shock, ards, or liver failure [4] . vincent and coworkers used sequential organ failure assessment (sofa) score criteria and the need for mechanical ventilation to define acute respiratory failure and estimated an overall icu mortality of 31 %. the mortality was much lower (7 %) when the lung was the only organ involved [5] . recently, flaatten and coworkers reported the mortality from acute respiratory failure at different time points after disease onset . mortality was again the lowest in single organ acute respiratory failure and rose with each add itional organ failure. higher mortality rates were found 90 days after the onset compared to at icu or hospital discharge [6] . mof following an icu admission , presence of circulatory shock on icu admission , older age, and pre-existing comorbidities (cirrhosis, malignancy and chronic renal failure) were independent risk factors for the mortality rate reported in several studies [4, [7] [8] [9] . acute lung injury (ali) ali and its more severe form, ards, are clinical syndromes defined as an acute onset of hypoxemic respiratory failure with diffuse pulmonary infiltrates in the absence of left atrial hypertension as the principal cause of acute pulmonary edema. ali is a major cause of acute respiratory failure in the icu and is associated with high morbidity and mortality. since it was first described by ashbaugh and colleagues [10] and than redefined in 1994 [11] , there have been significant advances in the understanding of etiology, pathophysiology, and the epidemiology of ali. clinical risk factors for ali are usually divided into direct (pulmonary) and indirect (extrapulmonary). pneumonia, aspiration, lung contusion; and inhalation injury are the principal pulmonary risk factors, while sepsis, shock, trauma, pancreatitis, and multiple transfusions represent the most important extrapulmonary risk factors. in recent years, transfusion-related ali (trali) and novel viral pathogens (severe acute respiratory syndrome [sarsj) have emerged as important risk factors for ali. the reported incidence of all has varied significantly. the 1972 report of the national heart and lung institute task force on respiratory diseases, estimated 150,000 cases of ards per year yielding the annual incidence of 75 per 100,000 person-years . subsequent studies reported an incidence of all ranging from 16 to 34 cases per 100,000 person-years in european countries [2, 12] and australia [13] , and a much higher incidences of ali in the usa, 78 per 100,000 persons-years (190,600 cases per year) [13] [14] [15] . while a significant minority of patients with all is treated with non-invasive ventilation (niv), the majority of studies included only patients treated with invasive ventilation. a recently completed, retrospective, community cohort study in olmsted county, minnesota included patients treated with niv and found an even higher incidence of ali, 156 per 100,000 person-years (personal communication, rodrigo cartin -ceba), mortality from ali varies greatly depending upon the age of the patient, underlying chronic illnesses, ali risk factors, and non-pulmonary organ dysfunctions [15] . two decades ago, the mortality rate from ali ranged from 50 -70 % [4, 8, 16, 17] , but has since declined and more recently has been estimated to be about 30-50 % [2, 13, 15, 23] . advances in general supportive care [9] and the use of new mechanical ventilation strategies [16] may account for most of the change. both the incidence of and mortality from all increase exponentially with age [1-3, 15, 18] . mof [7, 19, 20] , liver failure, severe sepsis [8, 9, 15, 17] , aspiration [15] , presence of infection and neurological failure on icu admission [7] , and pree-xisting cirrhosis [2, 8, 17, 21] , bone marrow transplantation, human immunodeficiency virus (hiv) [17] , hematologic [7, 22] or active malignancy [17, 22] , and charlson comorbidity score [23] have been associated with a higher mortality. persistent severe hypoxemia and cardiovascular failure also predict poor outcomes [21, 23] . non-survivors of ali die predominantly of moe a landmark study published in 1985 reported that only 16 % of deaths were caused by respiratory failure [24] . similar results (16 % and 9 %) were reported by two stud ies conducted in recent years [13, 25] . mof, septic shock , and underlying comorbidities are the most common causes of death in patients with ali. survivors of ali often have a prolonged recovery and significant short and longterm disability. while lung function usually returns to normal within several months [9] , neuromuscular and neurocognitive sequelae may persist much longer [26, 27] . the most important predictors of prolonged disability are the use of systemic steroids during the icu stay, presence of a complicating illness acquired during the icu stay, and the rate of resolut ion of ali and mof [27] . neuropsychological sequelae are also common and about 27 % of long-term survivors develop posttraumatic stress disorder [28] . with a decline in mortality from ali, more survivors are at risk of prolonged morbidity ('chronic critical illness') contributing to substantial increases in the utilization of health care resources. cardiogenic pulmonary edema is a common cause of acute respiratory failure. in about 10 % of the mechanically ventilated patients in an international cohort study, cardiogenic pulmonary edema was the principal reason for instituting mechanical ventilation [4] . other epidemiologic studies reported similar rates of cardiogenic pulmonary edema [2, 29] with mortality ranging from 28 -48 % [1, 4] . in the past two decades, niv, both continuous positive airway pressure (cpap) and bilevel positive airway pressure (bipap) ventilation, have received a great deal of interest in the management of patients presenting with acute cardiogenic pulmonary edema . randomized trials comparing either cpap or bipap with standard medical therapy, found similar improvements in arterial blood gases and breathing rates, reduced need for intubation, and improved outcome [30] . according to the world health organization, chronic obstructive pulmonary disease (copd) ranks fourth among all causes of death with an age-adjusted mortality rate of 39.9 per 100,000 person-years. the 20 th century pandemic of cigarette smoking is taking its toll, evident by the increase in the annual hospitalization rate for acute exacerbation of copd from 9.7 in 1988 to 24.4 % in 2005 . moreover, about 10 % of all hospitalizations are directly or indirectly attributable to copd [31] . many patients with acute exacerbation of copd require admission to the icu for acute respiratory failure. in an international cohort study [4] , acute exacerbation of copd was a principal indication for initiating mechanical ventilation in 13 % of patients with acute respiratory failure. the hospital mortality rate of copd patients admitted with acute exacerbation varies between 2.5 -30 %, depending on the methodology of the data collection and the patient population. seneff et al. [32] reported a hospital mortality rate of 24 % in 362 admissions for acute exacerbation of copd selected from the acute physiology chronic health evaluation (apache) iii database of 17,440 admissions in a prospective multicenter trial. mortalities rose to 30 % at hospital discharge and doubled to 59 % at the l-year follow-up. invasive mechanical ventilation was instituted in 170 of 362 patients with a mortality rate of 47 %. after controlling for the severity of illness , mechanical ventilation at icu admission was not asso ciated with either hospital mortality or subsequent survival. development of non-respiratory organ dysfunction was the most important predictor of hospital mortality, while the abnormalities in gas exchange (pac0 2 , ph , pa0 2) indicative of advanced dysfunction were strongly associated with six month mortality. esteban et al. [4] reported a hospital mortality of 28 % in patients receiving mechanical ventilation for acute exacerbation of copd. liu et al. [33] retrospectively studied a cohort of 138 patients with copd requiring invasive mechanical ventilation for acute respiratory failure. the cause of acute respiratory failure was acute exacerbation of copd in 55 % and pneumonia in 44 % of patients. the hospital mortality rate was 39.9 % in all patients and 31.1 % in the acute exacerbation of copd subgroup. respiratory acidosis was corrected (ph> 7.30) in 69.9 % of survivors but only in 21.8 % of non-survivors. in a recent study, 205 conse cutive patients hospitalized with acute exacerbation of copd were followed prospectively for 3 years [31] . the in-hospital mortality rate was 8.3 %. the overa1l6-month mortality was 24 %, with 1-,2-, and 3-year mortality rates of 33 %, 39 %, and 49 %, respectively. more severe gas exchange abnormalities and longer hospital stays were associated with the hospital mortalities. long-term mortality was associated with longer disease duration, lower serum albumin, low body mass index, and lower pa02' mof and sepsis were the most common immediate causes of death in patients with acute exacerbation of copd admitted to the icu. in another prospective study of 250 patients with acute exacerbation of copd [34] , invasive mechanical ventilation was started in 60 % and niv was tried in 40 % of patients and was successful in 54 % of them. median duration of ventilation was 6 days. after several clinical tr ials reported improved outcomes [35] , niv has become the principal init ial mode for providing mechanical ventilation to patients with acute exacerbation of copd [36] . since the indications for niv are more liberal than those of invasive ventilation, it is difficult to directly compare the outcomes of mechanically ventilated patients treated with the two modes. in a study by girou et al., however, adjusted odds of death (0.37; 95 % confidence interval [ci], 0.18-0.78) suggested that the mortality in patients with similar severity of illness treated with niv was significantly lower. severe status asthmaticus is a rare cause of acute respiratory failure requmng mechanical ventilation (1.5 % of patients in the international cohort study) [4] . patients in status asthmaticus who require invasive mechanical ventilation are at high risk of severe complications (pneumothorax, cardiopulmonary arrest) and mortality. afessa et al. reported the incidence and outcomes of status asthmaticus in a us inner city hospital, from 1995 to 1998 [37] . forty-eight out of 132 hospital admissions required mechanical ventilation (36 %). mechanically ventilated patients had significant mortality (21 %) and high complication rates. sixteen patients developed non-pulmonary organ failure and four developed pneumothorax requiring chest tube drainage. interestingly, all patients who died in this study were female . pneumonia is a common cause of hypoxemic acute respiratory failure. approximately 14-23 % of acute respiratory failure episodes requiring mechanical ventilation are due to pneumonia. icu mortality rates from acute respiratory failure due to pneumonia range from 37-44 % [1, 2, 4, 38] . in many patients with pneumonia, however, complications such as septic shock and ali, or acute worsening of underlying chronic lung disease (copd) are the principal reasons for instituting mechanical ventilation . compared to other ali risk factors, pneumonia is associated with higher mortality (see ali paragraph above). the majority of patients with interstitial lung disease and acute respiratory failure admitted to the icu require invasive mechanical ventilation . interstitial lung disease is, however, an uncommon cause of acute respiratory failure (less than 2 % of patients in the international cohort study [4] ). in a retrospective review [39] of 75 patients with interstitial lung disease who were mechanically ventilated at mayo clinic from 2003 to 2005, acute respiratory failure was the most common cause of icu admiss ion (77 %), followed by sepsis (11 %) and cardiopulmonary arrest (4 %). seventeen patients were initially treated with niv but eventually all patients required invasive mechanical ventilation. hospital mortality was 49 %. patients with idiopathic pulmonary fibrosis tended to have a higher mortality rate than non -idiopathic pulmonary fibrosis forms of interstitial lung disease. conventional lung protective mechanical ventilation was not associated with improved outcome. worsening hypoxemia and higher positive end-expiratory pressure (peep) settings were associated with increased mortality. in an earlier study, saydain and coworkers observed the clinical course of 38 patients with idiopathic pulmonary fibrosis admitted to the icu. acute respiratory failure was the most common reason for icu admission. while 49 % of the patients survived to hospital discharge, 12 of 13 survivors (92 %) died within 2 months after hospital discharge [40] . patients with neuromuscular disease are frequently treated with both acute and chronic mechanical ventilation. neuromuscular disease accounted for 2 % of patients receiving mechanical ventilation in the international cohort study [4] . compared to other causes of acute respiratory failure, patients with neuromuscular disease had higher costs and length of icu stay and 68 % required tracheostomy [4] . hospital mortality was 15 %. epidemiologic studies looking at the outcomes of acute respiratory failure due to specific forms of neuromuscular disease are scarce. recently, ali et al. [41] reported on the outcomes of 54 patients with guillain-barre syndrome who required mechanical ventilation. all but six patients (89 %) required tracheostomy. forty-six patients (85 %) survived to hospital discharge, and 39 (72 %) were alive at the l-year follow-up according to the international cohort study [4] , in 7.3 % of patients mechanical ventilation was employed because of trauma. hospital mortality for these patients was 20 %. in a retrospective incident study of acute respiratory failure in the usa [3] , acute respiratory failure related to trauma was more common in the younger age group and trauma without mof was associated with a very low mortality rate. in a scandinavian study, approximately 9 % of cases of acute respiratory failure were caused by trauma [2] . of the 508 cases of acute respiratory failure in the berlin study, 19 were due to trauma with mortality of 20 % [1] . complicating coma, shock, and ali are common indications for mechanical ventilation in patients with trauma. about 8-15 % of cases of ali are related to trauma [2, 4, 15] with mortality lower than that for other ali risk factors (24 %, see above) [15] . shock is characterized by global hypoperfusion leading to lactic acidosis, hyperventilation and hypoperfusion of respiratory muscles, resulting in type iv respiratory failure. up to 30 % of oxygen consumption in shock may be used by the respiratory muscles contributing to a global imbalance between oxygen delivery and consumption . pulmonary edema, ali, and anemia often contribute towards respiratory distress. work of breathing may ultimately overcome respiratory reserve leading to the development of acute respiratory failure. early use of mechanical ventilation in severe shock may be justified to limit the work of breathing and decrease oxygen consumption by respiratory muscles. septic shock, in particular, is commonly associated with acute respiratory failure and ali. in the international cohort study, septic shock was a primary indication for mechanical ventilation in 9 % of patients with mortality of 55 % [4] . coma is a non-specific syndrome of widespread central nervous system impairment resulting from various metabolic and structural etiologies. it usually results in type ii respiratory failure due to upper airway dysfunction and hypoventilation. intubation and invasive mechanical ventilation are usually required to protect the airway and maintain gas exchange. in the study by esteban et al. [4] , 16% of patients required mechanical ventilation because of coma. reported icu mortality was 36 % in patients with coma who received mechanical ventilation. advances in mechanical ventilation have dramatically changed the management and outcome of patients with acute respiratory failure. with increased access to mechanical ventilation , the burden of acute respiratory failure may grow beyond the health care budget of even the richest societies. inconsistent use of standardized definitions for acute respiratory failure and, in particular, indications for mechanical ventilation, present the major impediment to the meaningful understanding of clinical research results and will have to be overcome in future studies. population studies are needed to determine the risk factors, prevalence, and the attributable outcomes of various forms of acute respiratory failure in the community. such studies will help identify the best strategies for the prevention and treatment of acute respiratory failure, will pinpoint important uncertainties that need to be tested in clinical trials, and will allow informed decisions regarding allocation of scarce resources so that bedside practitioners may best improve the quality-adjusted survival of their patients . incidence, severity, and mortality of acute respiratory failure in incidence and mortality after acute respiratory failure and acute respiratory distre ss syndrome in sweden, denmark, and iceland. the arf study group acute respiratory failure in the united states: incidence and 31-day survival character istics and outcomes in adult patients receiving mechanical ventilation: a 28-day international study epidemiology and outcome of acute respiratory failure in intensive care un it pat ients outcome after acute respiratory failure is more dependent on dysfunct ion in other vital organs than on the severity of the respiratory failure the epidemiology of acute respiratory failure in critically ill pat ients identification of patients with acute lung injury. predictors of mortality the changing face of organ failure in ards acute respiratory distress in adults the american-european consensus conference on ards. definitions, mechan isms, relevant outcomes, and clinical trial coordination acute respiratory distress syndrome : an audit of incidence and outcome in scottish intensive care units incidence and mortality of acute lung injur y and the acute respiratory distress syndrome in three australian states incidence of acute lung injury in the united states incidence and outcomes of acute lung injury ventilation with lower tidal volumes as compared with traditional tidal volumes for acute lung injury and the acute respiratory distress syndrome acute lung injury in the medical icu: comorbid conditions, age, etiology, and hospital outcome survival following mechanical ventilation for acute respiratory failure in adult men acute lung injury at baragwanath icu. an eightmonth audit and call for consensus for other organ failure in the adult respiratory distress syndrome hemodynamic profile in severe ards: results of the european collaborative ards study early predictive factors of survival in the acute respiratory distress syndrome. a multivariate analysis outcome of respiratory failure in hematologic malignancy six-month survival of patients with acute lung injury: prospective cohort study causes of mortality in patients with the adult respiratory distress syndrome is outcome from ards related to the severity of respiratory failure? neuropsychological sequelae and impaired health status in surv ivors of severe acute respiratory distress syndrome one-year outcomes in survivors of the acute respiratory distress syndrome health-related quality of life and posttraumatic stress disorder in survivors of the acute respiratory distress syndrome how is mechanical ventilation employed in the intensive care unit? an international utilization review noninvasive ventilation in acute cardiogenic pulmonary edema: systematic review and meta -analysis factors affecting survival of hospitalized patients with copd hospital and i-year survival of patients admitted to intensive care units with acute exacerbation of chronic obstructive pulmonary disease analysis of risk factors for hospital mortali ty in patients with chronic obstructive pulmonary diseases requiring invasive mechanical ventilation prognostic factors, clinical course, and hospital outcome of patients with chronic obstructive pulmonary disease admitted to an intensive care unit for acute respiratory failure non-invasive positive pressure ventilation for treatment of respiratory failure due to exacerbations of chronic obstructive pulmonary disease secular trends in nosocomial infections and mortality associated with noninvasive ventilation in patients with exacerbation of copd and pulmonary edema clinical course and outcome of patien ts admitted to an icu for status asthmaticus hospital survival rates of patients with acute respiratory failure in modern respiratory intensive care units. an international, multicenter, prospective survey gajic 0 (2006) ventilator settings and outcome in pat ients with interstitial lung disease requiring mechanical ventilation in the intensive care unit outcome of patients with idiopathic pulmonary fibrosis admitted to the intensive care unit gajic 0 (2006) mechanical ventilation in patients with guiilain-barre syndrome key: cord-011095-79ce5900 authors: meskill, sarah d.; o’bryant, shelease c. title: respiratory virus co-infection in acute respiratory infections in children date: 2020-01-24 journal: curr infect dis rep doi: 10.1007/s11908-020-0711-8 sha: doc_id: 11095 cord_uid: 79ce5900 purpose of review: this investigation aims to understand the role and burden of viral co-infections for acute respiratory illnesses in children. co-infection can be either viral-viral or viral-bacterial and with new technology there is more information on the role they play on the health of children. recent findings: with the proliferation of multiplex pcr for rapid diagnosis of multiple viruses as well as innovations on identification of bacterial infections, research has been attempting to discover how these co-infections affect each other and the host. studies are aiming to discern if the epidemiology of viruses seen at a population level is related to the interaction between different viruses on a host level. studies are also attempting to discover the burden of morbidity and mortality of these viral-viral co-infections on the pediatric population. it is also becoming important to understand the interplay of certain viruses with specific bacteria and understanding the impact of viral-bacterial co-infections. summary: rsv continues to contribute to a large burden of disease for pediatric patients with acute respiratory illnesses. however, recent literature suggests that viral-viral co-infections do not add to this burden and might, in some cases, be protective of severe disease. viral-bacterial co-infections, on the other hand, are most likely adding to the burden of morbidity in pediatric patients because of the synergistic way they can infect the nasopharyngeal space. future research needs to focus on confirming these conclusions as it could affect hospital cohorting, role of molecular testing, and therapeutic interventions. acute respiratory illnesses are the most common cause of under-5-year-old mortality worldwide [1] . in particular, pneumonia is responsible for approximately 1.4-1.8 million fatal cases in children under age five globally [2] . beyond the mortality burden, there is significant morbidity with symptomatic viral infections estimated at more than five episodes per year in children under age three [3] . while single viral infections are relatively straightforward, there is interest in understanding the role of dual respiratory infections in pediatric patients. dual infections could either be viral-viral or viral-bacterial as the human respiratory tract is a reservoir for many organisms. molecular diagnostics have increased the ability to diagnose causative agents in patients with respiratory illnesses. real-time polymerase chain reaction (rt-pcr) allows for the detection of multiple viruses at once and is found to be more reliable and expedient than viral culture [4] . furthermore, newer methods, such as multiplex pcr and next-generation sequencing are producing more accurate and quicker results towards identifying these organisms [5] . given this ability, studies have attempted to discern if the positive test for pathogen is actually causative or if it is asymptomatic shedding leading to a positive test result. one study evaluated adult visitors to a tourist attraction taking clinical history of symptoms as well as testing pcr for common respiratory illnesses [6] . of those participants who tested negative for respiratory viral infections, 81.9-99.6% reported to be asymptomatic depending on the definition applied. only 6.2% of patients tested positive for a respiratory virus. of these positive results, up to 48.1% were considered symptomatic. another study focused on respiratory viral infection positivity specifically in the pediatric population [7] . testing children biweekly whether symptomatic or not, 56% of symptomatic episodes had detection of a viral pathogen compared to 40% of asymptomatic episodes. the younger the patient, the less likely a pathogen positive episode was asymptomatic (p = 0.01); in addition, multiple pathogens were found in significantly less asymptomatic episodes (p = 0.02). further studies attempted to discern which viruses were more likely to cause symptoms. one study in children under age 5 years old found that respiratory syncytial virus (rsv), human metapneumovirus (hmpv), and parainfluenza viruses (piv) were more likely to be causative of disease [8] . another study still found high rates of asymptomatic shedding of rsv and piv with more than half of positive tests associated with an asymptomatic patient [9] . however, within these data, younger patients were associated with higher rates of positive test results correlating with a symptomatic event demonstrating the importance of age in clinical manifestation of viral infections. rsv is responsible for an extremely high burden of disease in children. infection with rsv is one of the leading causes of death in children under 1 year of age worldwide, second only to malaria [10] . in a surveillance of acute respiratory infections in children under age 5, rsv was responsible for 20% of annual hospitalizations, 18% of emergency department visits, and 15% of office visits [11] . in an evaluation of children under 18 years old admitted to the hospital with a diagnosis of pneumonia, the most common cause of infection, whether bacterial or viral, was rsv quickly followed by rhinovirus [12] . rhinovirus is the most common cause of respiratory viral illness during all seasons except winter when rsv is predominant [13] . rhinovirus is second only to rsv in causing bronchiolitis in hospitalized patients [14] . unfortunately, the true burden of rhinovirus is under-reported as many studies do not include this virus in there molecular diagnostic testing as the rates of asymptomatic shedding are quite high and it is difficult to differentiate shedding from actual cause of disease [13] . while some studies do have rsv and rhinovirus as the leading cause of pneumonia in children, another important common viral contributor is influenza [15] . influenza and its complications are the leading cause of morbidity and mortality [15] . despite antiviral medications and vaccines, it is estimated by the world health organization (who) that the annual influenza epidemics cause 3 to 5 million severe infections and 250,000-500,000 deaths each year in developed countries and 300,000 hospitalizations and 35,000 deaths in the usa [16, 17] . influenza has recently been recognized as having a higher burden of disease than previously thought because of poor recognition and diagnosis even with molecular testing [18] . although vaccination against influenza decreases the risk of infection, now with less vaccination we are witnessing less herd immunity and those more vulnerable are becoming severely ill [19] . in my practice, i've witnessed a 2-month-old infant-too young for the influenza vaccine and caregivers opted out of the influenza vaccination for themselves-develop respiratory failure requiring endotracheal intubation due to influenza a epiglottitis [20] . human metapneumovirus (hmpv) also causes acute respiratory infections globally. hmpv has been shown to cause high hospitalization rates of 1 per 1000 in children 5 years old or younger, with an estimated 20,000 hospitalizations annually which is similar to the rates seen with influenza [21] . outpatient visits were estimated at one million clinic visits and 263,000 emergency department visits annually in the same population [21] . admitted pediatric patients with hmpv were more likely than those without the infection to require supplemental oxygen and had longer intensive care stays [21] . at the host level, the outcome of dual infection is commonly viral interference, such as when one virus competitively inhibits the replication of another virus, but it can also enhance replication in some cases [22] . it is postulated that the sequence of infections, the time interval between viral exposure, and the route of infection affect the pathogenicity of the co-infection [22] . one example of this used mice models. when the mice cell models that were infected with rhinovirus were then infected with influenza a virus 2 days later, there was an attenuated response to the influenza infection with less severe manifestation of disease [23] . this demonstrates that the preceding infection altered the host response and the timing and order of infection of the host are crucial in outward disease manifestation. co-infections can also alter the epidemiology of viral infections. for example, rhinovirus is a rapidly replicating virus and can interfere with the replication of other viruses while piv is an extremely slow replicator and its replication can be interrupted by the presence of other viruses [22] . viral loads can be compared among patients with one or multiple infections. one study found that viral loads were consistently high regardless of co-infection status (such as rsv, influenza a, and hmpv) but others had viral load vary based on coinfection status (such as piv1 and adenovirus) [24] . of these viruses, influenza a is least likely to be identified in coinfected patients. in mathematical modeling, the idea of resource competition can explain viral loads as the fasterreplicating viruses overcome slower replicating viruses by leaving no resources [25] . in this model, influenza a replicates faster than rsv and therefore keeps the rsv viral load below detection level. the pathophysiology behind dual viral infections can explain some of the epidemiology of viral-viral co-infections seen at the population level. given the high rates of rsv and rhinovirus infections, it makes sense that they are the most commonly identified viruses in co-infected patients across multiple studies [14, [49] [50] [51] [52] [53] [54] [55] . however, one study did conclude that the odds of rhinovirus detection were lower when rsv was present and the odds of rhinovirus were significantly higher in those patients who received rsv immunoprophylaxis [56] . this indicates that despite the high prevalence of co-infection with these viruses, there is still viral interference occurring. there is also evidence of viral competition when it comes to rsv and influenza. in terms of viral competition, when rsv infection rates are high, influenza rates of infection are low and the converse is true [57] . in addition, when both viruses are circulating in a small population the rates of co-infection of rsv and influenza are over 6 times less than expected [58] . on review of influenza interactions with other viral pathogens, there is evidence of competitive interference with rsv, rhinovirus, other influenza strains, and hmpv virus by evaluating population incidence and co-infection detection [59] . multiple studies have attempted to evaluate the clinical importance of viral co-infections. it would seem intuitive to think that having more than one virus causing disease in a person would lead to more severe symptoms and sequelae. one study did support this conclusion. in it the authors looked at all pediatric patients who had a respiratory viral panel sent, they found an unadjusted increase in the risk of moderate-severe illness, non-invasive ventilation, ecmo, and death in coinfected patients however in the adjusted analysis only the risk moderate-severe disease continued to be increased [60] . some studies found certain clinical outcomes to be at increased risk but not a persistence of high risk in all outcomes. one study evaluated patients under 12 months in france and found that viral co-infections were 2.7 times more likely to be in the picu however once in the picu there was no difference in length of stay, duration of mechanical ventilation, duration of supplemental oxygen [52] . another study supported this finding by evaluating children in the picu retrospectively comparing one virus vs co-infected viral status and found that the co-infected patients had longer average length of stay in the picu and longer time of intubation [61] . however, there was no difference in rates of needing highflow nasal cannula, mechanical ventilation, or having cardiovascular dysfunction. it is possible that the prolonged intubation times could be because patients who were co-infected with viruses had higher odds of bacterial co-infection. there are some studies that also found increased risk of clinical outcomes but these actually were based on specific viral combinations. one study found that specifically influenza viral co-infection with another influenza strain significantly increased the risk of icu admission or death but this did not carry through to any other co-infected status [62] . another study did a multicenter evaluation of bronchiolitis over 3 years and found longer length of stay in those with rsv/rv coinfection but no proof that these patients were sicker (as in no difference in icu admission or support with cpap or mechanical ventilation) [14] . another study also found an increase in length of stay and oxygen use in those patients with specifically rsv/rv co-infected status [63] . there are a few studies that found no difference in clinical outcomes on co-infected patients. the study looking at tourists did not find any association between viral co-infections and the likelihood of being symptomatic or presenting with more severe symptoms [9] . another one looking at hospitalized pediatric patients found no increased risk of icu admissions in those with multiple viruses identified [64] . beyond no difference in clinical outcomes, quite a few found less severity in those patients with multiple viral infections. one study found age to be important in understanding the risk of severe disease. in this study, they evaluated children with bronchiolitis in the netherlands and found those patients with dual infection had no difference in severity under 3 months of age but significantly less severe disease in patients over 3 months [65] . another study in the netherlands looking at pediatric patients had shorter mean length of stay and no difference in oxygen supply needs or intensive care (icu) admission [53] . another found that viral co-infected patients were significantly less likely on the inpatient ward (or 0.55), icu stay (or 0.32), require oxygen supplementation (or 0.55), or have length of stay greater than 3 days (or 0.32) [24] . one study was actually able to see a linear response to length of stay with a shorter length of stay the more viruses detected when evaluating all pediatric patients with hospitalized respiratory infections [66] . the best evaluation of the literature on viral co-infections and clinical severity is going to be in systematic reviews. there are three looking at viral co-infections that warrant discussion. scotta et al. [67• ] did a large systematic review of respiratory viral coinfections with illness severity in children with over 17,000 patients in the combined evaluation. in this review, viral co-infections did not influence risks of all outcomes assessed: mean length of stay, length of supplemental oxygen, need for hospitalization, need for intensive care admission, mechanical ventilation, or death. they also looked at sub-analyses of specific viral combinations and did not see any influence on outcomes. lim et al. [68] did a systemic review for children under age 5 years with respiratory illness and found insufficient evidence to suggest a difference in any clinical outcome based on co-infected status. in a very small subset of patients, they found a suggestion that children without co-morbidities actually did worse when only a single virus was identified. goka et al. [55] did a systemic review of patients of all ages with respiratory illnesses and found that studies that recruited young children were more likely to report high rates of co-infection and that there were inconclusive results on risk of hospitalization or icu admission. to cause respiratory illnesses, bacterial pathogens first need to colonize the nasopharyngeal space [26] . organisms achieve this colonization via positive and/or negative associations: positive association exists through mutualism, symbiosis or helping to evade the host's immune system; negative association exists through ammensalism, predation, or the host immune system disproportionally affecting one organism over the other [26] . overall, there are multiple mechanisms, for viruses and bacteria, to aid in the success of invasion and colonization of the human body. 1) viral pre-disposition to bacterial adherence: alteration of the host's respiratory epithelium causes viruses to increase the susceptibility of bacterial colonization during a simultaneous infection and after full recovery of a viral illness [26, 27] . examples include: influenza and streptococcus pneumoniae and adenovirus and streptococcus pneumoniae [26, 28] . 2) disruption of the epithelium barrier: viruses can intracellularly disarrange cellular processes or destroy infected cells through metabolic exhaustion or lysis [26] . the destruction of cells leads to the denuding of the epithelial layer, exposing the basement membrane; therefore, causing introduction of bacterial organisms [29, 30] . examples of this mechanism are s. pneumoniae binding to fibronectin after the denudation of the epithelium layer, and staphylococcus aeurus and morexella catarrhalis binding to extracellular matrix protein after destruction to the epithelium [31] [32] [33] . loss of the epithelium integrity and promotion of bacterial translocation are also seen in rhinovirus-induced paracellular migration of haemophilus influenzae [34] . 3) upregulation of adhesion proteins: viral infected cells may decrease the innate immune response by altering the expression of antimicrobial peptides (defensins), which are secreted in the respiratory mucosa [35] . during viral infections, there are cascades of proinflammatory responses leading to the upregulation of adhesion proteins found on epithelial cells, which leads to the cellular invasion of pathogenic organisms [26] . for example, rsvand parainfluenza viruses upregulate intracellular and outer membranes proteins such as intracellular adhesion molecule 1 (icam-1), p5-homologous fimbriae (p5 fimbriae), carcinoembryonic adhesion molecule-1 (ceacam-1), and platelet-activating factor receptor (pafr) [36, 37] . with the expression of these proteins, several bacterial organisms, s. pneumoniae and h. influenzae, are able to adhere to these molecules leading to invasion of the host's cells [36, 37] . 4) production of viral factors: production of viral components such as neuramindase (na), a glycoprotein produced by influenza and parainfluenza, and protein-gexpressed on rsv cells-destroy the integrity of infected cells. this destruction exposes bacterial receptors and aids in bacterial co-infections [38] [39] [40] [41] . 5) dysfunction of immune system components: respiratory viruses may affect the immune system by impairing neutrophil function, decreasing oxidative burst, and enhancing neutrophil apoptosis, thus increasing the susceptibility to bacterial superinfection [42, 43] . also, viruses can predispose to bacteria superinfection by rendering natural killer (nk) cells recruitment and activation ineffective, which is seen with influenza and s. pneumoniae [44] . viruses also alter monocyte function, decrease production and activity of cytokines, and prevent appropriate immune response routing, leading to enhanced bacterial colonization and increasing risk for mortality [45] [46] [47] [48] . because of the interaction between viruses and bacteria, there are many known specific virus-bacteria relationships ( table 1) . for example, influenza interacts with both streptococcus pneumoniae and staphylococcus aureus. the most common bacteria found in viral, secondary bacterial infections is s. pneumoniae [15] . s. pneumoniae has over 97 distinct serotypes and is a common cause of acute otitis media (aom), pneumonia, sepsis, and bacterial meningitis [85] [86] [87] . one of the most common complications of influenza infection is aom from either s. pneumonia or s. aureus [15, 88] . since influenza is one of the common viral contributors to pneumonia, it also increases the rates of bacterial pneumonia from s. pneumoniae and s. aureus. this is because influenza with both of these bacteria has synergistic relationships using the mechanisms listed above [15, 26] . in addition, the bacteria increase influenza's infectivity by activating hemagglutinin on the membrane, thus neutralizing antibodies and allowing more virions to be replicated inside the host cell [15, 89] . table 1 reviews the most common viral-bacterial co-infections and their associated complications. there are also reports of preceding bacterial infections leading to increase viral susceptibilities [26] . for example, s. pneumoniae and hmpv have a unidirectional synergistic relationship where s. pneumoniae predisposes children less than 2 years old to hmpv infections [90] . similarly, h. influenza stimulates adhesion proteins on human epithelial cells, creating an entry point for rhinovirus [91] . rhinovirus also has a bi-directional relationship with s. aureus: rhinovirus aids in the bacterial adhesion and engulfment into the epithelium cell and s. aureus promotes the replication of rhinovirus [70] . rhinovirus can actually increase the nasal load of s. aureus by 39% over baseline [70] . the evaluation of viral-bacterial co-infection on disease severity is an advancing field however no study reports a decrease in severity with viral-bacterial co-infection. one study [92] evaluated children presenting with bronchiolitis using nasal swabs to identify a plethora of viruses in addition to s. pneumoniae, m. catarrhalis, and h. influenzae. in this study, rsv and s. pneumoniae co-detection was significantly associated with severe disease in the regression analysis. in a cohort study of children admitted with acute respiratory disease, bacterial superinfections were significantly associated with higher illness severity scores (or = 2.12), more severe respiratory distress (or = 4.4), required more respiratory support (or 3.4), and have longer hospital length of stay [93] . it is encouraging to note that the children who received the pneumococcal vaccine had lower illness severity, less respiratory distress, required less respiratory support, and had less admissions to the picu [93] . in a review of the clinical significance of viral-bacterial coinfections in pediatric patients [94•] , 16 studies were found to evaluate clinical severity in this co-infected group. only four of these studies did not observe increased clinical severity, such as more frequent and longer picu admissions or longer ventilation requirements. it is also important to place this discussion in a historical context and discuss the influenza a pandemic of 1918. this pandemic had a mortality rate of 40-50 million people worldwide [15] . it is now known that secondary bacterial pneumonia caused the majority of the deaths during this time [95] . this further underscores that the viral infection alone was not as detrimental as the viral-bacterial co-infection. parainfluenza s. pneumoniae aom, acute rhinosinuisitis [78] a invasive pneumococcal disease: pneumococcus is isolated from a sterile site, i.e., sepsis, meningitis [84] cap community acquired pneumonia, aom acute otitis media, copd chronic obstructive pulmonary disease, lrti lower respiratory tract infection there is still a lot to learn about pediatric respiratory illness co-infections, as the interplay is intricate and not fully understood. looking at viral-viral co-infections, it seems that ultimately rsv seems to be the major decider of severity of infection whether or not the child has one or multiple viruses identified [96] . this information could influence the role of molecular testing in routine hospitalized patients. viralbacterial co-infections, on the other hand, usually lead to more severe diseases. overall, being able to better understand coinfection relationships can aid in the development of therapeutic methods as well as potentially affect the role of molecular testing. conflict of interest all authors declare no conflict of interest. human and animal rights and informed consent this article does not contain any studies with human or animal subjects performed by any of the authors. papers of particular interest, published recently, have been highlighted as: • of importance •• of major importance global, regional, and national causes of under-5 mortality in 2000-15: an updated systematic analysis with implications for the sustainable development goals viral-bacterial co-infections in the respiratory tract viral upper respiratory tract infection and otitis media complication in young children comparison of quantitative reverse transcription-pcr to viral culture for assessment of respiratory syncytial virus shedding virological and immunological outcomes of coinfections asymptomatic shedding of respiratory virus among an ambulatory population across seasons. msphere respiratory pathogens in children with and without respiratory symptoms clinical utility of pcr for common viruses in acute respiratory illness rates of asymptomatic respiratory virus infection across age groups respiratory syncytial virus: infection, detection, and new options for prevention and treatment the burden of respiratory syncytial virus infection in young children community-acquired pneumonia requiring hospitalization among u.s. children human rhinoviruses prospective multicenter study of viral etiology and hospital length of stay in children with severe bronchiolitis insights into the interaction between influenza virus and pneumococcus preventing and treating influenza quantitative review of antibody response to inactivated seasonal influenza vaccines. viruses: influenza other respi underdetection of laboratory-confirmed influenza-associated hospital admissions among infants: a multicentre, prospective study vaccines for preventing influenza in healthy children influenza aassociated epiglottitis and compensatory pursed lip breathing in an infant burden of human metapneumovirus infection in young children virological and immunological outcomes of coinfections attenuation of influenza a virus disease severity by viral coinfection in a mouse model multiple versus single virus respiratory infections: viral load and clinical disease severity in hospitalized children. influenza other respir viruses coinfections of the respiratory tract: viral competition for resources viral and bacterial interactions in the upper respiratory tract dynamics of nasopharyngeal colonisation: the key to pneumococcal disease adenovirus infection enhances in vitro adherence of streptococcus pneumoniae synergistic effect of adenovirus type 1 and nontypeable haemophilus influenzae in a chinchilla model of experimental otitis media adherence of type i streptococcus pneumoniae to tracheal epithelium of mice infected with influenza a/pr8 virus adherence of streptococcus pneumoniae to immobilized fibronectin adhesion mechanisms of staphylococci the respiratory pathogen moraxella catarrhalis adheres to epithelial cells by interacting with fibronectin through ubiquitous surface proteins a1 and a2 rhinovirus disrupts the barrier function of polarized airway epithelial cells defensins: antimicrobial peptides of innate immunity respiratory viruses augment the adhesion of bacterial pathogens to respiratory epithelium in a viral species-and cell type-dependent manner fimbria-mediated enhanced attachment of nontypeable haemophilus influenzae to respiratory syncytial virus-infected respiratory epithelial cells nontypeable haemophilus influenzae and streptococcus pneumoniae bind respiratory syncytial virus glycoprotein respiratory viruses predisposing to bacterial infections: role of neuraminidase the novel parainfluenza virus hemagglutininneuraminidase inhibitor bcx 2798 prevents lethal synergism between a paramyxovirus and streptococcus pneumoniae role of neuraminidase in lethal synergism between influenza virus and streptococcus pneumoniae decreased bacterial clearance from the lungs of mice following primary respiratory syncytial virus infection both influenza-induced neutrophil dysfunction and neutrophil-independent mechanisms contribute to increased susceptibility to a secondary streptococcus pneumoniae infection influenza infection leads to increased susceptibility to subsequent bacterial superinfection by impairing nk cell responses in the lung bactericidal activity of a monocytic cell line (thp-1) against common respiratory tract bacterial pathogens is depressed after infection with respiratory syncytial virus infection with human metapneumovirus predisposes mice to severe pneumococcal pneumonia il-10 is an important mediator of the enhanced susceptibility to pneumococcal pneumonia after influenza infection sustained desensitization to bacterial toll-like receptor ligands after resolution of respiratory influenza infection viral co-infections in pediatric patients hospitalized with lower tract acute respiratory infections virologic study of acute lower respiratory tract infections in children admitted to the paediatric department of blida university hospital frequent detection of viral coinfection in children hospitalized with acute respiratory tract infection using a realtime polymerase chain reaction the impact of dual viral infection in infants admitted to a pediatric intensive care unit associated with severe bronchiolitis diagnostic value of respiratory virus detection in symptomatic children using real-time pcr single, dual and multiple respiratory virus infections and risk of hospitalization and mortality single and multiple respiratory virus infections and severity of respiratory disease: a systematic review interference between respiratory syncytial virus and human rhinovirus infection in infancy epidemiological characteristics of influenza virus and respiratory syncytial virus among children in wuhan area from prevalence of co-infection between respiratory syncytial virus and influenza in children influenza interaction with cocirculating pathogens and its impact on surveillance, pathogenesis, and epidemic profile: a key role for mathematical modelling detection of multiple respiratory viruses associated with mortality and severity of illness in children the impact of multiple viral respiratory infections on outcomes for critically ill children influenza a viruses dual and multiple infections with other respiratory viruses and risk of hospitalisation and mortality. influenza other respir viruses severe lower respiratory tract infection in infants and toddlers from a non-affluent population: viral etiology and codetection as risk factors polymicrobial acute respiratory infections in a hospitalbased pediatric population infection with multiple viruses is not associated with increased disease severity in children with bronchiolitis coinfección vírica en las infecciones respiratorias infantiles large systematic review and meta-analysis on viral-viral co-infections and their role in severity of disease systematic review and meta-analysis of respiratory viral coinfections in children secondary bacterial infections associated with influenza pandemics. front microbiol staphylococcus aureus colonization and non-influenza respiratory viruses: interactions and synergism mechanisms. virulence the role of nasal carriage in staphylococcus aureus infections microbiology of sinusitis invasive disease caused by nontypeable haemophilus influenzae. emerg infect dis a mouse model of lethal synergism between influenza virus and haemophilus influenzae pneumococcal bacterial load colonization as a marker of mixed infection in children with alveolar communityacquired pneumonia and respiratory syncytial virus or rhinovirus infection presence of viral and bacterial pathogens in the nasopharynx of otitis-prone children: a prospective study temporal association between rhinovirus circulation in the community and invasive pneumococcal disease in children virus-induced secondary bacterial infection: a concise review. ther clin risk manag importance of viruses in acute otitis media outgrowth of the bacterial airway microbiome after rhinovirus exacerbation of chronic obstructive pulmonary disease bacterial coinfections in children with viral wheezing high incidence of pulmonary bacterial co-infection in children with severe respiratory syncytial virus (rsv) bronchiolitis rsv mediates pseudomonas aeruginosa binding to cystic fibrosis and normal epithelial cells invasive pneumococcal disease streptococcus pneumoniae: description of the pathogen, disease epidemiology, treatment, and prevention effect of clonal and serotype-specific properties on the invasive capacity of streptococcus pneumoniae use of a whole genome approach to identify vaccine molecules affording protection against streptococcus pneumoniae infection complications and associated bacterial coinfections among children hospitalized with seasonal or pandemic influenza synergistic role of staphylococcal proteases in the induction of influenza virus pathogenicity streptococcus pneumoniae exposure is associated with human metapneumovirus seroconversion and increased susceptibility to in vitro hmpv infection influenzae potentiates airway epithelial cell responses to rhinovirus by increasing icam-1 and tlr3 expression streptococcus pneumoniae colonization of the nasopharynx is associated with increased severity during respiratory syncytial virus infection in young children does viral co-infection influence the severity of acute respiratory infection in children? review of the literature on the role of viral-bacterial co-infections in pediatric respiratory illnesses predominant role of bacterial pneumonia as a cause of death in pandemic influenza: implications for pandemic influenza preparedness respiratory syncytial virus coinfections with rhinovirus and human bocavirus in hospitalized children publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations key: cord-260225-bc1hr0fr authors: sirpilla, olivia; bauss, jacob; gupta, ruchir; underwood, adam; qutob, dinah; freeland, tom; bupp, caleb; carcillo, joseph; hartog, nicholas; rajasekaran, surender; prokop, jeremy w. title: sars-cov-2-encoded proteome and human genetics: from interaction-based to ribosomal biology impact on disease and risk processes date: 2020-07-20 journal: j proteome res doi: 10.1021/acs.jproteome.0c00421 sha: doc_id: 260225 cord_uid: bc1hr0fr [image: see text] sars-cov-2 (covid-19) has infected millions of people worldwide, with lethality in hundreds of thousands. the rapid publication of information, both regarding the clinical course and the viral biology, has yielded incredible knowledge of the virus. in this review, we address the insights gained for the sars-cov-2 proteome, which we have integrated into the viral integrated structural evolution dynamic database, a publicly available resource. integrating evolutionary, structural, and interaction data with human proteins, we present how the sars-cov-2 proteome interacts with human disorders and risk factors ranging from cytokine storm, hyperferritinemic septic, coagulopathic, cardiac, immune, and rare disease-based genetics. the most noteworthy human genetic potential of sars-cov-2 is that of the nucleocapsid protein, where it is known to contribute to the inhibition of the biological process known as nonsense-mediated decay. this inhibition has the potential to not only regulate about 10% of all biological transcripts through altered ribosomal biology but also associate with viral-induced genetics, where suppressed human variants are activated to drive dominant, negative outcomes within cells. as we understand more of the dynamic and complex biological pathways that the proteome of sars-cov-2 utilizes for entry into cells, for replication, and for release from human cells, we can understand more risk factors for severe/lethal outcomes in patients and novel pharmaceutical interventions that may mitigate future pandemics. the sars-cov-2 (covid-19) pandemic has impacted every component of life, including research and medicine. in just a few months from the onset of infections to writing of this review, 10573 papers/objects have been published on sars-cov-2 ( figure 1 ). this body of literature primarily focuses on infectious diseases, the respiratory system, public environmental occupational health, biochemistry molecular biology, virology, immunology, pharmacology, microbiology, and healthcare science services, to name a few fields ( figure 1a ). title extraction of these papers reveals mainly clinically connected terms ( figure 1b) . the extensive infectious disease and clinical base of this literature has yielded knowledge of viral entry, replication, immune response, and transmission. however, in a short window of time, biochemical and molecular biology insights into sars-cov-2 have yielded a smaller body of literature that continues to grow (1267 out of the 10573 items), taking more time for data generation than clinical descriptions. of these 1267 biochemistry/molecular biology items, 934 are primary articles ( figure 1c ). title and abstract word extraction from these biochemistry/molecular biology items, followed by counting mentions of all human (20368) or sars-cov-2 proteins, shows a heavy focus on ace2 and spike (s) proteins ( figure 1d ). the virus primarily enters cells through the interaction of the sars-cov-2 surface glycoprotein, spike (s), interacting with the human encoded ace2, similar to that of the sars virus. 1,2 from the abstract/title terms, we identified 51/346 usages of ace2 and 76/295 of spike. other human proteins with repeated mentions include tmprss2 (13 titles/49 abstract), ace (1/32), furin (2/ 14), dpp4 (2/11), and c3 (2/2). additional sars-cov-2 proteins with mentions include nsp12 (rna-directed rna polymerase, 20/71), nucleocapsid (n, 17/71), membrane (m, 5/48), envelope (e, 4/31), nsp5 (3clpro/mpro, 7/26), nsp8 (3/19), nsp16 (2′-o-methyltransferase, 3/14), orf8 (1/10), nsp10 (3/9), nsp14 (guanine-n7 methyltransferase, 1/8), nsp3 (papain-like protease, 16/6), and nsp15 (uridylate-specific endoribonuclease, 16/4). only nsp6 and nsp11 for sars-cov-2 have no mentions within any of these titles or abstracts for biochemical linked papers on sars-cov-2. overall this suggests a few papers specifically related to sars-cov-2 proteins have been published; however, a large body of literature exists for the original sars and other coronaviruses that can give interpretation of the diverse functions performed by the viral-coded proteins and how they interact with human biology. the advancement of knowledge of the sars-cov-2 proteome has been slower than clinical insights due to the need for experimental work that is slow and that is being hampered by social isolation. the 29903 base-pair single-stranded rna genome of sars-cov-2 (ncbi nc_045512.2) has a 265 base-pair 5′ utr, multiple protein-coding segments, and a 228 base-pair 3′ utr. sars-cov-2 has a 79% genomic similarity with sars-cov, a known human pathogen, with both known to enter cells through the binding of human ace2. 3, 4 in addition to sars-cov and sars-cov-2, five other coronaviruses are capable of human-to-human transmission and infection (hku1, nl63, oc43, 229e, and mers-cov). 5 hundreds of coronaviridae family member genomes have been sequenced in human and other vertebrate hosts, 6,7 and many structures have been solved for coronaviridae species proteins, allowing for systematic assessments of the knowledge base. our group implemented a sequence-to-structure-to-function analysis 8, 9 to understand sars-cov-2 proteins, developing a robust understanding of protein conservation, structure, and molecular dynamics. 10 the data generated for each protein was then developed into the viral integrated structural evolution dynamic database (vistedd), a publicly released database of multiple tools for the virus. the database can be accessed at https://prokoplab.com/vistedd/. these tools consist of educational resources for the proteins coded by sars-cov-2 (molecular videos, 3d protein model prints, amino acid details of conservation, and dynamics), the mapping of critical sites to each protein, and the insights into how sars-cov-2 interacts with human proteins. generating this database has given our team a diverse understanding of sars-cov-2, particularly for host protein interactions of each of the viral proteins. multiple studies have begun building systemic insights for sars-cov-2 infections. multiple groups have performed systematic data assessment of ace2 expression and protein staining, suggesting the physiological cell types that can be targeted by the virus. they have shown expression in many tissues throughout humans, with expression within the lung found on the apical surface of polarized bronchial secretory epithelia cells. 11−14 once the virus enters the cells, it results in the alteration of broad biological pathways, including translation, splicing, protein homeostasis, and nucleic acid metabolism. 15 epithelial organoid cultures exposed to the virus produce a robust change in rna expression patterns for cytokine and interferon intracellular immune responses that give rise to tissue signals. 16 single cell profiling within the lungs of patients shows the intracellular cytokine/interferon response results in the recruitment of macrophages in severe cases and t-cells in moderate cases, with a high potential for therapeutic intervention. 17, 18 over activation of the cytokine/interferon response is connected to poor outcomes within patients, correlating with macrophage activation syndrome. 19 additional adverse outcomes for the activation of apoptosis within lymphocytes have been observed and may contribute to the noted lymphopenia. 20 proteomics and metabolomics of patient sera show the same macrophage dysfunction, while also elucidating platelet and complement dysregulation with the identification of severity classifiers. 21−23 in totality, the physiological response to the virus is likely mediated by a combination of immune system activation and the direct human interaction partners, altering cellular processes. an understanding of these detailed biological interactions can shed light on potential therapeutic opportunities while building a fundamental knowledge of viral biology. to date, few studies have been performed that systematically look at mapping how the sars or coronavirus proteins physically interact with human proteins. structural level insights for coronavirus proteins are surprisingly deficient of human interaction partners. 10 a few of these proteins have been targeted for interaction assessments, such as the nucleocapsid protein 24,25 (shown below). it has been speculated that the understanding of virus−host interactions represents a major untapped potential of viral inhibitors. 26 a 2018 review highlights the literature of viral−host interactions for coronaviruses, focused on synergizing the knowledge of independent experiments for virus receptors, translation, membrane dynamics, immune regulation, cell cycle control, and replication. 27 the more recent work by gordon et al. 28 covering the systematic affinity purification of 26 different sars-cov-2 proteins within human cells has elucidated many mechanisms and drug compounds for the regulation of viral processes. 28 bringing this data together with our vistedd tools, we provide a current snapshot of sars-cov-2 viral proteins ( figure 2 ). orf1ab is a large protein that is proteolytically cleaved to produce 16 different proteins, many involved in rna replication. the nmr structure of 2gdt has been solved, 29 and 250 sequences have been identified by basic local alignment search tool (blast). nsp1 interacts with proteins of the alpha dna polymerase ( figure 2 ) and is involved in regulating endonucleolytic rna cleavage of mrna, allowing the virus to enrich viral rna within a cell. 30, 31 nsp1 has been shown to interact with ribosomal subunits, resulting in the inhibition of translation, 5′ mrna capping changes, and mrna destabilization. 32−34 from a sars-cov yeast two hybrid screen, nsp1 was identified to interact with immunophilins, showing that it alters the intracellular immune response. 35 expression of nsp1 drives changes in interferon signaling. 36 these processes make nsp1 a potential virulence factor. 36−38 see prokoplab.com/ nsp1 for additional information. the protein has no solved protein structure, with itassergenerated predictions 39 that are mostly (67%) coiled, and 246 sequences have been identified by blast. all of the protein interaction partners are acetylated ( figure 2 ). the protein has been suggested to be dispensable to viral replication but does impact rates of replication. 40 see prokoplab.com/nsp2 for additional information. the protein has hundreds of solved x-ray crystal structures with a c4 zinc finger, and 3180 sequences have been identified by blast. the papain-like proteinase cleaves the first four nsp proteins, 41 where inhibition can block viral replication. 42 the proteinase can cleave proteins and has been shown to have deubiquitinase activity. 43−45 this deubiquitinase function has been linked to the regulation of immune system cytokine response, 46,47 specifically the type-i interferon signaling pathway, 48 and has connection to virulence. 49 see prokoplab. com/papain_like_proteinase for additional information. the protein has no solved protein structure, with itassergenerated predictions 39 that are mostly (58%) coiled, and 3325 sequences have been identified by blast. nsp4 interacts with several proteins involved in mitochondrial import for inner membrane insertion (figure 2 ). nsp4 and nsp3 interact and form within the membrane and are involved in transcription complex assembly anchoring. 50, 51 the complex is involved in the double membrane secretory vesicle formation 52, 53 in the endoplasmic reticulum, 54 conferring with human protein interaction partners. 28 see prokoplab.com/nsp4 for additional information. nsp5 has hundreds of solved x-ray crystal structures, with the protein found in a dimer form with a cysteine protease function, 55, 56 and 3397 sequences have been identified by blast. the enzyme cleaves most of the proteins of the larger rep protein with a highly conserved specificity, where inhibition is one of the most studied interventions. 57−59 see prokoplab.com/3c-like_proteinase for additional information. the protein has no solved protein structure, with itassergenerated predictions 39 that are mostly (63%) coiled, and 2558 sequences have been identified by blast. nsp6 interacts with multiple proteins involved in atp hydrolysis-coupled cation transmembrane transport ( figure 2 ). the protein is likely transmembrane-localized, along with nsp3/nsp4, 60 and is involved in autophagosome formations. 61−63 the few papers discussing nsp6 suggest a major future area of understanding and pharmaceutical intervention potential. see prokoplab. com/nsp6 for additional information. there are several solved structures for nsp7 that interact with nsp12/nsp8 (6nur, 2ahm, and 3ub0), 64−66 and 3256 sequences have been identified by blast. the nsp7 protein interacts with multiple small gtpases of the ras complex, many of which are prenylation-regulated ( figure 2 ). the nsp7/nsp8/nsp12 complex is a viral rna-directed rna polymerase unit, where nsp12 is enhanced through the binding of nsp7/nsp8. 67 see prokoplab.com/nsp7 for additional information. there are several solved structures for nsp8 that interact with nsp12/nsp7 (6nur and 3ub0), 64−66 and 3339 sequences have been identified by blast. the nsp8 protein interacts with proteins involved in translation, snrna 3′-end processing, 7s rna binding, and ribonucleoproteins ( figure 2 ). in addition to the information provided for nsp7, nsp8 has been suggested to also interact with the orf6 protein. 68 see prokoplab.com/nsp8 for additional information. nsp9 has many known protein structures, with the protein requiring dimerization to function, 69 and 3386 sequences have been identified by blast. nsp9 interacts with multiple proteins of structural constituents of the nuclear pore ( figure 2 ). nsp9 and nsp10 interact with the nuclear factor-κb repressing factor (nkrf) and may cause an interleukin (il)-8/il-6-mediated chemotaxis of neutrophils and an overexuberant host inflammatory response. 70 nsp9 is involved in viral rna synthesis and rna binding, which likely evolved from a protease. 71, 72 see prokoplab.com/nsp9 for additional information. nsp10 has many known protein structures, including those interacting with nsp14 and nsp16, and contains two zinc binding motifs; 73 3344 sequences have been identified by blast. nsp10 stimulates nsp14 3′−5′ exoribonuclease/ mismatch excision 74, 75 and nsp16 2′-o-methyltransferase activities. 76, 77 the interface of interaction with nsp14 and nsp16 overlaps, suggesting a dynamic regulation process 78 that may involve the linkage of functions through a spherical dodecameric structure. 79 a peptide-based inhibition of the nsp10 interaction has been proposed as a potential viral regulator. 80 see prokoplab.com/nsp10 for additional information. nsp11 is a little-known small 1.3 kda peptide with few interaction partners. 28 nsp12 has multiple known protein structures with a zinc active site and a structure that interacts with nsp7/nsp8, 64−66 and 5086 sequences have been identified by blast. nsp12 is involved in the replication of plus-strand rna through complement strand synthesis and then viral rna synthesis. 81 the enzyme is highly targeted for therapeutic inhibition of viruses. it is also known as rdrp and is the target of the drug remdesivir. see prokoplab.com/rna-directed_rna_polymerase for additional information. nsp13 has multiple known protein structures with a zinc active site, and 5598 sequences have been identified by blast. nsp13 interacts with multiple proteins involved in the centrosome−golgi apparatus and centrosome ( figure 2 ) and has a rna and a dna duplex-unwinding ability to separate strands with 5′ to 3′ polarity. 82−84 see prokoplab.com/helicase for additional information. nsp14 has multiple known protein structures with a zinc active site, and 2794 sequences have been identified by blast. nsp14 has an s-adenosyl-l-methionine (sam)-binding pocket and an exoribonuclease function that is involved in rna capping, 85−87 and it interacts with nsp10 and is known to interact with the human ddx1 rna helicase to enhance the virus replication. 88 see prokoplab.com/guanine-n7_ methyltransferase for additional information. nsp15 has multiple known protein structures, and 2489 sequences have been identified by blast. nsp15 is a mn 2+dependent toric monomer to the hexamer enzyme involved in uridylate-specific cleavage 89,90 that may be regulated by nsp7/ nsp8, 91 and it interacts with the retinoblastoma protein to impact the cell cycle 92 and is also known as nendou. see prokoplab.com/uridylate-specific_endoribonuclease for additional information. nsp16 has multiple known protein structures with na, mg, and s-adenosyl-l-methionine (sam), and 2495 sequences have been identified by blast. nsp16 is an sam-based enzyme for the methylation of ribose 2′-oh in viral rna capping 93, 94 and interacts with nsp10. 77 the protein is a critical component in the inhibition of the host type-i interferon response 95 and is also known as 2′-o-mtase. see prokoplab.com/2-omethyltransferase for additional information. the spike surface glycoprotein has multiple known protein structures that are heavily glycosylated and form a trimer complex 96, 97 and is a known structure of the interaction with the dimer of heterodimers ace2/slc6a19 (6m17); 3 6612 sequences have been identified by blast. s is a class-i viral fusion protein 98 and drives the specificity of cell targets through the interaction with ace2 to enter human cells. 99, 100 following binding to the receptor, s undergoes a conformational change to allow viral entry. 101 for the protein to function correctly, it must be proteolytically cleaved by trypsin and, upon cell-binding proteases such as tmprss2, elevate entry through the mediating tropism. 102 s is of interest to the development of immunizations and rapid detection of coronaviruses, as its surface is exposed. 103, 104 see prokoplab. com/spike for additional information. orf3a has no solved protein structure, with itassergenerated predictions, 39 and 65 sequences have been identified by blast. orf3a is a three transmembrane helix protein where the extracellular component localizes the protein to the golgi apparatus with a caveolin-1 binding potential 105 and is involved in the formation of viral particles. 106, 107 orf3a has been shown to impact the cell cycle. 108 see prokoplab.com/3a for additional information. the envelope protein (e) has no solved protein structure, with itasser-generated predictions, 39 and 94 sequences have been identified by blast. e is required for viral particle formation 109 with transmembrane helix-forming pentameric αhelical bundles with channel activity 110 that can contribute to the membrane permeability. 111 see prokoplab.com/e for additional information. the membrane protein (m) has no solved protein structure, with itasser-generated predictions, 39 and 1507 sequences have been identified by blast. m has human interaction partners that are involved in the mitochondrial matrix ( figure 2 ) and is a critical component of viral membranes that are involved in viral budding. 112 see prokoplab.com/m for additional information. orf6 has no solved protein structure, with itassergenerated predictions, 39 and 31 sequences have been identified by blast. two of the interaction partners are involved in the transcription-dependent tethering of rna polymerase ( figure 2 ). orf6 can function toward the inhibition of beta interferons 113 through the regulation of the signal transducer and activator of transcription 1 (stat1) 114 and endoplasmic reticulum (er) stress 115 and can interact with nsp8. 68 see prokoplab.com/orf6 for additional information. orf7a has multiple known protein structures, and 42 sequences have been identified by blast. orf7a has protein interaction partners involved in ribosomal large subunit biogenesis ( figure 2 ) and localizes to the er and golgi network. 116 it can regulate the cell cycle in g0/g1 progression. 117 see prokoplab.com/7a for additional information. orf8 has no solved protein structure, with itassergenerated predictions, 39 and 35 sequences have been identified by blast. orf8 has multiple interaction partners involved in the er lumen (figure 2) and is a protein shared with sarsr-batcov, with a high positive selection. 118 see prokoplab.com/ orf8 for additional information. the nucleocapsid protein (n) has multiple known protein structures, and 2261 sequences have been identified by blast. n has protein interaction partners involved in mrna binding, the ribonucleoprotein complex, and the mrna surveillance pathway (figure 2 ) and is critical for the viral replication 119 in multiple processes, including viral rna stability, replication, and packaging. 120 the protein is modified within the cell, including phosphorylation and adpribosylation. 121, 122 the protein consists of three domains, with the n-terminal domain involved in rna binding, the internal dynamic multimer structured unit, and the c-terminal domain, an acidic dimerization region. 123−125 the protein can interact with rna by serving as a rna chaperone 126 while also interacting with the m protein and human hnrnpa1 through the internal multimerization domain. 127, 128 see prokoplab.com/n for additional information. orf10 has no solved protein structure, with itassergenerated predictions, 39 and is unique to sars-cov-2. very little is known of its molecular function or cellular expression. see prokoplab.com/orf10 for additional information. ■ sars-cov-2 risk factors and genetics based on human protein interactions sars-cov-2 infection exhibits more adverse effects and outcomes in those with other comorbidities, including hypertension, diabetes mellitus, and coronary heart disease. the other risk factors for mortality include older age, elevated d-dimer levels, and a higher sequential organ failure assessment (sofa) score. 129 the mortality associated with sars-cov-2 infection is tied to the patient's progression to multiorgan dysfunction. the elderly are particularly susceptible to severe sars-cov-2 infection, which is most likely due to the immunosuppression and underlying comorbidities associated with advanced age. advanced age has been shown to have a depressive effect on both the innate and the adaptive immune system, known as immunosenescence. this is associated with decreased phagocytosis and the bactericidal effects of neutrophils 130 and is also associated with the downregulation of cytokine signaling 131,132 and innate immune receptors. 133 with sars-cov-2 infection, emphasis is placed on the adaptive immune system to aid in clearing virally infected cells. the elderly population has been shown to have a shift toward inhibitory pathways, particularly in cd8+ t cells and to a lesser degree in cd4+ t cells, 134 which may play a role in allowing disseminated viral spread. this reduction of t cell activity is also joined by the involution of the thymus with age, leading to less naive t cell output, 135 which further depresses immune functions. these accumulative effects on the immune system render the elderly population particularly susceptible to dispersed viral infection at baseline levels, which may ultimately result in viral sepsis. with the immunosenescence and increased prevalence of comorbidities associated with older age, it makes sense that this population is being hit the hardest by sars-cov-2; however, many younger adults who lack the above immunosenescence have also been killed from the infection, some of whom displayed no prior medical history. this aspect points to the idea that genetics may play a role in determining the severity of sars-cov-2 infection. the immune response to sars-cov-2 infection in severe cases characteristically induces lymphopenia, particularly of cd-8+ t cells, and increases il-2, il-6, il-10, and interferon (ifn)-γ levels. 136 this work is backed by multiple proteomic studies identifying biomarkers of severity that connect to the immune system. 21, 22 the cytokine storm induced by sars-cov-2 is not a new phenomenon and has been demonstrated in the pathogenesis of other novel human coronaviruses, including mers and sars-cov-1. 137 similar consequences in severe coronavirus infections appear to stem from the cytokine storm of proinflammatory chemokines and cytokines, eventually resulting in acute respiratory distress syndrome (ards) and multiorgan dysfunction. 138 a previous study on sepsis and cytokine storm indicates the presence of genetic variants in multiple pathways that have a polygenetic contribution. 139 in many patients with sars-cov-2 that have severe infection, the identification of hyperferritinemic sepsis often occurs. fever developed at day 1, sepsis developed at day 10, admission to the intensive care unit occurred at day 12 (for acute respiratory distress syndrome), and death occurred at day 19. critically ill patients, defined as those with septic shock, multiple organ dysfunction/failure, and/or respiratory failure, accounted for approximately 5% of the study population, yet the study population displayed a case fatality rate of 49.0% in early reports from wuhan, china. 129 hyperferritinemia on day 4 and day 7 predicts mortality long before the development of sepsis and intensive care unit admission. hyperferritinemia has been suggested to have genetic associations through pathogenic variants in genes targetable by il1rap and anti-c5 antibodies. 140 type-1 interferonopathies, like heterozygous null variants in irf7, have been shown to result in severe manifestations of seasonal influenza virus. 141 similar monogenetic variants likely exist that lead to the individual risk of severe disease onset from sars-cov-2 in previously healthy patients. much of the genetics around the immune activation leading to a cytokine storm and hyperferritinemic sepsis remains poorly defined and requires future initiatives and cohorts to define these genetic contributions adequately. initial sars-cov-2 infection is commonly associated with fever, cough, malaise, and fatigue. 142 in more severe cases, disseminated intravascular coagulation has been noted with elevated d-dimer levels in the serum of severe covid-19 patients, placing them into thromboembolic risk. 143 recent recommendations have been made to utilize thromboprophylaxis or full-anticoagulation therapy for patients in the thromboembolic risk category. 144 a specific protein−protein interaction was discovered between sars-cov-2's orf8 and the tissue plasminogen activator (tpa) protein of hosts. 28 the tpa, which is encoded by the plat gene, plays a crucial role in thrombolysis by catalyzing the conversion of plasminogen to plasmin, the major enzyme involved in lysis of blood clots. increased the activity of tpa can lead to excessive bleeding, whereas decreased activity is associated with thromboembolus formation, 145 increasing the chances of pulmonary embolism, stroke, and myocardial infarction. the extent to which orf8 interacts with tpa is not well understood, but its involvement may render a patient at risk for thromboembolism, as has been seen in the clinical setting. in a study by ladenvall et al., it was found that the discovered eight single nucleotide polymorphisms and the alu insertion polymorphism at the plat locus were not significant contributors to plasma tpa levels. 146 this finding indicates that inherited variants of the plat gene may not be directly involved with the coagulopathy in sars-cov-2 patients; however, the polymorphisms may render the host's tpa protein to a tighter binding by orf8, yielding greater repression during infection, placing the patient at higher risk for thromboembolism. it has also been shown that sepsis involves upregulation of platelet adhesion molecules and increased circulation of platelet−leukocyte aggregates. 147 this may point toward more of an immune-system-catalyzed coagulopathy, resulting in the presentation of strokes, 148 pulmonary embolisms, 149 myocardial infarctions, 150 and microvascular injury, 151 which impact severe sars-cov-2 patients. as coagulopathy has mainly been investigated in both viral and bacterial sepsis, there may be a dual effect of both the immune-mediated response and the protein−protein interaction of orf8 with the host tpa in cases of sars-cov-2 infection. further investigation is warranted to determine the extent of the interaction of orf8 with the host tpa to determine if it plays into the pathogenesis of sars-cov-2-related coagulopathy. sars-cov-2 has been associated with cardiac dysfunction, including myocardial infarction and heart failure. the underlying mechanisms for cardiac injury currently being hypothesized are indirect cardiac injury from the cytokine storm and inflammatory response, 152 severe hypoxia as a result of ards, 153 and direct viral invasion of cardiomyocytes. 154 interestingly, ace2, the host receptor for sars-cov-2, is expressed in the heart, 155 indicating direct viral invasion could be a potential cause of myocardial dysfunction. sars-cov-2's nonstructural protein 9 (nsp9) was found to interact with the e3-ubiquitin ligase mindbomb homologue 1 (mib1). 28 this ubiquitin ligase is a positive regulator of the delta-mediated notch signaling pathway, which is involved in multiple processes during cardiac development. 156 mutations in the mib1 have been associated with left ventricular noncompaction (lvnc) characterized by left ventricular trabeculations and reductions in cardiac systolic function. lvnc can range from being asymptomatic to presenting heart failure, depending on the extent the mutation has on the notch pathway. 157 the prevalence of lvnc in the general population is estimated to be around 1/5000 to 1/ 30000. patients with the asymptomatic form of lvnc may be at higher risk for exacerbation of cardiac dysfunction following sars-cov-2 due to involvement of this pathway, especially if they are unaware that they have this mutation. this may play a role in the cardiac dysfunction seen in younger sars-cov-2 patients who lack underlying comorbidities. aside from cardiac development, the notch pathway has also been implemented in cardiac repair, which was demonstrated in rat models where the notch 3 and notch 4 pathways were upregulated, thereby reducing postmyocardial infarctions in the setting of heart failure. 158 the mechanism behind the repair process is still under investigation; however, the disruption of the notch signaling pathway by the interaction of nsp9 with mib1 may prove to play a role in the cardiac involvement of sars-cov-2 infection. furthermore, although vertical transmission of sars-cov-2 has not been seen, 159 neonates who have tested positive for the infection may need to have their cardiac function assessed over time due to mib1's role in cardiogenesis and repair. while we present a detailed assessment of two interaction partners' connections to pathology and risk factors, many more likely exist. we postulate that if function of any protein diverges from normal biology to contribute to sars-cov-2 biology, it could result in a similar disease state within the cell as a loss of function or deleterious genetic mutation. thus, to journal of proteome research pubs.acs.org/jpr reviews understand the sars-cov-2-connected diseases through the human protein interactions, we assessed clinvar, a database of clinically identified variants. a query of the 332 sars-cov-2 human interaction partners through clinvar reveals 8311 protein-based variants within the list ( figure 3a ). in total, 188 of the queried 332 genes have a clinvar submission. of these clinvar-connected genes, there are a total of 111 that have a clinical annotation of pathogenic (pathogenic or likely pathogenic), with a total of 2386 different variants ( figure 3b ). the gene with the most pathogenic variants is fbn1, which is known to interact with sars-cov-2 nsp9 and is involved in autosomal dominant familial thoracic aortic aneurysms and aortic dissections and marfan syndrome. a further analysis of clinical disorders connected with those genes with 10 or more pathogenic-associated variants, excluding fbn1 (pkp2, acadm, ppt1, wfs1, col6a1, pcnt, fbn2, bcs1l, ngly1, cyb5r3, acad9, neu1, gnb1, nars2, tcf12, npc2, pigo, cdk5rap2, cenpf, ggcx, fkbp10, tbk1, fbln5, exosc3 , por, gpaa1, and rhoa), reveals a high connection to cardiac, neurological, diabetic, and syndromic biology. the sars-cov-2 orf8 has the most genes connected by interactions to pathogenic clinvar returns from queried genes, followed by protein m, nsp13, nsp7, and orf9c ( figure 3c ). orf8 is connected to 18 genes associated to human genetic diseases (col6a1, ngly1, neu1, npc2, fkbp10, dnmt1, plod2, smoc1, il17ra, adam9, sil1, lox, pofut1, tor1a, hyou1 , edem3, emc1, and hs6st2), with significant enrichment of these genes to protein folding (false discovery rate (fdr) = 0.00095) and endoplasmic reticulum lumen (fdr = 2.99 × 10 −5 ). while only associated with 3 pathogenic interaction partners, the nucleocapsid (n) protein has interesting disease genetics based on previous observations of a process known as viral-induced genetics. 160 the nucleocapsid (n) protein has the potential to impact and change cellular landscapes through the direct regulation of ribosomal biology. 161 the protein−protein interaction map by gordon et al. 28 supports the hypothesis that sars-cov-2 n proteins interact with multiple mrna-binding proteins and ribonucleoprotein complex proteins ( figure 2 ). in multiple viruses, proteins have been shown to interact with these complexes to regulate a process known as nonsense-mediated decay (nmd). 162 nmd is a cellular process involved in the removal of mrna that does not conform to the bulk of cellular mrna, where proteins accumulate on the transcript and direct the cellular degradation of the mrna. 163 the process is primarily used within cells to degrade mrna molecules with nonsense and frameshift genetic variants and those with improper splicing to prevent the cell from producing truncated proteins that can drive dominant-negative or deleterious gain of function outcomes. 164 viral rna is usually suppressed and degraded within cells through nmd, acting as a cellular immune system process. 165−167 thus, an evolutionary arms race has arisen where a virus can propagate more efficiently if it has a protein that can suppress nmd, keeping its rna levels elevated. 168−171 multiple lines of evidence for both sars-cov-2 and sars-cov suggested that the n protein is used to suppress nmd and evade cellular immune processes. nearly all of the coronaviruses and the larger nidovirales order genomes contain the n protein, which has been shown to interact with multiple ribosomal proteins, including crucial nmd factors. 28 directly inhibited by nmd, with the n protein expression blocking this inhibition. 162 positive-sense single-stranded rna viruses, including coronaviruses, are likely targets of nmd due to their many overlapping reading frames, retained introns, and long 3′ utrs present within the cytoplasm of human cells. the n protein interacts with three proteins annotated to the mrna surveillance pathway (upf1, pabpc1, and pabpc4) and several proteins involved in mrna binding and the ribonucleoprotein complex that are all known to have cellular interactions (figure 2 ). while the fine details of the n protein interaction on the factors are poorly understood, the three mrna surveillance genes are well-connected to nmd biology. pabpc1 is known to be critical for nmd, with its removal suppressing nmd. 175, 176 from plants to humans, upf1 is considered a key regulator of nmd through its recruitment of multiple proteins to rna. 177−180 the regulation switch of upf1 is known to be regulated/activated through phosphorylation at various sites to allow its protein interactions, 181−183 while the n protein has been shown in multiple viral species to be phosphorylated 184−188 and likely dynamic in modifications throughout the rna replication and viral lifecycle. 122, 189 these phosphorylation switches and the interaction of n to nmd proteins are potential sites of pharmaceutical or biological regulation that have been undervalued to this point. other notable interactions of the n protein are ras gtpaseactivating protein-binding protein homologues (g3bp1/2) and casein kinase 2 alpha (csnk2a2), 28 suggesting the regulation of stress granule formation. nmd is found at the intersection of a variety of cellular pathways beyond mrna surveillance and viral control. notably, it is closely associated with the integrated stress response requiring translation initiation factor eif2s1 for function. 190 cellular stresses such as hypoxia and er stress lead to the inhibition of nmd via phosphorylation of eif2s1. 190 this phosphorylation typically induces stress granule formation as well, which has been cited to aid viral replication in some cases and weaken them in others. 191 when g3bp1 is depleted within cells, there is a significant impairment of the replication for coronaviruses and respiratory syncytial viruses (rsvs). 191, 192 multiple viruses have been shown to regulate phosphorylation of eif2s1 at varying time points of infection with connection into nmd regulation. 193 stress granule formation can enhance nmd inhibition, such as hypoxic conditions modulating upf1 and eif2s1. 194, 195 the interaction of sars-cov-2 n protein human interactors promotes the inhibition of nmd and enhancement of both viral replication and truncated host polypeptides that can enhance viral pathogenicity (figure 4) . the regulation of nmd by viral proteins is crucial for allowing the viral rna to survive, but nmd processes within cells also regulate multiple endogenous transcripts, several in normal biology, and some based on genetic disease regulation. many genes, including isoforms with early truncation (frameshifts and nonsense codons) and genes involved in amino acid homeostasis, tumorigenesis, and cell cycle control, are activated when nmd is inhibited within a cell. 196−199 in total, this amounts to about 10% of transcripts within a cell that are regulated by nmd processes and could be altered within the cell by sars-cov-2. 196 on top of this, most individuals contain at least one gene where a nonsense or frameshift variant within the genome is being suppressed, being either inherited or somatic. assessments of human genomes reveal that every person has at least one variant regulated by nmd. 200 recently, our group has shown a complex involvement of this regulation with rare human variants, driving adverse outcomes through a process we have termed viral-induced genetics (vig). 160 in a patient with an epstein−barr virus (ebv) infection, they had an adverse immune response of classical hyperferritinemic sepsis like that of severe sars-cov-2 patients. this individual has both whole-exome sequencing in addition to multiple bloodbased rnaseq experiments performed throughout their clinical course. sequencing revealed a heterozygous splicing variant in the gene rnaseh2b, which is associated with recessive aicardi−goutieres syndrome 201 and has been connected to type-i ifn-mediated autoimmune disease. 202, 203 rnaseq of the patient, when healthy, showed that the splicing variant was present at very low levels, suggesting that the copy was being inhibited through nmd. while the patient was healthy for 16 years of life, the ebv was shown by rnaseq to inhibit nmd, resulting in the presence of the splice variant, which resulted in a dominant-negative rnaseh2b protein that drove cell dysfunction. this suggests that many human variants within genes connected to the immune system and viral response, which are usually suppressed by nmd and result in no cellular dysfunction, are activated by the virus through the inhibition of nmd and can give rise to severe viral outcomes. just like in a computer virus, the antivirus of the computer is often targeted. when additional computer code contains a risk to system failure that is inhibited by the antivirus, if the antivirus is shut down, the other system vulnerabilities become present and often contribute to the computer failure. the full extent of these variants and the disease process remain to be elucidated but is a promising avenue for exploration of viral-induced outcomes in sars-cov-2. the sars-cov-2 pandemic represents a unique challenge to scientists. unlike previous pandemics, our knowledge of the genome and its coded proteins was gleaned within weeks of the outbreak, now with thousands of sequences within a short window. this level of insight has allowed for a pivot to a more robust insight into the viral proteome and how it interacts with host proteins. the advancement of protein-based bioinformatics and previous coronavirus research studies have proved useful in defining the function of each protein coded by the virus. here, we show how many of these viral proteins interact with human proteins connected to biological pathways and disease connections, including numerous risk factors from immune to cardiovascular systems. most notably, we highlight literature on the role of the viral nucleocapsid (n) protein in nmd regulation, where the inhibition of nmd allows for viral rna stability while simultaneously activating genetics of cellular processes and viral-induced genetics. while we have seen thousands of publications on sars-cov-2 and other coronaviruses, the details of a proteome-wide knowledge base of sars-cov-2-coded proteins limit our ability to expand into the incredible potential of preventing and mitigating the current pandemic and future pandemics with a larger therapeutic toolset. characterization of coding/noncoding variants for shroom3 in patients with ckd molecular modeling in the age of clinical genomics, the enterprise of the next generation sars-cov-2 (covid-19) structural and evolutionary dynamicome: insights into functional evolution and human genomics expression of the sars-cov-2 cell receptor gene ace2 in a wide variety of human tissues ace2 receptor expression and severe acute respiratory syndrome coronavirus infection depend on differentiation of human airway epithelia receptor ace2 and tmprss2 are primarily expressed in bronchial transient secretory cells hca lung biological network. sars-cov-2 entry factors are highly expressed in nasal epithelial cells together with innate immune genes proteomics of sars-cov-2-infected host cells reveals therapy targets single-cell landscape of bronchoalveolar immune cells in patients with covid-19 eils, r. covid-19 severity correlates with airway epithelium-immune cell interactions identified by single-cell analysis imbalanced host response to sars-cov-2 drives development of covid-19 transcriptomic characteristics of bronchoalveolar lavage fluid and peripheral blood mononuclear cells in covid-19 patients proteomic and metabolomic characterization of covid-19 proteomic fingerprints for potential application to early diagnosis of severe acute respiratory syndrome the sars coronavirus nucleocapsid protein-forms and functions nucleocapsid phosphorylation and rna helicase ddx1 recruitment enables coronavirus transition from discontinuous to continuous transcription virus-host interactomes-antiviral drug discovery host factors in coronavirus replication novel beta-barrel fold in the nuclear magnetic resonance structure of the replicase nonstructural protein 1 from the severe acute respiratory syndrome coronavirus sars coronavirus nsp1 protein induces template-dependent endonucleolytic cleavage of mrnas: viral mrnas are resistant to nsp1-induced rna cleavage suppression of host gene expression by nsp1 proteins of group 2 bat coronaviruses a two-pronged strategy to suppress host protein synthesis by sars coronavirus nsp1 protein severe acute respiratory syndrome coronavirus protein nsp1 is a novel eukaryotic translation inhibitor that represses multiple steps of translation initiation severe acute respiratory syndrome coronavirus evades antiviral signaling: role of nsp1 and rational design of an attenuated strain unique sars-cov protein nsp1: bioinformatics, biochemistry and potential effects on virulence coronavirus nonstructural protein 1: common and distinct functions in the regulation of host and viral gene expression protein structure and sequence reanalysis of 2019-ncov genome refutes snakes as its intermediate host and the unique similarity between its spike protein insertions and hiv-1 the nsp2 proteins of mouse hepatitis virus and sars coronavirus are dispensable for viral replication identification of severe acute respiratory syndrome coronavirus replicase products and characterization of papain-like protease activity a noncovalent class of papain-like protease/deubiquitinase inhibitors blocks sars virus replication deubiquitinating activity of the sars-cov papainlike protease the papain-like protease from the severe acute respiratory syndrome coronavirus is a deubiquitinating enzyme severe acute respiratory syndrome coronavirus papain-like protease: structure of a viral deubiquitinating enzyme sars coronavirus papain-like protease inhibits the tlr7 signaling pathway through removing lys63-linked polyubiquitination of traf3 and traf6 coronavirus papain-like proteases negatively regulate antiviral innate immune response through disruption of sting-mediated signaling sars coronavirus papain-like protease inhibits the type i interferon signaling pathway through interaction with the sting-traf3-tbk1 complex the papain-like protease determines a virulence trait that varies among members of the sars-coronavirus species structure and cleavage specificity of the chymotrypsin-like serine protease (3clsp/nsp4) of porcine reproductive and respiratory syndrome virus (prrsv) mutation in murine coronavirus replication protein nsp4 alters assembly of double membrane vesicles localization and membrane topology of coronavirus nonstructural protein 4: involvement of the early secretory pathway in replication replication is supported by a reticulovesicular network of modified endoplasmic reticulum 3c-like proteinase from sars coronavirus catalyzes substrate hydrolysis by a general base mechanism dissection study on the severe acute respiratory syndrome 3c-like protease reveals the critical role of the extra domain in dimerization of the enzyme: defining the extra domain as a new target for design of highly specific protease inhibitors coronavirus main proteinase (3clpro) structure: basis for design of anti-sars drugs biosynthesis, purification, and substrate specificity of severe acute respiratory syndrome coronavirus 3c-like proteinase the substrate specificity of sars coronavirus 3c-like proteinase topology and membrane anchoring of the coronavirus replication complex: not all hydrophobic domains of nsp3 and nsp6 are membrane spanning coronavirus nsp6 proteins generate autophagosomes from the endoplasmic reticulum via an omegasome intermediate evolutionary analysis of sars-cov-2: how mutation of non-structural protein 6 could affect viral autophagy coronavirus nsp6 restricts autophagosome expansion structure of the sars-cov nsp12 polymerase bound to nsp7 and nsp8 co-factors insights into sars-cov transcription and replication from the structure of the nsp7-nsp8 hexadecamer nonstructural proteins 7 and 8 of feline coronavirus form a 2:1 heterotrimer that exhibits primer-independent rna polymerase activity the sars-coronavirus nsp7+nsp8 complex is a unique multimeric rna polymerase capable of both de novo initiation and primer extension the nonstructural protein 8 (nsp8) of the sars coronavirus interacts with its orf6 accessory protein severe acute respiratory syndrome coronavirus nsp9 dimerization is essential for efficient viral growth virus-host interactome and proteomic survey of pmbcs from covid-19 patients reveal potential virulence factors influencing sars-cov-2 the nsp9 replicase protein of sars-coronavirus, structure and functional insights the severe acute respiratory syndrome-coronavirus replicative protein nsp9 is a single-stranded rna-binding subunit unique in the rna virus world crystal structure of nonstructural protein 10 from the severe acute respiratory syndrome coronavirus reveals a novel fold with two zinc-binding motifs rna 3′-end mismatch excision by the severe acute respiratory syndrome coronavirus nonstructural protein nsp10/ nsp14 exoribonuclease complex structural basis and functional analysis of the sars coronavirus nsp14-nsp10 complex in vitro reconstitution of sars-coronavirus mrna cap methylation crystal structure and functional analysis of the sars-coronavirus rna cap 2′-o-methyltransferase nsp10/nsp16 complex coronavirus nsp10, a critical co-factor for activation of multiple replicative enzymes dodecamer structure of severe acute respiratory syndrome coronavirus nonstructural protein nsp10 coronavirus nsp10/nsp16 methyltransferase can be targeted by nsp10-derived peptide in vitro and in vivo to reduce replication and pathogenesis molecular anatomy of viral rna-directed rna polymerases multiple enzymatic activities associated with severe acute respiratory syndrome coronavirus helicase the severe acute respiratory syndrome (sars) coronavirus ntpase/helicase belongs to a distinct class of 5′ to 3′ viral helicases the human coronavirus 229e superfamily 1 helicase has rna and dna duplex-unwinding activities with 5′-to-3′ polarity structure-function analysis of severe acute respiratory syndrome coronavirus rna cap guanine-n7-methyltransferase functional screen reveals sars coronavirus nonstructural protein nsp14 as a novel cap n7 methyltransferase characterization of the guanine-n7 methyltransferase activity of coronavirus nsp14 on nucleotide gtp the cellular rna helicase ddx1 interacts with coronavirus nonstructural protein 14 and enhances viral replication crystal structure and mechanistic determinants of sars coronavirus nonstructural protein 15 define an endoribonuclease family crystal structure of a monomeric form of severe acute respiratory syndrome coronavirus endonuclease nsp15 suggests a role for hexamerization as an allosteric switch structural and biochemical characterization of endoribonuclease nsp15 encoded by middle east respiratory syndrome coronavirus binding of the methyl donor s-adenosyl-l-methionine to middle east respiratory syndrome coronavirus 2′-o-methyltransferase nsp16 promotes recruitment of the allosteric activator nsp10 coronavirus nonstructural protein 16 is a cap-0 binding enzyme possessing (nucleoside-2′o)-methyltransferase activity ribose 2′-o-methylation provides a molecular signature for the distinction of self and non-self mrna dependent on the rna sensor mda5 assembly of coronavirus spike protein into trimers and its role in epitope expression cryo-electron microscopy structure of a coronavirus spike glycoprotein trimer the coronavirus spike protein is a class i virus fusion protein: structural and functional characterization of the fusion core complex coronavirus spike proteins in viral entry and pathogenesis structure of sars coronavirus spike receptor-binding domain complexed with receptor characterization of severe acute respiratory syndrome-associated coronavirus (sars-cov) spike glycoprotein-mediated viral entry mechanisms of coronavirus cell entry mediated by the viral spike protein severe acute respiratory syndrome coronavirus spike protein expressed by attenuated vaccinia virus protectively immunizes mice potent binding of 2019 novel coronavirus spike protein by a sars coronavirus-specific human monoclonal antibody severe acute respiratory syndrome coronavirus orf3a protein interacts with caveolin severe acute respiratory syndrome coronavirus 3a protein is a viral structural protein the severe acute respiratory syndrome coronavirus 3a is a novel structural protein g1 phase cell cycle arrest induced by sars-cov 3a protein via the cyclin d3/prb pathway nucleocapsid-independent assembly of coronavirus-like particles by co-expression of viral envelope protein genes structure and inhibition of the sars coronavirus envelope protein ion channel expression of sars-coronavirus envelope protein in escherichia coli cells alters membrane permeability coronavirus particle assembly: primary structure requirements of the membrane protein severe acute respiratory syndrome coronavirus open reading frame (orf) 3b, orf 6, and nucleocapsid proteins function as interferon antagonists severe acute respiratory syndrome coronavirus orf6 antagonizes stat1 function by sequestering nuclear import factors on the rough endoplasmic reticulum/golgi membrane orf-6, induces caspase-3 mediated, er stress and jnk-dependent apoptosis structure and intracellular targeting of the sars-coronavirus orf7a accessory protein sars coronavirus 7a protein blocks cell cycle progression at g0/g1 phase via the cyclin d3/prb pathway severe acute respiratory syndrome (sars) coronavirus orf8 protein is acquired from sars-related coronavirus from greater horseshoe bats through recombination selective replication of coronavirus genomes that express nucleocapsid protein the coronavirus nucleocapsid is a multifunctional protein the severe acute respiratory syndrome coronavirus nucleocapsid protein is phosphorylated and localizes in the cytoplasm by 14−3-3-mediated translocation sequence comparison of the n genes of five strains of the coronavirus mouse hepatitis virus suggests a three domain structure for the nucleocapsid protein the nucleocapsid protein of coronavirus infectious bronchitis virus: crystal structure of its n-terminal domain and multimerization properties modular organization of sars coronavirus nucleocapsid protein coronavirus nucleocapsid protein is an rna chaperone characterization of protein-protein interactions between the nucleocapsid protein and membrane protein of the sars coronavirus the sars-cov nucleocapsid protein: a protein with multifarious activities clinical course and risk factors for mortality of adult inpatients with covid-19 in wuhan, china: a retrospective cohort study effect of age on human neutrophil function innate immunosenescence: effect of aging on cells and receptors of the innate immune system in humans altered cytokine production in the elderly cutting edge: impaired toll-like receptor expression and function in aging signaling pathways in aged t cells -a reflection of t cell differentiation the effect of age on thymic function human coronavirus infections: causes and consequences of cytokine storm and immunopathology molecular immune pathogenesis and diagnosis of covid-19 understanding the inflammatory cytokine response in pneumonia and sepsis: results of the genetic and inflammatory markers of sepsis (genims) study adults with septic shock and extreme hyperferritinemia exhibit pathogenic immune variation understanding human autoimmunity and autoinflammation through transcriptomics covid-19) in china: a systematic review and meta-analysis changes in blood coagulation in patients with severe coronavirus disease 2019 (covid-19): a meta-analysis thromboembolic risk and anticoagulant therapy in covid-19 patients: emerging evidence and call for action tissue plasminogen activator genetic variation at the human tissue-type plasminogen activator (tpa) locus: haplotypes and analysis of association to plasma levels of tpa the coagulopathy of acute sepsis covid-19 presenting as stroke pulmonary embolism in patients with covid-19: time to change the paradigm of computed tomography covid-19 and the cardiovascular system: implications for risk assessment complement associated microvascular injury and thrombosis in the pathogenesis of severe covid-19 infection: a report of five cases novel coronavirus outbreak research team. epidemiologic features and clinical course of patients infected with sars-cov-2 in the cardiovascular burden of coronavirus disease 2019 (covid-19) with a focus on congenital heart disease associated with acute respiratory distress syndrome receptor recognition by the novel coronavirus from wuhan: an analysis based on decade-long structural studies of sars coronavirus notch signaling in cardiac development and disease mutations in the notch pathway regulator mib1 cause left ventricular noncompaction cardiomyopathy activation of notch signaling in cardiomyocytes during post-infarction remodeling vertical transmission of coronavirus disease 19 (covid-19) from infected pregnant mothers to neonates: a review viral-induced genetics revealed by multi-dimensional precision medicine transcriptional workflow applicable to covid-19 nucleocapsid protein recruitment to replication-transcription complexes plays a crucial role in coronaviral life cycle interplay between coronavirus, a cytoplasmic rna virus, and nonsense-mediated mrna decay pathway nonsense-mediated mrna decay (nmd) mechanisms the nonsense-mediated decay rna surveillance pathway the host nonsense-mediated mrna decay pathway restricts mammalian rna virus replication nonsense-mediated mrna decay: novel mechanistic insights and biological impact beyond quality control: the role of nonsense-mediated mrna decay (nmd) in a combined proteomics/genomics approach links hepatitis c virus infection with nonsense-mediated mrna decay virus escape and manipulation of cellular nonsense-mediated mrna decay rna virus evasion of nonsense-mediated decay how retroviruses escape the nonsense-mediated mrna decay an interactome map of the nucleocapsid protein from a highly pathogenic north american porcine reproductive and respiratory syndrome virus strain generated using silac-based quantitative proteomics function of a retrotransposon nucleocapsid protein the cellular interactome of the coronavirus infectious bronchitis virus nucleocapsid protein and functional implications for virus biology interaction of pabpc1 with the translation initiation complex is critical to the nmd resistance of aug-proximal nonsense mutations a conserved role for cytoplasmic poly(a)-binding protein 1 (pabpc1) in nonsense-mediated mrna decay upf1 is required for nonsense-mediated mrna decay (nmd) and rnai in arabidopsis mammalian staufen1 recruits upf1 to specific mrna 3′utrs so as to elicit mrna decay nmd factors upf2 and upf3 bridge upf1 to the exon junction complex and stimulate its rna helicase activity interactions between upf1, erfs, pabp and the exon junction complex suggest an integrated model for mammalian nmd pathways upf1 phosphorylations create binding platforms for smg-6 and smg-5:smg-7 during nmd binding of a novel smg-1-upf1-erf1-erf3 complex (surf) to the exon junction complex triggers upf1 phosphorylation and nonsense-mediated mrna decay a post-translational regulatory switch on upf1 controls targeted mrna degradation phosphorylation of the porcine reproductive and respiratory syndrome virus nucleocapsid protein phosphorylation and subcellular localization of transmissible gastroenteritis virus nucleocapsid protein in infected cells severe acute respiratory syndrome coronavirus nucleocapsid protein expressed by an adenovirus vector is phosphorylated and immunogenic in mice phosphorylation of the mouse hepatitis virus nucleocapsid protein effects of phosphorylation of avian retrovirus nucleocapsid protein pp12 on binding of viral rna regulation of hepadnavirus reverse transcription by dynamic nucleocapsid phosphorylation nonsense-mediated mrna decay at the crossroads of many cellular pathways valiente-echeverría, f. strategies for success. viral infections and membraneless organelles innate immune evasion by human respiratory rna viruses mouse hepatitis coronavirus replication induces host translational shutoff and mrna decay, with concomitant formation of stress granules and processing bodies hypoxic inhibition of nonsense-mediated rna decay regulates gene expression and the integrated stress response possible roles in the control of translation and mrna degradation. cold spring harbor perspect nonsense surveillance regulates expression of diverse classes of mammalian transcripts and mutes genomic noise nonsense-mediated rna decay regulation by cellular stress: implications for tumorigenesis nonsense-mediated mrna decay factors act in concert to regulate common mrna targets nonsense-mediated mrna decay in health and disease the rules and impact of nonsense-mediated mrna decay in human cancers a novel rnaseh2b splice site mutation responsible for aicardi-goutieres syndrome in the faroe islands genetically defined autoinflammatory diseases rare adar and rnaseh2b variants and a type i interferon signature in glioma and prostate carcinoma risk and tumorigenesis key: cord-027550-yyqsatqw authors: mammas, ioannis n.; drysdale, simon b.; rath, barbara; theodoridou, maria; papaioannou, georgia; papatheodoropoulou, alexia; koutsounaki, eirini; koutsaftiki, chryssie; kozanidou, eleftheria; achtsidis, vassilis; korovessi, paraskevi; chrousos, george p.; spandidos, demetrios a. title: update on current views and advances on rsv infection (review) date: 2020-06-15 journal: int j mol med doi: 10.3892/ijmm.2020.4641 sha: doc_id: 27550 cord_uid: yyqsatqw respiratory syncytial virus (rsv) infection represents an excellent paradigm of precision medicine in modern paediatrics and several clinical trials are currently performed in the prevention and management of rsv infection. a new taxonomic terminology for rsv was recently adopted, while the diagnostic and omics techniques have revealed new modalities in the early identification of rsv infections and for better understanding of the disease pathogenesis. coordinated clinical and research efforts constitute an important step in limiting rsv global predominance, improving epidemiological surveillance, and advancing neonatal and paediatric care. this review article presents the key messages of the plenary lectures, oral presentations and posters of the '5th workshop on paediatric virology' (sparta, greece, 12th october 2019) organized by the paediatric virology study group, focusing on recent advances in the epidemiology, pathogenesis, diagnosis, prognosis, clinical management and prevention of rsv infection in childhood. precision medicine has evolved in recent years allowing the incorporation of novel taxonomies and stratification of patients, and using standardized clinical endpoints, genetic and other biomarker information (1) . its role in paediatric healthcare involves the selection of targeted diagnostic, therapeutic and prevention strategies matched to precise molecular, epidemiological and clinical profile of each patient; the management of respiratory syncytial virus (rsv) infection represents a good paradigm of precision medicine (2) . rsv is a single-stranded rna virus (figs. 1 and 2), which represents the most frequent viral cause of acute lower respiratory tract infection (alrti) in infants, with a worldwide distribution and seasonal occurrence (2) (3) (4) (5) . it was first isolated in 1956 from nasal secretions of chimpanzees with rhinorrhea and coryza; the novel virus was initially named 'chimpanzee coryza agent' (cca) (6, 7) . in the following year, when cca was also isolated from children with alrti, it gained its final name due to the syncytia observed on electron microscopy; syncytia are formed by fusion of infected host cells with neighboring cells leading to the formation of multi-nucleate enlarged cells. although the formation of syncytia is the hallmark of the cytopathic effect of rsv that is associated with host cellular membrane merging, syncytia are not pathognomonic of rsv (8) . syncytia are also observed in cell culture with several other viruses, such as parainfluenza, hsv-1, hiv and mev. recently, the international committee on taxonomy of viruses (ictv), which authorizes and organizes the classification and naming of viral species, grouped rsv under the genus orthopneumovirus within the family pneumoviridae (9, 10) . bronchiolitis is the most common clinical manifestation of rsv infection in infants and although it is usually self-limiting, in infancy it accounts for a significant number of hospitalizations and paediatric intensive care unit (picu) admissions (3) . despite its association with relatively high morbidity and mortality in premature neonates and in certain paediatric populations with underlying conditions, such as immunodeficiency and congenital heart disease, rsv infection may also lead to hospitalization of previously healthy, full-term infants (11, 12) . rsv-positive bronchiolitis is characterized by airway inflammation and oedema, mucus production and debris leading to airway obstruction and turbulent gas flow. even though various therapeutic interventions have been tried, such as bronchodilators, hypertonic saline and corticosteroids, supportive care remains the mainstay in most settings, with gentle suctioning of nasal secretions, prone position, fluid replacement and oxygen or respiratory support, as necessary. several clinical trials on the management and prevention of rsv-positive bronchiolitis have been recently completed, are underway, or in development (3) . currently, there is rapid expansion of rsv vaccine candidate development and there is hope that one will become available in the near future. of course, the safety of vaccines proposed for primary immunization in an antigen naïve child remains the top priority. this review article summarizes the key messages of the plenary lectures, oral presentations and posters of the '5th workshop on paediatric virology' held in sparta (greece) on october 12th, 2019, which was focused on rsv (table i) understanding the burden of rsv infections in real-time. rsv poses significant disease burden in infants and children worldwide (13) , and the international paediatric community is only beginning to appreciate its global impact in both high and low resource settings (4, 14, 15) . the clinical presentation of an rsv infection depends on the patient's age and individual risk factors (16, 17) . the concept of measuring individual-level differences in disease severity has drawn the attention of both public health stakeholders and regulatory agencies in recent years (18) (19) (20) . the impact of rsv infections of course should be differentiated from the disease caused by influenza and other viral respiratory infections (21, 22) . however, a recent extensive literature review and prospective cohort have shown that in infants and children this cannot be done based on clinical symptoms alone; distinguishing rsv from other viral respiratory infections requires laboratory confirmation (23, 24) . with rsv vaccines and antiviral agents in development, it will be important to: a) diagnose rsv infections in a timely manner; b) differentiate rsv from other forms of acute respiratory infections; and c) communicate the test results back to patients and parents/caregivers along with information on the individual disease risk and severity. the pedsidea programme. the vienna vaccine safety initiative (vivi, https://www.vi-vi.org) is an international non-profit research organization which, in collaboration with academic institutions and public health agencies in europe and the united states, has developed digital tools and programs to improve the quality of care for children and adults with alrti or influenza-like illnesses (ili) (13, 25, 26) . taking a person-centered approach, the vivi disease severity score ('vivi score') is a mobile application enabling healthcare professionals to measure disease severity at the point of care within minutes. it was designed to provide a uniform approach to define ad hoc severity at any given time point, based on extensive literature review as well as who criteria for uncomplicated and complicated ili (27) . in collaboration with the robert koch institute, the vivi score was validated in a cohort of 6,000 children (age 0-18) in berlin, germany and subsequently used in a european pilot project entititled 'partnering for enhanced digital surveillance of influenza-like disease and the effectiveness of antivirals and vaccines' (pedsidea) (27, 28) . since then, the pedsidea programme has been implemented in community clinic networks and adult intensive care units in the unites states for the real-time digital surveillance of influenza and rsv disease incidence and severity at the point of care (29) . the programme is expected to continue monitoring, in great detail, the clinical outcomes of 'natural' rsv and other viral respiratory infections in children and adults, including patients at the extremes of age. understanding the real-world disease burden may help facilitate the study of the effectiveness of novel influenza and rsv antiviral agents and vaccines, once they become available (30) . rsv infection: not for children only. rsv was not recognized as a potentially serious problem in older adults until the 1970s, when outbreaks of the virus occurred in long-term care facilities for the elderly (31) (32) (33) . since then, additional studies in hospitalized adults have suggested that rsv may be an important cause of illness in adults. molecular diagnostics suggest that rsv positive specimens are commonly identified in elderly and high-risk adults, in a frequency similar to that of seasonal influenza (31, 34) . even though a positive rsv respiratory panel does not equate pathogenesis, it has been suggested that rsv may account for as much as 10,000 deaths annually in the united states among individuals above the age of 65 years (31) (32) (33) 35) . this, in addition to the morbidity in infants, has stimulated interest in rsv vaccines and antiviral agents. additional natural history studies are needed to better understand the actual burden of rsv infection among the elderly and high-risk adults. the immune response to rsv infection. maternal antibodies may be able to mitigate rsv disease severity in young infants (17, (36) (37) (38) . it is assumed that the transplacental passage of rsv-specific antibodies occurs predominately during the third trimester of pregnancy. high titers can potentially protect term infants up to four months of age (38) . in premature infants, passive immunity may be 'compromised' (39) , but still has a role to play. the degree to which breast feeding may also contribute to passive immunity and to priming of immune system is currently under investigation (40) . once infection is established, the innate immune system plays a dual role in lowering the viral load and in mounting a secondary immune response. prematurity and other conditions that compromise the immune response may lead to reduced levels of antiviral cytokines, such as the interferons (41) . in infants, reduced signaling by tlrs and altered antigen-presenting cell functions, including low interleukin (il)-12 and enhanced il-6 and il-23 production, coupled with reduced activation of regulatory t cells, may result in an adaptive response that is skewed toward th2 and th17 and away from protective th1 and ctl responses (42) . the potential specific role(s) of certain pattern recognition receptors in humans has/ve been suggested by the fact that certain tlr missense mutations are associated to a phenotype with propensity to wheezing (43) . apart from the individual genetics (44), the stage of lung maturation among term and premature infants also impacts on the th1 to th2 switch. in the case of prematurity, the mucosa prior to alveolarization is being deluged with th2 inflammatory responses, even in the earlier stages of bronchopulmonary dysplasia (45) . impaired th1 activation, coupled with little or no b cell memory, and inhibition of antibody production by ifnγ, produces low-titer, low-affinity antibody (46) . the result may be a poorly protective and dysregulated defense mechanism that leads to bronchiolitis in susceptible infants (43) . later, the host immune response is permanently oriented to the direction of wheezing exacerbations, specifically triggered by rsv (47) . micrornas (mirnas) are involved in post-transcriptional gene regulation and play significant roles in the maintenance of the airway epithelial barrier of the respiratory tract (48) (49) (50) (51) (52) (53) (54) (55) (56) . mirnas have been implicated in the modulation of antiviral defense mounted by host innate and adaptive immunity, involving not only immune effector and inflammatory cells, but also parenchymal cells (50) . respiratory viruses, including rsv, attack, as a primary target, the epithelial cells of the respiratory tract causing an altered expression of distinct mirnas in the airway cells. the human innate immune response inhibits rsv replication early after inoculation, mainly through the action of interferons (53) . multiple mirnas are induced by infection in a cell-type-specific fashion (51) . rsv appears to alter host cell gene expression also through regulation of expression of mirnas related to the interferon response (50, 53) . abnormal expression of mirnas has been detected in both peripheral blood cells and airway epithelial cells in rsv-infected infants (54) . understanding alterations in mirna expression profiles and identifying mirna target genes in relation to the pathogenesis of rsv may help clarify the mechanisms of virus-host interactions, and immune dysfunction leading to airway hyper-reactivity and chronic respiratory diseases, such as asthma (49,50,53,54). there are several methods for the purification, quantification and being able to compare severity over time and/or across cohorts is useful in hospital-based qi programmes but also in multi-centre networks, such as pedsidea understanding the real-world disease burden caused by rsv will facilitate the study of the effectiveness of antivirals and vaccines, once they become available recent epidemiological data indicate that rsv infection is an important illness in elderly and high-risk adults, with a disease burden similar to that of non-pandemic influenza rsv and immune response maternal rsv-specific antibodies transmitted transplacentally during the third trimester of pregnancy are related to rsv disease severity in young infants rsv and mirnas a greater understanding of mirnas may enable them to be used as biomarkers of severe rsv infection and as novel targets for treatment or prophylaxis of rsv infection rsv and thrombocytosis thrombocytosis in rsv-positive bronchiolitis does not require routine prophylactic anti-platelet treatment or further investigations rsv and asthma there is compelling evidence that severe respiratory infection induced by rsv is associated with subsequent development of asthma later in childhood further understanding of the role of rsv in asthma pathogenesis will enable our understanding of the impact of future vaccines against rsv in asthma prevention rsv as a cause of pibo there are only few reports in the literature of children with pibo secondary to rsv as a single infection further research is required in order to investigate the potential impact of rsv co-infection in the severity and worse outcome in children with pibo imaging of rsv infection although imaging cannot diagnose rsv infection, it is important to identify the possible pattern of viral disease, in order to avoid unnecessary administration of antibiotic therapy and predict possible late effects standard radiological techniques, including ct, are unable to distinguish between acute bronchiolitis caused by rsv versus that caused by other respiratory viruses hrct of the lungs may be required to assess possible bronchial thickening and remodeling, the development of bronchiectasis and air-trapping antivirals against rsv ribavirin is currently the only licensed antiviral medication used to treat rsv infection; it has very limited efficacy and multiple toxicities, which means its use is usually reserved for severely immunocompromised children due to ethical and technical constraints human challenge models are only undertaken in adults, but if a product is shown to be efficacious in this setting it allows a faster move to trials in children than traditional trials which often take much longer to do a greater understanding of individual data in newly developed pharmaceutical agents against rsv will potentially lead to future personalized treatment regimens rsv and picu hfnc might have a role as a rescue therapy for children with rsv-positive bronchiolitis admitted to picu to reduce their requirement for high-cost intensive care heliox could be useful in addition to standard medical care in the management of children with rsv-positive bronchiolitis admitted to picu characterization of mirna expression profiles in biofluids, whole blood samples and tissue samples obtained in in vivo studies (55) . further research on mirnas is expected to clarify their value as biomarkers of rsv infections and their sequelae (i.e., recurrent wheezing and asthma). thrombocytosis and rsv infection. the most frequent causes of secondary thrombocytosis in childhood are acute respiratory tract infections (57) (58) (59) (60) (61) . to date, several authors have reported significantly higher mean platelet counts in patients with rsv than in patients with other acute respiratory tract infections (61) (62) (63) (64) . thrombocytosis is more likely to occur in younger patients, who have clinical manifestations of wheezing and dyspnoea (62, 64) . moreover, thrombocytosis has been suggested as an early marker of rsv infection (62) . excessive thrombocytosis has also been detected at an early stage in cases of rsv-positive bronchiolitis (65) . it has been proposed that thrombocytotic patients have a more severe clinical course and longer duration of hospitalization and that the platelet count may be a useful clinical marker associated with alrti severity (61, 64, 66, 67) . conversely, other authors have found that platelet counts do not correlate with disease severity and clinical outcome (68, 69) . routine prophylactic anti-platelet treatment or further investigations are not necessary in children with rsv-positive bronchiolitis and thrombocytosis (61, 68, 70) . there is compelling evidence that infants with severe rsv infection in the early months of life have a subsequent increased risk of developing recurrent wheezing and/or asthma, with a prevalence of up to 30% compared with non-rsv groups (71) (72) (73) (74) (75) . whether this association is causal has been the subject of considerable debate on the potential role of rsv infection in the pathogenesis of asthma as well as the impact of asthma predisposition (genetic, environmental exposure, etc.) on the clinical course of rsv infection. a recent large retrospective cohort analysis of australian children born between 2000 and 2010 suggested that different subgroups of high risk children, who developed rsv disease within the first 2 years of life, continued to be at elevated risk of having a first asthma hospitalization beyond the age of 7 years (76). on the other hand, large epidemiological observational studies demonstrate that the vast majority of infants hospitalized for rsv bronchiolitis do not fit into an 'at-risk' group (atopy, family history, etc.), suggesting that viral or host factors not thought of as classical risk factors, may play a role in disease severity (74, 77) . prospective studies with rsv-immunoprophylaxis (e.g., palivizumab) suggest that long-term effects of rsv prophylaxis appear less efficacious in infants with a family history of atopy. in addition, palivizumab decreases parent-reported recurrent wheeze, however the incidence of physician-diagnosed asthma is similar (78) . a recent single-blind, randomized, placebo-controlled trial showed that rsv prevention in otherwise healthy preterm infants, did not have a major effect on asthma or lung function at the age of 6 years (79). considering the above findings, perhaps a more appropriate conclusion would be that rsv infection is important in the mechanism of wheezing development, at least in the first few years of life (80) . rsv possesses the ability to counteract host defense systems through complex mechanisms that facilitate viral replication. this significant increase in asthma frequency seems to be predominantly related to long-term changes in neuroimmune control of airway tone rather than to allergic sensitization. in contrast to rsv bronchiolitis, atopy has been clearly associated with childhood asthma development after rsv-induced early wheezing (81, 82) . high-risk (parental atopy or asthma) birth cohort studies from wisconsin, united states, and australia have shown that young children suffering from rsv-induced wheezing episodes are at high risk of developing school-age asthma (81, 82) . further prospective, follow-up studies are needed to clarify individual and environmental factors that promote more severe viral illnesses and long-term adverse respiratory outcome of children hospitalized for severe rsv infection. developing a greater understanding of the pathophysiological mechanisms through which rsv causes recurrent wheezing/asthma, will lead to an evidence-based prevention strategy and perhaps reduce the subsequent risk for asthma (71) . the biomarker ccl5 (previously known as rantes, a β-chemoattractant for inflammatory cells including t-lymphocyte subsets), in the nasal epithelium during rsv bronchiolitis, is strongly predictive of physician-diagnosed asthma (83, 87) . furthermore, it has been suggested that prematurely born infants have a predisposition to rsv infection-related respiratory morbidity, including subsequent respiratory dysfunction (44,85). single-nucleotide polymorphisms in genes coding for il-8, il-19, il-20, il-13, mannose-binding lectin, ifng and rantes, have been associated with wheezing following rsv lrti in term-born infants (85) . the site of infection might be another important factor related to asthma risk, thus viral alrti in infancy indicates an increased risk of subsequent asthma, while gastrointestinal infections might be protective (86) . asthma after severe rsv bronchiolitis is positively correlated with maternal asthma, exposure to high levels of dog allergen, aeroallergen sensitization and recurrent wheezing; day care attendance and white race have been associated with decreased asthma risk (87) . several host factors, including respiratory allergy and virus-induced interferon responses, viral virulence factors, individual risk factors (e.g., young age, especially the first 6 months of life, small lung size and genetics), and environmental exposures (e.g., exposure to tobacco smoke, airway microbiome) modify the risk of virus-induced wheezing and promote more severe wheezing illnesses and the risk for progression to asthma (86, 88) . the anti-rsv mab palivizumab decreases the risk of severe rsv-induced illness and subsequent recurrent wheeze in prematurely born infants (89) . further understanding of the role of rsv in asthma pathogenesis may help develop vaccines against rsv as a way of asthma prevention. bronchiolitis obliterans (bo) is a chronic and irreversible lung disease leading to the obstruction and/or obliteration of the small airways (90) . most cases of bo in children are post infectious (pibo) and are mainly associated with adenovirus infections, although other viruses may also be implicated, including measles, influenza, parainfluenza and rsv (91) (92) (93) . an extensive search of the current literature in the context of the workshop demonstrated that rsv is detected in children with pibo with an incidence ranging from 4.3 to 30% (94-103); however, there are only a few reports of children with pibo secondary to rsv as a single infection. this creates skepticism about the aetiological role of rsv in pibo. further research is required to investigate the potential impact of rsv co-infection in the severity and outcome of children with pibo. chest radiography and rsv-positive bronchiolitis. although imaging cannot confirm the diagnosis of rsv infection, it is important to identify the possible pattern of viral disease, in order to avoid unnecessary administration of antibiotic therapy and to predict possible late effects (figs. 3-5) (104). the clinical syndrome of bronchiolitis is commonly diagnosed based on the patient's history and physical examination; chest radiography is not routinely recommended to reach the diagnosis due to recommended restriction of radiation exposure in the paediatric age group (104) (105) (106) . chest imaging, however, may be considered when a child with rsv infection and severe alrti is admitted to intensive care to better understand the extent of lung involvement and atelectasis, which is common in acute rsv infection (106). it is important to note that chest radiographs in children with rsv infection may be entirely normal or reveal non-specific findings, which are also encountered in other viral infections: most commonly, perihilar opacities and hyperinflation, atelectasis and rarely consolidation and bronchial cuffing or air-leak (107) . radiography is commonly obtained to rule out atelectasis and foreign body aspiration. guidelines suggest performing a chest radiograph in the presence of significant respiratory distress or hospitalization (108) . in newborns with rsv infection, the radiological pattern on chest radiography may be a predictor of clinical outcome (109) . however, it is highlighted that chest radiographs should not be routinely performed in children with bronchiolitis to avoid radiation exposure (106). it is also important to emphasize that chest radiograph is not the right way to rule out bacterial infection (106); the correct diagnostic approach for bacterial or ventilator-associated pneumonia in children in the picu is to perform respiratory culture or matrix-assisted laser desorption ionization time-of-flight (maldi-tof) mass spectrometry from sputum/aspirate or bronchoalveolar lavage (bal) specimens. chest ct and rsv-positive bronchiolitis. several studies have revealed that standard radiological techniques, including computed tomography (ct), are frequently unable to distinguish between acute bronchiolitis changes caused by rsv vs. those caused by other respiratory viruses (104, 108) . it is interesting that the radiographic findings, especially in high-resolution computed tomography (hrct), reflect the histopathologic changes that rsv infection provokes: plugging or occlusion of the bronchiolar airway lumens by sloughed necrotic and irregular epithelium and exudate, combined with peri-bronchiolar infiltration and reaction with inflammatory cells and submucosal oedema. the infiltration is a combination of neutrophils entering the airway submucosa and epithelial cell debris in the airway lumens. these cellular accumulations are likely to result in acute obstruction of the distal airways, an outcome much more likely to occur in the extremely narrow bronchioles of infants. because this is combined with the inherent loss of mechanical clearance of these small airways, it likely leads to increased spread of infection, augmented inflammation and clinical signs of wheezing/obstruction (110) . consistent with obstruction, the most common ct findings in rsv pneumonia include centrilobular nodules, ground-glass opacities, air-space consolidation, and peribronchial thickening (111) . these findings have a bilateral, usually asymmetric, central and peripheral distribution. up to 40% of children with bronchiolitis will develop further wheezing episodes in the first five years of life. in very severe or atypical cases, hrct of the lungs may be required to assess the extent of bronchial thickening and remodeling, the development of brochiectases and air-trapping (105) . restricted to the airways. case reports have also described clinical pictures resembling viral encephalitis and/or encephalopathic syndromes with severe sequelae in isolated cases (112) . the mechanism of the spread of the rsv infection to the cns compartment remains unclear (112) . brain magnetic resonance imaging (mri) in infants with cns involvement has shown predominantly non-specific findings similar to those also encountered in other viral and/or limbic system encephalitides (113) . in very rare instances, extra-pulmonary findings in rsv infection have also included acute necrotizing encephalopathy (ane) and acute hepatic failure with encephalopathy (114) . clinicians should have a high suspicion of ane in cases of children with a respiratory infection and acute neurological manifestations. antivirals against rsv. thus far, ribavirin is the only antiviral agent that has ever been licensed for the treatment of rsv infection (115) . however, its efficacy is not proven and due to significant toxicity its use has been primarily restricted to severe cases in immunocompromised patients with severe rsv-positive alrti (116) . several other antiviral candidates have been developed since, but none have been licensed as yet. types of molecules being tested include influenza antivirals, such as baloxavir, cc-42344, vis410, immunoglobulin, hyperimmune plasma, mhaa4549a, pimodivir (jnj-63623872), umifenovir, and ha minibinders, rsv antivirals including presatovir (gs-5806), ziresovir (ak0529), lumicitabine (als-008176), jnj-53718678, jnj-64417184, and edp-938, broad spectrum antivirals such as favipiravir, vh244, remdesivir, and eidd-1931/eidd-2801, as well as host directed strategies including nitazoxanide, eritoran, and diltiazem (117-119). novel molecules disrupt various stages of the virus life cycle, including cell entry, viral replication, and polymerization as well as after virus release through rsv neutralizing anti-or nanobodies (120) . the human challenge models. one method used occasionally in phase 2 clinical testing is human challenge models (115) . this method was used in phase 2a clinical testing of the non-fusion inhibitor edp-938 (clinicaltrials.gov identifier: nct03691623). all participants were inoculated with a known strain of rsv and were then randomized to receive the medication or placebo. the advantage of this methodology is the removal of the variability in exposure with natural infection and the collection of samples at precise, known times after infection, which can aid with the understanding of the biological mechanisms of the infection and development of antiviral agents or vaccines (121, 122) . due to ethical and technical constraints, experimental studies are only undertaken in adults, but if a product is shown to be efficacious at this setting, a faster move to trials in paediatrics takes place than in traditional childhood trials. rsv therapeutics and personalized medicine. the current clinical data indicate that rsv disease dynamics may not be identical in all patients (2, 3, 115, 119) . more research is needed to identify uniform clinical endpoints reflecting how patients function, thrive, and survive (us food and drug administration) and to understand inter-individual differences in disease presentation, with the goal of ultimately selecting the right treatment for the right patient. a greater understanding of individual differences may ultimately lead to future personalized treatment strategies. individualized approaches and a well-standardized methodology to assess disease severity at the time of enrolment, as well as during follow-up visits, will require integration of diagnostic, clinical and laboratory markers at the point of care (115) . individualized targeted treatment will constitute an important step in improving outcomes in patients with rsv infection while minimizing toxicity. a greater understanding of individual data in newly developed pharmaceutical agents against rsv will potentially lead to future personalized treatment regimens. applying such co-ordinated diagnostic, clinical and research efforts constitutes an important step in advancing paediatric care, improving outcomes and limiting global rsv morbidity and mortality. over the last decade, high-flow nasal cannula (hfnc) therapy has emerged as a new method to provide respiratory support in children with rsv-positive bronchiolitis (12, (123) (124) (125) (126) . its main advantages include its ease to set up and the fact that it is well tolerated, leading to better compliance, especially in comparison to other devices of non-invasive ventilation (125, 127) . initially, hfnc was trialed in infants with moderate to severe bronchiolitis admitted to picus, but nowadays its application has expanded to paediatric wards, even to emergency departments, in order to avoid a picu admission (127, 128) . recent data have shown that it does not significantly reduce time on oxygen compared with standard therapy, suggesting that early use of hfnc does not modify the underlying disease process (129) . however, the proportion of children who experi-ence treatment failure is lower in hfnc and many of those who experience treatment failure on standard therapy can be rescued by hfnc. additional studies comparing hfnc with continuous positive airway pressure (cpap) in the picu setting led to the same conclusion (130, 131) . consequently, hfnc may reduce the need for intubation and invasive respiratory support, thus potentially lowering costs and adverse effects of mechanical ventilation, such as ventilator-induced lung injury, infections and exposure to sedatives. in addition to effectiveness, most studies have shown no adverse events with hfnc and have concluded that it is a relative safe method for use even in general wards or emergency departments (123) . few cases of pneumothorax have been reported, abdominal distension has been less significant compared with cpap, and the majority of infants have been able to be fed orally or by nasogastric tube (123, 127) . heliox and rsv-positive bronchiolitis. since rsv-positive bronchiolitis is associated with airway obstruction and turbulent gas flow, its clinical course can be improved by heliox, which facilitates gas flow through high-resistance airways (132) (133) (134) (135) (136) (137) (138) . heliox is a mixture of helium-oxygen, which can be administered by all modes of ventilation in spontaneously breathing patients by face mask, hfnc or cpap, and can be adjusted to specific ventilators in intubated children. current evidence suggests that the addition of heliox may significantly reduce clinical scores evaluating respiratory distress and the respiratory rate, and may enhance co 2 elimination in the first hour after starting treatment in infants with acute refractory rsv bronchiolitis (134, 139) . recently, seliem and sultan (140) reported that heliox results in improvement of oxygenation when used with high flow nasal cannula in infants with acute rsv bronchiolitis, during the initial phase of therapy. the combination of heliox with cpap also seems to be beneficial, as the application of cpap may reduce the fio 2 needed in these infants. however, no benefit has been observed in terms of need for intubation and mechanical ventilation, length of treatment or picu stay. in addition, its application in the emergency department does not change the discharge rate (138, 139) . more clinical trials are needed to define the population that may respond to heliox and its place in the therapeutic regimens of rsv bronchiolitis. passive immunization and palivizumab. the prevention of rsv morbidity and mortality remains a global healthcare priority (115, 141) . according to the world health organization (who), the strategic focus for the prevention of rsv infection in children and adults includes the passive administration of immunoglobulins, as well as active immunization. passive immunization is currently the only option available to infants less than 6 months of age, which can be achieved through administration of antibodies to the infant or through active immunization of the mother during pregnancy. passive immunity wanes fast over time, thus, active immunization is the preferred approach for infants above six months of age, as well as older children and adults, including the elderly (141). to date, there is only one product available for prevention of rsv infection, palivizumab, the monoclonal antibody (mab) that has been shown to reduce hospital admission due to rsv infection in some high-risk infants by up to 80% (142) . it is expensive and, thus, reserved for high risk infants, mainly in high income countries. active immunization against rsv: looking back to the past. while antibodies are costly and transitory in their effect, active vaccination would represent the most cost-effective approach for the prevention of rsv infections and their transmission to high-risk individuals (115, 141) . up to date, several vaccine candidates are in development, but none have reached licensure yet (143, 144) . one of the main barriers for the development of rsv vaccines has been the fact that the majority of severe cases in infants occur within the first three months of life, i.e. at a time when active immunization is not really possible (145) . additional caution has been employed during vaccine design because of the failure of a historical vaccine [formalin inactivated rsv (fi-rsv)], which triggered a severe adverse effect, enhanced respiratory disease (erd) (146) . for more than 50 years live-attenuated vaccine approaches have been unsuccessful because of the difficulty in balancing immunogenicity and vaccine safety. it is worth noting that only live-attenuated vaccines have been tested for active infant immunization. active immunization against rsv: perspectives. recent breakthroughs in determining the structure and antigenic content of the rsv fusion (f) glycoprotein has enhanced interest in vaccine development research (115, 141, 147, 148) . the general approaches to vaccine development include engineered viruses that use knowledge of rsv gene function, naturally attenuated chimeric virus combining genes from rsv-related viruses, viral vectors encoding rsv surface antigens, and nucleic acid vaccines using plasmid dna or messenger rna encoding rsv antigens (149, 150) . as of august 2019, 43 rsv vaccines were in development (151). of these, 21 are in clinical trials in humans; 14 in phase 1, five in phase 2 and two (one just completed) in phase 3. twelve vaccines are in trials in children, four in pregnant women and 10 in older adults (some products are undergoing trials in more than one target population). vaccine types under investigation include live-attenuated/chimeric, particle-based, subunit and recombinant vector vaccines. this highlights the variety and breadth of immunization types and different populations that are being investigated to find an answer to the 60-year-old problem of producing a safe and effective rsv prophylactic agent. the resvax. the most advanced candidate vaccine, resvax, is an rsv fusion protein recombinant nanoparticle with aluminum phosphate as an adjuvant (115, 152, 153) . the new approach to develop this vaccine is based on engineering small particles that carry altered rsv proteins. the nanoparticles sensitize the immune system to the virus so that when a person comes in contact with it the immune system delivers a robust response. the phase 3 clinical trial included more than 4,600 pregnant women examining the efficacy of prevention of rsv disease in infants through maternal immunization. although the trial narrowly missed its primary end point of a reduction in medically attended rsv-positive alrti, it showed a 44% vaccine efficacy against rsv hospitalization, 25% efficacy against all respiratory hospitalizations and 39% efficacy against all-cause severe hypoxaemia (152) . a possible route to licensure is currently being sought with the us food and drug administration (fda) and european licensing agencies, bringing hope of a vaccine that could save the lives of countless young infants worldwide. we would like to thank the participants of the '5th workshop on paediatric virology' (sparta, greece, october 12th, 2019) for their comments, corrections and feedback. we would also like to thank the organizing committee the '24th world congress on advances in oncology' and the '24th international symposium on molecular medicine' for the outstanding hosting of the workshop, as well as all members of the pvsg and the newly founded institute of paediatric virology (ipv) based on the island of euboea for their valuable contribution in the preparation of the manuscript. no funding was received. not applicable. all authors (inm, sbd, br, mt, gp, ap, eik, ck, elk, va, pk, gpc and das) contributed to the conception and design of the study, wrote the original draft, edited and critically revised the manuscript, read and approved the final manuscript. not applicable. not applicable. das is the editor-in-chief for the journal, but had no personal involvement in the reviewing process, or any influence in terms of adjudicating on the final decision, for this article. the other authors declare that they have no competing interests. what is precision medicine? time for a more precise approach to diagnosis, treatment and prevention respiratory syncytial virus: diagnosis, prevention and management rsv global epidemiology network; resceu investigators: global patterns in monthly activity of influenza virus, respiratory syncytial virus, parainfluenza virus, and metapneumovirus: a systematic analysis are we missing respiratory viral infections in infants and children? comparison of a hospital-based quality management system with standard of care recovery of cytopathogenic agent from chimpanzees with coryza syncytia induction by clinical isolates of human respiratory syncytial virus a updating taxonomy changes: rsv is now known as orthopneumovirus ictv taxonomy history: human or thopneumovir us. international committee on taxonomy of viruses (ictv) bronchiolitis: an update on management and prophylaxis respiratory support in bronchiolitis: trial evidence pedsidea network: partnering for enhanced digital surveillance of influenza-like disease and the effect of antivirals and vaccines (pedsidea). influenza other respir viruses global burden of respiratory infections due to seasonal influenza in young children: a systematic review and meta-analysis hospitalizations associated with influenza and respiratory syncytial virus in the united states risk factors associated with rsv hospitalisation in the first 2 years of life, among different subgroups of children in nsw: a whole-of-population-based cohort study rsv global epidemiology network: global, regional, and national disease burden estimates of acute lower respiratory infections due to respiratory syncytial virus in young children in 2015: a systematic review and modelling study european influenza surveillance network: seasonality and geographical spread of respiratory syncytial virus epidemics in 15 european countries advancing drug development for respiratory syncytial virus. duke european centre for disease prevention and control (ecdc): technical workshop: burden of rsv disease in europe are we missing respiratory viral infections in infants and children? comparison of a hospital-based quality management system with standard of care incidence, severity and impact of influenza: a joint meeting organised by the isirv epidemiology group and ecdc can we distinguish respiratory viral infections based on clinical symptoms? lessons from a pediatric inception cohort evaluation of novel second-generation rsv and influenza rapid tests at the point of care vienna vaccine safety initiative promoting evidence-based vaccine safety research and communication -the vienna vaccine safety initiative influenza and other respiratory viruses: standardizing disease severity in surveillance and clinical trials pedsidea network: partnering for enhanced digital surveillance of influenza-like disease and the effect of antivirals and vaccines (pedsidea) the vivi disease severity score: enabling real-time surveillance of influenza disease severity for multi-dimensional mapping respiratory syncytial virus: infection, detection, and new options for prevention and treatment rsv infection: not for children only respiratory syncytial virus infection in elderly and high-risk adults respiratory syncytial virus infection in older adults: an under-recognized problem a multifaceted approach to rsv vaccination overlap between rsv, influenza viruses and human metapneumovirus in childhood the neonatal immune response to rsv infection: advances in our understanding of viral and host cellular interactions viral and host factors in human respiratory syncytial virus pathogenesis potential impact of maternal vaccination on life-threatening respiratory syncytial virus infection during infancy paediatric virology and its interaction between basic science and clinical practice (review) prevention of pediatric respiratory syncytial virus lower respiratory tract illness: perspectives for the next decade alpha/beta interferon receptor signaling amplifies early proinflammatory cytokine production in the lung during respiratory syncytial virus infection the innate immune response to rsv: advances in our understanding of critical viral and host factors genetic predisposition of rsv infection-related respiratory morbidity in preterm infants neonatal immunity, respiratory virus infections, and the development of asthma t follicular helper (tfh) cells in normal immune responses and in allergic disorders dutch rsv neonatal network: respiratory syncytial virus and recurrent wheeze in healthy preterm infants koutsaftiki c: micrornas as potential bio-markers in children with rsv infection micrornas and the immune response to respiratory virus infections respiratory syncytial virus infection of airway cells: role of micrornas annotating high confidence micrornas using deep sequencing data targeting microrna function in respiratory diseases: mini-review respiratory syncytial virus regulates human micrornas by using mechanisms involving beta interferon and nf-κb micrornas: mediators and therapeutic targets to airway hyper reactivity after respiratory syncytial virus infection microrna profiling from rsv-infected biofluids, whole blood, and tissue samples targeting mirnas to treat hepatitis c virus infections and liver pathology: inhibiting the virus and altering the host thrombocytosis and rsv infection in hospitalized children with bronchiolitis thrombocytosis in childhood a cross-sectional retrospective study to analyze the underlying causes and clinical characteristics of children with reactive thrombocytosis at a korean tertiary medical center thrombocytosis at an early stage of respiratory tract viral infection respiratory syncytial virus-positive bronchiolitis in hospitalized infants is associated with thrombocytosis association between secondary thrombocytosis and viral respiratory tract infections in children respiratory syncytial virus (rsv) bronchiolitis and excessive thrombocytosis epidemiology, clinical characteristics, laboratory findings and severity of respiratory syncytial virus acute lower respiratory infection in malaysian children thrombocytosis in pediatric patients is associated with severe lower respiratory tract inflammation reactive thrombocytosis in children with viral respiratory tract infections incidence and clinical significance of reactive thrombocytosis in children aged 1 to 24 months, hospitalized for community-acquired infections extreme thrombocytosis in admissions to paediatric intensive care: no requirement for treatment korovessi p: rsv bronchiolitis and paediatric asthma association between respiratory syncytial virus infection in infancy and subsequent asthma: a meta-analysis of observational studies association between respiratory syncytial virus hospitalizations in infants and respiratory sequelae: systematic review and meta-analysis eaaci task force on clinical practice recommendations on preschool wheeze: bronchiolitis needs a revisit: distinguishing between virus entities and their treatments the burden and long-term respiratory morbidity associated with respiratory syncytial virus infection in early childhood association between respiratory syncytial viral disease and the subsequent risk of the first episode of severe asthma in different subgroups of high-risk australian children: a whole-of-population-based cohort study exploring the association between severe respiratory syncytial virus infection and asthma: a registry-based twin study scientific committee for elucidation of infantile asthma: palivizumab prophylaxis in preterm infants and subsequent recurrent wheezing. six-year follow-up study respiratory syncytial virus prevention and asthma in healthy preterm infants: a randomised controlled trial iris (infección respiratoria infantil por virus respiratorio sincitial) study group: long-term burden and respiratory effects of respiratory syncytial virus hospitalization in preterm infants -the spring study rhinovirus-induced first wheezing episode predicts atopic but not nonatopic asthma at school age early life rhinovirus wheezing, allergic sensitization, and asthma risk at adolescence koutsaftiki c: predicting asthma following rsv-positive bronchiolitis in early childhood viral lower respiratory tract infections and preterm infants' healthcare utilisation respiratory syncytial virus infection and chronic respiratory morbidity -is there a functional or genetic predisposition? viral respiratory infection and the link to asthma determinants of asthma after severe respiratory syncytial virus bronchiolitis predictors of asthma following severe respiratory syncytial virus (rsv) bronchiolitis in early childhood role of viral infections in the development and exacerbation of asthma in children post infectious bronchiolitis obliterans caused by respiratory syncytial virus (rsv) in children diagnosing and managing bronchiolitis obliterans in children post-infectious bronchiolitis obliterans in children predictors of severity and mortality in children hospitalized with respiratory syncytial virus infection in a tropical region clinical analysis of 28 cases of bronchiolitis obliterans successful lung volume reduction surgery in an infant with emphysema after respiratory syncytial virus-induced obliterative bronchiolitis clinical characteristics of bronchiolitis obliterans in pediatric patients work of breathing to optimize noninvasive ventilation in bronchiolitis obliterans bronchiolitis obliterans: outcome in the medium term clinical studies of children with bronchiolitis obliterans pos-infectious bronchiolitis obliterans in children simultaneous viral infection and childhood bronchiolitis obliterans bronchiectasis and bronchiolitis obliterans post respiratory syncytial virus infection: think again postinfectious bronchiolitis obliterans in children: clinical and radiological profile and prognostic factors imaging in children with rsv infection national institute for health and care excellence (nice): bronchiolitis in children: diagnosis and management, nice guideline respiratory syncytial virus infection of the lower respiratory tract: radiological findings in 108 children chest radiographic features of human metapneumovirus infection in pediatric patients is radiological appearance of lower respiratory tract infection due to respiratory syncytial virus a predictor of clinical outcome? psv and its propensity for causing bronchioloitis imaging of pulmonary viral pneumonia a fatal case associated with respiratory syncytial virus infection in a young child respiratory syncytial virus-related encephalitis: magnetic resonance imaging findings with diffusion-weighted study atypical presentations of respiratory syncytial virus infection: case series management of rsv infection in children: new advances and challenges safety issues related to the administration of ribavirin overview of current therapeutics and novel candidates against influenza, respiratory syncytial virus, and middle east respiratory syndrome coronavirus infections new therapies for acute rsv infections: where are we? advances in respiratory virus therapeutics -a meeting report from the 6th isirv antiviral group conference a review of therapeutics in clinical development for respiratory syncytial virus and influenza in children the future of flu: a review of the human challenge model and systems biology for advancement of influenza vaccinology rsv-specific airway resident memory cd8+ t cells and differential disease severity after experimental human infection high-flow warm humidified oxygen via nasal cannula and rsv-positive bronchiolitis among children admitted to picu high-flow nasal cannula therapy for infants with bronchiolitis paris and predict: early high flow nasal cannula therapy in bronchiolitis, a prospective randomised control trial (protocol): a paediatric acute respiratory intervention study (paris) non-invasive ventilation for the management of children with bronchiolitis (novembr): a feasibility study and core outcome set development protocol high-flow warm humidified oxygen versus standard low-flow nasal cannula oxygen for moderate bronchiolitis (hfwho rct): an open, phase 4, randomised controlled trial a randomized trial of high-flow oxygen therapy in infants with bronchiolitis humidified high-flow nasal cannula oxygen in bronchiolitis reduces need for invasive ventilation but not intensive care admission outcomes of children with bronchiolitis treated with high-flow nasal cannula or noninvasive positive pressure ventilation groupe francophone de réanimation et d'urgences pédiatriques (gfrup): high flow nasal cannula (hfnc) versus nasal continuous positive airway pressure (ncpap) for the initial respiratory management of acute viral bronchiolitis in young infants: a multicenter randomized controlled trial (tramontane study) heliox and rsv-positive bronchiolitis the history and physics of heliox helium-oxygen mixture: clinical applicability in an intensive care unit the therapeutic use of helium use of heliox delivered via high-flow nasal cannula to treat an infant with coronavirus-related respiratory infection and severe acute air-flow obstruction noninvasive ventilation with helium-oxygen in children heliox inhalation therapy for bronchiolitis in infants heliox delivered by high flow nasal cannula improves oxygenation in infants with respiratory syncytial virus acute bronchiolitis prevention of rsv infection: what is new with the vaccines? immunoprophylaxis against respiratory syncytial virus (rsv) with palivizumab in children: a systematic review and economic evaluation who rsv vaccine consultation expert group: who consultation on respiratory syncytial virus vaccine development report from a world health organization meeting global burden of acute lower respiratory infections due to respiratory syncytial virus in young children: a systematic review and meta-analysis development of respiratory syncytial virus (rsv) vaccines for infants enhancement of respiratory syncytial virus pulmonary pathology in cotton rats by prior intramuscular inoculation of formalin-inactivated virus vaccine development for respiratory syncytial virus vaccines against respiratory syncytial virus: the time has finally come recombinant low-seroprevalent adenoviral vectors ad26 and ad35 expressing the respiratory syncytial virus (rsv) fusion protein induce protective immunity against rsv infection in cotton rats nucleoside modified mrna vaccines for infections diseases will the resvax vaccine be key revenue driver for novavax. market realist key: cord-253223-us0ey8dq authors: chow, brian d.w.; esper, frank p. title: the human bocaviruses: a review and discussion of their role in infection date: 2009-11-03 journal: clin lab med doi: 10.1016/j.cll.2009.07.010 sha: doc_id: 253223 cord_uid: us0ey8dq respiratory tract infections are a leading cause of morbidity and mortality worldwide. the human bocavirus (hbov) is a newly recognized human parvovirus first reported in 2005. since its discovery, this virus has been associated with upper and lower respiratory tract disease and gastroenteritis worldwide. this article is a comprehensive review of what is known about hbov. it includes an evaluation of diagnostic modalities, symptoms occurring in affected patients, and a discussion as to whether hbov is responsible for identified clinical manifestations. the article reviews the incidence and effect of coinfection and updates on related members (hbov-2 and hbov-3) recently reported. understanding of respiratory viruses such as hbov remains vitally important to the health of adult and pediatric patients. associated with upper and lower respiratory tract disease and gastrointestinal illness in adult and pediatric patients throughout the world. recently, two viruses closely related to the human bocavirus have been reported, provisionally named human bocavirus 2 (hbov-2) and human bocavirus 3 (hbov-3). 12, 13 for the sake of clarity, the remainder of this article refers to the original human bocavirus strain described by allander and colleagues as human bocavirus 1 (hbov-1). 11 it remains unclear whether hbov-1, hbov-2, and hbov-3 represent unique viral entities or distant genotypes of a single virus. because hbov-2 and hbov-3 have only been recognized over the last several months, the epidemiology and clinical manifestations associated with these viruses are only now being realized. the incidence of human bocaviruses varies widely, 14, 15 with various clinical presentations, and they are often found in association with other potential pathogens. this phenomenon has led to debate over the role of human bocaviruses as true pathogens. this article reviews the published studies and discusses the virologic, clinical, and diagnostic aspects of these newly recognized human viruses. hbov-1 was first described by allander and colleagues 11 in 2005. pooled, cell-free supernatant from respiratory samples was examined using a random polymerase chain reaction (pcr) technique. amplified pcr products were separated, ligated to a vector, and introduced into escherichia coli. clones were sequenced and evaluated through an automated protocol and compared against known sequences. the majority of the clones were determined to be human or recognized bacterial and viral pathogens. of the remaining sequences, a parvoviruslike sequence was identified with no known correlation to recognized human parvoviruses. phylogenetic analysis showed this to be a novel parvovirus most closely related to bovine parvovirus and canine minute virus (fig. 1) . it is now named the human bocavirus. in january 2009, kapoor and colleagues 12 reported the identification of a related parvovirus, termed hbov-2, from pediatric stool samples by random pcr analysis of nuclease-resistant virus particles. the resulting amplicons were subcloned into a plasmid vector, sequenced, and compared with genome entries on genbank. phylogenetic analysis of hbov-2 shows that it is most closely related to hbov-1. the amino acid identity of hbov-2 to hbov-1 is 78%, 67%, and 80% for the ns1, np1, and vp1/ vp2 proteins, respectively. there is significant divergence of the genomic targets used for detection of hbov-1, which explains why hbov-2 has remained unidentified to this point. a third bocavirus species was described in april 2009 from australia, and has been provisionally named human bocavirus 3 (hbov-3). similar to the methods used in discovery of hbov-1 and hbov-2, arthur and colleagues 13 digested filtered fecal samples with nucleases, then pelleted virions and evaluated using degenerate oligonucleotide primed-pcr. during these investigations, the group simultaneously discovered hbov-2 and a third species of bocavirus, hbov-3. analysis of the genome shows close homology to hbov-1 in the nonstructural protein encoding regions (ns1, np1), but hbov-3 is more similar to hbov-2 in the structural protein encoding regions (vp1/vp2). this finding suggests that hbov3 may be the product of recombination between hbov1 and hbov2. arthur and colleagues identified a recombination site that supports this hypothesis. as with hbov-2, the hbov-3 genome is divergent enough from hbov-1 to explain lack of detection using currently published primer sets ( table 1) . clinical presentation, seasonal distribution, and prevalence of hbov3 have yet to be characterized in full. further analysis of the phylogenetic relationships will be required to determine the correct taxonomy of this virus family. human bocaviruses are parvoviruses belonging to the family parvoviridae, subfamily parvovirinae, and are most closely related to bovine parvovirus and canine minute virus, in the genus betaparvovirus (see fig. 1 ). parvoviruses are single-stranded dna viruses with a small genome size between 4 kilobases (kb) and 6 kb that have three open reading frames that encode two nonstructural proteins, ns1 and np1, and the structural vp1 and vp2 proteins. the hbov genome does not encode a polymerase and depends on host cell dna polymerases for replication. parvoviruses have unenveloped capsids with icosahedral symmetry. 16 based on electron micrographs, hbov-1 appears structurally similar to other members of the parvoviridae family and is approximately 25 nm in diameter. 17 the vp2 gene is nested in the reading frame of vp1 and uses an alternate initiator codon. 11 the sequence of vp1 and vp2 differ only in the n-terminal extension of vp1. this region, referred to as the vp1 unique region, plays a critical role in virus infectivity in some parvoviruses. 18 in several parvoviruses, vp3 arises from posttranslational proteolytic cleavage of vp2 proteins. it remains unclear if vp3 exists in human bocavirus. the capsid of parvoviruses consists of about 60 copies of each viral capsid protein. the role of ns1 and np1 proteins in human bocavirus species remain unknown. zhi and colleagues 19 created an infectious clone of the related parvovirus b19 to determine the functions of these genes. their findings showed that ns and vp1 knockout mutants abolished the viral infectivity. the ns protein also seemed to be vital in genome transcription and to play a role in the regulation of capsid gene expression. parvoviruses have been shown to be transmitted by various routes, including respiratory, urine, and fecal-oral contact. 16 the mode of bocavirus transmission of hbov is unknown. most studies have reported the presence of hbov-1 dna in respiratory secretions and fecal samples. hbov-1 dna has also been detected in addition to serum, urine, and lymph nodes. [20] [21] [22] so far, hbov-2 and hbov-3 have only been identified in stool samples. similar to other respiratory viruses, transmission may occur through several routes, including inhalation, contact with infected secretions (through direct contact or possibly contaminated surfaces), and fecal-oral from the standpoint of gastrointestinal illness. although some parvoviruses cannot replicate without helper viruses, the mechanisms of cell entry and replication have not been described for human bocavirus. this lack of information is largely because of the inability to grow human bocaviruses in standard cell lines. following allander's initial description of hbov-1 in the human respiratory tract 11 , more than 60 studies have been published investigating the role of this virus in respiratory tract illness. clinical findings reported from hbov-1-positive patients from 9 representative studies are summarized in table 2 . most patients present with symptoms of upper respiratory tract illness, including cough, fever, and rhinorrhea. pharyngitis and rash are less common (11%-13%). additionally, patients tended to report earache. 14, 23 several studies raise the possibility that hbov-1 is associated with severe respiratory disease. reports describe this virus in children hospitalized for bronchiolitis, 24 asthma exacerbations, 15,25 and first-time episodes of wheezing. 26 wheezing is described in numerous studies. in one prospective study comparing 16 respiratory viruses in children admitted with respiratory infection, wheezing was seen in more than 50% of children in whom hbov-1 was recognized as the sole agent. surprisingly, children who had coinfections had less wheezing. 27 other common findings include tachypnea, fever, and hypoxia. one report from jordan found as many of 18% of children hospitalized with pneumonia were positive for hbov-1. 28 in thailand, fry and colleagues found that patients who had hbov-1 were more likely to be hospitalized with pneumonia (odds ratio of 3.56) compared with controls. most hbov-1 reports are retrospective analyses based from hospital settings in these studies, up to 6.6% of patients required stays in intensive care units and up to 40% required oxygen therapy at some point during their hospitalization. 20, 29 these studies likely overestimate the severity of disease associated with hbov-1 because of selection bias. in studies based on community samples, disease is mild and only rarely requires admission. to date, there have been no reported deaths associated with hbov-1. few studies list underlying comorbidities in hbov-1-infected patients. when reported, more than half of patients had an underlying medical condition. 20 the most common underlying conditions involve primary cardiac or pulmonary disease, including congenital heart lesions, heart failure, asthma, chronic obstructive pulmonary disease, and prematurity with chronic lung disease. 11, 20, 29 hbov-1 has been reported in immunocompromised patients who have respiratory and gastrointestinal illness. schenk and colleagues 22 reported a child coinfected with rhinovirus and hbov-1 following hematopoietic stem cell transplant. this patient developed fever, dyspnea, wheezing, and infiltrates on chest radiograph. in this patient, hbov-1 dna was isolated from nasopharyngeal, stool, and blood samples. koskenvuo and colleagues 30 reported a series of children who had acute lymphoblastic leukemia in whom hbov-1 was identified. presenting symptoms were febrile upper respiratory infection with otitis media, fever with vomiting and diarrhea, and isolated fever. garbino and colleagues 31 reported a case of hbov-1 in a hospitalized adult infected with hiv. all patients eventually recovered from their acute illness. although few studies have systematically examined immunocompromised patients for hbov-1, the incidence seems to be low. one study by muller and colleagues 32 examined bronchoalveolar lavage specimens from immunocompromised patients suspected of having pneumocystis jiroveci pneumonia. only 1 specimen of 128 (0.8%) was found to have hbov-1. miyakis and colleagues 33 prospectively examined bronchoalveolar lavage specimens from 53 adult lung transplant recipients and 67 symptomatic nontransplant patients and failed to find hbov-1 in any sample. these studies argue against hbov-1 as a pathogen of lower respiratory tract disease in these vulnerable populations. because many of these reports observe high coinfection rates, it is difficult to draw conclusions as to whether hbov-1 plays a role in pathogenicity of infected patients. to answer this several studies have compared the presence of hbov-1 in asymptomatic control groups to symptomatic individuals. [34] [35] [36] [37] all but one found hbov-1 more often in symptomatic children than in healthy controls. in the remaining study, asymptomatic carriage of hbov-1 was as frequent in asymptomatic infants as those who had acute respiratory tract disease. in a recent report by allander and colleagues 15 hbov-1 dna is also present in the serum of acutely ill patients suggesting that dissemination of this virus is possible. these findings provide support for hbov-1 as a cause for respiratory illness. when in vitro culture systems and animal models are available, a better understanding of the role of hbov-1 in human respiratory disease can be established. gastrointestinal symptoms are reported in up to 25% of patients who have hbov-1 respiratory infection (see table 2 ) suggesting hbov-1 may not be limited to the respiratory tract alone. the closely related canine and bovine parvoviruses are well described as both respiratory and enteric pathogens. 16 subsequent investigations have identified hbov-1 dna in stool samples from patients with gastrointestinal illness. the most common gastrointestinal symptoms include nausea, vomiting, and diarrhea. both watery and bloody diarrhea have been reported in conjunction with hbov-1. [38] [39] [40] [41] [42] [43] [44] [45] hbov-1 has been identified in 0.8% to 9.1% of stool samples from patients presenting with gastrointestinal illness. [38] [39] [40] [41] [42] [43] hbov-1 viral load in stool is significantly less than viral loads found in respiratory tract specimens of symptomatic patients. 44 similar to studies of respiratory illness, hbov-1 is commonly codetected with recognized enteric pathogens, with coinfection rates ranging from 21% to 77.6%. 41, 43 identified copathogens include norovirus, rotavirus, human calicivirus, astrovirus, adenovirus, campylobacter, salmonella, and clostridium difficile. [38] [39] [40] [42] [43] [44] in a recent casecontrol study on acute gastroenteritis, arthur and colleagues 13 examined stool specimens for potential pathogens, including all three species of human bocavirus. dna from human bocaviruses 1, 2, or 3 was isolated from 54 (27.4%) stool samples, making these human bocaviruses the second most common viral agents identified after rotavirus (37.1%). clinical illness associated with hbov-1 and rotavirus coinfection did not seem to be significantly different than that of rotavirus illness alone, suggesting the presence of hbov-1 did not worsen disease. 42 hbov-1 is infrequently reported in stools originating from immunocompromised patients. in a 4-year-old patient who had diarrhea, bocavirus was recognized on day 21 and again on day 75 following stem cell transplant. this patient went on to develop disseminated disease. 22 one patient who had a t cell deficiency was reported to have hepatitis associated with hbov-1. 46 hbov-1 was also associated with gastroenteritis in one patient who had b cell immunodeficiency. 39 unlike hbov-1-associated respiratory disease, large investigations for hbov-1-associated gastrointestinal illness have not been performed for the immunocompromised population. as with respiratory disease, the role of hbov-1 as a cause for gastrointestinal disease is unclear. several reports have identified hbov-1 in stool samples from asymptomatic patients. 42 , 44 arthur and colleagues compared stool samples positive for hbov-1, hbov-2, and hbov-3 from symptomatic patients and age-matched asymptomatic controls. the presence of hbov-1 failed to achieve statistical significance, supporting the hypothesis that hbov-1 may not be a cause of acute gastroenteritis. 13 the authors note that the lack of hbov-1 in symptomatic individuals may be because symptoms may appear before viral particles are shed in stool. similar findings were made for astrovirus and adenovirus, which are widely accepted causes of gastroenteritis. 13 also, hbov-2 was significantly found more often in patients with gastroenteritis. because of the low incidence in this study, the clinical significance of hbov-3 remains unclear. further data on higher viral load in stool samples corresponding with active disease would support causality. most studies identify coinfecting viral and bacterial pathogens in a substantial percentage of hbov-1-positive samples. although all studies did not examine samples for all pathogens, commonly reported pathogens include enterovirus, rhinovirus, rsv, parainfluenza, influenza, adenovirus, and hmpv ( table 3) . hbov-1 coinfection was a common occurrence in all studies with a median rate of 42.5%. hbov-1 coinfection is described with numerous pathogens; however, those that screen positive for rhinovirus, enteroviruses, and influenza have the highest incidence of hbov-1 coinfection. similarly, in samples found positive for hbov-1, a high rate of enterovirus, influenza, and rsv is seen. although this likely reflects the high frequency of these viruses in the population at large, one may speculate that these particular coinfecting viruses may provide a biologic benefit to hbov. hbov-1 has been recognized worldwide in association with upper and lower respiratory disease. most hbov-1-positive samples originate from children predominantly younger than 2 years of age. 11, 20, 27, 29, [34] [35] [36] [47] [48] [49] [50] [51] [52] [53] within this age group, children less than 6 months of age seem to be less frequently affected, perhaps because of passive maternal immunity. several studies have detected hbov-1 in adults. 20, 54, 55 in one study screening 1539 children and 273 adults, hbov-1 occurred at a similar frequency in both groups. 20 most studies report circulation of hbov-1 predominantly during the winter season. 20,47 seasonal peaks vary but most often are described in the early winter. most of these studies are retrospective, using archived respiratory samples submitted for analysis of common respiratory pathogens. this approach may lead to misrepresentation of when this virus circulates. several reports show increased frequency of hbov-1 infections during the spring. choi and colleagues 56 screened samples over a 5-year period from patients who had lower respiratory tract illness (lrti) in korea and found a higher frequency of hbov-1 between may and july. bastien and colleagues 14 studied samples originating from patients in canada and reported no apparent seasonal prevalence. only a few prospective studies of hbov-1 have been performed. in a 1-year prospective investigation in children less than 5 years of age hospitalized with respiratory tract disease, hbov-1 was detected in every month except august. 51 in this study hbov-1 peaked in the month of december, with 80% of hbov-1 isolates occurring between november and march. several seroepidemiology studies have been performed to assess the prevalence of hbov-1 infection during childhood. kahn and colleagues reported anti-hbov-1 antibodies are common in children. 57 screening serum samples using a viruslike particlebased elisa, kahn and colleagues found evidence of prior hbov-1 infection in 195 of 270 (72.2%) serum samples. 57 children between 4 and 8 months of age had the lowest prevalence of antibodies against hbov-1, whereas children younger than 2 months of age and those older than 5 years had the highest. 57, 58 the high proportion of seropositive children younger than 2 months of age suggests vertical transfer of antibodies. the subsequent decline in the percentage of seropositive children over the first 4 to 6 months of life likely represents waning maternal immunity. the low percentage of anti-hbov antibodies in children younger than 12 months of age correlates with population-based studies that demonstrate children younger than 1 year of age have a higher occurrence of infection. 20, 29, 34 by 5 years of age, most people have circulating antibodies against hbov-1, similar to other respiratory viruses, such as rsv, 59 rhinovirus, 60 and hmpv. 61 because of the difficulties of growing hbov-1 in vitro, it has yet to be determined which antibodies offer protection. also it is unclear if antibodies against hbov-1 will confer protection against hbov-2 or hbov-3 and vice versa. most protective antibodies are speculated to target the two viral capsid proteins vp1 and vp2. hbov-2 and hbov-3 both have approximately 80% aa identity with hbov-1 in this region with most sequence diversion occurring in the n terminal portion. the high similarity of viral capsid proteins suggest cross-reactive antibodies between bocavirus species may occur. studies on hbov-2 and hbov-3 seroepidemiology have not been performed thus far and are warranted. as more bocavirus species are recognized, our understanding of this family of viruses grows. based on the available sequence data of hbov-1, there seem to be two closely 68 58 42 46 30 28 ---59 21 14 -65d 14 -10 --7 11 --14 0 0 0 11 30 23 13 27 --6 6 --6 7 -73 9 9 9 0 5 -2 2 -----70 95 81 75 72 ----11 24 28 7 11 26d 18 12 12 6 ----7 -2 --14d 32 28 21 28 12 ----26 4 5 -49d 33 13 -9 12 --10 1 23 --3 42 total e 250 200 210 69 45 30 28 19 147 69 38 related genotypes. the two prototype strains (st1, dq000495.1; st2, nc_007455.1) have 99.5% identity at the nucleotide and protein level. because the amino acid identity between both genotypes differs by so little, it may be assumed they represent one serotype. analysis of 24 hbov-1 unique isolates (12 originating from respiratory samples and 12 from fecal samples) demonstrated little sequence variation from the prototypic strains. 39 there was also no difference in the proportion of each hbov-1 genotype identified in nasopharyngeal and fecal isolates, suggesting that each lineage infects both respiratory and enteric systems. the recent recognition of hbov-2 and hbov-3 demonstrates that the bocavirus genus has substantial diversity. hbov-2 and hbov-3 have similar genomic organization of the putative open reading frames (orfs) to hbov-1. phylogenetic analysis of hbov-2 orfs show that it is more closely related to hbov-1 (pairwise distance 22.2%-26.2%) than to the animal bocaviruses (pairwise distance 46.2%-55.8%). 12 hbov-3 nonstructural proteins ns1 and np1 are closely related to hbov-1, whereas the structural viral capsid proteins are more similar to hbov-2 (see fig. 1 ). arthur and colleagues 13 hypothesize that hbov-3 may be a result of a distant recombination event between hbov-1 and hbov-2. because only several strains of hbov-2 and hbov-3 have been recognized from a handful of regions worldwide, however, the true diversity is unclear. following the discovery of human bocavirus, numerous studies have used conventional pcr to detect hbov-1. 14, 20, 24, 28, 29, 34, 35, [38] [39] [40] [41] 43, 48, 49, 56, 58, 60, primers, target genes, and predicted amplicon sizes are listed in table 1 . most primer sets used for hbov-1 screening target the np1 gene. although there is a wide variation in detection of hbov-1 between study sites with detection rates as high as 45.2% reported in one study, 72 primers targeting the vp1/vp2 region have a slightly higher detection rate (median 7.5%, see table 1 ). this difference is not surprising because the vp1/vp2 is more conserved than ns or np. in addition, nested and real-time pcr have been used to detect hbov-1. real-time pcr yields a slightly higher median frequency of detection of hbov-1. 15, 17, 21, 26, 30, 36, 37, 44, 51, 52, 54, 67, 68, [83] [84] [85] [86] [87] [88] [89] [90] [91] [92] [93] the variation in detection of hbov-1 in these studies is likely attributable to multiple factors, including sensitivity of primer sets, laboratory technique, true variation in incidence of hbov-1, and methods used in sample collection. ziegler and colleagues 94 reported using nucleic acid sequence based amplification (nasba) to screen for hbov-1 in stool samples. nasba is an isothermic nucleic acid amplification technique used as an alternative to pcr for the detection of viruses. 95, 96 in the study by ziegler and coworkers, 94 no hbov-1 was detected using nasba. it is unclear if this represents a failure of the method, lack of hbov-1 in the samples, reaction inhibitors to stool samples, or other variables. an increasing number of studies are screening stool samples for the presence of hbov-1. 38, 39, [41] [42] [43] [44] based on fecal and respiratory hbov sequence similarities reported by lau and colleagues genomic targets used for respiratory samples should be as successful in stool samples. 39 the current literature finds that hbov-1 is found in stool samples less often than in respiratory samples (median 2% versus 7.5%). it is unclear whether this lower rate of detection is due to a truly lower incidence of hbov-1 in stool, decreased viral loads, or presence of reaction inhibitors commonly found in feces. to date, hbov-1 has not been successfully propagated in standard cell lines or animal models. this failure has been one of the major impediments to our understanding of this virus. parvoviruses have a large host range and tissue tropism, even among closely related strains. the viral capsid proteins seem to be the major determinant of host specificity. 16 the related human parvovirus b19 causes viremia and replicates in the bone marrow of children and adults and in the liver of the fetus. 16 two studies identified hbov-1 dna in serum of affected patients. fry and colleagues 36 demonstrated hbov-1 in acute serum from hbov-1-positive children who had respiratory disease. allander and colleagues 15 detected hbov-1 dna in more than half of acute-phase serum samples and in 19% of the convalescent-phase serum specimens. detection of hbov-1 in serum was associated with a high viral load in the nasopharynx. even with the detection of hbov-1 dna in the blood of affected patients, however, it remains unclear if hbov-1 viremia truly occurs. among healthy children, viral respiratory disease is often a self-limited and uncomplicated disease. therapy against most viral respiratory pathogens has not been shown to be effective. treatment of viral pathogens, such as influenza, may shorten the duration of illness in low-risk patients but has not been shown to reduce the duration of illness in the high-risk population. 97 treatment with ribavirin or intravenous immune globulin (ivig) remain controversial in the setting of viral respiratory disease and are only used in select populations with severe rsv and human parainfluenza virus (hpiv) disease. 98, 99 the relative ineffectiveness of antiviral compounds is, in part, because host inflammatory reaction leads to a substantial amount of pathology. in addition, many patients present for medical attention at a time past peak viral replication. for these reasons, it is difficult to demonstrate effectiveness of most antiviral compounds. in several case reports, patients have received antiviral treatment either as prophylaxis or as treatment of confirmed or presumed viral infections. schenck and colleagues 22 reported a child who had undergone hematopoietic stem cell transplant who was receiving acyclovir prophylaxis; however, he subsequently developed symptomatic hbov-1 disease with hbov-1 dna identified in respiratory, blood, and stool samples. during this patient's complicated hospital course, he was placed on ganciclovir and eventually foscarnet for cytomegalovirus (cmv) viremia. both the cmv viral load and the hbov-1 serum viral load became undetectable after initiating foscarnet. this patient continued to have hbov-1 dna detectable in stool samples, however. kupfer and colleagues 100 reported a patient undergoing treatment of b-cell lymphoma who received ganciclovir for cmv viremia who was also found to have hbov-1 in respiratory secretions. there was no clinical improvement in symptoms after initiating ganciclovir. supportive care, even in the intensive care setting, plays a large role in the management of patients who have severe viral illness. supplemental oxygen 22 and mechanical ventilation 101 have been used to support patients who have hbov-1-associated disease. in one study, up to 28% of hbov-1-positive children required intensive care, although not all required respiratory assistance. 20 one patient, coinfected with rsv and hbov-1, required support through extracorporeal membrane oxygenation until underlying cardiac physiology could be repaired. there have been no comparative studies using antiviral agents for the treatment of human bocaviruses. the large percentage of coinfections associated with human bocavirus infections suggests that evaluation for further pathogens should be undertaken for any patient diagnosed with hbov-1. treatment of copathogens (ie, influenza) may lead to patient improvement. future therapeutic strategies may include vaccines, hbov-neutralizing antibodies, and small interfering rna. three human bocavirus species are now recognized and are reported in association with respiratory and gastrointestinal diseases. because of wide variation in clinical presentations, severity of disease, and a high rate of coinfection, the role of the bocaviruses in causing disease has been debated. one consideration is that bocaviruses are passenger viruses with little or no clinical effect. another possibility is that bocaviruses require the presence of a copathogen for infection. several studies suggest that this is not the case, however. bocavirus is identified in patients who have disease despite the absence of copathogens. case-control studies have shown an increased risk for pneumonia in patients who have hbov-1-positive respiratory secretions and increased risk for gastroenteritis in patients who have hbov-2-positive fecal samples. two studies found no increase in hbov-1 frequency in symptomatic versus asymptomatic stool samples. one report finds hbov-1 did not worsen gastrointestinal disease when coinfected with known pathogens such as rotavirus. 13 the question of whether hbov-2 or hbov-3 is associated with respiratory infection has not been answered. little is known about the recently described bocaviruses hbov-2 and hbov-3. clinical epidemiology and seroepidemiology have yet to be described in detail. although antibodies to hbov-1 are common in human sera, cross-reactivity with hbov-2 and hbov-3 has not been investigated. furthermore, although hbov-2 and hbov-3 have been described in stool, their presence in the respiratory tract should be investigated. areas for further research on hbov-1 include examining objective measures of disease severity in patients who have coinfections and examining hbov-1 for evidence of disease distant to the respiratory and gastrointestinal tracts. presence of hbov-1 has been described in serum, 22 lymphatic tissue, 21 and in conjunction with elevated hepatic enzymes, 46 the clinical significance of which has yet to be determined. in summary, although the evidence for pathogenicity of human bocaviruses is increasing, more research is needed to determine severity of hbov-associated disease and clinical outcomes. rockville (md): agency for healthcare research and quality economic burden of respiratory infections in an employed population a newly discovered human pneumovirus isolated from young children with respiratory tract disease identification of a new human coronavirus a previously undescribed coronavirus associated with respiratory disease in humans evidence of a novel human coronavirus that is associated with respiratory tract disease in infants and young children characterization and complete genome sequence of a novel coronavirus, coronavirus hku1, from patients with pneumonia identification of a novel coronavirus in patients with severe acute respiratory syndrome identification of a third human polyomavirus identification of a novel polyomavirus from patients with acute respiratory tract infections cloning of a human parvovirus by molecular screening of respiratory tract samples a newly identified bocavirus species in human stool a novel bocavirus associated with acute gastroenteritis in australian children human bocavirus infection human bocavirus and acute wheezing in children fields' virology electron microscopy observation of human bocavirus (hbov) in nasopharyngeal samples from hbov-infected children human bocavirus-a novel parvovirus to infect humans molecular and functional analyses of a human parvovirus b19 infectious clone demonstrates essential roles for ns1, vp1, and the 11-kilodalton protein in virus replication and infectivity evidence of human bocavirus circulating in children and adults human bocavirus in tonsillar lymphocytes disseminated bocavirus infection after stem cell transplant human bocavirus in febrile children human bocavirus quantitative dna detection in french children hospitalized for acute bronchiolitis human bocavirus in children with acute asthma role of respiratory pathogens in infants hospitalized for a first episode of wheezing and their impact on recurrences detection of human bocavirus in ill and healthy spanish children: a 2-year study human bocavirus infection among children human bocavirus: prevalence and clinical spectrum at a children's hospital human bocavirus in children with acute lymphoblastic leukemia respiratory viruses in hiv-infected patients with suspected respiratory opportunistic infection low prevalence of human metapneumovirus and human bocavirus in adult immunocompromised high risk patients suspected to suffer from pneumocystis pneumonia absence of human bocavirus in bronchoalveolar lavage fluid of lung transplant patients human bocavirus infection in young children in the united states: molecular epidemiological profile and clinical characteristics of a newly emerging respiratory virus human bocavirus in italian patients with respiratory diseases human bocavirus: a novel parvovirus epidemiologically associated with pneumonia requiring hospitalization in thailand clinical and epidemiologic characteristics of human bocavirus in danish infants: results from a prospective birth cohort study human bocavirus infection in children with acute gastroenteritis and healthy controls clinical and molecular epidemiology of human bocavirus in respiratory and fecal samples from children in hong kong detection of human bocavirus in children hospitalized because of acute gastroenteritis human bocavirus infection in children with gastroenteritis human bocavirus in children hospitalized for acute gastroenteritis: a case-control study human bocavirus infection in children hospitalized with acute gastroenteritis in china role of human bocavirus infections in outbreaks of gastroenteritis human bocavirus and gastroenteritis hepatitis and human bocavirus primary infection in a child with t-cell deficiency detection of human bocavirus in canadian children in a 1-year study two-year prospective study of single infections and co-infections by respiratory syncytial virus and viruses identified recently in infants with acute respiratory disease bocavirus infection in hospitalized children epidemiological profile and clinical associations of human bocavirus and other human parvoviruses human bocavirus infection in children with respiratory tract disease human bocavirus infection in hospitalized children during winter human bocavirus detection in nasopharyngeal aspirates of children without clinical symptoms of respiratory infection human bocavirus infections in hospitalized children and adults the association of newly identified respiratory viruses with lower respiratory tract infections in korean children seroepidemiology of human bocavirus defined using recombinant virus-like particles seroepidemiology of human bocavirus in hokkaido prefecture national surveillance for respiratory syncytial virus frequent detection of human rhinoviruses, paramyxoviruses, coronaviruses, and bocavirus during acute respiratory tract infections a 1-year experience with human metapneumovirus in children aged <5 years detection of bocavirus dna in nasopharyngeal aspirates of a child with bronchiolitis prospective study of human bocavirus (hbov) infection in a pediatric university hospital in germany complete coding sequences and phylogenetic analysis of human bocavirus (hbov) human bocavirus (hbov) in thailand: clinical manifestations in a hospitalized pediatric patient and molecular virus characterization viruses in community-acquired pneumonia in children aged less than 3 years old: high rate of viral coinfection human bocavirus in french children human bocavirus in children pediatric hospitalization of acute respiratory tract infections with human bocavirus in hong kong detection of human bocavirus in japanese children with lower respiratory tract infections human bocavirus in iranian children with acute respiratory infections real-time pcr for diagnosis of human bocavirus infections and phylogenetic analysis human bocavirus in infants human bocavirus, a respiratory and enteric virus frequent detection of bocavirus dna in german children with respiratory tract infections human bocavirus detection in an atopic child affected by pneumonia associated with wheezing human bocavirus in children suffering from acute lower respiratory tract infection in beijing children's hospital human bocavirus commonly involved in multiple viral airway infections high prevalence of human bocavirus detected in young children with severe acute lower respiratory tract disease by use of a standard pcr protocol and a novel real-time pcr protocol detection of viruses identified recently in children with acute wheezing evidence of human coronavirus hku1 and human bocavirus in australian children the incidence of human bocavirus infection among children admitted to hospital in singapore frequent detection of viral coinfection in children hospitalized with acute respiratory tract infection using a real-time polymerase chain reaction detection of human bocavirus in children with kawasaki disease real-time pcr assays for detection of bocavirus in human specimens high rate of human bocavirus and adenovirus coinfection in hospitalized israeli children comprehensive detection of causative pathogens using real-time pcr to diagnose pediatric community-acquired pneumonia respiratory viral infections in children with leukemia isolation of human bocavirus from swiss infants with respiratory infections human bocavirus infection, people's republic of china human bocavirus dna detected by quantitative real-time pcr in two children hospitalized for lower respiratory tract infection viral etiology of acute respiratory infections with cough in infancy: a community-based birth cohort study impact of human bocavirus on children and their families no gastroenteric bocavirus in high risk patients stool samples sensitive commercial nasba assay for the detection of respiratory syncytial virus in clinical specimen dry cotton or flocked respiratory swabs as a simple collection technique for the molecular detection of respiratory viruses using real-time nasba update in prevention, evaluation, and outpatient treatment of influenza respiratory syncytial virus (rsv) immune globulin intravenous therapy for rsv lower respiratory tract infection in infants and young children at high risk for severe rsv infections: respiratory syncytial virus immune globulin study group use of intravenous immunoglobulin in human disease: a review of evidence by members of the primary immunodeficiency committee of the american academy of allergy, asthma and immunology severe pneumonia and human bocavirus in adult human bocavirus infection in a neonatal intensive care unit undiagnosed respiratory viruses in children epidemiological and clinical study of viral respiratory tract infections in children from italy clinical and genetic analysis of human bocavirus in children with lower respiratory tract infection in taiwan no evidence for an association between infections with wu and ki polyomaviruses and respiratory disease detection of human bocavirus in asturias, northern spain human bocavirus in hospitalized children role of human metapneumovirus, human coronavirus nl63 and human bocavirus in infants and young children with acute wheezing key: cord-294933-oc2glu4a authors: cinesi gómez, césar; peñuelas rodríguez, óscar; luján torné, manel; egea santaolalla, carlos; masa jiménez, juan fernando; garcía fernández, javier; carratalá perales, josé manuel; heili-frades, sarah béatrice; ferrer monreal, miquel; de andrés nilsson, josé m.; lista arias, eva; sánchez rocamora, juan luis; garrote, josé ignacio; zamorano serrano, miguel j.; gonzález martínez, mónica; farrero muñoz, eva; mediano san andrés, olga; rialp cervera, gemma; mas serra, arantxa; hernández martínez, gonzalo; de haro lópez, candelaria; roca gas, oriol; ferrer roca, ricard; romero berrocal, antonio; ferrando ortola, carlos title: clinical consensus recommendations regarding non-invasive respiratory support in the adult patient with acute respiratory failure secondary to sars-cov-2 infection date: 2020-06-19 journal: nan doi: 10.1016/j.medine.2020.03.002 sha: doc_id: 294933 cord_uid: oc2glu4a abstract coronavirus disease 2019 (covid-19) is a respiratory tract infection caused by a newly emergent coronavirus, that was first recognized in wuhan, china, in december 2019. currently, the world health organization (who) has defined the infection as a global pandemic and there is a health and social emergency for the management of this new infection. while most people with covid-19 develop only mild or uncomplicated illness, approximately 14% develop severe disease that requires hospitalization and oxygen support, and 5% require admission to an intensive care unit. in severe cases, covid-19 can be complicated by the acute respiratory distress syndrome (ards), sepsis and septic shock, and multiorgan failure. this consensus document has been prepared on evidence-informed guidelines developed by a multidisciplinary panel of health care providers from four spanish scientific societies (spanish society of intensive care medicine [semicyuc], spanish society of pulmonologists [separ], spanish society of emergency [semes], spanish society of anesthesiology, reanimation, and pain [sedar]) with experience in the clinical management of patients with covid-19 and other viral infections, including sars, as well as sepsis and ards. the document provides clinical recommendations for the noninvasive respiratory support (noninvasive ventilation, high flow oxygen therapy with nasal cannula) in any patient with suspected or confirmed presentation of covid-19 with acute respiratory failure. this consensus guidance should serve as a foundation for optimized supportive care to ensure the best possible chance for survival and to allow for reliable comparison of investigational therapeutic interventions as part of randomized controlled trials. non-invasive mechanical ventilation; high-flow nasal therapy; aerosol-generating procedures; infection control abstract: coronavirus disease 2019 (covid-19) is a respiratory tract infection caused by a newly emergent coronavirus, that was first recognized in wuhan, china, in december 2019. currently, the world health organization (who) has defined the infection as a global pandemic and there is a health and social emergency for the management of this new infection. while most people with covid-19 develop only mild or uncomplicated illness, approximately 14% develop severe disease that requires hospitalization and oxygen support, and 5% require admission to an intensive care unit. in severe cases, covid-19 can be complicated by the acute respiratory distress syndrome (ards), sepsis and septic shock, and multiorgan failure. this consensus document has been prepared on evidence-informed guidelines developed by a multidisciplinary panel of health care providers from four spanish scientific societies (spanish society of intensive care medicine [semicyuc] , spanish society of pulmonologists [separ] , spanish society of emergency [semes] , spanish society of anesthesiology, reanimation, and pain [sedar] ) with experience in the clinical management of patients with covid-19 and other viral infections, including sars, as well as sepsis and ards. the document provides clinical recommendations for the noninvasive respiratory support (noninvasive ventilation, high flow oxygen therapy with nasal cannula) in any patient with suspected or confirmed presentation of covid-19 with acute respiratory failure. this consensus guidance should serve as a foundation for optimized supportive care to ensure the best possible chance for survival and to allow for reliable comparison of investigational therapeutic interventions as part of randomized controlled trials. © 2020 elsevier españa, s.l.u. and semicyuc. all rights reserved. ventilación mecánica no invasiva; terapia nasal de alto flujo; procedimientos generadores de aerosoles; control de infección resumen la enfermedad por coronavirus 2019 (covid-19) es una infección del tracto respiratorio causada por un nuevo coronavirus emergente que se reconoció por primera vez en wuhan, china, en diciembre de 2019. actualmente la organización mundial de la salud (oms) ha definido la infección como pandemia y existe una situación de emergencia sanitaria y social para el manejo de esta nueva infección. mientras que la mayoría de las personas con covid-19 desarrollan solo una enfermedad leve o no complicada, aproximadamente el 14% desarrollan una enfermedad grave que requiere hospitalización y oxígeno, y el 5% pueden requerir ingreso en una unidad de cuidados intensivos. en casos severos, covid-19 puede complicarse por el síndrome de dificultad respiratoria aguda (sdra), sepsis y shock séptico y fracaso multiorgánico. este documento de consenso se ha preparado sobre directrices basadas en evidencia desarrolladas por un panel multidisciplinario de profesionales médicos de cuatro sociedades científicas españolas (sociedad española de medicina intensiva y unidades coronarias [semicyuc], sociedad española de neumología y cirugía torácica [separ], sociedad española de urgencias y emergencias [semes], sociedad española de anestesiología, reanimación y terapéutica del dolor [sedar]) con experiencia en el manejo clínico de pacientes con covid-19 y otras infecciones virales, incluido el sars, así como en sepsis y sdra. el documento proporciona recomendaciones clínicas para el soporte respiratorio no invasivo (ventilación no invasiva, oxigenoterapia de alto flujo con cánula nasal) en cualquier paciente con presentación sospechada o confirmada de covid-19 con insuficiencia respiratoria aguda. esta guía de consenso debe servir como base para una atención optimizada y garantizar la mejor posibilidad de supervivencia, así como permitir una comparación fiable de las futuras intervenciones terapéuticas de investigación que formen parte de futuros estudios observacionales o de ensayos clínicos. © 2020 elsevier españa, s.l.u. y semicyuc. todos los derechos reservados. the present document has been developed by consensus among the scientific societies involved in acute respiratory failure in adult patients, and seeks to provide a more detailed description of the recommendations on the use of non-invasive respiratory support (nirs) in the management of acute respiratory failure (arf) secondary to infection by the newly emergent sars-cov-2 coronavirus, which causes so-called covid-19 disease, as a complement to the information emitted by the spanish ministry of health, consumer affairs and social wellbeing (ministerio de sanidad, consumo y bienestar social [msc]), 1,2 which is frequently updated and establishes a series of general recommendations. the world health organization (who) has recently declared sars-cov-2 (covid-19) disease 3 as an international alert and public health emergency. this ongoing covid-19 pandemic is devastating, despite the widespread adoption of control measures. in fact, there are important regional differences in the availability and accessibility of medical resources among the more than 70 currently affected countries. these differences could in part explain the low mortality rates despite the high incidence of cases. in this regard, the different health authorities and governments have developed contingency plans to deal with the local outbreaks of the disease. 4 such measures are essential to control the epidemic, protect the healthcare professionals at the frontline, and mitigate the seriousness of the patient outcomes. the results of a recent analysis of the clinical characteristics of a selected cohort of 1099 patients with covid-19 throughout china 5 have evidenced that up to 15% (173/1099) developed serious disease according to the clinical criteria of severe community-acquired pneumonia of the american thoracic society. 6 of these patients with serious disease, 24.8% (43/173) had the composite outcome of admission to the intensive care unit (icu) or the use of mechanical ventilation (both invasive and non-invasive), or death. on the other hand, 2.9% (5/173) required extracorporeal oxygenation measures. this scenario, when extrapolated to the situation of the current disease outbreak in spain, points to the need to anticipate and demand a contingency plan from the national and regional health authorities for the management of healthcare resources and the safety of the professionals ---including the use of specific areas in hospital centers, such as expert units capable of assisting ventilated patients (icu, intermediate respiratory care units [ircus]), emergency care departments, hospitalization wards equipped with means and healthcare professionals capable of safely and efficiently dealing with the epidemiological challenge of controlling and treating the covid-10 disease outbreak in spain. furthermore, during the previous viral epidemics in the form of sars (severe acute respiratory syndrome) and mers (middle east respiratory syndrome), the healthcare workers suffered high infection rates of 18.6% during the mers outbreak and 21% during the sars outbreak. 7, 8 therefore, from the working groups and supervising scientific societies, contingency plans must be established, contemplating eventual critical situations in order to optimize the human and material resources and be prepared in advance for potential and unexpected scenarios of overwhelming demand. specifically, from the consensus group, and within this contingency plan, we feel that the following must be included: 1. guarantees that the hospital management bodies have an established protocol referred to personal protective equipment (ppe) for healthcare workers. verification that the healthcare staff is trained in fitting and removing ppe. 2. utilization of education, capacitation and the simulation of scenarios to assess response capacity in different outbreak scenarios in the healthcare centers. optimization and anticipation of human healthcare resources to improve the safety of the healthcare staff, and contemplation of possible sick leaves in the event of confirmed disease cases. 3. identification of reference hospital centers that can safely manage a possible increase in the number of cases. 4. boosting of the total capacity of the icus and ircus, with preparation in advance of the material resources, healthcare staff and equipment requirements, and hospitalization and emergency care areas where to group covid-19-positive patients in each hospital, if needed. 5. definition of a classification protocol for the identification of suspected, probable and confirmed cases, in order to guide correct management circuits within the hospital. 6. generation of a pre-established protocol for the management of cases of suspected covid-19 with serious disease criteria. 7. definition of clear criteria for the care of patients and their families, and of admitted patient visiting policies. in conclusion, we cannot predict how many seriously ill patients with covid-19 we are going to receive, but we need to anticipate the possible scenarios, adjust the resources as rationally as possible in concordance with the experiences of other affected countries, do the best we can to be prepared, and work jointly to overcome the epidemic. ), are based on the studies of other viral pandemics (influenza, sars, mers) and on the latest publications referred to covid-19. therefore, the current level of evidence is low, given the lack of methodological robustness and the inherent nature of the disease. this document will be brought up to date as knowledge evolves and with the changes in the recommendations of the national and international organisms and societies. • to adequately identify patients with arf amenable to the start of noninvasive respiratory support (nirs). • to know the processes underlying high risk of failure of nirs. acute respiratory failure is the most common cause of death among patients with influenza and viral infections in general. 9 with regard to covid-19 (the infectious disease caused by sars-cov-2), 3.4% of the patients infected in china presented acute respiratory distress syndrome (ards), representing 40.3% of the patients with a serious disease. 5 it is therefore very important to adopt a therapeutic strategy for arf secondary to covid-19 infection. a fundamental element in the management of arf in covid-19 patients in monitoring ---preferably of a non-invasive nature. these patients need to be located in individual rooms, ideally with negative pressure. this implies the need for centralized monitoring control (pulsioxymetry, respiratory frequency), preferably with video monitoring. in the same way as in other processes that cause arf, in patients with sars-cov-2 we can use the respiratory management strategy proposed by scala and heunks. 10 conventional oxygen therapy involves the administration of oxygen at different concentrations, and constitutes the basis of the therapeutic pyramid. the next step is high-flow nasal cannula (tafcn) therapy. this technique involves the use of a gas mixture at high flow rates (up to 60 lpm), with variable proportions (fio 2 ) of air and oxygen administered through a nasal cannula. the administered gas must be warm and with 100% humidification. 11 the advantages with respect to conventional oxygen are a constant fio 2 , dead space reduction, and the generation of a positive pressure that implies redistribution of intraalveolar fluid and alveolar recruitment. 12 the next step is non-invasive mechanical ventilation (nimv). the main characteristic of nimv is its use under spontaneous breathing conditions; sedation is therefore null or low. 13 the penultimate step is invasive mechanical ventilation (imv). in this case ventilation in controlled mode is usually used, and tracheal intubation is required. 10 the last step is extracorporeal membrane oxygenation (ecmo). 14 at present, the respiratory therapies that define nirs and hfnc and nimv. as an orientation and taking the above into account, the criteria for starting respiratory support in arf secondary to covid-19 would be the following 15 : clinical criteria: • moderate to severe dyspnea with signs of labored breathing and the use of accessory muscles, or paradoxical abdominal motion. • tachypnea in excess of 30 rpm. • pao 2 /fio 2 <200 (or the need to administer fio 2 >0.4 to secure spo 2 of at least 92%). • acute ventilatory failure (ph <7.35 with paco 2 >45 mmhg). in patients without any of the above criteria, the initially indicated treatment would be conventional oxygen therapy. in the presence of any of the above criteria, respiratory support would be indicated ---whether invasive or non-invasive. the use of nirs for sars and other viral pandemics is subject to controversy, with nimv failure rates of about 30%. 16 more recently, nimv has also been used in patients with arf due to influenza a h1n1, with failure rates of between 13 and 77%. 17---19 despite the uncertainty of the evidence and the lack of randomized clinical trials, the positive data obtained by most of the observational studies suggest that its use can be considered in carefully selected patients in experienced centers and in a protected environment (ideally rooms with negative pressure). in the present chinese epidemic, 5.1% of the patients required nimv, 2.3% imv and 0.5% ecmo. 5 accordingly, the treatment choice will also depend on the patient background disease (mainly of a respiratory nature), the location of the patient (room with negative pressure, closed individual room with air renewal), and the possibility of an eventual need for aerosol generating procedures. thus, globally speaking, we may encounter three different clinical scenarios: 1. patients without prior disease (de novo arf) with hypoxemic respiratory failure, and therefore amenable to escalate treatment up to ecmo. the nirs failure rate in this clinical scenario ---mainly in relation to nimv ---is extremely high. furthermore, there is evidence of increased mortality if the start of imv is delayed. we therefore do not recommend the use of nirs in these patients. 1, 20, 21 it could only be contemplated in carefully selected patients, and provided all the following criteria are met 19,21---27 : • pao 2 /fio 2 >100 despite conventional oxygen therapy. • absence of multiorgan failure (apache score <20). • a team with expertise in continuous monitoring is required. it is therefore advisable to perform the technique in special units with a pre-specified nursing staff ratio as in icus and ircus. • early orotracheal intubation (oti) in the following hour, in the absence of criteria of improvement. in this regard, and in addition to the traditional intubation criteria, we could consider intubation in patients treated with hfnc presenting a respiratory rate-oxygenation (rox) index ([spo 2 /fio 2 ]/respiratory frequency) of <3, <3.5 and <4 at 2, 6 and 12 hours after the start of hfnc treatment. likewise, intubation could be considered in patients with a heart rate, acidosis, consciousness, oxygenation, and respiratory rate (hacor) index of >5 one hour after the start of nimv therapy. extrapolating the evidence referred to de novo arf, the use of hfnc would be the first choice. 21, 28 non-invasive mechanical ventilation is defined as the second option in the event of insufficient patient response and without immediate criteria for imv. and therapeutic ceiling in nimv. the start of nirs would be indicated in these patients, provided the preventive measures are ensured. in this context it is essential to establish the treatment objectives with the patient and family, delimiting the therapeutic ceiling. in general, it is advisable to start treatment with hfnc before resorting to nimv. 21, 28 we recommend the following considerations for the use of nirs 29,30 : • adjust fio 2 to secure a target spo 2 of about 95%. 31 • if hfnc is used, it is advisable to administer flows of over 50 lpm; if possible, start with 60 lpm. • if nimv is decided, it is advisable to use positive endexpiratory pressure (peep) values and low assist pressures (in order to obtain a high end-tidal volume (vte) of <9 ml/kg ideal body weight). 31, 32 3. patients with severe exacerbation of chronic obstructive pulmonary disease (copd) with acute or exacerbated hypercapnic respiratory failure: perform a nirs therapeutic test, especially with nimv. high-flow nasal cannulas may be useful in these patients in the event of intolerance to nimv, or for the lowering of nimv. 33---36 to limit transmission of the infection to both the healthcare staff and to other patients. the use of nirs is a practice of special risk of sars-cov-2 transmission. 37--40 the capacity of sars-cov-2 to infect healthcare workers has been confirmed, though it is still too soon to establish comparisons with mers and sars. 40 thus, the spanish ministry of health, consumer affairs and social wellbeing has defined a series of recommendations for management of the disease, and this document seeks to disclose the measures that limit transmission of the infection to the healthcare staff. 2, 39 in order to be able to apply mirs to a patient with suspected or confirmed sars-cov-2 infection, it is crucial to follow the general preventive recommendations. in this regard, the public administrations must be in possession of the materials required for safe nirs use. 1. the healthcare staff assisting cases under investigation for covid-19 infection or with already confirmed infection subjected to nirs must wear personal protective equipment (ppe) for the prevention of infection 2,22,37,39,41 during aerosol-generating procedures that have been associated to an increased risk of air transmission of pathogens. the preventive measures should be targeted to microorganisms that are transmitted via droplets and through contact, and are to include: • a high-efficacy ffp2 mask or preferably an ffp3 mask (if available). • integral-frame protective goggles. • retraction of long hair into a knot or tail; a surgical cap can be used in this regard. • gloves. • long-sleeved, impermeable microbiological protective gowns. results of studies selected in the systematic review that measured the risk of sars transmission to healthcare professionals exposed to the cited procedures versus those not exposed to the same procedures. 48, 49 2. patient location within the hospital setting is related to the possibility of performing aerosol-generating procedures. in this respect, the available evidence on the use of nirs devices as invasive procedures and the risk of pathogen transmission to the healthcare professionals is stronger in cases of orotracheal intubation and patients with an artificial airway, as well as in noninvasive ventilation ---though these data come from limited studies of very low quality. interpretation of the data is therefore difficult (table 1) . therefore, in mild cases it is advisable to keep the patient in a room with negative pressure. if no such rooms are available, the patient can be located in an individual room with bathroom. the door to the room should be kept closed at all times. 39 patients with severe hypoxemia are to be admitted to special units. 3. when in-hospital transfer is required, both the patient and the professional in charge of moving the patient are to wear surgical masks. during transfer, the patient bed is to be covered with a disposable clean sheet that should be eliminated afterwards as group iii waste. 39 although the spanish ministry of health, consumer affairs and social wellbeing recommends the administered of oxygen using masks equipped with an exhaled air filter, such masks are not universally available in our setting. if these masks are not available, a surgical mask may be safely used over the nasal cannula or oxygen mask in order to limit viral dispersion. no studies have compared the safety in reducing sars-cov-2 dispersion between the former type of mask and the surgical mask, 39 but oxygen administration is considered to be a low-risk aerosol-generating procedure. 39, 40 therapy with high-flow nasal cannulas it is advisable to follow the mentioned general recommendations and keep a distance of at least two meters with respect to other patients and healthcare staff lacking adequate protection. 42 although there is still uncertainty regarding particle dispersion with this therapeutic modality, placing a surgical mask over the nasal cannula ---although not investigated to date ---could be an option in extreme situations. it is advisable to keep a distance of at least two meters with respect to other patients and healthcare staff lacking adequate protection. in general, and according to the available evidence, there are no contraindications to the use of nirs in patients with covid-19. however, the respiratory therapy used depends not only on the severity of respiratory failure but also on the availability of an area or location that meets the isolation and safety recommendations of the who. probably the most serious cases, in which probable and rapid intubation is anticipated, should be admitted to the icu in order to avoid possible delays in intubation that are negative for the patient outcome (fig. 1) . choice of respirator configuration. • although there is still uncertainty regarding particle dispersion in covid-19, during the sars epidemic a number of articles demonstrated particle dispersion with singlecircuit and expiratory port nimv of no more than four feet (1.25 m). 8, 43, 44 • dual-arm configurations are to be preferred, since they afford sealing of the circuit in both the inspiratory and the expiratory phase. high-efficiency antimicrobial filters are to be positioned in the expiratory arm in order to avoid inverse infection from the patient to the respirator. 39 • if dual-arm systems are not available and single-arm ventilators must be used, the expiratory orifice should be placed in the single tubing, with the fitting of a high-efficiency and low-resistance antimicrobial filter to minimize dispersion of the exhaled gas that could contaminate the environment. it likewise seems feasible to interposition a t-piece in the circuit to place the filter and intentional escape valve distal to the latter ---though the resulting increase in dead space must be taken into account. • if a high-efficiency antimicrobial filter cannot be fitted to the expiratory orifice, a high-efficiency antimicrobial filter should be placed between the patient/ventilator interface (without expiratory orifices) and the circuit. in this regard, the increase in resistance may need a change in the ventilator parameters, incrementing the level of pressure support. • a feasible alternative to dual-or single-arm systems with escape valve is to use single-arm systems with an active valve and the placement of an antimicrobial filter at the outlet of the active valve. • we do not recommend the use of heat and moisture exchangers (hmes). 45 choice of the type of interface. the interface is the connecting device that facilitates the physical but also the functional relationship between two independent elements: the ventilator and the patient. it is a key element in nimv, serving to vehiculize the negative pressure toward the patient with no artificial component positioned in the airway. the recommendations for use of the interface in sars-cov-2 infection are 40, 41, 46, 47 : • the recommended interface comes without an expiratory orifice, with no use of accessory ports, if any. • use of the helmet should be a priority if the interface is available, with correct knowledge of the pertinent fitting and maintenance technique. • in general terms, it is advisable to use a full face mask as first alternative, or an oronasal mask if not available. • strict monitoring of leakage points around the mask is required, particularly at the oronasal interfaces, in order to avoid skin damage to patients with adequate protection, and also to maintain the circuit airtight in order to avoid exhalation of the infected air. protective patches are to be avoided, due to the risk of increased leakage; repeated application of hyperoxygenated oils is advised. • use of the nasal interface is not advised, since it generates more aerosols, and furthermore in general in sars-cov-2 infection the problem is acute hypoxemic respiratory failure. choice of the type of elbow 46 . • we recommend the use of an elbow without anti-asphyxia valve. these are generally of a blue color. the use of elbows of this kind requires close patient monitoring due to possible ventilation system malfunction. considering the risk/benefit ratio (asphyxia versus dispersion), and the fact that these patients are located in very complex rooms under the continuous care of specialized medical staff, it is unlikely for accidental and undetected or uncorrected disconnection to occur, but a minimum safe nursing ratio is needed. • we misadvise the use of an anti-rebreathing elbow (which also houses an anti-asphyxia valve), due to the risk of increased expired air dispersion. monitoring to identify early failure and proceed to elective orotracheal intubation. the current covid-19 pandemic requires greater infection control precautions. nebulizers generate aerosol particles measuring 1---5 (m in size that can carry bacteria and viruses into the lungs. the risk of infection transmission through droplets and aerosols may increase during nebulizer treatment, due to the potential for generating a large volume of respiratory aerosols that can be ejected over a greater distance than in the natural dispersion pattern. furthermore, the larger particles may stimulate cough in patients and by-passers, and thus increase the risk of disease spread. nebulizer therapy in patients with pandemic covid-19 infection may transmit potentially viable viruses to susceptible individuals. in recent years some centers have experienced a change from the use of nebulizers to metered-dose inhalers (mdis) with valved-holding chambers (vhcs). the administration of inhalatory therapy preferably should be made using an mdi device and vhc. however, it is important to underscore that it is unlikely for patients with acute respiratory failure to be able to receive the medication in an effective manner with these devices. nebulization therapy should only be used under the following conditions: -serious and potentially fatal respiratory disease (e.g., patients with hypoventilation or impaired ventilation, severe copd or cystic fibrosis). -uncooperative patients or individuals unable to follow the required instructions for an mdi with vhc. -patients with a poor response to mdi with vhc. however, despite the important body of evidence suggesting an absence of either superiority or inferiority in comparison to mdi with vhc, the open nebulizer is still widely used in hospital centers. during the current covid-19 outbreak, and in order to reduce the risk of transmission of all infectious respiratory processes, we encourage all healthcare professionals to seriously consider avoiding the use of open nebulizers in patients under spontaneous breathing if the aforementioned devices are not available. preserving the safety of the patients and healthcare staff must be our priority concern. therefore, if aerosol therapy is needed, we recommend the use of vibrating mesh nebulizer devices with a mask or buccal pipette, limiting dispersion and fitting a surgical mask over the device. it must be taken into account that if a buccal pipette with anti-dispersion system is used, the deposited drug dose (particularly important in the case of beta 2 -adrenergic bronchodilators) may be greater, and require dose adjustment therefore may prove necessary. jet systems are disadvised due to the greater capacity to disperse particles into the environment. if such systems are necessary, it is essential to fit a surgical mask on the patient during nebulization. observation of the following points is recommended for the use of inhalatory therapy together with nirs: • the general recommendation for administering inhalatory therapy is to use pressurized cartridges with vhc or adaptor. if nimv is used, it should be placed in the inspiratory arm of the circuit, coordinating pulsation with patient inspiration. • if aerosol therapy is used, vibrating mesh nebulizers with adaptation to the elbow of the interface is the option of choice. as a second option we can use a vibrating mesh nebulizer with a t-piece fitted to the nimv circuit. since this is a ''closed system'', there is no dispersion into the environment provided leakage at the mask periphery is well controlled. • jet-type nebulizers with a t-tube generate greater turbulence and particles of larger size, as well as increased ease of particle dispersion. • if hfnc is used, we ideally should employ pressurized cartridges with vhc, a pipette with a vibrating mesh nebulizer or mesh device fitted to the dry arm of the water reservoir chamber. • in general, we should reduce the pressure support used in nimv and the temperature if hfnc is used. this consensus document arises from a situation of social and healthcare alarm, and from the demands of the professionals for management guidelines; it therefore focuses on specific situations that are outside the context of routine clinical practice in hospital centers. it therefore should be adapted to the particular circumstances found in each concrete scenario. manejo clínico de pacientes con enfermedad por el nuevo coronavirus (covid-19) procedimiento de actuación frente a casos de infección por el nuevo coronavirus (sars-cov-2) world health organization. coronavirus disease (covid-19) outbreak potential association between covid-19 mortality and healthcare resource availability clinical characteristics of coronavirus disease 2019 in china diagnosis and treatment of adults with communityacquired pneumonia. an official clinical practice guideline of the severe acute respiratory syndrome (sars) ---lessons for future pandemics mers transmission and risk factors: a systematic review clinical practice guidelines by the infectious diseases society of america: 2018 update on diagnosis, treatment, chemoprophylaxis, and institutional outbreak management of seasonal influenza highlights in acute respiratory failure high-flow nasal cannula oxygen therapy is superior to conventional oxygen therapy but not to noninvasive mechanical ventilation on intubation rate: a systematic review and meta-analysis current evidence for the effectiveness of heated and humidified high flow nasal cannula supportive therapy in adult patients with respiratory failure british thoracic society/intensive care society guideline for the ventilatory management of acute hypercapnic respiratory failure in adults extracorporeal membrane oxygenation for critically ill adults noninvasive ventilation in acute hypercapnic respiratory failure effectiveness of noninvasive positive pressure ventilation in the treatment of acute respiratory failure in severe acute respiratory syndrome noninvasive mechanical ventilation in severe pneumonia due to h1n1 virus pandemic 2009 influenza a in argentina: a study of 337 patients on mechanical ventilation early non-invasive ventilation treatment for severe influenza pneumonia official ers/ats clinical practice guidelines: noninvasive ventilation for acute respiratory failure clinical management of severe acute respiratory infection when novel coronavirus (2019-ncov) infection is suspected severe sars-cov-2 infections: practical considerations and management strategy for intensivists. intensive care med critically ill patients with 2009 influenza a(h1n1) infection in canada assessment of heart rate, acidosis, consciousness, oxygenation, and respiratory rate to predict noninvasive ventilation failure in hypoxemic patients clinical characteristics and outcomes of patients with 2009 influenza a(h1n1) virus infection with respiratory failure requiring mechanical ventilation an index combining respiratory rate and oxygenation to predict outcome of nasal high-flow therapy risk factors for noninvasive ventilation failure in critically ill subjects with confirmed influenza infection high-flow oxygen through nasal cannula in acute hypoxemic respiratory failure clinical course and outcomes of critically ill patients with sars-cov-2 pneumonia in wuhan, china: a single-centered, retrospective, observational study challenges on non-invasive ventilation to treat acute respiratory failure in the elderly lung-protective ventilation strategies and adjunctive treatments for the emergency medicine patient with acute respiratory failure driving pressure and mechanical power: new targets for vili prevention high-flow nasal therapy versus noninvasive ventilation in copd patients with mild-to-moderate hypercapnic acute respiratory failure: study protocol for a noninferiority randomized clinical trial efficacy and safety of high-flow nasal cannula oxygen therapy in moderate acute hypercapnic respiratory failure. rev bras ter intensiva high flow nasal cannula oxygen therapy versus non-invasive ventilation for chronic obstructive pulmonary disease with acute-moderate hypercapnic respiratory failure: an observational cohort study reva network comparison of high flow nasal cannula oxygen and conventional oxygen therapy on ventilatory support duration during acute-on-chronic respiratory failure: study protocol of a multicentre, randomised, controlled trial. the ''high-flow acrf'' study expert consensus on preventing nosocomial transmission during respiratory care for critically ill patients infected by 2019 novel coronavirus pneumonia the sars mers and novel coronavirus (covid-19) epidemics, the newest and biggest global health threats: what lessons have we learned? prevención y control de la infección en el manejo de pacientes con covid-19 rational use of personal protective equipment for coronavirus disease 2019 (covid-19) centers for disease control and prevention comparison of high-flow nasal cannula versus oxygen face mask for environmental bacterial contamination in critically ill pneumonia patients: a randomized controlled crossover trial exhaled air dispersion distances during noninvasive ventilation via different respironics face masks noninvasive positive-pressure ventilation impact of the humidification device on intubation rate during noninvasive ventilation with icu ventilators: results of a multicenter randomized controlled trial staff safety during emergency airway management for covid-19 in hong kong emergency committee regarding the outbreak of novel coronavirus (2019-ncov) physical interventions to interrupt or reduce the spread of respiratory viruses aerosol-generating procedures and risk of transmission of acute respiratory infections: a systematic review. ottawa: canadian agency for drugs and technologies in health key: cord-021894-lq8yr710 authors: cunningham, steve title: bronchiolitis date: 2018-03-13 journal: kendig's disorders of the respiratory tract in children doi: 10.1016/b978-0-323-44887-1.00024-9 sha: doc_id: 21894 cord_uid: lq8yr710 acute viral bronchiolitis is a common viral lower respiratory tract infection in young children. most typically caused by respiratory syncytial virus in 70% of cases, the condition lasts for 4 to 7 days, with a prolonged cough in many. children with comorbidity, particularly those born prematurely or with significant congenital heart disease, are at risk of more severe disease. nasal obstruction progresses over 3 to 4 days to difficulty with feeding and increased work of breathing with hypoxemia. crackles and/or wheeze may be auscultated. apnoea may be a presenting sign in those less than 3 months of age. viral load is highest at peak of symptoms and in those with more severe disease. approximately 2% to 3% of all children are admitted to hospital with bronchiolitis. the differential diagnosis may include bacterial pneumonia, congenital lesions of the lung or heart, or an interstitial lung disease. there are no effective treatments, and admission is for feeding support (by nasogastric or intravenous fluids) or treatment of hypoxemia. critical care support is required for some infants experiencing respiratory failure, though mortality rates remain unchanged. practice within and between countries varies significantly and alignment of practice is a common goal of guidelines. vaccines for rsv are in advanced development, as are several antiviral therapies for rsv. in most children, acute symptoms improve within 5 to 7 days and cough by 2 weeks. recurrent wheeze is common following acute bronchiolitis and a good association with a diagnosis of asthma in childhood. rsv infects 69% to 98% of infants in the first year of life. 18, 19 the rapid development of vaccines and treatment therapies for rsv has added impetus to the need to better define the burden of rsv disease. globally there are an estimated 33.8 million cases of rsv lower respiratory tract infection each year in children under 5 years of age, resulting in 3.4 million admissions to the hospital and 66 to 199 thousand deaths (with the majority in low-and middle-income countries). 20 in the united states an estimated 20% of children will attend primary care each year with rsv bronchiolitis, and up to 7% attend an emergency department (ed). 21 admission to hospital with rsv bronchiolitis is typically around 2.4% of all infants, 15, 22 though in previously healthy term infants, the admission rate to hospital with rsv bronchiolitis can be as low as 0.7%. 23 in infants who are born preterm at 32 to 35 weeks' gestation, 48% will develop bronchiolitis and 6% require admission to the hospital. 24 the risk of bronchiolitis is increased in a range of conditions compared with term infants, including preterm birth (respiratory rate [rr] 1. 89) , cystic fibrosis (rr 2.45), congenital heart disease (rr 3.35), chronic lung disease (rr 1.61), immunodeficiency (rr 1.73), down syndrome (rr 2.53), and cerebral palsy (rr 2.43). 25, 26 epidemiology bronchiolitis is the most common lower respiratory tract infection in children. the condition forms part of the spectrum of viral lower respiratory tract infection that includes bronchiolitis, viral pneumonia, and viral-induced wheeze. in polar hemispheres (north and south), bronchiolitis is a seasonal disease, dominating winter months, with a peak over 6 to 8 weeks around the winter solstice. in tropical climates, the disease is associated with rainy months and is seasonally more dispersed. 1 climate and environment appear to influence both season and severity. 2, 3 bronchiolitis is diagnosed clinically by integrating characteristic but variable signs and symptoms across a broad age range, though the majority of cases occur in children under 1 year of age. the condition can be caused by any respiratory virus and has a wide spectrum of disease severity. 4 a "classic" case would be an infant aged 3 to 5 months of age 5 who develops coryza and over the subsequent 3 to 4 days has increased difficulty with breathing, and consequent inability to maintain adequate oral feeding. wheeze or crackles can be heard on auscultation. improvement occurs by days 5 to 7, though a characteristic harsh cough may persist for 21 days or more. 6, 7 while the diagnosis often appears straightforward, the wide range of disease severity across a skewed but broad age range and the need for clinical diagnosis (with associated inconsistency) creates difficulty in establishing precise data. 8 in addition, while bronchiolitis is a clinical diagnosis applied to any infecting agent, the majority of data available relate to bronchiolitis caused by respiratory syncytial virus (rsv) infection; and within rsv bronchiolitis is a focus on those at high risk, in particular, those born prematurely. reference to these groups synonymously with bronchiolitis can make interpretation of epidemiological data difficult and may reduce the understanding of bronchiolitis caused by non-rsv and in lower risk patients (particularly children born at term). there are only limited estimates of population risk for bronchiolitis associated with all respiratory virus infections, but approximately 40% of infants are affected by bronchiolitis in the first year of life. 9 in the united kingdom, using primary care databases, the 1 year incidence of children given a specific diagnosis of bronchiolitis is 58 to 65 per 1000 children, 8, 10 rising to 204 per 1000 when a broader definition of bronchiolitis was used to capture potential cases. 8 this study highlights that in children with typical lower respiratory tract signs and symptoms, clinicians may not ascribe the discrete diagnosis of bronchiolitis; a finding also found keywords bronchiolitis viral lower respiratory tract infection wheeze respiratory syncytial virus abstract acute viral bronchiolitis is a common viral lower respiratory tract infection in young children. most typically caused by respiratory syncytial virus in 70% of cases, the condition lasts for 4 to 7 days, with a prolonged cough in many. children with comorbidity, particularly those born prematurely or with significant congenital heart disease, are at risk of more severe disease. nasal obstruction progresses over 3 to 4 days to difficulty with feeding and increased work of breathing with hypoxemia. crackles and/or wheeze may be auscultated. apnoea may be a presenting sign in those less than 3 months of age. viral load is highest at peak of symptoms and in those with more severe disease. approximately 2% to 3% of all children are admitted to hospital with bronchiolitis. the differential diagnosis may include bacterial pneumonia, congenital lesions of the lung or heart, or an interstitial lung disease. there are no effective treatments, and admission is for feeding support (by nasogastric or intravenous fluids) or treatment of hypoxemia. critical care support is required for some infants experiencing respiratory failure, though mortality rates remain unchanged. practice within and between countries varies significantly and alignment of practice is a common goal of guidelines. vaccines for rsv are in advanced development, as are several antiviral therapies for rsv. in most children, acute symptoms improve within 5 to 7 days and cough by 2 weeks. recurrent wheeze is common following acute bronchiolitis and a good association with a diagnosis of asthma in childhood. for infants born ≤ 32 weeks gestation, 75% of infants will have a lower respiratory tract infection in the first year of life, with 35% rsv positive and 40% rsv negative; of these infants, 41% of rsv positive will be admitted to the hospital versus 18% of rsv negative. 27 recent studies suggest that hospitalization rates for high-risk infants due to rsv are reducing over time and are now similar to those for rsv negative, possibly as a result of improvements in neonatal care or immunoprophylaxis in high-risk groups ( fig. 24 .1). 28, 29 risk of death is much higher amongst high-risk groups who are rsv positive, including preterm (1.2%), congenital heart disease (5.2%), and bronchopulmonary dysplasia (7.0%). 17 bronchiolitis has a viral etiology, with rsv the most common cause, reported in 43% to 75% 30,31 of cases. other viruses associated with bronchiolitis are human rhinovirus (18%), influenza, coronavirus, human metapneumovirus, adenovirus, parainfluenza virus and human boca virus 30, 31 ; that is, "any respiratory virus." rsv has two strains, a and b, with rsv a associated with more severe disease. 32, 33 reinfection in the same season with the same or different strain is possible. 34 as a sole infecting agent, rsv is associated with more severe bronchiolitis than other single respiratory virus infections. 5 coinfection of rsv with rhinovirus can produce even more severe disease. 35 rsv is the most common infectious agent in children admitted to the hospital with radiological features consistent with pneumonia (occurring in 28% of children-most commonly those under 5 years of age). 36 in young children who are well immunized, rsv represents the most common cause of lower respiratory tract infection. 37 what commences as an upper respiratory tract infection becomes a lower respiratory tract infection over the course of 2 to 5 days. infants are particularly susceptible as they have small bronchi that are more likely to become blocked by secretions and edema, and a less well-developed ability to respond to and clear viral infection. 38 histopathology is naturally limited to the most severe cases who have died, where the bronchioles are edematous and blocked by necrotic epithelium and neutrophils, with some mucus binding this debris together. 39 airway obstruction is intensified by poor airway clearance associated with loss of cilia function occurring within 24 hours and persisting for up to 3 months after the illness. 40 destruction of cilia is considered to be caused by virus replication and not mediated by inflammation. 38 rsv is associated with more severe airway pathology than that found in children dying from other respiratory viruses, even in those not mechanically ventilated. 39 viral shedding is higher and more prolonged in younger infants and those with more severe disease. 41 increased disease severity, longer hospital stay and use of intensive care is associated with higher viral load for rsv in nasopharyngeal secretions. 42, 43 severity of disease is associated with both infant risk factors (including lack of adaptive t cell response), 26,44 but also rsv virus specific factors (viral antigen load and direct cytotoxic effects). 45 determining the relative contribution of both of these to disease severity will be important; if the latter is dominant, antiviral agents provided early in the course of the disease may reduce severity, whereas dominance of the former might need additional immunomodulators. 4 biomarkers are now sought to better characterize those at risk of greater disease severity and to indicate recovery. infants hospitalized with rsv bronchiolitis have increased interleukin (il)-33 and il-13 in secretions. 46 polymorphisms of surfactant protein a are associated with increased risk of intensive care admission. 47 cysteinyl leukotrienes are increased in infants with rsv bronchiolitis and are still increased 1 month following infection. 48 more severe disease is also associated with increased serum cathelicidin, 49 lactate dehydrogenase, caspase 50 and il-15. 51 there is some evidence that more severe disease may be associated with an insufficient inflammatory response. 52 the interrelationship of the microbiome in bronchiolitis is also being actively explored. 53 bronchiolitis is diagnosed clinically. variance in the clinical interpretation of symptoms and physical findings lead to inconsistency in diagnosis, particularly in milder cases and children over 1 year of age. typical symptoms are rhinorrhea, proceeding over 2 to 4 days to a characteristic harsh moist cough with pyrexia that is typically below 39°c, although fever above 38.5°c is seen in 50% of infants. 54 ability to achieve adequate oral feeding declines as nasal obstruction with secretions develops and work of breathing increases. the time to peak symptoms of 4 days is associated with the peak in viral load, 42, 55 varying from infant to infant. in younger children (particularly <6 weeks of age), apnea may be a presenting sign, sometimes in the absence of other features of bronchiolitis. apnea may be temporarily improved by nasal suctioning, but it is most likely a direct viral effect in young infants. 56 apnea is a "red flag" sign in bronchiolitis that warrants a period of review in a supervised clinical setting to ensure that it has resolved. patients more likely to require intensive care include preterm infants and those with apnea, low birth weight, or a respiratory rate greater than 70/min. 57, 58 children tend not to relapse during the improving phase of the illness, which should give confidence to clinicians when considering discharge from ed or hospital. 58, 59 physical findings include an increased respiratory rate, chest recession, use of accessory muscles, hyperinflation, wheezing, crackles, and reduced arterial oxygen saturations. 60 physical findings vary depending on sleep state (and associated changes in tidal volume). respiratory rate is a key marker of disease severity, with ≥60/min considered severe and ≥70/ min critical. 26, 61 oxygen saturation may be improved (at least temporarily) by removal of nasal secretions. 62 bronchiolitis is a highly variable disease that requires assessment of disease severity by clinicians for decision making, some of which is subjective. clinical scoring systems have been developed in an attempt to standardize care and minimize variance. many early scores derived from asthma scores. the most commonly applied scores for bronchiolitis are outlined in 65 ) and to improve the ability to identify those at risk of deterioration. the ability of clinical scores to retain precision and reliability, when scoring is performed by larger numbers of health care professionals in the context of multicenter phase iii trials, is of current interest. symptoms in bronchiolitis vary across a wide but skewed continuum from mildly increased work of breathing with cough to respiratory failure and death. often divided into mild, moderate, and severe disease, the perspective on these gradations varies across health care systems. a world health organization (who) workshop has provided candidate definitions differentiating a diagnosis of rsv lower respiratory tract infection (spo 2 <95%) from severe (<93%) and very severe rsv disease (spo 2 <90%, inability to feed orally, or reduced level of consciousness). 66 infants can display variance in spo 2 within this range (90% to 95%) over short periods of observation without significant change in clinical status, 58,62,67 which may limit the discriminatory reliability of these definitions. from a secondary care perspective, moderate severity is often considered a need for admission to hospital and severe by need for critical care (positive pressure support). clinical scores are often designed to identify transition points in the level of care required. 64 the currently available evidence concerning transition points in level of care is poor. treatment guidance, particularly benefit from use of interventions at the ed/ward (i.e., spo 2 ) 68 and ward/critical care floor interface (i.e., highflow nasal cannula [hfnc] oxygen and continuous positive airway pressure [cpap]) is much needed. guidelines have provided signs and symptoms that should alert clinicians to a child at risk of deterioration and suggested criteria for admission to the hospital. 26 in hospitals, those most likely to deteriorate to the extent of being provided with critical care support are of lower birth weight (<5 lbs, 2.25 kg) and/or have a respiratory rate ≥70/min on day 1 of admission. 57 chest radiography is not required to confirm a diagnosis of bronchiolitis. a chest radiograph often leads to increased diagnostic uncertainty as the features may be similar to those of pneumonia (atelectasis, mucous plugging, and loss of volume) and consequently lead to greater inappropriate use of antibiotics. 69 chest radiography should be reserved for a child who is atypical, for example, showing persistently focal crackles, a temperature remaining above 39°c despite antipyretics, or respiratory failure requiring critical care support. 26, 70 laboratory tests do not aid in the clinical diagnosis of bronchiolitis. serious bacterial infection is unusual and complete blood counts and blood cultures are unhelpful (though recent evidence suggests that although still uncommon, it may be more frequent than previously considered). 65 dehydration is usually mild and best assessed clinically without electrolyte measurement. approximately 6% of infants with bronchiolitis can have concurrent urinary tract infection, so urine culture may be of value in persistently febrile infants, particularly those under 3 months of age. 71 measurement of arterial/capillary carbon dioxide is commonly performed, but can be restricted to those children with increased respiratory rate and work of breathing despite oxygen supplementation. 72 the clinical interpretation of signs and symptoms is difficult in a condition where age boundaries are loose (and skew to older ages in those with comorbidity) and symptoms vary from patient to patient and time to time. this naturally leads to variation in diagnosis and differential diagnosis. there is a common understanding that a clearer diagnosis is possible in those under 1 year of age and most guidelines reflect this. however, constraining a diagnosis of bronchiolitis to those less than 1 year of age may reduce the ability to identify the whole population of children with bronchiolitis who could benefit from potential interventions. in general, a broader bronchiolitis) is often performed to aid cohorting of patients within hospitals. the increasing recognition that multiple viruses may be identified in those with acute bronchiolitis has called into question the benefit of cohorting based on rsv status. 78 pcr diagnostics may sometimes be considered oversensitive to the detection of virus fragments postinfection, and multiplex pcr results should be interpreted with this understanding. differential diagnosis includes bacterial pneumonia or an alternative cause of crackles, wheeze, and increased work of breathing in a young child. persisting crackles (crepitations) in one lung zone, fixed focal wheeze, persistent pyrexia (>39°c) or persistently increased work of breathing in a child who appears otherwise recovered warrant further evaluation. prescient in the mind of most clinicians is that a bacterial pneumonia may be missed. chest radiographs have similar appearances and are poor discriminators. we can assume that bacterial coinfection risk is low, as the use of antibiotics in bronchiolitis is not associated with faster recovery. 79 further investigation could be limited to those with the persisting clinical features noted above. in children with more severe disease, there may be a role for antibiotics as bacteria are isolated in 33% to 44% of lavage samples in children with severe bronchiolitis who are intubated and ventilated. [80] [81] [82] definition of bronchiolitis is used in north america and asia that captures a higher percentage of older children with wheezing, where rhinovirus is the dominant infecting agent. 73 such children may be given a diagnosis of viral induced wheeze in other countries. there is most likely a continuum of viral lower respiratory tract infection across age ranges that moves from current diagnoses of viral bronchiolitis to viral pneumonia and viral-induced wheeze/wheezy bronchitis. 74, 75 the clinical features consistent with a diagnosis of bronchiolitis across different guidelines are presented in table 24 .2. diagnosis has a typical onset of a viral respiratory tract prodrome proceeding to lower respiratory symptoms over 3 to 4 days. the south african guideline (2010) considers hyperinflation the most reliable clinical sign in bronchiolitis. 76 the uk guideline (2015) provides a more proscriptive definition. 26 testing of nasal secretions for virus may help consolidate the clinical diagnosis of bronchiolitis and inform health care logistics. most commonly used, and with highest precision, are polymerase chain reaction (pcr) diagnostics for a range of respiratory viruses, but point of care (poc) testing for a more limited range of viruses (most often rsv) is increasingly precise and cost effective. 77 testing for rsv (as the most common infecting agent in therapies in addition to supplemental oxygen and hydration are poorly supported by current evidence. there is some evidence that infants handled less get better quicker, 87 and the use of additional therapies should be considered with that in mind. there is widespread variation across hospitals and countries in the management and treatment of bronchiolitis reflecting local custom and individual clinician practice. 88 reducing variation and associated health care costs is a key aim of bronchiolitis management presented through guidelines. guidelines for the care of infants with bronchiolitis based on systematic review and published in english are available from the united kingdom (2015), 89 92 and south africa (2010), 76 which has been updated as a critical review 2016. 93 no therapies receive support across all guidelines for use with the exception of supplemental oxygen. chest physiotherapy does not speed recovery. antibiotics, though still widely used, are of no benefit in bronchiolitis. 79 in addition, bronchodilators are less likely to be recommended in more recent guidelines, and the theory that they may be of greater benefit in infants more likely to develop asthma has been refuted. 94 nebulized hypertonic saline has been of benefit in cystic fibrosis and in early trials in bronchiolitis, 95 but larger well-designed trials have not demonstrated a persuasive benefit. [96] [97] [98] [99] [100] recent years have seen the increasing use of hfnc oxygen in acute bronchiolitis. 101 though clinical trials have not yet demonstrated important clinical or physiological benefits, 102 large well-designed trials are in progress and are beginning to report. 102a,103 cpap has some benefit in bronchiolitis, and may prevent deterioration when used early. 104 as with all management in bronchiolitis, the use of hfnc oxygen, cpap, and intubation varies across sites irrespective of disease severity, 105 and better understanding of the risks and benefits of these interventions is required. 106 there are no current effective pharmacological treatments for rsv. while ribavirin was previously used as an antiviral treatment for rsv, it is now considered ineffective. 107 novel treatments for acute infection are in development: antivirals and nebulized immunoglobulin. in this rapidly moving field, it seems probable that a treatment for rsv will become available in the next 5 years. 108 reduction in viral load has been demonstrated in adult challenge models of rsv treated with the antivirals als-8176 109 and gs-5806 110 prevention of spread of rsv depends on good hygiene, in particular, hand washing, as rsv may survive for up to 6 hours on surfaces contaminated by droplets. 111 similar precautions are appropriate for other respiratory virus infections associated with bronchiolitis. many hospitals use poc testing for rsv to determine cohorting of infants as inpatients. 107 while this is still common, the practice is called into question by the range of coinfection with other respiratory viruses revealed by pcr panel testing; up to 62% of children with viral respiratory tract infection have more than one virus detected. 112 prevention of rsv (as the most common cause of bronchiolitis) has been a long-term goal. early formalin inactivated there are no trials of outcome for antibiotic use in children with bronchiolitis receiving intensive care. though uncommon, congenital lesions may masquerade as bronchiolitis, and this should be borne in mind for children with atypical clinical features or those slow to recover. congenital heart disease may present as bronchiolitis when pulmonary vascular resistance falls increasing left to right shunt. more difficult to differentiate are children with congenital (or less commonly acquired) pulmonary malformations. fixed focal wheeze may be a sign of tracheomalacia or bronchomalacia, stenosis, or compression from lobar emphysema or a bronchogenic cyst and would warrant a chest radiograph. a slow recovering course with persistent chest signs could be an infected congenital pulmonary malformation (such as a congenital cystic adenomatoid malformation [ccam] or sequestration). children with persistent fine crackles, tachypnea, and low (often borderline) oxygen saturation may have an interstitial lung disease, particularly neuroendocrine cell hyperplasia (nehi) presenting as recurrent "bronchiolitis." young children with persistent, sometimes focal, crackles postadenovirus (though may also be other respiratory viruses and mycoplasma pneumonia) should be evaluated for postinfectious bronchiolitis obliterans (pibo); see section 6 of the book. management of bronchiolitis is supportive, assisting hydration and hypoxemia until improvement. with increased respiratory rate and nasal secretions, oral feeding is challenged, and those with severe disease require assistance with feeding by enteral or parenteral means. the threshold for supporting hydration is typically when an infant's intake is reduced to 50% to 75% of usual volume. the chosen percentage of intake depends on the child's status: an expreterm 10-week-old infant on day 3 of illness may be supported at 75% understanding that they most likely will deteriorate, whereas a robust 8-month-old term infant may be able to tolerate 50% feed volume for a couple of days until disease resolution. nasogastric feeding is easier to administer than intravenous fluids but has no advantage in recovery from acute disease. 83 oxygen may be used to treat hypoxemia. the threshold oxygen saturation at which to use supplemental oxygen varies across guidelines and is typically set between 90% and 94% at sea level. in children admitted to hospital with bronchiolitis, management at a threshold of 90% spo 2 is safe and as clinically effective as a 94% target. 84 the threshold oxygen saturation for admission to hospital is often 92%, as some data suggest that infants have a higher risk of desaturating further at this oxygen saturation. 85 oxygen desaturation may however have a disproportionate influence on decisions to admit children to the hospital, 68 and much like hydration status (previously mentioned), the context of the measurement should be considered. many infants discharged home from ed with bronchiolitis experience desaturation events subsequently that are not associated with clinical deterioration. 67 in hospitals, the use of intermittent oxygen saturation monitoring is much discussed, and though the benefit below 90% spo 2 is not established, once stable above 90% spo 2 , oxygen saturation monitoring should be stopped. 86 use of bronchiolitis occurs in 62% of those who are rsv positive and 32% of those who are rsv negative. 120 recurrent postinfectious wheeze is not reduced by montelukast 121 or inhaled corticosteroids, 122, 123 but there is good evidence of benefit from palivizumab, 124 and potentially azithromycin, 125 though the latter requires further study. in the longer term, there is good evidence that children who have had an admission to the hospital for rsv bronchiolitis are 3 times more likely to have a diagnosis of asthma and lower lung function at age 6 years 23 and a higher incidence of asthma at age 13 126 and 18 127 years. the question remains whether such children are predisposed to bronchiolitis because of premorbid anatomy 119 and the consequent interrelationship between host and virus specific effects on the development of asthma. vaccines were associated with more severe enhanced rsv disease and deaths, possibly resulting from inadequate t cell priming. 113 subsequent vaccine development has been cautious in view of this experience. in the 1990s, rsv intravenous immunoglobulin was developed 114, 115 but was rapidly superseded by palivizumab, a monoclonal antibody delivered by monthly intramuscular injection. when administered over the rsv season, it reduces hospital admission in high-risk infants. 116 palivizumab's monthly injections and limited efficacy have prompted the development of extended life monoclonal antibodies that are undergoing licensing trials in preterm infants. 117 they will hopefully be evaluated in the future for high-risk infants born at term. rsv vaccine development has gained significant impetus over the last 15 years with a wide range of candidate vaccines in development both for pediatric and maternal use; maternal immunization could provide passive transplacental protection to infants in the first 3 to 6 months of life (http:// www.path.org). a phase iii trial of maternal immunization by novovax is expected to conclude in 2020. for most children, bronchiolitis is a self-limiting disease, with cough as the most persistent symptom resolving at a median of 12 to 15 days. 84, 118 many children, however, develop recurrent respiratory symptoms. in the first few months following illness, this is considered in part to result from loss of cilia from the airway epithelial surfaces during the acute illness. 40 for those who experience chronic symptoms, the debate continues on whether children with more severe bronchiolitis and recurrent postinfectious wheezing have premorbid susceptibility, with some evidence suggesting poorer preexisting lung function. 119 recurrent wheeze in the year following oxygen saturation targets in infants with bronchiolitis (bids): a double-blind, randomised, equivalence trial sabre: a multicentre randomised control trial of nebulised hypertonic saline in infants hospitalised with acute bronchiolitis nebulized hypertonic saline for bronchiolitis in the emergency department: a randomized clinical trial 7% hypertonic saline in acute bronchiolitis: a randomized controlled trial 3% hypertonic saline versus normal saline in inpatient bronchiolitis: a randomized controlled trial the effect of 3% and 6% hypertonic saline in viral bronchiolitis: a randomised controlled trial nebulized hypertonic saline for bronchiolitis: a randomized clinical trial risk factors for bronchiolitis, recurrent wheezing, and related hospitalization in preterm infants during the first year of life chronic diseases, chromosomal abnormalities, and congenital malformations as risk factors for respiratory syncytial virus hospitalization: a population-based cohort study bronchiolitis: diagnosis and management in children prospective study of healthcare utilisation and respiratory morbidity due to rsv infection in prematurely born infants trends in respiratory syncytial virus and bronchiolitis hospitalization rates in high-risk infants in a united states nationally representative database respiratory syncytial virus hospitalization trends in infants with chronic lung disease of infancy viral etiologies of infant bronchiolitis, croup and upper respiratory illness during 4 consecutive years viral etiology of bronchiolitis among pediatric inpatients in northern taiwan with emphasis on newly identified respiratory viruses occurrence of groups a and b of respiratory syncytial virus over 15 years: associated epidemiologic and clinical characteristics in hospitalized and ambulatory children severity of respiratory syncytial virus infection is related to virus strain immunity to and frequency of reinfection with respiratory syncytial virus association of rhinovirus infection with increased disease severity in acute bronchiolitis community-acquired pneumonia requiring hospitalization among u.s. adults aetiology of childhood pneumonia in a well vaccinated south african birth cohort: a nested casecontrol study of the drakenstein child health study respiratory syncytial virus (rsv) and its propensity for causing bronchiolitis pecularities of lesions in viral and mycoplasma infections of the respiratory tract recovery of the ciliated epithelium following acute bronchiolitis in infancy respiratory syncytial virus infections in infants: quantitation and duration of shedding respiratory syncytial virus genomic load and disease severity among children hospitalized with bronchiolitis: multicenter cohort studies in the united states and finland respiratory syncytial virus load, viral dynamics, and disease severity in previously healthy naturally infected children type 1 and type 2 cytokine imbalance in acute respiratory syncytial virus bronchiolitis viral specific factors contribute to clinical respiratory syncytial virus disease severity differences in infants respiratory syncytial virus disease is mediated by age-variable il-33 surfactant protein a2 polymorphisms and disease severity in a respiratory syncytial virus-infected population latitudinal variations in seasonal activity of influenza and respiratory syncytial virus (rsv): a global comparative review altitude and environmental climate effects on bronchiolitis severity among children presenting to the emergency department environmental drivers of the spatiotemporal dynamics of respiratory syncytial virus in the united states viral bronchiolitis in children epidemiology of respiratory syncytial virus bronchiolitis in hospitalized infants in greece duration of illness in ambulatory children diagnosed with bronchiolitis duration of illness in infants with bronchiolitis evaluated in the emergency department the clinical burden of respiratory syncytial virus (rsv) bronchiolitis among infants in the united kingdom (uk) [phd]: imperial college epidemiology of respiratory syncytial virus infection in washington, dc. ii. infection and disease with respect to age, immunologic status, race and sex a comparison of the recording of 30 common childhood conditions in the doctor's independent network and general practice research databases viral respiratory tract infections in the first six months of life evaluation of hospitalized infants and young children with bronchiolitis-a multi centre study trends in bronchiolitis hospitalizations in the united states admission to hospital for bronchiolitis in england: trends over five decades, geographical variation and association with perinatal characteristics and subsequent asthma risk factors for hospital admission with rsv bronchiolitis in england: a population-based birth cohort study international variation in the management of infants hospitalized with respiratory syncytial virus. international rsv study group the risk of mortality among young children hospitalized for severe respiratory syncytial virus infection risk of primary infection and reinfection with respiratory syncytial virus respiratory-syncytialvirus infections, reinfections and immunity. a prospective, longitudinal study in young children global burden of acute lower respiratory infections due to respiratory syncytial virus in young children: a systematic review and meta-analysis the burden of respiratory syncytial virus infection in young children hospitalizations associated with influenza and respiratory syncytial virus in the united states increased risk of wheeze and decreased lung function after respiratory syncytial virus infection g489 blood gas analysis in acute bronchiolitis-who and when? a clustering approach to identify severe bronchiolitis profiles in children deciphering clinical phenotypes in acute viral lower respiratory tract infection: bronchiolitis is not an island variability in the diagnostic labeling of nonbacterial lower respiratory tract infections: a multicenter study of children who presented to the emergency department south african guideline for the diagnosis, management and prevention of acute viral bronchiolitis in children rapid point of care diagnostic tests for viral and bacterial respiratory tract infections-needs, advances, and future prospects co-infections in children hospitalised for bronchiolitis: role of roomsharing antibiotics for bronchiolitis in children under two years of age high incidence of pulmonary bacterial co-infection in children with severe respiratory syncytial virus (rsv) bronchiolitis pulmonary and systemic bacterial co-infections in severe rsv bronchiolitis concurrent bacterial infection and prolonged mechanical ventilation in infants with respiratory syncytial virus lower respiratory tract disease nasogastric hydration versus intravenous hydration for infants with bronchiolitis: a randomised trial oxygen saturation targets in infants with bronchiolitis (bids): a double-blind, randomised, equivalence trial predictors of major intervention in infants with bronchiolitis intermittent monitoring of oxygen saturation in infants and children with acute bronchiolitis: peekaboo pediatrics or good clinical care? racemic adrenaline and inhalation strategies in acute bronchiolitis variability in inpatient management of children hospitalized with bronchiolitis bronchiolitis in children: summary of nice guidance clinical practice guideline: the diagnosis, management, and prevention of bronchiolitis clinical practice guideline on acute bronchiolitis. clinical practice guidelines in the spanish national healthcare system finnish guidelines for the treatment of laryngitis, wheezing bronchitis and bronchiolitis in children acute viral bronchiolitis in south africa: strategies for management and prevention allergic diseases and the effect of inhaled epinephrine in children with acute bronchiolitis: follow-up from the randomised, controlled, double-blind, bronchiolitis all trial nebulized hypertonic saline for acute bronchiolitis: a systematic review 3% hypertonic saline versus normal saline in inpatient bronchiolitis: a randomized controlled trial nasal lavage leukotrienes in infants with rsv bronchiolitis serum cathelicidin level is associated with viral etiology and severity of bronchiolitis lactate dehydrogenase and caspase activity in nasopharyngeal secretions are predictors of bronchiolitis severity. influenza other respir viruses interleukin-15 is associated with disease severity in viral bronchiolitis a robust cytokine and chemokine response in nasopharyngeal secretions is associated with decreased severity in children with physician diagnosed bronchiolitis nasopharyngeal bacterial burden and antibiotics: influence on inflammatory markers and disease severity in infants with respiratory syncytial virus bronchiolitis north ryde: nsw ministry of health virus type and genomic load in acute bronchiolitis: severity and treatment response with inhaled adrenaline the g glycoprotein of respiratory syncytial virus depresses respiratory rates through the cx3c motif and substance p risk factors for requiring intensive care among children admitted to ward with bronchiolitis effect of oxygen supplementation on length of stay for infants hospitalized with acute viral bronchiolitis hospital course and discharge criteria for children hospitalized with bronchiolitis inter-observer agreement between physicians, nurses, and respiratory therapists for respiratory clinical evaluation in bronchiolitis bronchiolitis: recommendations for diagnosis, monitoring and management of children one to 24 months of age is nasal suctioning warranted before measuring o2 saturation in infants with bronchiolitis? wheezing in infants: the response to epinephrine measuring clinical severity in infants with bronchiolitis bacteremia in children hospitalized with respiratory syncytial virus infection who consultation on respiratory syncytial virus vaccine development report from a world health organization meeting held on 23-24 effect of oxygen desaturations on subsequent medical visits in infants discharged from the emergency department with bronchiolitis effect of oximetry on hospitalization in bronchiolitis: a randomized clinical trial variation in inpatient diagnostic testing and management of bronchiolitis predictors of airspace disease on chest x-ray in emergency department patients with clinical bronchiolitis: a systematic review and meta-analysis assessing the utility of urine testing in febrile infants aged 2 to 12 months with bronchiolitis respiratory syncytial virus (rsv) immune globulin intravenous therapy for rsv lower respiratory tract infection in infants and young children at high risk for severe rsv infections: respiratory syncytial virus immune globulin study group reduction of respiratory syncytial virus hospitalization among premature infants and infants with bronchopulmonary dysplasia using respiratory syncytial virus immune globulin prophylaxis palivizumab, a humanized respiratory syncytial virus monoclonal antibody, reduces hospitalization from respiratory syncytial virus infection in high-risk infants lower respiratory tract infection caused by respiratory syncytial virus: current management and new therapeutics epinephrine and dexamethasone in children with bronchiolitis decreased lung function precedes severe respiratory syncytial virus infection and post-respiratory syncytial virus wheeze in term infants clinical prediction rule for rsv bronchiolitis in healthy newborns: prognostic birth cohort study study of montelukast for the treatment of respiratory symptoms of post-respiratory syncytial virus bronchiolitis in children randomised placebo controlled trial of nebulised corticosteroids in acute respiratory syncytial viral bronchiolitis on behalf of the rsvcsg. lack of long term effects of high dose inhaled beclomethasone for rsv bronchiolitis-a randomized placebo-controlled trial respiratory syncytial virus and recurrent wheeze randomized trial to evaluate azithromycin's effects on serum and upper airway il-8 levels and recurrent wheezing in infants with respiratory syncytial virus bronchiolitis severe respiratory syncytial virus bronchiolitis in infancy and asthma and allergy at age 13 asthma and allergy patterns over 18 years after severe rsv bronchiolitis in the first year of life nebulized hypertonic saline for bronchiolitis: a randomized clinical trial the effect of 3% and 6% hypertonic saline in viral bronchiolitis: a randomised controlled trial 7% hypertonic saline in acute bronchiolitis: a randomized controlled trial sabre: a multicentre randomised control trial of nebulised hypertonic saline in infants hospitalised with acute bronchiolitis high-flow nasal cannula oxygen for bronchiolitis in a pediatric ward: a pilot study cpap and high-flow nasal cannula oxygen in bronchiolitis high-flow warm humidified oxygen versus low-flow nasal cannula oxygen for moderate bronchiolitis (hfwho rct): an open, phase 4, randomised controlled trial early high flow nasal cannula therapy in bronchiolitis, a prospective randomised control trial (protocol): a paediatric acute respiratory intervention study (paris) improved clinical and economic outcomes in severe bronchiolitis with pre-emptive ncpap ventilatory strategy variability of intensive care management for children with bronchiolitis mechanical ventilation drives inflammation in severe viral bronchiolitis bronchiolitis in children (sign 91). nhs quality improvement: scotland respiratory syncytial virus, an ongoing medical dilemma: an expert commentary on respiratory syncytial virus prophylactic and therapeutic pharmaceuticals currently in clinical trials. influenza other respir viruses activity of oral als-008176 in a respiratory syncytial virus challenge study oral gs-5806 activity in a respiratory syncytial virus challenge study possible transmission by fomites of respiratory syncytial virus viral coinfection in childhood respiratory tract infections respiratory syncytial virus disease in infants despite prior administration of antigenic inactivated vaccine key: cord-254265-8i86c8kt authors: camps, marta; ricart, sílvia; dimova, veselka; rovira, núria; muñoz‐almagro, carmen; garcia, juan jose; pons‐odena, martí; marcos, mª angeles; pumarola, tomàs title: prevalence of human metapneumovirus among hospitalized children younger than 1 year in catalonia, spain date: 2008-06-12 journal: j med virol doi: 10.1002/jmv.21209 sha: doc_id: 254265 cord_uid: 8i86c8kt human metapneumovirus was discovered recently respiratory virus implicated in both upper and lower respiratory tract infection. in children, the clinical symptoms of human metapneumovirus are similar to those produced by respiratory syncytial virus, ranging from mild to severe diseases such as bronchiolitis and pneumonia. the aim of the present study was to describe the prevalence of human metapneumovirus and other common respiratory viruses among admitted to hospital infants. from january 2006 to june 2006, 99 nasopharyngeal aspirates were collected from hospitalized children younger than 12 months in order to study respiratory viruses. human metapneumovirus detection was performed by cell culture and two rt‐pcr targeting on polymerase and fusion genes. the latter gene was used for phylogenetic analysis. in 67/99 children (67%) at least one viral pathogen was identified, the viruses detected most frequently were respiratory syncytial virus (35%), human metapneumovirus (25%) and rhinovirus (19%). the results obtained in this study, show that: (1) human metapneumovirus is one of the most important viruses among children less than 12 months; (2) children infected with human metapneumovirus were significantly older than those infected by respiratory syncytial virus; (3) human metapneumovirus was associated more frequently with pneumonia whereas respiratory syncytial virus was only detected in patients with bronchiolitis; (4) there was a clear epidemiological succession pattern with only a small overlap among the viruses detected most frequently; (5) all human metapneumovirus samples were clustered within sublineage a2. j. med. virol. 80:1452–1460, 2008. © 2008 wiley‐liss, inc. human metapneumovirus was discovered recently as a respiratory virus implicated in both upper and lower respiratory tract infection ranging from mild to severe disease in all age groups [van den hoogen et al., 2001] . since then, it has been reported in europe, asia, australia, south africa and america [peret et al., 2002; bastien et al., 2003; jpma et al., 2004; rao et al., 2004; sloots et al., 2006; brooks et al., 2007] , suggesting worldwide distribution. seroprevalence studies have shown that this virus has been present among humans for over five decades [van den hoogen et al., 2001; hamelin et al., 2004] . human metapneumovirus was not discovered previously due to its difficulty to grow in traditional cell cultures, although it has slow growth in the rhesus monkey kidney (llc-mk2) cell line [deffrasnes et al., 2005] . although monoclonal antibodies (mabs) are being developed [landry et al., 2005; percivalle et al., 2005] , these are not available commercially for direct antigen detection in nasopharyngeal aspirate (npa), and molecular assays remain the main approach available currently for human metapneumovirus identification. human metapneumovirus has a negative-strand rna genome of approximately 13 kb encapsidated by a helicoidal nucleocapsid and covered with a lipid bilayer [van den hoogen et al., 2002; biacchesi et al., 2003] . it has been classified in the pneumovirus subfamily of the paramyxovirus family, which also contains the respiratory syncytial virus and metapneumovirus genus. currently, two main genotypes a and b with the subtypes a1, a2, b1, and b2 are identified by molecular methods [biacchesi et al., 2003] . the clinical symptoms of human metapneumovirus are similar to those caused by respiratory syncytial virus, ranging from wheeze, cough, fever, bronchiolitis, and even pneumonia [wolf et al., 2006] . although some studies have detected human metapneumovirus in all age groups, human metapneumovirus infections could be more severe in children, elderly, and immunocompromised patients [pelletier et al., 2002; bastien et al., 2003; cane et al., 2003; falsey et al., 2003] . however, several epidemiological and clinical features have yet to be established firmly. the aim of the present study was to describe the role of human metapneumovirus and other common respiratory viruses including: influenza virus a, b, and c, parainfluenza 1-4 viruses, adenoviruses, respiratory syncytial virus a and b, rhinovirus, coronavirus 229e and oc43 and enterovirus as bronchiolitis, and bronchopneumonia pathogens among hospitalized children younger than 1 year, taking into account that in this age group respiratory viruses are the main etiological agents of lower respiratory tract infections [shay et al., 1999; smyth and openshaw, 2006; bush and thomson, 2007] . additionally, in human metapneumovirus positive samples, a comparative phylogenetic analysis of fusion (f) gene sequences was carried out to study the potential circulation of distinct human metapneumovirus genotypes among patients included. the present study falls within the framework of the ''study of prevalence, clinical and epidemiological features associated to human metapneumovirus infection among infants and adult population'' awarded by the fondo de investigaciones sanitarias (fis), spain. from january 2006 to june 2006, children younger than 12 months hospitalized in a 345-bed children's hospital (hospital sant joan de déu, barcelona, spain) with bronchiolitis or bronchopneumonia, were studied prospectively. within the first 24 hr of admission, a nasopharyngeal aspirate was collected and delivered to the laboratory of microbiology at the hospital clínic of barcelona, where it was processed to study respiratory viruses. a questionnaire about clinical and epidemiological features was also completed during the initial consultation. routine bacteriological testing (of blood and urine) was only performed in all febrile infants who have toxic manifestations, defined as a clinical appearance consistent with the sepsis syndrome [baraff et al., 1993] . other respiratory samples were obtained according to clinical indication or the decision of the attending physician. bronchiolitis was defined as an acute infection of the lower airway, characterized by increased respiratory effort (tachypnea) and expiratory wheezing and/or crackles associated with common viral infection symp-toms (rhinorrhea, cough) in infants younger than 1 year. the presence of lower respiratory infection (wheezing and/or crackles) with a focal infiltrate in the chest x-ray was considered as a bronchopneumonia. exclusion criteria were underlying medical conditions (cystic fibrosis, metabolic diseases, neurological diseases) and patients with two or more previous episodes of wheeze without the presence of radiological infiltrates. criteria for pediatric intensive care unit (picu) admission were: acute respiratory failure with hemoglobin saturation <90% and >40% fraction of inspired oxygen (fio 2 ) supplementation, common episodes of apnea and sepsis according to the international consensus conference on pediatric sepsis [goldstein et al., 2005] . informed consent was obtained from the patients' parents. the ethical committees of the hospital clínic and hospital sant joan de déu approved the study protocol. specimens for immunofluorescence assay (ifa) were suspended in pbs (phosphate buffered saline), when necessary, and spotted onto a slide that was air-dried and fixed with cold acetone and then stained with a pool of fluorescein-conjugated antibody to influenza virus a, influenza virus b, human parainfluenza virus 1-3, adenovirus, and respiratory syncytial virus (respiratory panel 1, viral screening and identification kit; light diagnostics, chemicon international temecula, usa). simultaneously, specimens were inoculated into mdck (madin darby canine kidney), hep-2 (human caucasian larynx carcinoma squamous cell) and llc-mk2 (rhesus monkey kidney epithelial cell) cell lines (vircell, granada, spain) for isolation of the viruses mentioned above and human metapneumovirus. mdck and llc-mk2 tube cultures were incubated at 33 and 378c, respectively, and maintained with growth essential medium [emem (ebss) þ 2 mm glutamine þ 1% non-essential amino acids (neaa)] adding 12 ml (500 mg/ml) of trypsin/edta for optimal growth of influenza viruses and human metapneumovirus. hep-2 cell line was maintained with the same medium plus 2% fetal bovine serum and incubated at 378c. cell cultures were examined twice weekly for the development of a cytopathic effect; positive cultures were harvested and stained for conventional virus identification (ifa). in ifa negative cases, a rt-pcr (reverse transcription-polymerase chain reaction) for common respiratory viruses and human metapneumovirus was undertaken. upon sample collection, an aliquot of each fresh specimen was collected to be used for rt-pcr analysis. nucleic acids from either dna/rna viruses present in the nasopharyngeal secretion or in infected cell cultures were extracted from 200 ml of specimen using nucli-sense easymag (biomérieux, nl-5281 rm boxtel, the netherlands) according to the manufacturer's instructions. the lysis buffer included 500 molecules of the cloned-amplified product used as an internal control in each reaction tube in order to exclude false negative results due to non-specific inhibitors or extraction failure. two independent multiplex nested rt-pcr assays able to detect from 1 to 10 copies of viral genomes were carried out using techniques described previously [coiras et al., 2003; coiras et al., 2004] . one rt-pcr assay detected influenza virus a, influenza virus b, influenza virus c, respiratory syncytial virus a, respiratory syncytial virus b, and adenovirus. another rt-pcr assay examined human parainfluenza virus 1-4a and 4b, human coronavirus 229e, human coronavirus oc43, and the generic detection of enterovirus and rhinovirus. in order to detect human metapneumovirus, extracted rna was used as a template for cdna synthesis by random primers according to the manufacturer's instructions (first strand cdna synthesis kit, roche diagnostics, mannheim, germany). amplification reaction was carried out using specific primers amplifying a conserved fragment of 170 bp in the polymerase gene (l): lf, 5 0 cat gcc cac tat aaa agg tca g 3 0 ; lr, 5 0 cac ccc agt ctt tct tga aa 3 0 , as described elsewhere . the pcr conditions comprised 45 cycles at 948c for 1 min (denaturalizaton), 508c for 50 sec (annealing), 728c for 1 min (extension) and a final extension at 728c for 10 min. to detect human metapneumovirus types a and b, in addition to the widely used primers of l gene that do not hybridize to type b [sarasini et al., 2006 ], a second nested pcr test using fusion gene (f) primers was performed. first round pcr used the following primers: ff1, 5 0 ttc gtt cta gga gca a 3 0 and fr, 5 0 gtc ttc ctg tcg taa ctt tg 3 0 . the second round used an inner forward primer ff 5 0 atg cca aca tct gca gga c 3 0 and the same reverse primer as in the first step, obtaining a final product of 450 pb. amplified products were analyzed by electrophoresis on 2% agarose gel, stained with ethidium bromide. fusion gene primers were either selected from published protocols or designed originally based on the sequence data of a canadian strain available from genbank, national center for biotechnology information (genbank) (accession no. ay297749) [biacchesi et al., 2003] . as an human metapneumovirus positive control, rna of human metapneumovirus (subtype a2, according to strain canada 97/83) was used in the pcr test. each set of rt-pcr reaction included a positive control for extraction and amplification handling obtained from our viral lysates, when available, and a negative control (viral transport medium containing no nucleic acid). all positive results were confirmed by two sequential assays. in addition, a second nested pcr targeting a fragment of the f gene was also used for further phylogenetic analysis and to confirm the previous l gene positive results and identify probable human metapneumovirus type b [bouscambert-duchamp et al., 2005; banerjee et al., 2007] . cdna synthesized previously was used as a template for the first f gene pcr amplification. the reaction mix contained ff1/fr primers and termocycler conditions were 35 cycles at 948c for 1 min (denaturation), 458c for 2 min (annealing), 728c for 2 min (extension) and a final extension step of 728c for 10 min. the second round pcr used an inner primer, ff, and the same reverse primer used before. in this pcr, annealing reaction was performed at 508c instead of 488c, and other conditions and reaction volumes were identical to those applied in the first round. pcr reactions that produced proper bands were purified on qiaquick gel extraction kit (qiagen, izasa, spain) and sequenced with bigdye terminator v3.1 (applied biosystems, foster city, usa) in an abi prism 3700 dna analyzer (applied biosystems, foster city, usa). sequences were aligned with different human metapneumovirus subtype strains from canada, netherlands, and japan (accession nos. ay145295, ay145298, ay145289, ay145292, ay145287, ay145294, ay145299, ay145297, ay145301, ay145296, dq362940.1, and ay312232) [bastien et al., 2003; galiano et al., 2006; huck et al., 2006] and avian metapneumovirus c sequence (accession no. ay590688) was used as an outgroup [govindarajan et al., 2004] . nucleotide sequence alignments were generated using the clustalw algorithm of the mega software [kumar et al., 2004] and the total length of each sequence was 302 nucleotides. phylogenetic and molecular analyses were conducted using mega version 3.0. phylogenetic trees were constructed by the phylip program package, version 3.66 (felsenstein, department of genetics, university of washington, seattle, wa), using the neighbor-joining method supplied by the treeview 32 program. prevalence of human metapneumovirus was calculated as the number of children positive by pcr for human metapneumovirus divided by the number of children in the same age group admitted during the same period and hospitalized with lower respiratory tract infection. quantitative variables were described with means ae standard deviation and significant differences between respiratory syncytial virus and human metapneumovirus groups were analyzed by using mann-whitney u-test. qualitative variables were reported as frequencies and percentages. comparison of proportions was determined by w 2 or fisher's exact test. probability values of p < 0.05 were considered significant. analysis was performed using the graph-pad prism3 statistical program (graphpad software, inc., san diego, ca). from january to june 2006, of the 120 children younger than 12 months who were hospitalized for lower respiratory tract infection, 99 with nasopharyngeal aspirate for respiratory viruses detection were included prospectively. the median age of the patients was 3.9 ae 4.1 months (range: 14 days to 12 months) and included 59 males (59%) and 40 females (41%). clinical diagnoses at the time of admission were bronchiolitis (74%) and (26%) bronchopneumonia, of which 67% and 72%, respectively, had an etiological diagnosis. in 67/99 children (67%), at least one viral pathogen was identified; 55/99 (55%) had an infection with one of the viruses investigated, whereas 12/99 (12%) had a coinfection of at least two viruses. a total of 80 viruses were identified, while 32/99 (33%) patients remained without an etiological diagnosis. the most frequent virus detected was respiratory syncytial virus (35%) followed by human metapneumovirus (25%) and rhino-virus (19%) (fig. 1) . viral co-infections were observed in 12 (12%) cases, with adenovirus and rhinovirus the most common viruses identified. in one patient with bronchopneumonia, a triple infection was detected (respiratory syncytial virus þ adenovirus þ rhinovirus). blood and urine cultures were obtained from 32 patients with criteria of toxic manifestations. none of the specimens of blood or urine grew a pathogen. three bronchial aspirates and one bronchoalveolar lavage were collected. an haemophilus influenzae and a streptococcus pneumoniae were recovered from bronchial aspirate and bronchoalveolar lavage, respectively. diagnosis of bronchiolitis was significantly more frequent in respiratory syncytial virus single-infected children (p < 0.001), whereas human metapneumovirus infection was significantly more common among infants with pneumonia (p < 0.001) ( table i) . human metapneumovirus infected children were significantly older (mean age: 6.2 ae 5.14 months) than those infected by respiratory syncytial virus (mean age: 2.5 ae 3.37 months) (p ¼ 0.009) (tables ii, iii) . thirteen patients required hospitalization to the pediatric intensive care unit (picu); 11 were first diagnosed as bronchiolitis and two as bronchopneumonia. eleven out of 13 patients admitted to the picu had an etiological diagnosis: 2 respiratory syncytial virus, 3 rhinovirus, 1 human metapneumovirus, 1 human metapneumovirus þ rhinovirus, 2 respiratory syncytial virus þ adenovirus, 1 h. influenzae and 1 s. pneumoniae þ respiratory syncytial virus. the duration of hospitalization was 2-14 days (mean 6.2 ae 3.8). although the differences were not statistically significant, patients with a dual viral infection required more days to recover (8.3 ae 4.9 days) than those with a single infection (5.28 ae 3.25 days). overall, 20 human metapneumovirus cases were detected by rt-pcr, of which 5 could also be recovered from the llc-mk2 cell line. all samples that were j. med. virol. doi 10.1002/jmv human metapneumovirus positive by rt-pcr targeting l gene were also confirmed by rt-pcr based on the f gene. sequencing the f amplicons of human metapneumovirus strains identified from npa and their subsequent phylogenetic analysis showed that only type a2 of human metapneumovirus circulated among patients included during the study period (fig. 2) . all human metapneumovirus samples tested were found in the same cluster sharing a nucleotide identity of 88.4% with a slightly higher amino acid similarity of 89%. notably, when the same sequences were examined leaving out p15 and p19 patients, the similarity increased to 91%. in the phylogenetic tree, both sequences seemed to form a different cluster within the subtype a2 (fig. 2) . the present study is based on the first half of the year, thus it is difficult to establish a distribution of viruses detected during the follow-up. nonetheless, during these 6 months, there were different distribution patterns among the viruses detected most frequently (fig. 3) . acute respiratory tract infections are an important cause of morbidity and mortality in children, and it is well known that a high percentage of these infections are caused by respiratory viruses [williams et al., 2002; jennings et al., 2004; meissner, 2005; pierangeli et al., 2007] . to date, respiratory syncytial virus has been an important trigger of acute respiratory tract infection, although the role of other respiratory viruses such as rhinovirus or human metapneumovirus [van den hoogen et al., 2001] , human coronavirus-nl63 [van der hoek et al., 2005] , human coronavirus-hku1 [woo et al., 2005] and human bocavirus [allander et al., 2007] are gaining attention increasingly. in the present report, respiratory viruses were involved in 67% of either bronchiolitis or bronchopneumonia patients admitted to hospital. despite the fact that both human metapneumovirus and respiratory syncytial virus are common pathogens in children, patients infected with human metapneumovirus were significantly older than respiratory syncytial virusinfected patients, a finding that has been reported elsewhere [williams et al., 2006; wolf et al., 2006; chung et al., 2007] . despite the fact that a limited range of age population has been selected, similar results to previous studies were found [wright et al., 1989; chung et al., 2007; weigl et al., 2007] , suggesting that during the first years of life respiratory viruses are the most important pathogens causing severe respiratory tract infections resulting in hospital admission. several studies have demonstrated that human metapneumovirus is more likely to be associated with bronchiolitis in early childhood [xepapadaki et al., 2004; garcia-garcia et al., 2007; do carmo debur et al., 2007] . however, other investigations including the present study found that human metapneumovirus was associated more frequently with bronchopneumonia compared to human metapneumovirus-bronchiolitis cases [choi et al., 2006; brooks et al., 2007] . these variable results may reflect that human metapneumovirus is not exclusively a bronchiolitis pathogen and can, also cause bronchopneumonia among infants. it probably indicates that human metapneumovirus causes a spectrum of lower respiratory tract illness, with a tendency toward the more severe end, namely pneumonia. in the study sample, human metapneumovirus was present in 25% of patients causing either bronchiolitis or broncopneumonia, a percentage second only to respiratory syncytial virus (35%) and surpassing rhinovirus (19%) . compared to other studies, a higher rate of human metapneumovirus infection was obtained, considering that it is responsible normally for 5-10% of hospitalizations of children suffering from acute respiratory tract infections [jpma et al., 2004; deffrasnes et al., 2007] . however, these rates reflect data from only the first half of the year, corresponding to the peak infection period and thus, increasing the proportion of human metapneumovirus cases. probably, it has led to an overestimate of the real prevalence. dual viral infections are frequent in childhood, mainly in infants, but to date they were not associated with an increased severity of illness [williams et al., 2004; wolf et al., 2006; van woensel et al., 2006 ]. in contrast, other studies have found an increase of 5-to 10-fold in the severity of disease in human metapneumovirus and respiratory syncytial virus dual infections [greensill et al., 2003; semple et al., 2005] . the present study showed 12% of viral coinfections, and although no human metapneumovirus plus respiratory syncytial virus cases were detected, coinfected patients required longer recovery. thirteen patients required hospitalization to the pediatric intensive care unit, and respiratory syncytial virus and rhinovirus were the main respiratory viruses involved. either as a single pathogen or in co-infection, they required more days in the pediatric intensive care unit. interestingly, one rhinovirus-infected patient spent 11 days at the pediatric intensive care unit, although further studies are needed to evaluate its role in the severity of illness. respiratory syncytial virus has been studied widely and in most cases involved in severe acute respiratory infections, however, it has been shown that rhinovirus can also be a life-threatening virus among children with lower respiratory infection [guittet et al., 2003; calvo et al., 2007] . currently, four distinct major human metapneumovirus phylogenetic lineages, a1, a2, b1, and b2 have been described . during the study period, 20 human metapneumovirus cases were detected, all belonging to type a2. maertzdorf et al. [2004] highlighted that some strains, particularly b1 and b2 sublineages, could not be detected by the widely used l6-l7 pair primers published by van den hoogen et al. [2001] . nonetheless, all samples were tested by either l and f genes rt-pcr, so it was considered that during the follow-up only human metapneumovirus subtype a2 circulated among patients included in this study. interestingly, in a study undertaken during the same year based on 171 children with upper respiratory infection attended in different primary care centers of catalonia, it was observed a cocirculation of both lineages among 10 human metapneumovirus identified (unpublished data). recently, huck et al. [2006] described a novel human metapneumovirus sublineage within a2 group, which they divided into a2a and a2b. in the present report, the sequences within the group a2 shared a nucleotide identity of 88.4%. when patients p15 and p19 were excluded, the identity of sequences reached 91%, suggesting that these patients may belong to another cluster. one limitation of this study is that follow-up was done during a short period of time, even though respiratory syncytial virus, human metapneumovirus and rhinovirus cases occurred in a defined succession with only small overlap between the viruses, as has been reported in other studies [choi et al., 2006; pierangeli et al., 2007] . further studies would be valuable to investigate how these seasonal patterns change, taking into account either the entire year and a wider age group, thus establishing a true pattern of respiratory viruses. overall, the results obtained in this study, show that: (1) human metapneumovirus is one of the most important viruses among hospitalized children less than 12 months, and therefore an important cause of severe lower respiratory tract infection in this age group; (2) children infected with human metapneumovirus were significantly older (mean 6.2 months) than those infected by respiratory syncytial virus (mean 2.5 months); (3) human metapneumovirus was associated more frequently to pneumonia whereas respiratory syncytial virus was only detected in bronchiolitis cases; (4) human metapneumovirus is associated with a spectrum of lower respiratory tract illness, with a greater predominace, in this population, with pneumonia; (5) although respiratory syncytial virus and human metapneumovirus circulate mainly during the winter season, there was a clear succession with a small overlap between viruses; (6) all human metapneumovirus sequences were clustered within sublineage a2. human bocavirus and acute wheezing in children human metapneumovirus infections among children with acute respiratory infections seen in a large referral hospital in india practice guideline for the management of infants and children 0 to 36 months of age with fever without source. agency for health care policy and research human metapneumovirus infection in the canadian population genetic diversity between human metapneumovirus subgroups detection of human metapneumovirus rna sequences in nasopharyngeal aspirates of young french children with acute bronchiolitis by real-time reverse transcriptase pcr and phylogenetic analysis human metapneumovirus infection among children acute bronchiolitis role of rhinovirus in hospitalized infants with respiratory tract infections in spain human metapneumovirus in a haematopoietic stem cell transplant recipient with fatal lower respiratory tract disease the association of newly identified respiratory viruses with lower respiratory tract infections in korean children detection of viruses identified recently in children with acute wheezing simultaneous detection of influenza a, b, and c viruses, respiratory syncytial virus, and adenoviruses in clinical samples by multiplex reverse transcription nested-pcr assay simultaneous detection of fourteen respiratory viruses in clinical specimens by two multiplex reverse transcription nested-pcr assays analysis of replication kinetics of the human metapneumovirus in different cell lines by real-time pcr human metapneumovirus acute respiratory infection by human metapneumovirus in children in southern brazil human metapneumovirus infections in young and elderly adults genetic heterogeneity of g and f protein genes from argentinean human metapneumovirus strains human metapneumovirus bronchiolitis in infancy is an important risk factor for asthma at age 5 international pediatric sepsis consensus conference: definitions for sepsis and organ dysfunction in pediatrics complete sequence of the g glycoprotein gene of avian metapneumovirus subgroup c and identification of a divergent domain in the predicted protein human metapneumovirus in severe respiratory syncytial virus bronchiolitis rhinovirus and acute respiratory infections in hospitalized children. retrospective study human metapneumovirus: a new player among respiratory viruses novel human metapneumovirus sublineage viral etiology of acute respiratory tract infections in children presenting to hospital: role of polymerase chain reaction and demonstration of multiple infections human metapneumovirus infection in hospital referred south african children mega3: integrated software for molecular evolutionary genetics analysis an sequence alignment detection of human metapneumovirus in clinical samples by immunofluorescence staining of shell vial centrifugation cultures prepared from three different cell lines realtime reverse transcriptase pcr assay for detection of human metapneumoviruses from all known genetic lineages reducing the impact of viral respiratory infections in children respiratory tract reinfections by the new human metapneumovirus in an immunocompromised child rapid detection of human metapneumovirus strains in nasopharyngeal aspirates and shell vial cultures by monoclonal antibodies characterization of human metapneumoviruses isolated from patients in north america detection and typing by molecular techniques of respiratory viruses in children hospitalized for acute respiratory infection in first detection of human metapneumovirus in children with acute respiratory infection in india: a preliminary report detection and pathogenicity of human metapneumovirus respiratory infection in pediatric italian patients during a winterspring season dual infection of infants by human metapneumovirus and human respiratory syncytial virus is strongly associated with severe bronchiolitis bronchiolitis-associated hospitalizations among us children a newly discovered human pneumovirus isolated from young children with respiratory tract disease absence of human metapneumovirus co-infection in cases of severe respiratory syncytial virus infection ten years' experience with year-round active surveillance of up to 19 respiratory pathogens in children estimates of world-wide distribution of child deaths from acute respiratory infections human metapneumovirus and lower respiratory tract disease in otherwise healthy infants and children the role of human metapneumovirus in upper respiratory tract infections in children: a 20-year experience comparison of human metapneumovirus, respiratory syncytial virus and influenza a virus lower respiratory tract infections in hospitalized young children clinical and molecular epidemiological features of coronavirus hku1-associated community-acquired pneumonia the tucson children's respiratory study. ii. lower respiratory tract illness in the first year of life human metapneumovirus as a causative agent of acute bronchiolitis in infants key: cord-003488-pfzy8p5v authors: cruces, pablo; gonzález-dambrauskas, sebastián; quilodrán, julio; valenzuela, jorge; martínez, javier; rivero, natalia; arias, pablo; díaz, franco title: respiratory mechanics in infants with severe bronchiolitis on controlled mechanical ventilation date: 2017-10-06 journal: bmc pulm med doi: 10.1186/s12890-017-0475-6 sha: doc_id: 3488 cord_uid: pfzy8p5v background: analysis of respiratory mechanics during mechanical ventilation (mv) is able to estimate resistive, elastic and inertial components of the working pressure of the respiratory system. our aim was to discriminate the components of the working pressure of the respiratory system in infants on mv with severe bronchiolitis admitted to two picu’s. methods: infants younger than 1 year old with acute respiratory failure caused by severe bronchiolitis underwent neuromuscular blockade, tracheal intubation and volume controlled mv. shortly after intubation studies of pulmonary mechanics were performed using inspiratory and expiratory breath hold. the maximum inspiratory and expiratory flow (qi and qe) as well as peak inspiratory (pip), plateau (ppl) and total expiratory pressures (tpeep) were measured. inspiratory and expiratory resistances (rawi and rawe) and time constants (k(ti) and k(te)) were calculated. results: we included 16 patients, of median age 2.5 (1–5.8) months. bronchiolitis due to respiratory syncytial virus was the main etiology (93.8%) and 31.3% had comorbidities. measured respiratory pressures were pip 29 (26–31), ppl 24 (20–26), tpeep 9 [8–11] cmh2o. elastic component of the working pressure was significantly higher than resistive and both higher than threshold (tpeep – peep) (p < 0.01). qi was significantly lower than qe [5 (4.27–6.75) v/s 16.5 (12–23.8) l/min. rawi and rawe were 38.8 (32–53) and 40.5 (22–55) cmh2o/l/s; k(ti) and k(te) [0.18 (0.12–0.30) v/s 0.18 (0.13–0.22) s], and k(ti):k(te) ratio was 1:1.04 (1:0.59–1.42). conclusions: analysis of respiratory mechanics of infants with severe bronchiolitis receiving mv shows that the elastic component of the working pressure of the respiratory system is the most important. the elastic and resistive components in conjunction with flow profile are characteristic of restrictive diseases. a better understanding of lung mechanics in this group of patients may lead to change the traditional ventilatory approach to severe bronchiolitis. electronic supplementary material: the online version of this article (10.1186/s12890-017-0475-6) contains supplementary material, which is available to authorized users. respiratory infections are the leading cause of childhood mortality and morbidity worldwide. bronchiolitis, the most common lower respiratory infection in infants, continues to be a major pediatric public health problem [1, 2] . research over the past 30 years has led to significant improvement in our understanding of its pathophysiology, in identifying high-risk populations, and in attenuating the severity of the disorder [3] [4] [5] [6] . bronchiolitis is usually a self-limited disease, but some children may develop respiratory failure with increased work of breathing (wob), hypoxemia and hypercarbia requiring mechanical ventilation (mv) in addition to usual supportive measures [7, 8] . research has mostly focused on gas exchange and criteria for respiratory failure, and the efficacy of various treatments to improve gas exchange, reduce wob, and hasten recovery [9] . surprisingly, little work has been done studying respiratory mechanics in children with severe bronchiolitis under mv [4, 5] . modern ventilators are thought to be not only a supportive machine, but also a bedside monitoring tool. respiratory mechanics are the expression of lung function trough measures of pressure and flow. when positive pressure is applied to the respiratory system, advance analysis of respiratory mechanics can discriminate the different elements of wob according to equation of motion: resistive component (the resistance to displacement of a determined gas flow), elastic component (elastic opposition to a determined change of volume) and a threshold load. the threshold load refers to the amount of work required to commence inspiratory flow and it is determined by autopeep [10] [11] [12] . interpretation of these data can help to tailor mv parameters to match the pathophysiology of the disease according to which component is predominantly compromised. for example, contemporary mv strategies for severe asthma totally differ from acute respiratory distress syndrome (ards). bronchiolitis is usually considered an airway obstructive disease based on physical exam, although bronchodilator therapy has been proven to be ineffective. groundbreaking studies 50 years ago described increased respiratory rate and decreased lung compliance in spontaneously breathing children with bronchiolitis [13] . a better characterization of respiratory mechanics in severe bronchiolitis is crucial to understand this disease to improve current ventilatory strategies and ultimately, it may improve usual picu outcomes; like mv duration, respiratory support requirements, picu and hospital length of stay. with these facts in mind, we designed this study to examine the analysis of respiratory mechanics in infants on mv due to severe bronchiolitis. our aim was to describe the work of breathing in this group of patients, analyzing the different components of the respiratory mechanics: the resistive and elastic forces, as well as threshold. this prospective observational study was conducted in 2 picu's: centro hospitalario pereira rossell is a 20 bed mixed medical surgical pediatric intensive care unit located in montevideo, uruguay, that covers all picu pathologies except cardiosurgery; hospital el carmen de maipú is a 6 bed polyvalent unit and a referral center for acute respiratory failure in santiago, chile. between may 1st and august 28th, 2015, children younger than 1 year old with clinical diagnosis of bronchiolitis requiring mv due to acute respiratory failure were screened for the study. patients were excluded if they had uncorrected congenital heart, pre-existing lung or airway disease, chronic respiratory failure requiring long-term mv and tracheostomy. additionally, patients with spontaneous breathing effort, endotracheal tube air leak >20% of tidal volume (v t ), consolidation or atelectasis greater than 2 quadrants on antero-posterior chest x-ray, bronchodilator administration 1 h before measurement at the time of measurement were excluded due to possible interference with data acquisition. we registered demographics at admission, clinical information, pediatric index of mortality 2 (pim 2) and outcome. patients were ventilated on volume control mode. ventilator parameters [peak inspiratory pressure (pip), plateau pressure (p pl ), extrinsic peep (peep), total peep (tpeep), driving pressure (δp = p pl -tpeep), expiratory v t (vte), inspiratory time (it), and respiratory rate (rr)], maximum inspiratory and expiratory flow (qi and qe), and arterial blood gases before the measurement were registered and p a o 2 /f i o 2 (pf) ratio and oxygenation index (oi) were calculated. each respiratory mechanics measurement was performed within 1 h after intubation by one of the investigators. patients were sedated and under the effect of neuromuscular blocker with no respiratory effort. in this setting, we estimated the components of the working pressure of the respiratory system. in absence of respiratory muscle activity, working pressure of the respiratory system is the pressure needed to overcome frictional forces, elastic forces and impedance and it can be calculated applying the equation of motion: an inspiratory hold followed by an expiratory hold was performed following the protocol described in fig. 1a . flow and pressure parameters in these quasi static conditions at the y piece (proximal flow sensor) were recorded in a ad-hoc microsoft excel 2010 (micro-soft®, ny, usa) database to calculate: respiratory system compliance (crs, ml·cmh2o −1 ·kg −1 ), inspiratory and expiratory airway resistance (rawi and rawe, cmh2o·l −1 ·s −1 ), inspiratory and expiratory time constants (k ti and k te , s) according to formulas described in (additional file 1: table s1 ). for each subject the components of working pressure, resistive, elastic and threshold were calculated and expressed as percentage of total working pressure of the respiratory system. figure 1b shows an illustration of different components of working pressure and how they are measured on the mechanical ventilator. each component of working pressure was expressed in cmh 2 o and as percentage of total working pressure of the respiratory system. calculations were performed as follows: resistive component: pip -p pl ; elastic component: p pl -tpeep; threshold tpeep -peep (or autopeep). mechanical ventilators used for measurements were engstrom® (ge datex, madison, wisconsin usa) and hamilton g5 or galileo® (hamilton, bonaduz, switzerland), depending on the availability. data are expressed as means ± standard deviation (sd) and continuous data were expressed as median and interquartile range (iqr). 95% confidence interval (ci95%) was calculated for proportions. normality was assessed with the anderson-darling test. kruskal-wallis test with post hoc dunns analysis was used for comparison of the working pressure components. t student and mann-whitney tests for comparison of flow, resistance and time constants were used. relation between variables was determined by pearson correlation test. significance was set at p < 0.05. all statistical analyses were performed using spss 20.0 (spss inc., chicago, il, usa). during the study period, 54 infants with bronchiolitis were screened. thirty-one subjects were not enrolled due to age older than 1 year old, 5 excluded due to time on mv greater than 24 h at the time of measurements, 1 excluded due to non-corrected congenital heart disease and 1 excluded due to chronic lung disease. sixteen patients were finally included in the study. fifty percent were male, median age was 2.5 months (1-5.8), weight 5.9 kg (4.5-7.4). respiratory syncytial virus (rsv) was the main etiology, identified in 15 cases. prematurity was main comorbidity, present in 5 patients. pim2 score was 7.5% (3.8-10.1), pf ratio was 195 mmhg (159-241) and oi was 7.1 (5.4-9.0). table 1 shows clinical and gas exchange characteristics of included patients. duration of mv was 5 days (iqr 2-7.25) and there was no mortality in this group. there were no complications related to the protocol. table 2 shows ventilatory parameters and respiratory system mechanics for the study population: vte 7.9 ± 1.4 ml·kg −1 ideal body weight, peep was 7.5 cmh2o (iqr 7-8. measurements of working pressure of the respiratory system parts were autopeep 1.5 (iqr 1-4.8) cmh 2 o, pip-p pl 5 cmh2o (iqr 0-11) and p pl -autopeep 22.5 . this accounts of resistive 21.5% (ci95% 18.4-26.8), elastic 72.7% (ci95% 62.4-77.4%) and threshold 4.7% (ci95% 0-10.9) of total working pressure respectively. elastic component of working pressure was significantly higher than resistive and both higher than threshold (p < 0.01). comparison between inspiratory and expiratory parameters showed that qi was significantly lower than qe [5 (4.27-6.75) l·min −1 v/s 16.5 (12-23.8) l·min −1 , p < 0.05], but no significant differences were found between k ti and k te [0. 18 there was a significant inverse relation between raw e and c rs (r = 0.71, p = 0.001) (additional file 2: figure s1 ), but not with tpeep (r = 0.36, p = 0.12), mean airway pressure (r = 0.45, p = 0.5) and raw i (r = 0.17, p = 0.48). in this study, we measured pulmonary mechanics at bedside in infants requiring mv due to severe bronchiolitis, using the measurements provided by two conventional contemporary mechanical ventilators. data obtained from these measurements show that the elastic component of the respiratory system (meaning the distal lung units and chest wall) and not the airway resistance is the main determinant of the work imposed upon mv. we calculated that almost three fourth parts of the total workload is generated to overcome the elastic component of the respiratory system. in addition, we found that the calculated resistance and time constant were similar during the expiratory and inspiratory phase, showing that an increase in expiratory resistance is not the main alteration in patients with severe bronchiolitis under mechanical ventilation. these findings may seem unexpected and contradictory with the current understanding of severe bronchiolitis as a primarily obstructive airway disease with an increase in abbreviations: mo months old, pf ratio pao 2 /fio 2 ratio, oi oxygenation index, pim 2 pediatric index of mortality 2, rsv respiratory syncytial virus, pnb preterm newborn, hmd hyaline membrane disease a viral studies were negative, but pneumococcal superinfection was diagnosed expiratory resistance, but they are supported by the observation done over a half century ago by krieger et al. [13] . they observed a decrease in c rs in spontaneous breathing infants with bronchiolitis when compared with healthy infants. more recent studies found the same alteration in c rs , most of the time attributed to significant air trapping and lung hyperinflation [14] . we were able to describe the threshold component in our patients, and autopeep was not clinically relevant, a finding comparable to that in previous studies [13, 14] . threshold or autopeep is a minor contributor to the total working pressure [15] . when compared to the few reports of c rs of infants without significant respiratory disease, patients of our study had consistently a decreased of c rs [16] . conflicting data exist with regard to the resistive airway component during severe bronchiolitis. krieger et al. found that there were not clinical significant differences between inspiration and expiration in infants during bronchiolitis; even more, the latter was shortened. in that study measured k ti :k te ratio in children with bronchiolitis was higher than normal subjects (mean 1:1.1). they stated that this finding could be explained by otis' theory of the equality of time constants (which are the product of compliance and resistance) in a system with different airway's caliber (unequally obstructed due to varying values of resistance), and flow is rapid in such a system, measured value of resistance would be the one of the larger airways [13] . it is important to realize that respiratory system mechanics measurements in patients under controlled positive pressure mv can be altered by the way the parameters are set. for instance, high ventilatory settings can elevate pip, without p pl changes [15] . a clinical study in 82 pediatric patients with bronchiolitis showed pip oscillations between 25 and 45 cmh 2 o when children were ventilated with moderate to high v t (10-15 ml·kg −1 ) [17] . an augmented v t can also require longer expiratory times to allow the passive expiration, increasing the risk of air trapping and decreasing compliance due to hyperinflation. one of the strengths of our study is that mv settings were standardized and compatible with current standard of care: v t and p pl were limited and low q i were applied. this could have caused the low resistance to expiratory flow observed in our cohort. normal values of raw e for this specific age group are scarce, but our patients' raw e was lower than data from premature newborns without significant respiratory disease on spontaneous breathing [18] . another consideration is the instrumentation used to measure the resistance in our study. when it is measured in a y-piece, the resistive component of instrumental airway is included. de la cruz et al. described in 21 patients that under these conditions the peak inspiratory pressure needed to ventilate the infant's lungs is overestimated compared with actual airway pressure. it would be difficult to correct this overestimation since measurements with a tracheal catheter would be really challenging (or even contraindicated) in small infants [19] . an interesting finding was the inverse correlation between c rs and raw e . as previously reported in patients with ards, this observation shows that lungs with lower c rs had higher elastic recoil [20] . this finding is concordant with our results, showing that during severe bronchiolitis the elastic component of working pressure is predominant, similar to ards pathophysiology. hammer et al. described that ten out of 37 patients with severe bronchiolitis fulfilled the aecc criteria for ards [21, 22] . it is important to note that these infants had consolidation or infiltrates on 4 quadrants on the chest x-ray with a murray's lung injury score greater than 2.7. in our view, this group of rsv-induced ards is different from the cases we are describing. we excluded patients with more than 2 quadrants of infiltrates on chest x-ray (or obvious x-ray patterns of ards), and also all the measurements were done within 1 h after intubation. hammer et al., don't describe ventilatory parameters and timing of measurements. because it was done more than 20 years ago, pre-low v t era and contemporary care of acute respiratory failure, it is difficult to compare both case series. surprisingly the duration of mv was greater than 14 days, compared to ours that was close to [23] definition includes a wide group conditions and pathologies under the brand ards, so some of our cases might be in the gray area of that ards definition, even when chest x-ray was not compatible with ards. on the other hand, bronchiolitis is a very heterogeneous condition including a wide spectrum of diseases. in our study, we aimed to describe a relatively homogenous group of ventilated infants with severe bronchiolitis with ad-hoc contemporary care. it is important to emphasize that most bronchiolitis, even the most severe forms, do not require invasive mechanical ventilation with the contemporary care [24] . in our centers about 2% of bronchiolitis are admitted to a critical care unit, and between 30% and 50% of them require finally invasive mechanical ventilation. our study is focus on that group of patients, infants with severe bronchiolitis requiring mechanical ventilation, so our pathophysiological findings may differ from moderate or mild disease. our study has some limitations. our infants had a single disorder and the age range was large, thus our findings cannot be generalized to infants or children who have other disorders such as asthma, chronic lung disease or congenital heart disease. reference values for healthy children in this age group has not been specifically reported and we did not include a control group, so comparisons and increments of the different components are only estimates. as previously commented, due to small size of patients we did not measure pleural pressure, so we could not determine the contribution of the chest to c rs . a single set of measurements, very close to intubation, was done, because we did not want to use long term or multiple doses of neuromuscular blockade. also, patients were on controlled mv without respiratory muscle activity, can influence in the low inspiratory resistance and measured autopeep, because patient efforts may increase the lung volume, facilitating hyperinflation [25] , being frequent the coexistence between intrinsic peep and active expiration. finally, we have to acknowledge that setting of mv parameters can directly modify the component of the equation of motion (i.e. q i , rr, i:e ratio), but we tried to standardize the ventilatory setting during measurements. despite these limitations, we consider our findings in infants under mechanical ventilation with severe bronchiolitis are important in terms of the pathophysiological approach to this condition. our analysis shows that the elastic component of the working pressure predominates rather than the airways resistance component. future epidemiologic multicentric studies should address the different components of working pressure in severe bronchiolitis. a better understanding of respiratory system mechanics during mechanical ventilation may lead to change the traditional pharmacological and ventilatory approach to severe bronchiolitis as a predominant airway resistance disease. acute viral bronchiolitis: physician perspectives on definition and clinically important outcomes viral bronchiolitis in children enfoque clínico de las enfermedades respiratorias del niño. santiago: universidad católica de chile clinical practice guideline: the diagnosis, management, and prevention of bronchiolitis acute bronchiolitis in infants, a review trends in bronchiolitis hospitalizations in the united states mechanical ventilatory support in infants with respiratory syncytial virus infection management of acute bronchiolitis role of ventilation in rsv disease: cpap, ventilation, hfo, ecmo respiratory mechanics in mechanically ventilated patients clinical review: respiratory monitoring in the icu -a consensus of 16 applied physiology in intensive care medicine mechanics of respiration in bronchiolitis pulmonary function testing in infants with respiratory syncytial virus bronchiolitis requiring mechanical ventilation peep does not improve pulmonary mechanics in infants with bronchiolitis noninvasive determination of total respiratory system compliance in infants by the weighted-spirometer method clinical observations on mechanical ventilation for respiratory failure in bronchiolitis comparison of dynamic and static measurements of respiratory mechanics in infants intratracheal pressure: a more accurate reflection of pulmonary airway pressure in pediatric patients with respiratory failure the effects of positive end-expiratory pressure on respiratory resistance in patients with the adult respiratory distress syndrome and in normal anesthetized subjects acute respiratory distress syndrome caused by respiratory syncytial virus the american-european consensus conference on ards. definition, mechanisms, relevant outcomes, and clinical trial coordination pediatric acute respiratory distress syndrome: consensus recommendations from the pediatric acute lung injury consensus conference high flow nasal cannula (hfnc) versus nasal continuous positive airway pressure (ncpap) for the initial respiratory management of acute viral bronchiolitis in young infants: a multicenter randomized controlled trial (tramontane study) tonic diaphragmatic activity in critically ill children with and without ventilatory support 'not applicable'. this work was supported by conicyt #1160631 (dr. cruces) and conicyt #11160463 (dr. diaz) grants. in accordance to irb recommendation, raw data will not be publicly shared due to absence of specific consent for publication from each patient's caregiver. informed consent was waived since respiratory mechanics measurements are part of routine care in the participating picu's. analysis of respiratory mechanics measurements of infants with severe bronchiolitis receiving controlled mv shows that the preponderant constituent of the working pressure of the respiratory system is the elastic component. the elastic and resistive components in conjunction with flow profile are characteristic of restrictive diseases. a better understanding of the pathophysiology may lead to improve the traditional pharmacological and ventilatory approach of severe bronchiolitis as a predominant airway resistance disease. additional file 1: table s1 . formulas for estimation of lung mechanics in quasi -static conditions. (docx 16 kb) additional file 2: figure s1 . correlation between respiratory system compliance and expiratory airway resistance measured in children on mechanical ventilation due to severe bronchiolitis. ethics approval and consent to participate institutional review board at centro hospitalario pereira rossell and hospital el carmen de maipú approved the study, and informed consent was waived since respiratory mechanics measurements are part of routine care in the participating picu's. 'not applicable'. the authors declare that they have no competing interests. • we accept pre-submission inquiries • our selector tool helps you to find the most relevant journal submit your next manuscript to biomed central and we will help you at every step: key: cord-003798-nki2sasr authors: vidaur, loreto; totorika, izarne; montes, milagrosa; vicente, diego; rello, jordi; cilla, gustavo title: human metapneumovirus as cause of severe community-acquired pneumonia in adults: insights from a ten-year molecular and epidemiological analysis date: 2019-07-24 journal: ann intensive care doi: 10.1186/s13613-019-0559-y sha: doc_id: 3798 cord_uid: nki2sasr background: information on the clinical, epidemiological and molecular characterization of human metapneumovirus in critically ill adult patients with severe community-acquired pneumonia (cap) and the role of biomarkers identifying bacterial coinfection is scarce. methods: this is a retrospective epidemiological study of adult patients with hmpv severe cap admitted to icu during a ten-year period with admission psi score ≥ 3. results: the 92.8% of the 28 patients with severe cap due to human metapneumovirus were detected during the first half of the year. median age was 62 years and 60.7% were male. the genotyping of isolated human metapneumovirus showed group b predominance (60.7%). all patients had acute respiratory failure. median apache ii and sofa score were 13 and 6.55, respectively. the 25% were coinfected with streptococcus pneumoniae. 60.7% of the patients had shock at admission and 50% underwent mechanical ventilation. seven patients developed ards, three of them younger than 60 years and without comorbidities. mortality in icu was 14.3%. among survivors, icu and hospital stay were 6.5 and 14 days, respectively. plasma levels of procalcitonin were higher in patients with bacterial coinfection (18.2 vs 0.54; p < 0.05). the levels of c-reactive protein, however, were similar. conclusion: human metapneumovirus was associated with severe cap requiring icu admission among elderly patients or patients with comorbidities, but also in healthy young subjects. these patients often underwent mechanical ventilation with elevated health resource consumption. while one out of four patients showed pneumococcal coinfection, plasma procalcitonin helped to implement antimicrobial stewardship. electronic supplementary material: the online version of this article (10.1186/s13613-019-0559-y) contains supplementary material, which is available to authorized users. human metapneumovirus (hmpv) is a worldwide distributed enveloped virus with a rna genome closely related to respiratory syncytial virus. hmpv belongs to the paramyxoviridae family, in the genus metapneumovirus, first identified in the netherlands in 2001 [1] . based on genetic and antigenic variability, hmpv strains have been classified in two groups or lineages (a and b) and four sublineages (a1, a2, b1 and b2) [1] [2] [3] . the virus has been reported as a common respiratory pathogen in childhood, associated mainly with upper but also with lower respiratory tract infections [2, 4] . during the annual epidemics, hmpv has been associated with a significant number of hospital admissions in young children [4] [5] [6] [7] . respiratory tract infections caused by hmpv during adulthood are less prevalent and less serious than those open access *correspondence: loretovidaurtello@gmail.com 1 critical care department, donostia university hospital-biodonostia health research institute, san sebastian, guipuzcoa, spain full list of author information is available at the end of the article in childhood. however, the presence of hmpv has been detected in 2-4% of adult patients admitted due to a community-acquired pneumonia (cap) [8, 9] and has been associated with asthma and chronic obstructive pulmonary disease exacerbation [10] [11] [12] . the same as with other common respiratory viruses, hmpv is usually associated with non-severe pneumonia, whereas risk factors like immunosuppression, specific comorbidities-chronic lung disease, heart disease, blood disorders-elderly and living in long-term care facilities are associated with a higher risk of severe viral pneumonia [13, 14] . nevertheless, recent studies suggest that hmpv infection is an underappreciated cause of critical illness, also in previously healthy patients [15] [16] [17] [18] . severe community-acquired pneumonia (scap) is a known infectious complication of respiratory viruses including hmpv. in these cases, clinical presentation, evolution and treatment differ depending on the pathogens involved, hmpv alone or hmpv coinfected with a bacteria. some biomarkers have been studied as diagnostic markers to discriminate between viral or bacterial pneumonias and help physicians to decide not to start or when to withdraw the antibiotic therapy [19, 20] . the main objective of this study was to describe the clinical and epidemiological characteristics of adults with severe pneumonia caused by hmpv who required intensive care unit (icu) admission, over a long period of time. secondary objectives were to characterize the epidemiological and molecular viral diversity and to compare the value of c-reactive protein (crp) and procalcitonin in identifying bacterial coinfections. this is a ten-year, retrospective epidemiological study with inclusion of patients with cap due to hmpv admitted in a 48-bed icu in the north of spain. in 2017, this icu assisted a referral population of 545,227 inhabitants older than 14 years. all patients older than 14 years from july 2007 to june 2017 admitted in the icu by cap with admission psi score ≥ 3 were considered eligible. during the first 2 years of the study, samples to detect respiratory viruses were obtained occasionally in patients with cap. however, it turned the standard of care in the icu after 2009 influenza pandemic. to be included, cases meet two of the following three criteria upon admission: (a) severe acute respiratory failure (pao2/fio2 < 250), (b) multilobar radiological involvement or (c) systolic arterial pressure < 90 mmhg. acute respiratory distress syndrome (ards) was diagnosed as an acute diffuse lung injury with increased vascular permeability, bilateral radiographic opacities and hypoxemia not fully explained by cardiac failure or fluid overload following the berlin criteria [21] . exclusion criteria: subjects with nosocomial pneumonia or admitted due to non-respiratory infection (non-severe coincidental infection) and patients with pneumonia during the preceding 2 months (persistence of viral rna in respiratory samples). patients were recruited from the computerized records of the microbiology department, and the medical records were revised by two clinical investigators (it, lv). the recorded clinical variables were socio-demographic (age and sex), comorbidities, the charlson comorbidity score and clinical symptoms at admission [22] . radiological and analytic findings at admission and during the evolution, coinfections, antibiotic therapy, the presence of shock or need of mechanical ventilation, icu and hospital stay were also recorded. the detection of hmpv in respiratory samples was made by reverse transcription polymerase chain reaction (rt-pcr), in house monoplex until july 2010 [5] , real-time commercial multiplex (luminex xtag respiratory viral panel [usa]) until july 2013 and seegene anyplex ™ ii rv16/allplex ™ respiratory panel [republic of korea] since then. the extraction of nucleic acids was made using an automatic biorobot1 m48 extractor (qiagen gmbh, hilden, germany) until july 2009 and the nuclisens ® easy-mag platform (bio-mèrieux sa, marcy l'etoile, france) from that date. the genotyping of hmpv was performed with a rt-pcr followed by sequencing [23] . blood cultures, streptococcus pneumoniae and legionella pneumophila antigenuria (alere binaxnow, scarborough, me, usa), and pharyngeal exudates with viral transport media to evaluate respiratory viruses were assessed in all the patients included in the study. coinfection was considered when hmpv was isolated with other viral or bacterial pathogens at the same time. discrete variables were expressed as counts (percentage) and continuous variables as medians and 25-75% interquartile ranges (iqrs). differences in continuous variables were analyzed by the mann-whitney u test or the kruskall-wallis test when appropriate. qualitative variables were analyzed by the chi-square test with yate's correction when necessary. the threshold for clinical significance was p < 0.05. data analysis was performed using spss for windows 21.0.0 (spss, chicago, il, usa). the obtained clinical samples and the medical intervention of the patients were ordered by the clinician attending each patient. the study was approved by the ethics committee for clinical research of the health area of gipuzkoa (spain). informed consent was waived due to the retrospective nature of the study. during the study period, 1942 respiratory samples were sent from the icu to the microbiology service to study viral etiology, hmpv being identified in 33 patients (1.7%). studied samples were mainly pharyngeal exudates (77.3%), but also tracheal aspirates (7.5%), bronchoaspirates (5.8%), bronchoalveolar lavages (5.6%) and sputum (4.2%), where bacterial culture was also performed. five patients with hmpv were excluded because admission causes were other than respiratory infection. cases were detected every year except in 2008 (n = 0-5). the highest prevalence was in 2015 and 2017 (fig. 1) . twenty-six of the 28 patients with respiratory infection due to hmpv (92.9%) were detected during the first half of the year and 16 (57.1%) in march-april. hmpv circulated every year later than influenza virus, being the epidemic peak of both infections separated by a period of 1-2 months. in fact, the 75% of cases (21/28) of hmpv infections in patients admitted to icu occurred out of the influenza epidemic period ( table 1) . genotyping of hmpv was performed in 27 cases, being ten cases of hmpv group a (39.3%) and 17 of hmpv group b (60.7%). the viral strains belonged to sublineages a2 (n = 10; 39.3%), b1 (n = 11; 39.3%) and b2 (n = 6; 21.4%). group a strains predominated until 2011 (72.7%), while later, the most frequent was genotype b (79%). after excluding seven patients with bacterial coinfection, there were not significant differences in the genotype of hmpv between six patients who developed ards (60% genotype b) and 15 who did not (67% genotype b). at icu admission, all patients had acute respiratory failure and received empiric antibiotic therapy. median apache ii score was 13 , and median saps iii and sofa scores were 58.5 [iqr 47.2-70.7] and 6.5 [iqr 3.5-9.5], respectively. median age of the included patients was 62 years [25-75% iqr 49.7-75.7], and 60.7% of them were under 65 years old (9 with less than two comorbidities). the 60.7% (n = 17) of the patients were male. main symptoms at admission were cough (89.3%), dyspnea (71.4%), fever (67.9%) and purulent respiratory secretions (67.8%). nineteen patients (67.9%) had major comorbidities such as immune compromise (n = 7), asthma (n = 3) or chronic respiratory disease (n = 3) ( table 2) . seven patients (none died) had coinfection with streptococcus pneumoniae. three episodes were coinfected with viral pathogens: human parainfluenza virus type 3 (hpiv3), human rhinovirus and cytomegalovirus (last one in an immunosuppressed patient). predominant radiologic pattern in patients with hmpv infection and without coinfection was the interstitial alveolar pattern (47.6%), while in the patients with streptococcus pneumoniae coinfection, the alveolar pattern was predominant (85.7%). eight patients had pleural effusion at admission, and two more developed it during the icu stay. pleural effusion was bilateral in four patients and massive (> 2 l) in three cases. seventeen (60.7%) patients had shock at admission, fourteen (50%) underwent invasive mechanical ventilation (median 5.5 days [iqr 5-14.2]) due to acute respiratory failure and four were tracheostomized due to prolonged mechanical ventilation. severe complications were frequent, highlighting acute renal failure in 12 patients (42.8%), of which two required renal replacement therapy; cardiac failure or cardiogenic shock in eight patients (28.5%); and ards in seven cases (25%) (two of them in patients with bacterial coinfection) ( table 2 ). three patients who developed ards were younger than 60 years (38, 47 and 54 years, respectively) without major comorbidities or bacterial coinfection. all of them underwent invasive mechanical ventilation due to acute respiratory failure (one had coinfection with hpiv3). the main clinical and epidemiological characteristics of the patients are summarized in the supplementary material (additional file 1: table s1 , additional file 2: table s2 ). the majority of the patients (83.3%) had lymphocytopenia (< 1000/ml) at admission ( our study gives new insights on the molecular epidemiology of hmpv pneumonia admitted to the icu over 10 years. hmpv was consistently detected in cap admitted to the icu, with an annual incidence ranging 0.5-1 case/100,000 inhabitants older than 14 years per year. molecular characterization of hmpv revealed group dominance of subgroup b. hmpv infection presented seasonal distribution, with 2/3 of cases detected in late winter-early spring each year. the 32% of the studied patients were younger than 65 years without comorbidities. hmpv cap often presented as acute respiratory failure with bilateral opacities and half of icu subjects underwent mechanical ventilation. lymphocytopenia and pleural effusion were common at admission. plasma procalcitonin was a sensitive tool to identify coinfection with bacteria (25%), which contributes to antimicrobial stewardship. these findings suggest the need to implement hmpv diagnosis tests in subjects with cap developing acute respiratory failure. two out of three patients of this study had shock at admission, half of them underwent mechanical ventilation, one out of four developed ards and one out of seven died during the clinical course, suggesting that hmpv is responsible for scap in adults. these data are concordant to that observed in the only study with a wide range of patients with hmpv infection in critically ill patients, in which 55% of the patients required mechanical ventilation, 48% developed ards and the mortality was 18% [18] . moreover, there are sporadic reports of 3-6 patients with hmpv infection acquired in the community and acute respiratory failure who required icu admission [15] [16] [17] . in a large prospective study of icu patients requiring invasive mechanical ventilation, hmpv was more frequently detected in patients admitted by severe respiratory infection than in patients with other causes, suggesting a causal role of hmpv in the development of severe respiratory infection [24] . most of the patients of this study had major comorbidities at admission, mainly chronic respiratory failure and immunosuppression, being those patients and the elderly the most susceptible to develop severe hmpv infections [2, 10, 14] . however, 60% of the patients were younger than 65 years old and one out of three did not have major comorbidities, being similar to cap related to other etiologies. interestingly, three patients (10.7%) were young adult patients without comorbidities and without bacterial coinfection that developed ards pointing out a main role of hmpv in the etiology of severe respiratory infections requiring mechanical ventilation. in the cohort of patients of hasvold et al. [18] , 15% of the patients had only minor comorbidities and were not immunosuppressed. one out of four episodes of severe acute respiratory infection was coinfected with bacteria, similar to that observed in other series [17, 18] . streptococcus pneumoniae, one of the bacterial species most frequently involved in post-viral super-infections [25] , was the main isolated bacterial pathogen. in these episodes, procalcitonin has been reported to discriminate between viral episodes and those with bacterial coinfection [26] , in contrast with crp. some studies have recommended different cutoff points of procalcitonin to discontinue early antibiotic therapy in patients with community-acquired therapy, being 0.25 ng/ml and, mainly 0.1 ng/ml the most recommended [27, 28] . none of the patients with documented bacterial coinfection in this study had a procalcitonin level lower than 1 ng/ml which supports the early discontinuation of antibiotic therapy in this group of patients with low plasma levels of procalcitonin. the results of this study, about procalcitonin plasma determinations, could help to develop personalized medicine in patients with cap, helping physicians to early discrimination between viral or bacterial pneumonia and antimicrobial stewardship [29] . three different genotypes of hmpv were associated with severe cap requiring icu admission, which supports that all of them are able to cause severe infections in adult patients. the low number of cases of the three different hmpv lineages, the presence of coinfections and the retrospective nature of the study made impossible to analyze the clinical pattern and the evolution of the patients based on the genotype of the infecting hmpv. however, to date, there are no significant differences in the evolution or clinical manifestation between different genotypes of hmpv in adults in the outpatient setting [30] . this study has some limitations and therefore, the results should be evaluated cautiously. the hmpv infection was diagnosed by oropharyngeal swab samples more than in low respiratory tract samples, mainly in non-intubated patients. the detection of a viral pathogen in respiratory samples of a patient with acute respiratory infection can be coincident and not related to icu admission. the retrospective design of the study can underestimate the actual incidence of hmpv infection because some patients admitted because of acute respiratory infection could not be investigated for viral etiology. however, from the 2009 influenza pandemics, nasopharyngeal swab samples with respiratory viral detection are routine of care being collected in the 90% of patients with scap admitted to icu. finally, three different molecular techniques were used, with potential selection bias due to the differences in sensitivity of these techniques. in conclusion, our study confirms that hmpv, a respiratory virus causing bronchiolitis and pneumonia in children, was associated with severe cap requiring icu admission among elderly patients or patients with comorbidities, but also in healthy young subjects. these patients often underwent mechanical ventilation with long icu and hospital stays, associated with elevated health resource consumption. the results of this study agree with recent observations [13] suggesting a shift in the paradigm of severe pneumonia, recommending that viral infection (and specifically hmpv) should be ruled out when complicated with acute respiratory failure. while one out of four patients showed pneumococcal coinfection, plasma procalcitonin levels helped to implement antimicrobial stewardship. additional file 1: table s1 . main characteristics of immunocompetent adult patients admitted to the intensive care unit due to a severe community-acquired pneumonia associated with human metapneumovirus infection (guipuzcoa, basque country, spain, 2007-2017). table s2 . main characteristics of immunosuppressed adult patients admitted to the intensive care unit due to a severe community-acquired pneumonia associated with human metapneumovirus infection (guipuzcoa, basque country, spain, 2007-2017). ards: acute respiratory distress syndrome; apache ii: acute physiology and chronic health evaluation ii; cap: community-acquired pneumonia; copd: chronic obstructive pulmonary disease; crp: c-reactive protein; hmpv: human metapneumovirus; icu: intensive care unit; psi score: pneumonia severity index; saps iii: simplified acute physiology score iii; scap: severe communityacquired pneumonia; sofa: sequential organ failure assessment. a newly discovered human pneumovirus isolated from young children with respiratory tract disease human metapneumovirus: lessons learned over the first decade genetic diversity of human metapneumovirus over 4 consecutive years in australia burden of human metapneumovirus infection in young children hospitalization rates for human metapneumovirus infection among 0 to 3 year olds in gipuzkoa human metapneumovirus and lower respiratory tract disease in otherwise healthy infants and children a 1-year experience with human metapneumovirus in children aged < 5 years systematic review of respiratory viral pathogens identified in adults with community-acquired pneumonia in europe community-acquired pneumonia requiring hospitalization among u.s. adults human metapneumovirus infections in adults: another piece of the puzzle human metapneumovirus infection plays an etiologic role in acute asthma exacerbations requiring hospitalization in adults human metapneumovirus infection in adults with community-acquired pneumonia and exacerbation of chronic obstructive pulmonary disease severe viral pneumonia in adults: what is important for the icu physician? an outbreak of severe respiratory tract infection due to human metapneumovirus in a long-term care facility linssen cf. human metapneumovirus in bronchoalveolar lavage fluid of critically ill patients with suspected pneumonia human metapneumovirus infections on the icu: a report of three cases viral infection in patients with severe pneumonia requiring intensive care unit admission the role of human metapneumovirus in the critically ill adult patient infection biomarkers in primary care patients with acute respiratory infections-comparison of procalcitonin and c-reactive protein serum procalcitonin measurement and viral testing to guide antibiotic use for respiratory infections in hospitalized adults: a randomized controlled trial acute respiratory distress syndrome: the berlin definition validation of a combined comorbidity index seasonal distribution and phylogenetic analysis of human metapneumovirus among children in osaka city respiratory viruses in invasively ventilated critically ill patients-a prospective multicenter observational study postviral complications. bacterial pneumonia procalcitonin (pct) levels for ruling-out bacterial coinfection in icu patients with influenza: a chaid decision-tree analysis effect of procalcitonin guided guidelines vs standard guidelines on antibiotic use in lower respiratory tract infections: the pro-hosp randomized controlled trial procalcitonin guidance of antibiotic therapy in community acquired pneumonia: a randomized trial towards precision medicine in sepsis: a position paper from the european society of clinical microbiology and infectious diseases the role of human metapneumovirus genetic diversity and nasopharyngeal load on symptom severity in adults lv made substantial contribution to the conception and design of the work, the acquisition, analysis and interpretation of the data and has drafted the work. it made substantial contribution to the conception and design of the study, analysis and interpretation of the data. dv, mm, jr and gc made substantial contributions to the interpretation of the data and substantively revised it. all authors read and approved the final manuscript. the authors declare that there has not been any source of funding for the research. the datasets supporting the conclusions of this article are included within the article (and its additional file). the study was approved by the ethics committee for clinical research of the health area of gipuzkoa (spain). informed consent was waived due to the retrospective nature of the study. not applicable. the authors declare that they have no competing interests. key: cord-020267-0axms5fp authors: nan title: ribavirin and respiratory syncytial virus date: 1986-02-15 journal: lancet doi: 10.1016/s0140-6736(86)92323-8 sha: doc_id: 20267 cord_uid: 0axms5fp nan experience in population medicine; management of the preventive health programme and information system described will require further training and a substantial time commitment. stocking1o argues that in the absence of extra resources it may be preferable for general practitioners to relinquish some patient contact for a role as "central manager and referral agency" within the practice. clearly there is considerable scope for substituting less expensive (and in some instances more appropriate) manpower resources for many tasks currently undertaken by general practitioners." none of the obstacles is insurmountable, and some should be addressed for other reasons. however, ploughing money into fpcs to create the sort of management structure and proficiency that already exists in (often coterminous) health authorities would appear to be both exceptionally wasteful and extremely unlikely in the present economic climate. reintegration of fpcs and health authorities would be the sensible option, not only to provide a viable management structure for general practice but also to secure the advantages of planned community health care which a closer relation between general practice and community medicine would allow. '2 even if a new contract was established linking remuneration to performance monitored by fpcs or district health authorities, would it lead to an improvement in the quality of care? preventive medicine lends itself to objective external assessment-it is possible to measure immunisation rates and screening coverage, for example, with the benefit of a computerised population register-and better care should follow. it is also probable that external audit of accessibility would be a major advance, especially in deprived urban areas. nevertheless, external audit of disease management is not without difficulty. lessons can be learned from the experience of recertification and peer review organisations in the usa.13 at best, external audit can monitor minimum standards, but the highest standards of care must ultimately depend on fostering enthusiasm and stimulating participation in continuing education and research in general practice. and good general practice demands qualities such as kindness and understanding which are not encompassed by performance indicators. the peer review procedure envisaged by the rcgp'4 appears to hold out the best chance of attaining this wider vision of quality of care, and would complement a more formal line of accountability. on balance, it seems likely that the people of newcastle will benefit if their general practitioners are contractually required to provide services to agreed 10 standards of minimum performance, especially if the organisation to which the practitioners are accountable is not only equipped with teeth but also is capable of making a rational decision about whom to bite. but if we must wait for the end of the tripartite structure in order to achieve this, then perhaps "waiting for greenot" is a more apposite description of where we stand. ribavirin is an investigational synthetic triazole nucleoside of value in the treatment of lassa feverl and certain viral respiratory infections. first synthesised in 1972, it possesses an unusually broad spectrum of antiviral activity, inhibiting under laboratory conditions a wide variety of rna and dna viruses, including among respiratory viruses influenza types a and b, parainfluenza types 1, 2, and 3, respiratory syncytial virus (rsv), and possibly coronavirus. its antiviral effect is expressed at various points in the replicative cycle but generally involves alterations of nucleotide pools and interference with the guanylation step required for 5' capping of viral messenger rna.2 2 ribavirin has low cellular toxicity and is well tolerated by human beings, the primary adverse effects being haematological with raised unconjugated bilirubin levels in 25% of subjects on 1 g orally per day and an occasional drop in haemoglobin on 4 g daily. these effects are rapidly reversible and may be related to the accumulation of drug or metabolites in red blood cells. ribavirin is embryotoxic and teratogenic in laboratory animals, though not in baboons. in temperate climates, rsv is the most frequent cause of acute lower respiratory tract disease in infants and young children. in britain rsv accounts for yearly hospitaladmission rates of 12 -5 to 24' 5 per 1000 among infants aged 1-3 months;3 and in north carolina it is responsible for 24-50% of all admissions for pneumonia in children under 5 years of age.4 in hospital roughly 14% of rsv-infected infants require intensive care and 5% need assisted ventilation.s although the mortality from rsv infection is generally low, it is especially high in infants with underlying congenital heart disease (37%, rising to 73% with concomitant pulmonary hypertension),5 and in the immunocompromised (23%),6 and is almost certainly raised in infants with bronchopulmonary dysplasia and cystic fibrosis. outbreaks among the elderly in nursing homes have also been associated with serious illness and a case fatality rate as high as 53&deg;7o has been reported. 7 the efficacy of aerosolised ribavirin in rsv-infected infants has been examined mostly in double-blind trials involving normal children and those with underlying disease. in normal infants with illness for several days, therapy over many -hours improved cough, rales, retractions, bronchiolitis, lethargy, overall severity scores, and arterial oxygen saturation by the second to fourth day.8-1o similarly rsv-infected infants with bronchopulmonary dysplasia and/or congenital heart disease improved more rapidly on ribavirin than on placebo and here benefit was most noticeable within the first 24 h of treatment and the improvements in arterial po2 were substantia1.11,12 in these studies, there were no fatalities among 30 ribavirin-treated infants with bronchopulmonary dysplasia and/or congenital heart disease or others requiring prolonged assisted ventilation, suggesting life-saving effects. a bonus of ribavirin is its effect against other important respiratory viruses. striking improvements were noted in 2 infants treated with ribavirin aerosol for parainfluenza virus type 3 infection complicating severe combined immunodeficiency disease13,14-a combination often causing respiratory failure and death. moreover, ribavirin aerosol therapy, when started within the first 24 h of symptoms, reduces fever and symptoms from influenza type a (h1n1) and type b in young adults, 11,16 but here the improvements are modest and insufficient to justify treatment in otherwise healthy subjects. the published work thus indicates the feasibility of broad-spectrum antiviral chemotherapy for three or possibly four potentially serious and often clinically indistinguishable repiratory pathogens. it is also noteworthy that resistance to ribavirin developed in none of the rsv strains isolated during treatment, and conceivably the modest reductions in viral shedding found in most investigations may lessen the incidence of nosocomially acquired infection. delivery of ribavirin via an infant oxygen hood, oxygen tent, inhalation tubing of a respirator, or face mask has the advantage of providing high drug concentrations at the site of viral replication and reduces the likelihood of systemic reactions. indeed no toxic or adverse effects of aerosol therapy were observed among any of 135 infants or adults studied, many receiving an estimated 0' 82 mg/kg per hourl 6 for periods of 100 h over 5 days. few otherwise healthy adults will feel that the accelerated clinical improvement is worth the inconvenience of many hours' confinement for this therapy, however safe. rather it should be considered for infants with bronchiolitis or pneumonia, and for high-risk patients with underlying cardiopulmonary disorders or immunodeficiency with probable rsv or influenza, and possibly parainfluenza infection. in high-risk patients early treatment seems indicated before onset of life-threatening disease or confirmation of the diagnosis by laboratory means. in britain, the drug is available on a named patient basis only. it is understandable that the idea of treating urinary incontinence by transferring it to another site should have been slow to gain acceptance, particularly when major surgery is involved. yet urologists, when faced with intractable cases of urethral leakage, have been making judicious use of uretero-ileo-cutaneous diversion for many years, fortified by the increasing safety of this operation and the improvement of collecting devices. the great majority of cases are women for whom no effective urinal is available. uncontrollable reflex detrusor activity due to vesicourethral neuropathy (especially in multiple sclerosis) is the commonest indication, followed by female stress incontinence persisting after multiple local operations. few surgeons deal with enough cases to make a convincing series and publications devoted to the subject are rare. in one report which appeared nine years ago, urinary diversion had been performed in 12 cases of multiple sclerosis (9 female)1 with very favourable results. it was concluded that urinary diversion should be considered much earlier in the treatment of incontinence in multiple sclerosis and not kept as a last resort. in a new report malone and co-workers2 again put urinary diversion in incontinence in a favourable light, stressing the great improvement in the patient's social acceptability that may be achieved. of the 13 patients 8 had uninhibited detrusor contractions associated with paraplegia (traumatic in 2 and due to multiple sclerosis in 6). in another case of multiple sclerosis an indwelling catheter had produced a patulous urethra. the remaining 4 patients were obstetric cases in all of whom several local operations for stress incontinence had failed. there was no operative mortality and complications such as wound and chest infection and paralytic ileus were limited to the patients with multiple sclerosis. in only one case of this disease was any deterioration of neurological status noted postoperatively. before the operation 8 of the patients had been unable to lead a reasonable social life, afterwards only 4; the remaining 4.were inhibited more by fear of the stoma leaking or appearing conspicuous than by episodes of appliance failure. in fact, 11 patients had either no leaks or only occasional leakage in the three months before interviews. 2 patients had troublesome dermatitis around the stoma and in 2 there was stomal ulceration with bleeding, but no one regretted having had the operation. the most common complication was pyocystis (in 4 patients) which was relieved by vaginal vesicostomy performed with the aid of a payr's crushing clamp.3 malone et al suggest that prophylactic use of this operation should be considered especially in elderly or infirm patients. they also stress the importance of a stomatherapy service for all the patients, and of specialised nursing care and physiotherapy for those with multiple sclerosis. the importance of longterm follow-up is emphasised, for early detection of urolithiasis as well as stomal or uretero-ileal stenosis. in the future fewer diversions may be required, thanks to advances in artificial sphincters and techniques of bladder denervation. particularly promising in women with multiple sclerosis is the injection of phenol into the vesical plexus bilaterally through the vagina and perineum. in one series of lassa fever. effective therapy with ribavirin mechanisms of action of ribavirin report to the medical council subcommittee on respiratory syncytial virus vaccines respiratory syncytial virus infection: admissions to hospital in industrial, urban, and rural areas pneumonia: an eleven-year study in a pediatric practice respiratory syncytial viral infection in infants with congenital heart disease respiratory syncytial virus in immunocompromised children communicable disease surveillance centre. respiratory syncytial virus infection in the elderly 1976-82 key: cord-284332-p4c1fneh authors: bosma, karen j.; taneja, ravi; lewis, james f. title: pharmacotherapy for prevention and treatment of acute respiratory distress syndrome: current and experimental approaches date: 2012-09-19 journal: drugs doi: 10.2165/10898570-000000000-00000 sha: doc_id: 284332 cord_uid: p4c1fneh the acute respiratory distress syndrome (ards) arises from direct and indirect injury to the lungs and results in a life-threatening form of respiratory failure in a heterogeneous, critically ill patient population. critical care technologies used to support patients with ards, including strategies for mechanical ventilation, have resulted in improved outcomes in the last decade. however, there is still a need for effective pharmacotherapies to treat ards, as mortality rates remain high. to date, no single pharmacotherapy has proven effective in decreasing mortality in adult patients with ards, although exogenous surfactant replacement has been shown to reduce mortality in the paediatric population with ards from direct causes. several promising therapies are currently being investigated in preclinical and clinical trials for treatment of ards in its acute and subacute, exudative phases. these include exogenous surfactant therapy, β(2)-adrenergic receptor agonists, antioxidants, immunomodulating agents and hmg-coa reductase inhibitors (statins). recent research has also focused on prevention of acute lung injury and acute respiratory distress in patients at risk. drugs such as captopril, rosiglitazone and incyclinide (col-3), a tetracycline derivative, have shown promising results in animal models, but have not yet been tested clinically. further research is needed to discover therapies to treat ards in its late, fibroproliferative phase. given the vast number of negative clinical trials to date, it is unlikely that a single pharmacotherapy will effectively treat all patients with ards from differing causes. future randomized controlled trials should target specific, more homogeneous subgroups of patients for single or combination therapy. the acute respiratory distress syndrome (ards) arises from direct and indirect injury to the lungs and results in a life-threatening form of respiratory failure in a heterogeneous, critically ill patient population. critical care technologies used to support patients with ards, including strategies for mechanical ventilation, have resulted in improved outcomes in the last decade. however, there is still a need for effective pharmacotherapies to treat ards, as mortality rates remain high. to date, no single pharmacotherapy has proven effective in decreasing mortality in adult patients with ards, although exogenous surfactant replacement has been shown to reduce mortality in the paediatric population with ards from direct causes. several promising therapies are currently being investigated in preclinical and clinical trials for treatment of ards in its acute and subacute, exudative phases. these include exogenous surfactant therapy, b 2 -adrenergic receptor agonists, antioxidants, immunomodulating agents and hmg-coa reductase inhibitors (statins). recent research has also focused on prevention of acute lung injury and acute respiratory distress in patients at risk. drugs such as captopril, rosiglitazone and incyclinide (col-3), a tetracycline derivative, have shown promising results in animal models, but have not yet been tested clinically. further research is needed to discover therapies to treat ards in its late, fibroproliferative phase. given the vast number of negative clinical trials to date, it is unlikely that a single pharmacotherapy will effectively treat all patients with ards from differing causes. future randomized controlled trials should target specific, more homogeneous subgroups of patients for single or combination therapy. acute lung injury (ali) and the acute respiratory distress syndrome (ards) arise from direct or indirect injury to the lungs, and results in a life-threatening form of respiratory failure. ali/ards is both common and serious: 6.5-8.5% of patients admitted to an intensive care unit (icu) will be diagnosed with ali or ards, [1] [2] [3] and approximately one-quarter to one-half of these patients will succumb to this disease process. [1, [4] [5] [6] over the past 40 years, ards has been the focus of extensive basic science and clinical research, although no single pharmacotherapy has been shown to reduce mortality in a large, randomized, controlled, multicentre trial of adult patients. the reasons for this are manifold, and include issues of dosing, route of administration and timing of the various interventions tested. more importantly, however, may be the nature of the disorder itself: the diagnosis of ards envelops a heterogeneous group of patients with varying causes and pathophysiological mechanisms at work. the notion that a therapeutic agent that can successfully alter a single biological target in an animal model of ali will reduce mortality in all patients with ards may be unrealistic. nonetheless, there is reason for hope on the scientific horizon. recent advances have been made in our understanding of the pathophysiological mechanisms underlying ali/ards, leading to the identification of potential novel targets for pharmacological intervention. some therapies are best aimed at preventing the development of ards, while others treat the syndrome as it unfolds or aid in its resolution. the challenge lays in identifying the subgroup of patients most likely to benefit from such focused therapy. this paper reviews the current experimental and existing approaches to managing ards, highlighting the pathophysiological basis for their use and potential for future clinical development. ali may occur following a direct insult to the pulmonary system such as aspiration of gastric contents, bacterial pneumonia or viral pneumonitis (e.g. h1n1 influenza virus), or an indirect insult such as the systemic inflammatory response associated with pancreatitis, sepsis or multiple trauma. table i shows common direct and indirect causes of ali/ards. whether this 'first hit' to the lung is direct or indirect, a pulmonary inflammatory response may occur, which often is adaptive and self-limited. however, when coupled with repeated 'hits' to the lung from insults such as injurious mechanical ventilation or other secondary processes such as hypotension, a cycle of intense inflammation and worsening pulmonary injury ensues. the 'multiple hit' theory of ards progression also provides a framework for studying the disease process (figure 1). clinically, ali manifests as bilateral airspace disease observed on chest radiograph and hypoxaemia, such that the ratio of partial pressure of arterial oxygen to fraction of inspired oxygen (pao 2 /fio 2 ) is greatly reduced. according to the 1994 american european consensus conference (aecc) definition, a chest radiograph consistent with pulmonary oedema and a pao 2 /fio 2 ratio <300 is sufficient to diagnose ali in the setting of an inciting pulmonary insult and the absence of congestive heart failure. the aforementioned criteria but with a pao 2 /fio 2 ratio <200 is classified as ards. [8] although differentiated by the aecc definition, ali and ards are often grouped together for the purpose of clinical trial enrolment and are treated as a single entity throughout this review. although not all patients follow the same clinical course, progression of ali/ards may be considered along a pathophysiological timeline of early, mid and late phases, with considerable overlap between these phases. table ii summarizes the pathogenetic mechanisms at work during each phase, linking each biological pathway to a potential drug therapy. a general overview of the pathophysiology of ards is provided here, with more detailed descriptions of the specific biologic pathways discussed in sections 3.1-3.3 as they pertain to each potential pharmacological therapy. the early phase, within the first 72 hours of the inciting lung injury, is characterized by inflammatory damage to the alveolar-capillary barrier. this results in increased vascular permeability, leading to interstitial and alveolar oedema as proteinaceous fluid fills the alveolar space. this inflammation-induced pulmonary oedema disrupts normal gas exchange and increases the work of breathing, leading to respiratory failure and the need for mechanical ventilation. mechanical ventilation itself may cause secondary insult to the already inflamed oedematous alveoli. during each tidal breath induced by mechanical ventilation, unstable alveoli undergo cyclical collapse and shearing open, termed 'atelectrauma'. furthermore, the non-collapsed alveolar units may receive a greater proportion of the delivered tidal volume, leading to damage due to overdistention or 'volutrauma'. further breakdown of the endothelial-epithelial barrier may occur with atelectrauma and volutrauma, along with the release of local proinflammatory mediators which further b see text for details. aa = arachidonic acid; arb = angiotensin receptor antagonist (blocker); fa = fatty acid; gm-csf = granulocyte macrophage colonystimulating factor; hne = human neutrophil elastase; icam-1 = intercellular adhesion molecule-1; il-8 = interleukin-8; mmps = matrix metalloproteinases; nf-kb = nuclear factor-kb; paf = platelet-activating factor; ppar-g = peroxisome proliferator activated receptor-g; rhpaf = recombinant human paf; tnfa = tumour necrosis factor-a. propagate this cycle of ventilator-exacerbated lung injury. [7] as inflammation ensues, neutrophils are recruited to the lung. damaged endothelial cells exhibit increased activity of the transcription factor nuclear factor-kb (nf-kb), which upregulates the surface expression of intercellular adhesion molecule (icam)-1. icam-1 mediates leukocyte adhesion and migration across the endothelium to the alveolar epithelium. activated neutrophils release proteases, such as matrix metalloproteinases (mmp) and neutrophil elastase (ne), which further damage the alveolarcapillary membrane. [9] activated neutrophils also contain high levels of arachidonic acid, [10] which is metabolized into leukotrienes, prostaglandins and thromboxanes. leukotrienes attract more neutrophils, prostaglandins are proinflammatory mediators, and thromboxanes play a role in vasoconstriction and platelet and leukocyte aggregation. neutrophil recruitment and activation may be an adaptive physiological response to injury, or may incite a vicious cycle of inflammation and further damage. [9] at this stage, patients may recover from the initial insult, with clearance of the pulmonary oedema and restoration of the barrier between capillary endothelial and alveolar epithelial cells, or may progress to the exudative or mid phase of ards. it is not fully understood why two patients exposed to the same insult may have completely different clinical courses; however, genetic factors, [11] co-morbid illnesses such as diabetes mellitus and alcohol addiction, [12] nutritional status, medications and exposure to further insults are all likely to play a role. understanding the host and environmental factors that place a patient at high risk of progressing to the exudative phase of ards will facilitate identification of targets for earlier intervention. the exudative or subacute phase typically occurs over the 3-7 days following the initial insult. pathologically, this mid phase is characterized by formation of intra-alveolar hyaline membranes rich in plasma proteins, fibrin and cellular debris. [13] a biopsy of the lungs at this stage will show diffuse alveolar damage and, clinically, the lungs have poor compliance with ongoing gas exchange problems including hypoxaemia and elevated dead space fraction. the inflammatory milieu within the alveoli, coupled with the cyclical opening, stretching and collapsing of alveoli via mechanical ventilation, initiates a number of pathogenic pathways in concert or in series. these include disruption of surfactant function and metabolism, ongoing neutrophil recruitment and activation, along with increased expression and release of inflammatory mediators, imbalance of oxidant and antioxidant activity, and activation of complement and coagulation cascades. each of these pathways is further discussed to provide context for the drugs or therapies aimed at ameliorating these various mechanisms (see section 3). interestingly, only a minority of patients will succumb to severe hypoxaemia or hypercarbia, as the major source of mortality is not the pulmonary injury per se, but rather the occurrence of multiple organ failure. in this setting, the injured lung may represent a rich source of inflammatory mediators that could contribute to the development of multi-organ failure. for example, stress failure and necrosis of the endothelial-epithelial barrier may allow various inflammatory mediators, bacteria and endotoxins to quickly spread from the lungs into the systemic circulation. indeed, it is this de-compartmentalization of inflammatory mediators from the lungs into the circulation that is felt to lead to cell apoptosis in distal organs, [14] and ultimately multiple organ dysfunction syndrome (mods) [ figure 2 ]. [15] once mods develops, disease is often irreversible and mortality may increase significantly to 60-98%, the latter occurring when three or more organs are involved for a period of more than 7 days. [15] [16] [17] thus, a key to developing novel therapies that will reduce mortality in ards will be identification of the cellular and molecular mechanisms by which ards leads to mods. survivors of the first week of ali/ards may enter the late phase of the disorder, known as the fibroproliferative phase. during days 8-28, the exudates and hyaline membranes become organized, and fibrosis may become apparent. type ii alveolar cells proliferate and line the alveolar walls, fibroblasts migrate and differentiate into myofibroblasts in the interstitial and alveolar spaces, and a collagen-rich extracellular matrix is laid down in the interstitium. [13] alveoli may be destroyed, pulmonary vascular area may be reduced and chronic inflammation is generally present. patients in the fibroproliferative phase of ards may slowly recover, or may fail to wean from mechanical ventilation and succumb to complications of a lengthy critical illness or pre-existing co-morbid illnesses. pharmaceutical interventions for late ards must interrupt the fibrosing alveolitis and aid in resolution, remodelling and repair of injured lungs. [13] often, therapies that might be beneficial during the early phase of lung injury are started too late in the course of the disease, when fibrosis is already established, muting their potential efficacy. when tested specifically for the late fibroproliferative phase of ards, anti-inflammatory therapies have yielded disappointing results. basic science research examining mechanisms of idiopathic pulmonary fibrosis may illuminate therapeutic pathways for fibroproliferative ards, but further work is required in this area. although no pharmacological therapies have been proven to reduce mortality in large, randomized controlled trials (rcts) involving adult patients, it appears that improvements in supportive care have reduced mortality to some extent. for example, mortality estimates ranged progression of acute respiratory distress syndrome (ards) to multi-organ failure (mof). initially, inflammatory damage to the alveolar-capillary barrier results in increased vascular permeability, leading to interstitial and alveolar oedema as proteinaceous fluid fills the alveolar space. there, the proteinaceous fluid interferes with the function and metabolism of the endogenous surfactant system. coupled with this, neutrophils that infiltrate lungs are subsequently activated and represent an important source of inflammatory mediators and oxygen free radicals, inducing further epithelial and endothelial cell damage and an altered host immune response. newly secreted mediators and/or spillover of inflammatory mediators from the lung into the systemic circulation ultimately contribute to the development of mof. inflammatory mediators released from organs such as the liver, heart and kidney return to the lung via the systemic circulation and may contribute to further pulmonary inflammation. thus, each new insult to the pulmonary system accelerates the acute lung injury cycle (reproduced from bosma et al. [7] [2007], with permission). from 55% to 65% as reported in the literature in the 1980s and early 1990s [18] [19] [20] [21] to more recent estimates of 32-46% in observational epidemiological studies [1, 2] and 25-31% in large clinical trials. [5, 6] although this mortality reduction may in part reflect differences in diagnostic criteria used post publication of the 1994 aecc definition, undoubtedly the largest impact has been the move to more 'protective' strategies of mechanical ventilation. in 2000, the national institutes of healthsponsored ards network (ardsnet) trial involving low tidal volume ventilation was published, and now constitutes the standard of care for patients with ali and ards. this trial compared a traditional tidal volume of 12 ml/kg with a lower tidal volume of 6 ml/kg in 861 patients and reported a mortality reduction from 40% in the control arm to 31% in the treatment arm. [5] these results definitively ended the debate fuelled by three previous inconclusive smaller trials regarding lower versus conventional tidal volumes. in terms of furthering ali/ards research, several lessons have been learned from this landmark study. first, ardsnet was set up to conduct well designed, large, phase iii studies with a concerted effort to optimize patient enrolment through involvement of many centres in an organized and cohesive group. [22] this enabled a study sufficiently powered to realize a mortality difference to be conducted within a reasonable timeframe, and pointed the way for other similarly structured ards research networks to become established. second, the treatment arm was associated with lower oxygenation values than the conventional arm, highlighting the potential danger of relying on oxygenation or other physiological parameters as surrogates for mortality. third, this study demonstrated that a nonpharmacological intervention could alter mortality, indicating that future rcts need to be carefully standardized in all aspects of supportive care in both treatment and control arms. one potential caveat ensuing from this study has been the assumption that any additional proven therapy would reduce mortality across a population as heterogeneous and diverse as that enrolled in the ardsnet low tidal volume trial. this approach may be misguided, as subsequent studies have demonstrated differences between patients with direct and indirect causes of ali/ards in responsiveness to specific therapies. [23, 24] research is ongoing to determine whether newer modes of mechanical ventilation, such as high-frequency oscillation (hfo), can further improve outcomes in ards relative to the ardsnet low tidal volume strategy. [25] in addition, other aspects of supportive care have been evaluated in large clinical trials, some conducted by ardsnet, and have proven effective in reducing morbidity associated with critical illness. these include cautious fluid management, [26, 27] adequate nutrition, [28] prevention of ventilator associated pneumonia, [29] [30] [31] [32] prophylaxis for deep venous thrombosis [33] and gastric ulcers, [34] weaning of sedation and mechanical ventilation as early as possible, [35] and physiotherapy and rehabilitation. [36] a recent review of all patients enrolled in ardsnet studies between 1996 and 2005 showed that these advancements in critical care (aside from lower tidal volume ventilation) are likely responsible for the improved survival in ali/ards patients in clinical trials noted over the last decade. [37] additional modalities used as 'rescue therapies' for the ards patient at risk of succumbing to severe hypoxaemia or respiratory acidosis have also been tested, including nitric oxide, prone positioning, hfo and extracorporeal membrane oxygenation (ecmo). nitric oxide [38] and prone positioning [39, 40] have not been shown to reduce mortality or duration of mechanical ventilation in patients with ali/ards, and are therefore not recommended for routine use. however, combined together, these therapies may provide a sustained improvement in oxygenation for patients with severe hypoxaemia and a mortality benefit for patients who are failing conventional mechanical ventilation strategies. [39] [40] [41] a clinical trial of hfo for routine care of patients with ards is currently underway, but existing evidence supports its use as salvage therapy if instituted early for patients failing conventional ventilation, [42] and may have additive benefits when combined with nitric oxide and prone positioning. [43] finally, ecmo has recently been studied in the cesar trial (see table iii for a list of trial acronyms). [44] this study showed that transferring adult patients with severe but potentially reversible respiratory failure, whose murray score exceeds 3.0 or who have a ph of <7.20 on optimum conventional management, to a centre with an ecmo-based management protocol, significantly improved survival without severe disability. recent evidence suggests ecmo is also useful for rescue therapy for adults with severe ards due to h1n1-influenza a virus infection. [45] pharmacological treatments for ali/ards may be employed prior to the onset of ards or in the early, mid or late phases of ards (table iv) . accordingly, their purpose may be to prevent ali in those at risk, mitigate the pathogenic mechanisms responsible for the cycle of lung injury and systemic inflammation in established ards, or aid in lung healing and repair. some therapies, such as corticosteroids, have been studied for prevention of ards, treatment of early ards and treatment of late ards, and are discussed within each context. the concept that ards may be prevented in those at high risk after an inciting insult is not new, but is one that is garnering greater attention in the scientific literature in recent years. since no pharmacological agent has proven effective in treating established ards in adults, attention has turned to prophylactic treatment to prevent the development of ards in those at highest risk. of course, any pharmacotherapy that is initiated prior to the diagnosis of disease must have a very high benefit to risk ratio and be cost effective. as such, it should have the following attributes: (i) be low risk, without serious adverse effects; (ii) be easily and widely applicable; and (iii) be relatively inexpensive. drug classes studied for ards prevention include imidazoles (e.g. ketoconazole), ace inhibitors, thiazolidinediones (e.g. rosiglitazone), chemically modified tetracycline derivatives, antioxidants, and corticosteroids and other immunomodulating agents. over 20 years ago, the first clinical trial examining prophylactic use of ketoconazole to prevent ards in patients at risk was published. [46] the rationale for using ketoconazole, an antifungal drug with anti-inflammatory properties, was as follows. as mentioned in section 1, patients with ards have increased levels of arachidonic acid metabolites in their bronchoalveolar fluid. [10, 87] metabolism of arachidonic acid leads to the production of leukotrienes, prostaglandins and thromboxanes. thromboxane a 2 is a potent vasoconstrictor, and is involved with platelet and leukocyte aggregation, while leukotrienes act as powerful chemokines to attract neutrophils. ketoconazole is an antifungal agent of the imidazole class which selectively blocks thromboxane synthetase. ketoconazole also inhibits 5-lipoxygenase, the enzyme necessary to generate leukotrienes, and inhibits procoagulant activity. [55] in addition to showing promise in preclinical animal studies, when given prophylactically to patients at risk of developing ards, ketoconazole has been shown to reduce the incidence of severe ards in three small trials. a 1988 study of 71 patients admitted to a surgical icu showed that in the group treated prophylactically with oral ketoconazole 200 mg/day, 2 of 35 patients (6%) ultimately developed ards, whereas 11 of 36 (31%) patients in the control group developed ards (p < 0.01). [46] similar results followed in a 1993 study of 54 patients with septic shock admitted to a surgical icu, where the incidence of ards in the group treated with ketoconazole 400 mg/day was 15% (4 of 26 patients) compared with 64% (18 of 28 patients) in the control group (p = 0.002), and mortality was 15% versus 39%, respectively (p = 0.05). [47] although both of these studies were conducted prior to the 1994 aecc definition, ards was strictly defined in the aforementioned studies, including a pao 2 /fio 2 ratio <150 or intrapulmonary shunt >20% in patients requiring mechanical ventilation and who had diffuse infiltrates on chest radiograph without clinical evidence of heart failure as pulmonary arterial occlusion pressures were <18 mmhg. building on the results of these two studies, sinuff and colleagues [48] developed practice guidelines for prophylactic ketoconazole use, and tested the implementation and efficacy of these guidelines in two icus (one control and one active comparator). they reported a significantly decreased incidence of ards in the icu population receiving ketoconazole prophylaxis, although mortality was equivalent within the two units. [48] in 2000, ardsnet published the karma study evaluating oral ketoconazole versus placebo for patients within 36 hours of an established diagnosis of ali or ards according to the 1994 aecc definition. [55] the study was stopped early after enrolment of 234 patients for failing to show a difference in mortality or ventilator-free days. of note, this study was designed to look at early treatment of ali/ards rather than prevention of ards in patients at risk, and therefore did not necessarily negate the findings of the three previous smaller studies. furthermore, a problem identified in the karma study was that even though blood ketoconazole concentrations were adequate, urinary metabolites of thromboxane were not affected, raising the possibility that the proper dose to achieve an anti-inflammatory effect was not given. however, since the karma f mortality reduction in subgroup of patients with ards, septic shock and relative adrenal insufficiency. g no mortality reduction in larger study, n = 180 (lasrs). il-8 = interleukin-8; ma = meta-analysis; mc = multicentre; ppar-c = peroxisome proliferator activated receptor-g; rhpaf = recombinant human platelet-activating factor; rsp-c = recombinant surfactant protein-c; sc = single-centre. study showed no difference in mortality, widely considered the most important endpoint to achieve, further research on ketoconazole for ali/ards ceased. [55] additionally, ketoconazole has numerous drug interactions and requires an acidic milieu to be absorbed via the enteral route, making routine use in the icu complicated. further research should examine whether other drugs in the imidazole class given intravenously have similar anti-inflammatory properties, and also establish the inflammatory dose-response curve for ali/ards. in addition, although the concept that prevention of ards will definitely lead to decreased mortality is intuitive, this still has to be proven in large multicentre clinical trials. the authors are unaware of any studies being conducted in this area presently. angiotensin-converting enzyme (ace) is produced in the lungs and is responsible for converting angiotensin i into angiotensin ii, a peptide active in vasoconstriction and sodiumfluid balance to maintain blood pressure homeostasis. ace inhibitors and angiotensin ii receptor antagonists (blockers; arbs) are classes of drugs commonly used to treat hypertension, and prevent progression of diabetic nephropathy in patients with diabetes. ace inhibitors also help to preserve vascular structure and function, by exerting a protective effect on endothelial cells. endothelial cell damage is the catalyst for the inflammatory and coagulation cascades activated in ali/ards. thus, the protection of endothelial cells offered by ace inhibitors may have a beneficial role in ards. [49] studies in transgenic mice have shown that ace, angiotensin ii and angiotensin ii receptor type 1a may promote lung injury, whereas ace2, a close homologue of ace, and angiotensin ii receptor type 2 may protect against severe lung dysfunction in models of ards. [88] the ace inhibitor captopril has been shown to prevent severe lung injury in an oleic acid-induced model in rats. in this model, captopril reduced expression of icam-1 in lung tissue, indicating a protective effect on endothelial cells, diminished activity of tissue plasminogen activator, involved in coagulation, and blocked nf-kb, the major signal transduction pathway that regulates the expression of multiple early-response genes related to inflammation. [49] in humans, two small cohort studies have demonstrated that polymorphism of the ace gene increases susceptibility to the development of ards and its outcome. [89, 90] two additional studies, published only in abstract form to date, have examined the association between ace inhibitor use and ards. a retrospective cohort study of 1423 adult critically ill patients found that 5.5% of patients developed ards after hospital admission, and that preexisting, long-term use of an ace inhibitor or arb was associated with decreased risk of ards development, after adjusting for predisposing conditions (odds ratio [or] 0.49; 95% ci 0.25, 0.94; p = 0.03). [91] the second abstract, a case-control study nested within a prospective cohort of 1553 critically ill patients at risk for ards, reported that patients on ace inhibitors had a lower prevalence of respiratory failure on admission to icu, but not lower incidence of ards after adjusting for confounders on multivariate analysis. however, among patients who developed ards, ace inhibitor use was associated with lower mortality (adjusted hazard ratio 0.66; 95% ci 0.45, 0.99). [92] the associations observed in these clinical studies is consistent with preclinical animal data, but requires further research prior to being applicable clinically. [90] peroxisome proliferator activated receptors (ppars) are ligand-activated transcription factors related to thyroid hormone, steroid and retinoid receptors. [50] there are three isoforms: ppar-g, ppar-a and ppar-b/d. ppar-g plays a central role in glucose homeostasis. thiazolidinediones, a class of oral antidiabetic drugs, are synthetic ligands for ppar-g. synthetic ppar-g agonists also have anti-inflammatory properties, inhibiting proinflammatory cytokine production and macrophage activation in vitro. [93, 94] this action is mediated in part by antagonizing the activity of transcription factor nf-kb. when activated, nf-kb induces overexpression of inflammatory cytokines such as tumour necrosis factor (tnf)-a, which in turn induces upregulation of icam-1 expression, as well as recruitment and activation of immune cells. icam-1, expressed on the surface of endothelial cells, mediates leukocyte adhesion and migration through endothelium into tissues. the anti-inflammatory properties of thiazolidinediones have been demonstrated in vivo in murine models of inflammatory bowel disease [95] and rheumatoid arthritis. [96] rosiglitazone is the most potent selective ppar-g of the thiazolidinediones. prophylactic administration of rosiglitazone has been shown to attenuate ali in an animal model of pancreatitis-associated ali. [50] in this study, rosiglitazone was dissolved and given intravenously to rats 30 minutes prior to induction of acute pancreatitis by sodium taurocholate. compared with control group rats with acute pancreatitis and its associated lung injury, prophylactic administration of rosiglitazone resulted in a significantly lower histological pulmonary injury score, reduced pulmonary expression of tnfa and icam-1 messenger rna, and decreased lung tissue myeloperoxidase activity, a measure of neutrophil infiltration in the lung. [50] this suggests that prophylactic rosiglitazone mitigates the ali associated with acute pancreatitis by its anti-inflammatory effect. unfortunately, the safety of rosiglitazone has recently been questioned due to its augmentation of sodium and water retention, leading to increased incidence of congestive heart failure in diabetic patients placed on this drug long-term. [97, 98] thus, further animal studies are needed to confirm the effects of rosiglitazone in acute pancreatitis and evaluate potential complications related to its use, prior to proceeding to human studies. during the early phase of lung injury, neutrophils are recruited into the pulmonary vasculature and activated to release proteases, such as mmps and ne, which damage the alveolarcapillary membrane, [51] resulting in further release of inflammatory mediators. a single laboratory in the state university of new york (new york, ny, usa) has demonstrated in various animal models that blocking the proteases ne, mmp-2 and mmp-9 with a unique modified tetracycline can prevent the increased pulmonary vascular permeability that ultimately leads to ards. the same group has developed a 'two-hit' porcine model of sepsis plus gut ischaemiareperfusion injury that parallels the insidious onset of sepsis-induced ards in humans. in this model, anaesthetized yorkshire pigs undergo cross-clamping of the superior mesenteric artery for 30 minutes to induce intestinal ischaemia, followed by intraperitoneal placement of a faecal blood clot. pigs are then awakened, extubated and taken to an animal icu for 48 hours of continuous observation, where they receive intravenous fluids, broad-spectrum antibacterials and pain control medications. when the pao 2 /fio 2 ratio falls below 250, pigs are anaesthetized and placed back on mechanical ventilation with tidal volumes of 10 ml/kg. in this model, they demonstrated that prophylactic administration of a synthetic, nonantimicrobial derivative of tetracycline called incyclinide (col-3; collagenex pharmaceuticals), prevented the development of both ards and septic shock. [51] incyclinide has not yet been tested in any human studies of ards prevention; however, the complex model developed by this group contains all the elements of a clinically relevant animal model and, therefore, these results show potential for phase ii studies. oxidative stress is associated with development of ards and mods via direct tissue injury. nathens and colleagues [52] examined the effect of antioxidant supplementation using atocopherol and ascorbic acid in critically ill surgical patients. in a prospective rct of 595 surgical icu patients (mainly victims of trauma), they found antioxidants did not reduce the risk of developing ards, but did decrease the risk of developing mods, and shortened duration of mechanical ventilation and length of icu stay. [52] antioxidants supplementation and nutritional strategies are now being studied for critically ill patients with early signs of mods, [99] but not specifically for ards prevention. antioxidants and nutrition have also been studied for treatment of ards, and are further discussed in this context in section 3.2.3. given that excessive and protracted inflammation is the overriding principle responsible for the various pathophysiological mechanisms leading to ards, broad and potent anti-inflammatory drugs, such as corticosteroids, would seem to be a rational choice for prevention. four rcts, published between 1985 and 1988, have examined the use of corticosteroids to prevent the onset of ards in patients at risk. a recent meta-analysis of these studies demonstrated that preventive corticosteroids may actually increase the risk of developing ards in critically ill adults. [53] furthermore, the meta-analysis suggested a weakly increased risk of death associated with preventive corticosteroid therapy in those patients who ultimately developed ards. thus, corticosteroid therapy is not recommended for preventing ards in those at risk. corticosteroid therapy has also been extensively studied for the treatment of established disease in the early and late phases, and is discussed further in these contexts (see the corticosteroids subsection of section 3.2.5 and section 3.3.1). platelet-activating factor (paf) is a potent proinflammatory mediator that is degraded by the enzyme paf acetylhydrolase. recombinant human paf acetylhydrolase (rhpaf-ah; epafipase) was studied in a phase iib rct to prevent ards in septic patients. [54] 127 patients with severe sepsis were randomized to receive rhpaf-ah 1 mg/kg, rhpaf-ah 5 mg/kg or placebo. the incidence of ards was not different amongst the three groups, but 28-day all-cause mortality was significantly decreased in the 1 mg/kg treatment group compared with placebo (21% vs 44%; p = 0.03). therefore, although rhpaf-ah does not appear to be an effective prophylactic treatment for ards, it may hold promise for treatment of severe sepsis. the majority of research to date has focused on treating ards once the diagnosis is established. although many studies are designed to treat 'early ards', with randomization occurring within 48 hours of diagnosis, these studies also likely capture many patients in the exudative phase of ards with intra-alveolar hyaline membranes and histological diffuse alveolar damage at the time of enrolment. this problem arises in part because the diagnostic criteria for ards are subjective and lack sensitivity and specificity when compared with pathological diagnosis. [100] thus, timing an intervention at a certain point after 'diagnosis' could result in the patient receiving treatment in the early, mid or even late pathophysiological stage of ali/ards. some more recent studies are now targeting time after intubation rather than time after diagnosis to achieve more uniform timing of intervention. however, since the acute and exudative phases occur along a continuum and are not generally distinguished clinically, therapies targeting these phases will be considered concomitantly. therapies currently under investigation for early and/or exudative ards include those targeting the disrupted surfactant system, oxidative stress and antioxidant activity, neutrophil recruitment and activation, expression and release of inflammatory mediators, activation of the coagulation cascade, and microvascular injury and leak. treatment of the overall inflammatory response with agents such as corticosteroids has also been studied and is discussed. finally, the only drugs specifically targeting resolution of the alveolar oedema of the acute phase are b 2 -adrenergic receptor agonists (b 2 -agonists). clearance of alveolar oedema depends on the balance between oedema formation and reabsorption. the rate of fluid reabsorption depends on the active transport of sodium and electrolytes; water follows in the direction of the transported electrolytes. the active transport of salt and water occurs via epithelial sodium channels induced via na + /k + adenosine triphosphatase (atpase). [101] b 2 -agonists are thought to increase alveolar fluid clearance via two possible mechanisms: (i) increasing the levels of intracellular cyclic adenosine monophosphate, which in turn upregulates na + /k + atpase, causing increased sodium transport across alveolar type ii cells; and (ii) reducing alveolar-capillary permeability, thereby decreasing oedema formation. preliminary animal and ex vivo studies demonstrated the potential of b 2 -agonists to accelerate the rate of alveolar fluid clearance. [102, 103] a small, single-centre rct randomized 40 patients with ali/ards to receive intravenous salbutamol (albuterol) 15 mg/kg/h or placebo for 7 days. [56] the primary endpoint of balti-1 was extravascular lung water measured by the singleindicator transpulmonary thermodilution system (picco ò ; pulsion medical systems) at day 7. patients in the salbutamol group had lower extravascular lung water and plateau pressures, although oxygenation did not differ between the treatment and placebo groups. this latter finding was perhaps due to the vasodilatory effects of b 2 -agonists contributing to shunting of oxygen in the capillary bed. there was no difference in 28-day mortality or ventilator-free days, although the study was not sufficiently powered to detect a difference in these endpoints. [56] funded by the medical research council, the same investigators in the uk are now conducting balti-2, using the same intravenous salbutamol protocol as in balti-1, but powered to detect clinically important outcomes. [104] it will be interesting to determine if the physiological benefits observed in balti-1 confer a reduction in 28-day allcause mortality in balti-2. aerosolized b 2 -agonists have fewer systemic adverse effects than intravenous preparations. the national heart, lung and blood institute (nhlbi), in conjunction with ardsnet, conducted a study of an aerosolized b 2 -agonist, the alta study. [105] the study was stopped for futility at the first interim analysis after enrolling 279 patients. [57] there was no difference in the primary outcome of ventilator-free days to day 28. this study may have been negative for the following reasons: (i) delivery of nebulized drug to lung injury sites may have been suboptimal, as was the case with aerosolized surfactant; and/or (ii) less severely ill patients with ali (rather than ards with more severe hypoxaemia) may retain adequate alveolar fluid clearance without the need for upregulation with b 2 -agonists. sixty-day mortality in the alta study was 19.7% compared with a 28-day mortality of 60% in the severely ill group of patients who received physiological benefit from intravenous salbutamol in balti-1. [106] exogenous surfactant administration has been very successful in treating and preventing neonatal respiratory distress syndrome (nrds). given the physiological and pathological similarities between nrds and ards, exogenous surfactant therapy has been under investigation for treatment of ali/ards for over a decade. although clinical trial results have been largely disappointing, recent studies show promise. the strong scientific rationale for targeting the disrupted surfactant system, as well as lessons learnt from previous trials, therefore merit further attention. endogenous surfactant is composed of 90% lipids (mainly phosphatidylcholine and phosphatidylglycerol) and 10% proteins. the role of endogenous surfactant in the healthy lung is to decrease surface tension and thereby prevent alveolar collapse. in addition, surfactant plays a role in suppressing inflammation and scavenging free oxygen radicals. four apoproteins have been identified, termed surfactant protein (sp)-a, -b, -c and -d. whereas the presence of either or both of the hydrophobic surfactant proteins sp-b and -c are important for the biophysical function of surfactant, the hydrophilic proteins sp-a and -d perform the various host defence roles, including modulation of leukocytes, enhancement of the function of phagocytic cells [107] and regulation of the host's immune system. [108, 109] in ali, disruption of the endogenous surfactant system occurs by a number of mechanisms: injury to alveolar type ii cells results in abnormal synthesis and secretion of surfactant, serum proteins that leak into the airspace interfere with surfactant function, serine endopeptidase and phospholipase a 2 cause degradation of surfactant, and, finally, mechanical ventilation, particularly with high tidal volumes, causes conversion of functional surfactant aggregate forms into dysfunctional forms. without optimal surfactant function, there is high surface tension at the alveolar surface in a non-uniform pattern within the lung leading to alveolar instability and collapse. the presence of bacteria within the airspace may also release and activate endotoxins, a process that is augmented in the presence of an abnormal surfactant system. based on the functional importance of the endogenous surfactant system in the normal lung and, more importantly, the consequences of an altered surfactant system in ali/ards, there is good rationale to consider exogenous surfactant administration as a therapeutic intervention in these patients. [109] in 1996, a phase iii, double-blind rct tested an aerosolized, synthetic surfactant called exosurf ò (glaxo wellcome) in 725 patients with sepsis-induced ards. [58] this study showed no significant difference in overall survival, duration of mechanical ventilation or oxygenation between the treatment groups and standard care. it was postulated that this lack of efficacy was due to a low level of alveolar deposition of the aerosolized preparation and/or due to the absence of surfactant proteins in the preparation. [23] currently, this surfactant preparation is not being evaluated for patients with ali/ards and is no longer marketed in the us. shortly afterwards, a smaller, open-label phase ii clinical trial evaluated tracheal instillation of a liquid bolus of the natural bovine extract surfactant, survanta ò (ross laboratories), in patients with severe ards. [59] there was a trend toward decreased mortality in the group of patients receiving up to four doses of phospholipids 100 mg/kg surfactant compared with the patients in the control group (18.8% vs 43.8%; p = 0.075), and no safety concerns were identified. however, survanta ò contains only very small amounts of sp-b. coupled with concerns regarding resource limitations, no further clinical trials of this exogenous surfactant preparation for adults with ards have been performed. recognizing the importance of surfactantspecific proteins brought progress to clinical surfactant research. in 2004, results were published for two phase iii clinical trials evaluating effect of a liquid, recombinant sp-c (rsp-c) surfactant, venticute ò (nycomed), instilled intratracheally in patients with established ards. [23] the two studies enrolled a total of 448 patients within 24 hours of diagnosis of ards and were powered to show a difference in ventilator-free days. although oxygenation was significantly better during the 24-hour treatment period in the surfactant group, there were no significant differences noted in the number of ventilator-free days or in 28-day survival. [23] a post hoc analysis demonstrated that patients with 'direct' causes of ards (i.e. pneumonia, witnessed aspiration of gastric contents or both) had a mortality benefit with surfactant treatment compared with standard care. a followup meta-analysis pooling results of five multicentre studies of rsp-c confirmed this finding: the subgroup of patients with severe ards due to pneumonia or aspiration had decreased mortality when treated with rsp-c (26.3% vs 39.3% in the usual care group; p = 0.018). [24] subsequently, a prospective phase iii rct evaluating effect of venticute ò in 1200 patients with pneumonia or aspiration of gastric contents was conducted. the study was terminated at 800 patients due to futility. neither these results nor the potential reasons for futility have been published to date. [60] calfactant (infasurf ò , ony inc.) is a modified natural surfactant produced by extracting the phospholipids, neutral lipids and surfactantspecific proteins sp-b and sp-c from newborn calf lungs. in in vivo animal lung studies, calfactant has shown greater surface activity than exosurf ò and survanta ò , [110] [111] [112] [113] and the highest level of resistance to inactivation due to its high ratio of protein sp-b to phospholipids. [114] [115] [116] from 2000 to 2003, calfactant was used in a multicentre study of ali/ards in the paediatric population 1 week (full-term infants) to 21 years of age. overall, calfactant significantly improved oxygenation and reduced mortality (19% vs 36%; p = 0.03), although the greatest impact was observed in the subgroup of patients with direct ali/ards while calfactant had little effect in patients with indirect ali or ards. [61] indeed, calfactant is the first and only pharmacological agent to demonstrate a mortality benefit for treatment of ali/ards. it is of note, however, that this study differs from other adult studies in that the majority of paediatric patients had direct causes of ards and the most common cause of death was respiratory failure, whereas adult studies have included a larger proportion of patients with indirect causes, such as sepsis, wherein the most common cause of death is multi-organ failure. based on those encouraging results, pneuma pharmaceuticals began conducting a large phase iii multicentre rct of calfactant for direct ards (origin of ards must be infectious pneumonia, aspiration, near drowning, smoke inhalation without pulmonary burn or inhaled industrial gas) in adults and children. a total of 880 patients in two consecutive studies of patients under 12 and over 12 years of age was planned. however, after the first interim analysis in january 2010, the paediatric arm of the study was stopped for futility due to an unexpectedly low mortality rate. recruitment in the adult arm (ages 12-85 years) is continuing as the interim analysis did not reveal futility or any safety concerns (wilson d, university of virginia health sciences center, charlottesville, va, usa, personal communication). [117] since reactive oxygen species also contribute to the tissue damage incurred in ali, antioxidant therapies have also been investigated as therapeutic options for established disease. n-acetylcysteine (nac) is a commercially available antioxidant approved for the treatment of paracetamol (acetaminophen) toxicity. nac is a precursor for glutathione, an antioxidant present in normal lungs and deficient in bronchoalveolar lavage fluid from ali/ards patients. additionally, because of its thiol group, nac can scavenge reactive oxygen species such as hydrogen peroxide and superoxide anion. in an rct of 46 patients, nac and oxothiazolidine carboxylate (procysteine ò , clintec technologies inc.), another glutathione precursor, were studied for their combined effect in ali/ards but failed to reduce mortality compared with placebo, [62] negating promising results of three prior small studies. [63] [64] [65] interestingly, recent evidence suggests that genetic diversity may explain variable responsiveness to nac. glutathione-s-transferases (gsts) are enzymes from a complex, multigene family with important roles in oxidative stress pathways. a study by moradi and co-workers [118] demonstrated that deletion of specific gst gene polymorphisms correlated with mortality and that treatment with nac significantly lowered mortality in these subgroups of patients. these results suggest that patients with gst gene deletions are more vulnerable to oxidative stress contributing to ards and may be in greater need of antioxidant therapy. [118] antioxidant supplementation to enteral nutrition rich in omega-3 fatty acids has also been investigated for patients with ali/ards. while the rationale for nutritional antioxidants such as vitamins e and c is to reduce the oxidative stress present in ali, the purpose of the omega-3 fatty acids is to reduce production of proinflammatory mediators. eicosanoids, such as prostaglandins, thromboxanes and leukotrienes, derived from omega-3 fatty acids are generally much less proinflammatory than those derived from omega-6 fatty acids. since omega-6 fatty acids compete with omega-3 fatty acids for the same rate-limiting enzymes in the production of eicosanoids, diets with a high proportion of omega-6 fats are thought to be proinflammatory and prothrombotic. examples of polyunsaturated omega-3 fatty acids are a-linolenic acid, eicosapentaenoic acid and docosahexaenoic acid. [119] a phase ii study enrolling 98 patients with ali compared an antioxidant enteral feeding formula containing eicosapentaenoic acid, g-linolenic acid and antioxidant vitamins with placebo, and observed improved oxygenation, reduced pulmonary inflammation, fewer days of mechanical ventilation and fewer non-pulmonary organ failures in the treatment arm, although there was no difference in mortality between this approach and the control group. [66] ardsnet proceeded to conduct the omega study, a phase iii study examining efficacy of omega-3 and antioxidant supplementation to enteral nutrition. the study was stopped for futility, but results have not yet been published. [67, 68] several therapies aimed at modulating neutrophil activity have been studied. to understand why previous clinical trials have been negative and highlight potential targets for novel therapies, it is important to understand the role of neutrophils in propagating lung injury and mods. polymorphonuclear neutrophils (pmns) form the first line of defence against invading pathogens, and neutropenia or defective neutrophil function predisposes the host to increased morbidity. extensive clinical and experimental data support the role of the activated neutrophil in the pathogenesis of organ injury in sepsis. the lung is particularly vulnerable. postmortem studies of patients with ards show massive pulmonary accumulation of neutrophils, with the highest counts in non-survivors. [120] the pathological impact of neutrophils may be due to their activation, transmigration or delayed apoptosis. however, neutrophil-independent mechanisms of ali must also exist, since ards has been described in neutropenic patients. neutrophil kinetics in the pulmonary circulation differ substantially from that of microvascular beds in the systemic circulation. the pulmonary circulation harbours a large intravascular reservoir of leukocytes, mainly neutrophils, referred to as the 'marginated pool'. [121] this marginated pool may equal or even exceed the pool of circulating neutrophils and exchanges with the latter as an ongoing phenomenon. thus, it is important to appreciate that circulating neutrophils, when isolated for experimental analysis, may not represent the characteristics of the entire population of neutrophils in the bloodstream. intravital microscopic studies have revealed that, in contrast to the systemic circulation where neutrophil sequestration is almost exclusively confined to the venular compartment, the major site of neutrophil retention in the lung is the alveolar capillary bed. [122] neutrophil activation can also lead to cytoskeletal changes that reduce cell deformability and slow their transit time through the alveolar capillaries. since one of the earliest manifestations of ards is accumulation of large numbers of neutrophils in the alveolar capillaries, it is possible that the accumulation of neutrophils may initiate selective capillary blockade and arteriovenous shunting leading to hypoxia seen in ards. activated neutrophils also produce human ne (hne), a protease capable of producing tissue damage by means of its degradation of elastin, fibronectin, laminin, collagen and proteoglycans. normally, protease inhibitors impede ne, but in the setting of an overwhelming inflammatory response, neutrophils generate reactive oxidants that inactivate endogenous protease inhibitors, leaving the activity of hne unchecked. this may lead to increased pulmonary inflammation and endothelial cell permeability. [9] sivelestat (elaspol ò , ono pharmaceuticals) is a competitive inhibitor of ne. it was launched in japan after a phase iii study demonstrated reduced icu stay and improved pulmonary function in patients with ali associated with the systemic inflammatory response syndrome (sirs). [70] however, the strive study [69] was terminated early after randomizing 492 patients from 105 sites in six countries, when the data and safety monitoring board found a trend to increased mortality at 180 days. final analysis revealed no difference in 28-day all-cause mortality (26% in both groups) or number of ventilator-free days between the treatment group and controls. epi-hne-4 or depelestat (debiopharm s.a.) is another hne inhibitor currently under development for treatment of inflammatory pulmonary diseases, including ali. in a repeated lung injury rat model depelestat administration afforded a significant protective effect on lung compliance and alveolar inflammation at day 14 compared with the control group. [71] a phase ii study examining safety and efficacy of intravenous depelestat for patients with ards has been completed, but results have not yet been published. [123] neutrophil transmigration neutrophil margination allows for a molecular interaction between the cell surfaces of the neutrophil and endothelial cell to occur. subsequently, as a consequence of cell surface integrins and their ligands, neutrophils undergo adhesion with endothelial cells. following adherence, neutrophils must pass through the endothelial monolayer, interstitial tissue and alveolar epithelium to reach the alveolar space. passage of large numbers of activated neutrophils can cause epithelial damage, sloughing and increased permeability both due to mechanical force exerted by neutrophil pseudopodia as well as due to release of toxic substances such as proteinases (e.g. elastases, cationic peptides, defensins, oxidants and mmps). [9] while there are conflicting reports on the effects of elastase on increased epithelial permeability, cationic peptides such as defensins can cause both epithelial and endothelial cell injury. defensin levels have been found to be greatly elevated in patients with ards and their levels correlate with the severity of lung injury. [124] neutralizing its effects could be important in the management of ards. ongoing research is examining if defensins can be used to identify patients with ali at an early stage. [125] delayed apoptosis of neutrophils once egressed into the extravascular space, neutrophils cannot return to the circulation and their elimination is dependant upon their clearance by apoptosis and subsequent recognition and elimination by macrophages and other phagocytic cells. normally, neutrophils are terminally differentiated cells with a terminal half-life of 5-6 hours in vivo. upon completion of their lifespan, neutrophils institute a programme of cell death known as 'apoptosis' and are then removed from the circulation by the liver and spleen. apoptosis, as opposed to necrosis, is believed to be crucial for resolution of inflammation as it does not result in loss of cell membrane integrity and bystander tissue damage by release of intracellular enzymes, proteases and reactive oxygen species. [126] expression of neutrophil apoptosis is delayed in ards. [127] this is not an unexpected finding, especially since pmn apoptosis is delayed in other critically ill patients with sepsis, trauma and burns. [128, 129] apoptosis of neutrophils may be an important consequence in determining the extent of lung injury. for example, it has been shown that the induction of neutrophil apoptosis by the administration of dead escherichia coli prior to reperfusion resulted in significant improvement in lung injury. [130] induction of neutrophil apoptosis in the alveolar space has the potential for resolution of inflammation in ards, and can be carried out in a number of ways that could include multiple strategies such as ligation of fas, activation of proapoptotic caspases and modulation of mitogen-activated protein kinases or transcription factors such as nf-kb. hastening neutrophil apoptosis in the alveolar space may also decrease the probability of secondary necrosis and further tissue damage in ards. it is intriguing that no significant differences were found between the expression of neutrophil apoptosis in patients at risk and those with established ards, nor did the extent of apoptotic inhibition correlate with overall outcome in ards. [131] therefore, while it is well established that ards is associated with accumulation of large numbers of neutrophils in alveolar spaces, their contribution to the severity of ards in humans remains uncertain. in summary, targeting neutrophil responses in ards may have therapeutic potential. however, as has been learnt from various ali and sepsis trials in the past, simple strategies to control dysregulated neutrophil behaviour may not be effective. rather, key stages of neutrophil function and kinetics may need to be identified in different clinical phases of ards, and selective immunomodulation strategies may need to be identified for individual patients. in addition to modulation of neutrophil function, there are other facets of the immune and inflammatory response currently under investigation as potential therapeutic targets for treatment of ards. these include modulation of macrophage activity with granulocyte macrophage colony-stimulating factor (gm-csf), inhibition of inflammatory mediators and broad suppression of the inflammatory response with corticosteroids. although most prostaglandins are proinflammatory mediators, prostaglandin e 1 (pge 1 ) has potential beneficial effects in ali, specifically due to its ability to modulate neutrophil activation. however, exogenous pge 1 is associated with several adverse effects and patient intolerance due to haemodynamic instability has been observed. tlc-c-53 (ventusô; the liposome company) is a liposomal dispersion of pge 1 . the development of pge 1 in liposomal form may potentiate its role in neutrophil downregulation, improve peripheral delivery of the drug to the lung and decrease systemic adverse effects, thus providing a good rationale for testing in humans. [73] a phase iii trial of 350 patients with ards randomized to intravenous tlc-c-53 at escalating doses for 7 days versus placebo found no difference in duration of mechanical ventilation or 28-day mortality between the treatment and control groups, although treatment was associated with accelerated improvement in oxygenation. [72] however, more than 50% of patients required a dose reduction due to hypotension or hypoxaemia. interestingly, those patients who tolerated and received at least 85% of the full dose had a shorter duration of mechanical ventilation. a subsequent multicentre phase iii trial of tlc-c-53 in 102 ards patients [73] demonstrated no differences in time to liberation from the ventilator or 28-day mortality; the trend to shorter duration of hypoxaemia in the treatment group failed to reach statistical significance. gm-csf has been shown to stimulate phagocytosis and oxidative functions of host defence neutrophils, monocytes and macrophages. [74] in addition to its systemic actions, gm-csf may also influence pulmonary host defence by modulating alveolar macrophage function and surfactant metabolism. as noted, apoptosis of neutrophils is an important mechanism by which these cells are cleared from inflamed lung regions, thereby facilitating resolution of inflammation. although both granulocyte colony-stimulating factor and gm-csf are thought to inhibit neutrophil apoptosis, in animal models of lung injury, gm-csf has been shown to help restore capillary barrier integrity, [132] preserve alveolar epithelial function and improve alveolar fluid clearance. [133] a pilot study of 45 patients with ards undergoing serial bronchoalveolar lavage found that patients who survived ards had higher concentrations of gm-csf in the bronchoalveolar lavage fluid on day 1 than patients who died. [134] the authors speculated that gm-csf might improve survival by prolonging the neutrophil lifespan in the alveoli and/or inducing proliferation of alveolar macrophages, thereby improving host defence and reducing infectious complications in this setting. in a phase ii trial, molgramostim (schering-plough), a recombinant human gm-csf, was given intravenously at a low dose (3 mg/kg) for 5 days to ten patients with severe sepsis and sepsis-related pulmonary dysfunction (defined as a pao 2 /fio 2 ratio of <287 with a pulmonary infiltrate on chest radiograph). [74] the primary outcome was 30-day survival, and secondary outcomes included oxygenation, occurrence of ards and degree of organ dysfunction at day 5. there was no difference in 30-day survival between the treatment and placebo groups, but oxygenation improved in the gm-csf group. ards was present in four of ten patients in the gm-csf group on study entry, but resolved in two of these patients by day 5, whereas in the placebo group ards was present in three patients on study entry and five patients on day 5. organ dysfunction was similar between the two groups, with no change between study entry and day 5. from july 2004 to july 2009, the nhlbi enrolled patients who had been diagnosed with ali/ards for at least 3 days into a phase ii rct of recombinant gm-csf (sargramostim [leukine ò ], genzyme corporation) versus placebo. [75] the primary outcome was the number of ventilator-free days during days 1-28. secondary outcomes included measures of lung epithelial cell integrity, alveolar macrophage function, changes in severity of respiratory gas exchange, non-respiratory organ failure and incidence of ventilator-associated pneumonia. this study has been completed, but results have not yet been published. [75] cytokine inhibitors cytokines are glycoproteins that act as messengers to cell surface receptors to promote or diminish the inflammatory cascade. specific cytokines are observed in high amounts in the bronchoalveolar lavage fluid of patients with ards, and are thought to play an important role in propagating lung injury. unsaturated phosphatidic acid plays an important role in intracellular signalling leading to neutrophil accumulation within the lungs, as well as proinflammatory cytokine expression and cell membrane oxidation, all of which leads to lung tissue damage. [135] lisofylline (cell therapeutics) is a cytokine inhibitor that impedes synthesis of phosphatidic acid-1a and, therefore, was thought to hold potential for treatment of ards. however, ardsnet stopped a phase ii/iii trial, the larma study, for futility after the first interim analysis failed to demonstrate any difference in 28-day mortality, ventilator-free days, organ failures or levels of circulating free fatty acids. [76] interleukin (il)-8 is another potent chemoattractant for neutrophils, observed in high levels in patients with early ards [136] and associated with increased mortality. [137] anti-il-8 monoclonal antibody has been shown to reduce pulmonary oedema and neutrophil accumulation in animal models of ards [77, 78] but has not yet been tested in humans. finally, tnfa has long been recognized as an important proinflammatory cytokine in ards, but more recent evidence suggests that it actually plays a dichotomous role in both contributing to permeability oedema but also increasing alveolar fluid clearance capacity. monoclonal anti-tnfa antibodies have been tested in patients with sepsis with disappointing results. [138] given its dual role in alveolar oedema formation and resorbtion, a more sophisticated approach than simply blocking all tnfa activity is likely to be required in ards. studies examining the efficacy of corticosteroids for acute exudative ards have shown conflicting results. in 1987, bernard et al. [80] published results of a study of 99 patients with ards randomized to high-dose pulse methylprednisolone (30 mg/kg every 6 hours for 24 hours) or placebo. there was no difference in 45-day mortality (60% vs 63%; p = nonsignificant) but the confidence intervals were wide, suggesting that the study may have been underpowered to detect a small difference in a population with heterogenous outcomes. in 2007, meduri and colleagues [79] published their results of 91 patients with severe early ards (<72 hours) from five hospitals randomized to methylprednisolone 1 mg/kg/day for 28 days versus placebo. they found corticosteroids significantly reduced icu mortality (21% vs 43%; p = 0.03), duration of mechanical ventilation and length of icu stay. [79] annane et al. [81] published a post hoc analysis of 177 ards patients enrolled in an rct of low-dose corticosteroids in septic shock. patients in the treatment group received hydrocortisone 50 mg every 6 hours plus fludrocortisone 50 mg/day for 7 days. although there was no mortality difference for ards patients overall, ards patients with relative adrenal insufficiency and septic shock had significantly reduced mortality when treated with low-dose hydrocortisone (53% vs 75% in the placebo group; p = 0.01). [81] the use of corticosteroids for acute exudative ards remains controversial, although the evidence is more definitive for corticosteroid treatment initiated late for fibroproliferative ards (see section 3.3.1). a study examining low doses of corticosteroids as adjuvant therapy for lung injury associated with h1n1 influenza virus (cortiflu) is planned. [139] 3.2.6 activated protein c microvascular injury and coagulation play critical roles in the pathogenesis of ali. plasma protein c levels are decreased in patients with ali, and are associated with higher mortality and fewer ventilator-free days. [82] recombinant human activated protein c (rhapc; drotrecogin alfa; eli lilly) was tested in a phase iii clinical trial of patients and demonstrated a significant mortality reduction from 30% to 24% in patients with severe sepsis. [140] a phase ii study was sponsored by the nhlbi to determine if drotrecogin alfa increased ventilator-free days in patients with ali (patients with severe sepsis were excluded). the study was terminated by the data safety monitoring board. although drotrecogin alfa significantly increased plasma protein c levels and decreased pulmonary dead space fraction, there was no significant difference in the number of ventilator-free days or in 60-day mortality (5 of 38 vs 5 of 37 patients, respectively; p = 1.0). [82] 3.2.7 hmg-coa reductase inhibitors (statins) hmg-coa reductase inhibitors, commonly known as statins, have recently been proposed as a treatment for ali/ards. the rationale for this is based on animal models suggesting that statins can attenuate organ dysfunction by reducing vascular leak and inflammation. [84] a prospective cohort study in ireland showed a nonsignificant trend towards lower odds of death in ards patients receiving a statin during their icu admission (or 0.27, 95% ci 0.06, 1.21; p = 0.09). [83] however, a recently published retrospective cohort study from the mayo clinic (rochester, mn, usa) showed no difference in mortality or organ dysfunction in ards patients treated with statins. [84] stip is currently enrolling patients admitted to an icu with respiratory distress and a pao 2 /fio 2 ratio <300 due to the h1n1 pandemic strain of influenza. [141] patients in this trial will be randomized to receive rosuvastatin 20 mg/day or placebo for 21 days. since this is a specific subpopulation of patients with ali, findings from this study may not be generalizable to other ali subgroups. the sails trial (also rosuvastatin 20 mg/day vs placebo) is also planned but not yet open for recruitment. [142] patients who survive the early and exudative phases of ards generally enter a period from week 1 to 3 consisting of fibroproliferation and organization of exudative debris within the airspace. this fibroproliferative relatively 'late' phase either slowly resolves or progresses to fibrosis. during this phase, patients are at risk of dying from other complications such as mods, or may fail to wean from mechanical ventilation due to severely impaired respiratory muscle and lung function. those who successfully wean off mechanical ventilation may have residual pulmonary fibrosis and reduced exercise capacity. for resolution to occur, removal of inflammatory cells, cellular debris, and soluble and insoluble proteins needs to take place. as noted in section 3.2.4, apoptosis of neutrophils facilitates resolution of inflammation. monocyte and macrophage phagocytic clearance of apoptotic cells appears to be an important mechanism by which neutrophils are cleared from inflamed lung regions. soluble proteins are likely to be primarily removed via paracellular diffusion, but removal of insoluble proteins appears to depend on the function of alveolar macrophages. mechanisms involved in remodelling of hyaline membranes and restoration of a functional alveolar-capillary barrier are incompletely understood at present, but therapeutic interventions aimed at modulation of phagocytosis/apoptosis are being evaluated. to date, far less research has targeted this later phase of the disease, as most trials have focused on earlier preventative processes. fibroproliferative ards is characterized by ongoing inflammation. in addition to being tested for prevention of ards, and treatment of the early and mid exudative phases, corticosteroids have also been tested for efficacy in reversing the fibrosing alveolitis of the late phase of ards. a study by meduri and colleagues [86] examined the effect of prolonged methylprednisolone therapy (2 mg/kg/day for 32 days) on 24 patients with severe ards that was unresolved after 7 days of respiratory failure. although this study demonstrated a significant hospital mortality benefit (2 of 16 patients [12%] in the corticosteroid group died vs 5 of 8 [62%] in the placebo group), the significance of these findings was controversial for two reasons: the calculated sample size to demonstrate a 30% absolute difference in mortality was 99 patients but the study was terminated early after enrolment of 24 patients, and the mortality in the placebo group was slightly higher than anticipated. [86] to shed further light on this issue, ardsnet specifically designed a study to focus on the late fibrotic stage of the disease, called lasrs. [85] this study examined the role of corticosteroids in 180 patients in the late phase (>7 days from onset) of persistent ards. methylprednisolone, dosed at 2 mg/kg/day for 14 days followed by tapering doses until day 25, was compared with placebo. there was no difference in 60-or 180-day mortality rates. methylprednisolone improved oxygenation, respiratory system compliance and blood pressure, resulting in an increased number of ventilator-free and shock-free days; however, a higher rate of neuromuscular weakness and, if initiated more than 14 days after the onset of ards, a significant increased mortality was observed in the methylprednisolone group. therefore, despite the improvement in cardiopulmonary physiology, methylprednisolone does not improve overall mortality in ards and is not recommended for treatment of late ards. given these results, the convincing lack of efficacy for prevention of ali prior to diagnosis and the lack of evidence of benefit in the early phase, corticosteroids cannot be recommended for routine treatment of ali/ards at any stage, at this time. furthermore, it may prove to be exceedingly difficult to determine which individual patient might benefit from corticosteroids and at what specific point to intervene. clearly, the current status of treatment options for patients with ali/ards is suboptimal. at this time, the clinical management of patients with ali/ards involves supportive therapy only. this primarily includes low stretch or 'lung protective' mechanical ventilation, conservative fluid management and adequate nutritional support. although the term 'supportive' may sound somewhat discouraging, these are important observations, not only because they impact on the outcome of patients with ali/ards but also because they should be embraced and implemented as 'standard care' for this patient population. furthermore, any new therapy being tested should be compared with optimal 'standard care'. other methods proposed to offer greater protection to the lungs while providing mechanical support to respiration include hfo and ecmo. studies into these modes are ongoing. although supportive therapies have reduced mortality, there is still significant need for improvements. previous studies have provided important insight into the pathophysiology of ali/ards. research is ongoing into therapies to prevent ali/ards in those at risk, treat it early in its course or aid in its resolution. each of these goals is associated with specific challenges. demonstrating that a prophylactic intervention reduces mortality, morbidity and is cost effective is challenging at best. this is most likely to occur when the risk of acquiring the disease is high, the outcome of the disease is uniformly devastating and treatment for the disease is nonexistent. for some critically ill patients at risk for ards, this may be the case. however, the diagnosis of ali/ards encompasses a very heterogeneous population, with incompletely understood risk factors and non-uniform, diverse outcomes. the greatest likelihood of success for prophylactic therapy will come when we have further delineated the subgroups at highest risk of dying from ali/ards and have accurate diagnostic tests to identify these patients. for ali/ards, specifically targeting the pathogenic mechanisms responsible for the increased risk of death in these patient subgroups would theoretically be high yield. basic science research identifying genetic polymorphisms of patients with highest mortality or greatest need for specific therapies shows great promise in this regard, but is not yet clinically applicable. until then, validating biomarkers and clinical indicators for poor prognosis in ali/ards should remain a primary research goal. finding therapies to treat ards in its late fibroproliferative phase is also in great need. too often patients survive the early and mid phase of ards only to succumb to complications in the late phase or undergo withdrawal of life support as they are unable to be weaned from mechanical ventilation. research into mechanisms of idiopathic pulmonary fibrosis may help identify common pathways to target for therapy. to date, the majority of research has focused on treating ali/ards in its earlier stages, in the hope that the disease process may be reversed prior to the patient entering the fibroproliferative phase. progress in finding therapies to treat established ards has been slow and hampered by a long series of negative clinical trials. however, there are several lessons to be learned from these rcts. first, a 'one-size-fits-all' approach has not worked for pharmacotherapy for ards. in this sense, the syndrome of ali/ards may be likened to cancer. cancer as a broad term signifies the uncontrolled replication of abnormal cells, but there are specific chemotherapeutic treatments for specific types of cancer, depending on its origin. some treatments may be effective for more than one type of cancer, but not for other types, and the magnitude of the benefit might vary according to the type and stage of disease. oncologists would not design a trial enrolling all patients with differing types of cancer and expect to find a single drug that shows a survival benefit. yet, that is what has been attempted with several large ards trials. recent studies have demonstrated that direct ards is likely to respond differently than indirect ards, and in fact within these broad categories, pathogenesis may differ. therefore, different therapies may need to be developed for specific aetiologies such as sepsis-related ards, sirs-related ards and various direct causes of ards. second, a well designed negative rct does not necessarily mean that the therapy tested should be abandoned. it means that the therapy is likely to not be appropriate for widespread application. however, just because a drug does not work for every ards patient does not necessarily mean it should not be used for anyone with ards. for example, there is no evidence for treating all patients with acute ards with corticosteroids, but there is evidence that treating ards patients with relative adrenal insufficiency and septic shock with low doses of hydrocortisone is likely to be beneficial. similarly, nitric oxide should not routinely be applied to all patients with ali/ards, but may be useful in refractory hypoxaemia, particularly in conjunction with other ventilation rescue strategies. third, a negative rct should potentially lead to further research so that we can gain further insight as to why the therapy failed to yield a clinical benefit. thomas edison, when asked why he pursued his quest to invent a functional and practical light bulb after innumerable failed attempts, is reported to have replied, ''i have not failed. i've just found 10 000 ways that won't work''. ards research should take us from bench to bedside and back to the bench again. basic science can help us understand basic mechanisms of disease, discover why a therapy failed, then provide new ideas to apply to the clinical realm. rcts are necessary to prove benefit and quantify risk prior to changing clinical practice. since we are in urgent need of therapies to treat ali/ ards, it is necessary that rcts continue to advance our clinical care. however, these rcts need to be well founded in basic biology and physiology research, and focused on specific hypotheses regarding mechanisms of disease. continuing to conduct large clinical trials on heterogeneous patients with ali/ards from multiple aetiologies will not only prove ineffective but also add enormous cost to the healthcare system. the most significant and promising finding from an rct to date is that calfactant, the natural bovine surfactant rich in sp-b and -c, reduces mortality in ali from 36% in the control arm to 19% in the paediatric population. indeed, calfactant is the first and only pharmacological agent to demonstrate a mortality benefit for treatment of ali/ards. the ongoing cards study will attempt to reproduce that finding in adults with direct causes of ards. [117] this trial is continuing enrolment after the first interim analysis, with a target completion date of march 2011. great gains have been made in providing supportive management to patients with ali/ards. ongoing and future research efforts will provide important insights into the complex pathophysiologies involved and may provide further rationale for patient-specific therapies and/or combination therapies targeting the various mechanisms contributing to this disorder. understanding who is at greatest risk of succumbing to ali/ards and establishing the optimal time to intervene will be essential to improving mortality for this syndrome. epidemiology and outcome of acute lung injury in european intensive care units: results from the alive study incidence and mortality of acute lung injury and the acute respiratory distress syndrome in three australian states characteristics and outcomes in adult patients receiving mechanical ventilation: a 28-day international study a simple clinical predictive index for objective estimates of mortality in acute lung injury the acute respiratory distress syndrome network. ventilation with lower tidal volumes as compared with traditional tidal volumes for acute lung injury and the acute respiratory distress syndrome higher versus lower positive end-expiratory pressures in patients with the acute respiratory distress syndrome emerging therapies for treatment of acute lung injury and acute respiratory distress syndrome the american-european consensus conference on ards: definitions, mechanisms, relevant outcomes, and clinical trial coordination transepithelial migration of neutrophils: mechanisms and implications for acute lung injury arachidonic acid remodeling in human inflammatory cells migrating to the lung in vivo recent advances in genetic predisposition to clinical acute lung injury chronic alcohol abuse is associated with an increased incidence of acute respiratory distress syndrome and severity of multiple organ dysfunction in patients with septic shock pharmacotherapy of acute lung injury and acute respiratory distress syndrome injurious mechanical ventilation and end-organ epithelial cell apoptosis and organ dysfunction in an experimental model of acute respiratory distress syndrome does programmed cell death (apoptosis) play a role in the development of multiple organ dysfunction in critically ill patients? a review and a theoretical framework an overview of mortality risk prediction in sepsis multiple organ dysfunction score: a reliable descriptor of a complex clinical outcome a multicenter registry of patients with acute respiratory distress syndrome: physiology and outcome adult respiratory distress syndrome: risk with common predispositions causes of mortality in patients with the adult respiratory distress syndrome improved survival of patients with acute respiratory distress syndrome (ards): 1983-1993 about the ards network effect of recombinant surfactant protein c-based surfactant on the acute respiratory distress syndrome a search for subgroups of patients with ards who may benefit from surfactant replacement therapy the oscillation for ards treated early (oscillate) trial pilot study review of a large clinical series: association of cumulative fluid balance on outcome in acute lung injury. a retrospective review of the ardsnet tidal volume study cohort pulmonaryartery versus central venous catheter to guide treatment of acute lung injury1 the use of an inflammation-modulating diet in patients with acute lung injury or acute respiratory distress syndrome: a metaanalysis of outcome data efficacy of an expanded ventilator bundle for the reduction of ventilatorassociated pneumonia in the medical intensive care unit continuous aspiration of subglottic secretions in the prevention of ventilatorassociated pneumonia in the postoperative period of major heart surgery oral decontamination for prevention of pneumonia in mechanically ventilated adults: systematic review and meta-analysis supine body position as a risk factor for nosocomial pneumonia in mechanically ventilated patients: a randomised trial thromboprophylaxis in medicalsurgical critically ill patients stress ulcer prophylaxis in critically ill patients: resolving discordant meta-analyses efficacy and safety of a paired sedation and ventilator weaning protocol for mechanically ventilated patients in intensive care (awakening and breathing controlled trial): a randomised controlled trial effects of physical training on functional status in patients with prolonged mechanical ventilation recent trends in acute lung injury mortality: 1996-2005 effect of nitric oxide on oxygenation and mortality in acute lung injury: systematic review and meta-analysis effect of prone positioning in patients with acute respiratory distress syndrome: a metaanalysis effect of mechanical ventilation in the prone position on clinical outcomes in patients with acute hypoxemic respiratory failure: a systematic review and meta-analysis1 hemodynamic and gas exchange response to inhaled nitric oxide and prone positioning in acute respiratory distress syndrome patients high-frequency oscillatory ventilation in adults: the toronto experience high-frequency oscillatory ventilation for adult patients with ards efficacy and economic assessment of conventional ventilatory support versus extracorporeal membrane oxygenation for severe adult respiratory failure (cesar): a multicentre randomised controlled trial extracorporeal membrane oxygenation for 2009 influenza a (h1n1) acute respiratory distress syndrome ketoconazole prevents acute respiratory failure in critically ill surgical patients a double-blind, prospective, randomized trial of ketoconazole, a thromboxane synthetase inhibitor, in the prophylaxis of the adult respiratory distress syndrome development, implementation, and evaluation of a ketoconazole practice guideline for ards prophylaxis angiotensin-converting enzyme inhibitor captopril prevents oleic acid-induced severe acute lung injury in rats rosiglitazone attenuates the severity of sodium taurocholate-induced acute pancreatitis and pancreatitis-associated lung injury chemically modified tetracycline prevents the development of septic shock and acute respiratory distress syndrome in a clinically applicable porcine model randomized, prospective trial of antioxidant supplementation in critically ill surgical patients corticosteroids in the prevention and treatment of acute respiratory distress syndrome (ards) in adults: meta-analysis recombinant platelet-activating factor acetylhydrolase to prevent acute respiratory distress syndrome and mortality in severe sepsis: phase iib, multicenter, randomized, placebo-controlled, clinical trial ketoconazole for early treatment of acute lung injury and acute respiratory distress syndrome: a randomized controlled trial the beta-agonist lung injury trial (balti): a randomized placebo-controlled clinical trial randomized, placebo-controlled trial of an aerosolized beta-2 adrenergic agonist (albuterol) for the treatment of acute lung injury aerosolized surfactant in adults with sepsis-induced acute respiratory distress syndrome: exosurf acute respiratory distress syndrome sepsis study group bovine surfactant therapy for patients with acute respiratory distress syndrome venticute in patients with pneumonia or aspiration of gastric contents and intubation/ventilation/oxygenation impairment (by2001/m1-007) [clinicaltrials.gov identifier nct00074906 effect of exogenous surfactant (calfactant) in pediatric acute lung injury: a randomized controlled trial a trial of antioxidants n-acetylcysteine and procysteine in ards: the antioxidant in ards study group n-acetylcysteine enhances recovery from acute lung injury in man: a randomized, double-blind, placebo-controlled clinical study glutathione (gsh) repletion by n-acetylcysteine (nac) in patients with the adult respiratory distress syndrome (ards) antioxidant treatment with n-acetylcysteine during adult respiratory distress syndrome: a prospective, randomized, placebocontrolled study effect of enteral feeding with eicosapentaenoic acid, gamma-linolenic acid, and antioxidants in patients with acute respiratory distress syndrome: enteral nutrition in ards study group omega study: information update early versus delayed enteral feeding and omega-3 fatty acid/antioxidant supplementation for treating people with acute lung injuryj or acute respiratory distress syndrome (the eden-omega study neutrophil elastase inhibition in acute lung injury: results of the strive study a phase iii clinical study of neutrophil elastase inhibitor ono-5046 na in sirs patients beneficial effect of an inhibitor of leukocyte elastase (epi-hne-4) in presence of repeated lung injuries liposomal prostaglandin e1 (tlc c-53) in acute respiratory distress syndrome: a controlled, randomized, double-blind, multicenter clinical trial. tlc c-53 ards study group a multi-centre, doubleblind, placebo-controlled study of liposomal prostaglandin e1 (tlc c-53) in patients with acute respiratory distress syndrome a randomized phase ii trial of granulocyte-macrophage colony-stimulating factor therapy in severe sepsis with respiratory dysfunction a randomized trial of gm-csf in patients with ali/ards [clinicaltrials.gov identifier nct00201409 national institutes of health. randomized, placebo-controlled trial of lisofylline for early treatment of acute lung injury and acute respiratory distress syndrome acid aspiration-induced lung injury in rabbits is mediated by interleukin-8-dependent mechanisms humanized monoclonal antibody against the chemokine cxcl-8 (il-8) effectively prevents acute lung injury methylprednisolone infusion in early severe ards: results of a randomized controlled trial high-dose corticosteroids in patients with the adult respiratory distress syndrome effect of low doses of corticosteroids in septic shock patients with or without early acute respiratory distress syndrome randomized clinical trial of activated protein c for the treatment of acute lung injury acute lung injury and the acute respiratory distress syndrome in ireland: a prospective audit of epidemiology and management statin administration did not influence the progression of lung injury or associated organ failures in a cohort of patients with acute lung injury efficacy and safety of corticosteroids for persistent acute respiratory distress syndrome effect of prolonged methylprednisolone therapy in unresolving acute respiratory distress syndrome: a randomized controlled trial prostacyclin and thromboxane a2 formation is increased in human sepsis syndrome: effects of cyclooxygenase inhibition angiotensin-converting enzyme 2 protects from severe acute lung injury angiotensin converting enzyme insertion/deletion polymorphism is associated with susceptibility and outcome in acute respiratory distress syndrome polymorphism of the angiotensin-converting enzyme gene affects the outcome of acute respiratory distress syndrome chronic use of angiotensin pathway inhibitors is associated with a decreased risk of acute respiratory distress syndrome use of ace inhibitors and development and outcome in ards the peroxisome proliferator-activated receptor-g is a negative regulator of macrophage activation ppar-gamma agonists inhibit production of monocyte inflammatory cytokines a novel therapy for colitis utilizing ppar-gamma ligands to inhibit the epithelial inflammatory response ppar y ligands inhibit nitrotyrosine formation and inflammatory mediator expressions in adjuvant-induced rheumatoid arthritis mice thiazolidinediones in type 2 diabetes: a cardiology perspective long-term risk of cardiovascular events with rosiglitazone: a meta-analysis trial of glutamine and antioxidant supplementation in critically ill patients (redoxs) [clinicaltrials.gov identifier nct00133978 comparison of clinical criteria for the acute respiratory distress syndrome with autopsy findings lung epithelial fluid transport and the resolution of pulmonary edema alveolar fluid clearance in the resected human lung upregulation of alveolar epithelial active na+ transport is dependent on beta2-adrenergic receptor signaling balti-2 (beta agonist lung injury trial-2) drug study of albuterol to treat acute lung injury (alta) [clinicaltrials.gov identifier nct00434993]. us national institutes of health, clinicaltrials.gov a role for b2 agonists in ards: the question remains unanswered surfactant proteins a and d and pulmonary host defense the future of surfactant therapy during ali/ards the role of exogenous surfactant in the treatment of acute lung injury a mutation in the surfactant protein b gene responsible for fatal neonatal respiratory disease in multiple kindreds target disruption of the surfactant protein b gene disrupts surfactant hemeostasis, causing respiratory failure in newborn mice differential activity and lack of synergy of lung surfactant proteins sp b and sp c in interactions with phospholipids surfactant inhibition by plasma proteins: differential sensitivity of various surfactant preparations importance of hydrophobic apoproteins as constituents of clinical exogenous surfactants is outcome from ards related to the severity of respiratory failure? calfactant for direct acute respiratory distress syndrome (cards) the role of glutathione-s-transferase polymorphisms on clinical outcome of ali/ards patient treated with n-acetylcysteine enteral omega-3 and acute respiratory distress syndrome evolution of bronchoalveolar cell populations in the adult respiratory distress syndrome the marginated pool physiological neutrophil sequestration in the lung: visual evidence for localization in capillaries safety and efficacy study of depelestat in acute respiratory distress syndrome (ards) patients [clinicaltrials.gov identifier nct00455767 high concentrations of alpha-defensins in plasma and bronchoalveolar lavage fluid of patients with acute respiratory distress syndrome biological markers to identify early sepsis and acute lung injury programmed cell death (apoptosis) and the resolution of systemic inflammation the role of apoptosis in acute lung injury delayed neutrophil apoptosis in sepsis is associated with maintenance of mitochondrial transmembrane potential and reduced caspase-9 activity inhibition of apoptosis in polymorphonuclear neutrophils from burn patients a novel therapeutic strategy for attenuating neutrophil-mediated lung injury in vivo neutrophil apoptosis in the acute respiratory distress syndrome granulocyte/ macrophage colony-stimulating factor treatment improves alveolar epithelial barrier function in alcoholic rat lung transgenic overexpression of granulocyte macrophage-colony stimulating factor in the lung prevents hyperoxic lung injury modulation of neutrophil apoptosis by granulocyte colony-stimulating factor and granulocyte/macrophage colony-stimulating factor during the course of acute respiratory distress syndrome phosphatidic acid signaling mediates lung cytokine expression and lung inflammatory injury after hemorrhage in mice role of interleukin 8 in the genesis of acute respiratory distress syndrome through an effect on neutrophil apoptosis increased interleukin-8 concentrations in the pulmonary edema fluid of patients with acute respiratory distress syndrome from sepsis regulators of endothelial and epithelial barrier integrity and function in acute lung injury low doses corticosteroids as adjuvant therapy for the treatment of severe h1n1 flu (cortiflu) safety and dose relationship of recombinant human activated protein c for coagulopathy in severe sepsis us national institutes of health, clinicaltrials.gov us national institutes of health, clinicaltrials.gov assistant professor, department of medicine, university hospital, 339 windermere road the authors thank jeanette mikulic for her assistance with preparation of the manuscript. no sources of funding were used to assist in the preparation of this review. the authors have no conflicts of interest that are directly relevant to the content of this review. key: cord-287156-3plpi6i9 authors: lassandro, giuseppe; palladino, valentina; amoruso, anna; palmieri, viviana valeria; russo, giovanna; giordano, paola title: children in coronaviruses’ wonderland: what clinicians need to know date: 2020-07-01 journal: mediterr j hematol infect dis doi: 10.4084/mjhid.2020.042 sha: doc_id: 287156 cord_uid: 3plpi6i9 human coronaviruses (hcovs) commonly cause mild upper-respiratory tract illnesses but can lead to more severe and diffusive diseases. a variety of signs and symptoms may be present, and infections can range in severity from the common cold and sore throat to more serious laryngeal or tracheal infections, bronchitis, and pneumonia. among the seven coronaviruses that affect humans (sars)-cov, the middle east respiratory syndrome (mers)-cov, and the most recent coronavirus disease 2019 (covid-19) represent potential life-threatening diseases worldwide. in adults, they may cause severe pneumonia that evolves in respiratory distress syndrome and multiorgan failure with a high mortality rate. children appear to be less susceptible to develop severe clinical disease and present usually with mild and aspecific symptoms similar to other respiratory infections typical of childhood. however, some children, such as infants, adolescents, or those with underlying diseases may be more at-risk categories and require greater caution from clinicians. available data on pediatric coronavirus infections are rare and scattered in the literature. the purpose of this review is to provide to clinicians a complete and updated panel useful to recognize and characterize the broad spectrum of clinical manifestations of coronavirus infections in the pediatric age. structural proteins, including the spike (s), envelope (e), membrane (m), nucleocapsid (n), and sometimes a hemagglutinin-esterase protein (he). the he protein binds to specific receptors and guides membrane fusion; the s protein is responsible for cell entry, the m and e proteins mediate viral assembly process, the inner n protein develops ribonucleoprotein complexes binding to viral rna. [1] [2] [3] [4] [5] to date, seven coronaviruses affect humans: in 1960s hcov-229e and hcov-oc43 were firstly reported; 6, 7 hcov-nl63 and hcov-hku1 were discovered subsequently in 2004 and 2005, respectively. 8, 9 additionally, three hcovs responsible for outbreaks involving high case fatality rates have been detected in humans in the last two decades: the severe acute respiratory syndrome (sars)-cov, the middle east respiratory syndrome (mers)-cov and the new coronavirus disease 2019 (covid-19) ( table 1) . table 1 . principal features of severe acute respiratory syndrome (sars)-cov, the middle east respiratory syndrome (mers)-cov and the most recent coronavirus disease 2019 (covid19) . classification beta-cov beta-cov beta-cov in several studies a similar prevalence in the detection of hcovs in patients with respiratory symptoms compared to healthy children has been found. [36] [37] [38] [39] moreover, patients with other underlying medical conditions or immunocompromised appear more susceptible to developing severe infections than healthy patients. [40] [41] [42] [43] additionally, human coronaviruses are responsible for other common childhood diseases such as acute otitis media [44] [45] [46] [47] , asthma exacerbations 48, and conjunctivitis 8 . they have also been involved in nosocomial infections, especially in the neonatal intensive care units (nicu). gagneur et al. in a prospective study determined the incidence of hcovrelated respiratory infections in newborns hospitalized in a nicu. among 64 neonates, seven positive nasal samples for hcovs (11%) were detected. all children were symptomatic. oxygen and ventilatory support were frequently needed. 49 sizun et al. evaluated the clinical role of coronaviruses respiratory infections in premature newborns. all premature infants infected had severe respiratory symptoms, including bradycardia, apnea, and hypoxemia, while chest x-ray revealed diffuse infiltrates. 50 it has also been shown that coronavirus infections are not only responsible for respiratory symptoms but can also affect other organs and systems in children. several studies have also reported that respiratory symptoms caused by coronavirus infection may be associated with central nervous system (cns) involvement. hcovs have an intrinsic capacity to affect neurons and diffuse centrifugally from cns via the transneuronal route. 51, 52 among neurological symptoms, febrile seizures, convulsions, loss of consciousness, encephalomyelitis, and encephalitis have been reported. [53] [54] [55] primarily in 1980, the viral genome was detected post-mortem in the cerebrospinal fluid of two patients with multiple sclerosis (ms). 56 subsequently, the hcovs neuroinvasion capacity was confirmed in a large panel of human brain autopsy samples affected by ms and other neurological diseases. 57 58 in 2017, a prospective study on 192 children with febrile seizures demonstrated that coronaviruses were frequently detected. 59 additionally, hcovs have been implicated as possible causes of many gastrointestinal disorders in children, and gastrointestinal symptoms have been reported in several studies in more than 50% of pediatric patients 28, 60, 61 . firstly, hcovs could be associated with neonatal necrotizing enterocolitis 62 all hcovs can also be detected in stool samples of patients affected by gastroenteritis. 60, 66 moreover, most of the hcovs found were coinfections with well-known gastroenteric viruses, including norovirus and rotavirus. hcovs may also be found occasionally in healthy children's stool samples. 67 although hcovs have always been associated with respiratory symptoms, these findings suggest that other systems may also be involved in children. the absence of serious symptoms may not be coupled with serological negativity. therefore, these viruses should be considered in the differential diagnoses of most of the common diseases of childhood. the 2002-2004 severe acute respiratory syndrome outbreak was a viral respiratory illness caused by sars-cov. the outbreak firstly emerged in the southern chinese province of guangdong in november 2002 and 68 then spread to 29 countries with 8,096 people infected and 774 died. 69 the sars global outbreak was contained in july 2003. since 2004, there have not been any known cases of sars reported anywhere in the world. 70 probably, civet cats or bats could be the initial step of the transmission to humans. humans to humans infection occurs by respiratory droplets or direct contact. healthcare or household contacts are critical routes of transmission. 71, 72 sars-cov infection cases were classified by the world health organization (who) into suspected, probable, and confirmed ( table 2) . 73 the median incubation period ranges between 2-11 days. sars causes atypical pneumonia, which may progress to respiratory failure. symptoms include fever, malaise, myalgia, headache, diarrhea, and rigors. adults are more likely to develop severe illness characterized by dyspnea, lymphopenia, acute respiratory distress syndrome (ards), and a fatal clinical course in 10% of cases. the exact number of children affected by sars worldwide is unknown. however, children appear to be less susceptible to sars with a lower incidence of the disease and no reported mortality. the majority of children had documented exposure to adults with sars, usually a family member. most infected children had previously attended school, but the spread of the infection in the school environment has not been demonstrated, and this could probably be linked to lower infectiousness of the virus among children. 74, 75 children have less severe symptoms than adults, and they rarely need intensive care. however, subclinical and asymptomatic infections appear uncommon. most children reported worldwide were healthy, previously and underlying conditions were infrequently reported. [75] [76] [77] usually, children require hospitalization after 3-4 days the onset of symptoms: fever (90-100%), dry cough (43-80%), sore throat (5-30%), rhinorrhea (33-60%), malaise and myalgia (10-40%), headache (14-40%) are common. dyspnea, tachypnea, and febrile seizures are infrequent. aspecific gastrointestinal symptoms, including abdominal pain, appetite lack, vomiting, and diarrhea, have been reported. physical examination at presentation is negative in the majority of children, and chest auscultation does not reveal significant findings. moreover, sometimes crackles or signs of lung consolidation can be detected. as well as the clinical examination, laboratory findings are not specific in children with sars and can be confused with those of other respiratory infections typical of childhood. commonly lymphopenia, the elevation of transaminases, lactic dehydrogenase, and creatine phosphokinase are detected. other hematological abnormalities such as leukopenia, thrombocytopenia, the elevation of d-dimer levels and mildly prolonged activated partial thromboplastin times are also observed. [78] [79] [80] circulating interleukin (il)-1β levels might be increased, resulting in caspase-1-dependent pathway activation responsible for an exaggerated and persistent inflammatory response and the consequent respiratory failure in severe cases. 81 in children, radiological findings are nonspecific and similar to other viral respiratory abnormalities. high fever (>38 °c) and cough or breathing difficulty and one or more of the following exposures during the 10 days prior to onset of symptoms: close contact with a person who is a suspect or probable case of sars cough or breathing difficulty history of travel, to an area with recent local transmission of sars residing in an area with recent local transmission of sars 1) a suspect case with radiographic evidence of infiltrates consistent with pneumonia or respiratory distress syndrome (rds) on chest x-ray (cxr). 2) a suspect case of sars that is positive for sars coronavirus by one or more assays. see use of laboratory methods for sars diagnosis. 3) a suspect case with autopsy findings consistent with the pathology of rds without an identifiable cause. www.mjhid.org mediterr j hematol infect dis 2020; 12; e2020042 commonly, the chest x-ray shows ground-glass opacity or focal consolidation. linear atelectasis and peribronchial thickening have also been reported. computed tomography (ct) shows more extensive airspace consolidation and ground-glass attenuation than chest x-ray, but it is performed in selective cases in pediatric age. [78] [79] [80] 82 usually, the clinical course is less severe in children compared to adults, and few patients require oxygen supplementation and assisted ventilation but preterm newborns, children younger than one year and older than 12 years of age have more severe symptoms and are likely to develop respiratory distress. [78] [79] [80] in pediatric age, sars infection commonly has a "biphasic" pattern. the first stage of the disease is characterized by virus replication and clinically by the onset of symptoms. the second phase is characterized by pulmonary involvement, which is typically less severe in children than in adults. most children will become afebrile within seven days, and they usually do not progress to respiratory distress, the adult third phase, that is only reported in a minimal number of cases, commonly among teenagers. 83, 84 in pregnant women, sars infection is associated with a high incidence of spontaneous miscarriage, prematurity, and intrauterine growth retardation (iugr). the increased morbidities during pregnancy are likely to be due to the hypoxic state and circulatory insufficiency that worsen placental blood flow and cause miscarriage or iugr. significantly, among pregnant women, mortality is 25%. 85 however, perinatal sars infections have not been documented. in none infants born from pregnant women affected, real-time pcr (rt-pcr) assays and viral cultures conducted on neonatal blood, body secretions and amniotic fluid were positive for sars. in infants, no congenital malformations have been reported. however, in premature newborns, severe gastrointestinal complications such as jejunal perforation and necrotizing enterocolitis have been described 86 . however, it is not known if these neonatal morbidities are related to prematurity or if maternal infection is a factor that increases their incidence. it is unclear why children develop a less serious disease than adults. recurrent viral respiratory infections typical of the pediatric age could be helpful to the immune system in promptly recognizing and defeating new viral pathogens. furthermore, the immaturity of the immune system could be protective because the inflammatory cascade that causes respiratory failure in adults is more difficult to activate. additionally, children generally have fewer comorbidities than adults. children recovered quickly from sars. li et al. assessed the radiological and clinical outcomes of fortyseven children with sars after 6 months from diagnosis. all children were asymptomatic while mild pulmonary abnormalities including ground-glass opacities and air trappings were found at ct in sixteen patients. 87 although clinical and laboratory findings of sars are aspecific in children, certain features can be useful to distinguish sars from other respiratory viral infections. children with sars have a lower incidence of rhinorrhea and productive cough and higher incidence of monocytopenia than children with influenza. 88 additionally, serum lactate dehydrogenase in the presence of a low neutrophil count and low serum creatine phosphokinase could be suggestive of sars infection. 89 sars infections in children appear to be a relatively mild and aspecific disease, and the diagnosis should be accompanied by laboratory assessment. although infants and teenagers are more likely to have a worse clinical course, usually, all pediatric patients recover entirely without significant long-term sequelae. the middle east respiratory syndrome (mers) is a viral respiratory infection caused by the mers-coronavirus (mers-cov). the first identified case occurred in 2012 in saudi arabia. 11, 90 subsequently, a total of 2494 confirmed cases of mers, including 858 associated deaths with a case-fatality rate of 34% were reported globally; the majority of these cases were reported from arabian peninsula, and in the middle east. 91 currently, mers is an extremely rare disease: in the last year mers was signaled only in saudi arabia. 92 mers-cov is a zoonotic virus: dromedary camels are the primary reservoir hosts. humans are infected through contact with infected dromedary camels, animal products, or humans, especially among close contact between family members and health care workers. mers-cov infection cases were classified by the who into suspected, probable, and confirmed ( table 3) . 93 usually, the mean incubation period ranges from 2 to 15 days. clinical severity of the disease varies from asymptomatic to fatal forms, and the impact of asymptomatic spread is unclear. the infection can cause severe pneumonia, which may progress to ards, respiratory failure, and death, particularly in older people, immunocompromised patients, and those with chronic diseases. common symptoms include fever, cough, and shortness of breath. gastrointestinal symptoms (including diarrhea, vomiting, abdominal pain), pericarditis, septic shock and disseminated intravascular coagulation have been reported. [94] [95] [96] [97] children appear to be less susceptible to mers-cov infection, and pediatric cases described in the literature are rare with a low proportion (0.1%-4%) of infected children. 98 age hospitalized with acute respiratory symptoms and/or fever. among these, none of 474 children tested resulted positive for mers-cov. 103 in pediatric age, few cases of mers cov infection have been described. most of the children were asymptomatic and positive during routine screening of mers-cov. al-tawfiq et al. reported a total of 31 pediatric mers-cov cases with a mean age of 10 years. overall, 42% were asymptomatic, while in symptomatic cases, fever and mild respiratory symptoms were common. 104 subsequently, alfaraj et al. reported a total of 7 pediatric mers-cov cases with a mean age of 8 years. in this case series, common symptoms were fever (57%), cough (14%), shortness of breath (14%), and gastrointestinal symptoms (28%). two (28.6%) patients had abnormal chest radiographic findings with bilateral infiltration, one (14.3%) required ventilatory support, and two (28.6%) required supplemental oxygen. 99 four with underlying conditions (cystic fibrosis, nephrotic syndrome, craniopharyngioma, and a right ventricular tumor) had a fatal outcome. these children developed a critical form of mers infection complicated by respiratory and multiorgan failure. frequently, clinical examination revealed bilateral rhonchi and crackles while chest x-ray showed diffuse bilateral infiltrates, ground-glass opacification and pleural effusion. [105] [106] [107] [108] [109] thrombocytopenia, leukopenia, increased creatinine and prolonged prothrombin time were the only laboratory findings reported in literature. 99, 105, 106 mers-cov in children is less frequent than adults and appears to be associated with low mortality unless the patients have underlying comorbidities. few cases of mers-cov have been reported during pregnancy. a pregnant woman, aged 39 years, had a stillbirth at approximately five months of gestation 110 and another woman gave birth to a healthy term baby, but she died after delivery. 107 in conclusion, although mers-cov represents a clinical concern for the adult population with a high fatality rate, it remains a sporadic disease in childhood. clinicians should learn to recognize and suspect mers-cov infection, as the symptoms and signs are nonspecific, based on epidemiological criteria to avoid the spread of the disease in patients at higher risk of worse clinical course. the outbreak of covid-19 infection (coronavirus disease 2019; previously 2019-ncov) began in wuhan, hubei, china, in december 2019, which then spread rapidly to other provinces of china and around the world. 111 on january 30, 2020, the who declared the outbreak of a public health emergency of international concern and, on march 11, 2020, a pandemic. 112 as of june 5, 2020, 188 other countries and regions, with more than 6.669.358 confirmed cases, are declared. among the confirmed cases, 2.904.828 are recovered, and 393.205 died. 113 recent genetic analysis suggests the covid-19 emerged from an animal source. the full genome sequences showed high homology between covid-19, bat coronavirus, and pangolin coronavirus, but further genetic study is required. moreover, according to current evidence, the principal route of transmission of covid-19 is from human to human. 114, 115 covid-19 spread between people through respiratory droplets and contact routes. droplet transmission occurs when there is close contact with a person with respiratory symptoms such as coughing or sneezing, who may spread potentially infectious droplets. transmission may also occur by direct contact with infected persons and indirect contact with infected surfaces or objects. covid-19 can persist on inanimate objects for days but can be efficiently inactivated by common disinfectants. airborne transmission may be possible when a high risk of aerosolization procedures are performed, such as endotracheal intubation and bronchoscopy. the virus is also detected in stool specimens, and consequently, the feco-oral transmission is also hypothesized. [116] [117] [118] [119] the high transmissibility of covid-19 may be explained by its demonstrated presence in the upper respiratory tract of asymptomatic or presymptomatic subjects with viral loads comparable to those detected from symptomatic patients. the real proportion of asymptomatic cases is unclear, ranging from 1% to 78% in different studies. transmission from asymptomatic patients infected with covid-19 most likely contributed to the rapid and extensive spread of pandemic but further studies are needed to more accurately estimate the proportion of genuinely asymptomatic cases and their risk of transmission. [120] [121] [122] [123] [124] [125] [126] covid-19 has been reported among all age groups. the median incubation period of covid-19 infection is 4-5 days with a range up to 24 days. 119, 127 covid-19 infection case is classified by the who into suspected, probable, and confirmed ( table 4) . 128 clinical severity of the infection varies, ranging from asymptomatic forms to critical diseases. common symptoms are fever, dry cough, malaise, lethargy, shortness of breath, sore throat, and myalgia. headache, conjunctivitis, productive cough, and diarrhea are also described. mild forms present as a common cold, and severe cases may worsen in pneumonia that may evolve to ards, shock, and multiple organ dysfunction. more severe clinical pictures are associated with stronger immune response and with the production of proinflammatory cytokines, including il-2, il-7, il-10, and tumor necrosis factor-α (tnf-α). adverse outcomes are common in elderly patients and those with underlying diseases. the need for intensive care admission is in 25-30% of patients. the fatality rate is estimated to range between 2 and 3%. [129] [130] [131] [132] [133] about 2% of covid-19 confirmed cases are children. [124] [125] [126] [127] [128] [129] [130] [131] [132] [133] [134] 135 generally, children appear to be less likely to develop a severe form of covid-19 infection, and commonly they have a mild clinical course with a good prognosis. few children may evolve into lower respiratory infections. probable reasons include having an immune system still immature, healthier respiratory tract, and less underlying conditions than adults. 136 most of them have an infected contact history with family members. moreover, children, especially those with asymptomatic or milder form, may represent significant spreaders. pediatric patients appear to be likely as adults to become infected but are less likely to develop symptoms. however, future studies are needed to understand the role of children in the transmission of the virus. [137] [138] [139] current researches show that the median age of infection in pediatric cases is 6-7 years. in symptomatic cases, symptoms are typical of acute respiratory infections and frequently included fever (59%) and cough (46%), which may be accompanied by nasal congestion, runny nose, conjunctivitis, pharyngitis, wheezing, myalgia, and expectoration. few children have an atypical presentation with gastrointestinal manifestations, including nausea, vomiting, and diarrhea. low oxygen saturation of less than 92%, dyspnea, cyanosis, and poor feeding, are less common than adults. among infants, symptoms such as irritability, reduced response, and poor feeding could be the main signs of infection. family clustering occurred for all infected infants. rarely infants require intensive care or mechanical ventilation or have any severe complications. common symptoms of pediatric age are summarized in figure 1 . the majority of children recovers 1-2 weeks after the onset of the disease. regarding biochemical results, leukopenia and lymphopenia are frequent in children. elevation of transaminases, myoglobin, muscle enzymes, and d-dimers might be seen in severe cases. [140] [141] [142] [143] [144] [145] [146] dong et al. reported that 94% of 2143 pediatric patients affected by covid-19 developed an asymptomatic, mild, or moderate form of infection. a severe disease characterized by dyspnea, central cyanosis, and oxygen saturation of less than 92% was reported in 5% of cases. a critical disease characterized by ards and multiple organs failure was reported in less than 1% of cases. 141 the prevalence of severe and critical disease appears higher in younger children, particularly in children aged <1-year-old and in children with underlying diseases. to date, death was an uncommon event reported in one 10month-old infant with intussusception and multiorgan failure and in one 14-year-old boy. 145, 147 other systemic symptoms appear to be related to the infection, but their link has not yet been demonstrated. since the outbreak of the pandemic, a large number of rashes, urticaria, and vasculitis affecting hands and feet of healthy children and adolescents have been reported as well as itching, burning, difficulty in joint movements and pain. 142 recently, the relationship between covid-19 infection and the development of cardiac diseases in children has been hypothesized. belhadjer et al. have reported a large number of febrile children resulted positive for covid-19 admitted in intensive care units for acute heart failure associated with a multisystem inflammatory state. in most of the children, clinical features appeared similar to those of kawasaki syndrome: lasting fever, cutaneous rash, lymphadenopathy, persistent activation of systemic inflammation and positive response to intravenous immunoglobulin. 148 similar clinical features have subsequently been reported in children with covid-19 positive serology. 149, 150 as in covid-19 infection, kawasaki syndrome is triggered by proinflammatory cascade activated primarily by innate immunity response. however, further studies are needed to establish the real pathogenetic relationship between emerging covid-19 and kawasaki-like syndromes. 151 dufort et al. 152 have recently reported the emergence of a multisystem inflammatory syndrome in children in new york state coincidental with widespread sars-cov-2 transmission, which can better clarify the relationship between kawasaki disease and covid-19. among 191 children admitted to the new york hospitals for multisystem inflammatory syndrome in children (mis-c), 95 patients had a laboratory-confirmed acute or recent severe acute respiratory syndrome coronavirus 2 [sars-cov-2] infection. this hyperinflammatory syndrome manifested with dermatologic, mucocutaneous, and gastrointestinal features associated with cardiac dysfunction. of these 95 patients, a total of 36 patients (37%) received a diagnosis of kawasaki's disease or atypical (or incomplete) kawasaki's disease; 7 of the 9 patients with coronary-artery aneurysms also received a diagnosis of kawasaki's disease. 152 covid-19 infection may also trigger the onset of other immune-mediated diseases such as immune thrombocytopenia, [153] [154] [155] [156] evans syndrome, 157 and autoimmune hemolytic anemia. 158 among radiological findings, ground-glass opacity, mono or bilateral infiltrates, mesh shadows, and tiny nodules are frequently detected. in severe cases, radiological alterations are diffused, presenting as a "white lung." however, radiologic evidence of pneumonia might be absent in 15-20% of children. 139, 140, [159] [160] [161] [162] [163] in selected cases, lung ultrasound might be useful in the managing and follow-up of covid-19 infection. this radiological technique can precociously identify abnormalities including small pleural effusion and subpleural consolidation and appear more available then x-ray and ct. [164] [165] clinical examination appears mostly negative for pulmonary signs, and in rare cases, rales and thoracic retractions have been reported. 161 whether pregnant women and children born to affected mothers are more likely to have a worse outcome is currently unclear. maternal-infant vertical transmission has not been documented. amniotic fluid, cord blood, neonatal throat swab, and breastmilk samples from newborns delivered by infected women were tested for covid-19, and all samples tested negative. 166 data on the maternal and perinatal outcomes of pregnant women infected with covid-19 is limited. most pregnant women with covid-19 present with fever and coughing. severe and critical maternal symptomatology have also been reported, but no women died. the most common adverse pregnancy outcome is preterm birth, occurring in 41% of cases while the rate of perinatal death is 7%, including one case of stillbirth and one neonatal death. there is no data on miscarriage for covid-19 occurring during the first trimester. in more than a third of cases, fetal distress and frequent admission neonatal intensive care units have been reported. 166, 167 rarely, cases of covid-19 positivity in newborns have been reported. common symptoms are fever, cough, lethargy, and vomiting milk. mottled skin and moderate respiratory distress presented with tachycardia, tachypnoea, subcostal retractions, and low oxygen saturation are also described in newborn babies. [169] [170] [171] [172] [173] although it can be severe in some cases, compared with sars-cov and mers-cov, covid-19 causes less severe disease in children. a recent meta-analysis shows that children infected with covid-19 have less fever than that other epidemic hcovs. 174 despite the rapid worldwide spread of covid-19 infection, additional data are needed to define the severity of the disease in children. the severity of the symptoms and the mortality rate will be better assessed in the future. differential diagnosis with common viral respiratory infections of childhood, such as influenza virus, adenovirus, respiratory syncytial virus, and metapneumovirus, should be considered. in the diagnosis of suspected cases, epidemiological and clinical criteria must be assessed. 73, 93, 138 rt-pcr represents the gold standard to confirm the diagnosis of hcovs infections performed on samples of respiratory secretions. [175] [176] [177] [178] [179] [180] [181] the viral load is higher in lower respiratory tract secretion samples than in upper respiratory tract samples. therefore, suspected cases resulted in firstly negative could be re-tested with a second swab, better if with a low respiratory sampling is performed as proved for sars and mers infection. 182, 183 currently, few data have been published about the sensitivity and specificity of rt-pcr nasopharyngeal swabs for covid-19. in vitro analyses suggest that the rt-pcr test is highly specific and sensitive. 184 in vivo, sensitivity is estimated to be higher than 70% but seems to be lower for "mild" cases while specificity is close to 100%. 185, 186 accuracy of rt-pcr swabs in clinical practice differs depending on the site and quality of the sample. taking swabs from children may be more difficult given the intrusive nature of the procedure and further reduce the specificity and sensitivity of the test. rt-pcr of bronchoalveolar lavage fluid appears the most accurate technique of virologic confirmation, but it may not always be easily collected in all patients, especially in pediatric age. although a negative test cannot currently rule out the disease, further studies are needed to define the exact specificity and sensitivity of rt-pcr nasopharyngeal swabs. 187, 188 moreover, rt-pcr appears to be useful in virus detection on stool samples. 116 to date, serology is not considered a diagnostic method. although most patients with covid-19 appear positive for immunoglobulin-g (igg) within 19 days while igm reaches a peak 20-22 days after symptom onset, the serological response is not useful for early individuation of positive patients. 189 additionally, numerous cross-reactions occur between covid-19 and common hcovs 190, and protective immunity against covid-19 is not proved. despite its potential role in supporting rt-pcr in the diagnosis of covid-19, the clinical and immunological meaning of serology is still unclear. 191 the spread of the infection can be prevented if children and family members were educated about proper hygienic practices and infection control measures, including regular hand washing, cover the mouth with napkin or towel when coughing or sneezing, avoid crowded places and contact with sick people. children with hcovs should receive early supportive therapy and continuous monitoring. additional oxygen, caloric, and hydro electrolytic support should be performed if necessary. frequent checks of oxygen saturation and hematological, urinary, and biochemical parameters, including liver, kidney, myocardial enzymes, and coagulation parameters should be analyzed. finally, blood gas analysis and radiological diagnostics of the chest should be done when necessary. this strategy could be useful in the prevention of ards, multiorgan failure, and other nosocomial infections possibly treated, if bacterial, with appropriate antibiotics. in critical cases, mechanical ventilation with endotracheal intubation and other more invasive interventions, such as blood purification and extracorporeal membrane oxygenation (emco), should be adopted. additionally, the use of antiviral drugs in children with severe hcovs infections may help to reduce viral load and the duration of symptoms. however, their safety and real effectiveness have not yet been proven. interferon alfa and beta, corticosteroids, lopinavir/ritonavir, and ribavirin, were used in the treatment of sars-cov and mers-cov in adults and children. 75, 76, 78, 192 however, ribavirin can cause hemolytic anemia and liver dysfunction, as well as corticosteroids, increase the risk of iatrogenic immune immunosuppression. 193 to date, there is no evidence regarding the management and treatment of covid-19 infection in children. in addition to supportive therapy, the use of nebulized interferonalpha-2b and oral lopinavir/ritonavir together with corticosteroids for complications and hydroxychloroquine or intravenous immunoglobulin for severe cases have been suggested. 145, 194, recently, a position paper of the italian society of pediatric infectious disease on the treatment of children with covid-19 infection has been published. 195 in asymptomatic or mild cases, only antipyretic therapy is recommended. in severe or critical cases, the use of hydroxychloroquine ± azithromycin or lopinavir/ritonavir must be considered. immunomodulating therapy with methylprednisolone or tocilizumab or anakinra must be considered in case of the simultaneous presence of ards or progressive deterioration of respiratory function, the elevation of proinflammatory biomarkers and an interval of at least seven days from symptoms onset. supportive therapy should include antipyretic therapy, inhalation therapy with topical steroids and/or bronchodilators and venous thromboembolism prophylaxis therapy. [196] [197] [198] [199] [200] [201] [202] [203] discharge from the hospital is recommended when the patient is without fever for almost three days, respiratory symptoms have improved, and rt-pcr samples are negative. 195 conclusions. most cases of hcovs infection in children have clinically mild symptoms and a relatively short time to resolution. children seem to have a better prognosis compared to adults, and death is a sporadic event. however, some children, such as infants, adolescents, or those with underlying diseases may be more at-risk categories and require greater caution from clinicians. learning to recognize pediatric clinical presentations often indefinite or similar to other typical infections of this age, allows clinicians to perform a correct and early diagnosis and prevent the spread of infections in the general population. furthermore, the psychological and social impact of the pandemic outbreak should be considered, especially in the pediatric age. moreover, we think it is necessary to implement innovative clinical tools, such as narrative medicine, to recognize the burden of disease in children and caregivers. [201] [202] [203] [204] references: update on human rhinovirus and coronavirus infections hosts and sources of endemic human coronaviruses mechanisms of coronavirus cell entry mediated by the viral spike protein discovery of seven novel mammalian and avian coronaviruses in the genus deltacoronavirus supports bat coronaviruses as the gene source of alphacoronavirus and betacoronavirus and avian coronaviruses as the gene source of gammacoronavirus and deltacoronavirus discovery of a novel coronavirus, china rattus coronavirus hku24, from norway rats supports the murine origin of betacoronavirus 1 and has implications for the ancestor of betacoronavirus lineage a a new virus isolated from the human respiratory tract recovery in tracheal organ cultures of novel viruses from patients with respiratory disease identification of a new human coronavirus characterization and complete genome sequence of a novel coronavirus, coronavirus hku1, from patients with pneumonia identification of a novel coronavirus in patients with severe acute respiratory syndrome isolation of a novel coronavirus from a man with pneumonia in saudi arabia coronaviruses: a paradigm of new emerging zoonotic diseases incubation periods of acute respiratory viral infections: a systematic review epidemiological and clinical features of human coronavirus infections among different subsets of patients. influenza other respir viruses epidemiology characteristics of human coronaviruses in patients with respiratory infection symptoms and phylogenetic analysis of hcov-oc43 during 2010-2015 in guangzhou human coronavirus infections in israel: epidemiology, clinical symptoms and summer seasonality of hcov-hku1. viruses the role of infections and coinfections with newly identified and emerging respiratory viruses in children species-specific clinical characteristics of human coronavirus infection among otherwise healthy adolescents and adults. influenza other respir viruses viral etiology of acute respiratory tract infections in children presenting to hospital: role of polymerase chain reaction and demonstration of multiple infections diagnostic value of real-time polymerase chain reaction to detect viruses in young children admitted to the paediatric intensive care unit with lower respiratory tract infection comparison of automated microarray detection with real-time pcr assays for detection of respiratory viruses in specimens obtained from children comparison of viral and epidemiological profiles of hospitalized children with severe acute respiratory infection in beijing and shanghai, china two-year prospective study of single infections and coinfections by respiratory syncytial virus and viruses identified recently in infants with acute respiratory disease human coronavirus nl63 infection and other coronavirus infections in children hospitalized with acute respiratory disease in hong kong, china phylogenetic analysis of human coronavirus nl63 circulating in italy human coronavirus infections in rural thailand: a comprehensive study using real-time reverse-transcription polymerase chain reaction assays epidemiology of human coronavirus nl63 infection among hospitalized patients with pneumonia in taiwan an outbreak of coronavirus oc43 respiratory infection in epidemiology and clinical presentations of the four human coronaviruses 229e, hku1, nl63, and oc43 detected over 3 years using a novel multiplex real-time pcr method human coronaviruses and other respiratory infections in young adults on a university campus: prevalence, symptoms, and shedding. influenza other respir viruses respiratory viruses associated with severe pneumonia in children under 2 years old in a rural community in pakistan croup is associated with the novel coronavirus nl63. version 2 croup is associated with the novel coronavirus nl63. version 2 detection of human coronavirus nl63 in young children with bronchiolitis coronavirus hku1 in an italian pre-term infant with bronchiolitis erdman dd; new vaccine surveillance network. human coronavirus in young children hospitalized for acute respiratory illness and asymptomatic controls viral respiratory infections in hospitalized and community control children in alaska role of respiratory viruses in acute upper and lower respiratory tract illness in the first year of life: a birth cohort study aetiological role of common respiratory viruses in acute lower respiratory infections in children under five years: a systematic review and meta-analysis genetic variability of human coronavirus oc43-, 229e-, and nl63-like strains and their association with lower respiratory tract infections of hospitalized infants and immunocompromised patients coronavirus 229e-related pneumonia in immunocompromised patients acute respiratory viral infections in pediatric cancer patients undergoing chemotherapy a multicenter consortium to define the epidemiology and outcomes of inpatient respiratory viral infections in pediatric hematopoietic stem cell transplant recipients acute otitis media and respiratory viruses etiology of acute otitis media and characterization of pneumococcal isolates after introduction of 13-valent pneumococcal conjugate vaccine in japanese children genetic characteristics and antibiotic resistance of haemophilus influenzae isolates from pediatric patients with acute otitis media after introduction of 13-valent pneumococcal conjugate vaccine in japan polymerase chain reaction-based detection of rhinovirus, respiratory syncytial virus, and coronavirus in otitis media with effusion regional, age and respiratorysecretion-specific prevalence of respiratory viruses associated with asthma exacerbation: a literature review coronavirusrelated nosocomial viral respiratory infections in a neonatal and paediatric intensive care unit: a prospective study neonatal nosocomial respiratory infection with coronavirus: a prospective study in a neonatal intensive care unit effect of olfactory bulb ablation on spread of a neurotropic coronavirus into the mouse brain susceptibility of laboratory mice to intranasal and contact infection with coronaviruses of other species detection of sars coronavirus rna in the cerebrospinal fluid of a patient with severe acute respiratory syndrome possible central nervous system infection by sars coronavirus detection of coronavirus in the central nervous system of a child with acute disseminated encephalomyelitis two coronaviruses isolated from central nervous system tissue of two multiple sclerosis patients neuroinvasion by human respiratory coronaviruses coronavirus infections in the central nervous system and respiratory tract show distinct features in hospitalized children respiratory and enteric virus detection in children human coronaviruses are uncommon in patients with gastrointestinal illness detection of the new human coronavirus hku1: a report of 6 cases association of coronavirus infection with neonatal necrotizing enterocolitis human coronavirus nl63, france impact of human coronavirus infections in otherwise healthy children who attended an emergency department new vaccine surveillance network. coronavirus infection and hospitalizations for acute respiratory illness in young children the role of human coronaviruses in children hospitalized for acute bronchiolitis, acute gastroenteritis, and febrile seizures: a 2-year prospective study detection of human coronaviruses in children with acute gastroenteritis comparative full-length genome sequence analysis of 14 sars coronavirus isolates and common mutations associated with putative origins of infection summary of probable sars cases with onset of illness from coronavirus as a possible cause of severe acute respiratory syndrome a major outbreak of severe acute respiratory syndrome in hong kong severe acute respiratory syndrome [internet]. world health organization (who) epidemiology of severe acute respiratory syndrome (sars): adults and children clinical presentations and outcome of severe acute respiratory syndrome in children other members of the hospital for sick children sars investigation team. children hospitalized with severe acute respiratory syndromerelated illness in toronto risk-stratified seroprevalence of sars coronavirus in children residing in a district with point-source outbreak compared to a low-risk area severe acute respiratory syndrome in children: experience in a regional hospital in hong kong severe acute respiratory syndrome among children severe acute respiratory syndrome-associated coronavirus infection in toronto children: a second look inflammatory cytokine profile in children with severe acute respiratory syndrome severe acute respiratory syndrome (sars): chest radiographic features in children serial analysis of the plasma concentration of sars coronavirus rna in pediatric patients with severe acute respiratory syndrome clinical progression and viral load in a community outbreak of coronavirusassociated sars pneumonia: a prospective study pregnancy and perinatal outcomes of women with severe acute respiratory syndrome infants born to mothers with severe acute respiratory syndrome long-term sequelae of sars in children childhood severe acute respiratory syndrome in taiwan and how to differentiate it from childhood influenza infection a case-control study of sars versus community acquired pneumonia middle east respiratory syndrome middle east respiratory syndrome coronavirus (mers-cov) available at: www.emro.who.int/pandemic-epidemic-diseases/mers-cov/merssituation-update middle east respiratory syndrome coronavirus (mers-cov). case definition ksa mers-cov investigation team. hospital outbreak of middle east respiratory syndrome coronavirus epidemiological, demographic, and clinical characteristics of 47 cases of middle east respiratory syndrome coronavirus disease from saudi arabia: a descriptive study middle east respiratory syndrome clinical features and viral diagnosis of two cases of infection with middle east respiratory syndrome coronavirus: a report of nosocomial transmission acute viral respiratory infections among children in mers-endemic riyadh, saudi arabia, 2012-2013 middle east respiratory syndrome coronavirus in pediatrics: a report of seven cases from saudi arabia el hadi mohamed ra. outbreak of middle east respiratory syndrome coronavirus in saudi arabia: a retrospective study correction: case characteristics among middle east respiratory syndrome coronavirus outbreak and non-outbreak cases in saudi arabia from 2012 to 2015 surveillance and testing for middle east respiratory syndrome coronavirus, saudi arabia middle east respiratory syndrome coronavirus not detected in children hospitalized with acute respiratory illness in middle east respiratory syndrome coronavirus disease is rare in children: an update from saudi arabia middle east respiratory syndrome coronavirus in children middle east respiratory syndrome coronavirus disease in children middle east respiratory syndrome coronavirus during pregnancy acute middle east respiratory syndrome coronavirus: temporal lung changes observed on the chest radiographs of 55 patients ct correlation with outcomes in 15 patients with acute middle east respiratory syndrome coronavirus stillbirth during infection with middle east respiratory syndrome coronavirus china novel coronavirus investigating and research team. a novel coronavirus from patients with pneumonia in china second-meeting-of-the-international-health-regulations-(2005)-emergencycommittee-regarding-the-outbreak-of-novel-coronavirus-(2019-ncov) coronavirus covid-19 global cases by the center for systems science and engineering covid-19 infection: origin, transmission, and characteristics of human coronaviruses probable pangolin origin of sars-cov-2 associated with the covid-19 outbreak characteristics of pediatric sars-cov-2 infection and potential evidence for persistent fecal viral shedding community transmission of severe acute respiratory syndrome coronavirus 2 modes of transmission of virus causing covid-19: implications for ipc precaution recommendations persistence of coronaviruses on inanimate surfaces and their inactivation with biocidal agents temporal profiles of viral load in posterior oropharyngeal saliva samples and serum antibody responses during infection by sars-cov-2: an observational cohort study public health-seattle and king county and cdc covid-19 investigation team. presymptomatic sars-cov-2 infections and transmission in a skilled nursing facility asymptomatic carrier state, acute respiratory disease, and pneumonia due to severe acute respiratory syndrome coronavirus 2 (sars-cov-2): facts and myths sars-cov-2 viral load in upper respiratory specimens of infected patients characteristics of and important lessons from the coronavirus disease 2019 (covid-19) outbreak in china: summary of a report of 72 314 cases from the chinese center for disease control and prevention estimation of the asymptomatic ratio of novel coronavirus infections (covid-19) the covid-19 task force of the department of infectious diseases and the it service istituto superiore di sanità. integrated surveillance of covid-19 in italy early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia global surveillance for covid-19 caused by human infection with covid-19 virus. case definition zhong ns; china medical treatment expert group for covid-19. clinical characteristics of coronavirus disease 2019 in china epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a descriptive study clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus-infected pneumonia in wuhan, china. jama. 2020:e201585 clinical features of patients infected with 2019 novel coronavirus in wuhan clinical findings in a group of patients infected with the 2019 novel coronavirus (sars-cov-2) outside of wuhan, china: retrospective case series mmwr morb mortal wkly rep are children less susceptible to covid-19? epidemiology and clinical features of coronavirus disease 2019 in children infection in children epidemiology and transmission of covid-19 in shenzhen china: analysis of 391 cases and 1,286 of their close contacts sars-cov-2 infection in children: transmission dynamics and clinical characteristics epidemiology of covid-19 among children in china. pediatrics. 2020:e20200702 a new vasculitis at the time of covid-19 a case series of children with 2019 novel coronavirus infection: clinical and epidemiological features diagnosis and treatment recommendations for pediatric respiratory infection caused by the 2019 novel coronavirus novel coronavirus infection in hospitalized infants under 1 year of age in china corona virus disease 2019, a growing threat to children? a 55-day-old female infant infected with covid 19: presenting with pneumonia, liver injury, and heart damage acute heart failure in multisystem inflammatory syndrome in children (mis-c) in the context of global sars-cov-2 pandemic multisystem inflammatory syndrome in children during the covid-19 pandemic: a case series features of covid-19 post-infectious cytokine release syndrome in children presenting to the emergency department sars-cov-2, which induces covid-19, causes kawasaki-like disease in children: role of proinflammatory and anti-inflammatory cytokines prevention multisystem inflammatory syndrome in children investigation team. multisystem inflammatory syndrome in children in covid-19-associated immune thrombocytopenia immune thrombocytopenia (itp) in a sars-cov-2 positive pediatric patient covid-19 and children with immune thrombocytopenia: emerging issues evans syndrome in a patient with covid-19 covid-19 infection associated with autoimmune hemolytic anemia chest computed tomography in children with covid-19 respiratory infection clinical and ct imaging features of the covid-19 pneumonia: focus on pregnant women and children clinical and ct features in pediatric patients with covid-19 infection: different points from adults ct features of novel coronavirus pneumonia (covid-19) in children radiographic and clinical features of children with 2019 novel coronavirus (covid-19) pneumonia lung ultrasound in the monitoring of covid-19 infection lung ultrasound can influence the clinical treatment of pregnant women with covid-19 clinical characteristics and intrauterine vertical transmission potential of covid-19 infection in nine pregnant women: a retrospective review of medical records maternal and perinatal outcomes with covid-19: a systematic review of 108 pregnancies outcome of coronavirus spectrum infections (sars, mers, covid 1 -19) during pregnancy: a systematic review and meta-analysis perinatal transmission of covid-19 associated sars-cov-2: should we worry? clin infect dis novel coronavirus in a 15-dayold neonate with clinical signs of sepsis, a case report a case report of neonatal covid-19 infection in china clinical characteristics and diagnostic challenges of pediatric covid-19: a systematic review and metaanalysis direct diagnosis of human respiratory coronaviruses 229e and oc43 by the polymerase chain reaction viral shedding patterns of coronavirus in patients with probable severe acute respiratory syndrome genomic sequencing of the severe acute respiratory syndrome-coronavirus genomic sequencing of a sars coronavirus isolate that predated the metropole hotel case www.mjhid.org evaluation of a real-time reverse transcription-pcr (rt-pcr) assay for detection of middle east respiratory syndrome coronavirus (mers-cov) in clinical samples from an outbreak in south korea in 2015 analytical and clinical validation of six commercial middle east respiratory syndrome coronavirus rna detection kits based on real-time reverse-transcription pcr mers-cov diagnosis: an update respiratory tract samples, viral load, and genome fraction yield in patients with middle east respiratory syndrome viral loads in clinical specimens and sars manifestations detection of 2019 novel coronavirus (2019-ncov) by realtime rt-pcr application and optimization of rt-pcr in diagnosis of sars-cov-2 infection false-negative results of initial rt-pcr assays for covid-19: a systematic review the appropriate use of testing for covid-19 interpreting a covid-19 test result antibody responses to sars-cov-2 in patients with covid-19 antigenic cross-reactivity between severe acute respiratory syndrome-associated coronavirus and human coronaviruses 229e and oc43 sars-cov-2 antibody testing -questions to be asked ribavirin and interferon alfa-2a for severe middle east respiratory syndrome coronavirus infection: a retrospective cohort study sars: systematic review of treatment effects. version 2 national health commission of people's republic of china. diagnosis and treatment of pneumonia caused by novel coronavirus china national clinical research center for respiratory diseases chinese medical doctor association committee on respirology pediatrics; china medicine education association committee on pediatrics; chinese research hospital association committee on pediatrics; chinese non-government medical institutions association committee on pediatrics; china association of traditional chinese medicine committee on children's safety medication; global pediatric pulmonology alliance. diagnosis, treatment, and prevention of 2019 novel coronavirus infection in children: experts' consensus statement treatment of children with covid-19: position paper of the italian society of pediatric infectious disease covid-19: ibuprofen should not be used for managing symptoms, say doctors and scientists ema gives advice on the use of nonsteroidal anti-inflammatories for covid-19 recommendations for inhaled asthma controller medications anticoagulant treatment is associated with decreased mortality in severe coronavirus disease 2019 patients with coagulopathy discovering drugs to treat coronavirus disease 2019 (covid-19) delle fave a. perceived well-being and mental health in haemophilia a narrative approach to describe qol in children with chronic itp. front pediatr itp-qol questionnaire for children with immune thrombocytopenia: italian version validation's key: cord-023528-z9rc0ubj authors: wilkins, pamela a. title: disorders of foals date: 2009-05-18 journal: equine internal medicine doi: 10.1016/b0-72-169777-1/50021-4 sha: doc_id: 23528 cord_uid: z9rc0ubj nan before the 1980s, intensive management of the compromised neonate was unusual and little was known regarding many of the problems of this special patient population. although some specific conditions had been described by astute clinician-researchers, most notably the "dummy" foal syndrome 1 and respiratory distress syndrome caused by primary surfactant deficiency, 2 little information regarding the diagnosis and management of conditions of the foal during the neonatal period was available, although at least one active group was investigating fetal and neonatal physiology of the horse in great britain. [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] when treatment of compromised foals was undertaken, the approach most commonly resembled treating them as small adults with little understanding of the different physiology of the equine neonate. the advent of improved management of reproductive efficiency of mares led naturally to increased interest in preservation of the conceptus to parturition and the foal thereafter. interested clinicians, taking their lessons from the field of human perinatology/neonatology and sometimes working hand-in-hand with their counterparts in the human field, pioneered investigations into these small patients and created the fields of equine perinatology and equine neonatal intensive care. [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] because of the foresight and energy of these early investigators, the field of veterinary perinatology/neonatology exploded in the 1980s, leading to the creation of equine neonatal intensive care units throughout the united sates and the world. from these units information about the normal and abnormal physiology of foals, the medical conditions affecting them, and methods for treatment and management of these problems has been developed through observational, retrospective, and prospective studies. this veritable explosion of information over the last 20 years has improved greatly the ability of all practitioners to provide appropriate care for these patients, whether in the field or at an equine neonatal intensive care unit. the ability not only to save the lives of these patients but also to treat them in such a manner as to allow them to fulfill their purposes, whether as pleasure animals or racing athletes, has improved almost exponentially from those early days. [20] [21] [22] [23] this chapter aims to provide the clinician with some of the most current information regarding the management of these patients, recognizing that much still remains unknown and that advances will continue to be made in this dynamic field. the reader is cautioned that much of this chapter is flavored by the experiences of the author and that variation in approach and treatment of specific problems exists between neonatal intensive care units (nicus) and between clinicians in the same nicu and that each year results in change. in some cases, information that is presented has been gleaned from human nicu studies, essentially using the critically ill infant as the experimental model. many of the problems of the newborn foal have their genesis in utero. identification of high-risk pregnancies is an important component of prenatal care of the foal, and some of the most commonly encountered problems of the dam resulting in abnormal foals include previous or concurrent disease, poor reproductive history, poor perineal or pelvic conformation, poor general health, poor nutritional condition, prolonged transport, history of previous abnormal foals, placental abnormalities, and twins. 24 some of the more common causes of abortion can result in the birth of severely compromised foals of variable gestation lengths (box 19-1). these include pa m e l a a . wi l k i n s infectious causes such as equine herpesvirus (ehv) types 1 (most commonly) and 4 (rarely), equine infectious anemia, equine arteritis virus, bacterial and fungal placentitis, leptospirosis, equine ehrlichiosis, and gram-negative septicemia/endotoxemia. [25] [26] [27] noninfectious causes of abortion include twinning and noninfectious placental abnormalities such as extensive endometrial fibrosis, body pregnancy, and abnormal length (long or short) of the umbilical cord. 24, 28 to the equine neonatologist opportunities for intervention may appear limited, and in the case of many of the aforementioned causes of fetal loss, this is true. however, one can do much in an attempt to preserve the pregnancy and in effect treat the fetus. when one is faced with a threatened pregnancy, one has various ways of evaluating the fetus and its environment and may use many potential therapies. once one identifies a pregnancy as high risk, one should evaluate the fetus for viability. evaluation should include as thorough an evaluation as possible of the reproductive tract, placenta, and fetal fluids. prepartum disorders in the mare usually are readily recognizable, but disorders of the fetus and placenta can be more subtle and difficult to determine. the first step is to take a thorough history of the mare. of particular interest is any history of previous abnormal foals, but the history taking should include questions regarding transportation; establishment of an accurate breeding date (sometimes more difficult than one would suspect); any pertinent medical history including any diagnostic testing performed for this pregnancy such as culture, endometrial biopsy, and cytologic results; and any rectal and ultrasound examination results. additionally, one should obtain information regarding possible ingestion of endophyte-infected fescue or exposure to potential infectious causes of abortion. 29, 30 a complete vaccination and deworming history is requisite, as is a complete history of any medications and supplements administered during pregnancy. after obtaining a history, one examines the mare per rectum. this examination should include palpation of the cervix, uterus, fetus, and all palpable abdominal contents. one should note any abnormalities. the cervix should be tight throughout gestation; the late gestation uterus will be large and distended with fluid and usually pulled craniad in the abdomen. palpation of the fetus frequently results in some fetal movement; however, one should interpret lack of movement with caution, for some normal fetuses do not respond. ultrasonographic evaluation of the uterus and conceptus per rectum can provide valuable information, particularly regarding placental thickness if placentitis is a concern. one may evaluate fetal fluids and estimate fetal size from the size of the eye later in gestation. 31 in the author's hospital the practitioners choose not to perform vaginal examinations or speculum examinations because of an association between these examinations and the subsequent development of placentitis. unless placentitis is recognized with ultrasonograhic evaluation per rectum and culture is desirable, these types of examinations are generally not necessary. following examination per rectum, one performs transabdominal ultrasonographic evaluation of the uterus and conceptus. 28 one can generate a biophysical profile of the fetus from this examination in the late-term fetus and readily determine viability. 32, 33 one also readily can determine the presence or absence of twins in the late pregnant mare in this manner. one performs the sonogram through the acoustic window from the udder to the xiphoid ventrally and laterally to the skinfolds of the flank. imaging of the fetus usually requires a lowfrequency (3.5-mhz) probe, whereas examination of the placenta and endometrium requires a higher-frequency (7.5-mhz) probe. a complete description of this examination is beyond the scope of this chapter, but the reader will find several complete descriptions of the technique and normal values for specific gestation lengths within the relevant veterinary literature. 33 the utility of this examination lies in its repeatability and low risk to the dam and fetus. sequential examinations over time allow the clinician to follow the pregnancy and to identify changes as they occur. a companion to transabdominal ultrasongraphy is evaluation of the fetal electrocardiogram (ecg). one can measure fetal ecgs continuously using telemetry or can obtain them using more conventional techniques several times throughout the day. 24, 28, 34 one places electrodes on the skin of the mare in locations aimed at maximizing the magnitude of the fetal ecg. because the fetus frequently changes position, multiple sites may be needed in any 24-hour period. to begin, one places an electrode dorsally in the area of the sacral prominence with two electrodes placed bilaterally in a transverse plane in the region of the flank. the fetal ecg maximal amplitude is low, usually 0.05 to 0.1 mv, and can be lost in artifact or background noise, so one commonly must move electrodes to new positions to maximize the appearance of the fetal ecg. the normal fetal heart rate during the last months of gestation ranges from 65 to 115 beats/min, a fairly wide distribution. the range of heart rate of an individual fetus can be narrow, however. bradycardia in the fetus is an adaptation to in utero stress, most commonly thought to be hypoxia. by slowing the heart rate, the fetus prolongs exposure of fetal blood to maternal blood, increasing the time for equilibration of dissolved gas across the placenta and improving the oxygen content of the fetal blood. the fetus also has altered the distribution of its cardiac output in response to hypoxia, centralizing blood distribution. 35, 36 tachycardia in the fetus can be associated with fetal movement, and brief periods of tachycardia should occur in the fetus in any 24-hour period. persistent tachycardia is a sign of fetal distress and represents more severe fetal compromise than bradycardia. the author has recognized dysrhythmias in the challenged fetus, most commonly as atrial fibrillation but also apparent runs of ventricular tachycardia. the ability to monitor the fetus in a high-risk pregnancy inevitably has led to questions of whether, how, and when to intervene. most equine neonatologists would agree that removal of the fetus from the uterus before its attainment of readiness for birth is not desirable. one of the difficulties in determining fetal preparedness for birth is that prediction of parturition is difficult in these mares. many of the parameters used in normal mares are unreliable in the high-risk pregnant mare. one must have an accurate history of any previous gestation length in terms of days for the specific mare in question to allow a more accurate estimate of her usual gestational length. evaluation of the usual mammary gland parameters, including size, the presence of "wax," and alteration of electrolyte concentrations, is not generally predictive in the high-risk mare, for in the author's experience many of these mares have changes predictive of parturition for weeks before actual parturition. 37, 38 this circumstance may be related to the observation that many high-risk pregnant mares, particularly those with placentitis, are presented for a primary complaint of early onset lactation. although pulmonary system maturity in human beings can be assessed with some degree of accuracy using measurement of lecithin/sphingomyelin ratios, this measurement-along with sphingomyelin, cortisol, and creatinine concentrations in the amnionic fluid-has proved to be of no benefit in the horse. [39] [40] [41] amniocentesis carries a high risk of abortion in the horse, even with ultrasound guidance, and is not a clinically useful technique at this time. 41 currently, no clear-cut guidelines are available as to when to intervene, but the presence of persistent fetal tachycardia or prolonged absence of fetal movements, including breathing movements, as determined by transabdominal ultrasound evaluation, should initiate discussion regarding the appropriateness of induction of parturition or elective cesarean section. the goal of induction or cesarean section is to remove a pregnancy that is threatening the survival of the dam with no thought to fetal survival or to remove the fetus from a threatening environment to improve its likelihood for survival. preterm induction is ill advised if fetal survival is desirable because of the limited ability to treat severely immature neonates. timing of intervention in these circumstances remains an art, not a science. the approach to management of the high-risk pregnancy is dictated to some degree by the exact cause for concern, but for many mares therapy is similar. many high-risk mares have placentitis, primarily caused by ascending bacterial or fungal infections originating in the region of the cervix. these infections can cause in utero sepsis or compromise the fetus by local elucidation of inflammatory mediators or altered placental function. 42, 43 premature udder development and vaginal discharge are common clinical signs. treatment consists of administration of broad-spectrum antimicrobial agents and nonsteroidal antiinflammatory drugs (table 19 -1). in the author's clinic, trimethoprim-sulfonamide drugs have been the antimicrobial of choice based on unpublished studies performed at the facility demonstrating increased concentration of these agents in the fetal fluids compared with penicillin and gentamicin. however, if culture and sensitivity results are available, one should institute directed therapy. nonsteroidal antiinflammatory agents such as flunixin meglumine are useful to combat alterations in prostaglandin balance that may be associated with infection and inflammation. although the efficacy of these agents is best when administered before the development of clinical signs, to date no detrimental effects have been reported in the fetus or dam when chronically used at low doses in well-hydrated patients. tocolytic agents and agents that promote uterine quiescence have been used and include altrenogest, isoxuprine, and clenbuterol. [44] [45] [46] [47] [48] altrenogest usually is administered, although its need in late gestation has been challenged. the efficacy of isoxuprine as a tocolytic in the horse is unproven, and bioavailability of orally administered isoxuprine appears to be highly variable. 48 the long-term use of clenbuterol is inadvisable because of receptor population changes associated with chronic use and its unknown effects on the fetus at this time. clenbuterol may be indicated during management of dystocia in preparation for assisted delivery or cesarean section. 46 the intravenous form of clenbuterol is not currently available in the united states. one can use three additional strategies in managing high-risk pregnancy patients. in mares with evidence of placental dysfunction, with or without signs of fetal distress, the author provides intranasal oxygen supplementation in the hope of improving oxygen delivery to the fetus. intranasal oxygen insufflation of 10 to 15 l/min to the mare significantly increases pao 2 and percent oxygen saturation of hemoglobin. 49 because of the placental vessel arrangement of the horse, improvement of these two arterial blood gas parameters should result in improved oxygen delivery to the fetus. blood gas transport is largely independent of diffusion distance in the equine placenta, particularly in late gestation, and depends more on blood flow. information from other species cannot be extrapolated to the equine placenta because of its diffuse epitheliochorial nature and the arrangement of the maternal and fetal blood vessels within the microcotyledons. 50,51 umbilical venous po 2 is 50 to 54 mm hg in the horse fetus, compared with 30 to 34 mm hg in the sheep, whereas the maternal uterine vein to umbilical vein po 2 difference is near 0. also unlike the sheep, the umbilical venous po 2 values decrease 5 to 10 mm hg in response to maternal hypoxemia and increase in response to maternal hyperoxia. [52] [53] [54] vitamin e (tocopherol) is administered orally to some high-risk mares as an antioxidant. administration of large doses of vitamin e before traumatic brain injury improves neurologic outcome in experimental models and has been examined as possible prophylaxis for human neonatal encephalopathy. [55] [56] [57] extrapolation of that information to the compromised equine fetus suggests that increased antioxidant concentrations in the fetus may mitigate some of the consequences of uterine and birth hypoxia, but no evidence is available to date demonstrating that protection occurs or that vitamin e accumulates in the fetus in response to supplementation of the mare. finally, many high-risk mares are anorectic or held off feed because of their medical condition. these mares are at particularly great risk for fetal loss because of their lack of feed intake, which alters prostaglandin metabolism. 58 therefore one should administer 2.5% to 5% dextrose in 0.45% saline or water (5% dextrose) intravenously at maintenance fluid rates to these patients. perhaps the most important aspect of managing high-risk pregnancy mares is frequent observation and development of a plan. one should observe mares at least hourly for evidence of early-stage labor and should put them under constant video surveillance if possible. depending on the primary problem, the team managing the mare should develop a plan for handling the parturition once labor begins and for fetal resuscitation following delivery. any equipment that might be needed should be readily available stallside, and a call sheet, listing contact numbers for all involved, should be posted on or near the stall. the plan should include a decision as to how to handle a complicated dystocia, should it occur, with permission for general anesthesia and cesarean section obtained before the event so that time is not wasted. an important question to be posed to the owner at the outset is which is most important to the owner, the mare or the foal, for the answer may dictate the direction of the decision tree once labor begins. 59 early recognition of abnormalities is of utmost importance for successful management of critically ill foals. to recognize the abnormal, one must know the normal. immediately following birth, foals effect several important physiologic and behavioral changes. chief among these changes is the adaptation of the cardiovascular and respiratory systems to extrauterine life. the normal transition of the respiratory tract involves opening closed 1384 part ii disorders of specific body systems alveoli and absorption of fluid from the airway, accomplished by a combination of breathing efforts, expiration against a closed glottis (grunting), and a change in sodium flux across the respiratory membrane from net secretion to net absorption. [60] [61] [62] [63] [64] the transition from fetal to neonatal circulatory patterns requires resolution of the pulmonary hypertension present in the fetus, normally shunting blood flow through the lower resistance ductus arteriosus in the fetal state, to direct cardiac output to the pulmonary vasculature for participation in gas exchange. this change is achieved by the opening of alveoli, decreasing airway resistance and providing radial support for pulmonary vessels, functional closure of the ductus arteriosus, and increasing the oxygen tension in the lung, reversing pulmonary vasoconstriction mediated by hypoxia. 65, 66 pulmonary tree vasodilators (prostacyclin, nitric oxide [no] ) and vasoconstrictors (endothelin-1, leukotrienes) play apparently well-coordinated, but as yet not fully elucidated, roles. in the normal newborn this change is smooth and rapid. these critical events are undermined by factors such as inadequate lung development, surfactant deficiency (primary or secondary), viral or bacterial infection, placental abnormalities, in utero hypoxia, and meconium aspiration. spontaneous breathing should begin in the neonate within 1 minute of birth, many foals attempt to breathe as their thorax clears the pelvic canal. during the first hour of life, the respiratory rate of a healthy foal can be as high as 80 breaths per minute but should decrease to 30 to 40 breaths per minute within a few hours. similarly, the heart rate of a healthy newborn foal has a regular rhythm and should be at least 60 beats/min at the first minute. 67, 68 one usually can auscultate a continuous murmur over the left side of the heart, although its loudness may vary with position. this murmur is thought to be associated with some shunting through the ductus arteriosus. one may auscultate variable systolic murmurs, thought to be flow murmurs, during the first week of life. 69 one should investigate more thoroughly murmurs that persist beyond the first week of life in an otherwise healthy foal, along with any murmur associated with persistent hypoxia. auscultation of the thorax shortly after birth reveals a cacophony of sounds as airways open and fluid is cleared. end-expiratory crackles are consistently audible in the dependent lung during and following lateral recumbency. for a normal newborn foal to appear slightly cyanotic during this initial adaptation period is not unusual, but this should resolve within minutes of birth. the equine fetus, as do all fetuses, exists in a moderately hypoxic environment, but the equine fetus has a greater partial pressure of oxygen, around 50 mm hg. 70 because the fetus is well adapted to low oxygen tensions, cyanosis is rarely present in newborn foals once adaption occurs, even those with low oxygen tensions. although in many species the fetal blood oxygen affinity is greater than the maternal blood, in the equine fetus the oxygen affinity of its hemoglobin is only about 2 mm hg greater than the maternal blood because of decreased levels of 2,3-diphosphoglycerate compared with other species. 71 the result is enhanced oxygen unloading in the equine fetus compared with others. 2,3-diphosphoglycerate concentration increases after birth in the foal and reaches mature levels by 3 to 5 days of age. the major blood adaptation of the equine fetus to chronic hypoxia is an increase in packed cell volume of up to 20%, increasing the oxygen content of the blood as compensation for decreased oxygen delivery at the placenta. 72 a larger than expected packed cell volume in any newborn foal should alert the clinician for possible sequelae from chronic hypoxia. the presence of significant cyanosis that persists should prompt the clinician to evaluate the foal thoroughly for cardiac anomalies resulting in significant right-to-left shunting or separated circulations, such as transposition of the great vessels. the chest wall of the foal is compliant, facilitating passage through the pelvic canal during parturition. this compliance requires that the foal actively participate in inspiration and expiration with several potential consequences. first, restriction of the thorax or the abdomen can result in impaired ventilation, which can occur easily when one restrains a foal and may result in spuriously abnormal arterial blood gas values (see the discussion on arterial blood gas evaluation, respiratory diseases associated with hypoxemia in the neonate). second, foals with primary pulmonary parenchymal disease resulting in poorly compliant lungs develop paradoxical chest wall motion, with the thorax moving inward during inspiration. [73] [74] [75] [76] the work of breathing can increase greatly, resulting in respiratory failure because of respiratory muscle fatigue. a foal that appears suddenly to improve a previously abnormal respiratory rate and pattern may in fact be in greater respiratory difficulty because of fatigue. one can observe a reduction in respiratory rate or abnormal breathing pattern in premature/dysmature foals or foals subjected to peripartum hypoxia/asphxia. although the genesis of these patterns is not understood fully, cheyne-stokes (lengthy periods of apnea interrupted by short breaths that wax and wane in depth), cluster (short periods of apnea interspersed with long periods of breathing), and biot's breathing (periods of apnea and breathing with no discernible pattern) may occur in these cases. foals attempting to maintain an adequate lung volume expire against a partially closed glottis, called valsalva's maneuver, producing an audible grunt. foals are normally nonresponsive while in the birth canal but should respond to stimulation immediately after birth. 67 the lack of responsiveness while in the birth canal has lead to presumption of fetal death during dystocia. because of this, one should attempt other tests before determining that a foal is dead intrapartum. one possibly may detect pulses in the tongue, neck, or any presented limbs or palpate the thorax for a heartbeat. in the author's facility, nasotracheal intubation of the foal combined with measurement of co 2 tensions in the exhaled gas aids practitioners in cases where they can reach the nose. nasotracheal intubation of foals under these circumstances actually can be performed readily with minimal practice. having long endotracheal tubes available of several different diameters (7 to 12 mm outer diameter) with an inflatable cuff is important. one can pass the tube blindly using a finger in one nostril for guidance and can check the position frequently by palpation of the throatlatch region. one inflates the cuff and begins manual ventilation with 100% oxygen or room air using an ambu-bag or equivalent. one can obtain continuous measurement of co 2 tension using a capnograph or single-use disposable end-tidal co 2 monitor attached to the ambu-bag or the nasotracheal tube. in a dead foal the end-tidal co 2 measurement will be negligible after the first 10 to 20 breaths. one must ensure tube placement and seal integrity and allow for multiple breaths. some co 2 will "wash out" with the first few breaths and can result in false hope initially. end-tidal co 2 varies in living intrapartum foals, depending on cardiac output and ventilation frequency, but should be consistently greater than 20 mm hg and is usually closer to 30 mm hg. once one establishes manual ventilation of a living foal, one must continue ventilation until the foal is delivered satisfactorily. the author has resuscitated and maintained many foals successfully in this manner throughout induction of general anesthesia in the mare and cesarean section delivery of the foal. the nasotracheal tube also provides a convenient site for administration of intratracheal medications such as epinephrine used for extrauterine intrapartum resuscitation of the foal. the reader is cautioned that intratracheal epinephrine increases endtidal co 2 measurements transiently, even in a dead foal, because of local actions on tissues. one should allow a washout period after intratracheal administration of epinephrine. the righting reflex is present as the foal exits the birth canal, as is the withdrawal reflex. cranial nerve responses are intact at birth, but the menace response may take as long as 2 weeks to develop fully. one should not consider lack of a menace reflex diagnostic of visual deficits in the newborn foal. within an hour of birth the normal foal will demonstrate auditory orientation with unilateral pinna control. the normal pupillary angle is ventromedial in the newborn foal; this angle gradually becomes dorsomedial over the first month of life. foals should begin attempting to stand shortly after birth and should be able to achieve this on their own within 2 hours of birth. 67 the normal newborn foal has a suck reflex shortly after birth and should be searching for an udder even before it stands. the expectation is that a normal foal will be sucking from the dam unaided by 3 hours post partum; many foals are overachievers and will be sucking well before this time. the normal foal may defecate shortly after standing but may not attempt defecation until after it first successfully sucks from the dam. urination varies more, with filly foals usually urinating before colt foals, but both usually do not urinate for several hours following birth, up to 12 hours for some colts. 67 for colt foals to fail to drop their penises when urinating over the first few days of life is not unusual. the gait of the newborn foal is hypermetric and the stance is base wide. extreme hypermetria of the forelimbs, usually bilateral but occasionally unilateral, has been observed in some foals and is associated with perinatal hypoxic/ischemic insults, but this gait abnormality usually resolves without specific therapy within a few days. spinal reflexes tend to be exaggerated, whereas the crossed extensor reflex may not be fully present until 3 weeks of age. 77 foals also exhibit an exaggerated response to external stimuli (noise, sudden visual changes, touch) for the first few weeks of life. foals are not bonded strongly to their mother for the first few weeks of life and will follow any large moving object, including other horses and human beings. orphan foals bond with surrogate mothers until they are several months of age; their primary motivation appears to be appetite. conversely, mares strongly bond with their foals shortly after parturition; the process begins once the chorioallantois ruptures and is driven more by olfaction and taste than by vision or hearing. interference with this process, by medical intervention or excessive owner manipulation of the foal, can disrupt normal bonding and result in foal rejection by the dam. 78 most newborn foals make the transition to extrauterine life easily. however, for those in difficulty, recognition of the condition immediately and institution of appropriate resuscitation is of utmost importance. a modified apgar scoring system has been developed as a guide for initiating resuscitation and assessing probable level of fetal compromise (table 19 -2). 79 one also must at least perform a cursory physical examination before initiating resuscitation, for issues of humaneness are associated with with serious problems such as severe limb contracture, microophthalmia, and hydrocephalus, among others. the initial assessment begins during presentation of the fetus. although the following applies primarily to attending the birth of a foal from a high-risk pregnancy, one can perform quiet and rapid evaluation during any attended birth. the goal in a normal birth with a normal foal is to disturb the bonding process minimally. this goal also applies to high-risk parturitions, but some disruption of normal bonding is inevitable. the lead clinician should control tightly the number of persons attending, and the degree of activity surrounding, the birth. one should evaluate the strength and rate of any palpable peripheral pulse and should evaluate the apical pulse as soon as the chest clears the birth canal. bradycardia (pulse <40 beats/min) is expected during forceful contractions, and the pulse rate should increase rapidly once the chest clears the birth canal. persistent bradycardia is an indication for rapid intervention. the fetus is normally hypoxemic compared with the newborn foal, and this hypoxemia is largely responsible for the maintenance of fetal circulation by generation of pulmonary hypertension. the fetus responds to conditions producing more severe in utero hypoxia by strengthening the fetal circulatory pattern, and the neonate responds to hypoxia by reverting to the fetal circulatory pattern. 80 during a normal parturition, mild asphyxia occurs and results in fetal responses that pave the way for a successful transition to extrauterine life. if more than mild transient asphyxia occurs, the fetus is stimulated to breathe in utero; this is known as primary asphyxia. 81 if the initial breathing effort resulting from the primary asphyxia does not correct the asphyxia, a second gasping period occurs in several minutes, known as the secondary asphyxia response. if no improvement in asphyxia occurs during this period, the foal enters secondary apnea, a state that is irreversible except with resuscitation. therefore the first priority of neonatal resuscitation is establishing an airway and breathing pattern. one should assume that foals not spontaneously breathing are in secondary apnea and should clear the airway of membranes as soon as the nose is presented. if meconium staining is present, one should suction the airway before delivery of the foal is completed and before the foal breathes spontaneously. one should continue to the trachea if aspiration of the nasopharynx is productive. overzealous suctioning worsens bradycardia as it worsens hypoxia. one should stop suctioning once the foal begins breathing spontaneously, as hypoxia will worsen with continued suction. if the foal does not breathe or move spontaneously within seconds of birth, one should begin tactile stimulation. if tactile stimulation fails to result in spontaneous breathing, one immediately should intubate the foal and manually ventilate the foal using an ambu-bag or equivalent. one can use mouth-to-nose ventilation if nasotracheal tubes and an ambu-bag are not available. the goal of this therapy is to reverse fetal circulation, and hyperventilation with 100% oxygen is the best choice for this purpose. however, recent evidence suggests that no clinical disadvantages are apparent in using room air for ventilation of asphyxiated human neonates rather than 100% oxygen. 82, 83 human infants resuscitated with room air recovered more quickly than those resuscitated with 100% oxygen in one study as assessed by apgar scores, time to the first cry, and the sustained pattern of breathing. 84 in addition, neonates resuscitated with 100% oxygen exhibited biochemical findings reflecting prolonged oxidative stress, present even after 4 weeks of postnatal life, which did not appear in the group resuscitated with room air. thus the current accepted recommendations for using 100% oxygen in the resuscitation of asphyxiated neonates needs further discussion and investigation. 85, 86 almost 90% of foals requiring resuscitation respond to hyperventilation alone and require no additional therapy. one can initiate nasotracheal intubation while the foal is in the birth canal if the foal will not be delivered rapidly, such as with a difficult dystocia. this technique is "blind" and requires some practice but may be beneficial and lifesaving. once spontaneous breathing is present, one apgar score in the foal should provide humidified oxygen via nasal insufflation at 8 to 10 l/min. one should initiate cardiovascular support in the form of chest compression if the foal remains bradycardic despite ventilation and a nonperfusing rhythm is present. one should make sure the foal is on a hard surface in right lateral recumbency with the topline against a wall or other support. approximately 5% of foals are born with fractured ribs and an assessment for the presence of rib fractures is in order before initiating chest compressions. 87 palpation of the ribs identifies many of these fractures, which usually are multiple and consecutive on one side of the thorax and located in a relatively straight line along the part of the rib with the greatest curvature dorsal to the costochondral junction. unfortunately, ribs 3 to 5 frequently are involved, and their location over the heart can make chest compression a potentially fatal exercise. auscultation over the ribs during breathing results in a recognizable click, identifying rib fractures that may have escaped detection by palpation. one should initiate drug therapy if a nonperfusing rhythm persists for more than 30 to 60 seconds in the face of chest compression. epinephrine is the first drug of choice (table 19-3) . practitioners pose various arguments regarding the best dose and the best frequency of administration for resuscitation. however, most of the data are acquired from human cardiac arrest studies and are not strictly applicable to the equine neonate because the genesis of the cardiovascular failure is different. 88, 89 vasopressin is gaining attention as a cardiovascular resuscitation drug, and although the author has used this drug in resuscitation and as a pressor, experience is limited at this time. 90 the author does not use atropine in bradycardic newborn foals because the bradycardia usually is caused by hypoxia, and if the hypoxia is not corrected, atropine can increase myocardial oxygen debt. 89 the author also does not use doxapram because it does not reverse secondary apnea, the most common apnea in newborns. because birthing areas are generally cold, one should dry the foal and place it on dry bedding once resuscitation is complete. the fetus has some homeothermic mechanisms, but its size in relation to its mother and its position within her body means that it is in effect a poikilotherm. the body temperature of the foal generally reflects that of its environment, namely its mother, although the human fetal temperature directly measured at cesarean section, induction of labor, or during labor is approximately 0.5â°c higher than the mothers. 91, 92 adaptation from poikilothermy to homeothermy normally takes place rapidly following birth. the fetus is capable of nonshivering thermogenesis, primarily through the oxidation of brown fat reserves, but this type of thermogenesis is inhibited in utero, probably by placental prostaglandin e 2 and adenosine. 93, 94 immediately after birth the foal must adapt to independent thermoregulation. local physical factors, including ambient temperature and humidity, act to induce cold stress, and the newborn must produce heat by metabolic activity. in response to the catecholamine surge associated with birth, uncoupling of oxidative phosphorylation occurs within mitochondria, releasing energy as heat. this nonshivering thermogenesis is impaired in newborns undergoing hypoxia or asphyxiation and in those that are ill at birth. infants born to mothers sedated with benzodiazepines are affected similarly, a consideration in the choice of sedative and preanesthetic medications in mares suffering dystocia or 1388 part ii disorders of specific body systems undergoing cesarean section. [95] [96] [97] heat losses by convection, radiation, and evaporation are high in most areas where foals are delivered, resuscitated ,and managed, and one must take care to minimize cold stress in the newborn and the critically ill foal. supplementary heat, in the form of radiant heat lamps or warm air circulating blankets, may be required. one should use fluid therapy conservatively during postpartum resuscitation, for the neonate is not volume depleted unless excessive bleeding has occurred. some compromised newborn foals are actually hypervolemic. fluid therapy of the neonate is discussed in more detail later in this chapter. because the renal function of the equine neonate is substantially different from the adult, one cannot simply scale down fluid therapy from adult therapy. [98] [99] [100] if intravenous fluids are required for resuscitation and blood loss is identified, administration of 20 ml/kg of a non-glucose-containing polyionic isotonic fluid over 20 minutes (about 1 l for a 50-kg foal) once intravenous access is established can be effective. the author stresses non-glucose-containing polyionic intravenous fluids because hyperglycemia, but not hypoglycemia, immediately after fetal or neonatal asphyxia interfered with the recovery of brain cell membrane function and energy metabolism in neonatal piglets in one recent study. 101 these findings suggest that post-hypoxic-ischemic hyperglycemia is not beneficial and might even be harmful in neonatal hypoxic-ischemic encephalopathy. indications for this shock bolus therapy include poor mentation, poorly palpable peripheral pulses, and the development of cold distal extremities, compatible with hemorrhagic shock. one should reassess the patient after the initial bolus and administer additional boluses as necessary. ideally, one should follow up on blood pressures and ecg readings and initiate appropriate pressor therapy if needed. again, these procedures are discussed in detail later in the chapter. one can administer glucose-containing fluids after resuscitation at a rate of 4 to 8 mg/kg/min (about 250 ml/hr of 5% dextrose or 125 ml/hr of 10% dextrose) to the average 50-kg foal, particularly in the obviously compromised foal. this therapy is indicated to help resolve metabolic acidosis, to support cardiac output because myocardial glycogen stores likely have been depleted, and to prevent postasphyxial hypoglycemia. under normal conditions, the fetal-to-maternal blood glucose concentration gradient is 50% to 60% in the horse, and glucose is the predominant source of energy during fetal development. 102, 103 glucose transport across the placenta is facilitated by carrier receptors (glucose transporter [glut] receptors), and a direct relationship exists between maternal and fetal blood glucose concentration when maternal glucose is in the normal range. 102 the glut receptors in the placenta are stereospecific, saturable, and energy independent. 104 although the enzyme kinetics for glut isoform 1 suggest that they are not saturable under conditions of euglycemia, equine maternal hyperglycemia results in increased fetal glucose concentration to a plateau point, likely caused by glut saturation. at term, the net umbilical uptake of glucose is 4 to 7 mg/kg/min, with most of the glucose being used by the brain and skeletal muscle. [105] [106] [107] the fetus only develops gluconeogenesis under conditions of severe maternal starvation. a certain percentage of the delivered glucose is used to develop large glycogen stores in the fetal liver and cardiac muscle in preparation for birth, and at birth the foal liver produces glucose at a rate of 4 to 8 mg/ kg/min by using these stores. fetal glycogen stores also are built using the substrates lactate, pyruvate, and alanine; fetal uptake of lactate across the placenta is about half that of glucose. 102, 108 the transition to gluconeogenesis, stimulated by increased circulating catecholamine concentration from birth and by stimulation of glucagon release at the time the umbilical cord breaks takes 2 to 4 hours in the normal foal, and glycogenolysis supplies needed glucose until feeding and glucose production are accomplished. 109 in the challenged foal, glycogen stores may have been depleted and gluconeogenesis delayed, so provision of glucose at rates similar to what the liver would normally produce during this period is requisite. persistent pulmonary hypertension (pph) also is known as reversion to fetal circulation or persistent fetal circulation, and its genesis lies in the failure of the fetus to make the respiratory and cardiac transition to extrauterine life successfully or reversion of the newborn to fetal circulatory patterns in response to hypoxia or acidosis. differentiating this problem from other causes of hypoxemia in the newborn requires some investigation, and multiple serial arterial blood gas analyses are necessary to confirm suspicion of this problem (see the section on arterial blood gas analysis, respiratory diseases associated with hypoxemia in the neonate). however, one should suspect the condition in any neonate with hypercapnic hypoxemia that persists and worsens; these foals are in hypoxemic respiratory failure. the fetal circulatory pattern, with pulmonary hypertension and right-to-left shunting of blood through the patent foramen ovale and ductus arteriosus, is maintained in these cases. pulmonary vascular resistance falls at delivery to about 10% of fetal values, while pulmonary blood flow increases accordingly. 110 early in the postnatal period these two changes balance each other, and mean pulmonary and systolic pressures remain increased for several hours. systolic pulmonary pressures can remain equivalent to systemic pressure for up to 6 hours of age in human infants, although diastolic pulmonary pressures are well below systemic diastolic pressures by 1 hour. 111 mean pulmonary artery pressures fall gradually over the first 48 hours. 112 the direct effects of lung expansion and increasing alveolar oxygen tension probably provide the initial stimulus for pulmonary arteriolar dilation and partly result from direct physical effects, but vasoactive substances are released in response to physical forces associated with ventilation, for example prostacyclin. 110 other vasoactive mediators thought to play a role in regulating pulmonary arteriolar tone include no, prostaglandins d 2 and e 2 , bradykinin, histamine, endothelin-1, angiotensin ii, and atrial natriuretic peptide. the increase in alveolar and arterial oxygen tensions at birth is required for completion of resolution of pulmonary hypertension. much of this increase is thought to be mediated by no, evidence for this being the parallel increase during gestation of the pulmonary vasodilation response to hyperoxia and the increase in no synthesis. 113 however, inhibition of no synthesis does not eliminate the initial decrease in pulmonary artery resistance occurring because of opening of the airways. 114 when these mechanisms fail, one can recognize pph. right-to-left shunting within the lungs and through patent fetal conduits occurs and can result from many factors, including asphyxia and meconium aspiration, but in many cases the precipitating trigger is unknown. inappropriately decreased levels of vasodilators (no) and inappropriately increased levels of vasoconstrictors (endothelin-1) currently are being examined as potential mechanisms. chronic in utero hypoxia and acidosis may result in hypertrophy of the pulmonary arteriolar smooth muscle. 115 in these cases, reversal of pph can be difficult and cannot be achieved rapidly. treatment of pph is twofold: abolishment of hypoxia and correction of the acidosis, for both abnormalities only bolster the fetal circulatory pattern. initial therapy is provision of oxygen intranasally at 8 to 10 l/min. some foals respond to this therapy and establish neonatal circulatory patterns within a few hours. failure to improve or worsening of hypoxemic respiratory failure following intranasal oxygen administration should prompt intubation and mechanical ventilation with 100% oxygen. this serves two purposes, one diagnostic and one therapeutic. ventilation with 100% oxygen may resolve pph and, if intrapulmonary shunt and altered ventilation-perfusion relationships are causing the hypoxic respiratory failure, arterial oxygen tension (pao 2 ) should exceed 100 mm hg under these conditions. failure to improve pao 2 suggests pph or large right-to-left extrapulmonary shunt caused by congenital cardiac anomaly. the vasodilators prostacyclin and telazoline (an î±-blocking vasodilator) cause pulmonary vasodilation in human infants with pph, but the effects on oxygenation vary and the sideeffects (tachycardia, severe systemic hypotension) are unacceptable. 116 recognition of no as a potent dilator of pulmonary vessels has created a significant step forward in the treatment of these patients, for inhaled no dilates vessels in ventilated portions of the lung while having minimal effects on the systemic circulation. 117 based on evidence presently available, use of inhaled no in an initial concentration of about 20 ppm in the ventilatory gas seems reasonable for term and near-term foals with hypoxic respiratory failure and pph that fails to respond to mechanical ventilation using 100% oxygen alone. 117, 118 the author has used this approach in the clinic, administering a range of 5 to 40 ppm no with success. hypoxic ischemic encephalopathy (hie), currently referred to as neonatal encephalopathy in the human literature, is one systemic manifestation of a broader syndrome of perinatal asphyxia syndrome (pas), and management of foals with signs consistent with a diagnosis of hie requires the clinician to examine other body systems fully and to provide therapy directed at treating other involved systems. 119 although pas primarily manifests as hie, the gastrointestinal tract and kidneys frequently are affected by peripartum hypoxia/ischemia/ asphyxia, and one should expect complications associated with these systems. hypoxic ischemic encephalopathy also may affect the cardiovascular and respiratory systems, and one also may encounter endocrine disorders in these patients. hypoxic ischemic encephalopathy has been recognized as one of the most common diseases of the equine neonate for generations. 1, 10, 12 in the past hie has been known as dummy foal syndrome and as neonatal maladjustment syndrome. the designation hie, although not perfect, attempts to describe the syndrome in terms of the suspected underlying pathophysiology. a wide spectrum of clinical signs is associated with hie and can range from mild depression with loss of the suck reflex to grand mal seizure activity. typically, affected foals are normal at birth but show signs of central nervous system abnormalities within a few hours after birth. some foals are obviously abnormal at birth, and some do not show signs until 24 hours of age. hypoxic ischemic encephalopathy commonly is associated with adverse peripartum events, including dystocia and premature placental separation, but a fair number of foals have no known peripartum period of hypoxia, suggesting that these foals result from unrecognized in utero hypoxia (box 19-2). severe maternal illness also may result in foals born with pas. in human beings, ascending placental infection now is suspected of being a major contributor to neonatal encephalopathy in infants, and the incidence of neonatal encephalopathy increases with the presence of maternal fever, suggesting a role for maternal inflammatory mediators. 120 the underlying pathophysiologic details of hie in the foal are unknown, and to date accurate experimental models of hie and pas in the foal have not been described. however, a great deal of attention has been paid to peripartum hypoxia/asphyxia by human counterparts because the effects of adverse peripartum events in the human neonate have far ranging implications for the affected human neonate and for society. therefore equine neonatologists have long looked to human studies and models of the human disease for understanding of the syndrome in the equine neonate. perinatal brain damage in the mature fetus usually results from severe uterine asphyxia caused by an acute reduction of uterine or umbilical circulation. the fetus responds to this challenge by activation of the sympathetic adrenergic nervous system, causing a redistribution of cardiac output that favors the central organs: brain, heart, and adrenal glands. 121, 122 if the hypoxic insult continues, the fetus reaches a point beyond which it cannot maintain this centralization of circulation, cardiac output falls, and cerebral circulation diminishes. 122 the loss of oxygen results in a substantial decrease in oxidative phosphorylation in the brain with concomitant decreased energy production. the na + /k + pump at the cell membrane cannot maintain the ionic gradients, and the membrane potential is lost in the brain cells. in the absence of the membrane potential, calcium flows down its large extracellular/intracellular concentration gradient through voltage-dependent ion channels into the cell. this calcium overload of the neuron leads to cell damage by activation of calcium-dependent proteases, lipases, and endonucleases. protein biosynthesis is halted. calcium also enters the cells by glutamate-regulated ion channels as glutamate, an excitatory neurotransmitter, is released from presynaptic vesicles following anoxic cellular depolarization. once the anoxic event is over, protein synthesis remains inhibited in specific areas of the brain and returns to normal in less vulnerable areas of the brain. loss of protein synthesis appears to be an early indicator of cell death caused by the primary hypoxic/anoxic event. 123 a second wave of neuronal cell death occurs during the reperfusion phase and is thought to be similar to classically described postischemic reperfusion injury in that damage is caused by production of and release of oxygen radicals, synthesis of no, and inflammatory reactions. 124 additionally, an imbalance between excitatory and inhibitory neurotransmitters occurs. 123 part of the secondary cell death that occurs is thought to be caused by apoptosis, a type of programmed cell death termed cellular suicide. secondary cell death also is thought be caused by the neurotoxicity of glutamate and aspartate resulting again from increased intracellular calcium levels. 125, 126 in human infants the distribution of lesions with hypoxic-ischemic brain damage following prenatal, perinatal, or postnatal asphyxia falls into distinct patterns depending on the type of hypoxia-ischemia rather than on postconceptual age at which the asphyxial event occurs. 126 periventricular leukomalacia was associated with chronic hypoxia-ischemia, whereas the basal ganglia and thalamus were affected primarily in patients experiencing acute profound asphyxia, providing direct evidence that the nature of the event determines the severity and distribution of neurologic damage in human beings. these remarkably selective patterns of injury in children, with differential variability in the damage caused to regions anatomically located within millimeters of each other, resulted in the hypothesis that location within neurotransmitter-specific circuitry loops is important. this hypothesis has important implications in the design of neuroprotective strategies and therapies for neonates experiencing hypoxic-ischemic-asphyxial events. now the evidence is overwhelming that the excitotoxic cascade that evolves during hie extends over several days from the time of insult and is modifiable. 125, 126 in brain injury, traumatic or hypoxic, the mechanisms underlying delayed tissue injury still are understood poorly. many believe that neurochemical changes, including excessive neurotransmitter release, are pivotal in the pathophysiology of secondary neuronal death. excitatory amino acid neurotransmitters and magnesium are known to play at least a minimal role in secondary cell death following brain injury; a fair body of literature regarding these factors has been generated over the last 10 years. the activation of the n-methyl-d-aspartate (nmda) subtype of glutamate receptors is implicated in the pathophysiology of traumatic brain injury and is suspected to play a role in hie. [125] [126] [127] mechanically injured neurons demonstrate a reduction of voltage-dependent mg 2+ blockade of nmda current that can be restored partially by increasing extracellular mg 2+ concentration or by pretreatment with calphostin c, a protein kinase c inhibitor. 128 this finding suggested that administration of mg 2+ to patients with brain injury could lead to improved outcome. subsequently, magnesium sulfate solution was shown to improve dramatically the immediate recovery of rats from hypoxia. 129 however, although pretreatment with magnesium sulfate protected against hypoxic ischemic brain injury, postasphyxial treatment worsened brain damage in 7-day-old rats, suggesting an age-related response in the rat. 130 delayed magnesium treatment of mature rats following severe traumatic axonal brain injury improved motor outcome when administered up to 24 hours after injury, with early treatments providing the most benefit. 131 maternal seizure in rats is associated with fetal histopathologic changes that are abolished by administration of magnesium sulfate to the mother, and magnesium sulfate has been demonstrated to protect the fetal brain from severe maternal hypoxia. 132 clinical trials investigating the efficacy of magnesium treatment following hypoxia in infants are under way, with few reports currently in the medical literature. magnesium sulfate was used to treat nine infants after perinatal asphyxia in one study (no control group), and all children were neurologically normal at 1 year of age. seizures did not occur in any of these children, nor were any adverse side effects noted. 133 magnesium sulfate administration failed to delay the global impairment in energy metabolism after hypoxia ischemia, characteristic of severe brain damage, in newborn piglets; at 48 hours after hypoxia ischemia, no difference could be found in the severity of injury in piglets treated with magnesium compared with piglets treated with placebo, suggesting magnesium may not be protective with severe acute injury. 134 in developing countries, birth hypoxia frequently is associated with hie, and although this finding is attributed most frequently to inadequate obstetric care, poor nutrition also may play a role. red blood cell magnesium levels were measured in more than 500 women in labor at a teaching hospital in south africa. 135 fifty five of the women delivered infants with hie and had significantly lower levels of magnesium than controls; the infants with hie also had significantly lower magnesium levels than controls. the large majority (54 of 55) of the women giving birth to hie infants were from poor social circumstances, suggesting nutrition might play a role in some cases of hie, with maternal magnesium levels affecting outcome in the infants. the authors suggested an early pregnancy intervention study may help determine the role of magnesium in the pathogenesis of hie in human infants born to at-risk mothers. therapy for the various manifestations of hypoxiaischemia involves control of seizures, general cerebral support, correction of metabolic abnormalities, maintenance of normal arterial blood gas values, maintenance of tissue perfusion, maintenance of renal function, treatment of gastrointestinal dysfunction, prevention and recognition and early treatment of secondary infections, and general supportive care. control of seizures is important because cerebral oxygen consumption increases fivefold during seizures. one can use diazepam for emergency control of seizures (table 19 -4). if diazepam does not stop seizures readily or one recognizes more than two seizures, then one should replace diazepam with phenobarbital given to effect. the half-life of phenobarbital can be long in the foal (100 hours), and one should keep this in mind when monitoring neurologic function in these cases after phenobarbital administration (j.e. palmer, personal communication, 1998). 136 earlystage, preseizure administration of phenobarbital has been advocated by some investigators for prevention of neonatal encephalopathy. however, one recent study in asphyxiated human infants demonstrated that early phenobarbital treatment was associated with a threefold increase in the incidence of subsequent seizures and consequently a trend toward increased mortality. seizures per se were associated with almost a twentyfold increase in mortality. their findings suggest that early phenobarbital administration may produce adverse rather than beneficial effects following asphyxia. because this was an observational study; the results need to be confirmed by appropriate randomized trials in similar clinical settings. 137 if phenobarbital fails to control seizures, one may attempt phenytoin therapy. in cases of hie, one should avoid ketamine and xylazine because of their association with increased intracranial pressure. one must protect the foal from injury during a seizure and also ensure the patency of the airway to prevent the onset of negative pressure pulmonary edema 138 or aspiration pneumonia. probably the most important therapeutic interventions are aimed at maintaining cerebral perfusion, which is achieved by careful titration of intravenous fluid support, neither too much nor too little (see fluid therapy in neonates) and judicious administration of inotropes and pressors to maintain adequate perfusion pressures (see pressor and inotrope therapy in neonates). cerebral interstitial edema is only truly present in the most severe cases 139, 140 ; in most cases the lesion is intracellular edema and most of the classic agents used to treat cerebral interstitial edema (e.g., mannitol) are minimally effective treating cellular edema. occasionally the author uses thiamine supplementation in the intravenous fluids to support metabolic processes, specifically mitochondrial metabolism and membrane na + ,k + -atpases, involved in maintaining cellular fluid balance. 141, 142 this therapy is rational and inexpensive but unproven in efficacy. only if cellular necrosis and vasogenic edema are present are drugs such as mannitol and dimethyl sulfoxide indicated, and again these cases are usually the most severely affected. in the author's clinic, practitioners rarely have used dimethyl sulfoxide in neonates for the last several years and have recognized no change in outcome by discontinuing its use. when the practitioners use intravenously administered dimethyl sulfoxide, they do so within the first hour after an acute asphyxial insult and use it primarily for its hydroxyl radical scavenging effects and its theoretical modulation of postischemic reperfusion injury. 143 naloxone has been advocated for treating hie in human beings and in foals, [144] [145] [146] perhaps based on a study suggesting that postasphyxia blood-brain barrier disruption was related causally to poor neurologic outcome in a lamb model of hie and that naloxone prevented disruption and neurologic dysfunction among those survivors with an intact blood-brain barrier. 145 however, other studies have demonstrated that naloxone exacerbates hypoxic-ischemic brain injury in 7-day-old rats subjected to unilateral common carotid artery ligation and hypoxia. moreover, systemic acidosis and cellular edema were no different in naloxone-treated animals compared with animals treated with saline solution. the authors concluded that high doses of naloxone in fact may reduce the resistance of the fetus to hypoxic stress. 146 the use of naloxone in human neonatal resuscitation remains controversial, for whether the contradictory effects are related to a reduction in acute neuronal swelling by osmotic effects or by a more direct receptor-mediated mechanism is currently unknown. 147 naloxone is most effective in resuscitation of compromised human infants born to mothers addicted to drugs. some practitioners are using î³-aminobutyric acid adrenergic agonists to manage hie in foals, based on evidence showing neuroprotection when used in ischemia alone and combined with nmda antagonists. [148] [149] [150] the author currently has no experience with these compounds and cannot comment regarding their efficacy in foals. regional hypothermia also is being investigated as a potential therapy for global hypoxia/ischemia; published data are consistent with the theory that cooling must be continued throughout the entire secondary phase of injury (about 3 days) to be effective. 151 experimentally, this approach has resulted in dramatic decreases in cellular edema and neuronal loss; its practical application remains to be demonstrated. despite a lack of consensus regarding the use of magnesium to treat infants with hie, the author has used magnesium sulfate infusion as part of the therapy for selected foals with hie for the past several years. the rationale is based primarily on the evidence demonstrating protection in some studies and a failure of any one study to demonstrate significant detrimental effects. the clinical impressions of the author to date suggest that the therapy is safe and may decrease the incidence of seizure in patients. the author administers magnesium sulfate as a constant rate infusion over 1 hour after giving a loading dose. the author has continued the infusion for up to 3 days without demonstrable negative effect beyond some possible trembling. given the current evidence, a 24-hour course of treatment may be effective and all that is necessary. postasphyxial treatment certainly may be beneficial in foals with hie, and maternal magnesium therapy may be beneficial in certain high-risk pregnancy patients. foals with pas often have a variety of metabolic problems including hypo-or hyperglycemia, hypo-or hypercalcemia, hypo-or hyperkalemia, hypo-or hyperchloremia, and varying degrees of metabolic acidosis. although one needs to address these problems, one should not forget the normal period of hypoglycemia that occurs postpartum and should not treat aggressively so as to avoid worsening the neurologic injury. foals suffering from pas also have frequent recurrent bouts of hypoxemia and occasional bouts of hypercapnia. intranasally administered oxygen is generally needed in these cases as a preventative therapy and as direct treatment, for the appearance of the abnormalities can be sporadic and unpredictable. additional respiratory support, particularly in those foals with centrally mediated hypoventilation and periods of apnea or abnormal breathing patterns, include caffeine (per os or per rectum) and positive pressure ventilation. caffeine is a central respiratory stimulant and has minimal side effects at the dosages used (10 mg/kg loading dose; 2.5 mg/kg as needed). 152 the author purchases whatever oral form of caffeine is available at the local convenience store or drug store and administers it dissolved in warm water per rectum. foals treated with caffeine have an increased level of arousal and are more reactive to the environment. adverse effects generally are limited to restlessness, hyperactivity, and mild to moderate tachycardia. mechanical ventilation of these patients can be rewarding and generally is required for less than 48 hours. one must monitor and maintain blood ph within the normal range. metabolic alkalosis can develop in some of these foals and requires clinician tolerance of some degree of hypercapnia. ph is important in evaluation and consideration of alternatives for treatment. if the respiratory acidosis is not so severe as to affect the patient adversely (generally >70 mm hg), and the ph is within normal limits, the foal may tolerate hypercapnia. 153 the goal is to normalize ph. foals with respiratory acidosis as compensation for metabolic alkalosis do not respond to caffeine. metabolic alkalosis in critically ill foals frequently is associated with electrolyte abnormalities, creating differences in strong ion balance. one handles this ph perturbation best by correcting the underlying electrolyte problem. maintaining tissue perfusion and oxygen delivery to tissues is a cornerstone of therapy for pas to avoid additional injury. one should maintain the oxygen-carrying capacity of the blood; some foals require transfusions to maintain a packed cell volume greater than 20%. adequate vascular volume is important, but one should take care to avoid fluid overload in the foal. early evidence of fluid overload is subtle accumulation of ventral edema between the front legs and over the distal limbs. fluid overload can result in cerebral edema, pulmonary edema, and edema of other tissues, including the gastrointestinal tract. this edema interferes with normal organ function and worsens the condition of the patient. one maintains perfusion by supporting cardiac output and blood pressure by judicious use of intravenous fluid support and inotrope/pressor support. the author does not target therapy to a specific systolic, mean, or diastolic pressure but monitors urine output, mentation, limb perfusion, gastrointestinal function, and respiratory function as indicators that perfusion is acceptable. for these patients to require pressor therapy is not unusual, but in some cases the hypoxic damage is sufficiently severe to blunt the response of the patient to the drugs. the kidney is a target for injury in patients with pph, and for renal compromise to play a significant role in the demise of these foals is not unusual. clinical signs of renal disease are generally referable to disruption of normal control of renal blood flow and tubular edema leading to tubular necrosis and renal failure. these foals have signs of fluid overload and generalized edema. one must balance urine output and fluid therapy in these cases to prevent additional organ dysfunction associated with edema. although evidence has accumulated that neither dopamine nor furosemide play a role in protecting the kidney or reversing acute renal failure, these agents can be useful in managing volume overload in these cases. [154] [155] [156] the aim is not to drive oliguric renal failure into a highoutput condition but rather to enhance urine output. overzealous use of diuretics and pressors in these cases can result in diuresis requiring increased intravenous fluid support and can be counterproductive. the author's approach is more conservative. low doses of dopamine administered as a constant rate infusion of 2 to 5 âµg/kg/min are usually effective in establishing diuresis by natriuresis. one should avoid large doses of dopamine (>20 âµg/kg/min) because high doses can produce systemic and pulmonary vasoconstriction, potentially exacerbating pph. 157 one can administer a bolus (0.25 to 1.0 mg/kg) or constant rate infusion (0.25 to 2.0 mg/kg/hr) of furosemide, but once furosemide diuresis is established, one must evaluate electrolyte concentrations and blood gas tensions frequently because potassium, chloride, and calcium losses can be considerable and because significant metabolic alkalosis can develop from strong ion imbalances. the author does not aim for urine production rates of 300 ml/hr, as has been presented by other authors as a urine output goal for critically ill equine neonates. 158 rather the author looks for urine output that is appropriate for fluid intake and does not attempt to drive urine output to an arbitrary goal by excessive fluid administration or pressor use. although the average urine output for a normal equine neonate is about 6 ml/kg/hr (~300 ml/hr for a 50-kg foal), these values were obtained from normal foals drinking a milk diet with a large free water component. [98] [99] [100] the urine of normal newborn foals is dilute, reflecting the large free water load they incur by their diet. expecting critically ill foals to produce such large volumes of urine, particularly those on restricted diets or receiving total parenteral nutrition, is an exercise in futility, and manipulating fluid, pressor, or diuretic therapy in attempt to meet an artificial goal is inappropriate. fluid therapy in the critically ill neonate is discussed later in this chapter. one final caveat regarding renal dysfunction in pas is that one should perform therapeutic drug monitoring when it is available. many antimicrobial agents used to manage these cases, most notably the aminoglycosides, depend on renal clearance. aminoglycoside toxicity occurs in the equine neonate and exacerbates or complicates the management of renal failure originally resulting from primary hemodynamic causes. the author monitors aminoglycoside concentrations for 30-minute peak and 23-to 24-hour trough values in these cases and adjusts dosage and frequency of drug administration based on these results. the author considers a trough value of less than 2 âµg/dl as desirable for gentamicin and amikacin. foals with pas suffer from a variety of problems associated with abnormalities within the gastrointestinal tract. 159 commonly they have ileus, recurrent excessive gastric reflux, and gas distention. these problems are exacerbated by constant feeding in the face of continued dysfunction and continued hypoxia. frequently, enteral feeding cannot meet their nutritional requirements, and partial or total parenteral nutrition is required. one must give special attention to passive transfer of immunity (see failure of passive transfer) and glucose homeostasis in these cases. although some practitioners use prokinetic agents as therapy for ileus in these cases, the author's approach is again more conservative. appearance of damage to the gastrointestinal tract can be subtle and lag behind other clinical abnormalities for days to weeks. low-grade colic, decreased gastrointestinal motility, decreased fecal output, and low weight gain are among the most common clinical signs of gastrointestinal dysfunction in these case, but more severe problems, including necrotizing enterocolitis and intussusception, have been associated with these cases. the return to enteral feeding must be slow in many of these cases. a currently debated topic is constant versus pulsed enteral feeding. [160] [161] [162] the author uses pulsed feeding through an indwelling small-gauge feeding tube. in many foals these tubes stay in place for weeks and cause no problems as the foals are returned to their dams for sucking or are trained to drink from a bottle or bucket. foals with pas are also susceptible to secondary infection. treatment of recognized infection is covered under sepsis in this chapter. if infection is recognized in these patients after hospitalization, one should give attention to the likelihood of nosocomial infection and should direct antimicrobial therapy based on known nosocomial pathogens in the nicu and their susceptibility patterns until culture and sensitivity results become available. one should make repeat determinations of immunoglobulin g (igg) concentration; additional intravenous plasma therapy may be required. nosocomial infections are often rapidly overwhelming, and acute deterioration in the condition of a foal with pas should prompt a search for nosocomial infection. the prognosis for foals with pas is good to excellent when the condition is recognized early and aggressively treated in term foals. up to 80% of these neonates survive and go on to lead productive and useful athletic lives. [20] [21] [22] [23] the prognosis decreases with delayed or insufficient treatment and concurrent problems such as prematurity and sepsis. in human nicus the survival rates of low-gestationlength infants has increased dramatically since the 1980s concurrent with improvements in obstetric and neonatal care. the now routine, well-validated use of antenatal steroid and artificial surfactant therapies has contributed greatly to the enhanced survival of this patient population, although the use of these particular therapies is not common or frequently indicated in the equine nicu. 163, 164 however, with improved care, outcomes in the equine nicu population have improved also, with survival of premature patients in many nicus exceeding 80%. 21 in the equine population, gestation length is much more flexible than in the human population; however, the definition of the term prematurity needs reexamination. traditionally, prematurity is defined as a preterm birth of less than 320 days of gestation in the horse. given the variability of gestation length in the horse, ranging from 310 days to more than 370 days in some mares, a mare with a usual gestation length of 315 days possibly could have a term foal at 313 days, whereas a mare with a usual gestation length of 365 days may have a premature foal at 340 days, considered the normal gestation length. foals that are born postterm but are small are termed dysmature; a postmature foal is a postterm foal that has a normal axial skeletal size but is thin to emaciated. dysmature foals may have been classified in the past as small for gestational age and are thought to have suffered placental insufficiency, whereas postmature foals are usually normal foals that have been retained too long in utero, perhaps because of an abnormal signaling of readiness for birth, and have outgrown their somewhat aged placenta. postmature foals become more abnormal the longer they are maintained, also may suffer from placental insufficiency, and are represented best by the classic foal born to a mare ingesting endophyteinfested fescue. 165 box 19-3 compares the characteristics of premature/dysmature foals with those of postmature foals. the causes of prematurity/dysmaturity/postmaturity include the causes of high-risk pregnancy presented in box 19-1. additional causes include iatrogenic causes such as early elective induction of labor based on inaccurate breeding dates or misinterpretation of late-term colic or uterine bleeding as ineffective labor. most causes remain in the category of idiopathic, with no discernible precipitating factor. despite lack of an obvious cause, premature labor and delivery does not just happen, and even if undetermined, the cause may continue to affect the foal in the postparturient period. all body systems may be affected by prematurity, dysmaturity, and postmaturity, and thorough evaluation of all body systems is necessary. respiratory failure is common in these foals, although the cause usually is not surfactant deficiency. immaturity of the respiratory tract, poor control of respiratory vessel tone, and weak respiratory muscles combined with poorly compliant lungs and a greatly compliant chest wall contribute to respiratory failure in these cases. most require oxygen supplementation and positional support for optimal oxygenation and ventilation. one must extend effort to maintain these "floppy foals" in sternal recumbency. some foals may require mechanical ventilation. these foals also require cardiovascular support but are frequently unresponsive to commonly used pressors and inotropes: dopamine, dobutamine, epinephrine, and vasopressin. careful use of these drugs and judicious intravenous fluid therapy are necessary. the goal should not be one of achieving specific pressure values (e.g., mean arterial pressure of 60 mm hg) but of adequate perfusion. renal function, reflected in low urine output, is frequently poor initially in these cases because of delay in making the transition from fetal to neonatal glomerular filtration rates. 166 the delay can result from true failure of transition or from hypoxic/ischemic insult. one should approach fluid therapy cautiously in these cases; initial fluid restriction may be in order to avoid fluid overload. many premature/dysmature/postmature foals have suffered a hypoxic insult and have all of the disorders associated with pas, including hie. treatment is similar to that of term foals with these problems. these foals also are predisposed to secondary bacterial infection and must be examined frequently for signs consistent with early sepsis or nosocomial infection. the gastrointestinal system of these foals is not usually functionally mature, which may result from a primary lack of maturity or from hypoxia. dysmotility and varying degrees of necrotizing enterocolitis are common. one commonly encounters hyperglycemia and hypoglycemia. hyperglycemia generally is related to stress, increased levels of circulating catecholamines, and rapid progression to gluconeogenesis, whereas hypoglycemia is associated with diminished glycogen stores, inability to engage gluconeongenesis, sepsis, and hypoxic damage. 167 immature endocrine function is present in many of these foals, particularly regarding the hypothalamic-pituitary-adrenal axis, and contributes to metabolic derangements. 168, 169 one should delay enteral feeding when possible until the foal is stable regarding metabolic and cardiorespiratory parameters. on intiating enteral feeding, one should provide small volumes initially and slowly increase the volume over several days. one frequently encounters musculoskeletal problems, particularly in premature foals, that include significant flexor laxity and decreased muscle tone. postmature foals frequently are affected by flexure contracture deformities, most likely because of decreased intrauterine movement as they increase in size. premature foals frequently exhibit decreased cuboidal bone ossification that predisposes them to crush injury of the carpal and tarsal bones if weight bearing is not strictly controlled. physical therapy in the form of standing and exercise is indicated in the management of all these problems, but one should take care to ensure that the patient does not fatigue or stand in abnormal positions. bandaging of the limbs is contraindicated because this only increases laxity, although light bandages over the fetlock may be necessary to prevent injury to that area if flexor laxity is severe. the foals are predisposed to angular limb deformity and must be observed closely and frequently for this problem as they mature. 170 the overall prognosis for premature/dysmature/ postmature foals remains good with intensive care and good attention to detail. many of these foals (up to 80%) survive and become productive athletes. 21 complications associated with sepsis and musculoskeletal abnormalities are the most significant indicators of poor athletic outcome. the last 20 years have seen an explosion of new therapeutic agents purportedly useful for treating sepsis. unfortunately, clinical trials investigating these new therapies have failed to demonstrate a positive effect, have shown negative results, or have resulted in diametrically opposed study results, one showing a benefit and another showing no benefit or a detrimental effect. on a positive note, the survival rate of foals being treated for sepsis has improved. work was done regarding foal diseases and their treatment in the 1960s, but the field did not attract much serious attention until the 1980s. since that time almost every major veterinary college and many large private referral practices have constructed nicus or their equivalent. next to hypoxic ischemic asphyxial syndromes, sepsis is the number one reason for presentation and treatment at these facilities. neonatal septicemia of the horse has been the subject of three international workshops, 171-173 and a perinatology lecture covering some aspect of neonatal sepsis has been presented at almost every large continuing education meeting attended by equine veterinarians. concensus criteria conferences 1 in the early 1990s defined sepsis and septic shock for human beings. 174, 175 sepsis was defined as the systemic response to infection manifested by two or more of the following conditions as a result of infection: a) temperature >38â°c or <36â°; b) heart rate >90 beats/min; c) respiratory rate >20 breaths per minute or paco 2 <32 torr; and d) white blood cell count >12,000 cell/âµl, <4,000 cell/âµl, or >10% immature (band) forms. septic shock was defined as sepsis induced hypotension or the requirement for vasopressors/ionotropes to maintain blood pressure despite adequate fluid resuscitation along with the presence of perfusion abnormalities that may include lactic acidosis, oliguria, or acute alteration in mental status. these definitions are broadly acceptable and applicable to neonatal sepsis in foals, and many of the treatment modalities in human medicine have been applied in some manner to the equine neonatal patient. additional definitions that have come into vogue that are actually useful at times, include the following: sirs, the systemic inflammatory response system; mods, multiple organ system dysfuction; and mofs, multiple organ failure syndrome. (sirs is sick, mods is sicker, and mofs is dying.) the compensatory response syndrome (cars) ideally balances sirs and keeps it from becoming detrimental. if balance is achieved, recovery is possible. imbalance progresses to septic shock, mods, and mofs. in horses, mods is manifested most commonly as renal failure, hepatic failure, central nervous system dysfunction, and disseminated intravascular coagulation. managing the septic patient involves early recognition of all the potential alphabet combinations and supporting the patient or intervening in the face of multiple clinical consequences, termed chaos (cardiovacular compromise; homeostasis; apoptosis; organ dysfunction; suppression of the immune system). 176 inflammatory mediators are involved in all these processes and can be beneficial or detrimental, depending on timing and opposing responses. neutrophils, platelets, lymphocytes, macrophages, and endothelial cells are involved, and the implicated inflammatory molecules grow daily in numbers. sepsis in the foal initially can be subtle, and the onset of clinical signs varies depending on the pathogen involved and the immune status of the foal. for the purposes here, the discussion is limited to bacterial sepsis, but the foal also is susceptible to viral and fungal sepsis, which can appear similar to bacterial sepsis. failure of passive transfer (fpt) of immunity can contribute to the development of sepsis in a foal at risk. 177, 178 testing for and treating fpt has received attention in the veterinary literature. it remains true, however, that foals presented to nicus that have an ultimate diagnosis of sepsis have fpt. 16, 19 the current recommendation is that foals have igg levels greater than or equal to 800 mg/dl for passive transfer to be considered adequate. other risk factors for the development of sepsis include any adverse advents at the time of birth, maternal illness, or any abnormalities in the foal. although the umbilicus frequently is implicated as a major portal of entry for infectious organisms in the foal, the gastrointestinal tract may be the primary site of entry. 179 other possible portals of entry include the respiratory tract and wounds. early signs of sepsis include depression, decreased suck reflex, increased recumbency, fever, hypothermia, weakness, dysphagia, failure to gain weight, increased respiratory rate, tachycardia, bradycardia, injected mucous membranes, decreased capillary refill time, shivering, lameness, aural petechia, and coronitis. if sepsis is recognized early, patients with sepsis may have a good outcome, depending on the pathogen involved. gram-negative sepsis remains the most commonly diagnosed, but increasingly gram-positive septicemia is being recognized. 180 foals in intensive care units and at referral hospitals have an additional risk of nosocomial infection. an attempt to isolate the organim involved early in the course of the disease becomes important. if possible, one should obtain blood cultures, and if localizing signs are present, one should obtain samples as deemed appropriate. cultures should be aerobic and anaerobic. recently, work has been done evaluating real-time polymerase chain reaction technology in sepsis in the foal as a means of identifying causative organisms. 181, 182 until one obtains antimicrobial sensitivity patterns for the pathogen involved, one should initiate broad-spectrum antimicrobial therapy (table 19 -5). intravenously administered amikacin and penicillin are good first-line choices, but one should monitor renal function closely. other first-line antimicrobial choices might include high-dose ceftiofur sodium or ticarcillin/clavulanic acid. one should treat failure of passive transfer if present. one should provide intranasal oxygen insufflation at 5 to 10 l/min even if hypoxemia is not present to decrease the work of breathing and provide support for the increased oxygen demands associated with sepsis. 183 should arterial blood gas analysis reveal significant hypoventilation, one may administer caffeine orally or per rectum to increase central respiratory drive. mechanical ventilation may be necessary in cases of severe respiratory involvement such as with acute lung injury or acute respiratory distress syndrome. if the foal is hypotensive, one may administer pressor agents or inotropes by constant rate infusion (table 19-6) . inotrope and pressor therapy generally is restricted to referral centers where these drugs can be given as constant rate infusions and blood pressure can be monitored closely. some practitioners use nonsteroidal antiinflammatory agents and, in specific circumstances, corticosteroids. use of these drugs should be judicious because they may have several negative consequences for the foal including renal failure and gastric/dunodenal ulceration. [184] [185] [186] nursing care is one of the most important aspects of treating septic foals. foals should be kept warm and dry. they should be turned at 2-hour intervals if they are recumbent. feeding septic foals can be a challenge if gastrointestinal function is abnormal, and total parenteral nutrition may be needed. if at all possible, foals should be weighed daily and blood glucose levels monitored frequently. some foals become persistently hyperglycemic on small glucose infusion rates. these foals may benefit from constant rate low-dose insulin infusions (table 19-7) . recumbent foals must be examined frequently for decubital sore development, the appearance of corneal ulcers, and for heat and swelling associated with joints and physis. the prognosis for foals in the early stages of sepsis is fair to good. once the disease has progressed to septic shock the prognosis decreases, although short-term botulism is a neuromuscular disease of foals characterized by flaccid paralysis. 187 although the disease is discussed in detail elsewhere in this text, the form most commonly observed in foals, the toxicoinfectious form, deserves some specific comments. the causative organism is clostridium botulinum, an anaerobic organism. although affected adults usually acquire the disease by ingestion of preformed toxin elucidated from the organism, in the foal less than 8 months of age the organism can survive and multiply in the gastointestinal tract and produce necrotic foci within the liver, giving the foal constant exposure to newly formed toxin. the horse is exquisitely sensitive to the toxin, and only small quantities of toxin are required to produce clinical signs and death in affected animals. the îµ-toxin of c. botulinum binds to the presynaptic membrane of motor neurons and prevents transmission of impulses by blocking the release of acetylcholine from the presynaptic vessicles. this block produces the clinical signs of muscle weakness, manifested in foals as trembling (shaker foals) or acute recumbency. 188 pupillary dilation, dysphagia, tremors, recumbency, and terminal respiratory distress caused by respiratory muscle paralysis occur. foals can be found acutely dead. in endemic areas (the northeast and mid-atlantic regions of united states), for these foals to be evaluated first as having colic is not unusual. treatment aims to neutralize the toxin by administration of botulinum antitoxin and to provide antimicrobial treatment of the infection with penicillin, metronidazole, and/or oxytetracycline. 189, 190 at a minimum, feeding of milk replacer via indwelling nasogastric tube at 20% of the body weight of the foal per day divided into every 2-hour meals is required. many of these foals require respiratory support (in the form of intranasal oxygen insufflation), because of respiratory muscle paralysis. respiratory acidosis is present on arterial blood gas analysis in most of these foals because of hypoventilation and lateral recumbency, but they can tolerate some degree of hypercapnia (paco 2~7 0 mm hg) if the ph is normal and oxygenation (pao 2 >70 mm hg; percent oxygen saturation of hemoglobin, >90%) is adequate. metabolic alkalosis can accompany the respiratory acidosis, but this is a compensatory change and resolves once gas exchange is normalized. some of these patients require mechanical ventilation, which may be lifesaving. one may discontinue mechanical ventilation as clinical signs resolve and the respiratory muscles gain strength. nursing care is important, and these foals should be turned every 2 hours. they should be maintained in sternal recumbency if possible and kept warm and dry. with good nursing care, good nutritional support, and adequate respiratory support, the prognosis for these foals is good. the limiting factor in the prognosis for life is often financial. 190 foals that recover from the acute stage of this disease eventually fully recover. botulism is an expensive disease to treat and is also an entirely preventable disease. 189, 190 all pregnant mares in endemic areas should be vaccinated against c. botulinum. vaccination does not prevent all cases of botulism, particularly if the foal has failure of passive transfer or acquires the disease after maternal immunity wanes and before its own vaccination. nutritional muscular dystrophy or white muscle disease is a vitamin e/selenium-responsive muscle disease of horses of all ages probably caused by a dietary deficiency of selenium and vitamin e. 191 the condition occurs most commonly in geographic areas with low selenium levels in the soil, generally the northeastern, northwestern, great lakes and mid-atlantic regions of the united states. two forms of the disease are described in foals: the fulminant form, in which the foal is found acutely dead, and the subacute form. in the fulminant form, death usually is attributed to myocardial lesions resulting in cardiovascular collapse. the subacute form is characterized by dysphagia and gait abnormalities primarily caused by stiffness of the muscles of locomotion. paralysis, if present, is not flaccid as in botulism. abnormal function of respiratory muscles may complicate the clinical situation. aspiration pneumonia may be present following problems associated with swallowing; the tongue and pharyngeal muscles frequently are affected in the early stages of disease. 191 foals with severe disease may have widespread muscle necrosis leading to hyperkalemia, which can be severe and result in death of the foal. serum activities of the muscle enzymes creatine kinase and aspartate aminotransferase may be greatly increased. diagnosis is confirmed at necropsy or ante mortem by determination of decreased vitamin e, selenium, and glutathione peroxidase concentrations in the blood of the foal before supplementation. myoglobinuria and acute renal failure are not uncommon in these foals. treatment of foals with nutritional muscular dystrophy is primarily supportive. one should address all metabolic abnormalities. some foals require intranasal oxygen insufflation. affected foals are unable to suck effectively, and one should provide enteral (via an indwelling nasogastric tube) or parenteral nutritional support. because of the high likelihood of aspiration pneumonia, one should administer broad-spectrum antimicrobial therapy parenterally. the patient should be kept quiet and should be stimulated minimally. affected foals should receive parenteral (intramuscular) vitamin e and selenium supplementation. selenium is toxic in large doses. the prognosis for severely affected foals is guarded. for less severely affected foals the prognosis is good with appropriate treatment. the disease is preventable by ensuring that mares receive sufficient vitamin e and selenium while pregnant and by supplementing foals with parenteral injections of vitamin e and selenium at birth in endemic areas. a more complete discussion of the pathophysiology of this disease and the nutritional management is presented elsewhere in this text. primary liver disease is uncommon in the foal and occurs primarily as a sequela to sepsis. clinical signs of severe liver disease may include depression, ataxia, and seizures. in affected foals, increases in serum liver enzyme activities and concentrations of ammonia and bile acids frequently can be identified. the mechanism(s) underlying hepatoencephalopathy are not delineated clearly, although increased excitatory neurotransmitters, or compounds that mimic their activity, are implicated. hepatoencephalopathy is discussed in more detail elsewhere in this text. tyzzer's disease (clostridium piliformis infection) rarely causes primary liver disease in foals from 4 to about 40 days of age. this disease is almost uniformly fatal. the incubation period is short, and the mare is thought to be the carrier. [192] [193] [194] [195] [196] clinical signs range from acute death to depression, fever, and pronounced icterus. the feces of affected foals may appear white to grey because of the lack of bile. clinicopathologic abnormalities include leukopenia, hyperfibrinogenemia, metabolic acidosis, and hypoglycemia. 197, 198 liver lesions at postmortem are characterized microscopically by multiple foci of necrosis. one usually can demonstrate variable numbers of elongated, slender intracytoplasmic bacilli within hepatocytes bordering the necrotic foci. infiltration of the portal triads with inflammatory cells and biliary duct hyperplasia and degeneration are observable. the bacillus also occurs in association with myocardial lesions. lesions in the intestine are characterised by mucosal necrosis with inflammatory cell infiltration, increased mucus production, submucosal lymphoid hyperplasia, and submucosal hemorrhage. necrosis of lymphoid follicles, congestion, and hemorrhage can be present in the spleen and mesenteric lymph nodes. 196 affected foals may have a profound metabolic acidosis that is unresponsive to treatment. the clinical course is short, and most affected foals die within a few hours of developing neurologic signs. primary liver disease has been reported in association with ferrous sulfate administration in a probiotic compound. 199 the lesion was massive hepatocellular necrosis and liver failure. the product is no longer commercially available. portosystemic shunt is rare in the foal but has been reported in foals as young as 3 months of age. [200] [201] [202] most infectious causes of neurologic abnormalities in foals are associated with sepsis. although rarely reported, halicephalobus gingivalis (deletrix) infection has been reported in three foals; in one case the foal was 3 weeks of age. 203, 204 possibly transmission in these cases was transmammary; the dam in one case died 1 year later with confirmed h. deletrix infestation of her udder. listeria monocytogenes has been reported as a cause of neurologic disease in foals. 205 recently, sarcocystis neurona was identified as the causative agent of central nervous system disease in a foal, and equine herpes myeloencephalitis has been diagnosed in individual foals and in herd outbreaks involving foals. 206, 207 neospora also was reported in one foal recently. 208 rhodococcus equi abscesses can form in the central nervous system or cause neurologic signs associated with compression, as with vertebral body abscesses. [209] [210] [211] cerebellar hypolasia, occipitoatlantoaxial malformation, and agenesis of the corpus callosum with cerebellar vermian hypoplasia have been reported in foals. [212] [213] [214] [215] [216] [217] ivermectin toxicity and moxidectin toxicity have been reported. 218, 219 electrolyte abnormalities such as extreme hypo-or hypernatremia may result in neurologic manifestations of disease. 220, 221 cervical stenotic myelopathy and degenerative myelopathy also have been reported in foals, although the age at onset is usually more than 4 months. 222 idiopathic epilepsy of arabian foals usually is associated with another infectious disease and is thought to be temporary and self-limiting. causes, diagnosis, and treatment of fpt of immunity are covered in detail elsewhere in this text. failure of passive transfer occurs when a foal fails to ingest a significant quantity of good-quality colostrum. failure of passive transfer may occur by several mechanisms: failure of the foal to suck from the dam for any reason and failure of the dam to produce sufficient quantity of quality colostrum. box 19-4 presents causes of fpt. several methods are available for measuring igg concentration in blood; the most reliable are enzyme-linked immunosorbent assay and single radial immunodiffusion technology-based tests. [223] [224] [225] [226] [227] [228] [229] foals usually are tested at 24 hours of age, but one may test the foal earlier if colostrum ingestion has occurred and a concern exists regarding the passive transfer of immunity status of the foal, recognizing that additional increases in igg concentration may occur with additional time. 230, 231 the concentration of igg in the blood of the foal has been used as an indicator of the adequacy of passive transfer, but the actual blood concentration at which fpt is diagnosed has been challenged in recent years. [232] [233] [234] foals with sepsis commonly have a serum igg concentration of less than 800 mg/dl. 16, 19 foals with fpt are more likely to die from sepsis. 177, 178, [235] [236] [237] one should consider the igg concentration only as a marker for adequacy of colostral absorption. all the measured igg is unlikely to be directed against the specific pathogen affecting any particular neonate, and igg is not the only immune protection afforded the foal by colostrum. many factors that confer local and more general immunity to the newborn are present in colostrum; these include growth factors, cytokines, lactoferrins, cd14, leukocytes, and other yet to be described proteins. [240] [241] [242] [243] [244] by considering igg a marker of adequacy for passive transfer, similar to î³-glutamyltransferase in calves, the clinician can make choices for replacement that are more beneficial to the patient. 245 after one identifies fpt in a foal, treatment depends on the current condition of the foal and its local environment. foals not presently ill and on well-managed farms with low population density and low prevalence of disease may not require treatment if their igg concentration is between 400 and 800 mg/dl. critically ill neonates with fpt in an equine nicu are by definition ill and in an environment with high disease prevalence. these patients require immediate treatment of fpt and frequent reassessment of their passive immunity status. critically ill foals often fail to demonstrate the expected increase in blood igg concentration based on grams of igg administered per kilogram of body mass compared with healthy, colostrum-deprived foals. 235, 246, 247 sick foals also demonstrate a more rapid decline in igg concentration than do healthy foals because they use and catabolize available protein. one may treat foals with fpt by oral or intravenous administration of various products containing igg. one can attempt oral administration of additional colostrum or igg-containing products such as plasma, serum, or lyophilized colostrum in foals less than 12 to 24 hours of age. [248] [249] [250] depending on the age of the foal and the maturity and function of the gastrointestinal tract, this treatment may be effective. many nicus and large breeding farms maintain colostrum banks for this purpose. one should administer plasma intravenously if the foal is not expected to absorb additional colostrum or if the enteral route is unavailable. commercially available hyperimmune plasma products designed for use in foals are available and can be stored frozen. plasma and banked colostrum should be stored in a non-frost-free freezer to minimize protein loss associated with freeze-thaw cycling. 251 one should administer plasma through special tubing with an in-line filter and should monitor patients closely for transfusion reactions. 252 one may use serum and concentrated igg products, but the practitioner should be aware that many of these products focus on igg retention and not on other factors associated with passive transfer of immunity. one should measure igg concentration after transfusion and provide additional plasma as necessary. administration of plasma to critically ill foals without fpt may be beneficial through provision of other factors present in the plasma. in these situations, fresh frozen plasma or fresh plasma may be best, particularly if transfusion of clotting proteins is desired. neonatal isoerythrolysis is a hemolytic syndrome in newborn foals caused by a blood group incompatibility between the foal and dam and is mediated by maternal antibodies against foal erythrocytes (alloantibodies) absorbed from the colostrum. the disease most often affects foals born to multiparous mares and should be suspected in foals less than 7 days of age with clinical signs of icterus, weakness, and tachycardia. a primiparous mare can produce a foal with neonatal isoerythrolysis if she has received a prior sensitizing blood transfusion or has developed placental abnormalities in early gestation that allowed leakage of fetal red blood cells into her circulation. many are the causes of jaundice in newborn foals, including sepsis, meconium impaction, and liver failure, but these usually can be differentiated readily from neonatal isoerythrolysis by measuring the packed cell volume, which is usually less than 20% in foals with neonatal isoerythrolysis. foals with neonatal isoerythrolysis are born clinically normal then become depressed and weak and have a reduced suckle response within 12 to 72 hours of birth. the rapidity of onset and severity of disease are determined by the quantity and activity of absorbed alloantibodies. affected foals have tachycardia, tachypnea, and dyspnea. the oral mucosa is initially pale and then becomes icteric in foals that survive 24 to 48 hours. hemoglobinuria may occur. seizures caused by cerebral hypoxia are a preterminal event. the salient laboratory findings are anemia and hyperbilirubinemia. most of the increased bilirubin is unconjugated, although the absolute concentration of conjugated bilirubin generally is increased well above normal. urine may be red to brown and is positive for occult blood. the natural development of neonatal isoerythrolysis has several prerequisites. first, the foal must inherit from the sire and express an erythrocyte antigen (alloantigen) that is not possessed by the mare. blood group incompatibility between the foal and dam is not particularly uncommon, but most blood group factors are not strongly antigenic under the conditions of exposure through previous parturition or placental leakage. factor aa of the a system and factor qa of the q system are highly immunogenic, however, and nearly all cases of neonatal isoerythrolysis are caused by antibodies to these alloantigens. the exception is in the case of mule foals in which a specific donkey factor has been implicated. [253] [254] [255] mares that are negative for aa or qa or both are considered to be at risk for producing a foal with neonatal isoerythrolysis. the risk involves approximately 19% and 17% of thoroughbred and standardbred mares, respectively. second, and perhaps most important, the mare must become sensitized to the incompatible alloantigen and produce antibodies to it. the mechanism for this is not known in many instances but generally is believed to result from transplacental hemorrhage during a previous pregnancy involving a foal with the same incompatible blood factor. 255 sensitization via transplacental contamination with fetal erythrocytes earlier in the current pregnancy is possible, but an anamnestic response is generally necessary to induce a pathogenic quantity of alloantibodies. 256 ten percent of thoroughbred mares and 20% of standardbred mares have antibodies to the ca blood group antigen without known exposure to erythrocytes. 255 some common environmental antigen is postulated possibly to lead to production of anti-ca antibodies. data suggest that these natural antibodies may suppress an immune response to other blood group antigens because mares negative for aa that have anti-ca antibodies often do not produce antibodies to aa of the erythrocytes in their foals that also contain ca antigen. this antibodymediated immunosuppression is thought to result from the destruction of fetal cells before the dam mounts an immune response to other cell surface antigens. natural alloantibodies have not been associated with neonatal isoerythrolysis in horses. after the mare becomes sensitized to the erythrocytes of her foal, alloantibodies are concentrated in the colostrum during the last month of gestation. unlike the human neonate, which acquires alloantibodies in utero and thus is born with hemolytic disease, the foal is protected from these antibodies before birth by the complex epitheliochorial placentation of the mare. thus the final criterion for foal development of neonatal isoerythrolysis is ingestion in the first 24 hours of life of colostrumcontaining alloantibodies specific for foal alloantigens. immunoglobulin-coated foal erythrocytes are removed prematurely from circulation by the mononuclear phagocyte system or are lysed intravascularly via complement. the rapidity of development and severity of clinical signs are determined by the amount of alloantibodies that was absorbed and their innate activity. alloantibodies against aa are potent hemolysins and generally are associated with a more severe clinical syndrome than antibodies against qa or other alloantigens. the highest alloantibody titers are likely to be produced by mares that were sensitized in a previous pregnancy and then subsequently reexposed to the same erythrocyte antigen during the last trimester of the current pregnancy. prior sensitization of a mare by blood transfusion or other exposure to equine blood products may predispose to neonatal isoerythrolysis. 256 one can make a tentative diagnosis of neonatal isoerythrolysis in any foal that has lethargy, anemia, and icterus during the first 4 days of life. blood loss anemia caused by birth trauma is attended by pallor. icterus caused by sepsis or liver dysfunction would not be associated with anemia. one must base the definitive diagnosis of neonatal isoerythrolysis on demonstration of alloantibodies in the serum or colostrum of the dam that are directed against foal erythrocytes. the most reliable serodiagnostic test for neonatal isoerythrolysis is the hemolytic cross-match using washed foal erythrocytes, mare serum, and an exogenous source of absorbed complement (usually from rabbits). 5 although this test is impractical in a practice setting, a number of qualified laboratories routinely perform this diagnostic service. the direct antiglobulin test (coombs' test) may demonstrate the presence of antibodies on foal erythrocytes; however, false negatives occur frequently. most human or veterinary hematology laboratories can perform routine saline agglutination cross-match between mare serum and foal cells. because some equine alloantibodies act only as hemolysins, agglutination tests may be falsely negative. most field screening tests of colostrum have not proved to be reliable enough for practical use. if one recognizes neonatal isoerythrolysis when the foal is less than 24 hours old, one must withhold the dam's milk and feed the foal an alternative source of milk during the first day of life. one can accomplish this by muzzling the foal and feeding it via nasogastric tube. the minimum necessary amount of milk is 1% of body mass every 2 hours (e.g., a 50-kg foal should receive 500 ml or 1 pint of mare's milk or milk replacer every 2 hours). the udder of the mare should be stripped regularly (at least every 4 hours) and the milk discarded. in most instances, clinical signs are not apparent until after the foal is 24 hours old, when colostral antibodies have been depleted or the absorptive capacity of the foal's intestine for immunoglobulin has diminished. withholding milk at this point is of minimal benefit. supportive care to ensure adequate warmth and hydration is paramount. the foal should not be stressed and exercise must be restricted. confining the mare and foal to a box stall is a best. intravenous fluids are indicated to promote and minimize the nephrotoxic effects of hemoglobin and to correct any fluid deficits and electrolyte and acid-base imbalances. antimicrobials may be necessary to prevent secondary infections. one should monitor foals carefully for the necessity of blood transfusion, although transfusion should be used only as a lifesaving measure. when the packed cell volume drops below 12%, blood transfusion is warranted to prevent life-threatening cerebral hypoxia. erythrocytes from the dam are perfect in terms of nonreactivity with the blood of the foal; however, the fluid portion of the blood of the mare has to be removed completely from the cells to prevent administration of additional harmful alloantibodies to the foal. one can pellet the erythrocytes of the dam from blood collected in acid-citrate-dextrose solution by centrifugation or gravity and then aseptically draw off the plasma by suction apparatus or syringe and replace it with sterile isotonic (0.9%) saline. one thoroughly mixes the cells with the saline and then repeats the centrifugation or sedimentation, followed by aspiration and discarding of the saline. one should perform this washing process at least three times. one then can suspend the packed erythrocytes in an equal volume of isotonic saline for administration. erythrocyte washing by centrifugation is more desirable than gravity sedimentation because antibody removal is more complete and packed cell preparations can be prepared more quickly (each gravity sedimentation requires 1 to 2 hours). packed red blood cells are advantageous in overcoming the problem of volume overload. when equipment or conditions do not allow the safe use of dam erythrocytes, an alternative donor is necessary. because the alloantibodies absorbed by the foal generally are directed against aa or qa and because the latter are highly prevalent among most breeds of horses, a compatible blood donor is difficult to identify. the odds of finding a donor without aa or qa are higher in quarter horses, morgans, and standardbreds than in thoroughbreds and arabians. previously blood-typed individuals negative for aa and qa and free of alloantibodies are optimal. one should give 2 to 4 l of blood or 1 to 2 l of packed erythrocytes over 2 to 4 hours. these allogeneic cells have a short life span and represent a large burden to the neonatal mononuclear phagocyte system, which may cause increased susceptibility to infection. in addition, these cells sensitize the foal to future transfusion reactions. one must measure all potential harm against the benefit in each situation. if a mule foal is the patient, one should not use blood from a female previously bred to a donkey. in cases in which transfusion will be delayed, one cannot identify a compatible donor, or the packed cell volume is so low as to be life-threatening (hemoglobin <5 mg/dl), one may administer polymerized bovine hemoglobin products at a dose of 5 to 15 ml/kg. 257 one may use dexamethasone (0.08 mg/kg) to treat peracute neonatal isoerythrolysis if the packed cell volume is less than 12% and transfusion may be delayed or is not fully compatible, but dexamethasone has detrimental effects on blood glucose regulation in the neonate, and because the antibody in question is of maternal origin, corticosteroid therapy in immunosuppressive doses probably is not indicated. intranasal oxygen insufflation (5 to 10 l/min) may be beneficial. most foals with neonatal isoerythrolysis have adequate passive transfer of immunity, but antimicrobial therapy is indicated to protect against secondary sepsis resulting from the compromised condition of the foal. supportive care and good nursing care, including keeping the foal warm and quiet are essential. one should expect the packed cell volume to decline again 4 to 7 days after transfusion. 258 the prognosis for neonatal isoerythrolysis in foals depends on the quantity and activity of absorbed antibodies and is indirectly proportional to the rate of onset of signs. in peracute cases the foal may die before the problem is recognized, whereas foals with slowly progressive signs often live with appropriate supportive care. like most diseases, neonatal isoerythrolysis is much more effectively prevented than treated. 259 any mare that has produced a foal with neonatal isoerythrolysis should be suspect for the production of another affected foal; thus one should provide all subsequent foals with an alternative colostrum source and discard the colostrum of the dam unless she is bred to a stallion with known blood type compatibility. mares negative for aa and qa alloantigens are most at risk of producing affected foals, thus they should be identified by blood-typing. subsequently, breeding of these mares may be restricted to aa-and qa-negative stallions, thus eliminating the possibility of producing an affected foal. in breeds with a high prevalence of aa or qa alloantigens (e.g., thoroughbreds and arabians), a stallion negative for these and suitable based on other criteria may be difficult to identify. if these "at risk" mares are bred as desired, their serum should be screened in the last month of pregnancy for the presence of erythrocyte alloantibodies. one must test mares with low or equivocal titers closer to the time of parturition. if one detects alloantibodies, the colostrum of the dam should be withheld and the foal then should be provided with an alternative colostrum source. maternal alloantibodies to ca do not appear to mediate neonatal isoerythrolysis in foals and actually may be preventive by removing potentially sensitizing cells from the circulation 56 ; therefore one should not deprive foals of colostrum from mares possessing anti-ca antibodies, even when ca is present on their erythrocytes. rarely, the antigens de, ua, pa, and ab have been associated with neonatal isoerythrolysis in foals; however, to consider mares without these alloantigens to be at risk for neonatal isoerythrolysis is not practical. these syndromes recently have been recognized and described within the veterinary literature, although they have been recognized widely in human neonatology for many years. [260] [261] [262] [263] affected foals demonstrate these hematologic abnormalities within the first week of life, and the mechanism is similar to neonatal isoerythrolysis following ingestion of maternal antibody directed against the platelet or the neutrophil. in general, affected foals are healthy but may demonstrate bleeding tendencies if thrombocytopenia is severe or they may be more susceptible to sepsis. one confirms the diagnosis by appropriate testing for platelet-and neutrophil-associated antibody. 264 one must rule out other causes of neonatal thrombocytopenia and neutropenia, particularly sepsis. foals born to the mare in the future seem likely to be at risk for developing similar problems, and one should treat future foals as one treats neonatal isoerythrolysis foals: prevent sucking from the dam and provide an alternate source of passive immunity in the form of banked colostrum or intravenous plasma. one should provide an alternative nutritional source, such as foal milk replacer, to the foal for the first 48 hours of life and should muzzle the foal while it is in the company of its dam for that period of time. treatment is primarily supportive, but in the case of severe thrombocytopenia, transfusion of platelet-enriched fresh plasma may be indicated. granulocyte colony-stimulating factor has been used in foals with neutropenia, but substantial efficacy has yet to be demonstrated. broad-spectrum antimicrobial therapy may be prudent in cases of alloantibody-associated neutropenia. treatment with immunosuppressive doses of corticosteroids is probably unwarranted, given the increased risk of infection, because the antibody in question is of maternal origin. other specific diseases of the immune system of foals, severe combined immunodeficiency, selective igm deficiency, transient hypogammaglobulinemia, agammaglobulinemia, and other unclassified immunodeficincies are covered in detail elsewhere in this text. the neonate can experience respiratory distress immediately after birth because of several congenital respiratory tract or cardiac anomalies. chief among these causes are bilateral choanal atresia, stenotic nares, dorsal displacement of the soft palate caused by anatomic deformity or neurologic impairment, accessory or ectopic lung lobes, lung lobe hypertrophy, lung lobe dysplasia, cardiac anomalies with right-to-left shunting, and miscellaneous causes such as subepiglotic cysts and severe edema of the larynx. [264] [265] [266] [267] [268] [269] [270] [271] one must evaluate and treat these situations immediately and should consider them true emergencies. one readily can recognize foals with airway occlusion by the lack of airflow through the nostrils despite obvious attempts to breathe and by respiratory stridor. these foals may demonstrate open-mouth breathing and their cheeks may puff outward when they exhale. one foal with congenital bilateral choanal atresia was recognized during extrauterine intrapartum resusucitation because of an inability to pass a nasotrancheal tube. one can establish an effective airway by orotracheal intubation in these cases under most circumstances, but some foals require an emergency tracheostomy. one diagnoses the underlying problem by endoscopy or radiography in most cases. treatment of choanal atresia and cystic structures is surgical, whereas severe laryngeal edema and laryngeal paralysis frequently respond to medical management. until the underlying problem is resolved in these cases, one should administer broad-spectrum antimicrobial therapy and feed the foal by intubation or total parenteral nutrition. one can give colostrum, but these foals frequently develop aspiration pneumonia if allowed to suck from their dams, so intravenously administered plasma also may be necessary to provide sufficient passive immunity. arterial blood gas determinations are the most sensitive indicator of respiratory function readily available to the clinician. the most readily available arteries for sampling are the metatarsal arteries and the brachial arteries. portable arterial/venous blood gas analyzers now are making arterial blood gas analysis more practical in the field, and the technique is no longer reserved for large referral practices. managing a critically ill equine neonate without knowledge of arterial blood gas parameters is veritably impossible. pulse oximetry is useful, but these monitors only measure oxygen saturation of hemoglobin. desaturation can occur rapidly in critically ill neonates. the utility of these monitors in the foal has yet to be demonstrated clearly, particularly in cases of poor peripheral perfusion. 272 the most common abnormalities recognized with arterial blood gas analysis are hypoxemia with normo-or hypocapnia and hypoxemia with hypercapnia. hypoxemia is defined as decreased oxygen tension of the arterial blood (decrease pao 2 ), and hypoxia is defined as decreased oxygen concentration at the level of the tissue, with or without hypoxemia. hypoxia results from hypoxemia, decreased perfusion of the tissue bed in question, or decreased oxygen-carrying capacity of the blood resulting from anemia or hemoglobin alteration. five primary means by which hypoxemia may develop are (1) low concentration of oxygen in the inspired air such as in high altitude or in an error mixing ventilator gas; (2) hypoventilation; (3) ventilation/perfusion mismatch; (4) diffusion limitation; and (5) intrapulmonary or intracardiac right-to-left shunting of blood. hypoxemia is not an uncommon finding in neonates but must be evaluated in terms of the current age of the foal and its position. 15, [273] [274] [275] [276] one also must consider the difficulty encountered in obtaining the sample because severe struggling can affect the arterial blood gas results. table 19 -8 presents normal arterial blood gas parameters for varying ages of foals. the normal foal has a small shunt fraction (~10%) that persists for the first few days of life and contributes slightly to a blunted response to breathing 100% oxygen compared with the adult. hypoxemia frequently occurs in foals with prematurity, pas, and sepsis, although other conditions also result in hypoxemia in the neonate. in the early stage of sepsis associated hypoxemia, paco 2 may be within normal limits or decreased if the foal is hyperventilating for any reason. if the lung is involved significantly in the underlying pathologic condition, such as with severe pneumonia, acute lung injury, or acute respiratory distress syndrome, increased paco 2 may well be present, representing respiratory failure. 277 hypoxemia usually is treated with intranasal humidified oxygen insufflation at 4 to 10 l/min. hypercapnia is not a simple matter to treat. one must try to distinguish between acute and chronic hypercapnia. acute hypercapnia usually is accompanied by a dramatic decrease in blood ph of 0.008 ph units for each 1 mm hg increase in paco 2 . this acidemia can promote circulatory collapse, particularly in the concurrently hypoxemic and/or hypovolemic patient. the effects of more chronic co 2 retention are less obvious because the time course allows for adaptation. the ph change is less, about 0.003 ph units per 1 mm hg increase in paco 2 , because it is balanced by enhanced renal absorption of bicarbonate by the proximal renal tubule. most foals with acute respiratory distress are in the acute stages of respiratory failure, but chronic adaptation begins to occur within 6 to 12 hours and is maximal in 3 to 5 days. one will note an increase in bicarbonate, particularly if the acidemia is primarily respiratory in origin. intravenous administration of sodium bicarbonate to correct respiratory acidosis/ acidemia should be done cautiously in these foals because co 2 retention may only be increased. also, one should remember that 1 meq of sodium is administered with each meq of bicarbonate and hypernatremia has been seen in foals treated exuberantly with sodium bicarbonate. foals with hypercapnia of several days' duration also may develop a blunted respiratory drive to increased co 2 . in these foals, oxygen administration, although essential to treat hypoxemia, may further depress ventilation and further decrease ph. this effect is caused by a loss of hypoxic drive following oxygen therapy. one should consider these foals candidates for mechanical ventilation if the paco 2 is greater than 70 mm hg or is contributing to the poor condition of the foal, such as causing significant ph changes. if hypercapnia is caused by central depression of ventilation, as frequently occurs in foals with pas, one can administer caffeine (10 mg/kg loading dose; then 2.5 mg/kg as needed) per rectum or orally in foals with normal gastrointestinal function. other clinicians may recommend continuous rate infusions of doxapram hydrochoride (dopram; 400 mg/total dose at 0.05 mg/ kg/min) for these foals. if this therapy fails, one should consider mechanical ventilation. mechanical ventilation of foals with central respiratory depression is rewarding and may be necessary only for a few hours to days. a special category is the foal with botulism exhibiting respiratory failure caused by respiratory muscle paralysis. these foals do well with mechanical ventilation, although the duration of mechanical ventilation is more prolonged, frequently more than 1 week. foals with primary metabolic alkalosis usually have compensatory respiratory acidosis. treatment of hypercapnia is not necessary in these cases because it is in response to the metabolic condition. these foals do not respond to caffeine, and they should not be ventilated mechanically if this is the only disorder present. in the neonate, bacterial pneumonia usually results from sepsis or aspiration during sucking. foals with sepsis can develop acute lung injury or acute respiratory distress syndrome as part of the systemic response to sepsis, and this is frequently a contributor to the demise of foals in septic shock. the best way to diagnose bacterial pneumonia is by cytologic examination and culture of a transtracheal aspirate, but blood culture may aid in early identification of the causative organism and allow for early institution of directed antimicrobial therapy. a second frequent cause of bacterial pneumonia in the neonate is aspiration caused by a poor suck reflex or dysphagia associated with pas, sepsis, or weakness. one must take care to ensure that aspiration is not iatrogenic in foals being bottle fed. auscultation over the trachea while the foal is sucking helps identify occult aspiration. one should suspect occult aspiration pneumonia in any critically ill neonate that is being bottle fed or is sucking on its own that has unexplained fever, fails to gain weight, or has a persistently increased fibrinogen level. older foals develop bacterial pneumonia, frequently following an earlier viral infection. 278 bacterial pneumonia is discussed in depth elsewhere in this text, but a few comments specific to the foal are necessary. one should auscultate and percuss the thorax of the foal, but results may not correlate closely with the severity of disease. the most commonly isolated bacterial organism in foal pneumonia is streptococcus zooepidemicus, and one may isolate it alone or as a component of a mixed infection. [278] [279] [280] transtracheal aspirate for culture and cytologic examination is recommended because mixed gram-positive and gram-negative infections are common, and antimicrobial susceptibility patterns can be unpredictable. one should split the obtained aspirate and submit samples for bacterial culture, virus isolation, and cytologic examination. additional diagnostics include radiography, ultrasonography, and serial determination of white blood cell counts (with differential) and blood fibrinogen concentrations. treatment includes administration of appropriate antimicrobial therapy. some foals may benefit from nebulization with saline or other local products. ascarid larval migration through the lung can mimic bacterial pneumonia. 281 in these cases the foal may not respond to antimicrobial therapy and should be dewormed with ivermectin. deworming the mare within 1 month of parturition and frequent deworming of the foal prevent ascarid migration pneumonia in most foals. a special category of bacterial pneumonia in foals is rhodococcus equi bronchopneumonia. this pneumonia of young foals was described first in 1923. 282 the organism originally was known as corynebacterium equi and is a gram-positive pleomorphic coccobacillus usually less than 1 âµm in diameter and 2 âµm in length. the organisms frequently are associated in l-and v-shaped clusters that have been termed chinese character formations. r. equi has an acid-fast staining characteristic under some growing circumstances because of the presence of mycolic acid in its cell wall, similar to mycobacterium and nocardia species. mycolic acid promotes granuloma formation. the organism is able to multiply in and destroy macrophages as it prevents phagosome lysosome fusion. 283, 284 much attention has been paid to this organism in recent years, given its propensity to produce enzootic and epizootic outbreaks of disease. the organism is thought to be primarily an opportunistic pathogen, and it lives in the soil of most geographic areas. foals are affected most frequently between the ages of 1 and 6 months, when maternally derived immunity has begun to wane. the disease is insidious, and foals may have significant pulmonary involvement before developing noticeable clinical signs. phagocytosis of r. equi by equine macrophages is not associated with a functional respiratory burst and, at least in human beings, the l-arginine-no pathway is not required for intracellular killing of this organism. 285, 286 optimal binding of r. equi to mouse macrophages in vitro requires complement and is mediated by mac-1, a leukocyte complement receptor type 3 (cr3, cd11b/ cd18). 287 opsonisation of r. equi with specific antibody is associated with increased phagosome-lysosome fusion and enhanced killing of r. equi, suggesting that the mechanism of cellular entry is important. 283 neutrophils from foals and adult horses are fully bactericidal, and killing of r. equi is enhanced considerably by specific opsonizing antibody. 288 the ability of r. equi to induce disease in foals likely depends on host and microbial factors. knowledge of the virulence mechanisms of r. equi was speculative until the discovery of the virulence plasmid. 289 as opposed to most environmental r. equi organisms, isolates from clinically affected foals typically contain 85-to 90-kb plasmids encoding an immunogenic virulence-associated protein (vapa) that is expressed on the bacterial surface in a temperature-regulated manner. 290 plasmid-cured bacteria lose their ability to replicate and survive in macrophages and are cleared from the lungs within 2 weeks of intrabronchial challenge without producing pneumonia. 291 however, expression of vapa alone is not sufficient to restore the virulence phenotype. six other genes have approximately 40% overall amino acid identity with vapa, and the identification of multiple genes with considerable homology suggests these genes constitute a virulence-associated gene family in r. equi. 292 other candidates for virulence factors include capsular polysaccharides and cholesterol oxidase, choline phosphohydrolase, and phospholipase c exoenzymes ("equi factors"), but their roles have not been defined clearly. the primary manifestation of disease caused by r. equi infection is severe bronchopneumonia with granuloma, abscess formation, or both. up to 50% of foals diagnosed with bronchopneumonia also have extrapulmonary sites of infection. 293 as the pneumonia progresses, clinical signs may include decreased appetite, lethargy, fever, tachypnea, and increased effort of breathing characterized by nostril flaring and increased abdominal effort. cough and bilateral nasal discharge are inconsistent findings. a smaller percentage of affected foals may have a more devastating, subacute form. these foals may be found dead or have acute respiratory distress with a high fever and no previous history of clinical respiratory disease. hyperfibrinogenemia is the most consistent laboratory abnormality in foals with r. equi pneumonia. neutrophilic leukocytosis (>12,000 cells/âµl), with or without monocytosis, is common. 294 thoracic radiography is a useful diagnostic aid, frequently revealing a prominent alveolar pattern with poorly defined regional consolidation and/or abscessation. ultrasonography is a helpful diagnostic tool when the disease involves peripheral lung tissue. although a number of serologic tests have been described, serologic diagnosis of r. equi infections is controversial and difficult because exposure of foals to this organism at a young age leads to production of antibody without necessarily producing clinical disease. 295, 296 serologic tests may be more useful at the farm level to detect overall exposure than at the individual level. bacteriologic culture combined with cytologic examination of a tracheobronchial aspirate remains the most definitive method for accurate diagnosis of r. equi pneumonia. however, foals without clinical disease exposed to contaminated environments may have r. equi in their tracheae from inhalation of contaminated dust; therefore one should interpret culture results in the context of the overall case presentation. 297 culture results in one study were as sensitive as polymerase chain reaction-based assays and offered the advantage of allowing in vitro antimicrobial susceptibility testing. 298 however, polymerase chain reaction is likely to be a useful tool, and results from a second trial suggest the assay is more sensitive and specific than culture of tracheobronchial aspirates for diagnosis. 299 the combination of erythromycin and rifampin has become the treatment of choice for r. equi infections in foals, and the combination reduces the likelihood of resistance to either drug. the recommended dosage regimen for rifampin is 5 mg/kg every 12 hours or 10 mg/kg every 24 hours orally. the recommended dose of estolate or ethylsuccinate esters of erythromycin is 25 mg/kg every 8 or 12 hours orally. 300 recently, azithromycin has been recommended for treatment of r. equi infection at a dosage of 10 mg/kg orally every 24 hours for 5 to 7 days and then every other day. 301 alternatively, clarithromycin at 7.5 mg/kg every 12 hours orally, in combination with rifampin, may be therapeutically effective. severely affected foals may require intranasal oxygen insufflation, intravenous fluid support, and nutritional support. treatment generally continues for 4 to 10 weeks until all clinical and laboratory evidence of infection is resolved. although well tolerated by most foals, erythromycin can result in soft feces. this diarrhea is generally self-limiting and does not require cessation of therapy, but one should monitor affected foals carefully. an idiosyncratic reaction characterized by severe hyperthermia and tachypnea has been described in foals treated with erythromycin during periods of hot weather. 302 affected foals should be moved to a colder environment and treated with antipyretic drugs and alcohol baths if necessary. clostridium difficile enterocolitis has been reported in the dams of nursing foals treated with erythromycin given orally. 303 the dam is exposed to active erythromycin by coprophagy or by drinking from a communal water source where the foal has "rinsed" its mouth. prevention of r. equi pneumonia on farms with recurrent problems is problematic. the most clearly demonstrated prophylactic measure to date has been the administration of plasma that is hyperimmune to r. equi to foals within the first week of life and then again when maternal immunity begins to wane at around 30 days of age. [304] [305] [306] [307] [308] [309] [310] [311] no effective vaccination protocols for the dam or foal have been described to date. farm management is important in preventing disease, and control measures include frequent manure removal, avoidance of overcrowded conditions, and planting of dusty or sandy soils. 304 the prognosis for r. equi bronchopneumonia is fair to good in foals with the more chronic form of the disease. foals with acute respiratory distress have a more guarded prognosis, as do foals with sites of significant extrapulmonary infection. the long-term prognosis for survival for foals with r. equi bronchopneumonia is good, and many foals perform as expected as athletes. 312 the most commonly identified causes of viral pneumonia in foals are equine herpesviruses 1 and 4 (ehv-1 and ehv-4), equine influenza, and equine arteritis virus (eva). equine herpesvirus 1 is probably the most clinically important, but outbreaks of eva in neonates have occurred and are devastating. 27, [313] [314] [315] [316] [317] [318] adenovirus is reported sporadically and as a problem in arabian foals with severe combined immunodeficiency. [319] [320] [321] in the neonate, infection with ehv-1 or eva is almost uniformly fatal and antemortem diagnosis is difficult, even once an outbreak on a particular farm is identified. several factors appear common to foals with ehv-1, including icterus, leukopenia, neutropenia, and petechial hemorrhage, but these problems also are identified in foals with severe sepsis. 315, 322, 323 the antiviral drug acyclovir (10 to 16 mg/kg orally or per rectum 4 to 5 times per day) has been used in cases of ehv-1 in neonates, with some evidence of efficacy in mildly affected foals or foals affected after birth. 323 if viral pneumonia is a possibility, one should collect blood and tracheal aspirates at presentation for bacterial and virus isolation. the lungs of foals with ehv-1 or eva are noncompliant, and pulmonary edema may be present. mechanical ventilation of these cases may prolong life, but death is generally inevitable because of the magnitude of damage to the lungs. foals suspected of having ehv-1 or eva should be isolated because they may be shedding large quantities of virus and pose a threat to other neonates and pregnant mares. foals with eva generally are born to seronegative mares, and intravenous treatment with plasma with a high titer against eva may prove beneficial because passive immunity appears to have a large role in protection against this disease in neonates. 318, 324 older foals and weanlings may be affected by herpesviruses. disease is usually mild, although a fatal pulmonary vasculotropic form of the disease has been described recently in young horses. 325, 326 the clinical signs of disease are indistinguishable from influenza and include a dry cough, fever, and serous to mucopurulent nasal discharge, particularly if secondary bacterial infection occurs. rhinitis, pharyngitis, and tracheitis may be present. treatment of affected foals is primarily supportive. foals also may become infected with ehv-2. the predominant clinical signs are fever and lymphoid hyperplasia with pharyngitis. 327, 328 diagnosis is by virus isolation. rib fractures have been recognized in 3% to 5% of all neonatal foals and can be associated with respiratory distress. 87 potential complications of rib fractures include fatal myocardial puncture, hemothorax, and pneumothorax. rib fractures frequently are found during physical examination by palpation of the ribs or by auscultation over the fracture sites. one can confirm the diagnosis by radiographic and ultrasonographic evaluation. often multiple ribs are affected on one side of the chest. specific treatment is generally unnecessary, but direct pressure on the thorax should be avoided in all cases. some specific patients may benefit from surgical stabilization of some fractures, particularly those fractures overlying the heart. pneumothorax can occur spontaneously or following excessive positive pressure ventilation 329 or following tracheostomy surgery or trauma. any foal being ventilated mechanically that suddenly has respiratory distress and hypoxemia should be evaluated for pneumothorax. diagnosis is by auscultation and percussion of the thorax, but one can confirm the diagnosis with radiographic and ultrasonographic evaluation of the thorax. needle aspiration of air from the pleural space also confirms the diagnosis. treatment is required in cases in which clinical signs are moderate to severe or progressive and involves closed suction of the pleural space. subcutaneous emphysema can complicate treatment of this problem. idiopathic or transient tachypnea has been observed in clydesdale, thoroughbred, and arabian breed foals. in human infants, transient tachypnea can be related to delayed absorption of fluid from the lung, perhaps because of immature sodium channels. 330 in foals, tachypnea generally occurs when conditions are warm and humid and is thought to result from immature or dysfunctional thermoregulatory mechanisms. clinical signs of increased respiratory rate and rectal temperature develop within a few days of birth and may persist for several weeks. treatment involves moving the foal to a cooler environment, body clipping, and provision of cool water or alcohol baths. these foals frequently are treated with broad-spectrum antimicrobial drugs until infectious pneumonia can be ruled out. a syndrome of bronchointerstitial pneumonia and acute respiratory distress has been described in older foals and appears to be a distinct entity from acute respiratory distress syndrome in neonatal foals in association with sepsis. 331 the underlying cause has not been identified, but the genesis is probably multifactorial with several potential pathogens being implicated. affected foals have acute respiratory distress with significant tachypnea, dyspnea, nostril flare, and increased inspiratory and expiratory effort. auscultation reveals a cacophony of abnormal sounds including crackles and polyphonic wheezes in all lung fields. loud bronchial sounds are audible over central airways, and bronchovesicular sounds are lost peripherally. affected foals are cyanotic, febrile, and unwilling to move or eat. foals may be found acutely dead. laboratory abnormalities include leukocytosis, hyperfibrinogenemia, and hypoxemia with hypercapneic acidosis. foals can be dehydrated severely and have coagulation changes consistent with disseminated intravascular coagulation. hypoxic injury to other organs, primarily the kidneys and liver, can occur. chest radiographs reveal a prominent interstitial pattern overlying a bronchoalveolar pattern that is distributed diffusely throughout the lung. this syndrome is a respiratory emergency. treatment is broad-based and includes administration of oxygen, nonsteroidal antiinflammatory agents, broad-spectrum antimicrobial therapy, nebulization, judicious intravenous fluid therapy, nutritional support, and corticosteroid therapy. one must manage hyperthermia in the foal. corticosteroid therapy appears to have been lifesaving in most of the reported surviving foals. because this syndrome is associated with high environmental temperatures in some areas, prevention involves control of ambient temperatures, not transporting foals during hot weather, and keeping foals out of direct sun on hot days, particularly foals being treated with erythromycin for suspected or confirmed r. equi infection. 332 uroperitoneum has been recognized as a syndrome in foals for more than 50 years. 333, 334 classically, affected foals are 24 to 36 hours old at the time clinical signs first are recognized. [334] [335] [336] previous reports had a proportionately larger affected male than female population. 334, 335, 337 the hypothesis was that colts were more at risk because their long, narrow, high-resistance urethra was less likely to allow bladder emptying, resulting in rupture of a full bladder during parturition when high pressures were applied focally or circumferentially around the bladder. 333 more recent reports suggest that such extreme sex bias may have been an artifact of small case numbers in the early reports. rupture or disruption of any structure of the urinary tract can occur. the dorsal wall of the bladder has been reported to be a frequent disruption site, with the ventral wall less likely to be involved. 336 the urachus appears to be the next most commonly affected structure. a few cases of ureteral and urethral defects have been reported. 336, 337 sepsis does not appear to favor one site over the others. 338 the pathophysiology of uroperitoneum is not yet understood fully. the high pressure exerted on a full bladder during parturition once was thought to be the main cause. full bladder and obstruction caused by a partial umbilical cord at parturition, strenuous exercise, and external trauma have been reported as causes. 339 a few reports describe smooth and noninflamed edges of torn tissue, suggesting the possibility of congenital bladder wall defects. 338, 340, 341 sepsis leading to urinary tract rupture and uroperitoneum may occur in foals hospitalized for a variety of unrelated problems. the onset of clinical signs of uroperitoneum may be insidious in these foals, and diagnosis may be less obvious. 338 clinical signs associated with uroperitoneum in the neonatal foal typically include straining to urinate, dribbling urine, and a stretched-out stance. weakness, tachycardia, tachypnea, and not sucking well are also common. a distended abdomen may be evident, and one may feel a fluid wave on ballottement of the abdomen. occasionally, urine accumulates in the scrotum and should not be confused with hernia. foals also may show signs of sepsis, including fever, injected mucous membranes, diarrhea, and disease of other body systems. laboratory findings vary depending on the duration of the uroperitoneum and on the presence and severity of sepsis. classic findings include hyperkalemia, hyponatremia, and hypochloremia arising from equilibration of urine electrolytes and water with blood across the peritoneal membrane. [335] [336] [337] the usual foal diet of milk, which is high in potassium and low in sodium, promotes the electrolyte abnormalities. foals that develop uroperitoneum while receiving intravenous fluids may not have classic electrolyte imbalances at the time clinical signs are recognized. 338 increased serum creatinine concentration is often present, whereas blood urea nitrogen concentrations occasionally, but not consistently, are increased. [335] [336] [337] metabolic acidosis and hypoxemia may be present. some patients also have serum hypoosmolality. 335 one should test foals for failure of passive transfer. one of the most sensitive laboratory tests for uroperitoneum is the ratio of peritoneal to serum creatinine. a ratio greater than or equal to 2:1 is considered diagnostic of uroperitoneum. one should collect peritoneal fluid and test it for creatinine concentration, as well as for cytologic findings, culture, and sensitivity. cytologic evaluation of peritoneal fluid is necessary to identify concurrent peritonitis or other gastrointestinal compromise. one should perform an electrocardiogram on initial evaluation of a foal with suspected uroperitoneum because hyperkalemia may result in bradycardia, increased duration of the qrs complex, a shortened q-t interval, increased p-wave duration, prolonged p-r interval, or atrioventricular conduction disturbances. other possible cardiac sequelae to hyperkalemia include cardiac arrest, third-degree atrioventricular block, ventricular premature contractions, and ventricular fibrillation. 337, 340 for any foal exhibiting signs of dypsnea, tachypnea, or hypoxemia, one should have thoracic radiographs taken before induction of anesthesia to rule out pleural effusion, pneumonia, or acute respiratory distress syndrome, which could complicate ventilation and oxygenation during anesthesia and the postoperative period. ultrasonography has become the tool of choice in the diagnosis of uroperitoneum and is a useful tool available to the practitioner. 342 one can image free peritoneal fluid readily, and tears within the bladder are readily visible. the empty bladder with a significant defect, in a fluid-filled abdomen, will collapse on itself and often have a u shape. one also can visualize urachal and urethral lesions. six of eight foals in one study had urinary tract lesions identified sonographically, and all 31 foals of another study underwent sonographic evaluation, and a significant correlation between ultrasonographic findings and location of the lesion at surgery existed. 336, 338 initial treatment aims to stabilize the patient and correct any electrolyte and acid-base abnormalities and provide fluid volume replacement. one should use 0.9% or 0.45% saline with 5% dextrose until laboratory data are available. a potassium concentration of greater than 5.5 meq/l can be life threatening. one can manage hyperkalemia by peritoneal drainage to decrease whole-body potassium stores using teat cannulae, foley catheters, large-gauge (16 or 14) intravenous catheters, or human peritoneal dialysis catheters. fluid replacement at least should equal the amount of fluid removed from the abdomen to prevent acute hypotension caused by expansion of previously collapsed capillary beds. abdominal drainage also helps ventilation and decreases the work of breathing by decreasing pressure on the diaphragm. one may administer calcium gluconate, glucose, sodium bicarbonate, or insulin intravenously to decrease serum potassium concentrations. these maneuvers do not correct the whole-body potassium overload, however, and once therapy is discontinued, hyperkalemia can reappear until the urine is removed from the abdomen. one should correct hyponatremia slowly. because of the real possibility of concurrent sepsis, one should obtain blood cultures before preoperative administration of antimicrobials. broad-spectrum coverage (penicillin and amikacin or ceftiofur sodium) is recommended until culture results become available. one should perform therapeutic drug monitoring when using aminoglycoside therapy. however, the peak value may be depressed because of the increased volume of distribution represented by the volume of urine in the abdomen, so one should not make dose adjustment based on a low peak until obtaining a new peak after surgical correction of the uroperitoneum. one should treat foals with failure of passive transfer with adequate volumes of intravenously administered plasma. after one has addressed the metabolic abnormalities, one may consider surgical management. medical management using an indwelling foley catheter has been described. 343 preoperative medical stabilization reduces anesthetic risk. safer inhalant agents such as isoflurane also have decreased risk. removal of the internal umbilical remnant at the time of surgery is usual. one should consider culturing any removed umbilical remnant and submitting the remnant for histopathologic evaluation. recurrence of urinary tract rupture can occur. sepsis, hypoxemia, pneumonia, peritonitis, and acute respiratory distress syndrome complicate the management of uroperitoneum. many affected foals are persistently oxygen dependent for several days following surgical correction, and one should perform serial arterial blood gas analyses before discontinuing intranasal oxygen supplementation. prognosis is associated closely with concurrent illness, especially septicemia. uncomplicated uroperitoneum from a defect in the bladder has a good prognosis. if the location of the lesion is other than the bladder, the prognosis is not as favorable. 337 foals with septicemia have a much poorer prognosis. 338, 339 acute renal failure most often occurs as a complication of prenatal asphyxial syndrome, sepsis, or aminoglycoside therapy. acute renal failure also has been reported following oxytetracycline administration in foals. 344 the dose of oxytetracycline commonly used to treat flexural deformities in foals is approximately 10 times the antimicrobial dose. many foals treated in this manner also have suffered some degree of perinatal asphxia, which also damages the kidney, because of prolonged parturition precipitated in part by the flexural deformity. evaluation of renal function in these foals before the administration of the first dose of oxytetracycline and continued monitoring of serum creatinine concentrations before administering subsequent doses of this nephrotoxic compound would seem reasonable. hemodialysis has been used as therapy in one of these cases, but prevention is important because these foals may fail to respond to usual therapy for oliguric renal failure and are euthanized. 344 the most commonly reported congenital deformity of the kidney of the foal is renal hypoplasia and dysplasia, which may have a heritable component. 345, 346 renal arteriovenous malformations have been reported also. 347 ectopic ureters and fenestrated ureters have been described in the foal. [348] [349] [350] congenital renal defects, among others, were reported in three weak, recumbent neonatal foals born to mares being treated for equine protozoal myeloencephalitis. 351 mares received sulfadiazine or sulfamethoxazoletrimethoprim, pyrimethamine, folic acid, and vitamin e orally. the foals were anemic, leukopenic, azotemic, hyponatremic, and hyperkalemic. serum folate concentrations were lower than those reported in the literature for clinically normal brood mares. treatment was unsuccessful. necropsy revealed lobulated kidneys with thin cortices and a pale medulla. the authors postulated that oral administration of sulfonamides, 2,4-diaminopyrimidines (pyrimethamine with or without trimethoprim), and folic acid to mares during pregnancy is related to congenital defects in newborn foals. the umbilicus serves as the conduit for nutrition and gas exchange between the dam and the fetal foal. the urine from the foal is expelled via this structure into the allantoic cavity. the author has recognized cases of in utero bladder distention in the fetus that were associated with multiple twists decreasing urine flow or focal stenosis creating the same effect. foals born with this condition did not have bladder rupture associated with parturition but did have other severe abnormalities that eventually resulted in their demise, primarily premature delivery with failure to adapt to extrauterine life (p.a. wilkins, j.e. palmer, and f.t. bain, unpublished data). at birth the umbilicus breaks, leaving a small external remnant and a large internal remnant. the umbilicus long has been regarded as the primary site of entry of pathogens into the neonate, although this has been challenged recently. treatment of the umbilicus after birth involves dipping it (preferably just the most distal component) with various caustic compounds. the most current recommendation is to treat the umbilicus with dilute chlorhexidine, povidone-iodine, or dilute iodine solutions for just a few times following birth. exhuberant treatment of the umbilical stump with caustic solutions can lead to scalding of the ventral abdomen and may promote patency of the urachus. the ultrasonographic appearance and measurements of the umbilical arteries, urachus, and umbilical vein of foals from 6 hours to 4 weeks of age have been described in detail. 342 a 7.5-mhz sector scanner transducer placed across the midline of the ventral portion of the abdominal wall of the foal works best because of the superficial location of these structures. the mean (â± sd) diameter of the umbilical vein was 0.61 â± 0.20 cm immediately cranial to the umbilical stalk, 0.52 â± 0.19 cm midway between the umbilicus and liver, and 0.6 â± 0.19 cm at the liver. the urachus and umbilical arteries of normal foals have a mean total diameter of 1.75 â± 0.37 cm at the bladder apex. the umbilical arteries scanned along either side of the bladder have a mean diameter of 0.85 â± 0.21 cm. one can use these measurements and the ultrasonographic appearance of the internal umbilical structures from clinically normal foals as references to diagnose abnormalities of the umbilical structures in neonatal foals. 352, 353 the most common abnormalities of these structures are focal abscess formation, hematoma, and urachal tear. herniae traditionally have been thought to develop from failure of closure at the umbilical stump after birth. however, the closure of the body wall defect at the umbilicus was studied in relation to the development of umbilical herniae in a large group of normal foals followed from birth until 5 months of age or from birth until 11 months of age. 354 at birth, approximately half of these foals had a defect in the body wall at the umbilicus that was termed a palpable umbilical ring. in 18 foals this defect disappeared within 4 days, but in one foal the ring did not close and a hernial sac with abdominal contents was palpable. this foal was considered to be the only foal to have a truly congenital umbilical hernia. twelve foals developed an umbilical hernia between 5 and 8 weeks of age. the prevalence of umbilical herniae was much higher than in other studies, possibly because of the prospective nature of the study. based on this study, the large majority of umbilical herniae would appear not to result from failure of closure but rather to be acquired after birth. one should consider the palpable ring structure within the body wall at the umbilicus a variant of normal in the foal and should not call it a hernia until the foal is at least 1 month of age. in one study of 147 horses treated for umbilical herniae over a 13 1 / 2 -year period, only 8.8% developed complications in association with umbilical defects. 355 six horses had intestinal incarceration; the incarceration was reduced manually in 3 horses before admission and resolved without treatment in 2 others. the hernia was surgically reduced in 1 horse. herniorrhaphy was performed on 4 of the 5 horses in which the incarceration did not require surgical reduction, and the fifth was managed conservatively. the study confirmed that complications of umbilical herniae are rare in horses; however, when they do develop, they may be one of various forms, some of which are insidious in onset. the primary differential diagnosis for an external swelling in the umbilical stump region is an external abdominal abscess, which will be firm, variably painful, warm, and nonreducible. ultrasonographic evaluation readily can confirm either possibility. one report describes a 3-day-old foal that died from intestinal strangulation caused by a remnant of vitelline vein that extended between the umbilicus and the portal vein. 356 patent urachus frequently is recognized in the abnormal neonate, probably because of the increased recumbency and decreased movement of these patients. cauterization of a patent urachus is no longer recommended except in cases that persist for long periods of time (>1 month) after the foal becomes more active. surgical resection may provide relief in some foals, but most cases resolve without treatment if given enough time. foals with a patent urachus may posture and strain frequently to urinate, some of this may be associated with irritation or local infection of the urachus. one can alleviate this by administration of broad-spectrum antimicrobial therapy such that the drug has a high concentration in the urine (e.g., trimethoprim-sulfa drug combinations) and by oral administration of phenazopyridine hydrochloride (pyridium), a dye that anesthetizes the urinary tract epithelial surfaces (see table 19 -7). this dye turns the urine orange and stains everything yellow-orange that it or the urine touches but can provide a great deal of relief to foals with this problem. the umbilicus has been considered the traditional point of entry of bacteria into the septic neonate, and septic foals have been referred to as having "navel ill" and "joint ill" in the past. although current thought suggests that the gastrointestinal tract may be the route of entry in most septic neonates, infection of the umbilicus-termed omphalitis, or omphalophlebitis if the vessels are involved-still occurs as a single focus of infection or along with more generalized infection. external signs, such as swelling, heat, pain, ventral edema, or purulent discharge may be present in some foals, but more usually external signs are minimal and one suspects infection because of infection in another site (e.g., an infected joint), fever, or otherwise unexplained increased blood fibrinogen concentration. one confirms the diagnosis by ultrasonographic evaluation of the internal umbilical remnant. any of the umbilical structures may be involved. a complete description of the evaluation is available within the relevant veterinary literature, but the examination is performed best with the foal standing using a 7.5-mhz probe with a standoff. 353 the usual finding is that the affected structure is larger than expected. a fluid-filled core and echogeneic shadows consistent with gas may be apparent in some cases. interpretation requires some experience, and the examiner should be familiar with variants of normal, such as gas shadows associated with a patent urachus and enlarged vessels caused by hematoma formation, so that treatment is not initiated inappropriately. two options for treatment are surgical and medical. medical treatment is preferable in cases in which the lesion is well localized and small and in foals with a medical condition that is not amenable to anesthesia and surgical intervention. one should institute broad-spectrum antimicrobial therapy, and one may need to continue therapy for 2 to 3 weeks. most affected foals respond to medical therapy. frequent reevaluation of the abnormality is necessary, every 5 to 7 days initially, and one should measure blood fibrinogen concentrations at reevaluation because they should stabilize and decrease with effective treatment. failure to respond to therapy within 10 days to 2 weeks suggests that an empiric change in the antimicrobial used may be necessary. in foals that are refractory to medical management or where the lesion is large, surgical excision of the entire umbilical remnant may be desirable. colic in the foal can be difficult to diagnose accurately because one cannot perform an examination per rectum. however, many diagnostic aids, most importantly ultrasonography, are available to help differentiate medical from surgical causes of abdominal discomfort in the foal. intestinal accidents of all types described in adult horses, with the possible exception of enteroliths, occur in foals. intussusception, volvulus, displacement, diaphragmatic hernia, and intra-and extraluminal obstruction have been reported in foals. abdominal ultrasonographic and radiographic evaluation greatly aids diagnosis. treatment is primarily surgical. foals with pas and intestinal dysmotility are at increased risk of intussusception and displacement, and miniature breed foals appear to be at increased risk for fecolith and enterolith formation. meconium retention or impaction is a common cause of abdominal discomfort in newborn foals. most foals defecate shortly after their first meal. the usual practice for most owners or veterinarians attending the birth of a foal is to administer an enema to aid this process. in the past, phosphate-based commercially available enemata (fleet) were used frequently, but if used excessively these types of enemata can create problems of their own, including rectal irritation and hyperphosphatemia. the best enema is warm soapy water made with a mild soap such as liquid ivory soap that can be administered through soft rubber tubing using gravity flow. foals with significant meconium retention become colicky within the first few hours of life as gas accumulates within their bowel. frequently, one can palpate the meconium through the abdominal wall. additional diagnostics can include abdominal ultrasonography and radiography, particularly if one must rule out other, more serious types of colic. these foals assume a classic stance with an arched back. one must differentiate this stance from the stance assumed by foals with uroperitoneum, which is more extended. foals with meconium retention have had simultaneous ruptured bladder, however, so the clinician must be sure to evaluate the foal fully for both problems. foals that do not respond rapidly to enema administration need additional treatment, which can include giving mineral oil (2 to 4 ounces) by nasogastric tube. one can treat persistent meconium retention resulting in significant abdominal distention by muzzling the foal to prevent further milk intake and administering intravenous fluids at an appropriate maintenance rate. if continuous rate infusion is possible, 5% to 10% dextrose is the preferred fluid to use to provide calories to the foal. one should not use dextrose as a bolus fluid. more aggressive treatment would include administration of retention enemata made using acetylcysteine, which serves as an irritant and increases secretion. extreme cases of meconium retention may require surgical intervention, but this is usually not necessary and most cases resolve with medical management alone within 12 to 24 hours. some foals require pain managment. one should avoid nonsteroidal antiinflammatory drugs in the neonate because of their effects on renal function and gastric mucosal blood flow (see gastric ulcers). many foals respond well to butorphanol administered intramuscularly at a dose of 3 to 5 mg to an average 50-kg foal. intranasal oxygen insufflation is beneficial in foals with significant abdominal distention. one should evaluate foals with meconium impaction/ retention for evidence of pas because intestinal dysmotility is common in pas. colostrum is a laxative, and these foals also may suffer from failure of passive transfer, with meconium retention resulting from the lack of adequate colostrum. these foals are also at risk of sepsis because the mucosal intestinal barrier probably has been disrupted and translocation of bacteria can occur. one should obtain blood cultures on these foals and should monitor them closely for signs of sepsis. atresia within the gastrointestinal system of the foal occurs infrequently, but clinical signs are characteristic. 357 acute colic occurs within the first few hours and is accompanied by abdominal distention similar to meconium retention. three primary types of atresia are described in the foal: membrane atresia, cord atresia, and blind-end atresia. antemortem diagnosis of atresia, short of abdominal exploratory surgery, is aided by the lack of meconium staining of the rectum or any administered enema fluids. additional diagnostic tests may include administration of a barium enema for a radiographic study, colonoscopy, and abdominal ultrasonography. abdominocentesis is usually normal until bowel rupture is imminent or has occurred. one can make affected foals more comfortable by muzzling them to prevent further milk intake and by supplying them with fluids and nutrition intravenously. if one attempts surgical correction, one first should initiate broad-spectrum antimicrobial therapy and determine passive transfer status. frequently, these foals are hypoxemic because of the abdominal distention, and oxygen supplementation is desirable. solid white foals born to overo-overo matings of american paint horses may suffer from congential aganglionosis of the ileum, cecum, and colon. these foals present similarly to foals with meconium impaction or atresia in that colic develops shortly after birth and involves progressive abdominal distention with feeding. the inherited defect is in the endothelin receptor gene. [358] [359] [360] [361] no effective treatment exists, but the clinician should be aware that not all white foals of this mating are affected, and some simply may have meconium retention, so a short period of treatment may be warranted. necrotizing enterocolitis is considered the most common acquired gastrointestinal emergency of human infants. 362, 363 the 1500 to 2000 infants that die every year from this disease in the united states and the large number of infants who develop short gut syndrome from this disease only represent the tip of the iceberg of the problems necrotizing enterocolitis causes. the widespread fear of necrotizing enterocolitis among neonatologists and pediatric surgeons has contributed in large part to the use of the intravenous route rather than the gastrointestinal tract for nourishing these infants for long periods. the pathogenesis of necrotizing enterocolitis is unknown but may result from a disturbance of the delicate balance among gastrointestinal perfusion, enteric organisms, and enteral feeding. risk factors for necrotizing enterocolitis in human infants include prematurity, hypoxic-ischemic insult, and formula or breast milk feedings. the clinical spectrum of necrotizing enterocolitis is multifactoral and ranges from temperature instability, apnea, lethargy, abdominal distention, bilious residuals, septic shock, disseminated intravascular coagulation, and death. medical management is usually adequate treatment for necrotizing enterocolitis. in the neonatal foal, necrotizing enterocolitis is probably one of the most underrecognized causes of gastrointestinal dysfunction and in the past has been attributed only to infection with anaerobic organisms including clostridium perfringens type c and c. difficile. 364 although a specific form of enteritis is associated with intestinal infection by these organisms, most necrotizing enterocolitis is associated with prematurity or pas in the infant and the foal. one should suspect necrotizing enterocolitis in any foal that is having difficulty tolerating oral feeding, demonstrating signs of ileus, or having episodes of colic and in any foal with occult blood or frank blood in the stool. foals exhibiting any of these clinical signs should not be fed orally if possible and should receive parenteral nutrition until gastrointestinal function returns to near normal. the mucosal barrier of the intestine is unlikely to be fully intact, and these foals are at risk for sepsis from bacterial translocation. one should institute broadspectrum antimicrobial therapy in these foals and, if any evidence of coordinated gastrointestinal motility is apparent, should administer sucralfate orally as a protectant. gastric ulcer disease has been recognized in foals, and lesions vary in anatomic distribution, severity, and cause. in clinically normal neonatal foals (<30 days of age), gastric ulcers and mucosal desquamation have been documented. [365] [366] [367] [368] because of these reports and other early reports of death following ruptured clinically silent ulcers in neonatal foals, for years many clinicians felt it necessary to treat critically ill neonates with antiulcer medication prophylactically. [369] [370] [371] recently, this paradigm has been challenged. the pathophysiology of gastric ulcer disease is described most reasonably as an imbalance in protective and aggressive factors. [372] [373] [374] these protective factors are responsible for maintaining a healthy gastrointestinal tract by promoting adequate mucosal blood flow, adequate mucus and bicarbonate production, prostaglandin e 2 production, epithelial growth factor production, gastric afferent innervation, epithelial cell restitution, and gastroduodenal motility. probably the most important factor is maintenance of mucosal blood flow. hypoxia, no, prostaglandins, and gastric afferent innervation influence mucosal blood flow. the aggressive factors include gastric acid, bile salts, pepsin, and enzymes. few specific causes have been found for gastric ulcer disease in foals. excessive administration of nonsteroidal antiinflammatory drugs can result in ulceration of the glandular and squamous epithelium because of an inhibition of prostaglandin production, which leads to a decrease in mucosal blood flow and an increase in acid production. nonsteroidal antiinflammatory drugs also can impair the healing of lesions and rarely are indicated in neonatal equine medicine. 372, 373 in the critically ill neonate the suspected cause of gastric ulcers has shifted away from an excessive amount of intraluminal gastric acid toward gastric mucosal ischemia caused by hypoxia, low blood flow conditions, or both. 375 perforating gastric ulcers are more likely a manifestation of necrotizing enterocolitis than of excessive gastric acid. shock, sepsis, or trauma can result in gastric mucosal ischemia, allowing for the disruption of epithelial cell integrity and permitting damage by aggressive factors or providing an environment suitable for the establishment of bacteria colonization. 375,376 impairment of mucosal blood flow also may result in reperfusion injury, allowing the formation of gastric ulcers. in the sick neonatal foal (<7 days of age) a wide variability in the intragastric ph has been documented depending on the type of disease, severity, and milk intake frequency and volume, suggesting that in the critically ill equine neonate, ulcer prophylaxis using histamine antagonists or proton pump inhibitors is not only unnecessary but unlikely to work. 377 clinically significant gastric ulcers can occur in the squamous, glandular, or both portions of the stomach as a primary problem or resulting from another problem. clinical signs include diarrhea, abdominal pain, restlessness, rolling, lying in dorsal recumbency, excessive salivation, and bruxism. in the neonatal foal the only clinical signs present may be depression or partial anorexia until a more catastrophic event, such as perforation, occurs. some lesions in the gastric mucosa extend from the pylorus into the proximal duodenum and can result in stricture of the pylorus and proximal duodenum. these foals are usually older (>1 month of age) and have a greater volume of reflux. bruxism and ptyalism are also more prominent in these older foals. the most sensitive and specific method for diagnosing gastric ulcers is visualization by endoscopic examination. 365 unfortunately, the use of gastric endoscopy has led to recognition of relative nonlesions and ulcers resulting from other problems and of clinically significant disease states. the clinician should not stop simply when ulceration of the stomach is recognized with endoscopy but should examine that patient fully for other potential sources of the clinical signs. other diagnostic tests may help in determining the severity of the ulcers, including fecal occult blood or gastric blood assessments, contrast radiography, abdominal ultrasound, and abdominocentesis. endoscopy of the foal stomach carries an additional risk of exacerbating colic in the short term, unless the examiner ensures that as much introduced air as possible is evacuated from the stomach at the end of the procedure. the presence of a brown gastric reflux fluid may indicate the presence of bleeding ulcers. blood in the feces of the neonate is more consistent with a diagnosis of necrotizing enterocolitis, which can be associated with gastric ulcers. contrast radiography is useful if one suspsects delayed gastric emptying or pyloric or duodenal stricture in older foals. if a stricture has occurred, one will note a delay in complete emptying of barium from the stomach (>2 hours). 367 abdominal ultrasound may be useful to visualize free abdominal fluid and gastric or small intestinal distention if one suspects a perforation. one can visualize portions of the descending duodenum, and a thickened duodenum should increase the index of suspicion for duondenal stricture. abdominocentesis also may confirm perforation. traditional therapy for gastric ulceration includes mucosal adherents, histamine type 2 receptor antagonists, proton pump inhibitors, and antacids. 378 the most widely used mucosal adherent is sucralfate, which is a hydroxy aluminum salt of sucrose. the main therapeutic action of sucralfate is to bind to the negatively charged particles in the ulcer crater. 378, 379 at a ph less than 2, sucralfate is converted to a sticky viscous gel, which adheres to the ulcer crater and remains adhered for 6 hours, but at a higher ph, sucralfate remains in a suspension. sucralfate is still effective because it inhibits pepsin and buffers hydrogen ions. other important actions of sucralfate include stimulating production of prostaglandin e, which maintains mucosal blood flow; increasing bicarbonate secretion; stimulating mucous secretion; decreasing peptic activity; and binding epidermal growth factor. the histamine type 2 receptor antagonists include cimetidine, ranitidine, and famotidine. these compounds block the interaction of histamine with the histamine type 2 receptor on the parietal cell, resulting in inhibition of gastric acid secretion. clinically normal neonatal foals have a highly acidic gastric fluid that is influenced by sucking. intravenous and oral administration of ranitidine increases intragastric ph in normal foals but critically ill neonatal foals have a blunted response to ranitidine administration. 377, 380 one possible conclusion reached from these studies is that in critically ill neonatal foals, gastric ulcers may not be caused by an increased intraluminal gastric acidity. the most commonly used proton pump inhibitor is omeprazole. this drug has not as yet been approved for use in foals under 30 days of age. omeprazole inhibits the secretion of hydrogen ions at the parietal cell by irreversibly binding to the h + ,k + -atpase proton pump of the cell. most of the lesions in older foals were healed after daily administration of omeprazole for 28 days according to one report. 381 table 19 -9 summarizes the therapeutic agents for treating gastric ulcers in foals. prophylactic treatment of critically ill neonates for gastric ulcers has been standard therapy for years because of the evidence of clinically silent ulcers. this approach may not be appropriate for several reasons. an increased incidence of nosocomial pneumonia and systemic sepsis is associated with high gastric ph in human patients in intensive care. [382] [383] [384] patients in intensive care units treated prophylactically with histamine type 2 receptor antagonists are more likely to develop pneumonia during ventilation therapy and gastric colonization with potentially pathogenic bacteria or yeast. 382, 385 an acidic environment appears to protect against airway colonization by bacteria of intestinal origin and bacteria translocated across the gastrointestinal tract. pathogenesis of ulcers in the neonatal foal most likely does not involve increased intraluminal gastric acid but instead may be caused by decreased mucosal perfusion associated with shock, hypoxia, and hypoxic/ischemic insult to the gastric mucosa. a recent report revealed that gastric ulcer disease in equine nicu patients is independent of pharmacologic prophylaxis. 386 in this study, despite decreased treatment, the incidence of gastric ulcers found in these foals at necropsy had decreased significantly. the decrease was attributed to overall improvement in management of these cases. similarly, in a human intensive care unit, the incidence of stress ulcers decreased independent of the use of prophylaxis. 375, 387 early treatment of sepsis, sufficient oxygenation, improved monitoring, institution of enteral feedings, and improved nursing care may contribute to the reduction in gastric ulcers in the neonatal patient. use of histamine type 2 receptor antagonist and proton pump inhibitors apparently may not be necessary; however, in some instances sucralfate may be useful. sucralfate reduced the rate of bacterial translocation in a rat model during hemorrhagic shock and also may prohibit the generation of acute gastric mucosal injury and progression to ulcer formation induced by ischemia-reperfusion. 388, 389 in a human medical intensive care unit, airway colonization by new pathogens occurred more frequently in patients receiving agents that increased gastric ph than in those receiving sucralfate. 382, 390 in the critically ill neonatal foal, risk factors for gastric ulceration have not been identified clearly, although foals treated routinely with nonsteroidal antiinflammatory drugs may be at increased risk for gastric lesions. prophylactic treatment for gastric ulcers in critically ill neonates may not be necessary, and one should consider carefully the pros and cons of their use before their administration. foal heat diarrhea is a mild, self-limiting form of diarrhea that occurs in foals between 5 and 14 days of age, about the time of the "foal heat" in the dam. the definitive cause of foal heat diarrhea has yet to be described, but the condition may be associated with dietary changes or changes in gastrointestinal function that occur around that time. this form of diarrhea is not caused by stongyloides westeri infestation as previously thought. 391 foals with foal heat diarrhea are not systemically ill and should not require therapy. one should evaluate fully any foals with diarrhea at this time for other possible causes of diarrhea, particularly if they are unwell or exhibit anorexia or dehydration. viral diarrhea occurs most commonly in large groups of mares and foals that are housed together. rotavirus is an isolate from the feces of up to 40% of foals with diarrhea worldwide, alone or with another pathogen. 392, 393 the virus infects and denudes the microvilli, resulting in increased secretion combined with decreased absorption. the virus interferes with disaccharidase function and alters the function of the intestinal sodium-glucose cotransport proteins. the initial clinical signs are anorexia and depression, with profuse watery diarrhea occurring shortly thereafter. severely affected foals may become significantly dehydrated and have electrolyte abnormalities, primarily hyponatremia and hypochloremia with metabolic acidosis. these foals generally require intravenous fluid support, whereas less severely affected foals may require only symptomatic therapy. definitive diagnosis is by detection of the virus in the feces of foals with diarrhea. however, none of the available tests are particularly sensitive, and the virus also may be found with other intestinal pathogens. recently, vaccination of pregnant mares has been suggested as a means of prevention, with preliminary results suggesting efficacy. 394,395 although a definitive role for adenovirus has not been established in the foal, adenovirus is a common co-isolate from foals with rotaviral diarrhea. 396 a specific equine coronavirus recently has been identified from an immunocompetent foal with diarrhea, and a second report of cornavirus diarrhea was published recently. 397,398 one case report suggests a parvovirus caused diarrhea in the foal. 399 treatment of viral diarrhea in foals is primarily supportive. intravenous fluid and parenteral nutritional support may be necessary in severe cases. very young foals may benefit from intravenous plasma administration and broad-spectrum antimicrobial coverage to limit bacterial translocation. one can administer sucralfate orally in these cases as a gastrointestinal protectant and to discourage bacterial translocation. foals with moderate to severe metabolic acidosis may benefit from sodium bicarbonate administration if their ventilatory function is normal. one administers sodium bicarbonate at half the calculated deficit (0.5 ã� standard base excess ã� body mass in kilograms) as an isotonic solution at the maintenance fluid rate. one should reevaluate sodium and bicarbonate (or standard base excess) concentrations regularly. nonspecific therapy of diarrhea is discussed elsewhere in this text. diarrhea is frequently the primary presenting complaint in foals with sepsis, so one should rule out this differential diagnosis in foals less than 1 week of age. one should evaluate all neonatal foals with diarrhea for possible sepsis and should include a blood culture whenever possible. clostridium perfringens and c. difficile are recognized increasingly as serious pathogens of the foal. 400-403 foals with either pathogen generally have abdominal pain, dehydration, and profuse watery diarrhea. some foals may have red-tinged or frankly bloody feces, which carries a poorer prognosis. most foals with this type of diarrhea require intensive care or, at the minimum, intravenous fluid administration. outbreaks of this type of diarrhea on farms occasionally occur, and the suggestion is that the dam has a role in transmission of the bacteria. diagnosis is by recognition of the offending organism by gram stain of the feces, by bacterial isolation from the feces, and by detecting the presence of toxins associated with the organisms. specific treatment includes oral administration of metronidazole and broad-spectrum antimicrobial coverage as prophylaxis for bacterial translocation associated sepsis in younger foals. foals with severe blood loss in their feces may require transfusion of whole blood. salmonella spp., escherichia coli, bacteroides fragilis, and aeromonas hydrophila have been implicated in diarrhea in foals. salmonella generally is associated with septicemia in foals, and although some convincing evidence exists for a role for e. coli in foal diarrheal disease, the extent of e. coli as a pathogen of the gastrointestinal tract in foals has yet to be described fully. 371, [404] [405] [406] [407] proliferative enteropathy is a transmissible enteric disease caused by lawsonia intracellulare. 408,409 most foals have been weaned before the appearance of clinical signs of depression, rapid and significant weight loss, subcutaneous edema, diarrhea, and colic. poor body condition with a rough hair coat and a pot-bellied appearance are common in affected foals. clinicopathologic abnormalities included hypoproteinemia, leukocytosis, anemia, and increased serum creatine kinase concentration. postmortem reveals characteristic intracellular bacteria within the apical cytoplasm of proliferating crypt epithelial cells of the intestinal mucosa. antemortem diagnosis of equine proliferative enteropathy is based on clinical signs, hypoproteinemia, and the exclusion of other common enteric pathogens. fecal polymerase chain reaction analysis may be positive for the presence of l. intracellulare, and affected foals develop antibodies against l. intracellulare. 410 treatment with erythromycin estolate alone or combined with rifampin for a minimum of 21 days is recommended with additional symptomatic treatment when indicated. cryptosporidium spp. cause gastroenteritis and diarrhea in many animal species and are not host-specific. cryptosporidium has been implicated as the casual agent of diarrhea in foals, but the organism is isolated from the feces of diarrheic foals and normal foals with the same frequency and concentration, making a clear role for the organism difficult to elucidate. 411-413 diarrhea caused by cryptosporidium in other species and that described for foals is generally self-limiting, with a clinical course of between 5 to 14 days. immunosuppressed patients, including foals compromised by concurrent disease, are thought to be at increased risk for complications resulting from infection with this organism. 411,412 treatment is symptomatic. cryptosporidiosis is a disease with zoonotic potential, and one should take appropriate precautions, including use of gloves and frequent hand washing, if organisms are identified in the feces of any patients so as to prevent spread to other patients and personnel. eimeria leukarti, trichomonas equi, and giardia equi have been identified in the feces of normal horses and horses with diarrhea. transmission studies have failed to produce reliable clinical signs, and the prevalence and significance of these organisms in the genesis of foal diarrhea remain unknown. strongyloides westeri is a common parasitic infection of foals. 392, 414 transmission is transmammary, and patent infection is recognizable in the foal by 8 to 12 days of age. this nematode previously was associated anecdotally with foal heat diarrhea, but the association has not been demonstrated clearly. the diarrhea is generally mild and is treated effectively by deworming with benzimidazole or ivermectin anthelmintics. 391 strongylus vulgaris fourth-stage larvae cause diarrhea in young foals during migration through the arterioles of the cecum and descending colon. clinical signs may resemble thromboembolic colic. 414 the prepatent period is about 6 months, and diagnosis is based on clinical examination, clinicopathologic changes, and farm deworming history. patients with diarrhea associated with this parasite may have peripheral leukocytosis, neutrophilia, eosinophilia, and hypoproteinemia. appropriate deworming with ivermectin (label dose), fenbendazole (10 mg/kg/day orally for 5 days), or thiabendazole (440 mg/kg/day orally for 2 days) is recommended, with the last two drug dosages being larger than the label dose. cyathostomiasis, or diarrhea resulting from the sudden emergence of encysted cyathostome larvae, is an unusual cause of diarrhea in the foal. the clinician managing critically ill neonates must recognize that intravenous fluid therapy simply cannot be scaled down from adult management approaches. fluid management of the ill neonate, particularly over the first few days of life, must take into consideration that the neonate is undergoing a large transition from the fetal to the neonatal state and that important physiologic changes are taking place. 166 these transitions include shifts in renal handling of free water and sodium and increased insensible losses because of evaporation from the body surface area and the respiratory tract. the newborn kidney has a limited ability to excrete excess free water and sodium, and the barrier between the vascular and interstitial space is more porous than that of adults. water and sodium overload, particularly in the first few days of life, can have disastrous long-term consequences for the neonate. 416, 417 in the equine neonate, excess fluid administration frequently manifests as generalized edema formation and excessive weight gain, frequently equivalent to the volume of excess fluid administered intravenously. in cases in which antidiuretic hormone secretion is inappropriate, as in some foals with pas, generalized edema may not form, but the excess free water is maintained in the vascular space. this syndrome of inappropriate antidiuretic hormone secretion is recognized in the foal that gains excessive weight not manifested as edema generally, with decreased urine output and electrolyte abnormalities such as hyponatremia and hypochloremia. 418 the foal manifests neurologic abnormalities associated with hyponatremia. the serum creatinine concentration varies in these cases, but urine always is concentrated compared with the normally dilute, copious amounts of urine produced by foals more than 24 hours of age on a milk diet. if measured, serum osmolarity is less than urine osmolarity. the treatment for this disorder is fluid restriction until weight loss occurs, electrolyte abnormalities normalize, and urine concentration decreases. if the clinician is unaware of this differential diagnosis, the neonate can be assumed mistakenly to be in renal failure, and the condition can be exacerbated by excessive intravenous fluid administration in an attempt to produce diuresis. the problem of appropriate fluid management in critically ill neonates has been recognized by medical physicians for years and has resulted in changes in fluid management of these patients. the approach taken has been one of fluid restriction, in particular sodium restriction but also free water restriction, and has resulted in improved outcome and fewer complications, such as patent ductus arteriosus and necrotizing enterocolitis. 416, 417 the calculations used for maintenance intravenous fluid support in these patients takes into consideration the ratio of surface area to volume and partially compensates for insensible water losses. maintenance fluids are provided as 5% dextrose to limit sodium overload and provide sufficient free water to restore intracellular and interstitial requirements. the calculation for maintenance fluid administration is as follows: first 10 kg body mass 100 ml/kg/day second 10 kg body mass 50 ml/kg/day all additional kilograms of body mass 25 ml/kg/day as an example, the average 50-kg foal would receive 1000 ml/day for the first 10 kg of body mass, 500 ml/day for the next 10 kg of body mass, and 750 ml/day for the remaining 30 kg of body mass for a total of 2250 ml/day. this translates to an hourly fluid rate of about 94 ml/hr. one should adjust the fluid and sodium requirements for ongoing losses exceeding the maintenance requirements. these losses can take the form of diarrheal losses and excessive urine output, such as those with glucose diuresis and renal damage resulting in an increased fractional excretion of sodium. the normal fractional excretion of sodium in neonatal foals is less than that of adult horses, usually less than 1% (j.e. palmer, unpublished data). in the critically ill foal the sodium requirement can be met with as little as 140 meq of sodium per day, about that administered in a single liter of normal equine plasma. one can address sodium deficits by separate infusion of sodium-containing fluids, although this may not be necessary if one considers the sodium being administered in other forms, including drugs administered as sodium salts and any constant rate infusions (pressors, inotropes, etc.) that are being provided as solutions made with 0.9% sodium chloride. the author has used this approach to fluid therapy in her nicu for the last few years and believes that the percentage of foals suffering from generalized edema and related problems has decreased. if one takes this approach to fluid therapy, one should take the weight of the patient once daily, or even twice daily, and monitor the fluid intake and output as closely as practical. one should evaluate any larger than anticipated weight gains or losses. one should not expect urine output to approach the reported normal of 300 ml/hr for a 50-kg foal because the free water administered is limited, unless the patient is experiencing diuresis (glucosuria, resolution of the syndrome of inappropriate antidiuretic hormone secretion, resolution of previous edematous state, renal disease). one should obtain the urine specific gravity several times daily and should determine fractional excretion of sodium at regular intervals. if the volume of urine produced by the patient is measured accurately, one can determine sodium losses accurately and can obtain creatinine clearance values. one should obtain blood pressure measurements at regular intervals throughout the day because hypotension can be a problem in these patients, particularly in septic foals and foals suffering from pas, and one may need to increase fluid therapy to maintain adequate vascular volume. patients with hypotension may need inotrope and pressor support. inotrope and pressor therapy generally is restricted to referral centers where these drugs can be administered as constant rate infusions and blood pressure can be monitored closely. blood pressure can be monitored directly or indirectly by the use of cuffs placed on the base of the tail. both techniques have advantages and disadvantages. although direct blood pressure measurements are considered the gold standard and are generally more accurate, the difficulty in placing and maintaining arterial catheters and lines in these patients severely restricts the utility of this method. indirect techniques can be inaccurate and are affected by cuff size and placement. however, indirect techniques are easier to use in the nicu and can be useful if trained staff are using the equipment. in the author's nicu, once practitioners identify the appropriate cuff size, they dedicate that cuff to that patient for the duration of the hospitalization to decrease variability caused by using different cuffs. one should monitor the blood pressure of all recumbent patients at regular intervals, and trends upward or downward should prompt the clinician to make necessary adjustments. foals suffering from pas and sepsis are the patients most at risk for significant hypotension and perfusion abnormalities. perfusion is maintained by supporting cardiac output and blood pressure with judicious use of intravenous fluid support and inotrope/pressor support. the author does not aim for any specific target systolic, mean, or diastolic pressure. instead the author monitors urine output, mentation, limb perfusion, gastrointestinal function, and respiratory function as indicators that perfusion is acceptable. for nicu patients to require inotrope and pressor therapy is not unusual, but in some cases hypoxic and septic damage is sufficiently severe to blunt the response of the patient to the drugs. one must approach each patient as an individual, and no single inotrope/pressor protocol will suffice for all patients. dobutamine is a î²-adrenergic inotrope that is frequently used as first choice therapy in nicu patients. its effects are î² 1 at the lower dose range. neonates have a limited ability to increase stroke volume in an effort to maintain cardiac output, and one may observe tachycardia in these patients as heart rate increases to maintain cardiac output and vascular pressure. dobutamine is useful after patients are volume replete for support of cardiac output. the dose range is between 2 to 20 âµg/kg/min provided as a constant rate infusion. dopamine has dopaminergic activity at low doses, î² 1 and î² 2 activity at moderate doses, and î± 1 activity at high doses. dopamine causes norepinephrine release, which has lead to the suggestion that this is its major mode of action at higher doses. at doses greater than 20 âµg/kg/min, intrapulmonary shunting, pulmonary venous vasoconstriction, and reduced splanchic perfusion may occur. dopamine also produces natriuresis at lower doses through a direct effect on renal tubules. for these reasons, dopamine has fallen out of favor at some referral institutions. norepinephrine has î± 1 and î² 1 activity but variable î² 2 activity, resulting in potent vasopressor effects; it has inotropic and chronotropic effects, but its chronotropic effect usually is blunted by vagal reflexes slowing the heart rate induced by the increase in blood pressure. in many critical care units, norepinephrine has become a pressor of choice and frequently is used along with dobutamine. evidence suggests that splanchic perfusion is maintained better with norepinephrine than with some other pressors. 419 the dose range is 0.2 to 2.0 âµg/kg/min, although larger doses have been used when necessary in certain patients. epinephrine has î± 1 , î± 2 , î² 1 , and î² 2 activity; î² activity predominates and results in increased cardiac output and decreased peripheral resistance at low doses. epinephrine has been associated with hyperglycemia, hypokalemia, lipolysis, increased lactate concentration, and increased platelet aggregation. the effect on renal function is controversial. use of epinephrine usually is limited to those patients not responding to other pressors. vasopressin (antidiuretic hormone) is a pressor gaining a great deal of attention in the critical care literature. vasopressin appears to be depleted from the neurohypophysis in septic shock, 420 and short-term administration of vasopressin spares conventional vasopressor use, in addition to improving some measures of renal function. 421 low-dose vasopressin infusion increases mean arterial pressure, systemic vascular resistance, and urine output in patients with vasodilatory septic shock that are hyporesponsive to catecholamines. these data indicate that low-dose vasopressin infusions may be useful in treating hypotension in patients with septic shock. 422 the author has been using low-dose vasopressin in patients in her nicu for the past few years and has the clinical impression that blood pressure is defended more readily using this agent in concert with other management strategies. the author commonly uses low-dose vasopressin constant rate infusion with dobutamine constant rate infusion as the initial inotrope/pressor therapy in cases requiring pressure defense, although no prospective studies are yet available regarding this drug in veterinary medicine. 20 for quality health care for animals, advances in medical science, and in some breeds the increasing value of the juvenile equine athlete. equine veterinarians that encounter pediatric orthopedic problems are only beginning to get the information needed to make appropriate treatment decisions. the equine neonate has specific differences in structure and physiology from adults that one must consider when designing an optimal therapeutic or management strategy. few investigations have focused on the equine neonatal musculoskeletal system, 1-6 but a large body of clinical information exists, and one can make cautious extrapolations from work in other species. 7 neonatal equine bones have accelerated modeling and remodeling processes 5 that result in accelerated fracture healing and an increased susceptibility to deformation caused by excessive loading. contralateral limb varus deformities of the growth centers (most commonly distal radius and metacarpus/ metatarsus) are common in overloaded limbs. the increased plasticity of the skeletal structure also is mirrored in the soft tissue support system, for these units become flaccid within 2 weeks of immobilization. 4 this laxity is important, because it further compromises the use of the fractured limb and can last as long as the coaptation was in place. additional divergences from adult physiology include musculoskeletal immaturity (generalized or focal) and immune system differences. finally, foals are lighter and can tolerate and will assume recumbency more readily than adults. the net results of these differences are that one must consider the use of external coaptation carefully, fractures heal quickly, one must consider damage to the contralateral limb from overstress, reducing weight bearing is possible, and infection is always lurking. stresses can affect the musculoskeletal system of the foal at any time, including in utero. although rare, reports describe in utero fractures (k. sprayberry, personal communication, 2003) that result in foal locomotor problems and even maternal uterine damage from sharp bone ends. the cause is presumably from vigorous muscular activity of the foal, but one cannot rule out direct trauma. the fractures result in foal lameness and can increase the likelihood of dystocia and caused colic in one mare when the broken bones damaged the uterus. treatment depends on how long the fracture has been present and on the fracture location and configuration, but if the fracture is repairable, internal fixation probably is necessary. fractures occurring during foaling result from aggressive obstetric manipulation (mandibles) or the advances in medical care of equine neonates in the last 20 years have resulted in the survival of many foals that previously would have died from sepsis, asphyxia, and prematurity; and the successful management of their musculoskeletal system can be a major challenge. major factors adding to the challenge are the immaturity of components of the musculoskeletal system and the demands placed on them by a growing and active foal. additional pressures to treat orthopedic conditions in foals have come from an overall increase in the demand chest compression. one should stabilize unstable mandibular fractures. appendicular fractures usually do not occur during parturition because of the robust character of the bones of the foal. after birth, foals are susceptible to external trauma from many sources. the dilemma is that younger foals with fractures are more likely to heal but also are more likely to develop contralateral limb problems because of excessive weight bearing and affected limb flexor tendon laxity if the limb is immobilized fully. as a result, internal fixation is often the best choice for neonatal fractures to keep the fractured limb in use. proximal sesamoid bone fractures result from hyperextension of the fetlock joint. foals are lame after the fracture, but the lameness can be mild and often diminishes quickly. soft tissue swelling occurs over the sesamoids. fractures are usually simple, can occur uniaxially or biaxially, and can be apical, midbody, or basilar. fractures can occur in any joint and can affect multiple sesamoids in one foal. however, they most commonly are single forelimb fractures 8 and in thoroughbreds are most frequent in the left front medial proximal sesamoid (j.p. morehead, personal communication, 2003). of particular interest to neonatologists is that proximal sesamoids fractures often occur in recovered neonatal patients that are allowed too much exercise too soon. foals from the nicu need a gradual introduction to pasture turnout to allow their musculoskeletal system to adjust. mares are often in need of turnout, but in the interest of their foals, they must wait. treatment of proximal sesamoid fractures in foals is stall confinement with support bandaging. healing occurs, albeit with some distortion of the shape of the sesamoid. severely displaced fragments result in large and misshapen sesamoids, and surgery may be considered for these foals, because restriction of fetlock flexion can occur after conservative therapy. third phalangeal fractures are also common in foals. these foals have a lameness that worsens with hoof compression. hoof abscesses are uncommon in young foals but should be considered. most commonly, radiographs reveal nonarticular small fractures on the wings on the third phalanx. the fractures are associated with hard ground and exercise. the fractures heal with stall confinement, and unlike adults, leave no discernable radiographic fibrous union. avulsion fractures of the proximal insertion of the peroneus tertius and the origin of the long digital extensor tendon have been reported. 9,10 both soft tissue structures attach to the extensor fossa of the distal femur. the two affected foals had lameness of a hindlimb associated with swelling, pain, and crepitation. radiographs revealed multiple avulsion fractures of the extensor fossa. because of the intraarticular fragments in the femoropatellar joint, and the fear of later degenerative joint disease, fragments were removed arthroscopically. both foals were juveniles at last follow-up; one foal was considered normal, and one had a mild residual lameness. tendon and ligament damage is uncommon in neonates probably because of their low body weight. extensor tendon damage following flexural deformities is the most common tendon problem and is discussed in congential flexural deformities of foals. gastrocnemius ruptures are one of the most devastating problems and have occurred after forced extraction because of a breech presentation, severe flexor tendon laxity, and tarsal contracture. loss of gastrocnemius function usually results in a non-weight-bearing limb, although an intact superficial digital flexor tendon may make some weight bearing possible. complete loss of support is difficult to treat successfully. coaptation of the limb is logical but difficult to obtain. schroeder-thomas splints have been used but are difficult to manage. tube casts also are used but must be changed frequently, and cast sores are inevitable (l.r. bramlage, personal communication, 2003) . the prognosis for athletic function is guarded. treatment for ligamentous injuries is usually some form of coaptation, although surgical repairs have been performed when coaptation was unworkable. 11 coaptation in proper limb alignment allows the ligaments to heal and should be used if the injury will destabilize a joint and cause damage to growing epiphyses or cuboidal bones. one can achieve coaptation with casts or splints under a bandage. casts are initially a greater expense, and cast sores and their resulting white hairs are a risk, but the rigid immobilization and the lack of the requirement for daily adjustment makes them preferable. important to musculotendinous health is some measure of weight bearing to avoid laxity after coaptation removal, which one can achieve by using tube casts and splints that allow weight bearing. following coaptation, bandaging and a gradual return to exercise are recommended for ligamentous injuries. patellar luxation can affect foals in one or both hindlimbs, and the luxation can vary from a laxity in the medial attachments to complete luxations that cannot be replaced in the patellar groove of the distal femur. 12, 13 medial luxations have not been reported. clinical signs vary from a slight discontinuous motion during stifle flexion to an inability to stand. many foals have a crouching stance on the affected limb because of an inability to extend the stifle. the pathophysiology of patellar luxations is unknown. congenital bilateral luxations are common in miniature horse foals and are believed to be genetic. luxations are rarer in other breeds and are occasionally traumatic. the affected limbs are usually not grossly abnormal except for effusion of the femoropatellar joint and the luxation. a shallow trochlear groove has been reported to be a cause of patellar luxation, but objective evidence is lacking. one should evaluate foals for the ability to stand. once the appropriate supportive care is provided, if a foal cannot stand, euthanasia is recommended. most bilateral luxations in horses fit in this category. however, miniature horse foals often can stand sufficiently to nurse despite bilateral luxations, and one may consider treatment. treatment consists of replacing and stabilizing the patella and sometimes surgically deepening the patellar groove. delaying surgical repair until the foal is approximately 30 days old is recommended to avoid neonatal problems, allow the musculoskeletal system to mature, and provide good anchors for suture. some surgeons worry that delay may cause further femoropatellar developmental abnormalities, but in a small number of cases, this has not been an issue. the prognosis for miniature horse foals appears to be good because of their low body weights and modest performance expectations. too few reports about the correction of unilateral luxations in light horses exist to make a definitive statement about prognosis except that success and failure have been experienced. 12, 13 congenital flexural deformities in foals can be classified as severe (rarely correctable), moderate (correctable with therapy), or mild (self-correctable). examples of severe flexural deformities include arthrogryposis (deformities of multiple limbs and often the head and neck), severe carpal deformities (flexor angle of the carpus less than 90 degrees), and tarsal contractures (rare). extraordinary methods have been used to correct severe deformities 14 but are often unsuccessful. mild flexural deformities are those that result in an upright conformation to the limb, but the foal can bear weight on the limb and load the flexor structures. these foals require no specific treatment and will self-correct with controlled exercise. moderate flexural deformities are those that make bearing weight on the limb and loading the flexor structures and ligaments difficult for the foal. when these deformities occur bilaterally (most common), the foals cannot rise to suckle or does so with great difficulty, and the lack of weight bearing worsens the flexural deformity. examples of moderate flexural deformities include carpal and forelimb fetlock flexural deformities that usually occur together, hindlimb fetlock flexural deformities with coronopedal flexion or hyperextension, and the uncommon coronopedal flexural deformity alone. treatment of moderate flexural deformities aims to place the solar surface of the foot on the ground so that the weight of the foal can stretch the flexor structures. splints are useful for restoring the limb to normal orientation but require attention to detail because the splints often exert an extreme amount of tension on the soft tissues, and the skin of the foal is thin. pressure sores are easy to create and at a minimum result in an extended convalescence. the first step in splint application is to apply a separate heavy bandage to the limb, which should be reapplied as necessary because the bandage can slip and cause focal constriction. commercial gauze over cotton bandage material works better than sheet cotton as a bandage. the splint is made of polyvinyl chloride pipe cut in half or thirds. using 50% of the diameter of the pipe results in less splint rotation but is bulkier and leaves more splint exposed to cause trauma. one cuts off the corners of the splint and pads the ends with gauze or roll cotton covered with tape. palmar or plantar placement of the splint is preferable, but severe deformities may require initial dorsal placement. as the limb straightens, one can bend the splint to tape the fetlock into the bend to extend it. one can tape the splint tightly to the limb over the bandage with nonelastic (white or duct) tape. this procedure requires at least two persons, one to extend the limb firmly and hold the limb and one to tape. one should leave the splint on for 8 to 12 hours and then remove it for 8 to 12 hours. one can reapply splints as necessary. in addition to splints, some medications are of value for treating flexural deformities. oxytetracycline (40 to 50 mg/kg) given intravenously appears to relax the soft tissues. 15 the mechanism of action is unknown, and the drug is most efficacious when given in the first 3 days of life. this dose is high but appears to be safe for healthy foals and can be repeated at 24-hour intervals. foals should be normovolemic during tetracycline administration. one should use the drug with caution in foals with renal impairment. foals should be urinating and have reasonable urinary parameters (serum urea nitrogen, creatinine, and urinalysis) before tetracycline use. diarrhea is an uncommon sequela to tetracycline use. one should monitor the unaffected limbs closely because all limbs experience a relaxation of the palmar/plantar support. 15, 16 discontinuation of tetracycline therapy before affected limbs are normal but after they can bear weight is common because of worsening laxity in the "normal" limbs. one also can use phenylbutazone (4 mg/kg) for a short time when the splints are used. some analgesia appears to help the foals use the limbs and stretch the soft tissues. one should not use phenylbutazone for long periods of time because of the potential of inducing gastric ulcers. surgical treatment of congenital flexural deformities rarely is indicated. severely affected foals rarely respond favorably to surgery, and mildly affected foals do not need it. surgery is most appropriate for foals with moderate flexural deformities that are neglected or have not responded to splinting and tetracycline. the most common surgical therapy performed for congenital flexural deformities is the inferior check ligament desmotomy for fetlock or coronopedal flexural deformities. ruptures of the extensor tendons commonly occur with congenital flexural deformities and result from the foal overloading the extensor tendons. no specific therapy for the ruptures is necessary. if the rupture is extensive, it can interfere with the ability to extend the fetlock and to place the foot flat. these foals then tend to knuckle over, even after correction of the flexural deformity. a firm fetlock bandage extends the digit and assists in foot placement until the extensor tendons heal. foals commonly are born with hyperextension deformities of the fetlock of varying degrees of severity. all but the worst deformities self-correct as muscle tone improves. a deeply bedded stall is all that is usually necessary to protect the soft tissues, but one can apply a light bandage to the coronary band and pastern if trauma is a problem. severe deformities are more problematic but rare, so therapeutic recommendations are not available. hyperextension of the carpus occasionally occurs and usually is treated conservatively. however, a tube cast to align the limb may be necessary to protect the dorsal surface of developing carpal bones. neonatal foals exhibit three categories of forelimb conformational deviations: angulation, rotation, and carpal offset. angular deviations most commonly are centered in the metaphysis and epiphysis, but their location is described by the closest joint, usually the carpus and fetlock. when the deviation of the distal limb is lateral to the long axis, the deviation is valgus, and when the deviation is medial, the deviation is varus. more than one joint can be affected, and although rare in neonates, valgus and varus can occur in different joints in one limb. rotational deformities appear to originate most commonly in the diaphysis or metaphysis of the radius or the metacarpus. in neonates the direction of rotation of the distal limb at both sites is almost exclusively outward. associated angular and rotational deviations occur. 17 in neonates, limb deviations occur in foals with narrower chests and less developed pectoral muscles than in straight foals, and they appear to have an initial greater overall weakness in the musculoskeletal system because it first interacts with gravity, body mass, and ground reaction forces. however, after the first few days of life, the asymmetric loading of the growth centers does affect limb deviations. angulation results from a compressive load that is asymmetric in a frontal plane but is uniform in the sagittal plane, and rotation occurs when the compressive load is asymmetric in both planes and the limb develops around an overloaded axis point. considered this way, valgus and outward rotation deviations in young foals are coupled, as are varus and inward rotation in older foals. the loading asymmetry for valgus/outward rotation foals is accentuated as foals assume a base-wide posture that is more stable side-to-side but promotes a lateralization of the limb load. the specific effects of intermittent versus static loads, strain magnitude versus strain rate, and shear and hydrostatic stress on growing bones is only beginning to be understood. however, clinical experience supports the general observation that excessive cartilage compression is deleterious to bone growth. offset carpal conformation describes a joint that appears to deviate outwardly and then inwardly, all within the carpus. the deformity is thought to be centered at the radiocarpal joint, but the specific structural cause of offset has not been determined. this conformation is more common in older foals but occasionally occurs in neonates. the deviation is particularly common when incomplete ossification of the carpal bones is present. the causes of conformational deviations are a matter of some debate. as always, the major factors are genetics or environment. genetic influences include the assortment of alleles that controls bone form and growth and the assortment that modulates bone remodeling. many in the horse industry believe that genetics is a strong determiner of limb conformation. environmental influences are many and include the intrauterine environment, the postnatal limb load, nutrition, and bad luck. suffice to say, the situation is complex, but one must consider biologic and mechanicobiologic influences when evaluating the growth of long bones. 18 several factors may contribute to the common occurrence of deviations in the carpus. first, the carpus is in the middle of the limb and is subject to the greatest bending forces. second, the carpal anatomy is complex and perhaps is not understood completely. the carpus has seven cuboidal bones, two long bones, and two epiphyses (distal radial and lateral styloid); and cartilage surrounds all. the ligamentous support includes collateral ligaments, innumerable intracarpal ligaments, and a palmar carpal soft tissue ligament. the distal radial physis is not flat transversely, but undulates in the frontal and sagittal planes. 3 a separate center of ossification for the lateral styloid process is found at its palmar-lateral aspect. because of this separate center of ossification, more cartilage and less bone are in the lateral aspect of the distal radial growth center, suggesting it may be more susceptible to growth alterations from load. less common conformation deformities in young foals include hindlimb deformities, windswept conformation, diaphyseal deviations (usually of the metacarpus/ metatarsus), gross congenital malformations such as agenesis and polydactyly, and acquired varus deformities of the carpus and fetlock. hindlimb conformational deviations can manifest as tarsal and fetlock angular deformities and external limb rotation, usually centered above the tarsus. windswept foals have limbs (usually both forelimb or both hindlimbs) that are curved in the same direction in the frontal plane. diaphyseal deviations, agenesis, and polydactyly are rare and have various presentations. acquired varus deformities are caused by excessive loading, which appears to be focused medially on the growth plates. one should evaluate the limbs to determine the location, extent, and potential cause of the deviation. evaluation consists of observation and then palpation for heat, swelling, or ligament laxity. ligamentous laxity of the medial carpal ligaments is an important cause of carpal valgus and should be evaluated carefully. lameness is not a characteristic of uncomplicated angular limb deformities and suggests further evaluations are necessary. radiography is indicated for foals with severe deviations (all tarsal valgus), ligamentous laxity, lameness, or joint effusions. ultrasonography may be valuable for selected soft tissue evaluations. conservative therapy is by far the most commonly used therapy in foals less than 30 days of age. 19 mild to moderate carpal valgus and external rotation of the carpus and fetlock are common and normal in neonates, particularly light breed horses. most congenital limb deviations improve with age, if the developing musculoskeletal system is protected from overuse and abnormal loads. approximately 90% of thoroughbred foals with congenital carpal valgus self-correct. those foals that do not most often have abnormal bone (incomplete ossification) with normal stress or normal bone with abnormal stress (ligamentous laxity or contralateral limb lameness). correction continues for several months, and on average, foals reach their straightest conformation (regarding angulation) at approximately 10 months of age (e.m. santschi, unpublished data). determination of the appropriate treatment for foals with angular limb deformities is based on the age of the foal, the severity and location of the deviation, and its causes. one must evaluate the entire foal and the affected limb. if the carpal collateral ligaments have no laxity and carpal incomplete ossification is not suspected, one may use an exercise program such as in table 19 -10, assuming that the foal has no contradicting additional problems. exercise is essential for the robust development of almost every body system for neonates, and fresh air and good ventilation reduce the occurrence of respiratory disease. appropriate limb loading along with growth and maturity is what straightens limbs, but excessive amounts of loading can be deleterious. for example, one should use exercise cautiously in foals with very asymmetric deviations. when one limb is much more deviated than the other, it appears to be loaded excessively and compromised more than if both limbs were affected similarly. and finally, limb deviations are additive. foals with external rotation and carpal valgus improve more slowly than those with one type of deviation. incomplete ossification of cuboidal bones and focal ligamentous laxity are complicating matters of great potential impact on adult conformation. they generally manifest as a moderate to severe limb deviation. physical examination indicates laxity because angular limb deviations are reducible. radiographs are the best way to evaluate the extent of carpal bone ossification. incomplete ossification of the cuboidal bones can be focal or widespread. focal immaturity is not common but can result in severe angulation. generalized immaturity is more frequent and initially often manifests as an offset conformation with valgus angulation. when the foal becomes heavier, assumes a base-wide stance, and is allowed exercise, crushing of the bones of the lateral carpus (usually the lateral styloid process of the radius, the ulnar, the fourth and the intermediate facet of the third carpal bone) results in a permanent intracarpal valgus deviation. the same result occurs when significant medial carpal ligament laxity goes untreated. in the forelimb, foals with collateral ligamentous laxity and moderate to severely immature cuboidal bones should have external coaptation placed on the affected limb to maintain axial orientation. tube casts that allow weight bearing on the digit are preferred to splints. ligamentous laxity in the carpus usually responds to tube casting for 7 to 10 days followed by bandaging and cautious exercise. the duration of similar coaptation necessary for immature carpal bones depends on the degree of immaturity and the speed with which the bones mature. because casts cannot be left on neonatal limbs for more than 7 to 10 days because of their fast growth, more than one cast may be necessary. treatment of tarsal valgus and rotational deformities is much less common than in the forelimbs because deviations are less common than in the forelimb, because some breeds prefer an outward position to the hindlimb, and perhaps because owners recognize it less frequently. 20 hindlimbs generally are unaffected by ligamentous laxity, but tarsal incomplete ossification is common and often is associated with tarsal valgus. treatment of tarsal incomplete ossification is important because tarsal crushing results in an unfavorable prognosis for athletic performance. 20, 21 hindlimbs require a slightly different approach to coaptation than forelimbs because of their anatomy. foals can rise to stand if their forelimbs are fixed in extension but cannot do so if their hindlimbs are extended. the multiple bony protuberances of the hock make cast sores more likely than in the forelimb, so casts are problematic. gutter splints are not useful because of the angle of the hock. severely limiting exercise is part of allowing the tarsus to mature without cartilage crushing, but foals cannot always be recumbent. extra small articulated anterior cruciate ligament splints for human beings (playmaker wraparound, dj orthopedics, vista, california) have given the best results. for small foals, a padded bandage is necessary under the splint, which is reversed to conform to the angle of the hock. the splints allow enough flexion in the hock for the foal to rise but appear sufficient when combined with stall rest to protect the cartilage from crushing. splints are left on the hocks until the cuboidal bones have ossified as shown by radiography. fetlock conformational deviations in neonates that are treated best conservatively are rare. outward rotation is the most common deviation but is thought to have minimal effect on the performance and improves with maturity. the only therapy used is to rasp the toe square to promote central breakover. severe outward rotation can promote a fetlock valgus conformation, so one can use a medial hoof wall extension of epoxy to bring the limb load medially. the most commonly treated fetlock deviations are inward but usually occur in foals older than 30 days. however, if the deviation is noticed in neonates, one can use small lateral hoof wall extensions that generally are made of epoxy with fiberglass cloth embedded to prevent chipping. windswept foals are born with multiple deviations. evaluating the foal as a whole is best rather than focusing on individual joints. most of these foals become straight over time with conservative therapy. no surgical procedures are commonly accepted for direct treatment of rotational or carpal offset deviations, so angular deviations are described. surgical procedures to correct carpal and fetlock valgus include periosteal transection and elevation and transphyseal bridging. periosteal elevation is thought to accelerate growth on the concave side of the metaphysis, and transphyseal bridging is used to restrict the growth on the convex side of the physis. studies indicate an approximately 80% improvement of carpal valgus foals after periosteal transection and elevation, but unfortunately they do not compare foals that had surgery with controls that did not. 22, 23 recently, some have suggested that most of the correction was unrelated to the surgery, 24 and one experimental study supports that conclusion. 25 as a result, at this time making firm recommendations about the indications for periosteal transection and elevation is difficult. however, periosteal transection and elevation has a low likelihood of complications and may be effective. the procedure is inexpensive and can be done in the field and therefore may be an option for clients with foals with carpal valgus in which a transphyseal bridging is undesirable or unnecessary. one indication is the very young foal born with a notably asymmetric epiphysis that results in a severe carpal valgus. this distal radial appearance is not particularly common, but the lack of ossification in the epiphysis can make a firm hold with a transphyseal bridging difficult to achieve. however, one can use distolateral radial periosteal elevation at an early age in an attempt to accelerate correction of the valgus and protect developing carpal bones. often a degree of anxiety exists about correction of fetlock angulations because of the much shorter time period for physeal growth. most fetlocks are in their final conformation by 60 days of age, so correction is best accomplished with earlier treatment, usually by 4 weeks of age. one can perform periosteal elevation on the medial (for varus deviations) or lateral (for valgus deviations) aspect of the distal metacarpus/metatarsus. the definitive treatment of limb angulation at a growth plate is transphyseal bridging. one should consider using the procedure at about 3 weeks of age for all moderate to severe fetlock deviations, at about 4 weeks for severe carpal deviations, and 6 to 8 weeks for mild fetlock deviations, moderate carpal deviations, and any worsening angular deformities. one must perform bridge removal when the limb straightens to prevent overcorrection. diaphyseal deviations are rare but can occur in varying degrees of severity. if the foal can bear weight on the limb, a conservative approach is indicated. one can consider periosteal elevation of the length of the concave surface of the long bone. if the foal cannot bear weight on the limb because of the severity of deviation, euthanasia is probably the best option. however, a revision osteotomy and internal fixation may be appropriate for selected foals. 26 polydactyly is also rare and sometimes can be corrected surgically. the outcome is based on the degree of articular involvement. bacteria may invade the foal musculoskeletal system and cause orthopedic infection after delivery by the circulation, by direct extension from another system, or by direct inoculation. hematogenous delivery is by far the most common and results in infection of synovial structures (joints, tendon sheaths, bursae) and bone. extension from another site without hematogenous delivery is rare. direct inoculation almost exclusively results from traumatic rather than surgical wounds. much is still to be learned about the pathophysiology of orthopedic infection, including the source of the infecting bacteria. the umbilicus commonly is accepted as a possible source of bacteria, 27 but many believe that the gastrointestinal and respiratory tracts are at least equally responsible. associated conditions in foals with septic arthritis include failure of passive transfer, pneumonia, and enteritis. 28 the classification of orthopedic sepsis in foals into infection of bones and joints is probably irrelevant because most foals with septic arthritis also have infectious osteitis or osteomyelitis. 27, 29 septic arthritis is more readily recognizable because the reactivity of the synovium to the bacteria causes joint effusion and lameness and because early radiographic signs of bone infection in foals are equivocal. also unclear are the reasons for the apparent site predilection for orthopedic infection in foals. the femoropatellar joint and the tarsocrural joint are affected most frequently, followed by the carpal and fetlock joints, and finally an assortment of miscellaneous joints such as the elbow, shoulder, and hip. 28 the common association of osteomyelitis of the distal femoral, tibial, and metacarpal/metatarsal physes with a newly recognized septic arthritis suggests that the infection in that area started at the growth center (epiphysis, physis, or metaphysis). the localization of the apparent initial site of infection to the growth center has been suggested to result from "looping" metaphyseal vessels with sluggish blood flow that allow pathogens more time to escape the circulation. 29, 30 however, transmission electron microscopy indicates that osteogenic cells and the vascular endothelium are a continuous network in developing embryos, 31 indicating that the relationship between circulation and bone is more intimate than previously suspected. a possible association between osteomyelitis and thickened or traumatized cartilage exists. focal osteomyelitis lesions occur commonly at the bone cartilage junction 27, 29 and particularly in areas where cartilage is attached at an angle to the long axis or where thickened. 29 an association also exists between incomplete ossification of the central and third tarsal bones and osteomyelitis. 32 trauma to the metaphysis is a known predisposing cause of osteomyelitis in young bacteremic rabbits. 33 a trend exists for foals with more than one joint affected to be affected bilaterally in the same joint, rather than in random joints. this trend suggests that a "window" exists when a joint may be more susceptible to infection and that trauma to the developing cartilage may be a contributing factor. in neonates, cartilage is vascular, 34 and possibly small traumatic cartilage lesions with associated hemorrhage and exposure of bacterial binding sites might be the inciting cause for the location of infection. the pathogens most commonly associated with septic arthritis in young foals are also those that frequently are implicated in neonatal sepsis. the most commonly isolated gram-negative organisms are escherichia coli and other enterobacteriaceae, actinobacillus equuli, and salmonella spp. frequently isolated gram-positive organisms include streptococcus spp., staphylococcus spp., and rhodococcus equi. 28 anaerobic bacteria and fungi are rare but should be considered in refractory cases. the diagnosis of orthopedic sepsis can be challenging. the most common clinical sign is lameness, followed by swelling around a joint or metaphysis. joint effusion alone may cause the swelling, but edema is also common, especially if metaphyseal osteomyelitis is present. but effusion and edema can be difficult to detect because of the tissue surrounding the focus of infection in the shoulder, elbow, hip, and coffin joints. one should evaluate lame foals carefully by palpation to localize pain and swelling. if one can find no pain or swelling, one should obtain a complete blood count and fibrinogen level. although a complete blood count is not always abnormal in foals with septic arthritis, abnormalities should raise the index of suspicion of infection. elevations in fibrinogen are fairly common in septic arthritis, 28 and fibrinogen almost always is elevated if the infection involves bone. if hematologic values are normal, the lameness could be caused by trauma, but the foal should be monitored closely for improvement, and closer evaluation is indicated if improvement is not rapid. an arthrocentesis is the diagnostic test of choice for confirmation of septic arthritis. one should perform joint puncture in a sterile fashion, and sedation is indicated to get an atraumatic tap. short-term anesthesia is preferable when joints have effusion because one may perform joint lavage at the same time. normal joint fluid should be clear to slightly yellow, should be viscous, and should contain less than 2500 nucleated cells per deciliter. the cell ratio should be roughly 50:50 polymorphonuclear and mononuclear. the total protein content should be less than 2.5 mg/dl. one should consider joints to be infected if the nucleated cell count is greater than 10,000 cells/dl. for joints falling between 2500 and 10,000 cells/dl, if the polymorphonuclear cell count is >90%, one should consider the joints infected. cytologists are often reluctant to diagnose infection when nuclear degeneration or bacteria are not visible. this is overly conservative and results in delay in treating infections because bacteria and nuclear degeneration are rare in early cases of joint infection. out of an abundance of caution, one should treat lame foals with suspicious joints as infected unless they are clearly normal. one should always culture joint fluid in an attempt to identify the offending organisms, but because of difficulties in culturing pathogens from joint fluid samples, absence of growth does not mean absence of infection. one obtains the best culture results if the foal has not been treated with antimicrobial agents beforehand. one should obtain as much joint fluid as possible for culture and should incubate it overnight in blood culture media before plate inoculation. as always in potentially septic foals, blood culture may assist in the isolation of the organism. other orthopedic infections that do not involve the joint may be more difficult to detect. often these are not apparent until infection breeches the joint and causes lameness. however, astute caretakers may notice early clinical signs such as mild lameness, fever, or edema centered at a growth center. radiography and advanced imaging modalities such as magnetic resonance imaging are the best diagnostic tools for the localization of areas of osteitis and osteomyelitis. one should examine the area of concern carefully, giving particular attention to the growth centers and subchondral bone. interpretation of radiographs may be difficult because these areas are complex and normally have irregular bone margins in the growing foal. if a normal contralateral joint is available, comparison radiographs may be useful. because of the high metabolic turnover in growing foal bone, changes occur faster than with adults, so radiographs at the earliest sign of potential infection of bone and joint are recommended. if evidence of osteolysis is clear, aspiration of the area may yield material for culture. the goals of treatment are to eliminate infection immediately and then resolve inflammation. bacteria and products of inflammation elicited by infection are responsible for destruction of bone and cartilage. the ultimate aim of treatment is to protect the structures critical to athletic performance such as subchondral bone and cartilage in weight-bearing areas. advances in the treatment of sepsis have resulted in hospital discharge rates of 78% for foals with septic arthritis, but their rate of high performers is 30%, 28 indicating a need for improvement. equine veterinarians cannot replace what has been destroyed, so early identification and aggressive therapies are presently the best methods to improve performance rates. one achieves the goals of treatment by physical removal of bacteria, products of inflammation, and debris and by medications to kill the bacteria and reduce inflammation. one should optimize the physiology and general health of the foal to assist this process; one should include other treatments and supportive therapies for septic foals, especially treatment of failure of passive transfer, in the therapeutic plan. intravenous administration of antimicrobials (see chapter 4) is the cornerstone of treatment of orthopedic infection, and if the drug is administered early in the course of infection and bacteria are susceptible, intravenous administration may be sufficient to eliminate the organisms. however, treatment of many foals does not begin until disease is advanced. if treatment begins after bacteria have had a chance to establish themselves, one should bring all appropriate methods to bear to end the infection. additional therapies for septic arthritis include joint lavage, arthrotomy (for drainage), 35,36 debridement (arthroscopically or arthrotomy), 37 intraarticular administration of antimicrobials, intravenous regional perfusion, 38 and antimicrobial beads. 39, 40 one can use any sterile isotonic solution to flush a joint, and additives do not appear to give significant additional benefit. if radiographs do not indicate osteomyelitis, lavage, intraarticular antibiotics, and if possible, regional perfusion are recommended. if osteitis or osteomyelitis is present, debridement is indicated arthroscopically or via arthrotomy (one should culture the debris if the pathogen is unknown). if the joint is closed, one may use antibiotics intraarticularly. if the joint is left open to drain, regional perfusion is useful. antimicrobial beads theoretically are best to use if the wound is closed, but they appear to give benefit even if the wound is open under a bandage. because of concerns about the use of beads in a joint, 41 beads often are used in tissue defects and the surrounding tissues. the major goal is to remove material that is compromising healthy tissues and to obtain high concentrations of antimicrobials in infected tissues. high antimicrobial concentrations are necessary because adhered bacteria are difficult to kill and may require many times the in vitro bacterial minimum inhibitory concentration. intraarticular administration of antimicrobials has been used for many years and has great value. 35 regional perfusion of diluted antimicrobials recently has come into use and may be administered intraosseously 42 or intravenously. intravenous perfusion is preferable because no special equipment is needed, but intraosseous perfusion may be valuable where intravenous access is impossible. the concept behind both procedures is to fill the venous vasculature in the area of the infection with antimicrobials diluted by a sterile balanced electrolyte solution. one isolates the anatomic area of interest using one or two tourniquets. the perfusate diffuses into all tissues and achieves much higher concentrations than are possible using intravenous therapy. this technique has shown excellent results as an adjunct therapy for orthopedic infection. 43 for foals, 12 to 20 ml total of perfusate containing 250 mg amikacin is useful for most single joint sites. amikacin has given consistently good results without complication and is a good choice based on its concentration-dependent activity. one may use a higher volume for the stifle, but the thigh musculature makes an effective tourniquet difficult to achieve. because of concerns that perfusion might dislodge bacteria and renew systemic sepsis, high concentrations of systemic antimicrobials are recommended at the time of the perfusion. if joint lavage and intraarticular administration of antimicrobials are not sufficient to resolve infection, one may perform arthrotomy to assist the joint to drain. passive and active drains add foreign material and so are not useful. maintaining the joint under a sterile bandage is critical and can be difficult to do in proximal joints such as the stifle and elbow. tie-over bandages can be useful in this application. the best measure of success is the resolution of lameness and local inflammation. radiographs may be helpful, but the most common sign of success is a failure of the infection to progress, rather than radiographic healing. one should continue intravenously administered antimicrobials for at least 1 week after the resolution of lameness. if an appropriate drug is available, one should give foals antimicrobials orally for at least 2 weeks more. a total of at least 4 weeks of antimicrobials is recommended for most foals with orthopedic infection. treatment failures usually result from an inability to kill bacteria adhered to isolated tissue (usually dead bone). sometimes this failure is caused by incomplete debridement or an inability to access a known site of infection, but more frequently it is because infection has flourished in an unknown site. for this reason, multiple imaging modalities (radiographs, ultrasound, computed tomography, and magnetic resonance imaging) used multiple times are recommended for all refractory cases of septic arthritis. osteomyelitis not associated with a joint still involves a growth center. the ideal treatment for these infections is surgical debridement, systemic antimicrobial therapy, and some form of local antibiotic delivery. 44, 45 even in the face of large initial osseous defects, infection may resolve, the defect may heal, and the foal may regain normal limb anatomy and function with appropriate therapy. clinical studies on 4 newborn throughbred foals suffering from convulsions with special reference to blood gas chemistry and pulmonary ventilation respiratory distress in a newborn foal with failure to form lung lining film clinical studies on the newborn thoroughbred foal. 2. heart rate, auscultation and electrocardiogram blood gas tensions and ph values in the normal thoroughbred foal at birth and in the following 42h measurements of pulmonary ventilation in normal newborn thoroughbred foals during the first three days of life some parameters of respiratory function in normal and abnormal newborn foals with special reference to levels of pao2 during air and oxygen inhalation modern concepts of neonatal disease in foals studies on fetal, neonatal and maternal cortisol metabolism in the mare surfactant studies in the fetal and neonatal foal neuropathology of the convulsive foal syndrome metabolic profiles of newborn foals neuropathological changes associated with the neonatal maladjustment syndrome in the thoroughbred foal blood gas and acid-base status in spontaneously delivered, term-induced and induced premature foals developments in management of the newborn foal in respiratory distress. 1. evaluation clinical and clinicopathological characteristics of the septicaemic neonatal foal: review of 38 cases intensive care of the neonatal foal nutritional support of the foal during intensive care comparison of empirically developed sepsis score with a computer generated and weighted scoring system for the identification of sepsis in the equine neonate ion transport properties of fetal sheep alveolar epithelial cells in monolayer culture pulmonary vascular biology during neonatal transition regulation of vasodilator synthesis during lung development morin fc 3rd: persistent pulmonary hypertension of the newborn: role of nitric oxide and endothelin in pathophysiology and treatment clinical studies on the newborn thoroughbred foal. 1. perinatal behavior the adaptive processes of the newborn foal blood pressure, electrocardiogram and echocardiogram measurements in the growing pony foal transfer of gases and metabolites in the equine placenta: a comparison with other species a comparative study of blood gas tensions, oxygen affinity and red cell 2,3 dpg concentrations in foetal and maternal blood in the mare, cow and sow neonatal polycythemia and hyperviscosity respiratory mechanics and breathing pattern in neonatal foals mechanics of ventilation: compliance respiratory studies in foals from birth to seven days old the distribution of ventilation-perfusion ratios in the lungs of newborn foals neurological examination of newborn foals maternal behavior equine medicine and surgery umbilical cord compression produces pulmonary hypertension in newborn lambs: a model to study the pathophysiology of persistent pulmonary hypertension in the newborn the sequence of events in neonatal apnoea resuscitation with room-air or oxygen supplementation resuscitation with room air instead of 100% oxygen prevents oxidative stress in moderately asphyxiated term neonates six years of experience with the use of room air for the resuscitation of asphyxiated newly born term infants resuscitation of newborn infants with room air or oxygen thoracic trauma in newborn foals an advisory statement from the pediatric working group of the international liaison committee on resuscitation pharmacology of pediatric resuscitation vasopressin and epinephrine for cardiac arrest temperature of the human fetus continuous monitoring of fetal temperature by noninvasive probe and its relationship to maternal temperature, fetal heart rate, and cord arterial oxygen and ph suppressive action of endogenous adenosine on ovine fetal nonshivering thermogenesis perinatal thermogenesis factors influencing the initiation of nonshivering thermogenesis reversible umbilical cord occlusion: effects on thermogenesis in utero diazepam in labour: its metabolism and effect on the clinical condition and thermogenesis of the newborn renal clearance, urinary excretion of endogenous substances, and urinary diagnostic indices in healthy neonatal foals indices of renal function: values in eight normal foals from birth to 56 days a comparison of inulin, para-aminohippuric acid, and endogenous creatinine clearances as measures of renal function in neonatal foals effects of hyperglycemia or hypoglycemia on brain cell membrane function and energy metabolism during the immediate reoxygenation-reperfusion period after acute transient global hypoxia-ischemia in the newborn piglet equine uteroplacental metabolism at mid-and late gestation glucose and oxygen metabolism in the fetal foal during late gestation mechanism of glucose transport across the human and rat placental barrier: a review glucose production in pregnant women at term gestation: sources of glucose for human fetus estimation of glucose turnover and 13c recycling in the human newborn by simultaneous [1-13c]glucose and [6,6-1h2]glucose tracers glucose disposal of low birth weight infants: steady state hyperglycemia produced by constant intravenous glucose infusion precursors to glycogen in ovine fetuses activation of glycogenolysis in neonatal liver the onset of breathing at birth stimulates pulmonary vascular prostacyclin synthesis pulmonary arterial pressure changes in human newborn infants from birth to 3 days of age postnatal circulatory adaptation in healthy term and preterm neonates pulmonary endothelial nitric oxide production is developmentally regulated in the fetus and newborn regulation of pulmonary vascular tone in the perinatal period the structural basis of persistent pulmonary hypertension of the newborn infant tolazoline hcl (priscoline) nitric oxide for respiratory failure in infants born at or near term effect of inhaled nitric oxide on experimentally induced pulmonary hypertension in neonatal foals systematic review of therapy after hypoxic-ischaemic brain injury in the perinatal period infection, inflammation and the risk of cerebral palsy the fetal circulation and its response to stress fetal adaptations to spontaneous hypoxemia and responses to maternal oxygen breathing pathophysiology of perinatal brain damage enhanced calcium uptake by ca1 pyramidal cell dendrites in the postischemic phase despite subnormal evoked field potentials: excitatory amino acid receptor dependency and relationship to neuronal damage activation of synaptic nmda receptors by action potential-dependent release of transmitter during hypoxia impairs recovery of synaptic transmission on reoxygenation molecular and biochemical mechanisms of perinatal brain injury glycine site of the excitatory amino acid n-methyl-d-aspartate receptor in neonatal and adult brain reduction of voltagedependent mg2+ blockade of nmda current in mechanically injured neurons magnesium attenuates a striatal dopamine increase induced by anoxia in the neonatal rat brain: an in vivo microdialysis study pretreatment with magnesium sulfate protects against hypoxic-ischemic brain injury but postasphyxial treatment worsens brain damage in seven-day-old rats improved motor outcome in response to magnesium therapy received up to 24 hours after traumatic diffuse axonal brain injury in rats fetal rat brain damage caused by maternal seizure activity: prevention by magnesium sulfate can magnesium sulfate reduce the risk of cerebral injury after perinatal asphyxia magnesium sulfate treatment after transient hypoxia-ischemia in the newborn piglet does not protect against cerebral damage concentrations of magnesium and ionized calcium in umbilical cord blood in distressed term newborn infants with hypoxic-ischemic encephalopathy preliminary study on the pharmacokinetics of phenobarbital in the neonatal foal adverse effects of early phenobarbital administration in term newborns with perinatal asphyxia negative pressure pulmonary edema as a post-anesthetic complication associated with upper airway obstruction in a horse cerebral oedema and cerebellar herniation in four equine neonates cerebral edema edematous necrosis in thiamine-deficient encephalopathy of the mouse a herd outbreak of equine leukoencephalomalacia dimethyl sulfoxide (dmso): a review naloxone reverses neonatal depression caused by fetal asphyxia the effects of naloxone on the post-asphyxic cerebral pathophysiology of newborn lambs naloxone exacerbates hypoxic-ischemic brain injury in the neonatal rat resuscitation of the newly born infant: an advisory statement from the pediatric working group of the international liaison committee on resuscitation neurologic disorders in foals other than hypoxicischemic encephalopathy combination therapy protects ischemic brain in rats: a glutamate antagonist plus a gamma-aminobutyric acid agonist effect of gamma-aminobutyric acid modulation on neuronal ischemia in rabbits cerebral hypothermia for prevention of brain injury following perinatal asphyxia current options in the management of apnea of prematurity hypocapnia and hypercapnia in respiratory management of newborn infants acute renal failure in children: aetiology and management low-dose dopamine in the intensive care unit: dnr or dnrx? use of dopamine in acute renal failure: a meta-analysis the effects of dopamine and epinephrine on hemodynamics and oxygen metabolism in hypoxic anesthetized piglets initial experience with norepinephrine infusion in hypotensive critically ill foal multiple organ involvement in perinatal asphyxia continuous enteral feeding impairs gallbladder emptying in infants trophic feeding of the preterm infant continuous nasogastric milk feeding versus intermittent bolus milk feeding for premature infants less than 1500 grams functional residual capacity and passive compliance measurements after antenatal steroid therapy in preterm infants current surfactant use in premature infants effects of the fungal endophyte acremonium coenophialum in fescue on pregnant mares and foal viability postnatal renal adaptation in preterm and term lambs risk factors of hypoglycemia in premature infants studies on equine prematurity. 4. effect of salt and water loss on the reninangiotensin-aldosterone system in the newborn foal studies on equine prematurity. 5. histology of the adrenal cortex of the premature newborn foal angular limb deformities in premature/ dysmature foals neonatal septicemia workshop 1, dorothy havemeyer foundation dorothy havemeyer foundation american college of chest physicians/society of critical care medicine consensus conference definitions of the systemic inflammatory response syndrome and allied disorders in relation to critically injured patient definition of sepsis to sirs with love failure of passive transfer in foals: incidence and outcome on four studs in new south wales a prospective study of septicaemia in colostrum-deprived foals care of the gut in the surgical intensive care unit: fact or fashion? bacterial isolates from blood and their susceptibility patterns in critically ill foals: 543 cases (1991-1998) development of real-time taqman pcr systems to facilitate the diagnosis and research of septicemia in foals expressed sequence tags (ests) isolated from blood of a septic thoroughbred foal early goal-directed therapy in the treatment of severe sepsis and septic shock chronic flunixin meglumine therapy in foals clinical and pathological effects of flunixin meglumine administration to neonatal foals gastric ulcers in foals experimentally induced toxicoinfectious botulism in horses and foals toxicoinfectious botulism in foals and adult horses botulism in the horse white muscle disease of foals naturally-occurring tyzzer's disease (bacillus piliformis infection) in horse foals suspected tyzzer's disease in two foals four cases of tyzzer's disease in foals in england bacillus piliformis infection (tyzzer's disease) in foals in northwestern united states: a retrospective study of 21 cases tyzzer's disease in a foal: light-and electron-microscopic observations clinical and clinicopathologic findings in two foals infected with bacillus piliformis serum biochemical and haematological findings in two foals with focal bacterial hepatitis (tyzzer's disease) toxic hepatopathy in neonatal foals the diagnosis and surgical correction of congenital portosystemic vascular anomalies in two calves and two foals clinical signs and radiographic diagnosis of a portosystemic shunt in a foal clinical and diagnostic features of portosystemic shunt in a foal evidence for transmission of halicephalobus deletrix (h gingivalis) from dam to foal halicephalobus (micronema) deletrix infection in two half-sibling foals listeriosis in an arabian foal with combined immunodeficiency suspected protozoal myeloencephalitis in a two-month-old colt pathological findings in horses dying during an outbreak of the paralytic form of equid herpesvirus type 1 (ehv-1) infection central nervous system neosporosis in a foal cauda equina syndrome, diskospondylitis, and a paravertebral abscess caused by rhodococcus equi in a foal vertebral body osteomyelitis due to rhodococcus equi in two arabian foals rhodococcus equi vertebral osteomyelitis in 3 quarter horse colts agenesis of the corpus callosum with cerebellar vermian hypoplasia in a foal resembling the dandy-walker syndrome: pre-mortem diagnosis by clinical evaluation and ct scanning cerebellar hypoplasia and degeneration in a foal cerebellar hypoplasia and degeneration in the young arab horse: clinical and neuropathological features imaging diagnosis: occipitoatlantoaxial malformation in a miniature horse foal occipitoatlantoaxial malformation with duplication of the atlas and axis in a half arabian foal occipitoatlantoaxial malformation in two non-arabian horses ivermectin toxicosis in a neonatal foal presumed moxidectin toxicosis in three foals hypovolemic hyponatremia and signs of neurologic disease associated with diarrhea in a foal extrapontine myelinolysis with involvement of the hippocampus in three children with severe hypernatremia relationships between radiography of cervical vertebrae and histopathology of the cervical cord in wobbling 19 foals assessment of colostral transfer and systemic availability of immunoglobulin g in new-born foals using a newly developed enzyme-linked immunosorbent assay (elisa) system evaluation of a test kit for determination of serum immunoglobulin g concentration in foals relationships among serum immunoglobulin concentration in foals, colostral specific gravity, and colostral immunoglobulin concentration measurement of igg in equine blood by immunoturbidimetry and latex agglutination a rapid, specific test for detecting absorption of colostral igg by the neonatal foal practical methods of determining serum immunoglobulin m and immunoglobulin g concentrations in foals passive immunity in the foal: measurement of immunoglobulin classes and specific antibody gammaglobulin and antibody variations associated with the maternal transfer of immunity and the onset of active immunity immunoglobulin metabolism in the neonatal foal prevalence (treatment days) and severity of illness in hypogammaglobulinemic and normogammaglobulinemic foals factors associated with failure of passive transfer of colostral antibodies in standardbred foals failure of passive transfer in foals failure of passive transfer of colostral immunity in the foal: incidence, and the effect of stud management and plasma transfusions the incidence and consequences of failure of passive transfer of immunity on a thoroughbred breeding farm failure of colostral immunoglobulin transfer as an explanation for most infections and deaths of neonatal foals secretion and composition of colostrum and milk immunoglobulin isotypes in sera and nasal mucosal secretions and their neonatal transfer and distribution in horses interleukin-18 in human milk improved recovery of insulin-like growth factors (igfs) from bovine colostrum using alkaline diafiltration cytokine-inducing activity of a proline-rich polypeptide complex (prp) from ovine colostrum and its active nonapeptide fragment analogs measurement of betacellulin levels in bovine serum, colostrum and milk in vivo antimicrobial and antiviral activity of components in bovine milk and colostrum involved in non-specific defence activities of gammaglutamyltransferase, alkaline phosphatase and aspartateaminotransferase in colostrum, milk and blood plasma of calves fed first colostrum at 0-2, 6-7, 12-13 and 24-25 h after birth efficacy of intravenous plasma to transfer passive immunity in clinically healthy and clinically ill equine neonates with failure of passive transfer evaluation of intravenous administration of concentrated immunoglobulin g to colostrumdeprived foals lyophilized hyperimmune equine serum as a source of antibodies for neonatal foals absorption of bovine colostral immunoglobulins g and m in newborn foals comparison of freezing and lyophilizing for preservation of colostrum as a source of immunoglobulins for calves a comparison of the reduction in immunoglobulin (igg) concentration of frozen equine plasma treated by three thawing techniques use of blood and blood products neonatal isoerythrolysis in mule foals characterization of a red blood cell antigen in donkeys and mules associated with neonatal isoerythrolysis prevalence of anti-red blood cell antibodies in the serum and colostrum of mares and its relationship to neonatal isoerythrolysis polymerized hemoglobin therapy in a foal with neonatal isoerythrolysis post-transfusion survival of 50cr-labeled erythrocytes in neonatal foals strategies for prevention of neonatal isoerythrolysis in horses and mules detection and effects on platelet function of anti-platelet antibody in mule foals with experimentally induced neonatal alloimmune thrombocytopenia neonatal alloimmune thrombocytopenia in a quarter horse foal neonatal thrombocytopenia: new insights into pathogenesis and implications for clinical management thrombocytopenia in horses detection and effects on platelet function of anti-platelet antibody in mule foals with experimentally induced neonatal alloimmune thrombocytopenia reef vb: cardiovascular disease in the equine neonate congenital polyalveolar lobe in three foals critical pulmonary stenosis in a newborn foal bilateral hypoplasia of the soft palate in a foal pulmonary lobar hypertrophy in a foal equine congenital defects congenital bilateral choanal atresia in a standardbred foal evaluation of pulse oximetry in anaesthetised foals using multiple combinations of transducer type and transducer attachment site electrocardiographic findings during parturition and blood gas tensions immediately after birth in thoroughbred foals blood gas and acid-base changes in the neonatal foal blood gas tensions and ph values in the normal thoroughbred foal at birth and in the following 42h mixed venous blood gases in recumbent and upright positions in foals from birth to 14 days of age ventilatory support of the neonatal foal report of foal pneumonia panel association of microbiologic flora with clinical, endoscopic, and pulmonary cytologic findings in foals with distal respiratory tract infection microbiologic changes during antimicrobial treatment and rate of relapse of distal respiratory tract infections in foals pathologic changes and pathogenesis of parascaris equorum infection in parasite-free pony foals spezifische infektiose pneumonie beim fohlen. ein neuer eiterreger beim pferd interaction of rhodococcus equi with phagocytic cells from rhodococcus equi-exposed and non-exposed foals electron microscopic investigation of intracellular events after ingestion of rhodococcus equi by foal alveolar macrophages influence of rhodococcus equi on the respiratory burst of resident alveolar macrophages from adult horses rhodococcus equi infection of monocytes/macrophages from human immunodeficiency (hiv)-infected patients and healthy individuals: evaluation of intracellular killing and nitric oxide production the intracellular bacterium rhodococcus equi requires mac-1 to bind to mammalian cells rhodococcus (corynebacterium) equi: bactericidal capacity of neutrophils from neonatal and adult horses identification of 15-to 17-kilodalton antigens associated with virulent rhodococcus equi virulence-associated 15-to 17 kilodalton antigens in rhodococcus equi: temperature-dependent expression and location of the antigens role of the 85-kilobase plasmid and plasmid-encoded virulence-associated protein a in intracellular survival and virulence of rhodococcus equi characterization of avirulence-associated gene family in rhodococcus equi corynebacterium equi infections in horses, 1958-1984: a review of 131 cases clinical manifestations, diagnosis, treatment, and prevention of rhodococcus equi infections in foals detection of corynebacterium equi-specific antibody in horses by enzyme-linked immunosorbent assay rhodococcus equi pneumonia in foals: comparison of elisa and agid serology on a commercial thoroughbred breeding farm studies of naturally occuring and experimental rhodococcus equi (corynebacterium equi) pneumonia in foals detection of virulent rhodococcus equi in tracheal aspirate samples by polymerase chain reaction for rapid diagnosis of r. equi pneumonia in foals comparison of nucleic acid amplification, serology, and microbiologic culture for diagnosis of rhodococcus equi pneumonia in foals use of erythromycin-rifampin combination in treatment of rhodococcus equi pneumonia pharmacokinetics of azithromycin and concentration in body fluids and bronchoalveolar cells in foals hyperthermia in foals treated with erythromycin alone or in combination with rifampin for respiratory disease during hot environmental conditions clostridium difficile associated with acute colitis in mares when their foals are treated with erythromycin and rifampicin for rhodococcus equi pneumonia strategies for the control of rhodococcus equi infections on enzootic farms immunoprophylaxis of rhodococcus equi pneumonia in foals failure of hyperimmune plasma to prevent pneumonia caused by rhodococcus equi in foals rhodococcus equi foal pneumonia: protective effects of immune plasma in experimentally infected foals protection against naturally acquired rhodococcus equi pneumonia in foals by administration of hyperimmune plasma methods of implementation of an immunoprophylaxis program for the prevention of rhodococcus equi pneumonia: results of a 5-year field study effect of prophylactic administration of hyperimmune plasma to prevent rhodococcus equi infection on foals from endemically affected farms valuation of equine immunoglobulin specific for rhodococcus equi virulenceassociated proteins a and c for use in protecting foals against rhodococcus equi-induced pneumonia associations between physical examination, laboratory, and radiographic findings and outcome and subsequent racing performance of foals with rhodococcus equi infection: 115 cases (1984-1992) endoscopic and virological observations on respiratory disease in a group of young thoroughbred horses in training epidemiology of ehv-1 and ehv-4 in the mare and foal populations on a hunter valley stud farm: are mares the source of ehv-1 for unweaned foals? clinical, serological and virological characteristics of an outbreak of paresis and neonatal foal disease due to equine herpesvirus-1 on a stud farm foetal and neonatal foal losses on equine herpesvirus type 1(ehv-1) infected farms before and after ehv-1 vaccination was introduced an outbreak of foal perinatal mortality due to equid herpesvirus type 1: pathological observations equine viral arteritis in newborn foals: clinical, pathological, serological, microbiological and immunohistochemical observations involvement of adenovirus in pneumonia in a thoroughbred foal adenoviral infection of arab foals with respiratory tract disease isolation of an adenovirus from an arab foal with a combined immunodeficiency disease clinical, haematological and biochemical findings in foals with neonatal equine herpesvirus-1 infection compared with septic and premature foals neonatal equine herpesvirus type 1 infection on a thoroughbred breeding farm passive transfer, rate of decay, and protein specificity of antibodies against equine arteritis virus in horses from a standardbred herd with high seroprevalence fatal nonneurological ehv-1 infection in a yearling filly pulmonary vasculotropic ehv-1 infection in equids virological and molecular biological investigations into equine herpes virus type 2 (ehv-2) experimental infections equine herpesvirus type 2: prevalence and seroepidemiology in foals subcutaneous emphysema in a neonatal foal immature epithelial na+ channel expression is one of the pathogenetic mechanisms leading to human neonatal respiratory distress syndrome bronchointerstitial pneumonia and respiratory distress in young horses: clinical, clinicopathologic, radiographic, and pathological findings in 23 cases (1984-1989) risk of adverse effects in pneumonic foals treated with erythromycin versus other antibiotics: 143 cases (1986-1996) disease of foals du plessis jl: rupture of the bladder in the newborn foal and its surgical correction metabolic abnormalities associated with rupture of the urinary bladder in neonatal foals exploratory celiotomy for suspected urinary tract disruption in neonatal foals: a review of 18 cases uroperitoneum in the foal uroperitoneum in the hospitalized equine neonate: retrospective study of 31 cases repair of a defect in the bladder of a foal rupture of the urinary bladder in neonatal foals bladder defects in newborn foals ultrasound of the urinary tract nonsurgical management of ruptured urinary bladder in a critically ill foal hemodialysis for treatment of oxytetracycline-induced acute renal failure in a neonatal foal bilateral renal hypoplasia in four young horses bilateral renal dysplasia and hypoplasia in a foal with an imperforate anus clinical vignette: renal arteriovenous malformation in a quarter horse foal percutaneous ultrasound-guided pyelography aided diagnosis of ectopic ureter and hydronephrosis in a 3-week-old filly bilateral ureteral tears in a foal multiple ureteral defects in a belgian foal congenital defects in newborn foals of mares treated for equine protozoal myeloencephalitis during pregnancy ultrasonography of umbilical structures in clinically normal foals clinical, ultrasonographic, and surgical findings in foals with umbilical remnant infections closure of the abdominal wall at the umbilicus and the development of umbilical hernias in a group of foals from birth to 11 months of age complications of umbilical hernias in horses: 13 cases persistent vitelline vein in a foal atresia coli in the foal: a review of six cases endothelin receptor b polymorphism associated with lethal white foal syndrome in horses a dinucleotide mutation in the endothelin-b receptor gene is associated with lethal white foal syndrome (lwfs): a horse variant of hirschsprung disease a missense mutation in the endothelin-b receptor gene is associated with lethal white foal syndrome: an equine version of hirschsprung disease incidence of the endothelin receptor b mutation that causes lethal white foal syndrome in white-patterned horses necrotizing enterocolitis presenting in the emergency department: case report and review of differential considerations for vomiting in the neonate new concepts in necrotizing enterocolitis hemorrhagic necrotizing enterocolitis associated with clostridium difficile infection in four foals endoscopic appearance of gastric lesions in foals: 94 cases endoscopic evaluation of changes in gastric lesions of thoroughbred foals gastroduodenal ulceration in foals prevalence of gastric lesions in foals without signs of gastric disease: an endoscopic survey gastric ulcers in foals exsanguination due to gastric ulceration in a foal gastrointestinal diseases of foals pathophysiology of peptic disorders in foals and horses: a review clinical syndromes of gastric ulceration in foals and mature horses gastroduodenal ulceration in foals stress ulcer: is routine prophylaxis necessary? peptic ulcer pathophysiology intragastric ph in critically ill neonatal foals and the effect of ranitidine a review of medical treatment for peptic ulcer disease effect of sucralfate on healing of subclinical gastric ulcers in foals effect of ranitidine on intragastric ph in clinically normal neonatal foals effects of omeprazole paste on healing of spontaneous gastric ulcers in horses and foals: a field trial incidence of pneumonia in mechanically ventilated patients treated with sucralfate or cimetidine as prophylaxis for stress bleeding: bacterial colonization of the stomach the protective role of gastric acidity in neonatal bacterial translocation upper gastrointestinal tract bleeding in critically ill pediatric patients gastric colonization as a consequence of stress ulcer prophylaxis: a prospective, randomized trial is prophylaxis for gastric ulcers necessary in critically ill equine neonates? a retrospective study of necropsy cases 1995-1999 stress ulcer prophylaxis in medical icu patients: annual utilization in relation to the incidence of endoscopically proven stress ulceration influence of stress ulcer prophylaxis on translocation of bacteria from the intestinal tract in rats effects of sucralfate on acute gastric mucosal injury and gastric ulcer induced by ischemiareperfusion in rats stress ulcer prophylaxis in medical icu patients: annual utilization in relation to the incidence of endoscopically proven stress ulceration efficacy of ivermectin in controlling strongyloides westeri infections in foals foal diarrhoea between 1991 and 1994 in the united kingdom associated with prevalence of and risk factors for fecal shedding of cryptosporidium parvum oocysts in horses overwhelming strongyloidosis in a foal verminous arteritis in a 3-month-old thoroughbred foal randomised trial of fluid restriction in ventilated very low birthweight infants restricted versus liberal water intake for preventing morbidity and mortality in preterm infants hyponatraemia in children with acute cns disease: siadh or cerebral salt wasting? comparison of norepinephrinedobutamine to dopamine alone for splanchnic perfusion in sheep with septic shock depletion of neurohypophyseal content of vasopressin in septic shock hemodynamic and metabolic effects of low-dose vasopressin infusions in vasodilatory septic shock references 1. firth ec, poulos pw: blood vessels in the developing growth plate of the equine distal radius and metacarpus microangiographic studies of metaphyseal vessels in young foals physeal form of the longbones of the foal comparison of splinting and casting on the degree of laxity induced in thoracic limbs in young horses histological features of the dorsal cortex of the third metacarpal bone mid-diaphysis during postnatal growth in thoroughbred horses effect of oxytetracycline on metacarpophalangeal and distal interphalageal joint angles in newborn foals the blood supply of the growth plate and the epiphysis: a comparative scanning electron microscopy and histological experimental study in growing sheep fractures of the proximal sesamoid bones in thoroughbred foals avulsion of the origin of the peroneus tertius tendon in a foal avulsion fracture of the origin of the extensor digitorum longus muscle in a foal surgical reconstruction of a ruptured medial collateral ligament in a foal correction of patellar luxation by recession sulcoplasty in three foals lateral patellar luxation in miniature horse foals anatomy and therapeutic resection of the peroneus tertius in a foal preliminary observations on oxytetracycline treatment of congenital flexural deformities in foals effect of oxytetracycline on metacarpophalangeal and distal interphalangal join angles in newborn foals torsion in quadrapeds and its impact on mammalian joints computer model of endochondral growth and ossification in long bones: biological and mechanobiological influences observations on the evaluation and selection of foal limb deformities for surgical treatment treatment response and athletic outcome of foals with tarsal valgus deformities: 39 cases incomplete ossification of the tarsal bones in foals: 22 cases periosteal transection and periosteal stripping for correction of angular limb deformities in foals periosteal transection and stripping for treatment of angular limb deformities in foals: clinical observations restricted exercise and transphyseal bridging for correction of angular limb deformities effect of hemicircumferential periosteal transection and elevation in foals with experimentally induced angular limb deformities diaphyseal angular limb deformities in three foals current concepts of infectious polyarthritis in foals factors associated with prognosis for survival and athletic use in foals with septic arthritis: 93 cases (1987-1994) the site of focal osteomyelitis lesions in foals pathological features of multiple bone infection in the foal stromal cell culture and relationships in perimedullary spaces of chick embryo shaft bones tarsal osteomyelitis in foals cartilage canals in equine articular/epiphyseal growth cartilage and a possible association with dyschondroplasia a histological study of acute hematogenous osteomyelitis following physeal injuries in rabbits open drainage, intra-articular and systemic antibiotics in the treatment of septic arthritis/tenosynovitis in horses comparison of various treatments for experimentally induced equine infectious arthritis a retrospective study of 192 horses affected with septic arthritis/tenosynovitis regional intravenous perfusion of the distal limbs of horses with amikacin sulfate treatment of sepsis in the small tarsal joints of 11 horses with gentamicin-impregnated polymethylmethacrylate beads use of antibioticimpregnated polymethyl-methacrylate in horses with open or infected fractures or joints: 19 cases (1987-1995) the effect of implanting gentamicin-impregnated polymethylmethacrylate beads in the tarsocrural joint of the horse regional perfusion of the equine carpus for antibiotic delivery how to perform equine digital intravascular perfusion surgical management of rhodococcus equi metaphysitis in a foal intraosseous regional perfusion for treatment of septic physitis in a 2-week-old foal key: cord-018319-tylkbh4h authors: chemaly, roy f.; rathod, dhanesh b.; couch, robert title: respiratory viruses date: 2011-01-04 journal: principles and practice of cancer infectious diseases doi: 10.1007/978-1-60761-644-3_32 sha: doc_id: 18319 cord_uid: tylkbh4h the respiratory viruses as a group are the most common cause of an acute infectious illness in developed societies. the immunocompromised state of many cancer patients constitutes the basis for the frequent failure of the host to promote a normal and rapid recovery from an acute respiratory viral infection and results in a more severe and prolonged infection that causes significant morbidity and mortality in these patients. those respiratory viruses that are most prevalent and most prone to produce lower respiratory illnesses and pneumonia in healthy hosts, rsv, influenza viruses, and parainfluenza viruses, are those most likely to cause severe illness and pneumonia leading to hospitalization in immunocompromised persons. however, viruses less prone to produce a lower respiratory illness but that are highly prevalent, such as rhinoviruses, may frequently be associated with severe illness. the limited availability of antivirals and vaccines for the acute respiratory viruses means that these infections will continue to be important for many years and dictate a need for utilizing infection control procedures as much as possible, particularly in hospitals and institutions, so as to minimize spread. efforts to develop specific vaccines are important as their use could prevent as well as reduce exposure of cancer patients to these viruses. development of specific antivirals is important for use in immunocompromised patients as normal recovery mechanisms may be seriously impaired. community respiratory virus infections were once primarily considered to be infections of children and generally nonserious. however, over the past two decades, it has become clear that these viruses can cause serious infections that require medical attention, particularly in infant, elderly, and immunocompromised patients. historically, the most common causes of respiratory infections in cancer patients were thought to be opportunistic bacteria and fungi, but newer diagnostic methods have revealed that respiratory viruses can cause serious morbidity and mortality in such patients, including leukemia patients and hematopoietic stem cell transplant (hsct) recipients. many viruses are known to cause respiratory tract infections, but the most common in hospitalized cancer patients are influenza viruses, respiratory syncytial virus (rsv), and parainfluenza viruses (piv) [1, 2] . however, all respiratory viruses can cause respiratory infections in cancer patients including rhinoviruses, enteroviruses, coronaviruses, human metapneumoviruses (hmpvs), adenoviruses, as well as cytomegaloviruses, herpes simplex, and varicella zoster viruses. in this chapter, we discuss the common clinical presentations, diagnostic methods, treatments, and prevention measures for respiratory virus infections in general and in cancer patients in particular (table 32. of these cases, most were in hsct recipients (45%). the summer [2] [3] [4] . rsv is a member of the paramyxoviridae family and the genus pneumovirus. it is an enveloped singlestranded rna virus approximately 150-300 nm in diameter that is contained in a helical nucleocapsid surrounded by a lipid envelope. this envelope bears two glycoproteins: the g protein, which attaches to the cell surface, and the f (fusion) protein, which enables the internal components to enter the cell after fusion of host and viral membranes and to initiate replication. both glycoproteins are integral to pathogenesis [2, 3, 5] . there are two groups of rsv viruses, groups a and b [2] [3] [4] [5] . rsv infections can present with a wide array of upper or lower respiratory symptoms. the incubation period is 4-6 days in adults. in infants and young children, the primary infection starts in the upper respiratory tract, with rhinorrhea, low-grade fever, and cough; it may progress to lower respiratory infection (lri) (bronchiolitis or pneumonia), at which stage most patients seek medical attention [3, 6] . it is also likely to involve the sinuses and the middle ear. rsv is known to cause apnea in infants, although the mechanism of this action remains unknown. older children and adults typically present with upper respiratory tract symptoms, but may also have constitutional symptoms such as fever and malaise [3, 7] . rsv upper respiratory infections (uris) can also progress to the lower respiratory tract, particularly in institutionalized adults and those with severe combined immunodeficiency, leukemia, hsct, and in lung transplant recipients [2, 8, 9] . these patients may present with wheezing and shortness of breath, with or without underlying comorbidities or known hyperactive airways. almost 35% of elderly patients with rsv infections report wheezing [8] [9] [10] [11] . patients with weakened immune systems because of malignancy, chemotherapy, steroid use, hsct, or solid organ transplantation are at high risk for developing severe illnesses. they also tend to have a longer duration of infection, with a varied presentation [12, 13] . leukemia patients and solid organ and stem cell transplant recipients are particularly at risk, with the latter being at high risk for pneumonia and death prior to engraftment [11] . rsv infections begin as uri, but progress to lri in 30-60% of hsct recipients, leading to respiratory failure with significant morbidity and mortality; some early studies described a mortality rate of 70-100% [14, 15] . in hsct recipients, the risk factors for progression to lri [16] [17] [18] [19] . torres et al. [20] found that a high apache ii score and not giving aerosolized ribavirin treatment were independent predictors of progression to pneumonia in leukemia patients; those with uri who were treated with aerosolized ribavirin were less likely than untreated patients to develop pneumonia (68 vs. 96%, p < 0.01) and die with rsv infection (6 vs. 36%, p = 0.1). the overall mortality rate in the study was 10% [20] . solid organ transplantation patients with rsv infection may present with dyspnea, cough, fever, and wheezing which progress to pneumonia in more than 70% of patients [21] . in lung transplant recipients, a higher frequency of rsv pneumonia has been reported, but the mortality rate is low; the mortality rate is also low in kidney transplant recipients [21] . in pediatric liver transplant patients, pohl et al. reported a mortality rate of 17% for rsv pneumonia with early infection onset and preexisting lung disease as predictors of severe disease [22] . in children, genetic polymorphisms in cytokine and chemokine-related genes (interleukin [il]-4, il-8, il-10, il-13, and ccr5) and genes related to potential virus-cell surface interactions or cell signaling (tlr-4, cx3cr1, sp-a, and sp-d) have been associated with severe rsv infections [23] . data on hiv patients with rsv infections are scarce; however, a cohort study by miller et al. [24] performed in the winter of 1994-1995 found no evidence of influenza, rsv, parainfluenza, adenovirus, or enterovirus infections in the bronchoscopic alveolar lavage fluids of 44 hiv-1-positive patients. rsv can be diagnosed on the basis of the clinical presentation in infants with lri during an outbreak period [3] . rsv infections in adults cannot be clinically differentiated from other viral infections that cause upper respiratory symptoms. for a specific diagnosis, rsv must be detected in respiratory secretions. nasal aspirates or washes are most likely to give a positive rsv test in young children. if a nasal aspirate or wash cannot be obtained, a nasopharyngeal swab or throat swab may be used. because immunocompromised or intubated patients are more likely to develop an lri due to rsv, tracheal aspiration or bronchoalveolar lavage should be performed [8, 25] . the gold standard for diagnosing rsv remains the identification of virus causing typical syncytia in cultures of hep-2 cells [25] . viral cultures may take 2 days (shell vial cultures) and up to 2 weeks (routine cultures) to become positive, making isolation less relevant in most clinical settings. other available methods include antigen detection by enzyme-linked immunosorbent assay (elisa), immunoflourescence assay, and polymerase chain reaction (pcr) tests. fan et al. [26] reported a sensitivity of 65-95% for rsv detection using rapid antigen testing with elisa. pcr tests have been shown to be more sensitive than direct antigen detection [27, 28] . for most immunocompromised patients, rsv treatment is focused on reducing symptom severity and preventing progression to lris. nonsteroidal anti-inflammatory drugs and antihistamines have been used in patients with a uri. ribavirin, a nucleoside analog, has in vitro activity against rsv and has been approved by the united states food and drug administration for the treatment of rsv infections in children. two schedules of aerosolized ribavirin have been used in immunocompromised patients: continuous and intermittent. on the continuous schedule, a daily dose of 6 g (concentration, 20 mg/ml) is delivered over 18 h via a small particle aerosol generator unit, administered via a face mask in a tent. on the intermittent schedule, a concentration of 60 mg/ml is delivered over 3 h every 8 h. aerosolized ribavirin may be beneficial in certain adults and children with lris. its early use has been shown in some retrospective studies to reduce morbidity and mortality in hsct recipients [3, 29] and patients with hematologic malignancies, particularly when the infection is treated early on [30]. however, its effectiveness in solid organ transplantation patients remains unknown [31] . an rsv-specific monoclonal antibody (palivizumab) did not demonstrate a therapeutic benefit in a major study conducted in children [32] . although the combination of ribavirin and intravenous immunoglobulin (ivig) or palivizumab has not been evaluated in a randomized trial, it is sometimes used in severely ill patients with rsv pneumonia, especially hsct recipients, given that they have high mortality rates from this infection [3, 11, 14] . in patients at risk for progression to lri, aerosolized ribavirin should be considered at an early stage. a recent trial demonstrated that both the intermittent and continuous schedules of aerosolized ribavirin were effective at preventing progression to lris in 91% and 80% of patients with rsv uris and hematologic malignancies (including hsct recipients), respectively [33]. rsv infection may be acquired nosocomially, thus specific infection control measures should be implemented when dealing with patients with known or suspected infections. briefly, patients should be isolated in private rooms when possible, and appropriate personnel protective equipments should be used (i.e., disposable gloves, masks, and gowns) [34] . no licensed vaccine is available for rsv; however, two agents, rsv-ivig and palivizumab, have been used to prevent rsv infection. rsv-ivig has been studied in children younger than 24 months with severe lung disease and those who were born prematurely [35], but it was removed from the market in 2003. a randomized double-blind placebo-controlled study in children found that palivizumab recipients had a 45% relative reduction in rsv hospitalizations ( p = 0.003). twenty-one children (3.3%) died in the palivizumab group vs. 27 (4.2%) in the placebo group with no deaths attributable to palivizumab [36] . palivizumab is easier to administer than regular ivig, which must be given over 4-6 h, and does not interfere with other vaccinations. however, rsv-ivig and ivig may provide additional protection against other respiratory viruses as well [35, 37, 38]. influenza viruses infect both the upper and lower respiratory tracts. outbreaks are common every winter, although the severity of the disease varies considerably. influenza viruses belong to the orthomyxoviridae family, with influenza types a, b, and c constituting one genus. these rna viruses are enveloped and measure about 80-120 nm in diameter. the designation of the viruses into types is based on the stable antigenic characteristics of the nucleoprotein and the matrix protein antigens. influenza a and b viruses have surface glycoproteins known as hemagglutinins (ha) and neuraminidase (na). three hemagglutinin subtypes (h1, h2, and h3) and two neuraminidase subtypes (n1 and n2) have been described for the influenza a viruses that infect humans. major antigenic changes in the glycoprotein (basis for new subtypes) are called antigenic shift and can cause pandemics; minor antigenic changes occur frequently, are called antigenic drift, and cause the annual epidemics. influenza b has only exhibited minor antigenic drift [3, 39] . the 2009 pandemic influenza a (h1n1) contains segments present in north american swine for years, but is a new reassortant virus that acquired m and na gene segments from a eurasian adamantane-resistant swine influenza virus [40] . influenza typically has a short incubation period and an abrupt onset of symptoms, such as headache, fever, chills, myalgia, and malaise, along with respiratory symptoms of runny nose, cough, and sore throat. it can also present as a febrile uri or with constitutional manifestations only or with few-to-no respiratory symptoms. influenza can progress to pneumonia in otherwise healthy persons, but particularly in patients with comorbidities such as lung diseases, heart disease, diabetes mellitus, renal diseases, and hemoglobinopathies, in immunocompromised individuals, residents of nursing homes or chronic care facilities, and in individuals over 65 years of age [41] . primary pneumonia occurs when the influenza virus directly involves the lung and should be suspected when clinical symptoms progress to high fever, dyspnea, and hypoxemia [42] . influenza virus infections also affect the epithelium of the tracheobronchial tree, leading to impaired defense and a secondary bacterial pneumonia. influenza infections can increase morbidity and mortality rates in cancer patients [2] . in a study of leukemia patients with influenza infections, 39% of patients developed pneumonia [43], with cough and dyspnea being the most common manifestations. half of the patients had lymphopenia [43] . the incidence of influenza infection in hsct recipients is reported as 0.4% [44], with a mortality rate up to 38% [43], mainly due to respiratory failure. in one study [44] , 17% of patients who developed influenza after hsct presented with pneumonia; of those who presented with uris, 14% experienced progression to lri. risk factors associated with progression to lri were lymphopenia and days from transplantation (i.e., pneumonia developed more commonly among those infected earlier after transplantation); whereas use of systemic steroids and autologous stem cell transplantation appeared to be protective. x-ray findings include a diffuse interstitial pattern or focal pulmonary infiltrates [45] . among patients with solid organ transplantation, lung transplant recipients are at the highest risk for influenza virus infection [46] . the initial presentation in these patients may not always be in the respiratory tract, as illness can present with nonspecific gastrointestinal symptoms; however, patients who required hospitalization always presented with pneumonia [47] . progression to bronchiolitis obliterans, which is a characteristic of chronic lung rejection after infection, was also reported. in patients postrenal transplantation, influenza pneumonia is often acute, with high fever, cough, dyspnea, cyanosis, leukopenia, and thrombocytopenia [48] . influenza infection can only be diagnosed clinically in persons exhibiting the classic syndrome during an epidemic. however, because other viruses can produce the same syndrome and influenza infection can produce other respiratory syndromes, a confirmatory test detecting the virus or viral antigens in nasal washes, throat swabs, respiratory tract secretions, or bronchoalveolar lavage specimens is needed in sporadic cases and in immunocompromised patients. viral culture remains the gold standard for diagnosis, but can take up to 72 h to yield results [49] . sputum and nasal washes or nose swabs are superior to throat swabs for diagnosis [50] . rapid antigen testing using immunoflourescence assays, enzyme immunoassays, and pcr-based testing are used frequently in clinical settings [51] . the results of these tests can be obtained in hours and can have good sensitivity (72-95%) and specificity (76-84%) if obtained early from those with more severe illnesses [52] . pcr-based assays are more sensitive than rapid antigen testing for diagnosing influenza a and b, but are not often used because of availability and cost [51] . samples should be obtained within 24-48 h of the appearance of symptoms for the most accurate results. two classes of drugs are available to treat influenza infections [53] : the neuraminidase inhibitors, zanamivir and oseltamivir, which are active against influenza a and b viruses; and the m2 inhibitors, amantadine and rimantadine, which are only active against influenza a viruses [54] . amantadines are not effective against influenza b and resistance has been reported for 2009 novel h1n1 virus; resistance to oseltamivir has increased for the seasonal h1n1 virus (table 32. 2) [55, 56] . therapy should be initiated as early as possible, preferably within 48 h after symptom onset [57] . zanamivir and oseltamivir have been used extensively to treat both influenza a and b; both were shown to reduce the mean duration of symptoms by 1 day when used within 48 h of symptom onset [58] . both neuraminidase inhibitors have been shown to significantly decrease the incidence of complications associated with influenza such as development of pneumonia when compared to placebo [59] . the most common side effects of oseltamivir are nausea and vomiting, although other toxicities have been reported. central nervous system side effects such as anxiety, insomnia, impaired thinking, confusion, lightheadedness, and hallucinations have been reported with amantadine use. newer ni inhibitors including parenteral preparations are under development [60] . the mainstay of influenza prophylaxis in the general population is the administration of influenza vaccine. annual vaccination has been recommended for many years for people at high risk for complications, including those older than 65 years, residents of nursing homes or other facilities, adults and children with chronic pulmonary or cardiovascular conditions, adults and children hospitalized during the previous year, women in the second and the third trimesters of pregnancy, immunocompromised patients, healthcare workers, and family member of those at high risk prior to the onset of influenza season [41] . the american committee on immunization practices has recently recommended vaccination for all persons for whom there is no contraindication [61] . currently, there are two types of vaccines available, an intramuscular (inactivated virus vaccine) and an intranasal (attenuated virus vaccine). the intranasal form is to be used only in healthy individuals between the ages of 2 and 49 years; it should not be used to vaccinate immunocompromised patients [62] . individuals with a significant allergy to eggs or those with acute febrile illness should not be given vaccine [63] . the advisory committee on immunization practices recommends that immunocompromised individuals be vaccinated prior to influenza season and receive daily chemoprophylaxis with an antiviral medication during community outbreaks [41] . influenza chemoprophylaxis may be used in patients at high risk for complications if the vaccine is contraindicated or not likely to be completely protective. antiviral drugs should be administered to patients within the first 6 months of hsct, those with documented graft-versus-host disease, unvaccinated healthcare workers who care for immunocompromised individuals, and residents of long-term care institutions during outbreak periods [64] . the american society for blood and marrow transplantation (asbmt) 2009 guidelines [65] for prevention of infection in hsct recipients recommend annual inactivated influenza vaccine before the beginning of the season and before stem cell transplant. the vaccine may be given 4-6 months after hsct. they also recommended prophylaxis and preemptive treatment during community and nosocomial outbreaks of influenza a for hsct recipients regardless of the vaccination status in those who are within 24 months of the transplant or in those with more than 24 months posttransplant, but have gvhd and/or are on immunosuppression. the drug to be used for chemoprophylaxis depends on the susceptibility pattern of the outbreak virus. piv are enveloped, single-stranded rna viruses that belong to the paramyxoviridae family. the envelope contains two glycoproteins, one with both ha and neuraminidase activity and the other with fusion activity. there are five types that share certain antigens with other members of the paramyxoviridae family [3, 66] . of the five, piv-3 is the most prevalent, with most adults demonstrating the presence of antibodies [67] . piv-3 infections may be epidemic in the spring and summer, but may also occur through the year; piv-3 is associated with pneumonia and bronchiolitis in infants. piv-1 and 2 are associated with croup in children which occurs as outbreaks every 2 years in the fall; piv-4a and 4b cause only mild illnesses [67, 68] . piv infections have a wide spectrum of presentations, from a simple uri to a life-threatening complication such as pneumonia. the incubation period is usually 2-6 days. young children may present with coryza, sore throat, hoarseness, and cough with chest x-rays revealing interstitial infiltrates. in adults, most infections are mild, but in immunocompromised individuals, the infection can progress to a lower respiratory tract illness including pneumonia and cause prolonged illness and even death [3, 67, 69] . immunocompromised individuals are at risk for a piv infection that can progress to pneumonia. a study at our institution found a higher rate of progression among leukemia patients than among hsct recipients [70] . piv usually presents as an uri. in a large study in hsct recipients over several years, piv infections were documented in 7.1% of cases, with 78% being communityacquired [71] . patients who have undergone hsct are at particular risk for developing severe piv-associated pneumonia [71] [72] [73] [74] ; wendt et al. [72] reported a mortality rate up to 30%. coinfections (aspergillus fumigatus being the most common) and mechanical ventilation were found to be significant risk factors for piv pneumonia-associated mortality in one study [71] . other factors associated with progression include neutropenia within 1 month prior to infection, an apache ii score higher than 15, and pulmonary coinfection; this study also found a mortality rate around 20%, with no difference between those treated and those not treated with aerosolized ribavirin [70] . patients who have undergone solid organ transplantation do not appear to be at increased risk for developing severe piv illness, but only a few studies have been reported [75, 76] . one of these studies in lung transplant patients found piv infections in 11% of patients, all of whom developed pneumonia, but the majority (74%) were treated with aerosolized ribavirin and all but one recovered [75] . except for croup in young children, the clinical pattern of piv infection is similar to that of other respiratory viruses and cannot be distinguished on the basis of symptoms alone. during a community outbreak, a presumptive diagnosis can be made; however, confirmation in laboratory tests may be appropriate in immunocompromised individuals. the virus can be detected in respiratory tract secretions, nasal washes, nasal swabs, throat swabs, and bronchoalveolar lavage specimens. viral culture remains the gold standard for diagnosis, but can take days to yield a result [3, 77] . rapid antigen detection by immunofluorescence or elisa is most commonly used and can have a sensitivity of as high as 75-95% [78] . recent pcr-based assays have sensitivities up to 100%, with high specificity [26, 79] . management of piv infection is mostly supportive as no piv-directed antiviral therapy has been licensed by the u.s. food and drug administration. ribavirin has been shown to be active against the virus in vitro and in animal models and has been used occasionally to treat immunocompromised patients with severe piv infections [80] . one case series reported a decrease in piv viral loads and clinical improvement after aerosolized ribavirin treatment in children with severe immunodeficiency [81] . nichols et al. [71] reported that aerosolized ribavirin did not reduce viral shedding or mortality rates in hsct recipients after the infection had progressed to the lower respiratory tract. our data also showed no apparent benefit of aerosolized ribavirin on the mortality rate in hsct recipients and leukemia patients [70] . on the other hand, a combination of methylprednisolone and intravenous or oral ribavirin has, apparently, been used successfully to treat piv pneumonia in a hsct and a heart transplant recipient, respectively [82, 83] . currently, no licensed vaccine is available for the prevention of piv infection. hence, infection control measures play an important role in containing the spread of the infection. patients with suspected or confirmed infections should be isolated, and personnel protective equipment should be used. human adenoviruses are dna viruses belonging to the mastadenovirus genus of the adenoviridae family which measure about 70-80 nm in diameter [3, 84] . there are at least 51 known human serotypes divided into subgenera a to f based on the dna genome and pattern of hemagglutination [84] . adenovirus infections are reported most frequently in infants and children and can occur throughout the year; however, they may cause serious infections in immunocompromised patients. after a primary infection in childhood, adenoviruses establish latency in adenoidal tissues along with lifelong persistence of specific antibodies [84, 85] . adenovirus infections are transmitted by either inhalation of aerosolized virus, inoculation of the virus into conjunctival sac, or through the fecal-oral route [3, 84] . subgroup a, type 31 and various types from subgroups b and c have been associated with pneumonia and hepatitis [86] . serotypes 4, 7, 14, and 21 are associated with outbreaks of acute respiratory disease in military recruits, mostly in winter and spring [3] . types 1, 2, 5, and 6 are most common in children and present as an acute upper respiratory tract illness which can progress to lower respiratory disease; types 3 and 7 are less common, but can cause severe disease. in adults, infections due to adenovirus are characterized by sore throat and gradual onset of fever. cough, coryza, and regional lymphadenopathy are commonly seen. the most common clinical symptoms besides respiratory symptoms are fever and diarrhea [3] . adenovirus infections are common after hsct and can occur as a localized illness or as part of a disseminated disease. it has been associated with delayed engraftment and graft failure. infections due to adenovirus in hsct recipients have a reported incidence of 0.5-3% [87, 88] and are more commonly reported in allogeneic hsct recipients than in autologous transplant recipients (6 vs. 0.92%) [87] ; however, the mortality rate can be as high as 75% in both groups [87, 89] . some reports also suggest that the incidence of adenovirus infection in patients after hsct may be rising due to transplantation practices [90, 91] . disseminated infection may occur without respiratory tract symptoms and disease can develop in almost any organ causing gastrointestinal disease, hepatitis, nephritis, pneumonia, conjunctivitis, thrombotic thrombocytopenic purpura, pancreatitis, or hemorrhagic cystitis. viremia may be present, but is not detected in all cases of disseminated disease [92] . adenovirus is known to be fatal even in the absence of any respiratory tract involvement; however, if pneumonia is present, the mortality has been reported to be higher (80 vs. 50%) [87] . coinfections with aspergillus spp. and bacteria such as nocardia, legionella spp., and mycobacterium tuberculous are frequently seen in this patient population [90, 93] . risk factors for adenovirus infections include gvhd, unrelated donor, total body irradiation, t-cell depletion, younger age (<7 years old), chronic disease, and recent transplantation [90, 91, 94, 95] ; the degree of t-cell depletion and posttransplant suppression of t-cell function are the most important ones [92] . adenovirus types 5 and 21 are associated with severe infections in hsct recipients [95] . there are a few reports of adenovirus infections in solid organ transplant patients in whom the virus involved the donor organ and led to pneumonia, hepatitis, hemorrhagic cystitis, nephritis, enterocolitis, or disseminated disease. in patients with previous liver transplantation, adenovirus pneumonia had a reported prevalence of 1.5% with a mortality rate of 66% [96] . serotype 5 is known to be associated with hepatitis [96, 97] , whereas serotypes 1 and 2 are more commonly associated with pneumonia. in lung transplant recipients, one study found adenovirus infection to be an early complication following surgery with a prevalence rate of 1.3% [98] . progressive adenovirus infections are known to be associated with graft loss, progression to bronchiolitis obliterans, or death [99] . adenovirus infection should be suspected in cases of acute respiratory disease in military recruits or during outbreaks. in most cases, infection caused by the virus cannot be differentiated from those caused by other respiratory viruses from the clinical presentation alone [3] . a definitive diagnosis can be established by viral culture or the detection of specific viral antigens. viral culture remains the gold standard for identification of adenovirus. nasopharyngeal aspirate or swab, throat swab, sputum samples, or bronchoalveolar lavage can be used, depending on the site of the infection. a cytopathic effect is seen in human cell lines such as hela (cervix), a549 (lung), hek (human embryonic kidney), and hep-2 (larynx) by strains of adenovirus except for types 40 and 41. adenovirus 40 and 41 grow well in hek 293 cells. adenovirus-specific enzyme immunoassay (elisa) or immunofluorescence assay can be used to detect the presence of virus in clinical samples. these rapid tests suffer from a sensitivity of only about 50%; pcr-based assays can detect adenovirus dna from a variety of clinical specimens and has better sensitivity [100, 101] . viral load quantification is a useful tool to measure prognosis and monitor clinical response [102, 103] . viral loads higher than 1 ã� 10 6 copies/ml have been associated with an increased likelihood of death [104, 105] . there have been no randomized clinical trials for the treatment of adenovirus infection in immunocompromised patients. most patients are managed using symptom-based treatment and supportive therapy. cidofovir is currently being used in immunocompromised patients since it has been shown to decrease plasma viral loads in hsct recipients [106] . although cidofovir is active against all strains of adenovirus in vitro, only retrospective data are available on the efficacy of cidofovir in hsct [106] [107] [108] [109] and solid organ transplant recipients [110] . nephrotoxicity is commonly encountered with this drug and it should be used with caution. there are two accepted regimens: 5 mg/kg every 1-2 weeks, or 1 mg/kg 3 times per week, with the latter being associated with less nephrotoxicity [110] . orally active ether lipid-ester prodrugs of cidofovir (s)-hpmpa are under development with some promising results in in vitro experiments and phase i trials [111] . when tested against five adenovirus serotypes, they were shown to be more active than the unmodified parent compounds [111] . intravenous ribavirin has been used in a few reported cases, but results were conflicting [112] [113] [114] . finally, another treatment option using adenovirus-specific donor t-cells infusion has been shown to be feasible and effective in protecting children from complications due to adenovirus infection and causing a significant decrease in viral loads [115] . currently, there are no vaccines available for adenovirus infection other than the oral partially attenuated vaccines contained in enteric-coated capsules with use restricted to the military [116] . only routine infection control practices are recommended for civilian populations where special precautions must be taken for contact and droplet exposure. in hsct recipients at high risk from adenovirus infection, weekly pcr surveillance of viremia and preemptive treatment with cidofovir can be used [92, 117] . rhinoviruses are members of the picornaviridae family. they are nonenveloped, single-stranded rna viruses that measure about 15-30 nm. the capsid of the virus is icosahedral and contains 60 copies of four polypeptides each. a canyon on the viral surface contains the attachment site for the host-cell receptor, with most rhinoviruses using this site to attach to the intercellular adhesion molecule-1 receptor expressed on the surface of host cells [3, 118] . more than 100 serotypes of the virus have been isolated, making a vaccine unlikely in the near future [3, 119, 120] . rhinoviruses are proven to cause 15-40% of common colds in adults [3] . each year, adults experience 2 or 3 colds, whereas children may experience 8-12 [3, 120] . children are the major reservoir for rhinoviruses with infection rates decreasing with age. although infections occur throughout the year, peaks may occur in the fall and spring. this infection is primarily due to the deposition of the virus on the nasal mucosa. this can occur via self-inoculation or contact with infected secretions such as small-and large-particle aerosols (respiratory droplets) [3, 118, 120] . individuals with rhinovirus infections may be asymptomatic. when symptoms occur, they are typically those of the common cold: most commonly rhinorrhea and sneezing, which are associated with nasal congestion. infections due to rhinovirus have an incubation period of 1-4 days. adults characteristically experience sneezing, nasal obstruction and discharge along with cough, and a sore or scratchy throat. sinuses are commonly involved so that the illness is rhinosinusitis. symptoms may last for 4-9 days and usually resolve with no complications. fever is not usually associated with adult illness [3, 121] . children, on the other hand, may experience fever, cough, and nasal discharge and obstruction. in addition, the duration of symptoms may be longer. although bronchitis, bronchiolitis, and bronchopneumonia have been reported in children, rhinovirus is not usually a major cause of lower respiratory illness [3, 122] . however, they are an important cause of exacerbations of asthma and chronic obstructive pulmonary disease in children and adults and of lri in the elderly [3, 119] . a retrospective study in adults with rhinovirus infections who had undergone hsct found that 32% of patients developed and eventually died of pneumonia [123] . one patient was found to have a coinfection with aspergillus spp. on autopsy; most of the other patients had interstitial pneumonitis and/or acute respiratory distress syndrome [123] . a study performed at another institution reported that 55% of hsct recipients with rhinovirus infections developed pneumonia and 33% died [124] . in a study of community-acquired pneumonia in immunocompromised patients (after hsct or solid organ transplantation, with hiv infection, or receiving steroids or chemotherapy), rhinovirus was responsible for about 12% of cases, with a mortality rate of 18% [125] . these findings suggest that rhinovirus may cause more severe complications in immunocompromised patients than previously thought. many viruses cause common cold symptoms, and a definitive diagnosis cannot be made only on the basis of the presenting symptoms but rhinoviruses are most commonly associated with colds. a definitive diagnosis can be made by isolating the virus from nasal washes or other nasal secretion specimens in tissue culture. newer diagnostic methods such as real-time reverse-transcription pcr (rt-pcr) have been used; however, these tests are not frequently performed given the self-limited nature of most infections. no specific antiviral treatment is available for rhinovirus infections. most cases are managed with supportive care using antihistamines, decongestants, and nonsteroidal antiinflammatory drugs. given the number of rhinovirus serotypes known to cause infection, an effective vaccine is unlikely to be developed in the near future. infection control measures such as hand washing and isolating patients who are known or suspected to have rhinovirus infections can help contain the spread of the virus. hmpv belongs to the subfamily pneumovirinae of the paramyxoviridae family. the virus was discovered in 2001 and is genetically similar to rsv. hmpv is an enveloped rna virus that is known to cause uris and lris in all age groups [126] . although hmpv infections are more common in children, a recent study revealed an infection rate of 4.5% in adults with acute upper respiratory illness; 11% of patients required hospitalization [127] . hmpv usually causes a mild infection of short duration (about 3-5 days) and is self-limiting. the incubation period is 4-6 days [128] . the most common presenting symptoms in adults are cough, nasal congestion, rhinorrhea, dyspnea, hoarseness, and wheezing [127] . children often present with cough, rhinitis, fever, and wheezing [129, 130] . a recent study of patients with hematologic malignancies revealed a 9% incidence of hmpv infection. nine of the 22 patients who had undergone hsct had pneumonia; three of these patients died [131] . another study in hsct recipients detected the presence of hmpv in 3% (in 5 out of 163) of bronchoalveolar lavage specimens. these 5 patients presented with fever, cough, nasal congestion, and sore throat within the first 40 days after transplantation. the infection progressed to respiratory failure, pulmonary hemorrhage, and culture-negative septic shock. the mortality rate was 80% (4 of 5 patients) [132] . hmpv's growth in culture is slow and unreliable, which makes this method of diagnosis impractical. the presenting symptoms are similar to those of other infections that cause an acute respiratory illness, making clinical diagnosis impossible. pcr-based methods have been used to diagnose the infection in some centers. no specific antiviral therapy exists for hmpv infections. in vitro studies have demonstrated that ribavirin has activity against the virus [133] ; however, no clinical studies have been reported. immune serum globulins may neutralize the virus as demonstrated in one in vitro study [133] . anecdotally, a lung transplant recipient with respiratory failure secondary to hmpv pneumonia was treated successfully with aerosolized ribavirin [134] . the use of general preventive measures for patients with known or suspected hmpv infections can help reduce the rate of transmission. no vaccine is currently available for this virus. coronaviruses are single-stranded rna viruses that measure about 80-160 nm in diameter. they are enveloped with clubshaped projections, giving them a crown-like appearancehence the name [3, 135] . coronaviruses can cause diarrhea in infants and may play a role in demyelinating diseases of the central nervous system [3, 136] . coronaviruses are difficult to grow in vitro; some strains can only be grown in human tracheal organ cultures. the major antigenic types that cause diseases in humans are 229e, oc43, hk, and nl63 viruses which cause common colds and may also cause lower respiratory tract illnesses in young children, and sars-cov, which causes a severe acute respiratory syndrome [3, 137, 138] . coronaviruses are found in all tropical, subtropical, and temperate climates. most infections occur in the late fall, winter, and early spring [3, 139] . cyclical outbreaks of these infections may occur every 2-4 years. respiratory infections due to hcov-229e and hcov-oc43 strains probably spread in a manner similar to rhinovirus, i.e., via direct contact with infected secretions or aerosol droplets [3] . the clinical manifestations of coronavirus infections are similar to those of rhinovirus infections. the most common symptoms are rhinorrhea, throat congestion, and fever. the middle ear may also be affected, leading to effusions and acute otitis media, especially in children [3, 135] . the incubation period is 1-3 days and the duration of the illness is shorter than that of rhinovirus infections, a mean of 6-7 days. the subtype sars-cov has a slightly longer incubation period of 4-5 days. in immunocompromised adults, coronaviruses can cause lower respiratory tract infections [3, 140] . a recent case series found that the infection rate among immunocompromised patients was significantly higher when compared to immunocompetent patients (8.8 vs. 4 .5%) [141] . no large studies have been conducted in cancer patients or other immunocompromised individuals, but a few cases have been reported. folz and elkordy [140] reported a patient who had undergone autologous hsct and was diagnosed with a coronavirus lri. the patient developed a fever, sore throat, cough, and severe hypoxia and was treated successfully using supportive measures. kumar et al. [142] described a patient who developed a fatal severe acute respiratory syndrome (sars) after liver transplantation. epidemiologically, these infections should be suspected during the late fall or winter or during an outbreak. however, no practical method is available to confirm the infection except pcr-based tests. these are used to diagnose coronavirus infections at some institutions [3, 143] . similar to rhinovirus, no specific antiviral therapy is available for coronavirus. most patients respond well to supportive treatment with nonsteroidal anti-inflammatory drugs, decongestants and antihistamines. general preventive measures must be used around patients with coronavirus infections; hand washing, disposing of infected material carefully, and proper disinfection can help prevent the spread of the virus. no vaccine against the virus has been developed. enteroviruses are single-stranded rna viruses that can multiply in the gastrointestinal tract. they belong to the picornaviridae family, but are relatively stable at a low ph. the virus is surrounded by an icosahedral capsid comprising four viral proteins. it has no lipid envelope and is not susceptible to alcohol, ether, or detergents. poliovirus is the prototype of this group, but nonpolio enteroviruses have a wide spectrum of manifestations including acute respiratory illnesses [3, 144] . enterovirus infections are more common in developing countries and socioeconomically depressed areas and are associated with poor hygiene and sanitation. they can occur throughout the year, but the peak occurs in the summer and fall [3, 145] . they can be transmitted by direct contact with feces during activities such as cleaning and diaper handling. the clinical picture of enterovirus infections is similar to that of rhinovirus infections; no specific symptoms are associated with the virus. immunocompromised patients have been reported to develop central nervous system infections, chronic disseminated infections, and a dermatomyositis-like syndrome with enterovirus infection. in a study of respiratory tract infections in hematologic malignancy patients, 3% of patients had enterovirus infections; most (66%) were hsct recipients with 33% of these developing pneumonia [131] . a study from spain reported on four hsct recipients who developed enterovirus infections, with a mortality rate of 75% (3 of 4) [146] . these findings demonstrate that immunocompromised patients may be at risk for lower respiratory tract infections and death from an enterovirus infection [3, 131, 146] . most enteroviruses can be isolated in cell cultures from nasopharyngeal or throat swabs. pcr-based testing is used to amplify the viral rna from throat swabs, cerebrospinal fluid, and tissues. for diagnosis, isolation of the virus from the throat is more clinically significant than from stool because this virus has a shorter duration of shedding from the throat. most enterovirus infections are self-limiting and do not require specific treatment. intensive care may be required for central nervous system, cardiac, and hepatic infections. ivigs have been used in some patients with severe infections, but no specific antiviral therapies are available [3, 147] . general infection control measures must be undertaken around patients with enterovirus infections. special attention must be paid to hand hygiene. material in contact with or soiled by feces should be handled carefully and discarded with proper precautions. the respiratory viruses as a group are the most common cause of an acute infectious illness in developed societies. the variety of viruses that can cause infection and illness in all age groups and their presence in high frequencies throughout the year describe the risk of exposure of all persons at all times including immunocompromised persons residing in the community. the immunocompromised state of many cancer patients constitutes the basis for the frequent failure of the host to promote a normal and rapid recovery from an acute respiratory viral infection and results in a more severe and prolonged infection that causes significant morbidity and mortality in these patients. those respiratory viruses that are most prevalent and most prone to produce lower respiratory illnesses and pneumonia in healthy hosts, rsv, influenza viruses and piv, are those most likely to cause severe illness and pneumonia leading to hospitalization in immunocompromised persons. however, viruses less prone to produce a lower respiratory illness but that are highly prevalent, such as rhinoviruses, may frequently be associated with severe illness. although not generally considered respiratory viruses, the herpes viruses are known to produce respiratory infection and disease, sometimes severe, in immunocompromised patients (table 32 .1). a historically important virus, measles virus, is now rarely encountered because of widespread vaccination. the limited availability of antivirals and vaccines for the acute respiratory viruses means that these infections will continue to be important for many years and dictate a need for utilizing infection control procedures as much as possible, particularly in hospitals and institutions, so as to minimize spread. efforts to develop specific vaccines are important as their use could prevent as well as reduce exposure of cancer patients to these viruses. development of specific antivirals is important for use in immunocompromised patients as normal recovery mechanisms may be seriously impaired. community respiratory virus infections in immunocompromised patients with cancer common community respiratory viruses in patients with cancer: more than just "common colds harrison's principles of internal medicine circulation patterns of genetically distinct group a and b strains of human respiratory syncytial virus in a community respiratory syncytial virus and parainfluenza virus risk of primary infection and reinfection with respiratory syncytial virus respiratory syncytial virus infections in previously healthy working adults respiratory syncytial and other virus infections in persons with chronic cardiopulmonary disease can respiratory syncytial virus and influenza a be distinguished clinically in institutionalized older persons? symptomatic respiratory syncytial virus infection in previously healthy young adults living in a crowded military environment respiratory syncytial virus and parainfluenza virus infections in the immunocompromised host respiratory virus infections in immunocompetent and immunocompromised persons respiratory syncytial virus infection in adults respiratory syncytial virusinduced acute lung injury in adult patients with bone marrow transplants combination therapy with aerosolized ribavirin and intravenous immunoglobulin for respiratory syncytial virus disease in adult bone marrow transplant recipients diagnosis and epidemiology of community-acquired respiratory virus infections in the immunocompromised host respiratory virus infections after stem cell transplantation: a prospective study from the infectious diseases working party of the european group for blood and marrow transplantation viral pneumonia in the immunocompromised adult with neoplastic disease: the role of common community respiratory viruses community respiratory virus infections among hospitalized adult bone marrow transplant recipients characteristics and outcome of respiratory syncytial virus infection in patients with leukemia community respiratory virus infections in immunocompromised patients: hematopoietic stem cell and solid organ transplant recipients and individuals with human immunodeficiency virus infection respiratory syncytial virus infections in pediatric liver transplant recipients viral and host factors in human respiratory syncytial virus pathogenesis community-based respiratory viral infections in hiv-positive patients with lower respiratory tract disease: a prospective bronchoscopic study rapid diagnosis of respiratory syncytial virus infections in immunocompromised adults rapid simultaneous diagnosis of infections with respiratory syncytial viruses a and b, influenza viruses a and b, and human parainfluenza virus types 1, 2, and 3 by multiplex quantitative reverse transcription-polymerase chain reaction-enzyme hybridization assay (hexaplex) lack of sensitivity of rapid antigen tests for the diagnosis of respiratory syncytial virus infection in adults enhanced clinical utility of the nuclisens easyq rsv a+b assay for rapid influenza pneumonia in lung transplant recipients: clinical features and association with bronchiolitis obliterans syndrome influenza virus pneumonia after renal transplant influenza virus comparison of four clinical specimen types for detection of influenza a and b viruses by optical immunoassay (flu oia test) and cell culture methods molecular diagnosis of influenza evaluation of diagnostic tests for influenza in a pediatric practice prevention and control of influenza enders lecture 2006: antivirals for influenza surveillance for neuraminidase inhibitor resistance among human influenza a and b viruses circulating worldwide from host cell selection of influenza neuraminidase variants: implications for drug resistance monitoring in a(h1n1) viruses updated interim recommendations. special considerations for clinicians regarding 2009 h1n1 influenza in severely immunosuppressed patients effectiveness of neuraminidase inhibitors in treatment and prevention of influenza a and b: systematic review and meta-analyses of randomised controlled trials efficacy and safety of oseltamivir in treatment of acute influenza: a randomized controlled trial current and future antiviral therapy of severe seasonal and avian influenza acip provisional recommendations for the use of influenza vaccines risk of transmission associated with live attenuated vaccines given to healthy persons caring for or residing with an immunocompromised patient antivirals for the chemoprophylaxis and treatment of influenza guidelines for preventing infectious complications among hematopoietic cell transplant recipients: a global perspective genetic variation and evolution of human parainfluenza virus type 1 hemagglutinin neuraminidase: analysis of 12 clinical isolates viral respiratory infections principles and practice of infectious diseases outbreak of pneumonia in a long-term care facility: antecedent human parainfluenza virus 1 infection may predispose to bacterial pneumonia the outcome of parainfluenza virus (piv) infection in patients with leukemia and recipients of hematopoietic stem cell transplantation (hsct) parainfluenza virus infections after hematopoietic stem cell transplantation: risk factors, response to antiviral therapy, and effect on transplant outcome parainfluenza virus respiratory infection after bone marrow transplantation parainfluenza virus and respiratory syncytial virus infection in infants undergoing bone marrow transplantation for severe combined immunodeficiency molecular epidemiology of two consecutive outbreaks of parainfluenza 3 in a bone marrow transplant unit paramyxovirus infection in lung transplant recipients influenza and parainfluenza respiratory viral infection requiring admission in adult lung transplant recipients comparison of different tissue cultures for isolation and quantitation of influenza and parainfluenza viruses efficiency of immunofluorescence for rapid detection of common respiratory viruses direct detection of respiratory syncytial virus, parainfluenza virus, and adenovirus in clinical respiratory specimens by a multiplex reverse transcription-pcr assay biochemistry and clinical applications of ribavirin treatment of respiratory viral infection in an immunodeficient infant with ribavirin aerosol treatment of parainfluenza virus 3 pneumonia in a cardiac transplant recipient with intravenous ribavirin and methylprednisolone successful treatment of parainfluenza virus 3 pneumonia with oral ribavirin and methylprednisolone in a bone marrow transplant recipient the challenge of respiratory virus infections in hematopoietic cell transplant recipients field virology fatal adenovirus hepatic necrosis in severe combined immune deficiency adenovirus infections in adult recipients of blood and marrow transplants respiratory tract viral infections in bone marrow transplant patients respiratory virus infections in bone marrow transplant recipients: the european perspective increasing incidence of adenovirus disease in bone marrow transplant recipients adenovirus infection in hematopoietic stem cell transplantation: effect of ganciclovir and impact on survival molecular monitoring of adenovirus in peripheral blood after allogeneic bone marrow transplantation permits early diagnosis of disseminated disease adenovirus infections in patients undergoing bone-marrow transplantation outcome and clinical course of 100 patients with adenovirus infection following bone marrow transplantation genotype prevalence and risk factors for severe clinical adenovirus infection, united states adenovirus infection in adult orthotopic liver transplant recipients: incidence and clinical significance adenovirus infection in pediatric liver transplant recipients adenovirus pneumonia in lung transplant recipients adenovirus infection in the lung results in graft failure after lung transplantation comparison of pcr, enzyme immunoassay and conventional culture for adenovirus detection in bone marrow transplant patients with hemorrhagic cystitis efficacy of pcr and other diagnostic methods for the detection of respiratory adenoviral infections effect of ribavirin on the plasma viral dna load in patients with disseminating adenovirus infection real-time blood plasma polymerase chain reaction for management of disseminated adenovirus infection internally controlled real-time pcr monitoring of adenovirus dna load in serum or plasma of transplant recipients high levels of adenovirus dna in serum correlate with fatal outcome of adenovirus infection in children after allogeneic stem-cell transplantation treatment of adenovirus disease in stem cell transplant recipients with cidofovir adenoviral infections and a prospective trial of cidofovir in pediatric hematopoietic stem cell transplantation early diagnosis of adenovirus infection and treatment with cidofovir after bone marrow transplantation in children cidofovir for adenovirus infections after allogeneic hematopoietic stem cell transplantation: a survey by the infectious diseases working party of the european group for blood and marrow transplantation treatment of adenovirus pneumonia with cidofovir in pediatric lung transplant recipients ether lipid-ester prodrugs of acyclic nucleoside phosphonates: activity against adenovirus replication in vitro severe adenoviral nephritis following bone marrow transplantation: successful treatment with intravenous ribavirin fulminant adenovirus hepatitis following unrelated bone marrow transplantation: failure of intravenous ribavirin therapy treatment of adenovirus infections in patients undergoing allogeneic hematopoietic stem cell transplantation safe adoptive transfer of virus-specific t-cell immunity for the treatment of systemic adenovirus infection after allogeneic stem cell transplantation adenovirus vaccines in the us military cidofovir for the treatment of adenoviral infection in pediatric hematopoietic stem cell transplant patients clinical virology of rhinoviruses role of viral infections, atopy and antiviral immunity in the etiology of wheezing exacerbations among children and young adults viral-induced rhinitis rhinovirus infections in an industrial population. ii. characteristics of illness and antibody response symptom profile of common colds in school-aged children rhinovirus infections in myelosuppressed adult blood and marrow transplant recipients respiratory viral infections after bone marrow/peripheral stem-cell transplantation: the christie hospital experience virological diagnosis in community-acquired pneumonia in immunocompromised patients a newly discovered human pneumovirus isolated from young children with respiratory tract disease human metapneumovirus infections in young and elderly adults human metapneumovirus: a newly described respiratory tract pathogen human metapneumovirus and lower respiratory tract disease in otherwise healthy infants and children a 1-year experience with human metapneumovirus in children aged <5 years a prospective study comparing human metapneumovirus with other respiratory viruses in adults with hematologic malignancies and respiratory tract infections brief communication: fatal human metapneumovirus infection in stem-cell transplant recipients comparison of the inhibition of human metapneumovirus and respiratory syncytial virus by ribavirin and immune serum globulin in vitro successful outcome of human metapneumovirus (hmpv) pneumonia in a lung transplant recipient treated with intravenous ribavirin polymerase chain reactionbased detection of rhinovirus, respiratory syncytial virus, and coronavirus in otitis media with effusion persistent infection of neural cell lines by human coronaviruses identification of residues critical for the human coronavirus 229e receptor function of human aminopeptidase n human and bovine coronaviruses recognize sialic acid-containing receptors similar to those of influenza c viruses surveillance of communityacquired viral infections due to respiratory viruses in rhone-alpes (france) during winter 1994 to 1995 coronavirus pneumonia following autologous bone marrow transplantation for breast cancer genetic variability of human coronavirus oc43-, 229e-, and nl63-like strains and their association with lower respiratory tract infections of hospitalized infants and immunocompromised patients severe acute respiratory syndrome (sars) in a liver transplant recipient and guidelines for donor sars screening community study using a polymerase chain reaction panel to determine the prevalence of common respiratory viruses in asthmatic and nonasthmatic children typing of human enteroviruses by partial sequencing of vp1 enteroviral disease in the united states pulmonary enterovirus infections in stem cell transplant recipients successful treatment of echovirus meningoencephalitis and myositis-fasciitis with intravenous immune globulin therapy in a patient with x-linked agammaglobulinemia key: cord-015893-e0fofgxq authors: ryhal, bruce title: viral disease, air pollutants, nanoparticles, and asthma date: 2011-05-03 journal: bronchial asthma doi: 10.1007/978-1-4419-6836-4_11 sha: doc_id: 15893 cord_uid: e0fofgxq health care providers who treat patients with respiratory disease are often asked by their patients, “what caused my asthma? and what causes my asthma suddenly to become worse?” these questions have always been difficult to answer, and moving directly to a discussion of the management of asthma is a much easier road to take. in recent years, though, enough information has accumulated about the causes of asthma that one can weave a story containing useful advice that may help patients participate in the management of their disease. and there are also recent studies that can provide answers to the questions posed by physicians who have watched in puzzlement as their previously well-controlled asthma patients have spiraled rapidly out of control. this story has been growing increasingly complex, with an ever-expanding cast of players that sometimes creates a tangled web of interactions. viral respiratory tract infections are the most common triggers of significant asthma • exacerbations. "upper respiratory tract infections" (uris) do not just involve the upper respiratory • tract. human rhinovirus (hrv), which causes the common cold, is the virus most likely to • trigger an asthma exacerbation. in contrast to the usual spring and summer temperate zone pollen season, viral infec-• tions begin to peak in the fall. the number, species, and typical course of viral respiratory tract infections that trigger • asthma vary with a person's age. both acute sinusitis and asthma exacerbations are associated with viral respiratory tract • infection and therefore antibiotics are rarely needed in uncomplicated cases. sulfur dioxide, nitrogen dioxide, ozone, and particulate matter in air pollution may • exacerbate asthma, and patients should be cautioned to stay indoors when levels of these irritants are high. indoor air pollution, especially from tobacco smoke, can be reduced with benefit to the • asthma patient. introduction health care providers who treat patients with respiratory disease are often asked by their patients, "what caused my asthma? and what causes my asthma suddenly to become worse?" these questions have always been difficult to answer, and moving directly to a discussion of the management of asthma is a much easier road to take. in recent years, though, enough information has accumulated about the causes of asthma that one can weave a story containing useful advice that may help patients participate in the management of their disease. and there are also recent studies that can provide answers to the questions posed by physicians who have watched in puzzlement as their previously well-controlled asthma patients have spiraled rapidly out of control. this story has been growing increasingly complex, with an ever-expanding cast of players that sometimes creates a tangled web of interactions. this chapter will look at how viral infections, air pollution, and possibly nanoparticles may act as causal agents of asthma. the concept of causal agent, though, has a variety of different interpretations. in general, agents may act on the respiratory tract to initiate asthma or to exacerbate it. initiation (or inception or development) of asthma refers to the start of asthma in a patient who was previously entirely free of this problem. an exacerbation (or trigger or precipitating event) means the significant and often sudden worsening of an established chronic asthmatic condition. avoidance of a proven initiating factor, if possible, could permit the primary prevention of asthma. in contrast, avoidance of triggering events will not halt the disease but only decrease the number of exacerbations in someone who already has chronic illness. in studying and treating asthma, identification of a specific trigger is usually much easier than trying to prove an initiating cause. viruses that affect asthma are acting on a complex and varied phenotype, and therefore the outcome of each infection can be quite varied. a simple linear cause-and-effect relationship between a viral infection and an asthmatic episode usually does not exist. koch's modified postulates for infection-caused disease are: the microorganism must be present in every case of the disease. • the microorganism must be isolated from a diseased organism and grown in pure • culture. the cultured microorganism should cause disease when introduced into a healthy • organism. the microorganism must be recovered from an inoculated, diseased experimental host. • this linear way of looking at viral-induced disease is not comprehensive enough to allow sufficient insight into the relationship between viral illness and asthma. no one viral infection consistently causes asthma in all or even most individuals. systems biology, though, can provide a conceptual framework for better understanding of the virus-asthma interaction. systems biology looks at the web of factors in the initial state of the individual patient and then examines how one or more external or internal influences perturb this state (1). in fig. 1 , the path taken by system a illustrates how one factor, for example a simple rhinovirus infection, may have very little long-term effect on mucosal inflammation in an individual with no atopic stressors and no genetic propensity toward asthma. this individual will return quickly to equilibrium and a low inflammatory state. the path of system b illustrates how multiple stressors, including genetic factors and atopic immune development, may interact with a viral infection to cause a long lasting or perhaps permanent change in the level of mucosal inflammation. some details of risk factors will be outlined and discussed in this chapter, but systems biology or systems medicine cannot yet specify each feature of the set of interactions in a way that leads to firm predictions about asthma. out of the complexity of the systems approach, though, some simple and compact principles do emerge, so that every precondition does not have to be known to predict the outcome of intervention or treatment. some general factors that appear to be important in the asthmatic response to viral infection include: though two-dimensional paper does not allow multidimensional maps, we can walk down a branching path in a narrative fashion to show the interaction of factors important in viral-caused asthma. in most of the twentieth century, the office or hospital diagnosis of viral respiratory infection was most often a good guess, a probability statement. common and more affordable viral molecular diagnostics, especially reverse transcriptase pcr (rt-pcr), and viral culture can now improve the accuracy of the guess when precision is needed. viruses may be detected in symptomatic or in asymptomatic patients. two thirds or more of acute respiratory tract infections (rtis) occurring in the community can be identified as viral. traditionally, these have been divided into upper and lower rti, but the difference between upper and lower infection seems to be more indistinct than previously believed. human rhinovirus (hrv), for example, replicates initially in the upper respiratory tract yet may cause extensive lower respiratory tract illness. the frequently used term viral upper rti (urti) is somewhat of a misnomer. the most commonly occurring respiratory virus is hrv, which accounts for nearly half of cases of viral respiratory illness, followed by influenza virus and coronavirus, with lesser contributions from parainfluenza virus, respiratory syncytial virus (rsv), adenovirus, metapneumovirus, and other miscellaneous viral species (2) (see table 1 ). the three main types of viruses that are known to affect asthma are hrv, rsv, and influenza. the peak periods of viral infection tend to vary from year to year, but generally in north america rhinovirus peaks in the fall and early spring, influenza in the early winter, and rsv in midwinter (fig. 2) . many communities can monitor the progress of these annual epidemics with viral culture and molecular diagnostics, thereby giving physicians a higher probability of knowing in advance what virus a patient may have. a molecular diagnostic panel is commercially available for identifying acute viral respiratory infection, though the cost-effectiveness of this type of testing for routine clinical use is yet to be determined. more details of the immunobiology of the major asthmogenic viral infection, hrv, have been revealed in the past several years (3) . the intercellular adhesion molecule icam-1 found on nasal epithelial cells is the attachment point for the majority of serotypes of hrv(4). hrv is divided into clades or strains hrv-a, hrv-b, and hrv-c. hrv-c has proven extremely difficult to culture. there are over 100 different serotypes (5). viral species influence asthma in the various age groups in different ways. age is a marker for the development and maturation of the immune system, which diversifies greatly over time. as the human body ages, the immune system molds itself to the environment to become a mirror image of specific, usually protein, molecules in the external local surroundings. age also has an important effect on the physics of scaling in the respiratory system. airway resistance is inversely proportional to the fourth power of diameter, which enlarges with age until young adulthood and then slowly declines. small increases in airway diameter therefore lead to huge reductions in airway resistance and give more "breathing room." about 80% of significant, prolonged wheezing episodes in children are triggered by respiratory viruses and hrv is most often involved (3). the common rhinovirus cold accounts in large part for the fall seasonal peak of asthma in school-age children. epidemiologic evidence combined with viral molecular diagnosis has suggested that this peak is a consequence of children returning to the classroom with the subsequent spread of respiratory viruses, mainly rhinoviruses (6) . viral exacerbations of asthma tend to be prolonged and severe. triggers such as a gust of pollen-laden breeze may be ameliorated by moving the young patient indoors, and exercise triggers can be removed by stopping the exercise, but a viral trigger is usually steady and persistent and replicates within the body. a study of children aged 6-8 years with asthma concluded that an asthma exacerbation was of a greater severity if a viral infection was present as opposed to a nonviral illness (7) . airway hyperreactivity and a corresponding cough and wheeze may be noted for well over 4 weeks after a rhinovirus infection in the asthmatic child. atopy confers additional risk on asthmatic children who become ill with respiratory virus infection (10) . school-aged children with atopic asthma, as opposed to those with nonatopic asthma, have been noted in a number of studies to experience more frequent symptomatic colds, more episodes of viral-triggered asthma, and more prolonged airway hyperreactivity after the colds (7) (8) (9) . the tendency to have higher numbers of symptomatic rtis and a longer duration of illness was also noted for allergic children in general, with and without asthma (9) . parents of children with atopy and asthma tend to be frustrated by the prolonged recovery time compared with their nonatopic siblings, and school absences are more problematic. inhaled corticosteroids and leukotriene receptor antagonists (ltras) are well known to control the number of wheezing exacerbations in school-age children with chronic persistent asthma, an effect that appears to encompass those episodes caused by viral illness. a survey of school children in ontario found that children presenting in september to the emergency department for asthma exacerbations, presumably mostly viral triggered, were less likely to have used preventive anti-inflammatory medications than their counterparts who did not have such severe exacerbations (12) . a retrospective study suggested that inhaled fluticasone and salmeterol administered prior to and during the fall could reduce the morbidity of the fall viral season in patients with asthma (13) . a trial of a montelukast added to usual asthma therapy was able to attenuate the fall asthma peak in one study (14) though this effect did not reach statistical significance in a later trial (15) . inhaled corticosteroids might be expected to prevent viral-induced wheezing in children with minimal chronic disease as well. a preventive effect, though, has not been consistently shown in clinical trials. a study conducted in school-aged children without persistent disease but with a history of viral-triggered wheezing demon strated that inhaled beclomethasone diproponate was not superior to placebo in reducing future episodes. the inhaled steroid failed to reduce days with symptoms, or the frequency, severity, or duration of episodes of upper or lower respiratory illness (11) . preventive medication should therefore be targeted especially to those patients with persistent chronic asthma. for acute treatment of a viral-provoked asthma exacerbation, oral systemic corticosteroids continue to be the most effective choice (16) and are part of the current therapy protocols (17). use of high-dose acute corticosteroid inhalers continues to be studied with varying success. whether vaccination can prevent asthma exacerbations is unclear. the expert panel report concluded that influenza vaccine does not reduce the frequency or severity of asthma exacerbations during the influenza season (17). many patients in the community with asthma experience severe complications from an influenza infection, so all reasonable means of prevention should still be taken, including vaccination. the influenza virus appears to be a less potent trigger of asthma than hrv, and influenza peaks are not as well correlated with childhood asthma peaks as in the case of hrv. an oral influenza antiviral (oseltamivir) improved pulmonary function and reduced exacerbation frequency in one randomized, placebo-controlled trial in school-age asthmatic children who had influenza (18) . unfortunately, increasing resistance of the influenza virus to antiviral agents limits their use as a long-term strategy to reduce illness in asthmatic children. the concept of using antivirals to reduce asthma morbidity in children seems theoretically promising. the preschool years can lay the groundwork for the later asthma issues of the type that have been discussed. diagnosing viral-triggered asthma in infants and preschool children, though, must be done with caution. asthma is defined as a chronic disease, and several, or even many, self-limited acute wheezing illnesses do not necessarily add up to a chronic illness. often children in this age group will experience wheezing in association with a variety of viral infections. parents are naturally anxious about treatment and prognosis in these children. preschool children who experience rsv-and hrv-induced wheezing are more likely to develop asthma in later years. the childhood origins of asthma study (coast) showed that viral wheezing illnesses in infancy and early childhood caused by hrv were the most significant predictors of the subsequent development of asthma at age 6 (19) . a bidirectional causation has been proposed with rsv: severe rsv was associated with a short-term increase in bronchial hyperresponsiveness, and, in turn, the presence of asthma was associated with long-term increased susceptibility for severe rsv disease (20) . whether early childhood viral infection initiates a series of events that lead to asthma has been an area of much interest and study. one analysis showed that infants reaching 4 months of age at the winter virus peak had a 29% increased risk of developing later asthma compared with those reaching age 1 year at the winter peak (21) . if viruses do initiate asthma in some patients, then prevention of rsv or hrv or a similar illness in a critical time period might prevent or reduce the frequency of asthma in later years. nonatopic infants who had received palivizumab (a humanized mab against rsv) for prevention of rsv infection showed an 80% reduction in risk of recurrent wheezing from ages 2 to 5 (22) , though no effect was noted in atopic children. the hypothesis that early viral infections lead to asthma is made less convincing by epidemiologic studies showing that frequent exposure to viral rtis throughout early childhood may actually decrease the risk of later asthma. studies in the united states and in the united kingdom have shown that day care attendance and other factors that increase the frequency of viral rtis reduce the risk of later (after 5-6 years) frequent wheezing (23, 24) . one interesting medical editorial on this topic was subtitled with tongue-in-cheek, "please sneeze on my child" (25) . that strategy may not be practical, but clinicians should be able to reassure worried parents that day care exposure does not seem to result in a long-term risk of asthma. while the factors that contribute to the development of asthma are still unclear, there is little doubt that viral infections act as potent triggers of asthma in preschool children. as noted, hrv is the most potent of triggers, though all hrvs do not seem to be alike. pathogenicity of hrv appears to vary among groups a, b, and c. hrv-c was found in a study of hospitalized preschool children to be associated with asthma more often than hrv-a (26), and hrv-c was noted to be the most frequent type found in patients presenting to the emergency department (27) . in contrast, experimental infection with a type of hrv-a resulted in no worse illness in allergic than in nonallergic subjects (28) . knowledge of a circulating virulent hrv strain in the community could put clinicians on the alert for more serious symptoms in their asthmatic patients with colds. there are several competing classification systems for the wheezing preschool child that aim to help with prognosis and treatment ( table 2) . as a conceptual model, one can create two opposing poles. at one pole is the small child who experiences rare mild wheezing with acute viral illness, has no wheezing or cough between episodes, and has no atopy or parental asthma. these children appear to benefit very little or not at all from acute or chronic corticosteroid therapy for viral-triggered wheezing illness (29) . at the other pole are children who wheeze daily or weekly, have an atopic history, have a parental history of asthma, and may be on chronic controller therapy. a viral infection in these children appears to be a trigger that requires a step up in asthma therapy, perhaps to a burst of oral corticosteroids. between these poles of severity are many children whose therapy must be individualized. the criteria from the national asthma education and prevention program help select preschool children who may benefit from acute and/ or chronic corticosteroids. these guidelines use the asthma predictive index (30) to specify which wheezy small children have or likely will have chronic asthma and could benefit from various forms of inhaled and oral corticosteroid therapy. owing to concerns about oral corticosteroids, other forms of treatment for viral wheezing have been examined in preschool children. a study in 1-to 6-year-old children showed a benefit of episodic high-dose inhaled steroids with viral rti and wheezing (31) , though some adverse effects on growth were noted. the effectiveness of a ltra, montelukast, was examined in a study of 2-5 year olds with a history of intermittent asthma. this study showed a reduction of typically viral-induced asthma exacerbations in children given the ltra as a daily controller (32) . both inhaled corticosteroids and ltras are options to control chronic asthmatic wheezing in this age group (17). prolonged or chronic cough after viral rti may be a problem. preschool children, whether asthmatic or not, spend a considerable percentage of the year with viral rti symptoms that are distressing to patient and parent. the years from teen through young adulthood tend to be the healthiest years of an individual's life. an expanded antiviral immunologic repertoire helps in reducing the number of annual viral rtis. while childhood is the time of most frequent viral rtis, young adults who are exposed to their own small children may have a secondary peak near their 30s. acute sinusitis is a common problem in this age group. sinusitis has been known to precede a worsening of asthma, and episodes of acute sinusitis have often been the occasion for a course of antibiotics. the entity of viral rhinosinusitis, though, is far more common than previously believed. a viral rti can produce a week or more of purulent discharge and radiographic abnormalities of the sinus cavities on ct scans (33) . most acute sinusitis is not predominantly initiated by bacteria nor, at least in the first week or so, antibiotic-responsive (34, 35) . the mechanism by which acute viral sinusitis becomes linked with worsening asthma is generally through the association of both diseases with viral infection (fig. 3) . the adult group of patients with asthma diverges into several different phenotypes, likely representing various diseases. asthma is often said to be a syndrome rather than one disease. different phenotypes may have varying responses to viral infection. a cluster analysis divided asthma patients into five different groups. one group, "benign asthma" seemed to have well-controlled symptoms regardless of triggers, viral or otherwise. another group that was female preponderant, "obese nonesosinophilic," had minimal atopy or eosinophilic inflammation yet a high level of symptoms in response to typical triggers (36) . chronic adult diseases of previously unknown cause have occasionally been found, in whole or in part, to have an infectious etiology. these include peptic ulcer disease (helicobacter pylori), polyarteritis nodosa (hepatitis b and c), reactive arthritis (shigella and chlamydia), and lyme arthritis (borrelia burgdorferi). a survey of asthma patients, of mean age 38, suggested that 45% of initial attacks started after an illness suggestive of a respiratory infection (37) . this subset tended to be nonatopic and may represent a distinct phenotype. viral and nonviral initiating infectious agents have been proposed for adult "infectious asthma," including mycoplasma, chlamydia, adenovirus, and adult rsv, but reasonable proof of an infectious mechanism is still pending. regardless of initiating cause, asthma is exacerbated in adults, as in children, by viral respiratory infections. respiratory virus is found at least 50% of the time in adults with asthma exacerbations, but not as frequently as in childhood acute significant wheezing episodes (3). older and elderly adults experience some degree of immune senescence but also have expanded specific antiviral immunity. the types of viral illness that exacerbate asthma are slightly different than in younger years. the peak of ed visits and asthma admissions for adults over 50 tends not to be in the fall but rather from december to january, suggesting a broader range of viral triggers than in the fall rhinovirus peak (38) . the contribution of influenza to excess morbidity in older adults is well known, but less generally appreciated is the contribution of rhinovirus to serious illness. concomitant heart disease, chronic obstruction pulmonary disease, and hypertension can make viralexacerbated asthma a more complicated and serious illness in older adults. a rhinovirus outbreak in a nursing home for elderly patients resulted in two thirds of the affected patients having lower respiratory tract symptoms, nearly one-third requiring corticosteroid or bronchodilator therapy, and three individuals having serious morbidity including one death (39) . a peak of rhinovirus rti may be seen in grandparents who care for small children. consistently effective treatments for viral-caused respiratory disease have been frustratingly slow to arrive in the modern pharmacopoeia. despite these obstacles, however, a proactive approach, including vaccination and respiratory hygiene, can improve the care of the patient at risk for viral illness and bronchospasm and avert complications. since the time of albert einstein, scientists have known to be wary of "spooky action at distance." particles that affect the respiratory tract must first be dispersed into the air and enter and contact the respiratory tissue to have an effect. these particles vary in size from molecules in the angstrom range (1 × 10 −10 m), to so-called nanoparticles (1-100 × 10 −9 m), to large pollen grains (50 × 10 −6 m), on up to the largest dust particles that can remain suspended in air (about 100 × 10 −6 m). air particles are divided into several common ranges for study purposes: • pm10 -particulates of an aerodynamic diameter of less than 10 mm or 10,000 nm • fine particles of diameters below 2.5 mm or 2,500 nm • ultrafine particles or nanoparticles of diameters below 0.1 mm or 100 nm study of the real-world, clinical effects of the individual components of air pollution is challenging since most or all components tend to be released into the air at about the same time. unwanted and/or unhealthy gases and particles make up the components of air pollution. outdoor air quality issues vary to great extent by specific location and depend on weather and climate, the level of vehicle traffic, and the type of fuel used for energy and manufacture. in the united states, the office of air quality planning and standards (oaqps) has established the national ambient air quality standard (naaqs) for each of the several pollutants. carbon monoxide, lead, nitrogen dioxide (no 2 ), ozone, sulfur dioxide (so 2 ), and particulate matter in the air have maximum exposure standards (fig. 4) . studies of the effect of air pollution on health attempt to use statistical analysis to separate the individual contribution of each component of pollution. additionally, provocation/exposure testing can be performed in the laboratory. many of the same questions that can be asked about viral disease can be asked about air pollution -does it initiate asthma and does it trigger asthma? clearly not everyone breathing air pollution gets asthma or wheezing, but exposure does seem to increase the risk. a population study in the netherlands found that children with higher exposure to traffic-related pollutants (no 2 , particulate matter) were more likely to develop asthma (40) . data from the taiwan children health study showed an increased prevalence of bronchitic symptoms among children with long-term exposure to outdoor air pollutants (41) . in addition to irritant properties, air pollution may contain immunologically active particles. nanoparticles, including particles of diesel exhaust, which are suspended in air are especially interesting to immunologists studying the development of asthma. they have been proposed to act as adjuvants and immunomodulators (42) . most diesel particulates have sizes of less than 1 mm and represent a mixture of fine particles and nanoparticles. acute wheezing may be triggered by exposure to high levels of pollutant gases including nitrogen dioxide, sulfur dioxide, and carbon monoxide. burning of fossil fuels is the main source of these pollutants in most locations. nitrogen dioxide and sulfur dioxide gases diffuse rapidly and impact upon the wet respiratory tract to produce highly irritating acids. sulfur dioxide can cause respiratory constriction in asthmatic patients at concentrations of 0.1 ppm when exercising (44) . healthy adults begin experiencing increased airway resistance at 5 ppm, and even nonasthmatic adults will develop bronchospasm at 20 ppm, though these levels would be highly unusual in outdoor air pollution. nitrogen oxides, and especially no 2 , are also irritating to the upper and lower respiratory tracts at low levels, and patients with asthma are more susceptible to these adverse effects. higher concentrations of outdoor no 2 were associated with more asthma symptoms in a study of inner city children (45) . though the mechanism of action is uncertain, exposure to carbon monoxide in city air was found in one european study to worsen lung function in adult patients with asthma (43) . ozone, while of critical importance to global health in the upper atmosphere, is an especially noxious chemical when generated at or near ground level. ozone (o 3 ) is not produced directly by traffic or by hydrocarbon burning. instead, the combination of no 2 and hydrocarbons with air and sunlight form the secondary pollutant ozone. high average ozone and airborne particulate matter were associated with more frequent asthma symptoms and ed visits and hospital stays in a study of asthma sufferers in the san joaquin valley in california (46) . ozone from air pollution has been shown to exacerbate asthma in children and adults, though this effect may be greater in children (47) . a study of over 90,000 emergency department visits in atlanta for pediatric asthma showed a relationship to ozone and primary pollutant concentrations from traffic sources. these pollutants increased ed visits even at relatively low concentrations (48) . the study of particulate matter in the air is quite complex, since the exact composition varies geographically. in general, high levels of particulate matter have long been associated in epidemiological studies with increased levels of respiratory disease. ongoing research is examining the importance of particle size, fine versus more coarse, in asthma and chronic respiratory illness. one study in turkey showed an 18% rise in asthma admissions when air contained high levels of coarse particles (49) . in contrast, a us study did not find increased hospitalizations for respiratory disease during those periods with high coarse particle levels (50) . the evidence for a negative effect on health from suspended fine particles is stronger (51) . genetic and phenotype differences may be important in the sensitivity of the asthma patient to air pollution. the risk of childhood asthma in mexico city was modulated in some children by genes controlling the response to oxidative stress, such as might occur while breathing ozone (52) . advice on how to avoid high concentrations of air pollutants is important for asthma patients. air pollution, like pollens and viruses, follows a seasonal pattern, and knowledge of the local pattern can help the primary physician with diagnosis and treatment. in the united states, a daily air quality index (aqi) is computed and distributed for most large population areas. the aqi, which is determined on the basis of the highest pollutant of the day, may be considered safe for patients with chronic respiratory disease if less than 50 (green zone). on days with poor air quality, asthma sufferers should come inside where pollutant levels are typically much lower. indoor ozone levels vary from 10 to 80% of outdoor concentrations, depending on the size of outdoor air flows into a building (53) . although asthma patients should continue their controller therapy during periods of high air pollution, pretreatment with controller medications may not always be successful. budesonide treatment in one study was not successful in preventing ozone-triggered functional airways impairment in test subjects with mild persistent asthma (54). while outdoor air pollution rightly receives a great deal of media and government attention, indoor air pollution can make living inside hazardous as well. fortunately, indoor air problems are usually amenable to personal control and behavioral advice. air quality issues may occur in both home and work environments. the field of occupational medicine examines workplace concerns and is reviewed in another chapter. home air quality is typically not regulated, though pollution may result from several indoor sources. hydrocarbon fuels are, of course, burned inside as well as outdoors. indoor gas cooking and heating stoves may produce no 2 , high levels of which have been associated with increased asthma symptoms in children (55) . good ventilation is essential if natural gas is to be burned indoors. indoor nitrogen oxides are also produced by wood-burning stoves, especially if not well vented. the most serious and prevalent type of home air pollution is secondhand or environmental tobacco smoke (ets). the risk from this indoor pollutant begins in utero. maternal smoking during pregnancy is associated with increased infant wheezing (56) . this risk of respiratory morbidity continues to increase with postnatal parental smoking (57) . laws regulating indoor tobacco smoking in one european country were followed by improved quality-of-life scores in a group of asthmatic indoor workers (58) and also by a reduction in the overall rate of hospitalizations for childhood asthma (59) . as noted previously, indoor ozone is usually much less than outdoor levels. in recent years, though, indoor ozone generators have been marketed to the public for odor control and purported heath benefits. a us epa review has warned the public about the potential hazards of adding an additional amount of a measured air pollutant to the indoor air. this chapter has examined some of the most significant initiating and exacerbating causes of asthma. viral respiratory infections, and to a lesser extent air pollution, are common triggers of exacerbations and may interact with individuals to affect the development of some forms of asthma. these causal factors do not exist in isolation but rather interact with the personal attributes of each patient, including his or her genetic and environmental background. the role of viruses and pollutants in asthma is important knowledge that has consequences for prevention, treatment, and avoidance of illness. helpful education may be given to patients and appropriate treatments selected, and health care providers can avoid the considerable human effort and resources wasted on interventions that are useless or harmful. viral and pollutant triggers demonstrate that the highly complex inflammatory asthmatic response is called forth on many more occasions than simply by the contact of pollen grains and other allergens with the respiratory tract. since so much of immune inflammation seems to have arisen from the need to combat infection, the interaction between asthmatic inflammation and viral infection is a natural topic for further investigation. some of the most significant advances in medical care have come through the treatment and prevention of viral illnesses, and furthering the understanding of respiratory viruses is a worthy priority for the future study of asthma prevention and treatment. in addition to natural harmful infectious particles, humans in recent decades have added many substances of their own creation, including the molecules and particles that constitute indoor and outdoor air pollution. control of this problem is very important for overall respiratory health. important action and advice is available for each asthma patient. by understanding and anticipating respiratory viral infections and air pollution as important causes of asthma, the health care provider can provide superior care for those who suffer from this chronic disease. thanks to albin leong md and denise ryhal bsn for helpful comments and for reviewing portions of this manuscript. the clinical applications of a systems approach a case-control study of acute respiratory tract infection in general practice patients in the netherlands the role of rhinovirus in asthma exacerbations expresssion of intercellular adhesion molecule-1 (icam-1) in nasal epithelial cells of atopic subjects: a mechanism for increased rhinovirus infection? common cold, uncommon variation understanding the september asthma epidemic weekly monitoring of children with asthma for infections and illness during common cold seasons duration of postviral airway hyperresponsiveness in children with asthma: effect of atopy allergic children have more numerous and severe respiratory infections than non-allergic children role of viral respiratory infections in asthma and asthma exacerbations effect of inhaled corticosteroids on episodes of wheezing associated with viral infection in school age children: randomised double blind placebo controlled trial the september epidemic of asthma exacerbations in children: a search for etiology dispensing of fluticasone propionate/salmeterol combination in the summer and asthma-related outcomes in the fall attenuation of the september epidemic of asthma exacerbations in children: a randomized, controlled trial of montelukast added to usual therapy the back to school asthma study: the effect of montelukast on asthma burden when initiated prophylactically at the start of the school year national asthma education and prevention programs expert panel report 3: guidelines for the diagnosis and management of asthma oral oseltamivir improves pulmonary function and reduces exacerbation frequency for influenza-infected children with asthma wheezing rhinovirus illnesses in early life predict asthma development in high-risk children the causal direction in the association between respiratory syncytial virus hospitalization and asthma evidence of a causal role of winter virus infection during infancy in early childhood asthma the effect of respiratory syncytial virus on subsequent recurrent wheezing in atopic and nonatopic children siblings, day-care attendance, and the risk of asthma and wheezing during childhood day-care attendance, position in sibship, and early childhood wheezing: a population-based birth cohort study day care, siblings, and asthma-please, sneeze on my child a novel group of rhinoviruses is associated with asthma hospitalizations association between human rhinovirus and severity of acute asthma in children effects of allergic inflammation of the nasal mucosa on the severity of rhinovirus 16 cold oral prednisolone for preschool children with acute virus-induced wheezing the asthma predictive index: a very useful tool for predicting asthma in young children preemptive use of high-dose fluticasone for virus induced wheezing in young children montelukast reduces asthma exacerbations in 2-to 5-year-old children with intermittent asthma computed tomographic study of the common cold effect of amoxicillin-clavulanate in clinically diagnosed acute rhinosinusitis antibiotics for acute maxillary sinusitis cluster analysis and clinical asthma phenotypes infectious asthma: a reemerging clinical entity? epidemiology of asthma exacerbations a rhinovirus outbreak among residents of a long-term care facility traffic-related air pollution and the development of asthma and allergies during the first 8 years of life air pollution and prevalence of bronchitic symptoms among children in taiwan the immune effects of naturally occurring and synthetic nanoparticles carbon monoxide pollution is associated with decreased lung function in asthmatic adults medical management guidelines for sulfur dioxide (so 2 ): agency for toxic substances and disease registry acute respiratory health effects of air pollution on children with asthma in us inner cities outdoor air pollution and uncontrolled asthma in the relationship between visits to emergency departments for asthma and ozone exposure in greater short-term associations between ambient air pollutants and pediatric emergency department visits particulate matter (pm(2.5), pm(10-2.5), and pm(10))and children's hospital admissions for asthma and respiratory diseases: a bidirectional casecrossover study coarse particulate matter air pollution and hospital admissions for cardiovascular and respiratory diseases among medicare patients epidemiological evidence of effects of coarse airborne particles on health nicotinamide adenine dinucleotide (phosphate) reduced:quinone oxidoreductase and glutathione s-transferase m1 polymorphisms and childhood asthma indoor ozone: north carolina public health epidemiology budesonide reduces neutrophilic but not functional airway response to ozone in mild asthmatics health effects of indoor nitrogen dioxide and passive smoking on urban asthmatic children international study of wheezing in infants:risk factors in affluent and non-affluent countries during the first year of life interactive effect of family history and environmental factors on respiratory tract-related morbidity in infancy respiratory symptoms, pulmonary function, and markers of inflammation among bar workers before and after a legislative ban on smoking in public places smoke-free legislation and hospitalizations for childhood asthma asthma and wheezing in the first 6 years of life definition, assessment, and treatment of wheezing disorders in preschool children: an evidence-based approach key: cord-007176-61e9obb3 authors: jackson, george gee; muldoon, robert lee title: viroses causing common respiratory infections in man. iii. respiratory syncytial viroses and coronavimses date: 1973-11-17 journal: j infect dis doi: 10.1093/infdis/128.5.674 sha: doc_id: 7176 cord_uid: 61e9obb3 nan 1. size. rs virus was estimated, from sucrose density gradient centrifugation studies, to be 90-120 nm in diameter [2] ; viral particles in infected cells measured 65 nm by electron microscopy. particles negatively stained with phosphotungstic acid measured 120-300 nm [23] . some viral particles were as large as 860 nm [25] . structure. in ultrathin sections of infected tissue culture, electron microscopy revealed viruslike particles in vacuoles or invaginations within the cytoplasm [14] , but complete particles were not found in the cell cytoplasm [21] . the enveloping membrane has been described as fringed and the nucleoprotein strand as having a herring-bone appearance, with a mean diameter of 13 cf antibody formed by infants during an rs infection has a higher antigen requirement than antibody present in sera of adults or than that found as maternal antibody [113] . neutralizing secretory antibody occurs in one-half or more of patients with lower respiratory disease and in 10%-20 % of those with milder infections [97, 116] . b. secondary. a rise in cf and neutralizing antibody was observed after reinfection in the presence of pre-existing serum antibody [18] . the cf antibody response in adults was always weak compared with that in children [65] . a. infection of tissue culture. isolates were made from 0.1-0.2 ml of specimen inoculated onto a monolayer consisting of 100,000-300,000 hep-2 cells. eagle's basal medium, containing 5 %inactivated chicken serum, is a suitable maintenance medium that should be changed every three to four days. stationary incubation at 36 c is satisfactory. inoculation of a culture of chang liver cells, four to six days old, maintained with a medium of eight parts eagle's basal medium, two parts inactivated horse serum, and 0.2 parts l-glutamine is satisfactory. the medium is changed every four days. virus can be propagated in kb cells in a medium consisting of eagle's basal medium with 2 %chicken serum. preferred cell line. the most sensitive cell cultures are hep-2, monkey kidney, human amnion, and human kidney, in decreasing order [5, 44] . i. the cytopathic effect begins with small syncytial areas randomly distributed early in infection. within one to four days, the entire cell sheet may be involved, with syncytial areas enlarging and becoming more numerous [2] . the time of appearance of cpe depends, within limits, on the number of serial passages of the virus [2] but is nearly always demonstrable within five days. ii. cytology. eosinophilic cytoplasmic inclusions are commonly found in infected cells, especially in the syncytia [15] . these inclusions are devoid of dna, rna, virions, and demonstrated specific antigens. chromosomal abnormalities are not observed. in infected vero cells, dense intracytoplasmic inclusions have diameters ranging from 90 to 130 nm [115] . iii. plaque formation. small macroscopic plaques developed after incubation for seven to nine days in hep-2 cells overlayed with agar [30] . four conditional-lethal, temperature-sensitive mutants of rs have been isolated and shown to be genetically stable. one of the mutants produced a typical, nonsyncytial plaques [93]. iv. hemadsorption. none demonstrated. no data available. one-day-old mice inoculated intracerebrally or intraperitoneally [1, 2] , weanling hamsters, rabbits, guinea pigs, mice, weighing 10 g, and young adult rats inoculated via the previously mentioned routes were refractory to infection [1] . a. natural infection 1. clinical. infection with rs virus has been observed every year since its recognition. it occurs in rather sharp epidemics, recurring at intervals of nine to 14 months, usually in the fall or spring. illness may be gradual or abrupt after an incubation period of three to five days. the symptoms associated with infection in children are cough (97 %), fever (93 %), rhinitis (57 %), pharyngitis (47%), malaise (38 %), vomiting (30 %), anorexia (27%), lymphadenopathy (22%), otitis media (17%), conjunctivitis (13 %), and abdominal pain (7 %) [13, 29, 49] . in an analysis of symptoms produced by infections with rs and by influenza a viruses, the two diseases could not be differentiated on a clinical basis [112] . in infants, as many as 60% the first isolations of virus were made one to two days before the onset of symptoms. isolates were more frequent and positive over a longer period from subjects with illness than from those who were infected but without symptoms. 3. immunity. all adults tested possessed detectable levels of neutralizing antibody to rs virus before challenge, but the titer of naturally acquired antibody had no significant effect on subsequent rs infection of volunteers and was poorly correlated with development of mild clinical illnesses. resistance to infection and illness appeared to be related to the level of nasal antibody but not to the level of serum antibody [121] . infection elicited an increase in the titer of serum antibody by cf and neutralization. immunity upon rechallenge was not tested. illnesses from infection are more severe in children than in adults [8, 10] . the cf test, with 8 units of antigen, will detect 90 %of infections among individuals older than six months and is as sensitive as neutralization. below six months, the cf test detects only 20 %of infections. the neutralization test is more sensitive than cf when serum from infants is used, but rises in neutralizing antibody have been detected in only half of the virus-positive infections in this age group. the use of unheated serum or the addition of antibody-free fresh serum increases the sensitivity of tests for neutralizing antibody [42, 60]. antiserum was produced in guinea pigs by three weekly ip inoculations of 1 ml of infected tissue culture harvest [2] . rabbits given three weekly iv inoculations of 1 ml each followed by two weekly im inoculations of 1 ml of infected tissue fluid combined with 2 ml of a mixture of mycobacterium butyricum, paraffin oil, and arlacel, produced cf and neutralizing antibody [2] . in adult rabbits, an alternative procedure is to give three injections at two-week intervals, the first two consisting of 8 ml of virus and adjuvant given im, and the third injection of virus alone administered iv [5] . respiratory syncytial virus is considered to be a paramyxovirus on the basis of its size, appearance by electron microscopy, and sensitivity to ether; it differs from other paramyxoviruses in that it has no known hemagglutinin. although minor antigenic variants have been found, they are not well discriminated by antibody in human sera. because there has been no sequential drift in the antigenic character of the prevalent strain, rs virus is considered to be a single type. epidemiologically, respiratory syncytial virus is very important, because it causes annual epidemics of acute respiratory diseases affecting infants, children, and adults. infection spreads rapidly from person to person and characteristically occurs as a discrete outbreak of acute respiratory illness in the winter or early spring. in infants and children, especially during the first six months of life, respiratory syncytial virus is the most important cause of bronchiolitis and a major cause of pneumonia. serum antibody acquired by transplacental passage does not provide immunity against infection and might possibly augment the local respiratory disease by an immunopathologic process. the virus may replicate in the middle ear, but its role in otitis is unproven. croup is an infrequent manifestation. pneumonia, as determined radiographically, is frequent, usually bilateral, and multilobar; it may be lobar with secondary bacterial infection. the pathologic lesion is one of necrosis of the epithelial mucosa of the trachea and bronchi and an interstitial inflammation. in adults, infection with rs virus usually causes upper respiratory symptoms; however, because of the prevalence of infection, it is an important cause of exacerbations of bronchitis, pneumonia, and "flu," requiring the hospitalization of adults. in some years, virus infection has given rise to an increase in secondary pneumonia due to diplococcus pneumoniae. infection is followed by an increase in serum cf and neutralizing antibody; also by secretory neutralizing antibody in the nasopharyngeal and tracheal secretions. primary infection does not establish complete immunity, and reinfection is common at all ages. inactivated vaccines of the type and potency previously produced have been detrimental because they have failed to prevent infections and they have induced a more severe disease with exaggerated pneumonia. attenuated live virus vaccines have not yet been successful, nor has any effective chemotherapy been developed. takano a virus isolated from wild cottontail rabbits was shown to possess chemical, physical, and biologic characteristics of the paramyxovirus family. although formation of syncytia was characteristic of its growth in tissue cultures, no antigenic relationship was detected by cf or neutralization tests with any known member of the paramyxovirus family [1] . isolates shown to be antigenically related to human rs virus were recovered from cattle with bronchopneumonia [2] . cytologic examination of bhk2 1 cells infected with bovine rs virus revealed intranuclear and intracytoplasmic viral components [4] . an a type particle with a diameter of 65 nm has been described in the cytoplasm of such cells [3] . the viral envelope was added as the virion passed through the cytoplasmic membrane in a budding process [4] . a. physical properties characterization 1. size. by gradocol filtration, the size of the virion was calculated to be 89 nm [2] . by electron microscopy, the usual diameter was measured as 80-160 nm [5] . 2. structure. the virions are pleomorphic. most particles are covered with projections (spikes) more densely packed than those seen on influenza viruses. these spikes are attached to the virion-by narrow stalks with a thickening (90-110 a) at the distal end [5, 11] . the internal component is a hollow, threadlike structure with a diameter of 70 nm and a definite structural pattern [21] . 3. heat stability. infectivity was destroyed at 56 c within 10 min. there was no loss of titer after 2 hr at 37 c or 10 days at 4 c. rates of thermal inactivation are dependent on the amount of particle aggregation. aggregation is dependent on the concentration of serum in the medium [ a. group antigen common cf antigens have been observed in known members of the coronavirus family, except in avian bronchitis virus. serologic data are still incomplete, but some observed interrelations are shown in table 1. table 1 . antigenic mosaic of coronavirus as determined by neutralization (n) and cf tests with animal serum [20, 25] . antigens produced in tissue culture or mouse brain cf antigens have been prepared from harvests of infected tissue culture and mouse brain, but attempts to prepare antigens from organ cultures have not been successful [2] . strains oc38 and oc43 cross-react, as shown by neutralization tests in mice or monkey-kidney cell culture. strains 229e and lp cross-react in neutralization tests but not to identical titers, indicating a close but not completely similar antigenic mosaic. a one-way cross exists between 229£ and oc32; antisera against the latter and b814 do not neutralize other coronaviruses. avian bronchitis virus reacts only with homologous virus (see table i ). in immunodiffusion tests, the number of detectable lines of precipitation varies from one (strain b814) to four (strain oc43). this may be a result of the procedure used for production of antibody [25] . c. antigenicity i. animals. in animals, specific antibody is elicited by the initial series of inoculations of cell-culture harvests. in neutralization tests with animal serum, no antigenic relationship has been detected between strains 229e and oc38-43 [20] . a. primary response. initial infection results in an increase of specific neutralizing antibody. about 50% of volunteers challenged with the oc strains of coronaviruses developed cf rises to mhv [20] . b. secondary response. the antigenic primacy of the initially infecting strain, heterologous interrelationships, and anamnestic responses are still to be worked out. [16] . coronaviruses of avian origin have phenotypic differences in the susceptible avian host cell range [4] and grow to a limited degree in nonavian tissues [32] . murine coronavirus has grown adequately only in tissues from mice [20] . coronavirus of rats has isolation propagation grown in only one of several cell lines studied [24] . coronaviruses of swine grow only in tissues of porcine origin [37] . a. infection of tissue culture. for growth of explants, a medium of 2 ml of eagle's medium with 0.2 % (wt/vol) bovine plasma albumin and incubation at 33 c in a humidified atmosphere containing 5 %(vol/vol) co 2 in air is satisfactory [5] . a ph of 7.0 is required for growth since inactivation is accelerated at ph 7.7 and 6.7 [25] . i. preferred cell line. human tracheal organ cultures. ii. growth cycle. after 1 hr at 33 c, only 18%of l132 cells wereinfected with strains 229e. virus structures were detected 6-8 hr later [17] .· infection of wi-38 cells with strain 229e resulted in a reorganization of the cytoplasm, as determined by electron microscopy. new viral structures were observed 6-12 hr after infection. clusters of virus were observed in intracytoplasmic vacuoles, called cisternae, 24-36 hr after challenge. strain oc43 in wi-38 matures in intracytoplasmic vesicles similar to those observed with strain 229e.. budding, such as that described for strain 229e was not observed [30] , and the budding process described for coronavirus is into cytoplasmic vesicles rather than from the plasma membrane, as has been observed with myxoviruses [6, 8, 28] . iii i. cpe. the cpe that gradually developed in human diploid cells gave them a stringy appearance. some intracytoplasmic vacuoles were observed [2] . strain b814 caused no cpe and could be detected only by electron microscopy or by interference with echovirus type 11 [1] . ii. cytology. no inclusions have been observed [2] . fluorescent antibody has been used for identification of viral antigen [15] . morphology, as observed by electron microscopy, is characteristic [5, 37] . the use of specific antisera, in combination with electron microscopy, can facilitate recognition of the virus in culture harvests [38] . iii. plaques. at 33 c, with a methyl cellulose overlay, plaques were formed in wi-38 cell cultures [8] . plaques can be produced in l132 cells infected with the 229e strain [17] . [4, 12, 24] . after four to five passages in tissue culture, strains oc38 and oc43 were administered intracranially to suckling mice. on the first passage in mice, illness, characterized by tremors, rigidity, and lethargy, was observed on days 11-15 after challenge. by the fourth passage in mice, these viruses were lethal for mice within 48-60 hr after challenge [9] . there is marked host specificity of different strains. a. natural infection 1. clinical. the use of explants of human embryonic nasal epithelium or trachea has resulted in the isolation of coronaviruses. serology has shown them to be associated with acute respiratory diseases of man. the exact importance of these viruses with accompanying epidemiologic data is unavailable because of the variability of strains and the difficult techniques required to establish diagnosis of the infection. in most studies, coronaviruses usually caused infections in the period from january through march [18, 33, 34] . only about one-half of naturally occurring infections cause clinical illness [26] . spread appears to be preferentially within families. in one study, secondary cases occurred in 17 of 26 families [22] . serum neutralizing antibody does not influence the occurrence of reinfection [33] . acquisition of infection with avian bronchitis virus from chickens is suggested by studies in poultry handlers [13] . nonsusceptible cells infection in man sponses occurred in 10%-20 % of these and other volunteers infected with coronaviruses [29] . strain 229e was recovered from sick as well as healthy volunteers in each of the four challenge passages [7] . volunteers challenged with the oc or b816 strain often showed cf rises to strains 229e and lp [20] . 3. immunity. incomplete. c. prevention 1. vaccine. none. none. diagnosis is based on electron microscopic examination of cell explants or, with some strains, detection of cpe in monolayers after serial blind passage. b. serology rises in the titer of cf antibody against strain 229e and hal antibody against strain oc43 are the most practical tests. neutralization is the serologic test of choice for specific identification. commercially unavailable. prevention laboratory diagnosis coronaviruses are a distinct group of viruses with common morphology and various degrees of antigenic similarity. the number of different coronaviruses that infect man and their exact interrelations are still unknown. their etiologic role in respiratory diseases has been established. also, they are associated with hepatitis in mice and avian bronchitis. data suggest that coronaviruses cause comment 3 %-4 % of acute respiratory illnesses in humans. the clinical syndrome is usually that of a common cold. asymptomatic infections also occur, possibly because of partial immunity to reinfection. coronavirus infections are most prevalent in the winter months and may occur in epidemic fashion, with the same strain being geographically widespread. recurrent epidemics of the same type do not seem to occur in sequential years. preliminary serologic investigations indicate that certain strains may infect children preferentially, whereas others are prevalent in adults. an alternative explanation of the findings is the emergence of new epidemic strains with disappearance of older strains for a period. transmission of the virus within families is frequent, and acquisition may be possible from poultry and other sources. no effective vaccines or chemotherapy have been developed. recovery of cytopathogenic agent from chimpanzees with coryza recovery from infants with respiratory illnesses of a virus related to chimpanzee coryza agent (cca). i. isolation, properties,and characterization recovery from infants with respiratory illness of virus related to chimpanzee coryza agent (cca). ii. epidemiologic aspects of infiltration in infants and young children association of the chimpanzee coryza agent with acute respiratory disease in children growth characteristic of chimpanzee coryza agent in tissue cultures role of respiratory syncytial virus in bronchiolitis, pneumonia, and minor respiratory disease. i. virus recovery and other observations during 1960 outbreak studies of acute respiratory illness caused by respiratory syncytial virus. i. laboratory findings in 109 cases respiratory syncytial virus infection in adult volunteers. ii. correlation of illness and clinical observations an outbreak of febrile illness and pneumonia associated with respiratory syncytial virus infection respiratory syncytial virus infection in adult volunteers. iii. prediction of illness and clinical observations studies on acute respiratory syncytial virus. 2. epidemiology and assessment of importance role of respiratory syncytial virus in bronchiolitis, pneumonia, and pharyngitis with bronchitis in children. ii. serologic studies over a 34 month period studies on acute respiratory illnesses caused by respiratory syncytial virus. 3. clinical and laboratory findings morphology and development of respiratory syncytial virus in cell culture growth and serologic characteristics of respiratory syncytial virus respiratory syncytial virus experimental cytial virus antigens by agar gel diffusion and immunoelectrophoresis interferon and respiratory syncytial virus speculation on pathogenesis in death from respiratory syncytial virus infection the late detection of respiratory syncytial virus in cells of respiratory tract by immunofluorescence double infection with rs virus and influenza virus infections in children. clinical comparison of overlapping outbreaks of influenza a2-hong kong-68 and respiratory syncytial virus infections differentiation of actively and passively acquired complementfixing antibodies in infants with respiratory syncytial virus infection the use of cough-nasal swabs in the rapid diagnosis of respiratory syncytial virus infection by the fluorescent antibody technique morphogenesis of respiratory syncytial virus in a green monkey kidney cell line (vero) respiratory syncytial virus neutralizing activity in nasopharyngeal secretions rsv infections and infant deaths respiratory syncytial virus tissue culture immunofluorescence as a laboratory aid rapid diagnosis of respiratory syncytial virus infection in children by the immunofluorescent technique experimental respiratory syncytial virus infection of adults. possible mechanisms of resistance to infection and illness recovery of a new syncytium virus from a cottontail rabbit a respiratory syncytial virus of bovine origin cultivation of a novel type of common cold virus in organ culture a new virus isolated from the human respiratory tract a new virus cultivated only in organ culture of human ciliated epithelium immunofluorescence of avian infectious bronchitis virus in primary chicken embryo kidney, liver, lung, and fibroblast cell cultures the morphology of three previously uncharacterized human respiratory viruses that grow in organ culture morphogenesis of avian infectious bronchitis virus and a related human virus (strain 229e) effects of a "new" human respiratory virus in volunteers growth and intracellular development of a new respiratory virus growth in suckling mouse brain of "ibv-like" viruses from patients with upper respiratory tract disease recovery in tracheal organ cultures of novel viruses from patients with respiratory disease direct electron microscopy of organ culture for detection and characterization of viruses neutralization of infectious bronchitis virus by human serum cultivation of "difficult" viruses from patients with common colds intracellular avian infectious bronchitis virus: detection by fluorescent antibody techniques in nonavian kidney cell cultures sensitivity of l132 cells to some "new" respiratory viruses the propagation of "coronaviruses" in tissue culture isolation from man of "avian infectious bronchitis virus-like" viruses (coronaviruses) similar to 229e virus with some epidemiological observations some characteristics of hemagglutinin of certain strains of "ibv-like" virus antigenic relationships among the coronaviruses of man and between human and animal coronaviruses symposium on the biology of large rna viruses community-wide outbreak of infection with 229e-like coronavirus in tecumseh, michigan seroepidemiologic studies of coronavirus infection in adults and children rat coronavirus (rcv); a prevalent naturally occurring pneumotropic virus of rats coronaviruses of man seroepidemiologic survey of coronavirus (strain 0c43) related infections in a children's population presence of neutralizing antibody against the 229e strain of coronavirus in the sera of residents of sendai electron microscopic studies of coronavirus coronavirus antibody titres in sera of healthy adults and experimentally infected volunteers intracellular development and mechanism of hemadsorption of a human coronavirus oc43 studies with human coronaviruses. ii. some properties of strains 229e and jackson and muldoon oc43 replication of avian infectious bronchitis virus in african green monkey kidney cell line vero virologic studies of acute respiratory disease in young adults. v. coronavirus 229e infections during six years of surveillance coronavirus infections in working adults: eight year study with 229e and oc43 protein composition of coronavirus oc43 hemadsorption by coronavirus strain oc43 characteristics of a coronavirus (strain 67n) of pigs detection of coronavirus strain 692 by immune electron microscopy of five subjects from whom coronaviruses were isolated, all developed a serologic response [3] .b. serologic. measurement of the cf antibody response was found to be twice as sensitive an index of infection as virus isolation. the cf antibody tends to be transitory, whereas titers of neutralizing antibody remain elevated for a longer period of time [13] . the observed prevalence of infection varies widely in different years [29, 33] .during a spring outbreak of respiratory disease in 1967, the infection rate in a community was 34 % [22] . from observations in a children's home, it was estimated (based on serology) that 19% of respiratory illness in a single season was caused by coronavirus strain oc43 [26] . among groups of adults during two of four winters when there were high rates of respiratory disease and infrequent virus recovery, infection with 229b occurred in 10%-24% of those with upper respiratory tract disease [18] .in three separate studies, each covering a seven-or eight-year period, coronavirus oc43 accounted for 3 % of 1,328 illnesses observed in children [26] ; coronaviruses 229b and oc43 accounted for 4 % of colds in an 'adult industrial population [34] , and 15%-35 % of students were infected during three seasons of high prevalence [33] .a serologic survey of adults and children showed that infection in infants below one year of age was infrequent. infection with oc38 and/ or oc43 occurred principally in the preschool years, whereas infection with 229b occurred later [23] . data from sera collected in 1966indicate that 30 % of adults and 15%-20 % of children had neutralizing antibody specific for strain 229e [7] . in a study of sera collected since 1967, 50 %-80 % of the population were found to have neutralizing antibody against 229e at a dilution of 1:20, as measured by plaque reduction in l132 cells. in cf tests (serum diluted 1: 20) between 50 %and 98 %of those tested had antibody [25] . sera collected from 139 adults between january 1969 and october 1970 showed that 8.6% had a neutralizing antibody titer 2:: 1: 8 against strain 229b [27] . 1. challenge. in an early study, harvest medium from human embryonic trachea containing strain b814 was used as inoculum. illness was observed in five of 11 volunteers [1] . after tissue and organ-culture passage, strain 229e was passed four times in volunteers and produced illness in each passage [7] . later, six coronaviruses isolated from persons with illness were given to volunteers. all six produced colds. heterologous antibody rekey: cord-008716-38sqkh9m authors: schmidt, alexander c; couch, robert b; galasso, george j; hayden, frederick g; mills, john; murphy, brian r; chanock, robert m title: current research on respiratory viral infections: third international symposium date: 2001-06-01 journal: antiviral res doi: 10.1016/s0166-3542(01)00136-x sha: doc_id: 8716 cord_uid: 38sqkh9m nan the third international symposium on respiratory viral infections was convened by the macrae group (new york, ny) in st. lucia, windward islands, on 1 -3 december 2000. for the third time, this symposium provided a forum for virologists, vaccinologists, clinicians, pharmacologists and public health specialists to discuss recent advances in respiratory virus research in an interdisciplinary fashion (kaiser et al., 1999; munoz et al., 2000) . the spectrum of discussion ranged from basic virology and pathogenesis to vaccinology, immunology, and management strategies for respiratory viral infections. epidemiology of respiratory viral disease and possible preparations for the next influenza pandemic were also an important part of the agenda. until 1953, influenza viruses were the only known filterable human respiratory tract pathogens. in 1931, shope recovered an influenza a h1n1 virus from swine, which probably was the first human influenza virus isolated. two years later smith, andrews and laidlaw recovered the first influenza isolate from humans, and soon after, efforts to develop an inactivated influenza a vaccine began. influenza b and c were isolated in 1940 and 1947 by francis and taylor, respectively. in 1953 in the laboratory of infectious diseases (lid) at the national institutes of health, and hilleman, then working at the walter reed army medical center, recovered the first human adenoviruses and established their importance in acute febrile respiratory tract disease (rowe et al., 1953; hilleman, 1954) . in the following years, robert chanock discovered most of the remaining respiratory viruses that are considered important lower respiratory tract pathogens today. the first of them, a croup-associated myxovirus, was discovered during an outbreak of croup in cincinatti in 1954, and it was later designated human parainfluenza virus type 2 (piv2). in 1956, morris and colleagues recovered the chimpanzee coryza agent (cca) during an outbreak of a cold-like illness in a chimpanzee colony, and a year later chanock and colleagues recovered two similar isolates from an infant with bronchopneumonia and from another infant with laryngotracheobronchitis, and characterized the human virus now known as respiratory syncytial virus (rsv) (chanock et al., 1957) . the discovery in 1958 of piv1 and piv3, the single most common cause of croup and the second most common cause of serious viral pediatric lower respiratory tract disease, respectively, broadened our understanding of the etiology of acute lower respiratory tract disease (chanock et al., 1958) . the discovery of piv4 in 1960 followed in short order. in 1961, a double-blind prospective study evaluating the use of tetracycline in the treatment of cold agglutinin-positive atypical pneumonia led to the identification of the etiologic agent of this disease. the agent, originally recovered by eaton from patients with this form of pneumonia, was known to be filterable and thought to be a virus but its role as an etiologic agent was heavily disputed. a large double-blind study by chanock, kingston and mufson, in which antibodies to the eaton 'virus' were used to define a subset of patients with pneumonia, showed that tetracycline therapy decreased duration of the disease, thereby excluding a virus etiology. subsequently, the eaton agent was shown by chanock, hayflick and barile to be a mycoplasma that grew in cell-free medium; later it was named mycoplasma pneumoniae (chanock et al., 1962) . serologic analyses and studies in adult volunteers confirmed the etiologic role of this organism in cold agglutinin-positive atypical pneumonia. renewed efforts in vaccine development against respiratory viruses began in the 1960s with the observation that infants and young children, after having recovered from respiratory tract infection with adenoviruses, shed virus from their gastrointestinal tract for an extended period of time without experiencing gastrointestinal symptoms. this led to the hypothesis that one could potentially use the gastrointestinal tract to vaccinate against respiratory tract disease caused by these viruses. wild-type adenovirus type 4 and 7 administered orally in enteric-coated capsules was found to protect military recruits against respiratory tract disease caused by these viruses. gastrointestinal symptoms were not observed, and although virus was shed from the intestine, it did not infect close contacts (couch et al., 1963) . the development of vaccines against respiratory viruses suffered a major setback in 1966 when formalin-inactivated rsv vaccine not only failed to protect infants against rsv infection but instead potentiated rsv disease upon subsequent rsv infection (kim et al., 1969) . the inactivated vaccine did not induce a potent neutralizing antibody response but it stimulated an exaggerated cd4 + t cell response without stimulating cytotoxic cd8 + t cells. this unanticipated failure of a non-living vaccine reoriented the research agenda of the laboratory of infectious diseases towards the development of live-attenuated virus vaccines. a cold-passaged rsv strain (cp52) was selected in 1966 as the first candidate live-attenuated rsv vaccine strain (friedewald et al., 1968) . this candidate vaccine was safe and immunogenic in adults and older children but was insufficiently attenuated in seronegative infants (kim et al., 1971) . since then, the search for a live rsv vaccine strain has been a central focus of the lid. developing a live rsv vaccine candidate that is attenuated yet immunogenic in seronegative infants has proved to be a formidable task. since incidence and morbidity of rsv are highest in the second and third month of life, a vaccine candidate has to be safe for administration to neonates, able to stimulate an immature immune system, and able to overcome the immunosuppressive and antiviral effects of passively acquired maternal rsv antibodies. initial vaccine candidates were derived in the late 1960s and early 1970s by passage of virus at low temperature (cold passage) or by chemical mutagenesis. several different lineages of mutants, such as temperature sensitive mutants generated by 5-fluorouracil (5fu) mutagenesis, were evaluated in infants and young children but were insufficiently attenuated or genetically unstable. the cold-passaged (cp) mutant that was subsequently further attenuated by the acquisition of two missense mutations that conferred temperature sensitivity (ts) to yield cpts rsv 248/404, has provided the most promising vaccine candidate tested thus far. this candidate vaccine virus was infectious, safe and immunogenic in 1-month-old seronegative infants, conferred protection against challenge with a second dose of vaccine virus 6 weeks later, and caused only mild upper respiratory tract symptoms (wright et al., 2000) . recent development of a method for rescue of infectious rsv from cdna by collins enhanced our ability to develop rsv vaccine candidates rapidly (collins et al., 1995) . site-directed mutagenesis can now be used for the first time to construct viruses with one or more additional attenuating mutations. using recombinant cdna technology, viable rsv mutants with deletion of the ns1, ns2, sh or m2-2 gene have been constructed as vaccine candidates that bear genetically stable attenuating mutations. for these reasons, it is likely that a live-attenuated vaccine that exhibits an acceptable balance between attenuation and immunogenicity can be developed within the next several years. this vaccine virus might possess one or more gene deletion mutations together with or without the earlier characterized cold-passaged (cp) and temperaturesensitive (ts) mutations. the first piv vaccine candidates were also prepared by formalin-inactivation. similar to the experience with formalin-inactivated rsv, these vaccines did not protect against piv disease. in the early 1980s, belshe attenuated a piv3 isolate by 45 passages at 20°c (cp45) (belshe and hissom, 1982) . in clinical studies, the piv3cp45 vaccine candidate has proved to be safe, genetically stable and immunogenic in seronegative in-fants (karron et al., 1995) . this vaccine candidate is currently being tested in phase ii clinical trials. a recombinant version of piv3cp45 has been rescued from cdna, and the genetic basis of its attenuation (att), temperature-sensitivity (ts) and cold-adaption (ca) phenotypes has been determined (skiadopoulos et al., 1999) . influenza a and b virus vaccine development followed this same path of serial passage at 20°c (cold-adaptation) to generate mutants with att, ts and ca phenotypes (maassab, 1969; maassab and bryant, 1999) . in contrast to piv and rsv, live attenuated influenza a vaccine strains were virus reassortants that were generated by mating the attenuated donor virus with an epidemic wild type virus so that the reassortant virus vaccine was a chimera that contained the attenuating genes of the donor virus, while the ha and na genes were derived from the current epidemic virus (murphy et al., 1980) . this strategy, developed by john maassab, of using the cold-adapted mutant virus a/aa/6/60 as the donor of the six attenuating internal and non-structural genes for construction of reassortant vaccine strains was validated by the large series of consecutive reassortants that have proven to be attenuated and immunogenic. analysis of the genetic basis of attenuation showed that the influenza a pb1 and pb2 genes each consistently specified the ts phenotype, and pa specified the ca phenotype. however, all three of these genes of the viral polymerase complex contribute to the attenuation of the trivalent influenza a (h1n1 and h3n2) and b vaccine viruses (murphy, 1993; maassab and bryant, 1999) . the safety, protective efficacy and phenotypic stability were confirmed in large phase iii trials, and licensure is expected in the near future (belshe et al., 1998) . evidence for the prophylactic effect of serum rsv neutralizing antibodies was demonstrated in the 1980s (prince et al., 1985b) . passive transfer of homologous rsv convalescent serum to cotton rats protected them against rsv replication in the lungs following subsequent intranasal challenge with wild type virus. a serum rsv neutralizing titer of 1:300 in recipient cotton rats conferred almost complete protection. this amount of neutralizing antibody necessary for protection was later confirmed in clinical trials and today forms the basis for passive rsv prophylaxis in high-risk infants (groothuis et al., 1993) . an increased incidence and severity of rsv disease is seen in preterm infants with or without chronic lung disease (cld), in children with congenital heart disease (chd), and in immunosuppressed children and adults. respiratory disease in general is a common cause for re-hospitalization of preterm infants. cunningham and colleagues compared a cohort of preterm infants (mean gestational age at birth 289 2 weeks) to a cohort of term infants and found a 10-fold increase in readmission for respiratory disease in preterm infants without cld, and a 18-fold increase for preterm infants with cld (2.5, 25 and 45%, respectively) (cunningham et al., 1991) . cld patients in a home oxygen program were at even higher risk (53% hospitalization, 13% icu admission) (groothuis et al., 1988) . children with pulmonary disorders such as cystic fibrosis, lung malformation or recurrent aspiration pneumonitis, when admitted for rsv disease, are as likely to require icu treatment (13-50%) and mechanical ventilation (up to 33%) as children with cld (35 and 28%, respectively) (arnold et al., 1999) . earlier studies of high-risk infants admitted for rsv disease yielded similar results, with icu treatment necessary in 13-34% and mechanical ventilation necessary in 12-16% (meert et al., 1990; navas et al., 1992) . apart from preterm infants with or without cld, children with congenital heart disease (chd) are a second group of high-risk patients, particularly when they suffer from pulmonary hypertension. in a prospective study of 699 children hospitalized in five consecutive rsv seasons (1976) (1977) (1978) (1979) (1980) , 63% of 27 patients with rsv disease and chd (compared with 14% of patients without chd) required icu treatment and 37% died (macdonald et al., 1982) . in a prospective study of 214 children with chd, the incidence of rsvrelated hospitalization during one rsv season was 15% for children younger than 4 years and 24% for infants younger than 6 months of age (simoes et al., 1998) . immunocompromised children are a very diverse population and not all of them are at equal risk for severe rsv infection. children receiving corticosteroid therapy have a much lower risk for rsv-related hospitalization and death than children receiving chemotherapy for malignancies or children with primary immunodeficiencies. rsvrelated mortality was 15% for chemotherapy-recipients and 40% for children with primary immunodeficiencies compared with 0% for steroid-treated children (hall et al., 1986) . rsv grew to very high titer in children receiving chemotherapy, and more than half the patients shed rsv for 3 weeks or longer. for immunocompromised adults, the picture is not much different from that described for children. infections are often acquired nosocomially, virus shedding is prolonged, and the incidence of pneumonia and death is high. rsv is the most important viral respiratory pathogen in these patients, followed by picornaviruses, influenza and parainfluenza viruses: 60% of confirmed rsv infections in leukemia and bone marrow transplant patients resulted in pneumonia, and the fatality rate was greater than 30% . whimbey and colleagues reported rsv case fatality rates for bone marrow transplant recipients as high as 31% when therapy was administered early and adequately (ribavirin and ivig), and 100%, when therapy was initiated late or inadequately . hiv-infected children in an urban setting in south africa also have an increased burden of viral lower respiratory tract illness (lri) although respiratory viruses are less frequently isolated from nasopharyngeal aspirates of hiv-infected children than from children without hiv infection. the relative risk for severe lri caused by rsv was twice as high in hiv-infected than in uninfected children two years of age or younger (madhi et al., 2000) . little information is available regarding genetic and environmental factors in susceptibility to rsv infections. most rsv epidemiologic studies are conducted in affluent countries and temperate climates although rsv is thought to be the leading cause of severe viral acute respiratory infections (ari) in infants around the globe (weber et al., 1998) . a 3-year prospective surveillance study was conducted in the yukon-kuskokwim (yk) delta of south-western alaska to determine the rate and severity of rsv infections requiring hospitalization for infants in this yupik eskimo population (karron et al., 1999) . the annual rate of rsv hospitalization for yk delta infants less than 1 year of age was unusually high, i.e. 53-249/1000. one in 125 children born in the yk delta, compared with between one in 550 to one in 11 000 infants in affluent countries (sims et al., 1976; martin et al., 1978; glezen et al., 1981) , required ventilatory support for rsv disease. rsv infection was the single most frequent cause of hospitalization of yk delta infants. as in temperate climates, rsv epidemics in the yk delta occur annually from november through june, with peak hospitalizations for rsv disease occurring between november and february. within sub-regions of the yk delta, epidemics were as brief as 1 month, probably because there is very limited traffic between villages in the winter months. most of the infants admitted to hospital were less than 1 year of age and had no medical risk factors for rsv disease. surprisingly, 9% of the admitted infants in the yk delta and 8% of infants in a comparison group admitted to johns hopkins hospital (jhh) were less than one month old. of children with severe disease, 34% in the yk delta and 24% at jhh were less than 2 months old. disease severity in non-high risk children did not differ between children admitted to jhh or yk delta regional hospital, suggesting that differences in hospitalization practices could not account for the high rates of hospitalization for rsv in yk delta infants. in the yk delta, 19% of those admitted for rsv disease were readmitted within a single rsv season. severity of rsv disease, age at first illness and receipt of ribavirin were all associated with readmission. in infants less than 6 months of age, a low neutralizing antibody titer in cord blood samples was strongly associated with severe disease. a questionnaire-based case control study that was matched for age and sub-region in the yk delta detected three risk factors influencing rsv hospital admission. medical risk factors (prematurity, chronic lung disease, congenital heart disease) increased the risk of admission 6.25-fold. more than eight people living in one household doubled the risk of hospitalization while breastfeeding had a protective effect. smoking, food-pre chewing and economic status were not significantly associated with rsv hospitalization. this study may be useful in the continued analysis of the impact of rsv in developing countries. as in the yk delta, rsv is the leading cause of viral lower respiratory tract disease in most developing countries, but lack of access to diagnostic reagents and hospital facilities has made it difficult to quantify the impact of rsv. also, the rate of severe rsv disease in term neonates was much higher than in earlier studies, both in the yk delta population and the comparison group in baltimore. these findings should be confirmed in other populations because they have important implications for rsv vaccine development. 2.4. respiratory syncytial 6irus (rsv) and human rhino6irus (hrv) infections in children with aids and lower respiratory tract illness (lrti) acute respiratory infections (ari) cause a great burden of disease in developing countries (de arruda et al., 1991) . although a growing number of children from developing nations are hiv infected, there is little knowledge about the frequency and severity of viral ari in hiv infected children. earlier studies of viral pathogens in immunocompromised adults indicated that cmv, herpes simplex, influenza, parainfluenza, rhinovirus, adenovirus, enterovirus, and rsv cause lower respiratory infection (connolly et al., 1994) . a recent study assessed the frequency of rsv and rhinoviruses (hrv) in hospitalized children with or without aids, who presented with lower respiratory tract infection (lrti). about 73 episodes of lrti in children with aids and 73 in children without hiv infection, matched by age and sample collection month, were studied in a rural area of southern brazil. the frequency of rsv infection was highest in the fall and winter, between february and july, whereas hrv was detected throughout the year. rsv was found in 8/73 (11%) and 9/73 (12%) of lrti episodes in aids and non-hiv infected children, respectively. hrv was found in 16/73 (22%) and 12/73 (16%) of the episodes in children with aids and hiv-uninfected children, respectively. no difference was detected in frequencies of hrv and rsv infections between the two groups. hrv infections, however, tended to be more frequently associated with pneumonia in children with aids (7/16, 44%) than in the control group (1/12, 8%) (p= 0.09). other clinical presentations of lrti were observed with equal frequency. these findings did not confirm hrv as a causative agent of pneumonia in children with aids, but suggest that further studies of lrti are desirable and that interventions for hrv could be considered for immunocompromised children with lrti. the human adenoviruses (ads) are a large family of over 50 serotypes, as well as numerous variants and intermediates. they are divided into six subgroups (a-f) that exhibit different tissue tropism. the clinical manifestations of adenoviral disease are protean. the most common are respiratory syndromes in both children and adults. it is estimated that ads cause 5-15% of all respiratory disease in children, including pharyngitis, tonsillitis and pertussis-like syndrome. in children and adults, ads are also associated with lower respiratory tract infections such as bronchitis and pneumonia. subgroup d ads are associated with both epidemic and sporadic ocular infections including conjunctivitis and keratoconjunctivitis. due to their stability in the environment, these viruses are highly transmissible, particularly in nosocomial settings. in adults, ads cause largescale epidemics of acute respiratory disease (ard) in closed populations of military recruits, dormitory residents and long-term care facility occupants, which are primarily associated with ad serotypes 4 and 7a, and to a lesser extent with serotypes 2, 3, 11, 21 and 35. while replication in the gastrointestinal tract is a feature of most ad infections, only ad40 and ad41 (subgroup f) are associated with gastroenteritis in infants and young children. ad infections in immunocompromised subjects are increasing as their numbers increase, with severe consequences. clinical manifestations of ad infection in immunocompromised patients include pneumonia, hepatitis, encephalitis, and systemic and disseminated disease, with case fatality rates from 18 to 60%, depending on the nature of the immunodeficiency. nearly all ad serotypes have been associated with these infections, but the higher numbered subgroup d serotypes that are usually not associated with clinical disease in the immunocompetent host have been especially common. a number of 'new' disease associations with ad infection have recently been reported, probably due to improved highly sensitive molecular diagnostic techniques that also increase the probability of laboratory contamination. detection of viral genome in the absence of positive viral culture has been described in cases of myocarditis and pericarditis in children and adults (martin et al., 1994; bowles et al., 1999; pauschinger et al., 1999) , sudden infant death (shimizu et al., 1995) , toxic shock-like syndrome (price, 2000) and 'unexplained death' (perkins et al., 1996) . isolation of ads from patients with central nervous system manifestations of fatal acute flaccid paralysis (cardosa et al., 1999) and encephalitis with cerebral edema (chatterjee et al., 2000) has also been reported. it is, perhaps, not surprising that the clinical manifestations of ad infection are evolving because the viruses themselves are in the process of continuous evolution. the ad mutational repertoire includes homologous recombination, illegitimate recombination, and single base mutation (sbm). homologous recombination occurs in conserved regions of the genome between closely related viruses within the same subgroup, and it requires regions of homology in the two parent strands. it is the primary mechanism responsible for intermediate ads, which are mosaic viruses with shared hexon characteristics, or with the hexon characteristics of one type and fiber of another. illegitimate recombination requires only short regions of homology of one to three nucleotides (short direct repeats), and is thought to be the result of polymerase stuttering or slippage. it causes deletions, insertions and duplications of short regions of dna. in ads it occurs in noncoding regions and hypervariable regions (hvrs) of hexon capsid proteins that tolerate structural variation. the hvrs of the hexon contain the viral neutralization epitopes, so that mutations in these regions result in deletion, formation or alteration of these epitopes, leading to antigenic shift. it is the primary mechanism by which new serotypes arise, particularly among the fastest growing group, the subgroup d ads. single base mutations accumulate gradually across the viral genome but can occur at a 30-fold higher rate in the hvrs, where they cause incremental antigenic drift and the creation of variant strains. ad evolution is compounded by all three mechanisms and perhaps others that have yet to be defined. as a result, serological identification of subgroup b2 and d ads has become extremely difficult. it seems reasonable that the time has come to consider a sequence-based ad classification system, similar to that in use for papillomaviruses and enteroviruses. adenovirus has re-emerged as a leading cause of febrile respiratory disease among military recruits. large and frequent epidemics were common at trainee camps before 1971 but were eliminated with the introduction of the live enteric type 4 and 7 vaccines. in 1996, the sole vaccine manufacturer discontinued production of these live enteric coated vaccines because of contractual issues. while limited vaccine stores were still available in 1997 and 1998, vaccine stores were completely depleted by early 1999. to monitor the effect of discontinued vaccination, weekly surveillance for febrile respiratory infections (fri), defined as oral temperature \100.5°f with respiratory disease symptoms, was conducted from october 1996-june 1998 at four military training camps. during this interval, 1814 (53%) of 3413 throat cultures yielded adenovirus. during the winter of 1997-1998, adenovirus infections caused more than 90% of fri at each of the four camps. ad 4, 7, 3, and 21 accounted for 57, 25, 9, and 7% of the isolates, respectively. three training camps experienced a high prevalence of adenovirus type 4 and the fourth camp experienced a type 7 outbreak. among symptomatic trainees, those who did not receive vaccine were 28 times more likely to be infected by ad 4 or 7 than vaccinated subjects (gray et al., 2000) . surveillance was extended to eight sites in june 1998 and virus isolation was attempted for adenovirus, influenza a and b, rsv, and parainfluenza 1-3. large ad 4 epidemics were observed in six training camps throughout the us, while rsv and influenza a and b viruses were isolated less frequently. the impact of adenovirus epidemics on basic training can hardly be overestimated. recruit camps were forced to convert barracks into special infirmaries to care for the ill, and hospitals were forced to halt elective surgeries. at one camp, the number of trainees that had to repeat their basic training because of extended illness increased 20-fold. this 'recycling' has an extremely negative effect on the morale of trainees and it impacts on the military's readiness. as many as 1800 preventable adenoviral trainee medical encounters occurred during the winter months of 1999 and in 2000; two military trainees died with molecular evidence of acute adenoviral infection, one with encephalomyelitis and another with acute respiratory distress syndrome (ards). vaccination against ad 4 and 7 has proven to be extremely safe and effective, and to prevent an enormous burden of disease in military trainees (howell et al., 1998) . it is urgent that a new manufacturer for adenoviral vaccines be identified, and vaccine production must resume as soon as possible. in recent years, much progress has been made in understanding virus-induced modulations of the host immune response to viral infections. for ad, more than 20 viral gene products are known to participate in the modulation of immune responses, and many of these gene products are expressed from genes clustered in the early region 3 (e3) (wold et al., 1999; horwitz, 2001) . the overall effect of ad e3 gene products on immune responses in vivo can be appreciated from the results of three studies in mice that investigated transplant rejection and development of autoimmune disease. in the first study, the expression of the complete ad e3 cassette in pancreatic islet cells as transgenes under the control of the rat insulin promoter (rip) enabled allogeneic islet donor cells containing the h-2 bxd class i mhc to be accepted long-term by h-2 d recipient mice (efrat et al., 1995) , indicating that ad e3 gene products could potentially be used as a powerful tool in the control of transplant rejection. the second study used the lymphocytic choriomeningitis virus (lcmv) model of autoimmune diabetes mellitus, in which the lcmv proteins np or gp are expressed on the surface of islet cells, and diabetes is induced by infection with lcmv that induces cd8+ (gp) or cd8 + and cd4 + (np) t-cell mediated immune responses. in this model, the co-expression of rip-e3 with lcmv-np or gp completely prevented the onset of diabetes after lcmv infection (von herrath et al., 1997) . similar protective effects of ad e3 transgenes were seen in a third study that used the non-obese diabetic mice (nod) model of diabetes mellitus, and the underlying mechanisms are currently being investigated (efrat et al., 2001) . thus, as the understanding of the mechanism of action of the ad e3 immunoregulatory genes are being pursued in various systems, they are being utilized to control selected immune reactions that might be involved in the genesis of autoimmune diabetes. some of the better-characterized gene products of the e3 region are (in order of increasing distance from the e3 promoter) gp19, 11.6, 10.4, 14.5 and 14.7k. only the functions of gp19 and 14.7k shall be discussed here; the other ad e3 gene products have been reviewed elsewhere (horwitz, 2001) . in vitro, gp19k reduces the expression of class i major histocompatibility complex (mhc) molecules by retaining the mhc heavy chain in the endoplasmic reticulum or retrieving it back from the golgi, and also by inhibiting peptide processing (bennett et al., 1999) . in the cotton rat model of adenovirus pneumonia, gp19k deletion mutants replicate like wild-type virus but they induce a much stronger inflammatory response (ginsberg et al., 1989) , whereas in c57bl mice an increase in pulmonary pathology is not seen (sparer et al., 1996) . the ad e3 14.7k protein inhibits tnfa-induced cell death by a process that does not involve down-regulation of the tnfa-receptor. in cotton rats (ginsberg et al., 1989) and c57bl mice (sparer et al., 1996) deletions of ad e3 14.7k modify the pulmonary inflammatory response, i.e. an increase of polymorphonuclear leukocytes in cotton rats and more pronounced alveolar infiltration in mice. in order to determine how the ad 14.7k protein prevents cell death, the cell proteins that interact with this viral protein were determined. using a yeast two-hybrid system, four 14.7k-interacting proteins (fips) were identified. three of them have been characterized and have been shown to participate in quite diverse cellular pathways (li et al., 1997 (li et al., , 1998 (li et al., , 1999b . fip-1 (also known as rag-a, a ras-related small gtpase) can bind to tctel, a component of the microtubule motor protein dynein, forming 14.7k-fip-1-tctel complexes (lukashok et al., 2000) , and 14.7k has been postulated to affect microtubule dependent macromolecular transport or even modulate the transport of virus. however, because 14.7k is not a structural protein of adenoviruses and must be made de novo from early viral transcripts, it is unlikely to play a role during viral entry, even though the process is known to be microtubule dependent. the role of 14.7k during viral exit from cells has not been studied. fip-1 also binds to a second gtpase (gip-2) that localizes in the centrosome and in addition to potential effects during mitosis may be involved in transporting macromolecules between the nucleus and the cytoplasm. fip-2 binds to abnormal huntingtin, and more specifically to the expanded polyglutamine tract that appears to be associated with cell death of neurons in huntington's disease (faber et al., 1998) . whether or not ad e3-14.7k and/or fip-2 can prevent huntingtin-induced cell death is currently being investigated. over-expression of fip-3, which is also called nf-kb essential modulator (nemo) or inhibitor of kappa kinase gamma (ikkg) causes morphologic changes and eventually apoptosis in a variety of cell lines. the amount of apoptosis induced by fip-3 can be reduced by 70% when ad e3-14.7k is present. apart from 14.7k, fip-3 seems to interact with a number of key molecules in the tnf receptor and nf-kb signaling pathways such as the receptor interacting protein (rip), the inhibitor of kappa b kinase beta (ikkb) and the nf-kb inducing kinase (nik) (li et al., 1999b) . these few examples of the effects ad e3 gene products have on the pathobiology of diseases as different as autoimmune diabetes, transplant rejection and huntington's disease indicate how much remains to be learned from studying adenovirus-host interactions. the rsv (strain a2) genome is a single stranded negative-sense rna of 15 222 nucleotides that is transcribed into 11 major subgenomic mrnas. three of the eleven encoded proteins are transmembrane proteins. the g protein mediates attachment to cell surface receptors, the f protein mediates virus-cell and cellcell fusion, and the function of the sh protein is unknown. other structural proteins are the m protein, which plays a role in virion assembly, the n, p and l proteins that make up the viral polymerase, and the m2 orf1 protein that functions as a transcription anti-termination factor. the other proteins include two non-structural species, ns1 and ns2, and the m2-2 protein encoded by the second orf of the m2 mrna. using a reverse genetics system to rescue infectious rsv from cdna, five of the 11 genes of rsv can be ablated individually and in some cases in combination without rendering the virus non-viable jin et al., 2000b) . these five non-essential genes are ns1, ns2, sh, g, and m2-2. since all of these genes confer a selective advantage to rsv in vitro and/or in vivo, they can be described as virulence factors -the deletion of which will lead to attenuation of the virus. deletion of the small hydrophobic (sh) transmembrane protein yields a recombinant rsv called rsva2dsh, which replicates in vitro as well as wild type (wt) rsv and induces plaques in hep-2 cells that are larger than wt rsv plaques. there is no reduction in synthesis of rna or protein associated with the deletion of sh. in chimpanzees, however, the virus is slightly attenuated (whitehead et al., 1999a) . deletion of the ns1 or ns2 gene results in a substantial reduction in replicative efficacy in vitro, and this reduction is more pronounced in hep-2 cells than in vero cells (which lack interferon a and b genes), suggesting that these two genes act as antagonists to type 1 interferon effects. deletion of ns1 and ns2 from bovine rsv provided direct evidence that ns1 and ns2 cooperatively antagonize a/b interferon-induced antiviral responses (schlender et al. 2000) . in chimpanzees, the level of replication of both rsva2dns1 and rsva2dns2 is reduced greater than 10 000-fold in the lower respiratory tract. in the upper respiratory tract, the dns1 virus is more attenuated than the dns2 virus (teng et al., 2000) . deletion of the m2 orf2 not only identifies a markedly attenuated rsv mutant but reveals an important role for this orf in the replicative cycle of rsv (bermingham and collins, 1999) . during infection with wildtype rsv, transcription appears to shut off at approximately 12-16 h post infection while rna replication increases concurrently. in contrast, this apparent switch from transcription to rna replication was not observed for the rsvdm2-2 virus, implying that m2-2 is a regulatory protein involved in the shift. instead, transcription continued to increase while rna replication remained low compared to wild-type rsv. overall, gene expression was increased 7-18 fold. the synthesis of the g and f proteins also was increased and resulted in increased syncytium formation. replication of the rsvdm2-2 virus in vitro was attenuated, probably due to reduced rna replication (bermingham and collins, 1999; jin et al., 2000a) . in chimpanzees, comparison of the four rsv gene deletion mutants mentioned above with wt rsv and the incompletely attenuated rsv cpts-248/ 404 vaccine candidate results in a hierarchy of increasingly more attenuated viruseswt rsv b dsh b dns2 b 248/404 b dns1 b dm2-2. the final rsv gene deletion mutant, rsva2dg, was not evaluated in chimpanzees because the absence of this major protective antigen would not be desirable in a vaccine virus. rsva2dg replicates as efficiently as wt rsv in vero cells, showing that g is not essential for efficient virus replication. however, the rsvdg virus is highly attenuated in balb/c mice, indicating the importance of the rsv g protein in vivo. it is evident from the above ranking that rsv reverse genetics is able to generate mutants exhibiting gradations in their level in attenuation ). this menu of viruses with different levels of attenuation is crucial in identifying an rsv vaccine that exhibits the desired balance between attenuation and immunogenicity in seronegative infants. since clinical data indicate that rsv 248/404 is just slightly under-attenuated in the 1-month-old target population, the dns1 mutant could be exactly what is needed. in order for rsv assembly to be an efficient process, viral structural proteins must be brought together in a coordinated fashion (peeples, 1991; lenard, 1996) . compared with other paramyxoviruses rsv exhibits several unique features. the g, m2-1 and m2-2 proteins are found only in the pneumo6irus genus of the paramyxo6iridae, and the role these proteins play in rsv assembly is much less well understood than the role of the f, hn and m proteins of other paramyxo6iridae (collins et al., 1996) . comparison of multiple human, bovine and ovine rsv strains shows that the cytoplasmic domains of the f and g proteins are well conserved amongst human rsv strains and subtypes, and conserved to some degree between the three species. in analogy to other paramyxoviruses, it is likely that the cytoplasmic domain of f and g interact in the process of virion assembly with cellular proteins involved in protein trafficking and in the polarized budding process, as well as with other viral proteins. the rsv g and m proteins co-localize in the golgi apparatus, not only in rsv-infected cells but also in cells transfected with only the g and m proteins, indicating that other viral proteins are not needed for this interaction (peroulis et al., 2001) . the g-m interaction is seen with fulllength g protein but not with a secreted form of g that lacks the conserved cytoplasmic domain and transmembrane domains. systematic deletion and substitution mutagenesis of the cytoplasmic domain of g has identified a sequence-specific, six amino acid motif that directly interacts with m (peroulis et al., 2001) . during rsv infection m protein can initially be detected in the nucleus, but later in the infectious cycle it is found in inclusions within the cytoplasm (ghildyal et al., unpublished) . the n and p proteins of rsv were earlier shown to be necessary and sufficient for these inclusions to form, and the rsv m2-1 and l proteins were also shown to be present in these inclusions (garcia et al., 1993) . these same investigators were unable to identify m protein in the inclusions (garcia et al., 1993) . using confocal immunofluorescent microscopy of infected and cotransfected cells the m protein was shown to be present in these inclusions . m protein does not localize to the inclusions unless m2-1 is also present . the m and m2-1 proteins not only co-localize by confocal microscopy but also interact in a protein overlay assay (ghildyal et al., unpublished) . taken together, these data suggest that, as with other single stranded negative-sense viruses (peeples, 1991; lenard, 1996) , the rsv m protein seems to play a crucial role in rsv assembly; bringing the nucleocapsid together with the envelope proteins by binding to the cytoplasmic domains of g and f and to the nucleocapsid proteins n and p together with or via m2-1. the interaction between m, m2-1 and the nucleocapsid proteins might be more complex than outlined here, and might involve additional cellular or viral proteins. to better understand rsv assembly, the domains in the m protein that interact with g, f and m2-1 will have to be defined. while influenza viruses readily develop resistance to older antivirals such as amantadine or rimantadine, resistance to neuraminidase inhibitors occurs much less frequently. nonetheless, resistant influenza a virus mutants can be isolated from patients treated with oseltamivir (treanor et al., 2000) . one of the resistant mutants carries an arginine to lysine mutation at position 292 (r292k) of the neuraminidase protein, causing a reduction in substrate binding and enzymatic activity, as well as resistance to oseltamivir (mckimm-breschkin, 2000) . the infectivity of influenza a viruses carrying the r292k mutation was earlier found to be markedly reduced in mice and ferrets. transmission of an influenza a h3n2 clinical isolate with a r292k mutation was studied in ferrets in comparison to transmission of the parent wt h3n2 virus that was isolated from the same patient. donor ferrets (four per group) were inoculated intranasally with the r292k mutant or wt virus and housed with three naïve contact ferrets per donor ferret. the four donors inoculated with wt virus were infected and transmitted virus to each of the 12 contacts. wild type virus replicated to between 10 4 and 10 5 pfu/ml nasal aspirate. only two of four donor ferrets inoculated with mutant virus became infected, and the level of replication of mutant virus was reduced 10-100-fold compared with that of wt virus. however, both infected ferrets transmitted virus to contacts. one of them transmitted the r292k virus to one of three contacts only, with virus detected at very low titer on 1 day only. the other donor transmitted virus to all three contacts, and the transmitted virus replicated to titers greater than 10 4 pfu/ml in the contact ferrets. sequence analysis showed that the donor virus was a mixed population of mutant and wt virus and that only wt virus was recovered from contacts. these data confirm the reduced infectivity of oseltamivir resistant r292k mutants. effective transmission of the r292k mutant virus in ferrets was not observed, suggesting that the transmission of oseltamivir-resistant virus from human to human will be unlikely, even during widespread use of na inhibitors in the treatment of influenza. respiratory virus infections in immunocompromised patients are characterized by persistence of viral infection, prolonged shedding of virus, a high rate of nosocomial acquisition, and a high frequency of pneumonia and death. similarly, respiratory virus infections in the elderly are responsible for a substantial amount of morbidity and mortality. detection of respiratory virus infections in these high-risk patients is important for several reasons. it enables the initiation of specific isolation procedures, initiation of specific antiviral therapy, cessation of unnecessary therapy, tests and procedures, and it can aid in identifying and preventing potential outbreaks. respiratory virus infections can be diagnosed using serology and culture techniques, as well as newer methods such as antigen-detection by enzyme-linked immunoassay (eia) or immunofluorescence (ifa), enzymatic detection by chemical reactions, or amplification of parts of the viral genome (pcr). serology is not useful in the acute phase of most illnesses and is of limited usefulness in immunocompromised patients, the elderly or those receiving blood products. recovery of virus in cell culture is still seen as the gold standard by many but the importance of obtaining a good specimen to ensure that the culture is not falsely negative is often overlooked. a good clinical specimen is the most critical factor in ensuring a correct diagnosis regardless of the diagnostic method used, although it is perhaps most important for culture. use of nasal wash to obtain a specimen for virus isolation is well-tolerated in cooperative adults and, compared to nasal swabs or throat swabs, increases the sensitivity of cell culture for virus detection. other factors critical to laboratory success are the use of appropriate transport media, temperature of transport/incubation and time until processing (atmar and englund, 1997) . this is particularly important in the case of rsv infections in immunocompromised or elderly patients, who often have a relatively low viral titer such as 10 2 -10 3 pfu/ml, whereas children often have a titer greater than 10 6 pfu/ml of nasal wash or bronchoalveolar lavage fluid (englund et al., 1996) . for rsv, parainfluenza and influenza virus there are a number of commercially available rapid detection kits. multiple simultaneous rt-pcr for detection of rsv, influenza a and b, and piv1, 2 and 3 (hexaplex ® ) was tested in 763 pediatric samples and yielded 100% sensitivity and 98% specificity as compared with culture (fan et al., 1998) , with only 8 h processing time. in a separate study, pcr for respiratory viruses in adult patients with hematologic malignancies was found to be as sensitive as culture (van kraaj and van elden, 2000) . for influenza, several antigen detection kits ((directigen, fluoia and quick-vue) and one neuraminidase (zstatflu) assay are available to detect virus, with sensitivities ranging from 50 to 96% and specificities ranging from 52 to 99%. rapid diagnosis of influenza in pediatric patients leads to a decrease in the frequency and duration of antibiotic use and an increase in the frequency of antiviral therapy (noyola and demmler, 2000) . rapid diagnosis kits for the detection of rsv in children range in sensitivity from 61 to 87% and in specificity from 93 to 97%, with some test kits performing better than others (dominguez et al., 1993) . in immunocompromised adults, however, the sensitivity of antigen detection kits from nasal wash or throat swab sample was only 15%, while endotracheal or bronchoalveolar sampling increased sensitivity to 70 or 80%, respectively, (englund et al., 1996) . several studies in recent years have highlighted the importance of upper respiratory tract infections in the exacerbation of asthma in children. freymuth and colleagues reported human rhinovirus (hrv) (46.9%) and rsv (21.2%) as the most frequent pathogens detected in patients with exacerbation of asthma, and noted that pcr increased detection rates 5.8-and 1.6-fold in hrv and rsv infections, respectively, over that of conventional assays (freymuth et al., 1999) . in a study of wheezing children between 2 months and 16 years of age, respiratory viruses were detected in 82%, with rsv in infants (detected in 68% of subjects) and hrv in older children (71%) as the predominant pathogens. both were strongly associated with wheezing (rakes et al., 1999) . in a community-based longitudinal study, respiratory viruses were detected in 80% of episodes of acute illness with reduced peak expiratory flow, 80% of episodes of wheezing, and in 85% of episodes of upper respiratory symptoms, cough, wheezing, and a fall in peak expiratory flow (johnston et al., 1995) . in these settings, rt-pcr provides a fast and sensitive method to detect rna viruses. real time quantitative pcr assays can be similar to conventional pcr in specificity and speed. however, real-time pcr can also quantify viral load (quantity of virus in respiratory secretions) during asthma exacerbations, and it is sometimes more sensitive than conventional pcr. real time taq-man quantitative pcr allows estimation of the input viral genome copy number by including a fluorescence reporter on one end and a quenching molecule on the other. the reporter does not fluoresce until the quencher has been cleaved off by the exonuclease activity of taq polymerase, permitting an estimate of the quantity of pcr product. fluorescence is being measured continuously every seven seconds, and quantification of the target is based on the number of pcr cycles it takes to produce detectable fluorescence. a retrospective study was conducted in 117 asthmatic children 9-11 years old to compare viral loads in quiescent and exacerbation periods of asthma using the taqman technology. nasal aspirates had been collected earlier and records of peak flow measurement and clinical scoring were available. a quiescent period was defined as absence of clinical symptoms for two weeks or longer. real time quantitative pcr detected respiratory viruses (mostly rhinoviruses) in 97% of the children with an asthma exacerbation and in 42% of children in quiescence. viral load was higher in children with exacerbation of asthma than in those with quiescent asthma and higher viral loads also correlated with more severe clinical disease. although the study showed that real time quantitative pcr is more sensitive than nonnested conventional pcr and also allowed esti-mation of viral load, there is the caveat that the study had a retrospective design. modalities of immunity to acute viral respiratory infections are both specific and nonspecific, humoral and cell mediated. fever, interferon (ifn), tumor necrosis factor (tnf), natural killer cells and activated macrophages are non-specific modalities stimulated by infection that are capable of mediating antiviral effects. lung collectins such as sp-a, have also been implicated in the control of viral respiratory infections (ghildyal et al., 1999) . specific modalities are antibody in serum and secretions, lymphocyte proliferation responses with cytokine release, and cytotoxic lymphocytes (ctls). all modalities participate, to some degree, in containing an infection and promoting recovery either via inactivation of free virus or elimination of infected cells. thus, there is considerable redundancy in the mechanisms controlling the virus infection and promoting recovery. on the other hand, protection against infection is primarily conveyed by specific antibody. sufficient data is available to conclude that serum igg neutralizing antibody is the primary mediator of resistance to influenza virus infection, presumably because infection is initiated in the lower respiratory tract and igg antibody derived from serum is the dominant antibody isotype at that site (couch and kasel, 1983) . in contrast, iga antibody is the primary mediator of resistance to rhinovirus and coronavirus infection because evidence indicates these infections are initiated in the nasopharynx where iga is the dominant antibody isotype (cate et al., 1966) . since primary infections with influenza virus, rsv, or piv induce disease in both the upper and lower respiratory tract, both igg and iga antibodies are correlates of immunity to infection and disease (crowe, 1999) . although adenoviruses also cause upper and lower respiratory disease, only serum antibody has been shown to correlate with immunity. reinfection with homologous rsv, piv, rhinovirus and coronavirus can occur but is generally confined to the upper respiratory tract, presumably because igg antibody in protective quantities is more durable in serum (and lower respiratory secretions) than is iga antibody in nasopharyngeal secretions. for rsv, the risk of infection declined from 74% for naïve subjects to 56 or 33% after one or two infections, respectively; the risk of lower respiratory disease in these groups declined from 18 to 15 to 3%, respectively, (glezen et al., 1986) . for a rhinovirus, resistance to reinfection of the nasopharynx correlated with the titer of iga antibody in secretions (cate et al., 1966) . rodent models of viral respiratory disease provided much of the early data on immune modalities conferring protection (crowe, 1999) . both cd4 + and cd8 + t cells can effect clearance of influenza a virus, rsv and piv in mice in the absence of the other cell type (lightman et al., 1987) . while cd8+ cytotoxic lymphocytes (ctl) mediate immunity through lysis of infected cells and expression of antiviral cytokines, cd4+ t cells exhibit limited direct antiviral activity but play a role in activating b cells and in inducing antiviral cytokine expression (epstein et al., 1998) . there is a general consensus that ctls contribute significantly to the resolution of primary infections with influenza, rsv and piv in rodents. while the correlation between antibody response and protection in humans was established decades ago, cellular immune responses were studied much later, and understanding of the development of these responses in infants and immunosenescence of them in the elderly is still incomplete. whether or not ctls in humans convey immunity to respiratory virus infection in the lower respiratory tract and whether they promote clearance of virus in the upper respiratory tract of humans remains to be elucidated. however, for rsv disease in humans, a correlation has been observed between the presence of rsvspecific ctls in year 1 and absence of severe rsv disease in year 2 (mbawuike, in press). morever, the level of influenza-specific ctls correlated inversely with the quantity of virus in nasal secretions after challenge of humans, and the ability of ctls to function at this site was demonstrated by a reduction in influenza virus titer in nasal turbinates of mice which were adoptively immunized with purified cd8 + ctls before intranasal challenge (mcmichael et al., 1983 mbawuike, personal communication). knowledge regarding viral-bacterial interactions in the respiratory tract goes back at least to the 1918 influenza pandemic, and interactions have been described for later influenza pandemics as well. epidemics and pandemics of influenza have been followed regularly by an increase in the incidence of bacterial pneumonia (schwarzmann et al., 1971; cartwright et al., 1991) . associations between rsv and haemophilus influenzae, bordetella pertussis, neisseria meningitidis and staphylococcus aureus infections have also been described (patel et al., 1992; jiang et al., 1999) . interactions between viral and bacterial diseases are fairly complex and the underlying mechanisms are only beginning to be elucidated. it is known that up-regulation of tnfa and il-1 increases adherence and uptake of pneumococci (cundell et al., 1995) , and that several cellular receptors for bacterial adherence are up-regulated by viral infections. s. pneumoniae and h. influenzae interact with paf receptors (swords et al., 2000) , and n. meningitidis interacts with cd14 and cd18 (raza et al., 1999) . not all interactions are regulated at the level of cell surface receptors. in the mouse influenza model, for instance, severe damage and desquamation of the respiratory epithelium enables access of s. pneumonia to the basal membrane and thereby increases the risk for invasive disease (plotkowski et al., 1986) . otitis media was traditionally thought to be a purely bacterial infection, with s. pneumoniae, h. influenzae and moraxella catarrhalis as the main pathogens. more recent studies, however, indicate that the majority of acute otitis media (aom) cases are a result of mixed bacterial and viral infection. heikkinen and colleagues reported an increase of rsv, parainfluenza virus, influenza or adenovirus infection in children with otitis media (heikkinen et al., 1999) . all respiratory viral infections of the nasopharynx are thought to predispose to aom but some viruses, e.g. rsv, are frequently found in the middle ear during aom. the mechanism underlying this respiratory tract infection-aom sequence involves eustachian tube obstruction, leading to negative middle ear pressure and inspissation of bacteria into the middle ear (giebink et al., 1980) . children with rsv, adenovirus or influenza virus infections have a 30% risk of developing aom within 2 weeks of the onset of the respiratory tract infection (henderson et al., 1982) , and coinfection with bacteria and viruses also adversely influences the outcome of aom. if aom does not respond to antibiotic therapy within 48 h, it is more likely to involve a viral infection (arola et al., 1990) . the effects of viral co-infection complicate the evaluation of clinical efficacy of anti-bacterial drugs in the treatment of aom (marchant et al., 1992) . with bacterial-viral co-infection in aom, there is also delayed clearance of bacteria from the middle ear during anti-bacterial therapy compared with disease attributable to bacterial infection alone (chonmaitree et al., 1990) . it is unclear whether the decrease in efficacy of antibiotics is due to impaired host responses (poor neutrophil function) or due to poor penetration of antibiotics into the middle ear. in a recent clinical trial heptavalent pneumococcal vaccine had 90% efficacy for prevention of bacteremia and 57% efficacy for prevention of aom caused by serotypes included in the vaccine. however, the frequency of aom caused by pneumococci not included in the vaccine increased (replacement phenomenon), so that the overall effect of the vaccine was reduced (eskola et al., 2001) . in contrast to the pneumococcal vaccine, viral vaccines seem very effective in preventing aom. belshe and colleagues reported considerable efficacy for an attenuated live influenza vaccine in preventing aom, and rsv prophylaxis with rsv antibodies was also associated with a marked decrease in otitis media (belshe et al., 1998; group, 1998a ). the human adenoviruses consists of over 51 known serotypes which have been divided into six subgroups (a-f) with distinctly different organ tropism. although other factors may influence infectivity and replication of these viruses, high affinity attachment of virions to host cell receptors represents a key determinant of tissue tropism. examples of this distinctly different organ tropism are a predominance of subgroup a adenoviruses (ad) such as ad31 in pneumonia in patients with primary immunodeficiencies; the preference of subgroup b viruses such as ad11, 34 and 35 for the urinary tract in patients with kidney transplants; and the predominance of subgroup c viruses in hepatitis in liver transplant patients. receptor binding is mediated by the ad fiber protein, a homotrimeric molecule composed of an amino terminal region that anchors the fiber to the penton base capsid protein, an elongated central shaft domain (van raaij et al., 1999) , and the carboxy-terminal receptor binding knob. a high resolution structure of the ad12 fiber knob bound to its receptor, the coxsackie-adenovirus receptor (car), has recently been obtained by x-ray diffraction (bewley et al., 1999) , and amino acid residues directly involved in receptor binding were defined for several adenoviruses through mutagenesis studies (roelvink et al., 1999) . adenoviruses differ remarkably in the isoelectric point of the fiber protein knob domain, e.g. ph 8.9 for ad8 versus ph 4.5 for ad35, suggesting that these fiber proteins cannot use the same receptor. besides car, heparin sulfate proteoglycans (dechecchi et al., 2000) and sialic acid (arnberg et al., 2000) mediate adenovirus attachment. since car is only expressed on the basolateral but not the luminal surface of epithelium, car can probably not be used to target adenoviral vectors in the therapy of cystic fibrosis. the important role that charge plays in virus-cell interactions can be deduced from the differential effect that removal of sialic acid from the cell surface by neuraminidase treatment has on adenovirus attachment. while ad19p attachment is not affected by neuraminidase treatment, ad5 attachment is increased and ad37 attachment is decreased. the difference in ph optima may well be a determinant of tissue tropism. the major pathogens in adenoviral eye infections, ad8, ad19 and ad37, belonging to subgroup d, are very similar in their knob charge. chimerization of fiber proteins, whether evolved in nature or generated by mutagenesis, can cause a significant change in adenovirus pathogenesis. in 1984, a new ad7 genotype (ad7h) appeared in argentina, uruguay and chile, where it caused severe respiratory tract infections in children. analysis of the ad7h fiber protein revealed that it was a chimera containing ad7 and ad3 sequences. in 1996 this new genotype and ad7d2 also emerged in japan, where ad7 had been absent for 30 years, and caused outbreaks of respiratory disease. the fiber protein, however, is not the only factor that determines adenovirus tropism. virus uptake is thought to also be mediated by an interaction between the penton base protein with integrin avß 3 or avß 5. adenoviruses also differ in their ability to induce inflammatory responses. while ad 7 is a potent inducer of interleukin 8 (il-8), a hallmark cytokine of viral pneumonia, ad5 is not; this might explain why ad7 but not ad5 causes significant respiratory disease. in summary, it can be concluded that neither subgroup classification alone nor fiber protein knob charge alone determine adenovirus tissue tropism. key amino acids in the knob, as well as certain motifs of the penton base protein and possibly other adenovirus proteins interact in virus attachment and internalization. avian influenza viruses of the h5n1 subtype were found to be transmitted directly from poultry to humans in 1997 in hong kong. these viruses were highly pathogenic in chickens and also caused severe clinical symptoms in humans, leading to the death of six of 18 infected individuals. in order to obtain a better understanding of the pathogenesis, tropism and kinetics of replication of these viruses in primates, cynomolgus monkeys (macaca fascicularis) were infected with the highly pathogenic h5n1 a/hong kong/156/ 97 isolate that was obtained from the index case. four monkeys were inoculated with 2.5× 10 4 tcid 50 in a 5 ml inoculum that was administered intratracheally, orally (tonsils) and onto the conjunctiva. two of the monkeys were euthanized on day 4 and the remaining two on day 7 post infection. the two monkeys that were euthanized on day 7, post infection developed a respiratory distress syndrome with high respiratory rate and fever on day 5. by day 7, one of the monkeys was lethargic and severely ill, with central cyanosis. the other monkey was also ill and developed fever. the virus replicated to a titer greater than 10 6 tcid 50 per g lung tissue on day 4 but could not be isolated on day 7. macroscopic lung pathology was dominated by peribronchial consolidation and necrotic lesions. histopathologic examination of the tissues collected 4 and 7 days post infection revealed extensive pathologic changes in the respiratory tract characteristic of a viral necrotizing interstitial pneumonia. although rt-pcr for h5n1 influenza virus was positive not only in the respiratory tract but in spleen, heart and also the cerebrum and cerebellum of one monkey, virus could only be demonstrated by immunoperoxidase staining in, and isolation from, the respiratory tract. the respiratory tract seems to be the major and probably the only target for the h5n1 influenza virus. although cynomologous monkeys have been used earlier as a model for h3n2 influenza disease (rimmelzwaan et al., 1997) , this is the first time a primate model for h5n1 viruses has been described. influenza h5n1 clinical symptoms observed in this study correlate well with what was seen in human disease caused by these highly virulent viruses, and was more severe than what is seen with h3n2 viruses. therefore, cynomologous monkeys may provide a useful model for studying influenza h5n1 pathogenesis and for developing h5n1 vaccines. 4.5. sb-242235, an orally acti6e, selecti6e p38 mitogen acti6ated protein (map) kinase inhibitor impro6es pulmonary functions in a murine influenza pneumonia model influenza infections are responsible for significant morbidity, especially in high-risk groups with underlying cardiopulmonary disease and in the elderly. the pathology results from a vigorous inflammatory response in the respiratory tract and damage to respiratory epithelial cells. in cells exposed to inflammatory cytokines, p38 mitogen activated kinase (map) activation leads to the upregulation of cytokines such as il-6, il-8 and tnfa (ono and han, 2000) . a recent study examined the effect of sb-242235, a highly selective orally bioavailable inhibitor of p38 map kinase, on pulmonary function in mice infected with influenza a virus. mice were infected intranasally with influenza a/pr8/34 and treated with sb-242235 at different time points post infection. initiation of treatment on day 0, 3 or 5 post-infection resulted in a 57% (p b0.01), 32% (pb 0.01) or 10% improvement in pulmonary capacity, respectively, compared with placebotreated control animals. no effect on virus clearance, survival, or antiviral immunity was observed. the efficacy of sb-242235 in reducing pulmonary resistance and increasing blood oxygenation was similar to that of the neuraminidase inhibitor oseltamivir, the steroid dexamethasone, and the cox-2 inhibitor nimesulide. sb-242235 was superior to non-specific nsaids indomethacin, naproxen, and ibuprofen. in mice and ferrets, sb-242235 reduced airway neutrophilia, and treatment was well-tolerated without any adverse effects. these data suggest that p38 map kinase is involved in influenza-induced cell signaling and that inhibition of this enzyme might reduce the severity of pulmonary disease. fortunately, the number of viruses that cause respiratory disease severe enough to require hospitalization is limited. in children younger than five years, rsv (subgroup a and b viruses), the parainfluenza viruses (piv1, -2 and -3), influenza a and b, and adenovirus types 1, 2, 3 and 5 are the major respiratory pathogens. influenza a and b remain, due to antigenic drift and shift, important agents in all age groups with very severe disease most commonly occurring in the elderly. rsv is the single most important cause of severe respiratory disease in infancy and childhood but it also causes significant morbidity in the elderly and in immunocompromised individuals. kim, chanock, brandt and parrott were the first to quantify the contribution of these viruses to the severe respiratory tract disease leading to hospitalization of infants and young children. rsv, piv3, piv1, piv2, adenoviruses and influenza b caused 23, 12, 6, 3, 6, and 1%, respectively, of respiratory disease leading to hospitalization, while influenza a was responsible for 3% between 1957 and 1968 (h2n2 era) and 7% between 1968 and 1976 (h3n2 era) (brandt et al., 1972; parrott et al., 1973; kim et al., 1979; murphy et al., 1988) . what are the general principles underlying vaccine development for these viruses? first, the protective antigens of the virus and mediators of immunity to reinfection are largely known. it is generally accepted that neutralizing antibodies to surface glycoproteins (g and f of rsv, hn and f of piv, and ha and na of influenza) or to capsid proteins (hexon and fiber proteins) of adenoviruses are the major mediators of resistance to reinfection. second, serum and mucosal antibodies make independent contributions to immunity against reinfection. whereas serum antibody is needed to mediate immunity in the lower respiratory tract, mucosal antibody is needed to protect the upper respiratory tract (with the exception of adenovirus, where serum antibody alone can prevent uri). third, live virus vaccines are generally more immunogenic than nonliving virus vaccines in immunologically naïve subjects, but identifying a live vaccine virus that is both safe and sufficiently immunogenic can prove to be a difficult task. fourth, different age groups might need different vaccines. infants younger than four months of age make less antibody than older children due to immunosuppression by maternal antibodies and immaturity of the immune system, and only potent immunogens, e.g. live virus vaccines, will be successful in this population (murphy et al., 1986) . on the other hand, a live virus vaccine that is sufficiently attenuated for use in infants might be over-attenuated in the elderly (gonzalez et al., 2000) . fifth, viruses differ in their ability to cause disease in the presence of maternal serum antibody. while mucosally delivered rsv, piv3 and influenza are infectious in the presence of maternal antibody, piv1, piv2 and adenovirus seem to be restricted in their replication in young infants by maternal antibodies. these five principles need to be considered when determining the optimal vaccination strategy (systemic versus mucosal, vectored antigen versus live attenuated virus) and the target age for vaccination. which vaccines are available? against influenza, the vaccine currently in use in the us is mostly a non-living, subvirion vaccine made from egg-grown purified virus disrupted with detergents. for practical purposes, the only relevant protein in the vaccine is the influenza hemagglutinin. the strains to be included in the vaccine for annual vaccination are selected by the public health service based on the antigenic profile of the currently circulating strains. subivirion or subunit vaccines plus adjuvant are also being developed (minutello et al., 1999; gluck et al., 2000) . live attenuated virus vaccines based on cold-adaptation of influenza a strain a/aa/6/60-h2n2 and influenza b strain b/aa/1/66 have been evaluated in phase iii clinical trials and should soon become available as a mucosal vaccine administered by nasal spray (maassab and bryant, 1999) . to date, an rsv vaccine has not been licensed. two non-living virus vaccines against rsv are being developed. one is a purified f protein subunit vaccine and the other consists of a g protein-specific peptide conjugated to the albumenbinding site of the streptococcus g protein (cano et al., 2000) . the purified f subunit vaccine is being evaluated for use in seropositive populations such as the elderly or high-risk older children (gonzalez et al., 2000) . used by itself, the f subunit vaccine has the disadvantage of not inducing a potent mucosal antibody response. also, this antigen induces a serum antibody response that is dominated by non-neutralizing antibodies (high ratio of titer of f protein-specific binding antibody to titer of neutralizing antibody). subgroup a and b live rsv vaccine candidates are being developed . one candidate vaccine bearing multiple attenuating mutations has been evaluated in 1-2 months old infants and was found to be attenuated and immunogenic but retained mild reactogenicity for the upper respiratory tract (wright et al., 2000) . it is likely that recombinant cdna technology will be used to develop or improve live attenuated rsv vaccines in the near future (whitehead et al., 1999a) . for example, a subgroup b rsv vaccine candidate was developed by substituting the g and f glycoprotein genes of rsv b for the corresponding genes in an attenuated recombinant subgroup a virus (whitehead et al., 1999b) , thereby rapidly generating a live attenuated subgroup b rsv vaccine candidate. to date, a piv vaccine has not been licensed. subunit and vectored vaccine candidates against piv3 have been developed and tested in animal models, but there are no reports of ongoing clinical trials. two live-attenuated piv3 vaccine candidates, a bovine piv3 (bpiv3) and a cold-adapted piv3 (cp45), have been evaluated in clinical trials, and both viruses were found to be safe and immunogenic in seronegative infants (karron et al., 1995 (karron et al., , 1996 . recombinant versions of these two viruses are also being evaluated as vaccines against piv3 and as vectors for the expression of foreign viral glycoproteins such as rsv g or measles ha (karron et al., 1995; durbin et al., 2000; schmidt et al., 2000) . the recombinant piv3cp45 vaccine candidate is currently being tested in phase ii trials in seronegative infants. for adenoviruses, a live virus vaccine against serotypes 4 and 7 has been used successfully in military recruits from 1971 to 1998. the vaccine made use of site-specific attenuation, i.e. a wt virus capable of inducing respiratory disease if administered to the respiratory tract was administered orally in an enteric coated capsule. the vaccine virus replicated only in the gastrointestinal tract and did not spread to the respiratory tract. the oral vaccine was found to be com-pletely attenuated and highly efficacious in inducing serum antibodies and protecting the upper and lower respiratory tract (couch et al., 1963; howell et al., 1998) . a similar tetravalent vaccine against serotypes 1, 2, 3 and 5 should be evaluated for use in young pediatric patients since these four viruses are responsible for over 80% of the adenovirus-related respiratory tract disease in this population. in virology, reverse genetics refers to the ability to rescue infectious virus from cdna. reverse genetic systems have been developed for all major viral respiratory pathogens, i.e. influenza a virus, paramyxoviruses, adenoviruses and most recently coronaviruses. this short review will only address the impact of reverse genetics on vaccine development for selected members of paramyxoviridae. for rsv, the generation of attenuated vaccine viruses started with conventional biological methods. first, wild-type rsv was passaged extensively at low temperature, and the resulting cold-passaged (cp)rsv was demonstrated to be attenuated in chimpanzees, as well as in seropositive adults and young children (friedewald et al., 1968 ). the cprsv virus was further modified by two rounds of chemical mutagenesis and biological selection for temperature-sensitivity (crowe et al., 1994a) . a panel of resulting mutant viruses was evaluated in mice and chimpanzees and ranked according to increasing attenuation. several promising candidate vaccines then entered clinical trials. rsv cpts248/404, a cold-passaged virus with two additional attenuating point mutations, is the most promising vaccine candidate tested so far, but it still causes transient nasal stuffiness in infants (wright et al., 2000) . the development of a reverse genetics system for rsv provided a method for expediting development of further attenuated viruses. as a first step, the biologically derived mutant viruses were sequenced completely and their mutations were identified and evaluated by introduction, individually or in combination, into wt recombinant (r)rsv. the direct identification of attenuating mutations made it possible, for example, to improve the above mentioned cpts248/404 mutation by incorporating one or more additional attenuating mutations from other vaccine candidates. furthermore, in many cases, the amino acid substitutions can be engineered to involve two nucleotide substitutions relative to wt, which should confer increased genetic stability. similarly, a reverse genetics system for piv3 was established and used to analyze all 15 mutations in the biologically derived cold passaged piv3cp45 (skiadopoulos et al., 1999) . however, the capabilities of reverse genetics go far beyond the analysis of existing biological mutants. a whole new set of methods for generating attenuated paramyxoviruses has been developed as described above. gene knock-out mutations, i.e. the deletion of non-essential genes, such as the rsv sh, ns1, ns2, m2-2 or g gene, generated a number of vaccine candidates with different levels of attenuation. as another example, in some cases an attenuating point mutation was found to involve a residue conserved, for example, between the l proteins of rsv and piv3 or between the c proteins of sendai virus and piv3. transfer of the attenuating mutation between the heterologous paramyxoviruses resulted in transfer of the attenuation phenotype. host range restriction could be employed as a basis of attenuation by creating antigenic chimeric viruses that use an animal virus as a platform for the expression of human rsv or piv glycoprotein protective antigen genes schmidt et al., 2000) . in this manner, an hpiv3 vaccine candidate was generated by replacing the bpiv3 f and hn glycoprotein genes in rbpiv3 with their hpiv3 counterparts (schmidt et al., 2000) . thus, the menu of available attenuating mutations for rsv and hpiv3 include numerous attenuating point mutations (which in some cases specify temperature sensitivity and in other cases do not), gene deletions, and host range restriction elements. the attenuating mutations or elements from each menu can be combined as desired to develop appropriately attenuated vaccine candidates. reverse genetics also expedites vaccine development because attenuated platforms can be used to make vaccines against additional viruses. for ex-ample, attenuated derivatives of rsv a2 (subgroup a) can be used to generate rsv subgroup b vaccine candidates by replacing the a2 f and g glycoprotein genes with their counterparts from the b1 strain of subgroup b (whitehead et al., 1999b) . as another example, a live-attenuated vaccine candidate was developed for hpiv1 by replacing the f and hn coding regions of hpiv3 with their hpiv1 counterparts. the resulting virus thus bears the antigenic determinants of hpiv1 in a wt hpiv3 backbone, which can then be attenuated by the introduction of mutations from the hpiv3 attenuation menu. reverse genetics can potentially help us to make vaccines that are satisfactorily attenuated but more immunogenic than wt virus. protective antigen genes can be moved closer to the genomic promoter and codon usage can be optimized for translation in mammalian hosts. cytokines and/or chemokines can be co-expressed from additional genes (bukreyev et al., 1999) , and reactogenicity can potentially be reduced, for example by ablating the secreted form of rsv g, which might be a decoy for antibody and might also perturb the t helper lymphocyte response. vaccine specificity can be broadened by adding additional genes to existing vaccine viruses, e.g. the measles hemagglutinin (ha) gene can be expressed from an additional orf in rhpiv3, generating a vaccine candidate that protects against both hpiv3 and measles (durbin et al., 2000) . this use of recombinant piv3 as vaccine and as a vector has several advantages over existing vector systems. it reduces the number of viruses to be administrated to infants and enables intranasal administration and thereby mitigates neutralization and immune suppression by maternal antibodies. the eradication of measles could be facilitated by a vaccine that does not include an infectious measles virus, which might cause prolonged infection in immunocompromised hosts. in summary, vaccines can be optimized through reverse genetics in a number of ways, such as the creation of novel combinations of mutations, the fine-tuning of the attenuation phenotype, the provision of a common attenuated platforms, and the generation of multivalent vaccines. 5.3. predicti6e 6alue of animal models in rsv 6accine de6elopment rsv was initially isolated from chimpanzees that developed a common cold-like disease in 1956. since then chimpanzees and other animal models for rsv disease have been studied extensively. principally, these higher primates have been used for three different purposes: the development of live virus vaccines, the recovery of rsv antibodies, and lastly to evaluate formalininactivated and subunit vaccines, and to study enhanced disease following vaccination with formalin-inactivated vaccine. a number of animal models are used to study rsv disease. among the apes, chimpanzees have been used most extensively for several reasons. they are permissive for rsv, their core body temperature is similar to that of humans, and symptomatic scoring of uri is possible (rhinorrhea score). the permissiveness of chimpanzees makes it possible to perform quantitative virologic studies in the upper and lower respiratory tract over the time course of an acute rsv infection. also, the restriction of rsv replication in the respiratory tract of chimpanzees correlates well with attenuation in seronegative human infants. in addition, human immunologic reagents can be used in chimpanzee studies. however, only young, carefully raised chimpanzees are rsv seronegative. other primates used include old world monkeys such as african greens, rhesus, cynomologous or bonnet monkeys and new world monkeys such as marmosets, tamarins and owl monkeys. although all these monkeys are semi-permissive for rsv, their core body temperature is higher than that of humans so that the level of attenuation of temperature-sensitive rsv mutants might be overestimated. although primate studies provide essential data for vaccine development, their use can be difficult. transport and care of primates, as well as sample collection are difficult and potentially dangerous, and they require well-trained personnel. primate research centers must provide an environment similar to that of a child-care facility and, therefore, maintenance costs are very high. lambs and calves are also used to study rsv infection, mostly because ovine and bovine rsv causes significant disease in their natural host. also, these viruses are important economically. cotton rats, mice and other rodents are used as small animal models. mice have the advantage of having a body temperature similar to that of humans and immunological reagents are readily available. also, gene knockout mice, transgenic mice and various inbred strains are available to study pathogenesis. the mouse model is not without difficulties, however. mice acquire a large portion of their maternal antibodies by suckling, and, therefore, they are not an optimal model for study of immunosuppression by maternal antibodies. peak rsv titers vary 100-fold among different mouse strains, and subgroup b rsv is poorly infectious and immunogenic in mice. immune mechanisms may also vary among different mice strains. rodent models can be used to study quantitative virology, immunology and airway pathophysiology (a model of wheezing), and weight loss can be used as a surrogate marker for rsv disease. whichever animal model one chooses to study respiratory syncytial virus, it is essential to clearly define whether the ultimate goal of the study is to prepare for clinical vaccine trials or to understand basic mechanisms of disease. several cold-passaged (cp) live attenuated rsv candidate vaccines have been evaluated in clinical trials. most of these candidate vaccines were derived by further attenuating the original cprsv through chemical mutagenesis and selection of ts mutants. the first two vaccines evaluated in the cpts lineage -cpts-248/955 and cpts-530/1009, were either insufficiently attenuated or were transmitted amongst seronegative children (karron et al., 1996) . a study of a more attenuated vaccine candidate, cpts-248/404, has recently been completed (wright et al., 2000) . this virus initially was evaluated in chimpanzees where it replicated to a peak titer of only 10 1.3 pfu/ml in the upper respiratory tract. due to this restriction in the respiratory tract, it was subsequently evaluated in clinical studies. cpts-248/404 did not replicate in adults or older children. in seronegative children 6-24 months of age, however, cpts-248/404 replicated to a peak titer of 10 4 pfu/ml in the upper respiratory tract and induced an antibody response against rsv f and g glycoproteins. the virus was well tolerated in this age group, and the frequency of upper or lower respiratory tract disease, otitis media or fever was not different from that of the placebo group. based on these data cpts-248/404 was selected to be the first rsv vaccine to be administered to 1-2-month-old infants, which represent the primary target group for vaccination. in this youngest age group, 17 of 24 vaccine recipients developed a clinical syndrome characterized by nasal congestion that occurred most typically between days 8 and 12 and lasted for approximately 24 h. since young infants are obligate nose breathers, this interfered with feeding and caused fussiness and difficulty in falling asleep. most vaccine recipients shed approximately 10 3 pfu of rsv per ml nasal wash. age of the infant or level of maternal antibodies against rsv did nor affect virus shedding, indicating that virus replication in the nasopharynx was independent of these variables. in these young infants, neutralizing antibody responses and igg elisa responses to rsv f or g were rarely detected, most likely because these responses were masked by the presence of maternally derived antibody. these young infants did, however, develop serum and mucosal iga responses preferentially to the rsv g glycoprotein and detection of serum iga correlated with protection from re-infection with a second dose of vaccine. although cpts-248/404 is not an acceptable vaccine in one to two month-old infants, several lessons were learned from the study of this vaccine candidate. first, seronegative infants are a much more susceptible host to rsv than chimpanzees. this means that for further attenuated vaccine candidates, the chimpanzee model of rsv infection will not be very useful. second, only viruses that do not replicate in adults and older children are attenuated enough for vaccination of infants. and third, reverse genetics is needed to further attenuate the existing biological rsv vaccine candidates. two of these recombinant rsv vaccine candidates are currently being evaluated in clinical trials. cpts-248/404dsh was generated by deleting the sh gene from the recombinant cpts-248/404 virus, and cpts-248/404/1030dsh was engineered to contain an additional (1030) mutation in the polymerase protein. preliminary data suggest that both recombinant viruses are well tolerated in 6-24-month-old children and are not associated with lower respiratory tract illness. whereas cpts-248/404dsh replicates as well as its parent biological virus without the sh gene deletion, the cpts-248/404/1030dsh mutant appears to be much more restricted in its replication in the nasopharynx. the more restricted cpts-248/ 404dsh induced both neutralizing and elisa igg antibodies in this age group. however, since this virus replicates, as well as cpts-248/404, it is likely not to be suitable for vaccination of one to two month old infants. cpts-248/404/1030?sh is currently being evaluated in one to two month old infants. in summary, although cpts-248/404 is close to an ideal vaccine candidate, further modifications of the virus through reverse genetics, such as addition of the 1030 mutation, are likely to be needed to generate an rsv vaccine that is immunogenic yet attenuated enough to be given to young infants. antibody preparations that neutralize free virus have been used as passive immunoprophylaxis to prevent a number of viral diseases, including hepatitis a and b, varicella and respiratory syncytial virus (rsv) disease. the efficacy of antibody preparations in preventing rsv disease was established first in cotton rat and chimpanzee models, and later in extended clinical trials. therapy of rsv infections with rsv antibodies, however, have so far failed to prove efficacious (malley et al., 1998; van woensel and kimpen, 2000) . the most important mechanism of action of antibody preparations is probably neutralization of free virus. this can occur via aggregation of free virus by bivalent or multivalent antibody, via receptor blockade, via antibody-complement lysis, or via fusion inhibition. receptor blockade is thought to result from steric inhibition of receptor binding rather than binding of antibody directly to the receptor-binding site itself. even if receptor binding does occur, virus infectivity can be neutralized through fusion inhibition. most rsv neutralizing antibodies are thought to use this mechanism of action. although complete antibody is most effective in neutralizing free virus, antibody binding fragments (fabs) alone can also neutralize. the minimal unit necessary to ablate infectivity is probably a peptide corresponding to one loop of the complementarity-determining region 3 (cdr3). although neutralizing and non-neutralizing antibodies against infectious virus are often distinguished, it is not clear whether there really are antibodies that bind glycoproteins in virions without neutralizing the virion (sakurai et al., 1999) . many so-called non-neutralizing antibodies bind purified (conformationally relaxed) rsv f glycoprotein but not the (conformationally correct) f protein on infected cells. different immunoglobulin subclasses neutralize virus with varying efficacy, and mouse and human igg subclasses also bind complement with varying efficacy. the replication of sendai virus (murine piv1) and influenza a virus can be inhibited intracellularly by iga and similar effects of iga on rsv replication may occur. a last but essential determinant of neutralizing activity is defined by the amount of antibody available to neutralize virus. to prevent rsv disease in the lower respiratory tract, a serum neutralizing antibody titer of approximately 1:300 is required (groothuis et al., 1993; top et al., 2000) . upper respiratory tract infections, however, can only be prevented with serum antibody titers as high as 1:5000-1:15 000. such titers can only be achieved in experimental settings in small rodent models. there is no single monoclonal antibody directed against rsv g protein that neutralizes completely on its own but cooperative neutralization occurs when multiple mabs are used. effective treatment for rsv illness is very limited. corticosteroids, bronchodilators, ribavirin, and, more recently, rsv mabs have been used but none of these therapeutic interventions are accepted as reproducibly efficacious. it seems that neither antivirals nor anti-inflammatory agents alone improve the outcome of rsv disease. a single dose of topically administered human fabs has been shown to clear free virus from the respiratory tract of rodents, but their effect is shortlived since infected cells release newly synthesized infectious virus within a day or two (crowe et al. 1994b ). whereas peak virus titers in humans usually occur around day 4, most patients with clinical rsv disease likely present no earlier than day 7. at this point, virus load is already in decline and it may be too late for antivirals, and antiinflammatory drugs such as map kinase inhibitors may be more effective in reducing cell injury. are there immunological consequences of passive immunoprophylaxis? in chimpanzees therapy with rsv antibodies suppresses the primary antibody response to rsv. this effect is mostly antigen-specific but may also have a non-specific aspect mediated by the fc portion of the antibody. the secondary antibody response to rsv in chimpanzees is enhanced by prior antibody therapy. however, this effect was not observed in mice or in clinical studies. t cell responses do not seem to be as affected by antibody therapy as humoral responses, and t cells might fill in for absent primary antibody responses. whether this is a desirable effect or not, remains uncertain. without a safe and effective rsv vaccine available, monthly infusion of rsv-igiv (respigam ® ) was the first effective measure to prevent rsv-induced lower respiratory tract infection (lri) and hospitalization in infants. the evaluation of rsv-ivig in cotton rats correctly predicted the serum concentrations necessary to protect against rsv-induced lri (prince et al., 1985a) . protective concentrations in children were achieved by monthly infusions of 750 mg/kg of rsv-ivig. in order to increase the potency of a rsv antibody preparation, and in order to be able to replace intravenous with intramuscular administration, a humanized monoclonal anti-rsv fusion protein antibody was developed. mab1129, a mouse monoclonal antibody developed in the laboratory of infectious diseases at nih against the rsv f protein antigenic site a, one of two antigenic sites that are conserved amongst different rsv strains, was selected for humanization based on in vitro and in vivo studies, and the complementarity determining regions (cdr) were transferred from the mouse antibody to a human igg. this humanized igg should have pharmacokinetics similar to human igg and permit repeated administration at monthly intervals. the chosen mab, medi-493, had no crossreactivity with adult or neonatal tissue, broadly neutralized rsv subgroup a and subgroup b isolates at concentrations of 20 ng/ml, and proved to be 50-100 times more active on a weight basis than rsv-ivig in the cotton rat model (johnson et al., 1997) . adult volunteers tolerated doses of the humanized antibody medi-493 from 1 to 30 mg/kg well, and only some volunteers developed a low-titer, transient anti-idiotypic antibody response. medi-493 had a half-life of 17 days, as is expected for igg, and serum concentration after iv and im administration were comparable except for the initial bioavailability. medi-493 was safe and well tolerated in phase i/ii studies in high-risk children, and had no specific immunogenicity in and of itself. monthly dosage of 15 mg/kg maintained serum concentrations greater than 40 mg/ml, and a single intravenous dose of 15 mg/kg reduced rsv titers in tracheal secretions of intubated children with rsv infection (malley et al., 1998) . the impact-rsv study that led to fda approval of medi-493, also called palivizumab, was a 2:1 randomized, double-blinded, placebo-controlled phase iii multicenter study conducted at 139 sites in the us, canada and the uk. study subjects received five doses of medi-493 or placebo at intervals of 30 days and were followed for a total of 150 days. the primary endpoint of this study, overall rsv-related hospitalization, was significantly reduced (pb 0.001) from 10.6% for placebo recipients (n= 500) to 4.9% for medi-493 recipients (n=1002). for premature infants with chronic lung disease, the incidence of rsv hospitalization was reduced from 12.8 to 7.9% (p= 0.038), and for premature infants without chronic lung disease, it was reduced from 8.1 to 1.8% (pb 0.001). total rsv hospitalization days, total days with increased oxygen requirement, total days with severe lri, rate of icu admission and total days in icu were secondary endpoints that occurred less frequently in the medi-493 treated group (p b 0.05). thus, administration of 15 mg/kg medi-493 by im injection was found to be safe, well-tolerated, and to lead to a 55% reduction of rsv hospitalization in high-risk children (group, 1998b) . this conclusion was confirmed in an outcome survey conducted in nine centers in 1998 and 1999, in which 1839 high-risk children received 15 mg/kg palivizumab. although direct comparison to the impact-rsv study is not possible, rsv hospitalization rates were low and similar to those observed in the impact-rsv study: 2.1% (vs. 1.8% in the impact-rsv study) of premature infants without cld and 4.0% (vs. 7.98%) of children with cld were hospitalized in the 1998-1999 rsv season (sorrentino and powers, 2000). an alternative strategy to protect neonates and young infants against severe rsv disease is vaccination of pregnant women. transfer of high concentrations of maternal rsv-specific igg to the fetus is expected to protect the infant against severe disease (glezen et al., 1981) . the safety and immunogenicity of respiratory syncytial virus purified fusion protein-2 (pfp-2 wyeth lederle vaccine and pediatrics, pearl river, ny) were recently evaluated for use in pregnancy. thirtyfive pregnant women were randomized at a ratio 2:1 to receive rsv pfp-2 or saline placebo at 30-34 weeks of gestation and were followed until the time of delivery. infants were followed during their first year of life and their first rsv season. rsv pfp-2 was safe and well tolerated by pregnant women, and there were no systemic reactions or serious adverse events associated with vaccine administration. all 35 infants were born healthy, and there were no differences in the frequencies and outcomes of neonatal events between the groups. during the first rsv season, there was no increase in the frequency or severity of respiratory tract illnesses in infants of vaccine recipients. there is ample material from which to draw lessons relevant to needed preparations for pandemic influenza. but, to date, the response, and specifically preparations for dealing with a serious pandemic of influenza remain more in the realm of academic reflection than meaningful action. although much time has been devoted to the development of a national response plan, we do not seem to be much better equipped to deal with a new pandemic of influenza than we were in the spring of 1957, when the h2n2 strain of influenza a virus emerged. at that time, we faced a burgeoning epidemic, whose virulence and propensity for spread were as yet unknown. a program of surveillance and field epidemiology to better define the epidemic had to be developed and, at the same time, preparations were needed for distribution and use of a new influenza vaccine, if it arrived in time. it did not. it appears today that not much progress has been made over the past 30 years, and still no real sense of urgency in dealing with the essentials of the problem can be felt. although much has been written about the 1918 epidemic, it is still perceived by most as an interesting but questionably relevant tale of death and disease during an earlier, pre-antibiotic era of medicine. many doubt that an epidemic of this severity would be possible today. but, here are a few of the facts. first, it is important to recognize that, for the us, it dwarfed all other outbreaks of the 20th century. at that time, more than 20 million died worldwide and in the united states, there were more than 500 000 registered deaths. from various studies, it is thought that, overall, perhaps 40% of the population became ill, of whom about 2% died. medical services were overwhelmed but, other than supportive care, there was little that curative medicine could offer. the deaths were so numerous that burials were greatly delayed because of the lack of morticians and grave diggers. pictures from the time provide at least a pale illumination of that catastrophic period. the effect of the disease was anything but uniform. surveys revealed morbidity rates ranging from 15% to more than 60% in different parts of the country. some remote and rural areas escaped the disease entirely but in some areas, it was remarkably lethal. western samoa, then a new zealand protectorate, registered 8500 deaths in a population of 38 000. this represented 22% of the entire population. curiously, american samoa, only 50 miles away but with a quarantine in effect, was one of the few political entities to escape the epidemic entirely. what was surprising and unique about the 1918 epidemic was that more than half of all deaths were in persons between 15 and 45 years of age -an age bracket in which death is a relatively uncommon phenomenon. pregnant women and those with cardiac problems were at highest risk but most, who died, were otherwise in good health. a substantial number in this age group died of a fulminant, rapidly progressive pneumonia marked by severe cyanosis with death occurring within a matter of 1-3 days, in brief, what seemed to be almost certainly, a primary influenza pneumonia that would have benefited little from antibiotics, had they been available. today, a better outcome might be foreseen with better ventilatory support in intensive care units; with the administration of antiviral agents; and with the administration of antibiotics for the treatment of secondary pulmonary infections. but under epidemic circumstances, the bulk of cases in a new pandemic would occur over a period of only 3-6 weeks and only a fraction of patients could be accommodated in suitable hospital settings, let alone appropriate intensive care units. what quantity of antiviral drugs is available for emergency use? where do we obtain the added quantities of the standard antibiotics that today, are produced on a just-in-time basis -when, during a period when clinical facilities are not stressed, we are regularly experiencing antibiotic shortages on a regular, rotating basis. a cogent question is whether under epidemic circumstances, our modern health care system could provide a standard of clinical care that would be better than it was in 1918. but there is still another dimension to the problem. do we appreciate that a 'pandemic', so characteristic of the emergence of a new influenza strain, means exactly that -a worldwide epidemic. many countries -developed and developing, have made no preparations and will inevitably turn to those with resources to help them in dealing with a catastrophe -a catastrophe that poses an international security threat. the rapid production and administration of large volumes of vaccine effective against the emergent new strain has been a basic building block of the strategy for dealing with pandemic influenza and, understandably so, given the limitations of curative medicine and the capacity of clinical services. the first real test of this strategy came during the 1957 epidemic. the first notice of a major outbreak occurred in mid april; specimens were received in the us on 13 may; and field testing of new lots of the vaccine began in july. not a bad record. it was foreseen that 60 million doses would be required. with good fortune in adapting the virus to grow at reasonably high titer on egg membrane and provided that sufficient fertilized eggs could be obtained, it was expected that a production target of 1 february could be met. even this was a problematical date given that the seasonal peak of epidemic influenza usually occurs between the end of december and the end of february. still, it was believed that substantial numbers would be able to be protected. however, 1957 was not a typical year, and such, one must note, was the case when the 1918 epidemic strain first appeared. widespread epidemics began occurring in mid to late september, two months before they were anticipated. more than half of all counties reported epidemics by mid-october and by the end of october, the peak incidence had past, long before any substantial quantity of vaccine was available. given these experiences, could we expect another new strain to behave differently today? over the past 30 years, extensive studies have been conducted in the search for a satisfactory live attenuated vaccine and for various approaches in production which would permit new antigenic variants to be produced rapidly and in quantity in tissue cell culture. however, today we are still producing influenza vaccine in the allantoic cavity of hens' eggs, as we were in 1957; procuring adequate supplies of fertile eggs in a timely manner remains a serious problem; and difficulties in adapting new strains to eggs remain. were we able to solve the vaccine production problem for our own country, this would not be the end to the practical quandary of dealing with the pandemic. few countries have influenza vaccine production capability and the international implications of the us having all or much of the vaccine supply are profound. following the 1976 swine influenza outbreak at fort dix, new jersey, the us had embarked on a program to rapidly produce an appropriate vaccine. vaccine production capability in europe and other countries was so marginal that a world health organization committee could only recommend that the fort dix situation be carefully monitored -a strategy of wait and hope. but as those at the who meeting commented in corridor conversations, they would not wish to face the ethical and political dilemma the united states would face were there a pandemic and the us was the only nation with a vaccine. in recent meetings with national and local hospital authorities, current capabilities of the medical system in us to deal with sudden surges in demand such as might follow release of a biological weapon were explored. such a release would result in an epidemic that would stress the system not unlike the way it would be stressed by a pandemic of severe influenza. from these meetings, it was evident that the elasticity of the nation's bed supply has been significantly reduced as drives for financial efficiency and the increasing use of out-patient procedures have sharply reduced the numbers of beds in all hospitals. meanwhile, managed care-driven market pressures and federal government reimbursement reductions have driven large numbers of hospitals into operating deficits. many of the municipal hospitals, once a primary source of care for the less prosperous and uninsured have been privatized. meanwhile, the hospitals are experiencing severe labor shortages, especially for nursing and technical personnel. few have either the resources or motivation to prepare to respond to the challenges posed by mass casualties due to any cause. reserves of antibiotics, as noted earlier, are marginal to nil; the public health infrastructure needed to deal with epidemic disease is grossly understaffed, underpaid and under trained; mechanisms for the development and implementation of community-wide plans are largely unexplored. it is not unreasonable to suggest that we are today less well-prepared to deal with an epidemic of influenza than we were 30 years ago in most parameters that one can identify. what might a new strain of influenza mean in terms of numbers of cases and deaths. estimates of past pandemics suggest that perhaps 40% of the population were affected in a first wave. with rising proportions of the population in urban areas, the greater and more rapid mixing of populations through travel, a figure of not less than 60% would seem more reasonable. thus, in a city of 3.0 million, one might expect 1.8 million cases of influenza. it is doubtful that either antibiotics or antiviral agents would be of much help given the number of cases and the dearth of reserve supplies. the number of deaths would vary greatly depending on the strain. in hong kong, a recent new strain, h5n1, resulted in death among six of 18 persons infected. it did not spread readily, however. it is estimated that the 1918 strain killed 2% of those who became ill, while the 1957 case-fatality rate was about one-tenth as large or 0.2%. thus, in a city of 3 million persons, one might anticipate between 3600 and 36 000 deaths over a period of 3-6 weeks. medical care would consist largely of supportive therapy given the numbers of those ill. public health measures, likewise, would consist of little more than reassurance given the likelihood that vaccine supplies would not be available and that stocks of antiviral drugs would be too small to be of significance. many would argue for the closing of schools, churches and other places of public gathering but experiences suggests that such actions produce little benefit. the wearing of masks was once a favored intervention but this, too, has been discredited. in brief, without vaccine, there would be little that could be done of practical benefit except to reassure the community that the epidemic would someday pass and to accept the criticism of the public and political leadership for failure to make reasonable preparations. at least four lessons can be derived from past experience. first, the threat of pandemic influenza caused by an especially virulent strain is a continuing threat that has to be taken seriously. second, adequate supplies of an effective vaccine, available in a timely manner, are absolutely critical to a preventive effort. solving this problem should command top priority for research and development funding. third, special plans, programs and funding are needed within the health care system to permit development of an adequate community-wide response to the occurrence of mass casualties whatever the cause. lastly, additional research in influenza is needed to better understand its pathogenesis and epidemiology with the expectation that better preventative measures might eventuate. pandemics are the most dramatic presentation of influenza a virus and they cause considerable excess mortality as a result of pneumonia and exacerbation of cardiopulmonary or other chronic diseases. the epidemiologic success of influenza a is in large part due to antigenic variation that takes place in the two surface glycoproteins of the virus, i.e. its hemagglutinin (ha) and neuraminidase (na) proteins. to date, 15 ha and 9 na subtypes have been defined and are used to classify influenza a viruses. antigenic variation occurs either gradually through accumulation of point mutations (antigenic drift) or more abruptly though introduction of a new ha gene into virus circulating in the human population, be it through reassortment of animal and human viruses or through a change in host-specificity of an animal virus (antigenic shift). a 'pandemic virus' can be defined as a virus with a new ha with or without a novel na gene, acquired through antigenic shift, that spreads readily from person-to-person in a population that is highly susceptible to infection. the 20th century saw three pandemics caused by new ha subtypes. influenza a h1n1 caused the 'spanish flu' in 1918, subtype h2n2 ('asian flu') was the causative agent of the 1957 pandemic and the h3n2 subtype ('hong kong flu') caused a pandemic in 1968. the excess mortality for the 1918, 1957 and 1968 pandemics in the us alone can be estimated to have been 500 000, 70 000, and 34 000 deaths, respectively. if one applies mathematical modeling to estimate the effects of a future pandemic, the us alone can expect between 89 000 and 207 000 excess deaths in the next pandemic (meltzer et al., 1999) . what are the viruses with pandemic potential? in 1997, 18 individuals in hong kong were infected with h5n1 influenza, an avian subtype earlier not known to infect humans (subbarao et al., 1998) . of the 18 patients 1-60 years of age, six died. molecular analysis established that all eight genes of the h5n1 virus were of avian origin and that reassortment with human viruses had not occurred (subbarao et al., 1998) . the reported 18 infections were most likely acquired from poultry; human-to-human spread, however, was a rare event. sequence analysis of the hong kong h5n1 virus suggests that it is a reassortant made up from two or three different avian parent viruses: ha from a goose h5n1 virus (xu et al., 1999) , na from a teal h6n1 virus (hoffmann et al., 2000) , and internal genes from a quail h9n2 (guan et al., 1999) or a teal h6n1 virus (hoffmann et al., 2000) . although this virus did not spread readily from person to person, deep concern was caused by the fact that this was the first known avian influenza a virus that caused disease in humans. until this outbreak it was thought that the receptor specificity of avian ha proteins limited their infectivity to avian species. this notion, however, needs to be revised. in march 1999, h9n2 influenza a viruses were isolated from two children with febrile upper respiratory tract illness (peiris et al., 1999) . this virus, again, was an avian virus that was able to infect humans without passage through an intermediate host (lin et al., 2000) . what are our options for vaccination against potentially pandemic viruses? in the case of the h5n1 viruses, there are four options for generating an effective influenza vaccine. a conventional inactivated vaccine can theoretically be generated by reassortment of internal genes from influenza a/pr/8/34 with h5n1 glycoprotein genes. there are, however, problems with incompatibility between certain gene segments. secondly, a cold adapted live attenuated vaccine approach could be taken. the influenza a/ann arbor/6/60 coldadapted strain has been used to generate an h5n1 vaccine in which the ha cleavage site was modified (li et al., 1999a) . this virus could also be used to produce an inactivated vaccine. thirdly, a surrogate virus that is nonpathogenic but antigenically related (takada et al., 1999) could be sought. one of the closest antigenically related viruses found to date was of the h5n3 subtype, but this virus was not highly immunogenic in humans. fourthly, purified protein could be used but again limited immunogenicity may be a problem. the development of vaccines against potential pandemic viruses poses a number of challenges. to begin with, most of the work has to be conducted in biosafety level 3+ laboratories. there is a large array of avian viruses that could potentially become pandemic viruses and those with a genotype that confers transmissibility have to be identified. h5n1, h9n2 and h6n1 viruses that were the genetic precursors of the h5n1 and h9n2 viruses that caused human infections in hong kong (guan et al., 1999; xu et al., 1999; hoffmann et al., 2000) and that continue to circulate among birds should certainly be given priority in vaccine development. an adequate strategy for vaccine development has to be selected, and safety testing must be expedited in mice, ferrets, chickens, and eventually humans. use of recently described plasmid based reverse genetics systems may lessen the technical challenges faced in generating vaccine candidates. assays to evaluate immunity have to be optimized (hemagglutination inhibition assays do not perform well with avian ha proteins, yet neutralization assays are time consuming and technically more difficult), and serologic and molecular diagnostic reagents must be made available. collaboration between veterinary and public health authorities must be maintained and new technologies such as plasmid based reverse genetics systems must be used. last but not least, alternative substrates for vaccine production and new adjuvants are urgently needed. although influenza epidemics occur every winter, we have only had three pandemics this past century -1918, 1957 and 1968 . while annual influenza epidemics generally cause an excess mortality of 20 000-40 000 people in the us, it is only the pandemics that remain in memory. of the three pandemics that occurred in the twentieth century, the 1918 pandemic was the most devastating and claimed the most victims. although there is wide spread public interest in the 1918 pandemic, the event is mostly remembered as an interesting event in the distant past. whether due to denial or not, few feel that an influenza pandemic is a current threat. most journalists see their role not only as educators and advocators but also as entertainers. the interest of the readership has to be captured, and it can only be maintained if their curiosity is satisfied. while the aids pandemic and its effect on people's lives found a wide audience through most of the early 1990s, the late 1990s were characterized by fatigue and a reduced interest in the pandemic (although it was still on the increase). as a result, editors were much less willing to include related stories. reportage on influenza pandemics suffers a similar fate: the disease itself is old, and the last pandemic occurred too long ago for most to remember. the mere warning of a coming pandemic is almost perceived as an unfulfilled promise if the event does not occur shortly after the article is published. breast cancer, in contrast, is one of a few examples, where interest in a disease process can be maintained over an extended period of time. the risk of suffering from breast cancer is felt much more acutely, it seems, and there is a sense that proactive behavior will lead to an improved outcome. whether the mass media will take on a role in changing the public's attitude toward influenza as a threat is very much in doubt as long as journalists themselves perceive the next pandemic as an event as anonymous and inevitable as an earthquake. rsv infection is initiated by g glycoprotein attachment to cell surface receptors and followed by virus-cell fusion that is mediated by the f protein. the cleavage-activated rsv f1 protein is thought to interact with the target cell membrane through its n-terminal fusion peptide, which is released from a shielded position within the f homotrimer through a major conformational change. insertion of the f1 n-terminus into the cell membrane destabilizes the cell membrane and induces lipid mixing that is followed by mixing of contents, thereby enabling the viral nucleocapsid to enter the cytoplasm. the rsv f protein was recently found to interact with a small gtpase called rhoa through a domain that lies just carboxyterminal to the f1 fusion peptide (pastey et al., 1999) . rhoa is a member of the ras superfamily that is expressed intracellularly in all cell types. it induces bundling of actin filaments into stress fibers, focal adhesion plaque formation, cell-to-cell adhesion and organization of integral membrane proteins; it has additional roles in cell morphology and motility as well as cell cycle transition from g1 to s. a series of overlapping rhoa peptides from the interaction domain were evaluated in rsv plaque reduction neutralization assays. rhoa peptide 77-95 has an ic 50 of about 1 mg/ml against an inoculum of 100 pfu. neutralizing activity is also seen against parainfluenza virus type 3 (piv3), but not against a variety of other paramyxo, orthmyxo, corona and filoviruses (pastey et al., 2000) . intranasal administration of 500 mg of peptide 77-95 prior to intranasal infection of mice with rsv reduces rsv replication more than 100-fold and also diminishes weight-loss. studies with recombinant rsv expressing green fluorescent protein indicated that rhoa peptide 77-95 inhibited rsv replication at a very early stage of infection. subsequently, it was shown that fusion of cells transfected with rsv f, g and sh was inhibited by this peptide, suggesting that it exerted an effect on rsv replication at the level of membrane fusion or entry. since rhoa is an intracellular molecule, one would have to hypothesize that the interaction of the n-terminal heptad repeat and fusion peptide with the target membrane causes enough membrane disruption to allow an interaction between rhoa and rsv f. alternatively, rsv f may not interact with rhoa during the entry process, and the rhoa-derived peptides may simply disrupt the f structure or function to render the virus noninfectious. the question of whether rhoa and rsv f interact during a natural infection is not yet resolved. rhoa can be found in the cytoplasm bound to gdp and a guanine nucleotide dissociation inhibitor, and, upon gtp exchange and isoprenylation, rhoa associates with the cell membrane. if the proposed model of rhoa-rsv f interaction at the membrane is correct, then inhibition of isoprenylation should inhibit rsv infection. indeed, inhibition of isoprenylation through hmg coa reductase inhibitors, such as lovastatin, inhibits rsv infection of hep-2 cells at an ic 50 of 3 mm and reduces peak rsv titers in lungs of mice greater than 100-fold with oral gavage of 1 mg per day (gower, t.l., graham, b.s., submitted). the antiviral effect of lovastatin is also seen with piv3 in vitro. although it has not been formally proven that rsv f interacts with rhoa in the infected cell membrane, rhoa signaling activity is triggered in rsv-infected cells (gower, t.l. et al., submitted) . while rhoa signaling is not required for rsv replication, syncytium formation is diminished when rhoa signaling activity is inhibited. in addition, rhoa signaling may play a role in other aspects of rsv pathogenesis. rhoa kinase inhibitors reduce the transcription of il-6 and il-8 mrna in rsv infected cells (cytokines abundant in the nasal secretions of rsv-infected patients), and obviate airway hyperresponsiveness, one of the cardinal symptoms of rsv disease (hashimoto, k. et al., submitted). the balb/c mouse model was used to explore whether mycophenolic acid (mpa), an inhibitor of de novo purine synthesis in t and b lymphocytes, could improve the outcome of rsv disease, which was monitored by clinical symptoms such as ruffling of fur, increased respiratory rate and weight loss. oral administration of 100 mg/kg mmf (the prodrug of mpa) daily from day 1 to 6 post-infection reduced weight loss on day 7 post-infection from 23% in untreated animals to 8% in mpa treated mice (p b 0.001). a reduction of weight loss (7.8%) was also observed when initiation of treatment was delayed until day 5 post-infection. virus titers in lungs of mmf treated mice were similar to those of untreated controls but histological changes were reduced. on day 7 post-infection, ifng levels were elevated 2.5-fold in the treatment group while il4, il5 and il10 levels were unchanged, indicating a shift toward a t helper cell type 1 response that is thought to correlate with improved rsv disease outcome. these data suggest that inflammatory responses contribute to rsv disease in mice and that an immunomodulatory approach to the treatment of human rs virus disease is worth further consideration. using a cell-based assay to identify compounds which are able to inhibit fusion of hela cells infected with rsv, more than 300 analogues of a lead compound were synthesized and one compound (termed r170591) was selected for further evaluation. the in vitro 50% inhibitory concentration (ic 50 ) of this benzimidazole derivative (mw 395) was150 pm, and thus its potency exceeds that of ribavirin almost 100 000-fold (ic 50 =10 mm). r170591 exhibits in vitro antiviral activity against human rsv (subgroup a and b) and bovine rsv but not against pneumovirus of mice or other paramyxoviruses. rsv-induced cytopathic effect was reduced by r170591 at 0.1 nm, and concentration of 10 nm reduced rsv titers 1000-fold in multi-cycle growth curves. time of addition studies indicated that both virus-cell fusion and cell-cell fusion were inhibited by this compound. selection of resistant viruses in vitro yielded two mutants with single point mutations in the f-protein: one upstream of the second heptad repeat motif and another within it (s398l and d486n). rsv titers, determined by quantitative rt-pcr, were reduced 10-fold in bronchoalveolar lavage fluid and in lung tissue of cotton rats treated once by inhalation with r170591 prior to rsv infection. rfi-641, a compound that was derived by chemical optimization of the earlier described antiviral cl-387626, is a small molecule antiviral drug that selectively inhibits rsv (wyde et al., 1998) . the molecule is water-soluble and not orally bioavailable, but it proves to be efficacious when administered intranasally or by inhalation. the in vitro ic 50 varies between 3 and 180 ng/ml for laboratory strains or clinical isolates of rsv subgroup a and b. viral specificity and the large therapeutic window of rfi-641 (\100 fold) indicate that the antiviral activity of the compound is not due to adverse effects on normal cells. addition of rfi-641 to cell culture prior to adsorption reduces rsv yield 1000-fold at 48 h post infection (wyde et al., 1998) . temperature shift experiments suggest that the rsv f protein is the target for rfi-641, and this observation is confirmed by the inhibitory effect that rfi-641 has on rsv b1 cp-52/2b5, a viable rsv mutant in which both the g and sh open reading frames are deleted. if rfi-641 is added to cell culture 5 h post infection, it inhibits syncytium formation indicating that fusion inhibition occurs both in the early and late phase of the infectious cycle. rfi-641-resistant viruses can be selected, albeit much less easily than amantadine resistant variants. resistance to rfi-641 is acquired by point mutations solely in the f protein, mostly upstream of the second heptad repeat motif, but not by mutations in the g or sh protein. when administered prophylactically by the intranasal route, rfi-641 inhibits rsv replication in vivo, with mice and cotton rats exhibiting a 10-1000-fold reduction in rsv replication on day 5 post infection. in african green monkeys, rfi-641 reduces peak rsv titers not only when administered prophylactically but also when therapy is initiated at 24 h post infection and continued for a total of 9 days. a nebulized form of rfi-641 has been shown to be active in monkeys. the preclinical profile of this drug supports its development for treatment and prophylaxis of rsv disease in pediatric, adult and geriatric populations. phase 1 clinical trials confirmed that rfi-641 is a potent antiviral, and that the safety profile of this drug is encouraging. 7.5. de6elopment of picona6irus 3c protease inhibitors ag7088 is a potent, irreversible inhibitor of human rhinovirus (hrv) 3c protease, the enzyme that is responsible for the cleavage of the viral polyprotein into its functional protein subunits (matthews et al., 1999) . ag7088 was discovered by protein structure based rational drug design, and the compound exhibited activity against a large set of different hrvs. the 50% effective concentration (ec 50 ) ranges between 3 and 81 mm. other piconaviruses such as coxsackievirus a21 or b3, enterovirus 70, and echovirus 11 are also sensitive to the compound. in a placebo controlled challenge study in which adult volunteers were infected with hrv39 and treated with ag7088 (8 mg per dose intranasally, 5 times per day), ag7088 reduced mean viral titers, as well as mucus weight, respiratory symptom score and total symptom score significantly. in this study viral titers were determined by culture and also by quantitative pcr (taqman) to exclude ex-vivo effects of ag7088 on the reduction of virus titers. a phase ii clinical trial with 868 subjects was conducted to determine the efficacy of ag7088 in naturally acquired piconavirus infections. patients selected for the study had to present within 36 h of onset of symptoms and had to suffer from at least two mild or one moderate cold symptom. a five step symptom score was used to record severity of rhinorrhea, cough, sneezing, sore throat, chills, headache, and malaise. the treatment group was stratified into two or four daily doses of 8 mg ag7088 per dose intranasally, and the mean respiratory symptom score for days 1-5 was chosen as the primary endpoint. only 29% of all enrolled patients were infected by picor-naviruses. no significant difference in respiratory or total mean symptom score for days 1-5 was detected. however, the drug was well tolerated and safe. the lack of efficacy in this particular study might have been due to a lower than expected frequency of picornavirus infections. in a retrospective analysis, stratification for start of therapy within 24 h detected a trend to fewer respiratory symptoms and fewer total symptoms. there was a trend to earlier onset of relief, but the difference between treatment and placebo group again did not reach significance. 7.6. pleconaril (p) treatment reduces the incidence of acute otitis media (om) in children with 6iral respiratory illness: a pilot study pleconaril, an orally bioavailable picornavirus 3c protease inhibitor, has in vitro antiviral activity against 93% of all rhinoviruses. to evaluate the efficacy of pleconaril in preventing acute otitis media (aom) in an outpatient population, a double-blind, placebo-controlled pilot study was conducted in children with viral respiratory illness. eighty-seven children with a median age of 3 years and a history of 2-3 episodes of aom were randomized to pleconaril treatment (5 mg/kg or 2.5 mg/kg) or placebo thrice daily for 7 days following presentation with upper respiratory symptoms. picornavirus rna was detected by rt-pcr in nasal samples in 51% of patients at baseline. the overall incidence of physician-diagnosed aom during the 14 day follow-up were 18.8% (9/48), 12.5% (3/24), and 0% (0/15) in the placebo, low dose and high dose groups, respectively. of the 87 children developing aom, 86 tested positive for picornavirus rna. pleconaril treatment reduced the frequency of nasal symptoms by 29% in the high dose (5.0 mg/kg, p= 0.006) and 9% in the low dose (2.5 mg/kg, p= 0.393) group. it reduced frequency of systemic symptoms by 40% in the 5.0 mg/kg group (p= 0.020) and 26% in the 2.5 mg/kg group (p= 0.096). pleconaril was well tolerated, and adverse events did not differ from the control group. these results encourage further expanded trials of pleconaril in children with viral respiratory infections. 7.7. oral oseltami6ir reduces influenza-related complications in all age groups influenza illness is associated with the development of secondary complications, often requiring antibiotic treatment or even hospitalization. insurance data indicate that up to 64% of influenza related hospital admissions occur in 15-64 year old individuals, often without recognized underlying disorders. influenza complications affect all age groups but the type of complication differs among them. while acute otitis media has been reported in over 20% of children with influenza, lower respiratory tract infections (lrti) such as bronchitis and less often pneumonia, are the most common complication in adults. in recent years six phase iii clinical trials were completed; two of them in pediatric populations, three in healthy adults and one in the elderly. the neuraminidase inhibitor oseltamivir (o) is already approved for the treatment of influenza in adults, and it was recently approved for treatment in children aged 1 year and older by the fda. in six phase iii trials, most of the patients were enrolled within 36 h of onset of symptoms, and oseltamivir was administered at 2 mg/kg twice daily for children and 75 mg twice daily for adults. subjects with febrile ( \37.8°c) influenza were randomized to a 5 day regimen of oseltamivir or placebo, and an important endpoint of all studies was the effect of oral oseltamivir (o) on the incidence of influenza-related complications requiring antibiotics. one thousand and twenty nine children between the age of 1 and 12 (mean age 6.4 years) were enrolled in the pediatric trials. in the adult and elderly populations, 953 patients with a mean age of 35 years and 736 elderly patients with a mean age of 73 years were enrolled. in the pediatric, adult and elderly trials 61, 64 and 65%, respectively, tested positive for influenza a or b by culture or fourfold increase in hai titers. physician-diagnosed secondary complications (bronchitis, pneumonia, lrti, sinusitis or otitis media) requiring antibiotics were assessed in patients with confirmed influenza infection. oseltamivir reduced the rate of complications compared with placebo by 40% in children (p=65/235, o =36/217; p =0.005), by 51% in adults (p =23/309, o = 11/301; p = 0.05), and by 28% in the elderly (p= 49/254, o= 31/223; p= 0.14). in the pediatric population, aom was the most common complication, followed by bronchitis, pneumonia and sinusitis. the frequency of aom was reduced from 17% in the placebo group to 10% in the oseltamivir group, a significant 40% reduction. in adults and the elderly, bronchitis and sinusitis were the most common complications of influenza. the frequency of bronchitis in the elderly was reduced from 15 to 11%. thus, oral oseltamivir reduced the incidence of secondary complications and associated antibiotic use in all age groups. when all age groups were combined, antibiotic use for any indication was also significantly reduced. twelve patients in the placebo group versus four patients in the oseltamivir group required hospitalization for probable or possible influenza-related complications, suggesting a possible effect on hospitalization. 7.8. neuraminidase inhibitors: clinical update 7.8.1. oseltami6ir oseltamivir was recently approved in the us for prophylaxis of influenza in adults and adolescents over 12 years of age after three successful phase three trials. one was conducted in healthy adults, one in a family setting with an infectious index case, and another evaluated seasonal prophylaxis in the elderly. the efficacy of oseltamivir in all three trials ranged between 70 and 90%. an application for the approval of oseltamivir for treatment of influenza in children over one year of age was recently approved by the fda. in otherwise healthy children of 1-12 years, treatment with oseltamivir reduced the median time to freedom from illness by 1.5 days (p= b 0.0001) and also reduced the relative risk of otitis media (aom) by 40%. in 6 -12 year old asthmatic children, oseltamivir treatment, started within 24 h of first symptoms, decreased the median illness duration by 1.7 days (p =0.07), and airway function was significantly improved (1 s forced expiratory volume on day 5 improved by 10.8% compared with baseline values among oseltamivir recipients and 4.7% among placebo recipients (p= 0.0147)). the incidence of development of drug resistant virus under therapy was evaluated in more than 1000 subjects. none of 542 subjects sampled on day 3 of treatment harbored resistant virus. on day 5, however, 4/451 subjects (1%) shed virus with a resistant phenotype. in children, the frequency of viral resistance was 4% (10/247). all of these subjects recovered normally despite this incidental finding. so far, resistant mutants have been less infectious than wild type virus; no evidence of transmission has been detected in an experimental infection model in ferrets. the impact study evaluated the benefit of early intervention in reducing the total time with symptoms starting from the time of first onset of fever. this reflects the total burden of illness from a patient's point of view. a total of 958 patients, 13-70 years of age were enrolled and treated within 48 h of the first onset of symptoms. treatment was initiated within 12 h in 25% of those recruited. using an accelerated time to failure model, earlier intervention was shown to strongly correlate with shorter duration of illness. the total duration of illness could be halved if treatment was started within 12 h of the onset of symptoms compared with intervention at 48 h. in phase iii trials, zanamivir reduced the duration of illness in adults over 12 years of age and in children between 5 and 12 years of age by 1.5 days (95% ci= 1-2.5 days). in high risk patients (525 subjects, 65% with asthma and 35% with chronic obstructive pulmonary disease) duration of illness amongst all influenza positive subjects was reduced by 1.5 days. duration of illness was reduced in both vaccinated (1.25 day) and nonvaccinated (2 day) subjects. during therapy, pulmonary function improved slightly between days 1 and 5. zanamivir was well tolerated, and there were no differences in adverse effects between treatment and placebo groups. with 9000 subjects studied in clinical trials, resistant mutants were not isolated by sampling on days 3 and 5. efficacy for treatment of influenza b was assessed in 300 subjects and found to be similar to that of influenza a. a prophylaxis study in a family setting showed 79% protective efficacy. finally, in a nursing home study, zanamivir exhibited a 60% in-creased efficacy in preventing influenza disease compared with rimantadine. rwj-270201, an orally available once daily neuraminidase inhibitor that is active against influenza a and b in vitro and in animals, was evaluated in phase ii trials of experimentally infected healthy adults. therapy was started approximately 24 h post infection with influenza a/texas h1n1 or influenza b/yamagata. viral load over time was the primary endpoint, and nasal wash titers were determined every 12 h on day 1-3 and daily thereafter. in the influenza a study, 90 subjects were enrolled into four treatment (100 to 400 mg per day) and one placebo arm. high dose treatment (400 mg once daily or 200 mg twice daily) reduced viral load by 68-73%, whereas 100 or 200 mg per day led to a reduction by 34%. shedding was reduced by 1-2 days in the high dose groups, and the incidence of fever declined from 25% in placebo controls to less than 5% in all treatment groups combined. to evaluate efficacy against influenza b, 56 subjects were enrolled into two treatments (800 or 400 mg per day) and one placebo group. a 61% reduction of viral load was shown for the high dose treatment (800 mg per day). no evidence for the emergence of resistant virus was detected, and oral rwj 270201 was well tolerated without excess gastrointestinal side effects. world-wide, acute respiratory infections (ari) are responsible for more than 4 million deaths per year in children under five years of age. viral respiratory infections contribute significantly to this burden of disease, not only in children but also in adults. although there is a myriad of viral respiratory pathogens, the number of virus families that cause significant burden of disease is limited. in children, the paramyxoviridae are the most important family of viruses, with respiratory syncytial virus (rsv) and the parainfluenza viruses (piv) causing most disease, followed by adeno-and influenza viruses. in adults, influenza a virus with its constantly changing antigenic composition surpasses other viral pathogens, but the impact of rsv and rhinoviruses are being increasingly appreciated. the epidemiology of respiratory virus infections very much depends on host and environmental factors. while adenovirus infections are a major concern in military training camps and in immunocompromised patients, they are not as important in the general adult population. the immunocompromised are not only at increased risk for viral respiratory disease but diagnosis and management are much more difficult in this group. newer diagnostic methods based on antigen detection or viral genome amplification make rapid diagnosis possible and thereby affect clinical management of viral respiratory disease. vaccines are still the most powerful tool in preventing viral respiratory disease but for many important pathogens there is still no vaccine available. the inactivated influenza vaccine, with its antigenic composition changing annually, is still the mainstay of influenza prevention. coldadapted live attenuated influenza virus vaccines are on the horizon but are not yet approved by the fda. adenovirus vaccines have been used successfully in the military for many years, and the recent resurgence of large outbreaks of respiratory disease after discontinuation of vaccination emphasizes the importance of maintaining a preventive strategy. live virus vaccine candidates against rsv and piv, derived biologically or by reverse genetics, are currently in clinical trials, and there is hope that safe and efficient vaccines will become available within this decade. new insight into virus-host and virus-bacteria interactions in viral respiratory illness may help us understand the complexity of these disease entities and allows a re-evaluation of management options. diseases earlier thought of as purely bacterial, such as otitis media, might be more effectively prevented by viral vaccines than by bacterial vaccines. populations at high risk for complications resulting from respiratory viral infections are now better defined and a more targeted prophylaxis is possible, be it passive prophylaxis against rsv disease with monoclonal antibody preparations or active prophylaxis with influenza-or adenovirus vaccines. in the management of influenza virus disease, much has changed for the better. neuraminidase inhibitors (ni) are now established as an effective intervention to decrease the severity and to shorten the duration of illness when therapy is initiated within 2 days of the onset of symptoms. zanamivir and oseltamivir are now licensed in the us and elsewhere for adults and adolescents twelve years of age and older, and a license for use in children over one year of age has recently been obtained for oseltamivir. both drugs are safe and well tolerated, and development of resistance occurs much less frequently than with older antivirals such as amantadine or rimantadine. rhinovirus infections, the cause of most colds, may now be amenable to treatment with the experimental 3c protease inhibitors pleconaril (given orally) and ag7088 (given intranasally), and initial studies indicate that pleconaril treatment of colds in children might reduce their risk of developing otitis media. antivirals effective against rsv and piv are still in an early phase of development, but inhibitors of the viral fusion protein look promising. despite all progress, severe deficiencies remain in the preventative efforts. our level of preparedness for an influenza pandemic does not seem much different from that 40 years ago. streamlining of hospital infrastructure and just-in-time production of antibiotics and antivirals limit our ability to respond effectively should a pandemic strike. although potential pandemic influenza viruses have been defined and novel techniques such as reverse genetics are being employed in vaccine development, the implementation of pandemic preparedness is still a daunting task. adenovirus type 37 uses sialic acid as a cellular receptor variable morbidity of respiratory syncytial virus infection in patients with underlying lung disease: a review of the picnic rsv database. pediatric investigators collaborative network on infections in canada respiratory virus infection as a cause of prolonged symptoms in acute otitis media viral infections of humans cold adaptation of parainfluenza virus type 3: induction of three phenotypic markers the efficacy of live attenuated, cold-adapted, trivalent, intranasal influenza virus vaccine in children cutting edge: adenovirus e19 has two mechanisms for affecting class i mhc expression the m2-2 protein of human respiratory syncytial virus is a regulatory factor involved in the balance between rna replication and transcription structural analysis of the mechanism of adenovirus binding to its human cellular receptor the detection of viral genomes by polymerase chain reaction in the myocardium of pediatric patients with advanced hiv disease infections in 18 000 infants and children in a controlled study of respiratory tract disease. ii. variation in adenovirus infections by year and season chimeric bovine respiratory syncytial virus with glycoprotein gene substitutions from human respiratory syncytial virus (hrsv): effects on host range and evaluation as a live-attenuated hrsv vaccine interferon gamma expressed by a recombinant respiratory syncytial virus attenuates virus replication in mice without compromising immunogenicity partial protection to respiratory syncytial virus (rsv) elicited in mice by intranasal immunization using live staphylococci with surface-displayed rsv-peptides isolation of subgenus b adenovirus during a fatal outbreak of enterovirus 71-associated hand, foot, and mouth disease in influenza a and meningococcal disease the role of nasal secretion and serum antibody in the rhinovirus common cold recovery from infants with respiratory illness of a virus related to chimpanzee coryza agent (cca). i. isolation, properties and characterization newly recognized myxoviruses from children with respiratory disease growth on artificial medium of an agent associated with atypical pneumonia and its identification as pplo isolation and characterization of adenovirus 5 from the brain of an infant with fatal cerebral edema respiratory viruses interfere with bacteriologic response to antibiotic in children with acute otitis media parainfluenza viruses rational design of live-attenuated recombinant vaccine virus for human respiratory syncytial virus by reverse genetics production of infectious human respiratory syncytial virus from cloned cdna confirms an essential role for the transcription elongation factor from the 5% proximal open reading frame of the m2 mrna in gene expression and provides a capability for vaccine development recovery of viruses other than cytomegalovirus from bronchoalveolar lavage fluid immunity to influenza in man immunization of types four and seven adenoviruses by selective infection of the intestinal tract respiratory viral infections in immunocompetent and immunocompromised persons host responses to respiratory virus infection and immunization satisfactorily attenuated and protective mutants derived from a partially attenuated cold-passaged respiratory syncytial virus mutant by introduction of additional attenuating mutations during chemical mutagenesis recombinant human respiratory syncytial virus (rsv) monoclonal antibody fab is effective therapeutically when introduced directly into the lungs of rsv-infected mice the molecular basis of pneumococcal infection: a hypothesis rehospitalization for respiratory illness in infants of less than 32 weeks' gestation acute respiratory viral infections in ambulatory children of urban northeast brazil heparan sulfate glycosaminoglycans are involved in adenovirus type 5 and 2-host cell interactions comparison of rapid diagnostic techniques for respiratory syncytial and influenza a virus respiratory infections in young children human parainfluenza virus type 3 (piv3) expressing the hemagglutinin protein of measles virus provides a potential method for immunization against measles virus and piv3 in early infancy prolonged survival of pancreatic islet allografts mediated by adenovirus immunoregulatory transgenes rapid diagnosis of respiratory syncytial virus infections in immunocompromised adults mechanism of protective immunity against influenza virus infection in mice without antibodies efficacy of a pneumococcal conjugate vaccine against acute otitis media. new engl huntingtin interacts with a family of ww domain proteins rapid simultaneous diagnosis of infections with respiratory syncytial viruses a and b, influenza viruses a and b, and human parainfluenza virus types 1, 2, and 3 by multiplex quantitative reverse transcription-polymerase chain reaction-enzyme hybridization assay (hexaplex) detection of viral, chlamydia pneumoniae and mycoplasma pneumoniae infections in exacerbations of asthma in children low-temperature-grown rs virus in adult volunteers cytoplasmic inclusions of respiratory syncytial virus-infected cells: formation of inclusion bodies in transfected cells that coexpress the nucleoprotein, the phosphoprotein, and the 22k protein surfactant protein a binds to the fusion glycoprotein of respiratory syncytial virus and neutralizes virion infectivity the respiratory syncytial virus matrix (m) protein localises to cytoplasmic inclusions in the presence of n, p, l and m2 proteins experimental otitis media after nasal inoculation of streptococcus pneumoniae and influenza a virus in chinchillas role of early region 3 (e3) in pathogenesis of adenovirus disease risk of respiratory syncytial virus infection for infants from low-income families in relationship to age, sex, ethnic group, and maternal antibody level risk of primary infection and reinfection with respiratory syncytial virus safety and immunogenicity of intranasally administered inactivated trivalent virosome-formulated influenza vaccine containing escherichia coli heat-labile toxin as a mucosal adjuvant evaluation of the live attenuated cpts 248/404 rsv vaccine in combination with a subunit rsv vaccine (pfp-2) in healthy young and older adults adult adenovirus infections: loss of orphaned vaccines precipitates military respiratory disease epidemics respiratory syncytial virus infection in children with bronchopulmonary dysplasia prophylactic administration of respiratory syncytial virus immune globulin to high-risk infants and young children. the respiratory syncytial virus immune globulin study group palivizumab, a humanized respiratory syncytial virus monoclonal antibody, reduces hospitalization from respiratory syncytial virus infection in high-risk infants palivizumab, a humanized respiratory syncytial virus monoclonal antibody, reduces hospitalization from respiratory syncytial virus infection in high-risk infants. the impact-rsv study group molecular characterization of h9n2 influenza viruses: were they the donors of the 'internal' genes of h5n1 viruses in hong kong? respiratory syncytial viral infection in children with compromised immune function prevalence of various respiratory viruses in the middle ear during acute otitis media a longitudinal study of respiratory viruses and bacteria in the etiology of acute otitis media with effusion recovery of a new agent from patients with acute respiratory disease characterization of the influenza a virus gene pool in avian species in southern china: was h6n1 a derivative or a precursor of h5n1? adenovirus immunoregulatory genes and their cellular targets prevention of adenoviral acute respiratory disease in army recruits: cost-effectiveness of a military vaccination policy fimbria-mediated enhanced attachment of nontypeable haemophilus influenzae to respiratory syncytial virus-infected respiratory epithelial cells respiratory syncytial virus that lacks open reading frame 2 of the m2 gene (m2-2) has altered growth characteristics and is attenuated in rodents recombinant respiratory syncytial viruses with deletions in the ns1, ns2, sh, and m2-2 genes are attenuated in vitro and in vivo community study of role of viral infections in exacerbations of asthma in 9 -11 year old children development of a humanized monoclonal antibody (medi-493) with potent in vitro and in vivo activity against respiratory syncytial virus first international symposium on influenza and other respiratory viruses: summary and overview evaluation of a live attenuated bovine parainfluenza type 3 vaccine in two-to six-month-old infants a live human parainfluenza type 3 virus vaccine is attenuated and immunogenic in healthy infants and children severe respiratory syncytial virus disease in alaska native children respiratory syncytial virus disease in infants despite prior administration of antigenic inactivated vaccine clinical and immunological response of infants and children to administration of low-temperature adapted respiratory syncytial virus influenza a and b virus infection in infants and young children during the years 1957 -1976 negative-strand virus m and retrovirus ma proteins: all in a family? interaction of an adenovirus 14.7-kilodalton protein inhibitor of tumor necrosis factor alpha cytolysis with a new member of the gtpase superfamily of signal transducers interaction of an adenovirus e3 14.7-kilodalton protein with a novel tumor necrosis factor alpha-inducible cellular protein containing leucine zipper domains recombinant influenza a virus vaccines for the pathogenic human a/hong kong/97 (h5n1) viruses identification of a cell protein (fip-3) as a modulator of nf-kb activity and as a target of an adenovirus inhibitor of tumor necrosis factor a-induced apoptosis do l3t4 + t cells act as effector cells in protection against influenza virus infection avian-to-human transmission of h9n2 subtype influenza a viruses: relationship between h9n2 and h5n1 human isolates an adenovirus inhibitor of tumor necrosis factor alpha-induced apoptosis complexes with dynein and a small gt-pase biologic and immunologic characteristics of cold-adapted influenza virus the development of live attenuated cold-adapted influenza virus vaccine for humans respiratory syncytial viral infection in infants with congenital heart disease increased burden of respiratory viral associated severe lower respiratory tract infections in children infected with human immunodeficiency virus type-1 reduction of respiratory syncytial virus (rsv) in tracheal aspirates in intubated infants by use of humanized monoclonal antibody to rsv f protein measuring the comparative efficacy of antibacterial agents for acute otitis media: the 'pollyanna phenomenon epidemiology of respiratory viral infection among paediatric inpatients over a 6 year period in north-east england acute myocarditis. rapid diagnosis by pcr in children structure-assisted design of mechanism-based irreversible inhibitors of human rhinovirus 3c protease with potent antiviral activity against multiple rhinovirus serotypes cytotoxic t-cell immunity to influenza does prematurity alter the course of respiratory syncytial virus infection? the economic impact of pandemic influenza in the united states: priorities for intervention safety and immunogenicity of an inactivated subunit influenza virus vaccine combined with mf59 adjuvant emulsion in elderly subjects, immunized for three consecutive influenza seasons current research on influenza and other respiratory viruses: ii international symposium. antiviral res use of live attenuated cold-adapted influenza a reassortant virus vaccines in infants, children, young adults and elderly adults temperature-sensitive mutants of influenza a virus. transfer of the two ts-1a2 ts lesions present in the udorn/ 72-ts-1a2 donor virus to the influenza a/alaska/6/77 (h3n2) wild type virus effect of age and preexisting antibody on serum antibody response of infants and children to the f and g glycoproteins during respiratory syncytial virus infection current approaches to the development of vaccines effective against parainfluenza and respiratory syncytial viruses improved outcome of respiratory syncytial virus infection in a high-risk hospitalized population of canadian children. pediatric investigators collaborative network on infections in canada effect of rapid diagnosis on management of influenza a infections the p38 signal transduction pathway: activation and function infection and disease with respect to age, immunologic status, race and sex rhoa interacts with the fusion glycoprotein of respiratory syncytial virus and facilitates virus-induced syncytium formation a rhoa-derived peptide inhibits syncytium formation induced by respiratory syncytial virus and parainfluenza virus type 3 effect of respiratory syncytial virus on adherence, colonization and immunity of non-typable haemophilus influenzae: implications for otitis media detection of adenoviral genome in the myocardium of adult patients with idiopathic left ventricular dysfunction paramyxovirus m proteins: pulling it all together and putting it on the road human infection with influenza h9n2 unexplained deaths due to possibly infectious causes in the united states: defining the problem and designing surveillance and laboratory approaches. the unexplained deaths working group the respiratory syncytial virus matrix protein interacts with the cytoplasmic domain of the g glycoprotein adherence of type i streptococcus pneumoniae to tracheal epithelium of mice infected with influenza a/pr8 virus immunoprophylaxis and immunotherapy of respiratory syncytial virus infection in the cotton rat quantitative aspects of passive immunity to respiratory syncytial virus infection in infant cotton rats rhinovirus and respiratory syncytial virus in wheezing children requiring emergency care. ige and eosinophil analyses infection with respiratory syncytial virus enhances expression of native receptors for non-pilate neisseria meningitidis on hep-2 cells induction of protective immunity against influenza virus in a macaque model: comparison of conventional and iscom vaccines identification of a conserved receptorbinding site on the fiber proteins of car-recognizing adenoviridae isolation of a cytopathogenic agent from human adenoids undergoing spontaneous degeneration in tissue culture human antibody responses to mature and immature forms of viral envelope in respiratory syncytial virus infection: significance for subunit vaccines bovine respiratory syncytial virus nonstructural proteins ns1 and ns2 cooperatively antagonize a/b interferon-induced antiviral response bovine parainfluenza virus type 3 (bpiv3) fusion and hemagglutinin-neuraminidase glycoproteins make an important contribution to the restricted replication of bpiv3 in primates bacterial pneumonia during the hong kong influenza epidemic of 1968 -1969 molecular identification of viruses in sudden infant death associated with myocarditis and pericarditis respiratory syncytial virus immune globulin for prophylaxis against respiratory syncytial virus disease in infants and children with congenital heart disease respiratory syncytial virus infection in north-east england identification of mutations contributing to the temperature-sensitive, cold-adapted, and attenuation phenotypes of the live-attenuated coldpassage 45 (cp45) human parainfluenza virus 3 candidate vaccine effectiveness of palivizumab: evaluation of outcomes from the 1998 to 1999 respiratory syncytial virus season the role of human adenovirus early region 3 proteins (gp19k, 10.4k, 14.5k, and 14.7k) in a murine pneumonia model characterization of an avian influenza a (h5n1) virus isolated from a child with a fatal respiratory illness non-typeable haemophilus influenzae adhere to and invade human bronchial epithelial cells via an interaction of lipooligosaccharide with the paf receptor avirulent avian influenza virus as a vaccine strain against a potential human pandemic recombinant respiratory syncytial virus that does not express the ns1 or m2-2 protein is highly attenuated and immunogenic in chimpanzees efficacy and safety of the oral neuraminidase inhibitor oseltamivir in treating acute influenza: a randomized controlled trial therapy for respiratory tract infections caused by respiratory syncytial virus expression of adenoviral e3 transgenes in beta cells prevents autoimmune diabetes respiratory syncytial virus infection in tropical and developing countries community respiratory virus infections in immunocompromised patients with cancer recombinant respiratory syncytial virus (rsv) bearing a deletion of either the ns2 or sh gene is attenuated in chimpanzees replacement of the f and g proteins of respiratory syncytial virus (rsv) subgroup a with those of subgroup b generates chimeric live attenuated rsv subgroup b vaccine candidates immune responses to adenoviruses: viral evasion mechanisms and their implications for the clinic evaluation of a live, cold-passaged, temperature-sensitive, respiratory syncytial virus vaccine candidate in infancy cl387626 exhibits marked and unusual antiviral activity against respiratory syncytial virus in tissue culture and in cotton rats genetic characterization of the pathogenic influenza a/goose/guangdong/ 1/96 (h5n1) virus: similarity of its hemagglutinin gene to those of h5n1 viruses from the 1997 outbreaks in hong kong key: cord-002227-x1ddi8wg authors: li, wanli; an, xinjiang; fu, mingyu; li, chunli title: emergency treatment and nursing of children with severe pneumonia complicated by heart failure and respiratory failure: 10 case reports date: 2016-07-29 journal: exp ther med doi: 10.3892/etm.2016.3558 sha: doc_id: 2227 cord_uid: x1ddi8wg pneumonia refers to lung inflammation caused by different pathogens or other factors, and is a common pediatric disease occurring in infants and young children. it is closely related to the anatomical and physiological characteristics of infants and young children and is more frequent during winter and spring, or sudden changes in temperature. pneumonia is a serious disease that poses a threat to children's health and its morbidity and mortality rank first, accounting for 24.5–65.2% of pediatric inpatients. due to juvenile age, severe illness and rapid changes, children often suffer acute heart failure, respiratory failure and even toxic encephalopathy at the same time. the concurrence in different stages of the process of emergency treatment tends to relapse, which directly places the lives of these children at risk. severe pneumonia constitutes one of the main causes of infant mortality. in the process of nursing children with severe pneumonia, intensive care was provided, including condition assessment and diagnosis, close observation of disease, keeping the airway unblocked, rational oxygen therapy, prevention and treatment of respiratory and circulatory failure, support of vital organs, complications, and health education. the inflammatory response was proactively controlled, to prevent suffocation and reduce mortality. in summary, positive and effective nursing can promote the rehabilitation of children patients, which can be reinforced with adequate communication with the parents and/or caretakers. severe pneumonia is a common life-threatening disease, particularly for children, and is more common in infants and young children (1, 2) . the estimated worldwide incidence of severe pneumonia in children less than 5 years of age is 50-80 per 1,000 person-years and the mortality is 1.0-5.5 per 1,000 person-years (3) (4) (5) . it often occurs in the winter and spring, with acute onset, complex clinical manifestations and fast-changing condition, which usually involves the circulation, nervous and digestive systems (1) (2) (3) . as a result, severe pneumonia produces corresponding clinical symptoms, such as respiratory failure, heart failure, toxic encephalopathy and intestinal paralysis, which endanger the lives of children in the short term, and is the first cause of death of pediatric inpatients (6, 7) . it is listed as the one of the four diseases requiring prevention and treatment in children by the ministry of health (8, 9) . pneumonia may occur at any point of the year, but is more common in the months of winter and spring or during the time of climate variability. the disease may be the primary illness, or secondary one after an acute infectious disease, such as bronchitis and measles, upper respiratory tract infections and whooping cough, and has a high morbidity and mortality rate in china (10) . as reported by several studies, the number of annual pneumonia patients in china is ≤21 million individuals, and among the children with pneumonia, 7-13% cases are of severe pneumonia, which ranks fifth amongst various diseases leading to death (11) (12) (13) (14) (15) (16) . the aim of this study was to conduct a retrospective analysis of the clinical data of 10 cases of children presenting with severe pneumonia at the xuzhou children's hospital between january 2009 and june 2012, retrieve and review the literature, and summarize emergency treatment and nursing experience. general information. ten cases of children diagnosed with severe pneumonia according to the guidelines of the world health organization were included in the present study (16) . in this group, there were 6 males and 4 females, aged 1 month to 6 years. the children patients were hospitalized due to fever, cough, asthma, dry and wet rales could be heard in lung auscultation, and a chest x-ray showed thickened lung markings with visible punctate and flake-like shadows. the children patients refused any intake of dairy products or food. there was one case of concurrent toxic encephalopathy, 1 case of gastrointestinal bleeding, and 1 case of toxic intestinal paralysis. the physical examination showed a body temperature of 38.5-40˚c, pulse rate of 120-190 beats/min, breathing of 30-60 beats/min, and dry and wet rales could be heard in lung. the x-ray examination showed thickened lung markings, and flake-like shadows, and abdominal palpation showed enlarged liver and spleen. respiratory or circulatory functions were at different levels of exhaustion. treatment. after admission, comprehensive emergency treatment measures were taken, such as improving the ventilation function, oxygen uptake, maintaining airway patency, administration of cardiac and diuretic drugs, reduction of intracranial pressure, and conducting anti-infection and symptomatic support measures. nine cases were successfully treated, 1 case succumbed to respiratory failure, and the success rate was 90%. condition assessment and diagnosis. a detailed inquiry of the medical history was carried out to determine whether the children patients had a history of recurrent respiratory tract infection, and whether they had measles, whooping cough and other respiratory diseases prior to onset, as well as whether the children patients had a full-term birth or asphyxia, and if the growth and development of children patients after birth was normal (16) (17) (18) . conditions such as fever, cough, extent of choked asthma, the presence or absence of breathing, faster heart rate, pulmonary rales, orthopnea, nose flap, three depression signs and cyanosis, as well as clinical manifestations of infected circulatory, digestive, and nervous system were assessed. evaluation of blood routine examination, chest x-ray, and etiology test results were conducted. psychological and social conditions of children and parents were also assessed (19) . common condition assessment and diagnostic criteria included: i) air exchange impairment, related to lung inflammation; ii) invalid airway clearance, related to excessive and sticky respiratory secretions, frail children patients, and inability to expectorate; iii) hyperthermia, related to lung infection; and iv) malnutrition, related to inadequate intake, increased consumption. close observation of the condition. attention was paid to changes in total peripheral resistance blood pressure (bp), the presence of double suction, such as nodding-like breathing, apnea and other conditions indicating respiratory failure. the presence of exacerbation of dyspnea, dysphoria, quickening heart rate, and an enlarged liver in a short period of time suggested heart failure (20) (21) (22) . children patients with severe wheezing often suffered respiratory acidosis due to retention of carbon dioxide. drowsiness, convulsions or coma in sick children suggested an occurrence of toxic encephalopathy. medicines such as antibiotics, antiasthmatic drugs, cardiac drugs and other medications were administered to patients at the appropriate dosage, the appropriate time and in an accurate manner. side effects of various drugs were observed, and if children patients showed signs of dysphoria, quickening heart rate, worsening asthma, or enlarged liver in a short period of time, indicating heart failure, then the infusion rate was reduced. in our study, close observation of consciousness, pupil changes and muscle tension was carried out, and if manifestations of intracranial pressure such as drowsiness, convulsions, irregular breathing and increased muscle tension appeared, immediate rescue steps were taken. abdominal distension and decreased or disappearing bowel sounds were observed, in order to detect toxic intestinal paralysis in time (23) . emergency treatment and therapy for respiratory failure. type i respiratory failure refers to the lone presence of hypoxemia and absence of hypercapnia, featuring ventilation dysfunctions, blood change of pao 2 ≤60 mmhg, and paco 2 which can be maintained at normal level or reduced (24) . type i respiratory failure also refers to the coexistence of hypoxemia and hypercapnia, impairment of ventilatory function and gas exchange functions, severe lung lesion, obstruction of trachea and bronchia caused by sticky secretions, blood change of pao 2 <60 mmhg, and paco 2 >50 mmhg. main clinical manifestations of children patients with type i pneumonia with respiratory failure include, poor mental state or dysphoria, polypnea, cyanosis of lips, dyspnea, nasal flaring and three depression signs. these symptoms are difficult to distinguish from type ii respiratory failure, and can be observed only by blood gas analysis, which shows a marked difference between type i and ii. type ii respiratory failure shows symptoms of type i respiratory failure and in addition more often than not, it also has symptoms such as shallow breathing, irregular rhythm, slow breathing, drowsiness or coma and even jaw breathing in some patients (25, 26) . in our study, changes in condition and changes in blood were closely observed, and where required, a tracheal intubation ventilator was employed to improve the cure rate. oxygen uptake. nasal catheter is easily blocked by secretions, leading to failure of effective uptake oxygen, and is therefore difficult to utilize. we employed the conventional oxygen mask inhalation method, which is comfortable and without stimulation, and was easily accepted by children (27, 28) . after using oxygen, once hypoxia was improved, timely adjustment of the oxygen flow or deactivation of oxygen was needed, because inadequate oxygen concentration such as excessive or overtime concentration may lead to changes such as lung tissue edema atelectasis and proliferation of alveolar capillary. the humidification bottle contained 50% alcohol, aiming to reduce the alveolar surface tension and help improve ventilation. maintenance of airway patency and administration of various aerosol inhalation therapies. i) this group of children patients took appropriate clinostatism according to the state of disease. under normal conditions, the children were placed in a horizontal position, with neck raised high, in order that airways would be unblocked. for children patients with severe heart failure and wheezing, semi-recumbent position was taken to reduce the burden on the heart and lungs (29, 30) . for children patients with increased respiratory secretions, the lateral position was useful to expectorate and prevent aspiration. ii) patients received daily routine aerosol inhalation, turning drugs into aerialfog-like fine particles, inhaled to the bronchioles or alveoli, which diluted secretions and had an anti-inflammatory and anti-allergic function and reduced local inflammatory exudates, in addition to reducing airway resistance and improving ventilation. aerosol inhalation time was 10-15 min and afterwards sputum suction was carried out immediately in order to clear respiratory secretions. commonly used drugs included: 20,000 units of gentamicin, 2-3 mg of α-chymotrypsin, 1-2 mg of dexamethasone, and 20 ml of saline. the dual role of the aforementioned ultrasonic aerosol inhalation was useful to reinforce treatment of children with severe pneumonia. according to the disease, aerosol inhalation was conducted every 4 or 6 h, with sputum suction ensuing, which produced a good suction effect for children patients with many sticky respiratory secretions. it should be noted that if patients had excessive sputum, the sputum suction was also carried out once prior to using ultrasound aerosol inhalation therapy, followed by thorough sputum suction. in the whole process of aerosol inhalation, providing oxygen inhalation or enhancing oxygen concentration effectively prevented the occurrence of hypoxemia (31) . iii) hypocalcemia may produce laryngospasm and tongue tenesmus, which causes obstruction and sudden suffocation. first, the child patient's tongue tip was pulled outside the mouth, and then artificial respiration was conducted, with most patients being able to relieve themselves spontaneously. pressurized oxygen was given, endotracheal intubation was carried out if necessary and calcium was replenished immediately. iv) when nasal obstruction caused breathing difficulties, 1% ephedrine drops were used after clearing away nasal secretions with a cotton swab, to maintain the airway patency. v) the diet of children with severe pneumonia was affected due to high fever, vomiting, diarrhea and other factors. when children patients were unable to eat, it was appropriate to supplement water, electrolytes, vitamins, and give them high-calorie, high protein, digestible food, that was eaten in small amounts but frequently, to prevent satiety from interfering with respiratory function. vi) previous findings showed that, ambroxol and low-dose heparin coupled with aerosol inhalation significantly shortened the time of the disappearance of all the clinical signs and improved overall efficiency of the clinical treatment (32, 33) . there was no bleeding and other adverse reactions involved in the clinical treatment process, which had advantages such as easy administration, safe use, inexpensiveness, a significant effect, and less adverse effects in the treatment of children with severe pneumonia, and was thus worthy of wider application. since the application of mechanical ventilation is prone to cause ventilator-related complications, to improve the successful rescue rate of children with severe pneumonia and shorten the course of treatment, the use of ncpap may be considered a priority. for severe pneumonia, ncpap can support cardiopulmonary function, and its early application can improve timely oxygenation, stabilize disease, prevent disease progression, reduce ventilator application and average hospitalization time in pediatric intensive care unit as well as avoid the adverse effects caused by intubation (34) . however, two points are important when applying it to infants: i) grasp indications and standard of respiratory failure that the children comply with; ii) over ncpap application process, nasal and oral secretions should be immediately removed, raising the neck high, to strengthen expectoration in order to maintain airway patency. operating parameters of using the ncpap oxygen therapy machine in our department: i) oxygen concentration inhaled (fio2) 40 or 90%; ii) the oxygen flow 4-12 l/min; and iii) the beginning pressure was maintained at 3-4 cm h 2 o. after observing 2-4 h, the adjustment of pressure was determined by clinical symptoms, signs and blood gas analysis. ncpap is an effective treatment for severe pneumonia with respiratory and heart failure, and can quickly correct hypoxemia, reduce endotracheal intubation and mechanical ventilation demand (35) . it has a positive effect and can reduce the stimulation of children, thus decreasing any occurrence of ventilator pneumonia. in addition, parents readily accepted this treatment (36) . short-term intubation ventilator was utilized when there was: i) respiratory arrest or respiratory and cardiac arrest; ii) sputum congestion, dyspnea, serious cyanosis; and iii) blood paco 2 >60 mmhg, in order to accelerate the discharge of paco 2 and reduce respiratory acidosis. emergency treatment and treatment of heart failure. when children patients coughed, their body temperature increased to ≤40˚c and was accompanied by dyspnea, dysphoria, pale face, cyanosis, drowsiness, fixed moist rales in lung, respiratory rate of ≥60 beats/min, heart rate of ≥160-180 beats/min in quiet state, low and blunt heart sound, gallop rate, and a progressively enlarged liver, 3 cm beyond the ribs or an increase of ≥1.5 cm in a short period of time. the above, as well as cold extremities and weak pulse, constituted clinical manifestations of a heart failure. in this situation, oxygen inhalation and tranquilizers were given immediately, 0.5 µg/(kg x min) and milrinone, a second generation of phosphodiesterase ⅲ (pde-ⅲ) inhibitor, which acts on the β receptor accessory pathway, was administered to maintain heart function. by selectively inhibiting pde-ⅲ in myocardial cells, increasing intracellular cyclic adenosine monophosphate levels, promoting calcium influx, enhancing calcium concentration in myocardial cytolymph, milrinone strengthens myocardial contractility, increases cardiac output, and exerts direct relaxation on vascular smooth muscle, dilates blood vessels and reduces cardiac preload and afterload. milrinone demonstrates a better clinical efficacy in the treatment of children with pneumonia complicated by heart failure than catecholamines (37) . emergency treatment and treatment of toxic encephalopathy and intestinal paralysis. when children show signs of exhaustion, dysphoria, coma, increased muscle tension, irregular breathing, and increased cerebrospinal fluid pressure, toxic encephalopathy should be considered. timely administration of sedatives, oxygen and mannitol are important in order to ease cerebral edema. decrease in intracranial pressure. in the present study, any changes including children' pupillary light reflex, headache, vomiting, consciousness, breathing, pulse, temperature, and fluctuation in bp were closely observed. the head was raised 15-30˚ to facilitate the intracranial venous return, leading to a corresponding reduction in intracranial pressure. precaution was taken when using dehydrating agent as well as to prevent it from leaking from blood vessels, and to prevent tissue necrosis. when administering furosemide, its effect and adverse drug reactions were observed. if intracranial pressure increased, lumbar puncture was avoided as much as possible, but if it had to be carried out then rescue preparation was performed. cerebrospinal fluid flow is not be in excess to avoid the formation of hernia. control of fever and convulsions. an increase in body temperature leads to an increased oxygen consumption in brain tissue, and aggravating cerebral hypoxia, which causes cerebral edema and nerve cell damage (38) . immediate measures must be taken to control hyperpyrexia by combining the use of artificial hibernation to decrease the temperature, lower metabolism and protect the central nervous system. the drug of choice is the application of cold compressors of chlorpromazine to the head, wearing ice caps and decreasing the temperature with drugs, thereby reducing hyperpyrexia-induced damage to brain tissue. for children patients in the study, when intracranial pressure increased, the motor cortex wass stimulated, causing convulsions and exacerbating cerebral hypoxia and brain edema, thereby aggravating intracranial pressure. the ward was kept quiet and visitors were avoided. treatment and care were performed simultaneously, as much as possible, and operating and any negative stimuli were reduced to prevent convulsions. seizures required the focus of intensive nursing, a dental pad was placed between the upper and lower teeth to prevent tongue bite, and extra gear was added to prevent any fall damage during sleep. drugs, such as phenobarbital and diazepam were administered to control fear. restless children were guarded by specially-assigned persons to prevent scratches and fall damage. heads of children in a coma were leaned to one side, to facilitate the discharge of secretions. should be given. for any child having a seizure, it was considered improper for the fright-checking agent to forcibly press the child's body, in order not to cause fractures. treatment of abdominal distention. abdominal distention often occurs in children with pneumonia, leading to children feeling uncomfortable and unrest, which hinders normal breathing. a hot water bag was placed on the abdomen, to facilitate venting. anal venting was also used and children became quiet after venting. if abdominal distention did not improve, it suggested the presence of toxic intestinal paralysis, and a poor prognosis. anti-infective therapy. after a clear diagnosis of severe pneumonia, collecting blood for bacterial culture and sensitivity test was performed prior to the use of antibiotics when conditions permitted. a separate venous access for antibiotics was established as much as possible, to rationalize the use of antimicrobial drugs and avoid side effects. in principle, the application of antimicrobial drugs was carried out on the basis of evidence-based medicine, but a majority of them are classified as empirical treatment. as the therapeutic window of children with severe pneumonia was very small, the initial drugs for treatment covered an antibacterial spectrum as broad as possible, as well as all the pathogens. there were sufficient data displaying that the inappropriate choice of anti-infective drugs for initial therapy and untimely appropriate treatment (effective therapy of antigens) had adverse consequences on the prognosis. the main indicators of efficacy were a decrease in body temperature, improvement in poisoning symptoms and ability to drink water or conduct breast-feeding or food intake. in general, 3-5 days after the body temperature became stable, the drug dosage was reduced and gradually discontinued. intravenous injection of immune globulin is considered a safe and effective method for the treatment of children with severe pneumonia. it can rapidly improve immunoglobulin g (igg) levels in the patient's blood; enhance the body's resistance to infection and immune function. by passively accepting lgg, the body acquires resistance to a variety of microbial infections. high-dose intravenous igg infusion can increase the igg concentration 3-to 6-fold in circulating blood compared to a normal person. therefore it has the ability to prevent infections. using igg infusion concurrently with antibiotics can be used to treat bacterial infections and has a broad anti-bacterial and viral spectrum, as well as a dual function of anti-bacterial antigens and viral antigens. in summary, positive and effective nursing can promote the rehabilitation of children patients, reduce the incidence of complications and children's mortality, thus play an important role in the rehabilitation of children with severe pneumonia. psychological counseling may also be strengthened. children feel a sense of inadaptation with regard to unfamiliar environment by instinct. they cried and even refused infusion in the face of examination and treatment by strangers. therefore, nurses must be careful and gentle to children patients. for older children, they were able to explain to them the importance of the infusion and blood tests for the treatment of the disease, and increase their sense of trust in the medical staff. health education is also essential, nurses should communicate more with parents and acquaint them with relevant knowledge and inform them regarding the prevention and treatment of diseases, such as that children usually need to do exercise, enhance nutrition, bask more in sunshine and engage in outdoor activities, ensure adequate sleep, pay attention to personal health, and get vaccinated for the prevention of pneumonia and influenza if necessary. global burden of childhood pneumonia and diarrhoea epidemiology and etiology of childhood pneumonia in 2010: estimates of incidence, severe morbidity, mortality, underlying risk factors and causative pathogens for 192 countries epidemiology and etiology of childhood pneumonia incidence and risk factors of childhood pneumonia-like episodes in biliran island, philippines -a community-based study global estimate of the incidence of clinical pneumonia among children under five years of age severe pneumonia caused by nocardia farcinica and complicated by staphylococcus haemoliticus superinfection trimethoprim-sulfamethoxazole-associated severe hypoglycaemia: a sulfonylurea-like effect in-hospital mortality due to infectious disease in an internal medicine department. epidemiology and risk factors people's medical publishing house respiratory rate and signs in roentgenographically confirmed pneumonia among children in china viral and mycoplasma pneumoniae community-acquired pneumonia and novel clinical outcome evaluation in ambulatory adult patients in china effect of corticosteroids on treatment failure among hospitalized patients with severe community-acquired pneumonia and high inflammatory response: a randomized clinical trial etiology of severe community-acquired pneumonia during the 2013 hajj-part of the mers-cov surveillance program management of severe community-acquired pneumonia: a survey on the attitudes of 468 physicians in iberia and south america severe pneumonia due to cytomegalovirus in chronic obstructive pulmonary disease poor adherence to the world health organization guidelines of treatment of severe pneumonia in children at khartoum, sudan childhood anemia at high altitude: risk factors for poor outcomes in severe pneumonia zinc as an adjunct to the treatment of severe pneumonia in ecuadorian children: a randomized controlled trial severe pneumonia in intensive care: cause, diagnosis, treatment and management: a review of the literature levels of serum brain natriuretic peptide in children with congestive heart failure or with severe pneumonia two severe cases of h7n9 pneumonia patients with immunoneuroendocrine axis dysfunction and vitamin d insufficiency severe forms of acute pneumonia with a protracted course accompanied by liver and intestinal dysfunction and dysbacteriosis in young children association of bacterial pneumonia and respiratory failure in children with community-acquired influenza infection chronic obstructive pulmonary disease severity is associated with severe pneumonia childhood very severe pneumonia and meningitis-related hospitalization and death in yemen, before and after introduction of h. influenzae type b (hib) vaccine severe pneumonia mortality in elderly patients is associated with downregulation of toll-like receptors 2 and 4 on monocytes hospital-acquired pneumonia is an independent predictor of poor global outcome in severe traumatic brain injury up to 5 years after discharge the management of severe community acquired pneumonia in the intensive care unit effect of semirecumbent sleep position on severity of obstructive sleep apnea in patients with heart failure oxygen-driving and atomized mucosolvan inhalation combined with holistic nursing in the treatment of children severe bronchial pneumonia atomization inhalation of ambroxol as an auxiliary therapy for severe pneumonia in neonates high incidence of pulmonary tuberculosis in children admitted with severe pneumonia in uganda moderate-dose glucocorticoids as salvage therapy for severe pneumonia in renal transplant recipients: a single-center feasibility study noninvasive ventilation in children: a review milrinone combined with dopamine for child pneumonia complicated with heart failure experimental study of relation of fever to cerebral edema key: cord-016882-c9ts2g7w authors: ribeiro, edna; leitão, céu; cristovam, elisabete; dias, ana title: viruses present indoors and analyses approaches date: 2017-06-12 journal: exposure to microbiological agents in indoor and occupational environments doi: 10.1007/978-3-319-61688-9_7 sha: doc_id: 16882 cord_uid: c9ts2g7w through human history viruses have shown enormous epidemiological and pandemic potential as the occurrence and spread of viruses in pandemic dimensions poses a threat to the health and lives of seven billion people worldwide. scientific evidence has associated harmful health effects to indoor air hazards recognizing the existence of a vital concern in public health sector. thus the assessment of human exposure to biological aerosols and droplets indoor became an imperative requirement of investigation. environmental bioburden assessment of viruses relies in both culture-dependent approaches that comprise classical methodologies, still prominent and vital in the field of modern biotechnology, and culture-independent approaches based on nucleic acid amplification techniques, which are considered the gold standard in clinical virology. the main factor influencing indoor microbiology is the human being and their activities. indoor environments to be considered are those regularly occupied by humans: residences, offices, schools, industrial buildings, health care facilities, farming activities and other settings occupied all the time, or in which occupant density is high. it’s well known that approximately 60% of total human respiratory and gastrointestinal infections are acquired indoor, since viruses have a rapid spread in the community and can be transmitted easily, especially in crowded and poorly ventilated environments, causing high morbidity and decline in quality of life and productivity. studies have shown that respiratory syncytial virus, rhinovirus, metapneumovirus, influenza and parainfluenza virus, and human enterovirus infections may be associated with virus-induced asthma, leading to diseases such as pneumonia. gastroenteritis infectious (about 30±40% of cases) is attributable to viruses. rotavirus, astrovirus, norwalk-like viruses and other caliciviruses are responsible for 48% of all reported outbreaks of infectious intestinal disease. safe working conditions are essential for healthy living, that’s why the programmes conceived as a result of strategic and preventive policy maintenance, in refrigeration and ventilation systems, are the determining factor for the control of biological pollutants. moreover, the development of highly sensitive and specific detection and identification methodologies with capacity to be used in diverse applications, such as diagnosis, public health risk assessment, research and for the implementation of preventive measures and protocols are imperative. indoor air pollution is a major global public health threat requiring increased hard work, linking research to policy-making. the evidence of the effects of physical and chemical pollutants on human health, present in the external and internal environment has no superior aggression than the existing bioaerosols. health effects from indoor air pollutants may be experienced soon after exposure or, possibly, years later, according to the nature of air contaminants, being classified, in an abbreviated approach, as physical, chemical or biological (nazaroff, 2016) . several studies have related hostile health effects to indoor air hazards and adequate assessment of human exposure to biological aerosols has been recognized as an imperative requirement and a very important concern in the area of public health (douwes et al., 2003) . scientific evidence has showed that the air within buildings can be more seriously polluted than the outdoor air, even in developed and industrialized countries, and it is well known that indoor environments occupied by humans, contain abundant material of microbial origin, consequently, the risks to human health may be greater due to exposure to air pollution indoors (fisk et al., 2007) . on the other hand, investigation indicates that people spend approximately 90% of their time indoors, with mechanical heating, cooling and ventilation systems, influencing irrefutably the quality of life (klepeis et al., 2001; nazaroff, 2016) . complex syndromes arise associated with indoor air quality, such as sick building syndrome, building-related disease leading to loss of productivity and absence at work. these syndromes are associated with increased incidence and prevalence of asthma and other chronic diseases worldwide. exposure to bioaerosol material can cause or can contribute to several relevant diseases. human occupancy and activities are major factors influencing indoor microbiology. humans are important primary sources of certain bacteria and viruses. the recent development of quantitative polymerase chain reaction (pcr) and other rna/dna-based measurement technologies has permitted studies that measure pathogenic material in indoor air (lax et al., 2014; nazaroff, 2016) . droplet transmission is not to be confused with airborne transmission. aerosols are suspensions in air (or in a gas) of solid or liquid particles, small enough to remain airborne for a prolonged period of time because of their low settling velocity. droplets do not remain suspended in the air. airborne transmission depends on viruses from evaporated droplets or dust particles that can remain suspended in the air for long periods. droplet transmission occurs when viruses travel on relatively large respiratory droplets (>10 μm) that people sneeze, cough, or exhale during conversation or breathing (primary aerosolization) (la rosa et al., 2013) . respiratory droplets initially all move forward with the exhaled air jet; very large droplets leave the jet quickly and fall on the ground and small droplets completely desiccate within the jet (tellier, 2009) . the transport and the settling of a bioaerosol are affected by its physical properties and the environmental parameters that it encounters. size, density and shape of droplets or particles, air currents, relative humidity and temperature, determine the capacity of generation of airborne bioaerosols from liquid suspensions, undergo desiccation, whereas those generated as dusts or powders partially rehydrate. the presence of moulds indicates a problem with water penetration or high humidity. bioaerosols can be transmitted at long distances. small particle aerosols, as shown during endotracheal intubation, are transmitted to persons in the immediate area near the patient. viruses' inductors of severe acute respiratory syndrome (sars), influenza and norovirus are transmitted from patients primarily by contact and/or droplet routes, while airborne transmission occurs over a limited distance (srikanth et al., 2008) . indoor-outdoor air exchange (mechanical ventilation), penetration (air filter), deposition, sources and aerosol resuspension, are extremely relevant for spread contamination. once aerosolized, aerosols viruses' particles may travel significant distances through buildings before being captured and retained by hvac filters, or they pass through as well, because most hvac filters are not 100% efficient in capturing particles. the rapidity with which airborne viruses are inactivated during transport or after filter capture is uncertain and merits additional study. analysis of ventilation filters certainly could play a role in the epidemiology of infectious diseases caused by pathogens released into the environment, suiting all the different situations of confinement (goyal et al., 2011) . the main source of indoor viruses is the human being. viruses are spread by air currents after resuspension of material scattered by aerosols droplets or saliva. we can say that viral infections are probably the most common acquired diseases indoor that affect man, knowing approximately a thousand types of different viruses involved. it is estimated about 60% of total human respiratory and gastrointestinal infections, with a rapid spread in the community, being a cause of high morbidity and decline in quality of life and productivity, since viruses can be easily transmitted, especially in crowded and poorly ventilated environments. it is well-known that viruses are shed in large numbers, with transmission routes extraordinary diverse, including direct contact with infected persons, faecal-oral transmission (through contaminated food and water), droplet and airborne transmission, and can survive for long periods on surfaces or fomites, emphasizing the possible role of surfaces in the transmission of viruses (barker et al., 2001; la rosa et al., 2013) . for instance, faeces can contain up to 10 12 viruses particles per gram and vomit up to 10 7 per millilitre, so the potential transfer contamination from hands to surfaces is frighteningly considerable. the most important source of potentially pathogenic viral aerosol is other humans and other means, as the flushing of a toilet that can aerosolize significant concentrations of airborne viruses. viruses' survival on fomites is influenced by temperature, humidity, ph and exposure to ultraviolet light. particle size, depth of penetration and the minimum dosage of the agent capable of causing disease are implicated in the infectivity. in addition, it is also important to be conscious of the risk groups, the most susceptible to contracting infection when exposed to microorganisms, conditioned by factors such as a weakened immune system, the children, the elderly, the pregnant women, the chronically ill, especially those suffering from respiratory or cardiovascular disease. chronic obstructive pulmonary disease (copd) and acute exacerbations are frequent complications, thought to be caused by interactions between host factors, bacteria, viruses and changes in air quality producing increased inflammation in the lower airway with a long-lasting adverse influence on health status (celli and macnee, 2004; celli and barnes, 2007) . approximately of 50% of acute exacerbations of copd are associated with symptoms of viral infections of the respiratory tract by rhinovirus, respiratory syncytial virus and influenza. studies have shown that respiratory syncytial virus (rsv), human rhinovirus (hrv), human metapneumovirus (hmpv), influenza and parainfluenza virus (hpiv), and human enterovirus infections may be associated with virus-induced asthma, leading to diseases such as pneumonia or death (tsukagoshi et al., 2013) . respiratory and enteric viruses are opportunistic pathogens transmitted mainly via other routes are able to spread via droplet nuclei or dust in certain circumstances (la rosa et al., 2013) . numerous studies identify the factors that are involved in the transmission of infection by aerosol indoor with the correlation between the pulmonary mechanism, the different human activities and the critical concentration of particles expelled. a virus (h3n2) had a higher transmissibility and uncontrollable potential than the a (h1n1) and b viruses (chen et al., 2009; chen and liao, 2010) . a major cause of morbidity and mortality, leading to diseases such as bronchiolitis, asthma and pneumonia (tsukagoshi et al., 2013; paba et al., 2014) . despite all the progress of the last decades in the prevention and care of health, respiratory infections represent one of the major causes of disease in humans. this group remains the leading cause of outpatient consultation as well as antibiotic prescription and work absence. the chronic infections, causing disability, have the most important impact on quality of life. the main effect of the inadequate quality of indoor air is in respiratory system. most studies focus on the influenza virus, but in general, we can say that a total concentration of viruses, in a variety of environments such as classrooms, health institutions, restaurants, offices and others corresponds to an average 4.7 ± 2.5 × 10 5 particles per cubic meter, without significant differences between the different indoor environments. also, is estimated that the viral particles inhaled daily indoor correspond approximately to 5 × 10 6 . respiratory viruses can be transported over considerable distances by air currents and be inhaled, penetrating deep into the respiratory system (prussin ii et al., 2015) . droplet transmission occurs when viruses travel on relatively large respiratory droplets (>10 μm) that people sneeze, cough, or exhale during conversation or breathing, primary aerosolization. a single cough can release hundreds of droplets, up to 40,000, at speeds of up to 50-200 miles per hour, each droplet containing millions of viral particles. aerosol droplets travel only short distances (1-2 m) before settlings on surfaces, where viruses can remain infectious for hours or days. secondary aerosolization can occur when air displacements disperse the viruses back into the air from contaminated surfaces (la rosa et al., 2013) . transmission occurs through air droplets, aerosol and fomites that may come into contact with nasal and conjunctival epithelium. the etiological viral agents involved include: influenza virus types a and b, parainfluenza viruses types 1, 2, 3, and 4, respiratory syncytial virus, adenovirus, and rhinoviruses/enteroviruses (paba et al., 2014) . the acute respiratory infections are the result of active multiplication of microbiological agents in the respiratory system when favourable conditions of the host exist. viral or bacterial ethology, predisposing factors related to anatomical factors, immune changes, colonization of the naso and oropharynx, and the spread of these infections is favoured by the continuity of the epithelia of the respiratory system and a continuum between upper and lower airways. the upper and lower respiratory infections are in most cases (60%) of viral origin. adenovirus (type 4), the first virus to be isolated from indoor aerosol, was identified in 1966 in aerosol samples from the quarters of military recruits infected with acute respiratory disease (artenstein and miller, 1966) . since then, human infections due to viral aerosol (or contact with contaminated surfaces) have been studied in various environments, including office building, hospitals, restaurants, transport systems and schools. in the last few years, other respiratory viruses have been discovered and linked to the upper and lower respiratory tract infections: human metapneumovirus, sars coronavirus, hku1 coronavirus, nl63 coronavirus, mers coronavirus and bocavirus. in 2007, two novel human polyomaviruses named kipyv and wupyv were discovered in the respiratory secretions of patients with acute respiratory symptoms. the outbreak of the influenza a virus (h1n1) infection in 2009 has reminded us again of the importance of monitoring and controlling airborne microorganisms in public facilities (lee et al., 2012) . unlike conventional viral cell cultures, with the introduction of the real-time pcr assay, the diagnosis of respiratory infections improved greatly. it is possible to search for up to 21 different respiratory pathogens including viruses and bacteria. in addition, it is possible to detect co-infections that may have implications on disease severity or therapeutic strategies (paba et al., 2014) . for bioaerosol particles, maybe the most important exposure pathway is inhalation followed by deposition in the respiratory tract. the probability of deposition varies with particle size, with lung morphology, and with breathing characteristics. airborne exposure indoors makes a meaningful contribution to the occurrence or spread of disease. rhinovirus (rv) is a small rna virus belonging to the picornaviridae family. more than 100 immunologically distinct serotypes have been identified and new serotypes are continuously emerging. this virus is responsible for more than 50% of cases of common cold, being the virus with the highest morbidity among respiratory diseases. in children causes bronchitis. although the method of transmission of rvs is in doubt, they are thought to be mainly transmitted via large droplets, but indirect contact with contaminated fomites has also shown to transmit infection or by aerosols. it's known that rv need only 10 s of contact (hand-to-hand) for infecting another person. the recurrence of viral infections is predominantly associated with an immune response to a serotype that does not provide immunity to reinfection with another serotype for the same virus (ballow, 2008) . rhinoviruses can survive on environmental surfaces for several hours. infectious viruses have been recovered from naturally contaminated objects in the surroundings of persons with rv colds. aerosols are generated by coughing, talking, sneezing and even simply breathing (huynh et al., 2008) . rhinovirus outbreaks in health care facilities, capable of determining severe infections and also death have been documented (macintyre et al., 2012) . rvs have also been detected in transport vehicles (la rosa et al., 2013) . even in the common cold, rhinoviruses and coronaviruses predominate (barker et al., 2001; heikkinen et al., 2003) , but the role of rhinoviruses is the most prominent, in particular are responsible for outbreaks of the common cold in the general community such as schools, day care centres and hospitals (barker et al., 2001; heikkinen et al., 2003; la rosa et al., 2013) . rhinoviruses and coronaviruses also cause a greater disease burden in elderly people living at home, compared with influenza or syncytial viruses. the infections by rhinovirus may be triggering factors of exacerbations of asthma, probably because they induce inflammation in airways that are already damaged and sensitized. rhinoviruses were detected by rt-pcr among infants (von mutius, 2004) , in the acute exacerbation of asthma when infection was prolonged (tsukagoshi et al., 2013) . some authors describe that bronchial epithelial patient cells with asthma are more effectively infected with rv than normal epithelia. it was shown that viral are upregulated in the epithelial cells of patients with allergies (canonica et al., 1995; tantilipikorn and auewarakul, 2011) . respiratory syncytial virus (rsv) is a single-stranded rna virus belonging to paramixoviridae family. rsv infections occur all over the world and outbreaks are common in the cold season in temperate climates and in the rainy season in tropical climates. is one of the most common viruses which can cause a type of potentially lethal pneumonia in older people and is a major cause of respiratory illness in young children producing bronchitis and pneumonia worldwide, affecting about 90% of children by the age of 2 years (24), inducting a preparative stage in the development of asthma (tsukagoshi et al., 2013) . school-aged children often carry rsv to their homes and extent infection to younger brothers as well as when admitted to hospital, tend to shed the virus abundantly and for prolonged periods permitting sufficient opportunity for spread in adults. sometimes this virus produces a flu-like syndrome indistinguishable from influenza (barker et al., 2001) . rsv is highly contagious and transmission can occur when infectious material comes into contact with mucous membranes of the eyes, mouth or nose, and possibly through the inhalation of droplets generated by a sneeze or cough. also result from contact with contaminated environmental surfaces, the commonest mode of transmission in school classrooms and day care centres. transmission with fomites predominates over droplet contact can be transmitted by the airborne route. rsv rna is detected in air samples from the hospital rooms of infected patients at large distances from the patient's bedside. particles containing rsv rna were detected in airborne throughout a health care facility, particles small enough to remain in the air for an extended period and to be inhaled deeply into the respiratory tract (6). evidence shows that direct and indirect contact is a key factor in transmission hands, touching surfaces contaminated with fresh secretions from rsv-infected infants (barker et al., 2001) . influenza virus (iv) is one of the most common and highly contagious infectious diseases and can occur in people of any age. influenza or flu produces an acute infection of the respiratory system with high transmissibility and global distribution. is a rna virus belonging to the orthomixoviridae family which is subdivided into three distinct antigenic serotypes: a, b and c, causing moderate to severe acute febrile illness, resulting in variable degrees of systemic symptoms, ranging from mild fatigue to respiratory failure and death. strains a and b have greatest potential epidemic, causing more versions of the flu. the flu vaccine only protects against viruses a and b. influenza c viruses are antigenically stable, cause subclinical disease and do not cause epidemics. the influenza a and b have multiple subtypes that still suffer mutations, emerging new strains with consequent increased risk of epidemics or pandemics. the continuing threat of pandemics by the human influenza virus suggests an urgent and constant surveillance. despite the vaccination continues to be considered the intervention of containment of infection, the viruses a (h1n1), a (h3n2) and influenza b persist with a global distribution and with such a power of infectivity that influenza activity management has been undertaken by the centres for diseases control. influenza a (h3n2) has a uncontrolled and potential infectivity demonstrating a much greater transmissibility than a (h1n1) and b viruses (chen and liao, 2010) . influenza affects all age groups, but it is the elderly and persons with underlying health problems who are at particular risk from complications of influenza and are more likely to require hospitalization. both influenza virus a and b revealed to survive on hard surfaces such as stainless steel and plastic for 24 ± 48 h and on absorbent surfaces such as cloth, paper and tissues for up to 12 h (barker et al., 2001) . asymptomatic patients shed virus and can transmit the disease, thus creating a reservoir for the virus. influenza virus is transmitted by droplets, through the coughing and sneezing of infected persons, but it can also be transmitted by airborne droplet nuclei as well as by contact, either through direct skin-to-skin contact or through indirect contact with contaminated environments (la rosa et al., 2013) . influenza viruses have been detected in different indoor environments, homes, schools, offices and others public buildings. places such as hospitals, where the presence of a susceptible population is often combined with a high population density, may harbour high concentrations of pathogens and therefore pose a considerable risk for the transmission of the virus, with potentially fatal consequences for hospitalized patients. schools are known to have an important role in influenza transmission in the community since children have a higher influenza attack rate than adults (children get the flu twice as often as adults) (zhao et al., 2007) . on buildings ventilation systems influenza a and b were detected (along with other groups of viruses), meaning that contamination exists in the surrounding environment. several reviews consider three modes of transmission of influenza, not mutually exclusive, by large droplets, self-inoculation of the nasal mucosa by contaminated hands and the aerosol transmission, the mode of the greatest impact for infection, since it requires specialized personal protective equipment. influenza virus rna was directly detected in aerosol particles generated by normal breathing in patients with influenza and collected through an orinasal facemask; particles of 5 mm or less have a significant penetration into the respiratory tract all the way to the alveolar region. increasing evidences point towards a role for aerosol transmission in the spread of influenza, at least over short distance where exposure to both aerosol and large droplets occurs. in most settings where there is adequate ventilation, long-range transmission does not appear to occur so frequently (tellier, 2009) . the relative importance of the aerosol transmission route for influenza remains controversial. to determine the potential for influenza to spread via the aerosol route, the authors 6x measured the size distribution of airborne influenza a virus. collected size-segregated aerosol samples during the 2009-2010 flu season in a health centre, a day-care facility and on board of aeroplanes. filter extracts were analysed using quantitative reverse transcriptase polymerase chain reaction. half of the 16 samples were positive, and their total virus concentrations ranged from 5800 to 37,000 genome copies m23. on average, 64% of the viral genome copies were associated with fine particles smaller than 2.5 mm, which can remain suspended for hours. modelling of virus concentrations indoors suggested source strength of 1.6 + 1.2 × 10 5 genome copies m23 air h21 and a deposition flux onto surfaces of 13 + 7 genome copies m22 h21 by brownian motion. over 1 h, the inhalation dose was estimated to be 30 +18 median tissue culture infectious dose (tcid50), adequate to induce infection. these results provide quantitative support for the idea that the aerosol route can be an important mode of influenza transmission (yang et al., 2011) . parainfluenza viruses (pivs) are a further major group of respiratory pathogens. they cause severe colds, croup, bronchitis and pneumonia in children and adults and in infants the virus can cause life-threatening disease. infection is probably spread by aerosols in addition to direct contact with contaminated surfaces. the persistence of piv on hospital surfaces contaminated with patients' secretions was noted as a potential source of transmission (barker et al., 2001) . goals for epidemiological surveillance of influenza include supervise the currently circulating virus subtypes and offer quick response to spread of new subtypes; follow the tendency of the morbidity and mortality to plan strategies to reduce the burden of the disease in public health; define strategies to reduce the occurrence of deaths; monitor the severity standard of the disease by detecting any virulence changes. severe acute respiratory syndrome (sars) is a respiratory illness caused by a type of coronavirus from the coronaviridae family, which can cause mild to moderate upper respiratory illness, such as the common cold or develop into potentially severe pneumonia. this virus is known as sars-cov. the sars epidemic broke out in 2002-2003 in southern china, and spread to other regions of asia and also to europe and north america. it caused more than 8000 infections worldwide with an approximately 10% fatality rate, along with enormous economic losses. one or a few trivial mutations at the receptor-binding surface of a virus may lead to dramatic epidemic outcomes by facilitating cross-species infections and human-to-human transmission of the virus (li, 2013). sars is a condition associated with substantial morbidity and mortality with patterns suggesting droplet or contact transmission (poutanen et al., 2003) . the earliest symptom is a sudden onset of high fever. some patients may also have chills and headaches followed by pneumonia; others showed respiratory distress (severe breathing difficulty) and sometimes death (la rosa et al., 2013) . the most common mode of transmission is contamination by warm air indoor, by water droplets generated by coughs or sneezes, but may be transmitted through the airborne route as well. transmission in an aircraft from an infected person to passengers located seven rows of seats ahead had been described. aerosol generated by the building's sewage systems is also responsible. many health care workers were infected after endotracheal intubation and bronchoscopy procedures which often involve aerosolization. these observations indicate the possible role of more remote modes of transmission, including airborne spread by small droplet nuclei, and emphasize the need for adequate respiratory protection in addition to strict contact and droplet precautions when managing sars patients. contaminated fomites or hospital surfaces might contribute to spread. it is known that these viruses may live on hands, tissues and other surfaces for up to 6 h and up to 3 h after droplets have dried. airborne spread of the virus appears to explain the happened large community outbreaks of sars (ignatius et al., 2004; hui and chan, 2010; la rosa et al., 2013) . a novel coronavirus, mers-cov (ncov, hcov-emc/2012), originating from the middle-east, has been discovered. incoming data reveal that the virus is highly virulent to humans. the members of this group (c) are likely to persist in the environment for a longer period of time and possess the highest oral-fecal components but relatively low respiratory transmission components. oral-urine and saliva transmission are also highly possible (goh et al., 2013) . human adenovirus (adv) is a non-enveloped, icosahedral virus of the genus mastadenovirus, family adenoviridae. there are more than 60 types classified into seven strains from a to g, defined via biological and molecular characteristics. clinical manifestations are highly heterogeneous, ranging from upper and lower respiratory tract infections to gastroenteritis, pneumonia, urinary tract infection, conjunctivitis, hepatitis, myocarditis and encephalitis. the burden of disease manifests as pneumonia, bronchiolitis, otitis media, conjunctivitis, and tonsillitis. the adenoviral detection rates indicate the potential contamination of the environment, with adverse effects on public health. adenoviruses can cause severe or lifethreatening illness, particularly in immunocompromised patients, children and the elderly. some types are capable of establishing persistent asymptomatic infections in tonsils, adenoids, and intestines of infected hosts, and shedding can occur for months or years (lessa et al., 2009; osuolale and okoh, 2015) . adenoviruses can occur anytime throughout the year but adenoviral respiratory infections are most common in the late winter, spring, and early summer. modes of transmission are also diverse, primarily spread by the respiratory route, person-to-person contact, fomites, and occasionally by airborne aerosols. since advs are able to infect a wide range of tissues, they can be excreted in large numbers in different body fluids during the acute illness, including oral secretions and faeces. the spread by the fecal-oral route happens through the ingestion of contaminated food or water. small doses of adv in aerosols resulted in infection accompanied by febrile acute respiratory disease, sometimes with pneumonia. humidity affects the viability and dispersal of advs in aerosol. these viruses tend to survive best at high relative humidities. aerosols seem to be very resistant to uv air disinfection. adenovirus outbreaks have been documented in different indoor environments, including health care facilities, schools, military hospitals and barracks, throughout the year (la rosa et al., 2013) . in developed countries, it is estimated that 30% ± 40% of infectious gastroenteritis cases are attributable to viruses. rotavirus, astrovirus, norwalk-like viruses, also known as small round structured viruses and other caliciviruses are responsible for 48% of all reported outbreaks of infectious intestinal disease. rotaviruses belong to the reoviridae family; they are segmented bicatenary rna viruses, which explain their genetic variability. is the most common cause of severe diarrhea among children, resulting in the death of over 500,000 children annually worldwide. rotaviral gastroenteritis is a serious public health problem in both developed and developing countries. the disease occurs most often in the winter, with annual epidemics occurring from december to june. the highest rates of illness occur among infants and young children. a large proportion of hospital admissions due to gastroenteritis in children less than 5 years old are caused by rotavirus. some adults acquired rotavirus infections a few days after their children's illnesses, suggesting that the children rather than the parents brought infection into the home, though disease tends to be mild. immunity after infection is incomplete, but repeated infections tend to be less severe than the original infection. rotavirus is shed in large numbers from an infected person by animate and non-porous inanimate surfaces. excretion of the virus can persist for up to 57 days after diarrhea has stopped in symptomatic patients, contributing to an increase of the number of environmental surfaces contaminated with rotavirus. it has been suggested that low humidity and people spending more time indoors contribute to the spread of rotavirus infections. handwashing is a very important means of preventing the spread of rotavirus (barker et al., 2001; anderson et al., 2004; bernstein, 2009) . norwalk-like viruses (nlvs), rna virus belonging to caliciviridae family, also known as small round structured viruses or caliciviruses, are an important cause of gastroenteritis outbreaks and are spread frequently through contaminated food or water. projectile vomiting associated with nlvs is probably a major source of cross-infection because it is estimated that 3×107 particles are distributed as an aerosol into the environment during a vomiting attack. carpets can also serve as reservoirs of infection. aerosols produced by vomiting can be inhaled or can contaminate hands or work surfaces, with the potential for subsequent transfer to foods or direct handto-mouth transfer. the importance of airborne transmission was demonstrated in a recent outbreak in a restaurant where no food source was detected but an analysis of the attack rate showed an inverse correlation with the distance from a person who had vomited. it was found that the risk of gastroenteritis amongst workers and customers who shared toilet facilities was twice of those who had a private bathroom (green et al., 2000; barker et al., 2001) . enteroviruses (evs) are members of the picornaviridae family, a large and diverse group of small rna viruses present worldwide. in humans, evs target a variety of different organs causing gastrointestinal, respiratory and myocardial and central nervous system diseases. in temperate climates, enteroviral infection occurs primarily in the summer and early fall. although the majority of infections are asymptomatic or result in a self-limited illness, fatalities do occur, especially in neonates or individuals with b-cell immunodeficiency. enterovirus outbreaks in neonatal units and school nurseries, reflects the susceptibility of infants to evs infection, leading to extensive discussion on control measures and interventions. faecal-oral transmission is the major mode of transmission. other important routes of transmission are person-to-person contact and the inhalation of airborne viruses in respiratory droplets. infectious coxsackievirus, a member of the ev genus, in large droplets and droplet nuclei generated by coughs and sneezes as well as in the air of rooms contaminated by such discharges, transmit this viral infection by the airborne route. aerosol transmission is suspected of having contributed significantly to the ev epidemic which infected up to 300,000 children and caused 78 deaths in taiwan in 1998 (chang et al., 2004; la rosa et al., 2013) . noroviruses (novs), emerging as the leading cause of epidemic gastroenteritis, are rna viruses belonging to the family caliciviridae, currently subdivided into five genogroups. novs are responsible for nearly half of all gastroenteritis cases and for more than 90% of non-bacterial infection epidemics worldwide. the illness can be severe and sometimes fatal, especially among vulnerable populationsyoung children, the elderly and the immunocompromisedand is a common cause of hospitalization. encephalopathy, disseminated intravascular coagulation, convulsions, necrotizing enterocolitis, post-infectious irritable bowel syndrome, and infantile seizures highly contagious with a low infectious dose, occurs repeatedly. faecal-oral spread is the primary transmission mode and the foodborne and waterborne transmission. airborne transmission of nov is also a cause of acute viral gastroenteritis. sources of contaminated aerosol are diverse. droplets being inhaled can be deposited in the upper respiratory tract, and subsequently be swallowed along with respiratory mucus. aerosol droplets produced during vomiting could settle onto indoor surfaces that might then be transferred to hands of exposed individuals through physical contact, or deposited on the floor from which they can be resuspended by human movement and turbulence. aerosol droplets can also be generated from toilet flushing. transmission via fomites is documented. the viruses were identified in indoor environments such as hospitals, schools, kindergartens, restaurants, care facilities, hotels and concert halls as well as airplanes, buses and cruise ships (morillo and timenetsky mdo, 2011; la rosa et al., 2013) . human adenoviruses (adv) are classified into 47 serotypes and six subgenera (a-f) with different tropisms are associated with outbreaks of gastroenteritis in schools, paediatric hospital and nursing homes. they may be second to rotavirus as a cause of gastroenteritis in young children, especially newborns, mainly caused by serotypes adv 40 and adv 41 of subgenus f. the clinical characteristics include watery diarrhea accompanied by vomiting, low grade fever and mild dehydration. institutionalized persons, immunocompromised persons and transplant recipients seem to be among the most severely affected, with mortality rates as high as 60%. respiratory symptoms are infrequent but some studies have suggested that adenovirus infections may be involved with chronic airway obstruction, pulmonary dysplasia, myocarditis and dilated cardiomyopathy, mononucleosis-like syndromes, sudden infant perinatal death and, perhaps most intriguingly, the development of obesity. although these associations may or may not be causal, understanding adenovirus transmission seems to be the key to their further study. adenoviruses are the most uv-resistant viruses, and their detection is now a key indicator of water quality (uhnoo et al., 1990; zlateva et al., 2005; gray, 2006) . indoor air quality proves to be of great importance in hospitals due to the spread of air microorganisms maximizing nosocomial infections. reports about infections correlated with the presence of viral aerosols in indoor air remain scarce. during and after illness, viruses are shed in large numbers in body secretions, including blood, faeces, urine, saliva, and nasal fluid (la rosa et al., 2013) . the microbial load in hospital indoor air is highline nuanced by the number of occupants, their activity and the ventilation. occupants are a potential source of microorganisms as they shed the microorganisms from the skin squamous and the respiratory tract. ventilation causes dilution thus reducing the microbial load. sinks, wash-basins and drains, nebulisers, humidiphiers, and cooling towers are the potential sources which colonize on the moist surfaces. sweeping of floors, changing of bed linens and entry into the hospital buildings through ventilation ducts also can be the sources of airborne microorganisms. since exposure levels are high, this may be an issue for immunocompromised patients (srikanth et al., 2008) . during the 2009-2010 flu seasons, size-resolved particle samples were collected on filters in a day care center and a health center. influenza a virus was identified in 50% of the samples with concentrations ranging from 5800 to 37,000 genomes per cubic meter and a substantial proportion of the detected viruses was associated with fine particles (<2.5 μm) that can remain airborne for extended periods and that can also penetrate and deposit deeply in the respiratory tract when inhaled (barker et al., 2001; nazaroff, 2016) . establishing how viruses are transmitted under different circumstances, and whether transmission requires close contact, is of great importance as such information will affect the choice of infection control measures in health-care settings (la rosa et al., 2013) . in a hospital paediatric unit when there was an increase in the number of children suffering from rotavirus gastroenteritis, on the surfaces in direct contact with children (thermometers, play mats and toys) rotavirus was detected in 63% of samples compared with 36% for surfaces without direct contact (telephones, door handles and washbasins). rotavirus was also found in hand washings of 19% of attendants of patients with non-rotavirus diarrhoea, indicating that they may have come into contact with other attendants and patients in adjacent beds. this highlights the potential for contaminated hands to spread the infection (soule et al., 1999) . norwalk-like virus gastroenteritis in an elderly care unit spread rapidly within and between wards, affecting both patients and staff. although infectious aerosols were probably the main route of dissemination of infection within a particular cohort of guests, contact with contaminated fomites was the most likely factor responsible for maintaining the outbreak by forming the link between successive cohorts (barker et al., 2001) . farming, one of the oldest professions of mankind, is by far the one that employs the largest number of individuals worldwide. although outdoor country work is supposedly healthy, farmers are at risk of respiratory diseases because of their work environment. it is very well established that chronic and acute respiratory diseases have been associated with work in confinement operations, like underfloor manure pits. respirable dust and asphyxiating gases such as hydrogen sulphide, carbon dioxide, methane, and ammonia in high concentrations were measured on these area stations (reeve et al., 2013; manbeck et al., 2016) . but bioaerosols are recognized as a serious threat on these environments. mice are prevalent on farms. faeces of deer mice can be contaminated with the hantavirus, which can cause a devastating infection in humans, an influenzalike syndrome that often leads to respiratory failure. farmers should wear protective respiratory equipment when cleaning building areas where mouse droppings are present. farms are also the usual sources of influenza outbreaks. influenza viruses infect pigs or poultry that can be transferred from animals to humans even without a mutation. this has so far been the case of the influenza a virus subtype h5n1 that, to date, only infected farmers in close contact with birds (cormier, 2007) . in animal slaughterhouses handling out cattle and sheep is hypothesized that these environments would contain significant amounts of bioaerosol due to the mechanical processes used to kill and process animals, a high degree of splashing and fluid handling, and also a high relative humidity of the environment. workers on these conditions and meatworkers having exposure to a number of significant zoonotic diseases including leptospirosis, parapoxvirus, human papillomavirus subtypes hpv2, hpv4 and hpv7 are known to be at occupational health risk. these workers are also affected by a higher-rate of malignancies of the lung compared to the general population (hall et al., 2013) . farmers and consumers of fresh farm products from farms irrigated with river water may be at risk of infection from adenoviruses. the findings highlight the lurking dangers of using contaminated surface water and the need for routine monitoring of such waters for protection of public health (sibanda and okoh, 2012) . workers in concentrated animal feeding operations are at risk of adverse respiratory outcomes from exposures to indoor contaminants. is indispensable an optimal management of indoor air quality, preventing the transmission of infectious respiratory disease to workers and animals (kim et al., 2005) . considering that viruses are obligate intracellular parasites, the use of culturedependent methods is achieved with the use of suitable hosts, such as whole animals or cultured cells. currently the most commonly used methods for virus cultures are inoculation of viruses into embryonated eggs and tissue cultures. virus started to be propagated in whole animals or embryonated chicken eggs before the use of cell culture methods. virus cultivation in embryonated eggs is intrinsically dependent on the utilized egg, which must be sterile and the shell should be intact and healthy. the inoculation of the samples is made by injection into the fluid of the egg through a hole drilled in the shell. viral growth and multiplication is revealed by embryo death, cell damage or through the formation of typical pocks or lesions on the membranes. the selection of the sites of viral inoculation in embryonated eggs is dependent of the studied virus, as each virus has different preferential location for growth and replication. chorioallantoic membrane (cam): virus growth and replication is indicated by visible lesions (pocks) derived, under optimal conditions, from a single virion and a grey white area in transparent cam. allantoic cavity: usually utilized for growth and replication of virus for vaccine production, provide a rich yield of influenza, some paramyxoviruses and avian viruses isolation. amniotic cavity: virus growth and replication of virus can be analyzed by haemagglutination assay. yolk sac: frequently utilized for growth, multiplication and isolation of mammalian viruses. currently, embryonated egg inoculation is conventionally considered the "gold standard" method for isolation and propagation of virus such as the influenza virus (jianqiang zhang, 2014) . mammalian cell culture technology has become a prominent and fundamental field in modern biotechnology, especially in the area of human health and has replaced embryonated eggs as the preferred methodology for virus growth and replication. cellular cultures rely on techniques such as media changes, passaging, and transfection under aseptic conditions to avoid contaminations (e.g. bacteria, yeast, among others). presently, numerous valuable cell monolayers are commercially available, and are regularly utilized in clinical laboratories for the diagnosis of virus infections. some of the most utilized cell are hela derivative (hep2), rhesus monkey kidney cells (rhmk), human lung fibroblasts (mrc-5), human lung carcinoma cells (a549), among others. these cell lines are selected for their ability to support the replication of a wide variety of clinically relevant viruses due to their ability to express cell type-specific factors that contribute to pathology during viral infection. for instance a549 cell line is considered representative of the alveolar type ii pneumocytes of the human lung as it exhibits features of an atii epithelial cell phenotype. applying such methodology enables the isolation of diverse viruses, such as adenovirus, cmv, rsv, influenza a & b, parainfluenza viruses types 1 to 3, vzv, as well as the ebola virus, severe acute respiratory coronavirus (sars-cov), and human metapneumovirus (hmpv). the recent adaptation of cellular cultures to shell vials with subsequent direct or indirect immunofluorescence technique has dramatically decreased the time of diagnostics for clinical samples from weeks to less than 48 h by staining for early antigens of viral infections. currently, this methodology is the most sensitive, non-molecular viral detection method, utilized for the identification of viruses such as respiratory viruses, enterovirus and adenovirus among others. although embryonated chicken egg inoculation is still considered the "gold standard" method for influenza virus isolation and propagation, several primary cells as well as continuous cell lines have also been developed for influenza virus isolation and replication (jianqiang zhang, 2014). the development of nucleic acid amplification techniques has endorsed the advance of molecular tools for virus identification using low specimen quantity with higher sensitivity and specificity, at the same time dramatically decreasing the time for identification. currently these methodologies are accepted as the gold standard for clinical virology and have been utilized for the identification of different viruses in environmental samples from diverse contexts. the fundamental principles of nucleic acid amplification techniques are based on the thermostable polymerase-based target nucleic acid amplification which results in the production of millions of copies of the targeted sequence. these amplification products are then analyzed through diverse techniques. the advances in molecular analysis techniques lead to the development of the real-time pcr where the target amplification and detection steps, using fluorescent dyes, occur concomitantly. this methodology uses copulated software that monitors the thermal cycler data at every cycle and produces a quantitative amplification plot for each reaction. thus, rt-pcr allows the performance of viral load assays to quantitatively assess the amount of virus in a sample. furthermore, a modification of polymerase chain reaction, nested polymerase chain reaction (nested pcr), has been developed in order to decrease non-specific binding in products. this methodology encompasses two sets of primers, used in two successive pcr runs, whereas the second set intended to amplify a secondary target within the first run product. currently, nucleic acid amplification techniques are considered the gold standard for rapid and accurate detection of viruses, compared to methodologies such as shell vial cell cultures. multiplex pcr assays were developed based on single target nucleic acid amplification methodologies with the aim to quantify multiple nucleic acid targets using specific probes to diverse viral targets in a single pcr reaction, allowing the assessment of viral co-infections. currently, for respiratory viruses three fdaapproved platforms for multiplex pcr assays are available. multiplex pcr combined with liquid-phase bead-based array technology. multiplex rt-pcr followed by electrochemical detection of hybridized capture probes on gold-plated electrodes. multiplex-pcr preceded by nested rt-pcr with detection trough melt curve analysis. currently, dna sequencing is considered one of the most valuable, accurate and consistent methodologies for microorganisms identification and is applied in contexts were quick and precise identifications are required. this method is used to determine the exact sequence of a certain stretch of dna. several new sequencing technologies have been developed (next generation sequencing) with the aim to provide fast/efficient techniques for analysis of microorganisms bioburden. genotyping assays utilize a combination of pcr and nucleic acid sequencing to identify viral genotypes. genotyping methodology allows for the identification of genetic variants and can be performed via genotyping chips or arrays, depending on the variants of interest and resources available. viral genotyping is mostly utilized to provide relevant clinical data to predict therapeutic responses to antiviral drugs and/or epidemiologic comparison. currently, it is a consensus among researchers that methodologies based on nucleic acid amplification techniques offer advantages compared with traditional methods such as inoculation on embryonated eggs and cell culture, due to higher sensitivity, specificity and fast results, which will be further discussed. a comparison between the different viral identification methods is presented in table 7 .1. the efficacy of sampling methodologies for viruses in environmental samples is still a matter of intense debate. a meta-analysis study performed with the aim to assess the efficacy of virus concentration methods associated to the molecular detection of adenovirus demonstrated that for detection in environmental samples qpcr or nested-pcr should be prioritized over pcr; in water samples (e.g. rivers or lakes) ultracentrifugation should be associated with nested-pcr and that microfiltration membrane, ultrafiltration, and qpcr must be associated for assessment of treated and untreated sewage samples (silva and melo, 2010) . it is currently acknowledged that viral contamination of recreational water presents a high risk of infection and is considered a significant public health hazard. published studies, summarized in table 7 .2, have addressed the assessment of virus bioburden in water environmental samples. the most commonly found viruses in aquatics environments are enteric viruses, such as enterovirus, rotavirus and norovirus, adenovirus and hepatitis virus a and c. begier and coworkers have correlated swimming in polluted seawater with enterovirus infection (begier et al., 2008) . for enteroviruses, the most common mode of transmission is the fecal-oral route, although aerosol transmission has also been reported. in a similar manner, drinking water contaminated with virus, such as norovirus, also presents a risk for human health, highlighting the importance of good hygiene practices with respect to water storage, shin and sobsey suggested that water chlorination could inactivate enteric viruses (shin & sobsey, 2008) . sample concentration is a critical step in viral diagnosis, since the number of viral particles in water is generally very low, which often results in false results if samples are tested directly using pcr (katayama et al., 2002) . therefore, some authors choose an adsorption-elution method, followed by ultrafiltration, to concentrate the viruses, before nested-pcr or quantitative real time pcr. in severally contaminated environments such as hospital wastewater treatment plants, prado and coworkers utilized pcr/rt-pcr, quantitative real-time pcr (qpcr) and genome sequencing, after sample concentrations techniques, to assess the presence of viruses associated with human pathologies such as acute gastroenteritis and (prado et al., 2011) . in intermediate contaminated environments, such as urban and bathing waters the assessment of microbial hazards with potential public health risk associated with viral contamination also preferentially utilizes nucleic acid amplification methodologies, after sample concentration. some authors chose to utilize the combination of culture dependent and independent methodologies for virus identification from environmental samples. in a study performed by roberto a. rodríguez and coworkers that aimed to assess the effects of sewer overflows to the viral contamination of receiving waters, concentrated samples were assayed for total culturable viruses using the plc/prf/5 cell line with associated confirmation by pcr/rt-pcr (rodríguez et al., 2012) . viruses can be transmitted directly from individual to individual via sneezing, coughing and touching, or indirectly via the environment. the prevalence of pathogenic viruses in healthcare settings potentially transmitted by airborne, droplet and contact represents a significant threat for both workers' and patients' health. thus, most of the presently performed studies regarding virus contamination of surfaces have focused on clinical settings, as demonstrated in table 7 .3. the data resultant from these studies is of foremost importance since it can allow the identification of critical locations and direct the selection of infection control measures for health-care settings to decrease viral associated nosocomial infections. protocols regarding transmission based precautions in both hospital personnel and patients are continuously updated at the international level (la rosa et al., 2013) . et al., 2014) in aerosol samples trough filtration method combined with real-time quantitative polymerase chain reaction (qpcr) technique (tseng et al., 2010) . in hospital settings, torque teno virus has been detected in samples collected with an impactor sampler with posterior culture in tryptone soy agar (tsa) and identification trough nested rt-pcr (carducci et al., 2011) or trough burkard c90m cyclone sampler and qrt-pcr (d'arcy et al., 2014) . nevertheless, culture-dependent methods and nucleic acid amplification techniques can also be used simultaneously. as an example, a study performed by goyal and coworkers utilized embryonated eggs and tissue cultures of vero, mdck, and rk-13 cell lines combined with nucleic acid extraction, pcr, rt-pcr and nucleic acid sequencing to assess contamination of respiratory viruses (vsr, influenza a and b, parainfluenza 1, 2 and 3, rinovirus, enterovirus, coronavirus, filoviruses, adenovirus and orthopoxvirus) and viruses with bioterrorism potential in ventilation filters from two large public buildings (goyal et al., 2011) . the assessment of viral contamination and/or infections play a key role in reducing global viral disease associated burden. it is currently acknowledged that the advances and development of molecular assays such as pcr, qrt-pcr, genotyping and multiplex assays endorsed important improvements in viral assessments regarding sensitivity, specificity, speed, simplicity and cost-effectiveness. nevertheless, the developments of new approaches that can provide alternatives to evade the limitations of nucleic acid amplification techniques are of foremost importance. in this context, currently nanobiosensors represent a new promising tool for virus detection. this methodology, still at research stage, encompasses the technology of viral disease biosensors with nanoparticles and nanomaterials, focused on the development of miniaturized biosensors with high sensitivity, specificity, and stability. this state of the art technology aims to deliver an alternative tool for effective and rapid viral disease diagnosis with no requirement of highly trained personnel or heightened laboratory facilities (kizek et al., 2015) . during the last two decades, there has been increasing concern within the scientific community over the effects of indoor air quality on human health. everything looked definitive when vaccines were produced, diseases had the end announced, but the truth is that infections insist on persisting in our common environment although the better knowledge. changes in building design formulated to improve energy efficiency have meant that modern homes and offices are frequently more airtight than older structures. indoor pollutants can emanate from a range of sources and we know much less about the health risks from indoor air pollution than we do about those attributable to the contamination of outdoor air (jones, 1999) . the indoor environments to be considered are those ordinarily and commonly occupied by humans: residences, offices, schools, industrial buildings, hospitals and other settings occupied a high proportion of the time, or in which occupant density is high (hanski et al., 2012) . biological hazards to man arise from exposure to high concentrations forms of bio-aerosols and three major groups of diseases associated with bio-aerosol exposure are infectious diseases, respiratory diseases and cancer. current knowledge is unclear regarding risk to cancer whether these excess risks occur from exposures to biological agents or are due to various chemicals used in industries (srikanth et al., 2008) . acclimated environments have an artificially multitude of chemical compounds (toxic, carcinogenic, radioactive) and biological (pathogenic) issued by a variety of sources, depending on the physical conditions (air humidity, air temperature, inadequate ventilation) of the environment. the air recirculation phenomenon is responsible for the increase of pathogenic microorganisms in the order of 1000 to 100,000 times in relation to the external air (lee et al., 2006) . incorrect cleaning filters and ducts of air conditioning provide the development of microbial particles including viruses that may lead the occupants of air-conditioned environments contracting respiratory infections or allergic diseases. viruses can persist in sufficient number to act as sources of infection for several hours, weeks or even months. the level of information and awareness of agricultural health and safety risks, disease, and injury prevention among the dairy farmers is low. training on health and safety in agriculture field is urgently needed. safe working conditions are essential for healthy living. the lack of a preventive policy maintenance programmes in refrigeration and ventilation systems is the determining factor for the occurrence of biological pollutants. take corrective actions preventing the spread of pathogens by the airborne route requires the use of special air handling and ventilation systems, such as airborne infection isolation rooms to contain and then safely remove the infectious agents. in addition, respiratory protection with validated and certified equipment is recommended (fisk et al., 2007) . environments have been studied more extensively than other issues due to their greater clinical significance. however, more work is still needed to provide a clearer picture regarding the rates of viral diseases transmission, airborne transmission in particular, in closed environments, and potential ways for reducing the levels of indoor viral pollution and transmission must be investigated. studies including homes, non-industrial workplaces and public buildings, are scarce (prussin ii et al., 2015) . ventilation filters of two large public buildings were sampled to determine the presence of human respiratory viruses by polymerase chain reaction and reversetranscription polymerase chain reaction. nine of the 64 filters tested were positive for influenza a virus, two filters were positive for influenza b virus, and one filter was positive for parainfluenza virus 1. filters are installed in hvac systems of buildings to protect ventilation equipment and maintain healthy indoor air quality. these filters process enormous volumes of air. building hvac filters may be used as a method of detection for airborne viruses. they may yield valuable information on the epidemiology and aerobiology of viruses in air that can report to the development of methods to prevent airborne transmission of viruses (goyal et al., 2011) . regrettably, several investigations have revealed that many hvac installations have a lot of operational and maintenance problems. numerous practical recommendations for design and operation of hvac systems are needed. following the recommendations will result in less pollution and increased indoor environmental quality (hanssen, 2004) . basic strategies of source control should not to make indoor air sterile but keep indoor environments dry, maintain good hygienic conditions in ventilation systems, apply effective filtration on mechanical supply ventilation, and use masks in the event of respiratory illness. the importance of hands in the transmission of viruses is well recognized and many of the studies relate specifically to handwashing. the number of asthma patients in most industrial countries has greatly increased, resulting in a morbidity rate of around 10−15% of the population. may be aerosol transmission is responsible for the most severe cases of disease involving viral infection of the lower respiratory tract (tellier, 2009; tsukagoshi et al., 2013) . rotavirus infection in adults air sampling for respiratory disease agents in army recruits approach to the patient with recurrent infections spread and prevention of some common viral infections in community facilities and domestic homes an outbreak of concurrent echovirus 30 and coxsackievirus a1 infections associated with sea swimming among a group of travelers to mexico rotavirus overview icam-1 on epithelial cells in allergic subjects: a hallmark of allergic inflammation environmental survey to assess viral contamination of air and surfaces in hospital settings standards for the diagnosis and treatment of patients with copd: a summary of the ats/ers position paper exacerbations of chronic obstructive pulmonary disease transmission and clinical features of enterovirus 71 infections in household contacts in taiwan viral kinetics and exhaled droplet size affect indoor transmission dynamics of influenza infection probabilistic indoor transmission modeling for influenza (sub) type viruses respiratory health and farming: an essay environmental viral contamination in a pediatric hospital outpatient waiting area: implications for infection control bioaerosol health effects and exposure assessment: progress and prospects meta-analyses of the associations of respiratory health effects with dampness and mold in homes surveillance of human viral contamination and physicochemical profiles in a surface water lagoon environmental monitoring for gastroenteric viruses in a pediatric primary immunodeficiency unit contamination of the hospital environment with gastroenteric viruses: comparison of two pediatric wards over a winter season group a rotavirus detection on environmental surfaces in a hospital intensive care unit prediction of intrinsic disorder in mers-cov/hcov-emc supports a high oral-fecal transmission detection of viruses in used ventilation filters from two large public buildings adenovirus transmission -worthy of our attention taxonomy of the caliciviruses metagenomic detection of viruses in aerosol samples from workers in animal slaughterhouses environmental biodiversity, human microbiota, and allergy are interrelated hvac -the importance of clean intake section and dry air filter in cold climate the common cold severe acute respiratory syndrome and coronavirus a new method for sampling and detection of exhaled respiratory virus aerosols effect of cleaning and disinfection of toys on infectious diseases and micro-organisms in daycare nurseries evidence of airborne transmission of the severe acute respiratory syndrome virus isolation of swine influenza virus in cell cultures and embryonated chicken eggs indoor air quality and health development of a virus concentration method and its application to detection of enterovirus and norwalk virus from coastal seawater temporal and spatial distributions of aerial contaminants in an enclosed pig building in winter nanoscale virus biosensors: state of the art the national human activity pattern survey (nhaps): a resource for assessing exposure to environmental pollutants viral infections acquired indoors through airborne, droplet or contact transmission longitudinal analysis of microbial interaction between humans and the indoor environment relationship between indoor and outdoor bioaerosols collected with a button inhalable aerosol sampler in urban homes health care transmission of a newly emergent adenovirus serotype in health care person nel at a military hospital in texas receptor recognition and cross-species infections of sars coronavirus viral contamination source in clinical microbiology laboratory respiratory viruses transmission from children to adults within a household online design aid for evaluating manure pit ventilation systems to reduce entry risk norovirus: an overview indoor bioaerosol dynamics incidence of human adenoviruses and hepatitis a virus in the final effluent of selected wastewater treatment plants in eastern cape province, south africa screening of respiratory pathogens by respiratory multi well system (mws) r-gene™ assay in hospitalized patients identification of severe acute respiratory syndrome in canada quantification and molecular characterization of enteric viruses detected in effluents from two hospital wastewater treatment plants total virus and bacteria concentrations in indoor and outdoor wintertime factors affecting contaminant distribution in a swine farrowing room the impact of combined sewage overflows on the viral contamination of receiving waters inactivation of norovirus by chlorine disinfection of water assessment of the incidence of enteric adenovirus species and serotypes in surface waters in the eastern cape province of south africa: tyume river as a case study artículo original avaliação de métodos de concentração e detecção molecular de adenovírus em águas não tratadas -uma metanálise monitoring rotavirus environmental contamination in a paediatric unit using polymerase chain reaction bio-aerosols in indoor environment: composition, health effects and analysis airway allergy and viral infection aerosol transmission of influenza a virus: a review of new studies detection of airborne viruses in a pediatrics department measured using real-time qpcr coupled to an air-sampling filter method molecular epidemiology of respiratory viruses in virus-induced asthma enteric adenoviruses influences in allergy: epidemiology and the environment limiting spread concentrations and size distributions of airborn e influenza a viruses measured indoors at a health centre, a day-care centre and on aeroplanes characteristics of bacterial and viral contamination of urban waters: a case study in outbreaks of influenza and influenza-like illness in schools in england and wales surveillance of viral contamination of invasive medical instruments in dentistry chromatography paper strip sampling of enteric adenoviruses type 40 and 41 positive stool specimens key: cord-294544-iutcduix authors: kesson, alison m. title: respiratory virus infections date: 2007-09-06 journal: paediatr respir rev doi: 10.1016/j.prrv.2007.07.003 sha: doc_id: 294544 cord_uid: iutcduix the respiratory tract is a frequent site of infection with a wide range of viruses. each family of viruses can cause differing clinical syndromes depending on the age of the patient and the immune response. as a corollary, different clinical syndromes can be caused by different families of viruses. respiratory virus infections represent a major public health problem because of their worldwide occurrence, ease of spread in the community and considerable morbidity and mortality. mortality from respiratory virus infections in healthy individuals in developed countries is rare; however, in less developed countries childhood mortality can be quite high with an estimated 5 million children globally under 5 years dying annually from respiratory virus infections. their worldwide occurrence results in people of all ages being susceptible to respiratory virus infections. however, on average, children are infected two to three times more frequently than adults, with acute respiratory virus infections being the most common infections experienced by healthy children and often resulting in loss of school time and a significant socioeconomic cost in medical visits, medications and parents' loss of work time. most viruses are transmitted by direct contact or droplets, although some are transmitted by aerosols. viruses that primarily infect the respiratory tract include influenza, adenoviruses, parainfluenza viruses, respiratory syncytial virus (rsv), coronaviruses, human metapneumovirus, rhinoviruses and enteroviruses. more recently, a new virus, bocavirus, has been identified. influenza, parain-fluenza, human metapneumovirus (hmpv) and rsv occur in epidemics while adenovirus, coronavirus and rhinoviruses occur endemically. other viruses which can cause respiratory diseases, more commonly in immunosuppressed people, include measles, varicella zoster virus (vzv), herpes simplex virus (hsv) and cytomegalovirus (cmv). aetiological diagnosis of viral respiratory tract illness requires laboratory confirmation as the symptoms and signs of the clinical illness lack specificity to permit aetiological recognition on clinical grounds alone. development of safe chemotherapeutic agents and highly immunogenetic and protective vaccines for each of the individual respiratory viruses is a continuing priority: except for influenza virus, this goal remains elusive. respiratory virus infections often have a seasonal distribution, especially in temperate climates, and while the peak incidence varies year to year there is often a predominant seasonal occurrence. rsv and influenza both have a peak incidence in winter and these peaks usually do not coincide but overlap. parainfluenza virus (piv) 3 usually peaks in winter with piv 1 and piv 2 peaking in autumn and early winter. the picornaviridae cause infections all year round with enteroviruses more common in summer and autumn while rhinoviruses are more common in winter and spring. adenoviruses tend to cause infections year round as do the herpesviridae, except for varicella which is more prevalent in the late winter and early spring. viral culture is the original method used for diagnosing respiratory virus infections and culture for viruses using primary and immortalized cells was expanded for wider diagnostic use in the 1950s. the main advantage of traditional cell culture methods is the ability to isolate and identify a wide range of viruses. 1 this has enabled the identification of many viruses, including those commonly causing respiratory infections -influenza, rsv, piv 1-4, adenoviruses, measles, enteroviruses, rhinoviruses, vzv, cmv and hsv. more recently recognized viruses such as severe acute respiratory syndrome coronavirus (sars-cov) and hmpv have also been grown in cell culture. for virus isolation to be successful careful attention needs to be paid to a number of issues. these include the selection of appropriate patients, collection of adequate specimens, transport of the specimen to the virology laboratory under optimal conditions and processing methods of the clinical samples. respiratory samples for virus isolation include nasopharyngeal aspirates, nasopharyngeal and/or oropharyngeal swabs, nasopharyngeal washes, bronchioalveolar lavages, sputum and lung biopsies. other than open lung biopsies all other specimens are contaminated with commensal respiratory flora. dacron or polyester swabs collected from the respiratory tract should be placed into viral transport medium containing antibiotics. the specimens should be kept cool at 2-8 8c or on wet ice until inoculation of the cell cultures to maintain virus infectivity, especially for labile viruses such as rsv. 2 detection of viruses in cell culture requires considerable expertise and is performed by microscopic examination looking for degenerative morphological changes in the cell monolayer called cytopathic effect (cpe). not all viruses grow in all cell types so clinical specimens are usually inoculated into several types of cells to provide an environment for the isolation of a suitable range of viruses. the use of a broad range of cell types potentially allows for the isolation of agents which are not expected rather than limiting detection to only a few virus types. this approach will also allow the detection of more than one virus in any given specimen which can occur in up to 5-10% of immunocompetent individuals and more frequently in immunocompromised patients especially with viruses which have latency such as cmv and hsv. recently, commercially produced co-cultivated cryopreserved cells have become available for the rapid identification of viruses. r-mix rapid cell culture (diagnostic hybrids) is a patented cell monolayer containing a mixture of a549 and mink lung (mv1lu) cells. 3 after 24-72 h of culture, using pooled or single fluorescein isothiocyanate (fitc)-labelled monoclonal antibodies directed against influenza a and b, rsv, parainfluenza 1-3 and adenoviruses, rapid identification of a respiratory virus infection can be established. growth of a virus in cell culture indicates the presence of viable, replication competent virus, a finding which cannot be determined using antigen or nucleic acid detection. in addition, the propagation of an isolate provides virus for antiviral susceptibility testing, serotyping and epidemiological studies. more recently newer viral culture formats have been developed which allow for more rapid detection of viruses, which is very useful for detection of those viruses which grow slowly in conventional cell culture. decreasing the time until cpe is detectable has been achieved for many viruses by centrifugation of the culture after the sample has been added. as viruses are unable to be centrifuged with conventional centrifuges, the enhanced detection may result from better contact between cells in the specimen and the cell culture thus allowing for earlier and more extensive infection of the cell culture sheet. furthermore, the detection of viral protein production (antigen detection) before there is evidence of cpe in conventional culture has also decreased the time to identify a virus and thus enhanced clinical utility of viral culture testing. the detection of viral protein production is usually performed using a fluorescent-labelled (e.g. fitc) monoclonal antibody directed against a viral protein. processing and reading these cultures is labour intensive; however, most common viruses can be detected in 24-48 h. alternatively enzyme-linked immunosorbent assay (elisa) detection of viral proteins in cell culture supernatants has also been performed. to activate the promoter. 4 this technology has been exploited to allow detection of hsv-1 or -2 using the baby hampster kidney (bhk) cell line stably transformed with the promoter of ul39 from hsv linked to the escherichia coli lacz gene. hsv can be detected within 24 h of infection using chromogenic substrate which turns infected cells from colourless to blue. hsv infected cells are easily identified and this technique could be expanded to determine viral titre in specimens if required. this system is marketed by diagnostic hybrids under the name elvis (enzyme-linked virus-inducible system). 5 the ability to detect viruses was significantly enhanced by the development of monoclonal antibodies (mab) directed against specific viral proteins for the viruses hsv-1, hsv-2, vzv, cmv, influenza a, influenza b, parainfluenza 1-3, and adenovirus, and linked to the fluorescent molecule fitc. these staining procedures are rapid: samples are batch run and results are usually available within 1-3 h depending on the number of specimens tested. rapid immunofluorescent (if) staining methods for the direct detection of viral antigens in respiratory specimens have excellent specificity and very good sensitivity. most respiratory viruses produce cpe in conventional cell cultures but often this is slow or very minimal in extent. the rapid detection of the respiratory viruses influenza a, influenza b, parainfluenza 1-3 and adenovirus by if is available in many laboratories and is extremely useful for the detection of these viruses, especially in paediatric patients who shed relatively large amounts of virus and for a longer period than do adults. the performance characteristics of the test depend on the type of specimen, age of the patient, duration of illness, mab reagent and level of expertise of the laboratory staff. generally, if methods for respiratory viruses are a little less sensitive than culture with the exception of rsv. however, the greater sensitivity of rsv if compared with rsv culture is due to the lability of rsv which is rapidly inactivated in samples that are not kept refrigerated and transported to the laboratory quickly for inoculation into cell cultures within a short period after collection of the specimen. the reported sensitivities of if detection of respiratory viruses varies but are broadly 90% for rsv, 80% for influenza a and b, 70% for parainfluenza 1-3 and 50% for adenoviruses. 6 mabs directed against hmpv have been developed and are currently under investigation to establish their clinical utility. membrane-based (cassette-based) enzyme immunoassays (eias), optical immunoassays and immunochromographic or lateral-flow systems have been introduced for less technically demanding detection of respiratory viruses. 7 some can now differentiate viruses, e.g. influenza a from influenza b, rsv from influenza a, and include a positive control to monitor performance of both the assay and the user. a significant aim of the manufacturers of these tests is to allow for testing to occur in the consulting physician's office. these techniques have the advantage of decreasing turn-around time for test results and have considerably simplified the technical component, but these tests are not as sensitive or specific as viral culture or direct if (dif) for detection of respiratory viruses. the positive predictive value of these assays is usually higher when there is a greater prevalence of disease in the respiratory virus season and the virus in question has been shown to be circulating in the community by viral culture or if. during periods of relatively high prevalence positive results can usually be accepted as correct; however, negative results should be confirmed by a second method, e.g. dif or culture. 8 viruses can be detected in clinical samples using highly specific nucleic acid probes that are complementary in sequence to viral rnas or viral dnas, or by using a nucleic acid amplification technique such as polymerase chain reaction (pcr), branch chain dna detection (bdna) or nucleic acid sequence based amplification (nasba). these techniques are becoming increasingly available in diagnostic laboratories but testing for multiple viruses using molecular methods is considerably more expensive than with conventional techniques and the methodology needs to be developed in house. evidence would suggest that testing using molecular methods is a little more sensitive than conventional approaches but the information available to date is variable. 9 influenza a virus was isolated in 1933, influenza b in 1940 and influenza c virus in 1951. influenza a and b viruses belonged to the family orthomyxoviridae and are in the genus influenzavirus. influenza c virus belongs to a separate genus. influenza virions are enveloped particles (i.e. surrounded by a lipid bilayer membrane of cellular origin) containing a single-stranded negative-sense segmented rna genome that is surrounded by a helical capsid, with influenza a and b containing eight segments of rna and influenza c containing seven segments. only influenza a and b are clinically important. influenza c infection occurs uncommonly and is usually associated with the mild upper respiratory tract illness; rarely it can cause bronchitis or pneumonia. the enveloped virion has peplomers or spikes consisting of two glycoproteins -haemagglutinin (h) which is involved in the attachment of the virus to cells and the initiation of infection, and neuraminidase (n) which facilitates release of newly formed virions from the cells. the two glycoproteins, h and n, exhibit substantial antigenic variation among influenza a viruses with 16 h subtypes and 9 n subtypes recognized. influenza b has only one type of h and n glycoproteins. the occurrence of annual influenza epidemics throughout the world is due to the high rate of antigenic variation from a stepwise mutation of the h and/ or n genes and reflected in variations of the antigenic characteristics of these proteins (and thus escape from immune memory) by the h and n glycoproteins of influenza a and b viruses. this stepwise mutation of h and n results in antigenic drift and the recurrent annual influenza epidemics seen each winter. the virus can infect and produce disease among populations who would otherwise possess immunity from previous infection because their antibodies fail to recognize the new antigenic variation(s). this is the reason for the necessity for annual influenza vaccination with differing serotypes of influenza a and b viruses. additionally, the segmented nature of the influenza genome allows for antigenic shift -the re-assortment of genome segments from two different influenza a viruses with major changes in the h or n proteins or both. it is this major variation in genetic make-up which gives the influenza a virus the potential for the development of global pandemics. during the past few years, new influenza viruses isolated in hong kong, h5n1, appear to have spread from poultry to a small number of persons, who have shown a high mortality rate. this has caused a high degree of global concern as spread of avian influenza a strains to humans is usually very uncommon but several hundred human cases of h5ni infection have been identified in countries across the world. 10 the major limitation to this virus causing a pandemic with a huge impact on health and the economy is the failure of the h5n1 influenza a to establish sustained human-to-human transmission. were this to occur a pandemic would be inevitable. commonly available routine diagnostic tests in laboratories can differentiate influenza a from influenza b; however, they are unable to identify the subtype. identification of the strain of influenza can be performed by a variety of methods at reference laboratories if required. 11 laboratory diagnosis of influenza virus infection by isolation of the virus in cell culture is the 'gold' standard technique. this technique however is slow and does not allow for diagnosis within the time frame for specific treatment. however, virus isolation remains critically important for epidemiological studies, the recognition of specific types of influenza circulating in the community and identification of new types and thus pandemics. the information on influenza types is used to design vaccines for the upcoming influenza season. influenza culture can be performed on nasopharyngeal aspirates, nasopharyngeal swabs, throat swabs and sputum, or lung biopsy tissue or bronchoalveolar lavages. specimens should be kept at 4 8c until processed. more recently, rapid cultures involving growth of influenza for up to 3 days and then labelling cells with antibodies directed against influenza a using an immunoperoxidase or fluorescence technique have been developed. these techniques are rapid and relatively inexpensive but do require a high degree of technical competency. they can accurately differentiate influenza a or b from other viruses but are not suitable for identifying the h type of influenza a. thus these tests would not be diagnostic in the early stages of a pandemic when the new h type of influenza a would be co-circulating with the old h type. direct detection of viral antigen in respiratory specimens can also be performed and is the technique of choice for a rapid sensitive assay. reverse transcription-polymerase chain reaction (rt-pcr) is now available in many laboratories and can differentiate influenza a and b and other viruses. it can also be used to identify differing h or n types of influenza a. detection of an immunological response, with a fourfold or greater rise in serum antibody titres using haemagglutination inhibition or complement fixation techniques when acute and convalescent samples are compared, also provides evidence of acute infection with the influenza a or b virus. serological tests are rarely diagnostic in individual cases within a timeframe that allows judicious use of anti-influenza drugs but they are very useful for tracking epidemics in the community. elisa tests for igm on a single sample are also available. igm and iga specific antibodies against influenza peak about 14 days after infection and igg specific antibody peaks at about 4-7 weeks. serological diagnosis and typing of influenza virus can be complicated by the fact that the anamnestic response to infection is highest for the strain causing the primary infection, even when there is subsequent infection by other strains. this has been termed the 'doctrine of original antigenic sin' and can lead to a lack of strain specificity with serological tests. parainfluenza virus types 1, 2 and 3 occur worldwide and among persons from all age groups. parainfluenza viruses 4a and 4b are much less frequent. parainfluenza 1 occurs in epidemics usually during autumn in alternate years, parainfluenza 2 occurs sporadically and parainfluenza 3 tends to cause annual winter epidemics in temperate climates. parainfluenza 1-3 are the main causes of croup in infants and young children under 5 years of age. parainfluenza 3 can also cause viral pneumonia and bronchiolitis in infants and small children. parainfluenza 4 occurs very infrequently and is usually associated with mild symptoms of upper respiratory tract illness (rhinorrhea, pharyngitis and cough). primary infection with parainfluenza provides some measure of immunity but this immunity is not complete or long lasting and reinfections occur commonly but they are rarely as severe as the illnesses seen with primary infection. laboratory diagnosis of parainfluenza infection is based on viral isolation in tissue culture and this remains the 'gold' standard. respiratory secretion specimens must be kept at 4 8c until processed. parainfluenza grows relatively slowly, taking 3-5 days, and as these viruses do not cause a direct cpe, they are detected using haemadsorption (adsorption of guinea pig red blood cells to infected cells). rapid diagnostic tests for detection of viral antigen in respiratory specimens using dif are specific for each of the parainfluenza virus types and show high sensitivity and specificity. rt-pcr procedures can either detect single viruses or parainfluenza as a group, with high sensitivity and specificity. a serological response with detection of a fourfold of greater rise in serum antibody levels between acute and convalescent samples, collected about 3 weeks after the onset of acute illness, may also provides evidence of infection. antibody can be measured using complement fixation, haemagglutination inhibition or neutralization. interpretation of a serological test relative to the parainfluenza type producing the infection may be complicated by heterotopic antibody responses seen in some infected individuals. rsv was first isolated in 1956 from a laboratory chimpanzee with an illness resembling the common cold, and shortly after it was demonstrated to be a human pathogen. epidemiological studies have shown that rsv represents the single most important cause of serious lower respiratory tract disease, especially bronchiolitis and pneumonia, in infants and children. rsv can cause severe pneumonia and death in persons with underlying immune deficiency. rsv belongs to the family paramyxoviridae and the genus pneumovirus. it is an enveloped virus with a single-stranded, negative-sense, non-segmented rna genome with at least 10 viral proteins. antigenic analysis of rsv has identified two subgroups, a and b, based on their reactivity to a panel of monoclonal antibodies with the b strain further differentiated into two variants, b1 and b2. annual epidemics of rsv occur during winter in temperate climates and the two subgroups usually co-circulate in the same geographical area, often with a predominance of subgroup a. diagnosis of rsv is commonly made by direct examination of respiratory secretions using if or an eia technique. a number of commercial rapid point-of-care diagnostic eia kits which identify rsv antigen in respiratory secretions are now available. these kits provide a high degree of sensitivity or specificity, although they are not as sensitive as dif. rsv can also be grown in cell cultures but the virus is extremely labile and great care must be taken in transport to ensure that is remains at 4 8c. rsv can also be diagnosed serologically using complement fixation by the detection of a fourfold rise in antibody titre. however, like most respiratory virus serology, this testing is not clinically useful because of the delay in diagnosis. furthermore, infants may possess maternal antibodies, thus complicating interpretation of serological data. adenoviruses were isolated from the primary cell cultures of adenoids from children in the early 1950s. adenoviruses are non-enveloped particles which contain linear doublestranded dna surrounded by an icosahedral capsid with fibre-like projections extending from each of the 12 capsid vertices. adenovirus infections occur worldwide and transmission varies from sporadic to epidemic. since adenoviruses are very stable they can be easily transmitted in the environment by fomites. adenoviruses are an important cause of upper and lower respiratory tract disease with types 1, 2, 3, 5 and 7 accounting for about 85% of all infections. rarely, adenoviruses can be isolated from the lungs, livers, kidneys and brains of patients with fatal infection. adenoviruses can be isolated in cell cultures and presumptively recognized by their characteristic cytopathic effect. currently 52 human adenovirus serotypes have been identified and they can be grouped into six subgenera, designated a through f, based on differing classification schemes concerned with the guanine (g) and cytosine (c) content of their dna, determined using dna restriction analysis procedures or pcr. 12 individual isolates can be serotyped using haemagglutination inhibition, neutralization or if. however, this is very time consuming and not routinely available. adenoviruses can be detected directly in respiratory secretions using if, pcr, elisa and immunochromatography. adenovirus infection can be diagnosed serologically using complement fixation by detection of a fourfold or greater rise in antibody titre during convalescence. type-specific antibody responses can be assayed by haemagglutination inhibition, elisa and neutralization assay. however, except for epidemiological investigations, antibody determination is a much less efficient means for determining infection than using direct methods such as pcr or antigen detection. rhinoviruses are a group of more than 100 serotypes and cause more common cold (minor upper respiratory tract) illness than any other virus that infects the respiratory tract. they account for about one half of common colds occurring in children and consequently cause substantial absenteeism from schools. in persons with underlying lung disease and immune disorders, rhinoviruses can cause pneumonia. rhinoviruses belong to the family picornaviridae, which are small, non-enveloped viruses with a single-stranded, positive-sense rna genome. unlike enteroviruses (also picornaviridae), rhinoviruses are inactivated when exposed to mild acid (ph <5), accounting for their failure to infect the gastrointestinal tract. rhinoviruses also caused otitis media in infants and children, being recovered from middle ear fluid in 10% of subacute or chronic cases that are negative for bacteria. recent studies associate rhinovirus infection with exacerbations of asthma and with acute lower respiratory tract disease, especially in persons with chronic obstructive airway disease and cystic fibrosis, and those who are immunocompromised. rhinoviruses prefer to grow at lower temperatures than many other respiratory viruses (33-34 8c), and grow less well at the higher temperature of the lungs than in the nose; however, they can establish infection in the lung. type-specific immunity develops following infection and is characterized by igg neutralizing antibodies in serum and secretions. long lasting immunity best correlates with the level and secretion of iga antibody from the nasal mucous membranes. diagnosis of rhinovirus infection rarely requires laboratory testing but virus isolation, detection of viral rna by rt-pcr, antigen detection by dif in cells from respiratory secretions or detection of a fourfold rise in antibody titres by neutralization test or eia can be performed if required. viral isolation in cell culture is very sensitive, especially when collected early in the illness. viral isolation is labour intensive and requires several days, with confirmation of the individual rhinovirus serotype requiring further antigenic characterization with type-specific antibody. therefore, it is not used as a routine diagnostic test. rt-pcr is unlikely to be available except as a research procedure. the human coronaviruses 229e and oc43 are an important cause of the common cold and belong to the family coronaviridae, genus coronavirus. they are enveloped with a helical nucleocapsid possessing a single-stranded, positivesense rna genome. coronavirus infections occur sporadically throughout winter and spring and are distributed worldwide. they are spread by large droplets via the respiratory route. children commonly experience one coronavirus infection per year, which is about three times more frequently than adults. coronaviruses are associated with 10-15% of upper respiratory tract illness, mainly causing common colds and otitis media. pneumonia rarely occurs with non-sars coronavirus infection. type-specific antibodies to single coronavirus types develop in response to infection and persist for about 4 months. circulating and mucosal antibody confers protection from illness and the possession of coronavirus antibodies increases with age, with circulating antibody found in most children older than 6 years. nevertheless, re-infections are common. laboratory diagnosis of coronavirus infections can be achieved using virus isolation, detection of viral antigen by if, detection of viral rna by rt-pcr and/or demonstration of a fourfold or greater rise in antibody titre during convalescence. virus isolation is complicated and is not generally recommended as a routine diagnostic test since most illnesses are minor common colds. procedures for antibody measurement include elisa, neutralization and hemagglutination inhibition. sars was first recognized in china in november 2002 and rapidly spread around the world with significant morbidity and mortality, and clusters of affected patients, e.g. healthcare workers and family contacts. 13 a coronavirus was identified as the cause. interestingly children and adolescents had significantly less severe disease than adults. 14 the sars coronavirus can be detected using rt-pcr or viral culture. due to the very high transmissibility of this agent and the severe outcomes, any patient considered to have sars should be isolated and laboratory specimens collected with high level precautions. the laboratory should be notified as culture of the sars coronavirus should only be attempted in high biocontainment facilities and specimens may need referral to external public health laboratories. dutch researchers reported the isolation of an agent that induced cytopathic effects on cultured cells, from 28 respiratory specimens collected over a 20 year period. 15 electron microscopy revealed filamentous viral-like particles, suggesting that the agent was a virus. using random arbitrarily primed-pcr, the virus discovered was found to be a member of the paramyxoviridae family and the first human pathogen of the genus metapneumovirus; it was named human metapneumovirus (hmpv). since its initial discovery, hmpv has been identified worldwide. in general, hmpv infection accounts for approximately 2-12% of paediatric lower respiratory illness. 16 rt-pcr detection is the only reliable method of detection at present. there are no standard methods for isolating hmpv in culture, although this is possible, and monoclonal antibodies to detect the virus by dif are being tested for clinical utility. human bocavirus is a newly discovered parvovirus that was first identified in sweden but which occurs globally. 17, 18 bocavirus has been detected in young children with respiratory distress, many with pneumonia with interstitial infil-trates noted on chest x-ray. however, whether bocavirus is a cause of respiratory disease is yet to be fully determined. common colds are very common and are usually selfdiagnosed. the term 'cold' does not constitute a single viral entity but rather a syndrome of nasal congestion, sneezing, rhinorrhoea and pharyngitis, with more severe clinical disease associated with high fever in children. colds are generally self-limiting with a median duration of 9-10 days. many viruses from a variety of viral families may be associated with this syndrome but the pattern of symptoms associated with colds does not appear to vary significantly between agents. approximately 40-50% of colds are found to be associated with rhinovirus infection with other frequently occurring agents, including coronaviruses, enteroviruses and non-primary infections with parainfluenza virus and rsv. transmission of most of the viruses responsible for the common cold is by direct contact and inoculation of virus into the upper respiratory tract. infection with rhinovirus results in the development of long lasting antibody which is protective against re-infection with the same serotype. this also produces a degree of resistance to challenge with heterologous rhinovirus serotypes but this protection is incomplete and short lived. recognized complications of colds include secondary bacterial infection of the paranasal sinuses and/or middle ear and exacerbations of asthma or bronchitis. viral otitis media commonly complicates colds in children, probably due to eustachian tube dysfunction, with rhinoviruses, 19 rsv, influenza virus and adenovirus 20 detected in the middle ear fluids in 20-40% of cases of otitis media with effusion in children. specific viral diagnosis is generally not attempted but if required, isolation of the aetiological agent in cell culture can be performed. rt-pcr may be more sensitive for detection of these agents, especially rhinoviruses, enteroviruses and coronaviruses. pharyngitis is a very common reason for seeking outpatient medical care. it can be divided into a syndrome with nasal symptoms, which has a predominately viral cause, and cases without nasal symptoms and caused by a diverse spectrum of aetiological agents, including group a and non-group a streptococci, chlamydia pneumoniae, mycoplasma pneumoniae and epstein-barr virus. croup or viral laryngotracheobronchitis is a clinically distinct illness that predominately affects children under 3 years of age. the characteristic physical finding in croup is inspiratory stridor. pivs account for about 75% of cases of croup 21 of which piv 1 and piv 2 are the most commonly associated. other causes of croup include rsv, influenza a and b, rhinoviruses, adenoviruses, as well as m. pneumoniae. m. pneumoniae and influenza tend to cause croup in older children. piv 2 and influenza a are associated with more severe disease, 22 but generally the clinical presentation of the croup syndrome due to individual agents is similar. measles, which is a rare cause of croup, can cause especially severe disease. 23 a specific viral cause for croup can be established with viral culture but is not routinely performed as the clinical syndrome is sufficient for diagnosis and management. bronchiolitis is due to inflammation of the bronchioles leading to a clinical syndrome characterised by obstruction of expiratory airflow usually proceeded by nasal congestion and rhinorrhea. there may often be history of exposure to an adult or sibling with a cold or minor respiratory illness or exposure to another infant with bronchiolitis within the day care setting. mild conjunctivitis may occur in about one third of cases and otitis media is recognized in 5-10%. the hospital course is variable but most infants improve within 3-4 days. 24 bronchiolitis is a disease predominately of infancy and the epidemiology of this disease closely parallels the incidence of the major infectious cause, rsv. the peak incident age is in infants between 2 and 6 months with over 80% of cases occurring in the first year of life. 25 rsv causes the majority of cases of bronchiolitis however other respiratory viruses include pivs, influenza and rhinoviruses. piv 3 and adenovirus types 3, 7 and 21 are relatively uncommon but may be associated with more severe disease including bronchiolitis obliterans. 26 rhinoviruses can cause bronchiolitis in infants with bronchopulmonary dysplasia. [26] [27] [28] pertussis is occasionally confused with bronchiolitis. anatomic defects such as vascular rings, foreign bodies and gastro-oesophageal reflux are additional differential diagnoses. bronchiolitis and pneumonia are both part of the spectrum of lower respiratory tract involvement, frequently co-exist and cannot be clearly distinguished. pneumonia is defined by the development of abnormalities in alveolar gas exchange accompanied by inflammation of the lung parenchyma, often associated with visible changes on chest xray, ct scanning or gallium scanning. there can be considerable variety to the presentation of viral pneumonia depending on the age and immunological competence of the host, as well as the specific viral pathogen. viral pneumonia is an important cause of morbidity and mortality in individuals with a compromised immune system. the clinical presentation varies considerably with the specific causative agent but typically includes fever and lower respiratory tract symptoms and signs, such as tachypnoea, non-productive cough, wheeze and increased breath sound. very young infants may present with apnoeic episodes with minimal fever. bacterial superinfection is a potential and serious complication of viral lower respiratory tract infection, particularly with influenza. underlying cardiopulmonary disease such as valvular heart disease or chronic obstructive pulmonary disease are well recognized risk factors for increased severity of disease with viral pneumonia. diagnosis of the specific cause of an acute pneumonia due to a particular viral agent is complicated by difficulty in obtaining appropriate lower respiratory tract samples for culture and in isolating or detecting certain pathogens, and additionally by the frequent asymptomatic shedding of some viruses, e.g. herpes simplex virus or adenoviruses. while viruses are clearly important and frequent causes of pneumonia in young children, they are less apparent in older children. rsv has been associated with the largest proportion of viral pneumonia in young children, particularly if accompanied by bronchiolitis. 29 piv 3 and influenza a and b are significant causes of pneumonia in children, especially during periods of epidemic prevalence. 30 adenoviruses are frequently isolated from children with respiratory disease and are implicated in about 10% of childhood pneumonias. however, their true impact is difficult to access because of the long intermittent asymptomatic respiratory shedding of adenoviruses in children. adenoviruses have been described as cause of significant outbreaks of viral pneumonia in institutionalized children. pneumonia is the most frequent serious complication of measles. rhinoviruses have been associated with community-acquired pneumonia in children despite their apparent temperature sensitivity. other occasional causes are enteroviruses, hsv and vsv. serological diagnosis essentially establishes a temporal but not a causal relationship between a viral infection and a clinical syndrome, and a positive serological result may be misleading during times of high prevalence of a particular viral agent. laboratory diagnosis of viral disease specimen collection, transport and processing: virology sensitivity of respiratory virus culture when screening with r-mix fresh cells transgenic cell lines for detection of animal viruses confirmation of low-titer, herpes simplex virus-positive specimen results by the enzyme-linked virus-inducible system (elvis) using pcr and repeat testing clinical virology manual evaluation of an acute point-of-care system screening for respiratory syncytial virus infection comparison of lateral-flow immunoassay and enzyme immunoassay with viral culture for rapid detection of influenza virus in nasal wash specimens from children role of cell culture for virus detection in the age of technology consultation on human influenza a/ h5. avian influenza (h5n1) infections in humans clinical features and rapid viral diagnosis of human disease associated with avian influenza a h5n1 virus coronavirus as a possible cause of severe acute respiratory syndrome clinical presentations and outcome of severe acute respiratory syndrome in children. research letters a newly discovered human pneumovirus isolated from young children with respiratory tract disease human metapneumovirus: a newly described respiratory tract pathogen cloning of a human parvovirus by molecular screening of respiratory tract samples human bocavirus in children detection of rhinovirus, respiratory syncytial virus and coronavirus infections in acute otitis media by reverse transcription polymerase chain reaction a longitudinal study of respiratory viruses and bacteria in the etiology of acute otitis media with effusion croup: an 11-year study in a pediatric practice influenza a and b virus infection in infants and young children during the years 1957-1976 laryngotracheo-bronchitis as a complication of measles during an urban epidemic clinical and physiological manifestations of bronchiolitis and pneumonia: outcome of respiratory syncytial virus epidemiology of respiratory syncytial virus infection in brochiolitis obliterans, bronchiolitis, and other sequelae of adenovirus type 21 infection in young children rhinovirus infection associated with serious illness among pediatric patients the tucson children's respiratory study: ii. lower respiratory tract illness in the first year of life rhinovirus infection associated with severe lower respiratory tract illness and worsening lung disease in infants with bronchopulmonary dysplasia impact of influenza virus infection as a cause of pediatric hospitalization key: cord-286337-qk90xb3a authors: hanada, shigeo; pirzadeh, mina; carver, kyle y.; deng, jane c. title: respiratory viral infection-induced microbiome alterations and secondary bacterial pneumonia date: 2018-11-16 journal: front immunol doi: 10.3389/fimmu.2018.02640 sha: doc_id: 286337 cord_uid: qk90xb3a influenza and other respiratory viral infections are the most common type of acute respiratory infection. viral infections predispose patients to secondary bacterial infections, which often have a more severe clinical course. the mechanisms underlying post-viral bacterial infections are complex, and include multifactorial processes mediated by interactions between viruses, bacteria, and the host immune system. studies over the past 15 years have demonstrated that unique microbial communities reside on the mucosal surfaces of the gastrointestinal tract and the respiratory tract, which have both direct and indirect effects on host defense against viral infections. in addition, antiviral immune responses induced by acute respiratory infections such as influenza are associated with changes in microbial composition and function (“dysbiosis”) in the respiratory and gastrointestinal tract, which in turn may alter subsequent immune function against secondary bacterial infection or alter the dynamics of inter-microbial interactions, thereby enhancing the proliferation of potentially pathogenic bacterial species. in this review, we summarize the literature on the interactions between host microbial communities and host defense, and how influenza, and other acute respiratory viral infections disrupt these interactions, thereby contributing to the pathogenesis of secondary bacterial infections. influenza and bacterial pneumonia are the leading cause of morbidity and mortality from infectious diseases worldwide. influenza and other respiratory viral infections predispose patients to secondary bacterial super-infections, which are frequently associated with a more severe clinical course. it is estimated that the so-called "spanish flu" pandemic of h1n1 influenza a virus from 1918 to 1919 resulted in more than 50 million deaths, with many caused by bacterial superinfection leading to secondary pneumonia (1) (2) (3) (4) (5) (6) (7) . even in the antibiotic era, over half of patients with severe infections in the 1957 h2n2 and 1968 h3n2 pandemics had bacterial complications (8) (9) (10) . bacterial co-infection was also detected in ∼30% of cases in the 2009 h1n1 pandemic, with high mortality rates despite administration of appropriate antibiotics (11) (12) (13) (14) (15) (16) (17) (18) . thus, it is evident that a better understanding of the pathogenesis of secondary bacterial pneumonia following viral infections is needed in order to make therapeutic strides for this devastating complication. the mechanisms of post-viral bacterial infection are complex, comprising multifactorial processes mediated by interactions between viruses, bacteria, and the host immune system. the pathogenesis of super-infection has been attributed to direct mucosal/epithelial damage by influenza virus, increased bacterial colonization of the upper and lower respiratory tracts (urt and lrt, respectively), and dysregulation of immune responses, which all lead to increased susceptibility to secondary bacterial infections. however, emerging evidence suggests that our microbial communities residing on our mucosal surfaces likely shape the rigor of our immune responses and shape the ecological relationships between host and pathogens. over the past 10 years, intense interest has focused on examining how the microbial communities which inhabit our bodies-which some consider to be a separate "organ system" given the sheer physical bulk, number of genes, and metabolic activities-govern the balance between health and susceptibility to diseases, including infections. this raises the possibility that disruptions in the normal microbial communities by an acute viral infection might contribute to the development of post-viral bacterial pneumonia. the recent development of culture-independent methods of microbial identification has enabled the study of microbial communities on mucosal surfaces of the human body, referred to as "microbiota." the microbial communities of mammalian hosts are diverse, comprised of bacterial, viruses, archaea, parasites, and fungi. the human microbiome project (hmp) and other similar large-scale sequencing projects worldwide have characterized the distinct microbial communities that have adapted to the unique environmental niches within our bodies, such as the gut, skin, airways, genitourinary tract, and oral cavity. the gut microbiome, in particular, has been shown to play an integral role in shaping the immune system starting early in life, with continued influence on priming the nature and robustness of immune responses throughout one's lifetime. the respiratory tract also harbors distinct communities of microbes, with multiple discrete ecological niches (e.g., nasal cavity, oropharynx, upper airways) that vary in terms of temperature, ph, oxygen tension, mucus production, and other factors. the effects of viral infections on both the gut and respiratory microbiome have recently undergone examination. surprisingly, influenza infection has been found to result in significant changes in the gut microbiome, despite the lack of detectable virions in the gi tract. by comparison, the effects of viral infection on the respiratory microbiome appear to be relatively modest, but detectable. while the effects of these alterations on risk of secondary bacterial pneumonia have not been studied, potential mechanisms by which these changes might modulate susceptibility to secondary bacterial infections include alterations in the nature and magnitude of the immune response in the host (microbiome on host effects) and facilitating growth of pathogens in the absence of normal commensals (inter-microbial effects). in this article, we review the current understanding of how alterations in the microbiome following viral infection might alter host immune responses and increase susceptibility to secondary bacterial infections. although the term "microbiome" encompasses all microbial communities, there is currently a paucity of studies on how the mycobiome (fungal microbiome) and the virome (viral microbiome) affect host defense against respiratory infections and vice-versa; thus, this review will focus on the bacterial microbiome literature. of the niches in the body, the gut microbial community has been the most intensively studied, with over 20,000 publications to date. while the virome and mycobiome (fungi) are also being analyzed, the bulk of the literature has focused on the bacterial component of the microbiome, and thus most of our understanding of the relation of the gut microbiome to host immunity and pathogenesis of chronic diseases comes largely from studies of the bacterial community. during health, the human gut bacterial community is diverse, with each individual harboring over 100 trillion bacteria, comprised of over 150 different species. the gastrointestinal microbiota is dominated by firmicutes (e.g., lactobacillus, bacillus, and clostridium) and bacteroidetes (e.g., bacteroides), with lower abundances of proteobacteria (e.g., escherichia) and actinobacteria (e.g., bifidobacterium) (19, 20) . wild-living mice exhibit more diverse microbiomes, with significant abundance of proteobacteria as well as firmicutes and bacteroidetes (21) . the gut microbiome, in addition to its metabolic functions in the host, plays an integral role in the development, instruction, and priming of the immune system. germ-free (gf) mice (which lack microbiota) have markedly underdeveloped gut-associated lymphoid tissues, decreased number and smaller-sized peyer's patches and mesenteric lymph nodes, and defects in antibody production, compared to specific pathogen free (spf) mice. not surprisingly, germ-free animals exhibit increased susceptibility to multiple types of infections, including viruses, bacteria, and parasites (22) (23) (24) (25) (26) (27) . however, compared to free-living mice or laboratory animals exposed to gut flora from wild mice, spf animals have a more limited microbial community and are also more susceptible to inflammatory diseases, with a reduced immune repertoire including deficits in memory responses (21, 28, 29) . although an extensive discussion of the healthy gut microbiome and its impact on host immunity is beyond the scope of this review, we will highlight a few important aspects of how the intestinal bacterial community microbiome maintains a healthy host immune environment. first, bacterial metabolites generated by gut commensals contribute to the maintenance of intact epithelial integrity, regulatory t-cell development, and a relatively anti-inflammatory immune state. in particular, short-chain fatty acids (scfas) such as acetate, propionate, and butyrate are fermentation products of dietary fiber and carbohydrates by large intestinal bacteria (30) . in addition to being a major energy source for intestinal epithelial cells, scfas promote the development of naive cd4 + t cells into regulatory t cells (31, 32) , induce "tolerogenic" dendritic cells in the intestinal mucosa (33), and limit autoimmity (34, 35) . at the same time, microbial metabolites are integral for promoting immune responses in the gut against pathogens, including inducing secretion of il-18 (36) and defensins (37, 38) . thus, the products of microbiome metabolism are integral to the appropriate regulation of mucosal barrier integrity and immune homeostasis. in addition, specific members of the bacterial community have been shown to foster the proper maturation and development of the immune system. while this is still an area undergoing intense investigation, one notable example is the discovery that segmented filamentous bacteria are critical promoters of intestinal mucosal iga production (39, 40) and th17 cell induction (41, 42) . dysbiosis, or an imbalance in the normal composition of the microbiome, is associated with a variety of chronic diseases, many of which are characterized by chronic inflammation or abnormal metabolism, including inflammatory bowel disease, cardiovascular disease, and diabetes. thus, fostering appropriate levels of diversity and composition of the gut microbial community is critical for promoting health and immune homeostasis. during health, the composition of the microbiome is governed by a number of selective pressures unique to each anatomic niche, including temperature, nutrient availability, ph, oxygen tension, and the local immune environment. shortterm perturbations in the gut microenvironment caused by illness, antibiotic usage, or dietary changes (e.g., starvation) can alter the gut microbiome and subsequently lead to transient alterations in immune responses. thus, investigating whether influenza and other respiratory viruses alter the gastrointestinal microbiome could have mechanistic implications for viralmediated suppression of antibacterial immune responses. although the composition of the gastrointestinal microbiome is largely influenced by dietary patterns, respiratory viral infections could also contribute, along with other stress inducers such as broad-spectrum antibiotics exposure and chronic inflammation. using animal models of pulmonary infections by influenza and respiratory syncytial virus (rsv), multiple groups have shown that the gut microbiome is clearly impacted by respiratory viral infections, despite the lack of detectable respiratory virus in the gut (43) (44) (45) (46) (47) . in a murine model of influenza infection, the investigators found that although the total numbers of bacteria in the gut did not decrease, there was a reduction in the quantities of segmented filamentous bacteria (sfb) and lactobacillus/lactococcus, accompanied by increases in enterobacteriaceae. interestingly, although sfb have previously been shown to induce th17 cells (41, 48) , flu-infected mice had increased il-17a levels and numbers of th17 cells in the small intestine and colon, which appeared to contribute to intestinal injury (43) . in this study, antibiotic treatment prior to influenza infection ameliorated the degree of intestinal injury, but not lung injury, suggesting that gut dysbiosis contributed to local but not systemic inflammation. other groups have similarly reported increased proteobacteria (the phylum of which enterobacteriaceae are members) (44, 45) , decreased firmicutes (which include sfb, lactobacillus and lactococcus species), and increased bacteroidetes (47) following infection by flu or rsvs but not after administration of live attenuated influenza vaccine (laiv), indicating that live viral infection is required for these changes (47) . the increase in proteobacteria appears to be mediated by type i interferons (ifns) (18) , which not only depleted anaerobic bacteria but also increased susceptibility to secondary salmonella colitis. however, caloric restriction also figure 1 | shifts in the mouse gut microbiome in the setting of influenza infection. during an acute respiratory viral infection, changes in the bacterial composition of the gut microbiome can be observed despite the absence of detectable virus in the gastrointestinal compartment. this suggests that systemic immune signals, physiologic changes (e.g., weight loss), and other still unknown factors are disrupting the normal ecology of the gut, thereby leading to dysbiosis. however, the majority of these studies have been conducted in laboratory animals housed under spf conditions. it remains to be determined whether human patients and mammalian hosts with more diverse baseline gut microbiota (i.e., mice in the wild), exhibit similar qualitative or quantitative changes. results in increased relative abundance of proteobacteria and increased bacteroidetes to firmicutes ratio, raising the possibility that decreased oral intake during influenza may contribute to changes in the microbiome (45, 47, 49, 50) . it has also been shown that influenza infection alters intestinal microbiota composition through type ii ifn produced by lung-derived t cells recruited to the intestine (43) . thus, changes in the gut microbiome appear to result not from direct viral effects but from systemic inflammatory signals that travel from the lung and trigger local inflammatory responses in the gut (figure 1 ). interactions between respiratory tract infections and the gut microbiome are bidirectional. while respiratory viral infections can change the gut microbiome, the gut microbiome also shapes the adaptive immune responses against respiratory pathogens. mice pretreated with an antibiotic cocktail showed increased morbidity and mortality during influenza infection (51, 52) . the severity of infection was associated with reductions in dendritic cell migration rate and the number of local t cells. mice given a 4 week oral course of broad-spectrum antibiotics before respiratory viral infection mounted an attenuated anti-pr8 antibody response, were incapable of inducing cd4 + t cell-mediated ifn-γ response to pr8 antigen, and had fewer influenza-specific cd8 + t cells (51, 52) . these mice also had higher viral titers in their lungs (51) . germ-free mice and antibiotic-treated mice also exhibit impaired antibody responses to seasonal influenza vaccination, which was restored by oral administration of flagellated e. coli, demonstrating a dependence on tlr5-mediated sensing of the host microbiota (53) . the gut microbiome is essential for priming innate immune responses against pulmonary infections as well. during viral infections, the degree of macrophage response to respiratory viruses depends on the presence of gut microbes. macrophages from animals treated with antibiotics exhibited defective responses to type i and ii ifns and impaired capacity to limit viral replication, suggesting that intestinal microbiota provide immune stimulation that establishes an "activation threshold" for innate antiviral immune responses (52) . a comparison of c57bl/6 mice from the jackson laboratory (which lack sfb in the stool) and taconic biosciences (which are sfb positive) revealed that sfb-deficient animals have increased lung bacterial burdens and more severe pneumonia when challenged with methicillin-resistant staphylococcus aureus (mrsa) (54) , which was associated with decreased il-17-mediated responses in the lung. another study using broad-spectrum antibiotic treatment followed by intranasal administration of s. pneumoniae in mice demonstrated that microbiome depletion led to decreased survival, increased lung bacterial burden, and increased systemic dissemination of bacteria (55) . antibiotic-pretreated animals displayed altered cytokine profiles in the lung compared to untreated controls following s. pneumoniae infection, including significantly decreased tnf-α levels at 6 and 24 h after infection. additionally, in the microbiota-depleted group, alveolar macrophages and blood neutrophils exhibited decreased phagocytic activity, and decreased inflammatory cytokine production following ex vivo stimulation by toll-like receptor (tlr) ligands such as lipoteichoic acid (lta) (55) . these effects might be mediated in part by decreased nod1 sensing of meso-dap (diaminopimelic acid)-containing peptidoglycan found in gut microbiota, which previously was shown to be essential for priming innate immune responses to s. pneumoniae (56) . thus, antibiotic-induced disruptions in the normal gut microbial community alter multiple aspects of normal host defense against acute respiratory pathogens (figure 2 ). collectively, the studies above suggest that modulation of the gastrointestinal tract microbiome plays an important role in acute respiratory infections, but precisely how the microbiome should be manipulated to promote appropriate immune responses during acute respiratory infections is unclear. currently, clinical studies have shown that although probiotics do not influence the incidence of respiratory tract infection, they do reduce the severity of symptoms and duration of the illness (57, 58) . pinpointing which members of the gut microbial community are essential for proper immune priming is challenging, but necessary for guiding further microbiome-based therapies. clostridium orbiscindens, a member of the human gut microbiome, has been found to produce desaminotyrosine (dat) from metabolism of flavonoids and amino acids. antibiotic-treated mice exhibited markedly decreased fecal and serum dat levels, which was associated with attenuated type i ifn responses to influenza infection and increased mortality (59) . thus, identification of dat-producing microbiota might serve as a modality for priming type i ifn responses against viral infections. another group demonstrated that oral administration of lactobacillus plantarum enhanced the type i ifn response and lowered viral titers in the lungs in a murine model of influenza infection (60) . other lactobacillus strains are known to enhance tnf-α and ifnγ production by nasal lymphocytes upon influenza infection (61) . oral administration of a probiotic cocktail containing lactobacillus restored the immune response and enhanced the activation of signaling pathways associated with recognition of single-stranded rna virus (62) . an alternative approach to administering probiotics is to alter the local metabolic environment to regulate immune responses. a recent report demonstrated that animals fed a high fiber diet had increased generation of scfas, leading to enhanced antiviral cd8 + t cell immune responses and attenuated neutrophil-mediated lung injury during influenza infection, resulting in improved survival (63) . thus, one strategy for decreasing the incidence of post-viral bacterial infections is to limit the severity of the primary viral infection. however, activation of antiviral immune responses, including type i and type ii ifns, have been associated with increased susceptibility to secondary bacterial pneumonia (64, 65) . thus, another strategy is to enhance immune responses against common bacterial causes of pneumonia. one group re-colonized antibiotic-treated or germ-free mice with groups of cultivatable commensal bacteria, and found that administration of lactobacillus reuteri, enterococcus faecalis, lactobacillus crispatus, and clostridium orbiscindens, which are strong stimulators of nod2 (i.e., cytosolic receptor for muramyl dipeptide, which is found in cell walls of certain bacteria), are able to protect against bacterial pneumonia by enhancing gm-csf production (66) . whether viral-induced changes in the gut microbiome is associated with immune defects that promote secondary bacterial pneumonia, or whether the impaired antibacterial defenses observed in virally-infected hosts can be restored by augmenting certain components of the microbiome are important areas to be investigated. the microbiome of the respiratory tract has also been investigated in the context of viral infections. its role in the development of secondary bacterial pneumonia following influenza and other acute respiratory viral infections is unclear. the respiratory tract is the main site of continuous contact with frontiers in immunology | www.frontiersin.org figure 2 | effects of antibiotic pre-treatment on immune responses to influenza, streptococcus pneumoniae, and lipoteichoic acid (lta). the effects of the gut microbiome on immune responses to respiratory pathogens have been investigated by administration of oral antibiotics to generate alterations in the gut flora, followed by acute infection, and analyzing host immune responses compared to non-antibiotic-pretreated animals. multiple aspects of innate and adaptive immune responses are altered in antibiotic treated animals, including decreased antibody production, decreased phagocytic activity, and decreased inflammatory cytokine production by innate immune cells (e.g., alveolar and peritoneal macrophages) following ex vivo stimulation with tlr ligands. exogenous microbes. as is the case with the gut, immunity at the mucosal interface of the respiratory tract is a constant balance of tolerance of commensal and non-invasive microbes and immune activation against pathogens. the urt and lrt have similar microbial community compositions, although microbe densities are much higher in the former in healthy hosts. several factors are known to influence airway microbiome composition including infection history, age, genetics, and structural lung disease. the urt is an interconnected system consisting of the anterior nares, nasal cavity, nasopharynx, sinuses, eustachian tube, middle ear cavity, oral cavity, oropharynx, and larynx, each of which serve as distinct niches with their own microbial communities. in healthy adults, bacteria present in the nasal cavity are typically those associated with skin, predominantly members of the actinobacteria (e.g., corynebacterium spp., propionibacterium spp.), followed by firmicutes (e.g., staphylococcus spp.), and proteobacteria (67) (68) (69) . the oropharynx contains members of firmicutes, proteobacteria, and bacteroidetes, including streptococcus, neisseria, haemophilus, and lachnospira spp. (68, 70, 71) . skin and oral cavity lineages are represented in the nasopharynxe.g., streptococcus, staphylococcus, corynebacterium, and prevotella (70, 72, 73) . a limited number of pathogens including streptococcus pneumoniae, neisseria meningitides, and haemophilus influenzae are commensal bacteria of the urt. in healthy individuals, the microbial community richness (i.e., the total number of bacterial taxa) is lower in the lrt than that in the urt (70, (74) (75) (76) . contrary to dogma that normal healthy lungs are a sterile environment, a distinct, and somewhat dynamic lung microbiome can be identified using sequencing technology, with microaspiration serving as the primary route of microbial immigration from the urt to the lrt (76, 77) . the major phyla in healthy lungs are bacteroidetes and firmicutes, which mainly include prevotella, veillonella, and streptococcus (78) (79) (80) . individuals with chronic airway diseases (e.g., cystic fibrosis, copd) have increased bacterial populations in the lungs (77) and differences in the relative abundance of certain species (81) . impaired airway clearance due to intrinsic or extrinsic factors leads to the proliferation of bacterial species that can exploit this growth opportunity (82) . how respiratory viral infection affects the diversity of microbial communities and whether viral-induced dysbiosis influences immune functions is being examined. nonetheless, bacterial colonization of the urt is generally considered as the first step in the development of invasive bacterial infections (83, 84) , including secondary bacterial infections following respiratory viral infection. bacterial abundance, species diversity, and factors that shape the immune response to subsequent infections are discussed in greater detail below. respiratory viruses enter the human body through the urt and are the most common type of acute infections of the respiratory tract. one possible mechanism by which influenza and other viral infections might predispose infected hosts to secondary bacterial pneumonia is by altering the microbial composition of the upper respiratory tract, fostering enhanced growth of pathogens, and facilitating the subsequent entry of large bacterial loads into the lrt (85) . this section will examine recent literature on how acute respiratory viral infections have changed the urt microbiome. given the effects of viruses on enhancing bacterial adherence to the epithelium (86) (87) (88) , it is perhaps not surprising that multiple studies of human subjects as well as in animal models have shown that viral infections are associated with increased colonization by potentially pathogenic bacteria (known as "pathobionts"). a comparative analysis using qpcr to detect specific bacteria in adult patients with or without influenza a infection showed that staphylococcus aureus, s. pneumoniae, and h. influenzae were present in 12, 24, and 32% of infected patients, respectively as compared to 5, 11, and 10% of uninfected patients (89) . in experimental in vitro models, viral infections increase the colonization rates of various bacteria in the urt (90-95), including s. pneumoniae and h. influenzae (86) (87) (88) . in children, influenza is associated with a 15-fold increase in nasopharyngeal titer of s. pneumoniae (96) . animal models have similarly confirmed that viral infection, particularly influenza, increases bacterial colonization rates in the urt, enhancing the risk of secondary bacterial infections (97) (98) (99) . higher pneumococcal colonization density has been linked to respiratory virus coinfection and invasive pneumococcal pneumonia, after adjusting for age and sex (85) . another case-control study comparing nasopharyngeal bacteria with and without pneumonia also found an association between nasopharyngeal load of s. pneumoniaebut not of h. influenza and m. catarrhalis-and viral coinfection and pneumonia (96) . in addition, viral infections potentially may enhance transmission of bacteria. in a study of mice colonized with s. pneumoniae and then infected with influenza a virus 3 days after, s. pneumoniae transmission occurred only when all mice were infected with influenza and was blocked by an influenza-neutralizing antibody (95) . however, while specific bacteria might gain a competitive advantage during viral infections, this does not universally translate to all bacterial taxa. a recent study of subjects with and without respiratory viral infections demonstrated lower overall bacterial reads from nasopharyngeal samples in virally-infected subjects compared with uninfected controls (100) . the relationship between acute viral infections and bacterial colonization appears to be bidirectional. bacterial carriage or their ligands can increase or decrease viral infectivity rate, thereby positively or negatively influencing the subsequent host immune response to viral infection. viral replication in the respiratory tract can be enhanced by exposure to s. pneumoniae (101) . patients harboring s. pneumoniae are more likely to experience subsequent acute respiratory illness episodes than those without colonization (102) . in addition, bacteria present in the airways can modulate host responses against viral infection. the presence of a nasopharyngeal commensal protected mice against rsv-induced airway hyperresponsiveness. rsv-infected mice who underwent antibiotic-mediated depletion of streptococcus viridans in the nasopharynx exhibited increases in number of inflammatory lymphocytes and airway hyperresponsiveness, and decreases in regulatory t cell number and transforming growth factor-β production (103) . others have shown that colonization of the urt with s. aureus drastically reduced influenza-induced acute lung injury and mortality in mice by recruiting a c-c chemokine receptor type 2 + cluster of differentiation (cd)11b + monocyte subset to the lungs and inducing an m2 macrophage phenotype (104) . with the availability of next-generation 16s rrna sequencing, microbiome-based studies have attempted to discern global patterns of change in the bacterial community of each anatomic niche during viral infections, such as changes in diversity. diversity can be assessed using a variety of indices, such as total number of unique species of the microbiome (i.e., richness) or other measures that account for both richness and the evenness of relative abundance of the members of the community (e.g., shannon index). results from microbiome analyses have not demonstrated consistent changes in diversity when comparing virally infected subjects with healthy controls. this is not surprising given the variability of the subjects sampled, differences in type and severity of viral infections, type and timing of sample collection, and analysis methodology. in some studies, increased bacterial diversity appeared to be associated with influenza severity. a french study of children admitted to the hospital with influenza revealed increased diversity of the nasopharyngeal microflora with increased influenza severity (105) . children with severe influenza showed decreased relative abundance of s. aureus and increased abundance of prevotella, streptobacillus, porphyromonas, granulicatella, veillonella, fusobacterium, and haemophilus. a recent chinese study in patients with h7n9 avian influenza demonstrated significantly increased diversity in the oropharyngeal microbiome of h7n9-infected patients compared to healthy controls, particularly h7n9 patients with secondary bacterial pneumonia (106) . conversely, a french study of nasopharyngeal samples and a south korean study of oropharyngeal samples from patients with acute respiratory viral infections both displayed decreases in diversity indices during viral infections compared to healthy controls (71, 100) . both studies included subjects ranging from infants to adults >80 years of age, limiting conclusions about age-related effects. longitudinal studies conducted in healthy volunteers who underwent experimental self-innoculation with rhinovirus also failed to demonstrate significant changes in diversity of the urt microbiome, while administration of laiv vaccine to healthy adults led to increases in diversity measures following viral challenge (73, 107) . thus, unlike other diseases where decreased diversity is considered deleterious to the host, the effects of viral infections on diversity per se are variable and not presently considered a good indicator of risk for complications, including secondary bacterial pneumonias. microbiome sequencing studies also enable investigators to identify changes in abundance among multiple bacterial taxa simultaneously, beyond just what can be cultured individually. this allows investigators to determine what groups of bacteria are changing in unison during viral infection and which are existing in competition with one another. this information may have implications for the development of probiotic therapies (as discussed below). a recent metagenomics-based study in france reported enrichment of s. aureus, s. pneumoniae, h. influenzae, moraxella catarrhalis and klebsiella pneumoniae in nasopharyngeal samples of subjects with confirmed respiratory viral infections compared to healthy controls (100) . an examination of the oropharyngeal microbiome of pneumonia patients with and without 2009 influenza a h1n1 pandemic viral infection showed that firmicutes (which include staphylococcus and streptococcus spp.) and proteobacteria (mainly pseudomonas amygdali, p. fluorescens, pseudomonas sp. uk4, acinetobacter baumanii and a. junii)-were significantly enriched in patients with influenza (108) . another study of patients with 2009 pandemic h1n1 influenza infection revealed that the predominant phyla of the upper respiratory tract (nasal and nasopharyngeal samples) in patients harboring pandemic h1n1 were actinobacteria, firmicutes, and proteobacteria although normal controls were not included; however, the authors suggested that flu is associated with an expansion of proteobacteria (109) which is generally less abundant in healthy hosts. these findings are supported by another group who found that moraxella and enterobacter spp. (which are classified as proteobacteria) were the most highly represented bacteria in nasopharyngeal samples obtained from patients with pandemic h1n1 influenza (110) . however, these studies demonstrated that there was considerable inter-subject variability, highlighting the need for longitudinal studies to decipher changes following viral infection. investigators have also sought to determine whether specific viruses are consistently linked to enrichment of certain bacterial taxa. in the nasopharyngeal compartment of aboriginal and non-aboriginal children in australia, positive associations were detected between hrv and s. pneumoniae, h. influenza, and moraxella catarrhalis carriage as well as between adenovirus and m. catarrhalis (111) . another study examining the presence of 20 respiratory viruses by pcr panel and prevalence of bacterial carriage in the nasopharynx of children found a strong positive association between s. aureus colonization and influenza virus (112). moreover, s. pneumoniae colonization was positively associated with the presence of hrv and enteroviruses; h. influenzae was positively associated with hrv and rsv; and m. catarrhalis colonization was positively associated with coronaviruses and adenoviruses. a 16s rrna sequencing-based study conducted in infants with acute rsv or hrv respiratory infections reported that infants with rsv had significantly higher abundance of staphylococcus spp. compared to hrv-infected infants (113) . an analysis of the urt bacterial content of 57 healthy asymptomatic individuals and 59 patients with influenza virus, parainfluenza, hrv, rsv, coronavirus, adenovirus, or metapneumovirus by culture-independent pyrosequencing revealed six distinct bacterial profiles-i.e., streptococcus + prevotella + veillonella, streptococcus + haemophilus + neisseria, streptococcus, moraxella, haemophilus, and klebsiella. these profiles, however, were not associated with virus type but were linked to the age of subjects (71) . given that many human studies are cross-sectional in nature, it remains unclear whether post-viral bacterial pneumonias might be the result of viral infections enhancing bacterial colonization or acquisition, colonizing bacteria influencing host susceptibility to respiratory viral infections, or a combination of both. another complicating factor particularly in cross-sectional studies examining the microbiome during viral infections is that the groups are not well-controlled and the sample numbers are relatively small considering the number of variables that could affect the respiratory tract microbiome-such as age, gender, oral hygiene and nose-picking habits, healthcare-based employment status, smoking status, medication use, exposure to small children, etc. the underlying type of viral infection, sampling timepoint after onset of infection, severity of infection, and concomittant antimicrobial usage are other confounding factors. this may underlie the highly variable and sometimes discrepant observations from microbiome studies in patients with viral infections. there have been few clinical studies comparing baseline pre-and post-infection microbiomes in otherwise healthy individuals with acute viral infections due to the difficulty of sampling before infection. however, the relatively few studies available provide insights into the dynamicity and stability of bacteria colonization patterns over time, and whether and how perturbations brought on by acute viral infections alter these patterns. in healthy children, the major phyla among nasopharyngeal microbiotas are proteobacteria, firmicutes, bacteroidetes, actinobacteria, and fusobacteria, with moraxella, haemophilus, streptococcus, flavobacteria, dolosigranulum, corynebacterium, and neisseria as predominant genera. changes in nasopharyngeal microbiome diversity were observed across seasons, with a predominance of proteobacteria and fusobacteria in fall-winter and bacteroidetes and firmicutes in spring; these differences were independent of recent antibiotics and viral co-infection (114) . however, another analysis of two nasopharyngeal washes collected 5.5-6.5 months apart from 40 children and adolescents with asthma showed no significant differences in nasopharyngeal microbiome diversity across seasons, although mean relative abundances of haemophilus, moraxella, staphylococcus, and corynebacterium varied significantly between summer and fall samples and between age groups. moreover, in 87.5% of patients, operational taxonomic units (otus) in patients varied significantly between time points (115) . an investigation of the frequency and seasonal variation in bacterial and viral load in asymptomatic healthcare professionals during the winter and summer months showed that of the 100 subjects tested during the winter, 34 were colonized with at least one bacterial species and 11 tested positive for at least one virus. the most frequently detected pathogens were methicillinresistant staphylococcus aureus (mrsa), m. catarrhalis, and coronavirus. in contrast, of the 100 subjects tested during the summer, 37 harbored at least one bacterium (mainly mrsa and k. pneumoniae) and four tested positive for one virus (116) . several larger scale surveillance studies of mainly pediatric populations have examined the natural temporal patterns in bacterial colonization during viral infections. one clinical investigation assessed the presence and density of s. pneumoniae, h. influenzae, and m. catarrhalis in the nasopharynx of children during urt infection and in the healthy state, and reported that the proportion of children colonized with these bacteria was higher during infection than during asymptomatic surveillance visits. mean density of all bacterial species was significantly higher at each visit when a virus was detected. interestingly, the percentage of colonized children and bacterial density were also higher at asymptomatic visits in which virus was detected than at those in which virus was not detected (117) . another study of 31 families with small children using longitudinal nasal swab sampling demonstrated that rhinovirus infection was associated with increased acquisition of s. pneumoniae from the community as well as increased transmission of s. pneumoniae within the family (118) . other groups have examined the effects of experimental innoculation of hrv into the urt (nares) (figure 3) . these studies reported no significant changes in total read counts or of the main phyla (e.g., actinobacteria, firmicutes, and proteobacteria) over time in nasopharyngeal samples (73) or throat swabs (119) . in the oropharyngeal compartment, rhinovirus infection was associated with a strong trend toward transient increases in the relative abundances of h. parainfluenzae, neisseria subflava and a weak trend toward an increase in s. aureus (119) . by 60 days, abundance of these bacteria had returned to baseline. nasopharyngeal sampling showed completely opposite results, with decreased relative abundance of haemophilus and neisseria spp., but an increase in the normal nasal commensal, propionibacterium, in subjects following hrv infection (73) . no differences in staphylococcus were observed. however, the number of subjects were small in both studies, limiting the power to detect changes over time. nasopharyngeal microbiota composition has been shown to be altered by influenza vaccination (figure 3) . administration of live attenuated influenza vaccine (laiv), which is nasally instilled, to healthy children increased the nasal colonization density of s. pneumoniae in subjects who harbored this bacterium at the time of vaccination, and transiently increased rates of colonization by h. influenza (120) . in healthy adult volunteers, it was demonstrated that intranasal laiv administration induced an increase in the diversity of the nasopharyngeal microbiome, figure 3 | changes in the human upper respiratory tract microbiome following viral exposure. given that bacterial pneumonia frequently arises as a result of aspirated bacterial pathogens, a potential mechanism by which viral infections might increase the risk of secondary bacterial infections is through increased colonization of the upper respiratory tract by bacterial pathogens. in human subjects, live attenuated influenza vaccine (laiv) and human rhinovirus (hrv) have been shown to disrupt the local host bacterial community, with increased relative abundance of potential pathogens (or pathobionts), such as staphylococcal and neisseria species. the major changes in the upper respiratory tract microbiome are highlighted here. as well as relative abundances of staphylococcus and bacteroides (107). these changes were not observed in subjects given saline nasal spray. in a mouse model, bacterial density in the nasopharynx after laiv administration was increased as much as 100,000 times compared to influenza-naive hosts, and the duration of carriage of s. pneumoniae or s. aureus was also increased 2 to 5-fold (121) . however, systemic vaccination can also alter the urt microbiome. a longitudinal study of healthy subjects found a significant association between the presence of lactobacillus helveticus, prevotella melaninogenica, streptococcus infantis, veillonella dispar, and bacteroides ovatus and influenzaspecific h1 and h3 iga antibody response (122) . thus, it is remarkable that a relatively mild viral stimulus such as flu vaccine can lead to detectable changes in the urt microbiome. although the data are still preliminary, animal studies have suggested that antiviral immune activation contributes to changes in the urt microbiome and facilitate colonization by potential pathogens, such as s. aureus. in a mouse model of s. aureus nasal colonization, the absence of type i ifn receptor was associated with decreased persistence of bacteria (107) . type iii ifn, which is also induced during influenza infections, led to changes in the nasal microbiome, including increased numbers of culturable bacteria. increased upper respiratory tract persistence of s. aureus as well as increased risk of s. aureus pneumonia was observed in flu-infected wildtype mice compared to mice lacking the type iii ifn receptor (123) . currently, however, is it unclear to what extent viral-induced changes in the urt microbiome alter subsequent immune responses against secondary bacterial infections. compared to studies of the urt microbiome, studies of the lrt microbiome following viral infections are relatively scarce due to the difficulty of obtaining uncontaminated samples from the lung. samples of convenience, such as sputum, suffer from oral contamination, but bronchoscopic samples are invasive and expensive to obtain on a regular basis. moreover, it is unclear whether outside of patients with chronic lung disease (e.g., copd), the lung microbial burden is of sufficient magnitude to exert robust effects on immune responses and risk of secondary bacterial infection during viral infection. data from a mouse model of influenza infection seem to indicate that flu infection has only a modest effect on bacterial counts, diversity and composition of the lung microbiome (46) . in subjects with chronic obstructive pulmonary disease (copd) after hrv infection but not in healthy individuals, there was an increase in bacterial burden and growth of bacteria present at baseline, particularly h. influenzae (124) . the researchers observed that the growth of bacteria seemed to arise from the existing community. s. pneumoniae intranasally inoculated into mice pre-infected with influenza virus first colonized the nose, followed by the trachea and lungs several days later with purulent inflammation. however, this effect was not observed in uninfected animals. this suggests that pneumococcal infection may sequentially develop from the urt to the lrt in influenza virus-infected subjects (97) . thus, it is possible that some individuals with influenza infection might develop changes in their lung microbiome as a result of changes in their urt microbial communities. respiratory viruses not only alter the bacterial community in the urt, but also promote bacterial colonization of the lrt by a variety of mechanisms that impair bacterial clearance. first, mucus production in the respiratory tract is increased to facilitate viral clearance during infections. however, excessive mucus production can lead to airway obstruction by impeding mucociliary clearance (125) . second, viral infections can also reduce ciliary beat frequency and the number of ciliated cells, disrupt the coordinated movement of cilia, and impede the repair of respiratory epithelial cells, further leading to reduced mucociliary clearance (126, 127) . third, respiratory viral infections impair innate immune responses against bacteria (128) (129) (130) . innate immune cells including macrophages and neutrophils are recruited to the lung by cytokines and chemokines for phagocytosis and bactericidal activity. prior viral infections dysregulate both alveolar macrophages (64, (131) (132) (133) (134) (135) (136) and neutrophils (65, 128, 129) , thereby inhibiting bactericidal activity. thus, with multiple aspects of pulmonary host defense impaired, it would not be entirely surprising if a subset of influenza infected patients developed secondary bacterial pneumonia as a result of being unable to clear aspirated pathobionts from the urt. in addition to enabling us to determine what is present during states of health, large-scale sequencing-based microbiome analyses have also revealed who is not present during disease. it has long been appreciated that mechanisms have evolved in bacteria that confer competitive advantages, permitting them to survive in an otherwise inhospitable host environment. however, interspecies competition also maintains homeostasis of the microbial community, either through their abilities to capture scare resources (e.g., iron), or targeted killing of other bacteria (e.g., bacteriocins), preventing one microbe from dominating the community. thus, it is possible that the immune response incited by acute viral infections, changes in the host epithelial surface caused by the virus, or the virus itself might lead to elimination of a host commensal that is responsible for keeping pathobionts in check. for example, s. epidermidis and propionibacterium acnes abundance in the nares has been shown to be negatively associated with s. aureus carriage (67) . understanding these interactions may create new avenues for therapeutic interventions aimed at reducing colonization by pathogenic bacteria during influenza epidemics or pandemics. one group of commensals that has been examined for its role in inhibiting nasal carriage by s. aureus and s. pneumoniae is corynebacterium spp. an early study in japan reported on the effects of introducing a corynebacterium strain into the nares of healthy adult hospital workers who were persistent carriers of s. aureus, with successful eradication in 71% of subjects (137). the mechanism appeared to be bacteriocin-independent. in comparison, s. epidermidis implantation did not have an effect. whether the s. epidermidis strain used expressed the serine protease esp, which inhibits biofilm formation by s. aureus and nasal colonization (138) , is unknown. subsequent studies by another group reported that c. pseudodiphtheriticum inhibited s. aureus growth, whereas c. accolens and s. aureus appeared to support each other's growth (139) . conversely, other investigators observed that corynebacterium spp. were enriched in children who were not nasally colonized with pneumococcus, and demonstrated that c. accolens inhibit s. pneumoniae growth in vitro by expressing a lipase that releases free fatty acids from skin surface triacylglycerols, which inhibit pneumococcal growth. thus, painstaking identification and mechanistic interrogation of interspecies competition between commensals might lead to novel insights as to how viral infections might confer competitive advantage to pathobionts, and how to exploit natural strategies employed by commensals to restore homeostasis to the host microbial niche. interestingly, a recent preclinical study using a murine model of rsv and s. pneumoniae superinfection employed nasal priming by a c. pseudodiphtheriticum strain to augment host defense against the viral infection, which enhanced clearance of secondary bacterial challenge and reduced lung injury measures (140) . finally, direct effects of the infecting virus on bacteria that comprise the microbiome may facilitate the transition from pathobiont to pathogen. a metagenomic analysis showed that ph1n1-associated airway microbiotas were enriched in genes associated with cell motility, transcriptional regulation, metabolism, and response to chemotaxis compared to the same bacteria in non-infected patients (108) . these data imply that influenza infection perturbs the respiratory microbiome, leading to the production of secondary metabolites including immune-modulating molecules. viruses have also been found to impair bacterial biofilm formation and disrupt existing biofilm (141) (142) (143) (144) . influenza has been shown to affect the s. pneumoniae transcriptome in terms of downregulating expression of genes associated with the colonizer state and upregulations of bacteriocins (142) . thus, direct effects of viruses on bacterial transcriptional patterns might be a mechanism by which colonizing bacteria acquire invasive potential, thereby leading to bacterial superinfections. there are several areas that must be addressed by future respiratory microbiome research. first, it is necessary to standardize protocols used to analyze the respiratory microbiome, including sampling, processing, and bioinformatics methodologies. for example, sputum may be an appropriate material for investigations of respiratory diseases since it contains components of the lrt and can be obtained easily. however, more reliable information on the lrt requires invasive samples such as bal or protected specimen brush frontiers in immunology | www.frontiersin.org or bronchial/lung biopsies. second, most studies are limited to experiments conducted in animal models. even in human studies, most analyses have been performed in a small number of patients and have described bacterial communities in the urt. the role of microbial communities outside of the lungs including gut, sinus, and skin should be considered in the context of airway diseases. third, most studies on the microbiome have focused on the bacterial component, and have largely omitted fungi and viruses. the role of viruses-including the vast number of phages that infect bacteria-and fungi in respiratory diseases cannot be examined through 16s rrna gene analyses, and there are no studies describing the composition and role of the respiratory virome due to the difficulty of comprehensive analyses for viruses. fourth, it is not sufficient to study microbial communities based on species composition; a functional characterization through transcriptome and proteasome analyses is necessary to understand mechanistic role of microbiome on outcomes of infection. finally, mucosal microbiome manipulations by vaccines, antibiotics, and probiotics in the gastrointestinal and respiratory tract niches represent novel approaches for the prevention, treatment, and management of acute and chronic lung diseases. however, given that antibiotic therapy could affect commensal bacteria and hasten the emergence of drug-resistant bacteria, more research is needed on the long-term effects of this therapy. animal models should be developed to study the influence of the urt and lrt microbiomes on immune responses to respiratory viral infections; only then will it be possible to consider the clinical application of microbiome modulation strategies. respiratory viral infections can initiate a cascade of host immune responses that alter microbial growth conditions in the urt, lrt, and the gut (supplemental table 1 ). activation of influenzainduced antiviral interferon pathways can lead to inadequate innate immune cell responses during host defense against secondary bacterial infections, resulting in the proliferation of potentially pathogenic bacterial species. concomitant changes in the gut microbiome caused by the initial viral infection may also alter immune cell priming against secondary bacterial challenge, although this has not been examined to date. although the picture is incomplete, recent microbiome literature provides additional insights into the pathogenesis of dysregulated immune responses following acute viral infections, that may promote the development of secondary bacterial pneumonias (figure 4) . clarifying the differences and dynamics of respiratory microbiota in healthy subjects and chronic lung diseases during acute respiratory viral infections can elucidate pathogenesis of viralbacterial interactions and provide a basis for developing novel approaches for the prevention, treatment, or management of acute respiratory infection and exacerbation of chronic lung diseases. sh and jd co-wrote the manuscript. sh, jd, and mp designed the figures and table. kc and mp edited and provided critical revisions of the manuscript. all authors approve the final version and agree to be accountable for the content of the manuscript. jd is supported by a research grant from the national institutes of health (grant no. r01hl108949). the views expressed in this article are those of the authors and do not necessarily reflect the position or policy of the department of veterans affairs or the us government. we thank cat meyer for her assistance with the figures. the supplementary material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fimmu. 2018.02640/full#supplementary-material interactions between influenza and bacterial respiratory pathogens: implications for pandemic preparedness bacterial pathogens and death during the 1918 influenza pandemic updating the accounts: global mortality of the 1918-1920 "spanish" influenza pandemic insights into the interaction between influenza virus and pneumococcus the 1918 influenza pandemic: insights for the 21st century predominant role of bacterial pneumonia as a cause of death in pandemic influenza: implications for pandemic influenza preparedness the origin and virulence of the 1918 "spanish" influenza virus pneumonia and hong kong influenza: a prospective study of the 1968-1969 epidemic bacteriology and histopathology of the respiratory tract and lungs in fatal asian influenza bacterial co-infection with h1n1 infection in patients admitted with community acquired pneumonia a/h1n1 viral infections hospitalized patients with 2009 h1n1 influenza in the united states critically ill patients with 2009 influenza a(h1n1) infection in canada community-acquired respiratory coinfection in critically ill patients with pandemic 2009 influenza a(h1n1) virus streptococcus pneumoniae coinfection is correlated with the severity of h1n1 pandemic influenza pandemic influenza a (h1n1): pathology and pathogenesis of 100 fatal cases in the united states critically ill patients with 2009 influenza a(h1n1) in mexico the microbiome in infectious disease and inflammation human microbiome project consortium. structure, function and diversity of the healthy human microbiome wild mouse gut microbiota promotes host fitness and improves disease resistance immunobiology of germfree mice infected with nocardia asteroides increased susceptibility to primary infection with listeria monocytogenes in germfree mice may be due to lack of accumulation of l-selectin+ cd44+ t cells in sites of inflammation germ-free mice produce high levels of interferon-gamma in response to infection with leishmania major but fail to heal lesions virus-helminth coinfection reveals a microbiotaindependent mechanism of immunomodulation susceptibility of germ-free pigs to challenge with protease mutants of salmonella enterica serovar typhimurium role of the indigenous microbiota in maintaining the virus-specific cd8 memory t cells in the lung of mice infected with murine cytomegalovirus sequential infection with common pathogens promotes human-like immune gene expression and altered vaccine response normalizing the environment recapitulates adult human immune traits in laboratory mice fermentation in the human large intestine: evidence and implications for health the microbial metabolites, short-chain fatty acids, regulate colonic treg cell homeostasis metabolites produced by commensal bacteria promote peripheral regulatory t-cell generation diet-derived short chain fatty acids stimulate intestinal epithelial cells to induce mucosal tolerogenic dendritic cells gut microbial metabolites limit the frequency of autoimmune t cells and protect against type 1 diabetes impacts of microbiome metabolites on immune regulation and autoimmunity enhanced production of il-18 in butyrate-treated intestinal epithelium by stimulation of the proximal promoter region expression of the cathelicidin ll-37 is modulated by short chain fatty acids in colonocytes: relevance of signalling pathways histone-deacetylase inhibitors induce the cathelicidin ll-37 in gastrointestinal cells apathogenic, intestinal, segmented, filamentous bacteria stimulate the mucosal immune system of mice segmented filamentous bacteria are potent stimuli of a physiologically normal state of the murine gut mucosal immune system induction of intestinal th17 cells by segmented filamentous bacteria the key role of segmented filamentous bacteria in the coordinated maturation of gut helper t cell responses respiratory influenza virus infection induces intestinal immune injury via microbiota-mediated th17 cell-dependent inflammation influenza virus affects intestinal microbiota and secondary salmonella infection in the gut through type i interferons impact of age, caloric restriction, and influenza infection on mouse gut microbiome: an exploratory study of the role of age-related microbiome changes on influenza responses influenza a virus infection impacts systemic microbiota dynamics and causes quantitative enteric dysbiosis respiratory disease following viral lung infection alters the murine gut microbiota tolllike receptor-induced innate immune responses in non-parenchymal liver cells are cell type-specific microbial ecology: human gut microbes associated with obesity one-year calorie restriction impacts gut microbial composition but not its metabolic performance in obese adolescents microbiota regulates immune defense against respiratory tract influenza a virus infection commensal bacteria calibrate the activation threshold of innate antiviral immunity tlr5-mediated sensing of gut microbiota is necessary for antibody responses to seasonal influenza vaccination intestinal microbiota of mice influences resistance to staphylococcus aureus pneumonia the gut microbiota plays a protective role in the host defence against pneumococcal pneumonia recognition of peptidoglycan from the microbiota by nod1 enhances systemic innate immunity probiotic bacteria reduced duration and severity but not the incidence of common cold episodes in a double blind, randomized, controlled trial probiotics for the prevention of respiratory tract infections: a systematic review the microbial metabolite desaminotyrosine protects from influenza through type i interferon oral administration of heat-killed lactobacillus plantarum l-137 enhances protection against influenza virus infection by stimulation of type i interferon production in mice augmentation of cellular immunity and reduction of influenza virus titer in aged mice fed lactobacillus casei strain shirota microbiota regulates the tlr7 signaling pathway against respiratory tract influenza a virus infection dietary fiber confers protection against flu by shaping ly6c-patrolling monocyte hematopoiesis and cd8+ t cell metabolism inhibition of pulmonary antibacterial defense by interferon-gamma during recovery from influenza infection type i ifns mediate development of postinfluenza bacterial pneumonia in mice the microbiota protects against respiratory infection via gm-csf signaling the human nasal microbiota and staphylococcus aureus carriage comparative analyses of the bacterial microbiota of the human nostril and oropharynx the nasal cavity microbiota of healthy adults topographical continuity of bacterial populations in the healthy human respiratory tract profiling bacterial community in upper respiratory tracts pyrosequencing analysis of the human microbiota of healthy chinese undergraduates characterization of the nasopharyngeal microbiota in health and during rhinovirus challenge bacterial topography of the healthy human lower respiratory tract sinus microbiome diversity depletion and corynebacterium tuberculostearicum enrichment mediates rhinosinusitis analysis of the upper respiratory tract microbiotas as the source of the lung and gastric microbiotas in healthy individuals application of a neutral community model to assess structuring of the human lung microbiome comparison of the respiratory microbiome in healthy nonsmokers and smokers enrichment of lung microbiome with supraglottic taxa is associated with increased pulmonary inflammation spatial variation in the healthy human lung microbiome and the adapted island model of lung biogeography respiratory microbiota: addressing clinical questions, informing clinical practice interpreting infective microbiota: the importance of an ecological perspective the role of nasal carriage in staphylococcus aureus infections streptococcus pneumoniae colonisation: the key to pneumococcal disease high nasopharyngeal pneumococcal density, increased by viral coinfection, is associated with invasive pneumococcal pneumonia effects of rhinovirus infection on the adherence of streptococcus pneumoniae to cultured human airway epithelial cells respiratory viruses augment the adhesion of bacterial pathogens to respiratory epithelium in a viral species-and cell type-dependent manner rhinovirus enhances various bacterial adhesions to nasal epithelial cells simultaneously quantitative detection of staphylococcus aureus, streptococcus pneumoniae and haemophilus influenzae in patients with new influenza a (h1n1)/2009 and influenza a/2010 virus infection effects of influenza a virus on lectin-binding patterns in murine nasopharyngeal mucosa and on bacterial colonization henriques normark b. influenza enhances susceptibility to natural acquisition of and disease due to streptococcus pneumoniae in ferrets effect of experimental influenza a virus infection on isolation of streptococcus pneumoniae and other aerobic bacteria from the oropharynges of allergic and nonallergic adult subjects influenza a virus alters pneumococcal nasal colonization and middle ear infection independently of phase variation synergistic stimulation of type i interferons during influenza virus coinfection promotes streptococcus pneumoniae colonization in mice influenza a virus facilitates streptococcus pneumoniae transmission and disease association between nasopharyngeal load of streptococcus pneumoniae, viral coinfection, and radiologically confirmed pneumonia in vietnamese children facilitated expansion of pneumococcal colonization from the nose to the lower respiratory tract in mice preinfected with influenza virus effect of adenovirus type 1 and influenza a virus on streptococcus pneumoniae nasopharyngeal colonization and otitis media in the chinchilla bacterial sinusitis and otitis media following influenza virus infection in ferrets the nasopharyngeal microbiota in patients with viral respiratory tract infections is enriched in bacterial pathogens streptococcus pneumoniae exposure is associated with human metapneumovirus seroconversion and increased susceptibility to in vitro hmpv infection co-colonization by streptococcus pneumoniae and staphylococcus aureus in the throat during acute respiratory illnesses pharyngeal microflora disruption by antibiotics promotes airway hyperresponsiveness after respiratory syncytial virus infection bacterial colonization dampens influenza-mediated acute lung injury via induction of m2 alveolar macrophages early nasopharyngeal microbial signature associated with severe influenza in children: a retrospective pilot study disordered oropharyngeal microbial communities in h7n9 patients with or without secondary bacterial lung infection the administration of intranasal live attenuated influenza vaccine induces changes in the nasal microbiota and nasal epithelium gene expression profiles modulation of potential respiratory pathogens by ph1n1 viral infection characterization of the upper respiratory tract microbiomes of patients with pandemic h1n1 influenza a metagenomic analysis of pandemic influenza a (2009 h1n1) infection in patients from north america kalgoorlie otitis media research project team. modelling the cooccurrence of streptococcus pneumoniae with other bacterial and viral pathogens in the upper respiratory tract associations between pathogens in the upper respiratory tract of young children: interplay between viruses and bacteria differences in the nasopharyngeal microbiome during acute respiratory tract infection with human rhinovirus and respiratory syncytial virus in infancy variability and diversity of nasopharyngeal microbiota in children: a metagenomic analysis nasopharyngeal microbiome diversity changes over time in children with asthma seasonal variation of respiratory pathogen colonization in asymptomatic health care professionals: a single-center, cross-sectional, 2-season observational study dynamics of bacterial colonization with streptococcus pneumoniae, haemophilus influenzae, and moraxella catarrhalis during symptomatic and asymptomatic viral upper respiratory tract infection acquisition and transmission of streptococcus pneumoniae are facilitated during rhinovirus infection in families with children changes in microbiota during experimental human rhinovirus infection the effects of live attenuated influenza vaccine on nasopharyngeal bacteria in healthy 2 to 4 year olds. a randomized controlled trial live attenuated influenza vaccine enhances colonization of streptococcus pneumoniae and staphylococcus aureus in mice kabat bfet al. taxa of the nasal microbiome are associated with influenza-specific iga response to live attenuated influenza vaccine lambda interferon restructures the nasal microbiome and increases susceptibility to staphylococcus aureus superinfection outgrowth of the bacterial airway microbiome after rhinovirus exacerbation of chronic obstructive pulmonary disease the airway epithelium: soldier in the fight against respiratory viruses influenza virus infection decreases tracheal mucociliary velocity and clearance of streptococcus pneumoniae ciliary dyskinesia is an early feature of respiratory syncytial virus infection depression of monocyte and polymorphonuclear leukocyte oxidative metabolism and bactericidal capacity by influenza a virus sustained desensitization to bacterial toll-like receptor ligands after resolution of respiratory influenza infection both influenza-induced neutrophil dysfunction and neutrophil-independent mechanisms contribute to increased susceptibility to a secondary streptococcus pneumoniae infection influenza virus-induced immune complexes suppress alveolar macrophage phagocytosis alteration of pulmonary macrophage function by respiratory syncytial virus infection in vitro depletion of alveolar macrophages during influenza infection facilitates bacterial superinfections immune impairment of alveolar macrophage phagocytosis during influenza virus pneumonia effect of virus infection on the inflammatory response. depression of macrophage accumulation in influenza-infected mice bacterial interference among nasal inhabitants: eradication of staphylococcus aureus from nasal cavities by artificial implantation of corynebacterium sp staphylococcus epidermidis esp inhibits staphylococcus aureus biofilm formation and nasal colonization nasal microenvironments and interspecific interactions influence nasal microbiota complexity and s. aureus carriage respiratory commensal bacteria corynebacterium pseudodiphtheriticum improves resistance of infant mice to respiratory syncytial virus and streptococcus pneumoniae superinfection. front microbiol host physiologic changes induced by influenza a virus lead to staphylococcus aureus biofilm dispersion and transition from asymptomatic colonization to invasive disease dynamic changes in the streptococcus pneumoniae transcriptome during transition from biofilm formation to invasive disease upon influenza a virus infection interkingdom signaling induces streptococcus pneumoniae biofilm dispersion and transition from asymptomatic colonization to disease streptococcus pneumoniae modulates staphylococcus aureus biofilm dispersion and the transition from colonization to invasive disease key: cord-019051-gtruu1op authors: weber, olaf title: the role of viruses in the etiology and pathogenesis of common cold date: 2009-11-10 journal: common cold doi: 10.1007/978-3-7643-9912-2_5 sha: doc_id: 19051 cord_uid: gtruu1op numerous viruses are able to cause respiratory tract infections. with the availability of new molecular techniques, the number of pathogens detected in specimens from the human respiratory tract has increased. some of these viral infections have the potential to lead to severe systemic disease. other viruses are limited to playing a role in the pathogenesis of the common cold syndrome. this chapter focuses on the viral pathogens that are linked to common cold. it is not the intention to comprehensively review all the viruses that are able to cause respiratory tract infections—this would go beyond the scope of this book. the list of viruses that are briefly reviewed here includes rhinoviruses, respiratory syncytial virus, parainfluenza virus, adenovirus, metapneumovirus and coronavirus. bocavirus is discussed as one example of a newly identified pathogen with a less established role in the etiology and pathogenesis of common cold. influenza virus does not cause what is defined as common cold. however, influenza viruses are associated with respiratory disease and the clinical picture of mild influenza and common cold frequently overlaps. therefore, influenza virus has been included in this chapter. it is important to note that a number of viruses are frequently co-detected with other viruses in humans with respiratory diseases. therefore, the viral etiology and the role of viruses in the pathogenesis of common cold is complex, and numberous questions remain to be answered. numerous viruses are able to cause respiratory tract infections. some of these may also cause severe diseases. others are limited to a role in the pathogenesis of the common cold syndrome. with the availability of new molecular techniques, the number of pathogens detected in specimens from the human respiratory tract has increased. the association of some of these agents with human respiratory disease is not always clear. it is not the intention of this chapter to comprehensively review the virology of all the viruses that cause or potentially cause respiratory tract infections. the chap-ter focuses on some pathogens that are linked to common cold. the list of viruses described includes rhinovirus, respiratory syncytial virus, parainfluenza virus, adenovirus, metapneumovirus and coronavirus. bocavirus is discussed as an example of a newly identified pathogen with a less established role in the etiology and pathogenesis of common cold. influenza virus does not cause what is defined as common cold, but is associated with respiratory disease and the clinical picture of mild influenza frequently overlaps with that of the common cold. therefore, influenza viruses are briefly described in this chapter. for details on the biology of the individual viruses and their role in pathogenesis of respiratory diseases further reading of standard literature and text books of virology is recommended. viruses with an established role in common cold are rhinoviruses, adenoviruses, parainfluenza viruses, coronaviruses and the respiratory syncytial virus, and these are reviewed in greater detail here. their structure and replication, the transmission and epidemiology and the clinical symptoms are described. in addition, some brief comments about current models of pathogenesis and animal models, respectively, complete the respective subchapters. table 1 provides an overview of the viruses that cause respiratory tract infections and that do, or may, play a role as a cause or in the pathogenesis of common cold. a number of viruses are frequently co-detected with other viruses in humans with respiratory diseases. therefore, the viral etiology and the role of viruses in the pathogenesis of common cold is complex and it is safe to say, not fully understood for each and every virus that is linked to respiratory tract infection. recent developments in the field of antivirals are described in the chapter by tom jefferson in this book. rhinoviruses cause the vast majority of the common colds in humans. although the infection usually is self limiting and the symptoms of the disease are mild in healthy adults, rhinovirus infections may cause serious illness in children or patients with pre-existing medical problems [1] . taxonomy, structure and replication rhinoviruses more than 100 serotypes, strains and isolates of rv have been isolated from humans. two human rv species have been described: human rhinovirus (hrv) a and b. eighteen serotypes and 2 subtypes (hrv 1a and 1b) belong to hrv a. five serotypes are assigned to hrv b and 82 serotypes are not yet assigned to a species including bovine rhinoviruses (brv) 1-3. rv are non-enveloped viruses with an icosahedral symmetry. the virus is small and has a diameter of approximately 30 nm. four capsid proteins, vp1-4 have been described. a protomer is composed of one copy of vp1, vp3 and vp0 (a precursor where vp4 and vp2 are covalently linked). cleavage of vp0 is the final step of the assembly process [3] . one or two copies of vp0 will remain uncleaved; no role for this uncleaved vp0 has been established yet. five protomers are arranged symmetrically about a fivefold axis, forming a pentamer that represents a corner of the icosahedron. the capsid is formed by 12 pentamers. rv have, similar to human enteroviruses, the same comparatively uneven surface with its characteristic canyon around the fivefold axis [4] . the canyon serves as an attachment site for the cell receptor [5] . in cscl, rv have a buoyant density of 1.38-1.42 g/ cm 3 . virions are unstable at a ph below 5-6, a feature, which distinguishes rv from other enteroviruses. however, as they are non-enveloped viruses, rv are stable against detergents and most organic solvents. on the other hand, alcohol and phenol are effective virucidal agents. the rv genome is organized as a single-stranded positive-sense rna of approximately 7100-7200 nucleotides in length with its 5' terminus covalently linked to a small protein, vpg. the 5'-untranslated region (utr) of approximately 0.65 kb is shorter than that of other enteroviruses, owing to a deletion of approximately 100 nucleotides between the internal ribosomal entry site (ires) and the translation start site. one open reading frame (orf) of about 2150 codons, a 3'-utr of approximately 40 nucleotides and a 3' poly(a) tail complete the structure of the genome. rv have a characteristic nucleotide composition with a preponderance of a and u, particularly in the third position of the codons. the genome is fully sequenced and has the accession number replication is initiated through attachment to the cell receptor. for most rv serotypes this is intercellular adhesion molecule-1 (icam-1) [6, 7] . it is hypothesized that the viral canyon structure releases a lipid moiety upon icam-1 binding, which, in turn, leads to a change in conformation, destabilization and the release of the viral rna into the cytoplasm [8] . rv shut off host cell protein synthesis by inactivating the cap binding complex. their ires allows them to replicate despite this inactivation. the rna serves as a messenger rna encoding a single polyprotein which is cleaved post-translationally by virus-encoded proteases. once the first round of translation and subsequent processing is complete a 3d pol rna-dependent rna polymerase produces negative-sense rna from the genomic template, which in turn serve as template for the production of positive-sense genomic rna. the synthesis of a single virus polyprotein requires post-translational processing to facilitate subsequent steps in viral replication. at least two proteolytic activities are encoded by the virus: 2a pro performs the first cleavage releasing capsid precursors and 3c pro catalyzes most other cleavage reactions. the protease has a trypsin-like structure but the active site of the enzyme is a cysteine sulfydryl [9] . rv are transmitted mainly by direct contact and less frequently through aerosols (for details see the chapter by diane pappas and owen hendley). virus can frequently be isolated from the hands of an infected individual and is transmitted to other individuals or to objects in the environment. during rv infection virus titers in nasal secretions are as high as 10 2 -10 3 tcid 50 /ml of nasal lavage fluid [10, 11] . infection of humans is very effective and less rv may be needed for infection of seronegative volunteers by nasal drops than needed for infection of a human embryonic fibroblast tissue culture [12] . in contrast, when the same virus was used for inhalation of aerosols a 20-fold disparity in infectious dose has been described [13] , suggesting that the lower respiratory tract is less susceptible for infection than the nasopharynx. after a short incubation period of 1-4 days virus is shed, peaks after another 2-3 days, and declines thereafter [13, 14] . the primary site of viral replication is ciliated epithelial cells as detected by in situ hybridization [15] . the histopathology of rv infection is not as yet very detailed. nasal mucosa biopsies reveal only few or no histopathological abnormalities despite active virus shedding. explant cultures inoculated with rhinovirus failed to develop cytopathic effects (cpe) [16] . biopsies showed marked edema of connective tissue, sparse infiltration of inflammatory cells, hyperemia and exudation of seromucous fluids [17] [18] [19] . infection of bovine tracheal organ cultures with brv leads to shedding of ciliated cells. it has been suggested that the immune response of the host contributes to the symptom complex. increased concentrations of the pro-inflammatory cytokines il-8, il-1 and il-6 [20] [21] [22] have been found in nasal secretions of subjects with symptomatic rv infection. a direct correlation between concentration in nasal fluids and symptom severity has been described for il-6 [22] . rv infection is usually accompanied by the typical common cold symptoms: nasal discharge and obstruction, sneezing, coughing, sore throat and, less frequently, fever. gastrointestinal symptoms are sometimes observed in children. the infection is usually limited to the upper respiratory tract. it is commonly believed that rv infection increases the risk for subsequent or secondary bacterial infections. in patients predisposed with existing under-lying diseases, like cystic fibrosis or chronic bronchitis, and in immunocompromised patients, elderly and infants, rv can cause serious infections of the lower respiratory tract. rv infection of the lower respiratory tract (lrti) was demonstrated by papadopoulos and co-workers [23] using in situ hybridization techniques. these authors demonstrated rv infection not only in epithelial cells but also in underlying submucosal cells. exacerbation of chronic bronchitis or asthma may be a consequence in these patients [24, 25] . indeed, approximately 80% of asthma exacerbations in children [26] and about 70% in adults [27] are associated with respiratory virus infections, and the vast majority of these are rv infections [28] . examination of the early innate immune responses to rv infection in asthmatic bronchial epithelia revealed profound impairment of virus-induced interferon (ifn)-expression leading to impaired apoptotic responses and enhanced rv replication [29] . rv infections lead to the production of type-specific iga, igg and igm antibodies. however, frequencies of response to natural infection have been reported to vary between 37% and 92% [30] . infected patients usually develop neutralizing antibodies to the infecting virus within 1-3 weeks after infection. iga is the dominant immunoglobulin in nasal secretions, has a protective role and may prevent re-infections with homotypic viruses or reduce the symptoms upon reinfection. the involvement in the viral clearance process is less clear and other mechanisms like the induction of an innate immune response are being discussed. both serum and secretory antibodies persist for several years after infection. in the absence of effective antiviral treatments, the diagnosis of rv infection for guiding an anti-rv therapy is not useful. the diagnosis of rv infection largely relies on the clinical symptoms. approximately 60-80% of the patients with a common cold syndrome of afebrile prominent nasal symptoms but minimal systemic disease that occurs between august and early november have rv infection [31] . however, the general method for identifying the etiological agent is the isolation and propagation in cell culture. in addition, polymerase chain reaction (pcr) can be used for rapid identification of rv in specimens. point-of-care diagnostics are under development. a major obstacle to understanding disease pathogenesis has been the lack of a small-animal model for rv infection. rv have shown a high degree of species specificity, limiting the use of experimental animal systems. therefore, aspects of pathogenesis have been studied in experimentally induced colds in human volunteers. infection of rabbits, guinea pigs or weanling mice by parenteral routes was not successful with certain strains of the virus [32] [33] [34] , but infection of mice was possible using a tissue-culture adapted hrv-2 [35] . chimpanzees or gibbons has been experimentally infected using specific strains of hrv [36, 37] . approximately 90% of the rv use human icam-1 as their cell receptor and do not bind mouse icam-1; the remaining 10% use a member of the low-density lipoprotein receptor family and can bind the mouse counterpart. recently, three novel mouse models of rv infection: minor-group rv infection of balb/c mice, major-group rv infection of transgenic balb/c mice expressing a mouse-human icam-1 chimera and rv-induced exacerbation of allergic airway inflammation were described by bartlett et al. [38] . these models have features similar to those observed in rv infection in humans, including augmentation of allergic airway inflammation, and may be useful in the development of future therapies for colds and asthma exacerbations. association between common cold symptoms and inflammatory mediators is an important aspect of understanding common cold and rv infection. although this association seems obvious, the exact mechanisms are less clear and one might expect a better understanding of the detailed mechanism(s) once inhibitors of viral replication are available for studies in humans. in addition, the new animal models developed by bartlett et al. [38] can be expected to support efforts to study pathogenesis of rv infection in vivo in greater detail. human respiratory syncytial virus (rsv) was first isolated from a laboratory chimpanzee with upper respiratory tract infection (urti) in 1956 [39] . rsv is today recognized as the leading viral agent in upper respiratory tract disease in infancy and childhood. the spectrum of rsv-caused diseases includes rhinitis, otitis media, pneumonia and bronchiolitis. the latter two diseases can be associated with a substantial morbidity and mortality. in addition, there is growing recognition for its importance as a causative agent for diseases in elderly and immunocompromised patients [40] . the world health organization estimates that rsv causes 64 million infections and 160 000 deaths annually [41] . a bovine rsv (brsv) has been described causing economically important respiratory diseases in cattle [42] . another animal rsv is the pneumonia virus of mice (pvm) [43] , suggesting that there was an interspecies spread in the evolution of these viruses. however, an animal reservoir for human rsv has not been described so far [44] . despite the importance of rsv as a leading cause for respiratory diseases, the pathogenesis of rsv infection is not fully understood and efficacious vaccines are not available. rsv is a member of the order mononegavirales, which includes several non-segmented negative-strand rna viruses. rsv is a member of the family of paramyxoviridae, subfamily pneumovirinae and represents the type species for the genus pneumovirus [43] . rsv virions consist of an envelope and a nucleocapsid. the viral gene expression and nucleic acid replication occur in the cytoplasm. the envelope is acquired by cell budding. virions are spherical to pleomorphic; filamentous and other forms are common. they measure 150-300 nm in diameter and up to 1000-10 000 nm in length [45] . the surface of the virion is covered by projections (spikes) formed by fusion (f) glycoproteins. the spikes are 11-20 nm long and are spaced 6-10 nm apart; they mediate attachment and penetration. the helical nucleocapsid is filamentous with a length of 600-800(1000) nm and a width of 12-15 nm [40] . the nucleocapsid does not enter the cells by surface fusion typical for paramyxoviruses but rather by membrane fusion which may involve clathrin-mediated endocytosis [46] . the unsegmented genome contains a single molecule of linear negativesense, single-stranded rna. virions occasionally contain a positive-sense single-stranded copy of the genome (partial self-annealing of extracted rna may occur). the complete genome is approximately 15 300 nucleotides long and fully sequenced. the genome has the accession number(s) [d00386] -[d00397] [43] . the rna genome has a 3'-extragenic leader region, followed by the ten viral genes and a 5' trailer region. each gene is transcribed into a separate mrna encoding for a single viral protein with the exception of the m2 mrna. this contains two overlapping orf, expressed by a ribosomal stop-restart mechanism into two proteins, m2-1 and m2-2 [47] . although gene expression is consistent with that of other members of the order of mononegavirales m2-1 and m2-2 have some regulatory features unique to rsv [44] . the five nucleocapsid-associated proteins are the n (nucleocapsid) protein, the phosphoprotein p (co-factor for rna synthesis), the l protein (large, a 2165-amino acid subunit of viral polymerase), the m2-1 (transcription processivity factor) and the m2-2 protein (which possesses regulatory functions) [44, 48] . the n protein binds the genomic and the antigenomic (positive-sense intermediate) rna and protects is against degradation. in addition, it reduces the detection and responses by the host's immune system (for instance toll-like receptors, tlrs) and intracellular rna recognition helicases, which initiate innate immune responses [44, 49, 50] . the viral envelope is formed by four rsv proteins that associate with the lipid bilayer: a matrix (m) protein that is located at the inner surface and is important for the assembly of the virion [51] , a glycosylated (g) protein, a fusion (f) protein and a small hydrophobic (sh) protein. two other rsv proteins, the ns1 and ns2 proteins are a minor part of the virion [44] . ns1 and ns2 are thought to modulate the host response to rsv infection. the g glycoprotein (~90 kda) has a peptide backbone with 24-25 side chains and is important for viral attachment to the host cell [52] . a second secretory form of the g protein exists that arises from a second initiation codon in the g orf. proteolytic trimming removes additional amino acids, and the final protein lacks the 65 n-terminal residues, including the membrane anchor [44, 52] . the ectodomain of the g protein has a mucin-like structure that differs from attachment proteins of other paramyxovirus. its function is not clear but it is thought to contribute to virus spread or to prevent trapping by mucus [44] . the f protein has two distinct functions: penetration into the host cell by membrane fusion and a syncytia-forming property. the f protein matures by activation through a furin-like intracellular protease that cleaves the precursor, f 0 into three fragments, f 1 , f 2 and p27. f 1 and f 2 are linked by a disulfide bond and represent the active form of f [53] . the hydrophobic n terminus of f 1 is conserved within the rsv and it is thought that this domain inserts into the host cell membrane when fusion occurs [44] . ns1 and ns2 are thought to modulate the host's immune response to rsv [44] . lack of m2-1 results in reduced expression of ns1 and ns2, and it is thought that this down-regulation of the host-defense antagonists may help to facilitate persistent rsv infection [44] . infection occurs through direct contact, through large respiratory droplets and, to a lesser extent, through small droplets. the site of the first replication is the nasopharynx. after an incubation period of 4-5 days the virus spreads to the lower respiratory tract [54, 55] . the clinical signs include cough, rhinitis, fever and signs of bronchiolitis like air trapping, wheezing and increased airway resistance. the most prominent clinical symptoms are cough and rhinorrhea, which occur in approximately 90% of primary rsv infections in infants and to some lesser extent in reinfected adults [56] . fever occurs in 30-40% of both infected infants and adults and otitis media is reported in approximately 20% of infected infants. ear and sinus pain is reported in 20-30% of infected adults. symptoms of lrti, including bronchiolitis, pneumonia, croup, wheeze and tracheobronchitis, are observed in 30-40% of infected infants and to some lesser extent in adults, with wheeze and tracheobronchitis being the most prominent disease symptoms. hospitalization is necessary in approximately 3% of infected children and below 0.1% of infected adults. rsv is the single most important agent in children younger than 3 years of age. importantly, children with a mild rsv disease have also been reported to have recurrent wheezing for up to 10 years after the primary acute disease [57] . extrapulmonary dissemination may occur in immunocompromised patients [58] . the virus may spread to kidneys, liver, the central nervous system and the heart. virus can be isolated from the nasopharynx of children for up to 14 days. in immunocompromised patients, virus recovery is possible for up to 1 month or even longer. in immunocompetent individuals the viral infection is usually restricted to the superficial cells of the epithelia and viral spread outside the respiratory tract is uncommon [54, 59] . an exception, however, is the middle ear: the virus frequently causes otitis media [60] . the typical pathological findings in rsv-infected tissue include epithelial necrosis and infiltrates of monocytes, t cells and neutrophils [61] . airways appear obstructed due to sloughed cells, mucus secretion, proliferation of bronchoalveolar epithelium or cellular infiltration. formation of syncytia in the bronchoalveolar epithelium is sometimes observed [61] . however, giant cell pneumonia or syncytia formation are related to severe t cell-deficient patients [44] . specific host factors that may influence the clinical signs and outcome of rsv infection including the general health status, the nutritional status [56, 62, 63] , gender, ethnic group, levels of maternal antibodies [64] , age of first rsv infection [65] and underlying cardiac or pulmonary diseases [66] . in addition, there are several environmental factors (e.g., tobacco use in household, stress, day care) that may influence the course of the disease or severity of symptoms. the role of inflammation and a bias of the host's immune response towards a humoral th2 response (the typical cytokines are il-4, il-5, il-10 and il-13) are discussed controversially in the literature. a strong inflammatory response does not seem to be determinative for the severity of clinical symptoms [67] , a finding supported by the fact that in many clinical studies patients receiving anti-inflammatory therapy did not significantly benefit from that treatment [68] . however, strong inflammatory responses also have been suggested to enhance the severity of clinical symptoms in rsv disease. the role of inflammatory chemokines in rsv pathogenesis has been supported by many preclinical and clinical studies (reviewed in [44] ). for example, genetic polymorphisms that increase il-8, a major chemoattractant for neutrophils, and ccr5 expression have been associated with increased rsv disease [69] . a link between an increased ratio of th2/th1 immune response (a bias toward the humoral vs the classical cytotoxic response) has been suggested by several authors. this discussion is based on evidence for elevated th2/th1 response ratios in clinical studies, the role of key th2 cytokines in the pathogenesis of asthma and the experience with a formalin-inactivated rsv vaccine in the 1960s [70] [71] [72] . this vaccine was poorly protective and vaccinated children and infants developed dramatically enhanced disease compared to naive patients upon natural rsv reinfection [48] . subsequent preclinical studies confirmed a bias toward a th2-specific cd4 + t cell response in animals treated with formalin-inactivated rsv vaccine vs naturally infected animals [73] . il-4 and il-13 support isotype switching to ige, which is bound to mast cells and eosinophils and, upon antigen contact, induces release of inflammatory mediators like histamine or leukotrienes by these cells. these mediators contribute to the development of the typical clinical symptoms associated with rsv disease. several viral proteins play an important role in the pathogenesis of rsv disease (reviewed in [44] ). the soluble g protein has been suggested to modulate the innate immune response by down-regulating inflammatory mediators such as il-6 or il-8 in epithelial cells as a response to rsv infection [74] . g protein also modulates inflammatory responses of monocytes by acting as a general antagonist for tlr activity [75] . the g-mediated suppression of tlr-4 signaling appears to be counteracted by the f protein, which has been described to induce signaling through this tlr [76] , although the significance of this activity is unclear. importantly, rsv infection can block the maturation of dendritic cells (dc), which serve as major antigen-presenting cells (reviewed in [44] ). this alteration of dc biology may support the shift of the th2/th1 balance towards th2, reduce antiviral interferon activity and limit the mobility of mature antigen-presenting cells, thus qualitatively altering the immune response to rsv infection (reviewed in [44] ). in summary, there are several host factors or viral factors that play roles in the pathogenesis of rsv infection. the picture, however, is highly complex and relative contributions of the various factors to rsv pathogenesis are not entirely understood. as mentioned above rsv is a leading cause of respiratory diseases in children and has increasing importance as a causative agent for respiratory diseases in elderly. in a prospective study of infants and children in the unites states, rsv was detected in 43% of pediatric hospitalizations for bronchiolitis, 25% for pneumonia, 11% for bronchitis and 10% for croup [54] . approximately 90% of infants have been infected at least once by 2 years of age [44, 77] . although rsv is represented by one serotype, a protective immunity against rsv is generally weak and reinfection occurs. virus-neutralizing antibodies, including secretory iga found in the respiratory tract, contribute to viral clearance and may play a role in protection against reinfection [54] . however, the iga response is short [44, 54] . in the lower respiratory tract, the igg response has been described as more efficient. the role of antibodies as a down-modulator of clinical symptoms has been confirmed by the clinical experience with palivizumab. in temperate regions, rsv circulates quickly during winter/early spring, but timing varies more elsewhere. effective vaccines are not available and effective therapies are not available. however, an rsv-neutralizing human-ized monoclonal antibody, palivizumab, reduces rsv-associated hospitalization if used as a passive immunoprophylaxis [78] . rsv infection is assumed to be frequently misdiagnosed, particularly in adults [56] , because the symptoms are similar to those caused by other respiratory viruses like influenza. laboratory diagnostic tests are usually performed on secretion samples obtained from the nasopharynx. novel rapid elisa-based or rt-pcr-based tests are useful particularly in a hospital setting to identify outbreaks, prevent further transmission, initiate therapies or reduce inappropriate use of antibiotics [56] . animal models are comprehensively reviewed by moore and stokes peebles [79] . rsv is species specific; however, some animal species exhibit semipermissive infection with rsv. chimpanzees were productively infected with rsv and exhibited upper respiratory tract illness, whereas adult squirrel monkeys, newborn rhesus monkeys, and infant cebus monkeys did not show symptoms but shed low levels of virus [80] . bonnet monkeys, which are more widely available than chimpanzees, can be infected with rsv [81] . non-human primate models of rsv infection, especially chimpanzee, have advantages but certainly limitations associated with the high cost and genetic variability, which limits the reproducibility of results. the cotton rat which is susceptible to both urti and lrti with rsv is seen as one of the best animal models of rsv infection and disease [79, 82] . in these animals, rsv infection led to histologically confirmed proliferative rhinitis, bronchiolitis, and the pulmonary infiltration of lymphocytes and neutrophils [82] [83] [84] . the cotton rat model was used to study the mechanism of antibody-mediated clearance of rsv [84] . the advantages of mouse models are obvious: they are inexpensive, inbred strains are available and a wealth of reagents (e.g., antibodies), arrays, probes or information (e.g., the genome sequence) is available. although there is variability between the strains regarding susceptibility and viral load, viral load does not vary much within the strains [79] . balb/c is the most widely used inbred mouse strain to study rsv infection [79] . rsv-infected balb/c mice show signs of clinical illness including weight loss, ruffled fur and ataxia peaking at day 8 after infection [85] . interestingly, the susceptibility to rsv replication in nose and lung increased with age. the predominant histological findings in rsv-infected 3-week-old balb/c mice were peribronchiolar and perivascular accumulations of mononuclear cells [86] . the role of th1 and th2 cell response to rsv infection has been the dominant focus of the balb/c mouse studies in this context [79] . rsv infection induces a th1 response dominated by high ifn-levels in the lungs of infected mice, abundant ifn--producing cells in the bronchoalveo-lar lavage fluid (balf) and rsv-specific cytotoxic t cell (ctl) response [87, 88] . stat1 (-/-) mice with a balb/c background have been described as having an excellent rsv disease phenotype (reviewed in [79] ). although this model has the limitation that it probably does not exactly mirror the complexity of a natural infection in humans, it is regarded as an attractive tool to study rsv pathogenesis and evaluating novel therapies. in addition to other mouse models and the infection in chinchillas [89] , infection of natural hosts has been studied in detail. bovine respiratory syncytial virus is a major cause of respiratory illness in calves and has been studied in this context. the clinical signs after experimental infection include cough, lung sound, dyspnea, fever, increased respiratory rate and pulmonary resistance. prominent histological findings include proliferative bronchiolitis, alveolitis, syncytia, and, to some extent, emphysema [90] . rsv infection of calves might be useful for evaluating vaccination strategies; however, it is not an animal model for the evaluation of novel therapies. the list of experimental models also includes pneumonia virus infection of mice (pvm) [91] . although the pvm model of respiratory disease is interesting because it shows the phenotype of a natural infection, pvm differs from rsv including the g and the ns1 proteins that fulfill important functions during rsv infection. human parainfluenza viruses (hpiv) are important causes of respiratory diseases in infants and children. they usually cause urti of which 30-50% may be accompanied by otitis media. hpiv may also cause lrti, about 0.3% of which require hospitalization. hpiv1-3 infections are second to rsv infections as the viral cause of serious acute respiratory infections in young children that occur primarily in the first 6 months of life [92, 93] . hpiv3 may cause severe diseases. approximately 80% of infants and children infected with hpiv3 developed febrile illness and one third of these infected individuals developed lrti, resulting in bronchitis or pneumonia [93] [94] [95] . most children have been infected with hpiv3 by the age of 2 years. croup is the main clinical manifestation of infection with parainfluenza viruses, especially hpiv1 and 2, and these infections may extend to the lower respiratory tract and result in pneumonia [94] . hpiv4 mainly causes mild urti in children and adults [93] . along with rsv, hpiv are also a leading causative agent of serious acute respiratory infections and community-acquired respiratory disease requiring hospitalization in adults. the proportions of hospitalizations associated with hpiv infection vary widely in hospital-based studies. according to the who, hpiv1 is estimated to account for 5800-28 900 annual hospitalizations in the usa, hpiv2 for 1800-15 600 hospitalizations, and hpiv3 for 8700-52 000 hospitalizations [92] . parainfluenza viruses belong to the order of mononegavirales, family paramyxoviridae, subfamily paramyxovirinae. hpiv1 and 3 belong to the genus respirovirus, and hpiv2 and 4 to the genus rubulavirus [96] . the virions are spherical enveloped particles of approximately 150-250 nm in diameter with an internal helical nucleocapsid. virions are enveloped by a lipid bilayer membrane that bears spike-like projections composed of hemagglutinin-neuraminidase (hn) and fusion (f) protein [97] . as for other paramyxoviruses, all hpivs contain a negative strand, ~15 500-nucleotide-long non-segmented rna genome [93] encoding two envelope glycoproteins, the hn, and the f protein, a matrix protein (m), a nucleocapsid protein (np) and several nonstructural proteins including a polymerase-associated protein (p/v) and the viral replicase (l) (reviewed in [93] ). the replication of piv is similar to that of other paramyxoviruses with the rna genome serving as a template for the transcription of the mrnas. binding of the hn glycoprotein to its cell receptor initiates the infection [98] . in addition to having this function, hn is thought to enhance the fusion activity of f, which, following virus attachment to the host cell, mediates the fusion of virus and subsequent penetration. f also mediates fusion of infected and uninfected cells, allowing virus to spread. f is synthesized as an inactive precursor (f 0 ) that is post-translationally cleaved by a host cell protease to yield two subunits, f 1 and f 2 that remain linked by a disulfide bond [93] . the mucous membranes of the upper respiratory tract are the common sites of infection. prominent clinical symptoms of hpiv infection can be characterized by rhinitis, pharyngitis and bronchitis. the incubation period is about 4 days [95] . coughing, hoarseness and fever that last for approximately 2-3 days are frequent. involvement of the trachea results in croup and extension to the lower respiratory tract may lead to pneumonia. severe disease characterized as bronchopneumonia or bronchiolitis has been observed with hpiv3 infections [93] [94] [95] . specific virus and host properties that determine the severity of hpivrelated disease are not yet understood. infection with piv induces an immune response to hn and f. neutralizing antibodies to piv correlate with partial resistance to infection or clinical symptoms but usually do not prevent re-infection [99] . secretory iga neutralizing antibodies are more important in adults than in children [100] . as mentioned above, the hpivs are important causes of respiratory tract diseases in infants and children. epidemics of hpiv3 usually occur in the early spring [101] . viruses do not persist for a long time in the environment [93] . the seasonal peak of hpiv1 and 2 infections is reflected in the seasonality of croup, which is the highest in autumn in the usa [102] . croup during the winter months is more likely to be caused by other viruses, such as influenza virus or rsv [93] . the diagnosis of piv infection is mainly clinical, and molecular diagnostic procedures are usually not performed. there is no specific antiviral treatment against piv available, and therapeutic intervention is mainly targeted against symptoms of croup. early treatment will reduce the severity of the symptoms, the rates at which patients return to a health care practitioner for additional medical attention, visits to the emergency department, and admission to the hospital [103] . effective vaccines are currently not available. attenuated strains have been studied and a virosomal formulation of an hpiv3 vaccine is currently under development [92] . the national institute of allergy and infectious diseases (niaid) is studying the safety and immunogenicity of a recombinant live-attenuated chimeric bovine/human parainfluenza type 3 virus, rb/ hpiv3, vaccine in a phase i study. the test vaccine is delivered as nose drops to adults 18-49 years of age, hpiv3-seropositive children 15-59 months of age, and hpiv3-seronegative infants and children 6-36 months of age [104] . hpiv vaccines are also developed by some companies [105]. adenoviruses cause infections of the respiratory and gastrointestinal tract, kidney, eye and other organs, the latter mostly as a consequence of immunosuppression [106] . they are known to frequently cause respiratory infections among people in institutional environments -outbreaks among children are reported at boarding schools and summer camps [107] . outbreaks have also been reported in military camps [108] . most infections with adenovirus result in infections of the upper respiratory tract. in addition, adenovirus infections may result in conjunctivitis, tonsillitis, ear infection or croup [106] [107] [108] [109] . adenoviruses are responsible for approximately 5% of acute respiratory infections in children under the age of 5 [107, 109] adenoviruses belong to the family adenoviridae, genus mastadenovirus. there are 6 species including human adenovirus a-f with 51 immunologically distinct human adenovirus serotypes [110] . the most common human adenoviral pathogens belong to the c adenoviruses and those mainly infect the upper respiratory tract [107] . the virions are not enveloped. they consist of a capsid and a core with proteins associated to it. the icosahedral capsid has a diameter of 70-100 nm [111, 112] . all capsids consist of 252 capsomers. the surface structure reveals a regular pattern with distinctive features. surface projections are often lost during preparation. distinct filaments protrude from the 12 vertices/ pentons [112] [113] [114] . the genome is not segmented and contains a single molecule of linear double-stranded dna with terminally redundant sequences, which have inverted terminal repetitions (itr). the complete genome of mastadenoviruses is approximately 31-36 kpb long and has a guanine + cytosine content of 48-61%. the genome has a terminal protein, which is covalently linked to the 5'-end of each dna strand [114, 115] . the viral genome encodes structural proteins and non-structural proteins. virions consist of 11 proteins located in the capsid, fibers, and core. the capsid is comprised of seven polypeptides, polypeptide ii which is the basis for the hexon (three tightly associated proteins). polypeptides vi, viii, ix are associated with the hexon, polypeptides vi and viii serve as bridge between the capsid and the core. five polypeptide iii copies are the basis for the penton. polypeptide iv forms the trimeric fiber [116] , which has a knob domain that serves as a viral receptor for the target host cell. the cell receptor is the receptor for coxsackie b virus and adenovirus (car) for adenoviruses a, c, d, e and f and cd46 for adenoviruses b with the exception of serotypes 3 and 7 [117] . the core consists of four proteins (v, vii, mu and the terminal protein, which is covalently linked to the 5´ end of the dna) and the dna (reviewed in [112] ). the replication cycle of adenoviruses is divided into two phases. the early phase includes adsorption of the virus to the host cell, penetration, transcription and translation of early genes. the early gene products mediate gene expression and dna replication, block apoptosis and promote the cell cycle progression. in addition, they possess potent immunomodulatory functions (reviewed in [112] ). the viral e1a gene product should briefly be mentioned: in the nucleus, e1a activates the expression of a number of genes by interacting with cellular transcription factors and other cellular regulatory proteins [112] . e1a has been postulated to play role in the pathogenesis of chronic obstructive pulmonary disease (copd) [118] [119] [120] . human adenovirus a-f can cause human infections ranging from respiratory disease, and conjunctivitis (b and d), to gastroenteritis (f serotypes 40 and 41) [106, 112] . the most common clinical picture after adenovirus infection of the respiratory tract is mild self-limiting upper respiratory disease with nasal congestion, coryza and cough [106, 113] . some patients develop exsudative tonsillitis that is clinically indistinguishable from streptococcus tonsillitis [120] . these infections are commonly caused by serotype 1, 2, 5 and 6 c adenoviruses and serotype 3 b adenovirus [107, 109] . respiratory symptoms may be accompanied by systemic manifestations including generalized malaise, chills, fever and headache [106] . however, adenoviruses may infect alveolar and bronchiolar epithelial cells [121] and cause pneumonia, bronchiolitis or bronchiolitis obliterans [122] [123] [124] . adenoviruses account for approximately 10% of pneumonias in children [106] . in contrast to many other respiratory viruses, both persistent and latent infections have been described, particularly in lymphocytes [106] . adenoviral dna may persist in the nuclei of infected cells or even integrate into the host dna. adenoviral e1a protein has been postulated to play a role in the pathogenesis of copd [121] . adenoviral dna was found in the lungs of copd patients and expression of e1a correlated with disease severity [125] [126] [127] . in response to inflammatory stimuli, e1a increases icam-1 and il-8 expression along with nuclear factor-b (nf-b) activation in lung epithelial cells ( [128] , reviewed in [113] ). while these factors support emphysema, e1a up-regulates transforming growth factor-1 (tgf-1) in bronchiolar epithelial cells [129] , supporting a role for e1a in airway remodeling [130] . in summary, adenoviruses are pathogens that frequently cause mild or severe acute infections of the respiratory tract. the importance of adenovirus infections, however, goes beyond acute airway disease. adenoviruses are non-enveloped pathogens and thus very stable to chemical or physical agents and adverse ph conditions. it is believed that respiratory adenoviruses are mainly spread via aerosols; however, other routes (fecal, waterborne) also frequently lead to infection. antibodies to one or more adenoviruses are found in approximately 50% of infants and nearly 100% of adults and since there are many different types of adenovirus, repeated adenoviral infections can occur [109, 131, 132] . although adenovirus infections can occur at any time of the year, respiratory tract disease caused by adenovirus is more common in late winter, spring, and early summer [106] . virological diagnosis can be performed using a variety of molecular or immunological approaches. this is, however, only important in the context of severe or epidemic diseases. development efforts for vaccines were discontinued [133] . antiviral therapy is only important in infected immunocompromised patients. in these patients, cidofovir has shown some promise [122] . human metapneumovirus (hmpv) was identified in 2001 [134] , and is today considered as a major cause of acute respiratory infections worldwide, especially in children. virtually all children have experienced an infection with hmpv by the age of 5-10 years (reviewed in [135] ). hmpv is a member of the order mononegavirales, family paramyxoviridae, subfamily pneumovirinae (as is rsv), genus metapneumovirus [136] . there are two major groups and at least four subgroups of hmpv [137] [138] [139] [140] . hmpv particles are enveloped, pleomorphic, filamenteous and spherical and have a mean diameter of approximately 210 nm [137] . the genome consists of a single-stranded negative rna of approximately 13.3 kb and contains eight genes in the order 3'n-p-m-f-m2-sh-g-l-5' coding for a nucleoprotein (n), a phosphoprotein (p), matrix protein (m), fusion protein (f), a transcription elongation factor (m2-1), a protein regulating rna synthesis (m2-2), a small hydrophobic protein (sh), attachment protein (g), a polymerase subunit (l) and probably additional proteins [141, 142] . the f protein is the major immunogenic viral protein [143] . replication is generally comparable to that of other members of mononegavirales. this virus infection occurs primarily during winter months or early spring and can manifest as both upper and lower respiratory tract disease [144] . after a severe hmpv infection, virus was detected in alveolar and airway epithelial cells [145] . it was also reported in this study that the virus caused acute organizing lung injury, tissue damage and, which is not observed in other paramyxovirus infections, induction of smudge cell formation. the clinical symptoms associated with hmpv infection are indistinguishable from those of rsv infection [146] and range from common cold to pneumonia. otitis media is observed in up to 50% of the infected individuals (reviewed in [135] ). as for other respiratory viruses, hmpv may cause exacerbation of underlying chronic diseases like asthma, congestive heart disease or chronic obstructive pulmonary disease. the importance of coinfections with rsv or influenza viruses is not clear (reviewed in [135] ). as for many other respiratory viruses, serious disease caused by hmpv is observed among immunosuppressed patients. results from a recent retrospective study suggested that hmpv infection may be an important cause of idiopathic pneumonia syndrome after stem cell transplantation [147] . hmpv is thought to be the second or third cause of severe acute respiratory tract infection in children, just ranking behind rsv and influenza virus [146, 148] . infections occur very early in life and up to 100% of children have experienced an hmpv infection by the age of 10 years. reinfections occur frequently. incidences in hospitalized children with acute respiratory tract infection range from 5% to 10%. incidence is up to 20% in patients consulting at an outpatient clinic (reviewed in [135] ). the routes of transmission are believed to be similar to those of rsv (respiratory droplets, hand-to mouth or hand-to eye contact) [149, 150] . the diagnosis of hmpv infection is mainly clinical. molecular diagnostic procedures are usually not performed routinely but are possible using standard technologies like rt-pcr or immunological techniques. there is no specific antiviral treatment available. however, ribavirin has shown some promise [151] and monoclonal antibodies are under development that could potentially be used to prevent hmpv infections [152] . several vaccines are being developed but are still in an early stage. human bocavirus (hbov), a parvovirus, was detected in children with lrti in 2005 using random amplification methods [153] . hbov infection is predominantly associated with respiratory and/or gastrointestinal symptoms in children at around 2 years of age [154] . seroprevalence reaches 95% in adults [155] . however, the role of hbov in the pathogenesis of human respiratory disorders is not yet fully understood -hbov infections are frequently accompanied by coinfections with other viral and bacterial pathogens [154] . hbov is classified into the parvoviridae family, subfamily parvovirinae, genus bocavirus. as other parvoviruses, hbov virions are icosahedral nonenveloped particles with a diameter of 21-25 nm [154] . the hbov genome (a linear single-stranded dna that encompasses approximately 5.2 kb) is organized like that of other parvoviruses: conserved genes encoding for the two non-structural proteins are located in the 5' region and genes for two structural proteins are located in the 3' region of the genome [156] . the structural proteins vp1 and vp2 are identical in sequence but differ in an n-terminal extension that is only present in vp1 (vp1 unique region, vp1u) and possesses a phospholipase a2-like activity (pla2) [157] . the functions of the two nonstructural proteins ns1 and np1 of hbov are not known; however, regulatory functions of ns1 of other parvoviruses have been described [154] . hbov has been detected in children with respiratory disease. the range of clinical manifestations is broad. diseases of the upper (rhinitis or coughing) and the lower respiratory tract (including pneumonia, bronchiolitis and wheezing) or even the gastrointestinal disease have been described (reviewed in [154] ). other symptoms include fever or rashes [158] . however, hbov infections are linked frequently with coinfections with viral and bacterial pathogens in up to approximately 69% of hbov dna-positive individuals [159] which makes it rather difficult to distinguish between symptoms that have been caused by hbov or by other pathogens. the role of vertical transmission, seen with other parvoviruses, is not known. antibodies against the viral structural protein vp1 have been detected in approximately 95% of children older than 2 years and adults [155] . in addition, igg1 subclass antibodies against hbov vp2-virus-like particles (vlp) were detected in approximately 98% of samples that were obtained from healthy adult blood donors [154] . the same authors found igm antibodies in 41.7% of sera from hbov dna-positive children but not in samples from dna-negative children. cellular immunity also plays a role in hbov infections and frequent cd4 + t helper cell reactions have been observed against hbov vlp [160] . many epidemiological aspects have been discussed above. however, it is noteworthy that the majority of the analyses have been performed in symptomatic individuals and more data from asymptomatic healthy chil-dren and adults would add to the understanding of the hbov epidemiology. diagnosis of hbov infection is mainly done by pcr amplification of viral dna or the detection of anti-hbov antibodies by elisa [154, 161] . at present, no specific treatment of hbov infections is available. coronaviruses are known to cause a variety of diseases in animals [161] . human coronaviruses are mainly associated with respiratory disorders; some may cause enteric infections [162] . the human coronaviruses hcov-229e and hcov-oc43 were identified in the 1960s [163] [164] [165] . a coronavirus causing a severe acute human respiratory syndrome (sars), the sars-cov, was first described in 2003 [166, 167] and two additional human coronaviruses, hcov-nl61 and hcov-hku1 that were both linked to respiratory disorders have been identified recently [168, 169] . because of the economic importance of coronaviruses in veterinary medicine (for instance in swine), development of vaccines is more advanced in veterinary medicine than in human medicine. however, with the appearance of the sars coronavirus, human coronaviruses gained a greater share of interest. coronaviruses belong to the order nidovirales, family coronaviridae, genus: coronavirus. in addition to the five human coronaviruses (hcov-229e, hcov-hku1, hcov-nl 63, hcov-oc43 and sars-cov), a specific human enteric coronavirus has been reported [170] . coronaviruses have been assigned to three groups based on antigenic relationships between species of different groups (reviewed in [171] ). hcov-229e and hcov-nl63 are included in group 1, hcov-hku1 and hcov-oc43 in group 2 and sars-cov represents an early split from group 2 [172] . coronaviruses are enveloped viruses, approximately 120 nm in diameter with large (20 nm) club-shaped surface projections (spike protein, s). the structure and function of the s protein have been reviewed elsewhere [173] . apart from s, coronaviruses have a smaller membrane protein, m (reviewed in [174] ). in addition, coronaviruses have a third envelope protein, the very small, non-glycosylated e protein [175] . e and m have been found to be essential for virus particle formation (reviewed in [176] ). group 2 coronaviruses also have an he (hemagglutinin esterase) protein that forms a layer of approximately 7 nm [175] . he is a neuraminic acetylesterase that hydrolyses the 9-o-acetylated sialic acid on erythrocytes, thus potentially destroying receptors [177] . another coronavirus protein, n, is closely linked to the rna genome (and forms a ribonucleoprotein, rnp). n, which may have a functional role in replication and transcription, is phosphorylated (reviewed in [178] ). coronaviruses have positive-sense single-stranded rna genomes of about 30 kb. the genome is generally organized in the following manner: 5'-utr-polymerase gene-structural protein genes-utr3' where the utr are untranslated regions each up to 500 nucleotides. the structural proteins are encoded in the following order: he (only group 2 coronaviruses) -s -e -m -n [171] . the infection is initiated by binding of the s protein to the cell receptor [179] . cd13 (human aminopeptidase n, apn), a metalloproteinase located on the surface of epithelial cells, has been identified as the cell receptor for hcov-229e [180] . a metallopeptidase, angiotensin converting enzyme 2 (ace 2) may be the cell receptor for sars-cov [181] . binding of the s protein to the cell receptor induces conformational changes in s that triggers fusogenic activity [182] . the coronavirus genome can only be released into the cytoplasm after fusion of the envelope with the cell membrane which is mediated through the s2 region of the s protein [173, 183] . replication occurs within the cytoplasm. early during infection the genomic rna is released and acts as an mrna for the translation of the first gene, the polymerase. mrnas for the other genes are generated subsequently. in general, coronaviruses have several 3' co-terminal subgenomic mrnas, socalled 'nested set'. the unique part of each mrna (which is not within the next smaller mrna) is translated during the replication cycle. at the 5' end of each gene there is a sequence that is common to all genes, the so-called 'transcription-associated sequence', which, as the name implies, is associated with the discontinuous transcription process (reviewed in [171] ). the various mechanisms that have been proposed for the production of subgenomic mrnas have been reviewed elsewhere [184] . human coronaviruses are generally thought of as common cold agents. this role was confirmed when healthy volunteers were infected with hcov-oc43 and hcov-229e and developed classical common cold symptoms [185] . whereas most infections result in mild disease or are even asymptomatic, additional factors like immunosuppression or coinfections might cause severe disease, even pneumonia [186, 187] . it is not clear whether hcov-229e and hcov-oc43 infect the lower respiratory tract in otherwise healthy people, because only urti was observed in this population. it has been suggested that the lower respiratory tract is more susceptible to hcov infection in children [187] . most studies were performed in healthy adults and there is less information about the most susceptible and vulnerable population, children and elderly. however, hcov-229e and hcov-oc43 nucleic acid were frequently detected in children with respiratory tract disease (11%) using rt-pcr, whereas in an otherwise healthy control group (asymptomatic bone marrow recipients) only one sample tested positive (0.37%, p < 0.01) [188] . this finding suggests that these coronaviruses cause upper and lower respiratory tract diseases in children that are more severe than in adults. it has been suggested that up to 30% of wheezing episodes in asthmatic children may be due to coronavirus infections [189] . hcov-nl63 was detected in young hospitalized children with severe lrti [190] . this virus has also been detected in elderly patients with fatal respiratory disease [191] . the risk of developing croup was about 6.6 times higher in children shown to be positive for hcov-nl63 than in those who tested negative [192, 193] . hcov-hku1 was first detected in elderly and children with underlying disease [194] . symptoms of hcov-hku1 include rhinorrhea, fever, coughing, and wheezing as well as bronchiolitis and pneumonia [195] . hcov-hku1 might also cause gastrointestinal disease [196] . hcov infection results in antibody titers in serum. secretory antibodies can be detected in the respiratory and enteric tracts (and in milk or colostrum) [197] . infections with hcov peak during winter season [198] . it is estimated that approximately 25% of common cold cases are caused by coronaviruses [162] . outbreaks of different human coronaviruses have found to alternate every 2-3 years [197] . although early studies demonstrated that antibodies against coronaviruses are frequently present in adults [199] , new studies suggest that there are differences with respect to the hcov species. hofmann et al. [200] demonstrated that infections with hcov-229e occur less frequently than with hcov-nl63 by measuring specific antibodies neutralizing either hcov-nl63 or hcov-229e. in addition, co-detection of coronaviruses with other viruses is common [187] . as with most coronaviruses, human coronaviruses are species specific. however, the sars coronavirus probably originated from an animal reservoir, presumably bats [201, 202] but was transmitted to humans by civet cats. the transmission of human coronaviruses from human to human occurs via secretions like aerosols and respiratory droplets (or, in case of enteric infection, feces) [197] . adults with acute symptomatic or inapparent infection transmit the virus to infants who develop clinical disease [162] . diagnosis of hcov infection is more clinical; an etiological diagnosis, however, can be performed using molecular or immunological techniques. vaccines against coronavirus diseases have been developed for domestic animals because of the economic importance [197] , but not for humans. recent antiviral strategies against coronavirus infection have been reviewed elsewhere [203] . these strategies explore small interfering rna, blocking of viral entry (e.g., using carbohydrate-binding agents) or neutralizing antibodies. in addition, viral enzymes like protease or helicase are studied as potential targets for novel antivirals (reviewed in [203] ). according to estimates from the who, the burden of influenza in the usa is currently estimated to be 25-50 million cases per year, leading to 150 000 hospitalizations and 30 000-40 000 deaths per year. if these numbers are extrapolated to the rest of the world, influenza virus would infect 5-15% of the world population, causing 3-5 million cases of severe disease and approximately 0.5 million deaths per year. although this number is high, it would only characterize inter-pandemic influenza [204] . epidemics and outbreaks of influenza follow a seasonal pattern, which differs according to the region in the world: in temperate climate zones, seasonal epidemics typically begin in the late fall with a peak in late winter. the seasonal pattern is less pronounced in tropical zones (reviewed in [205] ). the focus of this book is on 'common cold'. for details on influenza and influenza viruses, further reading of standard literature is recommended. influenza viruses are members of the orthomyxoviridae, genus influenzavirus and include influenza virus types a, b and c. the viruses are enveloped pleomorphic particles with a size ranging from 100 to > 300 nm, their genome is organized on eight (influenza virus a and b) or seven (influenza virus c) negative-sense single-stranded rna segments [206] . spikes consist of hemagglutinin (ha) and neuraminidase (na). the nomenclature for human influenza virus includes type, geographic location of the first isolation, isolate number and year of isolation. in addition, subtypes of influenza a are described by their ha and na designations. to date, 16 ha and 9 na types have been described. the viral envelope is also associated with a matrix protein (m) that, after infection, forms a tetrameric ion channel. several polymerase proteins (pb1, pb2, pa) form, together with the nucleoprotein (np) and the rna, a ribonucleoprotein (rnp) complex (reviewed in [206] ). influenza viruses are transmitted via the respiratory route and bind to a cell receptor that consists of oligosaccharides and that is present on the surface of respiratory epithelial cells. the sialic acid-2,6-galactose linkage (sa 2,6gal) that is associated with binding to human influenza virus ha is present in the human respiratory tract (reviewed in [207] ). after binding, the virus enters the host cell by endocytosis [208] . further steps include fusion of the virus to the endosome [206] and the release of the rnp into the nucleus through an ion channel that is formed by m2 [209] . the viral rna is a template for complementary rna and the mrna. the nonstructural nep/ns2 as well as m1 play a role in the nuclear export of novel rna. assembly occurs at the apical surface of the cell, budding occurs and the novel virus is released (reviewed in [206] ). influenza viruses are transmitted via the respiratory route [206] . host specificity is largely determined by the availability of host cell receptors on the surface of epithelial cells [210] . usually, influenza in humans is an urti that is characterized by cough, headache, malaise and fever [211] . however, complications are frequent, and encephalitis, reye's syndrome, myelitis [212] as well as muscular manifestations of the infection including myocarditis [213] , disseminated intravascular coagulation and toxic and septic shock [214] may occur. major airway congestion, inflammation and necrosis have been found in histopathological examinations [215] . although much progress in the understanding of the pathogenesis of human influenza has been made during the past decade, the molecular mechanisms responsible for the virulence of particular strains of influenza virus are not yet understood. influenza a virus can infect humans as well as waterfowl and chickens, swine, horses, and other species. influenza b virus, on the other hand, has a restricted host range and circulates predominantly in humans. however, influenza b virus was recently isolated from seals [216] . different types of ha mediate species-specific binding of the virus [217, 218] . it would be beyond the scope of this book on common cold to review all the recent literature that has been published regarding the epidemiology of influenza viruses. however, one important aspect should be mentioned here. influenza virus is a changing virus and the repetitive occurrence of the yearly epidemic is supported by 'antigenic drift', an accumulation of point mutations in the viral receptors (ha and na). the drift is attributed to a low fidelity of the viral rna polymerase [219] . these new variants then infect a population without pre-existing immunity (reviewed in [206] ). a second process that contributes to the emergence of new influenza variants is called 'antigenic shift'. an antigenic shift may occur during co-infection with other influenza viruses. such a co-infection may lead to a viral reassortment (an exchange of genome segments between different virus strains) [206] . if this process leads to a new virus strain that is able to effectively spread from individual to individual, a worldwide outbreak, a pandemic, may occur [207] . there have been several pandemics in the past century including the so-called "spanish flu" in 1918/1919, infecting about the half of the world population at that time and killing approximately 20-50 million people [204] . current pandemic concerns are focused on the highly pathogenic variants of the strain a/h5n1 or h1n1. the epidemiology of influenza has been comprehensively reviewed [220, 221] . influenza is typically diagnosed clinically, but laboratory diagnosis has been established and organized according to standardized procedures as part of national and global pandemic plans. several effective vaccines exist today (reviewed in [222] ) and a prepandemic vaccine has been licensed recently. international committee on taxonomy of viruses rhinoviruses (picornaviridae) structure of a human common cold virus and functional relationship to other picornaviruses structure of a human rhinovirus complexed with its receptor molecule many rhinovirus serotypes share the same cellular receptor a cell adhesion molecule, icam-1 is the major surface receptor for rhinoviruses cell recognition and entry by rhino-and enteroviruses structure of human rhinovirus 3c protease reveals a trypsin-like polypeptide fold, rnabinding site, and means for cleaving precursor polyprotein transmission of rhinovirus colds by self-inoculation an investigation of possible transmission of rhinovirus colds through indirect contact hand-to-hand transmission of rhinovirus colds effect of route of inoculation on experimental respiratory viral disease in volunteers and evidence for airborne transmission quantitative rhinovirus shedding patterns in volunteers localization of rhinovirus replication in vitro with in situ hybridization respiratory virus infection of monolayer cultures of human nasal epithelial cells histopathologic examination and enumeration of polymorphonuclear leukocytes in the nasal mucosa during experimental rhinovirus colds localization of human rhinovirus replication in the upper respiratory tract by in situ hybridization shedding of infected ciliated epithelial cells in rhinovirus colds association between nasal secretion interleukin-8 concentration and symptom severity in experimental rhinovirus colds increased levels of interleukin-1 are detected in nasal secretions of volunteers during experimental rhinovirus colds rhinovirus stimulation of interleukin-6 in vivo and in vitro: evidence for nuclear factor kb-dependent transcriptional activation rhinoviruses infect the lower airways recurrent wheezy bronchitis and viral respiratory infections rhinovirus and influenza type a infections as precipitants of asthma frequency, severity, and duration of rhinovirus infections in asthmatic and non-asthmatic individuals: a longitudinal cohort study neutrophil degranulation and cell lysis is associated with clinical severity in virus-induced asthma respiratory viruses and exacerbations of asthma in adults asthmatic bronchial epithelial cells have a deficient innate immune response to infection with rhinovirus rhinoviruses in seattle families, 1975-1979 viral infections and treatment. marcel dekker recovery of new viruses (coryzavirus) from cases of common cold in human adults viruses causing common respiratory infections in man further properties of five new organized picornaviruses (rhinoviruses) establishment of a mouse model for human rhinovirus infection experimental infections of chimpanzees with human rhinovirus types 14 and 43 experimental infection of gibbons with rhinovirus mouse models of rhinovirus-induced disease and exacerbation of allergic airway inflammation recovery of cytopathogenic agent from chimpanzees with coryza respiratory syncytial virus respiratory syncytial virus. brief review international committee on taxonomy of viruses viral and host factors in human respiratory syncytial virus pathogenesis morphogenesis and ultrastructure of respiratory syncytial virus sirna profiling reveals key role of clathrin-mediated endocytosis and early endosome formation for infection by respiratory syncytial virus coupled translation of the second orf of the m2 mrna is sequence dependent and differs significantly in the subfamily pneumovirinae respiratory syncytial virus and metapneumovirus pathogen recognition and innate immunity retinoic acid-inducible gene i mediates early antiviral response and toll-like receptor 3 expression in respiratory syncytial virus-infected airway epithelial cells identification of the respiratory syncytial virus proteins required for formation and passage of helper-dependent infectious particles the central conserved cystine noose of the attachment g protein of human respiratory syncytial virus is not required for efficient viral infection in vitro or in vivo cleavage of the human respiratory syncytial virus fusion protein at two distinct sites is required for activation of membrane fusion respiratory syncytial virus and metapneumovirus the pathogenesis of respiratory syncytial virus disease in childhood respiratory syncytial virus respiratory syncytial virus in early life and risk of wheeze and allergy by age of 13 respiratory syncytial virus infections in immunocompromised adults speculation on pathogenesis in death from respiratory syncytial virus infection prevalence of various respiratory viruses in the middle ear during acute otitis media pathological changes in virus infections of the lower respiratory tract in children respiratory syncytial virus respiratory syncytial virus: update on infection, treatment and prevention the epidemiology of acute respiratory tract infection in young children risk of respiratory syncytial virus infection for infants from low-income families in relationship to age, sex, ethnic group and maternal antibody level respiratory syncytial virus differential production of inflammatory cytokines in primary infection with human metapneumovirus and with other common respiratory viruses of infancy effectiveness of drug therapies to treat or prevent respiratory syncytial virus infection-related morbidity genetic susceptibility to rsv disease bronchoalveolar lavage cytokine profiles in acute asthma and acute bronchiolitis human infant respiratory syncytial virus (rsv)-specific type 1 and 2 cytokine responses ex vivo during primary rsv infection type 1 and type 2 cytokine imbalance in acute respiratory syncytial virus bronchiolitis priming immunization determines t helper cytokine mrna expression patterns in lungs of mice challenged with respiratory syncytial virus respiratory syncytial virus deficient in soluble g protein induced an increased proinflammatory response in human lung epithelial cells the cysteinerich region of respiratory syncytial virus attachment protein inhibits innate immunity elicited by the virus and endotoxin pattern recognition receptors tlr4 and cd14 mediate response to respiratory syncytial virus severe respiratory syncytial virus disease in alaska native children palivizumab in the prophylaxis of respiratory syncytial virus infection respiratory syncytial virus disease mechanisms implicated by human, animal model, and in vitro data facilitate vaccine strategies and new therapeutics experimental respiratory syncytial virus infection of four species of primates respiratory syncytial virus infects the bonnet monkey, macaca radiata the pathogenesis of respiratory syncytial virus infection in cotton rats enhancement of respiratory syncytial virus pulmonary pathology in cotton rats by prior intramuscular inoculation of formalin-inactivated virus mechanism of antibody-mediated viral clearance in immunotherapy of respiratory syncytial virus infection of cotton rats primary respiratory syncytial virus infection in mice respiratory syncytial virus infection in mice intracellular ifn-gamma expression in natural killer cells precedes lung cd8 + t cell recruitment during respiratory syncytial virus infection immune-mediated disease pathogenesis in respiratory syncytial virus infection chinchilla and murine models of upper respiratory tract infections with respiratory syncytial virus evaluation of severe disease induced by aerosol inoculation of calves with bovine respiratory syncytial virus complete sequence of the rna genome of pneumonia virus of mice (pvm) parainfluenza viruses parainfluenza viruses myxoviruses: parainfluenza international committee on taxonomy of viruses the role of viral glycoproteins in adsorption, penetration, and pathogenicity of viruses new frontiers opened by the exploration of host cell receptors acquisition of serum antibodies to specific glycoproteins of parainfluenza virus 3 in children ) secretory immunological response in infants and children to parainfluenza virus types 1 and 2 brief report: parainfluenza virus type 3 infections: finding in sydney and some observations on variations in seasonality world-wide croup: an 11 year study in pediatric practice viral croup: a current perspective adenoviruses worldwide epidemiology of human adenovirus infections recovery of new agents from patients with acute respiratory illness infections in 18,000 infants and children in a controlled study of respiratory tract disease. i. adenovirus pathogenicity in relation to serologic type and illness syndrome international committee on taxonomy of viruses the icosahedral form of an adenovirus adenoviruses. the viruses and their replication adenovirus: from foe to friend a proposed terminology for the adenovirus antigens and virion morphological subunits human adenovirus c the molecular composition of the adenovirus type 2 virion cd46 is a cellular receptor for all species b adenoviruses except types 3 and 7 transcriptional and transforming activities of the adenovirus e1a proteins latent adenovirus infection in copd relations of the new respiratory agents to acute respiratory diseases adenovirus infections and lung disease in situ hybridization studies of adenoviral infections of the lung and their relationship to follicular bronchiectasis histopathology of adenovirus infection of the respiratory tract in young children severe adenovirus bronchiolitis in children latent adenoviral infection in the pathogenesis of chronic airways obstruction immunodetection of adenoviral e1a proteins in human lung tissue amplification of inflammation in emphysema and its association with latent adenoviral infection latent adenoviral infection induces production of growth factors relevant to airway remodeling in copd the nature of small airway obstruction in chronic obstructive pulmonary disease adenoidalpharyngeal-conjunctival agents: a newly recognized group of common viruses of the respiratory system a study of illness in a group of cleveland families. x. the occurrence of adenovirus infections immunization with live types 7 and 4 adenovirus vaccines. ii. antibody response and protective effect against acute respiratory disease due to adenovirus type 7 a newly discovered human pneumovirus isolated from young children with respiratory tract disease human metapneumovirus international committee on taxonomy of viruses characterization of human metapneumoviruses isolated from patients in north america global genetic diversity of human metapneumovirus fusion gene use of the p gene to genotype human metapneumovirus identifies 4 viral subtypes analysis of the genomic sequence of a human metapneumovirus respiratory syncytial virus and parainfluenzavirus individual contributions of the human metapneumovirus f, g, and sh surface glycoproteins to the induction of neutralizing antibodies and protective immunity human metapneumovirus and lower respiratory tract disease in otherwise healthy infants and children detection of severe human metapneumovirus infection by real-time polymerase chain reaction and histopathological assessment human metapneumovirus infections in hospitalized children brief communication: fatal human metapneumovirus infection in stem-cell transplant recipients human metapneumovirus perspective on the host response to human metapneumovirus infection: what can we learn from respiratory syncytial virus infections? epidemiology of human metapneumovirus comparison of the inhibition of human metapneumovirus and human respiratory syncytial virus by ribavirin and immune serum globulin in vitro isolation and characterization of monoclonal antibodies which neutralize human metapneumovirus in vitro and in vivo cloning of a human parvovirus by molecular screening of respiratory tract samples human bocavirus -a novel parvovirus to infect humans seroepidemiology of human bocavirus in hokkaido prefecture complete coding sequences and phylogenetic analysis of human bocavirus (hbov) human bocavirus infection, people's republic of china human bocavirus: prevalence and clinical spectrum at a children's hospital high rate of human bocavirus and adenovirus co-infection in hospitalized israeli children cd4 + t helper cell responses against human bocavirus vp2 virus-like particles in healthy adults development of real time pcr assays for detection and quantification of human bocavirus coronaviruses. in: bn fields cultivation of a novel type of common-cold virus in organ cultures a new virus isolated from the human respiratory tract recovery in tracheal organ cultures of novel viruses from patients with respiratory disease identification of a novel coronavirus in patients with acute respiratory syndrome a novel coronavirus associated with severe acute respiratory syndrome identification of a new human coronavirus characterization and complete genome sequence of a novel coronavirus, coronavirus hku1, from patients with pneumonia international committee on taxonomy of viruses coronaviridae: a review of coronaviruses and torovirus the coronavirus surface glycoprotein the coronavirus membrane glycoprotein the small membrane protein the coronavirus hemagglutinin esterase glycoprotein human and bovine coronaviruses recognize sialic acid-containing receptors similar to those of influenza c viruses the coronavirus nucleocapsid protein isolated he protein from hemagglutinating encephalomyelitis virus and bovine coronavirus has receptor-destroying and receptor-binding activity feline aminopeptidase n serves as a receptor for feline, canine, porcine and human coronaviruses in serogroup 1 angiotensin-converting enzyme 2 is a functional receptor for the sars coronavirus conformational changes in the spike glycoprotein of murine coronavirus are induced at 37 degrees c either by soluble murine ceacam1 receptors or by ph 8 coronavirus spike proteins in viral entry and pathogenesis the molecular biology of coronaviruses effects of a "new" human respiratory virus in volunteers coronavirus 229e-related pneumonia in immunocompromised patients human coronavirus nl 63, a new respiratory virus frequent detection of human coronaviruses in clinical specimens from patients with respiratory tract infection by use of a novel real-time reverse-transcriptase polymerase chain reaction a model of viral wheeze in nonasthmatic adults: symptoms and physiology a previously undescribed coronavirus associated with respiratory disease in humans human coronavirus nl63 infection in canada prospective population-based study of viral lower respiratory tract infections in children under 3 years of age (the pri.de study) prospective study of human metapneumovirus infection in children less than 3 years of age clinical and molecular epidemiological features of coronavirus hku1-associated community-acquired pneumonia evidence of human coronavirus hku1 and human bocavirus in australian children detection of the new human coronavirus hku1: a report of 6 cases encyclopedia of virology coronaviruses and toroviruses coronative antibody titers in sera of healthy adults and experimentally infected volunteers human coronavirus nl63 employs the severe acute respiratory syndrome coronavirus receptor for cellular entry bats are natural reservoirs of sars-like coronaviruses severe acute respiratory syncytial virus in chinese horseshoe bats recent antiviral strategies against coronavirus-related respiratory illnesses influenza virus influenza seasonality: underlying causes and modeling theories orthomyxoviridae: the viruses and their replication influenza virus: the biology of a changing virus caveolae as an additional route for influenza virus endocytosis in mdck cells influenza a virusm2 ion channel activity is essential for efficient replication in tissue culture sialic acid species as a determinant of the host range of influenza a viruses does this patient have influenza influenza virus and cns manifestations influenza-associated deaths among children in the united states influenza b virus infection associated with non-bacterial septic shock-like illness histopathologic and immunohistochemical features of fatal influenza virus infection in children during the 2003-2004 season influenza b virus in seals differential sensitivity of human, avian, and equine influenza a viruses to a glycoprotein inhibitor of infection: selection of receptor specific variants receptor determinants of human and animal influenza virus isolates: differences in receptor specificity of the h3 hemagglutinin based on species of origin positive darwinian evolution in human influenza a viruses the epidemiology of influenza and its control influenza and influenza vaccination waiting for a pandemic key: cord-290505-omszep7u authors: pochon, cécile; voigt, sebastian title: respiratory virus infections in hematopoietic cell transplant recipients date: 2019-01-09 journal: front microbiol doi: 10.3389/fmicb.2018.03294 sha: doc_id: 290505 cord_uid: omszep7u highly immunocompromised pediatric and adult hematopoietic cell transplant (hct) recipients frequently experience respiratory infections caused by viruses that are less virulent in immunocompetent individuals. most of these infections, with the exception of rhinovirus as well as adenovirus and parainfluenza virus in tropical areas, are seasonal variable and occur before and after hct. infectious disease management includes sampling of respiratory specimens from nasopharyngeal washes or swabs as well as sputum and tracheal or tracheobronchial lavages. these are subjected to improved diagnostic tools including multiplex pcr assays that are routinely used allowing for expedient detection of all respiratory viruses. disease progression along with high mortality is frequently associated with respiratory syncytial virus, parainfluenza virus, influenza virus, and metapneumovirus infections. in this review, we discuss clinical findings and the appropriate use of diagnostic measures. additionally, we also discuss treatment options and suggest new drug formulations that might prove useful in treating respiratory viral infections. finally, we shed light on the role of the state of immune reconstitution and on the use of immunosuppressive drugs on the outcome of infection. infections and still contribute to significant mortality with rates ranging between 10 and 50% if infection progresses to the lower respiratory tract (boeckh, 2008; englund et al., 2011; renaud and campbell, 2011; protheroe et al., 2012; chemaly et al., 2014; spahr et al., 2018) . human pathogenic viruses frequently causing respiratory infections in the allogeneic hct setting include rsv, adv, ifv, piv, hmpv, rhv, hcov, and hbov (renaud and englund, 2012; abbas et al., 2017) . such respiratory virus infections contribute to morbidity and mortality for a number of reasons. firstly, hct recipients suffer from prolonged immunosuppression and a lack of both humoral and t cell-mediated immunity impairs viral clearance (kohlmeier and woodland, 2009; schmidt and varga, 2018) . in addition, these patients are usually antibody-depleted and the strength of the conditioning regimen and the ongoing immunosuppressive therapy diminishes the antiviral immune response further. moreover, the type of graft may have an impact on the immune response and pediatric hct recipients may also be immunologically naïve because of their age. secondly, viral factors shape the outcome. respiratory viruses vary in their pathogenicity, i.e., they cause more severe disease compared with other viruses. also, there is the capacity for certain strains of a given respiratory virus to display increased virulence as is the case with ifv and it is therefore important to identify viral and host genes that determine virulence. thirdly, a limited number of effective antivirals or the emergence of drug resistance also hamper successful treatment. finally, factors like persisting infections, co-infections and co-morbidities will also influence the outcome. in contrast to immunocompetent individuals, hct recipients present with prolonged viral shedding and have a higher rate of progression from uri to lri (chemaly et al., 2014; de lima et al., 2014; kim et al., 2014) . increased viral loads and protracted infections raise the likelihood of progression that differs between viruses. uri has been commonly defined as laboratory-confirmed viral nasal wash or swab with pharyngitis, cough, otitis, nasal discharge and/or congestion in the absence of infiltrates on chest x-ray or computed tomography (ct) scan and hypoxemia, and lri has been defined as laboratory-confirmed viral nasal wash or swab in the presence of infiltrates on chest x-ray or ct scan suggestive of a viral respiratory infection but not necessarily detection of viral nucleic acid in samples obtained from bal . in hct recipients, rsv is most frequently detected, followed by hmpv and piv. in 5-50% of cases, these viruses progress to lri, often complicate the transplant course by causing airflow obstruction or bronchiolitis obliterans and also increase the rate of mortality up to 50% (chemaly et al., 2014) . several respiratory viruses share a number of risk factors associated with lri occurrence. it is worth noting that cmv also represents an opportunistic pathogen that is causally related to pneumonia and lri. however, cmv will not be discussed in this review since it is not primarily considered a respiratory virus. that said, cmv is a pathogen that frequently occurs as part of a simultaneous infection -indeed a respiratory viral infection can often precede bacterial and fungal infections -thus further complications associated with respiratory virus infections need to be acknowledged. in this review, we will focus on respiratory virus infections and, particularly, those that are commonly detected after hct. we highlight risk factors that facilitate progression from uri to lri and discuss diagnostic approaches. current treatment strategies along with new treatment options will be discussed. adenovirus is a member of the adenoviridae that circulates throughout the year. currently, 90 human types are known which are further divided into seven species a-g 1 (accessed on 20 august 2018). alongside conjunctivitis and diarrhea, adv can cause pharyngitis, bronchitis and pneumonia but also lethal hepatitis or severe bloody colitis. pertinent to this review, adv is a pathogen associated with severe complications in immunosuppressed pediatric hct recipients including increased mortality (leen et al., 2006; feuchtinger et al., 2007; lion, 2014; feucht et al., 2015; hiwarkar et al., 2018) . in adult patients, adv infections are less commonly reported. however, it is possible this perception might be biased by reduced frequency of screening in adults. a study in adult allogeneic hct recipients reported an infection rate of 2.5%. pneumonia occurred in 24% of cases and was the most common cause of death associated with adv (yilmaz et al., 2013) . an important consideration is that adv infections infrequently present with respiratory symptoms at the onset of infection; instead they are commonly detected by monitoring stool (lo et al., 2013; lion, 2014) . indeed, gastrointestinal shedding pre-transplant has been demonstrated to reflect increased risk of viremia after hct (kosulin et al., 2018a) . human bocavirus was identified in 2005 as a human pathogen that causes respiratory tract infections in infants. it has been assigned to the parvoviridae and received its name because of sequence homology to two other members in the genus bocaparvovirus, bovine parvovirus infecting cattle and canine minute virus infecting dogs (allander et al., 2005) . currently, four hbov variants have been described. like hbov3, hbov1 can enter an episomal state, allowing the establishment of a persistent infection (kapoor et al., 2011) . whereas hbov2, hbov3, and hbov4 cause gastroenteritis and are genetically diverse, hbov1 exhibits a stronger association with lri than enteritis, often in conjunction with other respiratory pathogens, and has limited genetic diversity although reinfections with different variants occur (arden et al., 2006; ditt et al., 2008; arthur et al., 2009; kapoor et al., 2010; wang et al., 2010; martin et al., 2015) . symptoms following infection include cough, dyspnea, pharyngitis, bronchitis, pneumonia, and diarrhea. several reports have suggested that hbov1 can cause severe infections on its own and is not a mere bystander virus ursic et al., 2011; edner et al., 2012) . likewise, hbov can cause life-threatening infections and prolonged shedding in immunocompromised patients, and for immunocompetent children a median shedding time of 50 days has been reported (kupfer et al., 2006; allander, 2008; koskenvuo et al., 2008; de vries et al., 2009; martin et al., 2015) . this long shedding period might lead to more severe disease in immunocompromised children (schenk et al., 2007) . importantly, hbov does not exhibit seasonality and thus remains a threat throughout the year. human coronavirus belongs to the coronaviridae that are endemic in humans. annually hcov are responsible for 15-30% of uri with pharyngitis and rhinitis in immunocompetent hosts. historically, two common hcov were known: hcov-229e and hcov-oc43. however, the emergence of severe acute respiratory syndrome-coronavirus (sars-cov) along with two further hcov (hcov-hku1 and hcov-nl63) has expanded the family (van der hoek et al., 2004; woo et al., 2005) . in contrast to the low incidence of bronchitis or pneumonia in healthy children, severe clinical features have been described in immunocompromised patients. both the presence of a respiratory co-pathogen (rsv) and host factors like young age < 5 years and an immunocompromised status were reported to contribute to lri. however, it should be noted that only 10 children with hct were included (ogimi et al., 2017b (ogimi et al., , 2018a . in separate studies, hcov have been associated with increased mortality and prolonged shedding in the hct setting (milano et al., 2010; renaud and campbell, 2011) . risk factors for prolonged shedding (at least 21 days) in the upper respiratory tract were determined in a cohort of 44 patients and included high viral load, myeloablative conditioning, and prior high-dose steroid use (ogimi et al., 2017a) . of 44 patients, 31 samples were analyzed shown to contain evidence of hcov-oc43 (35%), hcov-nl63 (32%), hcov-hku1 (19%), and hcov-229e (13%) infection. analysis for duration of shedding showed that none of the strains appeared to cause longer shedding compared with others. in addition, genomic approaches investigated whether viral genome evolution could identify genetic changes associated with prolonged shedding. identification of such changes could aid the development of new antiviral agents. single nucleotide polymorphisms could not be identified until day thirty after the onset of viral shedding. this finding might not be surprising given the protracted evolution rate of hcov. however, overt viral genome changes might occur at a later time point, and changes in genome composition might result in a modification of the treatment strategy (ogimi et al., 2017a) . in another study, hcov were examined in bal (ogimi et al., 2017b) . the median time to hcov lri occurrence was 302 days after hct. among 23 bal samples analyzed, 48% were hcov-oc43, hcov-nl63 was detected in 22%, hcov-229e in 17% and hcov-hku1 in 13%. although somewhat limited because of sample size, these data show a similar frequency and order of detected viral strains compared to those detected from nasal samples with the exception of hcov-hku1 which was predominant in nasal samples (ogimi et al., 2017a) . the detection of rsvs did not change clinical outcome. influenza virus, an orthomyxoviridae member, exhibits high genomic variability due to an absence of proofreading activity in its rna -a process that contributes to antigenic drift. as some ifv strains are more virulent than others, the strain of ifv impacts on severity, depending on circulating strains, and this can be impacted upon by the composition of the vaccine and also the uptake of vaccine in the population. individuals with a compromised immune system such as the elderly and hct recipients are at increased risk of complications and death, and outbreaks in hct units have been reported (lalayanni et al., 2010; suyani et al., 2011) . in hct recipients, fever maybe absent in about 19% of cases as might classical symptoms such as sore throat, nasal congestion and discharge, cough, chills and myalgia . as with other respiratory viruses, ifv shedding in hct patients has been reported to be prolonged with a median of 12 days; lymphopenia correlated with the duration of shedding, and steroid use > 1 mg/kg increased ifv secretion (khanna et al., 2009; boudreault et al., 2011; engelhard et al., 2013) . infants may present with sepsis-like symptoms and pneumonia. complications in adults include pneumonia, myocarditis, encephalitis and guillain-barré syndrome. as with piv infections (see below), bacterial and fungal co-infections with ifv are known (nichols et al., 2004b; engelhard et al., 2013) . disease progression to lri takes place in up to 35% and mortality ranges between 15 and 28% in patients diagnosed with pneumonia. therefore, early treatment within 2 days of presenting with symptoms has been favored (whimbey et al., 1994; nichols et al., 2004b; chemaly et al., 2006; kmeid et al., 2016) . while risk factors have been validated for rsv and piv, equivalent quality indicators are limited for ifv. potential risk factors that might result in lri are lymphopenia and neutropenia as well as increased age (>65 years) while steroid use is unclear (ljungman et al., 2001; chemaly et al., 2006; choi et al., 2012) . ifv genome detection in blood was associated with hypoxemia, respiratory failure, and overall mortality (choi et al., 2012) . kmeid et al. (2016) applied an isi that had been developed for rsv to identify patients with ifv infection who might run at risk to develop progression to lri . according to variables such as age, neutrophil and lymphocyte counts, conditioning regimen, steroid use and time from transplantation, hct recipients were grouped into low, moderate, and high risk immunodeficiency categories. a high risk score was applied if the score was between 7 and 12 and was likely to include anc < 500/mm 3 (score 3) and/or alc < 200/mm 3 (3), age ≥ 40 years (2), myeloablative regimen (1), gvhd (1), steroid use within the last 30 days (1) and recent (within the last 30 days) or pre-engraftment allogeneic hct (1). patients with a high risk score had a significantly higher probability of developing lri compared to the low risk group (kmeid et al., 2016) . human metapneumovirus, formerly a member of the paramyxoviridae now belonging to the pneumoviridae, was identified in 2001 as an agent capable of causing respiratory disease in children (van den hoogen et al., 2001) . in immunocompetent individuals, hmpv causes symptomatic uri and lri predominantly in children or adults above 65 years of age (boivin et al., 2002) . no specific symptoms that distinguish hmpv from other viral respiratory infections exist. it has been reported that disease is more severe in case of a rsv co-infection but these data are considered controversial. in immunocompetent children, one study revealed that hmpv with rsv co-infection increased bronchiolitis (semple et al., 2005) , however, this could not be substantiated in other studies that investigated single infections and co-infections for hmpv and rsv (moe et al., 2017; yan et al., 2017) . for both hmpv and rsv, a and b types occur that can be distinguished by different primer sets in pcr (renaud et al., 2013) . immunocompromised children are at higher risk of developing lri with higher risk of requiring intensive care treatment with increased mortality (chu et al., 2014) . in a study of 21 hct recipients, hpmv infection was documented in the majority of patients but did not cause any symptoms or disease (debiaggi et al., 2006) . however, in a study that included 251 episodes of uri and lri, 16 of the 22 episodes in which hpmv was detected occurred in hct recipients, resulting in an infection rate of approximately 6% of all hct patients . another study revealed hpmv in bal samples from 5 of 163 patients who became symptomatic within a month after engraftment, and fatality reached up to 80% if bal was positive for hmpv (englund et al., 2006) . progression to lri has been reported to take place between 21 and 40% (renaud and campbell, 2011) . for hmpv, risk factors for mortality include length between hct and infection; neutropenia, lymphopenia, low monocyte count at diagnosis, and a steroid dose before diagnosis of ≥1 mg/kg (seo et al., 2016) . in the latter study, the presence of co-pathogens contributed to higher risk. glucocorticoid application and lymphopenia have been identified as risk factors that contribute to progression from uri to lri (seo et al., 2016) . in a systematic review, hmpv progressed to lri in 34% of cases and the mortality rate increased from 6 to 27% when lri was present but no hmpv-associated risk factors for increased mortality could be identified (shah et al., 2016b) . parainfluenza virus is a member of the paramyxoviridae that circulates throughout the year. piv causes lri in approximately 20% of healthy children (bicer et al., 2013) and is also responsible for life-threatening lri in hct recipients (srinivasan et al., 2011; shah et al., 2016a) . four piv serotypes are known. piv serotype 3 (piv-3) is the most frequent occurring serotype and has been responsible for infections after hct (cortez et al., 2001; nichols et al., 2004a; maziarz et al., 2010; hodson et al., 2011) . in one study comprising 3577 patients with piv infection, 6.4% had an infection with piv-3, and 24% of these patients developed pneumonia (nichols et al., 2001a) . another study reported piv infections in up to 18% of cases during the first 3 months post hct . in 20-40% of patients who experience uri, infection progresses to lri with a median time of 78 days. virus-associated mortality is around 10% after piv infection but increases to 27% if infection progresses to lri (nichols et al., 2001a; srinivasan et al., 2011; chemaly et al., 2012; ustun et al., 2012; seo et al., 2014a; shah et al., 2016a) . piv risk factors for progression to lri are lymphopenia, steroid use and co-infections with other respiratory agents, however, myeloablation and infections in the early post-transplant period were variables with unclear association (seo et al., 2014b; shah et al., 2016a) . pulmonary co-pathogens, e.g., aspergillus fumigatus, are often present in patients with pneumonia and highly contribute to mortality (nichols et al., 2001a; ustun et al., 2012) . respiratory syncytial virus, now also belonging to the pneumoviridae, is a frequent pathogen in infants and children that causes uri with rhinitis and laryngitis but also bronchitis and pneumonia if progression to lri occurs. in hct recipients, rsv infections occur up to 30% khanna et al., 2008; avetisyan et al., 2009; shah and chemaly, 2011; waghmare et al., 2013) . in approximately half of the hct recipients with uri caused by rsv, infection will progress and result in pneumonia that is associated with average mortality rates around 30% without effective treatment (nichols et al., 2001b; boeckh et al., 2007; khanna et al., 2008; shah and chemaly, 2011; seo et al., 2013; waghmare et al., 2013) . risk factors for progression to lri include advanced age, lymphopenia, myeloablative regimen, steroid use, gvhd and pre-engraftment infection and occur at a mean of 38% (nichols et al., 2001b; martino et al., 2005; khanna et al., 2008; shah and chemaly, 2011; hirsch et al., 2013) . further, smoking history, conditioning with high-dose total body irradiation with 1200-1575 cgy and specifically an absolute lymphocyte count ≤ 100/mm 3 at uri onset were significantly associated with progression to lri. in contrast, lung function, steroid use, lymphocyte engraftment dynamics, rsv subtypes and subtypespecific neutralizing antibody levels were not associated with progression, revealing transplant-related rather than viral factors which might put a focus on the characterization of rsv-specific t cells (kim et al., 2014) . in addition, absolute lymphocyte counts of > 1000/mm 3 at uri onset were protective and no advance to lri was observed. another study that examined risk factors for rsv rna detection in serum or plasma identified mechanical ventilation, neutropenia, monocytopenia as well as thrombocytopenia as associated factors, however, lymphopenia and steroid use did not increase the risk of rsv rna plasma detection (waghmare et al., 2013) . for that study, a median time of 52 days to lri onset was described. as described for ifv, an isi had been originally proposed to stratify rsv-infected patients into groups based on their risk of progression from uri to lri and to evaluate rsv-related mortality . this stratification should help to identify patients who might profit from antiviral therapy such as aerosolized ribavirin. this index is based on age, absolute neutrophil and lymphocyte counts, acute or chronic gvhd, myeloablative conditioning, corticosteroid use and time of infection. however, validation of this index needs to be performed in multicenter studies. rhinovirus, a member of the picornaviridae, comprises more than 100 serotypes and are divided into the three species a, b and c. there is no seasonal preference for rhv, however, more infections might occur during spring and autumn. rhv are the most frequently detected community-associated respiratory viruses that cause cough, a runny nose but also bronchitis or pneumonia in hct recipients (ison et al., 2003; milano et al., 2010) . symptomatic rhv infections occur in 10-30% of hct recipients and initial high viral loads are associated with prolonged shedding (parody et al., 2007; milano et al., 2010; ogimi et al., 2018b) . a recent study showed that median shedding time among patients with rhv infection was 9.5 days, and 18/38 (47%) of hct recipients had prolonged rhv shedding (at least 21 days; range, 2-89 days) that was similar for all three species but the majority of patients (69%) shed rhv species a. risk factors for prolonged rhv shedding pointed to a high initial viral load as defined by a ct value below the median (30.3) (ogimi et al., 2018b) . infection control measures should consider these long infection periods. progression to lri is increasingly being detected and fatal outcomes due to rhv infections have been described (gutman et al., 2007; jacobs et al., 2013; ogimi et al., 2018b) . patients who reveal respiratory tract infection symptoms before hct should be tested for respiratory pathogens by multiplex pcr. campbell et al. analyzed data from 458 adult and pediatric patients and identified 25% of patients to be positive for at least one respiratory virus pre-transplant. symptomatic patients had lower survival 100 days after hct compared with patients with negative test results and increased overall mortality. since this risk was increased even if only rhv was detected, the authors proposed to consider deferral of hct in symptomatic patients with any respiratory virus positive, not just only in case of rsv, ifv, piv, or hmpv detection. asymptomatic patients with a virus detected did not exhibit higher mortality and hct might proceed as planned; however, it is unclear if these patients actually shed replicating virus or if only viral nucleic acid is being detected. there was no higher incidence of bronchoscopy in the symptomatic versus the asymptomatic group. any decision to delay transplant has to consider hct factors such as underlying disease, the conditioning regimen associated with it and donor logistics (campbell et al., 2015) . in a follow-up study that focused on pediatric hct recipients, kim et al. (2017) detected respiratory viruses pre-transplant in 81 of 218 patients. rhv were detected in 24% of cases, followed by a group of viruses consisting of adv, rsv, hmpv pfv, and piv (11%). pre-transplant detection was associated with increased hospitalization during the first 100 days, and hcov was only detected pre-transplant in 2% of cases. since the conditioning regimen might have an impact on the incidence of respiratory virus infections pre-transplant, patients with myeloablative and non-myeloablative conditioning were compared. the incidences for respiratory virus infections were similar among both groups. however, in contrast to patients receiving non-myeloablative conditioning, lri are significantly increased during the first 100 days post hct when myeloablative regimen was given (schiffer et al., 2009) . immediately after hct in the neutropenic phase, patients frequently develop febrile episodes and are especially prone to bacterial infections. therefore, they are often treated with antibiotics to protect them from life-threatening bacterial infections. this treatment alters microbiota composition and has an impact on immune responses (ichinohe et al., 2011; abt et al., 2012; dyer et al., 2016 ). an immunodulatory compound associated with microbiota is butyrate, a short chain fatty acid that is important in maintaining gut microbiome equilibrium as well as in controlling immune responses in organs such as the lung (donohoe et al., 2011; chang et al., 2014) . in a recent study, haak et al. (2018) investigated the influence of butyrate on microbiota composition and its influence on respiratory viral infections and the associated risk to develop lri in hct recipients. patients who received antibiotic treatment resulting in altered microbiota colonization and diminished butyrate-producing bacteria had a higher risk of developing lri indicating that the presence of butyrate-producing bacteria appears to be protective (haak et al., 2018) . a recent analysis from the seattle group has shown that antibiotic exposure before the onset of viral respiratory infection increases the risk of progression from uri to lri in case of hmpv, piv and rsv infection (ogimi et al., 2018a) . however, a certain class of antibiotics that poses a particular risk could not be identified. another recent study described significant lower overall survival for patients with respiratory virus infection accompanied by bacterial co-infection that contributed to increased mortality (pinana et al., 2018) . risk factors for developing bacterial co-infection following a respiratory viral infection were steroid use ≥ 1mg/kg/d and cmv dnaemia requiring antiviral therapy. mortality-associated risk factors such as lymphopenia < 0.5 × 109/ml, cmv dnaemia requiring antiviral therapy at the time of viral lri diagnosis and oxygen support at the time of bal were used to establish a risk score in order to stratify patients to help predict mortality, no matter if a co-infection was present or not. this new risk score was compared to other scores including the isi described above, however, the application of isi criteria did not prove useful to predict mortality in this cohort. currently, isi does not include co-infections as a variable, and future studies are needed to validate and compare different risk scores (pinana et al., 2018) . the profound lymphopenia after hct has been described as a risk factor in the study above and multiple other studies, and it has recently been suggested that cd8 t cells may protect against a secondary infection (schmidt and varga, 2018) . the fourth european conference on infections in leukemia (ecil-4) developed guidelines for diagnosis and treatment on several respiratory viruses (hirsch et al., 2013) . hct candidates and recipients with uri or lri should be tested for respiratory viruses to guide infection management and possible deferral of transplantation. specimens should be taken from the site of infection; for uri, pooled swabs should be analyzed and for lri, a tracheal aspirate or ideally a bal should be performed. however, patient circumstances often do not permit invasive procedures. these guidelines favored a first-line screening for ifv a and b, rsv, and piv, however, with more advanced multiplex pcr assays becoming available, all agents might be examined at once (waghmare et al., 2016) . over the past years, more respiratory infections in hct recipients have been reported due to the development and use of new diagnostic methods. routine molecular diagnostics of respiratory viruses nowadays includes multiplex pcr approaches that have improved detection of respiratory agents in hct recipients (leber et al., 2018; sam et al., 2018) . multiplex assays covering the relevant respiratory viral agents are preferred over laborious and time-consuming viral culture and direct fluorescence antibody assays because of its sensitivity, specificity and rapid turnaround time. in addition, viral nucleic acid quantification is important because determination of a viral load might indicate prolonged viral shedding in case of rhv (ogimi et al., 2018b) . however, this does not provide information about active replication and viral culture techniques are not well established, e.g., for hcov. a problem with virus detection is a lack of standardization among assays since different primers, probes and techniques are used (huang et al., 2017) . nasal respiratory swabs are routinely performed pre-transplant and should be collected in case of suspected respiratory infection. a large prospective study associated respiratory virus detection before transplantation with prolonged hospitalization and decreased survival at day 100 (campbell et al., 2015) . rhv detection after a routine pretransplant screening can result in fatal infection, even if the patient is asymptomatic and presents with regular findings on chest ct (milano et al., 2010; campbell et al., 2015; waghmare et al., 2016) . if any respiratory virus is identified by multiplex pcr pre-transplant, hct should be postponed peck et al., 2004; hirsch et al., 2013) . however, as occurs in leukemia relapse cases, the time window from remission to hct can be narrow, thus making a decision difficult. irrespective of uri or lri, transplant should be delayed if possible, probably even in cases where data are scarce as is the case in hcov and hbov infections. while episodes of both uri and lri have shown a higher mortality risk in symptomatic adults in contrast to asymptomatic patients (abandeh et al., 2013) , transplant delay should be recommended in symptomatic adults. however, in pediatric patients who tend to shed virus more frequently than adults and acquire viruses more frequently, transplant delay should be considered on a case by case basis in asymptomatic patients. in aiding to determine progression from uri to lri, radiologic signs are considered an important component although inter-observer variability is high and radiologic signs are sometimes difficult to interpret (elemraid et al., 2014; franquet, 2018) . it is important to perform useful radiologic diagnostics in symptomatic patients with suspected lri. chest x-rays are generally not recommended because lack of sensitivity but ct should be performed to evaluate lri. in children, chest x-rays do not provide specific information in case of hmpv infection where common findings include hyperinflation, atelectasis and perihilar opacities (hilmes et al., 2017) . in an attempt to try and provide a standardized framework a scoring tool named the rsi has been established to quantify severity of radiologic findings and correlate them with outcome in piv lri. infiltrate expansion, when longitudinally assessed by rsi, was predictive of mortality in patients with piv-associated disease and discrepant results were obtained when ct and chest x-ray findings were compared. early signs suggest that the rsi appears to be a useful tool to predict mortality but needs to be validated in future studies (sheshadri et al., 2018) . computed tomography findings associated with lri include bronchial wall thickening and diffuse or patch-like ground glass opacities as a sign of interstitial infiltrates (franquet et al., 2006; herbst et al., 2013) . however, radiologic approaches often do not show clear signs of infection in symptomatic patients. additionally, the occurrence of simultaneous infections means correlating a specific virus with ct findings is difficult. to address this, a retrospective study by kim et al. (2016) examined ct scans from lri patients with bal specimens and single infections of either rsv, ifv or piv in order to identify viruscharacteristic ct lesions. patch consolidations of at least one centimeter or more than one segmental level were only observed in piv infections. ground-glass opacities were identified in all ct scans when ifv was detected and were less frequently seen with piv (detected in 71% of cases) and rsv (67%). bronchial wall thickening was prominent with ifv and rsv in about two thirds of ct scans analyzed whereas it was seen in only one third in piv infections. anatomically, rsv infection was localized in the upper and middle lobes (44%), piv preferentially in the lower lobes (69%) and ifv infection resulted in a diffuse pattern (kim et al., 2016) . however, due to the retrospective nature of the study by kim et al., no distinction between early and late infection stage ct findings could be made which might have revealed different patterns. other studies similarly identified ground-glass opacities for ifv infections (franquet et al., 2006; kanne et al., 2007) and bronchial wall thickening combined with nodules and tree-in-bud in rsv infections (mayer et al., 2014) . another small study also detected bronchial wall thickening in patients with confirmed rsv infections but also groundglass opacities, and these findings were equally seen in hmpv infections where lesions appeared to be more asymmetrical (syha et al., 2012) . although ct findings appear to be non-specific, they might help to differentiate between pathogens causing lri if a thorough diagnostic workup including multiplex pcr with representative specimens is employed to largely rule out simultaneous infections. infection control measures are of high importance and should be in place in case of suspected or confirmed respiratory virus infection (boeckh, 2008) . since these viruses are transmitted by close contact with infected individuals or contaminated material, hand hygiene is of utmost importance since nosocomial infections have occurred that must be avoided (hoellein et al., 2016) . the use of universal surgical mask might also be useful to prevent respiratory viral infections after hct (sung et al., 2016) . patients with documented respiratory virus infection should be isolated with restricted contact and strict protection measures should be applied (gloves, gowning, masks, eye protection) to visitors and healthcare workers (hirsch et al., 2013) . since hct recipients might shed these viruses for a long time because of their impaired immunity, precautions should be taken if a patient is discharged. for example, a patient should be escorted through the outpatient clinic to avoid direct contact with and spread to other patients, and prolonged isolation might be necessary (tomblyn et al., 2009; hirsch et al., 2013) . this might be especially important for rhv in case high viral loads are detected since shedding time in these patients exceeds 21 days (ogimi et al., 2018b) . however, piv-3 and hmpv infections might go unrecognized in immunocompetent persons, thus making it difficult to prevent infection (nichols et al., 2001b; debiaggi et al., 2007; shah et al., 2016b; birger et al., 2018) . each time a respiratory tract infection is proven after hct, a reduction of immunosuppressive treatment should be the response. a steroid dose greater than 1 or 2 mg/kg/day has been proven to be an independent risk factor for overall mortality in lri with rhv (seo et al., 2017) , rsv (shah et al., 2016a) , hmpv, and piv (waghmare et al., 2016) . however, no correlation between progression or mortality and steroid dose was found for ifv (waghmare et al., 2016) . adenovirus pneumonia is usually observed in adv disseminated disease. preemptive treatment is recommended as soon as viremia is higher or equal to 1000 copies/ml coupled with lymphocyte counts below 300/mm 3 and cd4 t lymphocyte counts below 25/mm 3 (hiwarkar et al., 2017) . this is particularly important in high risk patients and those who received cord blood graft or are under steroid treatment (lindemans et al., 2010) . high adv levels in stool have also been associated with gastrointestinal adv disease (feghoul et al., 2015) -an observation that underpins a current clinical trial addressing the usefulness of a preemptive treatment based on stool adv detection 2 . so far, no recommendation has been made for adv detection in upper airways. specific treatment of adv pneumonia should be given as early as possible after diagnosis (neofytos et al., 2007; lindemans et al., 2010) . several studies have reported successful cidofovir treatment of adv infections in immunocompromised hosts after hct, combined with withdrawal of immunosuppression (e.g., at 5 mg/kg once every week for 2 weeks, followed by a maintenance dose of 5 mg/kg once every fortnight, or three times weekly at 1 mg/kg) (lindemans et al., 2010; wy ip and qasim, 2013) . cidofovir inhibits incorporation of deoxycytidine triphosphate 2 https://clinicaltrials.gov/ct2/show/nct03481244 into viral dna by the viral dna polymerase, leading to viral dna chain termination. probenecid co-prescription is useful to prevent or diminish cidofovir nephrotoxicity due to its accumulation in renal tubules. cidofovir is the current standard of care treatment for adv infections and diseases. however, cidofovir does not clear the virus in the absence of t cell immune reconstitution (hiwarkar et al., 2017) . another novel compound is cmx001 (hexadecyloxypropyl cidofovir, brincidofovir, chimerix), an orally bioavailable lipid conjugate of cidofovir with good oral bioavailability which achieves higher intracellular levels of active drug compared with cidofovir. brincidofovir lacks nephrotoxicity making it an attractive alternative. in phase i and phase ii trials as well as in retrospective studies, brincidofovir has been shown to be highly efficacious in controlling and clearing adenoviraemia (grimley et al., 2017; hiwarkar et al., 2017; ramsay et al., 2017; averbuch et al., 2018) . gastrointestinal toxicity is the major side effect observed and might be associated with epithelial apoptosis and crypt injury, as might be observed with gvhd (detweiler et al., 2018) . further, a small compound inhibitor named hbx has been reported to be effective against adv infections. a cellular component important for adv replication is ubiquitin-specific protease 7 (usp-7), a deubiquitinating enzyme of the ubiquitin proteasome pathway. usp-7 interacts with e1b-55k, a regulator of adv replication. since usp-7 promotes adv replication, it represents a potential drug target because blocking its activity could aid in the treatment of adv infections (ching et al., 2013) . hbx and a derivative were generated that were able to block adv (c5 type) replication. with the exception of adv types a12 and a31, hbx effectively blocked in vitro replication of all adv tested, making it a promising new antiviral candidate (kosulin et al., 2018b) . for adv, there is no proven role for ganciclovir, foscarnet or immunoglobulin therapy in immunocompromised patients. importantly, donor lymphocyte infusions and more recently specific anti adv cytototoxic t cells should be considered in case of adv disease after hct (see below). there is anecdotal evidence of successful treatment of adv with ribavirin (schleuning et al., 2004) but most studies have not been supportive; (lankester et al., 2004) and conclude that cidofovir combined with immunotherapy is more efficient and should be preferred as the first treatment option. of note, ribavirin is active in vitro on species c isolates (morfin et al., 2005) . there is currently no recommendation of specific antiviral therapy due to the lack of effective agents against these viruses and the lack of clinical trials (hirsch et al., 2013) . several agents against rhv have been described in the context of immunosuppression: oral or intranasal pleconaril, a capsid binder, was effective in 2 randomized trials to mildly reduce the duration and severity of colds in immunocompetent adults but its development was halted after fda rejection partly due to concerns regarding virus resistance (senior, 2002) . vapendavir is another capsid binder under development 3 for the treatment of rhv infections of asthmatic adults (waghmare et al., 2016) . to avoid transmission of ifv, vaccination of family members, household contacts and hct recipients is recommended. however, hct recipients might not mount an adequate immune response if vaccination takes place during immunosuppression. available antivirals for ifv infections include the m2 ion channel inhibitors such as amantadine that exclusively act on ifv-a. however, neuraminidase inhibitors (nai) are preferred for prophylaxis and treatment of ifv infections since resistance to m2 inhibitors is frequent (englund et al., 1998; fiore et al., 2011; von lilienfeld-toal et al., 2016) . post exposure prophylaxis with oral oseltamivir, 75 mg bid for 10 days for adults and children whose weight is above 40 kg, is the current treatment of choice. oral oseltamivir, inhaled zanamivir and iv peramivir are nais that were shown to be effective in hct recipients (vu et al., 2007; tomblyn et al., 2009; casper et al., 2010; choi et al., 2011; engelhard et al., 2013) . treatment within 48 h after the occurrence of symptoms will result in better outcome (ljungman et al., 2001; nichols et al., 2004b; tomblyn et al., 2009; choi et al., 2011; engelhard et al., 2013) ; although treatment should be initiated as early as possible, protracted and also prolonged treatment have been shown to have favorable effects. the duration of therapy should extend the treatment period recommended for immunocompetent hosts to circumvent reoccurrence and might last for 10 days in hct recipients. also, longer therapy in case of unsuccessful clearance might be necessary (engelhard et al., 2013) . the most commonly described mutation resulting in oseltamivir and peramivir resistance is conferred by the h275y mutation in influenza a h1n1 that can be treated with inhaled zanamivir (memoli et al., 2010; engelhard et al., 2013) . in europe during the 2007-2008 winter season, high resistance rates up to 68% of ifv-a (h1n1) to oseltamivir have been reported (meijer et al., 2009) . new agents with potential efficacy against oseltamivir-resistant viruses are being tested like inhaled laninamivir which is available in japan, jnj-63623872, a non-nucleoside inhibitor of the rna polymerase protein pb2, nitazoxanide, an antiparasitic agent with activity against ifv, and antibodies (medi8852, vis410) (shahani et al., 2017) . in a retrospective analysis of 200 patients including 120 hct recipients, one-third of patients with piv infection had lower respiratory tract disease and independent risk factors of progression were neutropenia, apache ii score ≥ 15, and respiratory co-infections within a month of piv infection. in this study, treatment with aerosolized ribavirin and/or ivig did not prevent progression to pneumonia and did not affect duration of illness or survival (chemaly et al., 2012) . a meta-analysis performed in 2016 reinforced this evidence of inefficiency of ribavirin for piv: indeed, pi-lri progression was not significantly 3 https://clinicaltrials.gov/ct2/show/nct02367313 different in hct recipients who were treated with ribavirin at uri stage, and piv-associated mortality rate was slightly higher in patients treated with ribavirin-based therapy at lri stage than in those who were not treated (shah et al., 2016a) . a recombinant sialidase fusion protein inhibitor named das181 has been used to treat severe piv infections after hct (waghmare et al., 2015; dhakal et al., 2016; salvatore et al., 2016) . das181 cleaves the neu5ac α(2,3)-gal and neu5ac α(2,6)-gal sialic acid linkages on the surface of respiratory cells that are used by ifv and piv for attachment and entry, thereby inhibiting the latter. the largest cohort included 16 patients after hct, 14 of who received das181 for piv-associated pneumonia but unfortunately lacked a control group (salvatore et al., 2016) . thirteen out of 16 patients responded to treatment, and the three patients who did not respond had a viral, bacterial, or fungal co-infection, respectively. the drug was well tolerated. piv loads were recorded in 7 of 16 patients using nasopharyngeal swabs. a > 1 log decrease in piv load was seen in 5 of 7 patients accompanied by a partial or complete clinical response. high-dose ivig had no effect on mortality after piv lri in a retrospective analysis of 544 patients with piv infection after hct (seo et al., 2014a) . therefore, no specific treatment for piv infection is currently strongly recommended. however, current ecil-4 guidelines suggest treatment with aerosolized ribavirin or off-label use with systemic ribavirin. for infections other than piv and rsv, ribavirin use is not recommended (hirsch et al., 2013) . in a study among 105 patients with uri from the fred hutchinson cancer center (seo et al., 2016) , the probability of progression to lri within 40 days was 16%, and approximately 75% of the patients with uri who progressed to lri did so within 2 weeks after uri. even if several small reports support the use of ribavirin with or without ivig for hmpv infection, a larger study showed no protective effect of ribavirin to reduce hmpv progression and mortality (renaud et al., 2013) . to date, antiviral therapy is not recommended to cure or to prevent infection progression even in patients at higher risk of hmpv progression. no licensed therapeutics or vaccines exist (wen and williams, 2015) . therefore, treatment for hmpv remains so far nonspecific and mainly supportive. mab 338, a monoclonal antibody against a fusion protein of hmpv, demonstrated interesting results in hamster and mice models, and further investigation in clinical trials is warranted (ulbrandt et al., 2006; hamelin et al., 2010) . in 2013, international guidelines recommended aerosolized or systemic (oral or iv) ribavirin with ivig in patients with rsv uri undergoing allogeneic hct, allogeneic hct recipients with risk factors for progression to lri, and allogeneic hct patients with lri (hirsch et al., 2013) . however, the additional benefit of ivig remains controversial. in a retrospective study that reviewed rsv uri or lri infections in 280 hct recipients, a combined ivig and ribavirin treatment was beneficial (shah et al., 2013) . further, given significant exponential cost increases in aerosolized ribavirin between 2013 and 2015 and lack of evidence that aerosolized ribavirin is more efficient, early oral ribavirin treatment is currently more frequently considered (waghmare et al., 2016) and is well tolerated (e.g., 15 mg/kg/day in three divided doses for 10 days) (gorcea et al., 2017) . a clinical trial comparing rsv treatment with oral versus inhaled ribavirin is ongoing 4 . however, in france, aerosolized ribavirin is currently not available. lymphopenia is a specific risk factor that has been associated with progression to lrti in several studies. other risk factors such as total body irradiation, smoking history, stem cell source other than peripheral blood stem cells and oxygen requirement might be considered waghmare et al., 2013) . a lower virulence of rsv-b compared to rsv-a has been reported (kelly et al., 2016) . as stated above, the isi might be helpful to identify patients who would benefit from antiviral therapy (shah et al., 2016a) . data from studies with the rsv-specific monoclonal antibody palivizumab have shown controversial efficacy, apart from the fact that it is very costly in adults. in larger studies of hct recipients with rsv lri, adjunctive palivizumab did not lead to outcome improvement waghmare et al., 2013) . therefore, rsv-specific monoclonal antibody is not recommended as a treatment option and might be only discussed 4 https://clinicaltrials.gov/ct2/show/nct01502072 for very young (age < 2 years) allogeneic hct recipients with lri or at high risk for progression to rsv lri (e.g., palivizumab 15 mg/kg body weight) (hirsch et al., 2013) . moreover, several agents are under development against rsv infections (shahani et al., 2017) . firstly, there are fusion inhibitors like gs-5806 which reduced the viral load and the severity of clinical disease in a study of healthy adults (devincenzo et al., 2014) , mdt-637 5 and alx-0171. secondly, agents targeting the rsv polymerase exist: al-8176 which had an interesting antiviral activity compared to placebo in 62 healthy adults inoculated with rsv (devincenzo et al., 2015) and favipiravir which is currently being tested in phase iii clinical trials in the united states, europe, and latin america. thirdly, substances targeting the viral nucleocapsid protein have been investigated. a small interfering rna called aln rsv01 inhibiting the synthesis of the rsv nucleocapsid protein has demonstrated a benefit in phase i to iib clinical trials by decreasing the infection rate, reducing symptoms and the incidence of bronchiolitis obliterans. another drug, rsv-604, targets the n terminal portion of the nucleocapsid protein. it was tested on hct recipients, however, no data have been published (simões et al., 2015) . finally, polyclonal high-titer anti rsv antibodies might be an option (ri-001) 6 . current strategies recommended for the treatment of respiratory viral disease after hct, and molecules that are under investigation, are summarized in table 1 . before the era of specific antiviral manufactured t cells, donor lymphocyte infusions (dli) were used and recommended in case of disseminated adv infections after hct (bordigoni et al., 2001; taniguchi et al., 2012) . the high incidence of gvhd after dlis limits its use in patients with previous history of gvhd. anecdotal use of dli has been reported for threatening rsv lri (kishi et al., 2000) . there are currently three strategies to manufacture viral specific t cells (vst) for clinical use: the first one is direct selection of antiviral t cells, either by multimers specific for a virus-derived peptide in the setting of class i hla molecule, or by column selection of ifn-γ expressing t cells (with immunomagnetic beads) after viral antigen stimulation (feuchtinger et al., 2006; peggs et al., 2011; icheva et al., 2013) . the second one is ex vivo expansion of t cells cocultured with antigen presenting cells pulsed, infected or transfected with viral peptide/protein/viral lysate or plasmid (peggs et al., 2003; gerdemann et al., 2012 gerdemann et al., , 2013 . the third one is genetic modification of t cells which incorporate high affinity vsts receptor or chimeric antigen receptor genes (schub et al., 2009; cruz et al., 2013; bollard and heslop, 2016) . so far, in the setting of community acquired viral respiratory infections, adoptive immune therapy has been developed and investigated in clinical trials only for adv infections. this strategy is promising as it has been reported to offer a way to cure severe adenoviral infections in case of resistance to antiviral drugs. adenoviral clearance has been obtained after infusions of adv-vst generated by interferon [ifn-γ-based immunomagnetic isolation (and further in vivo expansion) from initial donor or third party donor] in 10 of 11 hematopoietic stem cells transplanted patients in a french phase i/ii trial (qian et al., 2017) . similar results have been published earlier (feucht et al., 2015) from 30 patients who received adv-vst for adv disease or viremia. 14 of 23 evaluable patients had in vivo expansion of th1-vst (median time 24 days); and 21 patients responded to vst infusions and adv clearance occurred in 14. of note, adv-vst infusions were not associated with acute toxicities or significant onset of gvhd. the main disadvantage of this technique is that high doses of immunosuppressive drugs and especially steroids should be avoided before infusions as this drug hampers in vivo expansion of specific t cells. currently, multiviral (including adv) t cell manufacturing and banking from a third-party donor is a promising strategy as it might offer an immediate way to prevent adv infections after hct or to cure 71% of adv infected patients after hct . ex vivo expanded cytotoxic t cells against piv-3 antigens should be further tested in clinical trials (mclaughlin et al., 2016; aguayo-hiraldo et al., 2017) . the outcome in patients who experience progression to lri is worse compared to patients with uri regardless of the virus involved (chemaly et al., 2014) . in a french cohort of 131 adult patients infected with viral respiratory infections, 16 (12%) died within 3 months. among these, eight patients (6%) died of viral pneumonia along with bacterial and/or fungal pneumonia. interestingly, the virus group and the time from hct had no impact on mortality. two factors were independently associated with increased overall mortality: steroid dose over 1 mg/kg body weight and a lymphocyte count lower than 0.5 g/l (wolfromm et al., 2014) . the fact that lri associated death risk does not depend on the virus type is supported by seo et al. (2017) who reported an overall mortality probability at day 90 as high as 41% in patients after hct who developed rhv lri, a rate that was comparable to rsv, piv and ifv lri in an adjusted model. among children, in a cohort of 58 patients, 3 (5%) died of respiratory failure (choi et al., 2013) . lri in the absence of uri and adenoviral infection has been described to be associated with a poorer outcome in another cohort of 166 patients with specific overall mortality of 7% (lo et al., 2013) . bronchiolitis obliterans syndrome and obstructive airflow decline have been associated with piv and rsv infections within the first 3 months after allogeneic hct which persisted at 1 year after transplant (versluys et al., 2010) . further, the occurrence of a lri within 100 days post-transplant was an independent predictive factor for late onset noninfectious pulmonary complications diagnosed in the first 36 months after transplantation in an observational prospective cohort study on 198 patients (bergeron et al., 2018) . therefore, functional pulmonary explorations and long-term surveillance are warranted after community acquired viral infections which occurred early after hct. to better understand risk factors of pulmonary complications in children after hct, a multicentric french trial is ongoing 7 . early treatment with β2 mimetics and inhaled steroids might be useful and should be proposed in case of persistent obstructive syndrome after respiratory viral infections resolution in hct recipients. this review has limitations because we present results from small case series that have restricted meaningfulness. further, it includes recommendations that were in part made before the era of t cell repleted haploidentical transplantations. this is an area of intense research and these new developments might impact the epidemiology and outcomes of viral infections after hct. respiratory virus infections continue to cause disease both in the pre-transplant as well as in the post-transplant period and should be taken very seriously, especially in children who 7 https://clinicaltrials.gov/ct2/show/nct02032381 tend to shed virus for longer periods. this long shedding time has implications for infection control. therefore, both personnel and patients and their families should be properly instructed in hand hygiene. in case of pre-transplant infections, hct should be deferred but the underlying disease, the conditioning regimen and donor availability need to be considered. in pediatric patients who also tend to acquire viruses more frequently, we favor transplant delay in elected cases, even in asymptomatic patients. without doubt, disease progression from uri to lri influences the outcome in hct recipients. there are a number of risk factors shared by respiratory viruses that are associated with lri occurrence; lymphopenia appears to be a specific risk factor in this case. other risk factors shared with increased mortality are steroid use at the time of lri diagnosis, oxygen requirement at the time of bal, and severity of disease based on apache ii score. in addition, myeloablative conditioning, gvhd, bacterial as well as fungal pulmonary co-infections (especially with ifv and piv infections) seem to worsen the outcome. is there a possibility to reduce the risk of progression from uri to lri? there might be a chance if co-infections can be reduced or diagnosed and treated promptly. these co-infections could be more easily avoided if they were nosocomially acquired by transfer from personnel or visitors. therefore, people who have access to the patient should be aware of this problem. also, shortening of the lymphopenic interval could reduce disease progression. this might be achieved with a stricter withdrawal of immunosuppressive drugs, e.g., steroids, or a milder form of conditioning. early and specific diagnostics are of the essence. it would be best to obtain diagnostic samples straight from the area of infection, i.e., from bal material and not a nasal swab in case of a lri, however, this might be sometimes impractical and not be tolerated by the patient. unless a specific viral agent is suspected and a single or duplex pcr are ordered, e.g., during an outbreak, novel multiplex assays should be employed that also identify a bacterial co-infection. what is more, novel multiplex assays that are able to quantify viral loads clearly have the potential to help identify which patients are at increased risk and who might need treatment urgently -particularly since supporting measures like radiological imaging techniques often do not point to a specific pathogen and provide non-specific results with regard to identification of the pathogen responsible. however, while chest x-rays discover inflammatory processes with delay, ct findings might help to differentiate between pathogens. all these diagnostic steps should be carried out after full evaluation of the circumstances and in an orderly fashion. the isi and the rsi (tables 2, 3 ) might be helpful to identify patients at risk but await validation in clinical trials. eventually, only few antivirals are available and licensed to treat respiratory virus infections. several drugs have been tested in hct recipients and promising results have been published. novel antiviral compounds are urgently required that might be used in combination with antiviral lymphocytes as currently being developed for cmv, ebv, adv, or bk antivirus ctls in us (nct01945814, nct01570283) and european international trials (the trace trial). the production of these cells should be confined to specialized laboratories, either low risk: 0-2 score, moderate risk 3-6 score, high risk 7-12 score anc, absolute neutrophil count; alc, absolute lymphocyte count; gvhd, graftversus-host disease; modified after shah et al. (2014) . to calculate the rsi score, the predominant pattern for each lung zone is multiplied with the extent of the volumetric radiologic involvement, e.g., if ground-glass opacities are detected in all six lung zones with all having a volumetric score of 3, the rsi score would be 36 (2 × 3 × 6). the maximum score is 72. modified after sheshadri et al. (2018). university-based or commercial, however, they should be easily accessible. the expansion of this approach to target respiratory viruses might be similarly useful to halt disease progression from uri to lri. finally, the application of car t cells to combat respiratory viral infections might be an option in the future, especially in combination with tumor targeting car t cells. however, pulmonary toxicity should be considered due to cross-reactivity of car t cells with non-targeted proteins, and also ards that often occurs in severe lri might be worsened by cytokine release syndrome. while viral pneumonia still remains a concern in hct patients, new drug developments, along with sophisticated t cell approaches, should diminish complications caused by respiratory viruses. cp and sv wrote and edited the manuscript. this work was supported by intramural funds from the robert koch institute. outcomes of hematopoietic sct recipients with rhinovirus infection: a matched, case-control study respiratory viruses in transplant recipients: more than just a cold. clinical syndromes and infection prevention principles commensal bacteria calibrate the activation threshold of innate antiviral immunity characterizing the cellular immune response to parainfluenza virus 3 human bocavirus cloning of a human parvovirus by molecular screening of respiratory tract samples frequent detection of human rhinoviruses, paramyxoviruses, coronaviruses, and bocavirus during acute respiratory tract infections a novel bocavirus associated with acute gastroenteritis in australian children successful brincidofovir treatment of metagenomics-detected adenovirus infection in a severely ill stat1-deficient patient respiratory syncytial virus infection in recipients of allogeneic stem-cell transplantation: a retrospective study of the incidence, clinical features, and outcome noninfectious lung complications after allogeneic haematopoietic stem cell transplantation virological and clinical characterizations of respiratory infections in hospitalized children asymptomatic shedding of respiratory virus among an ambulatory population across seasons the challenge of respiratory virus infections in hematopoietic cell transplant recipients randomized controlled multicenter trial of aerosolized ribavirin for respiratory syncytial virus upper respiratory tract infection in hematopoietic cell transplant recipients virological features and clinical manifestations associated with human metapneumovirus: a new paramyxovirus responsible for acute respiratory-tract infections in all age groups t cells for viral infections after allogeneic hematopoietic stem cell transplant treatment of adenovirus infections in patients undergoing allogeneic hematopoietic stem cell transplantation impact of corticosteroid treatment and antiviral therapy on clinical outcomes in hematopoietic cell transplant patients infected with influenza virus prospective study of respiratory viral infections in pediatric hemopoietic stem cell transplantation patients clinical outcomes associated with respiratory virus detection before allogeneic hematopoietic stem cell transplant how i treat influenza in patients with hematologic malignancies the microbial metabolite butyrate regulates intestinal macrophage function via histone deacetylase inhibition respiratory viral infections in adults with hematologic malignancies and human stem cell transplantation recipients: a retrospective study at a major cancer center the characteristics and outcomes of parainfluenza virus infections in 200 patients with leukemia or recipients of hematopoietic stem cell transplantation management of respiratory viral infections in hematopoietic cell transplant recipients and patients with hematologic malignancies a ubiquitin-specific protease possesses a decisive role for adenovirus replication and oncogene-mediated transformation respiratory viral infections after hematopoietic stem cell transplantation in children differences in clinical outcomes after 2009 influenza a/h1n1 and seasonal influenza among hematopoietic cell transplant recipients influenza viral rna detection in blood as a marker to predict disease severity in hematopoietic cell transplant recipients respiratory tract infections due to human metapneumovirus in immunocompromised children outbreak of human parainfluenza virus 3 infections in a hematopoietic stem cell transplant population infusion of donor-derived cd19-redirected virus-specific t cells for b-cell malignancies relapsed after allogeneic stem cell transplant: a phase 1 study prolonged respiratory viral shedding in transplant patients human bocavirus in an immunocompromised child presenting with severe diarrhea persistent symptomless human metapneumovirus infection in hematopoietic stem cell transplant recipients long-term study on symptomless human metapneumovirus infection in hematopoietic stem cell transplant recipients brincidofovir (cmx001) toxicity associated with epithelial apoptosis and crypt drop out in a hematopoietic cell transplant patient: challenges in distinguishing drug toxicity from gvhd activity of oral als-008176 in a respiratory syncytial virus challenge study oral gs-5806 activity in a respiratory syncytial virus challenge study das181 treatment of severe parainfluenza virus 3 pneumonia in allogeneic hematopoietic stem cell transplant recipients requiring mechanical ventilation. case rep genotyping of human bocavirus using a restriction length polymorphism the microbiome and butyrate regulate energy metabolism and autophagy in the mammalian colon priming of the respiratory tract with immunobiotic lactobacillus plantarum limits infection of alveolar macrophages with recombinant pneumonia virus of mice (rk2-pvm) life-threatening respiratory tract disease with human bocavirus-1 infection in a 4-year-old child accuracy of the interpretation of chest radiographs for the diagnosis of paediatric pneumonia european guidelines for prevention and management of influenza in hematopoietic stem cell transplantation and leukemia patients: summary of ecil-4 viral infections in immunocompromised patients brief communication: fatal human metapneumovirus infection in stem-cell transplant recipients common emergence of amantadine-and rimantadineresistant influenza a viruses in symptomatic immunocompromised adults adenovirus infection and disease in paediatric haematopoietic stem cell transplant patients: clues for antiviral pre-emptive treatment adoptive t-cell therapy with hexon-specific th1 cells as a treatment of refractory adenovirus infection after hsct adenovirus infection after allogeneic stem cell transplantation safe adoptive transfer of virus-specific t-cell immunity for the treatment of systemic adenovirus infection after allogeneic stem cell transplantation antiviral agents for the treatment and chemoprophylaxis of influenzarecommendations of the advisory committee on immunization practices (acip) imaging of community-acquired pneumonia thin-section ct findings in hematopoietic stem cell transplantation recipients with respiratory virus pneumonia safety and clinical efficacy of rapidly-generated trivirusdirected t cells as treatment for adenovirus, ebv, and cmv infections after allogeneic hematopoietic stem cell transplant rapidly generated multivirus-specific cytotoxic t lymphocytes for the prophylaxis and treatment of viral infections effective use of oral ribavirin for respiratory syncytial viral infections in allogeneic haematopoietic stem cell transplant recipients brincidofovir for asymptomatic adenovirus viremia in pediatric and adult allogeneic hematopoietic cell transplant recipients: a randomized placebo-controlled phase ii trial rhinovirus as a cause of fatal lower respiratory tract infection in adult stem cell transplantation patients: a report of two cases impact of gut colonization with butyrate-producing microbiota on respiratory viral infection following allo-hct prophylactic and therapeutic benefits of a monoclonal antibody against the fusion protein of human metapneumovirus in a mouse model the ct appearance of lower respiratory infection due to parainfluenza virus in adults chest radiographic features of human metapneumovirus infection in pediatric patients fourth european conference on infections in leukaemia (ecil-4): guidelines for diagnosis and treatment of human respiratory syncytial virus, parainfluenza virus, metapneumovirus, rhinovirus, and coronavirus brincidofovir is highly efficacious in controlling adenoviremia in pediatric recipients of hematopoietic cell transplant management of adenovirus infection in patients after haematopoietic stem cell transplantation: state-of-the-art and real-life current approach: a position statement on behalf of the infectious diseases working party of the european society of blood and marrow transplantation a parainfluenza-3 outbreak in a sct unit: sepsis with multi-organ failure and multiple co-pathogens are associated with increased mortality serious outbreak of human metapneumovirus in patients with hematologic malignancies multiplex pcr system for the rapid diagnosis of respiratory virus infection: systematic review and meta-analysis adoptive transfer of epstein-barr virus (ebv) nuclear antigen 1-specific t cells as treatment for ebv reactivation and lymphoproliferative disorders after allogeneic stem-cell transplantation microbiota regulates immune defense against respiratory tract influenza a virus infection rhinovirus infections in hematopoietic stem cell transplant recipients with pneumonia human rhinovirus infections of the lower respiratory tract in hematopoietic stem cell transplant recipients viral pneumonia after hematopoietic stem cell transplantation: highresolution ct findings bocavirus episome in infected human tissue contains non-identical termini human bocaviruses are highly diverse, dispersed, recombination prone, and prevalent in enteric infections respiratory syncytial virus outbreak on an adult stem cell transplant unit outcome of influenza infections in outpatients after allogeneic hematopoietic stem cell transplantation respiratory syncytial virus infection in patients with hematological diseases: single-center study and review of the literature ct findings in viral lower respiratory tract infections caused by parainfluenza virus, influenza virus and respiratory syncytial virus respiratory syncytial virus in hematopoietic cell transplant recipients: factors determining progression to lower respiratory tract disease impact of pretransplant respiratory virus detection through universal screening in children undergoing hematopoietic cell transplantation (hct) donor lymphocyte infusion for treatment of life-threatening respiratory syncytial virus infection following bone marrow transplantation outcomes of influenza infections in hematopoietic cell transplant recipients: application of an immunodeficiency scoring index immunity to respiratory viruses human bocavirus in children with acute lymphoblastic leukemia intestinal adenovirus shedding before allogeneic stem cell transplantation is a risk factor for invasive infection post-transplant broadspectrum antiviral activity of the deubiquitinase inhibitor hbx against human adenoviruses severe pneumonia and human bocavirus in adult outbreak of novel influenza a (h1n1) in an adult haematology department and haematopoietic cell transplantation unit: clinical presentation and outcome effect of ribavirin on the plasma viral dna load in patients with disseminating adenovirus infection multicenter evaluation of biofire filmarray respiratory panel 2 for detection of viruses and bacteria in nasopharyngeal swab samples adenoviral infections in hematopoietic stem cell transplantation how i treat adenovirus in hematopoietic stem cell transplant recipients adenovirus infections in immunocompetent and immunocompromised patients respiratory virus infections in bone marrow transplant recipients: the european perspective outcome of pandemic h1n1 infections in hematopoietic stem cell transplant recipients respiratory virus infections after stem cell transplantation: a prospective study from the infectious diseases working party of the european group for blood and marrow transplantation the impact of rsv, adenovirus, influenza, and parainfluenza infection in pediatric patients receiving stem cell transplant, solid organ transplant, or cancer chemotherapy human bocavirus 1 primary infection and shedding in infants prospective study of the incidence, clinical features, and outcome of symptomatic upper and lower respiratory tract infections by respiratory viruses in adult recipients of hematopoietic stem cell transplants for hematologic malignancies ctmorphological characterization of respiratory syncytial virus (rsv) pneumonia in immune-compromised adults control of an outbreak of human parainfluenza virus 3 in hematopoietic stem cell transplant recipients human parainfluenza virus-3 can be targeted by rapidly ex vivo expanded t lymphocytes oseltamivir-resistant influenza virus a (h1n1), europe, 2007-08 season rapid selection of oseltamivir-and peramivirresistant pandemic h1n1 virus during therapy in 2 immunocompromised hosts human rhinovirus and coronavirus detection among allogeneic hematopoietic stem cell transplantation recipients comparing human metapneumovirus and respiratory syncytial virus: viral co-detections, genotypes and risk factors for severe disease in vitro susceptibility of adenovirus to antiviral drugs is species-dependent treatment of adenovirus disease in stem cell transplant recipients with cidofovir parainfluenza virus infections after hematopoietic stem cell transplantation: risk factors, response to antiviral therapy, and effect on transplant outcome community-acquired respiratory syncytial virus and parainfluenza virus infections after hematopoietic stem cell transplantation: the fred hutchinson cancer research center experience prolonged outbreak of human parainfluenza virus 3 infection in a stem cell transplant outpatient department: insights from molecular epidemiologic analysis influenza infections after hematopoietic stem cell transplantation: risk factors, mortality, and the effect of antiviral therapy prolonged shedding of human coronavirus in hematopoietic cell transplant recipients: risk factors and viral genome evolution clinical significance of human coronavirus in bronchoalveolar lavage samples from hematopoietic cell transplant recipients and patients with hematologic malignancies antibiotic exposure prior to respiratory viral infection is associated with progression to lower respiratory tract disease in allogeneic hematopoietic cell transplant recipients initial high viral load is associated with prolonged shedding of human rhinovirus in allogeneic hematopoietic cell transplant recipients upper and lower respiratory tract infections by human enterovirus and rhinovirus in adult patients with hematological malignancies pretransplantation respiratory syncytial virus infection: impact of a strategy to delay transplantation respiratory virus infection among hematopoietic cell transplant recipients: evidence for asymptomatic parainfluenza virus infection directly selected cytomegalovirus-reactive donor t cells confer rapid and safe systemic reconstitution of virus-specific immunity following stem cell transplantation community acquired respiratory virus lower respiratory tract disease in allogeneic stem cell transplantation recipient: risk factors and mortality from pulmonary virus-bacterial mixed infections the clinical features and outcome of 2009 h1n1 influenza infection in allo-sct patients: a british society of blood and marrow transplantation study curative or pre-emptive adenovirus-specific t cell transfer from matched unrelated or third party haploidentical donors after hsct, including ucb transplantations: a successful phase i/ii multicenter clinical trial disseminated adenovirus infection after allogeneic stem cell transplant and the potential role of brincidofovir -case series and 10 year experience of management in an adult transplant cohort changing epidemiology of respiratory viral infections in hematopoietic cell transplant recipients and solid organ transplant recipients antiviral therapy of respiratory viruses in haematopoietic stem cell transplant recipients mortality rates of human metapneumovirus and respiratory syncytial virus lower respiratory tract infections in hematopoietic cell transplant recipients das181 for treatment of parainfluenza virus infections in hematopoietic stem cell transplant recipients at a single center evaluation of performance characteristics of panther fusion assays for detection of respiratory viruses from nasopharyngeal and lower respiratory tract specimens disseminated bocavirus infection after stem cell transplant timing and severity of community acquired respiratory virus infections after myeloablative versus non-myeloablative hematopoietic stem cell transplantation human bocavirus: passenger or pathogen in acute respiratory tract infections? intravenous ribavirin for eradication of respiratory syncytial virus (rsv) and adenovirus isolates from the respiratory and/or gastrointestinal tract in recipients of allogeneic hematopoietic stem cell transplants the cd8 t cell response to respiratory virus infections cmvspecific tcr-transgenic t cells for immunotherapy dual infection of infants by human metapneumovirus and human respiratory syncytial virus is strongly associated with severe bronchiolitis fda panel rejects common cold treatment outcome of respiratory syncytial virus lower respiratory tract disease in hematopoietic cell transplant recipients receiving aerosolized ribavirin: significance of stem cell source and oxygen requirement human metapneumovirus infections following hematopoietic cell transplantation: factors associated with disease progression human rhinovirus detection in the lower respiratory tract of hematopoietic cell transplant recipients: association with mortality parainfluenza virus lower respiratory tract disease after hematopoietic cell transplant: viral detection in the lung predicts outcome parainfluenza virus type 3 ab in allogeneic hematopoietic cell transplant recipients: factors influencing post-transplant ab titers and associated outcomes immunodeficiency scoring index to predict poor outcomes in hematopoietic cell transplant recipients with rsv infections impact of aerosolized ribavirin on mortality in 280 allogeneic haematopoietic stem cell transplant recipients with respiratory syncytial virus infections parainfluenza virus infections in hematopoietic cell transplant recipients and hematologic malignancy patients: a systematic review human metapneumovirus infections in hematopoietic cell transplant recipients and hematologic malignancy patients: a systematic review management of rsv infections in adult recipients of hematopoietic stem cell transplantation antiviral therapy for respiratory viral infections in immunocompromised patients progression of the radiologic severity index predicts mortality in patients with parainfluenza virus-associated lower respiratory infections challenges and opportunities in developing respiratory syncytial virus therapeutics community-acquired respiratory paramyxovirus infection after allogeneic hematopoietic cell transplantation: a single-center experience symptomatic parainfluenza virus infections in children undergoing hematopoietic stem cell transplantation universal mask usage for reduction of respiratory viral infections after stem cell transplant: a prospective trial h1n1 infection in a cohort of hematopoietic stem cell transplant recipients: prompt antiviral therapy might be life saving human metapneumovirus (hmpv) associated pulmonary infections in immunocompromised adults-initial ct findings, disease course and comparison to respiratory-syncytial-virus (rsv) induced pulmonary infections incidence and treatment strategy for disseminated adenovirus disease after haploidentical stem cell transplantation guidelines for preventing infectious complications among hematopoietic cell transplantation recipients: a global perspective off-the-shelf virus-specific t cells to treat bk virus, human herpesvirus 6, cytomegalovirus, epstein-barr virus, and adenovirus infections after allogeneic hematopoietic stem-cell transplantation isolation and characterization of monoclonal antibodies which neutralize human metapneumovirus in vitro and in vivo human bocavirus as the cause of a life-threatening infection human parainfluenza virus infection after hematopoietic stem cell transplantation: risk factors, management, mortality, and changes over time identification of a new human coronavirus strong association between respiratory viral infection early after hematopoietic stem cell transplantation and the development of lifethreatening acute and chronic alloimmune lung syndromes community acquired respiratory virus infections in cancer patients-guideline on diagnosis and management by the infectious diseases working party of the german society for haematology and medical oncology safety and tolerability of oseltamivir prophylaxis in hematopoietic stem cell transplant recipients: a retrospective case-control study respiratory syncytial virus lower respiratory disease in hematopoietic cell transplant recipients: viral rna detection in blood, antiviral treatment, and clinical outcomes how i treat respiratory viral infections in the setting of intensive chemotherapy or hematopoietic cell transplantation successful treatment of parainfluenza virus respiratory tract infection with das181 in 4 immunocompromised children correlation between bocavirus infection and humoral response, and co-infection with other respiratory viruses in children with acute respiratory infection new approaches for immunization and therapy against human metapneumovirus influenza a virus infections among hospitalized adult bone marrow transplant recipients a prospective study comparing human metapneumovirus with other respiratory viruses in adults with hematologic malignancies and respiratory tract infections viral respiratory infections diagnosed by multiplex pcr after allogeneic hematopoietic stem cell transplantation: long-term incidence and outcome characterization and complete genome sequence of a novel coronavirus, coronavirus hku1, from patients with pneumonia management of adenovirus in children after allogeneic hematopoietic stem cell transplantation clinical characteristics and viral load of respiratory syncytial virus and human metapneumovirus in children hospitaled for acute lower respiratory tract infection adenoviral infections in adult allogeneic hematopoietic sct recipients: a single center experience the authors thank matthew reeves for comments on the manuscript. key: cord-292836-1o2ynvy3 authors: ogimi, chikara; kim, yae jean; martin, emily t; huh, hee jae; chiu, cheng-hsun; englund, janet a title: what’s new with the old coronaviruses? date: 2020-04-21 journal: j pediatric infect dis soc doi: 10.1093/jpids/piaa037 sha: doc_id: 292836 cord_uid: 1o2ynvy3 coronaviruses contribute to the burden of respiratory diseases in children, frequently manifesting in upper respiratory symptoms considered to be part of the “common cold.” recent epidemics of novel coronaviruses recognized in the 21st century have highlighted issues of zoonotic origins of transmissible respiratory viruses and potential transmission, disease, and mortality related to these viruses. in this review, we discuss what is known about the virology, epidemiology, and disease associated with pediatric infection with the common community-acquired human coronaviruses, including species 229e, oc43, nl63, and hku1, and the coronaviruses responsible for past world-wide epidemics due to severe acute respiratory syndrome and middle east respiratory syndrome coronavirus. understanding the history and epidemiology of the common community human coronaviruses (hcovs) and those responsible for recent past epidemics is crucial to the control and treatment of novel coronaviruses. the epidemiology of previously described community coronaviruses in children is relatively well known from surveillance studies. however, important clinical and laboratory details of these viruses, such as duration of shedding, transmission rates, viral load over time, immunity, and morbidity in high-risk populations, are not well characterized. such information becomes more important and clinically relevant as we consider world-wide pandemics such as the ongoing severe acute respiratory syndrome coronavirus 2 (sars-cov-2) outbreak. coronaviruses are already known to be ubiquitous viruses and recognized as pathogens in both humans and animals. a coronavirus was first isolated as a causative agent of bronchitis in birds in 1937 [1] and was originally discovered in humans during studies that evaluated the common cold. the history of the discovery of hcovs is shown in table 1 [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] . the common human coronaviruses known today include the species 229e, oc43, nl63, and hku1. these 4 viruses are primarily viewed as relatively benign respiratory pathogens in humans, typically causing upper respiratory tract disease and common cold symptoms. by contrast sars-cov and middle east respiratory syndrome coronavirus (mers-cov) are highly pathogenic in humans, with high rates of severe pneumonia and fatal outcomes [21] . currently, the novel coronavirus sars-cov-2 is spreading worldwide, causing anxiety, disease, and mortality [22, 23] . despite the heightened interest, limited pediatric data are available regarding either the community hcovs or the newer pathogenic species in children. here, we review what is known about hcov infections in children in the precoronavirus disease 2019 (covid-19) era. coronaviruses belong to the coronaviridae family in the nidovirales order. the coronavirus subfamily is further classified into 4 genera known as the alpha, beta, gamma, and delta coronaviruses. these viruses cause a wide variety of generally species-specific illness in mammals and birds, including chickens, turkeys, bats, rats, dogs, cats, piglets, and whales [21] . coronaviruses were named for their characteristic crown-like surface projections seen in electron micrographs that correspond to large surface spike proteins. these viruses are enveloped, nonsegmented, positive-sense rna viruses and have the largest identified viral rna genomes, with an approximate length of 30 kilobases [24] . only alpha and beta coronaviruses are known to infect humans. both hcovs 229e and nl63 are alpha coronaviruses and hcov-hku1, hcov-oc43, mers-cov, and sars-cov are beta coronaviruses (table 1) [24, 25] . phylogenetic analysis has revealed that sars-cov-2 also falls within the beta coronavirus in the same subgenus as the sars-cov, but in a different clade [26] . all 7 coronaviruses that infect humans are believed to have originated in bats [27, 28] . based on modeling, it is speculated that hcov-oc43 was transmitted to humans around 1890 [29] . bats also appear to be the original host of sars-cov-2, and it is hypothesized that currently unknown wild animal(s) sold at the huanan seafood market in wuhan, china, might have played a role as an intermediate host to humans [26] . hcovs oc43 and 229e were isolated from nasal cavities of people with the common cold in the 1960s. in the 1970s, studies that used serology and viral culture linked hcovs 229e and oc43 with 8% of cases of lower respiratory tract infection (lrti) in hospitalized infants [30, 31] . poor replication in tissue culture and a lack of cytopathic effect in early attempts at culture were major obstacles in making further progress in the field. the development and widespread use of molecular diagnostics as well as the emergence of sars-cov in 2003 significantly accelerated coronavirus research. subsequently, new hcovs nl63 and hku1 were identified and are frequently seen in children. additional pathogenic coronavirus species and outbreaks with mers-cov and, recently, sars-cov-2 have led to increased interest, research, and concern [21, 23] . community-acquired hcovs species oc43, nl63, hku1, and 229e are found worldwide in temperate and tropical countries, as well as low-, middle-, and high-income countries [32, 33] . hcov infection can occur at any time of the year, with unpredictable year-to-year patterns, and outbreaks are reported in some years and in certain parts of the world. all 4 species may circulate yearly and even simultaneously [34] , with the highest rates of hcov infection seen in winter and spring months in temperate climates [21] . almost all surveillance data indicate hcov-oc43 is the most common species. a large prospective surveillance study conducted in norway from 2006 to 2015 that enrolled all hospitalized children aged ≤16 years with respiratory tract infections revealed that hcovs oc43 and nl63 were detected most frequently and were epidemic every second winter [35] . hcov-hku1 was prevalent every second winter during the year when detection rates for hcovs oc43 and nl63 were low; hcov-229e was the least common. seroprevalence data suggest that exposure is common in early childhood [36] . more than 90% of adults are seropositive for at least 1 hcov species [37] . a recent prospective active surveillance study of young infants in nepal that used multiplex polymerase chain reaction (pcr) showed that hcovs are less common in neonates compared with older infants [32] . outbreaks of variable sizes have been reported sporadically in medical students, young asthmatic children, and neonatal intensive care units [38] [39] [40] [41] . for example, an outbreak of hcov-229e was reported in a neonatal intensive care unit where 92% of infected preterm neonates developed symptoms, including worsening respiratory conditions and bradycardia [42] . potential nosocomial transmission by staff members was speculated. large surveillance studies of children and adults to evaluate the prevalence of all major respiratory viruses using multiplex pcr have been conducted in many settings, showing that hcov infections are the fourth or sixth most common virus detected overall and across all age groups [33, 43] . coinfections are relatively common, especially in children aged ≤5 years. a prospective surveillance study in norway demonstrated that annual hospitalization rates of children with lrti associated with hcov detection were 1.5 and 2.8 per 1000 children aged <5 years and aged <1 years, respectively [35] . the mode(s) of hcov transmission is not known [21, 25] . it is assumed that transmission occurs via a combination of droplet and direct/indirect contact, similar to other respiratory viruses. hcovs oc43 and 229e appear to be primarily transmitted during the first few days of illness when symptoms and viral load in the respiratory tract are highest. the incubation period of hcov-229e was 2 to 5 days (median, 3 days) in adults in challenge studies and has not yet been clearly defined for other hcovs or in children [25, 44] . the transmission of hcov in community epidemics is impacted by the characteristics of hcov infection and shedding in children. children have higher attack rates, as high as 78% within households, compared with adults, as shown in an active surveillance study in kenya [45] . these data complement community studies in the 1960s that showed a higher frequency of hcov-229e in families with children aged <15 years [36] , a higher rate of hcov-oc43 infection in children aged <5 years [46] , and frequent clustering of infections within families [46] . the attack rate of hcov in childcare settings has also been shown to be higher with younger age [47] . children may shed hcovs for extended periods of time after infection, potentially leading to additional transmissions within close-contact settings. there are very limited data regarding duration of shedding in children. in prospective childcare studies, 34% of children with hcov had detectable virus at 1 week or more following symptom onset, with shedding documented for up to 18 days ( figure 1 ) [47, 48] . a longitudinal study of weekly nasal swabs taken from symptomatic and asymptomatic adults and children similarly found that viral detection extended beyond 1 week [49] . children aged <5 years with hcov detection were frequently asymptomatic, especially with hcov-229e. these findings reinforce serologic-based findings of asymptomatic infection in 7% of children with hcov-229e in the 1960s [50] . clinical symptoms associated with the 4 common hcovs generally appear to be indistinguishable from cold symptoms or influenza-like illness (rhinorrhea, sore throat, cough, wheezing, and fever) associated with other respiratory viruses [21, 25, 51] and generally are similar in children and adults. hcovs 229e and oc43 were shown to be pathogenic in adult human volunteer studies. cold symptoms (eg, sore throat, rhinorrhea, cough) are very similar to those caused by other respiratory viruses [39, 44] . it is assumed that hcovs nl63 and hku1 have similar pathogenicity, although this has not been proven in challenge studies. the frequent detection of hcovs in asymptomatic patients as well as coinfection with other respiratory viruses make the interpretation of hcov pathogenesis and clinical findings challenging. a multicenter, prospective surveillance study conducted in the united states evaluated the prevalence of hcovs among hospitalized children with acute respiratory illness and demonstrated that the prevalence of hcovs was similar in both asymptomatic and symptomatic groups of children [52] . in contrast, a prospective community surveillance study of respiratory viruses in utah showed hcov detection was often associated with symptoms in all age groups, suggesting hcov infection might have been previously underestimated as an etiology of respiratory tract infection [49] . an analysis of new and old data reveals, not surprisingly, that viral coinfections are less common in children with influenza virus (9% of influenza in seattle, washington; 27% in seoul, south korea) than in children infected with a community hcovs (43% of hcov infections in seattle and 42% in seoul; table 2 ). a norwegian prospective surveillance study compared children hospitalized with respiratory tract [47, 48] . children attending group childcare were tested prospectively for respiratory viruses at each acute respiratory illness (ari). swabs were collected weekly from ari onset until symptoms were nonworsening and swabs were negative by real-time-polymerase chain reaction [47] . human coronavirus (hcov)-positive swabs (for all species) are represented by filled circles, and negative swabs are represented by open circles. weeks on x-axis were calculated as weeks since symptom onset with an hcov (+) acute respiratory illness. the lower limit of positivity was set at 1000 copies/ml for this analysis. sequencing of hcov isolates was not performed. infections vs asymptomatic children admitted for elective surgery [35] . multivariable analyses suggest that higher viral load (cycle threshold <28 by pcr), codetection of generally symptomatic viruses (respiratory syncytial virus [rsv], human metapneumovirus, influenza, and parainfluenza virus), younger age (<2 years), being female, and high-risk underlying conditions (lung, heart, or neurologic disorder) were associated with symptomatic children. children with only hcov detected were more likely to have a higher viral load compared with those with viral coinfection. associations of hcovs with lrti or asthma exacerbation in children have been reported [21, 31, 34, 38, 53, 54] . clearly, respiratory distress and pneumonia have been well documented in children with only hcov detected as a potential pathogen [34, 55] . specific hcov species and associated risk factors for disease severity have been evaluated, although some studies did not address potential confounders, such as the presence of coinfection [56] . one retrospective study of 212 hospitalized children with hcov (54 with and 158 without viral coinfection) showed that similar numbers of children received respiratory support and intensive care [57] . bivariate analyses showed that younger age (<2 years) and chronic complex medical conditions (cardiovascular, respiratory, genetic, or congenital) were associated with increased disease severity. no specific species were associated with severity of illness. although the presence of viral coinfection was not associated with increased disease severity, the analysis did not assess the impact of specific types of coinfections. a retrospective analysis of 1237 children who presented to seattle children's hospital (washington) for acute care with detected hcovs showed that disease severity did not vary by hcov species [58] . younger age, presence of underlying pulmonary disorder, and presence of coinfection, particularly rsv, were associated with increased likelihood of lrti in multivariable models. the impact of rsv coinfection with hcov has been described in other studies [59] . like other respiratory viruses, hcovs have been detected in middle ear effusions and nasal secretions in children with otitis media, and its possible etiology of acute otitis media has been implicated [60, 61] . the significant association between hcov-nl63 and croup has been reported, with evidence suggesting that hcov-nl63 is the second most common etiology of croup following parainfluenza virus type 1 [62, 63] . hcov has been described as a possible etiology of severe pneumonia in immunocompromised hosts [64] [65] [66] [67] . data are limited for this high-risk population and particularly are lacking in pediatric hematopoietic cell transplantation (hct) recipients [68] . among 404 children aged <18 years who underwent allogeneic hct from april 2008 to september 2018 at seattle children's hospital, hcovs were the third most common respiratory viruses detected post-hct following rhinovirus and parainfluenza virus (preliminary data). the cumulative incidence of hcovs in children during the first 365 days following hct are shown in figure 2 . hcovs were detected in bronchoalveolar lavage (bal) specimens from 2 young children in this cohort. the detection of hcov in bal specimens among hct recipients and patients with hematologic malignancy was also evaluated, but only 1 of 35 patients was a child [69] . among 16 patients in that study (15 adults, 1 child) with hcov and without other coinfections identified by bal, 10 required oxygen support, suggesting a role of hcov as a significant respiratory pathogen. in a retrospective study of immunocompromised and nonimmunocompromised children with hcov detected in nasal specimens in seattle, the prevalence of lrti that required oxygen supplementation (severe lrti) was 15% (13/85) and 11% (122/1152), respectively [58] . multivariable models showed that immunocompromised state, presence of rsv, and underlying pulmonary disorder were associated with increased risk of severe lrti. a prospective surveillance study with weekly nasal sampling in 215 allogeneic hct recipients of all ages demonstrated that the median shedding duration of hcovs was 3 weeks (range, 0-22 weeks) [70] . our follow-up study suggests that high viral load, high-dose steroids, and myeloablative conditioning are associated with prolonged shedding (≥21 days) of hcov in allogeneic hct recipients (2 pediatric and 42 adult patients) [71] . using available nasal samples from patients with prolonged shedding, we performed whole-genome sequencing, which revealed only small and slow genomic changes, consistent with previously estimated evolution rates. this is in contrast to more rapid genomic changes associated with influenza infections [72] . in addition, an 18-year-old pediatric patient had 3 hcov species (oc43, hku1, 229e) detected sequentially over a 5-month period without an associated poor outcome. the role of hcovs as enteric pathogens in humans has been debated, perhaps in part because of the known enteric disease associated with coronavirus in animals, including dogs. risku et al investigated the presence of hcovs in stool of children with and without gastroenteritis. all 4 species were found in 2.5% of stool samples (22/878) from children with gastroenteritis, whereas 1.7 % of stool samples (2/112) from controls were positive for hcovs. however, other known enteric pathogens, such as rotavirus or norovirus, were also found in 18 of 22 stool samples in children with gastroenteritis. among 4 patients with only hcov detected in stool, 3 had respiratory symptoms [73] . another study examined both stool and nasopharyngeal swabs in hospitalized children with acute gastroenteritis and in controls [74] . hcovs were more frequently detected in patients with gastroenteritis than in controls (23/260, 8.8% vs 4/151, 2.6%, respectively). interestingly, in patients with gastroenteritis, more than half (13/23) had respiratory symptoms and hcovs were more frequently found in nasopharyngeal samples than in stool samples (22/256, 8 .6%, vs 6/260, 2.3%, respectively). based on these studies, the significance of hcovs as enteric pathogens appears minor. hcov-oc43 has been detected in the central nervous system of children with acute disseminated encephalomyelitis or fatal encephalitis [75, 76] . a prospective study investigated associations between hcovs detection and various clinical manifestations [77] . this latter study proposed an etiological role of hcovs in febrile seizures given that children with febrile seizures had higher rates of hcovs detection with higher viral load in nasopharyngeal swabs than those with bronchiolitis or gastroenteritis and healthy controls. however, an etiologic connection between hcovs and neurologic diseases remains unproven [78] . carefully conducted epidemiological studies have not demonstrated an association between hcovs and kawasaki disease [21, 25] . an outbreak of sars-cov occurred in east and southeast asia in early spring of 2003. this international outbreak began in a hotel in hong kong and ultimately spread to more than 20 countries. the etiologic agent of sars-cov is a novel coronavirus identified by multiple investigators [10] [11] [12] 79] . the virus was classified as a beta-coronavirus, lineage b. this virus uses angiotensin-converting enzyme 2 as a functional cellular receptor. the virus also binds to the c-type lectin cd209l (also known as l-sign) and dc-sign [80] [81] [82] . during the outbreak, approximately 8098 cases occurred with 774 deaths, resulting in an overall mortality rate of 9% [24] . in hong kong, about 5% of the cases were children and adolescents [83] . young children appeared to have a milder form of the disease [84] [85] [86] [87] . among laboratory-confirmed and probable pediatric sars-cov cases, the most common symptoms included fever (98%), cough (60%), nausea or vomiting (41%), and constitutional symptoms such as myalgia (29%), chills (28%), and headache (28%) [84] . clinical manifestations of sars-cov in children are nonspecific, and it can be very difficult to differentiate sars-cov from other respiratory tract infections without laboratory testing in the outbreak setting. however, certain features may provide a clue. a comparison of 15 pediatric patients with laboratory-confirmed sars-cov and 15 age-and sexmatched patients with culture-confirmed influenza in taiwan revealed that rates of fever, cough, and constitutional symptoms such as chills and myalgia were similar between the 2 groups, but patients with sars-cov had significantly less rhinorrhea, sputum production, and sore throat than those with influenza [88] . in general, respiratory and constitutional symptoms are similar in the beginning of the illness in children and adults. however, a much higher proportion of adult patients progress to severe pneumonia, even acute respiratory distress syndrome. in fact, despite a high mortality rate in adults (9.6%-16.7%) [83] , there were no fatalities documented in the pediatric population [84] [85] [86] [87] . a study reported outcomes of 5 pregnant women infected with sars-cov [89] ; the gestational ages of their infants ranged from 26 weeks to 32 weeks. in 3 of these 5 pregnant women, cesarean section was needed because of maternal conditions, including hypotension and worsening pulmonary function. however, a systematic search for perinatal transmission of the sars-cov did not detect the virus in any of the 5 babies. the first case of mers-cov was reported in a man hospitalized in jeddah, saudi arabia, in june 2012. he died of severe pneumonia and renal failure, and a novel coronavirus was isolated from his sputum [14] . this new beta-coronavirus was named mers-cov [13] . it belongs to lineage c and is closely related to tylonycteris bat coronavirus hku4 (ty-batcov hku4) and pipistrellus bat coronavirus hku5 (pi-batcov hku5) [13] . the dromedary camel is known to be the intermediate host of mers-cov. the widely expressed cell-surface protease dipeptidyl-peptidase 4 (also known as cd26) was identified as a functional receptor for host cell entry [90] . as of november 2019, the world health organization reported 2494 laboratory-confirmed cases of mers-cov infection in 27 countries with 858 deaths globally, resulting in an approximate 34% mortality rate [91] . all known mers-cov infections can be traced to countries in the middle east, primarily saudi arabia. most cases have been in adults with underlying chronic diseases or immunosuppression who live or travel in the arabian peninsula. among 1351 confirmed cases between 2012 and 2019, cases aged <18 years were less than 5% of all confirmed cases [92] . mers-cov infection in adults usually occurs as sporadic cases, healthcare-associated infection, or transmission within families [93] . however, most confirmed pediatric cases were secondary cases after exposures to others within the same family [94] . the proportion of children among those infected with mers-cov has consistently been reported as being relatively low compared with the general population. from june 2012 to april 2016, the proportion of pediatric patients was 1.6% (9 of 552) of all positive cases in saudi arabia [94] . among patients hospitalized in riyadh, saudi arabia, from april 2014 to november 2016, the proportion of 295 confirmed pediatric patients (aged <18 years) was 2.4%, with age ranging from 9 months to 17 years (supplementary table 1 ). clinical manifestations in pediatric patients have not been systematically described. among the 31 pediatric patients with mers-cov infection documented from june 2012 to april 19, 2016, 13 patients (42%) were asymptomatic [94] . in another study, among 7 pediatric patients identified from april 2014 to november 2016, 3 were asymptomatic; fever, cough, shortness of breath, diarrhea, and vomiting were observed in 4 patients [95] . although pediatric patients typically have mild disease, high-risk children with underlying conditions, including cystic fibrosis, nephrotic syndrome, and unidentified underlying conditions, died of mers-cov infection, with a fatality rate of 12% (4/33) (see supplementary table 1 ) [96, 97] . a recent study summarized mers-cov infection in 11 pregnant women [98] . the gestational ages ranged from 6 weeks to 38 weeks. among those 11 pregnant women, 7 (64%) were admitted to intensive care units and 3 (27%) died. of the 11 births, 3 (27%) died, 2 had documented intrauterine death at 34 weeks and 5 months of gestation, and 1 was delivered at 24 weeks but did not survive. recognition of the importance of community coronavirus disease due to 4 established hcovs has increased over the past 20 years, with widespread availability of molecular diagnostic methods. however, detailed information on pathogenesis, immunity, and viral characteristics of disease in children remains limited. recent and ongoing epidemics of novel coronaviruses in the 21st century have highlighted issues of zoonotic origins of transmissible respiratory viruses and potential transmission, disease, and mortality related to these viruses. the role of children in the spread of disease with these novel viruses remains unclear. as the current pandemic with sars-cov-2 unfolds, more information regarding the role of children in viral transmission and their clinical presentation and outcome will become more evident. further study of coronaviruses in humans is imperative. supplementary materials are available at journal of the pediatric infectious diseases society online. discovery, diversity and evolution of novel coronaviruses sampled from rodents in china cultivation of a novel type of common-cold virus in organ cultures a new virus isolated from the human respiratory tract identification of a new human coronavirus a previously undescribed coronavirus associated with respiratory disease in humans growth in suckling-mouse brain of "ibvlike" viruses from patients with upper respiratory tract disease some characteristics of hemagglutination of certain strains of "ibv-like" virus characterization and complete genome sequence of a novel coronavirus, coronavirus hku1, from patients with pneumonia clinical and molecular epidemiological features of coronavirus hku1-associated community-acquired pneumonia identification of a novel coronavirus in patients with severe acute respiratory syndrome sars working group. a novel coronavirus associated with severe acute respiratory syndrome coronavirus as a possible cause of severe acute respiratory syndrome middle east respiratory syndrome coronavirus (mers-cov): announcement of the coronavirus study group isolation of a novel coronavirus from a man with pneumonia in saudi arabia a novel coronavirus genome identified in a cluster of pneumonia cases-wuhan chinese researchers reveal draft genome of virus implicated in wuhan pneumonia outbreak molecular basis of binding between middle east respiratory syndrome coronavirus and cd26 from seven bat species viral infections of humans epidemiology and control molecular evolution of the sars coronavirus during the course of the sars epidemic in china sars-cov infection in a restaurant from palm civet human coronaviruses, including middle east respiratory syndrome coronavirus. in: feigin and cherry's textbook of pediatric infectious diseases first case of 2019 novel coronavirus in the united states a novel coronavirus from patients with pneumonia in china coronaviruses: an overview of their replication and pathogenesis human coronaviruses. in: principles and practice of pediatric infectious diseases genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding tracing the sars-coronavirus middle east respiratory syndrome coronavirus in bats, saudi arabia complete genomic sequence of human coronavirus oc43: molecular clock analysis suggests a relatively recent zoonotic coronavirus transmission event coronavirus infection in acute lower respiratory tract disease of infants coronavirus infection in acute lower respiratory tract disease of infants burden and risk factors for coronavirus infections in infants in rural nepal respiratory viruses and influenza-like illness: epidemiology and outcomes in children aged 6 months to 10 years in a multicountry population sample clinical disease in children associated with newly described coronavirus subtypes human coronavirus in hospitalized children with respiratory tract infections: a 9-year population-based study from norway community-wide outbreak of infection with a 229e-like coronavirus in tecumseh, michigan development of a nucleocapsid-based human coronavirus immunoassay and estimates of individuals exposed to coronavirus in a u.s. metropolitan population the association of viral and bacterial respiratory infections with exacerbations of wheezing in young asthmatic children virologic studies of acute respiratory disease in young adults. v. coronavirus 229e infections during six years of surveillance neonatal nosocomial respiratory infection with coronavirus: a prospective study in a neonatal intensive care unit coronavirus-related nosocomial viral respiratory infections in a neonatal and paediatric intensive care unit: a prospective study outbreaks of human coronavirus in a pediatric and neonatal intensive care unit extensive multiplex pcr diagnostics reveal new insights into the epidemiology of viral respiratory infections effects of a "new" human respiratory virus in volunteers viral etiology of severe pneumonia among kenyan infants and children the tecumseh study of respiratory illness. vi. frequency of and relationship between outbreaks of coronavirus infection epidemiology of viral respiratory tract infections in a prospective cohort of infants and toddlers attending daycare epidemiology of multiple respiratory viruses in childcare attendees community surveillance of respiratory viruses among families in the utah better identification of germs-longitudinal viral epidemiology (big-love) study seroepidemiologic survey of coronavirus (strain 229e) infections in a population of children severity and outcome associated with human coronavirus oc43 infections among children new vaccine surveillance network. human coronavirus in young children hospitalized for acute respiratory illness and asymptomatic controls community study of role of viral infections in exacerbations of asthma in 9-11 year old children the pediatric burden of human coronaviruses evaluated for twenty years coronavirus-associated pneumonia in previously healthy children epidemiology and clinical characteristics of human coronaviruses oc43, 229e, nl63, and hku1: a study of hospitalized children with acute respiratory tract infection in guangzhou, china epidemiology and clinical features of human coronaviruses in the pediatric population characteristics and outcomes of coronavirus infection in children: the role of viral factors and an immunocompromised state genetic variability of human coronavirus oc43-, 229e-, and nl63-like strains and their association with lower respiratory tract infections of hospitalized infants and immunocompromised patients detection of rhinovirus, respiratory syncytial virus, and coronavirus infections in acute otitis media by reverse transcriptase polymerase chain reaction viral upper respiratory tract infection and otitis media complication in young children croup is associated with the novel coronavirus nl63 role of human coronavirus nl63 in hospitalized children with croup human coronavirus oc43 pneumonia in a pediatric cancer patient with down syndrome and acute lymphoblastic leukemia fatal respiratory distress syndrome due to coronavirus infection in a child with severe combined immunodeficiency coronavirus 229e-related pneumonia in immunocompromised patients fatal lower respiratory tract disease with human corona virus nl63 in an adult haematopoietic cell transplant recipient a multicenter consortium to define the epidemiology and outcomes of inpatient respiratory viral infections in pediatric hematopoietic stem cell transplant recipients clinical significance of human coronavirus in bronchoalveolar lavage samples from hematopoietic cell transplant recipients and patients with hematologic malignancies human rhinovirus and coronavirus detection among allogeneic hematopoietic stem cell transplantation recipients prolonged shedding of human coronavirus in hematopoietic cell transplant recipients: risk factors and viral genome evolution parallel evolution of influenza across multiple spatiotemporal scales detection of human coronaviruses in children with acute gastroenteritis detection of human coronaviruses in simultaneously collected stool samples and nasopharyngeal swabs from hospitalized children with acute gastroenteritis human coronavirus oc43 associated with fatal encephalitis detection of coronavirus in the central nervous system of a child with acute disseminated encephalomyelitis the role of human coronaviruses in children hospitalized for acute bronchiolitis, acute gastroenteritis, and febrile seizures: a 2-year prospective study human coronaviruses and other respiratory viruses: underestimated opportunistic pathogens of the central nervous system? canadian severe acute respiratory syndrome study team. identification of severe acute respiratory syndrome in canada angiotensin-converting enzyme 2 is a functional receptor for the sars coronavirus pathogenesis of severe acute respiratory syndrome human coronavirus nl63 employs the severe acute respiratory syndrome coronavirus receptor for cellular entry probable secondary infections in households of sars patients in hong kong severe acute respiratory syndrome in children other members of the hospital for sick children sars investigation team. children hospitalized with severe acute respiratory syndrome-related illness in toronto severe acute respiratory syndrome in children: experience in a regional hospital in hong kong clinical presentations and outcome of severe acute respiratory syndrome in children childhood severe acute respiratory syndrome in taiwan and how to differentiate it from childhood influenza infection infants born to mothers with severe acute respiratory syndrome dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-emc middle east respiratory syndrome coronavirus (mers-cov) mers situation update managing mers-cov in the healthcare setting middle east respiratory syndrome coronavirus disease is rare in children: an update from saudi arabia middle east respiratory syndrome coronavirus in pediatrics: a report of seven cases from saudi arabia middle east respiratory syndrome coronavirus disease in children middle east respiratory syndrome coronavirus in children middle east respiratory syndrome coronavirus (mers-cov) infection during pregnancy: report of two cases & review of the literature key: cord-016990-ot1wi3xi authors: zaki, sherif r.; paddock, christopher d. title: viral infections of the lung date: 2008 journal: dail and hammar&#x02019;s pulmonary pathology doi: 10.1007/978-0-387-68792-6_11 sha: doc_id: 16990 cord_uid: ot1wi3xi the lungs are among the most vulnerable to microbial assault of all organs in the body. from a contemporary vantage, lower respiratory tract infections are the greatest cause of infection-related mortality in the united states, and rank seventh among all causes of deaths in the united states.2,3 from a global and historic perspective, the scope and scale of lower respiratory tract infection is greater than any other infectious syndrome, and viral pneumonias have proven to be some of the most lethal and dramatic of human diseases. the 1918–1919 influenza pandemic, perhaps the most devastating infectious disease pandemic in recorded history, resulted in an estimated 40 million deaths worldwide, including 700,000 deaths in the u.s.4 the global outbreak of severe acute respiratory syndrome (sars) during 2003, although considerably smaller in scale, resulted in 8098 cases and 774 deaths5 and is a dramatic contemporary example of the ability of viral pneumonias to rapidly disseminate and cause severe disease in human populations. the lungs are among the most vulnerable to microbial assault of all organs in the body. from a contemporary vantage, lower respiratory tract infections are the greatest cause of infection-related mortality in the united states, and rank seventh among all causes of deaths in the united states. 2 ,3 from a global and historic perspective, the scope and scale of lower respiratory tract infection is greater than any other infectious syndrome, and viral pneumonias have proven to be some of the most lethal and dramatic of human diseases. the 1918-1919 influenza pandemic, perhaps the most devastating infectious disease pandemic in recorded history, resulted in an estimated 40 million deaths worldwide, including 700,000 deaths in the us. 4 the global outbreak of severe acute respiratory syndrome (sars) during 2003, although considerably smaller in scale, resulted in 8098 cases and 774 deaths s and is a dramatic contemporary example of the ability of viral pneumonias to rapidly disseminate and cause severe disease in human populations. although viruses are commonly identified causes of pneumonia of infants and young children, they are relatively infrequently recognized as agents of communityacquired pneumonia in adults. 6 in several large series that investigated a microbiologic cause of community-acquired pneumonia, viral etiologies were identified in only 9% to 18% of cases (table 11 .1).7-12 however, it is likely that viral pneumonias are underrecognized and underdiagnosed for various reasons. although some agents may cause distinct cytopathology or inclusions (e.g., adenoviruses, herpesviruses, and paramyxoviruses), many important pathogens (e.g., influenza viruses) do not, and none of these agents are resolved specifically in tissue by routine histologic stains. viruses require live cells for cultivation, and are generally more difficult than bacteria to isolate from clinical samples. for some viral pneumonias, 426 the pathogen appears to initiate a cascade of destructive host responses that continue or progress even in the absence of the specific infectious agent, and in these patients the etiologic agent may be absent from host tissues at the time of autopsy.13 thirty to sixty percent of community-acquired pneumonias are etiologically undetermined, 14 and it is entirely possible that viruses directly cause more episodes of pneumonia than currently appreciated. because viral infections of the lower respiratory tract often precede bacterial pneumonias, viruses may indirectly exert considerable influence on the cumulative morbidity and mortality of infectious pneumonias. 15 , 16 the mechanisms by which viruses may facilitate bacterial invasion of the respiratory tract are complex and varied. certain viruses cause the death of ciliated respiratory epithelium and thereby disrupt normal ciliary activity. viruses may also inhibit the phagocytic or bactericidal activities of neutrophils, t lymphocytes, and alveolar macrophages, and predispose the host to secondary infections. certain gram-positive and gram-negative bacteria adhere to and colonize virus-infected epithelium more readily than to noninfected cells by various hypothetical mechanisms, including alteration and induction of receptors at the host-cell surface and changes in the extracellular environmenty-19 finally, viral infections of the lung may exacerbate noninfectious pulmonary conditions (e.g., asthma and chronic obstructive pulmonary disease) and indirectly contribute to aggregate morbidity and mortality associated with these conditions. 20 although influenza viruses remain the most frequently identified cause of viral pneumonia in adults (table 11 .1), the diversity of agents identified as causes of viral pneumonias has expanded considerably. several newly recognized viral pneumonias have been identified since 1992 that are among the most feared and lethal of all emerging infections, including those caused by hantaviruses, nipah virus, and sars corona virus (co v). !3,21-23 certain causes "rubella (2), rhinovirus (1) , mixed viral infections (3) . source: adapted from greenberg. 6 of viral pneumonia, particularly those that occur in vulnerable patient cohorts, have diminished during this same interval. by example, the u.s. incidence of varicella pneumonia has dropped by two thirds since universal childhood vaccination for varicella was implemented in 1995, 24, 25 and advances in the clinical management of transplant recipients have reduced the incidence of cytomegalovirus (cmv) pneumonia?6 also occurring during the last decade has been the development and use of powerful molecular techniques that have unveiled the identity of historic pathogens (e.g., the hini "spanish" influenza a virus),27.28 and have facilitated the rapid characterization of emerging agents (e.g., sars-co v). 13 it should be noted that the disease manifestations of several of these agents (e.g., human parainfluenza virus [hpiv] , respiratory syncytial virus [rsv] , and influenza) are often confined to the upper airway and are not invariaorb rsv hpiv adeno cmv varicella other' 10 0 3 0 0 3 27 1 4 3 0 0 0 7 2 0 0 1 0 19 5 5 0 0 1 7 8 13 15 0 0 0 37 5 11 5 0 0 3 21 (11) 32 (16) 30 (15) 1 (0.5) 1 (0.5) 6 (3) ably associated with pneumonia. with some of these pathogens (e.g., influenza viruses, rsv, hantaviruses, and hpiv viruses) respiratory disease is the primary manifestation. for other agents, such as measles, nipah virus, and herpesviruses, typically the lungs are involved as part of a multisystem syndrome. the diagnosis of viral pneumonia, suspected by patient history and clinical manifestations, also can be supported histopathologically, and the general pattern of histopathologic lesions may suggest a specific diagnosis. many viruses can be identified in lung by examining the tissue response and cytopathic changes. some of these viruses cause recognizable tissue reaction patterns including necrotizing tracheobronchitis, bronchiolitis, and interstitial pneumonia. a summary of the key diagnostic histopathologic and ultrastructural features for the most common viral pathogens that cause a majority of pulmonary infections is provided in tables 11.2 and 11.3. yes (cytoplasmic; ill-defined) yes (nuclear and cytoplasmic) pulmonary tissue reaction necrotizing bronchiolitis; smudge cells; dad severe edema, early dad interstitial pneumonia; dad; occasional multinucleation interstitial pneumonia; dad; cytomegaly dad; necrosis and rare multinucleation dad; necrosis and rare multinucleation dad; necrotizing bronchiolitis interstitial pneumonia with multi nucleation dad dad; interstitial pneumonia with occasional multinucleation necrotizing bronchiolitis, interstitial pneumonia with occasional multinucleation recently recognized; human pathology not well described interstitial pneumonia with multinucleation; dad only certain viruses can cause cytopathic changes that are sufficiently distinct to enable the pathologist to recognize a specific diagnosis on routine histologic examination of lung specimens. with the availability of special figure 11 .1. negative stain electron microscopic images of different viruses that can cause pulmonary infections. a. adenovirus. adenoviruses are protein-shelled icosahedral-shaped nonenveloped viruses that measure approximately 70 to 90nm in diameter. two of the viruses are stain penetrated revealing the dna-containing nucleoprotein. b. sin nombre virus. sin nombre virus, the causative agent of hantavirus pulmonary syndrome, belongs to the genus hantavirus in the family bunyaviridae. the envelopes of hantaviruses are checkerboard in appearance, and particles measure 90 to 150nm in diameter. c. herpes simplex virus. the stain has penetrated the envelope of several of these herpesvirus particles, delineating the icosahedral-shaped nucleocapsids, which measure 90 to 100nm in diameter. d. cytomegalovirus. another herpesvirus; one virus particle (left) is intact while two nearby particles are stainpenetrated and show the viral nucleocapsids. the nucleocapsid of the upper right particle shows a central core that harbors the dna of the virus. e. influenza virus. influenzaviruses belong to the family orthomyxoviridae; viral particles are pleomorphic s.r. zaki and cd. paddock diagnostic techniques, such as immunohistochemistry (ihc) and in-situ hybridization (ish), many viruses can be detected in formalin-fixed, paraffin-embedded tissue samples even if specific viral inclusions cannot be found in histologic examination of tissue sections. among the techniques, ihc utilizing specific antibodies can be routinely performed on formalin-fixed tissue and can enhance the pathologist's accuracy in identifying organisms in tissue specimens. in addition to histologic pattern recognition, ihc, and ish in tissue, several other diagnostic tests are available to aid the pathologist. cell culture techniques, serology, polymerase chain reaction (pcr), and electron microscopy (em) all play vital roles in the diagnosis of these infections. while histologic techniques can be an excellent means of demonstrating organisms, tissue culture isolation remains essential for definitive identification of the virus. when a viral pneumonia is suspected, samples of lung tissues should be evaluated by cell culture, which has the advantage of being a nonbiased method for screening purposes that does not rely on the availability of specific antibodies or probes. electron microscopy offers the same utility as a broad scope diagnostic tool and has been especially critical in outbreaks of unknown etiology. it played a critical role during the hendra and nipah virus outbreaks in 1994 and 1999, respectively, and more recently, in the early recognition of a novel coronavirus associated with sars in 2003 and in the diagnosis of emerging transplant-associated infections. 13.22.29-33 the advantage of this approach is that viral particles may be demonstrated by negative stain or thin section em, either directly in clinical material or after amplification in cell culture. like culture, em is not limited by narrow specificity of reagents or by prior clinical bias ( fig. 11 .1). and can be filamentous or spherical in shape. the evenly spaced spikes cover the entire virus surface and contain both the hemagglutinin and neuraminidase surface glycoproteins. f. human metapneumovirus. these paramyxoviruses are heterogeneous in size and shape, and range in size from 150nm to 1 j.lm in diameter. g. parainfluenza virus. another paramyxovirus, the viral nucleocapsid, with its typical herringbone appearance, can be seen both within the stain-penetrated particle as well as partially extruded from the virion. an important feature that can help distinguish between paramyxovirinae (parainfluenza and measles viruses) and pneumovirinae (human metapneumovirus and respiratory syncytial virus) is the diameter of the nucleocapsids, which measure 18nm and 14nm, respectively. h. severe acute respiratory syndrome (sars) coronavirus. these 80-to 100-nm particles are named for the characteristic crown-like fringe on the surface. scale bars, 100nm. (a,b,d,f,h: courtesy of c. humphrey; c,g: courtesy of e. palmer; e: courtesy of ea. murphy, all at centers for disease control and prevention, atlanta, ga.) adenoviruses were first cultured and identified during the early 1950s by investigators searching for etiologic agents of acute respiratory infections. the initial adenovirus isolate was made serendipitously from adenoid tissues obtained from children during efforts to establish a primary human adenoid cell line. 34 a related virus was identified the following year by investigators studying respiratory disease in military recruits?5 these agents were subsequently named adenoviruses after the original source of tissue from which the prototype strain was identified. 36 adenoviruses are non enveloped viruses with a single, linear, double-stranded dna genome that is contained within an icosahedral capsid that measures 70 to 90nm in diameter (fig. ilia) . the capsid is comprised of seven known polypeptides, including the hexon capsomere, which contains group-specific antigenic determinants. 37 adenoviruses are a ubiquitous and diverse group of viruses found naturally in the upper respiratory tracts and gastrointestinal systems of humans, other mammals, and birds. most adenoviruses infect mucosal epithelium, although some pathogens of animals are trophic for endothelial cells, and endothelial infection has been identified in some immunocompromised humans. 38 adenoviruses are represented by at least 51 serotypes on the basis of resistance to neutralization by antisera to other known adenovirus serotypes, and comprise six subgroups or subgenera (a through f) that are distinguished by differential hemagglutination with erythrocytes from various animal species. 37 ,39ao more than 50% of the known adenovirus serotypes are associated with human diseases of the upper and lower respiratory tract, conjunctiva, urinary tract, intestine, and occasionally heart, liver, and central nervous system. the others are rarely encountered and mayor may not act as pathogens in recognizable disease. 37 it is estimated that approximately 5% to 10% of all pneumonias in infants and young children are caused by adenoviruses. 41 ,42 most pediatric cases of adenovirus pneumonia occur between 6 months and 5 years of age, and serotypes 3, 7, and 21 (all members of the b subgenus), are the most common causes of pneumonia in this patient cohort. 43 -45 serotypes 3 and 7 are particularly pathogenic adenoviruses that can cause disseminated and often fatal disease in previously healthy children. 46 in adults, pneumonia is generally associated with serotypes 3,4, and 7.47 periodic epidemics of adenovirus pneumonia in young adults have been identified, particularly among military recruits. 48 ,49 in a manner similar to other pathogens, adenoviruses take advantage of impaired or destroyed immune systems to establish persistent and disseminated infections in immunocompromised hosts. in this patient cohort, the s.r. zaki and cd. paddock case fatality rate of adenoviral pneumonia approaches 60%, compared with an approximately 15% mortality in immunocompetent patients. 37 immunocompromised patients are also susceptible to a broader range of different adenovirus serotypes. by example, the commonly recognized serotypes in normal children account for only about 50% of the adenovirus serotypes reported for children with congenital immune deficiencies. 37 .4 6 because some adenoviruses establish latency in lymphoid tissues and the kidneys of their host, it is believed that many, possibly most, cases of clinical disease caused by adenoviruses in immunocompromised patients are reactivated infections. 37 the lungs of patients with adenovirus pneumonia are typically heavy and edematous, and the bronchi are generally filled with mucoid, fibrinous, or purulent exudates. the histopathologic findings ( fig. 11 .2) include necrotizing bronchitis and bronchiolitis with extensive denudation ofthe surface epithelium,particularly in medium-sized (1 to 2mm in diameter) intrapulmonary bronchi ( fig. 11 .2a). affected airways may be occluded by homogeneous eosinophilic material, mixed inflammatory cells, detached epithelium, and cellular debris. the lamina propria of bronchi and bronchioles is typically congested and infiltrated by predominantly mononuclear inflammatory cell infiltrates. bronchial serous and mucous glands are also often involved and show necrosis and mixed infiltrates. 5o as the infection progresses, there is involvement of the pulmonary parenchyma, forming bronchocentric necrosis with hemorrhage, neutrophilic and mononuclear cell infiltrates, and karyorrhexis. these findings generally occur against a background of exudative diffuse alveolar damage, with filling of the air space by macrophages, fibrin, and detached pneumocytes, and hyaline membrane formation. 51 patients with fatal pneumonia may develop disseminated intravascular coagulopathy and demonstrate fibrin thrombi in vessels of the lungs, kidney, heart, adrenals, and central nervous system (see fig. 4 .20 in chapter 4). 48 adenoviruses form intranuclear inclusions in respiratory epithelial cells of the trachea, bronchi, and bronchioles, in the acinar cells of bronchial glands, and in alveolar pneumocytes, and are generally most abundant at the viable edges of necrotic foci. by using the hematoxylin and eosin (h&e) stain, early inclusions appear as small, dense, amphophilic structures surrounded by a cleared zone and peripherally marginated chromatin, similar to herpetic inclusions. as the cellular infection progresses, the inclusion becomes larger (as large as 14/-lm in some cells) and more basophilic, and the margins of the nuclear membrane become blurred to form the characteristic "smudge cell" (fig. 11 .2b,c).5o,51 tracheal aspirates of patients with adenovirus pneumonia may show distinctive features on cytologic preparations that include cells with fine strands of chromatin that radiate from a central inclusion to the marginated chromatin at the nuclear membrane ("rosette cells"), and cells with foamy, "honeycomb" nuclei, as well as typical smudge cells (see fig. 7 .45 in chapter 7). 52 various methods can be used to diagnose adenovirus infections that include antigen detection, cell culture, electron microscopy, molecular assays, and serology. direct detection techniques that identify the common group-reactive hexon antigen in tissues or body fluids include fluorescence antibody assays and enzyme immunoassays.53 immunohistochemistry staining methods have been used successfully to detect adenovirus-infected cells in formalin-fixed, paraffinembedded tissues using various commercially available, adenovirus group-specific antibodies ( fig.11 .2e,f).38,51 electron microscopy of adenovirus-infected tissues reveals a paracrystalline array of virions ( fig. 11 .2d ). 54, 55 most adenoviruses can be isolated in cell culture from bronchial washings, tracheal aspirates, or lung biopsy specimens during the early stage of the illness and grow well in various cell lines, including human embryonic kidney, hela, and hep-2 cells. 47 cell cultures infected with adenoviruses exhibit a relatively characteristic cytopathologic effect, described as a "cluster of grapes," within 3 to 5 days after inoculation. serotyping of the isolate is accomplished by using hemagglutination inhibition and neutralization tests with hyperimmune type-specific animal antisera. 56 molecular assays, particularly gene amplification using pcr, and ish methods, have been developed to detect adenovirus nucleic acid in respiratory secretions and in formalin-fixed, paraffin-embedded tissues. 51 ,57,58 broad-range, sensitive assays that can detect any adenovirus amplify common genomic sequences (e.g., the hexon gene region). other more specific assays detect specific adenovirus types with unique genomic sequences. 59 serologic assays include tests for groupspecific antibodies (e,g., complement fixation and enzyme immunoassays), or type-specific antibodies (e.g., neutralization and hemagglutination-inhibition assays). pitfalls associated with serologic testing for adenoviruses include occasional rises in heterotypic antibodies when typespecific assays are used, and relatively low sensitivity demonstrated by complement fixation assays. 59 human hantaviral diseases are caused by a group of closely related, trisegmented, negative-sense rna viruses of the genus hantavirus, of the family bunyaviridae. 60 -62 members of the genus hantavirus have similar morphologic features. 63 ,64 virus particles are 70 to 130nm in diameter and generally appear spherical to ovoid, although pleomorphic forms may be seen. a lipid envelope containing glycoprotein spikes surrounds a core consisting of the genome and its associated proteins (nucleocapsids) s.r. zaki and cd. paddock arranged in delicate tangles of filaments showing occasional granulation. the presence of characteristic inclusion bodies in thin section electron microscopy and a unique grid-like pattern on negative-stain electron microscopy differentiate these viruses from other members of the family bunyaviridae ( fig. 11.1b) . 65, 66 the severity and disease type largely depends on the viral serotype. two categories of hantavirus-associated illnesses are described: hemorrhagic fever with renal syndrome (hfrs) for disease in which the kidneys are primarily involved, and hantavirus pulmonary syndrome (hps) for disease in which the lungs are primarily involved. 67 -69 the isolation of the first recognized hantavirus (hantaan virus, named for the river in south korea), and its subsequent identification as the causative agent of hfrs was reported in 1978. 70 in 1993, the deaths of several previously healthy individuals due to a rapidly progressive respiratory disease in the southwestern united states were etiologically linked to a previously unrecognized hantavirus. clinically, the disease differs from hfrs in its pronounced pulmonary involvement and higher mortality rates and is known as hps. 23.69,71.72 hantavirus-associated diseases primarily affect blood vessels and result in different degrees of generalized capillary dilatation and edema. 73 in contrast to severe hfrs where abundant protein-rich, gelatinous retroperitoneal edema fluid is found, all hps patients have large bilateral pleural effusions and heavy edematous lungs. 7 in fatal far eastern hfrs, a distinctive triad of hemorrhagic necrosis can be seen in the renal medullary junctional zone, cardiac right atrium, and anterior pituitary. 75, 78 however, in patients with hps, hemorrhages are rare, and ischemic necrotic lesions, except those attributed to shock, are not seen. 72, 74 histologically, morphologic changes of the endothelium are uncommon but, when seen, consist of prominent and swollen endothelial cells. vascular thrombi and endothelial cell necrosis are rare. in hfrs, the most severe and characteristic microscopic lesions involve the kidney; however, an interstitial pneumonitis can also be seen in some fatal cases. in contrast, the microscopic changes of north and south american hps are principally seen in the lung and spleen. 72 ,74 the lungs show a mild to moderate interstitial pneumonitis characterized by variable degrees of edema and an interstitial mononuclear cell infiltrate comprised of a mixture of small and enlarged mononuclear cells with the appearance of immunoblasts ( fig.11 .3a). focal hyaline membranes composed of condensed proteinaceous intraalveolar edema fluid, fibrin, and variable numbers of inflammatory cells are observed ( fig. 11 .3b). typically, neutrophils are scanty and the alveolar pneumocytes are intact with no evidence of cellular debris, nuclear fragmentation, or hyperplasia. in fatal cases, with a prolonged survival interval, tissues show features more characteristic of the exudative and proliferative stages of diffuse alveolar damage. lung biopsies taken from patients who survive their illness appear similar with proliferated reparative type ii pneumocytes, severe edematous and fibroblastic thickening of the alveolar septa, and severe air-space disorganization with distorted lung architecture (see chapter 4) . other characteristic microscopic findings in hps cases include variable numbers of immunoblasts within the splenic red pulp and periarteriolar white pulp, lymph nodal paracortical zones, hepatic portal triads, and peripheral blood ( fig. 11.3d,e) . similarly, in severe hfrs cases, large mononuclear cells can be present in the spleen, lymph nodes, blood, and hepatic portal triads. 75 . 76 electron microscopic studies of hps lung tissue demonstrate infection of endothelial cells and macrophages. 69 . 72 the virus or virus-like particles observed are infrequent and extremely difficult to identify in autopsy tissues because of the considerable degree of viral pleomorphism and the postmortem deterioration of tissues. however, typical hantaviral inclusions are seen more frequently and their identity can be confirmed by immunolabeling ( fig. 11 .3f,g). similar inclusions are observed in epithelial cells in hfrs and are considered to be ultrastructural markers of hantavirus-infected cells. 63 .79.8o using immunohistochemistry, viral antigens are found primarily within capillary endothelium throughout various tissues in both hps and hfrs. in hps, marked accumulations of hantaviral antigens are in the pulmonary microvasculature and in splenic and lymph nodal follicular dendritic cells (fig. 11 .3c).72 despite the extensive endothelial cell accumulations of hantaviral antigens, there is little ultrastructural evidence of cytopathic effect. hantavirus pulmonary syndrome should be suspected in cases of adult respiratory distress syndrome (ards) without a known precipitating cause among previously healthy individuals. the level of suspicion should be particularly high when patients have a known exposure to rodents in areas where peromyscus maniculatus or other reservoirs of hantavirus are found. physicians need to differentiate hps from other common acute respiratory diseases, such as pneumococcal pneumonia, influenza virus, and unexplained ards. the diagnosis of hps, suspected by patient history and clinical manifestations, can also be supported histopathologically. although there is no single pathognomonic lesion that would permit certain histopathologic diagnosis of hps, the overall constellation of histopathologic hematologic findings suggests the diagnosis.72.7 4 diseases that need to be distinguished pathologically from hps include a relatively large number of different viral, rickettsial, and bacterial infections, as well as various noninfectious disease processes. virus-specific diagnosis and confirmation can be achieved through serology, pcr for hantavirus rna, or ihc for hantaviral antigens. 2 1,72 serologic testing can detect hantavirus-specific immunoglobulin m or rising s.r. zaki and cd. paddock titers of immunoglobulin g in patient sera and is considered the method of choice for laboratory confirmation of hps. immunofluorescent assays and enzyme-linked immunosorbent assays (elisas), which demonstrate the presence of specific antihantaviral antibodies, are currently used as rapid diagnostic tests and provide results within a few hours. recently, synthetic hantaviral nucleocapsid proteins have been used to improve the sensitivity and specificity of serologic assays. these proteins are more available than inactivated hantaviral antigens. 81 . 82 polymerase chain reaction detects viral rna in blood and tissues and is extremely useful for diagnostic and epidemiologic purposes. hantaviral rna can also be detected in formalin-fixed, paraffin-embedded archival tissue by reverse-transcriptase (rt)-pcr.83 immunohistochemistry testing of formalin-fixed tissues can be used to detect hantavirus antigens, and is a sensitive method to confirm hantaviral infections. 72 it has a unique role in the diagnosis of fatal hps cases when serum samples and frozen tissues are unavailable but formalinfixed autopsy tissues are obtainable. 84 • 85 severe acute respiratory syndrome the causative agent of sars is an enveloped, positivestranded rna virus that is a member of the genus coronavirus, of the family coronaviridae. corona viruses have the largest genomes of all rna viruses and replicate by a unique mechanism that results in a high frequency of recombination. maturation of sars coronavirus (sars-co v) is similar to features previously described for other coronaviruses. 8 6-9 0virions form by alignment of the helical nucleocapsids along the membranes of the endoplasmic reticulum or golgi complex and acquire an envelope by budding into the cisternae. the cellular vesicles become filled with virions and progress to the cell surface for release of the virus particles; large numbers of particles remain adherent to the plasma membrane at the cell surface. severe acute respiratory syndrome was recognized during a global outbreak of severe pneumonia that began in late 2002 in guangdong province, china, and gained prominence in early 2003 as cases were identified in more than two dozen countries in asia, europe, north america, and south america. the disease causes an influenza-like illness with fever, cough, dyspnea, and headache, and in severe cases it can cause death in humans. person-toperson transmission, combined with international travel of infected persons, accelerated the worldwide spread of the illness. 5 • 13 . 91 several reports have described diffuse alveolar damage with various levels of progression and severity as the main histopathologic findings in sars patients ( fig. 11 .4a,b)y·92-98 lungs typically show changes described for the proliferative phase of diffuse alveolar damage, with hyaline-membrane formation, desquamation of epithelial cells, fibrin deposit in the alveolar space, and hyperplasia of type 2 pneumocytes. increased mononuclear infiltrate in the interstitium can be seen in some cases. other findings identified in some patients included focal intra alveolar hemorrhage, necrotic inflammatory debris in small airways, and organizing pneumonia. in addition, multinucleated syncytial cells may be seen in the intraalveolar spaces of some patients who died 14 days or more after onset of illness ( fig. 1l .4c). infection with some coronaviruses, including sars-co v, is known to induce cell fusion in culture producing syncytial cells similar to those sometimes observed in lungs of patients who die from sars. these cells contain abundant vacuolated cytoplasm with cleaved and convoluted nuclei, but without obvious intranuclear or intracytoplasmic viral inclusions. the ish and ihc studies of tissues from sars patients have identified corona virus infection of upper airway bronchiolar epithelium ( fig. 11 .4d_f).92,99-101 infected ciliated columnar epithelial cells can be seen focally in lining epithelium of trachea and larger bronchi ( fig. 11 .4e). many of these infected cells slough from the epithelium and can be observed by using ish within the bronchial lumen. abundant viral antigens can also be found distributed focally in parenchyma of lungs of some patients and are seen predominantly in the cytoplasm of pneumocytes, in occasional macrophages, and in association with intra alveolar necrotic debris and fibrin (fig. ll.4d). double-stain studies indicate that most sars-co v-infected cells are type 2 pneumocytes. double-stain studies also detected viral nucleic acids with a distribution similar to that seen in ihc studies, mainly in pneumocytes and in some macrophages.looelectron microscopic examination of lung tissues selected from areas with abundant ihc staining shows numerous coronavirus particles and nucleocapsid inclusions. virions are seen in cytoplasmic vesicles and along the cell membranes of pneumocytes, in phagosomes of macrophages, and associated with fibrin in alveolar spaces (fig.ll.4g-i). because corona virus particles may be confused morphologically with other non viral cellular components, definitive ultrastructural identification can be achieved by using immunogold labeling electron microscopy. the primary histopathologic lesions seen in the lungs of patients who die from sars are somewhat nonspecific and can also be seen in acute lung injury cases caused by infectious agents, trauma, drugs, or toxic chemicals. 102 multinucleated syncytial cells similar to those seen in some sars patients can also be found in a number of virus infections, including measles, parainfluenza viruses, rsv, and nipah virus infections. 102 -104 in an early study of four human sars patients,13 viral antigens were not detected in the lung by ihe. the most likely explanation is that all patients in the study had a clinical course aver-s.r. zaki and cd. paddock aging more than 2 weeks. for many virus infections, viral antigens and nucleic acids are cleared within 2 weeks of disease onset by the host immune response. it is also possible that the pulmonary damage associated with sars is not caused directly by the virus, but represents a secondary effect of cytokines or other factors induced by the virus infection. similarly, in influenza virus infections, viral antigens are seen predominantly in respiratory epithelial cells of large airways and are only rarely identified in pulmonary parenchyma despite concomitant and occasionally severe interstitial pneumonitis. !os in recent reports by shieh et al. 100 and chong et al.,92 the temporal relationship between the duration of illness and clearance of sars-co v in human lung tissue was examined. viral antigens and nucleic acids were detected only in pulmonary tissues of patients who died early in the disease. the development of specific ihc, ish, and immunoelectron microscopy (iem) assays to identify sars-co v in formalin-fixed, paraffin-embedded samples has facilitated the assessment of the cellular tropism of sars-co v infection in human lung tissues. localization of sars-co v in the lung occurs mainly in the cytoplasm of pneumocytes, primarily type 2, and occasionally in alveolar macrophages ( fig. ll.4f ). type 2 pneumocytes are known to secrete pulmonary surfactant, resulting in reduced surface tension and preservation of the integrity of the alveolar space. these cells also play an important role in tissue restitution following lung damage. moreover, there is mounting evidence to support their contribution to the development of acute inflammatory lung injury following exposure to biological or chemical agents. additional studies are needed to further define the role of type 2 pneumocytes and alveolar macrophages in sars-co v infection. cynomolgus macaques inoculated with sars-cov develop pathologic findings of pneumonia and have been proposed as an animal model. 106 haagmans et a1. 107 showed extensive sars-co v antigen expression in experimentally infected macaques 4 days after infection. the antigens were mainly in alveolar lining epithelial cells with morphologic characteristics of type 1 pneumocytes, indicating type 1 pneumocytes are the primary target for sars-co v infection early in the disease. type 1 pneumocytes normally represent 90% of the alveolar epithelial cell volume and are easily damaged during pulmonary infections or other types of injury. in a more recent study on nonhuman primates,108 evidence of infection of type 1 pneumocytes in addition to some type 2 pneumocytes and macrophages was found. small animal models, such as rodents, would be very useful for evaluating vaccines, immunotherapies, and antiviral drugs, and we have recently identified the mouse as an animal model for this purpose. 10 9 in those studies, microscopic examination of trachea, bronchus, lung, thymus, and heart on day 2 after infection revealed mild and focal peri bronchiolar mononuclear inflammatory infiltrates with no significant histopathologic change in other organs. viral antigens and nucleic acids were focally distributed in bronchiolar epithelial cells, and virions were found in these same areas by ultrastructural analysis. data suggest that sars-co v replicates in mice to a titer sufficient to evaluate vaccines and antiviral agents. the mouse and other small animal models l1o might also be used to test the ability of the virus to replicate and cause disease and facilitate identification of host-immune mechanisms that contribute to the resolution of sars-co v infection. cytomegaloviruses (cmv) comprise a distinct and ancient group of herpesviruses that are widely distributed in nature, share similar growth characteristics in cell culture, and cause cellular enlargement and form distinctive inclusions in infected cells. these cytopathic changes, identified by early pathologists in the salivary glands of children dying from various unrelated diseases,111-113 led to the early designation of cytomegalic inclusion disease many years before the causative agent was isolated in the mid-1950s. the name cytomegalovirus was proposed in 1960 to reflect the cytopathic changes caused by these viruses. 114 cytomegaloviruses are highly host-specific, and various mammalian hosts, including nonhuman primates, rodents, and domesticated animals, are infected with their own distinct cmy. in this context, human cmv is stringently species-specific and, with rare exception, only infects cells of human origin. ll5 several cell types are permissive for cmv replication, including alveolar pneumocytes, vascular endothelium, fibroblasts, monocytes, dendritic cells, and exocrine and endocrine glandular epithelial cells.ll6 cytomegalovirus is a ~-herpesvirus with the largest genome (230 kilobase pair [kbp]) of all the herpesviruses known to infect humans. the double-stranded linear dna genome is contained within a 90 to 100nm icosahedral capsid and is surrounded by an amorphous material known as the tegument. these components are enclosed in a lipid bilayer envelope that is derived from the host cell nuclear or golgi membranes and contains several vir ally encoded glycoproteins necessary for infection of other cells. mature enveloped virions range from 150 to 200nm, making cmv one of the largest viruses that infect humans (fig. i1.1d ).ll7 the structure of cmv is typical of other human herpesviruses, but demonstrates some subtle ultrastructural differences from other viruses in this group including greater pleomorphism of the lipid envelope and dense body inclusions in the cytoplasm of infected cells.ll8 437 cytomegalovirus is a ubiquitous human pathogen, and in north america infects approximately 50% to 90% of the population.ll7 most of these infections are inapparent, although some cases of primary infection in otherwise healthy individuals result in a self-limited mononucleosis syndrome similar to that caused by epstein-barr virus; it is estimated that 20% to 50% of cases of heterophile-negative mononucleosis, and 8% of all cases of mononucleosis, are caused by cmy' 119 pulmonary involvement in cmv mononucleosis is infrequent and occurs in approximately 6% of these casesyo congenitally acquired cmv infection has various deleterious effects on the fetus, including mental retardation, neurologic abnormalities, sensorineural hearing loss, and retinitis, and in one series pulmonary involvement occurred in 64 % of symptomatic infants. 121 like all herpesviruses, cmv remains with its host for life after primary infection and establishes latency in various cell types, including vascular endothelial cells, monocytes and macrophages, neutrophils, and renal and pulmonary epithelial cells. 122 activation of viral replication occurs in persons with severely compromised immunity. patients with advanced hiv disease and recipients of hematopoietic stem cell or lung transplants are particularly at risk of developing cmv pneumonia. before the use of cmv screening and effective antiviral prophylaxis regimens, 10% to 30% of all patients undergoing allogeneic bone marrow transplantation for leukemia, and 15% to 55% of solid organ transplants, developed cmv pneumonia with case fatality rates of greater than 80% in some series. 26 ,123,124 the relatively high frequency of cmv pneumonia in lung transplant recipients may be a correlate of animal model data that indicate the lungs are a major site of latent cmv infection. 125 before the use of ganciclovir as therapy for cmv disease in aids patients, the case fatality rate for cmv pneumonia in this patient cohort was 75% when cmv was the only pathogen identified. mixed infections with cmv and another pathogen had an even worse prognosis, and the case fatality rate for patients with pulmonary disease caused by cmv and pneumocystis jiroveci (formerly carinii) was 92%. 126 cytomegalovirus pneumonia can show various histopathologic patterns (fig. 11 .5). extensive intra alveolar hemorrhage with scattered cytomegalic cells and relatively few inflammatory cell infiltrates may occur ( fig. 11 .5a).127 in a similar manner, extensive involvement of the alveolar epithelium with minimal inflammation or overt evidence of parenchymal injury has also been described. 128 other patterns include multifocal lesions with mixed inflammatory cell infiltrates, hemorrhage, necrosis, and cytomegalic cells, or a diffuse, predominantly mononuclear cell infiltrate, interstitial pneumonitis with intraalveolar edema and fibrin deposition, and diffusely distributed cytomegalic cells (fig. 11 .5e).129-131 the cytomegalic changes of cmv-infected cells are evident by standard h&e staining and are virtually pathognomonic of active cmv infection. the cells are enlarged (25 to 40!j,m) and contain amphophilic to deeply basophilic intranuclear and intracytoplasmic inclusions ( fig. 11 .5b,e). the single intranuclear inclusion is composed of viral nucleoprotein and assembled caps ids, and is a large (up to 20 !j,m), round to ovoid body with a smoothly contoured border that is generally surrounded by a clear halo that gives the inclusion a distinctive owl'seye appearance. the host cell nucleolus is often retained in the inclusion. 131 cytoplasmic inclusions are small (1 to 3!j,m), granular bodies that appear after the intranuclear inclusion is well developed and are not uniformly present in all cmv-infected cells (fig. 11 .5b). these inclusions represent a mixture of virions and various cellular organelles, and increase in size and number as the infection progresses. 132 unlike the intranuclear inclusion, the cytoplasmic inclusions stain with periodic acid-schiff stain and are deeply argyrophilic with gomori's methenamine silver stain.!33 cytomegalovirus pneumonia is defined by the presence of signs or symptoms of pulmonary disease combined with the detection of cmv in bronchoalveolar lavage (bal) fluid or lung tissue samples. in this context, detection methods that support this definition include virus isolation, histopathologic observation of cytomegalic cells, ish, or ihc stains (fig. 11 .5f). detection by pcr alone is considered too sensitive for the diagnosis of cmv pneumonia and is insufficient for this purpose. 134 cytomegalovirus is most often cultured in human diploid fibroblasts such as human embryonic lung and human foreskin fibroblasts. it grows slowly in conventional cell culture, and the cytopathic effect is generally not detected until the second week or longer after inoculation. for this reason, a shell vial method using centrifugation to enhance infectivity has become the standard isolation technique and can usually yield diagnostic results within 48 hours. 135 for centuries, the term herpes, derived from the greek erpein (to crawl), was used in medicine to describe any spreading cutaneous lesion. by the end of the 19th century, investigators surmised that the herpetic lesions of the lips and genitalia were manifestations of a single infectious agent, and recognized that it was a disease distinct from herpes zoster. 136.137 as with all human herpesviruses, hsv is a large, enveloped, double-stranded dna-containing virion with an icoshedral nucleocapsid approximately 100 to 1l0nm in diameter, composed of 162 cap somers. the nucleocapsid is surrounded by an amorphous, sometimes asymmetric material (the tegument) that is surrounded by a thin, trilaminar envelope that contains numerous glycoprotein spikes (fig. 11.1c ). the assembly of hsv begins in the nucleus of its host cell and the virus acquires its envelope as the capsid buds through the inner lamella of the nuclear membrane.120.l38 two serologic types are recognized and each is most frequently associated with particular disease syndromes; however, either serotype may cause any of the aggregate clinical syndromes. herpes simplex virus-1 causes gingivostomatitis, pharyngitis, esophagitis, keratoconjunctivitis, and encephalitis, and is the serotype most commonly associated with adult hsv pneumonia. herpes simplex virus-2 typically infects genital sites such as the penis, urethra, vulva, vagina, and cervix, and is the serotype associated with approximately 80% of disseminated disease and pulmonary infections in newborn infants. 136 all herpesviruses have the ability to persist in an inactive state for varying periods of time and then recur spontaneously following undefined stimuli associated with physical or emotional stress, trauma to nerve roots or ganglia, fever, immunosuppression, or exposure to ultraviolet radiation. 138 during the primary infection, hsv replicates at the portal of entry (typically oral or genital mucosae), and infects sensory nerve endings. the virus is transported centripetally along peripheral sensory nerves to central axons and finally to nerve cell bodies in the trigeminal, sacral, and vagal ganglia, where it replicates briefly before becoming latent.139-141 antiviral drugs have no effect on latent infection with hsv. following cues that initiate viral reactivation, hsv replicates in sensory ganglia and is transported centrifugally along sensory nerves to epithelial cells on mucosal surfaces. 138 reactivation of hsv from the trigeminal ganglion is associated with asymptomatic excretion of virus in saliva, and with the development of herpetic ulcers on the vermillion border of the lip, oral mucosa, or external facial skin. 142 newborn infants, severely immunosuppressed or burned patients, and patients with severe trauma are at greatest risk of developing hsv pneumonia.143-146 lower respiratory tract disease in neonates is most commonly associated with disseminated herpetic infections. disseminated hsv infection in the newborn was first described in 1935 as "hepatoadrenal necrosis"147 because of the prominent and frequent necroses that occur in the livers and adrenal glands of affected neonates. 148 most cases of neonatal disease represent primary hsv infections and are acquired during parturition from hsvinfected mothers. the incidence of neonatal hsv infection is approximately 1 in 3200 deliveries, and disseminated disease develops in approximately 25% of infected neonates. 149 in disseminated infections, signs and symptoms appear a mean of 5 days after birth (range, 0 to 12 days), and approximately 40% to 50% of these patients develop pneumonia. in the pre antiviral era, 85 % of neonates with disseminated disease died from the infection. with early diagnosis and high-dose acyclovir therapy, mortality has been reduced to approximately 30%?6,138,149 the disease can be exceedingly difficult to diagnose in a timely manner as only 10% of mothers of affected infants have clinically apparent hsv infection at the time of delivery.15o neonates that survive severe disseminated disease may develop hepatic and adrenal calcifications evident on abdominal radiographs. 151 in adults, infection of the respiratory tract with hsv may be associated with disseminated herpetic infection, but is more commonly identified as an isolated disease manifestation resulting from reactivation of latent herpetic infections in the oropharynx. herpetic tracheobronchitis is an ulcerative process characterized by large areas of denuded mucosal epithelium and fibrinopurulent exudate containing necrotic cells with densely eosinophilic cytoplasm. despite extensive tissue damage, cells with intranuclear inclusions may be sparse, and, when identified, are found most often at the margins of the ulcerated epithelium or occasionally in the mucous glands subjacent to ulcerated surfaces. ls2 aspiration of viruscontaining secretions into the lower respiratory tract is believed to be the most frequent cause of pulmonary infection with hsv; however, oral lesions may be absent in patients with herpetic laryngotracheobronchitis and bronchopneumonia. 153 disease can be also associated with airway trauma caused by tracheal intubation or from hematogenous dissemination of hsv. 144 ,1s2,1s4 chest radiographs of hsv pneumonia generally show ill-defined nodular or reticular densities of various sizes scattered in both lung fields. during the early stages of disease, these nodules measure 2 to 5 mm and are best seen in the periphery of the lungs. as the disease progresses, these lesions coalesce and enlarge to form more extensive infiltrates. 137 herpetic infections of the airways and lung are characteristically difficult to diagnose clinically and hsv pneumonia was not described as a distinct clinical entity until 1949.155 several studies attest to the relative infrequency with which this diagnosis is considered in patients with respiratory disease. for example, none of the 15 cases of hsv disease of the middle and lower respiratory tract identified in a review of autopsies at brooke army medical center during 1965 to 1968 were suspected prior to autopsy. 143 in a 1982 review of 20 culture-confirmed cases of hsv pneumonia, none of the patients had been diagnosed prior to death and all 16 had oral herpetic lesions at the time of death.144 recent investigations also suggest that lower respiratory tract disease caused by hsv may be more common than currently appreciated. in a study from sweden, hsv was cultured from proa diagnosis of tracheobronchitis and pneumonia is best established histologically (fig. 11.6 ). because lower respiratory tract hsv infections are often focused in the tracheobronchial tree, open lung biopsy may be less sensitive than bronchoscopy.154 herpetic lesions show extensive necrosis and karyorrhectic debris and are associated with hemorrhage and a sparse-to-moderate neutrophilic infiltrate (fig. 11.6a,b) . intranuclear inclusions are best appreciated in cells at the leading edge of necrotic foci ( fig. 11 .6b,c). inclusions appear either as homogeneous, amphophilic, and glassy (cowdry type b inclusions), or as eosinophilic with a halo separating the inclusion from the nuclear membrane (i.e., cow dry type a inclusions). 158 cowdry type b inclusions contain actively replicating virus. type a inclusions, considered noninfectious and devoid of viral nucleic acid or protein, represent the nuclear "scar" of hsv infection. 136 ,141 other changes associated with hsv, including multi nucleation and nuclear molding, and ballooning degeneration of the cytoplasm, are more frequently associated with squamous epithelium and are seldom encountered in the lung. because of the high frequency of hepatic and adrenal involvement with disseminated hsv infection in young children ( fig. 11 .6f,g), liver biopsy has been suggested as a diagnostic technique in this patient cohort. 148 commercially available antibodies exist for ihc detection of hsv in tissues (fig. 11.6d ).ls9 virus isolation remains an important diagnostic method; however, because hsv can be isolated from oropharyngeal secretions and occasionally from the lower respiratory tract of patients who lack overt pulmonary disease, virologic cultures must be interpreted in the context of complementary clinical, radiographic, and histopathologic findings as much as possible. cell culture systems susceptible to hsv include vero cells and foreskin fibroblasts. cytopathic effects generally develop within 24 to 48 hours after cultures are inoculated with infectious specimens. suitable specimens include scrapings made from mucocutaneous lesions, tracheobronchial aspirates, or bal specimens. in infants with evidence of hepatitis, it may also be useful to obtain duodenal aspirates for hsv isolation. 138 polymerase chain reaction methods that amplify hsv dna from clinical specimens, including tissue and blood, can be particularly useful by specifically distinguishing between hsv-l or hsv-2 infections.138,149 varicella-zoster virus (vzv), also known as human herpesvirus 3 (hhv-3) , is a human a-herpesvirus most closely related to hsv. it has a linear, double-stranded dna genome with approximately 125 kbp that encodes more than 70 proteins. the icosahedral nucleocapsid is indistinguishable in appearance from other herpes viruses. the nucleocapsid and the tegument are surrounded by a lipoprotein envelope derived from the host cytoplasmic membranes. the enveloped viral particle is pleomorphic to spherical in shape and 180 to 200nm in diameter. 16o the primary infection is initiated by inoculation of respiratory mucosa by the virus through infectious aerosols or by direct contact of skin lesions of patients with varicella or herpes zoster. after a primary viremia in the reticuloendothelial system, and secondary viremia in circulating mononuclear cells, the virus is disseminated to the skin, where it initiates a vesicular rash, and back to mucosal sites in the lungs. the release of infectious virus into respiratory droplets is a pathogenic characteristic that distinguishes vzv from other human herpesviruses. the attack rate for previously uninfected household contacts exposed to varicella is approximately 90%. for less sustained exposure, it is estimated to be approximately 10% to 30%. 160 during primary infection with vzv, viral replication in keratinocytes (fig. 11.7 a) and vascular and lymphatic endothelial cells of the superficial dermis produces the generalized, pruritic, vesicular rash of varicella commonly known as chickenpox. varicella-zoster virus also establishes latent infection within satellite cells and neurons of the trigeminal and dorsal root ganglia and can reactivate under various conditions to cause herpes zoster, a painful vesicular rash commonly referred to as shingles. 161 the origin of the term chickenpox is speculative, but believed to derive from gican, an old english term for itching. the term shingles originates from the medieval latin cinguls, or girdle, and alludes to the partial encircling of the trunk by the rash of herpes zoster. 137 ,160 varicella-zoster virus is ubiquitous in human populations around the world, and humans are the only known host. during the prevaccine era in the u.s., approximately 4 million cases, 4000 to 9000 hospitalizations, and 50 to 140 deaths were reported annually.25.162 the risk of severe illness during primary or recurrent vzv infection appears to depend more on host factors rather than a particular viral strain. chickenpox is considered a relatively benign infection in children, but adult patients are approximately 25 times more likely than children to develop pneumonia. the greatest risk of severe disease and pneumonia occurs in those patients with chronic lung disease, immunesuppressing conditions, neonates, and pregnant women. 163 varicella-zoster virus-related deaths have declined sharply in the u.s. since universal childhood vaccination was implemented in 1995. 24 ,25 s.r. zaki and cd. paddock varicella pneumonia was first described in the medical literature in 1942 164 and is the most frequently reported complication of chickenpox in adult patients. 24 ,165 pneumonia occurs in approximately 10% to 15% of adults infected with vzv. 166 ,167 the occurrence of pneumonia during herpes zoster is rare, and limited primarily to profoundly immunosuppressed patients, particularly bone marrow transplant recipients. m varicella-zoster virus pneumonia generally develops within 2 to 7 days following the onset of rash and may be characterized by fever, cough, tachypnea, chest pain, and hemoptysis. 26 .168 hypoxemia is common and may be severe. radiographically, the lungs show multiple, scattered, defined, nodular densities. untreated adult varicella pneumonia is fatal in approximately 10% of cases, but mortality is as high as 25% to 40% in certain high-risk cohorts, including pregnant women, transplant recipients, and neonates. 137 ,lfj9-171 massive pulmonary hemorrhage is a frequent terminal event. gross examination reveals lungs that are generally two to three times heavier than normal, firm, and plumcolored. there are often multiple necrotic and hemorrhagic lesions on the visceral pleura that resemble the pox lesions of skin. 169 ,172,173 pox may also be seen on the parietal pleura, although pleural effusions are uncommon and rarely prominent. 163 ,173 the trachea and bronchi are generally edematous and erythematous with occasional vesicles on the mucosal surfaces, and there may be lobular consolidation of the lungs. microscopically, pulmonary involvement consists primarily of interstitial pneumonitis and diffuse miliary foci of necrosis and hemorrhage in the pulmonary parenchyma that involve alveolar walls, blood vessels, and bronchioles ( fig. 11.7b,d) . 172 other findings may include intraalveolar collections of edema, fibrin, or hemorrhage, diffuse alveolar damage, and septal edema. 1m ,168.173 virally infected cells with intranuclear inclusions may be identified in respiratory epithelial cells, pneumocytes, interstitial fibroblasts, or capillary endothelium ( fig. 11 .7e,f).167 eosinophilic intranuclear inclusions and multinucleated syncytial cells may be difficult to locate but are best identified at the edges of necrotic foci (fig. 11.7c ). in cases of disseminated disease, similar necrotizing hemorrhagic lesions and occasional viral cytopathic changes in epithelial cells or fibroblasts may be observed in other tissues and organs, including esophagus, pancreas, liver, renal pelves, ureters, urinary bladder, spleen, bone marrow, thymus, lymph nodes, adrenal glands, and brain. 172,174 in those patients who recover from severe vzv pneumonia, some necrotic parenchymal foci may mineralize, and can be identified by chest radiograph years later as miliary, 1 to 5 mm, nodular opacities. microscopically, the lesions are characterized as discrete collections of dense fibrous connective tissue that surround multiple, small, calcified bodies. the periphery may include a cellular zone of fibroblasts and occasional giant cells.175 a radiographic survey of 16,894 persons identified pulmonary calcifications in eight (1.7%) of 463 patients who had chickenpox during adulthood compared with only eight (0.05%) of the remaining 16,431 who did not report having varicella as an adult. 176 a positive history of varicella predicts immunity in >95% of persons. 160 because pulmonary symptoms most often occur several days following the onset of the characteristic rash of varicella, a pathologic diagnosis is seldom required for a real-time diagnosis of vzv pneumonia. however, hematopoietic cell transplant recipients may present with signs of visceral dissemination and pneumonia 1 to 4 days before the localized cutaneous eruption of herpes zoster appears, and lower respiratory tract disease has been described in the absence of skin lesions, particularly in neonates and bone marrow transplant recipients. 26 ,137,177 commercially available antigen detection kits can be used for rapid diagnosis of cutaneous vzv infection. epithelial cells are scraped from the base of a newly formed vesicle, applied to a slide, and stained by using fluorescein-conjugated vzv monoclonal antibodies to detect specific viral proteins in the specimen. in a similar manner, the tzanck test uses wright-giemsa stain to demonstrate multinucleated giant cells in these specimens; however, this test does not differentiate between hsv and vzv, and false-negative results are common. commercially available antibodies are also available for ihc detection of vzv in tissue specimens ( fig. 11 .7e,f); however, relatively few laboratories are able to provide well-validated assays. some commercial laboratories offer pcr amplification to detect viral nucleic acid in clinical specimens. isolation of the virus in cell culture remains the reference standard for the diagnosis of vzv. in human melanoma cells, an excellent substrate for vzv isolation, the average time for visible cytopathic effect from the virus is 3 to 5 days.178 infectious vzv is usually recoverable from the clear fluid of cutaneous vesicles of varicella for approximately 3 days after the appearance of these lesions and for approximately 1 week from herpes zoster lesions. the lungs are the most common organ from which vzv is isolated at autopsy, but isolates have also been obtained from heart, liver, pancreas, gastrointestinal tract, brain, and eyes. 160 influenza is derived from the term influentia, meaning epidemic in the italian form of latin, originally used because epidemics were thought to result from astrologic or other occult influences. influenza is a highly contagious, acute respiratory illness with a spectrum of clinical illness ranging from asymptomatic or mild disease with rhinitis or pharyngitis to primary viral pneumonia with s.r. zaki and cd. paddock fatal outcome. influenza may also be associated with a broad range of other disorders affecting the heart, brain, kidneys, and muscle. influenzaviruses belong to the orthomyxoviridae family, which consists of four genera that include the two important influenza viruses types a and b associated with significant human disease. 179 ,180 influenza a viruses are further classified into subtypes based on the antigenicity of their hemagglutinin (ha) and neuraminidase (na) surface glycoproteins. only one type of ha and one type of na are recognized for influenza b. influenza a occurs in both pandemic and interpandemic forms. fortunately, pandemics, defined as worldwide outbreaks of severe disease, occur infrequently. interpandemic influenza, although less extensive in its impact, occurs virtually every year. the epidemiologic pattern of influenza in humans is related to two types of antigenic variation of its envelope glycoproteins, namely antigenic drift and antigenic shift. during antigenic drift, new strains related to those circulating in previous epidemics evolve by accumulation of point mutations in the surface glycoproteins. this enables the virus to evade the immune system leading to repeated outbreaks during interpandemic years. antigenic shift occurs with the emergence of a "new" potentially pandemic, influenza a virus that possesses a novel ha alone or in combination with a novel na. there are 16 recognized ha subtypes and nine na subtypes of influenza a virus. viruses from all ha and na subtypes have been recovered from aquatic birds, but only three ha subtypes (hi, h2, and h3) and two na subtypes (nl and n2) have established stable lineages in the human population since 1918. since 1997, widespread avian infection with influenza a (h5n1) and associated clusters of human disease have aroused concern about the threat of a pandemic, and attention has been appropriately focused on control measures to deal with such an event. all influenzaviruses have a segmented, negative-sense rna core surrounded by a lipid envelope. influenzavirus particles are pleomorphic. among isolates that have undergone a limited number of passages in cell culture or eggs, more filamentous than spherical particles are seen. spherical morphology becomes dominant when the virus is extensively passaged in the laboratory. a 10-to 12-nm layer of ha (rod-shaped) and na (mushroomshaped) spikes project radially on the surface of the influenza a and b viruses. hemagglutinin facilitates entry of virus into host cells through its attachment to sialic-acid receptors. because neutralizing antibodies are directed against this antigen, it is a critical component of current influenza vaccines. neuraminidase, the second major antigenic determinant, catalyzes the cleavage of glycosidic linkages to sialic acid and the release of progeny virions from infected cells. accordingly, it has become an important target for drug inhibitors such as oseltamivir and zanamivir. the m2 surface component and channel of influenza a (not present in influenza b virus) regulates the internal ph of the virus and is blocked by the antiviral drug amantadine. influenzaviruses are spread person-to-person primarily through the coughing and sneezing of infected persons. the typical incubation period for influenza is 1 to 4 days, with an average of 2 days. adults can be infectious from the day before symptoms begin through approximately 5 days after illness onset. children can be infectious for ~1o days, and young children can shed virus for several days before their illness onset. severely immunocompromised persons can shed virus for weeks or months. uncomplicated influenza illness is characterized by the abrupt onset of constitutional and respiratory signs and symptoms (e.g., fever, myalgia, headache, malaise, nonproductive cough, sore throat, and rhinitis). among children, otitis media,nausea,and vomiting are also commonly reported with influenza illness. respiratory illness caused by influenza is difficult to distinguish from illnesses caused by other respiratory pathogens on the basis of symptoms alone. influenza typically resolves after 3 to 7 days in most patients, although cough and malaise can persist for >2 weeks. among certain persons, influenza can exacerbate underlying medical conditions (e.g., pulmonary or cardiac disease), lead to secondary bacterial pneumonia or primary influenza viral pneumonia, or occur as part of a co-infection with other viral or bacterial pathogens. young children with influenza infection can have initial symptoms that mimic bacterial sepsis. more than 20% of children hospitalized with influenza can have febrile seizures. influenza has also been associated with encephalopathy, transverse myelitis, reye syndrome, myositis, myocarditis, and pericarditis. the risks for complications, hospitalizations, and deaths from influenza are higher among persons aged ~65 years, young children, and persons of any age with certain underlying health conditions than among healthy older children and younger adults. influenza-related deaths can result from pneumonia or from exacerbations of cardiopulmonary conditions and other chronic diseases. the histopathologic features of nonfatal and fatal influenza have been well described and include necrotizing bronchitis, thrombosis, interstitial inflammation, hemorrhage, hyaline membrane formation, and intra alveolar edema (figs. 11.8a,b, 11 .9a,c, and 11.10a). 105, [181] [182] [183] [184] [185] [186] [187] [188] [189] [190] [191] the pathology is more prominent in larger bronchi, and inflammation may vary in intensity in individual patients, viral inclusions cannot be identified by light microscopy (fig, 11 .8d), secondary bacterial infections with organisms such as streptococcus pneumoniae (group a streptococcus [gas]), staphylococcus aureus, and haemophilus influenzae may occur as a complication in about 50% to 75% of fatal cases and make it difficult to recognize the pathologic changes associated with the primary viral infec-445 tion ,190,192,193 the histopathologic features in other organs may include myocarditis, cerebral edema, rhabdomyolysis, and hemophagocytosis (figs, 11.8h and 11.9e,f), immunohistochemistry and ish assays demonstrate that viral antigens and nucleic acids are usually sparse and are primarily seen in the bronchioepithelial cells of larger bronchioles (figs. 11.8c,e,f and 11.9d) . 105.189,190 antigens are more readily identified in patients who die within 3 to 4 days of onset of illness. recent studies suggest that unlike human influenza viruses, avian virus h5n1 preferentially infects cells in the lower respiratory tract of humans, resulting in extensive damage of the lungs with minimal pathology in the upper respiratory tract (fig. 11.10a,c) . this may help explain why the h5n1 avian influenza virus is so lethal to humans but so difficult to spread from person to person. these studies show that the avian virus preferentially binds to the a-2,3-galactose receptors, which are found only in and around the alveoli. this is in contrast to the human influenzaviruses that preferentially bind to the a-2,6-receptors, which are found throughout the respiratory tract from the nose to the lungs. 194.195 in birds and other animals, viral antigens can be detected in the lung as well as a variety of extrapulmonary tissues (fig. 11.10b) . the diagnosis of influenza, suspected by history and clinical manifestations, can also be supported histopathologically. however, because of the absence of any characteristic viral inclusions and because the overall pathologic features of influenza may resemble other viral, rickettsial, and certain bacterial infections, an unequivocal diagnosis can be made only by laboratory tests such as viral culture, direct fluorescent antibody and rapid antigen assays, serology, and ihc. 190 ,196,197 measles measles (rubeola) is an infectious, acute febrile viral illness characterized by upper respiratory tract symptoms, fever, and a maculopapular rash. the causative agent, a member of the genus morbillivirus, of the family paramyxoviridae,198,199 is an enveloped virus that contains a negative sense, single-stranded rna genome of 16,000 nucleotides. other human pathogens in this family include parainfluenza, mumps, and respiratory syncytial viruses. measles virions are pleomorphic, generally spherical, enveloped particles from 120 to 250 nm in diameter. the virus is morphologically indistinguishable from other members of the paramyxoviridae family when viewed by negative contrast electron microscopy. a lipid envelope surrounds a helical nucleocapsid composed of rna and protein. two transmembrane glycoproteins, hemagglutinin (h) and fusion (f), are present in the envelope and appear as surface projections. these proteins mediate viral attachment and figure 11.8. influenza a. a. alveolar damage in a patient with fatal influenza a showing prominent congestion with intraalveolar macrophages and fibrin deposits. b. extensive ulceration of respiratory epithelium in a large airway of a patient with fatal influenza a. the lamina propria shows florid vascular congestion and focal hemorrhage, and predominantly mononuclear inflammatory cell infiltrates. c-e. immunohistochemical staining (c,e) of influenza virus a hemagglutinin antigens in the cytoplasm of residual respiratory epithelial cells of a large airway. this same focus of extensively infected respiratory epithelium (d) demonstrates that, unlike many other viral respiratory pathogens, influenza viruses do not elicit specific cytopathic effects in infected cells. f. in-situ hybridization assay demon-• fusion with respiratory epithelium. they are also believed to playa role in virus maturation through their interaction with the matrix (m) protein, which, in turn, is thought to interact with the nucleocapsid structure. 2oo measles is a highly communicable disease of worldwide distribution. before the introduction of measles vaccines, epidemics occurred about every 2 to 5 years when the percentage of nonimmune members of a population reached critical levels. recently, epidemics have occurred in cycles of about 10 years. in small and isolated communities, measles circulation may cease altogether unless it is reintroduced. if introduced in non immune populations, the disease tends to be more severe and may involve more than 90% of the population because of the highly infectious nature of the virus. although still a significant problem in underdeveloped countries, measles infection became uncommon in the us. after the development and widespread use of an effective measles vaccine. however, a recrudescence of measles infection occurred in several large us. urban centers in recent years, associated with reduced use of the vaccine among children and young adults. during the peak of this activity (i.e., between 1989 and 1991), greater than 50,000 measles cases and approximately 150 measlesassociated deaths were reported. 20l measles virus is highly contagious, spread by aerosols and droplets from respiratory secretions of acute cases.202-204 less frequently, contaminated fomites are involved in transmission. a person with acute measles is infective from just before onset of symptoms to defervescence of fever. in developed countries, likely settings for exposure to measles virus are infectious disease clinics, pediatric emergency rooms, and physicians' offices. 20s children are usually infected by 6 years of age, resulting in lifelong immunity, and almost all adults are immune. clinical infection in children younger than 9 months of age is generally uncommon because of passive protection afforded the infant by the transfer of maternal antibodies. however, with the resurgence of measles in the us. came the realization that most women of childbearing age 447 strating active replication of influenza a in respiratory epithelium in a large airway. g. electron micrograph of influenza a particles (arrows) attached to the cilia of a rodent tracheal epithelial cell. viral ribonucleoproteins are evident in the central aspect of the particles. the hemagglutinin and neuraminidase surface glycoproteins make up the peripheral spike layer. (courtesy of f.a. murphy.) h. rhabdomyolysis in a patient with fatal influenza a. bar, 100nm. a,b,d,h, h&e; c,e, immunoalkaline phosphatase stain, naphthol fast-red, and hematoxylin counterstain; f, digoxigenin-iabeled probe followed by immunoalkaline phosphatase staining, naphthol fast-red. and hematoxylin counterstain; g, uranyl acetate, lead citrate stain. acquired immunity to measles through vaccination and not through natural infection. lower levels of maternal antibodies were found to be transferred from an immunized mother to her infant; thus, a substantial number of measles cases occurred in children younger than 1 year of age in the us. in recent years. after an incubation period of about 1 to 2 weeks, the prodromal phase of measles begins with fever, rhinorrhea, cough, and conjunctivitis. koplik's spots, which are small, irregular red spots with a bluish-white speck in the center, appear on the buccal mucosa in 50% to 90% of cases shortly before rash onset. an erythematous maculopapular rash begins on the face 3 to 4 days after prodromal symptoms and usually spreads to the trunk and extremities. the symptoms gradually resolve, with the rash lasting for approximately 6 days, fading in the same order as it appeared. although recovery is rapid and complete in most cases, complications can arise as a result of continued and progressive virus replication, bacterial or viral superinfections, or abnormal host immune response. 204.206.207 the most common complications are secondary bacterial pneumonia and otitis media. 206 other complications include febrile convulsions, encephalitis, liver function abnormalities, chronic diarrhea, and sinusitis. several pulmonary and central nervous system (cns) syndromes that are often fatal have been described. death occurs in about 1 of every 1000 measles cases; however, the risk of death and other complications is substantially increased in infants, adults, malnourished and immunocompromised individuals, persons with underlying illnesses,and nonimmunized populations in underdeveloped countries.208-212 the first step in measles infection is attachment of the virus to cd46 cell surface receptors on the respiratory epithelium.213 adhesion and fusion of the virus to the respiratory epithelium is mediated by both the hand f viral glycoproteins. 214 this stage is followed by local replication in respiratory mucosa and draining lymph nodes. a primary viremia follows, with dissemination two types of multinucleated giant cells have been described in patient tissues during measles infection. 215 ,216 the reticuloendothelial giant cell (warthin-finkeldey) appears first during the incubation period and is seen in 449 the nucleus of a pneumocyte in the lung of a patient with fatal avian influenza. unlike other influenza viruses that cause disease in humans, h5n1 preferentially infects alveolar epithelial cells, and causes relatively minimal pathology in the upper airway. a, h&e; b,c, immunoalkaline phosphatase stain, naphthol fast-red, and hematoxylin counterstain. different lymphoid tissues throughout the body. the second type is the epithelial giant cell, which has been observed in the epithelium of essentially every major organ. the onset of the rash temporally coincides with the appearance of detectable serum antibody to measles virus. interestingly, virus replication and giant cell formation cease with the appearance of rash. t-cell immunity is essential in the process of viral clearance from lymphoid tissue and respiratory tract. while children with congenital agammaglobulinemia respond normally to measles virus infection, patients with cell-mediated immunodeficiency develop severe disease that presents as giant-cell pneumonia or encephalopathy in the absence of an exanthem. [208] [209] [210] [211] 217, 218 immunity to the f surface glycoprotein is necessary to prevent the spread of measles infection. 219 an atypical measles syndrome characterized by pulmonary consolidation with pleural effusions and hilar adenopathy has been reported in children exposed to wild-type measles virus who had previously received the killed-measles virus vaccine.220-222 recipients of the formalin-inactivated vaccine have a good antibody response to the h protein, but antibodies to the functional region of the f protein and to the nucleoprotein are weak or absent. it has been suggested that the lack of a functional f antibody response may playa role in virus spread. 223 the pathologic features of measles have been well described and several references containing detailed morphologic descriptions are recommended. 13 1,209.224-230 the typical morbilliform skin lesions, koplik's spots, and measles lymphadenitis are seldom seen by the surgical pathologist since the clinical diagnosis is usually apparent. histopathologic changes in the skin include mild congestion, edema, and a predominantly mononuclear infiltrate surrounding small vessels of the dermis, as well as other nonspecific features. occasional diagnostic multinucleated epithelial giant cells with eosinophilic cytoplasmic and nuclear inclusions are observed. 228 .231 pathognomonic reticuloendothelial multinucleated giant cells can be observed in appendix specimens from patients mistakenly operated on for acute appendicitis before the emergence of diagnostic koplik's spots and rash. these cells, which have been reported in various lymphoreticular tissues throughout the body, are typically large and contain from a few to occasionally up to 100 nuclei. these cells do not usually contain viral inclusions. the lymphoid tissues are typically hyperplastic, and the architecture is partially or totally obliterated by diffuse proliferation of immunoblasts. [232] [233] [234] a focal or generalized interstitial pneumonitis, similar to that seen in many other viral infections, is seen in the lungs of measles patients. histopathologic features seen include various degrees of peribronchial and interstitial mononuclear cell infiltrates, squamous metaplasia of bronchial endothelium, proliferation of type ii pneumocyte alveolar lining cells, and intraalveolar edema with or without mononuclear cell exudates and hyaline membranes. secondary changes, such as bacterial or viral superinfection, or organizational changes may alter the original pathology. the hallmark of the disease is the formation of multinucleated epithelial giant cells (fig. ll.lla,b) . these cells, which are often numerous, are formed by fusion of bronchiolar or alveolar lining epithelial cells (fig.ll.l1a) . in contrast to the reticuloendothelial giant cells, these cells generally contain characteristic nuclear and cytoplasmic inclusions. the intranuclear inclusions are homogeneous, eosinophilic, and surrounded by a slight indistinct halo (fig. 11.11c,d) . the cytoplasmic inclusions are deeply eosinophilic, vary in size, and some form large masses with a "melted tallow" appearance ( fig. 11.11d ). these giant cells may undergo degenerative changes with progressive loss of cytoplasm, increasing basophilia, and shrinkage of nuclei. the presence of measles virus in these giant cells may be demonstrated by immunofluorescent,235.236 ihc,21o.237 and ish techniques (fig.ll.lle,f) . these giant cells can also be seen in extra pulmonary tissues (fig. 11.11 g,h) . the diagnosis of typical cases of measles can usually be made on the basis of clinical signs and symptoms. other causes of a similar rash, but without other features of measles, include rubella, dengue virus, enteroviruses, and drug reactions, especially to ampicillin. the typical case of measles giant-cell pneumonia generally presents little diagnostic difficulty for the surgical pathologist. the presence of giant cells with both intranuclear and intracytoplasmic inclusions in a setting of interstitial pneumonitis is highly specific for measles infection. however, multinucleated giant cells are not seen in all cases of measles pneumonia and their absence should not exclude the j diagnosis. other viral and rickettsial agents may also cause a similar interstitial pneumonitis, but without the typical giant cells, and should be differentiated. 229 as previously noted, the histopathologic features in measles pneumonia can be somewhat variable,226 and secondary bacterial and viral infections may modify the histology, further complicating the pathologic diagnosis ( fig. 11.5d ).207.229 other viral pathogens, such as respiratory syncytial virus,238 parainfluenza,239.240 vzv,241 and a recently discovered hendra virus,242 as well as granulomatous diseases of the lung, may give rise to pneumonia with giant cells and should also be considered in the differential diagnosis. however, these clinical entities can be distinguished by history, histopathologic features, and laboratory tests. immunohistochemistry237.243,244 or ish 243 ,244 tests demonstrate viral antigens or nucleic acids in the majority of cases. laboratory confirmation is useful to avoid possible confusion with other rash-causing illnesses. diagnostic laboratory procedures consist of direct detection of either the virus or the viral antigens, usually by indirect immunofluorescence or by serologic methods using hemagglutination inhibition, neutralization, or enzyme immunoassay. specimens for serologic testing consist of acute-and convalescent-phase serum pairs. antibody appears within 1 to 2 days after onset of rash, and titers peak approximately 2 weeks later. alternatively, the presence of specific immunoglobulin m (igm) antibody can be used to diagnose recent infection. 245 human parainfluenza viruses (hpivs) are second only to rsv as a cause of lower respiratory tract disease in young children. human parainfluenza viruses are negative-sense, nonsegmented, single-stranded, enveloped rna viruses that possess fusion and hemagglutinin-neuraminidase glycoprotein "spikes" on their surface (fig. 11 .ig). the four serotypes of hpiv belong in the family paramyxoviridae, subfamily paramyxovirinae, and genera respirovirus (hpiv-l and -3) and rubulavirus (hpiv-2 and -4). the virions are variable in shape and size, ranging from 150 to 300nm. 246 human parainfluenza viruses are spread from respiratory secretions through close contact with infected persons or contact with contaminated surfaces or objects. infection can occur when infectious material contacts mucous membranes of the eyes, mouth, or nose, and possibly through the inhalation of droplets generated by a sneeze or cough. human parainfluenza viruses are unstable in the environment (surviving a few hours on environmental surfaces), and are readily inactivated with soap and water. they are ubiquitous, and infect most s,r. zaki and cd. paddock people during childhood. the highest rates of serious hpiv illnesses occur among young children. serologic surveys have shown that 90% to 100% of children aged 5 years and older have antibodies to hpiv-3, and about 75% have antibodies to hpiv-l and -2. the different hpiv serotypes differ in their seasonality, with hpiv-l causing biennial outbreaks of croup in the fall and hpiv-2 causing annual or biennial fall outbreaks. human parainfluenza virus-3 peak activity occurs during the spring and early summer months each year, but the virus can be isolated throughout the year. similar to rsv, hpivs can cause repeated infections throughout life, usually manifested by an upper respiratory tract illness (e.g., cold and sore throat). human parainfluenza viruses can also cause serious lower respiratory tract disease with repeat infection (e.g., pneumonia, bronchitis, and bronchiolitis), especially among the elderly, and among patients with compromised immune systems. each of the four hpivs has different clinical and epidemiologic features. the most distinctive clinical feature of hpiv-l and hpiv-2 is croup (i.e., laryngotracheobronchit is ); hpiv-l is the leading cause of croup in children, whereas hpiv-2 is less frequently detected. both hpiv-l and -2 can cause other upper and lower respiratory tract illnesses. human parainfluenza virus-3 is more often associated with bronchiolitis and pneumonia. human parainfluenza virus-4 is infrequently detected, possibly because it is less likely to cause severe disease. the incubation period for hpivs is generally from 1 to 7 days. 247 most hpiv infections cause a mild, self-limited illness; however, hpiv-3 infections are an important cause of bronchiolitis, croup, and pneumonia that may be lifethreatening in infants and newborns. 247 human parainfluenza virus infections are also increasingly being recognized as an important cause of severe morbidity and mortality in immunocompromised patients. 20 ,248--252 the mortality of bone marrow transplant patients with hpiv-3 infection has been reported to be as high as 60%. 249, 253, 254 in patients with severe hpiv infection, multinucleated giant cells derived from the respiratory epithelium may be seen in association with an interstitial pneumonitis and organizing changes (fig. 11.12a,b ) . 239,240.249,255-260 these giant cells, which may contain intracytoplasmic eosinophilic inclusions, (fig. 11.12c) , have also been reported in extrapulmonary tissues such as kidney, bladder, and pancreas. 258 other viral causes of giant cell pneumonia, including measks, rsv, vzv, and hsv, should be considered in the histopathologic differential and laboratory testing, including ihe, can be useful in making this differentiation possible ( fig. 11.12d) . diagnosis of infection with hpiv scan also be made by virus isolation, direct detection of viral antigens py enzyme-linked immunoassay (eia) or immunofluorescent assay (ifa) in clinical specimens, detection of viral rna by rt-pcr, demonstration of a rise in respiratory syncytial virus (rsv) is the most common cause of bronchiolitis and pneumonia among infants and children under 1 year of age. the causative agent is a negative-sense, nonsegmented, single-stranded, enveloped rna virus. the virion is variable in shape and size and ranges from 120 to 300 nm. respiratory syncytial virus is a member of the family paramyxoviridae, subfamily pneumovirinae, in the genus pneumovirus, and can be 453 ing poorly defined cytoplasmic eosinophilic inclusions. d. parainfluenza virus antigens in giant cells localized by using immunohistochemistry. a-c, h&e; d, immunoalkaline phosphatase staining, naphthol fast-red, and hematoxylin counterstain. further distinguished genetically and antigenically into two subgroups, a and b. the subgroup a strains are usually associated with more severe infections. two surface glycoproteins, g and f, are present in the envelope and mediate attachment and fusion with respiratory epithelium. the f protein also mediates coalescence of neighboring cells to form the characteristic multinucleated syncytial giant cells for which the virus name is derived. 265 respiratory syncytial virus is spread from respiratory secretions through close contact with infected persons or contact with contaminated surfaces or objects?66 infection can occur when infectious material contacts mucous membranes of the eyes, mouth, or nose, and possibly through the inhalation of droplets generated by a sneeze or cough. respiratory syncytial virus is unstable in the environment, surviving a few hours on environmental surfaces, and is readily inactivated with soap and water. in temperate climates, rsv infections usually occur during annual community outbreaks, often lasting several months, during the late fall, winter, or early spring months. the timing and severity of outbreaks in a community vary from year to year. respiratory syncytial virus spreads efficiently among children during the annual outbreaks, and most children will have serologic evidence of rsv infection by 2 years of age. illness begins most frequently with fever, runny nose, cough, and sometimes wheezing. during their first rsv infection, between 25% and 40% of infants and young children have signs or symptoms of bronchiolitis or pneumonia, and 0.5% to 2% require hospitalization. most children recover from illness in 8 to 15 days. the majority of children hospitalized for rsv infection are under 6 months of age. respiratory syncytial virus also causes repeated infections throughout life, usually associated with moderate-to-severe cold-like symptoms; however, severe lower respiratory tract disease may occur at any age, especially among the elderly or among those with compromised cardiac, pulmonary, or immune systems. the major histopathologic changes described in fatal rsv infections include necrotizing bronchiolitis and interstitial pneumonia (fig. 11.13a,b) .238,267-272 bronchiallumina and airways are usually filled with necrotic debris and inflammatory cells. these findings may be accompanied by various degrees of diffuse alveolar damage (fig. 11.13e ), organizational changes, and secondary bacterial superinfection. giant cell pneumonia is a feature seen in some cases ( fig. 11.13c,d) . the multinucleated giant cells contain irregular, intracytoplasmic, eosinophilic inclusions surrounded by a clear halo. these inclusions are extremely difficult to identify with any degree of certainty and are only seen in about half the cases (fig. 11.13d) . other viral causes of giant cell pneumonia should be considered in the histopathologic differential and laboratory testing, including ihc,271,273,274 can be useful in making this differentiation possible ( fig. 11.13b,f) . diagnosis of rsv infection can also be made by virus isolation, direct detection of viral antigens in clinical specimens by eia or ifa, detection of viral rna by rt-pcr, demonstration of a rise in rsvspecific serum antibodies, or a combination of these approaches (see also fig. 7.43, chapter 7) .264,275-281 in 2001, van den hoogen et a1. 282 described the identification of this new viral agent from clinical specimens obtained from patients with respiratory illness, which 455 they designated human metapneumovirus (hmpv). it is a negative-sense, nonsegmented, single-stranded, enveloped rna virus. the virion is variable in shape and size, ranging from 150 to 300nm (fig. 11.1f ). it has been categorized in the family paramyxoviridae, subfamily pneumovirinae, genus metapneumovirus, based on genomic sequence and gene constellation. human metapneumovirus can be further distinguished genetically and antigenically into two subgroups, a and b. similar to rsv, hmpv infection is ubiquitous and occurs during infancy and early childhood, with annual epidemic peaks occurring in the winter and spring months in temperate regions. seroprevalence studies reveal that 25% of all children aged 6 to 12 months have antibodies to hmpv; by age 5 years, 100% of patients have evidence of past infection. like rsv, hmpv has been associated with a wide spectrum of respiratory illnesses.283-287 the patient may be asymptomatic, or symptoms may range from mild upper respiratory tract illness to severe bronchiolitis and pneumonia. although rsv, hpiv-l, and hpiv-3 have been definitively linked to cases of lower respiratory tract disease in infants and young children, the relative contribution of hmpv remains undetermined. like rsv and the hpivs, studies suggest that hmpv may also contribute to respiratory disease in elderly adults and the immunocompromised. 288-29o histopathologic descriptions of features of hmpv infections are few and have not been well described. [291] [292] [293] [294] this is partly related to interpreting the clinical significance of virus detection in context with the ubiquitous nature of the virus. virus detection in such cases is usually made by culture isolation of the virus from upper airways or by pcr assays performed on nasopharyngeal aspirates or bal washings. in nonhuman primates viral antigens are observed in ciliated epithelial cells, type 1 pneumocytes, and alveolar macrophages. this distribution is associated with mild, multifocal, erosive, and inflammatory changes in airways, and an increased number of foamy macrophages in alveoli. 291 the bal specimens collected from patients within a few days of a positive hmpv assay show degenerative changes and cytoplasmic inclusions within epithelial cells, multinucleated giant cells, and histiocytes. the intracytoplasmic inclusions are ill-defined, eosinophilic structures that measure 3 to 4 ~m. 293 lung biopsy or autopsy tissue obtained and examined later in the disease show chronic airway inflammation, intraalveolar foamy and hemosiderin-laden macrophages, and acute and organizing lung injury including areas of diffuse alveolar damage with hyaline membrane formation and foci of a bronchiolitis obliterans/organizing pneumonia like reaction ( fig. 11 .14). in such cases, typical multinucleated giant cells or viral inclusion cannot be identified. 292 -294 in-situ hybridization studies on limited number of human cases human metapneumovirus is difficult to identify with commonly used viral diagnostic procedures. the virus replicates slowly in primary and tertiary monkey kidney cell lines, and the cytopathic effect can be difficult to discern. commercial monoclonal antibody reagents to hmpv are not widely available. most hmpv studies have been conducted using rt-pcr assays or by demonstration of a rise in hmpv-specific serum antibodies. two novel paramyxoviruses, hendra and nipah, have been recently identified in australia and malaysia; these viruses have been associated with acute febrile encephalitis and respiratory tract disease. both infections are zoonotic. hendra was first identified in 1994 when patients who came in close contact with sick horses developed an influenza-like illness. two patients died with pneumonitis and multiorgan failure. 30 the closely related nipah virus was identified during an outbreak in malaysia and singapore during 1998-1999 that included more than 250 patients. patients presented with a severe acute encephalitic syndrome, but some also had significant pulmonary manifestations. 22 ,295-302 most of the patients had a history of contact with pigs, most of them being pig farmers. in bangladesh in 2001 and 2003, outbreaks of nipah encephalitis occurred. 303 ,304 similar to the malaysian outbreak, the most prominent symptoms were fever, headache, vomiting, and an altered level of consciousness. respiratory illness was much more common in the bangladesh cases, however, with 64 % having cough and dyspnea. the reason for increased involvement of the respiratory tract in this outbreak is not known. epidemiologic and laboratory investigations identified fruit bats of the pteropus genus as asymptomatic carriers of hendra and nipah viruses and possible animal reservoirs. [305] [306] [307] [308] [309] hendra and nipah viruses belong to the recently designated genus henipavirus within the family paramyxoviridae, subfamily paramyxovirinae. both viruses are nonsegmented, negative-stranded rna viruses composed of helical nucleocapsids enclosed within an envelope to form roughly spherical, pleomorphic virus particles.3io-312 the structure of their genome is consistent with the other members of the subfamily. 30 histopathologic findings in fatal cases of hendra and nipah infections are similar with varying degrees of cns and respiratory tract involvement. 104 ,313-315 findings include a systemic vasculitis with extensive thrombosis, endothelial cell damage, necrosis, and syncytial giant cell formation in affected vessels ( fig. 11.15a ,b,f). plaques with various degrees of necrosis, in association with inclusion-bearing neurons, can be found in both the gray and white matter of the cns (fig. 11.15e ). multinucleated giant cells with intranuclear inclusions can occasionally be seen in lung, spleen, lymph nodes, and kidneys ( fig. 11 .15c,g). in the lung, vasculitis and fibrinoid necrosis can be seen in majority of cases. fibrinoid necrosis often involves several adjacent alveoli and is frequently associated with small vessel vasculitis. the multinucleated giant cells with intranuclear inclusions are usually noted in alveolar spaces adjacent to necrotic areas. histopathologic changes of bronchiolar epithelium are uncommon; rarely, the large bronchi may show transmural inflammation and ulceration. widespread presence of nipah virus antigens can be seen by ihc in endothelial and smooth muscle cells of blood vessels as well as in various parenchymal cells (fig. 11.15d ). the diagnosis of nipah virus infection, suspected by patient history and clinical manifestations, can be supported by characteristic histopathologic findings. from a diagnostic standpoint, perhaps the most unique histopathologic finding is the presence of syncytial and parenchymal multinucleated endothelial cells. this feature 457 occurs in only approximately one fourth of the cases and cannot be used as a sensitive criterion for the diagnosis of henipah virus infections; furthermore, these cells can also be seen in measles virus, rsv, hpiv, herpesviruses, and other infections. unequivocal diagnosis can be made only by laboratory tests such as ihc, cell culture isolation, pcr, or serology.l04,304,316-319 the parvoviruses are small (18 to 26nm) naked viruses that possess a single-stranded dna genome and require actively dividing cells to complete the viral replication cycle. in 2005, allander et a1. 320 identified a new parvovirus (genus bocavirus) associated with lower respiratory tract infections in children; however, there is no information as yet that describes specific pulmonary pathology attributed to this newly identified agent. on the other hand, human parvovirus b19, a member of the genus erythrovirus, has long been known to cause human disease and has been well studied. 321 ,322 the most commonly recognized manifestation of b19 infections is erythema infectiosum, and approximately one third of the cases of maternal parvovirus infections result in intrauterine parvovirus b19 infections. this places the fetus at increased risk for severe anemia, hydrops, and death. hydrops fetalis is the most commonly recognized complication of intrauterine parvovirus infection, accounting for 4% to 18% of all cases of nonimmune hydrops. cases tend to be clustered during community outbreaks of erythema infectiosum. 321 . 3 22 pathophysiologic effects and histopathologic findings are a result of the tropism of b19 parvovirus for erythroid precursor cells. villi from placentas from patients with bl9-associated nonimmune hydrops are edematous, and fetal capillaries show numerous nucleated erythroid precursors, some containing parvovirus inclusions. the infected cells with eosinophilic "ground glass" intranuclear inclusions and ring-like margination of nuclear chromatin are easily recognized and in the context of a hydropic fetus are pathognomonic of b19 infection. the liver is the major site of blood production in the fetus and the principal organ affected by intrauterine b19 infection. inclusion-bearing nucleated erythrocytes can also be frequently identified in the lung, making histopathologic examination of this organ a worthwhile endeavor for confirming the diagnosis of intrauterine bi9 infection ( fig. il.i6a,b) .323-332 however, these cells may be infrequent and irregularly distributed, requiring examination of multiple sections and use of ihc to confirm the diagnosis (fig.l1.16c,d) . the combination of fever and hemorrhage can be caused by different viruses, rickettsiae, bacteria, protozoa, and fungi. however, the term viral hemorrhagic fever (vhf) is usually reserved for systemic infections characterized by fever and hemorrhage caused by a special group of viruses transmitted to humans by arthropods and rodents. the vhfs are febrile illnesses characterized by abnormal vascular regulation and vascular damage and are caused by small, lipid-enveloped rna viruses. this syndrome can be caused by viruses belonging to four different families that differ in their genomic structure, replication strategy, and morphologic features (table 11 .4). arenaviruses, bunyaviruses, and filoviruses are negative-stranded, 459 in the nucleus and cytoplasm of erythroid precursors by using immunohistochemistry (ihe) (same patient as in a,b). a,b, h&e; c,d, immunoalkaline phosphatase staining, naphthol fast-red, and hematoxylin counterstain. whereas fiaviviruses are positive-stranded rna viruses. hemorrhagic fever viruses are distributed worldwide, and the diseases they cause are traditionally named according to the location where they were first described. the oldest and best known is yellow fever virus; others include lassa fever, lymphocytic choriomeningitis, ebola, and dengue viruses. viral hemorrhagic fevers share many common pathologic features, although the overall changes vary among the different diseases. the similar pathologic and immunopathologic findings in cases of vhf suggest that microvascular involvement and instability is an important common pathogenic pathway leading to shock and bleeding in many instances. infection of the mononuclear phagocytic system and endothelium are thought to play c s.r. zaki and cd. paddock 11 .17. ebola virus hemorrhagic fever. a. pulmonary congestion and lack of inflammation. b. numerous filamentous ebola virus inclusions are seen within hepatocytes in association with hepatocellular necrosis. c. ebola virus-infected intra alveolar macrophages as seen by colorimetric in-situ hybridization using digoxigenin-labeled probes. d. viral anti-gens are seen in endothelial cells and other interstitial cells in this lung section. a,b, h&e; c, digoxigenin-labeled probes followed by immunoalkaline phosphatase staining, naphthol fast-red, and hematoxylin counterstain; d, immunoalkaline phosphatase staining, naphthol fast-red, and hematoxylin counterstain. figure 11.18 . yellow fever. a. pulmonary congestion in fatal case of yellow fever associated with vaccination. b. yellow fever antigens in the pulmonary interstitium of the same patient. in natural infections, yellow fever antigens are usually not seen outside the liver in fatal cases. in contrast, in vaccine-associated cases viral antigens can be found in a variety of extrapulmonary a critical role in the pathogenesis of vhfs through the secretion of physiologically active substances, including cytokines and other inflammatory mediators (figs. 11.17c,d, 11.18b, 11.19b,c, and 11.20b). at autopsy common findings include widespread petechial hemorrhages and ecchymoses involving skin, mucous membranes, and internal organs. however, in many hf patients manifestations of bleeding may be minimal or absent. effusions, occasionally hemorrhagic, are also frequently seen. widespread, focal, and sometimes massive necrosis can be commonly observed in all organ systems and is often ischemic in nature. necrosis is usually most prominent in the liver and lymphoid tissues. the most con-461 organs, including heart, lung, and spleen. c. extensive midzonal hepatic necrosis characteristic of yellow fever. d. abundant antigens of yellow fever virus are observed in midzonal area of hepatic lobule in this immunohistochemical preparation. a,c, h&e; b,d, immunoalkaline phosphatase staining, naphthol fast-red, and hematoxylin counterstain. sistent microscopic feature is found in the liver and consists of multifocal hepatocellular necrosis with cytoplasmic eosinophilia, councilman bodies, nuclear pyknosis, and cytolysis (figs. 11.17b and 11.18c). inflammatory cell infiltrates and necrotic areas are usually mild and, when present, consist of neutrophils and mononuclear cells. commonly observed histopathologic changes in the lung include various degrees of hemorrhage, intra alveolar edema, interstitial pneumonitis, and diffuse alveolar damage (figs. 11.17 a, 11.18a, 11.19a, 11.20a, and 11.21a). several references containing detailed pathologic descriptions in human cases are recommended. 29.72.333-35o .' a. pulmonary hemorrhage and diffuse alveolar damage in a patient who was infected through organ transplantation. note that, unlike this case, which occurred due to immunosuppression, lcmv infections are rarely fatal and usually resolve with no specific treatment. b. abundant lcmv antigens in areas of the diagnosis of vhf should be suspected in patients with appropriate clinical manifestations returning from an endemic area, particularly if there is travel to rural areas during seasonal or epidemic disease activity. the diagnosis suspected by history and clinical manifestations can also be supported histopathologically, and the overall pattern of histopathologic lesions may suggest a specific diagnosis. however, because of similar pathologic features seen in vhf and a variety of other viral, rickettsial, and bacterial infections, unequivocal diagnosis can only be made by s.r. zaki and cd. paddock lung showing diffuse alveolar damage. c. using immunohistochemistry, abundant lcmv antigens are seen within cytoplasm of hepatocytes and sinusoidal lining cells in association with areas of hepatocellular necrosis. a, h&e; b,c, immunoalkaline phosphatase staining, naphthol fast-red, and hematoxylin counterstain. laboratory tests such as cell culture isolation, serology, pcr, and lhc (figs. 11.18d, 11.20c, and 11.2ib,c). the principles and practice of medicine unexplained deaths due to possibly infectious causes in the united states: defining the problem and designing surveillance and laboratory approaches. the unexplained deaths working group deaths: preliminary data for the origin of the 1918 pandemic influenza virus: a continuing enigma update: outbreak of severe acute respiratory syndromeworldwide viral pneumonia outcome and prognostic factors in community-acquired pneumonia requiring hospitalization hospital study of adult community-acquired pneumonia adult pneumonia in a general hospital. etiology and host risk factors prospective study of the aetiology and outcome of pneumonia in the community bacteria, viruses, and mycoplasmas in acute pneumonia in adults viral communityacquired pneumonia in nonimmunocompromised adults a novel coronavirus associated with severe acute respiratory syndrome approach to the patient with pneumonia how do viral infections predispose patients to bacterial infections? preceding respiratory infection predisposing for primary and secondary invasive haemophilus influenzae type b disease viral-bacterial synergistic interaction in respiratory disease immune impairment of alveolar macrophage phagocytosis during influenza virus pneumonia mechanisms of bacterial superinfect ions in viral pneumonias impact of respiratory virus infections on persons with chronic underlying conditions identification of a new north american hantavirus that causes acute pulmonary insufficiency nipah virus: a recently emergent deadly paramyxovirus genetic identification of a hantavirus associated with an outbreak of acute respiratory illness varicella-related mortality in california decline in mortality due to varicella after implementation of varicella vaccination in the united states pneumonia caused by herpesviruses in recipients of hematopoietic cell transplants initial genetic characterization of the 1918 "spanish" influenza virus characterization of the reconstructed 1918 spanish influenza pandemic virus transmission of lymphocytic choriomeningitis virus by organ transplantation a morbillivirus that caused fatal disease in horses and humans fatal west nile virus encephalitis in a renal transplant recipient transmission of west nile virus from an organ donor to four transplant recipients transmission of rabies virus from an organ donor to four transplant recipients isolation of a cytopathogenic agent from human adenoids undergoing spontaneous degeneration in tissue culture recovery of new agent from patients with acute respiratory illness adenoviruses: group name proposed for new respiratory-tract viruses adenoviruses in the immunocompromised host fatal disseminated adenovirus infections in immunocompromised patients respiratory disease and the adenoviruses adenoviruses from human immunodeficiency virus-infected individuals, including two strains that represent new candidate serotypes ad50 and ad51 of species bl and d, respectively infections in 18,000 infants and children in a controlled study of respiratory tract disease. 1. adenovirus pathogenicity in relation to serologic type and illness syndrome adenoviral diseases in children: a study of 105 hospital cases adenovirus type 7 associated with severe and fatal acute lower respiratory infections in argentine children wasz-hockert 0. type 7 adenovirus pneumonia bronchopneumonia with serious sequelae in children with evidence of adenovirus type 21 infection disseminated adenovirus disease in immunocompromised and immunocompetent children adenovirus pneumonia fatal pneumonia associated with adenovirus type 7 in three military trainees two fatal cases of adenovirus-related illness in previously healthy young adults-illinois histopathology of fatal adenovirus infection of the respiratory tract in young children adenovirus pneumonia in lung transplant recipients cytologic diagnosis of adenovirus bronchopneumonia adenoviral infections in children: the impact of rapid diagnosis report of one case and s.r. zaki and c.d. paddock review of the literature morphological and virological studies in six autopsies of children with adenovirus pneumonia antigenic relationships among the 47 human adenoviruses determined in reference horse antisera the polymerase chain reaction for detecting adenovirus dna in formalin-fixed, paraffin-embedded tissue obtained post mortem prevention of bleomycin-induced pulmonary fibrosis after adenovirusmediated transfer of the bacterial bleomycin resistance gene manual of clinical microbiology nucleotide sequence of the l genome segment of hantaan virus analysis of hantaan virus rna: evidence for a new genus of bunyaviridae phylogenetic analyses of virus isolates in the genus hantavirus, family bunyaviridae morphology and morphogenesis of viruses of hemorrhagic fever with renal syndrome: inclusion bodies-ultrastructure marker of hantavirus-infected cells ultrastructural characteristics of sin nombre virus, causative agent of hantavirus pulmonary syndrome distinction between bunyaviridae genera by surface structure and comparison with hantaan virus using negative stain electron microscopy isolation of the causative agent of hantavirus pulmonary syndrome update: hantavirus pulmonary syndrome-united states world health organization (who) collaborating center for virus reference and research hantavirus pulmonary syndrome: a clinical description of 17 patients with a newly recognized disease. the hantavirus study group isolation of the etiologic agent of korean hemorrhagic fever centers for disease control and prevention. outbreak of acute illness-southwestern united states hantavirus pulmonary syndrome. pathogenesis of an emerging infectious disease hantavirus-associated diseases hantavirus pulmonary syndrome in the united states: a pathological description of a disease caused by a new agent the pathology of thirty-nine fatal cases of epidemic hemorrhagic fever pathology of epidemic hemorrhagic fever kessler who gross anatomic features found in 27 autopsies of epidemic hemorrhagic fever pathology of hemorrhagic fever: a comparison of the findings, 1951 and 1952 identification of hantaan virus-related structures in kidneys of cadavers with haemorrhagic fever with renal syndrome an autopsy case of epidemic hemorrhagic fever (korean hemorrhagic fever) baculovirus expression of the small genome segment of hantaan virus and potential use of the expressed nucleocapsid protein as a diagnostic antigen utilization of autopsy rna for the synthesis of the nucleocapsid antigen of a newly recognized virus associated with hantavirus pulmonary syndrome detection and sequence confirmation of sin nombre virus rna in paraffin-embedded human tissues using one-step rt-pcr retrospective diagnosis of hantavirus pulmonary syndrome, 1978-1993: implications for emerging infectious diseases retrospective diagnosis of a 1983 case of fatal hantavirus pulmonary syndrome ultrastructural characterization of sars coronavirus proliferative growth of sars corona virus in vero e6 cells morphogenesis of avian infectious bronchitis virus and a related human virus (strain 229e) assembly of enveloped rna viruses electron microscopic studies of coronavirus coronavirus as a possible cause of severe acute respiratory syndrome analysis of deaths during the severe acute respiratory syndrome (sars) epidemic in singapore: challenges in determining a sars diagnosis the clinical pathology of severe acute respiratory syndrome (sars): a report from china lung pathology of severe acute respiratory syndrome (sars): a study of 8 autopsy cases from singapore a clinicopathological study on 3 cases of severe acute respiratory syndrome enteric involvement of severe acute respiratory syndrome-associated coronavirus infection lung pathology of fatal severe acute respiratory syndrome butany 1. pulmonary pathology of severe acute respiratory syndrome in toronto sars coronavirus-infected cells in lung detected by new in situ hybridization technique immunohistochemical, in situ hybridization, and ultrastructurallocalization of sars-associated coronavirus in lung of a fatal case of severe acute respiratory syndrome in taiwan tissue and cellular tropism of the coronavirus associated with severe acute respiratory syndrome: an in-situ hybridization study of fatal cases acute lung injury patterns: diffuse alveolar damage and bronchiolitis obliterans-organizing pneumonia pathology of infectious diseases nipah virus infection: pathology and pathogenesis of an emerging paramyxoviral zoonosis immunohistochemical and in situ hybridization studies of influenza a virus infection in human lungs newly discovered coronavirus as the primary cause of severe acute respiratory syndrome pegylated interferon-alpha protects type 1 pneumocytes against sars coronavirus infection in macaques replication of sars corona virus administered into the respiratory tract of african green, rhesus and cynomolgus monkeys prior infection and passive transfer of neutralizing antibody prevent replication of severe acute respiratory syndrome coronavirus in the respiratory tract of mice severe acute respiratory syndrome corona virus infection of golden syrian hamsters uber protozoenartige zellen in der niere eines syphilitischen neugoborenen und in der parotis von kindern concerning the nature of "protozoan-like" cells in certain lesions of infancy intranuclear and cytoplasmic inclusions ("protozoan-like bodies") in the salivary glands and other organs of infants serologic differentiation of viruses responsible for cytomegalic inclusion disease primary chimpanzee skin fibroblast cells are fully permissive for human cytomegalovirus replication monocyte-derived dendritic cells are permissive to the complete replicative cycle of human cytomegalovirus cytomegaloviruses and their replication human cytomegalovirus. morphology by negative staining infectiousmononucleosis-like disease with negative heterophil agglutination test. clinical features in relation to epstein-barr virus and cytomegalovirus antibodies cytomegalovirus infection in the normal host cytomegalic inclusion disease; an analysis of the clinical features based on the literature and six additional cases cytomegalovirus pneumonia in transplant recipients nonbacterial pneumonia after allogeneic marrow transplantation: a review of ten years' experience pulmonary complications of solid organ and hematopoietic stem cell transplantation lungs are a major organ site of cytomegalovirus latency and recurrence spectrum of pulmonary diseases associated with the acquired immune deficiency syndrome cytomegalovirus-induced alveolar hemorrhage in patients with aids: a new clinical entity? widespread presence of histologically occult cytomegalovirus cytomegalovirus pneumonia in bone marrow transplant recipients: miliary and diffuse patterns cytomegalovirus pneumonitis in patients with aids. findings in an autopsy series light microscopy of selected viral diseases (morphology of viral inclusion bodies) human cytomegalovirus infection of cultured fibroblasts. ii. viral replicative sequence of a wild and an adapted strain staining qualities of cytomegalovirus inclusions in the lungs of patients with the acquired immunodeficiency syndrome: a potential source of diagnostic misinterpretation definitions of cytomegalovirus infection and disease in transplant recipients direct detection of cytomegalovirus from bronchoalveolar lavage samples by using a rapid in situ dna hybridization assay pathology of neonatal herpes simplex virus infection varicella zoster and herpes simplex virus pneumonias viral infections of the lung herpes simplex viruses recovery of herpes simplex virus from human sacral ganglions herpesvirus hominis: isolation from human trigeminal ganglion infections with herpes simplex viruses (1) infections with herpes simplex viruses (2) herpetic infection of the middle and lower respiratory tract herpes simplex virus pneumonia: clinical, virologic, and pathologic features in 20 patients herpes simplex virus pneumonia in trauma patients herpes simplex virus pneumonia during standard induction chemotherapy for acute leukemia: case report and review of literature hepato-adrenal necrosis with intranuclear inclusion bodies usefulness of clinical features and liver biopsy in diagnosis of disseminated herpes simplex infection neonatal herpes simplex infection neonatal herpes simplex pneumonia calcification in the adrenal glands associated with disseminated herpes simplex infection necrotizing tracheobronchitis and bronchopneumonia consistent with herpetic infection herpetic tracheobronchitis herpes simplex virus infection of the adult lower respiratory tract isolation of herpes virus from a case of atypical pneumonia and erythema multiforme exudativum herpes simplex virus-the most frequently isolated pathogen in the lungs of patients with severe respiratory distress herpes simplex virus from the lower respiratory tract in adult respiratory distress syndrome the problem of intranuclear inclusions in virus diseases comparison of in situ hybridization and immunohistochemistry for detection of cytomegalovirus and herpes simplex virus varicella-zoster virus varicella-zoster virus: pathogenesis of latency and reactivation varicella-related deaths among adults-united states varicella pneumonia in adults severe forms of chickenpox in adults the epidemiology of varicella and its complications varicella pneumonia: study of prevalence in adult men report of seven cases and a review of literature varicella-zoster virus pneumonitis disseminated varicella infection in adult renal allograft recipients: four cases and a review of the literature risk factors and outcome of varicella-zoster virus pneumonia in pregnant women disseminated varicella at autopsy in children with cancer roentgenographic manifestations of varicella pneumonia with postmortem correlation visceral lesions associated with varicella calcification in chickenpox pneumonia a survey of pulmonary calcification following adult chicken-pox fulminant disseminated varicella zoster virus infection without skin involvement cell-free varicella-zoster virus in cultured human melanoma cells topley and wilson's microbiology and microbial infections changes in the respiratory mucosa resulting from infection with influenza virus bacteriology and histopathology of the respiratory tract and lungs in fatal asian influenza asian influenza a in boston, 1957-1958. i. observations in thirty-two influenza-associated fatal cases asian influenza a in boston, 1957-1958. ii. severe staphylococcal pneumonia complicating influenza fatal diffuse influenzal pneumonia: premortem diagnosis by lung biopsy clinicopathologic study of thirty-three fatal cases of asian influenza pathologic features of lung biopsy specimens from influenza pneumonia cases bronchotracheal response in human influenza. type a, asian strain, as studied by light and electron microscopic examination of bronchoscopic biopsies influenza a virus infection complicated by fatal myocarditis histopathologic and immunohistochemical features of fatal influenza virus infections in children during the 2003-2004 season probable person-to-person transmission of avian influenza a (h5n1) influenza-associated deaths among children in the united states h5n1 virus attachment to lower respiratory tract avian flu: influenza virus receptors in the human airway influenza diagnosis and treatment in children: a review of studies on clinically useful tests and antiviral treatment for influenza comparison of the directigen flu a+b test, the quickvue influenza test, and clinical case definition to viral culture and reverse transcription-pcr for rapid diagnosis of influenza virus infection paromyxoviridae and their replication electron microscopy in viral diagnosis measles surveillance-united states modem measles an experimental demonstration of the presence of the virus of measles in the mixed buccal and nasal secretions measles virus measles pneumonia in young adults. an analysis of 106 cases virus pneumonia following measles: a virological and histological study of autopsy material measles pneumonia and the nature of the inclusion-bearing giant cells: a light-and electron-microscope study fatal measles infection in children with leukemia disseminated measles infection after vaccination in a child with a congenital immunodeficiency measles giant cell pneumonia in an adult following long-term chemotherapy an epidemic of measles in southern greenland, 1951; measles in virgin soil. ii. the epidemic proper human membrane cofactor protein (cd46) acts as a cellular receptor for measles virus viral infections of the lung measles virus: both the haemagglutinin and fusion glycoproteins are required for fusion measles: clinical features. pathogenesis, pathology and complications in vivo and in vitro cellular changes specific for measles giant-cell pneumonia caused by measles and methotrexate in childhood leukaemia in remission measles giant cell pneumonia without rash in a case of lymphocytic lymphosarcoma. an electron microscopic study importance of antibodies to the fusion glycoprotein of paramyxoviruses in the prevention of spread of infection measles immunization with killed virus vaccine. serum antibody titers and experience with exposure to measles epidemic altered reactivity to measles virus. atypical measles in children previously immunized with inactivated measles virus vaccines pulmonary lesions in atypical measles difference in the appearance of antibodies to structural components of measles virus after immunization with inactivated and live virus the skin lesion in measles isolation of measles virus at autopsy in cases of giant-cell pneumonia without rash patterns of measles pneumonitis fatal measles (rubeola) pneumonia in adults role of measles virus in skin lesions and koplik's spots pathology of tropical and extraordinary diseases the lungs in fatal measles infection in childhood: pathological, radiological and immunological correlations measles rash. light and electron microscopic study of skin eruptions atypical lymphnode hyperplasia after administration of attenuated, live measles vaccine lymphadenopathy simulating the malignant lymphomas giant cell pneumonia; fluorescent antibody and histochemical studies on alveolar giant cells application of immunofluorescence to a study of measles analysis of viral antigens in giant cells of measles pneumonia by immunoperoxidase method. virchows arch a giant cell pneumonia due to respiratory syncytial virus. occurrence in severe combined immunodeficiency syndrome giant cell pneumonia associated with parainfluenza virus type 3 infection giant cell pneumonia caused by parainfluenza type 3 in a patient with acute myelomonocytic leukemia giant cell pneumonia caused by varicella zoster virus in a neonate a novel morbilli virus pneumonia of horses and its transmission to humans use of immunocytochemistry and biotinylated in situ hybridization for detecting measles virus in central nervous system tissue delayed acute measles inclusion body encephalitis in a 9-year-old girl: ultrastructural, immunohistochemical, and in situ hybridization studies baculovirus expression of the nucleoprotein gene of measles virus and utility of the recombinant protein in diagnostic enzyme immunoassays parainfluenza viruses parainfluenza viruses parainfluenza virus respiratory infection after bone marrow transplantation respiratory disease due to parainfluenza virus in adult bone marrow transplant recipients respiratory virus infections in bone marrow transplant recipients: the european perspective respiratory viral infections in lung transplant recipients: radiologic findings with clinical correlation outbreak of human parainfluenza virus 3 infections in a hematopoietic stem cell transplant population parainfluenza virus 3 infection after stem cell transplant: relevance to outcome of rapid diagnosis and ribavirin treatment pre-emptive oral ribavirin therapy of paramyxovirus infections after haematopoietic stem cell transplantation: a pilot study parainfluenza virus pneumonitis in an adult parainfluenza and influenza virus infections in pediatric organ transplant recipients human parainfluenza virus giant cell pneumonia following cord blood transplant associated with pulmonary alveolar proteinosis pathology of parainfluenza virus infection in patients with congenital immunodeficiency syndromes parainfluenza pneumonia in severe combined immunodeficiency disease disseminated parainfluenza infection in a child with severe combined immunodeficiency observations on clinical and immunofluorescent diagnosis of parainfluenza virus infections parainfluenza virus bronchiolitis. epidemiology and pathogenesis production of monoclonal antibodies against parainfluenza 3 virus and their use in diagnosis by immunofluorescence comparison of direct immunofluorescent staining of clinical specimens for respiratory virus antigens with conventional isolation techniques respiratory syncytial virus respiratory syncytial virus primary virus infection with cytoplasmic inclusion bodies. study of an epidemic involving thirtytwo infants with nine deaths mcquillin 1. pathological changes in virus infections of the lower respiratory tract in children respiratory syncytial virus infection in immunocompromised adults neonatal respiratory syncytial virus infection fatal respiratory syncytial virus pneumonitis in a previously healthy child fatal pneumonia in an adult due to respiratory syncytial virus demonstration of respiratory syncytial virus in an autopsy series pathology of infectious diseases comparison of cell culture and three enzyme-linked immunosorbent assays for the rapid diagnosis of respiratory syncytial virus from nasopharyngeal aspirate and tracheal secretion specimens molecular detection of respiratory syncytial virus in postmortem lung tissue samples from mexican children deceased with pneumonia clinically useful method for the isolation of respiratory syncytial virus respiratory syncytial virus infections in infants: quantitation and duration of shedding viral infections of the lung monoclonal antibodies for the rapid diagnosis of respiratory syncytial virus infection by immunofluorescence respiratory syncytial virus detection by immunofluorescence in nasal secretions with monoclonal antibodies against selected surface and internal proteins respiratory syncytial virus illnesses in human immunodeficiency virusand noninfected children a newly discovered human pneumovirus isolated from young children with respiratory tract disease prevalence and clinical symptoms of human metapneumovirus infection in hospitalized patients virological features and clinical manifestations associated with human metapneumovirus: a new paramyxovirus responsible for acute respiratory-tract infections in all age groups human metapneumovirus infection in the united states: clinical manifestations associated with a newly emerging respiratory infection in children children with respiratory disease associated with metapneumovirus in hong kong human metapneumovirus and lower respiratory tract disease in otherwise healthy infants and children a prospective study comparing human metapneumovirus with other respiratory viruses in adults with hematologic malignancies and respiratory tract infections human metapneumovirus in a haematopoietic stem cell transplant recipient with fatal lower respiratory tract disease respiratory tract reinfections by the new human metapneumovirus in an immunocompromised child experimental human metapneumovirus infection of cynomolgus macaques (macaca fascicularis) results in virus replication in ciliated epithelial cells and pneumocytes with associated lesions throughout the respiratory tract detection of severe human metapneumovirus infection by real-time 473 polymerase chain reaction and histopathological assessment mcadam al pathology of human metapneumovirus infection: insights into the pathogenesis of a newly identified respiratory virus human metapneumovirusassociated atypical pneumonia and sars from the centers for disease control and prevention. outbreak of hendra-like virus-malaysia and singapore, 1998-1999 fatal encephalitis due to nipah virus among pig-farmers in malaysia outbreak of nipah-virus infection among abattoir workers in singapore first case of nipah virus encephalitis in singapore case-control study of risk factors for human infection with a new zoonotic para myxovirus, nipah virus, during a 1998-1999 outbreak of severe encephalitis in malaysia clinical features of nipah virus encephalitis among pig farmers in malaysia the neurological manifestations of nipah virus encephalitis, a novel paramyxovirus molecular characterization of nipah virus, a newly emergent paramyxovirus nipah virus encephalitis reemergence nipah virusassociated encephalitis outbreak nipah virus infection in bats (order chiroptera) in peninsular malaysia isolation of hendra virus from pteropid bats: a natural reservoir of hendra virus antibodies to nipah-like virus in bats nipah virus in lyle's fiying foxes the natural history of hendra and nipah viruses elucidation of nipah virus morphogenesis and replication using ultrastructural and molecular approaches ultrastructure of hendra virus and nipah virus within cultured cells and host animals infection of humans and horses by a newly described morbillivirus fatal encephalitis due to novel paramyxovirus transmitted from horses comparative pathology of the diseases caused by hendra and nipah viruses reactivity of anti-nipah virus monoclonal antibodies to formalin-fixed, paraffin-embedded lung tissues from experimental nipah and hendra virus infections genetic characterization of nipah virus development of a fluorogenic rt-pcr assay (taqman) for the detection of hendra virus specific detection of nipah virus using real-time rt-pcr (taqman) cloning of a human parvovirus by molecular screening of respiratory tract samples human parvovirus b19 torok ti parvovirus infections (erythema infectiosum). in: wilfert cm parvovirus b19 infection of the fetus. histology and in situ hybridization cohen bi human parvovirus infection in pregnancy and hydrops fetalis intrauterine infection with human parvovirus b19: a light and electron microscopy study morphological diagnosis of parvovirus b19 infection. a cytopathic effect easily recognized in air-dried, formalin-fixed bone marrow smears stained with hematoxylin-eosin or wright-giemsa clinical and histopathological features of parvovirus b19 infection in the human fetus detection of parvovirus b19 dna in fetal tissues by in situ hybridisation and polymerase chain reaction immunohistological detection of human parvovirus b19 in formalin-fixed, paraffin-embedded tissues combined immunocytochemistry and non-isotopic in situ hybridization for the ultrastructural investigation of human parvovirus b19 infection intracellular localization of parvovirus b19 nucleic acid at the ultrastructural level by in situ hybridization with digoxigenin-labelled probes detection of parvovirus b19 dna in bone marrow cells by chemiluminescence in situ hybridization viral hemorrhagic fevers the pathology of lassa fever lassa virus hepatitis: a study of fatal lassa fever in humans pathologic and virologic study of fatal lassa fever in man pathology of 12 fatal cases of argentine hemorrhagic fever bolivian hemorrhagic fever. a pathologic description congo-crimean haemorrhagic fever in dubai: histopathological studies a nosocomial outbreak of crimean-congo haemorrhagic fever at tygerberg hospital. part iii. clinical pathology and pathogenesis rift valley fever virus infections in egypt: pathological and virological findings in man immunohistochemical and in situ localization of crimean-congo hemorrhagic fever (cchf) virus in human tissues and implications for cchf pathogenesis strano al yellow fever viral infections of the lung dengue haemorrhagic fever: a pathological study ultrastructural and immunohistochemical studies in five cases of argentine hemorrhagic fever venezuelan haemorrhagic fever rift valley fever affecting humans in south africa: a clinicopathological study pathology of thailand haemorrhagic fever: a study of 100 autopsy cases dengue and other viral hemorrhagic fevers fatal hemorrhagic fever caused by west nile virus in the united states acknowledgments. the authors gratefully acknowledge the invaluable assistance of gillian genrich, of the cdc, for her comments and help with organization of the references, cynthia goldsmith for her comments, and mitesh patel, of the cdc, for his help with preparation and assembly of the images. key: cord-007417-az8xd66p authors: hansbro, nicole g.; horvat, jay c.; wark, peter a.; hansbro, philip m. title: understanding the mechanisms of viral induced asthma: new therapeutic directions date: 2008-01-29 journal: pharmacol ther doi: 10.1016/j.pharmthera.2007.11.002 sha: doc_id: 7417 cord_uid: az8xd66p asthma is a common and debilitating disease that has substantially increased in prevalence in western societies in the last 2 decades. respiratory tract infections by respiratory syncytial virus (rsv) and rhinovirus (rv) are widely implicated as common causes of the induction and exacerbation of asthma. these infections in early life are associated with the induction of wheeze that may progress to the development of asthma. infections may also promote airway inflammation and enhance t helper type 2 lymphocyte (th2 cell) responses that result in exacerbations of established asthma. the mechanisms of how rsv and rv induce and exacerbate asthma are currently being elucidated by clinical studies, in vitro work with human cells and animal models of disease. this research has led to many potential therapeutic strategies and, although none are yet part of clinical practise, they show much promise for the prevention and treatment of viral disease and subsequent asthma. 1. introduction asthma is thought to affect at least 300 million people of all ages and ethnic backgrounds worldwide (global strategy for asthma management and prevention, 1995) . between 1 in 5 and 1 in 10 people are affected in western societies and the prevalence has doubled since 1980 (umetsu et al., 2002; aihw, 2005) . it is now considered to be an epidemic and results in a massive economic burden to communities. exacerbations are typically caused by exposure to environmental factors to which the individual is allergic. although asthma is clearly recognised as an inflammatory condition, our understanding of the mechanisms of pathogenesis remains rudimentary. clinically asthma is characterised by airway obstruction, wheezing and episodic breathlessness in association with increased sensitivity of the airways to non-specific stimuli (termed airway hyperresponsiveness (ahr)) (bousquet et al., 2000) . wheezing is a high-pitched whistling or squeaking, which originates from the chest and is made during breathing (michel et al., 2006) . a predominant feature of disease is the acute-on-chronic infiltration of pro-inflammatory activated cd4+ th2 cells and eosinophils into the airways, which are critical regulators of pathogenesis (robinson et al., 1993; kay, 2005) . typical pathogenic features include: ige production; airway smooth table 1 important factors released by respiratory epithelium upon viral infection (dakhama et al., 2005a) nitric oxide muscle (asm) and goblet cell hypertrophy/hyperplasia; mucus hypersecretion; eosinophil, neutrophil and mononuclear cell infiltration into submucosal layer of the airways; mast cell and macrophage activation; sloughing of airway epithelial cells; and ahr (foster et al., 1996; kumar, 2001; cohn et al., 2004) . th2 cells and activated inflammatory cells release a range of mediators that damage the mucosal epithelial lining and promote an exaggerated repair response that leads to airway remodelling and chronic disease. remodelling is the result of structural changes of the epithelium, submucosal layer, asm and vasculature (angiogenesis) (bousquet et al., 2000; vignola et al., 2003) . it is thought to be a major contributing factor to the development of ahr, and its progression may lead to fixed airflow obstruction and irreversible loss of lung function (li & wilson, 1997; vignola et al., 2003) . thus, airway inflammation is closely linked to ahr and airflow obstruction and recurring inflammatory insults may result in changes that lead to airway remodelling. the mechanisms responsible for the generation of inflammation and remodelling remain poorly understood but may be induced or exacerbated by respiratory viral infection. respiratory infections by rsv, rv, influenza and parainfluenza and metapneumovirus (mpv) have all been implicated in the development of asthma as well as exacerbations. infection with rsv and rv are by far the most widely and commonly associated with bronchiolitis and childhood wheeze and the induction and exacerbation of asthma (papadopoulos et al., 2002a; xepapadaki et al., 2004) . rsv may cause earlier and more severe exacerbations and is more frequently linked to the induction of asthma whereas rv is the most common cause of exacerbations in later life zhao et al., 2002) . whether an infection induces disease depends on viral (type (e.g. rsv, rv)), host (genetic susceptibility, age, immune responses) and environmental (allergen exposure, season) factors. initial infection occurs by inhalation and spreads to the lower respiratory tract (lrt). infection is largely restricted to the respiratory epithelium, which induces the release of a wide range of mediators (table 1 ) (dakhama et al., 2005a ) that drive subsequent immune and physiological responses specific for each virus (fig. 1) . rsv and rv are the most important causes of lrt infections in infants under 2 years, causing bronchiolitis that results in wheezing, and breathing difficulties, which may in severe cases result in hospitalization (sigurs et al., 2000; kotaniemi-syrjanen et al., 2003; henderson et al., 2005; sigurs et al., 2005) . asthmatics may be more susceptible to viral infections, which lead to more severe lrt symptoms and are associated with increased hospitalization (corne et al., 2002) . notably the commonest cause of asthma related-death is respiratory viral infection (mccann & imani, 2007) . severe bronchiolitis resulting in hospitalization has been shown to be associated in fig. 1 . rsv, rv, asthma and therapy. rsv attaches to and invades the respiratory epithelium through the attachment (g) and fusion (f) proteins. major and minor group rvs bind to icam-1 and ldlprs on respiratory epithelium, respectively, which induces viral internalisation and upregulation of additional receptors. upon invasion viral proliferation leads to the induction of inflammatory cells and mediators that enhance allergen penetrance and hallmark features of asthma including inflammation, wheezing, airway obstruction and ahr. this may induce the development and exacerbation of asthma. various processes in virus-associated asthma may be targeted therapeutically. some case control studies with a history of recurrent wheeze and a diagnosis of asthma in later childhood (sigurs et al., 2000 (sigurs et al., , 2005 . rsv is the most widely implicated precipitant but recent studies suggest that rv may also be important, particularly after the age of 2 (heymann et al., 2004) . methods of detection are constantly been improved which will facilitate the elucidation of the role of these viruses in asthma (pierangeli et al., 2007) . the mechanisms that underpin virus-induced induction or exacerbation of asthma or the factors responsible for predisposing an individual remain poorly understood. it is unclear whether virus-induced bronchiolitis promotes the development of asthma or if those individuals that suffer more severely from infection are also more susceptible to asthma development as a result of genetic susceptibility (including atopy) or aberrant lung function. understanding the mechanisms of pathogenicity of respiratory viral infections and their association with asthma will be pivotal in the development of prevention and treatment strategies for asthma. indeed it may be possible to identify at risk individuals and delay infection until later in life and to develop novel therapeutic agents or targets. in asthma triggers of chronic inflammatory processes (airway inflammation, mucus hypersecretion) are overzealous responses of the asthmatic immune system to normally innocuous antigens or infections and recurrent stimulation leads to airway remodelling. respiratory viral infections induce immune responses that may have the potential to both initiate and exacerbate asthma. collectively evidence strongly implicates respiratory viral infections in the development of an asthmatic phenotype in children, although a directly causative role has still not been proven. it is possible that either 1. respiratory infection in early life induces the development of chronic airway inflammation and airway wall remodelling, resulting in persistent wheeze or 2. that some infants have pre-existing th2 responses that induces susceptibility to virus-induced wheeze (legg et al., 2003; stensballe et al., 2006) . the evidence for virus-induced asthma exacerbations is stronger than for causation and infections are responsible for the majority of acute exacerbations (80% in children, 50-76% in adults) inducing worsened airflow obstruction and symptoms wark et al., 2001; murray et al., 2004; tan, 2005) . to determine if virus infection directly influences disease in acute asthma exacerbations wark et al., recruited adults presenting to emergency with acute asthma and determined that 76% of these subjects had evidence of a viral infection (wark et al., 2002) . when compared to subjects with noninfective acute asthma, those with acute asthma and infection had evidence of more severe clinical disease, had a lower mean forced expiratory volume (fev 1 % predicted), were more likely to be admitted to hospital and had a longer median length of stay. an acute neutrophilic infiltrate was observed in induced sputum, with evidence of neutrophil degranulation, unlike the eosinophilic inflammation associated with non-infective asthma (wark et al., 2002) . in addition lactate dehydrogenase was measured as a marker of asthmatic airway necrosis. those with virus-associated acute asthma had significantly elevated lactate dehydrogenase activity, which correlated closely with the degree of neutrophil influx and airflow obstruction and was an independent predictor of the severity of the acute illness. these results strongly implicate viral infection as a trigger of exacerbations with increased severity and indicate an important role for viruses in modifying inflammation in acute asthma (wark et al., 2002) . there are many different categories and phenotypes of asthma including mild, moderate and severe as well as clinical, allergic and pathophysiologic phenotypes (wenzel, 2004) . recently simpson et al., described different inflammatory (neutrophilic, eosinophilic and paucigranulocytic) subtypes of asthma based on the predominant granulocytic cell in induced sputum (simpson et al., 2006) . the precise roles of respiratory viral infection in the development and exacerbation of the different phenotypes of asthma remain largely unknown, however, there is the potential that viral infection may be particularly important in certain phenotypes. different and customised prevention and treatment strategies may be required for different phenotypes depending on their causes. targeted anti-viral strategies may be more effective in certain asthma phenotypes for example in severe and neutrophilic asthma, where infectious agents may play a substantial role in pathogenesis (wark et al., 2002) . many factors may be involved in susceptibility to virusinduced asthma particularly virus and host factors. virus infection is the commonest cause of wheeze in children that may lead to the development of asthma (heymann et al., 2004) . the time of year also plays a role with winter the dominant period for viral infections and wheeze (heymann et al., 2004) . genetic predisposition may be important and many genes are implicated in susceptibility to asthma including those involved in inflammatory responses, ige regulation, cytokine and chemokine production, airway function and remodelling (umetsu et al., 2002) . atopy may lead to adverse responses to infection and childhood wheezing is linked to elevated ige and sensitivity to at least one inhaled allergen. however, the genetics of western populations that are experiencing the asthma epidemic remain unchanged and the focus of this review will be on other important factors that are associated with rsvand rv-associated asthma. these include the age of infection and timing of infection relative to allergen exposure, increased innate susceptibility and adaptive immune, cytokine and chemokine responses, as well as environmental conditions and inhaled bacterial endotoxin. rsv is a lipid-enveloped single stranded (ss) negative sense rna pneumovirus and is a member of the paramyxoviridae family. the virus causes the majority of cases of bronchiolitis in 317 n.g. hansbro et al. / pharmacology & therapeutics 117 (2008) to examine effect of different clinical characteristics and treatments on hospitalization of infants for bronchiolitis in outpatient clinic infants (320) b2 years presenting with 1st episode of wheezing retrospective analysis of medical records 38% of patients with rsv were hospitalized vs 10% without rsv. children exposed to tobacco smoke hospitalized more often (24%) vs not exposed (12%). treatment with oral corticosteroids associated with fewer hospitalizations in those with family history of asthma/allergic rhinitis (9.7% vs 24%) and without rsv (2.5% vs 16.7%). alshawwa and rao, 2007 early life, which may induce wheeze that may develop into asthma and is also a major precipitant of asthma exacerbations. epidemiology -rsv is the most important respiratory pathogen of children under the age of 2 years and primary infections are a common cause of lrt disease (hall, 1999; henrickson et al., 2004) . the majority of infants are infected during the first year of life, and the incidence of exposure approaches 100% by age 3 (parrott et al., 1973; glezen et al., 1986; hall et al., 1995) . these infections are the most frequent cause (50-90%) of bronchiolitis and also induce pneumonia and tracheobronchitis (10-30%) and there are annual epidemics, primarily in infants (hall, 2001) . around 100,000 children are hospitalized as a result of bronchitis annually in the usa with 50% less than 6 months and 80% less than 1 year of age with an estimated cost of $ud300 million per year (shay et al., 1999) . hospitalization for bronchiolitis has dramatically increased over the last 20 years (shay et al., 2001) , which may result from changes in childcare practises or a generalized decrease in th1 immunity in the population. mortality rates from primary infection are 0.005-0.02% for healthy or 1-3% for hospitalized children (ruuskanen & ogra, 1993; chanock et al., 1957) . most children that contract severe rsv disease have no identifiable risk factors, with the exception of premature birth. infections also cause severe disease in the elderly and immunocompromised and mild upper rt (urt) symptoms (rhinorrhea, nasal blockage, pharyngitis and cough) can occur at any age (falsey & walsh, 1998; englund et al., 1988) . re-infection is also common, occurring every 2-3 years throughout life usually resulting in mild urt symptoms. importantly this results from the lack of development of long-term resistance to rsv infection by the immune system (bont et al., 2002) . the majority of symptoms result from the host's immune and inflammatory responses to infection (openshaw, 1995) and re-infection induces sustained and exacerbated inflammatory reactions. pathogenesis -upon rsv infection and interaction of the virus with the respiratory mucosal surface the viral g protein mediates attachment and the f protein induces the fusion of the viral envelope with the cytoplasmic membrane of the host cell resulting in internalisation (fig. 1) . after invasion the viral ssrna is released into the cytoplasm and induces the production of viral rna and proteins that induce inflammatory responses. the rsv proteins and their functions have recently been reviewed by meyer et al. (2007) . the outcome of these inflammatory responses is the development of symptoms of pathogenic infection. typically, rsv infections in humans are restricted to the mucosal epithelial cells of the urt, causing runny nose, nasal congestion and cough (hall et al., 1978) . during severe rsv infections, the virus spreads to the lrt resulting in more severe symptoms. in vitro studies of human infection, as well as autopsy samples from infants and children with acute rsv infections, show that viral replication in airway epithelial cells, particularly in the superficial layer of the bronchiolar epithelium, as well as types 1 and 2 pneumocytes. infection induces the generation of inflammatory mediators and a mononuclear inflammatory response, plugging of the bronchioles with mucus, cellular debris and fibrin strands, as well as necrosis of the bronchiolar epithelium (piedra et al., 1997; johnson et al., 2007) . the lack of cytopathology during infection implicates inflammatory responses as the pivotal driver of rsv-induced disease (zhang et al., 2002) . inflammatory cells consist mainly of monocytes, t cells and neutrophils and accumulate around bronchial and pulmonary arterioles, airways and parenchyma and are associated with edema, mucus production, wheezing, airway obstruction and ahr . the induction of these disease processes may be involved in the development and exacerbation of asthma. clinical and epidemiological studies have shown that rsv infections are associated with a rapid increase in the incidence of asthma in paediatric and adult populations worldwide (wang & forsyth, 1998; tan, 2005) . these infections are also one of the commonest causes of asthma exacerbations. recent epidemiological studies that link rsv with asthma are shown in table 2 and older studies are reviewed in ogra (2004) . rsv infections are the most important risk factor for the development of bronchiolitis leading to recurrent wheezing and respiratory symptoms (decreased lung function, recurrent wheezing, allergic rhinoconjunctivitis). however, it has not yet been conclusively demonstrated that these virus-induced symptoms then progress to the development of asthma. a link between infection, atopy and sensitization to common inhaled allergens (skin prick tests (spts), ige) has also been investigated but the results are inconclusive. asthmatics may be prone to more severe infections, which may be a prognostic indicator of allergic susceptibility. between 50 and 90% of all cases of childhood hospitalizations for bronchiolitis have been attributed to rsv infections (holberg et al., 1991) . collectively studies show that rsv-induced bronchiolitis and diseases of the small airways often lead to wheezing which frequently progresses to asthma (reviewed in heymann et al. (2004) ). many studies have reported that up to 75% of subjects with rsv bronchiolitis suffer from subsequent recurrent wheeze or respiratory symptoms years later (reviewed in ogra (2004) ). an important prospective study by stein et al., showed that children with more than one lrt rsv infection were 4 times more likely to have frequent wheeze by ages 6 and 11, however the association decreased thereafter and was non-significant by age 13 (stein et al., 1999) . another smaller prospective study demonstrated that severe rsv bronchiolitis was linked to current wheezing (38%) and reactive airway disease (rad, 30%) compared to uninfected controls (2% and 3%, respectively) matched for age, sex family, history and environment (sigurs et al., 2000) . controlled retrospective and prospective studies indicate a link between rsv and bronchial obstruction and decreased lung function, particularly for children with severe rsv disease that results in hospitalization (reviewed in sigurs (2002b) ). indeed bronchiolitis is linked to chronic reductions in lung function for at least 10 years after infection (pullan & hey, 1982) and children hospitalized with rsv infection before 2 years of age have reduced lung function (but not asthma) 20 years later (korppi et al., 2004b) . furthermore airway obstruction and ahr is increased in those with rsv bronchiolitis compared to controls (sigurs, 2002b) . a nested case-controlled study also showed that rsv-induced hospitalization correlated with wheezing, lrt infections and asthma during first 4 years of life. the correlation decreased with age and was not significant at 5 years but the association held for increased respiratory symptoms and chronic productive cough at 5-8 years in alaska native children (singleton et al., 2003) . in the stein study wheezing subjects were significantly more responsive to bronchodilators, which indicates that reduced lung function results from an abnormality in airway tone (stein et al., 1999) . rsv bronchiolitis, particularly in early life, is strongly linked to the development of asthma. indeed 50-92% of children with bronchiolitis develop wheezing and asthma 3 (23% versus (vs) 1%), 5, 7.5 (30% vs 3%) or even 10 or more years later (sly & hibbert, 1989; sigurs et al., 1995; larouch et al., 2000; sigurs et al., 2000; ogra, 2004) . recent studies have found that children hospitalized with rsv have an increased risk of recurrent wheeze up to the age of 13 years, independent of atopy and other asthma risk factors, identifying rsv infection as a potential inducer of asthma. indeed these subjects have significantly more respiratory symptoms, (43% vs 8% for asthma/recurrent wheezing), sensitization to common allergens (50% vs 28% spt, 45% vs 26% serum ige), airway obstruction and ahr at age 13 years compared with controls (sigurs et al., 2005) . other recent reports have also shown that rsv infection is associated with concomitant respiratory symptoms (wheezing) and subsequent asthma at 2 years of age and that high rates of rsv are isolated from children with recurrent wheeze or asthma (lazzaro et al., 2007; lee et al., 2007) . the association was not confirmed in other longitudinal or cohort studies and may depend on individual airway structure, genetic predisposition and environmental factors (reviewed in martinez (2003) ). there may be a link between atopy, rsv infection and wheezing (sigurs et al., 2000) and risk factors may include the severity of rsv infection encountered. atopic children have low th1 responses in cord blood and elevated th2 responses compared to non-atopics. atopic adults also have th2 responses to allergens whereas non-atopics have low-level th1 responses (ogra, 2004) . elevated expression of th2 responses may promote susceptibility to rsv infection and may also lead to exaggerated airway inflammation and reduced lung function. however, the link between rsv infections and atopy is controversial and was not found in other studies (stein et al., 1999; kneyber et al., 2000) . early rsv infections (first 2 years) may induce allergic sensitization to unrelated antigens in genetically predisposed individuals (sigurs et al., 1995; forster et al., 1996) . indeed rsv bronchiolitis in the first year of life leads to increased ige levels (33% vs 2% in controls) and is the most important risk factor for allergic sensitization and recurrent wheeze (16% vs 4% controls) (schauer et al., 2002) . furthermore allergic sensitization and asthma are significantly more common in all age groups in children with a prior rsv bronchiolitis (23% vs 2% and 41% vs 22% of controls, respectively) (sigurs, 2002a ). it appears that the severity of infection may be important and severe infection has been associated with the development of allergic sensitization 3, 6 or 7.5 years after hospitalization (sigurs et al., 1995 (sigurs et al., , 2000 . mild infection, does not appear to be a risk factor for allergic sensitization (stein et al., 1999; sigurs et al., 2000) . however, the data are conflicting and severe infection has been shown not to induce sensitization (at age 2-10 years) by other investigators (pullan & hey, 1982; carlsen et al., 1987; noble et al., 1997) . pre-existing th2 or impaired th1 responses and asthmatic predisposition may promote susceptibility to acute bronchiolitis and hospitalization for rsv infection (legg et al., 2003; stensballe et al., 2006) . furthermore, individuals with damaged airway epithelium or that are otherwise immunologically predisposed may be additionally susceptible to infection enhancing epithelial damage and causing a vicious cycle of disease perpetuation. although rsv infection has been extensively linked with inducing asthma and reduced lung function it is possible that infection is also a marker of susceptibility to allergic and/or infectious respiratory disease. it has been shown that although rsv infection is the greatest risk factor for wheezing and asthma in subjects with a family history of asthma, asthma does not develop in those without infection or a family history (sigurs et al., 2000; sigurs, 2001) . this indicates that rsv bronchiolitis is a marker of increased risk of asthma susceptibility. more recently it has been suggested that the host response to infection rather than the nature of the infection itself is the best prognostic indictor for subsequent allergic disease (everard, 2006a (everard, , 2006b . atopy may also be a risk factor for susceptibility to more severe rsv infections and exacerbations, which result in higher rates of mortality and hospitalization (jhawar, 2003) . a large nested case-controlled study demonstrated that maternal atopic dermatitis and maternal and paternal asthma (rr 1.72 and 1.23, respectively) were risk factors for rsv hospitalization in infants b1.5 years (stensballe et al., 2006) . taken together studies show that rsv infection in early life results in bronchiolitis that leads to wheezing and decreased lung function, which may progress to asthma. these processes may be enhanced in atopic individuals with elevated th2 responses. moreover there is evidence, although not conclusive, that severe infection may also promote sensitization to allergens, which may further increase the risk of wheeze and asthma. asthmatics may be susceptible to more severe infections, which may be used as an indictor of susceptibility to the development of asthma. experimental rsv infection of humans has been used to investigate the mechanisms of pathogenesis of infectious disease and the association with asthma. these studies have demonstrated that the virus persists in nasal washes for 5-14 days (noah & becker, 2000) and that infection induces bronchiolitis and airway inflammation. this promotes epithelial sloughing, mucus hypersecretion and an increase in viscosity and edema which in turn lead to hyperinflation of the lungs, airflow obstruction, cough and wheeze (holt & sly, 2002a) . symptoms may persist and resolution of tissue damage may take several weeks or results in structural remodelling of the airway wall and airway narrowing (pare et al., 1997; holt & sly, 2002a) . the mechanisms of rsv pathogenesis that lead to wheezing, ahr, allergy and asthma are still not well understood and many studies have implicated a range of different factors. these include: the age of first infection; type of innate and adaptive responses elicited; mucosal damage and repair involving remodelling (including angiogenesis) and; enhanced neurogenic stimulation leading to asm spasm and bronchoconstriction. according to the hygiene hypothesis (strachan, 2000) , the th2-biased immune system of the newborn must encounter th1-inducing agents during childhood in order to develop the ability to mount a th1 response. however, whether infections in infancy induce beneficial th1 responses depend upon the type of infection and the mechanisms responsible for these effects have not been characterised. thus the immaturity of the developing immune system during early life and the nature of immune responses to infections may be significant determining factors in the development of persistent wheeze and asthma. whether rsv infection induces immunological and pathological processes that may lead to asthma may depend on the age at which an individual is first infected. the immune phenotype of early life may lead to enhanced viral replication and th2-dominated inflammation induced by infection or allergen exposure. increased levels of il-4 were detected in rsv infected infants less than 3 months old compared to more than 3 months but the converse was true for eotaxin and there was no difference in mip-1î² or eosinophil cationic protein (ecp) (kristjansson et al., 2005) . in mothers infection induced high levels of ifn-î³ and low levels of il-4 whereas newborn infants produced 4-7 times less ifn-î³ and higher levels of il-4 but at age 3 these levels approached those of their mothers (mbawuike et al., 2001) . the immune response in early life may be involved in the induction of more severe lrt pathology in response to primary infection but infection of older children is not as severe and involves largely urt symptoms. an additional pathological consequence is that viral infections in early life may generate pulmonary inflammation during the development of the lung, small and large airways and immune, inflammatory and neuronal programing (reviewed in gern et al. (2005) ). this may result in altered pulmonary structure and immune responses leading to enhanced pro-inflammatory and th2-biased immune responses that may precipitate deleterious changes in lung structure and function. furthermore, the small size of neonatal bronchioles determines that they may become obstructed more readily, which may result in reduced clearance and confer enhanced severity of pathogenic infection in this age group. the combination of these events may have longterm effects on lung function, chronic respiratory inflammation, remodelling, alveolarization and epithelial dysfunction (gern et al., 2005) and consequently may promote the development of asthma. however, whether rsv infection induces th1 or th2 responses in humans in early life is debateable. another alternative explanation for the association of rsv infection in infancy with asthma is that early life infection induces th1 memory and therefore cd8+ ctl responses, which have the potential to induce pathologic immune responses to reinfection. rsv infection induces viral specific cd8+ cells in infants and the levels of cytotoxic lymphocytes (ctls) are inversely proportional to il-4 responses. ctl responses are directly linked to protection against infection, ctl memory is initiated upon reinfection and mhc i cd8+ levels correlate directly with ifn-î³ levels (mbawuike et al., 2001) . immune responses induced to rsv enhance viral clearance but are also implicated in disease pathogenesis. ineffective or aberrant innate and adaptive immune responses against rsv have been widely linked to more severe and recurring infections and the development and exacerbation of asthma in both adults and children (glezen et al., 1986; hall et al., 1991) . primary infection, particularly early in life, leads to an incomplete immune response, which does not elicit the development of sustained memory immunity. with respect to allergy rsv infection might only trigger defective immunity in genetically susceptible individuals or that allergic inflammatory and immune responses may promote the influx of virus-specific cells into the airways increasing inflammation and ahr (schwarze et al., 1999c) . elucidation of the pivotal immune responses that are protective against rsv will lead to a better understanding of the processes that result in bronchiolitis, wheezing and progression to asthma. innate responses to rsv infection have not been widely studied but may play an important role in rsv-induced asthma. rsv infection of the respiratory epithelium induces innate cellular and cytokine responses, which have substantial effects on adaptive t cell development and cell-mediated immune responses. neutrophils are the main immune cell in the bronchoalveolar lavage fluid (balf) of patients with severe rsv lrt disease and increased il-8 levels, which is a potent chemoattractor of neutrophils, are also a prominent feature. macrophages internalise and process virus and present antigens to adaptive immune cells and also release il-12 and il-10. monocytes release il-1, -6, -8, -10, platelet-activating factor (paf) and pge2 on exposure to rsv that further promote proinflammatory responses (schaller et al., 2006) . eosinophils are also recruited to the airways during primary rsv infection, which may contribute to the development of allergic airways disease (schwarze et al., 1999a) . infected patients also have more plasmacytoid dendritic cells (pdcs) and myeloid dcs (mdcs) in the rt and reduced numbers of these cells circulating in blood (gill et al., 2005) . this suggests that they are recruited to the lung during infection and may be an important target in rsv vaccine development. understanding the interplays between these different cells types will be crucial in elucidating the effects of infection on the development of asthma. ifn-î± is an important type i ifn innate cytokine that is released upon viral infection and induces innate and adaptive cellular responses. blood cultures of asthmatic children and adults release significantly reduced amounts of ifn-î± upon rsv infection, which indicates a systemic innate deficiency in asthmatics that may lead to heightened susceptibility to infection (gehlhar et al., 2006) . 2.2.4.1. th1/th2 responses. rsv has several t cell epitopes and infection induces cd4+ and cd8+ and th1 and th2 adaptive cellular responses as well as pro-and anti-inflammatory cytokines and chemokines in humans both in vivo and in vitro (meyer et al., 2007) . established infections are primarily cleared by a combination of th1 and th2 cell responses and the balance between the two may be crucial in determining the outcome of infection, the severity of rsv-induced disease and predisposition to asthma. aberrant responses of either of these subsets may induce pathology. nevertheless most studies suggest that th1 responses may result in viral clearance and mild symptoms whereas an aberrant bias towards a th2 phenotype may lead to more intense rsv-induced disease and promote the development of asthma . indeed individuals with elevated th2 responses are predisposed to reduced viral clearance and more severe disease compared to subjects with only urt infection independently of age or viral load (roman et al., 1997; aberle et al., 1999; legg et al., 2003; gern et al., 2006) . early life rsv bronchiolitis results in enhanced il-4 and ifn-î³ responses to rsv in later childhood (pala et al., 2002) and il-4 is the most important stimulus for th2 development and ige production. th2 responses are enhanced during severe disease that develops upon natural infection of rsv-vaccinated infants (kim et al., 1969; kapikian et al., 1969) and severe rsv bronchiolitis correlates with elevated humoral th2 responses, and may be linked with atopy, increased il-4:ifn-î³ and reduced th1 (ifn-î³, il-12 and il-18) responses. rsv infections may also have persistent immunological effects and induce long-term th2 memory responses during sensitization to inhaled allergens in childhood (holt & sly, 2002a ; van rijt et al., 2005) . other studies have demonstrated that mild bronchiolitis is associated with a shift towards th1 responses and is usual in most individuals, and also that there is no increase in th2 responses in severe bron-chiolitis . these contrasting results may be attributable to differences in the timing of sampling during infection, the lack of definitive detection of rsv or determination of virus load, age and atopic status of individuals. 2.2.4.2. cytokines and immunomodulatory molecules. cytokines of the th1, th2 or regulatory type are implicated in rsvinduced asthma and the effect of pro-inflammatory cytokines and chemokines released in response to infection may have particularly important roles. th2 (il-5) cytokines are upregulated in children with acute asthma and children with acute bronchiolitis or il-5 levels have higher numbers of eosinophils (martinez, 2003) . these responses may play key roles in the progression from bronchiolitis to asthma, however, the importance of eosinophils and il-5 in rsv-induced asthma has not yet been confirmed. il-12 is a th1 cytokine that promotes the development of th1 cells, the release of ifn-î³ and il-2 from th1 and natural killer (nk) cells and suppresses th2 responses. levels of il-12 may be reduced in subjects that are more susceptible to infection. non-specifically stimulated whole blood cultures from patients with rsv disease released significantly less il-12 than controls and il-12 levels inversely correlated with disease severity (bont et al., 2000b) . il-12 and ifn-î³ responses were suppressed during acute rsv disease but returned to control levels during convalescence and ifn-î³ (and il-4) levels were not different in subsequently wheezing infants (bont et al., 2000a) . reduced il-12 levels may lead to elevated susceptibility to th2 responses to rsv infection and predisposition to asthma. il-10 is a regulatory cytokine, which may promote an asthma phenotype by suppressing th1 cytokine production and antigen presentation promoting enhanced susceptibility to infection and th2-dominated responses that induce wheezing and pro-asthmatic responses to subsequent antigen challenge (bont et al., 2000a (bont et al., , 2000b . il-10 levels did not change during acute rsv disease but increased during convalescence and were significantly higher in subsequent wheezers and those that went on to develop recurrent wheeze and asthma. thus, the enhancement of il-10 and inhibition of il-12 production upon rsv infection may suppress immune function and permit more severe infection and disease progression. it has also been suggested that rsv infection may change and enhance the profile of pro-inflammatory cytokine and chemokine release that promotes more severe infection and alters the nature of the t cell response promoting allergy and inflammation. patients with rsv-induced bronchiolitis had elevated levels of mip-1î± (but not rantes), which correlated with disease severity in nasopharyngeal secretions and rantes, icam-1, il-4 and -5 and ige in serum (sung et al., 2001; garofalo et al., 2001 garofalo et al., , 2005 . furthermore treatment of human epithelial cells with tgf-î², which plays a pivotal role in airway remodelling and asthma, increased rsv replication and tnf-î± secretion and p38 mitogen-activated protein kinase activation. this may contribute to the elevated inflammatory responses in virus-associated asthma (mccann & imani, 2007). by contrast rsv-induced wheezing does not correlate with il-8 levels in nasal lavage fluid but is elevated with influenza or rv infection (gern et al., 2002) . thus it is possible that infection induces regulatory mechanisms that suppress immune responses allowing viral replication resulting in increased inflammatory responses. alternatively infection may induce inflammatory responses that enhance infection and allergic inflammation. this may subsequently promote the induction of regulatory mechanisms to limit inflammation-induced damage of host tissue. 2.2.4.3. other immune factors. in vivo and in vitro studies have shown that rsv infection of the respiratory epithelium causes the release of other factors that have immune functions including arachidonic acid metabolites and lts, mediators released by eosinophils and chemokines (reviewed in ogra (2004)). cell adhesion and homing molecules such as cd11b, icam-1 and e-selectin and antigen-presenting molecules including human leucocyte antigen classes i and ii are also upregulated upon infection. several transcription factors are also activated, which may induce the expression of a range of genes such as nk-il-6 and nuclear factor (nf)-kb. these factors regulate immunomodulatory mediators (tnf-î±, il-1î², il-2, -6, -11 and gm-csf), adhesion molecules (icam-1, vcam-1 and e-selectin) and chemokines (il-8, mip-1î±, mcp-1, eotaxin and rantes) (garofalo et al., 1996; oh et al., 2002; john et al., 2003; makela et al., 2003; schaller et al., 2006) . in combination these mediators and molecules induce the influx of inflammatory cells and may contribute to the development of infection-induced inflammatory and immune responses, ahr and asthma. ineffective or aberrant humoral responses may also have a role in rsv-induced asthma. infection induces increases in b cell numbers (roman et al., 1997) and the production of serum and mucosal igm, iga and igg antibodies. these are important in protection and not disease but occur at lower levels in infants. antibody responses to primary infection are ineffective and involve the production of partially neutralising antibodies against the g and f proteins but these responses are reinforced (especially igg and iga) upon reinfection. rsv-specific ige antibodies are also produced (welliver et al., 1980) in the majority of children and increased amounts and persistence promote the development of wheezing (ogra, 2004) . increased vascularity (angiogenesis) surrounding the airway wall is associated with chronic and fatal asthma but also with mild-to-moderate asthma in both children and adults (li & wilson, 1997; vrugt et al., 2000; barbato et al., 2006) , suggesting a pathogenetic role at all stages of asthma development. angiogenesis is regulated by a balance between pro-angiogenic (vascular endothelial growth factor (vegf), basic fibroblast growth factor (bfgf), angiogenins, chemokines) and antiangiogenic (endostatin, canstatin, tumstatin, arresten) factors (cohen, 2002) . vasodilation of the increased number of blood vessels in response to inflammatory stimuli may lead to edema and inflammation of the bronchial wall (influx of inflammatory cells, including eosinophils and release of mediators), airway narrowing and ahr (black & page, 1994; wilson, 2000; salvato, 2001; nomura et al., 2005) . elevated levels of vegf, bfgf and angiogenins occur in the airways of asthmatics and correlate with increased vascularity, vessel permeability and ahr (hoshino et al., 2001; kanazawa et al., 2004; nomura et al., 2005; feltis et al., 2006) . vegf influences vascular permeability through the formation of blood vessel fenestrations and vasiculo-vasculo organelles (dvorak et al., 1996; esser et al., 1998; neufeld et al., 1999) . vegf also stimulates endothelial cell proliferation and migration, matrix remodelling, and vasodilation, as well as inhibiting endothelial cell apoptosis and all of these processes are involved in angiogenesis (neufeld et al., 1999) . vegf also enhances sensitization of the rt to allergens and promotes th2 inflammation . rsv infection may contribute to the development of asthma by inducing the production of vegf and angiogenesis. vegf has been detected in nasal washings from rsv infected patients, indicating that infection stimulates vegf production, which may have a role in disease (lee et al., 2000) . furthermore, features of rsv bronchiolitis and pneumonia such as submucosal, adventitial, and interstitial edema are due to alterations in blood vessel permeability brought about by vegf activity (dvorak et al., 1996; esser et al., 1998; neufeld et al., 1999) . the specific role of vegf in rsv infection has not been intensively investigated. it is released from airway epithelial cells, th2 cells and mucosal fibroblasts upon infection (lee et al., 2000) but is also produced by monocytes, macrophages, t cells, keratinocytes, granulocytes, eosinophils and smooth muscle cells (gaudry et al., 1997; horiuchi & weller, 1997; neufeld et al., 1999) . thus a novel mechanism may be involved in rsv-induced asthma whereby the induction of vegf upon infection may lead to the development of angiogenesis that can enhance the inflammatory response. furthermore, rsv-induced secretion of vegf may contribute to exacerbations by increasing vascular permeability, and recurring infections may contribute to cycles of vegf production that promote angiogenesis and remodelling. neurological and immunological interactions that occur as a result of rsv infection have also been linked to the generation of airway inflammation, ahr and rad in children. excitatory non-adrenergic non-cholinergic nerves (nance) release neurotransmitters including substance p that participate in the early phase of the inflammatory response and also have an immunomodulatory role (piedimonte, 2002) . sloughing of the epithelium during infection may lead to exposure of neurogenic receptors (nk1), which enhances the pro-inflammatory effects of substance p leading to asm spasm and bronchoconstriction and contributing to respiratory symptoms. nerve growth factor participates in neuronal development and has beneficial effects on inflammation, repair and remodelling. however, it has been suggested that these effects may become pathogenetic during rsv infection and allergic inflammation and exacerbate inflammation and ahr (nassenstein et al., 2006) . the effect of co-infection of with rsv and other viruses has been little studied, however, notably the risk of developing bronchiolitis is 5 times greater in infants with co-infection with rv (papadopoulos et al., 2002a) . animal models of infection and allergic airways disease (aad) have been developed and used extensively to substantially contribute to the understanding of the mechanisms that underpin rsv-induced asthma and exacerbations. in particular rodent (mouse and to a lesser extent rat) and bovine models have been used to elucidate the mechanisms of the associations and to trial therapeutic agents and vaccines. chimpanzees are permissive to human rsv and are the best animal model but their availability and cost limits all but the most advanced clinical tests (whitehead et al., 1998) . using these models important factors have been identified that may play key roles in rsv-induced asthma and include; age of first infection, timing of infection relative to allergen exposure, induction of asthma, endotoxin exposure, innate factors and adaptive immunity, suppression of immunity, angiogenesis, neural networks and latent infections. the importance of the different viral proteins in infection has also been investigated. the use of animal models enables experimental protocols to be conducted that are not possible in humans. in particular the precise investigation of the different ages of infection, combinations of the timing of infection relative to allergic sensitization and collection of invasive tissue samples can only be achieved in animals. although there are problems with these models of rsv challenge including that rsv does not replicate in mice and does not induce the recruitment of granulocytes that is observed in human disease, such models have been used extensively to gain valuable insights into the mechanisms of rsv-induced disease. in most mouse models rsv infection induces significant acute respiratory inflammation and changes in lung function. high doses (10 7-8 plaque forming units (pfu)) induce severe alveolitis and pneumonia and low doses (10 5-6 pfu) result in bronchiolitis without these effects (dakhama et al., 2005a) . five days after infection mice develop acute airway obstruction, which correlates with the progression of inflammation and histopathology that is characterised by intense perivascular and peri-bronchial/bronchiolar influx of monocytes/ macrophages and some neutrophils and lymphocytes. these cells also occur in the alveoli during the peak of inflammation but leakage into the airway lumen is absent (jafri et al., 2004) . infection peaks at days 4-5 and resolves between days 7 and 9 and cytokines and chemokines (il-8, mips and rantes) are released by the respiratory epithelium and alveolar macro-phages in response to infection. after 3 days nk cells influx into the balf and are replaced between 4 and 8 days by cd4+ and cd8+ cells which return to pre-infection levels at 21 days (hussell & openshaw, 1998) . ctls induced in response to infection contribute to extensive peri-bronchiolar and -alveolar inflammation and are associated with disease symptoms (ostler et al., 2002) . ifn-î³ is important in viral clearance and contributes to pathology and is primarily released by nk cells with levels peaking at day 4. only low levels of il-4 and il-5 are present, however, il-4, -5, -10, -12, -13 and ifn-î³ are produced in the lungs within the first few days and their relative levels determines the course of disease (kalina & gershwin, 2004) . il-10 plays a pivotal role in the development of infectioninduced ahr in the absence of allergen exposure (makela et al., 2002) . acute infection develops into chronic disease with features of chronic inflammation (histopathology score) and ahr (in terms of enhanced pause (penh)) after the clearance of virus and up to 22 weeks after infection . penh is a noninvasive method of whole body plethysmography and uses a single exposure to a spasmogen to determine the overall level of ahr in all airways. jafri et al., used penh to show that airway obstruction and ahr remained for 42 and 154 days after infection, respectively, and that ahr correlated with chronic inflammation which persisted but not in alveoli (jafri et al., 2004) . this supports the concept that rsv disease may induce long-term respiratory changes in children (stein et al., 1999; sigurs et al., 2005) . however, these studies need to be confirmed by the measurement of lower airway resistance using invasive methods that precisely measure changes in airway and tissue specific function and employ dose responses to spasmogens. studies using these methods (measurement of respiratory impedance and airway resistance) have shown a lack of longterm effects of rsv infection on lung function (dakhama et al., 2005c; collins et al., 2007) . infection also induces mucus hypersecretion in the central and peripheral airways in the acute and chronic phases and severe and progressive pneumonia develops with increases in histopathology and chronic inflammatory changes. these processes contribute to airway obstruction (penh) that develops in the acute phase and progresses but does not correlate with viral load in balf, and this agrees with observations made in children (jafri et al., 2004) . the intensity of inflammation declines over time but remains around airways and vessels. neutralising monoclonal antibodies (mabs) against rsv substantially decrease inflammation and disease severity . using mouse models it has been shown that the age of first infection plays a key role in shaping dominant immune responses later in life (reviewed in hansbro et al. (2004) ) and establishes the subsequent pattern of th cell responses and the nature and severity of ensuing respiratory diseases (culley et al., 2002; holt & sly, 2002a; horvat et al., 2007) . primary rsv infection in neonatal mice has the same profile as in adults, however, it is associated with a slower and diminished ifn-î³ response and the development and persistence of th2 cytokine release by cd4+ t cells. these effects are substantial and can reverse the protective effect of mycobacterial exposure on aad . neonatal rsv infection results in early tnf-î± release and has long-term adverse effects on the respiratory system. these effects include the induction of ahr, peri-vascular and -bronchial inflammation and subepithelial fibrosis, which are exacerbated by subsequent allergen exposure and involve persistent il-13 expression and mucus hypersecretion (you et al., 2006) . the generation of th2 responses during immunological development has profound modulatory effects on the balance of subsequent th1/th2 responses and promote a marked increase in the th2 phenotype in adulthood (chen et al., 2001; walzl et al., 2001; culley et al., 2002) . the neonatal th2 bias may result from the types of t cells or dendritic cells present, their environment or a combination of these effects (nelson et al., 1994; goriely et al., 2001; white et al., 2002; bartz et al., 2003) . subsequent re-infection later in life may reinforce aberrant th2 responses (cytokines (il-13) and cd4+/cd8+ cells) and alter responses to allergens resulting in enhanced inflammation, mucus hypersecretion, ahr and allergy (enhanced weight loss and th2 cell and eosinophil recruitment to the airways) (chen et al., 2001; walzl et al., 2001; culley et al., 2002; dakhama et al., 2005b) . this suggests that aad of adults primed with an infection as neonates is likely to be caused by factors that promote th2 responses (culley et al., 2002; holt & sly, 2002a) . if initial infection is delayed until mice are 3 weeks of age ifn-î³ production increases and upon reinfection, although airway inflammation still occurs, there is a subsequent reduction in the severity of disease with no mucus production or ahr (culley et al., 2002; dakhama et al., 2005b) . thus delaying the age of infection until later in life may be an effective strategy for the prevention of rsv-associated asthma. animal models have been used to determine if rsv can induce the development of asthma by triggering pro-asthmatic immune responses that lead to variable airflow obstruction and airway inflammation. chavez-bueno et al. (2005) demonstrated that rsv induces acute and chronic disease with features of aad independently of allergic sensitization or genetic background in mice. rsv infection of balb/c or c57bl/6 mice in the absence of allergic sensitization led to similar levels of acute airway inflammation, airflow obstruction and ahr and the degree of airway inflammation correlated with ahr. the immune response was surprisingly similar between the two strains and was characterised by virus-dependent release of ifn-î³. importantly infection and inflammatory responses were not short lived. acute infection developed into persistent infection that correlated with airway inflammation, which were present 77 days after infection. while virus load and airway inflammation were greatest during the acute phase, chronic infection correlated with chronic inflammation and airflow obstruction. thus rsv infection can initiate acute events that result in airflow obstruction and possibly ahr and these changes can persist beyond the initial inflammatory response. other investigators have also shown that rsv infections in early life act synergistically with atopy to drive the development of allergic asthma (holt & sly, 2002b; kusel et al., 2007) . however, the immunological processes involved in the induction of allergic sensitization by rsv infection are not likely to be the same as those involved in exacerbation of allergic asthma by rsv. the effect of rsv infection on aads may be critically dependent on the relative timing of infection, allergic sensitization and challenge (peebles et al., 2001; barends et al., 2002 barends et al., , 2004 . the majority of studies in mice show that rsv augments aad, however, conversely some investigators suggest that rsv infection prevents atopy. indeed rsv induces a strong th1 and ifn-î³ mediated response that may modulate responses to allergens (peebles et al., 2001; juntti et al., 2003) . these contrasting observations can be explained as two competing immune responses are occurring simultaneously, which may subtly differ in different protocols. the development of allergy may depend on the phenotype of the immune response to allergens and rsv at the time of exposure. 2.3.4.1. allergen exposure prior to infection. prior exposure of the airways to allergen independently of allergen type predisposes to increased severity of virus-induced aad in mice and is likely to play a role in humans (peebles et al., 2001; makela et al., 2003; kalina & gershwin, 2004) . infection enhances th2 cytokine responses, mucus secreting cell hypertrophy, eosinophil influx into the lung and ahr in response to allergen and increases the severity of aad. the potency of the th2 responses elicited overrides the counterregulatory effects of th1 responses that are typically induced by infection (randolph et al., 1999) . 2.3.4.2. infection prior to allergen exposure. exposure of rsv infected mice or rats to allergens increases inflammation, mucus production and ahr and prolongs rsv replication (peebles et al., 1999; kalina & gershwin, 2004; hassantoufighi et al., 2007) . in particular cd4+ and cd8+, inflammatory responses in the lung are enhanced (lukacs et al., 2001; barends et al., 2004; schaller et al., 2006) . blocking il-13 during infection reduces chemokine expression and ahr and blocking rantes removes the effects of infection upon later allergen sensitization and challenge (lukacs et al., 2001) . the absence of the cc chemokine receptor 1 (ccr1) in deficient mice leads to reductions in t cells, il-13, eosinophils, mucus and ahr (john et al., 2005) . these observations suggest that chemokines and their receptors play roles in rsv-induced aberrant responses to allergens and may be important in mobilisation of virus-and allergen-specific t cells and allergic inflammation. it is possible that rsv infection may contribute to the development of allergy by damaging the respiratory mucosa, which exposes apcs and t cells to allergen, which may break tolerance and induce systemic th2 sensitization. excessive infection-induced respiratory damage may occur in predisposed individuals as a result of defects in t cell mobilisation, activation or activity. if allergen challenge of sensitized mice occurs concomitant with infection the inflammatory response is again enhanced and leads to th2 responses to rsvand promotes chronic infection (makela et al., 2002) . il-5 production leads to eosinophilia, il-13 to the release of mcp and further th2mediated inflammation but il-10 does not further enhance ahr. if rats are sensitized to extracts of the common household mould aspergillus fumigatus, which induces eosinophilia and th2 cytokine release, rsv infection before allergen challenge exacerbates the inflammatory response and ahr that is dependent on viral replication. infection causes increased expression of mhc ii on alveolar macrophages, which may be involved in initiating immune responses to allergens. persistence of infection is induced and is related to reduced ifn-î³ expression again suggesting that allergic sensitization can affect the progression of rsv infection (kalina & gershwin, 2004) . treatment of infected a. fumigatus challenged animals with recombinant ifn-î³ reduced allergic responses and th1 and th2 cytokines but not ahr. this suggests that pathological and physiological factors of disease are independent (hassantoufighi et al., 2007) . taken together these studies suggest that in general rsv induces increased severity of th2-mediated aad and that the development of aad increases the severity and persistence of rsv infection. some reports have suggested that exposure to endotoxin or environmental pollution can affect the progression of rsv infection (gurkan et al., 2000; monick et al., 2003) . exposure to such factors alters the relative proportions of cytokines produced upon infection and affects disease progression, however, the cellular and molecular processes involved are not understood. tlr-4 expression may provide a link between rsv, endotoxin and asthma. tlr-4 expression is not usual in resting airway epithelium and requires high endotoxin exposure to be upregulated. rsv infection induces increased expression of tlr-4 and responsiveness to endotoxin and initiates potent inflammatory responses (monick et al., 2003) . this may be linked to human asthma as endotoxin also induces asthma exacerbations in children with rsv-induced asthma (park et al., 2001) . animal models have been used extensively to elucidate the host immune responses that are induced by rsv rt infection and how these responses may contribute to the development and exacerbation of asthma. infection may inhibit or modulate the activity of both innate and adaptive (particularly t cell) immune responses during the development of disease, which may play a crucial role in the induction of pathology and aad. primary rsv infection induces innate responses that involve eosinophil and neutrophil influx into the lung that results in ahr and a cytokine response that is dominated by ifn-î³ (schwarze et al., 1999b) . eosinophil infiltration is il-5 but not il-4 or ifn-î³ dependent and is critical for the development of ahr. the innate response (first 3 days) is also characterised by an influx of nk cells producing ifn-î³, which are replaced by adaptive cd4+ and cd8+ cells and the release of il-12 with low levels of il-4, -5 and -13 (openshaw, 1995; boelen et al., 2000; van schaik et al., 2000; openshaw, 2001) . t cell responses facilitate viral clearance but also induce host tissue damage and pathology. infection induces innate responses involving tlrs, cytokines, chemokines and dcs that ultimately direct the development of adaptive t cell and antibody responses (durbin & durbin, 2004; krishnan et al., 2004) . 2.3.7.1. tlrs. rsv may use a variety of host cell factors to bind to and enter cells including tlr-4, cx3r1, heparin and caveolin (kalina & gershwin, 2004) . binding to tlr-4 initiates the production of il-6, -8 and -1î² and tnf-î± and may be responsible for the initial response to infection. however, the role of tlrs in virus-induced asthma is controversial. some studies report that the innate immune response to rsv infection in mice is dependent on the expression of cd14 and tlr-4 (kurt-jones et al., 2000; haeberle et al., 2002) , which may interact with the viral f protein (openshaw et al., 2003) . another report argues the opposite saying there is no significant role for tlr-4 in infection (ehl et al., 2004) . tlr-3 recognises double stranded (ds) rna and is constitutively expressed on respiratory epithelial cells and dcs. tlr-3 signals independently of myeloid differentiation factor 88 to induce nf-îºb activation and the expression of ifnî² activation of tlr-3 leads to apoptosis and elimination of infected cells and virus. recently it has been shown that tlr-3 and protein kinase r (pkr) are upregulated in human airway epithelial cells by rsv infection, which enhances epithelial responsiveness by activation of nf-îºb and il-8 and may sensitize these cells to subsequent viral or bacterial infection (groskreutz et al., 2006) . 2.3.7.2. chemokines. chemokines are produced by stromal, epithelial and immune cells and regulate immune responses (chemoattraction of leukocytes into the lung), inflammation, mucus production and angiogenesis. although cellular inflammation is often similar in response to different viral infections the types of chemokines and the levels that are released that drive subsequent immune responses differ substantially (schaller et al., 2006) . primary rsv infections induce the expression of chemokines belonging to the cxc (il-8, mip-2 and ip-10), cc (rantes, eotaxin, mip-1î±, mcp-1 and t cell activation gene-3) and c (lymphotactin) families in the lung. mip-1î± expression is high and mip-1î± deficient mice have reduced lung inflammation, but rsv titres that are the same as in wildtype mice. thus rsv-associated lung inflammation may be mediated by early production of inflammatory chemokines (haeberle et al., 2001) . 2.3.7.3. dcs. dcs are the most important antigen-presenting cell (apc) and take up viral antigens, traffic to local lymph nodes and present antigen to naã¯ve t cells causing their differentiation into effector cells. dcs direct innate and adaptive immune responses to viruses and allergens and are essential for allergic sensitization. different subsets of dcs exist with different functions. mdcs present viral antigens and promote th2 cell expansion to a great extent than pdcs, which are more important in the development of tolerance (van rijt et al., 2005) . dc networks are less active in infant animals but can be enhanced and mdcs are increased upon viral infection (holt & sly, 2002b; zuniga et al., 2004) . it is possible that the reduction of pdcs by conversion into mdcs during early life viral infection may inhibit the development of tolerance and exacerbate allergic responses. the outcome of rsv infection may depend on the nature of the adaptive response and the balance of th1 and th2 immunity. primary infection of balb/c mice induces a mixed th1/th2 response with an early burst of ifn-î³ release that is important in determining the phenotype of the subsequent response openshaw & tregoning, 2005) . other adaptive responses involving cd8+ cells and b cell/antibody responses also play significant roles in responses to rsv. 2.3.8.1. th1 cd4+ t cell responses. rsv infection typically induces a robust th1 response with elevated levels of ifn-î³ and il-12 in mice. ifn-î³ is the archetypal th1 cytokine and its release and signalling through the ifn-î³ receptor during infection are pivotal in controlling the th1/th2 response to infection. these processes are essential in moderating eosinophil migration and ifn-î³ and cd8+ cells are crucial for viral clearance. in the absence of ifn-î³ a dominant th2 response induces eosinophil influx of the lung and ahr (barends et al., 2003) . by contrast, the absence of il-12 and il-18 has little effect . il-12 does, however, induce th1 and suppresses th2 responses, promotes ifn-î³ production from nk and cd8+ cells, reduces il-4 and il-5 production from cd4+ and cd8+ cells and can prevent but is not essential for inhibiting virus-induced eosinophil influx to the lung (hussell & openshaw, 2000) . il-12 does not function through cd4+ or b cells and exacerbates disease in mice sensitized to allergen (openshaw et al., 2003) . the removal of cd4+ or the induction of cd8+ cells also eliminates eosinophil influx (hussell et al., 1997) . tnf-î± is another th1 cytokine and is over-produced during viral rt infections, which exacerbates inflammation by promoting neutrophil and eosinophil influx. anti-tnf-î± treatment of mice leads to ablation of weight loss and illness without affecting viral clearance and does not induce adverse side-effects, which indicates a potential for use in therapy (hussell et al., 2001 ). 2.3.8.2. th2 responses. th2 responses are induced by rsv infection in a variety of animal models or in the absence of ifn-î³ , which may contribute to the development of aad. these responses are potent and are similar to th2 responses observed after allergen exposure of allergic individuals (braciale, 2005) . rsv-induced ifn-î³ and il-12 responses do not diminish the th2 response during the development of rsv-induced allergy although the response is even greater in the absence of ifn-î³ (barends et al., 2003) . it is a specific set of t cells, a cd4+vî²14+ subpopulation, that induces a superantigen type response to rsv and is pivotal in inducing th2 mediated pathology (varga et al., 2001) . t1/st2 is a surface receptor of the il-1 family expressed on th2 but not th1 cells. t1/st2 was present on a subset of cd4+ t cells from mice with rsvinduced eosinophilia and t1/st2 mab treatment reduced th2 but not th1 pathology (walzl et al., 2001) . these studies suggest that under some circumstances infection may induce strong th2 responses but the mechanisms of how this leads to aad are unknown. il-4, -5, -10, -11 and -13 are th2 cytokines that are released during th2 responses and are likely to be involved in rsv-induced aad. in mice, primary rsv infection results in increased il-4 levels, however, it's importance in the development of th2 responses and aad is unclear. il-4 deficient mice or mice treated with a neutralising anti-il-4 and immunized with vaccinia virus expressing protein g had no reduction in pulmonary eosinophils or th2 cytokine secretion. furthermore infection of il-4 deficient mice resulted in enhanced numbers of eosinophils in the lung and ahr johnson et al., 2003) . by contrast il-5 release may be pivotal in rsv-induced th2 responses and aad. infection of mice results in il-5-mediated ahr and eosinophil influx into the lung in association with strong ifn-î³ responses. il-5 but not il-4 or ifn-î³ is the critical mediator of eosinophil influx, ahr and allergy (schwarze et al., 1999a) . il-5 deficiency leads to a reduction in pulmonary eosinophils and ahr, which can be reversed by replenishment with il-5. treatment with anti-very late antigen-4 prevents the influx of eosinophils into the lubg and ahr in response to rsv infection or il-5 replenishment. rsv infection induces the expression of il-10 in mouse pulmonary t cells (hussell et al., 1996) . the absence of il-10 inhibits the development of ahr in response to allergen sensitization and challenge. rsv infection overcomes this deficiency and induces eosinophil infiltration of the lung, airway mucus production and ahr, which are associated with increased th2 responses (makela et al., 2002) . il-11 is associated with the development of rsv-induced ahr and may promote the release of other th2 cytokines (einarsson et al., 1996) . although il-13 is required for the development of mucus hypersecretion and ahr in mouse models of aad and after secondary rsv infection of infected neonates (kuperman et al., 2002; dakhama et al., 2005b) it does not appear to play an important role in the induction of these responses after primary infection . this has not yet been investigated in humans. it is likely that as well as inducing a th2 phenotype rsv may take advantage of th2 responses that predominate in asthmatics that are ineffectual against infection. however, surprisingly rsv clearance can occur under th2 conditions whereby eosinophils take up rsv and inactivate the virus with ecp (soukup & becker, 2003) . il-10 increases fasl expression on macrophages and cd8+ cells and therefore also has an anti-viral effect (ruan et al., 2001) . cd8+ t cells target several rsv proteins and are sufficient to clear rsv from infected mice (cannon et al., 1988; cherrie et al., 1992) , however, clearance and immunopathology still occurs in cd8-deficient mice (graham et al., 1991) . adoptive transfer of virus-specific cd8+ ctls which home to the lung eliminates rsv. these cells induce viral clearance through perforin/ granzyme-mediated lysis of virus-infected cells (aung et al., 2001) . however, perforin (which is also produced by nk cells), cd95l and tnf are not necessary but ifn-î³ release is crucial for cd8+ t cell-mediated clearance. rsv was eliminated with unchanged kinetics from perforin deficient mice (aung et al., 2001; ostler et al., 2002) , whereas treatment of mice with neutralising antibody to ifn-î³ or transfusion of ifn-î³-deficient effector ctls abolished virus control and induced cd8+ t cellmediated pathology (ostler et al., 2002) . by contrast, high dose primary infection in ifn-î³ deficient mice led to attenuated immunopathology, but only slightly delayed clearance. this suggests that other cells and molecules can partially substitute for ctl-derived ifn-î³ driven virus clearance and further implicates ifn-î³ as a pivotal immune factor in rsv-induced immunopathology and cd8+ t cell-mediated control (ostler et al., 2002) . cd8+ cells may also suppress the development of virusspecific th2-dominated immune responses and eosinophilia although the mechanism of these effects remains unknown. hussell et al., showed that early ifn-î³ release by cd8+ cells in response to rsv infection of mice resulted in suppression of th2 responses (hussell et al., 1997) and the suppression of th2 responses by cd8+ cells is by other studies (srikiatkhachorn & braciale, 1997a) . interestingly the th2-inducing effects of the rsv g protein can be nullified by incorporating a cd8+ epitope onto the g protein (srikiatkhachorn & braciale, 1997a ). 2.3.10. cd4+/cd8+ interactions cd4+ and cd8+ t cells are exposed to presented viral antigens in the lymph nodes of the respiratory tract, which induces differentiation, activation and mobilisation of both effector and memory t cells. memory cd4+ t cells move to the rsv infected rt and proliferate and differentiate into cytokine releasing effector cells and induce their effects in situ (varga et al., 2000 (varga et al., , 2001 . during differentiation the cells are subject to infectioninduced modulation and it is likely that the cd8+/cd4+ interaction is occurring concurrently. similar interactions may take place during allergen provocation in the rt of asthmatics that have the potential to substantially affect t cell phenotype, an effect that may be enhanced upon infection. thus the complex interactions of cd4+ and cd8+ with infectious stimuli and allergens may be pivotally important in the development of rsvinduced aad. these processes may be targeted therapeutically to suppress allergen specific th2 responses in the lung. differential immune responses are induced by different rsv proteins, which may be important in the development or exacerbation of asthma. protein g vaccination of mice induces cd4+ but not cd8+ memory populations and leads to reduced nk cell influx and ifn-î³ production. this results in the induction of th2 responses, in the absence of a ctl response, with il-4 and il-5 release by th2 cells which promotes eosinophilia upon subsequent infection (alwan et al., 1994; srikiatkhachorn & braciale, 1997a; walzl et al., 2001; openshaw et al., 2003) . g protein defective rsv has been used to demonstrate that this protein is crucial in promoting airway inflammation and reduced lung function (schwarze & schauer, 2004) . f or m2 protein immunization induces a mixture of nk, th1 type cd4+ and mhc i cd8+ cells, leading to ifn-î³ production and reduced disease (alwan et al., 1994; srikiatkhachorn & braciale, 1997a; openshaw et al., 2003) . nk cells and the ifn-î³ they release regulate the induction and proliferation of cd8+ cells, which then clear the virus. primary rsv infection induces the expansion of activated m2 82-90 (h-2k d restricted peptide epitope in rsv m2 protein)-specific cd8+ t cells in lung. this implies that activation and proliferation of m2-specific cd8+ t cell precursors is normal. taken together these results suggest that natural infection that induces th2 responses may be dominated by responses to the g protein exacerbates infectious and allergic disease, whereas responses to the f and m2 proteins may be th1 mediated and reduce disease. antibody responses may have several roles in rsv-associated aad in mice. rsv infection may induce the development of proallergic ige antibodies and their receptors in the lung which may induce mast cell degranulation and ahr (dakhama et al., 2004) . the activation of anti-viral protein kinase upon infection may lead to isotype switching of b cells to produce ige (rager et al., 1998) . other studies have shown that exposure to allergen during acute rsv infection of mice results in the production of antigen specific igg1 responses, which are characteristic of th2 immunity (o'donnell & openshaw, 1998) . moreover non-neutralising antibody produced in response to formalin-inactivated (fi) virus may induce immune complex formation in the lung (openshaw et al., 2003) , which may be involved in pathogenesis. infants have poor t cell independent antibody responses and produce different types of antibodies compared to adults. early life infection with rsv may fix the nature of antibody production as well as t cell responses to subsequent infection throughout life and promote the development of ahr (openshaw et al., 2003). studies in mice have suggested that rsv may promote its own infection by suppressing host immunity resulting in enhanced inflammation. the rsv g protein may attenuate innate responses by binding to tnf-î± (valarcher & taylor, 2007) and the induction of cytokine production from monocytes and macrophages through the inhibition of tlr-4-nf-îºb mediated signalling (polack et al., 2005) . rsv-specific t cells may play a crucial role in viral clearance and limited evidence suggests that infection may suppress the activation and activity of t cells leading to enhanced viral replication and disease (harcourt et al., 2006) . during suppression the f protein of rsv may downregulate the activity of t cells and cytokine production including the release of ifn-î³ (kondo et al., 2004; schauer et al., 2004) . rt infection also suppresses ctl immunity through the rapid loss of virus-specific cd8+ memory cells and ifn-î³ release (chang & braciale, 2002) , which is mirrored in humans by the lack of the induction of immunological memory. this effect may occur during antigen receptor signalling and varies to different extents in different t cell functions (ctl activity, cytokine release). this may result in reduced ctl activity and allow viral persistence but may also enable the maintenance of cytokine responsiveness facilitating further virus-or allergen-associated inflammation promoting enhanced disease. despite this evidence immune suppression during infection has not yet been observed in humans. these effects may only be present in the lung and are not detectable in the blood (braciale, 2005) and it is unknown if cd8+ t cells with this phenotype exist in the rt of humans with severe rsv infection. rsv infection may also attenuate protective immune responses by suppression of type i ifn (ifn-î±,î²), modulation of dc activity, g protein mimicry of the cx3c chemokine, which may inhibit t cell migration and by producing viral variants that are not recognised by neutralising antibodies (tripp, 2004; meyer et al., 2007) . the viral elements responsible for dysregulated immune responses are not known. persistent rsv, other viral or bacterial infections, which are local or systemic, or underlying chronic lung disease may promote susceptibility to rsv-induced asthma. emerging evidence from both mouse and human studies suggests that rsv infections may persist at low levels, by mechanisms involving suppression of immunity and avoidance of immune detection and total clearance (seemungal et al., 2001; . this occurs in mice with functional neutralising antibody and ctl responses even though the virus possesses a cd8+ epitope and productive infection can be reactivated by depletion of cd4+ and cd8+ cells . in mouse models rsv is detectable by culture up to just 7 days after infection but by pcr for up to 77 days independently of genetic background and viral copy number correlates with ahr (tripp, 2004; chavez-bueno et al., 2005) . this association has not yet been proven but treatment with neutralising antibody reduces viral numbers and disease severity . however, it is possible that viral detection by pcr under these conditions may represent the persistence of viral debris following the administration of high viral inocula and may not be part of the disease process. it remains unknown whether viral persistence occurs in children following bronchiolitis and the development of wheezing and long-term pulmonary abnormalities. if persistent infection does occur chronic inflammation and altered immune (cytokine/ chemokine) responses may be induced in individuals with rsvinduced aad. thus persistent infection may be important in long-term morbidity and may provide a new therapeutic target. limited mouse and primate studies of acute and chronic asthma also link vegf with pathophysiological features of rsv infection and asthma suzaki et al., 2005; avdalovic et al., 2006; lee et al., 2006) . over-expression of vegf in the lungs of mice to levels found in asthma or during rsv infection, induces angiogenesis, oedema, inflammation, vascular remodelling, mucus cell hyperplasia/metaplasia and ahr . rat models of rsv-induced bronchiolitis have also been developed, where rats rapidly clear the virus in a similar manner to the self-limiting infection of human infants (piedimonte et al., 1999) . this model has been used to investigate the alteration of neural networks by rsv infection and infection of infant rats has enabled the analysis of the effects of early life infection (king et al., 2001) . a stronger neurogenic inflammatory response develops in the lrt of infant rats than in adult rats during infection, which may explain why bronchiolitis presents in infants but as an urt infection in older subjects. induction of inflammation involves the upregulation of nk1 in infected lungs (but not nk2 that is expressed on asm fibres) that are activated by nance nerves and mediate substance p induced immunomodulation and neurogenic and cellular inflammation that may lead to edema and obstruction (king et al., 2001; piedimonte, 2001; tripp et al., 2002) . nk1 receptors are also upregulated on t cells in bronchus-associated lymphoid tissue in response to infection, which may then be attracted into the airways and release pro-inflammatory cytokines in response to neurogenic stimulation by airborne irritants. recurrent stimulation may lead to persistent cycles of airway inflammation and obstruction. inhibition of nk1 or the administration of cgrp prevents rsv-induced ahr and may be potential therapeutic targets (dakhama et al., 2005c) . neurogenic stimulation by rsv infection also induces the release of lts from mast cells that induce mucus secretion (wedde-beer et al., 2002) . another alternative is that infection-induced damage may expose nerve endings and substance p and neurokinin a may then mediate asm contraction (jacoby, 2002) . other investigators have shown that rsv infection of rats and ferrets results in increases in cholinergic mediated contraction of asm and reduced inhibitory nance responses (larsen & colasurdo, 1999) . despite the vast array of literature describing studies of rsv infections in animal models, there is no single model which duplicates the pathological features of disease observed in human infection. the major drawback in studies of rsvinduced disease in mice is that rsv is not a natural mouse pathogen. symptoms induced upon infection are minimal compared to those observed in human infants, the virus has limited replication and infected animals show few if any signs of respiratory illness (domachowske et al., 2001) . furthermore, large doses of the virus are required and primary infection is rapidly aborted . such discrepancies between human disease and mouse models have severely hampered the investigation of rsv-induced disease. the pneumonia virus of mice (pvm) is the closest genetic relative of rsv and is a natural mouse pathogen (easton et al., 2004) . unlike rsv, pvm infection of mice reproduces many of the acute inflammatory responses described for rsv infection in humans. pvm replicates rapidly inducing inflammation leading to mucus plugging of the airways and overt signs of disease from urt symptoms to fatal pneumonia, which is dependent on the administered dose easton et al., 2004) . virus replication is associated with an influx of granulocytes and severe inflammatory bronchiolitis. we have recently established mouse models of rsv-like disease using pvm infection of neonates and adults . importantly neonatally infected mice also exhibit many of the symptoms observed during rsv disease of human infants harrison et al., 1999; rosenberg et al., 2005) . these models are now being utilised to more precisely elucidate the host pathogen relationships that result in rsvinduced asthma. the importance of inflammation in inducing disease has been demonstrated and inflammatory responses remain active after the cessation of viral replication. mip-1î± and its receptor ccr1 play pivotal roles in these inflammatory responses. a recent study using pvm in wild-type and tlr-4 deficient mice showed that there is no difference in the clinical, functional, histological and virological parameters investigated indicating that pvm infection is independent of tlr-4 signalling (faisca et al., 2006) . anti-viral therapy with ribavirin alone has little effect, however, treatment with ribavirin in combination with the anti-inflammatory agent and the ccr1 antagonist met-rantes substantially reduces morbidity and mortality following infection (rosenberg et al., 2005) . bovine rsv, pathogenesis and vaccine development have recently been reviewed (meyer et al., 2007; valarcher & taylor, 2007) . bovine rsv is closely related to human rsv and is the most common cause of lrt disease and the major single health problem in calves worldwide, particularly in winter (stott et al., 1980; valarcher & taylor, 2007) . indeed 60-70% of epizootic respiratory diseases in the first year of life are attributable to bovine rsv with mortality typically between 2 and 3% but reaches 20% in some outbreaks (meyer et al., 2007) . models of bovine lrt infection have been developed and used to investigate responses to infection and evaluate bovine vaccines. bovine rsv is a natural pathogen and pathogenic infection of cattle shares many similarities with rsv infection in humans (van der poel et al., 1994) . infection is largely restricted to respiratory epithelial cells but causes little cytotoxicity and pathology is mediated by inflammatory responses to infection (viuff et al., 2002; valarcher & taylor, 2007) . these responses also involve innate (neutrophil and macrophage recruitment) and adaptive (pro-inflammatory cytokine and chemokine release) immune responses that result in respiratory damage. the mechanisms of pathology have been investigated using genetic manipulation of the virus and have implicated the g and sh proteins in infection and the f protein and non-structural (ns) proteins in inflammatory responses (reviewed in valarcher & taylor (2007) ). 2.3.18.1. pathogenesis. bovine rsv is transmitted by direct contact, in airborne droplets or is possibly transferred passively by humans (hall et al., 1980; mars et al., 1999) . pathogenic infection of the rt is much more common in calves than adults (stott et al., 1980) , which results from a lack of specific immunity in naã¯ve animals. maternally-derived antibodies afford some protection but primary infection induces the most effective immunity (kimman et al., 1987) . infectious disease takes 2-5 days to develop and has similar clinical features to humans and may possibly result in persistent infection (valarcher et al., 2001) . disease of the urt induces mild symptoms of coughing and mucus production. in the lrt bronchiolitis and bronchopneumonia occur in association with edema, wheezing and dyspnea (verhoeff et al., 1984; belknap, 1993) . infection of cattle induces inflammation involving the influx of mononuclear cells, neutrophils, cd4+ (of mixed th1/th2 phenotype) and cd8+ cells and sometimes eosinophils into peri-bronchial regions along with necrosis and apoptosis of epithelial cells (viuff et al., 2002; antonis et al., 2006) . cd8+ ctls have a major role in the clearance of primary infection in calves and lymphocyte proliferation is attenuated by infection in vitro (keles et al., 1998; antonis et al., 2006) , whereas antibody-mediated responses are important in protection against secondary infection. the lumen of the airways become occluded with mucus and inflammatory debris and remodelling events occur that lead to breathing difficulties (kimman et al., 1989; viuff et al., 2002) . it is likely that similar immune responses are elicited as in human infection with rsv and involve the induction of nf-kb and the generation of pro-inflammatory cytokines and chemokines. the interaction of viral components (f protein and dsrna) with tlrs is known to drive the development of the immune response in cattle (reviewed in valarcher & taylor (2007) ). as in humans and mice there are age-related differences in immune responses to infection in cattle with reduced protective and enhanced inflammatory responses in younger animals. in response to primary infection younger calves have enhanced fever, virus-specific tnf-î±, il-6 and ifn-î³ release from pbmcs and reduced peripheral blood mononuclear and b cells and virus-specific iga and neutralising antibody responses (grell et al., 2005) . the immunology and pathogenesis of bovine rsv infection has been intensively studied using reverse genetic engineering of the virus, which is less variable that human rsv and the results may be extrapolated to human disease (collins et al., 1995) . the importance and roles of different bovine rsv proteins in the induction of pathogenesis and immune responses have been elucidated using these genetically manipulated viruses. the g protein of rsv is the major attachment protein and deletion mutants do not replicate in the absence of the g proteins in vivo, although replication is unaffected in vitro (karger et al., 2001; schmidt et al., 2002) . bovine viruses expressing only the secreted segment of the g protein also have 100 fold attenuated replication, which is 10 fold higher than mutants missing the entire g protein (teng et al., 2001) . mutants expressing the membrane-anchored g protein segment have no impairment of replication in the urt but were attenuated at least 10 fold in the lrt and did not induce pathogenic responses in the lung (maher et al., 2004) . this suggests that both fragments of the g protein are important in infection and pathogenesis with the secreted portion playing a particularly significant role. cleavage of the bovine f protein is required for activation and results in the production of the peptide and tachykinin, virokinin that induces the production of nks, substance p and other neurogenic pro-inflammatory mediators. virokinin itself induces asm contraction and therefore directly contributes to airflow restriction but does not have chemotactic properties (zimmer et al., 2003) . by contrast cleavage of human rsv does not produce tachykinins (valarcher et al., 2006) . deletion of various parts of the bovine f protein has shown that it is not necessary for proliferation in vivo but deficient mutants promote substantially reduced airway inflammation and eosinophil influx (valarcher et al., 2006) . the f protein is responsible for suppressing lymphocyte proliferation (schlender et al., 2002) , however, there is no effect on the expression of the eosinophil attractants rantes or mip-1î± in the absence of the f protein suggesting that other mechanisms may be involved in the suppression of eosinophil influx in cattle (valarcher et al., 2006) . ns proteins, particularly ns2, of bovine rsv downregulates the ifn-î±/î² response involving the attenuation of irf3 and stat-2 (schlender et al., 2000) , which has subsequently also been shown with human rsv (spann et al., 2004 (spann et al., , 2005 lo et al., 2005) . the lack of ns proteins results in highly attenuated replication and a lack of pathogenesis in vitro and in vivo (whitehead et al., 1999; valarcher et al., 2003) . the sh protein is not required for replication of bovine rsv in vitro (karger et al., 2001) but replication is attenuated in the lrt of chimpanzees and calves in vivo (whitehead et al., 1999; valarcher & taylor, 2007) . epidemiological studies have widely linked rsv infection to the development of bronchiolitis, wheeze, decreased lung function and possibly asthma in childhood. this association is equivocal later in life but may depend on the severity of the infection. there may also be a role for rsv in the induction of sensitization to allergens and allergy may predispose to more severe infection. enhanced severity of symptoms that result from infection may be a useful prognostic marker for the future development of allergic disease. both human and animal studies have investigated the mechanisms of how infection induces pathogenesis and how this is associated with asthma. the age of first infection is crucial in determining clinical outcomes and early infection may promote the development of a pro-allergic phenotype. this indicates that delaying the age of infection until later in life may be an effective therapeutic strategy. infection induces both innate and adaptive immune responses that promote th2 immunity and eosinophil influx into the lung that may contribute to the development of ahr. the timing of infection relative to allergen exposure and the immune phenotype of the individual may be significant in determining the effects of infection. an interesting novel concept is that rsv infection may induce the development of angiogenesis, which might exacerbate inflammatory exudation, edema and bronchial obstruction in asthma. other new hypotheses are that rsv may induce immune suppression allowing viral persistence and latent infections that can be reactivated. neural networks also have the potential to be involved through the induction of neurogenic inflammation. rvs are small non-enveloped ssrna viruses of the picornaviridae family and to date over 100 different serotypes have been identified (savolainen et al., 2002) . epidemiology -rvs are responsible for the majority of cases of urt infectious disease in humans particularly colds and have recently also been implicated as the cause of significant numbers of lrt infections. rv is also the most common respiratory viral pathogen that induces wheeze at all ages (kusel et al., 2006) . a third of all episodes of acute respiratory illness involve wheezing in both children and adults and rv infections are implicated in 3 times as many cases as other viruses. wheezing may then progress to the development of asthma. the mechanisms of pathogenesis of rv are much less well understood than for rsv. pathogenesis -rvs are divided into major and minor classes based on receptor binding properties. major group rvs bind to icam-1 while minor group viruses bind to very lowdensity lipoprotein receptors (ldlpr) (fig. 1) (dreschers et al., 2007) . binding of rv to ldlprs induces a conformational change in the capsid that is required for viral uptake. intracellular internalisation occurs through the activation of sphingomyelinase that generates ceramide-rich membrane rafts, which facilitate uptake. rv is not thought to replicate in lrt, however, it is possible that rv can infect the lrt to a certain extent or alternatively that immune responses to urt infection has knock-on effects on inflammation in the lrt. infection of the urt typically causes coryza and pharyngitis and virus can be detected in fluids from the nasopharynx, tonsils and the middle ear (holgate, 2006) . recent epidemiological studies correlating rv with asthma are shown in table 3 . rv is more strongly linked with exacerbations of asthma although it is emerging that rv may also be important in asthma induction. there is a strong association between rv infection and childhood wheeze and exacerbation of asthma that may lead to hospitalization (el-sahly et al., 2000; jartti et al., 2006) . rv is implicated in wheeze in 80% of children n3 years of age and 45-80% of adults and is responsible for 60% of cases of asthma exacerbations nicholson et al., 1997; atmar et al., 1998; freymuth et al., 1999; tan, 2005) . interestingly the peak of severe asthma exacerbations in children occurs shortly after their return to school after breaks and in adults one week later and this coincides with peaks in rv infections in the spring and fall (longini et al., 1984) . indeed in the fall rv was present in 29% and 52% of children that were asymptomatic or were having asthma exacerbations, respectively . while rsv may be an important factor in early childhood wheeze, the majority of children become infected with rsv, but most do not go on and develop asthma, suggesting that other factors are important in this process. to investigate this lemanske et al. (2005) prospectively recruited a cohort of children (285) at risk of asthma based on family history and followed them for 3 years to determine the relationship between viral infections and symptoms of asthma, confirming infective episodes and etiology by pcr on nasopharyngeal aspirates. at 3 years non-infective factors such as exposure to environmental tobacco smoke, the presence of asthma in an older sibling and ige-mediated food allergies were associated with persistent wheeze. rsv infections were also linked with an increased risk, however the strongest independent predictor of persistent childhood wheeze was an episode of rv infection associated with wheeze (or = 10, 63%). this suggests that rv infection in lemanske et al., 2005 infancy is associated with the development of asthma later in life. others agree and have shown that rv infection is the primary risk factor for wheezing in children under 3 years but that atopy is more important thereafter (heymann et al., 2004) . other recent studies have also indicated that recurrent wheezing in infants is promoted by rv and possibly to a greater extent than rsv (lehtinen et al., 2007) . a pathophysiological link between rv and asthma is indicated by the tendency of children infected with rv to be older than those infected with rsv (13 months vs 5 months) and an increased frequency to present with atopic dermatitis and blood eosinophilia (korppi et al., 2004a) . in the study by korppi et al., children (81) hospitalized for wheezing before the age of 2 were assessed by bronchoscopy and had a prior median symptomatic period of 6 months. of these children 40% were found to be asthmatics by the age of 3 years, and 90% of these were atopic. early predictors of asthma were atopic dermatitis, specific ige and inhalant allergy and rv was common during wheezing (58%). another recent study of infants (192) under the age of 3 hospitalized with bronchiolitis identified rv and rsv in 21% and 30% of cases, respectively (jacques et al., 2006) . it is unknown if asthmatics are more likely to develop colds, or if they are at a greater risk of more severe colds. corne et al., attempted to investigate this by recruiting couples, one of whom had asthma and determining if the asthmatics were more at risk of developing colds. they found that those with asthma were no more likely to develop colds (10% vs 9% of controls), but they were substantially more likely to develop lrt symptoms associated with these colds (43% vs 17%). this implies that viral infections in asthmatics are more likely to increase lrt inflammation (corne et al., 2002) . other investigators have shown that asthmatic infants hospitalized with wheezing have a greater chance of testing positive for rv than non-asthmatics, which suggests that asthma sufferers may also be more susceptible to infection (xatzipsalti et al., 2005) . furthermore, rv infection may be more long lasting in asthmatics and may persists for up to 6 weeks after hospitalization for acute asthma exacerbation and rv-induced ahr may also last for several weeks (kling et al., 2005) . experimental rv infection of human volunteers has enabled the study of the effects of infection on immune responses, lung function and asthma as well as the role of atopy and timing of infection on disease outcomes . infection upon binding of rv to icam-1 (greve et al., 1989) not only contributes to pathogenesis but also to the induction of allergic inflammation (canonica et al., 1995) . rv also induces the upregulation of icam-1 by activation of nf-îºb, which promotes further infection (papi & johnston, 1999) . rv infects the bronchial epithelial layer and some mononuclear cells and fibroblasts and increases airway inflammation, with changes observed in induced sputum and endobronchial biopsy. the bronchial epithelium of asthmatics is particularly susceptible to the cytotoxic effects of viruses (papadopoulos et al., 2000; mosser et al., 2005) . initial infection initiates rv-induced exacerbations and cytotoxicity leads to increases in the penetrance and effects of allergens. 3.2.1.1. immune responses. in both asthmatics and non-atopic non-asthmatics inflammatory changes involving almost all types of inflammatory cells are observed upon rv infection. in mild to moderate asthma, infection induces increased levels of il-8 and neutrophils in sputum, promotes the influx of eosinophils, mast cells, cd4+ and cd8+ cells into the airway wall and enhances ahr (fraenkel et al., 1995; grunberg et al., 1997a; van benten et al., 2001) . de kluijver et al., showed the inflammation upon infection may be less intense in the nonasthmatic, although these differences were small and no direct comparison with asthmatics was made at the time (de kluijver et al., 2002) . nevertheless reduced ifn-î³ responses in subjects with asthma may promote susceptibility to infection and there is an inverse correlation between ifn-î³ levels and rv persistence and symptoms in asthmatics . this deficiency may result from either the lack of recruitment of appropriate inflammatory cells or a reduced response from these cells. other differences in immune responses to rv infection have also been observed that may be involved in increased susceptibility of asthmatics to infection or enhanced inflammation. in subjects with acute rv-induced asthma wark et al., found that increased serum ip-10 levels but not rantes or il-8 was specifically associated with infection and correlated with the degree of airflow obstruction (wark et al., 2007) . these subjects also had less bronchodilatory response (increase in fev 1 ) to salbutamol compared to non-infective acute asthma, which correlated with levels of ip-10 in serum. ip-10 is a chemokine that binds to cxcr3 and is important in the recruitment of t cells to infected tissues. increased levels of ip-10 can activate mast cells and cause their migration. mast cells that are resident in the asm are specific for asthma and also express cxcr3. therefore, rv infection of the bronchial epithelium may lead to an early release of ip-10 that sets off a chain of events that enhance pre-existing asthmatic airway inflammation and promotes the migration and activation of mast cells into the asm thereby worsening bronchoconstriction and reducing the response to bronchodilators. nasal secretions of experimentally infected healthy volunteers contain increased levels of il-1î², a pro-inflammatory cytokine (proud et al., 1994) , while rv infection of subjects with allergic rhinitis or asthma, or in children with virusinduced asthma, resulted in elevated levels of g-csf and il-8 as well as increased blood and nasal neutrophilia (teran et al., 1997; gern et al., 2000) . have demonstrated that rv infection worsens airway inflammation and obstruction and enhances ahr to non-specific bronchoconstrictors in subjects with asthma or atopy. however, despite showing an ability to infect these subjects and recovery of virus, significant exacerbations of disease are not observed (grunberg et al., 1997a) . while treatment with inhaled corticosteroids (icss) effectively reduces ahr and even airway eosinophilia, it has no effect on virus-induced airway inflammation. this suggests that in the acute phase there is a disparity between these features of disease. epidemiological studies also link rv infection with reduced lung function. for example, a recent study detected rv in the respiratory epithelium in 45% of infants with recurring respiratory symptoms (201, 3-26 months) which was associated with abnormal lung function (decreased airways conductance, 86%) compared with rv negative infants (58%) (malmstrom et al., 2006) . it is possible that the serotype of rv involved may be important and some strains such as rv16 are particularly associated with reduced lung function and increased ahr and symptoms of asthma and rhinitis (peebles & hartert, 2000) . to determine whether atopy influences infection and worsening of ahr, xepadaki et al., studied a group of asthmatic children dividing them by atopy (xepapadaki et al., 2005) . in both groups ahr increased 10 days after a cold and remained elevated for 5-11 weeks. where the groups differed was that atopic children experienced more symptomatic colds and acute exacerbations, leading to a cumulative affect that resulted in the persistence of ahr. therefore, increased susceptibility of atopics to infection was demonstrated and recurring inflammatory insults were implicated as an indirect cause of more severe ahr. 3.2.1.4. timing of infection. as with rsv the timing of infection relative to allergen exposure may also be important in determining the outcome of rv infection in asthma. the severity of asthma exacerbations increases if infection occurs simultaneously with allergen exposure in asthmatics (green et al., 2002) . however, infection after allergen exposure has little effect on ahr (de kluijver et al., 2003) . numerous important studies have used a variety of in vitro cell culture systems to provide valuable insights into the pathogenesis of rv and its association with asthma. specifically the role of cytotoxicity, immune responses, ahr and remodelling involving angiogenesis have all been implicated. importantly it has now become clear that viral infection directly influences lower airway inflammation in asthma and that asthmatics may have a defective innate response in bronchoepithelial cells (becs) to rv. the bec is the first point of contact between infecting viruses and the host and plays an important early role in initiating innate and adaptive immune responses and inflammation (bals & hiemstra, 2004 ). there is conflicting evidence surrounding the nature of the cpe of rv infection on epithelial cells. while some studies have found that rv induces considerable cpe albeit with serotype dependent variation (schroth et al., 1999; papadopoulos et al., 2000) , others report none or very little cpe in vitro or in vivo (winther et al., 1984 (winther et al., , 1990 ). the magnitude of immune responses as well as differential regulation of different innate and adaptive components has been implicated in the increased susceptibility of asthmatics to rv and in rv-induced asthma and exacerbations. upon rv infection of asthmatics more intense inflammatory reactions involving mucus production, the influx of neutrophils, eosinophils, activated macrophages, cd4+ and cd8+ t cells into the rt and pro-inflammatory mediator release are associated with more severe and persistent lrt involvement and symptoms and delayed repair edwards et al., 2006; inoue et al., 2006) . although there are similarities in the cellular immune responses to rsv and rv infection there are significant differences in the levels and types of cytokines and chemokines released and the cell types that produce them. ineffective presentation of viral antigens also occurs in asthmatics, which may impair immunity and lead to asthma symptoms (parry et al., 2000) . many authors have used epithelial cell culture models to demonstrate that rv induces the release of pro-inflammatory mediators and it is possible that differential regulation of these factors may be important in rv-associated asthma. rv infection promotes the production of oxygen radicals in the bronchial epithelium and leads to the increased expression of nf-îºb and enhanced pro-inflammatory responses (contoli et al., 2005) . the activation of p38 mitogen-activated protein kinase by rho a has been shown to play an important role in these processes (dumitru et al., 2006) . rv infection of human cell lines relevant to the rt and asthma has demonstrated that the production of many different pro-inflammatory cytokines, chemokines and molecules is induced including; eotaxins 1 and 2, il-1î², -4, -6, -8, -16, ena-78, ip-10, gm-csf, tnf-î±, rantes, mcp-1, mip-1î± and icam-1 (subauste et al., 1995; grunberg et al., 1997a; johnston et al., 1998; yamaya et al., 1999; gern et al., 2000; papadopoulos et al., 2000; suzuki et al., 2001; papadopoulos et al., 2001; hosoda et al., 2002; konno et al., 2002; gern et al., 2003; hall et al., 2005; edwards et al., 2006) . of particular importance is the increased production of il-6, -8 and -16 and eotaxin, ip-10 and rantes, which are released upon rv infection of becs . il-6 and il-16 participate in the maturation of b and t cells and eosinophil and macrophage chemotaxis, respectively. rantes and eotaxin are also powerful chemoattractors of eosinophils and drive airway eosinophilic inflammation . il-8 and ena-78 and rantes and ip-10 are potent neutrophil and t cell chemoattractants, respectively . il-8 is also linked with reduced lung function in experimental rv infection, suggesting that chemokines may be involved in asthma exacerbations (grunberg et al., 1997b) . importantly ip-10 and rantes are the chemokines released in the greatest quantities from bec infected with rv (wark unpublished). moreover, when becs were treated with dexamethasone ip-10 was only inhibited at the maximum dose, although were inhibited at all doses. this is in keeping with experimental findings that moderate doses of icss are effective in reducing ahr and infiltration of eosinophils, but do not prevent the accumulation of cytotoxic t cells. therefore rv infection may induce neutrophil and t cell influx that may exacerbate allergic inflammation and which may be refractive to ics treatment. 3.2.2.4. ifns. the release of type i and iii ifns from becs are important innate responses to infection. they have anti-viral properties and play roles in apoptosis of infected cells and directing adaptive immune responses to clear the virus (takaoka et al., 2003; wark et al., 2005) . asthmatics have recently been shown to be particularly susceptible to the development of rv lrt infections as a result of reduced ifn-î± responses. this was shown in vitro by demonstrating that peripheral blood mononuclear cells (pbmcs) from both children and adults with asthma responded to rv inoculation with a reduced release of ifn-î± (corne et al., 2002; gehlhar et al., 2006) . wark et al. , have established a model of acute rv infection of the airway epithelium, using primary becs (pbecs) acquired by bronchoscopy and cultured in vitro. asthmatic pbecs allow a significantly greater replication of rv compared to controls. the innate response of asthmatic pbecs to rv is deficient in the release of ifn-î² (wark et al., 2005) , which promotes susceptibility to infection. this defect directly impairs the ability of the infected host cell to undergo early apoptosis and allows increased virus replication and ultimately cytolysis of infected cells. the defect is the result of altered intracellular signalling and not icam-1 expression or viral internalisation. the administration of exogenous ifn-î² induces apoptosis and suppresses viral replication, which identifies the potential for therapeutic intervention (wark et al., 2005) . as ifn-î² is released mostly by becs, little ifn-î² was detected in the balf of individuals experimentally infected with rv. further confirmation of the importance of these pathways was provided when microarray analysis of rv-infected differentiated becs identified 48 genes that were upregulated and most of which are involved in type i ifn pathways. the expression of these genes could be blocked by anti-ifn-î² mab or a dsrna inhibitor (chen et al., 2006) . rv infection of pbecs also induces the release of the novel immune mediator and type iii interferon, ifn-î», which also has anti-viral effects on rv replication (contoli et al., 2006) . again this group demonstrated that asthmatic pbecs had a markedly deficient ifn-î» response to infection with rv compared to healthy volunteers. the importance of the early response to infection was confirmed in vivo by experimental rv infection of volunteers; those with the greatest impairment of early release of ifn-î» to rv had significantly increased virus replication, severity of cold symptoms, airway inflammation and a more severe fall in lung function (contoli et al., 2006) . it has been suggested that defective innate immune responses result in deficient adaptive th1 responses. in support of this pbmcs from asthmatics have been shown to have deficient ifn-î³ responses to rv (wark et al., 2005) . others have made similar observations and demonstrated time and dose dependent increases in ifn-î³, il-12 and il-10 following rv infection of pbmc (papadopoulos et al., 2002b) . cells from normal subjects produced higher levels of ifn-î³ and il-12, while those from atopic asthmatics predominantly produced il-10 (papadopoulos et al., 2002b) . 3.2.2.5. tlrs. tlr-3 recognises rv and is upregulated upon infection, which leads to nf-îºb expression (groskreutz et al., 2006) . if tlr-3 is blocked anti-viral responses are suppressed and rv replicates and is released (hewson et al., 2005) . pkr is released and promotes the generation of pro-inflammatory il-6, -8 and rantes. asthmatics may be deficient in tlr-3, which may predispose to reduced immune responses and increased and persistent rv infection that may lead to enhanced viral cytotoxicity (hewson et al., 2005) . 3.2.2.6. th2 responses. th2 responses that are typically elevated in asthmatics including those involving il-4, increase the expression of icam-1 on airway epithelium, which promotes increased rv infection (bianco et al., 1998) . icam-1 may participate in eosinophil and t cell influx into the lrt in asthma. elevated th2 along with reduced th1 responses may promote susceptibility to rv infection and more frequent viremia in subjects with acute asthma exacerbations (xatzipsalti et al., 2005) . 3.2.2.7. ahr/remodeling. while viral infections initiate lower airway inflammation and asthmatics appear more susceptible these observations do not provide a link that infections increase the severity of ahr or long-term airway remodelling. infection of pbecs clearly will initiate the release of pro-inflammatory mediators that will enhance the recruitment and trafficking of inflammatory cells to the airways. however, it is unknown whether this will affect remodelling, either directly by influencing asm or indirectly by affecting other features of remodelling in chronic asthma. to determine if rv infection could directly influence remodelling, in vitro culture models of pbecs and fibroblasts from subjects with mild or moderate asthma and healthy controls have been employed. fibroblasts have the potential to play a critical role in remodelling and changes in the airway matrix (holgate et al., 2004) . infection of pbecs and fibroblasts from these subjects does not lead to the release of the known mitogenic factors tgf-î² or endothelin-1. culture of fibroblasts with media taken from pbecs that had been infected with rv also did not induce the release of these mediators. there was also no increase in the expression of the contractile protein alpha smooth muscle actin, which may be expressed if these cells develop a contractile phenotype and there was no change in cellular morphology. these results suggest that rv infection does not induce the release of proremodelling factors from these cells. however, exposure of fibroblasts to both rv directly and rv conditioned pbec media did promote inflammation with the induction of inflammatory mediators, similar to those observed in becs. this observation agrees with the results of oliver et al., who demonstrated that asthmatic asm cells released significantly more il-6 upon infection even though the virus does not replicate in these cells (oliver et al., 2006) . limited data suggest that rv may be involved in the generation of angiogenesis and remodeling. infection induces the release of vegf and fgf and other fibrosis and angiogenesis inducing factors from primary airway epithelial cells, epithelium and fibroblast cell lines and these factors have also been detected in rv-infected nasal secretions (ghildyal et al., 2005; de silva et al., 2006; psarras et al., 2006; volonaki et al., 2006) . psarras et al., demonstrated that rv infection of becs led to the induction of vegf and that when endothelial cells were treated with the medium of infected becs they began to form tubules and to proliferate. these effects were inhibited by anti-vegf and enhanced when the endothelium was exposed to th2 cytokines . rv research and the development of therapeutic strategies have been severely hampered by the lack of a small animal model with which to investigate disease pathogenesis and the induction and exacerbation of asthma by rv. mice do not express icam-1 that can be used as a receptor by major group human rvs and these viruses do not establish infections in mice. minor group rvs, however, can infect airway epithelial cells of mice by binding to ldlprs (dreschers et al., 2007) . a recent report (newcomb et al., 2006) , describes a mouse model of minor group rv infection with rv1b. virus persists in mouse lungs and induces neutrophilic inflammation in the airways involving mip-2 expression whereas major group rv does not. this model has since been used to demonstrate that rv infection induces mucin production in vivo and that mucin production in a model of murine aad is also increased by rv infection (bartlett et al., 2007) . rvs are the most common cause of common colds and urt illness and have recently been implicated in many lrt episodes. rv infections are also the commonest cause of wheezing and asthma exacerbations, particularly in individuals n 3 years of age. rv-induced wheezing may lead to the development of asthma and recent evidence suggests that rv may be the most important risk factor for wheeze and asthma in early life. as is the case with rsv, asthmatics may be more susceptible to rv and are more likely to suffer from more severe rv disease. in vivo and in vitro studies have been used to demonstrate that rv infection exacerbates airway inflammation, obstruction and ahr and atopic status and timing of infection may have important effects. these studies show that the magnitude and phenotype of the immune response to rv has a major impact on asthmatic outcomes. importantly in vitro studies have identified defects in innate (type i and iii ifns) and adaptive responses from cells from asthmatics to rv infection. these defects may promote susceptibility to infection and contribute to the asso-ciation between rv infection and the induction and exacerbation of asthma. in order to prevent virus-induced asthma and exacerbations we require a better understanding of predisposing factors for viral diseases and their association with the causation and exacerbation of asthma. the development of effective anti-viral agents and the conduct of large-scale prospective and intervention trials are also required to assess the potential of such strategies to prevent these diseases (wennergren & kristjansson, 2001) . in asthmatics rsv and rv infection directly enhanced airway inflammation and worsened airflow obstruction. much of this occurs as a result of the immune responses, which develop rapidly upon infection. for example, responses to naturally occurring colds due to rv infection results in the development of symptoms within 12 h which peak at 2-3 days but have resolved within a week (gwaltney et al., 1996) . the rapid response to infection, the often relatively mild symptoms that occur and brisk resolution determine that it is difficult to devise interventions that will be of substantial clinical benefit. in the case of asthmatics where acute exacerbations are triggered by virus infection the clinical consequences are more severe with increased asthma symptoms, medication use, emergency presentations and hospital admissions (johnston et al., 1996) . wark et al., have shown that virus-induced acute asthma worsens airway inflammation and the degree of this inflammatory response is directly related to the severity of acute clinical symptoms (wark et al., 2002) . therefore, effective therapeutic interventions would need to alter the natural history of virus-induced asthma, prevent the worsening of airway inflammation and lead to significant clinical improvement. potential therapeutic strategies to this problem have been discussed but the approaches with the most promise include: prevention of infection from occurring; anti-viral agents that eliminate infection while reducing the host's immune response; treatments that specifically target enhanced inflammatory responses that are induced during virus-induced asthma exacerbations and supplementation of protective anti-viral responses. anti-oxidants and angiogenesis inhibitors may also have beneficial effects (fig. 1) . research into the inhibition of rsv and rv infection may have important implications for the prevention of lrt infections and the development and exacerbations of asthma. there are no human vaccines available and further research is required to develop effective anti-viral preventative strategies, specifically anti-viral mabs, passive immunization and vaccines. although the evidence that anti-viral regimes reduce the risk of developing rad later in life is scarce, the use of antivirals to delay rsv infection in particular to later in life may reduce subsequent virus-related illnesses including asthma (culley et al., 2002) . genetic factors are fixed but investigations of such interventions may be developed to reduce asthma incidence have become areas of intense interest. mabs have demonstrated efficacy in preventing rsv infection and inflammation in animal models and a humanised mab (palivizumab) against the rsv f protein is effective against rsv and wheezing in children and reduces hospitalization in high-risk individuals. studies in mice show that palivizumab reduces viral load, acute disease and long-term lung function abnormalities . in the rat model palivizumab inhibits the invasion of virus particles into the lrt epithelium and the upregulation of the nk1 receptor. this prevents acute neurogenic lrt inflammation and longterm susceptibility to inflammation that is induced by infection (piedimonte et al., 2000; piedimonte, 2002) . a randomised controlled trial with palivizumab in children achieved a 55% reduction in rsv hospitalization. reductions were observed in children with (39%) and without (78%) chronic lung disease (impact study, 1998) . a recent large prospective casecontrolled study of at risk pre-term infants showed that palivizumab also protected against recurrent wheezing (13% vs 26% controls) and physician-diagnosed recurrent wheezing (8% vs 26% controls) independently of confounding variables (simoes et al., 2007) . one drawback is that escape mutants have been detected both in vitro and in vivo (zhao et al., 2004; zhao & sullender, 2005) , indicating that preventative strategies should be targeted at multiple epitopes, because palivizumab is effective in preventing hospitalization in high-risk groups, optimisation of its pharmacokinetic profile, extension of its half-life and increasing its neutralising abilities would produce an even more efficacious preventative strategy for rsv-associated disease. motavizumab is a derivative of palivizumab and is now in clinical trials. this mab has a 20 fold increase in neutralising abilities in vitro and its use reduces viral titres by 100 fold and inhibits viral replication in the rt compared to palivizumab in rats (wu et al., 2007) . further studies are required to establish the application of such mabs for the prevention of asthma. other mabs have also been used in mice as treatments of features of rsv-induced disease. rsv infection of mice leads to eosinophil and neutrophil influx into the lung and ahr, which are inhibited by anti-il-5 (schwarze et al., 2000) . administration of fi-rsv and anti-il-4 leads to a decrease in disease features and il-4 production following challenge, but ifn-î³ release is increased (tang & graham, 1994) . t1/st2 mab treatment reduced th2 but not th1 pathology and may also be a good treatment (walzl et al., 2001) . all of these mabs require further assessment. currently the only option for preventative treatment of rsv is passive immunization, which has been in use for many years. a randomised placebo controlled trial investigated the applicability of rsv immunoglobulin to prevent rsv-associated hospitalization (prevent study, 1997) . treatment reduced hospitalizations for lrt infections but required monthly administration of high levels of fluids and proteins. in another small study, children with chronic lung disease who were treated with immunoglobulin 7-10 years previously had improved lung function, reduced atopy and rad events (wenzel et al., 2002) . this indicates that prophylaxis may be able to prevent rad in children. studies in rats also show that passive transfer of antibodies against rsv induces protection in the lrt but not the urt and that serum titres of â�¥ 1:380 are protective whereas titres of â�¤ 1:100 are not (prince et al., 1985) . infants who are the most susceptible to virus infections are protected by maternal anti-viral antibodies (largely igg1) that are passively transferred, however these antibodies inhibit the induction of protective responses against primary infection or vaccination. as a result primary or recurrent infections do not induce protective immunity until later in life (henderson et al., 1979; glezen et al., 1986) . natural protection against viral disease results from a combination of antibody, cell-mediated and t-helper cell immunity. this combination of responses target the virus before infection of host cells as well as destroying cells that have already been infected with virus. therefore, potentially an ideal vaccine would need to stimulate humoral, cellular and t-helper responses similar to those induced by natural infection. vaccine development has been hampered by the requirement for early-life vaccination, the confounding effects of poor neonatal immune responses, the presence of maternal antibodies, the difficulties in balancing immunogenicity and attenuation and the risk of vaccineinduced disease. mucosal immunization may overcome the immune suppressive effects of maternal antibodies. 4.1.3.1. fi-vaccines. the widely recognised need for an effective rsv vaccine has also been held back by the adverse events following the implementation of the fi-rsv vaccine. this vaccine was developed in the 1960s but not only was it poorly effective it infamously induced more severe rsv-related disease upon natural infection in clinical trials (kapikian et al., 1969; kim et al., 1969) . the vaccine induced high serum antibody titres that were of low neutralising activity, lymphocytes with an enhanced rsv-specific proliferative response and blood eosinophilia . these observations were repeated in mice given the same vaccine or the g protein of rsv. enhanced disease involved eosinophilia of the lung (srikiatkhachorn & braciale, 1997b; tripp et al., 2000) and aberrant cd4+ t cells releasing th2 cytokines (il-4, -5, -10 and -13) (waris et al., 1996; srikiatkhachorn & braciale, 1997b; tripp et al., 2000) . reduced il-12 and cd8+ responses were also detected (waris et al., 1996; aung et al., 1999) . fibovine rsv has also been tested in calves and also induces the development of immunopathology, ineffective neutralising antibodies and induced inflammatory responses leading to eosinophil influx into the lung and enhanced ige production upon subsequent infection (gershwin et al., 1998; antonis et al., 2003; kalina & gershwin, 2004) . as is the case with rsv in mice the fi-bovine rsv did not induce long-term t cell memory . thus it has been proposed that vaccines did not possess sufficient neutralising antibody or ctl responses and on subsequent infection the virus was not cleared but induced a potent th2 response that exacerbated disease. 4.1.3.2. subunit vaccines. subunit vaccines are not associated with enhancing disease and may have potential applicability. however, the variability of human viruses is an issue that must be taken into account in the development of subunit vaccines. for rsv, vaccination with the f protein confers cross-protective immunity whereas only group-specific immunity is developed when the g protein is used (sullender et al., 1990; simard et al., 1997) . intranasal followed by parenteral immunization with the rsv f protein protects against both upper and lower rt infection in mice but may induce pulmonary pathology (murphy et al., 1990; connors et al., 1992; walsh, 1994) . a chimeric parainfluenza viral vaccine expressing the f protein has also been developed and is protective against infectious challenge in primates (tang et al., 2004) . in a variety of different human studies f protein vaccines have now been shown to be immunogenic and safe in children with or without chronic respiratory disease and pregnant women and reduce the prevalence of infections but not lrt infections (groothuis et al., 1998; munoz et al., 2003; piedra et al., 2003; simoes & carbonell-estrany, 2003; meyer et al., 2007) . largescale trials are now required to confirm efficacy. protein g-based subunit vaccines have also been developed but may initially induce detrimental th2 responses. immune responses involving il-4 and il-13 activity following immunization with vaccinia virus expressing rsv-protein g or fi-rsv must be attenuated to modulate protein g-specific responses resulting in severe rsv-induced disease. however, inhibition of il-4 or -13 alone has minimal impact on disease (johnson et al., 2003) . the co-administration of cytokines or th1-inducing adjuvants may abolish initial th2 responses (neuzil et al., 1997) . other novel subunit vaccines in development include the n protein, other chimeric f and g fusion proteins, synthetic peptides, recombinant proteins, recombinant vaccinia and parainfluenza viruses expressing other viral components and dna vaccines (reviewed in meyer et al. (2007) ). subunit vaccines require co-administration with adjuvants to enhance immunogenicity and induce the most desirable immune response. currently quillaja saponi or its component fractions or cpg oligodeoxynucleotides administered with whole or subunit vaccines induce potent neutralising antibodies and desirable immune responses in mice and reduce disease and infection in calves. this occurs even in the presence of maternal antibodies and particularly when presented as immunostimulating complexes (meyer et al., 2007) . however the applicability of these approaches to humans remains unknown. 4.1.3.3. live attenuated vaccines. natural infection does not predispose to enhanced disease upon subsequent infection. therefore, experimental live attenuated viruses are being developed for rsv (karron et al., 2005) for intranasal administration during the first days of life but the immunology underlying their activity is poorly understood. intranasal delivery would induce systemic and local responses and has the potential to promote protective responses in both the upper and lower airways. infants (6-9 months) have limited immune responses to viral glycoproteins and only highly attenuated virus strains with minimal adverse side-effects can be used in this age group which are particularly susceptible to vaccine-related illnesses. these agents do however induce protective immunity even in the presence of passively acquired antibodies in mice and chimpanzees, which is dependent on cd4+ and cd8+ responses (crowe et al., 2001) . several cold passaged, temperature sensitive attenuated viruses have been developed that protect against challenge in chimpanzees and induce protective local and systemic immunity in children (crowe et al., 1995; karron et al., 1997) . these viruses have since been genetically engineered so that they are sufficiently attenuated for use in infants, although their efficacy against infection has not yet been determined (karron et al., 2005) . genetic engineering is also currently in use to delete non-essential viral genes to produce other live attenuated vaccines or to insert genes to enhance immune responses, for example the insertion of gm-csf to increase the production of pdcs and macrophages (reviewed in mayer et al. (2007) ). the assessment of attenuated viruses that induce potent protective immune responses with negligible side-effects requires extensive time consuming and expensive clinical trials. novel prime-boost vaccination strategies with live attenuated and subunit vaccines could be used in combination to further enhance protective immunity. 4.1.3.4. bovine vaccines. the same problems that apply to human rsv vaccines may also occur in the development of vaccines for calves in the farming industry, but the level of risk of trials is more acceptable. vaccine development is also less complex as bovine rsv is less variable than human rsv and there is only 1 major antigenic group (prozzi et al., 1997) . this has led to the commercialization of several vaccines, which are protective against infection in calves and their development may have applicability to human vaccines. further, cattle may be used as the animal model in preclinical human vaccine studies. however, different bovine models have been used with different concentrations of viral inocula and routes of inoculation with passaged virus that does not induce severe disease. thus results are often not comparable or significant between experimental and control groups (mayer et al., 2007) . inactivated bovine rsv vaccines have been available for many years and reduce the prevalence and severity of subsequent infection without enhancing disease (west et al., 1999) . nevertheless, the longevity of induced protection and efficacy in the background of maternal antibodies could be improved. these vaccines are co-administered with cd8+ and th1 stimulating adjuvants in order to dampen th2 responses that may be induced by the vaccine and lead to protective th1 responses (ellis et al., 2005) . live attenuated bovine rsv vaccines have been developed by passaging virus in cell culture and have recently been commercialized (vangeel et al., 2006) . unlike fi-vaccines, live attenuated vaccines induce a primed memory t cell response and reduce pulmonary inflammation upon subsequent infection (miao et al., 2004) . they are administered parenterally, have equivalent efficacy as inactivated vaccines and induce protective immunity in calves ellis et al., 2007) . these vaccines are now used in quadrivalent therapies to protect against a number of bovine viruses (salt et al., 2007) . although effective in cattle inactivated or attenuated bovine rsv are not suitable as vaccines for human rsv as they do not induce protection in chimpanzees . thus there are currently no safe and effective human vaccines for rsvor rvand it is yet to be determined if delay or prevention of infection with neutralising antibodies or vaccination can reduce wheezing, long-term lung function abnormalities and asthma. nevertheless there are many promising approaches and candidates are being used to develop such vaccines, some of which are in human clinical trials to prevent infection, which if successful may be used in attempts to inhibit the development of asthma. the balance of th1/th2 immunity to infection determines the severity of disease where th1 responses mediate clearance and recovery but th2 responses induce eosinophilia and more severe symptoms. strategies to augment th1 responses during vaccination in early life may be useful in preventing the development of virus-induced asthma. recent efforts have concentrated on the development of the combination of subunit vaccines with th1inducing adjuvants and on the development of live attenuated vaccines. the most promising approaches for developing vaccines in young infants are the use of live attenuated vaccines and recombinant viruses expressing components of target viruses. there are no effective treatments for rsv/rv-induced disease and currently the optimal therapy for bronchiolitis involves intensive fluid and nutritional support, while the immune response of the subject clears the infection (jhawar, 2003) . in order to maximize the chances of successful treatment an anti-viral agent would need to be taken early during the course of infection in order to modulate the development of the host's immune response. it would have to be highly effective at controlling infection and the virus would need to have no or at least a limited ability to develop resistance. finally the agent must be acceptable to patients in terms of ease of administration, have a reasonable medication frequency and limited side-effects. currently the agents of most interest are those that inhibit viral attachment, including those that bind to viral capsids, and inhibitors of viral proteases, which are required for viral replication. to cause infection all respiratory viruses need to enter the host's becs and replicate. ninety percent of rvs use icam-1 as a receptor to enter cells. a soluble icam-1 molecule (tremacamra) has been developed and tested as a competitive binding inhibitor in 4 randomised controlled trials of experimental rv (rv39) colds, used as either a dry powder or nasal spray (turner et al., 1999) . treatment results in small reductions in symptoms, virus replication and the development of clinical colds. there were no serious adverse events, but medication had to be taken six times a day and was only effective if used within 12 h of infection. it has not been used as a therapy for acute asthma. the rv outer capsid consists of 4 viral proteins (vp1-4) (rossmann et al., 1985) . several agents have been devised that bind to vp1 and prevent virus attachment to the host cell. the first agents that were produced, the oxazolinyl isoaxoles, have small clinical effects but unacceptable sideeffects . further developments led to pleconaril, an agent with broad spectrum activity against rv as an oral preparation to be taken twice daily (hayden et al., 2003) . pleconaril has been assessed in two phase ii clinical trials to determine its efficacy against natural colds (hayden et al., 2003) . subjects began treatment 1-1.5 days after cold symptoms commenced. those taking pleconaril had reduced cold symptoms and duration of colds (0.5-1.5 days) with few side-effects. an application in the us for fda approval for use in clinical colds was unsuccessful as it was considered that there was a lack of substantial clinical benefit and concerns about the development of resistant rv mutants. at this stage there have been no clinical trials specifically assessing the efficacy of pleconaril in acute asthma. rv relies upon proteases to cleave the viral polyprotein for replication to occur. rupintrivir is a 3c protease inhibitor that targets this process (dragovich et al., 2002) and has high-level anti-viral activity against all rvs (dragovich et al., 2002; binford et al., 2005) . unfortunately in a natural infection study ruprintrivir failed to improve symptoms or reduce viral load and clinical development was ceased (patick et al., 2005) . anti-inflammatory treatments have therapeutic potential in virus-associated diseases including asthma by moderating inflammatory responses in response to infection. long-term treatment of asthma with icss controls airway inflammation (juniper et al., 1990) , improves lung function and symptoms and reduces the risk of acute exacerbations (adams et al., 2005) . while icss are effective at controlling chronic asthma they do not completely prevent acute exacerbations even in those who achieve good control and are compliant with therapy, with the majority of episodes triggered by viral infection (reddel et al., 1999) . in an attempt to prevent the recurrence of virusinduced wheeze, infants were treated intermittently with ics or placebo when these symptoms occurred (bisgaard et al., 2006) . treatment had no clinical effect on acute episodes of wheeze and did not influence whether children went on to develop persistent wheeze (bisgaard et al., 2006) . another study showed that treatment of acute asthma by increasing ics dose also did not prevent worsening of disease (fitzgerald et al., 2004) . the incomplete effect of ics in controlling virus-induced asthma was demonstrated best in adult asthmatics using experimental rv infection (grunberg et al., 2001) . treatment reduced ahr and airway inflammation prior to infection but had no effect on the development of inflammatory changes that were associated with infection (grunberg et al., 2001) . in a systematic review glucocorticoids were suggested to have little effect in rsv disease (black, 2003) and they also enhance viral replication and mortality in pvm infection (rosenberg et al., 2005) . oral corticosteroids have been used to treat acute viral bronchiolitis in children. however, meta-analysis of seven randomised controlled trials found that there was no effect on length of stay or clinical symptoms and complications (patel et al., 2004) . in asthmatics with persistent symptoms despite ics use, treatment with combination therapy with icss and labas effectively controls chronic symptoms and reduces the frequency of exacerbations (walters et al., 2003) . it has recently been shown that early treatment of symptoms of worsening asthma with ics/ laba, where the laba has a rapid onset of action and is used to relieve symptoms, does prevent deterioration leading to severe exacerbations (o'byrne et al., 2005) . while these studies did not identify the cause of the acute triggers it is likely that the majority of these were related to virus infection and the combination of ics/labas appear more effective than icss alone in preventing exacerbations. in vitro studies demonstrate that treatment of airway epithelial cells with ics/labas has synergistic and additive effects in reducing the release of inflammatory mediators . these observations may explain the efficacy of combination therapy and indicate an important role for their use. this may be particularly relevant to children where their value in controlling chronic asthma is not well defined but where ahr is known to persist following viral infection (xepapadaki et al., 2005) . it remains unknown what effect ics/laba treatments have on specifically preventing or treating virus-induced asthma. it has recently been recognised that macrolide antibiotics in addition to their anti-microbial action have important immune modulating effects and reduce the release of inflammatory mediators from airway epithelial cells (takizawa et al., 1997) . epithelial cells infected with rv and treated with erythromycin, showed reduced expression of icam-1, il-6 and -8 (jang et al., 2006) . initial clinical studies were, however, not promising; using experimental rv infection, treatment of healthy subjects with clarithromycin had little or no effect on the development of cold symptoms or nasal inflammation (abisheganaden et al., 2000) . however, unlike colds where the host inflammatory response in the airways is relatively mild, in conditions characterised by more intense neutrophilic inflammation, such as cystic fibrosis, macrolides appear to be effective (ferrara et al., 2005) , which has encouraged their use in asthma. in a small randomised controlled trial of infants with rsv bronchiolitis treatment with clarithromycin reduced systemic inflammation acutely and led to few wheezing episodes in the following 6 months (tahan et al., 2007) . in a large multicentre study of adults (278) with asthma, subjects were randomised to receive the ketolide, telithromycin (800 mg/d) or placebo (johnston, 2006) . those treated with telithromycin had a greater reduction in asthma symptom scores, but there was no difference between the groups in terms of improvement in pef rate. the trial did not document the presence of virus infection but the results suggest the effect is due to an ability to reduce inflammation in acute asthma. further studies are required to determine if there is a specific effect on virus-induced acute asthma. type i ifns are known to play crucial roles in defence against viruses by inducing anti-viral proteins such as pkr and rnase l in infected cells, apoptosis preventing virus replication and adaptive immune responses to infection (malmgaard, 2004) . the recent observations by wark et al., that asthmatic becs are more susceptible to infection with rv, which involves a deficient ifn-î² response, suggests that this may be an important therapeutic target to limit the affects of virus-associated acute asthma (wark et al., 2005) . several trials have also assessed the role of ifn-î±2 treatment (1.5 ã� 10 6 -4.5 ã� 10 7 iu/d intranasally) in experimental rv colds in healthy volunteers (scott et al., 1982; hayden & gwaltney, 1984) . these studies consistently showed a dose dependent improvement in symptom scores and reduced virus shedding, but treatment was associated with nasal bleeding and a lymphocytic nasal infiltrate. a similar response was observed with recombinant ifn-î² (sperber et al., 1988) . when used to treat natural colds ifn-î±2 reduced the frequency of colds but had at best modest effects on symptoms (monto et al., 1986) , while serine ifn-î² failed to reduce colds or improve symptoms. these modest improvements provide a proof of concept but the clinical benefit is difficult to justify in healthy individuals with mild colds. however, as is the case with the use of macrolides in asthma where the clinical effects of colds are much greater (corne et al., 2002) , efficacy may be more evident. a single case series exists for the use of ifn-î± (3 ã� 10 6 iu/d) to treat 10 stable but corticosteroid resistant asthmatics (simon et al., 2003) . treatment resulted in improved lung function and allowed a reduction in oral corticosteroid use. however, side-effects were also common, in the first 4 weeks all subjects experienced a 'flu-like' illness, 9 had nausea and 5 developed headaches. by 5-10 months side-effects were less though 1 subject developed leukopenia severe enough to necessitate temporarily ceasing treatment. the benefits of type i ifns now need to be assessed in the context of treatment of virusassociated acute asthma. important issues will need to be resolved including the optimum delivery of medication, whether administration should be to the nose or the airway, how soon treatment needs to commence following the development of cold symptoms and how this may influence airway inflammation and ahr in acute asthma. oxidative stress may plan an important role in asthma but it is unknown whether it is important in virus-induced disease. the anti-oxidant butylated hydroxyanisole has been used in a mouse model of rsv lung infection to reduce rsv-induced oxidative stress, disease symptoms, weight loss and ahr. the effects were mediated by suppression of neutrophil infiltration and cytokine and chemokine release in the lung (castro et al., 2006) . thus anti-oxidants may prevent rsv-induced inflammation and have long-term beneficial effects, which may ameliorate the consequences of infection in asthma. the use of vegf and angiogenesis inhibitors may have the potential to elucidate the specific roles of these factors in asthma and identify their potential as therapeutic targets. no efficacious vaccines yet exist for the prevention of rsv or rv infections in humans. effective bovine rsv vaccines are available and the formulation of these vaccines may be applicable to the development of human vaccines. combination therapy with icss and labas effectively controls persistent symptoms and numbers of exacerbations. this is the current best treatment option but does not treat the cause of the disease and cannot be used to prevent the development of disease in the first instance. a variety of novel options for the prevention of virus-induced asthma need to be fully assessed for their efficacy and applicability. rsv and rv infection of respiratory epithelium may play an important role in the development and exacerbation of asthma, however the pivotal mechanisms underpinning these relationships remain unresolved. infection is associated with persistent wheeze and decreases in lower lung function and worsens airway inflammation and airflow obstruction acutely in asthma, with other factors contributing to severity. asthmatics are more predisposed to th2 responses, may be more susceptible to infection and experience more severe lrt symptoms upon infection. the development of more severe disease may identify those people more at risk of developing wheeze and asthma. recurring infections may lead to a cycle of inflammation and repair that worsens airway remodelling. rsv infections are strongly linked to both development and exacerbation of asthma. early-life rsv infections, particularly those that induce severe disease, induce recurrent wheeze and bronchial obstruction and predispose to rad and potentially asthma that persists into later life. it is possible that persistent infection affects the developing lung and immune system and predisposes to recurring rsv infections, ahr, reduced lung function and respiratory symptoms. another alternative is that children with reduced lung function, genetic susceptibility or aberrant immune responses may be at increased risk of infection and that rsv disease is a marker of susceptibility to increased respiratory symptoms. both of these processes may occur under different circumstances. the association with rsv is inconclusive for allergic sensitization, which may involve igemediated allergy and randomised intervention studies are required to confirm this link. the mechanisms involved in rsv-induced asthma include; age of first infection, the timing of infection relative to allergic sensitization, the nature of innate and adaptive immune responses induced upon infection, latent infections and the potential involvement of pathogenetic processes such as angiogenesis and neurogenic inflammation. a combination of human studies and mouse models has been widely used to elucidate the mechanisms of how rsv infection are linked with asthma and novel models of pvm infection may be particularly useful in the future. rvs are widely recognised as the commonest cause of clinically significant rt infections and are strongly implicated in the exacerbation of asthma and more recently in the induction of asthma. the mechanisms of these associations have been investigated in human studies both in vivo and in vitro and have been shown to involve deficient immune responses, which may be different to those associated with rsv, that may be influenced by atopy and reduced lung function, ahr and remodelling. nevertheless more work is needed to elucidate role of rv in lrt diseases. it remains unknown if rv induces the development of wheeze and asthma or if asthmatics are more susceptible to rv infection. recently a novel mouse model has been described which may be used to substantially contribute to our understanding of rv and rv-associated asthma pathogenesis. despite the scope of the problem caused by respiratory viruses in acute asthma no specific vaccines or treatments exist that have been shown to modify the clinical outcome of rsv or rv infection. the optimal approach would be to develop safe and effective prevention strategies, such as efficacious vaccines, which induce immune responses that prevent viral infection. in the absence of a vaccine the different processes involved in the generation of virus-associated asthma and exacerbations could be targeted by specific treatments for individual patients. several therapeutic options are available and more are emerging. studies should focus on how established treatments such as with ics/laba may modify virus-associated acute asthma and assess novel anti-inflammatory strategies including macrolides or the use of ifns either alone or in combination to influence the course of disease. the benefits of these strategies now need to be assessed in the context of treatment of virusassociated acute asthma. important issues will need to be resolved including the optimum delivery of medication, including whether this should be intranasally or directly to the airway. it will be necessary to determine how soon treatment needs to commence following the development of cold symptoms and how this may influence airway inflammation and ahr in acute asthma. in summary, although rsv and rv have been implicated in initiating inflammation and asthma, whether previous infections modulate the immunological response or damage the airway epithelium, and promote the progression to chronic disease, or susceptibility to later development of exacerbations, remains unknown. asthmatics may have impaired anti-viral innate responses and defective interactions between innate and adaptive immune responses may promote infection and enhance allergic inflammation and ahr or decrease lung function. further studies are required to elucidate the links between infection, immune responses and susceptibility to chronic respiratory diseases and why some individuals but not others develop persistent wheeze and asthma. it is likely that there is a primary deficiency in the respiratory epithelium that predisposes to infection and asthma, which may be exacerbated by recurring exposure to environmental insults. there is a widely recognised need for further understanding of the mechanisms that induce disease and the development of effective vaccines and treatments. reduced interferon-gamma expression in peripheral blood mononuclear cells of infants with severe respiratory syncytial virus disease effect of clarithromycin on experimental rhinovirus-16 colds: a randomized, double-blind, controlled trial inhaled beclomethasone versus placebo for chronic asthma australian institute of health and welfare, canberra. asthma in australia cystic fibrosis and renal disease: a case report distinct types of lung disease caused by functional subsets of antiviral t cells vaccine-induced immunopathology during bovine respiratory syncytial virus infection: exploring the parameters of pathogenesis kinetics of antiviral cd8 t cell responses during primary and post-vaccination secondary bovine respiratory syncytial virus infection respiratory tract viral infections in inner-city asthmatic adults interleukin-4 diminishes cd8(+) respiratory syncytial virus-specific cytotoxic t-lymphocyte activity in vivo alternative mechanisms of respiratory syncytial virus clearance in perforin knockout mice lead to enhanced disease vascular remodeling is airway generation-specific in a primate model of chronic asthma innate immunity in the lung: how epithelial cells fight against respiratory pathogens epithelial damage and angiogenesis in the airways of children with asthma influence of respiratory syncytial virus infection on cytokine and inflammatory responses in allergic mice respiratory syncytial virus enhances respiratory allergy in mice despite the inhibitory effect of virus-induced interferon-gamma timing of infection and prior immunization with respiratory syncytial virus (rsv) in rsv-enhanced allergic inflammation rhinovirus induces muc5b production in a novel mouse infection and asthma exacerbation model respiratory syncytial virus decreases the capacity of myeloid dendritic cells to induce interferon-gamma in naive t cells recognising the clinical signs of brsv infection th2 cytokines exert a dominant influence on epithelial cell expression of the major group human rhinovirus receptor, icam-1 conservation of amino acids in human rhinovirus 3c protease correlates with broad-spectrum antiviral activity of rupintrivir, a novel human rhinovirus 3c protease inhibitor intermittent inhaled corticosteroids in infants with episodic wheezing systematic review of the biology and medical management of respiratory syncytial virus infection airways and vascular remodelling in asthma and cardiovascular disease: implications for therapeutic intervention both immunisation with a formalin-inactivated respiratory syncytial virus (rsv) vaccine and a mock antigen vaccine induce severe lung pathology and a th2 cytokine profile in rsv-challenged mice effect of lack of interleukin-4, interleukin-12, interleukin-18, or the interferon-gamma receptor on virus replication, cytokine response, and lung pathology during respiratory syncytial virus infection in mice monocyte il-10 production during respiratory syncytial virus bronchiolitis is associated with recurrent wheezing in a one-year follow-up study monocyte interleukin-12 production is inversely related to duration of respiratory failure in respiratory syncytial virus bronchiolitis natural reinfection with respiratory syncytial virus does not boost virus-specific t-cell immunity macrophage inflammatory protein-1alpha and rantes are present in nasal secretions during ongoing upper respiratory tract infection rhinovirus infection induces cytotoxicity and delays wound healing in bronchial epithelial cells from bronchoconstriction to airways inflammation and remodeling respiratory syncytial virus and t cells: interplay between the virus and the host adaptive immune system chimeric bovine respiratory syncytial virus with glycoprotein gene substitutions from human respiratory syncytial virus (hrsv): effects on host range and evaluation as a live-attenuated hrsv vaccine cytotoxic t cells clear virus but augment lung pathology in mice infected with respiratory syncytial virus icam-1 on epithelial cells in allergic subjects: a hallmark of allergic inflammation acute bronchiolitis: predisposing factors and characterization of infants at risk antioxidant treatment ameliorates respiratory syncytial virus-induced disease and lung inflammation respiratory syncytial virus infection suppresses lung cd8+ t-cell effector activity and peripheral cd8+ t-cell memory in the respiratory tract recovery from infants with respiratory illness of a virus related to chimpanzee coryza agent (cca). i. isolation, properties and characterization respiratory syncytial virus-induced acute and chronic airway disease is independent of genetic background: an experimental murine model memory cd8+ t cells in heterologous antiviral immunity and immunopathology in the lung rhinovirus induces airway epithelial gene expression through double-stranded rna and ifn-dependent pathways human cytotoxic t cells stimulated by antigen on dendritic cells recognize the n, sh, f, m, 22k, and 1b proteins of respiratory syncytial virus vasculogenesis, angiogenesis, hemangiomas, and vascular malformations asthma: mechanisms of disease persistence and progression production of infectious human respiratory syncytial virus from cloned cdna confirms an essential role for the transcription elongation factor from the 5â�² proximal open reading frame of the m2 mrna in gene expression and provides a capability for vaccine development respiratory syncytial virus lack of long-term effects of respiratory syncytial virus infection on airway function in mice cotton rats previously immunized with a chimeric rsv fg glycoprotein develop enhanced pulmonary pathology when infected with rsv, a phenomenon not encountered following immunization with vaccinia-rsv recombinants or rsv mechanisms of respiratory virus-induced asthma exacerbations role of deficient type iii interferon-lambda production in asthma exacerbations frequency, severity, and duration of rhinovirus infections in asthmatic and non-asthmatic individuals: a longitudinal cohort study cold-passaged, temperature-sensitive mutants of human respiratory syncytial virus (rsv) are highly attenuated, immunogenic, and protective in seronegative chimpanzees, even when rsv antibodies are infused shortly before immunization passively acquired antibodies suppress humoral but not cell-mediated immunity in mice immunized with live attenuated respiratory syncytial virus vaccines age at first viral infection determines the pattern of t cell-mediated disease during reinfection in adulthood the role of virus-specific immunoglobulin e in airway hyperresponsiveness virus-induced airway dysfunction: pathogenesis and biomechanisms the enhancement or prevention of airway hyperresponsiveness during reinfection with respiratory syncytial virus is critically dependent on the age at first infection and il-13 production alteration of airway neuropeptide expression and development of airway hyperresponsiveness following respiratory syncytial virus infection epidemiology and clinical outcome of virus-positive respiratory samples in ventilated patients: a prospective cohort study are rhinovirus-induced airway responses in asthma aggravated by chronic allergen exposure? vascular endothelial growth factor induction by rhinovirus infection 5-dihydro-2-oxazolyl)phenoxy]alkyl]isoxazoles. inhibitors of picornavirus uncoating gene expression in epithelial cells in response to pneumovirus infection animal models for studying respiratory syncytial virus infection and its long term effects on lung function structure-based design, synthesis, and biological evaluation of irreversible human rhinovirus 3c protease inhibitors. 6. structure-activity studies of orally bioavailable, 2-pyridone-containing peptidomimetics the cold case: are rhinoviruses perfectly adapted pathogens? rhinoviral infections activate p38map-kinases via membrane rafts and rhoa respiratory syncytial virus-induced immunoprotection and immunopathology the vesiculo-vacuolar organelle (vvo): a distinct endothelial cell structure that provides a transcellular pathway for macromolecular extravasation animal pneumoviruses: molecular genetics and pathogenesis combination therapy: synergistic suppression of virus-induced chemokines in airway epithelial cells the role of toll-like receptor 4 versus interleukin-12 in immunity to respiratory syncytial virus interleukin-11: stimulation in vivo and in vitro by respiratory viruses and induction of airways hyperresponsiveness spectrum of clinical illness in hospitalized patients with "common cold" virus infections efficacy of a saponin-adjuvanted inactivated respiratory syncytial virus vaccine in calves response of calves to challenge exposure with virulent bovine respiratory syncytial virus following intranasal administration of vaccines formulated for parenteral administration respiratory syncytial virus infection in immunocompromised adults vascular endothelial growth factor induces endothelial fenestrations in vitro the role of the respiratory syncytial virus in airway syndromes in childhood the relationship between respiratory syncytial virus infections and the development of wheezing and asthma in children suppression of pattern-recognition receptor tlr4 sensing does not alter lung responses to pneumovirus infection relationship of serum antibody to risk of respiratory syncytial virus infection in elderly adults respiratory syncytial virus infection in elderly and high-risk adults increased vascular endothelial growth factor and receptors: relationship to angiogenesis in asthma macrolides in the treatment of asthma and cystic fibrosis doubling the dose of budesonide versus maintenance treatment in asthma exacerbations acute bronchiolitis in infancy as risk factor for wheezing and reduced pulmonary function by seven years in akershus county respiratory illness associated with influenza and respiratory syncytial virus infection respiratory syncytial virus infection: its role in aeroallergen sensitization during the first two years of life interleukin 5 deficiency abolishes eosinophilia, airways hyperreactivity, and lung damage in a mouse asthma model lower airways inflammation during rhinovirus colds in normal and in asthmatic subjects detection of viral, chlamydia pneumoniae and mycoplasma pneumoniae infections in exacerbations of asthma in children transcriptional activation of the interleukin-8 gene by respiratory syncytial virus infection in alveolar epithelial cells: nuclear translocation of the rela transcription factor as a mechanism producing airway mucosal inflammation macrophage inflammatory protein-1alpha (not t helper type 2 cytokines) is associated with severe forms of respiratory syncytial virus bronchiolitis a comparison of epidemiologic and immunologic features of bronchiolitis caused by influenza virus and respiratory syncytial virus intracellular pool of vascular endothelial growth factor in human neutrophils impaired virus-induced interferon-alpha2 release in adult asthmatic patients relationship of upper and lower airway cytokines to outcome of experimental rhinovirus infection relationships among specific viral pathogens, virus-induced interleukin-8, and respiratory symptoms in infancy double-stranded rna induces the synthesis of specific chemokines by bronchial epithelial cells effects of viral respiratory infections on lung development and childhood asthma bidirectional interactions between viral respiratory illnesses and cytokine responses in the first year of life a bovine model of vaccine enhanced respiratory syncytial virus pathophysiology rhinovirus infects primary human airway fibroblasts and induces a neutrophil chemokine and a permeability factor mobilization of plasmacytoid and myeloid dendritic cells to mucosal sites in children with respiratory syncytial virus and other viral respiratory infections 543â��546. global strategy for asthma management and prevention (1995). global initiative for asthma deficient il-12(p35) gene expression by dendritic cells derived from neonatal monocytes role of t lymphocyte subsets in the pathogenesis of primary infection and rechallenge with respiratory syncytial virus in mice synergism between allergens and viruses and risk of hospital admission with asthma: case-control study age-dependent differences in cytokine and antibody responses after experimental rsv infection in a bovine model the major human rhinovirus receptor is icam-1 safety and immunogenicity of a purified f protein respiratory syncytial virus (pfp-2) vaccine in seropositive children with bronchopulmonary dysplasia respiratory syncytial virus induces tlr3 protein and protein kinase r, leading to increased double-stranded rna responsiveness in airway epithelial cells experimental rhinovirus 16 infection. effects on cell differentials and soluble markers in sputum in asthmatic subjects effect of experimental rhinovirus 16 colds on airway hyperresponsiveness to histamine and interleukin-8 in nasal lavage in asthmatic subjects in vivo rhinovirus-induced airway inflammation in asthma: effect of treatment with inhaled corticosteroids before and during experimental infection the effect of passive smoking on the development of respiratory syncytial virus bronchiolitis the influence of signal variation, bias, noise and effect size on statistical significance in treatment studies of the common cold inducible expression of inflammatory chemokines in respiratory syncytial virus-infected mice: role of mip-1alpha in lung pathology respiratory syncytial virus-induced activation of nuclear factor-kappab in the lung involves alveolar macrophages and tolllike receptor 4-dependent pathways respiratory syncytial virus: a continuing culprit and conundrum respiratory syncytial virus and parainfluenza virus principles and practice of infectious diseases respiratory syncytial virus infection in adults: clinical, virologic, and serial pulmonary function studies possible transmission by fomites of respiratory syncytial virus immunity to and frequency of reinfection with respiratory syncytial virus the role of p38 mapk in rhinovirus-induced monocyte chemoattractant protein-1 production by monocytic-lineage cells viral-induced angiogenesis rio de janiero role of atypical bacterial infection of the lung in predisposition/protection of asthma respiratory syncytial virus g protein and g protein cx3c motif adversely affect cx3cr1+ t cell responses pathogenic bacteria and viruses in induced sputum or pharyngeal secretions of adults with stable asthma efficacy of a live bovine respiratory syncytial virus vaccine in seropositive calves respiratory syncytical virus-induced chemokine expression in the lower airways: eosinophil recruitment and degranulation respiratory syncytial virus replication is prolonged by a concomitant allergic response intranasal interferon-alpha 2 treatment of experimental rhinoviral colds efficacy and safety of oral pleconaril for treatment of colds due to picornaviruses in adults: results of 2 double-blind, randomized, placebo-controlled trials respiratory-syncytial-virus infections, reinfections and immunity. a prospective, longitudinal study in young children hospitalization for rsv bronchiolitis before 12 months of age and subsequent asthma, atopy and wheeze: a longitudinal birth cohort study national disease burden of respiratory viruses detected in children by polymerase chain reaction toll-like receptor 3 is induced by and mediates antiviral activity against rhinovirus infection of human bronchial epithelial cells viral infections in relation to age, atopy, and season of admission among children hospitalized for wheezing risk factors for respiratory syncytial virusassociated lower respiratory illnesses in the first year of life rhinoviruses in the pathogenesis of asthma: the bronchial epithelium as a major disease target epithelial-mesenchymal communication in the pathogenesis of chronic asthma interactions between rsv infection, asthma, and atopy: unraveling the complexities interactions between respiratory tract infections and atopy in the aetiology of asthma expression of vascular endothelial growth factor by human eosinophils: upregulation by granulocyte macrophage colony-stimulating factor and interleukin-5 neonatal chlamydial infection induces mixed t cell responses that drive allergic airways disease gene expression of vascular endothelial growth factor and its receptors and angiogenesis in bronchial asthma effects of rhinovirus infection on histamine and cytokine production by cell lines from human mast cells and basophils intracellular ifn-gamma expression in natural killer cells precedes lung cd8+ t cell recruitment during respiratory syncytial virus infection il-12-activated nk cells reduce lung eosinophilia to the attachment protein of respiratory syncytial virus but do not enhance the severity of illness in cd8 t cell-immunodeficient conditions th1 and th2 cytokine induction in pulmonary t cells during infection with respiratory syncytial virus cd8+ t cells control th2-driven pathology during pulmonary respiratory syncytial virus infection inhibition of tumor necrosis factor reduces the severity of virus-specific lung immunopathology palivizumab, a humanized respiratory syncytial virus monoclonal antibody, reduces hospitalization from respiratory syncytial virus infection in high-risk infants. the impact-rsv study group mechanisms of mucin production by rhinovirus infection in cultured human airway epithelial cells virus-induced asthma attacks association of respiratory picornaviruses with acute bronchiolitis in french infants respiratory syncytial virus induces pneumonia, cytokine response, airway obstruction, and chronic inflammatory infiltrates associated with long-term airway hyperresponsiveness in mice effect of clarithromycin on rhinovirus-16 infection in a549 cells evaluation of the efficacy of prednisolone in early wheezing induced by rhinovirus or respiratory syncytial virus severe bronchiolitis in children respiratory syncytial virus-induced ccl5/rantes contributes to exacerbation of allergic airway inflammation respiratory syncytial virus-induced exaggeration of allergic airway disease is dependent upon ccr1-associated immune responses secreted respiratory syncytial virus g glycoprotein induces interleukin-5 (il-5), il-13, and eosinophilia by an il-4-independent mechanism il-13 is sufficient for respiratory syncytial virus g glycoprotein-induced eosinophilia after respiratory syncytial virus challenge the histopathology of fatal untreated human respiratory syncytial virus infection macrolide antibiotics and asthma treatment community study of role of viral infections in exacerbations of asthma in 9-11 year old children the relationship between upper respiratory infections and hospital admissions for asthma: a time-trend analysis low grade rhinovirus infection induces a prolonged release of il-8 in pulmonary epithelium the september epidemic of asthma exacerbations in children: a search for etiology effect of long-term treatment with an inhaled corticosteroid (budesonide) on airway hyperresponsiveness and clinical asthma in nonsteroid-dependent asthmatics association of an early respiratory syncytial virus infection and atopic allergy progress in defining the role of rsv in allergy and asthma: from clinical observations to animal models effects of pranlukast administration on vascular endothelial growth factor levels in asthmatic patients an epidemiologic study of altered clinical reactivity to respiratory syncytial (rs) virus infection in children previously vaccinated with an inactivated rs virus vaccine recombinant bovine respiratory syncytial virus with deletions of the g or sh genes: g and f proteins bind heparin evaluation of two live, coldpassaged, temperature-sensitive respiratory syncytial virus vaccines in chimpanzees and in human adults, infants, and children identification of a recombinant live attenuated respiratory syncytial virus vaccine candidate that is highly attenuated in infants the role of eosinophils in the pathogenesis of asthma in-vitro studies on mechanisms of immunosuppression associated with bovine respiratory syncytial virus prevalence of viral respiratory tract infections in children with asthma respiratory syncytial virus disease in infants despite prior administration of antigenic inactivated vaccine local and systemic antibody response to bovine respiratory syncytial virus infection and reinfection in calves with and without maternal antibodies pathogenesis of naturally acquired bovine respiratory syncytial virus infection in calves: morphologic and serologic findings exaggerated neurogenic inflammation and substance p receptor upregulation in rsv-infected weanling rats persistence of rhinovirus rna after asthma exacerbation in children long-term effects of respiratory syncytial virus (rsv) bronchiolitis in infants and young children: a quantitative review regulation of mite allergen-pulsed murine dendritic cells by respiratory syncytial virus interferon-gamma enhances rhinovirusinduced rantes secretion by airway epithelial cells rhinovirus-associated wheezing in infancy: comparison with respiratory syncytial virus bronchiolitis respiratory morbidity 20 years after rsv infection in infancy rhinovirus-induced wheezing in infancy-the first sign of childhood asthma? respiratory syncytial virus infection in children hospitalized for wheezing: virus-specific studies from infancy to preschool years innate immune responses in respiratory syncytial virus infections respiratory syncytial virus and other respiratory viruses during the first 3 months of life promote a local th2-like response understanding airway wall remodeling in asthma: a basis for improvements in therapy direct effects of interleukin-13 on epithelial cells cause airway hyperreactivity and mucus overproduction in asthma pattern recognition receptors tlr4 and cd14 mediate response to respiratory syncytial virus role of respiratory viruses in acute upper and lower respiratory tract illness in the first year of life: a birth cohort study early-life respiratory viral infections, atopic sensitization, and risk of subsequent development of persistent asthma asthma and airway hyper-responsiveness in adults who required hospital admission for bronchiolitis in early childhood neural control mechanisms within airways: disruption by respiratory syncytial virus respiratory syncytial virus infection and recurrent wheeze/asthma in children under five years: an epidemiological survey respiratory syncytial virus stimulation of vascular endothelial cell growth factor/vascular permeability factor contribution of vascular endothelial growth factor to airway hyperresponsiveness and inflammation in a murine model of toluene diisocyanate-induced asthma vascular endothelial growth factor (vegf) induces remodeling and enhances th2-mediated sensitization and inflammation in the lung immunostimulatory dna inhibits allergen-induced peribronchial angiogenesis in mice relationship of early childhood viral exposures to respiratory symptoms, onset of possible asthma and atopy in high risk children: the canadian asthma primary prevention study type 1 and type 2 cytokine imbalance in acute respiratory syncytial virus bronchiolitis prednisolone reduces recurrent wheezing after a first wheezing episode associated with rhinovirus infection or eczema rhinovirus illnesses during infancy predict subsequent childhood wheezing increased vascularity of the bronchial mucosa in mild asthma respiratory syncytial virus infection reversed anti-asthma effect of neonatal bacillus calmette-guerin vaccination in balb/c mice respiratory syncytial virus nonstructural proteins ns1 and ns2 mediate inhibition of stat2 expression and alpha/beta interferon responsiveness statistical procedures for estimating the community probability of illness in family studies: rhinovirus and influenza respiratory syncytial virus predisposes mice to augmented allergic airway responses via il-13-mediated mechanisms recombinant respiratory syncytial virus lacking secreted glycoprotein g is attenuated, non-pathogenic but induces protective immunity the failure of interleukin-10-deficient mice to develop airway hyperresponsiveness is overcome by respiratory syncytial virus infection in allergen-sensitized/challenged mice prior airway exposure to allergen increases virus-induced airway hyperresponsiveness induction and regulation of ifns during viral infections human rhinovirus in bronchial epithelium of infants with recurrent respiratory symptoms epidemiological and clinical features of hmpv, rsv and rvs infections in young children airborne transmission of bhv1, brsv, and bvdv among cattle is possible under experimental conditions respiratory syncytial virus bronchiolitis and the pathogenesis of childhood asthma differential recognition of tlr-dependent microbial ligands in human bronchial epithelial cells hla-restricted cd8+ cytotoxic t lymphocyte, interferon-gamma, and interleukin-4 responses to respiratory syncytial virus infection in infants and children transforming growth factor {beta} enhances respiratory syncytial virus replication and tumor necrosis factor alpha induction in human epithelial cells anti-respiratory syncytial virus (rsv) neutralizing antibody decreases lung inflammation, airway obstruction, and airway hyperresponsiveness in a murine rsv model respiratory syncytial virus infections: old challenges and new opportunities human and bovine respiratory syncytial virus vaccine research and development effects of a single intranasal dose of modifiedlive bovine respiratory syncytial virus vaccine on cytokine messenger rna expression following viral challenge in calves parental understanding of wheeze and its impact on asthma prevalence estimates respiratory syncytial virus up-regulates tlr4 and sensitizes airway epithelial cells to endotoxin intranasal interferon-alpha 2b for seasonal prophylaxis of respiratory infection quantitative and qualitative analysis of rhinovirus infection in bronchial tissues safety and immunogenicity of respiratory syncytial virus purified fusion protein-2 vaccine in pregnant women enhanced pulmonary histopathology is observed in cotton rats immunized with formalin-inactivated respiratory syncytial virus (rsv) or purified f glycoprotein and challenged with rsv 3-6 months after immunization allergens, viruses, and asthma exacerbations gender analysis in acute bronchiolitis due to respiratory syncytial virus age and sex as factors of response to rsv infections among those with previous history of wheezing nerve growth factor: the central hub in the development of allergic asthma? development of the airway intraepithelial dendritic cell network in the rat from class ii major histocompatibility (ia)-negative precursors: differential regulation of ia expression at different levels of the respiratory tract vascular endothelial growth factor (vegf) and its receptors adjuvants influence the quantitative and qualitative immune response in balb/c mice immunized with respiratory syncytial virus fg subunit vaccine murine rhinovirus 1b infection causes neutrophilic airway inflammation acute viral infections of upper respiratory tract in elderly people living in the community: comparative, prospective, population based study of disease burden chemokines in nasal secretions of normal adults experimentally infected with respiratory syncytial virus respiratory status and allergy nine to 10 years after acute bronchiolitis relationship between vascular endothelial growth factor and angiopoietin-2 in asthmatics before and after inhaled beclomethasone therapy budesonide/formoterol combination therapy as both maintenance and reliever medication in asthma anaphylactic sensitization to aeroantigen during respiratory virus infection respiratory syncytial virus: the virus, the disease and the immune response interleukin-6, interleukin-8, interleukin-11, and interferon-gamma levels in nasopharyngeal aspirates from wheezing children with respiratory syncytial virus or influenza a virus infection increased proinflammatory responses from asthmatic human airway smooth muscle cells in response to rhinovirus infection immunopathological mechanisms in respiratory syncytial virus disease potential mechanisms causing delayed effects of respiratory syncytial virus infection immune responses and disease enhancement during respiratory syncytial virus infection immunopathogenesis of vaccine-enhanced rsv disease links between respiratory syncytial virus bronchiolitis and childhood asthma: clinical and research approaches virus clearance and immunopathology by cd8(+) t cells during infection with respiratory syncytial virus are mediated by ifn-gamma in vitro activity of win 51711, a new broad-spectrum antipicornavirus drug enhanced il-4 responses in children with a history of respiratory syncytial virus bronchiolitis in infancy rhinoviruses infect the lower airways rhinovirus infection up-regulates eotaxin and eotaxin-2 expression in bronchial epithelial cells association of rhinovirus infection with increased disease severity in acute bronchiolitis a defective type 1 response to rhinovirus in atopic asthma mechanisms of rhinovirus-induced asthma rhinovirus infection induces expression of its own receptor intercellular adhesion molecule 1 (icam-1) via increased nf-kappab-mediated transcription the functional consequences of airway remodeling in asthma house dust endotoxin and wheeze in the first year of life respiratory syncytial virus-induced airway hyperresponsiveness is independent of il-13 compared with that induced by allergen infection and disease with respect to age, immunologic status, race and sex rhinovirus-induced pbmc responses and outcome of experimental infection in allergic subjects glucocorticoids for acute viral bronchiolitis in infants and young children in vitro antiviral activity and single-dose pharmacokinetics in humans of a novel, orally bioavailable inhibitor of human rhinovirus 3c protease respiratory viruses and asthma respiratory syncytial virus infection prolongs methacholine-induced airway hyperresponsiveness in ovalbumin-sensitized mice immune interaction between respiratory syncytial virus infection and allergen sensitization critically depends on timing of challenges neural mechanisms of respiratory syncytial virusinduced inflammation and prevention of respiratory syncytial virus sequelae the association between respiratory syncytial virus infection and reactive airway disease respiratory syncytial virus upregulates expression of the substance p receptor in rat lungs a humanized monoclonal antibody against respiratory syncytial virus (palivizumab) inhibits rsv-induced neurogenic-mediated inflammation in rat airways leukotriene synthesis during respiratory syncytial virus bronchiolitis: influence of age and atopy immunogenicity of a new purified fusion protein vaccine to respiratory syncytial virus: a multi-center trial in children with cystic fibrosis respiratory syncytial virus and parainfluenza viruses detection and typing by molecular techniques of respiratory viruses in children hospitalized for acute respiratory infection in reduction of respiratory syncytial virus hospitalization among premature infants and infants with bronchopulmonary dysplasia using respiratory syncytial virus immune globulin prophylaxis. the prevent study group immunoprophylaxis and immunotherapy of respiratory syncytial virus infection in the cotton rat increased levels of interleukin-1 are detected in nasal secretions of volunteers during experimental rhinovirus colds antigenic and molecular analyses of the variability of bovine respiratory syncytial virus g glycoprotein pathogenesis of respiratory syncytial virus bronchiolitis-related wheezing vascular endothelial growth factor-mediated induction of angiogenesis by human rhinoviruses wheezing, asthma, and pulmonary dysfunction 10 years after infection with respiratory syncytial virus in infancy activation of antiviral protein kinase leads to immunoglobulin e class switching in human b cells modulation of airway inflammation by passive transfer of allergenspecific th1 and th2 cells in a mouse model of asthma differences between asthma exacerbations and poor asthma control activation of cd4+ t cells, increased th2-type cytokine mrna expression, and eosinophil recruitment in bronchoalveolar lavage after allergen inhalation challenge in patients with atopic asthma respiratory syncytial virus infection in infants is associated with predominant th-2-like response the pneumonia virus of mice infection model for severe respiratory syncytial virus infection: identifying novel targets for therapeutic intervention structure of a human common cold virus and functional relationship to other picornaviruses suppressive effect of locally produced interleukin-10 on respiratory syncytial virus infection respiratory syncytial virus efficacy of a quadrivalent vaccine against respiratory diseases caused by bhv-1 brsv in experimentally infected calves quantitative and morphological analysis of the vascular bed in bronchial biopsy specimens from asthmatic and non-asthmatic subjects genetic clustering of all 102 human rhinovirus prototype strains: serotype 87 is close to human enterovirus 70 respiratory viral infections drive chemokine expression and exacerbate the asthmatic response rsv bronchiolitis and risk of wheeze and allergic sensitization in the first year of life severe respiratory syncytial virus infections and reduced interferongamma generation in vitro bovine respiratory syncytial virus nonstructural proteins ns1 and ns2 cooperatively antagonize alpha/beta interferon-induced antiviral response respiratory syncytial virus fusion protein mediates inhibition of mitogen-induced t-cell proliferation by contact mucosal immunization of rhesus monkeys against respiratory syncytial virus subgroups a and b and human parainfluenza virus type 3 by using a live cdna-derived vaccine based on a host range-attenuated bovine parainfluenza virus type 3 vector backbone rhinovirus replication causes rantes production in primary bronchial epithelial cells enhanced virulence, airway inflammation and impaired lung function induced by respiratory syncytial virus deficient in secreted g protein il-5 and eosinophils are essential for the development of airway hyperresponsiveness following acute respiratory syncytial virus infection cd8 t cells are essential in the development of respiratory syncytial virus-induced lung eosinophilia and airway hyperresponsiveness transfer of the enhancing effect of respiratory syncytial virus infection on subsequent allergic airway sensitization by t lymphocytes critical roles for interleukin-4 and interleukin-5 during respiratory syncytial virus infection in the development of airway hyperresponsiveness after airway sensitization latency and persistence of respiratory syncytial virus despite t cell immunity prevention of rhinovirus colds by human interferon alpha-2 from escherichia coli respiratory viruses, symptoms, and inflammatory markers in acute exacerbations and stable chronic obstructive pulmonary disease bronchiolitis-associated hospitalizations among us children bronchiolitis-associated mortality and estimates of respiratory syncytial virus-associated deaths among us children, 1979-1997 epidemiologic and clinical evidence of a respiratory syncytial virus-reactive airway disease link a cohort of children hospitalized with acute rsv bronchiolitis: impact on later respiratory disease clinical perspectives on the association between respiratory syncytial virus and reactive airway disease respiratory syncytial virus bronchiolitis in infancy is an important risk factor for asthma and allergy at age 7 asthma and immunoglobulin e antibodies after respiratory syncytial virus bronchiolitis: a prospective cohort study with matched controls severe respiratory syncytial virus bronchiolitis in infancy and asthma and allergy at age 13 subgroup specific protection of mice from respiratory syncytial virus infection with peptides encompassing the amino acid region 174-187 from the g glycoprotein: the role of cysteinyl residues in protection impact of severe disease caused by respiratory syncytial virus in children living in developed countries palivizumab prophylaxis, respiratory syncytial virus, and subsequent recurrent wheezing clinical and immunological effects of low-dose ifn-alpha treatment in patients with corticosteroid-resistant asthma inflammatory subtypes in asthma: assessment and identification using induced sputum sequelae of severe respiratory syncytial virus infection in infancy and early childhood among alaska native children childhood asthma following hospitalization with acute viral bronchiolitis in infancy role of monocytes and eosinophils in human respiratory syncytial virus infection in vitro suppression of the induction of alpha, beta, and lambda interferons by the ns1 and ns2 proteins of human respiratory syncytial virus in human epithelial cells and macrophages effects of nonstructural proteins ns1 and ns2 of human respiratory syncytial virus on interferon regulatory factor 3, nf-kappab, and proinflammatory cytokines tolerance and efficacy of intranasal administration of recombinant beta serine interferon in healthy adults virus-specific cd8+ t lymphocytes downregulate t helper cell type 2 cytokine secretion and pulmonary eosinophilia during experimental murine respiratory syncytial virus infection virus-specific memory and effector t lymphocytes exhibit different cytokine responses to antigens during experimental murine respiratory syncytial virus infection respiratory syncytial virus in early life and risk of wheeze and allergy by age 13 years atopic disposition, wheezing, and subsequent respiratory syncytial virus hospitalization in danish children younger than 18 months: a nested case-control study a survey of virus infections of the respiratory tract of cattle and their association with disease family size, infection and atopy: the first decade of the "hygiene hypothesis infection of a human respiratory epithelial cell line with rhinovirus. induction of cytokine release and modulation of susceptibility to infection by cytokine exposure the respiratory syncytial virus subgroup b attachment glycoprotein: analysis of sequence, expression from a recombinant vector, and evaluation as an immunogen against homologous and heterologous subgroup virus challenge a comparison of cytokine responses in respiratory syncytial virus and influenza a infections in infants bafilomycin a(1) inhibits rhinovirus infection in human airway epithelium: effects on endosome and icam-1 a potent antiangiogenic factor, endostatin prevents the development of asthma in a murine model respiratory development of 5-to 6-year-old children experiencing a first bronchiolitis episode before age one clarithromycin in the treatment of rsv bronchiolitis: a double-blind, randomised, placebo-controlled trial integration of interferon-alpha/beta signalling to p53 responses in tumour suppression and antiviral defence erythromycin modulates il-8 expression in normal and inflamed human bronchial epithelial cells viruses in asthma exacerbations anti-il-4 treatment at immunization modulates cytokine expression, reduces illness, and increases cytotoxic t lymphocyte activity in mice challenged with respiratory syncytial virus parainfluenza virus type 3 expressing the native or soluble fusion (f) protein of respiratory syncytial virus (rsv) confers protection from rsv infection in african green monkeys contribution of the respiratory syncytial virus g glycoprotein and its secreted and membranebound forms to virus replication in vitro and in vivo role of nasal interleukin-8 in neutrophil recruitment and activation in children with virus-induced asthma the brume surrounding respiratory syncytial virus persistence cd40 ligand (cd154) enhances the th1 and antibody responses to respiratory syncytial virus in the balb/c mouse substance p receptor expression on lymphocytes is associated with the immune response to respiratory syncytial virus infection efficacy of tremacamra, a soluble intercellular adhesion molecule 1, for experimental rhinovirus infection: a randomized clinical trial asthma: an epidemic of dysregulated immunity bovine respiratory syncytial virus infection persistent infection of b lymphocytes by bovine respiratory syncytial virus role of alpha/beta interferons in the attenuation and immunogenicity of recombinant bovine respiratory syncytial viruses lacking ns proteins bovine respiratory syncytial virus lacking the virokinin or with a mutation in furin cleavage site ra(r/k)r109 induces less pulmonary inflammation without impeding the induction of protective immunity in calves prolonged nasal eosinophilia in allergic patients after common cold respiratory syncytial virus infections in human beings and in cattle respiratory viral infections and asthma pathogenesis: a critical role for dendritic cells? role of interferon gamma in the pathogenesis of primary respiratory syncytial virus infection in balb/c mice efficacy of a modified live intranasal bovine respiratory syncytial virus vaccine in 3-week-old calves experimentally challenged with brsv immunopathology in rsv infection is mediated by a discrete oligoclonal subset of antigen-specific cd4(+) t cells the attachment (g) glycoprotein of respiratory syncytial virus contains a single immunodominant epitope that elicits both th1 and th2 cd4+ t cell responses bovine respiratory syncytial virus infections in young dairy cattle: clinical and haematological findings airway remodeling in asthma replication and clearance of respiratory syncytial virus: apoptosis is an important pathway of virus clearance after experimental infection with bovine respiratory syncytial virus synergistic effects of fluticasone propionate and salmeterol on inhibiting rhinovirus-induced epithelial production of remodelling-associated growth factors bronchial angiogenesis in severe glucocorticoid-dependent asthma humoral, mucosal, and cellular immune response to topical immunization with a subunit respiratory syncytial virus vaccine inhaled long acting beta agonists for stable chronic asthma inhibition of t1/st2 during respiratory syncytial virus infection prevents t helper cell type 2 (th2)-but not th1-driven immunopathology asthma and respiratory syncytial virus infection in infancy: is there a link? respiratory synctial virus infection in balb/c mice previously immunized with formalin-inactivated virus induces enhanced pulmonary inflammatory response with a predominant th2-like cytokine pattern exacerbations of asthma: addressing the triggers and treatments neutrophil degranulation and cell lysis is associated with clinical severity in virus-induced asthma asthmatic bronchial epithelial cells have a deficient innate immune response to infection with rhinovirus ifn-gamma-induced protein 10 is a novel biomarker of rhinovirus-induced asthma exacerbations leukotrienes mediate neurogenic inflammation in lungs of young rats infected with respiratory syncytial virus the antibody response to primary and secondary infection with respiratory syncytial virus: kinetics of class-specific responses relationship between respiratory syncytial virus bronchiolitis and future obstructive airway diseases phenotypes in asthma: useful guides for therapy, distinct biological processes, or both? respiratory outcomes in high-risk children 7 to 10 years after prophylaxis with respiratory syncytial virus immune globulin the effect of formalin-inactivated vaccine on respiratory disease 352 n differential patterns of methylation of the ifn-gamma promoter at cpg and non-cpg sites underlie differences in ifn-gamma gene expression between human neonatal and adult cd45ro-t cells recombinant respiratory syncytial virus (rsv) bearing a set of mutations from cold-passaged rsv is attenuated in chimpanzees recombinant respiratory syncytial virus bearing a deletion of either the ns2 or sh gene is attenuated in chimpanzees the bronchial microcirculation in asthma histopathologic examination and enumeration of polymorphonuclear leukocytes in the nasal mucosa during experimental rhinovirus colds respiratory virus infection of monolayer cultures of human nasal epithelial cells comparison of human metapneumovirus, respiratory syncytial virus and influenza a virus lower respiratory tract infections in hospitalized young children development of motavizumab, an ultra-potent antibody for the prevention of respiratory syncytial virus infection in the upper and lower respiratory tract rhinovirus viremia in children with respiratory infections human metapneumovirus as a causative agent of acute bronchiolitis in infants duration of postviral airway hyperresponsiveness in children with asthma: effect of atopy infection of human respiratory submucosal glands with rhinovirus: effects on cytokine and icam-1 production exposure of neonates to respiratory syncytial virus is critical in determining subsequent airway response in adults respiratory syncytial virus infection of human airway epithelial cells is polarized, specific to ciliated cells, and without obvious cytopathology in vivo selection of respiratory syncytial viruses resistant to palivizumab altered eosinophil levels as a result of viral infection in asthma exacerbation in childhood respiratory syncytial virus escape mutant derived in vitro resists palivizumab prophylaxis in cotton rats virokinin, a bioactive peptide of the tachykinin family, is released from the fusion protein of bovine respiratory syncytial virus bone marrow plasmacytoid dendritic cells can differentiate into myeloid dendritic cells upon virus infection key: cord-003492-rodqdtfj authors: montaner-tarbes, sergio; del portillo, hernando a.; montoya, maría; fraile, lorenzo title: key gaps in the knowledge of the porcine respiratory reproductive syndrome virus (prrsv) date: 2019-02-20 journal: front vet sci doi: 10.3389/fvets.2019.00038 sha: doc_id: 3492 cord_uid: rodqdtfj the porcine reproductive and respiratory syndrome virus (prrsv) is one of the most important swine diseases in the world. it is causing an enormous economic burden due to reproductive failure in sows and a complex respiratory syndrome in pigs of all ages, with mortality varying from 2 to 100% in the most extreme cases of emergent highly pathogenic strains. prrsv displays complex interactions with the immune system and a high mutation rate, making the development, and implementation of control strategies a major challenge. in this review, the biology of the virus will be addressed focusing on newly discovered functions of non-structural proteins and novel dissemination mechanisms. secondly, the role of different cell types and viral proteins will be reviewed in natural and vaccine-induced immune response together with the role of different immune evasion mechanisms focusing on those gaps of knowledge that are critical to generate more efficacious vaccines. finally, novel strategies for antigen discovery and vaccine development will be discussed, in particular the use of exosomes (extracellular vesicles of endocytic origin). as nanocarriers of lipids, proteins and nucleic acids, exosomes have potential effects on cell activation, modulation of immune responses and antigen presentation. thus, representing a novel vaccination approach against this devastating disease. the porcine reproductive and respiratory syndrome virus (prrsv) is one of the most important swine diseases in the world. it is causing an enormous economic burden due to reproductive failure in sows and a complex respiratory syndrome in pigs of all ages, with mortality varying from 2 to 100% in the most extreme cases of emergent highly pathogenic strains. prrsv displays complex interactions with the immune system and a high mutation rate, making the development, and implementation of control strategies a major challenge. in this review, the biology of the virus will be addressed focusing on newly discovered functions of non-structural proteins and novel dissemination mechanisms. secondly, the role of different cell types and viral proteins will be reviewed in natural and vaccine-induced immune response together with the role of different immune evasion mechanisms focusing on those gaps of knowledge that are critical to generate more efficacious vaccines. finally, novel strategies for antigen discovery and vaccine development will be discussed, in particular the use of exosomes (extracellular vesicles of endocytic origin). as nanocarriers of lipids, proteins and nucleic acids, exosomes have potential effects on cell activation, modulation of immune responses and antigen presentation. thus, representing a novel vaccination approach against this devastating disease. keywords: porcine reproductive and respiratory syndrome virus, prrsv, virus biology, immunology, vaccinology, extracellular vesicles economic impact prrsv is responsible for respiratory disease in weaned and growing pigs, as well as reproductive failures in sows. it is considered one of the most important swine diseases worldwide, with an economic impact estimated at $664 million in losses every year to u.s. producers, representing an increase of 18.5% in the last 8 years (1, 2) . in europe, the situation is similar and economic disease models have been carried out to determine the economic burden in the best and worst case scenario combining reproductive failure and respiratory disease, estimating annual losses from a median of e75,724, if the farm was slightly affected during nursing and fattening, to a median of e650,090 if a farm of 1,000 sows is severely affected in all productive phases (3) . nevertheless, there is scarce of information about the economic impact of this disease as a consequence of multiple factors (vaccination, treatment, respiratory symptoms, reproductive failure, and other prrsv-related diseases) making a difficult task to quantify exactly this parameter under field conditions. thus, the exact economic impact of prrsv remains a key gap in the knowledge for this disease. the porcine reproductive and respiratory syndrome virus (prrsv) was first isolated in the early 1990s in europe and north america (4, 5) . it is an enveloped single-stranded positivesense rna virus of the family arteriviridae, genus porarterivirus according to the international committee of taxonomy of viruses (6) . presently, there are four distinct species included in this genus (porarterivirus), prrsv-1 and prrsv-2 (with 30-45% variation in nucleotide sequences), along with other two viruses that do not affect pigs (lactate dehydrogenaseelevating virus and rat arterivirus 1) (7) . the genome size of prrsv is about 15 kb with 10 open reading frames (orfs), with replicase genes located at the 5 ′ -end followed by the genes encoding structural proteins toward the 3 ′ -end (8) . the majority of the genome (∼60-70%) encodes non-structural figure 1 | genome structure and mature viral particle of prrsv virus. (a) non-structural proteins are located in the 5 ′ end of the genome, codifying for two different polyproteins pp1a and pp1ab that are cleaved into at least 14 nsps (nsp1 to nsp12 and nsp1α and nsp1β, and nsp7α, and nsp7β). structural proteins located near the 3 ′ end, are associated to the viral envelope and rna packaging. (b) prrsv mature viral particle, composed of a lipid bilayer envelop with viral receptor glycoproteins involved on infection and cell internalization. single stranded positive rna is associated with nucleocapsid protein in the internal layer of the virus. proteins involved in replication (orf1a and orf1ab), whereas orfs 2-7 encodes structural proteins (n, m, gp2-gp5, e) ( figures 1a,b) (9) . using orf5 in molecular epidemiological studies, an enormous genetic variability has been described (10) . yet, data on whole genome sequencing is scarce and constitute another important gap in the knowledge of this virus and its evolution (box 1). prrsv replicase genes consist of two orfs, orf1a and orf1b, which occupy the 5 ′ proximal three-quarters of the genome ( figure 1a) . both are expressed from the viral genome, with expression of orf1b depending on a conserved ribosomal frameshifting mechanism. subsequently, extensive proteolytic processing of the resulting pp1a and pp1ab polyproteins yields at least 14 functional non-structural proteins (nsps), specifically nsp1 to nsp12, with both the nsp1 and nsp7 parts being subject to internal cleavage (giving origin to nsp1α and nsp1β, and nsp7α, and nsp7β, respectively), most of which assemble into a membrane-associated replication and transcription complex (11) . recently, a programmed ribosomal frameshift encoding an alternative orf that generates two extra proteins, nsp2tf and nsp2n, was discovered in prrsv and other arteriviruses (12, 13). these nsps, described for prrsv, have proven to box 1 | gaps in knowledge in prrsv. be necessary and sufficient for the induction of membrane modifications resembling those found in infected cells (14) . most importantly, all positive rna viruses seem to induce one of two basic morphotypes of membrane modifications: invaginations or double-membrane vesicles. prrsv also has a set of 8 structural proteins, including a small non-glycosylated protein and a set of glycosylated ones: gp2ab, gp3, gp4, gp5, and gp5a, m and n proteins (15) . however, nsp2, traditionally classified as a non-structural protein, has been found to be incorporated in multiple isoforms within the viral envelope (ovarian tumor domain protease region, hypervariable region and c-terminal region) (16) , giving new insights into the structure of this virus ( figure 1b) . first, the nucleocapsid protein (n), as one of the most important parts of the mature viral particle, has been deeply characterized on prrsv, finding important features shared in most nonsegmented rna viruses. the n protein consists of 123 amino acids for genotype 2 and 128 amino acids for genotype 1. the viral envelope glycoproteins (gp2 to gp5) are the first interactors with host cell receptors to initiate infection and are exposed to the immune system when viral particles are in blood and lymphoid tissue circulation (figure 2 ). there is also another protein that contribute to virion structure, m protein, that is required during viral entry to interact with heparan sulfate cell receptor on macrophages. later, gp5 is thought to bind to sialoadhesin and virus internalization and uncoating is triggered by a formation of a viral heterotrimer (gp2a, gp3, and gp4) with scavenger receptor cd163 (figure 2 ) (17, 18) . gp5 is the most abundant glycoprotein. first, it interacts with two cell entry mediators, heparan sulfate glycosaminoglycans and sialoadhesin/cd169 (17, 18) to favor viral entry and then possibly with the n protein and its mhc-like domain to carry n-viral rna complex to the budding site (figure 2) . gp2, gp3, and gp4 are protected with glycan shields, like most prrsv membrane proteins, to avoid antibody recognition and neutralization. gp2 has two glycosylation sites, gp3 have seven and gp4 have four, figure 2 | interactions between viral proteins and cell receptors for virus attachment, entry, uncoating and release of genetic ssrna to cell cytoplasm. blocking cd163, cd151 tetraspanin or vimentin seems to inhibit viral replication or infection in the host cell, but reduced replication or no effect is seen when receptors such as heparan-sulfate or siglec-1 are blocked, demonstrating that some viral proteins and cell receptors are indispensable in terms of production of infectious viral progeny and dissemination in the host. three of which are directly related to virus survival, causing lethal damage in virus production when more than two of these sites are mutated (19) (figure 2 ). viral replication starts by interaction of viral glycoproteins with different cellular receptors (figure 2 ) (17) . cd163 and cd169 play a main role during infection, uncoating of the viral particle, activation of clathrin-mediated endocytosis and release of viral genome in the cytoplasm (20) . cd163 has been defined as the main receptor for viral infection by evaluating the effect of prrsv on cd163 knockout pigs, where there is complete resistance to infection (21) . cysteine-rich domain 5 in this receptor seems to be necessary to establish interactions with prrsv-1 species, since its deletion by crispr/cas9 system (exon 7 of the gene encoding this region) implies protection for a large panel of these viruses demonstrated by in vitro challenge of edited-pig macrophages and in vivo experiments with srcr5 animals (22) (23) (24) . more important, edited pigs show no side effects when kept under standard husbandry conditions and cd163 seems to maintain its biological function (hemoglobin-haptoglobin scavenger) regardless the lacking cysteine-rich 5 domain, nevertheless, other unknown functions could be impaired by this modification. in conclusion, gene-edited pigs lacking srcr5 region of cd163 could be an important asset to confront prrsv epidemics with the final goal of eradication. cd169 seems to be related only to co-interactions with sialic acid in the virion surface, however, knockout pigs for either exon 1, 2, or 3 of cd169 were not protected from infection and viral load as well as antibody responses were similar to heterozygous (cd169 +/− ) or wild type pigs (cd169 +/+ ) (25) . the former experiments suggested that other unknown mechanisms could be involved in prrsv infection such as other receptors, new unknown susceptible cell types different from macrophages or possible leaking of cd169 expression in the knockout model. other molecules are also involved in viral entry, such as cd151 (26) and vimentin (27) ; blocking of any of these four molecules (cd163, cd169, cd151, and vimentin) had an effect on viral infection, either on internalization or complete inhibition of viral replication (17) . after cell entry, prrsv causes a series of intracellular modifications to complete its replication cycle, which includes rearrangements of intracellular membrane organelles to generate the replication complex. these include the formation of perinuclear double membrane vesicles apparently derived from endoplasmic reticulum, synthesis of genomic rna (grna), transcription of segmented rna (sgrna) and expression of viral proteins (20, 28) . at late stages of replication, the mature virions accumulate in the intracellular membrane compartments and they are then released into the extracellular space through exocytosis (29) . a non-classical spread pathway has been detected in several viruses including prrsv where virus dissemination is mediated by cell to cell nanotubules (30) . it was reported that almost all prrsv proteins interact with myosin and actin (especially f-actin and myosin iia) where nanotubules connected cells allowing the movement of structural proteins and rna, infecting naïve cells in a non-classical way even in the presence of neutralizing antibodies in the cell media. in addition, this non-classical pathway demonstrated that prrsv cell entry receptors were not necessary to establish infection, as nonpermissive cells became infected when were contacted by infected cells via nanotubes. this spreading strategy has been proposed as a mechanism to facilitate infection either by surfing of viral particles between adjacent cell membranes or as a receptor-independent mechanism for infection (31) ; importantly, has been reported for other viruses such as hiv-1 where nanotube number on macrophages increases after infection (32) and herpesvirus transmission between bovine fibroblasts (33) . interestingly, although several viral proteins were detected in nanotubules (nsp1β, nsp2, nsp2tf, nsp4, nsp7, and nsp8, gp5 and n), gp4 was detected in only a few nanotubes. in particular, the role of gp4 in this nonclassical spread pathway is not fully understood and it will be interesting to further evaluate gp4 interaction with other cellular components to elucidate the reason why gp4 is not transported to new recipient naïve cells. altogether these data indicate that prrsv has evolved different pathways to spread even though, in vivo, the virus shows narrow cell tropism for monocytes and macrophages (34, 35) (box 1). the innate immune response is the first system any given pathogen encounters, specially to prevent viral replication and invasion into mucosal tissues (respiratory tract in the case of prrsv) and, importantly, to initiate the strong adaptive immune response to fight against intracellular infectious agents (7). type i interferons (ifn α/β) comprise one of the most potent mechanisms against invading viruses in the first stages of infection, triggering an array of ifn-stimulated genes (isg) (36) . generally speaking, all nucleated cells have the ability to produce ifn α/β, but plasmacytoid dc (pdc) are the most potent producers of this family of cytokines (37) . prrsv has evolved a set of mechanisms for suppressing ifn α/β in vivo, maintaining low expression levels of this cytokines on infected pigs (38) during almost all time-course of infection shortly after transient elevation in the lungs (39) . suppression of ifn α/β also takes place in vitro in prrsv infected marc-145 and porcine alveolar macrophages (38, 40, 41) . further studies have shown that ifn type i suppression is a major strategy of prrsv to modulate host antiviral defense. in fact, several viral proteins have been identified as ifn antagonists (nsp1α, nsp1β, nsp2, nsp4, nsp11, and n) (7, (42) (43) (44) . as an example for n protein, upon dsrna stimulation, ifn-β production was shown to decrease proportionally with increasing levels of n expression and additionally it was found to downregulate ifn-dependent gene production by dsrna interfering with dsrna-induced phosphorylation and nuclear translocation of irf3 (45) . among prrsv non-structural proteins with type i ifn modulation capacity, nsp1 has been considered as the strongest antagonist of ifn-β production by acting on interferon regulatory factor 3 (irf3) phosphorylation and nuclear translocation. almost all nsps, excepting nsp1, have been related to the perinuclear region, associated with intracellular membranes, supposedly derived from the endoplasmic reticulum (er), which are modified into vesicular double-membrane structures with which the viral replication and transcription complex (rtc) is thought to be associated with (14, 46, 47) . nsp1 translocates to the nucleus during the first hours of infection, where it is capable of inhibiting irf3 association with creb-binding protein (cbp), promoting cbp degradation by a proteasome-dependent mechanism, without which the transcription enhanceosome may not assemble the transcription machinery for the interferon expression (15, 46) . recently, post-transcription protein expression of ifn β was shown to be regulated by prrsv by means of upregulating cellular mirna in porcine alveolar macrophages (48) nsp2 is the largest (mature) prrsv protein and contains at least four distinct domains: the n-terminal cp/otu domain, a central hypervariable region, a putative transmembrane domain, and a c-terminal region of unknown function that is rich in conserved cysteine residues. this protein is unique in the context of prrsv due to its genetic heterogeneity, its participation in diverse roles supporting the viral replication cycle, and its packaging within the prrsv virion (16, 49) . previous studies suggest that nsp2 has different roles related to immune evasion mechanisms. it has been determined that nsp2 otu domain (thiol-dependent deubiquitinating domain) inhibits the nuclear factor kappa-light-chain-enhancer of activated b cells (nf-kb) by interfering with the polyubiquitination process of ikbα (nuclear factor of kappa light polypeptide gene enhancer in b-cells inhibitor) and, subsequently, preventing the degradation of the ikbα protein (50) . moreover, viable deletion mutants in nsp2, when infecting cells, caused a downregulation of cytokines (il-1β and tnf-α) mrna expression, in comparison with that of parental virus, suggesting that certain regions of nsp2 might contribute to the induction of a virus-specific host immune response and that deletion of such a region could produce a more virulent virus (51) . there are several isoforms of nsp2, sharing a consistent core set between viral strains, which are integrated into mature virion at the final stage of replication (figure 1b) , although some of them could be strain-specific. inclusion of nsp2 within the prrsv virion suggests that it may function in previously unknown roles related to extracellular function, entry, or immediate-early viral replication events (16) . truncated forms of nsp2 have also been identified, named nsp2tf and nsp2n, with apparent roles in modulation of immune evasion. when deletion mutants for those forms were used to infect cells, there was a significant change in gene expression, a strong activation of those involved in cytokine-cytokine receptor interaction, tnf signaling, toll-like receptor signaling, nodlike receptor signaling, nf-κb signaling, rig-i-like receptor signaling, chemokine signaling, jak-stat signaling, cytosolic dna-sensing, and nk cell mediated cytotoxicity (13) , suggesting that an active role (direct or indirect) is played by these truncated forms in modulating host cells innate immune response, making prrsv infectious cycle more complicated than it was initially thought. nsp11, is a nidovirus conserved endoribonuclease with an uridylate-specific endonuclease (nendou). it has been demonstrated in vitro that overexpression of nsp11 enhanced viral titter (52) . moreover, nsp11 antagonizes type i ifn, specifically ifnβ production, activated by the retinoic acid inducible gene 1 like receptor, showing substrate specificity toward mitochondrial antiviral signaling proteins (mavs) and rig-i (transcripts and proteins), and demonstrating that this activity was associated to the endoribonuclease activity of this protein in which transfection mutant viruses were unable to degrade mavs mrna and impair ifnβ production (53) . another mechanism whereby this protein limits antiviral response is related to inflammasome and synthesis of il-1β, due to its important role in both the innate and adaptive immune response and in pathological mechanisms. it has been shown that prrsv could activate nlrp3 inflammasome in early stages of infection but induce host's immunosuppression later as measured by determining the levels of pro-il-1β and procaspase-1 mrna and the mature il-1β protein in porcine alveolar macrophages (pam) (54) . it is not surprising that nsp11 also interacts with the rna-silencing complex (risc), as it has been demonstrated in vitro in a marc-145 cell line that this protein and nsp1α are responsible for inhibiting risc and downregulating argonaute-2 protein expression increasing viral titter significantly, which demonstrates a direct relationship between this silencing complex and viral replication at least in vitro (55) . other non-structural proteins have been studied but there is an important gap on information about in vivo and in vitro functions and interaction in signaling pathways. additionally, the enormous variation among strains makes it difficult to characterize all protein variants and interactions with cell systems (macrophages, dendritic cells "dcs, " monocytes and others) (box 1) . recently, a body of evidence associates host genetics with different outcomes following prrsv infection in the respiratory and reproductive form of the disease (56) (57) (58) (59) (60) . although pathways and mechanisms involved in specific disease-resistance traits have not yet been fully characterized, it is clear that the genetic variation in disease resilience is polygenic, regulating aspects of both innate resistance and acquired immunity (56) . in connection with innate response, the average daily gain (adg) after prrsv infection was associated with a single genomic region in chromosome 4 (ssc4) which is best represented by the snp tag marker wur, located in the 3 ′ non-coding region of the interferon-inducible guanylate-binding protein 1 (gbp1) gene (61) . the pig genetic resistance to prrsv infection has been historically overlooked in prrsv research probably generating a confounding factor in immune response studies. a key gap in the knowledge of prrsv is linked the pig genetic variability after prrsv infection with the enormous variability of the virus itself (box 1) . in pigs, prrsv replicates in cells belonging to the innate immune system. pams are the primary cells to be infected in the lungs as well as other cells of the monocyte/macrophage lineage, which later could disseminate the virus to other tissues or support replication to release viral particles into the bloodstream (17) (figure 2) . moreover, prrsv is thought to be able to infect professional antigen presenting cells such as dcs and monocyte derived dendritic cells, (modc) impairing their normal antigen presentation ability by inducing apoptosis, down-regulating the expression of ifn-α, mhc class i, mhc class ii, cd11b/c and cd14, upregulating the expression of il-10 and inducing minimal th1 cytokine secretion (62) (63) (64) (65) . nevertheless, new evidence suggest by in vivo and in vitro experiments that specifically lung cdc1, cdc2, and modcs are not infected by prrsv-1 viruses from subtypes 1 and 3 and one possible explanation is the lower expression of cd163 and cd169 in those 3 dc subtypes, associating previous results of infection in dcs to culture conditions of monocytes in vitro that could cause a sensibilization to infection by certain strains as lena (66) . in addition, these findings were also tested in tonsil cdc and tracheal cdc1 and cdc2 observing that those cell populations are not infected by prrsv virus (67, 68) . moreover, a new type of pam has been characterized and named porcine intravascular macrophages (pim) due to its association to endothelial lung capillaries and not to the alveoli, presenting strong capacity to phagocytised bloodrelated particles (69) . importantly, when infected pim cells gave similar results of viral load to those derived from infected pam, but significantly upregulates of tnfα and non-significantly il-6 and il-8 expression after infection when compared to normal alveolar macrophages, indicating that these cells have an important pro-inflammatory role during prrsv infection in the lungs (69) . new interactions between cells and the virus need to be further explored to unravel possible immunological features that leads to correlates of protection. recently, it has been shown that a domain within nsp1α is able to stimulate the secretion of cd83, which in turn inhibits modc function in vitro, impairing the ability of modc to stimulate t cell proliferation (70) . production of ifn α/β and the mechanisms for cell activation by pdc are severely suppressed during prrsv infection, although these cells are not permissive to prrsv infection (71, 72) . however, this phenomenon is strain dependent, as other prrsv strains are able to stimulate pdc for ifn α/β production in large quantities (73) . again, there is an enormous variability between prrsv strains in relation with their effect on antigen presenting cells which prevent scientists from finding common mechanisms. it might be of interest to link this key gap of knowledge for prrsv with host genetics (box 1) . moreover, in prrsvinfected cells, n is abundantly expressed benefiting from the discontinuous transcription mechanism (74) . this protein is also distributed in the nucleus, induced by two nuclear localization signals called cryptic nls or nls-1 and functional nls or nls-2 (positions 10-13 and 41-47, respectively) (75). the effect of n protein has been examined in pams and modcs using transfection, finding a significant upregulation of il-10 gene expression. natural killer (nk) cells constitute another powerful arm of the innate immune system against prrsv, particularly when considering the high percentage of circulating nk cells in pigs (76) . the cytotoxic function of nk cells is reduced in prrsv infected pigs from day 2 after infection up to 3-4 weeks (38, 77, 78) . initial studies using in vitro systems demonstrated that the stimulation of porcine nk cells with proinflammatory cytokines (il-2 and il-15) was capable of activating nk cells and inducing them to express high levels of ifn-γ and perforins to cause lysis of infected cells, but a different scenario appears if cells are evaluated post-infection, indicating that a virus such as prrsv is capable of impairing nk cell cytotoxicity (79) . in vitro, the nk cytotoxicity against prrsv-infected pams was decreased and degranulation of nk cells inhibited (80) . in vivo, the immune response is the same as that observed in vitro, with some studies reporting that approximately half of viremic pigs had a reduction >50% in nk cell-mediated cytotoxicity and enhanced secretion of il-4, il-12, and il-10 and reduced frequency of cytotoxic t-cells (cd4 − cd8 + t) and double positive t cells (cd4 + cd8 + t) and upregulated frequency regulatory t-cells (tregs) (81). innate immune responses against prrsv are obstructed by different mechanisms as are adaptive responses. the modest and delayed b cell mediated neutralizing antibody response is one of the main characteristics associated to prrsv acquired immune responses. even though prrsv specific antibodies appear early at 7-9 days post-infection, the efficacy of those antibodies remains unclear. neutralizing antibodies take longer, appearing nearly 1 month after infection (34) . however, passive transfer of these neutralizing antibodies conferred almost full protection in a prrsv reproductive model (95% of offspring alive after challenging pregnant sows with high neutralizing antibody titter). nevertheless, in another experiment using the reproductive model, when the presence of prrsv was examined after the transfer of neutralizing antibodies, lungs, tonsils, buffy coat cells, and peripheral lymph nodes contained replicating prrsv similar to infected controls, although pigs were apparently protected against infection. in summary, passive transfer of high neutralizing antibody titter conferred protection to gilts and offspring (not detectable viremia), but did not eliminate the presence of viral particles in peripheral tissues nor transmission to animals they were in contact with (82) (83) (84) . curiously, the role of neutralizing antibodies in the protection against the respiratory form of the disease is a key gap of knowledge for prrsv. this point is critical to define precisely targets for improved vaccines based on the humoral immune response against this virus (box 1). n protein is involved in several mechanisms for immune evasion and is also one of the most immunogenic structural proteins (75) . antibodies against n appear early during acute infection, together with those against m and gp5 proteins, but are non-neutralizing and could be involved in antibody dependent enhancement (85, 86) . there are other "antibody-related mechanisms" that do not necessarily involve neutralizing activity. antibody-dependent cell-mediated cytotoxicity (adcc), antibody-dependent complement-mediated cytotoxicity (cdc) and antibodydependent complement mediated virolysis (adcv) have been examined in the context of prrsv, although none of these mechanisms were evident during infection or have not been deeply investigated on in vitro and in vivo models of this virus (87) . it is important to note that neutralizing antibodies appear late in prrsv infection and other immune mechanisms (cellular or antibody mediated immune response) might be acting to suppress viral replication in blood, causing the virus to be isolated in lymphoid tissues and maintaining suboptimal replication that will finally end in viral clearance. for type prrsv-2 it has been demonstrated that immunization of pigs with ectodomain peptides from gp5/m complex did not induce neutralizing antibodies (88) although those ectodomain-specific antibodies generated were capable of binding virus. an important feature that makes difficult to validate the location of neutralizing epitopes is the number of glycosylations in or around it. for prrsv-1 strains, up to 3 glycosylations may be found in, or flanking the gp5 neutralizing epitope that is located between amino acids 37-45 (89) , whereas for prrsv-2 strains there are four potential glycosylation sites (90) . when tested, prrsv with mutations in gp5 glycosylation sites (either at n44 or in the hypervariable region, upstream the neutralizing epitope) enhanced immunogenicity with increased concentration of antibodies directed to this epitope 5-10 fold higher compared with those induced by the wild type strains (89) . same results were obtained when administering another deglycosylation mutant (double deglycosylation in the putative glycosylation moieties on gp5) twice, which conferred better protection against homologous challenge (91) . in addition, when this protein is expressed early during infection, it stimulates production of early neutralizing antibodies and ifn-β, two main antiviral mechanisms, demonstrating its role in induction of self-protection mechanisms from the host (92) . available data about neutralizing antibodies induced by this protein are controversial, which may be due to the high variation among prrsv strains (93) and, as previously commented, the host genetics. orf5 is also complemented by a small frameshift of the subgenomic mrna called orf5a, encoding a type i membrane protein consisting primarily of alpha helix with a membrane-spanning domain (called gp5a) that is incorporated into virions as a very minor component, playing a role in viral replication, as mutation in the initiation codon or premature termination related to expression for this protein leads to non-efficient viral replication and lower titter (94, 95) . this protein is capable of eliciting specific antibody immune response in natural infections and after immunizations, although those are not neutralizing neither protective in a challenge trial after infection, making difficult to define the role of this particular small protein in the whole immune response and viral clearance of prrsv infection (96) . in summary, the role of humoral immunity remains elusive in prrsv infection (neutralizing and non-neutralizing antibodies) and a better characterization will be required to overcome this relevant gap of knowledge (box 1) . treg typically increase in number in chronic viral diseases to prevent a persistent inflammatory response and pathological damage associated to viral infections. conversely, tregs are described as key contributors in modulating the host immune response to viral infection. this cell population is an important component in regulating the magnitude of the immune response to infection (in viruses such as hiv and hcv), thus preventing excessive inflammation and tissue damage. however, they can also be inappropriately induced by viruses to switch the balance of the immune response in favor of maintaining viral replication (97) . in prrsv, the role of tregs remains unclear and appears to be a consequence of il-10 induction of some strains as early as 2 days post infection (81) . in some experiments, in vitro infected dcs with prrsv-1 exhibited an unbalanced ability to stimulate t cell immune responses in a strain-dependent manner, but no tregs were detected, at least in vitro, as measured by expression of cd25 and foxp3 markers (98) . when using prrsv-2 strains, the case seems to be different, as the virus was capable of stimulating il-10 production with concomitant generation of tregs (99) which was associated to nucleocapsid protein expression in the in vitro system. this group also suggested that il-10 production and treg could be related to impaired gamma interferon (ifn-γ) production and altered development of protective t-cell response by inhibiting t-cell proliferation as seen in the early stage of infection with viruses such as hcv. vaccine strains currently in use in the united states do not provide adequate heterologous protection, one possible explanation could lay on their inability to induce an adequate ifn-γ response due to their ability to stimulate tregs, at least in vitro (100). structural conformation, but not nuclear localization, of the expressed n protein was suggested as essential for the ability to induce il-10 that, in consequence, causes induction of tregs as measured by markers cd4+cd25+foxp3+ (99) . it should be noted that when the role of the nuclear localization signal was evaluated using deletion mutants, results suggested that nls-2 was not essential for virus survival, although pigs developed a significantly shorter duration of viremia and higher neutralizing antibodies than those of wild-type prrsv-infected pigs (101) . the role of tregs cells in the immune response against prrsv is a key gap of knowledge in order to develop more efficacious prrsv vaccines (box 1) . moreover, reports have highlighted the impact of prrsv infection on thymic cellularity mainly as a loss of cd4 + /cd8 + cells in the thymus of prrsv-infected pigs. acute lymphopenia, thymic atrophy, and lymphadenopathy associated with the presence of prrsv antigen in the thymus are some of the mechanisms whereby prrsv suppresses the immune response. in addition, presence of prrsv antigens in the thymus could also induce tolerance and presents a mechanism that could explain the presence of tregs during prrsv infection (93) . nevertheless, the picture is not complete and basic knowledge about the effect of prrsv on cell development in the thymus would be of great interest to understand the effect of this viruses in the host. prrsv immunology thus remains an unsolved puzzle due to complex interactions between different viral strains and the host. similar immune responses could be the key feature of this virus, such as persistence viremia, a strong inhibition of innate cytokines (ifn-α/β, tnf-α, il-1β, ifn-γ), dysregulation of nk cell function (cytotoxicity and degranulation), rapid induction of non-neutralizing antibodies, delayed appearance of neutralizing antibody, late and low cd8+ t-cell response, and induction of regulatory t cells (tregs) (102) . as a whole, neutralizing antibodies and prrsv-specific ifn-γ secreting cells do not fully depict the immune effector functions related to protective immunity, as the viral targets related to them are unknown. as a consequence, correlates of protection remain elusive for this infection due to the laborious work in vitro and in vivo and the enormous genetic diversity that causes confusion and makes it difficult to predict how immune responses against one isolate or strain could be applied to another in a cross-protective immune prediction model (103, 104) . without any doubt, the most important gap of knowledge for prrsv is the lack of correlates of protection that makes extremely difficult to have robust models to check vaccines efficacy against this disease (box 1). since the beginning of prrsv outbreaks in europe and the usa, the development of efficacious prrsv vaccines has been a challenge. classical approaches are not working properly for several reasons: viral mutation can lead to more pathogenic strains, there is a lack of knowledge on how the porcine immune system interacts with all prrsv proteins, and most importantly, there is no robust parameter (surrogate marker) that can be unequivocally linked with viral clearance. thus, there is no relationship between complete homologous or heterologous protection and classic immunological parameters such as an increase/decrease in particular cell population (105) , ifnγ production, neutralizing antibodies (106), non-neutralizing antibodies and clinical outcome (107) . in addition, highly divergent strains make it more difficult to develop a universal vaccine for this virus (28) . several different vaccines against prrsv have reached the market and have been reviewed recently (108) . most of these vaccines rely upon modified live virus (porcilis prrs from merck, ingelvac prrsflex eu from boehringer ingelheim, amervac-prrs from hypra, pyrsvac-183 from syva) against prrsv-1, as well as some to control prrsv-2 (fostera prrs from zoetis, ingelvac prrs mlv/ingelvac prrsatp from boehringer ingelheim). there is also evidence that most mlv vaccines of both prrsv-1 and prrsv-2 species elicit specific humoral and cell-mediated immune (cmi) responses, as they confer protection to homologous parental strains and partial protection to heterologous strains. although it is possible to control some prrsv outbreaks by use of mlv in combination with good practices, there are major safety issues such as a high mutation rate leading to reversion to virulence and recombination among vaccine and wild type strains. cases have been reported in which new viruses have been introduced as a consequence of mlv vaccines. for example, nucleotide sequence identities of atypical danish isolates were between 99.2 and 99.5% with the vaccine virus respprrs and 99.0-99.3% with vr2332, which is the parental virus to the vaccine virus, supporting the conclusion that the introduction of prrsv-2 in denmark was due to the spread of vaccine virus (109) . in china a recombination event was reported in which a prrsv variant with nucleotide deletions and insertions in the non-structural protein 2 (nsp2) gene also showed a possible recombination event between a mlv strain and a prototype chinese field strain (110) . current inactivated vaccine approaches are not highly effective since elicited immune responses are not enough to prevent spreading of the virus. however, this type of vaccine can augment anamnestic virus neutralizing antibodies and virusspecific ifn-γ responses following a wild-type virus infection or prrsv-mlv vaccination which can contribute to viral clearance (111, 112) . thus, the combination of modified live vaccines with inactivated ones can be a reasonable approach to control the disease under field conditions (113) but unfortunately, there is no robust data comparing this approach with other options available on the market. on the other hand, most inactivated vaccines are not approved for use in the united states due to the poor efficacy showed in challenge trials (114) as measured by production of prrsv specific neutralizing and non-neutralizing antibodies and low cellular immune responses leading to their failure in the porcine market. according to the centre for food security and public health of iowa state university, only box 2 | exosomes and therapeutic applications in prrsv. frontiers in veterinary science | www.frontiersin.org "biosuis prrs inact eu+am" is approved to be used in the us. however, new strategies are being evaluated to overcome these problems (115) , including nanoparticle entrapped antigens (116) (117) (118) (119) , plant based approaches (120) or vectored vaccines (121) . several attempts have been made to use structural proteins to develop vaccines against prrsv because they are specific targets of neutralizing antibodies. for this reason, one may hypothesize that antibodies against those proteins could be the main key to inhibit viral replication and spread as it is common for many viruses. approaches such as vlps combining different structural proteins have been tested (122) (123) (124) , finding that anamnestic response is possible (boosted igg and ifn-γ producing cells) in previously vaccinated or infected pigs but not in the prechallenge period. these structural proteins are able to prime the immune system, but no reduction of viremia was observed after challenge (123) . those results suggest that other viral proteins may be targeted to induce a protective response in pigs. a plausible explanation for this finding may be based on the presence of few neutralizing epitopes in their sequences, most of which are located in variable regions of the proteins, to the phenomena of glycan shielding for epitopes and to the high variability observed between prrsv virus strains. again, a critical gap of knowledge for prrsv is to precisely characterize common epitopes that are present in all prrsv strains. epitopes responsible for generating an efficient immune response eliciting cross-protective immunity remained elusive. taken together, this evidence points to the need for new vaccination approaches that comply with a pathogen free strategy, capable of eliciting effective cellular and antibody responses with mid to long term protection against homologous strains and preferable to heterologous challenge as well. extracellular vesicles(evs) are gaining increased scientific attention as novel vaccines against infectious diseases, including animal diseases of veterinary importance by its capacity of self-antigen presentation, activation of host cell and antibody immune responses and more important, to induce protection in lethal challenge trials (125-131) (box 2). in the case of prrsv, artificial micrornas (amirna) were initially synthetized to try suppressing expression of sialoadhesin (sn) or cd163 by recombinant adenoviral vectors to be contained in exosomes, causing a subexpression of sn and cd163 at mrna and protein level, and reducing viral titter when porcine macrophages were pre-treated with amirna thus providing new evidence supporting the hypothesis that evs can also serve as an efficient small rna transfer vehicle for pig cells (132) . more recently, prrsv viral proteins associated to extracellular vesicles (evs) in the size range of exosomes, were reported (129) . moreover, a targeted-pig trial using evs from sera of infected pigs who had overcome the disease, demonstrated that evs are capable of inducing specific ifn-γ secreting cells after a prime-boost strategy, are safe, free-of-virus and can differentiate infected from vaccinated animals (133) , moreover, it was demonstrated that those evs contained antigenic viral proteins recognized by pig immune sera and not by the pre-immune one. of interest, however, a recent article indicated that prrsv derived evs are capable of transmitting the virus from one cell to another (134) . whether these discrepancies are due to in vivo vs. in vitro experimental work and methods applied to isolate evs from serum samples or culture supernatant, remains to be determined. evs have also been explored as novel control strategies in other viral diseases. for example, in respiratory syncytial virus infection, evs are released with a selected modified cargo when compared with uninfected epithelial cells. when analyzed in detail, several viral proteins and diverse species of rna were detected and capable of activating innate immune responses through induction of cytokine and chemokine release (135) . similar scenarios of viral proteins exported in evs have been observed and extensively reviewed for hiv/hcv/htlv-1 (136), ebv (137) , and other viral diseases. moreover, viral products of various origin and size including ebola virus vp24, vp40, and np, influenza virus np, crimean-congo haemorrhagic fever np, west nile virus ns3, and hepatitis c virus ns3, when fused with nef c-terminal domain through dna vectors, were directed to the evs membrane or packaged into them and remained stable after fusion. more importantly, when injected in mice, dna vectors expressing the diverse fusion products elicited a well detectable antigen-specific cd8+ t cell response associating with a cytotoxic activity potent enough to kill peptide-loaded and/or antigen-expressing syngeneic cells, proving its promising results as a cytotoxic t lymphocyte vaccine (138) . prrsv is a complex disease and several gaps in the knowledge of its economic impact, biology and evolution, genetic polymorphism, mechanism of viral infections, elicitation of protective immune responses and novel control strategies, have been reviewed here (box 1). since the late 1980's, different approaches have permitted to examine more closely this virus allowing the discovery of new features of the complex replication cycle, the identification of proteins and nucleic acids playing a role together with extracellular vesicles and nanotubules in facilitating spreading, and a better understanding of immune evasion (non-neutralizing antibodies, glycan shielding, mutation, recombination events, among others) to further vaccine development. presently available prrsv vaccines have many limitations in terms of heterologous protection, but some efforts have been made by combining new adjuvant formulations with modified live viruses, dna and peptide vaccines, as well as extracellular vesicles a new vaccination approach. advancing in all these gaps in knowledge, will eventually accelerate eliminating and eventually eradicating this devastating veterinary disease of such huge economic importance. sm-t: wrote the first draft of the manuscript. mm, hdp, and lf: wrote sections of the manuscript. all authors contributed to manuscript revision, read, and approved the submitted version. this review received support from the feder project (comrdi16-1-0035-03). assessment of the economic impact of porcine reproductive and respiratory syndrome virus on united states pork producers assessment of the economic impact of porcine reproductive and respiratory syndrome on swine production in the united states cost of porcine reproductive and respiratory syndrome virus at individual farm level -an economic disease model isolation of swine infertility and respiratory syndrome virus (isolate atcc vr-2332) in north america and experimental reproduction of the disease in gnotobiotic pigs mystery swine disease in the netherlands: the isolation of lelystad virus ratification vote on taxonomic proposals to the international committee on taxonomy of viruses porcine reproductive and respiratory syndrome virus (prrsv): pathogenesis and interaction with the immune system the molecular biology of arteriviruses the structural biology of prrsv a bayesian phylogeographical analysis of type 1 porcine reproductive and respiratory syndrome virus (prrsv) identification of porcine reproductive and respiratory syndrome virus orf1a-encoded non-structural proteins in virus-infected cells efficient−2 frameshifting by mammalian ribosomes to synthesize an additional arterivirus protein nonstructural proteins nsp2tf and nsp2n of porcine reproductive and respiratory syndrome virus (prrsv) play important roles in suppressing host innate immune responses biogenesis and architecture of arterivirus replication organelles regulation and evasion of antiviral immune responses by porcine reproductive and respiratory syndrome virus highly divergent strains of porcine reproductive and respiratory syndrome virus incorporate multiple isoforms of nonstructural protein 2 into virions prrsv receptors and their roles in virus infection membrane proteins of arterivirus particles: structure, topology, processing and function influence of n-linked glycosylation of minor proteins of porcine reproductive and respiratory syndrome virus on infectious virus recovery and receptor interaction overview: replication of porcine reproductive and respiratory syndrome virus cd163 knockout pigs are fully resistant to highly pathogenic porcine reproductive and respiratory syndrome virus precision engineering for prrsv resistance in pigs: macrophages from genome edited pigs lacking cd163 srcr5 domain are fully resistant to both prrsv genotypes while maintaining biological function pigs lacking the scavenger receptor cysteine-rich domain 5 of cd163 are resistant to porcine reproductive and respiratory syndrome virus 1 infection replacement of porcine cd163 scavenger receptor cysteine-rich domain 5 with a cd163-like homolog confers resistance of pigs to genotype 1 but not genotype 2 porcine reproductive and respiratory syndrome virus an intact sialoadhesin (sn/siglec1/cd169) is not required for attachment/internalization of the porcine reproductive and respiratory syndrome virus role of cd151, a tetraspanin, in porcine reproductive and respiratory syndrome virus infection prrsv structure, replication and recombination: origin of phenotype and genotype diversity effect of porcine reproductive and respiratory syndrome virus (prrsv) (isolate atcc vr-2385) infection on bactericidal activity of porcine pulmonary intravascular macrophages (pims): in vitro comparisons with pulmonary alveolar macrophages (pams) porcine reproductive and respiratory syndrome virus utilizes nanotubes for intercellular spread effect of malaria on hiv/aids transmission and progression tunneling nanotubes (tnt) are induced by hiv-infection of macrophages: a potential mechanism for intercellular hiv trafficking tunneling nanotubes as a novel route of cell-to-cell spread of herpesviruses innate and adaptive immunity against porcine reproductive and respiratory syndrome virus arterivirus molecular biology and pathogenesis interferon-stimulated genes: a complex web of host defenses production of type i interferons: plasmacytoid dendritic cells and beyond immune responses in pigs infected with porcine reproductive and respiratory syndrome virus (prrsv) differential production of proinflammatory cytokines in the pig lung during different respiratory virus infections: correlations with pathogenicity in vivo and in vitro interferon (ifn) studies with the porcine reproductive and respiratory syndrome virus (prrsv) interferon type i response in porcine reproductive and respiratory syndrome virus-infected marc-145 cells modulation of innate immune signaling by nonstructural protein 1 (nsp1) in the family arteriviridae interplay between interferonmediated innate immunity and porcine reproductive and respiratory syndrome virus modulation of host cell responses and evasion strategies for porcine reproductive and respiratory syndrome virus porcine reproductive and respiratory syndrome virus nucleocapsid protein modulates interferon-β production by inhibiting irf3 activation in immortalized porcine alveolar macrophages the prrsv replicase: exploring the multifunctionality of an intriguing set of nonstructural proteins open reading frame 1a-encoded subunits of the arterivirus replicase induce endoplasmic reticulumderived double-membrane vesicles which carry the viral replication complex porcine alveolar macrophage polarization is involved in inhibition of porcine reproductive and respiratory syndrome virus (prrsv) replication porcine reproductive and respiratory syndrome virus nonstructural protein 2 (nsp2) topology and selective isoform integration in artificial membranes the cysteine protease domain of porcine reproductive and respiratory syndrome virus nonstructural protein 2 possesses deubiquitinating and interferon antagonism functions immunodominant epitopes in nsp2 of porcine reproductive and respiratory syndrome virus are dispensable for replication, but play an important role in modulation of the host immune response nonstructural protein 11 (nsp11) of porcine reproductive and respiratory syndrome virus (prrsv) promotes prrsv infection in marc-145 cells nonstructural protein 11 of porcine reproductive and respiratory syndrome virus suppresses both mavs and rig-i expression as one of the mechanisms to antagonize type i interferon production the endoribonuclease activity essential for the nonstructural protein 11 of porcine reproductive and respiratory syndrome virus to inhibit nlrp3 inflammasome-mediated il-1β induction porcine reproductive and respiratory syndrome virus (prrsv) inhibits rnamediated gene silencing by targeting ago-2 novel insights into host responses and reproductive pathophysiology of porcine reproductive and respiratory syndrome caused by prrsv-2 comparison of host genetic factors influencing pig response to infection with two north american isolates of porcine reproductive and respiratory syndrome virus variation among sows in response to porcine reproductive and respiratory syndrome 1 genetic resistance -an alternative for controlling prrs? porc heal manag genetic analysis of reproductive traits and antibody response in a prrs outbreak herd 1 validation and further characterization of a major quantitative trait locus associated with host response to experimental infection with porcine reproductive and respiratory syndrome virus north american porcine reproductive and respiratory syndrome viruses inhibit type i interferon production by plasmacytoid dendritic cells porcine reproductive and respiratory syndrome virus infects mature porcine dendritic cells and up-regulates interleukin-10 production cytokine profiles and phenotype regulation of antigen presenting cells by genotype-i porcine reproductive and respiratory syndrome virus isolates differential type i interferon activation and susceptibility of dendritic cell populations to porcine arterivirus porcine reproductive and respiratory syndrome virus type 1.3 lena triggers conventional dendritic cells 1 activation and t helper 1 immune response without infecting dendritic cells tonsil conventional dendritic cells are not infected by porcine reproductive and respiratory syndrome virus response of the cdc1 and cdc2 subtypes of tracheal dendritic cells to porcine reproductive and respiratory syndrome virus porcine alveolar macrophage-like cells are pro-inflammatory pulmonary intravascular macrophages that produce large titers of porcine reproductive and respiratory syndrome virus nsp1α of porcine reproductive and respiratory syndrome virus strain bb0907 impairs the function of monocyte-derived dendritic cells via the release of soluble cd83 impact of genotype 1 and 2 of porcine reproductive and respiratory syndrome viruses on interferon-α responses by plasmacytoid dendritic cells characterization of the cytokine and maturation responses of pure populations of porcine plasmacytoid dendritic cells to porcine viruses and toll-like receptor agonists sensing of porcine reproductive and respiratory syndrome virus-infected macrophages by plasmacytoid dendritic cells the viral innate immune antagonism and an alternative vaccine design for prrs virus the role of porcine reproductive and respiratory syndrome (prrs) virus structural and non-structural proteins in virus pathogenesis perforin expression can define cd8 positive lymphocyte subsets in pigs allowing phenotypic and functional analysis of natural killer, cytotoxic t, natural killer t and mhc un-restricted cytotoxic t-cells cross-protective immunity to porcine reproductive and respiratory syndrome virus by intranasal delivery of a live virus vaccine with a potent adjuvant porcine reproductive and respiratory syndrome virus-induced immunosuppression exacerbates the inflammatory response to porcine respiratory coronavirus in pigs natural killer cells in host defense against veterinary pathogens suppression of nk cell-mediated cytotoxicity against prrsv-infected porcine alveolar macrophages in vitro evaluation of immune responses to porcine reproductive and respiratory syndrome virus in pigs during early stage of infection under farm conditions protection against porcine reproductive and respiratory syndrome virus (prrsv) infection through passive transfer of prrsv-neutralizing antibodies is dose dependent role of neutralizing antibodies in prrsv protective immunity passive transfer of virus-specific antibodies confers protection against reproductive failure induced by a virulent strain of porcine reproductive and respiratory syndrome virus and establishes sterilizing immunity the challenge of prrs immunology immunological responses of swine to porcine reproductive and respiratory syndrome virus infection mechanisms of adaptive immunity to porcine reproductive and respiratory syndrome virus dissociation of porcine reproductive and respiratory syndrome virus neutralization from antibodies specific to major envelope protein surface epitopes neutralizing antibody responses of pigs infected with natural gp5 n-glycan mutants of porcine reproductive and respiratory syndrome virus certainties, doubts and hypotheses in porcine reproductive and respiratory syndrome virus immunobiology protective humoral immune response induced by an inactivated porcine reproductive and respiratory syndrome virus expressing the hypo-glycosylated glycoprotein 5 gp5 expression in marc-145 cells inhibits porcine reproductive and respiratory syndrome virus infection by inducing beta interferon activity porcine reproductive and respiratory syndrome (prrs): an immune dysregulatory pandemic discovery of a small arterivirus gene that overlaps the gp5 coding sequence and is important for virus production novel structural protein in porcine reproductive and respiratory syndrome virus encoded by an alternative orf5 present in all arteriviruses immune response to orf5a protein immunization is not protective against porcine reproductive and respiratory syndrome virus infection regulatory t cells and infection: a dangerous necessity european genotype of porcine reproductive and respiratory syndrome (prrsv) infects monocyte-derived dendritic cells but does not induce treg cells role of porcine reproductive and respiratory syndrome virus nucleocapsid protein in induction of interleukin-10 and regulatory t-lymphocytes (treg) regulatory t cells in arterivirus and coronavirus infections: do they protect against disease or enhance it? mutations within the nuclear localization signal of the porcine reproductive and respiratory syndrome virus nucleocapsid protein attenuate virus replication immune control of prrs: lessons to be learned and possible ways forward immunological solutions for treatment and prevention of porcine reproductive and respiratory syndrome (prrs) antiviral regulation in porcine monocytic cells at different activation states porcine reproductive and respiratory syndrome virus isolates differ in their susceptibility to neutralization effector mechanisms of humoral immunity to porcine reproductive and respiratory syndrome virus improved vaccine against prrsv: current progress and future perspective. front microbiol sequence analysis of porcine reproductive and respiratory syndrome virus of the american type collected from danish swine herds complete genome sequence of a novel variant porcine reproductive and respiratory syndrome virus (prrsv) strain: evidence for recombination between vaccine and wild-type prrsv strains porcine reproductive and respiratory syndrome (prrs) virus-specific interferon-γ + t-cell responses after prrs virus infection or vaccination with an inactivated prrs vaccine failure of an inactivated vaccine against porcine reproductive and respiratory syndrome to protect gilts against a heterologous challenge with prrsv comparison of different vaccination schedules for sustaining the immune response against porcine reproductive and respiratory syndrome virus porcine reproductive and respiratory syndrome virus vaccines: immunogenicity, efficacy and safety aspects inactivated and subunit vaccines against porcine reproductive and respiratory syndrome: current status and future direction biodegradable nanoparticle-entrapped vaccine induces cross-protective immune response against a virulent heterologous respiratory viral infection in pigs plga nanoparticle entrapped killed porcine reproductive and respiratory syndrome virus vaccine helps in viral clearance in pigs evaluation of hydrophobic chitosan-based particulate formulations of porcine reproductive and respiratory syndrome virus vaccine candidate t cell antigens an innovative approach to induce cross-protective immunity against porcine reproductive and respiratory syndrome virus in the lungs of pigs through adjuvanted nanotechnology-based vaccination plant-based porcine reproductive and respiratory syndrome virus vlps induce an immune response in mice vectored vaccines to protect against prrsv development of a porcine reproductive and respiratory syndrome virus-like-particle-based vaccine and evaluation of its immunogenicity in pigs virus replicon particles expressing porcine reproductive and respiratory syndrome virus proteins elicit immune priming but do not confer protection from viremia in pigs generation of porcine reproductive and respiratory syndrome (prrs) viruslike-particles (vlps) with different protein composition exosomes as potent cell-free peptide-based vaccine. i dendritic cellderived exosomes transfer functional mhc class i/peptide complexes to dendritic cells induction of protective immunity against experimental eimeria tenella infection using serum exosomes extracellular vesicles in parasitic diseases exosomes from plasmodium yoelii-infected reticulocytes protect mice from lethal infections serumderived exosomes from non-viremic animals previously exposed to the porcine respiratory and reproductive virus contain antigenic viral proteins b lymphocytes secrete antigen-presenting vesicles biological properties of extracellular vesicles and their physiological functions inhibition of porcine reproductive and respiratory syndrome virus infection by recombinant adenovirusand/or exosome-delivered the artificial micrornas targeting sialoadhesin and cd163 receptors targeted-pig trial on safety and immunogenicity of serumderived extracellular vesicles enriched fractions obtained from porcine respiratory and reproductive virus infections exosomes mediate intercellular transmission of porcine reproductive and respiratory syndrome virus (prrsv) respiratory syncytial virus infection changes cargo composition of exosome released from airway epithelial cells exosomes and their role in the life cycle and pathogenesis of rna viruses pathogenic role of exosomes in epstein-barr virus (ebv)-associated cancers an exosome-based vaccine platform imparts cytotoxic t lymphocyte immunity against viral antigens we are particularly grateful to christa helwig for english editorial assistance. the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.the reviewer sg declared a past supervisory role with one of the authors mm to the handling editor.copyright © 2019 montaner-tarbes, del portillo, montoya and fraile. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord-151024-qe7c2uks authors: koca, caglar; civas, meltem; sahin, selin m.; ergonul, onder; akan, ozgur b. title: molecular communication theoretical modeling and analysis of sars-cov2 transmission in human respiratory system date: 2020-11-07 journal: nan doi: nan sha: doc_id: 151024 cord_uid: qe7c2uks severe acute respiratory syndrome-coronavirus 2 (sars-cov2) caused the ongoing pandemic. this pandemic devastated the world by killing more than a million people, as of october 2020. it is imperative to understand the transmission dynamics of sars-cov2 so that novel and interdisciplinary prevention, diagnostic, and therapeutic techniques could be developed. in this work, we model and analyze the transmission of sars-cov2 through the human respiratory tract from a molecular communication perspective. we consider that virus diffusion occurs in the mucus layer so that the shape of the tract does not have a significant effect on the transmission. hence, this model reduces the inherent complexity of the human respiratory system. we further provide the impulse response of sars-cov2-ace2 receptor binding event to determine the proportion of the virus population reaching different regions of the respiratory tract. our findings confirm the results in the experimental literature on higher mucus flow rate causing virus migration to the lower respiratory tract. these results are especially important to understand the effect of sars-cov2 on the different human populations at different ages who have different mucus flow rates and ace2 receptor concentrations in the different regions of the respiratory tract. information and communication technology (ict) framework provides a novel perspective to fight human diseases [1] [3] . in this respect, molecular communication could pave the way for a solution to develop therapeutic and diagnostic platforms. recent severe acute respiratory syndrome-coronavirus 2 (sars-cov2) pandemic have resulted in a [4] , [5] . in [4] , the authors model dengue virus transmission inside the body from its entrance to the host to the transmission to affected organs. the channel considered, which is from skin to the receiver organs, is characterized in terms of noise sources and path loss. aerosol transmission, in which droplets carry virus, is the another means of virus transport mechanism. in [5] , the authors determine the aerosol channel impulse response and find the response of their system for the sources such as breathing, coughing and sneezing. on the other hand, a study considering the sars-cov2 transmission process through the human respiratory tract from molecular communication perspective is is yet to be studied in the literature. sars-cov2 enters the host human through the nose, mouth and eyes. we consider the case that droplets carrying viruses enter the host human from the nose. viruses travel via mucus flow in the respiratory tract and reach host cells as illustrated in fig. 1 . sars-cov2 virus binds a special receptor on the host cell called angiotensin-converting enzyme or ace2. binding is followed by a time delay, τ , which is due to the mechanisms needed for virus replication. in this study, we consider this system and accordingly develop a model for the human respiratory tract by separating the respiratory tract into seven segments. our aim is to determine the impulse response of the sars-cov2-ace2 binding process to investigate the probability distribution of binding locations. the binding location distribution, which depends on several system parameters including ace2 density and mucus flow rate offers vital information on the course of disease. our contributions can be summarized as follows: • proposing a novel model of human respiratory tract that reduces complexities of the original system: we model human respiratory tract by partitioning the tract into seven segments from nasal cavity to alveoli. • determining impulse response of sars-cov2 infection process for the first time in literature • calculating ace2 receptor densities in the different regions of the respiratory tract: based on the available data on surface parameters, we calculate ace2 receptor density crudely. • investigating the effects of mucus layer thickness, mucus flow rate and ace2 density on the virus population reaching the different regions of the respiratory tract: our results shows that mucus flow rate and ace2 densities affect the respiratory regions where the viruses reach drastically. the rest of the paper is organized as follows. in section ii, we provide a brief background about sars-cov2. in section iii, the developed system model is outlined. in section iii, the diffusion model for viruses diffusing through the mucus layer is derived. next, in section v, the impulse response of the system for different receptor and virus concentration is determined. in section vi, markov chain model of the events following the binding process are stated. in section vii, the simulation results are presented. finally, conclusions are stated in section viii. severe acute respiratory syndrome -coronavirus 2 (sars-cov2), also named novel-coronavirus (2019-n-cov), has been identified as the causative infectious agent of coronavirus disease, responsible for the current pandemic. covid-19 has turned from a local pneumonia outbreak, which originated in wuhan, china in december 2019, into a global pandemic in a matter of months, which has as of now, october 2020, caused more than a million deaths worldwide and spread to more than 200 countries [6] . belonging to the family of coronaviruses, sars-cov2 is the third and the newest coronavirus in the family to cause an epidemic, just as sars-cov in 2003 and mers-cov in 2012, and the only one to cause a pandemic. sars-cov2 is reported to be a zoonotic viral disease. bats, snakes, and pangolins have been cited as potential reservoirs based on genome sequencing studies [7] [9] . although it predominantly causes pneumonia and associated comorbidities, covid-19 is considered to be a syndrome, given that it affects multiple different organs and systems within the human body. typical clinical symptoms of the patients include fever, dry cough, difficulty of breathing (dyspnea), fatigue, joint pain (arthralgia), muscle pain (myalgia), and loss of sense of smell (anosmia) [10] [12] . the presence of high variety of pathological events are attributed to different pathophysiological mechanisms involved in sars-cov2 and proves that it is more than a respiratory syndrome. current epidemiological data suggests that sars-cov2 is an airborne viral disease, meaning that it is transmitted through respiratory droplets and droplet nuclei, which are mostly spread during human-to-human contact [13] [15] . respiratory droplets (> 5−10µm in diameter) and droplet nuclei (aerosols) (< 5µm in diameter ), are generated and expelled/disseminated from an infected person during speaking, shouting, coughing, or sneezing [16] . indirect surface transmission, i.e., fomite transmission, and orofecal transmission have also been reported [17] [19] . some studies have detected stable sars-cov2 viral rna on solid surfaces such as plastic, aluminum, and stainless steel, yet the significance of fomite transmission is still debated with contradicting views [17] , [18] . the main pathway of sars-cov2 inside the human host is reported to be the respiratory tract. mucosal openings such as the nose, eyes, or mouth have been identified as the principal sites, where the initial viral entry takes place [20] . although there are numerous possibilities for viral entry, one pathway a virus particle could take on the macroscopic level is as follows. a virus laden particle enters through the nasal cavity, with the help of the downstream flow of mucosal secretions and gravity, it travels down through the pharynx, larynx, and trachea, enters a bronchi, passes down to bronchioles and finally reaches alveoli. on a microscopic level, once the virus laden droplets reach mucosal membranes, they diffuse through the mucosa (consisting of mucus, periciliary layer, and midlayer) and attach to certain membrane receptors on host cell surfaces, the most prominent one being ace2, which has been identified as the primary functional receptor for sars-cov2, just as for sars-cov [21] [24] . the current knowledge on sars-cov2 infection indicates that the elderly are more susceptible and vulnerable to the infection, while children seem to be the least affected group. numerous studies report lower rates of sars-cov2 infection with milder symptoms in children compared to adults [25] [27] . some studies attribute these results to their findings that ace2 expression in children is lower than that of adults. [28][30] . other possible reasons held responsible for lower rates of morbidity and mortality from sars-cov2 in children include: the differences in immune responses between children and adults, differences in ace2 receptor distribution patterns, and lower rates of testing in children due to abundance of asymptomatic cases [31] . the morphological structure of the virus comes to prominence when discussing viral binding processes. sars-cov2 is an enveloped, positive-sense, single-stranded rna virus and similar to its prior relatives sars-cov and mers-cov, it belongs to betacoronavirus genus of the coronavirinae family. sars-cov2 viral genome contains four major structural proteins: the s (spike) protein, the m (membrane) protein, the n (nucleocapsid) protein, and the e (envelope) protein [32] . the s protein has a trimeric structure, consisting of an s1 receptor binding subunit and an s2 fusion subunit. during viral infection, s1 and s2 subunits are cleaved by a metalloprotease, tmprss-2 (transmembrane protease serine 2), which facilitates viral entry. the s1 subunit functions as the part, which directly binds to the host cell receptor, i.e., ace2 receptor, creating a receptor binding domain (rbd). the s2 subunit takes role in membrane fusion [33] . following viral binding, there are two possible pathways of viral entry for enveloped viruses into host cells: either cytoplasmic fusion in which their envelope fuses with plasma membrane and they release their genome into cytosol, or endosomal membrane fusion (endocytosis) in which they are engulfed by an endosome, and their membrane is fused with the endosomal membrane [34] , [35] . there are multiple mechanisms of endocytic entry suggested by various studies, involving clathrin dependent, caveolae dependent endocytosis [36] , [37] , and clathrin independent, caveolae independent endocytosis [38] , [39] . in section ii, we presented physio-morphological structure and behavior of the virus, regarding its entry mechanisms into human body and target cells. here, we will present our system model. we assumed that the virus carrying aerosol particles enter the human host through the nose, and diffuse through the mucus layer in the nasal cavity, where ace2 receptors are found most abundantly [40] . the diffusion of the virus takes place in the mucus layer, which renders the shape of the respiratory tract insignificant. given the fact that the mucus layer is continuous within the respiratory tract [41] , we assume a cylindrical tube with radius r(y) and length l. the change in the radius throughout the tract has limited effect, unless it also modulates the properties of mucus, especially the mucus thickness. for a large portion of the respiratory tract, mucus layer covers the periciliary layer and a thin surfactant layer separates the two [42] . in our work, we assume that the surfactant layer reduces the surface tension between these two layers to a negligible value, and consequently ignored. furthermore, we assume that the diffusion coefficient, d, of the virus in periciliary and mucus layers to be the same. in a healthy respiratory system, the mucus inflow to the alveoli is countered by the mucus outflow due to the pericilliary layer. we ignored the mucus outflow mechanism as it may turn the mucus flow into a very complex turbulent fluid model. in other words, we treat it as if it is one single layer. the existing works studying ace2 distribution and mucus flow do not comment on differentiations within a region, i.e., ace2 are homogeneously distributed. hence, our model assumes cylindrical symmetry. the virus moves under the influence of the mucus flow from nasal cavity to the alveoli. we partition the respiratory system into seven parts, namely nasal cavity, larynx, pharynx, trachea, bronchi, bronchiole and alveoli. our model is presented in fig. 2 . due to the complicated structure of the tracheobronchial tree, we assign transition regions to the closest region. furthermore, since after each branching, the individual branches become narrower but more numerous, we used the surface area, s i , of each of the seven regions, i ∈ {1, 2, ..., 7}, to calculate its corresponding radii values, r i as where l i is the length of the i th region. the resultant the respiratory tract is shown in fig. 3 . note that fig. 3 is not to scale, as the corresponding radii for alveol region is two orders larger than the next region, i.e., bronchiole region. due to the cylindrical symmetry assumption, we can make a longitudinal upon entering the mucus and periciliary layer, viruses use their viral s-spike proteins to bind to ace2 receptors on host cell surfaces [43] . we will use the binding rate, λ, to describe the binding process. due to the spherical shape of the coronavirus, we safely ignore the effect of the orientation of the virus at the time when it makes contact with the ace2 receptor. as viruses bind to ace2 receptors on the host cell's membrane surface, ace2 receptors are downregulated. therefore, the number of surface receptors decreases [44] , [45] , making it less likely for other viruses to bind. we consider two scenarios depending on the ace2 receptor concentration and the virus population: • large virus and large receptor concentration • only large virus concentration as stated in section iii we assume a constant mucus flow rate, v from the nasal cavity to pharynx, larynx, trachea, bronchi, bronchiole and finally to the alveoli. furthermore, the viruses also diffuse with a diffusion coefficient, d, in the mucus layer. the virus concentration is derived using brownian motion with drift. we assign y axis for the distance from the entrance of nasal cavity, x axis for the distance from a longitudinal cutting point and z axis as the depth in the mucus layer. due to the assumption of cylindrical symmetry, the reference point for x coordinate is arbitrary. if a droplet containing n viruses is incident to the mucus level at the location (x 0 , y 0 , z 0 ), the virus concentration at time t is the standard deviation for brownian motion is given as σ = √ 2dt. therefore, 95.45% of the population of viruses falls into a sphere of radius 2 √ 2dt, centred at (x 0 , y 0 +vt, z 0 ), while 99.7% into a sphere with the same centre and radius of 3 √ 2dt. hence, for vt 3 √ 2dt, drift dominates the diffusion and diffusion along the y-axis can be ignored. similarly, for vt 3 √ 2dt, drift is dwarfed by diffusion and can be ignored. fig. 4 shows the dominating trends for brownian motion with drift. for v = 50µms −1 [46] and d = 1.5 × 10 −11 m 2 s −1 [47] , the diffusion of the virus in the respiratory tract is shown in fig. 5 . clearly, the effects of the diffusion is only visible for large t. the ace2-virus binding can be modelled by obtaining the virus population distribution over the respiratory tract. to achieve this, we start with modelling the kinematics of a single virus incident on the mucus layer. later, we use our findings as stepping stones to reach impulse response for different scenarios as described in section iii. we begin our analysis by considering a single virus is moving under the influence of mucus flow. the mucus layer has a thickness of h(y) and a velocity of v, while the respiratory tract radius is r(y), where y lies in the direction of the respiratory tract from nose to lungs. then, at any segment dy, the concentration of the virus due to a single virus is given by the time ∆t that the virus spends in a segment of length ∆y is the probability that it binds to a single ace2 receptor in the segment with length ∆y becomes where λ is the molar association constant, n a is the avogadro's constant and λ 1 = λ/n a is the association constant for a single virus. note that in the last step, we used first order taylor series expansion, i.e., e x = 1 + x for small x. then, p nb , the probability of not binding during ∆t is if the ace2 concentration per unit area at y is f (y), then number of ace2 receptors, n(y), in the patch of length ∆y becomes n(y) = 2πr(y)f (y)∆y, (11) and the probability of the virus evading all ace2 receptors in the same patch, p e (y), is expressed as p e (y) = p n(y) nb where from (13) to (14) we use the first order truncation of the binomial expansion, i.e., (1 + x) n = 1 + nx for |nx| 1, which holds due to n a being much larger than any other value in (14) . this assumption is especially effective for ∆y → dy. from (15), we reach the rate of binding in the patch of length dy as then, we find to the number of viruses at y, n (y) using an initial value problem with rate p b (y) where we used the fact that dy = vdt by definition. an important observation is that n (y) obtained in (21) does not necessarily normalise. definingv as where l is the total length of the respiratory tract,v gives us the rate of the viruses that reach the end of the respiratory tract, i.e., alveoli. here, since the viruses cannot travel neither forward nor backward, we assume that they will eventually bind to an ace2 in alveoli. so far, we only assumed the existence of a single virus to reach (21) . as stated in section iii, there are several scenarios depending on • n (y) = 2c(y)πr(y)h(y)dy, the total number of viruses number on a dy thick strip, • n(y) = 2πr(y)f (y)dy, total number of ace2 receptors in the same strip, • e b , the expected number of virus bindings in the same strip. e b is loosely calculated by replacing c 1 with c and carrying out (3) to (15) . hence, (3) becomes and replacing f (y) with n(y)/2πr(y)dy, a(y)n (y)n(y), where a(y) does not depend on ace2-virus bindings. since each ace2-virus binding destroys both a virus and a receptor, both the virus and the receptor concentrations are affected. hence, our model must incorporate variations in the concentrations. each binding causes the number of viruses, n (y) and number of receptorsn(y) to change as using (25), (26) and (27) we reach two assumptions: 1) large n (y): if n (y) is large and e b n (y), the total virus concentration remains constant within the same segment. 2) large n(y): if n(y) is large and e b n(y), the total ace2 concentration remains constant within the same segment. since a(y) is quite low, i.e., on the order of 10 −10 , for some cases, both of these assumptions hold. fig. 6 illustrates under which conditions these assumptions hold. as fig. 6 shows, when both n (y) and n(y) are large, the assumptions may not hold. since a(y) depends on r(y) and h(y), the boundaries may change. note that changes in n(y) causes a change in the system. as the system parameters change with the input, the system is no longer linear time-invariant. as a result, obtaining the impulse response when (27) does not hold is of no practical use. 1) large n(y): for large n(y), the virus-ace2 bindings do not change the receptor number in the same segment. hence, regardless of how many bindings happen in a given segment, the binding probability of any virus in the same segment is constant. as a result, (17) , multiplied by n (y) gives the expected number of binding on the segment. furthermore, (21) multiplied by the incident virus count, n 0 , gives us the virus population reaching to any location y of the respiratory tract, i.e., hence, the virus concentration is simply the derivative of (28), i.e., . we proceed to obtain impulse response, i(y, t), by adding the unbounded or free virus distribution,v (y). the free virus population is situated at the location y = vt of the respiratory tract, due to the fact that virus movement on the respiratory tract is solely under the influence of mucus flow. the total number of free viruses is equal to the difference between the initial number of viruses and the total number of bound viruses.v where δ(.) is the dirac delta function. thus, the impulse response becomes 2) large n (y) only: in case e b n (y), viruses outnumber the ace2 receptors. this causes all ace2 receptors to bind to a virus. bound virus distribution in the respiratory tract is the same as the ace2 receptor concentration. note that, since bound ace2 receptors downregulate, large n (y) only case cannot be modelled as a linear time invariant system. n(y) = 2πr(y)f (y)dy, and where u(.) is the step function and is used to assured that virus distribution is limited to the region 0 − vt, i.e., the range we find the impulse response by adding (33) and (34), i.e., 3) no limiting case: in case both assumptions fail, active number of ace2 receptors constantly changes due to the binding viruses. therefore, no assumption can be made for this case, and neither (31) nor (34) holds. as a result, there is no closed form expression for this case. in the host cell, the virus replicate and new virions are released out of the cell via exocytosis. we can model this process with a stationary markov chain with six states, namely, binding (b), endocytosis (en d), release of viral rna (c), replication (r), degradation (d), exocytosis (exc) as illustrated in fig. 7 . the bound virus, can enter the cell via endocytosis, which is mediated by ace2 receptors. in the state-b, the virus is found bounded to the ace2 receptor. covid-19 is an rna virus, i.e., virus can replicate in the cytoplasm. thus, in the state-c viral rna is released to the cytoplasm. in the host cell, the virus can be degraded by lysosomes [48] , which is represented by the state-d. the transition matrix of the markov chain representing the life cycle of the virus in the host cell, q, is given by where we set g 1 = −r be , g 2 = −(r ec + r ed ), g 3 = −(r cr + r cd ), and g 4 = −(r rd + r re ). the transition rates are provided in table ii . represents corresponding state occupancy probabilities. the relation between the states can be expressed as [49] which has a solution of (38) is in the form of using eigenvalue decomposition, we can express q as where v i is an eigenvector of the matrix and λ i the corresponding eigenvalue. as a result, we can express (39) as thus, the probability of transition from the state j to the state k in time t is given by morphometric measurements of the respiratory tract such as length, diameter, surface area, and mucosal thickness were obtained from estimation studies, journals, databases, and anatomy literature. given the non-uniform shape and the continuously narrowing nature of the respiratory tract, as in the tracheobronchial tree, we use median values for branching or narrowing structures. divided into 23 generations of dichotomous sections, the tracheobronchial tree designates a generation for each divided branch starting from trachea, generation 0, and ending at alveoli, (generation 23). the first 16 generations, from generation 0 to generation 16, are defined as the conducting zone, i.e., no gas exchange takes place in this region. from generation 17 to generation 23 is called the transitional and respiratory zone, where gas is exchanged within functional units [50] . the generation 0 directly gives the dimensions for the trachea. generations 1 to 4 are assumed to be bronchi, 5 to 16, bronchiole and 17 to 23 alveoli respectively. for our parameter data, we mainly use weibel's "dimensions of human airway model a" and gehr's "annexe a. anatomy and morphology of the respiratory tract" [51] . although there are studies investigating ace2 receptor gene and protein expressions across different tissues and in specific cell types using single cell rna sequencing gene expression profiling datasets, mass spectrometry and immune assay techniques, [24] , [52] [54] , to the best of our knowledge, data on the number of ace2 receptors on different tissues is not explicitly stated in studies. most studies provide relative expressions of the receptor in different tissues, shown as proportions, percentages, or plots with no numeric values. some studies address circulating ace2 levels, which we cannot directly utilize as we need tissue-specific values. there exist some studies which report ace2 expression data in animals, which are not compatible with our work either [55] . the primary challenge of this work is to obtain the ace2 receptor densities in different tissues of respiratory tract. the lack of studies giving these values is mostly due to the difficulty of measuring ace2 receptor concentration in a diverse population of all ages. to address this challenge, we exhaustively search among various literature to calculate our estimated values. the specific works that we use are referenced in table iii . therefore, we first gather data on the percentage of ace2 expressing cells for the seven region model described in section iii. then, we search for the total number of cells in each region. for tissues in which there is no sufficient quantitative data on the percentage of ace2 expressing cells, the relative proportions of ace2 expressions of two or more tissues, one of which we have previously calculated are used. then, we calculate the number of ace2 expressing cells in each tissue accordingly. note that these preliminary calculations are the estimates based on the currently available data in the literature. due to the lack of data, the effect of age in sars-cov2 susceptibility cannot be directly analyzed. however, we investigate the effects of thicker mucus as seen more in elderly and effects of higher ace2 concentration in nasal cavity as observed in smokers. in this section, we first present the impulse response simulation and then continue with simulating the effect of mucus flow rate, v, ace2 receptor density, f (y) and mucus thickness, h(y), on the virus-ace2 binding. 1) impulse response of unobstructed viral progression: in section. v, we find an analytic expression for the impulse response of unobstructed viral progression through the respiratory tract. here, we confirm our analytic expression with a monte carlo simulation in fig. 8 . the physiological parameters that we use in the simulations are presented in table iii . for the monte carlo simulation, we divide the respiratory tract to ∆y = 5µm patches. the initial number of viruses are n 0 = 50000. each virus is independent of each other, i.e., a new number is generated using the marsenne twister for each virus in each segment. we see that our analytical solution is in full agreement with the monte carlo simulation of the system for large n 0 . 2) mucus flow rate: as it can be seen in fig. 9 , mucus flow rate has a significant impact on the reach of the virus population. if the patient suffers from another condition causing nasal drip or any other faster mucus flow, the virus spends less time in the upper respiratory system. therefore, ace2virus bindings in the upper respiratory tract is limited, causing the bulk of the virus population to migrate to the lower parts of the respiratory tract, especially bronchioles and alveoli. the virus population in the alveoli is 20-folds more if the mucus drop rate is v = 100µms −1 compared to v = 25µms −1 . this causes the virus to take hold in the alveoli before an immune response can be launched. 3) nasal ace2 receptor density: fig. 10 shows us the impact of the ace2 receptor concentration in the nasal cavity. assuming distribution of the ace2 receptors in the other parts of the respiratory tract is the same for different age groups, the difference in the ace2 levels in nasal cavity has a significant effect on the virus population reaching the lower respiratory tract. the impact of ten-fold increase in ace2 receptor concentration is six-fold increase in virus concentration in the lower respiratory system. 4) mucus thickness: our model suggests an impact of the mucus thickness. since we assume that the virus can move freely in the mucus layer via diffusion, thicker mucus implies that there is less chance for the ace2-virus binding. fig. 11 shows the effect of the mucus thickness. the virus population in the alveoli is 4.45 times more in the four times thicker mucus compared to the base mucus level. in this study, we analyze sars-cov-2-ace2 receptor binding event to determine bound virus population at the different regions of respiratory system. to this end, we develop a molecular communication model of sars-cov-2 transmission through the human respiratory tract, which reduces the inherent complexity of the respiratory system. we perform an analysis of the developed model using mucus flow rate and ace2 receptor densities, which are calculated based on the realistic data of respiratory tract surface parameters. based on the analysis, we reach that higher mucus flow rate results in virus migration to the lower respiratory tract, which is compatible with the experimental results found in the literature. our model will be useful to describe the travel of the virus through the respiratory tract and to simulate the effect of interventions (e.g. antivirals) to decrease the viral load. information and communication theoretical understanding and treatment of spinal cord injuries: state-of-the-art and research challenges rate of information flow across layered neuro-spike network in the spinal cord an information theoretical analysis of multi-terminal neuro-spike communication network in spinal cord in vivo channel characterization for dengue virus infection modeling of viral aerosol transmission and detection a pneumonia outbreak associated with a new coronavirus of probable bat origin probable pangolin origin of sars-cov-2 associated with the covid-19 outbreak cross-species transmission of the newly identified coronavirus 2019-ncov pathological findings of covid-19 associated with acute respiratory distress syndrome clinical characteristics of coronavirus disease 2019 in china virological assessment of hospitalized patients with covid-2019 airborne transmission of sars-cov-2: the world should face the reality community transmission of severe acute respiratory syndrome coronavirus 2, shenzhen, china, 2020 early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia infection prevention and control of epidemic-and pandemic-prone acute respiratory infections in health care aerosol and surface stability of sars-cov-2 as compared with sars-cov-1 prolonged infectivity of sars-cov-2 in fomites sars-cov-2 and the role of orofecal transmission: systematic review from sars to mers: evidence and speculation angiotensin-converting enzyme 2 is a functional receptor for the sars coronavirus pathology and pathogenesis of severe acute respiratory syndrome structure, function, and antigenicity of the sars-cov-2 spike glycoprotein single-cell rna expression profiling of ace2, the putative receptor of wuhan 2019-ncov coronavirus disease 2019 in children, united states severe acute respiratory syndrome coronavirus 2 (sars-cov-2) infection in children and adolescents: a systematic review human and novel coronavirus infections in children: a review nasal gene expression of angiotensin-converting enzyme 2 in children and adults single nucleus multiomic profiling reveals agedynamic regulation of host genes associated with sars-cov-2 infection expression of sars-cov-2 receptor, ace2, and tmprss2 in the lung airways is lower in children compared to adults and increases due to smoking and copd sars-cov-2 in children: spectrum of disease, transmission and immunopathological underpinnings coronavirus envelope protein: current knowledge structural basis for the recognition of sars-cov-2 by full-length human ace2 fusion of enveloped viruses in endosomes targeting the endocytic pathway and autophagy process as a novel therapeutic strategy in covid-19 sars coronavirus entry into host cells through a novel clathrin-and caveolae-independent endocytic pathway entry of human coronavirus nl63 into the cell clathrin-dependent entry of severe acute respiratory syndrome coronavirus into target cells expressing ace2 with the cytoplasmic tail deleted understanding sars-cov-2 endocytosis for covid-19 drug repurposing sars-cov-2 entry factors are highly expressed in nasal epithelial cells together with innate immune genes guyton and hall textbook of medical physiology physiology of airway mucus clearance the origin, transmission and clinical therapies on coronavirus disease 2019 (covid-19) outbreak-an update on the status downregulation of ace2 induces overstimulation of the renin-angiotensin system in covid-19: should we block the renin-angiotensin system sars-cov-2 pandemic and research gaps: understanding sars-cov-2 interaction with the ace2 receptor and implications for therapy mucus viscoelasticity and mucociliary transport rate quantification of multivalent interactions by tracking single biological nanoparticle mobility on a lipid membrane singlecell analysis and stochastic modelling unveil large cell-to-cell variability in influenza a virus infection information theory of intercellular signal transduction anatomy and physiology of respiratory system relevant to anaesthesia annexe a. anatomy and morphology of the respiratory tract single-cell rna analysis on ace2 expression provides insight into sars-cov-2 blood entry and heart injury expression of the sars-cov-2 cell receptor gene ace2 in a wide variety of human tissues ace2 protein landscape in the head and neck region: the conundrum of sars-cov-2 infection age-and gender-related difference of ace2 expression in rat lung sars-cov-2 reverse genetics reveals a variable infection gradient in the respiratory tract cell number and distribution in human and rat airways lung volumes in healthy nonsmoking adults a cellular census of human lungs identifies novel cell states in health and in asthma single-cell transcriptomic analysis of human lung provides insights into the pathobiology of pulmonary fibrosis essentials of respiratory care anatomical and histological factors affecting intranasal drug and vaccine delivery histology of the human nasopharyngeal mucosa estimating in vivo airway surface liquid concentration in trials of inhaled antibiotics key: cord-010368-plpghewn authors: kenmoe, sebastien; kengne-nde, cyprien; modiyinji, abdou fatawou; bigna, jean joel; njouom, richard title: association of early viral lower respiratory infections and subsequent development of atopy, a systematic review and meta-analysis of cohort studies date: 2020-04-24 journal: plos one doi: 10.1371/journal.pone.0231816 sha: doc_id: 10368 cord_uid: plpghewn introduction: existing evidence on the relationship between childhood lower respiratory tract infections (lrti) and the subsequent atopy development is controversial. we aimed to investigate an association between viral lrti at <5 years and the development of atopy at > 2 years. methods: we conducted a search at embase, pubmed, web of science, and global index medicus. we collected data from the included articles. we estimated the odds ratio and the 95% confidence intervals with a random effect model. we determined factors associated with atopy development after childhood lrti using univariate and multivariate meta-regression analyses. we recorded this systematic review at prospero with the number crd42018116955. results: we included 24 studies. there was no relationship between viral lrti at <5 years and skin prick test-diagnosed-atopy (or = 1.2, [95% ci = 0.7–2.0]), unknown diagnosed-atopy (or = 0.7, [95% ci = 0.4–1.3]), atopic dermatitis (or = 1.2, [95% ci = 0.9–1.6]), hyperreactivity to pollen (or = 0.8, [95% ci = 0.3–2.7]), food (or = 0.8, [95% ci = 0.3–2.5]), or house dust mite (or = 1.1, [95% ci = 0.6–2.2]). although not confirmed in all studies with a symmetric distribution of the 23 confounding factors investigated, the overall analyses showed that there was a relationship between childhood viral lrti at < 5 years and serum test diagnosed-atopy (or = 2.0, [95% ci = 1.0–4.1]), allergic rhinoconjunctivitis (or = 1.7, [95% ci = 1.1–2.9]), hyperreactivity diagnosed by serum tests with food (or = 5.3, [1.7–16.7]) or inhaled allergens (or = 4.2, [95% ci = 2.1–8.5]), or furred animals (or = 0.6, [95% ci = 0.5–0.9]). conclusion: these results suggest that there is no association between viral lrti at < 5 years and the majority of categories of atopy studied during this work. these results, however, are not confirmed for the remaining categories of atopy and more particularly those diagnosed by serum tests. there is a real need to develop more accurate atopy diagnostic tools. a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 atopy is a genetic predisposition to the development of allergic diseases such as atopic dermatitis, atopic eczema, atopic asthma, atopic conjunctivitis or allergic rhinitis [1] . atopy also includes increased hypersensitivity to inhaled or food allergens, with the development of ige mediated by th2 cells [2] . atopic diseases is associated to a significant morbidity and a very important economic burden for society [3] . atopic disease prevalence has experienced in recent decades an exponential increase in the world [4, 5] . common viruses associated with lower respiratory tract infections (lrti) include influenza, rhinovirus, respiratory syncytial virus (hrsv), metapneumovirus, parainfluenzavirus, enterovirus, adenovirus, bocavirus, and coronavirus [6, 7] . data have showed the association between hrsv lrti and subsequent wheezing or asthma [8] [9] [10] [11] . with the advent of molecular assays, the description of childhood infections caused by non-hrsv has further demonstrated the implication of these diseases in long-term sequelae [12, 13] . a meta-analysis by liu et al., have demonstrated the association between childhood rv infections and the subsequent development of asthma [13] . a systematic review showed that pneumonia mainly due to adenovirus was linked to sequelae including obstructive pulmonary disease or chronic bronchitis [12] . the subgroup analyses in this latter however found that the 3 included studies with mycoplasma pneumoniae pneumonia were not associated with long-term sequelae [12] . studies on the prevalence of atopy among people presenting with viral lrti in childhood have shown divergent results [14] [15] [16] . some studies have reported an increased risk of allergic sensitization after viral lrti in childhood [14, 17] . protection against allergic sensitization through the stimulation of th-1 cytokine production has been suggested by other studies [16] . maximum confusion has been demonstrated by other studies that have shown no influence of childhood viral lrti in the development risk of subsequent atopy [15, 18, 19] . the resolution of the question of the association between viral lrti in childhood and the subsequent development of atopy could serve as a basis for preventive measures and management of atopic diseases [20] [21] [22] . the purpose of this systematic review and meta-analysis of long-term sequelae of lower respiratory tract infections in early childhood (a learned study) was to investigate the association between viral lrti at <5 years and the atopy development at > 2 years. dissemination guidelines [23] and reported according to the prisma (preferred reporting items for systematic review and meta-analysis) guidelines (s1 table) [24] . study participants were children with a history of laboratory confirmed viral lrti before 5 years. viral lrti was considered using the definition proposed by the authors of the included studies. children with viral lrti, as reference cases, were compared to control children who had no history of lrti in childhood. studies including only participants with medical conditions (premature birth, immunodeficiency or other comorbidities) were excluded. the exposure in this systematic review was a viral lrti in children at <5 years. the outcome of this systematic review was the development of atopy at> 2 years including atopic diseases and sensitization to food and atmospheric allergens. atopic status was determined by skin prick tests (spt) and total or allergen-specific serum ige antibody assessed by immunoassays. included studies were prospective and retrospective cohorts with a minimum follow-up duration of one year. atopic diseases were diagnosed clinically. we considered atopy category whose data on outcomes were available in three or more studies. the pubmed, excerpta medica database, web of science, and global index medicus databases were queried for articles published from inception through july 15, 2019. all languages and geographic areas were considered for this systematic review. the combination of terms used for the bibliographic search is listed in table 1 . we manually screened the included studies and relevant review reference lists to locate additional articles. two independent investigators (ks and afm) reviewed the titles and abstracts of the articles found by the electronic and manual search [25] . we summarized the selection process of potentially relevant articles on a prisma flowchart. the disagreements between the two investigators were resolved by discussion and consensus. two researchers (ks and afm) independently extracted data from full text of included articles. the following data was collected (title, first author, year of publication, time of data collection, country, participants interview period, lrti type, lrti rank, lrti period, age at lrti, virus associated with the lrti, control age, control gender, total number of cases and controls, numbers with atopy at follow up, and data on confounders). the discrepancies were resolved by discussion and a consultation of a third arbitrator (rn) if necessary. in accordance with newcastle-ottawa scale (nos) criteria including patient selection, comparability of groups, and outcome evaluation, two independent researchers (ks and afm) assessed the quality of all included studies [26] (s2 table) . to reach consensus, all the differences were discussed between the two researchers. we estimated odds ratio (or) as a measure of the association between childhood viral lrti and subsequent atopy development. we evaluated the publication bias by visual inspection of funnel diagram and egger test. we estimated heterogeneity between studies by the q test and the i 2 statistic [27, 28] . we considered heterogeneity as significant between studies for p-value <0.1 or i 2 > 50%. we conducted sensitivity analyses with studies with a low risk of bias, studies including only inpatients or the first episode of viral lrti. we performed subgroup analyses on the basis of the type of lrti, who region, age at lrti, age at follow up, and type of virus detected in lrti. we applied a multivariate metaregression with a stepwise manual selection procedure to identify factors associated with the variation of overall risk of atopy. we successively removed from the model variables by considering the signification of the p-value and information criteria for the model like log-likelihood, deviance, akaike information criterion (aic), bayesian information criterion (bic), and corrected akaike information criterion (aicc). we reported the explained heterogeneity (r2) by variables included in models. a variable with p value < 0.05 was considered statistically significant in the final model. we assessed the influence of confounding factors by conducting a sensitivity study that included only equitably distributed studies for each risk factor between reference cases and controls. for each potential confounding factor, we assessed the distribution between reference cases and controls by recalculating the p values using the exact tests of fisher and chi-2. the two p values> 0.05 of fisher's exact test and chi-2 indicated a symmetric distribution of the confounding factor between reference cases and controls. we synthesized the study selection process in fig 1. the electronic (4634 articles) and manual (23 articles) searches identified 4657 articles. the first selection by titles and abstracts resulted in the exclusion of 4249 irrelevant articles. we read and fully reviewed the complete texts of the remaining 330 articles. we excluded a total of 309 articles for multiple reasons including mismatch of the study population (no control group, inclusion of non-viral lrti and non-lrti infections, and inclusion of only patients with underlying medical conditions), the type of study not appropriate (case report, comment on study, editorial, and review), lack of data on outcomes, conference abstract or complete texts not found, and irrelevance of the article-supplementary. we finally included 22 articles (24 studies) in the qualitative and quantitative synthesis of this systematic review . we showed the individual characteristics of the publications included in s3 table. most studies were conducted in europe, detected hrsv, had a low risk of bias, included hospitalized children under 1 year of age with their first episode of bronchiolitis, had followed children between 5-10 years old, and were prospective. children with a history of viral lrti in childhood were recruited between 1960 and 2014 and articles were published between 1981 and 2017.the individual nos score from the included studies are presented in s4 table. comparison of reference cases with controls infant post viral lrti atopy rhinoconjunctivitis was significantly more frequently reported in reference cases than in controls (or = 1.7, 95% ci = 1.0-2.9). positivity for furred animals was significantly more frequent in controls compared to reference cases (or = 0.6, 95% ci = 0.5-0.9). the overall effect remained unchanged for the majority of our results when assessed by sensitivity analyses of the impact of lrti rank, hospitalization, and study quality ( table 2 ). the effect observed in the main analysis of the positive serum test was lost for studies reporting the first episodes of lrti (or = 2.0, 95% ci = 0.6-6.7) and hospitalized children (or = 2.5, 95% ci = 0.9-6.5). in contrast to the association observed between lrti history and development of allergic rhinoconjunctivitis (or = 1.7, 95% ci = 1.1-2.9), no effect was observed for studies reporting the first episode of lrti (or = 1.4, 95% ci = 0.5-3.6). the significant preponderance of furred animal positivity in controls compared to reference cases was lost in studies including only the first lrti episodes (or = 0.5, 95% ci = 0.3-1.0). in the subgroup analyses (s5 table) , a statistically significant association between childhood viral lrti and subsequent atopy was observed only in the bronchiolitis subgroup for the catepositivity to atopy by serum tests was not significantly associated only for patients 5 to 10 years (or = 1.0, 95% ci = 0.5-2.1). positivity to house dust mite allergen was only associated with patients aged 15 to 20 years (or = 2.8, 95% ci = 1.0-7.6). the positivity to food allergens by serum test was inversely proportional to the age of the patients and the association was lost between 5 and 10 years (or = 2.0, 95% ci = 0.7-5.3). there was no statistically significant difference by who region and viruses screened subgroups. in metaregression analyses, only the type of lrti was admitted in the best multivariate model for the type of lrti in the atopy diagnosed by spt, atopy diagnosed by serum test, and allergic rhinoconjunctivitis (s6 table) . follow-up delay of participants was positively associated with house dust mite and negatively associated with atopy diagnosed by serum test for food. a total of 84.8% (89/105) of the 23 confounding factors collected in the included studies had a symmetric distribution between reference cases and control participants (s7 table) . we conducted a sensitivity analysis that included only studies with symmetric distribution for these confounding factors for atopy categories with a significantly different distribution between reference cases and controls (atopy diagnosed by serum tests, positivity to food and inhalant allergens by serum tests, allergic rhinoconjunctivitis, and furred animals). the significant difference observed in the overall analysis was lost in the majority of these categories of atopy (s8 table) . there was no heterogeneity in overall and sensitivity analyses for atopy with unknown or not reported diagnosis method, atopic dermatitis, food allergy, furred animals, and positive serum tests for inhalants ( table 2 ). the analyses of atopy diagnosed by spt showed a publication bias (p egger = 0.085). the funnel diagrams of the main analysis are presented in the s2-s12 figs. our results highlight that there is no relationship between a history of lrti at < 5 years and atopy diagnosed by spt, atopy diagnosed unknown/not reported, atopic dermatitis, and hyperresponsiveness to common allergens including pollen, food allergens or house dust mites. our results on atopy diagnosed by serum tests, allergic rhinoconjunctivitis, and positivity by serum tests to food or inhaled allergens cannot be definite with an increased risk observed in the global analysis and not confirmed by the analyses in studies reporting confounding factors. the increased risks of developing atopy observed in some subgroup analyses were more frequent in case of bronchiolitis due to hrsv between 9-12 months and in prospective studies conducted in europe. our results are consistent with the quantitative analysis by knyber et al. who concluded that there was no relationship between hospitalization for hrsv bronchiolitis at < 1 year and subsequent allergic sensitization [10] . similar to the findings of this review, kneyber et al. also concluded that hrsv infection in childhood was associated with allergic sensitization to food or inhaled allergens tested with serum tests. however, we have no definitive conclusion on this point since our analyses, taking into account studies with confounders, such as family history of atopy [51] , did not confirm this finding. similar to the findings of the present work, several studies have also shown divergent results between serum and skin tests [52] [53] [54] [55] . there are many hypothetical reasons that may explain these observed differences between serum and skin test results. first, differences in the composition and/or concentration of skin and serum tests targets may lead to differences in the results of both tests [52] . in a context of immune immaturity, for example, insufficient migration of mast cells to the epidermis could lead to positive results for serum tests and false negative for skin tests. serum tests also involve false positive results due to nonspecific binding with the antibodies used [56] . technical differences in the handling of skin and serum tests may also be involved in the differences observed between the two methods [57] . the systematic review by fauroux et al. reported for studies conducted between 1995 and 2015 in industrialized countries the controversial nature of the results on the association between infantile hospitalizations for hrsv lrti and subsequent atopy [58] . pérez-yarza in a systematic review including children younger than 3 years with hrsv respiratory infection from 1985 to 2006 also suggested controversial findings about the subsequent risk of allergic sensitization development defined by positive skin or serum tests specific for common allergens [11] . while this systematic review may help clarify the relationship between lrti in childhood and subsequent atopy, the weaknesses of the work must be emphasized. more than three quarters of the included studies in this systematic review were from europe. this suggests an important problem in the external validity of our results on a global scale, with the absence of america, south east asia and eastern mediterranean. this systematic review is the only one to date to address this topic with a strict atopy definition with the consideration of 11 different categories depending on the type of diagnosis used, allergic diseases and sensitization to common allergens. this systematic review includes a multitude of sensitivity analyses with studies reporting their first episode of lrti, studies reporting children hospitalized for lrti, and quality of studies. other special strengths of this systematic review include the large size of the participants included, 5294 reference cases and 27091 controls, the long follow-up period of more than half a century of children from birth to about 30 years old and with several points of follow-up including all age groups. the data was carefully extracted from a structured questionnaire and we used an appropriate data analysis to consider 23 important confounding variables. no relationship was found in this systematic review between viral lrti at <5 years and the subsequent development of a spt-diagnosed atopy, sensitization to common allergens or the development of atopic dermatitis. this conclusion was not confirmed for the association between viral lrti at < 5 years and the subsequent development of serum test diagnosed atopy, serum test positive for food or inhaled allergens, allergic rhinoconjunctivitis, and sensitization to furred animals. thus, more longitudinal investigations adjusted to confounding factors are important to elucidate the implication of childhood lrti in the development of atopy or allergy to food or inhalant assessed by serum tests, allergic rhinoconjunctivitis, and sensitization to furred animals. these findings should encourage research on the long-term burden of viral lrti in childhood in non-european regions and non-hrsv viruses. prospective randomized studies including intervention against the development of the lrti would be ideal to rule out the residual confusion about the causal relationship between infantile lrtis and the development of subsequent atopy. the imminent arrival of the vaccine against hrsv on the market or the prophylactic means such as palivizumab could be a way to carry out these interventional studies. to reduce the burden of atopy, there a real need of more accurate diagnosis tools and efforts should focus on other major risk factors including genetic predisposition, diet habits, air pollution, family size, and the use of vaccines or antibiotics. supporting information s1 allergy and allergic diseases. first of two parts t-cell subsets (th1 versus th2) economic burden of adult patients with moderate to severe atopic dermatitis indicated for systemic treatment investigating international time trends in the incidence and prevalence of atopic eczema 1990-2010: a systematic review of epidemiological studies the changing prevalence of asthma, allergic rhinitis and atopic eczema in african adolescents from etiology and risk factors for mortality in an adult community-acquired pneumonia cohort in malawi demographic and seasonal characteristics of respiratory pathogens in neonates and infants aged 0 to 12 months in the central-east region of tunisia association between respiratory syncytial virus-associated acute lower respiratory infection in early life and recurrent wheeze and asthma in later childhood association between respiratory syncytial virus hospitalizations in infants and respiratory sequelae: systematic review and meta-analysis. the pediatric infectious disease journal long-term effects of respiratory syncytial virus (rsv) bronchiolitis in infants and young children: a quantitative review the association between respiratory syncytial virus infection and the development of childhood asthma: a systematic review of the literature long term sequelae from childhood pneumonia; systematic review and meta-analysis association between rhinovirus wheezing illness and the development of childhood asthma: a meta-analysis allergic predisposition among infants with bronchiolitis acute bronchiolitis: predisposing factors and characterization of infants at risk atopic versus infectious diseases in childhood: a question of balance? patterns of allergic respiratory disease in children with a past history of bronchiolitis respiratory syncytial virus in early life and risk of wheeze and allergy by age 13 years symptoms, atopy, and bronchial reactivity after lower respiratory infection in infancy effect of palivizumab prophylaxis on subsequent recurrent wheezing in preterm infants early ribavarin treatment of bronchiolitis: effect on long-term respiratory morbidity interventions to prevent long-term consequences of centers for reviews and dissemination. crd's guidance for undertaking reviews in 301 healthcare: centers for reviews and dissemination preferred reporting items for systematic reviews and meta-analyses: the prisma statement rayyan-a web and mobile app for systematic reviews assessing risk of bias in prevalence studies: modification of an existing tool and evidence of interrater agreement quantifying heterogeneity in a meta-analysis the combination of estimates from different experiments acute bronchiolitis in infancy as risk factor for wheezing and reduced pulmonary function by seven years in akershus county human metapneumovirus bronchiolitis in infancy is an important risk factor for asthma at age 5 hospitalization for rsv bronchiolitis before 12 months of age and subsequent asthma, atopy and wheeze: a longitudinal birth cohort study association of an early respiratory syncytial virus infection and atopic allergy respiratory morbidity 20 years after rsv infection in infancy respiratory status and allergy after bronchiolitis risk factors for virusinduced acute respiratory tract infections in children younger than 3 years and recurrent wheezing at 36 months follow-up after discharge: the pediatric infectious disease journal exhaled nitric oxide in acute phase of bronchiolitis and its relation with episodes of subsequent wheezing in children of preschool age causal direction between respiratory syncytial virus bronchiolitis and asthma studied in monozygotic twins long-term consequences of respiratory syncytial virus acute lower respiratory tract infection in early wheezing, asthma, and pulmonary dysfunction 10 years after infection with respiratory syncytial virus in infancy adolescent asthma after rhinovirus and respiratory syncytial virus bronchiolitis rsv bronchiolitis and risk of wheeze and allergic sensitisation in the first year of life asthma and immunoglobulin e antibodies after respiratory syncytial virus bronchiolitis: a prospective cohort study with matched controls respiratory syncytial virus bronchiolitis in infancy is an important risk factor for asthma and allergy at age 7 severe respiratory syncytial virus bronchiolitis in infancy and asthma and allergy at age 13 asthma and allergy patterns over 18 years after severe rsv bronchiolitis in the first year of life atopy does not predispose to rsv bronchiolitis or postbronchiolitic wheezing factors predisposing to abnormal pulmonary function after adenovirus type 7 pneumonia association between pronounced iga response in rsv bronchiolitis and development of allergic sensitization respiratory morbidity in adulthood after respiratory syncytial virus hospitalization in infancy the outcome after severe bronchiolitis is related to gender and virus development of allergy in children. i. association with virus infections discordance between aeroallergen specific serum ige and skin testing in children < 4 years of age comparison of skin-prick test and specific serum ige determination for the diagnosis of latex allergy comparison of serum-specific ige (immunocap) and skin-prick test results for 53 inhalant allergens in patients with chronic rhinitis skin prick test and specific serum ige in the diagnostic evaluation of suspected cow's milk and hen's egg allergy in children: does one replace the other? allergic rhinitis and its impact on asthma (aria) 2008 update (in collaboration with the world health organization, ga(2)len and allergen) comparison of the multi-test ii and comforten allergy skin test devices the burden and long-term respiratory morbidity associated with respiratory syncytial virus infection in early childhood key: cord-294568-12eyo13f authors: fernandes-matano, larissa; monroy-muñoz, irma eloísa; angeles-martínez, javier; sarquiz-martinez, brenda; palomec-nava, iliana donají; pardavé-alejandre, hector daniel; santos coy-arechavaleta, andrea; santacruz-tinoco, clara esperanza; gonzález-ibarra, joaquín; gonzález-bonilla, cesar raúl; muñoz-medina, josé esteban title: prevalence of non-influenza respiratory viruses in acute respiratory infection cases in mexico date: 2017-05-03 journal: plos one doi: 10.1371/journal.pone.0176298 sha: doc_id: 294568 cord_uid: 12eyo13f background: acute respiratory infections are the leading cause of morbidity and mortality worldwide. although a viral aetiological agent is estimated to be involved in up to 80% of cases, the majority of these agents have never been specifically identified. since 2009, diagnostic and surveillance efforts for influenza virus have been applied worldwide. however, insufficient epidemiological information is available for the many other respiratory viruses that can cause acute respiratory infections. methods: this study evaluated the presence of 14 non-influenza respiratory viruses in 872 pharyngeal exudate samples using rt-qpcr. all samples met the operational definition of a probable case of an influenza-like illness or severe acute respiratory infection and had a previous negative result for influenza by rt-qpcr. results: the presence of at least one non-influenza virus was observed in 312 samples (35.8%). the most frequent viruses were rhinovirus (rv; 33.0%), human respiratory syncytial virus (hrsv; 30.8%) and human metapneumovirus (hmpv; 10.6%). a total of 56 cases of co-infection (17.9%) caused by 2, 3, or 4 viruses were identified. approximately 62.5% of all positive cases were in children under 9 years of age. conclusion: in this study, we identified 13 non-influenza respiratory viruses that could occur in any season of the year. this study provides evidence for the prevalence and seasonality of a wide range of respiratory viruses that circulate in mexico and constitute a risk for the population. additionally, our data suggest that including these tests more widely in the diagnostic algorithm for influenza may reduce the use of unnecessary antibiotics, reduce the hospitalisation time, and enrich national epidemiological data with respect to the infections caused by these viruses. introduction acute respiratory infections (aris) represent the leading cause of morbidity and mortality worldwide [1] [2] , are the most common cause of outpatient care in adult patients [3] , and are responsible for 70% of hospitalisations due to respiratory diseases in child populations aged 1-4 years and up to 90% in infants under 1 year of age [4] . aris are a group of diseases with normally less than 15 days of evolution that are caused by different microorganisms. a viral aetiological agent is estimated to be present in up to 80% of cases [5] [6] [7] . these infections can occur in the upper and lower respiratory tract. upper aris may include one or more of the following conditions: rhinopharyngitis, pharyngoamygdalitis, sinusitis, and acute otitis media [8] [9] . lower aris include epiglottitis, laryngitis, laryngotracheobronchitis (croup), bronchitis, bronchiolitis, and pneumonia [9] . these infections are easily transmitted via coughing or sneezing. contagion occurs through the inhalation of aerosols and microdroplets that contain the causative agent. another important form of contagion is through direct contact of hands with objects contaminated with respiratory secretions from infected individuals, which can be self-inoculated into the nasal and buccal mucosae and/or into the ocular cavity [10] . a large amount of information is available concerning the timing and distribution of influenza viruses in the population following the reappearance of avian influenza a subtype h5n1 in 2003 and 2004 and the influenza a subtype h1n1 pandemic in 2009 [11] . influenza viruses are one of the main causative agents of aris worldwide; however, many other respiratory viruses for which insufficient epidemiological information is available can also cause aris. studies performed at the international level have frequently identified human respiratory syncytial virus (hrsv), human parainfluenza virus (hpiv), influenza virus (flu), human mastadenovirus (hmdv), rhinovirus (rv), and enterovirus (ev) and less frequently identified human metapneumovirus (hmpv), primate bocaparvovirus (pbpv), and human coronavirus (hcov) [12] . these viruses can serve as the causative agents for aris that occur outside of influenza season, when the rate of positivity can drop below 10%. although a large percentage of aris are caused by viral infections, the causative viruses have not been specifically identified in the majority of cases due to difficulties such as the high number of possible aetiological agents, similar symptoms among aris caused by different aetiological agents, the emergence of new viruses or new variants of previously described viruses, and the high cost of the detection tests [4] . thus, little information is available concerning the prevalence and seasonality of these viruses, mainly in undeveloped countries, where the possibilities of carrying out this type of study on a regular basis is unusual. the investigation of the causative agents of acute respiratory infections is important in order to lead the development of vaccines to target the most prevalent viruses and to reduce the unnecessary prescription of antibiotics and of antiviral oseltamivir phosphate. in addition, analysis like this can help to identify the age groups more susceptible to infection by each virus, in order to take the necessary actions for prevention and treatment. therefore, our aim was to determine the viral aetiology of aris in samples from patients who presented respiratory symptomatology but were negative for influenza by rt-qpcr testing. to evaluate the presence of noninfluenza respiratory viruses circulating in mexican population in the different seasons of the year, we analized every pharyngeal exudate sample received by the laboratorio central de epidemiología (lce) (central epidemiology laboratory) between the epidemiological week 40 of 2014 and the week 39 of 2015, according to the following criteria. all samples had a previous negative result for influenza by rt-qpcr and met one of the following operational case definitions: influenza-like illness (ili): a person of any age who presents a fever greater than or equal to 38˚c, a cough, and cephalalgia accompanied by one or more of the following symptoms: rhinorrhoea, coryza, arthralgias, myalgias, prostration, odynophagia, thoracic pain, abdominal pain, or nasal congestion. in patients under five years of age, irritability is considered a cardinal sign in place of cephalalgia. in patients older than 65 years, fever is not required as a cardinal symptom. severe acute respiratory infection (sari): a person of any age who presents difficulty breathing accompanied by a fever greater than or equal to 38˚c and a cough with one or more of the following symptoms: poor general condition, thoracic pain, polypnoea or acute respiratory distress syndrome (ards) or any death associated with ili or sari. a total of 872 samples were selected. (s1 table) total nucleic acid extraction the superscript™ iii platinum 1 one-step rt-qpcr system kit (invitrogen, carlsbad, califórnia, eua. catalog: 12574035) was used in a 7500 fast real-time pcr system (applied biosystems 1 , foster city, califórnia, eua) to amplify viral genetic material. primers and probes were used for each of the following viruses: hmpv, hrsv, hpiv 1-4, betacoronavirus 1 (βcov1), human coronavirus (hku1, 229e, nl63) (hcov), hmdv, rv, ev, and pbpv. the human rnase p (rp) gene was used as an internal control ( table 1 ). the viruses were evaluated in uniplex reactions with the following reaction mixture: 12.5 μl of 2x reaction mix, 0.5 μl of each primer and probe, 1 μl of enzyme, 5.5 μl of rnase-free water, and 5 μl of total dna or rna lyophilizates (amplirun1 vircell; granada-spain) were used as the positive controls for all evaluated viruses. all samples that presented amplification for any of the viral markers plus the internal control were considered positive results. all samples without amplification of the viral markers but with amplification of the internal control were considered negative results. samples that did not present amplification of the internal control were considered inadequate. descriptive statistics were used to analyse the prevalence of the viruses included in the study, with the percentages given with a 95% confidence interval. the chi-square test of homogeneity and independence and fisher's exact test were used to compare categorical variables (p-values <0.05 were considered significant). anova and student's t-test were used for hypothesis testing of the quantitative variables. the analyses were performed with the spss statistics version 24.0 software, and the graphics were generated with the statgraphics 1 centurion xvi.ii and microsoft 1 excel 1 2010 software. the information of the biological specimens used in the present study is not traceable to the patient data identity. all the samples were used in an anonymous way. the study was approved by the ethics committee and the research committee of the national committee of scientific research of the instituto mexicano del seguro social with the registration number r-2016-785-041. of the 872 samples analysed, 451 were from males (51.7%) and 421 were from females (48.3%). the average age was 41 years, with minimum and maximum extremes of 0 and 101 years, respectively. the population was divided into 4 age groups based on the age ranges included on the instituto mexicano del seguro social (imss; mexican social security institute) health cards. according to this stratification, the samples were distributed as follows: 265 samples corresponded to children 0-9 years of age (30.4%), 28 to young people 10-19 years of age (3.2%), 263 to adults 20-59 years of age (30.2%), and 316 to adults 60 years and older (36.2%). (table 2) . unintentionallya, the analysed samples were collected from patients from three geographical areas of the country as follows: the north zone (baja california, baja california sur, 25.9% of the samples (fig 1) . additionally, these data were analysed based on the age groups (s2 table and s1 file) . of the total samples analysed, 312 were positive for at least 1 virus (35.8%), with 47.1% from female patients (n = 147) and 52.9% from male patients (n = 165); there was no significant difference between the groups (p>0.05). of the total positive samples, rv had the highest number of detections (33.0%), followed by hrsv (30.8%), hmpv (10.6%), hmdv (9.6%), hpiv3 (8.7%), βcov1 (8.7%), ev (5.8%), and pbpv (4.5%). the other viruses presented percentages below 4%, although together they accounted for 10.3% of all positive cases. only hpiv2 was not detected (table 3 ) the 3 viruses most often identified in cases of co-infection were rv in 48.3% of the cases, followed by hrsv (35.7%) and hmdv (32.1%). interestingly, in the case of pbpv, despite it being the causal agent of only 19.6% of co-infections, these co-infections represented 78.6% of the pbpv cases detected. the behaviour of hmdv was similar, with 60% of all cases in which this virus was present involving a combination with other viruses. both of these cases represent a significant association (p<0.05) with co-infection (table 4) . the most commonly observed viral combination was hmdv + rv (16.1%), followed by hpiv3 + rv and hrsv + βcov1 (both 7.1%). although it occurred only 2 times, the co-infection with 4 viruses involved the same agents in both cases (hrsv + hmdv + βcov1 + pbpv). a comparison was performed between the days of hospitalization required by the patients with negative samples and those with samples positive for a single virus or in whom 2 or more viruses were detected. the analysis of this clinical data showed that the hospitalization was significantly higher in the negative samples than in the positive ones for a virus or with coinfections: 7.5, 5.1 and 4.5 days on average, respectively (p <0.05). we observed the same tendency with the average number of comorbidities and symptoms: 0.65 in the negative samples versus 0.35 in positive samples (p <0.05) and 7.7 in the negative samples against 7.4 in positive samples (p > 0.05), respectively. nevertheless, the number of clinical symptoms and comorbidities were greater in the aris positive for a single virus than in the patients with co-infections (p <0.05) ( table 5 ). the highest number of positive cases occurred in the group aged 0 to 9 years (62.5%) and the second highest in the group aged 60 years or older (20.5%), followed by the 20-to 59-year-old group (14.4%) and finally the 10-to 19-year-old age group (2.6%), which represented a significant difference (p<0.05). consistently, the 0-to 9-year-old age group comprised 91.1% of the co-infections. interestingly this group contained 92.9% of all pbpv infections, 86.7% of the hmdv infections, and 86.5% of the hrsv cases (fig 3) . when conducting the analysis of the overall behaviour of the viruses in the period covered by the study, we observed that the months with high and low positivity differed significantly table 4 . viruses participation in co-infections. (p<0.05). fig 4b-4e shows the monthly behaviour of each virus. the month of november presented the highest percentage of cases during the year ( fig 4a) . when the analysis was performed by season, we observed differences between the four seasons of the year (p<0.05). generally, summer had a significantly lower proportion of positive samples (24.5%; p<0.05), whereas fall had the highest proportion (44.9%; p<0.05). however, the individual trends of the viruses were not the same. for instance, spring (march 21 to june 20) accounted for the greatest proportions of rv, hmdv, hpiv4, and hpiv1 cases (29.5%, 38.5%, 46.8%, and 48.0%, respectively). in contrast, most hrsv (48.2%) and ev (72.6%) cases were detected in the fall (september 21 to december 20), and winter (december 21 to march 20) presented the highest prevalence of hmpv (47.3%) and βcov1 (60.5%). the other viruses (hcov 229e, hcov hku1, hpiv 3, pbpv, and hcov nl63) were detected throughout the year, with no seasonal trends observed (p>0.05). approximately 27 million ari cases occur annually in mexico [14] . these cases can be caused by a large variety of aetiological agents. however, the main purpose of epidemiological surveillance in the country is to detect the antigenic variations of influenza that are presented each season, which determine the changes in the vaccine composition. in 2013, the instituto de diagnóstico y referencia epidemiológicos (indre; institute of epidemiological diagnosis and reference) implemented a differential diagnosis of influenza that included 14 other respiratory viruses [15] . due to this, at this moment mexico does not have enough epidemiological information about the great diversity of viruses causing acute respiratory infection. this lack of information, makes physicians search only for influenza, it is therefore the most requested confirmatory test in the laboratory during the whole year, even when we are out of influenza season (season comprises from october to may), leading to useless negative influenza results in most of the cases, without identifying the real causal agent of ari in mexico. this problem have direct implications for each patient, as the clinician does not know the causal agent, the patient does not receive the suitable treatment. it also has implications that affect the whole society, because the ignorance of the circulation patterns and incidence of other respiratory viruses limit preventive actions by health institutions. according to data from the secretary of health, approximately 80% of the samples from patients with aris that are received for confirmation of influenza virus infection outside of flu season are negative for the different strains of this virus and remain without a defined aetiology. therefore, the objective of this study was to determine the viral aetiology of these infections and to analyse the behaviour of non-influenza respiratory viruses in the mexican population. beginning and ending with the 2015 influenza season, 872 samples collected over one year were evaluated to determine the presence of hmpv, hrsv, hpiv 1-4, βcov1, hcov, hmdv, rv, ev, and pbpv. these 14 respiratory viruses share symptoms with influenza but are rarely suspected or can be confused with influenza. in contrast to other studies in other countries investigating this issue, this study included samples corresponding to all age groups and regions of the country. therefore, the study population best represents the demographic distribution of aris in the country. in contrast to most of the prevalence studies of aris, in which all age groups are not normally analysed and data is limited to a single region of the country, in this study, nor the age of the patient, nor the region of the country from which the sample came were included as inclusion or exclusion criteria. therefore, the population analysed in this study represents better the demographic distribution of aris in our country. this type of study helps provide relevant data for the development of treatment and prevention strategies because the currently available antiviral agents and vaccines are primarily directed at influenza infection. however, the proportion of aris caused by different influenza viral agents is not negligible, with the detection range of non-influenza respiratory viruses spanning from 16.5 to 72.7% in studies conducted worldwide [4;16-20] , depending on factors such as the study design, study population, detection technique used, and period covered by the study. in our case, the prevalence of the analysed viruses was 35.8%, which was within the cited range. the most prevalent viruses were rv, hrsv, and hmpv, and the only virus that was not detected was hpiv2. this result was consistent with other studies in which type 2 parainfluenza virus had the lowest detection frequency [21-24]. however, there is evidence that its prevalence increases when hpiv-3 and hpiv-1 are reduced [25] [26] . consistent with our results, rv was identified as the most common virus in ari cases in several studies [17;20;22;27] in which the presence of influenza and other respiratory viruses from throat swabs is determined by molecular techniques of own design or using commercial panels. equal data were observed in a study of mexican children [28] . this virus is the major aetiological agent of the common cold [29], however, it may also be involved in serious aris [30] [31] [32] , which are primarily observed in patients with underlying comorbidities, such as asthma [33] [34] [35] or other chronic pulmonary diseases [36] , indicating a certain degree of opportunism. similar to rv, hrsv was identified in high proportions in other studies [4;37] , conducted in poland in which 380 individuals were analysed without age selection, and in mexico in which 383 children up to 5 years of age were studied. this trend is also observed in studies where a higher prevalence of influenza was observed [19;21;23;38] , and hrsv has been associated with the pathogenesis of asthma. more importantly, hrsv is the leading cause of child mortality caused by viruses [39] . the third most prevalent virus was hmpv. in mexico, the first study in which the presence of this virus was determined was published in 2005 [40] . subsequently, other mexican studies demonstrated its importance in the child population [16;41-42] . studies such as that of diaz et al. [16] demonstrated that hmpv was mostly associated with severe acute respiratory infections instead of mild and moderate infections. the importance of the differential diagnosis of other respiratory viruses in samples with negative influenza results becomes apparent when we observe the prevalence of the three main viruses identified in this study as well as their associations with severe cases and deaths, especially in the child population. co-infections represented 17.9% of the positive samples. this percentage ranged between 6.9 and 18.6% in other studies that also included various age groups [20;43-44] . however, much higher percentages have been found (above 30%) in study populations composed of children under 5 years of age [16;22-23;45] . our results confirmed that viral co-infection was common, especially in the child population because 91.1% of all co-infections occurred in the 0 to 9 year age group. this result could be attributed to slower viral elimination due to a still-developing immune system [46] . the virus most involved in co-infections was pbpv, as in 78.6% of the cases in which it was identified, it was found together with another virus. studies have reported identification percentages of this virus in conjunction with other agents ranging from 47.4 up to 90% [47] [48] . however, its prevalence in asymptomatic patients is also high (44% or 43%) [49] , which calls into question whether pbpv by itself is capable of generating disease [50] or if it only participates as a facilitator for another agent to infect the host. at present, cellular and animal models are still being developed for this virus [51] , therefore, the evidence needed to confirm this hypothesis does not yet exist. from a clinical perspective, whether the presence of a co-infection results in a more serious case or is a poor prognostic factor is a matter of controversy within the scientific community. although some studies have reported cases with these characteristics [52] [53] [54] , reports by other authors have suggested that co-infections are not synonymous with clinical differences or greater severity [55] [56] . in the study of martínez-roig et al. [57] , an inverse relationship was found between the number of viruses detected and the hospitalization time as well as the need for oxygen therapy. a similar relationship was observed in the study of canducci et al. [58] , where there was a greater hypoxia and lengthier hospitalization time for infections caused purely by hrsv compared to coinfections. in our study, the aris caused by a single virus also presented lengthier hospitalization times than aris caused by 2 or more viruses, although the differences were not statistically significant. notably, similar to the estimation of prevalence, differences in the reported ranges of and discrepancies in the severity associated with viral co-infections can be attributed to the detection techniques employed, the population, the time period of the study, and the viruses studied. because of the short period of time comprised by this study and the limited number of samples, it is not possible to state, but it can be suggested certain seasonal behaviors of some studied viruses. generally, the peak of respiratory infections occurs in the period between november and april in countries of the northern hemisphere [59] [60] . the influenza season is well known in mexico and worldwide; however, the seasons of other respiratory viruses are not well known. according to our results, the highest prevalence of these viruses in mexico appears to occur from october to march (autumn and winter), which coincides with the influenza season. detection was significantly higher in november of 2014; during this month, there was a marked decrease in the national mean temperature from 23.3 to 18.3˚c. according to cui et al. [61] , mean temperature is the key climatic parameter associated with the prevalence of many respiratory viruses because some viruses survive and/or replicate better at low temperatures [61] . on the other hand, viegas et al [62] proposed that mucus release by cilia was reduced when the temperature of the respiratory tract was lowered; consequently, the local innate immune response (neutrophil and natural killer (nk) cell migration) was also reduced, thereby promoting viral infection. climatic factors may also indirectly favour the transmission efficiency because low temperatures induce a change in social behaviour that favours interior overcrowding and increases the likelihood of close contact and transmission of infections [63] . for this reason, it is a common suggestion to avoid going to school or work when you are undergoing an infectious process. our results suggest that some viruses have a more marked seasonal trend than other viruses. in winter, the prevalences of hmpv, hpiv3, and bcov1 were higher than in the other seasons of the year. the circulation of hmpv in particular predominates in the colder seasons because a marked reduction in its prevalence is observed in the spring until it disappears completely in the summer. furthermore, in a study conducted in mexico city, in which 525 children between 1 and 15 years of age were analysed and in other study performed in china with 607 hospitalized patients was reported that the highest prevalence of hmpv occurs in the winter and that its seasonality overlaps with that of other viruses [19;43] . similar to hmpv, some authors agree that hrsv is a virus that circulates preferentially in colder seasons [19;43;64] . this study demonstrates that hrsv can cause aris in all seasons of the year but is consistent with other studies that report that its prevalence is highest during the fall and winter. conversely, rv seemed to be the virus best suited to the climatic conditions of the country because there were no significant differences in its prevalence in the different seasons. according to the results of a study conducted in china [61] , the optimal circulation temperature range of this virus is 15 to 25˚c, which is similar to the range of the mean mexican temperature and may justify its high prevalence in all months of the year. however, despite all of the information reported to date, factors affecting the circulation of different viruses remain unclear. annual research is needed to establish the seasonality of these viruses with more accuracy and precision. although influenza is one of the main causative agents of respiratory infections worldwide, it is of vital importance to determine the prevalence and timing of other causal agents. in this study, we identified 13 non-influenza respiratory viruses that occurred in any season of the year. this study provides evidence for the prevalence and seasonality of a wide range of respiratory viruses circulating in mexico that constitute a risk for the population. additionally, our data suggest that including these tests more widely in the diagnostic algorithm for influenza could reduce the use of unnecessary antibiotics, reduce the hospitalisation time, and enrich national epidemiological data regarding infections caused by these viruses. supporting information s1 human metapneumovirus in adults human metapneumovirus: review of an important respiratory pathogen reforzamiento pulmonar: su relación con la infección respiratoria aguda y la prescripción inadecuada de antibioticos non-influenza viruses in acute respiratory infections among young children. high prevalence of hmpv during the h1n1v.2009 pandemic in poland summary health statistics for u.s. children: national health interview survey viruses and bacteria in the etiology of the common cold bronchiolitis-associated hospitalizations among us children infecciones respiratorias agudas en menores de 5 años intervención educativa sibre infecciones respiratorias agudas preparación y respuesta ante una pandemia de influenza coronavirus infections in hospitalized pediatric patients with acute respiratory tract disease centers for disease control and prevention. real-time rt-pcr assay for non-influenza respiratory viruses anuario de morbilidad lineamientos para la vigilancia de influenza por laboratorio viral coinfection in acute respiratory infection in mexican children treated by the emergency service: a cross-sectional study the frequency and seasonality of influenza and other respiratory viruses in tennessee: two influenza seasons of surveillance data surveillance for severe acute respiratory infections in southern arizona prevalence of respiratory virus in symptomatic children in private physician office settings in five communities of the state of veracruz, mexico influenza and other respiratory virus infections in outpatients with medically attended acute respiratory infection during human rhinovirus and disease severity in children rhinovirus infection in children hospitalized with acute bronchiolitis and its impact on subsequent wheezing or asthma: a comparison of etiologies the role of rhinovirus in asthma exacerbations the abcs of rhinoviruses, wheezing, and asthma rhinovirus-induced lower respiratory illness is increased in asthma and related to virus load and th1/2 cytokine and il-10 production pathogenesis of rhinovirus infection frequency of viruses associated with acute respiratory infections in children younger than five years of age at a locality of mexico city comparative viral frequency in mexican children under 5 years of age with and without upper respiratory symptoms the impact of viral genotype on pathogenesis and disease severity: respiratory syncytial virus and human rhinoviruses human metapneumovirus infections in mexico: epidemiological and clinical characteristics human metapneumovirus and other respiratory viral infections in children attending a day care center human metapneumovirus in children with influenza-like illness in yucatan, mexico viral etiology of acute respiratory tract infections in hospitalized children and adults in shandong province, china respiratory viral infections during the 2009-2010 winter season in central england, uk: incidence and patterns of multiple virus co-infections detection of multiple respiratory pathogens during primary respiratory infection: nasal swab versus nasopharyngeal aspirate using real-time polymerase chain reaction diagnostic value of respiratory virus detection in symptomatic children using real-time pcr human bocavirus isolated from children with acute respiratory tract infections in korea human bocavirus: a novel parvovirus epidemiologically associated with pneumonia requiring hospitalization in thailand human bocavirus infections in hospitalized children and adults frequent and prolonged shedding of bocavirus in young children attending daycare human bocavirus: lessons learned to date is virus coinfection a predictor of severity in children with viral respiratory infections? human bocavirus in chile: clinical characteristics and epidemiological profile in children with acute respiratory tract infections does respiratory virus coinfection increases the clinical severity of acute respiratory infection among children infected with respiratory syncytial virus? multipathogen infections in hospitalized children with acute respiratory infections human respiratory syncytial virus in children with lower respiratory tract infections or influenza-like illness and its co-infection characteristics with viruses and atypical bacteria in hangzhou viral coinfection in childhood respiratory tract infections two-year prospective study of single infections and co-infections by respiratory syncytial virus and viruses identified recently in infants with acute respiratory disease health protection agency. respiratory pathogen circulation public health agency of canada. the respiratory virus detection surveillance viral aetiology of acute respiratory infections among children and associated meteorological factors in southern china respiratory viruses seasonality in children under five years of age in seasonality and the dynamics of infectious diseases seasonal patterns of viral and bacterial infections among children hospitalized with community-acquired pneumonia in a tropical region we thank the san angel laboratory for their support for covering the costs of the publication of this manuscript. key: cord-017499-51yy7y9n authors: freye, enno; levy, joseph victor title: mechanism of action of opioids and clinical effects date: 2008 journal: opioids in medicine doi: 10.1007/978-1-4020-5947-6_2 sha: doc_id: 17499 cord_uid: 51yy7y9n nan the opium poppy probably reached china about the seventh century a.d. through the efforts of arab traders who advocated its use for medicinal purposes. in chinese literature, however, there are earlier references to its use. the noted chinese surgeon hua to of the three kingdoms (220-264 a.d.) used opium preparations and cannabis indica for his patients to swallow before undergoing major surgery. the opium poppy, papaver somniferum, is an annual plant, i.e., the plant matures one time, and does not regenerate itself. new seed must be planted each season. from a small seed, it grows, flowers, and bears fruit (a pod) only once. the entire growth cycle for most varieties of this plant takes about 120 days. the tiny seeds (like the seeds on a poppy seed roll) germinate quickly in warm air and sufficient soil moisture. in less than 6 weeks, the young plant emerges from the soil, grows a set of four leaves, and resembles a small cabbage in appearance. the lobed, dentate (jagged-edged) leaves are bluish-green with a dull gray or blue tint. the major legal opium production areas in the world today are in governmentregulated opium farms in india, turkey, and tasmania (australia). the major illegal growing areas are in southwest asia (afghanistan, pakistan, and iran) and in the highlands of mainland southeast asia (burma, laos, vietnam, and thailand)popularly known as the golden triangle ( figure ii-3) . opium poppy is also grown in colombia, mexico, and lebanon. opium poppies containing small amounts of opium alkaloids were, at one time, widely grown as an ornamental plant and for seeds in the united states. the opium poppy control act of 1942 declared the possession of this plant illegal. from the cut capsule latex is exuded, which is collected and further processed in order to gain the different ingredients ( figure ii-2) . within the secreted latex collectors will find the major constituents of opium poppy, which are as follows: 1. morphine (10%-17%), the most important alkaloid, which was discovered by the pharmacist sertürner in a small town of einbeck, located in lower saxonia in germany in 1803. he decided to name the extract from the opium poppy morphine ( figure 3. thebaine (0.5%-2%) a precursor of many of semi-synthetic opioid agonists (i.e. etorphine, oxymorphone) and antagonists (naloxone, naltrexone, diprenorphine, cyprenorphine), mixed agonist/antagonists (nalbuphine) as well as the partial agonist buprenorphine ( figure c ). 4. benzylisoquinolines are a group of agents, which do not interact with the opioid receptor. the most important one is papaverine (0.5%-1%) a phosphodiestrase inhibitor, which relaxes the smooth muscle, and noscapine (2%-9%), which is used as a cough suppressant ( figure d) . raw or cooked opium contains more than 35 different alkaloids, including morphine, codeine, and thebaine ( figure ii-4) . in mainland southeast asia, the morphine alkaloid alone accounts for approximately 10% of the total weight of opium. heroin manufacturers must first extract the morphine from the opium, before converting the morphine to heroin. the extraction is a simple process, requiring only a few chemicals and a supply of water. morphine sometimes is extracted from opium in small clandestine laboratories, which are typically set up near the opium poppy fields. since the morphine base is about one-tenth the weight and volume of raw opium, it is desirable to reduce the opium to morphine before transporting the product from the field to a heroin laboratory. the following is a step-by-step description of morphine extraction in a typical mainland southeast asian laboratory. an empty 55-gallon oil drum is placed on bricks about a foot above the ground and a fire is built under the drum. thirty gallons of water are added to the drum and brought to a boil. ten to fifteen kilograms of raw opium are added to the boiling water. with stirring, the raw opium eventually dissolves in the boiling water, while soil, leaves, twigs, and other non-soluble materials float in the solution. most of these materials are scooped out of the clear, dark brown liquid opium solution. slaked lime (calcium hydroxide) or, more often, a readily available chemical fertilizer with a high content of lime, is added to the solution. lime will convert the water-insoluble morphine alkaloid into water-soluble calcium morphenate. (other opium alkaloids do not react with lime to form water-soluble calcium salts, as does morphine.) codeine is an opium alkaloid that is slightly water-soluble and some codeine will be carried over with the calcium morphenate in the liquid. otherwise, for the most part, the other alkaloids will become a part of the sludge. as the solution cools, the morphine solution is scooped from the drum and poured through a filter. cloth rice sacks are often used as filters and can then be squeezed in a press to remove most of the solution from the wet sacks. liquid saponated cresol (lysol) is commonly added to the solution to facilitate filtering. the morphine-rich solution is then poured into large cooking pots and reheated but, this time, not boiled. ammonium chloride (a powder) is added to the heated calcium morphenate solution to adjust the alkalinity to a ph of 8 to 9, and the solution is then allowed to cool. within 1 or 2 h, morphine base precipitates (crashes) out of the solution and settles to the bottom of the cooking pot. the solution is then poured off through cloth filters. any solid morphine base chunks in the solution will remain on the cloth. the morphine base is removed from both the cooking pot and from the filter cloths, wrapped and squeezed in cloth, and then dried in the sun. when dry, the crude morphine base is a coffee-colored coarse powder. this form of morphine is commonly known by the chinese term pi-tzu in mainland southeast asia. if morphine base is to be stored or transported to another location, it may be pressed into blocks. crude morphine base is generally 50%-70% morphine, and is an intermediate product in the heroin process. addicts do generally not use this morphine base. this crude morphine base may be further purified (and changed to morphine hydrochloride) by dissolution in hot water and hydrochloric acid, then adding activated charcoal, reheating, and filtering. the solution is filtered several times before being allowed to cool. as the solution cools, morphine hydrochloride precipitates out of the solution and settles to the bottom. the precipitate is trapped (or captured) by filtration. if the morphine hydrochloride is to be stored or transported to another location, it may be pressed into bricks. morphine hydrochloride (often tainted with codeine hydrochloride) is usually pressed into brick-sized blocks in a press and wrapped in paper or cloth. the most common block size is 2 in. by 4 in. by 5 in., and weighs about 3 lb (1.3 kg). it takes a full day to extract morphine from opium. as described in the preceding paragraphs, the chemicals used to isolate morphine from opium (known as extraction) include calcium hydroxide (slaked lime) and ammonium chloride. the precursor chemical normally used in the conversion of morphine to heroin (known as acetylation) is acetic anhydride. chemical reagents used in the conversion process include sodium carbonate and activated charcoal. chemical solvents needed are chloroform, ethyl alcohol (ethanol), and ethyl ether. other chemicals may be substituted for these preferred chemicals, but most or all of these preferred chemicals are readily available from smugglers and suppliers. laboratory equipment includes large chinese cooking woks, measuring cups, funnels, filter paper, litmus paper, and enamel (or stainless steel) pots. only the most sophisticated heroin laboratories use glass flasks, propane gas ovens, vacuum pumps, autoclaves, electric blenders, venting hoods, centrifuges, reflux condensers, electric drying ovens, and elaborate exhaust systems. it is common to find portable, gasoline-powered generators at clandestine heroin conversion laboratories. generators are used to power various electrical devices. heroin synthesis from morphine (either morphine base or morphine hydrochloride) is a two-step process that requires between 4 and 6 h to complete . heroin base is the intermediate product. typically, morphine hydrochloride bricks are pulverized and the dried powder is then placed in an enamel pot. acetic anhydride is added, which then reacts with the morphine to form heroin acetate. (this acetylation process will work either with morphine hydrochloride or morphine base.) the pot lid is tied or clamped on, using a damp towel for a gasket. the pot is carefully heated for about 2 h, below boiling, at a constant temperature of 85 c (185 f). it is never allowed to boil or to become so hot as to vent fumes into the room. tilting and rotation agitate the mixture until all of the morphine has dissolved. when cooking is completed, the pot is cooled and opened. during this step, morphine and the anhydride become chemically bonded, creating an impure form of diacetylmorphine (heroin). water is added to the thick, soupy mixture and the mixture is stirred as the heroin dissolves in the solution. sodium carbonate (a crystalline powder) is dissolved in hot water and then added slowly to the heroin solution until effervescence stops. this precipitates heroin base, which is then filtered and dried by heating in a steam bath. for each kilogram of morphine, 685 g-937 g of crude heroin base is formed, depending on the quality of morphine. the tan-colored heroin base (about 70% pure heroin) may be dried, packed, and transported to a heroin-refining laboratory, or it may be purified further before conversion to heroin hydrochloride (a water-soluble salt form of heroin) at the same site. mainland southeast asian heroin base is an intermediate product that can be further converted to either smoking heroin (heroin no. 3) or injectable heroin (heroin no. 4). to make heroin no. 3, the crude base is mixed with hydrochloric acid, resulting in heroin hydrochloride (hcl). adulterants, including caffeine, are added after this conversion. for each kilogram of crude heroin base, about 1 kg of caffeine is used. various flavorings such as quinine hydrochloride or strychnine hydrochloride are sometimes added to heroin no. 3. next, the wet paste mix is stirred to dryness over a steam bath. the resulting dry heroin no. 3 will be in the form of coarse lumps. the lumps are crushed and passed through a mesh sieve, and the grains (pieces) are then packaged for sale. the entire process takes about 8 h and requires only minimal skill. while extra attention to stirring is required to assure dryness, one person can prepare 1 kg of heroin no. 3 during this time. the reaction of morphine with acetic anhydride produces heroin acetate. to the heroin acetate mixture in the pot, water is added and mixed by stirring. a small amount of chloroform is added. the mixture is stirred and then allowed to stand for 20 min. doing so dissolves highly colored impurities and a red, greasy liquid is formed at the bottom of the container. the water layer is carefully poured off and saved in a clean pot, leaving the red grease in the pot. in a clean pot, activated charcoal is stirred into the aqueous solution and is filtered to remove solid impurities. the decolorizing effects of the charcoal, combined with the chloroform treatment, will leave a light yellow solution. the use of charcoal is repeated one or more times, until the solution is colorless. sodium carbonate (a crystalline powder) is dissolved in hot water and then added slowly to the heroin solution until effervescence stops. this precipitates the heroin base, which is then filtered and dried by heating on a steam bath. the heroin base is heated until dried. the powder should be very white at this stage. if not white, the base is redissolved in diluted acid, treated repeatedly with activated charcoal, re-precipitated, and dried. the ultimate purity and color of the resulting heroin hcl will depend largely on the quality of the heroin base. the heroin base is then dissolved in ethyl ether. conversion to the hydrochloride salt is achieved by adding hydrochloric acid in ethanol to the heroin mixture. the heroin then precipitates. the process of extracting morphine from opium involves dissolving opium in boiling water, adding lime (calcium oxide), or slaked lime (calcium hydroxide), or limestone (calcium carbonate) to precipitate non-morphine alkaloids, and then pouring off the morphine in solution. ammonium chloride is then added to the solution to precipitate morphine from the solution. the chemicals used to process opium to morphine have a number of legitimate purposes and are widely available on the open market. an empty oil drum, some cooking pots, and filter cloths or filter paper are needed. in the united states, opium preparations became widely available in the nineteenth century and morphine was used extensively as a painkiller for wounded soldiers during the civil war. the inevitable result was opium addiction, contemporarily called the army disease or soldier's disease. these opium and morphine abuse problems prompted a scientific search for potent, but nonaddictive, painkillers. in the 1870s, chemists developed an opium-based and supposedly nonaddictive substitute for morphine. the bayer pharmaceutical company of germany was the first to produce the new drug in large quantities under the brand name heroin. this product was obtained by acetylation of morphine. soon thereafter studies showed heroin to have narcotic and addictive properties far exceeding those of morphine. although heroin has been used in the united kingdom in the treatment of the terminally ill, its medical value is a subject of intense controversy. among the commonly known classes of opioids/opiates being used in practice are morphine, codeine, heroin, and the antagonist naloxone ( figure ii-6 ). morphine by itself is still made from opium and although there is a major first-pass effect (i.e. degradation by liver enzymes), oral administration is still possible, but requires substantial dosage increase. codeine, which is also taken orally, has a strong ability to inhibit coughing, but it induces less analgesia. among the phenylpiperidines a number of synthetic compounds have entered the market. the most known is meperidine/pethidine (demerol®), which is very similar to morphine, but is more efficacious when given orally for the control of pain. another derivative is loperamide (imodium®), an agent being used as a common antidiarrheal, which does not enter the brain, as it is incapable of crossing the blood-brain barrier. hence it is not abused and therefore is sold as a doc (drug over the counter). contrary, fentanyl (sublimaze®) is an opioid, which is at least 200 times as potent as morphine. this agent is used with nitrous oxide or droperidol (a neuroleptic) in intravenous anesthesia (neuroleptanesthesia), but it is also a used in a transdermal patch for the control of chronic pain. another known opioid is methadone, which has a good oral efficacy, a much longer half-life than morphine (8-12 h) , and in regard to its effect much like morphine. it is used for treatment of heroin addiction and for the control of chronic pain. a methadone congener, which is being used solely in the methadone substitution programs is laam ( -levoacetylmethadol), only needs to be taken once every 72 h. the opioid propoxyphene (darvon®) has figure ii-6. molecular structure of different opioid ligands with agonistic or antagonistic properties the lowest analgesic potency (0.02 times morphine). it is almost always given together with aspirin for the control of mild to moderate pain. it is very popular clinically due to misplaced concerns about the abuse potential of codeine. it is interesting to note that all commonly used opioids have a similar structure in regard to their terminal morphine ring and the distance between the ring and the n-substitution. such common traits suggests that opioids must have a common structure in order to interact with a specific receptor site ( figure ii-7) . thus, central analgesics mediate their action by means of an interaction with specific opioid receptor sites located within specific areas of the central nervous system, which are engaged in the transmission of nociceptive afferences and the identification of pain. there, opioids act as agonists at highly definite receptor sites, and there is general agreement on the existence of at least three types of opioid receptor sites (table ii-1). 1. the morphine mu receptor ( ) at which the prototype morphine binds, 2. the kappa receptor ( ) at which the prototype agonist is ketocyclazocine, and 3. the delta receptor ( ) at which the prototype endogenous opioid ligand enkephalin binds. opioid receptors are distributed widely in brain and found in spinal cord and peripheral sensory and autonomic nerves. there are the three well-characterized members of the opioid receptor family, designated by the greek symbols , and . the more recently discovered orl1 receptor is placed with this family due to its high degree of structural homology. these receptors were renamed op1, op2, op3 and op4, respectively, by an international union of pharmacology (iuphar) nomenclature committee in 1996 [1] . this nomenclature has proved unpopular. the nomenclature (x-opioid peptide receptor) has been proposed giving , mu or mop; , delta or dop; , kappa or kop and orl1 or nop receptors. in order to keep matters straightforward the original nomenclature is used in the following chapters. the products of endogenous opioid peptide genes activate opioid receptors physiologically: proenkephalin (giving methionine-and leucine-enkephalin; metenk and leu-enk, respectively; figure ii-8), prodynorphin (dynorphins a and b and -neo-endorphin) pro-opiomelanocortin ( -endorphin) and pronociceptin (nociceptin, also known as orphanin fq). met-enk and leu-enk have highest affinity for -receptors, less affinity for , and very low affinity for -receptors; the dynorphins have preferential affinity for -receptors, but bind to the and types with high affinity; -endorphin binds with high affinity to and receptors, but has little affinity for receptors. all the peptides are full agonists at their cognate receptors. endomorphin-1 and -2, derived from an unknown precursor, are endogenous peptides with high selectivity for -receptors. these peptides are unusual in that they are partial agonists. none of the proenkephalin, prodynorphin or pro-opiomelanocortin peptide products or the endomorphins displays affinity for the orl1 receptor. similarly, the orl1 receptor agonist nociceptin has no appreciable affinity for , or receptors. the four receptor types have been cloned and shown to be 7-transmembrane receptors activating g proteins of the pertussis-toxin insensitive g i/o family, but including g z. evidence for subtypes of , and opioid receptors exists, but the molecular basis for the observed functional and pharmacological differences is unclear. putative 1 and 2 receptors are differentiated by several agonist and antagonist ligands. however, there is only one receptor gene, the protein product of which has properties of the putative 2 receptor. the distinction between the proposed 2 and 2 receptors is based largely on the apparent preferential blockade of the 1 type by the antagonist, naloxonazine [2] . there is only one cloned receptor gene, corresponding to the putative 1 receptor, but several forms of the -receptor mrna arising from alternative splicing have been reported. the receptors these encode differ at the end of the c-terminal tail and show subtle differences in the binding profile of opioid ligands; a role for the variants is not known. the cloned -receptor, with high affinity for u69593 is the 1 subtype. the proposed 2 and 3 subtypes are poorly defined in both molecular and pharmacological terms (table ii-2) . a recent explanation for subtypes has evolved with the identification of opioid receptor heterodimers or hetero-oligomers that appear to have properties different from the monomeric receptors. an interesting addition to ligands that bind to the 1 receptor is the hallucinogen salvinorin-a. this is a highly efficacious and potent agonist, but is most unusual in that it has no nitrogen atom. endogenous opioid systems have a functional role in modulating pain perception; opioid agonists are therefore potent analgesics. opioid receptors are also present in hypothalamus (figure ii-9), where they influence temperature regulation and control of hormonal secretion. in the forebrain, endogenous opioids are involved in behavioral reinforcement and appear to play a role in anxiety and in the expression of emotions. in addition, opioids influence gastrointestinal and autonomic nervous system function. originally, a fifth binding site, the sigma receptor, was included in this group. however, actions mediated through this receptor are not reversed by naloxone so it is not a true opioid receptor. the -receptors have been further sub-classified into two distinct subtypes (1 and 2), as have the -receptors. kappa receptors have been divided into 1, 2, and 3 sub-types. recently, several of these receptors have been cloned successfully. in animal models, some laboratories have cloned up to 10 -receptor subtypes [4] . however, the functional significance of these "spliced variants" remains unclear at present. originally suggested by martin and coworkers [5] , all three opioid receptor types mediate different opiate effects as they figure ii-9. difference in topographic density of the three opioid receptor sites within the central nervous system. adapted from [3] normally serve endogenous opiates (the endorphins, dynorphins, and enkephalins; table ii-1): 1. activation of the mu receptors ( ) results in analgesia, euphoria, respiratory depression, nausea, gi slowdown, and miosis. receptors of this type are mostly located in periaquaductal gray (pag), spinal trigeminal nucleus, caudate and geniculate nuclei, thalamus, and spinal cord. 2. binding at the kappa receptors ( ) induces modest analgesia, dysphoria, feelings of depersonalization and disorientation, miosis, and mild respiratory depression. these receptors are mainly found in basal ganglia, nucleus accumbens, ventral tegmentum, cortex, hypothalamus, periaqueductal grey, the spinal cord, and in the periphery. 3. occupation of the delta receptors ( ) results in anxiolysis and central pain relief, although its overall significance is not all that well understood. they are mainly found in the nucleus accumbens and the limbic system (table ii-2) . molecular biology techniques have enabled the primary amino acid sequence of the human -, -, and -opioid receptors to be determined. the pharmacological and functional properties of the cloned receptors, the development of "knockout" animals, which are deficient in a receptor or part of a receptor, and the manipulation and substitution of various amino acids in critical domains of the various opioid receptors have provided new insights in opioid action. in this regard, the three opioid receptor genes, encoding mu (mor), delta (dor), and kappa (kor) have been cloned. the binding affinities of a range of opioids to the -, -, and -opioid receptors and also to the cloned orphinan receptor have been examined in animals. the animal data indicate that while the commonly prescribed opioids (agonists and antagonists) bind preferentially to the -receptor, they also interact with all three receptor types. morphine and normorphine (a minor metabolite of morphine) show the greatest relative preference for the -receptor. methadone (which also has some nmda-receptor blocking activity) shows significant binding to -receptors, while buprenorphine, and to a lesser extent naloxone, avidly binds to all three receptor types. there is evidence (albeit inconsistent) that the d-enantiomer of methadone blocks the nmda receptor [6] . the binding affinity of buprenorphine to the receptor is much greater than that of naloxone, which explains why the latter only partially reverses buprenorphine overdose. animal data also indicate that codeine and diamorphine have very poor binding to opioid receptors, which reinforces the possibility that both are prodrugs where the pharmacologically active species are morphine [7] and 6-monoacetyl morphine, respectively [8] . oxycodone may also act through an active metabolite, though there are some data, which suggest that this is not the case [9] . pethidine is considered to be a potent -receptor agonist, but it does bind weakly to all three opioid receptors (table ii-1) . ketobemidone has a lower affinity for the -receptor than does morphine, but it shows greater discrimination for this receptor compared to -receptors. the binding of both of these opioids to the -receptor is similar [10] . this difference in opioid action is also mirrored in the difference in affinity of various narcotic ligands interacting with the three relevant opioid receptor sites (table ii-3) . it should be noted that some of those ligands, either pure antagonists, mixed agonist/antagonists or partial agonists, are characterized by displacement potency at a specific receptor site. from the above binding and displacement values it can be seen, that opioid practically bind to all three receptor sites, however with different affinity. the preference in binding to one receptor site manifests itself in the visible clinical effect, which may either be of agonistic or of antagonistic nature. the binding of morphine, methadone, buprenorphine, and naloxone to the cloned human -receptor shows excellent congruence with the animal data [16] . fentanyl shows a similar binding affinity, while codeine demonstrates greater binding affinity to the cloned human receptor (table ii-3; figure ii -10). thus, for these commonly administered opioids, there is no great variability in their affinity for the humanreceptor. the clinical relevance of these data is that different opioids act in different ways. from anecdotal clinical experience there is considerable interindividual adapted from [11, 12, 13, 14, 15] variability in response to each opioid and this reinforces the need to assess an individual's response to opioid analgesia carefully. it also is premature to extrapolate from laboratory data, which in many instances have not yet been replicated, to the clinic. however, data increasingly inform the clinical use of these drugs and will be particularly relevant to new approaches to their use such as "opioid switching". figure ii-10 shows the putative analgesic effect mediated by the main -opioid receptor depicting that higher affinity also correlates closely with analgesic potency. but aside from -receptor interaction, analgesia can also be mediated through a -receptor and a -receptor site. the classification of different opioid receptor types is based on the original description by martin and coworkers from 1976 [5] . the effects presumed to be mediated at -receptors have been defined as a result of both human and animal studies, while the effects mediated at -receptors derive predominantly from animal models. receptors mediate analgesia that persists in animals made tolerant to -agonists. the -agonists produce less respiratory depression and miosis than -agonists. it is assumed that opioid receptors mediate opioid receptors are found in several areas of the brain, particularly in the periaqueductal grey matter, and throughout the spinal cord ( figure ii-9 ). supraspinal systems have been described for -, -, and -receptors, whereas -and -receptors modulate pain at the spinal level [3, 17, 18] . the different distribution of the various opioid subsites suggests different mechanisms of action in the mediation of analgesia. thus, -selective opioids like morphine, fentanyl and sufentanil, due to the high density of binding sites, mediate their main action within the brain stem and the midbrain. due to their close vicinity to respiratory and cardiovascular regulating centers in the brain stem, selective -opioids accordingly induce a marked depression of respiration and blood pressure. on the other hand, due to the main distribution of the -receptors within the cortex (lamina v, vi) [19] it is conceivable that these ligands induce a lesser respiratory and cardiovascular depressive effect. as a consequence and contrary to -ligands, -ligands induce a marked sedative appearance. in addition, there is a lesser addiction liability with -ligands, which is easily derived from the fact that the relevant areas in the limbic system show a low concentration of -binding sites. also, the lesser analgesic potency of -ligands is enlightened by the fact that most of the -selective receptors can be found in the deep lamina vi of the cortex. since their dendrites retrograde descend to the thalamus, all ascending nociceptive input is modified, resulting in a depression of nociceptive afferences and a reduction in arousal. certain dendrites of petrosal cells of the cortex also descend down to the brain stem, whereby the activating, ascending reticular system (ars) is affected resulting in a reduction of vigilance [20] . in summary, due to the dissimilarity of distribution of the three opioid receptor subtypes with the spinal cord and the supraspinal areas of the cns, a functional differentiation can be expected. this effect is reflected in difference of binding affinities with the brain where 22% of all receptor sites are referred to the -, 36% to the -and 42% to the -opioid receptor [20, 21] . the present understanding of the effect profiles of opioid receptors, however, remains incomplete, as new advances make it clear that their disposition and structure are extremely complex. opioids inhibit pain signals by different mode of actions: • inhibition of ca ++ -influx into the buttons of the presynaptic cell (e.g. the one releasing substance p; figure ii -11) . this is because ca ++ -influx is necessary for neurotransmitter release, whereby opioids reduce or prevent substance p from being released. • acting as an inhibitory neurotransmitter, since the opioid hyperpolarizes the postsynaptic cell by enhancing k + -flow out of the neuron, which makes it more difficult for all incoming nociceptive afferences to stimulate the next neuron, and thus more difficult to send painful information. • moderation of central perception of painful information in the limbic system so as to make it less aversive when it is perceived. several facts have led to the assumption that opioids interact with specific binding sites in the cns. a slight molecular substitution at the side chain of the morphine molecule structure results in considerable changes of potency (table ii-5) . whereas pethidine (meperidine, usp), a piperidine derivative, may be considered a weak analgesic, fentanyl, a piperidine derivative, is about 100-300 times more potent than morphine. the opioid antagonists levallorphan and naloxone are noted for a low and a analgesic effect, respectively. furthermore, only the levorotator (levo-) isomers of opioids, which appear in their natural form (i.e. compounds which, when in solution, rotate plane-polarised light to the left) are pharmacologically active (e.g. levorphanol). their dextrorotatory (dextro-) isomers, which can be synthesized in the laboratory (e.g. dextrophane), shows a negligible pharmacological effect. both substances are structurally the mirror image of each other ( figure ii-12) . in this context it is important to note that only the levo-stereoisomer of the racemic mixture is the pharmacologically active ingredient. this observation supports the part ii figure ii-11. mechanism of action of opioids at the central nervous system. by binding at the same receptor site as the endogenous opioids (i.e. enkephalins, endorphins), the release of excitatory neurotransmitters such as acetylcholine and glutamate is decreased thereby reducing the receiving cells excitatory input. the degree of opiate receptor binding is proportionally to the net release of excitatory transmitters and the reduction of depolarization produced by the arriving nociceptive nerve impulse. this enkephalin inhibitory system normally modulates the activity of the ascending pain pathways within the spinal cord and the brain. opioid agents act by binding to unoccupied enkephalin receptors, thereby potentiating the analgesic effects of the system notion that stereroselectivity of an opioid analgesic is a prerequisite in order to bind to the opiate receptor site, thus inducing analgesia. based on their interactions with the various receptor subtypes, opioid compounds can be divided into agonist, agonist/antagonist, and antagonist classes (table ii-4) . by definition an agonist is a drug that has affinity for and binds to cell receptors to induce changes in the cell that stimulate physiological activity. the agonist opioid drugs have no clinically relevant ceiling effect to analgesia. as the dose is raised, analgesic effects increase in a log linear function, until either analgesia is achieved or dose-limiting adverse effects supervene. efficacy is defined by the generally opioids exist in optical isomers, which are a mirror image in the molecular form. only the levorotatory (levo)-isomer, which in solution rotates plane-polarized light to the left, produces the characteristic analgesic effect of an agent. the dextrorotatory isomer is totally inactive. this sterospecificity of opiate action supports the concept of selective receptor binding to a site, which is able to distinguish in "handedness or goodness of fit" of an opioid molecule maximal response induced by administration of the active agent. in practice, this is determined by the degree of analgesia produced following dose escalation through a range limited by the development of adverse effects. potency, in contrast, reflects the dose-response relationship. potency is influenced by pharmacokinetic factors (i.e. how much of the drug enters the body systemic circulation and then reaches the receptors) and by affinity to drug receptors. the concepts of efficacy and potency are illustrated in the following figure, which shows the dose-response curves for two drugs with a full agonistic and a partial agonistic action. if the logarithm of dose is plotted against response an agonist will produce an s-shaped or sigmoid curve. the efficacy of the two drugs, defined by maximum response is the same. the full agonist produces the same response as a partial agonist but at a lower dose, and therefore is described as more potent ( figure ii-13 ). an antagonist by definition is an agent that has no intrinsic pharmacological action but can interfere with the action of an agonist. competitive antagonists bind to the same receptor and compete for receptor sites, whereas non-competitive antagonists block the effects of the agonist in some other way. contrary the mixed agonist/antagonists analgesics can, in turn, be subdivided into the mixed agonist/antagonists and the partial agonists, a distinction also based on specific patterns of drug-receptor interaction. both the partial agonist and the agonist/antagonist drugs have a ceiling effect for analgesia, and although they produce analgesia in the opioid-naive patient, in theory they can precipitate withdrawal in patients who are physically dependent on morphine-like drugs. for these reasons, they have been considered generally to have a limited role in the management of patients with cancer pain. the mixed agonist/antagonist drugs produce agonist effects at one receptor and antagonist effect at another. pentazocine is the prototype agonist/antagonist: it has agonist effects at the -receptors and weak to medium antagonistic action at the figure ii-13. typical dose-response curves of a full agonist, a partial agonist and an antagonist on opioid-related effects -receptor thus in addition to analgesia, pentazocine may produce -mediated psychotomimetic effects not seen with full or partial agonists. when a mixed agonist/antagonist is administered together with an agonist, the antagonist effect at the -receptor can generate an acute withdrawal syndrome. a partial agonist has low intrinsic activity (efficacy) so that its dose-response curve exhibits a ceiling effect at less than the maximum effect produced by a full agonist ( figure ii-11) . buprenorphine is the main example of a partial agonist opioid. increasing the dose of such a drug above its ceiling does not result in any further increase in response. this phenomenon is illustrated in the figure in which buprenorphine is the partial agonist. the full agonist is more potent than the partial agonist (in the lower part of the curve it will produce the same response at a lower dose), but is less effective than both coadministered ligands because of its ceiling effect. when a partial agonist is administered together with an agonist, displacement of the agonist can cause a net reduction in pharmacological action, which may be sufficient to generate an acute withdrawal syndrome. while this is a theoretical possibility with morphine and buprenorphine, no such interaction has been reported clinically. similarly, it has been suggested that the effects of morphine may be blocked in a patient switched from buprenorphine, because of the prolonged action of buprenorphine and the assumption that it will "antagonize" the effect of morphine. this has been one of the reasons, why buprenorphine has not been in cancer pain management. however, the recent development of a transdermal formulation of buprenorphine may encourage its use in chronic cancer pain (and chronic noncancer pain). an analgesic ceiling with buprenorphine is only reached at doses of 8-16 mg or more in 24 h [22, 23] . when used in usual recommended doses (e.g., two patches of 70 g/h of transdermal buprenorphine, equivalent to 3-4 mg per 24 h) buprenorphine can be considered a full -agonist since at these doses its effect will lie on the linear part of the dose-response curve [24] . relative potency is the ratio of the doses of two analgesics required to produce the same analgesic effect. by convention the relative potency of each of the commonly used opioids is based upon a comparison with 10 mg of parenteral morphine. data from single-and repeated-dose studies in patients with acute or chronic pain have been used to develop an equianalgesic dose table (table ii-5) that provides guidelines for dose selection when the drug or route of administration is changed. the information contained in the equianalgesic dose table does not represent standard doses, nor is it intended as an absolute guideline for dose selection. many variables may influence the appropriate dose for an individual patient, including intensity of pain, prior opioid exposure in terms of drug, duration, and dose (and the degree of cross-tolerance that this confers), age, route of administration, level of consciousness, metabolic abnormalities (see below), and genetic polymorphism in the expression of relevant enzymes or receptors. in addition, a substitution at the side chain, for example the substitution of a methyl-group by an allyl-group or the substitution by a cyclopropyl-group results in the new opioid antagonist naloxone, diprenorphine and naltrexone respectably, or mixed agonists/antagonists (nalorphine, levallorphane), which have the capability of antagonizing the effect of the parent compound ( figure ii-14) . similarly, when the n-methyl group of the highly potent opioid oxymorphone or the pure agonist etorphine (1000 times of morphine) is replaced by a cyclopropylmethyl group, the highly potent antagonists naltrexone and diprenorphine are derived. these compounds are 2.5 times as potent as naloxone and while the former is used as an oral preparation in the rehabilitation of the earlier opiate addict, the latter is used in veterinary medicine for the reversal of immobilization of wild animals. in addition, diprenorphine is also the original substance of buprenorphine where additional three methyl groups are incorporated in the molecule ( figure ii-15 ). such minor changes in the molecular structure and their resultant major pharmacological effect suggest, that similar to hormones and catecholamines, opioids bind with specific receptor sites which results in the characteristic effects such as analgesia. various research groups corroborated this hypothesis almost simultaneously. pert and snyder [17] , terenius [25] and kosterlitz [26] were the first research group to identify selective binding sites in the cns using radioactive labeled opioids. these so-called opiate binding sites were found mainly in neuronal structures and nervous pathways involved in the transmission of nociceptive signals such as the first relay station of pain transmission, the substantia gelatinosa of the spinal column. in the posterior horn the impulse is passed over to the second neuron while, simultaneously, descending nerve fibers from the reticular system (the cortico-and reticulo-spinal tract) induce either a facilitation or an attenuation of pain transmission, which results in a modulation of pain impulses at the spinal level ( figure ii-11 ). the course of pain transmission is to the contralateral side of the spinal cord where impulses have already undergone a distinct separation. it is the paleospinothalamic pathway, consisting of nonmyelinated c-fibers, which mediate the excruciating, dull pain component, which is difficult to localize as it ends in the nonspecific nuclei of the medial thalamus [27] . en route, the paleospinothalamic tract sends off afferent fibers to the midbrain area, such as the periaqueductal grey matter and the reticular formation [28] . the pathway ends in intralaminar nuclei of the thalamus and the nucleus limitans, a patch of pigmented nerve cells border the mesencephalon ( figure ii-16) . from there, subcortical pain pathways link with the pallidum, the alleged psychomotoric center that sends fibers to all areas of the brain hemisphere. the neospinothalamic pathway, in contrast, consists of myelinated a 2 -fibres, which transfer impulses to the nucleus ventrocaudalis-parvocellularis (n.v-c parvocellularis). from there pain sensations are projected to the postcentral gyrus, which enables the patient to localize the source of pain ( figure ii-16) . both, the central grey matter and the pallidum are characterized by a dense accumulation of opiate binding sites [29, 28] . it is worth noting that nervous pathways transmitting the dull, chronic and less pinpointed pain components are more affected by opioids, while , is an important relay station in the mediation of nociceptive afferents to higher pain modulating and discriminating centers of the cns, which is necessary for the nonspecific feeling of pain and is closely coupled with emotions the neospinothalamic pathway conveys the sharp and well localized pain components which accompany any injury and are always the first to be perceived. the indefinable, dull, emotional component is perceived later, giving pain its negative character. this separation in pain pathways is of special importance. impulses from the fast pathway usually antagonize the mediation of slow afferent impulses on all levels in the cns: substantia gelatinosa and reticular formation, as well as the specific and the nonspecific projecting nuclei of the thalamus [30] . opioid binding sites, as they are visualized with receptor-binding techniques, strikingly map the paleospinothalamic pain pathway ( figure ii-17) . furthermore, there is a high density of opioid binding sites in various other parts of the brain [3, 17, 18, 31]: 1. the corpus striatum, being part of the limbic and the extrapyramidal motor system, is responsible for triggering opioid-induced muscular rigidity. it is not only the regulatory center for locomotion but it is also the center for the regulation of attention and perception. 4. the nucleus solitary tract in the brain stern, which is the origin of the noradrenergic dorsal pathway bundle, which is in command of vigilance and the cough reflex. 5. the nuleus amygdala, being part of the limbic system, is in charge of the mediation of euphoria (or "kick") when opioids are used for other purposes than pain. 6. the locus coeruleus being the origin of the neurosympathetic system in the brain stem, regulates peripheral vasodilatation. 7. lastly, a dense accumulation of opioid binding sites is found in the substantia gelatinosa at the dorsal horn of the spinal cord. -current thinking is that effective opioid analgesics work through stimulating mu ( ) receptors, which also produce euphoria. -euphoria is mediated by the actions of opiates at a cluster of brain areas that include the nucleus accumbens and ventral segmental area. dopamine influx seems to cause subjective pleasure, or euphoria. -opioids may have a "disinhibiting" (inhibition of inhibitory neurons) effect that allows greater dopamine influx. because the main property of opioids is the blockade of nociceptive transmission in the mesencephalon (i.e. the nucleus limitans and the limbic system) the following effects can be observed: 1. no pain (analgesia), since any sensation is not identified as painful. 2. a lack of the negative emotional component of pain. on the contrary, euphoria may result and pain is no longer experienced as an emotional distress, even though pain impulses are transmitted via the ventrocaudal-parvocellular nucleus to the postcentral cortex. 3. during the opioid-induced pain-free state, the site of pain, however, still can be localized. as a consequence pain has lost its negative character and is no longer experienced or perceived as uncomfortable and distressing. in contrast to the analgesics that have a peripheral site of action (e.g. acetylsalicylic acid; asa), opioids act at the relay station of nociceptive-propagating pathways at the synapse of nerve conduction. within the nerve, pain impulses are transmitted as a change in electric conduction. and in order to guarantee maintenance of the nociceptive impulse, the excitatory impulse releases a neurotransmitter at the terminal nerve. due to its chemical configuration, the transmitter fits exactly into a binding site at the opposite nerve ending resulting in an increase of excitability and a change in the electrical nerve conduction. opioids have the property of binding to specific receptor sites at pre-and post terminal nerve endings resulting in an inhibition of a release of the excitatory neurotransmitter. the continuity of the impulse is interrupted, the nociceptive signal is no longer transmitted and thus can no longer be perceived as such ( figure ii-11 ). due to the difference in stereoconfiguration, opioids differ in their affinity (i.e. goodness of fit) at these binding sites ( figure ii-18 ). this explains why different opioids are characterized by a large variety in potency. in addition, opioids also differ in their intrinsic activity (i.e. the degree of conformational change of the receptor site) resulting in different intracellular effects. taken together affinity and intrinsic activity results in the efficacy of a drug within the system ( figure ii-19) . thus, binding properties are reflected in varying analgesic potencies. contrary, the intensity of binding with the receptor site (i.e. the intensity with which the opioid adheres to the binding site) is reflected in the duration of effects (table ii-6 and figure ii-20) [32, 14, 33] . for instance opioid analgesics such as sufentanil or lofentanil have an exceptional goodness of fit to the opioid receptor site, which results in high potency. on the other hand, the low dissociation coefficient from the receptor of buprenorphine or lofentanil is characterized by a long duration of action, while the high association coefficient demonstrates increase of affinity to the binding site. contrary to agonists, antagonists are able to displace an opioid from its receptor binding site and take up his position. displacement is only possible because the antagonist has a greater affinity to the binding site. therefore, affinity of an opioid antagonist is expressed in its antagonistic potency. naloxone or naltrexone have a very high affinity to the receptor and easily displace an opioid whereas levallorphan is five times weaker ( figure ii-21) . in order to induce a similar antagonistic effect, a higher dose of levallorphan is necessary. in order to induce increasing effects with opioids, the goodness of fit not only is a prerequisite. of additional importance is the conformational change the receptor undergoes after binding, which is expressed in the "the intrinsic activity". an opioid must, therefore, not only fit to the receptor; it must also induce a chain reaction in the transmembrane receptor domain resulting in a net effect ( figure ii-19) . the reaction after opioid binding seems to depend on the side chain of the molecule. thus it appears that one portion of the opioid molecule provides the binding to the receptor whereas another portion is responsible for the induction of a conformational change (i.e. intrinsic activity), which will either be of agonistic or antagonistic nature. in a sensitive and specific opiate-receptor assay, the guinea pig ileum with its dense accumulation of receptor binding sites, it was possible to demonstrate receptor affinity and pharmacological efficacy ( figure ii-22) . this assumption is underlined by the effects induced by "pure" opioid antagonists such as naloxone or naltrexone, which also have a good fit with the receptor site, however when given on their own do not induce an analgesic effect. for instance, if naloxone is given by itself, the compound does not induce effects similar to its parent compound oxymorphone ( figure ii-14) . also, in contrast to a potent opioid like fentanyl, the antagonist naloxone has a lower dissociation coefficient resulting in a shorter duration of action, which may result in a reoccurrence of an opioid-like effects such as respiratory depression. however, due to its high source: adapted from [33, 34, 35] part ii figure ii-20. difference in affinity and intrinsic activity of various opioids. note, that codeine has a similar intrinsic activity as sufentanil. however, due to the higher affinity of the latter the net analgesic potency is much larger association coefficient (i.e. affinity), it induces a rapid displacement of the agonist and a reversal of all opioid effects. on the other hand mixed agonist/antagonists, such as pentazocine, nalorphine, levallorphan, nalbuphine and butorphanol, demonstrate characteristics, which enable them to displace a pure agonist at the receptor site (antagonistic effect), but at the same time when administered by themselves, they induce opioid related effects such as analgesia and respiratory depression (agonistic effects; table ii-7) . such dual activity is only possible by means of their intrinsic activity at two distinct and different receptor sites: one the antagonistic activity at the -and its agonistic action art the -receptor site. and lastly, partial agonists like meptazinol and buprenorphine induce their analgesic potency via the -opioid receptor. although having a high affinity, their analgesic ceiling effect at the higher dose range is due to a lesser intrinsic activity, resulting in a lesser net analgesic appearance than pure agonists. such difference in the characteristic traits of opioids can be summarized as follows: 1. the affinity to the receptor (displacement properties or extrinsic activity) 2. the intensity of binding to the receptor (duration of effect) 3. the ability to change the conformation of the receptor (intrinsic activity) 4. the competitive potency (antagonism) 5. the degree of metabolism (duration of effect) note the relatively high antagonistic potency of buprenorphine, however, is due to its high affinity to the receptor site resulting in the displacement of a ligand at the preoccupied receptor site. similarly like a hormone or other extracellular "first messengers" that bind to its receptor on a cell surface, a signal is transmitted or "transduced" to the cells interior, thus setting a series of events that produce a biological response. such "events" include both chemical reactions and physical reactions like a conformational change in the protein molecules. the biological responses include cell differentiation, altered metabolism and cell growth and division. there are three signaling pathways that share many of the same intracellular events. each pathway is characterized by its receptor and by the cascade of intracellular events that lead to a biological response. each receptor has an extracellular, transmembrane, and intracellular component and the binding of a ligand to the receptor represents the "primary message". the term "secondary messenger" is used for those mediators that diffuse from one part of the intracellular space to its spatially removed target. among these secondary messengers are adenosine-3,-5-cyclic phosphate (c-amp). many integral membrane glycoprotein membranes share a seven transmembrane alpha-helix motif ( figure ii-23) . the ß-adrenergic receptor, whose natural ligands are epinephrine and norepinephrine, is an example of such receptors. similarly in the opiate receptor, binding of a ligand presumably initiates a conformational change in the membrane protein that is transmitted to the cell interior. this physical reaction can then facilitate other physical or chemical reactions, which are conveyed to ion channels, resulting in a change of transmembrane ion flow. the transduction of the signals from external messengers, including opiate ligands involves intracellular heterotrimeric g-proteins, which are bound to the inner cell (plasma) membrane, a secondary messenger system, involving cyclic amp, and a target response. as the name implies, these proteins are trimers, consisting of an , , and subunit. they are bound to the inner membrane and the subunit can bind the guanine nucleotides, gtp and gdp. g-proteins are involved in vision, smell, cognition, hormone secretion and muscle contraction in humans, and in mating in yeast. there are more than 100 receptors (not including odor receptors) that utilize g-proteins, and there are at least 20 members of the g-protein family, with each member having its characteristic , , and subunits. while the subunit is different for each g-protein, the ß/ pair can be the same. however, all of the g-proteins share a similar structure. in regard to the opioid receptor, specifically the g-proteins transmit the signal from the intracellular part of the receptor to the effector. adenylyl cyclase (ac), which is an inner membrane-bound enzyme, regulates the production of the secondary messenger, adenylyl cyclase. other effectors that are g-proteindependent include additional enzymes, like cyclic gmp phosphodiesterase, and transmembrane ion channels ( figure ii in its resting conformation, the g-protein consists of a complex of the three subunit chains and a gdp molecule bound to the alpha subunit. the alpha subunit is in close proximity to the intracellular part of the transmembrane receptor and, when a ligand binds to the receptor, the change in its conformation causes it to bind to the g-protein at the alpha subunit. this results in an exchange of bound gdp for gtp, which is more abundant in the cell than gdp. gtp causes a conformational change in the alpha subunit, thus "activating" it so that the alpha subunit dissociates from the -pair. the alpha subunit diffuses along the membrane until it binds to an effector, thereby activating it. the alpha subunit is also a gtpase, so the signal transduction is regulated at this level by hydrolysis of gtp to gdp and inorganic phosphate. such hydrolysis can occur spontaneously or upon interaction with a gtpase activating protein, "gap". the gdp-alpha subunit complex then binds to the ß/ complex to reform the original trimeric protein. since the stimulation of the external receptor can activate a number of g-proteins, signal amplification can occur. while this is a desired response in many instances, control at this level is needed to modulate it. g-proteins, then, are nano-switches when they turn on the effector by binding of the alpha subunit and turning it off when the gtp is hydrolyzed. the duration of production of secondary messenger, like cyclic amp, is determined by the rate of hydrolysis. in this sense, the g-protein acts as a nano-timer. although there is controversy over the role of the ß/ subunits in modulation of signals, it is likely that there are both inhibitory and stimulatory effects. if different receptors act on the same g-protein, or if different g-proteins act on the same effector, the potential exists for a "graded" response to an extracellular signal. if the same receptor acts on many g-proteins, or if one g-protein acts on many effectors, then there may be many simultaneous responses to the primary messenger. following binding the g-proteins activates the membrane-bound effector, adenylyl cyclase (ac). this enzyme catalyzes the synthesis of cyclic amp resulting in the formation of atp, camp and pyrophosphate. because this molecule is freely diffusing through the cytoplasm, it is a "secondary messenger" (figure ii-25). the reverse reaction, the formation of atp from camp and pyrophosphate, is catalyzed by a specific phosphodiesterase. camp is involved in a number of physiologic processes. for the breakdown of glycogen, stimulation of the ß-adrenergic receptor involves activation of adenylyl cyclase and synthesis of cyclic amp. the activity of camp-dependent protein kinase (capk) requires camp in order to phosphorylate ser and thr residues on other cellular proteins. glycogen phosphorylase is activated by capk, making glucose-6-phosphate available for glycolysis. adenylyl cyclase activity is regulated at a number of levels, including modulation of gtpase activity of ga, phosphodiesterase activity, and protein phosphatases. inhibitory g proteins, gi, are analogous to the stimulatory g proteins, gs, except for the exchange of gtp by gdp by the -subunit and the subsequent inhibitory action of gia on adenylyl cyclase. rather, it has been determined that ligand induced dimerization is the mechanism through which the receptor ptks are activated. this dimerization brings the tyrosine kinase catalytic domain on each receptor into close enough arrangement so that each kinase can phosphorylate tyr residues in the other's tyrosine kinase domain. such activated catalytic domains can then phosphorylate tyrosines outside of the catalytic domains, which can then modify other intracytoplasmic proteins, either by phosphorylation or by other means. all these changes are reversed with an overexpression of activation when an opioid is antagonized by a specific antagonist such as naloxone with activation of the excitatory nmda-(n-methyl-d-aspartate) receptor, resulting in a rebound with an increase in transmission of stimuli ( figure ii-27) . the next step in the signaling pathway involves activation of an inner membranebound monomeric g protein known as ras, which initiates a series of kinase reactions that ultimately carry the signal to the transcriptional apparatus of the nucleus. ras, being a g protein, is activated when its bound gdp in the resting state is replaced by gtp. it, too, has gtpase activity, but the half-life is too slow to allow for effective regulation of a signal. another gtpase activating protein, gap, increases the rate of gtp hydrolysis by ras. a "kinase cascade" ensues, involving raf (a ser/thr kinase), map kinase (also known as mek, which is both a tyr kinase and ser/thr kinase, and a family of proteins known as mapks or erks. opioids induce a variety of clinical relevant effects, which can be subdivided in being advantageous and/or even detrimental. one of the major consequences following opioid administration is that of analgesia, or antinociception. and while nsaids induce their antiniociceptive effect via cyclooxygenase (cox) inhibition, local anesthetics selectively block ion-channels, thus inhibiting the transmission of nociceptive efferent to the higher pain modulating centers in the cns. contrary, opioids bind to those areas, which not only are involved in transduction but also in the modulation and identification of painful afferences. although the majority of opioids are able to induce a maximal analgesic effect, the dosages necessary to induce such a result differ significantly. for instance, an opioid like sufentanil part ii needs a much lower dosage than the less potent opioid morphine. this is due to the higher affinity and intrinsic activity of sufentanil, suggesting that only a lesser portion of receptors needs to be occupied in order to induce the desired effect. however, a high analgesic potency necessarily does not reflect a better efficacy. this is because in certain painful conditions, some opioids are more efficacious than others. on the other hand, not all painful conditions, as the patient expresses them, can be treated successfully with an opioid. therefore, before starting an opioid therapy it is mandatory to evaluate the kind of painful condition the patient has, use the specific opioids as indicated, and avoid those painful states where opioids are contraindicated or result in a lesser therapeutic effect. however, there is the general position: in intense to severe, excruciating pain, opioids are the sole agents, which are able to induce sufficient analgesia -pain from muscular dysfunction. in patients who present pain of myofacial nature, opioids are contraindicated since they will not result in an alleviation of nociception. due to muscle spasm or an increase in tension physical therapy presents the first defense line in the therapeutic approach. this is accompanied by the administration of a benzodiazepine, which induces a muscle relaxant effect and/or the injection of a corticoid together with a local anesthetic (0.5% bupivacaine) in so-called trigger points ( figure ii-28) . trigger points are typical points which are sore and from which the pain radiates to referred areas. such points can be felt as knots or bumps under the palpating finger, which can be moved over the underlying musculature. following the in injection of the local anesthetic the circulus vituosus of increased muscle tension and myofacial pain is interrupted. local ischemia is alleviated and local accumulation of pain mediating substances is flushed out. a typical example is tension-type headache, which is the moist common type of headache. originating from increased stress, it is accompanied by emotional factors and fear. thus the painful condition can be considered of psychosomatic nature. -pain of neurogenic or deafferentiation origin, also termed as complex regional pain syndrome (crps), this type of pain is mostly seen after injury of peripheral nerves leading to spontaneous and paroxysmal discharges. such pain typically is seen as post-herpetic pain, central pain after stroke, diabetic peripheral neuropathy, phantom limb pain, traumatic nerve avulsion, trigeminal neuralgia, lumbosacral plexopathy, all being circumscribed as neuropathic pain. it originates proximal of the peripheral nociceptor ( figure ii-29) , and characteristically is due to a dysfunction or lesion of the peripheral nerve fibers and/or centrally located nervous structures. typically this type of pain is accompanied by a sensory deficit: it is of a burning, shooting, stabbing, piercing, tearing or electricshock-like, paroxysmal and vice-like nature. this pain is of paresthetic, hypoor hyperesthetic quality often is refractory to any opioid therapy. the causes for such painful conditions may be quite different: compared to a normal situation (top), there is spontaneous ectopic firing from increased sodium-channel activity after peripheral nerve injury (middle), which eventually results in central sensitization with stimulus independent pain (bottom). modified from [36] ectopic spontaneous discharges originating from a lesioned or a cut nerve, this results in an upregulation of sodium-channels being the source of spontaneous discharge. continuous nociceptive barrage later results in central sensitisation and "wind-up" (figure ii-29 ). partial denervation with spontaneous discharge of nerve activity is followed by an induced release of a nerve growth factor (ngf). such release induces sprouting of fibers into adjacent afferent somatic nerve fibers resulting in an enlargement of the receptive field and an increased conduction of nociceptive impulses to higher pain centers ( figure ii-30 other topical formulations with capscaicin or emla cream present additional therapeutic options for treatment of neuropathic pain. in addition, transcutaneous electrical stimulation (tens) or even spinal cord stimulation (scs) may present an alternative and effective strategy, resulting in the attenuation of pain. in the latter technique, analgesia is induced by the electrically induced release of endogenous opioids (enkephalins, endorphins, dynorphin) in the spinal cord and within the hypothalamus activating the descending serotoninergic and noradrengergic pathways. -opioids in visceral painful condition. another type of pain, which cannot be treated sufficiently with an opioid, is visceral pain. such a painful condition may arise from the intestine (e.g. the irritable bowel syndrome or ibs) or pain emerging from other internal organs such as the gall bladder, the urinary tract or pain following an appendectomy, cholecystectomy or hysterectomy ( figure ii-33 ). due to the participation of smooth muscles in such a condition a peripheral analgesic with a muscle relaxant effect can be of advantage. because the sympathetic nervous system to a major part is involved in such a condition, therapeutic implications incorporate a ganglionic blocker, surgical sympathectomy, or intravenous conduction anesthesia with guanethidine. -psychosomatic painful conditions, if treated with an opioid, in the long run are bound to end in opioid resistance. such a painful condition is mainly seen in the depressive patient or it may even be a premonitoring sign of schizophrenia. however, pain can also be part of a conversion-neurotic syndrome [37, 38] , where aside from pain fear, phobia, and obsessive-compulsive symptoms are the dominant elements. painful conditions being sensitive to opioids all agonizing painful states, which are due to • posttraumatic • postoperative • ischemic, or of • tumor origin, can be treated sufficiently with an opioid. the rational for the therapy with a central analgesic is related to the fact, that the nociceptive impulses are transmitted via specific pain afferents reaching supraspinal areas. by blockade of specific receptor sites within the brain, opioids induce a reduction and/or result in a total blockade of nociceptive impulses. opioids therefore present the main line of defense in all medium to severe painful conditions. when using opioids one has to realize that this group of ligands, besides their beneficial analgesic effect at the same time also induces a detrimental respiratory depression. this is a mayor drawback when using opioids in acute pain and is directly proportional to their analgesic potency. for instance a potent opioid such as fentanyl already is able to induce respiratory depression in the lower dose range. however, a less potent opioid like codeine or tramadol, even when given in dosages higher than their therapeutic margin, will not induce a clinically relevant respiratory depressive effect ( figure ii-34) . because opioids given for alleviation of chronic pain are given orally and in a controlled release formulation, there is no acute rise in opioid plasma level, which otherwise would induce respiratory impairment. in addition, chronic pain patients cannot be considered as being opioid naïve. their respiratory center already has developed some degree of habituation, being less sensitive to the opioid agent. those opioid ligands, which inherit a lesser respiratory depressive effect, however, are characterized by a comparable reduced analgesic potency. also, a pure -type ligand such as morphine, fentanyl or sufentanil is characterized by a doserelated decrease in respiration until total apnea becomes apparent. contrary, the potent partial agonist buprenorphine with increasing doses demonstrates a ceiling effect, which is seen at a dose of 2 g/kg ( figure ii-35) . typically, when administering high dosages of potent -type ligands such as fentanyl or alfentanil, a time related sequence of effects on respiration can be observed. the progression of respiratory depression is a characteristic trait, which develops within seconds to minutes: 1. a reduction in respiratory rate (bradypnea) with a partial compensation of tidal volume. 2. a respiration, which is only kicked off by external stimuli, such as noise or pain. changes in tidal volume (liter/min) in subjects being exposed to increasing dosages of the pure -type ligand fentanyl and the partial agonist buprenorphine. note, the ceiling effect in respiratory depression at higher doses of buprenorphine. adapted from [22] 3. a short period where respiration is forgotten, originally termed in europe as "oublie respiratoire", as it was observed in the early times of neuroleptanesthesia when using fentanyl together with a neuroleptic agent for the induction and maintenance of anesthesia. at this stage, however, the patient can be ordered to take deep breaths. 4. the total apnea, where in spite of any external stimuli or the command to breathe the patient spontaneously will not be able to take a deep breath. he needs immediate respiratory assistance. this centrally-induced opioid-related respiratory depression is due to a blockade of the respiratory regulating centers within the brain stem (pons and medulla), resulting in a lesser sensitivity to an increase in arterial pco 2 and/or a reduction of arterial po 2 [39, 40] . in addition the activating reticular system (ars), which descends down into the brain stem, acts as a regulatory pacemaker for the inspiratory center, by which respiratory depressive effect of opioids are affected. this is reflected in the clinics when in addition to an opioid a benzodiazepine is added, which by depressing vigilance, results in an immediate cessation of respiration [41] . any opioid-induced respiratory depression instantaneously and effectively can be reversed by the administration of a specific opioid antagonist such as naloxone. because of the higher affinity of the antagonist, naloxone displaces the agonist from the receptor site (competitive antagonism), and after binding respiratory depression is reversed and normal ventilation is instigated ( figure ii-36) . clinically, an opioid-related respiratory depression is reversed by titrating the dose of naloxone necessary to • initiate a sufficient spontaneous respiration, however • avoiding an acute abstinence syndrome with tachycardia and hypertension, and at the same time • remaining a sufficient level of analgesia during reversal one should consider the half-life of naloxone, which is between 20 and 30 min [42, 43] . therefore "remorphinisation" with a reoccurrence of respiratory depression may appear if the half-life of the agonist is longer than the antagonist, or if high concentrations of the agonist are still circulating in the blood plasma [44] . following successful reversal it therefore is mandatory to administer an additional dose of naloxone intramuscularly, which acts like a depot, or hook the patient up to a continuous intravenous drip of a naloxone solution, sufficient for long-term receptor occupation by the antagonist. all these procedures, however, do not replace the need for a continuous surveillance, which is necessary in order to detect any possible gradual development of respiratory impairment. respiratory depression can also be reversed by a mixed agonist/antagonist such as nalbuphine. although being less potent than naloxone, it however is one of the mixed ligands having a sufficient antagonistic potency (table ii-8) . at the same time the ligand has a 3-fold longer duration of action than [47, 48] , while the lesser antagonistic potency results in a more gradual and not in abrupt displacement, resulting in lesser sympathetic overdrive [49] ( figure ii-37) . another pure antagonist, nalmefene shows the longest duration of antagonism with up to 8 h of action [51] . because of its high antagonistic potency (2.5-fold of naloxone) an acute abstinence syndrome can be induced if the necessary dose is not titrated to patients need [52] . it had been proposed that different receptor sites in the cns mediate opioid-related analgesia and respiratory impairment [53] . such difference has also been demonstrated for fentanyl-analogues in the animal [54] , suggesting a clinical relevance for reversal of respiratory impairment, however at the same time maintaining antinociception. such connotation was further corroborated by experimental data where the selective antagonist naloxonazine was able to reverse opioid induced analgesia, but not respiratory impairment. this led to the assumption that -opioid subreceptors are involved in the mediation of opioid-induced respiratory depression (i.e. 1 and 2 ) [55, 56] . clinical data seem to underline this assumption, as low doses of sufentanil demonstrated a lesser respiratory depressive effect and a higher analgesic potency when compared to fentanyl. such difference in action reportedly is due to a disparity in receptor affinity to -subsites, with a higher affinity to the 1 -and a lesser affinity to the 2 -receptor [57] . other experiments, however, suggest that the co-binding of -selective ligands to the -receptor results in respiratory impairment. subanalgesic doses of the -selective ligand d-ala 2 -d-leu-enkephalin, when co-administered with morphine, induced a potentiation of analgesia, while another -ligand d-ala 2 -met-enkephalinamid produced a reduction in analgesia [58] . such -related differentiation in efficacy was also seen with the potent ligand sufentanil. there respiratory impairment was reversed while at the same time maintaining antinociception using the highly selective -antagonists naltrindol and naltribene respectively [59] ( figure ii-38) . the implication of / -receptor interaction is further supported by receptor binding studies, where sufentanil demonstrates higher -selectivity than fentanyl (table ii-9) . such putative interaction between -and -receptors is further corroborated when co-administering intrathecally a -and a -selective ligand resulted in a potentiation of effects [61] . from such data it can be concluded that a coupling mechanism between the -and -opioid receptor not only seems to result in an increase in analgesia, but at the same time also seems to cause respiratory depression. such coupling mechanisms may result in a modulation to potentiation of effects whereby it is still uncertain whether both sites independently operate from each other or whether the -receptor only accentuates the effects induced by -binding. besides a direct action of opioids on the sensitivity of the respiratory center to changes of arterial po 2 and pco 2 , also centrally-induced sedative effects very likely influence respiration. such sedative effects can be derived with the aid of the electroencephalogram where clinically different potent opioids qualitatively induce a different in the eeg pattern. since such eeg changes are dose-related, one is able to derive a dose-relationship. at the same time such eeg-changes reflect the bioavailability of centrally active agents acting on nervous structures of the cns, depicting the effect-concentration site [62, 63] . thus, following intravenous administration of an agent, it is not the plasma concentration, which is responsible for a centrally induced effect. more importantly, it is the actual concentration of the opioid at the receptor site, which is affected significantly by issues such as distribution of an agent, its lipophilicity, or the present brain perfusion. therefore vigilance changes can be considered as important aspects in an opioidrelated respiratory impairment being derived in two relevant experiments: 1. wakefulness by itself already is a fact resulting in sufficient respiration. this could be demonstrated nicely in volunteers where hyperventilation and the resultant hypocapnia resulted in a rhythmic respiratory pattern. if however, the same volunteers were asleep or in anesthesia, hypocapnia was followed by apnea [64] . 2. in the animal laryngeal stimulation during anesthesia resulted in apnae, without, however, initiating a cough reflex. being awake, a cough reflex without apnae was induced following laryngeal stimulation [65] . 3. there is a close exponential correlation of the physiologic regulatory mechanism affecting respiration. this had been demonstrated after sufentanil application in the canine, whereby increasing dosages of a selective antagonist not only dose-related reversal of sufentanil-induced hypercarbia and hypoxia with the two selective -antagonists naltrindol (nti) and naltribene (ntb) respectively, in the canine. due to the higher lipophilicity of naltribene being able to pass through the blood-brain barrier, there is a superior reversal effect. in spite of increasing doses of the antagonist there is a blockade of response to the electrically induced evoked potential, which is only reversed by the highly specific -antagonist cyprodime. adapted from [60] reversed the depressed respiratory drive but at the same time induced an increase of power in the high frequency beta band (13-30 hz) of the eeg ( figure ii-39) , reflecting increase in vigilance [66] . 4. clinically such sedative related respiratory depression can also be derived in patients, when cumulative dosages of an opioid reach a point where the respiratory center "forgets" to respond adequately by initiating deep breaths (oublie respiratoire). this is seen in classical neuroleptanalgsia where the patient's vigilance can be increased to a point by external stimuli (e.g. pain, auditory stimuli) resulting in the initiation of an inspiratory effort [67] . from all these data it can be derived that the simultaneous binding of opioid within the activating reticular system (ars) in the brain stem, vigilance is depressed, which secondarily affects the response of the respiratory center following hypercapnia. at such instances the overall mesencephalic reticular control mechanism is no longer able to adequately respond to a stimulus and only with an increase in vigilance there is an accelerated reactivity, being able to sufficiently respond to an increase in arterial pco2. since the reticular mechanism is coupled with reticulo-cortical afferences, such changes can be derived from cortical changes in the eeg. such a "forgotten" reaction to sufficiently respond to a given stimulus [65] is also seen in the clinical environment when a benzodiazepine is given on-top of an opioid resulting in a further deterioration of respiratory drive. this is because a benzodiazepine depresses the reaction of the ars, and the concomittant reduction in vigilance results in a lessened reaction to external stimuli, producing a clinically relevant suppression of respiration. in general prolongation of respiratory depression after opioid administration has to be expected when the following agents are coadministered: 1. all agents that inhibit the biotransformation of opioids such as contraceptives, anti-tumor agents, anti-arrhythmics, antidepressants, systemically administered antimycotics, neuroleptic drugs, and volatile anesthetics [68, 69, 70, 71, 72] . by inhibition of conjugation of glucoronide and oxidative dealkylation, the necessary metabolic pathways for degradation and termination of activity of most agents, a prolongation of action has to be expected. 2. all agents, which are able to displace the opioid from protein binding within the plasma, resulting in a higher portion of the pharmacologically active agent. preparations such as cumarine derivatives, and phenylbutazone, which when coadminstered are prone to result in a prolongation of effects [73, 74, 75, 76] . 3. in addition, hypoproteinemia and acidosis of the blood, both of which result in lesser protein binding, cause a higher concentration of non-bound opioid in the blood plasma. such increase in plasma concentration now is able to bind to the receptor site with an increase of efficacy and a longer duration of action [77] . following opioid-based anesthesia, several factors cause an overhang of opioid action, which may even result in a "re-morphinisation" and the re-occurrence of respiratory impairment: 1. the excessive intramuscular premedication with an opioid, which may act like a depot. 2. the premedication with a long-acting benzodiazepine, which is able to induce a reduction in vigilance lasting into the postoperative period. 3. the uncritical intraoperative use of high concentrations of a volatile anesthetic, which results in a lesser biodegradation of the opioid. 4. the intraoperative administration of fractional doses of an opioid, which results in an accumulation. due to the fact that a portion of each dose of an intravenously administered opioid is also taken up by peripheral sites (e.g. fatty tissue, musculature, skin, internal organs) there is an accumulation of the agent, which act like a depot. from there the drug later diffuses into the blood-stream, resulting in a prolongation of effects ( figure ii-40 ). 5. an insufficient loading dose of the opioid, which may result in the necessity of re-administration of small amounts of the drug intraoperatively with consequent peripheral accumulation. 6. long-term intravenous administration of an opioid by drip, resulting in the increase of the agent in the peripheral compartment with later recirculation into the blood stream. 7. the combination of opioids with different half-lifes, which may result in an unforeseen potentiation of effects. 8. uncritical administration of bicarbonate resulting in alkalosis of the blood, which induces a faster release of the opioid from the peripheral compartment. 9. a non-corrected hypovolemia, which coincides with lesser protein binding of the agent and a higher portion of the free active compound. 10. uncritical use of a selective antagonist such as naloxone, not considering that its half-life is shorter than the agonist, resulting in a later reoccurrence of respiratory impairment. the sedative effect of opioids goes in hand with their capability to induce sleep (lat. hypnos). such an effect is mostly seen with the mixed agonist/antagonists, while morphine takes a medium position ( figure ii-41) . the hypno-sedative effect of opioids is useful in premedication and during postoperative analgesia, where a sedated status of the patient is advantageous. in contrast to a potent sedative nature of mixed agonist/antagonists, the pure -type ligand fentanyl is characterized by a very low sedative potency. when in the beginning of use of neuroleptic analgesia for anesthesia, fentanyl was used together with the neuroleptic agent droperidol, often patients reported of intraoperative "awareness". although being an obligatory part of anesthesia, sleep, was not sufficiently maintained throughout the whole procedure. therefore in order to guarantee a sufficient level of sleep in patients receiving a fentanyl-based anesthetic technique, an additional hypnotic (propofol), a benzodiazepine (midazolam), a neuroleptic agent (i.e. dehydrobenzperidol), or a volatile anesthetic (sevoflurane, desflurane, or enflurane) has to be given on-top the opioid. nowadays the problem of awareness again has gained much attention [78] , since the technique of total intravenous anesthesia (tiva) with remifentanil and [84] propofol, completely omitting nitrous oxide (n 2 o), often results in an insufficient level of sleep with awareness. typically pure -ligands such as bremacozine and tifluadom (table ii-3) , which in comparison to morphine have a 2-fold analgesic potency [79, 80] , do not induce a respiratory depressive effect [81] . their lack in respiratory impairment is due to the selective binding in deep layers of the cortex [19, 82] , where in comparison to the brain-stem, a more than 50% higher concentration of -binding sites is found [3, 83] . their predominant sedative effect is due to centripetal fibers descending down from deep layers of the cortex to the thalamus, thus decreasing the nociceptive input [20] . although having the advantage of an increased sedative effect combined with the lack in respiratory impairment, clinically, the use of -ligands had to be abandoned. this is because of their intense dysphoric side effects, which lasts for several hours. in addition, their analgesic potency, in comparison to pure -ligands is much lower. therefore such agents cannot be regarded as suitable for intraoperative use, where an intense nociceptive barrage can only be blocked by a potent -opioid. only the mixed agonist/antagonists (e.g. nalbuphine, butorphanol), which exert their analgesic action through binding at the -site, currently are in clinical use mainly for postoperative analgesia [85, 86] . this is because cumulative dosages, contrary to a typical -ligand like morphine, result in a ceiling effect for respiratory depression ( figure ii-42 ). in addition, because of their wide margin of safety, high dosages part ii figure ii-42. ceiling effect of respiratory depression following cumulative doses of the mixed agonist/antagonist nalbuphine. in contrast to morphine, at a certain dose there is no further increase in the degree of respiratory impairment. adapted from [89] have been advocated in balanced anesthesia where the opioid resulted in an up to 70% reduction in mac (minimal alveolar concentration) of the volatile agent [87, 88] . opioids in general induce a dose-related hyposedative component, which is mirrored in the electroencephalogram by an increase of activity in the slow -with concomitant decrease of power in the fast -domain. however, when giving a large bolus dose of fentanyl (7-10 g/kg body weight) alfentanil (50 g/kg body weight) morphine (3-10 mg/kg body weight) or sufentanil (2-3 g/kg body weight) an immediate dominance of delta-waves in the eeg becomes evident, being accompanied by sleep. for instance, such effects clinically are seen when high-dose opioid anesthesia is used in cardiac patients for the induction of anesthesia. such a sleepinducing effect is due to a short-term blockade of all afferences being switched in the activating reticular system (ars) of the mesencephalon. aside from a blockade within the nucleus limitans a deep level of analgesia is initiated [90] . such a "narcotic component", with dominance of delta-activity in the eeg, and contrary to equi-analgesic doses of fentanyl, it is more apparent after sufentanil [91] , which makes this agent more suitable for the induction of cardiac patients ( figure ii-43) . following induction with a potent -ligand such as fentanyl or sufentanil the initial "narcotic component" later transforms into a "pure analgesic component". this is because the opioid is redistributed, which results in a lesser concentration within the cns and a lesser binding in areas within the ars. at this stage there is a note, a pronounced delta activation after injection and a lesser arousal reaction induced by laryngoscopy and intubation, which in the sufentanil group reflected in a lesser decline of power in the slow delta-domain of the eeg. adapted from [92] dominance in the -band (7-13 hz) of the eeg, which is stable, not being affected by any nociceptive stimuli [93, 94] . clinically, such an effect has been described for the precursor of fentanyl, the opioid phenoperidine [95] and for fentanyl [96] . after a period of 10-15 min the deep narcotic component changes into a sedative state, which is stable and cannot be reversed to desynchronization by any nociceptive stimulus. during such "analgesic state" the patient again is able to respond to verbal commands, while at the same time having a deep analgesic level ( figure ii-44) . without the addition of nitrous oxide, such patients are awake, however, nociceptive afferents are not able to modulate the ars, the endotracheal tube is tolerated while at the same time nociception is only sensed as a touch. such phenomena are due to afferents ascending along the spinothalamic tract, which directly ascend to the postcentral cortical area by which the impulse can be localized. collaterals, which ascend through the nucleus limitans within the limbic system and convey nociception, are sufficiently blocked by the opioid and the patient does not perceive pain ( figure ii-45) . the opioid receptor system bordering the fourth cerebral ventricle and the underlying activating reticular system is the relevant anatomical structure in mediating sedation. selective perfusion of increasing concentrations of the opioid fentanyl in the awake canine induced a dose-related enlargement of slow-wave high amplitude delta-activity within the eeg, characterized by a sleep-like behavior ( figure ii-46) . this effect was reversed by the levo-isomer of naloxone inducing an arousal reaction. it, however, was not reversible by the dextro-isomer of the antagonist [98] . the physiological significance of opioid receptors in the control of vigilance is also reflected in the high density of opioid binding sites in the mesencephalon [99] . physiologically this is mirrored by an arousal reaction following intense acoustic or a nociceptive stimulus, both of which induce a reversal from the low frequency delta-to high frequency beta-activity in the eeg ( figure ii-47) . in summary, it is concluded that opioids primarily affect the limbic system, the specific site for inducing the negative component of nociception. lastly such assumption is underlined by the result from mc kenzie and coworkers in the animal where the opioids morphine and pethidine were not able to sufficiently block any pain related nervous transmission from the mesencephalon to the higher cortical areas [100] . in contrast, both ligands were able to block nociceptive transmission from the mesencephalon to hippocampal areas of the limbic system, the part of the cns, which is responsible for the identification of pain, causing the negative, grief, stinging and an intense emotional feeling associated with pain ( figure ii-48) . such differences in pain modulation were corroborated in patients undergoing stereotactic, painful stimulation within specific areas of the cns [101] . nociceptive afferents of the spinothalamic tract end in the nucleus ventrocaudalis parvo-cellularis part ii figure ii-46. selective perfusion of increasing doses of fentanyl through the fourth cerebral ventricle of the awake canine. note, the direct sedative (delta-synchronisation in the eeg) effect via the underlying ars. this effect is mediated through opioid receptors located on the floor of the ventricle, since it was reversible with naloxone (desynchronisation with beta activation in the eeg). adapted from [98] thalami, from where they further ascend to different cortical areas. since these nuclei reflect a specific somatotopic differentiation, electrical stimulation within this area induced painful sensations in different parts of the body. decoding of painful afferents was only possible when the stimulating electrode was placed within the nucleus limitans, where collaterals of the spinothalamic tract switch to the limbic system. there stimulation induced a less well-localized, however, intense unspecific displeasure [102] . following the administration of high dosages of opioids with different potency, epileptogenic activities in the eeg with tonic-clonic seizures can be induced in the animal. pethidine (meperidine), morphine, alfentanil, fentanyl and sufentanil when administered in doses above 20, 180, 5, 4 and 4 mg/kg body weight respectively, induced epileptogenic discharges [103] . because such massive dosages are never used in anesthesia or for analgesia in acute or chronic pain, epileptogenic effects, although being cited in the literature after fentanyl [104, 105] and sufentanil [106] are of insignificant nature. this is because the clinical picture resembles tonicclonic seizures, however, in the eeg no such discharges could be derived [107] . therefore, those high doses of opioids, which induced epileptogenic activity in the rat [108] or the canine [103] are far off from therapeutic range. thus, in general, an epileptogenic activity of opioids can be canceled out. one exception is the use of high dosages of pethidine (meperidine), where the metabolic product norpethidine is a potent epileptogenic compound, which especially in the newborn is able to induce epileptogenic activity [109] . the cause for the few observations of a pseudoepileptogenic activity of opioids when being administered within the therapeutic range very likely is due to a desinhibition of the cortical motor center within the cns, as this phenomenon was observed during the induction of anesthesia or following a decline in plasma concentration. such assumption is underlined by part ii figure ii-48 . two main components of painful afferents: localization of nociception (cortical area) and the initiation of the negative component (i.e. the limbic system within the mesencephalon). opioids mainly modify the latter area "epileptogenic activity" during the induction of anesthesia with the pure hypnotic etomidate, where desinhibition of the motor cortex activity was the cause of cortical discharges [110] . each cough involves a complex reflex arc beginning with the stimulation of sensory nerves that function as cough receptors. there is evidence, primarily clinical, that the sensory limb of the reflex exists in and outside of the lower respiratory tract. although myelinated, rapidly adapting pulmonary stretch receptors (rars), also known as irritant receptors, are the most likely type of sensory nerve that stimulates the cough center in the brain, afferent c-fibers and slowly adapting pulmonary stretch receptors (sars) also may modulate cough. rars, c-fibers, and sars have been identified in the distal esophageal mucosa; however, studies have not been performed to determine whether they can participate in the cough reflex. although gastroesophageal reflux disease can potentially stimulate the afferent limb of the cough reflex by irritating the upper respiratory tract without aspiration and by irritating the lower respiratory tract by micro-or macroaspiration, there is evidence that strongly suggests that reflux commonly provokes cough by stimulating an esophageal-bronchial reflex. each involuntary cough involves a complex reflex arch beginning with the stimulation of sensory nerves in the airway epithelium that function as "cough receptors." efferent impulses from these receptors are conducted by means of the vagus nerve to the "cough center" in the brain stem. because cough can be voluntarily suppressed, controlled, or initiated, there also can be afferent input from the cerebral cortex. the function of this "cough center" is to receive these impulses and produce a cough by activating efferent nervous pathways to the diaphragm and laryngeal, thoracic, and abdominal musculature. the possibility that there might be afferent input other than the vagus nerve and cerebral cortex was based on clinical observations described in case reports and a few animal studies [111] . histologic studies of the respiratory tract in both animals and humans have revealed sensory nerve endings within the basal layer of the epithelium and between epithelial cells of the larynx, trachea, and bronchi [111] . these nerve endings are thought to be cough receptors. they contain neuropeptides, such as substance p and calcitonin-gene related peptide (cgrp), which mediate neurogenic inflammatory events in the airways. these sensory nerve endings have been found to be most numerous in the posterior wall of the trachea, at branching points of large airways, and less numerous in the more distal, smaller airways. none have been found beyond terminal bronchioles. it is not known for certain which type of afferent nerve mediates cough. a model that summarizes the current understanding of cough is schematically depicted in the figure. it shows the myelinated, rapidly adapting pulmonary stretch receptors (rars), also referred to as irritant receptors, as the most likely type of sensory nerve that stimulates the cough center in the brain. both mechanical and chemical stimulation of rars have been shown experimentally to cause cough. another type of sensory nerve in the airways, c-fibers, may also participate in regulating cough. they are unmyelinated, vagal afferent fibers that may be activated by the same triggers as rars. their activation releases neuropeptides locally that may secondarily stimulate cough by activating rar nerves. however; impulses transmitted by c-fibers alone probably do not stimulate cough, because experimental evidence has shown that they inhibit cough centrally in the brain. a third type of sensory nerve, the slowly adapting pulmonary stretch receptors (sars), may modulate cough. although these nerves do not directly respond to chemical and mechanical triggers, they do appear to be activated by the deep breath of a cough and may enhance cough by making the expiratory effort more forceful. in addition to mechanical and chemical stimuli, cough has been caused in animals by thermal and electrical provocation. the sites most sensitive to all stimuli are the larynx, trachea, and cannulae of the larger airways. outside of the lower respiratory tract, cough receptors have been demonstrated histologically only in the hypopharynx [111] . however, it has been inferred from clinical studies that sensory nerve endings subserving the cough reflex via the vagus nerve probably exist in the extemal auditory canals and eardrums, hypopharynx, pericardium, stomach, and esophagus, because stimulation of these sites has been reported to cause cough [111] . based on the fact that cough can be voluntarily initiated, postponed, and/or suppressed, this provides evidence that there also can be afferent input from the cerebral cortex. in addition to directly stimulating cough by carrying impulses from cough receptors to the cough center, vagal afferents may indirectly provoke cough by another mechanism. they may stimulate neurotransmitter release or mucus secretion from airway submucosal glands that, in turn, stimulate the cough reflex [111] . the existence of a discrete central cough center is controversial. what is known is that afferent pathways first relay impulses to an area in or near the nuc1eus tractus solitarius. these impulses then are integrated into a coordinated cough response in the medulla oblongata of the brain stem, probably separate from the medullary centers, which control breathing. although electrical stimulation studies of different areas in the medulla have evoked cough in animals, suggesting that the cough center is diffusely located [111] a discrete cough center still may exist, because these electrical stimulations may have activated afferent pathways of the cough reflex. the motor outputs trom the cough center are in the ventral respiratory group, with the nucleus retro-ambigualis sending impulses via motoneurons to the respiratory skeletal muscles and the nucleus ambiguus sending impulses to the larynx and bronchial tree. more specifically, the efferent impulses of the cough reflex are transmitted from the medulla to the expiratory musculature, through the phrenic nerve and other spinal motor nerves, and to the larynx through the recurrent laryngeal branches of the vagus nerves ( figure ii-49) . vagal efferents also innervate the tracheobronchial tree and mediate bronchoconstriction [111] . although stimulation of cough and bronchoconstriction can be experimentally separated using nonpermeant anions to stimulate cough without bronchoconstriction, these two phenomena normally are activated simultaneously to facilitate the most effective cough. bronchoconstriction may improve clearance of secretions by narrowing the cross-sectional area of the airways, thereby increasing the velocity of air leaving the patients lower respiratory tract during the expiratory phase of coughing [111] . experimentally, it has been shown in animals that the efferent pathways of the cough reflex are anatomically distinct and separate from the efferent pathways of normal spontaneous ventilation. blockade of the cough reflex arch by means of opioids, known as the antitussive action, refers to the fact that they suppress this protective reflex. this is of benefit during anesthesia and/or in patients being artificially ventilated in the intensive care unit (icu) because it results in the tolerance of an endotracheal tube. however, the antitussive potency differs significantly among the various opioids ( figure ii-50) . the action is not related to a specific receptor site, because a stereoselective action of opioids in regard to their antitussive effect could not be demonstrated. in addition, reversal with the selective antagonist naloxone is less selective [112] . the mode of action is a blockade of the cough center within the brainstem. three of the most commonly used suppressors of the cough reflex are hydrocodone, codeine and hydromorphone. all of them are characterized by low analgesic potency, they model for afferent limb of cough reflex in airways. myelinated. rapidly adapting pulmonary stretch receptors (rars) and unmyelinated c-fibers are sensory nerves that participate in the afferent limb of the cough reflex. mechanical and chemical stimuli activate sensory nerve enelings in the epithelial layer. rars appear to be the main type of sensory nerve stimulating cough centrally. although c-fibers may inhibit cough centrally, neuropeptides released in the periphery upon stimulation of c-fibers may indirectly stimulate cough by activating rars. slowly adapting pulmonary stretch receptors (sars) do not respond to irritant stimuli that initiate cough but may enhance cough centrally by making expiratory muscular effort more forceful demonstrate a negligible dependence liability, and they are common components in doc (drugs over the counter) cough medicine. a similar antitussive potency, however, is also seen with the more potent opioids such as diamorphine, fentanyl or sufentanil. the latter are used in an opioid-based anesthetic regimen or in icu patients who are in need of ventilatory support. when a potent opioid such as sufentanil is used, the patient is adapted much easier to the respiratory cycle of the ventilator resulting in lesser doses of additional sedative agents. morphine in this regard has a much weaker antitussive activity while pethidine (meperidine) and all mixed agonist/antagonists are characterized by a negligible antitussive action ( figure ii-50) . it can be summarized that potent opioids also inherit a marked antitussive effect, while weak opioids and especially the mixed opioid analgesics are unable to sufficiently suppress the cough reflex. during the induction of anesthesia, while injecting an intravenous bolus dose of a potent opioid such as fentanyl or sufentanil, often a cough reflex is initiated. such part ii dependence or addictive liability (how addictive a drug is likely to be) depends upon how quickly the drug enters/leaves the brain. also, it is directly proportional to the analgesic potency and it depends on the opioid receptor sites with which the ligand interacts. for instance, members of mixed agonist/antagonists (i.e. nalbuphine, pentazocine, butorphanbol) demonstrate a predominant interaction with the -opioid receptor, which is characterized by a low dependence liability ( figure ii-51 ). in addition, development of dependence also is related to the speed for instance, an opioid like buprenorphine is characterized by a low dissociation constant reflecting long binding to the receptor, a long duration of action and a slow separation from the receptor. the definition of addiction is based in two different terms: 1. physiological dependence produced by repeated drug-taking that is characterized by a withdrawal syndrome, when drug is removed (e.g. alcohol, opiates). 2. psychological dependence produced by repeated drug taking that is characterized by obsessions and compulsive drug-seeking behaviors; results in a detrimental impairment in physical, mental or social functioning. there are five classes of abused psychoactive drugs 1. opioids produce a dream-like state; effects include: analgesic (reduction in pain), hypnotic (sleep inducing), euphoria (sense of happiness or ecstasy) using morphine, heroin, or the cough suppressant codeine. 2. depressants produce feelings of relaxation/sedation and a dream-like state, anxiolytic (anxiety-reducing) and hypnotic effects; reduce central nervous system activity. members of the class are alcohol, barbiturates, and benzodiazepines. 3. stimulants increase alertness, arousal, and elevated mood; activate central nervous system (sympathomimetic = mimic the activation of the sympathetic nervous system). members of this class are cocaine, amphetamines, nicotine, caffeine. 4. psychedelics produce distortions of perception and an altered sense of reality. typical representatives are lsd, psilocybine, mescaline, mdma (ecstasy), and pcp (phencyclidine). 5. marijuana with one of its major active ingredient thc produces feelings of well-being and sense of acuity (sharpness). it also can produce feelings of relaxation. also, it is recognized, that opioids, which demonstrate a fast onset of action or due to their galenical preparation (injection, smoking) result in an immediate high plasma concentration, followed by an instantaneous receptor binding, results in a "kick" with an euphoric feeling. such preparations therefore are more prone to induce a behavior pattern of abuse. therefore addictive liability or how addictive a drug is likely to be, depends upon how quickly the drug enters/leaves the brain (table ii-10) . most importantly, the tendency of opioids to result in an abuse is very much linked to the fact of why and when the opioid is ingested. for instance, an opioid taken only for the mere pleasure will rapidly result in the development of dependency. contrarily, if an opioid is taken for the attenuation of pain, the likelihood to develop an abuse behavior is very low. aberrant drug related behavior has to be suggested when the following signs of abuse are obvious: in physical examination. a routine physical examination can elucidate common complications of heroin use or assist in diagnosing opioid dependence. chronic intravenous use can be confirmed by the presence of "track" marks, which are callouses that follow the course of a subcutaneous vein. these are caused by repeated injections into adjacent sites over an accessible vein. tracks are often found in easily accessible body areas, such as the backs of the hands, antecubital fossae, on the legs, or in the neck. signs of recent injection may be found in unusual places in patients attempting to hide their sites of injection. a thorough examination for tracks or recent injection sites should include looking between the table ii-10. factors that influence how quickly a drug will enter the brain 1. chemical structure: how fatty is the drug (i.e. its lipophilicity)? does the drug have nutrients that our brain uses? 2. how fast does the drug cross the blood brain barrier (bbb) lipophilic drugs (i.e. heroin, fentanyl) cross the bbb much faster than hydrophilic agents (i.e., morphine); they mimic nutrients our brain needs and can "slip" through transporters in the bbb. 3. route of administration: is the drug entering directly into the blood stream or is it entering first into the stomach? the routes of administration increase the likelihood that a drug enters the blood stream whereby it increases the addictive liability of the drug. intravenous injection > smoking/snorting > sublingual application > inhalation via nostrils and lungs because they have abundant blood capillaries and more blood supply under the tongue and the lung respectively. fingers and toes, under the fingernails and toenails, in the axillae, breast veins, and the dorsal vein of the penis. one complication of drug use that can be found on examination is nasal septal perforation from repeated intranasal insufflation (especially when cocaine is mixed with heroin and snorted). a heart murmur may indicate subacute bacterial endocarditis, a complication of intravenous injection without using good sterile technique. posterior cervical lymphadenopathy may suggest early viral infection, especially with hiv. hepatic enlargement may indicate acute hepatitis; a small, hard liver is consistent with chronic viral hepatitis due to hepatitis b or c virus, which are common among injection drug users who share needles. signs of opioid intoxication may include pinpoint pupils, drowsiness, slurred speech, and impaired cognition. signs of acute opioid withdrawal syndrome include watering eyes, runny nose, yawning, muscle twitching, hyperactive bowel sounds, and piloerection. on the other hand the following behaviors are more suggestive of an addiction disorder: • selling prescription drugs • prescription forgery • stealing or "borrowing" drugs from others • injecting oral formulations • obtaining prescription drugs from nonmedical sources • concurrent abuse of alcohol or illicit drugs • multiple dose escalations or other non-compliance with therapy despite warnings • multiple episodes of prescription "loss" • repeatedly seeking prescriptions from other clinicians or from emergency rooms without informing the prescriber or after warnings to desist • evidence of deterioration in the ability to function at work, in the family, or socially that appear to be related to drug use • repeated resistance to changes in therapy despite clear evidence of adverse physical or psychological effects from the drug the following behavior pattern is less suggestive of an addiction disorder. • aggressive complaining about the need for more drugs. • drug hoarding during periods of reduced symptoms. • requesting specific drugs. • openly acquiring similar drugs from other medical sources. • unsanctioned dose escalation or other noncompliance with therapy on one or two occasions. • unapproved use of the drug to treat another symptom. • reporting psychic effects not intended by the clinician. • resistance to a change in therapy associated with "tolerable" adverse effects with expressions of anxiety related to the return of severe symptoms. and while addiction is characterized by a compulsive drug-using and drug-seeking behavior that interferes with normal functioning and causes use of the drug despite increasingly damaging consequences, there are different mechanisms from dependence as addicts can experience intense cravings for drugs even years after sobriety (after body set-points, etc. should have adjusted back to normal). the nervous pathways involved in the development of dependence is the mesolimbic-dopaminergic reward system, where direct activation (i.e. cocaine, nicotine, alcohol) or inhibition of the inhibitory gabaergic neurons in the ventral tegmental area (vta) directly project to dopaminergic neurons in the nucleus accumbens (i.e. opioids such as heroin) results in an increased release of dopamine in the nucleus accumbens and stimulation of the prefrontal area resulting in euphoria. on the other hand, insufficient release of dopamine in this area results in dysphoria, which is seen in abstinence ( figure ii-52 ). in the acute phase, opioids inhibit adenylyl cyclase and camp. over time, the downstream transcription factor (creb) is activated which increases adenlyly cyclase production ( figure ii-53) . chronic opioid use leads to upregulation of camp and creb, directing to tolerance, dependence and withdrawal symptoms. the significance of intracellular changes in creb is supported by data in mice without creb, which are less likely to develop addiction. also, recent research has also implicated an opioid peptide, dynorphin, in this pathway. besides initial changes in creb, chronic administration of an opioid results in the induced increased formation of peptides syntheses fosb within the nucleus accumbens. such overexpression of fosb increases the sensitivity to cocaine and opioids resulting in an increased likelihood of relapse. in general an opioid-related dependency has to be distinguished from a dependency of the barbiturate-, alcohol-or cocaine-type. this is because they all result in different psychopathological and withdrawal symptoms. the latter is a set of physiological reactions that occur in response to removal of a drug following repeated treatment; often (although not always), the reactions are opposite those produced by the drug itself. in the beginning it is the pleasure seeking behavior that results in repetitive drug administration until finally, the drugs of abuse that produce physical dependence (e.g., opiates or alcohol), results in the avoidance of the unpleasant withdrawal syndrome can contribute to repeated drug-taking ( figure ii-54 ). the main physiological consequence of nausea and emesis is the removal of toxins, which is an important protective reflex mechanism being induced during food intoxication. it however, is also seen after radiation or chemotherapy, after the administration of an opioid-based anesthetic regimen or during long-term therapy for alleviation of chronic pain. about 20% of all patients experience nausea and/or emesis after opioid anesthesia. the cause of such reaction is a stimulation of the chemoreceptor biochemical changes induced during addiction and the development of tolerance, which is followed by withdrawal when an antagonist is administered or by lack in maintenance dosages trigger zone (ctz), which lies in close vicinity to the emetic center, bordering the fourth cerebral ventricle, above the area postrema ( figure ii-55 ). this area is richly supplied with dopaminergic, histaminergic, serotonergic (5-ht 3 ), and cholinergic receptor sites, being the origin of metabolic or drug induced vomiting [113] . contrary to the other areas within the cns, the ctz is characterized by leaking capillaries (windowed capillaries), through which opioids as well as toxins can disseminate. such anatomical difference indicates that this area does not contain the usual blood-brain barrier (bbb). being located within the dorsal part of the activating reticular formation (ars), all visual, cortical and limbic efferences, as well as efferences of nearby nuclei of the vasomotor center and the center for salivation and respiratory control are switched, resulting in a controlled succession during vomiting. once the vomiting center is stimulated by any of the efferent stimuli, a coordinated sequence of events is commenced: • stop of rhythmical contractions of the stomach, followed by an accumulation of food in the abdomen, resulting in. • retroperistaltic action. • contraction of the cardia with increase of pressure in the stomach. • due to the coordinated contractions of diaphragm, intercostal muscles and the rectus abdominis muscle, food is being expelled forcefully via the opened orifice of the stomach, the dilated esophagus and the opened glottis. because the ctz shows a dense accumulation of serotonin receptors, the serotoninantagonist ondansetron (zofran®) is able to induce an antiemetic effect [114, 115] . other agents, which are given for reversal of emesis, are metoclopramide, and/or the neuroleptic agents haloperidol, triflupromazine, or alizapride-hcl, all of which interact through direct binding with the dopaminergic d 2 -receptor. another antiemetic is diphenhydramine, which exerts its action via binding at the cholinergic and histaminergic receptors ( figure ii-56) . postoperative nausea and emesis (ponv), however, still present a problem specifically related to anesthesia. in a large survey with over 2000 patients and using multivariance analysis, the following main risk factors for ponv were identified: • female sex • young age • history of ponv/motion sickness • nonsmoking status • long duration of anesthesia each 30 min increase in duration increases ponv risk by 60%, and a baseline risk of 10% is increased to 16% after 30 min [116] . the type of operation, the addition of nitrous oxide (n 2 o), high age and/or the addition of an opioid to the anesthetic regimen, in comparison to the above risk factors, had a lesser impact on the incidence on ponv [117, 118] . commonly the key strategic antiemetic agents for reducing patients ponv are as follows (figure ii-56): the 5-ht 3 -receptor antagonists are used for both the prevention and treatment of ponv and have a low side-effect profile. they are given toward the end of surgery for greatest efficacy, and are more effective in preventing vomiting than in preventing nausea. dolasetron, granisetron, and ondansetron all have favorable side-effect profiles. no evidence has revealed differences in efficacy and safety among the 5-ht 3 -receptor antagonists used for the prophylaxis of ponv. a recent study demonstrated the equivalent efficacy and safety of granisetron and ondansetron when these agents were used in combination antiemetic therapy. in this study, low-dose granisetron (0.1 mg) plus dexamethasone 8 mg was found to be not inferior to ondansetron 4 mg plus dexamethasone 8 mg in patients undergoing abdominal hysterectomy with general anesthesia. the combinations prevented vomiting in 94% and 97% of patients, respectively, in the first 2 h after tracheal extubation, and in 83% and 87% of patients, respectively, in the 24 h after extubation. dexamethasone has been found to be effective for the management of ponv and their proposed mechanism of action is that of membrane stabilization and inhibition of inflammation. use of this agent is controversial because of its alleged association with delayed wound healing. it has a slow onset but a prolonged duration of action, and therefore it is advised that dexamethasone be administered upon induction of anesthesia. the most commonly used dose for adults is 8-10 mg i.v. smaller doses of 2.5-5 mg have also been used and found to be as effective. based on a quantitative, systematic review of the data, no adverse side effects, especially delayed wound healing, have been noted following a single antiemetic dose of dexamethasone [119] . the neuroleptic drug droperidol, a butyrephenone derivative, is widely used for ponv prophylaxis and is comparable with ondansetron as a prophylactic antiemetic. similar to haloperidol it acts as a dopamine antgonist at the ctz and the area postrema. for greatest efficacy, droperidol is administered at the end of surgery or concomitantly with morphine via patient-controlled analgesia systems. the use of low doses (0.625-1.25 mg) of droperidol has not been associated with the typical side effects of higher doses of this drug (hypotension, extrapyramidal symptoms, sedation, akathisia, dysphoria). in 2001, the food and drug administration began requiring that droperidol labeling include a "black box" warning stating that the drug may cause death or life-threatening events resulting from qtc prolongation and the possibility of life-threatening torsades de pointes. the labeling requirement was based on 10 reported cases associated with droperidol use (at doses of 1.25 mg) during its approximately 30 years on the market [120] . however, no case reports in peer-reviewed journals have linked droperidol with qtc prolongation, cardiac arrhythmias, or death at the doses used for the management of ponv. also, in a randomized, doubleblind, placebo controlled trial, droperidol was not associated with a significant increase in the qtc interval in comparison with saline solution [121] . in another recent study, droperidol did not increase the qtc interval any more than did ondansetron [122] . transdermal scopolamine (transderm scop® 1.5 mg), an antimuscarinic ganet, works by blocking the cholinergic receptor. it has an antiemetic effect when applied the evening before surgery or 4 h before the end of anesthesia preventing the patient from post-discharge nausea, vomiting and retching. the phenothiazines, promethazine, and prochlorperazine act both as d 2 -and the h 1 -receptor antagonist. they also inhibit histamine receptors and possibly cholinergic receptors in the gut. both have been shown to be effective antiemetics when administered intravenously at the end of surgery. all three drugs may cause sedation, dry mouth, and dizziness. metoclopramide is benzamide that blocks d 2 -receptors both centrally and peripherally in the gastrointestinal tract increasing gastric emptying. the antihistamines, especially diphenhydramine act on both the ctz and the vestibular pathways of the inner ear. at higher doses however, they can prolong general anesthesia and recovery times. consensus guidelines agree that patients at high or moderate risk for ponv are most likely to benefit from prophylaxis. patients at low risk for ponv are usually not candidates for prophylaxis unless their condition may be compromised by the medical sequelae or vomiting. those at moderate risk for ponv should receive antiemetic monotherapy or combination therapy. those at high risk should receive combination therapy with two or three antiemetics from different classes. drugs with different mechanisms of action can be combined for optimal efficacy. for example, the 5-ht 3 -receptor antagonists (more effective against vomiting) can be combined with droperidol (more effective against nausea). a multimodal approach that incorporates both baseline risk reduction and antiemetic therapy should be adopted for ponv prophylaxis. a recent prospective, double blind, randomized, controlled trial compared three strategies for the prevention of ponv in patients undergoing laparoscopic cholecystectomy: (1) a multimodal approach using ondansetron, droperidol, and total intravenous anesthesia (tiva) with propofol; (2) a combination of ondansetron and droperidol, with isoflurane and nitrous oxide-based anesthesia; and (3) tiva with propofol alone. the complete response rate was higher in the multimodal group (90%) than in the combination group (63%) or tiva-only group (66%), as was the degree of patient satisfaction. nausea and vomiting may persist in some patients after they leave the postanesthesia care unit (pacu). after medication and mechanical causes of ponv have been excluded, rescue therapy with antiemetics can be initiated. for patients who received no prophylaxis, low-dose therapy with 5-ht 3 -receptor antagonists may be initiated. consensus guidelines also recommend low-dose therapy with a 5-ht 3 -receptor antagonist for patients in whom dexamethasone prophylaxis has failed. for patients in whom initial 5-ht 3 -receptor antagonist prophylaxis has failed, a 5-ht 3 -receptor antagonist rescue therapy should not be given within the first 6 h after surgery. similarly, patients in whom prophylactic combination therapy with a 5-ht 3 -receptor antagonist plus dexamethasone has failed should be treated with an antiemetic from a different class. as a general guideline, patients who experience ponv within 6 h after surgery should be treated with an antiemetic other than the one used for prophylaxis. for the treatment of patients who experience ponv > 6 h after surgery, drugs from the prophylactic antiemetic regimen may be repeated, except for dexamethasone and transdermal scopolamine, which have a longer duration of action. also, propofol may be used in small doses (20 mg as needed) for the treatment of ponv in a supervised environment. the preliminary results of a recent analysis support the recommendation that a rescue antiemetic should be from a class other than that of the original antiemetic agent [123] . this analysis of a previous trial reported that in patients who failed prophylaxis with ondansetron or droperidol, promethazine was significantly more effective in controlling ponv than the original agent. dimenhydrinate was also more effective than droperidol in patients who failed prophylaxis with droperidol. in summary, the first step in the management of ponv is to identify surgical patients at high or moderate risk for ponv, then reduce baseline risk factors in these patients. combination antiemetic therapy is recommended for patients at high risk for ponv for patients at moderate risk, monotherapy or combination therapy may be considered. a multi modal approach for the prevention of ponv including the use of antimetics with different mode of action, hydration and tiva with propofol has been shown to be most effective. patients who have not received prophylaxis and experience ponv can be treated with a low dose of a 5-ht 3 -receptor antagonist. in patients who fail prophylaxis treatment with an antiemetic, another agent than the one used for prophylaxis is recommended. opioids can induce muscular rigidity, which is due to an increased tone of the striatal muscle. especially, the muscles of the thoracic cage and of the abdomen show this rigidity, a phenomenon, which is observed after the bolus injection of a potent opioid, such as the fentanyl series (i.e. fentanyl, sufentanil, alfentanil and remifentanil; figure ii-57) . increase in muscle tone is directly correlated to the -receptor interaction, because mixed agonist/antagonists and highly selective -opioid antagonists (i.e. ctap), but not -(e.g. nor-binaltorphine) nor -antagonists (e.g. naltrindole) were able to reverse such muscular rigidity [124, 125] . in addition, administration of the selective antagonist methylnaltrexone in the nucleus raphe pontis was able to reverse increased muscle tone after alfentanil in the animal [126] suggesting this nucleus is an additional important site of action of opioids to induce rigidity. clinically this rigidity is a disadvantage because it results in an insufficient ventilation of the patients and is characterized by the following features [127, 128] : • it appears shortly after intravenous injection of a potent opioid. • it can be induced especially in the elderly patient population. • it is potentiated by nitrous oxide (n 2 o). • it is more likely to develop in patients with parkinson's disease. the anatomical correlate by which opioids induce muscular rigidity is the striatum, and, being part of the basal ganglion system, it has the task to control locomotion ( figure ii-58) . within the striatum there is a dense accumulation of opioid binding sites, which interact with dopaminergic d 2 -receptors. similar as in parkinson's disease, there is a reduction in the dopamine level with an ensuing imbalance of the cholinergic transmitter system, both of which are in balance with each other and a necessary prerequisite for the control of muscle tone [129] . while in parkinson's disease, increased muscle tone is induced by decrease of dopaminergic neurons in the striatum, opioid-induced rigidity is due to an enhanced degradation of the transmitter dopamine resulting in a functional deficit of a sufficient level in the nigro-striatal pathway [130] . the exact mode of action of opioids to reduce dopamine level within the nigrostriatal system and induce muscular rigidity, very likely is induced by inhibition of tyrosine hydroxylase, the necessary enzyme for the synthesis of dopamine [131] . due to the interconnection with the inhibitory gabaminergic system, output of gaba in the pallidum declines ( figure ii-58) . this, in turn, causes an overactivity of cholinergic neurons projecting to thalamic neurons. from here the area 6a of the cortical premotor center is activated and the corticospinal tract leads efferents to the anterior horn of the spinal cord [132] . although opioids do not directly affect muscle tone, rigidity rapidly can be reversed by the injection of a competitive or non-competitive muscle relaxant [133] . although the increased efferent output at the neuromuscular junction is not reduced, muscle relaxants induce their action by inhibiting the binding of acetylcholine at the motor endplate ( figure ii-59) . because the gabaminergic system in the putamen is involved in the mediation of opioid-induced muscular rigidity, any increase in gabaminergic transmission can also ease this side effect. such a notion has been supported by results, where a benzodiazepine reduced the increased muscle tone, an effect that could be reversed by the specific benzodiazepine antagonist flumazenil [125] . in addition, significance of the neurotransmitter dopamine in basal ganglia of the cns, which are involved in the regulation of muscle tone 1 = pallidum externum; 2 = putamen; 3 = nucleus caudatus; 4 = thalamus; 5 = hypothalamus; 6 = lobus parietalis; 7 = central grey; 8 = corticospinal tract; 9 = inhibitory dopaminergic pathway; 10 = thalamo-cortical neurons; 11 = substantia nigra since neighboring 2 -receptors interact with the substantia nigra, opioid-related rigidity could be attenuated by the additional administration of the 2 -agonist dexmedetomidine [135] . the miotic action of opioids on the pupil is an easily recognizable and quantifiable effect in man. the neural pathways responsible for regulating pupil size are reasonably well defined. yet, the mechanisms behind this and related effects of opioids on the eye in humans and laboratory animals have just begun to be explored. opioid-induced miosis in the human, dog and rabbit is thought to be mediated through the central nervous system. this action is a specific opioid effect as demonstrated by its antagonism by naloxone. theories have been advanced suggesting comparison between groups p < 0.05 p < 0.01 figure ii-59. alfentanil-induced truncal rigidity in patients following induction of anesthesia. in comparison to the rapid bolus injection of the drug, slow injection over a period of 2 min resulted in a significant lesser reduction of thoracic compliance. the increase in truncal rigidity was instantly reversed by a low dose (25 mg/70 kg body weight) of the fast acting muscle relaxant succinylcholine. adapted from [134] that morphine produces its effects by direct stimulation of the edinger-westphal (preganglionic parasympathetic) nucleus [181] . an alternative view has been postulated that morphine depresses cortical centers, which normally inhibit the edinger-westphal nucleus. others have suggested that miosis is caused by stimulation of opioid receptors located on the iris sphincter, although this opinion seems to be in the minority. the exact site, or sites, of action within the cns, which are responsible for opioid-induced miosis remain obscure. it is generally accepted, however, that sympathetic innervation is not essential, the miotic effect being entirely dependent on the integrity of the parasympathetic system. for example, lee and wang [182] have shown that dogs with a sectioned oculomotor nerve fail to show miosis even with a 30 fold increase in the dose of morphine. in contrast, dogs show normal responses following sympathectomy. while local application of a muscarinic antagonist (scopolamine) that blocks the pupil sphincter completely abolishes the pupillary effects of morphine in the rabbit, application of a sympathetic neuronal blocker (guanethidine) or of an alpha-adrenergic antagonist (phentolamine) that block the pupil dilator had no effect in those experiments. thus, pharmacologic dissection of the autonomic innervation of the pupil suggests that opioid-induced miosis is mediated solely through the parasympathetic system. other cns structures may also be involved in opioid-induced miosis. lee and wang [182] have shown that removal of the cerebral hemispheres potentiates the miotic action of morphine in the dog. they interpreted this effect as being a reflection of the loss of tonic inhibition originating in the occipital lobes. the latter are known to play a regulatory role in pupillary function, particularly with respect to the near-response (accommodation, convergence and miosis) and, hence, their removal might be expected to alter the pupillary response to drugs. the same authors also observed that acute or chronic optic nerve section did not alter the miotic response to morphine in dogs. in humans, it was shown that morphine produced a dose-related miosis under conditions of low ambient light. taken together, these findings suggest that morphine may cause miosis through more than one mechanism. the main neural structures, which are thought to regulate pupillary size are found in the midbrain, mainly the pretectal area and the edinger-westphal nucleus of the oculomotor complex. because neuronal unit activity in the edinger-westphal nucleus has been shown to correlate with light-induced pupillary constriction. opioids therefore depress or abolish spontaneous and light-induced firing of pupilloconstrictor neurons in the pretectal area, while the opposite effect is observed in the edinger-westphal nucleus where a marked increase in spontaneous firing rate resulting in madriasis. it is because of this increase in activity that certain animal species (rat, cat, monkey) demonstrate an opioid-induced mydriasis. in addition, the brain stem region regulating pupil size is known to have multiple inputs, including the cortex and midbrain, and several others can be assumed to exist. depression by morphine of tonic inhibitory input trom the cortex may partially account for the miosis observed by lee and wang [182] . these findings suggest that opioids may act directly on the neurons subserving the parasympathetic light reflex. also, in contrast to other workers, morphine has no local action on the iris. for example, lee and wang [182] could not produce miosis by injecting 20% of an effective systemic dose of morphine (1 mg) directly into the anterior chamber of the eye in dogs. although opioid binding sites have been found in the retina of the rat, cow, toad and skate, opioids injected into the anterior chamber may stimulate retinal receptors in some species, causing miosis via reflex parasympathetic output. following oral ingestion, but also after the systemic administration, opioids also bind to selective receptors located within the intestinal tract. the physiological significance of peripherally located opioid binding sites within the intestine is that of regulation of the propulsive transit. the intestine with a total surface of nearly 400 m 2 is an underestimated important anatomical site as it has a high accumulation of neuronal tissue, which has been termed the enteral nervous system (ens), which acts like a second brain. since there is a close interconnection of the ens with the cns via the vagus nerve, regular impulses to and from the ens are being exchanged. anatomically the intestine is surrounded by two separate syncytial, netlike nervous structures. one is the myentericus plexus (auerbach) located between the longitudinal and the circulatory muscle fibers ( figure ii-60) . the second is the submucosal meissner plexus, located between circulatory and the submucosal muscle fibers. within the auerbach plexus of the intestinal tract, there is a balance between the cholinergic and enkephalinergic neurons: binding of systemically applied opioids to enkaphalinergic receptor sites results in an inhibition of transit followed by constipation. contrarily, cholinesterase inhibitors induce an accumulation of acetylcholine at ach-receptors with an increase in motility and an enhancement of transit. presently, however, not very much is known of the long-term effect of central analgesics on opioid-receptors within the myenteric plexus, and if opioid ligands induce only a constipating effect, whether they also depress the immune system in the intestine or result in a distress the neuroregulatory and endocrine function. while analgesia, respiratory depression, bradycardia, antitussive action and miosis all are centrally induced opioid effects, the most relevant peripheral opiod action is that of constipation [109] [136] . this is most relevant in patients with chronic pain taking an opioid for its attenuation. being one of the major side effects, it often results in the necessity to take a laxative on a routine basis. the cause for such constipation is the constriction of the pylorus resulting in a delay in emptying of the stomach [137] . however, most important, opioids induce a constriction of the small intestine resulting in a delay of the propulsive transit. because selective opioid binding sites are mainly located in the small intestine, opioids inhibit the release of local acetylcholine [136, 137, 138] , followed by a concomitant loss of coordinated propulsive movements of the gut. a constipating effect of opioids on the large intestine is of significantly lesser degree, because this part of the intestinal tract contributes to a much lesser extent to the overall constipating effect. this is because continuous propulsive movements are not seen on this area, and contrary to the small intestine, the percentage of enkephalinergic neurons is significantly lower [139, 140] . in addition, enkephalin derivatives are able to inhibit transit in the small while at the same time increasing contractions in the large intestine [141] . systemicallyselective applied opioids therefore primarily interact with enkephalinergic neurons in the antrum, the duodenum, and the small intestine, all of which results in a delay of transit [142, 143, 144, 145] . the constipating effect of an opioid can be reversed by a selective peripheral acting antagonists such as methylnaltrexone [146, 147] or alvimopane [148] . opioids, which interact primarily with the -opioid receptor induce a lesser constipating effect [148, 149] , while -selective ligands induce no effect on gastrointestinal transit [150] . comparable to a ketamine-or a volatile anesthetic based regimen, an opioid-based anesthesia results in a longer delay of gastrointestinal emptying in the postoperative period [151, 152] (figure ii-61) . this, however, is clinically of little significance, as the potential constipating effect does not last longer than 48 h after anesthesia. contrarily to many other anesthetics, opioids in general do not depress the cardiovascular system. this is also reflected in the higher therapeutic range (ld 50 /ed 50 ) being derived in the animal (table ii11) . such data can also be conveyed to the human, since a wide therapeutic margin of safety is directly correlated with a lack in cardiovascular impairment. while carfentanil, with a potency twice that of sufentanil, is solely used in veterinary medicine for the immobilization of wild animals [153] , lofentanil (20fold potency of fentanyl), due to its intensive receptor binding, is characterized by a duration of action of 24 h [154] . both fentanyl derivates are not in clinical use, because the high potency and the intense receptor binding would be difficult to handle in patients. from the table, however, it is obvious that the higher the selectivity to the receptor site, and the higher the potency, the lesser the amount of cardiovascular depression [33, 35, 103, 155, 156, 157] . following the injection of potent opioids, bradycardia is the most prominent cardiovascular effect seen in patients. this is due to a direct central stimulation of the nucleus nervi vagi and a typical effect of -ligands. thereafter, a reduction of the sympathetic drive is initiated resulting in an overexpression of parasympathetic activity. also, a direct peripheral negative inotropic activity with a potentiation of acetylcholine release at the sinus node of the heart is discussed [158] . the increase in vagal tone and the reduction of sympathetic drive on the peripheral vasculature results in a decline of mean arterial pressure. a reduction of sympathetic tone on vessel tone and a reduction of resistance is also termed as "pooling" of circulating blood volume. such a reduction of peripheral resistance in certain cases may be of benefit for the patient, as it is accompanied by a reduction in afterload of the heart [159, 160] . bradycardia, the reduced peripheral resistance (i.e. afterload of heart) as well as the pooling effect with a reduction of preload of the heart, can be of benefit for a patient with myocardial infarction. this is because those three variables are major determinants in myocardial oxygen consumption (mvo 2 ) [160, 161] . it, however, should be noted that the sympatholytic action with pooling of blood volume induced by potent opioids might demask a previously compensated hypovolemic condition in a patient resulting in significant hypotension. for instance, in patients with multiple trauma a reduced dose of the opioid should be given, either diluted or slowly injected while measuring blood pressure continuously. in general, however, especially in polytraumatized patients, opioids are of benefit, as they reduce the stress-related release of hormones and particularly of angiotensin ii, maintaining the effect of circulating catecholamines on the vasculature. opioid-related bradycardia with an accompanying hypotension can rapidly be reversed with increasing doses of the vagolytic agent atropine (0.25-05-1.0 mg/kg body weight). the incidence and the severity of such a drop in blood pressure cannot be foreseen. it is related to the autonomic basal tone of the patient and the dose of the injected potent -ligand ( figure ii-62) . depending on the product, the autonomic basal tone of, and the applied dosages to the patient, either parasympathetic (inhibitory) and/or a sympathetic (excitatory) symptoms are induced (table ii12) . such clinical effects can be diminished by atropine, an -blocker (e.g. phenoxybenzamine), a ß-blocker (e.g. propranolol), and a ganglionic blocker (e.g. hexamethonium) respectively [103] . the stimulatory effects of opioids can also be explained in the laboratory where stimulation of cyclic amp formation, phosphinoside hydrolysis, and the elevation of intracellular calcium, resulting from mobilization of calcium stores and by stimulating influx, which leads to an increased neurotransmitter release and neurotransmission [163] . thus, at the cellular level these changes may underlie the opioid stimulatory effect. in addition, such stimulation is also discussed as playing a part in the development of tolerance to opioid drugs [164] . the effect of increasing doses (mg/kg body weight) of potent opioids on the cardiovascular system of the canine, where low amounts result in parasympathetic activation, and high to massive doses induce an increase of sympathetic drive. adapted from [103] in opioid-based anesthesia, vagal-or sympathetic-induced side effects can be reduced or eliminated by the following techniques: 1. the preliminary administration of atropine (up to 1 mg/kg body weight). 2. the simultaneous administration of a volatile anesthetic (n 2 o, enflurane, desflurane, sevoflurane). 3. the simultaneous use of a neuroleptic agent (e.g. droperidol, haloperidol). 4. the simultaneous use of a benzodiazepine (e.g. diazepam, midazolam, lorazepam). 5. the simultaneous use of a hypnotic (e.g. barbiturate, etomidate, propofol). adapted from [103, 162] all these agents induce a depression of cns activity in different areas of the central nervous system, which results in equilibrium of the autonomic nervous system discharge, thus, reducing the overshoot of sympathetic and/or parasympathetic tone ( figure ii-63) . mixed agonist/antagonists, when given in dosages above the therapeutic range, induce a cardiostimulatory sympathomimetic effect, which purportedly is induced via stimulation of -receptor sites [165] . as a result, tachycardia, an increase in peripheral vascular resistance, and an increase in pulmonary artery pressure are induced (table ii13) , all of which increase myocardial oxygen consumption (mvo2). therefore agonist/antagonists should not be given above their therapeutic range in patients with mi or with a preexistent cardiovascular disease [166] . a malfunction at the atrio-ventricular node in the myocardium, followed by prolongation of the p-q interval is a phenomenon, which can be induced in patients demonstrating a preexisting abnormal conduction system in the heart. such prolongation manifests itself especially when potent opioids are being administered (fentanyl, sufentanil), whereby the opioid-induced acetylcholine release induces a stimulation of vagal activity. thus, patients already having a prolongation of p-q time or who present a sick-sinus syndrome, extreme bradycardia has to be anticipated, which could result in concomitant cardiac arrest. in order to prevent such a scenario, the opioid should not be given as a bolus, but rather as a diluted solution. in addition, the solution should be injected slowly over a long period of time neuroleptics block afferents from entering the ascending reticular formation, which increase vigilance; tranquillizers protect the hippocampus from an excitatory activation, while barbiturates, hypnotics and volatile anesthetics primarily block the cerebral cortex from arousal table ii13 . different cardiovascular effects of -ligands, mixed agonist/antagonists, and partial agonists resulting in a decrease (⇓) or an increase (⇑) blood pressure heart rate pulmonary artery pressure adapted from [166, 167, 168] of at least 2 min. if, however, extreme bradycardia is recognized on the monitor, atropine is the agent of choice (0.5-1.0 mg/kg body weight) for rapid reversal. in very extreme cases, the antiarrythmic agent metaproterenol may become necessary, as it is able to increase atrio-ventricular conduction. high doses of methadone or its derivative -levoacetylmethadol (laam) may result in life threatening torsades de points with the potential of ensuing ventricular fibrillation. predisposing factors for the development of such a situation are a prolongation of atrio-ventricular conduction time, hypopotassemia, and/or the simultaneous intake of agents, which inhibit metabolism of the opioid (e.g. tricyclic antidepressants, imidazol derivatives, antimalaria agents, or antihistaminics). a direct negative inotropic effect on the myocardium has been demonstrated in the isolated papillary muscle and in the langendorff preparation of the heart for a variety of opioids [169, 170] . such direct effects, however, are not of clinical significance, because such a depression is only evident in concentrations above the therapeutic range. in addition, compensatory cardiovascular and the autonomic regulatory mechanisms come into play when an opioid is given to a subject. following the intravenous injection of pethidine (meperidine, usp), hypotonia and syncope may result. because of the atropine-like molecular structure of this agent, tachycardia, as well as reflex bradycardia can be observed [171] . for the reason of these potential side effects pethidine should not be given to patients with myocardial infarction [109] . in addition, it is observed that in a shock-like situation, due to the release of endogenous opioids (enkephalins, endorphins), the additional administration of an exogenous opioid results in an additional occupation of opioid binding sites within the myocardium. this aspect is followed by a negative inotropic effect with an unfavorable consequence on hemodynamics [172] . some experimental work has postulated a putative direct negative inotropic effect of n 2 o in an opioid-based anesthetic regimen [173] . since this is mainly seen when n 2 o is given in concentrations above 50% with a resultant drop in fio 2 , this very both agents dose-dependently reduce adrenaline-induced ventricular extrasystoles. adapted from [180] pvc-premature ventricular countraction likely is due to an insufficient myocardial oxygen supply. in addition, high concentrations of n 2 o have a direct vasodilatory effect, resulting in a reduction of venous return to the heart and a drop in blood pressure [174] . it therefore is advocated that in patients receiving opioid anesthesia with a preexisting cardiovascular disease, the optimal concentration in fio 2 should be around 0.5. opioids also have been demonstrated to induce an anti-arrhythmic effect. this has been shown in the animal for meptazinol [175] and in experimental coronary artery occlusion, using fentanyl, sufentanil and carfentanil respectively [176, 177, 178] (figure ii-64 ). the reason for such an antifibrillatory effect seems to be due to the increase in vagal tone [179] . international union of pharmacology. xii. classification of opioid receptors separation of opioid analgesia from respiratory depression: evidence of different receptor mechanism opiate receptors: different ligand affinity in various brain regions identification and characterization of three new alternative spliced mu-opioid receptors the effects of morphine and nalorphine-like drugs in the nondependant and morphine-dependant chronic spinal dog the d-and the l-isomers of methadone bind to the non-competitive site on the nmda receptor in the rat forebrain and spinal cord plasma concentrations of codeine and its metabolite morphine, after single and repeated oral administration novel receptor mechanisms for heroin and morphine-6ß-glucoronide analgesia the intrinsic antinociceptive effects of oxycodone appear to be k-opioid receptor mediated ketobemidone, methadone and pethidine are non-comptitve n-methyl-d-aspartate (nmda) antagonists in the rat cortex and spinal cord the binding spectrum of narcotic analgesic drugs with different agonist and antagonist properties selectivity of ligands for opioid receptors, in opioids i. handbook of experimental pharmacology comparison between epidural fentanyl, sufentanil, carfentanil, lofentanil and alfentanil in rats: analgesia and other in vivo effects interaction of -opioid receptor agonists and antagonists with the analgesic effect of buprenorphine in mice european federation of the international association for the study of pain chapters opiate receptor: demonstration in nervous tissue multiple opiate receptors: support for unique mu, delta and kappa sites autoradiographic localization of opiate k-receptors in the guinea pig brain autoradiographic localization of kappa opiate receptors to deep layers of the cerebral cortex may explain unique sedative and analgesic effects characterization of opioid receptors in nervous tissue buprenorphine causes ceiling effect in respiratory depression but not in analgesic effect the clinical pharmacology of buprenorphine: extrapolating from the laboratory to the clinic clinical pharmacology of buprenorphine: ceiling effects at high doses specific uptake of narcotic analgesics by subcellular fractions of the guinea pig ileum in vitro models in the study of structure-activity relationships of narcotic analgesics über die antagonistischen systeme der schmerzempfindung und des schmerzgefühls im peripheren und zentralen nervensystem a morphine-like factor "enkephalin" in rat brain: subcellular localization determination of methionine enkephalin in discrete regions of rat brain pain mechanisms: a new theory neurotransmitter receptor binding in the brain interaction of naloxone with mu-and delta-opioid agonists on respiration of rats 3h-sufentanil, a superior ligand for the mu-opiate receptor: binding properties and regional distribution in rat brain and spinal cord alfentanil (r 39 209) -a particularly shortacting narcotic analgesic in rats n-4-substituted 1-(2arylethyl)-4-piperidinyl-nphenylpropanamides, a novel series of extremely potenet analgesics with unusually high safety margin neuropathic pain: aetiology, symptoms, mechanisms, and management current views on the role of psychiatrists in the management of the chronic pain psychodynamics and psychotherapy in the treatment of patients with chronic pain the effects of morphine and meperidine on the central respiratory mechanisms in the cat: the action of levallorphan in antagonizing these effects respiratory and cardiovascular effects of met-enkephalin applied to the ventral surface of the brain stem fentanyl in the fourth cerebral ventricle causes respiratory depression in the anesthetized but not in the awake dog pharmacokinetics of naloxone in rats and man. basis for its potency and short duration of action prevention of late fentanyl-induced respiratory depression after the injection of the opiate antagonists naltrexone and s-20682 as compared to naloxone pharmacokinetics of fentanyl as a possible explanation for recurrence of respiratory depression analgesic effectiveness of the narcotic agonist-antagonists opioid agonists, antagonists and mixed narcotic analgesics: their use in postoperative and chronic pain management reversal of narcotic-induced respiratory depression with nalbuphine hydrochloride reversal of fentanyl-related respiratory depression with nalbuphine; effects on the co2-response curve of man antagonism of postoperative opioid-induced respiratory depression: nalbuphine versus naloxone nalbuphine reverses fentanyl-related eeg changes in man prolonged antagonism of opioid action with intravenous nalmefene in man antagonism of fentanyl-induced respiratory depression with nalmefene enkephalins: isolation, distribution and function multiple opioid receptors mediate the respiratory depressant effect of fentanyl-like drugs in the rat minireview: multiple mu opiate receptors multiple morphine and enkephalin receptors and the relief of pain differences in magnitude and duration of opioid induced respiratory depression and analgesia with fentanyl and sufentanil mu and delta receptors: their role in analgesia and in the differential effects of opioid peptides on analgesia opioid-induced respiratory depression and analgesia may be mediated by different subreceptors the delta receptor is envolved in sufentanilinduced respiratory depression delta opid receptor enhancement of mu opioid receptor-induced antinociception in spinal cord the pharmacokinetics and pharmacodynamics of gi87084b. anesthesiology context-sensitive half-time in multicompartment pharmacokinetic models for intravenous anesthetic drugs influence of cerebral activity in wakefulness on regulation of breathing waking and ventilatory responses to laryngeal stimulation in sleeping dogs respiratory safety association des analgésiques centraux et des neuroleptiques en cours d'intervention part ii drug interactions of clinical significance with opioid analgesics drug interaction and side effects index™ biotransformation von fentanyl. ii. akute arzneimittelinteraktion -untersuchungen bei ratte und mensch the magnitude and the duration of respiratory depression produced by fentanyl and fentanyl plus droperidol in man a quick guide to common drug interaction, in patient care plasma protein binding of phenytoin after cholecystectomy and neurosurgical operations intravenous fentanyl kinetics morphine and phenytoin binding to human plasma protein in renal and hepatic failure plasma concentrations of fentanyl in normal surgical patients and those with severe renal and hepatic disease reduction in the incidence of awareness using bis monitoring bremazocine: a potent, long-acting opiate kappa-agonist tifluadom (kc-5103) induces suppression and latency changes of somatosensory-evoked potentials which are reversed by opioid antagonists bremazocine: an opiate which induces sedation and analgesia but no respiratory depression cellular distribution of the mrna for the k-opiod receptor in the human neocortex: a non-isotopic in situ hybridization study opiate receptor binding sites in human brain utilisation pratique des analgesiques centraux en anesthesie et reanimation patient-controlled analgesia using butorphanol for postoperative pain control: an open label study nalbuphine versus pentazocine in postoperative pain after orthopedic surgery the enflurane sparing effect of morphine, butorphanol, and nalbuphine mac reduction of enflurane and isoflurane and postoperative findings with nalbuphine hcl and fentanyl: a retrospective study ceiling effect for respiratory depression by nalbuphine the "narcotic" component of fentanyl. l'anesthese vigile et subvigile die hypnotische wirkung von fentanyl und sufentanil kardiovaskuläre und zentralnervöse effekte unter fentanyl versus sufentanil bei der intubation herzchirurgischer patienten eeg findings in neuroleptanalgesia central nervous effects of neuroleptanalgesia as induced by haloperidol and phenoperidine exzitatorische und inhibitorische phänomene am zentralnervensystem, verursacht durch fentanyl perfusion of the fourth cerebral ventricle with fentanyl induces naloxone reversible hypotension, bradycardia, baroreflex depression and sleep in unanaesthetized dogs regional distribution of opiate receptor binding in monkey and human brain the effects of morphine and pethidine on somatic evoked responses in the midbrain of the cat, and their relevance to analgesia wechselwirkungen zwischen dem system der schnellen schmerzempfindung und dem des langsamen über die zweiteilung der schmerzempfindung und des schmerzgefühl, in schmerz comparative study of cardiovascular, neurological, and metabolic side effects of eight narcotics in dogs genralized grand mal seizure after recovery from uncomplicated fentanyl-etomidate anesthesia another case of grand mal seizure after fentanyl aministration tonic-clonic activity after sufentanil seizure-like movements during fentanyl infusion with absence of seizure activity in a simultaneous eeg recording the effects of high-dose fentanyl on cerebral circulation and metabolism in rats opioid analgesics and antagonists, in the pharmacological basis of therapeutics diagnose and treatment of symptoms of the respiratory tract 3 h-codeine binding in the guinea pig lower brain stem physiology and pharmacology of vomiting treatment of postoperative nausea and vomiting after outpatient surgery with the 5-ht3 antagonist ondansetron comparison of ondansetron versus placebo to prevent postoperative nausea and vomiting in women undergoing ambulatory gynecologic surgery coinsensus guidelines for managing postoperative nausea and vomiting postoperatives erbrechen -ein score zur voraussage des a meta-analysis of nausea and vomiting following maintenance of anaesthesia with propofol or inhational agents dexamethasone for the prevention of postoperative nausea and emesis-a quantitative systemic review food and drug admistration black box warning on the perioperative use of deoperidol: a review of the cases effect of low-dose droperidol on the qt interval during and after general anesthesia: a placebo-controlled study prolongation of qtc interval after postoperative nausea and vomiting treatment by droperidol or ondansetron the effectiveness of rescue antiemetics after failure of prophylaxis with ondansetron or droperidol: a preliminary report role of opioid receptors in the substantia nigra in morphine-induced muscular rigidity antagonism of dermorphin-induced catalepsy with naloxone, trh-analog cg3703 and the benzodiazepine antagonist, ro 15-1788 catatonia" produced by alfentanil is reversed by methylnaloxonium microinjections into the brain abdominal muscular rigidity induced by morphine and nitrous oxide studies in muscular rigidity, nitrous oxide and narcotic analgesic agents the effect of fentanyl and droperidol on the dopamine metabolism of the rat striatum morphine katalepsy in the rat: relation to striatal dopamine metabolism tyrosine hydroxylation in the rat striatum after fentanyl and droperidol in vivo decrease of neocortical choline acetyltransferase after lesion of the globus pallidum in the rat attenuation of fentanyl-induced truncal rigidity die lungencompliance wird durch die rasche injektion von alfentanil beeinträchtigt dexemedetomidine, acting through central alpha2-adrenoceptors, prevents opiate-induced muscle rigidity in the rat gastrointestinal effects of opioids naloxone and morphine inhibit gastric emptying of solids effect of synaptic transmission blockade on morphine action in the guinea pig myenteric plexus enkephalin-like immunoreactivity in the human gastrointestinal tract autoradiographic localisation of opiate receptors in rat small intestine influence of opiates on colonic motility relative involvement of mu, kappa, and delta receptor mechanisms of opiate-mediated antinociception in mice inhibition of gastrointestinal transit by morphine in rats results primarely from direct drug action on gut opioid sites effect of morphine on gastric emptying a comparison of the effect of oral controlled release morphine and intramuscular morphine on gastric emptying methylnaltrexone prevents morphine-induced delay on oral-cecal transit time without affecting analgesia: a double-blind randomized placebo-controlled trial oral methylnaltrexone for opioid-induced constipation effects of adl 8-2698, a peripherally restricted mu opioid anntagost, on gut motility in methadone and laam-dependent patients with opioidinduced constipation. a dose-ranging study the effects of morphine and nalbuphine on intestinal transit in mice peptide opioid antagonist seperates peripheral and central opioid antitransit effects die gastro-coekale transitzeit nach fentanyl/midazolam-im vergleich zur enfluran-und ketamin/midazolam-narkose keine hemmung der intestinalen motilität nach ketamin-/midazolamnarkose immobilization of free-ranging wild animals using a new drug carfentanil and lofentanil sufentanil, a very potent and extremely safe intravenous morphine-like compound in mice, rats and dogs the development of new synthetic narcotics pharmacotherapy of opioids: present and future developments hämodynamische wirkungen hoher dosen von fenanyl, meperidine und naloxon beim hund cardiovascular effects of high doses of fentanyl, meperidine and naloxone in dogs filling pressures of the heart and pulmonary circulation of the patient with coronary artery disease after large doses of morphine control of myocardial oxygen consumption utilisation pratique des analgésiques centraux en anesthésie et réanimation determinants of the stimulatory opioid effect on transmitter release and possible cellular mechanisms: overview and original results stimulatory effects of opioids sigma receptor ligands: function and activity. neurotransmissions comparative effects and analgesic efficacy of the agonist-antagonist opioids a review of its pharmacological properties and therapeutical uses, in new drug series contractile responses to morphine, piritramid and fentanyl: a comparative study of effects on the isolated myocardium myocardial opiate receptor activity is stereospecific, independent of muscarinic receptor antagonism, and may play a role in depressing myocardial function utilisation de la pentazocine comme analgesique pour le traitement des douleurs post-operatoires. etude comparative entre le pethidine, la piritramide et la pentazocine, in utilisation de la pentazocine en anesthesie et reanimation hemodynamic changes following corticosteroid and naloxone infusion in dogs subjected to hypovolemic shock without resuscitation nitrous oxide as an adjunct to narcotic anesthesia does nitrous oxide or a reduced fi02 alter the hemodanymic function during high dose sufentanil anesthesia? the antiarrhythmic effect of meptazinol effects of high doses of fentanyl on myocardial infarction and cardiogenic shock in the dog the antifibrillatory effect of fentanyl, sufentanil, and carfentanbil in the acute phase of local myocardial ischemia in the dog antifibrillatory action of the narcotic agent fentanyl protective effect of the vagotonic action of morphine sulfate on ventricular vulnerability les effets anti-arrythmiques des opiaces. comparison avec un beta-bloqueur chez le chien. cah d'anesthesiol opposite pupillary effects in the cat and the dog after microinjection of morphine, normorphine and clonioline in the edinger-westphal nucleus mechanism of morphine -induced miosis in the dog key: cord-018302-lmly43rd authors: renaud, christian; englund, janet title: respiratory syncytial virus and human metapneumovirus infection in transplant recipients date: 2016-02-15 journal: transplant infections doi: 10.1007/978-3-319-28797-3_31 sha: doc_id: 18302 cord_uid: lmly43rd respiratory viral infections due to respiratory syncytial virus (rsv) and human metapneumovirus (hmpv) cause infections in immunocompromised transplant patients ranging from mild upper respiratory infections to severe lower respiratory tract disease with respiratory failure. these viruses are more readily diagnosed due to improvements in sensitive molecular diagnostic methods. the epidemiology of rsv and hmpv is similarly becoming more readily appreciated in hematopoietic stem cell transplant (hsct) patients of all ages as well as solid organ transplant (sot) patients, with lung transplant recipients having evidence of more frequent and severe complications related to these viruses. rsv and hmpv infection typically but not always present with upper respiratory signs and symptoms that progress to lower respiratory tract disease. treatment options for rsv are limited, with aerosolized, intravenous, and oral ribavirin all studied in hsct and lung transplant patients. no antiviral therapy for the treatment of hmpv is available, although ribavirin has shown some effectiveness in vitro. new antiviral agents including rsv fusion inhibitors and nucleoside analogs are being developed, with some under clinical evaluation. respiratory viruses may cause serious morbidity and mortality in the immunocompromised host, and the transplant recipient appears particularly vulnerable. the impact of infection with respiratory viruses and the subsequent development of severe lower respiratory tract disease has been increasingly appreciated as respiratory viruses become more readily detectable. respiratory syncytial virus (rsv) and human metapneumovirus (hmpv) are among the best-documented respiratory viruses causing a wide range of respiratory disease in transplant recipients, ranging from asymptomatic shedding to fatal respiratory failure. understanding the epidemiology and clinical characteristics of rsv and hmpv permits clinicians to intervene pretransplant, provide appropriate infection control and prevention measures, potentially treat patients, and manage immunosuppressive therapy. severe respiratory disease in the seriously immunocompromised host in the mid-twentieth century was originally attributed to infection with opportunistic pathogens such as gram-negative bacteria, fungi, pneumocystis jiroveci , and mycobacteria, as well as cytomegalovirus (cmv) and adenovirus. in the later twentieth century, episodes of acute upper respiratory infection (uri) and lrti without an identifi ed etiology were often considered to be "idiopathic" pneumonia or attributed to regimen-related toxicity or acute respiratory distress syndrome (ards). in the classic 1982 study of 215 non-bacterial, non-fungal pneumonias in 525 allogeneic hematopoietic stem cell transplant (hsct) recipients, 44% of the episodes of pneumonia remained undiagnosed. the overall mortality rate associated with idiopathic pneumonia in these hsct recipients was 60%, a strikingly high fi gure [ 1 ] . the potential morbidity of rsv infections was fi rst recognized in immunocompromised children in the 1970s, more than a decade earlier than in immunocompromised adults [ 2 -6 ] . the recognition of respiratory viruses as an important clinical problem likely refl ects the increasing number of severely immunodefi cient patients, more aggressive attempts to identify the cause of respiratory illness in high-risk patients, and the increasing ability of clinical virology and pathology laboratories to identify respiratory viruses in clinical specimens. although uncommon or atypical pathogens may be responsible for respiratory disease in the transplant recipient, the same viruses that cause typically mild but acute respiratory illness in the general population are responsible for hospitalizations in persons of all ages with underlying medical conditions [ 7 ] . these same viruses are also a common cause of respiratory disease in transplant recipients [ 8 -15 ] . with the widespread availability of sensitive and reliable molecular diagnostic methods, rsv and hmpv have been detected worldwide in transplant recipients and shown to be common causes of respiratory disease in the immunocompromised host [ 16 , 17 ] . in both the general population as well as in transplant recipients, rsv and hmpv may produce a wide constellation of clinical syndromes ranging from the common cold to bronchiolitis to severe pneumonia, but in contrast to the general population, rsv and hmpv may signifi cantly impact the morbidity and mortality of the transplant recipient. rsv was fi rst identifi ed in 1956 and became appreciated as a major cause of epidemic bronchiolitis and pneumonia in young children in the 1960s [ 18 , 19 ] . hmpv was fi rst identifi ed in 2001 by molecular techniques in symptomatic children by van den hoogen et al. as a paramyxovirus causing bronchiolitis and uri in children [ 20 ] . both viruses are classifi ed within the pneumovirinae subfamily of the paramyxoviridae family of non-segmented, negative-strand, enveloped rna viruses [ 21 ] . hmpv belongs to the metapneumovirus genus whereas rsv is a member of the pneumovirus genus. both viruses are highly pleomorphic and their sizes vary from 150 to 600 nm. the rsv and hmpv genomes are approximately 13-15 kb in length and closely resemble each other, excluding a few differences in the order of the genes and the absence of the non-structural genes (ns1 and ns2) from hmpv genome. the remaining eight genes code for nine proteins present in both viruses: the nucleoprotein (n protein), the phosphoprotein (p protein), the matrix protein (m protein), the fusion glycoprotein (f protein), the putative transcription factor (m2-1 protein), the rna synthesis regulatory factor (the m2-2 protein), the small hydrophobic glycoprotein (sh protein), the attachment glycoprotein (g protein) and the viral polymerase (l protein). the rna core of the virion is associated with p, n, l, m2-1, m2-2 proteins, surrounded by m protein and covered by a lipid envelope. f is the most highly conserved of the envelope glycoproteins within each virus and between rsv and hmpv. the fusion glycoprotein is essential in promoting attachment and fusion of the virus with the cell membrane during viral entry. the fusion protein is the target of many vaccines under development as well as that of monoclonal antibodies such as palivizumab, which is used to prevent rsv disease in preterm infants. by contrast, the g gene is the most variable. whole genome analysis of both rsv and hmpv has shown the existence of two genotypes, a and b. in hmpv, those two major genetic groups are further divided into subgroups a1, a2, b1, b2 based upon the sequence variability of the g and f genes. subgroup a2 is again divided into a2a and a2b. rsv and hmpv infections produce both humoral and cellular immune responses. humoral immunity protects against reinfection while cellular immunity controls established infection and terminates viral shedding. protective immunity in immunocompetent hosts is thought to be relatively shortlived. both viruses interfere with the host's innate immune system resulting into incomplete clearance and partial immunity. prompt and accurate identifi cation of respiratory viral pathogen is critically important in the transplant recipient because it enables specifi c infection control precautions to be instituted, the initiation of specifi c antiviral therapy, impacts the use of immunosuppressive therapy, and potentially affects whether transplantation should proceed [ 22 -24 ] . furthermore, identifi cation of a respiratory viral pathogen can assist in avoiding unnecessary therapy, procedures, and surgical procedures (such as open lung biopsy), as well as assist in the identifi cation of a potential cluster or epidemic of infections within the medical unit, hospital, or community. in hospitalized adults (both immunocompromised and immunocompetent), rapid viral diagnosis has been shown to reduce mortality and decrease the length of hospital stay and total cost [ 25 , 26 ] . laboratory diagnosis of respiratory viruses including rsv and hmpv has evolved considerably; adequate specimen collection is still essential for the successful identifi cation of viruses in clinical samples. newer types of nasopharyngeal swabs have shown improved viral diagnostic sensitivity compared to previous swabs, with similar sensitivity to nasal washes when using sensitive molecular methods in patients [ 27 , 28 ] . for example, nylon fl ocked swabs and foam swabs increase cell capture within the swab and then release into the transport media, increasing viral recovery [ 29 , 30 ] . nasal wash or aspiration methods are superior for isolation of viruses by culture and increase the sensitivity of culture, antigenic assays and quantitative molecular assays [ 31 ] . nasal washes are well tolerated in cooperative adults and offer the advantage of visualizing the quality of the specimen. bronchoalveolar lavage remains the specimen of choice to diagnose lower respiratory tract infections because of the ability to simultaneously test for potential co-pathogens such as fungi, pneumocystis jiroveci , and bacteria, as well as to document viral infection in the lower airways. discordance in viral detection between upper and lower respiratory tract samples have been described with both viruses; negative upper tract and positive lower tract specimens in immunocompromised patients are possible but more discordance has been noted for hmpv compared with rsv. molecular diagnosis of rsv and hmpv is faster and more sensitive than viral culture or antigen detection, and most laboratories currently use commercial or in-house molecular assays to detect rsv and hmpv (table 31-1 ) . many genes have been targeted to detect rsv, including the n, f and l genes, with similar genes also targeted to detect hmpv. many rapid assays have been approved including some highly multiplexed respiratory panels allowing detection of rsv and hmpv as well as many other respiratory viruses and bacteria. several different primer sets may be utilized simultaneously in the reaction mix, and the virus identifi ed by the size of the amplicon or following hybridization with a virus-specifi c probe. some commercial assays are very rapid and require minimal technical expertise, with only 1-2 h of turn around time [ 32 , 33 ] . some laboratories have developed quantitative assays using hydrolysis probe technology with standard curves to help understand the signifi cance of positive results and to follow viral loads under therapeutic management [ 34 -36 ] . no quantitative commercial assays are yet available. molecular assays have also been used to detect viral rna from blood/serum as a prognostic marker [ 37 ] . unlike molecular methods, isolation of virus by culture confi rms the presence of a complete infectious unit capable of further multiplication. positive culture results may be obtained with as little as a single infectious virion, below the threshold of detection for most other detection methods, including some nucleic acid amplifi cation test (naat) methods . another advantage of viral culture is that multiple viruses may be identifi ed from a single sample and viruses can grow independently of point mutations that could potentially create false negative results by naat. the major limitations of viral isolation include the time, expense, and expertise required for virus isolation. a variety of cell lines can be used to grow rsv (hep-2, a549, rhmk) or hmpv (llc-mk2, vero), and detection by cell culture can also be accomplished using several types of cells together, such as the r-mix cells (mixture of mink lung cells and a549 cells) [ 38 ] (diagnostic hybrids, athens, oh). centrifugation combined with viral antigen detection methods permits more rapid diagnosis [ 39 ] . rsv-and hmpv-specifi c monoclonal antibodies have been used for immunofl uorescence (ifa) techniques either directly on respiratory specimens or in cell culture [ 40 , 41 ] . the sensitivity of ifa is lower than that of naat for detection of rsv and hmpv. however, results can be reasonably fast, the method is relatively inexpensive, and importantly, this method also confi rms that an appropriate specimen has been properly obtained by looking at ciliated epithelial cells. sensitivity of ifa when performed by an experienced laboratory is as high as 70-90% of the samples positive by pcr-at least in children [ 34 ] . ifa can detect viruses that would be missed by naat because of point mutations. ifa positivity also has good clinical correlation while low grade naat positivity can be detected for longer periods of time with unclear signifi cance and transmissibility. enzyme immunoassays (eias) and rapid antigenic diagnostic tests ( radts) are commercially available for rsv and to a lesser extent for hmpv. these assays lack sensitivity and/or specifi city and are not recommended in transplant populations. more than one diagnostic method should be used, since no method is perfect. molecular assays have the greatest sensitivity-although perhaps at times can perhaps be too sensitive. paradoxically, rare point mutations causing mismatches have been described causing false negative results of naat in transplant units. viral cell culture requires time, is becoming less available in laboratories, and is more expensive and less sensitive, although it can catch those strains with mismatches and provide information on viral replicative nature while receiving treatment. further characterization of rsv and hmpv strains obtained from culture or directly from the clinical specimen is frequently desirable. antigenic differences among virus strains isolated from different geographic locations or at different times may also be examined. pools of monoclonal antibodies and "rna fi ngerprinting" have been used in the analysis of rsv strains in nosocomial outbreaks [ 42 , 43 ] but direct sequencing of the f and g glycoproteins is more commonly utilized [ 44 , 45 ] . next-generation sequencing is a very promising tool to characterize rsv or hmpv strains during severe infection. this could provide information on phylogenicity to identify outbreaks and also detect mutations that could be associated with antiviral or monoclonal resistance as well as increased virulence. some human gene alleles in the human genome may increase rsv severity in infants, but little is known yet on these genomic variations in transplant recipients. next-generation sequencing has the potential to provide information on the virus and the human genomes simultaneously. rsv is well known to cause annual winter outbreaks in the community ( figure 31-1 ). surveillance studies of respiratory viruses from transplant centers have established the high frequency and the signifi cant clinical impact of respiratory viral infections in hsct recipients overall [ 8 -15 , 46 , 47 ] as well as the relative importance of rsv in terms of morbidity and mortality (table 31 -2 ). a 1988 retrospective review conducted at the children's hospital of philadelphia revealed a [ 47 ] . one of the earliest studies demonstrated fatal rsv pneumonia in four of 11 immunocompromised adult hsct and solid organ transplant recipients with rsv infection [ 5 ] . most studies conducted in the twentieth century utilized classical virological methods that were relatively insensitive for the detection of rsv and/or did not detect recently described respiratory viruses such as hmpv. thus, these studies likely underestimated the true frequency of rsv overall. nonetheless, early studies from large transplant centers reported serious sequelae in small numbers of patients who developed pneumonia where rsv was detected [ 5 , 9 , 13 ] . at the fred hutchinson cancer research center (hutch), a prospective surveillance study conducted in 1987 documented respiratory viral infections in 15 (19%) of 78 immunocompromised patients who were followed until hospital discharge [ 13 ] ; fi ve (33%) patients developed pneumonia and two (13%) patients died (one with rsv, the other with adenovirus). a subsequent prospective surveillance study described 127 viral infections and revealed an overall frequency of viral infections of approximately 4% [ 11 ] ; 49% of rsv isolates were from bal. this study demonstrated the relatively high numbers of patients with rsv lower respiratory tract disease, which was higher than rates of lrti caused by piv (22%) infl uenza (10%), or rhinovirus (3%). results of viral surveillance in transplant units vary depending on the type of surveillance protocols utilized, time of year surveillance was conducted, type of clinical samples evaluated, and laboratory tests utilized. at the huddinge university hospital, stockholm, a prospective surveillance study conducted among hsct recipients between 1989 and 1996 detected 39 (7.1%) respiratory viral infections in 545 patients, including rsv in 21% [ 14 ] . at m.d. anderson cancer center (mdacc), prospective surveillance conducted using culture techniques among hospitalized adult hsct recipients during two 6-month winter periods detected 67 (31%) respiratory viral infections in 217 hospitalized patients with acute respiratory symptom. nearly half of these were due to rsv (33 patients, 49%). the impact of these illnesses was considerable: 20 (61%) patients had rsv infection progressing to pneumonia, and 12 patients with rsv pneumonia died. during the winter period when community viruses were frequent and nosocomial transmission was high, the frequency and mortality of respiratory viral-associated pneumonias was more than four times as high as cmv-associated pneumonia. recent prospective studies conducted in transplant centers worldwide continue to demonstrate the importance of rsv and report incidence of infections and risk factors for fatal disease (table 31 -2 ). a 2005 study from barcelona, spain, described the 2-year incidence of symptomatic respiratory viral infections in over 400 patients followed for up to several years posttransplant. altogether, 29% of allogeneic hsct recipients and 14% of autologous hsct recipients had respiratory viruses detected, with 19 patients (4.6%) receiving either autologous or allogeneic transplants having symptomatic rsv disease [ 55 ] . risk factors associated with having a respiratory virus identifi ed included close household contacts with children under the age of 12 years and chronic graft versus host disease. lymphopenia was identifi ed as a major risk for uri progression. similar rates have been demonstrated in other centers in the usa [ 15 ] , south america [ 56 ] , and europe [ 57 ] . less data is available documenting the impact of rsv in sot recipients (table 31 -2 ), rsv has been shown to cause lower respiratory tract disease and has been associated with other complications, such as organ rejection and bronchiolitis obliterans. lung transplant recipients have the highest rates of rsv lower respiratory tract disease in diverse types of organ transplant. rsv infections in lung transplant recipients have also been associated with organ rejection and progressive bronchiolitis obliterans, both of which have been observed to be seasonally related [ 58 , 59 ] . adult renal transplant recipients have also been reported to develop rsvassociated lower respiratory tract disease. the mortality has been low, and most patients have recovered without specifi c antiviral therapy [ 5 , 60 ] . rsv infections in pediatric liver transplant recipients have been associated with signifi cant morbidity and some but relatively low mortality [ 51 ] . among 483 pediatric liver transplant recipients cared for at the university of pittsburgh between 1985 and 1991, 17 (3.4%) children developed rsv infections, three-quarters of which were nosocomially acquired. the majority of the children had lower respiratory tract involvement, and two (12%) children died. specifi c antiviral therapy was not administered. the risk factors for more severe disease included onset of infection early after transplant, preexisting lung pathology, augmented immunosuppression prompted by rejection, and younger age. infections occurring late after transplantation in the absence of rejection were usually not severe. an intensive prospective approach to determining the incidence and risk factors for respiratory viruses was carried out at the hutch among 122 hsct recipients, who were pro-spectively enrolled between 2000-2004 and tested weekly through 100 days posttransplant using both culture and rt-pcr detection methods [ 50 ] . the cumulative incidence estimates of hmpv and rsv at day 100 were similar, at 6.2% and 5.8%, respectively. multivariable analysis demonstrated that only recipient cmv seropositivity was associated with increased risk for acquisition of a respiratory virus (hazard ratio = 4.1, ci 1.7-10.1, p = 0.002). the frequency of rsv infections and associated morbidity and mortality differs substantially, potentially accounting for the variability reported by different institutions. these differences refl ect the intensity of viral surveillances, the time of surveillance, the viruses prevalent in the community, the degree of immunosuppression of the patients, infection control policies, the inclusion of potential as well as actual transplant recipients, surveillance in outpatients as well as inpatients, the types of laboratory assays utilized, and the case defi nition utilized (i.e., both clinical and laboratory defi nitions). hmpv has been detected worldwide with a seasonal distribution. community outbreaks occur yearly mainly in winter and spring (january to may in the northern hemisphere; june to july in the southern hemisphere) (figure 31-1 ). often hmpv outbreaks will be concomitant with or subsequent to rsv outbreaks. hmpv most commonly affects young children less than 2 years old and is second only to rsv as a cause of bronchiolitis. seroprevalence studies have shown a high percentage of children have contracted the virus by age 5-10 years. however, reinfection can occur later secondary to insuffi cient immunity or infection with different genotypes. predominant hmpv strains can vary from location to location and from year to year. vicente et al. have reported higher virulence by genotype a [ 61 ] , while papenburg et al. reported higher virulence by genotype b [ 62 ] . however, the interaction or impact of hmpv with other viruses or bacteria remains unclear, particularly in immunocompromised patients. the importance of hmpv in transplant recipients has not been as well studied as rsv (table 31 -2 ). it was fi rst reported shortly after the detection of hmpv by boivin et al. [ 17 ] . an early prospective longitudinal study from spain documented hmpv in both autologous and allogeneic hsct recipients, with the incidence and clinical impact of hmpv and rsv disease documented to be quite similar [ 55 ] . one early prospective study documented hmpv infections in 22 adults with hematologic malignancies that progressed from upper respiratory infection to pneumonia, with a case-fatality rate close to 14% [ 63 ] . lower respiratory tract disease and pneumonia due to hmpv infection in hsct recipients has been reported to have an overall incidence of 1-4% [ 48 , 55 , 64 , 65 ] . a single case series described hmpv-positive nasal aspirate samples in 86% of 21 adults following hsct, many of whom were asymptomatic [ 66 ] . this study demonstrated very high rates of genetically similar viruses and differs from most other studies due to high rates of genetically identical viruses. these authors suggested that these hmpv infections may have originated in the hospital nosocomially; nonetheless, this study is an outlier compared to other reports of hmpv in transplant centers. pneumonia rates following hmpv infection have been reported at 20-28% with mortality rates of 0-4% [ 67 -69 ] . among 163 hsct recipients who underwent bal for investigation of lower respiratory tract disease with pulmonary infi ltrates by radiographic imaging, hmpv was detected in bal samples from 5 of 163 (3%) patients; four of these fi ve died with acute respiratory failure highlighting the potential severity of hmpv pneumonia [ 64 ] . a retrospective cohort study at the hutch described a high mortality rate of 43% among patients with hmpv pneumonia, a rate similar to rsv pneumonia mortality [ 49 ] . studies from other transplant units continue to document the potential of hmpv to cause severe lower respiratory tract disease, with clinical presentations and outcomes generally similar to rsv [ 49 , 70 ] . the signifi cance of hmpv infection in sot recipients remains less well defi ned, with the exception of hmpv disease in lung transplant recipients [ 70 ] . case reports of severe disease have been described following liver and renal transplantation [ 71 , 72 ] . rates of hmpv infection in lung transplant recipients have been reported to be similar to those seen in studies of hsct recipients, varying from 4-6% [ 57 , 73 ] . in most of these patients, hmpv appears to frequently be the sole pathogen detected. detection of hmpv in lung transplant recipients may not necessarily signify disease, as was noted in a study of 93 lung transplant recipients undergoing bal mainly for surveillance purposes; four cases of hmpv was detected in asymptomatic patients [ 74 ] . hmpv infection has been found in 4-6% of lung transplant recipients, but prevalence may be higher during nosocomial outbreaks [ 39 , 75 ] . one study in the setting of a community outbreak identifi ed hmpv in bal samples from 9 of 26 (35%) patients; their clinical presentation varied from asymptomatic infection to severe disease [ 39 ] . acute allograft rejection was more frequent in the hmpvinfected group than in the non-hmpv-infected group (33% vs. 6%, respectively; p = 0.0257); and overall mortality was also higher (33% vs. 0%, respectively; p < 0.0025) [ 39 ] . another prospective study found hmpv infection as frequent as rsv after lung transplantation, and to cause as much pneumonia and acute allograft dysfunction (63% vs. 72%, respectively), but only rsv was associated with chronic allograft dysfunction at 6 months [ 76 ] . in another study, 25% of hmpv infections in lung transplant recipients were associated with acute allograft dysfunction compared with 88% for rsv [ 49 ] . a meta-analysis of hmpv respiratory infections and allograft rejection, among lung transplantation recipients indicated that detection of hmpv from airway secretions may be a signifi cant posttransplantation occurrence. a total of 2883 samples from 1007 lung transplant recipients, were analyzed for virus detection; 337 samples had viruses identifi ed and 57 (17%) were positive for hmpv. twenty of these 57 (35%) cases of hmpv had acute rejection within 3 months of viral detection. there were fi ve (9.4%) cases of chronic rejection in association with hmpv. all studies included in the meta-analysis, with the exception of one, identifi ed rejection within 3 months. another study has also described cases of chronic rejection within 6 months [ 77 ] . disease manifestations of rsv are dependent on many factors including the immunity and immune competence of the host, the time of infection related to transplant, the type of transplant, the age and underlying health of the patient, and the degree and duration of immunodefi ciency. rsv infections in hsct recipients typically follow the same clinical sequence as rsv infections in previously healthy children: signs and symptoms of a uri such as rhinorrhea, sinus congestion, sore throat, or otitis media frequently precede signs of lower respiratory tract disease including cough, wheezing, hypoxia, and pneumonia [ 5 , 6 , 9 ] (tables 31-3 and 31-4 ). the presence of wheezing with respiratory symptoms during the respiratory virus season may provide the clue that rsv may be present. progression of uri to lri has been associated with patients who are early (<1 month) posttransplant, risk factors for the progression of rsv upper respiratory disease to lower tract disease or pneumonia and factors relating to fatal disease have been evaluated. the most common risk factor described in multiple centers using different methods of case ascertainment and viral detection remains lymphopenia. in a prospective multicenter study carried out by the european group for blood and marrow transplantation, lymphopenia but not neutropenia significantly increased the risk for lower respiratory tract disease [ 80 ] . older age and donor status are also signifi cant risk factors in some studies, whereas cmv serostatus, acute graft versus host disease, time relative to engraftment, and preemptive aerosolized ribavirin at a low dose of 2 h daily were not signifi cant [ 81 ] . investigators from mdacc have demonstrated that season of year, relapse of malignancy, presence of graft versus host disease, increasing age, and lack of engraftment are inpatient risk factors for the development of rsv pneumonia [ 9 , 12 , 22 , 82 ] . recently, large retrospective studies have identifi ed graft source including cord or marrow (adjusted hazard ratio (hr), 4.1, 95% ci 1.8-9.0) and oxygen requirement (adjusted hr 3.3, 98% ci, 15-6.7) to be independently associated with death due to respiratory failure in hsct recipients [ 83 ] . smoking history, conditioning with high-dose total body irradiation, and an absolute lymphocyte count < 100/mm 3 at the time of uri onset are also signifi cantly associated with disease progression [ 84 ] . since initiation of therapy hinges on prompt diagnosis, the possibility of false negative laboratory tests must be considered in individual patients and the diagnosis should be aggressively pursued by other means, such as bal. regardless of the therapeutic intervention, high rates of mortality due to rsv pneumonia are documented in seriously immunocompromised patients when therapy has been initiated after the development of respiratory failure (figure 31-2a ) . survival rates remain low for severely immunosuppressed patients who are intubated due to progressive rsv pneumonia unrelated to super-imposed pulmonary hemorrhage, pulmonary edema, or bacterial superinfection [ 5 , 50 , 55 , 81 , 83 ]. hmpv may cause upper or lower respiratory tract infections in hsct recipients. asymptomatic shedding from upper respiratory tract has been reported, indicating that not all hmpv infections result in severe lower tract disease [ 50 , 66 , 85 ] . hsct recipients with hmpv disease in the immediately posttransplant period typically present with respiratory symptoms including nasal congestion, sore throat, cough, or fever. once lower respiratory tract disease develops, rapidly progressive pulmonary infi ltrates frequently accompanied by hypotension, septic shock, or both may be present [ 64 ] ( figure 31-2b and c ) . in a prospective viral surveillance in hsct recipients where samples for respiratory viruses were obtained weekly, regardless of the presence of respiratory symptoms, a cumulative incidence estimate of hmpv of 6.2% (95% ci, 1.3-11.2) over 1 year was determined; all cases of hmpv had clinical respiratory symptoms identifi ed ranging from mild to more severe disease with single or multiple symptoms [ 50 ] (table 31-4 ) . among 15 hsct recipients with hmpv detected by rt-pcr in bal, 10 (67%) had positive hmpv rt-pcr in nasal wash sampled within 11 days prior to or following the bal [ 49 ] . viral rna was detected in the serum of one of these severely immunocompromised hsct recipients at two time points, 4 days apart, with a viral load of ~8 log10 copies/ml in each sample. this patient died of severe respiratory disease. in assessing risk factors associated with overall mortality at day 100 posttransplant, the use of bone marrow as the stem cell source, steroid treatment and oxygen use have been associated with overall mortality [ 49 ] . radiographic fi ndings associated with hmpv infection in the hsct recipient may consist of centrilobular nodules, ground glass opacities, tree-in-bud to diffuse bilateral alveolar infi ltrates [ 86 , 87 ] (figure 31 -2b-f ). centrilobular nodules have been associated with less mechanical ventilation while ground glass opacities tended to be associated with increased rates of hypoxemia [ 49 ] . alveolar consolidation corresponds to more extensive damage on histological examination. histological evaluation has also shown hyaline membrane formation, foci of bronchiolitis obliterans and organizing pneumonia and diffuse alveolar hemorrhage. variable disease severity has been reported following hmpv infection in sot recipients. in one study of 114 lung transplant recipients, hmpv was detected in four symptomatic and one asymptomatic patients (4.3%), but viral infection was not persistent and resolved without major complications [ 88 ] . in another study, nine lung transplant recipients with hmpv were compared with 17 transplant recipients without hmpv infection; hmpv infection was associated with signs of acute graft rejection and increase overall mortality (three of nine with hmpv-infected patients died and none died in the hmpv-negative group) [ 54 ] . outbreaks of respiratory viruses occur annually in the community, with potential for patients, families, or health care workers to become infected following exposure outside the hospital. however, nosocomial transmission within the hospital setting becomes a serious concern because of the high rates of morbidity and mortality in immunocompromised patients documented in nosocomial outbreaks. hospitalbased oubreaks of rsv infection in hsct recipients can occur through introduction of circulating community strains as well as transmission of identical viral strains among patients [ 42 , 44 ] . outbreaks of rsv have been associated with high mortality rates ranging up to 45% of infected patients [ 89 , 90 ] . the transmission of identical strains of rsv within the outpatient setting into the hospital setting has also been shown [ 91 ] , demonstrating the importance of infection control measures in both the inpatient and outpatient setting. nosocomial transmission of hmpv can also occur, with outbreaks possible in both inpatient and outpatient units. in one study, 15 patients were diagnosed with hmpv within 7 weeks in a tertiary care cancer unit [ 92 ] . molecular subtyping revealed infection with genotype a2a virus, implicating nosocomial transmission. four patients (26.6%) died from hmpv-associated pneumonia and consequent multi-organ failure. current options for the antiviral therapy of rsv disease in immunocompromised hosts are limited. large, controlled, therapeutic trials for rsv pneumonia or lower tract disease in immunocompromised patients have not been conducted. aerosolized ribavirin, licensed in the usa for the therapy of rsv bronchiolitis and pneumonia in infants and young children in 1986, is the antiviral agent currently utilized for the treatment of rsv disease in immunocompromised patients. in general, ribavirin is given as an inhaled aerosolized solution via a face mask in a protective environment, such as a "scavenging tent," to protect health care workers from potential drug contamination. ribavirin was initially licensed for use when given for 12-18 h/day, but the use of intermittent aerosolized ribavirin given over 2 h, three times daily, was found to have similar effectiveness to 12-16 h/day continuous infusion ribavirin in healthy children [ 93 -95 ] and has been utilized in adults because of ease of administration and enhanced tolerability. a randomized trial in hsct patients at risk for lrti evaluated intermittent dosing of ribavirin given over 2 h three times daily versus continuous ribavirin administration using an adaptive randomized trial design in 50 hsct patients, with the authors concluding that the intermittent schedule was preferable because of ease of administration and evidence of higher effi cacy [ 96 ] . only one randomized, controlled, multicenter clinical trial of aerosolized ribavirin for the prevention of rsv disease progression to lri has been conducted in hsct recipients early posttransplant, and despite an enrollment period of several years, only 15 patients were enrolled [ 97 ] . none of the ten patients randomized to high dose, short duration aerosolized ribavirin (administered as 2 g/100 ml water given over 2 h three times daily) had disease progression compared to 2/5 control patients, a trend that was not statistically significant ( p = 0.08). viral loads appeared to be reduced during the ribavirin treatment, but did rebound after cessation of therapy. data demonstrating effectiveness of ribavirin is mainly retrospective. in an open trial in adult hsct recipients with "rsv-induced acute lung injury," monotherapy with aerosolized ribavirin was reported to be of benefi t if initiated prior to the development of radiographic infi ltrates [ 6 ] . in another open trial in adult hsct recipients with radiographically proven rsv pneumonia, combination therapy with aerosolized ribavirin and high rsv-titered ivig was reported to be of benefi t only if initiated prior to the onset of respiratory failure [ 8 , 98 ] . a retrospective mdacc study of confi rmed rsv infections in 280 allogeneic hsct recipients from 1996 to 2009 utilized multivariable logistic regression to demonstrate that lack of ribavirin aerosol therapy at the upper respiratory tract disease stage was an important risk factor associated with rsv lrti and all-cause mortality [ 99 ] . in a retrospective study of hsct recipients with confi rmed lower respiratory tract rsv infection based on analysis of bronchoalveolar lavage fl uid at the hutch, viral rna detection in the blood was detected in 30% of 92 patients at a median of 2 days following diagnosis of lower respiratory tract disease [ 37 ] . neutropenia, thrombocytopenia, and mechanical ventilation increased the risk of rsv rna detection in the plasma or serum but lymphopenia and steroid use did not. the detection of rsv rna in the serum or plasma increased the risk of overall mortality with an adjusted hazard ratio (ahr) of 2.09 ( p = 0.02). data in solid organ transplant recipients is even more limited. favorable responses have been reported in an open trial of lung transplant recipients with lower respiratory tract disease who received monotherapy with aerosolized ribavirin [ 58 ] , as well as open trials with oral ribavirin [ 100 -102 ] , although controlled studies have not been performed. oral ribavirin was found to be well-tolerated, result in less hospitalization, and be less expensive than intravenous or inhaled ribavirin in a retrospective study of 52 lung transplant recipients [ 102 ] . the treatment of rsv disease with a combination of antiviral therapy and passively administered immunoglobulin has been investigated in animal models and in children [ 103 -106 ] . therapy with ivig containing high levels of rsvspecifi c antibodies alone does not seem to be effective in placebo-controlled trials in children who were not immunocompromised [ 104 , 107 ] . in small open trials at mdacc, combination therapy with aerosolized ribavirin (18 h/day) and high rsv-titered ivig (0.5 g/kg every other day) was associated with a favorable response in adult hsct recipients and patients undergoing induction chemotherapy for leukemia who had rsv lower respiratory tract disease in whom therapy was initiated prior to respiratory failure [ 8 , 98 ] . at the dana farber cancer institute, combination therapy with aerosolized ribavirin (18 h/day) and rsv-ivig (1.5 g/kg for one dose) was similarly associated with a favorable response in 2 hsct recipients with clinically severe rsv pneumonia occurring early following transplant [ 105 ] . in subsequent years, mdacc has utilized a combined regimen with similar response, although standard ivig in frequent and large doses (500 mg/kg qod) has been substituted for high-titered ivig. other therapeutic options for the treatment of rsv include the use of oral ribavirin, which has been studied in hsct recipients and found to be safe and less expensive than intravenous or aerosolized ribavirin [ 100 , 108 , 109 ] , and is recommended as an option in addition to intravenous and inhaled ribavirin by the european conference on infections in leukemia [ 94 ] . other options include iv ribavirin (an investigational drug, icn) and topical immunoglobulins administered with aerosolized or intravenous ribavirin [ 105 , 110 , 111 ] . the relative ease of administration of iv ribavirin is attractive, but high rates of mortality (80%) and signifi cant cases of hemolytic anemia (20%) make this option currently problematic. although the european experience with combination aerosolized/intravenous ribavirin has been favorable [ 112 ] and intravenous ribavirin is relatively simple to administer, the high rates of mortality and signifi cant cases of hemolytic anemia make this approach controversial. monotherapy with iv ribavirin may be more toxic in these patients than has been previously reported in patients with hemorrhagic fevers [ 113 , 114 ] . the decision to initiate therapy with aerosolized ribavirin with or without immunotherapy for a rsv-uri must take into consideration many factors, including the patient's risk of developing serious lower respiratory tract disease (and specifi cally, the degree of anticipated lymphopenia), the potential exposure of health care workers to the medication, the psychological and physical discomfort to the patients of aerosol therapy, the adverse effects of aerosolized ribavirin such as bronchospasm, the high cost of these drugs as well as the intensive respiratory therapy needed to safely administer aerosolized ribavirin, and the need for hospitalization with more frequent or prolonged ribavirin dosing regimens. in patients who have already undergone conditioning therapy and stem cell infusion but have not yet engrafted, the initiation of antiviral therapy at the uri stage may be beneficial. early studies conducted in the 1990s were small and uncontrolled. one study conducted at fhcrc treated 25 hsct recipients with upper tract rsv disease with low dose aerosolized ribavirin administered at a high concentration (60 mg/ml) for 2 h each day (total: 2 g/day [ 8 ] ). unfortunately, 8/25 patients developed pneumonia, and seven of these died. another study evaluated combination therapy with aerosolized ribavirin and 500 mg/kg ivig every other day in 12 patients, two of whom developed pneumonia and died [ 8 , 115 ] . this "preemptive" strategy, similar to that used in the prevention of cmv disease and cmv pneumonia, is used at some transplant centers for those patients at highest risk of rsv disease progression, such as pre-engrafted patients with rsv detected in the fi rst weeks following transplantation. other options for preemptive therapy include immunotherapy with ivig or rsv-specifi c monoclonal antibodies, although little data on effi cacy of immunotherapy is available. no antivirals for the therapy of hmpv are currently available or routinely utilized. ribavirin is active in vitro and in vivo against hmpv, although there are no controlled studies or evidence from large retrospective reviews for the treatment of hmpv pneumonia in humans and no drug has yet demonstrated clinical effectiveness in humans [ 116 , 117 ] . intravenous, oral or inhaled ribavirin alone or in combination with ivig has been reported as potentially successful therapeutic options. a retrospective analysis compared the outcome between 13 immunocompromised patients with hmpv pneumonia treated with ribavirin â± ivig and ten untreated patients. ribavirin treatment was associated with more hypoxemia and similar mortality, possibly related to late initiation of therapy [ 49 ] . a seattle study describing hmpv lower respiratory tract disease in 55 immunocompromised children, including nine undergoing hsct and eight sot recipients, demonstrated that hsct recipients had more evidence of severe disease [ 91 ] . five of eight hsct recipients but no sot recipients had lower tract disease and were treated with aerosolized ribavirin; three had been diagnosed with hmpv pretransplant and during the posttransplant period received both ribavirin and ivig. two additional children received aerosolized ribavirin only. ribavirin was generally administered at a dose of 2 g given three times daily for 5-11 days. two of the three patients diagnosed with hmpv pretransplant who received ribavirin and ivig died [ 91 ] . an aggressive infection control strategy can be effective in reducing the nosocomial acquisition of rsv by transplant recipients [ 91 , 118 ] . infection control strategies play a crucial role in the prevention of respiratory viral infection [ 89 , 118 -120 ] . an effective strategy is based on understanding the potential seriousness of these infections in transplant recipients, knowledge of the viruses circulating in the community, and ongoing surveillance in high-risk patients. continuing education of patients, family members, visitors, and staff regarding the potential seriousness of these infections must be repeatedly emphasized. frequent and routine clinical screening of high-risk patients for acute upper and/or lower respiratory tract illness or fl u-like illness must be conducted, with sampling of respiratory secretions from symptomatic high-risk patients routinely performed both pretransplant and posttransplant. each health care-acquired infection should be viewed as a sentinel event warranting an investigation and reaffi rmation or modifi cation of the preventative strategy. infection control strategies should be designed to prevent spread by multiple modes of transmission [ 90 , 91 ] . multiple respiratory viruses may circulate in the community concurrently and can be spread by different means. infection control measures may need to be intensifi ed during community or hospital outbreaks, and the intensity and duration of infection control measures should be tailored to the risk of serious disease in different subsets of transplant recipients, and to what works in the "real world." guidelines for the prevention of opportunistic infections among hsct recipients have been issued by the centers for disease control and prevention (cdc), the infectious diseases society of america, and european and american societies for blood and marrow transplantation. the guidelines clearly present evidencebased recommendations rated by the strength of the recommendation and the quality of the supporting evidence, similar to guidelines previously issued for the prevention of opportunistic infections in those with human immunodefi ciency virus [ 120 ] . preventive strategies for hsct recipients, their household contacts and other close contacts, and health care workers are clearly outlined in this document. the prevention of nosocomial acquisition of respiratory viral infections in hsct recipients has been demonstrated in one prospective study comparing rates of infection in patients cared for in a "protected environment" with patients cared for on a transplant unit where infection control measures were strongly encouraged, but not rigidly enforced [ 9 ] . the effectiveness of infection control interventions has also been demonstrated by the dramatic decline in the frequency of nosocomial crv infections among hsct recipients cared for in this transplant unit after the implementation of an aggressive, multifaceted infection control strategy [ 118 ] . although this intensive multifaceted approach has been effective, modifi ed versions of this strategy have also been effective. for instance, the seattle cancer care alliance (scca ) adult inpatient transplant unit uses a similar strategy with the exception that health care workers and other visitors do not wear masks when entering the patient room [ 91 ] . however, these workers are intensively screened for signs and symptoms of respiratory illnesses prior to entering the unit, and restricted from entering the unit if they are symptomatic. similarly, hsct recipients with respiratory symptoms are not transferred to other units, but are cared for on the transplant unit using modifi ed droplet precautions with all persons entering the room wear gloves, gown, and mask, and the door to the room is kept closed. it may not be feasible to so intensively protect patients for the duration of increased susceptibility to respiratory viral diseases. protective strategies are costly and cumbersome, and pose unpleasant restrictions on the freedom and quality of life of the patient and their families. this problem is further compounded by the growing trend to discharge patients early from the hospital and to perform outpatient hsct or posttransplant care. transplant recipients residing in the community and followed frequently in the outpatient setting are another group in which infection control practices must become priority [ 91 ] . the prevention of exposure to respiratory viruses is particularly challenging among high-risk transplant recipients living in the community because respiratory infections are so prevalent and so contagious. examples of protective measures for outpatients include washing hands frequently and thoroughly, avoiding close contact with individuals suffering from respiratory illnesses, and encouraging close contacts to vigorously practice respiratory hygiene. in many cases, such as individuals living with children, such efforts may be nearly impossible. consideration of removing day care exposures for young children or decreasing exposure to transplant recipients to children (including siblings), can and should be discussed with families. the rigor and duration of prophylactic measures need to be individualized based on the immunologic status of the patient and the risk for serious disease, the needs of the patient, and quality-of-life issues. passive immunization with immunoglobulin, immunoglobulin products, and humanized monoclonal antibodies have been actively studied in the pediatric infant population. palivizumab , a humanized monoclonal antibody directed against the rsv f protein, is licensed for the prevention of rsv disease in premature infants and infants with congenital heart disease, and is administered as a monthly injection during the 4-5 months of rsv season. the cost of this therapy has led to new guidelines for use in the pediatric population [ 121 ] . similar interventions have been utilized to attempt to decrease the morbidity of serious rsv disease in hsct recipients but direct proof of effectiveness has not yet been demonstrated [ 115 ] . for example, passive immunoprophylaxis in immunocompromised patients has been evaluated in an open trial conducted [ 122 ] using high-titered human rsvig. in this adult study, signifi cant antibody titer increases to other respiratory viruses were extremely variable, although the subset of patients with the lowest titers appeared to receive the greatest increase in viral-specifi c antibody. the cost of this potential therapy remains quite high. the monoclonal rsv antibody palivizumab (synagis) has been studied in an open label study in adult hsct recipients [ 123 ] . immunoprophylaxis with monoclonal rsv antibodies would be prohibitively expensive in older children and adults, but may have the potential to protect against rsv infection, based on pediatric studies. new antibody products, including long-lasting monoclonal antibodies with enhanced activity against rsv, are under development and if available at a less expensive price, could potentially provide protection against rsv for patients in the pretransplant and immediate posttransplant phase. newer monoclonal antibodies that have the potential to neutralize against both rsv and hmpv have also been described, offering hope for newer preventive modalities that may protect against both these viruses [ 124 ] . screening of transplant recipients for respiratory viruses prior to transplant is not routinely recommended based on current us and international transplant guidelines [ 94 , 125 ] . however, the assessment of viral shedding or infection in symptomatic transplant candidates prior to transplant is recommended. delay of transplant based on detection of rsv or hmpv may be warranted depending on the evaluation of the risk and benefi ts of continuing on with transplantation. consequences of postponing transplantation should be considered, including progression of underlying malignancy, logistical issues regarding the donor availability, and accessibility of services for the patient. an early study of rsv diagnosed prior to hsct demonstrated that delaying transplant reduced the risk of pneumonia following transplant [ 23 ] . more recently, a large prospective study conducted in 458 patients at the hutch demonstrated that nearly 25% of subjects had respiratory viruses detected pretransplant [ 24 ] . overall, patients with a virus detected prior to transplant had fewer days alive and lower survival at day 100 (ahr 2.4; 95% ci 1.3-4.5) compared with patients who did not have viruses. in hsct recipients who had respiratory symptoms and a virus detected prior to transplant (mainly adults), an increased overall mortality was seen compared with patients without respiratory viruses detected. higher rates of pretransplant infection and sequelae of infection were seen in pediatric patients. detection of respiratory viruses in asymptomatic patients was not associated with increased mortality. this data strengthens current guidelines that recommend patients with respiratory symptoms prior to transplant should be tested for respiratory viruses and transplant delayed, when feasible. however, this study was performed chiefl y in adults. the higher rates of respiratory viruses documented in children pretransplant make routine screening of pediatric patients worthy of further investigation. experimental approaches to the therapy of rsv antiviral therapy include novel fusion inhibitors [ 126 ] , nucleoside agents, small rna inhibitory molecules [ 109 ] , and high-titered monoclonal antibody preparations. two promising rsv antivirals that have shown effi cacy in challenge studies in healthy adults include the alios compound al8176 (alios biopharma, south san francisco, ca), an oral anti-rsv nucleoside designed to inhibit rsv replication by acting on the viral polymerase, and the gilead sciences compound gs5806 (gilead sciences, foster city, ca), an orally bioavailable rsv fusion inhibitor [ 126 , 127 ] . clinical trials of these agents in healthy young children with rsv have been proposed [ 128 ] . an international multicenter, placebo-controlled clinical trial of gs5806 was initiated in july 2014, and remains ongoing in adult hsct recipients with documented rsv infections (clinica ltrials.gov identifi er nct02135614). other antiviral agents are under development. no rsv or hmpv vaccine is currently available. in general, active immunization of transplant recipients will be unlikely to prevent severe disease seen in the fi rst several months posttransplant. however, prevention of rsv infection in families, staff, and nosocomial spread of virus by the use of vaccines holds true promise to benefi t transplant recipients themselves. promising advances in new vaccines directed against both rsv and hmpv have been reported over the past decade, with progress evident in both rsv fusionprotein based vaccines and live attenuated vaccines. advances in the understanding of the pre-and post-fusion nature of the rsv f protein [ 129 ] has led to increased work in developing protein-based vaccines appropriate for older children, adults, and pregnant women. advances in technology and better molecular understanding of rsv and hmpv have resulted in new potential rsv candidate vaccines [ 130 ] . at least 12 rsv vaccines are in clinical studies in phase 1 or 2 clinical studies, with one rsv f vaccine under study in pregnant women. examples of live rsv vaccine candidates under study include live-attenuated vaccines relying on genetic manipulation of the rsv genome [ 131 ] , vectored virus vaccines utilizing the chimpanzee adenovirus or vaccinia virus ankara [ 132 ] , or chimeric viruses containing a backbone of attenuated parainfl uenza with the f gene of rsv added [ 133 , 134 ] . a chimeric hmpv vaccine containing a backbone of avian hmpv and f genes of the hmpv [ 135 ] . although live viral vaccines are unlikely to be given to transplant recipients pretransplant or early posttransplant, they offer hope for the potential control of rsv and hmpv disease in family members and health care workers the future. nonbacterial pneumonia after allogeneic marrow transplantation: a review of ten years' experience disseminated parainfl uenza infection in a child with severe combined immunodefi ciency cellular response to respiratory viruses with particular reference to children with disorders of cell-mediated immunity giantcell pneumonia caused by parainfl uenza virus respiratory syncytial virus infection in immunocompromised adults respiratory syncytial virus-induced acute lung injury in adult patients with bone marrow transplants: a clinical approach and review of the literature impact of respiratory virus infections on persons with chronic underlying conditions community respiratory virus infections in immunocompromised patients with cancer community respiratory virus infections among hospitalized adult bone marrow transplant recipients orthomyxoviral and paramyxoviral infections in transplant patients respiratory virus infections after marrow transplant: the fred hutchinson cancer research center experience viral pneumonia in the immunocompromised adult with neoplastic disease: the role of common community respiratory viruses respiratory virus infection in immunocompromised patients respiratory virus infections in bone marrow transplant recipients: the european perspective community respiratory virus infections in immunocompromised patients: hematopoietic stem cell and solid organ transplant recipients, and individuals with human immunodefi ciency virus infection characterization of human metapneumoviruses isolated from patients in north america virological features and clinical manifestations associated with human metapneumovirus: a new paramyxovirus responsible for acute respiratorytract infections in all age groups recovery of cytopathogenic agent from chimpanzees with coryza epidemic bronchiolitis and pneumonitis related to respiratory syncytial virus a newly discovered human pneumovirus isolated from young children with respiratory tract disease the distinguishing features of human metapneumovirus and respiratory syncytial virus diagnosis and epidemiology of communityacquired respiratory virus infections in the immunocompromised host pretransplantation respiratory syncytial virus infection: impact of a strategy to delay transplantation clinical outcomes associated with respiratory virus detection before allogeneic hematopoietic stem cell transplant clinical and fi nancial benefi ts of rapid detection of respiratory viruses: an outcomes study clinical and economical impact of multiplex respiratory virus assays comparison of respiratory virus detection rates for infants and toddlers by use of fl ocked swabs, saline aspirates, and saline aspirates mixed in universal transport medium for room temperature storage and shipping swabbing for respiratory viral infections in older patients: a comparison of rayon and nylon fl ocked swabs comparison of nasopharyngeal nylon fl ocked swabs with universal transport medium and rayon-bud swabs with a sponge reservoir of viral transport medium in the diagnosis of paediatric infl uenza self-collection of foam nasal swabs for respiratory virus detection by pcr among immunocompetent subjects and hematopoietic cell transplant recipients comparison of nasopharyngeal fl ocked swabs and nasopharyngeal wash collection methods for respiratory virus detection in hospitalized children using real-time polymerase chain reaction implementation of fi lmarray respiratory viral panel in a core laboratory improves testing turnaround time and patient care comparison of three commercial rt-pcr systems for the detection of respiratory viruses evaluation of quantitative and type-specifi c real-time rt-pcr assays for detection of respiratory syncytial virus in respiratory specimens from children application of taqman low-density arrays for simultaneous detection of multiple respiratory pathogens detection and quantifi cation of human metapneumovirus in pediatric specimens by real-time rt-pcr respiratory syncytial virus lower respiratory disease in hematopoietic cell transplant recipients: viral rna detection in blood, antiviral treatment, and clinical outcomes evaluation of r-mix freshcells in shell vials for detection of respiratory viruses rapid detection of respiratory viruses by shell vial culture and direct staining by using pooled and individual monoclonal antibodies clinically useful method for the isolation of respiratory syncytial virus detection of human metapneumovirus antigens in nasopharyngeal secretions by an immunofl uorescent-antibody test nosocomial transmission of respiratory syncytial virus in immunocompromised adults antigenic and nucleic acid analysis of nosocomial isolates of respiratory syncytial virus molecular characterization of strains of respiratory syncytial virus identifi ed in a hematopoietic stem cell transplant outpatient unit over 2 years: community or nosocomial infection? respiratory tract infections due to human metapneumovirus in immunocompromised children respiratory viral infections in immunocompetent and immunocompromised persons viral infections in pediatric bone marrow transplant patients changing epidemiology of respiratory viral infections in hematopoietic cell transplant recipients and solid organ transplant recipients mortality rates of human metapneumovirus and respiratory syncytial virus lower respiratory tract infections in hematopoietic cell transplantation recipients respiratory virus infection among hematopoietic cell transplant recipients: evidence for asymptomatic parainfl uenza virus infection respiratory syncytial virus infections in pediatric liver transplant recipients incidence and outcomes of respiratory viral infections in lung transplant recipients: a prospective study the impact of rsv, adenovirus, infl uenza, and parainfl uenza infection in pediatric patients receiving stem cell transplant, solid organ transplant, or cancer chemotherapy human metapneumovirus infection in lung transplant recipients: clinical presentation and epidemiology prospective study of the incidence, clinical features, and outcome of symptomatic upper and lower respiratory tract infections by respiratory viruses in adult recipients of hematopoietic stem cell transplants for hematologic malignancies respiratory tract viral infections in bone marrow transplant patients respiratory viruses in bronchoalveolar lavage: a hospital-based cohort study in adults paramyxovirus infection in lung transplant recipients community respiratory viruses: organ transplant recipients severe respiratory syncytial virus pneumonia in an adult renal transplant recipient: successful treatment with ribavirin differences in clinical severity between genotype a and genotype b human metapneumovirus infection in children comparison of risk factors for human metapneumovirus and respiratory syncytial virus disease severity in young children a prospective study comparing human metapneumovirus with other respiratory viruses in adults with hematologic malignancies and respiratory tract infections brief communication: fatal human metapneumovirus infection in stem-cell transplant recipients detection of severe human metapneumovirus infection by real-time polymerase chain reaction and histopathological assessment persistent symptomless human metapneumovirus infection in hematopoietic stem cell transplant recipients frequency of human metapneumovirus infection in hematopoietic sct recipients during 3 consecutive years clinical characterization of human metapneumovirus infection among patients with cancer human metapneumovirus infection in hematopoietic stem cell transplant recipients respiratory viral infections in hematopoietic stem cell and solid organ transplant recipients physical and chemical methods for enhancing rapid detection of viruses and other agents the effect of centrifugation on the rapid detection of adenovirus in shell vials incidence and morbidity of human metapneumovirus and other community-acquired respiratory viruses in lung transplant recipients a prospective molecular surveillance study evaluating the clinical impact of communityacquired respiratory viruses in lung transplant recipients immunofl uorescence with respiratory syncytial virus comparison of fl ocked and rayon swabs for collection of respiratory epithelial cells from uninfected volunteers and symptomatic patients respiratory metapneumoviral infection without co-infection in association with acute and chronic lung allograft dysfunction infl uenza in children and young adults with cancer: 20 cases respiratory syncytial virus pneumonia in hospitalized adult patients with leukemia respiratory virus infections after stem cell transplantation: a prospective study from the infectious diseases working party of the european group for blood and marrow transplantation community-acquired respiratory syncytial virus and parainfl uenza virus infections after hematopoietic stem cell transplantation: the fred hutchinson cancer research center experience respiratory viral infections in adults with hematologic malignancies and human stem cell transplantation recipients: a retrospective study at a major cancer center outcome of respiratory syncytial virus lower respiratory tract disease in hematopoietic cell transplant recipients receiving aerosolized ribavirin: signifi cance of stem cell source and oxygen requirement respiratory syncytial virus in hematopoietic cell transplant recipients: factors determining progression to lower respiratory tract disease long-term study on symptomless human metapneumovirus infection in hematopoietic stem cell transplant recipients viral pneumonia after hematopoietic stem cell transplantation: high-resolution ct fi ndings highresolution ct and pathologic fi ndings of noninfectious pulmonary complications after hematopoietic stem cell transplantation diagnosis of human metapneumovirus infection in immunosuppressed lung transplant recipients and children evaluated for pertussis an outbreak of respiratory syncytial virus in a bone marrow transplant center detection and control of a nosocomial respiratory syncytial virus outbreak in a stem cell transplantation unit: the role of palivizumab nosocomial transmission of respiratory syncytial virus in an outpatient cancer center serious outbreak of human metapneumovirus in patients with hematologic malignancies high-dose, short-duration ribavirin aerosol therapy in children with suspected respiratory syncytial virus infection ecil-4): guidelines for diagnosis and treatment of human respiratory syncytial virus, parainfl uenza virus, metapneumovirus, rhinovirus, and coronavirus management of respiratory viral infections in hematopoietic cell transplant recipients and patients with hematologic malignancies an adaptive randomized trial of an intermittent dosing schedule of aerosolized ribavirin in patients with cancer and respiratory syncytial virus infection randomized controlled multicenter trial of aerosolized ribavirin for respiratory syncytial virus upper respiratory tract infection in hematopoietic cell transplant recipients combination therapy with aerosolized ribavirin and intravenous immunoglobulin for respiratory syncytial virus disease in adult bone marrow transplant recipients impact of aerosolized ribavirin on mortality in 280 allogeneic haematopoietic stem cell transplant recipients with respiratory syncytial virus infections mayo clinic ho and transplant infectious diseases s. oral ribavirin therapy for respiratory syncytial virus infections in moderately to severely immunocompromised patients oral versus inhaled ribavirin therapy for respiratory syncytial virus infection after lung transplantation oral ribavirin for respiratory syncytial virus infection after lung transplantation: effi cacy and cost-effi ciency respiratory syncytial virus and human metapneumovirus infection in transplant recipients immunoglobulin administration and ribavirin therapy: effi cacy in respiratory syncytial virus infection of the cotton rat intravenous immunoglobulin treatment of respiratory syncytial virus infections in infants and young children immunotherapy of respiratory syncytial virus pneumonia following bone marrow transplantation respiratory syncytial virus immune globulin treatment of rsv lower respiratory tract infection in previously healthy children respiratory syncytial virus infections in adult bone marrow transplant recipients respiratory syncytial virus infection in recipients of allogeneic stem-cell transplantation: a retrospective study of the incidence, clinical features, and outcome a randomized, double-blind, placebo-controlled study of an rnaibased therapy directed against respiratory syncytial virus phase i study of intravenous ribavirin treatment of respiratory syncytial virus pneumonia after marrow transplantation prevention and treatment of respiratory syncytial virus and parainfl uenza viruses in immunocompromised patients ribavirin therapy in bone marrow transplant recipients with viral respiratory tract infections lassa fever. effective therapy with ribavirin prospective, double-blind, concurrent, placebo-controlled clinical trial of intravenous ribavirin therapy of hemorrhagic fever with renal syndrome respiratory syncytial virus upper respiratory tract illnesses in adult blood and marrow transplant recipients: combination therapy with aerosolized ribavirin and intravenous immunoglobulin comparison of the inhibition of human metapneumovirus and respiratory syncytial virus by ribavirin and immune serum globulin in vitro effect of ribavirin and glucocorticoid treatment in a mouse model of human metapneumovirus infection infection control of nosocomial respiratory viral disease in the immunocompromised host seasonal infl uenza in adults and children-diagnosis, treatment, chemoprophylaxis, and institutional outbreak management: clinical practice guidelines of the infectious diseases society of america infectious disease society of a, american society of b and marrow t. guidelines for preventing opportunistic infections among hematopoietic stem cell transplant recipients updated guidance for palivizumab prophylaxis among infants and young children at increased risk of hospitalization for respiratory syncytial virus infection immune-globulin prophylaxis of respiratory syncytial virus infection in patients undergoing stem-cell transplantation phase 1 evaluation of the respiratory syncytial virus-specifi c monoclonal antibody palivizumab in recipients of hematopoietic stem cell transplants a broadly neutralizing human monoclonal antibody exhibits in vivo effi cacy against both human metapneumovirus and respiratory syncytial virus guidelines for preventing infectious complications among hematopoietic cell transplantation recipients: a global perspective oral gs-5806 activity in a respiratory syncytial virus challenge study chemotherapy of respiratory syncytial virus infections: the fi nal breakthrough preventing severe respiratory syncytial virus disease: passive, active immunisation and new antivirals structural basis of respiratory syncytial virus neutralization by motavizumab the changing landscape of respiratory syncytial virus live-attenuated respiratory syncytial virus vaccines chimpanzee adenovirus-and mva-vectored respiratory syncytial virus vaccine is safe and immunogenic in adults phase 1 study of the safety and immunogenicity of a live, attenuated respiratory syncytial virus and parainfl uenza virus type 3 vaccine in seronegative children attenuated human parainfl uenza virus type 1 (hpiv1) expressing the fusion glycoprotein of human respiratory syncytial virus (rsv) as a bivalent hpiv1/rsv vaccine new approaches for immunization and therapy against human metapneumovirus key: cord-016009-qa7bcsbu authors: starkel, julie l.; stapke, christina; stanley-o’malley, abigail; noland, diana title: respiratory date: 2019-10-07 journal: integrative and functional medical nutrition therapy doi: 10.1007/978-3-030-30730-1_51 sha: doc_id: 16009 cord_uid: qa7bcsbu lung disease rivals the position for the top cause of death worldwide. causes and pathology of the myriad lung diseases are varied, yet nutrition can either affect the outcome or support treatment in the majority of cases. this chapter explores the modifiable risk factors, from lifestyle changes to dietary intake to specific nutrients, anti-nutrients, and toxins helpful for the nutritionist or dietitian working with lung disease patients. general lung health is discussed, and three major disease states are explored in detail, including alpha-1 antitrypsin deficiency, asthma, and idiopathic pulmonary fibrosis. although all lung diseases have diverse causes, many integrative and functional medical nutrition therapies are available and are not being utilized in practice today. this chapter begins the path toward better nutrition education for the integrative and functional medicine professional. anti nutrients and inhibitors of lung physiology -942 51 lung disease is far more prevalent worldwide than commonly thought. in fact, death from chronic lung disease is increasing, and as of 2017, chronic obstructive pulmonary disease (copd) has become the third leading cause of death in the united states in the past decade, disproportionately affecting the elderly [1] . another lung disease, asthma, affects 1 in 13, or about 25 million americans, according to the centers for disease control and prevention and the national center for health statistics [2] . this is 7.6% of adults, more women than men, and 8.4% of children. asthma is the leading chronic disease in children [3] . this disease has been increasing since the early 1980s in all age, sex, and racial groups. in europe, lung disease represents 15% of all deaths -the fourth leading cause. according to the world health organization (who), in 2008, 9.5 million people died from acute or chronic lung disease, representing one sixth of the global total deaths [4] . worldwide, four respiratory disease categories appear in the top ten leading causes of death in 2010 [5] . specifically, copd was the third leading cause of death, followed by lower respiratory infections as the fourth, lung cancer as the fifth, and tuberculosis as the tenth [4] . the major risk factor is smoking, leading to 50% of all lung disease-related deaths in europe, where smoking is more prevalent (28% prevalence) than in the united states (15% prevalence) by nearly twofold [6, 7] . lung cancer, particularly non-small-cell lung cancer (nsclc) subtype, is the leading cause of cancer-related death worldwide [8] . added together, lung disease rivals the position for the top cause of death. throughout the life cycle, diet and lifestyle are important modifiable risk factors in the development, progression, and management of obstructive lung diseases, such as asthma and copd [9] , as well as restrictive lung diseases such as pulmonary fibrosis and sarcoidosis. inflammation, in particular, seems to be the leading contributor toward the progression of lung diseases. as with many diseases, maintaining a healthy lifestyle, including sufficient sleep, low stress, regular exercise, a whole foods diet rich in phytonutrients from plants (fruits and vegetables), and potential anti-inflammatory supplements, is beneficial in supporting the body during these difficult diseases. inflammation, in particular, seems to be the leading contributor toward the progression of lung diseases. high inflammatory foods should be avoided, such as fried foods and foods disproportionately high in carbohydrates, sugar, alcohol, and excessive protein. a healthier suggestion would be a diet with more than half of all food consumed as vegetables, about one third as protein, and the remainder (one sixth) as other foods, such as fruits, dairy, grains, or starches. some dietary supplements may also be recommended for their anti-inflammatory benefits, which will be discussed later in the chapter. as human life expectancy increases, we can expect to see more chronic disease. the world health organization estimates that by 2030, chronic lung disease will account for 20% (one fifth) of all deaths [10] , up from one sixth in 2008. despite these growing numbers, relatively little human nutrition research exists for respiratory health, compared to other, less prevalent, diseases. investigators in the areas of aging and lung biology suggest some hope, using genetics and animal models, as well as epidemiological research, to further the general medical approach to lung disease. the pulmonary system is composed of the upper and lower respiratory tracts. air flows in through the nose or mouth, past the frontal and maxillary sinuses, down the pharynx (throat), past the larynx (voice box), and then down the trachea. this makes up the upper respiratory tract. once past the trachea, the air divides into the left and right bronchi, which supply the left and right lungs, each divided into five sections called lobes. the bronchi then divide into smaller bronchioles, at the end of which are air sacs called the alveoli. this makes up the lower respiratory tract [11] (. fig. 51 .1). the diaphragm is the central muscle that is used for breathing. the intercostal muscles, located between the ribs, and the abdominal muscles are helpful for breathing out when the breath becomes labored, such as during exercise. the neck muscles and the muscles in the collarbone area help with breathing when the other muscles are compromised or impaired. in some neurological diseases, such as amyotrophic lateral sclerosis (als) [see 7 chap. 51, newton], nerve damage from the brain to breathing muscles can result in impaired movement of these muscles and thus impaired breathing. in certain cases, such as in lung cancer when a lobectomy, removal of part of the lung, is required, there is an expected decrease in short-and long-term pulmonary function and oxygenation. however, respiratory muscle strength may be preserved [13] . in a pneumonectomy, removal of the entire lung, dramatic changes in thoracic anatomy take place, such as elevation of the hemidiaphragm, hyperinflation of the remaining lung, and influx of fluid into the postpneumonectomy space [14, 15] . there are phagocytic macrophages on the cellular surface of the alveoli, type i epithelial cells and type ii epithelial cells. phagocytic macrophages destroy inhaled bacteria and serve an important role in suppressing or activating the immune response to antigens and pathogens, similar to dendritic cells discussed below. macrophage function has been shown to be inhibited by cigarette smoke [16] . alveolar macrophages also secrete enzymes, arachidonic acid metabolites, growth factors, immune response components, cytokines, and lymphocytes [17] . type i cells are responsible for maintaining the structure of the alveolar wall, whereas type ii cells and clara cells are responsible for the production of pulmonary surfactant (composed of 85-90% lipid and 10-15% protein as lecithin and myelin), which is essential for lung function. the surfactant reduces surface tension, facilitating easier stretching and collapsing of alveoli during respiration [18] . diseases associated with inadequate surfactant production are acute/adult respiratory distress syndrome (ards) and infant respiratory distress syndrome (irds) [19] . irds is seen in premature babies born prior to 32 weeks of gestation due to immature development of pulmonary surfactant, which only begins to develop around the 20th week of gestation [18] . dipalmitoylphosphatidylcholine, phosphatidylglycerol, and cholesterol compose the lipid portion of the surfactant, where apoproteins and proteins found in blood plasma compose the protein portion [18, 20] . the importance of cholesterol is minimized in today's medical community. those with higher levels of cholesterol tend to have more in their fatty cell membranes which resist pathogenesis at a cellular level. low cholesterol predicts a greater risk of dying from gastrointestinal, neoplastic, or respiratory diseases. it occupies 30-40% of our cell membranes, enhances the mechanical strength of the membrane, and reduces permeability [21] . it suppresses main-phase transition of the lipid bilayer [22] . collagen, a fibrous protein, along with elastin and proteoglycans, is a fundamental component of the connective tissue that composes the lungs, and collagen is present in the blood vessels, bronchi, and alveolar interstitium [23] . connective tissue in the lung is key for the passive diffusion of oxygen and carbon dioxide that characterizes alveolar-capillary gas [18] . collagen homeostasis is vital to maintaining respiratory function, where collagen production and degradation are balanced. dysregulated collagen homeostasis that favors collagen production over degradation can lead to pulmonary fibrosis and compromised lung function [24] . some key nutrients to consider for collagen synthesis and crosslinking to maintain connective tissue integrity are vitamin c, vitamin b6, iron, copper, zinc [25] , riboflavin, thiamin, and pantothenic acid [11] . the airways of the respiratory system (with the exception of parts of the nose and mouth) have cilia, special hairs coated with mucus that trap pathogens and other particles that enter with the air that is inhaled. cilia are responsible for triggering this mucus upward toward the pharynx where these particles or bacteria can be coughed out or swallowed. mucus present in the lungs can also trap inhaled particles such as viruses, bacteria, and smoke particulates [11, 12] . along the lining of the respiratory tract, there are several types of cells that are involved in immune response, such as secretory cells (i.e., goblet cells and clara cells) and mast cells. ciliated epithelium and mucus secreted by glands present on airways, goblet cells, and the secretory products of clara cells serve an important mechanism for lung protection. however, excessive goblet cells or hypertrophy of mucous glands may result in increased viscosity of mucus seen in pathologies like bronchitis [16] . ciliary function is also impaired by cigarette smoke [16] . dendritic cells are also found in the airway lining from the trachea to the alveoli. immature dendritic cells phagocytize bacteria or other antigens, where they then mature and travel to lymphoid tissues to communicate with the immune system. this delivery of antigens can promote tolerance of the antigen by releasing anti-inflammatory cytokines. conversely, this delivery can also trigger the opposite response if the antigen is recognized as a pathogen, where t lymphocytes are activated and inflammatory cytokines are released [16] . one potential cause of infections in the upper respiratory tract or bronchial tubes, such as bronchitis, or deep in the lungs, such as pneumonia, is when cilia become damaged and do not trap inhaled germs and particles as effectively. in diseases such as cystic fibrosis, thick mucus secretions can accumulate in the airways and lungs, making it hard to clear and thus increasing risk for infection. in asthma, specific inhaled particles can trigger a reaction causing the airways to narrow, restricting breathing [12] . surface enzymes and factors can also be found in the lining of the airways that compose the majority of the innate immune system of the respiratory tract. these include: 5 lysozymes: found in leukocytes with bactericidal properties 5 lactoferrin: a bacteriostatic agent (inhibits bacterial reproduction) synthesized by lymphocytes and glandular mucosal cells 5 alpha-1 antitrypsin: an antiprotease to protect lung tissue from excessive enzymatic activity 5 interferon: an antiviral substance that may be produced by lymphocytes and macrophages 5 complement: participates as a cofactor in antigenantibody reactions [16] gas exchange takes place in the alveoli so oxygen can enter the body to support metabolic function and the carbon dioxide product from these functions can be removed. this is accomplished through millions of capillaries in the alveoli. these capillaries in the alveoli then connect to arteries and veins that move blood throughout the body. the pulmonary artery supplies carbon dioxide-rich blood to these capillaries within the alveoli to remove carbon dioxide, and the oxygen-rich blood then gets delivered to the heart through the pulmonary vein. the lungs also serve the vital function of maintaining acid-base balance through changes in minute ventilation. these changes affect the ph of the blood by either retaining or excreting carbon dioxide [11] . poor physiologic management of co 2 and bicarbonate can lead to the conditions of respiratory acidosis and respiratory alkalosis. respiratory acidosis is characterized by higher blood concentrations of co 2 and h + , caused by hypoventilation or decreased rate of breathing. hypoventilation can have acute or chronic etiologies, resulting from copd, interstitial lung diseases, respiratory muscle fatigue (i.e., extended asthma attack), or mechanical abnormalities (i.e., deformities). respiratory alkalosis is characterized by lower blood concentrations of co 2 and h + due to hyperventilation, or increased rate of breathing. possible causes of hyperventilation can also be chronic or acute, such as pneumonia and fever, increased stress and anxiety, liver disease, stroke or meningitis, pregnancy, overuse of aspirin and/or caffeine, excessive mechanical ventilation, or increases in altitude [18] . a pulse oximeter tool can be used to measure the percentage of oxygenated hemoglobin in an individual's blood to determine their overall respiratory status. typically, oxygen saturations of 92% or less are indicative of central hypoxia [26] . pulse oximetry is especially useful for assessing individuals with asthma and copd [26] . oral health must also be considered as a contributing factor to respiratory health [27] . for example, in patients affected with periodontal disease, 1 mm of dental plaque could contain around 10 9 of bacteria. one potential mechanism of this connection is aspiration of bacteria from the oropharynx into the upper or lower respiratory tracts, leading to their adherence to the alveolar and bronchial lining, potentially colonizing respiratory ducts and causing respiratory infections. in addition, cytokines and enzymes associated with inflammation of periodontal tissues can be transferred into the lungs, potentially triggering or exacerbating lung infections [27] (. fig. 51 . a systematic review done in 2013 examined oral health in the elderly and its association with risk of aspiration pneumonia. this review suggested that maintaining oral health, such as brushing after each meal, cleaning dentures once per day, and professional oral healthcare, potentially reduced the amount of potential respiratory pathogens that resulted in lower incidence of aspiration pneumonia [28] . several other systematic reviews have found that adequate oral hygiene plays an important role in preventing pneumonia, particularly in clinical settings where there is increased risk for hospital-acquired pneumonia (hap) and ventilatorassociated pneumonia (vap), as well as in older populations [29] . in addition, associations have been made between copd and the risk of periodontitis, although systematic reviews have established that these associations are preliminary and further studies are needed [29] . another important consideration in respiratory health is orofacial development and structure. anatomical obstructions at the level of the nose and pharynx, such as those caused by allergic rhinitis and hypertrophy of the tonsils, pose an increased risk for obstructive sleep apnea syndrome and respiratory infections due to lack of airflow through the upper respiratory system [30] . it has been established that the lung has a microbiome of its own that may have a large impact on health and disease [31] . the fungal microbiome, or mycobiome, may also have a significant impact on respiratory health, although more research is needed to determine definitive associations [31] . dysbiosis may occur in the lungs with a bacterial infection. a few specific bacterial strains have been studied, and one, in particular, pseudomonas aeruginosa, seems to grow in inflammatory conditions. it then seems to encode inflammatory components causing further inflammation. anti-inflammatory nutrients could help stop the cycle, and vitamin d use has some research supporting this. recurrent bacterial respiratory infections may damage lungs and lead to worse outcomes in future lung disease [32] . an increased interest in research of the relationship of the airway and gut microbiome is indicating potentially positive results regarding the use of probiotics in pediatric populations that may aid in asthma prevention and intervention [33, 34] . the gut-lung axis has also been established, where the microbiomes of the lung and gut have been immunologically linked and are thought to have an impact on respiratory disease [35, 36] . the autophagy mechanism within our microenvironment provides a constant "cleanup" system to recycle cell debris from microscopic biowaste generated by dynamic cellular biochemistry [37] . enzymes such as neutrophil elastase function like garbage disposals recycling waste molecules. alpha-1 antitrypsin is a thermostat-like control factor that signals the proteolytic enzymes to stop and protect healthy tissue from being affected. antiproteases in the lung, such as alpha-1 antitrypsin, are required to prevent the overactivity of neutrophil elastase to prevent the degradation of healthy lung tissue. those with the genetic mutations of a1at deficiency are at disadvantage, and subsequent lung tissue damage can occur promoting lung diseases like copd, asthma, bronchitis, and emphysema. key components of lung structure are elastin and collagen, which provide support for the bronchioles and clusters of alveoli (acini). the key enzyme present in these cells is neutrophil elastase, which is responsible for the destruction of respiratory bacteria. protease and antiprotease imbalance in the lung resulting in emphysema can be caused by alpha-1 antitrypsin deficiency and nicotine in cigarette smoke or polluted inhalant exposure [18] . ifmnt approaches to the a1at-deficient patient assess for nutrient insufficiencies for some of the important connective tissue, collagen, and elastin system key nutrients: vitamin c, vitamin d, biotin, balanced fatty acids, and gut microbiome. when insufficiencies or deficiencies are identified, appropriate food and dietary supplementation interventions can be recommended. it should be noted that if an individual is identified with a1at deficiency genotype, the status of liver health should also be assessed, as a1at pathophysiology can express in liver cirrhosis. more recent studies of respiratory disease [38] have revealed the relationship with bacterial or viral infections exacerbating the individual's genotype eliciting expression of the associated diseases. one of the most recognized inherited conditions of altered autophagy mechanisms is alpha-1 antitrypsin deficiency, with 80-100 genetic variants affecting severity of lung expression. low levels of circulating a1at allow potentially harmful enzymes like neutrophil elastase to remain in the lungs unchecked. low levels of a1at, and the consequent proliferation of neutrophil elastase, leave lung tissue vulnerable to destruction, resulting in a decline in lung function. there are several categories of lung disease and many diseases within those categories (. table 51 .1). some micronutrients and phytonutrients have important antioxidant and methyl-donating properties important for the lungs and therefore have great role in a nutritional approach to lung health. iron's interaction with the lungs is essential. it carries oxygen from the lungs to the peripheral parts of the body, as well as carbon dioxide back to the lungs to be exhaled. however, too little or too much iron can pose a problem for the lungs. before iron administration, it is important to rule out hemochromatosis, or iron overload, for an individual. iron-deficiency anemia often presents in many chronic diseases including those of the lung, such as copd, lung cancer, and ipf [57] . increased mortality, decreased quality of life, increased hospital admissions, and cost of treatment have been reported for those with chronic disease and low iron [58] . anemia of chronic disease (acd) is usually at the root of this. acd is often the result of inflammation. inflammatory proteins, including il-6, stimulate the production of hepcidin in the liver, which inhibits absorption and increases storage of iron resulting in a functional iron deficiency. typical iron markers, such as transferrin saturation, total iron binding capacity (tibc), and ferritin, are also affected by inflammation and are less useful markers in chronic disease. soluble transferrin receptor (stfr) seems to be a lesser known marker that is less affected by inflammation [59] . because of the difficulty with iron absorption, intravenous iron is often used to replete deficiencies. as iron is a pro-oxidant, researchers studied any negative repercussions. there does not seem to be any increased oxidative stress with intravenous iron, but glutathione, the body's endogenous super antioxidant, does seem to decrease, likely in response to the pro-oxidative activity of iron. in a recent study, administration with vitamin e was seen to eliminate these negative effects [57] . excessive iron can also be problematic for lung health for those with the genetic mutation for hemochromatosis (hfe). disorders of iron overload are increasingly being recognized as risk factors for most of the chronic diseases like cardiovascular, alzheimer's, and cancer [60] . high iron can catalyze the formation of highly reactive hydroxyl radicals, oxidative stress, and programmed cell death. in the instance of lung cancer and other cancers affecting the lungs, tumors sequester iron for their own growth, usually leaving the patient with iron-deficiency anemia. in fact, 90% of cancer patients undergoing chemotherapy are iron deficient. inflammation also plays a role in iron homeostasis. the pro-inflammatory cytokines cascade down to affect the proteins that regulate . chronic obstructive pulmonary disease (copd) disease that restricts airflow through either inflammation of the lining of the bronchial tubes or destruction of alveoli increased risk of emphysema if genetic variant of alpha-1 antitrypsin deficiency and smoking or exposed to high levels of air pollution [11] bronchiectasis a disorder of the airways that leads to airway dilation and destruction, chronic sputum production, and a tendency toward recurrent infection [39] bronchiolitis airway injury that can be caused by infections, irritants, toxic fumes, drug exposures, pneumonitis (typically viral), organ transplants, connective tissue disorders, vasculitis, or other insults [40] dyspnea shortness of breath or difficulty breathing [11] emphysema thinning and destruction of the alveoli, resulting in decreased oxygen transfer into the bloodstream and shortness of breath. increased risk of emphysema if genetic variant of alpha-1 antitrypsin deficiency and smoking or exposed to high levels of air pollution [11] alpha-1 antitrypsin deficiency a deficiency of a1at, a protein produced in the liver that protects the lungs from excessive neutrophil elastase, an autophagic enzyme. a1at may also accumulate in liver and cause liver disease [55] obstructive asbestosis fibrotic lung disease resulting from extensive inhalation of asbestos fibers [42] desquamative interstitial pneumonitis (dip) form of idiopathic interstitial pneumonia that is more common in cigarette smokers but may be seen in nonsmokers, in patients with underlying connective tissue diseases or those exposed to inorganic dust/particles [43] sarcoidosis immune-mediated systemic disorder that is characterized by granuloma formation of the lung parenchyma and the skin [44] restrictive pathophysiologyneuromuscular weakness amyotrophic lateral sclerosis (als) progressive neurological disease that affects the motor neurons of the nervous system [11] guillain-barre syndrome progressive immune system attack on the peripheral nerves, usually following an infectious illness such as a respiratory infection. may eventually cause respiratory distress syndrome [11] restrictive pathophysiologychest wall/pleural disease kyphoscoliosis kyphoscoliosis: a deformity of the thoracic cage that results in restriction of the lungs and impairs pulmonary function [45] ankylosing spondylitis autoimmune inflammatory disorder characterized by inflammation of the axial skeleton and peripheral joints [46] chronic pleural effusions chronic accumulation of fluid between the two outer membranes surrounding the lungs [11] pulmonary vascular disease pulmonary embolism blood clot that typically originates from thrombi in the deep venous system of the legs and travels to the lungs pulmonary arterial hypertension (pah) progressive disorder of primary pulmonary arterial vasculopathy characterized by a mean pulmonary arterial pressure >25 mm hg at rest (>30 mmhg during exercise) [48] iron homeostasis [61] . iron can also impair cytokine secretion, which can leave those with an iron overload much more susceptible to infection, increasing the morbidity and mortality of infectious diseases, including those of the lung [59] . oxidative stress may contribute to injury of lung tissue, causing further fibrosing in those lung diseases with that characteristic. allele variants in the genes associated with iron homeostasis (c282y, s65c, and h63d hfe) are significantly more common in those with idiopathic pulmonary fibrosis (ipf) than those without ipf (40.4% ipf patients vs 22.4% non-ipf) and are associated with higher irondependent oxygen radical generation [62] . iron is implicated in lung pathology. monitoring iron status and using supplements or diet to aid the body in increasing or decreasing the iron load are imperative for the nutritionist working with lung disease patients. choosing a good non-constipating form of iron is important, such as iron glycinate. the b vitamins are also important to monitor for lung health. vitamin b6 and its bioactive form, p-5-p, are typically known to protect dna from mutation or damage [63] . however, there is mixed evidence on its role for lung cancer. some research has shown that it is helpful for lung cancer patients as it is important for apoptosis when using chemotherapy, because it sensitizes cancer cells to apoptosis [63] . however, research in 2017 showed that adult male smokers taking greater than 20 mg vitamin b6/day for long periods tended to have a greater risk for lung cancer. many variables, including genetic variants, form of b6, and the status of other co-nutrients may be at play [64] . other studies showed that men in the top quintile of vitamin b6 serum concentration had about one half the risk of lung cancer, and specifically, vitamin b6 and folate were inversely associated with risk of lung cancer [65] . . squamous cell (epidermoid) carcinoma about 25-30% of all lung cancers. these start in early versions of squamous cells, which are flat cells that line the inside of the airways in the lungs. often linked to a history of smoking and tend to be found in the central part of the lungs, near the bronchus [50] large cell (undifferentiated) carcinoma about 10-15% of lung cancers. it can appear in any part of the lung and tends to grow and spread quickly. a subtype of large cell carcinoma, known as large cell neuroendocrine carcinoma, is a fast-growing cancer that is very similar to small-cell lung cancer [51] small-cell lung cancer (sclc) about 10-15% of lung cancers are sclc. typically start in the cells lining the bronchi and parts of the lung such as the bronchioles or alveoli [52] infectious diseases pneumonia inflammation of the lungs, usually caused by bacteria, viruses, or fungi [11] bronchitis inflammation and eventual scarring of the lining of the bronchial tubes accompanied by restricted airflow, excessive mucus production, and persistent cough [11] tracheitis bacterial infection that can develop in the trachea [53] infant respiratory distress syndrome also known as hyaline membrane disease (hmd) or respiratory distress syndrome, this condition affects the alveolar ducts and terminal bronchioles in which the hyaline membrane is a fibrinous material composed of blood and cellular debris, caused by the absence of proper surfactant production due to an immature or poorly developed lung [54] upper respiratory infection (uri) acute infections involving the nose, sinuses, pharynx, larynx, trachea, and bronchi, referred to as the common cold [11] bronchopulmonary dysplasia (bpd) chronic lung disorder which may affect infants who have been exposed to high levels of oxygen therapy and ventilator support [11] other cystic fibrosis disease characterized by abnormally thick mucus secretions from the epithelial surfaces of many organ systems, including the respiratory tract, the gastrointestinal tract, the liver, the genitourinary system, and the sweat glands [11] acute lung injury clinical and radiographic changes in lung function associated with critical illness (acute respiratory distress syndrome is most severe form) [11] respiratory because of disagreement in research, particularly with smokers or former smokers, using food first for b vitamins may be a prudent way forward. good sources of vitamin b6 are fish, chickpeas, chicken, potatoes, turkey, bananas, ground beef, and winter squash. pyridoxal kinase (pdxk) is the enzyme that converts pyridoxine and other vitamin b6 precursors to its bioactive form of p-5-p. dysfunction of this enzyme is a good prognostic for lung cancer and other lung diseases. mthfr 1298aa genotype is associated with a higher risk of lung cancer in women but not in men. the mthfr 677tt genotype was associated with a significantly decreased risk of lung cancer in women but not in men. in contrast, the mthfr c677t and a1298c polymorphisms interacted with smoking status in men but not in women [66] . methylation gene testing is imperative to understand the patient's status. some studies suggest that a higher intake of riboflavin (vitamin b2) may protect against lung cancer in smokers [67] . folate deficiency was also associated with asthma and attacks of shortness of breath [8] . correcting acidosis may preserve muscle mass in diseases where wasting is an issue, such as copd or ipf. for those receiving chemotherapy, a higher ph (more alkaline status) is helpful for muscle mass protection. high alkaline diets contain more fruits and vegetables, and those supply more magnesium, which is needed to activate vitamin d. as discussed below, vitamin d is extremely helpful for lung health. sleep quality involves maintaining adequate 7-8 hours with good sleep hygiene (see 7 chap. 34). good rem cycling, feeling refreshed upon awakening, and other characteristics of good sleep play significant roles in maintaining healthy acid-base balance. dietary intake of the minerals magnesium, potassium, sodium, chloride, and calcium promotes the balance of acidbase microenvironment. after exposure and tissue retention of toxic minerals and metals, these substances can contribute to perturbations in the acid-base metabolic milieu. some conditions reduce oxygen intake and should be addressed. one of the most common oxygen-impairing conditions is sleep apnea, altered sleep with random halting of breathing during sleep that is often accompanied by snoring. other limiting conditions are respiratory diseases like copd, a1at deficiency, asthma, cystic fibrosis, etc. vitamin a is an important antioxidant and a general umbrella term for several fat-soluble retinoids, including retinol, retinal, and retinyl esters. there are also other substances that are provitamin a carotenoids or precursors to vitamin a. two forms are found in foods, the preformed forms of retinol or retinyl esters, which are found in dairy, fish, caviar, and meats (especially liver), and the provitamin a carotenoids, including the most important and common provitamin a carotenoid, beta-carotene, as well as others including alpha-carotenes and cryptoxanthin, which are found in plant-based foods. our bodies must convert these two forms within our cells to retinal and retinoic acid, the active forms of vitamin a in the body. new studies of the gene, β-carotene 15,15′-monooxygenase (bcmo1), which is responsible for the enzymatic conversion of β-carotene to vitamin a, are revealing that individuals with heterozygous or homozygous bcmo1 snps have 30-60% less efficient conversion than those with normal gene function (see 7 chap. 17) [68] . other carotenoids found in food, such as lycopene, lutein, and zeaxanthin, are not converted to vitamin a but have other antioxidant benefits in the body. most vitamin a is stored in the liver as retinyl esters, and deficiency is not visible until these stores are nearly depleted. vitamin a's role as an antioxidant helps the lungs in several ways, including maintaining alveolar epithelium cells and preventing development of respiratory tract infections. most of the developed world's population does not have a risk of deficiency due to sufficient vitamin a intake. however, most people with cystic fibrosis have pancreatic insufficiency, which reduces the ability to absorb fat and therefore the fat-soluble vitamins a, d, e, and k. according to a study in 2002, between 15% and 40% of people with cystic fibrosis had a vitamin d deficiency, also a fat-soluble vitamin. with the addition of pancreatic replacement treatments, better nutrition, and vitamin a supplementation, deficiency has become rare. however, improved vitamin a status has not been thoroughly studied as of 2018, and therefore it is largely unknown if an improved vitamin a status has any effect on cystic fibrosis [69] . vitamin a deficiency has been shown to be associated with emphysema in rats. smoke exposure significantly decreases vitamin a concentration in lung tissue, significantly more in those with copd [70] . retinoic acid seems to play a beneficial role in the treatment of ipf. a review showed that in all studies, retinoic acid decreased fibrosing, the formation of collagen, and reduced the expression of alpha-smooth muscle actin (alpha-sma), all hallmarks of ipf [71] . it is important to not take large doses of vitamin a if one is in a malnourished state as it can cause toxicity and should be monitored with blood testing of vitamin a retinol. nourish the body with all foods and all nutrients slowly. the non-provitamin a carotenoids have also shown some benefit. lycopene, found in high amounts in guavas, watermelon, tomatoes, papaya, grapefruit, sweet red peppers, asparagus, purple cabbage, mangos, and carrots, slowed forced expiratory volume (fev) decline in former smokers [70] . vitamin d's importance with lung health cannot be understated. vitamin d deficiency, or even insufficiency, is linked to accelerated decline in lung function, increased inflammation, and reduced immunity in chronic lung diseases. vitamin d has a role in the regulation of inflammation, immunity, cellular proliferation, senescence, differentiation, and apoptosis. sufficient vitamin d levels are correlated with better asthma control, better immune response related to respiratory infections, and reduced severity of exacerbations with copd and asthma when exposed to inflammation-causing pathogenic activity [72] . vitamin d is obtained through sunlight on the skin (without sunscreen) and very few dietary sources. therefore, supplementation is generally recommended. higher vitamin d levels are shown to be protective in many lung disease states. sufficient levels improve treatment response with medications and reduce asthma severity [68] . with infectious diseases of the lung, higher vitamin d concentrations are shown to have a protective action [6] . vitamin d has a protective effect on lungs of smokers, and higher levels of vitamin d inhibit the pro-fibrotic phenotype of lung fibroblasts and epithelial cells. current data suggest an inverse association between serum vitamin d and lung cancer risk, and vitamin d deficiency at 16-20 weeks' gestation is associated with impaired lung function and asthma at 6 years of age [73] . lower levels of vitamin d are associated with an increased risk for respiratory infections, cystic fibrosis, chronic obstructive pulmonary disease, and interstitial lung disease [74] . vitamin c is an important antioxidant that helps decrease oxidative damage in the body, including in lung tissue. it is also essential for lipid metabolism. it is present in the airway surface liquid and creates an interface between the epithelial cells and the external environment. vitamin c is a cofactor in collagen synthesis, which can aid in repair of bronchial and alveolar tissue when damaged. it also provides beneficial control of lipid peroxidation of cellular membranes, including those surrounding as well as those within intracellular organelles. vitamin c has some of the best lung protective capabilities, according to current research. vitamin c may also diminish oxidative attack on nonlipid nuclear material and is an antioxidant component of plasma and extracellular fluids surrounding the lungs. it is an antioxidant that not only fights oxidative stress but also reduces oxidized vitamin e and glutathione, allowing them to become active as antioxidants again. vitamin c is antiinflammatory and is helpful in all inflammatory states of the lung, even allergies. there are many ways in which vitamin c, along with its antioxidant partners, glutathione, vitamin e, vitamin a, and plant-based phytonutrients, affects lung health. it is well established that increased levels of vitamin c in the diet improve health outcomes for smokers and their offspring, as smoking depletes vitamin c [75, 76] . vitamin c is also helpful in fighting infectious diseases such as respiratory infections and pneumonia, copd regardless of smoking status, asthma, and lung cancer [77] . specifically, in certain lung cancers, vitamin c, along with other nutrients such as lysine, proline, epigallocatechin gallate, and zinc, can inhibit the proliferation of certain carcinoma lines and induce apoptosis, as well as inhibit lung cancer metastasis [78] . even in lung transplants, vitamin c is helpful against oxidative stress by reducing glutathione and lowering lipid peroxidation, along with vitamins a and e [79, 80] . the literature suggests these benefits can be achieved at 500-3000 mg/day. check iron status before administering vitamin c supplementation as vitamin c doubles iron absorption from foods. vitamin e's primary role is as an antioxidant, breaking free radical chain damage and preventing peroxidation of lipid molecules. this vitamin also is promising with regard to beneficial effects on lung function preservation. oxidative stress and inflammation are key features in many lung diseases; therefore nutrients with antioxidant capacity can be useful. a few studies suggest that alpha-tocopherol found in sunflower and olive oils has a beneficial effect on fev (forced expiratory volume), whereas gamma-tocopherol found in canola, soybean, and corn oils has a negative effect on fev [81] . however, from these authors' perspective, this is likely due to the source and type of the oils, which can be inflammatory, rather than the form of vitamin e. for example, a recent study showed that gamma-tocopherol was protective in allergic asthma [82] . in addition, sufficient levels of vitamin e, in the alpha-tocopherol form, were found to reduce susceptibility of the elderly to acquiring pneumonia. some of the positive effects of vitamin e are synergistic with vitamin c [83] . phytonutrients have been found to have two effects with respect to lung disease: one is a symptom-improving pattern, and the other is a rate-reducing pattern [84] . idiopathic pulmonary fibrosis (ipf) is largely characterized by reduced antioxidant and increased inflammatory action. recent literature is showing the ability of certain flavonoids, in particular quercetin, to reduce inflammation and act as a strong antioxidant countering the pro-oxidant environment of ipf. quercetin is recognized as the most potent ros scavenger. taken together with glutathione, the impact is even greater, and it seems to help improve the antioxidant and inflammatory status more for those with ipf than non-diseased controls [85] . curcumin has been shown to slow or limit fibrosing in murine studies related to lung, liver, or kidney fibrosing [86] [87] [88] [89] . it has also been shown to attenuate metastatic melanoma in the lungs when delivered in a nanoparticle [90] . the potential for curcumin is interesting and hopeful. fisetin and fenugreek have also been studied as useful phytonutrients that help combat inflammation in lungs [91, 92] . fisetin is found in apples, strawberries, persimmons, cucumbers, and onions, among many other fruits and vegetables. fenugreek is a plant used frequently in south and central asian cooking, where both the seeds and leaves are used. there are now supplements available for both of these phytonutrients. this is a reminder to eat a primarily plantbased diet when combating inflammation and to broaden our palates to include healthy foods and ingredients from other cultures than our own. lastly, the powerful antioxidant cannabidiol (cbd), from the cannabis and closely related hemp plants, is a powerful shield against oxidative stress, prevalent in lung disease [93] . the research is not robust regarding lung function and minerals, and most has been done with regard to cystic fibrosis where bone density is associated with general nutritional status, including minerals. there have also been many studies trying to determine a correlation between mineral status and copd, where, again, the research shows that mineral status is not predictive but overall nutrient status may fall if not monitored. in contrast, one study in japan showed an inverse association between dietary calcium and the risk for copd [94] . in an nih-aarp diet and health study, magnesium, iron, selenium, zinc, and copper intakes, both dietary and supplemental, were studied with respect to lung cancer. mineral supplementation did not affect lung cancer risk, yet dietary intake of calcium, along with vitamin d, and iron reduced the risk, and dietary intake of magnesium increased risk [95] . boron has been shown to be protective against lung cancer, along with other nutrients, at levels of 3 mg/day [96] . there is some research showing that selenium is helpful, particularly for smokers, for improved fev. higher magnesium status is correlated to better fev but is not yet seen as an association. this may be due to magnesium's role as the vitamin d activator. there have been a few studies showing increased copper levels are related to decreased fev. some recent research has also shown that dietary zinc and iron are associated with reduced lung cancer, but the same was not seen with calcium, copper, magnesium, or selenium [97] . low mineral bone density is prevalent at a higher rate among cystic fibrosis patients, and therefore supplementation with vitamin d, vitamin k2, magnesium, calcium, and the trace minerals can be helpful [98] . alpha-lipoic acid (ala) is a powerful antioxidant endogenously produced in the human body from foods such as yeast, organ meats, spinach, broccoli, and potatoes and is both water-and fat-soluble. ala, along with n-acetyl cysteine (nac), glycine, and vitamin c, is an important precursor to glutathione, which is a powerful endogenous antioxidant and the primary antioxidant in the lungs. ala has been shown to be anti-inflammatory in lung tissue in those with acute lung injury, and the proposed action is via inhibition of the nf-kappab signaling pathway [99] . ala has also been shown to downregulate some cancerpromoting actions prevalent in lung cancer, likely by this same pathway [100] . it also may alleviate nicotine-induced lung oxidative stress [101] . n-acetyl cysteine (nac), another precursor to glutathione, is a powerful antioxidant on its own as well. in relation to the lungs, nac helps the clearance of mucus in the lungs by pulmonary cilia. this has been shown to be effective at 400-600 mg/day in divided doses [102] . there is significant research on nac and lung health, showing improvement with nearly all lung issues, including nearly 40 studies showing improvement for bronchitis [103] , infectious diseases by reducing the bacterial count [104] , smokers, and people with asthma and copd, through both its antioxidant effects and by reducing the viscosity of sputum and mucus. at an oral dose of 1800 mg/day, the mean glutathione concentration in lung tissue increased by 49% on one study [105] . there are additional studies showing improvement for those with copd, asthma, cystic fibrosis, pulmonary fibrosis, and symptoms related to allergies or other infections. the dose that has been studied and has been shown to be most useful is 600 mg twice daily and more effective if nebulized [106, 107] . both ala and nac supplementation should be accompanied by vitamin b6 and the complex of b vitamins to prevent an elevation in liver enzymes (. fig. 51 .3). there are several specialty labs that conduct micronutrient analysis and functional testing, such as genova diagnostics and spectracell. these tests can be useful for evaluating levels of individual nutrients as they function in the body, rather than just in serum, which is not an accurate indicator of tissue or functional status. patients suffering from copd, interstitial lung disease, and other diseases tend to have muscle and weight loss related to respiratory acidosis, and increasing weight and muscle mass helps with quality of life. respiratory acidosis occurs with co 2 buildup where the lungs are no longer able to effectively exchange o 2 and co 2 . nutritional supplementation should attempt to reduce metabolic co 2 production. fat metabolism produces less co 2 than carbohydrate metabolism, so emphasizing a higher fat, lower carbohydrate diet can be helpful [110] . in general, a high intake of omega-6 fatty acids is associated with poorer forced expiratory volume (fev) in patients with lung disease because of their pro-inflammatory nature. however, a complete fatty acid panel or a red blood cell membrane fatty acid test would reveal more details about the status of an individual's omega-6 pathway. certain omega-6s and the work of their corresponding metabolizing enzymes such as elongase and delta-5 or delta-6-desaturase may allow healthful omega-6s (linoleic (la), gamma-linolenic (gla), lipoxins [111] , prostaglandin 1 series metabolites) to flow down an anti-inflammatory pathway instead. important cofactors for this pathway are vitamin b2, vitamin b3, vitamin b5, vitamin b6, biotin, vitamin c, zinc, and magnesium. lipid metabolism dysregulation is understood to be part of the pathogenesis of idiopathic pulmonary fibrosis. in ipf, free fatty acids play a role in the proliferation of fibroblasts. certain fats, in particular palmitic acid, oleic acid, and linoleic acid, are elevated in the lungs of those with ipf, whereas stearic acid is low. stearic acid is found in meat, poultry, fish, grain products, and milk and milk products. the palmitic, oleic, and linoleic acids enhance the tgf-ß1-induced expression of α-smooth muscle actin (sma) and collagen type 1 in mrc-5 cells, which can lead to fibrosis. stearic acid inhibits the levels of these fibrosing cells. stearic acid also improves the thrombogenic and atherogenic risk factor profiles [112] . in one study on patients with copd, omega-3 fatty acids were found to reduce inflammation in bacterial infections of the lungs without suppressing the ability to clear the bacteria. those taking epa, dha, ala, and gla had improved exercise capacity and had lower risk of developing copd [113] . although results have been mixed over the years possibly due to doses used in studies, a recent 2018 prospective study showed that pufas (omega-3s) from fish help prevent lung cancer and can be part of treatment during lung cancer. in general, the strongest evidence for improved lung function and slowing decline is with the epa and dha forms of omega-3 fatty acids [114] . because of toxicity issues in fish, increasing quality supplements vs fish intake may be more prudent. protein is essential for all lung conditions, and lack of it can result in poorer pulmonary function, decreased exercise capacity, and increased risk exacerbations. since many lung diseases have oxidative stress as a characteristic, it can cause protein carbonylation which may negatively affect dna expression and lipid membranes. nutritional supplementation with added protein and healthy carbohydrates can increase body weight and muscle strength and improve quality of life. those with copd, interstitial lung diseases, and others that affect oxygen absorption and co 2 exhalation have greater levels of hypoxia and sometimes respiratory acidosis, which exacerbates the loss of muscle through oxidative stress and inflammation. supplementation of free essential amino acids versus complete proteins has been shown to help prevent muscle wasting among copd patients. muscle-building exercise is often prescribed for those with copd and interstitial lung diseases [115] . supplemental l-carnitine at 2-6 g/day for 1-2 weeks increased the capacity of copd patients to rehabilitate and build muscle and helped inspiratory muscle strength. carbohydrates should be monitored for sufficient but not excessive levels. more co 2 is produced with the utilization of carbs versus fats for energy. therefore, with gas exchange being an issue with most lung disorders, a slightly higher fat and lower carbohydrate diet may be indicated. it is worth mentioning fiber for a moment, as it is mostly delivered in carbohydrate-rich foods. there is evidence that consuming whole fruits and vegetables higher in dietary fiber is associated with reduced severity of asthma and copd [116] . a diet that derives its carbohydrates from vegetables and fruits rather than from processed carbohydrates such as grains, breads, pasta, or added sugars will deliver fewer carbohydrate grams. glutathione (gsh), a tripeptide composed of cysteine, glutamine, and glycine and produced from methionine, is in every cell in the body. it is the most powerful and abundant endogenous antioxidant in the airway epithelial lining and is responsible for detoxification of electrophilic compounds, the scavenging of free radicals, and modulation of cellular processes such as dna synthesis and repair, differentiation, apoptosis, and immune function [117] . it is also a heavy metal chelator. it is more effective than some other antioxidants because it is intracellular and extracellular. in isolated type ii alveolar epithelial cells, extracellular glutathione inhibits hyperoxia-induced injury, inhibits pro-inflammatory cytokine release, and promotes cell growth. it is obviously very important to maintaining lung function as this is the inflammatory process that begins lung cell or tissue damage, as mentioned above. the highest levels of glutathione concentrations in the body are in the lungs, liver, and brain. gsh depletion leads to activation of nf-kb (pro-inflammatory signaling) and increased pro-inflammatory gene transcription and cytokine release from histone deacetylase suppression in epithelial cells. total and reduced gsh concentrations are much lower in people with ards, pulmonary fibrosis, and hypersensitivity pneumonitis than observed in healthy adults. alterations in alveolar and lung gsh metabolism are widely recognized as a central feature of many inflammatory lung diseases such as idiopathic pulmonary fibrosis, acute respiratory distress syndrome, cystic fibrosis, and asthma [118] . we make glutathione in the body with cysteine and methionine, and it is difficult to take exogenously because digestion can destroy it. the precursors of cysteine (essential), glutamine, and glycine and cofactors (vitamin c, vitamin e, vitamins b1, b2, b6, and b12, folate (b9), minerals selenium, magnesium, and zinc, and alpha-lipoic acid, see below) are therefore recommended so that the body can produce it on its own. the two enzymes necessary to produce it, gamma-glutamylcysteine synthetase and glutathione synthetase, must also be functioning well. we also recycle glutathione if the precursors and cofactors are available. cysteine is usually the most rate-limiting precursor, and many people supplement with n-acetylcysteine to provide the body with this nutrient. although glutathione is produced in every cell of the body, the greatest production is in the liver, so focusing on liver health is important to maintain good glutathione production. production declines with age and with lung disease, as well as other conditions. there are very few foods containing glutathione; they are raw or very rare meat, especially liver, unpasteurized milk and other unpasteurized dairy products, and freshly picked fruits and vegetables, such as avocado and asparagus. however, as mentioned earlier, it may be destroyed during digestion. glutathione contains sulfur molecules, which may be why foods high in sulfur help to boost its natural production in the body. these foods include: 5 cruciferous vegetables, such as broccoli, cauliflower, brussels sprouts, and bok choy 5 allium vegetables, such as garlic and onions 5 eggs 5 nuts 5 legumes 5 lean protein, such as fish and chicken other foods and herbs that help to naturally boost glutathione levels include: 5 milk thistle (a liver-regenerating herb) 5 flaxseed 5 guso seaweed 5 whey glutathione is also negatively affected by insomnia. getting enough rest on a regular basis can help increase levels. addressing a drop in glutathione for lung health involves maintaining good levels of the precursors and cofactors mentioned above. a good way to bring in the less abundant amino acid cysteine is to take n-acetylcysteine (nac). doses of 400-600 mg were more effective than placebo in reducing symptoms [117] . supplemental selenium can also help with glutathione production. glutathione supplementation has also become more effective. there are several forms, from capsules to topical liposomal, which have shown good absorption. inhaled gsh has good research for use in cystic fibrosis (cf), chronic otitis media with effusion (ome), hiv seropositive individuals, idiopathic pulmonary fibrosis (ipf), and chronic rhinitis. it is not recommended for asthma due to significant side effects, and additional evidence is needed to determine if use with emphysema is recommended although theoretically it should be useful. it is also not recommended to use inhaled gsh during cancer chemotherapy treatment as it may interfere with the medication's actions. the mechanism of action of inhaled glutathione is limited to the upper airways and lungs and does not seem to affect serum levels. before considering inhaled gsh treatment, the patient should undergo urine sulfite sensitivity testing using a readily available special test strip called "em-quant 10013 sulfite test. " if positive, inhaled gsh should not be used as bronchoconstriction may occur. the recommended dose is 600-5000 mg per day, depending on response, and whether inhaled gsh is considered safe. efficacy should be tested using a baseline pulmonary function test and a follow-up test after a prescribed time later [119] (. fig. 51 .4, 7 box 51.1). there are also serum tests for glutathione levels. these cofactors are vitamin c; vitamin e; vitamins b1, b2, b6, and b12; folate (b9); minerals selenium, magnesium, and zinc; and alpha-lipoic acid. what do the glutathione cofactors do that makes them so important? 5 direct cysteine toward glutathione production and increase cellular uptake of cysteine 5 help form the glutathione molecule out of the three precursor amino acids 5 help recycle glutathione from its oxidized gssg form back to its reduced (active) gsh form 5 help maintain glutathione levels and keep the gssg-gsh ratio balanced 5 recycle each other, improving overall antioxidant activity 5 stimulate the activity of the whole glutathione enzymatic system co10 is a fat-soluble compound produced endogenously and also available through food and supplementation. it is required in the production of atp, is a powerful antioxidant, and therefore is helpful against oxidative stress, an important issue in lung disease. coq10 achieves its strong effects through a set of different mechanisms. it influences genes through its epigenetic effect to reduce inflammation, helps with the immune system, and even reduces aging by reducing systemic oxidative stress and mitochondrial aging [120] . lungs are the most susceptible organ to oxidant damage because they interact directly with oxygen. therefore, it makes sense that antioxidants, and those that especially affect the lungs, are helpful in tissue and lung cell preservation [121] . coq10 levels are significantly lower in those with copd and asthma with insignificant amounts of research on the levels of coq10 with other lung issues. it has been shown that supplementing patients with coq10 resulted in measurable benefits. in one study, patients with copd using steroids to reduce inflammation were able to reduce their steroid dosage when using coq10 [122] . in another study, benefits were shown for copd patients during exercise, measuring performance, tissue oxygenation, and heart rate at a low dosage of 90 mg/day [123] . the levels of coq10 in the blood have been shown to indicate the degree of systemic oxidative stress, which implies it could be used as a marker to assess copd [121] . several studies confirm the beneficial role of coq10 in decreasing oxidative stress, cardiovascular risk, and modulating inflammation during aging. dosage levels of 1200 mg/ day of coq10 have been shown to be therapeutic. however, in the reduced, more absorbable form, ubiquinol, 400 mg/ day, was shown to be as effective. there is a wide range of toxins and anti-nutrients that can significantly impact the respiratory system. this can occur through acute or chronic exposure to these agents. the earth's air is the source of oxygen, and the lungs provide access to that oxygen to support life. the human need for oxygen is precarious because humans can only survive for about 6 minutes without the precious gas. from about 1760 to sometime between 1820 and 1840 in europe and the united states, the ramp-up of new industrial revolution manufacturing processes opened a new era of increasing chemical and heavy metal atmospheric contamination. these pollutants can enter the body through breathing the polluted air. the more concentrated atmospheric pollutant densities cluster around areas of dense population. the dirty air provides a serious direct threat to those with respiratory diseases. an integrative and functional approach to assessing an individual with respiratory disease needs to include consideration of potential environmental contributors to the etiology of a condition. . table 51 .2 lists environmental pollutants that are known to promote lung pathology. a 2016 study published in the canadian respiratory journal examined exhaled fractional nitric oxide (feno)an indicator of inflammation in the lungs -in school children at three different schools located three different distances from a large steel mill [127] . steel processing is known to be a source of ambient iron, nickel, lead, copper, vanadium, and zinc. the study found statistically significant differences in feno between the two closer schools compared to the farthest school from the mill, indicating potential increased lung inflammation caused by heavy metals and/or air pollutants [127] . although acute metal toxicity is possible, chronic, low-grade exposure is more common and may contribute to respiratory complications and disease. an individual's ability to vitamin c -as an antioxidant, it assists glutathione in this function and has been shown scientifically to raise glutathione levels short term; it is recycled by glutathione from its oxidized state back to its active state, thus strengthening antioxidant defenses; vitamin c also recycles vitamin e and alpha-lipoic acid vitamin e -as an antioxidant it also assists glutathione in eliminating free radicals much like vitamin c; it is also required for the proper functioning of glutathione enzymes; it recycles vitamin c and alpha-lipoic acid b vitamins -vitamins b1 and b2 maintain glutathione and its enzymes in their active forms; vitamin b2 participates in the formation of a glutathione molecule; vitamin b6 influences glutathione synthesis indirectly as it is important for the proper functioning of amino acids including gsh precursors; vitamin b6 increases the amount of magnesium (a vital cofactor) that can enter cells; folate (b9) pushes cysteine toward glutathione production rather than homocysteine production; folate and vitamin b12 work together in amino acid metabolism and protein synthesis. you can read more vitamin b12 deficiency and its effect on immune health at 7 http://www. immunehealthscience. com/vitamin-b12-deficiency. html selenium -part of the enzyme glutathione peroxidase (gpx). glutathione peroxidases, also known as selenoproteins, are a family of antioxidant enzymes that speed up the reaction between glutathione and free radicals magnesium -required for the proper functioning of the enzyme gamma-glutamyl transpeptidase (ggt) involved in the synthesis of glutathione zinc -zinc deficiency reduces glutathione levels, especially in red blood cells. however, zinc levels above normal have pro-oxidant properties and reduce glutathione too alpha-lipoic acid -an antioxidant produced by the body; it has been scientifically proven to enhance and maintain glutathione levels by stimulating enzymes involved in the synthesis of glutathione; it also helps increase the cellular uptake of cysteine, the crucial building block of glutathione; in addition, alpha-lipoic acid recycles vitamins c and e based on data from ref. [124] eliminate these metals via detoxification in conjunction with gastrointestinal health and other factors can serve as important factors in whether or not these metals accumulate in the body. chronic arsenic exposure may be linked to respiratory complications [128] . chronic arsenic ingestion via contaminated drinking water may be connected to respiratory symptoms such as chronic cough, shortness of breath, blood in sputum, and abnormal breath sounds [129] . arsenic can also be ingested through foods such as rice and rice products, shellfish, and seaweeds, which have been shown to have high levels of inorganic arsenic (more toxic than organic arsenic found in fish) [120] . however, ingested inorganic arsenic is typically biotransformed and excreted in the urine [130] . that said, altered biotransformation has been observed depending on an individual's age, gender, nutritional status, and genetic polymorphisms responsible for the biotransformation of inorganic arsenic [130] . chronic inhalation versus ingestion may result in irritation of the throat and respiratory tract [131] . individuals most affected by arsenic exposure are children, nursing children, and infants of exposed pregnant mothers [132] . acute inhalation of cadmium may lead to dyspnea and coughing [133] . long-term exposure to cadmium has been reported to contribute to emphysema, dyspnea, and inflammation of the nose, pharynx, and larynx [123] . individuals most affected by cadmium toxicity are those with occupations with cadmium exposure, such as those who work in certain types of factories, women, due to higher intestinal absorption because of low iron stores, and residents of asia due to high intake of rice grown in contaminated soil [134] . the 2013 us national health and nutrition examination survey (nhanes) demonstrated an association between obstructive lung disease and serum lead and cadmium concentrations in the blood, where cadmium was shown to partially mediate the association between smoking and obstructive lung disease [135] . in the 2015 korean nhanes, obstructive lung function was found to be associated with higher serum blood levels of cadmium and lead as well [136] . the specific mechanism of heavy metal burden and its effects on respiratory health must be further investigated. although testing and treatment of heavy metal burden have its limitations, it is worth considering as heavy metal accumulation can wreak havoc on the body. an example of heavy metal testing that can be used in practice is urine provocation testing with a chelating agent, such as fda-approved dmsa. eliminating heavy metals from the body can be potentially harmful and requires careful monitoring and guidance by an experienced healthcare professional. air pollutants that are used as indicators of air quality are carbon monoxide, lead, nitrogen dioxide, ozone, particles, and sulfur dioxide [137] . air pollution has been shown to have adverse effects on human health [138] . a 2017 systematic review and meta-analysis done in china showed an association between respiratory disease and ambient nitrogen dioxide, which is increased through fuel combustion, industrial production, and fuel exhaust [129] . diesel exhaust particles in particular have been associated with an increase in cytokines such as il-2, il-6, and ige in nasal mucosa [139] . nitrogen dioxide in particular can potentially contribute to respiratory disease as it is a free radical that is highly reactive and poorly water-soluble and can be deposited in the lungs when inhaled [138] . in another study performed in england, air concentration of nitrogen dioxide was significantly associated with respiratory hospital admissions [140] . other pollutants, such as fine particulate matter and ozone, have been shown to significantly affect respiratory function in copd patients [141] . increased ozone exposure has also been associated with increased airway inflammation and respiratory symptoms along with decreased respiratory function in children [142] . optimization of nutrition and antioxidant status is essential to combating the potential health effects of air pollutants. . [143] . it would be reasonable to assume having adequate stores and ability to utilize these nutrients may protect against other insults to the respiratory system as discussed in this section through their anti-inflammatory properties. acute and chronic exposure to certain chemicals can also pose a risk to respiratory health. obtaining a full occupational and social history when assessing individuals is important in order to identify any potential exposure to chemicals. one of the most well-known and common toxic chemical exposures that affects respiratory health is cigarette smoke. smoking cigarettes has been identified as a main cause of copd [144] . increased oxidative stress from inhaling cigarette smoke appears to activate the nf-kb inflammatory pathway, increasing the production of pro-inflammatory cytokines such as interleukin (il)-1, il-6, and il-8 and tumor necrosis factor-ɑ (tnf-ɑ) [144] . it also appears to reduce anti-inflammatory cytokines such as il-10 [145] . electronic cigarettes, or e-cigs, have been increasing in popularity in recent years and are marketed as a better alternative to tobacco cigarettes. however, recent evidence suggests that the vapor and associated chemicals produced by e-cigs may be harmful to the respiratory system, although further research is needed to determine the mechanism [146, 147] . exposure to metalworking fluid aerosols has been associated with asthma, hypersensitivity pneumonitis, impaired lung function, allergic alveolitis, and sinusitis [148] . a 2015 review also identified an association between occupational exposure to pesticides and increased risk of asthma and chronic bronchitis [149] . there are many chemicals that are toxic when inhaled. for example, inhalation of chlorine is toxic to the lungs, where low doses can cause airway injury and high doses can cause both airway and alveolar injury [150] . these injuries can manifest as dyspnea, hypoxemia, pulmonary edema, and pneumonitis [150] . high doses of carbon dioxide, such as that released from dry ice, can also induce respiratory failure. stress may also play a role in respiratory health and the body's ability to combat insults imposed on the respiratory system. from a physiological standpoint, it is worth noting that acute stress via activation of the sympathetic nervous system increases ventilation through the production of glucocorticoids [139] . repeated acute stress may also affect growth and repair mechanisms [139] . chronic biological stress in the form of infections can also be inflammatory and negatively affect the immune system and may affect an individual's susceptibility to respiratory complications. see the chronic infections and respiratory health section on page # below for further information on this association. however, appropriate amounts of physical stress, such as in the form of exercise, can be beneficial to respiratory health. some research has indicated a benefit of aerobic exercise to respiratory muscle strength in cystic fibrosis patients [151] . chronic stress can be defined as recurrent acute stress or inability to moderate acute stress responses [139] . this can be in the form of physical or emotional stress. chronic stress and negative emotions such as depression, anxiety, and anger may be linked to endocrine and immune processes [152] . immunoglobulin e (ige) and cytokine production, as well as respiratory inflammation, are markers that characterize the asthma response and have been shown to respond to stress in some capacity [139] . it has been hypothesized that increased stress may increase susceptibility to air pollution given its effects on the inflammatory response [139] . another connection between emotions and respiratory health is acknowledged in east asian medicine, noting the association between the lungs and feelings of sadness, grief, and anxiety [153] (. table 51 .3). asthma is a chronic inflammatory lung disease, triggered by either an ige allergic reaction or nonallergic factors, and results in reversible airway obstruction and inflammation of the airway [11] . it is characterized by recurrent episodes of wheezing, breathlessness, coughing, and chest tightness [11] . severe asthma or asthma that is chronic or poorly controlled may lead to airway and lung remodeling that involves deposition of fibrotic tissue which leads to constriction of the bronchi [18] . although the exact mechanisms have not yet been identified, compromised nutritional status, such as deficiencies in selenium, zinc, and vitamins a, c, d, and e, has been connected to asthma [155] . the pathophysiology of asthma, nutrition considerations, genotypic characteristics, and lifestyle influences will be discussed in this section. there are numerous potential triggers to the development and/or exacerbation of asthma which can be summarized in 7 box 51.2. the various causes of asthma have led to the classification of several different subtypes and endotypes of asthma in hopes of choosing more targeted treatments. the pathophysiology of asthma is complex and not fully understood, due in part to its heterogeneous nature, which necessitates its organization into individual phenotypes and endotypes. this organization is important to be able to utilize targeted treatments by identifying the root causes of the symptoms. however, more research is needed to more clearly identify the specific pathological mechanisms of each phenotype and particular treatment responses [156] . two of the most common asthma phenotypes are allergic and nonallergic asthma [147] ; allergic is characterized by increased th2 immunity (th2 high) and nonallergic defined by varying mechanisms depending on the trigger (th2 low) [157] (see also 7 chap. 19) . allergic asthma involves the ingestion of typically harmless environmental triggers (listed in . table 51 .3) by antigenpresenting cells in the bronchi, which interact with immature helper t cells that, in turn, trigger an unwarranted allergic response [18] . this reaction occurs from repeated exposure to a trigger and is referred to as the type 1 hypersensitivity response [18] . this increased th2 immunity upregulates eosinophilic inflammation, tissue damage, airway hyperresponsiveness, and bronchoconstriction [113] . mast cell activation disorders, which is characterized by diseases and conditions related to mast cell mediators and the activation of mast cells, must also be considered when addressing allergic asthma [158] . in contrast, nonallergic asthma can be caused by other factors such as anxiety, exercise, stress, dry air, cold air, viruses, hyperventilation, smoke, or other irritants [11] . . inhaled cadmium (cd) is deposited in the alveoli where it is then absorbed into the bloodstream cd is transported to erythrocytes or bound to albumin, where it is then taken up by the liver to form a complex with metallothionein (mt) cd interferes with the absorption of zinc and competes for the same enzyme binding sites enzymatic activity of zinc-dependent enzymes reduces preferential binding of cd to mt can cause zinc deficiency altered biotransformation and excretion of ingested arsenic via contaminated water are linked to respiratory complications chronic inhalation of arsenic may result in irritation of respiratory tract diesel exhaust particles in particular have been associated with increase in cytokines such as il-2, il-6, and ige in nasal mucosa [139] nitrogen dioxide is a free radical that is highly reactive and poorly water-soluble and can be deposited in the lungs when inhaled [102] rising pollen and mold counts [154] increasing ozone [154] chemicals increased oxidative stress from inhaling cigarette smoke may activate the nf-kb inflammatory pathway, increasing the production of pro-inflammatory cytokines such as interleukin (il)-1, il-6, il-8, tumor necrosis factor-ɑ(tnf-ɑ) cigarette smoke may reduce anti-inflammatory cytokines such as il-10 [145] stress repeated acute stress may also affect growth and repair mechanisms [139] ige and cytokine production, as well as respiratory inflammation, are markers that characterize the asthma response and have been shown to respond to stress in some capacity [139] increased stress may increase susceptibility to air pollution given its effects on the inflammatory response [139] ( individuals suffering from nonallergic asthma will tend to be less responsive to th2-targeted treatments due to a differing immune response at play [157] . some of the additional proposed phenotypes are eosinophilic, exacerbation-prone, exercise-induced, fixed obstruction/airflow limitation, poorly steroid-responsive, and adult-onset obesity-related [159] . several of the proposed endotypes are summarized in . the american partnership for eosinophilic disorders defines eosinophilic asthma as a type of asthma characterized by especially high levels of eosinophils, more commonly developed later in adulthood, although may occur in some children [160] . many with eosinophilic asthma do not have underlying allergies or history of allergic conditions such as eczema, food allergy, and hay fever, which are thought to be seen more in people with allergic asthma [160] . in contrast to allergic asthma, the cause of eosinophilic asthma is still unknown. histamine intolerance must also be considered in assessing the root cause of asthma. ingesting histamine-rich foods and beverages such as bananas, grapes, strawberries, citrus fruits, tomatoes, nuts, chocolate, pineapples, fish, spinach, fermented foods, and beverages [161] has been shown to provoke a histamine response that may result in asthma exacerbations, among many other potential signs and symptoms [162] . disruptions in redox, or oxidation/reduction, reactions in addition to hindered antioxidant defense have been . usually not responsive to glucocorticoids [159] based on data from ref. [157] found to be a risk factor for asthma severity and development [163] . the levels of glutathione, one of the lung's most predominant antioxidants in both reduced and unreduced forms, are thought to be important for lung homeostasis and tied to asthma [163] . more research is needed to determine the exact differences in the pathophysiologies of the various subtypes of asthma in order to develop more targeted treatments. minerals such as zinc, selenium, copper, and manganese may serve as cofactors to major enzymes with antioxidant activity in the lung, such as superoxide dismutase, catalase, and glutathione peroxidase [164] . asthma has been associated with decreased activity of these enzymes [165] . low selenium intake has been associated with multiple chronic diseases including asthma [163] . selenium serves as a cofactor to glutathione peroxidase, an enzyme with antioxidant activity in the lung that is responsible for maintaining gsh/gssg redox balance [163] . imbalance between oxidants and antioxidants seems to serve an important role in asthma. levels of nonenzymatic antioxidants glutathione, ascorbic acid, alpha-tocopherol, lycopene, and beta-carotene, in addition to antioxidant enzymes superoxide dismutase (sod) and glutathione peroxidase, were significantly lower in asthmatic children compared to healthy controls [165] . the amino acids glycine and glutamine, which are important in glutathione synthesis, were also found to be significantly lower in children with asthma [165] . dha has also been found to be abundant in airway mucosa, where it is decreased in individuals with asthma and cystic fibrosis [166] . magnesium is known to elicit the relaxation of bronchial smooth muscle, decrease responsiveness to histamine, have an anti-inflammatory effect, and decrease the susceptibility of animals to developing anaphylactic reactions [25] . it is estimated that two-thirds of the population in the western world is not consuming the recommended daily allowance of magnesium [167] . magnesium can be used intravenously as an effective treatment of acute asthma attacks. one double-blind controlled trial that used 1.2 g of magnesium sulfate when patients did not respond to treatment with beta-agonists found decreased likelihood of hospitalization and improved lung function [168] . magnesium sulfate as an adjunct therapy with bronchodilators and steroids has also been shown to have a benefit in children with moderate to severe asthma [168] . although the exact mechanism is not yet known, magnesium is thought to increase glutathione concentrations in the lung [169] . more research is needed to determine additional associations between specific nutrients and asthma. however, optimization of the nutrients discussed in this section has the potential to reduce the severity and/or progression of asthma (. fig. 51.5) . asthma has a strong genetic component, with more than 100 genes associated with it in varying degrees across many populations [18] . more recent potential genetic associations include filaggrin, which encodes for the epithelial barrier; ormdl3, which encodes transmembrane protein; beta-2 adrenergic receptor gene, expressed throughout smooth muscle and epithelial cells of the lung; and interleukin-4 receptor gene, which has a variant associated with elevated ige [171] . a 2011 systematic review and meta-analysis showed that deficiencies in selenium, zinc, vitamins a, c, d, and e, and low fruit and vegetable intake could be associated with the development of asthma [155] . although this data is tenuous due to lack of randomized controlled trials, it does give some indication of the relationship between nutrition status and dietary patterns with respect to asthma development. more research needs to be done to isolate the impact of these nutrients and dietary patterns on asthma prevention and development. a 2015 review conducted by berthon and wood noted the protective effects of the mediterranean diet for allergic respiratory diseases as evidenced by epidemiological studies. this diet emphasizes minimally processed plant foods in the form of fruit, vegetables, cereals, beans, breads, nuts, seeds, and olive oil and low to moderate intake of dairy, poultry, fish, and wine, as well as low intake of red meat [172] . this association was the strongest in children, where the mediterranean diet had a protective effect on atopy, wheezing, and asthma symptoms [172] . however, there is less data available to support this pattern in adults. the same review noted an association between the "western" diet, which emphasizes refined grains, red and cured meats, french fries, sweets and desserts, and highfat dairy products and increased risk of asthma in children [172] . a meta-analysis and systematic review done in 2014 showed a reduction of risk in childhood wheezing with high fruit and vegetable intake and also showed negative association between fruit and vegetable intake and asthma risk in adults and children [173] . in contrast, food allergy has been especially linked with allergic asthma in children [161] . a study examining food allergy in asthmatic children identified higher serum levels of ige in asthmatic children compared to healthy controls, where all asthmatic children in the study were also identified as having a positive skin prick test (spt) to various food allergens [174] . a study done on 322 children under the age of 1 diagnosed with asthma, with or without allergic rhinitis, was placed on a meat-based formula of carrots, beef, broccoli, and apricots for 6 weeks. it was found that 61% had nearly complete resolution of symptoms [25] . this same study also found that the most common food triggers were milk, egg, chocolate, soy, legumes, and grains [25] . while food allergy as a cause of asthma is more common in children, hidden food allergy has been reported to be the root cause of asthma in around 40% of adults [25] . improvement in respiratory symptoms was also seen in a small study of adults given an antigen-free elemental diet in a hospital setting [25] . removal of food triggers has also been linked to improvement in exercise-induced asthma [25] . identifying food allergies can be a complicated process because many of the testing methodologies such as skin prick tests (spts) and blood tests can yield false-positive results for up to 50-60% of cases, according to the food allergy research & education organization [175] . a food elimination diet and/or oral food challenge can be a powerful tool in determining food allergy specific to asthma symptoms, where a dietitian or nutritionist in conjunction with physician and/or allergist can serve an important role through this process to support the individual. oxidative stress may play a key role in the development of asthma, which can also be true for the development of chronic diseases such as cardiovascular disease, diabetes, and cancer [117] . it has been shown that obesity may be a risk factor for people with and without allergy and may worsen pre-existing asthma [159] . individuals with asthma are twice as likely to have gastroesophageal reflux disease (gerd) than people who do not have asthma, especially those resistant to treatment [159] . celiac disease and asthma have also been linked. an italian cohort study was done that showed a significant association between treated asthma and celiac disease, where antibiotic exposure in the first year of life was controlled for and not found to contribute to this association [176] . it has also been found that individuals with celiac disease following a glutenfree diet experienced improvement in asthma symptoms [25] . it is well-known that toxic exposure to particulate matter, airborne pollutants, or cigarette smoke can trigger asthma symptoms [165] . more specifically, a dose-dependent relationship between cigarette smoke exposure and rates of asthma has been shown [165] . traffic density and asthma exacerbations have also been clearly demonstrated [165] . certain medications may also serve as triggers to asthma. aspirin-exacerbated respiratory disease (aerd) is considered another asthma subtype caused by nonsteroidal anti-inflammatory drugs (nsaids) and is characterized by asthma, chronic rhinosinusitis, and acute respiratory reactions [159] . in addition, overuse of antibiotics in childhood has been linked to asthma [18] , indicating a connection between the microbiome and asthma development. allergic bronchopulmonary mycosis (abpm) noted in . table 51 .4 is caused by a hypersensitivity reaction to fungal colonization of the airways [159] . this is typically caused by the fungus aspergillus fumigatus. without treatment, this may lead to fixed airflow obstruction and bronchiectasis [159] . the progression of asthma is complex and multifaceted, from preconception through childhood and adulthood. research suggests that early life events are largely predictive for regulatory mechanisms within the pulmonary immune system [177] . for example, prenatal exposure to a farming environment, one rich in microbial compounds, is thought to influence innate immune patterning in the mother which may affect the development of the neonatal immune system [177] . this influence in immune patterning can be seen through higher expression of toll-like receptors 2 and 4 and cd14 on peripheral blood cells, which implies possible desensitization to allergens in children [178] . t regulatory cells, which serve an important role in immune regulation and are thought to play an important role in asthma by suppressing the th2 inflammatory response to harmless air particles, have been shown to be impaired in the cord blood of neonates at hereditary risk for allergy [179] . in the 2017 study performed by singh et al. looking at serum ige and cutaneous sensitivity to food allergens in asthmatic children here was a negative correlation of total ige and duration of breastfeeding, indicating a connection between breastfeeding and the immune response [174] . additionally, reduced maternal intake of vitamins d and e and zinc during pregnancy has been associated with increased asthma symptoms in children [180, 181] . vitamin d has been associated with the maintenance and/or development of the t regulatory cells stated earlier in mice; however more research is needed to determine a definitive association in humans [177] . a clinical trial performed on non-smoking asthmatic patients showed higher vitamin d levels were associated with greater lung function; furthermore, supplementation with vitamin d showed improved treatment response to glucocorticoids [182] . vitamin d may also directly increase the antiinflammatory cytokine, interleukin (il)-10 and also enhance steroid-induced il-10 production (see . fig. 51.6) [177] . more research is needed to determine the exact mechanism of vitamin d in asthma and respiratory disease. beta-agonists, combined with corticosteroids, serve as the primary conventional therapy [183] . typically, a short-acting beta-agonist will first be prescribed to manage symptoms as needed, where low-dose inhaled corticosteroids may also be prescribed [156] . if symptoms persist, it is recommended to evaluate problems such as adherence to use, inhaler technique, or persistent allergen exposure and comorbidities [156] . once these are ruled out, the step-up treatment is a combination of an inhaled corticosteroid with a long-acting beta-agonist [156] . a summary of other conventional treatments and their mechanisms can be found in . table 51 .5 below. unfortunately, conventional methods for the treatment of asthma may have harmful side effects. for example, the use of . fig. 51.6 effect of asthma treatments on regulatory pathways. (reprinted from lloyd and hawrylowicz [177] . with permission from elsevier) systemic glucocorticoids may lead to immunosuppression, cataracts, and osteoporosis, where long-acting beta-agonists have the potential of increasing asthma exacerbation risk and death [25] . beta-agonist desensitization is thought to be one of the reasons for increasing asthma exacerbation risk and death [184] . related to several subtypes of asthma and their differing pathophysiologies, it is important to first determine the subtype before deciding on treatment. for example, in an individual with allergic asthma, this could be a potentially simple fix once the allergen that exacerbates symptoms is identified. a more conventional approach may involve starting the individual on an inhaled corticosteroid or an ige antagonist (i.e., omalizumab) [159] , rather than identifying the root cause of the patient's symptoms. while medications may be warranted until the trigger is identified, finding the underlying causes may not be common practice in many conventional settings. in contrast, the ifmnt assessment takes a much deeper dive into identifying triggers and any nutrient insufficiencies, inflammation or immune dysregulation, biochemical individuality, lifestyle, energy dysfunction, toxic load, sleep, and stress issues are taken into account. with this information, the practitioner can make more targeted dietary, lifestyle, and supplement recommendations to obtain sustained resolution of symptoms by treating the root cause (. table 51 a 26-year-old female presented with a complaint of reactive airway disease, which was diagnosed as asthma and had been prescribed inhalers. she reported that she felt like she had difficulty breathing most of her life, especially when exercising. however, her condition was not severe enough to seek help until she was 25 years of age. she reported a lot of stress during this time related to applying for a postgraduate training position. she also reported 1 year prior to diagnosis developing new allergic symptoms. her past medical history was significant for conditions related to airways, including chronic sinus infections, strep throat, bronchitis, and recurrent pneumonia. she could not remember the last time she felt well but assumed it was sometime as a young child. her nutrition and health goals were to breathe better and to not have to rely on inhalers. the following data was collected on her initial visit. . methyl xanthine found in tea used less commonly due to side effects relaxes airways due to inhibition of phosphodiesterases; acts as a functional antagonist in airway smooth muscle [171] based on data from ref. [18] . table 51 .6 summary of an integrative and functional medical nutrition therapy assessment adequacy of nutrient-dense foods to begin to assess nutritional status organic or nonorganic to assess toxic load and nutrient intake food preparation and processing to assess nutrient content and identify potential contaminants (e.g., plastic endocrine disruptors) assess food sensitivities or intolerances to identify potential triggers microbiome status: assess comprehensive digestive stool analysis for microbiology and fermented food intake; history of antibiotics or microbiota agonists (medications, toxins, stress, etc.) toxin intake via plastics or inhalation and skin absorption which may affect immune response assess flavonoids intake as they are antioxidant and anti-inflammatory compounds with mast cell inhibitory action; adequacy may reduce airway reactivity consider celiac disease and gluten intake as potential inflammatory antigens mineral assess and restore zinc, selenium, magnesium, manganese, iron, and iodine status to normal reference. caution to not supplement or intake of food sources higher than reference antioxidants assess and restore antioxidant balance; vitamins a, c, d, and e and glutathione assess quercetin intake (leafy vegetables, broccoli, red onions, peppers, apples, grapes, black and green tea, red wine) as it may act as mast-cell stabilizing agent inhibiting release of histamine, tnf-alpha release, formation of prostaglandin d2, reducing interleukin production consider supplementation of quercetin if quercetin intake is low [185] protein status assess and restore to support connective tissue and immune status ensure adequate glutamine and glycine intake oils/lipid/fatty acids assess fatty acid balance as dha important in lung tissue integrity assess adequate serum cholesterol and fat intake to support lipid bilayer important for cellular function in lung (epithelial cells, surfactant production, etc.) methylation assess methylation status and detoxification capacity of toxins related to asthma exacerbation; important assessment biomarkers suggested: mcv/mch, homocysteine, methylmalonic acid, rbc folate, genomic methylation snps inflammation/immune dysregulation assess asthma biomarkers to help identify root cause (see . fig. 51 extreme exhaustion, depression, add, anxiety (accompanied by panic attacks), constipation, pain in legs, neuropathy in feet (numbness and tingling), rapid heartbeat, and a very severe rash on feet known as chilblains. 5 utis -recurrent as a child. 5 poor immune function (frequent infections). 5 antibiotic use (very frequent from childhood into adulthood). 5 sinus infections, strep throat, and bronchitis -she had recurrent sinus infections and strep throat about once a year every year and often this would lead to bronchitis, she could not remember if she had these issues before middle school. 5 depression, anxiety, add. 5 acne. 5 peptic ulcers. 5 yeast infections -multiple throughout college. 5 eczema. 5 two recent episodes of pneumonia the last episode resulted in her asthma diagnosis. 5 asthma. evaluate exposure to fungus to identify allergic bronchopulmonary mycosis assess individual's medication history, considering short-and long-term use of conventional treatments evaluate exposure to particulate matter, airborne pollutants, cigarette smoke, or toxic metals such as cadmium and arsenic sleep and stress assess sleep adequacy (7-9 hours with 5-hour rem sleep) and quality (good sleep hygiene with little light/ sound/emf disturbance) to support detoxification of toxins that may worsen respiratory status and aid in repair of damaged lung tissue . periostin plays a role in the pathogenesis of allergic diseases, including asthma, as it is associated as a downstream molecule of the cytokine, il-13. periostin is used as a biomarker for type 2 immunity and can be used to determine the potential effectiveness of medications used to treat asthma, such as anti-ige antibodies and anti-il-13 antibodies. asthmatic patients with high serum periostin tend to be aspirin intolerant, eosinophilic, late asthma onset, and have a high nitric oxide fraction. high periostin can also indicate a reduced response to inhaled corticosteroids [186, 224] . periostin in the right panel is stained brown and is localized in the thickened basement membrane in asthmatic patients. (reprinted from izuhara et al. [186] . with permission from the korean academy of asthma, allergy and clinical immunology) for several weeks, led to being immobile for almost 2 weeks 5 8th-12th grade: was often sick (strep, sinus infections, bronchitis); described it as being constantly sick from fall through winter every year; also developed eating disorder during this time; had severe menstrual cramps (induced vomiting) accompanied by acne, which led to being put on birth control at age 17 as a precursor to accutane (never prescribed); chronic constipation starting during this time university 5 freshman -sophomore year: eating disorder was most severe during this time. 5 first semester of freshman year: developed digestion issues, after eating certain foods (especially mexican or salsas), stomach would become distended, experienced pain, and often would result in vomiting. pain so severe during finals week she was admitted to er with no diagnosis. ct scan revealed possible peptic ulcers. 5 junior-senior year: depression, anxiety, and inability to focus were most severe during this time which resulted in missing a lot of class and struggling as a student; suffered multiple panic attacks; gained a lot of weight (from 120 to 180 lb); end of senior year became engaged to be married -moved to dallas, tx. 5 lived in dallas for 6 months, continued to experience depression and anxiety and weight gain, and moved back to home state 5 initially started running (~2 miles a day) and experiencing inability to breathe, diagnosed with pneumonia, prescribed inhaler to help with running; other symptoms: eczema around the eyes and neck (after running outside), pain in calves, numbness and poor circulation in feet (pulse not detected by several health professionals), and development of chilblain rash (very painful, itching, lasts about 3-4 weeks from development to resolution); increased running -ran a half-marathon. visited pcp and several specialists for help with chilblain rash with no resolution or diagnosis; lost a lot of weight (from 170 to 140 lb). 5 ongoing increased depression, anxiety, and inability to focus; pcp rx cymbalta (depression and anxiety); cymbalta discontinued after ~2 months (did not tolerate side effects), continued psychological therapy for several months; chilblain rash continued. stopped running long distances. gained weight back (from 140 to 170 lb); subsequently saw blog for integrative rd and followed suggestion to eliminate gluten and focusing on whole foods diet. 5 chilblains and eczema began to resolve while following integrative rd recommendations of gluten-free diet with some improvements. however, difficulty breathing got worse, and diagnosis of asthma was made with fast-acting inhaler used for exercise; as time progressed, breathing continued to worsen, led to daily inhaler use. weight at this time is still at around 170 lb. high dairy diet (consumed dairy products at most meals and snacks), consumed three smaller meals with three snacks in between 5 meals and snacks balanced with protein, fat, and carbs, with carbs coming from fruits and vegetables and fat mainly from full fat cheese, greek yogurt, and butter 5 mostly nonorganic produce and commercially raised meats digestion, assimilation, and elimination 5 hx of peptic ulcers and chronic constipation (bm ~1-2 times a month) 5 bms currently at about 2 × per week on encounter utilization, cellular, and molecular (mapdom) 5 hx of likely gluten sensitivity. 5 presented symptoms of possible dairy sensitivity (bloating, acne, asthma). 5 evidence for compromised intestinal barrier. 5 minerals: infrequent bms could indicate low fiber or low mineral status (mg); when bms do occur, they are hard and dry (low mg); severe menstrual cramps (low mg); labs showed low k and na, on yaz birth control (low zinc and low b vitamins). 5 antioxidants: consumed adequate fruits and vegetables each day. 5 protein: has some evidence of poor/slowed wound healing as evidenced by sore on leg that has not completely healed after a year; cuts that take months to heal. 5 d and fat-soluble a, e, and k vitamins -hx of poor immune function (low d), vdr +/+ (low d and possibly a). 5 oils/fatty acids: high omega-6/omega-3 ratio, higher intake of damaged fats, very low intake of omega-3. 5 methylation: symptoms of depression, anxiety, add combined with mthfr c677t snp and on yaz (low b6 and folate). 5 eicosanoid fatty acids status -suspect issues with pge1 series pathway to control inflammation due to following signs and symptoms: allergies, autoimmune condition (asthma), peptic ulcers, eczema, and severe menstrual cramps 5 immune function -suspect gut dysbiosis due to following s&s: poor immune function, yeast infections, hx frequent antibiotic use, cyst, and constipation body composition 5 genetic makeup that indicated prone to gluten and dairy sensitivity, low vitamin d status, and impairment in methylation 5 broad spectrum probiotic + fermented foods 5 bioactive b complex (includes 50 mg p5p b6 and 800 mcg 5 thf) 5 260 mg gla evening primrose oil and zinc 3. aim to eat three larger meals a day, allowing space in between of ~ 5 hours; increase omega-3 intake by adding in small fatty fish, such as sardines or anchovies, once per week and taking fish oil; decrease omega-6 intake, switch from conventionally raised meats to organic, pasture-raised; and replace fat in diet from dairy with coconut sources, more nuts, and avocados. 5 patient presented ~6 months after the initial visit (september 2015). her breathing had improved immensely. she was able to stop taking her albuterol inhaler before exercise, recently stopped daily inhaler. 5 after dairy-free diet for 3 months, reintroduced dairy (cheese, butter, yogurt). asthmatic symptoms returned about 2-3 days after the addition of each. noticed the more dairy consumed, the worse her symptoms became. 5 at time of appointment, diet whole foods, gluten-free, and dairy-free. weight loss 10 lb within the first month of going dairy-free, continued to lose some weight. when reintroduced dairy symptoms of bloating and increase in weight, which resolved returning to dairyfree diet. 5 bms are regular now at ~ 2 × daily. this patient case followed some common patterns in the development of chronic disease and the comorbidities that are common, especially autoimmune conditions like asthma. the first is the genetic susceptibility of the individual; several snps are prone to dairy sensitivity. second, significant evidence for gut dysbiosis, promoted compromised gut barrier, can contribute to the development of dairy sensitivity. third is the exposure to dairy protein antigen. diet history evidenced trigger for asthmatic condition. additionally, inflammation, immune dysfunction, and methylation issues present. signs and symptoms significant for decrease in pge1 series anti-inflammatory pathways. low dietary omega-3s potential contributor to asthma. immune dysfunction evidenced by extensive history of infection-antibiotic use. genomic snp mthfr c667t gene, which indicated a greater need for folate. the use of yaz birth control and symptoms of depression, anxiety, and add known further to deplete b6 and folate. the diet and supplements recommended targeted control of inflammation, restore gut ecology, promote proper methylation, and replete nutrient insufficiencies. results from 6-month follow-up showed successful outcome in helping improve breathing and wean her off of inhalers. this case is an example of the ifmnt approach able to address the complexity of the whole patient story and bring the metabolic priorities into a manageable intervention program for the individual. one study found that the composition of the nasopharyngeal microbiota in children was linked to the frequency of upper respiratory tract infections and acute sinusitis [189] . a study that intranasally inoculated mice with lactobacillus fermentum reduced the amount of s. pneumoniae in the respiratory tract and increased the number of macrophages in the lung and lymphocytes in the trachea [189] . these findings may indicate a benefit of manipulating the upper respiratory tract microbiota with orally or nasally administered probiotics in the prevention and/or treatment of upper respiratory tract infections. allergic bronchopulmonary mycosis (abpm) is caused by a hypersensitivity reaction to fungal colonization of the airways. this is typically caused by the fungus aspergillus fumigatus. without treatment this may lead to fixed airflow obstruction and bronchiectasis [159] . guillain-barre syndrome (gbs) is a rare neurological disorder in which the body's immune system attacks the peripheral nervous system, known as the network of nerves located outside of the brain and spinal cord [190] . it is often preceded by a bacterial or viral infection. there are several potential mechanisms in which these infections trigger gbs. if an individual contracts a campylobacter jejuni bacterial infection, antibodies made to fight this infection can attack axons in motor nerves, which can potentially cause paralysis and respiratory failure [190] . campylobacter can be ingested via contaminated food or other exposures [190] . pérez-guzmán 2005 states that hypocholesterolemia is common among tuberculosis patients and suggests that cholesterol should be used as a complementary measure in antitubercular treatment [8] . alpha-1 antitrypsin (a1at) deficiency is an underrecognized disease in the united states, with around documented 100,000 people suffering from it, according to the alpha-1 foundation. this deficiency is inherited through autosomal codominant transmission, meaning affected individuals have inherited an abnormal aat gene from each parent [191] . individuals with this deficient allele present with aat levels at less than 35% to low-end normal levels [191] . however, it is also possible for individuals with a variant of this allele to be asymptomatic given different environmental conditions or lifestyle factors, such as refraining from smoking to reduce lung disease development risk [191] (7 box 51.4). a1at deficiency most often manifests in the lungs as chronic obstructive pulmonary disease (copd) (i.e., emphysema or bronchiectasis or "genetic copd"). a1at deficiency is often undiagnosed because people with genetic copd experience the same symptoms as people with copd, such as [191] : 5 shortness of breath 5 wheezing the only way you will know for sure if you have genetic copd due to alpha-1 is to get tested. ________ a1at deficiency can manifest in the liver as cirrhosis. symptoms related to the liver 5 unexplained liver disease or elevated liver enzymes 5 eyes and skin turning yellow (jaundice) 5 swelling of the abdomen (ascites) or legs 5 vomiting blood (from enlarged veins in the esophagus or stomach) a1at expresses sometimes in the skin as panniculitis [191] . panniculitis typically appears as raised red spots on the skin, which may break down and give off an oily discharge. while panniculitis spots (called nodules) may appear anywhere on the body, the most common places are the thighs, buttocks, and areas subject to injury or pressure. normal genotype m m 5 most common abnormal genes are called s and z 5 abnormal variant combinations: 5 zz (highest risk) 5 sz (lower risk increasing if smoker, inhalant pollutants) 5 mz (lower risk of carrying an a1at gene variant; considered "carriers") 5 alpha-1 is the most commonly known genetic risk factor for emphysema 5 up to 3% of all people diagnosed with copd may have undetected alpha-1 5 alpha-1 can also lead to liver disease. the most serious liver diseases are cirrhosis and liver cancer 5 the world health organization (who), american thoracic society (ats), and the european respiratory society (ers) recommend that everyone with copd be tested for alpha-1 alpha-1 is a progressive disease that benefits from early detection. it can cause serious lung diseases, such as copd and emphysema when undiagnosed. in some cases, alpha-1 can also cause liver disease [225] symptoms related to the lung [225] : 5 shortness of breath 5 wheezing 5 chronic bronchitis, which is cough and sputum (phlegm) production that lasts for a long time 5 recurring chest colds 5 less exercise tolerance 5 year-round allergies 5 bronchiectasis the alpha-1 antitrypsin (a1at) protein protects the body, especially fragile lung tissues, from the damaging effects of a powerful enzyme called neutrophil elastase that is released from white blood cells. in a1at deficiency, a genetic mutation reduces levels of the protective protein in the bloodstream. a1at deficiency can lead to chronic obstructive pulmonary disease (copd), specifically emphysema, and liver disease. smoking, which can inhibit what little a1at protein an affected person does have, increases the risk of lung disease. alpha-1 antitrypsin deficiency is completely determined by mutations in a single gene. the severity of symptoms is mostly a function of which mutations a person has and how many copies. however, smoking can greatly increase the risk of lung disease due to aat mutations. 23andme reports data only for the pi * m, pi * s, and pi * z versions of the gene that encodes aat. keep in mind that it is possible to have another mutation that causes this condition that is not included in this report [192] . a1at deficiency is a genetic disorder that reduces circulating levels of a protein that protects the lungs by trapping a1at in the liver, where the protein is produced, and prevents a1at from entering circulation. a1at deficiency can lead to chronic obstructive pulmonary disease (copd), specifically emphysema, and liver disease. when a disease-causing mutation is fairly common, as the pi * s and pi * z mutations are in europeans, it suggests that the mutation actually conferred an evolutionary advantage at one time. some researchers have suggested that several thousand years ago when the pi * z and pi * s mutations first arose, these versions of the gene for a1at gave people a survival advantage by creating an environment in their lungs that helped fight off infections. the scientists theorize that the antimicrobial benefits of the aat mutations outweighed the cost of an increased risk of copd and liver disease in the era before antibiotics were available [193] . in contrast to lung disease, manifestation of liver disease related to a1at can be referred to as a "toxic gain of function, " due to accumulation of mutant a1at protein rather than protease deficiency within the liver [144] . when taken together, fibrotic lung diseases are the leading cause of mortality worldwide. under the umbrella of interstitial lung disease (ild), pulmonary fibrosis (pf) is the most common. any ild that involves scarring of the lungs falls in the pulmonary fibrosis category. pulmonary fibrosis is the scarring of lungs, which destroys tissue over time, making it impossible to transfer oxygen from inhaled air into the bloodstream. there are more than 200 different diseases under the pulmonary fibrosis umbrella. because pf is often misdiagnosed or goes undiagnosed, there is not an accurate count of those with these diseases. however, it is estimated that as many as 1 in 200 adults over 60, or 200,000 people in the united states, are affected [184] . there are more than 50,000 deaths from ipf every year in the united states. more people die each year from idiopathic pulmonary fibrosis than from breast cancer [194] . there are other forms of interstitial lung disease including the newly identified pleuroparenchymal fibroelastosis, cryptogenic organizing pneumonia (cop), desquamative interstitial pneumonitis, nonspecific interstitial pneumonitis, hypersensitivity pneumonitis, acute interstitial pneumonitis, interstitial pneumonia, sarcoidosis, and asbestosis [195] . symptoms include cough and dyspnea, restrictive pulmonary function tests with impaired gas exchange, and progressive lung scarring. the disease progresses with an initiation of inflammation. fibrosing starts with the action of transforming growth factor-β (tgf-β)-dependent differentiation of fibroblasts to myofibroblasts, which then express α-sma (smooth muscle actin) [196] . after the tgf-β-dependent differentiation of fibroblasts to myofibroblasts, which express α-sma, there is sustained, excessive deposition of collagen by the myofibroblasts in the lung interstitium leading to the progressive lung damage in patients with pf [185] . research published in 2011 supported the idea that dysfunctional type ii aecs (alveolar epithelial cells) facilitate lung fibrosis through increased susceptibility to injury, leading to excessive and dysregulated remodeling [197] . the disease seems to progress in steps, and inflammation is not typically present continuously, except during certain periodic episodes of deterioration (. fig. 51.8 ). there are five main categories of pf causes: drug-induced, radiation-induced, environmental, autoimmune, and occupational. of these five, four have identifiable causes. some of the autoimmune diseases that can lead to pf are rheumatoid arthritis, scleroderma, sjogren's syndrome, polymyositis, dermatomyositis, and antisynthetase syndrome. idiopathic pulmonary fibrosis (ilp) is defined as pf with an unknown cause, including a genetic cause for some families [see . fig. 51.9 ]. the symptoms of ilp are a dry, hacking cough, shortness of breath, fatigue, chest discomfort, loss of appetite, and unexplained weight loss, all caused by the fibrosing of the lungs. diagnosis can be difficult, and pf is often misdiagnosed as copd or other more common lung diseases. in addition, in the recent past, path to a true diagnosis was invasive. since damage to the lungs, even through a diagnostic biopsy, can trigger further lung damage or a period of fibrosis, many physicians or patients are cautious with a biopsy approach to diagnosis. since the current treatments are limited, one must evaluate whether defining the exact form of pf is necessary for treatment and follow-up. difficulty breathing, crackling sounds while breathing, and low oxygen levels are the first indicators. clubbed fingernails may also be a symptom. high-resolution ct scans are performed, which can show scarring. the pulmonologist will ask many questions and order more blood tests to try to distinguish between the 200 forms of pf. the future is pointing to molecular endotyping as a more accurate way to diagnose. molecular endotyping includes genetic, metabolic, transcriptional, and environmental factors to help determine the pathophysiology [199] . genetic research has been progressing for a couple decades with illuminating results. there are more than a dozen genetic variants that have been associated with this family of diseases. researchers now believe at least 20% of idiopathic pulmonary fibrosis (ipf) patients with multiple family members suffering from ipf have some common familial genetic variants, which may allow researchers to eventually drop the term idiopathic and further define various forms or categories, with differing progression or outcome. the name given to this version of interstitial pneumonias is familial interstitial pneumonia (fip) [200] [see . currently two categories of genetic focus have been defined: those genes related to telomere biology (shorter telomeres) and those related to surfactant protein processing. the genes related to shorter telomeres are tert, terc, htr, dkc1, and rtel1. more mutations have been found in the tert gene, which encodes the protein component of telomerase, than any other gene. further research may allow targeted therapies to affect the genetic expression associated with the development of ipf [201, 202] . a common variant within the promoter of the muc5b gene is the most replicated single-nucleotide polymorphism related to familial and sporadic forms of ipf as well as early radiographic findings of ipf [203] (. figs. 51.9 and 51.10). wound contraction and re-epithelialization . fig. 51.8 the cellular and molecular mechanisms of fibrosis in multiple organs. the cellular and molecular mechanisms of fibrosis in multiple organs. once an injury occurs in an organ, epithelial and/or endothelial cells are impaired, which results in the release of chemokines and growth factors, including il-13 and tgf-b1. macrophages and monocytes are recruited and activated, both of which further release cytokines and chemokines and further induce fibroblast activation. activated fibroblasts transform into a-sma-expressing myofibroblasts and migrate into the wound along the fibrin lattice. ecm is excessively accumulated, and some parenchymal cells (hepatic stellate cells in the liver, tubular epithelial cells in the kidney, alveolar epithelial cells in the lung, or cardiomyocytes in the heart) are further differentiated into myofibroblasts or fibroblasts by the stimulation of cytokines and chemokines, especially for tgf-b1. after the inflammatory phase, two events occur. one is the regeneration of injured tissues followed by wound contraction and reepithelialization. in contrast, once chronic injury, inflammation, and necrosis occur, myofibroblasts are perpetually activated, and excessive ecm is deposited, finally resulting in fibrosis formation. ctgf, connective tissue growth factor; ecm, extracellular matrix; egf, epidermal growth factor; emt, epithelial-mesenchymal transition; hsc, hepatic stellate cell; il, interleukin; mmp, matrix metalloproteinase; tgf, transforming growth factor; timp, tissue inhibitors of metalloproteinase. (reprinted from chen et al. [198] . with permission from elsevier) conventional treatment is typically palliative. the american thoracic society recognizes that supplemental oxygen and transplantation are the only suggested treatments for ipf. supplemental oxygen is prescribed, and the need for oxygen increases over the progression of the disease. keeping the oxygen saturation level over 90% (normal is in the upper 90s) is ideal and is how healthcare providers determine the level of supplemental oxygen to be used. cardiovascular exercise, in this case called pulmonary rehabilitation, is recommended to maintain as much use of the lungs as possible. infrequently, nutrition and counseling are recommended and are placed into the category of symptom management. nutrition can have a significant role in the management of this disease, but little implementation exists in some of the proposed protocols. there are currently two medications available in the united states with minor impact on the disease progression: nintedanib (commonly called ofev) and pirfenidone (esbriet). histopathological quantification showed similar amounts of dense collagen fibrosis, fibroblast foci, and alveolar macrophages in untreated or pirfenidone-or nintedanibtreated ipf patients [204] . both have significant side effects, including fatigue and gi issues, and patients may have to evaluate their quality of life versus length of life. other antiinflammatories or immune-suppressing medications used are corticosteroids, mycophenolate mofetil/mycophenolic acid (cellcept®), or azathioprine (imuran®). immunesuppressing drugs may be harmful for those with short telomeres, and researchers are exploring this potentially contradictory recommendation [205] . lung transplantation is a final effort. about is half of all transplants. with the prevalence of this disease closer to 200,000, this is a small fraction of those with the disease. some of those with the transplant go on to live productive lives, while others develop pf again, in the transplanted lungs. overall, there is a shorter life expectancy in those with pf, because of telomere shortening. bone marrow or immune response abnormalities have been found in some ipf cases before and after lung transplantation, which increases the associated morbidity. as stated above, inflammation occurs at the beginning and throughout the progression of all fibrosing diseases, including those of the lungs. therefore, reducing inflammation is one wise strategy to slow fibrosing. there are several nutrients that can help slow or reverse the inflammation involved in the fibrosing process. the following two-part diagram shows where in the fibrosing pathogenesis each phytonutrient acts [198] (. fig. 51 .11). a few of those compounds are discussed in more detail here. curcumin, the active constituent in the common spice turmeric, has been shown to reduce fibrotic activity in several studies. in mice, curcumin inhibited collagen secretion of ipf fibroblasts. it affects the signaling of tgf-β, in a dosespecific manner, resulting in reduced expression of α-sma, which is responsible for inappropriate fibrosing. this was shown in vitro and in vivo in mice, with intraperitoneal, but not oral, administration. at the time of the study, oral ingestion of curcumin was not adequately absorbed into plasma, and there was greater than ten times plasma concentration of curcumin following an intraperitoneal injection [88] . however, some new oral products on the market are showing greater absorption. the results of this study suggest more research into curcumin, including improved delivery into patients. for example, some delivery options may include nebulized curcumin directly into the lungs, binding it to highly absorbable agents for oral use or liposome-encapsulated curcumin suitable for intravenous use (already shown to be effective in an animal model). according to manufacturers of curcumin products, some are more readily absorbed than others. one study on fibrosing suggested that a dose of around 2200 mg curcumin split into three doses taken with meals including pepper (bioperine) achieved doses that were sufficient to exert the desired therapeutic effect. research into using quercetin also has some promising results in slowing the progression of ipf. quercetin reversed lung fibrosing in mice and reversed the disease progression normally caused by typical pulmonary senescence markers [206] . it is worth mentioning that n-acetylcysteine (nac), a long-used therapeutic agent for breaking down mucus in the lungs, has not been found to be effective in those with ipf. in fact, due to its acidic nature, it has even been shown to be harmful when used in the inhaled form [207] . several of the drugs being developed have a natural product as a model or foundation. until a drug or gene therapy is developed that stops or reverses this disease, it may make sense for the patient to focus on anti-inflammation and reducing myofibroblast activation, the extracellular matrix (ecm) accumulation, and the epithelial-mesenchymal transition (emt) process. the phytochemicals listed in . fig. 51 .11 would be good ones to investigate. with the recent identification of genes associated with ild, a call for gene-related therapies both related to telomere lengthening and connective tissue disease has been initiated, and this type of therapy, as with any disease, could be personalized [208] . one recent study looked at various biomarker values as a more precise way of diagnosing. the biomarker molecules were classified according to their involvement into alveolar epithelial cell injury, fibroproliferation, and matrix remodeling as well as immune regulation. furthermore, genetic variants of tollip, muc-5b, and other genes associated with a differential response to treatment and with the development and/or the prognosis of ipf were identified. research into personalized medicine for treatment is starting [209] . although controversial, because of the lack of research on interpretation of the results, telomere length testing is available directly to consumers and through healthcare . fig. 51.11 antifibrosis therapy. the molecular mechanisms and therapeutic targets of natural products against fibrosis. a tgf-b exerts a profibrotic effect through smad-dependent [target (1)] and smadindependent pathways [target (2) ]. in the smad-dependent pathway, tgf-b1 directly phosphorylates and activates the downstream mediator smad2 and smad3 through tgf-b receptor i, and then smad2 and smad3 bind smad4, which forms a complex that moves into the nucleus and initiates gene transcription. smad7, transcribed by smad3, is a negative regulator of tgf-b/smad signaling, and the imbalance between smad3 and smad7 contributes to fibrosis. pi3k, erk, and p38 mapk are downstream mediators of the smad-independent tgf-b pathway. pparg [target (3)] could inhibit tgf-b to reduce fibrosis, while ctgf [target (4)], a matricellular protein, contributes to wound healing and virtually all fibrotic pathology. additionally, gas6 contributes to fibrosis through the tam receptor, which further activates the pi3k/akt pathway. similarly, lpa triggers fibrosis through the lpa1 receptor [target (5) ] that stimulates b-catenin to induce fibrogenesis. the activation of the hedgehog pathway [target (6) ] induces the transcriptional activity of gli to express target genes, which have an important role in interstitial fibrosis, undergoing myofibroblast transformation and proliferation. il pathway [target (7) ] stimulates nf-kb [target (8) ] to activate tgf-b to induce fibrogenesis, while nrf2 [target (9) ] antagonizes nf-kb activity to protect against fibrosis. b the chemical structures of isolated compounds and their therapeutic targets are presented. ctgf, connective tissue growth factor; il, interleukin; lrp, low-density lipoprotein receptor-related protein; ri, transforming growth factor-b receptor i; rii, transforming growth factor-b receptor ii; sara, smad anchor for receptor activation; stat, signal transducer and activator of transcription; tcf, t-cell factor; tgf, transforming growth factor. (reprinted from chen et al. [198] practitioners. there are a few different methods: quantitative polymerase chain reaction, or qpcr, which has a 20% variability rate, and flow cytometry and fluorescent in situ hybridization, or flow-fish, which has a 5% variability rate. most research labs use flow-fish for research. telomere length is a hot topic in research, the antiaging industry, and with popular health blogs. shorter-thanaverage telomeres have also been linked to heart disease and heart failure [163, 210, 211] , cancer [212] , diabetes [213] , and osteoporosis [214] . research has shown ways to slow telomere shortening. some include reducing stress, meditation, practicing loving kindness (a technique encouraging compassion) [215] , reducing exposure to air pollution and toxins [216] , cardiovascular exercise [217] , and a healthy fat and high vegetable diet [218, 219] . one study showed that 45 minutes of cardiovascular exercise three times per week resulted in longer telomeres representing 10 years of biological age, similar to those of marathon runners, compared to those who didn't exercise much or at all [220] . intermittent fasting, which reduces oxidative stress and keeps weight in check, has exploded in the scientific literature as a way to increase longevity and slow telomere shortening [221, 222] . nicotinamide adenine dinucleotide (nad+) supplements may also help maintain telomere length by activating sirtuins, the antiaging enzymes; parps, which are involved in dna repair; and cd38, which plays a role in insulin production. another supplement, cycloastragenol, derived from the herb astragalus, has also been shown to activate telomerase in mice. an ingredient called ta-65 has been derived and is used in supplements [223] . overall, a healthy lifestyle and diet seem to delay the shortening of telomeres. with relation to pf, the gene mutations involved in telomere shortening may or may not be influenced by the above interventions. more research is needed for this. pulmonary fibrosis is a devastating disease with no management or a known cure. the integrative and functional medicine nutritionist can help her/his patient by managing weight, encouraging a healthy diet full of anti-inflammatory foods and encouraging a healthy lifestyle with exercise and stress reduction. there is some promising research into natural supplement use to target the different areas of progression within the disease process and some ongoing drug and gene therapy development to follow. the prevalence of lung disease in the united states and worldwide is growing and will continue to grow rapidly with the deterioration of earth's atmosphere, which is caused by pollutants such as industrial and construction toxins and volcanic and wildfire particulates. poor maternal, childhood, and adult nutrition from micronutrient-poor diets resulting in nutrient insufficiencies, not necessarily nutrient deficiencies, is also contributing to increased lung disease diagnoses or poorer results during treatment [226, 227] . lifestyle choices and habits also play a role in the development of many of the lung diseases in today's world, such as smoking or vaping, which uses chemicals that are poorly studied to date. other lung diseases have their roots in genetics. some key processes drive many lung diseases, with the inflammatory process being the most important, according to current literature. nutrition can be of great help with inflammation, using a diet rich in whole foods providing micronutrients and phytonutrients. understanding genetics is also key to unraveling the causes and potential future treatments for many lung diseases. those patients with both genetic and environmental determinants, such as in those who smoke and have genes associated with copd, are at the greatest risk [228] . despite the prevalence of lung disease, there is a general lack of nutrition knowledge among practitioners, including familiarity with the research about the use of nutrition for prevention, slowing disease progression, or as a treatment of lung disease. historically, nutrition has been used in a supportive role, primarily monitoring macronutrients to prevent weight loss, muscle atrophy, and acid/alkaline balance. although this is extremely important, more attention needs to be directed toward emphasizing micronutrients and phytonutrients. research is strong regarding the benefits of vitamins, minerals, and pre-and probiotics, and indeed, some integrative and functional practitioners are using vitamin and mineral nutritional therapy in oral, intramuscular, and intravenous applications, when allowed, in practice. a newer area of research is around nutraceuticals, including targeted vitamins, minerals, and plantderived constituents concentrated to therapeutic doses. some exciting research around the use of curcumin and quercetin, for example, has been shown to dampen inflammation to the point of disrupting the disease process (see above). the expanding knowledge of the microbiome is identifying the importance of the lung and airway microbiome in respiratory health. more research, and indeed more education for nutritionists around the existing research, is needed to fully understand the best opportunities for the use of nutrition in the treatment or prevention of lung disease. the intersection of aging biology and the pathobiology of lung diseases: a joint nhlbi/nia workshop chronic respiratory disease | gateway to health communication | cdc global status report on noncommunicable diseases the top 10 leading causes of death in the united states vitamin d status has a linear association with seasonal infections and lung function in british adults respiratory health and disease in europe: the new european lung white book impact of dna repair, folate and glutathione gene polymorphisms on risk of non-small cell lung cancer nutrition and respiratory health-feature review measuring the global burden of disease and epidemiological transitions: 2002-2030 nutrition therapy & pathophysiology decrease in pulmonary function and oxygenation after lung resection sequelae and complications of pneumonectomy surgery for tumor recurrence in a pneumonectomy space function and structure of the respiratory system function and structure of the respiratory system the respiratory system chapter 2. function and structure of the respiratory system cholesterol interactions with fluid-phase phospholipids: effect on the lateral organization of the bilayer the effect of membrane lipid composition on the formation of lipid ultrananodomains physical studies of cholesterolphospholipid interactions lung collagen composition and synthesis. characterization and changes with age always cleave up your mess: targeting collagen degradation to treat tissue fibrosis concord: fritz perlberg publishing the use (or otherwise) of pulse in general practice the two-way association of periodontal infection with systemic disorders: an overview oral health care and aspiration pneumonia in frail older people: a systematic literature review periodontal systemic associations: review of the evidence association between oral habits, mouth breathing and malocclusion the microbiome and the lung vitamin d supplementation to prevent acute respiratory tract infections: systematic review and meta-analysis of individual participant data probiotics supplementation in children with asthma: a systematic review and meta-analysis probiotics in asthma and allergy prevention gut-lung axis: the microbial contributions and clinical implications the gut-lung axis in respiratory disease the role of autophagy in allergic inflammation: a new target for severe asthma the lung microbiome in moderate and severe chronic obstructive pulmonary disease learn about bronchiectasis american lung association learn about bronchiolitis american lung association idiopathic pulmonary fibrosis | national heart, lung, and blood institute (nhlbi) american lung association desquamative interstitial pneumonia american lung association kyphoscoliosis -an overview diagnosis and treatment of inflammatory joint disease. hip pelvis pulmonary arterial hypertension (pah) lung carcinoid tumor what is small cell lung cancer? cell lung cancer bacterial tracheitis alpha-1 foundation | alpha1. org obstructive sleep apnea (osa) in adults anemia in chronic obstructive pulmonary disease and the potential role of iron deficiency disruption of iron homeostasis and lung disease iron homeostasis in health and disease out of balance -systemic iron homeostasis in iron-related disorders inflammation and neurodegeneration laboratory, chronic diseases research center (cedoc), nova medical school (nms)/faculdade de ciências médicas hfe gene variants and iron-induced oxygen radical generation in idiopathic pulmonary fibrosis prognostic impact of vitamin b6 in lung cancer effects of vitamin b6 metabolism on oncogenesis, tumor progression and therapeutic responses association of the b-vitamins pyridoxal 5′-phosphate (b6), b12, and folate with lung cancer risk in older men sex differences in risk of lung cancer associated with methylene-tetrahydrofolate reductase polymorphisms dietary intake of b vitamins and methionine and risk of lung cancer two common single nucleotide polymorphisms in the gene encoding β-carotene 15,15′-monoxygenase alter β-carotene metabolism in female volunteers consensus report on nutrition for pediatric patients with cystic fibrosis potential micronutrients and phytochemicals against the pathogenesis of chronic obstructive pulmonary disease and lung cancer the controversial role of retinoic acid in fibrotic diseases: analysis of involved signaling pathways vitamin d and susceptibility of chronic lung diseases: role of epigenetics vitamin d deficiency at 16 to 20 weeks' gestation is associated with impaired lung function and asthma at 6 years of age vitamin d and lung disease dietary vitamin c intake protects against copd: the korea national health and nutrition examination survey in 2012 smokers with adequate vitamin c intake show a preferable pulmonary function test vitamin c mitigates oxidative stress and tumor necrosis factor-alpha in severe community-acquired pneumonia and lps-induced macrophages inhibitory effect of a mixture containing vitamin c, lysine, proline, epigallocatechin gallate, zinc and alpha-1-antitrypsin on lung carcinogenesis induced by benzo(a) pyrene in mice thoracic transplantation: oxidative stress and nutritional intakes in lung patients with bronchiolitis obliterans syndrome dual role of vitamin c utilization in no2-induced oxidative stress in lung tissues of mice two faces of vitamin e in the lung vitamin e isoform γ-tocotrienol downregulates house dust mite-induced asthma serum tocopherol levels and vitamin e intake are associated with lung function in the normative aging study dietary nutrients associated with preservation of lung function in hispanic and non-hispanic white smokers from new mexico the disturbed redox-balance in pulmonary fibrosis is modulated by the plant flavonoid quercetin curcumin inhibits transforming growth factor β induced differentiation of mouse lung fibroblasts to myofibroblasts directory of open access journals curcumin attenuates radiationinduced inflammation and fibrosis in rat lungs curcumin effect on bleomycininduced pulmonary fibrosis in mus musculus antifibrotic effects of curcumin are associated with overexpression of cathepsins k and l in bleomycin treated mice and human fibroblasts orally administered chitosan-coated polycaprolactone nanoparticles containing curcumin attenuate metastatic melanoma in the lungs effect of glycosides based standardized fenugreek seed extract in bleomycin-induced pulmonary fibrosis in rats: decisive role of bax, nrf2, nf-κb, muc5ac, tnf-α and il-1β evaluating the ameliorative potential of plant flavonoids and their nanocomposites in bleomycin induced idiopathic pulmonary fibrosis cannabidiol and (-)delta9-tetrahydrocannabinol are neuroprotective antioxidants dietary intake of six minerals in relation to the risk of chronic obstructive pulmonary disease mineral intake and lung cancer risk in the nih-american association of retired persons diet and health study nothing boring about boron dietary mineral intake and lung cancer risk: the rotterdam study bone mineral density, lung function, vitamin d and body composition in children and adolescents with cystic fibrosis: a multicenter study the alpha-lipoic acid derivative dhlhzn: a new therapeutic agent for acute lung injury in vivo suppression of a cancer stem-like phenotype mediated by alpha-lipoic acid in human lung cancer cells through down-regulation of β-catenin and oct-4 influence of alpha-lipoic acid on nicotine-induced lung and liver damage in experimental rats effect of n-acetylcysteine in subjects with slow pulmonary mucociliary clearance the effect of oral n-acetylcysteine in chronic bronchitis: a quantitative systematic review the intrabronchial microbial flora in chronic bronchitis patients: a target for n-acetylcysteine therapy? the effect of oral n-acetylcysteine on lung glutathione levels in idiopathic pulmonary fibrosis attenuation of influenza-like symptomatology and improvement of cell-mediated immunity with long-term n-acetylcysteine treatment high-dose oral n-acetylcysteine, a glutathione prodrug, modulates inflammation in cystic fibrosis national institutes of health potential micronutrients and phytochemicals against the pathogenesis of chronic obstructive pulmonary disease and lung cancer the relation between dietary intake of individual fatty acids, fev1 and respiratory disease in dutch adults lipoxins: nature's way to resolve inflammation the role of free fatty acids in idiopathic pulmonary fibrosis association between ω3 and ω6 fatty acid intakes and serum inflammatory markers in copd a prospective study of dietary polyunsaturated fatty acids intake and lung cancer risk effectiveness of essential amino acid supplementation in stimulating whole body net protein anabolism is comparable between copd patients and healthy older adults whole fruits and fruit fiber emerging health effects glutathione redox control of asthma: from molecular mechanisms to therapeutic opportunities is there any relationship between plasma antioxidant capacity and lung function in smokers and in patients with chronic obstructive pulmonary disease? the treatment of pulmonary diseases and respiratoryrelated conditions with inhaled (nebulized or aerosolized) glutathione. evid based modifications of plasma proteome in long-lived rats fed on a coenzyme q10-supplemented diet increased oxidative stress in patients with chronic obstructive pulmonary disease (copd) as measured by redox status of plasma coenzyme q10. pathophysiology coenzyme q10 supplementation reduces corticosteroids dosage in patients with bronchial asthma effects of coenzymeq 10 administration on pulmonary function and exercise performance in patients with chronic lung diseases glutathione cofactors. available at geology and health: closing the gap pulmonary fluorosis: a review comparison of the fractional exhaled nitric oxide levels in adolescents at three schools located three different distances from a large steel mill inorganic arsenic and respiratory health, from early life exposure to sex-specific effects: a systematic review the broad scope of health effects from chronic arsenic exposure: update on a worldwide public health problem chronic arsenic toxicity & human health human exposure to dietary inorganic arsenic and other arsenic species: state of knowledge, gaps and uncertainties toxic substances portal -arsenic dynamed plus record no. t114977, chronic arsenic poisoning toxicological aspects of cadmium and occupational health activities to prevent workplace exposure in japan: a narrative review current status of cadmium as an environmental health problem serum heavy metals and obstructive lung disease: results from the national health and nutrition examination survey relationship between blood levels of heavy metals and lung function based on the korean national health and nutrition examination survey iv-v department of the environment and energy systematic review and meta-analysis of the association between ambient nitrogen dioxide and respiratory disease in china a framework for examining social stress and susceptibility to air pollution in respiratory health quantifying the impact of current and future concentrations of air pollutants on respiratory disease risk in england short-and long-term effects of ambient ozone and fine particulate matter on the respiratory health of chronic obstructive pulmonary disease subjects weekly personal ozone exposure and respiratory health in a panel of greek school children. environ health perspect nutritional solutions to reduce risks of negative health impacts of air pollution cigarette smoking and inflammation revisited effects of tobacco smoke on immunity, inflammation and autoimmunity electronic cigarette, effective or harmful for quitting smoking and respiratory health: a quantitative review papers review: the toxicity of e-cigarettes and children's respiratory health respiratory health and allergies from chemical exposures among machining industry workers in selangor, malaysia occupational pesticide exposures and respiratory health persistent effects of chlorine inhalation on respiratory health respiratory muscle function in patients with cystic fibrosis. pediatr pulmonol emotions, morbidity, and mortality: new perspectives from psychoneuroimmunology understanding mindbody interaction from the perspective of east asian medicine. evid based complement alternat med climate change and respiratory disease: european respiratory society position statement nutrients and foods for the primary prevention of asthma and allergy: systematic review and meta-analysis global strategy for asthma management and prevention. glob initiat asthma tests for assessing asthma pediatric expression of mast cell activation disorders asthma phenotypes and endotypes: an evolving paradigm for classification a histamine-free diet is helpful for treatment of adult patients with chronic spontaneous urticaria serum diamine oxidase activity in patients with histamine intolerance leukocyte telomere length and cardiovascular disease in the cardiovascular health study regulation of antioxidant enzymes in lung after oxidant injury oxidative stress in asthma resolution of acute inflammation in the lung the alkaline diet: is there evidence that an alkaline ph diet benefits health? magnesium in prevention and therapy effects of magnesium supplementation on the glutathione redox system in atopic asthmatic children diet and asthma: vitamins and methyl donors drugs for asthma dietary inflammatory index is related to asthma risk, lung function and systemic inflammation in asthma fruit and vegetable intake and risk of wheezing and asthma: a systematic review and meta-analysis correlation of cutaneous sensitivity and cytokine response in children with asthma coeliac disease and asthma association in children: the role of antibiotic consumption regulatory t cells in asthma prenatal farm exposure is related to the expression of receptors of the innate immunity and to atopic sensitization in school-age children characterization of regulatory t cells in urban newborns is vitamin d deficiency to blame for the asthma epidemic? maternal food consumption during pregnancy and the longitudinal development of childhood asthma vitamin d levels, lung function, and steroid response in adult asthma camp regulation of airway smooth muscle function quercetin acutely relaxes airway smooth muscle and potentiates β-agonist-induced relaxation via dual phosphodiesterase inhibition of plcβ and pde4 using periostin as a biomarker in the treatment of asthma biomarkers in asthma | aaaai developmental bisphenol a exposure modulates immune-related diseases nasopharyngeal microbiota composition of children is related to the frequency of upper respiratory infection and acute sinusitis disorders/patient-caregiver-education/fact-sheets/guillain-barré-syndrome-fact-sheet clinical manifestations, diagnosis, and natural history of alpha-1 antitrypsin deficiency let's talk about alpha-1 antitrypsin deficiency alpha1-antitrypsin deficiency what is pulmonary fibrosis | pulmonary fibrosis foundation home page -ipf foundation ipf foundation a clinical evaluation of baofeikang granule in combined pulmonary fibrosis and emphysema treatment -pulmonary fibrosis foundation endoplasmic reticulum stress enhances fibrotic remodeling in the lungs natural products as a source for antifibrosis therapy idiopathic pulmonary fibrosis: phenotypes and comorbidities the genetic basis of idiopathic pulmonary fibrosis telomerase mutations in families with idiopathic pulmonary fibrosis telomerase and idiopathic pulmonary fibrosis genetics of pulmonary fibrosis wolters pj. histopathological and molecular analysis of idiopathic pulmonary fibrosis lungs from patients treated with pirfenidone or nintedanib telomere shortening is behind the harm of immunosuppressive therapy in idiopathic pulmonary fibrosis quercetin enhances ligand-induced apoptosis in senescent idiopathic pulmonary fibrosis fibroblasts and reduces lung fibrosis in vivo application of n-acetylcysteine in pulmonary disorders. in: the therapeutic use of n-acetylcysteine (nac) in medicine integrating genomics into management of fibrotic interstitial lung disease existing and emerging biomarkers for disease progression in idiopathic pulmonary fibrosis association of shorter mean telomere length with risk of incident myocardial infarction: a prospective, nested case-control approach telomere length of circulating leukocytes is decreased in patients with chronic heart failure de vivo i. telomere length, cigarette smoking, and bladder cancer risk in men and women monocyte telomere shortening and oxidative dna damage in type 2 diabetes telomere length in leukocytes correlates with bone mineral density and is shorter in women with osteoporosis loving-kindness meditation practice associated with longer telomeres in women association between leukocyte telomere shortening and exposure to traffic pollution: a cross-sectional study on traffic officers and indoor office workers physical exercise prevents cellular senescence in circulating leukocytes and in the vessel wall association of marine omega-3 fatty acid levels with telomeric aging in patients with coronary heart disease telomere length, oxidative damage, antioxidants and breast cancer risk physical activity and telomere length: impact of aging and potential mechanisms of action protein restriction, epigenetic diet, intermittent fasting as new approaches for preventing age-associated diseases fasting mimicking diet is very relevant for health and longevity cycloastragenol is a potent telomerase activator in neuronal cells: implications for depression management periostin: an emerging biomarker for allergic diseases zemaira® | alpha-1 antitrypsin deficiency diagnosis assessment of quality of life in children suffered from asthma identifying maternal conditions affecting altered embryologic development. neonatal advanced practice nursing: a case-based learning approach susceptibility for cigarette smoke-induced damp release and damp-induced inflammation in copd key: cord-016020-awanrm9u authors: fox, julie d.; tilley, peter a. title: respiratory pathogens date: 2007 journal: molecular pathology in clinical practice doi: 10.1007/978-0-387-33227-7_41 sha: doc_id: 16020 cord_uid: awanrm9u respiratory tract infections are among the most common presenting complaints of patients in both hospital and community settings. they are a considerable burden in terms of both patient morbidity and public health interventions. laboratory diagnosis of respiratory tract infections should provide guidance in therapy and prognosis, as well as useful epidemiological information reflecting trends in the community. understanding and monitoring such trends facilitates early recognition of new infectious agents in a population. a summary of the common viruses and bacteria causing respiratory tract infections and their clinical relevance is given in tables 41–1 and 41–2, respectively. respiratory tract infections are among the most common presenting complaints of patients in both hospital and community settings. they are a considerable burden in terms of both patient morbidity and public health interventions. laboratory diagnosis of respiratory tract infections should provide guidance in therapy and prognosis, as well as useful epidemiological information reflecting trends in the community. understanding and monitoring such trends facilitates early recognition of new infectious agents in a population. a summary of the common viruses and bacteria causing respiratory tract infections and their clinical relevance is given in tables 41-1 and 41-2, respectively. even with a significant clinical effort and analysis of multiple specimens, current laboratory methods fail to diagnose approximately half of lower respiratory tract infections. in fact, laboratory diagnosis of communityacquired pneumonia (cap) is so poor that current clinical practice guidelines do not recommend testing for all but the most severely affected patients and advise use of empiric therapy. 1 this pragmatic approach fails to address issues of antimicrobial overuse and resistance, public health surveillance, and advancement of medical knowledge. many "atypical" bacteria are known to cause severe respiratory symptoms, but lack of good diagnostic procedures has hampered the measurement of the real impact of such infections in the community. despite vaccination policies, bordetella pertussis infection remains relatively common in children and adults and is associated with chronic cough in adults. 2 mycoplasma pneumoniae, legionella pneumophila, and chlamydophilia (previously chlamydia) pneumoniae are all recognized causes of lower respiratory tract infections, but again, their impact has not been studied in detail. in addition, despite the well-recognized association of viral infections with upper and lower respiratory tract infections, the current diagnostic virology procedures do not provide an answer rapidly enough to with parainfluenza virus type 4, human coronaviruses, rhinoviruses, and some enteroviruses would not ordinarily be identified without rna detection methods. the impact of such infections is only just being realized, and there is probably an underestimation of their clinical importance, particularly for immunocompromised individuals, the elderly, or those with underlying conditions such as asthma. 5, 6 human metapneumovirus has been confirmed as an important cause of severe lower respiratory tract infection. the virus has been circulating for more than 50 years, and studies using molecular assays have confirmed its wide distribution, 7,8 but many laboratories have not been successful in isolating this virus. the recent identification of the agent causing severe acute respiratory syndrome (sars), known as sars-cov, has illustrated the need for expansion of diagnostic testing to encompass new emerging viruses and the limitations of conventional virological laboratory approaches to respiratory pathogen diagnosis. 9, 10 even if it is possible to culture viruses efficiently, isolation and confirmation of the cause of a cytopathic effect can take days to weeks, depending on the pathogen. waiting for a culture-positive result can take many days, during which time the patient may be inappropriately treated and, if hospitalized, infection control measures may not be initiated. also, the use of primary primate cells in culture (which gives the best yields of influenza and parainfluenza viruses) is unlikely to be sustainable in the long term. when available, monoclonal antibodies are useful in direct virus-specific antigen detection tests, and these can be used for rapid diagnosis. many laboratories are able to provide diagnostic testing for influenza, parainfluenza (types 1-3), respiratory syncytial virus (rsv), and respiratory adenoviruses. a respiratory specimen containing cells is necessary for sensitive detection of viruses by immunofluorescence or other antigen-detection methods. good-quality diagnostic samples (often lavage or aspirate samples) can usually be obtained from young, immunocompetent, hospitalized individuals, but in other circumstances the ideal sample may not be available. delays in transportation may reduce specimen quality and compromise assay results. the most-difficult (and least-efficient) specimens for diagnostic testing are swab samples containing minimal cellular material taken from largely asymptomatic individuals in the community. smears from these samples can be difficult to interpret in a direct antigen test and thus culture of the sample is usually required for pathogen identification. bacterial antigen detection by a direct fluorescent antibody (dfa) test is similarly compromised by poor sensitivity and has the added concern of artifacts leading to false-positive results, particularly for b. pertussis and l. pneumophila. serological assays of an antibody response to infection are available for some respiratory pathogens. although useful for retrospective evidence of infection in a community, the results are not timely enough for patient management. for some cell-associated or intracellular bacteria and viruses, antibody responses develop slowly, if they develop at all, and convalescent sera taken many weeks after disease onset are required to make a definitive diagnosis (e.g., for c. pneumoniae, legionella species, rsv). for other infections such as influenza, antibody responses are brisk, but frequent reinfection reduces the igm response and convalescent sera are required to demonstrate changing titers. the limitations of conventional testing are well recognized for viral 11, 12 and bacterial 13,14 pathogens, and some of the key differences between nucleic acid and culture or direct antigen testing methods are summarized in table 41 -3. utilization of rapid viral diagnostic procedures, such as may be provided by molecular amplification methods, could help to reduce the emergence of antibiotic-resistant bacteria. one study demonstrated that a 52% reduction in antibiotic use was possible using molecular methods for viral diagnosis. 15 despite the obvious economic burden of cap, we do not have accurate data on how many of these infections are viral in origin or caused by the atypical bacteria for which routine diagnostic testing is not readily available. the identification of emerging human viral infections (such as h5n1 influenza and sars-cov) has heightened awareness of the gaps in respiratory pathogen diagnosis. nucleic acid detection assays are likely to be utilized more widely to identify novel emerging pathogens that could result in worldwide outbreaks. molecular amplification assays are being used successfully for the identification of organisms associated with pneumonia. 16 the potential for nosocomial spread of respiratory pathogens is well recognized for patients admitted to the hospital with rsv, parainfluenza, or other infections, serving as a reservoir for transmission to vulnerable patients and leading to possible outbreaks. 17 previously, available diagnostic methods were not sensitive enough to identify sources of outbreaks, but the advent of molecular amplification methods has allowed even environmental sampling to be helpful in confirming outbreak sources and linking clustered cases of infection. 18 samples used for detection of respiratory pathogens include swabs (usually nasopharyngeal or throat), aspirates (nasopharyngeal or tracheal), sputum (usually from individuals presenting with pneumonia), or bronchoalveolar lavage specimens. for infections involving the entire respiratory tract, nasopharyngeal specimens are practical for diagnosis. for other infections, which are more focal, melting-curve analysis of pcr products using the intercalating dye sybr green differentiates between specific and nonspecific products without further manipulation of the pcr products. specific sequence information required for "catch-all" approach, which may be advantageous when novel design; triage of testing can be difficult; pathogens need to be identified generic primers may be used to identify novel pathogens sensitivity exquisite sensitivity but can be prone to generally less sensitive than nucleic acid detection methods cross-contamination problems specificity careful handling required to avoid careful handling required to avoid contamination, but contamination (common problem); less-common problem than for molecular methods; dfa primer/probe design crucial subject to over interpretation strain typing most definitive method limited serotyping (e.g., influenza, legionella) automation automated extraction equipment becoming difficult to automate available; automated detection commonplace safety inactivated before analysis, but antimicrobial isolates useful for antimicrobial sensitivity testing and sensitivity information requires knowledge of phenotyping, but specialized safety requirements needed for genotypic mutation culture of category 3/4 pathogens quality assurance proficiency and validation of methods not well culture depends critically on cell line or medium quality; established maintaining quality can be difficult lower respiratory tract specimens are required (e.g., for legionella). sample preparation is a critical step for the detection and analysis of organisms. numerous methods, from simple boiling to sophisticated automated protocols, are available for disruption of the organism and purification of the nucleic acids. many studies have demonstrated inhibitors in respiratory specimens, making some form of extraction (with or without freezing) necessary to avoid frequent false negative results. commercial kits for preparation of samples are available and should reduce interlaboratory variation in results. simultaneous extraction of rna and dna facilitates assays for both viruses and bacteria. molecular techniques for the detection and analysis of pathogens associated with respiratory infection provide specific diagnoses for individual cases and for outbreaks. currently, fda-cleared molecular tests are not available for the detection of respiratory pathogens, with the exception of mycobacterium tuberculosis (see chapter 43). molecular tests are performed using either validated laboratorydeveloped procedures or commercial testing reagents. published diagnostic methods for detection of respiratory pathogen dna or rna directly from clinical specimens utilize target amplification procedures such as polymerase chain reaction (pcr) or nucleic acid sequence-based amplification (nasba).although direct detection methods based on nucleic acid hybridization would be theoretically possible, the amount of target nucleic acid in specimens may be minimal and such methods would lack sensitivity compared to amplification methods, unless the organism was propagated before analysis. thus, the molecular amplification procedures reported for direct detection of respiratory pathogens in clinical samples include pcr (e.g., reference 19 and figure 41 assays have utilized bacterial ribosomal rna (rrna; e.g., reference 22 ). for cellular samples tested for respiratory pathogens, targeting messenger rna (mrna) or genomic antisense rna may enhance diagnostic sensitivity. a variety of formats have been utilized for the detection of amplified products. procedures that separate target amplification from the detection phase (agarose gel analysis or endpoint hybridization) are well established 24, 25 and may allow multiple targets to be analyzed in a single reaction, providing added typing information. for ease of use and incorporation into diagnostic laboratories, most laboratory-developed assays for detection of respiratory pathogens utilize real-time amplification methods in which the amplification and detection steps are combined. some methods use intercalating dyes with the analysis of pcr product melting temperatures (e.g., as described previously 26 and illustrated in figure 41 -1), whereas others use fluorogenic primers or probes (e.g., taqman, hybridization format, and molecular beacons 19, [21] [22] [23] 27 ) to ensure the specificity of the reaction. figure 41 -2 illustrates a real-time rt-pcr assay for sars-cov using probe-specific detection of amplified products (one fluorescence measurement per cycle). the range of real-time pcr or rt-pcr and nasba methodologies used for respiratory targets is diverse given the fact that assays are laboratory developed. 28, 29 the difficulty with diagnosis of respiratory infections is the wide range of pathogens with similar presentations. the nucleic acid technologies utilized currently in the majority of diagnostic laboratories are real-time pcr single-target assays. in some cases, generic primers can be designed to pick up several related pathogens. such generic primers may be based on conserved protein coding sequences (such as those essential for enzyme function) or noncoding regions for which variation is limited because of the need for maintenance of secondary structure. the use of primer sets to pick up genera or even families of organisms has shown promise in limited studies, including analysis of legionella and mycobacteria. generic assays have the ability to detect many related organisms and may be used to characterize previously undescribed species in respiratory infection. limited multiplex procedures have been reported for detection of related organisms using real-time pcr or nasba procedures, but such assays are difficult to set up and control. for many respiratory pathogens, there is sufficient variation that multiplex approaches have been developed to detect, for example, all possible respiratory adenovirus, 19 influenza, 20 or parainfluenza 21 types. a simple, dual-labeled multiplex nasba assay is shown in figure 41 -5 that uses separate primer sets and specific molecular beacon probes for parainfluenza types 2 and 3 (hpiv2 and hpiv3) in the same reaction mix. each probe is labeled with a different fluorophore, allowing detection and differentiation of both viruses in a single reaction. an ambitious multiplex nested rt-pcr procedure with gel analysis of the amplicons detects influenza a, b, and c viruses, rsv (a and b subtypes) , and adenoviruses in a single assay. 30 the procedure, while complex to set up and validate, was reported to have good specificity and better sensitivity than antigen/culture procedures. interpretation and validation of a negative result are important parts of diagnostic tests based on nucleic acid amplification. some assays incorporate an internal control system to distinguish true-negative from false-negative results. the internal control may amplify with the pathogen-specific primers but result in an amplicon with a different size or internal sequence from the pathogen amplicon. alternatively, the internal control may be an external sequence spiked into the reaction (heterologous control) and amplified with a primer set different from the pathogen primers. in the example shown in figure 41 -3, amplification of the rna heterologous control is consistent across many clinical samples and ensures that there are no gross inhibitors present in the reactions. for cellular samples obtained for detection of respiratory pathogens, the internal control reaction can utilize human dna, rrna, or mrna detection. such approaches have the added value of assessing for sample collection and integrity, as well as amplification inhibitors. the relative merits of commercial versus laboratorydeveloped tests depend on the laboratory facilities, the technical expertise available, and the clinical need for expanded diagnosis. commercial testing reagents provide quality controls and procedure standardization that facilitate clinical studies. 24, 32 many companies are focusing on providing analyte-specific reagents (asrs) for respiratory pathogen assays. asrs will provide the laboratory with quality-controlled primers and probes, while allowing them the flexibility to test for currently known circulating pathogens or according to a local testing algorithm. microarray-based detection of multiplexed pcr products also has been reported. 31 classically, typing of bacteria or viruses has used serological techniques that rely on antibody-antigen interactions. one benefit of approaches based on dna or rna detection is the more-detailed, quantitative assessment of the relationship between organisms, providing valuable data relevant to outbreak investigations and community health. variation at the nucleic acid sequence level is not necessarily reflected in altered protein sequence or function; thus, additional sequence variation information may not correlate with conventional typing methods. restriction fragment length polymorphism (rflp) analysis by pulsed field gel electrophoresis (pfge), either with or without blot hybridization, has been utilized for analysis of complex dna genomes from a variety of respiratory pathogens. rflp analysis also has been applied to pcr or rt-pcr products from respiratory bacteria and viruses. in general, such methods can provide resolution down to the subtype level and have proven useful in outbreak investigation, as illustrated in figure 41-6 for b. pertussis isolates. the difficulty with gel-based typing assays, such as pfge, is standardizing results and sharing data between laboratories. amplified fragment length polymorphism (aflp) analysis represents an alternative method with better discriminatory power and portability, but this approach has not been used extensively for respiratory isolates to date. for respiratory viruses, other methods have been used for typing, including heteroduplex mobility assay (hma), single-strand conformation polymorphism (sscp), and rflp analysis of amplified pcr products. in general, hma is considered technically complex but has the capacity to distinguish viral quasispecies with >3% nucleotide differences. sscp and rflp, while technically easier, generally can resolve viruses only to the subtype level, and rflp has the added constraint of assessing only sequence differences in restriction sites. the use of sequencing to assess the relationship among viruses is well established, and molecular phylogenetic knowledge is expanding, allowing modeling of viral populations and prediction of new outbreaks. 33 the level of resolution using primary sequence is at one genome, and point mutations can be identified. sometimes this provides more information than originally sought and creates problems in interpretation; while in other circumstances even small sequence variations can confer important changes in viral transmissibility and disease outcome. identification of emerging viruses, which may have been recently introduced into the human population (e.g., sars-cov and influenza a h5n1 types), is critical to public health. analysis of such novel viruses has relied heavily on sequencing of isolates or amplicons. 9, 10, 34 human influenza a viruses are associated with enhanced morbidity and mortality compared with influenza b or influenza c viruses. differences in pathogenicity for subtypes of influenza a also have been reported; for example, h3n2 is associated with more severe infection than h1n1. such types and subtypes of influenza a can circulate independently, and their identification is important for assessment of current vaccine efficacy. reassortment of the two predominant influenza subtypes infecting humans in recent times has been reported, and analysis of main hemagglutinin (ha) types has been undertaken by a range of molecular and nonmolecular methods. 35 (1997-1998 and 2003-2004 34,38 ) emphasize the need for detailed surveillance of influenza viruses and vigilance in identification of emerging viruses of importance to public health. detailed analysis of avian h5n1 viruses that have infected humans to date have confirmed that all genes are of avian origin and are associated with minimal or very inefficient humanto-human spread. the potential for reassorted viruses that could more easily spread among humans is clear, and molecular methods are now an important part of influenza surveillance. recent studies of human metapneumovirus have identified two main lineages, with sequence diversity within each group (figure 41-8 40 ) , thus displaying a similar pattern to rsv isolates that are classified into two major groups,a and b. 39 further studies will confirm whether this distinction is associated with differences in virulence. sequence analysis for typing of bacteria has been slower to develop than that for viruses but has been utilized for investigation of some atypical bacteria associated with outbreaks of respiratory infection (e.g., legionella 41 ). due to the problem of recombination, characterization of a single bacterial gene often does not reflect the organism as a whole. multilocus sequence typing is a strategy that addresses this and appears useful for analysis of b. pertussis. 42 microarray hybridization methods have been used to identify and differentiate related pathogens. 31 such an approach was useful in first identifying the agent of sars as a coronavirus. 43 molecular methods provide additional information about the virulence and type of infectious organism, as illustrated by recent experience with sars-cov and influenza types. molecular tests have advantages over conventional procedures, but the sensitivity of molecular amplification methods can lead to problems with interpretation of results. for many organisms, a gold standard method is not available that accurately reflects the enhanced sensitivity of molecular methods, as has been seen with pcr testing for b. pertussis or c. pneumoniae in clinical samples. studies confirm that pcr tests are very sensitive, and pcr-positive individuals may be culture negative or asymptomatic, so that results must always be interpreted in the clinical context. inhibitors of amplification are common in respiratory specimens, so a negative result must be interpreted in the context of the nucleic acid extraction method and the control results to monitor for nucleic acid degradation and amplification inhibition. when assays for the detection of respiratory pathogens are designed, primers and probes should not cross-react with normal respiratory flora or other respiratory pathogens. the triage of molecular testing for respiratory infection diagnosis is difficult. currently, a single respiratory pathogen test detects only one or a few related pathogens. also, bacterial testing and viral testing are not combined. thus, many molecular tests must be used to screen for all appropriate pathogens, which increases testing costs. thus, a laboratory that embarks on using molecular methods for the diagnosis of respiratory infections may require a complex testing algorithm. one approach is use a multiplex amplification procedure to identify multiple pathogens in a single assay, with certain assays now commercially available. 24, 32 unfortunately, such tests tend to be expensive and, if developed by the laboratory, are very difficult to control and ensure equal sensitivity and specificity for all pathogens. thus, despite the potential for replacement of many culture and antigen procedures with nucleic acid amplification assays,such a molecular diagnostic revolution has not yet happened. the exception is for new pathogens when nucleic acid amplification and detection methods are clearly far superior to alternatives (e.g., metapneumovirus, sars-cov) or for testing of samples that are suboptimal for routine procedures (e.g., in surveillance situations). respiratory infections are currently underdiagnosed, despite the fact that accurate pathogen identification is important to ensure appropriate patient management and monitor infectious trends in the community. the major stumbling blocks in the diagnosis and investigation of respiratory infections are the complexity of testing algorithms and the number of potential targets that cause both upper and lower respiratory tract symptoms. real-time pcr methods have vastly improved the sensitivity for detection and recognition of some difficult-to-culture organisms, and will likely become standard practice in the clinical laboratory in the next few years. there is, however, a limit to how many organisms can be "multiplexed" in a single test. microarray hybridization of randomly amplified pcr products from respiratory cultures and clinical samples has shown some success. 31 if the promise of early experiments is maintained when applied to large-scale clinical studies, this could answer some of the technical problems surrounding the use of multiplex systems. microarray hybridization, while not currently as convenient as realtime pcr detection methods, potentially has the benefit of being able to resolve complex product mixtures and provide clinically valuable information. the use of molecular methods for typing and outbreak investigation of respiratory pathogens of public health importance is well established and is likely to expand. future directions will incorporate the use of microarray systems for respiratory pathogen detection and analysis to allow crossing of the barriers between conventional virology and bacteriology (and mycology/parasitology). once microarray systems have been developed and validated, the costs of this enhanced technology may be reduced and justifiable. identification of novel viruses, which have presumably only recently been introduced to humans, has reinforced the need for careful surveillance of emerging respiratory pathogens and institution of appropriate infection control measures. lessons should be learned from the continuous sensitive surveillance and typing of organisms such as influenza and b. pertussis to direct the use and efficacy of available vaccines. molecular techniques developed for detection and analysis of the microbes responsible for respiratory infections will be vital to our understanding of pathogenic mechanisms, appropriate management, and prevention of outbreaks in the future. gel-based typing procedures (pfge, hma, sscp) slowly will be replaced by sequencebased alternatives (e.g., multilocus sequence typing or mlst), which are more amenable to standardization and sharing of data among laboratories. phylogenetic tree illustrating the relationships among fusion gene sequences for 12 human metapneumovirus strains (from 2002). af371337 is the genbank accession number for the prototype human metapneumovirus strain, and hmpv 35 is a canadian strain canadian guidelines for the initial management of community-acquired pneumonia: an evidence-based update by the canadian infectious diseases society and the canadian thoracic society. the canadian community-acquired pneumonia working group bordetella pertussis in the aetiology of chronic cough in adults. diagnostic methods and clinic clinical and financial benefits of rapid detection of respiratory viruses: an outcomes study contribution of influenza and respiratory syncytial virus to community cases of influenza-like illness: an observational study respiratory tract viral infections in inner-city asthmatic adults polymerase chain reaction is more sensitive than viral culture and antigen testing for the detection of respiratory viruses in adults with hematological cancer and pneumonia human metapneumovirus infections in young and elderly adults human metapneumovirus as a cause of community-acquired respiratory illness identification of severe acute respiratory syndrome in canada identification of a novel coronavirus in patients with severe acute respiratory syndrome clinical assessment of a generic dna amplification assay for the identification of respiratory adenovirus infections a comparison of nested polymerase chain reaction and immunofluorescence for the diagnosis of respiratory infections in children with bronchiolitis, and the implications for a cohorting strategy detection of bordetella pertussis in a clinical laboratory by culture, polymerase chain reaction, and direct fluorescent antibody staining; accuracy, and cost polymerase chain reaction as a sensitive and rapid method for specific detection of mycoplasma pneumoniae in clinical samples cost-effectiveness of rapid diagnosis of viral respiratory tract infections in pediatric patients nucleic acid amplification tests for the diagnosis of pneumonia molecular epidemiology of two consecutive outbreaks of parainfluenza 3 in a bone marrow transplant unit detection of adenoviruses (adv) in culture-negative environmental samples by pcr during an adv-associated respiratory disease outbreak multiplexed, real-time pcr for quantitative detection of human adenovirus rapid detection of influenza a and b viruses in clinical specimens by light cycler real time rt-pcr development and evaluation of nuclisens basic kit nasba for diagnosis of parainfluenza virus infection with "end-point" and "real-time" detection detection of mycoplasma pneumoniae by real-time nucleic acid sequence-based amplification development and evaluation of nucleic acid sequence based amplification (nasba) for diagnosis of enterovirus infections using the nuclisens basic kit rapid simultaneous diagnosis of infections with respiratory syncytial viruses a and b, influenza viruses a and b, and human parainfluenza virus types 1, 2, and 3 by multiplex quantitative reverse transcription-polymerase chain reaction-enzyme hybridization assay (hexaplex) multiplex pcr for typing and subtyping influenza and respiratory syncytial viruses multiplex real-time pcr assay for detection of influenza and human respiratory syncytial viruses development and clinical evaluation of an internally controlled, single-tube multiplex real-time pcr assay for detection of legionella pneumophila and other legionella species real-time pcr in virology nasba and other transcription-based amplification methods for research and diagnostic microbiology simultaneous detection of influenza a, b, and c viruses, respiratory syncytial virus, and adenoviruses in clinical samples by multiplex reverse transcription nested-pcr assay microarray-based detection and genotyping of viral pathogens evaluation of the prodesse hexaplex multiplex pcr assay for direct detection of seven respiratory viruses in clinical specimens the application of molecular phylogenetics to the analysis of viral genome diversity and evolution molecular aspects of avian influenza (h5n1) viruses isolated from humans molecular diagnosis of influenza influenza virus types and subtypes detection by single step single tube multiplex reverse transcription-polymerase chain reaction (rt-pcr) and agarose gel electrophoresis influenza ah1n2 viruses, united kingdom, 2001-02 influenza season molecular changes associated with the transmission of avian influenza a h5n1 and h9n2 viruses to humans circulation patterns of genetically distinct group a and b strains of human respiratory syncytial virus in a community human metapneumovirus infections in hospitalized children detection of legionella species in respiratory specimens using pcr with sequencing confirmation multilocus sequence typing of bordetella pertussis based on surface protein genes characterization of a novel coronavirus associated with severe acute respiratory syndrome key: cord-002590-24o2viv3 authors: rahe, michael c.; murtaugh, michael p. title: mechanisms of adaptive immunity to porcine reproductive and respiratory syndrome virus date: 2017-06-13 journal: viruses doi: 10.3390/v9060148 sha: doc_id: 2590 cord_uid: 24o2viv3 the adaptive immune response is necessary for the development of protective immunity against infectious diseases. porcine reproductive and respiratory syndrome virus (prrsv), a genetically heterogeneous and rapidly evolving rna virus, is the most burdensome pathogen of swine health and wellbeing worldwide. viral infection induces antigen-specific immunity that ultimately clears the infection. however, the resulting immune memory, induced by virulent or attenuated vaccine viruses, is inconsistently protective against diverse viral strains. the immunological mechanisms by which primary and memory protection are generated and used are not well understood. here, we summarize current knowledge regarding cellular and humoral components of the adaptive immune response to prrsv infection that mediate primary and memory immune protection against viruses. porcine reproductive and respiratory syndrome virus (prrsv) is the most severe enemy of porcine health and wellbeing. the highly mutable, enveloped, rna virus was discovered nearly 30 years ago but, while extensive research has been carried out and many vaccines have been developed, there is still no reproducible immunological intervention that develops a broadly protective immune response against virulent prrsv. prrs disease was first described on farms in north carolina in the usa at the end of the 1980s. outbreaks were marked by reproductive losses, post-weaning pneumonia, and increased mortality in growing pigs. initial efforts to identify an etiological agent responsible for the new disease syndrome were unsuccessful, leading to the disease being temporarily designated mystery swine disease (msd) in north america. koch's postulates for msd were fulfilled in 1991 with a previously unidentified rna virus discovered in europe, named lelystad virus [1, 2] . the discovery was quickly followed by isolation of the virus, initially referred to as swine infertility and respiratory syndrome virus or sirs virus, in north america [3] . the name prrsv was introduced in 1992 and encompasses prrsv-1 (genotypes first isolated in europe) and prrsv-2 (genotypes first isolated in north america) [4, 5] . today, both virus types are globally distributed, with prrsv-1 viruses predominantly in europe and prrsv-2 viruses largely in north america, asia and south america [6] . recent discovery of multiple arteriviral nucleotide sequences in nonhuman primates has led to a reclassification of prrsv as two distinct viruses, prrsv-1 and prrsv-2 [7] . here, we use the generic prrsv to refer broadly to both viruses when evidence indicates that are equivalent, and the specific prrsv-1 and prrsv-2 is used when a distinction is desired. the reasoning is based on the many similarities of the two viruses in fine details of genome structure and organization, transcriptional strategy, host preference, clinical signs of disease, and prrsv infects cells of the macrophage/monocyte lineage, including dendritic cells [20] [21] [22] [23] . permissive cells express cluster of differentiation (cd)163, a hemoglobin-haptoglobin scavenger, which is the necessary receptor for prrsv infection and replication [24] [25] [26] . macrophages and dendritic cells are common members of the mononuclear phagocyte system that plays a varied, and important, role in many aspects of tissue remodeling, development, immunity and immunopathology [27] . classically designated as part of the innate immune system, these leukocytes are critical for the development of a productive adaptive immune response. macrophages and, particularly, dendritic cells take up and present antigen to t cells and b cells, thus initiating an adaptive immune response against the presented antigen [28,29]. if a pathogen is able to infect and destroy, manipulate, or maintain itself within macrophages or dendritic cells, it then has the potential to modulate the immune response into a favorable situation for its own replication and survival. therefore, many pathogens employ strategies for macrophage infection as a way to make the host more amenable to infection. recent research into mycobacterium tuberculosis (mtb) has shown that, after phagocytosis, the bacterium arrests phagosome maturation and intra-phagosome lipolysis resulting in mtb survival and an increased supply of nutrients for growth [30, 31] . human immunodeficiency virus (hiv) infects macrophages to establish reservoirs within the host for the chronic stage of the disease when cd4 + t cells are largely depleted and neutralizing antibodies may be present [32] [33] [34] . leishmania major is a protozoan which infects phagocytes to subvert the immune system. the parasite expresses glycoprotein (gp)63, a multifaceted surface-expressed pathogenicity factor that is responsible for preventing antigen presentation and killing by natural killer (nk) cells [35] [36] [37] . indeed, there are many more examples of burdensome pathogens which target phagocytic cells, especially macrophages and dendritic cells, in an attempt to gain a foothold within the immune system and allow for unchecked survival and replication [38] [39] [40] . prrsv is one of these pathogens. the ability of prrsv to subvert the immune system has not been investigated as extensively as more prominent pathogens of humans, such as hiv. prrsv has been shown to inhibit the production, or the downstream effects, of type 1 interferons, particularly interferon (ifn)-α, on intracellular signaling [41] [42] [43] [44] [45] [46] [47] [48] . interestingly, multiple prrsv proteins (nonstructural protein (nsp) 1, nsp2, nsp4, nsp5, nsp11 and nucleocapsid) have been reported to possess interferon inhibiting abilities. in addition, a number of in vivo experiments have reproduced earlier in vitro findings showing that interferon-α is inhibited during the early stages of prrsv infection [47, 49, 50] . while the impact of type 1 interferon suppression is likely to create a favorable environment for the virus to replicate and survive in phagocytic cells, it is still unclear what effect, if any, suppression of type 1 interferon activity has on the adaptive immune response to infection [51] . future investigations could clarify the relative contributions of viral proteins on modulation of interferon production and their impacts on viral growth, survival, and the subsequent development of the adaptive immune response. apart from interfering with interferon expression, prrsv has also displayed the in vitro ability to subvert the immune system by spreading from cell to cell. recent work has uncovered the ability of the virus to spread infectious viral rna, several replicases, and certain structural proteins between cells via intercellular nanotubules [52, 53] . while this activity theoretically allows for prrsv to avoid neutralizing antibodies, the presence and significance of this mechanism in prrsv pathogenesis has yet to be fully elucidated. future studies are needed to determine if this process operates in naturally permissive macrophages and dendritic cells, if it can be interrupted, for example by intracellular antibodies, and what effect it might have on viral propagation [54, 55] . vaccines depend upon innate immune stimulation to promote effective adaptive immune response to antigen, resulting in production of antibodies and cytotoxic t cell responses. the ability of a pathogen to successfully infect and replicate within innate immune cells makes the development of a protective immune response more difficult. as a result, the generation of effective vaccines against pathogens that target immune cells is fraught with challenges. extensive variation in viral genetics, primary immune responses, and cross-protection indicates that much remains to be learned about cellular pathogenesis in order to arrive at better immunological solutions. immunosuppression refers to suppression of the immune system and its ability to fight infection. hiv and infectious bursal disease virus are examples of viral infections that destroy entire lymphoid cell populations that ablate or disable adaptive immune responses. lymphoproliferative cancers block cellular differentiation and deprive the body of mature, effector lymphocytes, thus causing immunosuppression in a different manner. prrsv does neither; infection does not lead to severe lymphoid depletion or ablation, and it does not interfere profoundly with lymphocyte differentiation or maturation. leukocyte perturbations in lymphoid tissues are associated with prrsv infection, suggesting that adaptive immunity might be weakened, though not destroyed [56] [57] [58] [59] [60] [61] . the immune system also maintains peripheral tolerance to self and commensal bacteria through immunosuppressive mechanisms that include regulatory t cells (tregs), characterized as cd4 + cd25 + forkhead box p3 (foxp3) + t lymphocytes [62] . treg suppressive properties were discovered when thymectomized or treg-depleted mice succumbed to autoimmune reactions [63, 64] . tregs suppress effector and effector memory t cell proliferation by cytokine deprivation leading to polyclonal apoptosis, and by suppression of antigen presenting cells by cytotoxic t lymphocyte-associated antigen-4 (ctla-4) and other mechanisms [62] . studies in prrsv infections give an ambiguous picture about the role of tregs. prrsv-2 strains are reported to induce a strong treg response which included transforming growth factor (tgf)β-1 secretion in vitro as well as in vivo [65, 66] . other studies did not show treg responses to infection with either prrsv-1 or prrsv-2 [67, 68] . interleukin-10 (il-10), an immunosuppressive cytokine expressed by various cell types including tregs, was induced by prrsv-2 vaccination in weaned pigs in one study, but was not induced in weaned or adult pigs in another study [69] . additional in vitro and in vivo studies reported il-10 mrna transcription and cytokine production after prrsv infection [70] [71] [72] . however, kinetic analysis in serum of viremic pigs of various ages showed that elevated il-10 levels were primarily a function of age and were not associated with infection status [69] . the only exception was in weaned pigs infected with a virulent virus, in which a transient increase was associated with viral pathogenesis [69] . on balance, the immunological evidence for prrsv inducing a state of immunosuppression does not appear to be compelling. secondary infections following prrs disease outbreak in swine herds, suggesting a reduced ability to fight infection, is an alternative indicator of immunosuppression. an early study showed concurrent pulmonary bacterial infections in 58% of 221 prrs cases [73] . however, the study did not determine if bacterial infections were present before the prrs outbreaks. the immunosuppression question also was addressed in more controlled settings using dual infection models with prrsv and various bacterial species. a summary of published literature in 2003 showed no predisposition to bacterial disease in 8 of 15 coinfection models, three ambiguous outcomes, and four cases in which severity of disease was increased [74] . more recent studies found a positive association between prrsv infection and replication of porcine circovirus 2 (pcv2) or swine influenza virus [75, 76] . it is possible that bacterial infections in swine herds increase following prrs outbreaks due an increased burden of viral infection on host resilience to pathogen burden. subclinical viral and bacterial infections are common, with pcv2, salmonella enterica, haemophilus parasuis, various mycoplasma species, leptospira, and escherichia coli being examples. control of infection is maintained by a combination of immune resistance to microbial replication and tissue tolerance to damage. in a coinfection model of influenza virus and legionella pneumophila, it was clearly demonstrated that l. pneumophila infection was subclinical in healthy mice, but was lethal in the presence of influenza virus [77] . overwhelming disease was due to loss of tissue resilience, since the bacterial load was unchanged [77] . this model might account for mortalities observed in experimental swine following prrsv exposure [78] . given the variable results of prrsv coinfection models in swine and an alternative mechanism for increased disease in prrsv-infected herds, generalized immunosuppression does not appear to be a key feature of prrsv pathogenesis. prrsv, like many viruses, has developed countermeasures to host immune responses that enable it to survive and replicate for extended periods of time before the infection is resolved. prrsv modulation of intracellular antiviral defense mechanisms has been reviewed extensively [79] . the effects of prrsv infection on adaptive immune response, i.e., antigen-specific t cell, b cell, and antibody responses, are less well characterized. the antiviral response of t cells to prrsv, examined primarily by the ifnγ enzyme-linked immunospot (elispot), appears to develop slowly over a period of weeks, and is not associated with changes in viral loads in blood or in infected lung and lymphoid tissues [80, 81] . peripheral blood mononuclear cells (pbmc) from young, weaned pigs show limited ifnγ responses even when stimulated by phytohemagluttinin, which might account for the low anti-prrsv responsiveness after re-stimulation in vitro [69] . however, pbmc from growing pigs and mature sows, which showed higher levels of ifnγ sensitivity, still showed limited responsiveness [69] . these findings indicate that prrsv may interfere with specific cell-mediated immunity, but more direct evidence is needed for a fuller understanding. by contrast, the interaction of prrsv with pigs does not appear to retard or attenuate the development of humoral immunity or b cell differentiation. induction of antibody responses to prrsv proteins, both structural and non-structural, occurred in the same time frame as antibody responses to keyhole limpet hemocyanin (klh), an irrelevant protein antigen [51] . the antibody response to klh was also the same in the presence or absence of prrsv infection [51] . similarly, prrsv infection did not inhibit cellular or humoral immune protection in response to pseudorabies virus vaccination [82] . thus, the adaptive b cell response is not delayed or suppressed by prrsv. an extended viremia and prolonged survival in lymphoid tissues is characteristic of prrsv infection. these features show that prrsv has mechanisms of immune avoidance that are not present in viruses such as influenza virus and foot and mouth disease virus, in which sterilizing immunity is achieved within 10-14 days. it appears from the findings of field observations and experimental investigations that some type of prrsv-specific t cell interference is present, whereas specific b cell inhibition or a generalized state of immunosuppression are not immunological hallmarks of prrsv infection. the antibody response to prrsv typically dominates discussions of prrsv immunity, as neutralizing antibodies are the crucial component of immune-mediated protection against most viral infections [83,84]. as a result, shortly after the identification of prrsv as the causative agent of mystery swine disease, there was a strong push to identify the presence and dynamic response of neutralizing antibodies against prrsv and then to characterize their specificity for prrsv variants. early work suggested that neutralizing antibodies against homologous prrsv could be found as early as 9-11 days after inoculation [85]. however, this was likely the non-affinity matured immunoglobulin (ig)m response, as anti-swine igm ablated the previously observed neutralizing activity. subsequent research showed that the high affinity neutralizing igg response, detected at around 28-42 days post-inoculation, is specific for the inoculating virus with partial neutralizing activity against heterologous viruses [86] [87] [88] [89] [90] . following the identification of prrsv neutralizing antibodies, the effectiveness of immunoglobulins in protecting against infection was evaluated with passive transfer studies. these experiments displayed the effectiveness of neutralizing antibodies at preventing clinical infection and disease against homologous challenge [91, 92] . however, these studies also showed that immune protection can be quite limited, especially between prrsv-1 and prrsv-2 [93]. within prrsv-1 or prrsv-2, protection against homologous inoculation is consistently solid, whereas protection against heterologous challenge is variable for unclear reasons [93-95]. however, genetic similarity, based primarily on orf5 sequence comparisons, shows no relationship with degree of protection [96]. these results appeared to explain the potential field problem, in which vaccinated or live virus inoculated animals become infected with a variant prrsv genetically different enough from the inoculating strain to evade the immune system, propagate, and then cause disease. hence, ever since the mutability, antigenic variability, and resultant immunological elusiveness of prrsv were first appreciated, a broadly neutralizing antibody response to prrsv has been coveted by immunologists and practitioners [97] . recent research shows that there are animals capable of developing a broadly neutralizing antibody response to genetically disparate viruses [9, 98] . however, this immune capability has only been found in a proportion of animals in groups of similar genetics age, sex, and exposure history [9] . the seemingly random ability of some animals to develop broadly neutralizing antibodies suggests that the inherent variation of the adaptive immune response may play a role in conferring broadly neutralizing capabilities to certain animals. investigations into this ability are needed at the lymphocyte level and while the obvious target is the b cell, t cells cannot be overlooked, as the induction of a humoral immune response requires antigen-specific t cell driven help [99, 100] . therefore, animals able to develop a strong neutralizing antibody response would require both b cells and t cells that are capable of recognizing neutralizing epitopes. the conditions needed to achieve cross-neutralizing antibody production are not known, but may involve multiple exposures to the same or different virus isolates. sows with high titered, broadly neutralizing antibodies were found in herds with multiple exposures to virulent field viruses [9] . in an experimental study, cross-neutralization was reported in animals exposed first to a prrsv vaccine strain followed by homologous or heterologous virus challenge [86] . however, the majority of data analyzed were below the neutralization assay cutoff. duration of viremia, up to 42 days, was linked with increased breadth of neutralizing antibodies following a single viral infection [101] . however, since cross-neutralization activity and titer data were not presented, it was not possible to further interpret the results. the animals were not subsequently challenged, so it is not known if the cross-neutralizing activity in serum was predictive of protection. other studies showed that significant neutralizing antibody responses are not commonly observed during viremic infection of young pigs, as well as in adult sows [69, [102] [103] [104] . recently, vaccinology research in hiv has shown that sequential immunizations, tailored for specific stages of the immune response, may be useful for inducing broadly neutralizing antibodies [105] [106] [107] . the approach is based on the finding that early immune responses to hiv resulted in neutralizing antibodies against the circulating virus which quickly led to immune escape of the virus and the ineffectiveness of generated antibodies. the antibody-resistant virus then stimulated a secondary antibody response which again selected for antibody resistant virus. this virus-antibody hide and seek continued, eventually resulting in the selection of several neutralization targets of the virus as well as the generation of broadly neutralizing antibodies [108] [109] [110] . cloning of the antibodies showed that somatic mutations are generally necessary for antibody neutralizing capabilities against hiv-1 [111, 112] . these findings have shown that the b cell response of the host adapts in the germinal center as the virus evolves, suggesting that tailored sequential immunization could lead to the development of a broadly neutralizing antibody response [113] . the consistent generation of a broadly neutralizing antibody response to prrsv on the herd level has evaded the swine health industry since the emergence of prrsv. there are multiple proposed mechanisms by which prrsv may evade or inhibit the development, or the effectiveness, of a neutralizing antibody response, such as glycan shielding of envelope glycoprotein (gp)3 or gp5 [114, 115] , the existence of decoy epitopes in gp5 [116] , lymphocyte dysregulation [79] , and inhibition of the innate immune response [117] . comprehension of defense mechanisms employed by prrsv makes the development of a broadly neutralizing immune response appear to be a daunting task. however, as previously shown, some animals are capable of developing such a response. simply, the key to adapting the immune phenomenon of some animals to a vaccine capable of inducing broadly protective immunity in many animals lies in identifying conserved epitopes on surface proteins which are necessary for infection. while the purported targets of neutralization have been extensively discussed in recent reviews, it is worth noting that several epitopes on the membrane (m) protein, gp5, gp2, gp3, and gp4, have been shown, or implicated, to harbor neutralizing activity [114, 116, [118] [119] [120] [121] [122] [123] [124] . however, knocking out only cd163 in the pig is sufficient to render animals non-susceptible to prrsv infection and replication [24, 25, 125] . it is proposed that following endocytosis, cd163 associates with the virus within the endosome, resulting in uncoating of the virus and the release of the viral genome into the cellular cytoplasm [126] . since cd163 is necessary for viral infection and replication, the logical next step is to identify the conserved regions of viral surface proteins, most likely the minor glycoproteins (gp2, gp3, and gp4), that interact with cd163 [124, 127] . traditionally, the non-neutralizing antibody response to prrsv has been considered useful only for its ability to identify if an animal had been exposed and seroconverted to virus. indeed, there are many structural and non-structural proteins of prrsv which make this possible through their ability to induce a robust humoral immune response [15, 80, 102] . however, recent research on other pathogens has shown that non-neutralizing antibodies may play a much larger role in immunity than was previously appreciated [128] [129] [130] [131] . alternative antibody functions, such as antibody dependent cell-mediated cytotoxicity (adcc), antibody-dependent complement-mediated cytotoxicity (cdc), and antibody-dependent complement-mediated virolysis may be important in the clearance of virus and virally infected cells from an animal. to our knowledge, there are only two published papers investigating non-neutralizing antibody functions in the context of prrsv infection [59, 132] . both of these in vitro studies utilized a prrsv-1 virus and failed to find an effect of adcc and cdc on infected cells. however, experiments focused on prrsv-2 viruses with extended time points beyond 12 h are warranted. a more extensive review of non-neutralizing antibody functions can be found in the cited review [133] . if antibodies are the most important effectors of the immune system against viral infection, then b cells that make the antibodies are the most important cells. previous research on the interaction between prrsv and the porcine b cell is contradictory. it has recently been suggested that prrsv infection results in lymphocyte apoptosis and immune impairment [61] . several sources have shown that prrsv largely or exclusively induces a specific humoral response to infection [51, 134] . other studies report that prrsv infection results primarily in polyclonal b cell activation leading to hypergammaglobulinemia and the development of immune complexes [135] [136] [137] [138] . the majority of work describing infection leading to polyclonal activation and hypergammaglobulinemia was performed in germ-free isolator piglets. this model is very effective for comparing b cell and antibody repertoire development in the fetus, as the germ-free status of the pigs removes many of the variables present when experiments are performed on conventionally reared animals [139] . however, these animals are deprived of the microflora and maternal antibodies to which conventional animals are exposed. as a result, the translation of immunological outcomes observed in isolator pigs to conventional pigs must be performed with caution. studies in mice show that the immune systems of specific-pathogen free laboratory mice are similar to neonatal human immune systems, whereas feral mice displayed immune systems more comparable to adult humans. effectively, the immune systems of germ-free animals may not display "normal" immune system phenotypes due to the lack of exposure to microflora [140, 141] . the development of protective humoral immunity, after vaccination or exposure to a pathogen, is dependent upon two lines of defense. the first immune defense is secreted antibodies, first from short-lived and then from long-lived, plasma cells residing somewhere in the body (figure 1 ). the second line of defense is memory b cells (figure 1 ). memory cells are sentinels against reinfection which are activated upon antigen recognition to proliferate and differentiate into antibody secreting plasma cells, thus rapidly boosting circulating antibody titers with high affinity class switched antibodies [142] . antibody repertoire development in the fetus, as the germ-free status of the pigs removes many of the variables present when experiments are performed on conventionally reared animals [139] . however, these animals are deprived of the microflora and maternal antibodies to which conventional animals are exposed. as a result, the translation of immunological outcomes observed in isolator pigs to conventional pigs must be performed with caution. studies in mice show that the immune systems of specific-pathogen free laboratory mice are similar to neonatal human immune systems, whereas feral mice displayed immune systems more comparable to adult humans. effectively, the immune systems of germ-free animals may not display "normal" immune system phenotypes due to the lack of exposure to microflora [140, 141] . the development of protective humoral immunity, after vaccination or exposure to a pathogen, is dependent upon two lines of defense. the first immune defense is secreted antibodies, first from short-lived and then from long-lived, plasma cells residing somewhere in the body (figure 1 ). the second line of defense is memory b cells (figure 1 ). memory cells are sentinels against reinfection which are activated upon antigen recognition to proliferate and differentiate into antibody secreting plasma cells, thus rapidly boosting circulating antibody titers with high affinity class switched antibodies [142] . currently, there is scant research on the memory b cell response to prrsv. strong memory responses have been shown against nsp2, nsp7, n, and the 3 end of gp5 [51, 144] . the specific memory b cells are abundant in tonsil, lymph nodes draining the lungs and reproductive tract, and spleen. unfortunately, there are many questions about the porcine memory response to prrsv which have yet to be answered, including if memory cell kinetics closely mimic antibody kinetics, the response of prrsv-specific memory pools upon homologous or heterologous viral challenge, and the importance of these cells in conferring protection against challenge. the development of sensitive and specific reagents, such as b cell tetramers, is a first step in being able to answer these critical questions. additionally, it is possible that the key to understanding the broadly neutralizing response to prrsv lies within circulating or lymphoid organ resident memory b cells. the potential to investigate these cells for identification of heavy and light chain antibody sequences is reviewed in rahe and murtaugh [133] . plasma cells are terminally differentiated b cells responsible for making antibodies. apart from the immature plasmablast, two types of plasma cells have been defined in the mouse and human [145, 146] . short-lived plasma cells quickly boost antibody titers while long-lived plasma cells maintain circulating antibody titers in the face of continual antibody degradation. mulupuri et al. identified prrsv-specific plasma cells in several secondary lymphoid organs, such as the spleen, tonsil, sternal lymph node, and inguinal lymph node [51] . interestingly, no prrsv-specific or klh-specific plasma cells were found in the bone marrow of immune pigs [51] . this was surprising, as the bone marrow has been long considered as the reservoir for long-lived plasma cells in both mice and humans [147] [148] [149] . it then begs the question, do pigs have long-lived plasma cells and, if so, where do they reside? mulupuri et al. found prrsv and klh specific plasma cells in secondary lymphoid organs 120 days after inoculation [51] . however, these cells may not be "long lived" as the prolonged viremia of prrsv may result in a somewhat continuous stimulation of memory b cells resulting in the appearance of this plasma cell population in secondary lymphoid organs. it seems unlikely that pigs do not have long lived plasma cells, as the half-life of porcine antibodies in serum is, on average, approximately nine days [150, 151] . therefore, without long lived plasma cells, pigs would quickly lose humoral protection as antibody titers waned. the identification of the anatomic location as well as the understanding of mechanisms for inducing a strong long lived plasma cell response may be important for future vaccine design as well as comprehending host-pathogen interactions. interestingly, even though neutralizing antibodies have historically garnered the majority of attention in prrsv immunology, it is well-known that pigs readily control infection in the absence of neutralizing antibodies. furthermore, viremia is reported in the presence of neutralizing antibodies [152, 153] . therefore, there must be other facets of the immune system which effectively function to control infection and eliminate prrsv from the host. while some of this activity may be attributed to non-neutralizing functions of antibodies, the t cell response to infection demands further investigation. a recent prrs immunity review summarized previous research on functional t cell subsets, and prrsv epitope targets, as well as gaps in t cell immunity [11] . here, we provide context for the understanding of novel results that have not been comprehensively reviewed. early research on the t cell response to prrsv identified a large, transient decrease in the cd4 + /cd8 + t cell ratio early, usually within the first week, in the course of infection [154] . the change in this ratio could have been due to a temporary loss of cd4 + cells through apoptosis or to an increase in cd8 + cells due to antigen-specific proliferation [154] . the importance of these findings for clearance of prrsv or protection from infection were not known at the time, and other explanations, such as fluxes in cell populations between spleen, other lymphoid tissues, and blood could not be discounted. experiments to address the helper t cell type 1/helper t cell type 2 (th1/th2) paradigm in the pig showed that prrsv induced a strong th1 response, as expected, identified in vivo by an increased expression of th1-specification factor tbx21(t-bet) in cd4 + cells [155] . however, the finding is at odds with previously reports indicating that prrsv infection results in the production of il-10, a cytokine classically associated with a th2 phenotype. similarly, monocyte-derived dendritic cells (mo-dcs) infected with prrsv down regulate swine leukocyte antigen (sla)-i, sla-ii, cd40 and cd80 as well as promote il-10 secretion over il-12 secretion [156] . delineation of the th1/th2 response to prrsv, elucidation of th1/th2-specific cytokine markers in swine, as well as identifying associated cytokine responses of dendritic cells within secondary lymphoid organs where t cell proliferation and differentiation is most likely to occur, would help to resolve these outstanding questions [157] . the th17 cell has classically been identified, in mouse and human, as playing an important role in extracellular bacterial immunity through the production of the pro-inflammatory cytokines, il-17a, il-17f, and il-22 [158, 159] . il-17 producing th17 cells are known to exist in the pig [160] . the importance of this t cell subset in the context of prrsv infection has recently been investigated. a strain of chinese highly pathogenic prrsv (hp-prrsv) appeared to suppress th17 cells in the peripheral blood and lungs of pigs, resulting in an increased susceptibility to secondary bacterial infections [56] . remarkably, the effect was prrsv strain-specific, as a non-hp prrsv strain failed to elicit the same response. future research into the t cell response to prrsv, especially with t cell tetramers and functional elispots, will be essential for the characterization of both cd4 + and cd8 + antigen specific t cells. understanding how antigen-specific t cells interact with both infected and uninfected antigen presenting macrophages and dendritic cells will be helpful for advancing the field of prrsv immunity. the natural killer cell is an innate lymphoid cell which can have a profound impact on adaptive immunity, but is also able to induce an early and rapid innate response against pathogens through a variety of mechanisms. nk cells produce cytokines, such as ifnγ, show cytotoxic activity against infected cells not expressing mhci, can induce dendritic cell maturation, and effect the destruction of infected cells in adcc [161] . however, nk cells may deploy even more extensive and important functions in porcine immunity than are currently realized. an early clue that nk cells were involved in innate responses to prrsv was a sharp peak in serum ifnγ shortly after infection [162] . the acute response was attributed to nk cells, as the result was deemed too early for a t cell response, and suggested that decreased viral burdens in the lung prior to humoral or t cell responses could be due to the function of nk cells. however, it is known that porcine macrophages are also capable of producing ifnγ in the presence of prrsv infection [163, 164] . furthermore, prrsv appears to suppress the nk cell response without significantly affecting nk cell numbers [165] [166] [167] [168] . the cause of this suppression has yet to be determined, although viral proteins, rather than soluble factors from cells, may be responsible [59] . potential roles of additional nk cell functions, such as adcc, in prrsv immunity are poorly understood [133] . prrsv has tormented the health and wellbeing of swine worldwide since its discovery in the late 1980s. unfortunately, after almost 30 years of research into the porcine immune response to prrsv, there is still no effective means for inducing a broadly protective immune response at the herd level. the reasons for this failure are not completely known, but presumably include mechanisms by which the virus subverts the immune system. the ability of the virus to rapidly mutate while not losing fitness challenges the host immune system to keep pace. at the same time, infection of macrophages, a key player in immunoregulation, challenges both innate and adaptive immune cell mobilization as well as induction of a coordinated response that is needed for effective control and elimination of the virus. fortunately, foundational advances in the understanding of viral pathogenesis and immunity are enabling more informative investigations. the identification of cd163 as the necessary and sufficient receptor for infection supports the implications of broadly neutralizing antibodies that a conserved target is present on all prrsv. understanding how prrsv surface glycoproteins interact with cd163 should lead to the identification of conserved epitopes which are necessary for infection. if, as appears to be the case, there is only one conserved way into the cell, then there must be a conserved viral sequence, or structure, which enables viral entry. furthermore, the knowledge that pigs eventually develop sterilizing immunity, if given enough time, supports the concept that conserved epitopes exist on the virus. therefore, the study of mature animals, which have cleared the virus, may provide the key to understanding how the immune system eventually gets the upper hand on the virus and cures infection. even with seminal advances in several aspects of the study of prrsv, there remains much to be understood and clarified. currently, the published literature presents conflicting views on many aspects of prrsv adaptive immunity, especially related to t and b cell responses and the production, or inhibition, of cytokines in the face of infection. the continued development of antigen-specific reagents, of high sensitivity and specificity, is needed for understanding how the host responds to prrsv infection. furthermore, it is important that future prrsv studies focus on the relevant host animal, the conventional pig. while the study of this outbred animal species is perhaps challenging at times, it affords the ability to study the host-pathogen interaction in the only species in which the virus naturally interacts. additionally, knowledge gained about the immunology of conventional pigs will accelerate immunological elucidation of other pig-pathogen interactions. in conclusion, prrsv continues to be the most burdensome pathogen of pigs worldwide, due to its propensity for immune evasion and manipulation. however, the continued study of the porcine immune response to infection, with improved reagents and methods, will illuminate those aspects of the host-pathogen interaction that are now hidden. it is through these discoveries that the complex question that is prrsv will finally be answered. mystery swine disease in the netherlands: the isolation of lelystad virus experimental reproduction of porcine epidemic abortion and respiratory syndrome (mystery swine disease) by infection with lelystad virus: koch's postulates fulfilled isolation of swine infertility and respiratory syndrome virus (isolate atcc vr-2332) in north america and experimental reproduction of the disease in gnotobiotic pigs porcine reproductive and respiratory syndrome virus comparison: divergent evolution on two continents north american and european porcine reproductive and respiratory syndrome viruses differ in non-structural protein coding regions porcine reproductive and respiratory syndrome virus (porcine arterivirus) reorganization and expansion of the nidoviral family arteriviridae prrsv, the virus broadly neutralizing antibodies against the rapidly evolving porcine reproductive and respiratory syndrome virus the ever-expanding diversity of porcine reproductive and respiratory syndrome virus innate and adaptive immunity against porcine reproductive and respiratory syndrome virus chimeric porcine reproductive and respiratory syndrome viruses reveal full function of genotype 1 envelope proteins in the backbone of genotype 2 the prrsv replicase: exploring the multifunctionality of an intriguing set of nonstructural proteins current knowledge on the structural proteins of porcine reproductive and respiratory syndrome (prrs) virus: comparison of the north american and european isolates novel structural protein in porcine reproductive and respiratory syndrome virus encoded by an alternative orf5 present in all arteriviruses localization and fine mapping of antigenic sites on the nucleocapsid protein n of porcine reproductive and respiratory syndrome virus with monoclonal antibodies evolutionary diversification of type 2 porcine reproductive and respiratory syndrome virus porcine reproductive and respiratory syndrome virus: description of persistence in individual pigs upon experimental infection porcine reproductive and respiratory syndrome virus (prrsv): pathogenesis and interaction with the immune system porcine reproductive and respiratory syndrome virus infection of gnotobiotic pigs: sites of virus replication and co-localization with mac-387 staining at 21 days post-infection virus quantification and identification of cellular targets in the lungs and lymphoid tissues of pigs at different time intervals after inoculation with porcine reproductive and respiratory syndrome virus (prrsv) enhanced replication of porcine reproductive and respiratory syndrome (prrs) virus in a homogeneous subpopulation of ma-104 cell line characterization of interaction between porcine reproductive and respiratory syndrome virus and porcine dendritic cells gene-edited pigs are protected from porcine reproductive and respiratory syndrome virus genetic engineering alveolar macrophages for host resistance to prrsv interferon-alpha response to swine arterivirus (poav), the porcine reproductive and respiratory syndrome virus pig immune response to general stimulus and to porcine reproductive and respiratory syndrome virus infection: a meta-analysis approach in vivo and in vitro interferon (ifn) studies with the porcine reproductive and respiratory syndrome virus (prrsv) differential production of proinflammatory cytokines in the pig lung during different respiratory virus infections: correlations with pathogenicity antigen-specific b-cell responses to porcine reproductive and respiratory syndrome virus infection porcine reproductive and respiratory syndrome virus utilizes nanotubes for intercellular spread porcine reproductive and respiratory syndrome virus (prrsv) infection spreads by cell-to-cell transfer in cultured marc-145 cells, is dependent on an intact cytoskeleton, and is suppressed by drug-targeting of cell permissiveness to virus infection schwartz-cornil, i. rotavirus anti-vp6 secretory immunoglobulin a contributes to protection via intracellular neutralization but not via immune exclusion intracellular neutralization of viral infection in polarized epithelial cells by neonatal fc receptor (fcrn)-mediated igg transport the chinese highly pathogenic porcine reproductive and respiratory syndrome virus infection suppresses th17 cells response in vivo thymic depletion of lymphocytes is associated with the virulence of prrsv-1 strains identification of apoptotic cells in the thymus of piglets infected with highly pathogenic porcine reproductive and respiratory syndrome virus suppression of nk cell-mediated cytotoxicity against prrsv-infected porcine alveolar macrophages in vitro the comparative profile of lymphoid cells and the t and b cell spectratype of germ-free piglets infected with viruses siv, prrsv or pcv2 type 2 porcine reproductive and respiratory syndrome virus infection mediated apoptosis in b-and t-cell areas in lymphoid organs of experimentally infected pigs the molecular mechanisms of regulatory t cell immunosuppression. front. immunol. 2011, 2, 60 study on cellular events in post-thymectomy autoimmune oophoritis in mice. ii. requirement of lyt-1 cells in normal female mice for the prevention of oophoritis t cell-mediated maintenance of natural self-tolerance: its breakdown as a possible cause of various autoimmune diseases induction of t helper 3 regulatory cells by dendritic cells infected with porcine reproductive and respiratory syndrome virus induction of inducible cd4+cd25+foxp3+ regulatory t lymphocytes by porcine reproductive and respiratory syndrome virus (prrsv) prrsv-infected monocyte-derived dendritic cells express high levels of sla-dr and cd80/86 but do not stimulate prrsv-naïve regulatory t cells to proliferate european genotype of porcine reproductive and respiratory syndrome (prrsv) infects monocyte-derived dendritic cells but does not induce treg cells age-dependent resistance to porcine reproductive and respiratory syndrome virus replication in swine upregulation of interleukin-10 gene expression in the leukocytes of pigs infected with porcine reproductive and respiratory syndrome virus cytokines transcript levels in lung and lymphoid organs during genotype 1 porcine reproductive and respiratory syndrome virus (prrsv) infection changes in lymphocyte subsets and cytokines during european porcine reproductive and respiratory syndrome: increased expression of il-12 and il-10 and proliferation of cd4-cd8 high concurrent respiratory infections in 221 cases of prrs virus pneumonia. j. swine health prod prrs plus"-prrsv infection in combination with other agents; national pork board porcine reproductive and respiratory syndrome virus (prrsv) influences infection dynamics of porcine circovirus type 2 (pcv2) subtypes pcv2a and pcv2b by prolonging pcv2 viremia and shedding in vitro and ex vivo analyses of co-infections with swine influenza and porcine reproductive and respiratory syndrome viruses role of tissue protection in lethal respiratory viral-bacterial coinfection pathogenic and humoral immune responses to porcine reproductive and respiratory syndrome virus (prrsv) are related to viral load in acute infection porcine reproductive and respiratory syndrome (prrs): an immune dysregulatory pandemic antibody response to porcine reproductive and respiratory syndrome virus (prrsv) nonstructural proteins and implications for diagnostic detection and differentiation of prrsv types i and ii the level of virus-specific t-cell and macrophage recruitment in porcine reproductive and respiratory syndrome virus infection in pigs is independent of virus load mechanisms of t cell-b cell interaction quantitative analysis of porcine reproductive and respiratory syndrome (prrs) viremia profiles from experimental infection: a statistical modelling approach immune response against porcine reproductive and respiratory syndrome virus during acute and chronic infection the challenge of prrs immunology serum immune responses to the proteins of porcine reproductive and respiratory syndrome (prrs) virus immunization for hiv-1 broadly neutralizing antibodies in human ig knockin mice hiv-1 vaccines. hiv-1 neutralizing antibodies induced by native-like envelope trimers hiv-1 broadly neutralizing antibody precursor b cells revealed by germline-targeting immunogen analysis of a clonal lineage of hiv-1 envelope v2/v3 conformational epitope-specific broadly neutralizing antibodies and their inferred unmutated common ancestors maturation pathway from germline to broad hiv-1 neutralizer of a cd4-mimic antibody early antibody lineage diversification and independent limb maturation lead to broad hiv-1 neutralization targeting the env high-mannose patch broad diversity of neutralizing antibodies isolated from memory b cells in hiv-infected individuals rational hiv immunogen design to target specific germline b cell receptors progress toward active or passive hiv-1 vaccination immune evasion of porcine reproductive and respiratory syndrome virus through glycan shielding involves both glycoprotein 5 as well as glycoprotein 3 influence of n-linked glycosylation of porcine reproductive and respiratory syndrome virus gp5 on virus infectivity, antigenicity, and ability to induce neutralizing antibodies identification of neutralizing and nonneutralizing epitopes in the porcine reproductive and respiratory syndrome virus gp5 ectodomain interaction between innate immunity and porcine reproductive and respiratory syndrome virus the primary neutralization epitope of porcine respiratory and reproductive syndrome virus strain vr-2332 is located in the middle of the gp5 ectodomain monoclonal antibody analysis of porcine reproductive and respiratory syndrome virus epitopes associated with antibody-dependent enhancement and neutralization of virus infection effect of virus-specific antibodies on attachment, internalization and infection of porcine reproductive and respiratory syndrome virus in primary macrophages characterization of antigenic regions in the porcine reproductive and respiratory syndrome virus by the use of peptide-specific serum antibodies gp4 of porcine reproductive and respiratory syndrome virus contains a neutralizing epitope that is susceptible to immunoselection in vitro dna shuffling of the gp3 genes of porcine reproductive and respiratory syndrome virus (prrsv) produces a chimeric virus with an improved cross-neutralizing ability against a heterologous prrsv strain the minor envelope glycoproteins gp2a and gp4 of porcine reproductive and respiratory syndrome virus interact with the receptor cd163 precision engineering for prrsv resistance in pigs: macrophages from genome edited pigs lacking cd163 srcr5 domain are fully resistant to both prrsv genotypes while maintaining biological function porcine reproductive and respiratory syndrome virus entry into the porcine macrophage arterivirus minor envelope proteins are a major determinant of viral tropism in cell culture comparison of antibody-dependent cell-mediated cytotoxicity and virus neutralization by hiv-1 env-specific monoclonal antibodies opportunities to exploit non-neutralizing hiv-specific antibody activity non-neutralizing epitopes induce robust hcv-specific antibody-dependent cd56 + nk cell responses in chronic hcv-infected patients the immune correlates of protection for an avian influenza h5n1 vaccine in the ferret model using oil-in-water adjuvants porcine reproductive and respiratory syndrome virus-infected alveolar macrophages contain no detectable levels of viral proteins in their plasma membrane and are protected against antibody-dependent, complement-mediated cell lysis effector mechanisms of humoral immunity to porcine reproductive and respiratory syndrome virus polyclonal activation of b cells occurs in lymphoid organs from porcine reproductive and respiratory syndrome virus (prrsv)-infected pigs lymphoid hyperplasia resulting in immune dysregulation is caused by porcine reproductive and respiratory syndrome virus infection in neonatal pigs porcine reproductive and respiratory syndrome virus subverts repertoire development by proliferation of germline-encoded b cells of all isotypes bearing hydrophobic heavy chain cdr3 polyclonal hypergammaglobulinemia and formation of hydrophobic immune complexes in porcine reproductive and respiratory syndrome virus-infected and uninfected pigs antibody repertoire development in fetal and neonatal piglets. xv. porcine circovirus type 2 infection differentially affects serum igg levels and antibodies to orf2 in piglets free from other environmental factors antibody repertoire development in fetal and neonatal piglets. iv. switch recombination, primarily in fetal thymus, occurs independent of environmental antigen and is only weakly associated with repertoire diversification normalizing the environment recapitulates adult human immune traits in laboratory mice dirty mice might make better models immunological memory and protective immunity: understanding their relation heterogeneity in the differentiation and function of memory b cells interleukin-21 drives proliferation and differentiation of porcine memory b cells into antibody secreting cells humoral immunity due to long-lived plasma cells competence and competition: the challenge of becoming a long-lived plasma cell immunoglobulin synthesis by human lymphoid tissues: normal bone marrow as a major site of igg production antibody formation in mouse bone marrow. i. evidence for the development of plaque-forming cells in situ bone marrow is a major site of long-term antibody production after acute viral infection half-lives of immunoglobulins igg, iga and igm in the serum of new-born pigs half-life of porcine antibodies absorbed from a colostrum supplement containing porcine immunoglobulins an interferon inducing porcine reproductive and respiratory syndrome virus vaccine candidate elicits protection against challenge with the heterologous virulent type 2 strain vr-2385 in pigs virological and immunological responses to porcine reproductive and respiratory syndrome virus in a large population of gilts changes of lymphocyte subpopulations in pigs infected with porcine reproductive and respiratory syndrome (prrs) virus a novel lineage transcription factor based analysis reveals differences in t helper cell subpopulation development in infected and intrauterine growth restricted (iugr) piglets the role of porcine reproductive and respiratory syndrome virus infection in immune phenotype and th1/th2 balance of dendritic cells species specialization in cytokine biology: is interleukin-4 central to the t h 1-t h 2 paradigm in swine? interleukin (il)-22 and il-17 are coexpressed by th17 cells and cooperatively enhance expression of antimicrobial peptides influenza a inhibits th17-mediated host defense against bacterial pneumonia in mice cd40 engagement strongly induces cd25 expression on porcine dendritic cells and polarizes the t cell immune response toward th1 natural killer cells in host defense against veterinary pathogens infection with porcine reproductive and respiratory syndrome virus stimulates an early gamma interferon response in the serum of pigs expression of interferon-gamma and tumour necrosis factor-alpha in pigs experimentally infected with porcine reproductive and respiratory syndrome virus immunohistochemical staining of ifn-γ positive cells in porcine reproductive and respiratory syndrome virus-infected lungs porcine reproductive and respiratory syndrome virus modifies innate immunity and alters disease outcome in pigs subsequently infected with porcine respiratory coronavirus: implications for respiratory viral co-infections porcine reproductive and respiratory syndrome virus-induced immunosuppression exacerbates the inflammatory response to porcine respiratory coronavirus in pigs evaluation of immune responses to porcine reproductive and respiratory syndrome virus in pigs during early stage of infection under farm conditions porcine reproductive and respiratory syndrome virus induces pronounced immune modulatory responses at mucosal tissues in the parental vaccine strain vr2332 infected pigs the authors declare no conflicts of interest. key: cord-016499-5iqpl23p authors: mackay, ian m.; arden, katherine e. title: rhinoviruses date: 2014-02-27 journal: viral infections of humans doi: 10.1007/978-1-4899-7448-8_29 sha: doc_id: 16499 cord_uid: 5iqpl23p picornaviruses, which include the human rhinoviruses (hrvs) and enteroviruses (evs), are the most frequent cause of acute human illness worldwide. hrvs are the most prevalent cause of acute respiratory tract illnesses (aris) which usually commence in the upper respiratory tract (urt). aris are the leading cause of morbidity in children under 5 years and occur in all seasons. aris linked to hrv infections are associated with excessive and perhaps inappropriate antibiotic prescribing and with significant direct and indirect healthcare expenditure. ari incidence is highest in the first 2 years of life, with up to thirteen episodes per year including up to six positive for an hrv, and it is not uncommon to average one infection per child-month. picornaviruses, which include the human rhinoviruses (hrvs) and enteroviruses (evs), are the most frequent cause of acute human illness worldwide [ 1 ] . hrvs are the most prevalent cause of acute respiratory tract illnesses (aris) which usually commence in the upper respiratory tract (urt). aris are the leading cause of morbidity in children under 5 years and occur in all seasons [ 2 , 3 ] . aris linked to hrv infections are associated with excessive and perhaps inappropriate antibiotic prescribing [ 4 ] and with signifi cant direct and indirect healthcare expenditure [ 5 , 6 ] . ari incidence is highest in the fi rst 2 years of life, with up to 13 episodes per year including up to six positive for an hrv, and it is not uncommon to average one infection per child-month [ 3 , 7 -9 ] . in preschool-aged children, nearly 50 % of general practitioner visits are for ari [ 10 ] , many of which are self-limiting. aris can often be managed in the community with supportive care from parents, but complications can arise that require a medical visit for management of asthma, otitis media, or sinusitis [ 11 ] . hrvs replicate in nasal cells, sinus cells, bronchial epithelial cells (becs) [ 12 , 13 ] , and smooth muscle cells [ 14 ] but not in monocytes [ 15 ] or dendritic cells (dcs) [ 16 ] . the infl ammatory immune response they trigger very soon after infection has its greatest impact in the young, the elderly, those with asthma or chronic obstructive pulmonary disease (copd), and in the immunocompromised. first infections usually elicit a stronger response. antiviral interventions have been under development for decades; to date most have met with varying degrees of failure or unacceptability. vaccines have been considered unachievable because of the large number of diverse and distinct viral types. there are 100 classically defi ned and recognized hrv serotypes grouped into two species, hrv-a and hrv-b, and a recently defi ned third species, hrv-c, containing more than 60 genotypes identifi ed and characterized entirely by molecular means. their cousins, the four enterovirus species (ev-a, ev-b, ev-c, and ev-d), are also found in the airways at times. most systematic and mechanistic studies of hrv etiology and pathogenesis have been informed by studies in adults, mostly prior to the discovery of hrv-cs. adults exhibit reduced symptoms from hrv infections because of prior exposure and the resultant protective immune memory which that imparts (see sect. 7.3 ). furthermore, many modern studies (1) draw conclusions about lower respiratory tract (lrt) disease using urt specimens and (2) infrequently sample, doing so across small cross sections of time. these limitations have hampered attempts to associate virus detection and disease. current thinking is that hrv-cs may be key players in asthma exacerbations although our inability to culture them routinely has hindered our progress in understanding their role. the impact of the hrvs has been underestimated for decades, and the concept of the hrvs as a very large assemblage of genetically, immunogenically, antigenically, and temporally distinct and stable viral entities remains rare; they are more commonly considered a single variable virus, a view that science does not support. the disease most commonly associated with the airways and resulting from hrv infection is the common cold, a selflimiting coryzal illness [ 17 -19 ] . the term dates back to ancient greece, but evidence that the syndrome and asthma, another disease most frequently due to hrv infection, has been with us since ancient times can be viewed in writings on the ebers papyrus, a medical document written in the sixteenth century bc [ 20 , 21 ] . in 1930 the common cold was considered either to be due to exposure to the elements or to infection by bacteria [ 22 ] . it was later understood to be largely due to something in bacteria-free fi ltrates, and so the search for viral causes began [ 23 , 24 ] . the common cold unit (ccu) was established in salisbury, uk, to seek solutions to the mysteries of the common cold, mostly through adult volunteer infection studies and careful systematic science [ 23 ] . the ccu functioned for 44 years (1946-1990) , and it was here in 1953 that the fi rst in vitro culture of an hrv was achieved using lung tissue from a particular embryo ( fig. 29 .1 ) [ 25 , 26 ] . propagation failed once this tissue was expended [ 22 , 33 ] . once hrv isolation was possible, viral serotyping developed and culture techniques were further refi ned. this leads to an international effort to characterize and name the hrvs [ 27 -30 ] . in 2006 renewed interest in hrv research was triggered by the description of a distinct clade of hrv types [ 31 ] found using molecular typing. the resultant fl urry of hrv research raised questions about many earlier paradigms of rhinovirology and of the role of established respiratory viruses in aris. the novel clade was proposed as a new species, hrv-c, which was taxonomically confi rmed in 2009 [ 32 -34 ] . prior to the discovery of the hrv-cs, the genus rhinovirus had been abolished and the hrv-a and hrv-b species assigned to the genus enterovirus within the family picornaviridae [ 35 ] . the hrv-cs have been assigned a new naming scheme based on genetic sequence in the absence of antigenic or serological data. while the sequencing of all serotyped hrv genomes was completed in 2009, few of the hrv-cs or apparently novel hrv-as or hrv-bs have been similarly characterized, so the full spectrum of hrv genomes, the rhinovirome, remains incomplete. in this chapter we have described individual serotyped hrvs as the "classical" types, a type being the description for a single, genetically stable, stand-alone hrv. methods for epidemiologic analysis the original clinical defi nition of an hrv infection was written using data from cell and tissue culture and adult human infection studies. after 1953 in vitro isolation methods employed a virus interference test to more easily determine successful isolation; cultures suspected of infection with an uncharacterized hrv prevented infection by another, readily titratable virus [ 36 ] . later, price (1956 ; the jh strain) and then pelon and co-workers (1957; 2,060 strain) developed culture systems that permitted hrv replication to be more easily identifi ed [ 37 , 38 ] . the early hrvs were initially classifi ed as echoviruses (echo 28; later hrv-1) [ 39 ] . at the same time, propagation of the hgp (hrv-2) strain resulted from using increased acidity, lowered cultivation temperatures, and constant motion (rotation) [ 40 , 41 ] . despite the challenges [ 42 ] , virus isolation was a more sensitive indicator of infection than an antibody rise in paired sera [ 43 ] . it was found that several cell lines and methods were required to encompass virus concentrations ranging from 10 1 to 10 5 tcid 50 /ml [ 44 -47 ] and growth differences among the different virus types. additionally, cell age after plating (<72 h), inoculum volume (relevant to the culture vessel), medium ph (6.8-7. 3), and cell density were important factors for the reproducible appearance of hrv-induced plaques and for higher virus yields [ 48 -51 ] . the hrvs can grow at temperatures above 35 °c (some prefer that under certain conditions) [ 52 ] , but rolling at 33 °c, preceded by a 2-4-h stationary incubation period [ 41 ] , has historically provided the highest yield and fastest in vitro hrv growth [ 36 , 50 , 53 , 54 ] . serodiagnosis grew increasingly impractical as the number of serotypes increased [ 49 , 55 ] . however, antibody-based methods were essential for type-specifi c neutralization of infection [ 56 ] from which early epidemiology data were derived and around which the hrv nomenclature system evolved in 1967 [ 28 ] . the fi rst classical strains were officially named in 1967 [ 57 ] , the last in 1987 [ 30 ] . today we know that cell culture-based methods are unreliable for accurately representing respiratory virus epidemiology; although enhanced by immunofl uorescence, they are still used [ 58 ] . the hrv-cs have not been successfully cultured in any cell lines or primary cell culture, although many attempts have been described [ 32 , 59 -62 ] . in 2011 hrv-c15 and w23 (another hrv-c) were shown to grow using organ culture [ 63 ] . sinus tissue hosted increasing levels of viral rna, as did adenoid, tonsil, and nasal polyp tissue, but much less effectively, as measured by in situ hybridization [ 63 ] . the sinus organ culture system also allowed testing of the fi rst reverse engineered hrv-c (pc15) [ 63 ] . isolation identifi ed hrvs in ~23 % of adults with aris, associated with 0.5 illnesses per year [ 64 ] . because culture is ineffi cient and subjective and requires expertise, even for the culturable hrv types, it is becoming an art lost to clinical laboratories the world over. it is unsurprising that pcr-based methods now prevail, providing a much improved understanding of the nature and scope of hrv infections. the virological and immunobiological cost of this improvement is a paucity of low passage "wild" hrv isolates to work with; thus, many research fi ndings from recent years have employed easy to grow highly passaged and adapted hrv isolates. the impact of virus adaptation on the reliability of data from use of such viruses is unknown. pcr-based assays have dramatically increased the frequency of hrv detection [ 65 -70 ] . the improved sensitivity and reduced turnaround time have shown that hrvs, as a group, are usually the predominant viruses in ari cases [ 71 -73 ] . with reliable detection levels that extend from as few as 10 2 tcid 50 /sample to well above clinically relevant loads, pcr can detect virus levels which are commonly shed during all stages of experimental infection studies [ 74 , 75 ] . the common understanding of the systemic [ 76 -78 ] or symptomatic [ 79 , 80 ] context of hrv detections was established during the era of culture detection, and pcr has challenged these paradigms by detecting virus more often than culture. hrvs are sometimes found in "healthy controls"; however, it is likely that with more thoughtful defi nitions of "healthy," these detections would reduce. it is not uncommon to experience a feeling that one is "coming down" with something that never develops further. this is likely due to a transient infection or reinfection by an hrv or other respiratory virus that is eliminated quickly by the host response. it is possible to correlate viral nucleic acid load at the sampling site with disease severity; however, this is made diffi cult by the highly variable sampling effi ciency of respiratory tract specimens which only permit the generation of reliable quantitative pcr (qpcr) data if serial specimens are available [ 81 ] . the 5′ untranslated region (utr; figs. 29.2 and 29.3 ) is the most common target for diagnostic oligonucleotides since the fi rst hrv rt-pcr in 1988 [ 82 ] , and the region has retained relevance for virus detection by its adaptation to reverse transcriptase real-time methods (rt-rtpcr) [ 53 , 65 , 66 , 69 , 74 -76 , 79 , 83 -103 ] . the 5′utr is comprised of a number of conserved sequence "islands" (fig. 29 .2 ) that permit the robust detection of the majority of hrvs and those "respiratory evs" which can be regularly detected in the respiratory tract [ 104 , 105 ] . the detection of respiratory evs in no way detracts from the importance of supporting clinical decision making using these assays. however, repositioning [ 68 ] . the pcr primers of broadly reactive conventional rt-pcr [ 82 , 113 ] and rt-rtpcr [ 75 , 114 ] assays are shown these primers or changing the method of employing them [ 106 -109 ] may undermine assay performance, as evidenced by predicted hybridization mismatches, uncommonly low detection frequencies [ 110 ] , and by comparison of multiple primer sets using the same specimens [ 111 ] . the addition of an oligoprobe rtpcr method increases amplicon detection sensitivity and specifi city, identifying 100-fold fewer tcid 50 /ml or 10 fold fewer genome copies than agarose gel detection of amplicon [ 75 , 79 , 112 ] . other molecular tools, capable of detecting multiple targets, have evolved in recent years [ 58 , 70 , 115 -121 ] , and some have gone on to be approved for clinical laboratory use [ 122 ] . microarrays can detect thousands of viral targets, but are expensive for routine use (usd30-300 per sample) and not sensitive enough to avoid a pre-hybridization pcr amplifi cation when using clinical specimens. at their most robust, microarrays, like pcr, rely on the existence of conserved regions of sequence to detect unknown viruses allowing them to detect previously unknown hrv types [ 123 ] . highthroughput or "deep" sequencing platforms have become less expensive and more readily available, and they have succeeded in fi nding new diversity within the hrv species [ 124 ] . the experiments remain costly so have not yet found a place for regular screening tasks and remain coupled to a need for pre-pcr steps. rapid protein-or virion-based assays are not (yet) adequately sensitive [ 125 , 126 ] . because of the high number of hrvs and the high frequency of infections, genotyping methods have become an essential accompaniment for understanding hrv epidemiology. nucleotide sequencing of the vp1, 5′utr+vp4+vp2 (called hereafter vp4/vp2), or 5′utr region has replaced traditional serological methods, because of its speed and need for fewer specialized reagents compared to serotyping. vp1 yields the most comprehensive subgenomic genotyping information and is essential for the minimal defi nition of a new hrv type [ 127 ] . the vp4/vp2 region (fig. 29. 3 ) is considered easier to use because it encompasses suffi cient genetic diversity to confi rm the identity of a clinical hrv type while also providing broad enough sensitivity to amplify the ~160 hrvs from a challenging biological substrate, clinical specimens [ 128 ] . screening of airway specimens for hrvs is not routine [ 111 ] due to factors including cost and the perceived low clinical relevance of detection. genotyping is mostly relegated to research facilities. because of this, hrv molecular epidemiology studies tend to be smaller and focused on a specifi c disease or research question. most in-depth molecular studies of hrv replication have focused on a single hrv type. generally, it is presumed that results can be extrapolated to the other hrv types and to the in vivo situation. hrvs replicate in the cytoplasm (fig. 29 .4 ) [ 129 ] with membrane-associated replication structures containing double-stranded rna (dsrna) replicative intermediates (ri) which are formed in cells 4 h after infection [ 52 , 130 ] . single-stranded infectious rna forms after ris start to accumulate [ 130 ] . genomic rna (plus strand) is the template for complementary minus strand synthesis which in turn is the template for new genomic plus strands that become incorporated into virions [ 131 ] . virions are synthesized from 4 to 7 h after infection and reach maximum release levels at 10-18 h [ 131 ] . hrv replication in epithelial cells may shut off host cell transcriptional activity via direct cleavage of transcription factors and nuclear pore complex components. protease 2a (2a pro ) of hrv-b2 may directly cleave eukaryotic initiation factor 4g (eif4g) when bound to eif4e [ 132 , 133 ] . the eifs have key roles in initiation and rate control of host cell translation [ 132 ] . host cellular protein production is virtually replaced by hrv-b14 proteins after only 6 h of infection [ 134 ] . hrv-b14-infected cells also display reduced nuclear importing and degraded nuclear pore complex (npc) components [ 135 ] . this may represent another hrv strategy for limiting the host response by preventing or reducing key signaling pathway molecules (e.g., irf-3, stat1, nf-κb) and shutting down host cell protein synthesis. protease 3c (3c pro ) from hrv-a16 targets the nucleus and can disrupt active and passive nucleocytoplasmic transport [ 129 , 136 ] . recombinant 2a pro protein from hrv-a16,hrv-a89, hrv-b4, hrv-b14, hrv-c2, and hrv-c6 exhibited differing specifi cities and kinetics against eif4g as well as npc components demonstrating functional diversity between hrv types [ 137 ] . this fi nding underscores the functional diversity within the hrv species and the risk of extrapolating too greatly from the study of single hrv types. it is apparent from a wealth of immunobiological data that hrvs still effi ciently trigger a proinfl ammatory immune response that has considerable clinical impact among at-risk groups, and that their putative interruption of host cell machinery does little to hinder this. the virion encapsulates an approximately 7 kb positive sense rna genome (fig. 29. 3 ), which tends to be more adenine and uracil (a+u) rich than the ev genome [ 139 ] . in particular, a+u more frequently occupies the third or "wobble" genome replication in association with membranes produces the viral polyprotein which is co-and posttranslationally processed by 2a pro and 3c pro into the proteins ( p1 -p3 ) and structural peptides ( vp1 -vp4 ; vp2 and vp4 derive from the vp0 precursor protein) that assemble into protomers, pentamers, and fi nally capsids. nonstructural proteins are also released in these cleavages as well as through autoproteolytic cleavage. mature hrv virions packaged with an ssrna genome escape by cell lysis (adapted with permission from arden et al. [ 138 ] ) codon position. the single rna "gene" acts as messenger rna to encode the single multi-domain, proteolytically processed "polyprotein." the coding region is bracketed by utrs which perform regulatory functions necessary for genome duplication [ 140 ] . these are very similar genomic, transcriptional, and translational features to those of their close cousins, the evs. most of the information currently required for virus identifi cation by the international committee on taxonomy of viruses (ictv) can be found through analysis of the genetic features of hrvs ( fig. 29.3 ). there are 158 complete hrv polyproteins on the genbank database ( fig. 29 .5 ). the fi rst complete hrv genome sequence (hrv-b14) was described in 1984 [ 141 ] followed by hrv-a2 in 1985 [ 142 ] and hrv-a1b in 1988 [ 143 ] (fig. 29 [ 145 ] . sequencing of the vp4/vp2 region was completed for all classical strains in 2002 [ 146 ] , and the complete set of 1d regions were available in 2004 [ 147 ] . currently there are at least 50 named hrv-c vp1 regions the current spectrum of 168 complete hrv complete polyprotein amino acid sequences available on the genbank database. the alignment was conducted using mafft within geneious pro v5.6 [ 148 ] . the phylogenetic and molecular evolutionary analyses were con-ducted using mega version 5 (poisson model, 500 bootstraps with consensus support shown at the nodes where space permitted [ 149 ] ) (reprinted with permission from miller and mackay [ 150 ]) available and 20 complete hrv-c genomes. many more genomes are appearing as part of the rhinovirus consortium's efforts to complete and study the rhinovirome using highthroughput sequencing technologies to genetically characterize hrvs from their combined clinical specimen stores ( http://www.international-rhinovirus-consortium.org/ ). many 5′utr and vp4/vp2 sequences reside on the genbank database, most of which are labeled using in-house laboratory schemes rather than an approved nomenclature. analysis of the full-length genomes supports the use of 5′utr, vp1, and vp4/vp2 subgenomic regions for useful representation of hrv species and types [ 144 , 147 ] . recombination, the process of genetic exchange which results in a chimeric genome [ 151 ] , can only be detected in mature viruses after the fact, and it must therefore be inferred indirectly through genomic analysis and comparison. predictions of infrequent recombination among the hrvs [ 83 ] have been made based on examination of the available set of hrv coding and noncoding regions [ 152 ] . intensive analyses reported that recombination is not a driving force for the evolution of hrv types [ 144 , 153 , 154 ] . some discrepancies are likely because of the different number of sequences used, the different origins of the viruses used for sequencing, and the analysis methods employed. hrv-c evolution seems to have been more affected by prior recombination, than is apparent for members of hrv-a or hrv-b. this is similar to the ev species but with far fewer predicted recombination events than for ev evolution [ 114 , 151 , 155 , 156 ] . most of the recombination proposed to have affected the hrvs occurred between hrv-c and hrv-a and is often found within the 5′utr or at the 5′utr/vp4 junction [ 83 , 157 , 158 ] but rarely in coding sequence (2a [ 158 ] or 3c [ 83 ] ). the high sequence diversity among the individual hrv polyprotein coding sequences may keep recombination events to a minimum in order to retain viral fi tness [ 158 ] . the ability of hrvs to recombine in practice awaits empirical evidence; the extent of recombination among all hrv or ev types and the frequency with which viable recombinants arise are entirely unquantifi ed. the 28-30 nm hrv virion has been visualized for only a handful of hrv-a and hrv-b types (including hrv-a1a, hrv-a2, hrv-b3, hrv-b14, and hrv-a16), but no hrv-c structures have been empirically determined to date. the fi rst, hrv-b14, was described in 1985 [ 159 ] followed by hrv-a1a in 1989 [ 160 ] , hrv-a16 in 1993 [ 161 ] , hrv-a3 in 1996 [ 162 ] , and hrv-a2 in 2000 [ 163 ] . hrv-c structure has only been predicted using computer modeling, but their basic structure seems to be that expected of an hrv ( fig. 29.6 ) [ 33 ] . the hrv capsid shell is composed of 60 protomers, each comprising one copy of the viral proteins vp4, vp3, vp2, and vp1. vp1, vp2, and vp3 (each ~30 kda) are to some extent exposed on the capsid surface, whereas vp4 (~7 kda) is internalized and associated with viral rna. five protomers come together at a point around a fi vefold axis, and this cluster is called the pentamer. the fi vefold axis is circumscribed by a cleft referred to as the "canyon." vp1, vp2, and vp3 are each formed by a convoluted set of protein sheets and loops [ 159 ] . the loops protrude beyond the external capsid surface and contain discontinuous antigenic sites. of the hrv types studied, four neutralizing antibody immunogenic (nim) regions have been identifi ed on hrv-b14 and hrv-a16: nim-1a (located in vp1), nim-1b (vp1), nim-ii (vp2 and vp1), and nim-iii (vp3 and vp1) [ 159 ] . antigenic sites identifi ed on hrv-a2 are called a, b, and c [ 164 ] . the scope and location of antigenic and immunogenic moieties among the hrv-cs is unknown. using known receptor binding sequence as a guide for computer modeling ( fig. 29 .6 ), it has been predicted that when discovered, the receptor for the hrv-cs will differ from the major and minor receptors defi ned for the hrv-as and hrv-bs [ 33 ] . the three hrv species within the genus enterovirus are a genetically, immunogenically, and antigenically diverse assemblage of >160 viral types (table 29 .1 ). this accounts for the combination of hrv-a1a and -a1b, exclusion of hrv-87, which is actually ev-d68 despite confusion over acid liability [ 169 -171 ] and combination of hrv-hanks which is actually hrv-a21 [ 147 ] . serological studies indicate that some hrv-a and hrv-b types may not be distinct enough to deserve a unique identity [ 147 ] . species within the genus share >70 % amino acid (aa) identity in the polyprotein and in 2c+3cd and >60 % aa identity in p1 ( fig. 29 .3 ) as well as their host cell receptors, a limited natural host range, a genome base composition (g+c) that varies by no more than 2.5 %, and a similar compatibility of proteolytic processing, replication, encapsidation, and genetic recombination [ 172 ] . a variant of the same hrv type shares 87-88 % aa identity or more in vp1 [ 129 ] . much of the nongenetic criteria remain undefi ned for the hrv-cs. in 2008 the genera enterovirus and rhinovirus were offi cially combined, retaining the former genus name enterovirus with the human enterovirus c as the prototype species. a genus in the order picornavirales , family picornaviridae , is at least 58 % different in its amino acid identity from any other genus. in 2009 a proposal establishing the species human rhinovirus c was ratifi ed by the ictv. formal hrv-c numbering commenced in 2010, and type numbers were initially assigned based on the date of submission of relevant sequences to genbank (hrv-c1, formerly nat001; hrv-c2, f. nat045; hrv-c3, f. qpm; hrv-c4, f. c024, etc.; table 29 .1 ) [ 127 ] . a clinical detection of an hrv-c can be considered a novel type principally based on its vp1 sequence or provisionally ("c_pat," table 29 .1 ) based on vp4/vp2 [ 146 ] and could be confi rmed as a variant of a previously characterized hrv-c by identity thresholds to either region. the 5′utr can be and still is used [ 173 , 174 ] for hrv genotyping, but it is a more problematic region than vp1 or vp4/vp2 because of the recombination activity that affects this region, especially among the hrv-cs [ 175 ] . this is presented as phylogenetic intermingling of some hrv-a and hrv-c types [ 114 ] . nonetheless, careful application of sequence identity thresholds when comparing clinical sequences to the genbank database (≥96 % identity required before assigning a clinical detection to a particular type) succeeds in characterizing hrv species and types [ 9 ] . there are currently 50 types within hrv-c (which includes the types once grouped together under hrv-"a2," hrv-x, and hrv-ny clades), 78 hrv-a types, and 25 hrv-bs. the most up-to-date information on current taxonomic trends can be found at the ictv picornaviridae study group website ( http://www.picornastudygroup.com/ ). ( c ) hrv-c3 versus hrv-a2 simplot data projected onto the hrv-c3 pentamer. the domains of interest are mostly shown within a single asymmetric unit. ( d ) a minor group pentamer (hrv-a2, gray ) including antigenic sites (sites a -c , green ) and very-low-density-lipoprotein receptor (vldlr) footprint ( red ) [ 167 ] . attachment of the vldl-r involves adjacent vp1 molecules. magnifi ed vp1 area represents one half of a vldl-r footprint [ 168 ] . amino acid substitutions ( arrowed ) contributed to the differences between minor group sites b and c (adapted with permission from mcerlean et al. [ 33 ] ) historically a key feature distinguishing the hrvs from the evs was the instability of the hrv capsid in the presence of acid and their lower preferred laboratory propagation temperature (33-34 °c versus 37 °c for evs). over time hrvs have been subclassifi ed in different ways. the fi rst was based on tissue tropism and host range. hrvs that preferred growth using monkey cells were called "m" strains and those (the majority) that grew only in human cell cultures, "h" strains [ 56 , 176 -180 ] . these two groups correlate with receptor usage [ 131 ] (table 29 .1 ) and possibly with the titer of the inoculum employed [ 181 ] . in 1962 it was proposed to abandon this terminology in favor of a sequential numbering system [ 177 ] . picornaviruses recognize a variety of cellular receptors [ 169 , 182 , 183 ] . hrv types are also subdivided into major and minor groups defi ned by use of one of the two main receptor molecules [ 184 , 185 ] . the capsid of the majority of classical hrvs ( n = 89) [ 184 ] interacts with the amino-terminal domain of the 90 kda intercellular adhesion molecule (icam-1; cd54) [ 186 -189 ] . receptor binding destabilizes the hrv capsid, probably by dislodging the "pocket factor," and initiates uncoating [ 164 , 182 , 190 ] . icam-1 interacts with its receptor, leukocyte function antigen-1 (lfa-1), and plays a role in recruitment and migration of immune effector cells [ 191 ] . the minor group [ 184 ] of classical viruses employ members of the low-density lipoprotein receptor (ldlr) family to attach to cells [ 167 ] . binding of vldl-r occurs outside of the canyon employing a different destabilizing and uncoating mechanism. heparan sulfate may act as a receptor under specifi c conditions [ 183 , 192 , 193 ] . in 1990 andries et al. defi ned, and laine et al. refi ned, two "antiviral groups" (a and b) based on their susceptibility to a panel of antiviral molecules [ 165 , 194 ] . these groupings refl ected the nature of the amino acid (and hence nucleotide) sequence of the region interacting with the antiviral molecules. these antiviral groups can also be visualized using phylogeny [ 194 ] . when sequences from other subgenomic regions, including p1, 2c, and 3cd, were examined by phylogeny, the species were found, in most cases, to inversely correlate with antiviral grouping labels (table 29 .1 ). m and h indicate early cell tropism-based classifi cation (monkey, human) abandoned in favor of a sequential numbering system [ 177 ] . hrv types were later divided into the major and minor groups defi ned by receptor tropism [ 184 , 185 ] . receptor-designated minor group hrv types are underlined, and major group types are shown in bold. antiviral groups (a and b) are labeled [ 165 , 194 ] . hrv-a8 and hrv-a95 are also likely the same serotype [ 147 ] . a full list of genetically close serotype pairings was presented by ledford et al. [ 147 ] hrv-c nomenclature was defi ned in 2010 and currently includes a number of p rovisionally a ssigned t ypes (pat) which are confi rmed once preliminary vp4/vp2 data can be confi rmed with vp1 sequence and the provisional number removed (e.g., c_pat1 to c_pat13 have already been reassigned) today, sequencing and phylogeny play a central role in species classifi cation within the genus, and together, they are surrogates for the important biological classifi cation criteria [ 146 , 147 , 165 , 195 -197 ] . for the hrv-cs, fi rst described as the "hrv-a2" clade (not to be confused with the single virus, hrv-a2, this naming scheme appeared after the hrv-c clade's name was proposed) of viruses in 2006 [ 31 ] , sequencing of 5′utr and vp4/vp2 has provided the bulk of hrv information from clinical studies. while culture in primary sinus tissue has been reported [ 63 ] , no receptor is yet defi ned. hrvs are the most numerous and frequently detected of all the "respiratory viruses," so-called because of their predominant detection in and tropism for the human urt or lrt ( fig. 29 .7 ). the circulation of hrvs varies with population age, underlying disease, immunocompromise, over time, and across distance. circulation is infl uenced by the nature, strength, distinctiveness, and memory of the immune response hrvs trigger and by the nature and prevalence of other concurrently circulating respiratory, and perhaps nonrespiratory, viruses. with the recent discovery of the unculturable hrv-cs came the realization that previous hrv epidemiology was only reliable if conducted by one or more suitably broad-spectrum hrv pcr assays [ 111 ] ; hence, prior to 1988, detection of the full spectrum of ≥160 hrvs did not occur. after 1988, the ability to detect all types very much depended on the nature of the pcr primers and detection methods used. the great number of distinct hrv types has burdened the search for answers to epidemiology-related questions. however, as for other important respiratory viruses including human respiratory syncytial virus (hrsv) and the infl uenza viruses (ifvs), the virus types within a species show evidence of being both distinct and discrete viruses that are independently recognized by their host and consequently independently infect their hosts. each hrv type is also genetically stable [ 144 ] . the hrv species circulate variably from year to year with evidence of epidemics of distinct types. a prospective longitudinal cohort study over 6 months examined hrv frequency and diversity in 272 specimens from 18 healthy children (0-7 years of age) [ 198 ] . a median of three hrvs and a maximum of six were detected per child. a similar outcome resulted from an australian cohort study [ 9 ] . genotyping reveals more of the hrv diversity at a single site than culture ever could with molecular studies fi nding between 34 and 70 distinct hrvs at a single location [ 9 , 128 , 199 ] . the number of additional hrv cases that occur in children outside of specifi cally defi ned symptomatic periods remain to be defi ned, with current studies indicating that a much higher number of hrv infections may occur. more comprehensive investigation of hrv type and illness will be undertaken during analysis of data from the australianbased observational research in childhood infectious diseases (orchid) study ( http://clinicaltrials.gov/show/ nct01304914 ). interestingly, the hrv-bs are often underrepresented, even when accounting for the smaller number of known hrv-b types [ 128 ] . a number of studies have not found any robust patterns between the circulating hrv types or species and clinical outcome, but the majority of studies seeking this information are short and sample infrequently, limiting their ability to fi nd the patterns they seek [ 128 ] . studies into the relative sensitivities of nasopharyngeal aspirates (npa) and swab sampling methods produce differing results, but generally, if seeking the best diagnostic yield for as many respiratory viruses as possible (i.e., seeking a laboratory diagnosis to support clinical decision making), npas are the sample of optimal choice. one study reported similar clinical sensitivities between swabs and npas for human coronaviruses (hcovs), ifvs, and hrsv, but reduced sensitivities using swabs for hrvs, human adenoviruses (hadvs), human metapneumovirus (hmpv), or parainfl uenza viruses (hpivs) [ 201 ] . a second study reported no difference in sensitivities for hrvs, hadvs, and hpivs but a reduced sensitivity for hrsv and ifvs when using swabs [ 202 ] . nasopharyngeal washes also yield more viral culture success than either nasal or pharyngeal swabs. nonetheless, many studies use nasal swabs as the sample of choice because they allow self-collection and involve much less discomfort than npas, and pcr has meant that infectious virus is not required, only viral nucleic acid which relaxes some limitations imposed by the need for rapid, careful, temperaturecontrolled, and expensive transport requirements [ 64 , 203 , 204 ] . bronchoalveolar lavage samples are best for seeking lrt etiologies, especially in adults where nasal wash viral loads can be low compared to those in children, but this is an invasive method with some risk attached [ 205 ] . hrvs infect all people, all around the globe. spread of hrvs is most obvious and frequent from child to child and from child to parent [ 206 ] . in populations of mixed age, the majority of hrv detections occur in children [ 128 ] . among 272 specimens from 18 healthy children, over a third (37 %) were hrv positive. children less than 5 years of age (44 % of whom were hrv positive) were shown to have more hrv infections and a wider diversity of hrv types than children more than 5 years old (28 % hrv positive) [ 198 ] . healthy adults in the military [ 54 , 207 ] , at university [ 208 ] , at home [ 209 -212 ] , and in the workplace [ 209 ] have also featured prominently in historical, culturebased, and volunteer infection studies and heavily infl uenced our view of hrv infection outcomes [ 64 , 206 ] . although studies of children in hospital-based populations usually report more signifi cant clinical outcomes (relating to the lrt) [ 213 ] than community-based studies, these data are still broadly applicable. hospital populations originate from the community and refl ect the more serious and perhaps fi rst exposures to the virus. hospital-based populations defi ne the potential of a virus to cause severe clinical outcomes. disease at this end of the spectrum has the strongest infl uence on future prioritization of therapeutic research and developments [ 214 ] . modern air travel contributes to the rapid spread of respiratory viruses as seen in their often frequent detection among travelers [ 215 ] including those with febrile illnesses [ 216 ] . apart from children, hrvs are found with the great clinical impact in the elderly (described as 60-90 years of age) with 50 % of aris positive for an hrv, sometimes with a greater burden of disease than ifvs [ 217 ] . those with asthma or copd are also affected by the ari triggering exacerbations of wheezing illness (see sect. 8.2 ). it is thought that this is not a different type of infection but rather a different response to infection by the host. wheezing can also result from infection in atopic people who do not have underlying asthma or copd. hrvs cause signifi cant impact in the immunocompromised, and this group is the only population to date that has been found to host truly persistent hrv infections (see sect. 5.7 ). because the hrvs are the largest group of viruses to infect humans, it is not surprising that they confuse differential diagnoses during pandemics and have key roles in co-detections and asymptomatic disease. the study of hrvs is the study of all respiratory viruses; while each can be considered in isolation, this will likely be detrimental to a greater understanding of respiratory virus pathogenesis. hrvs circulate throughout the year but usually with a bimodal peak in temperate locations in both hemispheres. the highest peaks, mostly defi ned using adult populations, are in the autumn (fall) and spring [ 64 , 66 , 211 ] (and, peculiarly, on a monday [ 218 ] ). the major winter dip in hrv prevalence closely coincides with the peaks of other respiratory viruses, particularly ifvs [ 219 ] and hrsv [ 66 ] . one hypothesis states that a miasma exists in the school classroom, of particular relevance to those who suffer asthma exacerbations, and this miasma maintains immune stimulation, which subsequently wanes among school children during holidays, to be challenged anew upon return to school [ 220 ] . it is clear that an interplay or interference takes place between viruses at the population level, particularly evident among rna viruses. there is a correlation between spiking spring and autumnal hrv case numbers and an asthma exacerbation "season" 10-24 days after return to school from holidays, in a range of climates [ 220 -223 ] . this was particularly obvious among asthma hospitalizations of children (5-15 years of age) in ontario, canada, which peaked at weeks 37-39 across a decade [ 223 ] . upon investigation, hrvs were the most prevalent of the viruses found in a 1-year analysis of emergency room presentations in ontario [ 223 ] . hrvs also predominate during "hay fever season" [ 172 ] . although a defi ned seasonality is not always found in the tropics [ 224 ] , this may sometimes be due to testing that does not include hrvs [ 222 , 225 ] or only some hrvs [ 226 ] . all the hrv types continue to circulate today, including those named in the earliest of the nomenclature assignments. at a single site during 12-24 months, 70 or more types can co-circulate [ 8 , 9 ] [ 174 ], dropping [ 198 , 227 ] if the study time frame at the site is shortened. a recurring hrv type, defi ned using molecular tools, accounted for 1.6 % of any virus detected in a birth cohort followed for 12 months [ 8 ] and, in another cohort, occurred twice in two children, within a 6-month period [ 198 ] . within a given year and across different years, it is apparent that hrv species exchange predominance [ 9 , 36 , 60 , 227 -229 ] . no evidence exists to satisfactorily explain this; however, herd immunity may be a factor. the use of cell and tissue culture underestimated the frequency of multiple infections in patients, most likely because the dominant virus out-replicated any others, or due to viral load differences, specimen quality issues, differing cell tropisms, or the triggering of an antiviral state by the fi rst virus. when the majority of respiratory viruses are sought using pcr techniques, multiple virus-positive specimens can comprise a third of those tested [ 230 ] , dropping to around a fi fth of ari episodes when fewer viruses are sought [ 217 ] . there is sometimes an emphasis on the high number of hrv cases that are identifi ed in the presence of another virus, and including hrv testing does raise the frequency of pathogen detection above one per sample [ 231 ] . coinfections, or, more correctly for pcr-based studies, co-detections (since pcr cannot determine infectivity), have been found to either increase [ 71 , 232 -236 ] or have no impact on the clinical outcome in their host [ 237 -241 ] , and so the issue of clinical relevance of co-detections is still uncertain. in extreme cases, half of all hrv detections can be found concurrently with another virus. on the surface, this is a signifi cant fraction, and yet 80 % or more of hrsv, hmpv, ev, and ifv detections and 71 % of hcov-nl63 detections can be found in the company of another virus [ 242 ] . other studies fi nd different, but still higher proportions of co-detections involving non-hrvs [ 217 ] . whether co-detections represent a particular synergism between the involved viruses, a differential capability to manipulate the host immune response, a sign of innocuousness for the most frequently involved virus [ 243 ] , or a chance due to overlapping seasons remains unclear. it is clear, however, that co-detections are not an anomaly or an error due to "overly sensitive" pcr tests; they are evidence of further biological complexity that, until recently, remained hidden from us. recent studies have shown that the initial impression of hrvs being overrepresented in these cases was incorrect. closer analysis of viral co-detections has revealed patterns [ 231 , 244 ] . these became clear when codetections were examined bidirectionally, not just how many hrvs were positive for virus x but also how many of virus x cases were positive for an hrv. whether in a hospital or a community setting, hrvs more often occur as the sole virus detected in aris [ 9 , 244 ] . considering their ubiquity, it is interesting that relatively low numbers of concurrent detections occur [ 245 , 246 ] , supporting the concept that hrvs have a direct role in the clinical outcome of their infection [ 247 ] . the hrv partnership with host immunity may be a mutualistic one, inadvertently imparting an advantage to the host by protecting against more cytopathic respiratory viral pathogens, while the host provides a vessel for hrv replication and transmission. studies of single respiratory viruses without being in the context of the respiratory virome are of limited value in drawing conclusions about clinical impact. much of the longitudinal epidemiology data previously relied upon to form assessments of hrv signifi cance was acquired using culture-based techniques. with improved and more comprehensive testing, patterns can be seen among the interactions of hrvs and other respiratory viruses. virus interference is a type of virus-virus interaction (vvi) that has been known for decades. vvi has recently been categorized into types [ 248 ] . at the population level, it has been noted that during trials of live attenuated ifv (laiv) vaccines, an interferon (ifn) response was triggered that protected vaccinees against off-target viruses for 7 days postvaccination [ 249 ] . this 1970 study went so far as to suggest such effects could be maintained for a prolonged period using a regime of consecutive schedule vaccinations, each separated by 7 days or more, during times of a prolonged epidemic [ 249 ] . a similar effect was produced using live ev vaccines (lev) to replace pathogenic ev types and interrupt outbreaks [ 250 ] . orally administered levs succeeded in their principal task but also reduced the incidence of aris during epidemics by 50 % overall [ 250 ] . this shows that immune activation in the gastrointestinal system generates an anatomically distinct protective effect and there may be a similar effect on the gut's infl ammatory status after respiratory virus infection. in contrast to the laiv results, the offtarget protective effect was reversed in a study using a trivalent inactivated ifv vaccine [ 251 ] . the mechanism underneath these opposing outcomes is unclear. during the heyday (1960s) of tissue culture for virus studies, a common biological assay for infection with hrv involved attempted infection of the culture with an enterovirus (ev) or hpiv-1 [ 252 , 253 ] . failure of the superinfecting virus to grow heralded the likely presence of a noncytopathogenic hrv. virus interference has been used to measure ifn in specimens through its inhibition of hrv growth [ 254 ] . more recently hrv-hadv dual pcr-positive cases were found less often than expected and harbored lower viral loads of hrv than did specimens from cases of sole hrv infections [ 255 ] . signifi cantly, the majority of these instances of vvi involve rna viruses [ 244 ] . it has been shown that dual infections of peripheral blood mononuclear cells (pbmcs) with viruses other than hrsv (including hrvs) induced immune responses similar to those of single infections, but coinfections including an hrsv resulted in reduced ifn-γ responses [ 71 ] . vvis are affected by the ability of each to moderate the host response against them. virus interference has also been identifi ed in virus positives as a series of patterns among respiratory specimens tested for up to 17 respiratory viruses (fig. 29.8 ) [ 9 , 244 ] . statistical analyses supported that many of the co-detections occurred in patterns, in particular that fewer co-detections involved an hrv than would have been expected by chance alone ( p ≤ 0.05). for some period, rna virus infection, especially the hrv group, may render the host less likely to be infected by other viruses and, by extrapolating to the community level, help constrict the epidemic periods of other viruses by reducing the number of fully susceptible hosts. virus interference as a feature of respiratory virus epidemiology can also be seen in results of other studies [ 256 ] . during an 8-week period that spanned peak 2009 h1n1 pandemic infl uenza season in wisconsin, it was infl uenza a virus (ifav) that seemed to dominate hrv in children with asthma who were sampled weekly [ 236 ] . whether this refl ects all ifv-hrv interactions or just those involving a novel ifv such as 2009 h1n is unclear. it was found that pbmcs from these children exhibited normal immune responses [ 236 ] . reports of subjects with continuous and extended (greater than 2-3 weeks) periods of hrv positivity [ 3 , 257 ] increased as pcr methods replaced cell culture for hrv detection. this had only rarely been recorded using culture [ 54 ] . hrv rna has been detected days prior to symptoms commencing and for as long as 5 or more weeks after they cease [ 3 , 258 -261 ] . studies that only defi ne the period between aris in children as that time when specimens are rt-pcr negative [ 3 ] will not detect overlapping serial infections (fig. 29.9 ). epidemiology that incorporates hrv typing generally does not fi nd chronic shedding [ 204 ] . hrv shedding normally ceases within 11-21 days, after signs and symptoms have stopped [ 3 , 9 , 44 , 75 , 85 , 260 ] . thus, the perception of persistence is probably due to serial or overlapping infections by multiple untyped strains [ 8 , 54 , 210 , 262 ] . few studies [ 263 ] have suitably addressed persistence in hrv infections involving healthy subjects since pre-and post-sampling clinical data are rarely described [ 80 , 264 ] . to date, true persistence-an ongoing detection of a single confi rmed hrv type-has been limited to individuals with underlying immunosuppression or immune dysfunction [ 260 ] . hrv-cs were detected more than three times longer in immunocompromised young patients than in immunocompetent children, with a mean of 16 versus 53 days [ 265 ] . multiple detection of the same hrv type (100 % identical hrv-1a sequence in each patient over time) extended to 4 months in hematopoietic stem cell transplant recipients. the proof of causality is as diffi cult to achieve as the proof of innocuousness when it comes to respiratory viruses and aris. the defi nition of "well" subjects prior to or at the time of sampling or inoculation is sometimes not clear, especially for young children who cannot reliably report symptoms [ 3 , 96 , 204 ] . often parents notice a symptomatic illness before an infection is detected in the laboratory [ 3 ] , supporting the importance of diaries in longitudinal home-based community studies. nonetheless, even with the support of telephone interviews and home visits, milder cold symptoms may be missed. it is not uncommon for an asymptomatic control to subsequently become symptomatic or have been symptomatic before sampling [ 8 , 266 ] . some studies employ sensitive symptom scoring systems [ 267 ] , but the criteria for being symptomatic are usually designed to describe and clearly discriminate overt or more "severe" illnesses, those with obvious and measurable signs. strict defi nitions help improve patient management and the commencement or better direction of treatment or cohorting. however, in research studies the arbitrary degree of severity required for reporting a symptomatic event often overlooks very simple changes in host biology due to a virus's replication. these changes to the norm are mild but nonetheless represent disease (a disorder of structure or function that produces specifi c symptoms or that affects a specifi c location and is not simply a direct result of physical injury) in the literal sense. such minor or short-lived, often unrecorded [ 3 ] , indications of infection include sinus pain, headache, sore throat, earache, watery eyes, fatigue, muscle aches and pains, and mood changes. within families, hrvs are frequently transmitted from vsig activated "shields up" a b children who are usually symptomatic [ 204 ] . infants frequently exposed to other children have more asymptomatic viral infections [ 8 ] . among infected adult family members, asymptomatic infections are more likely [ 204 ] . among older parents, whether their children live at home or not, asymptomatic infections are more frequent following hrv challenge than among adults without children or in younger parents [ 268 ] . in a study of viral species in age-stratifi ed cases and controls, signifi cantly lower viral loads were found in those without the required symptoms [ 269 ] . qpcr may prove useful to determine viral load cutoffs to address this issue in the future, although the respiratory tract is a diffi cult tissue for qpcr [ 200 ] . the high sensitivity of pcr-based methods has raised concerns over the clinical relevance of a virus-positive result [ 269 ] . it is clear that a proportion, around fi ve to 28 % of study-defi ned asymptomatic control populations [ 90 , 91 , 269 ] , are virus positive using sensitive pcr-based methods. this may vary up to nearly 50 % of cases when stratifi ed by age, virus, and season or when including highrisk populations [ 8 , 269 ] . every respiratory virus, even ifvs and hrsv, can be found in cases without symptoms at the time of specimen collection even after specifi c inoculation of adults [ 137 , 269 , 270 ] . this is a complex and incomplete story in need of more research, and so it is frustrating that positivity in asymptomatic people is often used to rank viral importance. better data are required from asymptomatic controls for any conclusion to be drawn about causality [ 266 ] , but this requirement often disregards the memory of a normal functioning protective host immunity. it is the host response that defi nes the degree of clinical severity for the infl ammatory disease that is the hallmark of an ari [ 271 ] . it is well known that previous exposure to a virus affords protection from the full clinical spectrum of disease upon repeat exposure to that virus. it should come as no surprise then that hrvs, which usually cause brief infection anyway, could well produce only minor signs and symptoms upon reinfection. the unique and extremely personal infection history of each member of a control group cannot be determined unless they are part of a longitudinal cohort. so, what do cohort studies, supported by comprehensive pcr-based testing, tell us about asymptomatic virus infections? some cohort studies do not look in asymptomatic children, seeking samples only at times of symptomatic illness [ 66 , 246 , 272 ] . a birth cohort of children enrolled and sampled when ill and every 6 months for 24 months identifi ed hrvs 14-28 % of infants and toddlers who had no nasal symptoms (defi ned solely by the presence of rhinorrhea) [ 273 ] . the childhood origins of asthma (coast) birth cohort followed 285 infants at high risk for allergies and asthma for 12 months and identifi ed hrv infections as preceding (mean age of fi rst detection, 4 months) those of hrsv (mean age at least 6 months), and hrvs were found in 35 % of asymptomatic versus 61 % of moderately to severely ill patients; the most frequently symptomatic children also had the greatest proportion of asymptomatic infections [ 8 ] . in a study of 58 children with asthma sampled weekly for 5 weeks during each of two peak hrv seasons, nearly two-thirds who were virus positive but not sensitized to at least one allergen showed no asthma symptoms, and nearly half showed no ari symptoms; in the children who were sensitized, less than one-third showed no asthma symptoms, and only a fi fth had no ari symptoms [ 227 ] . a convenience population of 15 healthy children (1-9 years old) without asthma were followed during at least three seasons, and picornaviruses were detected in 5 % of 740 specimens (21 % of infections) not associated with symptoms, the impact of hrv typing and of sampling based only on symptoms. the example provided here diagrammatically represents a single, hypothetical monitoring period, starting at time = 0, for a single individual. the period of potentially detectable hrv is indicated by an open box. if sampling occurred at each time point ( 0 -6 ) and hrv positives were genotyped, it would be apparent that three different strains infected the individual, although discerning hrv-x from hrv-z at time point 3 would require a molecular cloning approach. illness, in different forms, may have continued over the entire period depending on the symptoms required/recorded and the period of time represented by the monitoring period. in this case a clinical diagnosis may record only a single symptomatic episode. genotyping may not be performed, and sampling may be intermittent, and so association between viral type or species and disease is impossible. in the study examples indicated by ( a ) start and fi nish sampling or ( b ) symptomatic sampling, ( asterisks mark sampling times in fi lled bars), the laboratory data would have made only one or two identifi cations, respectively. in the third example, ( c ) frequent sampling of this type has previously led to conclusions of hrv persistence or chronic shedding; when combined with genotyping, it becomes apparent that different hrv types are present although 9 of the 25 infections came from households with an infected sibling [ 3 ] . in summary, there is clear evidence for the presence of hrvs in asymptomatic controls. a precise proportion cannot yet be defi ned. some study controls show signs of a "lead-in" period where rna positivity precedes an ari defi ned on follow-up, while others may have been defi ned as symptomatic if more symptoms had been accounted for. mechanisms and routes of transmission hrvs have been found at extra-respiratory sites. viremia was determined in the blood of children with lrt infection or pericarditis [ 274 , 275 ] , and hrv-c was more commonly associated with viremia than was hrv-a, supporting possible increased pathogenicity [ 274 ] . blood was also positive for hrv rna and infectious virus from infants at necropsy [ 276 , 277 ] , and hrv rna was detected in the plasma of children with asthma, bronchiolitis, or common cold [ 76 ] . an hrv was once isolated from feces [ 203 ] , and more recently higher than expected loads of hrvs were detected in fecal specimens from children with suspected meningitis and fever of unknown origin [ 77 ] , with gastroenteritis [ 278 ] , and in a child with pericarditis [ 275 ] . nonetheless, the nasopharynx is still considered the main site of focal virus production [ 279 ] , regardless of inoculation route [ 280 ] , and most studies of transmission routes have centered on the urt. in contrast to ifv and hrsv, hrv infection involves less destruction of tissue. ciliated epithelial cells are sloughed off in proportion to the severity of an hrv ari, but this damage is minimal and does not occur during the viral incubation period or with subclinical infections [ 137 , 281 ] . the incubation period between infection and onset of virus shedding into nasal secretions is 1-4 days with shed viral titers peaking in adults between days 2 and 10 [ 44 , 282 ] . the time until successful hrv transmission among adults in a childless family setting is usually 5-8 days and requires the donor to be shedding at least 10 3 tcid 50 at some stage, to have recoverable virus on the hands and in the nares, enough shared time, and a moderate to severe ari [ 283 ] . the lungs have been shown to host replicating hrv [ 260 ] , and the reader of such reports may be left with the perception that detection of hrv replication in the lrt explains all lrt symptoms. however, relatively few studies seek or identify true hrv replication in the lrt. while the overwhelming majority of lrt cases detect hrv from the urt, a correlation between urt positivity and lrt disease does exist [ 284 ] . it is well known from experimental inoculation studies that hrv infection can result from inoculation of the conjunctival sac after virus is moved through the nasolacrimal duct [ 280 ] . in these studies virus was commonly delivered by aerosol or intranasal instillation of 0.25 ml to 5 ml of suspension [ 43 -46 , 280 , 285 -287 ] . in the laboratory, hrvs can retain infectivity for hours to days on suitable, nonporous solid surfaces, especially if the inoculum remains damp [ 47 , 287 ] , which supports direct self-inoculation especially in the family setting and indirect inoculation via fomites [ 288 ] . in a trial to defi ne the movement of virus from a contaminated donor to a recipient via multiple surfaces or by hand-to-hand contact, 13 % (donor to objects to recipient) and 6 % (donor to recipient fi ngers) of the virus recoverable from the donor's fi ngertips were recoverable from the recipients' [ 289 ] . even under observation, eye rubbing (0.37 h −1 -2.5 h −1 ) and nosepicking (0.33 h −1 -5.3 h −1 ) occur frequently [ 47 , 290 ] , suggesting self-inoculation could outpace personal hygiene, particularly in the young. it was once thought strange that aris were so common, but isolation rates for the expected viruses were so low [ 36 , 291 ] . with a better understanding of the importance of preexisting antibody (something common among the predominantly adult volunteers used by many studies), the discovery of a third, unculturable species of hrv (still causing aris but impossible to isolate or detect using antibody-based systems for which no reagents existed), and a vastly improved diagnostic sensitivity, this is much less confounding. in the past, household cross infection, determined by ari, was low, about fi ve exposures to infected members required for infection [ 17 ] despite viral loads in nasal washings peaking at 1.6 × 10 5 tcid 50 /ml [ 44 ] . experimental transmission was also reportedly ineffi cient [ 45 ] . in contrast, "naturally" close-quartered military populations, interacting over 1-4 weeks, experienced rapid spread of hrvs to >50 % of the group [ 54 ] . the use of pcr recently clarifi ed this discrepancy, confi rming that frequent transmission in families is more common than culture-based studies had identifi ed, often resulting in asymptomatic infection among older siblings and parents [ 204 ] . pcr has helped defi ne the scope of viral rna, if not actual infectious virus, survival, and spread. transmission studies require infectious hrv, and so the hrv-cs do not contribute to the historical data. under crowded or intimate conditions and with more severe colds, transmission reaches 38-100 % [ 283 , 292 ] . in some studies, both large-and small-particle aerosols proved ineffi cient, supported by a low isolation rate from saliva (39 % compared to 65 % of hand washes and 50 % of nasal swabs) [ 44 , 47 , 293 ] and from only 8.3 % of participants exposed to large-particle aerosols [ 293 ] . in other human donor-recipient model studies however, aerosol proved to be the main transmission route among antibody-free adults [ 46 , 282 ] . the discrepancy may have been due to insuffi ciently long or intense exposure in the earlier aerosol experiments [ 45 , 267 ] . apart from particle size, spread of virus by aerosol is affected by existing nasal obstruction which can divert secretions from the nares to contaminate saliva, the presumptive source of virus in coughs and sneezes [ 44 ] . when exposed to 10 liters of a small-particle aerosol, 10 1 tcid 50 of hrv-15 was associated with fever and prominent tracheobronchitis in antibody-free (<1:2) adult volunteers but not when delivered via nasal drops or a coarse aerosol [ 46 ] . it has also been found that simple breathing releases hrv rna (the same type was also identifi ed from nasal mucous) from at least a third of adults and children with symptomatic aris and infectious hrv could be isolated from a fi fth [ 294 , 295 ] . it is apparent that hrvs accumulate at sites with heavy human traffi c, potentially forming a secondary source of infection. hrv rna can be detected from 32 % of ~47-hourold fi lters placed to sample air in offi ce buildings [ 296 ] . in aircraft, high effi ciency particulate air (hepa) fi lters have been found to harbor hrv rna more than 10 days after they were removed for servicing [ 297 ] . hrv infections trigger a vigorous proinfl ammatory immune response that is thought to drive the symptoms experienced as illness [ 271 , 298 , 299 ] , but they do not seem to actively prevent or interfere with the host's immune response the way most other viruses have evolved to do. there may be a role for repeated challenge by hrvs and other respiratory viruses leading to infl ammation and tissue remodeling. the host response to hrv infection can be broadly broken into the innate (very fast, encoded in the germ line, nonadaptive) and adaptive (slower to develop, reliant on t cells, b cells, and the generation of antibody) responses. while the innate system is "always watching," it is signifi cantly amplifi ed by virus infection. the adaptive response is initiated by the host's fi rst infection with a particular virus and then functions to limit subsequent infections through the production of neutralizing antibodies and amplifi cation of existing cell-mediated immunity. after virus-receptor binding and internalization, the earliest host cell immune response to an hrv infection is elicited by the innate immune system (fig. 29.10 ). epithelial cells represent the front line against hrv invasion although alveolar macrophages and dcs are better equipped to respond [ 300 ] and do so despite not hosting hrv replication directly [ 16 ] . virus detection is mediated by pattern recognition receptors (prrs) that have evolved to recognize conserved molecular structures shared among diverse pathogens. internal-or surface-mounted prrs include sentinels that specifi cally recognize picornavirus rna and protein and, in doing so, trigger an immune circuit that results in the production of ifns and subsequently hundreds of ifn-stimulated gene products. the innate response to viral infection hinges on inducing two type i ifns (initially ifn-ß then ifn-α), secreted cytokines that produce antiviral, antiproliferative, and immunomodulatory outcomes [ 301 ] . the type iii ifns (ifn-λ1 or il-29, ifn-λ2 or il-28a, and ifn-λ3 or il-28b) are also produced in response to viral infection in a range of cells, although their receptor is not as widespread [ 302 ] . the type ii ifn, ifn-γ, is produced by activated t cells and natural killer cells rather than in direct response to virus [ 303 ] . detection of viral components triggers protein signaling cascades that regulate ifn synthesis through the activation of viral stress-inducible genes (vsigs) [ 301 , 304 ] . these are sometimes expressed constitutively but upregulated after ifn induction following hrv infection [ 305 ] . released ifn-ß binds to the ifn-α/ifn-ß receptor in an autocrine (the same cell) and paracrine (neighboring cells) manner, starting a positive feedback loop for type i ifn production, the "second wave." vsigs include the antiviral proteins protein kinase r (pkr), 2′5′oas/rnasel, and the mx proteins [ 306 ] . ifn-α upregulates expression of mxa, 2′4′-oas, and pkr [ 307 ] . the mx pathway is also induced after virus infection but is not constitutively expressed [ 307 ] . depending on the sentinel system stimulated, there are different pathways to vsig activation. those vsigs with antiviral properties (e.g., mxa, pkr, 2′5′oas/rnasel) inhibit different stages of virus replication and strengthen an antiviral state in the host. while this state is well known, the nature of its induction by different respiratory viruses and the impact of induction upon the replication of other respiratory viruses are topics for considerable ongoing research. one pathway to ifn induction relies on the ifn-upregulated cytosolic sentinels retinoic acid inducible gene rig-i-like receptors (rlrs) rig-i (specifi c for ifav and others) and melanoma differentiation-associated gene 5 (mda5, specifi c for picornaviruses and others) [ 306 , 308 ] . these rna helicases recognize either rna with a 5′-triphosphate or distinct dsrnas, which results in activation of nf-κb leading to "classical" type i ifn induction [ 301 , 306 ] . studies into the innate response to hrv infection have been limited to the use of a very few easily cultured types. it is presumed that the result can be extrapolated to most if not all types. this is yet to be tested. rig-i is degraded by hrv-a16 [ 309 ] , ifn regulatory factor (irf)-3 homodimerization is interfered with hrv-b14 which limits ifn-β induction [ 310 , 311 ] , and mda5 is degraded by hrv-a1a but not hrv-a16 [ 312 ] . another pathway for recognizing hrv infection involves the toll-like receptors (tlrs), transmembrane prrs that terminate in an intracellular signaling region. the endosomally localized tlr3, tlr7, tlr8, and tlr9 recognize nucleic acids and are also involved in innate antiviral responses. tlr7 and tlr8 identify g/u-rich ssrna from endocytosed viruses, while tlr9 recognizes unmethylated cpg dna present in dna viruses [ 301 , 318 ] . tlr2 and tlr4 are found on the cell surface and recognize hrv or hrsv proteins, respectively [ 318 , 319 ] , and tlr3 recognizes dsrna. tlrs operate mainly, but not exclusively, in plasmacytoid dc [ 301 ] . the particular tlr that notifi es of an hrv incursion may depend on the method of virus approach [ 319 ] . tlr7 activation can reduce 2′5′oas and mxa mrna expression and ip10 protein in adolescents with asthma compared to healthy controls [ 320 ] . tlr3 activation did not result in a similar disparity [ 320 ] . it has been suggested that hrvs may have evolved with humans to such an extent that their symbiotic relationship serves to help train the human immune system [ 321 ] . intriguingly, within the hrv species, there are differences in the type and level of host response induced [ 322 ] which may refl ect receptor usage, route of entry and cell type infected, hrv species, or the degree of laboratory-adapted virus used during in vitro studies. after initial hrv infection, the innate response results in production of proinfl ammatory cytokines, vasoactive peptides, and chemokines that attract leukocytes, granulocytes, dcs, and monocytes (table 29. 2 ) [ 321 , 323 , 324 ] . the t-lymphocyte response to viral intrusion can be broadly categorized as t h -1-like and t h-2-like. other t-cell subsets exist, but most work in relation to hrv has been conducted on the earliest defi ned subsets. the t h -1 cellular response is important in managing cellular immunity and producing interleukin (il)-2 and ifn-γ. the t h -2 cellular response manages humoral immunity and stimulates b cells via il4 (initiating production of ige), il5 (infl uencing eosinophils), and il13 (crucial component of allergen-induced asthma). these two t-cell responses act in concert with epithelialderived chemokines (e.g., eotaxin) to promote the recruitment and activation of eosinophils and mast cells, contributing to chronic airway infl ammation and the hyperresponsiveness of airways to a variety of nonspecifi c stimuli [ 325 ] . t h-2 lymphocytes, opposing t h-1 lymphocytes, contribute to an allergic infl ammatory cascade, akin to what occurs to rid humans of parasites [ 326 ] . the t h-1 response can also be repressed by binding of microrna, which leads to an altered balance favoring a t h-2 state in mice and probably in humans [ 327 ] . regulatory t cells (t reg ) suppress allergic infl ammatory pathways and are therefore fundamental in protecting the airway from allergen sensitization [ 326 ] . considerable immunobiological research has focused on asthma exacerbation, with which hrvs are intimately involved. although upregulated by hrv infection, the t h -1 response is comparatively defi cient in people with asthma [ 328 , 329 ] . this is problematic as an increased t h -1-like cytokine response, deduced from higher sputum mrna ifn-γ/il5 values, speeds clearance of hrv and symptom amelioration [ 85 ] . one possible cause of the t h -1 defi ciency in people with asthma is inadequate maturation of type i and iii ifn responses due to reduced exposure to infections early in life [ 330 ] . the "hygiene hypothesis" [ 331 , 332 ] posits a pathway for an asthma etiology described [ 325 ] in terms of the young, unchallenged immune system, dependent on infections to stimulate the development of its t h-1-like functions. one theory suggests that hrvs play a central role in developing that effi cacious antiviral immunity, particularly in infancy, via their frequent, usually mild self-limiting infections [ 333 ] . genome-wide expression analysis of becs from healthy and asthmatic adult subjects after hrv-a1a infection revealed some signifi cant differences that were found between cell types and response to infection [ 61 ] . these included immune response genes (il1b, il6, il8, il1f9, il24) and airway remodeling genes (loxl2, mmp10, fn1) and an overall proinfl ammatory response and metabolic slowdown consistent with proteolytic cleavage of transcription factors by some hrvs [ 133 , 334 -336 ] in the infected cells. this study further noted some similarities to gene expression changes observed in brushings from people with mild asthma after allergen exposure and in bal cells from subjects with corticosteroidresistant asthma [ 61 ] . overall, hrv replication and the host transcriptional response to it were similar in normal or asthmatic bec cells [ 61 ] . this indicated, at least in adults, that something beyond the epithelial cell is an important contributor to more severe clinical outcomes in asthma. the application of inactivated hrv-b14 was found to promote release of il10 from monocytes (an immunosuppressive cytokine) and to inhibit the stimulation of il12 (drives t h -1 development) [ 337 ] . however, neither il10 nor il12 was signifi cantly induced in asthmatic adult volunteers in response to hrv-a16 compared to healthy subjects [ 338 ] . while ifn-α was detected after transfection of dcs with hrv-b14 ssrna, low tnf-α and il12 levels were also noted [ 16 ] . it was posited that the reduced il12 could indicate negatively affected local immunity possibly predisposing to secondary infections [ 337 ] . infection of stromal lung cells by hrv-b14 triggered exaggerated levels of the pleiotropic il11 (an il6type cytokine), akin to those triggered by hrsv, which were also detected in nasal secretions from children with wheezing [ 339 ] . other cytokine changes have been identifi ed in atopic adult volunteers challenged with hrv-a16. g-csf and il8 (chemo-attractant for neutrophils) levels rose in the urt (as examined by protein detection in nasal lavage) and lrt (mrna detection in sputum) with concomitant rises in blood and nasal neutrophil numbers [ 85 , 203 , 340 ] . the nasal epithelial cells of atopic individuals, especially in season, express more icam-1 than those of nonatopic adults [ 341 ] as do normal subjects infected by the major group hrv-b14 [ 341 ] . by contrast, ifn-γ and il8, which appear later postinfection, downregulate icam-1 expression in infected cells [ 191 ] and encourage infi ltration of neutrophils [ 342 ] , respectively. changes in icam-1 levels may modify participates in creation of an antiviral state; produced by and infl uences the maturation of dcs il-1β proinfl ammatory properties; enhances adhesion molecule expression including icam-1; induces il-2 receptor gm-csf a granulocyte and monocyte growth factor il-2 stimulates growth and differentiation of t and b lymphocytes and cytotoxic activity of nk cells and monocytes il-4 t h 2 differentiation, promotes ige synthesis il-6 activation, differentiation, and proliferation effects on t and b lymphocytes; induces c-reactive protein stimulating pyrexia il-8/cxcl-8 neutrophil chemoattractant resulting in neutrophilic, monocytic, and lymphocytic recruitment and degranulation activity il-10 anti-infl ammatory factor produced by monocytes that acts by inhibiting proinfl ammatory cytokines il-1, il-6, and tnf-α irf7 a master hub, regulating antiviral immunity ip10/cxcl10 chemoattractant for activated t h 1 and nk cells tnf-α proinfl ammatory activity similar to il-1 β; activates neutrophils; induces vascular permeability mpc-1 a monocyte attractant bradykinin potent infl ammatory mediator, increases vascular permeability tslp 3 an il-7-like cytokine that activates myeloid dcs to induce naive t cells into t h 2 cells producing il-4, il-13, and tnf-α; induced by hrv in the presence of il-4 bec bronchial epithelial cells, dc dendritic cell, irf interferon regulatory factor, ifn-γ inducible cytokine protein, nk natural killer, pbmc peripheral blood mononuclear cells, il interleukin, tnf tissue necrosis factor, tslp thymic stromal lymphopoietin t-lymphocyte-mediated cytotoxic or t h interactions with hrv-infected cells, upregulating receptor expression and encouraging eosinophil and t-cell infi ltration into the lower airways of asthmatic individuals [ 187 , 343 ] . before an hrv can enter a cell, it must pass through a defensive barrier of secreted anti-hrv antibody, mostly iga. the ease with which this passage occurs is proportional to the progression of clinical disease. healthy adult volunteers were found to develop iga by at least 3 days to 2 weeks after inoculation-about the same time as serum antibody-and retain peak levels for at least 8 weeks [ 178 , 344 -346 ] , falling faster than serum levels [ 347 , 348 ] . there is also some evidence for a degree of nasal immune memory [ 347 ] . volunteers with pre-study serum antibody could still be infected in some studies [ 46 , 210 , 292 ] , but not in others [ 349 ] . infection is more clear in volunteers without preexisting nasal antibody to experimental challenge virus; they become infected, exhibit more severe ari, and shed more virus for longer [ 292 , 347 ] . iga does not seem to modify illness severity or virus shedding, but high levels prevent reinfection by the initiating virus type. low levels or absence of iga does not prevent reinfection by the same hrv type, which may manifest as symptomatic or asymptomatic disease [ 349 ] . older children, adolescents, and adults have greater amounts of hrv-neutralizing antibody than young children [ 42 ] , accompanying a trend toward decreasing numbers of symptomatic aris with increasing age [ 218 , 350 ] . this feature raises an issue: did the use of older subjects in many common cold studies underplay the pathogenic potential of the hrvs because protective or partially cross-protective antibodies moderated the impact of infection? consequently, quantifying levels of type-specifi c serum antibody became routine practice prior to some studies. adult volunteer studies determined that no infections resulted if preexisting neutralizing antibody titers ≥1:16 existed; as levels grew from 0, so did levels of resistance to infection [ 43 , 210 ] . adults were protected by serum titers of 1:3-1:8 [ 209 , 210 ] . the trend was interrupted by adults in the 20-39 year age group, presumably because they had begun families and their young children acquired and amplifi ed currently circulating types from the community and transmitted them into a household that was either immune naïve or lacking suffi cient antibody or cell-mediated memory for protection [ 351 ] . traditional vaccine strategies were quickly ruled out as a prophylactic intervention for hrv illness because of the extensive antigenic variability that is a hallmark of the genus enterovirus [ 178 , 325 ] . however, if it were possible to identify "master" strains [ 352 ] that exhibit suffi cient antigenic cross-reactivity to induce broad heterotypic responses against many other hrv strains, then an effective vaccine could still be possible. in fact, boosting host immunity to an hrv type by repeat infection does heighten immunity to one or more other types [ 44 , 353 ] . the highest of these heterotypic antibody titers develop against those types with the highest preexisting antibody levels [ 352 ] . the fi rst description of a unifying hrv numbering system recounted the appearance of minor serological cross-reactions, which were removed by modifi cation of the technique [ 57 ] . subsequently, cross-reactions were better defi ned during experimental inoculation when multiple hrv immunogens and antigens were used to deduce the extent of heterotypic responses [ 56 , 210 , 352 , 354 ] . less promising for hrv vaccinology was the description of antigenic variation within hrv types which suggested that immunity to one variant of the type might not protect against infection by other variants [ 355 , 356 ] . the "prime strain" is a specifi c antigenic variant of a prototype hrv type that is neutralized to a lesser extent by antisera from the prototype, while yielding antisera that effectively neutralize both itself and the prototype [ 357 ] . another form of this cross-neutralization is ascribed to the "intertypes," which are hrv isolates that share a lower-level serological relationship with a pair of hrv strains, which themselves share neutralizing reactivity, e.g., hrv-a36 and hrv-a58 [ 358 ] . the low-level reciprocal neutralizing activity was not equivalent in both directions; anti-hrv-a36 sera had a higher titer for hrv-a58 than anti-hrv-a58 sera did for hrv-a36 [ 358 ] . over 40 strains were linked directly by such one-or two-way cross-reactions or indirectly through two or more strains. hrv-a67 and hrv-a28 are linked via hrv-a11, hrv-a13, and hrv-a32 (anti-a11 serum neutralizes hrv-a28, anti-a13 neutralizes hrv-a11, and anti-a32 neutralizes both hrv-a13 and hrva-a67 [ 358 , 359 ] ). a surrogate molecular method which provided insight into these interrelationships, perhaps expanding upon them to identify useful patterns for vaccine immunology purposes, would be most welcome. in summary, heterotypic immunity and hrv intertypes might be exploitable features of hrv immunobiology that could confer maximum protection upon the host from the minimum number of hrv types [ 358 ] . hrvs circulate in great numbers, and any specifi c roles for distinct hrv types in initiating disease remain to be defi ned. the relatively inconsequential common cold is the most frequent manifestation of viral infection in humans, with 30 to >80 % of colds positive for an hrv [ 69 , 360 , 361 ] . furthermore, aris due to hrv infection can exacerbate or result in a much greater burden of disease in those with asthma, copd, or cystic fi brosis. other complications include otitis media, pharyngitis, and wheeze in atopic people without asthma. the role of viruses in the origin of some of these diseases or their exacerbation is still unresolved. the lrt disease may mask the urt nature of the infection, favoring clinical diagnosis of an lrt illness. interestingly, during the 2009 h1n1 pandemic, much of the parentinitiated healthcare visits from a birth cohort in the united states were not due to pandemic virus but hrv and hrsv [ 362 ] . there is no known natural murine rhinovirus on which to base a small animal model of hrv infection, and mice are not natural hosts for hrvs. a recently developed model of airway disease using mengovirus (a picornavirus infecting rodents) may yield valuable in vivo airway infection and infl ammation data [ 363 ] . hrvs are often detected in neonates and infants with lrt signs and symptoms because the very young have narrow, immature airways and are more signifi cantly affected by airway swelling, excessive secretions, and smooth muscle contraction [ 364 ] . this may also be due to the relatively naive immunity of very young children. much of the more severe disease in hrv-positive children occurs in the youngest of them. some key examples are addressed below. for the common cold, as for any illness, accurate epidemiology and burden of disease data underpin the prioritization of preventing, treating, and further researching the etiological agent. to assign funds for researching the agent, health policy makers also need to understand how effi cacious and costeffective the development of an intervention will be [ 365 ] . the host immune response to hrv replication is the main cause of the signs (quantifi able fever, rhinorrhea) and symptoms (feeling of fever, myalgia, headache, fatigue, and mood change) of a cold that the host experiences [ 321 , 366 , 367 ] . a feature of common colds is increased vascular permeability which, enhanced by kinins, results in increased plasma protein (albumin and immunoglobulin [ig] g) levels in mucus, approaching the levels in serum [ 368 ] . histamine levels do not rise in nasal secretions of otherwise healthy cold sufferers [ 368 ] . during the resolving phase of the ari, glandular proteins (lysozyme, siga) predominate [ 346 ] . the common cold syndrome is also described as rhinosinusitis (the agglomeration of rhinitis and sinusitis since they frequently clinically coexist) [ 369 , 370 ] . this consists of nasal discharge or rhinorrhea, nasal obstruction, sore throat, sinus pain, headache, sneezing, watery eyes, cough, fever, fatigue, muscle aches and pains, and mood changes [ 367 , 371 ] . these are caused directly or indirectly by viral infection; cough is the result of vagus nerve irritation by mucus; sneeze results from trigeminal nerve irritation; sore throat is likely due to the action of prostaglandins and bradykinins; and fever, psychological effects, fatigue, and myalgia are mediated by cytokines [ 367 ] . hypertrophic adenoids have also been found to have a high proportion of viral, especially hrv, occupation regardless of host symptomatic state [ 372 ] . observation of natural culture-confi rmed hrv colds in adults noted that cough usually started by day 1 and was more persistent up to 9 days later [ 264 , 373 ] . rhinorrhea, sneezing, and sore throat were reported by half or more of patients and headache by at least a quarter of cases [ 207 , 373 ] . as neutrophils accumulate at the site of primary urt infection, the myeloperoxidase in their azurophilic granules creates the yellow-green coloration of nasal mucus that was once considered a sign of bacterial superinfection [ 271 , 367 ] . a common cold caused by an hrv cannot be clinically distinguished from one that caused by any of the other respiratory viruses [ 207 , 371 ] . as is likely for a single hrv type, once the host has been infected by an hmpv, hpiv, ifv, etc., a secondary exposure to that same virus type will produce less severe clinical outcomes due to pre-primed host immunity. asthma is a clinical diagnosis made on the basis of patient history, physical examination, assessment of airway obstruction or reversibility, and response to bronchodilators [ 374 ] . it is a complex chronic respiratory disease involving airway infl ammation, airfl ow obstruction, and airway hyperresponsiveness, which manifests as recurrent reversible attacks with deteriorating asthma control that are generated by interactions between infectious agents and other environmental and genetic factors that remain incompletely characterized [ 375 ] . the mechanistic role for hrvs in asthma inception and exacerbation is not yet defi ned [ 364 , 376 ] but is being revealed as the extremely complex interplay between infl ammation due to virus versus that due to atopy is explored [ 315 ] . possible virus-host interactions include (i) severe hrv infection of healthy infants which may result in subsequent development of asthma; (ii) hrvs may trigger asthma in children with a genetic predisposition toward atopy; (iii) repeated mild infections may protect against more asthmogenic/cytopathic viruses or the overdevelopment of the t h 2type response; and (iv) hrvs may simply exacerbate that which already exists [ 377 ] . it is unclear if the risk of atopic asthma during infancy is increased by aris which affect the development of the immune system, or whether aris lead to asthma development in children with a genetic predisposition to more severe responses to infection [ 325 , 343 , 378 ] , or a mix of both. in children with asthma, viruses have been detected in at least 77 % of exacerbations (65 % picornaviruses, probably hrvs [ 379 ] ) and in 50 % of adults [ 380 ] . acute wheezing episodes (including bronchiolitis and acute asthma) are a frequent, epidemic, and seasonal lrt manifestation of urt respiratory virus infection of children from all ages, especially during the fi rst year of life [ 214 , 381 -385 ] . bacteria are not major factors in wheezing exacerbations [ 386 ] . wheezing is blamed for high socioeconomic and healthcare costs, overuse of antibiotics, being the primary cause of hospitalization among children, and, rarely, for death [ 109 , 387 , 388 ] . traditionally hrsv infection has most often been the virus causally associated with expiratory wheezing, wheezy bronchitis, or asthma exacerbations because of the virus's well-known ability to infect the lrt, its more frequent detection in some studies [ 386 ] , and the low perceived likelihood of urt viruses such as hrvs replicating in the warmer lrt. nonetheless, periods of epidemic wheezing in the absence of high rates of hrsv detection are common [ 383 , 389 ] . hrvs even predominated in some culture-based studies of wheeze [ 384 , 390 ] . the coast study used sampling criteria that were intentionally designed to investigate the role of hrsv in illness, but instead indicated that hrvs were the most important predictor of subsequent wheezing in early childhood, and this is supported worldwide [ 224 , 391 , 392 ] . the asthmatic airway is characterized by an infi ltration of eosinophils and th2-type t cells (th2 cells) [ 393 ] . in those with an atopic background, eosinophilia was more common, and the virus isolation rate was higher than in the nonatopic group [ 394 ] . the cytokine and eosinophil activation profi les for hrsv-induced wheezing differ from those induced by hrv in which il5 is signifi cantly higher in serum and nasal aspirates than for hrsv [ 118 ] . ip10 was the only cytokine signifi cantly elevated in all symptomatic wheezing groups [ 118 ] . signifi cantly higher rates of hrv detection with more obvious lrt symptoms are more common in children with asthma than in non-asthmatic populations [ 380 , 388 , 395 , 396 ] . exacerbations of asthma are often preceded by a symptomatic rather than asymptomatic hrv infection [ 17 , 379 , 388 , 397 -399 ] although, in some instances, an exacerbation is the only sign of infection [ 400 ] . reduced peak expiratory volume in children is especially associated with detection of respiratory picornaviruses [ 379 ] . severe "wheezy bronchitis," a historical term describing an acute illness with preceding ari and characterized by cough, wheezing, breathlessness, and mucous production, was more often positive for a virus than mild disease [ 394 ] . even the use of culture found that hrvs predominated in both urt and lrt (sputum containing becs) or combined respiratory tract samples [ 394 ] . bacteria were often present with ifv, but not with hrvs [ 394 ] . the airway epithelial cells form a physical barrier in addition to their roles in immune surveillance and regulatory control [ 393 ] . however, the asthmatic bronchial epithelium is compromised by incomplete tight junctions that are more sensitive to airborne pollutants [ 401 ] and most likely to allergens and respiratory viral infections. this is further specifically disturbed by hrv infection which reduces expression levels of tight and adherens junction proteins [ 402 ] . in those with asthma, the presence of an hrv can induce illness that, while often more severe than in non-asthmatics, has been associated with signifi cantly different hrv load or duration of hrv rna detection in people with asthma compared to those without [ 403 ] . hrv-c types are often detected in more serious clinical outcomes than hrv-a or -b [ 265 ] although hospitalizations may be fewer for hrv-cs than the other species [ 404 ] . aom is diagnosed by middle ear effusion (otorrhea) with simultaneous signs and symptoms of ari including fever, earache, rhinitis, cough, sore throat, chest wheeze, nocturnal restlessness, irritability, poor appetite, diarrhea, and vomiting. transient abnormal (negative) ear pressure upon tympanometry occurs in two-thirds to three quarters of uncomplicated colds among healthy children [ 405 , 406 ] . aom is a frequent reason for outpatient antibiotic therapy which can reduce the time to resolution of symptoms in infants and has been attributed to reducing the overall hospital burden of aom [ 407 -412 ] . since a longitudinal day-care study in 1982, the association between aom and viral urt infection has been coalescing, and it is now clear that aom often occurs with or shortly after a viral ari, most frequently in the young and occurring more often during winter than summer [ 413 , 414 ] . the use of infl uenza vaccines reduced aom occurrence by a third during an epidemic period [ 415 ] , but the use of pneumococcal vaccine did not reduce the occurrence of aom overall, just that relatively small fraction (6 %) due to the target bacteria [ 416 ] . the isolation by culture and pcr detection of viruses from middle ear fl uids and the refractory nature of some aom cases to antibiotic therapies confi rmed that viruses play an important role in this illness [ 409 , 414 , 417 ] . studies relying on underperforming culture-based techniques underestimated the role for viral aris [ 414 , 418 ] , but other studies using pcr techniques and including hrvs found them to be the most frequently detected virus in middle ear fl uids and nasopharyngeal secretions [ 409 , 417 ] . the use of pcr has identifi ed respiratory viruses, most often hrvs, in nasal secretions of 50-70 % of children with aom [ 66 , 413 ] . because virus is often detected in the nasopharynx at the same time as the middle ear fl uid, the question of the relevance of a pcr positive is a valid one [ 419 ] . picornaviruses have been detected in 30 % of nasopharyngeal swabs taken during cold season from aom-prone infants and young children, and large quantities of hrv rna have been detected by in situ hybridization of adenoid tissues from 65 % of children with recurrent aom and/or adenoid hyperplasia [ 259 , 420 ] . in a cohort of children followed from 2 to 24 months and using culture-rt-pcr, hrvs in the urt were the second most frequent pathogens associated with aom, after hrsv [ 66 ] . viruses, most often hrvs (30.8 % of aom with ari), were also detected concurrently with non-ari periods associated with aom episodes (14 % of aom without ari) [ 418 ] suggesting that aom may be the only manifestation of some hrv aris, just as wheezing sometimes is. in the united states, 180 subjects were enrolled and followed in a birth cohort until the fi rst aom episode or between 6 and 12 months of age 362 ]. hrvs accounted for 55 % of viruses detected and 69 % of specimens with a single virus detected. this dominance was maintained even through the 2009 h1n1 infl uenza pandemic [ 362 ] . in the day-care aom study mentioned above, primary acquisition of streptococcus pneumoniae or haemophilus infl uenzae had minimal importance as an initiation factor for aom with effusion, but nasopharyngeal colonization was important [ 413 ] . animal studies have shown that virusbacteria interactions have a role in nasopharyngeal colonization and aom development [ 414 ] . positive correlation has been made between hrv detection in aom-prone children and moraxella catarrhalis infection as well as a tendency toward the copresence of streptococcus pneumoniae [ 259 ] . the presence of hrv-b14 was shown to increase adherence of s. pneumoniae in human tracheal epithelial cell cultures [ 421 ] . it is believed that these three bacterial pathogens can colonize without symptoms until a viral ari shifts the balance toward a cytokine-mediated infl ammatory state [ 419 ] . other diseases in which hrvs are often detected this disorder of older patients encompasses emphysema (alveolar destruction) and chronic bronchitis (large airway infl ammation with chronic mucous production) and describes a long-term obstruction to airfl ow in the lung (compared to asthma which is a reversible obstruction with normal fl ow between exacerbations). while bacteria are found in half of all exacerbations, antibiotic therapies have often yielded poor outcomes [ 422 ] . hrv infections result in more copd exacerbations (~66 % of cases [ 92 ] ) than any other virus identifi ed to date [ 422 , 423 ] . an experimental human model of hrv infection in copd provided preliminary evidence that hrvs cause exacerbations [ 286 ] . viral culture associated symptomatic hrv infections with exacerbations among chronic bronchitics, including cases of isolation from sputum (lrt sample) in the absence of hrv in the urt [ 424 ] . adding the measurement of an infl ammatory marker in the serum, like il-6, further improves the speed of predicting an infectious etiology for exacerbations of copd [ 425 ] . pneumonia is a disease that often occurs early in life, is responsible for millions of deaths each year [ 426 ] , and is caused by viral and/or bacterial infections. a diagnosis of pneumonia requires a radiologically confi rmed infl ammatory infi ltration of the lung tissue. childhood communityacquired pneumonia (cap) is common in developing countries [ 427 ] . cap also complicates existing chronic medical conditions and takes advantage of immunosenesence [ 428 ] . the role of hrvs in contributing to the development of bacterial pneumonia is likely underestimated [ 426 , 429 ] . determining an etiology is confounded by the rarity of obtaining lrt specimens, by short-term studies, and by the complex milieu of viruses and bacteria involved. less invasive sampling of the urt permits more routine sampling and screening, and so convenience and reduced risk have led to the detection of putative pathogens in the urt with the general assumption that they account for lrt disease, especially in children under the age of 5 years [ 430 ] . pneumonia studies are complicated by the lack of a suitable control group; sputum is not produced from the healthy lower airway and needle aspiration, while a gold standard is also a hospital procedure with some risk [ 431 ] . studies that are comprehensive and use sensitive molecular testing are also rare for the study of cap etiology. when used for cap investigations, pcr methods almost double the microbiological diagnoses over conventional culture and serology techniques, especially improving the identifi cation of mixed infections and fastidious viruses [ 432 ] . rapid diagnosis aids management and helps make decisions about treatment, while prolonged searching for an etiological agent leads to further invasive testing [ 256 , 433 ] . at least a quarter of clinical cap cases remain unsupported by microbiological fi ndings [ 241 , 432 ] . infections causing pneumonia vary with age and vaccination status [ 433 ] . viruses can be detected in up to 90 % of infants (1-12 months of age) with pneumonia, and these cases follow a seasonal pattern [ 427 , 433 ] . bacteria can also be detected in over 90 % of infants and older children, the elderly, and those with severe cap [ 432 , 434 ] . studies that predated the use of pcr pronounced hrsv, followed by hrvs, the major viral contributors to cap, with viruses comprising 27-72 % of childhood pneumonia cases [ 429 , 434 ] . in the pcr age, the role of hrvs has received increasing attention, and they are increasingly the major viral group detected from both urt and lrt (sputum) specimens of children with cap. this holds true even when studies extend across 1 or more years, which presumably would account for seasonal variation in virus prevalence [ 241 , 256 , 426 , 434 ] . it is suspected that viruses such as hrv prepare the way for subsequent bacterial infection in some direct or indirect fashion [ 65 , 259 , 420 , 435 ] . there are laboratory data which support this [ 436 ] as well as observational data showing a high proportion of hrvbacterial co-detections [ 256 , 437 ] . mixed infections including viruses are a possible cause of antibacterial treatment failure and sometimes a puzzle for physicians. mixed infections occur frequently in lrt diseases such as pneumonia, which is not surprising since new techniques make it clear that the lungs are not the sterile environments we once thought [ 427 , 434 , 438 , 439 ] . viral-bacterial coinfections can comprise 15 % of patients, while viral-viral (2-30 %) and bacterial-bacterial (1-7 %) are much less common [ 230 , 241 , 256 , 434 , 437 ] . hrsv or hrv is often co-detected with s. pneumoniae in urt samples [ 256 , 437 , 440 ] . hrv detections dominate in younger children with pneumonia during peak hrv seasons, although frequently in co-detections with other viruses [ 230 ] . acute bronchitis (less than 4-week duration in children) is defi ned as a sudden cough that often results from large airway infection and frequently involves viruses. croup or laryngotracheobronchitis (viral or recurrent [ 441 ] ) is a common lrt illness in children that includes the trachea and larynx as well as the larger airways, resulting in a barking cough. patients with croup most often have a viral infection with some role for hrvs, although the extent of this is unclear [ 441 , 442 ] . despite testing, a third of cases remain without a viral etiology [ 441 ] . tracheobronchitis resulted from some hrv-a15 infection of volunteers [ 46 ] . chest pain and cough have been reported in half or more of adults with hrv infection [ 207 ] as well as in children and adults with hrvs detected during exacerbations of bronchitis, with or without an associated ari [ 443 , 444 ] . bronchiolitis occurs seasonally, especially in winter, in infants (1-12 months of age), affecting the small peripheral bronchioles. winter is the peak season for hrsv circulation, but not usually for hrv. bronchiolitis is a clinical diagnosis encompassing various disease entities and is most often reported in association with detection of hrsv, a winter virus [ 445 , 446 ] . however, hrvs make up the majority of hrsv-negative bronchiolitis cases [ 128 ] , and hrvs are co-detected with hrsv for which hospitalization is prolonged compared to cases positive for either virus alone [ 447 ] . those children positive for an hrv during a clinically diagnosed bout of bronchiolitis have a signifi cantly higher risk of recurrent wheezing in the subsequent year than those in whom another virus is detected [ 446 ] . hrvs were reported in over fi vefold more cases of bronchiolitis than hrsv among patients in a 2-year prospective cohort of very low birth weight infants in buenos aires, argentina [ 448 ] . after a viral ari, some proportion of infections may be complicated by sinusitis (infl ammation of the sinus mucosa), the extent of which may be underestimated in children if the ari is mild and unattended by parents [ 11 ] . symptoms may include sinus pain, headache, facial pain, discolored nasal discharge, postnasal drip, cough, sore throat, malaise, and sometimes fever (more so in children) [ 367 , 449 ] . the precise role for viruses and bacteria in sinusitis is still unclear [ 450 ] . sinusitis is a common comorbidity in those with asthma [ 451 ] . the in situ presence of hrv-b14 rna in maxillary sinus epithelium was reported in seven of 14 adults with acute sinusitis [ 452 ] . hrvs were also detected by pcr in half of adults with acute maxillary sinusitis; half of the hrv positives were negative for any bacteria [ 65 ] . the common cold is often associated with computed tomographically confi rmed sinus cavity occlusion or abnormality in adults with self-diagnosed aris [ 367 , 453 ] . magnetic resonance imaging identifi ed reversible abnormalities of the paranasal sinuses in a third of healthy adult volunteers following challenge with hrv-a39 [ 454 ] . further evidence of the tropism of hrvs for sinus tissue comes from it being, so far, the only successful host for in vitro hrv-c replication [ 63 ] . culture-and serology-based testing has shown that virus infections in cystic fi brosis (cf) patients occur with the same prevalence as the general community, but the consequences of infection are more obvious or severe. these include deterioration of lung function, cough, increased expectoration and weight loss, and a synergistic increase in bacterial growth or acquisition of new bacterial infections [ 107 , 455 -458 ] . the mechanism behind the acquisition of new bacteria is still unknown and not always observed [ 459 ] , but may involve a reduction in the host's immune response or viral damage to the respiratory epithelium. there is circumstantial evidence that hrv infections have been associated with respiratory exacerbations in cystic fi brosis patients [ 459 , 460 ] , albeit in very low numbers by nonmolecular studies [ 461 ] and without a significantly different clinical outcome from non-hrv aris in these patients [ 107 ] . molecular methods have not yet been applied regularly, thoroughly, and systematically, but they generally fi nd hrvs to be prominent among cf children with ari-associated respiratory exacerbation and involved in mixed viral-bacterial infections [ 459 ] . hand washing and disinfectant wipes have been shown to be effective methods of interrupting transfer from fomites to the nose or to conjunctivae [ 87 , 288 , 293 ] . however, with eye rubbing, face touching, and nose-picking occurring frequently [ 47 , 290 ] , self-inoculation often outpaces personal hygiene, particularly in the young. hand disinfection is frequently recommended for prevention of hrv infection but has not been supported by controlled clinical trials in a natural setting [ 462 ] despite good results in experimental tests [ 463 ] . ethanol-containing disinfectants were more effective than simple hand washing with soap and water for removal of hrv-a39 inoculum, as assessed by culture, and the inclusion of organic acids afforded a residual antiviral effect [ 463 -465 ] . however, continual hand washing with extra ingredients resulted in skin irritation [ 462 ] . the experimental testing [ 463 , 464 ] may have been biased by short study periods, the absence of a mucus carrier to mimic natural surface deposition and overly stringent control over virus application/hand disinfection compared with the natural study. additionally, the natural setting study used pcr [ 462 ] which detects hrvs more often than culture. the disparity between outcomes may also refl ect the contribution of airborne hrv transmission. because of the absence of a vaccine or specifi c antiviral, the most popular method of intervention in uncomplicated hrv aris is treatment of the symptoms. this is achieved using analgesics, decongestants, antihistamines, and antitussives. due to a lack of studies, data are limited on the effectiveness of over-the-counter common cold medications for children [ 466 ] . anticholinergic agents have proven useful to reduce rhinorrhea [ 467 ] . for controlling symptoms in those with exacerbated asthma, most of which do not require hospitalization, bronchodilators and oral corticosteroids are the main treatments [ 468 ] . the interruption of proinfl ammatory immune responses or specifi c signaling pathways using steroids, or other novel therapeutics, may prove to be a more robust approach for treating hrv infections; they have not been successful for hrsv [ 325 ] . when initiated early in the illness, a combination of antiviral (ifn-α2b) and anti-infl ammatory (chlorpheniramine) components showed promise for interrupting nasal viral replication and symptoms [ 469 ] . antiviral agents (table 29. 3 ) require early application to effectively precede the pathogenic immune response to hrv infection [ 325 ] , but they often fail to reproduce their in vitro successes in vivo. most antirhinoviral drugs are based on capsid-binding agents (fig. 29.11 ) . additionally, oral delivery can complicate drug safety because this route increases the risk of systemic side effects compared to a nasal or topical route, but these risks must be considered alongside the disease to be treated; drug side effects are disproportionately severe compared to a common cold than to a severe asthma exacerbation. a systemic route is benefi cial if an effect is sought on hrv replication sites that are otherwise inaccessible, such as those not associated with respiratory tract illness [ 470 ] . the recent discovery of the new species, hrv-c, has shone a bright light on how little was known about the hrvs. the hrv-cs and also the newly discovered hrv-as and hrv-bs are fastidious in culture, with a single report of hrv-c growth in primary sinus tissue, and the identity of a cellular receptor still unknown. thus, it is diffi cult to proceed in many areas, including basic virology, seroepidemiology, immunobiology, and antiviral testing. determination of the receptors for these new hrvs would aid the search for a more accessible culture system. there would be great interest in a vaccine for some or all of the hrvs, but with increasing evidence of the interactions between hrvs, their hosts, and other respiratory viruses, it may not be wise to interfere before we fully understand what the impact of losing a constantly circulating hrv challenge would be. antivirals specifi cally targeting the hrvs may be a better bet, but routine hrv testing and genotyping will fi rst need to be more widespread as surveillance for antiviral resistance will be an important component of monitoring the success of any intervention. studies to determine whether there are differences in clinical and immunobiological impact between the many different types are lacking but would greatly improve our ability to plan future routine testing, understand all the clinical responses to the diverse hrvs and to outbreaks of ari, and improve hrv epidemiology. it is interesting to note that the hrv-bs are signifi cantly underrepresented in hrv detections. we do not yet know their niche or clinical impact. it may be possible that hrv-bs are the most well adapted of the hrvs, causing little to no detectable clinical impact, or they may create a different impact than that which we expect, or they may be a species in decline. the jury remains out on whether hrvs cause or are involved in the development of asthma or merely trigger exacerbations once asthma is established. with a very high healthcare impact from asthma around the world and atopic conditions that may be exacerbated by hrvs on the rise, this is an important area for further investigations. picornavirus infections: a primer for the practitioner who estimates of the causes of death in children picornavirus infections in children diagnosed by rt-pcr during longitudinal surveillance with weekly sampling: association with symptomatic illness and effect of season cost burden of viral respiratory infections: issues for formulary decision makers the cost of communitymanaged viral respiratory illnesses in a cohort of healthy preschoolaged children epidemiology of viral respiratory infections serial viral infections in infants with recurrent respiratory illnesses community-wide, contemporaneous circulation of a broad spectrum of human rhinoviruses in healthy australian preschool-aged children during a 12-month period morbidity and treatment in general practice in australia incidence of acute otitis media and sinusitis complicating upper respiratory tract infection: the effect of age rhinovirus replication causes rantes production in primary bronchial epithelial cells rhinoviruses infect the lower airways mechanism of rhinovirusinduced changes in airway smooth muscle responsiveness rhinovirus enters but does not replicate inside monocytes or airway macrophages the ssrna genome of human rhinovirus induces a type i ifn response but fails to induce maturation in human monocyte-derived dendritic cells observations on the incidence and distribution of the common cold in a rural community during 1948 and 1949 transmission of the common cold to volunteers under controlled conditions. iv. specifi c immunity to the common cold the common cold a short history of the common cold the medical features of the papyrus ebers the common cold cold wars: the fi ght against the common cold in pursuit of the common cold. london: william heinemann medical books limited propagation of common-cold virus in tissue cultures the cultivation in human-embryo cells of a virus (d.c.) causing colds in man rhinoviruses: basis for a numbering system. i. hela cells for propagation and serologic procedures rhinoviruses: basis for a numbering system. ii serologic characterization of prototype strains a collaborative report: rhinoviruses-extension of the numbering system a collaborative report: rhinoviruses -extension of the numbering system from 89 to 100 frequent detection of human rhinoviruses, paramyxoviruses, coronaviruses, and bocavirus during acute respiratory tract infections clinical features and complete genome characterization of a distinct human rhinovirus genetic cluster, probably representing a previously undetected hrv species, hrv-c, associated with acute respiratory illness in children distinguishing molecular features and clinical characteristics of a putative new rhinovirus species, human rhinovirus c (hrv c) ratifi cation vote on taxonomic proposals to the international committee on taxonomy of viruses ratifi cation vote on taxonomic proposals to the international committee on taxonomy of viruses rhinoviruses and common colds a cytopathogenic agent isolated from naval recruits with mild respiratory illnesses the isolation of a new virus associated with respiratory clinical disease in humans classifi cation of the "2060" virus as echo 28 and further study of its properties some virus isolations from common colds. i. experiments employing human volunteers a plaque method for assaying some viruses isolated from common colds problems in characterizing and identifying an apparently new virus found in association with mild respiratory disease in recruits relation between naturally acquired immunity and infectivity of two rhinoviruses in volunteers quantitative rhinovirus shedding patterns in volunteers short-duration exposure and the transmission of rhinoviral colds production of tracheobronchitis in volunteers with rhinovirus in a small-particle aerosol transmission of rhinovirus colds by self-inoculation reproducible plaquing system for rhinovirus serotypes in hela cells -agarose suspension micro-neutralization test for identifi cation of rhinovirus serotypes growth, plaque production and cationic stabilization of rhinovirus type 1 (echovirus 28) enhancement of rhinovirus plaque formation in human heteroploid cell cultures by magnesium and calcium an rna replication-center assay for high content image-based quantifi cations of human rhinovirus and coxsackievirus infections rhinoviruses replicate effectively at lower airway temperatures epidemiology of the common cold in military recruits with emphasis on infections by rhinovirus types 1a, 2, and two unclassifi ed rhinoviruses viruses as precipitants of asthma symptoms iii. rhinoviruses: molecular biology and prospects for future intervention characterization and classifi cation of echo 28-rhinovirus-coryzavirus agents rhinoviruses: a numbering system immunofl uorescence versus xtag multiplex pcr for the detection of respiratory picornavirus infections in children characterisation of a newly identifi ed human rhinovirus, hrv-qpm, discovered in infants with bronchiolitis a novel group of rhinoviruses is associated with asthma hospitalizations rhinovirus-induced modulation of gene expression in bronchial epithelial cells from subjects with asthma a diverse group of previously unrecognized human rhinoviruses are common causes of respiratory illness in infants molecular modeling, organ culture and reverse genetics for a newly identifi ed human rhinovirus c rhinovirus infections in an industrial population i. the occurrence of illness detection of rhinovirus in sinus brushings of patients with acute community-acquired sinusitis by reverse transcription-pcr epidemiology of documented viral respiratory infections and acute otitis media in a cohort of children followed from two to twenty-four months of age nested pcr for specifi c detection and rapid identifi cation of human picornaviruses improved detection of rhinoviruses in clinical samples by using a newly developed nested reverse transcription-pcr assay frequency and natural history of rhinovirus infections in adults during autumn a recently identifi ed rhinovirus genotype is associated with severe respiratory-tract infection in children in germany single versus dual respiratory virus infections in hospitalized infants: impact on clinical course of disease and interferon-gamma response communityacquired pathogens associated with prolonged coughing in children: a prospective cohort study a communitybased, time-matched, case-control study of respiratory viruses and exacerbations of copd detection of human rhinovirus rna in nasal washings by pcr real-time reverse transcription-pcr assay for comprehensive detection of human rhinoviruses rhinovirus viremia in children with respiratory infections high detection frequency and viral loads of human rhinovirus species a to c in fecal samples; diagnostic and clinical implications metagenomic analyses of viruses in stool samples from children with acute fl accid paralysis use of polymerase chain reaction for diagnosis of picornavirus infection in subjects with and without respiratory symptoms rhinovirus/enterovirus rna in tonsillar tissue of children with tonsillar disease quantifi cation of microorganisms: not human, not simple, not quick polymerase chain reaction amplifi cation of rhinovirus nucleic acids from clinical material new respiratory enterovirus and recombinant rhinoviruses among circulating strains typing of human rhinoviruses based on sequence variations in the 5′ non-coding region relationship of upper and lower airway cytokines to outcome of experimental rhinovirus infection incidence, etiology, and symptomatology of upper respiratory illness in elite athletes environmental contamination with rhinovirus and transfer to fi ngers of healthy individuals by daily life activity prevalence of viral respiratory tract infections in children with asthma rapid detection of human rhinoviruses in nasopharyngeal aspirates by a microwell reverse transcription-pcr-hybridization assay human picornavirus and coronavirus rna in nasopharynx of children without concurrent respiratory symptoms role of respiratory viruses in acute upper and lower respiratory tract illness in the fi rst year of life respiratory viruses, symptoms, and infl ammatory markers in acute exacerbations and stable chronic obstructive pulmonary disease rhinovirus identifi cation by bgl i digestion of picornavirus rt-pcr amplicons detection of rhinovirus, respiratory syncytial virus and coronavirus infections in acute otitis media by reverse-transcriptase polymerase chain reaction effi cacy and safety of oral pleconaril for treatment of colds due to picornaviruses in adults: results of 2 double-blind, randomized, placebo-controlled trials comparison of results of detection of rhinovirus by pcr and viral culture in human nasal wash specimens from subjects with and without clinical symptoms of respiratory illness early detection of acute rhinovirus infections by a rapid reverse transcription-pcr assay detection of rhinovirus and enterovirus in upper respiratory tract samples using a multiplex nested pcr polymerase chain reaction for human picornaviruses detection of respiratory syncytial virus, parainfl uenzavirus 3, adenovirus and rhinovirus sequences in respiratory tract of infants by polymerase chain reaction and hybridization detection of enteroviruses and rhinoviruses in clinical specimens by pcr and liquid-phase hybridization virological and serological analysis of rhinovirus infections during the fi rst two years of life in a cohort of children simultaneous detection of fourteen respiratory viruses in clinical specimens by two multiplex reverse transcription nested-pcr assays acute, chronic and persistent enterovirus and poliovirus infections: detection of viral genome by seminested pcr amplifi cation in culture-negative samples multicenter evaluation of a commercially available pcr assay for diagnosing enterovirus infection in a panel of cerebrospinal fl uid specimens improved detection of rhinoviruses in nasal and throat swabs by seminested rt-pcr effects of upper respiratory tract infections in patients with cystic fi brosis amplicon sequencing and improved detection of human rhinovirus in respiratory samples respiratory picornaviruses and respiratory syncytial virus as causative agents of acute expiratory wheezing in children detection of 12 respiratory viruses with two-set multiplex reverse transcriptase-pcr assay using a dual priming oligonucleotide system usefulness of published pcr primers in detecting human rhinovirus infection differential detection of rhinoviruses and enteroviruses rna sequences associated with classical immunofl uorescence assay detection of respiratory virus antigens in nasopharyngeal swabs from infants with bronchiolitis amplifi cation of rhinovirus specifi c nucleic acids from clinical samples using the polymerase chain reaction newly identifi ed human rhinoviruses: molecular methods heat up the cold viruses multicode-plx system for multiplexed detection of seventeen respiratory viruses diagnostic system for rapid and sensitive differential detection of pathogens masstag polymerasechain-reaction detection of respiratory pathogens, including a new rhinovirus genotype, that caused infl uenza-like illness in new york state during different cytokine profi le and eosinophil activation are involved in rhinovirus-and rs virus-induced acute exacerbation of childhood wheezing development of a respiratory virus panel test for detection of twenty human respiratory viruses by use of multiplex pcr and a fl uid microbead-based assay comparison of three multiplex pcr assays for the detection of respiratory viral infections: evaluation of xtag respiratory virus panel fast assay, respifinder 19 assay and respifinder smart 22 assay diagnostic performance of two highly multiplexed respiratory virus assays in a pediatric cohort molecular diagnosis of respiratory virus infections microarray-based detection and genotyping of viral pathogens characterization of the viral microbiome in patients with severe lower respiratory tract infections, using metagenomic sequencing rapid multiserotype detection of human rhinoviruses on optically coated silicon surfaces rapid and sensitive detection of respiratory virus molecular signatures using a silver nanorod array sers substrate proposals for the classifi cation of human rhinovirus species c into genotypically-assigned types prospective genotyping of human rhinoviruses in children and adults during the winter of 2009-2010 differential processing of nuclear pore complex proteins by rhinovirus 2a proteases from different species and serotypes replication of rhinovirus rna the structure and replication of rhinoviruses the eif4g-eif4e complex is the target for direct cleavage by the rhinovirus 2a proteinase eukaryotic initiation factor 4gii (eif4gii), but not eif4gi, cleavage correlates with inhibition of host cell protein synthesis after human rhinovirus infection human rhinovirus 14 infection of hela cells results in the proteolytic cleavage of the p220 cap-binding complex subunit and inactivates globin mrna translation in vitro inhibition of nuclear import and alteration of nuclear pore complex composition by rhinovirus rhinovirus 3c protease can localize in the nucleus and alter active and passive nucleocytoplasmic transport upper respiratory tract viral infection and mucociliary clearance els the complete nucleotide sequence of coxsackievirus a21 systematic nomenclature of picornavirus proteins the complete nucleotide sequence of a common cold virus: human rhinovirus 14 human rhinovirus 2: complete nucleotide sequence and proteolytic processing signals in the capsid protein region the nucleotide sequence of human rhinovirus 1b: molecular relationships within the rhinovirus genus genome-wide diversity and selective pressure in the human rhinovirus new complete genome sequences of human rhinoviruses shed light on their phylogeny and genomic features genetic clustering of all 102 human rhinovirus prototype strains: serotype 87 is close to human enterovirus 70 vp1 sequencing of all human rhinovirus serotypes: insights into genus phylogeny and susceptibility to antiviral capsid-binding compounds ref type: computer program) mega5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods from sneeze to wheeze: what we know about rhinovirus cs recombination in the evolution of picornaviruses molecular characterization of human rhinovirus fi eld strains isolated during surveillance of enteroviruses phylogenetic relationships and molecular adaptation dynamics of human rhinoviruses recombination and selection in the evolution of picornaviruses and other mammalian positive-stranded rna viruses recombination of poliovirus rna proceeds in mixed replication complexes originating from distinct replication start sites the mechanism of rna recombination in poliovirus sequencing and analyses of all known human rhinovirus genomes reveals structure and evolution analysis of genetic diversity and sites of recombination in human rhinovirus species c structure of a human common cold virus and functional relationship to other picornaviruses crystal structure of human rhinovirus serotype 1a (hrv1a) the structure of human rhinovirus 16 human rhinovirus 3 at 3.0 a resolution crystallization and preliminary x-ray analysis of human rhinovirus serotype 2 (hrv2) picornavirus-receptor interactions alignment of capsid protein vp1 sequences of all human rhinovirus prototype strains: conserved motifs and functional domains full-length human immunodefi ciency virus type 1 genomes from subtype c-infected seroconverters in india, with evidence of intersubtype recombination members of the low density lipoprotein receptor family mediate cell entry of a minor group common cold virus virus taxonomy: eighth report of the international committee in taxonomy of viruses the major and minor group receptor families contain all but one human rhinovirus serotype a probable new human picornavirus associated with respiratory diseases human rhinovirus 87 identifi ed as human enterovirus 68 by vp4-based molecular diagnosis classifi cation and nomenclature of viruses: ninth report of the international committee for the taxonomy of viruses clinical characteristics and genetic variability of human rhinovirus in mexico prevalence of and risk factors for human rhinovirus infection in health aboriginal and non-aboriginal western australian children 5′ noncoding region alone does not unequivocally determine genetic type of human rhinovirus strains some further virus isolations from common colds serotypes of viruses (rhinoviruses) isolated from common colds demonstration of dual rhinovirus infection in humans by isolation of different serotypes in human heteroploid (hela) and human diploid fi broblast cell cultures relationship of rhinovirus infection to mild upper respiratory disease rhinoviruses and respiratory disease viral cell recognition and entry human rhinovirus type 54 infection via heparan sulfate is less effi cient and strictly dependent on low endosomal ph many rhinovirus serotypes share the same cellular receptor isolation of a monoclonal antibody that blocks attachment of the major group of human rhinoviruses the major human rhinovirus receptor is icam-1 a cell adhesion molecule, icam-1, is the major surface receptor for rhinoviruses cell recognition and entry by rhino-and enteroviruses cdna cloning reveals that the major group rhinovirus receptor on hela cells is intercellular adhesion molecule 1 structural studies of two rhinovirus serotypes complexed with fragments of their cellular receptor interferon-gamma (ifngamma) down-regulates the rhinovirus-induced expression of intercellular adhesion molecule-1 (icam-1) on human airway epithelial cells human rhinovirus type 89 variants use heparan sulfate proteoglycan for cell attachment entry of a heparan sulphate-binding hrv8 variant strictly depends on dynamin but not on clathrin, caveolin, and fl otillin two groups of rhinoviruses revealed by a panel of antiviral compounds present sequence divergence and differential pathogenicity sequence analysis of human rhinoviruses in the rna-dependent rna polymerase coding region reveals within-species variation phylogenetic analysis of human rhinovirus capsid protein vp1 and 2a protease coding sequences confi rms shared genus-like relationships with human enteroviruses virus taxonomy. seventh report of the international committee for the taxonomy of viruses highly frequent infections with human rhinovirus in healthy young children: a longitudinal cohort study association between human rhinovirus c and severity of acute asthma in children challenges facing real-time pcr characterization of acute respiratory tract infections non-invasive sample collection for respiratory virus testing by multiplex pcr nasal swab versus nasopharyngeal aspirate for isolation of respiratory viruses studies with rhinoviruses in volunteers: production of illness, effect of naturally acquired antibody, and demonstration of a protective effect not associated with serum antibody rhinovirus transmission within families with children: incidence of symptomatic and asymptomatic infections rapid diagnosis of respiratory syncytial virus infections in immunocompromised adults epidemiology of the common cold patterns of illness in rhinovirus infections of military personnel rhinoviruses and respiratory illnesses in university students rhinovirus infections in an industrial population. iv. infections within families of employees during two fall peaks of respiratory illness epidemiology of infections with rhinovirus types 43 and 55 in a group of university of wisconsin student families the seattle virus watch. v. epidemiologic observation of rhinovirus infections, 1965-1969 in families with young children viruses in families spectrum of clinical illness in hospitalized patients with "common cold" virus infections epidemiology of acute lower respiratory disease in children spectrum of viruses and atypical bacteria in intercontinental air travelers with symptoms of acute respiratory infection incidence of respiratory viruses among travelers with a febrile syndrome returning from tropical and subtropical areas acute viral infections of upper respiratory tract in elderly people living in the community: comparative, prospective, population based study of disease burden acute respiratory illness in an american community: the tecumseh study etiology and clinical characterization of respiratory virus infections in adult patients attending an emergency department in beijing a modern miasma hypothesis and back-to-school asthma exacerbations seasonal variation in childhood asthma hospitalisations in finland, 1972-1992 descriptive epidemiology of asthma in trinidad, west indies the september epidemic of asthma hospitalization: school children as disease vectors distribution and seasonality of rhinovirus and other respiratory viruses in a crosssection of asthmatic children in trinidad, west indies respiratory infections in the american tropics viral etiologies of acute respiratory infections among hospitalized vietnamese children in ho chi minh city weekly monitoring of children with asthma for infections and illness during common cold seasons host and viral factors associated with severity of human rhinovirus-associated infant respiratory tract illness rhinovirus transmission one if by air, two if by hand viruses in communityacquired pneumonia in children aged less than 3 years old: high rate of viral coinfection evidence from multiplex molecular assays for complex multipathogen interactions in acute respiratory infections human metapneumovirus in severe respiratory syncytial virus bronchiolitis dual infection of infants by human metapneumovirus and human respiratory syncytial virus is strongly associated with severe bronchiolitis dual respiratory virus infections human metapneumovirus infection in young children hospitalized with respiratory tract disease increased h1n1 infection rate in children with asthma the association of newly identifi ed respiratory viruses with lower respiratory tract infections in korean children comparison of human metapneumovirus, respiratory syncytial virus and infl uenza a virus lower respiratory tract infections in hospitalized young children prevalence and clinical characteristics of human metapneumovirus infections in hospitalized infants in spain hmpv infections are frequently accompanied by co-infections etiology of communityacquired pneumonia in hospitalized school-aged children: evidence for high prevalence of viral infections the impact of dual viral infection in infants admitted to a pediatric intensive care unit associated with severe bronchiolitis rhinovirus infections of children in hospital; isolation of three possibly new rhinovirus serotypes do rhinoviruses reduce the probability of viral co-detection during acute respiratory tract infections? human bocavirus; multisystem detection raises questions about infection community epidemiology of human metapneumovirus, human coronavirus nl63, and other respiratory viruses in healthy preschool-aged children using parent-collected specimens rhinovirus-associated hospitalizations in young children a systematic approach to virus-virus interactions induction of endogenous interferon by use of standard live vaccines for prevention of respiratory viral infections potential use of nonpathogenic enteroviruses for control of human disease increased risk of noninfl uenza respiratory virus infections associated with receipt of inactivated infl uenza vaccine some virus isolations from common colds. ii. virus interference in tissue cultures recovery and characterization of non-cytopathogenic rhinoviruses the formation of interferon during acute respiratory virus infection of volunteers broad spectrum respiratory pathogen analysis of throat swabs from military recruits reveals interference between rhinoviruses and adenoviruses comprehensive detection of causative pathogens using real-time pcr to diagnose pediatric community-acquired pneumonia persistence of rhinovirus and enterovirus rna after acute respiratory illness in children prolonged shedding of rhinovirus and re-infection in adults with respiratory tract illness presence of viral and bacterial pathogens in the nasopharynx of otitis-prone children. a prospective study chronic rhinoviral infection in lung transplant recipients persistence of rhinovirus rna after asthma exacerbation in children human rhinoviruses: coming in from the cold rhinoviruses are a major cause of wheezing and hospitalization in children less than 2 years of age detection of viruses in human adenoid tissues by use of multiplex pcr clinical severity and molecular typing of human rhinovirus c strains during a fall outbreak affecting hospitalized patients human bocavirus detection in nasopharyngeal aspirates of children without clinical symptoms of respiratory infection a model for obtaining predictable natural transmission of rhinoviruses in human volunteers parenthood and host resistance to the common cold frequent detection of respiratory viruses without symptoms: toward defi ning clinically relevant cutoff values time lines of infection and disease in human infl uenza: a review of volunteer challenge studies the host response, not the virus, causes the symptoms of the common cold respiratory illness during winter: a cohort study of urban children from temperate australia predominance of rhinovirus in the nose of symptomatic and asymptomatic infants detection of human rhinovirus c viral genome in blood among children with severe respiratory infections in the philippines pneumonia and pericarditis in a child with hrv-c infection: a case report virological studies of sudden, unexplained infant deaths in glasgow 1967-1970 detection of human rhinovirus c in fecal samples of children with gastroenteritis localization of human rhinovirus replication in the upper respiratory tract by in situ hybridization sites of rhinovirus recovery after point inoculation of the upper airway shedding of infected ciliated epithelial cells in rhinovirus colds aerosol transmission of rhinovirus colds transmission of experimental rhinovirus colds in volunteer married couples correlation of rhinovirus load in the respiratory tract and clinical symptoms in hospitalized immunocompetent and immunocompromised patients association between interleukin-8 concentration in nasal secretions and severity of experimental rhinovirus colds an experimental model of rhinovirus induced chronic obstructive pulmonary disease exacerbations: a pilot study an investigation of the possible transmission of rhinovirus colds through indirect contact mechanisms of transmission of rhinovirus infections role of infectious secretions in the transmission of rhinovirus a study quantifying the hand-to-face contact rate and its potential application to predicting respiratory tract infection the complex epidemiology of respiratory virus infections studies of experimental rhinovirus type 2 infections in polar isolation and in england hand-to-hand transmission of rhinovirus colds exhalation of respiratory viruses by breathing, coughing, and talking a new method for sampling and detection of exhaled respiratory virus aerosols detection of airborne rhinovirus and its relation to outdoor air supply in offi ce environments detection of respiratory viruses on air fi lters from aircraft the cold case: are rhinoviruses perfectly adapted pathogens? ifn-g-induced protein 10 is a novel biomarker of rhinovirus-induced asthma exacerbations the type i interferon response during viral infections: a "swot" analysis antiviral innate immunity pathways type iii interferon (ifn) induces a type i ifn-like response in a restricted subset of cells through signaling pathways involving both the jak-stat pathway and the mitogen-activated protein kinases interferons and viruses: an interplay between induction, signalling, antiviral responses and virus countermeasures viral stress-inducible genes rhinovirus induces airway epithelial gene expression through double-stranded rna and ifndependent pathways the interferon response circuit: induction and suppression by pathogenic viruses il-29 and ifn-alpha regulate the expression of mxa, 2′,5′-oas and pkr genes in association with the activation of raf-mek-erk and pi3k-akt signal pathways in hepg2.2.15 cells the interferon-inducible rna helicase, mda-5, is involved in measles virus-induced expression of antiviral cytokines rig-i is cleaved during picornavirus infection human rhinovirus attenuates the type i interferon response by disrupting activation of interferon regulatory factor 3 attenuation of the type i interferon response in cells infected with human rhinovirus mda-5 is cleaved in poliovirus-infected cells interferons: signaling, antiviral and viral evasion sensing of rna viruses: a review of innate immune receptors involved in recognizing rna virus invasion viral infections and atopy in asthma pathogenesis: new rationales for asthma prevention and treatment innate and adaptive immune responses to viral infection and vaccination principles of intracellular viral recognition viruses and toll-like receptors human rhinovirus recognition in non-immune cells is mediated by toll-like receptors and mda-5, which trigger a synergetic pro-infl ammatory immune response toll-like receptor 7 function is reduced in adolescents with asthma modulation of the immune system by human rhinoviruses diversity in the bronchial epithelial cell response to infection with different rhinovirus strains host defense function of the airway epithelium in health and disease: clinical background the common cold at the turn of the millennium relationship of viral infections to wheezing illnesses and asthma innate and adaptive immune responses in asthma microrna-21 limits in vivo immune response-mediated activation of the il-12/ifn-gamma pathway, th1 polarization, and the severity of delayed-type hypersensitivity a defective type 1 response to rhinovirus in atopic asthma acute severe asthma innate immunity in the pathogenesis of virusinduced asthma exacerbations hay fever, hygiene, and household size family size, infection and atopy: the fi rst decade of the "hygiene hypothesis microbial manipulation of immune function for asthma prevention rhinovirus 3c protease precursors 3cd and 3cd' localize to the nuclei of infected cells human rhinovirus 2a proteinase cleavage sites in eukaryotic initiation factors (eif) 4gi and eif4gii are different rhinovirus 2a proteinase mediated stimulation of rhinovirus rna translation is additive to the stimulation effected by cellular rna binding proteins human major group rhinoviruses downmodulate the accessory function of monocytes by inducing il-10 rhinovirus-induced lower respiratory illness is increased in asthma and related to virus load and th1/2 cytokine and il-10 production interleukin-11: stimulation in vivo and in vitro by respiratory viruses and induction of airways hyperresponsiveness nasal-secretion leukocyte populations determined by fl ow cytometry during acute rhinovirus infection expression of intercellular adhesion molecule-1 (icam-1) in nasal epithelial cells of atopic subjects: a mechanism for increased rhinovirus infection? rhinovirus infection of human embryonic lung fi broblasts induces the production of a chemoattractant for polymorphonuclear leukocytes immune responses to viral infections: relevance for asthma the role of nasal secretion and serum antibody in the rhinovirus common cold an elisa for the detection of rhinovirus specifi c antibody in serum and nasal secretion analysis of nasal secretions during experimental rhinovirus upper respiratory infections further characterization of the local respiratory tract antibody response induced by intranasal instillation of inactivated rhinovirus 13 vaccine the time course of the humoral immune response to rhinovirus infection evaluation of an enzyme-linked immunosorbent assay that measures rhinovirusspecifi c antibodies in human sera and nasal secretions effect of rhinovirus 39 infection on cellular immune parameters in allergic and nonallergic subjects rhinovirus infections in tecumseh, michigan: frequency of illness and number of serotypes antibody to rhinovirus in human sera. ii. heterotypic responses homologous and heterologous resistance to rhinovirus common cold antigenic groupings of 90 rhinovirus serotypes antigenic variation among strains of rhinovirus type 51 hyper-antigenic variation occurs with human rhinovirus type 17 antigenic variation of rhinovirus type 22 isolation of rhinovirus intertypes related to either rhinoviruses 12 and 78 or 36 and 58 is a rhinovirus vaccine possible? allergens, viruses, and asthma exacerbations viruses and bacteria in the etiology of the common cold effect of the 2009 infl uenza a/h1n1 pandemic on viral respiratory infections in the fi rst year of life lower respiratory tract infection induced by a genetically modifi ed picornavirus in its natural murine host viruses as precipitants of asthma symptoms ii. physiology and mechanisms infl uenza vaccination: policy versus evidence: no gap between policy and evidence mechanisms of symptoms of the common cold and infl uenza mechanisms of the symptoms of rhinosinusitis kinins are generated in nasal secretions during natural rhinovirus colds european position paper on rhinosinusitis and nasal polyps european position paper on rhinosinusitis and nasal polyps 2012. a summary for otorhinolaryngologists development of a predictive index for picornavirus infections frequent detection of respiratory viruses by real-time pcr in adenoid samples from asymptomatic children rhinovirus infections in an industrial population. ii. characteristics of illness and antibody response prevalence of asthma symptoms and atopic disorders in preschool children and the trend over a decade acute exacerbations of asthma: epidemiology, biology and the exacerbation-prone phenotype gene-environment interactions in asthma viral infections and asthma inception rhinovirus infections more than a common cold community study of role of viral infections in exacerbations of asthma in 9-11 year old children respiratory viruses and exacerbations of asthma in adults rhinovirus and respiratory syncytial virus in wheezing children requiring emergency care virus-induced eosinophil mediator release requires antigen-presenting and cd4+ t cells the etiologic and epidemiologic spectrum of bronchiolitis in pediatric practice role of viral infection and host factors in acute episodes of asthma and chronic bronchitis the association of viral and bacterial respiratory infections with exacerbations of wheezing in young asthmatic children how viral infections cause exacerbation of airway diseases viruses as precipitants of asthma symptoms. i. epidemiology the childhood origins of asthma (coast) study viral infection in wheezy bronchitis and asthma in children viral specimen collection by parents increases response rate in populationbased virus studies rhinovirus illnesses during infancy predict subsequent childhood wheezing tlr3-and th2 cytokinedependent production of thymic stromal lymphopoietin in human airway epithelial cells role of viruses and bacteria in acute wheezy bronchitis in childhood: a study of sputum greater frequency of viral respiratory infections in asthmatic children as compared with their nonasthmatic siblings asthma exacerbations in children are associated with rhinovirus but not human metapneumovirus infection role of viral infections, atopy and antiviral immunity in the etiology of wheezing exacerbations among children and young adults synergism between allergens and viruses and risk of hospital admission with asthma: case-control study viruses as precipitants of asthmatic attacks in children viral respiratory infections in asthmatic children staying in a mountain resort defective epithelial barrier function in asthma rhinovirus infection-induced alteration of tight junction and adherens junction components in human nasal epithelial cells enhanced severity of virus associated lower respiratory tract disease in asthma patients may not be associated with delayed viral clearance and increased viral load in the upper respiratory tract prevalence and clinical characterization of a newly identifi ed human rhinovirus c species in children with acute respiratory tract infection effect of upper respiratory tract infection on eustachian tube ventilatory function in the preschool child the antiviral compound enviroxime targets the 3a coding region of rhinovirus and poliovirus importance of respiratory viruses in acute otitis media prevalence of various respiratory viruses in the middle ear during acute otitis media respiratory virus infection as a cause of prolonged symptoms in acute otitis media is acute otitis media a treatable disease? antimicrobials for acute otitis media? a review from the international primary care network a placebo-controlled trial of antimicrobial treatment for acute otitis media a longitudinal study of respiratory viruses and bacteria in the etiology of acute otitis media with effusion role of viruses in middle-ear disease infl uenza vaccination in the prevention of acute otitis media in children effi cacy of a pneumococcal conjugate vaccine against acute otitis media rhinovirus in acute otitis media temporal relationships between colds, upper respiratory viruses detected by polymerase chain reaction, and otitis media in young children followed through a typical cold season presence of viral nucleic acids in the middle ear: acute otitis media pathogen or bystander? rhinovirus in adenoid tissue effects of rhinovirus infection on the adherence of streptococcus pneumoniae to cultured human airway epithelial cells diagnosis of pathogens in exacerbations of chronic obstructive pulmonary disease overview of virus-induced airway disease virological studies in chronic bronchitis identifying viral infections in vaccinated chronic obstructive pulmonary disease (copd) patients using clinical features and infl ammatory markers. infl uenza other respi viruses viral pneumonia viral pneumoniae in children: incidence and aetiology community-acquired viral pneumonia childhood community-acquired pneumonia community-acquired pneumonia in children etiological diagnosis of childhood pneumonia by use of transthoracic needle aspiration and modern microbiological methods improved diagnosis of the etiology of community-acquired pneumonia with real-time polymerase chain reaction diagnosis and management of pneumonia in children viruses and bacteria in sputum samples of children with community-acquired pneumonia a study of pneumonia in a rural area in southern alabama respiratory viruses augment the adhesion of bacterial pathogens to respiratory epithelium in a viral species-and cell type-dependent manner etiology of communityacquired pneumonia in 254 hospitalized children atypical bacterial infections explained by a concomitant virus infection metagenomic analysis of respiratory tract dna viral communities in cystic fi brosis and non-cystic fi brosis individuals evolution of the df508 cftr mutation the viral aetiology of croup and recurrent croup croup: an 11-year study in a pediatric practice rhinovirus neutralizing antibody responses and their measurement rhinovirus infection in acute exacerbations of chronic bronchitis: a controlled prospective study respiratory syncytial virus, human bocavirus and rhinovirus bronchiolitis in infants rhinovirus bronchiolitis and recurrent wheezing: 1-year follow-up prospective multicenter study of viral etiology and hospital length of stay in children with severe bronchiolitis human rhinoviruses in severe respiratory disease in very low birth weight infants intranasal fl unisolide spray as an adjunct to oral antibiotic therapy for sinusitis respiratory illness caused by picornavirus infection: a review of clinical outcomes rhinitis, sinusitis, and asthma rhinovirus rna in the maxillary sinus epithelium of adult patients with acute sinusitis computed tomographic study of the common cold physiologic abnormalities in the paranasal sinuses during experimental rhinovirus colds viral infections of the respiratory tract in patients with cystic fi brosis respiratory infections in cystic fi brosis patients caused by virus, chlamydia and mycoplasma -possible synergism with pseudomonas aeruginosa clinical manifestations of exacerbations of cystic fi brosis associated with nonbacterial infections association of respiratory viral infections with pulmonary deterioration in patients with cystic fi brosis the role of respiratory viruses in cystic fi brosis rhinovirus c and respiratory exacerbations in children with cystic fi brosis infective respiratory exacerbations in young adults with cystic fi brosis: role of viruses and atypical microorganisms a randomized trial of the effi cacy of hand disinfection for prevention of rhinovirus infection effectiveness of hand sanitizers with and without organic acids for removal of rhinovirus from hands effi cacy of organic acids in hand cleansers for prevention of rhinovirus infections virucidal hand treatments for prevention of rhinovirus infection over-the-counter cold medications: a critical review of clinical trials between 1950 and 1991 ipratropium nasal spray: a new treatment for rhinorrhea in the common cold a 5-versus 3-day course of oral corticosteroids for children with asthma exacerbations who are not hospitalised: a randomised controlled trial combined antiviralantimediator treatment for the common cold in vitro antiviral activity and single-dose pharmacokinetics in humans of a novel, orally bioavailable inhibitor of human rhinovirus 3c protease treatment of picornavirus infections the common cold: control? uncommon(ly considered) manifestations of infection with rhinovirus, agent of the common cold intranasal interferon-a treatment of experimental rhinoviral colds human tolerance and histopathologic effects of long-term administration of intranasal interferon-a2 safety and effi cacy of intranasal pirodavir (r77975) in experimental rhinovirus infection combating enterovirus replication: state-of-the-art on antiviral research rhinovirus chemotherapy a comparison of the anti-rhinoviral drug binding pocket in hrv14 and hrv1a a new oral rhinovirus inhibitor bta798 human rhinovirus 3c protease as a potential target for the development of antiviral agents in vitro resistance studies of rupintrivir, a novel inhibitor of human rhinovirus 3c protease effi cacy of tremacamra, a soluble intercellular adhesion molecule 1, for experimental rhinovirus infection inhibitory effects of tiotropium on rhinovirus infection in human airway epithelial cells levofl oxacin inhibits rhinovirus infection in primary cultures of human tracheal epithelial cells pellino-1 selectively regulates epithelial cell responses to rhinovirus azithromycin induces antiviral responses in bronchial epithelial cells suggested reading newly identifi ed human rhinoviruses: molecular methods heat up the cold viruses do rhinoviruses reduce the probability of viral codetection during acute respiratory tract infections? human rhinoviruses: the cold wars resume proposals for the classifi cation of human rhinovirus species c into genotypically-assigned types cold wars: the fi ght against the common cold acknowledgments we wish to sincerely thank the following for valuable discussions: john upham, anne chang, danielle wurzel, michael nissen, ron turner james gern, and stephen b. liggett. we are grateful for the extreme patience of corin and ronan mackay, slightly less so for their frequent provision of our fi rsthand experience in hrv clinical symptoms. key: cord-003357-4qrg6lqu authors: wang, yingchen; dong, tuo; qi, guiyun; qu, lixin; liang, wei; qi, binbin; zhang, zhe; shang, lei; gao, hong; du, xiqiao; lu, bing; guo, yan; liu, zhenwei; yu, huisong; cui, qi; wang, xiaocen; li, ye; guo, weiyuan; qu, zhangyi title: prevalence of common respiratory viral infections and identification of adenovirus in hospitalized adults in harbin, china 2014 to 2017 date: 2018-11-27 journal: front microbiol doi: 10.3389/fmicb.2018.02919 sha: doc_id: 3357 cord_uid: 4qrg6lqu background: respiratory infections pose a great challenge in global health, and the prevalence of viral infection in adult patients has been poorly understood in northeast china. harbin is one of the major cities in northeast china, and more than half of any given year in harbin is occupied by winter. to reveal the viral etiology and seasonality in adult patients from harbin, a 4-year consecutive survey was conducted in harbin, china. methods: from january 2014 to december 2017, specimens were obtained from adult patients admitted to the second affiliated hospital of harbin medical university with lower respiratory tract infections. sputum samples were examined by direct immunofluorescence assays to detect seven common respiratory viruses, including influenza virus (type a and b), parainfluenza virus (type 1 to 3), respiratory syncytial virus and adenovirus. adenovirus positive samples were seeded onto a549 cells to isolate viral strains. phylogenetic analysis was conducted on the highly variable region of adenoviral hexon gene. results: a total of 1,300 hospitalized adult patients with lower respiratory tract infections were enrolled, in which 189 patients (14.5%) were detected as having at least one viral infection. the co-infection rate in this study was 25.9% (49/189). the dominant viral pathogen from 2014 to 2017 was parainfluenza virus, with a detection rate of 7.2%, followed by influenza virus, respiratory syncytial virus and adenovirus. based on the climate seasons determined by daily average temperature, the highest overall viral detection rate was detected in spring (22.0%, 52/236), followed by winter (13.4%, 109/813), autumn (11.4%, 13/114) and summer (10.9%, 15/137). adenovirus type 3 strains with slight variations were isolated from positive cases, which were closely related to the gb strain from the united states, as well as the harbin04b strain isolated locally. conclusion: this study demonstrated that common respiratory viruses were partially responsible for hospitalized lower respiratory tract infections in adult patients from harbin, china, with parainfluenza virus as the dominant viral pathogen. climate seasons could be rational indicators for the seasonality analysis of airborne viral infections. future surveillance on viral mutations would be necessary to reveal the evolutionary history of respiratory viruses. background: respiratory infections pose a great challenge in global health, and the prevalence of viral infection in adult patients has been poorly understood in northeast china. harbin is one of the major cities in northeast china, and more than half of any given year in harbin is occupied by winter. to reveal the viral etiology and seasonality in adult patients from harbin, a 4-year consecutive survey was conducted in harbin, china. methods: from january 2014 to december 2017, specimens were obtained from adult patients admitted to the second affiliated hospital of harbin medical university with lower respiratory tract infections. sputum samples were examined by direct immunofluorescence assays to detect seven common respiratory viruses, including influenza virus (type a and b), parainfluenza virus (type 1 to 3), respiratory syncytial virus and adenovirus. adenovirus positive samples were seeded onto a549 cells to isolate viral strains. phylogenetic analysis was conducted on the highly variable region of adenoviral hexon gene. results: a total of 1,300 hospitalized adult patients with lower respiratory tract infections were enrolled, in which 189 patients (14.5%) were detected as having at least one viral infection. the co-infection rate in this study was 25.9% (49/189). the dominant viral pathogen from 2014 to 2017 was parainfluenza virus, with a detection rate of 7.2%, followed by influenza virus, respiratory syncytial virus and adenovirus. based on the climate seasons determined by daily average temperature, the highest overall viral detection rate was detected in spring (22.0%, 52/236), followed by winter (13.4%, 109/813), autumn (11.4%, 13/114) and summer (10.9%, 15/137). adenovirus type 3 strains with slight variations were isolated from positive cases, which were closely related to the gb strain from the united states, as well as the harbin04b strain isolated locally. lower respiratory tract infections are a persistent public health problem, causing more than two million deaths per year worldwide, with a rate of 36 deaths per 100,000 population (gbd 2016 causes of death collaborators, 2017 . the morbidity and mortality of respiratory infections could be even worse in developing countries, including china. viral infections played an important role in pediatric lower respiratory tract infections, and the corresponding common viral pathogens were influenza a and b virus (iav and ibv), parainfluenza virus (piv, type 1 to 3), respiratory syncytial virus (rsv) and human adenovirus (adv) (pavia, 2011) . these seven viruses were also common in respiratory infections of the adult population in shandong province, china (liu et al., 2015) . to the best of our knowledge, there were no commercial vaccines available for most of these viruses, except for influenza a and b viruses (lambkin-williams et al., 2018) . the etiology of respiratory infection in the adult population has been overlooked, at least in china, although there are plenty of reports on the epidemiology of respiratory viral infection in the pediatric population (wang et al., 2016) . china is a vast country with notable variations in climate characteristics among different regions. due to the differences in socioeconomic, geographic or climate factors, the epidemiological features of viral infection in the respiratory tract show dramatic variation among different study populations (sloan et al., 2011; lu et al., 2013; he et al., 2014; rehder et al., 2015) . harbin is one of the major metropolises in northeast china, hosting more than eight million people. the winter in harbin lasts for more than half a year, while the summer is short, resulting in the length of four seasons being unequal. viral respiratory infections in the pediatric population from harbin were reported by the authors' team in 2009 (zhang et al., 2009) , in which the adult population was not included. a clear picture of the viral etiology in hospitalized adults with respiratory infections ought to be critical for both public health policy makers and clinical practitioners. phylogenetic history of several respiratory viruses isolated from china in recent years have been well established, including the influenza virus yang et al., 2018) , the piv (pan et al., 2017) and the rsv zou et al., 2016) . the information about adenoviral evolutionary history in china was limited to outbreak reports and novel strains identification (lu et al., 2014; li et al., 2018) , leaving a gap for circulating stains among adult patients. human adenovirus is one of the common pathogens in respiratory infections and could be divided into seven species (ismail et al., 2018) . the most recent reported human adenovirus type is type 85 in species d, which was isolated from japan (hashimoto et al., 2018) . human adenovirus species b type 55 (hadv b55) was reported to be responsible for adult pneumonia outbreaks in beijing china during 2011 with the genome identity as 99.87%, comparing to the prototype strain of hadv b55 in 2006 (cheng et al., 2018) . adenoviral infections in children from harbin were found to be adv species b, as reported by the authors, while the viral type among adult patients is still poorly understood. the adenoviral virion was composed of 252 capsomeres, containing 240 hexons and 12 pentons, while each penton was anchored by one fiber (or two fibers in several types) (nemerow et al., 2012) . the knob region in adenoviral fiber was responsible for cellular attachment through host receptors including car, cd46, dsg-1 and sialic acids (baker et al., 2018; lenman et al., 2018) . the rgd loop of penton could be recognized by the host cell's integrin (veesler et al., 2014) . the hexon protein is the major neutralizing antigen of adenoviruses (madisch et al., 2005) , while the hyper variable region (hvr) in hexon protein hosts its type specific epitopes reported by the authors' team (yuan et al., 2009) . experiments on chimeric adenoviral capsids showed that the replacement of hvrs in hexon could alter the viral serotypes (qiu et al., 2012; tian et al., 2018) . the hexon specific cd4 + and cd8 + t cells have been proved to be an effective treatment for human adenoviral infections in clinical settings (zandvliet et al., 2010; feucht et al., 2015) . recent reports indicated that antibodies against fiber knob or penton base may also be involved in the neutralization of adenoviruses tomita et al., 2018) . to the best of our knowledge, the hypervariable region of adenoviral hexon protein plays an essential role in viral type-specific immunity. in this report, the prevalence of common viruses in the lower respiratory tract infection of hospitalized adult patients from harbin, china was explored in hopes of revealing the clinical and pathogenic features of respiratory viruses. adenoviral strains were isolated from positive cases to identify potential molecular variations within its hypervariable regions. from january 2014 to december 2017, sputum samples were collected from 1,300 adult patients hospitalized for lower respiratory tract infections in the second affiliated hospital of harbin medical university. all patients enrolled in this study lived in urban and suburban areas of harbin without travel histories within 3 months before admission and sampling. all patients were older than 18 years and suffering from at least one complaint, including fever with body temperature ≥ 38 • c, cough, expectoration, hemoptysis, chest tightness (chest pain), or dyspnea. the sputum samples were expectorated spontaneously into sterile containers and delivered to the laboratory within 2 h, tested immediately or stored at −80 • c prior to use. patients' clinical data, including symptoms, history of illness, clinical diagnoses and laboratory test results were surveyed through the hospital's health information system. this study had been approved by the ethical review committees of harbin medical university in accordance with the declaration of helsinki. both informed and written consents were obtained from each participant who provided specimens. the presence of common respiratory viruses, including piv type 1 to 3, influenza virus a and b virus (iav and ibv), rsv, and human adv was determined by the d 3 ultra tm dfa respiratory virus screening kit (diagnostic hybrids, inc., athens, oh, united states). virus isolation for the adv-positive specimens was performed using the a549 cell line (provided by the cancer hospital of harbin medical university) following the protocol described previously (smith et al., 1986; huang et al., 2013) . in brief, cells seeded with clinical samples were incubated at 37 • c for 4 days. if there was no observed cytopathic effect, two additional blind passages of the culture were performed to avoid false negative results. the cultures with adv-like cytopathic effects were passaged again to confirm viral presence. the viral dna of human adv was extracted from infected cells using the axyprep multisource genomic dna miniprep kit (axygen biosciences) according to the manufacturer's instructions and eluted in 100 µl elution buffer. the highly variable region in the adenoviral hexon gene was amplified using the following primer pairs: forward, 5 -caattcactcgctccta-3 and reverse, 5 -gtggaaaggcacataacg-3 . all primers were synthesized by comate bioscience co., ltd. pcr was performed with the platinum pcr supermix (invitrogen) following the manufacturer's instructions. a total reaction volume of 50 µl contained 5 µl of 10 × pcr buffer (takara bio premix ex taq tm ), 200 nm of each primer, 200 nm of each dntps, 2 µl of each dna sample, 0.25 µl taq enzyme (takara bio premix ex taq tm ), and sterile water. the cycling conditions were 5 min at 95 • c, followed by 35 cycles of 30 s at 95 • c, 30 s at 55 • c, and 30 s at 72 • c, with a final 5-min extension at 72 • c. dna extracted from the a549 cell culture and sterile water were used as negative controls. the amplicons were bi-directionally sequenced using the sanger sequencing method with an abi prism 3130 genetic analyzer (comate bioscience co., ltd., jilin, china). the daily average air temperature from january 1st, 2014 to december 31st, 2017 in harbin city was kindly provided by the harbin meteorological observatory. historical temperature datasets covering 1981-2010 in harbin were accessible from the china meteorological data service center. climate seasons were determined based on the smoothed daily average temperature curve according to the chinese national standard no: qx/t 152-2012 (administration, 2012) . in brief, spring starts when the daily average temperature permanently rose above 10 • c, while summer comes when it rose above 22 • c permanently. the autumn starts when the daily temperature drops to 22 • c permanently and the winter begins at 10 • c. "permanently" means the daily temperature has remained above or below the threshold for at least five consecutive days. the details can be found in the supplementary table s1. statistical analysis was conducted using the r language (version 3.4.2). the prevalence (detection percentage) of viruses was calculated by dividing the sum of positive cases by the number of cases in total. chi-square tests or fisher's exact tests were selected for comparing the cross tables of categorical variables. the wilcoxon rank-sum or kruskal-wallis tests were chosen for continuous variable comparisons as appropriate. datasets were visualized by an excel spreadsheet. the quality of sequencing data was checked manually via chromas software version 2.4 (technelysium pty ltd., south brisbane, qld, australia), before contigs for each isolate were assembled using ugene (protsyuk et al., 2015) software suite (version 1.9) guided by the published hexon gene sequence of human adenoviruses. similar strains to the isolates in this report were found from the genbank database via the blast search method (boratyn et al., 2013) . fifty-eight nucleotide sequences of the hexon gene covering all seven species of human adv were selected as the reference in this study. before the tree building process, multiple sequences were aligned by muscle (edgar, 2004) software (version 3.6). the unrooted neighbor-joining tree was reconstructed using the kimura 2 parameters distance, whose robustness was tested by the bootstrap method with 1,000 replications implemented in mega (kumar et al., 2018) software (version 10.0.1). the parsimony tree was also built on the same dataset. in total, 1,300 hospitalized adult patients with respiratory infection were enrolled from january 2014 to december 2017 in harbin city. among these patients, 710 were male and 590 were female, as shown in bronchiectasis, 20 with asthma and 51 with other infections. some clinical signs were more frequent among cases with certain diagnoses. in all, 370 (66.5%) pneumonia cases complained of fever, while only 185 (37.3%) cases with bronchitis had fever (p < 0.001). cough was the chief complaint of the bronchial infections (p < 0.001), including bronchitis (56.0%), copd (55.5%) and asthma (60.0%). out of 1,300 enrolled patients and 583 were diagnosed with chronic diseases, including chronic respiratory disease, cancer, diabetes mellitus, chronic cardiac diseases, cerebrovascular disease, chronic kidney disease, and other chronic diseases lasting more than 1 year. among the 1,300 total patients, 189 (14.5%) cases were determined to be positive with at least one of seven viruses, including influenza virus (a and b), piv (type 1, 2, and 3), rsv and adv. one hundred and forty patients were infected with only one virus as the single infection, and 49 patients were co-infected with more than one virus. double infections were observed in 43 cases, while triple infection was found in six cases. as shown in table 2 , the most frequent single infection was piv (59 cases) and the major double infection was combined influenza and parainfluenza viruses (17 cases). the details were accessible from the supplementary table s2 . the median age of the viral positive group was 63 (iqr 54-69) and the negative group was 61 (iqr 51-71). the age difference between the viral infection positive and negative group was not statistically significant (p = 0.730). the viral detection rate varied from 16.6% (108/652) in the middle age group to 11.9% (43/360) in the senior citizen group, but the difference was not statistically significant (p = 0.104). there was no significant difference in the viral infections between genders (p = 0.902). the virus detection rate among the study years varied significantly (p = 0.017) from 18.6% (52/280) in 2015 to 9.7% (29/299) in 2014, as shown in table 1 . the detection rate was not equal among calendar seasons (p = 0.002) or climate seasons (p = 0.003). based on the climate seasons, the highest overall detection rate was in spring (22.0%, 52/236), followed by winter (13.4%, 109/813), autumn (11.4%, 13/114), and summer (10.9%, 15/137). for individual virus positive samples, certain viruses were more frequently detected in spring than in other seasons, including influenza virus (8.9%, 21/236), piv (9.7%, 23/236), and rsv (9.3%, 22/236), as shown in table 3 . adv was more frequently detected in winter, with a detection rate of 1.8% (15/813). piv was the dominant viral pathogen during the study period, accounting for 65.5% (19/29) of the viral infection cases in 2014, 40.3% (21/52) in 2015, 54.7% (29/53) in 2016, and 43.6% (24/55) in 2017. as shown in table 3 , viral co-infections were more not equally distributed among seasons, with the highest ratio in spring at 7.2% (17/236, p = 0.022). flu piv rsv adv cases total 189 overall detected respiratory viral positive cases were plotted against daily average air temperature in figure 1 . the low temperature over days was a strong indicator for respiratory viral infections. positive cases were rare in summer, which began late in june and ended in mid of august. one exception was found in the summer in 2017 which was related to a sudden drop of air temperature from 27.2 • c on 18th july to 18.8 • c on 27th july 2017. the overall percentage of viral infection was significantly connected with clinical diagnoses (p = 0.025), as shown in table 1 . the highest detection rate was observed in the pneumonia group (17.8%, 99/556), followed by bronchitis (13.9%, 69/496), while the lowest detection rate was found in the asthma group (5%, 1/20). chest tightness was the only symptom that showed statistically significant difference (p = 0.011) between the positive (38.1%, 72/189) and negative (48.1%, 534/1111) group of viral infections. the respiratory viral infection did not prolong hospital stay statistically (p = 0.306), although the median days in the hospital for the positive viral infection group was 12 (iqr 9-17), which was shorter than 13 in the negative group (iqr 9-17). overall, the viral detection rate was relatively lower in the chronic diseases group (11.7% vs. 16.9%), with statistical significance (p = 0.008). detection rates varied among different chronic diseases, but the difference was not significant (p = 0.467). the viral co-infection ratio among different diagnosed groups was not significant (p = 0.372). two strains of human adv were isolated from two patients diagnosed with pneumonia, respectively. the harbin17b strain ( came from a male patient aged 69 whose samples were collected in december 2017. the other strain, named harbin17a, was isolated from a male patient aged 24 whose samples were collected in january 2017. based on the evolutionary tree reconstruction of the hexon gene sequencing data, both strains isolated in this study were identified as human adv species b type 3. these two strains were nearly identical, with the closest evolutionary distance to the strain gb (genbank: ay599834; atcc: vr-3) from the united states as well as the harbin04b strain from a previous local epidemic, as shown in figure 2 . the parsimony tree was similar in topology with neighborjoining tree (figure not shown in the article). multiple sequence alignments indicated that, compared with the gb strain, the harbin17a and harbin17b strain shared a variable site in the 482nd codon (gb numbering) of the hexon gene hosting an a to c mutation, resulting in threonine (t) to proline (p) mutation of the amino acid sequences. the harbin04b strain had a threonine on the same site in the hexon protein encoded by the codon acu, which was identical to the harbin17a and harbin17b strain. adult populations with respiratory infections bear heavy burdens of hospitalization due to severe respiratory illness, especially for senior citizens (muller-pebody et al., 2006) . the constantly changing nature of viral pathogens has made the surveillance of these infections critical to public health authorities (zou et al., 2016; li et al., 2017) . the key indicators for viral infections from this report and recent published surveys in china has been summarized in table 4 . the overall detection rate of viral infection among hospitalized adult patients in this report is 14.5%, which was consistent with the result of 16.8% in the age group above 14 years old by a national survey from 2009 to 2013 in china (feng et al., 2014) . the dominant viral pathogen throughout this study period was piv, with a detection rate of 7.2%, followed by influenza virus. the national survey on hospitalized patients with respiratory infection in china from 2009 to 2013 showed that influenza virus had the highest detection rate of 8.0% in adult patients. the detection rate of respiratory viruses in the over 16 years age group was 24.2% in shandong province of china (liu et al., 2015) and was dominated by influenza virus (8.7%) from 2011 to 2013. from 2012 to 2015, the respiratory viral detection rate in hospitalized patients above 14 years old within beijing and shandong china was reported to be 38.9%, in which influenza virus had the highest frequency of 8.0% (yu et al., 2018) . compared with recent surveys in beijing, shandong and other regions in china, this report showed that the piv dominated epidemic seasons from 2014 to 2017 in adult patients in harbin, which should be important for preparing future epidemic control strategies in public health. the viral infection rate within asthma subgroup of this report was 5%, and it was one patient with rsv among 20 asthma patients in total as shown in table 3 . asthma is related to viral infection, while the detection rates varies among different pathogens. a recently published meta-analysis showed that the mean prevalence of rsv, influenza virus, piv and adv was 13.6%, 10%, 5.6%, and 3.8%, respectively (zheng et al., 2018) . the apparently low detection rate within asthma group in this report could be possibly caused by the sampling bias due to the limited subgroup size. air temperature was a major meteorological variable associated with respiratory viral infection (chan et al., 2015) . harbin city is located at 46 degrees north latitude with an annual average temperature of 4.9 degrees celsius. climate seasons are not in equal length in harbin, according to historical statistics from 1981 to 2010. the longest season in harbin is winter, which starts on the 4th of october and lasts 201 days, followed by spring on the 23rd of april with 61 days. starting on the 23rd of june, summers in harbin are relatively short at 53 days, while autumn is 50 days and is the shortest season, starting on the 15th of august. in routine epidemiological analysis, 12 months in a year were usually divided into four seasons equally, which is not the case in harbin and possibly compromises contagious disease seasonality prediction. based on climate seasons defined by the daily average air temperature, influenza, parainfluenza and rsv detected in this report all presented significant seasonal peaks in spring, although similar phenomena were absent within the calendar seasons. sputum samples were acceptable both in viral culture and antigen detection in respiratory infections compared with nasopharyngeal swabs and aspirates (covalciuc et al., 1999) , although they were not frequently collected in recent surveys on lower respiratory tract infected adult patients from china. detection of respiratory virus in sputum samples has been proven effective on a small scale, either by the immunofluorescence method (honkinen et al., 2012) or the pcr method (branche et al., 2014) . this report provided a medium scale test on the validity or effectiveness of respiratory viral detection from sputum samples with 1,300 cases. in this report, two nearly identical strains were isolated from pneumonia patients in harbin, possessing high similarity with the local strain harbin04b and the strain gb. the harbin04b strain was isolated from a pediatric patient in harbin (zhang et al., 2009) . adv strain gb was named vr-3 in the atcc database, which was isolated from an adult patient with common cold in maryland, united states in 1953 (huebner et al., 1954) . the similarity of these strains across asian and american continents through several decades may result from the globalization movement in modern china. it could also be explained by another hypothesis based on the evolution history of adenoviruses. on the geological scale, homo sapiens inherit human adenoviruses from our ancestors within the hominidae, resulting in an evolutionary rate of human adv as low as 2.8e-8 substitution per site per year (hoppe et al., 2015) . considering its 36 kbp genome, we would have to wait a millennium (or 992 years exactly) before a single site mutation within an adenoviral genome occurred naturally. there were a few limitations to this study. first, the 4 years of investigation on a single city in this present report was relatively short and much more localized than other long-term or national surveillance, which may lead to a bias of the viral etiology. second, the enrolled population in this study consisted of adult patients only, while the samples from pediatric patients were not collected simultaneously. the comparison of the viral prevalence status between adult and pediatric populations in this study was difficult. third, the bacterial culture results of the clinical samples were not included in this report, which prevents us from drawing a comprehensive picture on the pathogens associated with lower respiratory tract infections. in this report, 14.5% of lower respiratory tract infections in hospitalized adults from harbin were found to be related with common respiratory viruses. the piv dominated viral infection from 2014 to 2017 with detection rates of 7.2%. climate seasons based on daily temperature records were found to be reliable in seasonality analysis. adv type 3 strains with slight variations were isolated from positive cases. further surveillance would be important to continuously monitoring the viral etiology in adult patients both local and abroad. zq, yw, and td designed this study and prepared the manuscript. gq, yg, and wg collected the specimens. yw, td, and yg conducted the epidemiological investigation. td, lq, wl, bq, zz, ls, hg, xd, bl, zl, hy, qc, xw, and yl performed the laboratory experiments. yw and td analyze the data and made the figures and tables. all authors reviewed the manuscript. division of climatic season designer oncolytic adenovirus: coming of age blast: a more efficient report with usability improvements detection of respiratory viruses in sputum from adults by use of automated multiplex pcr hospitalization incidence, mortality, and seasonality of common respiratory viruses over a period of 15 years in a developed subtropical city comparative genomic analysis of re-emergent human adenovirus type 55 pathogens associated with adult severe community-acquired pneumonia reveals conserved genomes and capsid proteins comparison of four clinical specimen types for detection of influenza a and b viruses by optical immunoassay (flu oia test) and cell culture methods muscle: multiple sequence alignment with high accuracy and high throughput viral etiologies of hospitalized acute lower respiratory infection patients in china adoptive t-cell therapy with hexon-specific th1 cells as a treatment of refractory adenovirus infection after hsct global, regional, and national age-sex specific mortality for 264 causes of death, 1980-2016: a systematic analysis for the global burden of disease study 2016 recombinant type human mastadenovirus d85 associated with epidemic keratoconjunctivitis since 2015 in japan a 3-year prospective study of the epidemiology of acute respiratory viral infections in hospitalized children in shenzhen viruses and bacteria in sputum samples of children with community-acquired pneumonia multiple cross-species transmission events of human adenoviruses (hadv) during hominine evolution outbreak of febrile respiratory illness associated with human adenovirus type 14p1 in gansu province adenoidal-pharyngeal-conjunctival agents: a newly recognized group of common viruses of the respiratory system genomic analysis of a large set of currently-and historically-important human adenovirus pathogens mega x: molecular evolutionary genetics analysis across computing platforms the human viral challenge model: accelerating the evaluation of respiratory antivirals, vaccines and novel diagnostics polysialic acid is a cellular receptor for human adenovirus 52 a swimming pool-associated outbreak of pharyngoconjunctival fever caused by human adenovirus type 4 in beijing, china genetic characterization of continually evolving highly pathogenic h5n6 influenza viruses in china genetic variation of human respiratory syncytial virus among children with fever and respiratory symptoms in shanghai viral etiology of acute respiratory tract infections in hospitalized children and adults in shandong province epidemiology of human adenovirus and molecular characterization of human adenovirus 55 in china epidemiology of human respiratory viruses in children with acute respiratory tract infections in jinan, china phylogenetic analysis of the main neutralization and hemagglutination determinants of all human adenovirus prototypes as a basis for molecular classification and taxonomy modelling hospital admissions for lower respiratory tract infections in the elderly in england structure of human adenovirus human parainfluenza virus infection in severe acute respiratory infection cases in beijing, 2014-2016: a molecular epidemiological study. influenza other respir viral infections of the lower respiratory tract: old viruses, new viruses, and the role of diagnosis shared bioinformatics databases within the unipro ugene platform serotype-specific neutralizing antibody epitopes of human adenovirus type 3 (hadv-3) and hadv-7 reside in multiple hexon hypervariable regions detection of multiple respiratory viruses associated with mortality and severity of illness in children impact of pollution, climate, and sociodemographic factors on spatiotemporal dynamics of seasonal respiratory viruses identification of a critical and conformational neutralizing epitope in human adenovirus type 4 hexon antibodies against adenovirus fiber and penton base proteins inhibit adenovirus vector-mediated transduction in the liver following systemic administration single-particle em reveals plasticity of interactions between the adenovirus penton base and integrin alphavbeta3 prevalence of respiratory viruses among children hospitalized from respiratory infections in shenzhen assessment of human-to-human transmissibility of avian influenza a(h7n9) virus across five waves by analyzing clusters of case-patients in mainland china variation in influenza b virus epidemiology by lineage characteristics of neutralizing antibodies to adenovirus capsid proteins in human and animal sera comparison of the prevalence of respiratory viruses in patients with acute respiratory infections at different hospital settings in north china molecular modeling and epitopes mapping of human adenovirus type 3 hexon protein combined cd8+ and cd4+ adenovirus hexon-specific t cells associated with viral clearance after stem cell transplantation as treatment for adenovirus infection respiratory viruses in hospitalized children with acute lower respiratory tract infections in harbin regional, age and respiratory-secretion-specific prevalence of respiratory viruses associated with asthma exacerbation: a literature review evolution and transmission of respiratory syncytial group a (rsv-a) viruses in guangdong we gratefully acknowledge the harbin meteorological observatory and the china meteorological data service center for providing air temperature datasets in this report. the supplementary material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmicb. 2018.02919/full#supplementary-material key: cord-003917-bswndfvk authors: lalle, eleonora; biava, mirella; nicastri, emanuele; colavita, francesca; di caro, antonino; vairo, francesco; lanini, simone; castilletti, concetta; langer, martin; zumla, alimuddin; kobinger, gary; capobianchi, maria r.; ippolito, giuseppe title: pulmonary involvement during the ebola virus disease date: 2019-08-24 journal: viruses doi: 10.3390/v11090780 sha: doc_id: 3917 cord_uid: bswndfvk filoviruses have become a worldwide public health concern, especially during the 2013–2016 western africa ebola virus disease (evd) outbreak—the largest outbreak, both by number of cases and geographical extension, recorded so far in medical history. evd is associated with pathologies in several organs, including the liver, kidney, and lung. during the 2013–2016 western africa outbreak, ebola virus (ebov) was detected in the lung of infected patients suggesting a role in lung pathogenesis. however, little is known about lung pathogenesis and the controversial issue of aerosol transmission in evd. this review highlights the pulmonary involvement in evd, with a special focus on the new data emerging from the 2013–2016 ebola outbreak. ebolavirus is part of the filoviridae family, which consists of three genera: marbugvirus, cuevavirus, and ebolavirus. there are currently six known, genetically distinct, species of ebolavirus-ebola virus (ebov), sudan ebolaviurs (sudv), tai forest ebolavirus (tafv), bundibugyo ebolavirus (bdbv), reston ebolavirus (restv), and bombali ebolavirus (bomv) [1, 2] . no virus has triggered fear in the general population more than the filovirus ebolavirus [3] . ebov is categorized among the deadliest viruses, with mortality rates up to 90%. the zoonotic origin of outbreaks are often the result of transmission from primates, although the suspected natural reservoir for ebov, bats, is still being questioned. since it was first identified in 1976 in zaire (the actual democratic republic of congo), 27 confirmed outbreaks, mainly in the central part of africa, have occurred, and each outbreak was accompanied by high case fatality rates up to 88%, including the new declared outbreak ongoing in the north kivu province of the democratic republic of the congo [4] [5] [6] . the 2013-2016 ebola outbreak is the largest (both by number of cases and geographical extension) ebolavirus outbreak ever reported, resulting . tlr3 stimulates irf-7 and nf-κb via myd88 activation, leading to the release of proinflammatory cytokines and the production of ifn-α, -β, and -λ, respectively. secretion of proinflammatory cytokines and chemokines activate the immune system, through recruitment of eosinophils, neutrophils, macrophages, dendritic cells, t cells, and nk cells. most respiratory viruses have developed strategies to escape antiviral defense, mainly by interfering with the ifn system or by affecting the epithelium barrier, with the consequence of a loss of integrity and protection. furthermore, respiratory viruses can perturb (skewed or exaggerated) inflammatory responses and production of soluble mediators. ebov infection is acquired through direct contact with bodily fluids. the virus enters blood circulation through breaks in the skin and mucosa and spread to different organs, causing systemic manifestation of cardiovascular, coagulation, or inflammatory disturbances [10] . the terminal stages of evd usually involve massive tissue injury and hemorrhage, resulting in multiorgan failure and shock, the main cause of exitus in evd patients [13] . during evd, respiratory symptoms such as chest pain, shortness of breath, cough, and nasal discharge are signals of the multisystem involvement, but, so far, lung damage has not been directly linked to ebov replication in the respiratory tract. however, new evidences collected during the recent 2013-2016 ebola outbreak hypothesized shedding of the virus in the lung and identified viral replication markers in sputum samples collected from ebov infected patients [14] . on the other hand, the high virulence of ebov is attributed in large part to the ability of this virus to interfere with the host immune response, and the high degree of variation in lung pathogenesis is usually linked to indirect damage due to endothelial and epithelial inflammation and the hyper-activation of the immune system subsequent to ebov infection. in fact, viral direct damages are always associated with indirect damages, caused by inflammatory and immune reactions elicited by the viruses through the activation of soluble mediators (cytokines and chemokines) as part of the immune response ( figure 1 ). the acute inflammation process is characterized by increasing blood flow, which enables plasma and leukocytes to reach extra-vascular sites of injury. even though inflammation may be often restored, in evd, severe inflammation is associated with a cytokine storm and more serious pathological changes are observed. for instance, ebov in vitro infection of monocytes and macrophages triggers the robust expression of inflammatory mediators, including il-1β, il-6, il-8, mip-1a, mip-1β, mcp-1, and tnf-α [15, 16] , whereas the dysregulation of immune mediators in humans has been associated with the secretion of other inflammatory mediators, such as il-1β, il-8, ccl2, ccl3, ccl13, cxcl1, cxcl10, cxcl11, cxcl12, il6, mif, spp [17] [18] [19] . in addition, severe inflammatory upon entrance into the cell, viruses are recognized by the toll-like receptor (tlr) on either cell membrane or in endosomes. tlrs activate interferon regulatory factors (irfs) leading to ifn-α and ifn-β release via the toll/il-1 receptor domain-containing adaptor (trif). tlr3 stimulates irf-7 and nf-κb via myd88 activation, leading to the release of proinflammatory cytokines and the production of ifn-α, -β, and -λ, respectively. secretion of proinflammatory cytokines and chemokines activate the immune system, through recruitment of eosinophils, neutrophils, macrophages, dendritic cells, t cells, and nk cells. most respiratory viruses have developed strategies to escape antiviral defense, mainly by interfering with the ifn system or by affecting the epithelium barrier, with the consequence of a loss of integrity and protection. furthermore, respiratory viruses can perturb (skewed or exaggerated) inflammatory responses and production of soluble mediators. ebov infection is acquired through direct contact with bodily fluids. the virus enters blood circulation through breaks in the skin and mucosa and spread to different organs, causing systemic manifestation of cardiovascular, coagulation, or inflammatory disturbances [10] . the terminal stages of evd usually involve massive tissue injury and hemorrhage, resulting in multiorgan failure and shock, the main cause of exitus in evd patients [13] . during evd, respiratory symptoms such as chest pain, shortness of breath, cough, and nasal discharge are signals of the multisystem involvement, but, so far, lung damage has not been directly linked to ebov replication in the respiratory tract. however, new evidences collected during the recent 2013-2016 ebola outbreak hypothesized shedding of the virus in the lung and identified viral replication markers in sputum samples collected from ebov infected patients [14] . on the other hand, the high virulence of ebov is attributed in large part to the ability of this virus to interfere with the host immune response, and the high degree of variation in lung pathogenesis is usually linked to indirect damage due to endothelial and epithelial inflammation and the hyper-activation of the immune system subsequent to ebov infection. in fact, viral direct damages are always associated with indirect damages, caused by inflammatory and immune reactions elicited by the viruses through the activation of soluble mediators (cytokines and chemokines) as part of the immune response ( figure 1 ). the acute inflammation process is characterized by increasing blood flow, which enables plasma and leukocytes to reach extra-vascular sites of injury. even though inflammation may be often restored, in evd, severe inflammation is associated with a cytokine storm and more serious pathological changes are observed. for instance, ebov in vitro infection of monocytes and macrophages triggers the robust expression of inflammatory mediators, including il-1β, il-6, il-8, mip-1a, mip-1β, mcp-1, and tnf-α [15, 16] , whereas the dysregulation of immune mediators in humans has been associated with the secretion of other inflammatory mediators, such as il-1β, il-8, ccl2, ccl3, ccl13, cxcl1, cxcl10, cxcl11, cxcl12, il6, mif, spp [17] [18] [19] . in addition, severe inflammatory cytokines/chemokines may spill over into the circulation and result in systemic cytokine storms, which are responsible for multi-organ dysfunction and for the impairment of the vascular system and disseminated intravascular coagulation [20, 21] . dendritic cells (dcs) play an essential role in the link between the innate and adaptive immune response, and their maturation is essential for the correct functionality of dcs, such as the migration, processing, and presentation of viral antigens to t-and b-cells for their activation and correct viral clearance [22, 23] . ebov infection has been shown to influence these mechanisms through impairment of dcs in upregulating co-stimulatory molecules (cd40, cd86, and cd80) and major histocompatibility complex (mhc) class ii, as well as soluble chemokines and cytokines [24] . ebov infection is also able to influence the adaptive immune response: severe lymphopenia and the destruction of lymphoid tissue is one of the hallmarks of ebov infection. fatal cases showed a more marked reduction of nk cells and γδ t-cell frequency, as well as a loss of peripheral blood cd4+ and cd8+ t cells [25, 26] . moreover, a recent study showed that patients with fatal outcome presented lower, or often absent, levels of both ebov-specific igm and igg, which, when detected, appeared later than in survivors [19] . overall, the alteration of the innate and adaptive response explains the paralysis of the immune system and its inability to initiate and maintain a protective immune response. at the pulmonary level, many of the pathological changes are, in fact, secondary to systemic alterations, correlating with general pathogenic mechanisms, which are the major causes of severe disease in humans, even at the respiratory level [19, 27] . evd is a viral hemorrhagic fever (vhf) characterized by acute systemic manifestations with vascular damage, plasma leakage, severe inflammation, and disruption of the immune system [28] . evd transmissibility seems to vary depending on the stage of disease [29] . a high-level of ebov replication, associated with systemic dissemination to multiple cell types, results in a complex pathogenesis, which is linked to an increased risk of infection transmission [29] . as stated above, these pathogenic mechanisms include detrimental immune suppression and over-activation of the immune response, disordered coagulation, and tissue damage due to direct viral and indirect host-mediated effectors. in the absence of adequate supportive care, these processes commonly result in multiple organ failure and death within about 10 days of symptom onset in humans. it is well recognized that ebov infection is acquired by direct contact with bodily fluids. notably, studies conducted in animal models have instilled doubts about possible airborne/droplet transmission (see section 3.1). however, this route of infection in humans is still debated. piercy and colleagues evaluated the actual stability of the virus particles in aerosol droplets [30] . they created ebola-containing aerosol droplets and, according to the decay rates, estimated that ebov and restv can survive in aerosols for roughly 100 and 160 min, respectively, at 50% to 55% relative humidity and 22 ± 3 • c [30] . therefore, a key additional question to ask is whether primary pulmonary infection of ebov could be a potential scenario for the future. a fair amount of studies, based on animal experiments (table 1) and clinical evidence collected during the outbreaks ( table 2 ), suggest that pulmonary infection may be a possibility. this possibility will be fully investigated below. after its first discovery in 1989 in cynomolgus macaques imported to reston, virginia, restv was detected in domestic swine in the philippines in a co-infection with the porcine reproductive and respiratory syndrome virus (prrsv, family arteriviridae, genus arterivirus) and porcine circovirus type 2 (pcv-2; family circoviridae) [32, 34] . later on, restv was identified to cause asymptomatic infections with mild respiratory symptoms, which may result in severe mortality in cases of co-infections with other viral pathogens like viruses in the families arteriviridae and circoviridae. the virus was first isolated in lung and lymphoid tissues in the original disease investigation [32] . however, the massive presence of the virus in the lungs may be due to the fact that restv infection in pigs has been mostly associated with other infections of the respiratory tract, which may contribute to the specific localization of the virus and the respiratory symptoms of the disease. marsh et al. [33] conducted an experimental study to rule out the effect of other pathogens affecting pigs, using a 2008 philippines swine isolate of restv. specifically, five-week-old pigs were exposed (via the oro-nasal or subcutaneous route) to the virus, and the subsequent viral replication in internal organs and shedding of the virus from the nasopharynx was observed. the researchers detected the highest levels of virus replication in lung and lymphoid tissues, confirming previous results [32] . the detection of restv in domestic swine raised important biosecurity concerns about the potential for the disease's emergence in humans and other livestock, mainly in animals for food consumption [32, 33] . the evidence of restv seropositive individuals further increased the concern for human infections and the worries of researchers, farm owners, and the public at large (world health organization. who, 2009, available online: https://www.who.int/csr/resources/publications/hse_ epr_2009_2.pdf). interestingly, so far restv has not been seen to result in any human disease, even if there is concern that its passage through swine may allow restv to diverge and shift its potential for pathogenicity [58] . on the other hand, several studies investigated if other ebola viruses may be transmitted through the aerosol route and may result in primary pulmonary infection [9, 10, 59] . researchers reviewed the different animal models and offered an overview regarding the possibilities of ebola viruses causing aerosol infections in non-human primates (nhps) and other animals. experimental studies analyzed the respiratory tract involvement in filovirus infections when the animals were exposed to the virus through different aerosol routes (artificially aerosolized virus or natural aerosol transmission) [36, 37, 39, 60] . in these experimental studies conducted on nhps and pigs, ebov was inoculated via the aerosol route, and, following mucosal exposure, ebov replicated, reaching high concentrations, mainly in the respiratory tract, with the development of severe lung pathology. interestingly, weingartl et al. demonstrated that piglets inoculated oro-nasally with ebov and then transferred to a different room housing macaques in an open inaccessible cage system resulted in ebov infection of all macaques, suggesting a need to revise prevention and control measures during outbreaks [37] . viral replication was observed within alveolar spaces [36, 37] , in type i pneumocytes and macrophages [36] , and in type ii pneumocytes, bronchiolar epithelial cells, and endothelial cells [38] , supporting the respiratory involvement. the upper and lower respiratory tract, the lymphoid tissues, and the mediastinal lymph nodes showed infection signs, as well [39] . similarly, in experiments on cynomolgus macaques placed separately in cages with experimentally infected piglets [37] , and on guinea pigs exposed via aerosols to a guinea pig-adapted ebov strain [39] , viral antigens were detected within alveolar and septal macrophages, pneumocytes, epithelial cells, endothelial cells, fibroblasts, and other interstitial cells of the respiratory tree [61] . considering the pathology of the respiratory system, the expression of disease in the lungs and the patterns of lesions seem to be influenced by the exposure routes (aerogenous or hematogenous). broncho-interstitial pneumonia, characterized by injury to both the bronchiolar and the alveolar epithelium, is commonly caused by aerogenous viral infections [62] . moreover, such pathological features were generally not evidenced following the inoculation of ebov by other routes in nhps and laboratory animals [9, 35] . as shown in animal studies, primary pulmonary infections could occur and cause active viral shedding from the respiratory tract, thus potentially setting up a cycle of ongoing respiratory transmission in humans [9, 63] . overall, experimental works conducted so far have shown that ebov infection induces respiratory complications, that the virus can be shed via the respiratory secretions, and that it can cause similar pulmonary lesions both in animals exposed to aerosols and in those kept nearby in separate cages with no close contact. the pathophysiological mechanism of pulmonary disease in patients with evd is unknown. notably, autopsies were performed on a limited number of humans (about 30 cases), primarily during the 1976 sudv and 1995 ebov evd outbreaks and revealed interesting characteristics at microscopic level. during the first known sudv outbreak, chest pain was almost universal (83% of patients), often accompanied by a dry cough. autopsies were further performed on two patients and thickening of the alveolar walls due to proliferative accumulations of alveolar cells was found [64] . furthermore, a possible pathogenetic role of the virus in the respiratory tract was suggested by the fact that viral inclusions within alveolar macrophages and free viral particles within alveolar space were found in the lungs from fatal evd cases who showed congestion, focal intra-alveolar edema, diffuse alveolar damage, and hemorrhaging. [9, 10] . one of the most common symptoms in evd patients is a cough (up to 49%), especially during the progression of the disease, when viral loads in serum significantly increase, and the virus is copiously emitted in most body fluids, as well as in aerosol particles of various sizes [65, 66] . among the reported evd cases in the literature, respiratory symptoms were commonly reported with a wide range of symptoms, such as a cough (from 3% [67, 68] to 60% [69] ), dyspnoea or breathless (detected from 0% [70] to 49% [57] ), and chest pain (from 7.5% [71] to 98.6% [72] ). moreover, a who study on the first 9 months of the epidemic in western africa found that nearly 30% (194 out of 665) of the patients experienced coughing and 2.4% (20 of 831) had a bloody cough [73, 74] . a study of 27 ebov-positive patients of the 2013-2016 outbreak in western africa, treated in europe and usa, reported that cough and dyspnoea were present at admission in seven (30%) and five (22%) evd patients, respectively. at symptom onset, only a cough was reported in one patient. furthermore, during hospitalization, 14 patients (52%) experienced hypoxemia while they were breathing ambient air, 12 patients (44%) had pulmonary oedema, seven patients (26%) had pneumonia, 39 patients (33%) had respiratory failure, and six patients (22%) had a diagnosis of acute respiratory distress syndrome (ards). of these patients, four patients (15%) received non-invasive mechanical ventilation, and seven patients (26%) received invasive mechanical ventilation [75] . notably, the first ebov-positive patient treated in italy, mechanically ventilated for respiratory insufficiency for 5 days, had high levels of ebov rna in the lower respiratory tract secretions. the authors concluded that the absence of other identified respiratory pathogens in broncho-alveolar lavage fluids and aspirates supported the hypothesis of a direct contribution to the lung tissues damage by ebov. notably, ebov rna was detected in bronchial aspirate fluids when the ebov rna concentration in the concomitant blood samples was barely detectable. furthermore, the blood ebov rna concentrations in the previous days were significantly lower than the concentrations detected in the bronchial aspirate samples. these findings suggest that this ebov infection is unlikely a spillover from the blood compartment, eventually accompanied by delayed clearance. instead, the most plausible explanation is that the virus actually replicated into the lower respiratory tract [76] . in the second ebov-patient treated in italy, our group investigated the presence of ebov genetic material in the lungs and blood during the patient's treatment and recovery. the patient showed a persistence of ebov replication markers within the respiratory tract, with a prolonged detection of ebov viral rnas (negative and positive sense rnas: neg-rna and pos-rna, respectively), known to be associated with ebov replication, in the lower respiratory tract for up to five days after the ebov viral load in blood was already undetectable. these results suggest that ebov may replicate in the lungs, although it is possible that the lungs simply provided a protective environment that allowed rna to linger longer than it did in the plasma. nevertheless, the detection of pos-rna together with neg-rna in the sputum (until day 9 and 10 of the hospital stay, respectively) supports the concept of active viral replication within the respiratory tract, rather than plasma spill-over or prolonged rna stability [14] . overall, the pathophysiological mechanisms of pulmonary disease in patients with evd are still uncertain, but there could be multiple contributing factors, including vascular leak from endothelial infection, cytokine dysregulation, or direct damage to ebov-infected cells (figure 2 ). viruses 2019, 11, x for peer review 12 of 21 of pos-rna together with neg-rna in the sputum (until day 9 and 10 of the hospital stay, respectively) supports the concept of active viral replication within the respiratory tract, rather than plasma spill-over or prolonged rna stability [14] . overall, the pathophysiological mechanisms of pulmonary disease in patients with evd are still uncertain, but there could be multiple contributing factors, including vascular leak from endothelial infection, cytokine dysregulation, or direct damage to ebov-infected cells (figure 2 ). our understanding of ebov transmission in humans mainly relies on epidemiological observations. contact with bodily fluids from evd patients remains the most likely route of transmission. notably, the number of past outbreaks and associated epidemiological studies hat carefully examine transmission patterns is small. therefore, conclusions about transmission are based on relatively limited data sets [10] . interestingly, 18 (6.6%) of the 2774 cases in the 1976 sudv outbreak in nzara, sudan, and 55 (17.4%) of the 316 cases during the 1995 ebov outbreak in kikwit, drc, had no direct or physical contact with an infected person or known infected dead body [77, 78] , thus pointing to other possible routes of transmission, e.g., human to human respiratory tract infection through droplet and aerosols. during the 2013-2016 western africa epidemic, more than 890 health care workers (hcw) were infected, with a case fatality rate of 57% [60] , whereas during our understanding of ebov transmission in humans mainly relies on epidemiological observations. contact with bodily fluids from evd patients remains the most likely route of transmission. notably, the number of past outbreaks and associated epidemiological studies hat carefully examine transmission patterns is small. therefore, conclusions about transmission are based on relatively limited data sets [10] . interestingly, 18 (6.6%) of the 2774 cases in the 1976 sudv outbreak in nzara, sudan, and 55 (17.4%) of the 316 cases during the 1995 ebov outbreak in kikwit, drc, had no direct or physical contact with an infected person or known infected dead body [77, 78] , thus pointing to other possible routes of transmission, e.g., human to human respiratory tract infection through droplet and aerosols. during the 2013-2016 western africa epidemic, more than 890 health care workers (hcw) were infected, with a case fatality rate of 57% [60] , whereas during the current 2018-2019 outbreak in drc, as of 22 july 2019, 140 hcw have been already affected (5.4% of total cases) [6] . currently, full body protection is recommend by who and cdc [79, 80] . all hcw involved in the care of evd patients must receive training and demonstrate competency in performing all ebola related infection control practices and procedures, specifically in proper donning and doffing ppe even if using an n95 mask or a powered air-purifying respirator (papr). the risk of infection via inhalation of contaminated aerosols from exposed individuals has not been documented. however, droplets containing ebov that have become aerosolized (e.g., from coughing sneezing, vomiting, invasive medical or surgical procedures, or surfaces) may have the potential to come into contact with a person's mucous membrane in their nose or mouth or non-intact skin. therefore, respiratory protection may be helpful in providing a barrier to help prevent infectious materials from contacting a wearer's mucous membranes. finally, the epidemiologic and viral evidence of ebov detection and replication in the respiratory tract raise concerns on the need of strict application of cough etiquette for patients and of droplet and/or respiratory precautions for all hcw involved in the clinical management of evd suspected and confirmed cases. acute respiratory tract infections (artis) remain a leading cause of mortality, morbidity, and economic loss, and viruses are one of the main causes of such disease. who estimates that artis cause nearly four million deaths per year, a rate of more than 60 deaths/100,000 people [81] . the microbial etiology of aris is varied, with viruses being the most common cause in humans [82] , leading to a high level of awareness and the necessity to develop countermeasures to control them (table 3) . filoviruses are not commonly considered to be viruses responsible for aris, even if respiratory symptoms may be present as a consequence of diffuse systemic alterations. interestingly, evidence collected in animal studies, in the epidemiological analysis of transmission chains, and in the most recent ebola outbreaks suggests that ebov may be able to cause primary pulmonary infection. this evidence highlights the ability of the virus to be shed in the lung, suggesting a role in lung pathogenesis. specifically, the relevant proportion of evd patients without any epidemiologic link to the exposure to contaminated biological samples or fomites, or to any contact with evd patients; the evidence of respiratory signs and symptoms commonly reported all over the clinical course; the abundance of viral antigens in the lungs in animal necropsies; the prolonged persistence of ebov detection and replication within the respiratory tract days after undetectable ebov viral load in plasma; and similar clinical patterns in several other viral respiratory tract infections are all different parameters with consistent evidence of a major role in the pathogenesis of evd in respiratory tissues [10] . on the other hand, there is no evidence of aerosol transmission in evd. however, different studies addressing this issue have been performed [30, 83] , and aerosol transmission was considered a possibility as a consequence of epidemiological observations in past outbreaks, where people showed signs of evd even in the absence of a direct or physical contact with an infected person or known infected dead body [78, 84] . this hypothesis was corroborated by other studies, in which the presence of free viral particles in alveoli and within intra-alveolar macrophages demonstrated a pulmonary involvement [10] . from a clinical point of view, the 2013-2016 ebov outbreak underlined the lung involvement in evd pathogenesis. in fact, only a few patients treated in europe and usa had a cough and difficulty breathing at admission. nevertheless, during the clinical progression, half of the patients experienced hypoxemia while breathing room air, one third had respiratory failure, and one fourth received invasive or non-invasive mechanical ventilation [75] . in the italian experience at the national institute for infectious diseases "l. spallanzani" (inmi), respiratory symptoms were present in both patients, in the absence of other common respiratory pathogens [14, 76] . one case required mechanical ventilation and the other presented ebov replication markers in the lungs even after clearance of the virus from the blood. the inmi experience suggests a direct role of the virus in lung pathogenesis. although lung pathogenesis in evd may be secondary to systemic alterations (correlating with general pathogenic mechanisms) the direct presence of the virus is undisputable in the lung, and its interaction with the immune system, whose hyper-activation may be the most likely explanation of the lung damage, is also indisputable. further research will be needed to better understand the potential role of pulmonary involvement in evd and whether it may be a factor in the transmission of the virus from one human to another. the discovery of bombali virus adds further support for bats as hosts of ebolaviruses new filovirus disease classification and nomenclature emergence of zaire ebola virus disease in guinea marburg and ebola viruses world health organization (who) assessing the potential role of pigs in the epidemiology of ebola virus in uganda assessment of the risk of ebola virus transmission from bodily fluids and fomites tissue and cellular tropism, pathology and pathogenesis of ebola and marburg viruses transmission of ebola viruses: what we know and what we do not know the airway epithelium: soldier in the fight against respiratory viruses epithelial antimicrobial peptides and proteins: their role in host defence and inflammation centers for disease control and prevention (cdc) cdc grand rounds: discovering new diseases via enhanced partnership between public health and pathology experts detection of viral rna in tissues following plasma clearance from an ebola virus infected patient proinflammatory response during ebola virus infection of primate models: possible involvement of the tumor necrosis factor receptor superfamily infection and activation of monocytes by marburg and ebola viruses kinetics of soluble mediators of the host response in ebola virus disease human ebola virus infection results in substantial immune activation inflammatory and humoral immune response during ebola virus infection in survivor and fatal cases occurred in sierra leone during the 2014-2016 outbreak in west africa into the eye of the human fatal zaire ebola virus infection is associated with an aberrant innate immunity and with massive lymphocyte apoptosis dendritic cells and the control of immunity the lack of maturation of ebola virus-infected dendritic cells results from the cooperative effect of at least two viral domains ebola and marburg viruses replicate in monocyte-derived dendritic cells without inducing the production of cytokines and full maturation immune barriers of ebola virus infection different features of vδ2 t and nk cells in fatal and non-fatal human ebola infections ebola haemorrhagic fever virus: pathogenesis, immune responses, potential prevention ebola virus disease the ebola transmission paradox the survival of filoviruses in liquids, on solid substrates and in a dynamic aerosol preliminary report: isolation of ebola virus from monkeys imported to usa discovery of swine as a host for the reston ebolavirus ebola reston virus infection of pigs: clinical significance and transmission potential reston virus in domestic pigs in china lethal experimental infections of rhesus monkeys by aerosolized ebola virus replication, pathogenicity, shedding, and transmission of zaire ebolavirus in pigs transmission of ebola virus from pigs to non-human primates immunopathogenesis of severe acute respiratory disease in zaire ebolavirus-infected pigs bulletin of the world health organization vols. 1 to 97 ebola hemorrhagic fever: tandala, zaire, 1977-1978 ebola virus disease in southern sudan: hospital dissemination and intrafamilial spread seroepidemiological study of filovirus related to ebola in the philippines epidemiologic notes and reports update: ebola-related filovirus infection in nonhuman primates and interim guidelines for handling nonhuman primates during transit and quarantine isolation and partial characterisation of a new strain of ebola virus ebola hemorrhagic fever outbreaks in gabon, 1994-1997: epidemiologic and health control issues the reemergence of ebola hemorrhagic fever, democratic republic of the congo an outbreak of ebola in uganda available online outbreak of ebola hemorrhagic fever in the republic of the congo ebola and marburg hemorrhagic fevers: neglected tropical diseases? global alert and response reemerging sudan ebola virus disease in uganda development of a reverse genetics system to generate recombinant marburg virus derived from a bat isolate ebola virus disease in the democratic republic of congo outbreak of ebola virus disease in northern democratic republic of congo clinical and virological characteristics of ebola virus disease patients treated with favipiravir (t-705)-sierra leone risks posed by reston, the forgotten ebolavirus the pathogenesis of ebola virus disease animal models of ebolavirus infection pathologic features of filovirus infections in humans kennedy and palmer's pathology of domestic animals ebola virus transmission who/international study team. ebola haemorrhagic fever in sudan ebola virus transmission via contact and aerosol-a new paradigm clinical illness and outcomes in patients with ebola in sierra leone ebola virus disease, democratic republic of the congo experimental treatment of ebola virus disease with tkm-130803: a single-arm phase 2 clinical trial clinical presentations and outcomes of patients with ebola virus disease in freetown endovascular treatment for acute ischemic stroke patients: implications and interpretation of ims iii, mr rescue, and synthesis expansion trials: a report from the working group of international congress of interventional neurology mortality among pcr negative admitted ebola suspects during the 2014/15 outbreak in conakry, guinea: a retrospective cohort study epidemiological characteristics, clinical manifestations, and treatment outcome of 139 paediatric ebola patients treated at a sierra leone ebola treatment center insights from clinical research completed during the west africa ebola virus disease epidemic electrolyte and metabolic disturbances in ebola patients during a clinical trial clinical management of ebola virus disease in the united states and europe ebola virus disease complicated with viral interstitial pneumonia: a case report antibody prevalence against haemorrhagic fever viruses in randomized representative central african populations risk factors for patients without a reported exposure cdc centers for disease control and prevention guidance for donning and doffing personal protective equipment (ppe) during management of patients with ebola virus disease interim infection prevention and control guidance for care of patients with suspected or confirmed filovirus haemorrhagic fever in health-care settings, with focus on ebola nucleic acid amplification-based diagnosis of respiratory virus infections understanding ebola virus transmission ebola and marburg virus antibody prevalence in selected populations of the central african republic the repertoire of adenovirus in human disease: the innocuous to the deadly respiratory syncytial virus infection in adults respiratory syncytial virus measles virus: cellular receptors, tropism and pathogenesis pathological and ultrastructural analysis of surgical lung biopsies in patients with swine-origin influenza type a/h1n1 and acute respiratory failure pathogenesis of severe acute respiratory syndrome pathogenesis of the viral hemorrhagic fevers dengue viruses can infect human primary lung epithelia as well as lung carcinoma cells, and can also induce the secretion of il-6 and rantes pathogenesis of lassa fever. viruses pathophysiology of hantavirus pulmonary syndrome in rhesus macaques this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license key: cord-009860-qebenhxz authors: falsey, ann r.; treanor, john j.; betts, robert f.; walsh, edward e. title: viral respiratory infections in the institutionalized elderly: clinical and epidemiologic findings date: 2015-04-27 journal: j am geriatr soc doi: 10.1111/j.1532-5415.1992.tb01929.x sha: doc_id: 9860 cord_uid: qebenhxz objective: to prospectively evaluate the incidence and impact of viral respiratory infection in the institutionalized elderly during a winter season. design: prospective descriptive study, without intervention. method: patients with respiratory illnesses were evaluated by a directed history and physical examination. nasopharyngeal secretions for viral culture were obtained, and acute and convalescent serum samples were obtained for analysis. serologic evidence of infection with respiratory syncytial virus (rsv) and parainfluenza were determined by enzyme immunoassay (eia), and influenza by hemagglutination‐inhibition assay and eia. setting: a 591‐bed nursing home. participants: residents with signs or symptoms of acute respiratory illness (nasal congestion, pharyngitis, cough, wheezing, or respiratory difficulty) were eligible for study. results: a viral etiology was documented in 62 out of 149 illnesses (42%). rsv was the most common virus associated with illness; it was documented in 27% of respiratory illnesses, followed by rhinovirus (9%), parainfluenza (6%), and influenza (1%). rsv was associated with significantly more severe disease when compared with rhinovirus. clustering of specific viral infections occurred, suggesting nosocomial transmission. conclusions: viruses are an important cause of acute respiratory infections in the institutionalized elderly during the winter months. cute respiratory tract infections are a major cause of acute morbidity in the united states. the a frequency of respiratory infections is high in young children and appears to decline with advancing age.' however, the impact on the elderly host may be of greater consequence due to an aging respiratory tract, a declining immune system, and multiple medical problems.' residents of long-term-care facilities represent a debilitated subgroup of the elderly population and thus are particularly prone to excess morbidity with respiratory infections. however, with the exception of influenza, comprehensive studies on viral respiratory infections in the institutionalized elderly are lacking. this is despite the fact that each year many residents of nursing homes become ill with respiratory infections not proven to be influenza, either by culture or serology. the purpose of this study was to identify all common respiratory viruses which cause symptomatic dsease during the winter months in nursing home patients, by utilizing both viral cultures and serology, and to assess their clinical impact. respiratory illness (nasal congestion, pharyngitis, cough, wheezing, or respiratory difficulty) were eligible for study. results: awiral &ology was documented in 62 out of 149 illnesses (42%). rsv was the most common virus associated with illness; it was documented in 27% of respiratory illnesses, followed by rhinovirus (9 %) , parainfluenza (6%), and influenza (1 %). rsv was associated with significantly more severe disease when compared with rhinovirus. clustering of specific viral infections occurred, suggesting nosocomial transmission. conclusions: viruses are an important cause of acute respiratory infections in the institutionalized elderly during the winter months. j am geriatr soc 40115-119,1992 health-related facility for more independent residents. each floor of the skilled nursing facility had a separate dining area, whereas residents of the health-related facility ate in a single large dining hall. medical care for residents was provided by three full-time staff physicians. infection control policies at the facility included quarantine of floors with 2 3 documented cases of influenza and amantadine prophylaxis for 2 weeks for residents of such floors. no specific isolation procedures were implemented for non-influenza respiratory illnesses. no attempt was made to influence either individual patient care or existing infection control policy during this study. subjects between december 11, 1989 and march 13, 1990 , the head nurse on each floor identified residents who had signs or symptoms of acute respiratory illness including nasal congestion, pharyngitis, cough, wheezing, or respiratory difficulty with or without fever. an institutionwide illness log was created and reviewed by a study nurse daily from monday through friday. each subject listed in the illness log was then evaluated by a directed history and physical examination. a global seventy of illness score was assigned by the study nurse, using an analog scoring system rangng from 0 to the analog score consisted of a horizontal line with one end representing mildest illness and the other end the most severely ill. this score represented the study nurses' overall assessment of the severity of an illness based on both the patient's symptoms and objective findings. in a separate analysis the analog scores were found to have a high correlation with objective indices of disease (falsey, j med virol, in press). arterial oxygen saturation (sa02) was measured percutaneously, and a nasopharyngeal swab for virus culture and serum were obtained on the initial visit. chest roentgenograms were ._ -obtained and antimicrobials prescribed at the discretion of the patient's staff physician. each patient was reevaluated daily for 3 days and convalescent sera obtained 4-8 weeks after the acute illness. viral cultures nasopharyngeal secretions were inoculated onto cell culture within 4 hours. each sample was placed on hep-2, madin-darby canine kidney (mdck), and human foreskin fibroblast (hff) cell lines. all terminal hep-2 cultures were examined for rsv by a direct immunofluorescent antibody test (genetic systems, seattle, wa). any cell line demonstrating cpe was examined by ifa for parainfluenza virus (piv) 1,2,3, adenovirus, influenza a and 8, and herpes simplex virus (hsv). rsv isolates were subgrouped a or b with group specific monoclonal antibodies.4 rhinovirus isolates were confirmed by acid lability testing. paired sera were analyzed for evidence of recent infection with rsv, influenza, and parainfluenza viruses. serum igg levels to the fusion (f) and attachment (g) glycoproteins of a and b rsv subgroups (designated ga and gb) were determined for all sera by enzyme immunoassay (eia) as previously r e p~r t e d .~ serologic evidence of rsv infection was defined as a ?$-fold rise in igg to any of the rsv antigens. sera was also tested for evidence of influence infection by hemaglutination-inhibition assay (hai) and hemagglutinin-specific eia.6 serum igg levels to parainfluenza virus, types 2 (piv-2) and 3 (piv-3), were determined by enzyme immunoassay using virus infected vero cells as antigen. briefly, vero cells in 96-well microtiter plates were infected with piv-2 or piv-3 (wild isolates) and when cpe was detectable, the cells were fixed in 80% acetone/pbs. paired sera were incubated in duplicate serial 2-fold dilutions in the wells for 1 hour at 37oc. plates were washed and then incubated with horseradish peroxidase conjugated goat anti-human igg for 1 hour. substrate was added and optical density was read on a dynatech spectrophotometer. control wells with uninfected vero cells were used to define background absorption which was substrated from the values of the antigen plates. because heterologous titer rises between piv types 1 and 3 are common and serologic differentiation of infecting type is not possible, specimens were screened using piv-2 and piv-3 antigens. evidence of parainfluenza infection was defined as a 24-fold nse in igg to either piv antigen. during the 3-month study period, 177 respiratory illnesses were identified in 164 residents. of these, 133 residents who experienced 149 illnesses agreed to be studied. eighty-four percent of the study patients were female, and the mean age was 88. most had underlying medical conditions, with neurologic (77%), cardiac (47%), pulmonary (25%), diabetes (12%), and malignancy (7%) being the most frequent. cultures and serology forty-one viruses were isolated during 39 of the 149 illnesses (26%) ( table 1) . rsv was isolated from 18 patients, followed by rhinovirus in 14, herpes simplex (hsv) in six, parainflu-enza-1 in two, and parainfluenza-2 in a single patient. influenza virus was not isolated. two patients had dual infections (hsv/rsv and hsv/rhinovirus). rsv group could be determined for 15 of the 18 rsv isolates. nine of these were group b, and six were group a. one-hundred-six of the 133 (80%) had acute and convalescent sera available for analysis. thirty-three (31 %) showed serologic evidence of rsv infection. forty rsv infections were documented either by culture alone (5), serology alone (22), or both (13). fifteen of the 18 individuals from whom rsv was isolated had acute and convalescent sera available. of these, 13 showed 14-fold rises in rsv titers. the other two patients had acute titers of igg greater than the highest dilution and, therefore, rises could not be detected. in addition, six patients (5.7%) demonstrated 24-fold rises in piv titers (3 to piv-2 and 3 to piv-1). two patients had a 24-fold rise in ha1 titers to influenza which was confirmed by ha-specific eia. overall, a viral etiology of the respiratory infection was documented by culture or serology in 62 of 149 illnesses (42%). epidemiology respiratory illnesses were reported throughout the study period from 12/11/89 to 3/13/ 90, with the majority reported in january (figure 1) . illnesses were noted on all nine floors of the skilled nursing facility and on 18 of 19 floors in the healthrelated facility. rsv was isolated throughout most of the study period with a peak occurrence in mid-january. although rsv infections were geographically scattered throughout the institution, eight and 14 cases occurred on the 7th and 8th floors of the skilled nursing facility, respectively. on the 8th floor, all five of the typeable rsv isolates were group b, while on the 7th floor four of five typeable isolates were group a. it was also noted that in three of the four wings on these two floors, cases of rsv tended to occur in patients residing on one side of the hallway. rhinovirus infections also occurred throughout the study period, although nine of the 14 cases occurred during a 4-day period in december. five of the 14 cases also occurred on the 8th floor, with the remainder scattered widely throughout the institution. all six cases of piv infection occurred during late december or early january. the three piv-1 infections occurred within a 5-day period in patients who lived on the same floor in adjacent rooms. clinical syndromes the clinical features of illness were compared in patients with the two most frequently documented viral infections, rsv and rhinovirus ( table 2 ). the spectrum of illness associated with rsv infection was broad, ranging from mild nasal congestion only to high fever and respiratory distress. the most prominent complaint was nasal congestion (92%), followed by cough (90%) and sputum production (60%). appearance on physical examination was also variable with analog scores ranging from 1 to 7 (mean analog score 3.7 -t-1.5). fifty-four percent had a score of 1 4 , suggesting moderately severe disease. nine had oral temperatures greater than 101of. sixtyfive percent of patients had abnormal lung findings including rales in 4096, wheezing in 35%, and rhonchi in 13%. the mean room air saoz was 95%, and two patients required supplemental oxygen during the acute illness. twelve patients had chest roentgenograms, four of whom had an infiltrate. antibiotics were prescribed in 49% of rsv-infected patients. one patient was hospitalized with pneumonia, and two others died, one during the acute rsv infection, the other 1 month later after a steady decline in health and with an ill-defined pulmonary process. most patients recovered without sequelae, although the functional status of two patients deteriorated after their illness. in contrast, illness associated with rhinovirus infection was generally mild. rhinorrhea was the most frequent complaint (79%). cough was also a prominent symptom (71%), although less common than in the rsv-infected group (90%). sputum production was significantly less frequent with rhinovirus infection compared to rsv (21% vs 60%, p = 0.05 by chi square). on physical examination patients generally did not appear acutely ill. only one person had an analog score >3, and the mean analog score was 1.6 -i-1.5 which was significantly lower than the mean illness score in rsv infection (p < .001 by t test). the most prominent sign was nasal discharge (28%). only two patients (14%) received antibiotics, and all recovered without sequelae. piv infection was documented in six individuals, three with piv-i, and three with piv-2. although the numbers are too small to permit comparison of clinical syndromes to other infecting agents, each type of piv infection appeared to produce a distinct illness. the three individuals with piv-1 had a mean analog score of 3.7. all complained of cough and constitutional symptoms. fever and abnormal chest exams were present in two of three patients, and all three received antibiotics during their illness. piv-2 illnesses were somewhat milder, with a mean analog score of 2.0. all three complained of sore throat, and two were hoarse. pharyngeal erythema was noted in two of three patients, and none had a temperature >99of or received antibiotics. no cases of pneumonia were documented in either group, and all recovered without sequelae. viruses are a major cause of acute respiratory infections in the general population. the groups of viruses responsible for most acute respiratory infections are rhinovirus, coronavirus, influenza, rsv, parainfluenza, and adeno~irus.~ an attempt was made to diagnose each of these pathogens, with the exception of coronavirus, utilizing either culture, serology, or both. several studies of "influenza-like" illnesses in nursing homes have shown that a variety of infectious agents can be found.*-" this study provides evidence of the importance of viruses as a cause of respiratory infection in the nursing home since 42% of acute respiratory illnesses during one winter season were viral in origin. the number and severity of respiratory illnesses during the study period was not unusual compared with previous winter months at this nursing home. however, the season was somewhat atypical since influenza was not isolated, despite its prevalence in other nursing homes in the community. it is possible that high rates of influenza vaccination among staff (37%) and residents (78%) may have influenced influenza infection within the institution." respiratory syncytial virus (rsv), the most common virus isolated in this study, is usually considered a pathogen of infants and young children. while less common in adults, rsv can lead to severe communityacquired pneumonias in elderly or immunocompromised a number of outbreaks of rsv have been reported from chronic care fa~ilities.'~-'~ it is noteworthy that, in our prospective analysis, rsv accounted for 27% of the acute respiratory infections. while it represents only 1 year of study, these data provide further evidence that rsv is an important pathogen in the institutionalized elderly. the clinical syndrome of nasal congestion, cough with expectoration, and fever is consistent with previous reports of rsv infection in the elderly. wheezing, which is common in infants with rsv, was also found in 35% of patients in this study, most of whom did not have preexisting pulmonary disease. one of the prominent features of rsv infection in this study was the wide spectrum of illness it produced, ranging from a mild coryza1 illness to high fever and respiratory distress. rates of pneumonia (5%-67%) and death (0%-20%) varied significantly among previously published outbreaks. factors determining the severity of illness are not completely understood but may be related to the status of the host and to the infecting group or strain of rsvs4 recovery of virus did not appear to be associated with more severe illness as the clinical course of culture-positive cases of rsv did not differ from those with only serologic evidence of infection. cases of rsv infection were widely distributed geographically, occurred over a 3-month period, and were of both a and b groups. the presence of both groups suggests, in contrast with most previous reports, that the present outbreak was not from a single nosocomial source. there was, however, epidemiologic evidence suggesting nosocomial transmission within the institution. clustering of a group infections on the seventh floor and b group infections on the 8th floor, as well as clustering of cases by sides of the hall on three of the four wings, is suggestive of nosocomial spread. rsv is thought to be transmitted by fomites and requires direct contact with respiratory secretions." although documentation of illness among staff members was not attempted during this study, nosocomial transmission of rsv by hospital personnel has been described." therefore, strict handwashing and the use of gowns and gloves, which have been shown to be effective measures for controlling nosocomial rsv infections in hospitalized infants, may be effective in the nursing home as well. rhinovirus was the second most frequent virus isolated in this study. in healthy adults rhinoviral infection is a self-limited illness characterized by sneezing, rhinorrhea, cough, and mild sore throat. 21 there are very limited data on rhinoviral infection in the elderly. analysis of the 14 culture-documented infections in this study indicates rhinovirus produces a relatively benign illness in the elderly as well. infection was characterized by rhinorrhea and cough, but without high fever or signs of lower respiratory tract involvement. generally, the patients did not appear acutely ill, and all recovered without sequelae. unlike other respiratory viruses, such as influenza or adenoviruses, rhinovirus infection produces relatively minor damage to the nasal epithelium and probably none to the tracheal mucosa. 22 since rhinovirus replication is markedly reduced at body temperature, direct invasion of the lower respiratory tract should be unusual at any age. rhinoviruses are transmitted primarily by contact with infected secretions followed by autoinoculation, and transmission can be decreased by handwashing and environmental di~infection.'~, 24 piv is a common cause of croup and bronchitis in young ~hildren.'~ reinfection in young healthy adults typically presents as a uri; however, pneumonia has rarely been described. 26 while piv infection in the elderly has not been well characterized, there have been several outbreaks of piv-1 and -3 infections reported in nursing homes.27, 28 the illness has been characterized by fever, rhinorrhea, pharyngitis, and cough, and pneumonia has been common, ranging from 17% to 29%. although piv infection was uncommon in the present study, we can state that patients with piv-1 infection were all febrile, moderately ill, and received antibiotics. the mode of transmission of piv appears to be direct person-to-person and/or droplet aerosol spread. therefore, infection control measures similar to those used in rsv outbreaks may be useful in piv outbreaks. in summary, viral respiratory tract infections are common in the nursing home during the winter months. both rsv and rhinovirus were prevalent during this study, with rsv being associated with a significantly more severe illness. frequently, the diagnosis of "a viral infection" is one of exclusion and of little use to the clinician. however, diagnosis of these agents is important for several reasons. first, infection control policies for rsv, rhinovirus, and piv are different from those for influenza. secondly, with specific viral diagnosis, the use of antibiotics and amantadine might be decreased, thereby reducing unnecessary side effects. and, finally, as new vaccines become available, the elderly may represent a potential target population. acute respiratory illness in an american community: the tecumseh study the effect of aging on susceptibility to infection studies with different types of visual analog scores for measurement of pain variation in seventy of respiratory syncytial virus with subtype strain-specific serum antibody responses in infants undergoing primary infection with respiratory syncytial virus serum antibody responses in naturally occurring influenza-a virus infection determined by enzyme-linked immunosabsorbent assay, hemagglutination-inhibition, and complement fixation the common cold epidemiology of acute respiratory illness during an influenza outbreak a prospective study upper respiratory tract infection and serum antibody responses in nursing home patients acute upper respiratory tract viral illness and influenza immunization in homes for the elderly risk factors for outbreaks of influenza in nursing homes: a case control study case report: respiratory syncytial virus infection-a cause of respiratory distress and pneumonia in adults fatal pneumonia in an adult due to respiratory syncytial virus an epidemic of respiratory syncytial virus in the elderly people: clinical and serological findings an outbreak of an influenza-like illness in a nursing an outbreak of respiratory syncytial virus in an old people's home an outbreak of respiratory syncytial virus pneumonia in a nursing home for the elderly concurrent respiratory syncytial virus and influenza a infections in the institutionalized elderly and chronically ill respiratory syncytial infections within families control of nosocomial respiratory syncytial viral infections rhinovirus infections in an industrial population. 1. the occurrence of illness principles and practices of infectious disease transmission of rhinovirus colds by self-inoculation interruption of experimental rhinovirus transmission viral respiratory illnesses parainfluenza pneumonia in adults centers for disease control. parainfluenza outbreaks in extended care facilities-united states public health laboratory science communicable disease surveillance centre. parainfluenza infections in the elderly we thank patricia hennessey, rn, for data collection, joanne prives for transcription assistance, and the staff of st. ann's home for their help in conducting the study. key: cord-022337-f3a349cb authors: busse, william w.; dick, elliot c.; lemanske, robert f.; gern, james e. title: infections date: 2007-05-09 journal: asthma doi: 10.1016/b978-012079027-2/50112-x sha: doc_id: 22337 cord_uid: f3a349cb wheezing with respiratory infections is extremely common in early childhood. it is estimated that the prevalence of wheezing during the first five years of life varies from 30–60%. in the majority of children who experience wheezing with respiratory infections, these episodes of wheezing become less frequent as the child grows older. however, determining whether the initial episode of wheezing with a viral respiratory illness is an important factor in the eventual development of asthma is an important question. although a significant body of information suggests an association between respiratory tract illnesses in early life and the later development of airway dysfunction, this relationship is difficult to establish and indicates the complexity of factors that surround the development of bronchial hyperresponsiveness and eventual expression of asthma. a similarly important issue to resolve is the relationship between respiratory infections and the pathogenesis of airway hyperresponsiveness. it is apparent that viral, not bacterial, upper respiratory infections (uris) trigger asthma attacks. with the use of more sensitive techniques to identify respiratory viruses, the relationship between respiratory infections, particularly viral uris, and asthma has become even more convincing and important. these episodes of wheezing become less frequent as the child grows older. however, a question still remains as to whether the initial episode of wheezing with a viral respiratory illness is an important factor in the eventual development of asthma. although a significant body of information suggests an association between respiratory tract illnesses in early life and the later development of airway dysfunction, this relationship is difficult to establish and indicates the complexity of factors that surround the development of bronchial hyperresponsiveness and eventual expression of asthma. furthermore, the source of subjects for study, i.e. follow-up of hospitalized patients vs. outpatients, contributes to the difficulty of understanding this problem. eisen and bacap found that children hospitalized for bronchiolitis prior to age 2 years had an increased risk for asthma. rooney and williams'^ also evaluated, retrospectively, the records of infants hospitalized for bronchiolitis at 18 months or younger; allergic manifestations and a family history of asthma were more frequent in children who eventually experienced one or more episodes of wheezing. finally, mcconnochie and roghmann^ identified 77 patients who had bronchiolitis at 25 months or younger and compared their outcome to children without a history of bronchiolitis. when these children were evaluated approximately 7 years later, only upper respiratory allergy, bronchiolitis and passive smoking exposure were found to be independent predictors of wheezing following bronchiolitis. consequently, it is apparent that the final conclusions on the relationship between respiratory infections in infancy and later asthma must consider a host of influences, including parental smoking, underlying airway responsiveness and gender. a similarly important issue to resolve is the relationship between respiratory infections and the pathogenesis of airway hyperresponsiveness. t o evaluate the effect of bronchiohtis on airway responsiveness, sims et al.^ identified 8-year-old children who had respiratory syncytial virus (rsv) respiratory infections and quantitated bronchial 'lability' by exercise tests. compared with appropriate controls, the fall in the peak flow with exercise was greater in children who had bronchiolitis; however, airway reactivity to exercise was not different between children with or without subsequent episodes of wheezing. since other variables confounded their study, sims et al.^ could not prove that respiratory infections led to the later development of asthma. other efforts have been made to ascertain if viral lower respiratory tract infections (lris) in early life cause persistent pulmonary function abnormalities. pullan and hey^ evaluated 130 children admitted to hospital during the first 5 years of life with rsv lris; 42% of the hospitalized children had future episodes of wheezing, while only 19% of control subjects experienced similar airway symptoms. however, few patients (6.2% vs. 4.5% of controls) had troublesome respiratory symptoms by 10 years of age. furthermore, although a three-fold increase in bronchial responsiveness was found in the children with bronchiolitis, atopy was not increased. analogous conclusions were reached by weiss et al^ when they assessed the outcome of an antecedent acute respiratory illness on airway responsiveness and atopy in young adults. airway responsiveness, evaluated by eucapnic hyperventilation to subfreezing air, was increased in children with a previous history of either croup or bronchiohtis, or greater than two acute lower respiratory illnesses. the possibility has also been raised that a predisposition to wheezing in infancy depends more on intrinsic airway structure than atopy.^ this position is supported by the high degree of airway responsiveness found in infancy in physiological evaluations^^~^^ and the incidence of wheezing with respiratory infections.^^ however, it is difficult to precisely assess airway responsiveness in young children due to limitation of lung size and other age-related factors. to help clarify the relationship between premorbid lung function and wheezing with respiratory illnesses, martinez et al}^ conducted a prospective study of respiratory illness in infancy and childhood. lung function values were determined prior to any lris. included in these measurements were tidal expiratory patterns, specifically the time to peak tidal expiratory flow (tme) divided by total expiratory time ( t e ) , or the tme/te ratio; morris and lane^^ had shown that decreasing tme/te ratios correlated with lower lung function in patients with progressive chronic obstructive lung disease. of the infants studied by martinez et al,^^ 36 developed an lri and 24 wheezed with at least one of these infections. there was no diflerence in preinfection lung function between those infants who did not have an lri and those with an infection but no wheezing (table 30 .1). however, infants who wheezed with the respiratory infection had diminished tme/te values and reduced expiratory system conductance when measured prior to wheezing with the infection. these data suggest that alterations in lung function are compatible with reduced airway conductance or a slow respiratory system time constant that precedes and predicts wheezing with respiratory infections in infants. furthermore, it appears that a given child's response to infection is determined not only by the infection but also by pre-existing lung function. taussig et al}^ also noted that lower levels of lung function predispose to wheezing with lri, as opposed to the infection per se. the precise nature of this predisposition remains to be defined but may lie in airway geometry, airway-parenchymal interaction, or mucosal and smooth muscle response. furthermore, this pulmonary-structural predisposition may be enhanced by an exaggerated ige response to viral infection,^^'^^ resulting in more inflammation and severe wheezing with hospitalization. since the majority of infants who develop wheezing with lris do not wheeze throughout life,^^ it is likely that pulmonary function abnormalities that favour wheezing with viral infections are modified with the growth and development of the lung. long-term outcome then seems to be more closely linked to the persistence of ongoing airway damage or bronchospasm associated with the development of atopy and true clinical asthma. further, and possibly definitive, insight into the relationship between wheezing with early respiratory infections and the later development of asthma has come from a unique they identified a number of factors that affect wheezing before the age of 3 years and their relationship to wheezing at 6 years of age. the study population has previously been reported^"^ and consists of newborns enrolled between 1980 and 1984 with follow-up information at 3 and 6 years of age. key assessments in infancy included cord-serum i g e levels, pulmonary function testing before any lower respiratory tract illness had occurred, measurement of serum i g e at 9 months of age, and a questionnaire completed by the children's parents when the child was 1 year old. the children were classified into four groups: no wheezing, transient wheezing, late-onset wheezing or persistent wheezing (table 30 .2, opposite). at 6 years of age, serum ige, pulmonary function testing and allergy skin testing were repeated and these factors were assessed in relationship to their history of wheezing. at 6 years of age, 20% of the children had at least one lower respiratory illness with wheezing during the first 3 years of life, but with no wheezing at age 6 years. these children had diminished airway function before the age of 1 year and, at 6 years of age, were more likely to have mothers who smoked but not mothers with asthma and did not have evidence of atopy, i.e. elevated serum i g e or skin test reactivity. in addition, 15% had no wheezing before the age of 3 years but had wheezing at age 6 years and 13.7% had wheezing both before 3 years of age and at 6 years of age. those with late-onset wheezing and persistent wheezing were more likely to have mothers with a history of asthma, elevated serum i g e levels and diminished lung function at 6 years of age (tables 30.2 -m g g <u g^ <u 0 g ^ qj on rg g i n c ( n (j <-i h i j 0 <u b o o <u < ! oj g toe g ' n (u <u (u w g o <u § 1 g <u 2 la o <u ^ •^ co g 0 co a 0 u vh o i n o d v gh g c3 th (u n <u (u vh > g 0 g <u 4-1 g c/3 g o co ' v h a. a o u a c« "g u g :3 co ' -' a ^ 0 u vh o c/^ o g n (u <u •»-> g co <u g 0 co "v1 a. a number of conclusions can be drawn from this prospective longitudinal study. most infants who wheeze early in life have a transient condition and this predilection to wheezing with respiratory infection is associated with diminished lung function; these children are not at enhanced risk for asthma or allergic disease later in life. the groups of particular interest regarding the role of respiratory infections and development of asthma are those infants with either persistent or late-onset wheezing. children with asthma at age 6 years appear to have a predisposition to asthma, as indicated by a maternal history of asthma and elevated serum i g e levels in the first years of life. moreover, these children have reduced lung function by 6 years of age. the role of respiratory infections in the actual development of asthma in those with a predisposition to asthma is less clear; that is, do respiratory infections have any relationship to the subsequent development of asthma? this issue remains to be fully elucidated. insight to factors that may determine a host's response to viral infections and its subsequent effects on lung function has been gained by animal studies. sorkness et al?^ infected neonatal rats with sendai virus. at 7 and 13-16 weeks afer infection, the rats that had been infected had lower p02 values and increased lung resistance when compared with controls. furthermore, the infected animals were more sensitive to aerosolized methacholine. lung histology in the infected animals revealed increased numbers of bronchiolar mast cells and eosinophils. in subsequent studies, these investigators have found some rat strains, i.e. brown norway, are more susceptible to the effects of the virus and more likely to have persistently altered lung function. such studies in an animal model are compatible with the observations by martinez et al?^ and indicate that many factors determine the eventual influence of respiratory infections on airway function, i.e. wheezing and bronchial hyperresponsiveness, including a predisposition to produce ige antibodies. however, what needs to be clarified are the mechanisms by which respiratory viruses interact with ige-dependent factors and promote an asthma-like picture. for decades clinicians associated asthma exacerbations with respiratory infections. with prospective studies, it became apparent that viral, not bacterial, upper respiratory infections (uris) triggered these asthma attacks. with the use of more sensitive techniques to identify respiratory viruses, the relationship between respiratory infections, particularly viral uris, and asthma has become even more convincing and important. in early studies, mcintosh and coworkers^^ prospectively studied 32 young children, aged 1-5 years, with severe asthma. the aetiology of each uri was carefully evaluated and confirmed by culture and serological tests for viral antibody titres. over 2 years, there were 102 confirmed viral respiratory infections and 139 episodes of wheezing; 58 episodes (42%) of wheezing occurred in relationship to viral respiratory infections, of which rsv was the most prevalent and likely to provoke asthma. respiratory bacteria, haemophilus influen:(ae. streptococcus pneumoniae, j?-haemolytic streptococcus and staphylococcus aureus, were also cultured but their presence did not correlate with an asthma attack. a prospective outpatient study from the university of wisconsin evaluated 16 children, aged 3-11 years, with histories of four or more asthma attacks associated with respiratory illnesses during the previous year.^^ detailed clinical records profiled each child's asthma severity, which was further substantiated with biweekly examinations. during an asthma exacerbation or apparent uri, additional bacteria and virus cultures were collected and asthma symptoms carefully quantitated. the 16 children experienced 61 episodes of asthma; 42 occurred in conjunction with a symptomatic respiratory infection and 24 were confirmed to be of viral aetiology by culture and/or serum haemagglutination titres. in this study, rhinovirus was the most frequently identified virus in association with wheezing. some patients had episodes of asymptomatic viral infection, but asthma was not worsened. only one episode of wheezing coincided with a bacterial infection. to extend these observations and to further determine if there was a relationship between different respiratory infections and wheezing, mertsola et al?^ identified 54 patients, aged 1-6 years, who had recurrent attacks of wheezing. over a 3-month observation period, these patients experienced 115 episodes of upper or lower respiratory symptoms. of these episodes, laboratory evidence of a viral or mycoplasma infection was found in 45%. more relevant to our discussion was the observation that wheezing occurred in 58% of the laboratory-confirmed viral respiratory infections. moreover, of the children with wheezing with respiratory infections, one-third were admitted to hospital because of severe dyspnoea. finally, of the agents isolated, coronavirus and rhinovirus were the most frequent isolates. there is also evidence that the viruses which cause wheezing with respiratory tract infections may be age dependent; for example, infants wheeze with rsv while older children have exacerbations of asthma with rhinovirus.^^ to extend these observations. duff and colleagues^^ examined the relationship of viral infections, passive smoke exposure and ige antibody to inhaled allergens in infants and children who were treated for acute episodes of wheezing in an emergency department. the investigators identified 99 subjects, aged 2 months to 16 years of age, and 57 control patients, aged 6 months to 16 years of age, who were seen in a paediatric emergency room. in acutely wheezing children less than 2 years of age, viruses were detected by culture of nasal washings in 70% of the patients; the most commonly identified virus was rsv. positive virus cultures for rsv were noted in only 20% of the appropriate age-matched controls. in contrast, nasal washes yielded positive cultures in only 31% of children over 2 years of age who presented with acute wheezing. however, the most frequently identified respiratory virus in this age group was rhinovirus. when these investigators evaluated risk factors associated with wheezing during the viral infections, interesting and insightful correlates were noted (table 30 .4). for children less than 2 years of age, coexisting allergic diseases were not a risk factor. rather, passive exposure to cigarette smoke, as indicated by urinary cotinine values ^1 0 jug/ml, was a risk factor for wheezing. the profile in children over 2 years of age with wheezing and viral uris was quite different. in these children, the presence of allergy, i.e. positive radioallergosorbant test (rast) values, and not passive cigarette smoke exposure, was the dominant risk factor for wheezing with viral respiratory infections. observations by duff et al}^ confirm previous information that rsv is the major viral infection causing wheezing in infants, whereas rhinovirus is the causative respiratory infection associated with wheezing in older children. moreover, wheezing in infants is not increased by the presence of allergic disease but rather made more probable if cigarette smoke exposure is present. in the older child, allergies are a major risk factor for wheezing with rhinovirus; these data also imply that a unique relationship may exist between rhinovirus and allergy, and this interaction may influence the development of wheezing with this particular respiratory virus. the detection of respiratory viruses, particularly rhinovirus, by culture is difficult because of the fastidiousness of viruses in culture. consequently, determination of the association between rhinovirus infections and provocation of asthma using virus culture may give an underestimation of the true frequency between colds and asthma. johnston ef al}^ developed a polymerase chain reaction (pcr) assay to detect picornavirus infections. this approach has an increased sensitivity and thus greater likelihood to detect causative viruses in relationship to clinical symptoms of a cold and eventual influence on asthma. using pcr technology, along with culture, johnston and colleagues^^ studied the association between upper and lower respiratory viral infections and acute exacerbations of asthma in school-age children; 108 children, 9-11 years of age, were followed over a 13-month period. children or their parents recorded the peak expiratory flow rate twice daily and daily upper and lower respiratory tract symptoms were scored as to severity. with the onset of respiratory symptoms, or a significant fall in peak expiratory flow values, the child was visited by an investigator and nasal aspirates were obtained for virus detection. there were a number of important observations by johnston and colleagues in this large study. first, there was a seasonal pattern to the appearance of respiratory symptoms and changes in peak flow values of the asthma patients; the months of n o v e m b e r -december and april appeared to be the most frequent times for colds and asthma exacerbations. second, respiratory viruses were detected in approximately 80% of episodes with respiratory tract symptoms or falls in the peak flow (table 30 .5). third, rhinovirus was the most frequently detected microorganism and constituted over 60% of the viruses detected (table 30, 6) . fourth, the severity of asthma symptoms in many of these subjects was severe. from these observations, and those of others, it is apparent that rhinovirus, the cause of the common cold, is the respiratory infection most likely to exacerbate wheezing in individuals with existing asthma. it is possible that this relationship simply reflects the fact that rhinovirus is the most common cause of infectious respiratory symptoms, i.e. the common cold. however, this association may also mean in contrast to observations in children, the relationship between respiratory infections and episodes of wheezing is not as striking in adults. when both children and adults were evaluated, respiratory viruses were more frequently identified during episodes of asthma in children under 10 years of age compared with adults. eight adult subjects evaluated by minor et alp had only three documented virus infections with increased wheezing. although an explanation for such findings is not clear, it is likely that asthma flares were more diflficult to sharply delineate and the pattern of wheezing often appeared chronic rather than episodic in adults. similarly, when hudgel and coworkers^^ evaluated 19 adult asthma patients over a 15-month period, 76 episodes of asthma were recorded but only eight could be documented with a viral url although clinically apparent viral respiratory infections precipitate asthma in adults, the relative frequency appears less than in children. in another study, huhti et al?^ evaluated adults admitted to hospital with asthma exacerbations. the viral detection rate was 19.0%, with rsv, parainfluenza and influenza the more frequent isolates. using virus culture and pcr techniques, nicholson and coworkers^^ evaluated the relationship of symptomatic colds and asthma exacerbations in 138 adult asthmatic subjects. these investigators found symptomatic colds were reported in 80% of episodes with symptoms of wheeze, chest tightness or breathlessness. in 24%) of laboratoryproved episodes of non-bacterial respiratory infections, there was a reduction in peak flow ^ 50 litre/min. infections with rhinoviruses, coronaviruses, influenza, rsv, parainfluenza and chlamydia were all associated with objective measures of airflow limitation. the findings of nicholson et al. are further evidence of the importance of respiratory viruses, particularly rhinovirus, to asthma exacerbations, and that these episodes can be severe and occur in adults. therefore, evidence exists to underscore the importance of viral respiratory infections to asthma exacerbations in patients of all ages. there is little evidence that bacterial respiratory infections, other than sinusitis, provoke asthma. berman et al?^ performed transtracheal aspirates on 27 adult patients with asthma during an infectious exacerbation. bacteria cultured from transtracheal aspirates were sparse and, more importantly, did not correlate with clinical illness. moreover, transtracheal aspirates from normal subjects, without respiratory infections, yielded a similar degree of bacterial colonization. a common complication of colds is the development of sinusitis, which has been proposed as a factor in the worsening of asthma. although the association between paranasal sinusitis and bronchial asthma has long been observed, it is conceivable that sinusitis and asthma may coexist as complications of the same respiratory infection. however, some feel that an aetiological relationship exists between sinusitis and asthma.^"^'-^^ although these observations and associations are intriguing and clinically important, additional studies are needed to clarify how sinusitis may influence asthma. a number of mechanisms have been proposed to explain how viral respiratory infections provoke asthma (table 30 .7). when evaluating these possibilities, it is important to realize that the effect respiratory viruses have on airway function will depend upon many factors, including the subject's gender, environmental factors such as cigarette smoke exposure, age, baseline lung function and atopic status, along with the particular respiratory virus. however, there is now convincing evidence that rhinoviruses are the predominant respiratory infection provoking wheezing in patients with asthma and the existence of allergic disease appears to be an important risk factor. therefore, it is proposed that respiratory viruses, particularly rhinoviruses, may interact with those factors that contribute to or determine existing allergic airway disease and enhance existing bronchial inflammation. the end-result of the eflects of the virus on underlying altered epithelial function altered autonomic nervous system regulation of airway smooth muscle function enhanced allergic inflammation enhanced eosinophilic recruitment eosinophilic infiltration of the airway generation of pro-inflammatory cytokines enhanced inflammatory mediator release altered j5-adrenergic function sinopulmonary reflex production of virus-specific ige antibodies allergic airway disease will be an enhanced likelihood for wheezing. although other factors likely participate in this process, the following discussion examines how respiratory viruses either influence ige-dependent events or promote the development of allergic inflammation. welliver et al}'^ tested 79 children, all less than 12 months of age and with documented rsv infection, for the presence of ige-specific antibody to the infecting virus. their clinical patterns of illness were divided into four groups: (a) upper respiratory tract illness; (b) pneumonia without wheezing; (c) pneumonia and wheezing; and (d) wheezing (bronchiolitis). nasal secretions from each patient were measured for ige-specific antibody to rsv, and rsv ige antibody titres were compared with the patient's clinical illness. i g e titres to rsv were highest in patients with evidence of airway obstruction, e.g. pneumonia/wheezing or bronchiolitis. parainfluenza virus-specific ige responses were also examined in individuals with upper respiratory illness alone or bronchiolitis by welliver et al}^ parainfluenza-specific ige antibody were detected in 8 of 12 with bronchiolitis but only 1 of 10 with upper respiratory disease alone. these studies suggest that some respiratory viruses stimulate ige-specific antibody responses and the degree to which this occurs is associated with the clinical manifestations of upper vs. lower airway disease. to further evaluate the significance of virus-specific i g e antibody, welliver et al?^ prospectively monitored 38 infants for 48 months after an initial episode of bronchiolitis. only 20% of infants with undetectable titres of rsv ige had subsequent episodes of documented wheezing. in contrast, 70% of those children with high rsv i g e antibody titres continued to experience wheezing when evaluated 4 years later. a portion of these same subjects was evaluated at 7-8 years of age^'^ and underwent skin testing to common inhalant allergens, pulmonary function testing and methacholine provocation to determine bronchial responsiveness. for the entire study groups, rsv i g e responses were evaluated in relationship to family history of asthma, passive smoke exposure, number of recurrent wheezing episodes, number of positive skin tests and the degree of airway responsiveness to methacholine. the investigators found that an association exists between rsv ige responses and the development of any wheezing following bronchiolitis; the nature, however, of the association between rsv i g e antibodies and any recurrent wheezing is unclear. therefore, the consequence of an initial i g e response to the infecting virus with regard to long-term influence on airway function and the development of asthma is unresolved. although rhinoviruses have emerged as the most common respiratory infection causing asthma exacerbations,^^ there are a number of paradoxes to this relationship. first, rhinoviruses are infections that appear localized to the upper airway; identification of rhinovirus by culture in the lower airway is unusuap^ and pneumonic infiltrates rare. second, even though rhinoviruses infect airway epithelium, the inflammatory response is often unimpressive. for example, fraenkel and coworkers^^ performed nasal biopsies before and after an experimental rhinovirus infection. in both normal and atopic groups, there were no significant changes in inflammatory cells during the cold or the convalescent period compared with baseline values. thus, immunohistochemical analysis of rhinovirus-infected upper airways does not reveal inflammation. third, rhinovirus infections tend to be self-limited and later sequelae are rare. therefore, the mechanisms of lower airway dysfunction are not readily explained by an overzealous inflammatory airway response to this particular virus. the frequent association of asthma with respiratory infections, and in particular rhinovirus illnesses, may relate to the frequency with which rhinoviruses are the cause of colds. this frequency, however, does not explain the apparently serious lower airway events that are associated with rhinovirus infections in subjects with asthma. a major goal of current research is to establish how this particular class of respiratory viruses affects lower airways so profoundly and how these effects can last for weeks beyond the acute respiratory illness. in an initial series of experiments to evaluate the effects of respiratory infection on airway responsiveness, we infected subjects with rhinovirus experimentally and determined its effect on a variety of airway functions."^^ the patients selected for study were evaluated on three separate occasions: at baseline, during acute infection and during recovery. of particular interest was the effect of the infection on the immediate and latephase allergic response (lar) to inhaled antigen. all 10 patients had a rhinovirus respiratory infection at time of study. during the acute rhinovirus respiratory infection, airway responsiveness to histamine significantly increased over baseline values. likewise, acute airway reactivity to inhaled antigen was increased. the change in airway responsiveness to histamine and antigen was similar, suggesting that the effects of the respiratory infection on responsiveness to antigen related to alterations in non-specific bronchial reactivity. prior to rhinovirus inoculation, only 1 of 10 patients had an lar to inhaled antigen. however, during the acute respiratory infection, 8 of 10 patients experienced late-phase airway obstruction to inhaled antigen challenge. furthermore, when evaluated during the recovery period (4 weeks after rhinovirus inoculation), five of the seven patients available for testing still had lars to inhaled antigen. these observations indicate that rhinovirus respiratory infection not only increased airway responsiveness but also changed the pattern of the airway response to inhaled antigen and promoted the development of allergic inflammation, as indicated by the development of the lar. the recovery pattern of airway hyperresponsiveness following the viral respiratory infection was also evaluated. although increased airway responsiveness was still detected 4 weeks after rhinovirus inoculation, there was a trend towards recovery in both the reaction to inhaled histamine and the response to antigen. however, as already discussed, the increased frequency of lars to antigen was still noted 4 weeks after the viral infection, suggesting that a viral respiratory infection has a greater, and possibly more lasting, effect on factors that participate in the development of lars. virus-associated airway hyperresponsiveness is a multifactorial process involving a complex interplay of ige-dependent reactions, epithelial activation or damage, autonomic nervous system dysfunction and, of particular interest and relevance to our discussion, enhanced allergic inflammation."^^ in ige-mediated reactions, the tissue response, be it the skin, nose or airway, is influenced by i g e sensitization of mast cells and basophils, release of bronchospastic and inflammatory mediators from sensitized cells, and the response of the target organ, which in asthma is bronchial smooth muscle. t o evaluate the effects of respiratory viruses on a facet of the immediate hypersensitivity response, i.e. mediator release, basophil histamine secretion was determined. in an in vitro model system, peripheral blood mononuclear cells were incubated with influenza a virus and the effect of this exposure upon ige-dependent basophil histamine release and leukotriene (lt)c4 was measured. following incubation with virus, both histamine and ltc4 secretion was enhanced. t o define the role of tcells in this process, t-cells were removed by magnetic head separation with anti-cd3 antibody. in the absence of t-cells, virus incubation did not enhance histamine release."^^ furthermore, analysis of virus-treated peripheral blood mononuclear cells revealed that the virus caused the generation of interferon (ifn)-y. these findings suggest that t-cells, and their cytokine products such as ifn-y, may play an integral role in the processes by which respiratory viruses enhance basophil histamine release, and are likely to affect other cell functions. the use of bronchoscopy with lavage and biopsy has provided direct assessment and analysis of airway mediators and the histology of inflammatory events in asthma. we have used fibreoptic bronchoscopy to perform segmental bronchoprovocation with antigen and bronchoalveolar lavage (bal) to assess immediate and late responses of the airway to antigen and to study the effects of a rhinovirus illness on these allergic events."^"^ t o conduct these experiments, a bronchoscope is introduced into the airways, bronchial segments are identified and an allergen is administered directly on to the airway mucosa. the immediate response to antigen is characterized by a brisk rise in mast cell mediators. when lavage of the previously allergen-challenged segments is performed 48 h later, there is a highly cellular, eosinophil-rich response detected. based on our previous observations that a rhinovirus 16 infection increases bronchial responsiveness and lars to inhaled antigen, we used segmental antigen challenge and lavage to test our hypothesis that the rhinovirus upper respiratory illness may enhance the lower airways allergic inflammatory response to antigen. histamine is released into the airway immediately following antigen challenge and represents mast cell activation. we found histamine release into the airway immediately following local antigen challenge to be significantly potentiated by rhinovirus infection (table 30 .8). these observations indicate that an acute rhinovirus illness can enhance airway mast cell mediator release. these findings are similar to earlier reports and indicate an effect of the infection on mast cell activation."^"^ interestingly, and relevant to earlier observations that rhinovirus effects on airway function are long-lasting, enhanced histamine release to antigen was still present 6 weeks after the acute infection; at this time, the individuals were symptom-free. our findings suggest that viral effects on allergic responses can persist for weeks beyond the acute illness and that such persistence can explain the long-lasting effects on airway function in asthma from a cold. when lavage is performed 48 h after antigen challenge, there is a characteristic and often dramatic influx of eosinophils into the lavage fluid. we found significant enhancement of eosinophil recruitment to the airway 48 h after antigen when the subjects had an acute rhinovirus infection (fig. 30.1) . like the observations with enhanced and persistent histamine release, not only was a greater eosinophil recruitment to antigen challenge noted during the acute infection, but this respiratory virus effect persisted into the recovery period 6 weeks later. these findings suggest a possible explanation for the increase in lars to antigen noted during acute rhinovirus infection; if allergen exposure during rhinovirus infection is associated with enhanced eosinophil recruitment to the airway, allergic inflammation and an lar are more likely to occur. fraenkel and colleagues'^^ provide additional insight into the mechanisms by which rhinovirus infections promote airway inflammation and the likelihood for increased asthma during colds. both normal and asthmatic subjects were recruited for their study. the investigators obtained bronchial mucosal biopsies before and during an acute experimental rhinovirus infection and evaluated the changes in inflammatory cell infiltrate due to the virus and airway responsiveness. an increase in the airway response to histamine during the cold was associated with increases in submucosal lymphocytes. further, there was an increase in epithelial eosinophils during the experimental cold ( fig. 30.2) . in asthmatic compared with normal subjects, the increase in mucosal eosinophils persisted into the convalescent period. although the changes in various features of immunohistology were not dramatic, they did follow a pattern that is consistent with our findings of enhanced eosinophil recruitment to the airways and were correlated with alterations in lung physiosiveness. collectively, there is evidence from airway physiology that rhinovirus infections enhance bronchial responsiveness and histological features of allergic inflammation, i.e. there is a greater likelihood for lars to occur. furthermore, from studies that directly sample the lower airway, there is evidence of increased eosinophil recruitment and mucosal eosinophilia at the time of rhinovirus infection, and these abnormalities persist for weeks beyond the acute illness. all these findings indicate that rhinovirus infections can perpetuate features of allergic inflammation and may thus explain an increase in the likelihood of asthma. the questions now remaining to be answered concern the mechanisms by which rhinovirus infections enhance allergic airway inflammation. using the nasal mucosal response to rhinovirus as a surrogate of the airway response to rhinovirus, fraenkel et al?^ biopsied nasal tissue during an acute rhinovirus infection. mast cells, eosinophils, lymphocytes and neutrophils were identified by appropriate monoclonal antibodies. there were no significant changes in the numbers of inflammatory cells present during the cold or the convalescent period compared with baseline values. although these observations contrast with biopsy findings in the lower airway,'^^ changes in airway 0.048 0028 100-, histology were not dramatic. these findings suggest that other mechanisms, i.e. enhanced mediator release or, more likely, cytokine generation, are responsible for changes in airway function during a cold. in our study with segmental antigen challenge,"^-^ the lavage fluid was analysed for tumour necrosis factor (tnf)-a. lavage samples obtained 48 h after antigen challenge had a significant rise in tnf-a; these changes were noted in both normal controls and allergic subjects. o u r findings raise the possibility that rhinovirus infection causes secretion of tnf-a and if enhanced secretion of cytokines were to occur in the allergic subject, features of allergic inflammation are more likely to occur. these findings also raise the possibility that rhinovirus can directly stimulate cytokine production. linden et a/.^^ measured ifn-y, interleukin (il)-1)s, granulocyte-macrophage colony-stimulating factor (gm-csf), il-4 and il-6 in nasal secretions of subjects with allergic rhinitis. during an experimental coronavirus cold, there was an increase in ifn-y but not the other cytokines. the increase in ifn-y is expected and represents an antiviral response to the virus. however, ifn-y can have pro-inflammatory effects, i.e. promotion of adhesion protein expression and enhanced survival of eosinophils."^^ moreover, these observations indicate that rhinovirus infections can stimulate cytokine production in vivo. t o extend these observations, live rhinovirus was incubated with human mononuclear cells and lung macrophages. gern et al.^'^ found that these cells took up the virus, but it did not replicate inside the cells. none the less, rhinovirus was able to activate monocytes and airway macrophages and cause secretion of tnf-a. these observations indicate that rhinovirus can cause synthesis and release of pro-inflammatory cytokines, which may occur independent of cell infection. to identify the mechanisms by which rhinovirus promotes allergic inflammation, it is essential that the immune response to this virus be established so that the factors, and cells, involved in the promotion of allergic inflammation are identified. in determining the effect of rhinovirus on the immune response, it has been established that the receptor for the majority (90%) of rhinovirus strains is intercellular adhesion molecule (icam)-!."^^ the other 10% of rhinovirus strains use the low-density lipoprotein receptor.^^ interaction of rhinovirus with these receptors is the first step in the host's response to this virus. with this information, we have found that a major rhinovirus, rhinovirus 16, binds to mononuclear cells via the icam-1 receptor, with binding occurring predominantly to monocytes but not lymphocytes. when rhinovirus 16 was incubated with a mixture of monocytes and lymphocytes, the following events occurred: there was a large increase in the number of cd3 "^ cells that express the early activation marker cd69; when the cd3 "^ /cd69 "^ cells were isolated and analysed, they were found to have increased expression of mrna for ifn-y. these in vitro observations suggest that rhinovirus activates monocytes to generate cytokines that stimulate lymphocytes. once activated, these lymphocytes (cds "^/cd69 "^) generate a variety of cytokines, including ifn-y. although ifn-y is normally important in antiviral activity, it also possesses a number of pro-inflammatory functions, including increased expression of endothelial and epithelial icam-1 (more receptors for rhinovirus) and the promotion of eosinophil survival. therefore, in addition to activation of a small number of rhinovirus-specific lymphocytes, rhinovirus can cause a 'non-specific' activation of lymphocytes through the generation of cytokines from monocytes; these cytokines can, in turn, promote either viral elimination or inflammation. perhaps the sequelae of this particular response are more likely if allergic inflammation already exists. to extend this possibility, einarsson and coworkers^^ evaluated the effect of respiratory viruses on stromal cell production of il-11. il-11 is an extremely cationic cytokine and has the capacity to induce airway hyperresponsiveness in the murine lung. these investigators found that rhinoviruses, along with rsv and parainfluenza, were potent stimulators of il-11. in contrast, cytomegalovirus and adenovirus were weaker activators and are viruses not associated with asthma exacerbations. furthermore, increased levels of il-11 were found in nasal secretions of children with uris and were more likely to appear in the nasal aspirates of those children with reactive airway disease. these observations are another example of the mechanism by which respiratory viruses can enhance airway inflammation through the generation of specific cytokines and thus increase the likelihood of asthma during a cold. using a mouse model, coyle et al?^ characterized the immune response to virus peptides and the resulting cytokine patterns. these investigators found that bystander cd4"^ th2 immune responses to ovalbumin switched peptide-specific cds"^ t-cells in the lung to il-5 producers. when these il-5-producing cds"^ t-cells were challenged, via the airways, with virus peptide, a significant eosinophil infiltration was induced. furthermore, in vitro studies indicated that il-4 could switch virus-specific cd8~^ t-cells to il-5 production. these results demonstrate a network by which allergic eosinophilic inflammation can occur during a respiratory viral infection and how this mechanism would impair cd8~^ t-cell responses (less ifn-y production) and thus delay virus clearance. such observations indicate not only a network to promote allergic inflammation but also a mechanism by which virus particles may persist for extended periods. our studies in humans, and those of coyle et al}^ in the mouse, suggest a possible link between eosinophils and airway changes during viral respiratory infections. when garofalo et alp sampled nasolaryngeal samples from children with rsv respiratory illnesses, the eosinophil protein, eosinophil cationic protein, was significantly increased in samples from children with bronchiolitis when compared with subjects with upper airway disease or rsv pneumonia but no wheezing. these data suggest that eosinophil activation by rsv may cause airway injury and the presence of wheezing. to extend these findings with in vitro experiments, kimpen et al}"^ incubated isolated human eosinophils with rsv. rsv directly activated eosinophils to release superoxide and ltc4. furthermore, incubation of eosinophils with rsv primed the cell for further activation and generation of inflammatory mediators. both observations indicate that an airway-active virus, in this case rsv, can interact with eosinophils and cause them to release proinflammatory mediators. as indicated by the authors, these effects of the virus could play a role in the pathogenesis of rsv bronchiolitis and may also be important for the development of more long-term bronchial hyperresponsiveness associated with viral infections. it is also possible that other respiratory viruses, such as rhino virus, may have similar effects during a cold. the mechanisms involved in the development of airway hyperresponsiveness, airway obstruction and recurrent wheezing with viral respiratory infections have yet to be fully appreciated. none the less, current evidence indicates that viruses, or products of virusinfected cells, influence the inflammatory property and potential of many cells. precisely how these effects of the virus translate into increased airway injury, responsiveness and obstruction will require further work. as the mechanisms of these interactions are established, so will improved understanding of asthma pathogenesis and treatment. the significance of early wheezing lung function, pre-and post-natal smoke exposure, and wheezing in the first year of life the relationship of acute bronchiohtis to bronchial asthma: a 4-to-14 year follow-up the relationship between proven viral bronchiolitis and subsequent wheezing bronchiolitis as a possible cause of wheezing in childhood study of 8-year-old children with a history of respiratory syncytial virus bronchiolitis in infancy wheezing, asthma, and pulmonary dysfunction 10 years after infection with respiratory syncytial virus in infancy the relationship of respiratory infections in early childhood to the occurrence of increased levels of bronchial responsiveness and atopy viral respiratory infection in infancy: provocation or propagation? semin respir m^^ airway reactivity in infants: a positive response to methacholine and metaproterenol airway response to cold, dry air in normal infants response of normal infants to inhaled histamine \ the tucson children's respiratory study. 11. lower respiratory tract illness in the first year of life diminished lung function as a predisposing factor for wheezing respiratory illnesses in infants tidal expiratory flow patterns in air-flow obstruction lung function in infants and young children: functional residual capacity, tidal volume, and respiratory rate the development of respiratory syncytial virus specific ige and the release of histamine in nasopharyngeal secretions after infection role of parainfluenza virus-specific ige in pathogenesis of croup and wheezing subsequent to infection lower respiratory illness in early childhood and lung function and bronchial reactivity in adolescent males asthma and wheezing in the first six years of life persistent airway hyperresponsiveness after neonatal viral bronchiolitis in rats the association of viral and bacterial respiratory infections with exacerbations of wheezing in young asthmatic children viruses as precipitants of asthmatic attacks in children recurrent wheezy bronchitis and viral respiratory infections viruses as precipitants of asthma symptoms. i. epidemiology risk factors for acute wheezing in infants and children: viruses, passive smoke, and ige antibodies to inhalant allergens use of polymerase chain reaction for diagnosis of picornavirus infection in subjects with and without respiratory symptoms. / clin microbiol (\99?>) community study of role of viral infections in exacerbations of asthma in 9-11 year old children rhinovirus and influenza a infections as precipitants of asthma viral and bacterial infections in adults with chronic asthma association of viral and mycoplasma infections with exacerbations of asthma respiratory viruses and exacerbations of asthma in adults transtracheal aspiration studies in asthmatic patients in relapse with ^infective' asthma and in subjects without respiratory disease chronic sinus disease associated with reactive airway disease in children predictive value of respiratory syncytial virusspecific ige response for recurrent wheezing following bronchiohtis the relationship of rsv-specific immunoglobulin e antibody responses in infancy, recurrent wheezing, and pulmonary function at age 7-8 years exacerbations of asthma in adults during experimental rhinovirus infection \ immunohistochemical analysis of nasal biopsies during rhinovirus experimental colds rhinovirus upper respiratory infection increases airway reactivity in late asthmatic reactions respiratory infections: their role in airway responsiveness and pathogenesis of asthma the effect of t cell depletion on enhanced basophil histamine release after in vitro incubation with live influenza virus a common cold virus, rhinovirus 16, potentiates airway inflammation after segmental antigen bronchoprovocation in allergic subjects experimental rhinovirus 16 infection potentiates histamine release after antigen bronchoprovocation in allergic subjects lower airways inflammation during rhinovirus colds in normal and in asthmatic subjects nasal cytokines in common cold and allergic rhinitis glucocorticoids inhibit cytokine-mediated eosinophil survival rhinovirus enters but does not replicate inside monocytes and airway macrophages a cell adhesion molecule, icam-1, is the major surface receptor for rhinovirus members of the low density lipoprotein receptor family mediate cell entry of a minor-group common cold virus interleukin-11: stimulation in vivo and in vitro by respiratory viruses and induction of airways hyperresponsiveness \ virus-specific cd8^ cells can switch to interleukin 5 production and induce airway eosinophilia eosinophil degranulation in the respiratory tract during naturally acquired respiratory syncytial virus infection activation of human eosinophils in vitro by respiratory syncytial virus acknowledgements support for this chapter has come from grants nih hl 44098, ai-26609, ko8-01828 and general clinical research grant rr-03186. key: cord-024183-1mrdjc39 authors: hutchison, alastair a.; leclerc, francis; nève, véronique; pillow, j. jane; robinson, paul d. title: the respiratory system date: 2013-10-08 journal: pediatric and neonatal mechanical ventilation doi: 10.1007/978-3-642-01219-8_4 sha: doc_id: 24183 cord_uid: 1mrdjc39 this chapter addresses upper airway physiology for the pediatric intensivist, focusing on functions that affect ventilation, with an emphasis on laryngeal physiology and control in breathing. effective control of breathing ensures that the airway is protected, maintains volume homeostasis, and provides ventilation. upper airway structures are effectors for all of these functions that affect the entire airway. nasal functions include air conditioning and protective reflexes that can be exaggerated and involve circulatory changes. oral cavity and pharyngeal patency enable airflow and feeding, but during sleep pharyngeal closure can result in apnea. coordination of breathing with sucking and nutritive swallowing alters during development, while nonnutritive swallowing at all ages limits aspiration. laryngeal functions in breathing include protection of the subglottic airway, active maintenance of its absolute volume, and control of tidal flow patterns. these are vital functions for normal lung growth in fetal life and during rapid adaptations to breathing challenges from birth through adulthood. active central control of breathing focuses on the coordination of laryngeal and diaphragmatic activities, which adapts according to the integration of central and peripheral inputs. for the intensivist, knowledge of upper airway physiology can be applied to improve respiratory support. in a second part the mechanical properties of the respiratory system as a critical component of the chain of events that result in translation of the output of the respiratory rhythm generator to ventilation are described. a comprehensive understanding of respiratory mechanics is essential to the delivery of optimized and individualized mechanical ventilation. the basic elements of respiratory mechanics will be described and developmental changes in the airways, lungs, and chest wall that impact on measurement of respiratory mechanics with advancing postnatal age are reviewed. this will be follwowed by two sections, the first on respiratory mechanics in various neonatal pathologies and the second in pediatric pathologies. the latter can be classified in three categories. first, restrictive diseases may be of pulmonary origin, such as chronic interstitial lung diseases or acute lung injury/acute respiratory distress syndrome, which are usually associated with reduced lung compliance. restrictive diseases may also be due to chest wall abnormalities such as obesity or scoliosis (idiopathic or secondary to neuromuscular diseases), which are associated with a reduction in chest wall compliance. second, obstructive diseases are represented by asthma and wheezing disorders, cystic fibrosis, long term sequelae of neonatal lung disease and bronchiolitis obliterans following hematopoietic stem cell transplantation. obstructive diseases are defined by a reduced fev1/vc ratio. third, neuromuscular diseases, mainly represented by dmd and sma, are associated with a decrease in vital capacity linked to respiratory muscle weakness that is better detected by pimax, pemax and snip measurements. • describe the expanded concept of breathing in terms of multiple motor functions that ensure airway protection, volume homeostasis, and ventilation. • describe the upper airway structures and how their functions affect the entire airway. • describe the nasal functions involved in air conditioning, in protection, in the maintenance of airway patency, and in the consequences of nasal bypass and obstruction. • describe the oral cavity and pharyngeal regions and control of airway patency. • describe factors involved in obstructive sleep apnea and the effects of cpap therapy. • describe the coordination of sucking, nutritive swallowing, and breathing during development and the relationship to apnea and bradycardia. • describe the importance of nonnutritive swallowing in pharyngeal clearance and how this may be influenced by cpap. • describe the three major functions of the larynx involving breathing. • describe the triple mechanism of laryngeal closure in airway protection. • describe how flow and subglottic volume are controlled during eupnea. • describe the integrative nature of the central control of laryngeal functions. • describe the roles of laryngeal muscles and their coordination with pump muscles during development and in eupnea, sighs, grunting, incremental breathing, and gasping. • describe how this information has been/ can be applied in respiratory support. • describe afferent inputs that alter laryngeal muscle functions and alter breathing and cardiovascular function. • describe the impact of central behavioral state and induced central depression upon laryngeal function and the interactions with detected changes in the gaseous environment. it is impossible for anyone to find the correct function of a part unless he is perfectly acquainted with the action of the whole instrument. galen, ad 120-200. breathing consists of multiple motor acts whose mechanical effects aim to ensure that the airway is protected, has optimal supra-and subglottic volumes, and provides vital ventilation. the upper airway extends from the nose or mouth to the larynx (seikel et al. 2005) , but its functions, in the context of ventilation, extend to the entire airway. maintenance of airway volume and ventilation is actively controlled by the centrally coordinated activities of nasal, oral, pharyngeal, and laryngeal upper airway muscles and those of the pump muscles. these act in concert with lower airway smooth muscle. effective breathing requires that the brain coordinates ventilatory functions with cardiovascular function and with other motor acts, e.g., swallowing, postural changes, and speech. adaptations in breathing must occur appropriately, rapidly, and constantly. protective and mechanical changes occur in milliseconds. changes in circulatory gas transport alter breathing within seconds. other adjustments optimize breathing in tune with changes in growth, aging, metabolism, and the environment. this chapter addresses how each upper airway structure plays critical roles in the dynamic processes of breathing. … we would do well to keep our mouths shut and reflect on the marvelous physiology of the nose. richard godfrey 1994 normal breathing occurs via the nose (fig. 4.1 ). inspired and expired air is conditioned by the nasal mucosa, whose surface area and blood supply are such that particles are filtered and temperature and humidity are modified. the nasal vascular system can achieve a 25 °c gradient between the nasal ostia and the nasopharynx (godfrey 1994) , a property that enables air breathing at very different environmental temperatures while maintaining a remarkable constancy of temperature and humidity in the lower airway, thereby optimizing cellular function and ciliary action. given its vascularity, it is not surprising that the nose is a source of nitric oxide, that trauma to the nasal passages can be induced by nasal continuous positive airway pressure (ncpap) prongs and other cannulae, and that epistaxis can require hospital admission. the nose can sniff out noxious substances and protect the airway from particles by sneezing. it can increase its resistance rapidly, switching on a watery secretion "like a tap" (godfrey 1994) , and can trigger laryngeal closure and reduced ventilatory drive (editorial 1992) . the extreme protective reflex response to nasal irritation is the dive reflex that is typified by apnea with glottic closure, bradycardia, and redistribution of blood flow to the brain, heart, and adrenals (see also sect. 47.3.1) (de burgh daly 1997; editorial 1992) . the dive reflex can be triggered by nasal water, tobacco smoke, or ice applied to the trigeminal area (angell james and de burgh daly 1969; de burgh daly 1997) . water entry into the nose is dramatic even for adults who "suffer a sense of impending suffocation and the almost impossibility of voluntarily making an inspiratory effort" (de burgh daly 1997) . exaggerated dive reflex responses can cause total upper airway closure and cardiac arrest and have been implicated in accidental and deliberately induced deaths (de burgh daly 1997). nasal breathing in the newborn and infant is the predominant but not obligatory form of breathing and is related to the high position of the epiglottis ( fig. 4 .1) (seikel et al. 2005 ). there is a nasal cycle whereby resistance alternates from one nostril to the other every 2-4 h in 80 % of humans (widdicombe 1986) . pressure in the axilla or on the lateral chest increases ipsilateral nasal resistance (widdicombe 1986) . resistance is highest in the regions adjacent to the external ostia, whose dimensions are altered by the alae nasi muscles that cause nasal flaring during exercise hyperpnea and respiratory distress, especially in newborns. newborn nasal resistance exceeds that of the adult in absolute terms but is less than that of their lower airways. nearly half of the adult's total airway resistance is nasal. thus, minor increases in nasal resistance can produce major overall effects on breathing (editorial ). in adult humans, nasal occlusion results in a switch to mouth breathing in <5 s (editorial 1992) . both this switch to oral breathing and the resumption of nasal breathing following release of the occlusion are delayed by nasal and pharyngeal anesthesia. occlusion of the nasal passages in infants results in mouth breathing in ~8 s (range <1-32 s) (editorial 1992; rodenstein 2004) . the response is quicker with advancing age but is decreased in rapid eye movement (rem) sleep, more so at 6 weeks than in the newborn period (editorial 1992) . these observations led to a proposal that the response may be related to the sudden infant death syndrome (sids) (editorial 1992) . in premature infants, the change to oral breathing results in a sixfold increase in pulmonary resistance, which over time might result in fatigue with hypoventilation (miller et al. 1987) . oral breathing places an increased burden on the lower airway to modify the temperature and humidity of inspired gas, presumably via changes in lower airway vascularity. preventing nasal breathing can decrease gas exchange and functional residual capacity (frc) (editorial 1992) . postoperative nasal packing in adults can lead to arterial hypoxemia, apnea, and sleep disturbance, with the latter also occurring in normal volunteers after local anesthesia of the nasal passages (editorial 1992) . in newborn lambs, nasal obstruction also affects gas exchange, with hypoxemia, hypercapnia, and acidosis being observed. these effects are aggravated by carotid body denervation. the impact of nasal obstruction diminishes with advancing age (editorial 1992) . blockage of the nose (choanal stenosis being an extreme example) can cause obstructive sleep apnea (osa) (marcus 2000) . human neonates whose nasal resistance is increased by birth trauma or trauma secondary to repeated suctioning breathe orally. they can have cyanotic episodes, stridor, and hypercapnia; these problems disappear with resolution of the nasal injury (miller et al. 1987) . in summary, the nose provides air conditioning, and its sensory receptors initiate reflexes that alter upper airway patency and mediate extended ventilatory changes in response to a challenge. functions in breathing …after relaxing, many sword swallowers are able to relax and breathe while swallowing a sword. from swordswallow.com 2009. the oral cavity extends from the lips to the plane of the faucial pillars, where it joins the pharynx ( fig. 4.1) . the three pharyngeal regions are the nasopharynx, from the posterior nares to the uvula; the oropharynx, from the uvula to the epiglottis; and the laryngopharynx, from the epiglottis to the esophagus. the pharynx is a conduit for air and/or food. the muscles of the tongue, especially the genioglossi, maintain patency of the oral cavity. pharyngeal patency is a function of the cavity dimensions (rodenstein 2004 ) and the balance between the opening forces exerted by pharyngeal muscle activities and the collapsing ones exerted by the tissue-intrapharyngeal pressure gradient and by pharyngeal mucosal adhesion (fig. 4.2) . patency is critically influenced by posture. narrowing occurs with neck flexion and hyperextension and can alter cavity pressures required to achieve flow. narrowing can also follow an inspiratory occlusion of the mouth/nose (a load) that augments diaphragmatic activity and thus negative pressure within the pharynx. upper and lower airway afferents induce reflex compensatory change in glossopharyngeal muscles to offset the load effect ( fig. 4 .2) (bailey and fregosi 2006) . obstructive apnea occurs during sleep in ~1-4 % of children and adults (marcus 2000) . in children, a persistent partial form of upper airway obstruction, obstructive hypoventilation, is seen (marcus 2000) . in adults, a spectrum of decreased inspiratory airflow is seen in snoring, upper airway resistance syndrome, and osa. pharyngeal osa occurs when a critical tissue pressure (p crit ) exceeds the intrapharyngeal opening pressure (fig. 4 .2) (marcus 2000; dempsey et al. 2010) and is influenced by factors that alter pharyngeal dimensions, namely, adenotonsillar hypertrophy, obesity (less common in children than adults), low subglottic airway volume, and craniofacial structural abnormalities. the major role played by the central motor output to the oral cavity and pharyngeal muscles that modify p crit is emphasized by the fact that osa occurs only in sleep, predominantly in rem sleep in children (marcus 2000) . the following factors also stress the importance of central control in osa. obstructive apnea occurring during mixed apnea in preterm newborns involves a direct central mechanism, as shown by the onset of pharyngeal and/or laryngeal closure before diaphragmatic activation (idiong et al. 1998 ) (see sect. 47.3.1) . in adults pharyngeal closure can accompany central apnea (marcus 2000) . central arousal patterns during osa differ between children and adults (marcus 2000) . other factors affecting the incidence of osa are gender, a familial tendency, and racial/ethnic factors (marcus 2000) . anti-inflammatory medications can benefit osa, suggesting a role for inflammation in its etiology (praud and dorion 2008) . therapy with ncpap improves osa by increas-ing intrapharyngeal pressure and its transverse diameter (rodenstein 2004) , by augmenting lower airway volume and probably by stimulating breathing via pharyngeal pressure sensors (angell james and de burgh daly 1969) . in children and adults with osa, cpap therapy can improve their metabolism. the abilities to suck, swallow, and breathe are developed during fetal life (harding 1986 ). at all ages, swallowing is accompanied by laryngeal closure. term newborns and infants up to 12-18 months of age can lock the larynx into the nasopharynx (figs. 4.1 and 4.3) . this separation of the nasopharyngeal-laryngeal air passage from the oropharyngeal nutritive passage enables the term infant to breathe and feed simultaneously but with decreased ventilatory drive. this ability is not developed in the preterm neonate whose sucking pattern is also immature (lau 2006) . coordination of sucking-swallowing-breathing is defined by the ability to feed "by mouth with no overt signs of aspiration, oxygen desaturation, apnea, or bradycardia" and when "a ratio of 1:1:1 or 2:2:1 suck: swallow: breathe" is attained (lau 2006) . safe swallowing occurs at end-expiration and end-inspiration (lau 2006) . in term newborns, 55 % of swallows occur at these points of the breathing cycle. in preterm infants, 55 % of swallows occur during deglutition apnea (lau 2006 nonnutritive swallowing (nns) is "essential for survival," since >2 l of oral and nasal secretions are produced daily. without nns, "the lungs rapidly fill with these secretions, producing death within a few days" (thach 2005) . when pharyngeal secretions reach a critical volume, neural receptors, in the interarytenoid notch, stimulate the laryngochemoreflex which triggers nns (thach 2005) . the critical pharyngeal volume for nns may be increased by applied positive pharyngeal pressure, providing an explanation for the decrease in nns frequency with cpap (thach 2005) . in adult humans nns occurs mainly during expiration. in infants nns occurs at any time in the respiratory cycle (thach 2005) . volitional swallowing in adults clears the pharynx and reduces the protective laryngeal adductor reflex. during sleep, the protection provided by nns and glottic closure is imperfect, and some aspiration is common in normal individuals (thach 2005) . swallow breaths are short inspirations that precede a swallow, occurring when the upper pharyngeal sphincters are closed (thach 2005) . these breaths are thought to result in pharyngeal air being inhaled as opposed to being swallowed (thach 2005) . the increased gastric air found when positive pharyngeal pressure is applied suggests that cpap can alter normal pharyngeal clearance mechanisms and infers the need to titrate the applied pressure carefully (thach 2005) . finally, swallowing may be related to sleep apnea and be part of recovery from sleep apnea (see sect. 47.3.1) (thach 2005) . in summary, patency of the oral cavity and the pharynx and the coordination with swallowing and sucking are critical for breathing. the term, but not preterm, newborn can suck and breathe simultaneously, although ventilatory drive is decreased. physiological control of breathing and swallowing involves important sensory input that limits aspiration and protects the lower airway. intensive care therapies can alter normal protective, breathing and swallowing functions. like a swiss watch, …our vocal tract depends on the precise functioning of many structures and muscles." jared diamond, 1992 (the third chimpanzee). the larynx has three major functions involving breathing. it serves as a protective structure guarding the subglottic airway; as a flow controller of subglottic absolute and tidal volumes during eupnea and complex coordinated movements; and as a vibratory modulator of flow that generates speech, song, and laughter ( fig. 4.4) . 4.1.4.1.1 laryngeal closure and airway protection protection of the subglottic airway is a centrally controlled major task of "breathing." laryngeal stimulation can trigger a clearing response, e.g., an expiration reflex or a cough, or can result in airway closure. laryngeal closure occurs at the level of the epiglottis, at the vestibular folds, and at the vocal folds. this triple mechanism has been likened to closure of a nutcracker, an analogy whose accuracy is all too evident to intensivists faced with laryngospasm (fink 1973) . and flow/volume control in adult humans the glottis opens maximally during inspiration, closes gradually to a minimum about three quarters through expiration, and then opens again in late expiration (england et al. 1982a ). glottic resistance is least at peak inspiration, increases as expiration progresses, and then decreases in late expiration to minimize inspiratory flow resistance with the onset of pump muscle activity (england et al. 1982a ). there is no sensation of expiratory glottic closure in eupnea. accurate, rapid laryngeal opening and closure maintains an optimal subglottic airway volume, ensures tidal volume changes that enable gas exchange, and enhances airway patency and surfactant secretion. the smooth tidal flow pattern, produced by laryngeal and diaphragmatic activities interacting with the mechanics of the respiratory system, avoids tissue exposure to acceleration or deceleration injuries. the same laryngeal intrinsic muscles are involved in all laryngeal functions emphasizing that the central coordinations of the motor acts of breathing are truly integrative, a fact also stressed by the impacts that laryngeal sensory inputs and the resultant vagal outputs have on the cardiovascular system. laryngeal closure is important for other motor acts, e.g., effective lifting. central control of breathing is linked to emotions. it is of note that in laughter the order of abductor-adductor activities seen in breathing is reversed (luschei et al. 2006 the intrinsic laryngeal muscles and their innervation are shown. the left panel shows from bottom to top the posterior cricoarytenoids, the sole abductors of the larynx, and the abductors: the thyroarytenoids, the lateral cricoarytenoids, the transverse and oblique arytenoids, and the aryepiglottic muscles. all of these muscles are supplied by the recurrent laryngeal nerve (x). the internal branch of the sln is sensory, while the external sln branch is motor for the cricothyroid muscles (right panel) (from o'connor, d. reproduced with permission from myer et al. (1995) . redrawn by agreement with springer-verlag) breathing and vocalizations in practices aiming to augment metabolic and mental well-being. understanding the actions of the laryngeal intrinsic muscles is a necessary first step in gaining insights into the nature of the central control of breathing. the posterior cricoarytenoids (pca) are the sole abductors of the vocal cords. there are several adductors, with the thyroarytenoids (ta) being the major ones ( fig. 4.4) . some adductors, the cricothyroid and the vocalis part of ta, enhance abduction by tensing the vocal cords. this restricts vertical movement, improving flow control in the more horizontal plane. the magnitude of abductor activity exceeds that of the adductors when glottic size is increased and vice versa, consistent with relationships between glottic size and flow resistance (england et al. 1982a; insalaco et al. 1990 ; kuna et al. 1988 ). animal studies indicate that, in general, glottic opening and closing are achieved by reciprocal laryngeal abductor and adductor activities ( fig. 4 .5) (bartlett 1989; harding 1986; hutchison et al. 1993) . concurrent actions of laryngeal abductor and adductor muscles during inspiration and expiration may occur in adult humans, but their nature is unknown kuna et al. 1988 kuna et al. , 1990 . during eupnea the inspiratory outline of the pca emg resembles the flow pattern, while the ramp shape of the diaphragm emg relates to the inspired volume trace . in animals little expiratory ta activity is seen, and trans-upper airway pressure is minimal (fig.4 .5). in eupneic expiration in adult humans, pca activity diminishes, but some ta activity is present "rounding off" the early expiratory volume the thyroarytenoid (ta) muscles, and those of the diaphragm (d), together with flow, volume, and trans-upper airway pressure changes (reproduced with permission from hutchison et al. (1993)) trace, but without the flow retardation or transupper airway resistance patterns typical of grunting (kuna et al. 1988) (fig. 4.5) . time delays exist between emg and mechanical flow changes . the timing delays differ in eupnea and grunting, reflecting different mechanoreceptor feedbacks and central outputs that affect function (fig. 4 .5) . clinically, grunting is the expiratory noise produced when air flows through a partially closed glottis (harrison et al. 1968) . total glottic closure is silent but sensed. physiologically, grunting is a breathing pattern seen throughout life. it consists of a spectrum of degrees and timings of expiratory laryngeal closure, associated with volume retention and increased subglottic pressure, followed by laryngeal opening with a rapid return to baseline end-expiratory volume (eev) (figs. 4.5 and 4.6) (see sect. 47.3.1) . in newborns with respiratory distress, harrison et al. showed that when grunting was prevented by endotracheal intubation, without positive end-expiratory pressure (peep), their oxygenation worsened (harrison et al. 1968 ). this finding established the role of airway peep and likely influenced the introduction of cpap therapy. harrison et al. also focused on venous return (harrison et al. 1968 ), stressing the importance of cardiac function in grunting. the outline of the volume-time trace in grunting (figs. 4.5 and 4.6 ) resembles that of a volume with a prolonged inspiratory time, as used in artificial ventilation to improve oxygenation. pre-surfactant, this strategy reduced the required peak inspiratory pressure and decreased the incidence of bronchopulmonary dysplasia. today, the maintenance of subglottic volume, the "open lung" approach, is a fundamental tenet of all ventilatory support. in keeping with the concept of alveolar interdependence, airway volume recruitment is achieved not by a maximal prolonged inflation but with an augmented breath (sigh) followed by an expiratory hold, mimicking the volume-time profile of grunting or of airway pressure release ventilation. spontaneous sighs can be biphasic, with inspiration being hutchison and bignall (2008)) interrupted briefly by laryngeal closure. this mechanism may reset vagal afferent feedback, avoiding a hering-breuer inflation reflex and possible loss of the acquired volume (see sect. 47.3.1) . in summary, human and animal data emphasize the importance of laryngeal control of expiratory subglottic airway volume. this central control strategy whereby airway pressure is dependent upon volume control has been shown by the study of bubble physics to produce a more stable mechanical system (hildebrandt 1974) . thus, coordinated central control of laryngeal and pump muscles is a major focus in the control of breathing. this knowledge can be applied in respiratory support (hutchison and bignall 2008) . behavioral state is a major controlling factor of breathing pattern. in fetal lambs, during the highvoltage electrocorticogram (ecog) state, apnea (no phasic laryngeal abductor or diaphragmatic activities) and tonic laryngeal adductor activity occur. laryngeal adductor activity can be enhanced by laryngeal contact with cooled lung liquid, by fetal movements, by "arousals," by swallowing, by uterine contractures, and by fetal hypoxemia. in the low-voltage ecog state, laryngeal and diaphragmatic activities are similar to those seen postnatally (harding 1986 ). hypercapnia mainly enhances fetal breathing in the low-voltage ecog state (harding 1986 ). in general, active laryngeal retardation of lung liquid egress is reported in the absence of fetal breathing movements and is related to lung growth (harding 1986 ). stimulation of the superior laryngeal nerve (sln) produces laryngeal closure (harding 1986 ) that will prevent aspiration of noxious material, e.g., meconium. the coordination of the acts of fetal swallowing (harding 1986 ) and "inspiratory" activities of the respiratory muscles stresses the developmental importance of the controller's ability to switch patterns rapidly between upper airway closure (laryngeal adduction) with lack of diaphragmatic activity (apnea) and laryngeal abductor and pump activities (see sect. 47.3.1). airway volume is high in the fetus compared to the newborn. it has been speculated that during different fetal behavioral states, the brain is setting the homeostatic limits for high and low airway volume to prepare the fetus for the major needs of subglottic volume control at birth (hutchison 2007 ) (see also sect. 47.3.1). life's greatest airway volume challenge, the establishment and maintenance of an air-filled airway at birth, is usually overcome without a hitch by breathing patterns whose complexities exceed those used during the majority of adult life. at birth, an incremental breathing pattern retains more inspired volume than is expired ( fig. 4 .6) (hutchison et al. 1994) . incremental breathing shares features with grunting with the addition of timing changes in laryngeal and pump muscle activities at end-expiration when the onset of pump muscle activity occurs before laryngeal muscles open the glottis fully. the result is a decrease in expired volume and an increase in eev (hutchison et al. 1994 ). this incremental mechanical process is aided by the airway stability accrued from the increased end-expiratory positive subglottic pressure that limits the development of a negative airway pressure during inspiration. given their tendency to chest wall distortion, it is not surprising that incremental breathing can occur in preterm neonates (eichenwald et al. 1992) . incremental breathing is also seen in gasping during acute cerebral hypoxia-ischemia (hutchison et al. 2002) . gasping is typified by brief laryngeal abductor and pump muscle activities followed by long periods of laryngeal adductor activity with apnea (hutchison et al. 2002) . subglottic pressure is increased, maintaining airway volume and likely promoting autoresuscitation (hutchison et al. 2002) . laryngeal and pump muscle activities nasal and laryngeal afferent inputs alter laryngeal abductor and adductor activities and thus glottic size. the laryngochemoreflex (lcr), whose afferent pathway is the sln, results in protective responses, including swallowing, apnea, obstructed respiratory efforts, cough, hypertension, and arousal from sleep (thach 2008 ). an obstructive apnea with bradycardia results if the lcr stimulus is persistent, especially in the immature or depressed brain. this lcr response alters with age -coughing becomes the major response. however, acquisition of a viral infection in infancy can rekindle its potency (thach 2008) . it is augmented by hypoxemia and anemia and has been implicated in apnea of prematurity and sids (thach 2008 ) (see sect. 47.3.1). other laryngeal sln afferents, including those from mechanoreceptors, drive receptors, and temperature receptors, affect laryngeal intrinsic muscle activities. although sln section does not alter the eupneic breathing pattern, these afferents are important. upper airway bypass can alter the inspiration-inhibition hering-breuer reflex. application of acid to the larynx in animals, mimicking chronic aspiration, alters the subsequent response to an applied airway load (see sect. 47.3.1). during noninvasive ventilation, nonsynchronous delivery of airflow to the upper airway in neural expiration results in laryngeal closure (rodenstein 2004; scharf et al. 1978) . thus, noninvasive ventilation and pacing of a paralyzed diaphragm should be applied synchronously with neural inspiration (rodenstein 2004; scharf et al. 1978) . in intact animals, compensatory glottic abduction follows single-breath total occlusion of inspiration or expiration at the mouth/nose. during the unimpeded expiration following a single inspiratory occlusive load, unopposed ta activity can produce laryngeal closure. thus when no air enters the lung, a compensatory reflex mechanism can prevent expiratory volume loss, maintaining absolute subglottic volume. lower airway afferent inputs affect glottic size (bailey and fregosi 2006) . stretch receptor discharges, induced by peep, decrease expiratory ta activity (harding 1986) . by contrast increased expiratory glottic adduction follows rapidly adapting receptor stimulation, e.g., with deflation or irritant stimulation secondary to a pneumothorax (bartlett 1989) , or follows c-fiber stimulation, e.g., with experimental pulmonary edema (bartlett 1989 ). direct and indirect stimulation of chest wall muscle afferents can invoke several flow patterns involving glottic closure (bartlett 1989; stecenko and hutchison 1991) . while pseudoasthma can be fabricated by partial glottic closure (rodenstein 2004) , matching laryngeal control of flow pattern to maintain optimal subglottic airway volume may explain changes reported with true increased airway resistance. subglottic inspiratory flow limitation in croup is associated with expiratory flow resistance, probably glottic in origin (argent et al. 2008) . increased lower airway resistance in adult asthmatic patients induces a breathing pattern characterized by laryngeal expiratory flow retardation (collett et al. 1983; sekizawa et al. 1987) . in adults, resistive loads applied at the mouth also decrease expiratory glottic size (brancatisano et al. 1985) . this change may occur immediately, suggesting that resistive loading, unlike total occlusive (elastic) loading, produces sudden flow changes that stimulate airway receptors to cause laryngeal adduction (brancatisano et al. 1985) . therapy with cpap reverses the expiratory laryngeal adduction in some asthmatic subjects, suggesting that airway pressure changes may alter the dynamics between stretch receptor and irritant receptor stimulations, thus changing the breathing pattern (collett et al. 1983) . in normal adults a voluntary deep breath can decrease laryngeal resistance and lower airway resistance (sekizawa et al. 1987) . by contrast, bronchoconstrictive stimulation in normal adults can result in inspiratory and expiratory glottic narrowing (higenbottam 1980 ). an increased resistance to inspiratory flow may be advantageous in that more transpulmonary pressure is applied to opening a constricted peripheral airway. if lower airway volume and resistance changes affect laryngeal function, can absence of laryngeal control affect lower airway resistance? laryngeal bypass during invasive ventilation is associated with atelectasis and with the development of increased lower airway resistance (hutchison and bignall 2008) . the latter effect may stiffen the conducting airways and thus stabilize total airway volume but demand increased effort. in summary, the data reflect the importance of laryngeal subglottic volume control as a determinant of lower airway resistance and point to interactions between lower airway afferents and coordinated laryngeal and pump muscle activities in this dynamic process (see also sect. 47.3.1). central control of coordinated laryngeal and diaphragmatic muscle activities is determined by developmental stage, behavioral states, metabolism (temperature), central inputs, and centrally acting chemicals. postnatally in lambs, increased expiratory ta activity with decreased pca activity occurs during the nrem state. by contrast, expiratory ta activity in rem is mainly absent, while pca activity is variable (harding 1986 ). in normal adult humans, expiratory ta activity is absent during stable nrem, while expiratory pca activity decreases in nrem and is variable in rem (kuna et al. 1988 . at all ages, expiratory ta activity occurs at arousal. thus behavioral state is a key factor in subglottic airway volume control that, in turn, is important in sleep apnea. increased central drive, with hyperventilation and/or hypercapnia, promotes laryngeal abduction in inspiration and expiration in adults and term newborns, in whom increased pca, diaphragmatic and intercostal muscle activities occur (bartlett 1989; insalaco et al. 1990; kuna et al. 1994; wozniak et al. 1993) . laryngeal adduction can occur during hypercapnic hyperventilation in preterm neonates, probably due to chest wall distortion (but see also sect. 47.3.1) (eichenwald et al. 1993) . this adduction also occurs in adults at the mechanical limits of airway volume ). in general, however, increased central drive decreases laryngeal resistance unless mechanical limitations are present. decreased central drive with hypocapnia diminishes expiratory glottic size and is associated with periodic breathing in adults (kuna et al. 1993; rodenstein 2004) . laryngeal closure has been noted in human newborns and infants with apnea or suspected apparent life-threatening events milner 1991, 1993) . during central apnea and periodic breathing in lambs, expiratory ta activity is noted (praud 1999) . in depressed human infants at birth, laryngeal closure can block intubation, and, in lambs, acute cerebral hypoxia-ischemia results in expiratory ta activity with laryngeal closure (hutchison et al. 2002) . centrally acting depressant drugs diminish central drive and, in lambs, produce laryngeal closure with apnea (praud 1999) . in former preterm infants, up to ~55-60 postmenstrual weeks, exposure to anesthesia and/or operative stress can result in apnea postoperatively (see sect. 47.3.1) . thus, the intensivist should avoid hypocapnia during noninvasive ventilation and be aware that central depression can result in apnea with glottic closure. hypoxia stimulates expiratory laryngeal abduction and increases ventilation in human adults and newborn lambs (england et al. 1982b; insalaco et al. 1990; praud et al. 1992) . in lambs, the increase in ventilation is dependent upon carotid body input -a sudden decrease in that input induces expiratory ta activity (praud et al. 1992 ). however, animal and human data indicate that the effects of carotid body input vary depending upon central state and the presence/absence of other inputs (de burgh daly et al. 1979 ). if animals are vagotomized and paralyzed, direct carotid body stimulation can induce expiratory ta activity, and exposure to hypoxia after airway vagal blockade or intrathoracic vagal section results in laryngeal adduction (bailey and fregosi 2006) . in adult humans, the degree of expiratory glottic adduction with hypoxia exceeds that during hypercapnia (england et al. 1982b) . thus, it is argued that the "pure" carotid body reflex response is laryngeal expiratory adduction that will retain airway volume and promote oxygenation. this pure response can be countered by the presence of ventilation which increases airway stretch receptor feedback that results in an overall expiratory laryngeal abduction response. in the author's view, a unifying explanation for the diverse findings with hypoxia is that the effect of carotid body input is to augment the central coordinated output to laryngeal and pump muscles that is selected under different conditions (see sect. 47.3.1). when central depression exists, protective inputs and/or absence of volume-related inputs produce an apneic response with glottic closure that is augmented by hypoxia. this can be seen in the "vagal" preterm infant who is sedated for a surgical procedure. at intubation laryngeal closure and apnea/bradycardia can be triggered. if hypoxemia ensues, the problems are aggravated. when tidal ventilation and airway volume feedback are restored, the impact of any ongoing hypoxemia is to augment breathing. hering and breuer found that their volumerelated reflexes were active even with hydrogen breathing. this fits the modern focus on ventilation for resuscitation from asphyxia. this chapter of upper airway physiology for the pediatric intensivist has focused on motor and specifically on laryngeal aspects relevant to the control of breathing patterns. the major message is that upper airway physiology is involved in all aspects of "breathing": protection, volume homeostasis, and ventilation; its functional impact covers the entire airway, from the nose to the alveolus. understanding upper airway physiology can guide and improve therapy, while therapy can aid a return to normal homeostasis or detract from it. current knowledge of upper airway physiology has had major implications for the application of invasive and noninvasive ventilatory support. much remains to be learned from the interactions between physics and biochemistry in the entire airway and how they affect breathing patterns. • the upper airway plays roles in all the extended functions of breathing, namely, in airway protection, in the control of supra-and subglottic volumes, and in tidal ventilation. • nasal functions include air conditioning and airway protection. stimulation can result in profuse secretions, altered patency, and the cardiorespiratory dive reflex. obstruction can affect gas exchange markedly; thus, a rapid switch to oral breathing is advantageous. • pharyngeal patency can be altered in sleep. nonnutritive swallowing is vital to minimize aspiration. therapy with cpap improves pharyngeal patency in osa but may alter pharyngeal clearing mechanisms and increase gastric air. • laryngeal closure can protect the airway rapidly. laryngospasm can be hard to treat. • controlled expiratory laryngeal closure modifies airflow and subglottic airway volume in normal breathing, at the establishment of airway volume at birth or when airway volume is threatened by mechanical, chemical, or central changes. • intrinsically or extrinsically induced changes in central state alter the laryngeal motor outputs in response to mechanical or chemical inputs. • understanding how laryngeal motor functions affect breathing patterns, airway pressures, and gas exchange is at the core of many technical and procedural advances in the provision of resuscitative measures and of invasive and noninvasive respiratory support. paul d. robinson and j. jane pillow the mechanical properties of the respiratory system are a critical component of the chain of events that result in translation of the output of the respiratory rhythm generator to ventilation. a comprehensive understanding of respiratory mechanics is therefore essential to the delivery of optimized and individualized mechanical ventilation. this chapter describes the basic elements of respiratory mechanics and reviews the developmental changes in the airways, lungs, and chest wall that impact on measurement of respiratory mechanics with advancing postnatal age. during spontaneous breathing, the forces driving inspiration are generated by the inspiratory muscles, whereas during mechanical ventilation, the forces are generated by the ventilator or by a combination of the ventilator with spontaneous inspiratory muscle contributions. the inspiratory muscle pressure needs to be sufficient to overcome three major mechanical properties of the respiratory system: elastance (e) (to overcome tissue forces required to change the lung volume (v)), resistance (r) (to overcome resistance to flow (v')), and inertance (i) (for acceleration (v'') of gas volumes). elastance is often considered in terms of its inverse, the compliance (c). the total pressure (p total ) generated is equal to the sum of the elastic (p el , proportional to volume and 1/compliance), resistive (p res , proportional to flow and resistance), and inertive (p in , proportional to acceleration and inertance) components: although pressure losses to inertia are considerable during high-frequency ventilation, the inertive component is normally a small portion of p total at normal breathing frequencies and can be effectively neglected. thus the equation driving flow during normal respiration can be simplified: nonetheless, the linearized relation of driving pressure to volume and flow does not fully characterize the pediatric respiratory system: resistance and compliance are both dependent on volume, volume history, and flow, particularly in the very small or premature neonate. further, the simplified equation does not fully incorporate the complexities of the respiratory system. almost all the tests used to measure respiratory mechanics in the infant and pediatric population have been developed in adults and then adapted for younger subjects. measurements of respiratory mechanics are most appropriately corrected to lung volume, such as functional residual capacity (frc). in the absence of a measure of frc, measurements of respiratory mechanics made in infancy are often related to body size (e.g., height/length or weight). measurements at the body surface are necessary to include the contributions of the chest wall. the chest wall contribution to respiratory mechanics can be isolated, by subtraction of the pleural measurements from those obtained at the body surface (e.g., airway opening). the respiratory system is in a passive state when there is no inspiratory muscle activity (p mus = 0). a passive condition is present in the paralyzed patient but can also occur without paralysis following hyperventilation (mortola 2004) . under passive conditions, resting lung volume is determined by to describe the principal components of respiratory mechanics of the airways, lungs, and the chest wall and the developmental and environmental considerations that impact on these measurements in the neonatal and young child two opposing forces. the outward recoil of the chest wall results from the elastic characteristics of the diaphragm/abdomen and of the rib cage. the outward recoil of the chest wall directly opposes the inward recoil of the lung. inward recoil is determined by the viscoelastic properties of the lung, including the mechanical properties of the lung tissues, and the collapsing pressure generated at the alveolar air-liquid interface. during spontaneous breathing, the passive respiratory mechanics can be measured using occlusion techniques. occlusion of the airways at lung volumes above the resting lung volume relaxes the respiratory muscles by stimulating the slowly adapting stretch receptors and the vagally mediated hering-breuer inflation reflex. passive tests assess the mechanical properties of the entire respiratory system. simultaneous measurement of transpulmonary pressure allows measurement of respiratory system compliance (c rs ) to be partitioned into lung (c l ) and chest wall (c w ) components. transpulmonary pressure (p tp ) is derived from the difference in pressure at the airway opening (p ao ) and pleural pressure (or elastic recoil pressure, p el ), measured using an esophageal balloon, liquid-filled catheter, or miniature pressure transducer. other passive measurements of respiratory mechanics include forced expiratory maneuvers to provide measures of maximal flow at frc (v′ max,frc ) (lum et al. 2006a ). partial forced expiratory flow-volume curves in infants are obtained by rapid compression of the chest through endinspiratory inflation of a jacket placed around the chest and abdomen. during tidal breathing, this rapid thoracoabdominal compression (rtc) technique provides a v′ max,frc that is similar to forced flows at low lung volumes (e.g., maximal expiratory flow mef 25 %,frc ). modification of the rtc technique by raising lung volume of the infant towards total lung capacity (so-called raised volume rtc or rvrtc) allows assessment of forced expiratory flows over a more extended range of volumes. reported measures include forced vital capacity (fvc) and forced expiratory volumes after defined time periods (e.g., 0.4, 0.5, or 1 s, termed fev 0.4 , fev 0.5 , and fev 1 , respectively) and the ratio of these values e.g., (fev t /fvc, where t is the time period chosen). in contrast, dynamic tests of respiratory mechanics allow the relative contributions of the airways and lung tissues to be partitioned, in addition to separately identifying the chest wall contribution to airway and tissue mechanical properties. dynamic mechanics derive mechanical variables from analysis of dynamic waveforms obtained either during normal (tidal) breathing or via imposed oscillatory waveforms. during tidal breathing, multilinear regression is used to determine dynamic respiratory system compliance (c dyn,rs ) and resistance (r rs ). kano and colleagues showed that consideration of volume dependence is important when using this approach to assess dynamic mechanics during mechanical ventilation. consideration of volume dependence is important because ventilatory settings may "push" the pressure-volume curve onto the flattened upper portion of the curve (kano et al. 1994 ). in the overdistended lung, fits obtained by multilinear regression are improved by the inclusion of a volume-dependent elastance term in the regression model: where p ao is the airway opening pressure, v is the tidal volume, e 1 + e 2 v is the total elastance over the cycle (e 1 and e 2 v representing the volumeindependent and volume-dependent portions of elastance, respectively), and the final term p a,ee represents the alveolar pressure at end-expiration. an important consideration during the interpretation of dynamic respiratory mechanics is the concept of frequency dependence. kano showed that increasing the ventilation rate from 30 to 80 breaths/min in a group of near-term newborn infants increased e rs and r rs by a mean of 8.3 and 17.5 %, respectively (kano et al. 1994) . frequency dependence and the mechanical properties of the respiratory system under normal tidal breathing conditions are also determined using forced oscillatory techniques (fot). these techniques are essentially noninvasive and are especially useful in infants and children as no active subject cooperation is required. the technique involves application of a pressure waveform (forcing function) to the airway opening and measurement of the resultant flow. the measured impedance reflects the complex viscoelastic resistance of the respiratory system (z rs ). simultaneous measurement of the transpulmonary pressure and hence chest wall impedance (z w ) allows determination of the lung impedance (z l ) by subtraction. the impedance (z) can be separated into the resistance (r rs ), the impedance component in phase with flow, and reactance (x rs ), representing the impedance components in phase with volume (elastance) and acceleration (inertance). the low-frequency forced oscillation technique (lfot) measures impedance over a range of frequencies (usually ~0.5-20 hz). lfot is obtained in infants by invoking the hering-breuer inflation reflex to inhibit temporarily any respiratory muscle activity, during which brief time (6-10 s) the lfot signal is applied. fitting the constant-phase model to the resultant pressure and flow data permits partitioning of respiratory mechanics into frequency-independent airway resistance (r aw ) and airway inertance (i aw ) and a constant-phase tissue component. this constant-phase tissue component is described by [(g − jh)/ωα, where g and h are coefficients for tissue damping and elastance, respectively, ω is angular frequency, and α determines the frequency dependence of the real (pressure change in phase with flow) and imaginary (pressure change in phase with volume) parts of the impedance] (hantos et al. 1992a ). the interrupter technique provides a further option to assess partitioned mechanics. the interrupter technique involves measurement of changes in airway opening pressure after a midexpiratory occlusion, which is used to calculate airway resistance (r aw ). after airway occlusion at mid-expiration, there is a biphasic change in p ao : the immediate rapid rise in p ao represents the resistive pressure drop across the conducting airways and is followed by a secondary slower increase in p ao (often referred to as p dif ) generally attributed to stress recovery in the respiratory tissues (lung and chest wall) and gas redistribution associated with ventilation inhomogeneity (bates et al. 1988) . a major limitation of this technique is that it requires mechanical ventilation and paralysis to obtain reliable data. particular challenges for lung function testing in infants and children include the marked developmental changes occurring over the first months and years of life. additional considerations include position, sleep state and sedation, and feasibility of individual tests given minimal capacity of these age groups for active cooperation and preferential nasal breathing (stocks and hislop 1996) . thus, in contrast to the adult and older child, tests in infants are performed using a mask instead of a mouthpiece and nose clips. lung development represents a period of considerable change in both lung architecture and volume and is affected by several factors including genetic, in utero and postnatal environmental exposures, somatic growth, puberty, changes in muscularity, and ongoing alveolarization. our understanding of the interactions between these factors is based on a mix of pathological and physiological data, which are predominantly cross-sectional in nature. consensus between these studies is lacking. while some of the lack of agreement is attributable to technical approaches used by different authors, a comprehensive understanding of factors influencing respiratory mechanics over time can only be answered by strong longitudinal data, which are urgently required. these tests of lung mechanics provide us with essential insight into how tissue properties change with development, and a full understanding of the changes that occur during healthy development is an important step to detect the subsequent effects of disease and response of the individual to therapeutic interventions. measurements need to be accompanied by accurate records of height (length) and weight to facilitate interpretation of longitudinal data. over the course of development, there is considerable remodelling of all of the structural components of the respiratory system, although the exact patterns of change in airways, alveoli, and blood vessels may differ with respect to each other. the term "dysanaptic growth" was originally introduced by green et al. (1974) and describes the disproportionate but physiologically normal growth of the lung relative to the airways. in infancy, airway size relative to lung volume is larger than in older children and adults. there is considerable debate in the pediatric literature about the degree, extent, and stage of development to which biological variability of airway size to lung volume occurs. the data outlined in this chapter suggest that dysanapsis is a feature of all stages of postnatal lung development. whether disproportionate growth along the length of the airway tree occurs is less clear. increased growth of the larger central airways, in relation to peripheral generations, has been described through the first year of life, after which point adult relative proportions are achieved and maintained through further growth (horsfield et al. 1987) . disproportionate central and peripheral airway growth, however, is not a consistent finding in the literature (hislop et al. 1972) . the most rapid period of tracheal growth appears to occur during the first 4 years of life (wailoo and emery 1982) . an elevated ratio of peripheral airway resistance to central airway resistance (and its contribution to total airway resistance) in young children compared to adults was originally proposed by hogg et al. (1970) , based on retrograde catheter measurements. the authors described relatively little change in the contribution of the central airways (those proximal to the 12th-15th airway generations) to conductance with age but marked increase in peripheral airway conductance from the age of 5 years of age. these changes were attributed to altered linear dimensions of the peripheral airways, based on morphometric data. the ratio of central to peripheral resistance can also be examined noninvasively by comparing measured forced expiratory flows (fef) breathing air to fef measured breathing a less-dense gas mixture of 80 % helium/20 % oxygen (heliox). fef measured breathing heliox is typically higher than during air breathing (e.g., 1.2-1.6 times at 50 % fvc). in older subjects with peripheral airway disease, this pattern is lost with ratios approaching parity (cooper et al. 1983; dosman et al. 1975) . density dependence data during infancy, in comparison to values reported in later life, appear to challenge the findings of hogg et al. (1970) . davis et al. examined the changes in density dependence through the first 2 years of life (davis et al. 1999) . fef 30-40 % higher were measured with heliox: although no relationship existed between density dependence and age, length, or fvc in the first 2 years of life (in keeping with hogg et al. (1970) ), the values obtained were similar to those reported across 16 other studies in older children ranging from school age to adulthood (summarized in table 2 of the original manuscript (davis et al. 1999) ). these equivalent cross-sectional density dependence values suggest that there is no difference in the convective accelerative and turbulent pressure loss with age, supporting the notion that the ratio of resistance of the peripheral to the central airways is similar in infants, older children, and adults. sex-specific differences in respiratory mechanics are well recognized in infants and young children and are a particularly important consideration in forced expiratory maneuvers in infants. airways are larger in females before puberty, and this difference is detectable from infancy (tepper et al. 1986a; hoo et al. 2002a) : v′ max,frc is approximately 20 % higher in girls over the first 9 months of life (hoo et al. 2002a ). similar sex-dependent increases in v′ max,frc are evident in preterm girls compared to preterm boys, suggesting such differences in lung function may be developmental rather than environmental in origin (stocks et al. 1997) . larger lungs have been reported in males between the ages of 6 weeks and 14 years (thurlbeck 1982) . presence and timing of the growth spurt is an important confounding factor, with lung growth occurring out of phase to somatic growth, especially in boys (degroodt et al. 1986; quanjer et al. 1989; schrader et al. 1984) . lung development in females is almost complete following menarche but continues throughout puberty in males (neve et al. 2002) . there is increasing evidence that the periconceptional and intrauterine exposures impact on development of the respiratory system and consequently impact on respiratory mechanics in infancy and childhood. factors known to influence lung mechanics after birth include maternal smoking, nutritional deprivation and intrauterine growth restriction, infection and/or inflammation, and maternal glucocorticoids. prenatal exposure to nicotine may cause abnormal airway branching and dimensions as well as increase airway smooth muscle and collagen deposition resulting in reduced lung function at birth that persists into early adulthood regardless of postnatal exposure (hayatbakhsh et al. 2009; svanes et al. 2004 ). lung function irregularities include airflow limitation, reduced fev 1 , and airway hyperreactivity wongtrakool et al. 2012) . chronic restriction of nutrients and/or oxygen in late pregnancy impairs development of the fetal small airways and lungs, including reduced numbers of alveoli that are also enlarged with increased septal wall thickness and basement membranes compared to nonexposed infants (pike et al. 2012) . such differences in alveolar number can influence the subsequent rate of disease progression, including an accelerated rate of decline in fev 1 with advancing age (stocks and sonnappa 2013). several cross-sectional studies have looked at the changes in airway resistance (reflecting airway size) during childhood. hall and colleagues using lfot described a quasilinear decrease in r aw with increasing length during infancy in 37 infants, including some with repeat measurements (hall et al. 2000) . lanteri and sly examined changes across the pediatric age range and also reported linear decreases in both r rs and r aw with increasing height (when plotted on a loglog plot) in 51 children (range 3 weeks-15 years) with healthy lungs (fig. 4 .7) (lanteri and sly 1993) . pooled data across the preschool age range reinforces the consistent relationship seen with increasing height (beydon et al. 2007a decreasing resistance of the respiratory system (r rs ) and airway resistance (r aw ) with increasing height. cross-sectional data taken from 51 subjects (aged 3 weeks to 15 years) (lanteri and sly 1993) . both resistance and height are plotted as natural log values but are labeled with absolute numbers for clarity examined (gerhardt et al. 1987a ). this increase occurred at a slower rate than the increase in frc, which led to a rapid drop in specific conductance ( fig. 4.8b ). this rapid decrease in specific airway resistance or conductance through early life has been described previously (doershuk et al. 1974; stocks and godfrey 1977) . tepper et al. measured maximal fef at frc, from partial expiratory flow-volume curves and corrected for changes in lung volume: they reported higher flows in the neonatal range which decreased to a steady value through the 2nd year of life, similar to those reported elsewhere in older children (tepper et al. 1986a) . differential increases in anatomic dead space volume (twofold), compared to frc (threefold), have also been shown in the pediatric population (wood et al. 1971) . rapid postnatal increases in lung volume are reported widely. frc increases by a magnitude of 40 times (from 80 ml in infants to 3,000 ml in adults) (dunnill 1982) and over ten times in total lung weight (polgar and weng 1979) . the rapid growth of the distal lung, in comparison to relatively steady changes in airway dimensions, is a major feature of respiratory system development beyond birth. after the appearance of primitive saccules at approximately 28 weeks gestation, mature alveoli with secondary septa appear from 34 weeks gestation onwards. alveolar multiplication was initially thought to cease at around 2 years of age (thurlbeck 1982) . more recent estimates indicate that increase in alveolar number continues until at least 8 years (dunnill 1982) . recent advanced imaging studies using hyperpolarized helium magnetic resonance imaging (mri) provide evidence of ongoing alveolar multiplication in adolescence (narayanan et al. 2012) . increase in alveolar size (expansion) is also evident during infancy and childhood, with recent epidemiology studies suggesting lung size increases into early adulthood, when chest wall development is complete (quanjer et al. 1989; reid 1977 ). based on cross-sectional studies, specific lung compliance falls during early infancy before remaining relatively constant from the early preschool years. tepper et al., based on measurements of compliance from the linear portion of the static pressure-volume (pv) curve in almost 50 infants, described an increase in lung compliance over the age range studied, but decreasing specific lung compliance with increasing body length in the first 2 years of life (tepper et al. 2001 ). gerhardt et al. examined similar changes in 40 infants and children over the first 5 years of life. lung compliance increased markedly (by ~20-25-fold) over the weight range examined (fig. 4 .8a) but in proportion to frc, resulting in a constant-specific compliance over the first 5 years of life. the same pattern of findings was reported in 63 children aged 2-7 years (greenough et al. 1986 ). this initial rapid period of alveolar growth is also supported by evidence from measurements using the interrupter technique and fot. lanteri and sly showed a biphasic change in p dif with age: p dif was highest in young infants, falling rapidly over the first 12 months, before increasing again after approximately 5 years of age (lanteri and sly 1993) . hall and colleagues, using lfot measures of tissue mechanics derived by fitting data to the constant-phase model, showed that both tissue parameters g and h decreased in a quasihyperbolic manner with increasing length between 7 weeks and 2 years of age (hall et al. 2000) . the difference in the pattern of change in g and h compared to the change in r aw with increasing length is further evidence of dysanapsis: tissue mechanical properties change more rapidly than airway mechanics over the first 2 years of life ( fig. 4.9 ). the chest wall of the neonate and infant, and to a lesser extent the growing child, is especially compliant compared to the compliance of the chest wall in the adult subject. a highly compliant chest wall in infancy results in less opposition to the tendency of the lung to collapse, promoting a low residual lung volume and also distortion of the chest wall, resulting in a loss of volume during inspiration. the disproportionately high compliance of the chest wall compared to the compliances of the lung means that the compliance of the respiratory system (c rs ) is approximately equal to the compliance of the lung (c l ) during childhood, especially in neonates and infancy (davis et al. 1988; gerhardt et al. 1987a) . several active (dynamic) mechanisms of respiratory mechanics serve to partially compensate for the instability in frc associated with a compliant chest wall and tendency for small airway closure during tidal breathing. infants acutely increase their end-expiratory lung volume (eelv) and maintain it above resting lung volume by modulating (abbreviating) their expiratory time (mortola and saetta 1987) ( fig. 4.10a ). in addition, they reduce expiratory flow through post-inspiratory activity of the diaphragm and inspiratory chest wall muscles (kosch and stark 1984) (fig. 4 .10b) (lopes et al. 1981) , stiffening the chest wall and by increasing laryngeal resistance (± glottic closure) briefly (kosch et al. 1988) . such dynamic modifications of respiratory mechanics occur via neural (vagal) receptor-mediated reflexes. restriction of lung function measurement to periods of quiet sleep is necessary to reduce the variability in measurements resulting from such dynamic processes (henschen and stocks 1999) . the changes in chest wall, lung, and respiratory system mechanics over the first 4 years of life were examined by papastamelos et al., in 40 subjects (papastamelos et al. 1995) . the authors used a modified mead-whittenberger technique (cook et al. 1957) : manual ventilation overrode respiratory drive and relaxed the respiratory muscles, avoiding the need for intubation or induction of a hering-breuer reflex. in this cohort, the ratio of chest wall to lung compliance fell from a mean (sd) of 2.9 (1.1) to 1.3 (0.4) (p < 0.005). chest wall to lung compliance ratios are even higher in preterm infants (gerhardt and bancalari 1980) . the stiffening of the chest wall, due to rapid rib ossification, increasing musculature, and altered chest wall configuration (bryan and wohl 1986; mead 1979; openshaw et al. 1984; leiter et al. 1986 ), is such that near-adult values, where lung and chest wall compliance are equal (mittman et al. 1965) , are reached after the first year of life. stiffening of the chest wall over the first year of life allows the infant to shift its maintenance of frc from dynamic elevation of eelv to a more passive mechanism achieved by the balance of outward recoil of the chest wall and inward recoil of the lung (colin et al. 1989 ). stiffening of the chest wall likely also explains the increase in p dif in children after age of 5 years observed by lanteri and sly (lanteri and sly 1993) . frc as a proportion of total lung capacity (tlc) increases with age through childhood, in the majority (engstrom et al. 1956; mansell et al. 1977; weng and levison 1969) , but not all (schrader et al. 1988 ), studies to date. residual volume (rv), as a proportion of tlc, also increases with age through childhood (merkus et al. 1993) , while closing capacity (measured using single-breath nitrogen washout) decreases significantly with age, as a proportion of tlc, converging towards rv (mansell et al. 1977) . this increased rv/ tlc ratio suggests that increased stiffness of the chest wall is not entirely compensated for by increase in expiratory muscle force over time (schrader et al. 1988 ). the study by merkus et al. also highlighted the need to use tlc as a measure of lung growth and not fvc, due to the fact that an increasing rv/tlc with age led to errors in maximal expired flows due to measurement at progressively higher lung volumes (merkus et al. 1993 ). the postnatal period is characterized by ongoing rapid lung development, with differing rates of growth seen in the airway, lung parenchyma, and chest wall. the dysanapsis that occurs between the components of the respiratory system has important consequences for measurements of lung mechanics performed during infancy and childhood. accounting for these developmental changes and dysanapsis is necessary to optimize disease detection and subsequent assessment of an individual's response to interventions. insights into these changes are largely derived from crosssectional studies, which have identified important sources of potential error in existing methodol. this is compared to the passive expiratory time constant (slope of the dotted line) measured after an end-inspiratory occlusion. the difference between the two slopes is generated due to active use of the respiratory muscles during expiration ("braking"). the mag-nitude of difference between the active flow-volume curvederived eelv (frc) and the passive relaxation volume (v r ) reflects the degree of active (dynamic) mechanisms employed to elevate eelv. this active use of respiratory muscles is also illustrated in (b) from data collected in a separate infant study wherein diaphragm emg activity of breaths three and four extends into expiration (both figures reproduced with permission from the publishers (mortola and saetta 1987; kosch and stark 1984) ) • the postnatal period is characterized by ongoing rapid lung development in all parts of the respiratory system (airways, lung parenchyma, and chest wall). the physiologically normal rate of change of the lung relative to the airways is disproportionate. this dysanaptic growth pattern appears to be a feature of all stages of postnatal lung development. ogy. strong longitudinal data are now urgently required to confirm these apparent patterns and answer the important questions that remain. j. jane pillow the predominant neonatal acute respiratory diseases include transient tachypnea of the newborn (ttn), respiratory distress syndrome (rds) due to surfactant deficiency and/or structural immaturity, pulmonary interstitial emphysema (pie), meconium aspiration syndrome (mas), persistent pulmonary hypertension of the newborn, pneumonia, and congenital diaphragmatic hernia (cdh). as there are no reported measurements of lung mechanics or lung function for infants with pie and pneumonia, they are not discussed further. transient tachypnea of the newborn (ttn) is characterized by delayed resorption of fetal lung fluid within the distal airspaces. the mechanical consequences of ttn include reduced lung volume (functional residual capacity, frc) and consequently also decreased lung compliance (increased elastance), tidal volume, and respiratory rate (benito zaballos et al. 1989) . although increased interstitial fluid might be expected to increase lung tissue resistance, there are no reported studies of tissue resistance in infants with ttn. • passive measures of lung mechanics provide information about the mechanics of the entire respiratory system. • dynamic measures of lung mechanics allow partitioning of the lung and chest wall components to separately identify the chest wall contribution to airway and tissue mechanical properties. • standardization of the measurement technique is essential. results generated are corrected for either lung volume (e.g., frc) or, in the absence of this, a measure of growth (e.g., weight). • a number of periconceptional, intrauterine, and postnatal factors impact on the development and mechanics of the respiratory system. important postnatal factors include maternal smoking, nutritional deprivation and intrauterine growth restriction, infection and/or inflammation, and maternal glucocorticoids. • the rate of airway growth (detected by increases in conductance or decreases in resistance) is not as rapid as the rate of frc increase with age. this greater relative increase in lung volume leads to a rapid decrease in specific resistance during early childhood. • postnatal alveolarization appears to now extend into adolescence. rapid alveolar growth occurs during infancy leading to an initial fall in specific lung compliance, which reaches a plateau during ongoing lung growth. these changes are also evidenced by the changes seen in lfot tissue parameters and interrupter technique-derived p dif . • the highly compliant chest wall of the infant leads to employment of active (dynamic) measures to maintain eelv above frc. as the ratio of chest wall to lung compliance falls and reaches the adult values of parity beyond infancy, these gradually transition to more passive mechanisms as the chest wall stiffens through early childhood. • to describe flow (f), lung volumes (v), and respiratory pressure (p) measurements together with resistance (r) and compliance (c) measurements in neonatal respiratory diseases like ttn, rds is predominantly a disease of the distal lung parenchyma. surfactant deficiency promotes atelectasis, typically characterized by low lung volumes and reduced lung compliance. lung function is also reflective of the degree of structural maturation, as surfactant deficiency is predominantly a disease of the premature infant. in the intubated infant, lung resistance measures are highly variable and not reproducible (22-32 %, icc <0.75), in contrast with less variable and more reproducible measurements of lung compliance (obtained from esophageal manometry). hence, the clinical relevance of dynamic mechanics measurements of resistance in intubated newborn infants with respiratory distress is questionable (gappa et al. 2006a) . measurements of impedance, obtained using forced oscillatory mechanics (a quasistatic lung mechanics measurement), may provide a more reliable assessment of lung mechanics. dorkin measured respiratory impedance in six paralyzed and intubated infants, three of whom also had pulmonary interstitial emphysema (dorkin et al. 1983 ). the tracheal tube contributed to almost all the inertance and approximately 50 % of the respiratory system resistance in the intubated infant. after subtraction of the impedance of the endotracheal tube, resistance ranged from 22 to 34 cm h 2 o/l/s, compliance from 0.22 to 0.68 ml/ cm h 2 o, and inertance from 0.0056 to 0.047 cm h 2 o/l/s (gappa et al. 2006a; dorkin et al. 1983) . the low-frequency forced oscillation technique (lfot) evaluates impedance simultaneously across a range of frequencies (usually ~0.5-14 hz). fitting of the resultant impedance data to the constant-phase model permits estimation of partitioned mechanical variables of the airways and the parenchymal tissues (hantos et al. 1992b ). using the lfot, impedance measurements in naïve (surfactant-deficient) newborn preterm lambs showed that respiratory system resistance (r rs ) and reactance (x rs ) are markedly frequency dependent (pillow et al. 2001a) . respiratory system resistance in the preterm infant is also dominated by the resistive properties of the tissues, contrasting sharply with the predominant airway contribution to respiratory system resistance in later life (pillow et al. 2005) . although compliance is also low (increased elastance), the tissue resistance is disproportionately greater, resulting in mechanical uncoupling of the parenchyma and increased hysteresivity (ratio of tissue resistance to tissue elastance) (pillow et al. 2001a (pillow et al. , 2005 . increased surfactant pool size (pillow et al. 2004a ) and lung volume recruitment (pillow et al. 2004b ) both enhance mechanical coupling of the tissues, evident as a lowering of the hysteresivity ratio in the lung. increased contribution of the tissues to respiratory system mechanics, and the associated increased frequency dependence, results in elevation of the resonance frequency of the lung (pillow et al. 2001a) . frequency dependence also becomes less marked with increasing postnatal age ( fig. 4 .11) (pillow et al. 2005) . variability in measurements of lung mechanics in newborn infants in part reflects lung maturation associated with advancing gestation: lung compliance increases 0.17 ml/cm h 2 o/week, to reach mean (sd) values of 2.50 (0.07) ml/cm h 2 o at term equivalent (bhutani et al. 2005) . other factors include in utero fetal exposures such as placental nutrition, inflammation, antenatal steroids, and postnatal treatments including caffeine, glucocorticoids, surfactant, and ventilation modality. early treatment with caffeine in surfactant-treated immature baboons increased c rs over the first day of life and increased ventilatory efficiency index (yoder et al. 2005) . the initial benefit of antenatal steroids on improved lung compliance at birth fades beyond 1 week after initial exposure (mcevoy et al. 2008 ). there is no evidence of that antenatal steroids have a persistent effect on lung mechanics: infants born at term after exposure to up to three courses of betamethasone at gestations between 25 and 33 weeks had no difference in gas mixing or lung volume/mechanics when compared to nonexposed term controls (hjalmarson and sandberg 2011) . like antenatal steroids, postnatal steroids also increase respiratory system compliance (durand et al. 1995) . low-dose and high-dose postnatal dexamethasone for cld achieve a similar increase in c rs suggesting equal effectiveness for improvement of lung volume (mcevoy et al. 2004) . early postnatal dexamethasone may be more effective than late dexamethasone in achieving lower r aw /r rs and higher specific conductance (sg aw ) (merz et al. 1999; vento et al. 2004) , which may indicate a degree of fixed change in the airway walls in infants receiving delayed glucocorticoid treatment. during administration, surfactant represents a fluid bolus in the airway that increases inspiratory and expiratory time constant, associated with an acute increase in respiratory system resistance. effective delivery of surfactant to the lung thus benefits from a transient increase in the inspiratory time and potentially brief increase in the delta p (peak inspiratory pressure-positive endexpiratory pressure) to overcome the increase resistance resulting from the fluid bolus. treatment with exogenous surfactant (da silva et al. 1994) or increases in positive end-expiratory pressure (peep) during recruitment from atelectasis increases lung volume: (dimitriou et al. 1999 ) lung volume increases by 61 ± 39 % within a median of 4 min after surfactant administration, with altered distribution of lung volume towards the dorsal rather than ventral compartment. the increase in lung volume after surfactant or volume recruitment maneuvers increases maximal compliance of the respiratory system, with higher tidal volumes achieved at lower pressures than required prior to treatment (miedema et al. 2011a; mcevoy et al. 2010) . importantly, the increase in compliance also impacts on the time constant (τ = r rs × c rs ) of the respiratory system. changes in the inspiratory and expiratory time constants need to be monitored as they necessitate adjustment of the inspiratory and expiratory times and influence the maximal frequency that can be used during ventilation with passive expiration. compared to treatment of rds with synchronized intermittent mandatory ventilation (simv), a small clinical trial showed that infants treated with high-frequency oscillatory ventilation (hfov) have early and sustained improvement in pulmonary mechanics and higher dynamic respiratory compliance (vento et al. 2005) . the improvement in pulmonary mechanics in patients treated with hfov is likely associated with enhanced lung volume recruitment. the neonatal lung with rds exhibits hysteresis (miedema et al. 2011b) . hence lung recruitment maneuvers that aim to recruit the lung and subsequently ventilate it on the most compliant part of the deflation pressure volume curve can achieve effective ventilation with minimum pressure and volume cost of ventilation. importantly, recruitment of the lung by increasing mean distending pressure during hfov is also associated with changes in compliance and the time constant for volume delivery during hfov (pillow 2012) . consequently, volume delivery can vary substantially over the time course of an hfov volume recruitment maneuver (miedema et al. 2012 ) and potentially result in rapid fluxes of the partial pressure of arterial carbon dioxide. the effect of compliance on volume delivery during hfov is more evident at lower frequencies (pillow 2012; pillow et al. 2001b ). the development of hfov ventilators that incorporate volume guarantee during hfov will provide protection from such rapid changes in ventilation during hfov. infants with rds often have their clinical course complicated by the persistence of a patent ductus arteriosus (pda), which presents as a leftright shunt. ligation of a pda (left-right shunt) increased dynamic compliance by 77 % in 16 newborn infants measured before and after ligation, but did not influence dynamic r rs or mean airway pressure (szymankiewicz et al. 2004a ). although meconium aspiration syndrome (mas) is often considered as a high airway resistance disease due to the presence of meconium in the airways, studies in rabbits show that the acute aspiration of meconium also causes a significant reduction in the lung-thorax compliance (sun et al. 1993 ). impaired compliance after mas is responsive to standard surfactant instillation (sun et al. 1993) . larger volume (15 ml/kg) surfactant lung lavage is an emerging treatment for mas: following surfactant lung lavage, dynamic compliance approximately doubled, and airway resistance nearly halved in a group of mas infants on mechanical ventilation (szymankiewicz et al. 2004b ). these changes in dynamic respiratory mechanics were associated with a decrease in mean (sd) airway pressure from 12.4 ± 3.6 to 5.4 ± 2.1 cm h 2 o within 48 h after surfactant lung lavage. meconium appears to have a differential effect on hyperreactivity of the airways and the lung tissue. whereas tracheal smooth muscle reactivity increases with increasing meconium concentration in response to histamine and acetylcholine, a negative correlation was observed in the lung tissue (mokry et al. 2007 ). airway hyperresponsiveness 2 weeks after birth (approximately 5 days after cessation of ecmo) is responsive to bronchodilator treatment (koumbourlis et al. 1995) as evidenced by percent change in mef 25 and the mef 25 /fvc ratio. airway hyperreactivity after mas is also reduced by budesonide (mokry et al. 2007 ). limited information is available for the longterm lung function outcomes after mas. neonates treated with ecmo for meconium aspiration have better long-term lung function outcomes than infants with diaphragmatic hernia treated with ecmo in the neonatal period (spoel et al. 2012a) , most likely reflecting differences in lung capacity and structure. at least 50 % of children who had mas in the neonatal period have evidence of trapped air on lung function during mid-late childhood. there are no published reports evaluating responsiveness to bronchodilators beyond infancy after neonatal mas. persistent pulmonary hypertension of the newborn (pphn) presents with profound hypoxia either in the absence of significant lung disease (primary pphn) or hypoxia out of proportion with the degree of respiratory disease (secondary pphn -complicating rds, meconium aspiration, pneumonia, etc.). lung function in 1-yearold infants who had pphn and who were treated with inhaled nitric oxide (ino) but not extracorporeal membrane oxygenation (ecmo) was compared with lung function outcomes of infants who were randomized to receive either ecmo or conventional management as part of the uk ecmo trial. v′ max,frc was lower than predicted in all three groups (p < 0.001). there were no statistical differences between the three groups in the z-scores for v′ max,frc (hoskote et al. 2008) . nonetheless, only 26 % of ino-treated infants had v′ max,frc z-scores below normal compared to 37 and 56 %, respectively, for ecmo and cm groups. a prospective evaluation of lung function in infants with pphn treated with/without ino was reported by dobyns and colleagues (1999) : compared to healthy control infants of the same age, there were no differences in lung volume or passive respiratory mechanics for infants with pphn, nor was there any effect of ino on later pulmonary function in the pphn group. together, these results suggest that ino treatment does not worsen outcome compared to ecmo or conventional management. infants with congenital diaphragmatic hernia have impaired airway function in the first year of life: maximal expiratory flows at frc were a mean z-score of −1.5 lower than healthy term infants (i.e., 1.5 standard deviations below the mean frc for healthy term infants) with no evidence of significant change between 6 and 12 months of age (spoel et al. 2012b) . conversely, and perhaps surprisingly, given the usual association of cdh with pulmonary hypoplasia in at least one lung, spoel noted that measured values of functional residual capacity were relatively high (47 % fell above the normal range) (spoel et al. 2012b ). as expected, mean z-score for fev 1 and fvc was negatively influenced by the presence of chronic lung disease, the duration of ventilation, and ecmo support (spoel et al. 2012a) . patients with cdh develop hyperinflation, with elevated plethysmographic frc at 1 year (hofhuis et al. 2011 ) that is still evident at 8-12 years (spoel et al. 2012a; hamutcu et al. 2004; majaesic et al. 2007 ). spoel showed that greater impairment was evident in infants with cdh who required extracorporeal membrane oxygenation (spoel et al. 2012b ). however, poorer outcome in ecmotreated cdh may reflect the initial severity of disease rather than ecmo: beardsmore showed that ecmo per se did not worsen respiratory function at 1 year of age compared to conventionally ventilated controls (beardsmore et al. 2000) . spoel and colleagues also noted deterioration in lung function over time (5-12 years) in patients with cdh who were treated with ecmo in the neonatal period (see table 4 .1) (spoel et al. 2012a ): mean (se) sds score for fev 1 after bronchodilation was higher at 5 years (−0.71 (0.40)) than at 8 years (−2.27 (0.36)) and 12 years (−2.73 (0.61)). deterioration over time may be related to maldevelopment of the alveoli and pulmonary vessels with disturbed lung growth. it is also possible that increased susceptibility to recurrent respiratory tract infections may contribute to deterioration in lung function over time. an active lifestyle and healthy eating pattern may be especially important for children with cdh to counteract the deterioration in lung function over time, given the known positive effect of participation in sports and the negative interaction of bpd is most commonly considered as the chronic respiratory condition complicating extreme prematurity and/or prolonged neonatal mechanical ventilation. factors that independently influence the tidal flow-volume-derived indices include variations in disease severity, degree of alteration to airway and lung mechanics, and also the requirement for oxygen supplementation ). there are, nonetheless, quantitative differences in tidal breathing function measured in bpd infants when compared with measurements obtained from healthy term and preterm controls. bpd infants are consistently more tachypneic than their healthy counterparts ), but differences in other lung function variables are less consistent. tidal volume (v t ) is either less than (paetow et al. 1999) or similar to (patzak et al. 1999; schmalisch et al. 2003) values obtained in healthy infants: v t relative to healthy infants may also decrease with advancing postnatal age (tepper et al. 1986b) , likely reflecting the altered maturational progression in lung structure and function. bpd infants have increased minute ventilation (mv). increased mv is primarily due to increased respiratory rate rather than increases in v, in part reflecting increased dead space ventilation. infants with bpd have an increased work of breathing. within the tidal flow-volume loop, increased work of breathing is evident from linear or concave expiratory limb morphology. bpd infants also have a less variable shape to the tidal flow-volume waveform, indicating that they are functioning near their maximum respiratory capability with minimal reserve capacity to adapt to changes in intrinsic (e.g., airway obstruction, infection, or aspiration) or extrinsic (e.g., cold stress) environment . tidal flow indices are difficult to interpret in part due to opposing effects of neurorespiratory control and lung mechanics on the morphology of the tidal flow waveform. whereas bpd infants have higher respiratory drive (quantified from the mean inspiratory flow: v t /t i where t i is inspiratory time), the ratio of the time to peak expiratory flow relative to expiratory time (t ptef :t e ) after methacholine-induced airway obstruction can either increase or decrease (clarke et al. 1994) . normal fluctuations in v t , t i , and mv seen in healthy infants in response to alternate hypoxic-normoxic breath testing are not evident in bpd infants, indicating reduced chemoreceptor sensitivity to hypoxic stimulus (calder et al. 1994) . lung and chest wall mechanics of infants with bronchopulmonary dysplasia (bpd) were extensively reviewed by gappa and colleagues (2006b) . changes are reflective of maldevelopment of the lungs and airways and remodelling of the lung parenchyma and airway walls associated with increased collagen content. in preterm infants, respiratory system compliance (c rs ) increases relative to body weight over the first 2 years of life, while an initially high respiratory system resistance (r rs ) decreases over the same period (baraldi et al. 1997a) . airway walls of preterm infants are more compliant than term infants as evidenced through measurements obtained using the high-speed interrupter technique (hit) (henschen et al. 2006) . following the nichd consensus conference in 2001 (jobe and bancalari 2001) , some investigators evaluated differences in lung function according to whether infants had mild, moderate, or severe chronic lung disease. using the single occlusion technique, hjalmarson and sandberg showed that infants with severe bronchopulmonary dysplasia (born at mean 25 weeks ga) had increased specific conductance and decreased specific compliance (sc rs ) compared to healthy preterm infants (born at mean 29 weeks gestation) (hjalmarson and sandberg 2005) . there were no differences in lung mechanics between the healthy preterm infants and those with mild or moderate bronchopulmonary dysplasia. the data published by tortorolo et al. indicate a similar trend towards lower c rs at day 7 in infants who later went on to develop mild bpd and to a greater extent in severe bpd (tortorolo et al. 2002) . their results contrast with the findings by shao and colleagues in infants of similar gestations that infants with bpd had decreased c rs (10.8 vs 16.0 ml/kpa/kg), but no difference in specific sc rs (0.69 vs 0.75 ml/kpa/kg) or r rs (6.6 vs 7.3 cm h 2 o/ml/s) compared to non-ventilated preterm infants. infants with bpd do not show an improvement in airway resistance (r aw , measured by plethysmography) over a 4-month period (shao et al. 1998) . several authors have attempted to use lung function as a predictor of bpd. the value of initial r rs for prediction of lung disease is unclear: some have found increased initial r rs is associated with respiratory outcome at 1 year (choukroun et al. 2003; lui et al. 2000; snepvangers et al. 2004) , whereas others observed that r rs is not predictive of bpd (tortorolo et al. 2002) . both lui (lui et al. 2000) and merth (merth et al. 1997) showed that early rds is not an important determinant of later lung function. serial measurements of compliance, however, show that lower c rs values after day 5 were evident in infants who later developed severe bpd. merth observed that bpd infants have reduced c rs at 1 year of age irrespective of rds (merth et al. 1997) . v′ max,frc obtained using the tidal rapid thoracic compression technique is reported consistently as being lower than healthy controls throughout the first 3 years of life, regardless of the bpd era or treatment strategies (lum et al. 2006b ). reduced expiratory flows are indicative of abnormal structural and functional development of the airways (fig. 4.12) . serial lung function measurements in infants with both bpd and "healthy" unsedated preterm infants indicate a decline over the first year of life, suggesting that factors other than bpd may contribute to abnormal airway function in preterm infants (hoo et al. 2002b) . raised volume rapid thoracic compression (rvrtc) measurements show similar mild-moderately severe airflow obstruction. however the predominance of airflow obstruction primarily at low lung volumes indicates the pathology is more likely to involve the small peripheral rather than larger central airways (lum et al. 2006b ). concurrent increased residual volume (rv), frc, and rv/ tlc (total lung capacity) ratios were more marked in infants with recurrent wheeze, indicative of a degree of hyperinflation and gas trapping. intermediate values (between healthy controls and wheezy bpd infants) were observed for rv, frc, and rv/tlc in non-wheezy preterm infants (robin et al. 2004) , likely reflective of abnormal airway growth or the effects of preterm delivery. forced deflation from frc showed that preterm infants with developing bpd have severe lower airway obstruction as early as 3-4 weeks postnatal age. increased flows and fvc following bronchodilation indicate that reopening of obstructed airways is achieved with bronchodilators (motoyama et al. 1987) . a recent systematic review and meta-analysis assessed the effect of preterm birth on later fev 1 : (kotecha et al. 2013 ) 59 studies were included in the meta-analysis, including 28 studies in children born preterm without bpd and 39 studies in children with bpd diagnosed at either 28 day of life (bpd 28 ) or 36 weeks postmenstrual age (bpd 36 ). the meta-analysis showed that just being born preterm decreased fev 1 compared to term-born controls, with further decrements in fev 1 evident for the bpd 28 and bpd 36 week groups. the mean differences (95 % ci) for % fev 1 compared with term-born controls were −7.2 % (−8.7 %, −5.6 %) for preterm group without bpd and −16.2 % (−19.9 to −12.4 %) and −18.9 % (−21.1 to −16.7 %) for the bpd 28 and bpd 36 groups, respectively. including data from preterm studies not including a control group resulted in a pooled % fev 1 estimate of 91.0 % (88.8-93.1 %) for preterm infants without bpd, 83.7 % (80.2-87.2 %) for bpd 28 , and 79.1 % (76.9-81.3 %) for bpd 36 , respectively. of interest, the authors noted that %fev 1 for bpd 28 has improved over the years. bpd survivors also have lower forced expiratory flows: 70 % of children with bpd had maximal flow at functional respiratory capacity (v max frc) below 40 % of the predicted value (baraldi et al. 1997a) . forced expiratory flow at 25 % of forced vital capacity was reduced in young adolescents born prematurely, regardless of prior bpd or level of respiratory support (anand et al. 2003) . of concern, doyle and colleagues showed that airflow limitation worsened between 8 and 18 years ). together, these data suggest that the preterm infant population is at risk of developing chronic obstructive disease and should receive long-term respiratory follow-up (baraldi and filippone 2007a; filippone et al. 2003; lum et al. 2011) . airway obstruction is accompanied by impaired diffusing capacity in children and adults born prematurely (hakulinen et al. 1996; vrijlandt et al. 2006) . similar findings in infants and toddlers with bpd in the face of normal alveolar volumes indicate parenchymal disease and impaired alveolarization (balinotti et al. 2010 ). several studies have evaluated the effect of bronchodilators on lung mechanics in infants with bpd. bronchodilators decrease r rs , without affecting c rs : ultrasonic nebulizers appear to be most effective (fok et al. 1998) . bronchial hyperresponsiveness at 2 years of age was related to mean neonatal c rs and r rs over the first 3 days in ventilated preterm neonates using breath occlusion test. c rs but not r rs was related to bronchial hyperresponsiveness at 2 years' age (snepvangers et al. 2004 ). fifty-six percent of children in the epicure study born at 25 weeks pma or less had abnormal baseline spirometry at 11 years of age, with 27 % showing a positive bronchodilator response: more marked responses were evident in the infants with prior bpd (fawke et al. 2010) . less than half of the epicure study children with abnormal lung function at 11 years were receiving treatment, suggesting more targeted respiratory follow-up is warranted. the published literature on persistence of bronchial hyperreactivity into early adulthood in the population of young adults born prior to routine antenatal glucocorticoid and postnatal surfactant treatment is conflicting: both persistence (halvorsen et al. 2004 ) and resolution (narang et al. 2008 ) of bronchial hyperresponsiveness are reported, although both studies report persistently abnormal baseline spirometry. • measurements of dynamic mechanics are most often obtained in intubated infants, but measurements of dynamic resistance are highly variable and may persistence of bronchial hyperreactivity into adulthood is unknown for the current population of premature infants. véronique nève and francis leclerc a restrictive ventilatory defect is observed when expansion of the lung is restricted because of alterations in lung parenchyma or as a consequence of extraparenchymal diseases affecting pleura, chest wall, or neuromuscular apparatus. chronic ild are characterized by derangements of the alveolar walls and loss of functional alveolar-capillary units. pediatric ild comprises a heterogeneous group of rare disorders with considerable mortality (clement 2004; fan et al. 2004; fan and langston 1993) . chronic ild in immune-competent children has been defined as the presence of respiratory symptoms and/or diffuse infiltrates on chest radiographs, abnormal pulmonary function tests (pft) with evidence of restrictive ventilatory defect and/or impaired gas exchange, and persistence of any of these findings for >3 months (clement 2004) . pft, in children over 6 years, show a reduced forced vc (fvc) and fev 1 and a normal or elevated fev 1 / fvc ratio (fan et al. 2004) (fig. 4.13) . the not be reliable. reasons for variability include the highly frequency-dependent nature of respiratory mechanics, rapidly changing nature of respiratory disease, maturation and the acute effects of antenatal exposures, and postnatal treatments and interventions. • rds is typically characterized by low lung volumes, reduced lung compliance, and elevated tissue resistance. • meconium aspiration is characterized by both increased resistance and decreased compliance. both resistance and compliance are improved by surfactant lavage. mas may be complicated by airway hyperreactivity and gas trapping beyond infancy. • infants with congenital diaphragmatic hernia have impaired airway function at 1 year of age, with evidence of continuing deterioration throughout childhood. worse lung function is likely associated with initial severity of disease and intensity/duration of mechanical ventilation. • bpd infants are typically more tachypneic than their healthy counterparts and have increased work of breathing. airway walls are more compliant and may be prone to collapse. while lung function outcomes for infants with bpd have improved over the last one to two decades, airflow limitation remains an issue for long-term respiratory function after bronchopulmonary dysplasia and may worsen with time. diffusing capacity is also impaired in children and adults born prematurely and may reflect parenchymal disease and impaired alveolarization. • although patients receiving ecmo generally have worse lung function than those treated without ecmo, this is likely due to the initial severity of lung disease and duration of mechanical ventilation rather than an effect of ecmo on long-term lung function per se. to describe flow (f), lung volumes (v), and respiratory pressure (p) measurements together with resistance (r) and compliance (c) measurements in restrictive lung diseases, obstructive lung diseases, and neuromuscular disorders (nmd) decrease in tlc, in general, is relatively less than that of vc because of normal chest wall recoil and inspiratory muscle function in most patients (martinez and flaherty 2006) . functional residual capacity (frc) and residual volume (rv) are normal or elevated resulting in increased frc/tlc and rv/tlc (gaultier et al. 1980; steinkamp et al. 1990; zapletal et al. 1985) . the latter finding suggests air trapping (fan et al. 2004) . airflow limitation has been demonstrated in some studies (fan and langston 1993) . diffusing capacity of carbon monoxide (dl co ), that evaluates the capacity of the lung to exchange gas across the alveolar capillary interface, is low in absolute term but normal when corrected for alveolar v (gaultier et al. 1980; zapletal et al. 1985) . the lung p/v curve is shifted down, and elastic recoil p at maximum inspiration is increased (fan and langston 1993; steinkamp et al. 1990; zapletal et al. 1985) . reduced c l is observed, with lower specific c l in children with fibrotic changes on transbronchial lung biopsy specimen (steinkamp et al. 1990) . the decreased c l and increased elastic recoil can increase the retractive force exerted on the walls of lung airways and reduce the airway r (r aw ) or increase fev 1 % and preserve the peak expiratory f (pef). however, once lung v becomes severely reduced, pef declines because it is then measured at a relatively small lung v (cotes et al. 2006) . pft can aid in establishing disease severity and prognosis. in nonspecific interstitial pneumonia and idiopathic fibrosis, dl co <40 % corresponds to advanced disease and predicts impaired survival. similarly, exertional desaturation <88 % at baseline testing and a decrease in fvc >10 % over the course of the short-term follow-up identify patients at particular risk of mortality (martinez and flaherty 2006) . ali/ards are characterized by an acute onset of respiratory failure, diffuse bilateral pulmonary infiltrates on the chest radiograph, the absence of clinical evidence of left atrial hypertension, and a ratio of pao 2 to fio 2 (p/f) of less than 200 mmhg (regardless of peep). acute lung injury (ali) is a subset of ards with less severe impairment in oxygenation (p/f <300) (bernard et al. 1994) . spo 2 /fio 2 (s/f) may be a good noninvasive surrogate marker for p/f in children: s/f ratios of 263 and 201 have been shown to correspond to p/f ratios of 300 and 200, respectively, the ards cutoff of 201 having 84 % sensitivity and 78 % specificity (bach and bianchi 2003) . decreased c l was associated to these items (ware and matthay 2000) . ali/ards are rare diseases in children and have mortality rates ranging from 18 to 75 % (flori et al. 2005; zimmerman et al. 2009 ). mechanical ventilation represents the main therapeutic support to maintain acceptable pulmonary gas exchange while treating the underlying disease. a "lung protective ventilation strategy" with limitation in airway p and tidal volume (vt) (the acute respiratory distress syndrome network 2000) led, in adult studies, to a reduction in mortality at day 28 and a reduction in hospital mortality (petrucci and iacovelli 2007; the acute respiratory distress syndrome network 2000) . no published data exist for children, but practice in picu is derived from adult patients (khemani and newth 2010 the most characteristic alteration in acute ards lung is marked fall in c l caused by loss of surfactant function, atelectatic lung regions, accumulation of interstitial/alveolar plasma leakage (ware and matthay 2000) , and an associated fall in frc (hammer 2001) . in adults, the decrease in c l was shown to be more important in early ards from pulmonary origin (and associated with normal chest wall c (c cw )) than in extrapulmonary ards. the latter was associated with low c cw (gattinoni et al. 1998) . a decrease in respiratory system compliance (c rs ) and a proportional decrease in tlc, fvc, and frc were also observed in children during the acute phase of ards (hammer et al. 1998; newth et al. 1997) . ali/ards may resolve completely in some patients, while in others it progresses to fibrosing alveolitis with persistent hypoxemia and a further decrease in c l (ware and matthay 2000) . fibroproliferation occurs early in ards, and its extent may be predictive of outcome (marshall et al. 2000) . the recovery phase is characterized by the gradual resolution of hypoxemia and improved c l (ware and matthay 2000) . pharmacologic interventions, such as corticosteroids, starting in the late course of ards may reduce fibrosis and may have a beneficial impact on pulmonary outcome (tang et al. 2009 ). several studies have demonstrated persistent impairment in pulmonary function of unknown long-term significance for children who required mechanical ventilation in pediatric intensive care units for respiratory failure (khemani and newth 2010) . most survivors of ards have persistent mild reductions of dl co even as long as a year after their episode. the lung v and f return to normal in most instances, although a subset of patients will have persistent impairment. both obstructive and restrictive deficits may be seen (alberts et al. 1983; elliott et al. 1981; ghio et al. 1989; hert and albert 1994; peters et al. 1989) . timing of recovery occurring in the first year after ards was described in two prospective studies of adult survivors of ards (herridge et al. 2003; mchugh et al. 1994) . after discharge from the icu, a restrictive ventilatory defect and reduced dl co were observed in almost all patients and an obstructive defect in only 5 %. improvement was observed until 6 months after discharge. a relatively static period was observed after that. by 6 months, abnormalities in fvc, tlc, and dl co were observed in 55, 45, and 26 %, respectively, of patients. patients with more severe ards, as determined by their higher ali scores (corresponding to patients ventilated for >2 weeks), were more impaired for fvc throughout the follow-up and did not return to normal pulmonary function levels (mchugh et al. 1994) . patients of herridge's study were shown to have a mild restrictive defect with mild to moderate reduction in dl co at 3 months. lung v and spirometric measurements were normal by 6 months, but dl co remained low throughout the 12-month follow-up (herridge et al. 2003) . for the pediatric age group, a few small-sized observational cohort studies have reported sequelae in 10-100 %, in mostly asymptomatic subjects (ben-abraham et al. 2002; fanconi et al. 1985; golder et al. 1998; lyrene and truog 1981; weiss et al. 1996) . obstructive (reversible and nonreversible airflow obstruction) and restrictive abnormalities have been observed after discharge. like in adults, recovery during the following months reached plateau levels at 12 months with no further improvement (golder et al. 1998) . decrease in c l was shown to persist in ≥50 % of children and adults, evaluated 1-2 months (aggarwal et al. 2000) , or 34 months after ards (klein et al. 1976) , and was associated with a restrictive defect in some patients. symptomatic upper airway obstruction as a result of laryngotracheal injury from long-term intubation has been described in minority (10 %) of adults after ards with evidence of upper airway obstruction on inspiratory and expiratory f/v curves (elliott et al. 1988 ). the prevalence of childhood obesity is increasing in developed countries. obese children have more respiratory symptoms than their normal-weighted peers, and respiratory-related disorders increase with increasing weight. the prevalence of asthma has also increased in children. both obesity and asthma have their beginnings in early childhood (litonjua and gold 2008) . wheezing, asthma, and obesity seem to be associated (guerra et al. 2002; hancox et al. 2005; schachter et al. 2001 ). however, three large studies (bibi et al. 2004; schachter et al. 2003; wickens et al. 2005) showed a clear increase in symptoms of asthma in association with obesity but not more frequent airway hyperresponsiveness in obese children (deane and thomson 2006) . c cw is reduced in obesity, because of increased adiposity in the abdomen and around the thoracic cage that restricts rib expansion and because the decreased total thoracic and pulmonary v may pull the chest wall below its resting level to a flatten portion of its p/v curve. c cw decrease correlated with co 2 retention, independent of body fat, and with shortness of breath (subramaian and strohl 2004) . c l may be decreased in some obese individuals because of a large pulmonary blood volume and intrinsic alterations of elastic characteristics of lung tissues (subramaian and strohl 2004) . as additional fat in the abdomen raises the diaphragm, the frc and expiratory reserve volume (erv) are reduced in the erected position and further reduced in the supine position (koenig 2001) . the lung bases are poorly ventilated which contributes to hypoxemia. tlc and vc may also be reduced, but rv is usually maintained (therefore rv/tlc ratio may be increased). in morbidly obese individuals, an increase in body mass correlates with reduced fev 1 (carey et al. 1999 ) and fvc (fev 1 / fvc ratio remains normal) (zerah et al. 1993) . data in children and adolescents confirm the reduced frc and static lung v (li et al. 2003) and the decrease in fvc and fev 1 with increasing proportions of body fat as a percentage of body weight (lazarus et al. 1997 ). all these effects were shown to be reversible in morbidly obese patients (with body mass index (bmi) >40) following weight loss (camargo et al. 1999; carey et al. 1999; deane and thomson 2006) . in seated patients with simple morbid obesity, inspiratory and expiratory muscle strength is normal (yap et al. 1995) . when patients are supine, maximal inspiratory p (pimax) decreases by about half as a result of overstretching of the diaphragm, causing it to operate on the descending limb of its length-tension curve (laghi and tobin 2003) . morbidly obese patients without hypoventilation (simple obesity) compensate for the respiratory load by increasing respiratory drive and diaphragmatic pressure output (pankow et al. 1998 ) and increasing rib cage contribution to tidal breathing. scoliosis is the most common abnormality of the spine with direct effect on the thoracic cage. idiopathic scoliosis (including infantile, juvenile, and adolescent) accounts for 80-85 % of scoliosis. neuromuscular scoliosis (as in duchenne muscular dystrophy (dmd) or spinal muscular atrophy (sma)) and scoliosis associated with congenital vertebral abnormalities account each for 5 % of scoliosis. infantile and congenital scoliosis, untreated, results in severe spinal deformities and is at risk of cardiopulmonary insufficiency before adulthood (canet and bureau 1990) . patients with scoliosis and a cobb angle <70° are usually asymptomatic, those with an angle between 70° and 100° often experience dyspnea on exertion, and those with the angle >100° are at risk for chronic respiratory failure (estenne et al. 1998) . scoliosis can produce severe reductions in lung v and restrictive ventilatory defect with decrease in fvc, fev 1 , and tlc as well as a normal fev 1 /fvc ratio (gagnon et al. 1989; muirhead and conner 1985; upadhyay et al. 1993; weber et al. 1975 ). among patients with moderate to severe scoliosis (35-100°), the vc decrease is related to the cobb angle. when the cobb angle exceeds 100° (severe scoliosis), vc falls to about 50 % of predicted (laghi and tobin 2003) . impaired lung growth (davies and reid 1971) , significant decrease in c l and c cw (at about 25-50 % of predicted in children and adults with scoliosis) (laghi and tobin 2003) , and/or impaired muscle strength as a consequence of inefficient coupling between the respiratory muscles and the thoracic cage account for the effect of scoliosis on lung function. in adults with scoliosis and cobb angles of 66-136°, pimax was about 50-76 % (estenne et al. 1998; laghi and tobin 2003) . decreased tlc is often associated with increased rv resulting in very high rv/tlc ratio (day et al. 1994) , probably reflecting the dysfunction of expiratory muscles, which do not allow full exhalation. significant gas trapping with elevated plethysmographic frc/helium frc ratio can occur with response to bronchodilators, indicating airway hyperresponsiveness that results from chronic airway inflammation secondary to the poor clearance of secretions (boyer et al. 1996) . the chest distortion causes airway distortion thereby contributing to a slight increase in airway r (r aw ) (canet and bureau 1990) . scoliosis, by inducing significant displacement/rotation of the intrathoracic trachea and/or main stem bronchi, can cause mechanical obstruction as shown on the f/v loop with flattening of the initial portion of the expiratory loop suggesting central airway obstruction (borowitz et al. 2001) . the decreased c rs may account for the increased work of breathing, with increased transdiaphragmatic pressure during tidal breathing; increased rib cage contribution to tidal breathing; transversus abdominis recruitment during exhalation, as observed in most patients with severe scoliosis (laghi and tobin 2003) ; and the associated increased risk of respiratory muscle fatigue and eventual respiratory failure. in contrast to idiopathic scoliosis which tends to stabilize (or progress at a lower rate) after the person reaches skeletal maturity, neuromuscular scoliosis (such as in dmd or sma) continues to worsen because of the progressive worsening of the muscle weakness. recurrent aspiration and pneumonias secondary to impaired clearance of airway secretions are potential complications. due to the severity of their primary muscle weakness, patients with neuromuscular scoliosis may not be able to maintain adequate ventilation and may develop severe atelectasis (koumbourlis 2006) . scoliosis is reported in 60-100 % of sma children and is due to the inability of the trunk muscles to support spine in the upright position. all children with type 2 sma develop scoliosis starting from around 3 years. the average curve is more than 54° at 10 years of age (mullender et al. 2008) . as the scoliosis progresses, respiratory function deteriorates, and the risk of life-threatening complications increases (rodillo et al. 1989) . spinal fusion is recommended when scoliosis progresses and reaches a cobb angle of between 40° and 60° in children ≥10 years of age (tsirikos and baker 2006) . in dmd, scoliosis incidence ranges from 75 to 95 % (muntoni et al. 2006) . dmd develop scoliosis after losing independent ambulation (in the second decade of life). once scoliosis reaches 30°, it progresses with age and growth. corticosteroids may delay the development and progression of scoliosis in dmd (muntoni et al. 2006) . optimal timing for surgical intervention is while lung function is satisfactory and before cardiomyopathy becomes severe enough to risk arrhythmia under anesthesia (cobb angle between 30° and 50°). best prognosis for recovery seems to be fvc >40 %, although others use the absolute vc of <1.9 l as an indicator of rapid progression of scoliosis and poor prognosis (finder et al. 2004) . comparison between preand postoperative pulmonary function reveals no improvement due to correction of scoliosis. long-term studies show that decline of pulmonary function in dmd patients is unchanged in operated patients compared to patients who had no surgery (muntoni et al. 2006 ). asthma is the most frequent chronic disease observed in children (prevalence ranging from 8 to 12 % (isaac 1998)). asthma is difficult to diagnose in children ≤5 years. children presenting with wheeze at an early age belong to a heterogeneous group: early transient, late onset, and persistent wheezers (martinez and helms 1998) . half of the "early" wheezing children become asymptomatic by school age. they may have diminished lung function early on, but by 6 years lung function is improved (martinez et al. 1995) . children who had wheezing that began in infancy and continued at 6 years demonstrated normal pulmonary function initially with reduced lung function by 6 years. those children with "persistent wheezing" have some ongoing chronic inflammatory process that results in airway alterations and some loss of lung function in early childhood, which extends to varying degrees in adulthood (oswald et al. 1997 ). respiratory syncytial virus (rsv) bronchiolitis in infancy is often associated with recurrent wheezing and asthma during subsequent years. however, wheezing tends to diminish by school age or adolescence. most follow-up studies of rsv bronchiolitis in infancy show that forced expiratory flow (fef) rates and fev 1 are lower at school age compared with control groups (hall et al. 1984; pullan and hey 1982) . children who had been hospitalized for rsv bronchiolitis as infants later had lung function abnormalities similar to those found in children with asthma (kattan et al. 1977; wennergren and kristjansson 2001) . increased r to airflow is the basis of the clinical manifestations of asthma, including dyspnea and wheeze. usually, airflow limitation is reversible. fixed airway obstruction may be seen in later disease stage (strachan et al. 1996) or be a component of a specific asthma phenotype (bush 2004) . airway obstruction in childhood is associated with a reduced fev 1 in adulthood (jenkins et al. 1994; roorda et al. 1994) . airflow obstruction (afo) can be assessed by body plethysmography, spirometry, forced oscillation technique (fot), and interrupter technique, but these are more difficult to perform in children <6 years. incentive spirometry can be performed in the majority of children ≥3 years (beydon et al. 2007b ) together with frc helium measurement and r measurement (using interrupter technique (r int )) (beydon et al. 2007b) , fot, or plethysmography for measurement specific r aw (sr aw ) (dab and alexander 1976) . infant pfts are mainly research tools. the fev 1 is the "gold standard" of measuring airway obstruction in children over 6 years (miller et al. 2005; pellegrino et al. 2005) . preschoolers often do not exhale for more than 1 s. therefore fev 1 may not be an accurate index of bronchial obstruction in this age group, and the utility of fev 0.5 or fev 0.75 as outcome measure in this age group has been explored (aurora et al. 2004; beydon et al. 2007b; neve et al. 2006; vilozni et al. 2005) . international guidelines (nhlbi 2002) recommend an initial evaluation and regular reeval-uation for the assessment of the severity and the control of asthma. asthma severity describes the underlying disease state as evaluated by fev 1 and daytime and nighttime symptoms, all measured before treatment. patients with moderate and severe persistent asthma are said to have values of 60-80 % and <60 % of predicted, respectively. fev 1 may not be the best measure of severity in childhood asthma because most asthmatic children have fev 1 values in the normal range independent of disease severity when clinically stable (spahn et al. 2004) . diminished fev 1 values in school-age children should identify children at risk of fixed airway obstruction at adulthood (rasmussen et al. 2002) . for children, fev1/fvc appears to be a more sensitive measure of severity in the impairment domain (nhlbi 2007) a concave shape on the f/v curve may be observed as a result of small airway obstruction (pellegrino et al. 2005) . it may be observed in asthmatic children with a normal fev 1 , together with a significant improvement following administration of a bronchodilator (basek et al. 2005; brand and roorda 2003) . a concave shape is associated with impairment in the fef between 25 and 75 % of fvc (fef 25-75 % ) that is believed to measure peripheral airway obstruction. fef 25-75 % is among the first parameters to be abnormal in pediatric asthma and often the most significantly impaired of all spirometric measures (paull et al. 2005; spahn and chipps 2006) . a fef 25-75 % ≤65 % is well correlated with bronchodilator responsiveness in asthmatic children with normal fev 1 and may suggest suboptimal asthma control (simon et al. 2010) . plethysmography may show lung distension/air trapping with larger rv and tlc in children than in adults (jenkins et al. 2003) . the rv is a sensitive parameter of airway obstruction in children, and a decrease in rv after bronchodilator administration appears to be specific for asthma diagnosis (walamies 1998) . air trapping may be observed when fvc and fev 1 values are normal in mild or moderate asthma (cooper et al. 1977) or in controlled asthma (vilozni et al. 2009 ). indeed, the principal event taking place in the asthmatic lung is the closure of small airways with increase in rv that is associated to a protective increase in tlc to preserve the functional range of fvc. once the limit of the chest wall expansion has been reached, further increase in rv will result in falls in both fvc and fev 1 (irvin and bates 2009) . frc may be elevated, due to airway closure and/or dynamically elevated with an increased expiratory time constant (stanescu 1999) . it is a compensatory mechanism that minimizes the increase in r aw and the expiratory flow limitation in obstructive airway disease. hence, at an early stage of the disease, an isolated increase in lung v may be the sole functional abnormality (landau et al. 1979; paton 2000) . in asthmatic children, frc increases with severity of asthma (greenough et al. 1987) and is more elevated in symptomatic children ). trapped air is associated with hypoxemia (wolf et al. 1983 ) but correlates poorly with f limitation and may be lacking in patients with severe asthma (basek et al. 2005; desmond et al. 1986 ). in preschool children with asthma, a 6-week inhaled corticosteroid therapy is associated with reduced hyperinflation as indicated by lower frc helium . bronchodilators were shown to decrease helium frc in 80 % of children aged 2-7 years, and the decrease was correlated to baseline frc. some children exhibited an increase in frc after bronchodilation when baseline value was low (greenough et al. 1989) . children higher baseline r int measurement was shown in young preschool asthmatics (beydon et al. 2003; nielsen and bisgaard 2001) and children with a history of wheeze (mckenzie et al. 2000) compared with healthy children. children with persistent wheeze were shown to have higher r int value than those with transient or no previous wheeze, but there was an overlap in r int values between the three groups (brussee et al. 2004 ). using fot, increased r of the respiratory system at 5 hz has been reported in asymptomatic asthmatic preschool children as compared with healthy subjects in some studies (nielsen and bisgaard 2001) . plethysmographic specific r aw (sr aw ), product of r aw , and plethysmographic thoracic gas volume (tgv) may be altered by both hyperinflation and decreased airway diameter. in preschool children, it was shown to be increased in children with a history of wheeze (lowe et al. 2002) ; it was able to identify responses to bronchodilators (nielsen and bisgaard 2001) and inhaled steroids (nielsen and bisgaard 2000) . sr aw was more strongly related to fef after 50 % of fvc has been exhaled (fef 50 % ) than to fev 1 and could be used in preschool children to predict mild airflow limitation (mahut et al. 2009 ). however, there is no established cutoff for sr aw to separate healthy subjects from asthmatics (marchal and schweitzer 2005) . in cf, the combination of mucus retention, bacterial infections, and inflammation results in obstructive lung disease primarily involving the small airways (bedrossian et al. 1976; fox et al. 1974; ratjen and grasemann 2005) . restrictive alterations develop secondary to the damage of lung parenchyma by inflammatory changes and by neutrophil degradation products. chronic airway infection, progressing to bronchiectasis, gas trapping, and hypoxemia and hypercarbia, is the hallmark of cf lung disease. pulmonary insufficiency is responsible for at least 80 % of cf-related death (cystic fibrosis foundation 2008). fev 1 is the gold standard for lung function in cf. fev 1 reflects the progression of pulmonary disease and correlates with mortality. an fev 1 of less than 30 % predicted in conjunction with other clinical indicators is used for selecting suitable candidates for lung transplantation (kerem et al. 1992) . in cf, obstruction primarily affects the small airways. an early change associated with airflow obstruction in small airways is a concave shape on the f/v curve and a reduction in the fef 25-75 % (pellegrino et al. 2005 ) such as reported in cf patients (corey et al. 1997; 1976 ). this fef often shows abnormalities in patients in whom fev 1 is in the normal range. however, the large interindividual variability of this fef must be taken into account in its interpretation. diminished fevs and fefs have also been reported in sedated infants with cf (including infants considered to be asymptomatic by their physician) using the raised volume rapid thoracoabdominal compression technique (ranganathan et al. 2002; and in preschool children using incentive spirometry (kozlowska et al. 2008; marostica et al. 2002; vilozni et al. 2007 ). other techniques detecting small airway obstruction afo in small airways is associated with lung hyperinflation as indicated by increased rv, rv/ tlc, and tlc. a small airway obstruction syndrome with decreased vc and fev 1 increased rv, but a normal fev 1 /vc ratio and tlc has also been described (stanescu 1999) . the condition is attributed to premature closing of airway leading to air trapping as demonstrated by high-resolution computed tomography (hrct) scans (cotes et al. 2006) . studies using body plethysmography have provided evidence for trapped gases in the majority of patients with cf (beier et al. 1966) . the ratio of rv to tlc that indicates hyperinflation, a key feature of cf lung disease, has been found to correlate with disease severity in numerous studies (beier et al. 1966; landau et al. 1979; landau and phelan 1973a) . percentage of children with hyperinflation and trapped gas increases with age, and children with severe hyperinflation at 6-8 years showed the most severe disease progression over time (kraemer et al. 2006) . in children <6 years, frc is the only lung volume that can be measured routinely. hyperinflation is one of the earliest features of cf (gappa et al. 2001) . elevated tgv has been found in cf infants (beardsmore et al. 1988) , and hyperinflation has been detected by the helium dilution technique in preschool children (beydon et al. 2002) . multiplebreath washout methods (mbw) may be particu-larly sensitive to changes in peripheral lung function and detect early functional abnormalities in infant and preschool children with cf (aurora et al. 2005; ranganathan et al. 2008) . frequency dependence of lung c as indicative afo in small airways has been observed in the majority of cf, but the invasive nature of this technique limits its clinical use (landau and phelan 1973a) . alterations in lung elastic recoil were found with variable frequency (cook et al. 1959; landau and phelan 1973a; mansell et al. 1974 ). as the airway disease becomes more advanced and/or more central airways become involved, fev 1 will be reduced out of proportion to the reduction in vc and an obstructive ventilatory defect be observed. severity of lung disease in cf is classified by the degree of impairment in fev 1 (with fev 1 < 50 % of predicted having severe disease) (pellegrino et al. 2005 ), (fig. 4.14) . the obstructive ventilatory defect is reversible if an improvement in fvc and/or fev 1 of at least 12 % of baseline after betaadrenergic agents is observed (pellegrino et al. 2005 ). significant reversibility can be observed in 50-60 % of cf patients, and 10-20 % show reduced lung function in response to short-acting inhaled bronchodilators (brand 2000; landau and phelan 1973b; shapiro et al. 1976 ). studies in cf have shown decreased c and increased r (cook et al. 1959 ). due to the large surface area of small airways, their contribution to r aw is relatively small. r aw measurements are therefore normal in many patients with cf until they develop disease involving the larger airways (landau and phelan 1973a) . as the disease progresses, bronchial obstruction leads to tissue destruction with bronchiectasis and the development of areas of pulmonary emphysema and fibrosis. in more advanced disease, the destruction of lung tissue leads to decreased c that parallels the decline in fev 1 (hart et al. 2002) . this increases respiratory load and favors a rapid and shallow breathing pattern that further impairs gas exchange in cf patients. the loss of lung v that develops in patients due to tissue destruction as well as the hyperinflation decreases fvc: therefore fev 1 /fvc ratio may be normal even in patients with severe disease (landau and phelan 1973a) . a small proportion of cf patients also develop a restrictive lung disease with decreased plethysmo(ries et al. 1988 ). pulmonary disease resulting from a neonatal respiratory disorder is called chronic lung disease of infancy (cld). bronchopulmonary dysplasia (bpd) (defined as the need for supplemental oxygen for at least 28 days after birth) accounts for the vast majority of cases of cld. what is now considered the "old" bpd was originally described in slightly preterm newborns who had been exposed to aggressive mechanical ventilation and high concentrations of inspired oxygen. diffuse airway damage, smooth muscle hypertrophy, neutrophilic inflammation, and parenchymal fibrosis reflected extensive disruption of relatively immature lung structures. the "new" form of bpd is interpreted as a developmental disorder: despite being delivered several weeks before alveolarization begins, these infants often have only mild respiratory distress syndrome at birth, but lung development is affected with interruption of alveolarization at a very early stage with subsequent alveolar-capillary hypoplasia (baraldi and filippone 2007b ). most of the information on long-term lung function in survivors of bpd refers to patients who had the condition before surfactant treatment was available ("old" form of bpd). in the first months of life, survivors of bpd are severely affected. infants with bpd at 28 days of age have increased total and expiratory r and severe f limitation especially at low lung v (hazinski 1990 ). neonates and young infants with cld have increased levels of frc by plethysmography and decreased frc values by nitrogen washout, suggesting the presence of trapped air (wauer et al. 1998) . specific dynamic c (c dyn ) may be reduced by more than 50 % (allen et al. 2003; gerhardt et al. 1987b ) by small airway narrowing but also by interstitial fibrosis, edema, and atelectasis (hazinski 1990 ). lung mechanics measurement that reflects the severity of neonatal disease (i.e., c rs at 10-20 days) has been shown to correlate with subsequent reduction in lung function in 2-year-old bdp (baraldi et al. 1997b; bhandari and panitch 2006) . childhood during infancy and early childhood, an improvement of airway physiology is observed, and c l improves over time (baraldi et al. 1997b; bhandari and panitch 2006; gerhardt et al. 1987b ). maximum flow rates at frc were reported to increase significantly within the first 2 years along with a reduction in r aw (farstad et al. 1995; trachsel and coates 2005) . however analysis of fef shows that substantial afo persists in numerous survivors of bdp and in preterm infants without bpd during the first 3 years of life (baraldi et al. 1997b; tepper et al. 1986b ). the degree of airflow limitation in the first years of life seems to predict later pulmonary function (at 8 years) suggesting tracking of lung function with time, negligible "catch-up" growth of the lung, and irreversible early airway-remodelling process (baraldi and filippone 2007b) . body plethysmography studies in school children and adolescents generally reveal normal tlc with hyperinflation as reflected by an increased rv and rv/tlc ratio. consistently children diagnosed with bpd are more prone to hyperinflation than prematurely born children without bpd. frc is normal or mildly increased. differences between frc determined by helium dilution technique and by plethysmography may indicate the presence of trapped air (trachsel and coates 2005) . at the age of 8 years, c dyn was reported to be 57 % of the control group, in bpd, and 74 % in prematurely born children without cld (parat et al. 1995; trachsel and coates 2005) . school-age children with a history of bpd have lower fev 1 than children born at term or those born prematurely without lung disease. they also have a lower fvc and fev 1 /fvc ratio than children born at term (allen et al. 2003; bhandari and panitch 2006) . data from 18 studies in children, adolescents, and young adults show that spirometric values are consistently lower in survivors of bpd, at any age between 6 and 20 years, than in controls born at term with fev 1 ranging from normal to severely decreased values (baraldi and filippone 2007b) . patients who were born prematurely but did not have bpd usually fare better, but they too may have airflow limitation at school age and later (baraldi and filippone 2007b) . asthma-like symptoms are often associated to spirometric evidence of airflow limitation in children who had bpd as infants. but airflow limitation is only partially reversed by β2-agonists in children suggesting a stabilized remodelling process. hrct studies have documented scattered parenchymal fibrosis and architectural distortion in many survivors of bpd, findings that are unusual in children with asthma (baraldi and filippone 2007b) . there is no evidence that children with bpd born since the introduction of surfactant-replacement therapy (i.e., "new" bpd) have better spirometric results at school age than those born in the pre-surfactant era (i.e., "old" bpd) as shown in studies evaluating cohort of infants weighting less than 1,000 g at birth or who were born before a gestational age of 29 weeks (doyle 2006; halvorsen et al. 2006) . these results suggest that prematurity itself has a very important independent influence on the long-term respiratory prognosis (baraldi and filippone 2007b) . hematopoietic stem cell transplantation (hsct) is an established therapy for many chemosensitive or radiosensitive malignancies in children. bo is the most common late noninfectious pulmonary complication (i.e., that presents after the first 100 days following transplantation) with 80 % of cases occurring between 6 and 12 months. the reported incidence range is 0-48 % (soubani and uberti 2007) . bo seems to be less common in children than in adults (cerveri et al. 2005) . the most important association with bo is chronic graft versus host disease (gvhd), especially progressive chronic gvhd which evolves without hiatus from active acute gvhd. most cases of bo are thought to be secondary to bronchial mucosal damage from gvhd with inflammation of the small airways and subsequent obliteration. the main symptoms associated with bo are dry cough, dyspnea, and wheezing. twenty percent of patients are asymptomatic. spirometry is the main tool to diagnose and follow up patients with bo following hsct. based on the experience of lung transplantation, a "bo syndrome" has been defined by pft rather than histology (estenne et al. 2002) . it was suggested that the diagnosis of bo is made when there is evidence of (1) new onset of afo with reduction in fev 1 < 75 % of predicted with a fev 1 /fvc < 0.70 not responsive to bronchodilators; (2) air trapping or small airway thickening or bronchiectasis on hrct of the chest with inspiratory and expiratory cuts with nonparenchymal involvement, rv on pft > 120 % of predicted or pathological confirmation of constrictive bronchiolitis; and (3) absence of infection in the respiratory tract documented by clinical symptoms, radiological studies, or microbiological cultures (soubani and uberti 2007) . there are some studies that suggested that a reduction in mean fef 25-75 % may precede the decline in fev 1 and is a sensitive but not specific indicator of subsequent development of bo (estenne et al. 2000; ouwens et al. 2002; patterson et al. 1996; reynaud-gaubert et al. 2000) . a pediatric study defined afo as fev 1 < 80 % and fef 25-75 % < 60% of predicted (schultz et al. 1994) . since the evolution of the disease despite treatment is particularly negative with a progressive worsening of respiratory function and a high mortality, early diagnosis is important. this can be achieved by systematic regular monitoring of the respiratory function starting from the onset of acute gvhd (cerveri et al. 2005 ). decrease in vc is closely linked to weakness of respiratory muscles and disease progression. vc below 40 % predicts nocturnal alveolar hypoventilation, and vc <25 % or <1 l, in duchenne patients, is strongly associated with respiratory failure. in mild respiratory muscle weakness, vc is less sensitive than maximum respiratory pressures. in patients with neuromuscular disorders, respiratory failure may present either acutely as a result of pneumonia or more slowly, as a result of progressive ventilatory decompensation. dmd and sma account for the majority of patients seen in pediatric practice. the most frequently noted abnormality of lung v in patients with respiratory muscle weakness is a reduction in vc. vc can be measured in cooperative children, usually older than 6 years. vc reflects the strength of both inspiratory and expiratory muscles but is not specific, as it can be reduced by other factors than muscle weakness, such as reduction in both c l (gibson et al. 1977) and c cw in patients with chronic respiratory muscle weakness (de troyer et al. 1980) . the decreased c cw probably results from stiffening of tendons and ligaments of the rib cage and ankylosis of the costosternal and thoracovertebral joints (laghi and tobin 2003) . a decrease in c l could be related to diffuse microatelectasis in a few patients (estenne et al. 1993) . in children younger than 4 years with nmd, higher c cw normalized to body weight than in controls has been reported (papastamelos et al. 1996) . such a high c cw results in chest wall deformation during tidal breathing and excessive work of breathing. it also predisposes to atelectasis and can result in fixed deformation of the chest wall (i.e., pectus excavatum). absence of lung stretch with sigh breaths and chest wall deformities can also result in reduced lung growth for children with nmd (bach and bianchi 2003; panitch 2009 ). in patients with nmd, a decrease in vc is an early sign of respiratory impairment. decrease in vc and tlc is closely linked to a weakness of respiratory muscles (braun et al. 1983; hahn et al. 1997; ragette et al. 2002) and disease progression (inkley et al. 1974; samaha et al. 1994 ). in patients with advanced nmd, a restrictive ventilatory defect is observed with tlc reduction. in dmd, a typical evolution of lung v is observed (hahn et al. 1997) . in ambulatory dmd the lung v increases with age (ascending phase), and predicted v is almost normal (tangsrud et al. 2001) . with loss of ambulation between 7 and 12 years (gozal 2000) , measured vc remains stable (plateau phase), but % predicted values begin to decline. in the following years, vc declines by about 200 ml or 6-8 % per year (descending phase) phillips et al. 2001; rideau et al. 1981) . in sma, there is a characteristic pattern of involvement with intercostal muscle weakness and relative sparing of the diaphragm (kuzuhara and chou 1981; perez et al. 1996) . the rib cage is neither stabilized nor expanded during inspiration. over a period of time, the retraction of the rib cage has a detrimental effect on alveolar development. in type 1 sma, with disease beginning before birth, pulmonary hypoplasia has been described (cunningham and stocks 1978) . the best parameter to monitor respiratory muscle strength in children with sma over 6 years is fvc (% predicted) (manzur et al. 2003) . in sma, lung v (% predicted) decreases with age, with a greater vc decrease in type 2 than in type 3 sma. a decline in vc from 87 to 73 % was shown to occur in type 3 sma from 12 to 18 years (souchon et al. 1996) . the fvc may therefore be normal or near normal in stronger ambulant type 3 sma (samaha et al. 1994) . in type 2 sma, fvc is more severely impaired and was shown to decline from 55 % predicted to 37 %, from 10 to 16 years, with a yearly average decline by 2-5 % between 7 and 15 years of age (barois et al. 2005; herridge et al. 2003; souchon et al. 1996) . a restrictive pattern was observed in a majority (70 %) of type 2 sma children followed longitudinally . in intermediate type 1 sma, severe impairment in fvc has also been described (barois et al. 2005) with progressive decrease in fvc from 60 % predicted (at 7 years) to 16 % (at 15 years) in children without tracheotomy and from 22 % predicted (at 7 years) to 14 % (at 15 years) in children with tracheotomy (ioos et al. 2004 ). progression of scoliosis seems to contribute to vc decline in children with nmd (miller et al. 1988 ), but treatment of scoliosis with spinal stabilization did not prevent further vc decline in patients with dmd (kennedy et al. 1995; miller et al. 1988 ). in type 2 sma, beneficial effects of spinal surgery on pulmonary function remain controversial (mullender et al. 2008; wang et al. 2007 ), but the rate of pulmonary function decline may be slowed (wang et al. 2007 ). static lung v may also be affected in some nmd patients by coexistent lung or airway disease (american thoracic society, european respiratory society 2002). measurement of postural change in vc gives a simple index of weakness of the diaphragm relative to the other inspiratory muscles. a fall of 30 % or more in the supine compared with the erect posture is generally associated with severe diaphragmatic weakness (american thoracic society, european respiratory society 2002). studies in adults with generalized nmd suggest that supine vc is a simple, sensitive, and specific test for diaphragm weakness and can replace invasive diagnostic tests (fromageot et al. 2001; lechtzin et al. 2002) . thresholds of vc have been identified to predict treatable complications and outcome in patients with nmd. a vc below 60 % is a sensitive and specific predictor of the onset of sleep-disordered breathing; vc below 40 % predicts nocturnal alveolar hypoventilation, and vc < 25 % or <1 l, in dmd, is strongly associated with respiratory failure and poor survival unless patients are treated with mechanical ventilation (baydur et al. 1990; canny et al. 1989; hukins and hillman 2000; mellies et al. 2003; phillips et al. 2001 phillips et al. , 1999 ragette et al. 2002; simonds et al. 1998; wallgren-pettersson et al. 2004) . rv is usually normal or increased, the latter, particularly with marked expiratory weakness (kreitzer et al. 1978) . consequently, tlc is less markedly reduced than vc, and the rv/tlc and frc/tlc ratios are often increased without necessarily implying airway obstruction (american thoracic society, european respiratory society 2002). a hypodynamic type of ventilatory defect with increased rv, low vc, and normal tlc can be observed early in the course of nmd. such a presentation has recently been described in patients with dmd (tangsrud et al. 2001) . these children may later exhibit chest wall deformities leading to a true restrictive syndrome. r aw is normal in uncomplicated respiratory muscle weakness. the maximum expiratory and maximum inspiratory f/v curves characteristically show a reduction in those f that are most effort dependent, that is, maximum expiratory f at large lung v (including pef) and maximum inspiratory f at all lung v. with severe expiratory weakness, an abrupt fall in maximum expiratory f is seen immediately before rv is reached (vincken et al. 1987) . oscillations of maximum expiratory and/or inspiratory f, the so-called sawtooth appearance, are seen particularly when the upper airway muscles are weak and in patients with extrapyramidal disorders (american thoracic society, european respiratory society 2002; vincken and cosio 1989) . in mild respiratory muscle weakness, vc is less sensitive than maximum respiratory p measurement. vc is normal, or only minimally reduced, if respiratory muscle strength is more than 50 % of predicted (laghi and tobin 2003) . this finding results from the sigmoid shape of the p/v relationship of the respiratory system. several invasive and noninvasive tests to assess respiratory muscle strength have been reported to be of value in testing respiratory muscle strength in patients with nmd. in children, normative data are only available for pimax, pemax, and sniff nasal inspiratory pressure (snip). pimax and pemax are tests of global inspiratory and expiratory muscle strength. they were found to be reduced in nmd patients (baydur 1991; black and hyatt 1971) . the pimax decline was related to the decline of inspiratory reserve volume and tlc, and the pemax decline was related to the decline of erv and the increase of rv in children and adults with dmd (hahn et al. 1997) . in dmd, earlier in the course of the disease, inspiratory muscle strength evaluated by pimax remained relatively well preserved (as compared with pemax) implying relative sparing of the diaphragm (hahn et al. 1997; mcdonald et al. 1995) . in patients with diaphragm weakness, a vc decline of 25 % or more following supine positioning is associated with a mean pimax decline of 18 ± 14 % (ragette et al. 2002) . sleepdisordered breathing usually develops when pimax is less than 45 cm h 2 o. diurnal hypercapnia is likely when respiratory muscle strength falls to 35 cm h 2 o (ragette et al. 2002) . the sniff is a natural maneuver which many children find much easier to perform than pimax. inspiratory muscle strength can easily be assessed by snip in children with nmd (stefanutti et al. 2000) . a recent report comparing snip and pimax in 241 patients with nmd found that the values of pimax were at least the same or even greater than the snip, particularly in patients with severe ventilatory restriction. this can be explained by the fact that patients with severe neuromuscular disorders may not be able to perform a rapid sniff maneuver owing to significant muscle atrophy (hart et al. 2003) . the reduction of pemax is the first sign of respiratory muscle dysfunction in dmd children (hahn et al. 1997; mcdonald et al. 1995) . expiratory muscles that contribute to pemax are primarily the abdominal muscles, and their strength normally exceeds that of inspiratory muscles (american thoracic society, european respiratory society 2002). therefore pemax < pimax indicates prevailing expiratory muscle weakness, a characteristic for children with type 2 and 3 sma . recurrent chest infections may therefore occur early in sma children due to a predominance of expiratory muscle weakness with insufficient cough and retention of airway secretions. in patients with muscular dystrophies, pemax >45 cm h 2 o has been found to be necessary for an effective cough (mellies and dohna-schwake 2005; mellies et al. 2001; szeinberg et al. 1988) . assessment tools to measure the different components of cough include also inspiratory vc (ivc) and the peak expiratory flow or peak cough flow (pcf). expiratory muscle weakness is often associated with a decrease in pcf and erv. the effect of coughing can be visualized on the maximum expiratory f/v curve in healthy subjects as a transient f exceeding the maximum achieved during forced expiration. the absence of such supramaximal f transients during coughing presumably results in impaired clearance of airway secretions and is associated with more severe expiratory muscle weakness (american thoracic society, european respiratory society 2002; polkey et al. 1998) . patients who could not generate peak f transients had significantly reduced pef, fvc, and pemax values ≤45 cm h 2 o (szeinberg et al. 1988) . impaired coughing leads to mucus retention, atelectasis, and recurrent pneumonia. in adults with nmd, pcf below 160-200 l/min is associated with insufficient clearance of airway secretions (bach and saporito 1996) . pcf (below 160 l/) and ivc <1.1 l seem also to be able to identify children with nmd at high risk for severe chest infections (dohna-schwake et al. 2006) . baseline pcf measurements above 160 l/ min, however, do not guarantee adequate airway clearance, because respiratory muscle function can deteriorate during respiratory infections (labanowski et al. 1996) . for this reason a peak cough expiratory flow rate of 270 l/min has been used to identify patients who would benefit from assisted cough techniques (finder et al. 2004; laroche et al. 1988) . in dmd, the likelihood of having a pcf value <270 l/min has been shown to rise significantly when fvc is < 2.1 l (gauld and boynton 2005) . the illustrations presented in this paper were captured with the jaeger program 5.20.0.52 (viasys healthcare, höchberg, germany). • a restrictive ventilatory defect of pulmonary origin is usually associated with a decrease in lung c (c l ) that can be due to an increase in the quantity of interstitial tissue in the lung, like in interstitial lung disease (ild), pulmonary fibrosis, infiltration, or edema. acute lung injury (ali) and acute respiratory distress syndrome (ards) result from pulmonary edema and inflammation. • restrictive defect that arises in the chest wall as a consequence of severe obesity or disease process affecting the ribs or the vertebral column, such as kyphoscoliosis, is associated with reduction in c cw . • in restrictive diseases, expansion of the lung is restricted because of alterations in lung parenchyma or as a consequence of extraparenchymal diseases affecting pleura, chest wall, or neuromuscular apparatus. a restrictive ventilatory defect is observed in these diseases with reduction in total lung capacity (tlc) and normal forced expiratory volume in 1 s (fev 1 )/ vital capacity (vc) ratio. • obstructive diseases of the lung are extremely common. airflow limitation is a functional consequence of asthma and chronic obstructive pulmonary disease (chronic bronchitis, emphysema), cystic fibrosis (cf), bronchiectasis, and bronchiolitis. airflow limitation is also observed in bronchiolitis obliterans (bo). an obstructive defect is defined by a reduced fev 1 / vc ratio. severity of lung function impairment is based on fev 1 % of predicted (fev 1 < 50 % corresponding to severe impairment). airflow obstruction (afo) is often reversible in asthma with improvement in fev 1 and/or fvc ≥12 % of baseline ( fig. 4.15) . afo in small airways is suspected from a concave shape on the fig. 4.15 in cystic fibrosis, as the airway disease becomes more advanced and/or more central airways become involved, fev 1 will be reduced out of proportion to the reduction in vital capacity (vc) and an obstructive ventilatory defect be observed with a reduced fev 1 /vc ratio. a fev 1 < 50 % predicted corresponds to severe impairement of lung function expiratory f/v curve. afo in small airways is associated with lung hyperinflation. in children with asthma an isolated increase in lung volume may be the sole functional abnormality. hyperinflation is one of the earliest features of cf. • decrease in vc is closely linked to weakness of respiratory muscles and disease progression. vc reflects the strength of both inspiratory and expiratory muscles, but it can be reduced by other factors than muscle weakness (as decreased c l and c cw ). a fall of 30 % or more in the supine compared with the erect posture is associated with severe diaphragmatic weakness. vc below 40 % predicts nocturnal alveolar hypoventilation, and vc <25 % or <1 l, in duchenne patients, is strongly associated with respiratory failure. in mild respiratory muscle weakness, vc is less sensitive than maximum respiratory pressures (pimax, pemax, sniff nasal inspiratory pressure (snip)). analysis of static pulmonary mechanics helps to identify functional defects in survivors of acute respiratory distress syndrome the outlook for survivors of ards statement on the care of the child with chronic lung disease of infancy and childhood ats/ers statement on respiratory muscle testing lung function and respiratory health in adolescents of very low birth weight nasal reflexes the mechanics of breathing in children with acute severe croup quality control for spirometry in preschool children with and without lung disease multiple-breath washout as a marker of lung disease in preschool children with cystic fibrosis prevention of pectus excavatum for children with spinal muscular atrophy type 1 criteria for extubation and tracheostomy tube removal for patients with ventilatory failure. a different approach to weaning modulation of upper airway muscle activities by bronchopulmonary afferents lungfunction tests in neonates and infants with chronic lung disease: tidal breathing and respiratory control growth of lung parenchyma in infants and toddlers with chronic lung disease of infancy chronic lung disease after premature birth pulmonary function until two years of life in infants with bronchopulmonary dysplasia spinal muscular atrophy. a 4-year prospective, multicenter, longitudinal study (168 cases) respiratory functions of the larynx interrupter resistance elucidated by alveolar pressure measurement in open-chest normal dogs respiratory muscle strength and control of ventilation in patients with neuromuscular disease decline in respiratory function and experience with long-term assisted ventilation in advanced duchenne's muscular dystrophy lung function in infants with cystic fibrosis respiratory function in survivors of the united kingdom extracorporeal membrane oxygenation trial the lung in cystic fibrosis. a quantitative study including prevalence of pathologic findings among different age groups pulmonary pathophysiology in cystic fibrosis long-term assessment of pulmonary function tests in pediatric survivors of acute respiratory distress syndrome pulmonary function in newborn infants with transitory tachypnea and pneumothorax definitions, mechanisms, relevant outcomes, and clinical trial coordination pulmonary function tests in preschool children with cystic fibrosis pulmonary function tests in preschool children with asthma statement: pulmonary function testing in preschool children pulmonary outcomes in bronchopulmonary dysplasia postnatal changes in pulmonary mechanics and energetics of infants with respiratory distress syndrome following surfactant treatment the relationship between asthma and obesity in children: is it real or a case of over diagnosis? maximal static respiratory pressures in generalized neuromuscular disease relief of central airways obstruction following spinal release in a patient with idiopathic scoliosis evidence of airway obstruction in children with idiopathic scoliosis factors influencing glottic dimensions during forced expiration effect of expiratory loading on glottic dimensions in humans bronchodilators in cystic fibrosis usefulness of monitoring lung function in asthma respiratory muscle and pulmonary function in polymyositis and other proximal myopathies interrupter resistance and wheezing phenotypes at 4 years of age respiratory mechanics in children phenotype specific treatment of asthma in childhood absence of ventilatory responses to alternating breaths of mild hypoxia and air in infants who have had bronchopulmonary dysplasia: implications for the risk of sudden infant death prospective study of body mass index, weight change, and risk of adult-onset asthma in women in: chernick v (ed) kendig's disorders of the respiratory tract in children. w.b. saunders company, philadelphia canny gj, szeinberg a, koreska j, levison h (1989) hypercapnia in relation to pulmonary function in duchenne muscular dystrophy the effects of adiposity and weight change on forced expiratory volume decline in a longitudinal study of adults profiles of neuromuscular diseases. spinal muscular atrophy early respiratory system mechanics and the prediction of chronic lung disease in ventilated preterm neonates requiring surfactant treatment evaluation of a tidal expiratory flow index in healthy and diseased infants task force on chronic interstitial lung disease in immunocompetent children transition from dynamically maintained to relaxed end-expiratory volume in human infants changes in the glottic aperture during bronchial asthma studies of respiratory physiology in the newborn infant. iii. measurements of mechanics of respiration studies of respiratory physiology in children. ii. lung volumes and mechanics of respiration in 64 patients with cystic fibrosis of the pancreas occult pulmonary abnormalities in asymptomatic asthmatic children ventilation distribution and density dependence of expiratory flow in asthmatic children five-to seven-year course of pulmonary function in cystic fibrosis longitudinal analysis of pulmonary function decline in patients with cystic fibrosis lung function: physiology, measurement and application in medicine werdnig-hoffmann disease. the effects of intrauterine onset on lung growth role of positive end-expiratory pressure changes on functional residual capacity in surfactant treated preterm infants a simplified approach to the measurement of specific airway resistance effect of scoliosis on growth of alveoli and pulmonary arteries and on right ventricle direct measurement of static chest wall compliance in animal and human neonates density dependence of forced expiratory flows in healthy infants and toddlers pulmonary functions in congenital scoliosis clinical implications of chemoreceptor reflexes role of carotid-body chemoreceptors and their reflex interactions in bradycardia and cardiac arrest analysis of lung volume restriction in patients with respiratory muscle weakness obesity and the pulmonologist changing relationships between stature and lung volumes during puberty pathophysiology of sleep apnea trapped gas and airflow limitation in children with cystic fibrosis and asthma appropriate positive end expiratory pressure level in surfactant-treated preterm infants infant lung function after inhaled nitric oxide therapy for persistent pulmonary hypertension of the newborn specific airway resistance from the perinatal period into adulthood. alterations in childhood pulmonary disease predictors of severe chest infections in pediatric neuromuscular disorders respiratory system impedance from 4 to 40 hz in paralyzed intubated infants with respiratory disease the use of a helium-oxygen mixture during maximum expiratory flow to demonstrate obstruction in small airways in smokers respiratory function at age 8-9 years in extremely low birthweight/very preterm children born in victoria in 1991-1992 bronchopulmonary dysplasia in very low birth weight subjects and lung function in late adolescence the problem of lung growth effects of early dexamethasone therapy on pulmonary mechanics and chronic lung disease in very low birth weight infants: a randomized, controlled trial editorial (1992) the nose and the respiratory system developmental changes in sequential activation of laryngeal abductor muscle and diaphragm in infants hypercapnia increases expiratory braking in preterm infants pulmonary function and exercise gas exchange in survivors of adult respiratory distress syndrome upper airway obstruction following adult respiratory distress syndrome. an analysis of 30 survivors influence of human vocal cord movements on airflow and resistance during eupnea comparison of human vocal cord movements during isocapnic hypoxia and hypercapnia respiratory studies in children. i. lung volumes in healthy children, 6-14 years of age lung volume restriction in patients with chronic respiratory muscle weakness: the role of microatelectasis neck and abdominal muscle activity in patients with severe thoracic scoliosis detection of obliterative bronchiolitis after lung transplantation by indexes of ventilation distribution bronchiolitis obliterans syndrome 2001: an update of the diagnostic criteria chronic interstitial lung disease in children pediatric interstitial lung disease revisited long-term sequelae in children surviving adult respiratory distress syndrome cardiopulmonary function in premature infants with bronchopulmonary dysplasia-a 2-year follow up lung function and respiratory symptoms at 11 years in children born extremely preterm: the epicure study flow limitation in infants with bronchopulmonary dysplasia and respiratory function at school age respiratory care of the patient with duchenne muscular dystrophy: ats consensus statement the curse of adam: effort closure of the larynx pediatric acute lung injury: prospective evaluation of risk factors associated with mortality randomised crossover trial of salbutamol aerosol delivered by metered dose inhaler, jet nebuliser, and ultrasonic nebuliser in chronic lung disease helium flow-volume curves in the detection of early small airway disease supine fall in lung volumes in the assessment of diaphragmatic weakness in neuromuscular disorders pulmonary function test study and after spinal fusion in young idiopathic scoliosis lung function testing in infants with cystic fibrosis: lessons from the past and future directions lung function testing in neonates and infants with chronic lung disease: lung and chest wall mechanics acute respiratory distress syndrome caused by pulmonary and extrapulmonary disease. different syndromes? relationship between peak cough flow and spirometry in duchenne muscular dystrophy lung function in interstitial lung diseases in children chestwall compliance in full-term and premature infants pulmonary mechanics in normal infants and young children during first 5 years of life serial determination of pulmonary function in infants with chronic lung disease impairment after adult respiratory distress syndrome. an evaluation based on pulmonary mechanics in patients with respiratory muscle weakness the nose and the lower airways timing of recovery of lung function after severe hypoxemic respiratory failure in children pulmonary manifestations of neuromuscular disease with special reference to duchenne muscular dystrophy and spinal muscular atrophy variability of maximum expiratory flow-volume curves total respiratory compliance and functional residual capacity in young children abnormalities of lung mechanics in young asthmatic children effect of budesonide on pulmonary hyperinflation in young asthmatic children changes in functional residual capacity in response to bronchodilator therapy among young asthmatic children the relation of body mass index to asthma, chronic bronchitis, and emphysema clinical implications of maximal respiratory pressure determinations for individuals with duchenne muscular dystrophy diffusing capacity of the lung in school-aged children born very preterm, with and without bronchopulmonary dysplasia long-term prospective study in children after respiratory syncytial virus infection airway and respiratory tissue mechanics in normal infants pulmonary outcome in adolescents of extreme preterm birth: a regional cohort study better care of immature infants; has it influenced long-term pulmonary outcome? acute lung injury: pathophysiology, assessment and current therapy total lung capacity by n2 washout from high and low lung volumes in ventilated infants and children long-term pulmonary sequelae in children who were treated with extracorporeal membrane oxygenation for neonatal respiratory failure sex differences in the relation between body mass index and asthma and atopy in a birth cohort input impedance and peripheral inhomogeneity of dog lungs the upper respiratory tract in perinatal life the significance of grunting in hyaline membrane disease changes in pulmonary mechanics with increasing disease severity in children and young adults with cystic fibrosis limitations of sniff nasal pressure in patients with severe neuromuscular weakness maternal smoking during and after pregnancy and lung function in early adulthood: a prospective study bronchopulmonary dysplasia. in: chernick v (ed) kendig's disorders of the respiratory tract in children assessment of airway function using partial expiratory flow-volume curves: how reliable are measurements of maximal expiratory flow at frc during early infancy? new aspects of airway mechanics in pre-term infants one-year outcomes in survivors of the acute respiratory distress syndrome sequelae of the adult respiratory distress syndrome narrowing of glottis opening in humans associated with experimentally induced bronchoconstriction anatomy and physics of respiration postnatal growth and function of the pre-acinar airways lung function at term reflects severity of bronchopulmonary dysplasia effect of antenatal corticosteroid treatment on lung function in full-term newborn infants prospective longitudinal evaluation of lung function during the first year of life after extracorporeal membrane oxygenation age as a factor in the distribution of lower-airway conductance and in the pathologic anatomy of obstructive lung disease sex-specific prediction equations for vmax(frc) in infancy: a multicenter collaborative study development of airway function in infancy after preterm delivery growth of the bronchial tree in man airway function in infants treated with inhaled nitric oxide for persistent pulmonary hypertension daytime predictors of sleep hypoventilation in duchenne muscular dystrophy world federation of pediatric intensive critical care societies (wficss) non-invasive positive pressure ventilation in the preterm neonate: reducing endotrauma and the incidence of bronchopulmonary dysplasia laryngeal and diaphragmatic muscle activities and airflow patterns after birth in premature lambs respiratory muscle activities after birth in asphyxiated preterm lambs laryngeal muscle activities with cerebral hypoxia-ischemia in newborn lambs airway closure during mixed apneas in preterm infants: is respiratory effort necessary? pulmonary function in duchenne muscular dystrophy related to stage of disease thyroarytenoid muscle activity during hypoxia, hypercapnia, and voluntary hyperventilation in humans respiratory capacity course in patients with infantile spinal muscular atrophy physiologic dysfunction of the asthmatic lung factors in childhood as predictors of asthma in adult life a comparison of the clinical characteristics of children and adults with severe asthma bronchopulmonary dysplasia influence of nonlinearities on estimates of respiratory mechanics using multilinear regression analysis pulmonary function abnormalities in symptom-free children after bronchiolitis effect of spinal surgery on lung function in duchenne muscular dystrophy prediction of mortality in patients with cystic fibrosis the design of future pediatric mechanical ventilation trials for acute lung injury pulmonary function after recovery from the adult respiratory distress syndrome pulmonary complications of obesity dynamic maintenance of end-expiratory lung volume in full-term infants posterior cricoarytenoid and diaphragm activities during tidal breathing in neonates effect of preterm birth on later fev1: a systematic review and meta-analysis scoliosis and the respiratory system contribution of airway hyperresponsiveness to lower airway obstruction after extracorporeal membrane oxygenation for meconium aspiration syndrome lung function from infancy to the preschool years after clinical diagnosis of cystic fibrosis progression of pulmonary hyperinflation and trapped gas associated with genetic and environmental factors in children with cystic fibrosis respiratory muscle function in amyotrophic lateral sclerosis thyroarytenoid muscle activity during wakefulness and sleep in normal adults posterior cricoarytenoid muscle activity during wakefulness and sleep in normal adults laryngeal response to passively induced hypocapnia during nrem sleep in normal adult humans effect of hypercapnia on laryngeal airway resistance in normal adult humans preservation of the phrenic motoneurons in werdnig-hoffmann disease sleep and neuromuscular disease: frequency of sleep-disordered breathing in a neuromuscular disease clinic population disorders of the respiratory muscles the spectrum of cystic fibrosis. a study of pulmonary mechanics in 46 patients the variable effect of a bronchodilating agent on pulmonary function in cystic fibrosis small airways disease" in children: no test is best changes in respiratory mechanics with age clinical significance of severe isolated diaphragm weakness oral feeding in the preterm infant effects of body fat on ventilatory function in children and adolescents: cross-sectional findings from a random population sample of school children spirometry in the supine position improves the detection of diaphragmatic weakness in patients with amyotrophic lateral sclerosis a comparative analysis of contractile characteristics of the diaphragm and of respiratory system mechanics the effects of obesity on pulmonary function asthma and obesity: common early-life influences in the inception of disease importance of inspiratory muscle tone in maintenance of frc in the newborn specific airway resistance in 3-year-old children: a prospective cohort study early changes in respiratory compliance and resistance during the development of bronchopulmonary dysplasia in the era of surfactant therapy lung function tests in neonates and infants with chronic lung disease: forced expiratory maneuvers nature and severity of lung function abnormalities in extremely pre-term children at 11 years of age patterns of laryngeal electromyography and the activity of the respiratory system during spontaneous laughter adult respiratory distress syndrome in a pediatric intensive care unit: predisposing conditions, clinical course, and outcome relationships between specific airway resistance and forced expiratory flows in asthmatic children clinical correlations and pulmonary function at 8 years of age after severe neonatal respiratory failure lung elastic recoil in cystic fibrosis relationship of lung recoil to lung volume and maximum expiratory flow in normal children muscular dystrophy campaign sponsored workshop: recommendation for respiratory care of children with spinal muscular atrophy type ii and iii pediatric pulmonary function testing: plethysmography and gas dilution techniques pathophysiology of osas in children spirometry in 3-to 6-year-old children with cystic fibrosis fibroproliferation occurs early in the acute respiratory distress syndrome and impacts on outcome pulmonary function testing in idiopathic interstitial pneumonias types of asthma and wheezing asthma and wheezing in the first six years of life. the group health medical associates profiles of neuromuscular diseases. duchenne muscular dystrophy randomized, double-blinded trial of lowdose dexamethasone: ii. functional residual capacity and pulmonary outcome in very low birth weight infants at risk for bronchopulmonary dysplasia decreased respiratory compliance in infants less than or equal to 32 weeks' gestation, delivered more than 7 days after antenatal steroid therapy respiratory compliance in preterm infants after a single rescue course of antenatal steroids: a randomized controlled trial recovery of function in survivors of the acute respiratory distress syndrome airway resistance and atopy in preschool children with wheeze and cough functional significance of the area of apposition of diaphragm to rib cage pediatric pulmonary function testing: neuromuscular disorders sleep-disordered breathing and respiratory failure in acid maltase deficiency daytime predictors of sleep disordered breathing in children and adolescents with neuromuscular disorders growth of airways and air spaces in teenagers is related to sex but not to symptoms pulmonary function in infants with neonatal chronic lung disease with or without hyaline membrane disease at birth early versus late dexamethasone treatment in preterm infants at risk for chronic lung disease: a randomized pilot study changes in lung volume and ventilation during surfactant treatment in ventilated preterm infants changes in lung volume and ventilation during lung recruitment in highfrequency ventilated preterm infants with respiratory distress syndrome the effect of airway pressure and oscillation amplitude on ventilation in preterm infants oral breathing in response to nasal trauma in term infants pulmonary function and scoliosis in duchenne dystrophy standardisation of spirometry relationship between chest wall and pulmonary compliance and age effects of meconium on airway reactivity to histamine and acetylcholine in vitro mechanics of breathing measurements of respiratory mechanics in the newborn: a simple approach early onset of airway reactivity in premature infants with bronchopulmonary dysplasia the assessment of lung function in children with scoliosis a dutch guideline for the treatment of scoliosis in neuromuscular disorders muscular dystrophy campaign funded workshop on management of scoliosis in duchenne muscular dystrophy 24 the pediatric airway: an interdisciplinary approach longitudinal evaluation of airway function 21 years after preterm birth alveolarization continues during childhood and adolescence: new evidence from helium-3 magnetic resonance lung and thorax development during adolescence: relationship with pubertal status spirometry in 3-5-year-old children with asthma assessment of pulmonary function in the early phase of ards in pediatric patients global strategy for asthma management and prevention. national institute for health expert panel report 3: guidelines for the diagnosis and management of asthma the effect of inhaled budesonide on symptoms, lung function, and cold air and methacholine responsiveness in 2-to 5-year-old asthmatic children discriminative capacity of bronchodilator response measured with three different lung function techniques in asthmatic and healthy children aged 2 to 5 years changes in rib cage geometry during childhood childhood asthma and lung function in mid-adult life bronchiolar airflow impairment after lung transplantation: an early and common manifestation variability of tidal breathing flow-volume loops in healthy and sick newborns the pathophysiology of respiratory impairment in pediatric neuromuscular diseases expiratory flow limitation and intrinsic positive end-expiratory pressure in obesity developmental changes in chest wall compliance in infancy and early childhood chest wall compliance in infants and children with neuromuscular disease long-term pulmonary functional outcome of bronchopulmonary dysplasia and premature birth adult obstructive sleep apnea: pathophysiology and diagnosis a practical approach to the interpretation of lung function testing in children physiologic definitions of obliterative bronchiolitis in heart-lung and double lung transplantation: a comparison of the forced expiratory flow between 25% and 75% of the forced vital capacity and forced expiratory volume in one second rhythms and complexity of respiration during sleep in pre-term infants do nhlbi lung function criteria apply to children? a cross-sectional evaluation of childhood asthma at national jewish medical and research center interpretative strategies for lung function tests thoracoabdominal pattern of breathing in neuromuscular disorders clinical determinants of abnormalities in pulmonary functions in survivors of the adult respiratory distress syndrome lung protective ventilation strategy for the acute respiratory distress syndrome nocturnal oxygenation and prognosis in duchenne muscular dystrophy changes in spirometry over time as a prognostic marker in patients with duchenne muscular dystrophy long term respiratory consequences of intrauterine growth restriction tidal volume, recruitment and compliance in hfov: same principles, different frequency effects of gestation and antenatal steroid on airway and tissue mechanics in newborn lambs in vitro performance characteristics of high-frequency oscillatory ventilators variability in preterm lamb lung mechanics after intra-amniotic endotoxin is associated with changes in surfactant pool size and morphometry monitoring of lung volume recruitment and derecruitment using oscillatory mechanics during high-frequency oscillatory ventilation in the preterm lamb partitioning of airway and parenchymal mechanics in unsedated newborn infants the functional development of the respiratory system from the period of gestation to adulthood expiratory muscle function in amyotrophic lateral sclerosis abnormalities of functional residual capacity in symptomatic and asymptomatic young asthmatics larynx and neonatal apneas obstructive sleep disordered breathing in children: beyond adenotonsillectomy chemoreceptor and vagal influences on thyroarytenoid muscle activity in awake lambs during hypoxia wheezing, asthma, and pulmonary dysfunction 10 years after infection with respiratory syncytial virus in infancy compilation of reference values for lung function measurements in children patterns and predictors of sleep disordered breathing in primary myopathies airway function in infants newly diagnosed with cystic fibrosis relative ability of full and partial forced expiratory maneuvers to identify diminished airway function in infants with cystic fibrosis early detection of lung disease in children with cystic fibrosis using lung function risk factors for airway remodeling in asthma manifested by a low postbronchodilator fev1/vital capacity ratio: a longitudinal population study from childhood to adulthood pediatric pulmonary function testing: cystic fibrosis the lung: its growth and remodelling in health and disease early detection of airway involvement in obliterative bronchiolitis after lung transplantation. functional and bronchoalveolar lavage cell findings respiratory function in the muscular dystrophies restricted pulmonary function in cystic fibrosis pulmonary function in bronchopulmonary dysplasia the travels of a pulmonologist through the upper airway scoliosis in spinal muscular atrophy: review of 63 cases follow-up of asthma from childhood to adulthood: influence of potential childhood risk factors on the outcome of pulmonary function and bronchial responsiveness in adulthood site of upper airway obstruction in preterm infants with problematical apnoea site of upper airway obstruction in infants following an acute lifethreatening event pulmonary function in spinal muscular atrophy fetal nicotine exposure increases airway responsiveness and alters airway wall composition in young lambs obesity is a risk for asthma and wheeze but not airway hyperresponsiveness asthma and atopy in overweight children vocal cord closure. a cause of upper airway obstruction during controlled ventilation influence of preterm onset of inspiration on tidal breathing parameters in infants with and without cld changes in the fev1-height relationship during pubertal growth respiratory muscle force and ventilatory function in adolescents obstructive lung disease in children after allogeneic bone marrow transplantation anatomy and physiology for speech, language, and hearing effect of a previous voluntary deep breath on laryngeal resistance in normal and asthmatic subjects impaired gas mixing and low lung volume in preterm infants with mild chronic lung disease the paradoxical effect of adrenergic and methylxanthine drugs in cystic fibrosis forced expiratory flow between 25% and 75% of vital capacity and fev1/forced vital capacity ratio in relation to clinical and physiological parameters in asthmatic children with normal fev1 values impact of nasal ventilation on survival in hypercapnic duchenne muscular dystrophy neonatal respiratory mechanics and development of bronchial hyperresponsiveness in preterm infants bronchiolitis obliterans following haematopoietic stem cell transplantation clinical and genetic study of chronic (types ii and iii) childhood onset spinal muscular atrophy office-based objective measures in childhood asthma is forced expiratory volume in one second the best measure of severity in childhood asthma? diagnosisrelated deterioration of lung function after extracorporeal membrane oxygenation prospective longitudinal evaluation of lung function during the first year of life after repair of congenital diaphragmatic hernia small airways obstruction syndrome phascinating physiology usefulness of sniff nasal pressure in patients with neuromuscular or skeletal disorders fibrosing alveolitis in childhood. a long-term follow-up specific airway conductance in relation to postconceptional age during infancy early life influences on the development of chronic obstructive pulmonary disease influence of ethnicity and gender on airway function in preterm infants ventilatory function in british adults after asthma or wheezing illness at ages 0-35 surfactant improves lung function and morphology in newborn rabbits with meconium aspiration parental smoking in childhood and adult obstructive lung disease: results from the european community respiratory health survey cough capacity in patients with muscular dystrophy mechanics of breathing after surgical ligation of patent ductus arteriosus in newborns with respiratory distress syndrome pulmonary function after surfactant lung lavage followed by surfactant administration in infants with severe meconium aspiration syndrome use of corticosteroids in acute lung injury and acute respiratory distress syndrome: a systematic review and meta-analysis lung function in children with duchenne's muscular dystrophy physiologic growth and development of the lung during the first year of life expiratory flow limitation in infants with bronchopulmonary dysplasia static compliance of the respiratory system in healthy infants can we breathe and swallow at the same time? some aspects of clinical relevance in the maturation of respiratory control in infants ventilation with lower tidal volumes as compared with traditional tidal volumes for acute lung injury and the acute respiratory distress syndrome the international study of asthma and allergies in childhood (isaac) steering committee. worldwide variation in prevalence of symptoms of asthma, allergic rhinoconjunctivitis, and atopic eczema postnatal human lung growth early changes of pulmonary mechanics to predict the severity of bronchopulmonary dysplasia in ventilated preterm infants pediatric pulmonary function testing: long-term sequelae of neonatal lung disease spinal muscular atrophy: classification, aetiology, and treatment of spinal deformity in children and adolescents changes in residual volume relative to vital capacity and total lung capacity after arthrodesis of the spine in patients who have adolescent idiopathic scoliosis effect of dexamethasone on tracheobronchial aspirate fluid cytology and pulmonary mechanics in preterm infants hfov in premature neonates: effects on pulmonary mechanics and epithelial lining fluid cytokines. a randomized controlled trial the role of computer games in measuring spirometry in healthy and "asthmatic" preschool children spirometry in early childhood in cystic fibrosis patients frc measurements using body plethysmography in young children flow oscillations on the flow-volume loop: clinical and physiological implications flowvolume loop changes reflecting respiratory muscle weakness in chronic neuromuscular disorders lung function and exercise capacity in young adults born prematurely normal growth and development of the trachea diagnostic role of residual volume in paediatric patients with chronic symptoms of the lower airways 117th enmc workshop: ventilatory support in congenital neuromuscular disorders -congenital myopathies, congenital muscular dystrophies, congenital myotonic dystrophy and sma (ii) consensus statement for standard of care in spinal muscular atrophy the acute respiratory distress syndrome assessment of functional residual capacity using nitrogen washout and plethysmographic techniques in infants with and without bronchopulmonary dysplasia pulmonary function in asymptomatic adolescents with idiopathic scoliosis respiratory and cardiac function in children after acute hypoxemic respiratory failure standards of pulmonary function in children relationship between respiratory syncytial virus bronchiolitis and future obstructive airway diseases obesity and asthma in 11-12 year old new zealand children in the physiology of the nose hypoxemia in attack free asthmatic children: relationship with lung volumes and lung mechanics prenatal nicotine exposure alters lung function and airway geometry through alpha7 nicotinic receptors relationship between anatomic dead space and body size in health, asthma, and cystic fibrosis laryngeal and pump muscle activities during co 2 breathing in neonates effects of posture on respiratory mechanics in obesity lung function in immature baboons with respiratory distress syndrome receiving early caffeine therapy: a pilot study lung function in children and adolescents with idiopathic interstitial pulmonary fibrosis effects of obesity on respiratory resistance incidence and outcomes of pediatric acute lung injury key: cord-026005-f2khcjdy authors: lópez, alfonso; martinson, shannon a. title: respiratory system, mediastinum, and pleurae date: 2017-02-17 journal: pathologic basis of veterinary disease doi: 10.1016/b978-0-323-35775-3.00009-6 sha: doc_id: 26005 cord_uid: f2khcjdy nan diseases of the respiratory system (respiratory apparatus) are some of the leading causes of morbidity and mortality in animals and a major source of economic losses. thus veterinarians are routinely called to diagnose, treat, and implement health management practices to reduce the impact of these diseases. in companion animals, diseases of the respiratory tract are also common and, although of little economic significance, are important to the health of the animals and thus to clinicians and pet owners. in the past few years, animal shelters have been recognized as a major risk factor for respiratory diseases in dogs and cats, a comparable situation to what is reported in human beings with nosocomial infections. to facilitate the understanding of the structure and function, it is convenient to arbitrarily divide the respiratory system into conducting, transitional, and gas exchange systems ( fig. 9-1 ). the conducting system includes nostrils, nasal cavity, paranasal sinuses, nasopharynx, larynx, trachea, and extrapulmonary and intrapulmonary bronchi, all of which are largely lined by pseudostratified, ciliated columnar cells, plus a variable proportion of secretory goblet (mucous) and serous cells (figs. 9-2 and 9-3 and efig. 9-1 ). the transitional system of the respiratory tract is composed of bronchioles, which are microscopic structures that serve as a transition zone between the conducting system (ciliated) and the gas exchange (alveolar) system (see fig. 9 -1). the disappearance of cilia in the transitional system is not abrupt; the ciliated cells in the proximal bronchiolar region become scarce and progressively attenuated, until the point where distal bronchioles no longer have ciliated cells. normal bronchioles also lack goblet cells but instead have other types of secretory cells, notably club cells (formerly clara cells) and neuroendocrine cells. club cells, also referred to as secretory bronchiolar cells, contain numerous biosynthetic organelles that play an active role in detoxification of xenobiotics (foreign substances), similar to the role of hepatocytes ( fig. 9-4) . club cells are also critical stem cells in the repair and remodeling of not only the bronchioles but also of most of the respiratory tract. in addition, club cells contribute to the innate immunity of the lung by secreting protective proteins (collectins) and pulmonary surfactant (see b) . in carnivores and monkeys, and to a much lesser extent in horses and human beings, the terminal portions of bronchioles are lined not only by cuboidal epithelium but also by segments of alveolar capillaries. these unique bronchioloalveolar structures are known as respiratory bronchioles ; also see fig. 9 -1). the gas exchange system of the respiratory tract in all mammals is formed by alveolar ducts and millions of alveoli ( fig. 9-6 ; also see fig. 9 -1). the surface of the alveoli is lined by two distinct types of epithelial cells known as type i (membranous) pneumonocytes and type ii (granular) pneumonocytes ( fig. 9-7) . all three-the conducting, transitional, and exchange systems of the respiratory system-are vulnerable to injury because of constant exposure to a myriad of microbes, particles and fibers, and toxic gases and vapors present in the air. vulnerability of the respiratory system to aerogenous (airborne) injury is primarily because of (1) the extensive area of the alveoli, which are the interface between the blood in alveolar capillaries and inspired air; (2) the large volume of air passing continuously into the lungs; and (3) the high concentration of noxious elements that can be present in the air (table 9 -1). for human beings, it has been estimated that the surface of the pulmonary alveoli is approximately 200 m 2 , roughly the area animal and cultured for microbes, yeasts, and fungi, many species of bacteria are recovered, such as mannheimia (pasteurella) haemolytica in cattle; pasteurella multocida in cats, cattle, and pigs; and bordetella bronchiseptica in dogs and pigs. the organisms that constitute the normal flora of the respiratory tract are restricted to the most proximal (rostral) region of the conducting system (nasal cavity, pharynx, and larynx). the thoracic portions of the trachea, bronchi, and lungs are considered to be essentially sterile. the types of bacteria present in the nasal flora vary considerably among animal species and in different geographic regions of the world. some present in the nasal flora are pathogens that can cause important respiratory infections under some circumstances. for instance, mannheimia (pasteurella) haemolytica is part of the bovine nasal flora, yet this bacterium causes a devastating disease in cattle-pneumonic mannheimiosis (shipping fever). experimental studies have established that microorganisms from the nasal flora are continuously carried into the lungs via tracheal air. despite this constant bacterial bombardment from the nasal flora and from contaminated air, normal lungs remain sterile because of their remarkably effective defense mechanisms. the conducting portion of the respiratory system is lined by pseudostratified columnar ciliated epithelium (most of the nasal cavity, paranasal sinuses, part of the larynx, and all of the trachea and bronchi), olfactory epithelium (part of the nasal cavity, particularly ethmoidal conchae), and squamous epithelium (nasal vestibulum and parts of the larynx). the pattern of injury, inflammation, and host response (wound healing) are characteristic for each of these three types of epithelium independent of its anatomic location. pseudostratified ciliated epithelium, which lines most of the nasal cavity and nasopharynx, part of the larynx, and all of the trachea and bronchi, is exquisitely sensitive to injury. when these cells are irreversibly injured, whether caused by viral infection, trauma, or inhalation of toxic gases, the ciliated cells swell, typically lose their attachment to underlying basement membrane, and rapidly exfoliate ( fig. 9-8) . a transient and mild exudate of fluid, plasma proteins, and neutrophils covers the ulcer. in the absence of of a tennis court. the alveolar surface of the equine lung is estimated to be approximately 2000 m 2 . it has also been estimated that the volume of air reaching the human lung every day is approximately 9000 l. lungs are also susceptible to blood-borne (hematogenous) microbes, toxins, and emboli. this fact is not surprising because the entire cardiac output of the right ventricle goes into the lungs, and approximately 9% of the total blood volume is within the pulmonary vasculature. the pulmonary capillary bed is the largest in the body, with a surface area of 70 m 2 in the adult human; this area is equivalent to a length of 2400 km of capillaries, with 1 ml of blood occupying up to 16 km of capillary bed. the respiratory system has its own normal flora (microbiota), as does any other body system in contact with the external environment. if a sterile swab is passed deep into the nasal cavity of any healthy (rhinoviruses), infectious bovine rhinotracheitis (bovine herpesvirus 1), feline rhinotracheitis (felid herpesvirus 1), and viruses of the canine infectious respiratory disease (cird) group such as canine adenovirus 2 (cav-2) and canine parainfluenza virus (cpiv) . if damage to the mucociliary blanket becomes chronic, goblet cell hyperplasia takes place, leading to excessive mucus production (hypersecretion) and reduced mucociliary clearance, and when there is loss of basement membrane, repair is by fibrosis and granulation tissue (scarring). in the most severe cases, prolonged injury causes squamous metaplasia, which together with scarring causes airway obstruction and an impediment to mucociliary clearance. in laboratory rodents, hyperplastic and metaplastic changes, such as those seen in nasal polyps and squamous metaplasia, are considered a prelude to neoplasia. the second type of epithelium lining the conducting system is the sensory olfactory epithelium, present in parts of the nasal mucosa, notably in the ethmoidal conchae. the patterns of degeneration, exfoliation, and inflammation in the olfactory epithelium are similar to those of the ciliated epithelium, except that olfactory complications or secondary bacterial infections, a specific type of progenitor cells known as basal cells or nonciliated secretory cells (preciliated cells) , which are normally present in the mucosa, migrate to cover the denuded basement membrane and undergoes mitosis, eventually differentiating into new ciliated epithelial cells (see fig. 9 -8). cellular migration, proliferation, and attachment are regulated by locally released interleukins (il-1β, il-2, il-4, and il-13), growth factors, integrins and extracellular matrix (ecm) proteins such as collagen, and fibronectin. the capacity of ciliated epithelium to repair itself is remarkably effective. for example, epithelial healing in an uncomplicated ulcer of the tracheal mucosa can be completed in only 10 days. this sequence of cell degeneration, exfoliation, ulceration, mitosis, and repair is typically present in many viral infections in which viruses replicate in nasal, tracheal, and bronchial epithelium, causing extensive mucosal ulceration. examples of transient infections of this type include human colds epithelium. neurons in the olfactory mucosa have the unique ability to regenerate, a fact that is being explored as a potential source of new neurons in the treatment of spinal cord injury. squamous epithelium, located in the vestibular region of the nose (mucocutaneous junction), is the third type of epithelium present in the nasal passages. compared with ciliated and olfactory epithelia, nasal squamous epithelium is quite resistant to all forms of injury. the pharyngeal mucosa, composed of squamous epithelium, has similar patterns of necrosis and inflammation as the oral mucosa (see chapter 7). the patterns of necrosis, inflammation, and repair in intrapulmonary bronchi are similar to those previously described for the nasal and tracheal epithelium. in brief, injury to ciliated bronchial epithelium may result in degeneration, detachment, and exfoliation of necrotic cells. under normal circumstances, cellular exfoliation is promptly followed by inflammation, mitosis, cell proliferation, cell differentiation, and finally by repair ( fig. 9-9 and see . depending on the type of exudate, bronchitis can be fibrinous, catarrhal, purulent, fibrinonecrotic (diphtheritic), and sometimes granulomatous. when epithelial injury becomes chronic, production of mucus is increased via goblet cell hyperplasia (chronic catarrhal inflammation). this form of chronic bronchitis is well illustrated in habitual smokers who continually need to cough out excessive mucus secretions (sputum). unfortunately, in some cases, excessive mucus cannot be effectively cleared from airways, which of the bronchial wall or cylindrical when destruction involves a large segment of a bronchus. grossly, bronchiectasis is manifested by prominent lumps in the lungs (bosselated appearance or having rounded eminences) resulting from distention of bronchi with exudate, which results in a concurrent obstructive atelectasis of surrounding parenchyma ( fig. 9-11 ). the cut surfaces of dilated bronchi are filled with purulent exudates; for this reason, bronchiectasis is often mistaken for pulmonary abscesses. careful inspection, usually requiring microscopic examination, confirms that exudate is contained and surrounded by remnants of a bronchial wall lined by squamous epithelium and not by a pyogenic membrane (connective tissue) as it is in the case of a pulmonary abscess. the squamous metaplasia further interferes with the normal function of the mucociliary escalator. the epithelial lining of the bronchiolar region (transitional zone) is exquisitely susceptible to injury, particularly to that caused by some respiratory viruses (bovine parainfluenza virus 3, bovine respiratory syncytial virus, adenovirus, or canine distemper virus), oxidant gases (nitrogen dioxide [no 2 ], sulfur dioxide [so 2 ], or ozone [o 3 ]), and toxic substances (3-methylindole or paraquat). the precise explanation as to why bronchiolar epithelium is so prone to injury is still not clear, but it is presumably due in part to (1) its high vulnerability to oxidants and free radicals; (2) the presence of leads to chronic obstructive bronchitis and emphysema (see . chronic bronchial irritation causes squamous metaplasia of highly functional but vulnerable ciliated epithelium to nonfunctional, but more resistant, squamous epithelium. squamous metaplasia has a calamitous effect on pulmonary clearance because it causes a structural loss and functional breakdown of portions of the mucociliary escalator. hyperplasia of bronchial glands occurs frequently in chronic bronchitis, which translates to an increase of the reid index (bronchial-gland to bronchial-wall ratio) (efig. 9 -2). this index is less than 30% in the healthy human lung and in the lungs of most domestic species, except for cats, which generally have an index higher than 40%. the term airway remodeling encompasses all the structural changes that accompany chronic bronchitis such as hypertrophy and hyperplasia of smooth muscle, submucosal glands, and goblet cells; fibrosis; and increased bronchial vascularity. bronchiectasis is one of the most devastating sequelae to chronic remodeling of the bronchi. it consists of a pathologic and permanent dilation of a bronchus with rupture of the bronchial wall as a result of obstruction or chronic inflammation. destruction of walls occurs in part when proteolytic enzymes and oxygen radicals released from phagocytic cells during chronic inflammation degrade and weaken the smooth muscle and cartilage (chondromalacia) that help to maintain normal bronchial diameter ( fig. 9-10 ). bronchiectasis may be saccular when destruction affects only a small localized portion . this same type of lesion is seen in viral or mechanical injury to the mucosa of the conducting system. two days after exposure, the basement membrane is lined by rapidly dividing preciliated cells, some of which exhibit mitotic activity (inset). ten days after injury, the nasal epithelium is completely repaired. h&e stain. b, schematic representation of the events of injury and repair in the respiratory mucosa of the conducting system. blue cell, ciliated mucosal epithelial cell; pink cell, goblet cell; red cell, neutrophil. (a from lópez a, prior m, yong s, et al: am j vet res 49:1107 -1111 , 1988 into well-organized, microscopic polyps inside the bronchiolar lumen. the external surface of the exudate eventually becomes covered by ciliated cells. this lesion is referred to as bronchiolitis obliterans, and the polyps may become so large as to cause airflow impairment ( fig. 9 -12 and see fig. 9 -9). in mild but persistent bronchiolar injury, goblet cells normally absent from bronchioles proliferate from basal cells, resulting in goblet cell metaplasia and causing a profound alteration in the physicochemical properties of bronchiolar secretions ( fig. 9-13 ). the normally serous bronchiolar fluid released by club (clara) cells becomes a tenacious material when mucus produced by goblet cells is added. as a result of increased viscoelasticity of the mucus, bronchiolar secretions cannot be removed effectively by ciliary action, leading to plugging and obstruction of distal airways. under such conditions, often grouped as chronic obstructive pulmonary disease, coughing is required to clear mucus from obstructed bronchioles. pulmonary emphysema and atelectasis are further sequelae to bronchiolar metaplasia and mucous hypersecretion blocking or partially blocking the lumens of these bronchioles. these two inflation abnormalities are characteristically present in chronic obstructive pulmonary disease (copd), which is called "recurrent airway obstruction (rao or "heaves") in horses (see recurrent airway obstruction, under disorders of horses). peribronchiolar club (clara) cells rich in mixed function oxidases, which locally generate toxic metabolites (see fig. 9 -4); and (3) the tendency for pulmonary alveolar macrophages and leukocytes to accumulate in this region of the lungs. depending on the types of injury and inflammatory response, bronchiolitis is classified as necrotizing, suppurative, catarrhal (mucous metaplasia), or granulomatous. once injury to bronchiolar ciliated cells becomes irreversible, the cells degenerate and exfoliate into the bronchiolar lumen, leaving a denuded basement membrane. repair in the bronchiolar region is similar to, but less effective than, that in the tracheal or nasal mucosa. under normal circumstances, recruited phagocytic cells remove exudate and cell debris from the lumina of affected bronchioles, thus preparing the basement membrane to be repopulated with new, undifferentiated cells originating from a rapidly dividing pool of club (clara) cells. after several days, these proliferating cells fully differentiate into normal bronchiolar cells. in severe acute injury, such as that caused by aspiration pneumonia or by highly pathogenic microorganisms, exudate attaches and cannot be removed from the basement membrane of bronchioles. the exudate becomes infiltrated by fibroblasts, which form small nodular masses of fibrovascular tissue that develop postviral bronchiolitis is associated with increased expression of tlrs and unusual susceptibility to inhaled endotoxin. hyperreactive animals typically have an increased number of mast cells, eosinophils, and t lymphocytes in the airway mucosa. clinically, airway hyperresponsiveness is characterized by an exaggerated bronchoconstriction after natural exposure to mild stimuli, such as cold air, or after animals are experimentally exposed to aerosols of histamine or methacholine. because of their extremely delicate structure, alveoli are quite vulnerable to injury once the local defense mechanisms have been overwhelmed. the alveolar wall is a thin membrane formed by a core of interstitium supporting an extensive network of alveolar capillaries. fibroblasts (septal cells), myofibroblasts, collagen, elastic fibers, and few interstitial macrophages and mast cells constitute the alveolar interstitium. the wall of the alveolar capillaries facing the airspace is remarkably thin and has three layers composed of vascular endothelium, basal lamina, and alveolar epithelium. these three layers of the alveolar capillaries constitute what is customarily referred to as the blood-air barrier (see fig. 9 -7). the epithelial side of the alveolus is primarily lined by rather thin type i proliferation of lymphocytes (balt hyperplasia) is also a common microscopic lesion seen in chronic bronchiolitis. airway hyperresponsiveness, or hyperreactive airway disease, is another sequela of bronchiolar injury arising from gene-environment interactions. it develops in human beings and animals (experimentally) after a transient and often innocuous viral infection of the lower respiratory tract or from exposure to certain allergens. experimental work has shown that airway hyperreactivity in club (clara) cell edema. alveolar repair is possible as long as the basement membrane remains intact and lesions are not complicated by further injury or infection. within 3 days, cuboidal type ii (granular) pneumonocytes, which are the precursor cells and more resistant to injury, undergo mitosis and provide a large pool of new undifferentiated cells . these new cells repave the denuded alveolar basement membrane and finally differentiate into type i pneumonocytes. when alveolar injury is diffuse, proliferation pneumonocytes, which are arranged as a very delicate continuous membrane extending along the alveolar surface (see fig. 9 -7). type i pneumonocytes are particularly susceptible to noxious agents that reach the alveolar region either aerogenously or hematogenously. injury to type i pneumonocytes rapidly causes swelling and vacuolation of these cells . when cellular damage has become irreversible, type i cells detach, resulting in denudation of the basement membrane, increased alveolar permeability, and alveolar figure 9-15 hyperplasia of type ii pneumonocytes. a, acute alveolar injury, crude oil aspiration, cow. note proliferation of cuboidal epithelial cells (type ii pneumonocytes) (arrows) along the luminal surface of the alveolar wall. during alveolar repair, type ii pneumonocytes are the precursor cell for necrotic and lost type i pneumonocytes. h&e stain. b, chronic alveolar injury, interstitial pneumonia, horse. note entire alveolar membrane lined with cuboidal type ii pneumonocytes (arrowheads). the alveolar interstitium is expanded with inflammatory cells, and the alveolar lumens contain cell debris mixed with leukocytes. h&e stain. ( (2). necrosis of these cells leads to transient alveolar edema (area that is pink) (3), which is followed by hyperplasia of type ii pneumonocytes (4), stem cells that differentiate (5) into type i pneumonocytes as part of alveolar repair and healing (6). (courtesy dr. a. lópez, atlantic veterinary college.) more information on postmortem examination of the lung can be found at www.expertconsult.com. information on this topic is available at www.expertconsult.com. information on this topic is available at www.expertconsult.com. microbes, toxins, and pneumotoxicants can gain access into the respiratory system by the following routes (table 9 -2; also see table 9 -1): aerogenous, hematogenous, direct extension, and by local production of free radicals and toxic metabolites. pathogens, such as bacteria, mycoplasmas, and viruses, along with toxic gases and foreign particles, including food, can gain access to the respiratory system via inspired air. this is the most common route in the transmission of most respiratory infections in domestic animals. some viruses, bacteria, parasites, and toxins can enter the respiratory system via the circulating blood. this portal of entry is commonly seen in septicemias, bacteremias, and with protozoa and viruses that target endothelial cells. also, circulating leukocytes may release infectious organisms such as retroviruses and listeria monocytogenes while traveling through the lungs. of type ii pneumonocytes becomes so spectacular that the microscopic appearance of the alveolus resembles that of a gland or fetal lung; this lesion has been termed epithelialization or fetalization. although it is part of the normal alveolar repair, hyperplasia of type ii pneumonocytes can interfere in gas exchange and cause hypoxemia. in uncomplicated cases, type ii pneumonocytes eventually differentiate into type i pneumonocytes, thus completing the last stage of alveolar repair (see fig. 9 -14). in some forms of chronic interstitial lung injury, the surface of the alveolar basement membrane could become populated with migrating bronchiolar cells, a process known as alveolar bronchiolization or lambertosis. in severe cases, lambertosis, a metaplastic change, can be mistaken microscopically with alveolar adenomas. type i pneumonocytes are one of the three structural components of the blood-air barrier, so when these epithelial cells are damaged, there is an increase in alveolar capillary permeability and transient leakage of plasma fluid, proteins, and fibrin into the alveolar lumen (see fig. 9-14) . under normal circumstances, these fluids are rapidly cleared from the alveolus by alveolar and lymphatic absorption, and necrotic pneumonocytes (type i) and fibrin strands are phagocytosed and removed by pulmonary alveolar macrophages. when there is persistent and severe injury, fibroblasts and myofibroblasts may proliferate in the alveolar walls (alveolar interstitium), causing alveolar septal fibrosis, whereas in other forms of severe injury, fibroblasts and myofibroblasts actively migrate from the interstitium into the alveolar spaces, causing intraalveolar fibrosis. these two types of alveolar fibrosis are most commonly seen in toxic and allergic pulmonary diseases and have a devastating effect on lung function. endothelial cells are also major players in the normal and abnormal physiology of the alveolus (see . these cells trap and share circulating antigens with intravascular and interstitial macrophages. the junction between alveolar endothelial cells is not as tight as that of the type i pneumonocytes, allowing some movement of fluid and small-size molecular weight proteins into the alveolar interstitium. endothelial cells maintain an intimate cell contact with erythrocytes and leukocytes passing through the lung, since the lumen of alveolar capillaries is slightly smaller (5.0 µm) than the diameter of red and white blood cells. erythrocytes are easily deformable, so their transit time through the alveolar capillaries is shorter than that of leukocytes, which are less deformable cells. this longer transit time of leukocytes and their close cellular contact with alveolar endothelial cells have major impacts in lung inflammation and acute respiratory distress syndrome (ards). on a minute-to-minute basis, the pulmonary defense mechanisms deal effectively with noxious stimuli and mild tissue injury without the need for an inflammatory response. however, if normal defense mechanisms are ineffective or insufficient (overwhelmed), the inflammatory process is rapidly turned on as a second line of defense. postmortem examination of the respiratory tract should always be conducted in a thorough and systematic manner and include the conducting system (trachea, bronchi, and bronchioles), the lungs, and the thoracic cavity and pleura. detailed record keeping and photographic documentation are essential elements of a thorough examination. normal lungs typically have a homogeneous pink color and are slightly deflated from loss of negative intrathoracic pressure. the e-sections that follow describe a systematic approach to this process. 480.e1 chapter 9 respiratory system, mediastinum, and pleurae the respiratory tract should always be examined in a systematic manner. to determine whether negative pressure is present in the thoracic cavity, the diaphragm is punctured through the abdominal cavity before the thoracic cavity has been opened. when the diaphragm is punctured in a fresh carcass, the loss of negative pressure in the thorax causes the diaphragmatic cupola to drop back caudally toward the abdominal cavity, and at the same time, there is an audible sound caused by the inrush of air into the thorax. lack of this movement may be an indication of advanced pneumothorax, pleural effusion, or the presence of uncollapsed lungs caused by pulmonary edema, pneumonia, fibrosis, or emphysema. in carcasses that have been dead for a long time, pulmonary air and gas produced by saprophytic bacteria leak into the pleural cavity, reducing the negative thoracic pressure and collapsing the lung. the rib cage must be removed by cutting along the costosternal joints and along the neck of the ribs (close to the costovertebral joints) in such a way that pleural adhesions and abnormal thoracic contents can be observed and grossly quantified (e.g., 200 ml of clear, yellow fluid). the tongue, pharynx, esophagus, larynx, trachea, and thoracic viscera (lungs, heart, and thymus) should be removed as a unit (often called the pluck) and placed on the necropsy table. the pharynx and esophagus are opened starting at the pharynx by a single cut with scissors along the dorsal midline and are inspected for ulcers, foreign bodies, and neoplasms. the larynx and trachea must be examined by opening both along the dorsal midline from cranial to caudal ends and then extending the incision into the large bronchi of the caudal lung lobes. normal tracheobronchial mucosa has a smooth and glistening pearl-colored surface with empty lumina in airways. the presence of foamy fluid in airways indicates pulmonary edema. feed particles may suggest aspiration; however, careful examination of the mucosa is required because aspiration of ingesta from stomach or rumen into the lungs commonly takes place agonally or can be displaced into these areas when the carcass is moved. the lungs should be examined before incision. normal lungs typically have a homogeneous pink color (see fig. 9 -16). external changes include the presence of rib imprints on the pleural surface when lungs fail to collapse. in addition, the lungs should be inspected for changes in color and texture and distribution of lesions. color changes can be various shades of red, indicating hypostatic congestion, hyperemia (acute pneumonia), and hemorrhage; dark blue collapsed lobules or areas are indicative of atelectasis; pale pink to white lungs indicate notable anemia, fibrosis, or emphysema; and uniformly or patchy yellow-brown lungs indicate chronic passive congestion and pulmonary fibrosis likely secondary to chronic heart failure. lungs from exsanguinated animals are generally paler than the normal pink color because of reduced blood in the pulmonary tissue. lungs with postmortem autolysis show green discoloration, a change that is also seen in other organs (efig. 9 -3). a covering of yellowish material on the pleural surface indicates accumulation of fibrin. because it is impossible to describe the texture of normal lungs, experience in palpation is required to appreciate the actual texture of a normal lung. texture is determined by gently palpating the surface and parenchyma of the lungs. normal texture can change to firm, hard, elastic (rubbery), or crepitus (with a crackling sound or feeling). for a detailed description of lung texture, see the section on classification of pneumonias in domestic animals. palpation of the lungs, which should be gentle, also permits detection of nonvisible nodules or abscesses in the parenchyma. knowing the distribution of a lesion in the lungs also facilitates diagnosis because particular etiologic agents cause lesions with specific distribution. distribution of lesions is generally described as focal, multifocal, locally extensive, or diffuse. according to their topography, pulmo-nary lesions can also be classified as cranioventral, dorsocaudal, and so on. necropsy reports must also contain an estimate of the extent of the pulmonary lesions, preferably expressed as a percentage of the volume of the lungs affected. for instance, a report may read "cranioventral consolidation involving 40% of the lungs." if the lungs have focal lesions, a rough estimate of the number should also be included in the report. for instance, "numerous (approximately 25), small (1 to 2 cm in diameter), hard nodules were randomly distributed in all lung lobes." two methods are used to examine the nasal structures. the first is making a midsagittal cut through the head and removing the nasal septum; the second is making several transverse sections of the nose at the level of the second premolar teeth. this latter method is preferred when examining pigs suspected of having atrophic rhinitis or animals suspected of having nasal neoplasms. microscopic examination of pulmonary tissue is routinely done in diagnostic laboratories. samples of normal and abnormal lungs, along with other appropriate tissue, should always be submitted in 10% buffered-neutral formalin for histopathologic evaluation. a minimum of four lung samples (left cranial, left caudal, right cranial, and right caudal) should be taken for histopathologic examination in animals with a history of respiratory signs. to improve fixation, a paper towel can be placed over the samples of lung floating in fixative. when detailed evaluation of the alveolar walls is required, lungs can be fixed by a gentle intratracheal injection of fixative; however, this technique displaces transudates and exudates and can artificially cause distention of the perivascular and peribronchial spaces. lung biopsy specimens are taken only sporadically because complications often outweigh the diagnostic value. however, the use of new techniques, such as endoscopic-directed biopsies, has notably reduced some of these complications. biopsies of the lungs are recommended in cases of chronic persistent pulmonary disease unresponsive to treatment or intrathoracic masses of undetermined origin. endoscopic-directed biopsies of the nasal and bronchial mucosa are routinely used in clinical practice and generally have a much better diagnostic value. two valuable diagnostic tools in human medicine, bronchoalveolar lavage (bal) and transtracheal wash (ttw), have in recent years become more widely used in veterinary clinical diagnosis of respiratory ailments, particularly in horses, dogs, and cats. the basis of bal and ttw is sampling the lung or trachea of a living animal by infusing sterile fluid into the trachea or deep lung (respectively) and retrieving it to determine the cellular and biochemical composition of this fluid. in other words, the composition of the fluid reflects what is present in the bronchioloalveolar spaces and trachea. these procedures are performed by inserting a tube directly through the larynx into the trachea or bronchus, or transtracheally by inserting a tube through a needle percutaneously into the cervical trachea. microscopic examination of properly collected, stored, and processed samples may reveal many erythrocytes and siderophages in pulmonary hemorrhage or left-sided heart failure; inclusion bodies or syncytial cells in viral pneumonias; increased number of leukocytes in pulmonary inflammation; abundant mucus in asthma or equine recurrent airway obstruction (rao); the presence of pulmonary pathogens, such as parasites, fungi, and bacteria; or tumor cells in cases of pulmonary neoplasia. in the healthy animal, 80% to 95% of the bal cells are pulmonary alveolar macrophages (see fig. 9-19, a) . clearance, or a combination of both is the underlying pathogenetic mechanism in many pulmonary diseases ( fig. 9-17) . the anatomic configuration of the nasal cavity and bronchi plays a unique role in preventing or reducing the penetration of noxious material into the lungs, especially into the alveoli, which is the most vulnerable portion of the respiratory system. the narrow nasal meatuses and the coiled arrangement of the nasal conchae generate enormous turbulence of airflow, and as a result, physical forces are created that forcefully impact particles larger than 10 µm onto the surface of the nasal mucosa ( fig. 9-18 ). although particles smaller than 10 µm could escape trapping in the nasal cavity, these mediumsized particles meet a second barrier at the tracheal and bronchial in some instances, pathogenic organisms can also reach the pleura and lungs through penetrating injuries, such as gunshot wounds, migrating awns, or bites, or by direct extension from a ruptured esophagus or perforated diaphragm. the lungs, particularly the bronchioles and alveoli, are vulnerable to endogenous injury caused by the local generation of free radicals during inflammation or by toxic metabolites generated by club (clara) cells (see fig. 9 -4, b). inflammatory processes in the respiratory system, particularly those caused by infectious organisms, can spread to contiguous or distant tissues. for instance, rhinitis may spread into the sinuses causing rhinosinusitis. similarly, laryngeal inflammation may spread into the lungs when exudate in the larynx is aspirated. lung disease can have profound systemic effects when cytokines, produced locally during necrosis or inflammation, are released into circulation. as a result of the enormous vascular bed present in the lung, sepsis and septic shock often develop when proinflammatory molecules overwhelm the antiinflammatory response during the so-called "cytokine storm." it is axiomatic that a particle, microbe, or toxic gas must first gain entry to a vulnerable region of the respiratory system before it can induce an adaptive immune response or have a pathologic effect. the characteristics of size, shape, dispersal, and deposition of particles present in inspired air are studied in aerobiology. it is important to recognize the difference between deposition, clearance, and retention of inhaled particles. deposition is the process by which particles of various sizes and shapes are trapped within specific regions of the respiratory tract. clearance is the process by which deposited particles are destroyed, neutralized, or removed from the mucosal surfaces. the difference between what is deposited and what is cleared from the respiratory tract is referred to as retention. the main mechanisms involved in clearance are sneezing, coughing, mucociliary transport, and phagocytosis (table 9 -3). abnormal retention of particles resulting from increased deposition, decreased same rate in all levels of a conducting system, a "bottleneck" effect would be created in major airways as the minor but more numerous airways enter the bronchi. for this reason, the mucociliary transport in proximal (rostral) airways is physiologically faster than that of the distal (caudal) ones. ciliary activity and mucus transport increase notably in response to stimuli such as in respiratory infections. the mucociliary blanket of the nasal cavity, trachea, and bronchi also plays an important role in preventing injury from toxic gases. if a soluble gas contacts the mucociliary blanket, it mixes with the mucus, thus reducing the concentration of gas reaching deep into the alveoli. in other words, mucus acts as a "scavenger system," whereby gases are solubilized and subsequently cleared from the respiratory tract via mucociliary transport. if ciliary transport is reduced (loss of cilia) or mucus production is excessive, coughing becomes an important mechanism for clearing the airways. in addition to the mechanical barrier and physical transport provided by the mucociliary escalator, other cells closely associated with ciliated epithelium contribute to the defense mechanism of the conducting and transitional systems. among the most notable are the microfold (m) cells, which are modified epithelial cells covering the bronchial-associated lymphoid tissue (balt), both of which are strategically situated at the corner of the bifurcation of bronchi and bronchioles, where inhaled particles often collide with the mucosa because of inertial forces. from here, inhaled particles and soluble antigens are phagocytosed and transported by macrophages, dendritic cells, and other professional antigen-presenting cells (apcs) into the balt, thus providing a unique opportunity for b and t lymphocytes to enter into close contact with inhaled pathogenic substances. pulmonary lymphocytes are not quiescent in the balt but are in continual traffic to other organs and contribute to both cellular (cytotoxic, helper, and suppressor t lymphocytes) and humoral immune responses. immunoglobulin a (iga), produced by mucosal plasma cells, and, to a lesser extent, immunoglobulin g (igg) and m (igm) play important roles in the local immunity of the conducting and transitional systems, especially with regard to preventing attachment of pathogens to the cilia. chronic airway diseases, especially those caused by infectious agents such as mycoplasmas or retroviruses, are often accompanied by severe hyperplasia of the balt. the mucociliary clearance terminates at the pharynx, where mucus, propelled caudally from the nasal cavity and cranially from the tracheobronchial tree, is eventually swallowed and thus eliminated from the conducting system of the respiratory tract. some respiratory pathogens, such as rhodococcus equi, can infect the intestines after having been removed and swallowed from the respiratory tract into the alimentary system. alveoli lack ciliated and mucus-producing cells; thus the defense mechanism against inhaled particles in the alveolar region cannot be provided by mucociliary clearance. instead, the main defense mechanisms of alveoli (exchange system) are phagocytosis provided by the pulmonary alveolar macrophages and antimicrobial molecules of the alveolar lining fluid ( fig. 9-19 ). pulmonary alveolar macrophages are highly phagocytic cells, which are not to be confused with pulmonary intravascular macrophages, and are derived largely from blood monocytes and, to a much lesser extent, from a slowly dividing population of interstitial macrophages. after a temporary adaptive stage within alveolar interstitium, blood monocytes reduce their glycolytic metabolism and increase their oxidative metabolism to function in an aerobic rather than an anaerobic environment. pulmonary alveolar macrophages contribute to the bifurcations. abrupt changes in the direction of air (inertia), which occurs at the branching of major airways, cause particles in the 2-to 10-µm size range to collide with the surface of bronchial mucosa (see fig. 9 -1). because the velocity of inspired air at the level of the small bronchi and bronchioles has become rather slow, inertial and centrifugal forces no longer play a significant role in the trapping of inhaled particles. here, in the transitional (bronchiolar) and exchange (alveolar) regions, particles 2 µm or smaller may come into contact with the mucosa by means of sedimentation because of gravitation or by diffusion as a result of brownian movement. infective aerosols containing bacteria and viruses are within the size range (0.01 to 2 µm) that can gain access to the bronchiolar and alveolar regions. in addition to size, other factors, such as shape, length, electrical charge, and humidity, play an important role in mucosal deposition, retention, and pathogenicity of inhaled particles. for example, particles longer than 200 µm may also reach the lower respiratory tract provided their mean aerodynamic diameter is less than 1 µm. asbestos is a good example of a large but slender fiber that can bypass the filtrating mechanisms by traveling parallel to the airstream. once in the terminal bronchioles and alveoli, asbestos fibers cause asbestosis, a serious pulmonary disease in human beings. in summary, the anatomic features of the nasal cavity and airways provide an effective barrier, preventing the penetration of most large particles into the lungs. once larger particles are trapped in the mucosa of conducting airways and small particles are deposited on the surface of the nasal, tracheal, or bronchoalveolar mucosa, it is crucial that these exogenous materials be promptly removed to prevent or minimize injury to the respiratory system. for these purposes, the respiratory system is equipped with several defense mechanisms, all of which are provided by specialized cells operating in a remarkably well-coordinated manner. conducting system (nose, trachea, and bronchi) mucociliary clearance is the physical unidirectional movement and removal of deposited particles and gases dissolved in the mucus from the respiratory tract. mucociliary clearance, also referred to as the waste disposal system, is provided by the mucociliary blanket (mucociliary escalator) and is the main defense mechanism of the conducting system (nasal cavity, trachea, and bronchi) (see figs. 9-2 and 9-3). mucus acts primarily as a barrier and a vehicle, and it is a complex mixture of water, glycoproteins, immunoglobulins, lipids, and electrolytes. these substances are produced by goblet (mucous) cells, serous cells, submucosal glands, and fluid from transepithelial ion and water transport. once serous fluid and mucus are secreted onto the surface of the respiratory mucosa, a thin, double-layer film of mucus is formed on top of the cells. the outer layer of this film is in a viscous gel phase, whereas the inner layer, which is in a fluid or sol phase, is directly in contact with cilia (see fig. 9 -3 and see efig. 9 -1). the respiratory system of a healthy human produces approximately 100 ml of mucus per day. each ciliated cell in the conducting system has approximately 100 to 200 motile and chemosensory cilia (6 µm long), beating metachronously (forming a wave) at a ciliary beat frequency of approximately 1000 strokes per minute, and in a horse, for example, mucus moves longitudinally at a rate of up to 20 mm per minute. rapid and powerful movement of cilia creates a series of waves that, in a continuous and synchronized manner, propel the mucus, exfoliated cells, and entrapped particles out of the respiratory tract to the pharynx. the mucus is finally swallowed or, when present in large amounts, is coughed up out of the conducting system. if mucus flow were to move at the activated alveolar macrophages. similarly, inhaled particles, such as dust, pollen, spores, carbon, or erythrocytes from intraalveolar hemorrhage, are all phagocytosed and eventually removed from alveoli by pulmonary alveolar macrophages. most alveolar macrophages leave the alveoli by migrating toward the bronchiolar (transitional) region until the mucociliary blanket is reached. once there, pulmonary macrophages are removed in the same way as any other particle: along the mucociliary flow to the pharynx and swallowed. in the cat, as many as 1 million macrophages per hour move out from the alveoli into the conducting system and pharynx. destruction and removal of inhaled microbes and particles by alveolar macrophages is a well-orchestrated mechanism that engages many cells, receptors (i.e., toll-like receptors [tlrs]), and pulmonary secretions in the lung. the cell-to-cell interactions are complex and involve pulmonary alveolar macrophages, pneumonocytes, endothelial cells, lymphocytes, plasma cells, natural killer (nk) cells, and dendritic cells. antibodies are also important in the protection (acquired immune response) of the respiratory tract against inhaled pathogens. iga is the most abundant antibody in the nasal and tracheal secretions and prevents the attachment and absorption of antigens (immune exclusion). igg and, to a lesser extent, ige and igm promote the uptake and destruction of inhaled pathogens by phagocytic cells (immune elimination). igg is the most abundant antibody in the alveolar surface and acts primarily as an opsonizing antibody for alveolar macrophages and neutrophils. in addition to antibodies, there are several secretory molecules locally released into the alveoli that constitute the alveolar lining material and contribute to the pulmonary defense mechanisms. the most important of these antimicrobial products are transferrin, anionic peptides, and pulmonary surfactant (table 9 -4). to facilitate phagocytosis and discriminate between "self" and "foreign" antigens, pulmonary alveolar macrophages are furnished with a wide variety of specific receptors on their cell surfaces. among the most important ones are fc receptors for antibodies; complement receptors (for c3b, c3a, and c5a); tumor necrosis factor (tnf) receptor; and cd40 receptors, which facilitate phagocytosis and destruction of opsonized particles. toll-like receptors (tlrs) recognize microbial components, and apoptosis stimulating fragment (fas) receptors are involved in apoptosis and in the phagocytosis of apoptotic cells in the lung. "scavenger receptors," which are responsible for the recognition and uptake of foreign particulates, such as dust and fibers, are also present on pulmonary alveolar macrophages. lungs are also susceptible to hematogenously borne microbes, toxins, or emboli. the hepatic (kupffer cells) and splenic macrophages are the primary phagocytic cells responsible for removing circulating bacteria and other particles from the blood of dogs, some rodents, and human beings. in contrast, the cell responsible for the removal of circulating particles, bacteria, and endotoxin from the blood of ruminants, cats, pigs, and horses is mainly the pulmonary intravascular macrophage, a distinct population of phagocytes normally residing within the pulmonary capillaries (see fig. 9 -7). in pigs, 16% of the pulmonary capillary surface is lined by pulmonary intravascular macrophages. in ruminants, 95% of intravenously injected tracer particles or bacteria are rapidly phagocytosed by these intravascular macrophages. studies have shown that an abnormally reduced number of kupffer cells in diseased liver results in a compensatory increase in pulmonary intravascular macrophages, even in animal species in which these phagocytic cells are normally absent from the lung. in some abnormal conditions, such as sepsis, pulmonary innate and adaptive immune response by rapidly attaching and phagocytosing bacteria and any other particles reaching the alveolar lumens. the number of free macrophages in the alveolar space is closely related to the number of inhaled particles reaching the lungs. this ability to increase, within hours, the number of available phagocytic cells is vital in protecting the distal lungs against foreign material, particularly when the inhaled particle load is high. unlike that of tissue macrophages, the life span of alveolar macrophages in the alveoli is notably short, only a few days, and thus they are continuously being replaced by newly migrated blood monocytes. alveolar phagocytosis plays a prominent role in the innate defense mechanism against inhaled bacteria without the need of an inflammatory reaction. bacteria reaching the alveoli are rapidly phagocytosed, and bactericidal enzymes present in lysosomes are discharged into the phagosome containing the bacteria (see b) . except for some facultative pathogens that are resistant to intracellular killing (e.g., mycobacterium tuberculosis, listeria monocytogenes, brucella abortus, rhodococcus equi, and some salmonella spp.), most bacteria reaching the lungs are rapidly destroyed by (ros) not only induce extensive pulmonary injury but also impair the defense and repair mechanisms in the lung. oxygen and free radical scavengers, such as catalase, superoxide dismutase, ubiquinone, and vitamins e and c, are largely responsible for protecting pulmonary cells against peroxidation. these scavengers are present in alveolar and bronchiolar epithelial cells and in the extracellular spaces of the pulmonary interstitium. in summary, the defense mechanisms are so effective in trapping, destroying, and removing bacteria that, under normal conditions, animals can be exposed to aerosols containing massive numbers of bacteria without any ill effects. if defense mechanisms are impaired, inhaled bacteria colonize and multiply in bronchi, bronchioles, and alveoli, and they produce infection, which can result in fatal pneumonia. similarly, when blood-borne pathogens, inhaled toxicants, or free radicals overwhelm the protective defense mechanisms, cells of the respiratory system are likely to be injured, often causing serious respiratory diseases. for many years, factors such as viral infections, toxic gases, stress, and pulmonary edema have been implicated in predisposing human beings and animals to secondary bacterial pneumonia. there are many pathways by which the defense mechanisms can be impaired; only those relevant to veterinary species are discussed. viral agents are notorious in predisposing human beings and animals to secondary bacterial pneumonias by what is known as viral-bacterial synergism. a good example of the synergistic effect of combined virus-bacterial infections is documented from epidemics of human beings with influenza virus in which the mortality rate has been significantly increased from secondary bacterial pneumonia. the most common viruses incriminated in predisposing animals to secondary bacterial pneumonia include influenza virus in pigs and horses; bovine herpesvirus 1 (bohv-1), bovine parainfluenza virus 3 (bpiv-3), and bovine respiratory syncytial virus (brsv) in cattle; canine distemper virus (cdv) in dogs; and felid herpesvirus 1 (fehv-1) and feline calicivirus (fcv) in cats. the mechanism of the synergistic effect of viral-bacterial infections was previously believed to be the destruction of the mucociliary blanket and a concurrent reduction of mucociliary clearance, but in experimental studies, viral infections did not significantly reduce the physical removal of particles or bacteria out of the lungs. now, it is known that 5 to 7 days after a viral infection, the phagocytic function of pulmonary alveolar macrophages and, to a lesser extent, the mucociliary clearance are notably impaired (see fig. 9 -8). other mechanisms by which viruses impair defense mechanisms are multiple and remain poorly understood (box 9-1). immunization against viral infections in many cases prevents or reduces the synergistic effect of viruses and thus the incidence of secondary bacterial pneumonia. certain gases also impair respiratory defense mechanisms, rendering animals more susceptible to secondary bacterial infections. for instance, hydrogen sulfide and ammonia, frequently encountered on farms, especially in buildings with poor ventilation, can impair pulmonary defense mechanisms and increase susceptibility to bacterial pneumonia. the effects of environmental pollutants on the defense mechanisms of human beings and animals living in crowded and polluted cities remain to be determined. excessive release of cytokines by pulmonary intravascular macrophages may result in acute lung injury. existing in an oxygen-rich environment and being the site of numerous metabolic reactions, the lungs also require an efficient defense mechanism against oxidant-induced cellular damage (oxidative stress). this form of damage is caused by inhaled oxidant gases (e.g., nitrogen dioxide, ozone, sulfur dioxide, or tobacco smoke), by xenobiotic toxic metabolites produced locally, by toxins reaching the lungs via the bloodstream (e.g., 3-methylindole and paraquat), or by free radicals (reactive oxygen species) released by phagocytic cells during inflammation. free radicals and reactive oxygen species anomalies 2 localized congenital anomalies of the nasal cavity are rare in domestic animals and are often merely part of a more extensive craniofacial deformity (e.g., cyclops) or a component of generalized malformation (e.g., chondrodysplasia). congenital anomalies involving the nasal cavity and sinuses, such as choanal atresia (lack of communication between the nasal cavity and pharynx), some types of chondrodysplasia, and osteopetrosis, are incompatible with life. examples of nonfatal congenital anomalies include cystic nasal conchae, deviation of the nasal septum, cleft upper lip (harelip and cheiloschisis), hypoplastic turbinates, and cleft palate (palatoschisis) (see fig. 7 -32). bronchoaspiration and aspiration pneumonia are common sequelae to cleft palate. nasal and paranasal sinus cysts are slowly growing and expansive lesions that mimic neoplasia and cause severe cranial deformation in horses. as in other organs or systems, it is extremely difficult to determine the actual cause (genetic vs. congenital) of anomalies based on pathologic evaluation. metabolic disturbances affecting the nasal cavity and sinuses are rare in domestic animals. immunodeficiency disorders, whether acquired or congenital, are often associated with increased susceptibility to viral, bacterial, and protozoal pneumonias. for example, human beings with acquired immunodeficiency syndrome (aids) are notably susceptible to pneumonia caused by proliferation of pneumocystis (carinii) jirovecii. a similar ubiquitous organism, which under normal circumstances is not pathogenic, is also found in the pneumonic lungs of immunosuppressed pigs, foals, dogs, and rodents. pigs infected with the porcine reproductive and respiratory syndrome (prrs) virus frequently develop pneumocystis carinii infection ( fig. 9-20) . arabian foals born with combined immunodeficiency disease easily succumb to infectious diseases, particularly adenoviral pneumonia. combined infections with two respiratory viruses, such as canine distemper virus (cdv) and canine adenovirus 2 (cav-2), are sporadically reported in immunosuppressed puppies. also, large doses of chemotherapeutic agents, such as steroids and alkylating agents, cause immunosuppression in dogs, cats, and other animals, increasing susceptibility to secondary viral and bacterial infections. stress, uremia, endotoxemia, dehydration, starvation, hypoxia, acidosis, pulmonary edema, anesthesia, and ciliary dyskinesia are only some of the many conditions that have been implicated in impairing respiratory defense mechanisms and consequently predisposing animals to develop secondary bacterial pneumonia. the mechanisms by which each of these factors suppresses pulmonary defenses are diverse and sometimes not well understood. for example, hypoxia and pulmonary edema decrease phagocytic function of pulmonary alveolar macrophages and alter the production of surfactant (abnormal head tilt and abnormal gait), which in severe cases may lead to emaciation. based on the nature of exudate, rhinitis can be classified as serous, fibrinous, catarrhal, purulent, or granulomatous. these types of inflammatory reactions can progress from one to another in the course of the disease (i.e., serous to catarrhal to purulent), or in some instances exudates can be mixed, such as those seen in mucopurulent, fibrinohemorrhagic, or pyogranulomatous rhinitis. microscopic examination of impression smears or nasal biopsy, and bacterial or fungal cultures are generally required in establishing the cause of inflammation. common sequelae of rhinitis are hemorrhage, ulcers, and, in some cases, nasopharyngeal polyps (hyperplasia) arising from inflamed mucosa. rhinitis also can be classified according to the age of the lesions as acute, subacute, or chronic; to the severity of the insult as mild, moderate, or severe; and to the etiologic agent as viral, allergic, bacterial, mycotic, parasitic, traumatic, or toxic. serous rhinitis. serous rhinitis is the mildest form of inflammation and is characterized by hyperemia and increased production of a clear fluid locally manufactured by serous glands present in the nasal submucosa. serous rhinitis is of clinical interest only. it is caused by mild irritants or cold air, and it occurs during the early stages of viral infections, such as the common cold in human beings, upper respiratory tract infections in animals, or in mild allergic reactions. catarrhal rhinitis. catarrhal rhinitis is a slightly more severe process and has, in addition to serous secretions, a substantial increase in mucus production by hypersecretion of goblet cells and mucous glands. a mucous exudate is a thick, translucent, or slightly turbid viscous fluid, sometimes containing a few exfoliated cells, leukocytes, and cellular debris. in chronic cases, catarrhal rhinitis is characterized microscopically by notable hyperplasia of goblet cells. as the inflammation becomes more severe, the mucus is infiltrated with neutrophils, giving the exudate a cloudy appearance. this exudate is referred to as mucopurulent. purulent (suppurative) rhinitis. purulent (suppurative) rhinitis is characterized by a neutrophilic exudate, which occurs when the nasal mucosa suffers a more severe injury that generally is accompanied by mucosal necrosis and secondary bacterial infection. cytokines, leukotrienes, complement activation, and bacterial products cause exudation of leukocytes, especially neutrophils, which mix with nasal secretions, including mucus. grossly, the exudate in suppurative rhinitis is thick and opaque, but it can vary from white to green to brown, depending on the types of bacteria and type of leukocytes (neutrophils or eosinophils) present in the exudate . in severe cases, the nasal passages are completely blocked by the exudate. microscopically, neutrophils can be seen in the submucosa and mucosa and form plaques of exudate on the mucosal surface. neutrophils are commonly found marginated in vessels, in the lamina propria, and in between epithelial cells in their migration to the surface of the mucosa. fibrinous rhinitis. fibrinous rhinitis is a reaction that occurs when nasal injury causes a severe increase in vascular permeability, resulting in abundant exudation of plasma fibrinogen, which coagulates into fibrin. grossly, fibrin appears as a yellow, tan, or gray rubbery mat on nasal mucosa. fibrin accumulates on the surface and forms a distinct film of exudate sometimes referred to as pseudomembrane ( fig. 9-22 ). if this fibrinous exudate can be removed, leaving an intact underlying mucosa, it is termed a croupous or pseudodiphtheritic rhinitis. conversely, if the pseudomembrane is difficult to remove and leaves an ulcerated mucosa, it is referred to as diphtheritic or fibrinonecrotic rhinitis. the term diphtheritic was derived from human diphtheria, which causes a severe and destructive inflammatory process of the nasal, tonsillar, pharyngeal, and laryngeal mucosa. nasal amyloidosis. amyloidosis, the deposition of amyloid protein (fibrils with a β-pleated configuration) in various tissues, has been sporadically reported as a localized lesion in the nasal cavity of horses and human beings (see nasal amyloidosis, in disorders of horses). congestion and hyperemia. the nasal mucosa is well vascularized and is capable of rather dramatic variation in blood flow, whether passively as a result of interference with venous return (congestion) or actively because of vasodilation (hyperemia). congestion of the mucosal vessels is a nonspecific lesion commonly found at necropsy and presumably associated with the circulatory failure preceding death (e.g., heart failure, bloat in ruminants in which the increased intraabdominal pressure causes increased intrathoracic pressure impeding the venous return from the head and neck). hyperemia of the nasal mucosa is seen in early stages of inflammation, whether caused by irritation (e.g., ammonia and regurgitated feed), viral infections, secondary bacterial infections, toxemia, allergy, or trauma. hemorrhage. epistaxis is the clinical term used to denote blood flow from the nose (nosebleed) regardless of whether the blood originates from the nasal mucosa or from deep in the lungs, such as in horses with "exercise-induced pulmonary hemorrhage." unlike blood in the digestive tract, where the approximate anatomic location of the bleeding can be estimated by the color the blood imparts to fecal material, blood in the respiratory tract is always red. this fact is due to the rapid transport of blood out of the respiratory tract by the mucociliary blanket and during breathing. hemorrhages into the nasal cavity can be the result of local trauma, can originate from erosions of submucosal vessels by inflammation (e.g., guttural pouch mycosis), or can be caused by neoplasms. hemoptysis refers to the presence of blood in sputum or saliva (coughing or spitting blood) and is most commonly the result of pneumonia, lung abscesses, ulcerative bronchitis, pulmonary thromboembolisms or hemorrhage, and pulmonary neoplasia. inflammation of the nasal mucosa is called rhinitis, and inflammation of the sinuses is called sinusitis. these conditions usually occur together, although mild sinusitis can be undetected. clinically, rhinosinusitis is characterized by nasal discharge. rhinitis. the occurrence of infectious rhinitis presupposes an upset in the balance of the normal microbial flora of the nasal cavity. innocuous bacteria present normally protect the host through a process called competitive exclusion, whereby potential pathogens are kept at a harmless level. disruption of this protective mechanism can be caused by respiratory viruses, pathogenic bacteria, fungi, irritant gases, environmental changes, immunosuppression, local trauma, stress, or prolonged antibacterial therapy. inflammatory processes in the nasal cavity are not life-threatening and usually resolve completely. however, some adverse sequelae in cases of infectious rhinitis include bronchoaspiration of exudate leading to bronchopneumonia. chronic rhinitis often leads to destruction of the nasal conchae (turbinates), deviation of the septum, and, eventually, craniofacial deformation. also, nasal inflammation may extend into the sinuses causing sinusitis; into facial bones causing osteomyelitis; through the cribriform plate causing meningitis; into the eustachian tubes causing otitis media or guttural pouch empyema (eustachitis) in horses; and even into the inner ear causing otitis interna and vestibular syndrome the nasal septum has been removed to expose nasal conchae. the nasal mucosa is hyperemic and covered by yellow-white purulent exudate (arrows). inset, histological section showing submucosal congestion and edema and also large aggregates of neutrophils on the superficial mucosa (asterisk). h&e stain. (courtesy dr. a. lópez, atlantic veterinary college.) microscopically, the lesions include a perivascular edema with fibrin, a few neutrophils infiltrating the mucosa, and superficial plaques of exudate consisting of fibrin strands mixed with leukocytes and cellular debris covering a necrotic and ulcerated epithelium. fungal infections, such as aspergillosis, can cause a severe fibrinonecrotizing rhinitis. granulomatous rhinitis. granulomatous rhinitis is a reaction in the nasal mucosa and submucosa that is characterized by infiltration of numerous activated macrophages mixed with a few lymphocytes and plasma cells (figs. 9-23 and 9-24). in some cases, chronic inflammation leads to the formation of polypoid nodules that in severe cases are large enough to cause obstruction of the nasal passages ( fig. 9-25 ). granulomatous rhinitis is generally associated with chronic allergic inflammation or infection with specific organisms, such as fungi (see fig. 9 -24), tuberculosis, systemic mycosis (see section on granulomatous pneumonia), and rhinosporidiosis ; also see fig. 9 -25). in some cases, the cause of granulomatous rhinitis cannot be determined. sinusitis. sinusitis occurs sporadically in domestic animals and is frequently combined with rhinitis (rhinosinusitis), or it occurs as extend into the adjacent bone (osteomyelitis) or through the ethmoidal conchae into the meninges and brain (meningitis and encephalitis). nasal amyloidosis. amyloidosis, the deposition of amyloid protein (fibrils with a β-pleated configuration) in various tissues, has been sporadically reported as a localized lesion in the nasal cavity of horses. unlike amyloidoses in other organs of domestic animals where amyloid is generally of the reactive type (amyloid aa), equine nasal amyloidosis appears to be of the immunocytic type (amyloid al). affected horses with large amyloid masses have difficulty breathing because of nasal obstruction and may exhibit epistaxis and reduced athletic performance; on clinical examination, large, firm nodules resembling neoplasms (amyloidoma) can be observed in the alar folds, rostral nasal septum, and floor of nasal cavity. microscopic lesions are similar to those seen in other organs and consist of a deposition of hyaline amyloid material in nasal mucosa that is confirmed by a histochemical stain, such as congo red. progressive ethmoidal hematoma. progressive ethmoidal hematoma (peh) is important in older horses and is characterized clinically by chronic, progressive, often unilateral nasal bleeding. grossly or endoscopically, an ethmoidal hematoma appears as a single, soft, tumor-like, pedunculated, expansive, dark red mass arising from the mucosa of the ethmoidal conchae ( fig. 9-28) . microscopic examination reveals a capsule lined by epithelium and hemorrhagic stromal tissue infiltrated with abundant macrophages, most of which are siderophages. viral infections. viruses, such as equine viral rhinopneumonitis virus, influenza virus, adenovirus, and equine picornavirus, cause mild and generally transient respiratory infections in horses. the route of infection for these respiratory viruses is typically aerogenous. all of these infections are indistinguishable clinically; signs consist mainly of malaise, fever, coughing, conjunctivitis, and nasal a sequela to penetrating or septic wounds of the nasal, frontal, maxillary, or palatine bones; improper dehorning in young cattle, which exposes the frontal sinus; or maxillary tooth infection in horses and dogs (maxillary sinus). based on the type of exudate, sinusitis is classified as serous, catarrhal, fibrinous (rare), purulent, or granulomatous. paranasal sinuses have poor drainage; therefore exudate tends to accumulate, causing mucocele (accumulation of mucus) or empyema (accumulation of pus) ( fig. 9-27 ). chronic sinusitis may promised horses, particularly in arabian foals with inherited combined immunodeficiency disease. bacterial infections. strangles, glanders, and melioidosis of horses are all systemic bacterial diseases that cause purulent rhinitis and suppuration in various organs. these diseases are grouped as upper respiratory diseases because nasal discharge is often the most notable clinical sign. strangles. strangles is an infectious and highly contagious disease of equidae that is caused by streptococcus equi ssp. equi (streptococcus equi) . it is characterized by suppurative rhinitis and lymphadenitis (mandibular and retropharyngeal lymph nodes) with occasional hematogenous dissemination to internal organs. unlike streptococcus equi ssp. zooepidemicus (streptococcus zooepidemicus) and streptococcus dysgalactiae ssp. equisimilis (streptococcus equisimilis), streptococcus equi is not part of the normal nasal flora. infection occurs when susceptible horses come into contact with feed, exudate, or air droplets containing the bacterium. after penetrating through the nasopharyngeal mucosa, streptococcus equi drains to the regional lymph nodes-mandibular and retropharyngeal lymph nodes-via lymphatic vessels. the gross lesions in horses with strangles (mucopurulent rhinitis) correlate with clinical findings and consist of copious amounts of mucopurulent exudate in the nasal passages with notable hyperemia of the nasal mucosa. affected lymph nodes are enlarged and may contain abscesses filled with thick purulent exudate (purulent lymphadenitis). the term bastard strangles is used in cases in which hematogenous dissemination of streptococcus equi results in metastatic abscesses in such organs as the lungs, liver, spleen, kidneys, or brain or in the joints. this form of strangles is often fatal. common sequelae to strangles include bronchopneumonia caused by aspiration of nasopharyngeal exudate; laryngeal hemiplegia ("roaring"), resulting from compression of the recurrent laryngeal nerves by enlarged retropharyngeal lymph nodes; facial paralysis and horner syndrome caused by compression of sympathetic nerves that run dorsal to the medial retropharyngeal lymph node; and purpura hemorrhagica as a result of vasculitis caused by deposition of streptococcus equi antigen-antibody complexes in arterioles, venules, and capillaries of the skin and mucosal membranes. in severe cases, nasal infection extends directly into the paranasal sinuses or to the guttural pouches via the eustachian tubes, causing inflammation and accumulation of pus (guttural pouch empyema). rupture of abscesses in the mandibular and retropharyngeal lymph nodes leads to suppurative inflammation of adjacent subcutaneous tissue (cellulitis), and in severe cases the exudate escapes through cutaneous fistulas. strangles can affect horses of all ages, but it is most commonly seen in foals and young horses. it is clinically characterized by cough, nasal discharge, conjunctivitis, and painful swelling of regional lymph nodes. some horses become carriers and a source of infection to other horses. glanders. glanders is an infectious world organization for animal health (oie)-notifiable disease of equidae caused by burkholderia mallei (pseudomonas mallei) that can be transmitted to carnivores by consumption of infected horsemeat. human beings are also susceptible, and untreated infection is often fatal. this gramnegative bacterium has been listed as a potential agent for biologic warfare and bioterrorism. in the past, burkholderia mallei was found throughout the world, but today, glanders has been eradicated from most countries, except for some areas in north africa, asia, and eastern europe. there also have been sporadic outbreaks reported in brazil. the pathogenesis of glanders is not fully understood. results from experimental infections suggest that infection occurs discharge varying from serous to purulent. viral respiratory infections are common medical problems in adult horses. equine viral rhinopneumonitis. equine viral rhinopneumonitis (evr) is caused by two ubiquitous equine herpesviruses (ehv-1 and ehv-4) and may be manifested as a mild respiratory disease in weanling foals and young racehorses, as a neurologic disease (myeloencephalopathy), or as abortion in mares. the portal of entry for the respiratory form is typically aerogenous, and the disease is generally transient; thus the primary viral-induced lesions in the nasal mucosa and lungs are rarely seen at necropsy unless complicated by secondary bacterial rhinitis, pharyngitis, or bronchopneumonia. studies with polymerase chain reaction (pcr) techniques have demonstrated that, like other herpesviruses, ehv-1 and ehv-4 persist in the trigeminal ganglia for long periods of time (latency). reactivation because of stress or immunosuppression and subsequent shedding of the virus are the typical source of infection for susceptible animals on the farm. equine influenza. equine influenza is a common, highly contagious, and self-limiting upper respiratory infection of horses caused by aerogenous exposure to type a strains of influenza virus (h7n7 [a/equi-1] and h3n8 [a/equi-2]). equine influenza has high morbidity (outbreaks) but low mortality, and it is clinically characterized by fever, conjunctivitis, and serous nasal discharge. it occurs mainly in 2-to 3-year-old horses at the racetrack. as with human influenza, equine influenza is usually a mild disease, but occasionally it can cause severe bronchointerstitial pneumonia with pulmonary edema. in some horses, impaired defense mechanisms caused by the viral infection are complicated by a secondary bacterial bronchopneumonia caused by opportunistic organisms (streptococcus zooepidemicus, staphylococcus aureus, or bacteroides sp.) found in the normal flora of the upper respiratory tract. uncomplicated cases of equine influenza are rarely seen in the postmortem room. equine influenza virus (h3n8) recently did an equine to canine "host-jump" causing extensive outbreaks of respiratory disease in dogs (see pneumonias of dogs). other equine respiratory viruses. equine picornavirus, adenovirus, and parainfluenza virus produce mild and transient upper respiratory infections (nasopharynx and trachea) in horses, unless complicated by secondary pathogens. in addition to reduced athletic performance, infected horses may have a temporary suppression of cell-mediated immunity leading to opportunistic infections such as pneumocystis carinii pneumonia. fatal adenoviral infections with severe pneumonia or enteritis occur commonly in immunocomdisorders of ruminants (cattle, sheep, and goats) infectious bovine rhinotracheitis. infectious bovine rhinotracheitis (ibr), or "rednose," occurs worldwide and is a disease of great importance to the cattle industry because of the synergism of the ibr virus with mannheimia haemolytica in producing pneumonia. the causative agent, bovine herpesvirus 1 (bohv-1), has probably existed as a mild venereal disease in cattle in europe since at least the mid-1800s, but the respiratory form was not reported until intensive management feedlot systems were first introduced in north america around the 1950s. typically, the disease is manifested as a transient, acute, febrile illness, which results in inspiratory dyspnea caused by obstruction of the airways by exudate only in very severe cases. other forms of bohv-1 infection include ulcerative rumenitis; enteritis; multifocal hepatitis in neonatal calves; nonsuppurative meningoencephalitis; infertility; and in experimental infections, mastitis, mammillitis, and ovarian necrosis. except for the encephalitic form, the type of disease caused by bohv-1 depends more on the site of entry than the viral strain. like other herpesviruses, bohv-1 also can remain latent in nerve ganglia, with recrudescence after stress or immunosuppression. this virus also causes bovine abortion, systemic infections of calves, and genital infections such as infectious pustular vulvovaginitis (ipv) and infectious balanoposthitis (ibp). the respiratory form of ibr is characterized by severe hyperemia and multifocal necrosis of nasal, pharyngeal, laryngeal, tracheal, and sometimes bronchial mucosa ( fig. 9 -29 and see fig. 9 -22). as in other respiratory viral infections, ibr lesions are microscopically characterized by necrosis and exfoliation of ciliated cells followed by repair. secondary bacterial infections of these areas of necrosis result in the formation of a thick layer of fibrinonecrotic material (diphtheritic) in the nasal, tracheal, and bronchial mucosa (see fig. 9 -22). intranuclear inclusion bodies, commonly seen in herpesvirus infections, are rarely seen in field cases because inclusion bodies occur only during the early stages of the disease. the most important sequela to ibr is bronchopneumonia, which is caused either by direct aspiration of exudate from airways or as a result of an impairment in pulmonary defense mechanisms, thus predisposing the animal to secondary bacterial infection, most frequently mannheimia haemolytica (see pneumonic mannheimiosis discussion). postmortem diagnosis of ibr is confirmed by isolation of the virus or its identification by immunohistochemistry or pcr in affected tissues. other causes of rhinitis. nasal granulomas occur in cattle presumably as a result of repeated exposure to an unidentified inhaled antigen. nasal granulomas (atopic rhinitis) are reported mainly in cattle in australia, south africa, and the united kingdom, where affected cattle develop multiple, small, pink or red, polypoid nodules, starting in the nasal vestibule that in time extend into the caudal aspect of the nasal septum (see fig. 9 -23). these nodules are composed of fibrovascular tissue mixed with lymphocytes (granulation tissue) superficially lined by hyperplastic epithelium with abundant mast cells and eosinophils in the lamina propria (nasal eosinophilia). the microscopic features suggest that hypersensitivity type i (immediate), type iii (immune complex), and type iv (delayed) may be involved in nasal granulomas of cattle. bovine (idiopathic) nasal granuloma must be differentiated from nasal mycetomas, nasal rhinosporidiosis, and nasal schistosomiasis, which also cause the formation of nodules in the nasal mucosa of cattle. an eosinophilic material consistent with the splendore-hoeppli phenomenon is occasionally observed in bovine mycotic granulomas. this phenomenon seen in some mycotic or bacterial infections is microscopically via the ingestion of contaminated feed and water and, very rarely, via inhalation of infectious droplets. the portals of entry are presumed to be the oropharynx or intestine, in which bacteria penetrate the mucosa and spread via lymph vessels to regional lymph nodes, then to the bloodstream, and thus hematogenously to the internal organs, particularly the lungs. lesions in the nasal cavity start as pyogranulomatous nodules in the submucosa; these lesions subsequently ulcerate, releasing copious amounts of burkholderia mallei-containing exudate into the nasal cavity (see fig. 4-25, a) . finally, ulcerative lesions in conchal mucosa heal and are replaced by typical stellate (star-shaped), fibrous scars. in some cases, the lungs also contain numerous gray, hard, small (2 to 10 mm), miliary nodules (resembling millet seeds) randomly distributed in one or more pulmonary lobes because of the hematogenous route. microscopically, these nodules are typical chronic granulomas composed of a necrotic center, with or without calcification, surrounded by a layer of macrophages enclosed by a thick band of connective tissue infiltrated with macrophages, fewer giant cells, lymphocytes, and plasma cells. cutaneous lesions, often referred to as equine farcy, are the result of severe suppurative lymphangitis characterized by nodular thickening of extended segments of lymph vessels in the subcutaneous tissue of the legs and ventral abdomen (see fig. 4 -25, c). eventually, affected lymph vessels rupture and release large amounts of purulent exudate through sinuses to the surface of the skin. melioidosis (pseudoglanders). melioidosis (pseudoglanders) is an important, life-threatening disease of human beings, horses, cattle, sheep, goats, pigs, dogs, cats, and rodents caused by burkholderia pseudomallei (pseudomonas pseudomallei). this disease in horses is clinically and pathologically similar to glanders, hence the name pseudoglanders. in human beings, this infection can cause severe sepsis and septic shock and has also been considered to have potential for biologic welfare. melioidosis is currently present in southeast asia and, to a much lesser extent, in northern australia and some european countries where the causative organism is frequently found in rodents, feces, soil, and water. ingestion of contaminated feed and water appears to be the main route of infection; direct transmission between infected animals and insect bites has also been postulated as a possible mechanism of infection. after gaining entrance to the animal, burkholderia pseudomallei is disseminated by the bloodstream and causes suppuration and abscesses in most internal organs, such as nasal mucosa, joints, brain and spinal cord, lungs, liver, kidneys, spleen, and lymph nodes. the exudate is creamy or caseous and yellow to green. the pulmonary lesions in melioidosis are those of an embolic bacterial infection with the formation of pulmonary abscesses, which can become confluent. focal adhesive pleuritis develops where abscesses rupture through the pleura and heal. rhinosporidiosis. the protistan parasite, rhinosporidium seeberi, causes nasal infection in human beings, horses, mules, cattle, dogs, and cats. gross lesions vary from barely visible granulomas to large expansive polypoid nodules that may be mistaken as tumors. these granulomatous nodules are detected by direct observation when present in the nasal mucosa close to the nares or by rhinoscopy when located in the deep nasal cavity. the offending organism, rhinosporidium seeberi, is readily visible in histologic preparations and in impression smears, appearing as a large (400 µm), oval sporangium containing thousands of endospores (see fig. 9 -26). rhinosporidium seeberi was once considered a mycotic agent, but recent phylogenetic investigations suggest that it is an aquatic protistan parasite of the class mesomycetozoea. giant, basophilic, intranuclear inclusion bodies in the nasal epithelium, particularly in the nasal glands ( fig. 9-32 ). immunosuppressed piglets can develop a systemic cytomegalovirus infection characterized by necrosis of the liver, lungs, adrenal glands, and brain with intralesional inclusion bodies. inclusion body rhinitis is clinically a characterized by a deeply eosinophilic homogeneous material surrounded by bacteria or mycelia. it is thought to result from a localized antigen-antibody response in tissue. oestrus ovis. oestrus ovis (diptera: oestridae; nasal bot) is a brownish fly about the size of a honeybee that deposits its first-stage larvae in the nostrils of sheep in most areas of the world. microscopic larvae mature into large bots (maggots), which spend most of their larval stages in nasal passages and sinuses, causing irritation, inflammation, and obstruction of airways. mature larvae drop to the ground and pupate into flies. this type of parasitism in which living tissues are invaded by larvae of flies is known as myiasis ( fig. 9-30 ). although oestrus ovis is a nasal myiasis primarily of sheep, it sporadically affects goats, dogs, and sometimes human beings (shepherds). the presence of the larvae in nasal passages and sinuses causes chronic irritation and erosive mucopurulent rhinitis and sinusitis; bots of oestrus ovis can be found easily if the head is cut to expose the nasal passages and paranasal sinuses. rarely, larvae of oestrus ovis penetrate the cranial vault through the ethmoidal plate, causing direct or secondary bacterial meningitis. other causes of rhinitis. infectious rhinitis is only sporadically reported in goats, and most of these cases are caused by pasteurella multocida or mannheimia haemolytica. the lesions range from a mild serous to catarrhal or mucopurulent inflammation. foreign body rhinitis caused by plant material is sporadically seen cattle, sheep, and goats ( fig. 9-31 ). inclusion body rhinitis. inclusion body rhinitis is a disease of young pigs with high morbidity and low mortality caused by a porcine cytomegalovirus (suid herpesvirus-2) and characterized by a mild rhinitis. this virus commonly infects the nasal epithelium of piglets younger than 5 weeks and causes a transient viremia. because this disease is seldom fatal, lesions are seen only incidentally or in euthanized animals. in uncomplicated cases, the gross lesion is hyperemia of the nasal mucosa, but with secondary bacterial infections, mucopurulent exudate can be abundant. microscopic lesions are typical and consist of a necrotizing, nonsuppurative rhinitis with toxigenic strains of pasteurella multocida. the only lesion associated with infection with bordetella bronchiseptica alone is a mild to moderate turbinate atrophy (nonprogressive atrophic rhinitis), but this bacterium actively promotes the colonization of the nasal cavity by pasteurella multocida. the toxigenic strains of pasteurella multocida produce potent cytotoxins that inhibit osteoblastic activity and promote osteoclastic reabsorption in nasal bones, particularly in the ventral nasal conchae, where abnormal bone remodeling results in progressive atrophy of conchae. the degree of conchal atrophy in pigs with atrophic rhinitis varies considerably, and in most pigs, the severity of the lesions does not correspond to the severity of the clinical signs. the best diagnostic method of evaluating this disease at necropsy is to make a transverse section of the snout between the first and second premolar teeth. in normal pigs, conchae are symmetric and fill most of the cavity, leaving only narrow airspaces (meatuses) between coiled conchae. the normal nasal septum is straight and divides the cavity into two mirror-image cavities. in contrast, the septum in pigs with atrophic rhinitis is generally deviated and the conchae appear smaller and asymmetric ( fig. 9-33 ). conchal atrophy causes dorsal and ventral meatuses to appear rather enlarged, and in the most advanced cases, the entire nasal conchae may be missing, leaving a large, empty space. it may seem logical to assume that after loss of conchae in an obligate nasal breather, such as the pig, the filtration defense mechanism of the nasal cavity would be impaired, thus enhancing the chances of aerogenous infections in the lung. however, the relationship between atrophic rhinitis, pneumonia, and growth rates in pigs is still controversial. osteoclastic hyperplasia and osteopenia of the conchae are the key microscopic lesions in atrophic rhinitis. depending on the stage of the disease, mucopurulent exudate may be found on the surface of the conchae. hyperplastic or metaplastic changes can occur in the nasal epithelium and glands, and infiltrates of lymphoplasmacytic cells can be present in the lamina propria. in summary, atrophic rhinitis is an important disease in pigs worldwide; morphologic characterized by a mild and transient rhinitis, causing sneezing, nasal discharge, and excessive lacrimation. atrophic rhinitis. a common worldwide disease of pigs, atrophic rhinitis (progressive atrophic rhinitis) is characterized by inflammation and atrophy of nasal conchae (turbinates). in severe cases, atrophy of the conchae may cause a striking facial deformity in growing pigs because of deviation of the nasal septum and nasal bones. the etiopathogenesis of atrophic rhinitis is complex and has been a matter of controversy for many years. pathogens historically associated with atrophic rhinitis include bordetella bronchiseptica, pasteurella multocida, haemophilus parasuis, and viral infections such as porcine cytomegalovirus (inclusion body rhinitis). in addition, predisposing factors have included genetic makeup, environment, and nutritional deficiencies. the cause of atrophic rhinitis is currently believed to be a combined infection by specific strains of bordetella bronchiseptica producing dermonecrotic toxin and linguatula serrata. linguatula serrata is a rare but highly specialized pentastomid parasite that shares some morphologic features with arthropods and annelids and causes infection when dogs consume uncooked ruminant meat containing infective larvae. it occurs primarily in carnivores, although sheep and goats may become aberrant hosts. human beings can also acquire the infection by ingesting raw ovine or caprine meat. the adult parasite is found throughout the nasal passages and sometimes can reach the sinuses and middle ear by moving through the exudate in the eustachian tubes. in common with other nasal parasites, linguatula serrata acts as an irritant, causing sneezing, catarrhal inflammation, and epistaxis. the eggs of this parasite leave the host in the exudate, which is coughed up or swallowed and eliminated in the feces. the nasal cavity and paranasal sinuses of dogs can occasionally be infested with other parasites, including mites (pneumonyssus caninum) and rhinosporidium seeberi (see figs. 9-25 and 9-26). allergic rhinitis. allergic rhinitis (hay fever; nasolacrimal urticaria), which is so common in human beings sensitized and reexposed to inhaled pollens or allergens, has been reported only sporadically in dogs and cats. hay fever in human beings and animals is a type i hypersensitivity reaction in which an ige-mediated degranulation of mast cells results in an acute rhinitis and conjunctivitis. microscopically, the nasal mucosa is edematous and infiltrated with numerous eosinophils, neutrophils, and some macrophages. clinically, allergic rhinitis is characterized by profuse serous nasal discharge and lacrimation. other causes of rhinitis. a nonspecific (idiopathic) chronic lymphoplasmacytic rhinitis is occasionally seen in dogs. immotile cilia syndrome (ciliary dyskinesia), a congenital disease, reduces mucociliary clearance and is an important factor in recurrent canine rhinosinusitis, bronchitis, bronchiectasis, and pneumonia. feline viral rhinotracheitis. feline viral rhinotracheitis (fvr) is a common, worldwide respiratory disease of cats caused by felid herpesvirus 1 (fehv-1). the disease causes an impairment of pulmonary defense mechanisms predisposing cats to secondary bacterial pneumonia or to a coinfection with feline calicivirus. the virus also can remain latent in ganglia. the vast majority of cats that recover from fvr become carriers and shed fehv-1, either spontaneously or following stress. susceptible animals, particularly kittens with low maternal immunity, become infected after exposure to a diseased or carrier cat. replication of fehv-1 in the nasal, conjunctival, pharyngeal, and, to a lesser extent, tracheal epithelium causes degeneration and exfoliation of cells. lesions caused by fehv-1 are fully reversible, but secondary infections with bacteria, such as pasteurella multocida, bordetella bronchiseptica, streptococcus spp., and mycoplasma felis, can cause a chronic, severe suppurative rhinitis and also conjunctivitis. intranuclear inclusion bodies are rarely seen in cats with fvr because inclusions are only present during the early stages of infection and have already disappeared by the time the cat is presented for diagnosis. respiratory sequelae to fvr can include chronic bacterial rhinitis and sinusitis with persistent purulent discharge; lysis of nasal bones, which can lead to conchal atrophy; permanent damage to the olfactory epithelium; and secondary bacterial pneumonia. in addition to rhinitis and interstitial pneumonia, fvr also causes ulcerative keratitis, hepatic necrosis, emaciation, abortion, and diagnosis is simple, but additional understanding of the pathogenesis will be necessary before effective preventive measures can be established. atrophic rhinitis is clinically characterized by sneezing, coughing, and nasal discharge. obstruction of the nasolacrimal duct is common and results in accumulation of dust and dried lacrimal secretions on the skin inferior to the medial canthus of the eye. viral infections. dogs have no specific viral infections affecting exclusively the nasal cavity or sinuses. acute rhinitis and sinusitis occurs as part of the canine infectious respiratory disease (cird) group caused by several distinct viruses, such as canine distemper virus, cav-1 and -2, canine parainfluenza virus, reovirus, and canine herpesvirus. the viral lesions in the respiratory tract are generally transient, but the effect of the virus on other tissues and cells can be fatal, as in distemper encephalitis in dogs. bacterial infections. as in other species, secondary bacterial rhinitis, sinusitis, and pneumonia are possible sequelae of respiratory viral infections; bordetella bronchiseptica, escherichia coli, and pasteurella multocida are the most common isolates in dogs with bacterial rhinitis. mycotic infections. aspergillus spp. and penicillium spp. cause mycotic rhinitis and sinusitis in dogs (canine nasal aspergillosis) ( fig. 9-34 ). nasal biopsies reveal extensive necrosis of the nasal epithelium and thick plaques of fibrinopurulent exudate mixed with many fungal hyphae. cryptococcus neoformans and blastomyces dermatitides infections of the nasal cavity occur sporadically in dogs ( fig. 9-35 ). lesions are characterized by mucosal granulomas containing periodic acid-schiff (pas)-positive fungal organisms, and the infection is clinically characterized by mucopurulent nasal discharge. fvr; these two viral infections account for 80% of all cases of feline respiratory diseases. a febrile systemic hemorrhagic syndrome with high mortality (up to 50%) has been reported in cats infected with virulent strains of fcv. feline chlamydiosis. feline chlamydiosis is a persistent respiratory infection of cats caused by chlamydophila felis. infection results in a conjunctivitis (similar to the conjunctivitis seen in human trachoma caused by chlamydia trachomatis) and serous or mucopurulent rhinitis. in the past, chlamydophila felis was incriminated as the agent responsible for "feline pneumonitis," but its role in causing bronchointerstitial pneumonia in cats has been seriously challenged in recent years (see pneumonias of cats). mycotic infections. the most common mycotic infection in the feline nasal cavity is caused by cryptococcus neoformans and cryptococcus gatti, but not all animals exposed to these fungi necessarily develop cryptococcosis unless they are immunosuppressed. stillbirths. clinical signs of fvr infection are characterized by lethargy, oculonasal discharge, severe rhinitis, and conjunctivitis. feline calicivirus. feline rhinitis can be caused by different strains of feline calicivirus (fcv). it is an important infection of the respiratory tract of cats, and depending on the virulence of the strain, lesions vary from a mild oculonasal discharge to severe rhinitis, mucopurulent conjunctivitis, and ulcerative gingivitis and stomatitis. the lesions, in addition to rhinitis and conjunctivitis, include acute, diffuse interstitial pneumonia with necrotizing bronchiolitis (see pneumonias of cats) and in some cases prominent ulcers of the tongue and hard palate. primary viral lesions are generally transient, but secondary bacterial infections (bordetella bronchiseptica, pasteurella multocida, or escherichia coli) are a common complication. some kittens develop lameness after infection or vaccination with calicivirus because of an acute and self-limiting arthritis ("limping kitten syndrome"). carrier state and virus shedding from oronasal secretions and feces are natural sequelae after recovery from the acute phase of the disease. clinical and pathologic features of fcv disease are strikingly similar but not identical to those of hemorrhage, increased lacrimation as a result of obstruction of nasolacrimal ducts, and sneezing. in some instances, it is not possible to clinically or grossly differentiate neoplasms from hyperplastic nodules or granulomatous rhinitis. some neoplasms may infiltrate adjacent bone structures and produce notable facial deformities, loss of teeth, exophthalmus, and nervous signs. large neoplasms also project into the meatuses, narrow the lumen, and interfere with airflow, causing stertorous breathing (see . biopsies, as well as brush and imprint cytology, have proven effective in the antemortem diagnosis of nasal neoplasms, particularly in those of epithelial lineage. a unique group of nasal carcinomas (enzootic nasal tumors, enzootic intranasal tumors, and enzootic nasal carcinoma) of sheep and goats arise from the surface epithelium and glands of the ethmoidal conchae. these types of carcinomas are caused by betaretroviruses in sheep (entv-1) and goats (entv-2). the enzootic nasal tumor has been successfully transmitted to susceptible animals by the lesions vary from discrete nasal granulomas to large confluent masses of mucopurulent exudate filling the entire nasal cavity and paranasal sinuses. microscopic examination of the exudate reveals the typical thick-walled pas-positive organisms (see fig. 9 -35). mycoplasma felis can also cause mucopurulent conjunctivitis and a mild upper respiratory infection, with clinical signs and lesions overlapping those seen with chlamydiosis, fvr, and fcr infections. respiratory infections and bronchopneumonia in cats may also be associated with the immunosuppressive effects of feline retroviruses such as feline leukemia virus (felv) and feline immunodeficiency virus (fiv). nasal aspergillosis and allergic rhinosinusitis are sporadically reported in cats (see disorders of the conducting system: species-specific diseases of the nasal cavity and paranasal sinuses: disorders of dogs: mycotic infections). neoplasms of the nasal cavity and paranasal sinuses may arise from any of the tissues forming these structures, including bone (osteoma or osteosarcoma), cartilage (chondroma or chondrosarcoma), connective tissue (fibroma or fibrosarcoma, myxoma or myxosarcoma), and blood vessels (hemangioma or hemangiosarcoma), and from all the different types of cells of glands and lining epithelium (adenoma, carcinoma, or adenocarcinoma). nasal tumors originating from stromal tissues, such as bone, cartilage, and connective tissue, are morphologically indistinguishable from those seen in other sites. in general, nasal neoplasms are rare in domestic animals, except for enzootic ethmoidal tumor (retroviral) in sheep and goats, which can occur in several animals in a herd (see the next section). in companion animals, nasal neoplasms are most common in dogs, particularly in medium to large breed dogs such as the collie, airedale terrier, basset hound, and german shepherd. the cat and the horse are less frequently affected. the main sites in order of frequency are the nasal passages and sinuses for dogs, the tip of the nose and nasal passages for cats, and the maxillary sinus and nasal passages for horses. the majority of neoplasms in the nasal cavity are malignant. benign nasal neoplasms (papilloma and adenoma) are rare and generally are either solitary or multiple, well-delineated nodules. in contrast, nasal carcinomas and nasal sarcomas are generally larger but vary in size and are often pale and multilobulated masses composed of fleshy to friable tissue (figs. 9-36 and 9-37). malignant neoplasms are locally invasive and tend to infiltrate sinuses, meninges, frontal brain, olfactory nerves, and vessels resulting in epistaxis. carcinomas vary from anaplastic (poorly differentiated) to well differentiated, in which cell and tissue morphology retains some glandular (adenocarcinoma) or squamous cell patterns. because nasal tumors in dogs and cats are usually large and invasive at the time of diagnosis, prognosis is usually poor and survival times are short. sarcomas originating in the nasal cavity and paranasal sinuses are less common than carcinomas. mesenchymal tumors can arise from bone (osteoma or osteosarcoma), cartilage (chondroma or chondrosarcoma), blood vessels (hemangioma or hemangiosarcoma), and connective tissue (fibroma or fibrosarcoma). overall, benign epithelial and mesenchymal tumors are less common than their malignant counterparts. secondary tumors in the nasal cavity are rare, with lymphoma being the most common secondary tumor in the nasal cavity of domestic animals ( fig. 9-38 ). nasal neoplasms become secondarily infected by bacteria, and clinical signs often overlap with those of infectious rhinitis and include catarrhal or mucopurulent nasal discharge, periodic anomalies 3 congenital anomalies of the pharynx, guttural pouches, larynx, and trachea are rare in all species. depending on their location and severity, they may be inconsistent with postnatal life, pose little or no problem, interfere with quality of life, or manifest themselves in later life. if clinical signs of respiratory distress, such as stridor, coughing, dyspnea, or gagging, do occur, they are usually exacerbated by excitement, heat, stress, or exercise. brachycephalic airway syndrome. see disorders of the conducting system: species-specific diseases of the pharynx, guttural inoculation of cell-free tumor filtrates. enzootic nasal tumors are typically invasive but do not metastasize ( fig. 9 -39). in some regions of the world, ethmoid tumors have been reported in horses and pigs, particularly in those farms where the endemic nasal tumors of ruminants are known to occur. nonneoplastic exophytic masses that resemble neoplasms are commonly found in horses, cats, and, to a lesser extent, other species. in horses, polyps tend to form in the ethmoidal region, whereas in cats, polyps are most frequently found in the nasopharynx and eustachian tubes. the pathogenesis of these benign growths is uncertain, although in many cases they follow chronic rhinitis or sinusitis. most recently, lymphatic obstruction secondary to inflammation has been postulated as the main culprit. grossly, polyps appear as firm, pedunculated nodules of various sizes protruding from the nasal mucosa into the nasal passages or nasopharynx ( fig. 9 -40); the surface may be smooth, ulcerated, secondarily infected, and hemorrhagic. microscopically, polyps are characterized by a core of wellvascularized stromal tissue that contains inflammatory cells and are covered by pseudostratified or squamous epithelium (see fig. 9 -40). nasal and paranasal sinus cysts are common idiopathic lesions in horses and are medically important because they clinically mimic table 1 -1 for potential, suspected, or known genetic disorders. tracheal collapse and tracheal stenosis. tracheal collapse with reduction in tracheal patency occurs in toy, miniature, and brachycephalic breeds of dogs, in which the condition is also called tracheobronchial collapse or central airway collapse. the defect also occurs in horses, cattle, and goats. by radiographic, endoscopic, or gross examination, there is dorsoventral flattening of the trachea with concomitant widening of the dorsal tracheal membrane, which may then prolapse ventrally into the lumen ( fig. 9-41 ). most commonly, the defect extends the entire length of the trachea and only rarely affects the cervical portion alone. affected segments with a reduced lumen contain froth and even are covered by a diphtheritic membrane. in horses, the so-called scabbard trachea is characterized allergic reactions. grossly, the mucosa of the epiglottis and vocal cords is thickened and swollen, often protrudes dorsally onto the epiglottic orifice, and has a gelatinous appearance ( fig. 9-43 ). laryngeal and tracheal hemorrhage. hemorrhages in these sites occur as mucosal petechiae and are most commonly seen in coagulopathies; inflammation; septicemia and sepsis, particularly in pigs with classical swine fever (hog cholera); african swine fever or salmonellosis; and horses with equine infectious anemia. severe dyspnea and asphyxia before death can cause congestion, ecchymosis, and petechiae in the laryngeal and tracheal mucosa; this lesion must be differentiated from postmortem imbibition of hemoglobin in autolyzed carcasses (see chapter 1). by lateral flattening so that the tracheal lumen is reduced to a narrow vertical slit. segmental tracheal collapse causing stenosis has been associated with congenital and acquired abnormalities. in severe cases, abnormal cartilaginous glycoproteins and loss of elasticity of tracheal rings causes the trachea to collapse. in some other cases, it is an acquired tracheal lesion that follows trauma, compression caused by extraluminal masses, peritracheal inflammation, and flawed tracheotomy or transtracheal aspirate techniques. other tracheal anomalies include tracheoesophageal fistula, which is most commonly found in human beings and sporadically in dogs and cattle. congenital fistulas can occur at any site of the cervical or thoracic segments of the trachea. acquired tracheoesophageal fistula can be a complication of improper intubation, tracheotomy, or esophageal foreign body. laryngeal hemiplegia. laryngeal hemiplegia (paralysis), sometimes called roaring in horses, is a common but obscure disease characterized by atrophy of the dorsal and lateral cricoarytenoid muscles (abductor and adductor of the arytenoid cartilage), particularly on the left side. muscular atrophy is most commonly caused by a primary denervation (recurrent laryngeal neuropathy) of unknown cause (idiopathic axonopathy) and, to a much lesser extent, secondary nerve damage (see the section on denervation atrophy in chapters 14 and 15). idiopathic laryngeal hemiplegia is an incurable axonal disease (axonopathy) of the cranial laryngeal nerve that affects mostly larger horses. secondary laryngeal hemiplegia is rare and occurs after nerve damage caused by other pathologic processes such as compression or inflammation of the left recurrent laryngeal nerve. the medial retropharyngeal lymph nodes are located immediately ventral to the floor of the guttural pouches. as a result of this close anatomic relationship, swelling or inflammation of the guttural pouches or retropharyngeal lymph nodes often results in secondary damage to the laryngeal nerve. common causes of secondary nerve damage (wallerian degeneration) include guttural pouch mycosis, retropharyngeal abscesses, inflammation because of iatrogenic injection into the nerves, neck injury, and metastatic neoplasms involving the retropharyngeal lymph nodes (e.g., lymphosarcoma). grossly, the affected laryngeal muscle in a horse with laryngeal hemiplegia is pale and smaller than normal (muscle atrophy) . microscopically, muscle fibers have lesions of denervation atrophy (see chapters 14 and 15). atrophy of laryngeal muscles also occurs in dogs as an inherited condition (siberian husky and bouvier des flanders), as a degenerative neuropathy in older dogs, secondary to laryngeal trauma in all species (e.g., choke chain damage), or secondary to hepatic encephalopathy in horses. the abnormal inspiratory sounds (roaring) during exercise in horses with laryngeal hemiplegia are caused by paralysis of the left dorsal and lateral cricoarytenoid muscles, which cause incomplete dilation of the larynx, obstruction of airflow, and vibration of vocal cords. laryngeal edema. laryngeal edema is a common feature of acute inflammation, but it is particularly important because swelling of the epiglottis and vocal cords can obstruct the laryngeal orifice, resulting in asphyxiation. laryngeal edema occurs in pigs with edema disease; in horses with purpura hemorrhagica; in cattle with acute interstitial pneumonia; in cats with systemic anaphylaxis; and in all species as a result of trauma, improper endotracheal tubing, inhalation of irritant gases (e.g., smoke), local inflammation, and animal species is classified as fibrinous, catarrhal, purulent, or granulomatous (figs. 9-44 and 9-45). chronic polypoid tracheitis occurs in dogs and cats, probably secondary to chronic infection. the most common causes of tracheitis are viral infections, such as those causing infectious bovine rhinotracheitis (see fig. 9 -29), equine viral rhinopneumonitis, canine distemper, and feline rhinotracheitis. viral lesions are generally mild and transient but often become complicated with secondary bacterial infections. at the early stages, the mucosa is notably hyperemic and can show white foci of necrosis. in the most severe cases, the affected mucosa detaches from the underlying basement membrane, causing extensive tracheal ulceration. chemical tracheitis is also commonly seen after aspiration (see fig. 9 -45). also, inhalation of fumes during barn fires can cause extensive injury and necrosis of the tracheal mucosa. in forensic cases, the presence of carbon pigment in the mucosal surface of trachea, bronchi, and bronchioles indicates that the burned animal was alive during the fire. parasitic infections of the larynx and trachea can cause obstruction with dramatic consequences, but burdens sufficient to cause such effects are not commonly seen in veterinary practice. besnoitiosis (besnoitia spp.) . besnoitiosis (besnoitia spp.) is caused by several species of this apicomplexan coccidian parasite, whose life cycle is still unknown. this parasite can cause pedunculated lesions on the skin, sclera, mucosa of the nasal cavity, and larynx of horses and donkeys, cattle, goats, and wild animals. besnoitiosis has been reported from africa, central and south america, north america, and europe. grossly, pale, round, exophytic nodules up to 2 cm in diameter can be observed protruding from mucosal surfaces. microscopically, these nodules consist of finger-like projections covered by hyperplastic and sometimes ulcerated epithelium containing numerous thick-walled parasitic cysts with little inflammatory response. cattle. see disorders of cattle. inflammation of the pharynx, larynx, and trachea are important because of their potential to obstruct airflow and to lead to aspiration pneumonia. the pharynx is commonly affected by infectious diseases of the upper respiratory and upper digestive tracts, and the trachea can be involved by extension from both the lungs and larynx. pharyngeal obstruction and perforation. intraluminal foreign bodies in the pharynx, such as medicament boluses, apples, or potatoes, can move down and obstruct the larynx and trachea. also, pharyngeal obstruction can be caused by masses in the surrounding tissue, such as neoplasms of the thyroid gland, thymus, and parathyroid glands. a number of nonspecific insults can cause lesions and clinical signs. trauma may take the form of penetrating wounds in any species: perforation of the caudodorsal wall of the pharynx from the improper use of drenching or balling guns in sheep, cattle, and pigs; choking injury because of the use of collars in dogs and cats; and the shearing forces of bite wounds. the results of the trauma may be minimal (local edema and inflammation) or as serious as complete luminal obstruction by exudate. foreign bodies may be lodged anywhere in the pharyngeal region; the location and size determine the occurrence of dysphagia, regurgitation, dyspnea, or asphyxiation. pigs have a unique structure known as the pharyngeal diverticulum (4 cm long in adult pigs), which is located in the pharyngeal wall rostral and dorsal to the esophageal entrance. it is important because barley awns may lodge in the diverticulum, causing an inflammatory swelling that affects swallowing. the diverticular wall may be perforated by awns or drenching syringes, which results in an exudate that can extend down the tissue planes between muscles of the neck and even into the mediastinum. the pharynx of the dog may also be damaged by trauma from chicken bones, sticks, and needles, resulting in the formation of a pharyngeal abscess. equine inflammation of the trachea (tracheitis). the types of injury and host inflammatory responses in the trachea are essentially the same as those described for the nasal mucosa. although tracheal mucosa is prone to aerogenous injury and necrosis, it has a remarkable capacity for repair. according to the exudate, tracheitis in all palate, are important causes of respiratory problems and reduced athletic performance in horses. an undersized epiglottis is prone to being entrapped below the arytenoepiglottic fold, causing an equine syndrome known as epiglottic entrapment. this syndrome also occurs in horses with lateral deviation and deformity of epiglottis, epiglottic cysts, or necrosis of the tip of the epiglottis. hypoplastic epiglottis also occurs in pigs. dorsal displacement of the soft palate, particularly during exercise, narrows the lumen of the nasopharynx and creates abnormal air turbulence in the conducting system of horses. epiglottic entrapment is clinically characterized by airway obstruction, exercise intolerance, respiratory noise, and cough. subepiglottic and pharyngeal cysts. anomalous lesions, such as subepiglottic and pharyngeal cysts, are occasionally seen in horses, particularly in standardbred and thoroughbred racehorses. these cysts vary in size (1 to 9 cm) and occur most commonly in the subepiglottic area and to a lesser extent in the dorsal pharynx, larynx, and soft palate. cysts are lined by squamous or pseudostratified epithelium and contain thick mucus. large cysts cause airway obstruction, reduced exercise tolerance, or dysphagia and predispose to bronchoaspiration of food. equine pharyngeal lymphoid hyperplasia. equine pharyngeal lymphoid hyperplasia, or pharyngitis with lymphoid follicular hyperplasia, is a common cause of partial upper airway obstruction in horses, particularly in 2-and 3-year-old racehorses. lymphoid hyperplasia is also seen in healthy horses as part of a response to mild chronic pharyngitis, which in many instances tends to regress with age in older animals. the cause is undetermined, but chronic bacterial infection combined with environmental factors may cause excessive antigenic stimulation and lymphoid hyperplasia. the gross lesions, visible endoscopically or at necropsy, consist of variably sized (1 to 5 mm) white foci located on the dorsolateral walls of the pharynx and extending into the openings of the guttural pouches and onto the soft palate. in severe cases, lesions may appear as pharyngeal polyps. microscopically, the lesions consist of large aggregates of lymphocytes and plasma cells in the pharyngeal mucosa. clinical signs consist of stertorous inspiration, expiration, or both. inflammation of guttural pouches. the guttural pouches of horses are large diverticula (300 to 500 ml) of the ventral portion of the auditory (eustachian) tubes. these diverticula are therefore exposed to the same pathogens as the pharynx and have drainage problems similar to the sinuses. although it is probable that various pathogens, including viruses, can infect them, the most common pathogens are fungi, which cause guttural pouch mycosis and guttural pouch empyema in the horse. eustachitis is the term used for inflammatory processes involving the eustachian (pharyngotympanic) tube. because of the close anatomic proximity of guttural pouches to the internal carotid arteries, cranial nerves (vii, ix, x, xi, and xii), and atlantooccipital joint, disease of these diverticula may involve these structures and cause a variety of clinical signs in horses. guttural pouch mycosis occurs primarily in stabled horses and is caused by aspergillus fumigatus and other aspergillus spp. infection is usually unilateral and presumably starts with the inhalation of spores from moldy hay. grossly, the mucosal surfaces of the dorsal and lateral walls of the guttural pouch mucosa are first covered by focal, rounded, raised plaques of diphtheritic (fibrinonecrotic) exudate, which with time can become confluent and grow into a large fibrinonecrotic mass ( fig. 9-46) . microscopically, the lesions are severe necrotic inflammation of the mucosa and submucosa with widespread vasculitis and intralesional fungal hyphae. necrosis of the mammomonogamus (syngamus) spp. mammomonogamus (syngamus) laryngeus is a nematode that is seen attached to the laryngeal mucosa of cattle in tropical asia and south america, and cats (gapeworm: mammomonogamus ierei) in the caribbean and southern united states. occasionally, human beings with a persistent cough or asthma-like symptoms have the parasite in the larynx or bronchi. oslerus (filaroides) osleri. see disorders of dogs. b a c empyema of guttural pouches is a sequela to suppurative inflammation of the nasal cavities, most commonly from streptococcus equi infection (strangles). in severe cases, the entire guttural pouch can be filled with purulent exudate ( fig. 9 -47). the sequelae are similar to those of guttural pouch mycosis except that there is no erosion of the internal carotid artery. it is clinically characterized by nasal discharge, enlarged retropharyngeal lymph nodes, painful swelling of the parotid region, dysphagia, and respiratory distress. guttural pouch tympany develops sporadically in young horses when excessive air accumulates in the pouch from the one-way valve effect caused by inflammation or malformation of the eustachian tube. arabian and german warm-blooded horses are particularly susceptible to develop guttural pouch tympany. it is generally unilateral and characterized by nonpainful swelling of the parotid region. cattle. tracheal edema and hemorrhage syndrome of feeder cattle, also known as the honker syndrome or tracheal stenosis of feedlot cattle, is a poorly documented acute disease of unknown cause, most often seen during the summer months. severe edema and a few hemorrhages are present in the mucosa and submucosa of the dorsal surface of the trachea, extending caudally from the midcervical area as far as the tracheal bifurcation. on section, the tracheal mucosa is diffusely thickened and gelatinous. clinical signs include inspiratory wall of the guttural pouches can extend into the wall of the adjacent internal carotid artery causing hemorrhage into the lumen of the guttural pouch and recurrent epistaxis. invasion of the internal carotid artery causes arteritis, which can also lead to formation of an aneurysm and fatal bleeding into the guttural pouches. in other cases, the fungi may be angioinvasive, leading to the release of mycotic emboli into the internal carotid artery, generally resulting in multiple cerebral infarcts. dysphagia, another clinical sign seen in guttural pouch mycosis, is associated with damage to the pharyngeal branches of the vagus and glossopharyngeal nerves, which lie on the ventral aspect of the pouches. horner's syndrome results from damage to the cranial cervical ganglion and sympathetic fibers located in the caudodorsal aspect of the pouches. finally, equine laryngeal paralysis (hemiplegia) can result from damage to the laryngeal nerves as previously described in the section on laryngeal hemiplegia. pekingese, and others. the defects are a result of a mismatch of the ratio of soft tissue to cranial bone and the obstruction of airflow by excessive length of the palatine soft tissue. secondary changes, such as nasal and laryngeal edema caused by forceful inspiration, eventually lead to severe upper airway obstruction, respiratory distress, and exercise intolerance. tracheal hypoplasia. tracheal hypoplasia occurs most often in english bulldogs and boston terriers; the tracheal lumen is decreased in diameter throughout its length. canine infectious respiratory disease. canine infectious respiratory disease (cird), formerly called canine tracheobronchitis or kennel cough, is a highly contagious group of infectious diseases characterized clinically by an acute onset of coughing notably exacerbated by exercise. the term is nonspecific, much like the dyspnea that can progress to oral breathing, recumbency, and death by asphyxiation in less than 24 hours. necrotic laryngitis. necrotic laryngitis (calf diphtheria, laryngeal necrobacillosis) is a common disease of feedlot cattle and cattle affected with other diseases, with nutritional deficiencies, or housed under unsanitary conditions. it also occurs sporadically in sheep and pigs. necrotic laryngitis, caused by fusobacterium necrophorum, is part of the syndrome termed necrotic stomatitis or laryngeal necrobacillosis, which can include lesions of the tongue, cheeks, palate, and pharynx. an opportunistic pathogen, fusobacterium necrophorum produces potent exotoxins and endotoxins after gaining entry either through lesions of viral infections, such as ibr and vesicular stomatitis in cattle, or after traumatic injury produced by feed or careless use of specula or balling guns. the gross lesions, regardless of location in the mouth or larynx (most common in the mucosa overlying the laryngeal cartilages), consist of well-demarcated, dry, yellow-gray, thick-crusted, and fibrinonecrotic exudate ( fig. 9 -48) that in the early stages is bounded by a zone of active hyperemia. deep ulceration develops, and if the lesion does not result in death, healing is by granulation tissue formation. microscopically, the necrotic foci are first surrounded by congested borders, then by a band of leukocytes, and finally the ulcers heal by granulation tissue and collagen (fibrosis). the lesions can extend deep into the submucosal tissue. numerous bacteria are evident at the advancing edge. there are numerous and important sequelae to calf diphtheria; the most serious is death from severe toxemia or overwhelming fusobacteremia. sometimes, the exudate may be copious enough to cause laryngeal obstruction and asphyxiation or be aspirated and cause bronchopneumonia. the clinical signs of necrotic laryngitis are fever, anorexia, depression, halitosis, moist painful cough, dysphagia, and inspiratory dyspnea and ventilatory failure because of fatigue of the respiratory muscles (diaphragmatic and intercostal). laryngeal contact ulcers. ulcerative lesions in the larynx are commonly found in feedlot cattle. grossly, the laryngeal mucosa reveals circular ulcers (up to 1 cm in diameter), which may be unilateral or bilateral and sometimes deep enough to expose the underlying arytenoid cartilages. the cause has not been established, but causal agents, such as viral, bacterial, and traumatic, have been proposed, along with increased frequency and rate of closure of the larynx (excessive swallowing and vocalization) when cattle are exposed to market and feedlot stresses such as dust, pathogens, and interruption of feeding. contact ulcers predispose a calf to diphtheria (fusobacterium necrophorum) and laryngeal papillomas. ulceration of the mucosa and necrosis of the laryngeal cartilages have also been described in calves, sheep, and horses under the term laryngeal chondritis. laryngeal abscesses involving the mucosa and underlying cartilage occur as a herd or flock problem in calves and sheep, presumably caused by a secondary infection with trueperella (arcanobacterium) pyogenes. anomalies 5 brachycephalic airway syndrome. brachycephalic airway syndrome is a clinical term that refers to increased airflow resistance caused by stenotic nostrils and nasal meatuses and an excessively long soft palate. these abnormalities are present in brachycephalic canine breeds such as bulldogs, boxers, boston terriers, pugs, a "common cold" in human beings or bovine respiratory disease complex (brdc) in cattle. the infection occurs commonly as a result of mixing dogs from different origins such as occurs at commercial kennels, animal shelters, and veterinary clinics. between bouts of coughing, most animals appear normal, although some have rhinitis, pharyngitis, tonsillitis, or conjunctivitis; some with secondary pneumonia become quite ill. the pathogenesis of cird is complex, and many pathogens and environmental factors have been incriminated. bordetella bronchiseptica, canine adenovirus-2 (cav-2), and canine parainfluenza virus-2 (cpiv-2) are most commonly implicated. the severity of the disease is increased when more than one agent is involved or if there are extreme environmental conditions (e.g., poor ventilation). for example, dogs asymptomatically infected with bordetella bronchiseptica are more severely affected by superinfection with cav-2 than those not carrying the bacterium. other agents are sometimes isolated but of lesser significance and include canine adenovirus-1 (cav-1: infectious canine hepatitis virus), reovirus type 1, canid herpesvirus-1 (cahv-1), canine respiratory coronavirus (crcov), and mycoplasma species. depending on the agents involved, gross and microscopic lesions are completely absent or they vary from catarrhal to mucopurulent tracheobronchitis, with enlargement of the tonsils and retropharyngeal and tracheobronchial lymph nodes. in dogs with bordetella bronchiseptica infection, the lesions are suppurative or mucopurulent rhinitis and tracheobronchitis, and suppurative bronchiolitis. in contrast, when lesions are purely viral, microscopic changes are focal necrosis of the tracheobronchial epithelium. sequelae can include spread either proximally or distally in the respiratory tract, the latter sometimes inducing chronic bronchitis and bronchopneumonia. oslerus (filaroides) osleri. oslerus (filaroides) osleri is a nematode parasite of dogs and other canidae that causes characteristic protruding nodules into the lumen at the tracheal bifurcation. they are readily seen on endoscopic examination or at necropsy. in severe cases, these nodules can extend 5 cm cranially or caudally from the tracheal bifurcation and even into primary and secondary bronchi. the disease occurs worldwide, and oslerus osleri is considered the most common respiratory nematode of dogs. the gross lesions are variably sized, up to 1 cm, submucosal nodules that extend up to 1 cm into the tracheal lumen ( fig. 9 -49, a). microscopically, nodules contain adult parasites with a mild mononuclear cell reaction; with the death of the parasite, an intense foreign body reaction develops with neutrophils and giant cells b) . clinically, it can be asymptomatic, although it most often causes a chronic cough that can be exacerbated by exercise or excitement. severe infestations can result in dyspnea, exercise intolerance, cyanosis, emaciation, and even death in young dogs. neoplasms of the guttural pouches occur rarely in horses and are usually squamous cell carcinomas. laryngeal neoplasms are rare in dogs and extremely so in other species, although they have been reported in cats and horses. the most common laryngeal neoplasms in dogs are papillomas and squamous cell carcinomas. other less common tumors are laryngeal rhabdomyoma, previously referred to as laryngeal oncocytoma, and chondromas and osteochondromas. lymphoma involving the laryngeal tissue is sporadically seen in cats. when large enough to be obstructive, neoplasms may cause a change or loss of voice, cough, or respiratory distress with cyanosis, collapse, and syncope. other signs include dysphagia, anorexia, and exercise intolerance. the neoplasm is sometimes visible from the oral cavity and causes swelling of the neck. the prognosis is poor because most lesions recur after excision. tracheal neoplasms are even more uncommon than those of the larynx. the tracheal cartilage or mucosa can be the site of an osteochondroma, leiomyoma, osteosarcoma, mast cell tumor, and carcinoma. lymphoma in cats can extend from the mediastinum to involve the trachea. each lung is subdivided into various numbers of pulmonary lobes (see fig. 9 -16). in the past, these were defined by anatomic fissures. however, in current anatomy, lobes are defined by the ramification of the bronchial tree. following this criterion, the left lung of all domestic species is composed of cranial and caudal lobes, whereas the right lung, depending on species, is composed of cranial, middle (absent in horse), caudal, and accessory lobes. each pulmonary lobe is further subdivided by connective tissue into pulmonary lobules, which in some species (cattle and pigs) are rather prominent and in others are much less conspicuous. from a practical standpoint, identification of the lungs among different species could be achieved by carefully observing the degree of lobation (external fissures) and the degree of lobulation (connective tissue between lobules). cattle and pigs have well-lobated and well-lobulated lungs; sheep and goats have well-lobated but poorly lobulated lungs; horses have both poorly lobated and poorly lobulated lungs and resemble human lungs; finally, dogs and cats have well-lobated but not well-lobulated lungs. the degree of lobulation determines the degree of air movement between the lobules. in pigs and cattle, movement of air between lobules is practically absent because of the thick connective tissue of the interlobular septa separating individual lobules. this movement of air between lobules and between adjacent alveoli (via the pores of kohn) constitutes what is referred to as collateral ventilation. this collateral ventilation is poor in cattle and pigs and good in dogs. the functional implications of collateral ventilation are discussed in the section on pulmonary emphysema. the lungs have an interconnecting network of interstitial stromal tissue supporting the blood and lymphatic vessels, nerves, bronchi, bronchioles, and alveoli. for purposes of simplicity, the pulmonary interstitium can be anatomically divided into three contiguous compartments: (1) bronchovascular interstitium, where main bronchi and pulmonary vessels are situated; (2) interlobular interstitium separating pulmonary lobules and supporting small blood and lymph vessels; and (3) alveolar interstitium supporting the alveolar walls that contain pulmonary capillaries and alveolar epithelial cells (no lymphatic vessels here) (see discussion on the blood-air barrier in the section on alveoli). pulmonary changes, such as edema, emphysema, and inflammation, may affect one or more of these interstitial compartments. anomalies 6 congenital anomalies of the lungs are rare in all species but are most commonly reported in cattle and sheep. compatibility with life largely depends on the type of structures involved and the proportion of functional tissue present at birth. accessory lungs are one of the most common anomalies and consist of distinctively lobulated masses of incompletely differentiated pulmonary tissue present in the thorax, abdominal cavity, or subcutaneous tissue virtually anywhere in the trunk. large accessory lungs can cause dystocia. ciliary dyskinesia (immotile cilia syndrome, kartagener's syndrome) is characterized by defective ciliary movement, which results in reduced mucociliary clearance because of a defect in the microtubules of all ciliated cells and, most important, in the ciliated respiratory epithelium and spermatozoa. primary ciliary dyskinesia often associated with situs inversus has been reported in dogs, which as a result usually have chronic recurrent rhinosinusitis, pneumonia, and infertility. pulmonary agenesis, pulmonary hypoplasia, abnormal lobulation, congenital emphysema, lung hamartoma, and congenital bronchiectasis are occasionally seen in domestic animals. congenital melanosis is a common incidental finding in pigs and ruminants and is usually seen at slaughter (fig. 9-50 ). it is characterized by black spots, often a few centimeters in diameter, in various organs, mainly the lungs, meninges, intima of the aorta, and caruncles of the uterus. melanosis has no clinical significance, and the texture of pigmented lungs remains unchanged. congenital emphysema is sporadically seen in dogs (efig. 9 -4). pulmonary calcification ("calcinosis"). calcification of the lungs occurs in some hypercalcemic states, generally secondary to hypervitaminosis d or from ingestion of toxic (hypercalcemic) plants, such as solanum malacoxylon (manchester wasting disease), that contain vitamin d analogs. it is also a common sequela to uremia and hyperadrenocorticism in dogs and to pulmonary necrosis (dystrophic calcification) in most species. calcified lungs may fail to collapse when the thoracic cavity is opened and have a characteristic "gritty" texture ( fig. 9-51) . microscopically, lesions vary from calcification of the alveolar basement membranes (see by pathologists. recent investigations suggest that excessive lipid originates from the breakdown products of neoplastic cells. bronchial and bronchiolar obstructions such as those caused by lungworms can also cause alveolar lipidosis. the pathogenesis relates to the inability of alveolar macrophages that normally remove part of the surfactant lipids to exit the lung via the mucociliary escalator. exogenous lipid pneumonia. another form of lipid pneumonia occurs accidentally in cats or horses given mineral oil by their owners in an attempt to remove hairballs or treat colic (aspiration pneumonia). to achieve gaseous exchange, a balanced ratio of the volumes of air to capillary blood must be present in the lungs (ventilation/perfusion ratio), and the air and capillary blood must be in close proximity across the alveolar wall. a ventilation-perfusion mismatch occurs if pulmonary tissue is either collapsed (atelectasis) or overinflated (hyperinflation and emphysema). the term atelectasis means incomplete distention of alveoli and is used to describe lungs that have failed to expand with air at the time of birth (congenital or neonatal atelectasis) or lungs that have collapsed after inflation has taken place (acquired atelectasis or alveolar collapse) (figs. 9-52 and 9-53). during fetal life, lungs are not fully distended, contain no air, and are partially filled with a locally produced fluid known as fetal lung fluid. not surprisingly, lungs of aborted and stillborn fetuses sink when placed in water, whereas those from animals that have breathed float. at the time of birth, fetal lung fluid is rapidly reabsorbed and replaced by inspired air, leading to the normal distention of alveoli. congenital atelectasis occurs in newborns that fail to inflate their lungs after taking their first few breaths of air; it is caused by obstruction of airways, often as a result of aspiration of amniotic fluid and meconium (described in the section on meconium aspiration syndrome) (see fig. 9 -52). congenital atelectasis also develops when alveoli cannot remain distended after initial aeration because of an alteration in quality and quantity of pulmonary surfactant produced by type ii pneumonocytes and club (clara) cells. this infant form of congenital atelectasis is referred to in human neonatology as infant respiratory distress syndrome (irds) or as hyaline membrane disease because of the clinical and microscopic features of the disease. it commonly occurs in babies who are premature or born to diabetic or alcoholic mothers and is occasionally found in animals, particularly foals and piglets. the pathetic, gasping attempts of affected foals and pigs to breathe have prompted the use of the name "barkers"; foals that survive may have brain damage from cerebral hypoxia (see chapter 14) and are referred to as "wanderers" due to their aimless behavior and lack of a normal sense of fear. acquired atelectasis is much more common and occurs in two main forms: compressive and obstructive (see fig. 9 -53). compressive atelectasis has two main causes: space-occupying masses in the pleural cavity, such as abscesses and tumors, or transferred pressures, such as that caused by bloat, hydrothorax, hemothorax, chylothorax, and empyema ( fig. 9-54 ). another form of compressive atelectasis occurs when the negative pressure in the thoracic cavity is lost because of pneumothorax. this form generally has massive atelectasis and thus is also referred to as lung collapse. obstructive (absorption) atelectasis occurs when there is a reduction in the diameter of the airways caused by mucosal edema and inflammation, or when the lumen of the airway is blocked by fig. 9 -51) to heterotopic ossification of the lungs (efig. 9 -5). in most cases, pulmonary calcification in itself has little clinical significance, although its cause (e.g., uremia or vitamin d toxicosis) may be very important. alveolar filling disorders are a heterogeneous group of lung diseases characterized by accumulation of various chemical compounds in the alveolar lumens. the most common are alveolar proteinosis, in which the alveoli are filled with finely granular eosinophilic material; pulmonary lipidosis, in which alveoli are filled with macrophages containing endogenous or exogenous lipid; and alveolar microlithiasis, in which the alveoli contain numerous concentric calcified "microliths" or "calcospherites." a similar but distinct concretion is known as corpora amylacea, which is an accumulation of laminated bodies composed of cellular debris, lipids, proteins, and possibly amyloid. for most alveolar filling disorders, there is little host response, and in many cases, it is an incidental finding. most of the alveolar filling disorders originate from inherited metabolic defects in which alveolar cells (epithelial or macrophages) cannot properly metabolize or remove lipids or proteins, whereas others result from an excessive synthesis of these substances in the lung. endogenous lipid (lipoid) pneumonia. endogenous lipid pneumonia is an obscure, subclinical pulmonary disease of cats and occasionally of dogs, which is unrelated to aspiration of foreign material. although the pathogenesis is not understood, it is presumed that lipids from pulmonary surfactant and from degenerated cells accumulate within alveolar macrophages. accumulation of surfactant lipids can occur in metabolic abnormalities of alveolar macrophages or in bronchial obstruction where surfactant-laden macrophages cannot exit the lungs via the mucociliary escalator. the gross lesions are multifocal, white, firm nodules scattered throughout the lungs (efig. 9-6) . microscopically, the alveoli are filled with foamy lipid-laden macrophages accompanied by interstitial infiltration of lymphocytes and plasma cells, fibrosis, alveolar epithelialization, and, in some cases, cholesterol clefts and lipid granulomas. lipid (lipoid) pneumonia occurs frequently in the vicinity of cancerous lung lesions in human beings, cats, and dogs. the reason for this association remains unknown and frequently unrecognized appearance of atelectasis is more common in species with poor collateral ventilation, such as cattle and pigs. the extent and location of obstructive atelectasis depends largely on the size of the affected airway (large vs. small) and on the degree of obstruction (partial vs. complete). atelectasis also occurs when large animals are kept recumbent for prolonged periods, such as during anesthesia (hypostatic atelectasis). the factors contributing to hypostatic atelectasis are a combination of blood-air imbalance, shallow breathing, airway obstruction because of mucus and fluid that has not been drained from bronchioles and alveoli, and from inadequate local production of surfactant. atelectasis can also be a sequel to paralysis of respiratory muscles and prolonged use of mechanical ventilation or general anesthesia in intensive care. in general, the lungs with atelectasis appear depressed below the surface of the normally inflated lung. the color is generally dark blue, and the texture is flabby or firm; they are firm if there is concurrent edema or other processes, such as can occur in ards or "shock" lungs (see the section on pulmonary edema). distribution and extent vary with the process, being patchy (multifocal) in congenital atelectasis, lobular in the obstructive type, and of various degrees in between in the compressive type. microscopically, the alveoli are collapsed or slitlike and the alveolar walls appear parallel and close together, giving prominence to the interstitial tissue even without any superimposed inflammation. pulmonary emphysema. pulmonary emphysema, often simply referred to as emphysema, is an extremely important primary disease in human beings, whereas in animals, it is always a secondary condition resulting from a variety of pulmonary lesions. in human medicine, emphysema is strictly defined as an abnormal permanent enlargement of airspaces distal to the terminal bronchiole, accompanied by destruction of alveolar walls (alveolar emphysema). this definition separates it from simple airspace enlargement or hyperinflation, in which there is no destruction of alveolar walls and which can occur congenitally (down syndrome) or be acquired with age (aging lung, sometimes misnamed "senile emphysema"). the pathogenesis of emphysema in human beings is still controversial, but current thinking overwhelmingly suggests that destruction of mucus plugs, exudate, aspirated foreign material, or lungworms (see fig. 9 -53). when the obstruction is complete, trapped air in the lung eventually becomes reabsorbed. unlike the compression type, obstructive atelectasis often has a lobular pattern as a result of blockage of the airway supplying that lobule. this lobular lungs are extremely well-vascularized organs with a dual circulation provided by pulmonary and bronchial arteries. disturbances in pulmonary circulation have a notable effect on gaseous exchange, which may result in life-threatening hypoxemia and acidosis. in addition, circulatory disturbances in the lungs can have an impact on other organs, such as the heart and liver. for example, impeded blood flow in the lungs because of chronic pulmonary disease results in cor pulmonale, which is caused by unremitting pulmonary hypertension followed by cardiac dilation, right heart failure, chronic passive congestion of the liver (nutmeg liver), and generalized edema (anasarca). hyperemia is an active process that is part of acute inflammation, whereas congestion is the passive process resulting from decreased outflow of venous blood, as occurs in congestive heart failure ( fig. 9-56 ). in the early acute stages of pneumonia, the lungs appear notably red, and microscopically, blood vessels and alveolar capillaries are engorged with blood from alveolar walls is largely the result of an imbalance between proteases released by phagocytes and antiproteases produced in the lung as a defense mechanism (the protease-antiprotease theory). the destructive process in human beings is markedly accelerated by defects in the synthesis of antiproteases or any factor, such as cigarette smoking or pollution, that increases the recruitment of macrophages and leukocytes in the lungs. this theory originated when it was found that human beings with homozygous α 1 -antitrypsin deficiency were remarkably susceptible to emphysema and that proteases (elastase) inoculated intratracheally into the lungs of laboratory animals produced lesions similar to those found in the disease. more than 90% of the problem relates to cigarette smoking, and airway obstruction is no longer considered to play a major role in the pathogenesis of emphysema in human beings. primary emphysema does not occur in animals, and thus no animal disease should be called simply emphysema. in animals, this lesion is always secondary to obstruction of outflow of air or is agonal at slaughter. secondary pulmonary emphysema occurs frequently in animals with bronchopneumonia, in which exudate plugging bronchi and bronchioles causes an airflow imbalance where the volume of air entering exceeds the volume leaving the lung. this airflow imbalance is often promoted by the so-called one-way valve effect caused by the exudate, which allows air into the lung during inspiration but prevents movement of air out of the lung during expiration. depending on the localization in the lung, emphysema can be classified as alveolar or interstitial. alveolar emphysema characterized by distention and rupture of the alveolar walls, forming variably sized air bubbles in pulmonary parenchyma, occurs in all species. interstitial emphysema occurs mainly in cattle, presumably because of their wide interlobular septa, and lack of collateral ventilation in these species does not permit air to move freely into adjacent pulmonary lobules. as a result, accumulated air penetrates the alveolar and bronchiolar walls and forces its way into the interlobular connective tissue, causing notable distention of the interlobular septa. it is also suspected that forced respiratory movements predispose to interstitial emphysema when air at high pressure breaks into the loose connective tissue of the interlobular septa ( fig. 9-55 ). sometimes these bubbles of trapped air in alveolar or interstitial emphysema become confluent, forming large (several centimeters in diameter) pockets of air that are referred to as bullae (singular: bulla) (see efig. 9 -4); the lesion is then called bullous emphysema. this lesion is not a specific type of emphysema and does not indicate a different disease process but, rather, is a larger accumulation of air at one focus. in the most severe cases, air moves from the interlobular septa into the connective tissue surrounding the main stem bronchi and major vessels (bronchovascular bundles), and from here it leaks into the mediastinum, causing pneumomediastinum first, and eventually exits via the thoracic inlet into the cervical and thoracic subcutaneous tissue causing subcutaneous emphysema. note that mild and even moderate alveolar emphysema is difficult to judge at necropsy and by light microscopy unless special techniques are used to prevent collapse of the lung when the thorax is opened. these techniques include plugging of the trachea or intratracheal perfusion of fixative (10% neutral-buffered formalin) before the thorax is opened to prevent collapse of the lungs. important diseases that cause secondary pulmonary emphysema in animals include small airway obstruction (e.g., heaves) in horses and pulmonary edema and emphysema (fog fever) in cattle (see fig. 9 -55) and exudates in bronchopneumonia. congenital emphysema occurring secondary to bronchial cartilage hypoplasia with subsequent bronchial collapse is occasionally reported in dogs. severe and persistent cases of heart failure, the lungs fail to collapse because of edema and pulmonary fibrosis. terminal pulmonary congestion (acute) is frequently seen in animals euthanized with barbiturates and should not be mistaken for an antemortem lesion. hypostatic congestion is another form of pulmonary congestion that results from the effects of gravity and poor circulation on a highly vascularized tissue, such as the lung. this type of gravitational congestion is characterized by the increase of blood in the lower side of the lung, particularly the lower lung of animals in lateral recumbency, and is most notable in horses and cattle. the affected portions of the lung appear dark red and can have a firmer texture. in animals and human beings who have been prostrated for extended periods of time, hypostatic congestion may be followed by hypostatic edema, and hypostatic pneumonia as edema interferes locally with the bacterial defense mechanisms. pulmonary hemorrhage. pulmonary hemorrhages can occur as a result of trauma, coagulopathies, and disseminated intravascular coagulation (dic), vasculitis, sepsis, and pulmonary thromboembolism from jugular thrombosis or from embolism of exudate from a hepatic abscess that has eroded the wall and ruptured into the caudal vena cava (cattle). a gross finding often confused with intravital pulmonary hemorrhage is the result of severing both the trachea and the carotid arteries simultaneously at slaughter. blood is aspirated from the transected trachea into the lungs, forming a random pattern of irregular red foci (1 to 10 mm) in one or more lobes. these red foci are readily visible on both the pleural and the cut surfaces of the lung, and free blood is visible in the lumens of bronchi and bronchioles. rupture of a major pulmonary vessel with resulting massive hemorrhage occurs occasionally in cattle when a growing abscess in a lung invades and disrupts the wall of a major pulmonary artery or vein ( fig. 9-58 ). in most cases, animals die rapidly, often with spectacular hemoptysis, and on postmortem examination, bronchi are filled with blood (see fig. 9 -58). pulmonary edema. in normal lungs, fluid from the vascular space slowly but continuously passes into the interstitial tissue, where it is rapidly drained by the pulmonary and pleural lymphatic vessels. clearance of alveolar fluid across the alveolar epithelium is also a major mechanism of fluid removal from the lung. edema develops when the rate of fluid transudation from pulmonary vessels into the interstitium or alveoli exceeds that of lymphatic and alveolar removal ( fig. 9-59 ). pulmonary edema can be physiologically classified as cardiogenic (hydrostatic; hemodynamic) and noncardiogenic (permeability) types. hydrostatic (cardiogenic) pulmonary edema develops when there is an elevated rate of fluid transudation because of increased hydrostatic pressure in the vascular compartment or decreased osmotic pressure in the blood. once the lymph drainage has been overwhelmed, fluid accumulates in the perivascular spaces, causing distention of the bronchovascular bundles and alveolar interstitium, and eventually leaks into the alveolar spaces. causes of hemodynamic pulmonary edema include congestive heart failure (increased hydrostatic pressure); iatrogenic fluid overload; and disorders in which blood osmotic pressure is reduced, such as with hypoalbuminemia seen in some hepatic diseases, nephrotic syndrome, and protein-losing enteropathy. hemodynamic pulmonary edema also occurs when lymph drainage is impaired, generally secondary to neoplastic invasion of lymphatic vessels. permeability edema (inflammatory) occurs when there is excessive opening of endothelial gaps or damage to the cells that constitute the blood-air barrier (endothelial cells or type i pneumonocytes). hyperemia. pulmonary congestion is most frequently caused by heart failure, which results in stagnation of blood in pulmonary vessels, leading to edema and egression of erythrocytes into the alveolar spaces. as with any other foreign particle, erythrocytes in alveolar spaces are rapidly phagocytosed (erythrophagocytosis) by pulmonary alveolar macrophages. when extravasation of erythrocytes is severe, large numbers of macrophages with brown cytoplasm may accumulate in the bronchoalveolar spaces. the brown cytoplasm is the result of accumulation of considerable amounts of hemosiderin; these macrophages filled with iron pigment (siderophages) are generally referred to as heart failure cells ( fig. 9-57 ). the lungs of animals with chronic heart failure usually have a patchy red appearance with foci of brown discoloration because of accumulated hemosiderin. in this type of edema is an integral and early part of the inflammatory response, primarily because of the effect of inflammatory mediators, such as leukotrienes, platelet-activating factor (paf), cytokines, and vasoactive amines released by neutrophils, macrophages, mast cells, lymphocytes, endothelial cells, and type ii pneumonocytes. these inflammatory mediators increase the permeability of the blood-air barrier. in other cases, permeability edema results from direct damage to the endothelium or type i pneumonocytes, allowing plasma fluids to move freely from the vascular space into the alveolar lumen ( fig. 9 -60 and see fig. 9 -14). because type i pneumonocytes are highly vulnerable to some pneumotropic viruses (influenza and brsv), toxicants (nitrogen dioxide [no 2 ], sulfur dioxide [so 2 ], hydrogen sulfide [h 2 s], and 3-methylindole), and particularly to free radicals, it is not surprising that permeability edema commonly accompanies many viral or toxic pulmonary diseases. a permeability edema also occurs when endothelial cells in the lung are injured by bacterial toxins, sepsis, ards, dic, anaphylactic shock, milk allergy, paraquat toxicity, adverse drug reactions, and smoke inhalation (efig. 9-7) . the concentration of protein in edematous fluid is greater in permeability edema (exudate) than in hemodynamic edema (transudate); this difference has been used clinically in human medicine to differentiate one type of pulmonary edema from another. microscopically, because of the higher concentration of protein, edema fluid in lungs with inflammation or damage to the blood-air barrier tends to stain more intensely eosinophilic than that of the hydrostatic edema from heart failure. grossly, the edematous lungs-independent of the cause-are wet and heavy. the color varies, depending on the degree of congestion or hemorrhage, and fluid may be present in the pleural cavity. if edema is severe, the bronchi and trachea contain considerable amounts of foamy fluid, which originates from the mixing of edema fluid and air ( fig. 9-61 ). on cut surfaces, the lung parenchyma oozes fluid like a wet sponge. in cattle and pigs that have distinct lobules, the lobular pattern becomes rather accentuated because of edematous distention of lymphatic vessels in the interlobular septa and the edematous interlobular septum itself ( fig. 9-62 ). severe pulmonary edema may be impossible to differentiate from peracute pneumonia; (h&e)-stained sections (see fig. 9 -60), particularly if a fixative such as zenker's solution, which precipitates protein, is used. acute respiratory distress syndrome. acute (adult) respiratory distress syndrome (ards; shock lung) is an important condition in human beings and animals characterized by pulmonary hypertension, intravascular aggregation of neutrophils in the lungs, acute lung injury, diffuse alveolar damage, permeability edema, and formation of hyaline membranes ( fig. 9-63) . these membranes are a mixture of plasma proteins, fibrin, surfactant, and cellular debris from necrotic pneumonocytes (see fig. 9-55, b) . the pathogenesis of ards is complex and multifactorial but in general terms can be defined as diffuse alveolar damage that results from lesions in distant organs, from generalized systemic diseases, or from direct injury to the lung. sepsis, major trauma, aspiration of gastric contents, extensive burns, and pancreatitis are some of the disease entities known to trigger ards. all these conditions provoke "hyperreactive macrophages" to directly or indirectly generate overwhelming amounts of cytokines causing what is known as a "cytokine storm." the main cytokines that trigger ards are tnf-α, interleukin (il)-1, il-6, and il-8, which prime neutrophils previously recruited in the lung capillaries and alveoli to release cytotoxic enzymes and free radicals. these substances cause severe and diffuse endothelial and alveolar damage that culminates in a fulminating pulmonary edema (see fig. 9 -63). ards occurs in domestic animals and explains why pulmonary edema is one of the most common lesions found in many animals dying of sepsis, toxemia, aspiration of gastric contents, and pancreatitis, for example. a familial form of ards has been reported in dalmatians. the pulmonary lesions in this syndrome are further discussed in the sections on interstitial pneumonia and aspiration pneumonia in dogs. neurogenic pulmonary edema is another distinctive but poorly understood form of life-threatening lung edema in human beings that follows cns injury and increased intracranial pressure (i.e., head injury, brain edema, brain tumors, or cerebral hemorrhage). this type of pulmonary edema can be experimentally reproduced in laboratory animals by injecting fibrin into the fourth ventricle. it involves both hemodynamic and permeability pathways presumably from massive sympathetic stimulation and overwhelming release of catecholamines. neurogenic pulmonary edema has sporadically been reported in animals with brain injury or severe seizures or after severe stress and excitement. pulmonary embolism. with its vast vascular bed and position in the circulation, the lung acts as a safety net to catch emboli before they reach the brain and other tissues. however, this positioning is often to its own detriment. the most common pulmonary emboli in domestic animals are thromboemboli, septic (bacterial) emboli, fat emboli, and tumor cell emboli. pulmonary thromboembolism (pte) refers to both local thrombus formation and translocation of a thrombus present elsewhere in the venous circulation ( fig. 9-64 ). fragments released inevitably reach the lungs and become trapped in the pulmonary vasculature ( fig. 9-65 and see fig. 9 -64). small sterile thromboemboli are generally of little clinical or pathologic significance because they can be rapidly degraded and disposed of by the fibrinolytic system. larger thromboemboli may cause small airway constriction, reduced surfactant production, pulmonary edema, and atelectasis resulting in hypoxemia, hyperventilation, and dyspnea. parasites (e.g., dirofilaria immitis and angiostrongylus vasorum), endocrinopathies (e.g., hyperadrenocorticism and hypothyroidism), glomerulopathies, and hypercoagulable states can be responsible for pulmonary arterial thrombosis and pulmonary thromboembolism in dogs (efig. 9-8) . pieces of this fact is not surprising because pulmonary edema occurs in the very early stages of inflammation (see efig. 9-7) . careful observation of the lungs at the time of necropsy is critical because diagnosis of pulmonary edema cannot be reliably performed microscopically. this is due in part to the loss of the edema fluid from the lungs during fixation with 10% neutral-buffered formalin and in part to the fact that the fluid itself stains very poorly or not at all with eosin because of its low protein content (hemodynamic edema). a protein-rich (permeability) edema is easier to visualize microscopically because it is deeply eosinophilic in hematoxylin and eosin fig. 9-104) . 1, normal alveolar capillary externally covered by type i and type ii pneumonocytes and internally by vascular endothelium (see fig. 9 -14 for more detail). 2, at the early stages of sepsis, proinflammatory cytokines (interleukin 1 [il-1] and tumor necrosis factor [tnf]) cause circulating neutrophils to adhere to the endothelial surface. following a "cytokine storm," the marginated neutrophils further activated by inflammatory mediators suddenly release their cytoplasmic granules (proteolytic enzymes and elastases myeloperoxidase) into the surrounding milieu (arrows). 3, enzymes released by these neutrophils cause injury to type i pneumonocytes (arrows) and endothelial cells (arrowheads), disrupting the blood-air barrier and causing permeability edema (curved arrows), alveolar hemorrhage (double-headed arrow), and exocytosis of neutrophils into the alveolar space (double-headed arrow). 4, extravasated plasma proteins admixed with surfactant and cell debris form thick hyaline membranes along the alveolar wall. 5, in the unlikely event that the animal survives, the healing process starts with alveolar macrophages removing cellular debris, reabsorption of edema, and hyperplasia of type ii pneumonocytes (double-headed curved arrow) that subsequently differentiate into type i pneumonocytes (see fig. 9 b a recognized in the bovine lung after strong pneumatic stunning at slaughter (captive bolt) ( fig. 9-66, a) . although obviously not important as an antemortem pulmonary lesion, brain emboli are intriguing as a potential risk for public health control of bovine spongiform encephalopathy (bse). fragments of hair can also embolize to the lung following intravenous injections (see fig. 9-66 , b). hepatic emboli formed by circulating pieces of fragmented liver occasionally become trapped in the pulmonary vasculature after severe abdominal trauma and hepatic rupture (see fig. 9-66, c) . megakaryocytes trapped in alveolar capillaries are a common but incidental microscopic finding in the lungs of all species, particularly dogs (see fig. 9-66, d) . tumor emboli (e.g., osteosarcoma and hemangiosarcoma in dogs and uterine carcinoma in cattle) can be numerous and striking and the ultimate cause of death in malignant neoplasia. in experimental studies, cytokines released during pulmonary inflammation are chemotactic for tumor cells and promote pulmonary metastasis. pulmonary infarcts. because of a dual arterial supply to the lung, pulmonary infarction is rare and generally asymptomatic. however, pulmonary infarcts can be readily caused when pulmonary thrombosis and embolism are superimposed on an already compromised pulmonary circulation such as occurs in congestive heart c d thrombi breaking free from a jugular, femoral, or uterine vein can cause pulmonary thromboembolism. pulmonary thromboembolisms occur in heavy horses after prolonged anesthesia (deep vein thrombosis), recumbent cows ("downer cow syndrome"), or in any animal undergoing long-term intravenous catheterization in which thrombi build up in the catheter and then break off (see fig. 9 -65). septic emboli, pieces of thrombi contaminated with bacteria or fungi and broken free from infected mural or valvular thrombi in the heart and vessels, eventually become entrapped in the pulmonary circulation. pulmonary emboli originate most commonly from bacterial endocarditis (right side) and jugular thrombophlebitis in all species, hepatic abscesses that have eroded and discharged their contents into the caudal vena cava in cattle, and septic arthritis and omphalitis in farm animals (see fig. 9 -64). when present in large numbers, septic emboli may cause unexpected death because of massive pulmonary edema; survivors generally develop pulmonary arteritis and thrombosis and embolic (suppurative) pneumonia, which may lead to pulmonary abscesses. bone marrow and bone emboli can form after bone fractures or surgical interventions of bone. these are not as significant a problem in domestic animals as they are in human beings. brain emboli (i.e., pieces of brain tissue) in the pulmonary vasculature reported in severe cases of head injury in human beings have recently been pulmonary macrophages (alveolar, intravascular, and interstitial), which have an immense biologic armamentarium, are the single most important effector cell and source of cytokines for all stages of pulmonary inflammation. these all-purpose phagocytic cells modulate the recruitment and trafficking of blood-borne leukocytes in the lung through the secretion of chemokines (see etable 9 -1). before reviewing how inflammatory cells are recruited in the lungs, three significant features in pulmonary injury must be remembered: (1) leukocytes can exit the vascular system through the alveolar capillaries, unlike in other tissues, where postcapillary venules are the sites of leukocytic diapedesis (extravasation); (2) the intact lung contains within alveolar capillaries a large pool of resident leukocytes (marginated pool); and (3) additional neutrophils are sequestered within alveolar capillaries within minutes of a local or systemic inflammatory response. these three pulmonary idiosyncrasies, along with the enormous length of the capillary network in the lung, explain why recruitment and migration of leukocytes into alveolar spaces develops so rapidly. experimental studies with aerosols of endotoxin or gram-negative bacteria have shown that within minutes of exposure, there is a significant increase in capillary leukocytes, and by 4 hours the alveolar lumen is filled with neutrophils. not surprisingly, the bal fluid collected from patients with acute pneumonia contains large amounts of inflammatory mediators such as tnf-α, il-1, and il-8. also, the capillary endothelium of patients with acute pneumonia has increased "expression" of adhesion molecules, which facilitate the migration of leukocytes from capillaries into the alveolar interstitium and from there into the alveolar lumen. in allergic pulmonary diseases, eotaxin and il-5 are primarily responsible for recruitment and trafficking of eosinophils in the lung. movement of plasma proteins into the pulmonary interstitium and alveolar lumen is a common but poorly understood phenomenon in pulmonary inflammation. leakage of fibrinogen and plasma proteins into the alveolar space occurs when there is structural damage to the blood-air barrier. this leakage is also promoted by some types of cytokines that enhance procoagulant activity, whereas others reduce fibrinolytic activity. excessive exudation of fibrin into the alveoli is particularly common in ruminants and pigs. the fibrinolytic system plays a major role in the resolution of pulmonary inflammatory diseases. in some cases, excessive plasma proteins leaked into alveoli mix with necrotic type i pneumonocytes and pulmonary surfactant, forming microscopic eosinophilic bands (membranes) along the lining of alveolar septa. these membranes, known as hyaline membranes, are found in specific types of pulmonary diseases, particularly in ards, and in cattle with acute interstitial pneumonias such as bovine pulmonary edema and emphysema and extrinsic allergic alveolitis (see pneumonias of cattle). in the past few years, nitric oxide has been identified as a major regulatory molecule of inflammation in a variety of tissues, including the lung. produced locally by macrophages, pulmonary endothelium, and pneumonocytes, nitric oxide regulates the vascular and bronchial tone, modulates the production of cytokines, controls the recruitment and trafficking of neutrophils in the lung, and switches on/off genes involved in inflammation and immunity. experimental work has also shown that pulmonary surfactant upregulates the production of nitric oxide in the lung, supporting the current view that pneumonocytes are also pivotal in amplifying and downregulating the inflammatory and immune responses in the lung (see etable 9 -1). as the inflammatory process becomes chronic, the types of cells making up cellular infiltrates in the lung change from mainly neutrophils to largely mononuclear cells. this shift in cellular composition is accompanied by an increase in specific cytokines, such as failure. it also occurs in dogs with torsion of a lung lobe (fig. 9-67) . the gross features of infarcts vary considerably, depending on the stage, and they can be red to black, swollen, firm, and cone or wedge shaped, particularly at the lung margins. in the early acute stage, microscopic lesions are severely hemorrhagic, and this is followed by necrosis. in 1 or 2 days, a border of inflammatory cells develops, and a few days later, a large number of siderophages are present in the necrotic lung. if sterile, pulmonary infarcts heal as fibrotic scars; if septic, an abscess may form surrounded by a thick fibrous capsule. in the past three decades, an information explosion has increased the overall understanding of pulmonary inflammation, with so many proinflammatory and antiinflammatory mediators described to date that it would be impossible to review them all here (see chapters 3 and 5). pulmonary inflammation is a highly regulated process that involves a complex interaction between cells imported from the blood (platelets, neutrophils, eosinophils, mast cells, and lymphocytes) and pulmonary cells (type i and ii pneumonocytes; endothelial and club [clara] cells; alveolar and intravascular macrophages; and stromal interstitial cells, such as mast cells, interstitial macrophages, fibroblasts, and myofibroblasts). blood-borne leukocytes, platelets, and plasma proteins are brought into the areas of inflammation by an elaborate network of chemical signals emitted by pulmonary cells and resident leukocytes. long-distance communication between pulmonary cells and blood cells is largely done by soluble cytokines; once in the lung, imported leukocytes communicate with pulmonary and vascular cells through adhesion and other inflammatory molecules. the best known inflammatory mediators are the complement system (c3a, c3b, and c5a), coagulation factors (factors v and vii), arachidonic acid metabolites (leukotrienes and prostaglandins), cytokines (interleukins, monokines, and chemokines), adhesion molecules (icam and vcam), neuropeptides (substance p, tachykinins, and neurokinins), enzymes and enzyme inhibitors (elastase and antitrypsin), oxygen metabolites (o 2 •, oh•, and h 2 o 2 ), antioxidants (glutathione), and nitric oxide (e -table 9 -1). acting in concert, these and many other molecules send positive or negative signals to initiate, maintain, and, it is hoped, resolve the inflammatory process without causing injury to the lung. chapter 9 respiratory system, mediastinum, and pleurae ewith several episodes of hemorrhage are characterized by large areas of dark brown discoloration, largely in the caudal lung lobes. microscopically, lesions are alveolar hemorrhages, abundant alveolar macrophages containing hemosiderin (siderophages), mild alveolar fibrosis, and occlusive remodeling of pulmonary veins. recurrent airway obstruction. recurrent airway obstruction (rao) of horses, also referred to as copd, heaves, chronic bronchiolitis-emphysema complex, chronic small airway disease, alveolar emphysema, and "broken wind," is a common clinically asthma-like syndrome of horses and ponies. rao is characterized by recurrent respiratory distress, chronic cough, poor athletic performance, airway neutrophilia, bronchoconstriction, mucus hypersecretion, and airway obstruction. the pathogenesis is still obscure, but genetic predisposition, t h 2 (allergic) immune response, and the exceptional sensitivity of airways to environmental allergens (hyperreactive airway disease) have been postulated as the basic underlying mechanisms. what makes small airways hyperreactive to allergens is still a matter of controversy. epidemiologic and experimental studies suggest that it could be the result of preceding bronchiolar damage caused by viral infections; ingestion of pneumotoxicants (3-methylindole); or prolonged exposure to organic dust, endotoxin, and environmental allergens (molds). it has been postulated that sustained inhalation of dust particles, whether antigenic or not, upregulates the production of cytokines (tnf-α, il-8, and monokine-inducible protein ) and neuropeptides (neurokinin a [nka], neurokinin b [nkb], and substance p), attracting neutrophils into the bronchioloalveolar region and promoting leukocyte-induced bronchiolar injury. summer pasture-associated obstructive pulmonary disease (spaopd) is a seasonal airway disease also reported in horses with similar clinical and pathologic findings. more recently, the term inflammatory airway disease (iad) has been introduced in equine medicine to describe rao-like syndrome in young horses 2 to 4 years old. the lungs of horses with heaves are grossly unremarkable, except for extreme cases in which alveolar emphysema may be present. microscopically, the lesions are often remarkable and include goblet cell metaplasia in bronchioles; plugging of bronchioles with mucus mixed with few eosinophils and neutrophils (see fig. 9 -13); peribronchiolar infiltration with lymphocytes, plasma cells, and variable numbers of eosinophils; and hypertrophy of smooth muscle in bronchi and bronchioles. in severe cases, accumulation of mucus leads to the complete obstruction of bronchioles and alveoli and resultant alveolar emphysema characterized by enlarged "alveoli" from the destruction of alveolar walls. feline asthma syndrome. feline asthma syndrome, also known as feline allergic bronchitis, is a clinical syndrome in cats of any age characterized by recurrent episodes of bronchoconstriction, cough, or dyspnea. the pathogenesis is not well understood but is presumed to originate, as in human asthma, as a type i hypersensitivity (igemast cell reaction) to inhaled allergens. dust, cigarette smoke, plant and household materials, and parasitic proteins have been incriminated as possible allergens. this self-limited allergic disease responds well to steroid therapy; thus it is rarely implicated as a primary cause of death except when suppressed defense mechanisms allow a secondary bacterial pneumonia. bronchial biopsies from affected cats at the early stages reveal mild to moderate inflammation characterized by mucosal edema and infiltration of leukocytes, particularly eosinophils. increased numbers of circulating eosinophils (blood eosinophilia) are present in some but not all cats with feline asthma. il-4, interferon-γ (ifn-γ), and interferon-inducible protein (ip-10), which are chemotactic for lymphocytes and macrophages. under appropriate conditions, these cytokines activate t lymphocytes, regulate granulomatous inflammation, and induce the formation of multinucleated giant cells such as in mycobacterial infections. inflammatory mediators locally released from inflamed lungs also have a biologic effect in other tissue. for example, pulmonary hypertension and right-sided heart failure (cor pulmonale) often follows chronic alveolar inflammation, not only as a result of increased pulmonary blood pressure but also from the effect of inflammatory mediators on the contractibility of smooth muscle of the pulmonary and systemic vasculature. cytokines, particularly tnf-α, that are released during inflammation are associated, both as cause and as effect, with the systemic inflammatory response syndrome (sirs), sepsis, severe sepsis with multiple organ dysfunction, and septic shock (cardiopulmonary collapse). as it occurs in any other sentinel system where many biologic promoters and inhibitors are involved (coagulation, the complement and immune systems), the inflammatory cascade could go into an "out-of-control" state, causing severe damage to the lungs. acute lung injury (ali), extrinsic allergic alveolitis, ards, pulmonary fibrosis, and asthma are archetypical diseases that ensue from an uncontrolled production and release of cytokines (cytokine storm). as long as acute alveolar injury is transient and there is no interference with the normal host response, the entire process of injury, degeneration, necrosis, inflammation, and repair can occur in less than 10 days. on the other hand, when acute alveolar injury becomes persistent or when the capacity of the host for repair is impaired, lesions can progress to an irreversible stage in which restoration of alveolar structure is no longer possible. in diseases, such as extrinsic allergic alveolitis, the constant release of proteolytic enzymes and free radicals by phagocytic cells perpetuates alveolar damage in a vicious circle. in other cases, such as in paraquat toxicity, the magnitude of alveolar injury can be so severe that type ii pneumonocytes, basement membranes, and alveolar interstitium are so disrupted that the capacity for alveolar repair is lost. fibronectins and transforming growth factors (tgfs) released from macrophages and other mononuclear cells at the site of chronic inflammation regulate the recruitment, attachment, and proliferation of fibroblasts. in turn, these cells synthesize and release considerable amounts of ecm (collagen, elastic fibers, or proteoglycans), eventually leading to fibrosis and total obliteration of normal alveolar architecture. in summary, in diseases in which there is chronic and irreversible alveolar damage, lesions invariably progress to a stage of terminal alveolar and interstitial fibrosis. for pneumonia, see section species-specific pneumonia of domestic animals. exercise-induced pulmonary hemorrhage. exercise-induced pulmonary hemorrhage (eiph) is a specific form of pulmonary hemorrhage in racehorses that occurs after exercise and clinically is characterized by epistaxis. because only a small percentage of horses with bronchoscopic evidence of hemorrhage have clinical epistaxis, it is likely that eiph goes undetected in many cases. the pathogenesis is still controversial, but current literature suggests laryngeal paralysis, bronchiolitis, and extremely high pulmonary vascular and alveolar pressures during exercise, alveolar hypoxia, and preexisting pulmonary injury as possible causes. eiph is seldom fatal; postmortem lesions in the lungs of horses that have been affected disease may be known by different names. in pigs, for instance, enzootic pneumonia and mycoplasma pneumonia refer to the same disease caused by mycoplasma hyopneumoniae. the word pneumonitis has been used by some as a synonym for pneumonia; however, others have restricted this term to chronic proliferative inflammation generally involving the alveolar interstitium and with little or no evidence of exudate. in this chapter, the word pneumonia is used for any inflammatory lesion in the lungs, regardless of whether it is exudative or proliferative, alveolar, or interstitial. on the basis of texture, distribution, appearance, and exudation, pneumonias can be grossly diagnosed into four morphologically distinct types: bronchopneumonia, interstitial pneumonia, embolic pneumonia, and granulomatous pneumonia. by using this classification, it is possible at the time of a necropsy to predict with some degree of certainty the likely cause (virus, bacteria, fungi, or parasites), routes of entry (aerogenous vs. hematogenous), and possible sequelae. these four morphologic types allow the clinician or pathologists to predict the most likely etiology and therefore facilitate the decision as to what samples need to be taken and which tests should be requested to the diagnostic laboratory (i.e., histopathology, bacteriology, virology, or toxicology). however, overlapping of these four types of pneumonias is possible, and sometimes two morphologic types may be present in the same lung. the criteria used to classify pneumonias grossly into bronchopneumonia, interstitial pneumonia, embolic pneumonia, and granulomatous pneumonia are based on morphologic changes, including distribution, texture, color, and general appearance of the affected lungs (table 9 -5). distribution of the inflammatory lesions in the lungs can be (1) cranioventral, as in most bronchopneumonias; (2) multifocal, as in embolic pneumonias; (3) diffuse, as in interstitial pneumonias; or (4) locally extensive, as in granulomatous in the most advanced cases, chronic bronchoconstriction and excess mucus production may result in smooth muscle hyperplasia and obstruction of the bronchi and bronchioles and infiltration of the airway mucosa by eosinophils. a syndrome known as canine asthma has been reported in dogs but is not as well characterized as the feline counterpart. few subjects in veterinary pathology have caused so much debate as the classification of pneumonias. historically, pneumonias in animals have been classified or named based on the following: 1. presumed cause, with names such as viral pneumonia, pasteurella pneumonia, distemper pneumonia, verminous pneumonia, chemical pneumonia, and hypersensitivity pneumonitis 2. type of exudation, with names such as suppurative pneumonia, fibrinous pneumonia, and pyogranulomatous pneumonia 3. morphologic features, with names such as gangrenous pneumonia, proliferative pneumonia, and embolic pneumonia 4. distribution of lesions, with names such as focal pneumonia, cranioventral pneumonia, diffuse pneumonia, and lobar pneumonia 5. epidemiologic attributes, with names such as enzootic pneumonia, contagious bovine pleuropneumonia, and "shipping fever" 6. geographic regions, with names such as montana progressive pneumonia 7. miscellaneous attributes, with names such as atypical pneumonia, cuffing pneumonia, progressive pneumonia, aspiration pneumonia, pneumonitis, farmer's lung, and extrinsic allergic alveolitis until a universal and systematic nomenclature for animal pneumonias is established, veterinarians should be acquainted with this heterogeneous list of names and should be well aware that one the parts of the face with the tip of your finger has been advocated by some pathologists. the texture of a normal lung is comparable to the texture of the center of the cheek. firm consolidation is comparable to the texture of the tip of the nose, and hard consolidation is comparable to the texture of the forehead. the term consolidation is frequently used to describe a firm or hard lung filled with exudate. pneumonias ( fig. 9-68) . texture of pneumonic lungs can be firmer or harder (bronchopneumonias), more elastic (rubbery) than normal lungs (interstitial pneumonias), or have a nodular feeling (granulomatous pneumonias). describing in words the palpable difference between the texture of a normal lung compared with the firm or hard texture of a consolidated lung can be a difficult undertaking. an analogy illustrating this difference based on touching changes in the gross appearance of pneumonic lungs include abnormal color, the presence of nodules or exudate, fibrinous or fibrous adhesions, and the presence of rib imprints on serosal surfaces (see fig. 9 -68). on cut surfaces, pneumonic lungs may have exudate, hemorrhage, edema, necrosis, abscesses, bronchiectasis, granulomas or pyogranulomas, and fibrosis, depending on the stage. palpation and careful observation of the lungs are essential in the diagnosis of pneumonia. (for details, see the section on examination of the respiratory tract.) bronchopneumonia refers to a particular morphologic type of pneumonia in which injury and the inflammatory process take place primarily in the bronchial, bronchiolar, and alveolar lumens. bronchopneumonia is undoubtedly the most common type of pneumonia seen in domestic animals and is with few exceptions characterized grossly by cranioventral consolidation of the lungs (fig. 9-69 and see fig. 9 -68). the reason why bronchopneumonias in animals are almost always restricted to the cranioventral portions of the lungs is not well understood. possible factors contributing to this topographic selectivity within the lungs include (1) gravitational sedimentation of the exudate, (2) greater deposition of infectious organisms, (3) inadequate defense mechanisms, (4) reduced vascular perfusion, (5) shortness and abrupt branching of airways, and (6) regional differences in ventilation. the term cranioventral in veterinary anatomy is the equivalent of "anterosuperior" in human anatomy. the latter is defined as "in front (ventral) and above (cranial)." thus, applied to the lung of animals, "cranioventral" means the ventral portion of the cranial lobe. however, by common usage in veterinary pathology, the term cranioventral used to describe the location of lesions in pneumonias has come to mean "cranial and ventral." thus it includes pneumonias affecting not only the ventral portion of the cranial lobe (true cranioventral) but also those cases in which the pneumonia has involved the ventral portions of adjacent lung lobes-initially the middle and then caudal on the right and the caudal lobe on the left side. bronchopneumonias are generally caused by bacteria and mycoplasmas, by bronchoaspiration of feed or gastric contents, or by improper tubing. as a rule, the pathogens causing bronchopneumonias arrive in the lungs via inspired air (aerogenous), either from infected aerosols or from the nasal flora. before establishing infection, pathogens must overwhelm or evade the pulmonary defense mechanisms. the initial injury in bronchopneumonias is centered on the mucosa of bronchioles; from there, the inflammatory process can spread downward to distal portions of the alveoli and upward to the bronchi. typically, for bronchopneumonias, the inflammatory exudates collect in the bronchial, bronchiolar, and alveolar lumina leaving the alveolar interstitium relatively unchanged, except for hyperemia and possibly edema. through the pores of kohn, the exudate can spread to adjacent alveoli until most or all of the alveoli in an individual lobule are involved. if the inflammatory process cannot control the inciting cause of injury, the lesions spread rapidly from lobule to lobule through alveolar pores and destroyed alveolar walls until an entire lobe or large portion of a lung is involved. the lesion tends to spread centrifugally, with the older lesions in the center, and exudate can be coughed up and then aspirated into other lobules, where the inflammatory process starts again. at the early stages of bronchopneumonia, the pulmonary vessels are engorged with blood (active hyperemia), and the bronchi, bronchioles, and alveoli contain some fluid (permeability edema). in cases in which pulmonary injury is mild to moderate, cytokines locally released in the lung cause rapid recruitment of neutrophils and alveolar macrophages into bronchioles and alveoli ( fig. 9-70 and see fig. 9 -69). when pulmonary injury is much more severe, proinflammatory cytokines induce more pronounced vascular changes by further opening endothelial gaps, thus increasing vascular permeability resulting in leakage of plasma fibrinogen (fibrinous exudates) and sometimes hemorrhage in the alveoli. alterations in permeability can be further exacerbated by structural damage to pulmonary capillaries and vessels directly caused by microbial toxins. filling of alveoli, bronchioles, and small bronchi with inflammatory exudate progressively obliterates airspaces, and as a consequence of this process, portions of severely affected (consolidated) lungs sink to the bottom of the container when placed in fixative. the replacement of air by exudate also changes the texture of the lungs, and depending on the severity of bronchopneumonia, the texture varies from firmer to harder than normal. the term consolidation is used at gross examination when the texture of pneumonic lung becomes firmer or harder than normal as a result of loss of airspaces because of exudation and atelectasis. (for details, see the discussion of lung texture in the section on classification of pneumonias in domestic animals). inflammatory consolidation of the lungs has been referred to in the past as hepatization because the affected lung had the appearance and texture of liver. the process was referred to as red hepatization in acute cases in which (1) congestion, (2) red hepatization (liver texture), (3) gray hepatization, and (4) resolution. because of the use of effective antibiotics and prevention, pneumococcal pneumonia and its four classic stages are rarely seen; thus this terminology has been largely abandoned. currently, the term bronchopneumonia is widely used for both suppurative and fibrinous consolidation of the lungs because both forms of inflammation have essentially the same pathogenesis in which the pathogens reach the lung by the aerogenous route, injury occurs initially in the bronchial and bronchiolar regions, and the inflammatory process extends centrifugally deep into the alveoli. it must be emphasized that it is the severity of pulmonary injury that largely determines whether bronchopneumonia becomes suppurative or fibrinous. in some instances, however, it is difficult to discriminate between suppurative and fibrinous bronchopneumonia because both types can coexist (fibrinosuppurative bronchopneumonia), and one type can progress to the other. suppurative bronchopneumonia. suppurative bronchopneumonia is characterized by cranioventral consolidation of lungs (see figs. 9-68 and 9-69), with typically purulent or mucopurulent exudate present in the airways. this exudate can be best demonstrated by expressing intrapulmonary bronchi, thus forcing exudate out of the bronchi (see fig. 9 -69). the inflammatory process in suppurative bronchopneumonia is generally confined to individual lobules, and as a result of this distribution, the lobular pattern of the lung becomes notably emphasized. this pattern is particularly obvious in cattle and pigs because these species have prominent lobulation of the lungs. the gross appearance often resembles an irregular checkerboard because of an admixture of normal and abnormal (consolidated) lobules (see fig. 9 -69). because of this typical lobular distribution, suppurative bronchopneumonias are also referred to as lobular pneumonias. different inflammatory phases occur in suppurative bronchopneumonia where the color and appearance of consolidated lungs varies considerably, depending on the virulence of offending organisms and chronicity of the lesion. the typical phases of suppurative bronchopneumonia can be summarized as follows: 1. during the first 12 hours when bacteria are rapidly multiplying, the lungs become hyperemic and edematous. 2. soon after, neutrophils start filling the airways, and by 48 hours the parenchyma starts to consolidate and becomes firm in texture. 3. three to 5 days later, hyperemic changes are less obvious, but the bronchial, bronchiolar, and alveolar spaces continue to fill with neutrophils and macrophages, and the affected lung sinks when placed in formalin. at this stage, the affected lung has a gray-pink color, and on cut surface, purulent exudate can be expressed from bronchi. 4. in favorable conditions where the infection is under control of the host defense mechanisms, the inflammatory processes begin to regress, a phase known as resolution. complete resolution in favorable conditions could take 1 to 2 weeks. 5. in animals in which the lung infection cannot be rapidly contained, inflammatory lesions can progress into a chronic phase. approximately 7 to 10 days after infection, the lungs become pale gray and take a "fish flesh" appearance. this appearance is the result of purulent and catarrhal inflammation, obstructive atelectasis, mononuclear cell infiltration, peribronchial and peribronchiolar lymphoid hyperplasia, and early alveolar fibrosis. complete resolution is unusual in chronic bronchopneumonia, and lung scars, such as pleural and pulmonary fibrosis; bronchiectasis as a consequence of chronic destructive bronchitis (see bronchiectasis [dysfunction/responses to injury and patterns of injury]); atelectasis; pleural adhesions; and lung abscesses may remain unresolved there was notable active hyperemia with little exudation of neutrophils; conversely, the process was referred to as gray hepatization in those chronic cases in which hyperemia was no longer present, but there was abundant exudation of neutrophils and macrophages. this terminology, although used for and applicable to human pneumonias, is rarely used in veterinary medicine primarily because the evolution of pneumonic processes in animals does not necessarily follow the red-to-gray hepatization pattern. bronchopneumonia can be subdivided into suppurative bronchopneumonia if the exudates are predominantly composed of neutrophils and fibrinous bronchopneumonia if fibrin is the predominant component of the exudates (see table 9 -5). it is important to note that some veterinarians use the term fibrinous pneumonia or lobar pneumonia as a synonym for fibrinous bronchopneumonia, and bronchopneumonia or lobular pneumonia as a synonym for suppurative bronchopneumonia. human pneumonias for many years have been classified based on their etiology and morphology, which explains why pneumococcal pneumonia (streptococcus pneumoniae) has been synonymous with lobar pneumonia. in the old literature, four distinct stages of pneumococcal pneumonia were described as lumen of bronchiole capillary pulmonary defense mechanisms to allow them to colonize the lungs and establish an infection. suppurative bronchopneumonia can also result from aspiration of bland material (e.g., milk). pulmonary gangrene may ensue when the bronchopneumonic lung is invaded by saprophytic bacteria (aspiration pneumonia). fibrinous bronchopneumonia. fibrinous bronchopneumonia is similar to suppurative bronchopneumonia except that the predominant exudate is fibrinous rather than neutrophilic. with only a few exceptions, fibrinous bronchopneumonias also have a cranioventral distribution (fig. 9-72 and see fig. 9-68) . however, exudation is not restricted to the boundaries of individual pulmonary lobules, as is the case in suppurative bronchopneumonias. instead, the inflammatory process in fibrinous pneumonias involves numerous contiguous lobules and the exudate moves quickly through pulmonary tissue until the entire pulmonary lobe is rapidly affected. because of the involvement of the entire lobe and pleural surface, fibrinous bronchopneumonias are also referred to as lobar pneumonias or pleuropneumonias. in general terms, fibrinous bronchopneumonias are the result of more severe pulmonary injury and thus cause death earlier in the sequence of the inflammatory process than suppurative bronchopneumonias. even in cases in which fibrinous bronchopneumonia involves 30% or less of the total area, clinical signs and death can occur as a result of severe toxemia and sepsis. the gross appearance of fibrinous bronchopneumonia depends on the age and severity of the lesion and on whether the pleural surface or the cut surface of the lung is viewed. externally, early stages of fibrinous bronchopneumonias are characterized by severe congestion and hemorrhage, giving the affected lungs a characteristically intense red discoloration. a few hours later, fibrin starts to permeate and accumulate on the pleural surface, giving the pleura a ground glass appearance and eventually forming plaques of fibrinous exudate over a red, dark lung (see fig. 9 -72). at this stage, a yellow fluid starts to accumulate in the thoracic cavity. the color of fibrin deposited over the pleural surface is also variable. it can be bright yellow when the exudate is formed primarily by fibrin, tan when fibrin is mixed with blood, and gray when a large number of leukocytes and fibroblasts are part of the fibrinous plaque in more chronic cases. because of the tendency of fibrin to deposit on the pleural surface, some pathologists use the term pleuropneumonia as a synonym for fibrinous bronchopneumonia. on the cut surface, early stages of fibrinous bronchopneumonia appear as simple red consolidation. in more advanced cases (24 hours), fibrinous bronchopneumonia is generally accompanied by notable dilation and thrombosis of lymph vessels and edema of interlobular septa (see fig. 9-72, b) . this distention of the interlobular septa gives affected lungs a typical marbled appearance. distinct focal areas of coagulative necrosis in the pulmonary parenchyma are also common in fibrinous bronchopneumonia such as in shipping fever pneumonia and contagious bovine pleuropneumonia. in animals that survive the early stage of fibrinous bronchopneumonia, pulmonary necrosis often develops into pulmonary "sequestra," which are isolated pieces of necrotic lung encapsulated by connective tissue. pulmonary sequestra result from extensive necrosis of lung tissue either from severe ischemia (infarct) caused by thrombosis of a major pulmonary vessel such as in contagious bovine pleuropneumonia or from the effect of necrotizing toxins released by pathogenic bacteria such as mannheimia haemolytica. sequestra in veterinary pathology should not be confused with "bronchopulmonary sequestration," a term used in human pathology to describe a congenital malformation in which whole lobes or parts of the lung develop without normal connections to the airway or vascular systems. for a long time. "enzootic pneumonias" of ruminants and pigs are typical examples of chronic suppurative bronchopneumonias. microscopically, acute suppurative bronchopneumonias are characterized by hyperemia, abundant neutrophils, macrophages, and cellular debris within the lumen of bronchi, bronchioles, and alveoli (see fig. 9 -70). recruitment of leukocytes is promoted by cytokines, complement, and other chemotactic factors that are released in response to alveolar injury or by the chemotactic effect of bacterial toxins, particularly endotoxin. in most severe cases, purulent or mucopurulent exudates completely obliterate the entire lumen of bronchi, bronchioles, and alveoli. if suppurative bronchopneumonia is merely the response to a transient pulmonary injury or a mild infection, lesions resolve uneventfully. within 7 to 10 days, cellular exudate can be removed from the lungs via the mucociliary escalator, and complete resolution may take place within 4 weeks. in other cases, if injury or infection is persistent, suppurative bronchopneumonia can become chronic with goblet cell hyperplasia, an important component of the inflammatory process. depending on the proportion of pus and mucus, the exudate in chronic suppurative bronchopneumonia varies from mucopurulent to mucoid. a mucoid exudate is found in the more chronic stages when the consolidated lung has a "fish flesh" appearance. hyperplasia of balt is another change commonly seen in chronic suppurative bronchopneumonias; it appears grossly as conspicuous white nodules (cuffs) around bronchial walls (cuffing pneumonia). this hyperplastic change merely indicates a normal reaction of lymphoid tissue to infection. further sequelae of chronic suppurative bronchopneumonia include bronchiectasis (see figs. 9-10 and 9-11), pulmonary abscesses, pleural adhesions (from pleuritis) ( fig. 9-71) , and atelectasis and emphysema from completely or partially obstructed bronchi or bronchioles (e.g., bronchiectasis). clinically, suppurative bronchopneumonias can be acute and fulminating but are often chronic, depending on the etiologic agent, stressors affecting the host, and immune status. the most common pathogens causing suppurative bronchopneumonia in domestic animals include pasteurella multocida, bordetella bronchiseptica, trueperella (arcanobacterium) pyogenes, streptococcus spp., escherichia coli, and several species of mycoplasmas. most of these organisms are secondary pathogens requiring a preceding impairment of the fulminating hemorrhagic bronchopneumonia can be caused by highly pathogenic bacteria such as bacillus anthracis. although the lesions in anthrax are primarily related to a severe septicemia and sepsis, anthrax should always be suspected in animals with sudden death and exhibiting severe acute fibrinohemorrhagic pneumonia, splenomegaly, and multisystemic hemorrhages. animals are considered good sentinels for anthrax in cases of bioterrorism. interstitial pneumonia refers to that type of pneumonia in which injury and the inflammatory process take place primarily in any microscopically, in the initial stage of fibrinous bronchopneumonia, there is massive exudation of plasma proteins into the bronchioles and alveoli, and as a result, most of the airspaces become obliterated by fluid and fibrin. leakage of fibrin and fluid into alveolar lumina is due to extensive disruption of the integrity and increased permeability of the blood-air barrier. fibrinous exudates can move from alveolus to alveolus through the pores of kohn. because fibrin is chemotactic for neutrophils, these types of leukocytes are always present a few hours after the onset of fibrinous inflammation. as inflammation progresses (3 to 5 days), fluid exudate is gradually replaced by fibrinocellular exudates composed of fibrin, neutrophils, macrophages, and necrotic debris ( fig. 9-73 ). in chronic cases (after 7 days), there is notable fibrosis of the interlobular septa and pleura. in contrast to suppurative bronchopneumonia, fibrinous bronchopneumonia rarely resolves completely, thus leaving noticeable scars in the form of pulmonary fibrosis and pleural adhesions. the most common sequelae found in animals surviving an acute episode of fibrinous bronchopneumonia include alveolar fibrosis and bronchiolitis obliterans, in which organized exudate becomes attached to the bronchiolar lumen (see fig. 9-12) . these changes are collectively referred to as bronchiolitis obliterans organizing pneumonia (boop), a common microscopic finding in animals with unresolved bronchopneumonia. other important sequelae include pulmonary gangrene, when saprophytic bacteria colonize necrotic lung; pulmonary sequestra; pulmonary fibrosis; abscesses; and chronic pleuritis with pleural adhesions. in some cases, pleuritis can be so extensive that fibrous adhesions extend onto the pericardial sac. pathogens causing fibrinous bronchopneumonias in domestic animals include mannheimia (pasteurella) haemolytica (pneumonic mannheimiosis), histophilus somni (formerly haemophilus somnus), actinobacillus pleuropneumoniae (porcine pleuropneumonia), mycoplasma bovis, and mycoplasma mycoides ssp. mycoides small colony type (contagious bovine pleuropneumonia). fibrinous bronchoa b n alveolar epithelium. inhaled antigens, such as fungal spores, combine with circulating antibodies and form deposits of antigen-antibody complexes (type iii hypersensitivity) in the alveolar wall, which initiate a cascade of inflammatory responses and injury (allergic alveolitis). hematogenous injury to the vascular endothelium occurs in septicemias, sepsis, dic, larva migrans (ascaris suum), toxins absorbed in the alimentary tract (endotoxin) or toxic metabolites locally generated in the lungs (3-methylindole and paraquat), release of free radicals in alveolar capillaries (ards), and infections with endotheliotropic viruses (canine adenovirus and classical swine fever [hog cholera]). interstitial pneumonias in domestic animals and human beings are subdivided based on morphologic features into acute and chronic. it should be kept in mind, however, that not all acute interstitial pneumonias are fatal and that they do not necessarily progress to the chronic form. acute interstitial pneumonias. acute interstitial pneumonias begin with injury to either type i pneumonocytes or alveolar capillary endothelium, which provokes a disruption of the blood-air barrier and a subsequent exudation of plasma proteins into the alveolar space (see fig. 9-14) . this leakage of proteinaceous fluid into the alveolar lumen constitutes the exudative phase of acute interstitial pneumonia. in some cases of diffuse alveolar damage, exuded plasma proteins mix with lipids and other components of pulmonary surfactant and form elongated membranes that become partially attached to the alveolar basement membrane and bronchiolar walls. these membranes are referred to as hyaline membranes because of their hyaline appearance (eosinophilic, homogeneous, and amorphous) microscopically (see figs. 9-55 and 9-63). in addition to intraalveolar exudation of fluid, inflammatory edema and neutrophils accumulate in the alveolar interstitium and cause thickening of the alveolar walls. this acute exudative phase is generally followed a few days later by the proliferative phase of acute interstitial pneumonia, which is characterized by hyperplasia of type ii pneumonocytes to replace the lost type i pneumonocytes (see fig. 9 -15). type ii pneumonocytes are in fact progenitor cells that differentiate and replace necrotic type i pneumonocytes (see fig. 9-14) . as a consequence, the alveolar walls become increasingly thickened. this process is in part the reason why lungs become rubbery on palpation, what prevents their normal collapse after the thorax is opened, and why the cut surface of the lung has a "meaty" appearance (see fig. 9 -74). of the three layers of the alveolar walls (endothelium, basement membrane, and alveolar epithelium) and the contiguous bronchiolar interstitium (see fig. 9-7) . this morphologic type of pneumonia is the most difficult to diagnose at necropsy and requires microscopic confirmation because it is easily mistaken in the lung showing congestion, edema, hyperinflation, or emphysema. in contrast to bronchopneumonias, in which distribution of lesions is generally cranioventral, in interstitial pneumonias, lesions are more diffusely distributed and generally involve all pulmonary lobes, or in some cases, they appear to be more pronounced in the dorsocaudal aspects of the lungs (see fig. 9 -68). three important gross features of interstitial pneumonia are (1) the failure of lungs to collapse when the thoracic cavity is opened, (2) the occasional presence of rib impressions on the pleural surface of the lung indicating poor deflation, and (3) the lack of visible exudates in airways unless complicated with secondary bacterial pneumonia. the color of affected lungs varies from diffusely red in acute cases to diffusely pale gray to a mottled red, pale appearance in chronic ones. pale lungs are caused by severe obliteration of alveolar capillaries (reduced blood-tissue ratio), especially evident when there is fibrosis of the alveolar walls. the texture of lungs with uncomplicated interstitial pneumonia is typically elastic or rubbery, but definitive diagnosis based on texture alone is difficult and requires histopathologic examination. on a cut surface, the lungs may appear and feel more "meaty" (having the texture of raw meat) and have no evidence of exudate in the bronchi or pleura (fig. 9-74 ). in acute interstitial pneumonias, particularly in cattle, there is frequently pulmonary edema (exudative phase) and interstitial emphysema secondary to partial obstruction of bronchioles by edema fluid and strenuous air gasping before death. because edema tends to gravitate into the cranioventral portions of the lungs, and emphysema is often more obvious in the dorsocaudal aspects, acute interstitial pneumonias in cattle occasionally have a gross cranioventral-like pattern that may resemble bronchopneumonia, although the texture is different. lungs are notably heavy because of the edema and the infiltrative and proliferative changes. the pathogenesis of interstitial pneumonia is complex and can result from aerogenous injury to the alveolar epithelium (type i and ii pneumonocytes) or from hematogenous injury to the alveolar capillary endothelium or alveolar basement membrane. aerogenous inhalation of toxic gases (i.e., ozone and no 2 ) or toxic fumes (smoke inhalation) and infection with pneumotropic viruses (influenza, herpesviruses, or canine distemper virus) can damage the figure 9 -74 interstitial pneumonia, lung, feeder pig. a, the lung is heavy, pale, and rubbery in texture. it also has prominent costal (rib) imprints (arrows), a result of hypercellularity of the interstitium and the failure of the lungs to collapse when the thorax was opened. b, transverse section. the pulmonary parenchyma has a "meaty" appearance and some edema, but no exudate is present in airways or on the pleural surface. this type of lung change in pigs is highly suggestive of a viral pneumonia. (courtesy dr. a. lópez, atlantic veterinary college.) pneumonia are centered in the alveolar wall and its interstitium, a mixture of desquamated epithelial cells, macrophages, and mononuclear cells are usually present in the lumens of bronchioles and alveoli. ovine progressive pneumonia, hypersensitivity pneumonitis in cattle and dogs, and silicosis in horses are good veterinary examples of chronic interstitial pneumonia. pneumoconioses (silicosis and asbestosis), paraquat toxicity, pneumotoxic antineoplastic drugs (bleomycin), and extrinsic allergic alveolitis (farmer's lung) are well-known examples of diseases that lead to chronic interstitial pneumonias in human beings. massive pulmonary migration of ascaris larvae in pigs also causes interstitial pneumonia ( fig. 9-77 ). there is an insidious and poorly understood group of chronic idiopathic interstitial diseases, both in human beings and in animals, that eventually progress to terminal interstitial fibrosis. these were originally thought to be the result of repeated cycles of alveolar injury, inflammation, and fibroblastic/myoblastic response to an unknown agent. however, aggressive antiinflammatory therapy generally fails to prevent or reduce the severity of fibrosis. now, it is acute interstitial pneumonias are often mild and transient, especially those caused by some respiratory viruses, such as those responsible for equine and porcine influenza. these mild forms of pneumonia are rarely seen in the postmortem room because they are not fatal and do not leave significant sequelae (see the section on defense mechanisms/barrier systems). in severe cases of acute interstitial pneumonias, animals may die of respiratory failure, usually as a result of diffuse alveolar damage, a profuse exudative phase (leakage of proteinaceous fluid) leading to a fatal pulmonary edema. examples of this type of fatal acute interstitial pneumonia are bovine pulmonary edema and emphysema, and ards in all species. chronic interstitial pneumonia. when the source of alveolar injury persists, the exudative and proliferative lesions of acute interstitial pneumonia can progress into a morphologic stage referred to as chronic interstitial pneumonia. the hallmark of chronic interstitial pneumonia is fibrosis of the alveolar walls (with or without intraalveolar fibrosis) and the presence of lymphocytes, macrophages, fibroblasts, and myofibroblasts in the alveolar interstitium (figs. 9-75 and 9-76). in other cases, these chronic changes are accompanied by hyperplasia and persistence of type ii pneumonocytes, squamous metaplasia of the alveolar epithelium, microscopic granulomas, and hyperplasia of smooth muscle in bronchioles and pulmonary arterioles. it should be emphasized that although the lesions in interstitial the term bronchointerstitial pneumonia is used in veterinary pathology to describe cases in which microscopic lesions share some histologic features of both bronchopneumonia and interstitial pneumonia (efig. 9-9 ). this combined type of pneumonia is in fact frequently seen in many viral infections in which viruses replicate and cause necrosis in bronchial, bronchiolar, and alveolar cells. damage to the bronchial and bronchiolar epithelium causes an influx of neutrophils similar to that in bronchopneumonias, and damage to alveolar walls causes proliferation of type ii pneumonocytes, similar to that which takes place in the proliferative phase of acute interstitial pneumonias. it is important to emphasize that bronchointerstitial pneumonia is a microscopic not a gross diagnosis. examples include uncomplicated cases of respiratory syncytial virus infections in cattle and lambs, canine distemper, and influenza in pigs and horses. embolic pneumonia refers to a particular type of pneumonia in which gross and microscopic lesions are multifocally distributed in all pulmonary lobes. by definition, lung injury is hematogenous, and the inflammatory response is typically centered in pulmonary arterioles and alveolar capillaries. lungs act as a biologic filter for circulating particulate matter. sterile thromboemboli, unless extremely large, are rapidly dissolved and removed from the pulmonary vasculature by fibrinolysis, causing little, if any, ill effects. experimental studies have confirmed that most types of bacteria when injected intravenously (bacteremia) are phagocytosed by pulmonary intravascular macrophages, or they bypass the lungs and are finally trapped by macrophages in the liver, spleen, joints, or other organs. to cause pulmonary infection, circulating bacteria must first attach to the pulmonary endothelium with specific binding proteins or simply attach to intravascular fibrin and then evade phagocytosis by intravascular macrophages or leukocytes. septic thrombi facilitate entrapment of bacteria in the pulmonary vessels and provide a favorable environment to escape phagocytosis. once trapped in the pulmonary vasculature, usually in small arterioles or alveolar capillaries, offending bacteria disrupt endothelium and basement membranes, spread from the vessels to the interstitium and then to the surrounding lung, finally forming a new nidus of infection. embolic pneumonia is characterized by multifocal lesions randomly distributed in all pulmonary lobes (see fig. 9 -68 and e-figs. 9-10 and 9-11). early lesions in embolic pneumonia are characterized grossly by the presence of very small (1 to 10 mm), white foci surrounded by discrete, red, hemorrhagic halos ( fig. 9-78 ). unless emboli arrive in massive numbers, causing fatal pulmonary edema, embolic pneumonia is seldom fatal; therefore these acute lesions are rarely seen at postmortem examination. in most instances, if unresolved, acute lesions rapidly progress to pulmonary abscesses. these are randomly distributed in all pulmonary lobes and are not restricted to the cranioventral aspects of the lungs, as is the case of abscesses developing from suppurative bronchopneumonia. the early microscopic lesions in embolic pneumonias are always focal or multifocal ( fig. 9-79) ; thus they differ from those of endotoxemia or septicemia, in which endothelial damage and interstitial reactions (interstitial pneumonia) are diffusely distributed in the lungs. when embolic pneumonia or its sequela (abscesses) is diagnosed at necropsy, an attempt should be made to locate the source of septic emboli. the most common sources are hepatic abscesses that have ruptured into the caudal vena cava in cattle, omphalophlebitis in farm animals, chronic bacterial skin or hoof infections, and a contaminated catheter in all species (see fig. 9-64) . valvular or mural endocarditis in the right heart is a common source of septic emboli and embolic pneumonia in all species. most frequently, bacterial proposed that a genetic mutation alters the cell-cell communication between epithelial and mesenchymal cells in the lung. this aberrant cellular communication leads to an overexpression of inflammatory and repair molecules (i.e., il-4, il-13, tgf-β1, and caveolin), leading to increased apoptosis and interstitial deposition of extracellular matrix (ecm). the chronic interstitial (restrictive) diseases in human medicine include "idiopathic pulmonary fibrosis," "nonspecific interstitial pneumonia," "unusual interstitial pneumonia," and "cryptogenic organizing pneumonia," also referred to as idiopathic bronchiolitis obliterans organizing pneumonia (idiopathic boop). feline idiopathic pulmonary fibrosis is an example of this type of progressive interstitial disease in veterinary medicine. it has been reported that in rare cases, chronic alveolar remodeling and interstitial fibrosis can progress to lung cancer. the lung has numerous circular areas of hemorrhage distributed randomly throughout all lung lobes (embolic pattern [see fig. 9 -68]). these foci arise from injury to the microvasculature in alveolar septa and the visceral pleura secondary to lodgment of bacterial or fungal emboli (septic emboli) from valvular or mural endocarditis in the right heart or from other bacterial or fungal diseases where the bacterium or fungus gains access to the circulatory system as occurs in many bacterial and fungal enteritides or pneumonias caused by salmonella spp., e. coli, or aspergillus spp. the pathogenesis of granulomatous pneumonia shares some similarities with that of interstitial and embolic pneumonias. not surprisingly, some pathologists group granulomatous pneumonias within one of these types of pneumonias (e.g., granulomatous interstitial pneumonia). what makes granulomatous pneumonia a distinctive type is not so much the portal of entry or site of initial injury in the lungs but, rather, the unique type of inflammatory response that results in the formation of granulomas, which can be easily recognized at gross and microscopic examination. as a rule, agents causing granulomatous pneumonias are resistant to intracellular killing by phagocytic cells and to the acute inflammatory response, allowing prolonged persistence of these agents in tissues. the most common causes of granulomatous pneumonia in animals include systemic fungal diseases, such as cryptococcosis (cryptococcus neoformans and cryptococcus gatti), coccidioidomycosis (coccidioides immitis), histoplasmosis (histoplasma capsulatum), and blastomycosis (blastomyces dermatitidis) (see fig. 9 -35). in most of these fungal diseases, the port of entry is aerogenous, and from the lungs the fungi disseminate systemically to other organs, particularly the lymph nodes, liver, and spleen. filamentous fungi such as aspergillus spp. or mucor spp. can also reach the lung by the hematogenous route. granulomatous pneumonia is also seen in some bacterial diseases, such as tuberculosis (mycobacterium bovis) in all species and rhodococcus equi in horses. sporadically, aberrant parasites such as fasciola hepatica in cattle and aspiration of foreign bodies can also cause granulomatous pneumonia (efig. 9-12 granulomatous pneumonia is characterized by the presence of variable numbers of caseous or noncaseous granulomas randomly distributed in the lungs (see fig. 9 -68). on palpation, lungs have a typical nodular character given by well-circumscribed, variably sized nodules that generally have a firm texture, especially if calcification has occurred ( fig. 9-80) . during postmortem examination, granulomas in the lungs occasionally can be mistaken for neoplasms. microscopically, pulmonary granulomas are composed of a center of necrotic tissue, surrounded by a rim of macrophages (epithelioid cells) and giant cells and an outer delineated layer of connective tissue commonly infiltrated by lymphocytes and plasma cells ( fig. 9-81 ). unlike other types of pneumonias, the causative agent in granulomatous pneumonia can, in many cases, be identified isolates from septic pulmonary emboli in domestic animals are trueperella (arcanobacterium) pyogenes (cattle), fusobacterium necrophorum (cattle, pigs, and human beings), erysipelothrix rhusiopathiae (pigs, cattle, dogs, and human beings), streptococcus suis (pigs), staphylococcus aureus (dogs and human beings), and streptococcus equi (horses). granulomatous pneumonia refers to a particular type of pneumonia in which aerogenous or hematogenous injury is caused by organisms or particles that cannot normally be eliminated by phagocytosis and that evoke a local inflammatory reaction with numerous alveolar and interstitial macrophages, lymphocytes, a few neutrophils, and sometimes giant cells. the term granulomatous is used here to describe an anatomic pattern of pneumonia typically characterized by the presence of granulomas. g g but yet unproven that viral infections may also predispose horses to airway hyperresponsiveness and recurrent airway obstruction (rao). equine influenza. equine influenza is an important and highly contagious flulike respiratory disease of horses characterized by high morbidity and low mortality and explosive outbreaks in susceptible populations. it is an oie-notifiable disease. two antigenically unrelated subtypes of equine influenza virus have been identified (h7n7 [a/equi-1] and h3n8 [a/equi-2]). the course of the disease is generally mild and transient, and its importance is primarily because of its economic impact on horse racing. the types of injury and host response in the conducting system are described in the section on disorders of the nasal cavity and paranasal sinuses of horses. uncomplicated lesions in the lungs are mild and self-limiting bronchointerstitial pneumonia. in fatal cases, the lungs are hyperinflated with coalescing areas of dark red discoloration. microscopically, there is a bronchointerstitial pneumonia characterized by necrotizing bronchiolitis that is followed by hyperplastic bronchiolitis, hyperplasia of type ii pneumonocytes, hyaline membranes in alveoli, and sporadic multinucleated giant cells. the microscopic changes are ards in severe and fatal cases. the influenza virus antigen can be readily demonstrated in ciliated cells and alveolar macrophages. clinical signs are characterized by fever, cough, abnormal lung sounds (crackles and wheezes), anorexia, and depression. secondary bacterial infections (streptococcus equi, streptococcus zooepidemicus, staphylococcus aureus, and escherichia coli) commonly complicate equine influenza. equine viral rhinopneumonitis. equine viral rhinopneumonitis (evr), or equine herpesvirus infection, is a respiratory disease of young horses that is particularly important in weanlings between 4 and 8 months of age and to a much lesser extent in young foals and adult horses. the causative agents are ubiquitous equine herpesviruses (ehv-1 and ehv-4) that in addition to respiratory disease can cause abortion in pregnant mares and neurologic disease (equine herpes myeloencephalopathy) (see the section on disorders of the nasal cavity and paranasal sinuses of horses). the respiratory form of evr is a mild and a transient bronchointerstitial pneumonia seen only by pathologists when complications with secondary bacterial infections cause a fatal bronchopneumonia microscopically in sections by pas reaction or grocott-gomori's methenamine silver (gms) stain for fungi or by an acid-fast stain for mycobacteria. viral infections of the respiratory tract, particularly equine viral rhinopneumonitis and equine influenza, are important diseases of horses throughout the world. the effects of these and other respiratory viruses on the horse can be manifested in three distinct ways. first, as pure viral infections, their severity may range from mild to severe, making them a frequent interfering factor in training and athletic performance. second, superimposed infections by opportunistic bacteria, such as streptococcus spp., escherichia coli, klebsiella pneumoniae, rhodococcus equi, and various anaerobes, can cause fibrinous or suppurative bronchopneumonias. third, it is possible equine henipavirus (hendra virus). fatal cases of a novel respiratory disease in horses and human beings suddenly appeared in approximately 1994 in hendra, a suburb of brisbane, australia. this outbreak was attributed to a newly recognized zoonotic virus that was tentatively named equine morbillivirus. now called hendra virus (hev), this emerging viral pathogen is currently classified as a member of the genus henipavirus (includes hendra virus and nipah virus), in the family paramyxoviridae. fruit bats (flying foxes) act as natural reservoirs and are involved in the transmission by poorly understood mechanisms. the lungs of affected horses are severely edematous with gelatinous distention of pleura and subpleural lymph vessels. microscopically, the lungs have diffuse alveolar edema associated with vasculitis, thrombosis, and the presence of multinucleated syncytial cells in the endothelium of small pulmonary blood vessels and alveolar capillaries. the lymphatic vessels are notably distended with fluid. the characteristic inclusion bodies seen in other paramyxovirus infections are not seen in horses; however, the virus can be easily detected by immunohistochemistry in pulmonary endothelial cells and alveolar epithelial cells (pneumonocytes). clinical signs are nonspecific and include fever, anorexia, respiratory distress, and nasal discharge. equine multinodular pulmonary fibrosis. equine multinodular pulmonary fibrosis is a lung disease characterized by well-demarcated fibrotic nodular lesions in the lung (efig. 9-13 ). until recently, the pathogenesis was unclear, but recent studies proposed equine herpesvirus 5 (ehv-5) as the putative etiology. grossly, the lungs show multifocal to coalescing, firm tan nodules scattered in all pulmonary lobes, which resemble pulmonary neoplasia. microscopically, alveolar walls are thickened due to collagen deposition, infiltration of lymphocytes and macrophages, and cuboidal cells lining the alveolar walls. the alveolar lumens contain neutrophils and macrophages, some of which may contain a large eosinophilic intranuclear inclusion body. typical clinical signs include weight loss, low-grade fever, and progressive exercise intolerance. this condition has a poor prognosis. rhodococcus equi. rhodococcus equi is an important cause of morbidity and mortality in foals throughout the world. this facultative intracellular gram-positive bacterium causes two major forms of disease: the first involves the intestine, causing ulcerative enterocolitis, and the second severe and often fatal bronchopneumonia. although half of foals with pneumonia have ulcerative enterocolitis, it is rare to find animals with intestinal lesions alone. occasionally, infection disseminates to lymph nodes, joints, bones, the genital tract, and other organs. because rhodococcus equi is present in soil and feces of herbivores (particularly foals), it is common for the disease to become enzootic on farms ("hot spots") where the organism has been shed earlier by infected foals. serologic evidence of infection in horses is widespread, yet clinical disease is sporadic and largely restricted to young foals or to adult horses with severe immunosuppression. virulence factors encoded by plasmids (virulenceassociated protein a [vapa gene]) are responsible for the survival and replication of rhodococcus equi in macrophages, thus determining the evolution of the disease. this bacterium has also been sporadically incriminated with infections in cattle, goats, pigs, dogs, and cats, and quite often in immunocompromised human beings, for example, those infected with the aids virus, after organ transplantation, or undergoing chemotherapy. it is still debatable whether natural infection starts as a bronchopneumonia (aerogenous route) from which rhodococcus equi reaches the intestine via swallowed sputum or whether infection starts as an enteritis (oral route) with a subsequent bacteremia into the lungs. (streptococcus equi, streptococcus zooepidemicus, or staphylococcus aureus) . uncomplicated lesions in evr are seen only in aborted fetuses or in foals that die within the first few days of life. they consist of focal areas of necrosis (0.5 to 2 mm) in various organs, including liver, adrenal glands, and lungs. in some cases, intranuclear inclusion bodies are microscopically observed in these organs. outbreaks of interstitial pneumonia in donkeys have been attributed to multiple strains of asinine herpesviruses (ahv-4 and -5). clinically, horses and donkeys affected with the respiratory form of evr exhibit fever, anorexia, conjunctivitis, cough, and nasal discharge. equine viral arteritis. equine viral arteritis (eva), a pansystemic disease of horses, donkeys, and mules caused by an arterivirus (equine arteritis virus [eav]), occurs sporadically throughout the world, sometimes as an outbreak. this virus infects and causes severe injury to macrophages and endothelial cells. gross lesions are hemorrhage and edema in many sites, including lungs, intestine, scrotum, and periorbital tissues and voluminous hydrothorax and hydroperitoneum. the basic lesion is fibrinoid necrosis and inflammation of the vessel walls (vasculitis), particularly the small muscular arteries (lymphocytic arteritis), which is responsible for the edema and hemorrhage that explain most of the clinical features. pulmonary lesions are those of interstitial pneumonia with hyperplasia of type ii pneumonocytes and vasculitis with abundant edema in the bronchoalveolar spaces and distended pulmonary lymphatic vessels. viral antigen can be detected by immunoperoxidase techniques in the walls and endothelial cells of affected pulmonary vessels and in alveolar macrophages. clinical signs are respiratory distress, fever, abortion, diarrhea, colic, and edema of the limbs and ventral abdomen. respiratory signs are frequent and consist of serous or mucopurulent rhinitis and conjunctivitis with palpebral edema. like most viral respiratory infections, eva can predispose horses to opportunistic bacterial pneumonias. african horse sickness. african horse sickness (ahs) is an arthropod-borne, oie-notifiable disease of horses, mules, donkeys, and zebras that is caused by an orbivirus (family reoviridae) and characterized by respiratory distress or cardiovascular failure. ahs has a high mortality rate-up to 95% in the native population of horses in africa, the middle east, india, pakistan, and, most recently, spain and portugal. although the ahs virus is transmitted primarily by insects (culicoides) to horses, other animals, such as dogs, can be infected by eating infected equine flesh. the pathogenesis of african horse sickness remains unclear, but this equine orbivirus has an obvious tropism for pulmonary and cardiac endothelial cells and, to a lesser extent, mononuclear cells. based on clinical signs (not pathogenesis), african horse sickness is arbitrarily divided into four different forms: pulmonary, cardiac, mixed, and mild. the pulmonary form is characterized by severe respiratory distress and rapid death because of massive pulmonary edema, presumably from viral injury to the pulmonary endothelial cells. grossly, large amounts of froth are present in the airways, lungs fail to collapse, subpleural lymph vessels are distended, and the ventral parts of the lungs are notably edematous (see fig. 4-40) . in the cardiac form, recurrent fever is detected, and heart failure results in subcutaneous and interfascial edema, most notably in the neck and supraorbital region. the mixed form is a combination of the respiratory and cardiac forms. finally, the mild form, rarely seen in postmortem rooms, is characterized by fever and clinical signs resembling those of equine influenza; it is in most cases transient and followed by a complete recovery. this mild form is most frequently seen in donkeys, mules, and zebras and in horses with some degree of immunity. detection of viral antigen for diagnostic purposes can be done by immunohistochemistry in paraffin-embedded tissues. chapter 9 respiratory system, mediastinum, and pleurae clinically, rhodococcus equi infection can be acute, with rapid death caused by severe bronchopneumonia, or chronic, with depression, cough, weight loss, and respiratory distress. in either form, there may be diarrhea, arthritis, osteomyelitis, or subcutaneous abscess formation. parascaris equorum. parascaris equorum is a large nematode (roundworm) of the small intestine of horses; the larval stages migrate through the lungs as ascarid larvae do in pigs. it is still unclear whether migration of parascaris equorum larvae can cause significant pulmonary lesions under natural conditions. experimentally, migration of larvae results in coughing, anorexia, weight loss, and small necrotic foci and petechial hemorrhages in the liver, hepatic and tracheobronchial lymph nodes, and lungs. microscopically, eosinophils are prominent in the interstitium and airway mucosa during the parasitic migration and in focal granulomas caused by dead larvae in the lung. dictyocaulus arnfieldi. dictyocaulus arnfieldi is not a very pathogenic nematode, but it should be considered if there are signs of coughing in horses that are pastured together with donkeys. donkeys are considered the natural hosts and can tolerate large numbers of parasites without ill effects. dictyocaulus arnfieldi does not usually become patent in horses, so examination of fecal samples is not useful; bal is only occasionally diagnostic because eosinophils (but not parasites) are typically found in the lavage fluid. mature parasites (up to 8 cm in length) cause obstructive bronchitis, edema, and atelectasis, particularly along the dorsocaudal lung. the microscopic lesion is an eosinophilic bronchitis similar to the less acute infestations seen in cattle and sheep with their dictyocaulus species. the results of experimental studies suggest that natural infection likely starts from inhalation of infected dust or aerosols. once in the lung, rhodococcus equi is rapidly phagocytosed by alveolar macrophages, but because of defective phagosome-lysosome fusion and premature lysosomal degranulation, bacteria survive and multiply intracellularly, eventually leading to the destruction of the macrophage. interestingly, rhodococcus equi appears to be easily killed by neutrophils but not macrophages. released cytokines and lysosomal enzymes and bacterial toxins are responsible for extensive caseous necrosis of the lungs and the recruitment of large numbers of neutrophils, macrophages, and giant cells containing intracellular gram-positive organisms in their cytoplasm. depending on the stage of infection and the immune status and age of affected horses, pulmonary lesions induced by rhodococcus equi can vary from pyogranulomatous to granulomatous pneumonia. in young foals, the infection starts as a suppurative cranioventral bronchopneumonia, which progresses within a few days into small variable-size pulmonary abscesses. these abscesses rapidly transform into pyogranulomatous nodules, some of which become confluent and form large masses of caseous exudate ( fig. 9-82 ). microscopically, the early lesion starts with neutrophilic infiltration, followed by an intense influx of alveolar macrophages into the bronchoalveolar spaces. this type of histiocytic inflammation persists for a long period of time because rhodococcus equi is a facultative intracellular organism that survives the bactericidal effects of equine alveolar macrophages. in the most chronic cases, the pulmonary lesions culminate with the formation of large caseonecrotic masses with extensive fibrosis of the surrounding pulmonary parenchyma. pcr analysis of tracheobronchial aspirates has successfully been used as an alternative to bacteriologic culture in the diagnosis of rhodococcus equi infection in live foals. b a rhinotracheitis (ibr)/bovine herpes virus 1 (bohv-1), bovine parainfluenza virus 3 (bpiv-3), and bovine respiratory syncytial virus (brsv); and noninfectious interstitial pneumonias, such as bovine pulmonary edema and emphysema, reinfection syndrome, and many others. bovine enzootic pneumonia. enzootic pneumonia, sometimes simply referred to as calf pneumonia, is a multifactorial disease caused by a variety of etiologic agents that produces an assortment of lung lesions in young, intensively housed calves. the hostmicrobial-environmental triad is central in the pathogenesis of this disease. morbidity is often high (up to 90%), but fatalities are uncommon (>5%) unless management is poor or unless new, virulent pathogens are introduced by additions to the herd. enzootic pneumonia is also called viral pneumonia because it often begins with an acute respiratory infection with bpiv-3, brsv, or possibly with one or more of several other viruses (adenovirus, bohv-1, reovirus, bovine coronavirus [bcov] , and bovine rhinitis virus). mycoplasmas, notably mycoplasma dispar, mycoplasma bovis, ureaplasma, and possibly chlamydophila, may also be primary agents. following infection with any of these agents, opportunistic bacteria, such as pasteurella multocida, trueperella (arcanobacterium) pyogenes, histophilus somni, mannheimia haemolytica, and escherichia coli, can cause a secondary suppurative bronchopneumonia, the most serious stage of enzootic pneumonia. the pathogenesis of the primary invasion and how it predisposes the host to invasion by the opportunists are poorly understood, but it is likely that there is impairment of pulmonary defense mechanisms. environmental factors, including air quality (poor ventilation), high relative humidity, and animal crowding, have been strongly incriminated. the immune status of the calf also plays an important role in the development and severity of enzootic pneumonia. calves with bovine leukocyte adhesion deficiency (blad), which prevents the migration of neutrophils from the capillaries, are highly susceptible to bronchopneumonia. lesions are variable and depend largely on the agents involved and on the duration of the inflammatory process. in the acute phases, lesions caused by viruses are those of bronchointerstitial pneumonia, which are generally mild and transient, and therefore are seen only sporadically at necropsy. microscopically, the lesions are necrotizing bronchiolitis, necrosis of type i pneumonocytes with hyperplasia of type ii pneumonocytes, and mild interstitial and alveolar edema. in the case of bpiv-3 and brsv infection, intracytoplasmic inclusion bodies and the formation of large multinucleated syncytia, resulting from the fusion of infected bronchiolar and alveolar epithelial cells, can also be observed in the lungs (fig. 9-83) . airway hyperreactivity has been described in calves after brsv infection; however, the significance of this syndrome in relation to enzootic pneumonia of calves is still under investigation. the mycoplasmas also can cause bronchiolitis, bronchiolar and alveolar necrosis, and an interstitial reaction, but in contrast to viral-induced pneumonias, mycoplasmal lesions tend to progress to a chronic stage characterized by striking peribronchiolar lymphoid hyperplasia (cuffing pneumonia). when complicated by secondary bacterial infections (e.g., pasteurella multocida and trueperella pyogenes), viral or mycoplasmal lesions change from a pure bronchointerstitial to a suppurative bronchopneumonia (fig. 9-84) . in late stages of bronchopneumonia, the lungs contain a creamy-mucoid exudate in the airways and later often have pulmonary abscesses and bronchiectasis (see fig. 9-11) . note that the same viruses and mycoplasmas involved in the enzootic pneumonia complex can also predispose cattle to other diseases, such as pneumonic mannheimiosis (mannheimia aspiration pneumonia. aspiration pneumonia is often a devastating sequela to improper gastric tubing of horses, particularly exogenous lipid pneumonia from mineral oil delivered into the trachea in treatment of colic. gross and microscopic lesions are described in detail in the section on aspiration pneumonias of cattle. opportunistic infections. chlamydophila (chlamydia) spp., obligatory intracellular zoonotic pathogens, can cause systemic infection in many mammalian and avian species; in horses, they can also cause keratoconjunctivitis, rhinitis, pneumonia, abortion, polyarthritis, enteritis, hepatitis, and encephalitis. serologic studies suggest that infection without apparent disease is common in horses. horses experimentally infected with chlamydophila psittaci develop mild and transient bronchointerstitial pneumonia. there are unconfirmed reports suggesting a possible association between these organisms and recurrent airway obstruction in horses. detection of chlamydial organisms in affected tissue is not easy and requires special laboratory techniques such as pcr, immunohistochemistry, and fluorescent antibody tests. horses are only sporadically affected with mycobacteriosis (mycobacterium avium complex, mycobacterium tuberculosis, and mycobacterium bovis). the intestinal tract and associated lymph nodes are generally affected, suggesting an oral route of infection with subsequent hematogenous dissemination to the lungs. the tubercles (granulomas) differ from those in ruminants and pigs, being smooth, gray, solid, sarcoma-like nodules without grossly visible caseous necrosis or calcification (efig. 9-14) . microscopically, the tubercles are composed of macrophages, epithelioid cells, and multinucleated giant cells. fibrosis increases with time, accounting in part for the sarcomatous appearance. adenovirus infections occur commonly in arabian foals with combined immunodeficiency (cid), a hereditary lack of b and t lymphocytes. in cases of adenoviral infection, large basophilic or amphophilic inclusions are present in the nuclei of tracheal, bronchial, bronchiolar, alveolar, renal, and intestinal epithelial cells. as it occurs in other species, infection with a unique fungal pathogen known as pneumocystis carinii typically occurs in immunosuppressed or immunoincompetent individuals such as arabian foals with cid (see fig. 9 -20). diagnosis of pneumocystis carinii requires microscopic examination of lungs and special stains. idiopathic interstitial pneumonia. interstitial and bronchointerstitial pneumonias of undetermined cause that can progress to severe pulmonary fibrosis have been reported in foals and young horses. the gross and microscopic lesions are reminiscent of those of bovine pulmonary edema and emphysema or ards. the lungs are notably congested and edematous and microscopically are characterized by necrosis of the bronchiolar epithelium, alveolar edema, hyperplasia of type ii pneumonocytes, and hyaline membranes. the cause of this form of equine interstitial pneumonia is not known, but toxic and particularly viral causes have been proposed. bovine respiratory disease complex (brdc) and acute undifferentiated respiratory disease are general terms often used by clinicians to describe acute and severe bovine respiratory illness of clinically undetermined cause. these terms do not imply any particular type of pneumonia and therefore should not be used in pathology reports. clinically, the brd complex includes bovine enzootic pneumonia (multifactorial etiology); pneumonic mannheimiosis (mannheimia haemolytica); respiratory histophilosis (histophilus somni), previously known as respiratory hemophilosis (haemophilus somnus); mycoplasma bovis; respiratory viral infections, such as infectious bovine 528.e1 chapter 9 respiratory system, mediastinum, and pleurae pneumonic mannheimiosis (shipping fever) is the most important respiratory disease of cattle in north america, particularly in feedlot animals that have been through the stressful marketing and assembly processes. mannheimia haemolytica biotype a, serotype 1 is the etiologic agent most commonly responsible for the severe pulmonary lesions. a few investigators still consider that pasteurella multocida and other serotypes of mannheimia haemolytica are also causes of this disease. even after many years of intense investigation, from the gross lesions to the molecular aspects of the disease, the pathogenesis of pneumonic mannheimiosis remains incompletely understood. experiments have established that mannheimia haemolytica a1 alone is usually incapable of causing disease because it is rapidly cleared by pulmonary defense mechanisms. these findings may explain why mannheimia haemolytica, despite being present in the nasal cavity of healthy animals, only sporadically causes disease. for mannheimia haemolytica to be established as a pulmonary infection, it is first required that stressors impair the defense mechanisms and allow the bacteria to colonize the lung (see section on impairment of defense haemolytica). clinically, enzootic pneumonia is usually mild, but fatal cases are occasionally seen even in farms with optimal health management. pneumonic mannheimiosis (shipping fever). shipping fever (transit fever) is a vague clinical term used to denote acute respiratory diseases that occur in cattle several days or weeks after shipment. the disease is characterized by a severe fibrinous bronchopneumonia, reflecting the fact that death generally occurs early or at an acute stage. because mannheimia haemolytica (formerly pasteurella haemolytica) is most frequently isolated from affected lungs, the names pneumonic mannheimiosis and pneumonic pasteurellosis have been used synonymously. it is known that pneumonic mannheimiosis can occur in animals that have not been shipped and that organisms other than mannheimia haemolytica can cause similar lesions. therefore the term shipping fever should be relinquished in favor of more specific names, such as pneumonic mannheimiosis or respiratory histophilosis. irregular areas of coagulative necrosis are typically bordered by a rim of elongated cells often referred to as oat-shaped cells or oat cells that are degenerating neutrophils mixed with a few alveolar macrophages (see fig. 9 -86). in the early stages of necrosis, there is no evidence of vascular thrombosis, suggesting that necrosis is primarily caused by the cytotoxin of mannheimia haemolytica and is not the result of an ischemic change. the interlobular septa become distended with protein-rich edematous fluid, and the lymphatic vessels contain fibrin thrombi. the trachea and bronchi can have considerable amounts of blood and exudate, which are transported by the mucociliary escalator or coughed up from deep within the lungs, but the walls of the trachea and major bronchi may or may not be involved. because of the necrotizing process, sequelae to pneumonic mannheimiosis can be serious and can include abscesses, encapsulated sequestra (isolated pieces of necrotic lung), chronic pleuritis, fibrous pleural adhesions, and bronchiectasis. clinically, pneumonic mannheimiosis is characterized by a severe toxemia that can kill animals even when considerable parts of the lungs remain functionally and structurally normal. cattle usually become depressed, febrile (104° to 106° f [40° to 41° c]), and anorexic and have a productive cough, encrusted nose, mucopurulent nasal exudate, shallow respiration, or an expiratory grunt. hemorrhagic septicemia. pneumonic mannheimiosis should not be confused with hemorrhagic septicemia (septicemic pasteurellosis) of cattle and water buffalo (bubalus bubalis) caused by inhalation or ingestion of serotypes 6:b and 6:e of pasteurella multocida. this oie-notifiable disease does not occur in north america and currently is reported only from some countries in asia, africa, and recently in germany. in contrast to pneumonic mannheimiosis, in which lesions are always confined to the lower respiratory tract, the bacteria of hemorrhagic septicemia always disseminates hematogenously to other organs. at necropsy, typically, generalized petechiae are present on the serosal surfaces of the intestine, heart, and lungs and in skeletal muscles. superficial and visceral lymph nodes are swollen and hemorrhagic. variable lesions include edematous and hemorrhagic lungs with or without consolidation; hemorrhagic enteritis; blood-tinged fluid in the thorax and abdomen; and subcutaneous edema of the head, neck, and ventral abdomen. bacteria can be cultured from blood, and animals have high fever and die rapidly (100% case fatality). respiratory histophilosis (haemophilosis). respiratory histophilosis is part of the histophilus somni (haemophilus somnus) disease complex, which has at least eight different clinicopathologic forms, each one involving different organs. this complex includes septicemia, encephalitis (known as thrombotic meningoencephalitis [tme]), pneumonia (respiratory histophilosis), pleuritis, myocarditis, arthritis, ophthalmitis, conjunctivitis, otitis, and abortion. the portals of entry for the different forms of histophilosis have not been properly established. the respiratory form of bovine histophilosis is the result of the capacity of the bacterium to induce both suppurative and fibrinous bronchopneumonia (efig. 9-15 ). the latter is in some cases indistinguishable from that of pneumonic mannheimiosis. the pathogenesis of respiratory histophilosis is still poorly understood, and the disease cannot be reproduced consistently by administration of histophilus somni alone. like mannheimia haemolytica, it requires predisposing factors such as stress or a preceding viral infection. histophilus somni is often isolated from the lungs of calves with enzootic pneumonia. the capacity of histophilus somni to cause septicemia and localized infections in the lungs, brain, eyes, ear, heart, mammary gland, male and female genital organs, or placenta is perhaps attributable to specific virulence factors, such as immunoglobulin-binding proteins (igbps) and lipooligosaccharide (los). also, histophilus mechanisms). these stressors include weaning, transport, fatigue, crowding, mixing of cattle from various sources, inclement weather, temporary starvation, and viral infections. horizontal transmission of viruses and mannheimia haemolytica occurs during crowding and transportation of cattle. viruses that most commonly predispose cattle to pneumonic mannheimiosis include bohv-1, bpiv-3, and brsv. once established in the lungs, mannheimia haemolytica causes lesions by means of different virulence factors, which include endotoxin, lipopolysaccharide, adhesins, and outer membrane proteins; however, the most important is probably the production of a leukotoxin (exotoxin), which binds and kills bovine macrophages and neutrophils. the fact that this toxin exclusively affects ruminant leukocytes probably explains why mannheimia haemolytica is a respiratory pathogen in cattle and sheep but not in other species. during mannheimia haemolytica infection, alveolar macrophages, neutrophils, and mast cells release maximum amounts of proinflammatory cytokines, particularly tnf-α, il-1, il-8, adhesion molecules, histamine, and leukotrienes. by locally releasing enzymes and free radicals, leukocytes further contribute to the injury and necrosis of bronchiolar and alveolar cells. the gross lesions of acute and subacute pneumonic mannheimiosis are the prototypic fibrinous bronchopneumonia, with prominent fibrinous pleuritis ( fig. 9-85 and see fig. 9 -72) and pleural effusion. lesions are always cranioventral and usually ventral to a horizontal line through the tracheal bifurcation. the interlobular septa are distended by yellow, gelatinous edema and fibrin. the "marbling" of lobules is the result of intermixing areas of coagulation necrosis, interlobular interstitial edema, and congestion ( fig. 9-86) . microscopically, lung lesions are evident 4 hours after experimental infection in which neutrophils fill the bronchial, bronchiolar, and alveolar spaces. within 24 to 48 hours, the cytotoxic effect of mannheimia haemolytica is manifested by necrosis of individual alveolar cells and fibrin begins to exude into the alveoli from increased permeability of the air-blood barrier. these changes are exacerbated by endothelial swelling, altered platelet function, increased procoagulant activity, and diminished profibrinolytic activity in the lungs. by 72 hours, alveolar macrophages start to appear in the bronchoalveolar space. at this time, large and the pulmonary defense mechanisms. lung lesions are typically those of a chronic bronchopneumonia with numerous well-delineated caseonecrotic nodules (fig. 9-87 and e-fig. 9-16) . microscopically, lesions are quite characteristic and consist of distinct areas of pulmonary necrosis centered on bronchi or bronchioles. the lesion is formed by a core of fine eosinophilic granular debris surrounded by a rim of neutrophils, macrophages, and fibroblasts (see fig. 9-87) . although the origin of the caseonecrotic lesions is under investigation, recent studies incriminate reactive oxygen species (ros) and reactive nitrogen species (rns) as the major contributors for cell injury in the lung. the diagnosis is confirmed by isolation or somni has the ability to undergo structural and antigenic variation, evade phagocytosis by promoting leukocytic apoptosis, inhibit intracellular killing, reduce transferrin concentrations, and induce endothelial apoptosis in the lungs of affected calves. mixed pulmonary infections of histophilus somni, mannheimia haemolytica, pasteurella multocida, trueperella pyogenes, and mycoplasmas are fairly common in calves. mycoplasma bovis pneumonia. mycoplasma bovis is the most common mycoplasma sp. isolated from pneumonic lungs of cattle in europe and north america. pulmonary infection is exacerbated by stress or any other adverse factor (e.g., viral infection) that depresses n control programs for infectious disease. it was eradicated from north america in 1892 and from australia in the 1970s, but it is still enzootic in large areas of africa, asia, and eastern europe. the etiologic agent, mycoplasma mycoides ssp. mycoides small colony type, was the first mycoplasma isolated and is one of the most pathogenic of those that infect domestic animals. natural infection occurs in cattle and asian buffalo. the portal of entry is aerogenous, and infections occur when a susceptible animal inhales infected droplets. the pathogenic mechanisms are still inadequately understood but are suspected to involve toxin and galactan production, unregulated production of tnf-α, ciliary dysfunction, immunosuppression, and immune-mediated vasculitis. vasculitis and thrombosis of pulmonary arteries, arterioles, veins, and lymphatic vessels lead to lobular infarction. the name of the disease is a good indication of the gross lesions. it is a severe, fibrinous bronchopneumonia (pleuropneumonia) similar to that of pneumonic mannheimiosis (see figs. 9-72 and 9-85) but having a more pronounced "marbling" of the lobules because of extensive interlobular edema and lymphatic thrombosis. typically, 60% to 79% of lesions are in the caudal lobes (not cranioventrally), and pulmonary sequestra (necrotic lung encapsulated by connective tissue) are more frequent and larger than pneumonic mannheimiosis. unilateral lesions are common in this disease. microscopically, the appearance again is like that of pneumonic mannheimiosis, except that vasculitis and thrombosis of pulmonary arteries, arterioles, and capillaries are much more obvious and are clearly the major cause of the infarction and thrombosis of lymphatic vessels in interlobular septa. mycoplasma mycoides ssp. mycoides small colony type remains viable in the sequestra for many years, and under stress (e.g., starvation), the fibrous capsule may break down releasing mycoplasma into the airways, thus becoming a source of infection for other animals. clinical signs are those of severe sepsis, including fever, depression, and anorexia followed by severe respiratory signs such as opened-mouth breathing, dyspnea and coughing, and crepitation and pleural friction on thoracic auscultation. vaccination is highly effective in preventing the disease. bovine tuberculosis. tuberculosis is an ancient, communicable, worldwide, chronic disease of human beings and domestic animals. it continues to be a major problem in human beings in underdeveloped countries, and it is on the rise in some industrialized nations, largely because of the immunosuppressive effects of aids, immigration, and movement of infected animals across borders. the world health organization (who) estimates that more than 1 million people die of tuberculosis and 8 million new cases appear each year, mostly in developing countries. mycobacterium tuberculosis is transmitted between human beings, but where unpasteurized milk is consumed, mycobacterium bovis from the milk of cattle with mammary tuberculosis is also an important cause of human tuberculosis. mycobacterium bovis infections have also been reported in a number of domestic and wild mammalian species; in some countries, wildlife reservoirs exist and may act as a source of infection for cattle. bovine tuberculosis is primarily caused by mycobacterium bovis, but infection with mycobacterium tuberculosis, the pathogen of human tuberculosis, and mycobacterium caprae (formerly mycobacterium bovis ssp. caprae/mycobacterium tuberculosis ssp. caprae) can occur sporadically. tuberculosis can be acquired by several routes, but infection of the lungs by inhalation of mycobacterium bovis is the most common in adult cattle, whereas ingestion of infected milk is more predominant in young animals. organisms belonging to the mycobacterium avium complex can also infect cattle, but for infection caused by these organisms, the term atypical mycobacteriosis (not tuberculosis) is currently preferred. immunohistochemical labeling of tissue sections for mycoplasma antigens. mycoplasma bovis is also incriminated in arthritis, otitis, mastitis, abortion, and keratoconjunctivitis. contagious bovine pleuropneumonia. contagious bovine pleuropneumonia is an oie-notifiable disease of historic interest in veterinary medicine because it was the object of early national a b c than 90% of bovine cases, a chronic, moist cough can progress to dyspnea. enlarged tracheobronchial lymph nodes can contribute to the dyspnea by impinging on airways, and the enlargement of caudal mediastinal nodes can compress the caudal thoracic esophagus and cause bloating. interstitial pneumonias. atypical interstitial pneumonia (aip) is a vague clinical term well entrenched in veterinary literature but one that has led to enormous confusion among veterinarians. it was first used to describe acute or chronic forms of bovine pneumonia that did not fit in any of the "classic" forms because of the lack of exudate and lack of productive cough. microscopically, the criteria for diagnosis of aip in cattle were based on the absence of obvious exudate and the presence of edema, interstitial emphysema (see the section on pulmonary emphysema), hyaline membranes, hyperplasia of type ii pneumonocytes, and alveolar fibrosis with interstitial cellular infiltrates. at that time, any pulmonary disease or pulmonary syndrome that had a few of the previously mentioned lesions was traditionally diagnosed as aip, and grouping all these different syndromes together was inconsequential because their etiopathogenesis were then unknown. field and laboratory investigations have demonstrated that most of the bovine syndromes previously grouped under aip have rather different causes and pathogeneses ( fig. 9-88) . furthermore, what was "atypical" in the past has become so common that it is fairly routine nowadays to find "typical cases" of aip. for all these reasons, investigators, largely from britain, proposed that all these syndromes previously clustered into aip should be named according to their specific cause or pathogenesis. the most common bovine syndromes characterized by edema, emphysema, hyaline membranes, and hyperplasia of type ii pneumonocytes include bovine pulmonary edema and emphysema (fog fever), "extrinsic allergic alveolitis" (hypersensitivity pneumonitis), "reinfection syndromes" (hypersensitivity to dictyocaulus sp. or brsv), milk allergy, ingestion of moldy potatoes, paraquat toxicity, toxic silo gases, mycotoxins, and others. acute bovine pulmonary edema and emphysema (fog fever). acute bovine pulmonary edema and emphysema (abpee), known in britain as fog fever (no association with atmospheric conditions), occurs in cattle usually grazing "fog" pastures (i.e., aftermath or foggage, regrowth after a hay or silage has been cut). epidemiologically, abpee usually occurs in adult beef cattle in the fall when there is a change in pasture from a short, dry grass to a lush, green grass. it is generally accepted that l-tryptophan present in the pasture is metabolized in the rumen to 3-methylindole, which in turn is absorbed into the bloodstream and carried to the lungs. mixed function oxidases present in the nonciliated bronchiolar epithelial (club) cells metabolize 3-methylindole into a highly pneumotoxic compound that causes extensive and selective necrosis of bronchiolar cells and type i pneumonocytes (fig. 9-89 and see fig. 9 -88) and increases alveolar permeability, leading to edema, thickening of the alveolar interstitium, and alveolar and interstitial emphysema. 3-methylindole also interferes with the lipid metabolism of type ii pneumonocytes. the gross lesions are those of a diffuse interstitial pneumonia with severe alveolar and interstitial edema and interlobular emphysema (see fig. 9-55, a) . the lungs are expanded, pale, and rubbery in texture, and the lesions are most notable in the caudal lobes. microscopically, the lesions are alveolar and interstitial edema and emphysema, formation of characteristic hyaline membranes within alveoli (see fig. 9-55, b) , and in those animals that survive for several days, hyperplasia of type ii pneumonocytes and alveolar interstitial fibrosis. respiratory infection usually starts when inhaled bacilli reach the alveoli and are phagocytosed by pulmonary alveolar macrophages. if these cells are successful in destroying the bacteria, infection is averted. however, mycobacterium bovis, being a facultative pathogen of the monocytic-macrophage system, may multiply intracellularly, kill the macrophage, and initiate infection. from this first nidus of infection, bacilli spread aerogenously via airways within the lungs and eventually via the lymph vessels to tracheobronchial and mediastinal lymph nodes. the initial focus of infection at the portal of entry (lungs) plus the involvement of regional lymph nodes is termed the primary (ghon) complex of tuberculosis. if the infection is not contained within this primary complex, bacilli disseminate via the lymph vessels to distant organs and other lymph nodes by the migration of infected macrophages. hematogenous dissemination occurs sporadically when a granuloma containing mycobacteria erodes the wall of a blood vessel, causes vasculitis, and allows the granuloma to discharge mycobacteria into the alveolar circulation. if dissemination is sudden and massive, mycobacteria are widely disseminated and numerous small foci of infection develop in many tissues and organs and the process is referred to as miliary tuberculosis (like millet seeds). the host becomes hypersensitive to the mycobacterium, which enhances the cell-mediated immune defenses in early or mild infections but can result in host-tissue destruction in the form of caseous necrosis. the evolution and dissemination of the pulmonary infection are closely regulated by cytokines and tnf-α production by alveolar macrophages. unlike abscesses that tend to grow rather fast, granulomas evolve slowly at the site of infection. the lesion starts with few macrophages and neutrophils ingesting the offending organism, but because mycobacterium organisms are resistant to phagocytosis, infected macrophages eventually die, releasing viable bacteria, lipids, and cell debris. cell debris accumulates in the center of the lesion, whereas viable bacteria and bacterial lipids attract additional macrophages and a few lymphocytes at the periphery of the lesion. some of these newly recruited macrophages are activated by local lymphocytes and become large phagocytic cells with abundant cytoplasm resembling epithelial cells, thus the term epithelioid macrophages. multinucleated giant cells (also macrophages) appear at the edges of the lesion, and finally the entire focus of inflammatory process becomes surrounded by fibroblasts and connective tissue (see fig. 9 -81). it may take weeks or months for a granuloma to be grossly visible. bovine tuberculosis, the prototype for granulomatous pneumonia, is characterized by the presence of a few or many caseated granulomas (see fig. 9 -80). the early gross changes are small foci (tubercles) most frequently seen in the dorsocaudal, subpleural areas. with progression, the lesions enlarge and become confluent with the formation of large areas of caseous necrosis. calcification of the granulomas is a typical finding in bovine tuberculosis. single nodules or clusters occur on the pleura and peritoneum, and this presentation has been termed pearl disease. microscopically, the tubercle is composed of mononuclear cells of various types. in young tubercles, which are noncaseous, epithelioid and langhans' giant cells are at the center, surrounded by lymphocytes, plasma cells, and macrophages. later, caseous necrosis develops at the center, secondary to the effects of cell-mediated hypersensitivity and enclosed by fibrosis at the periphery. acid-fast organisms may be numerous but more often are difficult to find in histologic section or smears. clinically, the signs of tuberculosis relate to the dysfunction of a particular organ system or to general debilitation, reduced milk production, and emaciation. in the pulmonary form, which is more grossly, the postmortem lesions vary from subtle, gray, subpleural foci (granulomatous inflammation) to severe lesions, in which the lungs are firm and heavy and have a "meaty appearance" because of interstitial pneumonia (efig. 9 -17) with type ii pneumonocyte hyperplasia, lymphocytic infiltration, and interstitial fibrosis. characteristically, discrete noncaseous granulomas formed in response to the deposition of antigen-antibody complexes are scattered throughout the lungs. chronic cases of extrinsic allergic alveolitis can eventually progress to diffuse fibrosing alveolitis. clinically, it can be acute or chronic; the latter has a cyclical pattern of exacerbation during winter months. weight loss, coughing, and poor exercise tolerance are clinical features. full recovery can occur if the disease is recognized and treated early. reinfection syndrome. hypersensitivity to reinfection with larvae of dictyocaulus viviparus is another allergic syndrome manifested in the lungs that causes signs and lesions indistinguishable from abpee, with the exception of eosinophils and possibly larvae in the alveolar exudate. the hypersensitivity reaction in the lung causes diffuse alveolar damage and edema, necrosis of type i pneumonocytes, and hyperplasia of type ii pneumonocytes. in the later stages of the disease, there is formation of small granulomas with interstitial infiltrates of mononuclear cells. it has been suggested but not confirmed that emphysema with diffuse proliferative alveolitis and formation of hyaline membranes can also occur sporadically in the late stages of brsv infection in cattle. presumably, this disease shares many similarities with "atypical" infections occasionally seen in children with respiratory syncytial virus (rsv human strain), in which a hypersensitivity to the virus or virus-induced augmentation of the immune response results in hypersensitivity pneumonitis (see fig. 9 -88). brsv infection is also known to enhance hypersensitivity to environmental allergens in cattle. other forms of bovine interstitial pneumonia. inhalation of manure ("pit") gases, such as nitrogen dioxide (no 2 ), hydrogen interstitial cell infiltrates, fibrosis, emphysema acute proliferation phase hyperplasia of type ii pneumonocytes clinically, severe respiratory distress develops within 10 days of the abrupt pasture change, and cattle develop expiratory dyspnea, oral breathing, and evidence of emphysema within the lungs and even subcutaneously along the back. experimentally, reducing ruminal conversion of l-tryptophan to 3-methylindole prevents the development of abpee. a number of other agents cause virtually the same clinical and pathologic syndrome as is seen in abpee. the pathogenesis is assumed to be similar, although presumably other toxic factors are specific for each syndrome. one of these pneumotoxic factors is 4-ipomeanol, which is found in moldy sweet potatoes contaminated with the fungus fusarium solani. mixed function oxidases in the lungs activate 4-ipomeanol into a potent pneumotoxicant capable of producing irreversible oxidative injury to type i pneumonocytes and bronchiolar epithelial cells, presumably through lipoperoxidation of cell membranes. similarly, purple mint (perilla frutescens), stinkwood (zieria arborescens), and rapeseed and kale (brassica species) also cause pulmonary edema, emphysema, and interstitial pneumonia. extrinsic allergic alveolitis. extrinsic allergic alveolitis (hypersensitivity pneumonitis), one of the most common allergic diseases in cattle, is seen mainly in housed adult dairy cows in the winter. this disease shares many similarities with its human counterpart known as farmer's lung, which results from a type iii hypersensitivity reaction to inhaled organic antigens, most commonly microbial spores, mainly of the thermophilic actinomycete, saccharopolyspora rectivirgula (micropolyspora faeni), commonly found in moldy hay. this is followed by an antibody response to inhaled spores and local deposition of antigen-antibody complexes (arthus reaction) in the lungs (see fig. 9 -88). because it affects only a few animals of the herd or the sporadic person working in a farm, it is presumed that intrinsic host factors, such as dysregulation of dendritic cells, t lymphocytes, igg, interleukins, ifn-γ, and surfactant, are involved in the pathogenesis of the disease. chapter 9 respiratory system, mediastinum, and pleurae efigure 9 -17 interstitial pneumonia, adult cow. note meaty appearance of the pulmonary parenchyma and mild edematous distention of the interlobular septa. inset, thick hyaline membranes (arrows) lining hypercellular alveolar walls. hypersensitivity pneumonia was suspected. (courtesy dr. a. lópez, atlantic veterinary college.) gases, inhalation of no 2 (silo gas) also causes bronchiolitis, edema, and interstitial pneumonia and, in survivors, bronchiolitis obliterans ("silo filler's disease"). smoke inhalation resulting from barn or house fires is sporadically seen by veterinarians and pathologists. in addition to skin burns, animals involved in fire accidents suffer extensive thermal injury produced by the heat on the nasal and laryngeal mucosa, and severe chemical irritation caused by inhalation of combustion gases and particles in the lung. animals that survive or are rescued from fires frequently develop nasal, laryngeal, and tracheal edema, and pulmonary hemorrhage and alveolar edema, which are caused by chemical injury to the blood-air barrier or by ards caused by the excessive production of free radicals during the pulmonary inflammatory response (see efig. 9-7) . microscopic examination of the lungs often reveals carbon particles (soot) on mucosal surfaces of the conducting system. verminous pneumonia (dictyocaulus viviparus). pulmonary lesions in parasitic pneumonias vary from interstitial pneumonia caused by migrating larvae to chronic bronchitis from intrabronchial adult parasites, to granulomatous pneumonia, which is caused by dead larvae, aberrant parasites, or eggs of parasites. in many cases, an "eosinophilic syndrome" in the lungs is characterized by infiltrates of eosinophils in the pulmonary interstitium and bronchoalveolar spaces and by blood eosinophilia. atelectasis and emphysema secondary to the obstruction of airways by parasites and mucous secretions are also common findings in parasitic pneumonias. the severity of these lesions relates to the numbers and size of the parasites and the nature of the host reaction, which sometimes includes hypersensitivity reactions (see section on reinfection syndrome). a common general term for all of these diseases is verminous pneumonia, and the adult nematodes are often visible grossly in the airways ( fig. 9-90) . dictyocaulus viviparus is an important pulmonary nematode (lungworm) responsible for a disease in cattle referred to as verminous pneumonia or verminous bronchitis. adult parasites live in the bronchi of cattle, mainly in the caudal lobes, and cause severe bronchial irritation, bronchitis, and pulmonary edema, which in turn are responsible for lobular atelectasis and interstitial emphysema. atelectasis is confined to the lobules of the lungs ventilated by the obstructed bronchi (dorsocaudal). interstitial emphysema (interlobular) is caused by forced expiratory movements against a partially obstructed single bronchus. in addition to the inflammation of bronchial mucosa, bronchoaspiration of larvae and eggs also causes an influx of leukocytes into the bronchoalveolar space (alveolitis). verminous pneumonia is most commonly seen in calves during their first summer grazing pastures that are repeatedly used from year to year, particularly in regions of europe that have a moist cool climate. the parasite can overwinter in pastures, even in climates as cold as canada's, and older animals may be carriers for a considerable length of time. at necropsy, lesions appear as dark or gray, depressed, wedgeshaped areas of atelectasis involving few or many lobules usually along the dorsocaudal aspect of the lungs. on cut surface, edematous foam and mucus mixed with white, slender (up to 80-mm long) nematodes are visible in the bronchi (see fig. 9 -90). in the most severe cases, massive numbers of nematodes fill the bronchial tree. microscopically, the bronchial lumens are filled with parasites admixed with mucus because of goblet cell hyperplasia, and there is squamous metaplasia of the bronchial and bronchiolar epithelium because of chronic irritation. there are also inflammatory infiltrates in the bronchial mucosa; alveolar edema; hyperplasia of balt sulfide (h 2 s), and ammonia (nh 3 ), from silos or sewage can be a serious hazard to animals and human beings. at toxic concentrations, these gases cause necrosis of bronchiolar cells and type i pneumonocytes and fulminating pulmonary edema that causes asphyxiation and rapid death (see fig. 9-60) . like other oxidant secretory granules released by club cells contain several proteins, such as surfactantlike protein, antiinflammatory protein (cc10), and bronchiolar lining proteins. b, ros produced by club cells are also absorbed into capillaries within the lamina propria and are transferred by the circulatory system to pulmonary capillaries where they disrupt the air-blood barrier, causing degeneration and necrosis of type i pneumonocytes. this process leads to leakage of plasma fluid (alveolar edema [pink color]) and extravasation of erythrocytes (alveolar hemorrhage) and neutrophils (inflammation). ingested pneumotoxicants can be metabolized by the liver, leading to release of ros into the circulatory system that then disrupts the air-blood barrier in a similar manner. fig. 9 -77). microscopically, there are focal intraalveolar hemorrhages caused by larvae migrating through the alveolar walls. some larvae admixed with edematous fluid and cellular exudate (including eosinophils) may be visible in bronchioles and alveoli. the alveolar walls are thickened because of edema and a few inflammatory cells. clinical signs include cough and expiratory dyspnea to the point of oral breathing. hydatid cysts, the intermediate stage of echinococcus granulosus, can be found in the lungs and liver and other viscera of sheep and to a lesser extent in cattle, pigs, goats, horses, and human beings. the adult stage is a tapeworm that parasitizes the intestine of canidae. hydatidosis is still an important zoonosis in some countries, and perpetuation of the parasite life cycle results from animals being fed uncooked offal from infected sheep and consumption of uninspected meat. hydatid cysts are generally 5 to 15 cm in diameter, and numerous cysts can be found in the viscera of affected animals ( fig. 9-91 ). each parasitic cyst is filled with clear fluid; numerous daughter cysts attach to the wall, each containing several "brood capsules" with protoscolices inside. hydatid cysts have little clinical significance in animals but are economically important because of carcass condemnation. aspiration pneumonias. the inhalation of regurgitated ruminal contents or iatrogenic deposition of medicines or milk into the trachea can cause severe and often fatal aspiration pneumonia. bland substances, such as mineral oil, may incite only a mild suppurative or histiocytic bronchopneumonia, whereas some "home remedies" or ruminal contents are highly irritating and cause a fibrinous, necrotizing bronchopneumonia. the right cranial lung lobe tends to be more severely affected because the right cranial bronchus is the most cranial branch and enters the ventrolateral aspect of the trachea. however, the distribution may vary when animals aspirate while in lateral recumbency. in some severe cases, pulmonary necrosis can be complicated by infection with saprophytic organisms present in ruminal contents, causing fatal gangrenous pneumonia. aspiration pneumonia should always be considered in animals whose swallowing has been compromised-for example, those with cleft palate or hypocalcemia (milk fever). on the other hand, neurological diseases such as encephalitis (e.g., rabies) or encephalopathy (e.g., lead poisoning) should be investigated in animals in which the cause of aspiration pneumonia could not be caused by persistent immunologic stimuli; hypertrophy and hyperplasia of bronchiolar smooth muscle because of increased contraction and decreased muscle relaxation; and a few eosinophilic granulomas around the eggs and dead larvae. these granulomas, grossly, are gray, noncaseated nodules (2 to 4 mm in diameter) and may be confused with those seen at the early stages of tuberculosis. the clinical signs (coughing) vary with the severity of infection, and severe cases can be confused clinically with interstitial pneumonias. expiratory dyspnea and death can occur with heavy parasitic infestations when there is massive obstruction of airways. a different form of bovine pneumonia, an acute allergic reaction known as reinfection syndrome, occurs when previously sensitized adult cattle are exposed to large numbers of larvae (dictyocaulus viviparus). lesions in this syndrome are those of a hypersensitivity pneumonia as previously described. other lung parasites. ascaris suum is the common intestinal roundworm of pigs; larvae cannot complete their life cycle in calves, but the larvae can migrate through the lungs and cause severe pneumonia and death of calves within 2 weeks of infection. infection is usually acquired from the soil on which infested pigs were previously kept. the gross lesions are a diffuse interstitial pneumonia with hemorrhagic foci, atelectasis, and interlobular edema and it also occurs in canada, europe, australia, and probably elsewhere. this disease has two major clinicopathologic forms: one involves the central nervous system of goat kids and young goats and is characterized by a nonsuppurative leukoencephalomyelitis; the other form involves the joints of adult goats and is characterized by a chronic, nonsuppurative arthritis-synovitis. in addition, infection with cae virus can cause chronic lymphocytic interstitial pneumonia. the lentivirus of cae, caprine arthritis and encephalitis virus (caev), is closely related to visna/maedi virus and, in fact, cross infection with cae virus in sheep has been achieved experimentally. similar to maedi, cae infection presumably occurs during the first weeks of life when the doe transmits the virus to her offspring through infected colostrum or milk. horizontal transmission between infected and susceptible goats via the respiratory route has also been described. after coming into contact with mucosal cells at the portal of entry, the virus is phagocytized by macrophages, which migrate to the regional lymph nodes. infected macrophages are disseminated hematogenously to the central nervous system, joints, lungs, and mammary glands. like maedi, there is some evidence that the recruitment of lymphocytic cells results from dysregulation of cytokine production by infected macrophages and lymphocytes in affected tissues. it can take several months before serum antibodies can be detected in infected goats. grossly, the interstitial pneumonia is diffuse and tends to be most severe in the caudal lobes. the lungs are gray-pink and firm in texture with numerous, 1-to 2-mm, gray-white foci on the cut surface. the tracheobronchial lymph nodes are consistently enlarged. microscopically, the alveolar walls are thickened by lymphocytes and conspicuous hyperplasia of type ii pneumonocytes ( fig. 9-93 ). one important difference between the pneumonias of cae and maedi is that in cae the alveoli are filled with proteinaceous eosinophilic material (alveolar proteinosis), which in electron micrographs has structural features of pulmonary surfactant. the pulmonary form of cae can be mistaken for parasitic pneumonia (muellerius capillaris) because these two diseases have lymphocytic interstitial pneumonia and can coexist in the same goat. explained otherwise. depending on the nature of the aspirated material, histopathologic evaluation generally reveals foreign particles such as vegetable cells, milk droplets, and large numbers of bacteria in bronchi, bronchioles, and alveoli (efig. 9-18 ). vegetable cells and milk typically induce an early neutrophilic response followed by a histiocytic reaction with "foreign body" multinucleated giant cells (see efig. 9-12 ). special stains are used for the microscopic confirmation of aspirated particles in the lung (e.g., pas for vegetable cells and oil red-o for oil or milk droplets). maedi (visna/maedi). maedi is an important, lifelong, and persistent viral disease of sheep and occurs in most countries, except australia and new zealand. maedi means "shortness of breath" in the icelandic language, and it is known as graaff-reinet disease in south africa, zwoegerziekte in the netherlands, la bouhite in france, and ovine progressive pneumonia (opp) in the united states. more recently, the disease has also been referred to as ovine lentivirusinduced lymphoid interstitial pneumonia or simply lymphoid interstitial pneumonia (lip). maedi is caused by visna/maedi virus (vmv), a nononcogenic small ruminant lentivirus (srlv) of the family retroviridae that is antigenically related to the lentivirus causing caprine arthritisencephalitis (cae). seroepidemiologic studies indicate that infection is widespread in the sheep population, yet the clinical disease seems to be rare. the pathogenesis is incompletely understood, but it is known that transmission occurs largely vertically, through ingestion of infected colostrum, and horizontally, via inhalation of infected respiratory secretions. once in the body, the ovine lentivirus causes lifelong infections within monocytes and macrophages, including alveolar and pulmonary intravascular macrophages; clinical signs do not develop until after a long incubation period of 2 years or more. pulmonary lesions at the time of death are severe interstitial pneumonia and failure of the lungs to collapse when the thorax is opened. notable rib imprints, indicators of uncollapsed lungs, are often present on the pleural surface ( fig. 9-92 ). the lungs are pale, mottled, and typically heavy (two or three times normal weight), and the tracheobronchial lymph nodes are enlarged. microscopically, the interstitial pneumonia is characterized by balt hyperplasia and thickening of alveolar walls and peribronchial interstitial tissue by heavy infiltration of lymphocytes, largely t lymphocytes (see fig. 9 -75). recruitment of mononuclear cells into the pulmonary interstitium is presumably the result of sustainable production of cytokines by retrovirus-infected pulmonary macrophages and lymphocytes. hyperplasia of type ii pneumonocytes is not a prominent feature of maedi, likely because in this disease there is no injury to type i pneumonocytes, but there is some alveolar fibrosis and smooth muscle hypertrophy in bronchioles. secondary bacterial infections often cause concomitant bronchopneumonia. enlargement of regional lymph nodes (tracheobronchial) is due to severe lymphoid hyperplasia, primarily of b lymphocytes. the virus can also infect many other tissues, causing nonsuppurative encephalitis (visna), lymphocytic arthritis, lymphofollicular mastitis, and vasculitis. maedi is clinically characterized by dyspnea and an insidious, slowly progressive emaciation despite good appetite. death is inevitable once clinical signs are present, but it may take many months. caprine arthritis-encephalitis. caprine arthritis-encephalitis (cae) is a retroviral disease of goats (small ruminant lentivirus) that has a pathogenesis remarkably similar to that of visna/maedi in sheep. it was first described in the united states in the 1970s, but such as pasteurella multocida, pneumonia may progress to fibrinous or suppurative bronchopneumonia. one might expect some specific evidence pointing to the infectious agents (e.g., large intranuclear inclusion bodies in epithelial cells with adenoviral infection), but this is often not the case, either because examination is seldom done at the acute stage when the lesions are still present or because secondary bacterial infections mask the primary lesions. in the late stages, chronic enzootic pneumonia is characterized by hyperplastic bronchitis, atelectasis, alveolar and peribronchiolar fibrosis, and marked peribronchial lymphoid hyperplasia (cuffing pneumonia). ovine pneumonic mannheimiosis. ovine pneumonic mannheimiosis is one of the most common and economically significant diseases in most areas where sheep are raised. it is caused by mannheimia haemolytica and has a pathogenesis and lesions similar to those of pneumonic mannheimiosis of cattle. colonization and infection of lungs are facilitated by stressors such as changes in weather; handling; deworming; dipping; viral infections such as parainfluenza virus 3 (piv3), respiratory syncytial virus (rsv), and adenovirus; and probably chlamydiae and bordetella parapertussis infections. lesions are characterized by a severe fibrinous bronchopneumonia (cranioventral) with pleuritis ( fig. 9-94 and e-fig. 9-19 ). subacute to chronic cases progress to purulent bronchopneumonia, and sequelae include abscesses and fibrous pleural adhesions. a similar form of pneumonic mannheimiosis has been reported with increased frequency in bighorn sheep. septicemic pasteurellosis. septicemic pasteurellosis, a common ovine disease, is caused by bibersteinia trehalosi (formerly pasteurella trehalosi or mannheimia haemolytica biotype t) in lambs 5 months of age or older or by mannheimia haemolytica (biotype a) in lambs younger than 2 months of age. both organisms are carried in the tonsils and oropharynx of clinically healthy sheep, and under abnormal circumstances (particularly under stress from dietary or environmental changes) bacteria can invade adjacent tissues, enter the bloodstream, and cause septicemia. gross lesions include a distinctive necrotizing pharyngitis and tonsillitis; ulcerative esophagitis (efig. 9-20) ; severe congestion and edema of the lungs; focal hepatic necrosis; and petechiae in the mucosa of the tongue, esophagus, and intestine and particularly in the lungs and pleura. clinically, goats are active and afebrile but progressively lose weight despite normal appetite. the encephalitic or arthritic signs tend to obscure the respiratory signs, which are only evident on exertion. secondary bacterial bronchopneumonia is common in affected animals. bacterial pneumonias. in the past, pasteurella haemolytica was incriminated in four major ovine diseases known as (1) acute ovine pneumonic pasteurellosis (shipping fever), (2) enzootic pneumonia (nonprogressive chronic pneumonia), (3) fulminating septicemia, and (4) mastitis. under the new nomenclature, mannheimia haemolytica is responsible for ovine pneumonia resembling shipping fever in cattle (ovine pneumonic mannheimiosis), septicemia in young lambs (younger than 3 months of age), and ovine enzootic pneumonia and sporadic severe gangrenous mastitis in ewes. bibersteinia (pasteurella) trehalosi (formerly pasteurella haemolytica biotype t) is the agent incriminated in septicemia in lambs 5 to 12 months old. chronic enzootic pneumonia. in sheep, this entity is a multifactorial disease complex that, in contrast to ovine pneumonic mannheimiosis, causes only a mild to moderate pneumonia and it is rarely fatal. it generally affects animals younger than 1 year of age. significant costs associated with chronic enzootic pneumonia include reduction of weight gain, labor costs, veterinary fees, and slaughterhouse waste. the modifier "chronic" is used here to avoid any confusion with pneumonic mannheimiosis ("acute enzootic pneumonia"). it is also sometimes called atypical pneumonia, chronic nonprogressive pneumonia, proliferative pneumonia, or other names. chronic enzootic pneumonia is a clinical epidemiologic term and does not imply a single causal agent but is the result of a combination of infectious, environmental, and managerial factors. the list of infectious agents involved in ovine enzootic pneumonia includes mannheimia haemolytica, pasteurella multocida, parainfluenza virus 3 (pi-3), adenovirus, reovirus, respiratory syncytial virus (rsv), chlamydiae, and mycoplasmas (mycoplasma ovipneumoniae). in the early stages of enzootic pneumonia, a cranioventral bronchointerstitial pneumonia is characterized by moderate thickening of alveolar walls because of hyperplasia of type ii pneumonocytes. in some cases, when lungs are infected with secondary pathogens, viviparus of cattle. as seen in cattle with dictyocaulus viviparus, areas of atelectasis secondary to bronchiolar obstruction are present, particularly along the dorsal caudal aspects of the caudal lung lobes. microscopically, affected lungs are characterized by a catarrhal, eosinophilic bronchitis, with peribronchial lymphoid hyperplasia and smooth muscle hyperplasia of bronchi and bronchioles. bronchioles and alveoli can contain edematous fluid, eosinophils, and parasitic larvae and eggs. microscopic granulomas caused by aspirated eggs can be observed in the distal lung. the clinical signs (cough, moderate dyspnea, and loss of condition) and lesions relate mainly to obstruction of the small bronchi by adult worms and filaria. anemia of undetermined pathogenesis and secondary bacterial pneumonia are common in small ruminants with this parasitic disease. muellerius capillaris. muellerius capillaris, also called the nodular lungworm, occurs in sheep and goats in most areas of the world and is the most common lung parasite of sheep in europe and northern africa. it requires slugs or snails as intermediate hosts. the lesions in sheep are typically multifocal, subpleural nodules that tend to be most numerous in the dorsal areas of the caudal lung lobes ( fig. 9-95, a) . these nodules are soft and hemorrhagic in the early stages but later become gray-green and hard or even calcified. microscopically, a focal, eosinophilic, and granulomatous reaction occurs in the microscopically, the hallmark lesion is a disseminated intravascular thrombosis often with bacterial colonies in the capillaries of affected tissues. the alveolar capillaries contain bacteria and microthrombi, and the alveolar lumens have fibrin and red blood cells. mannheimia haemolytica and bibersteinia trehalosi are readily isolated from many organs. affected animals usually die within a few hours of infection, and these animals only rarely have clinical signs such as dullness, recumbency, and dyspnea. contagious caprine pleuropneumonia. a number of mycoplasma spp., often referred to as the "mycoides cluster," can produce respiratory tract infections in goats; however, only mycoplasma capricolum ssp. capripneumoniae is considered to cause contagious caprine pleuropneumonia. this disease is the goat counterpart of contagious bovine pleuropneumonia in cattle; sheep do not have a corresponding disease. this oie-notifiable disease is important in africa, the middle east, and areas of asia, but it is also seen elsewhere. the gross lesions caused by mycoplasma capricolum ssp. capripneumoniae are similar to those of the bovine disease and consist of a severe, often unilateral fibrinous bronchopneumonia and pleuritis; however, distention of the interlobular septa (which are normally not as well developed in goats as in cattle) and formation of pulmonary sequestra are less obvious than in the bovine disease. clinically, contagious caprine pleuropneumonia is similar to contagious bovine pleuropneumonia, with high morbidity and mortality, fever, cough, dyspnea, and increasing distress and weakness. other small ruminant mycoplasmas. pneumonia, fibrinous polyarthritis, septicemia, meningitis, mastitis, peritonitis, and abortion are possible manifestations of disease caused by mycoplasma mycoides ssp. mycoides large colony type and mycoplasma mycoides ssp. capri. the pathogenicity of other mycoplasmas, such as mycoplasma ovipneumoniae, mycoplasma arginini, and mycoplasma capricolum ssp. capricolum, in sheep and goats is still being defined and specific description of the lesions would be premature. these organisms probably cause disease only in circumstances similar to those for enzootic pneumonia, where host, infectious, and environmental factors create a complex interaction in the pathogenesis of the disease. it has been suggested that igg antibodies directed against ovine mycoplasmal antigens cross-react with ciliary proteins, causing inflammation and ciliary dysfunction, a condition in lambs referred to as coughing syndrome. tuberculosis. although tuberculosis has generally been considered uncommon in sheep and goats, caprine tuberculosis has become a significant disease in areas of spain and europe. mycobacterium caprae (formerly mycobacterium bovis ssp. caprae/mycobacterium tuberculosis ssp. caprae) is the most common cause, but infection with mycobacterium bovis or with the mycobacterium avium complex does occur when the disease is prevalent in other species in the locality. the pulmonary form, similar to that seen in cattle, is characterized by a granulomatous pneumonia with multiple, large, caseous, calcified, and well-encapsulated granulomas scattered throughout the lungs. intralesional acid-fast organisms within macrophages are not as abundant as in bovine tuberculosis. staphylococcus aureus. young sheep (2 to 12 weeks old) are susceptible to staphylococcus aureus septicemia (tick pyemia). this bacterium causes disseminated inflammation and abscesses in the joints, heart, liver, kidneys, and cns, and in the lung it can also produce bronchopneumonia and pulmonary abscesses (efig. 9-21 ). dictyocaulus filaria. dictyocaulus filaria, also called the large lungworm, is a serious, worldwide, parasitic disease of the lungs, most commonly of lambs and goat kids but occurring in adults as well. the life cycle and lesions are similar to those of dictyocaulus epithelial cells spreads rapidly throughout the nasal, tracheal, and bronchial mucosa, with the more severe outbreaks reflecting more involvement of intrapulmonary airways and secondary infection with pasteurella multocida, trueperella (arcanobacterium) pyogenes, or haemophilus spp. although uncommon, human beings infected with swine influenza (h1n1) can transmit the virus to pigs; therefore it is important that veterinarians or workers with influenza-like illness stay away from pig farms. natural transmission of h1n1 and h5n1 from human beings to ferrets (mustela putorius furo) and from human beings to cats and dogs has also been reported. pulmonary lesions caused by influenza virus alone are rarely seen in the postmortem room because this disease has a very low mortality rate unless complicated with secondary bacterial infections. grossly, a copious catarrhal to mucopurulent inflammation extends from the nasal passages to the bronchioles, with the volume of mucus being sufficient to plug small airways and cause a lobular or multilobular atelectasis in the cranioventral regions of the lungs. the appearance can be similar grossly, although not microscopically, to that of mycoplasma hyopneumoniae. fatal cases have severe alveolar and interstitial pulmonary edema. microscopically, the lesions in uncomplicated cases are typical of a virus-induced, necrotizing bronchitis-bronchiolitis, which in severe cases extends into the alveoli as bronchointerstitial pneumonia. it is characterized by necrosis of the bronchial/bronchiolar epithelium, thickening and infiltration of the alveolar wall with mononuclear cells and aggregates of macrophages, neutrophils, mucus, and some necrotic cells within the alveolar lumen. if these changes are extensive enough, the lumen of bronchioles can be occluded by exudate, causing lobular atelectasis. viral antigen can be demonstrated in infected epithelial cells by immunoperoxidase techniques. in the later stages of alveolar inflammation, neutrophils are progressively replaced by intraalveolar macrophages, unless the pneumonia is complicated by secondary bacterial infections. recent serologic surveys indicate that infection is also prevalent in wild pigs. clinically, a sudden onset of fever, nasal discharge, stiffness, labored breathing, weakness or even prostration, followed by painful and often paroxysmal coughing, is seen in animals of all age groups and may affect most of the herd. the outbreak subsides virtually without mortality within 1 or 2 weeks; the clinical appearance is much more alarming than the pathologic changes, unless the pigs have secondary infection with bacteria. infection can be confirmed using pcr in secretions collected with nasal swabs. the most important effect of most outbreaks of influenza is severe weight loss, but pregnant sows may abort or give birth to weak piglets. porcine reproductive and respiratory syndrome. a disease originally named mystery swine disease was first recognized in the united states in 1987. in 1990, it was seen in europe, and the disease now occurs worldwide in most major pig-raising countries. in 1991, dutch investigators isolated a virus as the etiologic agent; porcine reproductive and respiratory syndrome virus (prrsv) is currently classified in the genus arterivirus of the family arteriviridae. as its name implies, prrs is characterized by late-term abortions and stillbirths and respiratory problems. the respiratory form is generally seen in nursery and grow/finish pigs. the pathogenesis has not been completely elucidated, but it is presumed that there is a mucosal portal of entry with virus replication in macrophages of the lymphoid tissue, followed by viremia and finally dissemination of infected macrophages to the lungs and other organs, such as the thymus, liver (kupffer cells), spleen, lymph nodes, and intestine. the pulmonary alveolar and intravascular macrophages are the major targets for prrs virus, which induces apoptosis of these cells. the virus also downregulates the innate immune response by subpleural alveoli where the adults, eggs, and coiled larvae reside ( fig. 9-95, b) . clinical signs are usually not apparent. goats differ from sheep by having diffuse interstitial rather than focal lesions, and the reaction to the parasites seen microscopically varies from almost no lesions to a severe interstitial pneumonia with heavy infiltrates of mononuclear cells in alveolar walls resembling cae or mycoplasmal infections. secondary effects of muellerius capillaris infection in sheep and goats include decreased weight gain and possibly secondary bacterial infections. protostrongylus rufescens. protostrongylus rufescens is a worldwide parasite of sheep, goats, and wild ruminants. it requires an intermediate snail as a host. infection is usually subclinical, but protostrongylus rufescens can be pathogenic for lambs and goat kids and can cause anorexia, diarrhea, weight loss, and mucopurulent nasal discharge. the adult parasite lives in bronchioles as dictyocaulus spp., but it causes pulmonary nodules similar to those of muellerius capillaris. porcine pneumonias are unequivocally a major obstacle for the contemporary swine industry. the incidence, prevalence, and mortality rates of pneumonias in pigs depend on a series of complex, multifactorial interactions. among the most commonly recognized elements linked to porcine pneumonias are the following: • host (age, genetic makeup, immune status) • infectious agents (viruses, bacteria) • environmental determinants (humidity, temperature, ammonia concentrations) • management practices (crowding, mixing of animals, air quality, nutrition, stress) because of the nature of these multifactorial interactions, it will become obvious in the following paragraphs that more often than not a specific type of pneumonia frequently progresses to or coexists with another. the term porcine respiratory disease complex (prdc) has been introduced in clinical practice to describe pigs with signs of respiratory infection involving combined bacterial and viral infections. commonly implicated microbes include porcine reproductive and respiratory syndrome virus (prrsv), swine influenza virus (siv), porcine circovirus 2 (pcv2), porcine respiratory coronavirus (prcov), mycoplasma hyopneumoniae, and pasteurella multocida. swine influenza (swine flu). swine influenza is a highly contagious acute respiratory viral disease of swine that is caused by swine influenza virus (siv), a type a influenza virus of the family orthomyxoviridae. it is generally accepted that swine influenza resulted from adaptation of the type a influenza virus that caused the human influenza pandemic during world war i. the most common subtypes of siv currently circulating in pigs are h1n1, h1n2, and h3n2. swine influenza is enzootic worldwide and is known to infect human beings who are in close contact with sick pigs. in 2009, an outbreak of swine-human influenza (h1n1), presumably transmitted from pigs to human beings, emerged in mexico and rapidly spread to many countries throughout the world. this new "pandemic" was attributed to a triple-reassortant of influenza a virus containing gene segments of swine, eurasian avian, and human strains. human infection with this novel strain affected mainly children and young adults, as well as individuals of any age with an underlying debilitating condition. transmission between influenza-infected and susceptible pigs occurs mainly by aerosol or oral route. siv attaches to and replicates within epithelial cells of the upper respiratory tract; the infection of similar inclusions are occasionally seen in bronchial glandular and renal epithelial cells. the lungs show thickening of the alveolar walls because of hyperplasia of type ii pneumonocytes and interstitial infiltrates of mononuclear cells, peribronchiolar fibrous hyperplasia, and necrotizing bronchitis/bronchiolitis. circovirus can be confirmed in affected tissue by immunohistochemical or pcr techniques. dual infections with pcv2 and prrsv frequently occur in pigs, and secondary infections with pneumocystis carinii are commonly seen in pigs with this coinfection. characteristically, alveoli are filled with a distinctive foamy exudate that contains the organism, which is not visible in h&e-stained sections but is easily demonstrated with gomori's methenamine silver stain (see fig. 9-20) . in human beings, pneumocystis (carinii) jirovecii pneumonia (pneumocystosis) is one of the most common and often fatal complications in aids patients. as in aids patients, abnormal populations of cd4 + and cd8 + t lymphocytes have been incriminated as the underlying mechanism leading to pneumocystosis in foals and pigs. nipah virus. nipah virus belongs to the paramyxoviridae family and shares a genus (henipavirus) with the closely related hendra virus (see section on pneumonias of horses). another emerging zoonotic disease, nipah virus caused a major epidemic with significant human mortality in southeast asia in 1998 and 1999. people handling pigs were primarily affected. similar to hendra virus, fruit bats (flying foxes) act as natural reservoir and are involved in the transmission to pigs by poorly understood mechanisms. in pigs, this virus infects the respiratory system resulting in pneumonia with syncytial cells occurring in the vascular endothelium and in the respiratory epithelium at all levels of the lung. disease is spread to human beings via the respiratory route. human-to-human transmission of this virus has been reported in more recent outbreaks. other viral pneumonias of pigs. porcine respiratory coronavirus (prcov) is sporadically incriminated in pneumonia in pigs. this viral pneumonia is generally mild, and most pigs fully recover if the pneumonia is not complicated with other infections. lesions in the lung are those of bronchointerstitial pneumonia with necrotizing bronchiolitis. interestingly, infections with porcine and other respiratory coronaviruses have been used to investigate the pathogenesis of severe acute respiratory syndrome (sars), an emerging and highly contagious condition in human beings that is attributed to a novel human coronavirus (sars-cov). the relationship between sars-cov and animal coronavirus is still under investigation. other viruses rarely incriminated in porcine respiratory disease complex (prdc) include paramyxovirus, encephalomyocarditis virus, hemagglutinating encephalomyocarditis virus, and adenovirus. petechial hemorrhages in the lung and pulmonary edema may be seen with african swine fever, classical swine fever, and pseudorabies virus infections. porcine enzootic pneumonia. porcine enzootic pneumonia, a highly contagious disease of pigs caused by mycoplasma hyopneumoniae, is grossly characterized by suppurative or catarrhal bronchopneumonia ( fig. 9-96 and efig. 9-23 ). when its worldwide prevalence and deleterious effect on feed conversion are taken into account, this disease is probably the most economically significant respiratory disease of pigs. although an infectious disease, it is very much influenced by immune status and management factors, such as crowding (airspace and floor space), ventilation (air exchange rate), concentrations of noxious gases in the air (ammonia and hydrogen sulfide), relative humidity, temperature fluctuations, and mixing of stock from various sources. it has been demonstrated with inhibiting interferons and deregulates the adaptive immune response, thus interfering with the normal defense mechanisms predisposing pigs to septicemia and bacterial pneumonia. the most common opportunistic organisms are streptococcus suis, salmonella choleraesuis, mycoplasma hyopneumoniae, haemophilus parasuis, bordetella bronchiseptica, pasteurella multocida, and pneumocystis carinii. dual viral infections with prrsv and porcine circovirus 2 (pcv2), siv, and porcine respiratory coronavirus (prcov) are commonly found in pigs, and such coinfections increase the severity of disease. on postmortem examination, pulmonary lesions vary from very mild changes characterized by failure of the lung to collapse when the thorax is opened and the presence of rib imprints (see fig. 9 -74) to severe changes manifested by consolidation of the lung in cases that have been complicated with bacterial pneumonia. tracheobronchial and mediastinal lymph nodes are typically enlarged. microscopically, pulmonary changes are those of interstitial pneumonia characterized by thickening of alveolar walls by infiltrating macrophages and lymphocytes and mild hyperplasia of type ii pneumonocytes. necrotic cells are scattered in the alveolar lumens. unlike some other viral infections, bronchiolar epithelium does not appear to be affected. diagnosis of prrs in tissue collected at necropsy can be confirmed by immunohistochemistry and pcr techniques. infected pigs may become carriers and transmit the infection through body fluids and semen. clinically, prrs in nursery and young growing animals is characterized by sneezing, fever, anorexia, dyspnea, cough, and occasional death. some piglets develop severe cyanosis of the abdomen and ears, which explains why this syndrome was named blue ear disease when first described in europe. porcine circovirus-associated disease. another emerging porcine syndrome, characterized clinically by progressive emaciation in weaned pigs, was originally described in the 1990s in canada, the united states, and europe. since then, it has disseminated to many countries, causing economic devastation in pig farms worldwide. because of the clinical signs and lesions in many organs, this syndrome was named postweaning multisystemic wasting syndrome (pmws). porcine circovirus 2 (pcv2) has been incriminated as the etiologic agent and is a member of the circoviridae family. pcv2 has been associated with a number of syndromes in pigs, including systemic pcv2 infection (the preferred term for pmws because it may also affect mature pigs), pcv2-associated pneumonia, pcv2-associated enteritis, porcine dermatitis and nephropathy syndrome (pdns), pcv2-associated reproductive failure, and, most recently, pcv2-associated cerebellar vasculitis. the diseases caused by pcv2 are now collectively known as porcine circovirus-associated disease (pcvad); the most common manifestations are systemic pcv2 infection (pmws) and pcv2-associated pneumonia as part of the porcine respiratory disease complex. all of these manifestations affect more than one organ, and there is substantial overlap between the syndromes. at necropsy, pigs with systemic pcv2 infection (pmws) and pcv2-associated pneumonia are often in poor body condition, and the most remarkable changes, not considering other possible secondary infections, are enlargement of the superficial and visceral lymph nodes and a mild interstitial pneumonia characterized by failure of the lungs to collapse when the thorax is opened. jaundice is occasionally observed. microscopically, the lymphoid tissues show lymphoid depletion, histiocytic replacement of follicles, and notable proliferation of parafollicular histiocytes, some of which fuse and form syncytial cells (granulomatous lymphadenitis); necrosis of the lymphoid follicles is seen less often. in some cases, large basophilic inclusion bodies are present singly or as grapelike clusters (botryoid inclusions) within the cytoplasm of macrophages, particularly in peyer's patches, spleen, and lymph nodes (efig. 9-22 ). chapter 9 respiratory system, mediastinum, and pleurae peribronchial, bronchiolar, and alveolar interstitium. additional virulence factors include the ability of mycoplasma hyopneumoniae to cause immunosuppression, reduce the phagocytic activity of neutrophils in the lung, and change the chemical composition of mucus. all of these functional alterations can predispose the lung to secondary bacterial infections. the lesions caused by mycoplasma hyopneumoniae start as a bronchointerstitial pneumonia and progress to a suppurative or mucopurulent bronchopneumonia once secondary pathogens are involved (commonly seen at necropsy). in most pigs, gross lesions affect only portions of the cranial lobes, but in more severely affected pigs, lesions involve 50% or more of the cranioventral portions of the lungs (see fig. 9 -96). the affected lungs are dark red in the early stages but have a homogeneous pale-gray ("fish flesh") appearance in the more chronic stages of the disease. on cut surface, exudate can easily be expressed from airways, and depending on the stage of the lesions and secondary infections, the exudate varies from purulent to mucopurulent to mucoid. microscopic lesions are characterized by an influx of macrophages and neutrophils into the bronchi, bronchioles, and alveoli, and with time there is also notable balt hyperplasia (see fig. 9-96, b) . in some cases, accumulation of exudate can be severe enough to cause occlusion of bronchioles and atelectasis of the corresponding lobules. the suppurative bronchopneumonia may be accompanied by a mild fibrinous pleuritis, which is often more severe if other organisms, such as mycoplasma hyorhinis, pasteurella multocida, or actinobacillus pleuropneumoniae, are also involved. abscesses and fibrous pleural adhesions are sequelae of chronic complicated infections. clinically, enzootic pneumonia occurs as a herd problem in two disease forms. a newly acquired infection of a previously clean herd causes disease in all age groups, resulting in acute respiratory distress and low mortality. in a chronically infected herd, the mature animals are immune and clinical signs are usually apparent only in growing pigs at times of particular stress such as at weaning. in such herds, coughing and reduced rate of weight gain are the most notable signs. porcine pasteurellosis. porcine pasteurellosis is an infectious disease complex with unclear pathogenesis that includes primary infections by pasteurella multocida alone (primary pasteurellosis) or, more frequently, after the defense mechanisms are impaired and a secondary bacterium colonizes the lung (porcine pneumonic pasteurellosis). in rare cases, pasteurella multocida causes acutely fatal septicemias in pigs (primary septicemic pasteurellosis). it is important to remember that pasteurella multocida serotypes a and d are both part of the normal nasal flora and are also causative agents of bronchopneumonia, pleuritis, and atrophic rhinitis in pigs. pasteurella multocida is one of the most common secondary pathogens isolated from the lungs of pigs with swine influenza virus (siv), porcine reproductive and respiratory syndrome virus (prrsv), porcine circovirus 2 (pcv2), pseudorabies (suhv-1), classical swine fever (hog cholera), enzootic pneumonia, and porcine pleuropneumonia. secondary infections with pasteurella multocida notably change the early and mild bronchointerstitial reaction of enzootic and viral pneumonias into a severe suppurative bronchopneumonia with multiple abscesses and sometimes pleuritis. the other important role of pasteurella multocida in porcine pneumonias is as a cause of a fulminating, cranioventral, fibrinous bronchopneumonia (pleuropneumonia) after influenza virus infection or stress from inadequate ventilation resulting in high levels of ammonia in the air. the nature of the lesion and the predisposing factors of poor management or coexisting viral infections suggest that fulminating porcine pasteurellosis has a pathogenesis similar to that of pneumonic mannheimiosis of cattle. pharyngitis with subcutaneous cervical edema, fibrinohemorrhagic polyarthritis, and focal lymphocytic pcr that mycoplasma hyopneumoniae is present in the air of infected farms. the causative agent, mycoplasma hyopneumoniae, is a fastidious organism and very difficult to grow; thus the final diagnosis is frequently based on interpretation of lesions alone or supported by ancillary tests to detect this mycoplasma in affected lungs by immunohistochemistry, immunofluorescence, or pcr. the bronchopneumonic lesions of porcine enzootic pneumonia are in most cases mild to moderate, and thus mortality is low unless complicated with secondary pathogens, such as pasteurella multocida, trueperella (arcanobacterium) pyogenes, bordetella bronchiseptica, haemophilus spp., mycoplasma hyorhinis, and other mycoplasmas and ureaplasmas. although the pathogenesis of porcine enzootic pneumonia is not completely elucidated, it is known that mycoplasma hyopneumoniae first adheres to the cilia of the bronchi by means of a unique adhesive protein, produces ciliostasis, and finally colonizes the respiratory system by firmly attaching to the ciliated epithelial cells of the trachea and the bronchi of the cranioventral regions of the lungs. once attached to the respiratory epithelium, it provokes an influx of neutrophils into the tracheobronchial mucosa; causes extensive loss of cilia (deciliation); stimulates an intense hyperplasia of lymphocytes in the balt; and attracts mononuclear cells into the factors have been identified. these factors allow actinobacillus pleuropneumoniae to attach to cells; produce pores in cell membranes; damage capillaries and alveolar walls, resulting in vascular leakage and thrombosis; impair phagocytic function; and elicit failure of clearance mechanisms. the gross lesions in the acute form consist of a fibrinous bronchopneumonia characterized by severe consolidation and a fibrinous exudate on the pleural surface. although all lobes can be affected, a common site is the dorsal area of the caudal lobes. in fact, a large area of fibrinous pleuropneumonia involving the caudal lobe of a pig's lung is considered almost diagnostic for this disease (fig. 9-97) . on cut surface, consolidated lungs have notably dilated interlobular septa and irregular but well-circumscribed areas of necrosis caused by potent cytotoxins produced by actinobacillus pleuropneumoniae. except for the distribution, pulmonary lesions of porcine pleuropneumonia are identical to those of pneumonic mannheimiosis of cattle. the microscopic lesions are also very similar and include areas of coagulative necrosis surrounded by a thick cluster of "streaming (oat-shaped/oat cell) leukocytes" and notable distention of the interlobular septa because of severe edema and lymphatic thrombosis. bronchioles and alveoli are filled with edematous fluid, fibrin, neutrophils, and few macrophages (see fig. 9 -97). pigs with the chronic form have multiple pulmonary abscesses and large (2 to 10 cm) pieces of necrotic lung encapsulated by connective tissue (sequestra)-changes frequently seen in slaughterhouses. interstitial nephritis are also present in porcine pneumonic pasteurellosis. sequelae of porcine pneumonic pasteurellosis include fibrous pleuritis and pericarditis, pulmonary abscesses, so-called sequestra, and usually death. in contrast to ruminants, mannheimia haemolytica is not a respiratory pathogen for pigs, but in some instances, it can cause abortion in sows. porcine pleuropneumonia. porcine pleuropneumonia is a highly contagious, worldwide disease of pigs caused by actinobacillus (haemophilus) pleuropneumoniae (app), which is characterized by a severe, often fatal, fibrinous bronchopneumonia with extensive pleuritis (pleuropneumonia). survivors generally develop notable residual lesions and become carriers of the organisms. porcine pleuropneumonia is an increasingly important cause of acute and chronic pneumonias, particularly in intensively raised pigs (2 to 5 months old). transmission of actinobacillus pleuropneumoniae occurs by the respiratory route, and the disease can be reproduced experimentally by intranasal inoculation of the bacterium. considered a primary pathogen, actinobacillus pleuropneumoniae can sporadically produce septicemia in young pigs and otitis media and otitis interna with vestibular syndrome in weaned pigs. two biovars and 15 serotypes of the organism have been identified; all serotypes can cause the disease, but differences in virulence exist. the pathogenesis is not yet well understood, but specific virulence factors, such as rtx toxins (hemolytic/cytolytic toxins apx i to apx iv), capsular factors, fimbriae and adhesins, lipopolysaccharide, and permeability tuberculosis. tuberculosis is an important disease in domestic and wild pigs that has a much greater prevalence in pigs than in cattle or other domestic mammals in many countries. porcine tuberculosis is attributed to infection with mycobacterium bovis and porcine mycobacteriosis to infection with mycobacterium avium complex. a common scenario in small mixed-farming operations is the diagnosis of avian tuberculosis at the time that pigs are slaughtered, and the source is ingestion of tuberculous chickens or contaminated litter. as would be expected, granulomas are found in the mesenteric, mandibular, and retropharyngeal lymph nodes; to a lesser extent in the intestine, liver, and spleen; and only in rare cases in the lung. the route of infection in pulmonary tuberculosis and mycobacteriosis of pigs is most often hematogenous after oral exposure and intestinal infection. lung lesions are those of a granulomatous pneumonia. the microscopic lesions are basically those of tubercles (granulomas), but the degree of encapsulation, caseation, and calcification varies with the type of mycobacterium, age of the lesion, and host immune response. other bacterial pneumonias of pigs. septicemias in pigs often cause petechial hemorrhages in the lung and pulmonary edema. salmonellae, escherichia coli, and listeria monocytogenes can cause severe interstitial pneumonia in very young animals. salmonella choleraesuis causes a necrotizing fibrinous pneumonia similar to porcine pleuropneumonia, and salmonella typhisuis causes a chronic suppurative bronchopneumonia. in high health herds, actinobacillus suis may cause fibrinohemorrhagic pleuropneumonia and is easily confused with porcine pleuropneumonia. metastrongylosis. metastrongylus apri (elongatus), metastrongylus salmi, and metastrongylus pudendotectus (lungworms) of domestic and feral pigs occur throughout most of the world and require earthworms as intermediate hosts for transmission. the incidence of disease has therefore decreased with development of confinement housing. the importance of pig lungworms is mainly because infection results in growth retardation of the host. clinical signs include coughing because of parasitic bronchitis. the gross lesions, when noticeable, consist of small gray nodules, particularly along the ventral borders of the caudal lobes. the adult worms are grossly visible in bronchi, and microscopically, the parasites cause a catarrhal bronchitis with infiltration of eosinophils and lobular atelectasis ( fig. 9-99) . ascaris suum. the larvae of ascaris suum can cause edema, focal subpleural hemorrhages, and interstitial inflammation (see fig. 9 -77). along their larval migration tracts, hemorrhages also occur in the liver and, after fibrosis, become the large white "milk spots" seen so frequently as incidental findings at necropsy. it has been reported that ascaris suum may cause immunosuppression in severely affected pigs. pigs can be killed if exposed to an overwhelming larval migration. other causes of pneumonia. foreign body granulomatous pneumonia occurs frequently in pigs after inhalation of vegetable material (starch pneumonia), presumably from dusty (nonpelleted) feed. lesions are clinically silent but are often mistaken for other pneumonic processes during inspection at slaughterhouses. microscopically, pulmonary changes are typical of foreign body granulomatous inflammation in which variably sized feed particles are surrounded by macrophages and neutrophils, and often have been phagocytosed by multinucleated giant cells. feed (vegetable) particles appear as thick-walled polygonal cells that stain positive with pas because of their rich carbohydrate (starch) content (see efig. 9-12) . clinically, porcine pleuropneumonia can vary from an acute form with unexpected death and blood-stained froth at the nostrils and mouth to a subacute form characterized by coughing and dyspnea accompanied by clinical signs of sepsis such as high fever, hypoxemia, anorexia, and lethargy (efig. 9-24) . a chronic form is characterized by decreased growth rate and persistent cough. animals that survive often carry the organism in the tonsils, shed the organism, and infect susceptible pigs. haemophilus pneumonia. in addition to glasser's disease characterized by polyserositis (pericarditis, pleuritis, peritonitis, polyarthritis, and meningitis) (efig. 9-25) , some serotypes of haemophilus parasuis (originally haemophilus influenzae suis) can also cause suppurative bronchopneumonia that in severe cases can be fatal. the causal organism, haemophilus parasuis, is usually carried in the nasopharynx of normal pigs and requires abnormal circumstances such as those following stress (weaning and cold weather) or viral infections (swine influenza or pcv2). specific pathogen-free (spf) pigs seem to be particularly susceptible to glasser's disease (arthritis and serositis) but not to pulmonary infection (bronchopneumonia). streptococcal pneumonia. streptococcus suis is a common cause of porcine disease worldwide and a serious zoonosis capable of causing death by septic shock or meningitis and residual deafness in butchers, veterinarians, and pig farmers. typically, streptococcus suis gains entrance to the susceptible young pig through the oropharyngeal mucosa and is carried in the tonsils, nasal mucosa, and mandibular lymph nodes of healthy animals, particularly in survivors of an outbreak. infected sows can abort or vertically transmit the infection to their offspring. some serotypes of streptococcus suis cause neonatal septicemia, and this can result in suppurative meningitis, otitis, arthritis, polyserositis, myocarditis, valvular endocarditis, and embolic pneumonia ( fig. 9-98 ). other serotypes of streptococcus suis can reach the lung by the aerogenous route and cause a suppurative bronchopneumonia, in combination with pasteurella multocida, escherichia coli, or mycoplasma hyopneumoniae, or in combination with actinobacillus pleuropneumoniae, which causes a fibrinous bronchopneumonia. coinfections of streptococcus suis with pcv2 and prrsv are also frequently seen in some farms. gross lesions in the acute stages include serous to catarrhal to mucopurulent nasopharyngitis and conjunctivitis. the lungs are edematous and have a diffuse interstitial pneumonia ( fig. 9 -100) microscopically characterized by necrotizing bronchiolitis, necrosis and exfoliation of pneumonocytes, mild alveolar edema, and, several hours later, thickening of the alveolar walls because of interstitial mononuclear cell infiltrates and hyperplasia of type ii pneumonocytes. secondary infections with bordetella bronchiseptica and mycoplasmas are common and induce life-threatening suppurative bronchopneumonia. the thymus may be small relative to the age of the animal because of viral-induced lymphocytolysis. microscopically, eosinophilic inclusions are present in the epithelial cells of many tissues, in the nuclei or cytoplasm, or in both (see fig. 9 -100). they appear early in the bronchiolar epithelium but are most prominent in the epithelium of the lung, stomach, renal pelvis, and urinary bladder, making these tissues good choices for diagnostic examination. viral inclusions are rarely seen in the later stages of this disease. the suppurative secondary bronchopneumonias often hinder the detection of viral lesions in the lung, particularly because bronchiolar cells containing inclusion bodies exfoliate and mix with the neutrophils recruited by the bacterial infection. distemper virus antigens can be readily demonstrated in infected cells by the immunoperoxidase technique (see fig. 9 -100), which can also be used in skin biopsies for the antemortem diagnosis of canine distemper. distemper virus also has a tendency to affect developing tooth buds and ameloblasts, causing enamel hypoplasia in dogs that recover from infection. of all distemper lesions, demyelinating encephalomyelitis, which develops late, is the most devastating (see chapter 14). sequelae to distemper include the nervous and pneumonic complications mentioned previously and various systemic infections, such as toxoplasmosis and sarcocystosis, because of depressed immunity. persistent viral infection occurs in some dogs that survive the disease, and they may become carriers and the source of infection for other susceptible animals. clinical signs consist of biphasic fever, diarrhea, vomiting, weight loss, mucopurulent oculonasal discharge, coughing, respiratory distress, and possible loss of vision. weeks later, hyperkeratosis of the foot pads ("hard pad") and the nose are observed, along with nervous signs, including ataxia, paralysis, convulsions, or residual myoclonus (muscle twitches, tremors, and "tics"). in general, inflammatory diseases of the lungs are less of a problem in dogs than in food-producing species and can be subdivided in two major groups, infectious and noninfectious pneumonias. "canine infectious respiratory disease" (cird) is the term currently used by clinicians to describe a heterogeneous group of respiratory infections in dogs; these diseases were previously clustered under the name of infectious tracheobronchitis or "kennel cough." cird is the canine counterpart of brd and prd complexes in cattle and pigs, respectively. the most common viruses in cird include canine parainfluenza virus (cpiv), canid herpesvirus 1 (cahv-1), canine adenovirus-2 (cav-2), canine respiratory coronavirus (crcov), canine distemper virus (cdv), and canine influenza virus (civ). bordetella bronchiseptica, streptococcus equi ssp. zooepidemicus, and mycoplasma spp. are the most frequent bacterial isolates in cird. it has been recently recognized that animal shelters are an important source of viral and bacterial infections for dogs and cats. uremia and paraquat toxicity are perhaps the two most notable noninfectious causes of canine respiratory disease. canine distemper. canine distemper is an important and ubiquitous infectious disease of dogs, other canidae, wild felidae, mustelidae, and marine mammals throughout the world. it is caused by a morbillivirus that is antigenically related to the human measles, rinderpest (officially eradicated in 2011), "peste de petit ruminants," and phocine distemper viruses. canine distemper virus (cdv) is transmitted to susceptible puppies through infected body fluids. the virus invades through the upper respiratory tract and conjunctiva, proliferates in regional lymph nodes, becomes viremic, and in dogs with an inadequate antibody response, infects nearly all body tissues (pantropic), particularly the epithelial cells. distemper virus hampers the immune response, downregulates cytokine production, and persists for a long time in some tissues. cdv can target the lungs either directly as a viral pneumonia or indirectly by its immunosuppressive effects rendering the lungs susceptible to secondary bacterial and protozoal infections, or as a coinfection with other viruses such as canine adenovirus-2 and canid herpesvirus 1. 15:292-294, 2003.) inclusion bodies occur within epithelial cells in early lesions. cahv-1 has also been identified as a cause of ulcerative keratoconjunctivitis in older dogs. canine influenza (canine flu). canine influenza is an emerging contagious respiratory infection of dogs that was first described in the united states and subsequently in other countries. it has a high morbidity (close to 100%), but the mortality, as with most other influenza infections, is relatively low (less than 8%). this disease, first diagnosed in greyhounds, is caused by a novel influenza-a virus (canine influenza virus or civ), a mutation from a previously recognized h3n8 strain of equine influenza virus. dog-to-dog transmission does occur and therefore this infection must be distinguished from other viruses of the canine infectious respiratory disease (cird) group. pulmonary lesions are generally mild and transient, but infected dogs are susceptible to secondary bacterial bronchopneumonia. the most relevant lesions in dogs dying unexpectedly from canine influenza are pleural and pulmonary hemorrhages. microscopically, there is necrotizing tracheitis, bronchitis, and bronchiolitis with exudation of neutrophils and macrophages. in severe cases, hemorrhagic interstitial or bronchointerstitial pneumonia may be accompanied by vasculitis and thrombosis. influenza antigen can be demonstrated by immunohistochemistry in airway epithelium and alveolar macrophages. clinically, dogs with canine influenza are lethargic, inappetent, and hyperthermic and frequently cough and show nasal discharge. these signs resemble those seen in dogs with kennel cough or secondary bacterial pneumonia. in addition, there are confirmed cases of canine influenza caused by the porcine h1n1 presumably transmitted from infected pet owners. bacterial pneumonias. dogs generally develop bacterial pneumonias when the pulmonary defense mechanisms have been impaired. pasteurella multocida, streptococcus spp., escherichia coli, klebsiella pneumoniae, and bordetella bronchiseptica can be involved in pneumonia secondary to distemper or after aspiration of gastric contents ( fig. 9-102 and efig. 9-27 ). streptococcus zooepidemicus can cause acute and fatal hemorrhagic pleuropneumonia with canine adenovirus type 2 infection. cav-2 infection is a common but transient contagious disease of the respiratory tract of dogs, causing mild fever, oculonasal discharge, coughing, and poor weight gain. the portal of entry is generally by inhalation of infected aerosols followed by viral replication in the surface cells of the upper respiratory tract, mucous cells of the trachea and bronchi, nonciliated bronchiolar epithelial cells, and type ii pneumonocytes. pulmonary lesions are initially those of bronchointerstitial pneumonia, with necrosis and exfoliation of bronchiolar and alveolar epithelium, edema, and, a few days later, proliferation of type ii pneumonocytes, mild infiltration of neutrophils and lymphocytes in the alveolar interstitium, and hyperplastic bronchitis and bronchiolitis. large basophilic intranuclear viral inclusions are typically seen in bronchiolar and alveolar cells ( fig. 9-101) . infection with cav-2 is clinically mild unless complicated with a secondary bacterial infection or coinfections with other viruses such as distemper virus. experimental work suggests cav-2 reinfection may lead to hyperreactive airways, a nonspecific condition in which the bronchial mucosa becomes highly "responsive" to irritation such as that caused by cold air, gases, or cigarette smoke. however, it is not clear if this outcome is true in natural infections. canid herpesvirus 1. canid herpesvirus 1 (cahv-1) can cause fatal systemic disease in newborn puppies and is probably a contributing factor in "fading puppy syndrome." hypothermia has been suggested as a pivotal component in the pathogenesis of fatal infections in puppies. many dogs are seropositive, suggesting that transient or subclinical infections are more common than realized; the virus remains latent in the trigeminal and other ganglia and can be reactivated after stress, resulting in asymptomatic transmission of cahv-1 virus to offspring via the placenta, thus resulting in abortion or stillbirths. in puppies, cahv-1 causes ulcerative tracheitis, interstitial pneumonia (efig. 9-26) , and focal necrosis and inflammation in the kidneys, liver, and brain. eosinophilic intranuclear aspiration pneumonia starts as an acute necrotizing bronchitis and bronchiolitis caused by aspiration of irritant materials such as gastric acid or a caustic material administered by mouth. the aspirate also contains potentially pathogenic bacteria, and because the mucociliary apparatus is damaged and these bacteria are not removed, they settle into the ventral portions of the lung (from gravity) and provoke a fibrinosuppurative and necrotizing bronchopneumonia. b, bronchoalveolar spaces are filled with neutrophils, macrophages, and bacteria (arrows). h&e stain. inset, large colonies of bacteria (arrows). h&e stain. (courtesy dr. a. lópez, atlantic veterinary college.) a b infection; thus it most frequently affects outdoor and hunting dogs. from the lung, infection is disseminated hematogenously to other organs, mainly bone, skin, brain, and eyes. pulmonary lesions are characterized by multifocal to coalescing pyogranulomatous pneumonia, generally with firm nodules scattered throughout the lungs (fig. 9-103) . microscopically, nodules are pyogranulomas with numerous macrophages (epithelioid cells), some neutrophils, multinucleated giant cells, and thick-walled yeasts (see fig. 9 -35, c). yeasts are 5 to 25 µm in diameter and are much better visualized when they are stained with pas reaction or gomori's methenamine silver stain. nodules can also be present in other tissues, chiefly lymph nodes, skin, spleen, liver, kidneys, bones, testes, prostate, and eyes. this fungus can be easily identified in properly prepared and stained transtracheal washes or lymph node aspirates. clinical signs can reflect involvement of virtually any body tissue; pulmonary effects include cough, decreased exercise tolerance, and terminal respiratory distress. coccidioidomycosis. coccidioidomycosis (san joaquin valley fever), caused by the dimorphic fungus coccidioides immitis, occurs mainly in animals living in arid regions of the southwestern united states, mexico, and central and south america. it is a primary respiratory tract (aerogenous) infection commonly seen at slaughterhouses in clinically normal feedlot cattle. in dogs, coccidioidomycosis also has an aerogenous portal of entry and then a b hemorrhagic pleural effusion in dogs. death is generally a consequence of severe sepsis and septic shock or from β-hemolytic streptococcal bacteremia causing emboli in the lungs, liver, brain, and lymph nodes. the primary source of the infection cannot be determined in most cases. dental disease in dogs may be a source of systemic and pulmonary infection, a concept wellrecognized in human medicine for many years. the role of mycoplasmas in canine pneumonia is still uncertain because these organisms are frequently isolated from normal nasopharyngeal flora. tuberculosis is uncommon in dogs because these animals appear to be quite resistant to infection; most cases occur in immunocompromised dogs or in dogs living with infected human beings. dogs are susceptible to the infection with mycobacterium tuberculosis, mycobacterium bovis, and mycobacterium avium complex, and therefore canine infection presupposes contact with human or animal tuberculosis. the clinicopathologic manifestation is pulmonary after inhalation or alimentary after oral exposure, but in most cases infection is disseminated to lymph nodes and visceral organs. the gross lesions are multifocal, firm nodules with necrotic centers, most often seen in the lungs, lymph nodes, kidneys, and liver. diffuse granulomatous pleuritis and pericarditis with copious serofibrinous or sanguineous effusion are common. microscopically, granulomas are formed by closely packed macrophages but with very little connective tissue. mycotic pneumonias. mycotic pneumonias are serious diseases seen commonly in animals in some regions. there are two main types: those caused by opportunistic fungi and those caused by a group of fungi associated with systemic "deep" mycoses. all of these fungi affect human beings and most domestic animals but are probably not transmitted between species. aspergillosis. opportunistic fungi, such as aspergillus spp. (particularly aspergillus fumigatus), are important in birds, but in domestic animals, they mainly affect immunosuppressed individuals or those on prolonged antibiotic therapy. the pulmonary lesion is a multifocal, nodular, pyogranulomatous, or granulomatous pneumonia. microscopically, there is necrosis and infiltrates of neutrophils, macrophages, and lymphocytes, with proliferation of fibroblasts eventually leading to encapsulation of the granuloma. fungal hyphae are generally visible in the core of the lesion and in the walls of blood vessels. systemic mycoses (dimorphic fungal infections) . systemic (deep) mycoses are caused by blastomyces dermatitidis, histoplasma capsulatum, coccidioides immitis, and cryptococcus neoformans/ cryptococcus gatti (see fig. 9 -35). blastomycosis mainly affects dogs and is discussed here, whereas cryptococcosis is discussed in the section on pneumonias of cats. in contrast to other fungi, such as aspergillus spp., organisms of the systemic mycosis group are all primary pathogens of human beings and animals and thus do not necessarily require a preceding immunosuppression to cause disease. these fungi have virulence factors that favor hematogenous dissemination and evasion of immune and phagocytic responses. systemic dissemination is often exacerbated by the administration of immunosuppressant drugs such as corticosteroids. these fungi are usually detected by cytological evaluation of affected tissues. blastomycosis. blastomycosis occurs in many countries of the north american continent, africa, the middle east, and occasionally in europe. in the united states, it is most prevalent in the atlantic, st. lawrence, and ohio-mississippi river valley states, compared with the mountain-pacific region. blastomyces dermatitidis is a dimorphic fungus (mycelia-yeast) seen mainly in young dogs and occasionally in cats and horses. this fungus is present in the soil, and inhalation of spores is considered the principal route of from the alveolar interstitium associated with larvae or dead worms because little reaction develops to the live adults. crenosoma vulpis. crenosoma vulpis is a lungworm seen commonly in foxes and sporadically in dogs with access to the intermediate hosts-slugs and snails. the adult lungworms live in small bronchi and bronchioles in the caudal lobes, causing eosinophilic and catarrhal bronchitis manifested grossly as gray areas of inflammation and atelectasis. in some animals, crenosoma vulpis causes bronchiolar goblet cell metaplasia and mucous obstruction, resulting in lobular atelectasis due to the valve effect of the mucous plug. eucoleus aerophilus. eucoleus aerophilus (capillaria aerophila) is a nematode parasite typically found in the trachea and bronchi of wild and domestic carnivores. in some cases, this parasite may also involve the nasal passages and sinuses. although generally asymptomatic, some dogs cough because of the local irritation caused by the parasites on the tracheal or bronchial mucosa. paragonimus spp. paragonimus kellicotti in north america and paragonimus westermani in asia are generally asymptomatic fluke infections in fish-eating species. the life cycle involves two intermediate hosts, the first a freshwater snail and the second a freshwater crab or crayfish; in north america, cats and dogs acquire infection by eating crayfish. gross lesions include pleural hemorrhages where the metacercariae migrate into the lungs. later, multifocal eosinophilic pleuritis, and subpleural cysts up to 7 mm long containing pairs of adult flukes, are found along with eosinophilic granulomas around clusters of eggs. like many other parasitic pneumonias, lesions and scars are more frequent in the caudal lobes. pneumothorax can occur if a cyst that communicates with an airway ruptures to the pleural surface. other parasitic infections. angiostrongylus vasorum and dirofilaria immitis are parasites of the pulmonary arteries and right ventricle and, depending on the stage, can produce different forms of pulmonary lesions. adult parasites can cause chronic arteritis that leads to pulmonary hypertension, pulmonary arterial thrombosis, interstitial (eosinophilic) granulomatous pneumonia, pulmonary interstitial fibrosis, congestive right-sided cardiac failure, and eventually caudal vena caval syndrome. other lesions include pleural petechial hemorrhages and, in later stages, diffuse pulmonary hemosiderosis and multifocal pulmonary infarcts. larvae and eggs also cause alveolar injury, thickening of the alveolar walls with eosinophils and lymphocytes (interstitial pneumonia), and multifocal or coalescing granulomas with giant cells (parasitic granulomas). pneumocystis carinii has been reported as a sporadic cause of chronic interstitial pneumonia in dogs with a compromised immune system (see pneumonias of horses; also see fig. 9 -20). aspiration pneumonia. aspiration pneumonia is an important form of pneumonia that occurs in dogs when vomit or regurgitated materials are aspirated into the lungs, or when drugs or radiographic contrast media are accidentally introduced into the airways (efig. 9-28) . as in other animal species, aspiration pneumonia may be unilateral or may more often affect the right cranial lobe ( fig. 9-104 ). the severity of lesions depends very much on the chemical and microbiologic composition of the aspirated material. in general, aspiration in monogastric animals, particularly in dogs and cats, is more severe because of the low ph of the gastric contents (chemical pneumonitis). in severe cases, dogs and cats die rapidly from septic shock and ards (see fig. 9 -63), which is microscopically characterized by diffuse alveolar damage, protein-rich pulmonary edema, neutrophilic alveolitis, and formation of typical hyaline membranes along the alveolar walls (see fig. 9-104 ). in animals that survive the acute stages of aspiration, pulmonary lesions progress to bronchopneumonia. aspiration pneumonia is a common sequela to disseminates systemically to other organs. clinical signs relate to the location of lesions, so there can be respiratory distress, lameness, generalized lymphadenopathy, or cutaneous lesions, among others. the lesions caused by coccidioides immitis consist of focal granulomas or pyogranulomas that can have suppurative or caseated centers. the fungal organisms are readily seen in histologic or cytologic preparation as large (10 to 80 µm in diameter), double-walled, and highly refractile spherules containing numerous endospores (see fig. 9-35, d) . histoplasmosis. histoplasmosis is a systemic infection that results from inhalation and, in dogs, possibly ingestion of another dimorphic fungus, histoplasma capsulatum. histoplasmosis occurs sporadically in dogs and human beings and, to a lesser extent, in cats and horses. bats often eliminate histoplasma capsulatum in the feces, and droppings from bats and birds, particularly pigeons, heavily promote the growth and survival of this fungus in the soil of enzootic areas. pulmonary lesions are grossly characterized by variably sized, firm, poorly encapsulated granulomas and, sometimes, more diffuse involvement of the lungs. microscopically, granulomatous lesions typically have many macrophages filled with small (1 to 3 µm), punctiform, intracytoplasmic, dark oval bodies (yeasts) (see fig. 9 -35, a) that are best demonstrated with pas reaction or gomori's methenamine silver stain. similar nodules or diffuse involvement can be present in other tissues, chiefly lymph nodes, spleen, intestine, and liver. toxoplasmosis. toxoplasmosis is a worldwide disease caused by the obligate intracellular, protozoal parasite toxoplasma gondii. cats and other felidae are the definitive hosts in which the mature parasite divides sexually in the intestinal mucosa. human beings, dogs, cats, and many wild mammals can become intermediate hosts after accidental ingestion of fertile oocysts shed in cat feces or ingestion of undercooked or raw meat containing tissue cysts, and fetuses can be infected transplacentally from an infected dam. in most instances, the parasite infects many cells of different tissues and induces an antibody response (seropositive animals) but does not cause clinical disease. toxoplasmosis is often triggered by immunosuppression, such as that caused by canine distemper virus. toxoplasmosis is characterized by focal necrosis around the protozoan. pulmonary lesions are severe, multifocal necrotizing interstitial pneumonia with notable proliferation of type ii pneumonocytes and infiltrates of macrophages and neutrophils. other lesions in disseminated toxoplasmosis include multifocal necrotizing hepatitis, myocarditis, splenitis, myositis, encephalitis, and ophthalmitis. the parasites appear microscopically as small (3 to 6 µm) basophilic cysts that can be found free in affected tissues or within the cytoplasm of many epithelial cells and macrophages (see efig. 8-8) . similar findings can be seen sporadically in dogs infected with neospora caninum and sarcocystis canis, and immunohistochemistry would be required to differentiate those protozoal organisms from toxoplasma gondii. filaroides hirthi. filaroides hirthi, a lungworm of the alveoli and bronchioles of dogs, has long been known as a cause of mild subclinical infection in large colonies of beagle dogs in the united states. however, it can on occasion cause severe and even fatal disease in individual pets, presumably as a result of immunosuppression. clinical signs may include coughing and terminal respiratory distress. grossly, the lesions are multifocal subpleural nodules, often with a green hue because of eosinophils, scattered throughout the lungs. microscopically, these nodules are eosinophilic granulomas arising 548.e1 chapter 9 respiratory system, mediastinum, and pleurae other pneumonias. idiopathic pulmonary fibrosis is a rare condition of uncertain etiology reported in the west highland white terrier breed that shares similarities with human and feline idiopathic pulmonary fibrosis. microscopically, there is diffuse interstitial pneumonia and progressive alveolar fibrosis with capillary obliteration, hyperplasia of type ii cells, some of which exhibit cellular atypia, and finally hypertrophy and hyperplasia of smooth muscle. the interstitial fibrosis eventually spills over alveolar spaces causing conspicuous intraalveolar fibrosis. although upper respiratory tract infections are common and important in cats, pneumonias are uncommon except when there is immunosuppression or aspiration of gastric contents. viral infections such as feline rhinotracheitis and calicivirus may cause lesions in the lungs, but unless there is secondary invasion by bacteria, they do not usually cause a fatal pneumonia. feline rhinotracheitis. feline rhinotracheitis is an important viral disease of cats caused by the ubiquitous felid herpesvirus 1 (fehv-1). this infection affects primarily young or debilitated cats causing inflammation in the nasal, ocular, and tracheal mucosa and, to a much lesser extent, the lung (see species-specific diseases of the nasal cavity and paranasal sinuses). when lungs are affected, fehv-1 causes bronchointerstitial pneumonia with necrosis of bronchiolar and alveolar epithelium, thickening of the alveolar walls, and extensive permeability edema. eosinophilic intranuclear inclusion bodies may be seen in infected epithelial cells early in infection. feline calicivirus. feline calicivirus (fcv) causes upper respiratory disease, stomatitis, conjunctivitis, and, to a lesser extent, interstitial pneumonia. microscopically, affected lungs exhibit the typical pattern of bronchointerstitial pneumonia with necrotizing bronchiolitis, thickening of alveolar walls, occasionally hyaline membranes, hyperplasia of type ii pneumonocytes, and macrophages admixed with cellular debris in the alveolar lumens. because pulmonary lesions are similar to those caused by fehv-1, isolation or in situ detection is required for final diagnosis. feline infectious peritonitis. feline infectious peritonitis (fip) is caused by fip virus (fipv), a mutated form of feline enteric cleft palate, and in dogs with megaesophagus secondary to either myasthenia gravis or persistent right aortic arch. it is also an important complication of general anesthesia or neurologic diseases affecting laryngeal function. paraquat. paraquat, a broad-spectrum herbicide widely used in gardening and agriculture, can cause severe and often fatal toxic interstitial pneumonia (pneumonitis) in dogs, cats, human beings, and other species. after ingestion or inhalation, this herbicide selectively accumulates in the lung where paraquat toxic metabolites are produced by club (clara) cells. these metabolites promote local release of free radicals in the lung, which causes extensive injury to club cells and to the blood-air barrier, presumably through lipid peroxidation of type i and ii pneumonocytes and alveolar endothelial cells (see fig. 9 -89). paraquat toxicity has been used experimentally as a model of oxidant-induced alveolar injury and pulmonary fibrosis. soon after poisoning, the lungs are heavy, edematous, and hemorrhagic because of extensive necrosis of epithelial and endothelial cells in the alveolar walls. the lungs of animals that survive acute paraquat toxicosis are pale, fail to collapse when the thorax is opened, and have interstitial emphysema, bullous emphysema, and occasionally pneumomediastinum. microscopic findings in the acute and subacute phases include necrosis of type i pneumonocytes, interstitial and alveolar edema, intraalveolar hemorrhages, and proliferation of type ii pneumonocytes. in the chronic stages (4 to 8 weeks later), the lesions are typically characterized by severe interstitial and intraalveolar fibrosis. uremic pneumopathy. uremic pneumonopathy (pneumonitis) is one of the many extrarenal lesions seen in dogs with chronic uremia. lesions are characterized by a combination of pulmonary edema and calcification of vascular smooth muscle and alveolar basement membranes. in severe cases, alveolar calcification prevents lung collapse when the thorax is opened. in the more advanced cases, the lungs appear diffusely distended, pale red or brown in color, and show a rough pleural surface with rib imprints (see fig. 9 -51). on palpation, the pulmonary parenchyma has a typical "gritty" texture because of mineralization of the alveolar and vascular walls, which are best visualized microscopically by using special stains such as von kossa (see fig. 9 -51). because this is not primarily an inflammatory lesion, the term pneumonitis should not be used. fig. 9-63) . a, note that the lungs did not collapse when the thorax was opened (loss of negative pressure) and as a result fill almost the entire thoracic cavity. the cranioventral aspects of the lung are consolidated with hemorrhage. b, alveolar capillary congestion, thick hyaline membranes along the alveolar septa (arrows), and intraalveolar hemorrhage. these microscopic changes are typical of the diffuse alveolar damage seen in lungs with ards. h&e stain. (courtesy dr. a. lópez, atlantic veterinary college.) feline calicivirus has removed chlamydophila felis from its previously overstated importance as a lung pathogen. tuberculosis. cats are susceptible to three types of mycobacterial infections: classic tuberculosis, feline leprosy, and atypical mycobacteriosis. classic tuberculosis in cats is rare and generally caused by mycobacterium bovis and mycobacterium microti but also, to a lesser extent, by mycobacterium tuberculosis. nosocomial tuberculosis (mycobacterium bovis) in cats has been reported with increased frequency. the usual route of infection for feline tuberculosis is oral, through infected rodents/meat or unpasteurized milk, so the granulomatous lesions are mainly in the intestine and mesenteric lymph nodes where they may disseminate through infected phagocytes to other organs. the solid and noncaseated appearance of tuberculous nodules is grossly similar to that of neoplasms, so they must be differentiated from pulmonary neoplasms (e.g., lymphoma). classic tuberculosis with dermal lesions in cats should be differentiated from feline leprosy (localized skin granulomas) caused by mycobacterium lepraemurium and other nonculturable species of acid-fast bacilli. atypical mycobacteriosis is caused by contamination of a skin wound with saprophytic and nonsaprophytic mycobacteria such as those of the mycobacterium avium complex. advances in pcr techniques have notably reduced the time required for etiologic diagnosis of mycobacteriosis in veterinary diagnostic laboratories. cryptococcosis. cryptococcosis (pulmonary cryptococcus neoformans or cryptococcus gatti) is the most frequent systemic mycosis in cats, and lesions are akin to those discussed in the section on mycotic pneumonias of dogs. it occurs worldwide in all species but is diagnosed most frequently in cats, horses, dogs, and human beings. some healthy dogs and cats harbor cryptococcus in the nasal cavity and become asymptomatic carriers. clinical infection may occur in immunocompetent cats and in cats that are immunologically compromised, such as by felv, fiv, malnutrition, or corticosteroid treatment. lesions can occur in nearly any tissue, resulting in a wide c coronavirus (fecv), and is one of a few viral infections of domestic animals that result in pyogranulomatous pneumonia. this disease is microscopically characterized by a vasculitis affecting many tissues and organs ( fig. 9-105) . other viral pneumonias. other viruses sporadically incriminated in feline interstitial pneumonia are cowpox virus (cpxv) and influenza a h1n1. pasteurellae. bacteria from the nasal flora such as pasteurella multocida and pasteurella-like organisms are occasionally associated with secondary bronchopneumonia in cats ( fig. 9-106) . pasteurella multocida also causes otitis media and meningitis, but its role as a respiratory pathogen is mainly associated with pyothorax. interestingly, there are reports of pasteurella multocida pneumonia in older or immunosuppressed human beings acquired through contact with domestic cats. mycoplasmas. mycoplasmas are often isolated from the lungs of cats with pulmonary lesions but are not definitively established as primary pathogens in feline pneumonias. feline pneumonitis. the term feline pneumonitis is a misnomer because the major lesions caused by chlamydophila felis (formerly chlamydia psittaci) are severe conjunctivitis and rhinitis (see species-specific diseases of the nasal cavity and paranasal sinuses). the elucidation of the importance of feline viral rhinotracheitis and organism infects erythrocytes in the erythrocytic stage of disease and multiplies in intravascular macrophages/monocytes, including those in the alveolar capillaries (efig. 9-29) , during the leukocytic stage of disease. aspiration pneumonia. aspiration pneumonias are common in cats as a result of vomiting, regurgitation, dysphagia, or anesthetic complication or after accidental administration of food, oral medicaments, or contrast media into the trachea (iatrogenic). pulmonary lesions are similar to those described for dogs, and the type of lung lesion depends on the chemical and bacterial composition of the aspirated material (see the section on aspiration pneumonia of dogs). feline idiopathic pulmonary fibrosis. feline idiopathic pulmonary fibrosis is a rare, progressive, and fatal disease of cats of uncertain etiology characterized by multifocal fibrotic nodules subpleurally and randomly in the lung making the pleural surface resemble nodular cirrhosis of the liver ( fig. 9-108) . microscopically, the affected alveolar and peribronchiolar interstitium is thickened by excessive fibrosis, abundant deposition of extracellular matrix, and hypertrophy of smooth muscle. some investigators suggest an intrinsic cellular defect in type ii pneumonocytes as the underlying cause. the alveolar walls are diffusely lined by cuboidal hyperplastic type ii pneumonocytes, and the alveolar lumens often contain exfoliated cells and necrotic debris. this feline condition has morphologic features similar to "equine multinodular pulmonary fibrosis" and "cryptogenic pulmonary fibrosis" in human beings. fetal pneumonias. pneumonia is one of the most frequent lesions found in fetuses submitted for postmortem examination, particularly in foals and food-producing animals. because of autolysis, lack of inflation, and the lungs being at various stages of development, fetal lesions are often missed or misdiagnosed. in the nonaerated fetal lung, the bronchoalveolar spaces are filled with a viscous, locally produced fluid known as lung fluid or lung liquid. it has been estimated that an ovine fetus produces approximately 2.5 ml of "lung fluid" per kilogram of body weight per hour. in the variety of clinical signs. however, granulomatous rhinitis, sinusitis, otitis media and interna, pneumonia, ulcerative dermatitis, and meningoencephalitis are most common. the pulmonary lesion in cryptococcosis is a multifocal granulomatous pneumonia and, like those occurring in other internal organs, they are small, gelatinous, white foci. the gelatinous appearance is due to the broad mucous capsule around the yeast (see fig. 9-35, b) . microscopically, lesions contain great numbers of fungal organisms (4 to 10 µm in diameter without the capsule) and only a few macrophages, lymphocytes, and multinucleated giant cells. this thick polysaccharide capsule does not stain well with h&e, and thus there is a large empty space or halo around the yeast. feline lungworm. aelurostrongylus abstrusus, known as feline lungworm, is a parasite that occurs in cats wherever the necessary slug and snail intermediate hosts are found. it can cause chronic respiratory disease with coughing and weight loss and, sometimes, severe dyspnea and death, particularly if there are secondary bacterial infections. the gross lesions are multifocal, amber, and subpleural granulomatous nodules up to 1 cm in diameter throughout the lungs. on incision, these nodules may contain viscous exudate. microscopically, the adult parasites, eggs, and coiled larvae are in the bronchioles and alveoli, where they cause catarrhal bronchiolitis, hyperplasia of submucosal glands, and, later, granulomatous alveolitis, alveolar fibrosis, and fibromuscular hyperplasia ( fig. 9-107) . during routine examination of feline lungs, it is quite common to find fibromuscular hyperplasia in bronchioles and arterioles in otherwise healthy cats. it was alleged in the past that this fibromuscular hyperplasia was a long-term sequela of subclinical infection with aelurostrongylus abstrusus. however, this view has been challenged; thus the pathogenesis and significance of pulmonary fibromuscular hyperplasia in healthy cats remains uncertain. in severe cases, fibromuscular hyperplasia is grossly visible in the lungs as white subpleural nodules. other parasitic pneumonias. toxoplasma gondii, paragonimus kellicotti, and dirofilaria immitis can also affect cats (see the section on parasitic pneumonias of dogs). cytauxzoon felis is an apicomplexan hemoparasite that affects domestic and wild felidae. the diseases that cause fetal pneumonia in farm animals. gross lesions in the lungs are generally undetected, but microscopic lesions include focal necrotizing interstitial pneumonia and focal necrosis in the liver, spleen, or brain. fetal bronchointerstitial pneumonia also occurs in some viral abortions, such as those caused by infectious bovine rhinotracheitis (ibr) virus and bovine parainfluenza virus 3 (bpiv-3) in cattle and equine viral rhinopneumonitis (evr) in horses. fetal pneumonias in dogs and cats are infrequently described, perhaps because aborted puppies and kittens are rarely submitted for postmortem examination. with advancements in molecular biology techniques, the etiologic diagnosis of abortions and their association with pulmonary fetal lesions is rapidly improving. neonatal pneumonias and septicemias. these entities are rather common in newborn animals lacking passive immunity because of the lack of either ingestion or absorption of maternal colostrum (failure of passive transfer or hypogammaglobulinemia). in addition to septicemias causing interstitial pneumonia, farm animals with hypogammaglobulinemia can develop bronchopneumonia by inhalation of bacterial pathogens. these include histophilus somni and pasteurella multocida in calves; streptococcus spp. in foals; and escherichia coli, listeria monocytogenes, and streptococcus suis in pigs. meconium aspiration syndrome. meconium aspiration syndrome (mas) is an important but preventable condition in human babies that originates when amniotic fluid contaminated with meconium is aspirated during labor or immediately after birth. the pathogenesis of mas is basically the same as in those of fetal bronchopneumonia (see fig. 9-109) . fetal hypoxia, a common event during dystocia or prolonged parturition, causes the fetus to relax the anal sphincter and release meconium into the amniotic fluid. aspiration of meconium can occur directly from aspirating contaminated amniotic fluid before delivery (respiratory movements with an open glottis) or immediately after delivery when the meconium lodged in the nasopharynx is carried into the lung with the first breath of air. this latter form of aspiration is prevented in delivery rooms by routine suction of the nasopharynx in meconium-stained babies. mas is well known in human babies, but the occurrence and significance in animals remains largely unknown. mas has been reported in calves, foals, piglets, and puppies. although pulmonary lesions are generally mild and transient, aspiration of meconium can be life-threatening for newborn babies and animals because it typically occurs in compromised neonates already suffering from intrauterine hypoxia and acidosis. neonatal acidosis is known to impair colostrum absorption in calves. common mas sequelae are lobular atelectasis, pulmonary hypertension, and possibly airway hyperreactivity. in the most severe cases of mas, focal (patchy) atelectasis can be observed grossly in the lung, indicating failure of the lungs to be fully aerated because of the mechanical obstruction and the chemical effect of meconium on pulmonary surfactant (see fig. 9 -52). microscopically, meconium and keratin exfoliated from skin of the fetus into the amniotic fluid are present in bronchi, bronchioles, and alveoli and accompanied by mild alveolitis characterized by infiltration of leukocytes followed by alveolar macrophages and occasional giant cells (efig. 9-30) . lung cancer in animals is rare, unlike in human beings, in which the incidence is alarming and continues to be the number one cause of death due to cancer in canada, the united states, and europe. interestingly, prostatic and breast cancers, so much feared by men fetus, this fluid normally moves along the tracheobronchial tree, reaching the oropharynx, where a fraction is swallowed into the gastrointestinal tract, and a small portion is released into the amniotic fluid. at the time of birth, the lung fluid is rapidly reabsorbed from the lungs by alveolar absorption and lymphatic drainage. aspiration of amniotic fluid contaminated with meconium and bacteria from placentitis is the most common route by which microbial pathogens reach the fetal lungs. this form of pneumonia is secondary to fetal hypoxia and acidosis ("fetal distress"), which cause the fetus to relax the anal sphincter, release meconium into the amniotic fluid, and, in the terminal stages, inspire deeply with open glottis, resulting in the aspiration of contaminated fluid ( fig. 9-109 ). gross lesions are only occasionally recognized, but microscopic changes are similar to those of a bronchopneumonia. microscopically, bronchoalveolar spaces contain variable numbers of neutrophils, macrophages, epidermal squames, and pieces of meconium that appear as bright yellow material because of its bile content. in contrast to postnatal bronchopneumonia, lesions in fetuses are not restricted to the cranioventral aspects of the lungs but typically involve all pulmonary lobes. in cattle, brucella abortus and trueperella (arcanobacterium) pyogenes are two of the most common bacteria isolated from the lungs of aborted fetuses. these bacteria are usually present in large numbers in the amniotic fluid of cows with bacterial placentitis. inflammation of the placenta interferes with oxygen exchange between fetal and maternal tissue, and the resultant fetal hypoxia induces the fetus to "breathe" with an open glottis and aspirate the amniotic fluid. aspergillus spp. (mycotic abortion) and ureaplasma diversum cause sporadic cases of placentitis, which results in fetal pneumonia and abortion. in addition to the respiratory route (aspiration), pathogens, such as bacteria and viruses, can also reach the lungs via fetal blood and cause interstitial pneumonia. listeriosis (listeria monocytogenes), salmonellosis (salmonella spp.), and chlamydiosis (chlamydophila abortus [c. psittaci]) are the best known examples of blood-borne primary benign neoplasms of the lungs, such as pulmonary adenomas, are highly unusual in domestic animals. most primary neoplasms are malignant and appear as solitary masses of variable size that, with time, can metastasize to other areas of the lungs and to distant organs. it is sometimes difficult on gross and microscopic examination to differentiate primary lung cancer from pulmonary metastasis resulting from malignant neoplasms elsewhere in the body. it is often difficult to determine the precise topographic origin of a neoplasm within the lungs-for example, whether it originates in the conducting system (bronchogenic carcinoma), transitional system (bronchiolar carcinoma), exchange system (alveolar carcinoma), or bronchial glands (bronchial gland carcinoma). according to the literature, pulmonary carcinomas in animals arise generally from club (clara) cells or type ii pneumonocytes of the bronchioloalveolar region, in contrast to those in human beings, which are mostly bronchogenic. tumors located at the hilus generally arise from major bronchi and tend to be a solitary large mass with occasional small metastasis to the periphery of the lung. in contrast, tumors arising from the bronchioloalveolar region are often multicentric with numerous peripheral metastases in the lung parenchyma. because of histologic architecture and irrespective of the site of origin, many malignant epithelial neoplasms are classified by the all-encompassing term of pulmonary adenocarcinomas. dogs and cats are the species most frequently affected with primary pulmonary neoplasms, largely carcinomas, generally in older animals. the mean age for primary lung tumors is 11 years for dogs and 12 years for cats. pulmonary carcinomas in other domestic animals, except for retrovirus-induced pulmonary carcinoma in sheep, are less common, possibly because fewer farm animals are allowed to reach their natural life span. these neoplasms can be invasive or expansive, vary in color (white, tan, or gray) and texture (soft or firm), and often have areas of necrosis and hemorrhage, which result in a "craterous" or "umbilicate" appearance. this umbilicate appearance is frequently seen in rapidly growing carcinomas in which the center of the tumoral mass undergoes necrosis as a result of ischemia. some lung neoplasms resemble pulmonary consolidation or large granulomas. cats with moderately differentiated neoplasms had significantly longer survival time (median, 698 days) than cats with poorly differentiated neoplasms (median, 75 days). dogs with primary lung neoplasms, grades i, ii, and iii, had survival times of 790, 251, and 5 days, respectively. ovine pulmonary adenocarcinoma (ovine pulmonary carcinoma). ovine pulmonary adenocarcinoma, also known as pulmonary adenomatosis and jaagsiekte (from the south african afrikaans word for "driving sickness"), is a transmissible, retrovirus-induced neoplasia of ovine lungs caused by jaagsiekte sheep retrovirus (jsrv). it occurs in sheep throughout the world, with the notable exception of australia and new zealand; its incidence is high in scotland, south africa, and peru and unknown but probably low in north america. this pulmonary carcinoma behaves very much like a chronic pneumonia, and jsrv shares many epidemiologic similarities with the ovine lentivirus responsible for maedi and the retrovirus responsible for enzootic nasal carcinoma in small ruminants. pulmonary adenomatosis has been transmitted to goats experimentally but is not known to be a spontaneous disease in that species. ovine pulmonary adenocarcinoma affects mainly mature sheep but can occasionally affect young stock. intensive husbandry probably facilitates horizontal transmission by the copious nasal discharge and explains why the disease occurs as devastating epizootics with 5% to 80% mortality when first introduced into a flock. differential diagnosis between maedi and pulmonary adenomatosis can prove difficult because both diseases often coexist in the same flock and women, are a distant second. to say that cigarette smoking is responsible for this epidemic of lung cancer is unnecessary. although dogs have been proposed as valuable "sentinels" for environmental hazards, such as exposure to passive smoking, asbestos, dyes, and insecticides, it is not known if the prevalence of canine lung tumors has increased in geographical areas with high contamination. alterations in genes (oncogenes) and chromosomes and changes in biologically active molecules have been linked to lung cancer in recent years. as with many other forms of cancer, epidemiologic studies indicate that the incidence of pulmonary neoplasms increases with age, but there are still insufficient data to confirm that particular canine or feline breeds have a higher predisposition to spontaneous lung neoplasms. a standard nomenclature of pulmonary neoplasms in domestic animals is lacking, and as a consequence, multiplicity of names and synonyms occur in the veterinary literature. some classifications are based on the primary site, whereas others emphasize more the histomorphologic type. the most common types of benign and malignant pulmonary neoplasms in domestic mammals are listed in box 9-2. clinically, the signs of pulmonary neoplasia vary with the degree of invasiveness, the amount of parenchyma involved, and locations of metastases. signs may be vague, such as cough, lethargy, anorexia, weight loss, and perhaps dyspnea. in addition, paraneoplastic syndromes, such as hypercalcemia, endocrinopathies, and pulmonary hypertrophic osteoarthropathy, have been associated with pulmonary neoplasms. primary neoplasms of the lungs. primary neoplasms of the lungs arise from cells normally present in the pulmonary tissue and can be epithelial or mesenchymal, although the latter are rare. any malignant tumor metastatic from another body location (e.g., osteosarcoma in dogs, uterine carcinoma in cows, and malignant melanoma in horses) box 9-2 classification of pulmonary neoplasms 553.e1 chapter 9 respiratory system, mediastinum, and pleurae abundant cytoplasm containing numerous acidophilic granules, which are positive for pas and for s-100 protein using immunohistochemistry. although this tumor can cause bronchial obstruction and respiratory signs, in most cases, it is an incidental finding in older horses submitted for postmortem examination. lymphomatoid granulomatosis. lymphomatoid granulomatosis is a rare but interesting pulmonary disease of human beings, dogs, cats, and possibly horses and donkeys characterized by nodules or large solid masses in one or more lung lobes. these frequently metastasize to lymph nodes, kidneys, and liver. microscopically, tumors are formed by large pleomorphic mononuclear (lymphomatoid) cells with a high mitotic rate and frequent formation of binucleated or multinucleated cells. tumor cells have a distinct tendency to grow around blood vessels and invade and destroy the vascular walls. lymphomatoid granulomatosis has some resemblance to lymphoma and is therefore also referred to as angiocentric lymphoma; phenotypic marking confirms that neoplastic cells are a mixed population of plasma cells, b and t lymphocytes, and histiocytes. cerebral and cutaneous forms of lymphomatoid granulomatosis have also reported in human beings, dogs, and cats. secondary neoplasms of the lungs. secondary neoplasms of the lungs are all malignant by definition because they are the result of metastasis to the lungs from malignant neoplasms elsewhere. because the pulmonary capillaries are the first filter met by tumor emboli released into the vena cava or pulmonary arteries, secondary neoplasms in the lung are relatively common in comparison to primary ones. also, secondary tumors can be epithelial or mesenchymal in origin. common metastatic tumors of epithelial origin are mammary, thyroid ( fig. 9-111) , and uterine carcinomas. tumors of mesenchymal origin are osteosarcoma ( fig. 9-112, a) ; hemangiosarcoma ( fig. 9-112, b) ; malignant melanoma in dogs; lymphoma in cows, pigs, dogs, and cats ( fig. 9-113) ; and vaccineassociated sarcoma in cats. usually, secondary pulmonary neoplasms are multiple; scattered throughout all pulmonary lobes (hematogenous dissemination); of variable size; and, according to the growth pattern, can be nodular, diffuse, or radiating (efig. 9-31) . the appearance of metastatic neoplasms differs according to the type of neoplasm. for example, dark red cystic nodules containing blood indicate hemangiosarcoma, dark black solid nodules indicate melanoma, and hard solid nodules (white, yellow, or tan color) with bone spicules indicate osteosarcoma. the gross appearances of or in the same animal. death is inevitable after several months of the initial onset of respiratory signs, and a specific humoral immune response to jsrv is undetectable in affected sheep. during the early stages of ovine pulmonary carcinoma, the lungs are enlarged, heavy, and wet and have several firm, gray, variably sized nodules that in some cases can be located in the cranioventral lobes mimicking a bronchopneumonic lesion ( fig. 9-110, a) . in the later stages, the nodules become confluent, and large segments of both lungs are diffusely, but not symmetrically, infiltrated by neoplastic cells. on cross section, edematous fluid and a copious mucoid secretion are present in the trachea and bronchi ( fig. 9-110, b) . microscopically, the nodules consist of cuboidal or columnar epithelial cells lining airways and alveoli and forming papillary or acinar (glandlike) structures (see fig. 9-110, a) . because the cells have been identified ultrastructurally as originating from both type ii alveolar epithelial cells and club (clara) cells, the neoplasm is considered a "bronchioloalveolar" carcinoma. sequelae often include secondary bronchopneumonia, abscesses, and fibrous pleural adhesions. metastases occur to tracheobronchial and mediastinal lymph nodes and, to a lesser extent, to other tissues such as pleura, muscle, liver, and kidneys. neoplastic cells stain strongly positive for jsrv using immunohistochemistry. clinically, ovine pulmonary adenocarcinoma is characterized by a gradual loss of condition, coughing, and respiratory distress, especially after exercise (e.g., herding or "driving"). appetite and temperature are normal, unless there are secondary bacterial infections. an important differentiating feature from maedi (interstitial pneumonia) can be observed if animals with pulmonary adenomatosis are raised by their hind limbs; copious, thin, mucoid fluid, produced by neoplastic cells in the lungs, pours from the nostrils of some animals. carcinoid (neuroendocrine) tumor of the lungs. carcinoid tumor of the lungs is a neoplasm presumably arising from neuroendocrine cells and is sporadically seen in dogs as multiple, large, firm pulmonary masses close to the mainstem bronchi. it has also been reported in the nasal cavity of horses. tumor cells are generally polygonal with finely granular, pale, or slightly eosinophilic cytoplasm. nuclei are small, and mitotic figures are absent or rare. granular cell tumor. granular cell tumor is a rare and locally invasive tumor that has been reported mainly in human beings and older horses. the cell origin of this tumor was thought to be the myoblast, but it is currently presumed to be schwann cells, which are normally present in the bronchovascular bundles of the lung. microscopically, neoplastic cells are large, polyhedron-shaped with metastatic carcinomas are generally similar to the primary neoplasm and sometimes have umbilicated centers. proper diagnoses of pulmonary neoplasms in live animals require history, clinical signs, radiographs, cytologic analysis of bal fluid, and, when necessary, a lung biopsy. identification of a specific lineage of neoplastic cells in biopsy or postmortem specimens is often difficult and requires electron microscopy or immunohistochemical techniques. electron microscopy allows identification of distinctive cellular components such as osmiophilic lamellar phospholipid nephritic bodies in alveolar type ii epithelial cells or melanosomes in melanomas. immunohistochemical staining is also helpful in identifying tumor cells. the thoracic wall, diaphragm, and mediastinum are lined by the parietal pleura, which reflects onto the lungs at the hilum and continues as the visceral pleura, covering the entire surface of the lungs, except at the hilus where the bronchi and blood vessels enter. the space between the parietal and visceral pleura (pleural space) is only minimal and under normal conditions contains only traces of clear fluid, which is a lubricant, and a few exfoliated cells. samples of this fluid are obtained by thoracocentesis, a simple procedure in which a needle is passed into the pleural cavity. volumetric, biochemical, and cytologic changes in this fluid are routinely used in veterinary diagnostics. anomalies 7 congenital defects are rare and generally of little clinical significance. cysts within the mediastinum of dogs and, less often, cats in severe cases, the amount of fluid present in the thoracic cavity can be considerable. for instance, a medium-size dog can have 2 l of fluid, and a cow may accumulate 25 l or more. excessive fluid in the thorax causes compressive atelectasis resulting in respiratory distress (see fig. 9 -54). hydrothorax is most commonly seen in cattle with right-sided heart failure or cor pulmonale (hydrostatic) (efig. 9 -32); dogs with congestive heart failure (hydrostatic), chronic hepatic disease (hepatic hydrothorax) ( fig. 9-114) , or nephrotic syndrome (hypoproteinemia); pigs with mulberry heart disease (increased vascular permeability); and horses with african horse sickness (increased vascular permeability). hemothorax. blood in the thoracic cavity is called hemothorax, but the term has been used for exudate with a sanguineous component. causes include rupture of a major blood vessel as a result of severe thoracic trauma (e.g., hit by car); erosion of a vascular wall by malignant cells or inflammation (e.g., aortitis caused by spirocerca lupi); ruptured aortic aneurysms; clotting defects, including coagulopathies; warfarin toxicity; disseminated intravascular coagulation (consumption coagulopathy); and thrombocytopenia. hemothorax is generally acute and fatal. on gross examination, the thoracic cavity can be filled with blood, and the lungs are partially or completely atelectatic ( fig. 9-115 ). chylothorax. the accumulation of chyle (lymph rich in triglycerides) in the thoracic cavity ( fig. 9-116 ) is a result of the rupture of major lymph vessels, usually the thoracic duct or the right lymphatic duct. the clinical and pathologic effects of chylothorax are similar to those of the other pleural effusions. causes include thoracic neoplasia (the most common cause in human beings but a distant second to idiopathic cases in dogs), trauma, congenital lymph vessel anomalies, lymphangitis, dirofilariasis, and iatrogenic rupture of the thoracic duct during surgery. the source of the leakage of chyle is rarely found at necropsy. when the leakage of chyle occurs in the abdominal cavity, the condition is referred to as chyloabdomen. cytologic and biochemical examination of fluid collected by thoracocentesis typically reveals large numbers of lymphocytes, lipid droplets, few neutrophils in chronic cases, and high triglyceride content. can be large enough to compromise pulmonary function or mimic neoplasia in thoracic radiographs. these cysts may arise from the thymus (thymic branchial cysts), bronchi (bronchogenic cysts), ectopic thyroid tissue (thyroglossal duct cysts), or from remnants of the branchial pouches, and they are generally lined by epithelium and surrounded by a capsule of stromal tissue. anomalies of the thoracic duct cause some cases of chylothorax. pleural calcification. pleural calcification is commonly found in dogs and less often in cats with chronic uremia. lesions appear as linear white streaks in parietal pleura, mainly over the intercostal muscles of the cranial part of the thoracic cavity. the lesions are not functionally significant but indicate a severe underlying renal problem. vitamin d toxicity (hypervitaminosis d) and ingestion of hypercalcemic substances, such as vitamin d analogs, can also cause calcification of the pleura and other organs. pneumothorax. pneumothorax is the presence of air in the thoracic cavity where there should normally be negative pressure to facilitate inspiration. human beings have a complete and strong mediastinum so that pneumothorax is generally unilateral and thus not a serious problem. in dogs, the barrier varies, but in general it is not complete, so often some communication exists between left and right sides. there are two main forms of pneumothorax. in spontaneous (idiopathic) pneumothorax, air leaking into the pleural cavity from the lungs occurs without any known underlying disease or trauma. in secondary pneumothorax, movement of air into the pleural cavity results from underlying pulmonary or thoracic wall disease. the most common causes of secondary pneumothorax in veterinary medicine are penetrating wounds to the thoracic wall, perforated esophagus, iatrogenic trauma to the thorax and lung during a transthoracic lung biopsy or thoracoscopy, tracheal rupture from improper intubation, and rupture of emphysematous bullae or parasitic pulmonary cysts (paragonimus spp.) that communicate with the thoracic cavity. pneumothorax and pneumomediastinum caused by high air pressure (barotrauma) are also well documented in cats after equipment failure during anesthesia. clinical signs of pneumothorax include respiratory distress, and the lesion is simply a collapsed, atelectatic lung. the air is readily reabsorbed from the cavity if the site of entry is sealed. pleural effusion. pleural effusion is a general term used to describe accumulation of any fluid (transudate, modified transudate, exudate, blood, lymph, or chyle) in the thoracic cavity. cytologic and biochemical evaluations of pleural effusions taken by thoracocentesis are helpful in determining the type of effusion and possible pathogenesis. based on protein concentration and total numbers of nucleated cells, pleural effusions are cytologically divided into transudates, modified transudates, and exudates. hydrothorax. when the fluid is serous, clear, and odorless and fails to coagulate when exposed to air, the condition is referred to as hydrothorax (transudate). causes of hydrothorax are the same as those involved in edema formation in other organs: increased hydrostatic pressure (heart failure), decreased oncotic pressure (hypoproteinemia, as in liver disease), alterations in vascular permeability (inflammation), or obstruction of lymph drainage (neoplasia). in cases in which the leakage is corrected, if the fluid is a transudate, it is rapidly reabsorbed. if the fluid persists, it irritates the pleura and causes mesothelial hyperplasia and fibrosis, which thickens the pleura. from a perforated esophagus. chronic injury typically results in serosal fibrosis and tight adhesions between visceral and parietal pleurae (see fig. 9 -71). when extensive, these adhesions can obliterate the pleural space. pleuritis or pleurisy. inflammation of the visceral or parietal pleurae is called pleuritis, and according to the type of exudate, it can be fibrinous, suppurative, granulomatous, hemorrhagic, or a combination of exudates. acute fibrinous pleuritis can progress with time to pleural fibrosis ( fig. 9-117 ). when suppurative pleuritis results in accumulation of purulent exudate in the cavity, the lesion is called pyothorax or thoracic empyema ( fig. 9-118) . clinically, pleuritis causes considerable pain, and in addition, empyema can result in severe toxemia. pleural fibrous adhesions (between parietal and visceral pleura) and fibrosis are the most common sequelae of chronic pleuritis and can significantly interfere with inflation of the lungs. pleuritis can occur as an extension of pneumonia, particularly in fibrinous bronchopneumonias (pleuropneumonia), or it can occur alone, without pulmonary involvement ( fig. 9-119 ). bovine and ovine pneumonic mannheimiosis and porcine and bovine pleuropneumonia are good examples of pleuritis associated with fibrinous bronchopneumonias. polyserositis in pigs and pleural empyema, particularly in cats and horses, are examples of pleural inflammation in pleural tissue is readily susceptible to injury caused by direct implantation of an organism through a penetrating thoracic or diaphragmatic wound; by hematogenous dissemination of infectious organisms in septicemias; or by direct extension from an adjacent inflammatory process, such as in fibrinous bronchopneumonia or in contrast to those with the effusive ("wet") form, in which thoracic involvement is primarily that of a pleural effusion. cytologic evaluation of the effusion typically shows a low to moderate cellularity with degenerated leukocytes, lymphocytes, macrophages, and mesothelial cells, and a pink granular background as a result of the high protein content. pleuritis is also an important problem in horses. nocardia spp. can cause fibrinopurulent pneumonia and pyothorax with characteristic sulfur granules. although mycoplasma felis can be isolated from the respiratory tract of normal horses, it is also isolated from horses with pleuritis and pleural effusion, particularly during the early stages of infection. the portal of entry of this infection is presumably aerogenous, first to the lung and subsequently to the pleura. the pleural surface of the lung is often involved in neoplasms that have metastasized from other organs to the pulmonary parenchyma and ruptured the visceral pleura to seed the pleural cavity. mesothelioma is the only primary neoplasm of the pleura. which involvement of the lungs may not accompany the pleuritis. pleural inflammation is most frequently caused by bacteria, which cause polyserositis reaching the pleura hematogenously. these bacteria include haemophilus parasuis (glasser's disease) (see , streptococcus suis, and some strains of pasteurella multocida in pigs; streptococcus equi ssp. equi and streptococcus equi ssp. zooepidemicus in horses; escherichia coli in calves; and mycoplasma spp. and haemophilus spp. in sheep and goats. contamination of pleural surfaces can be the result of extension of a septic process (e.g., puncture wounds of the thoracic wall and, in cattle, traumatic reticulopericarditis) and ruptured pulmonary abscesses (e.g., trueperella pyogenes). in dogs and cats, bacteria (e.g., nocardia, actinomyces, and bacteroides) can cause pyogranulomatous pleuritis, characterized by accumulation of blood-stained pus ("tomato soup") in the thoracic cavity. this exudate usually contains yellowish flecks called sulfur granules ( fig. 9-120 ), although these are less common in nocardial empyema in cats. many species of bacteria, such as escherichia coli, trueperella pyogenes, pasteurella multocida, and fusobacterium necrophorum, can be present in pyothorax of dogs and cats. these bacteria occur alone or in mixed infections. the pathogenesis of pleural empyema in cats is still debatable, but bite wounds or penetration of foreign material (migrating grass awns) are likely. pyogranulomatous pleuritis with empyema occurs occasionally in dogs, presumably associated with inhaled small plant material and penetrating (migrating) grass awns. because of their physical shape (barbed) and assisted by the respiratory movement, aspirated grass awns can penetrate airways, move through the pulmonary parenchyma, and eventually perforate the visceral pleura causing pyogranulomatous pleuritis. cats with the noneffusive ("dry") form of feline infectious peritonitis (fip) frequently have focal pyogranulomatous pleuritis, mesothelioma is a rare neoplasm of the thoracic, pericardial, and peritoneal mesothelium of human beings that is seen most commonly in calves, in which it can be congenital. in human beings, it has long been associated with inhalation of certain types of asbestos fibers (asbestos mining and ship building) alone or with cigarette smoking as a probable cocarcinogen; no convincing association between the incidence of mesothelioma and exposure to asbestos has been made in domestic animals. in animals, there may be pleural effusion with resulting respiratory distress, cough, and weight loss. mesothelioma initially causes a thoracic effusion, but cytologic diagnosis can be difficult because of the morphologic resemblance of malignant and reactive mesothelial cells. during inflammation, mesothelial cells become reactive and not only increase in number but also become pleomorphic and form multinucleated cells that may be cytologically mistaken for those of a carcinoma. grossly, mesothelioma appears as multiple, discrete nodules or arborescent, spreading growths on the pleural surface ( fig. 9-121) . microscopically, either the mesothelial covering cells or the supporting tissue can be the predominant malignant component, so the neoplasm can microscopically resemble a carcinoma or a sarcoma. figure 9 -120 nocardiosis. a, chronic pleuritis (nocardia asteroides), pleural cavity, cat. the pleural cavity is covered with abundant red-brown ("tomato soup") exudate" (syringe). once considered to be pathognomonic of nocardia spp. infection, it is no longer regarded as being diagnostic of nocardiosis. the fluid contains abundant protein, erythrocytes, granulomatous inflammatory cells, and sulfur granules. b, chronic pleuritis (nocardia asteroides), visceral pleura, dog. the thickened pleura has a granular pink-gray appearance because of granulomatous inflammation and the proliferation of fibrovascular tissue of the pleura. c, chronic pleuritis (nocardia asteroides), dog. the pleura has been thrown up into villous-like projections composed of abundant fibrovascular tissue and granulomatous inflammation. leakage from the neocapillaries of the fibrovascular tissue is responsible for the hemorrhagic appearance of the pleural exudate. h&e stain. d, chronic pleuritis (nocardia asteroides), thoracic cage, parietal pleura, cat. large pieces of exudate, which contain yellow sulfur granules, are present on the thickened pleura. although considered malignant, mesotheliomas rarely metastasize to distant organs. secondary neoplasms of the pleura. secondary tumors may also spread into the visceral and parietal pleura. thymomas are rare neoplasms that grow in the cranial mediastinum of adult or aged dogs, cats, pigs, cattle, and sheep. thymomas are composed of thymic epithelium and lymphocytes (see chapter 13). old age, both in human beings and in animals, is known to be a risk factor for pulmonary infections, but the precise mechanisms involved in this increased susceptibility are still under investigation. some studies have shown that in aged individuals the antibacterial properties provided by surfactant proteins, proinflammatory cytokines, and complement are altered. pulmonary hyperinflation (often referred to as senile emphysema) has been reported as an age-related change in human and canine lungs. other age-related changes described in canine lungs include mineralization of bronchial cartilage, pleural and alveolar fibrosis, and heterotopic bone formation (so-called "pulmonary osteomas"). we thank all pathologists at the atlantic veterinary college, university of prince edward island for providing case material. suggested readings are available at www.expertconsult.com. lung section showing a distended and partially occluded blood vessel (center of figure) containing large granular cells. these large cells are macrophages, and their cytoplasm is filled with myriad merozoites isolation of porcine circoviruslike viruses from pigs with a wasting disease in the usa and europe exercise-induced pulmonary hemorrhage effect of mucociliary transport relies on efficient regulation of ciliary beating epidemiology, diagnosis, and treatment of blastomycosis in dogs and cats canine h3n8 influenza virus infection in dogs and mice failure of respiratory defenses in the pathogenesis of bacterial pneumonia in cattle the respiratory system advances in diagnosis of respiratory diseases of small ruminants canine nasal disease transmission of equine influenza virus to dogs dear jd: bacterial pneumonia in dogs and cats acute respiratory distress syndrome in dogs and cats: a review of clinical findings and pathophysiology inflammatory response to infectious pulmonary injury laryngeal paralysis: a study of 375 cases in a mixed-breed population of horses stem cells of the respiratory tract exudative pleural disease in small animals bovine respiratory disease research pulmonary thromboembolism coccidioidomycosis in dogs and cats: a review cousens c: pathology and pathogenesis of ovine pulmonary adenocarcinoma prognosis factors for survival in cats after removal of a primary lung tumor: 21 cases (1979-1994) the acute respiratory distress syndrome: from mechanism to translation endogenous lipid pneumonia in cats: 24 cases (1985-1998) retroviral infections in sheep and goats: small ruminant lentiviruses and host interaction canine and feline nasal neoplasia the acute respiratory distress syndrome equine respiratory medicine and surgery canine pleural and mediastinal effusions: a retrospective study of 81 cases a review of histiocytic diseases of dogs and cats estimation of nasal shedding and seroprevalence of organisms known to be associated with bovine respiratory disease in australian live export cattle polymicrobial respiratory disease in pigs current state of knowledge on porcine circovirus type 2-associated lesions common and emerging infectious diseases in the animal shelter chronic rhinitis in the cat advances in the understanding of pathogenesis, and diagnosis and therapeutics of feline allergic asthma mannheimia haemolytica and bovine respiratory disease detection of respiratory viruses and bordetella bronchiseptica in dogs with acute respiratory tract infections current perspectives on the diagnosis and epidemiology of mycoplasma hyopneumoniae infection mannheimia haemolytica: bacterialhost interactions in bovine pneumonia acute lung injury review rhodococcus equi: the many facets of a pathogenic actinomycete tumors of the respiratory system key: cord-023509-tvqpv6fp authors: corrin, bryan; nicholson, andrew g. title: occupational, environmental and iatrogenic lung disease date: 2011-03-02 journal: pathology of the lungs doi: 10.1016/b978-0-7020-3369-8.00007-0 sha: doc_id: 23509 cord_uid: tvqpv6fp nan in practice the term is confined to the effects of mineral dust on the lungs. diseases caused by organic dusts are not included among the pneumoconioses and, in medicolegal practice at least, the presence of dust alone is insufficient to indicate pneumoconiosis: for compensation to be considered, the mineral dust must alter the structure of the lung and cause disability. the british industrial injuries advisory council defined pneumoconiosis as 'permanent alteration of lung structure due to the inhalation of mineral dust and the tissue reactions of the lung to its presence, excluding bronchitis and emphysema' . 4 parkes recommends that cancer and asthma caused by mineral dust should also be excluded from the definition, an opinion with which we concur. 5 to reach the lung, dust particles have to be very small. particle density and shape also affect the aerodynamic properties of dust. host factors such as airflow characteristics, airway branching patterns and airway disease also affect dust deposition. three deposition mechanisms are recognised ( fig. 7.1.1 ): 1. inertial impaction: when air streams change direction or velocity, the inertia of the entrained particles causes them to maintain their original direction for a distance that depends upon their density and the square of their diameter. the same rules govern a car approaching a bend too fast: the car crashes into the outside of the bend. 2. sedimentation (gravitational settlement): under the influence of gravity, particles settle with a speed that is proportional to their density and the square of their diameter. 3. diffusion: very small airborne particles acquire a random motion as a result of bombardment by the surrounding gas molecules. inhaled dust particles are liable to sediment out in the alveoli if they have a diameter in the range of 1-5 µm, are roughly spherical in shape, and in density approximate to that of water. larger or denser particles impact or precipitate on the walls of the conductive airways and are rapidly removed by ciliary action. smaller particles may reach the alveoli but do not sediment so readily and many are therefore exhaled. very small particles are deposited on the walls of alveoli by diffusion but because they are so small the total amount of dust deposited in this way is insignificant compared with that deposited by sedimentation ( fig. 7.1. 2). direct measurement shows that most lung dust (96%) has a particle diameter less than 2.5 µm. 6 fibrous dust particles behave differently. fibres over 100 µm in length may reach the alveoli if they are very thin and remain aligned with the air stream. fibre penetration is inversely related to path length and the number of bifurcations. 7 tall people have longer conductive airways and experience more deposition in these sites than short people who have greater alveolar deposition for the same level of exposure. 8 slightly more dust is deposited in the right lung than the left, probably because the right main bronchus is more in line with the trachea, and is broader and shorter than the left, and carries 55% of the inhaled air. 9, 10 dust clearance from the lung inhaled dust that settles in the conductive airways is removed within a day or two by ciliary action. only dust that reaches the alveoli is liable to cause pneumoconiosis and much of this is also removed, but the clearance rate here is much slower: many coalminers continue to expectorate mine dust years after retirement. alveolar clearance is gravity largely effected by macrophages, principally via the airways to the pharynx but also via lymphatics to the regional lymph nodes. the airway and interstitial routes interconnect at the bronchiolar level 11 where some dust-laden macrophages leave the interstitium for the air space. 12 this interconnection is probably the route utilised by circulating macrophages clearing other parts of the body of endogenous or exogenous particulate matter via the lung. 13 long asbestos fibres present a particular problem to macrophage clearance. some minerals, notably chrysotile asbestos, undergo slow physicochemical dissolution in the lungs. only a small fraction of the inhaled dust gains access to the interstitium, a necessary step if it is to cause pneumoconiosis. some free dust enters through the bronchus-associated lymphoid tissue 11, 12 and some is taken up by, or pierces, the alveolar epithelium ( fig. 1.34 , p. 21). [14] [15] [16] some of this is transported within hours to the hilar lymph nodes. 17 so rapid is this translocation that it is thought not to involve most, the lesions are more numerous and better developed in the upper lobes than the bases but the reverse is true of asbestosis. the reasons for this are complex but undoubtedly involve the dust deposition:clearance ratio for the effect of the dust will depend upon both its amount and the duration of its stay in the lungs. there are well-recognised regional differences in the distribution and clearance of inhaled material, which in turn are dependent upon man's upright posture, the consequent gravitational forces being maximal at the apices. 35 when standing at rest, the apices of the lungs are hardly perfused, so that lymph formation and clearance are much better at the bases. [36] [37] [38] similarly, the apices are relatively less well aerated; alveoli in the lower lobes receive more air than those in the upper lobes. 37, 39 the greater respiratory excursions at the bases are thought to promote macrophage mobility there. it is to be expected therefore that the bases would both receive and clear more dust than the apices, rendering it difficult to predict on theoretical grounds which parts of the lungs carry the heaviest dust burden. in fact, more dust of all types is found in the upper lobes, the part most severely affected by every type of pneumoconiosis except asbestosis. 40, 41 the predilection of asbestos to affect the periphery of the lower lobes is attributed to the dangerous long asbestos fibres preponderating there. 41, 42 pulmonary reactions to mineral dust the main tissue reaction to mineral dust is fibrosis. silica is highly fibrogenic and is therefore very likely to cause pneumoconiosis. carbon is non-fibrogenic and therefore, unless there are complications, coal pneumoconiosis causes little disability. tin too is harmless, and stannosis therefore unimportant, although the chest radiograph is highly abnormal because tin is very radiopaque. stannosis is one of several terms that specify pneumoconiosis due to a particular mineral, the best known being silicosis, asbestosis and anthracosis. the blackness of carbon and red-brown colour of iron give ample evidence, both naked-eye and microscopically, of the type and amount of these dusts when they are present in the lung ( fig. 7.1.3 ), but other inorganic dusts may be more difficult to identify. however, a flick-out substage condenser and polaroid filters to test for refractility and birefringence respectively are useful adjuncts that are too often neglected by the histopathologist. crystalline silica is traditionally regarded as being only weakly birefringent, in contrast to silicates which generally show up brightly with simple crossed polaroid filters. 43 however, with modern microscope lamps, if the light source is set at high intensity when using polaroid filters, both silica and silicates are birefringent. 44 mineralogists use polarising microscopy for analysis, but only by studying large polished crystals with controlled orientation of the light. the small dust particles found in tissue sections are too small to permit analysis by this technique but it is nevertheless very useful for detecting their presence ( fig. 7.1.4) . particle shape gives a useful indication of mineral type but appearances are sometimes deceptive: the plate-like crystals of talc are seldom observed as such, usually being viewed edge-on, when they appear to be needle-shaped. occasionally, stains can be used to identify minerals, e.g. a modified perls' reaction for inhaled iron, and irwin's aluminon stain for aluminium, but these too have largely been replaced by modern analytical techniques. ultrafine dust particles are particularly liable to be transported across the alveolar epithelium. 11 the integrity of the alveolar epithelium is very important to dust translocation from the air spaces to the interstitium. much more dust reaches the interstitium if the epithelium is damaged. 18, 19 it is widely thought that macrophages that have left the interstitium for the alveolar space never return, 17, 20 but this is probably untrue. 21 heavily laden macrophages accumulate in alveoli bordering the terminal and respiratory bronchioles, eventually filling them completely. erosion of the alveolar epithelium permits re-entry of these macrophages into the interstitium, 22 very close to foci of bronchial mucosaassociated lymphoid tissue (malt), which are found near the terminal bronchioles. 23 these aggregates guard the mouths of lymphatics, which commence at this point; alveoli are devoid of lymphatics. dustladen interstitial macrophages accumulate in and around the bronchial malt, which macklin therefore referred to as dust sumps. 24 most pneumoconiotic lesions are found in the region of the dust sumps and are therefore focal. asbestosis is diffuse rather than focal because the long asbestos fibres are not readily mobilised and cannot be concentrated in the centriacinar dust sumps. this is also seen on occasion with platy non-fibrous dusts such as talc, mica, kaolinite and feldspar. [25] [26] [27] [28] [29] [30] [31] [32] [33] within the dust sumps the dust particles are not static. they are constantly being freed and reingested by interstitial macrophages and, because these cells are mobile, successively inhaled dusts soon become intimately mixed. 34 macrophages play an important role in pneumoconiosis and if the dust is fibrogenic the repeated phagocytosis of indestructible mineral particles results in constant fibroblast stimulation. the zonal distribution of pneumoconiosis pneumoconiosis affects both lungs but seldom evenly and some pneumoconioses show characteristic patterns of lung involvement. in microincineration combined with dark-field microscopy can also be used to demonstrate small particles. incombustible mineral particles that cannot be seen with bright-field or polarising microscopy are rendered visible by this technique and their position on the slide can be compared with tissue reactions evident in a serial section that has not been incinerated. microincineration has, however, also been largely replaced by modern analytical techniques that will now be considered. analytical electron microscopy is very helpful in identifying minerals, whether applied to lung digests or tissue sections. [45] [46] [47] [48] scanning electron microscopy permits the examination of thicker sections than transmission electron microscopy but does not detect very small particles. however, scanning electron microscopy allows more tissue to be examined and avoids the difficulty of cutting mineral particles with an ultramicrotome. mineral particles in a 5-µm thick deparaffinised section can be recognised in a scanning electron microscope set to collect the back-scattered electrons. 46 the instrument can then be focused on points of potential interest and switched to x-ray diffraction, which provides information on crystal structure (fig. 7.1.5) . alternatively, elemental analysis may be undertaken with either energy-dispersive or wavelength-dispersive x-ray spectroscopy. with energy-dispersive x-ray spectroscopy, all elements of atomic number above 11 are identified, whilst with wavelength-dispersive x-ray spectroscopy the section can be scanned for one particular element. with the former technique different elements are shown graphically as individual peaks, the heights of which are proportional to the amounts of the different elements within the particle studied, thereby giving information on probable molecular formula ( fig. 7.1.6) . thus, different silicates can be distinguished from each other and also from silica, which registers as pure silicon, oxygen (atomic number 8) not being detected. the fact that the elements of low atomic number that constitute organic chemicals are not detected means that any minerals present (except beryllium, atomic number 4) can be recognised easily in tissue sections. only particles can be analysed however: elements present in only molecular amounts cannot be detected by x-ray analysis. the detection of trace amounts of substances such as beryllium requires bulk chemical analysis or techniques that are not widely available such as atomic absorption spectrometry, neutron activation analysis and microprobe mass spectrometry. 49, 50 the last of these techniques can also provide molecular (as opposed to elemental) analysis of organic as well as inorganic particles. 51 another analytical technique of interest is microscopic infrared spectroscopy which provides data on the compound nature of microscopic particles in tissue sections ( fig. 7.1.7) . micro-raman spectroscopy is also useful in this respect. some metals cause hypersensitivity, which can be identified by exposing the patient's lymphocytes to metals and measuring their reaction in vitro. 52 radiological grading of pneumoconiosis a scheme for grading pneumoconiosis radiologically by comparison with standard radiographs has been adopted by the international labour organisation (ilo) and is widely used. 53 small opacities (up to 1 cm diameter) are graded by their profusion, 1, 2 and 3 indicating increasing numbers, and by their size, increasing through p, q and r if rounded and s, t and u if irregular. type p opacities are described as punctiform and measure up to 1.5 mm in diameter; larger lesions up to 3 mm in diameter (type q) are described as micronodular or miliary; and those over 3 mm and up to 1 cm in diameter (type r) are described as nodular. irregular opacities cannot be sized so accurately, s, t and u indicating fine, medium and coarse respectively. large opacities (over 1 cm diameter) are graded by their combined size, increasing through a, an opacity measuring between 1 and 5 cm in diameter; b, one or more opacities whose combined area does not exceed the equivalent of one-third of the area of the right lung field (when they are regrouped in the mind's eye or measured with a transparent ruler); and c, one or more opacities whose combined area exceeds one-third of the area of the right lung field (when similarly regrouped). in coalworkers, small opacities (up to 1 cm diameter) correspond to simple coalworker's pneumoconiosis and large opacities (over 1 cm diameter) to complicated coalworker's pneumoconiosis, which is also known as progressive massive fibrosis. silicotic lesions have been identified in the lungs of egyptian mummies, and the injurious effects on the lungs of inhaling mine dust have been recognised for more than 400 years. as long ago as the sixteenth century in joachimsthal, bohemia (now jachymov, czech republic), diseases of miners' lungs were attributed to the dust the miners breathed. silicosis, tuberculosis and lung cancer are all now known to have been prevalent among the miners in this region, the cancer being largely attributable to the high level of radioactivity in the mines. silicosis was recognised in the uk soon after the discovery in 1720 that the addition of calcined flint to the clay from which china is made produced a finer, whiter and tougher ware. the preparation and use of this flint powder were highly dangerous, causing the condition known as potter's rot, one of the first of the many trade names by which silicosis has since been known. aluminium oxide (alumina) now provides a safe, effective substitute for flint in this industry. in 1830 it was noted that sheffield fork grinders who used a dry grindstone died early, and amongst other preventive measures it was recommended that the occupation should be confined to criminals: fortunately for them, the substitution of carborundum (silicon carbide) for sandstone was effective enough. however, silicosis still occurs in some miners, tunnellers, quarrymen, stone dressers and metal workers. silica in one form or another is used in many trades -in the manufacture of glass and pottery, in the moulds used in iron foundries, as an abrasive in grinding and sandblasting, and as a furnace lining that is refractory to high temperatures. rocks such as granite and sandstone are siliceous and their dusts are encountered in many mining and quarrying operations. in coal mining in the uk the highest incidence of the disease was in pits where the thinness of the coal seams required the removal of a large amount of siliceous rock, a process known as 'hard heading' . in south africa, silicosis causes a high mortality among the gold miners on the witwatersrand, where the metallic ore is embedded in quartz. slate is a metamorphic rock that contains both silica and silicates, and slateworkers develop both silicosis and mixed-dust pneumoconiosis. 55, 56 nor are rural industries immune from the disease, particularly if ventilation is inadequate, as it is in certain african huts where stone implements are used to pound meal and the occupants develop mixed-dust pneumoconiosis. 57 silicosis and mixed-dust pneumoconiosis have also been reported in dental technicians. 58 desert sand is practically pure silica but the particles are generally too large to reach the lungs. however, silicosis has been reported in inhabitants of the sahara, libyan and negev deserts and those living in windy valleys high in the himalayan mountains, [59] [60] [61] [62] [63] [64] [65] whilst in california the inhalation of dust raised from earth has led to silicate pneumoconiosis in farm workers, 66 horses 67 and a variety of zoo animals. 68 the silica in rocks such as granite, slate and sandstone is largely in the form of quartz and this is therefore the type of silica encountered in most of the industries considered above. cristobalite and tridymite, which are possibly even more fibrogenic than quartz, are more likely to be encountered in the ceramic, refractory and diatomaceous earth industries where processing involves high temperatures. many workers with silicosis are asymptomatic. as a general rule, exposure to silica dust extends over many years, often 20 or more, before the symptoms of silicosis first appear: by the time the disease becomes overt clinically, much irreparable damage has been inflicted on the lungs. the initial symptoms are cough and breathlessness. from then onwards, respiratory disability progresses, even if the patient is no longer exposed to silica dust. ultimately, there may be distressing dyspnoea with even the slightest exercise. silicosis sometimes develops more rapidly, perhaps within a year or so of first exposure. such 'acute silicosis' was observed in the scouring powder industry in the 1930s when these cleansing agents consisted of ground sandstone mixed with a little soap and washing soda. 69, 70 the additives were considered to have rendered the silica in the sandstone more dangerous but it is possible that the rapidity of onset of the disease merely reflected the intensity of the dust cloud to which the packers were exposed. confusingly, the term 'acute silicosis' has since been applied to a further effect of heavy dust exposure in tunnellers, sand blasters and silica flour workers, namely pulmonary alveolar lipoproteinosis (see below), 71, 72 whilst the terms 'accelerated silicosis' or 'cellular phase silicosis' have been substituted for 'acute silicosis' in referring to the rapid development of early cellular lesions. 43, 73 the time from first exposure to the development of symptoms (the latency period) is inversely proportional to the exposure level. however, it is evident that a certain amount of silica can be tolerated in the lungs without fibrosis developing, indicating either a time factor in the pathogenetic process or a threshold dust load that has to be reached before fibrosis develops. silica particles that are roughly spherical in shape and of a diameter in the range of 1-5 µm sediment out in the alveoli and are concentrated within macrophages at macklin's dust sumps, as explained previously (see p. 27). early lesions, as seen in so-called accelerated or cellular phase silicosis, consist of collections of macrophages separated by only an occasional wisp of collagen. the early lesions have been likened to granulomas and on occasion have been mistaken for langerhans cell histiocytosis or a storage disorder, but langerhans cells are scanty and the histiocytes contain dust particles rather than accumulated lipid or polysaccharide. the macrophages of the early lesion are gradually replaced by fibroblasts and collagen is laid down in a characteristic pattern. the mature silicotic nodule is largely acellular and consists of hyaline collagen arranged in a whorled pattern, the whole lesion being well demarcated ( fig. 7.1.8 ) and sometimes calcified. small numbers of birefringent crystals are generally evident within the nodules when polarising filters are used, but these mainly represent silicates such as mica and talc, inhaled with the silica. silica particles are generally considered to be only weakly birefringent, 43 but fairly strong birefringence is evident in strong light (see above). 44 silicotic nodules develop first in the hilar lymph nodes and are generally better developed there than in the lungs. [74] [75] [76] indeed, silicotic nodules are occasionally found in the hilar lymph nodes of persons who have no occupational history of exposure to silica and whose lungs are free of such lesions, the silica in the nodes being presumed to represent inhaled particles derived from quartz-rich soil. 77 severely affected lymph nodes often calcify peripherally, giving a characteristic eggshell-like radiographic pattern. this is sometimes the only radiological abnormality. 76 such enlarged lymph nodes may occasionally press upon and obstruct adjacent large bronchi 78 or result in a left recurrent laryngeal nerve palsy, 79 so simulating malignancy. 80 sometimes the nodules develop within the walls of major bronchi, occasionally causing a middle-lobe syndrome (see p. 92). 81 silicotic nodules are also found along the lines of the pleural lymphatics 75, 82 where they have been likened to drops of candle wax on the visceral pleura. very rarely, silica-induced fibrosis is more pronounced in the pleura than in the lungs. 83 lung tissue between the nodules is often quite normal and not until the process is very advanced is there any disability ( fig. 7. 1.9b). in severe cases large masses of fibrous tissue are formed, which may undergo central necrosis and cavitation ( fig. 7.1.10 ). 84 on close inspection it is evident that these consist of conglomerations of many silicotic nodules closely packed together. in such severe cases cor pulmonale develops. occasionally, silicotic nodules develop in the abdominal as well as the thoracic lymph nodes, and in the liver, spleen, peritoneum and bone marrow. [85] [86] [87] [88] [89] in about 10% of cases, the typical pulmonary nodules that predominantly affect the upper lobes are accompanied by diffuse fibrosis that is maximal in the lower lobes. 27, 33, [90] [91] [92] the latter may show 'honeycombing' and closely resemble idiopathic pulmonary fibrosis. the association is too common to be explained by chance and the diffuse fibrosis is therefore regarded as a further manifestation of the pneumoconiosis, possibly due to an interaction between the dust and the immunological factors discussed below. the pathogenesis of silicosis has excited much interest and many different theories have been advanced over the years. an early theory held that the hardness of the silica was responsible, but this was discounted by the observation that silicon carbide (carborundum) is harder than silica but is non-fibrogenic. theories based on the piezoelectric property and on the solubility of silica were successively abandoned although the latter had a long period of popularity. it gained support from kettle's experiments which showed that fibrosis developed about chambers placed in an animal's peritoneal cavity if the chambers contained silica powder sealed in by a collodion membrane through which solutes such as silicic acid could pass. however, it was later shown that the pores in a collodion membrane are quite irregular in size and when the experiments were repeated using chambers guarded by millipore membranes, no fibrosis developed, despite solutes being able to diffuse out. 93 the solubility theory also fails to take account of the differing fibrogenicity of the various forms of silica despite them being of similar solubility. 54 furthermore, if the outer, more soluble layer of the particles is removed by etching, fibrogenicity is increased although solubility is decreased. in line with this, freshly fractured crystalline silica is more pathogenic in every respect than its aged equivalent, 94 which may partly explain the severity of silicosis in trades such as sandblasting. these observations suggest that the fibrogenicity of silica is connected with its surface configuration. it is now known that uptake of the silica by macrophages is necessary for silicosis to develop. if silica and macrophages are enclosed together in peritoneal millipore chambers, a soluble product of the macrophages diffuses out and causes fibrosis. this observation led to the realisation that the fibrogenicity of the various crystalline forms of silica correlated well with their toxicity to macrophages and for a time macrophage death was thought to be necessary. 95 it is now considered that before the macrophages are killed by the ingested silica, they are stimulated to secrete factors that both damage other con stituents of the lung and promote fibrosis. [96] [97] [98] [99] [100] [101] [102] transforming growth factor-β is one fibrogenic factor that has been implicated in the pathogenesis of silicosis. [103] [104] [105] toxic damage to macrophages is due to silica particles injuring the phagolysosomal membranes, so releasing acid hydrolases into the cytoplasm. 95 it is important in the pathogenesis of the disease indirectly because when the macrophage crumbles, the silica particles are taken up by fresh macrophages and the fibrogenic process continues. it has been suggested that early involvement of the hilar lymph nodes in the fibrogenic process promotes the development of the disease in the lung by delaying dust clearance. 74 immunological factors have been implicated in the pathogenesis of silicosis because many patients with silicosis have polyclonal hypergammaglobulinaemia, rheumatoid factor or antinuclear antibodies, and because there is a well-recognised association between autoimmune diseases such as systemic sclerosis and rheumatoid disease and exposure to silica. 43, [106] [107] [108] [109] the relation of immunity to dust exposure appears to be a reciprocal one: on the one hand, the presence of dust results in rheumatoid lesions in the lungs being more florid (see caplan's syndrome, p. 341), whilst on the other, non-specific immunisation of rabbits with horse serum results in experimental silicotic lesions being larger and more collagenous. 110 it is doubtful whether pneumoconiosis and autoimmune disease play a causative role in each other but one seems to aggravate the other and may lead to its earlier development. one of the commonest and most feared complications of silicosis is chronic respiratory tuberculosis. 109 once this infection has been added to the silicosis, the prognosis rapidly worsens. it is thought that in the presence of silica, the tubercle bacilli proliferate more rapidly because the ingested silica particles damage phagolysosomal membranes and thereby interfere with the defensive activity of the macrophages. the synergistic action of silica dust has long been held responsible for the inordinately high incidence of respiratory tuberculosis in mining communities. many former south african gold miners now have acquired immunodeficiency syndrome (aids) as well as silicosis and tuberculosis has consequently reached almost epidemic proportions amongst these men. phagocyte damage by ingested dust particles may also cause some cases of chronic necrotising aspergillosis complicating pneumoconiosis. 111 a series of studies suggesting that there might be a link between silica inhalation and lung cancer was reviewed by the international agency for research on cancer in 1987, leading to the conclusion that the evidence for carcinogenicity of crystalline silica in experimental animals was sufficient, while in humans it was limited. 112 subsequent epidemiological publications were reviewed in 1996, when it was concluded that the epidemiological evidence linking exposure to silica to the risk of lung cancer had become somewhat stronger 113 but that in the absence of lung fibrosis remained scanty. 113 the pathological evidence in humans is also weak in that premalignant changes around silicotic nodules are seldom evident. 114 nevertheless, on this rather insubstantial evidence, lung cancer in the presence of silicosis (but not coal or mixed-dust pneumoconiosis) has been accepted as a prescribed industrial disease in the uk since 1992. 115 some subsequent studies have provided support for this decision. 116 in contrast to the sparse data on classic silicosis, the evidence linking carcinoma of the lung to the rare diffuse pattern of fibrosis attributed to silica and mixed dusts is much stronger and appears incontrovertible. 33, 92 alveolar lipoproteinosis in response to heavy dust exposure a further complication of exposure to silica is the development of alveolar lipoproteinosis (see p. 317). 71, 72, 117, 118 very heavy experimental exposure to silica, and indeed other dusts, stimulates hypersecretion of alveolar surfactant to such an extent that the normal clearance mechanism is overwhelmed. [119] [120] [121] [122] [123] [124] [125] alveolar macrophages are enlarged by numerous phagolysosomes distended by lamellar bodies that represent ingested surfactant. the alveoli are filled by such cells and, having a foamy cytoplasm, they produce the appearances of endogenous lipid pneumonia, similar to that more usually encountered as part of an obstructive pneumonitis distal to a bronchial tumour. the macrophages gradually disintegrate and the free denatured surfactant slowly becomes compacted, during which time its staining with both eosin and the periodic acid-schiff reagents intensifies until the appearances are finally those of alveolar lipoproteinosis. this process prevents the aggregation and concentration of the dust in the usual foci and thereby hinders the development of silicosis. lipoproteinosis and silicosis may be seen in conjunction but, more often, different areas of the lung show one or the other. the lipoproteinosis has its own severe impact on lung function, but, unlike silicosis, is potentially reversible (by massive alveolar lavage). occasional patients exposed to silica develop renal disease. [126] [127] [128] [129] two mechanisms appear to operate. first, translocation of silica particles from the lungs leads to their deposition in the renal interstitium with resultant nephrotoxity. second, silica stimulates an autoimmune response characterised by the formation of various antibodies, notably rheumatoid factor and antinuclear antibodies, which leads to the development of immune complex-mediated glomerulonephritis. 128, 129 amorphous silica manmade submicron forms of silica, variously known as amorphous, vitreous, colloidal, synthetic or precipitated silica, are widely used in industry. they consist of pure non-crystalline silicon dioxide. particle size ranges from 5 to 200 nm but aggregates of the particles measure from 1 to 10 µm. industrial surveys suggest that inhalation of such dust is harmless, observations that are in accord with the results of animal experiments. 54 an amorphous silica is the principal component of the fossilised remains of diatoms that constitute the sedimentary rock, diatomite ( fig. 7.1.11 ). this is generally obtained by open-cast mining, following which the rock is crushed and calcined. the calcined product is used in filters, insulation material and as a filler. being amorphous, the silica in diatomite is harmless, but calcining (>1000°c) results in its conversion to crystalline forms of silica. diatomaceous earth pneumoconiosis is unusual and its risk appears to be related to the amount of cristobalite and tridymite (two forms of crystalline silica) produced in the calcining process. 130 the silicates are complex compounds in which silicon and oxygen form an anion combined with cations such as aluminium and magnesium: talc, for example, is a hydrated magnesium silicate with the formula mg 3 si 4 o 10 (oh) 2 . silicates include fibrous forms (asbestos and the zeolites), plate-like forms (talc and mica) and clays (kaolinite and fuller's earth). in histological sections, the platy talc and mica particles are generally cut tangentially and therefore appear needleshaped (see fig. 7 .1.4). they are strongly birefringent whereas the clays are only weakly so. talc particles in the lung exceeding 5 µm in length should arouse suspicion of intravenous drug abuse. 131 of the fibrous silicates, zeolite is used as a building material in certain communities, notably in central turkey. pneumoconiosis is not a problem but zeolites are of medical interest because, like asbestos, they present a mesothelioma risk. asbestos is dealt with separately (see below). pneumoconiosis has been described with various non-fibrous silicates, notably in the rubber industry, which uses talc and, less commonly, mica as lubricants. other occupations posing a risk include the extraction of kaolinite from china clay (kaolin), 27, 132, 133 and in the open-cast and underground mining of fuller's earth (montmorillonite, bentonite and attapulgite clays, which were originally used in 'fulling' (degreasing) wool). 134, 135 however, all these substances are commonly contaminated with silica, asbestos or both, and it has been questioned whether in pure state they are at all fibrogenic. the modifying effect of inert substances such as iron on that of silica is well known (see mixed-dust pneumoconiosis, below) and it has been suggested that talc, mica and fuller's earth act in a similar way in regard to their more fibrogenic contaminants, the pneumoconioses attributed to them in reality representing mixed-dust pneumoconiosis or asbestosis. contrary evidence comes from reports of pulmonary fibrosis in persons heavily exposed to pure talc, mica or kaolin. 32 all these silicates are evident in the tissues as plate-like birefringent crystals which often provoke a foreign-body giant cell reaction (see fig. 7 .1.3) and may result in fibrotic nodules. large focal lesions resembling the progressive massive fibrosis of coalworkers may be produced, and also a diffuse 'asbestosis-like' form of pneumoconiosis, the latter attributed to poor macrophage mobilisation of the plate-like particles. [27] [28] [29] [30] [31] [32] [132] [133] [134] [135] [136] [137] [138] it would appear therefore that silicates are indeed fibrogenic if enough is inhaled; they appear to vary in fibrogenicity but in all cases they are less fibrogenic than silica. inert dusts are non-fibrogenic and therefore of little clinical consequence, although elements of high atomic number can give rise to a striking chest radiograph. 139 it should be noted however that inert or lowly fibrogenic materials may be associated with substances of medical importance, for example, kaolin, bentonite and barytes (barite) may all be contaminated with silica 27, 134, 140 and talc may be contaminated with asbestos. the best known of the inhaled inert mineral dusts is carbon while, of the remainder, iron is the most widespread. others include tin and barium. with all these dusts, particles retained in the lung are gathered at macklin's dust sumps by heavily laden macrophages which are lightly bound together there by a few reticulin fibres. collagen is not formed and the worker suffers no ill-effects. the lungs take on the colour of the dust and in siderosis assume a deep brick-red hue. carbon deposition is commonly found in the lungs, particularly those of city dwellers and tobacco smokers. it is also the principal constituent of coal, which is dealt with separately below, and large amounts of pure carbon may be inhaled by workers involved in the manufacture of carbon black, carbon electrodes and charcoal. [141] [142] [143] [144] although carbon is regarded as being non-fibrogenic, the very heavy lung burdens encountered in industries such as these may lead to the complicated form of pneumonconiosis known as progressive massive fibrosis that is more commonly encountered in coal workers (see p. 340). heavy pure carbon deposition may also be acquired domestically when wood is burnt in buildings devoid of a chimney, so-called 'hut lung', 145 a term that is also applied to the domestic acquisition of carbon mixed with silica or silicates, resulting in forms of mixeddust pneumoconiosis. 57, 64, 65 anthracofibrosis is a term introduced by chinese bronchoscopists for bronchial stenosis or obliteration associated with carbon pigmentation of the mucosa. 146 although the original description incriminated tuberculosis, mixtures of various mineral dusts acquired at work or domestically are a more likely cause. [147] [148] [149] [150] iron dust in the lungs was first described by zenker in 1867, when he also introduced the terms siderosis and pneumonokoniosis. 3 zenker was describing a woman who coloured paper with iron oxide powder ('rouge'), a substance which is still encountered by some workers engaged in polishing silver, glass, stone and cutlery. siderosis is also found in welders, iron foundry fettlers, steel workers, boiler scalers and haematite miners and crushers. iron dust particles are reddish-brown but in the lung may be masked by carbon 151 : when evident, or revealed by microincineration, they resemble haemosiderin and generally give a positive perls' reaction, but particularly with haematite, heat (60-80 o c) and concentrated (12n) hydrochloric acid may be necessary. 152 haematite miners in both the uk (cumbria) and france (lorraine) have an increased risk of bronchial carcinoma, but radon gas rather than haematite is the suspected carcinogen. radon is a decay product of uranium. minute amounts are present in all rocks but local concentrations occur and these are liable to build up in mines if ventilation is limited. silver, as well as iron, is found in the lungs of silver polishers, where it stains elastin in alveolar walls and pulmonary vessels grey. such argyrosiderosis is as harmless as siderosis. tin miners are subject to silicosis but not stannosis because the ore, which is found in association with siliceous rocks, contains only low concentrations of the metal. tin smelters, on the other hand, and factory workers exposed to high concentrations of tin dust or fume, are liable to inhale large amounts of this inert metal and develop the striking chest radiograph of stannosis. they remain in good health however for tin is completely non-fibrogenic. tin particles in the lung resemble carbon but are strongly birefringent and remain after microincineration: microprobe analysis provides positive identification. other inert dusts include barium, which also has a high atomic number and is therefore radiopaque, 139 and minerals of low radiodensity such as limestone, marble and cement (all chiefly composed of calcium carbonate) and gypsum (hydrated calcium sulphate). however, the extraction of barium ore (almost entirely in the form of barium sulphate, which is known as barytes in europe and barite in the usa) may entail exposure to silica and silicates. pure baritosis resembles stannosis and siderosis. the term 'mixed-dust pneumoconiosis' refers to the changes brought about by inhaling a mixture of silica and some other less fibrogenic substance such as iron, carbon, kaolin or mica. 33, 151, [153] [154] [155] the proportion of silica is usually less than 10%. typical occupations include foundry work and welding and the mining of coal, haematite, slate, shale and china clay. the action of the silica is modified and, although fibrotic nodules are formed, they lack the well-demarcated outline and concentric pattern of classic silicosis. the lesions are found in a centriacinar position and are stellate in outline with adjacent scar emphysema. they are firm and generally measure no more than 5 mm in diameter. they closely resemble the fibrotic nodules of simple coal pneumoconiosis (see below). confluent lesions also occur on occasions. these resemble the progressive massive fibrosis of coalworkers and appear to represent a single large lesion rather than a conglomeration of individual nodules, as in advanced silicosis. abundant dust is generally evident in lesions of all sizes; this consists of black carbon or brown iron mixed with crystals of varying degrees of birefringence, silicates generally being strongly birefringent and silica weakly so. calcification is unusual. mixed-dust pneumoconiosis carries an increased risk of pulmonary tuberculosis, but not to the same degree as silicosis. in some cases the stellate nodules are accompanied by diffuse fibrosis, as in silicosis and again possibly involving interactions between the dust and immunological factors. involvement of the bronchi with consequent stenosis (so-called anthracofibrosis) is described above. the term 'anthracosis' was initially applied to changes observed in a coalminer's lung 157 but is now often extended to include the common carbon pigmentation of city dwellers' lungs, and the term 'coal pneumoconiosis' is more appropriate to a special form of pneumoconiosis to which coalworkers are subject, particularly those who work underground. the principal constituent of coal, carbon, is non-fibrogenic, so suspicion has naturally fallen on the ash content of mine dust, some of which derives from the coal, some from adjacent rock strata and some from stone dust laid in the roadways to minimise the risk of coal dust explosions. coal itself appears to be the responsible agent because coal-trimmers, working in the docks and not exposed to rock dust, also develop the disease. 158 coalminers encountering siliceous rock are, of course, also liable to develop silicosis like other underground workers. coal consists largely of elemental carbon, oxygen and hydrogen with traces of iron ore and clays such kaolinite, muscovite and illite, but no silica. the mineral content varies with the type and rank (calorific value) of the coal. all coal derives from peat, the youngest type being lignite and the oldest anthracite, with bituminous (house) coal in between. as it ages, the oxygen and mineral constituents diminish and the coal hardens. lignite is soft and said to be of low rank, anthracite hard and of high rank, with bituminous coal intermediate. although high-rank coal is of low mineral content, its dust is more toxic to macrophages in vitro and is cleared more slowly in vivo. this observation may explain why, in the uk, high-rank coal is associated with a higher prevalence of coal pneumoconiosis. the low mineral content of high-rank coal is reflected in the mineral content of the lungs of those who hew such coal in the uk, but in the ruhr, in germany, and in pennsylvania, in the usa, anthracite miners' lungs contain more silica than those who hew bituminous coal, the silica presumably deriving from other sources. not surprisingly, the presence of silica is reflected in the tissue reaction to the inhaled dust, resulting in a more fibrotic reaction very analogous to mixed-dust pneumoconiosis. a spectrum of changes is therefore encountered in coalminers' lungs, ranging from coal pneumoconiosis through mixeddust pneumoconiosis to silicosis; the findings in any individual depend upon the nature of the coal being mined and the type of work undertaken. in high-rank british collieries the development of coal pneumoconiosis appears to depend on the total mass of dust inhaled, whereas in low-rank british collieries the mineral content of the lung dust appears to be more important. 159 this may explain apparently contrary data drawn from different coalfields -data based on coals of different composition that are not strictly comparable. some workers have stressed the importance of silica in the dust whereas others, particularly in the high-rank coalfields of south wales, have been unable to detect any association between silica and the level of pneumoconiosis. both findings may be correct, but only for the particular group of miners examined in each case. 160 the lesions of coal pneumoconiosis are generally focal and fall into one or other of two major types, simple and complicated, depending upon whether the lesions measure up to or over 1 cm; simple corresponds to categories 1-3 of the ilo grading system (see p. 331) and complicated, which is also known as progressive massive fibrosis, to ilo categories a-c. more diffuse interstitial fibrosis has been reported in about 16% of welsh and west virginian coalminers, usually involving those carrying a particularly heavy dust burden; it runs a more benign course than non-occupational interstitial fibrosis (idiopathic pulmonary fibrosis). 161 similar findings have been reported from france. 162 simple coal pneumoconiosis consists of focal dust pigmentation of the lungs, which may be associated with a little fibrosis and varying degrees of emphysema. its clinical effects are relatively minor. some degree of black pigmentation (anthracosis) of the lungs is common in the general urban population, especially in industrial areas, but much denser pigmentation is seen in coalminers, whose lungs at necropsy are black or slate-grey. black pigment is evident in the visceral pleura along the lines of the lymphatics and on the cut surface where it outlines the interlobular septa and is concentrated in macklin's centriacinar dust sumps ( fig. 7.1.12 ). the dust is generally more plentiful in the upper parts of the lungs and in the hilar lymph nodes, possibly due to poorer perfusion and consequently poorer lymphatic drainage there (see p. 21). 162 two forms of coal dust foci are recognised, macules and nodules, the former being soft and impalpable and the latter hard due to substantial amounts of collagen. both lesions are typically stellate but the more fibrotic the nodules, the more rounded they become, until it is difficult to distinguish them macroscopically from those of silicosis. in these circumstances reliance has to be placed on the whorled pattern of the collagen that is evident microscopically in silicosis. the stellate nodules are analogous to those seen in mixed-dust pneumoconiosis caused by mixtures of silica and inert dusts other than carbon (see above). with polarising filters, small numbers of birefringent crystals may be seen in both macules and nodules, usually representing mica or kaolinite derived from rock that bordered the coal. macules consist of closely packed dust particles, free or within heavily laden macrophages, so that the lesion appears black throughout ( fig. 7.1.13 ). appropriate stains show that the dust-laden macrophages and free dust are lightly bound by reticulin. very little collagen is evident. although striking in their appearance, dust macules are thought to have little effect on lung function. nodules contain substantial amounts of collagen and are thought to have an adverse, but limited, effect on respiration. they vary from a heavily pigmented, stellate lesion, which apart from its collagen content resembles the dust macule ( fig. 7. 1.14), to one that is less pigmented and more circumscribed. the stellate, heavily pigmented type of nodule is seen in lungs that have a relatively low ash content whilst the more rounded and less pigmented nodule is seen in lungs with relatively high ash loads. 163 radiologically (see p. 321), p-type opacities correspond to macules, q-type opacities to the stellate nodules that resemble those of mixeddust pneumoconiosis and r-type opacities to the rounded nodules that resemble those of silicosis. 53, 164 thus, the radiological changes of simple coalworker's pneumoconiosis are due to the dust and the small amount of collagen present and do not reflect any emphysema that may also be present. however, pulmonary dust foci are often associated with emphysema ( fig. 7.1.15 ) and the severity of the emphysema appears to correlate with the dust load. the prevalence of chronic bronchitis and emphysema is high in the coal industry and it has long been debated whether occupation or cigarette smoking is the major factor contributing to emphysema in coalminers. [165] [166] [167] [168] as well as mineral dust, nitrous fumes from shot-firing form another occupational hazard of coal mining. heppleston made a special study of the emphysema found in coalminers, claiming that it differs from centriacinar emphysema, as seen in smokers in the general population, and attributing it to the dust. 169 he introduced the term 'focal emphysema of coalworkers' to describe this special process. others find it very difficult to identify any convincing difference between the emphysema of coalworkers and that encountered outside the industry but heppleston based his claims on the study of serial sections. by this means he showed that, although both forms affect respiratory bronchioles, the focal emphysema of coalworkers affects more proximal orders of these airways and is not associated with the bronchiolitis seen with centriacinar emphysema. furthermore, focal emphysema is a dilatation lesion whereas coniosis, also known as progressive massive fibrosis, can have very serious consequences. particularly when the lesions are large, it is associated with productive cough, breathlessness, significant impairment of lung function and premature death. the major factor accounting for the development of progressive massive fibrosis appears to be the sheer bulk of coal dust in the lung, rather than coal rank or the silica content of the mine dust. 179 progressive massive fibrosis has occasionally been recorded in dockers loading silica-free coal into the holds of ships 158 and in workers exposed to pure carbon in the manufacture of carbon black and carbon electrodes. [141] [142] [143] progressive massive fibrosis is characterised by large (over 1 cm) black masses, situated anywhere in the lungs but most common in the upper lobes. the lesions may be solitary or multiple and very large, occupying most of the lobe and even crossing an interlobar fissure to involve an adjacent lobe (figs 7.1.3b, 7.1.16). they cut fairly easily, often with the release from a central cavity of black fluid flecked by cholesterol crystals. for many years it was believed that the condition was the result of synergism between mycobacterial infection and dust but the failure of the attack rate to decrease as tuberculosis declined negated this view. 180 today, more emphasis is placed on total dust load for the lesions tend to affect lungs that carry an unduly heavy dust burden. if the remainder of the lung shows little evidence of dust accumulation, the possibility of the masses representing caplantype lesions (see below) should be considered. centriacinar emphysema involves destruction of adjacent alveolar walls. by definition, therefore, focal emphysema is not a true emphysema at all (see p. 102). however, it has been shown that mineral dusts cause elastin and collagen breakdown in the rat lung. 170 focal emphysema may progress to the destructive centriacinar form and this has strengthened claims that mine dust plays a causal role in centriacinar emphysema. 1, [171] [172] [173] [174] [175] [176] in the uk, these claims have been accepted and chronic bronchitis and emphysema in coalminers and metal production workers have been accepted as prescribed industrial diseases since 1992. 177 in germany too, chronic obstructive pulmonary disease is now compensatable as an occupational disease. the conditions for compensation in the uk were initially: • underground coal mining for a minimum of 20 years in aggregate • forced expiratory volume in 1 second at least 1 litre below that expected or less than 1 litre in total • radiological category of at least 1/1. however the last of these criteria has now been dropped. the inclusion of a time element and the omission of some estimate of dust load (such as radiological category) have been criticised, with some justification. 178 as with lung cancer caused by chromates benefit is paid irrespective of smoking habits. whereas simple coal pneumoconiosis, particularly the macular variety, has little effect on lung function, complicated coal pneumomicroscopically, the lesions consist of dust and connective tissue intermixed in a random fashion. central necrosis and cavitation commonly occur. the necrosis is thought to be ischaemic. 181 it is amorphous or finely granular, and eosinophilic apart from abundant dust particles and cholesterol crystal clefts. the fibrotic component in a complicated pneumoconiotic lesion is rich in fibronectin, with collagen only more abundant at the periphery. 182 two types of progressive massive fibrosis are recognisable, corresponding to the two types of nodule described in simple coal pneumoconiosis. 163 the first appears to have arisen by enlargement of a single nodule, whereas the second is a conglomeration of individual lesions, each of which corresponds to the more circumscribed type of nodule seen in simple coal pneumoconiosis. the ash content of the lungs bearing these two types of progressive massive fibrosis varies in the same way as with the two types of simple pneumoconiotic nodules, the enlarged single lesion being found in lungs with a relatively low ash content, and the conglomerate lesion in lungs with a relatively high ash content. the second type resembles the conglomerate nodules of large silicotic lesions but lacks the characteristic whorled pattern of the latter. the diffuse interstitial fibrosis found in a minority of coalworkers is associated with heavy dust deposition. it may progress to honeycombing but, as with the focal forms and unlike idiopathic interstitial fibrosis, it is better developed in the upper zones, the reasons for which are discussed above (see the zonal distribution of pneumoconiosis, p. 329). the pathogenesis of coal pneumoconiosis has much in common with that of silicosis, and indeed many other pneumoconioses. it involves the promotion of fibrogenic factor synthesis and release by cells phagocytosing the inhaled dust. several such factors have now been identified, the degree of fibrosis produced varying with the amount of dust inhaled and the ability of its constituents to promote the production of the responsible cytokines. these include plateletderived growth factor, insulin-like growth factors 1 and 6, transforming growth factor-β and tumour necrosis factor-α. 101, 183, 184 as with other minerals, the indestructability of the dust perpetuates the process. as in silicosis, immunological factors appear to be involved, for there is an increased prevalence of rheumatoid arthritis 185 and of circulating autoantibodies [186] [187] [188] in miners with coal pneumoconiosis. rheumatoid factor has also been demonstrated within the lung lesions. 189 these abnormalities are generally more pronounced in miners with complicated pneumoconiosis but are also found in those with the simple variety. it is also possibly pertinent to the immunological basis of coal pneumoconiosis that some of the pulmonary manifestations of rheumatoid disease are more pronounced in coalminers. this was first pointed out by caplan and will be considered next. caplan described distinctive radiographic opacities in the lungs of coalminers with rheumatoid disease, 190 and it is now recognised that similar lesions may develop in rheumatoid patients exposed to siliceous dusts. the development of such rheumatoid pneumoconiosis does not correlate with the extrapulmonary or serological activity of the rheumatoid process. nor is there a strong relation to dust burden: caplan lesions are characteristically seen in chest radiographs that show little evidence of simple coal pneumoconiosis. pathologists recognise the lesions as particularly large necrobiotic nodules similar to those seen in rheumatoid patients who are not exposed to dust (fig. 7.1.17) . however, because of their large size (up to 5 cm diameter) they may be confused with progressive massive fibrosis undergoing central ischaemic necrosis (see above) or silicosis complicated by caseating tuberculosis. such errors will be less likely if the radiological evolution of the lesions is considered for they tend to cavitate and undergo rapid remission, only to be succeeded by others. they are also well demarcated radiologically. pathologically, they resemble rheumatoid nodules in showing peripheral palisading but differ in their large size and the presence of dust. 191 the dust accumulates in circumferential bands or arcs within the necrotic centres of the lesion (fig. 7.1.18 ), an arrangement that suggests periodic episodes of inflammatory activity. caplan lesions differ from tuberculosis in lacking satellite lesions and tubercle bacilli, and from progressive massive fibrosis in showing characteristic bands of dust pigmentation (table 7. asbestosis is defined as diffuse interstitial fibrosis of the lung caused by exposure to asbestos dust. 192, 193 it does not cover asbestos-induced carcinoma of the lung or asbestos-induced pleural disease. the development of asbestosis depends on the presence of fairly large dust burdens: this is in contrast to mesothelioma and other forms of asbestos-induced pleural disease, which, although also dose-related, occur following the inhalation of far smaller amounts of asbestos dust. asbestos is a generic term for more than 30 naturally occurring fibrous silicates, fibre being defined as an elongated particle with a length-tobreadth (aspect) ratio of at least 3. asbestos fibres have a high aspect ratio, generally over 8. based on their physical configuration they can be divided into two major groups, serpentine and amphibole. the physical dimensions and configuration of asbestos fibres are strongly linked to their pathogenicity. chrysotile (white asbestos) is the only important serpentine form. it accounts for most of the world production of asbestos of all types ( 194 being a serpentine mineral, chrysotile consists of long, curly fibres that can be carded, spun and woven like cotton ( fig. 7.1.19 ). the curly chrysotile fibres are carried into the lungs less readily than the straight amphibole asbestos fibres, and once there undergo physicochemical dissolution and are cleared more readily. they readily fragment into short particles that are easily ingested by macrophages and in the acidic environment of the macrophage phagolysosome they are particularly unstable. the half-life of chrysotile in the lungs is estimated to be in the order of only a few months. 195, 196 not surprisingly therefore chrysotile is the least harmful type of asbestos in respect of all forms of asbestos-induced pleuropulmonary disease. [197] [198] [199] it may nevertheless cause pulmonary fibrosis if sufficient is inhaled. 200, 201 in contrast to chrysotile, amphibole forms of asbestos consist of straight rigid fibres that are stable within the lung. they do not fragment, they are insensitive to chemical attack and their clearance halflives are in the order of decades rather than months. 196 the main amphibole forms of asbestos of commercial importance are crocidolite (blue asbestos) and amosite (brown asbestos). crocidolite, reputedly the most dangerous in regard to all forms of asbestos-related disease, was formerly mined in western australia (wittenoom) and south africa (cape province and the transvaal); it was the principal amphibole used in the uk. amosite, the name of which derives from the acronym for the former asbestos mines of south africa company in the transvaal, was the principal amphibole used in north america. amphiboles are no longer imported by the developed countries but much remains in old lagging and presents a considerable dust hazard when this is removed. tremolite, a further amphibole asbestos, contaminates quebec chrysotile deposits, montana vermiculite and many forms of commercial (non-cosmetic) talc and is responsible for much of the asbestos-related disease in chrysotile miners and millers. 202 another amphibole asbestos, anthophyllite, was formerly mined in finland. it causes pleural plaques (see p. 716) but not lung disease, possibly because its fibres are relatively thick ( fig. 7.1.20) . 203 erionite is a zeolite rather than a type of asbestos but is comparable in form to amphibole asbestos and is also biopersistent. it is found . these coated structures are termed 'asbestos bodies' . because other fibres may gain a similar coat, the non-specific term 'ferruginous body' has been advocated. however, coated carbon fibres (so-called coal bodies) are easily recognised as such by their black core. 206 in practice, ferruginous bodies with the appearance of asbestos bodies almost always prove to have an asbestos core. 207, 208 long fibres are more likely to be coated than short ones, which are cleared more quickly: in one study few fibres less than 5 µm in length were coated and few fibres over 40 µm in length were uncoated. 209 amphiboles form bodies more readily than chrysotile. a comparison of light and electron microscopic fibre counts found that 0.14% of chrysotile, 5% of crocidolite and 26.5% of amosite formed bodies. 210 nevertheless, sufficient chrysotile fibres are coated to permit recognition of asbestosis by standard histological criteria (diffuse fibrosis and asbestos bodies), even if chrysotile is the only asbestos present. 211 despite the biodegradability of chrysotile, asbestos body numbers do not materially diminish with time. 212 very occasionally however a patient with diffuse pulmonary fibrosis and a history of asbestos exposure has no evident asbestos bodies but analysis shows a fibre burden within the range found in asbestosis, justifying fibre analysis in such cases. 212a there is evidence that alveolar macrophages are involved in the coating of asbestos fibres to form asbestos bodies and that the bodies are less harmful to the macrophages than uncoated fibres. 213 asbestos bodies give a prussian blue reaction for iron when stained by perls' method and their yellow-brown colour makes them easily recognisable in unstained films of sputum or in unstained histological sections. sections may be cut 30 µm thick to increase the yield and help identify bodies that lie at an angle to the microtome blade. there is a good correlation between the numbers of asbestos bodies seen in lung sections and those in tissue digests. 214, 215 the bodies may be found singly or in irregular clumps or stellate clusters. they are unevenly distributed but in well-established asbestosis they are easily found. if they are not evident, asbestos burden may be assessed quantitatively in tissue digests (see below). their presence in lung tissue, sputum or bronchoalveolar lavage fluid merely confirms exposure, not the presence of disease. however, the number of asbestos bodies in lavage fluid correlates well with lung asbestos burden 216, 217 and the number in sputum correlates with the duration and intensity of exposure. [216] [217] [218] fibre counts 193, 208, [219] [220] [221] [222] [223] quantitation is desirable in certain circumstances (box 7.1.1), in which case it is best effected on 2-cm 3 blocks of fixed or fresh lung tissue obtained from three different sites, avoiding tumour and thickened pleura. the tissue blocks are digested with caustic soda or bleach, following which the fibres may be collected on a millipore membrane or viewed in suspension in a red blood cell-counting chamber. if phase contrast optics are used both coated and uncoated fibres can be assessed. 219 alternatively, dark ground illumination can be used to demonstrate uncoated fibres. 221 however, electron microscopy is to be preferred as it detects far more fibres than are visible by light microscopy and can also provide information on fibre type. it is important that the laboratory is well practised in fibre analysis and has established its own control range for the general population as well as asbestosis as most lungs contain some asbestos. ambient fibres are generally shorter than 5 µm and some workers therefore confine their counts to fibres that are at least as long as this. 224 justification for this comes from animal experiments demonstrating that long fibres cause more inflammation, chromosomal damage, fibrosis, lung tumours and mesotheliomas than short fibres, [225] [226] [227] [228] and from studies in humans suggesting that long fibres in parts of central turkey where it causes both mesothelioma and a pattern of interstitial pulmonary fibrosis that is comparable to asbestosis. 204, 205 asbestos use and exposure exposure to asbestos occurs in countries where it is extracted ( asbestos is used particularly for fireproofing, in heat and sound insulation and for strengthening plastics and cement. thus, unless adequate precautions are taken, exposure is experienced by dockers unloading asbestos in the close confines of a ship's hold, by thermal insulation workers (laggers and strippers) in shipyards, power stations, train maintenance depots, factories and other large buildings, by construction workers such as carpenters cutting asbestos building panels, and by workers making asbestos products such as fireproof textiles, brake and clutch linings, and specialised cement. as well as such direct exposure, exposure may also be: • indirect, as experienced by the families of asbestos workers • paraoccupational, as experienced by those working alongside an asbestos worker • neighbourhood, as experienced by those living downwind of an asbestos works or mine • ambient, as experienced by those living or working in a building containing asbestos. exposure to asbestos incorporated in the structure of a building carries a negligible health risk if the asbestos material is well maintained to prevent shedding of dust. stripping asbestos out is more dangerous than maintaining it in situ, but maintenance is sometimes neglected. the near indestructibility of asbestos accentuates the health problems that its ubiquity poses. because of their aerodynamic properties, fibres of 100 µm or more in length may reach the finer bronchioles and alveoli. once impacted, the sharp asbestos fibres become coated with a film of protein that is rich in iron. the coating is thickest at the ends of the fibres, giving a 229 other human studies have shown that, although asbestos load is maximal in the upper lobes, more long fibres are found at the bases, where fibrosis is most marked. 41,230 a further reason for limiting attention to the longer fibres is that the shorter ones are cleared more easily and their number therefore varies with the time lapsed since last exposure. for these reasons asbestos regulations in many countries now limit attention to fibres that are over 5 µm in length and have a length-to-diameter (aspect) ratio greater than 3: such fibres have become known as regulatory or world health organization (who) fibres. values are best expressed as fibres/g dry lung. by light microscopy, normal values range up to 50 000: over 20 000 is seen with mesotheliomas, and over 1 000 000 in asbestosis (table 7 .1.5). 216, 224, 231, 232 however, compared with electron microscopy, light microscopy is relatively insensitive, showing only 26.5% of the amosite, 5% of the crocidolite and 0.14% of the chrysotile. 210 light microscopic counts correlate poorly with severity of asbestosis 219 and electron microscopy non-asbestos fibres commonly found in the lung include mullite, which derives from fly ash. this may constitute up to 70% of the total fibre burden (see table 7 .1.6) and is thought to be harmless. there is no firm evidence that manmade fibres present a health hazard 237 but in certain localities natural non-asbestos mineral fibres, zeolites for example, are important causes of mesothelioma (see p. 718) and also cause interstitial pulmonary fibrosis. 204 in contrast to the first half of the twentieth century, much of the asbestosis encountered today is asymptomatic, identified radiologically or histologically in lungs resected for carcinoma or removed at autopsy. symptomatic cases are characterised by an insidious onset of breathlessness, a dry cough and crackles over the lower lung fields. finger clubbing is a variable feature. lung function tests show a restrictive respiratory defect. radiology initially shows small irregular basal opacities that gradually coalesce to become linear, coarsen and eventually progress to a honeycomb pattern of small cysts. the principal differential diagnosis, both clinically and pathologically, is from idiopathic pulmonary fibrosis. this is aided by the slow progression of asbestosis, which often extends over 20 years, as opposed to an average course of 2-3 years from presentation to death for the idiopathic condition. most cases of asbestosis are diagnosed solely on the occupational history and these clinicoradiological features. recourse to histology is unusual but biopsy (preferably as a wedge of lung) may be undertaken if the clinical features are atypical. histology also arises when the pathologist samples lung parenchyma remote from a resected carcinoma (the universal importance of which cannot be overemphasised). asbestosis (established) over 1 000 000 over 100 000 000 the light microscopic counts include total fibres (coated and uncoated). the electron microscopic counts include only amphibole asbestos. results from different laboratories vary and these figures, derived from several sources, 216, 231, 232 provide only a general guide. reliable results depend upon counts being made regularly and the normal range from that laboratory being ascertained. ratios of counts obtained by electron and light microscopy vary greatly but approximate to 100. is better in this respect. [197] [198] [199] by transmission electron microscopy, values may range up to 5 000 000 in controls, with asbestosis generally above 100 000 000 and mesotheliomas found at any level down to 1 000 000, all these figures representing amphibole fibres/g dried lung (see table 7 .1.5). 216, 231, 232 it should be noted that counts from different parts of the same lung may vary widely; 42,231-235 caution should therefore be exercised in interpreting a count obtained on a single sample. there is also wide discrepancy between laboratories, even when analysing the same sample. 234 results obtained in an individual case therefore have to be evaluated against a standard set of values unique to that laboratory. electron microscopy also provides valuable information on the type of fibre. chrysotile differs physically from the amphiboles in two respects: its fibres are both curved and hollow (figs 7.1.20 and 7.1.22). with an electron microscope equipped for microprobe analysis, the various forms of asbestos may also be distinguished from other fibres and from each other (box 7.1.2), 230, 236 an important point as the amphibole forms of asbestos are far more dangerous than chrysotile (table 7 .1.6). [197] [198] [199] coroners require autopsy verification of the diagnosis in all suspected cases and this also necessitates hystology. when the lungs from a patient with asbestosis are seen at autopsy, pleural fibrosis is often found, and although this may also be attributable to asbestos exposure it is to be regarded as an independent process and not part of the asbestosis: it is dealt with separately on page 715. slicing the lung affected by asbestosis shows a fine subpleural fibrosis, especially of the lower lobes ( fig. 7 .1.23). in severe cases the fibrosis often extends upwards to involve the middle lobe and lingula, and sometimes the upper lobes also. microcystic change associated with the fibrosis develops in advanced cases and in severe disease there may be cysts over 1 cm in diameter. however, these classic changes are seldom seen in developed countries today. following decades of dust suppression in asbestos factories, current patients have mild to moderate asbestosis and are dying of related cancer or of non-pulmonary disease. 238 in some of these cases the asbestosis is only detectable microscopically. fixation of the lungs through the bronchi and the use of heard's barium sulphate impregnation technique facilitate demonstration of the fibrosis (see p. 757 and fig. 7.1.23 ). the mild degree of asbestosis currently encountered is of little functional significance but is often critical in determining whether an associated carcinoma of the lung should be attributed to asbestos exposure (see below). the histological diagnosis of asbestosis requires an appropriate pattern of interstitial fibrosis associated with the presence of asbestos bodies. both components must be present. the fibrosis is paucicellular, lacking any significant degree of inflammation and being collagenous rather than fibroblastic. it is generally considered that asbestosis begins about the respiratory bronchioles and alveolar ducts where most of the asbestos fibres impact. 15 alveolar walls attached to these bronchioles show fine interstitial fibrosis. however, this early lesion has to be interpreted with caution because it is not specific to asbestos, being found with other inhaled mineral dusts 239, 240 and even in many cigarette smokers who have not been so exposed. 241 it more likely represents a non-specific reaction to a variety of inhaled particles. it may cause mild airflow obstruction but is not associated with the radiographic, clinical or restrictive changes of classic asbestosis. as the disease progresses, the focal changes join up so that the basal subpleural regions show widespread interstitial fibrosis and eventually complete destruction of the alveolar architecture. in severe cases there may be honeycombing and metaplastic changes in the alveolar and bronchiolar epithelium. apart from the presence of asbestos bodies the changes resemble those of non-specific interstitial pneumonia, or more rarely usual interstitial pneumonia. fibroblastic foci may be found but they are uncommon. there is often an increase in alveolar macrophages but the desquamative interstitial pneumonia that has been reported in association with asbestos 242, 243 is not to be regarded as a variant of asbestosis 192 ; concomitant smoking is a more likely cause. 241 a variety of other non-specific inflammatory processes such as organising pneumonia have been reported in asbestos workers and if localised some have been suspected of representing malignancy until biopsied. 244 several schemes have been proposed for grading the extent and severity of asbestosis. these are of value in epidemiological studies but should only be applied to cases meeting the histopathological criteria for a diagnosis of asbestosis. one such scheme is shown in box 7.1. 3. 193,245,246 in well-established asbestosis asbestos bodies are numerous and easy to find, aggregates of them sometimes forming clumps ( fig. 7.1.24) . in earlier lesions a detailed search may be necessary, in which fibrosis confined to the walls of respiratory bronchioles and the first tier of adjacent alveoli 2 b extension of fibrosis to involve alveolar ducts and/or two or more tiers of alveoli adjacent to the respiratory bronchiole, with sparing of at least some alveoli between adjacent bronchioles 3 fibrotic thickening of the walls of all alveoli between at least two adjacent respiratory bronchioles 4 honeycomb change a an average score is obtained for an individual case by adding the scores for each slide (0-4), then dividing by the number of slides examined b grade 1 and, to a lesser extent, grade 2 need to be distinguished from smoking-induced peribronchiolar fibrosis and mixed-dust pneumoconiosis. case the examination of unstained or perls-stained sections facilitates their identification. minimum criteria for the diagnosis of asbestosis require the identification of diffuse interstitial fibrosis in well-inflated lung tissue remote from a lung cancer or other mass lesion and the presence of either two or more asbestos bodies in tissue with a section area of 1 cm 2 or a count of uncoated asbestos fibres that falls in the range recorded for asbestosis by the same laboratory. 192, 193 there are marked variations in the concentration of asbestos fibres between samples from the same lung 216, 235 and it is therefore recommended that at least three areas be sampled, the apices of the upper and lower lobes and the base of the lower lobe. 223 the equivalent of mallory's alcoholic hyalin of the liver has been described in the lungs in asbestosis, 193, 247 and subsequently in other [248] [249] [250] [251] it is seen as small eosinophilic cytoplasmic inclusions within hyperplastic type ii alveolar epithelial cells (fig. 7.1.25a ). electron microscopy shows that the inclusions consist of a tangle of tonofilaments ( fig. 7.1.25b ) and by immunocytochemistry a positive reaction is obtained with antibodies to cytokeratin, both these features being typical of mallory's hyalin in the liver. the inclusions also react for ubiquitin, the accumulation of which is indicative of cellular damage, in particular faulty proteinolysis. 251 the differential diagnosis of asbestosis includes pulmonary fibrosis due to many other causes, any of which may of course affect an asbes-tos worker as much as members of the general population. the proportion of diffuse pulmonary fibrosis in asbestos workers that is not attributable to asbestos has been estimated to be as high as 5% and likely to rise as the risk of asbestosis diminishes with better industrial hygiene. 252 the principal differential diagnosis of asbestosis is from idiopathic pulmonary fibrosis. both diseases affect the bases and periphery of the lungs predominantly. in the late stages, cystic change is more evident in idiopathic pulmonary fibrosis but this criterion is not totally reliable. nor is the presence of pleural fibrosis, although it is usually present in asbestosis and is seldom found in idiopathic pulmonary fibrosis. asbestosis seldom progresses or does so very slowly after exposure ceases 253, 254 whereas idiopathic pulmonary fibrosis typically proves fatal within 3-4 years from onset. the fibrosis of asbestosis is generally paucicellular: inflammation is not a feature and the fibroblastic foci that characterise the usual interstitial pneumonia pattern of fibrosing alveolitis are seldom observed in asbestosis. very often the distinction from idiopathic pulmonary fibrosis has to be based on the amount of asbestos in the lung and, if asbestos bodies are not readily identifiable, this has to depend on fibre counts. errors are made both by overlooking substantial numbers of asbestos bodies completely and by ascribing undue importance to scanty bodies. if considering the possibility of minimal asbestosis (to justify attributing carcinoma of the lung to asbestos, for example) it should be remembered that a little peribronchiolar fibrosis is also characteristic of smokers' lungs, centriacinar emphysema and early mixeddust pneumoconiosis. [239] [240] [241] 255 as described above, at least two asbestos bodies/cm 2 in the presence of interstitial fibrosis distant from any lung cancer or other mass lesion is required for a diagnosis of asbestosis. 192 although the causes of asbestosis and idiopathic pulmonary fibrosis are very different, they resemble each other in several ways, suggesting that similar pathogenetic mechanisms may operate. 105, [256] [257] [258] in both these diseases there is degeneration of the alveolar epithelium and capillary endothelium, with patchy loss of the former, 256 and bronchoalveolar lavage shows an increase in macrophages that might perpetuate the damage by releasing lysosomal enzymes, nitric oxide and hydroxyl radicals. 257, [259] [260] [261] both diseases are also characterised by an increased prevalence of circulating non-organ-specific autoantibodies. 262 experimentally, asbestos exposure leads to the activation of a variety of fibrogenic cytokines at sites of lung injury. 105, [263] [264] [265] [266] [267] [268] inhaled asbestos activates a complement-dependent chemoattractant for macrophages 269 and macrophage stimulation involves the secretion of fibroblast stimulating factors, [270] [271] [272] asbestos being intermediate between haematite and silica in regard to macrophage-mediated fibrogenicity. 273 the epithelial damage could be mediated directly by the needle-like asbestos fibres or indirectly through enhanced phagocyte generation of free radicals (which is much greater with amphibole asbestos than with either chrysotile or silica). 268, 274 fibrogenic cytokines released by activated pulmonary phagocytes and regenerating alveolar epithelial cells in asbestosis include tumour necrosis factor-α and transforming growth factor-β, 268 as in idiopathic pulmonary fibrosis. as a result of better industrial hygiene, asbestosis is less severe today than in earlier years when it followed much heavier exposure, with the consequence that death from respiratory failure and cor pulmonale is less common and sufferers are surviving longer. there is therefore now a greater risk of asbestos-related cancer eventually developing. asbestos exposure predisposes to two varieties of malignant neoplasm, carcinoma of the lung and mesothelioma of the pleura and peritoneum. the risk of malignancy increases with dose but the relative risk of carcinoma is much smaller than that of mesothelioma. for example, with heavy exposure, as in lagging, the risk of mesothelioma is increased 1000-fold whereas it is increased only fivefold for lung cancer. hence, with light exposure there is a substantial risk of mesothelioma but only a small risk of lung cancer. asbestosis requires heavy exposure and in one group of patients with asbestosis, 39% died of pulmonary carcinoma, 10% of mesothelioma and 19% of other respiratory diseases. 238 although there were many earlier reports, the link with carcinoma of the lung may be considered to have been firmly established by 1955, 275 that between crocidolite asbestos and mesothelioma by 1960, 276 and that between amosite asbestos and mesothelioma by 1972. 277 mesothelioma is considered on page 717. in regard to carcinoma of the lung, asbestos is not such a potent pulmonary carcinogen as cigarette smoke but together their effects are multiplicative rather than additive (table 7 .1.7). 278, 279 however, the risk attributable to asbestos is the same regardless of smoking history, being increased fivefold in both smokers and non-smokers. there is usually a latent period in excess of 20 years between first exposure to asbestos and the development of lung cancer and the risk increases the greater the cumulative exposure. the increased risk involves carcinomas of all the histological types encountered in the lung, although adenocarcinoma has been disproportionately overrepresented. 245, [280] [281] [282] [283] [284] [285] it is uncertain 114 whether the increased risk of carcinoma is caused by the asbestos 192, [286] [287] [288] [289] [290] [291] or the asbestosis. [292] [293] [294] [295] [296] [297] [298] [299] the latter view envisages the carcinoma arising in the foci of alveolar epithelial hyperplasia and dysplasia that commonly accompany any interstitial fibrosis (see carcinoma complicating idiopathic pulmonary fibrosis, p. 275). however, most carcinomas complicating asbestosis arise in the bronchi rather than the alveolar tissue. on the other hand, more arise in the sites worst affected by asbestosis, the lower lobes and the periphery of the lung, than in the general population ( fig. 7.1.26) . 238, 245, [281] [282] [283] [284] [285] the majority view has been that asbestosis is a necessary precursor of the carcinoma but evidence to the contrary is finding increasing support (table 7 .1.8). 192 in the uk, industrial compensation was formerly only awarded to an asbestos worker for carcinoma of the lung if there was also asbestosis or diffuse pleural fibrosis but new rules were introduced in 2006. asbestosis remains a sufficient criterion but diffuse pleural thickening is not and asbestosis is no longer a necessary criterion: asbestos is deemed to have been responsible if the patient worked in asbestos textile manufacture, spraying, lagging or gas mask manufacture for at least 5 years before 1975 or 10 years after 1975. the basis for these changes is the premise that heavy asbestos exposure is sufficient in itself to account for carcinoma of the lung. 278 together, these factors have a multiplicative rather than additive effect mortality ratio non-smoking controls 1 non-smoking asbestos workers 5 cigarette-smoking controls 11 cigarette-smoking asbestos workers 53 asbestosis associated with carcinoma of the lung. the asbestosis has been highlighted by barium sulphate impregnation and is seen as a grey subpleural band to the right of the picture. although the carcinoma has arisen in the same lobe as the asbestosis it has not obviously arisen in an area affected by asbestosis. 1. asbestosis diagnosed clinically, radiologically or histologically or a minimum count of 5000 asbestos bodies per gram dry lung tissue (/g dry), or an uncoated asbestos fibre burden of 2 million amphibole fibres more than 5 µm in length/g dry, or 5 million amphibole fibres more than 1 µm in length/g dry or estimated cumulative exposure to asbestos of at least 25 fibres/ ml-years or an occupational history of 1 year of heavy exposure to asbestos (e.g. manufacture of asbestos products, asbestos spraying) or 5-10 years of moderate exposure (e.g. construction or ship-building) and 2. a minimum lag time of 10 years lung fibre counts in the asbestosis range (see table 7 .1.5) provide valuable evidence of such exposure. compensation standards for asbestos-associated lung cancer in different countries are shown in box 7. 1.4. 192,289 asbestos-induced airway disease although asbestosis causes a restrictive respiratory defect, airflow limitation is also seen in this disease. much of the airflow limitation is attributable to cigarette smoking but it is also seen in non-smoking asbestos workers and is worse in those with asbestosis. 300 the pathological basis of this appears to be small-airways disease (see p. 123). 301 it is possibly a non-specific reaction to inhaled dust or cigarette smoke. 302 because it is not established that this lesion progresses to interstitial alveolar fibrosis (asbestosis) the term 'asbestos airways disease' is suggested. 302 fibrosis limited to the bronchioles is specifically excluded from the definition of asbestosis in the latest guidelines (although these retain grade 0 for fibrosis limited to the bronchiolar walls). 193 it should also be noted that, although emphysema is considered to be a destructive rather than fibrotic condition, a little focal the presence of asbestos-related bilateral pleural plaques or asbestos-related bilateral pleural thickening and occupational exposure and a lag time of at least 12 years the presence of asbestosis or pleural plaques or diffuse pleural thickening or fibre-years of exposure only fibre-years of exposure are taken into account exposure, at least 10 years' latency and asbestos-related pleural or parenchymal changes asbestosis is not required but smoking is taken into consideration attempts are made to quantify separately the attributability to asbestos, smoking and other factors (e.g. radon) fibrosis is generally evident in this common condition 255 and does not necessarily indicate early asbestosis. aluminium has been implicated in the development of respiratory disease during the refining of its principal ore, bauxite, to yield various aluminium oxides (aluminas), in the preparation of the metal by smelting alumina, in the production of corundum abrasive and in the production of special aluminium powders used in explosives. bauxite is a mixture of various aluminium oxides, hydroxides and silicates, iron oxide and titanium dioxide. the oxides of aluminium are obtained by differential heating of the ore and the respiratory effects of this work appear to be no more than mild airway irritation. it is generally accepted that aluminium oxide is inert. aluminium is prepared by the electrolytic reduction of its oxide dissolved in sodium aluminium fluoride (cryolite), a process releasing a considerable amount of fluoride-rich effluent. exposed workers have complained of what is termed pot-room asthma. the pathology of this condition is not well described but the pathogenesis is thought to involve irritation rather than allergy. 303 the abrasive corundum is formed from bauxite mixed with coke and iron heated in an electric arc furnace, a process in which workers may be exposed to the fumes of alumina and free silica. in the past some of these workers developed diffuse pulmonary fibrosis (shaver's disease) 369 and, although this was initially attributed to the aluminium, it is now agreed that the free silica was the responsible agent. the exposure to free silica has been reduced and the disease is now regarded as historic. aluminium powder holds a paradoxical position in regard to lung disease. in certain industries it has caused very severe pulmonary fibrosis, yet in others it has proved harmless. indeed, at one time canadian miners breathed aluminium dust before work, in the belief that this would reduce the danger of silica in the mine dust 304 and more recently silicosis has been treated by such means in france. 305 it is questionable whether this practice is effective but it at least appears to cause no harm. the explanation for these contradictory observations probably lies in differing methods of manufacture of aluminium powder. aluminium metal appears to be an inert substance but this is only because it has a high affinity for oxygen and the surface layer of aluminium oxide so formed is firmly bound to the underlying metal, unlike ferric oxide which permits further rusting of iron. granular aluminium powders, produced in a ball mill or from a jet of molten aluminium, therefore acquire a protective coat of surface oxide and are inert. with stamped aluminium powders, however, surface oxidation is prevented by lubricants added to aid the separation of these flake-like particles. the usual lubricant (stearin) contains stearic acid and this polar compound combines with the underlying metal, which is thereby protected from both atmospheric oxidation and the action of body fluids when such dust is inhaled. in certain circumstances, however, non-polar lubricants in the form of mineral oils have been substituted for stearin. this happened in germany during the second world war when munition production was stepped up but stearin was difficult to obtain, 306, 307 and in the uk in the 1950s to make the powder darker for purely commercial reasons. 308 in vitro, oil-coated stamped aluminium powder reacts with water to produce aluminium hydroxide, which affords the underlying metal no protection against further attack, so that aluminium hydroxide continues to be formed. 309 this substance is a protein denaturant, once used in the tanning industry, and it is believed that this property underlies the very ex ceptional cases of severe pulmonary fibrosis that have occurred in connection with stamped aluminium powder produced with mineral oil rather than stearin. 309, 310 the fibrosis has a very characteristic pattern, affecting the upper lobes and progressing rapidly, the interval from onset of symptoms to death being as short as 2 years. 308 there is marked shrinkage of the lungs with gross elevation of the diaphragm and buckling of the trachea (fig. 7.1.27 ). the lungs are grey ( fig. 7.1.28) and microscopically, numerous small black jagged particles are seen. these can be shown to contain aluminium with irwin's aluminon stain or by microprobe analysis. 311 what appears to be a different pathological effect of aluminium dust on the lungs is the rare development of granulomatous disease resembling sarcoidosis and berylliosis. 312, 313 this represents hypersensitivity to the metal, amenable to confirmation with a lymphocyte transformation test similar to that used to diagnosis berylliosis (see below). rare cases of desquamative interstitial pneumonia and pulmonary fibrosis have been reported in aluminium welders. 311, 314 elements with atomic numbers from 57 (lanthanum) to 71 (lutetium) are known as the lanthanides or rare earth metals. they are used in many manufacturing processes, including the production of hightemperature ceramics and the grinding of optical lenses. carbon arc lamps used in reproduction photography emit appreciable quantities of oxidised lanthanides, particularly cerium oxide, and there are reports of pneumoconiosis in exposed individuals. 315 the pathological changes reported have varied from granulomatous nodules to diffuse interstitial fibrosis indistinguishable from the idiopathic variety except for the presence of rare earth elements (usually cerium) detected by polarising light microscopy and electron microprobe analysis. 315 hard metal is a tungsten alloy containing small amounts of cobalt, titanium, molybdenum and nickel. it is exceptionally tough and once formed can only be worked with diamond. it is used in the tips of drill bits, on abrasive wheels and discs, and in armaments. interstitial lung disease is liable to arise in its manufacture or in those using hard metal as an abrasive. 316 experimental work suggests that cobalt is the dangerous constituent 317 but this element is soluble and, unless industrial contact has been recent, analysis of lung tissue usually shows tungsten and titanium but no cobalt. the role of cobalt is also indicated by the development of similar interstitial lung disease in diamond polishers using high-speed polishing discs made with a diamond-cobalt surface that lacked tungsten carbide and the other constituents of hard metal. 318, 319 hard-metal lung disease and cobalt lung take two forms, an industrial asthma and interstitial fibrosis. the latter has a diffuse lower zonal distribution and the appearances mimic idiopathic pulmonary fibrosis. however, an unusual feature is the presence of moderate, or perhaps only small numbers, of giant cells (fig. 7.1.29a, b) . 316, 320 not only are there multinucleate alveolar macrophages but syncytial cell forms develop in the alveolar epithelium. electron microscopy confirms that these are multinucleate type ii pneumocytes (fig. 7.1.29c ). 316 such epithelial changes are well known in measles pneumonia but the viral inclusion bodies that characterise this infection are not found in hard-metal pneumoconiosis. the changes are those initially described as a particular pattern of idiopathic interstitial pneumonia termed giant cell interstitial pneumonia or gip (see p. 264). elemental analysis shows that many, but not all, cases of gip represent hardmetal disease. the exceptions seldom give a history of cobalt exposure and must be presumed to represent true idiopathic cases. conversely, epithelial giant cells are not always found in hard-metal pneumoconiosis and so their presence, although highly characteristic, is neither totally specific nor totally sensitive. beryllium is the lightest of metals. it has an atomic weight of 4 and special properties that make it especially useful in many applications. it is more rigid than steel, has a high melting point and is an excellent conductor of heat and elecricity. unfortunately, the inhalation of beryllium dust or fume is exceedingly dangerous. 321, 322 those who worked with beryllium compounds before precautionary measures were taken suffered a high morbidity and mortality. sometimes, the escape of dangerous fumes from the factories was on such a scale that people living in nearby houses, downwind from the places in which these materials were being worked, contracted and occasionally died from berylliosis ('neighbourhood cases'). 323 alternatively, contamination of a beryllium worker's clothes might lead to berylliosis in a temperatures. the alloys of beryllium are also now widely used, especially those with copper, on which it confers elasticity and resistance to fatigue. alloy manufacture and the machining of beryllium alloys are therefore further activities that entail a risk of berylliosis, as is the recovery of the metal in the recycling of scrapped electronic and computer parts. seemingly innocuous occupations such as dental laboratory technician are not without risk of chronic berylliosis. 329 there are good grounds for regarding chronic berylliosis as being an allergic condition. many of those affected react strongly to skin tests with dilute solutions of beryllium salts, although these must be undertaken with care: occasionally in a highly sensitised person even so small an exposure may evoke a systemic reaction. the skin reaction is of the delayed type, occurs in only 5% of exposed individuals, is not associated with a clear-cut dose-response curve and represents a granulomatous response. further evidence for the disease having an allergic basis derives from bronchoalveolar lavage, which demonstrates an excess of t-helper lymphocytes that proliferate in vitro on exposure to beryllium salts. 330 a positive transformation test given by these lymphocytes is a more reliable indicator of disease than in vitro blood lymphocyte transformation testing, 52 which is safe but not wholly reliable and indicates only sensitization, rather than berylliosis. susceptibility to berylliosis varies widely from person to person and it is notable that chronic pulmonary disease is strongly associated with the hla antigen dpβ1 and the glu69 gene. 331, 332 the importance of genetic factors is supported by a report of the disease in identical twins. 333 chronic berylliosis is thought to be initiated by the metal binding to tissue proteins and acting as a hapten to initiate a delayed hypersensitivity response characterised by a proliferation of t-helper lymphocytes. these sensitised cells in turn secrete a variety of cytokines (e.g. interleukin-2, tumour necrosis factor-α and interferon-γ) that recruit and activate macrophages, which mature into epithelioid cells. the resultant epithelioid cell granulomas destroy the lung tissue and lead to pulmonary fibrosis. if beryllium enters the subcutaneous tissues through a cut or abrasion, as often happened in the earlier days of fluorescent lamp manufacture, a sarcoid-like granuloma soon appears at the site; in time, the overlying epidermis may break down to form an ulcer. even more serious are the lesions produced by the inhalation of beryllium. chronic pulmonary berylliosis takes the form of a widespread granulomatous pneumonia with a histological picture identical to that of sarcoidosis (fig. 7.1.30a ). both berylliosis and sarcoidosis affect the upper lobes more than the lower (fig. 7.1.30b ) and in both diseases the granulomas are preferentially distributed along lymphatics and may involve adjacent blood vessels. in neither condition is there widespread necrosis but in both diseases the granulomas occasionally display a little central necrosis or hyalinisation. as in sarcoidosis, the hilar lymph nodes may be involved but, unlike sarcoidosis, not in isolation. over a period of many years, the sarcoid-like granulomas gradually undergo progressive fibrosis, with consequent impairment of pulmonary function. in the later stages, when the disease has become chronic, dispersal of beryllium from its site of initial absorption may lead to generalisation of the disease and to the appearance of similar granulomas elsewhere, particularly in the liver, kidneys, spleen and skin, but this is unusual. relative. 324 beryllium compounds may also cause contact dermatitis and conjunctivitis. 325 beryllium is also classified as a probable pulmonary carcinogen, 326 but this is controversial. two forms of berylliosis are recognised, acute and chronic. acute berylliosis was first reported in germany in 1933 327 and is now largely of historical interest, being only encountered as a result of rare accidental or unexpected exposure. it follows the inhalation of a soluble beryllium salt and represents chemical injury, the pathology being that of diffuse alveolar damage (see p. 136). further consideration will be confined to chronic berylliosis, which is allergic in nature. chronic berylliosis was first reported in 1946 in the fluorescent lamp industry. 328 beryllium has now been replaced in this application but it has since proved to be of great value in the nuclear, electronic, computer and aerospace industries and the production of refractory materials and crucibles that are to be subjected to particularly high and there is a lifelong risk of disease. progression often entails alternating exacerbations and remissions, long after exposure has ceased. in keeping with the view that berylliosis is a hypersensitivity reaction, very little beryllium is necessary to cause the disease. particulate beryllium is so scanty in the affected tissues and the atomic number of beryllium so low that electron microprobe analysis is generally unsuitable for its detection. furthermore conventional detectors are protected by a beryllium window. however, the substitution of a polymeric window has enabled beryllium to be detected by electron microprobe analysis, presumably in a patient with fairly heavy exposure. 334 ion or laser microprobe mass spectroscopy can also detect very small amounts of beryllium in tissue sections but these techniques are not widely available. the differential diagnosis of chronic berylliosis is from sarcoidosis, to which it is identical morphologically. [335] [336] [337] however, as noted above, it is unusual for berylliosis to cause significant hilar lymphadenopathy in the absence of pulmonary disease, which is a common feature of sarcoidosis. extrathoracic granulomas, erythema nodosum and uveitis, which are all common in sarcoidosis, are unusual in berylliosis. however, one group found that 6% of patients initially diagnosed as having sarcoidosis actually had chronic berylliosis. 338 similar findings have been reported by others. 324, 339 any patient thought to have sarcoidosis who has worked with or near metals should be offered a beryllium lymphocyte transformation test. a list of laboratories performing this test can be found at www.dimensional. com/~mhj/medical_testing.html. although polyvinyl is not a mineral and the reaction of the lungs to its presence is therefore not a true pneumoconiosis, it is generally so termed and is dealt with here for convenience. workers are exposed to polyvinyl chloride dust in the milling and bagging of this plastic and micronodular opacities may be detected in their lungs radiologically. however, the material is non-fibrogenic and histology merely shows a foreign-body reaction to the dust particles. 340 the radiological opacities may abate when exposure ceases. 341 nevertheless, one polyvinyl chloride worker developed systemic sclerosis, 342 which is a recognised complication of silicosis (see p. 335). polyvinyl chloride is produced from vinyl chloride monomer, which has a causal association with angiosarcoma of the liver and probably other forms of cancer, including carcinoma of the lung (see p. 534). in the late 1990s a characteristic lung disease was identified in workers at several factories producing plush material by spraying nylon flock on to an adhesive backing material. [343] [344] [345] [346] the flock fibres are too large to be inspired but may be mixed with smaller nylon shards of respirable size. the workers complained of cough and breathlessness and were found to have a restrictive ventilatory defect with interstitial markings on radiography. their symptoms improved on removal from the workplace but relapsed on return to work. pathologically, there was lymphocytic bronchiolitis and peribronchiolitis with widespread lymphoid hyperplasia represented by lymphoid aggregates. chronic berylliosis is characterised by the gradual onset of cough, shortness of breath, chest pain, night sweats and fatigue. these symptoms may develop within a few weeks of exposure or many years later. once the worker is exposed, the beryllium is retained in the tissues granulomas were not identified. the histological appearances suggest a severe immunological reaction and raise possibilities such as rheumatoid disease and sjögren's syndrome but consideration of the clinical and serological setting and the occupation should permit recognition of the cause. the industrial production of popcorn and other foodstuffs appears to carry a risk of obstructive airway disease. [347] [348] [349] [350] biopsy of affected workers has shown peribronchiolar fibrosis and granulomas and air sampling has identified many volatile organic compounds, of which the flavouring agent diacetyl (2,3-butanedione) is suspected of being responsible for the bronchiolitis. it is difficult to continue paint spraying (air brushing, aerographics) without adequate respiratory protection but in the early 1990s several small aerographic factories operated in the neighbourhood of alicante, southeastern spain without any concern for the workers' health. the workers were required to paint patterns on textiles using a hand-held spray gun. the atmospheric pollution was intense but complaints of respiratory difficulties were met with reassurances and the workers urged to continue. this they did because of the otherwise poor economy, often returning to work when disabling breathlessness had settled down. a change of paint (to acramin f) may have contributed because the worst-affected workers were employed at two plants that had made this switch. their illness has been described as the 'ardystil syndrome' after the name of one of these factories. some workers were left with permanent respiratory disability. one required a lung transplant and 6 others died. [351] [352] [353] [354] transbronchial biopsy showed organising pneumonia, which in the fatal cases had progressed to irreversible interstitial fibrosis. a similar outbreak of respiratory disease was subsequently reported in algerian textile factories where acromin f was applied by the same technique. 355, 356 acromin f is marketed as a paste and used as such without ill-effect. its use in heavy spray form appears to be responsible for the 'ardystil syndrome' . workers in engineering workshops may be exposed to the prolonged inhalation of fine sprays or mists of the longer-chain hydrocarbons that constitute many mineral oils. this may result in exogenous lipid pneumonia, 357 which is described on page 314, or extrinsic allergic alveolitis. [358] [359] [360] the vapour of shorter-chain hydrocarbons such as paraffin oil (kerosene: c 10-16 ) and petrol (gasoline: c 4-12 ) and gaseous hydrocarbons such as propane may act as acute asphyxiants or central nervous system depressants but have negligible pulmonary toxicity. however, if they are ingested or aspirated in their liquid form they are acutely toxic to the lungs, producing a chemical pneumonitis with the features of diffuse alveolar damage. ingestion may be accidental or deliberate (see fig. 4 .19, p. 142) whereas aspiration is generally inadvertent, occurring in siphoning accidents, such as those experienced by fairground operatives who 'breath or eat fire' ('fire-eater's lung'). 361, 362 animal experiments involving the intratracheal injection of kerosene resulted in acute pulmonary exudates, which cleared except for residual bronchiolitis. 363 welder's pneumoconiosis, first recognised in 1936, 364 essentially represents the fairly harmless deposition of iron in the lungs (siderosis -see p. 337). however, welders may suffer various ill-effects from the inhalation of substances other than iron (table 7.1.9). some of these are para-occupational risks, that is, encountered by welders because they work near another process and are inadvertently exposed: thus, shipyard welders may be exposed to asbestos, 365 and those in foundries to silica. welders may therefore develop a mixed-dust pneumoconiosis (see p. 337), rather than just siderosis. however, one analytical investigation identified excess amounts of iron alone in association with pulmonary fibrosis; the silicon content did not differ from that in controls. 366 more directly, welders may be exposed to asbestos insulation that they themselves use, while welders of special steel alloys run the risk of metal-induced asthma, metal fume fever, polymer fume fever and the consequences of toxic metal fume inhalation, 367 all of which are described separately in this chapter, as is lung disease in aluminium welders. chronic bronchitis has been attributed to the inhalation of low concentrations of irritants such as ozone and nitrogen dioxide by welders but this risk is unproven and the subject of much controversy. welders may also inhale carcinogenic hexavalent chromium compounds in the course of their work and therefore develop lung cancer. the term 'welder's lung' is often applied indiscriminately to any of these diseases and, as it has no specific meaning, is best avoided. dust, fume and gas are some of the terms used to describe different physical forms of respirable agents. they are defined in table 7 .2.1 on the finely divided fume of several metals is highly toxic to the lungs and capable of producing severe acute and chronic damage to both the conductive airways and the alveoli, resulting in acute tracheobronchitis and bronchiolitis, diffuse alveolar damage, obliterative bronchiolitis and pulmonary fibrosis. important metal fumes in this respect include aluminium, which is released together with silica fume in bauxite smelting (see shaver's disease, 369 above), cadmium from welding or cutting special steels, chromium from cutting its alloys or in the manufacture of chromates, cobalt released in the production and use of its alloys (see hard-metal disease, above), mercury released in various industries and in the home, 370 nickel carbonyl released during the purification of metallic nickel or the manufacture of nickel alloys 371 and beryllium (see above). many irritant gases cause severe acute and chronic damage to both the conductive airways and alveoli. the changes are non-specific and similar to those wrought by toxic metal fumes (see above) and viruses amongst other agents. they consist of acute tracheobronchitis and bronchiolitis, obliterative bronchiolitis, diffuse alveolar damage and pulmonary fibrosis. the gases liable to produce such damage include oxides of nitrogen, sulphur dioxide, ozone, phosgene, chlorine, ammonia and various constituents of smoke, notably acrolein. some of these are also touched upon in chapter 7.2 because they are of general as well as occupational importance, although there is no rigid difference between general and occupational pollution. ozone, sulphur dioxide and nitrogen dioxide are oxidising gases that may be found together as industrial atmospheric pollutants. each is capable of producing diffuse alveolar damage by means of its oxidising properties and the release of free active radicals. in addition, they cause damage to distal airways, particularly terminal and respiratory bronchioles, with resulting bronchiolitis. oxides of nitrogen may be encountered with fatal consequences by farmhands seeking to free a blockage in a silo when they encounter pockets of this gas that have accumulated on top of the fermenting silage: the term 'silo-filler's disease' is generally applied to the initial haemorrhagic oedema or the obliterative bronchiolitis that develops in those who survive the initial chemical injury. [372] [373] [374] [375] asphyxia due to the farmhand encountering pockets of carbon dioxide is a further hazard within agricultural silos. other farmhands have suffered from the inhalation of toxic gases or bacteria when handling liquid manure. [376] [377] [378] [379] welding, which is considered below, may also involve exposure to toxic gases such as oxides of nitrogen. ozone, the principal oxidant gas of photochemical smog, produces pulmonary changes at relatively low levels and may be encountered at higher concentrations in various industries. potentially dangerous levels of ozone are produced from atmospheric oxygen by ultraviolet radiation given off in welding while ozone is used in industry to sterilise water, bleach paper, flour and oils, and mask the odour of organic effluents. the damage wrought by ozone is predominantly centriacinar in distribution, affecting terminal and respiratory bronchiolar epithelium and proximal alveolar epithelium. [380] [381] [382] there is loss of cilia and necrosis of centriacinar alveolar type i epithelial cells. the changes are dose-dependent and, in one study, the youngest animals were most sensitive. 383 in long-term experiments, hyperplastic bronchiolar clara and ciliated cells extended peripherally to line alveolar ducts. 384 the role of granulocytes is stressed in some experimental studies 385 and it is notable that neutrophil migration is prominent when the human lungs are damaged by ozone. 386 aldehydes such as acetaldehyde, formaldehyde and acrylic aldehyde (acrolein) are widely used in the plastics and chemical industries. the first is a liquid and the others are water-soluble gases. pathologists are of course familiar with formaldehyde solution from its use as a disinfectant and histological fixative. all these aldehydes are intensely irritant and their acute effects generally prevent prolonged exposure to high concentrations. chronic effects include skin sensitivity and asthma, and in rats nasal carcinoma. however, the doses to which these experimental animals were exposed far exceed any that are likely to be encountered by humans, in whom there is no convincing evidence of aldehyde-induced cancer. 387 ammonia gas is extensively used in industry as a raw material, notably in the manufacture of nitrogenous products such as fertilisers and plastics. it is highly soluble and its acute irritative effects are mainly felt in the eyes, nose and throat, but high levels affect the major airways, possibly leading to them being blocked by exudates. survival usually brings full recovery but bronchiectasis and obliterative bronchiolitis have been described. chlorine gas is widely used in the chemical industry. it is transported and stored under pressure in liquid form. heavy exposure through its accidental release or use as a war gas has proved fatal through its acute toxicity causing exudative airway occlusion and pulmonary oedema. survivors usually recover completely but, as with nitrogen dioxide and ammonia, there is a risk of obliterative bronchiolitis. phosgene (carbonyl chloride, cocl 2 ) is a poisonous, colourless gas that was responsible for thousands of deaths during world war i, when it was used in chemical warfare. it is used industrially in the preparation of some organic chemical compounds and is formed, perhaps inadvertently, 388 by the combustion of methylene chloride in products such as paint strippers. phosgene causes injury to terminal bronchioles and alveoli, with resulting oedema and hyaline membrane formation. the mechanism of cell damage is uncertain but it may depend on inactivation of intracellular enzymes by the gas. longterm problems are rare but chronic bronchitis and emphysema have been described in survivors. mustard gas (bichloroethyl sulphide, c 4 h 8 cl 2 s) is a further agent that has been used in chemical warfare. it is primarily a skin vesicant but when inhaled it results in widespread epithelial destruction and pulmonary oedema. survivors may be left with irritant-induced asthma (reactive airways dysfunction), chronic bronchitis, tracheobronchomalacia, bronchiectasis and bronchiolitis obliterans. [389] [390] [391] thionyl chloride is used in the manufacture of lithium batteries where it is liable to result in the release of sulphur dioxide and hydrochloric acid fumes. workers in such factories have developed lung injury varying from mild, reversible interstitial disease to severe obliterative bronchiolitis. 392 hydrogen sulphide is the principal chemical hazard of natural gas production. high levels of the gas also buid up in sheds housing large numbers of pigs, the source here being the pig manure. once inhaled the gas is rapidly absorbed into the blood stream. the effects are therefore widespread but include the usual respiratory effects of irritant gases, varying from sneezing to pulmonary oedema and acute respiratory distress, depending upon the exposure. in alberta 221 cases were identified over a 5-year period. the overall mortality was 6%; 5% of victims were dead on arrival at hospital. most required admission to hospital but the survivors experienced no long-term adverse effects. 392a the danger of asphyxia from the inhalation of gases devoid of oxygen is fairly widespread in industry. 393 it generally arises from the use of inert gases, which, being non-toxic, give a false sense of security. pockets of these gases tend to form in confined spaces. anoxic death from the accumulation of methane is well known in mines and has also occurred in slurry pits and sewers. anoxic asphyxia in diving (and anaesthesia) has resulted from the incorrect connection of gas cylinders or failure to notice that a mixed gas contains insufficient oxygen. deaths have occurred in welding when argon or carbon dioxide has been used to shield the weld and prevent oxidation of the metals at the high temperatures employed. deaths have also resulted from inadvertent entry to discharged oil tanks filled with nitrogen to reduce the risk of explosions, or from the formation of pockets of nitrogen gas applied in liquid form to freeze the contents of damaged pipes so that they can be repaired without the necessity to drain down. the respiration of a gas devoid of oxygen causes loss of consciousness within seconds because it not only fails to provide oxygen but removes that present in the pulmonary arterial blood. the changes at autopsy are those common to cellular hypoxia. they include cerebral and serosal petechiae and pulmonary congestion and haemorrhage but these features are not specific and are not always present. the cause of death can generally only be surmised from the circumstances surrounding the death. occupational asthma is the commonest cause of work-related respiratory disease in many western countries (table 7 .1.10). [394] [395] [396] the reported incidence ranges from 13 per million workers in south africa to 174 per million workers in finland. 395, 396 it occurs in many industries (table 7 .1.11) 397 and occupational factors can be identified as contributing to asthma in about 15% of adult cases. 398 over 250 aetio399 in the uk a third are organic, a third chemical, 6% metallic and the rest miscellaneous. the commonest, in descending order, are isocyanates, flour and grain, laboratory animals, glutaraldehyde, solder or colophony and hardening agents. 400 atopy appears to predispose to occupational asthma when the allergen is of high molecular weight but not when it is of low molecular weight. for example, atopic individuals are particularly prone to develop asthma if employed in the manufacture of biological detergents, whereas atopy does not increase the risk of asthma from sensitisation to toluene di-isocyanate, which is a serious health problem in the manufacture of polyurethane. similarly, platinum salts are such potent sensitising agents that nearly all those exposed to them develop asthma. asthma-provoking metals other than platinum include chromium, cobalt, nickel and vanadium, all of which are used in steel alloys, and possibly aluminium (see pot-room asthma, p. 357). other asthma-inducing factors encountered in industry include grain and flour dust, certain wood dusts, soldering fluxes containing colophony (pine resin), epoxy resin hardeners such as phthallic anhydride, isocyanate-containing foams and paints, formaldehyde and the excreta of laboratory animals. contaminated humidifiers may cause occupational asthma as well as humidifier fever and extrinsic allergic alveolitis. 401 pathologically, occupational asthma is identical to nonoccupational asthma (see p. 109). byssinosis is a further form of occupational asthma, 402 one encountered in the cotton industry. the sensitising agent is a component of the cotton bract, which is the part of the cotton harvest other than the cotton fibre. bract consists of dried leaf, other plant debris and soil particles and contains a variety of fungal and bacterial residues, including lipopolysaccharide endotoxin, but the exact nature of the sensitising agent remains unknown. 403 the endotoxin is unlikely to be responsible for byssinosis but may be the cause of so-called mill fever, a self-limiting illness characterised by malaise, fever and leukocytosis that is experienced by many people on first visiting a cotton mill. dust levels and the risk of byssinosis are particularly high in the carding rooms where the raw cotton is teased out before it is spun. affected workers are worse when they return to work after the weekend break, a feature attributed to antibody levels having built up during this brief respite from the cotton dust. there is no link with atopy and the fluctuating antibodies are precipitins of the immunoglobulin g class. complement activation by both arms of the complement cascade has been reported. 404, 405 when the lancashire economy was largely cotton-based, necropsies on workers suffering from byssinosis generally showed gross emphysema, and this came to be accepted as evidence of byssinosis. however, it is now realised that in this heavily industrialised part of the uk, emphysema is as common in the general population as in cotton workers and it can no longer be considered a component of byssinosis. other findings in byssinosis are more commensurate with asthma, namely an increase in bronchial muscle and mucous cells. 406 no granulomas or other evidence of extrinsic allergic alveolitis are found. fever may be the predominant feature in a variety of occupational illnesses and the unifying term 'inhalation fever' has been proposed. 407 however, the individual occupations are of interest and these conditions will therefore be considered separately. mill fever has been mentioned above under byssinosis. humidifier fever is an acute illness characterised by malaise, fever, myalgia, cough, tightness in the chest and breathlessness, all of which are worse on monday mornings if the humidifier responsible is at work rather than home. the chest complaints, and their aggravation on return to work after the weekend, are features shared with byssinosis (see above) but the general complaints fit better with extrinsic allergic alveolitis (see p. 279). humidifier fever develops in circum-stances that also lead to the development of a form of extrinsic allergic alveolitis, and not surprisingly the same name has been extended to this latter condition, with inevitable confusion. both diseases are caused by microbiological contamination of humidifiers or air conditioners so that a fine spray of microorganisms is emitted into the office, factory or home. investigations have generally shown the baffle plates of the air conditioner to be covered with a slime of bacteria, fungi or protozoa (mainly amoeba and ciliates), and extracts of this have been used to identify precipitins in the patient's sera, as in extrinsic allergic alveolitis. however, unlike extrinsic allergic alveolitis, humidifier fever resolves within a day and leaves no permanent injury. for this reason there is seldom the opportunity to study the tissue changes, and partly for this reason it remains unclear whether the disease is mediated by immune complexes, as in extrinsic allergic alveolitis, or by endotoxins derived from the contaminants. a febrile illness occurring in precipitin-negative farm-workers after heavy exposure to fungi in their silos was attributed to inhaled fungal toxins and named pulmonary mycotoxicosis. 409 it is also known as precipitin test-negative farmer's lung and organic dust toxic syndrome. 410 the condition is generally self-limiting and is seldom biopsied but desquamative interstitial pneumonia and diffuse alveolar damage have been reported. 411, 412 metal fume fever this is a self-limiting acute illness characterised by fever, sweating, myalgia, chest pain, headache and nausea, that comes on monday mornings when occupational exposure is experienced after a weekend's respite, as with bysinnosis and humidifier fever; during the week tolerance develops. 368, 413 the disease involves the release of cytokines such as tumour necrosis factor and is presumed to have an allergic basis. 414 the metals involved are chiefly zinc, copper and magnesium, and, to a lesser extent, aluminium, antimony, iron, manganese and nickel. occupations at risk include any that generate such metal fumes, but particularly welding. it is most commonly associated with welding zinc-coated surfaces. if the symptoms persist, alternative diagnoses, such as acute cadmium poisoning and other specific toxic metal fume diseases, should be suspected: these are not self-limiting and may cause severe bronchiolitis or diffuse alveolar damage (see above). this illness resembles metal fume fever except that it occurs without regard to previous exposure: no tolerance develops and there is therefore no particular susceptibility on mondays. the polymers concerned are quite inert, except when heated to produce fume: polytetrafluorethylene (ptfe, teflon, fluon, halon) is a notable example. as with other self-limiting diseases, little is known of the tissue changes. 54 environmental irradiation chiefly affects the skin but in some parts of the world rocks near the surface release significant amounts of radon gas. this carcinogen is liable to accumulate in buildings and be inhaled, so subjecting the occupants to an increased risk of lung cancer. the installation of underfloor ventilation is therefore advocated in such areas. this subject is explored more fully on page 533. the body is vulnerable to both increases and decreases in pressure and it is the lungs that often bear the brunt of the damage. increased pressure may result in blast injury or crushing of the chest while decreased pressure may result in the lungs literally bursting or dissolved gases being released within the blood (caisson disease), or the vascular alterations that underlie mountain sickness developing. some of these pressure changes entail a risk of pneumothorax and it is essential that this is properly investigated postmortem by the chest being opened under a water seal. loud music has been incriminated as a specific form of air pressure change causing pneumothorax and metereologists have shown that 'spontaneous' pneumothoraces tend to occur in clusters associated with natural drops in atmospheric pressure. 1,2 explosions may cause injury by the body being violently thrown against a less moveable object, by objects being thrown against the body or by the blast wave hitting the body. these mechanisms often act together but sometimes there is only blast injury, to which the lungs are particularly vulnerable. for a time it was considered that the damage was direct, the blast wave travelling down the airways to injure the lungs. however, at the start of the second world war, experiments conducted in the uk showed that the lungs were injured indirectly, the blast wave being transmitted to them through the chest wall: pulmonary blast injury is worst on the side of the body towards the explosion, and can be reduced by protective clothing. 3 underwater explosions are particularly dangerous because water is incompressible. there may be severe internal injury but no external evidence of damage other than a trickle of blood from the mouth or nose. this is because the injury is rate-dependent. quite small thoracic deform-ation may produce severe pulmonary damage if peak compression is attained very quickly, typically in less than 5 ms. conversely, severe chest wall distortion may produce only minor pulmonary contusion if this time is extended beyond 20 ms. 4 at necropsy, the lungs are contused, with blood evident in the airways and parenchyma. depending on the force of the blast, the haemorrhage may be pinpoint, patchy or confluent. it tends to follow the lines of the ribs and may be accompanied by pleuropulmonary lacerations having the same distribution. in this case there will also be haemothorax, pneumothorax and possibly air embolism. patchy pulmonary haemorrhages cuff the blood vessels. 5, 6 in patients who survive for a few days, the lungs resemble the liver macroscopically and histologically show chronic interstitial inflammation and fibrosis as well as haemorrhage. 7 other injuries are often present and fat embolism, aspiration pneumonia, fluid overload and infection may all be added to the effects of the blast wave. 'chest squeeze' is another form of barotrauma caused by high pressure but here the body is compressed rather than subject to a sudden wave of pressure as in blast injury. it is experienced by divers who descend very deeply, thereby subjecting their bodies to such high pressure that their chest walls are literally crushed, so that their ribs break and their lungs are severely compressed. more common mishaps experienced by divers include drowning and decompression sickness, both of which are dealt with below, and neurological syndromes such as nitrogen narcosis, which will not be considered further. 8 'burst lung' is the most acute form of decompression sickness. 9 it is experienced by divers and submariners making rapid ascents from depth and by aviators who ascend too rapidly in unpressurised aeroplanes, experience failure of a plane's pressure system or have to eject at high altitudes. injury to the lung is caused by trapped alveolar gas expanding so rapidly that it exceeds total lung capacity before it can escape through the trachea. the lungs literally burst: the alveolar walls rupture and blood mixes directly with alveolar air. the victim experiences chest pain and there may be blood-stained froth at the mouth or frank haemoptysis. air may enter the alveolar walls to cause interstitial emphysema or air embolism. asthmatics may be at particular risk because of regional air-trapping. 10 . diving mammals such as porpoises and whales are protected from such dangers of peripheral air-trapping by cartilage extending far out into the finest conductive airways so that these passages never close, even at the end of full expiration (fig. 7.2.1) . 11, 12 patients requiring positive-pressure artificial respiration are also at risk of burst lung, but the complications of the resultant interstitial emphysema differ from those experienced by divers. in divers, the chest wall is buttressed by the surrounding water and air in the interstitium is liable to track towards the hilum of the lungs and enter pulmonary veins, with resultant cerebral and coronary air embolism, either of which may prove fatal. 13 iatrogenic burst lung, on the other hand, takes place in patients whose chest wall is not so buttressed, and then outward rupture of the interstitial air is more likely, resulting in pneumothorax. extension of the interstitial emphysema to the mediastinum, neck and chest wall is also more likely in such patients, resulting in surgical emphysema at these sites. however, there are exceptional cases marked by both cerebral embolism and extensive air tracking. 14 the same circumstances that lead to burst lung may also cause decompression sickness, which is also known as caisson disease. 9 in this condition there is a sudden release of nitrogen gas that has gone into solution in the lipids of adipose tissue and of myelinated nervous tissue at the higher pressure: the released nitrogen gains access to the blood stream in which it forms bubbles. 15 doppler ultrasound techniques show that this is quite customary when divers ascend from depth, 16 but the lungs generally provide an effective filter so that there are no untoward systemic effects, although there may be sudden chest pain on deep inspiration ('the chokes'). gradual decompression permits the nitrogen to diffuse across the alveolar membranes and be exhaled. if, however, substantial amounts of nitrogen are released from solution, sufficient pulmonary arteries may be blocked to cause pulmonary hypertension, with resultant opening of arteriovenous communications or a patent foraman ovale, so permitting the gas to enter the systemic circulation. this is often followed by limb pains ('the bends') and perhaps cerebral symptoms ('the staggers'). fatal cases are characterised by gas bubbles within blood vessels throughout the body and froth in the heart chambers. delayed effects include ischaemic necrosis of bones and other tissues. 17 deep-diving mammals are protected by the same mechanism that prevents them suffering from burst lung. they exhale before diving and during the dive the chest is compressed to the extent that virtually all the gas in the lungs passes into the cartilage-buttressed nonrespiratory airways (see fig. 7.2.1) , resulting in very little to be absorbed by the blood. the pulmonary collapse also serves to reduce buoyancy. the distribution of the little gas that is absorbed is minimised by bradycardia. 18 many viscera experience anaerobic respiration but hypoxia is minimised in the heart and musculature by high levels of haemoglobin and myoglobin. the brain is further protected by the supplying arteries drawing on oxygen stored in an unusual spongelike cervical organ known as the rete mirabilis. mountain sickness is due to reduced atmospheric pressure brought about more slowly than that responsible for decompression sickness .19,20 it may be acute or chronic. acute mountain sickness is likely to be experienced by anyone who ascends above 3000-4000 m without a period of acclimatisation at intermediate levels. symptoms are as liable to occur in people born at high altitude who return after a few weeks spent at sea level as in those who go to the mountains for the first time: acclimatisation is obviously short-lived and is therefore necessary whenever an ascent is to be made. the ill-effects are commonly precipitated by exercise. in the susceptible, acute mountain sickness commonly appears within 3 days of ascent. the basis of acute mountain sickness is tissue hypoxia. it results in deteriorating intellectual and psychological function, headache, nausea, vomiting, and more rarely pulmonary and cerebral oedema. high-altitude pulmonary oedema is characterised by increasing dyspnoea, cyanosis and a dry cough, and later the production of copious, frothy sputum, which sometimes becomes blood-stained. 21 the pulmonary artery pressure is markedly raised but wedge pressures are normal, indicating that the left side of the heart is unaffected and that pulmonary venous constriction is unlikely to be an important contributory factor. the pulmonary oedema fluid has a high protein content 22 and the condition has been characterised as a non-cardiogenic high-permeability oedema associated with excessive pulmonary hypertension. 23, 24 hypoxia is a well-known cause of pulmonary arteriolar constriction but in acute mountain sickness the vascular response appears to be exaggerated for the pulmonary artery pressure is considerably higher than is usual for the altitude. an association with certain hla complexes (hla-dr6 and hla-dq4) suggests that this has a genetic basis. 25 although arteriolar constriction only tends to protect the pulmonary capillaries, it could explain the oedema if the process was patchy -as is the resultant oedema -for patchy arteriolar constriction would subject the rest of the lung to abnormally high pressures and lead to capillary stress failure in these areas (see pp. 402 and 448). 26, 27 measurements of capillary pressure suggest that this is indeed the case. 28 furthermore, vasodilators such as calcium channel blocking agents and inhaled nitric oxide gas 23, 29, 30 have been used with success to counter acute mountain sickness, supporting the idea that hypoxic vasoconstriction plays a central role. autopsy shows the lungs to be heavy and firm. the cut surface weeps oedema fluid, which is often blood-stained, but a striking feature is the patchy distribution of the changes. areas of haemorrhagic oedema alternate with others that contain clear oedema fluid and others that are normal apart from overinflation. pulmonary arterial thrombi are commonly found. microscopy confirms the presence of haemorrhagic oedema and may show neutrophils and hyaline membranes in the alveoli. the alveolar capillaries are congested and may contain thrombi. there may also be an increase in mast cells and rarely pulmonary infarction. the right ventricle is commonly dilated whereas the left ventricle is normal. highlanders generally show right ventricular hypertrophy and increased muscle in their pulmonary arteriesm, changes that are not apparent in lowlanders. 31, 32 chronic mountain sickness prolonged residence at high altitude leads to hypoxic pulmonary hypertension (see p. 424), an increase in red cell mass and cor pulmonale. livestock taken from lowland plains to high-altitude pastures suffer similarly but the natural stock of the himalayas and ethiopian highlands are apparently immune. so too are other species long established at high altitude such as the llama and yak. these species are said to have adapted to their climate, that is, the forces of natural selection have bred out the pulmonary vasoconstrictive response to hypoxia. cattle of european origin and humans acclimatise to high altitude by processes such as increasing their red cell mass but generally they are not adapted like native species and suffer hypoxic pulmonary hypertension at altitudes in excess of 3000 m. certain himalayan highlanders may be an exception to this in that their small pulmonary arteries are reported not to show the muscularisation that characterises hypoxic pulmonary hypertension. 33 in cattle of european origin, the dependent oedema of right-sided cardiac failure caused by hypoxic pulmonary hypertension affects the breast (brisket) particularly and in the rocky mountains of north america such cattle are said to have 'brisket disease' . 34 a human counterpart of this has been described in children of chinese ancestry who have been taken to reside in tibet and who have developed a fatal form of subacute infantile mountain sickness. 35 a small minority of permanent residents in the andes develop the changes of chronic mountain sickness to a marked degree and are said to suffer from monge's disease. 36 the basis of this is alveolar hypoventilation, which leads to a progressive fall in systemic arterial oxygen saturation and elevation of haemoglobin concentration to an unusually severe degree. the latter averages about 25 g/dl, which exceeds even the 20 g/dl found in healthy high-altitude residents. patients with monge's disease are so deeply cyanosed that their lips are virtually black. their pulmonary artery resistance is also markedly raised. the cause of the alveolar hypoventilation is uncertain but the only cases of monge's disease that have come to necropsy had conditions such as kyphoscoliosis that predispose to alveolar hypoxia. drowning is defined as suffocation by submersion, and usually occurs in water. it is the commonest cause of accidental death among divers but 96% of drowning accidents do not involve deep descents. falling into quite shallow water is a particularly common cause of drowning in young children. in adults, men outnumber women by 4 to 1. more die in fresh water than the sea, not because it is more hazardous to the lungs than sea water, but because unguarded inland waters and swimming pools are visited more frequently. alcohol consumption contributes to many deaths by drowning. drowning is not simply a matter of being unable to keep one's head above water. this may be merely a secondary event. for example, the entry dive may result in underwater head injury, or the exertion of swimming may precipitate a heart attack. furthermore, the struggling swimmer going down for the third time ('drowning not waving') is the exception: most drowning is characterised by the swimmer failing to surface or quietly dropping beneath the surface without anyone noticing. swimming underwater can be extremely hazardous if it is preceded by hyperventilation, a danger that needs to be more widely appreciated. hyperventilation results in undue loss of carbon dioxide so that instead of hypercapnia forcing the swimmer to surface to breathe, progress under water may be continued until hypoxia causes sudden loss of consciousness. panic contributes to many swimming accidents and is often precipitated by the inadvertent aspiration of just a little water. most people are naturally buoyant, but only slightly so. with the lungs fully expanded the average adult has a positive buoyancy of about 2.5 kg, which is sufficient to keep the head out of the water if the rest of the body is submerged. if an arm (weight about 3 kg) is raised to wave for help, the head will go down. if the swimmer shouts, exhalation reduces buoyancy to neutral at normal end-expiration and to negative at residual volume. buoyancy cannot be regained when the head is submerged and unless able to swim to the surface, the person will continue to sink. autopsy generally shows that the lungs are full of water, but some victims die of 'dry drowning' due to laryngospasm. events may also be modified by the temperature of the water. sudden immersion in cold water may result in tachycardia, hypertension and hyperventilation, making it difficult for the victim to keep the airways free of water. it may also result in sudden death due to ventricular fibrillation. even a good swimmer loses consciousness within an hour of immersion in very cold water. drowning is then inevitable unless a correctly fitted life jacket is worn, in which case there is a danger of death from hypothermia. however, as in open heart surgery, cold prolongs the interval before there is irreversible brain damage. if the person is rescued, water in the lungs is quickly absorbed, even if it is saline, and therefore hyperosmolar:aspirated sea water is quickly equilibrated by pure water joining it from the blood but the alveolar epithelial barrier remains impermeable to protein and once osmotic equilibrium is reached, all is quickly reabsorbed. [37] [38] [39] fresh water is absorbed even more quickly. it is unnecessary to tip the patient to hasten this process. any water recovered in this way comes from the stomach and time that should be devoted to mouth-to-mouth breathing and cardiac massage is lost. these resuscitative efforts may need to be prolonged as fresh water in particular inactivates alveolar surfactant, leading to alveolar collapse which persists until the surfactant is replenished. very few victims who are resuscitated on site fail to survive, and very few who cannot be resuscitated on site recover later. interchange of fluid between the blood and air spaces may cause major fluctuations in plasma volume with consequent changes in ionic concentrations and haemolysis. hypervolaemia may cause circulatory problems but hyperkalaemia consequent upon the haemolysis is not thought to be as important as was formerly believed: ventricular fibrillation following submersion is more likely to be a complication of hypothermia than of electrolyte imbalance. circulatory collapse may ensue shortly after rescue. this is due to loss of the circulatory support provided by the pressure the water exerts on the body, which results in a considerable increase in cardiac output while the body is immersed. on leaving the water the loss of this support results in a tendency to venous pooling. although this is countered by baroreceptor responses, these are reduced by prolonged immersion in cold water. circulatory collapse is believed to be the cause of death in many persons who perish within minutes of rescue. to counter this effect, patients should be lifted out of the water in the prone position. it can be seen that, in fatal cases, the pathologist is faced with several possibilities. thus, death may have been due to: • natural causes before the body entered the water • unnatural causes before entry, the body merely being disposed of in the water • natural causes in the water • injuries received in the water from impact with rocks, a boat or a ship's propeller, or in tropical waters from predators such as a crocodile or a shark (any of which may also be incurred after death, as may disfigurement by fish and rats) • 'dry drowning' • true drowning • hypothermia • circulatory failure after rescue. true drowning is indicated by froth in the airways and heavy waterfilled lungs. both fresh and salt water contain numerous microscopic algae known as diatoms and those representative of the water in which the drowning occurred are found in the lungs. unless death occurred before submersion, diatoms are also found in other viscera because these tiny life forms easily enter the circulation. thus, the presence of diatoms in digests of organs such as the kidneys, liver, brain and bone marrow suggests that death was due to drowning. because they have a siliceous capsule, diatoms are resistant to putrefaction as well as digestion and can be identified in the body long after death. however, a positive test is not always accepted as proof of drowning and a negative test does not exclude drowning. the various physical forms in which respirable environmental agents may be encountered are defined in table 7 .2.1. some effects of inhalant lung injury are recognised as distinct disease entities and are dealt with elsewhere: for example, the pneumoconioses on page 327, extrinsic allergic alveolitis on page 279, chronic bronchitis on page 98 and lung cancer on page 532. other respirable agents, such as lead fume and carbon monoxide gas, exert their harmful effects elsewhere in the body and will not be considered further. this section is concerned with toxic substances that may be inhaled by the general public. those that are more likely to be encountered in the workplace or in war zones are considered on page 355. the lungs have a rather stereotyped pattern of response to inhaled toxins, displaying degenerative changes and inflammation of varying degree, the former sometimes amounting to necrosis. in general, the site of maximal absorption or injury is related to solubility (for gases and vapours) and particle size (for aerosols such as dusts, fog, fumes, mists, smog and smoke): the less water-soluble and the smaller the particle size, the further down the respiratory tract the agent will penetrate ( fig. 7 [40] [41] [42] thus, ammonia produces intense congestion of the upper respiratory passages and laryngeal oedema whereas phosgene has little effect on these sites but causes pulmonary oedema. 40 air pollution [43] [44] [45] [46] [47] the toxic (as opposed to allergenic) air pollutants thought to pose the greatest threat to the lungs comprise smoke particles, sulphur dioxide, oxides of nitrogen, various aldehydes and ozone. smoke and sulphur dioxide derive particularly from the combustion of fossil fuels in domestic fires and power stations, nitrogen dioxide is an important car exhaust and domestic gas appliance pollutant and ozone is the principal photochemical product of smog. aldehydes such as formaldehyde and acrylic aldehyde (acrolein) also contribute to general air pollution because they are released in the combustion of diesel oil and petrol. collectively, these pollutants have been incriminated in the exacerbation (rather than causation) of asthma. they also predispose to respiratory infection and result in airway inflammation and hypersecretion. 48, 49 their effect on children is of particular concern because development of the lungs is known to continue well into childhood and damage to the lungs before their growth is complete is likely to be irreparable. at the other extreme of life episodes of severe air pollution are known to hasten the deaths of many patients with chronic airway disease. particularly high concentrations of the agents responsible for air pollution may be encountered in industry and their effects are therefore also considered in chapter 7.1, on occupational diseases of the lung. many of the polycyclic hydrocarbons found in polluted air are carcinogenic (see p. 532) and it is therefore not surprising that urban air pollution has been found to be associated with excess mortality from lung cancer. 50 domestic air pollution is rife in many of the poorer parts of the world due to the burning of biomass (wood, dried cow dung, bagasse, straw) in unventilated living rooms for heating and cooking. the women are particularly at risk of developing chronic bronchitis while their children have an increased incidence of acute respiratory infections. 51,52,52a volcanic ash (tephra) irritates the eyes, skin and respiratory tract and in some eruptions may contain much free silica (e.g. montserrat in 1995 and mount st helens, washington state, usa in 1980) or be associated with the release of radon gas (e.g. the azores in 1957). 53 the destruction of the world trade center in 2001 caused massive air pollution of new york city that had lasting respiratory effects on survivors, rescue workers and local residents. [54] [55] [56] at the time of the disaster there was much smoke from combustion of aeroplane fuel and flammable materials in the building while the collapse of the twin towers released dust from cement and dry-wall partitions that was highly alkaline. [57] [58] [59] this caused considerable irritation of the eyes and the conductive airways. a year later many victims were still suffering from bronchial hyperreactivity and poor ventilatory function, in a so-called reactive airways dysfunction syndrome 54, 55 and there was continuing spirometric decline 5 years later. 60 the respirable portion of the dust formed only a small fraction of the whole but given the level of exposure its future effects cannot be discounted, particularly as it contained substances such as asbestos. unusual effects attributed to the disaster include acute eosinophilic pneumonia and granulomatous pneumonitis. 61, 62 allergenic air pollutants are dealt with in detail in the sections on asthma (see p. 109) and extrinsic allergic alveolitis (see p. 279). allergenic air pollution is generally occupational or domestic but periodic widespread air pollution was responsible for the epidemics of asthma seen in barcelona in the 1980s, which were eventually traced to ships discharging cargoes of soya flour (see p. 114). smoking-related diseases figure large throughout this book and in this section they are merely summarised collectively. of the greatest importance, both in the number of patients they affect and in their clinical effects on the individual, are the various forms of chronic obstructive lung disease and lung cancer, but there are many other respiratory diseases associated with smoking, and a few that are less common in smokers (box 7.2.1). 63 not surprisingly, these diseases are often encountered in combination and sometimes one may obscure another. for example, a cigarette smoker may have emphysema in the upper lobes and idiopathic pulmonary fibrosis in the lower lobes. 64, 65 alternatively, langerhans cell histiocytosis and desquamative interstitial pneumonia may affect the same parts of the lungs, in which case the focal lesions of the former may be masked by the latter condition. 63 the term 'smoking-related interstitial lung disease' has been introduced to cover a spectrum of interstitial diseases related to smoking 63, 66, 67 as well as being used in a more restricted sense to describe a combination of air space enlargement and interstitial fibrosis predominantly affecting the lower lobes. 68,69,69a quite advanced interstitial fibrosis has been reported in smokers with no clinical evidence of interstitial lung disease. 69b early changes detectable in smokers include chronic bronchiolitis, fibrosis of the bronchiolar wall and mild peribronchiolar interstitial fibrosis. 70, 71 even earlier changes are detectable at the molecular level: as many as 152 smoking-responsive genes that are significantly up-regulated or down-regulated have been identified in normal cigarette smokers. 72 there is marked individual variation, which may explain why many lifelong heavy smokers experience no respiratory problems. histological evidence that a patient smokes is provided by an increase in the number of alveolar macrophages and a characteristic brown discoloration of cytoplasm due to the phagocytosis of tar and other particulate matter derived from tobacco smoke (fig. 7.2.3) . cigarette smokers are at greater risk of lung disease than cigar and pipe smokers, probably because they inhale more deeply. they do this because cigarette smoke is more acid than cigar and pipe smoke and its nicotine content is therefore absorbed more easily through the lungs than the buccal mucosa. smokers obviously put their own health at greatest risk but the lesser hazards of passive smoking are now well recognized (see p. 532). passive smoking involves both the smoke exhaled by others and that coming from smouldering tobacco between puffs, the latter being known as sidestream smoke. the harmful effects of maternal smoking on the unborn child also come in this category. they include increased airway responsiveness and reduced lung function during the neonatal period and an increased risk of sudden infant death syndrome. reduced numbers of alveolar attachments to the bronchioles have been demonstrated in such infants. 73 smoking is also associated with disease of other organs (e.g. carcinoma of the oesophagus and bladder) but these are outwith the remit of this text. tobacco smoking by waterpipe (shisha, hubble-bubble) is enjoying a rise in popularity, both in its heartland, the middle east, and western countries, and wherever it is practised it is widely perceived as being less dangerous than smoking cigarettes. 74 this is probably a mis conception. what evidence there is suggests that waterpipe tobacco smoking is just as harmful as cigarette smoking, if not more so. 75 the lungs may be injured in burned patients in many ways (box 7.2.2) 76,77 but an important consideration when a body is recovered from a fire is whether death was due to the fire or took place beforehand, the latter raising the possibility of foul play. a vital reaction to the skin burns and the presence of soot in the lower airways provide evidence that death occurred in the fire but an absence of soot from the airways may be due to death occurring rapidly, from asphyxia or poisoning by gases released in the conflagration. soot is cleared rapidly and if the patient survives a few days an absence of soot from the airways is to be expected. 76 lung injury may result directly from heat and smoke inhalation or indirectly from the release of mediators associated with blast injury or shock. although air temperature in a fire may reach very high temperatures thermal injury seldom extends beyond the carina but more extensive injury from heat alone was seen in men exposed to steam escaping from a fractured boiler pipe. 78 those dying immediately showed coagulative necrosis of the respiratory mucosa down to the level of the alveolar ducts and alveolar congestion and oedema, while those surviving a little longer exhibited diffuse alveolar damage. the diffuse alveolar damage probably represented a manifestation of shock from their extensive cutaneous scalding whereas the mucosal necrosis is directly attributable to heat. diffuse alveolar damage is extrinsic allergic alveolitis sarcoidosis blast injury asphyxia poisoning by combustion products (e.g. carbon monoxide, cyanide) direct thermal injury (largely limited to the trachea) irritant smoke, fume and gas (e.g. oxides of nitrogen, ammonia, acrolein, sulphur dioxide) hypovolaemic shock secondary to skin loss septicaemic shock from: infected skin burns infected central lines secondary viral and bacterial pneumonia fluid overload tracheostomy complications, including tracheobronchitis, pneumonia and barotrauma oxygen toxicity absorption of toxic topical disinfectants thromboembolism uraemia usually part of systemic multiorgan failure in these patients, and is the leading cause of death in burns. 79 the ubiquity of plastics today means that smoke contains numerous irritants, including isocyanates, aldehydes and fluorinated organic chemicals. irritant smoke products have two principal effects. firstly, they cause an immediate painful stimulation of the eyes and respiratory tract which at low concentrations may prevent escape and at high concentrations may cause laryngeal spasm and death. secondly, they cause bronchopulmonary injury some hours after exposure. burned patients dying within 4-12 days often show tracheobronchial necrosis and diffuse alveolar damage with prominent hyaline membranes. 76, 77, 80 secondary herpesvirus infection is often present. 81, 82 the respiratory changes caused by heat and smoke are non-specific and careful consideration of the many causes of lung injury in burned patients listed in box 7.2.2 and of the clinical circumstances and management is generally required. often it will be concluded that the cause of the lung injury is multifactorial. long-term consequences of smoke inhalation include bronchiectasis and obliterative bronchiolitis. 83 methyl isocyanate, the chemical released at bhopal the bhopal catastrophe of 1984 was caused by the accidental release of 30 tons of methyl isocyanate gas (ch 3 -n=c=o) from a pesticide plant. 84 over 200 000 people were exposed, of whom 2500 died, mostly within hours of exposure, and 60 000 were seriously injured. the victims complained of intense ocular and respiratory irritation. some survivors were left with persistent respiratory impairment, which was thought to be due to obliterative bronchiolitis. 85, 86 methyl isocyanate is an extremely potent respiratory irritant, destroying the epithelium throughout the conducting airways, with comparatively less parenchymal injury. in survivors, epithelial regeneration, often involving squamous metaplasia, quickly commences, but not before endobronchial granulation tissue projections have developed, resulting in obliterative bronchiolitis. tear gases are chemical irritants delivered as an aerosol for the purpose of riot control. they react with mucocutaneous sensory nerve receptors causing intense irritation of the eyes, mucous membranes and skin. the respiratory effects are mainly concentrated on the upper tract so that there is violent sneezing, severe rhinorrhoea and cough but there may also be tracheobronchitis and rarely pulmonary oedema. 87 patients with pre-existent asthma or chronic obstructive pulmonary disease are most severely affected while others may be left with reactive airways dysfunction. toxins reaching the lungs via the blood stream may be drugs, food contaminants, metabolites produced elsewhere in the body, or chemicals ingested intentionally or accidentally, either in the home or the workplace. the lungs are selectively damaged by certain blood-borne toxins for a variety of reasons. for example, the herbicide paraquat is preferentially taken up by the lungs because of its molecular homology with certain endogenous substances. as detailed below, the type i alveolar epithelial cells are the cells that bear the brunt of the damage in paraquat poisoning. on the other hand, the alveolar capillary endothelium has its own selective uptake mechanisms (see metabolic functions of the pulmonary endothelium, p. 23) which may be responsible for it being selectively damaged by other chemicals. the bronchiolar clara cells are selectively injured by some ingested chemicals because they are equipped to deal with inhaled xenobiotics, but occasionally this activity results in metabolites that are extremely toxic. an example of this from veterinary medicine is provided by the furan-derivative 4-ipomeanol, which is found in mouldy sweet potatoes and results in acute pulmonary oedema in cattle fed such a diet. when this chemical is injected into mice, the bronchioles are denuded of clara cells whereas the intervening ciliated cells are completely unaffected. the selective damage to the bronchiolar clara cells appears to stem from the oxidative efficiency of their p-450 cytochromes, 88 which is much higher than those of the liver. chemicals having a similarly selective effect on bronchiolar clara cells include 3-methylfuran, carbon tetrachloride, naphthalene and 1,1-dichloroethylene, the last of which is a volatile compound that is widely used in the plastics industry. procarcinogens may be activated in the airways by similar mechanisms. paraquat is a dipyridylium compound that is widely used in agriculture as a herbicide. it kills all green plants but is inactivated on contact with the soil. it is applied as a spray and if the manufacturer's instructions are followed there is no danger to health. most fatal cases of paraquat poisoning, both accidental and suicidal, have been due to ingestion of the 20% aqueous solution gramoxone. the less concentrated granular form weedol is unlikely to be ingested accidentally but may be taken suicidally. 89 paraquat is not absorbed by the intact skin but repeated or prolonged application damages the epidermis so that absorption into the blood stream with consequent systemic effects is possible, but rare. 90 although paraquat has toxic effects on the liver, kidneys and myocardium, these are transient and attention has centred on the pulmonary changes, which are usually fatal. following suicidal ingestion of large amounts of paraquat, death from multiorgan failure and pulmonary haemorrhage occurs within a few days, whereas most victims of accidental paraquat poisoning die from progressive pulmonary fibrosis between 10 and 14 days after ingestion. in those who survive longer, a honeycomb pattern of pulmonary fibrosis may be apparent. 91 paraquat is a powerful oxidant and owes its toxicity to the production of active oxygen radicals. the lungs are particularly susceptible because paraquat is concentrated there by an active uptake mechanism in the alveolar epithelium. the inadvertent uptake of paraquat probably stems from a similarity between the molecular arrangement of its quaternary nitrogen atoms and the amine groups of endogenous oligoamines such as putrescine, spermidine and spermine, which are concerned in alveolar epithelial cell division and differentiation (fig. 7.2.4) . 92 this results in paraquat levels being 6-10 times higher in the lung than in the plasma. once taken up by the lung, paraquat is not metabolised but participates in redox cycling so that superoxide radicals are constantly produced. epithelial injury is proportional to the concentration of paraquat, while it is lessened by hypoxia and antioxidants such as superoxide dismutase, and potentiated by increased concentrations of oxygen. [93] [94] [95] [96] the high concentration of oxygen in the alveoli is a further reason why the lungs are particularly vulnerable to paraquat. knowledge of the toxic effects of paraquat comes from observations on autopsy series 89, 97, 98 and from experimental studies that have enabled the sequence of pulmonary changes to be observed. [99] [100] [101] [102] in accordance with paraquat being taken up by the alveolar epithelium, electron microscopy shows that these cells suffer more profound damage than the endothelium. 99 type i epithelial cells swell and undergo necrosis (fig. 7. 2.5), 103 whilst type ii cells, although remaining capable of proliferation, show ultrastructural evidence of damage with derangement of cell organelles. 99, 100 histological changes in the lungs follow the pattern of diffuse alveolar damage, with a characteristic feature of the early exudative phase being intense vascular congestion and alveolar haemorrhage. 89, 97, 104 hyaline membranes are most clearly seen by about 5 days (fig. 7.2.6 ) and epithelial proliferation and fibrosis are conspicuous by about 14 days. the pattern of pulmonary fibrosis in paraquat poisoning has been disputed. some authors have stressed its interstitial position, whereas others have clearly demonstrated that it is intra-alveolar. 98, 102, [104] [105] [106] [107] however, as described on page 148, it generally assumes an obliterative pattern of intra-alveolar fibrosis in which the lumina of several adjacent alveoli are totally effaced, rendering them completely airless (see fig. 4 .24, p. 148). a new multisystem disease appeared abruptly in the environs of madrid in 1981. [108] [109] [110] over 20 000 people were affected and about 1 in 60 died. the disease was initially thought to be mycoplasma pneumonia but was soon found to be associated with the use of adulterated oil sold illicitly by door-to-door salesmen. although it was sold for culinary purposes the oil had been produced for industrial use in steel manufacture. it consisted of rapeseed and olive oil mixed with liquified animal fat, aniline and other organic chemicals. it has not been possible to identify the exact chemical responsible for the disease or to reproduce the changes in other species but the later induction of similar pathological changes by another substance contaminated with an aniline derivative is possibly relevant (see l-tryptophan-induced eosinophilia-myalgia syndrome, p. 389). 111 some clinical and pathological features of the disease suggest that immune mechanisms may also be involved. the initial clinical features included fever, respiratory distress, cough, haemoptysis, skin eruptions and marked eosinophilia. radiographs suggested pulmonary oedema and sometimes showed pleural effusion. about 5% of patients died at this stage but most recovered quickly. however, within a few weeks many were readmitted to hospital with nausea, vomiting, diarrhoea and abdominal pain. about a quarter then proceeded to develop weakness, myalgia, weight loss, scleroderma-like skin signs and pulmonary hypertension. 112, 113 many of these patients died after a long, wasting illness or are permanently disabled with neurological and hepatic disorders. in the early phase the lungs showed the most severe changes, which consisted of a combination of diffuse alveolar damage, eosinophilic infiltrates and arterial luminal narrowing by endothelial swelling and vacuolation, intimal foam cell infiltration and a non-necrotising vasculitis. 109, 112, 114 there was also capillary thrombosis, which later extended into arteries and veins, culminating in fibrosing obliteration of these blood vessels. in some patients dying of haemoptysis, dilated thin-walled blood vessels were identified in the mucosa of major blood-filled airways. late features in the lungs included plexogenic arteriopathy (see p. 420), possibly secondary to changes in the liver. similar inflammatory and vascular changes were seen in many other tissues. notable extrapulmonary features included fasciitis, vasculitis, neuronal degeneration, perineuritis, hepatic injury and tissue eosinophilia. sauropus androgynus is a vegetable that is widely cultivated for the table in many south-eastern asian countries. it is apparently harmless when cooked but recently there has been a vogue in taiwan for consuming large amounts of its unprocessed juice, blended with that of guavas or pineapple, because of its supposed efficacy as a slimming aid and in blood pressure control. coincident with this fad there has been an upsurge in patients with symptoms of obstructive lung disease. within a 4-month period more than 60 such patients were seen at one hospital. [115] [116] [117] they had four features in common: recent consumption of uncooked s. androgynus juice, fixed ventilatory obstruction, radiological evidence of bilateral bronchiectasis and an absence of any previous chronic respiratory disease. four patients agreed to undergo open-lung biopsy. this showed chronic bronchiolitis or obliterative bronchiolitis of constrictive pattern. the lymphocytes were mainly t cells but immunofluorescent and electron microscopy showed no evidence of an immune process. four patients underwent single-lung transplantation. the excised lungs showed sclerotic obliteration of bronchial arteries in the walls of bronchi 4-5 mm in diameter with segmental necrosis of bronchi 2-4 mm in diameter. the changes were considered to fit best with segmental ischaemic necrosis of bronchi at the watershed zone of the bronchial and pulmonary vasculature. 118 further patients have required lung transplantation but public education of the dangers of this herbal medicine now appears to have been successful. 119 alcohol and nicotine outstrip all other recreational drugs in popularity and their effects are of course well known. those of tobacco smoking are summarised above and dealt with in detail in the chapters on obstructive lung disease (chapter 3) and carcinoma of the lung (chapter 12.1). less well known is the lung disease that results from smoking blackfat tobacco, a practice popular with guyanese indians. blackfat is the trade name of a type of tobacco that is flavoured with mineral oil, some of which vaporises and is inhaled when the tobacco is smoked, to cause exogenous lipid pneumonia (see p. 314 [ch8] ). 120 in recent years the smoking of two other substances, marijuana and cocaine, has gained in popularity. it would not be surprising if the long-term effects of smoking these substances were similar to those of cigarette smoking but as yet it is too early to judge. however, the short-term effects are similar to those of tobacco smoking and this bodes badly for their ultimate effects. marijuana consists of the dried leaves of the cannabis plant, also known as hemp, as opposed to hashish, which is the plant's resin, and a further extract known as 'weed oil' . all these substances are smoked because they contain cannabis alkaloids which have psychoactive effects. however, this habit also exposes the lungs to many of the same respiratory irritants that are found in tobacco smoke. initial exposure to marijuana smoke often results in coughing while habitual smokers produce black sputum. bronchial biopsy shows inflammation and squamous metaplasia and bronchoalveolar lavage demonstrates increased numbers of cells, which are predominantly macrophages but also include neutrophils. [121] [122] [123] [124] [125] these changes are virtually identical to the short-term effects of tobacco smoke and are therefore likely to be similarly followed by the development of chronic obstructive lung disease and lung cancer. indeed, the dangers of smoking marijuana are probably greater than those of smoking tobacco as compared with tobacco smoking it is associated with a fivefold greater increase in blood carboxyhaemoglobin and a threefold increase in the amount of tar inhaled. 126 it is estimated that three cannabis cigarettes result in the same degree of bronchial damage as 20 tobacco cigarettes. 127 there is also evidence that the effects of smoking marijuana and tobacco are additive. 128 not surprisingly therefore, epidemiological studies report a doserelated impairment of large-airway function in marijuana smokers. 129 there are also several reports attributing pneumothorax to marijuana smoking ( fig. 7.2.7) . 130, 131 the pneumothorax may be spontaneous or develop during the deep, sustained inspiratory effort involved in smoking marijuana (or cocaine), which may be enhanced by a partner applying positive ventilatory pressure by mouth-to-mouth contact. thoracoscopy in such cases has shown predominantly apical, irregular bullous emphysema, while lung biopsy has demonstrated widespread alveolar filling by heavily pigmented macrophages. 131, 132 evidence is also beginning to accumulate that long-term cannabis use increases the risk of lung cancer. 133 smoking cannabis in the form of weed oil is also reported to result in exogenous lipid pneumonia. 134 cocaine cocaine hydrochloride is a fine white powder derived from the leaves of the plant erythroxolon coca by a complex chemical process. it is heat-labile and therefore cannot be smoked. users inject it intravenously or inhale it unheated through the nose, the latter practice being known as 'snorting' . however, a heat-stable free-base form that can be smoked is easily prepared from the hydrochloride with baking powder and a solvent such as ether. this process results in a crystalline deposit that is known as 'rock' because of its appearance or 'crack' because of the crackling sound it emits when heated. when smoked, the cocaine is readily absorbed and an intense surge of euphoria is experienced within 8 seconds. the intravenous route takes twice as long and 'snorting' several minutes. the hard addict therefore prefers to smoke 'crack' . a variety of pulmonary complications of smoking free-base cocaine has been reported. 128, [135] [136] [137] [138] [139] [140] [141] [142] [143] [144] acute effects include cough, shortness of breath, chest pain and haemoptysis. asthma may be aggravated, black sputum is produced, and pneumothorax and interstitial emphysema have resulted from valsalva manoeuvres undertaken in the belief that they promote even more rapid absorption. biopsy has shown pulmonary congestion and oedema, organising pneumonia, haemorrhage, haemosiderosis, diffuse alveolar damage and interstitial pneumonia or fibrosis. less common effects include eosinophilic pneumonia, extrapulmonary eosinophilic angiitis, medial thickening of pulmonary arteries and the barotrauma described above (see fig. 7.2.7) . severe burning of the airways has also been seen due to 'crack' being smoked before all the ether used in its preparation has evaporated. 'snorting' unheated cocaine has its own complications: substances such as cellulose or talc with which the drug is 'cut' (mixed as a diluent) are liable to provoke a foreign-body giant cell reaction in the lungs (fig. 7.2.8 ). 145 however, particles of foreign material larger than those in the usual respirable range (allowing for the fibrous shape of substances such as cellulose) should suggest intravenous use (see 'filler embolism', below). heroin is usually injected, but it may be smoked, when, as with marijuana, it is liable to lead to a very pronounced macrophage response. intravenous heroin abuse sometimes causes the sudden onset of a potentially fatal high-permeability pulmonary oedema (fig. 7.2.9 ). intravenous abuse of heroin and other drugs is also liable to cause 'filler embolism', which will now be considered. 'filler embolism' is the result of illicit drug usage in which compounds designed for oral use are injected intravenously to heighten their effects. oral preparations consist largely of fillers such as talc or starch and this insoluble particulate matter accumulates in the pulmonary capillaries. it provokes a foreign-body giant cell reaction, thrombosis and fibrosis and may cause pulmonary hypertension ( fig. 7 this 'designer' drug, taken for its central stimulant activity (street names 'ice' or 'u-4-e-uh', pronounced euphoria), is related to the appetite suppressor aminorex, discussed on page 424, and has similarly been associated with pulmonary hypertension. assessing whether a particular clinical manifestation represents an adverse drug reaction considers previous experience with the drug, alternative aetiological agents, the timing of events, drug levels, and the effect of withdrawing the drug and rechallenge with the drug. 7 it is worth bearing in mind that: • one drug may cause several patterns of disease. • one pattern of disease may be produced by a variety of drugs. • a drug reaction may develop long after the drug has been withdrawn. • a drug reaction may develop suddenly even though the dose of the drug has not been altered. • drug effects may be augmented by factors such as age, previous radiotherapy and elevated oxygen levels. • drug reactions may be localised. • many drugs cross the placenta to affect the fetus. an alternative classification of adverse drug reactions, which is more appropriate to pathology practice and which will be followed here, is one based on the pattern of disease. some pathological patterns of drug-induced lung disease are shown in table 7 central depression of respiration occurs as a side-effect of barbiturates, morphine and its derivatives, and even mild sedatives, and may be particularly troublesome in patients suffering from chronic obstructive lung disease. ventilation in such patients may be largely dependent on hypoxic respiratory drive and treatment with oxygen may therefore also have an adverse effect on respiration by lowering the degree of hypoxia and so diminishing the stimulation of the respiratory centre. peripheral impairment of the respiratory drive may be brought about by aminosides and other antibiotics, while corticosteroids may result in a myopathy affecting the respiratory muscles. other iatrogenic hazards affecting the peripheral nerves controlling respiration include nerve root disease complicating immunisation and surgical damage to the spinal and phrenic nerves. asthmatic patients are particularly susceptible to exacerbations of their disease by drugs (box 7.3.2). this effect may occur either as a predictable pharmacological side-effect of the drug or as an idiosyncratic response. examples of the former include β-adrenergicic antagonists and cholinergic agents while examples of the latter include sensitivity to the colouring agent tartrazine, for which reason many manufacturers have eliminated tartrazine from their red, orange and yellow tablets. allergic bronchoconstriction also forms part of generalised anaphylactic reactions induced by vaccines and antisera and occurs as a localised response to penicillin, iodine-containing contrast media, iron dextran and other medicaments. bronchospasm may also be initiated by the non-specific irritant effect of inhaling nebulised drugs if they are prepared as a hypotonic solution, a side-effect that is prevented by using isotonic solutions. aspirin and other non-steroidal anti-inflammatory agents aspirin-induced asthma has been recognised for many years and more recently several of the newer anti-inflammatory drugs have been found to exacerbate asthma in certain sensitive individuals. the basis for this is uncertain but the likelihood of an individual antiinflammatory drug provoking an asthmatic response is related to its potency as an inhibitor of prostaglandin cyclooxygenase pathway, resulting in the production of leukotrienes. [8] [9] [10] as well as asthma being exacerbated by drugs, the disease has been caused by occupational exposure in the pharmaceutical industry to certain drugs which can be inhaled during manufacture, notably penicillin, cephalosporin, methyldopa, cimetidine and piperazine. obliterative bronchiolitis of the constrictive type has been reported with penicillamine 11,12 and gold 13, 14 but in many cases it is possibly the underlying condition rather than the drug that is res ponsible (see p. 123). this is often rheumatoid disease, which is sometimes complicated by bronchiolitis obliterans whether the patient is under treatment or not. 15 organising pneumonia extending into peripheral bronchioles (see p. 120) may be seen with a variety of drugs but results in a restrictive rather than obstructive lung defect and is to be regarded as a cytotoxic effect of the drug acting primarily at the alveolar level (see below). raw sancropus androgyns taken as a slimming aid causes severe obliterative bronchiolitis (see p. 376). the cytotoxic effects of drugs may be acute or chronic, leading to changes as varied as pulmonary oedema, diffuse alveolar damage, pulmonary haemorrhage and haemosiderosis, organising pneumonia, interstitial pneumonitis and interstitial fibrosis. 16, 17 some of the most severe acute effects are seen with the chemotherapeutic agents used in malignant disease 18 but they are also recorded with drugs that are not traditionally thought to be cytotoxic, e.g. desferrioxamine administered as a prolonged intravenous infusion in acute iron poisoning. 19 pulmonary toxicity due to busulphan was first described in 1961, 20 and has been the subject of several subsequent studies. [21] [22] [23] [24] it remains the mainstay of treatment for chronic myeloid leukaemia. like other alkylating agents, it acts by cross-linking dna strands. clinical estimates of the incidence of pulmonary toxicity vary around 4% but subclinical damage is thought to be much more common. although not strictly dose-dependent, toxicity is rarely seen with a total cumulative dose of less than 500 mg. synergy with radiation and other cytotoxic drugs occurs. 25 similar effects have been reported for most cytotoxic agents, particularly bleomycin. 26 pulmonary toxicity is seen less commonly with other alkylating agents, such as cyclophosphamide and melphalan. [27] [28] [29] [30] bleomycin is a cytotoxic antibiotic derived from streptomyces species. it is widely used in the treatment of neoplasms such as lymphomas and germ cell tumours, and is thought to produce its therapeutic and toxic effects by altering the normal balance between oxidants (active oxygen radicals) and antioxidant systems. 26 bleomycin produces superoxide radicals when incubated with oxygen and iron in vitro. oxygen enhances its effects, 31 a fact well known to anaesthetists who accordingly take care to limit concentrations of inspired oxygen to 30% in patients on bleomycin who are undergoing surgery. [32] [33] [34] radiotherapy and cytotoxic agents such as bleomycin are also synergistic. bleomycin is preferentially concentrated in the lungs and pulmonary fibrosis can be produced in animals when it is administered intravenously, intraperitoneally or by intratracheal instillation. electron microscopy shows that the early changes consist of swelling and vesiculation of endothelial cells, interstitial oedema and type i epithelial cell necrosis. 35, 36 the reported incidence of bleomycin toxicity varies from 2 to 40% depending on the type of patient being treated and on dosage. in general, toxic effects increase with age and cumulative dose: above a total dose of about 500 units they rise significantly. the acute morphological changes attributable to drugs include pulmonary oedema and diffuse alveolar damage. acute pulmonary oedema is seen in heroin addicts who die while injecting themselves intravenously but it is also seen in patients administered a variety of drugs therapeutically, for example hydrochlorothiazide, salicylate, opiates, vinorelbine,and desferrioxamine. the oedema is of the high permeability type (see p. 402), rich in protein, and is occasionally haemorrhagic or accompanied by the hyaline membranes of diffuse alveolar damage. diffuse alveolar damage has alveolar epithelial necrosis as its basis (figs 7.3.1 and 7.3.2). however, the continuing action of many cytotoxic drugs affects the regeneration process so that atypical type ii epithelial cells develop, a characteristic feature that was first described with busulphan and subsequently with bleomycin. 21, 37 these two drugs differ chemically but both act (by different mechanisms) on dna. the atypical cells have abundant deeply eosinophilic or amphophilic cytoplasm and large nuclei, which may be multiple but are usually single. the nuclei measure up to 12 µm and are densely stained throughout or contain either large homogeneous deeply eosinophilic inclusions or clear vacuoles (fig. 7.3.3) . electron microscopy distinguishes the inclusions from nucleoli and shows them to consist of tubular aggregates derived from the internal nuclear membrane. 37 airway epithelium shows similar nuclear changes and often undergoes squamous metaplasia. the presence of such cells in sputum specimens submitted for cytology can lead to a misdiagnosis of malignancy. fibrosis may follow diffuse alveolar damage or develop insidiously, perhaps many years after drug therapy ceased (fig. 7.3.4) . 38 it may be both interstitial and intra-alveolar. the interstitial component is often accompanied by a non-specific chronic inflammatory infiltrate. the proportions of inflammation, which is potentially reversible, and fibrosis, which when collagenous is irreversible, obviously bear on the prognosis. however, most case reports antedate the recent classification of interstitial pneumonia described in chapter 6 and it is uncertain how their pathological appearances would now be classified. the majority lack the classic features of usual interstitial pneumonia and fibrotic non-specific interstitial pneumonia. many show overlapping patterns of intersitital pneumonia and this alone should arouse suspicion that a drug may have been responsible. however, some cytotoxic drugs result in pulmonary changes by more than one mechanism: for example, methotrexate may produce hypersensitivity reactions with granuloma formation [41] [42] [43] [44] or pulmonary eosinophilia 45 as well as diffuse alveolar damage. pulmonary toxicity is also occasionally seen in patients undergoing treatment with gold salts for rheumatoid disease: in addition to diffuse alveolar damage, there may be eosinophilia and dermatitis in these cases, again indicating possible hypersensitivity. 46 nitrofurantoin is another example of a drug resulting in a variety of patterns of alveolar injury: diffuse alveolar damage, desquamative interstitial pneumonia, giant cell interstitial pneumonia, organising pneumonia and eosinophilic pneumonia have all been recorded in association with this drug. [47] [48] [49] it should also be noted that in patients with neoplastic disease, clinical features suggestive of a pulmonary drug reaction may be due to factors other than drugs. in leukaemic patients, for example, these include direct infiltration of the lungs by leukaemic cells, opportunist infection and, if bone marrow transplantation has been undertaken, the effects of irradiation and possibly graft-versus-host disease. phospholipidosis is encountered with drugs such as the antidysrhythmic agent amiodarone, 50 which block lysosomal enzymes involved in the breakdown of complex lipids. this leads to their accumulation throughout the body but the effect is most marked in tissues that take up the drug and contain cells rich in lysosomes. the lung fulfils both these requirements through its rich complement of alveolar macrophages. these cells accumulate the enzyme substrate (phospholipid) in their cytoplasm with the result that large foam cells fill the alveoli (fig. 7.3.6 ). the appearances are those of endogenous lipid pneumonia, similar to that seen in obstructive pneumonitis. however, with amiodarone cytoplasmic vacuolation is also seen in epithelial and interstitial cells. the phospholipid inclusions contained within the vacuoles are particularly well seen in unstained frozen sections viewed by polarised light. 51 identical changes to those induced by amiodarone were seen in the lungs of rats exposed to very high levels of the antidepressant drug iprindole 52 and the anorectic drug chlorphentermine. 53 these three compounds, iprindole, chlorphentermine and amiodarone, all belong to the amphiphilic group of drugs which block lysosomal phospholipase and sphingomyelinase. although their pharmacological actions are very different, a molecular homology is apparent (fig. 7.3.7) . it is likely that all patients receiving substantial amounts of amiodarone develop phospholipidosis throughout the body, but this is generally well tolerated. only a minority experience respiratory impairment and in these there is also evidence of pulmonary inflammation and fibrosis, which is possibly mediated immunologically. 54 these patients generally have a restrictive lung deficit, the onset of which may be acute or chronic. bronchoalveolar lavage shows foamy macrophages but these cells indicate exposure to the drug rather than drug toxicity; nor are they specific to amiodarone, being observed on occasion with other drugs. lymphocytes of suppressor type may also be detected on lavage. 54 histologically, amiodarone toxicity is diagnosed on a combination of phospholipidosis and interstitial pneumonia and fibrosis. occasionally the hyaline membranes of diffuse alveolar damage are superimposed on the interstitial changes (see fig. 7.3.2) . [55] [56] [57] in some patients the fibrosis is intraalveolar rather than interstitial and the appearances are those of organising pneumonia. 58 the process may be localised and mimic a neoplasm radiologically. 59, 60 amiodarone toxicity is probably dose-dependent but there is considerable individual variation in the amount required, 61 ,62 which appears to be under genetic control. 63 amiodarone toxicity is uncommon in patients taking daily doses of 200 mg or less whereas the there are drugs that undoubtedly cause a usual interstitial pneumonia pattern, for example the chemotherapeutic agents and nitrofurantoin (fig. 7. 3.5), while others, for example the statins, are recorded as having induced a non-specific interstitial pneumonia pattern. 39 a drug history is therefore imperative when assessing any patient with diffuse parenchymal lung disease. organising pneumonia similar to the cryptogenic condition described on page 308, and probably similarly reversible with steroids, has been encountered with a variety of drugs, including amiodarone, sulphasalazine and pencillamine. 40 penicillamine has also been incriminated in the development of both diffuse alveolitis and bronchiolitis obliterans, but both these changes could well be due to the underlying rheumatoid disease for which the pencillamine is administered. 15 in busulphan lung there may be an organising intraalveolar fibrinous exudate, 21 which at its most extreme results in irreversible effacement of the alveolar architecture by sheets of loose connective tissue (see p. 148). with continued experimental administration of the drug iprindole mentioned above, the phospholipidosis it produced gradually evolved into alveolar proteinosis (more properly called lipoproteinosis; see p. 316), 65 but this has not been reported as a drug effect in humans. alveolar proteinosis has however been recognised in a number of patients receiving chemotherapy for conditions such as leukaemia. the mechanism here is probably based on the cytotoxic action of the drug and the material filling the alveoli may represent the detritus of degenerate alveolar cells rather than excess pulmonary surfactant, as in the primary auto-immune form of alveolar proteinosis. eosinophilic pneumonia, the pathology of which is described on page 461, may be caused by several drugs, including nitrofurantoin, para-aminosalicylic acid, sulphasalazine, phenylbutazone, gold compounds, aspirin and penicillin (see box 9.3, p. 460). 66, 67 it may also follow radiation to the chest. 68 the tissue eosinophilia is generally accompanied by a rise in the number of eosinophils in the blood. the clinical picture varies from transient asymptomatic opacities on a chest radiograph to a life-threatening illness with severe respiratory distress and hypoxaemia, so-called acute eosinophilic pneumonia (see p. 462). the reaction is often associated with a florid rash. withdrawal of the drug may be all that is required to effect resolution but corticosteroids are usually given as they produce a marked improvement. this syndrome of necrotising granulomatosis, vasculitis and eosinophilia in asthmatic patients, which is described more fully on page 465, has been reported when leukotriene receptor antagonists have been used to treat asthma. however, it is likely that the syndrome has been merely unmasked by the antileukotriene permitting a reduction in corticosteroid dose rather than representing a direct effect of the antileukotriene. 69, 70 mesalazine has also been implicated in inducing a vasculitis during treatment for inflammatory bowel disease. 71 the eosinophilia-myalgia syndrome was identified in the usa in 1989 and quickly identified as being due to the ingestion of ltryptophan from one particular japanese supplier. withdrawal of this substance led to the virtual elimination of the disease, but not before 2000 patients had been affected, 1 in 60 fatally. [72] [73] [74] [75] [76] cases were subsequently described in europe where there were further fatalities. l-tryptophan is an essential amino acid that is freely available to the public: its purchase does not require a medical prescription. it has been promoted as a dietary supplement and as an agent against insomnia and premenstrual tension. women in the reproductive years preponderated in the patients affected by the resultant eosinophiliamyalgia. the clinicopathological features of the syndrome are similar to those of the spanish toxic oil syndrome (see p. 375) and differ more in degree than type. the discovery of an aniline-derived contaminant in the tryptophan-induced condition is a further link connecting these two syndromes. 77 an immune basis is suggested by the identification of t lymphocytes activated against fibroblasts in the eosinophilia-myalgia syndrome. 78 the illness is a multisystem disorder and besides blood eosinophilia and myalgia there may be arthralgia, fever, rash and involvement of the lungs, liver and central nervous system. as in the toxic oil syndrome, there is fasciitis, wasting and muscle pain associated with blood and tissue eosinophilia. the lungs are affected in 60% of cases. pulmonary symptoms have included cough, dyspnoea and chest pain. radiographs have shown diffuse bilateral infiltrates and pulmonary hypertension has been documented in a few cases. 79 histology of the lungs shows an oedematous myxoid intimal thickening affecting small pulmonary blood vessels and a diffuse interstitial lymphocytic and eosinophilic infiltrate. 72, 73, 75, 76, 80 these cells may also be seen within the walls of the thickened blood vessels (fig. 7.3.8) . 72, 76 massive ingestion of l-tryptophan has resulted in the appearances of an organising pneumonia. 81 as an adverse drug reaction, granulomatous alveolitis is best exemplified by the extrinsic allergic alveolitis of pituitary snuff-takers, but it is also encountered on rare occasions with cytotoxic and other drugs, including methotrexate, bacille calmette-guérin (bcg) immunisation, interferons, ciprofloxacin, antiviral therapy and tumour necrosis factor antagonists. [42] [43] [44] 48, [82] [83] [84] [85] [86] [87] [88] [89] [90] the histological appearances may suggest extrinsic allergic alveolitis or sarcoidosis but the centri-acinar or lymphangitic concentration of these conditions is usually lacking. however, unless an infective agent can be demonstrated the diagnosis generally requires consideration of the clinical and environmental details, including any drug regimen. exogenous lipid pneumonia may result from the unintentional aspiration of various fat-based medicaments such as liquid paraffin, oily nose drops and petroleum jelly or of fat-rich dietary supplements in the form of ghee. [91] [92] [93] [94] [95] [96] [97] the consumption of liquid paraffin as an aperient is common in some countries and may be taking place without the knowledge of the patient's medical practitioner. regurgitation and aspiration of ingested oil are especially likely to happen during sleep in the presence of a hiatus hernia or when the oesophagus fails to empty completely into the stomach because of achalasia of the cardia. the aspiration of vegetable oil occurred in the past from the use of menthol in olive oil for the treatment of tuberculous laryngitis, and occasionally from the use of iodinated vegetable oils for bronchography. [98] [99] [100] [101] more recently exogenous lipid pneumonia has developed from the constant sucking of lollipops formulated for the administration of the analgesic fentanyl but also containing a stearate component. 102 the treatment of epistaxis by nasal packing with paraffin gauze has also led to exogenous lipid pneumonia. the pathology of exogenous lipid pneumonia is described on page 314. other medicines may also be aspirated unwittingly, for example a ferrous sulphate tablet may cause brown iron staining and necrosis of the bronchus at the point of impact, progressing to bronchial stenosis. [103] [104] [105] distal infection is then likely, as with any foreign body. barium sulphate aspiration may complicate gastrointestinal radiography. 106 large amounts may impair ventilation but being inert there is no permanent injury to the lungs, although the striking changes are evident on the chest radiograph. an outbreak of pulmonary hypertension affecting many swiss, austrian and german patients in the period 1966-68 was probably due to the anorectic drug aminorex, 107 which was accordingly withdrawn with regression in the number of new cases. the pathology in these patients was identical to that of primary pulmonary hypertension (see p. 420) and it proved impossible to reproduce the condition in laboratory animals but the epidemiological evidence that aminorex was to blame is very strong. fenfluramine and phentermine, further anorectic drugs that are chemically similar to aminorex, have also been associated with such plexogenic pulmonary hypertension, [108] [109] [110] [111] [112] and with fibroproliferative plaque on the tricuspid valve and pulmonary arteries. 113 pulmonary hypertension due to pulmonary veno-occlusive disease has sometimes complicated the use of cytotoxic chemotherapeutic agents 114 or followed bone marrow transplantation. 115 non-steroidal anti-inflammatory agents such as indomethacin and diclofenac cross the placenta and, if given in late pregnancy, may cause premature closure of the ductus arteriosus, resulting in severe neonatal pulmonary hypertension. 116, 117 pulmonary hypertension is a well-recognised association of human immunodeficiency virus (hiv) infection but until recently has been unexplained. now, however, evidence is emerging that the highly active antiretroviral therapy administered to hiv-positive patients might be responsible for the pulmonary hypertension. 118 the older high-oestrogen contraceptive drugs carried a slight risk of thromboembolism but this is not seen with the newer preparations. pulmonary thromboembolism has also occurred with a drug-induced lupus syndrome associated with anticardiolipin antibodies. chemotherapeutic drugs such as mitomycin may cause widespread smallvessel thrombosis resulting in the haemolytic-uraemic (thrombotic microangiopathic) syndrome. there is prominent involvement of pulmonary vessels and patients often suffer from respiratory as well as renal insufficiency, and pulmonary hypertension. the syndrome can develop during treatment or up to several months after the drug has been withdrawn. pulmonary thromboembolism is also recorded as a complication of immunoglobulin infusion. 119 non-traumatic fat embolism has resulted from the agglutination or 'creaming' of fat emulsions administered intravenously as a source of calories to debilitated patients. [120] [121] [122] [123] [124] [125] the agglutinated liposomes occlude fine blood vessels throughout the body, causing effects such as priapism, osteonecrosis and pancreatitis. they may be demonstrated in the pulmonary capillaries but the lungs have considerable vascular reserve and it is uncertain what effect the vascular occlusion has on pulmonary function. agglutination of these fat emulsions is particularly common in severely ill patients and this has been attributed to the elevated blood levels of acute-phase proteins, especially c-reactive protein, that are found in the very ill. the agglutination is also induced by calcium and may be brought about by administering calcium and other mineral supplements through the same venous line as the fat. once agglutinated, the fat is less soluble and may be demonstrated in paraffin sections. sudan black is especially useful for this purpose (fig. 7.3.9 ). microvascular crystal embolism is a further risk of parenteral nutrition, the crystals representing various calcium salts that may precipitate in the circulation. 126 transient diffusion abnormalities attributed to oil embolism are very common in patients undergoing lymphangiography but serious respiratory impairment is limited to those patients with pre-existing lung disease or in whom substantial amounts of contrast medium are injected rapidly. [127] [128] [129] [130] other emboli of an iatrogenic nature described in pulmonary arteries include the broken-off ends of intravenous catheters and cannulas, particles from dialysis tubing, 131 prosthetic implants of substances such as teflon and silicone 88, [132] [133] [134] [135] and various materials injected to occlude abnormal blood vessels. 136, 137 diffuse pulmonary haemorrhage diffuse pulmonary haemorrhage may result from interference with the clotting mechanism by anticoagulants 138 or from widespread pulmonary capillaritis, the latter reported in leukaemic patients treated with retinoic acid. 139 pulmonary haemorrhage has also been reported as an idiosyncratic reaction to lymphangiography media 140 and as a complication of immunoglobulin infusion, 141 while the development of anti-basement membrane antibodies resulting in goodpasture's syndrome has been attributed to penicillamine. 141a infection is a common pulmonary hazard in any patient receiving corticosteroids, chemotherapy or any other immunosuppressant drug. viral, bacterial, fungal and protozoal infections, often in combination, may all develop in the lungs of such patients and tissue reactions may be atypical. pneumocystis jiroveci, for example, may elicit a granulomatous reaction or cause diffuse alveolar damage rather than the usual foamy alveolar exudate (see p. 226). metastatic calcification, described on page 489, may result from any drug causing hypercalcaemia, e.g. high doses of vitamin d, calcium and inorganic phosphate or excessive alkali intake in the treatment of peptic ulceration. carcinoma of the lung may be promoted by drugs. arsenicals cause squamous metaplasia of the bronchi and occasionally squamous carcinoma, while peripheral scar cancers, usually adenocarcinomas, have developed in lungs showing fibrosis due to drugs such as busulphan. drugs may result in a variety of pleural diseases. 142 common examples include effusions, chronic inflammation and fibrosis. these are usually encountered in isolation but may be associated with chronic interstitial pneumonia or fibrosis. sometimes there is also serological evidence of systemic lupus erythematosus: many drugs, including hydantoin, practolol, procainamide, hydralazine and sulphonamides, are associated with the development of a syndrome resembling systemic lupus erythematosus that includes pleural disease. whether the drugs are directly responsible for the syndrome or merely promote the development of latent natural disease is uncertain. ergotamine derivatives such as methysergide and bromocriptine are notable for the production of pleural fibrosis, which is sometimes associated with mediastinal and retroperitoneal fibrosis large amounts or prolonged treatment are generally required to produce this effect. [143] [144] [145] in patients given practolol, pleural thickening has become evident several years after the drug was discontinued. this shows the need for a careful drug history in any patient with unexplained pleural fibrosis. reports of radiation-induced lung damage began to appear soon after ionising radiation became widely used in the treatment of malignant disease. [146] [147] [148] despite refinements in radiotherapy techniques it is often impossible to avoid irradiating small areas of lung when treating cancer of the lung, breast, spine, thymus and oesophagus. parts of the lungs are also included in 'mantle' irradiation of mediastinal lymph nodes affected by lymphoma. occasionally, the whole of both lungs is irradiated, as in the treatment of widespread pulmonary metastases or as part of whole-body irradiation prior to marrow transplantation for the treatment of leukaemia. radiation pneumonitis, usually localised, is estimated to affect about 8% of patients. 149 therapeutic irradiation is given as divided doses over several weeks in order to minimise damage to adjacent tissue. the effects of such fractionated treatment are cumulative. in the lungs an early exudative phase soon passes and progressive damage becomes apparent only after months or even years. 150, 151 the changes are generally confined to the area of lung that is irradiated but are widespread when the whole body is irradiated prior to bone marrow transplantation or there is accidental whole-body irradiation. however, localised irradiation of the lung has been followed by abnormalities in non-irradiated areas. these include bilateral alveolar exudates, 152 migratory organising pneumonia affecting both lungs 153, 154 and fulminant bilateral interstitial pneumonia. 155 the likelihood of lung injury is increased by the simultaneous use of cytotoxic drugs and oxygen therapy. 156 furthermore, chemotherapy following irradiation may result in exacerbation of the injury in areas previously irradiated, a phenomenon termed 'recall pneumonitis' . 157, 158 in the long term, irradiation also results in an increased incidence of lung carcinoma. this was seen in patients given therapeutic irradiation to the spine for ankylosing spondylitis 159 and is still encountered on occasion following irra diation for breast cancer. 160 the pathogenesis of radiation injury is described on page 146. radiation damage to the lung is traditionally separated into fulminant acute injury coming on within days, subacute pneumonitis developing within several weeks (typically 2-3 months) and interstitial fibrosis slowly evolving from the subacute stage or making itself apparent years later. the migratory organising pneumonia referred to above is an unusual further effect, as is chronic eosinophilic pneumonia. 68 in the pleura, radiation causes fibrinous effusions and adhesions. pleural effusion and pulmonary oedema may be augmented by the long-term effects of radiation on the heart. fulminant acute injury is an unusual and unexpected effect of therapeutic radiation but one that is likely to come to the attention of the pathologist as an autopsy is often requested. the clinical features are those of acute lung injury and the pathological changes are those of diffuse alveolar damage. the cause is likely to be accidental overdosage, augmentation of the radiation damage by accompanying oxygen therapy or treatment with cytotoxic drugs. occasionally however these factors can be excluded, in which case the damage has to be ascribed to 'hypersensitivity' . subacute radiation pneumonitis is encountered more commonly. after an interval of about 2-3 months the patient complains of shortness of breath and a non-productive cough. the chest radiograph shows hazy opacification proceeding to more dense consolidation. lung biopsy shows alveolar and interstitial oedema, possibly with residual hyaline membranes, proliferation of atypical alveolar epithelial cells and interstitial fibroblasts and organising thrombosis. later, as the process advances, there is widespread fibrosis comparable to that illustrated in figure 4 .24 on page 148 and ultimately dense scarring (fig. 7.3.10) . tracheal and aortic injury may complicate radiation treatment of tracheal lesions, sometimes resulting in an aortotracheal fistula. 161 patients requiring mechanical ventilation are liable to suffer lung injury in a number of ways. in addition to effects of barotrauma such as pneumothorax and surgical emphysema, they often develop diffuse alveolar damage. the high oxygen tension that is often combined with mechanical ventilation is a major factor 162-164 but mechanical forces other than the high pressures responsible for barotrauma can also contribute to this form of lung injury, notably by resulting in excessive end-expiratory stretch and repeated collapse/recruitment of the alveolar walls. 165, 166 low tidal volume ventilation is therefore a fundamental part of the management of diffuse alveolar damage. although oxygen is necessary to life, it is cytotoxic in high concentrations. severe hyperoxia damages dna, inhibits cellular proliferation and ultimately kills cells. its toxicity is thought to be due to the intracellular production of active oxygen radicals, some of which derive from activated neutrophils attracted to the site of injury. [167] [168] [169] [170] under normal conditions most of the oxygen is reduced to water by cytochrome oxidase, and any active radicals produced are eliminated by superoxide dismutase, catalase and other antioxidants. however, these defence mechanisms may prove inadequate when active radicals are produced in excess. 171 problems are likely to arise in clinical practice when lung disease necessitates the concentration of oxygen in the inspired air being raised in order to maintain normal blood levels of oxygen and prevent cerebral hypoxia. [172] [173] [174] a 'safe' level for oxygen administration is not firmly established and, because of species differences in susceptibility to oxygen, caution is needed in extrapolating from animal studies. however, animal experiments have shown that previous damage to the lungs renders them unduly sensitive to oxygen 175,176 and conversely that prior exposure to high levels of oxygen confers some resistance to subsequent oxygen exposure. 177 clinical studies suggest that less than 50% oxygen (at atmospheric pressure) can be tolerated for long periods without ill effect. little, if any, serious lung damage results from administration of 100% oxygen for up to 48 hours but concentrations between 50% and 100% carry a risk of damage if this period is exceeded. 171, 178 extracorporeal oxygenation of the blood circumvents the problem but if it is to be prolonged it becomes a major undertaking that poses its own hazards; it is therefore generally reserved for patients who remain hypoxaemic despite other measures. 179 intravenous blood oxygenators are employed to minimise the supplementation of inspired oxygen and partial liquid ventilation utilising perfluorocarbon has also been used. 180 experimentally, disruption of cd40 binding to reduce the release of proinflammatory cytokines has shown promising results in blunting oxygen-induced lung injury. 181 none of the morphological changes attributable to oxygen toxicity is specific. 174 the earliest ultrastructural change in experimental oxygen poisoning is swelling of endothelial cells, the cytoplasm of which becomes grossly oedematous and vacuolated. swelling and fragmentation of type i epithelial cells follow and these cells become separated from their basement membrane, which is then coated by thin strands of protein. 178 this coating is replaced by proliferating type ii cells by the 12th day. with recovery in room air the lungs practically return to normal. 182 the full clinical picture of oxygen poisoning is the acute respiratory distress syndrome and the corresponding pathological changes are those of diffuse alveolar damage, 174 as described on page 136. patients with hypovolaemic shock or undergoing major surgery often require massive blood transfusions and this provides another possible cause of pulmonary damage. although hypervolaemia is the commonest cause of pulmonary oedema after blood transfusion, transfusion-related acute lung injury is more often fatal. platelet and white cell aggregates are known to develop in stored blood, but a relationship between the number of microaggregates transfused and the degree of respiratory impairment has not been convincingly demonstrated. leukocyte antibodies are a more likely cause of lung injury in these patients. such antibodies are often found in multiparous female donors as a result of sensitisation by fetal white cells during pregnancy. alternatively, the recipient may have developed them during pregnancy or as a result of previous blood transfusions. the implicated antibodies are thought to initiate alveolar capillary damage within hours of transfusion by stimulating granulocyte aggregation. 183, 184 electron microscopy has shown capillary endothelial damage with activated granulocytes in contact with alveolar basement membranes. 185 cardiopulmonary bypass entails oxygenation and circulation of the blood by extracorporeal devices, so permitting major heart surgery. in the early days of such surgery it was not unusual for patients to develop fatal respiratory insufficiency in the postoperative period. this led to the term 'postperfusion lung' . electron microscopic studies showed alveolar damage with degranulation of neutrophils in pulmonary capillaries. 186, 187 the syndrome is now less common but infants remain susceptible. 188 the most likely explanation is that the synthetic materials with which blood comes into contact during the bypass procedure are able to activate complement. this is mediated by hageman factor (factor xii) and the alternative pathway. aggregation of neutrophils leads to their sequestration in the lungs and damage results from their release of lysosomal enzymes and active radicals. [188] [189] [190] the process is delayed by hypothermia. 189 a postcardiac injury syndrome develops after a variety of myocardial or pericardial injuries: it has been described after cardiac surgery (postpericardiotomy syndrome), myocardial infarction (dressler's syndrome), blunt trauma to the chest, percutaneous puncture of the heart and implantation of a pacemaker. 191 there is a delay of anything between a few days and a few months between the cardiac injury and the onset of symptoms, which comprise chest pain, breathlessness, dyspnoea and fever. examination usually reveals haemorrhagic pleural or pericardial effusions and pulmonary infiltrates. the syndrome usually resolves spontaneously and few pathological studies have therefore been conducted. however, the changes of diffuse alveolar damage have been reported, principally hyaline membrane formation and type ii pneumocyte hyperplasia. 192 the pathogenesis is obscure. antibodies reacting with myocardial antigens often develop after cardiac surgery but there is no relationship between these and the development of the syndrome. [192] [193] [194] this minimally invasive technique is used to destroy lesions as varied as pulmonary metastases and the connection between the left atrium and ectopic foci in the muscular sleeves that surround the terminations of the pulmonary veins (see p. 76). the former may be complicated by pneumothorax and the latter by pulmonary vein stenosis. 195, 196 central venous cannulation (synonym: catheterisation) is widely used in treating seriously ill patients and may give rise to serious complications. the commonest early complications related to the respiratory tract are caused by local trauma: they include pneumothorax, subcutaneous emphysema, haemothorax and air embolism. infection occurs later, causing endocarditis, septic emboli and lung abscesses. 197 thrombosis is another common late complication: one autopsy study of patients with central venous lines showed that 15% had major pulmonary emboli and 65% had microscopic emboli in their pulmonary arteries. 198 pulmonary artery cannulation, for example with a swan-ganz catheter, may result in pulmonary infarction or any of the traumatic complications of central venous catheterisation mentioned above. 199, 200 tracheotomy entails a small immediate risk of haemorrhage from damaged subthyroidal arteries, while an endotracheal tube predisposes to infection, as with all foreign bodies. infection is also promoted by the filtering action of the upper respiratory air passages being bypassed. the latter factor also necessitates humidification of the inspired air and on occasion the humidifier or ventilator has become contaminated so that an aerosol of bacteria is introduced directly into the lower respiratory tract. 201 high-pressure ventilation may also lead to interstitial emphysema, pneumothorax and surgical emphysema. asphyxia may follow an endotracheal tube becoming blocked by secretions or through it being badly positioned. secretions need to be constantly removed yet repeated suctioning to achieve this has led to cardiac dysrrythmia and even cardiac arrest. 202 if the balloon on the endotracheal tube is too near the tracheostomy it may act as a fulcrum, causing the tip of the tube to press into the tracheal wall. pressure necrosis and perforation may follow, leading to mediastinitis, tracheo-oesophageal fistula or erosion of a large blood vessel. these are also complications of tracheobronchial laser therapy. pressure from the balloon may lead to a tracheal diverticulum and after the tube is withdrawn the trachea may become narrowed at either the site of the incision or further down where the balloon on the tracheal tube causes pressure. small, shallow ulcers generally heal quickly but deeper ulcers cause necrosis of the tracheal cartilage, and healing is then often accompanied by fibrous stenosis (fig. 7.3 .11) or web formation. this results in wheezing and dyspnoea but not before the trachea has narrowed to 30% of its original size, which may take months. earlier narrowing may be caused by oedema or a fibrinous pseudomembrane. 203, 204 sometimes the stenosis takes the form of a large mass of granulation tissue at the tracheostomy site, a so-called granuloma ball. in children especially, intubation may lead to tracheomalacia so that after the tube is removed the airway collapses. 205 necrotising sialometaplasia is a further complication of prolonged intubation. 206 the incidence of such posttracheostomy complications can be minimised by careful placement of the stoma and tube, avoidance of large apertures and high cuff pressures, elimination of heavy connecting equipment and meticulous care of the tracheostomy. nasogastric feeding tubes may of course lead to aspiration lesions in the lungs and even fatal asphyxia if they are inadvertently allowed to enter the trachea rather than the oesophagus. bronchoscopy is generally a safe, almost routine procedure. a review of 23 862 patients who underwent bronchoscopy identified severe complications in 152 (0.637%), of whom three died. 207 the fatal cases comprised a 78-year-old with coronary heart disease who developed cardiac arrest and two patients who had had tracheal transplantation for oesophageal cancer and required bronchoscopic laser treatment but died of airway obstruction. the pleural cavity is intubated in the treatment of pneumothorax and pleural effusions the tube being placed anteriorly to drain air and posteriorly to drain fluid. complications include laceration of an intercostal artery or vein, the lung, the diaphragm and the heart. pneumonectomy has been practised since the 1930s, since when the mortality associated with this operation has dropped from over 50% to near zero in the best hospitals. risk factors include underlying lung disease, other medical conditions and more extensive procedures such as pleuropneumonectomy and pneumonectomy combined with chest wall resection. the anatomical changes that take place soon after pneumonectomy have been extensively studied by radiologists who describe the air-filled postpneumonectomy space gradually filling with fluid and contracting as the mediastinum shifts and the ipsilateral dome of the diaphragm rises. 209 much of the space is filled by fluid within 2 weeks but complete opacification may take up to 6 months. rapid filling in the immediate postoperative period suggests haemorrhage or chylothorax. however, fluid accumulation is normally rapid after pleuro-pneumonectomy and may compromise the function of the other lung. pathologists conducting autopsies long after the operation may find complete fibrous obliteration of the postpneumonectomy space, coupled with mediastinal shift and elevation of the hemidiaphragm, but often there is persistent brown fluid, which may be clear, cloudy or occasionally purulent. 210 the remaining lung is generally enlarged, with its volume greater than predicted. animal studies have shown that if one lung is excised early in life the enlargement is partly due to enhanced growth but later it represents only dilatation of existing air spaces. hepatocyte growth factor is thought to be involved in the proliferation of residual lung cells following pneumonectomy. 211 pulmonary complications include those typically seen after other thoracic procedures, such as haemorrhage and infection, and those unique to the postpneumonectomy state, namely anastomotic dehiscence and postpneumonectomy pulmonary oedema. the latter presents as the acute respiratory distress syndrome and represents the early stages of diffuse alveolar damage. it follows severe shift of the heart and mediastinum, which is commoner in children and young adults, in whom the tissues are more compliant. [212] [213] [214] [215] the condition complicates up to 4% of lung resections 216, 217 and is commoner following excision of the right lung when severe herniation of the left lung into the postpneumonectomy space stretches the trachea and left main bronchus and the latter is compressed between the left pulmonary artery in front and the arch of the aorta behind. in the long term the compression can result in bronchomalacia and postobstructive bronchiectasis. if postpneumonectomy oedema develops the immediate postoperative mortality is high -50% following pneumonectomy, 42% following lobectomy and 22% following sublobar resections. 217, 218 the pathogenesis is probably multifactorial but apart from factors such as fluid overload and high inspired oxygen concentrations there is probably an element of alveolar wall injury, induced by oxidant generation secondary to lung stretching and general surgical trauma. 219, 220 occupational dust exposure and chronic obstructive pulmonary disease -a systematic overview of the evidence occupational exposures and the risk of copd: dusty trades revisited the origin of the term 'pneumonokoniosis' industrial injuries advisory council. pneumoconiosis and byssinosis. london: hmso; 1973 aerosols: their deposition and clearance human lung parenchyma retains pm2.5 airway branching patterns influence asbestos fiber location and the extent of tissue injury in the pulmonary parenchyma and lymph nodes in infants' lungs pulmonary sumps, dust accumulations, alveolar fluid and lymph vessels candida lung abscesses complicating parenteral nutrition a diffuse form of pulmonary silicosis in foundry workers inhalation of china stone and china clay dusts: relationship between mineralogy of dust retained in the lungs and pathological changes talc pneumoconiosis. a report of six patients with postmortem findings diffuse 'asbestosis-like' interstitial fibrosis of the lung pulmonary pathology in workers exposed to nonasbestiform talc talc pneumoconiosis -a pathologic and mineralogic study diffuse interstitial fibrosis in nonasbestos pneumoconiosis -a pathological study the disposal of coal and haematite dusts inhaled successively vertical gradient of alveolar size in lungs of dogs frozen intact apical localization of phthisis distribution of blood flow and ventilation perfusion ratio in the lung, measured with radioactive co 2 apical localization of pulmonary tuberculosis, chronic pulmonary histoplasmosis, and progressive massive fibrosis of the lung effect of inspiratory flow rate on the regional distribution of inspired gas apical scars and their relationship to siliceous dust accumulation in non-silicotic lungs topographic distribution of asbestos fibres lung structure as a risk factor in adverse pulmonary responses to asbestos exposure. a case-referent study in quebec chrysotile miners and millers particle deposition in casts of the human upper tracheobronchial tree differences in particle deposition between the two lungs pulmonary retention of ultrafine and fine particles in rats experimental study of the dust-clearance mechanism of the lung the lung: an excretory route for macromolecules and particles phagocytic potential of pulmonary alveolar epithelium with particular reference to surfactant metabolism chrysotile asbestos inhalation in rats: deposition pattern and reaction of alveolar epithelium and pulmonary macrophages the uptake of mineral particles by pulmonary epithelial cells translocation of particles to the tracheobronchial lymph nodes after lung deposition: kinetics and particle-cell relationships patterns of particle deposition and retention after instillation to mouse lung during acute injury and fibrotic repair silica deposition in the lung during epithelial injury potentiates fibrosis and increases particle translocation to lymph nodes the permeability of lung parenchyma to particulate matter the migration of bronchoalveolar macrophages into hilar lymph nodes etude au microscope electronique du granulome pulmonaire silicotique experimental the postnatal development of lymphoreticular aggregates in human lung in relation to occupational and non-occupational exposure the distribution of amosite asbestos in the periphery of the normal human lung diseases associated with exposure to silica and nonfibrous silicate minerals detection of silica particles in lung tissue by polarizing light microscopy a practical method for the identification of particulate and crystalline material in paraffin-embedded tissue specimens particles in paraffin sections demonstrated in the backscattered electron image quantitative analysis of inorganic particulate burden in situ in tissue sections microanalysis in histopathology ion microprobe mass analysis of beryllium in situ in human lung: preliminary results laser probe mass spectrometry (lamms) analysis of beryllium, sarcoidosis and other granulomatous diseases molecular identification of foreign inclusions in inflammatory tissue surrounding metal implants by fourier transform laser microprobe mass spectrometry value of beryllium lymphocyte transformation test in chronic beryllium disease and in potentially exposed workers radiographic and pathologic correlation of coal workers' pneumoconiosis slateworker's pneumoconiosis transkei silicosis three cases of dental technician's pneumoconiosis related to cobaltchromium-molybdenum dust exposure: diagnosis and follow-up deposition of silicosis dust in the lungs of the inhabitants of the sahara regions sulla possibilita e sulla frequenza della silicosi pulmonare tra gli abitanti del deserto libico simple siliceous pneumoconiosis of bedouin females in the negev desert simple siliceous pneumoconiosis in negev bedouins silicosis in a pakistani farmer silicosis in a himalayan village population -role of environmental dust non-occupational pneumoconiosis at high altitude villages in central ladakh silicate pneumoconiosis of farm workers silicate pneumoconiosis and pulmonary fibrosis in horses from the monterey-carmel peninsula comparative pathology of silicate pneumoconiosis two cases of acute silicosis -with a suggested theory of causation acute silicosis silicose aigue fatale par inhalation volontaire de poudre à recurer acute silicosis in tombstone sandblasters accelerated silicosis in scottish stonemasons the relation between fibrosis of hilar lymph glands and the development of parenchymal silicosis distribution of silicotic collagenization in relation to smoking habits silicosis presenting as bilateral hilar lymphadenopathy silicotic lymph node lesions in nonoccupationally exposed lung carcinoma patients obliterative central bronchitis due to mineral dust in patients with pneumoconiosis left recurrent laryngeal nerve palsy associated with silicosis anthracotic and anthracosilicotic spindle cell pseudotumors of mediastinal lymph nodes: report of five cases of a reactive lesion that simulates malignancy right middle lobe atelectasis associated with endobronchial silicotic lesions pleural pearls following silicosis: a histological and electronmicroscopic study silicainduced pleural disease: an unusual case mimicking malignant mesothelioma factors associated with massive fibrosis in silicosis silicosis of systemic distribution hepatosplenic silicosis hepatic silicosis silicotic lesions of the bone marrow: histopathology and microanalysis peritoneal silicosis pulmonary fibrosis in non-ferrous foundry workers irregular opacities in coalworkers' pneumoconiosis -correlation with pulmonary function and pathology pneumoconiosis-related lung cancers -preferential occurrence from diffuse interstitial fibrosis-type pneumoconiosis the effects of dusts on peritoneal cells within diffusion chambers freshly fractured quartz inhalation leads to enhanced lung injury and inflammation: potential role of free radicals an examination of the cytotoxic effects of silica on macrophages activity of macrophage factor in collagen formation by silica fractionation of connective-tissue-activating factors from the culture medium of silica-treated macrophages effect of sio 2 -liberated macrophage factor on protein synthesis in connective tissue in vitro the regulation of lung fibroblast proliferation by alveolar macrophages in experimental silicosis the ability of inflammatory bronchoalveolar leucocyte populations elicited with microbes or mineral dust to injure alveolar epithelial cells and degrade extracellular matrix in vitro cytokines and cytokine network in silicosis and coal workers' pneumoconiosis activation of murine macrophages by silica particles in vitro is a process independent of silica-induced cell death localization of type i procollagen gene expression in silica-induced granulomatous lung disease and implication of transforming growth factor-beta as a mediator of fibrosis transforming growth factor-beta (tgfbeta) in silicosis mechanisms in the pathogenesis of asbestosis and silicosis changes in the composition of lung lipids and the 'turnover' of dipalmitoyl lecithin in experimental alveolar lipo-proteinosis induced by inhaled quartz case report: pulmonary alveolar proteinosis and aluminum dust exposure induction of surfactant protein (sp-a) biosynthesis and sp-a mrna in activated type ii cells during acute silicosis in rats silicon nephropathy: a possible occupational hazard rapidly progressive silicon nephropathy silica and glomerulonephritis: case report and review of the literature rapidly progressive crescenteric glomerulonephritis in a sandblaster with silicosis diatomaceous earth pneumoconiosis particle size for differentiation between inhalation and injection pulmonary talcosis kaolin pneumoconiosis. a case report kaolin pneumoconiosis. radiologic, pathologic and mineralogic findings silicosis in wyoming bentonite workers pneumoconiosis due to fuller's earth pulmonary disease caused by the inhalation of cosmetic talcum powder talc dust pneumoconiosis mica-associated pulmonary interstitial fibrosis baritosis: a benign pneumoconiosis silicosis in barium miners scleroderma in gold-miners on the witwatersrand with particular reference to pulmonary manifestations immunological factors in the pathogenesis of the hyaline tissue of silicosis mini-review -immunologic aspects of pneumoconiosis adverse effects of crystalline silica exposure chronic necrotizing pulmonary aspergillosis in pneumoconiosis -clinical and radiologic findings in 10 patients silica and some silicates. monographs on the evaluation of the carcinogenic risk of chemicals to humans occupational lung disease: 1. exposure to crystalline silica and risk of lung cancer: the epidemiological evidence do silica and asbestos cause lung cancer? report by the industrial injuries advisory council in accordance with section 171 of the social security administration act 1992 on the question whether lung cancer in relation to occupational exposure to silica should be prescribed. london: hmso; 1992 crystalline silica exposure, radiological silicosis, and lung cancer mortality in diatomaceous earth industry workers acute silicoproteinosis acute silicolipoproteinosis atypical reaction to inhaled silica alveolar proteinosis. its experimental production in rodents experimental endogenous lipid pneumonia and silicosis pathogenesis of experimental pulmonary alveolar proteinosis pneumoconiosis in carbon electrode makers graphite pneumoconiosis of electrotypers case report: a case of wood-smoke-related pulmonary disease hut lung. a domestically acquired particulate lung disease bronchial stenosis due to anthracofibrosis anthracofibrosis attributed to mixed mineral dust exposure: report of three cases anthracofibrosis, bronchial stenosis with overlying anthracotic mucosa: possibly a new occupational lung disorder occupational anthracofibrosis pneumoconiosis and other causes of death in iron and steel foundry workers histochemical study of certain iron ore dusts mixed-dust pneumoconiosis proposed criteria for mixed-dust pneumoconiosis: definition, descriptions, and guidelines for pathologic diagnosis and clinical correlation chronic interstitial pneumonia in silicosis and mix-dust pneumoconiosis: its prevalence and comparison of ct findings with idiopathic pulmonary fibrosis proposed criteria for mixed-dust pneumoconiosis: definition, descriptions, and guidelines for pathologic diagnosis and clinical correlation coal pneumoconiosis coal workers' pneumoconiosis pneumoconiosis in coal trimmers dust exposure, dust recovered from the lung, and associated pathology in a group of british coalminers the relationship between the mass and composition of coal mine dust and the development of pneumoconiosis interstitial fibrosis in coal miners -experience in wales and west virginia coal worker's pneumoconiosis: ct assessment in exposed workers and correlation with radiographic findings variations in the histological patterns of the lesions of coal workers' pneumoconiosis in britain and their relationship to lung dust content comparison of radiographic appearances with associated pathology and lung dust content in a group of coalworkers small airways disease and mineral dust exposure. prevalence, structure and function coal mining, emphysema, and compensation revisited coal mining, emphysema, and compensation revisited -reply coal mining and chronic obstructive pulmonary disease: a review of the evidence the pathogenesis of simple pneumokoniosis in coal workers mineral dusts cause elastin and collagen breakdown in the rat lung: a potential mechanism of dust-induced emphysema emphysema in coal workers' pneumoconiosis pulmonary disability in coal workers' pneumoconiosis quantified pathology of emphysema, pneumoconiosis, and chronic bronchitis in coal workers emphysema and dust exposure in a group of coal workers coal worker's lung: not only black, but also full of holes contributions of dust exposure and cigarette smoking to emphysema severity in coal miners in the united states report by the industrial injuries advisory council in accordance with section 171 of the social security administration act 1992 on the question whether bronchitis and emphysema in coal miners and metal production workers should be prescribed. london: hmso; 1992 the new prescription: industrial injuries benefits for smokers? exposure to respirable coalmine dust and incidence of progressive massive fibrosis the attack rate of progressive massive fibrosis ischemic necrosis in anthracosilicosis the composition of massive lesions in coal miners mechanisms of fibrosis in coal workers' pneumoconiosis: increased production of platelet-derived growth factor, insulin-like growth factor type i, and transforming growth factor beta and relationship to disease severity secretion and mrna expression of tnf alpha and il-6 in the lungs of pneumoconiosis patients a broader concept of caplan's syndrome related to rheumatoid factors differential agglutination test in rheumatoid arthritis complicated by pneumoconiosis circulating antinuclear antibody and rheumatoid factor in coal pneumoconiosis serologic changes in pneumoconiosis and progressive massive fibrosis of coal workers immunological investigations of coalworkers' disease certain unusual radiological appearances in the chest of coal-miners suffering from rheumatoid arthritis pathological studies of modified pneumoconiosis in coal-miners with rheumatoid arthritis (caplan's syndrome) asbestos, asbestosis, and cancer: the helsinki criteria for diagnosis and attribution report of the asbestosis committee of the college of american pathologists and pulmonary pathology society the asbestos cancer epidemic rapid short-term clearance of chrysotile compared with amosite asbestos in the guinea pig persistence of natural mineral fibers in human lungs: an overview a pathological and mineralogical study of asbestos-related deaths in the united kingdom in 1977 correlation between fibre content of the lungs and disease in naval dockyard workers correlation between fibre content of the lung and disease in east london asbestos factory workers the effects of the inhalation of asbestos in rats mass and number of fibres in the pathogenesis of asbestos related lung disease in rats fiber burden and patterns of asbestos-related disease in chrysotile miners and millers carcinogenesis and mineral fibres lung diseases due to environmental exposures to erionite and asbestos in turkey prospective study of mesothelioma mortality in turkish villages with exposure to fibrous zeolite analysis of ferruginous bodies in bronchoalveolar lavage from foundry workers analysis of the cores of ferruginous (asbestos) bodies from the general population fiber counting and analysis in the diagnosis of asbestos-related disease concentrations and dimensions of coated and uncoated asbestos fibres in the human lung comparison of the results of asbestos fibre dust counts in lung tissue obtained by analytical electron microscopy and light microscopy asbestos bodies and the diagnosis of asbestosis in chrysotile workers asbestos fiber content of lungs with diffuse interstitial fibrosis: an analytical scanning electron microscopic analysis of 249 cases comparison of phagocytosis of uncoated versus coated asbestos fibers by cultured human pulmonary alveolar macrophages numbers of asbestos bodies on iron-stained tissue sections in relation to asbestos body counts in lung tissue digests asbestos content of lung tissue in asbestos associated diseases: a study of 110 cases analysis and interpretation of inorganic mineral particles in 'lung' tissues asbestos bodies in bronchoalveolar lavage in relation to asbestos bodies and asbestos fibres in lung parenchyma asbestos bodies in the sputum of asbestos workers: correlation with occupational exposure the optical and electron microscopic determination of pulmonary asbestos fibre concentration and its relation to human pathology reaction assessment of mineral fibres from human lung tissue detection of asbestos fibres by dark ground microscopy analysis of lung asbestos content guidelines for mineral fibre analyses in biological samples: report of the ers working group relationship between occupations and asbestos-fibre content of the lungs in patients with pleural mesothelioma, lung cancer, and other diseases inflammation-generating potential of long and short-fibre amosite asbestos samples asbestos-stimulated tumour necrosis factor release from alveolar macrophages depends on fibre length and opsonization the pathogenicity of long versus short fibre amosite asbestos administered to rats by inhalation and intra-peritoneal injection cytogenetic and pathogenic effects of long and short amosite asbestos exposure and mineralogical correlates of pulmonary fibrosis in chrysotile asbestos workers quantitative assessment of inorganic fibrous particulates in dust samples with an analytical transmission electron microscope fibre distribution in the lungs and pleura of subjects with asbestos related diffuse pleural fibrosis malignant mesothelioma in women observations on the distribution of asbestos fibres in human lungs analysis of asbestos fibres and asbestos bodies in tissue samples from human lung: an international interlaboratory trial distribution and characteristics of amphibole asbestos fibres in the left lung of an insulation worker measured with the light microscope the identification of asbestos dust with an electron microprobe analyser symposium on man-made mineral fibres mortality in cases of asbestosis diagnosed by a pneumoconiosis medical panel small-airway lesions in patients exposed to nonasbestos mineral dusts small airways disease and mineral dust exposure relation of smoking and age to findings in lung parenchyma: a microscopic study electron microscopic studies in desquamative interstitial pneumonia associated with asbestos desquamative interstitial pneumonia associated with chrysotile asbestos fibres localized inflammatory pulmonary disease in subjects occupationally exposed to asbestos criteria for the diagnosis and grading of asbestosis report of the pneumoconiosis committee of the college of american pathologists and the national institute for occupational safety and health cytoplasmic hyalin in asbestosis further observations on cytoplasmic hyaline in the lung lung pathology of severe acute respiratory syndrome (sars): a study of 8 autopsy cases from singapore lung pathology of fatal severe acute respiratory syndrome immunohistochemical detection of ubiquitin-positive intracytoplasmic eosinophilic inclusion bodies in diffuse alveolar damage idiopathic pulmonary fibrosis in asbestosexposed workers mortality of workers certified by pneumoconiosis medical panels as having asbestosis radiographic evidence of asbestos effects in american marine engineers chronic airflow obstruction in lung disease. philadelphia: saunders fine structural changes in cryptogenic fibrosing alveolitis and asbestosis pulmonary asbestosis and idiopathic pulmonary fibrosis: pathogenetic parallels crocidoliteinduced pulmonary fibrosis in mice hydroxyl radicals are formed in the rat lung after asbestos instillation invivo asbestos fibers and interferon-gamma up-regulate nitric oxide production in rat alveolar macrophages oxidant sand antioxidant mechanisms of lung disease caused by asbestos fibres antibodies in some chronic fibrosing lung diseases asbestos causes translocation of p65 protein and increases nf-kappa b dna binding activity in rat lung epithelial and pleural mesothelial cells rapid activation of pdgf-a and -b expression at sites of lung injury in asbestos-exposed rats upregulation of the pdgf-alpha receptor precedes asbestos-induced lung fibrosis in rats tnf-alpha receptor knockout mice are protected from the fibroproliferative effects of inhaled asbestos fibers alveolar cell stretching in the presence of fibrous particles induces interleukin-8 responses the molecular basis of asbestos induced lung injury inhaled asbestos activates a complementdependent chemoattractant for macrophages alveolar macrophage stimulation of lung fibroblast growth in asbestos-induced pulmonary fibrosis cellular and molecular basis of the asbestos-related diseases up-regulated expression of transforming growth factor-α in the bronchiolar-alveolar duct regions of asbestos-exposed rats a study of macrophage-mediated initiation of fibrosis by asbestos and silica using a diffusion chamber technique enhanced generation of free radicals from phagocytes induced by mineral dusts mortality from lung cancer in asbestos workers diffuse pleural mesotheliomas and asbestos exposure in northwestern cape province carcinogenicity of amosite asbestos asbestos exposure, cigarette smoking and death rates smoking, exposure to crocidolite, and the incidence of lung cancer and asbestosis pathology of carcinoma of the lung associated with asbestos exposure a study of the histological cell types of lung cancer in workers suffering from asbestosis in the united kingdom lung cancer cell type and asbestos exposure histological type of lung carcinoma in asbestos cement workers and matched controls incidence of lung cancer by histological type among asbestos cement workers in denmark lung cancer in asbestos cement workers in denmark asbestos and lung cancer in glasgow and the west of scotland exposure to crocidolite and the incidence of different histological types of lung cancer asbestos and lung cancer asbestos and lung cancer: is it attributable to asbestosis or to asbestos fibre burden? in: corrin b, editor. pathology of lung tumours asbestos fibreyears and lung cancer: a two phase case-control study with expert exposure assessment the quantitative risks of mesothelioma and lung cancer in relation to asbestos exposure is asbestos or asbestosis the cause of the increased risk of lung cancer in asbestos workers? relation between asbestosis and bronchial cancer in amphibole asbestos miners asbestosis as a precursor of asbestos related lung cancer -results of a prospective mortality study asbestos exposure, asbestosis, and asbestosattributable lung cancer asbestos and lung cancer -reply criteria for attributing lung cancer to asbestos exposure asbestosis: a marker for the increased risk of lung cancer among workers exposed to asbestos asbestos, asbestosis, and lung cancer: a critical assessment of the epidemiological evidence airway function in lifetime-nonsmoking older asbestos workers severe diffuse small airways abnormalities in long term chrysotile asbestos miners morphology of small-airway lesions in patients with asbestos exposure does aluminum smelting cause lung disease? the treatment of silicosis by aluminum powder silicose aigue. caractéristiques cliniques, radiologiques, fonctionnelles et cytologiques du liquide bronchoalveolaire. a propos de 6 observations pathologie and pathogenesis der aluminiumlunge die aluminiumlunge -eine neue gewerbeerkrankung. z f d ges pulmonary fibrosis in workers exposed to finely powdered aluminium aluminium pneumoconiosis i. in vitro comparison of stamped aluminium powders containing different lubricating agents and a granular aluminium powder effect on the rat lung of intratracheal injections of stamped aluminium powders containing different lubricating agents and of a granular aluminium powder desquamative interstitial pneumonia in an aluminum welder sarcoidlike lung granulomatosis induced by aluminum dusts aluminum induced pulmonary granulomatosis aluminum welding fume-induced pneumoconiosis rare earth (cerium) pneumoconiosis rare earth (cerium oxide) pneumoconiosis: analytical scanning electron microscopy and literature review hard metal disease interstitial lung disease and asthma in hard-metal workers: bronchoalveolar lavage, ultrastructural, and analytical findings and results of bronchial provocation tests the biological action of tungsten carbide and cobalt: studies on experimental pulmonary histopathology cobalt lung in diamond polishers rapidly fatal progression of cobalt lung in a diamond polisher hard metal pneumoconiosis and the association of tumor necrosis factor-alpha the pulmonary toxicity of beryllium beryllium and lung disease recent chronic beryllium disease in residents surrounding a beryllium facility nonoccupational beryllium disease masquerading as sarcoidosis -identification by blood lymphocyte proliferative response to beryllium beryllium disease international agency for research on cancer. an evaluation of carcinogenic risk to humans. overall evaluation of carcinogenicity: an updating of iarc monographs vols 1-42 uber eine apparatur zur erzeugung niedriger staubkonzentrationen von grosser konstanz und eine methode zur mikrogravinctrischen staubbestemmung. anwendung bei der untersuchung von stauben aus der berylliumsgewinnung delayed chenical pneumonitis occurring in workers exposed to beryllium compounds chronic beryllium disease in a dental laboratory technician maintenance of alveolitis in patients with chronic beryllium disease by berylliumspecific helper t cells hla-dpb1 glutamate 69: a genetic marker of beryllium disease immunogenetic basis of environmental lung disease: lessons from the berylliosis model beryllium disease in identical twins beryllium detection in human lung tissue using electron probe x-ray microanalysis diagnostic criteria for chronic beryllium disease (cbd) based on the uk registry 1945-1991 united kingdom beryllium registry: mortality and autopsy study lung disease and the lightest of metals misdiagnosis of sarcoidosis in patients with chronic beryllium disease diagnoses of chronic beryllium disease within cohorts of sarcoidosis patients the pathology of interstitial lung disease in nylon flock workers flock worker's lung: broadening the spectrum of clinicopathology, narrowing the spectrum of suspected etiologies polyethylene flock-associated interstitial lung disease in a spanish female popcorn workers' lung clinical bronchiolitis obliterans in workers at a microwave-popcorn plant clinical bronchiolitis obliterans in workers at a microwave-popcorn plant bronchiolitis obliterans syndrome in popcorn production plant workers popcorn worker's lung' in britain in a man making potato crisp flavouring toxicity in textile airbrushing in spain outbreak of organising pneumonia in textile printing sprayers epidemic outbreak of interstitial lung disease in aerographics textile workersthe 'ardystil syndrome': a first year follow up organizing pneumonia in textile printing workers: a clinical description outbreak of pulmonary disease in textile dye sprayers in algeria five-year follow-up of algerian victims of the ''ardystil syndrome mineral oils and petroleum 357. skorodin ms, chandrasekhar aj. an occupational cause of exogenous lipoid pneumonia machine operator's lung. a hypersensitivity pneumonitis disorder associated with exposure to metalworking fluid aerosols metalworking fluid-associated hypersensitivity pneumonitis: a workshop summary an outbreak of hypersensitivity pneumonitis at a metalworking plant: a longitudinal assessment of intervention effectiveness stenius-aarniala b. fire-eater's lung fire-eater's lung: seventeen cases and a review of the literature pulmonary mechanics in dogs given different doses of kerosene intratracheally x-ray appearance of the lungs of electric arc welders the health of welders in naval dockyards: the risk of asbestosrelated diseases occurring in welders welders' pneumoconiosis: tissue elemental microanalysis by energy dispersive x ray analysis the respiratory health of welders toxic fumes and gases 368. nemery b. metal toxicity and the respiratory tract lung changes associated with the manufacture of alumina abrasives acute inorganic mercury vapor inhalation poisoning nickel poisoning ii. studies on patients suffering from acute exposure to vapors of nickel carbonyl silo-filler's disease -a syndrome caused by nitrogen dioxide silo-filler's disease: nitrogen dioxide-induced lung injury silo filler's disease silo-filler's disease in new york state dung lung: a report of toxic exposure to liquid manure death caused by fermenting manure acute toxic exposure to gases from liquid manure slurry lung': a report of three cases pulmonary lesions in rats exposed to ozone pathology of adult respiratory distress syndrome response of macaque bronchiolar epithelium to ambient concentrations of ozone ultrastructural alterations of alveolar tissue of mice differentiated bronchiolar epithelium in alveolar ducts of rats exposed to ozone for 20 months ozone-induced acute tracheobronchial epithelial injury -relationship to granulocyte emigration in the lung ozone-induced airway inflammation in human subjects as determined by airway lavage and biopsy a cohort study of workers exposed to formaldehyde in the british chemical industry -an update pulmonary toxicity following exposure to methylene chloride and its combustion product, phosgene the diversity of the effects of sulfur mustard gas inhalation on respiratory system 10 years after a single, heavy exposure: analysis of 197 cases tracheobronchomalacia and air trapping after mustard gas exposure an international collaborative pathologic study of surgical lung biopsies from mustard gas-exposed patients thionyl-chloride-induced lung injury and bronchiolitis obliterans hydrogen sulfide poisoning: review of 5 years' experience anoxic asphyxia anoxic asphyxia -a cause of industrial fatalities: a review sword '94: surveillance of work-related and occupational respiratory disease in the uk occupational asthma as identified by the surveillance of work-related and occupational respiratory diseases programme in south africa incidence of occupational asthma by occupation and industry in finland sword '93: surveillance of work-related and occupational respiratory disease in the uk american thoracic society statement: occupational contribution to the burden of airway disease aetiological agents in occupational asthma reported incidence of occupational asthma in the united kingdom, 1989-97 occupational asthma in a factory with a contaminated humidifier the pathology of byssinosis byssinosis: a review in vitro alternative and classical activation of complement by extracts of cotton mill dust: a possible mechanism in the pathogenesis of byssinosis antibody independent complement activation by card-room cotton dust occupational fevers 407. raskandersen a, pratt ds. inhalation fever -a proposed unifying term for febrile reactions to inhalation of noxious substances humidifier fever pulmonary mycotoxicosis organic dust toxicity (pulmonary mycotoxicosis) associated with silo unloading desquamative interstitial pneumonitis and diffuse alveolar damage in textile workers: potential role of mycotoxins pulmonary mycotoxicosis: a clinicopathologic study of three cases pulmonary involvement in zinc fume fever cytokines in metal fume fever music: a new cause of primary spontaneous pneumothorax atmospheric pressure influences the risk of pneumothorax: beware of the storm experimental study of blast injuries to the lungs biophysics of impact injury to the chest and abdomen histologic, immunohistochemical, and ultrastructural findings in human blast lung injury histologic, immunohistochemical, and ultrastructural findings in human blast lung injury the ultrastructure of rat lung following acute primary blast injury disorders associated with diving decompression disorders in divers intrapulmonary air trapping in submarine escape training casualties airway structure and alveolar emptying in the lungs of sea lions and dogs the structure and function of the small airways in pinniped and sea otter lungs mechanisms of death in shallow-water scuba diving pulmonary barotrauma in submarine escape training the pathophysiology of decompression sickness hemodynamic changes induced by recreational scuba diving dysbaric osteonecrosis: aseptic necrosis of bone deep diving mammals: dive behavior and circulatory adjustments contribute to bends avoidance current concepts: high-altitude illness illnesses at high altitude high altitude pulmonary edema high altitude pulmonary oedema; characteristics of lung lavage fluid prevention of high-altitude pulmonary edema by nifedipine pulmonary circulation at high altitude association of high-altitude pulmonary edema with the major histocompatibility complex pathogenesis of high-altitude pulmonary oedema: direct evidence of stress failure of pulmonary capillaries high-altitude pulmonary edema: current concepts high-altitude pulmonary edema is initially caused by an increase in capillary pressure when lungs on mountains leak -studying pulmonary edema at high altitudes inhaled nitric oxide for high-altitude pulmonary edema lung pathology in high-altitude pulmonary edema pathological features of the lung in fatal high altitude pulmonary edema occurring at moderate altitude in japan lack of smooth muscle in the small pulmonary arteries of the native ladakhi -is the himalayan highlander adapted? aspects of hypoxia pulmonary blood vessels and endocrine cells in subacute infantile mountain sickness the human pulmonary circulation: its form and function in health and disease pulmonary edema associated with salt water near-drowning -new insights the effect of salt water on alveolar epithelial barrier function near-drowning: clinical course of lung injury in adults haggard hw. action of irritant gases upon the respiratory tract regional respiratory tract absorption of inhaled reactive gases respiratory irritants encountered at work health effects of outdoor air pollution committee of the environmental and occupational health assembly of the committee of the environmental and occupational health assembly of the fine particulate air pollution and mortality in 20 us cities, 1987-1994 health effects of diesel exhaust emissions respiratory changes due to long-term exposure to urban levels of air pollution: a histopathologic study in humans chronic glandular bronchitis in young individuals residing in a metropolitan area an association between air pollution and mortality in 6 united states cities chronic bronchitis in women using solid biomass fuel in rural peshawar indoor air pollution: a poverty-related cause of mortality among the children of the world risk of copd from exposure to biomass smoke human health impacts of volcanic activity persistent hyperreactivity and reactive airway dysfunction in firefighters at the world trade center the world trade center residents' respiratory health study: new-onset respiratory symptoms and pulmonary function pulmonary function after exposure to the world trade center collapse in the new york city fire department characterization of the dust/smoke aerosol that settled east of the world trade center (wtc) in lower manhattan after the collapse of the wtc 11 chemical analysis of world trade center fine particulate matter for use in toxicologic assessment longitudinal assessment of spirometry in the world trade center medical monitoring program acute eosinophilic pneumonia in a new york city firefighter exposed to world trade center dust granulomatous pneumonitis following exposure to the world trade center collapse smoking-related interstitial lung diseases: a concise review concomitant upper-lobe bullous emphysema, lower-lobe interstitial fibrosis and pulmonary hypertension in heavy smokers: report of eight cases and review of the literature heterogeneity in combined pulmonary fibrosis and emphysema smoking-related interstitial lung diseases challenges in pulmonary fibrosis 4: smoking-induced diffuse interstitial lung diseases combined pulmonary fibrosis and emphysema: a distinct underrecognised entity smoking-related changes in the background lung of specimens resected for lung cancer: a semiquantitative study with correlation to postoperative course clinical assembly contribution to the celebration of 20 years of the ers. smoking-related lung diseases: a clinical perspective clinically occult interstitial fibrosis in smokers: classification and significance of a surprisingly common finding in lobectomy specimens bronchiolar inflammation and fibrosis associated with smoking -a morphologic cross-sectional population analysis relation of smoking and age to findings in lung parenchyma: a microscopic study variability in small airway epithelial gene expression among normal smokers airway alveolar attachment points and exposure to cigarette smoke in utero patterns of global tobacco use in young people and implications for future chronic disease burden in adults tobacco smoking using a waterpipe: a re-emerging strain in a global epidemic pathology of the lung in fatally burned patients respiratory tract pathology in patients with severe burns heat injuries to the respiratory system death in the burn unit: sterile multiple organ failure pulmonary interstitial edema and hyaline membranes in adult burn patients herpesvirus infections in burn patients pulmonary herpes simplex in burns patients long-term course of bronchiectasis and bronchiolitis obliterans as late complication of smoke inhalation disaster at bhopal: the accident, early findings and respiratory health outlook in those injured chronic lung inflammation in victims of toxic gas leak at bhopal respiratory morbidity 10 years after the union carbide gas leak at bhopal: a cross sectional survey management of the effects of exposure to tear gas ingested toxic agents 88. boyd mr. evidence for the clara cell as a site of cytochrome p450-dependent mixed-function oxidase activity in lung pulmonary histological appearances in fatal paraquat poisoning pulmonary damage due to paraquat poisoning through skin absorption paraquat induced pulmonary fibrosis in three survivors kinetics and cellular localisation of putrescine uptake in human lung tissue enhancement of oxygen toxicity by the herbicide paraquat hypoxic protection in paraquat poisoning the effects of variable o 2 tension and of exogenous superoxide dismutase on type ii pneumocytes exposed to paraquat paraquat-induced injury of type ii alveolar cells the changing pattern of paraquat poisoning in man remodeling of the alveolar structure in the paraquat lung of humans: a morphometric study experimental paraquat poisoning the pathogenesis and structure of paraquatinduced pulmonary fibrosis in rats pulmonary ultrastructure after oral and intravenous dosage of paraquat to rats patterns of pulmonary structural remodeling after experimental paraquat toxicity experimental models of interstitial pneumonia: paraquat, iprindole fine structure of the lung lesion in a case of paraquat poisoning fatal pulmonary intra-alveolar fibrosis after paraquat ingestion paraquat lung: a reappraisal an immunohistochemical study of the fibrosing process in paraquat lung injury toxic-allergic syndrome caused by ingestion of rapeseed oil denatured with aniline pathology of a new toxic syndrome caused by ingestion of adulterated oil in spain clinical epidemiology of toxic oil syndrome 3-(phenylamino)alanine, a novel aniline-derived amino acid associated with the eosinophilia-myalgia syndrome -a link to the toxic oil syndrome pulmonary hypertension due to toxic oil syndrome. a clinicopathologic study pulmonary hypertension in patients with eosinophilia-myalgia syndrome or toxic oil syndrome pulmonary vascular lesions in the toxic oil syndrome in spain outbreak of bronchiolitis obliterans associated with consumption of sauropus androgynus in taiwan histopathological study of sauropus androgynus-associated constrictive bronchiolitis obliterans: a new cause of constrictive bronchiolitis obliterans sauropus androgynus-constrictive obliterative bronchitis/bronchiolitishistopathological study of pneumonectomy and biopsy specimens with emphasis on the inflammatory process and disease progression segmental necrosis of small bronchi after prolonged intakes of sauropus androgynus in taiwan sauropus bronchiolitis -reply the lipoid pneumonia of blackfat tobacco smokers in guyana a morphometric analysis of the male and female tracheal epithelium after experimental exposure to marijuana smoke differential examination of bronchoalveolar lavage cells in tobacco cigarette and marijuana smokers tracheobronchial changes in habitual, heavy smokers of marijuana with and without tobacco airway inflammation in young marijuana and tobacco smokers an unusual cause of patchy ground-glass opacity pulmonary hazards of smoking marijuana as compared with tobacco british lung foundation. a smoking gun? tracheobronchial histopathology in habitual smokers of cocaine, marijuana, and/or tobacco effects of cannabis on pulmonary structure, function and symptoms large lung bullae in marijuana smokers the pulmonary pathology of illicit drug and substance abuse bong lung: regular smokers of cannabis show relatively distinctive histologic changes that predispose to pneumothorax cannabis use and risk of lung cancer: a case-control study exogenous lipid pneumonia related to smoking weed oil following cadaveric renal transplantation a review of the respiratory effects of smoking cocaine medical complications of cocaine abuse blackened bronchoalveolar lavage fluid in crack smokers -a preliminary study pulmonary histopathology in cocaine abusers pulmonary complications of crack cocaine: a comprehensive review cocaine-induced churg-strauss vasculitis pulmonary artery medial hypertrophy in cocaine users without foreign particle microembolization crack lung: an acute pulmonary syndrome with a spectrum of clinical and histopathologic findings effects of 'crack' cocaine on pulmonary alveolar permeability pulmonary hemorrhage and antiglomerular basement membrane antibody-mediated glomerulonephritis after exposure to smoked cocaine (crack): a case report and review of the literature cellulose granulomas in the lungs of a cocaine sniffer intravenous injection of talc-containing drugs intended for oral use. a cause of pulmonary granulomatosis and pulmonary hypertension autopsy findings in drug addicts pulmonary 'mainline' granulomatosis: talcosis of intravenous methadone abuse self induced pulmonary granulomatosis. a consequence of intravenous injection of drugs intended for oral use the pulmonary vascular lesions of intravenous drug abuse microcrystalline cellulose pulmonary embolism and granulomatosis talc granulomatosis: laboratory findings similar to sarcoidosis postmortem findings of pulmonary lesions of older datum in intravenous drug addicts. a forensic-pathologic study recreational use of aminorex and pulmonary hypertension fatal drug reactions among medical inpatients drug-related hospital admissions: a review of australian studies published 1988-1996 incidence of adverse drug reactions in hospitalized patients: a meta-analysis of prospective studies (see comments) the quality in australian health care study adverse reactions to drugs drug-induced pulmonary disease -an update an algorithm for the operational assessment of adverse drug reactions. ii. demonstration of reproducibility and validity aspirin idiosyncrasy and tolerance aspirin-sensitive asthma: abnormal platelet response to drugs inducing asthmatic attacks; diagnostic and physiopathological implications mechanism of aspirin sensitivity bronchiolitis and bronchitis in connective tissue disease. a possible relationship to the use of penicillamine bronchiolitis obliterans in a patient with localized scleroderma treated with d-penicillamine constrictive bronchiolitis obliterans following gold therapy for psoriatic arthritis gold-induced pulmonary disease: clinical features, outcome, and differentiation from rheumatoid lung disease progressive airway obliteration in adults and its association with rheumatoid disease drug-induced infiltrative lung disease drug-induced and iatrogenic infiltrative lung disease interstitial lung disease associated with drug therapy pulmonary toxic effects of continuous desferrioxamine administration in acute iron poisoning interstitial pulmonary fibrosis following busulfan therapy busulphan lung report of a case and review of the literature busulfan lung: report of two cases and review of the literature a 60-year-old man with pulmonary infiltrates after a bone marrow transplantation -busulfan pneumonitis drug-induced pulmonary disease lung parenchymal injury induced by bleomycin pathology of high dose intermittent cyclophosphamide therapy cyclophosphamide pneumonitis pulmonary histopathologic changes associated with melphalan therapy melphalan-induced pulmonary interstitial fibrosis potentiation of bleomycin-induced lung injury by exposure to 70% oxygen factors influencing postoperative morbidity and mortality in patients treated with bleomycin bleomycin therapy and anaesthesia risk factors of anesthesia and surgery in bleomycin-treated patients the pathogenesis of bleomycin-induced pulmonary damage in mice experimentally induced bleomycin sulfate toxicity origin and significance of intranuclear tubular inclusions in type ii pulmonary alveolar epithelial cells of patients with bleomycin and busulfan toxicity late bcnu lung -a light and ultrastructural study on the delayed effect of bcnu on the lung parenchyma statin-induced fibrotic nonspecific interstitial pneumonia bronchiolitis obliterans organizing pneumonia during treatment with acebutolol and amiodarone methotrexate-induced pneumonitis (baltimore) bronchoalveolar lavage findings suggest an immunologic disorder pneumonitis complicating low-dose methotrexate therapy for rheumatoid arthritis -discrepancies between lung biopsy and bronchoalveolar lavage findings methotrexate pneumonitis: review of the literature and histopathological findings in nine patients the pulmonary toxicity of antineoplastic agents relationship of gold and penicillamine therapy to diffuse interstitial lung disease desquamative interstitial pneumonia following long-term nitrofurantoin therapy two unusual pathological reactions to nitrofurantoin: case reports bronchiolitis obliterans organising pneumonia associated with the use of nitrofurantoin amiodarone lung toxicity: a human and experimental study effect of amiodarone on the lung shown by polarized light microscopy fine structural alterations in the lungs of iprindole-treated rats effects of chlorphentermine on the rat lung amiodarone-induced hypersensitivity pneumonitis. evidence of an immunological cell-mediated mechanism amiodarone pneumonitis: three further cases with a review of published reports amiodarone-associated pulmonary toxicity. a clinical and pathologic study of eleven cases amiodarone lung: pathologic findings in clinically toxic patients amiodarone-induced bronchiolitis obliterans organizing pneumonia (boop) amiodarone-related pulmonary mass and unique membranous glomerulonephritis in a patient with valvular heart disease: diagnostic pitfall and new findings nodular amiodarone lung disease amiodarone pneumonitis: no safe dose amiodarone pneumonitis: no safe dose susceptibility to amiodarone-induced pulmonary toxicity: relationship to the uptake of amiodarone by isolated lung cells acute amiodarone-induced pulmonary toxicity following lung resection pulmonary alveolar proteinosis developing from desquamative interstitial pneumonia in long term toxicity studies of iprindole in the rat eosinophilic lung diseases mesalazine-induced eosinophilic pneumonia chronic eosinophilic pneumonia after radiation therapy for breast cancer churg-strauss syndrome in patients receiving montelukast as treatment for asthma inhaled corticosteroids and churg-strauss syndrome: a report of five cases pulmonary migratory infiltrates and pachypleuritis in a patient with crohn's disease immunodeficiency virus-infected patients receiving antiretroviral therapy ciprofloxacin-induced acute interstitial pneumonitis pulmonary granulomas after tumour necrosis factor alpha antagonist therapy pulmonary sarcoidosis developing during infliximab therapy sarcoid-like granulomatous disease following etanercept treatment for ra pneumonia due to liquid paraffin: with chemical analysis a case of chronic paraffin pneumonitis liquid paraffin pneumonia -with chemical analysis and electron microscopy paraffinoma confirmed by infrared spectrophotometry foreign body granulomata of the lungs due to liquid paraffin exogenous lipoid pneumonia due to nasal application of petroleum jelly reaction of human lungs to aspirated animal fat (ghee): a clinicopathological study clinical reactions following bronchography the reaction of pulmonary tissue to lipiodol experimental study of bronchographic media on lung a method for the identification of lipiodol in tissue sections of lungs, lipids, and lollipops bronchial necrosis and granuloma induced by the aspiration of a tablet of ferrous sulphate syndrome of iron pill aspiration pulmonary disease associated with l-tryptophan-induced eosinophilic myalgia syndrome. clinical and pathologic features a case of the eosinophilia-myalgia syndrome associated with use of an l-tryptophan product an investigation of the cause of the eosinophilia-myalgia syndrome associated with tryptophan use l-tryptophan and the eosinophiliamyalgia syndrome: pathologic findings in eight patients histopathologic features of the l-tryptophan-related eosinophilia-myalgia (fasciitis) syndrome 3-(phenylamino)alanine, a novel aniline-derived amino acid associated with the eosinophilia-myalgia syndrome -a link to the toxic oil syndrome immunemediated mechanisms and immune activation of fibroblasts in the pathogenesis of eosinophilia-myalgia syndrome induced by l-tryptophan pulmonary hypertension in patients with eosinophilia-myalgia syndrome or toxic oil syndrome tryptophan-induced lung disease -an immunophenotypic, immunofluorescent, and electron microscopic study bronchiolitis obliterans organizing pneumonia associated with massive l-tryptophan ingestion pulmonary disease complicating intermittent therapy with methotrexate case records of the massachusetts general hospital. a 28-year-old man with increasing dyspnea, dry cough, and fever after chemotherapy for lymphoma interferon-alpha therapy associated with the development of sarcoidosis proliferation and differentiation in mammalian airway epithelium sarcoid-like pulmonary disorder in human review of the literature severe barium sulfate aspiration into the lung: clinical presentation, prognosis and therapy aminorex and the pulmonary circulation pulmonary hypertension and fenfluramine irreversible pulmonary hypertension after treatment with fenfluramine dietary pulmonary hypertension appetite-suppressant drugs and the risk of primary pulmonary hypertension fatal pulmonary hypertension associated with short-term use of fenfluramine and phentermine autopsy findings of heart and lungs in a patient with primary pulmonary hypertension associated with use of fenfluramine and phentermine pulmonary veno-occlusive disease following therapy for malignant neoplasms pulmonary veno-occlusive disease in an adult following bone marrow transplantation: case report and review of the literature indomethacin in the treatment of premature labor. effects on the fetal ductus arteriosus prenatal diagnosis of intrauterine premature closure of the ductus arteriosus following maternal diclofenac application roles and mechanisms of human immunodeficiency virus protease inhibitor ritonavir and other anti-human immunodeficiency virus drugs in endothelial dysfunction of porcine pulmonary arteries and human pulmonary artery endothelial cells pulmonary embolism after intravenous immunoglobulin fat embolism in infancy after intravenous fat infusions pulmonary fat accumulation after intralipid infusion in the preterm infant intralipid microemboli the pathogenesis of fat embolism pulmonary lipid emboli in association with long-term hyperalimentation the impact of intravenous fat emulsion administration in acute lung injury microvascular pulmonary emboli secondary to precipitated crystals in a patient receiving total parenteral nutrition -a case report and description of the high-resolution ct findings pulmonary complications following lymphangiography with a note on technique changes in pulmonary function due to lymphangiography pulmonary complications of lymphangiography respiratory distress syndrome from lymphangiography contrast medium spallation and migration of silicone from blood-pump tubing in patients on hemodialysis acute pneumonitis after subcutaneous injections of silicone in transsexual men pulmonary granulomas secondary to embolic prosthetic valve material pulmonary teflon granulomas following periurethral teflon injection for urinary incontinence acute pneumonitis after subcutaneous injections of silicone for augmentation mammaplasty a pathological study following bronchial artery embolization for haemoptysis in cystic fibrosis isobutyl-2-cyanoacrylate pulmonary emboli associated with occlusive embolotherapy of cerebral arteriovenous malformations hemorragie alveolaire diffuse secondaire a l'utilisation d'anticoagulants oraux diffuse alveolar hemorrhage with underlying pulmonary capillaritis in the retinoic acid syndrome intrapulmonary hemorrhage with anemia after lymphangiography alveolar hemorrhage as a complication of treatment with abciximab d-penicillamine induced goodpasture's syndrome in wilson's disease drugs and the pleura pleuropulmonary changes induced by ergoline drugs pleuropulmonary disease as a side-effect of treatment with bromcriptine pleuropulmonary disease due to pergolide use for restless legs syndrome fibrosis of the lung following roentgen-ray treatments for tumor radiation reaction in the lung radiation pneumonitis: experimental and pathologic observations radiation pneumonitis following combined modality therapy for lung cancer: analysis of prognostic factors the pathogenesis of radiationinduced lung damage radiation pneumonitis: a review adult respiratory distress syndrome after limited thoracic radiotherapy migratory bronchiolitis obliterans organizing pneumonia after unilateral radiation therapy for breast carcinoma migratory organizing pneumonitis 'primed' by radiation therapy hamman-rich syndrome 'primed' by radiation? recall' pneumonitis: adriamycin potentiation of radiation pneumonitis in two children recall lung pneumonitis due to carmustine after radiotherapy pulmonary radiation injury mortality from cancer and other causes after radiotherapy for ankylosing spondylitis increased risk of lung cancer after breast cancer radiation therapy in cigarette smokers aortotracheal fistula secondary to bacterial aortitis respirator lung -a misnomer pathology of adult respiratory distress syndrome pulmonary morphology in a multihospital collaborative extracorporeal membrane oxygenation project lung injury caused by mechanical ventilation ventilator-induced lung injury oxygen radicals mediate endothelial cell damage by complement-stimulated granulocytes in vitro damage of rat lungs by oxygen metabolites intercellular adhesion molecule-1 contributes to pulmonary oxygen toxicity in mice -role of leukocytes revised intercellular adhesion molecule-1 expression on the alveolar epithelium and its modification by hyperoxia normobaric oxygen toxicity of the lung oxygen pneumonitis in man pathology of pulmonary oxygen toxicity diffuse alveolar damage -the role of oxygen, shock and related factors diffuse interstitial pulmonary fibrosis. pulmonary fibrosis in mice induced by treatment with butylated hydroxytoluene and oxygen potentiation of diffuse lung damage by oxygen: determining values resistance and susceptibility to oxygen toxicity by cell types of the gas-blood barrier of the rat lung ultrastructural observations on the development of the alveolar lesions extracorporeal membrane oxygenation for adult respiratory failure pulmonary pathology of patients treated with partial liquid ventilation disruption of the cd40-cd40 ligand system prevents an oxygen-induced respiratory distress syndrome pathogenesis and reversibility of the pulmonary lesions of oxygen toxicity in monkeys. ii ultrastructural and morphometric studies diffuse alveolar damage, respiratory failure and blood transfusion pulmonary injury -secondary to extracorporeal circulation fine structural changes in the lungs following cardiopulmonary bypass complement and the damaging effects of cardiopulmonary bypass acute lung injury during cardiopulmonary bypass: are the neutrophils responsible? inflammatory response to cardiopulmonary bypass: mechanisms involved and possible therapeutic strategies the pleuropulmonary manifestations of the postcardiac injury syndrome the postmyocardial infarction syndrome. the nonspecificity of the pulmonary manifestations the postcardiac injury syndromes antiheart antibodies following open heart surgery: incidence and correlation with postpericardiotomy syndrome analysis of the factors associated with radiofrequency ablation-induced pneumothorax irreversible intrapulmonary vascular changes after pulmonary vein stenosis complicating catheter ablation for atrial fibrillation pulmonary venous stenosis after treatment for atrial fibrillation right sided infective endocarditis as a consequence of flow directed pulmonary artery catheterisation complications and consequences of endotracheal intubation and tracheotomy. a prospective study of 150 critically ill adult patients pathologic changes of the trachea after percutaneous dilatational tracheotomy pseudomonas aeruginosa respiratory tract infections in patients receiving mechanical ventilation cardiac arrhythmias resulting from tracheal suctioning obstructive fibrinous tracheal pseudomembrane -a potentially fatal complication of tracheal intubation 12-h pretreatment with methylprednisolone versus placebo for prevention of postextubation laryngeal oedema: a randomised double-blind trial tracheobronchomalacia in children necrotizing sialometaplasia (adenometaplasia) of the trachea severe complications of bronchoscopy the postpneumonectomy state evaluation of post-pneumonectomy space by computed tomography the postpneumonectomy space: factors influencing its obliteration hepatocyte growth factor stimulates proliferation of respiratory epithelial cells during postpneumonectomy compensatory lung growth in mice severe airway obstruction caused by mediastinal displacement after right pneumonectomy in a child. a case report postpneumonectomy syndrome: diagnosis, management, and results treatment of left pneumonectomy syndrome with an expandable endobronchial prosthesis postpneumonectomy syndrome: another twist acute lung injury and acute respiratory distress syndrome after pulmonary resection the mortality from acute respiratory distress syndrome after pulmonary resection is reducing: a 10-year single institutional experience prevalence and mortality of acute lung injury and ards after lung resection lung injury following pulmonary resection in the isolated, blood-perfused rat lung the pathogenesis of lung injury following pulmonary resection it is estimated that 5% of all hospital admissions are due to effects of therapeutic drugs, that 10-18% of inpatients experience a drug reaction and that 3% of deaths in hospital may be related to drug therapy. [1] [2] [3] [4] the lungs are often involved in these adverse reactions. the mechanism of an adverse drug reaction may be based on:• overdosage: toxicity linked to excess dose, or impaired excretion, one classification of adverse drug reactions is that based upon the type of drug (box 7.3.1). 6 this is not adopted here but in passing it is worth noting that pharmacists are generally very helpful in supplying details of adverse reactions to specific drugs. alternatively, information on the long list of potentially pneumotoxic drugs may be obtained at http://www.pneumotox.com. a useful scheme for chapter key: cord-255166-sar50ej0 authors: stone, sophia; nelson-piercy, catherine title: respiratory disease in pregnancy date: 2007-05-07 journal: obstet gynaecol reprod med doi: 10.1016/j.ogrm.2007.03.006 sha: doc_id: 255166 cord_uid: sar50ej0 breathlessness in the absence of an underlying pathology is common in pregnancy. asthma affects about 7% of women of childbearing age. treatment is the same as for the non-pregnant population and most drugs are safe in pregnancy. educating women to continue preventer inhaled corticosteroid therapy will reduce the risk of attacks. respiratory infections are associated with a higher morbidity in pregnancy and should be treated aggressively. most chronic pulmonary diseases do not alter fertility. large reserves in respiratory function allow the fetus and mother to survive without compromise in most cases. the use of chest x-rays should not be avoided in pregnancy. women with a chronic respiratory disease should receive pre-pregnancy counselling and education. women should be managed in a multidisciplinary setting with the respiratory team. the presence of pulmonary hypertension and cor pulmonale is associated with a high risk of death in pregnancy. severe chest disease leading to respiratory failure is uncommon in women of childbearing age but respiratory symptoms are extremely common in pregnancy. this is in part an effect of the physiological adaptation of the respiratory and vascular systems to pregnancy and in part because of limited functional capacity and mobility from diaphragmatic elevation by the gravid uterus in late gestation. oxygen consumption is increased by 20% from early pregnancy, minute ventilation by 40-50% secondary to tidal volume increase, and maternal hyperventilation results in a mild fully compensated respiratory alkalosis (increased arterial po 2 and decreased arterial pco 2 , compensatory fall in serum bicarbonate to 18-22 mmol/l; arterial ph 7.44). the commonest respiratory symptom in pregnancy is breathlessness. although not the subject of this article, pulmonary embolus and cardiac causes are important differential diagnoses, but it is most commonly the increased awareness of the physiological hyperventilation of pregnancy which leads to a subjective feeling of breathlessness. women most often present in the third trimester but may become symptomatic at any gestation. classically physiological breathlessness of pregnancy is present at rest or while talking and paradoxically improves with activity. table 1 summarises other causes. other presenting symptoms of respiratory disease include chest pain, cough, sputum production, haemoptysis, fever or cyanosis. on examination, respiratory rate is unchanged by pregnancy. presence of raised temperature should be noted. expansion on inspiration reflects tidal volume and is reduced in many respiratory diseases. percussion note may be dull in the presence of pleural effusion, consolidation, collapse or fibrosis. it is enhanced in the presence of a pneumothorax. auscultation may reveal wheezes indicative of asthma, fine crepitations in the presence of pulmonary oedema and pleural rubs indicative of inflammatory conditions of the pleura in pneumonia or pulmonary infarction; and bronchial breathing+coarse crackles suggest consolidation. absent breath sounds occur with pneumothorax or extensive collapse. a simple non-invasive investigation is transcutaneous oxygen saturation. the normal oxygen saturation is 495%. a fall in saturation on exercise, e.g. climbing stairs, indicates some form of cardiopulmonary disease and should be investigated further. measurement of arterial blood gases should be reserved for those who are markedly breathless, those whose oxygen saturations are low at rest or which drop on exercise, and those who appear unwell. reluctance to perform a chest x-ray in pregnancy may delay a diagnosis. ionising radiation from a chest x-ray is approximately 0.2 rad (o5% of the maximum recommended exposure in pregnancy) and abdominal shielding will reduce fetal exposure further. sputum should be sent for microbiological examination. asthma is a common chronic inflammatory condition of the lung airways characterised by episodes of reversible bronchoconstriction as a result of various stimuli. it affects up to 7% of women of childbearing age. it may present with episodes of wheeze, shortness of breath, chest tightness or cough. signs classically include a diffuse, bilateral, expiratory (7inspiratory) wheeze and tachypnoea but are often absent. there may be an associated personal or family history of atopy and recognised triggers include pollen, dust, animals and infections. objectively the diagnosis may be confirmed by measuring the peak expiratory flow rate (pefr) or forced expiratory volume in 1 s (fev 1 ) as an indication of the degree of bronchoconstriction. a 420% diurnal variation on x3 days in a week for 2 weeks on pef diary or fev 1 x15% increase after a trial of a b 2 agonist or steroid tablets or a similar decrease after 6 minutes of exercise are diagnostic. pefr and fev 1 are unaffected by pregnancy. atopy is often associated with asthma. it has no effect on pregnancy and drugs used in the treatment of hayfever (the antihistamines most commonly used with best safety profile are chlorpheniramine and intranasal beclomethasone) may be safely prescribed. effect of asthma on pregnancy: although a number of small studies have suggested an association of asthma with the development of pre-eclampsia, intrauterine growth retardation (iugr), preterm birth and low birthweights, most pregnancies are unaffected by the effects of asthma. severe poorly controlled asthma resulting in episodes of maternal hypoxaemia could, however, give rise to such complications. it is therefore of concern that many women stop their medication at the start of the pregnancy because of concerns regarding the safety profile of these drugs for the fetus. in a recent us study, between 5 and 13 weeks' gestation there was a 23% reduction in use of inhaled corticosteroids, 13% reduction in short-acting b 2 -agonists and a 54% reduction in rescue steroids. effect of pregnancy on asthma: asthma can worsen, improve or remain unchanged in pregnancy. endogenous steroids in labour ensure that acute asthma attacks are very uncommon during labour and delivery. there may be a deterioration postpartum. management: pregnancy is an ideal opportunity to optimise therapy. ideally, this should occur pre-pregnancy. a pre-conceptual visit would allow time to optimise therapy and educate women regarding the importance and safety of continuing their medication to ensure good asthma control throughout pregnancy. inhaler techniques should be checked and home peak flow monitoring encouraged. pregnant women should be monitored closely so that any change in course can be matched by an appropriate change in therapy. smoking should be discouraged. drug therapy in pregnancy is essentially the same as for the non-pregnant population and should follow the british thoracic society guidelines on the management of asthma. a stepwise approach is recommended (figure 1 ). inhaled b 2 -agonists, inhaled and oral steroids, oral and intravenous theophyllines and intravenous magnesium sulphate should be used as normal. only leukotriene antagonists are not recommended because of limited data, although the guidelines suggest that it is safer to continue these in women who are already taking them and who are resistant to the other therapies. acute asthma attacks should be managed aggressively as an emergency in hospital. multidisciplinary care from obstetricians, respiratory physicians and, in severe cases, itu staff will ensure that appropriate treatment as for the non-pregnant patients is administered (table 2) . continuous oxygen to maintain saturation above 95% and continuous fetal monitoring should be instigated. a pefr of 33-50% best/predicted indicates acute severe asthma attack;o33% is life-threatening. other concerning signs are spo 2 o92%, po 2 o8 kpa with normal pco 2 , silent chest, cyanosis, feeble respiratory effort, hypotension, altered consciousness and exhaustion. if caesarean section is required, regional blockade is preferable to general anaesthesia in women with asthma and opiates should be avoided for pain relief, especially in the presence of an acute physiological diagnosis by exclusion and typical history anaemia full blood count asthma pefr -response to bronchodilators pulmonary embolus arterial blood gases (kpo 2 and kpco 2 )/ vq scan ctpa mitral stenosis, cardiomyopathy ecg, echocardiogram, chest x-ray chest x-ray, sputum culture, serology pneumothorax chest x-ray hyperventilation/ anxiety arterial blood gases (kpco 2 but not kpo 2 ) table 1 obstetrics, gynaecology and reproductive medicine 17:5 attack. prostaglandin e 2 does not cause bronchoconstriction and may used for induction of labour. prostaglandin f 2a should be used only in extreme circumstances in the management of postpartum haemorrhage and with caution since it may precipitate an acute attack. likewise aspirin and non-steroidal anti-inflammatory drugs should be avoided, although if aspirin is indicated for pre-eclampsia prophylaxis, and there is a history of aspirin exposure without problems, then it should be safe to use. encourage women to continue their regular medication in labour. those women taking 47.5 mg prednisolone tablets for 42 weeks prior to the onset of labour require parenteral hydrocortisone 100 mg 6-8 hourly during labour. ergometrine has been reported to cause bronchospasm but syntometrine routinely used for active management of the third stage does not. postpartum, breastfeeding may reduce the incidence of asthma in the child and should be encouraged. none of the medications for asthma is contraindicated in breastfeeding. pneumonia occurs in the pregnant population with a frequency equal to that in the general population. effects of pregnancy on pneumonia: pneumonia in pregnancy is often more virulent and mortality is higher than in the nonpregnant population. the spectrum of pathogens is similar to that in the non-pregnant population and the management does not differ. coexisting maternal disease, including asthma and anaemia, and immunosuppressive therapy increase the risk of contracting pneumonia. effect of pneumonia on pregnancy: severe pneumonia may precipitate preterm delivery and result in low birthweight infants. there is an increased risk of serious maternal complications, including respiratory failure. clinical features: women may present with cough, fever, rigors, breathlessness and pleuritic pain. signs include fever, purulent sputum, coarse crackles on auscultation and signs of consolidation. diagnosis may be confirmed with a chest x-ray. blood and sputum cultures should be taken. bacterial pneumonia is associated with a raised white blood cell count, although mycoplasma pneumonia is not. knowledge of the increased mycoplasma activity in the community during an epidemic period may help guide the clinician to the increased likelihood of mycoplasma infection. mycoplasma and chlamydial pneumonias are diagnosed on serological assays with complement fixation tests. management: supportive measures in the management of pregnant women with pneumonia include oxygen administration if there is evidence of hypoxia, and rehydration especially in the presence of fever. chest physiotherapy will help clear secretions and aid oxygenation. oral antibiotic therapy should be commenced for community acquired bacterial infections -oral amoxicillin (0.5-1 g t.d.s.) and clarithromycin (500 mg b.d.) are the current recommended strategies. for severe community acquired or hospital acquired infections intravenous cefuroxime and clarithromycin should be used. tetracyclines cause discolouration of the teeth in the fetus and should be avoided after 20 weeks' gestation. community-acquired pneumonia is most commonly caused by streptococcus pneumoniae, haemophilus influenzae and mycoplasma pneumoniae. b-lactam and macrolide antibiotics are safe in pregnancy and are effective in treating community acquired infections. viral pneumonia viral respiratory infections, including varicella, influenza a and severe acute respiratory syndrome (sars), may present in pregnancy and are associated with a worse outcome than in the non-pregnant population. current antiviral and respiratory therapies can reduce maternal morbidity and mortality. the royal college of obstetricians and gynaecologists' guidelines on the treatment of varicella infection in pregnancy recommend that all non-immune pregnant women exposed to varicella should be given zoster immunoglobulin (zig) as soon as possible. those who develop clinical varicella should be treated with aciclovir if within 24 hours of the onset of the rash and after 20 weeks' gestation since mortality and morbidity is higher at advanced gestations. hospital admission should be arranged in the event of respiratory deterioration, neurological symptoms, haemorrhagic rash and bleeding, or in the presence of a dense rash with mucosal lesions. delivery during a viraemic period is hazardous with maternal risks of bleeding, thrombocytopenia, disseminated intravascular coagulation and hepatitis. supportive treatment and intravenous aciclovir may be necessary. there is a high risk of varicella transmission to the newborn with significant morbidity and mortality. therefore, vzig should be given to the newborn after delivery (if maternal infection occurred within 5 days pre and 2 days post-delivery). if the neonate develops varicella, aciclovir should be commenced. influenza vaccination can reduce the prevalence of hospitalisations among pregnant women during the influenza season and is not contraindicated in pregnancy. give high flow oxygen b 2 -agonist bronchodilators high-dose inhaled b 2 -agonists by oxygen-driven nebuliser ipratropium bromide add nebulised ipratropium bromide (0.5 mg 4-6 hourly) if poor response to bronchodilators or severe acute asthma steroid therapy systemic steroids (40-50 mg/day) in all cases for at least 5 days or until recovery other therapies consider single dose intravenous magnesium sulphate (1.2-2 g infusion over 20 minutes) routine antibiotics are not recommended pneumocystis pneumonia in association with hiv pneumocystis pneumonia (pcp) is the most common opportunistic infection in patients progressing to acquired immunodeficiency syndrome (aids) and continues to carry significant maternal risk to the immunocompromised population. it should be suspected in the presence of profound hypoxia out of proportion to the chest x-ray findings, and bronchoscopy should be considered. treatment is with high-dose co-trimoxazole (septrin)7pentamidine, usually contraindicated in pregnancy because of the theoretical risks of neonatal kernicterus and haemolysis. however, in the presence of pcp the benefits of treatment outweigh these risks and women with hiv and a past history of pcp or with a cd4+ cell count ofo200 cells/ml should receive prophylactic septrin or nebulised pentamidine when embarking on pregnancy. this is an infection caused by mycobacterium tuberculosis which characteristically causes caseating granulomas, usually involving the lungs as the primary site. the incidence of tuberculosis (tb) worldwide is rising, in part due to the susceptibility to tb of hivinfected patients, and in the uk is more prevalent amongst asian and african immigrants. in pregnancy recent studies have suggested a high prevalence (50%) of extrapulmonary tb (at sites such as lymph nodes, bone, liver, spleen, bone marrow, caecum, nervous system and eye). the patient is often asymptomatic but typically can present with a cough, haemoptysis, weight loss and night sweats. the diagnosis is confirmed with sputum examination for acid-fast bacilli (ziehl-neelsen stain). although pregnancy and tb have little effect on each other, treatment should not be delayed in pregnancy. because of a rising incidence of drug-resistant tb, treatment is often with prolonged courses of multiple chemotherapeutic agents at the advice of the respiratory physicians. the usual drugs given are rifampicin (no proven adverse fetal effects but risk of maternal hepatotoxicity), isoniazid (in combination with pyridoxine 50 mg/day to reduce the risk of peripheral neuritis), pyrazinamide and/or ethambutol. liver function should be monitored monthly. streptomycin is associated with eighth nerve damage and should therefore be avoided. after delivery the neonate should be given prophylactic isoniazid treatment if the mother is sputum positive. the infant should also be vaccinated as soon as possible. breastfeeding is not contraindicated since very little of the drugs are excreted in breast milk. this is a complication of gastric regurgitation often at the time of a general anaesthetic in late pregnancy (usually at caesarean section). it may also occur in the event of an excessively high regional block. increased intra-abdominal pressure, delayed gastric emptying and reduced gastro-oesophageal sphincter tone contribute to the risk. ranitidine, an h 2 antagonist, which reduces gastric acid secretion and an antacid which neutralises the gastric acid are now routinely prescribed for women in labour with risk factors for caesarean section or prior to an elective procedure. the last confidential enquiry into maternal deaths (2000) (2001) (2002) reported one death from gastric aspiration in an obese woman (bmi 435) following failure to intubate the trachea after induction for general anaesthesia. cystic fibrosis (cf) is an autosomal-recessive disorder affecting the body's exocrine glands, including the pancreas, sweat glands and lungs. it results from a gene deletion in 70% of cases and has a carrier frequency of 1 in 25 caucasians. a resultant defect in the cf transmembrane conductance regulator protein causes impaired movement of water and electrolytes across epithelial surfaces. formerly, cf patients rarely lived to adulthood, but improvements in treatment have meant more women with cf are now reaching reproductive age and wish to become pregnant. the signs and symptoms of cf vary, depending on the severity of the disease and the degree of bacterial infection. some cases are very mild and may not be diagnosed until adulthood. cf is characterised by the production of very thick and sticky mucus. the ducts leading from the pancreas become obstructed, causing pancreatic insufficiency, diabetes and malnutrition. about 90% of cf cases involve the lungs with recurrent or persistent infections, development of bronchiectasis and respiratory failure. a productive cough is almost always present, and very often patients appear barrel-chested. frequent hospitalisations are a result of recurring respiratory, gastrointestinal and nutritional problems. effect of cystic fibrosis on pregnancy: female fertility may be impaired because of malnutrition or thickened cervical mucus but is usually normal. men are usually sterile. most reported series of pregnancies in women with cf have shown that with careful planning and close monitoring by a dedicated cf team, pregnancy outcomes are favourable. commonly reported adverse events are iugr and prematurity, which includes iatrogenic early delivery in unwell women. effect of pregnancy on cystic fibrosis: pregnancy does not affect disease severity or cause deterioration in the patient's condition. maternal mortality is not significantly greater than non-pregnant age-matched women with cf except in the presence of pulmonary hypertension, cyanosis, arterial hypoxaemia (oxygen saturationo90%), severe lung disease (fev 1 o60% predicted) and/or malnutrition when both maternal and fetal outcome may be poor. management: the risk of a child being born with cf is 2-2.5% if the carrier status of the father is unknown (based on the uk carrier status of 1 in 25) and 50% if the father is heterozygous for the gene. therefore pre-conceptual genetic counselling is important. during the pregnancy, regular assessment of fetal growth should be planned to detect early signs of fetal growth restriction. ideally women with cf planning a pregnancy should be seen and assessed pre-conceptually. women with mild disease can then be reassured that pregnancy is safe and liaison may be planned ahead between a cf centre and an obstetrician with a special interest in cf. screening for diabetes and baseline lung function tests can be performed. dietary supplementation and enzyme supplements can be commenced to optimise the patient's condition prior to conception and continued throughout pregnancy. women with cf are at increased risk of developing gestational diabetes. chest physiotherapy and prophylactic antibiotics may be implemented prior to conception and continued throughout the pregnancy. infective chest exacerbations should be treated aggressively with antibiotic therapy. conversely, if pre-conceptually the patient is found to have pulmonary hypertension complicating the cf, cor pulmonale or an fev 1 o30-40%, she may be counselled regarding the associated high risk and be strongly advised against pregnancy. effective contraception should also be discussed in such cases. in order to achieve a successful outcome, multidisciplinary management aims to ensure adequate maternal nutrition, control of pulmonary infection and avoidance of prolonged hypoxia. women may develop symptoms of breathlessness or evidence of hypoxia in late pregnancy. admission for bed rest and oxygen therapy should be arranged. delivery should be planned if their clinical condition deteriorates further. there is no contraindication to vaginal delivery but a prolonged second stage should be avoided because of the susceptibility of these patients to pneumothoraces. general anaesthesia should be avoided. there is no contraindication to breastfeeding but women may need to continue nutritional supplements postpartum. bronchiectasis is a sequela of cf, pneumonias and rarer causes such as kartagener's syndrome. it is characterised by irreversibly dilated damaged bronchi predisposing to persistently infected mucus and bacterial infections. a cough productive of large amounts of sputum is characteristic. the condition is uncommon in the childbearing years but there is some evidence to suggest an association with iugr when present in pregnancy. women should be managed jointly with respiratory physicians and their condition can deteriorate in pregnancy. close attention to postural drainage and physiotherapy will help. regular sputum cultures and treatment of chest infections are necessary intermittently or continuously if recurrent infections are frequent. the use of bronchodilators may help. as in cf, the presence of pulmonary hypertension and/or hypoxaemia adversely affects prognosis. there are few data regarding pregnancy outcome in women following lung transplant. of a series of 10 pregnancies in 10 women who were cf lung transplant recipients, five with a long stable interval of 43 years between transplant and the pregnancy had favourable outcomes; the remaining five had preterm deliveries. all 10, however, showed progressive decline in lung function and all died of chronic rejection within 38 months of delivery. restrictive ventilatory defects characterised by a reduction in lung volumes and an increase in the ratio of fev 1 to forced vital capacity (fvc) occur when lung expansion is limited because of alterations in the lung parenchyma or because of abnormalities in the pleura, chest wall or neuromuscular apparatus. the majority of pulmonary diseases do not alter fertility. a large reserve in respiratory function allows fetus and mother to survive without compromise in most cases. fvc of 41 l or 50% of predicted have been suggested as a cut-off for successful pregnancies and although more severe cases can negotiate pregnancy, patients with severe restrictive lung disease should be advised to avoid pregnancy or consider a therapeutic termination. associated polycythaemia causing hyperviscosity may add to the thrombotic risk during a pregnancy. women with an associated kyphoscoliosis are often delivered prematurely because of deterioration in lung function. mode of delivery tends to be caesarean section because of abnormal fetal presentation. each case should be assessed individually. sarcoidosis is characterised by non-caseating epithelioid granulomas that may affect any organ system. the aetiology of the disease remains unknown. the disease most commonly involves granuloma formation in the lungs. other commonly involved organ systems include the lymph nodes (especially the intrathoracic nodes), skin, eyes, liver, heart, and nervous, musculoskeletal, renal and endocrine systems. the course of sarcoidosis is variable, ranging from self-limited acute disease to a chronic debilitating disease that may result in death. spontaneous remissions occur in nearly two-thirds of patients, but 10-30% of patients have a more chronic or progressive course. because sarcoidosis can involve any organ system, the clinical presentation is often variable. many patients are asymptomatic but there may be chest symptoms. other features include erythema nodosum, anterior uveitis, hypercalcaemia, arthropathy, fever or central nervous system symptoms. effect of pregnancy on sarcoidosis: according to reported series, sarcoidosis either does not change in pregnancy or if it changes, improves, possibly because of the increased circulating cortisol. however, there is a tendency to relapse in the puerperium. this should not be a contraindication to pregnancy except in severely affected cases pre-pregnancy. factors indicating a poor prognosis include parenchymal lesions on chest x-ray, advanced radiographic staging, advanced maternal age, low inflammatory activity, requirement for drugs other than steroids and presence of extrapulmonary sarcoidosis. effect of sarcoidosis on pregnancy: sarcoidosis does not adversely affect pregnancy and is not transmitted to the fetus. there is one report of sarcoid granulomata found in the placenta of one patient. management: ideally patients require evaluation before pregnancy to establish chronicity, baseline pulmonary function, inflammatory activity, staging and response to treatment. systemic steroids should be continued in pregnancy. angiotensin converting enzyme levels, used as a marker of disease activity, are unreliable in pregnancy. women should be advised to avoid vitamin d because of the risk of hypercalcaemia. intravenous hydrocortisone should be administered in labour in women taking 47.5 mg/day of prednisolone. wegener's granulomatosis this is a rare form of systemic vasculitis in which necrotising granulomatous lesions affect the upper respiratory tract, lungs and kidneys. without treatment the condition has a poor prognosis. remission may be achieved with prednisolone and cyclophosphamide, a teratogenic drug. women should be advised to avoid pregnancy until remission has been achieved and to wait at least 3 months following cessation of cyclophosphamide before conceiving. cyclophosphamide may also cause ovarian failure. pregnancy has been associated with exacerbation of disease activity and cyclophosphamide has been used in the latter half of pregnancy, causing a fetal leucopenia. antineutrophil cytoplasmic antibody (anca) titres are markers of disease activity but their reliability in pregnancy has not been evaluated. amniotic fluid embolism is fortunately rare (1 in 80 000) but should be suspected when a woman presents with a sudden onset of hypoxia (with dysnoea, cyanosis or respiratory arrest), collapse, an acute hypotensive crises or disseminated intravascular coagulation or haemorrhage in the absence of any other explanation. onset is usually during labour, caesarean section or within 30 minutes of delivery. it has also been described during dilatation and curettage procedures. management is supportive with early involvement of the intensive care team. the aim is to achieve adequate oxygenation with ventilatory support, invasive monitoring to maintain good cardiac output and correction of a coagulopathy. mortality rate is 480%. the pathogenesis is not entirely clear. it is thought to be due to the passage of amniotic fluid and debris into the maternal circulation, precipitating an anaphylactic-like reaction. fetal squames are however present in the circulation of healthy pregnant women without any consequences. bronchial carcinoma accounts for 95% of all primary tumours of the lungs. although the incidence continues to rise in women, few cases have been reported in pregnancy. the commonest presenting symptoms are cough, chest discomfort and haemoptysis and chest x-ray may demonstrate round shadows. metastasis has been reported in the pericardium and placenta. lymphomas may also present with respiratory symptoms. the strength of association between cigarette smoking and bronchial carcinoma overshadows any other aetiological risk factors and 20-30% of pregnant women report smoking in the developed world. few will stop on becoming pregnant but focused counselling by specialist staff has been shown to be more effective than other measures, with more than doubling of cessation rates. the effectiveness and safety of nicotine replacement therapy in pregnancy have yet to be established. besides maternal morbidity, smoking adversely affects the pregnancy by doubling the risk of premature delivery, both spontaneous and iatrogenic, as a result of placental abruption and lowers the mean birth weight. reduced ovarian and tubal function and implantation predispose to subfertility, miscarriage and ectopic pregnancy. breathlessness in the absence of an underlying pathology is common in pregnancy, especially in the second and third trimesters. the use of chest x-rays when underlying disease is suspected should not be avoided in pregnancy and most drugs can be used safely in pregnancy. it is important that women with a chronic respiratory disease should receive pre-pregnancy counselling and education regarding the risks of pregnancy and the importance of continuing their medications. it also provides the opportunity to optimise their condition and so reduce adverse pregnancy outcomes. women should be managed in a multidisciplinary setting with regular review by chest physicians and access to chest physiotherapy if necessary. liaison with obstetric anaesthetists in the antenatal period will optimise care at delivery with regard to pain relief and reduce anaesthetic risks. respiratory diseases complicated by pulmonary hypertension and cor pulmonale have a poor prognosis in pregnancy.f pneumonia in pregnancy is often more virulent and therefore should be treated aggressively all non-immune pregnant women exposed to varicella should be given zoster immunoglobulin ( british thoracic society: scottish intercollegiate guidelines network. british guideline on the management of asthma british thoracic society: guidelines for the management of community acquired pneumonia respiratory disease cessation of asthma medication in early pregnancy pregnancy in cystic fibrosis lung transplant recipients: case series and review restrictive lung disease in pregnancy tuberculosis and pregnancy-results of a study in a high prevalence area in london asthma exacerbations during pregnancy: incidence and association with adverse pregnancy outcomes handbook of obstetric medicine key: cord-005646-xhx9pzhj authors: nan title: 2nd world congress on pediatric intensive care 1996 rotterdam, the netherlands, 23–26 june 1996 abstracts of oral presentations, posters and nursing programme date: 1996 journal: intensive care med doi: 10.1007/bf02316512 sha: doc_id: 5646 cord_uid: xhx9pzhj nan we present the results of a prospective population-based audit of paediatric intensive care activity in two comparable communities with markedly different delivery systems. in the trent region of the uk (4.2 million people), children receive intensive care largely without the supervision of a paediatric intensivist in a variety of hospitals, few of which have designated paediatric intensive care units (picus). critically ill children otherwise receive intensive care in children's wards, special care baby units (scbus) or adult intensive care units. in the australian state of victoria (4.5 million people), children receive intensive care almost exclusively in one centre -a picu staffed by full time paediatric intensivists. the two regions are otherwise demographically comparable. in both groups, data were collected on all children admitted to an intensive care unit between 1/4/94 and 31/3/95 and children who received intensive care (defined by levels of intervention and nurse dependency) in other sites during the same period. values of each variable at first contact with the icu, and the highest and lowest values over the first 24 hours were recorded. the principal outcome was survival to discharge from the intensive care unit. severity of illness was assessed using pim (paediatric index of mortality) and prism. risk-adjusted mortality was compared using flora's z test and logistic regression. the rate of utilisation of intensive care (>1000 admissions in each region) were similar. there was some variation in case mix between the two groups, but crude mortality rates were similar (7.4% in trent and 6.6% in victoria). however severity corrected data and other measures of picu performance were dramatically better in' the centralised delivery system. the substantial excess mortality in the trent region provides strong evidence for the benefits of centralisation of paediatric intensive care services. there are considerable difficulties in evaluating the efficiency and effectiveness oflcare in children presenting with respiratory failure during acute medical illness. optimal outcomes for such episodes include survival and the shortest length of stay (los) in intensive care with negligible risk of readmission. we have tried to determine whether or not the time course of acute severe medical illness with respiratory failure is predictable. study i (n=1000): a retrospective study of intubated and mechanically ventilated children (>28 days, <17 years) with acute severe medical illness. measures: diagnosis, intensive care los in calender days, and survival. results: the underlying diagnosis fell within one of three broad categories: respiratory disease (n=521, mortality 19.2%), central nervous system (cns) disease (n=342, mortality 38.7%), and systemic inflammation or multisystem (sims) disease (n=137, mortality 47.5%. the los in survivors was: respiratory -median (interquartile range) 8(4-16) days, cns 4(3-8) days, £p,4£ 5(7-g) days. 5:i'~'-+cen diag~,~is-rc!ated-grnnp~ (drgs) were identified (8 respiratory, 5 cns, 3 sims disease) and each have been characterised by mortality and los. study ii (n=300): a prospective study of patients supported by the hypothesis that los for the above drgs was predictable (compared with study i data). in certain instances attributable causes for variances in los were identified: e.g. disease severity, timing ofdrug therapy, and associated disease. with daily paediatric risk of morality scoring within each drg, four profiles of instability were identified. discussion: the time course of acute severe medical illness with respiratory failure is predictable and variance may be attributable to specific care or diagnostic factors. we are now developing a means of linking drg-specific clinical care pathways with an integrated computerised decision support and education facility at the bedside. the objective of this open, prospective study was to assess the relation between basic patient characteristics as well as effectiveness of treatment on the one hand and resource utilization in pediatric intensive care on the other. as universal, non-monetary indicators of resource utilization we used the therapeutic intervention score system (tiss) and length-ofstay (los), from which indicators for total resource utilization per admission (tisstot) and average daily resource utilization (tiss-mean = tisstot/los) were obtained. overall 593 admissions, totalling 3130 days, were included. mortality was 8.4%; non-survivors accounted for 14.1% of overall resource utilization. in non-survivors, both total resource utilization per admission and average daily resource utilization were higher, whereas los was not different from survivors'. severity of illness, surgical status, the presence of substantial chronic comorbidity, emergency admission and transfer from another hospital constituted the major predictive determinants of tisstot (r:=0.19) and tissmean (ra=0.45) in multiple regression analysis (p<0.0001). hence these indicators are appropriate non-monetary measures of resource utilization, a considerable proportion of which are determined by a concise set of basic clinical characteristics. subsequently we analysed the relation between effectiveness of care and resource utilization by assessing severity of illness corrected mortality in low, medium and high resource users, respectively. these 3 categories were delineated by percer/tiles of resource utilization (< p20, p20-ps0, > ps0). despite on average long los and high resource utilization in the high risk group, a relatively low standardized mortality was found, probably warranting prolonged intensive treatment in this patient category. summary: objective:the primary purposes of intensive care are to provide treatments to patients with life-threatening physiological dysfunction or to monitor and observe patients perceived to be at significant risk of dying. this collaborative study was performed to describe our patients and their outcome. in order to improve our results we tried to identit~ high risk groups, patients and methods: 13 picus entered the study, the data included all the admissions with >12 hs. during a 60 days period between the l°june and the 30th september 1993. the records included: age, sex, weight, mechanical ventilation (mv), post-operative condition (p.op), malnutrition, diagnosis, length of stay, prism score and outcome. student test, mann-whitney or wileoxon were performed for univariate analysis. fisher exact test or chi square for dicotomic variables. risk group analysis was performed by logistic regression, odds ratio and 95% confidence interval. results: 650 patients entered the study. mean age was 47.6 months (ds hh¢# 60) and median 18 months. we found significant statistical differences in calculated ,is observed mortality rate comparing malnourished with euthrofic patients; mechanical ventilated (mv) with non mv patients. no differences in ter ~,h of stay or di~ noses were found. effect of the un sanctions on the morbidity rate araong the iraqi small children ( below 3 years old of age ) in bagdad. abdulsamad a.abood / institute of medical technology, bagdad. meningitis is essentially a childhood disease (i). the risk of infection are increased by powerty and overcrowding (7). the impah'ed immunity may be an important pathogenic factor underlying the susceptibility to infections in undernourished subjects (5). in general, malnutrition is a man made disease and it begins quite in the womb and ends in the grave (i). 1918 small children, below 3 years of age were admitted to the pediatric hospital in washash with meningitis over 4 cold months in i994, in contrast to only 176 child admitted with meningitis over the same period in1989. all of the children who admitted in 1994 were frankly undernourished, 45% of them were infected with enterobacteriae, because they were exposed to faulty hygiene and lack of asepsis. these facts showed precisely that our small children had suffered at most from the un_ sanctions against iraq, because of food, milk and drug shortage, since 4 years which had resulted a severe undernutrition among them, which impaired their immune status. m wells, of riera-fanego, j lipman. baragwanath intensive care unit, university of the witwatersrand, south africa. background the use of prism or other scoring systems in the icu is of great importance for evaluating the efficacy and efficiency of a particular icu, the prism score was developed and validated in the usa and europe but has recently been shown to be inaccurate in a south american population, a south african population as well as several european studies. part of the poor performance of the prism score is as a result of differences in the case mix between the reference population and other paediatric icus. since scoring systems should generally be used only in populations similar to the reference population from which the prediction model was developed, a modification of the prism score is necessary to improve its discriminatory ability in a wide range of patient groups, aim to improve the predictive power of the prism score in a south african paediatdc icu population. patients & methods we analysed prism, demographic and clinical data collected prospectively from 1528 consecutive paediatrie icu admissions. the prediction of actual mortality by prism was evaluated by standard statistical methodology (goodness-of-fit test and receiver operating characteristic (roc) analysis), the components of the prism logistic regression equation (prism score, operative status and age) and the 14 physiological variables making up the prism score in addition 10 new variables analysed (nutritional index, the need for inotropes and institution of mechanical ventilation) were subjected to discriminant analysis to determine their association with outcome. results the goodness-of-fit test showed a significant failure of prism to accurately predict mortality over a wide range of expected mortality (chi2[8] = 195, p = 0). prism underpredicted mortality at lower prism scores, but overpredicted mortality in patients with high prisms. similarly roc annysis indicated apoor predic~jve power (az = 0.73 ± 0.01), with an area under the curve significantly less than that for the prism reference population (p = 0), prism showed equally poor discriminatory function at all age groups and diagnosfic categories. '~mth the addition of an index of nutrifional status (proportional weight-far-age), and indicators of early respiratory and cardiovascular failure to the logistic regression formula, and a recalibration of the acute physiological score component, the roc can be improved to 0.83 ± 0.02, with a good fit described by the goodness-of-fit test (cn218] = 3, p = 0.89). discussion the prism score is not accurate in our patient population has been recalibrated in view of the poor discriminatory function that we have shown. part of the inaccuracy derives from the different demographic characteristics of our icu population and a different pattern of diseases. in addition to assessments of acute physiological aberrations, an assessment of nutritional status and early respiratory and cardiovascular failure significantly improve the discriminatory ability of the prism score, these parameters have been devised with a view to improving the accuracy of prism in our population, while not decreasing its accuracy in icus similar to the reference population. in interviewing parents regarding how physicians have communicated bad news, the response i have received is that it has not infrequently been done without appropriate care, understanding and compassion. personal experience and the lessons learned from parents, chaplains and others who deal extensively with these situations have provided me with an approach that has been supportive, compassionate, and caring. an especially difficult communication situation for the intensivist occurs when the parents have to be informed of the death of their child. for the parent, death is the hardest loss of all -the ultimate unalterable loss. circumstances surrounding the death are an important consideration (e.g., a fatal crash caused by a drunken driver, a prolonged illness, a suicide, aids). each produces a different grief reaction. the physician needs to inform parents of their child's death sympathetically coming right out with the news and leaving details until later. allow pauses and time for the paren~ to express sorrow and grief, the best communication may be thoughtful silence and a tender touch. there is disbelief that this happened. it is necessary to repeat oneself. acknowledgment of the parent's "feeling terrible" and the physician's acknowledgment of how terrible he/she feels that the life of the child could not be saved is an important first step in the parent's dealing with this tragic loss. with prolonged resuscitation, it is helpful to have a member of the icu team talk to the parents while the resuscitative efforts are ongoing so that the parents are not left unsupported at this time. a progress report should be delivered in a caring, lucid, and sensitive.manner, indicating that every effort is being made to save the life of their desperately injured child. after a child has died, it is helpful to the family if the physician maintains some contact with them. this should take the form of follow-up telephone calls at approximately 6, 12, and 24 months. this can help to screen for depression in the parents. in giving bad news to the family and making every effort to support them through this tragic time, it is necessary to remind oneself that the intensivist has personal needs for dealing with grief and will also require support to pass through this stage. direct evidence that child mortality is lower in specialist pediatric icus comes from 3 studies. a study in oregon (ccm 1981; 19:150-9) found that mortality adjusted for severity of illness was 102% of expected in 3 pediatric units and 139% of expected in 71 general units (p<0.05). a study in holland (ccm 1995; 23:238-45) found that mortality in high risk patients was 85% of expected in 6 tertiary pediatric units, and 143% of expected in 4 nontertiary units (p<0.05). a third unpublished study, has found that children in victoria (who almost all receive intensive care in a pediatric icu) have a much lower standardised mortality rate than children in the trent region of the uk (where many children receive intensive care in adult icus). there is indirect evidence that icus looking after many children are likely, on average, to perform better than icus looking after few children: numerous studies in many specialities have found that units looking after many cases of a particular disease have better results than units with few cases. see luft hs, "hospital volume, physician volume, and patient outcomes", happ, 1990; and farley d, medical care 1992; 30:77-94. compared to general icus, medical and nursing staff in pediatric icus are likely to be better at looking ~fter children, and plcu rmos have greater skills in pediatric intubation, ventilation, iv drip insertion and drug doses. picus are more likely to have appropriate equipment to manage children -especially for uncommon but life-threatening situations. icus in pediatric hospitals are more likely to have physicians and surgeons with pediatric expertise available for consultation at all times. the american academy of pediatrics, the society of critical care medicine, the british paediatric association and the australian nh&mrc have all said that children should receive intensive care in'specialist pediatric units. the weight of authoritative opinion, and direct and indirect evidence is strongly in favour of looking after children in dedicated pediatric icus. neurological deficit showed higher cbf values (125.7/115.2 ml/100g/ rain.) than the 11 patients with good outcome (mean cbf 1 17.5 sd +8.1; cbf 2 19.9 sd _+9.1 ml/100g/rain}. discussion: in asphyxia decrease of ph is due to reduced tissue oxygenation and indicates the severity of metabolic derangements. co2reactivity in newborns with perinatal asphyxia correlates with the lowest ph and therefore may reflect severity of asphyxia. continuous monitoring of cerebral activity is carried out in our unit on all admissions at risk of cerebral dysfunction, a number of monitors are commercially available and we report our experience with the cfam2 which provides in addition to amplitude integrated eeg analysis, continuous raw eeg display and frequency distribution. bilateral recordings are commenced as soon as possible and continued while clinically indicated. forty one children ranging in ages from 4 weeks to 16 years were monitored for periods from 3 hours to i0 days, diagnoses included traumatic brain injury (11), sepsis/meningitis/encephalitis (1 t), status epilepticus (8) and miscellanous others (11). results are tabulated below. patients 13 12 16 status epilepticus 10 4 1 * beta activity 1 8 15 * background voltage 10 3 1 * < i o/zv 2 or more of above 12 2 1 * (*z2 p < 0,001) asymmetry developed in 4 children, all of whom died. positive predictors of good outcome included a mean background activity of >10zzv, the presence of faster frequencies (usually 13) in response to sedative drugs and the absence of seizures. all monitoring is performed by the picu staff and increasing expertise in interpretation has resulted in earlier therapeutic and diagnostic interventions. regional it was previously found that histamine, a vasoactive mediator, accumulated in brain compartments (kov~ics et al 1995 neurosci lett 195:25) , and antihistamines prevented brain edema formation (dux et al. 1987 neuroscience 22:317) in asphyxiated newborn pigs. in the present study we investigated the effect of intracarotid histamine injection on the blood-brain barrier (bbb) permeability, left internal carotid artery of 30 newborn pigs (4-8 h; 1,180-1,530g; ketamine anesthesia, 10 mg x kg 4) was catheterized through the external branch and different doses of histamine (0, 10 -6, 5xi0 -6, 10 -5, 5x104, 104 m, respectively, in 6 groups of animals; n=5 in each) diluted in 1.0 ml isotonic saline was injected into the vessel through 1 rain. bbb permeability was determined for a small (sodium fluorescein, sf, 376 da) and a large (evans blue/albumin, eba, 67 kda) tracer (2%, 5 mlxkg 4, 30 rain circulation time for both dyes) concomitantly in frontal, parietal and occipital cortex, hippocampus, and periventrieular white matter both on left and right sides 1 h after the challenge. then, intravascular dyes were removed by perfusion and bbb permeability for both tracers was quantified by fluorescence spectrophotometry (wavelengths for excitation and emission were 440 nm and 525 nm for sf; and 620 nm and 680 nm for eba, respectively). histamine injection, in doses higher than 10 .6 m, significantly (p<0.05; kruskal-wallis one way anova on ranks followed by dunn's test) increased bbb permeability for both tracers in each brain region. changes in left hemisphere were more intense (p<0.05) than those in right one after the doses of 5xi0 -6 and 10 -5 m in each region, i0 4 m histamine administration induced similar edema in both sides. increased intracarotid histamine levels resulted in a dose-dependent vasogenic brain edema formation. histamine might have a pathogenetic role in neonatal hypoxicischemic cerebral injuries. supported by otka f-12722 and h-u.s,-jfno.392, $162 in coma caused by traumatic brain jnjury, an indication of the likely outcome is provided by the best motor response to pain in the first .$ hours after the insult. in a study in our picu, the proportion of children who died or had a severe disability was 100% in 35 who had no response to pain, 40% in 47 with an extensor response, 14% in 64 with a flexor response, and 1% in 61 who localized in response to pain. the long term outcome of traumatic brain injury appears to be worse in children <4 years old. other risk factors in traumatic brain injury are absent basal cisterns, midline shift or subdural haemorrhage on ct scan (or loss of grey-white differentiation in nontraumatic injury); or an intracranial pressure >30 mmhg despite hyperventilation, mannitol and barbiturate infusion. apart from brain death, there are two findings implying such a poor prognosis that consideration should be given to stopping treatment: first, after traumatic injury, the absence of any motor response to painful stimulus in the cranial nerve distribution (providing drug effects and a post-ictal state have been excluded); and second, in acute brain injury from trauma, infection, hypoxia, or ischaemia, the b{lateral absence of short-latency somatosensory evoked potentials (providing brain stem haemorrhage, subdural and extradural effusions, and decompressive craniectomy have been excluded). in children over 2 months of age, recovery from prolonged coma or a vegetative state is exceedingly rare when more than 12 months have elapsed after traumatic brain injury, and when more than 3 months have elapsed after nontraumatic injury. overproduction of nitric oxide (no) via an inducible isoform of" no synthasc (inos) produces profound vasodilatation in adult septic shock. high nitrate levels have been reported in hypotensive children with sepsis syndrome ]. cardiovascular collapse is a prominent feature of severe meningocoecai disease (mcd). however, systemic vascular resistance (svr) was slightly higher in a group of non-survivors ~ and the rote of no in ivicd remains unclear. children with a presumptive diagnosis of mcd were enrolled. parental consent was obtained. blood was drawn on admission and 12hrly thereafter. plasma was separated immediately and stored at -80°c. the final concentrations reported represent the product of nitrite and nitrate (nox). nox was measured spectrophotometrically using the greiss reaction. 21 children were studied (median age (range); 27m (5-203)). the diagnosis of mcd was confirmed in 18 children, 12 of whom had a glasgow meningococcal score (gms) of" ~8. in this group with severe mcd there were 3 deaths. peak nox was significantly higher (,.54(27-78) vs 96(50-363)nmol/ml, median) and systolic btood pressure was significantly lower in children with severe mcd than mild mcd (p<0.05. wilcoxon rank test). there was a significant correlation between peak nox and gms (spearman's rank correlation r=0.6 (p=0.01)) and prism (r=0.6 (p:0.01)). nox production from adm.ission onwards was also higher in the severe mcd group (p:0.002, kmskal ~wallis). we have demonstrated that plasma nox levels are elevated in children with mcd, correlate directly with the severit 3' of disease and are inversly related to systolic blood presssure. similar to hypotensive septic syndrome, mcd appears to be associated with an up-regulation of the l-arginine-no pathway.. non-survivors with mcd have higher svrs and may be relatively hypovolaemic. in our group of severe mcd there was a significantly lower systolic pressure and increased no formation. excess inos expression at different stages in mcd may contribute to the pathology of the disease. the identification of agents which can boost and/or inhibit no reiease may therefore represent different treatment strategies for mcd. u. merz, th. peschgens, g. kusenbach, m. b6hle, h. h6rnchen in this controlled, prospective study 30 ventilated premature infants with a birth weight < 1250 g were randomized to receive treatment with dexamethasone (dex) either on day 7 of life or on day 14 of life. dex was given over 16 days tapering from 0.5 mg/kg/day to 0.1 mg/kg/day. the infants treated with dex on day 7 of life could be weaned earlier from the ventilator -in median after 14 days (range 10 -34) versus 24 days (range 8 -44) in the [ate treatment group (p = 0.01). the need for supplemental oxygen was shorter in the early treatment group -in median 24 days (range 10 -50) versus 40 days (range 10 -70) (p = 0.2, ns). the incidence of chronic lung disease was lower in the early treatment group -6 of 14 infants (42.9%) versus 10 of 16 patients (62.5%) (ns). to evaluate the long-term efficacy of early dex treatment we performed a respiratory function test in the age of 3 -6 months using an infant whole body-plethysmograph. the intrathoracic gas volume (itgv), the airway resistance (r.w) and the airway conductance (gaw) were measured and no significant differences could be detected between the groups. the frequency of adverse effects due to dex therapy was found to be without significant differences between the early and the late treatment group. we conclude that early dex treatment had short-term improvements in pulmonary outcome in our study population, long-term efficacy however, remained unproven. several factors contribute to the development of chronic lung disease (cld) in premature infants including structural immaturity of the lung, mechanical ventilation, and oxidative stress. reactive oxygen species are formed during normal cellular metabolism but they are generated in higher concentrations during inflammation or inhalation of high oxygen concentrations. to study the relationship between increased oxidative stress, antioxidants and the development of cld we examined 102 ventilated premature infants with birth weights below t500g. 32 infants developed severe chronic lung disease of prematurity (cld), defined by radiological signs of cld and an increased oxygen requirement at a postconceptional age of 36 weeks, and 29 infants had moderate cld with an increased oxygen requirement on day 28 but not at an age of 36 weeks. ventilator settings (fio2, peak inspiratory and mean airway pressure) and the incidence of early-onset-sepsis were significantly higher in the severe cld group than in infants with moderate cld or without cld (n=41) during the first week of life. plasma concentrations of the two antioxidative substances bilirubin and uric acid (ua) were comparable in all groups during the first days of life. however, on day seven bilirubin and ua were significantly decreased in the plasma of infants with severe and moderate cld compared to the non cld group (p<o.001). there was no difference between both cld groups. in serially obtained tracheal aspirate fluids (taf) malondialdehyde (mda) was measured by hplc as an indicator of lipid peroxidation reflecting oxygen injury of the lung. ua was found in higher concentrations in taf than bilirubin. ua has been proven to be a potent antioxidant, capable to react especially with hydroxyl radicals and hydrochlerous acid, which play an important role in the induction of lipid peroxidation. infants with severe cld had significantly higher mdnua ratios in taf from day 5 to 14 compared to the non-cld group (p<o.01). mdnua ratios in taf of infants with moderate cld were significantly higher from day 7 on but still lower than in the severe cld group. in addition, the ratio oxidized / reduced glutathione was significantly increased in taf of infants with cld compared to infants without cld between day 5 and 14. the data support the hypothesis that an imbalance of oxidative stress and antioxidant defense contributs to the onset of cld at the end of the first week of life. introduction. rocuronium bromide (roe), a neuromuscular blocking agent with an onset time dose to that of succinyleholine l'z, has been studied in american society of anesthesiologists' physical status (asa) i or ii pedlatrie patients under non-critical elective conditions. there is a paucity of studies looking at the pharmacodynamics of roc under emergent conditions, involving pediatric patients who are asa iii or iv, without the use of adjuvant inhaled anesthetics ~. methods. with approval of our committee on clinical investigations (irb), roe 1.2 mg/kg was administered to 15 asa iii/iv pediatric intensive care patients (age 40 days to 23 months; m:f=8:7) on intravenous sedation. the time to reach 25% a~d 10% of b~elinc tetanie stimulus response was recorded using a r.elaxogra'ph/qmt-100 nerve stimulator/recorder (datex, helsiaki). depth of clinical relaxation at the 25% level was recorded. results. the time from rapid bolus to 25% was 25.4+12.6 see with a range of 10-50 see; and to 10% was 30.1+17.6 see, with a range of 10-69 see. at the 25% level, excellent clinical relaxation was seen. conduslon. in critically ill children, use of roe at 1.2 mg/kg produces rapid onset of neuromuscular blockade and excellent clinical relaxation, such that roe should be considered for rapid sequence intubation. int anesth clin. 1995;33(1):39-60. anesthesiology. 1994;81 (5) computed tomography of the chest (ct) in mechanically ventilated adults can detect intrathoracic pathology not appreciated on chest radiographs (cxr) and influence intensive care management. no such data exists for ventilated children. aims: 1) to assess if ct provides additional information over cxr regarding extent and nature of intrathoracic disease in ventilated children. 2) to determine whether such information alters clinical management, inclusion criteria :10 ventilated children with cxr changes inconsistent with their respiratory status underwent ct if either a) pao2:fio2 ratio <30 and/or mean paw>15 cm h20 or b) there was an unexplained increase in ventilatory requirement. methods : high resolution ct was performed in 3 patients and spiral ct in 7 patierits, to ensure minimal transport related morbidity, patients were transferred to the ct scanner by a specialised mobile intensive care team. results: in 2/10 patients ct demonstrated greater extent of disease than appreciated on cxr but did not significantly alter clinical management. in 7/10 patients ct provided additional information regarding the nature of disease present, in 2/7 children this involved a further diagnosis and in 5/7 children the exclusion of a suspected pathology. new information led to a positive therapeutic intervention in 2 children, prevented inappropriate manoeuvres in 3, and had no significant effect on acute management in 2 children. conclusions: initial data suggests that in a selected group of mechanically ventilated children chest ct can add to the sensitivity and specificity of intrathoracic diagnosis provided by the chest radiograph and directly influence acute management. case selection criteria and choice of the most appropriate protocol requires further study. pressure control ventilation (pcv) utilizes a decelerating flow pattern which may improve gas distribution and lead to alveolar recruitment. in contrast, volume control ventilation (vcv) employs a constant flow. in children, the effects of pcv as compared to vcv are unclear. the purpose of this study was to determine how these two modes compare in terms of dynamic compliance (cdyn). peak iaspiratory pressure (pip), and mean airway pressure (paw) at equivalent minute ventilation. methods: sixteen infants and pediatric patients ranging in age from 1 day to 13 years were studied. diagnoses included ards (6), postoperative cardiac surgery (7), head trauma (1), and resfrictive lung disease (2). patients were randomized to pcv (9) or vcv (7). initial measurements of gas exchange (abg's) and respiratory mechanics (ventrak, novametrix medical systems) were obtained after a 20 minute stabilizadon period. respiratory mechanics included pip, peep, paw, delivered tidal volume, and cdyn (avolume/apressure). the patients were then crossed over to the alternate mode of ventilation holding delivered tidal volume, peep, inspiratory time, minute ventilation, and fio2 constant. data were collected after 20 minutes, in each mode the absence of intrinsic peep was confirmed. to assure that the measurements were not affected by changes in clinical status, the patients were returned to the initial mode of ventilation and measurements repeated (final) . patients were ventilated with a siemens 900c or sv300. reselts: data were analyzed using 2-way analysis of variance with repeated measures. ~ <0.05 vs. vcv) vcv pcv ~ initial ] final ! cdljn 3.5_+0.7 4.3_+0.8 * 3.7_+0.6 3.9_+0.7 i , pip 32+1.0 30l-_t.0 * 31_+1,0 31+-1,0 paw 9.2_+0.6 10.9i-_0.7 * 9.7+0.7 10.0-!-_0.8 pao2 97_+14 92+-10 87_+9 97_+14 discussion: at the same minute ventilation, the decelerating flow pattern of pcv resulted in a 23% increase in cdyn and an 18% increase in paw while decreasing pip by 6%. the lack of a significant change in oxygenation may be a result of the limited time in each ventilator mode as well as the inclusion of patients with both normal and abnormal lungs. there was no significant difference in initial and final measurements indicating patient stability. the beneficial effects of iecre~l~iug cdyn and paw while decreasing pip indicate that pcv may be a preferable mode of ventilation in patients with lung injury. further randomized studies examining the effect of pcv on respiratory outcome measures in pediatrics are indicated. prolonged positive pressure ventilation following repair of cdh is associated with a high prevalence of iatrogenic lung injury, in our unit dudng 1981-1990 314 late deaths after repair of cdh were due to chronic lung disease. since 1990 babies requiring assisted ventilation for more than 7days following surgery were transferred to a cnep chamber to limit lung injury. cnep of -6cm of h20 was combined with positive pressure ventilation via an endotracheal tube dudng the transition phase. immediate reduction of peak inspiratory and positive end pressures were possible and following extubation respiratory support was maintained by cnep v~th appropriate inspired oxygen. overall outcome: [1981] [1982] [1983] [1984] [1985] [1986] [1987] [1988] [1989] [1990] n=68 deaths before surgery (%) 11 ( ecmo during 1990 -1995 /16 who were ventilated for more than 7 days received cnep and there were no deaths and no chronic lung disease in that group. cnep assisted ventilation may be an important management option for babies who require prolonged respiratory support to avoid the adverse effects of chronic positive pressure ventilation, introduction so far 2 modes of liquid ventilation (lv) have been used in experimental animals and, exceptionally, in humans: 1. total liquid ventilation (tlv)-functional residual capacity (frc) is filled by perfluorocarbons (pfc), and slow tidal volume (tv) breathing is performed by pfc. 2. partial liquid ve,0ti,la~ion (page) -only frc is filled by pfc. gas tv is delivered by conventional mechanical ventilation (cmv), high frequency jet ventilation (hfjv) or high frequency oscillation (hfo). the aim of our study is to present our limited experience with page in newborns and infants. page was used in two groups of infants: 1, in 2 infants with brain death before disconnection from cmv, because recipients for organ transplantation were not available. these infants have relatively normal lungs (fio~ less than 0.4). infants stayed on page for 1 hour, during that period no ventdator manipulations were made. after page, infant were switched to cmv for next 6 hours. 2. very critically diseased infants with ards (rds) -2 on ecmo more than 5 days, 1 before cannulation for ecmo, 4 on hfo because of intractable respiratory failure, preoxygenated rm 101 (miteni, italy) was used in the doses up to 40 ml/kg intratrachealy. blood gases and parameters of pulmonary mechanics were followed (dynamic compliance -c dyn, airway resistance -raw, bicore monitor). page was combined with no inhalation (5-80 p.p.m, in 2 infants). in both groups ad hoc an approvement from e local ethical commission and informed parental consent were obtained. in the first qroud with relatively normal lung parameters of oxygenation drops after pfc instilation intratracheally and stayed depressed for 4-6 hours. slight pco2 retention occured in both cases during page. c dyn increased almost double during page period, raw drops transitorily after pfc instilation but in 10 minutes they were identical like in prepage period, parameters of oxygenation (peo2/fio2) after 4-6 hours after page improved and were better than in prepage period. after that time infants were disconnected and died. in the second group no improvement of oxygenation was seen in one ecmo baby, in spite ()f transient improvement of c dyn. in the second ecmo baby, oxygenation improved and flow of pump could be decreased by more than 20%. none of these babies, however, survived, improvement was only transient in spite of repeated dosis of pfc. in these babies serious problems were to maintain the adequate frc by liquid, because of severe air leak, in 5 babies on hfo/hfjv with severe ards/rds the improvement of oxygenation were seen in all the cases immediately after pfc instiletion for the period of 4-5 hours. after that period, pfc dose had to be repeated. two babies of this group survived. conclusion. page is going steadily from tabs to clinical practice. it is simple, could be performed anywhere, cheaper than tlv. however, because liquivent -perflubren (aliance pharmaceutical) is not available in europe, rm 101 of 82 (mitenti, italy) is the only solution, which could be currently used here. before the widespread use of page in clinics, liquid network among most nicus and picus must be built up, the criteria for page must be defined and ethinal-legal problems resolved as well. after resolution of these particular problems page can be life saving procedure for very special part of critically ill newborns end infants. catherine caronia, peter silver, laura nimkoff, cad quinn, jack gorvoy, and mayer san. division of pediartic critical care, medici,, schneider children's hospital, new hyde park, ny 11040, imroduetiun: cystic fibrosis (cf) patients awaiting lung transplantation present a therapeutic dilenuna when severe respir, aory decompemalion occurs, endotracheal intubation and mechanical ventilation is known to have no long term benefits and is associated with high morbidity and mortality. noninvasive respiratory support appears to be a beneficial alternative. methods: we instituted bipap (respironics, inc,, murrayville, pa) in 9 end-stage cf patients who were admitted to the pediatric icu with severe respiratory decompeusation. all patients were awaiting tung transplantation. after a control period, bipap was applied via a tight fitting nasal or facial mask, using the spo~aneous breathing mode, expiratory pressures were set at 4-8 cm hhzo. inspiratory pressures were started at 8 cm ~i o and increased in 2 cm i-i20 increments until the patient's respiratory comfort was achieved and substantiated by non-invasive monitoring. patients were instructed to use bipap during night sleep and whenever subjectively required, data are reported as mean _+ s.d. results: all 9 patiems utilized nocturnal bipap for 6-10 hours/day during a follow-up period of 2-19 months. compared to their pre-bipap status, the patiems' oxygen requirement and respiratory rate both oz~ cundusion: bipap tl~rapy improves the respiratory status of decompeusatir!g end-stage cf paacnts. it is well tolerated for long term use at home, and provides an extended period of respiratory comfort and stability for cf patients awaiting lung transplantation. l. bindl*, g. kiihl**, p. lasch***, appel**, j.m611er**** and the "arbeitsgemeinschaft ards im kindesalter" background acute respiratory distress syndrome (ards) is a therapeutic challenge in pediatric intensive care in view of the high mortality, in 1992 about 50 german paediatlic hospitals founded a working group aiming on collaborative clinical research in this field. aims and methods the aim of both a prospective and retrospective survey conducted in german pediatric intensive care units in 1993 was to accumulate data on the epidemiology, risk factors, natural history and treatment strategies in a large group of pediatric ards patients who were treated in the tt~ee year period from 1991 to 1993.all patients had acute bilateral alveolar infiltration of noncardiogenic origin and a po2~io2 ratio < 150mmhg. the influence of sex, underlying disease and single organ failure was analyzed using the fischer's exact test, the influence of additional organ failure on mortality was tested with the cochran-mantel-haenszet statistics. results 112 patients were reported giving an incidence of 7 cases per 1000 admissions to pediatric icus. median age was 24 month. in 43% of the cases, ards was associated with a pulmonary, in 39% with a systemic underlying disease. in 20% immunocompetence was impaired. mortality was 46% and not dependent on age, sex and triggering event. the number of associated organ failures, however, strongly influenced mortalib,. mortafity in immuno-compromised patients was 8 t %. the analysis of treatment modalifies employed in the patients revealed a lack of uniform therapeutic strategies. on the other hand, the patients were exposed to interventions not yet supported by controlled trials. conclusions the observation of the lack of uniform treatment strategies led to the elaboration of recommendations on ventilator therapy and patient monitoring within the working group. the data gathered in this survey provide the basis for the design of prospective multicenter studies urgently needed to evaluate innovative treatment modafities in pediatric ards. recurrent apnea and respiratory failnre due to severe lower respiratory tract disorders such as bronchiolitis or pneumonia are the most common reasons for mechanical ventilation during respiratory syncytial virus (rsv) infection. acute respiratory distress syndrome (ards) has been described as a complication of severe rsv infectionj in contrast to the low mortality rates associated with rsv infection (< 5 %), mortality rates in the range of 40-70 % have been reported in pediatric patients with ards. however, studies on ards are usually lumped in respect to causation and the disease course of rsv induced ards has not been previously studied. we examined the lung function abnormalities of 37 infants with rsv induced respiratory failure requiring assisted ventilation, measurements included respiratory mechanics, maximal expiratory flow-volume curves and lung volumes, ards was defined clinically using the criteria which were recently proposed by the american-european consensus conference on ards~: acute disease onset, pao2/fio~ ratio _< 200 mrn hg, bilateral infiltrates on chest radiograph and absence of clinical evidence of left atrial hypertension. we calculated the murray lung injury scores modified for use in pediatric patients 3 from total respiratory system compliance, radiographic findings, ventilator settings and blood gas results. we identified 10 infants with severe restrictive lung disease that fialfilled the clinical criteria fbr classification as ards. all had lung injury scores above 2.5 which is the recommended cut-off for a diagnosis of ards, twenty-seven infants had obstructive disease consistent with a clinical diagnosis of bronchiolitis. the ards patients were significantly younger, had a longer time of assisted ventilation (p <0.05) and a greater proportion of infants with preexisting illnesses (p=0.023, odds ratio =6.67) when compared to the patients with obstructive disease. with the exception of one immunodeficient patient, none of these infants died. given the low mortality despite a clinical picture of severe lung injury, there is evidence that rsv induced respiratory failure may represent a relatively benign cause of ards in pediatric patients, bachmann an audit of patients with severe acute bypoxic respiratory failure (ahrf) receiving highfrequency oscillatory ventilation (hfov) in our unit ( n=32, mortality 75%) revealed that sub-groups with severe underlying disease (n=14, mortality 100%)and those with mu~pie organ failure ( > 2 systems failing, n=7 mortality 100%) accounted for all the deaths beyond the neonatal period. v~ therefore hypothesized that in a modem paedistric intensive care unit (picu): a) children greater than one month of age with ahrf do not die in the absence of severe, pre-existing disease or multi-organ dysfunction syndrome, b) respiratory parameters alone will predict outcome poorly in ahrf. method prospect~/e sty/of all adm~ns to our tertiary picu. data it, citing the respiratory parameters (oxygena~n index [ol] , aiveolar-artedal oxygen tension gradient , pao2/fio2 ratio) were collected hourly from the bedside charts throughout admission. patients were included in the study if ahrf was present at admission either none or in combination with other organ dysfun~on. ahrf was defined as the acute (<48hour) onset of respiratory dysfunctk:~l with a pao2/fio2 ratio.< 200 for six consecutive hours dunng the first 24 hours of admission (with no evidence of left anal hypertension), x-ray review defined a sub-group of patients with acute respiratory distress syndrome (ards) by the presence of bilateral interstitial infiltrates. results to date 59 children (ages 1-168 months, weight 1.2-70 kg) have been admitted in ahrf. 18 of these also had ards. the overall mortality was 23.7% (14/59), and greater in the ards group than the non-ards group (10t18, 55.5% vs, 4141, 9.7%, p< o.01) . it was not possible to predict survivors from non-survivors on the basis of the seventy of the respiratory failure alone, the a-ado2 on the day of admission (best in 24 hours) was not significantly different between survivors and non-survivors: (mean, + sd)(174 mmhg +_108, vs 304 mmhg _+_156). kdl non-survivors were immunodeficient (n=8), previously extmrnsly premature infants (<28140),(n=3) or suffedng fcom chronic metabolic or gastrointestinal disease (n=3). no previously normal child died. conclusion the severity of respiratory failure does not allow predioljon of outcome in our patients. we believe that this reflects that modem picu is so effective at providing respiratory support that pre-existing pathology alone de~ prognosis. this suggests that an abnormally regulated host response or abnormal persistence of a pathogen may be required to induce lung injury of sufficient severity that the resulting respiratory failure cannot be supported in a modem picu. introduction: postural changes (supine to prone) is a therapeutic intervention that could be useful in children with adult respiratory distress syndrome. objective: to determine the effects of postural changes in the oxygenation of young children with ards. method,s: a prospective stud3," was performed in eleven subjects aged 6 to 120 months (mean=33) with the diagnosis of ardsreceiving vendlatory support. (mean peep and fio2 of 9 and 0.75 respectively). postural changes was performed every 8-12 hours, during a period of time ranging from 5 to 16 days. arterial blood gases were determined before and 30-60 n~n after the postural change, no modification in the mechattical ventilation other that changes in the fio2 were performed. the oxygenation was determined by the index pao2/fi02 (p/f). to study the differences between the oxygenation mean, before and after the postural changes the wilcoxon test for paired samples was used, results: 184 changes were performed (104 from supine to prone and 80 from prone to supine). a9% increased p/f ratio was obtained after the change from supine to prune. although, not all the patients receiving postural changes improved their p/f. six of them (group i) showed an improve in the p/f when changed from supine to prone, returning to their base line when positioned from prone to supine. no improvement on the p/f was observed in the remaining 5 subjects (group ii)after postural changes (table 1) . during the maneuver no complications were observed. two patients had a pneumothorax, not related with the postural change. conclusions: postural changes (supine to prone) is an easy way to improve oxygenation in some children with ards. change to prone change to supine introduction: the common noninvasive diagnostic efforts to identify possible obstruction of the intrathorucic airway, are of limited value. invasive procedures such as bronchoscopy and bronchography may also be noncontributory and entail risks. we evaluated the usefulness of 3d-ct in the diagnosis and management of pediatric patients with suspected intrathoracic airway obstruction (itao). methods: we used a diagnostic algorithm (see diagram) in patients with suspected itao resulting in respiratory distress. three-dimensioual imaging of the tracheobronchial tree was reconstructed, following high speed spiral ct scan, by specific computer software (advantage window computer work station, general electric, milwaukee, wisconsin). non-ionic contrast medium was injected, in some patients, to delineate the intrathoracie large vessels.. results: eight patients were studied. in 5 patients the 3d-ct revealed intrathoracic airway abnormalities. these patients underwent further invesive studies which confirmed the following diagnoses: 2 patients had bronchomalacia, 1 had bronchial stennsis due to a dilated pulmonary artery mad 2 patients had subglottie stenosis extending to the thoracic cavity. three patients had no significant disruption in the configuration of the tracheobronchial tree and thus did not require invasive diagnostic procedures. conclusion: computer reconstruction of three dimensional images of the tracheobronehial tree is a safe and reliable diagnostic tool for itao. ards and ecmo; preliminary data from a randomized clinical trial. j fackler, c steinhart, d nichols, d bohn, m heulitt, t green, l martin, k newth, m klein, j ware. many suggest ecmo be considered experimental for ards and undertaken only with careful data collection and reporting. a mtflticenter pediatric rct is in progress to determine whether 1) ecmo and/or 2) permissive hypercapnia, offer significant advantage for the treatment of ards. methods: all patients aged 2 wk to 18 yr (without congenital heart disease) are eligible for study. data collection begins when a patient receives at least 50% oxygen and a peep of 6 cm h20 for 12 hours (stage t). if the predicted mortality reaches 60% within 7 days (stage 2), eligible patients are asked for written consent for randomization. patients are excluded from randomization with significant chronic lung disease, immune compromise, cardiac disease; or profound acute central nervous system damage. the prime outcome variable is survival. at the studies onset, 400 pts were estimated to be required so that 65 pts were randomized per arm. results: 131 patients are enrolled from 9 centers. data are complete on 85. 66 patients never reached stage 2 (i.e. 60% mortality). 47 patients improved and 19 died. of the latter, 13 had randomization exclusion criteria even if stage 2 was reached. 19 patients reached stage 2. 11 had exclusions from randomization and all died. eight patients (4 survivors were eligible for randomization; consent was obtained in no case. two patients received ecmo. overall survival is 60% (51/85). in patients without randomization exclusions, survival is 77% (34/44). morbidity m survivors (discharge -admission popc or pcpc score >_2) was seen in none of the 4 stage 2 surviviors and 15% (7/41) of those who reached only stage !. conclusion: the rct requires completion. the records of hospital in-patients at king faisal specialist hospital and research center who received external cardiac massage as part of their cardiopulmonary resuscitation were reviewed. success of resuscitation was analyzed as (1) short term (restoration of spontaneous circulation), and (2) long term (discharge from hospital). of 234 such patients, 171 (73.1%) survived the initial resuscitation, and 66 (28.2%) were discharged. success of outcome was not related to age, location of patient, time of day, or rhythm at arrest, including asystole. longer resuscitation time was associated with less chance of restoration of spontaneous circulation (p<0.001), but not associated with hospital discharge rate. results for patients with congenital heart disease were similar to those with other medical or surgical conditions. in this series, 36.7% of ward in-patients survived to discharge, compared to two 5"*;'~r ~r;~'9 ,.,.'her,, the r-e~ult~ were 0c/ "'~d ~, ~,°(. overall, 39 7% of patients who survived the initial resuscitation were discharged from hospital. where resuscitation continued for more than 30 minutes, 18.9% of patients had tong term survival. outcome from asystole was no worse than for other cardiac rhythms, we believe that previous reports of poor outcome from asystole in pediatric cardiac arrest should noi influence decisions to stop resuscitation for pediatric in-patients prematurely. successful restoration of spontaneous circulation with long term survival can be achieved after prolonged resuscitation. abdelmoniem~ lindsey jahusou~,mariano fiallos, university of florida, 820 prudential drive, suite 203 jacksonville, florida 32207 usa central acidosis is well recognized as a marker of inadequate tissue perfusiou, and ventilation. however, obtaining central venous blcod is difficult and fraught with complications in the child undergoing cardiopuimonary resuscitation. intraosseous blood may be used instead of central venous blood to judge ph and pcoz during short durations of cardiopulmonary resuscitation and during hemorrhagic shock. the purpose of this study is to compare the ph and pcoz status of intraosseous and central venous during prolonged cardiopulmonary resuscitation after fluid and drug infusion. we hypotbesized that there would be no difference in ph and pco2 values of simultanecusly obtained intraosseous and central venous blood samples. eighteen (18) introduction: cardiopulmonary arrest (cpa) in children is usually preceded by a deterioration of cardiac or respiratory function due to sepsis, dehydration and hypovolemia. early recognition of clinical and laboratory signs followed by immediate intervention are essential for prevention of cpa. the purpose of the present study was to identify factors which contributed to high rates of mortality from cpa in patients admitted to a paediatric intensive care unit (p1cu). methods: a prospective study was done of all non-surgical patients with cpa who were admitted to the picu, hospital baca ortiz, quito ecuador from january to october 1995. clinical and laboratory variables before and after admission to the picu, time from hospital admission to picu admission and the pediatric risk of mortality score (prism) were recorded on a questionnaire designed specifically for this study. results: of the 70 non-surgical patients admitted to the picu, 14 (20%) were admitted after developing cpa on the general pediatric wards. mean age was 16 + 19.1 months, with 13 of 14 patients under 20 months of age. initial diagnoses upon picu admission included meningitis (n=3), respiratory failure (n=2), congenital heart disease (n=2), severe neurological impairment (n=2), end stage neoplastic disease (n=2), hypovolaemic shock (n=l), peritonitis (n=l) and sepsis (n=l). mean time from hospital admission to p1cu admission was 16 _+ 19.2 hours. the mean prism score upon hospital admission was 30+ 13.7 (score > 20 = > 50% mortality). 79% (11/14) of the patients died. one of the three survivors had severe neurologie injury. prior to picu admission, patients experienced tac~,cardia (n=9), hypotension (n=8), neurological deterioration (n=8), respiratory, distress (n=7), oliguria (n=5), bradycardia (n=3), metabolic acidosis (n=7), hyponatremia (n=4), hypokalemia (n=2), hypocalcemia (n=2) and severe hypoglycemia (n=2). there were serious delays from the time of development of clinical and laboratory abnormalities to the time of admission to picu. conclusion: in the critically ill pediatric patient, rapid recognition of clinical and laboratory signs of deterioration, followed by immediate intervention, are required to prevent end stage shock and cpa. we found serious delays in intervention following development of important premonitory clinical and laboratory abnormalities in patients less than 20 months of age on the general pediatric wards, which iikely contributed to the dismal 79% mortality rate. hospitals throughout ecuador should institute immediate improvements in ctinical supervision, and provide training in paediatric advanced life support (pals) to decrease excessively high rates of and mortality from cpa. intraosscous access is recommended by the american heart association and american academy of pediatries as a means of rapid access to the vascular system for childhood emergencies. bone marrow and fat embolism is a concern and has been reported post intraosseous infusion in stable animals but has never been studied in animals subjected to cardiopuimonary resuscitation. we undertook this study to investigate the incidence and magnitude of lat and bone marrow embolism with the use of intraosseous infusion during prolonged cardiopuhaonary resuscitation and after fluid and drug infusion. we hypothesized that there will be no difference in the magnitude of fat embolism between cardiopulmonary resuscitation only and other cxperirnental conditions. thirty-one (31) piglets were anesthetized, mechanically ventilated, and instrumented (carotid artery, pulmonary artery and intraosseous earmulas ). the animals then underwent bypoxic cardiac arrest followed by chest compressions with the mechanical thumper (michigan insmunents) and mechanical ventilation for a minimum of 45 minutes. the animals were divided in groups: a (n=5) which had no intraosseous, ~'oup b (n=6) had intraosscous with no infi~ion, and groups c (n=6), d (n=6), e (n=8) had intraosseous with infusion of adrenaline, normal saline and sodium bicarbonate, at cessation ofcardiopulmonary resuscitation, representative lung samples were collected fi'om upper and lower lobes of each lung, embedded in ocp and firozen immediately. ltmg specimens were stained using oil red-o dye and observed for fat globules and bone marrow elements. the amount of emboli present was rated as a percentage in relationship to iung tissue, by a pathologist blinded to the experimental groups. buffy coat specimens were collected before and at cessation of cardiopuimonary resuscitation, stained with oil red-o dye and observed for fat globules. percentage of fat present were compared using analysis of variance. fat globules were seen in the prebronchial blood vessels and in intravascular areas throughout all lung fields. there was no difference in appearance or distribution of fat globules between groups. quantity varied in the different groups[(a) 45%, (b) 44%, (c) 30% (d) 23%, (e) 25%], but were not statistically significant (p = .097). fat globules in the buffy coat were few and inconsistent with lung findings. fat and bone marrow emboli were present in all experimental conditions, the use of the intraosseous cannula does not increase the magnitude of embolization during cardiopuimonary resuscitation. the decision to use the intraosscous route should not be influenced by the risk of embolization. tzareva iv/,, md*, nedialkova r, md**, *dept. of pathophysiol, *~dept. of child surg. and icu, emergency medical institute pirogov, sofia, among 566 children with blunt abdominal trauma, treated in emi pirogov during the last five years, 79 children had serious disturbances of the basic vital functions, connected with the trauma, and most often with massive haemorrhage, for this reason being an object of reanimation and intensive care. in the group of children who survived -37, predominated the trauma of only one abdominal organ (mainly the spleen, rarely the kidneys, the intestine) and only 15 children had injuries of more than one abdominal organ. in the same group, in 15 children the abdominal trauma was combined with chest or head trauma or bone fractures. in the group of children who died -12, a profound combined trauma was present. the haemodynamic parameters in all children showed a characteristically significant tachycardia along with normal or even high blood pressure, while hypotonia was present in only 64% of the children on the first trauma day. despite the fact that only 13.4% of the children had direct chest injury as well, the gas exchange was considerably disturbed -899'0 of the children were hypoxemic during the first, and 100% during the third trauma day -in 25% significant -below 8.0 kpa (60 mmhg). together with the markable decrease in haemoglobin levels, this determines the pronounced disturbance in oxygen transport. during the first trauma day all the children were acldo~c, and a metabolic alkalosis was present during the following days. twelve of the children with severe combined trauma died within several hours, with the symptoms of irreversible haemorrhagic shock, or in the next 2-3 days, developing multiple organ failure. in conclusion, the intensive therapy of children with severe abdominal and combined trauma, should take in consideration the special haemodynamical trauma answer in children, and requires dynamic monitoring of the most influenced homeostatic parameters -blood gases, acid-base metabolism, haemostasis. introduction: endocrine emergencies, other than diabetic ketoacidosis, are uncommon causes of pediatric intensive care unit (picu) admissions. we report our experience of children diagnosed of adrenal insuficiency (ai) admitted in the picu, during the last four years. subjects: five eases of ai requiring 7 intensive care unit admissions are presented. four females anna 1 male, with ages ranging from 11 days to 7 years, none of them had a previous systemic or endocrine diseases that could suggest al the initial clinical manifestations were: dehydration (5), vomits (3), abdominal pain (2), seizures (2), lethargy (2) and hyperpigmentation in the muco-genitat area in a newborn male and ambigna genitalia in a newborn female. the reason for their admission in the p1cu were: shock in two subjects; three because of hyperkalemia and hyponatremia (k/na: 5.6/123; 9/126; 7,1/134 meq/l); and two with severe hyponatremia (na: 117; 113 meq/l). laboratory findings: severe hyponatremia (5), increased concentration of urinary sodium and chloride (4); metabolic acidosis (4); hyperkalemia (3); increased levels of urea (3) and hypoglycemia (2). in all of them, the electrolytes abnormalities did not normalize with replacement and only normalized after the administration of hydrocortisone. tile ai was due to: autoimmtme disease in two subjects, congenital adrenal hypoplasia, congenital adrenal hyperplasia secondary to 21 alia hydroxylase deficiency and in one no etiology was found, at the present time, comments: aiis an uncommon disease in the pediatric age. anearly diagnosis is crucial, as if the treatment is delayed could lead to patients death. in subjects with arterial hypotension and electrolytes abnormalities refractory to the usual treatment, they should be treated with corticosteroids, if no etiology is found. although, previously samples must be obtained to make the diagnosis, 0: denotes the number of cases. gerbaka b; hakme c; akatcherian c. toxics are frequently involved in domestic accidents during childhood; among non medical products ingestion, carbohydrate poisoning is a serious injury often made possible by inadequate stocking. over 10 years, 43 children aged 10 years and less were examined in the emergency department of hotel-dieu de france hospital for carbohydrate ingestion. 62,8% are boys; age goes from 13 months to 6 years (moan = 2,5years). kerosene is found in 35,8% of cases; all were admitted (mean = 2,8 days). 79,1% were symptomatic on first examination but 93% of all children presented signs of gastric (58%) or respiratory (69,8%) irritation sometime during their history; 37,2% had neurological signs and 41,9% presented some fever. leucocytosis is found in 65% of cases; 25,6% of the children received antibiotics. chest x ray was abnormal in 48,8% of cases: mainly parahilar infiltrates were found, all children survived; 76,7% with a normal course (1,9 days of hospital stay) whereas those who presented complications (severe pneumonia, coma) stayed in the hospital for 6 days (mean) with short course of assisted ventilation for two of them; long term follow up was not possible. we fonnd nick's criteria for hospital admission to be of value: -symptomatic children with normal x ray } 6 to 8 hours monitoring -asymptomatie children with x ray abnormality } -symptomatic children with x ray abnormality: hospital admission -asymptomatic children with normal x ray : no admission. these criteria would have helped to avoid admission in 8 children and would have allowed a short t2 hours stay for 6 more. we found chest x ray to be mandatory in carbohydrate ingestion; other tests were not helpful, aside arterial blood gases measurement in case of respiratory involvement; we now also advocate more restriction in antibiotic use. prevention remains efficient and should be stressed on. severe liver failure [slf] is a rare but severe condition in infants. we report our experience. patients: slf was defined as liver insufficiency with hepatic encephalopathy and a decrease in the level of factor v to below 25 %. between 1984 and 1996, 29 infants (mean : 4 mo) were admitted for slf (neonates excluded). main causes were metabolic disorders (41.3%) (tyrosinemian=5, hemochromatosis n=2, reye's syndrome n=2, other n=3), virus-induced flf (20.6%) and hematologic diseases (13.7%). in 4 cases, the causes remained undetermined. results: olt was contraindicated in 12 cases because of multiple organ failure (n=10), or underlying disease. all of them died within 6 days after admission. 7 patients had no indications for olt, all but one are alive. (1 of them was transplanted later for tyrosinemia and 1 died lately (virus induced-slf). among the t0 infants who underwent emergency olt, 6 are alive and 4 died because of primary non function of the graft. conclusion: slf in infants admitted before their first birthday is a severe condition with an overall mortality rate reaching 60%. inherited metabolic disorders are the first cause of slf at this age. contraindications for olt are frequent because of underlying disease or multiple organ failure. a number of children undergo primary graft failure after liver transplantation. it is unknown if there is any increased morbidity or mortality following retransplantation. this study seeks to explore these issues. methods: a pediatric intensive care/iiver transplant database is in formation. records of all liver transplant patients are reviewed and abstracted. this data is then computerized to allow analysis. this data provides the source for this study. statistical analysis was performed via student's t-test where appropriate. results: of the 350 patients who have thus far received at our center orthotopic liver ransplants, the records of 112 who underwent 140 transplants form the basis for this review. twenty-three patients underwent multiple transplants, 19 required one additional, three required 3 organs, and one patient survived after a fourth organ transplant, there was no significant difference in age at first transplant between those who received multiple organs and those who did not (40 vs, 44 months, p=ns). the anesthesia time for the procedure did not significantly increase tbr subsequent transplants (8.3 vs, 7,3 hours), nor did time in the intensive care unit (t6.6 vs. 22.2 days), nor did time on the ventilator (8.4 vs. 15.3 days) subsequent transplants did not predispose to having more bleeding in the intensive care unit for usage of packed red blood cells or platalets was not significantly altered (299 vs 306 ml and 127 vs 207 ml respectively). patients who required retransplantatior~ did receive mere fresh frozen plasma (ffp)daring their first transplant than in the subsequent ones (275 vs 81 ec, p < 0.05). however ffp use was not significantly different than patients who did not require retransplant. patients who underwent retransplant had a markedly increased mortality (47%) than the overall mortality for liver transplants at our center (20%), conclusion: children who require another liver transplant have a markedly increased mortality. bleeding and prolonged icu stay is not significantly different between the first and subsequent transplants, fulminant hepatic failure and ortothopic liver transplantation.dr.sasb6n,j;centeno,m;entin,e;acarenza,m;ciocca, m:gofii,j;bianco,g;weller, g;imventarza,o. unidad de cuidados intensivos.hospital de pediatria "dr.j.p. garrahan"1245.buenos aires.argentina. introduction:fulminant hepatic failure (fhf) is a clinical syndrome, defined by the development of hepatic encefalopathy within 8 weeks from onset of illness in a previously healthy person.by far,the most comun cause of pediatric fhf in all series, is acute viral hepatitis.we report our experiences with the pediatric fhf and ortothopic liver transplantation (olt) as attemative of treatment. patients:30 childrens with fhf diagnosis were admitted at the picu from 1/1/1993 to 1/12/1995.symptomatic treatment was given to all children and all were put on list for olt,) following the king's college criterion (protrombina time,age,atiologies,bilirrubin,and encefalopathy state). results:etiologic causes corresponded to the 30 childrens were:23, hav (76%); 6, noa nob (20%);1 ,autoinmune (4%).the age was mean:4 years (range:16 month-10 years).seventeen patients were transplanted,13 chidmn were discarded because:no donors:5;withdrow of the list:3,because sepsis in 2 and bleeding of cns 1;and no admission at list:5 because genetic syndrome 1 ,massive intestinal necrosis, 1 ,mitral valvulopathy 1 and sepsis,2. 25 patients (86%) had at least one complication dudng the post operative period.the most frequent was the acute renal insufficiency(ari) and 4 patients requiered continuos hemofiltration.the gtobal mortality rate was 75%.the mortality of patients without olt was 100% and the mortality of patients with olt was 41%,4 patients dayed because sepsis, (2 candidiasis) and the others 3 because mof.the actuarial survival at 1 year is 54% and the follow up of 8 months. conclusions:the fhf is a very severe and frequent disease at picu. supportive treatment only is associated with a very poor prognosis and high mortality rate.the most frequent etiology in our country is the hav. the olt is applicable in this cases and is a valid alternative of treatment (mortality in our series 41%).the ari is the most frequent complication during the post opeative period.in argentina,due the high prevalence of hav,prevention must be considered the main and only way to avoid this catastrophic illness.to assess the efficacy of gastric intramucosal ph (phi) for evaluation of tissular perfusion and prediction of hemodynamic complications m critically ill children. patients and methods: thirty critically ill children (16 boys and 14 girls) whose age ranged from 3 month and 12 years old were studied. a tonometry catheter was placed in the stomach of all patients at their °admission in pediatric icu. intramucosal ph measures were made at the admission and each 6-12 hours during the study: a total of 202 determinations were made. the catheter was removed after extubation and/or checking of hemodyrmmic stability of the patient. the intramucosal ph was derived from application of the henderson-hasselbaeh formula using the pco2 value from the tonometer and the arterial bicarbonate. values of phi between 7.30 and 7.45 were considered normal. the relationship between phi and severity of patient measured through prism, presence of major (cardiorespiratory arrest, shock) and minor (hypotension, hypovolemia or arrhytlmtias) hemodynamic complications, mortality and stay in the picu, was analysed. results: the admission value of phi was 7.48 -t-0.15 (range 7.04-7.68). five patients (16%) had an admission phi < 7.30. no relationship was found between an admission phi < 7.30 and a higher incidence of hemodynamic complications. sixteen patients (53%) showed some values of phi < 730 during their evolution. patients with phi < 7.30 had a higher number of hemodynanuc complications than the rest (p< 0.0001). every cardiorespiratory arrest (cra) and shock cases were related to a phi < 7.30. patients with major complications (cra and shock) had a phi lower (p= 0.03), as well as a higher number of measurements of low phi (p= 0.003) than patients with minor hemodynamie complications. the value of phi lower than 730 presented a 90% of sensibility and 98% of specificity with regard to hemodymanic complications. there was no relationship between phi < 7.30 and prims score and stay in picu. patients with phi < 7.20 presented a prims higher than the rest of patients (p< 0.05). conclusions: the phi value may be an early sign of presence of hem0dyaaimc complications in the critically ill child. we tested the hypothesis that gastric intramural ph (phi) can be used as an early sign of failure m weaning pediatric patients because the blood flow from nonvital areas is diverted to meet the increased demands of respiratory muscles. methods: 24 children (mean age (4.2_+0.3) years + sd) who were thought by their physicians to be weanable from mechanical ventilation (mv.). these patients were ventilated on serve 900c ventilators, receiving ranitidine, and had intestinal tonometer (tonometrics, inc.) 60 minutes before obtaining a sample.. all children were placed on pressure support (ps) at levels judged to overcome the resistance of the endotracheal tube and ventilatory circuit (2 em h.,o). a sample of arterial blood and a sample oftonometer were obtained during vm and weaning (ps). phi, hemodynamic and respiratory data were recorded during vm and weaning we did not interfere with the primary caretaker's decisions regarding extubation. patients were considered to be successfully weaned if they were able to sustain spontaneous ventilation for more than 24 hours after extubation. paired t-test were used to compare the values obtained during mechanical ventilation with those obtained during weaning trials. unpaired ttest were used to compare values from the group that was successfully weaned (a=i5) with those from the group that were not (b=9). results: we did not find statistical differences in any of those variables mesured during mv for patients who were successfully weaned(group a) and those who were not (group b). gastric phi was in group a: 7.35 + 0.03 (vm) and 739 + 0.02 (weaning); in group b: 7.40 _+ 0.04 (vm) and 7.4t _+ 0.02 (weaning). discussion: although we did not find differences in gastric phi during vm, the group a had a lower value than group b because of the number of cardiac patients (70%) and transfusion therapy, in fins group. in group b 75% of patients showed a problem in upper airway (subglottic edema, and enlarged tonsils). we found it after extubation. conclusion: 1) gastric phi is a good predictor of risk in critically ill patients but maybe because of the small size of the sample, in our study is not of practical value as a predictor of failure in weaning pediatric patients from vm. 2) this test is not a predictor of problems in upper airway~ important etiology of failure weaning in children. objectives: i-to determine the prognostic value of the gastric intramueesal phi in mortality and multiple organ dysfunction (sdmo) in critically ill children. 2-to compare this value, with the pediatrics risk index mortality score (prims). methods: aprospective study was performed with 51 critically illcbildren, aged from 1 mouth to 16 years. the athnittiug diagnosis was: 26 post-surgery (13 neurosurgery, 9 spinal fusion and 4 thoracic or abdominal surgery), 7 sepsis, 6 polytraumatism, 5 adult respiratory distress syndrome and 8 with miscellaneous. all the subjects were monitorized on picu admission and treated for their underlying condition. gastric intramucnsal pt{ was measured following the tonometric method, ou admission and every 4-8 hours depending on the patients state. the severity of the clinical condition was evaluated using the the prims, on admission (prims-i) and during the first 24 hours, when the clinical condition deteriorate, the worse score was utilized for the statistical analysis (prims-2). to perform the statistical analysis the subjects were divided in two groups, one with the phi<7.30and the other with phi>7.30.aunivariate analysis (student's tand wilcoxon two tailed test, chi-square) and multivariate analysis were used. results: 12 out of the 51 subjects dyed. of 14 children developing multiorgan failure (mof) 9 expired. 50% of the patients admitted to the picu with sepsis, ards and miscellaneous had a phi < 7.30. in contrast, with 27 % of post-surgical and none of the postqraan~atism. the mortaliry rate, in children with a phi<7.30was 47% (ci 95%:26.16; 69,04) and 11.76% (ci 95%:4,67; 26.62) in children with phi>7.30 (p=0.011). mofwas observed in41,18% of children withphi<7.30v.s, 20.6% with phi >7.30.no relatiouship was observed between the phi and the score of prims-i and 2. perforating an unconditional logistic regression analysis, two independent variables have mortality predictive value: the phi and the prism-2. (table i) following induction of anaesthesia, a laser doppler probe (moorsoft instruments ltd) was inserted 7cm into the patient's rectum, the probe's special design ensuring that the optical prism lay against the mucosa. continuous monitoring of rectal mucosal perfusion ("flux") was continued throughout the operation. after 10 rain cpb at 35°c, "steady state" readings of nasopharyngeal temperature, mean femoral arterial pressure (map) and flux were recorded over a further 5 min before cpbinduced core cooling to 14-24°c. steady state was defined as a 5 rain period with no change in core temperatures or map. other 5 rain steady state recordings were taken immediately prior to low flow, immediately prior to rewarming and after rewarming to 35°c, before initiation of any vasoactive drugs. the cpb flow rate was kept at 100 m l k g -1 min q, the pcv at 25_+3%, the p~co 2 at 5.3+0.5 kpa and the pro2 at 20+5 kpa. results: initial warm and rewarm map (both 46 mmhg) were significantly lower (19=0.008) than during the 2 cold cpb periods (63 & 64 mmhg). the mean cold flux before (152) and after (159) low flow were both significantly lower (p=0.001) than the mean initial warm cpb flux (211). the mean rewarm cpb flux (127) was significantly lower than all other flux values (p=0.001). there were no siglaificant correlations between map and flux except at the first warm cpb period (r=0,33, p=0.04). conclusions: although hypothermia significantly reduces rectal mucosal perfusion, rewarming produces an even greater reduction in gut perfusion which, considering that mucosal oxygen constmaption is highest during this time, may prove crucial in the postoperative development of mof. therapy aimed at improving gut perfusion during cpb should be directed at the rewanning period in particular. abstract this work is aimed at establishing a clinical procedure for the diagnosis of enteritis necroticans (en), even at the communal level, and to define criteria for diagnosis able to distinguish between acute forms. subjects and method : 100 cases admitted at the institute for protection of children's health dpch), having characteristic symptoms, were examined clinically, by roentgenography of the abdominal cavity, with the analysis of the blood (total protein, electrolytes, hematocrite) and cultures of intestinal fluid and faeces. through surgical operations, the pathological lesions were observed and recorded. results: common epidemiological features: the average age is 6-8 years old (3-15) ; male/female : 1.85; in 70% of the cases, the disease occurred after a meal rich in protides. the acute toxic form accounted for 15% : severe shock appearing early, with very severe dehydration associated with profoundly decreased blood protein concentration and lowered natriemia as well. the lesions of the small intestine were expanded, all of them were necrotic. in the surgical form (20%), the predominant feature was an obstruction -peritonitis syndrome, the peritoneal fluid showed a characteristic inflammatory reaction. for the rest of cases 65% were the internal form, the shock syndrome was less severe, the abdominal distention was light and disappears gradually, the inflammatory reaction of the peritoneal fluid was not so characteristic. conclusion (ino) is a selective pulmonary vesodilator that is rapidly inactivated compared to intravenous vasodilators. these qualities make ino an attractive agent for the treatment of pulmonary hypertension (pittn). the efficacy of ino has been studied in persistent fetal circulation, acute respiratory distress syndrome (ards), and congenital heart disease (chd). potential adverse effects oflno include: nitrogen dioxide (no0 toxicity, methemoglobinemia, and platelet dysfimction. our objective was to evaluate the safety of ino in pediatric patients (pts). methods: pediatric pts. with phtn from ards or chd were studied under an established, approved protocol conforming to fda guidelines tbr an investigational new drug. informed consent was obtained for each child prior to treatment. 1no was sequentially titratad from 10 parts per million (ppm) to 20, 40, 60, and 80 ppm at ten minute intervals. parameters monitored before and during therapy included nitric oxide (no) and no~ concentrations (cone.), mean arterial blood pressure (map), and percent methemoglobin (mhg). no and noz levels were continuously monitored using an inline dr~ger electrochemical detection device. ~,litp was continuously measured with an indwelling arterial catheter. mhg was measured by co-oximetry. a mhg level e 5% or no2 cone. ~ 5 ppm were considered adverse effects by study criteria. pretreatment map was compared to map at 40 and 80 ppm ino using paired t-tests. ap value < 0.05 was considered statistically significant. results: thirty-two mechanically ventilated children with phtn (16 with ards, 16 with chd) were studied. five pts. were treated following cardiopulmonary bypass. methemoglobin (met-hb) levels were routinely measured in two prospective clinical studies on no inhalation in 25 pediatric patients with pulmonary hypertension following heart surgery with extracorporeal circulation and in 19 pediatric and neonatal ards patients, the observed differences between the groups prompted in an in vitro study, red blood cells (rbc) of 20 patients sampled before and after surgery with and without extracorporeal circulation (ecc), respectively, were incubated with 32 ppm no for 100 rain, met-hb, atp, and nadht nadph concentrations were compared, during therapeutic exposure no increased met-hb from 0.2 -2-_ 0.1 to 1.2 _+ 0.7 % in cardiac surgery patients and from 0.2 ± 0,1 to 0.5 ± 0.4 % in ards patients (p < 0.01 ). rbc's having undergone ecc were more susceptible to met-hb formation (p< 0,001 ) whereas intracellular coenzymes did not differ neither between the groups (table) nor before and after no exposure. ecc predisposes to increased methemoglobinemia upon exposure to no both in vivo and in vitro. our data suggest a reduced activity of met-hb reducing enzymes rather than diminished availability of energetic substrates, variation of the inhaled nitric oxide concentration with the use of a continuous flow ventilator. anne pmc de jaegere ~, frans im jacobs 2, nico gc laheij 2, john n van den anker t . dept. of paediatrics ~, central instrumentation 2, sophia children's hospital, erasmus university rotterdam, rotterdam, the netherlands. objective: to investigate the homogeneity of nitric oxide (no) concentration in a delivery system with a continuous flow ventilator. design: bench study, setting: biomedical laboratory. interventions: a nitrogen/nitric oxide (njno) gas mixture was injected at three different sites in the patient circuit: just before and just behind the humidifier, and 20 centimetres before the y-connector. ventilator flow (12, 15, 20 l/rain), ventilator rate (40 to 110, increments of 10) and compliance of the testlung (0.36; 0.5; 1.0 ml/cm h20) were changed. carbon dioxide (co2) instead of n2/no was injected at the same points in the circuit. measurements and main results: a) though the flow ratio of the njno and the ventilator gas were kept constant, the no concentration ([no]) raised with increasing ventilator rates. the increase in [no] was up to 40% when the n2/no injection site was close to the y-connector of the ventilator circuit. minimal changes in [no] were noticed when the n~/no was mixed to the ventilator gas before the humidifier. b) analysis of the ventilator flow pattern showed variations at different places in the ventilator circuit. the magnitude cf the p, ow change depended on the meas~:rement site. the closer to the expiratory valve the highest the flow change was. the duration of the flow change was inversely proportional to the adjusted ventilator flow. c) real time measurements of the co 2 concentration ([coz]) showed variations during tile respiratory cycle. these [co2] variations were higher when the co2 gas was blended closer to the yconnector. conclusions: the ventilator flow variations in relation to the fixed side flow of the n2/no gasmixture result in changes of the inhaled [no] during the respiratory cycle. the no concentration during inspiration is always higher then during expiration. this could not be detected with the available monitoring system. to ensure a constant [no] by blending a njno gas balance in a continuous flow ventilator, the site of injection should be as close as possible to the inspiratory outlet. nitric oxide, a potent and selective pulmonary vasodilator, has recently been successfully used to treat pulmonary hypertension of variable etiology in infants and children. side-effects and complications in infants are so far not well known. we describe here two cases in which prolonged (5 and-7 days respectively) high-dose (50 -80 ppm) nitric oxide was used to treat refractor~¢ pulmonary hypertension. one patient was a newborn infant with pulmonary hypertension secondary to a large leftsided diaphragmatic hernia. nitric oxide was begun under conventional ventilation (babylog 8000) at 7 hours of life with a slight initial improvement in oxygenation. he was then placed on oscillation with the same nitric oxide concentration due to worsening respiratory failure. he died on 5th day of life. monitored nitric dioxide concentration never exceeded 4 ppm. the other patient was a 3 months old infant with severe pulmonary hypertension due to a complete atrioventricular septal defect. he required high-dose nitric oxide to come off cardiopulmonary bypass after surgical repair of his heart defect. he slowly improved over the week following surgery but developped suddenly respiratory failure due to massive pulmonary hemorrhage and died. surprisingly, a particular autopsy finding in both infants was a massive acute necrotizing tracheobronchitis. we conclude that nitric oxide is an excellent and sometimes lifesaving treatment of pulmonary hypertension in infants. tracheobronchitis has not yet been reported as a possible complication of nitric oxide administration. we suggest that caution needs to be taken with prolonged high-dose administration and this possible complication to be looked for at autopsy. introduction: permissive hypereapnia (ph) is a beneficial strategy for patients with acute respiratory distress syndrome (ards) to minimize barotrauma by decreasing the peak inspiratory pressure (pip). hypercapnia and hypoxia cause pulmonary vasoconstriction, pulmonary artery (pa) hypertension, and, thus, an increased afterload to the right ventricle. this increased afterload may result in increased right ventricular (rv) work load and subsequent rv dysfunction. one therapeutic approach is the use of inhaled nitric oxide (inn), a selective pa vasodilator. the objectives of this study were to test the hypothesis that in a swine model of ards with ph, inn would improve rv work load and not change intrinsic rv contractility. methods: in 11 swine (25-35 kg), ards was induced by surfactant depletion. hypercapnia was achieved by decreasing the pip while increasing the peep to maintain a constant mean airway pressure, inn was administered in concentrations of 2, 5, and 10 ppm in a random order. pulmonary blood flow (qpa) was determined by an ultrasonic flow probe. rv total power (tp) and stroke work (sw) were calculated by fourier transformation of the pa pressure (ppa) and qpa data. preload recruitable stroke work (prsw), a preload and afterload independent measure of ventriculur contractility, was determined by a shen-subtraction method and vena caval occlusion. respiratory failure with pulmonary hypertension in piglets gerfried zobel*, bernd urlesberger*, drago dacar**, siegfried rtdl*, fritz reiterer* and ingeborg friehs** depamnents of pediatrics* and cardiac surgery**, university of graz,austria objective: to evaluate gas exchange, pulmonary mechanics and bemodynamic data during partial liquid ventilation (plv) combined with inhaled nitric oxide (no) in acute respiratory failure with pulmonary hypertension. design: prospecfive~ randomized, controlled study. setting: university research laboratory. subjects: twelve piglets weighing 9 to 13 kg. interventions: acute respiratory failure with pulmonary hypertension was induced by repented lung lavages and a continuous infusion of the stable endoperoxane analogue of thromboxane. thereafter the animals were randomly assigned either for plv or conventional mechanical ventilation. initially perfhiorocarbon liquid (30ml/kg) was instilled into the endotracheal tube over 5 min followed by 5-10ml/kg~. all animals were treated with different concentrations of no ( 1-10-20 ppm) inhaled in random order. measurements and results: continuous monitoring included ecg, cvp, mpap, map, san2 and svo2 measurements. during plv pao2/fio2 increased significantly from 62_+3.2 mmhg to 193±44 mmhg (p<0.01) within 10 rain, while pao2]fio2 remained constant at 61 -+3.3mmhg. qs/qt decreased significantly from 48-+4% to 25-+5% (p<0.01) during plv and did not change during conventional mechanical ventilation. static pulmonary compliance (cstat) increased significantly ff~m 0.4r±0.07 to 0.75_+0.03 ml/cmh20/kg (p<0.01) during plv and decreased slightly from 0.58_+0.08 to 0.46e0.04 ml/cmh20/kg during conventional mechanical ventilation. the infusion of the endoperoxane analogue resulted in a sudden decrease of pao2/fio2 from 262_+44 to 106_+8.0 mmhg in the plv group and from 71±7 to 52+_2.0 mmhg in the control group. inhaled no significandy improved oxygenation in both groups (pao2/fio2:344_+38 mmhg during plv and 196+_.56 mmhg during conventional mechanical ventilation). during inhalation of no mpap decreased significantly from 57-+2 m 35±2 mmhg (p<0.01) in both groups. there was no significant change in oxygenation and mpap during inhalation of 1 and 20 ppm no. conclusions : plv significantly improves oxygenation and pulmonary compliance in acute respiratory failure. the additional application of inhaled no further improves oxygenation and pulmonary hemodynamics when acute respiratory failure is associated with severe pulmonary hypertension. inhaled no is very effective in improving oxygenation and pulmonary blood flow even at low doses. the work was supported in part by grants of the austrian nationalbank nr 5545. as in neonates, severe respiratory failure in infants and children can be aggravated by pulmonary hypertension, resulting in further deterioration of oxygenation due to increasing intrapulmonary shunting. we analysed the influence of inhalational nitric oxide (ino) in treatment, course and outcome of severe ards in a pediatric population. since 1993 20 infants and children (age: 1-107 months) with ards and oi > 15 (mean value: 32.5± 11) underwent a trial with ino (concentration: 3, 10, 30, 60 and 100 ppm) to prevent further respiratory failure. 11 patients had a significant improvement of their oxygenation (rise of pa09 > 15 mm hg) for at least 24 hours (responders); mean best ~fficient no dose: 24.6 ppm. the non-responders had only a short-term improvement or ino had no effect. in responders and nonresponders there was no significant difference with regard to age, underlying disease, ards severity, time on mechanical ventilation, blood gases and ventilator settings before notrial, nor was there a different grade of pulmonary hypertension (estimated by echocardiography). the only difference was an higher ol in the group of the non-responders: 40.9 ± 9.i vs. 25.6 ~ 6.7, p < 0.002. in the group of the 11 respenders there was a secondary deterioration of lung function after i -6 days on ino in 5 children (transient responders): in these patients, as well as in the group of the non-responders, alternative modalities of treatment (hfov and/or ecmo) became necessary. 6 children (30 %) died: 2 transient respenders and 4 non-responders. in infants and children with ards due to different underlying diseases ino can acutely lead to a significant improvement of oxygenation in about 50 % of the cases. the right selection of patients for no therapy and the influence of ino on the survival rate of ards in childhood has to be evaluated in further studies. and pediatric cardiology, university of graz, a-8036 graz purpose: after fontan procedure cardiac output is critically dependent on the pulmonary vascular resistance. even minor elevations of the pulmonary vascular resistance may significantly decrease cardiac output. inhaled no is an effective, selective pulmonary vasodilator in experimental and clinical situations of pulmonary hypertension. the aim of this study is to evaluate the effects of inhaled no on oxygenation and pulmonm 3, circulation in children after a bidirectional glenn-anastomosis (n-~) or a fontan-like operation (n=9). material and methods: from june t993 to january 1996 13 children with a mean age of 7.1+~2.1 (sem) yrs and a mean body weight of 24.3-+5.8 (sem) kg were treated with inhaled no after glenn-or fontan-like operations. all but one had complex cardiac malformations with single ventricle. all children were mechanically ventilated with an fin2 >0.75. inhaled (no) was applied using a rrdcrdproeessor based system which additionally allowed measurement of no/nox using the chemihimniscence method. methemogtobin concentrations were determined 3 times a day. the major indication for postoperative inhalation of no was a high (>10mmhg) transpulmonary pressure gradient (tpg--cvp-lap). severe myocardial dysfunction of the single ventricle was excluded by echocardiography. results: the mean duration of mechanical ventilation was 8.1_+2.2 (sem) days the. mean dose of inhaled no was 4.4-+0.8 (sem) ppm, the mean duration of no-inhalation was 106_+19 (sem) hours. the mean methemoglobin concentration was 1.2-+0.2 (sem)%. hemodynamic data and arterial oxygen saturation before inhaling no and 15 minutes later are given in table 1 acute hypoxaemic respiratory failure (ahrf) in children occurs in a heterogenous group of diseases with pulmonary pathophysiological processes ranging from reversible physiological intrapulmonary shunting to fixed structural lung damage. we hypothesized that inhaled nitric oxide (ino), a selective pulmonary vasodilator, might identify those patients with potentially reversible disease, i,e, large response may indicate a greater likelihood ef reversibility and thus survival. a retrospective review of the early response to ino in 30 infants and children (aged 1 month to 13 years, median 7 months) with severe ahrf(18 with ards). the mean p(a-a)o2, pao2 / fio2, oxygenation index (oi) and acute lung injury (all) score prior to the commencement of ino were 568 +_9.3, 56 +_2.3, 41 _+3,8 and 2.8 +_0.1 respectively, the magnitude of response to ino was quantified as the % change in oi occurring within 60 minutes of 20 ppm ino therapy. this response was compared to patient outcome data. results. there was a significant correlation between response to ino and patient outcome, kendall tau b r=0,43, p<0.02 (table) conclusion. in ahrf response to ino appears te define a subgroup of patients with improved outcome compared to nonresponders. we speculate that response to ino may be useful in selecting patients with potentially reversible lung disease for special support therapies such as ecmo. randomised controlled trials are needed to define the role of ino in paediatric ahrf. between may 1994 and december 1995, 22 patients (pts) were treated for mas. treatment groups were: group i only 02:6 pts; group i1 conventional mechanioal ventilation (cmv): 11 pts; group ii1 hfo: 1 pt; group iv hfo+no: 4 pts. therapy was stepwise intensified until oxygenation improved ( i -) ii -) iii --) iv). "high volume strategy" was used with hfo (mawp 18-24 cm h20). the initial no-concentration was 20-30 ppm, with rapid reduction down to 5-10 ppm once oxygenation improved. results: one pt (group it) died of hypoxic-ischemic encephaiopathy (termination of therapy); all other newborn babies survived. in group iv pt 1 and 2 showed barotrauma prior to hfo. pt 1,2 and 4 were treated with additional mgci2 (max. mg serum concentration 2.8 -6.5 mmol/i). following the identification of inhaled nitric oxide 0"no) as a selective pulmonary vasodilator (frostell et al 1992) [ 617 .+6,3 626+6.3 data are compared to baseline values within each group. *=p<0.05, **=p<0.03, ***=p<0.0l among 12 patients who fulfilled ecmo criteria, 6 improved with no and did not required extracorporeal life support. tltree out of 6 ecmo patients eventually survived. conclusions: m our study low-dose of irthaled no showed a variable effect on oxygenation in newborns with acute respiratory failure. an acute response to no appeared to be correlated with a better short-term outcome and the avoidance of extracorporeal support in ecmo candidates. differently, lack of acute and/or sustained response was associated with death or need for ecmo. although the nature and severity of the underlying disease or the degree of prematurity may play an important role in these patients, we believe lack of acute response to no may be an early predictor of bad outcome, prompting toward alternative treatments such as ecmo or liquid ventilation. *picea s., °bartuli a.,°dionisi-vici c., *dello strologo l., §villani a., §bianchi r., ^salvatori g.,*rizzoni g, °sabetta g. *div. of nephrology, °div. of metabolism, §intensive care unit, ^div. of neonatology. "bambino gesfl" children research hospital. rome, italy. successful prevention of handicaps or death in newborns with ~ depends on rapidity and efficiency of treatment. poor response to nutritional and/or pharmacological treatment requires extracorporeal removal of nh4. efficiency and cardiovascular tolerance are often difficult to obtain with peritoneal or hemodialysis in neonates. we report the results of cavhd in 3 newborns with hc. methods: vascular access: femoral vessels. blood flow: 10-35 ml/min, dialysate flow: 200-500 ml/h. filter: amicon minifilter plusrm(polysulfone membrane; 0.08 sq.m.). no ultrafiltrate(uf) production, patients: case 1 with carbamoytphosphate synthetase deficiency (body weight -bw-: 3.2 kg) showed hc at day 4, a relapse of hc occurred at day 14 due to an infectious event. case 2 and 3 (bw: 3.0 and 2.8 kg), both affected by propionic aeidemia, showed hc at day 5 and day 7, respectively. plasma nh4 (~tg/dl) decrease is shown in the complications: transitory ischemia of arterial cannulation limb and transitory thrombocytopenia occurred in case 1; surgical repairing of artery after cavt-id was necessary in case 3; no cardiovascular instability was observed during cavhd . outcome,'all patients recovered from hc in less than 1 day: case 1: alive, mild b)iootonia at 34 mos; case 2: dead after 10 days from cavhd withdrawal for pulmonary hemorrhage; case 3: alive, normal development at 7 mos. conclusions: 1) in newborns with hc, ca~q-id provides good cardiovascular tolerance,high efficiency and quick removal of nh4, even without uf production (i.e. only by diffusion). this allows easier management (no need of fluid and electrolyte balance). 2) arterial complications seem frequent in neonates treated by cavhd. venovenous circulation could overcome this problem. vb nguyen, m jokie, c leeaeheux paediatric intensive case service, hospital university centre, avenue c6te de nacre, 14033 caen cedex, france background, the implication of polymorphonuclear neutrophils (pmns) in the physiopathology of children's haemolytic.uraemie syndrome (hus) becomes more and more evident. the purpose of the present study is to role out their impact among other pronostie elements during the course of the disease. patients and methods. diarrheal prodrome and its duration, patient's age, maximal blood nitrogen level, anuria and dialysis time, extra.renal involvements, white enll and pmn counts and thrombopenia duration have been retrospectively analysed in 18 infants with good outcome and in 8 another children with unfavorable outcome. results. neither diarrhoea or its duration, nor children's age, nor blood nitrogen level, nor anuria or dialysis time had any predictive value for the disease evolution in the acute phase of our patients. adversely, extra-nenal involvements was accompanied by severe and complicated courses of the disease (p<0,02). the elevation of white cells and pmns (heyon 20 x 109/i) and pmns (more than 15 x 109/1) as well as its persistence beyon a week were most frequently observed in complicated forms (p<0,001, p<0,001 and p<0,01, respectively). a transient thrombopenia (less than 5 day@ in patients with elevated counts of white cells may be a filrther obvious sign of an unfavorable course of the disease (13<0,02). conclusion. the elevated count of white cells and pmns, either alone or associated to one rapid regeneration of platelets, seems enabled to predict an unfavorable evolution of the hus in children. msud results from an inherited impairement of catabolic pathway of branch chain amino-acids. high leucine blood levels may induce acute brain dysfunction. this dramatic complication led us to propose leucine removal procedures as continuous hemofiltration. patients and methods three newborns in acute msud onset were treated by hf, hdf and hd. extracorporeal circulation was performed through a 6.5 fr catheter, a circuit with a blood pump (priming volume = 40 ml). patients and procedures characteristics are summarized below in the sucralfate (an aluminium salt of sucrose octa sulfate) is used to prevent and treat upper gastrointestinal bleeding in critically ill patients. with minimal absorption, the potential for side effects is thought to be limited, though aluminium toxicity has been reported in patients with chronic renal failure. these patients may already have had high body stores of aluminium. we report 5 critically ill children with high serum concentrations of aluminium following sucralfate therapy. all 5 had renal impairment. the normal aluminium level is < 0.4 gmol/l and in patients with chronic renal failure < 2.2 ].tmol/l. none of these patients had known preexisting chronic renal disease. cpb was conducted under deep hypothermia (t,°16°c) and cardiocirculatory arrest (cca) or under hypothermia (t,°24°c) and low-flow perfusion. continuous holter-electrocardiograms (h-ecg) were recorded from the ilranediate postoperative (po) period on for 72 hours. h-ecg were also recorded prior to the operation and before discharge. following dr were observed: snpraventricutar (sv) and ventricular (v) extrasystoles (es) (>50/24h), sv and v tachycardia (svt and vt), accelerated junctional rhythm (ajr) and junctional ectopic tachycardja (jet), and 2nd and 3rd degree atrioventricular block (avb2 and avb3). the incidence of po dr was 20% in the pre-op h-ecg, 74% on the 1st, 33% on the 2rid, 34% on the 3rd po day and 21% befbre discharge. compared to the pre-op findings, an increased incidence of sves, ves, svt and avb3 on the 1st po day was observed, whereas vt and a jr or jet were exclusively observed po. all types of dr were observed up to the 3rd po day. ty23e of dr before discharge was similar to pre-op findings and there was no definitive avb3. considering patient groups according to the most frequent isolated op-procedure, the incidence of dr on the first po day was 56% after asd ii-closure (n=23), 74% after stthaortal vsd-closure (n=lg), 75% after correction of a complete avsd (n=8), 80% after correction of a tetralogy of fallot (n=20) and 100% after fontan-operation (n=10). incidence and type of dr were not significantly different between groups. longer cpb-dttration and use of cca were risk factors for po ves and vt (p<0,005 and p<0,05, respectively) whereas use of cca and degree of hypothermia were risk factors for the development of a jr and jet (p<0,02 and p<0,0001, respectively). -our results indicate that po dr after cpb in children m'e frequent but mainly transient. in our series, specific cpb-related parameters are of greater influence than surgical procedure itseif for the development of dr and are discriminant risk factors for particular types of dr. the course of anp, cgmp/anp (as indicator for atrial natriurefic peptide biological activity), and no2 and no3 (as indicator for endogenous nitric oxide (no) synthesis) was investigated in i9 infants (median age 4 months) undergoing cardiopulmonary bypass (cpb). patients were divided into 2 groups according to whether they had (group 1, n=13) or not (group 2, n=6) preoperative heart failure (hf) and pulmonary hypertension (pht). group 1 patients had preoperatively significantly higher levels of anp (p<0.005), cgmp (p<0.02) and no2 and no3 (,p<0.02) but had significantly lower cgmp/anp (i0<0.05) than group 2 patients. during cpb, anp was significantly higher in group 1 patients ~<0.02). as compared with prebypass values, cgmp/anp was reduced in both groups during cpb (p<0.0001). cgmp/anp inversely correlated with duration of cpb and aortic clamping time (p<0.001, respectively). no2 and no3 were significantly higher in group 1 than in group 2 patients (p<0.05) without any intraindividual change during cpb. from the early postoperative period on anp, cgmp/anp and no2 and no3 were similar in both groups. after cpb, anp correlated in both groups with blood pressure (p<0,001) and diuresis (p<0.05). no2 and no3 inversely correlated with pulmonary arterial pressure immediately after cpb (19<0.05 patients after a fontan-type of procedure have elevated central venous pressures (cvp) leading to congestion in the gastrointestinal system and often ascites. purpose of this study was to evaluate whether this causes a different postoperative gastric mucosal ph (phi). methods: we evaluated a series of 35 patients, who underwent cardiac surgery with cardiopulmonary bypass (age: 5 days to 16 years (mean 2,2 yrs), weight: 3.2 to 37kg (mean 10.2 kg). a commercially available tonometer (tonometics®) for sigmoidal use in adults was inserted into the stomach after induction of anesthesia. the phi measurements were done according to manufacturer recommendations we compared three groups of patients: 1) aeyanotic (n=20), among them 9 p with vsd and 5 p with avsd; 2) cyanotic (n=10): tof: 6p, tga: 4p; 3) cyanotic after a fontan-type procedure (n=5). phi were measured at picu arrival and after 6h. fudhermore we compared lactat levels at these time points. differences between the groups were evaluated with one way anova on ranks with pairwaise multiple comparisons (dunn's method). the relationship between cvp and phi was investigated by regression analysis. results: the median phi for groups i, 2 and 3 were 7.28, 7.27 and 7.13 at ardval and 7.30, 7.25 and 7.21 after 6h respectively. at picu arrival group 3 was significantly (p<0.05) different from groups 1 and 2. there was no significant difference between the latter two groups, after 6h group 1 was different from group 3, there were no other significant differences. the median lactate levels for groups t, 2 and 3 were 2.2, 3,2 and 4.1 at ardval and 1.6, 3.1 and 3.3 after 6h respectively. at ptcu arrival group 3 was significantly (p<0.05) different from group 1, after 6h there were no significant differences. there was a weak negative correlation between cvp and phi: r= -0.21; p<0.05. conclusion: patients after a fontan-type of procedure have lower phi than patients after other cardiac surgical procedures, however, this is only in part due to the elevated cvp and venous congestion. eleven children were investigated 32 months (median) after postoperative mof. iviof was defined as the failure of at least two vital organ systems (kidney, liver, lung, central nervous system) in addition to cardiac insufficiency and high fever. underlying surgical procedure was repair of tetralogy of fallot (n=3), fontan-(n=7) or seuning procedure (n=l). all patients fulfilled criteria for mof in the 3 first postoperative (po) days. six patients needed peritoneal or hemodialysis for 31 days (median) during the po period. one patient showed cerebral infarction due to thromboembolism in the territory of the right internal carotid artery immediately after the operation. the follow-up protocol consisted of extensive investigations of heart-, renalliver-, and lung functions as well as complete neurological and psychological examinations. all patients had adequate cardiac examination. lung function was normal in all but 2 patients who had an obstructive syndrome. only 1 patient showed an isolated decreased creatinine clearance. abnormalities of the liver ftmction tests were only noticed in patients after fontan procedure. severe neurological sequels such as paraplegia (n = 1) and diplegia (n-i) were observed in 2 of the 11 patients. the remaining 9 children presented with a delayed graphomotorical and speech development associated with normal intelligence. -in our series the most frequent and severe sequels after postoperative mof were neurological. -abnormal liver fimction tests are more likely to be a consequence of the fontan hemodynamics than a sequel of mof. the optimal dosing schedule of surfactant therapy for the treatment of neonatal respiratory distress syndrome (rds) remains unclear. goal: surfaetant function and the concentration of phospholipids (pl) in tracheal aspirates are compared in a prospective randomized trial involving neonates with rds who received either two or more (3 or 4) doses of survanta. methods; ventilated neonates <35w with rds were treated with survanta 1oo mg/kg if fio 2 >_40% or mean airway pressure _>7,5 cm hzo, after 6h a 2nd dose was given (same criteria), if the support still exceeded the criteria 12h after the 2nd dose, the patient was randomized to no extra dose (two}, or to an extra dose of survanta (morel (and a 4th dose 12h later; same criteria), pl was measured in tracheal aspirates and corrected for dilution with the urea method. "active" large aggregates and "non-active" small aggregates of surfactant were separated by centrifugation and quantified. surface tension of the large aggregate fraction was measured by pulsating bubble surfactometer, results: 13 neonates were randomized, 6x two and 7x more (5x3 and 2x4 doses), gestational age was 31,7±2,4w and birth weight 1582±568g. most patients had severe rds with initial ventilation: rate 63.1_+11,1, peak inspiratory pressure (pip) 24,3-+6.4 cm hzo, fio 2 75.3±21.0%. at randomization: rate 63.5±6.9, pip 20.3-+2.5 cm hzo, fio 2 29.5±15.7%, and 24 h after randomization: rate 45.9±17.1, pip 18.7_+2.2 cm hzo, fio 2 26.8±6.6%, without signif, differences between the groups. there was 1 relapse (again fio2_>60% within 72h) in group two and t bpd in group more. in total, 112 tracheal aspirates were analyzed. pl was not signif, different before randomization (two 27.5 ± 15.7 vs more 24.5 ± 11.4 /jmol/ml), but neither after randomization (two 21.2-+ 11.0 vs more 19.3±7,o /~mol/ml). there was no difference in the % small aggregates (two 4.2±1.9 vs more 6.9±5.5%), the surface tensions (ran/m) were not signif, different (each time two vs more): before randomization 10.0±2,3 vs 14.2-+7.2, in the 24h after randomization 12.6±5.0 vs 11.2-+3,8, or 24-48h after randomization 17.0-+5.5 vs 12.8±9.8, or 48-72h after randomization 15.7_+0.4 vs 13.7-+5.6. conclusion: neonates who received more than two doses of survanta did not have higher pl, nor a better surfactant function than neonates who received only two doses of survanta. continuation of the trial is necessary to evaluate clinical outcome. may not indicate need for treatment p.c. clemens s.j. neumann university of hamburg, department of pediatrics, klinikum schwerin, wismarsche str.. 397, d-19049 schwerin. aim of the study: the finding of elevated tsh and decreased t4 in the newborn usually is classified as "transient hypothyroidism", thus the elevation of tsh is classified as consequence of the lowered t4. but on the other hand several data sets show that tsh elevation as well as low t4, one independently of the other one, are associated with different kinds of perinatal stress. each of these laboratory deviations, if not associated with the other value being abnormal too, is generally accepted not to be an indication for treatment. from this we conclude, that more pefinatal stress, as in intensive care neonates, may produce tsh elevation as well as low t4, but only coincidentially, not the tsh elevation being the consequence of low t4, thus not to be classified as "hypothyroidism", thus not indicating treatment. if this hypothesis is right, we should find an association of increasing pefinatal stress with an increasing number of neonates from tsh and t4 normal via tsh or t4 abnormal to high tsh and low t4. method: in the newborn screening program in germa w we determine primarily tsh, and only in the neonates with elevated tsh, in addition we determine t4. thus in our study we asked whether we find an association of increasing perinatal stress with an increasing number of neonates from tsh normal via tsh abnormal while t4 normal to high tsh and low t4. definitions for this study were: tsh elevation = >20 mu/1 (as usual in the german screening programs), t4 lowered = < 6 p_g/dl perinatal stress score was 0 or 1 or 2 or 3 in dependency of the neonate having stress in none to all of the following three categories: (a) forceps or vacuum extraction or sectio co) birth weight below 2500 g (c) at the 5th day existence of a relevant neonatal disorder (rds, ictems gravis, infection/sepsis, vitium cordis with hemodynamic relevance, severe malformation). results: our data of 1131 neonates show a high significant association (chi2 = 84, p <0.001) of, on one hand, perinatal stress score 0 with normal tsh, versus, on the other hand, perinatal stress score 2 or 3 with high tsh and low t4. discussion: facing the background given above, in the intensive care newborn, the constellation of high tsh and low t4 may be only a coincidential addition of two independent abnormalities. in tbese cases -the high tsh not being the consequence of low t4 -the classification as "hypothyroidism" is not justified, thus a therapy not indicated. on the other hand of course there exist rare cases with high tsh as consequence of low t4 thus with hypothyroidism tlms with indication for therapy. unfortunately we have no criteria, that enable a certain discrimination of these two categories thus in respect to the question of therapy or not. conclusion: further research has to be done to learn how to discriminate the coincidential high tsh and low t4 from the causal constellation of high tsh and low t4. until we have certain discrimination criteria we have to treat both groups of neonates. few studies have focused on fa composition of surfactant pc in preterm infants before and after surfactant therapy. methods: tracheal aspirates were collected in 7 venttlated mfants from birth until extubatlon (27/7_1 /twk ga, 859.+ 155g bw). after lipid extraction, t.l.c,, and methylation, fas of pc were quantified by gaschromatography. intralipid a (53.2 % linoleic acid,18:2•6) was started 48h after birth. results: six infants developed respiratory distress syndrome (rds) and received survanta r i00mg/kg (sr), all doses within 18h after birth (ix s r n=l, 2x s r~ n=3, 3x s r n=2). one child did not develop rds. in alt patients, the patmitate % in pc was ~ 65% (before sr<=natural composition), increased to ~ 85% after s r, and remained >80% for i5h after lx s a, 22.3.+i1.8h after 2x, and 38.5.+3.3h after 3 doses. in 4 patients, intubated long enough, the palmitate % decreased with a half-life of 78.7_+42.8h to a new plateau which was still higher than baseline after 1 week. linoleic acid % was 5.85_+2.3 (with rds), decreased after s r~ and returned to baseline due to the decrease in patmitate %. thereafter the linoleic acid % increased linearly with 0.021% per h, in 1 patient even up to 15.1%. other fas did not increase after return to baseline. in neonatal medicine the current parameters, arterial oxygen saturation and arterial oxygen pressure, are poor indicators for oxygen delivery and oxygen demand. the purpose of this study was to obtain venous blood samples from the inferior vena cava in stable neonates with respiratory failure and to determine a parameter that reflects more adequately the balance between oxygen delivery and oxygen demand. "l~e study included 22 neonates requiring mechanical ventilation tbr severe respiratory insufficiency. an umbilical venous and arterial catheter were inserted in the inferior vena cava and in the aorta respectively. paired blood samples were obtained at the time that the patients were hemodynamically stable. fifty paired arterial and mixed venous blood samples were analyzed. 1jnear regression analysis showed the following correlations: in a neonatal intensive care unit adjacent to a delivery room caring for 4000 mothers per year, (with a referral of 400 mostly for preterm delivery), virtually every neonate <ls00g was seen by the same ophtalmologist, j.o. the incidence of retinopathy of prematurity (rop) was in 1976-80 of ii cases (464 preterms <ls00g with 58% surviving to discharge, and 4% rop in survivors; 8% if less than 30 weeks). in 1987-1993, 57 cases of rop were diagnosed by the same ophtalmologist. 679 were born at less than 30 weeks, of which 452 (66%) survived to discharge, and among those survivors, rop was present in 53 (12%), with rates varying from 58% at 24 weeks to 5% at 29. objective: to compare a method of teicoplanine (teico) infusion using an electrical syringe-pump with a manual injection. methods: infusion of teico was simulated through a standart neonatal i.v. system. the infusion system consisted of an life care 4 infusion pump (abbo'it lab.) with its i.v. set for maintenance intravenous fluid (flow < 6 mi/h) connected to a 3-way stopcock. an 1 meter extension tubing (vygon lab.) was placed between the stopcock and a neonatal catheter, an another 1 meter tubing (injection tubing) was connected to the stopcock. the volume of the injection circuit was 2.6 ml. simulations were realised for 2 weights (1 or 3 kg), with a doses of 8 mg/kg and a injected volume of 0,8 ml to 3 ml. our goal was to perform a complete infusion in 10 minutes. we compare one method of infusion with an electrical syringe-pump with 2 manual methods: a: teico was infused with a syringe-pump pilot c (becton & dickinson lab.) according to a protocol using 1) a priming before connecting the syringe to the tubing (for immediate starting of infusion), 2) a programmed volume injected in 5 minutes (the drug volume was greater than the dose prescribed to avoid loss of teico into the syringe), 3) a 5 ml wash out was performed over 5 minutes with the syringe pump. b: teico was manually injected in a few seconds in the tubing followed by a 5 ml wash out over 10 minutes. c: idem as b but a nu site valve (medex) was connected to the proximal end of injection tubing to avoid leakage between removal of the teico syringe and connection of the wash out syringe. during each run, serial samples were collected every ten minutes over a one hour period. the samples were assessed using hplc method. results: the amount of drug delivred at 10 minutes was calculated and expressed as a perventage of the total amount of teico prescribed. results are a mean of 5 to 8 rons. a b c 1kg 94.2+17,9% 86,8+9.1% 94,4-+6.4% 3kg 95-+4,9% 72-+13,4% 90.9-+6% conclusion: with a drug volume injected < circuit volume and a valve, a direct i.v. administration of the drug in the circuit followed by a wash out seems an accurate alternative to drug infusion conbroled by a syringe pump and is easier to perform. doxapram hydrochloride is a central and respiratory stimulant which is used in neonatal intensive care units to treat caffeine-resistant apnoes of the newborn. routinely, doxapram is administered intravenously. oral administration would be much easier but data on bioavailability after oral administration are limited (two studies) and show a large variation. we therefore studied the oral bioavailability of doxapram in 8 preterm infants. doxapram treatment was started with an intravenous loading dose of 2,5 mg/kg during 15 minutes, followed by a continuous infusion of 1 mg/kg/h. after 24 h the intravenous administration was changed to continuous oral administration with the same dosage, blood samples were collected 24 h after both the intravenous and oral administration and analyzed by hplc-assay. apnoea rates per 6 hours were listed before and after starting doxapram treatment. the median apnoea rate per 6 hours declined from 9 to 2 after both the intravenous and oral administration. the oral bioavailability of doxapram is between 43 and 65%, indicating that the oral route can be used effectively in these infants 2. there were no differences in the decline of the apnoea rate after intravenous versus oral administration of doxapram, infasurf r has been comnared with exosurf r in two studies: one as drodhvlaxis and the other a rescue trial. similar. althouah non-sianificant benefits were found for the natural surfactant. in 6 trials there was a sianificant reduction in dulmonarv air leaks for 0.53: 95% ci 00.41-0.641 for babies treated with natural comnared to svnthetic surfactants. for 7 trials (3554 babies~ comdarina natural and synthetic surfactants for rds {6 comdarina survanta r and exosurf r. and one infasurf r and exesurfr% neonatal mortalitv was sianificantlv reduced (or 0.80: 95% ci 0.66-0.971 with natural compared to synthetic surfactant treatment. in two further trials different natural surfactant drenarations were compared. reduced oxvaen needs for 24 hours after treatment were found for r r infasurf and curosurf respectively when each was compared to survanta r. andarent lonaer-term benefits from these surfactants were not statisticallv sianificant. further trials will be needed to ascertain differences between various surfactant preparations. introduction bacterial meningitis is a common cause of admission in the paediatric age group, and one which still carries a high morbidity and mortality. these complications are strongly associated with a delay in antibacterial therapy. a significant proportion of these patients require mechanical ventilation; it is in this particular group of patients that survival is greatly diminished. the identification of risk factors for mechanical ventilation associated with bacterial meninges may help improving survival by shortening the delay to icu referral. the paediatdc risk of mortality score (prism) is the most widely used scoring system in the paediatric icu literature; the accuracy of prism on predicting outcome in meningitis has been shown to be poor. aim we aimed to describe our population of patients with bacterial meningitis requiring ventilation, and then to identify predictors of survival. methods this study was conducted at the baragwanath hospital in south africa which is an universityaffiliated institution with end average annual paediathc admission of 4500 patients. baragwanath icu admits 250 non-neonatal paediatdc patients per year. a retrospective chart review from january 1991 to december 1995 of 42 consecutive paedistdc icu admissions with bacterial meningitis was performed. results approximately 150 cases of bacterial meningitis are admitted to the general paediatric wards every year; this constitutes ± 3% of all admissions. the mortality of these patients is + 14%. during this time 42 (23m, 19f) patients (7%) were accepted for icu admission, with a mortality of 42.2%. the median age was 9 months (range 15 days to 17 years). the median delay to hospital admission was 48 hours, and the median delay to icu admission was a further 4 hours. the median duration of tcu stay was 48 hours (40 for non-survivors, 120 for survivors). the main presenting features were convulsions (34%), altered mental state (40%), fever (50%), respiratory symptoms (24%), headache (18%), and diarrhoea and vomiting (32%). the most common indications for icu admission were seizures (55%), coma (36%), shock (26%), respiratory failure (29%) and acidosis (21%). in 29% of patients there was a cardiorespirstory arrest prior to admission. the most common organisms in the csf was pneumococcus (57%), haemophilus influenza (15%) and e coli (12%). the presence of leucopenia (wcc < 5), a low platelat count (<100), acidaemia (ph<7.2), and the need for inotropic support were strongly associated with nonsurvival. tachycardia and hypotension were not significantly essorjsted with poor outcome, as has been previously reported. discussion early diagnosis of meningitis and prompt institution of antibiotic therapy requires a high level of clinical suspicion. paediatric patients with bacterial meningitis that require mechanical ventilation have a poor prognosis. there is little evidence to suggest that nomsurviva[ can be predicted prior to icu admission, but a rapid deterioration requiring ventilation and inotrepic support with evidence of severe sepsis (low platelet count, leucopenia) is nmost invariably associated with a fatal outcome. the long term functional outcome of these patients make this disease even more devastating; the rote of icu in the management of these patients has yet to be fully estanished. objective and study design: a retrospective study was pertbrmed for all patients diagnosed with hemorrhagic shock and encephalopathy syndrome (hse) during 1984 to 1994. soroka medical center is the only medical facility in the southern negev region of israel serving a population of -400,000 residents, consisting primarily of jews and bedouins. results: 20 patients (17 bedouins and 3 jews) were diagnosed with hse. main features on arrival included profound shock and coma with con~alsions. active bleeding and/or disturbing coagulation tests with falling heuroglobin levels and thrombocytopenia was noted in every case. all infants developed diarrhea shortly after arrival. elevated urea, creatinine and liver enzymes was noted in all cases. annual incidence tbr inlhnts <1 year of age was 5:10,000 for bedouins and 0.6:10,000 for jews. patients ranged in age from 6-32 wks and arrived at the hospital late night/early morning (2:00am-ll:00am), during winter/early spring (november-april). all were healthy prior to admission, with short prodromal symptoms of upper respiratory tract or gastrointestinal infection noted in 10 cases. most infants had markedly elevated body temperature on arrival. a history of overwrapping and/or excessive hearing was obtained in 4 of 20 infants. bacteriological and virological cultures were negative in all infants. one infant died and neurological sequelae were observed in all survivors. the high prevalence of hyperpyrexia during sleep in the presence of negative microbiological results with no evidence of excessive hearing, and the high incidence of rise among a closed, culturally isolated society known to have a high incidence of congenital malformations, may support previous assumptions that hse results from hyqperpyrexia, originating in most cases from a "physiologic" heat induced trigger which starts and peaks during the night in previously healthy infants with susceptible, predisposing genetic underlie. approximately 10% of hospitalised infants with respiratory syaacytial virus (rsv) infection require mechanical ventilation. our general practice is not to use specific antiviral therapy. we have undertaken studies of rsv-infected mechanically ventilated patients without underlying congenital heart disease or immunodeficiency. study i (n=45): a retrospective review of 45 infants (mean post-conceptual age 48 weeks; median duration of intubation was 8 days (interquartile range 5-11 ). blinded review of chest x-rays during the first three days of mechanical ventilation revealed a spectrmn of lower respiratory tract findings from marked diffiase consolidation in all zones without hyper-inflation (n=10) to gross hyperinfiation without consolidation (n=5), with the remaining patients have an intermediate picture (n=30). death occurred only in patients who were at the poles of this continuum (4/10 consolidation and 1/5 hyperinflation). patients at these poles could be differentiated by gender predominance, birth gestation, alveolar-artarial (a-a) oxygen gradient (p<0.0 l), oxygenation index (p<0.0 l), peak inspiratory pressure (p<0.05), and intubation days (p<0.01). study ii (n=28): prospective audit of infants with the aim of verifying the above differentiation. separating patients based on their early x-rays and a-a gradient we confirmed the above predictable difference in duration of supportive therapy (consolidation v intermediate: 6(4-7) v 14( 12-33 ) p<0.01 ). severe lower respiratory tract rsv-infecrion in young infants results in different distinctive partems of disease with characteristic radiological and clinical features, and each has a predictable timecot~se. conflicting reports on special therapy should be interpreted with respect to these observations before making conclusions about the efficacy of any specific treatment. the clinical data of 186 children wich suffered from a severe mycosis between jan.1990-dac.1994 and wich were treated with amphotericin b in 10 spanish hospitals were collected. mean age was 6+3.9 years; 157 were mate (84%) and 29 female (16%). the most common underlying diseases were leukemimymphoma (31%) and congenital heart defects (20%). the major pathogens isolated were candida albieans (61%), other candida (18%) and aspergillus (17%). and liposomal amphotericin (la) in 68 (37%). in the ca group, the starting dose was 0,3~0,1 mg/kg, reaching a maximal dose of 0.9+0,3 mg/kg after 4.7+3 days. maintenance time was 14+10 days and the cumulative dose: 12~17 rag. in the la group, the starting dose was 1.1±1 mg/kg, reaching a maximal dose of 2.9_+2.4 mg/kg (p<0.01) after 5+5 days. maintenance time was 19~16 days (p<0.05) and the cumulative dose 42_+37 mg (p<0.05). the reasons for la use were: elective in 66% of cases, previous renal failure in 29%, and ca inefficacy in 4%. hepatic or renal function impairment was two times more frequent in la group than in ca group, the antifungal efficacy was 62% for ca and 67% for la. therapeutic failure or toxicity induced withdraw of ca in 7% of cases, vs, 1,5% for la (p<0,01). mortality was not different in both groups (21 vs. 22%), adverse effects were related to ca in 28 events vs, only one in la group (p<0,01). the commonest side effects in ca patients were fever (18%), chilis (10%) and nausea (9%). in ca group, 44 cases (37%) developped analitical anormalities related with amphotericin, vs. 10 cases (15%) in la patients (p<0.001). in the first group, the commonest serum alterations were hypokaliemia (25%), raised creatinine (14%) and bilirrubin (6%). in the second one, hypokaliemia presented in 7% of cases (p<0.01), raised bilirrubin in 6% and creatinine in 3% (p<0,05), the antifungal efficacy of la is at least the same of ca. from the clinical and analitical points of view, la is much less toxic than ca. la can be used at a greater dose than ca and is safe in children with renal failure. laurence desplanques -serge gottot -christian dageville d~artement de sant~ publique -facult~ xavier bichat -16 rue henri huchard 75018 paris -france -the french << reaped ~> network was created to implement a nosecomial infections (ni) quality care program in nicu and picu, the first objective was to describe the annual ni incidence rate in each icu population : all patients stayed more than 48 hours in icu. methods : n] criteria were defined by the reaped group according to cdc criteria. all data were collected by a medical and nursing team. all infection data were validated by an external investigator. results : 4525 patients were admitted over a 14 months period. 68% were newborns. 371 ni were identified among 311 patients. the overall ni incidence rate (ir) was 8.2% and 5.9°/00 person day (from 5.0 to 8.2°/00 according to age, lowest rate for newborns). septicemia (50% of ni) and pneumonia (41% of ni) were the two main ni. according to age, the septicemia ir varied from 6.8 to 10.9°/oo catheter day (lowest rate for newborns) and the pneumonia ir from 3.9 to 7.4°/00 ventilator day (lowest rate for newborns). there were very few other infections (uti : 4%, ir : 7.4°/00 catheter day). gram positive cocci were isolated in 73% of septicemia ( 70% of them were coagulase negative staphylococcal). gram negative bacilli were isolated in 53% of pneumonia (40% of them were pseudomonas). 5% of ni were caused by candida, mostly septicemia. the septicemia and pneumonia ir varied according to unit even after adjustment for age. discussion the aminoglycoside antibiotics are frequently used in newborns for the treatment of severe infection and sepsis due to gram-negative microorganisms. the currently recommended dosage schedule for tobra (2.5 mg/kg q18h) does not take into account differences in gestational or postnatal age during the first 4 weeks of life. we questioned the validity of these recommendations and studied the population kinetics of tobramycin to establish predictive equations that enables the clinician to select the appropriate initial dosing schedule. methods tobra trough (t=0) and peak values (t= 1) were taken on day 2-4 after birth in 460 newborns. tobra was administered as a 30-minute intravenous infusion already in an adapted dosage schedule: 3.5 mg/kg q24h in infants with gas < 28 weeks; 2.5 mg/kg q18h in infants with gas between 28-36 weeks and 2.5 mg/kg q12h in infants with gas > 36 wks, tobra concentrations were analyzed by tdx-assay, a one-compartment model was assumed and non-linear mixed effect modelling (using nonmem) was applied to the data, a trough level < 2 mg/l and a peak level between 6 and 10 mg/l was required, with the present dosage scheme 40% of the trough levels were too high and almost 60% of the peak levels too low. calculations showed that the following dosage schedule should result in optimal levels of tobra. preterm infants gas < 28 wks: 6 mg q48h preterm infants gas 28-36 wks: 4.5 mg q36h preterm infants gas > 36 wks: the currently recommended dosage schedules for toeira result in high trough and low peak levels. prolongation of the dosing interval and increasing the amount of drug per dose according to the above scheme will improve tobra level control. since january 1993 british clinicians have been conducting a randomized controlled trial of neonatal ecmo. mature infants (>-35 weeks gestation and birthweight 2 2 kg) with severe cardiopulmonary failure have been randomized to receive continued care in their referring institution or referral to a designated ecmo centre for further management. we now present the preliminary results which have prompted closure of recruitment to this trial. the final outcome will be assessed as intact survival against death or severe disability at one year of age for all the recruited patients. patients were categorised by diagnosis such as isolated persistent fetal circulation, secondary persistent pulmonary hypertension of the newborn or congenital diaphragmatic hernia and by severity of illness at the point of first contact with the clinical coordinators of the trial -judged primarily by the oxygenation index (240 before randomization). 180 patients were randomized (90 in each arm). hospital outcome data are reported for all patients and 1 year outcomes on t18 (65 survivors). at this stage 26 of the babies allocated to ecmo are known to have died compared to 52 of those allocated to conventional management (rr 0.5; 95% ci 0.35-0.72; p=0.0002). fewer deaths have been obsea-ved amongst ecmo allocated babies in all the diagnostic categories used. a 28% incidence of disability and impah~nent has been observed amongst survivors. this rate is similar in both groups and the survival advantage is not offset by an increased rate of disability or impairment following allocation to ecmo. we consider that these data combined with those available from other studies provide conclusive evidence that the survival to discharge from hospital is substantially higher in patients allocated to ecmo than in comparable infants not so allocated. therefore recruitment to this trial has been closed whist awaiting complete one year outcome data. sigston pe, goldman ap. #keating j. crook r. ~e dj~. great ormond street hospital for children nhs trust, and ~biochemistry department, kings college hospital, london, united kingdom. isoflurane is a safe and effective means of long term sedation in both children and adults in the intensive care setting. the use of isoflurane, by adding it to the sweep gas allows the use of this volatile anaesthetic agent in patients on ecmo, enabling rapid control and weaning of sedation. a potential problem with the long term use of isoflurane is fluoride ion accumulation with the possibility of renal toxicity, the purpose of this study was to assess plasma fluoride levels in patients receiving prolonged isoflurane on ecmo. method: fifteen infants and children (aged 1 day -10 years, median 2 weeks) receiving ecmo support for either cardiac or respiratory failure were recruited to this study. the patients were sedated with isoflurane as well as intravenous agents (morphine and midazolam). isoflurane was administered (0% -3%) via a calibrated vaporiser to the sweep gas, adjusting the level to maintain adequate sedation. blood samples were obtained on a daily basis for plasma inorganic fluoride assay. the relationship between plasma fluoride and amount of isoflurane administered, as %-hours (vaporiser setting in % x hours) was calculated by linear regression. results: the duration of ecmo ranged from 42 to 532 (mean 207) hours, during which the amount of isoflurane administered varied from 7 to 418 (mean 168) %-hours. 75 blood samples were anaiysed, demonstrating individual peak plasma fluoride levels of 2.7 to 16.5#mol/1, mean 7,1p.molli (toxic threshold = 50gruel/f). the plasma fluoride positively co;related with the %-hours of isoflurane (r = 0.65, p = < 0.001). conclusion: this study shows that although there is a dose related accumulation of inorganic fluoride ions in patients sedated with isoflurane on ecmq, the peak fluoride levels are well below the suggested toxic threshold. merzel y, lev a, bar yosef g, halbertal m, lorber a ecmo center, picu, emek medical center, israel. the mortality rate of pediatric patients with acute myocarditis is 20-60% according to the severity of myocardial damage. a 15 month old gzrl presented with high fever, respiratory and cardiac failure. diagnosis of acute myocarditis was made and the patient was ventilated with high pressures and fio2 of 1.0. she required high doses of inotropes. echocardiography revealed a dilated la and lv with severe mr. lvedd was 41 mm and lvsf 9%. calculated oxygenation index was 55. she was resuscitated after a cardiac arrest. she was commenced on ecmo (using biomedicus centrifugal pump and avecor 800 oxygenator) at a flow of 100 ml/kg/mm with immediate improvement of hemodynamlcs, oxygenation and pc02. resptratory assistance and vasoactive drugs were reduced. the patient was transported by air, on ecmo, to the ecmo cevter. she developed arf and cvvh-d was performed. cardiac fimction started to improve after 12 days. ecmo was discontinued on day 18. echo revealed lvedd 34 mm and lvsf 24%. ippv was discontinued on day 20. on discharge, a month later, her lvedd was 29 mm and lvsf 28%. she behaves normally for age without neurologic or other medical sequellae. literature search revealed no case of acute myocarditis, as severe, that was treated successfully. survavors of disease this severe usually suffer dilated cardiomyopathy and permanent disability. the use of ecmo allows myocardial rest which prevents long term myocardial damage. introduction ecmo is increasingly used in the care of critically ill newborns. despite the frequent use of betalactam antibiotics in the treatment of these infants there are no data available on the dispbsition of cefotaxime (ctx) and amoxicilfin (am) d0ring ecmo. the purposes of this study were to determine the pharmacokinetics of these two drugs in infants on ecmo and consequently formulate appropriate dosing regimens. we therefore studied the pharmacokinetics of ctx (100 mg/kg ql 2h) and am (50 mg/kg q6h) in 8 term infants on day 3 after birth, blood samples were taken before (t-o) and 0.5,1,2,4,6 (am) and t2 h (ctx) after the intravenous bolus injection and analyzed by hplc-assays. 2. ctx 100 mg/kg q12h results in adequate serum levels of ctx in fullterm infants on ecmo, am 50 mg/kg q6h results in very high serum trough levels. recalculation based on the known volume of distribution and elimination serum half-life of these infants resulted in the following dosage recommendation: 50 mg/kg q12h. persistent pulmonary hypertension of the new-born (pphn) is characterised by rapid fluctuations in pulmonary artery pressure (pap) and a clinical impression of stifflungs. lung mechanics were measured in 35 term infants, mean age 1.5 +_ 0.7 days who were paralysed and ventilated within the first three days of life. fourteen infants had pphn with systemic or suprasystemic pap measured by echocardiography. in these patients, the respiratory system resistance was 29.4% higher (p < 0.001) and compliance 22.4 % lower (p = 0.03) during systemic or suprasystemic pap compared to when the pulmonary hypertension had resolved. in contrast, there were no changes in resistance in the 14 infants with respiratory distress syndrome (rds) and no pulmonary hypertension or in the seven infants with normal lungs, where two readings were taken 24 hours apart. the changes in lung mechanics interfered with mechanical ventilation, resulting in a 12.5 mmhg rise in paco2 (p=0.007) during pulmonary hypertension. inhalation of nitric oxide 10 ppm resulted in a 16% decrease in respiratory system resistance and an improvement in oxygenation. the bronchial and vascular smooth muscle was increased by 120% in postmortem lung samples from eight infants with pphn compared to six age matched post-mortem controls with normal lungs (p<0.001). these findings suggest a co-constriction and co-hypertrophy of bronchial and vascular smooth muscle during pphn. anatomically the pulmonary vasculature and bronchi lie in close proximity to each other. thus mediators such as endothelin-1 released locally may act on both vascular and bronchial smooth muscle to produce the observed vasoconstriction, bronchoconstriction and smooth muscle hypertrophy. prince of wales children's hospital university of new south wales, randwick, n.s.w. australia. introduction an increasing mortality in asthmatic children has been reported. the increased severity of asthmatic illness leads to an increased demand for icu admission, and a corresponding increased need for mechanical ventilation. geographic end environmental factors are thought to be partly responsible for differences in disease sevedty throughout the wodd. for this reason, epidemiological studies from diverse areas are important, risk factors for icu admission, and for the institution of mechanical ventilation should be identified, to optimise icu admission criteria and to avoid unnecessary delays in admitting at-risk patients. aim to document the clinical characteristics of ventilated and non-ventilated asthmatic patients admitted to icu. methods this is a retrospective study of all paediatric asthma icu admissions from january 1990 to december 1995. results there were 65 patients admitted to the icu for acute severe asthma in the study period. the male:female ratio was 33:32, the mean age 76.1 • 57.3 months, the mean prism 8.5 4-11.1%, and the mean duration of admission 135 4. 129 hours. there was no seasonal variation in admissions. only 40% (26/65) patients required mechanical ventilation. in 22% of all patients this was the first presentation with asthma. there were some significant differences between ventilated and non-ventilated patients (see table) . there was a significantly higher incidence of concomitant and nosocomial pneumonias in the ventilated patients (84.0% vs 21.1%) as well as segmental lung collapse (68.0% vs 26.3%). there were no deaths. discussion the need of mechanical ventilation significantly increases the morbidity of and duration of icu stay of asthmatic patients. younger asthmatic paediatdc patients have a significantly higher risk of ventilation. the need for ventilation is predicted principally from a worsening pco2 and respiratory acidaemia, which is often independently interpreted by the clinician as respira4ory exhaustion. this study has shown that icu admission is important in the management of young paediatdc patients with acute severe asthma and respiratgry fa!!ure. intravenous salbutamoi in the emergency, department management of severe asthma in children. g.j.browne,a. perma,x. phung,m.soo westmead hospital, sydney, australia. it is postulate that if an initial intravenous loading dose of salbutamol is given in severe asthma, a more rapid clinical response will occur, reducing requirements for continued high doses of nebulised salbutamoi with fewer side effects. this double blinded study was conducted in the emergency department of westmead hospital a university hospital in sydney, australia. all children with severe asthma had initial nebuliser therapy (5rag of salbutamol with 4ml of saline). if asthma remained severe 20 minutes later, they were given a dose of intravenous hydrocortisone (5mg/kg) and either normal saline or salbutamol 15microgm/kg intravenously. frequent nebulised salbutamoi therapy continued during the initial first hour if clinically indicated. continuous respiratory and haemodynamic monitoring occurred in the first 2 hours. serum potassium and glucose determinations were made at study commencement and 1 hour after intravenous therapy. salbutamol determination was made at study commencement. children remained clinically monitored for the next 22 hours, with their ongoing treatment determined by clinical response. 29 children with severe asthma 12 months to 12 years of age were studied, with 14 given intravenous salbutamol and 15 given intravenous saline. the intravenous satbutamol group (ivsg) showed rapid reduction in asthma severity scale in the first 2 hours, with reduced need for high frequency nebuliser therapy ( _<2 hourly), occurring 8.78 hours.earlier. no clinically significant side-effects were found in either group, although, tremor more frequent in the [vsg. biochemistry and salbutamol concentrations were similar in both groups. the use of intravenous salbutamol (i5 microgm/kg) in the management of severe childhood asthma is a safe and effective therapy with no significant side-effects and the potential to abort severe asthma attacks in the emergency department. intravenous terbutaline in picu piva j., amantra s, rosso a., zambonato s, giugno k, maia t. introduction: the admission to a picu of children with respiratory failure secondary to an acute obstructive lower airway disease is a common event, especially during winter seasons. these diseases have several causes, but most of them (especially asthma and chronic airway disease) have a good response to the administration of b2-adrenergic drugs. objective: to find the dosis of intravenous terbutaline that is safe, efficient and with minimal adverse effects when used in children admitted to a picu with acute obstructive lower airway disease and respiratory failure. material and methods: we study the records of all children that were admitted to our picu during the winter of 1995. only the patients that had respiratory failure and acute lower airway disease and who needed the use of iv terbutaline were selected. the records were divided in two groups: less than 12 months and more than a year old these two groups were compared in the following aspects: the minimal and maximal dosis, and the length of time of use of iv terbutaline, frequency of tachycardia, hypokalemia, and mechanical ventilation. to establish any difference in the two groups we use the t exact test of fisher and x2, with p< 0.05, results: during the period of study were admitted 367 patients to the picu, and 38 (10,3%) of them used of iv terbutaline. the mean age was 14.2 +12.2 month, used iv terbutaline during 7.24 +3.75 days (0.5 to 17 days), the initial rate was 0.55 +0.26p~g/kg/min, and the means of therapeutic dosis was 2.48 +l.181~g/kg/min (ranged from 0.5 to 4.4). twelve (31.5%) patients had tachycardia art obstacle to the increases in the rate of use of iv terbutaline during any time. mechanical ventilation was necessary in 22 patients (57.8%) and 11 (28.9%) patients died. the children under 1 year of age used initial dosis of iv terbutaline lower than the children up of 1 year old (0.45 p.g/ kghnin x 0.57 ~tg &g/rain, p<0.001), but without difference in the length of use, the maximal dosis, the rate of mechanical ventilation and tachycardia. the frequency of hypokalemia was most common in the group of children under year of age. acute respiratory failure during status asthmaticus may require mechanical ventilation. current therapy includes paralysis, pressure control ventilation (pcv) and permissive hypercapnia to limit pulmonary barotranma and its hemodynamic consequences. asthmatic children exert a significant amount of respiratory effort during exhalation. with paralysis, this expiratory effort is lost. unloading the inspiratory work of breathing while maintaining the patient's expiratory eftbrt using pressure support ventilation (psv), may be beneficial. methods: children receiving pcv (peak inspiratory pressure (pip) = 4 kpa. rate 10 breaths/min) and pco2 > 8 kpa were switched to psv. children were initially ventilated with psv 3.7 kpa and peep = 0.3 kpa (servo 900c). all children received beta agonist therapy, ipratropium and anesthesia with ketamine or inhalational anesthesia, and were breathing spontaneously. respiratory parameters and blood gases are shown be~bre psv, within 30 minutes (start) and when the ph had normalized (during). data are presented as median and range, * p < 0.03 compared to before psv. results: children with hypercarbia during pcv responded to psv, normalizing pcos and ph within 6 hours. the mean respiratory rate decreased from a median of 45 (31-46) to 35 (22-35) while the pip was decreased to 3.2 (2.5-4.0) kpa within 6 hours. the i:e ratio also significantly decreased. conclusion: psv permitted patients to active/y exhale while unloading the inspiratory work of breathing. perhaps this strategy shifts the patient's respiratory effort from inspiration to exhalation, thus permitting the child to meet the excess work of breathing caused by bronchoconstriction. maged z. youssef, peter silver, laura nimkoff, and mayer sagv. division of pediatric critical care medicine, schneider children's hospital, new hyde park, ny 11040. introduction: mechanical vemiladon of patients with severe bronchospasm can be difficult, due to poor chest compliance and increased airway resistance. ketarmne is a cormnonly used anesthetic agent that has been shown to have bronchodilator properties. the purpose of this study was to determine ifa continuous infusion of ketamine had an effect on the oxygenation and chest compliance of children with severe lironchospasm who were mechanically ventilated. methods: a retrospective chart review was conducted of pediatric patients in severe bronchospasm who were mechanically ventilated in our picu and treated with a continuous ketamine infusion. all patients were receiving aggressive bronchodilator therapy and adequate sedation prior to keramine. patients were excluded if any new bronchodilator or sedative agents were started within 24 hours of initiation of ketamine treatment. all patients were simultaneously treated with benzodiazepines. for each patient, the pao2/fio ~ ratio and dynamic compliance [tidal volume/(peak imp. pressure -peep)] was determined immediately prior to ketamine, and at 1, 8, and 24 hours post-ketsmine initiation. data are presented as mean ± s.d., and were a~yzed using one way anova and the multiple comparison method of bonferroni. patients (age 6.0 ± 5.7 yrs.) received * p<0.05 ketamine for severe bronchospastu during mechanical ventilation in our picu. both . .xto-* * the pao2/fio2 ratio and dynamic . . -.... . compliance increased significantly following initiation of the ketamine 200infusion (see figure) . the mean ketamine dose was 32 ± 10 mcg/kg/min, and the -, mean infusion duration was 40 ± 31 too-[/ hours. one patient required glycopyrrotate 6 ~' to control excessive airway secretions, and " one patient required an additional dose of o--j i ~-~4 ~/me diazepam to control hallucinations after i 8 cessation of ketamine. all patients were t~n~,mr~ *~am~ successfully weaned off mechanical ~l~s ~,~s~on ventilation and discharged from the picu. conclusion: continuous ketamine infusion to mechanically ventilated pediatric patients with refractory broncliospasm results in a significant improvement in oxygenation and dynamic compliance of the chest. reports of adults with status nsthraaticus document significant morbidity and mortality, whereas studies in children have had more varied results. different centers report mechanical ventilation (mv) in 10 to 33% of admissions, occurrence of pneumothoraces or paeutuomediastinums in 2 to 11%, and mortality in up to 7% of patients ~'t3. we retrospectively reviewed 113 status asthmaticus admissions to the pediatric intensive care unit (picu) between january 1993 and december 1995. seventy-five of these patients were admitted fr~an the emergency department of chla (er admit). the mean length of stay in the picu was 2.1 days and the mean length of stay in the hospital was 4.6 days. based on 95 patients who had arterial blood analyses, 36 patients had hyperoapnia (pco2 > 45). all patients received oxygen, inhaled albuterol (alb), and cortieosteroid therapy. ninety-five percent of patients also received methylxanthine (mx) therapy. of the 113 admissions, 12 patients (11%) required mv. only 4 of these patients were admitted through our emergency department, whereas the remaining 8 patients were intuhated at outside facilities. twenty-three cases required intr:wenous beta-agonist therapy, either isoproterenol osop) or terbutaline (terb). h~ff of the ea.~es re~%wed were complicated with hypokalemia (k+< 3.5). c,', ,~lications ofpoeumothoraces or pneumomediastinums were seen in 10% of ,'r:u~ported patients, but in only 4% of er admit patients. only 2% of these were in mechanic.all, )atients. there were no deaths in the review. respiratory mechanics measurements 'are useful in mechanically ventilated children to optimize ventilator settings. nevertheless, the transducers used to measure flow (f) and pressure (p) remain expensive. objective. to evaluate the performances of piezoelectric p transducers (350 us dollar) in measuring f and p. methods. we used a previously described monitoring system measuring respiratory parameters [ 1] . in this study f was obtained by a differential piezoelectric p transducer (_+ 12.7 cmi-i20, honeywell) whose sensitivity has been reduced to +_ 2 cmh20 by an electronic amplification equipment and p by a piezoelectric p transducer (_+ 7().3 cmhzo, honeywell) connected to a grid pneumotachymeter &nt) ffleisch 0 or 1 ). volume (v) (5 to 400 ml) obtained by numeric integration off (0.125 to 10 l/rnin ) and p (2 to 70 cmh20) were respectively delivered through a calibrated seringe and an electronical manometer (pic 400 premier) and calculated by the computer. bland and altman analysis was used for assessment of results bias. coefficient of repeatability (cr) was estimated by the standard deviation of repeated measurements of the parameters as calculated in a oneway analysis of variance. results. mean difference (mdi 0 between injected v (5 to 50 ml) and measured v using pnt 0 was 0.15 ml, sd = 0.13 ml. difference and mean v were not correlated. sd of repeated v measurements were not correlated to v. cr was 0.4 ml. mdif between injected v (25 to 400 ml) and measured v using pnt 1 was 3 lrd, sd = 6 ml sd of repeated v measurements were not correlated to mean v. cr was 6 ml. mdif between injected p and measured p was 0.3 cmi-i20, sd 0.4 cm h20 sd of repeated p measurements were not correlated to mean p. cr was 0.3 cmh20. conclusion. inexpensive piezoelectrical transducers can be used to measure f and p and evaluate respiratory mechanics in ventilated children. previous studies have already shown the problem of the reproducibility of pft in preterm ventilated babies. were studied 10 preterm ventilated babies {mean weight 1128 gr) in the first week of life in clinically stable condition, measuring flow, airway pressure and esophageal pressure simultaneously. each baby was studied twice with an interval of one hour and each study was done increasing the rate till 60 to inhibit spontaneous breaths. none sedative has been used. only mechanical breaths were analyzed. compliance and resistence were calculated with a computer system using the linear regression method. we expressed quantitatively the intrapatient variability as the percentage of variation of tidal volume, compliance and resistence between the two studies in each baby. then intraclass correlation coefficient test (icc) was applied to confirm qualitatively our results (total agreement =1, good reproducibjtity > 0.75). we h~£ed, an a6eept~ble ~efiabirl¢, ~-~r;= '~ . during mechanical ventilation, an air leak (al) and plateau phase duration (pl) may influence dynamic and static compliance (cdy and cst, respectively). this study evaluated the effect of al and pl on two methods of measuring c.dy and est. methods. 13 intubated, ventilated patients in a pediatric intensive care unit were evaluated after obtaining informed consent. patients were intuhated with a cuffed endotracheal tube and ventilated with a serve 900(2 ventilator. cdy and cst were determined using the serve ands~rmedics 2600. objective: evaluate the repercussion in respiratory mechanics and arterial blood gases and the impact of the ventilator adjustments on the auto-peep magnitude. material and methods: the measurement of the auto-peep was performed using an eletronic-pneumatic controlled device with a oclasion valve installed between endotracheal canutla and the ventilator circuit. the d~'ice was connected to a solenoid to detecte the end of inspiratuo phase and thus, the activation of the oclusion valve. the signs of pressure and flow were monitorized using a diferential transducer and it was processed using a pc computer and tmeumoview® software. the stud3 were divided in 2 phases: phase a. where the ventilator adjustments was performed using the routine of the unit and phase b, where the targets of mechanical ventilation were to minimize the auto-peep. static compliance (crs) was ineasured by the single-breath occlusion technique, using a mean of ten occlusions for analysis. passive respiratory resistance measurements and the tidal breathing flow-volume loops were also obtained., while the ventilatory settings were siguificantly reduced soon atier ecmo was started. before ecmo crs measured in all patienls was 0.23_+t).03 ml/cmh20/kg (mean_+sem). for each patient the ecmo course was divided into four periods, proportional to the duration of the treatment, and the best ~alue of crs in each period was chosen for analysis. as shown on the figure. crs significantly improved (*p<0,05) from the second half of the ecmo course in the group of patient that finally were successfidly weaned from ecmo. no change ill compliance was measured in the group of patients who failed to respond to the extracorporeal hmg support our data suggest that compliance measurements during ecmo can be useful togelher with overall clinical evaluation to predict both outcome and duration of cxtracorporeai support in the neonatal and pediatric population. objectives: brain temperature determines the amount of neuronal damage caused by hypoxic insults. thus measuring brain temperature at standardised conditions is in request. we investigated whether brain temperature of neonates varies with head insulation environmental temperature, body activity and time course. patients and methods: we investigated non-invasive brain temperature analogues in 19 healthy prematures tess than two weeks of age in an incubator (gestational age 31.5 + 2.1 wks; x + sd, weight 1653 + 370 g). we measured nasopharyngeal temperature (tnasoph) by a thermistor placed in the nasopharynx via a feeding tube, zero-heatflux temperature (zht) at the temple by a thermistor and healflux transducer, insulated by two pads, as well as rectal and incubator temperatures. patient activity was documented by video taping. measurements were performed during periods of increased insulation 1) by turning the head with its measuring site on to the mattress ( (5) 3 (5) -2 (6) 4 (5) 0 (6) 4 (3) 2.5 2 4(5) 25(10) 20{12) 8(5) 5(10) -2(7) 5 6 38 (22) 112(34)i70 (48)51(27) 20 (18) 5(15) 7.5 3 38 (19) 125(21) t85(29) 120(30) 70(30) 30(20) 10 4 53 (30) 133(28) 182(33) 157(24) 154(34) 110(45) web 2170 (lmg/kg) 5 at 30 rain 3 3 (5) -4 (6) 5 (6) 4 (3) 5 (4) -3 (6) the vehicle had no effect. paf caused dose dependent rise in ao and pa pressure and reduction in flow to lpa (up to 80% like the vascular endothelium, the endocardial endothelium (ee) has a significant impact on adjacent myocytes, and may critically alter myocardial function.~ we have previously shown that ee cells are capable of sensing and responding to hypoxia by the release of prostacyclin (pgl). 2 potassium channels in other cell types have been reported to be oxygen sensitive. to determine whether potassium channels modulate the ee hypoxic response, we investigated the effects of three potassium channel inhibitors on hypoxia-induced pg] 2 release from ee cells. methods: ovine endothelial cells were harvested and passaged onto 30 ,~ microcarriers. cells were constantly perfused with normoxic and hypoxic kreb's solution, and with three potassium channel blockers: glibenclamide (gb, 3 #g/ml), tetraethyl-antmonium (tea, 10 ram) and 4 aminopyridine (4ap, i0 mm), perfusate was assayed for prostacyclin (ria). data were compared by analysis of variance. * p<.05 compared to 3normoxic control; # p< .05 compared to hypoxic control. adrenaline is extensively used for resuscitation in neonates with rds. however, effects of adrenaline on systemic, pulmonary and cerebral hemodynamics have not been defined in newborns with rds. thirteen anesthetized, and ventilated newborn piglets were subjected to repeated saline lung-lavage series while mean systemic arterial pressure (abp), mean pulmonary arteriat pressure (pap), mean left atrial pressure (lap) and mean central venous pressure (cvp), cardiac output and blood flow in the internal carotid artery (ica) were measured. systemic vascular resistance (s~), pulmonary vascular resistance (pvr) and cardiac index (ci) were calculated. sixty minutes after luug-lavage, the adrenaline group (a) (n=6) received adrenaline as a continuous infusion of 1.2 lag/kg/mi, while the control group (c) (n=7) received saline. none of the varlables were changed by saline. however, significant increases in abp (p<0.0001), pap (p<0.0001), ci (p<0.001) and svr (p<0.01) were observed after administration of adrenaline, whiie pvr and ica were not modified. mean±sd for abp/pap (p/a), fvr/svr (p/s) and ci (ml/mirdkg) were: ratios of pap/abp and pvpjsvr significantly increased following infusion of adrenaline. these data suggest: 1) the cerebral perfusion is preserved during the infusion of adrenaline; 2) effect of the adrenaline infusion on the systemic circulation is more pronounced than its effect on the pulmonary circulation in newborn piglets with surfactant deficiency. s demirak~a, ch knothe, kj hagel, j bauer department of pediatrics, justus-liebig-university giessen, frg inhaled no is a short acting selective pulmonary vasodilator. we studied the effects of 80 ppm no and 100% oxygen during heart catheterization in 16 children (age 1 -6 years, median 9 years) with heart defects and elevated pulmonary vascular resistance index (pvri) in order to asses the value of no as a tool of decision making for corrective cardiac surgery. patients were eligible for testing when they were more than one year old and had a pathologically elevated pvri in a previous heart catheterization. intubation, 'anesthesia and muscle paralysis were performed in all patients during testing of pulmonary reagibility. calculations of pulmonary vascular resistance and flow were based on the fick method. response to no was assumed when pvri declined more than 30%, 9 of the 16 patients were responders to no. effects of no and oxygen on pvri, mean pulmonary arterial pressure (mpap) and pulmonary vascular flow (qp) in all responders are described in the table below. cardiac surgery was offered to all responders, and 5 of them were successfully operated. surgery is planned in another 3 patients and parental consent for surgery was not given in one patient. in ebstein disease, during the first days of life, the ability of right ventricle to propel blood to the pulmonary artery is impaired due to high pulmonary vascular resistances. the flow is mainly directed to left atrium through tricuspid insufficiency, right atrium and foramen ovale. to decrease pulmonary resistances and increase pulmonary blood flow, high frequency oscillations, mechanical ventilation, nitric oxide and prostaglandin are required. after few days, a forward circulation is normally established. we cared two newborns with ebstein disease where this approach was hindered by a large pulmonary valve insufficiency. both of them were diagnosed in utero, showing a large tricuspid insufficiency with a non opened pulmonary valve and a ductal left to right shunt. one fetus was hydropic. at birth, blood stream from the ductus arteriosus was directed to the right ventricle through the pulmonary valve insufficiency then to right atrium, left atrium and ventricle, aorta and ductus arteriosus. a low pulmonary blood flow was demonstrated by low mean velocities (10cm/sec). a high reverse flow was seen in descending aorta with a negative flow in the renal artery. both of these newborns were oliguric because of ductus arteriosus steal. pulmonary blood flow doppler evaluation allowed different strategies of ventilation, switching between hfo and conventional ventilation, modulation of pge1 doses, inhaled pulmonary vasodilators (nitric oxide) and surfactant. the hydropic baby died, the other survived after 3 weeks of intensive care complicated by supraventricular arythmia (wpw). in conclusion, during neonatal period, in ebstein disease, a large pulmonary insufficiency leads to a vicious circle where lungs are excluded, inducing severe asphyxia and high pulmonary resistances. the blood is backward propeled from the aorta through the ductus arteriosus to the right ventricle and atria, then left cavities to aorta. arec must be considered when pulmonary blood flow does not increase despite optimal therapy. guti~rrez-larraya f*, mandoza a*, velasco jm*, zavaneua (3**, gatindo a ~, s&nchez-andrede r, s&nchez jl***, mellon a***, mar f***. pediatric cardiology*, pediatric cardiac surgery**, pediatric intensive care unit***. hospital 12 de octubre. madrid. background: transesophageal pacing (tp) is effective and sate both for diagnosis and treatment of pediatric arrhythmias. material and methods. eleven consecutive patients are included. a tri or quaddpolar 6 or 7f temporal transvenous catheter with an interpolar distance of 13 to 22 mm was advanced through the nares and positioned to the point with the largest amplitude of atrial deflection, surface ecg and a bi or monopolar electregram were recorded simultaneously, selecting filters when needed (5 to 100 mhz). pacing was performed with a programmable stimulator (medtronic 5328) beginning with 2 ms and increasing ma to 10 and then increasing up to 9.9 ms. narula method was selected to diagnose sinusal node disfunction (snd) and overdrive pacing to treat tachyarrhythmias. results. tp was useful in all the 11 patients and no complications were observed: in 3 patients a snd was diagnosed (one needing a definitive pacemaker), in two patients with atrial ratter (ripe 1) sinus rhythm was recovered, in one patient with a postoperative junctional ectopic tachycadia we were able to get atrial synchrony with marked bemodinamic improvement, and 5 patients with paroxysmal supraventricular tachycardia sinus rhythm was easily and quickly restored (2 of them recquirad repited episodes of tp until pharmacelogycal levels of antiarrhythmic drugs were raised). mean age and weight were 31 months and 12.7 kg (one patient had 2.1 kg). there was a close relation between height and depht insertion (r= 0.98). mean stimulation parameters were 9,1 ms and 13.5 ma. discussion. in experiencied hands tp is an effective and safe way to treat and diagnose cardiac arrhythmias even in newborns. it should be tried before endovenous pacing is stablished and it is faster than pharmacologycal treatment. bailing g., eicken a., sebening w., vogt m., schumacher g., bl~hlmeyer k.; kinderkardiologie, deutsches herzzentrum m0nchen, germany to assess the outcome of balloon valvuloplasty in infants with cardiac failure caused by critical aortic stenosis a retrospective study was performed. between 1986 and 1995 33 neonates, aged 1 -28 days (median 9 d), weight 2.t -4,1 kg (median 3,3 kg) with critical valvar aortic stenosis were dilated by balloon (aovp) as the first line treatment. 21 patients received prostaglandin el, 18 needed inotropic drugs and 16 mechanical ventilation. associated cardiac lesions : persistent ductus arteriosus (pda) in 27 patients (restrictive pda in 8 cases), a mitral regurgitation (mivr) in 27 cases (15 severe and 12 moderate or mild mivr), angiographic findings of endocardial fibroelastosis (efe) in 12 patients, mitral stenosis (mivs) in 8, coarctation of the aorta (coa) in 2, and finally a small musculary ventricular septum defect (vsd) in i patient. vascular approach for ballooning : a. axitfaris in 20 cases (61%) a. femoralis in t 0 (30%) and v. femoralis in 3 cases (9%). the median ratio between inflated balloon and aortic valve diameter was 0,99. dilatation was achieved in all 33 cases. the peak systolic gradient across the aortic valve (pre aovp) ranged from 0 to 137 mmhg (median 50 mmhg) and was reduced to 0 to 55 mmhg (median 15; gradient reduction is significant (p < 0,01)). aortic regurgitation (aovr) was absent or mild in 30, moderate in 2 and severe in 1 patient after aovp. 23 children survived (actual suwival rate: 70%; early mortalffy: n = 3; late mortality: n = 7). mid term follow up (0-8,8 years; mean 2,7 years) showed an increase of the systolic peak doppler gradient across the aortic valve (median 41 mmhg) but no increase of aovr. 10 re-interventions (re-aovp: n = 3, commissurotomy: n = 2, mitral valve replacement n = 2, resection of subaortic stenosis: n = 1, resection of coarctation: n = 2,vsd-closura: n = 1 ) were performed in 6 patients. rv contractility and pulmonary vascular mechanics(pvm) in immature animal models are poorly underslood. we developed an acute rv injury model to measure rv contractility and pvm in response to commonly used cateehalamines. ten anesthetized piglets (9-12kg) were instrumented with micromanometers in the lv, rv, pa, and la. a pulmonary artery flow probe was placed to measure cardiac output(qpa). ultrasonic dimension crystals were sutured to the myocardium and dynamic chamber volumes estimated using shell subtraction methodology. rv injury was induced with 3-7 cryoprobe injuries at -50 to -70°c for 3-4 minmes each. da at 10mg/kg/min, db at 10mg/kg/min, and ep at 0.1 mg/kg/min were infused in random order. rv contractility was evaluated by calculating a load independent measure of contractility, the preload recmitable stroke work(prsw), during vena caval occlusions. to describe pvm, input resistances), characteristic impedance(z0), total pewer(tp), and efficieacy 03f=qimo"p) were measured. measurements were made pre-and post-injury, during infusions, and between infusions. clyoablation decreased prsw (22.8_+7.8 to 13.8+4.1, p<0.001). at the end of the experiment, prsw remained depressed to this level indicating stability of the model. one factor contributing to organ dysfunction for infants undergoing repair of congenital heart defects (chd) is their "inflammatory response" to cardiopulmonary bypass (cpb). this response is characterized by an increase in cytokine release, complement activation and endothelial injury. modified ultrafiltration (muf) is a method for removing tissue water and inflammatory mediators by rapid ultrafiltration followin~ cpb, muf may acutely improve post-operative end organ function. in this study, we evaluated the effects of muf on the pulmonary and cerebral function of infants undergoing cpb for repair of chd. we prosnecrivety randomized 30 infants (.~ 5 mos) to either muf (n=16) or no muf (n=14)(control) following correction for chd. the study intervals were 1) before cpb, 2) immediately after cpb, and 3) 20minutes after cpb. pulmonary function was evaluated by measuring dynamic compliance (cdyn) and airway resistance (raw). for 13 pts (mue=6 pts; control=7 pts) exposed to a period of deep hypothermie circulatory arrest (dhca), cerebral metabolism (cmro2) was calculated at each interval using the xe 133 clearance technique for cerebral blood flow measurements and arterial and jugular bulb saturation measurements to calculate cmro2. a reduction in cmro2 has been consistently demonstrated after dhca. the effects of muf on cdyn and on cmro2 are shown below: p<0.05 vs pre-cpb; # p<0.05 vs post-cpb • p--o.06 vs. post-cpb this study demonstrates that immediately following exposure to cpb, muf will improve pulmonary compliance. raw was not different between groups. there was no significant difference in hours of post-op ventilation for either group. in those pts exposed to dhca a trend towards better cerebral metabolic recovery compared to control was demonstrated. this is the first technique applied to infants undergoing dhca where cmro2 after cpb was greater than precpb measm~s. although this may be beneficial to postoperative hemodynamics, ventilatory management and long-term neurologic recovery, more patients and longer follow up will be necessary to verify such an effect. the effects of conventional mechanical ventilation (cmv) on left ventricular (lv). diastolic filling in neonates are not well established. one approach to improve lv filling is the use of cmv to provide a phasic increase in airway pressure {thoracic augmentation). this phasic increase in airway pressure may result in an increase in lv filling similar to that which occurs with cpr. thoracic augmentation has not been evaluated in neonates with ventricular dysfunction who frequently demonstrate increased heart rates. attempts to maintain low peak airway pressures during cmv may result in a prolonged inspiratory time that occurs over multiple cardiac cycles. this may alter lv filling in the later cardiac cycles. to determine the effects of inspiratory time on lv diastolic filling, 10 infants were examined with doppler echocardiography less than 24 hrs after surgery for the arterial switch procedtme. pulsed doppler recordings of the millal valve (mv) were obtained with the inspiratory time adjusted to occur over 3 cardiac cycles (21 sec.). a pressure transducer was placed in line with the ventilator, and the respiratory cycle was recorded superimposed on the doppler tracing to provide accurate determination of inspiration and expiration. doppler recordings were obtained from the apical 4-chamber view and the following measurements were made: peak e and peak a velocities, eia ratio, and deceleration time. compared to the expiratory phase of cmv, the initial beat during the iuspiratory phase of cmv resulted in an increase in mv peak e (.53 +-.06 vs .65 -+ .08 m/s, p<0.05) and peak a (.47 + .07 vs .63 -+ .09 m/s, p<0.05) velocities with no change in mv deceleration times (p<.01). compared to the initial beat during tile inspiratory phase, the third beat during the inspiratory phase resulted in decreased peak e (.65 + .08 vs .40 + .05 m/s, p<0.05) and peak a (.63 + .09 vs .40 + .05 m/s, p<0.05) velocities with no difference in deceleration times. thus, cmv augments lv filling during the initial phase of inspiration. however, as the increase in airway pressure is distributed over multiple cardiac cycles, lv filling falls below baseline levels. these observations indicate that while thoracic augmentation may be beneficial, to optimize lv filling the inspiratory time of cmv must be < 3 cardiac cycles. energy expenditure in pediatric orthotopic liver tranaplantat~on, to determine the actual calorie requirements of critically ill children and evniuate the correlations between measured, stress-p~lictod and repleted energy exponditttm and the severity of illness. des/gn: a prospective, dinlcal study. se~ng: tertiary care pediatric icu in a university hospital. patients: ten patients aged 6 to 210 months with disorders prompting picu admission, including sepsis, respiratory failure, solid organ transplantation, and cardiovascular surgery. inta~entions: all patients were studied within 24 hrs of major surgery or transplantation, or following acute illness. all patienls were severely stressed clinically and all but two were intubated by cuffed tubes, in three of them, still in a stress state, the study repeated on the third day of the disease, energy expenditure mensurements (mee), as well as illness seventy scoring systems, mtfltisystern organ failure scores and various anthropemetric and clinical indices of nutritional status, the stress-predicted energy expenditure (s-pee), the basal metabufie rote (pbmr), the repleted energy (re) and the recommended dietary allowances (rda) were measured or calculated in each patient. multiple regression analysis was used to analyze the data. measurements and main results: although the mean mee was significantly lower than the mean s-pee (37.6+11 kcal/kg/day vs. 50.35:16 kcal/kg/day, p<.002), it did not differ significantly from the pbmr (mean difference -2.62 kcal/kg/day, range -10.07 to +9.06 kcal/kg/day). the s-pee/mee ratio ranged from 1.04 to 2.07, while the re/rda ratio (21.25:4 kcal/kg/day)/(75.85:7 kcal/kg/dny) ranged from only .1 to .5. the prism/tiss ratio was not correlated better with mee than the diagnostic category (r~=.36 vs..38, respectively). the re was positively correlated withthe mee (rz=.65, i)=.07) while negative oarrelatian has been found between mee and age, mid-arm circumference, triceps skinfotd and the use of vaseactive agents (r~.81, -88, -.67, p<.005 and -.71 resp~lively). concl.m~: if s-pee is used for caloric repletion in the stressed oritic~ly fll el~d, these patients will be substantially overfed by as much as 100%. although pbmr appears to approximate the mee by ±10%, other clinical and nutritional indices should also be ennsidered. objective: to deter .mine..t.he metabpli.c and.nutritional state of mechanically ventilated intants and children m relatmn wlm severity or msease. patients and methods: 37 mechanically ventilated infants and children, median age 7 months (range 3 days to 13years), were studied. severity of illness was assessed using prism, prism-ii~ and fiss-scores. oxygen consumption (vo2), energy expenditure (mee) and respiratory quotient (rq) were determmed by mdirect calorimetry. total urinary nitroger(tun) and creatinine excretion, levels of albumin and crp were aetermmed in 16 patients. in these patients daily caloric intake and substrate utilization were assessed. they were categorized in subgroups: a partial feeding (recent admission to p1cu); b complete feeding. results: mee of the total group (n=37) 0a) i=intake g/kg/day (% total intake); u=utilization g/kg/day (% total production). nitrogenba]ance was negative in all patients in group a (mean -227.7 --176:4 mffkg/day) and positive in all but one patient in group b (.mean 84.9±109.d n~g/..kg/day;p=0.001). no significant correlations were round between creatinine height index, crp, albumine, jun vs v u2/kg conclusions: the mean measured energy expenditure does not exceed predicted resting energy expenditure, but ~ere is a wide range. in a majority ot patients with complete feeding h.igh carbohydrate intake resulted, in high kq and lipogenesis. in patients witla partial teeding the highly negatwe nitrogen'balance suggests that in the early phase of diseasean higher protein intake should be provided. severity of illness scores ann oiocnemicm markers of physiologic stress correlatedpoorly with oxygen consumption. leite,hp; iglesias, s; faria, c; ikeda, a; albuquerque, mp; carvalho, wb pediatric icu -s~o paulo federal university -s~o paulo, brazil objectives: 1 ) to evaluate patterns of use and monitoring of nutritional support in critically ill children; 2) to evaluate an education program in nutrition support given throughout the resident physician training in the pediatric icu. patients and methods: records of 37 patients receiving nutritional support during 1993 were reviewed. aider this first phase, knowledge and understanding of the role of nutrition support was conveyed to the residents through didactic lectures. in a second phase thedata were reevaluated in 35 children who were given nutrition support in 1995. results: from a total of 425 days ofthempy, the single parenteral route was utilized in 80,5%, the digestive route (tube feeding or oral route) in 19,5%. of this time. a previous nutr~ional assessment was performed in 3 children; no patient had the nutr~on goals set. the nitrogen to nonprotein calories ratio ranged among 1:80 and 1:250. only 29,7% of the patients had their estimated caloric needs supplied and this goal was achieved only in those patients who were on enteral tube feeding. patients did not achieved their goals for vitamins. the supply ofoligonleme~s was adequate except the zinc. nutritional monitoring parameters including weight, serum albumin and serum triglycerides were performed in almost all the patients but without uniformity. the reevaluation ofthase parameters showed adequacy of protein and micronutrients supply; however deficiency in nutritional monitoring and infrequent enteral feeding were still detected. conclusion: there were lacks in the implementation of nutritional support, which were partially corrected in the 2rid phase of the study, although the training of residents may have contributed to give them cognitive skills, it didn't changed policies and procedures as desired. we recommend reinforcement of the education program concerning basic nutritional aspects, and the organization ofa multidisciplinary team in charge of coordinating the providing of nutritional support. plasme free fatty acids (ffa) are the meier energy source for mast tissues. during fasting ffa are released from the breakdown af triglycefides in edipose lissue (at). lipalysis, le. the rote of release o/ ffa, has been megsured in humans by means of stable isotope techniques using labeled pa or glyeerd as traces. no information is avoilob!e io dale on the ro of la. we infused albumin hound u13c-pa and u13c-la in 7 critically ill infants, receiving 20 kcel/kg/doy of iv glucose end na oral feeding (weight 3.6,i.,3 kg;, range 1.9-5.8; ego 57:64 days, range 1 149) and measured simultaneously the ra of pa and la from (he isotopic enrichment of plasma fea by gas chromatography-mass speclrome|ry ai 1:50, 2:00 and 2:10 hours from tile shod of the infusion. a subcutaneous gluted at biopsy was obtained far fatty acid (fa) composition. we intended to (1) in fie infants sbjdied atipa ~'os hi9her than attla (~p<o.o01)' and ihe ra of pa was higher then thai of la (~p=0.00 §). however the ratio el the re's to the respeclive fa in at was higher for la than far pa (*p=0.02). in conclusion the ra af la was higher then expected from ~he fa composition of at. we speculate [hat la is preferentially released during tipolysis and its contributuion to the energy metnbolism of the sick infonl could io !orger than what: is normai!y assumed from ihe fa composilion of at and of plasma fea. preoperative starvation can be prevented in infants undergoing non gi surgery by continuing feeds till as long as it is safe to do so. some gi disorders require withholding of feeds for a long period. for these and for infants already suffering from pem~ parenteral feeds are given preoperatively. peroperatively nutrition is maintained by ensuring adequate glucose supply in the infusion fluid and its smooth metabolism. proper placement of feeding tubes are also carried out peroperatively. postoperative early resumption of oral feeds is ensured by new techniques of anaesthesia and pain relief and methods to ensure early return of peristalsis. enteral feeds of pre digested substances and feeds through transanastomotic tubes and through gastrostomies and jejunostomies can prevent starvation in many situations. parenteral feeding is carried out only when enteral feeding is not possible, for example in necrotising enterocolitis~ gastroschisis, short gut syndrome, high enteric fistula and acute pancreatltls. the artedal ketone body ratio (akbr) is established as a valuable tool in the management of adult patients undergoing surgery for severe liver disease.the redox theory emphasises the central role of the liver in preserving homeostasis and the importance of the hepatic mitochonddal redox potential (nad+/nadh) in monitoring liver function and integrity.the akbr (plasma acetoacetate/3hydroxybutyrate) is a measure of hepatic mitochonddal redox status, reduced ratios associated with poorer outcome. the venous ketone body ratio (vkbr) is influenced by peripheral ketone utilisation and therefore thought unreliable in assessing hepatic redox its use has been dismissed as no correlation with the ratio in hepatic venous blood was found. inspite of this. vkbr is used in the diagnosis of lactic acidoses and respiratory chain defects. the objective of this study was to determine the relationship between the akbr and vkbr in a paediatnc intensive care population. 31 children admitted to the paediatdc intensive care unit, with indwelling arterial and central venous lines as part of their routine care, were recruited for the study. the median (range) age was 2.0 years (0-16.6 years) with prism score 10 (0-36), simultaneous akbr and vkbr were measured. suppression of ketogenesis was confirmed using a rapid 3-hydroxybutyrate strip test (ketofilm, genzyme diagnostics) pdor to sampling. the median vkbr 0.50 (range 0.13-1.46) was lower than median akbr 0.56 (range 0.11-1,33), there was good agreement between artedal and venous ratios, bland-altman analysis giving a 95% confidence interval for relative bias of -0.13 to -0.03. there was a significant correlation between vkbr and akbr r 2 0,8271, p,0.001 (intercept 0.02, slope 0,85) conclusions; values for akbr tend to be higher than vkbr, however there is a consistent relationship in critically ill children. in paediatdc patients in whom ketogenesis is suppressed, vkbr may be considered equivalent to akbr. this simple test may prove to be a useful adjunct in predicting mortality in cdt{cally ill children. to understand the present status of pediatric intensive care in china, we conducted a survey between october and december, 1993, involving 20 hospitals in 14 provinces and municipalities. the results showed that there were totally 41 icus for pediatric patients, including 19 pediatric icus (picu), 18 neonatal icus (nicu) and 4 pediatric surgical icus (sicu), with total of 403 beds. the physicians to bed and nurses to bed ratios were 1:1.5 and 1:0.91 respectively. the average number of equipment per bed was 0.47 ventilator, 0.34 multi-fnnction monitor and 0.47 infusion pump. very few of the icus had portable x-ray machine, biochemical and blood gas analyzers, and hemodialysis machines. the most frequently treated diseases/conditions were pneumonia, intracraniat infections, post-operation and sepsis in picus, and neonatal pneumonia, hypoxic ischemic encephalopathy and sclerema in nicus. pneumonia and respiratory failure accounted for 33.29% and 26.50% of all the diseases/conditions treated in the icus and the mean case fatality rate of respiratory failure was 24.50%. the results of the survey suggest that there is shortage of tcu beds and modern equipment, and treatment is often delayed due to excessively strict criteria for mechanical ventilation. a set of simple, and nationally acceptable criteria for evaluation of severity and cure of the diseases/conditions is urgently required. the medical reports of 286 children were reviewed and each admission was analysed in terms of age, sex, diagnosis, management within the hosp~al, length of hospital stay and type of poisoning. the youngest age was 23 days and the oldest was 16 years. hundred and twelve (39.5%) of the children were girls and 174 (60.5%) were boys. the most common (26.5%) reason of admission was intoxication among all of the cases and the greatest group (73.7%) of the poisoned children had ingested medication which was followed by another group of patients (9.2%) who had eaten mushrooms. the peak incidence of drug ingestion was salicylate intoxication (20%). forty drug poisoninigs were accidental while 16 was intentional. the majority (82.4%) of the cases that committed suicide was between 12 and t7 years old, and the main cause of suicide was being unsuccessful at school. we conclude that poisoning -especially salicylate intoxication -is still a major problem in turkey and we believe that emphasis on the need to stere all kinds of drugs in a secure place and re-examination of a chin resistant packaging should help to reduce childhood poisoning significantly which is a preventable condition. included were all patients who died in the hospital, except those treated in the neonatal icu (predominantly premature and sga newborns) and those who died in the emergency room. among a total of 7179 hospital admissions (excluding the neonatal icu and emergency room), 99 patients died (1.4%). of these 99 patients 73 (74%) died in the pediatric icu, 18 (18%) in the ward and 8 (8%) in the operating room. a chronic underlying disease was present in 66 (67%). withholding or withdrawal of therapy was implemented in 48 (48%) children, 27 (27%) died due to failed cpr, 20 (20%) were brain dead and 4 (4%) died following a dnr order. justification for therapeutic restrictions in the 52 patients of the dnr and w/w groups was imminent death in 32 (62%), lack of future relational potential in 12 (23%) and excessive health burden in 8 (15%). withdrawal or withholding of therapy was carried out by extubation in 46%, vasoactive drugs were stopped in 25 %, and mechanical ventilation was withdrawn in 21%, analgetics and sedatives were frequently used (in 73% and 79%, respectively). hence decisions concerning restrictions of treatment are common in pediatric practice, mostly due to imminent death. patients in which treatment was restricted were characterised by a longer hospital stay, a worse prognosis, a higher frequency of chronic underlying diseases and a lower acute mortality risk. despite restrictions of intensive therapy, most patients were allowed to die in the pediatric icu. background microalbuminuria (mca), a subclinical increase in urinary albumin, reflects glomerular and overall vascular permeability 1"2. an increase in urinary excretion of albumin occurs after burns and trauma. transcanillary albumin escape rate is also increased in response to elective surgery 3 and in critically ill adult patients 4. we investigated a possible relationship between urinary albumin levels and clinical instability, as measured by pediatric risk of mortality (prism) scores. method we studied 26 consecutive patients (median age of 13 months, range 2-100). patients whith nephropathies or any abnormality of urine analysis were excluded from the analysis. prism scores, mca (mg.1-1) (immunonepbelometric) and urinary creatinine (cu) (mmol.l -!) (jaffb) (48 hour collection sample) were determined within 48 hours from admission to the picu. the mca/cu ratio (mg.mmol.1 "l) was used to correct for urine output variability. diagnoses included respiratory failure (6), postoperative (5), neurologic (5), sepsis (4), trauma (3) and miscellaneous cases (3). pearson's correlation was performed to correlate data. results and conclusions. mean prism score and mca/cu were 16.9 ± 5.9 sd and 1004-39 sd, respectively. a significant correlation was found between mca/cu and prism scores (r=0.80, p<0.001). our observations show that, independently of the initial insult, the paediatric unstable patient may have increased capillary permeability, which is correlated with the degree of physiological derangement, as measured by prism scores. microalbuminuria can be rapidly determined since it is routinely used in the management of diabetic patients, it is inexpensive, simple to measure and blood-spearing. therefore, it might have a role in the clinical assessment of capillary permeability and transcapillary albumin escape worldwide the demand for paediatric intensive care services exceeds the supply. in developing countries sporadic access to such services alters expectation of care and can lead to children being either mechanically or handventilated in general paediatric wards. the outcome of children requiring ventilation in a major teaching hospital in india was reviewed.children were ventilated on an adult intensive care unit (aicu) if a bed was available, otherwise in the general paediatric wards. over a 4 year period 109 children were ventilated on aicu with 54 deaths. yearly mortality rates varied between 43-58%. over a 3 month period 37 patients were ventilated on the paediatric wards. of the 15 p~tients over 4 weeks of age 11 died (chi squared 0.t >p>0.05) reasons for the higher mortality rate on the paediatric ward likely include the higher patient:nurse ratio, and more limited resources. a predictor of mortality based on simple physiological observations without the need for expensive blood tests and including chronic health status would be a useful tool. the establishment of a paediatric intensive care unit is proposed to redress the balance of care. to assess the performance of the pediatric intensive care unit of hospital dona estef~nia by an international standard score, the authors did a prospective study of 1149 consecutive admissions to the unit during a period of 29 months. mean age was 50.63 _+ 54.07 months; mean lengh of stay was 3.16 + 5.59 days. the effectiveness and efficiency were determined by the admission prism. admission efficiency was defined by two criteria: a) mortality risk > 1% or b) the administration of at least one intensive care unit-dependent therapy. the cumulative observed mortality was 5.57% and the expected mortality was 5.97%, with a standardized mortality ratio (smr) = 0.933. the overall performance of the prism score-based predictive model was found to be good (goodness-of-fit test x2 [5] = 6.387;p=0.271). of 1149 patients admitted, combining the two criteria (icudependent therapy and mortality risk) an admission efficiency of 825 (71.8%) was found, equating to 3263 (89.94%) of 3628 1cu days. conclusion: in our study the assessment of the admission efficiency and of the effectiveness of the unit was possible by using the prism score of admission. there was no significant difference between mean values for otiss and ntiss)in level l patients (p=0.12 paired t-test).for level 2 and 3 patients mean value of ntiss was greater than otiss (p<0.0001). there was a significant correlation between levels using either ntiss or otiss (mean difference level 1 and 2, level 2 and 3, ( p < o.oool). conclusions: a new tiss has been developed and used in a picu. nurses were able to accurately score the interventions on their shift. the assignment of patients to intensive care levels correlates with tiss values allowing a quantitative measure of severity. objective : to compare the rate of cerebral palsy (cp) between monochorionic-twins, dichorionic-twins and singletons born at 25 to 32 weeks' gestation. design : two-year prospective cohort study. setting : geographically defined study (region of franche-comt~., france). main outcome measures : type of plasentation was obtained by anatomopathological, or macroscopic examination of placenta and comparison of 6 twins' blood-groups. neurological assessment was performed at two years of age (uncorrected for gestational age) by family doctor (pediatrician or physician), or neonatologist of the icu at tertiary center. sample : 167 of 17i survivors aged of two years (98% follow-up rate), born between 09/30/90 and 10/01192. triplets and chromosomic malformation were non included. results : thirteen (11%) of the 119 singletons had cp.vs 3/29 (10%) of dichorionic twins and 6/19 (32%) of monochorionic twins (p=0.04). four of the 19 monochorionic twins (21%), 2/29 dichorionic twins (10%) and 4/119 (3%) nngletons suffer from quadriplegia (p<0.01).in a multivariate approach, monochorionic twin placentation was the strongest risk-factor of cerebral palsy (or=9.7, ic 95% = 2a-39, p<6.01). others risk-factors of cp were : lack of father's profession (or 11, p<0 .03), maternal antecedent of abortion (or 3.2, 1-10, p<0.04), vaginal delivery (or 3.4, 1-11, p<0.03), hyaline membrane disease (or 3.4, 1.2-t0, ~0.02). discussion : this is the first population-based study to uplight the role of monochorial twin-placentation as a strong risk factor of cp for premature infants. cp is more severe in monochodonic twins than in other infants. mecanism of cerebrat deficiency is not clear since none of our infants with cp was survivor of an in utero cotwin's death, and none of these infants was exposed to twin to twin transfusion syndrome. were these monochorionic-twins affected by an undiagnosed neurological structural defect that could lead both to prematurity and handicap remains an open question, a vital role of the intensivist is to ensure that knowledge and practice are imparted to trainees in the icu so that patients receive optimal care. teaching effectiveness varies widely leaving gaps in knowledge and practice in the trainee. being an effective teacher should not be a "gift" of a privileged few. the icu provides a fertile ground for using a variety of methods for teaching, e.g. didactic, at the bedside, emergencies, and in the performance ofproeeaures. in this environment, much can be learned. we have embarked upon a program to facilitate this learning process. i) teaching needs to be recognized as the foundation of good clinical care, i.e., patient related, and in its ability to generate discussion and research investigation. 2) teaching structurally has many components including the speaker, audience, varying situations, and the message delivered. 3) establishment of a program using these components to enhance teaching abilities at all levels, a) evaluate base-line teaching skills initially, b) individualize interventions to improve teaching skills, e) demonstration of learned skills with re-evaluation. this process is analogous to the analysis of a clinical disorder in a patient which, once recognized, interventions are then instituted and then re-evaluated. 4) instill the desire to use these attained skills to teach and interest others to teach. teaching excellence should be recognized through awards, honors, and academic advancement. a major emphasis of this program is to provide participants with skills necessary to teach thought processes, decision-making skills (what to do, what to avoid) and implementing appropriate management during stressful emergency situations common to the picu. introduction: many" e-mail based discussion groups exist on the internet to provide medical professionals with a rapidly responsive medium for the international exchange of ideas relating to patient care. several such lists each serve more than a thousand professionals in more than 30 countries, each distributing a dozen or more messages each day to every subscriber. there is very little known about the time being spent by professionals interacting with these lists, and very little known about the impact of the discussions on patient care. we wished to test the hypothesis that these discussion groups provide infortuation which is being used to change the care of individual patients and the general approach to patient problems. methods: in early january 1996 a pilot electronic survey was sent to a small fraction (n=63) of the memberships of 2 e-mail discussion groups, picu@its.mew.edu, and nicu-net@u.washington.edu (the full memberships of both. groups (n=t439 for nicu-net, n=1045 for picu) will be surveyed in early february of 1996). participants were asked for demographic information, experience and skill level relating to e-mail, time spent with the discussion groups, perceived usefulness of different types of discussions, and the ways in which the discussions were used clinically. the pilot study was analyzed for construct validity by correlating an overall assessment question with a summary of the specific questions. scale reliability was measured by cronbach's alpha statistic. results: the pilot survey response rate was 30163 (48%). the majority of respondents were male physicians, with an average age of 39+_5 years, who had completed subspecialty training in intensive care, and were working at a university-affiliated hospital. most had been using e-malt for more than 6 months, and considered themselves moderately adept in that use. 63% felt that the list helped weekly to keep them informed about current issues and practices in their field(s), and 57% felt that, at least monthly, they used information from the list(s) that was not readily available in medical journals. overall, 75% agreed that the list improved their professional competency. when asked to compare the value of 6 months of membership on an e-mail discussion group with more traditional educational media, 34% compared it with attending a national conference, and 26% compared it to a journal subscription. cronbach's alpha was .76, construct validity testing yielded coeff=.50, p <.05. conclusior~: internet-based e-mail discussion groups for health care professionals can be an important part of a strategy for maintaining professional competency. despite the very low cost of this medium for most, the value is felt to be comparable to that of t~r more expensive forums for education. further study will include distribution of the full survey in early february of 1996. fronk shann, tony slater, gale pearson and the pim study group we have developed a new score for predicting the risk of mortality in children admitted to intensive care. the score is calculated from only seven variables collected at the time of admission to icu: mechanical ventilation (yes/no), booked admission after elective surgery (yes/no), the presence of any one of 14 specified underlying conditions, both pupils fixed to light (yes/no), the base excess, the pao 2 divided by the fio2, and the systolic blood pressure. most scores used to predict outcome in intensive care require the collection of a large number of variables (so many icus do not calculate them routinely), and they use the worst value of each variable in the first 24 hours in intensive care. this means they appear to be more accurate than they really are (about 40% of child deaths in icu occur in the first 24 hours -so they are diagnosing these deaths rather than predicting them), and they blurr the differences between traits (a child admitted to a good unit who recovers will have a low score; but the same child who is mismanaged in a bad unit will have a high score -the bad unit's high mortality rate will be incorrectly attributed to its having sicker patients). pim was developed in the picu at the royal children's hospital in melbourne, and has been tested in six other picus in australia and one in the uk. objectives: to study the characteristics of the muhiorgan dysfunction syndrome (mds) in children. methods: a retrospective study with all the children with mds diagnosed from january 1990 to june 1995 is presented. 173 children fulfilled the wilkinson criteria (i). in all of them the number of organs affected and the prims score were determined during the first 24 hours. several groups were performed according to the clinical diagnosis, the hospital of origin and the order of organs affected. results: the 173 subjects studied were an 8% of the pediatric intensive care unit admissions. 100 of them expired (58%). no differences in age, sex and weight were observed between the children dying and the survivals. the most common causes of mds were sepsis, both nosocomial (25%) and medingococcal (i4%) and acute respiratory failure. sixty-fivepercent of the patients were from the hospital wards and the remaining were directly admitted to the pigu from the emergency room. the systems affected were: respiratory (93%), cardiovascular (92%), hematologic (61%), central nervous system (52%), renal (43%) and (hepatic) liver (28%). the organs initially failing were: heart (39%), tung (28%) and central nervous system (18%). the children dying had a larger number of organs with failure than the survivors (3.89 v,s. 3.34, p<0.001).the prmis score was higher in the children expiring than in the survivors (22.4 v.s. 17, p <0.001). s.mmary: the mds is a common pathology in picu, with a high mortality, the mortality is higher in children with a larger number of organs affected and a higher prism score. sepsis is the most common etiulogy. methods : from june ist to july 15th 1995, all patients admitted to the pediatric icu were included. the score was measured at day 1 (d1) and day 3 (d3) and we used 10 variables. for each organ system, we defined 2 categories : dysfunction or failure, which we respectively confered 1 or 4 points. results : 56 patients were admitted : 22 newborns, 34 children. 23 were medical and 33 were surgical patients. 36 (64 %) patients had two or more organ failure at the admission, 12 (21,4 %) patients died, which 6 (50 %) in the first 48 hours. the mortality rate was the same for children with two or more organ faiiure at d1 and d3 : 6/36 (16,6 %) at d1, 4/22 (18,2 %) at d3. the mean score is different for children who survived or who died : 8,6 versus 17,9 at d1 ; 10,6 versus 18,2 at 133. when the score is > 15, the mortality rate is significant. conclusion : in this study, there is a good correlation between the score of severity and the mortality rate but we have few included patients. we need a prospective multicentric study to assess these results and we must compare this score to other scores of severity used in picu. back.qround: injury to the central nervous system is the cause of death in the majority of pediatric trauma victims, studies have identified a wide range of factors associated with poor outcome from brain injury. however, when single features are analyzed, they are not sufficiently accurate predictors. few studies have used a multivariate analysis of these factors and pediatric outcome, methods: clinical and radiographic features of 164 comatose children after traumatic brain injury were analyzed, clinical parameters, the initial cranial ct scan, and demographic characteristics were analyzed for an association with death or vegetative survival at 6 months. a tree diagram in which risk factors may differ within the study subpopulations was constructed using recursive partitioning. results: chitdren with a motor score _<2 had an 11-fold increased risk of poor outcome compared to those with motor scores >2. among patients with scores of _<2, those with abnormal pupillary reflexes experienced a 13-fold increased risk of death compared to those with normal pupillary reflexes. among patients with a motor score >2, an intracranial diagnosis code (no pathology, mild shift _<5 mm, swelling, shift >5 mm, surgical mass lesions, or non-operative mass lesions) was highly predicative of poor outcome at 6 months. children with ct findings other than normal or mild swelling had a 4-fold increased risk of poor outcome. of children with swelling, shift or mass lesions, the pupillary light reflex was associated with outcome. children with abnormal pupils had a 6-fold increased risk of poor outcome. discussion: a few clinical and radiographic features stratified comatose children into fairly distinct risk groups. information available early after traumatic brain injury in comatose children provides useful prognostic information on the likelihood of death or devastating injury. a retrospective study of 70 children with the diagnosis of epidural hematoma was made during 1990-1995 period. ages ranged between 7 days and 17 years (18% less than 1 year, 40% between 1 and 10 years, and 42% older than 10 years), 82% of them were admitted at the picu. 51% of the cases were due to falls, 35% to road traffic accident and 14% to other causes. on admission gcs was less than 8 in 19% of the cases and more than 14 in 53%. diagnosis was made during first 4 hours in 63% of patients and delayed more than 12 hours in 28% of them. neurologic impairment was present at admission in 33% of patients, and delayed in 30%. even so,27% remained without impairment. radiological findings at first ct were skull fracture (68%); epidural hematoma localization was: in the right side (63%), frontal area (24%), temporoparietal (66%) and occipital (t0%). associated lesions were: several (13%) or unilateral (51%) cerebral contusions, diffuse brain oedema (10%), unilateral hemispheric oedema (14%) and 38% showed shifted middle line. four patients died, half of them during the first 24 hours. 41 fully recovered (58.6%) and 25 have sequelae of different nature :7 were left with severe motor disability (10%); at the follow-up t3 have some degree of neurodisability. next datas keep correlation with death or neurosurgical impairment: only were significative multiple cerebral contusion (p=0.002) and brain oedema (p=0.05), gcs less than 8 at the admission (p--0.002), shock (p=0.003) and remaining cerebral contusion in control ct correlated with death or diasability at discharge. on the other hand, neither surgical drainage volume nor first or highest levels of icp (12 cases),nor pupillary abnormalities (10 cases) correlated with worse prognosis. conclusion: gcs equal or less than 8 an shock are main factors related to worse prognosis, also multiple cerebral contusions in ct and diffuse brain oedema. the results of a modified gcs were compared to outcome and intensive therapy in 78 children (mean age 8,5t4,7 years) with head and associated injuries (53,6% of all cases) of different causes (traffic accidents, falls). the gcs was regularly used inn the course of intensive therapy. according to our own and other experiences the gcs was divided in 3 stages: stage 1 (4-8 points), stage 2 (9-12 points) und stage 3 (13-19 points) palhuiugy wile sp, tdhlg c~'lcb1al blood ~0w. sabgcqucntl}. rhc slat,: rerltncd to t1011tl,91 iiltlils. the p0st,~pem~v~ b}i~g wij!!,:q1! ,:_a!~p!ica!j0n~:. 4 ri~;¢ ill the level of sensibflizatjou lo tile cerebn~ anhgrns up to 1t.4-o7 was flofcd iu 9 i,alicnts. there wa.~ al~ iuclt~a~e ill cerebral vdociij,. ~m d~;'ati0a il~ p¢fiphc~ai re~ista/isc of the large ce~'bral ve~ds. neur0h;~c ~:yn'.pt,m~at0!a~, (s::mno!en~', _r_uscu!~r l~:pot0ni& !ryper*'flema) was nbserwed tu lt~ese pal~enls o. cbruc~l ~0nnds. rile ple~c.ut abse~vafion~ suggesl ihal die ~tttdy at" ihe stale ~f hematocr~chcplm/itic bm~ic~ in ckil&en with 31110on emergensy is of abviou.~ !?ece~sib; in co~.te ctin g severe pa~0lo2~-i~mnediately f0u0wing ne ,:~per,'~fion. background: reconstruction of the heart by three-dimensional (3d) echocardiography provided new information on anatomy of complex congenital heart defects, we assessed the utility of 3d ultrasound in detecting morphological changes in cerebral anatomy in newborns before and after cardiac surgery. methods: transfontanel cross-sectional ultrasound, scans were obtained in standardized coronal and median sagittal planes. subsequently, rotational scanning was used to acquire the multiple sequential crosssections of the brain. for rotational scanning, a conventional 5 mhz transducer was rotated 180 degrees.scanning took less than one minute and required no sedation, data was stored in the image processing computer which allowed for off-line three dimensional reconstruction of different brain regions.twelve infants aged 3 -21 (median 7) days were assessed before and after cardiac surgery, results: cavity of lateral ventricle, choroid plexus and the periventricular brain parenchyma could be reconstructed in all. accurate estimation of size and volume of lateral ventricle, aqueduct, and other ultrasonographic visible pathological brain lesions could be performed. reconstruction of various brain areas was accomplished in 3-10 minutes. the localisation and extension of severe periventricular hemorrhage which was detected preoperatively in one infants was better visualized than in conventional ultrasonography. epicortical and subarachnoidal space could be reconstructed in all and allowed detection of hemorrhage in one case which was not detected by conventional ultrasound. conclusion: 3d reconstruction of different areas of the brain may provide additional quantitative information on size and volume of the internal ventricle and choroid plexus, and better understanding of the topographical aspects and the extension of intra-and periventricular hemorrhage than conventional cross-sectional ultrasound. introduction: intracranial cerebral blood has been estimated to be 70% venous, the invasive measurment of venous blood saturation in the jugular bulb provides quantitative information on cerebral oxygen supply and consumption. however, routine oxymetric measurement of blood saturation in the jugular bulb by insertion of a catheter line into the internal jugtdar vein is an invasive procedure which has limited use especially in infants and young children. thus the aim of this study was to investigate the correlation between the non-invasive spectroscopic measurement of rso2 and the oxymetric determination of the blood saturation in the jugular bulb in infants and children undergoing routine cardiac catheterization.. methods: during routine cardiac catheterization 30 infants and children (age 5 day-16 year, median 4,5 year) the rso2 was measured continuously using a two chanel cerebral oxymeter (invos 3100a). the sensor was placed in standardized location at the left temporal head side. after the routine oxymetric blood sampling in the superior vena cava the oxymetric catheter was manupilated into the left jugular bulb. after control of the catheter position simultenuous values of the rso2 were documented. results: over a range of (33-87%) sjo2, a significant linear correlation was found between the spectroscopic measurement of rso2 and the oxymetric determination of venous blood saturation in the jugular bulb (r=0,83, p<0,001) and the superior vena cava (r=0,65, p<0,05). no significant correlation was found between rso2 and the arterial blood saturation in the descending aorta and as well as to the standared hemodynamic parameters. conclusion: meusurement of rso2 by mrs may provide continuous non-invasive information on cerebral venous blood saturation and thereby possibly on cerebral oxygen supply and consumption in infants and children. these may be of clinical value particulary during and immediately after heart surgery by means of non-pulsatile cardiopulmonary bypass. information on refractory status epilepticus (rse) from developing countries is scarce. we analysed 43 cases of rse admitted over last 2 yrs. the objective was to study etiology end evaluate efficacy of diezepam infusion. median age of the patients was 1.25 years irange 1.5 months to t 1.5 yrs); 70% were boys. onset of seizures was 1-t44 hours (median 24 hours) prior to hespitalisation. the glasgow coma scale score ranged from 3.11 (mean+sd 5 + 2). the commonest underlying causes were acute cns infections (26/43, 60%; bacterial meningitis, 16, encephalitis, 10) and epilepsy (8/43, 10%). oiazepam infusion in incremental dose (range 0.01-0.025 mg/kg/min) was used in 38 patients over 3.4_+2.1 days. seizures were controlled n 31 (82%), mechanical ventilation was required in 10 (26%)only, while none had hypotension; 84% patients survived. thiopental infusion (holus 5 mg/kg followed by 0.2 mglkg/min, and increments of 0.1 mg/kg/min till seizure control) was used in 8 patients over 1.7_+0.7 days; seizure were controlled in all, but five patients needed mechanical ventilation, six developed hypotension needing infusion of vasopressoi drugs, 3 out of 8 (38%) died, overall mortality was 26%, mainly due to acute cns infections (n-6) and prolonged se. the patient was a 2-year-old gift di~aosed of dov,~'s s~drom¢, tetralogy of fallot. (t.f.) before admission a vasovagal crisis after coughing and vomiting was seen, and she was taken to the emergency room. mother said she had eyanosis in the mucous membranes of the mouth with exercise.on physical examination, she ~as afebrile, normal fundi and neurologic examination was normal. a harsh systolic murmur was hear~ with decrased intensity during bradycardia. chest rx disclosed a decreased pulmonary vascular markings. ecg: synus rhythm, with bradycardia and nodal escape rhyflmas. she was transferred to our picu because of severe h3,pertomc seizure, lost conciousness, and deeembrate poslamng~ ~t cyancx~is. the episode lasted for ~weral seconds, and ceased v~th diazepam. on admission she was lethargy, and neurologlc exammation showed weakness of left leg without babinski, and normal funduscopic. the patient had two episodes of bradycardia and isoproterenol was begun. during those episodes the patient was cyanotic, and the murmur was heard with the same intensity. act scan disclosed a tight parieto-temporai abscess with midline shift, lnmediately after the diagnostic ct, we administered antibiotics, antiedema treatment and it was drained. the abscess culture was negative. a ct control disclosed air and midlme shift. ~ the next two days she had three episodes of h39oxia and c'yauosis ceased with o@gen, morphine and propanolol the patient died during a fourth episode. discussion: arrhytmias are uncommon in patients with tetralogy of fallot before surgery. in our case the first diagnosis was sick sinus syndrome vs bradycardia secondary to cyanotic episodes. the incidence of cerebral abscess in children with congenital heart disease (chd) is approximately 5%. tetralogy of fallot is the most common associated lesion, and is unusual in children under 2 years of age. conclusion: 1) brain abscess is a rare complication of patients with cyanotic chd, but should be suggested in patients with °'apparent" sick sinus syndrome. in patients with down's syndrome, t.f.,with cyanotic episodes, and difficult neurologic exploration, a brain ct scan is recommended. guillain-ba~re syndrome (gbs) is an acute autoimmune reaction, directed primarily toward the myelin encasing the peripheral motor nerves= this reaction causes a delay or block in nerve conduction. the presentation often can be very subtle but is followed by rapid loss of neuromuscular power, leading to acute respiratory distress, resulting from weakness of muscles and aspiration pneumonia. there were 3 boys -4, 8, and i i years old with gbs, treated in our icu. two of them due to the respiratory distress were intubated nasotracheally and ventilated mechanically with servo-9ooc (siemens-elema, sweden) ventilator. duration of ventilation was i i and 34 days, respectively. plasma exchange was performed in all cases. the numbers of plasma exchange sessions were 2-4 in each case. mean amount of plasma exchanged per session was 28,24 ml/kg. plasma was substituted with albumin, plasma or saline. the most important aspect of the management of patients with gbs in the icu involves the airway care, prevention and treatment of aspiration pneumonia and the mechanical ventilation if respiratory distress presents. endotracheal intubation should be performed whenever there is evidence of retention of pulmonary secretions, refractory to chest physical therapy, weakness of protective reflexes of the airway, leading to aspiration pneumonia and (or) atelecr~sis. cardiac arrhithmias too, is a main threat to the circulatory stability in gbs. therapeutic plasmapharesis has been shown to be beneficial, reducing the time for weaning from the ventilator and for achieving independent ambulation. however, plasma exchange is expensive and not without significant risks for the patient. some authors find that plasmapheresis is not effective for patients with fulminant course of gbs and blocking of nerve conduction. recent studies have demonstrated that intravenous high-dose immunoglobulin can be equally effective. there were no significant complications associated with plasma exchange. all presented patients survived without residual disability. tetraparesis associated with long-term paneuronium use in an infant. paneuronium is a muscle relaxant used in ventilatory management of patients with respiratory distress in intensive care unit. after the end of sedation some patients were found to have severe tetraparesis. paresis was accompanied by complete areflexia and diffuse atrophy of alt extremity muscles. this neuromuscular complication is caused by prolonged high-dosage pancuronium treatment. in the last 5 years, numerous reports have linked the use of pancuronium bromide with prolonged paralysis, disuse atrophy and areflexia. this side-effect is well known in adults patients but rare in a pediatric intensive care unit. we describe one pediatric observation of tetraparesis after prolonged pancuronium treatment in a 9-month-old girl, this female infant developed respiratory distress syndrome and was intubated and mechanically ventilated. to decrease chest wall rigidity pancuronium bromide was administered during 11 days. (she received approximately 120 mg of pancuronium bromide). on day 12 the drug was discontinued and the patient had severe tetraplegia and areflexia with normal head movements. electromyograpliy showed absence of any disorder of neuromuscular transmission. this infant showed a recovely of muscles after 3 months. the other causes of peripheral neuropathies were eliminated. electroencephalograms and head scans were normal. the recovery pattern observed in our patient correspond to the process of regeneration after axonal degeneration. it is suggested that these neuromuscular complications were caused by prolonged high-dosage pancuronium treatment (associated with cortieoid and aminoglucosides). polyneuropathy syndrome in adult lc.u. appeared in literature in 1984 and is extremely common in long stay cases. the etiology of these disorders remains elusive. it is tempting to ascribe them to administration of drugs (muscle relaxants, steroids, aminoglycosidea), plolonged immobility, malutrition, sepsis and ischemia associated with reperfusion injury. to our knowledge there is only one case report of similar condition in a children i.c.u. (pascucci 1990) we present a serie of 16 previously healthy children, aged 9 months to 13 years, who admitted in i.c.u with respiratory failure and who following weaning from m.v, remained in profound diffuse hypotonia with proximal and distal muscle weakness for various length of time, recovery of muscle strength occured in a week or months {the longest i0 months), all children, except one, 3-4 days before admission developed symptoms of either respiratory or upper airway infection with fever. on admission viral and bacterial cultures were positive in 2 cases (haemophilus influenze, herpes virus). during treatment 9 patients became septic. muscle histological and neurophusiological investigations have not been done. considering the multifactorial nature of the aquired nmd in adult critically ill pts, is impossible to attribute the muscle weakness of our pts to any specific cause, in conclusion, our findings suggest the need for further investigation of nmd in critically ill children treated in i.c.u. a van esch, ha van steen~l-m011, ir ramtal, g derksen-lubsen, idf habbema. febrile status epilepticus (fse) is a prolonged and serious febrile seizure. little is known about the outcome of fse in neurologically normal children. this survey involved patients between 6 months and 6 years of age who had visited due to their first fse, the sophia children's hospital during the period of january 1981 till december 1991. patients with a history of neurologic disorders were excluded. 57 patients were identified, 65% were male. the cause of the fever remained unknown in 51% of the cases. in all case the fse was generalized and it most frequently occurred at night (47%). the mean age at fse was t.6 years (0.5-4.7), the mean temperature 39.6°c (38.5-40°c). the mean follow up time was 1.7 year. twelve children (21%) had neurologic sequelea. the neurologic sequelae varied from speech deficit (4 case mild, v2 -1 year delayed; 4 case moderate > 1 year delayed) to severe retardation and epilepsy (4 cases). speech deficit was detected after a mean period of 6 months (range 0-18), age, gender, temperature, family history and time of onset were no significant risk factors for neurologic sequelae. duration of seizure [rr 3.0 (0.8-11.3)] and more than two drugs to treat fse (rr 5.2 (t.5-18.1) were related to neurologic sequelae. we recommend that fse children should be followed for at least a year to detect possible speech disorders properly and start early intervention. unusual presentation of myasthenlg gra%qs ibtza e. modesto ,v~ abe~gochea a, sanch]s 1l all, go l varas k folgado s, garcia e. p.1.c.u. la fe, valencia. spain case report: the patient was a 2-year-o!d gift transferred to our pic because of severe respiratory failure. the patient, convaleseem of ehiekenpox, came into contact with horse manure previous afternoon. in the morning, she was lethargy, and irritability, with poor finding, and ~ an episode of coughing, cyanosis and acute respiratory failure after mucous vomiting when she was drinking milk. on admission she had severe respiratory distress, respiratory acidosis, and the sat 02 was 86%. she was mtubated without difficulty, and was transferred to our p.i.c.u. physical examination reveals stable hemodynamies, pupils equal, round, reactive to light, normal fandi, and muscle relaxation. crusted vesicles diseminats~d. rhonehi over both lungs. hepatomegaly (+) and splenomegaly (+). ~lhe urine, hematologic, and c.s.f. laboratory findings were normal. c.t. scan of the brain, e.e.g., and ekg. revealed no'abnormalities. rx chest disclosed a retrocardiac atelectasis. speci~ts of stool and blood were obtained for cultures and study of c. botul#num toxins. pending receipt of these results, a broad-speotmm antibiotic and acyctovir was begun. the initial differennal diagnosis consisted of laryngospasm associated with aspiraqlon, botulism, and postmfecfious varicella encephalitis. after 15 hours, weatm~ was begun. the neurologic examination showed a low modified glasgow coma ~ale (mgcs), generalized hypotouia and muscle weakness. these data suggested three diagnoses, posfnfecfious encephalitis, residual neuroumsoaar blockade, and excessive doses of sedative and analgesic drugs. after 20 hours she regained skeletal muscle poxver and ufltlcient respiratory effort, the mcgs was acceptable, and blood gases were normal. she was given n~-tigmine and atropine, and her tr~ma was extubated. an acute respiratory failure ocurrs 120 ram. after. chest radioga'aph disclosed a left inferior lobe atelectasis. after 20 hours weaning begun~and the same episode w~as seen. at this point her mother stated that the girl showed weakness of the eyelids or extraneular muscles. it suggested myasthenic syndrome vs ~-barr6 syndrome. c. botul#num toxins were negative, chotinesterase level ~as normal. edrofoinum test ~as positive. anti-acetyleholine receptor antibodies were negatives. e.m.g. confirmed myasthenia gravis (congenital vs juvenile serenegative). pyridostigmine was begun and the trachea was extubated without complications. conclusion: din the differential diagnosis of weamng failure we must consider ~c gravis~ 2)myasthenia gravis could resemble encephalitis, because of low ocs, overall if is triggered by viral infection. 3)in some diseases (this case) gcs could not he an aemuate index of mental state. a burguet*, a menget*, e monnet**, a gasca-avanzi*, c fromentin*, h allemand**, jy pauchard*, ml dalphin*. * r4animation infantile potyvaiente chu st jacques 25030 besancon cedex. ** d~padement de sant6 publique 25030 besancon cedex, france, objective : to point out that strabism is) of one-year-old premature is a good predictor of a poor neurological outcome at two years of age. design and setting : two-year prospective cohort study and geographically defined study (region of franche-comte, france). main outcome measures : neurological assessment was performed at one and two years of age (uncorrected for gestationnal age). a mailing questionnaire was sent to the famity and fuu-filled by thefamily doctor (pediatrician or physician), or neonatologist of the icu at tertiary center, s was diagnosed at one year of age by the examinator but s was not used to diagnose cerebral palsy (cp). sample : 161 of 171 survivors (94%) evaluated at one and two years of age. results : correlation of one and two years neurological evaluation is weak (kappa=0.5). correlation of s at one year and cp at two year is fair (kappa=0,72). the goal of this paper is to review evidence related to hypothesis that the "waiting" axons and cells of the transient subplate zone may participate in the structural plasticity of the human cerebral cortex after perinatai brain damage (kostovic et al, metabot brain res4:17, t989) and to correlate this phenomenon with different forms and mechanisms of structural plasticity. it is our basic assumption that all lesions occuring during cortical histogenesis will lead to more or less pronounced structural reorganization. here we show that various components of the subplate zone participate in several forms of the structural "plastic" responses in the human cortex: modification of convolutional pattern, changes in size of cytoarchitecturat areas~ columnar reorganization, dendritic and synaptic plasticity. the etiological factors which induce lesions and subsequent plastic changes act via the following pathogenetic mechanisms: * disturbances of radial unit formation (rakic); * changes in ingrowth of afferent fibres; * changes in the rate of normally occuring reorganisational events, depending on the critical period for a given histogenetic event. in the present study developmental lesions (localized perlventricular leukomalacia and haemorrhages) were demonstrated by ultrasound in live-born infants ranging between 26 to 40 weeks of gestation. in younger infants (24-34 w) who died shortly after birth, examination revealed lesions of the white matter with the preservation of the subplate zone. in infants who died one week of more after the lesion, we have observed localized micropolygyria, cavities, condensed layer vi -subplate zone, and columnations of the cortical plate. these changes are less prominent if the lesion occurs after diminishment of the subplate zone (after 34 w). since in the fetal cortex the subplate zone serves as predominant source of growing fibers, transient neurons, trophic factors and contains cellular substrata for migration, this zone is the most likely candidate for major types of structural plasticity. in conclusion, cerebral cortex of the low -birthweight infants is more susceptible to the various lesions but shows vigorous structural plasticity and conspicuous functional recovery due to the growing, transiently located neuron at elements. the mortality due to meningoccocal sepsis is high in spite of important progress in emergency and intensive care medicine. during the last decade multiple scoring-systems have been developed in order to establish a therapeutic approach and to evaluate the final outcome of a meningococcal infection. different clinical and biological data (shock, ecchymosis, peripheral wbc and platelet count, coagulopathy, acidosis, meningism, etc) are taken into consideration and the importance given to these data depends on the scoring-system used. a review of the different scoring-systems is given and a clinical case is presented. we report the case of a 4 year old male, who was transfered to our icu 12 hours after onset of temperature and skin rash. the parents described a fast deterioration of his condition. the boy presented wide spread ecchymosis, high temperature, no signs of meningism, circulatory insufficiency and shock, coagulopathy and low peripheral wbc and platetet count. disseminated intravascular coagulopathy developed promptly. the glasgow meningococcal septicemia prognostic score (gmss) was used and the obtained score reached the highest level (15/15). this corresponds to a 100% mortality. the patient required mechanical ventilation for 5 days. at admission he received human albumine, fresh frozen plasma, dexamethason, dopamine, dobutamine and a continuous infusion of adrenaline. antibiotical treatment consisted of ceftdaxone. the evolution was favorable and the infant fully recovered. retrospectively the gmss was compared to other meningococcal scoring scales which gave the same mortality (100%). we conclude that the scoring-systems are important to evaluate the seriousness and to assess the therapeutic approach, but they should be used cautiously even when 100% mortality is predicted by several risk evaluations scoring-systems. the aim of this study was to assess the haemodynamic status on admission and the critical care management of children presenting with meningococcat infection. this was a retrospective study of the charts of 46 consecutive admissions. mean age was 3.43 years (+/-3.46). the average duration of symptoms prior to admission was 20.4 hours (+/-14.09). on admission 17.4% were hypotensive, 45.6% had clinical signs of haemodynamic instability and 54.8% of cases that had a blood gas analysis on admission had a metabolic acidosis (bases excess < -5.q): the mortality rate was 10.9%. 80% of patients that died were hypotensive on admission and all had a metabolic acidosis. of the 41 survivors 9.7% were hypotensive on admission, 39% had clinical signs of haemodynamic instability, 25% required invasive pressure monitoring and 7.3% were ventilated and received inotropic support. this study demonstrates that at the time of presentation with meningococcal infection children had a high incidence of established haemodynamic instability. successful management of this infection is dependent on early presentation and initiation of therapy and on aggressive support of the cardiovascular and vital organ systems. dept. of intensive care medicine and dept of infectious diseases, our lady's hospital for sick children, crumlin, dublinl2, ireland. jude. pediatric intensive care unit, ch&u, 59037 lille-france. more than 10% of children surviving sip (defined as purpura with shock) have snli. objective. to search for a specific hemostatic profile in children with snli. patients and methods. between may 1989 and march 1995, 34 children with sip were admitted to our picu : 6 (17.6%) died and 28 (82.4%) ranged in age from 1 to 185 months (mean : 29) survived, 5 of them (17.8%) with snli (defined as the need of a surgical procedure). in survivors, two hemostasis studies (between h0 and h12, and 24 h later) included the determination of coagulation factors (routine tests), protein c (pc : amidolytic activity, biogenic), total protein s (ps : elisa, stago), c4b binding protein (c4bbp : laurell's technique, stago), antithrombin3 (at3 : chomogenic test, stago), and plasminogen activator inhibitorl (pail : chromogenic test, biopool). three severity scores were determined at admission : french group of pediatric intensive care, gedde-dahl, and crp. statistical analysis used the wilcoxon's test. results. at admission (lst sample) severity scores and at3, pc, ps, c4bbp levels were not different between the group with snli and the group without snli ; quick time (22 4-5% vs 35 ± 14% ; p = .025), vti+x (20 4. 3% vs 30 4-10% ; p = .04i) and pall (105 4-157 ui/m! vs 580 4. 570 ui/ml ; p = .028) were lower in the group with snli. on the 2nd sample there was no difference between the two groups. kinetics of hemostatic abnormalities was not different between the two groups. conclusion. in the literature, intravascular coagulation (dic), low fibronectin and at3 were identified as predictors of snli, and a negative correlation was found between the mean size of the skin lesions and pc activity, at3, and total ps. in this series, apart from dic, there were no specific hemostatic abnormalities that support the use of treatments such as pc, at3, and pail antibodies administration to prevent snli. further studies including more children are needed. the aim of study was to investigate the efficacy of intravenous immunglobulin with enriched igm content pentaglob/n /biotest/. in our pediatric intensive care unit ten septic children /group i/-their average age 2,6 years /sd:o,6/, 7 of them with gramm negative and one with gramm positive blood cultures, and two with unindentified bacteria-were treated with basis sepsis therapy and pentaglobin. the application of pentaglobin was as follows: 1,5 ml/kg loading dose for one hour, followed by a continuous intravenous infusion 0,1-0,4 ml/kg/hour depending on body temperatura /lanser scheme/ for 72-96 hours. another ten septic patients /control-group ii/the mean age 2,5 years/sd:o,65/, their blood cultures were gramm negative bacteria 6, positive 2, and the bacteria was not indentified in two cases -were treated with only the basis therapy. results: the duration of intensive treatment decreased from an average 22,7 days /sd:8, min 12-max 38 days/ to 19,5 days /sd:5,2 min 9-max 25 days/ in the group treated wit pentaglobin. the difference was significant /x 2 p<0,01/. in the group i nobody died, but three in the group ii. conclusion: the pentaglobin therapy can improve the efficacy of the basis therapy of sepsis. sinus bradycardia after an episode of sepsis is a rare symptom complex decribed in children with hematologic malignancies. we present a case of postsepsis bradycardia following severe typhlitis and septic shock in a 12 year old boy with relapse common all. blood and ascitic fluid specimen grew clostridium species and pseudomonas aeruginosa. at surgery there was a necrotic gangrenous terminal ileum and cecum, requiring ileocecal bowel resection with ileostoma. while clinically recovering from sepsis he developed bradycardia for 120 hours. extensive diagnositic procedures was given and the heart rate slowly increased to normal range of age. postsepsis bradycardia in children with hematologic malignancies after an episode of sepsis is self-limiting and after careful differential diagnostics warrants an expectative attitude. nitrate level is known to be enhanced during sepsis. serum nitrate is the stable metabolic end-product of endogenous nitric oxide generation. nitric oxide has demonstrated to be a powerful anti microbial final mediator and also a key molecule driving to the lethality of one of the most common complication of sepsis; the endotoxic shock. such facts prompted us to investigate the possible diagnostic and/or prognostic value of monitoring serum level in high risk, presumptive and confirmed sepsis patients. additionally we have explored the usefulness of this mediator as index of therapeutic response. in our study it is demonstrated that there is an important relationship between nitrate level and the occurrence of neonatal sepsis. septic newborn group showed 6 fold higher nitrate level than that of healthy control group. in addition, the group of patients with high risk of sepsis which finally became septics, exhibited 3 fold higher nitrate level at 24-72 hours before the first symptoms appeared, when compare with those who did not develop sepsis. however in the presumptive sepsis group, there was no difference between the patients which finaliy ,&'ere considered septics and those which not. in all septic cases, after 7 days of a successful therapy with antibiotics, the level of nitrate diminish 3 fold. our results suggest the utility of monitoring nitrate as index for the diagnosis of neonatal sepsis. the potential benefits of exchange transfusion, plasma exchange, and haemofiltration have all been described in children with overwhelming sepsis. however, little hard evidence exists to prove the benefits of any of these techniques. i have treated five patients with plasma exchange (pe), having been asked to see all these patients at a point when it was felt death was inevitable. two of the patients had staphylococcal, two meningococcal and one enterococcal septicaemia. all patients showed a dramatic haemodynamic improvement following pe with improvement in blood pressure, reduction in inotrope requirement and improvement in tissue perfusion. three patients survived. one of the patients with staphylococcal sepsis and both of the patients with meningococeal sepsis had developing gangrene of the limbs which showed remarkable reperfusion with pe. in two of the patients measurements of cardiac output (co) and systemic vascular resistance (svr) showed ~a reduction in co and a rise in svr over the course of a pe despite the reduction or cessation of vasoconstricting inotropes. many believe haemofiltration is of value in septic shock. a trial with a no treatment limb is difficult to achieve. i believe we now have enough evidence to justify a controlled trial of haemofiltration versus plasma exchange in patients with septic shock and unstable haemodynamic status whilst on inotropic support. during the next several days, cough and chest pain suggested pulmonary embolism confirmed by radiologic evaluation. echocardiographic examination showed multiple thrombosis of the superior vena cava, right atrium and ventricle and pulmonary artery. estimated protein c level was 50.7 % (normal range 70-140%); identical deficiency was found in patient's mother and elder sister. cvc was removed, and alter 2-month heparin therapy and supstitution of protein c with fresh frozen plasma, there was almost complete thrombolysis of the great vessels and cardiac chambers. we conclude that invasive diagnostic and therapeutic procedures in such patients may result in higher risk for severe thrombosis at unusual sites, and numeuos further complications bronchopulmonary dysptasia (bdp) is a chronic pulmonary disease of preterm and term babies treated with mechanical ventilation for respiratory problems of different origin and requiring oxygen therapy 28 days after birth. bpd is a disease affecting the growth and development of pulmonary tissue. such pulmonary }esions heal by squamous metaplasia leading to scar formation and fibrous tkssue r~growth, the pediatric intensive care unit makes the survival of babies w~h very low birth weight (500 -999 g) possible. with the increase in their aulyival, the number of complications in low birth weight babies increases as well. bdp is a very serious complication. therefore the importance of early diagnosis and treatment of bdp must be stressed in order to reduce the consequences. babies with bdp must be under medical suveillance for at least 3 years as the disease needs at least that long for complete resolution. tn the icu of pediatric department at madbor teaching hospital: during the past two years (1994-95) 154 newborns were treated with mechanical ventilation. the neonatal and postnatal death rate of all newborns admitted to our icu was 7,1%o.ln the two years from 1994 to 1995, 16 newborns were admitted to our icu (2 %~ of all newborn babies at maribor teaching hospital), with birth weight 500-999 g. in the icu, the survival of these babies and parallel to it the number of complications is increasing. during the mentioned 2-year period, 8 babies with very low birth weight (500-999 g) survived: 5 in 1994 and 3 in t995. in 45-50 %, first or second stage bdp was treated,there was no case of third of fourth stage bdp. the treatment consisted of eary removal from mechanical ventilation, oxygen therapy~ intensive treatment of infection, volume and caloric intake contro}, corticosteroid treatment throught 6 weeks with decreasing doses, diuretic end antioxydant therapy. the children are to be reevaluated at the age of 3 and 6 months and again at i and 3 years. oeure j van der, markhorst do, haasnoot k department of pediatrics, pediatric intensive care unit, free university hospital, amsterdam, the netherlands. case summary a 4%-month 6.5 kg girl of african origin was admitted to the pedfatric irtensive care unit with pneumonia and progressive respiratory irlsuffjderey. she was intubated and ventilated by pressure regulated volume controijed ventilation (servo 300c, siemens, soma, sweden). maximum conditions were inspiratory minute volume 3.2 l, peep 10 cm h~o ahd 100% 0~. chest x-ray showed bilateral interstitial consolidation. material obtained by broncho-alveolar lavage showed preumocystis car}nil htv-serology (elisa and westerll blott) and p24-antigerl were positive, confirming the diagnosis of pediatric aids. she was then treated with high dose co-tllmoxazoie, penthamldine, z{(~ovudire and steroids iv. because of thee x-ray features, high need for o 2 (100%, pad 2 56 mm hg), not responding to elevatiofi of peep (max 10 cm h=o) and pao2/fio = <200 (s6). m acute respiratory distress syhdrome (ards) was diagnosed. because conventional ventilation (cv) failure, hfo-v (31ooa, serisor medics,yorba linda, ca) was initiated. starting mean airway pressure (map) of 19 cm h~o was based or map of the cv, oscillatory pressure amplitude (dp) of 47 was, at ii~itial frequency of 7.5 hz, adjusted ur~til chest wall vibrations were visible, it was required to raise map to 26 cm h20 and dp to 66 before optimal lung volume and ventilation were achieved and need for o 2 reduced within hours, this was monitored by frequent blood-gas analysis and chest x-rays. map and dp could slowly be reduced, after a good response the first day, gradually 02demand reduced and the patient could be weaned from the ventilation. map, dp, fi02 and oxygenation index (map x pa0~jfio 2) are shown in table i. chest x-ray follow-up showed gradually improving lung features, with marked improvement of aereation. after 10 days hf0-v she could be succesfully detubated when a map of 10 cm h20 was acmeved. results : sianificant increase in ventilato~ rate and mean airway pressure was noticed after the change to savi. no differences in oxygenation, co 2 partial pressure and systolic, diastolic or mean blood pressure between imv and savi periods were noted. in 6 infants however an improvement in pao2/p43.ol/ and decrease in paco 2 was observed after the switch to savi. these babies had a lower initial a/a oxygen tension ratio and required higher initial ventilator rate /p<o.05/ in comparison to the rest of the studied patients. conclusion:infants with low a/a po2ratio who need high initial ventilator rate are likely to obtain benefit from savi.. fumimare hatori, haruo uchida, masao katayama, and rika muto department of anesthesia and critical care medicine chiba children's hospital, chiba city, japan. purpose: this study was made to find out whether the heat and moisture exchangers (hme) was effective enough to humidify in the respiratory management for children. patients: the study population consisted of 21 patients who were provided with artificial airway. they were divided into the two groups: ii with heated humidifier (a) and i0 with hme (b). methods: hme could be used for those without pulmonary complication and with less leakage around the endotracheal tube. in both a and b groups, (i) we have measured relative humidity, temperature, and absolute humidity at the endotracheal tube connector. for these measurements, humidity sensor system was utilized. (2) any troubles and complications caused by the artificial airway were investigated. results: (i) in group a, we confirmed the mean values as the relative humidity of 96.5%, the temperature of 32.8~c, and the absolute humidity of 34.0mg/l. in group b, they were 92.7%, 29.5°c, and 28.7mg/l respectively. no difference was noted between the two groups as to the relative humidity, but the temperature and absolute humidity were lower in group b with a significant difference (p<0.0001). (2) in neither of the groups, any respiratory complications such as obstruction or stenosis of the artificial airway were noted. in pediatric icu, hme can be used without any problems under given conditions. titei: objective: evaluate the precision, reproductibility and aplicability of a new device to measure auto-peep in pediatric patients during mechanical ventilation. material and methods: it's an electromc-pneumatic eontolled device with an oclusion valve and a pressure monitor installed between endotracheal canulla and the ventilator circuit. the measurements were performed with a sollenoid conected with to the ventilator to detect the end of inspiration phase and the actwation of the oclusion valve. the signs of pressure and flow were moniturized using a difere~rtial transducer and it was processed using a pc computer and a pneumoviaw® software. the auto-peep also displayed in the ventilator pressure monitor. results: we submitted 17 children with neuromuscular disease or respiratory distress in this study. the incidence of auto-peep was 76%, with variability between 1,5 to 13 cmh20. all the measurements were performed 3 times to verify the precision and the reproduetibility and evaluation of the pressure and flow curves. conclusion: the device is low cost, easy to use and can be used without the pneumotacograph. the auto-peep measurement can be done during mechanical ventilation with time cycled, pressure limited and continuous flow, the most commom ventilator used in pediatric patient. resistance was modeled by 3 endotracheal tube (ett) sizes (2.5, 3, 3,5), and compliance by 2 test lungs (1 and 5 ml/crnh20), for each resistance -compliance arrangement, we measured tidal volume (vt) at various frequencies, peak to peak (p-p) and mean airway pressures. results vt increased when ett size increased with all 3 ventilators in accordance with fredberg results (jap, 1987; 62: 2485-90) . the maximum vt delivered was smaller with the hfv-babyiog 8000 than with the ohf 1 or the sm 3100a at a given frequency (figure on the left). increasing p-p resulted in a linear increase in vt delivery in the 0-30% range of maximum p-p (figure on the right). hfv-babylog 8000 didn't provide significant vt increase when p-p was set above 50% and vt deliven/decreased when mean airway pressure decreased. --i 5~ sm 3100a a number of ventilatory parameters had been analyzed with russia-produced monitor humitemp htm 902 (servisinstrument j,-s. co) in 24 newborn infants with rdsn of t-iv stages on cmv, cppv, imv, and cpap steps using respirators of various kinds (stephan-staxel, bear-2001, newport 8rezee, sechrist). parameters investigated were: temperature of inspired mixture (t), relative and absolute humidity (rh and ah, respectively), static compliance (c), expired tidital volume (vte), expired minute volume of ventilation (mve). the monitor read these parameters in following ranges; rh-from 10 to 100%; ah-up to 55mg/i; mve-up to 3.0 i/mini vte-from 3 to 130 ml; t-accurate to 3%; c-from 0.1 to t0 ml/cm h20. continuous monitoring time ranged from 2 to 96 hours, it was noted, that during this time the monitor and transducers had no malfunctions, and it's using didn't require additional calibration, it was found, that humidifiers mr 498 and mr 410 (fisher, paykel) couldn't maintain parameters t and ah continuously (these values varied from 34,3 to 38.2 c and from 37,5 to 47.1 mg/i, respectively, when heating regulator position was unchanged). changes of parameters c depended clearly on cppv conditions and rdsn severity (with rdsn of stages i-ii c value ranged from 2.4 to 1.2 ml/cm h20; and with stages iit-tv-from 0.3 to 1.1). tn 6 newborn suffering from rdsn of stages ii-ltl and ill-lv with pip>25 mbar, fi02>0,7, peep=4-7 mber, c-from 0.3 to 1.2 ml/cm h20, effectivity of exosurf therapy was studied. in 4 newborns in 4-12 hours of therapy pip decreased to 0.3-0.4, and c increased to 1,7-2.4 ml/cm h20. in 2 newborn infants with aad02>500 mmhg and c from 0,3 to 0.8 mltcm h20 positive effects of exosurf on lung compliance were not observed. in 3 newborns the monitor had revealed decreased of c (from 3.4-2.9 to 1,8-1.3 ml/cm h20), manifested clinically by pneumothorax. in general, monitor htm 902 made possible; 1), to estimate the adequacy of cmv-parameters and regimes in newborn infants; 2). to select optimal t and ah values in the respiratory outline in dependence on lung damage severity and infused volume; 3). to reveal rdsn severity; 4), to optimize indications and adequacy of surfactaot therapy; 5). to diagnostieate the air leakage syndrome; 6). to effects to some agents (broncholytics, spasmolytics); 7). to obtain objective indications for imv/simv and cpap regimes. albano communication is an important aspect of human development and existence, and an inability to vocalise can be a problem in ventilatordependent patients. we present our experience with speaking aids as a means of enhancing verbal communication in four ventilatordependent children in our paediatric intensive care unit. the age of the children ranged from 7 months to 5 years, and the period of ventilation ranged from 3 months to 21 months via a tracheostnmy. they require continuous flow generated pressure limited or control ventilation at rates of 13-20 bpm. the reasons for ventilation include tetraptegia following a shrapnel injury; tetraplegia following congenital cervical spine damage; tetraplegia following atlanto-axial subluxation; and critical illness polyneuropathy following adult respiratory distress syndrome from prolonged ventilation for a severe head injury. the first three patients have passy-mnir one-way speaking valves and the final patient has a bivona foam cuffed tmcheostomy tube with a talk attachment in view of recurrent aspiration. an improvement in quaiity of speech has been shown by independent assessment. we will review the present literature on this subject and discuss the advantages and disadvantages of these two types of speaking aids in the light of our experience. the prognosis of antenatally diagnosed cdh is closely related to the degree of ph. there have been attempts to correlate antenatal or postnatal criteria to mortality: none have been demonstrated to be predictive of lethal ph. the aim of this retrospective study was to determine whether antenatal or early postnatal data could correlate with the findings of post-mortem examinations. patients and methods: between july 1990 and july 1994, 32 cdh patients have been antenatally and postnatally managed at our institution. twentythree infants underwent a post-mortem examination. ph was assessed by using the lung weight to body weight ratio (lw/bw) and the radial alveolar count (rac). antenatal results: cdh diagnosis was made at 24 weeks of gestation (wg) (15-37). twenty-eight patients had a left sided cdh, 3 had a right sided cdh, and one had a bilateral cdh. herniated organs were stomach none (n=21), or liver alone (n=4), or both stomach and liver (n=5 the patient was a 3-yenr-old girl with chronic renal insufficiency see~ to renal dysptasm, two months before admission a kidney trar~ptant was performed. one morah later she showed acute graft rejection with serum ereafinine (cr) level of 0.7 mg%. the rejection was unreslxmsive to an increased steroid dosage, and okt3 was begun with resolution of the rejection. one week arer, new rejection episode was seen marestxmsive to an increased steroid dosage, and transp~ ~s performed five days before admission to our ptc. hemedialysis and peritoneal dialysis (p.d.) each other day, was indicated (g.r.f.< 10 ml/rnin). four days before admission t ~ rose to 38°c. "lhe diagnosis of opporttmistic pneumoma was made on the basis of tach3,pr',e~ hypoxi~ and diffuse interstitial infiltrates. senma ~ was positive for cytomegaloviras (cmv), and stool culture for c albicans. pentamidine, ganciclovir (dhpg), arai-cmv gamma globulin, eritromicine and amphotericin b was administered. on admission in our picu, trachea was mmbated, (a-a) o2 gradient was 600, paofffio~: 65, lung injury score > 3 with peep level of 8 cm hzo. she had normal fiver function. during te next days she had fever and developed ards. bal was negative. p.d. was of little efficiency. we adjusted pentanfdine, and dhpg doses for severe renal failure, with supplements after hero, sis, and at~rp.d.. during ~ next days she was afebrile, and the chest became radiologlcally normal. after ten days on menhani~al ventilation (mv.), the patient was extubated. cr. level was 3.2 rag%, (a-a) oz gradient was 20, and paoyfioz was 375, the patiem was discharged with chronic ambulatory p.d. discussion: opportunistic pneumonia is a major complicalaou in imm~romised children, specially after kidney tvansplaraafion. c m.v. infection can result at~r okt3 administration. in the treatment dhik} dose must be adapted to the degree of renal insu~cieney, with supplements after hemedialysis, and after pd. pneu~y~tis cann# tmeumov~ is ehemeterized by ventilafion-perfusion mistmaeh, decreased pulmonary compliance, hypoxia arld elevated (a-a) oz gradient, with diffuse interstitial infiltrates. in our ease bal was negative. although we did not find the etiology the prevoclons eombh~ation of arairmcrobiat therapy, along with m.v., and supportive measures were the most effective trealme~. conclusion: 1) in patients with severe renal failure and life-threatening infections, we must co~ider drug adjuslments. 2) in our patient we gave dhpg supplements at~r pd. with excett~at results, although p.d. was of little effiele~. introduction: endotracheal intubation and mechanical ventilation have become an important treatmem for many diseases accompanied by respiratory failure. with the frequent use of this treatment modality, an increasing number of complications associated with endotracheal intubation have gained clinical significance. material and methods: a transversal study was realized to find the prevalence of pulmonary aspiration with endotracheat tubes in 36 infants and children. aspiration was assessed by applying two dyes (evans blue, er)¢rosine sodic) on the tongue and searching for the dye during suctioning in the endotracheal aspirate. the factors, that potentially have influenced the aspiration, including weight, age, sex, cause of respiratory failure, main pressure airway (map), level of consciousness, presence of swallowing and body position were evaluated. all the variables studied had their association with aspiration tested by chi-square method with relative risk considering a confidence interval of 95%. the results were adjusted by multivariate analysis. results: the overall prevalence of aspiration was 36.1%. among all children who aspirated, compared to those who did not, there was a statistically significant difference in the presence of swallowing (p=0.005). the odds ratio to aspiration in the presence of swallowing was 38.4 (t.75 -100 c.i.95%) and the relative risk 55.5. aspiration was not significantly affected by sex, weight, age, cause of respiratory failure, map, level of consciousness and position of the body during the ventilation. conclusion: the endotracheal intubated children frequently aspirate as intubated adults and that preventive measures are ineffective. the presence of swallowing movements is the main risk factor to aspiration of oropharingeal content in intubated patients. clinical features and shortterm outcome skling, rp gie pneumonia is the second most important cause of death in young south african children. the clinical features, intensive care course and outcome of children being ventilated for pneumonia in the developing world is unreported. aim: to describe the clinical findings, aetiology and shortterm outcome of children younger than 6 months with pneumonia requiring ventilation. the data of all babies under the age of six months with a lower respiratory tract infection admitted to the paediatric icu for ventilation were prospectively collected over a period of 14 months. tracheal aspirates and blood specimens were submitted for viral and bacterial cultures. results: forty-seven babies aged 14 to 174 days were ventilated for pneumonia. twenty-six infants had been born prematurely; t2 had been ventilated during the neonatal period and 4 had bpd. the median duration of symptoms was 1 day, the most common being cough, tachypnoea, apnoea and cyanosis. five babies (10%) died. the mean duration of ventilation was 8 days (range 1-85 days) and of ward stay after icu discharge 19 days (range 1-161 days), blood euttures were positive in 7 children (15%). viruses were cultured in 14 children (30%). conclusion: 1) fifty-five percent of children below 6 months requiring ventilation for pneumonia were premature infants, of whom 46% had been ventilated during the neonatal period. 2) the median duration of symptoms prior to admission was 1 day. 3) ninety percent of the children survived and were discharged from hospital. 4) viral pneumonia was responsible for 30% of the admissions. mechanical ventilation and atrial natriuretic factor release ulloa santamarfa, e, p6rez navero jl, ibarra de la rosa i, espino hernladez m, velasco jabalquinto mj, frfas p6rez m. picu. reina sofia children's llospital. c6rdoba. spain. mechanical ventilation effects on renal function decreased diuresis and natriuresis due several factors including anf. several studies have demostrated anf released due increaasing pressure in right atrium. on the other hand, mechanical ventilation, overall peep modality, inhibits peptide release althougt cvp increased is found. this study was designed to demostrate anf stimulation is due rigth atrium stretch which be higher during mechanical ventilation instead of atrium pressure. we desing a prospective study including 14 patients, age range 16 months-13 years with congenital heart disease. all of them were admitted at pediatric intensive care unit after extracorporeal surgery and were assisted by mechanical ventilation. hemodinamic state was stabilized in all patients and nor renal neither neurological diseases were found. after 24 hours with mechanical ventilation, plasmatic levels of anf were measurement, pvc, pericardical pressure were assessment; all patient were sedated with midazolan and paralized with neuromuscular blocking agent; mechanical ventilation technique was as follow: imv between 20 and 30, tidal volume and fi o2 enough to mantain respiratory parameters in normal range. afterwards, at least twentyfour hours in spontaneous breathing, the study was made again in each patient. atrial stretch was assesssment according to following equation: transmural pressure= cvp -pericardial pressure. cvp were significantly higher with mechanical ventilation than when the patient was breathing by himself. (5.4+__ 2.2 vs 3.8 + 1.8 mm hg; p<0.01). however, transmural pressure during mechanical ventilation were lower than during spontaneous breathing (8.92 +__ 3.86 vs 11.76 +__ 3.32 mm hg; p < 0.01) equal, plasmatic anf levels were lower during mechanical ventilation ( 87.77 + 46.55 vs 108.92 + 49.06 pg/rnl; p<0.01). in conclusion, anf secretion decreases during mechanical ventilation, even with cvp higher. anf release would depend on atrial stretch meassured by transmural pressure, lower in patients with mechanical ventilation and it would not depend on atrial pressure. the paediatric intensive care unit shaikh zayed hospital, lahore is an acute care area devoted to the care of critically sick children upto the age of 13 years. in a 6 bedded unit with limited equipment, constant care is ensured by the presence of at least one nurse aed one doctor round the clock. in this setup we have the facility to ventilate 2-3 children at one time, between sep. 93 and dec. 95, out of 885 patients admitted to icu, 171 (19.32%) were below 1 yr of age, while 48 (28%) were below 1 month of age. life support was discontinued in 17 (9.9%). total mortality was 56 (32.7%), major mortality was in 0-1 month age group 22 (12.8%), and 1 month to 6 month 15 (8.7%). majority of the patients were of sepsis (36.2%), cns disorder (22,2%) followed by respiratory problems (14.6%). it seems therefore that the major indicatiou for ventilation was overwhelming septicemia leading to multiple organ failure, rather than purely respiratory problems. high frequency oscillation (hfo) in the therapy for ards in pediatric patients requiring aggressive conventional mechanical ventilation (cmv) -routine or experimental mode ef pre ecmo therapy. fedora m., nekvasi~ r, vobruba v., srnsky p,, zapadlo m. dpt. critical care medicine, nicu and ecmo center, university children's hospita! brne, nicu of university hospital prague, czech republic. introduction: 9 pediatric patients (8 males, 1 female, average age 4.7 months, average body weight 5,8 kg) with severe ards ventilated with aggressive regimen of pcv or prvc were connected to hfo (sensormedics 3100) as the last "rescue" therapy due to uncontrollable respiratory failure before intended ecmo. in the course of hfo 2 of them were given no in the concentrations of 5-80 p.p.m., 3 were subjected repeatedly to surfactant replacement therapy (alveofact). results: ecmo was needed in no patient, 8 patients survived, 1 patient was disconnected from the ventilator because of brain death in spite of conspicuous improvement of oxygenation and other parameters, some relevant parameters 48 hours before and 48 hours after starting hfo are given in table 1~ in all the cases, the disconnection from hfo was carried out through the simv regimen, never directly to cpap. table 1 : the levels of blood gases, oxygenation index (oi), aado2,map,fio2 and pao2/fio2 ratio 48 hours before and 48 hours after starting hfo. conclusion: although none of the patient had to be subjected to pediatric ecmo, hfo should be carried out only in workplaces having the immediate possibility of using this method in the case of hfo failure. speculation: should not hfo be used ir pediatric patients with ards earlier than aggressive cmv? can hfo ce considered standard, not experimental method of therapy? refractory hypoxemia in premature patients is characterized in a persistent elevation of pulmonary vascular resistance, with right to left shunt through the ductus arteriosus and or foramen oval. we report the case of a vlbw patient (ga 27w, bw 1010g) who present a severe hypoxemia related to hyaline membrane disease and a pulmonary and systemic infection to group b streptococcus, refractory to conventional ventilatory support and surfactant therapy, associated to hemodynamic failure falling in ecmo criteria used for term infants. a rescue therapy with hfov (sensor medics 3100a) is decided at 5 h of live, the table resume the patient's evolution before and after hfov. at 36w of postgestational age the patient present a fio2 of 0.23 with a chest x ray compatible with a cld type l at discharge no oxygen requirements was needed and actually he's doing well. conclusion: hfov, using an adequate alveolar recruitment strategy, was effective in the rescue of a severe hypoxemic respiratory failure with a rapid off of ecmo criteria entry in our vlbw premature patient, during the united nmioffs embargo ~nst yugoslavia the prevalence of the ast}nnafic ~acks in c~dldren aratsed. the mo~t common causes have beem dramm~e worsening of life standard, ecom~c disaster in global community, gr~ number of refugees from the other parts of former yugodavia. it wm obviom that mcio-ecoumnical conditions took a part in the exacerbations of previously known cldldhood asthra~, ~av~ of micro-and m~mclimaflc changes, psychosocis] and emotional cryses, lack of medics-m~nts for p~ve~on and tl~rspy of acute asflanatic attacks. about 10% of d-dldv~ tmslod in our picu for these year~ exp~dvncod ~vcr~ attack for the flint time iu ~jzeir lifts. it has been cu~ 1~%~ children in mspir~ry picu of our hos~mt. the scut~ revere attack (more ~asn ~/o of hight clinical score) was detected in 62% of all children admitted with respirak~ problems. from tl~ mmlysss we exclu&d: bmncldolifis, ~i anomalies, ~eve~ i~ccqions. concerning our drug supplies (which wc~e reduced), we started our therapy by administration of oxygen, ~ta2-ago~dst inhalations (but sometimes we had the solution for jet nebulizcm only for o~e inhalation per p~cnt), mwinophyllin and mefl~ylpr~ini~done in/ravenously. 48% of ih~ asthmatics needed repea~ doses of muinophyl~n pinch.ally, tnch.,ding the fluids. the bronchodilak)r msponm was poor ~r~cl slow, hospital stay in picu was for 4 days and for 14 days in other units sl~rwsvds. tim ~ of their stable condifio~ was hard at borne (or refugees camps), without p~ventkm, so they came bsvk to hospital for morn than 3 times in 27% of cases, dtrdng ~e4je last motlfl~s file dtustion improved, concerning tim drugs supply for prevention, and we hope that these lifc~restening conditions wouldd~ introduction: the incidence of ards is increasing as survival of critically ill patients is higher. the application of new therapeutic modalities have increased the survival rates in (ards) adult patients. objective: to study the therapeutic efficacy of new tleamlents in children with ards material and methods: a retros~ctive study was conducted from 1990 to 1995. 17 children with severe ards, (lung severity score > 2,5) (r), aged 15 days to 16 years, were included. the diagnosis were as follows: 9 interstitial pneumonitis, 5 non interstitial lung infection, 2 with lung aspiration and 1 with clinical sepsis. 5 patients had different tipes of cancer and 4 to suffer inmunodeficiency disease, the first 8 subjects (group t) were treated with conventional measures. from october of 1994new therapeutic modalities were introduced, including: less agressive ventilatory support, postural changes (prone to supine) in 9 subjects, administration of corticosteroids in 8 patients, rfitric oxide in 3, pe~ssive hypercapnia and administration of exogeans sarfactant in one, pao2/fio2, d(a-a)o2, oxigenation index (oi) and the score of respirator), severity disease were similar in both groups. the two groups evolntiou was compared. results: -ten patients died, 6 from group i and 4 from group ii (75% v.s.44:4%,ns). -the evolution time, either to exitus or weaning from ventilatory support was higher in group ii (22.9 v.s. 13.6days in group i, ns), -the incidence of barotrauma was observed in 12 subjects (70.6%), 6 from group i and 6 from ii. of these patients 75% expired. -during the course of the disease, 15 (88%) patients had more than one damaged organ. only in one subjet mof was considered to be the main cause of death. the majority of the patients expired because of their respiratory disease, although, 80% of them met criteria of mof. -fifty percent of the subjects were infected at the time of death. stmmry: a trend toward a higher survival rate is observed in the subjects receiving the new modalifies therapeutic intervention (corticosteroides, postural changes and permissive hypercapnia). our results are not significative,probably because of the small number of subjects studied. a new doubleaurae~t two-stage et-tube (dl-ett) was desig~aed and tested in the rabbits with acute king injury under conventional mechanical ~entilation_ ventilation efficiency of dl-ett was emrrpared with that of canveniionally t~sed single lumen et-tube (sl-ett). meth~s: dl-ett was specially made out of two sl-ett. vertical crosssections at the distal end of two et-tube (td 3_0 rmn portax) were adhered with each other to form a tracheal stage lumen wifu id 3.0mm the two remained uncut parts of the tubes corlntithted the oval s~ge with two separate imnens. dl-ett and sl-ett were randomly applied to five adult paralyzed rabbits with acute lung injury (by 0.1 nffkg oleic acid. iv). a bird inter 3 vetffttator (bird products corporation) was used for time-cycled pressure-limited ventilation at 40/min of respiratory rate, 10 ern h20 of peak i_~piratory pressure, l: 1 of ire ratio, 6 ljmin. of flow rate and 0.21 of fich. peak inspirntory pressure, mean mrway pressure, posi6ve end-expiratory pressure at tip of et-mbe and bemodynamics were measured and recorded continuously. arterial blood and expired gas were measured ~by avl 993 blood gas analyzer) after each stabilization t.~iod of 30 minntes. _analysis w~as by prated t test. result: dl-ett acaltety improve cos removal at all amman. pa(?oz was decreased by t0.6+_t.5 (p<0.0l) and physiologic dead space fraction (v~zvt) reduced by 22% +-1.8% (p<0.0t), compared with dl-ett. there were no significant change in arterial oxygenation. conelus|on: the double-lumen two-stage et-tabe significantly increases ventilation effmiency with simple operation in rabbits v, ith acute hmg injury, lts availability may influence future clinical management of ~ennated patient~. this ~muly was fimded by the science and technology. commiuee of beijing municipality. analis of hemostasis alterations on different coagulation cascades in 46 children with septic shock has shown that coagulation disorder character is dependent on lung affection rate. the initial manifestation of the respiratory distress-syndrome (rds) are characterized by the obvious activation of blood thrombin potential, moderate coagulopathy and not sharply marked endoteliosis, the witlebrand's factor (wf) increase tot 140-220%. progress in the clinical picture of "shock lung" leads to chronometric and structural hypocoagulation with potential hypercoagulation in "mix-test", high level of firbin derivative, thrombocytopenia with thrombocytopaty and the wf increase to 210~315%, terminal stages of the rds, as a rule, are characterized by potential hypercoaguletion absense, depletion of at-lit and plasminogen, prevalence of antithrombin and antiaggregating activity, obvious endoteliosis (the wf to increase250-540%). the arteriowenous difference according to index of the thromboelastography (teg) in the rds ill-iv rates was 69,8% less than in the 1-11 rates, disorder of lung filtering ability in severe rds is confimed also by minimal arterio-venous difference of activated euglobulin lyses (ael) in children with the rds ill-iv rates is only 11,4%, while the patients whit rds i-i1 rates have the ael-activity in arterial blood 2,1 times as much than in venous blood. the use of then allows to determine the potential hypercoagulation rate, the at-ill level and fibrinogen quantity during the anticoagulant therapy and also the character of the x-factor activation and thrombocytic hemostasis. the effective therapy component of septic genesis rds in children is the controled coagulation method with the use of the individual selected heparin doses in according to desagregants, kryoplasma, proteolisis inhibitors and trombolytics. it is necessary to avoid the heparintherapy for children with the rds complicated with producting coagulopaties and termal phases of blood disseminated intravascular coagulation (dic). bronchoseopy has been used for evaluation of the potential problems of the airways and for investigation the bronchial specimens for diagnostic purposes. regent technical advances result in performing this procedure at the bedside manner and in critically ill patients. we have performed 150 hronehoaeopy during last three years on 1362 pediatric patients with respiratory problems, in 90% of cases the opentube hroneh0seopy was performed (for diagnostic as well as for therapeutic reasons) and collected secretions or bioptic material were examined. the indieatiuns were: acute upper respiratory problems, chronic wheezing, inspiratory strider, tracheal or bronchial bleeding, chronic eongh, retractable atelectssis, severe pulmonary infections, lymph node perforation in lung tuberculosis and soquells like bronehiectssis and fibrosis. our results were: anatomical malformations in 10%, mueosal oedema with chronic inflammation and thick secretions in 56%, easuos masses in 11%, granulation tissue and purulent secretions in foreign bodies and bronehieetasis in 16%, and only 7% of eases were normal finding. our exlxdenees pointed that this invasive procedure in carefully selected patients has important role in establishing the diagnosis and in theintroduction: tbg has been a useful investigation in the management of ventilator-dependent infants in our experience. one ml of contrast was hand ventilated into the respiratory tree via their nasotracheal tubes and their anatomy and dynamics demonstrated on radiological screening. case descriptions: three infants who were difficult to ventilate requiring high airway pressures, high peep and a significant oxygen requirement had tbgs. the ages ranged from 3 to 9 months. two cases were complicated by complex cardiac lesions. in all cases there were frequent episodes of desaturation, where hand ventilation proved difficult and various intermittent lobar collapses occurred. microlaryngobronchoscopies (mlb) performed on the infants by experienced paediatric ent surgeons failed to identify the airway problems. more than one mlb was frequently done. concern about introducing contrast into the airways of infants with limited cardiorespiratory reserve combined with an uncertainty about how much extra intbrmafion would be gained often led to a delay in investigation. when performed these fears proved groundless, the anatomy and pathology of the airways were demonstrated in full and the correct therapeutic plan started. in two cases tracheostomy and peep producing patency of bronchomalacic segments allowed weaning to low levels of ventitatory support. in one case tracheal reconstruction was undertaken and in the cardiac cases the respiratory component of the ventilatory dependence was fully assessed. at the age of 4 months, a baby boy with a history of minor respiratory problems, was admitted to hospital with an upper airway infection and severe dyspnoea. shortly after arrival at the icu he had a total airway obstruction. after intubation there were still difficulties to establish a normal gas exchange, and he was tranferred to the regional picu. ct scan and bronchoscopy verified a congenital tracheal stenosis affecting the whole trachea except the upper 15 mm below the vocal cords. the diameter was estimated to less than 2 ram. an unsuccessful attempt was made to dilate the extremely rigid stenosis with a balloon. after the procedure he had a respiratory and circulatory arrest, and he was put on ecmo as a bridge to surgical correction. after 4 stable days on ecmo, surgery was performed during ecmo with a tracheal homograft transplantation. immediately after surgery, ecmo was discontinued. a silastic dumont type stcnt was inserted inside the homogra~, and a nasotracheal tube was placed inside the stent for assisted intermittent mechanical ventilation. repeated bronchoscopies were performed to remove granulation tissue and secretions. at 9 months of age, the stem was removed with an endoscopic procedure. however, the trachea was still soft and collapsable, and another silicon stent was placed inside the trachea for another 4 months period, after removal he had some respiratory problems and he was treated with nebulized salbutamol, mcemic epinephrine and steroids. he was discharged from the hospital at 14 months of age and his condition is now stable. this is the first procedure of its kind in sweden. it was accomplished by international and multidisciplinary collaboration. ecmo may be a bridge to corrective surgery and long time stenting may be necessary in the postoperative period. post mtubation laryngitis ( pil ) is still a frequent complication, occurmg in l -6 % of intubated patients. inhaled racemic epinephrine has for long been used as an accepted therapy, but this drug is not always available. the authors undertook a randomized, double-blind, placebo-controlled trial to determine the efficacy of inhaled l-epinephrine(le) in the treatment of plu in the period between july/93 and may/95, 289 patients were submitted to endotracheal intubation for ventilatory support. atter the extubation procedure patients were considered for enrollement if they met the following criteria: clinical signs of laryngeal estridor and a downes and rafaelly score for upper respiratory obstruction equal to or higher than 4 patients with primary upper respiratory disease were excluded all patients enrolled reeieved either inhaled l-epinephrine 1% or normal saline. dexametasene ( 0,6 mg/kg/day) was given to all patients in both groups. after 2 inhalations, au patients were monitored for a period of 1-20 minutes and monitoring included cardiac and respiratory rate, mean arterial blood pressure, arterial blood gases and the dowries and rafaelly score. statistical analysis included, qui-square with the fisher correction test and the z-test for paired variables. thirty eight patients ( 13,1% ) met the criteria for enrollment, 18 to the le group and 20 to the placebo group.there were no significant differences in both groups in regard to age, sex, initial score ( 5,05 x 5,1 ) and endotracheal tube diameter. the period of ventilatory support and tracheal intubation was significantly higher in the le group (8,06 x 4,54, p = 0,01). the follow-up score showed a significant drop only at 30 minutes after the inhalations (p = 0,03). re-intubation due to laryngitis, occured in 1 patient of the le group and in 4 of the placebo group with no statistical sxgnificance (p = 0,2). no difference was observed on the monitored hemodynamic variables during the 120 minutes, except for the mean arterial pressure at 60 minutes, being heighar on the placebo group (p = 0,05). we concluded that, although the l-epinephrine group showed a trend in better scores post-inhalation and fewer re-intubations due to laryngitis, the results were not statistically significant. we especulate that the period of intubation may have affected our results. similarlly there were no differences in the incidence of adverse effects between both groups. objectives:to evaluate the complications of endotracheal intubation in children with upper airway obstruction due to epiglottitis or croup. methodes: during a 5 year period (1991 -1995) all patients with epiglottifis or croup were reviewed to determine the complications of endotracheal intubation, especially upper airway obstruction due to granulomas. results: 33 patients were reviewed. in 17 children (mean age 2.5 years) with epiglottitis the mean duration of intubation was 4.0 days (3 -5). no complications were seen. in 16 patients (mean age 2.3 years) with croup the mean duration of intubation until the first extubation was 8.1 days (1 -15 days). elective extubation was performed if an airleak was present or after 7 days without airleak but in the absence of fever and obvious secretion. reintubation was not necessary in 10 children (62.5%). in this group the mean duration of intubation was 6.4 days (1 -12). in 6 patients (37.5%) reintubation was necessary because of severe upper airway obstruction due to granulomas. mean duration of intubation until the first extubation was 10.8 days (6) (7) (8) (9) (10) (11) (12) (13) (14) (15) (16) (17) (18) (19) . there seems to be a difference in duration of intubation between these two groups with croup, however it is not significant (p > 0.1). all the patients with granulomas could be successfully extubated after microlaryngeal surgery, with a mean intubation period of 35.3 days (21 -47). revealed no complications, where as endotracheal intubation in children suffering from croup showed a high incidence (37.5%) of granulomas. however1 laryngeal steepsis and other serious complications were not sesn~ 3 patients (42 days averagely] was obviously seen in ~he peak =one of fl, f2 resonance and in the zone of high freq,-~ncy :r, ~;~e composition while 12 cases(3 day~ average;y] :~bowed no abnormality both clinically and isryngoscopica!~y. 7/10 patients with catheter placement for more than 6 week~ end 1126 p~tie,~ts for less than 5 weeks had t;~ryngeal abnormal change in their larynges,abnormal changes of sound spectrogram were all seen in 3 patients with placement for mope than 5 weeks. our data suggest= ca] the complication of endotracheal intubation was increases with increasing length of time of catheter placsm. entjbut aeriuoa complication is rare i (b] the time limit of pernasal endotraoheal catheter placement is 5 weeks within which the procedure is • comparatively safe and effective means for maintaining e tong term artificial airway. in a 6-year period (1986) (1987) (1988) (1989) (1990) (1991) (1992) we diagnosed tbm as an apparent dilatation of the trachea and main bronchi ih four premature infants on continued mv for respiratory distress syndrome (rds). the infants were three boys and one girl with gestational age (ga) 26-33 weeks and body weight (bw) 1100-1965 g. mv was provided by bourns 2001 cub time-cycled and pressure-limited ventilator to attain normal gas tensions. no jet ventilation was used. chest radiographs were reviewed for a complete evaluation, and for the evaluation of the airway. after the intial subjective diagnosis of tbm, the width of the tracheal and main bronchial air column was measured at the lower level of the first and the third thoracic vertebal body it1, t3) and near the carina; the width of the main bronchi below the carina was also measured. in all infants, tbm became apparent close to the 20lh day, that is, after 2-3 weeks of mv. therefore, for the time period from birth to the 20th day the following ventilatory parameters were reviewed and analyzed: (1) the percentage of total ventilation time when more than 40% o2 concentration was required, (2) the peak inspiratory pressure, (3) the positive end-expiratory pressure, and (4) the duration of high frequency ventilation (80-160 breaths per minute). also noted were the apgar scores (1 and 5 min after birth), the duration of hypotension (systolic bp below 40 mmhg) and circulatory instability, the presence of systemic or tracheal conatal or later infection, the duration of mv, and the final clinical outcome. the records were also reviewed for other possible pertinent data. rigid respiratory endoscopy in children fraga j, amant6a s, piva j, nogueira a, palombini b. introduction: the respiratory endoscopy is an important procedure to diagnose and treat many airway's diseases in children. although have had advances in radiologic investigation exams and pulmonary function tests, the direct anatomic visualization of airway is important to the management of many respiratory problems. objective: evaluation the respiratory endoscopies performed with a rigid bronchoscope in a pediatric reference hospital. material and methods: we study the records of all children that were submitted to respiratory endoscopy under general anesthesia from march 1989 to march 1992. age, sex, clinical to indicate the procedure, diagnosis and complications of endoscopy were registered. results: three hundred and fifty six respiratory endoscopies were performed. the most common indications for endoscopy were strider (52%), suspected foreign body (16%), atelectasis (16%) and difficult tracheal extubation (8%). the most frequent diagnosis were laryngomalacia (36%) and subglottic stenosis (6%) in the glottic and subglottic areas, and foreign body (9%) and tracheomalacia (7%) in the tracheobronchial area. normal endoscopy was performed in 54 (21%) of the children. only three slight complications of the endoscopy were observed. two patients presented bradycardia during the exam, and the third need tracheal intubation due to post-endoscopic subglottic edema. conclusion: the rigid endoscopy in children is efficient and has no serious complications. near drowning; indicators of acute and long term prognosis bernardien t.mj. thunnissen t, reinoud j.b.j. gemke 1, loes veenhuizer?, krijn haasnoot 3, a.johannes van vugh0 department of pediatrics, ~wilhelmina children's hospital, utrecht, 2sophia hospital, zwolle, and ~free university hospital, amsterdam, the netherlands. in this retrospective study factors that affect short and long term prognosis after submersion were analysed. all patients that were admitted to a tertiary pediatric icu between january i, 1986 and january i, 1992 were included. of 34 patients, aged 0-13 years, 8 died in the icu, one after hospital discharge. survivors and non-survivors showed significant differences with respect to central temperature, pupillary reactions, arterial ph, pediatric risk of mortality (prism) score and therapeutic intervention scoring system (tiss) upon admission (p < 0.05). non-survivors more frequently required mechanical ventilation, bicarbonate administration and active reheating. ards was seen in 22 patients (65 %), invariably within 6 hours after admission. no patients with cardiac arrest on" admission snrvived without sequelae. hypothermia appeared to have no protective effect on hypoxic damage. survivors with persistent sequelae _> 6 months after discharge had significantly higher prism and t1ss scores (mean 27 and 34, respectively) than those with complete recovery (mean 14 and 23, respectively). long term cognitive problems were present in 7/25 survivors (28%) and emotional disturbances in 5/25 (20%). in conclusion, a concise number of clinical and laboratory parameters, representing acute severity of illness, are important prognostic indicators for survival and health status of children after submersion. there were 59 (91%) bronchoscopies, and 6 (9%) were oesophagoscopies.the average age was 2,8 years for bronchoscopies, and 4 years for oesophagoscopies. the outcome of the patients was good. no complications were observed. extraction is recomended in every symptomatic patient. orphenadrine is an anticholinergic drug mainly used to decrease symptoms of parkinson disease. orphenadrine has a peripheral and central effect and overdose can result in athetoid movements, convulsions, cyanosis, coma, arrhythmias, shock and cardiac arrest. physostigmine is a specific antagonist of the peripheral and central effects and can be a useful antidote. we report the case of a two and a half year old female who was transfered to our icu for general convulsions. the little girl had, three hours before admission, accidently ingested 400rag of orphenadrinehydrochlodde (disipal®), which was her grandmothers anti-parkinson medication. three hours after ingestion she presented neurological signs: confusion, unstable walking, and periods of aggression. generalized tonic-clonic seizures appeared who were rebel to administration of multiple anti epileptica but ceased after iv administration of diazepam and endotracheal intubation and ventilation. an episode of ventdcular tachycardia responded well to the iv administration of tidocaine. the levels of orphenaddne in the serum were high at admission (3550pg/l) and were present in the blood up to 96 hours after ingestion. high serum levels are, in the literature, associated to a high mortality rate. physostigmine was administered three times at a 0.02mg/kg dose in the first 24 hours. we decribe the noted effects of physostigmine on the different symptoms. the patient survived and could leave the icu after one week. in conclusion: orphenadrine poisoning is a very complicated medical problem associated with high mortality. in severe intoxication, the benefit of physostigmine more than counterbalances its side effects. objective: to define the optimal volume of dilution for endotracheal (et) administration of epinephrine (epi) design: prospective, randomized, laboratory comparison of four different volumes of dilution of endotracheal epinephrine (1.2, 5, and 10 ml of saline) setting large animal research facility ofa universi~ medical center subjects and interventions: epinephrine (0.02 mg/kg) diluted with four different volumes ( 1, 2.5. and i 0 rot) of normal saline was injected into the et tube of five anesthehzed dogs. each dog served as its own control and received all four volumes in different sequences at ieast one week apart. arterial blood samples for plasma epinephrine concentration and blood gases.were collected before and 0.25, 0.5. 0.75_ 1.2.3, 4. 5. 10, 15.20, 25.30 , and 60 minutes after drug administration. heart rate and arterial blood pressure were continuously monitored. measurements and main results: higher volumes of diluent (5 and i0 ml) caused a significant decrease of pao2, from 147:!:8 tort to 106±i0 torr, compared to the tower volumes of diluent (1 and 2 ml), from 136±10 torr tu135+_7 torr (p<0.05). these effects persisted for over 30 minutes. mean plasma epinephrine concentrations significantly increased within 15 seconds following administration for all the volumes of diluent. mean plasma epinephrine concentrations, maximal epinephrine concentration (cmax), and the coefficient of absorption (ka) were higher in the 5 ml and 10 ml groups. the time interval to reach maximal concentration (tmax) was shorter in the 5 ml and 10 ml groups. yet these results were not significantly different. heart rate. systolic and diastolic blood pressures did not differ significantly between the groups throughout the study. conclusions: dilution of endotracheal epinephrine into a 5 ml volume with saline optimizes drug uptake and delivery, without adversely affecting oxygenation and ventilation. the aetiology and outcome of paediatric out-of-hospital cardiac arrest was studied during a 10-year period in southern finland served by physician staffed emergency care units. the files of 100 prehospital patients less than 16 years old without palpable pulse and spontaneous respiration were analysed retrospectively. fifty patients were declared dead on the scene (dos) and resuscitation (cpr) was initiated in 50 patients. the sudden infant death syndrome was the most common cause of arrest (68%) in the dos patients as well as in patients receiving cpr (36%). asystole was the initial cardiac rhythm in 70% of the patients in whom cpr was attempted. eight of the 13 hospitalised patients were discharged, 6 of them with mild or no disability, 1 with moderate disability and one in vegetative state. in multivariate analysis the short duration of cpr (<16 minutes) was the only factor significantly associated with better survival. due to various aetiologies the survival rate from prehospital paediatric cardiac arrest is quite low. on the other hand, hypothermic near-drowning victims seem to have a relatively good prognosis. duration of cpr less than 16 minutes was the best predictor of intact survival, our study supports the previous findings of the importance of early and effective resuscitation efforts for establishing ventilation and perfusion on the scene. in our system well trained physician staffed emergency care units are able to provide immediate and effective als on the scene. on the other hand, these units also appear to be able to refrain from resuscitation when the prognosis is pessimistic. objective: to assess the normal ,gastric intramucosal ph ~hi) by tonometry in healthy children patients and methods: twelve healthy children (6 males and 6 females) with age rmaged from 6 months to 12 years scheduled for minor plastic or urologic surgery. children were previously medicated with midazolam (0.25 mg/kg) and atropine (0.02 mg~) both i.m.. anaesthetic induction was standardized with 02 -n20 (75%) administered via facial mask and increased halotane concentrations (up to 2%). all patients got an endotraeheal tube after iv. administration of femanile (2 mcg:jkg) and vecuronium (0.1 mg/kg) or suxametonio (1 mg/kg), pmaesthesia was maintained with o 2 -n20 (60-75%) and isofluorane (0.5-1%). during surgery, 8 children needed mechanical ventilation and the others maintained spontaneous breathing. ekg, heart rate, blood pressure, and pulse oximetry were moniterized. after anaesthesia, a sigmoid tenometry catheter (tonometrics, inc.) was inserted in the stomach of the patients by direct visualization with laryngoscope and magyll clamps. children were all maintained normoventilated and with normal cardiorespiratery variables. cadet's balloon was £~led with 2.5 ml of saline. thirty minutes after the insertion 1 rrd was extracted and rejected, just afterwards the remanent 1.5 ml was extracted and immediately analyzed. simultaneously an arterial gasometry by puncture was performed. gastric phi was calculated by the henderson-hasselbalch's equation using the pco 2 obtained from the tenometry catheter and the bicarbonate value obtained from the arterial gasometry. results: average gastric phi was 7.34 -i-0.027, range (7.29-7.46). objective: demons~ating intramucesai ph (phi) alterations during transport of patients from operative room to pediatric intensive care unit (picu), material and methods: phi measurements were performed with gastric tonometer catheter in t4 patients undergoing cardiac surgery with cardiopulmona d" bypass (cpb), there was 9 mate and 5 female, the average age = 3yl0ra, average weight = 12,5 kg, average time of cpb = 70 rain. the measurements were made at the end of the surged' and when the patients had arrived in the picu statistical aualysis: average and ~andart deviation and test "t" student. objetive: to asses the efficacy of gastric iatramucosad ptt (phi) and arterial lactate levels to evaluate splacalc tissular perfusion in an experimental model of intestinal ischemia. suneets ~nd methods: twelve piglets weights t3-20 kgs. undergoing orthot~ie liver trasplantation. the intestinal ischemia was induced by aortic damping. tonometry catheter (tonometrics inc.) w~s placed in the stomach after artaesthesia and ot intubation. phi ~s determined 7 times and lactate levels was determined fi times in 3 stages: i) pre-ae~hepatic stage (twice: before surgery and before aortic clamping ); ii) end anhepatic stage (only phi): iii) reperfusion stage (a 30, 90, 120 and 180 minutes). the phi was derived from application of the henderson-hassdbach formula using the pco2 value from the tonometer and the arterial bic~rbonate. all pipets received raaitidiila before sttrgery. values of phi above 7,35 and lactate levels between 6 and 15 mg/dl were considered nortrm. the results were statistically anaj.izated with anova and bonferroni tests. results: the phi was normal on pre anhepatic stage (> 7,35) and lactate levels were slightly increased (21, 5 +_ 8, 9 and 19, 5 ±5, 9mg/dl ns) . in relalion to we-anhepatics values, phi decreased signncatly at the mid of anhevatic stage (7,39_+0,14 vs 6,94_+0,1 p<0,001), phi remain low in stage iii, at 30 rain (6,86+0,12 p< 0,001) and 90 min(g94-+o, 12 p< 0,001). arterial lactate levels increased significatly in relation to levels in stage i, at 30 rain (63,6_+9,7 p<0,o01) arid 90 rain (65,8±9,9 p<0,001) of reperfusion stage. there is a slight improvement on phi and lactate ievels at 120 and t80 rain althought the differences did not reach significance. cnmments: phi and arterial lactate levels propperly reflect hypoperfusion on the experimental model of acute intestinal isdlemia. b~kground : the paediatrie gallbladder diseases generally described are calculous ¢hol~tstitis, cystic duct obstruction, congenital anomaly of the biliary tract, and inflammation. in the neonatal period, noulithogenie gallbladder disease could be also due to erythroblastosis or hyperalimentation. obieetive : we describe an other type of disease affecting the gallbladder in neonates thought to be related to their vascular vulnerability. methods : four patients with abnormal gallbladder ultrasound not related to classical observations were included. we have studied and reviewed the biological and clinical data, the ultrasound findings and their evolutions. results : four patients, 30 to 32 ~.k-old neonates ~ffth a birthweight be~,een 1,3 and 1,9 kg, were intubated and under total parenteral nutrition for 10 to 35 days. none of them were symptomatic on repeated clinical evaluations. one newborn developped hypotensien on umbilical bleeding at 3 hours of life. in two cases, signs of cholestasis were discovered : the total bilirubin level has risen to 5 mg/dl; the direct bilirubin level was 1,5 mg/dl while the urina were dark and the ~o~,ls :mcolour~. the c~mplct~ ~crology as a!! the culvare~ remained negative. the ultrasound explorations were atypical : in the four eases, an initial increasing broad and thickness of the wall of the gallbladder with an hyperecbogenie inside content, which was not sludge, was discovered. in three eases the images resolved in ten to fifteen days. in one ease, an asymptomatie thrombosis of the vena portu which remained patent was discovered. in this case, at one month, the ultrasound showed images encountered in chronic ebolecystitis and, at one year, the gallbladder appeared atrophic. none of them underwent surgery. conelusiou : the gallbladder diseases are multifactorial. besides the prematurity, the infections, the total parenteral nutrition, the premature neonate is exposed to vascular vulnerability affecting also the gallbladder and this may explain our findings. progress in prognosis of pts with b-nhl had followed the use of multimodality chemotherapy (ct). with the prolonged survival, there are comlications due to myetosupression & desease process. the syndrome of neutropenic enterocolitis (ne) is one of the ominous problems because ofpts increased susceptibility to infection & overwhelming sepsis. this material included 25 neutropenic pts (4-14 years) with the stages iil& iv of b-nhl who were treated with the modifired bfm-90 (mtx 1 g/m 2 in 24-h inf.); 22 males, 3 females. seventeen episodes of ne were observed & only after the first 2 courses of ct (13 of 25 after tst, 53%; 4 of 24 after 2nd, 17%). the symptoms existed 3 to 14 days. wbc ranged from 50 to 600 in l~tl (median, 100). the first signs of ne were directly correlated to the beginning of the neutropenia & the recovery of neutrophils led to the disappearance of abdominal recovery of neutrophils led to the disappearance of abdominal pain. the conservative treatment included gastrointestinal tract decompression, broad spectrum antibiotics initially, volume & electrolyte substitution, nutritional support, correction of acid-base balance, symptomatic treatment. sixteen pts were treated nonoperatively, 1 died. on autopsy the transmural bowel necrosis due to thrombosis of branches of a.mes.sup, was found. the bowel perforation occurred in one patient, he was undergone laparotomy & hemicolonectomy & survived. we conclude that ne is a frequent complication in neutropenic pts with the st. lii& iv of b-nhl. it occurs after the induction courses of ct. close observation by surgeons, oncologists & pediatric intensivists is mandatory. conservative treatment is effective & more preferable until leucopenia resolves. operation is necessary only for those.with perforation. near infrared spectroscopy as a tool for evaluation of intestinal perfusionpresentation of an animal model. c. scheibenpflug, p. buxbaum and a.m. rokitansky the recent development of and investigations in the so called near infrared spectroscopy ( nirs --transcutanous emission and simultaneous registration of intensity of spectralcolours depending upon modulations of tissue perfusion ) enable physicians to measure and qualify organ perfusion and nowadays is mainly used to control cerebral as well as skeleton muscular blood flow in trauma patients at intensive care units ( icu ). today intestinal perfusion, hypoperfusion , cell damage caused by reperfusion injury, bacterial and toxin translocation are serious problems in critically ill patients at an icu. paediatric intensive care physicians put major concern on intestinal perfusion, which for. instance gains more and more importance, especially in the neonatal period for example as an etiologic factor for necrotizing enterocolitis. we established an animal model, in which we measured intestinal perfusion by nirs under various invasive and noninvasive conditions. methods and results will be referred. for preliminary conclusion we propose near infrared spectroscopy ( nirs ) also as a potent diagnostic tool to determine early intestinal malperfusion in order to prevent lethal outcome. fm'ther investigations in animals as well in paediatric iritensive care patients should be done to estimate our efforts. introduction: following the acute phase of necrotising enterocolitis (nec) starvation of the gut for a period up to 3 weeks is a generally accepted treatment modality in many centres. objective criteria to refeed these patients are hardly available. recently the double sugar test has become available as a parameter for (ab)normal gut permeability ~'2. aim of the study: to evaluate the changes in permeability of the small bowel in patients with nec and controls before introduction of enteral feeding. methods: a lactulose! rbarrmose (i/r) test was performed in two groups. group 1 was studied 2-3 times within a 5-week period of starvation (n=5, mean gest. age 35, range 31-40 weeks). in group 2 seven different control patients were studied (mean gest.age 33, range 28-38 weeks). the test was performed by giving a patient after at least a 4 hour fast 1 ml/kg bodyweight l/r solution and determination of the 1/r ratio in a 4-hour urine sample by chromatography. results: objective: to evaluate the prognostic factors in the response to nitric oxide (no) in children with acute respirator/ distress syndrome (ards) and/or pulmonary hypertension (pht). patients and methods: 23 critically ill children received no inhaled for ands and/or pht treatment. 14 patient before and after cardiac surgery (2 cardiac transplants), 5 with bronchopneu~onia, 2 multiple trauma, 1 sepsis and 1 cardiorespiratory arrest. 15 patients showed /j~ds and 8 pht, in 4 with associated ards. we analyzed age, sex, diagnosis, pao2, pa02/fi02, oxygenation index, pht, shock, and sepsis as prognostic factors and response factors to n0. results : after no administration oxygenation did not improve in 2 patients (8.6 %) and pht did not diminishe in one children (12 %). 12 patients survived (52 %), 8/15 (53.3 % with /d%ds) and 4 /8 (50 %) with pht. the four patients with isolated pht survived , and the 4 patients with pht and ards dead. patients after cardiac surgery presented less mortality (35.7 %) than the rest of patients (66.6 %). patients with shock presented higher mortality (64.2 %) than the rest of patients (22.2 %). there are no differences in response to no in respect of sex, age, diagnosis, shock, and sepsis. survivors showed higher increase of pao2/fi02 64.3 ± 58.4 to no than non-survivors 48.4 ± 51.1 (n.s). patients with pht showed higher increase in pa02/fi02 to no administration ( 88 ± 47.1) than patients with ards (43.4 ± 50.8), (n.s), but patients with ards showed a higher increase in 0!, 15 ± 6.7, than patients with pht 4.8 ± 4 (p < 0.05). patients with pa02/fi02 < i00 showed less increase in pa02/fi02, 47.8 ± 46.3, than the rest of patients 82.8 ± 65.5 (n.s) conclusions: i. mortality of isolated pht treated with no is less than patients with ap~s. patients with shock and those with pht and ards showed higher mortality. 2. we have not found any clinical or analytical factor to predict clinical response to no administration. 14 patients showed ards, and 9 severe pht after cardiovascular surgery, in 5 with associated ards. we registered respiratory assistance, blood gases, pao2/fi02, the oxygenation index (oil, and mean pulmonary pressure/ mean systemic pressure (pap/sap) before and after no inhalation. we measured continuous concentration of no and no2 by electrochemical method (noxbox, bedfont, airliquide). results: no administration improved oxygenation mean pao2 from 74 ± 17 tm~g to i19 ± 54 ~g (p < 0.01), mean pa02/fi02 fr<xa 83 ~ 30 to 13£± 72 (p < 0.01)and 0i from 28 ± 14 to 20 ± 12 (p < 0.01). 2 patients did not improved with no administration. the oxygenation improved in the first five minutes and the best oxygenation was achieved with 5 -15 ppm. there is no significant change in pac02. pap/sap diminished from 62 ± 17 % to 42 ± 9 % ( p< 0.05), in one patient there is no response. the no administration was maintained between ~5 minutes to 47 days. the effect of no on pulmonary pressure and oxygenation was maintained without change during all the time it was administrated. n02 concentration were always less than 2 ppm y metahemoglobinaemia less than 3.5 %. there are no side effects secondary to no administration. 12 patients survived (52 %). concluaions:l.administration of no improves oxygenation in children with ~/eds and diminishes pht after cardiac surgery. 2.no effect is fast and maintained during the evolution. high frequency oscillatory ventilation in combination with inhaled nitric oxide g~thberg sylvia, md, edberg k e md, phi), dept of paediatric intensive care, children's hospital, s-416 85 g6teborg, sweden background: for man), years severely sick infants with congenital heart malformations or acute lung disorders have died due to pulmonary hypertension, hypoxia and multiple organ failure. today there are several therapeutic facilities coming up for these infants. ecmo progranunes have been introduced in severul centres as well as improved venlllatory techniques, as high frequency oscillatory ventilatior~ (hfov), which provides adequate ventilation with less risk for hmg injury.. in 1987, the endothelium derived relaxing factor was identified as nitric oxide (no), and extensive studies have sho~-a that inhaled no reduces pnimonm3 o vascular resistance and improves oxygenation in hypoxic infants with pulmona~ hypertension. material.. from july 1994 to january 1996, we have treated 13 severely hypoxic children with combined ttfov-no. seven were newborn, 3 with cdh, one with mas, one with irds, one with paediatric ards due to rsv infection and one with poor oxygenation atler open heart surgery. 6 were between 1 month and 9 years and all had ards of differentorigin but one who was hypoxic after open heart surgery. method: hfov was given by means of sensormedics 3100a oscillatory ventilator to 12 patients, and dr~ger babylog 8000 was used in one case. mean airway pwssures varied from 10-34 cm h20. oscillatory pressures varied from 25-85 cm h20 and ventilatory rates from 6-15 hz. fi o2 varied from 0,21 -1,0. no was administered by nomius classic. no and no2 was measured in the inspiratory limb with an electrcchemieal device. noconcentration varied from 2 to 20 ppm and the no2 -concentration between 0-0,2 ppm. methemoglobin was never more than 3,7 % (average 1,7). duration of treatment varied from 1 to 20 days. combined trealment with hfov and inhaled no improved oxygenation and carbon dioxide elimination in 12 out of 13 treated childretl 5 patients died, 3 due to their underlying congenital heart disease, one of asphyxia due to rsv-infection and one of cdh with progressive hypoxia and multiple organ failure. combined treatment with hfov and inhaled no improves oxygenation in severely hypoxic children. treatment should be slarted early to reduce the risk for chronic lung injury following barotranma and high oxygen concentrations. we speculate that the combined hfov-no may reduce the use of ecmo, and that it may improve outcome in ceotres where ecmo programmes are not introduced. apart from its vasodilative properties nitric oxide appears to act also in physiologic immune defense. intracetlular concentrations of no synthesized by macrophages are in the range of 103 above those produced by vascular endothelium. we investigated the bacteriostatic effect of nitric oxide at concentrations used during inhaled therapy for pulmonary vasodilation in neonates. ten different strains of five species were used (staph. anrens, staph. epidermidis, strop. group b [gbs], e. coil and pseudomenas aeruginosa), which are ~e most often tracheally isolated bacteria in mechanically ventilated premature infants and neonates. we compared bacterial growth of cultures applying three different concentrations of nitric oxide (40 ppm. 80 ppm, 120 ppm) m the growth of the same strains in room air for a duration of 24 hours. no bacteriostatic effect was demonstrable at no concentratioes of 40 ppm. e. coil showed decreased bacterial growth at 80 ppm and 120 ppm, however without reaching statistical significance (p=0.058). at nitric oxide concentrations of i20 ppm staph. epidermidis and gbs grew significantly less as compared to colonies of the same strains in room air. no effects were found regarding the growth of staph. aureus and pseudomonas aneruginosa. we conclude that nitric oxide has a selective bacteriostatic effect on some of the most often trachealb, isolated bacteria in premature infants and neonates. this effect appears to be dose-dependant and occurs in the upper range of dosages used with inhaled no therapy. further research is required in order to examine the mechanisms of action as well as specific interactions between different strains of bacteria and nitric oxide. the no,box monitor (bedfont, kent, uk) is used to monitor nitric oxide (no) and nitdc dioxide (no 2) during no administration in ventilated neonates. according to the manufacturers specifications the monitor can be applied in cases where airway pressures range from 5 to 50 mbar. we investigated in-vitro the accuracy of the no monitor in a range of ventilatory conditions. using a dr~ger babylog 8000 we ventilated an artificial lung. no was administered {800 ppm no in 100% nitrogen) with a mass flow controller to the inspiratory tube, at a distance of 20 cm from the y-piece. the no target value was set to 10 ppm. the ventilator was operated in both cpap and ippv modes at different settings. the no sampling tube was placed on two different locations; in the inspiratory tube dose to the y-piece; in the expiratory tube haft way between the y-piece and the ventilator. the no-monitor was ca)ibrated with 84 ppm no at 30 mbar. fig. 1 and 2 show the sensitivity of the no-monitor for respectively static pressure (cpap) and pulsatile pressure (ippv) with the sampling tube located in the inspiratory tube. fig. 3 shows the resur for pulsatile pressure with sampling in the expiratory tube. we conclude that the accuracy of the bedfont no-monitor is dependent upon airway pressure and sampling site, the latter possibly caused by incomplete mixing, pressure and no are linearly related when sampling occured further away from the administration site. based on this study we suggest to place the sampling tube in the expiratory tube. during neonatal care the clinician should be aware of varying inaccuracies depending on the respiratory pressures. hypothesis: availability of therapy with inhaled nitric oxide (ino) decreases ecmo use in patients referred .to a tertiary care hospital for treatment of respiratory, failure unresponsive to conventional therapies. background: at children's hospital of wisconsin (chw) treatment for patients who have failed conventional treatment for respiratory failure, sometimes called "rescue" therapy, has included ecmo since 1986 and high frequency oscillatory ventilation since 1992. ino has been in experimental use at chw since may of i994 and has resulted in the perception of a decreased need for ecmo therapy in those patients referred for rescue therapy. this study was designed to tbst the validity of that perception. study type: retrospective cohort. methods: the medical records of all 105 patients referred to chw from 1/lj93 to 4/1/95 for rescue therapy for severe respiratory failure were included in the chart review. data were collected regarding diagnoses, illness severity, hospital course including interventions and complications, and outcomes. exclusion criteria w~ere the finding of structural heart disease or congenital diaphragmatic hernia as the basis for hypoxemia. qualification for ino treatment included an a-ado2 -600 or oi ~-40 for two hours or -> 25 for twelve hours and echocardiographic demonstration of persistent pulmonary hypertension of the newborn. patients were classified into two groups based on the availability of ino at the time of their hospitalization. results: in the time period of the study, 105 patients were referred for possible ecmo therapy. twelve patients greater than 4 weeks old, 31 with congenital diaphragmatic hernia and 12 with congenital heart disease were excluded from this analysis, leaving 50 patients for study, ino availability reduced ecmo use from 16 of 34 (47%) patients in the ~ino unavailable" group to 2 out of 16 (12.5%) patients in the "ino available" group, p=&026 by fisher's exact test. the fact that the two groups were composed of patients of similar severity of illness is reflected by comparable rates of ecmo and ino rescue therapy (47% vs. 56%). conclusion: by providing an alternative rescue therapy, ino has reduced the need for ecmo in this group of neonates referred for respiratory failure. introduction: true hepatnrenal syndrome (his) is defined an acute renal failure {arf) in the presence of severe liver disease without other known causes of renal failure. hrs is frequently seen in the course of hepatic cirrhosis• in children, cirrhosis is rare; however, arf can be seen in combination with aseites and liver dysfunction• we describe 3 patients with hepatic dysfunction and aseites in combination with ar~ and abnormal sodium-water handling, leading to the diagnosis of hrs. pathophysiology: three factors are considered in the pathogenesis of hr~: i) hepatic dysfunction, 2) deranged hemodynamics, including abnormal blood pressure, reduced effective arterial blood volume and abnormal blood flew distribution, and 3) neuro-humoral dysrsgulatiom, including elevated levels of aldosteron, renin, angiotensin-ll, ade, vasodilatim 9 nitric oxide and vasoconstrictor peptide endothelin-l. the main pathogenetic feature is decreased cortical renal blood flow, decrease of glomerulur filtration rate (gfr), vastly increased sodium retention, uliguria, and azotemia. treatment: therapy is based on counteracting sodium and fluid retention by highdose aldosteron antagonists and loop diuretics, improving renal perfusion by lowdose dopamin, and strict restriction of fluid and sodium. interventions as paracenteals of aacites or n peritoneo-systemic shunt are associated with high morbidity and poor outcome in children. reversal of hem by conservative measures can only be attained at early stages of hrl liver transplantation is the only definitive treatment that can reverse ere at advanced stages. patients: the described patients developed severe ascites with insidious renal dysfunction and abnormal sodium-water handling during admission at picu and fullfilled clinical criteria fur hrs. treated according to the cited principles, all patients showed improvement of gfr, with increased natriuresis and gradual decrease of ascites. eventually, renal function normalised completly. conclusion: ere deserves greater recogmitimn in the picu population; diagnosis can be suspected on clinical criteria. with this increased awareness, therapy tun be instituted at an early phase, with better prospects for recovery. positive outcome of hem depends on early recognition of the clinical picture, understanding of the pathophysiology, and early institution of consistent treatment. mtx is an antimetatxflite widely used as chemotherapeutic agents. high dose ivitx (i to 30~m2) administered as a prolonged intravenous infusion (over 4-42 hours), is often used to treat malignant paediatric diseases. major complications of this treatment are myelosuppression, orointestinal mucositis, dermatitis and impairment of anal function. we report two cases of mtx overd~age occurred in two children (5-year-old. 14 month-old) t~ted for acute lymphoblastic leukaemia. they were treated by cavh and the mtx bhk~d levels rapidly decreasedavoiding multisystemic involvement. establishment of alkaline diuresis and monitoring of plasma mtx levels during treatment is essential to prevent nephrotoxicity. however. leuco',cnn rescue may not prevent the development of potentially lethal toxicities in patients with mtx concentrations persistantl} exceeding t0mm. in theses cases, em'ly treatment of mtx intoxication may pm~cnt myelosuppression and reducerenal damage. the goal is to lower the concentration to below 10 mmoll, at which time rescue agents aleme would be expected to be cllcctive. respective indications of these remo',at mctny.:is are still discussed : hacmt~ialysis t~ eharc(~l haemoperfusion should be prolx',sed for massive and acute intoxication. however, rebound has been reported after combined hcmodialysis and hemoperfusion. exchange transfusion may be proposed as a treatment for prolonged and moderate intoxication. peritoneal dialysis is an incflbedve method for remo~ al of mtx. cavh was used in our icu. cavh is a simple method for blood purification and n':dy iluid control. use of cavh was never be reported in this indication to our knowledge. simplicity, rap~d application and gco.l clinical tolerance are the main advantages of this technique. the technique presents ~peclal advantages in terms of low priming volume of extracorporeal circuit, low blood flow, low rate heparinisation. our results show a decreaseof plasma mtx concentration and a rapid reduction of halfqite of elimination (t5 hours over the period of cavh). moreover, we didn't delec~d rebound after stopping prc,xedure. small size of the i:ratients may present sometime special problems, but these technical problems can be overcome, no severe complication (needing, inlection) were observed during filtration, in summary, aggressive intravenous fluid hydration and alkaliniaation of the urine coupled with careful monitoring of renal function and plasma mtx concentrations during and al'tcr infusion along with lem~overin rescue has reduced the inndcace of life-threatening toxicity after highdose mtx. however, some mtx inu>xication still occurred, leading to se~em toxicity, particularly nephrotoxicity. in these cases, we think that cavh (or cavhd) is a reliable, rapid method without rcix~und increase in plasma mtx concentration or important adverses effects compared to other procedure removal. gouyon jb, germain jf, semama d, pr6vot a, desgres j preliminary limited data suggested that hemofiltration and hemodiafiltration may be valuable in some neonates with decompensation of maple syrup urine disease (msud). venovenous hemofiltration (vvhf) and hemodiafiltration (vvhdf) were performed with a new neonatal hemo(dia)filter (miniflow 10, hospal) on 8 anesthetized rabbits infused with branched-chain amino acids (leucine, isoleucine and valine) and c~-keto-isocaproate. the bcaa and aketo-isocaproate blood levels were close to those previously observed in neonates with msud when extracorporeal blood purification was required. vvhf and vvhdf performances were assessed with two different blood flows (qb = 8.3 and 16.6 ml/min). vvhdf was performed with 4 dialysate flow rates (qd = 0,5, 1.0, 2.0 and 3.0 l/h). thus, each animal was submitted to 10 successive procedures. within each studied period, clearances of the 3 bcaa were strictly similar. bcaa clearances obtained by vvhf were similar to ultrafiltrate rates (respectively, 0.78 4-0.14 and 1.79 4-0.28 ml/min at high and low qb ; p < 0.05). the ~x-keto-isocaproate clearances obtained by vvhf were 0.39 4-0.17 and 0.92 4-0.43 ml/min at low and high qb (not significantly different). whatever qd value, the vvhdf procedures always allowed higher bcaa and c~-keto-isocaproate clearances as compared with the corresponding v'~hf period with similar qb. bcaa clearances obtained by vvhdf with a 0.5 l/h dialysate flow, were 4.1 4-0.5 mljmin and 5.4 4-0.5 ml/min at iow and high qb, respectively. the concurrent a-keto-isocaproate clearances were 2.5 4-,. 0,8 ml/min and 2.9 _+ 1,0 ml/min. at both qb regimens, bcaa clearances provided by vvhdf were markedly higher than values previously obtained with peritoneal dialysis in human neonates with msud. the management of renal failure in the newborn is difficult. when dialysis is instituted peritoneal dialysis (pd) is usually the technique of choice. this is can be problematic and impossible in some patients with pre-existing intra-abdominal pathology. continuous arterio-venous haemofiltration (cavh) has been described in infants but sick preterm infants are not able to support the circuit. i have devised a means of having pumped haemofiltration in small/preterm infants (phis/pi) and describe its use in nine patients ranging in size from 750 to 3000gms for periods of 1 to 7 days. vascular access was achieved through 24 or 22 guage cannulae in either a peripheral artery and a central vein or through two central veins. blood was pumped out using an ivac 572 infusion pump and through a gambro fh22 haemofilter. a second ivac pump was used to remove haemofiltrate from the filter and a third to infuse replacement solution. removal rate was set to give a clearance of 15mls/min/1.73sq.m and blood flow rate set to between 5 and 10 times the removal rate. heparin was infused into the circuit to prevent clotting of the filter. biochemical and fluid balance control was achieved in all infants. guaranteed fluid removal allowed the administration of full nutritional support. four patients died when treatment was withdrawn because of an untreatable underlying problem. one recovered renal function but died some weeks later from unrelated problems, three survived and recovered renal function and one patient is still on treatment. this system allows a secure means of achieving fluid and electrolyte control in the preterm infant. the use of this technique may allow haemofiltration to become as applicable to preterm infants as it is to older children and adults. unibrtunately, children often receive no treatment, or inadequate treatment for pain and painful procedures. this prospective, multicentric study focuses on the efficacy, safety and side effects of novalgin (metamizol sodium) for this indication. patients and method: novalgin was administered to 56 children, aged between 6-16 years, with acute, postoperative or procedural pain. novalgin (10-15 mg/kg) was given 6-8 hourly iv or im respectively, in some cases (15) in combination with opioids (tramadol 10, piritramid 3, butorphanol 2). the pain relief was assessed by six-step verbal rating scale (vrs) from 0 to 5, vital signs were monitored, the side effects, that occured were recorded. results: pain relief was good (vrs less 2) in 53 children -94.6 % of study patients. novalgin was very well tolerated, only one patient had adverse reaction -hyperpyrexia following intravenous application of the drug. discussion: novalgin (metamizol sodium) is safe and effective drug in the management of acute pain in children with low incidence of side effects. obie~qve: a prostx~tive study comparing simultaneous, indepeadent ratings conducted by intensi~4sts using an american (comfort) and an european chartwig) sedation scale for mechanically ventilated pediatric patients. measurements and results: the study comprised 30 observations in 18 mechanically ventilated pediatric patients (aged 16 days to 5 years) in a pediatric intensive care unit (from march 1995 to january 1996 . each patient was sedated by his/her managing physician with opiates, benzodiazepines, barbiturates, used isolated or in combination. each observation consisted of a 3-mid period of oly~ervatien of the patient in his or her pediatric icu bed, after each observation, the comfort (analyses 8 dimensional physiologic and behavioral subscores -range 8 to 40 paints) and hartwig (analyses 4 dimensional behavioral subsenres -range 8 to 25 points) were performed by the intensivist. we established the comfort scores ~ correspanding to adequate (range 17 to 26), excessive (range 8 to 16), and inadequate (range 27 to 40) sedation; and, hartwlg scores z correslxmding to adequate (range 15 to t8), excessive (range 8 to 141, and inadequate (range 19 to 25). statistical mmlysisj: agreement rate (kappa) and p <.01 was considere d s!l~nificant. comfort 18 (60.9%) 2 (6,6%/ 10 (33.4%) hap, twig , 17 (56.6%) 7 (23.4%) 6 (20.0%) to the comfort score, the average for adequately sedated, inadequately sedated, and too sedated was 20.28+-2.78, 2z5_+0.70, and 15a.+_l10, respectively. and to the ha~twig scorn, the average for adequately sedated, inadequately sedated, and too sedated was 16.35:k-'0.77, 20.85-&l57, and 13.0l-0.89, respectively. conclnsion: in our study there were no significantly statistical difference when you apply a more complex scale (conff'ort) or a less complex scale (hartwig) to assess the sedation of mechanically vemilated pediatric patients. the application of local and intravenous morphine infusion after surgery of urinary tract eva nemeth , m.d. semmelweis medical university , first oepartment of paediatrics , budapest , hungary in±roduction:continuous analgesia with morphine may be ~egaroed as a safe and effective method of pain relief during postoperative period. subjects and methods: 24 children /mean age 2.3 years/ underwent elective ureteroneoimplanta±ion were randomly selected to receive either morphin intravenously of lo ug/kg/h /group one/ or bladder morphineinfusion 50 ug/kg/h /group two/ after surgery. all patients were prospectively evaluated during their s±ay in the postanaesthetic care unit. cardiac and respirafory rates,blood pressure,sa 02 ~,degree of alertness,pain perception and complaints of the paticnto ~cr~ recorded hourly. pruritus,nausea and vomiting,voiding difficul-±ies,sedation,dysphoria were systematically sough and quoted. statistical analysis was performed by chi square test. results:postoperative analgesia was the same in the two groups,but side effects were less in the bladder morphine group,because of the lower se morphine concentration.the differentes weren't significant in two groups. conclusions:the administration of bladder morphine infusion is a safe and effective method in children. objetive: compare the evaluations of sedation level made by physicians and nurses with the visual analog scale (vas) and the comfort scale (cs) in pediatrics patients receiving difforents modes of intravenous sedation. material ~ method." file evaluations were made by an attending physician and nurse with the vas and by another physician (always the same) using the cs. the observations were divided following the sedation mode: one drug (fentanyl or midazolan), two continuous drugs, one continuous and one intermi~ent drug and two intermittent drug (fentanyl and midazolan). the groups were compared using the t-student test. the groups also were compared between the percentual of agreement of the evaluations of sedation level made by physicians and nurses with the cs and vas using the x 2 . results: we didnk find any statistical difference between the observations made by physicians and nurses with the vas in the differmts modes of intravenous sedation, the average of the observations using the cs betwom one drug and two drugs modes didnk exhibit also statistical difference. the observations made by physicians mad nurses using the the vas when compared with the cs didn't show statistical difference between the sedation level. we found statistical difference only in percentual of concordance of sedation level between physicians and nurses when compared the one and two drugs modes of sedation. conclusion: we didn't find differences in the observations made by physicians and nurses in the sedation level, only in concordance pereentua/ of observations when compared two modes of sedation. the observations using the cs (more complex) didnk show differences when compared with the vas. effects of age, concurrent administration of other pharmacologic agents, and disease [cardiac(n=31) & pulmonary(n=22)] on the pk & pd of b were evaluated in volume overloaded infants aged 4 days-6 mo (n=53). single doses of 0.005,0.01,0.015,0.02,0.025, 0,03, 0.035,0.05 & 0.10 mg/kg iv were given over 1-2 min after baseline evaluation. age was used as a continuous vadable to determine its effects on the variability in the pk & pd of b. values for pk parameters were compared between patients in cardiac and pulmonary disease groups. hierarchical multiple regression analyses were used to determine the effects of age, disease and other pharmacologic agents on the variability of bumetanide excretion rate (ber) and pd responses, e.g. urine flow rate (ufr) & electrolyte excretion. cit, cir & cinr increased with age (p<0.05) while t,2decreased markedly in the first monthe of life (p<0.05). ber normalized for dose increased with increasing age. patients with pulmonary disease exhibited significantly greater clearance and shorter t~= (p<0.05) than those with cardiac disease whereas vd~ was similar in both groups. the administered dose of b was the primary determinant of ber but increasing age also contributed. penicillin antibiotics decreased ber. dose response curves for ufr and electrolyte excretion were similar between disease groups. more of the variability in ber and pd responses could be accounted for in the pulmonary group than the cardiac group but this was not statistically significant. conclusion: the pk of bumetanide were influenced significantly by age and disease. differences in pk between patients with pulmonary and cardiac disease were primarily due to differences in total clearance. age and the administered dose of b were positive determinants of ber and pd responses while penicillin antibiotics had a negative impact on both, once b reached its site of action, no differences in pd responses were detected between disease groups. the pharmacodynamic effects of bumetanide were evaluated in volume overloaded infants (n=56) aged 4 days-6 months. single doses of 0.005, 0.01,0.015, 0.02, 0,025, 0.03, 0.035, 0.05 & 0.10 mg/kg iv were given over 1-2 rain. bumetanide concentration in blood (n=l 0) & urine (n=6) samples were quantified by hplc. baseline urine samples were collected over 2-4 hours prior to drug administration. determinations of urine volume, electrolytes (na ", k +, ci, ca ++ and mg++), creatinine and osmolality were performed before and at 0-1, 1-2, 2-3, 3-4, 4-6 and 6-12 hours after bumetanide dosing. changes in urine flow rate and electrolyte excretion were plotted as a function of bumetanide excretion rate which was considered the effective dose of the drug. peak bumetanide excretion rate increased linearly with increasing doses of drug and showed no evidence of approaching a maximum. time course patterns for urine flow rate and electrolyte excretion were similar for all dosage groups. urine flow rate and electrolyte excretion increased lineady up to a bumetanide excretion rate of approximately 7 #g/kg/hr and either plateaued (urine flow rate) or declined at bumetanide excretion rates > 10 #g/kg/hr. bumetanide had no detectable effect on serum electrolyte concentrations, conclusion: maximal diuretic responses occurred at a bumetanide excretion rate of about 7 ;~g/kg/hr. higher bumetanide excretion rates produced no increased diuretic effect. peak bumetanide excretion rate of about 7 #g/kg/hr corresponded to bumetanide doses of 0.035-0.050 mg/kg. neonates using an electrical syringe-pump. authors: tr~luyer j.m., sertin a., bastard v., settegrana, c., bourget p., hubert p. background and objective: many problems can be observed with drug administration by i.v. route, especially in neonates. so we evaluate different protocols of teico delivery using an electrical syringe-pump. methods: we simulate infusion of teico with a syrlnge-pump (pilot c, becton & dickinson lab.) trough d standart neonatal i.v. system. for 2 weights (1 or 3 kg) we used 2 doses of teico (8 mg and 16 mg/kg) and a dose volume _<4.2 ml. our goal was to perform a complete infusion in 10 minutes. the infusion system consisted of an life care 4 infusion pump (abbott lab.) with its lv. set for maintenance intravenous fluid (flow _< 6 ml/h) connected to a 3-way stopcock. an 1 meter extension tubing was placed between the stopcock and a neonatal catheter. an another 1 meter tubing (injection tubing) connected the teicoplanine syringe to the stopc, ock. the volume of the injection circuit (from the syringe to the distal part of the catheter was 2.6 ml 4 methods of injections were assessed: a: injection of the predetermined volume of teico in 10 minutes with no wash out. b: idem as a but the teico was injected in 5 minutes, followed by a wash out (5 ml / 5 minutes). c: twice the required volume was introduced in the syringe and the volume to infuse was programed in 5 minutes, followed by a wash out (5 ml/5 minutes). d: ]dem as c but a priming was performed before connecting theteico syringe to the tubing. during each run, serial samples were collected every ten minutes over a one hour period. the samples were assessed using hplc method. results: the amount of drug delivred at 10 minutes were calculated. the results are a mean of 2 to 6 runs and expressed as the percentage of the total amount of teico prescribed. a 2,8 % 6,4 % b 47 % 62,3 % c 82a % 86,8 % d 94,2% 95 % conclusiom for accurate and reliable intermittent drug infusion with a syringe pump it is mandatory to use a precise protocol of administration and to take in account 1) a priming (for immediate starting of infusion), 2) a drug volume greater than the dose prescribed and a programmed volume injected, 3) a wash out of the tubing (with a volume ~ 1,5 x volume of tubing injection) caz is an antibiotic with activity against the major pathogens responsible for neonatal bacterial infections. we previously reported the pharmacokinetics of caz in 136 preterm infants on day 3 of life which showed that the clearance of caz increased with increasing gestationat age (ga). mean serum half-life of infants with gas < 32 wks was 8.7 h. we wanted to investigate the effect of postnatal age on caz pharmacokinetics, we therefore studied caz pharmacokinetics on day 19-21 of life in 10 preterm infants with gas < 32 wks. caz (25 mg/kg) was administered as an intravenous bolus injection. blood samples were coilected before (t =0), and 0.5,1,2,4,8 and 12 h after the caz dose and analyzed by hplcassay, the pharmacokinetics of caz followed a one-compartment open model. during 1995 11 newborns with complex congenital heart defects requiering either htx or palliative staged single ventricle repair were admitted to our hospital: hlh n=8, unbalanced cavsd, tga with hypopl. rv and hypoplastic aoa. tga with hypopl. rv, sas and dextrocardia. 8/i 1 children had been admitted with cardiogenic shuck and mukiorgan failure due to intermittend closure ofductus arteriosus; in 3/8 stabilization failed. parents were informed about the known and unknown risks of the always palliative surgery; in 2 cases parents denied further therapy. one pafiem with hlh underwent orthotopic htx at the age of 5 month after the ducms art. had been stunted in the newborn period. 9 month later he is still in favourable condition and without any sign of acute organ rejection. 5/11 underwent first stage of palliative single ventricle repair: norwood -op. ( 3 ) ( n=3 ), damus-kaye-stansel -procedure ( 2 ). the clue to adequate postoperative management was to archieve a balanced distribution of flow to systemic and pulm circulation, that is to protect the single ventricle from volume overload and to guarantee sufficient oxygenation and pulmonary development as well. with the centralvenous sato2 at about 50% provided maintaining the arterial sato2 at about 75_+5% is corresponding with a qp/qs of 1:1. using modified bt-shunts of3.5mm resp. a central anrtopulm, shunt of 4mm in one case l severe puim. hypertension, surgery at 6 weeks of age ) there was no excessive pulm. blood flow and no need to increase pvr with inspired co2. one child ( norwood at 5 weeks, preexisting pnim_ edema ) developed severe pulur hypertension and parenchymal pulm. dysfunction after prolonged bypass and multiple transfusions due to intraoperative bleeding: hypoxemia could be managed successfully by implanting a second shunt of4mm 18hh later and temporarily using prostacyclin and no; at sternum closure 6 dd later the second shtmt was banded to 3ram. follow-up ranges 5-5 month: all 5 children are at home being assigned for second stage operation at about 6 month of age. establishing clinical practice guidelines has become increasingly important in the current health care environment. significant effort has been focused upon development of post-operetive critical care pathways. however, benchmark data upon which such pathways should be based has not been well reported. length of mechanical ventilation (lmv) and length of stay (los) for children following cardiac surgery, for example, is poorly described. we prospectively recorded the lmv and los in 168 patients who underwent cardiothoracic surgery between 9/1/93 to 6/30/95. only patients who belonged in any one of five categories of congenital heart disease (ventricular septal defect _+ other septal defects (vsd), atrioventricular (av) canal, tetralogy of fallot (tof), transposition of great arteries (tga), and single ventricle physiology (fontan)) were included. eight non-survivors were excluded from the analysis. all patients were admitted to an intensive care unit 0cu) post-operatively where mechanical ventilation was managed by 4 pediatric intensivists. lmv was defined as the period from post-operative admission to planned extubation. length of stay (los) was defined to be from le from the icu. cytokine patterns during and after cardiac surgery in young children. especially in children, cardiac surgery with cardiopulmonary bypass (cpb) can cause a systemic inflammatory response. this process is thought to be mainly a result of inflammation induced by surgery and exposure of blood to an artificial surface, and of reperfusion injury during weaning of bypass. complement activation, degranulation of granulocytes, induction of free oxygen radicals, endotoxemia and release of cytokines, are important contributing factors. we studied cytokine patterns before, during and after cpb in young children admitted for complex surgery or for septal defect correction. in the first group, significant amounts of il-6 and il-lra could be detected preoperatively. these findings could reflect the already existing hemodynamic dysregutation. in both groups, cpb procedure upregulated the circulating pro-inflammatory cytokines il-6/8, but not il-1b. at the same time, il-lra became detectable. therefore, we suggest that in these patients the production of the anti-inflammatory cytokine il-ira was not induced by the preceding acnvity ot pro-inflammatory cytoidnes. during cpb, we noticed a sharp decline in the capacity of the leucocytes to secrete il-6/8. the ex-vivo production of il-lra however, was only slightly attenuated. we conclude that there is a differential regulatory pathway for the induction of il-6/8 and il-lra. in addition, we studied the influence of dexamethasone administration on the cytokine pattern. administration delayed the appearance of il-6/8 and il-ira in the plasma, interestingly, it did only interfere with the ex-vivo production of pro-inflammatory cytokines. the latter supports our hypothesis that production of il-6/8 and of il-lra is regulated by two independent pathways, (60%) of 43 pts. 82% ofpts < 12 months of age developed metabolic alkalosis as compared with 38% ofpts > 12 months of age.the infants with metabolic alkalosis received more citrated blood products and furosemide. following cardiac pulmonary bypass the highest ph-values and be-values were observed 24-48 hours and 48-72 hours, respectively. ii. prospective study: metabolic alkalosis was registerd in 2t children (70%), 8 of those <12 month (75%) developed metabolic alkalosis and 67% of those elder than 12 monms.durmg the postoperative course patients younger than 12 months developed the highest ph-and base excess values after 102 and t05 hours, in the subset of the older patients maximum ph and base excess was found after 48 and 81 hours, respectively. in one case the top level ofph-value exceeded 7.6, the base excess +20 mvalb. conclusion: children undergoing cardiac surgery with cardiopulmonary bypass often develop metabolic alkalosis.in contrast to previous reports, we did not observe an association between metabolic alkalosis and mortality, nor greater frequency of cardiac arrythmias or prolonged mechanical ventilation. in context with decreasing serum lactate levels, our data show positive correlation of metabolic alkalosis with postoperative improvement of liver function. respirator, mechanics and weaning outcome in children undergoing cardipvascular surgery. vassallo j., cernadas c., saporiti a., landry l., rivello g., buamsha d., rufach d., magliola r. mechanical ventilation (mv) and acute respiratory failure are common events in children unergolg cardiovascular surgery (cvs), the development of new techniques helped to measure some of the main respiratory mechanics (rm) in a non invasive fashion. our goal was to evaluate the predictive value of these measurements in weaning (w) outcome in these patients, patients and methods: we prospectively evaluated children considered clinically to be ready for w with < 20 kg and > 24 hs mv. patients with diaphragm paralysis and those who failed w because of upper airway obstruction were excluded. before patient extubation the following measurements were recorded during spontaneous ventilation (cpap/t piece) using the cp 100 neonatal pulmonary monitor bicore (lrvine, ca): total respiratory system static compliance (cssr) and resistance (rts), rapid shallow breathing index (rsbi). maximal inspiratory negative pressure (pi max) was measured using an unidirectional expiratory valve. threshold values predicting w success (ws) were: cssr > 0.5 ml/cm h20, rts < 75 cm h20 /l/sec, rsbi 160 and pi max > -30 cm t120. w failures (wf) -patient reintubation within the following 48 hs, these values were compared between w success and failures using fisher exact test. an apriori level of statistical significance was chosen at p < 0.05. 4 considered, an increase in tnf-a levels is observed after cardiac surgery (p<0.001) with a return to previous values after 24 hours (p<0.005). 72 hours after cpb, similar values are observed in groups ii and ill, but there is a further increase in serum tnf-a levels in group i when compared with both other groups (p<0.03). we found no statistically differences in any other moment. there was a significant correlation between serum tnf-o levels determined 72 hours after surgery and cpb duration (p<0,003). conclusions: cpb in childhood provokes a significant increase in serum tnfa levels, in newborns the inflammatory response is maintained 72 hours after surgery. this enhancement of serum tnf-e levels indicates the existence of a relevant inflammatory response in these patients. introduction: cardiac surgery appears to induce a systemic inflammatory response. we have investigated the behaviour of il-1 i~ and il-6 before and after cardiac surgery. patients and methods: we studied serum il-1 6 and il-6 levels from 20 children with congenital heart disease (10 boys and 10 girls), aged from 7 days to 14 years, undergoing open heart surgery, before cpb (d we found no statistically differences in the il-i levels in the different groups and moments. there is a significant increase in il-6 immediately after surgery (p<0,01) with similar levels 24 hours after cpb and a significant decrease (p<0.01) 72 hours after cpb. preoperatory il-6 levels were higher in the groups i and tl than in group i11 (p<0.05). 24 hours after cpb serum il-6 levels in group 1 were significantly higher when compared with group 111 (p<0.05). conclusions: cpb in childhood induces a significant transient increase in serum il-6 levels, strongly relevant in newborns. cpb was not associated to a significant modification in serum il-1 6 levels. thus, cpb in childhood induces a dissociated behaviour in the proinflammatory il-6 and il-1 & pathways. obiective, to evaluate the effects of amg receipt on the clinical condition during the first 12 hours after birth (t2), the morbidity and mortality in immature outborn neonates. methods. we studied 44 outborn neonates with ga 26 to 29 wks, admitted during the years 1993 to 1995. eighteen neonates exposed to amg (ga:27,6+lwks, bw: 1066_+195g) and 26 neonates did not (ga: 27,7_+1wks, bw: 1042_+187g). results. amg-exposed neonates compared to those not exposed had lower incidence of apgar score at 5 min _< 3 (6% vs 35%, p<.05), lower incidence of ph t2 <7.20 (11% vs 48%, p<.05), decrease need of bicarbonate 12 (22% vs 54%, p<.05), lower fio212 (fio212min>40: 17% vs 48%, p<.05 and fio212max >80: 17% vs 52%, p<.05), lower incidence of intubation (67% vs 92%, p<.05), lower requirements of surfactant (50% vs 79%, p<.05) and lower mortality (11% vs 50% p<.01). there were no differences between the two groups for the following parameters: type of delivery, hypothermia hypoglycemia and anemia during admission, hypernatremia, hypotension 12 (map<30mmhg), need of dopamine and or plasma 12, incidences of ptx pda sepsis nec severe rop major ivh (plus pvl) and bpd and duration of intubation. conclusions. the main beneficial effects of amg receipt on the immature outborn neonates were the decrease of mortality and the decrease of surfactant need. there was no effect of amg receipt upon other severe morbidity in this high risk group of neonates. premature babies are very sensitive on homeostatic disturbances, and often develope intracranial haemorrhage (ich). ultrasound scan of the bram shows four grades of ich: -grade i -only periventricular hyperechogenic areas -grade ii -haemorrhage ham the lateral ventricles -grade ili-dilated lateral ventricles -gtrade iv -intracerebral haemorrhage. the purposes of this study were: 1 to show the incidence of ich in premature babies and its correlation with the gestational age, 2. to determine the severity of ich 3. to present the outcome &those babies. in the study were included 393 premature babies successively-born at the department of gynecology and obstetrics before 37 gestational week (g.w.) and grouped in three groups: less than 28 g.w., 28-32 g.w., 33-36 g.w. to all of them was performed ultrasound scan of the brain. results : 1. the incidence of ich hi premature babies is 49 % and there is ingh level of correlation with the gestational age: -babies born before 28 t~ g.w. have 100% incidence of ich and graduated : i grade -5%, ii grade -65%, iii grade -25%, iv grade -5% -babies old between 28-32 g.w. have incidence of 61% : i grade -24%, i[ grade -62%, iii grade -14%. -babies older than 32 g.w. have incidence of 33%: i grade -46%, ii grade 48%, iii grade -6% 2. sixty of 393 premature babies have died and it is 15.2% lethality. in all died ilffant was confirmed the grade of ich diagnosed by ultrasotmd scan of the brain. d. maksimo~5c. z.braiko~ic, n.vunjak. p. ivanovski (5~iversi~, children's hospital. belgrade, yugosla~, ia infantile intracranial hemorrhage is the most frequent and serious manifestation of late hemorrhagic disease of the newborn caused by ,,~tamm k deficiency in earl?,, ti~fancy. in the last two years, we recorded five cases of infantile intracranial hemorrhage due to "dtamin k deficiency, despite routine prophylax~s (intramuscular vitamin k, 1 mg) , with bpieal clinical presentation: age was 18 -65 days (average 40 days): vomiting, poor feeding, lethar~'irritabiljty, palor, bulging t0ntanelle and convatsiones were present in most cases.two patients developed signs of hemorrhagic shock, with hemoglobin level less than 70 g.1. in 3~5 f \qi level was less than 30 % of predicted value. there was no evidence of head trauma or liver disease in none of patients. four inlants were breast fed, while one, who had diarrheal disea.se, was on adapted milk formula. routine therapy wa.s given (including vitamin k and fresh frozen plasma). two patients were discharged with no sequellae, one developed posthemon'hagic hydrocephalus as a complication and two patients died. late hemorrhagic diseo.se of the newborn is sill/ a significant cause of morbidib' and mortality in earl3' infancy, despite different approaches to prophylaxis developed in recent years. background: neonatal hearing screening in at risk newborns can detect 50% of the children with a congenital hearing loss. automated abr hearing screening (algo-1) has been introduced for healthy newborns. the aim of this study is to test the validity of this algo-1 screener in at risk newborns in a neonatal intensive care unit. subjects: 250 at risk newborns (median gest.age: 30.0 wks, median birthweight 1350 g) selected according to the criteria of the american joint committee on infant hearing. interventions: algo-i automated abr-hearing screening at a level of 35 db was performed in the neonatal intensive care unit. when bilaterally referred, further audiologic screening and/or therapeutic intervention took place. when passed uni-or bilaterally, children enrolled in a) a nation wide screening programme (ewlng) at the age of 9 months and b) in a half yearly follow-up programme in which hearing and speech-and language development were observed according to egan an illingworth. results: screening without disturbance from ambient noise or from routine technical equipment was possible in the incubator, even during nasal cpap therapy. 245 (98%) newborns passed algo-1 screening. 5 (2%) did not pass bilaterally. 1 of 5 with a congenital rubella died shortly after screening.in 4 of 5 bilateral congenital hearing loss of ->35 db was confirmed. 235 of the newborns passed were still alive at the age of 1 year. ewing screening was performed in 183 of 235 (77,9%). 161/183 passed, 15 of 183 had passagere conductive hearing loss, in 7/183 no further investigation was performed. all 235 children enrolled in the i/2 yearly follow-up programme had normal speech-and language development. in this study all 4 at risk newborns with bilateral congeni "tai hearing loss were detected with algo-1 screening. screening results showed no false negatives at follow-up. the algo-1 infant hearing screener can be used as an valid automated abr-screener to detect hearing loss in at risk newborns in a neonatal intensive care unit. gancia gp, bruschi l pnlito e, ferrari g, rondini g -divisione di patologia nc~matate e turapia intensiva -irccs policlinico s. mattco -pavia, italy latrogenic esophageal perforations (iep) in preterm and term infants are seldom reported in litteraturc, in association with difficult endotracheal (et) intubation (with or without stylets), insertion of gastric tube, and pharyngeal suctioning with stiff catheters. crieopharyngeal muscle spasm caused by instrumentation may also lead m a narrowing of lumen, with increased risk of local injury. we report 4 iep observed in intubatcd, mechanically ventilated newborn infants (2 male, 2 female, all outborn). a common feature of iep was inability to pass a nasogastric (ng) tube into the stomach, mimicking e~)phageal atresia.~se 1: birth weight (bw) 185(i g, gestational age (ga) 37 wk, sepsis. before admission to n1cu, the baby underwent multiple et inmbations, because of inappropriate securing of et robe. bloody secretions in pharynx were observed. the endoscopy showed a large lesion at the end of proximal third of the esophagus, case 2: bw 1080 g, ga 32 wk, rds. chest x-ray (cxr) showed a retrostcrnal air leak: the ng tube was stopped }~etwcen d8 and d9 and soluble contrast was seen in upper mediastinum.case 3: bw 76(/g, ga 26 wk, rds. the endo~opy showed an esophageal lesion. cxr showed a paravertebral route of ng tube and a right pneumothorax.case 4: bw 102(i g, cz 22 ,.v!:. rd c. ~!,'.::;;: ::':'_'rvt!~'2s l" ~k':.rvrx. cwr, d,,,,vs ~,,mr~e, ~n rhe upper mediastinum and abnormal route of ng tube through a false passage. surgical intervention is needed in case of mediastinitis or mediastinal abscess: conservative management included broad spectrum antibiotics, total parenteral nutrition, antireflux therapy and, if necessary, drainage of air leaks. enteral feeding has been stopped lor 15 days and cautiously resumed after radiographic study. [x~cal sequelae and death are uncommon, but iep occur in newborns with high risk of death due to prematurity and other diseases. in our patients, et intubation has been performed by experienced personnel: therefore the lack of skills in resu~itative procedures is not always the main factor of iep. prevention of iep requires appropriate materials (et tubes, laryngoscope blades, suction catheters), and procedures (positioning of the infant with correct neck estension, firm et placement). sedation and pain control may help to prevent the muscle spasm. aggressive treatment has improved the tong-term outcome of extremely low birth weight neonates (elbw) but it has also increased the chances of iatrogenic lesions. reviewing the charts of our neonates we observed a high number of vascular injuries. from 1987 to 1994, 2898 neonates were admitted to the neonatal intensive care unit (nicu); 335 of them were elbw (11.5%). studying the charts of these elbw we observed 9 cases (4 m -5 f) with vascular lesions (2.6%). mean gestational age of these patients was 28.7 weeks (rain 24-max33). mean weight at birth was 880g . mean weight at diagnosis was 1825g (1230-2700). in the same period 10 patients with vascular injuries were reported in the 2563 neonates over 1500g (0.3%). the injuries observed in elbw group were: 6 arteriovenous fistula (2 bilateral) at femoral,level, 1 carotid lesion and 2 limb ischemic lesions. aetiology was in 7 cases by venipuncture, in one case umbilical catheter and in the case of carotid lesion a wrong surgical maneuver. no general simptoms were observed. the vessels were repaired with microsurgical technique in six cases: the carotid lesion and five arteriovenous fistula; one case was solved with thrombolitic drugs; an amputation at knee level was required in one case after a long period of medical treatment. the last neonate with an arteriovenous fistula was only observed for parent's will. at follow-up (clinical and by ecodoppler) 7 out of 9 neonates presented normal vascular function without sequelae. from our experience elbw neonates have more chances than older neonates to develop iatrogenic vascular lesions. we advocate an aggressive microsurgery and/or medical treatment to obtain good results and prevent late sequelae. a retrospective comparison between 2 natural surfactants l.j.i.zimmermang m.c.m,van oosten. dept. pediatrics, div. neonatofogy, sophia children's hospital/erasmus university, rotterdam, the netherlands. aim: retrospective comparison of alvofact (in 1993) versus survanta (in 1994) as rescue treatment for neonatal respiratory distress syndrome (rds). methods: both surfactants were given at an initial dose of 100 mg/kg (except for alvofact 50 mg/kg for mild rds grade mi). repeat doses were attowed (survanta 100 mg/kg, alvofact 50 mg/kg) up to a maximum of 200 mg/kg, all parameters and outcome criteria were strictly defined beforehand. the initial response (good,mild,no response,relapse) to surfactam therapy was defined on the basis of the decrease in fio 2. results: there were no signif. differences in patient population and initial parameters: ga (29.9+_2.2 vs 29a _+2,6 wks), birth weight (t332_+431 vs 1227-+444 g), severity of rds (grade ill-iv: 78.6% vs 80.3%), apgar scores, cord blood gases, initial ventilatory settings. in '93 however, the initial surfactant dose was administered earlier than in '94 (14.4-+ 17.4 vs 6.5_+7.8 hrs postpartum, p= 0.025). although the average total cumulative dose was equal in '93 and '94 (169.3-+65,8 vs 167.4_+69 .4 mg/kg), more doses of alvofact were given compared to survanta {2.3_+1.1 vs 1.7_+0.6, p=o.o01) and more patients in '93 received more than two doses than in '94 (46% vs 18 % of patients). there was no difference in the incidence of non-putmonarycomplications. aivofact ( there was a better initial response to survanta and a better respiratory outcome in 1994: in the group < 1250g the duration of ventilation was half in 1994, and in the group >~125og the duration of extra o 2 need was half in 1994 as compared to 1993. we speculate that the main reason for this difference is the earlier and initially higher dosing used with survanta compared to that used with alvofact which was given in the same total cumulative dose but over a larger time span. background: e×ogerlous sur&ct~t raplacem~t treatmem has become rou~ne k~ the t~eatme~t of respira~"¢ dim'~ syndrome (i~ds) of pr~e~tur~, wh~eas its effica w th odi~ respiratory diseoses is sdi1 being wader mvesugatio~. objective: "eac~ mt ereat isto report ottr results of prospect/re, non-randomized "re~-o.e" study oe suffact~t replacement in outhom premamae infa~t~ with rds reruirmg me~aical ventilatioa (nfv). p~tien~ and metho0.s: from j-aly 1993 to june 1995, 18/58; (31%) out~ ~¢ infaats, at a mesa age of 22 z 2,7 horn's ( 13 boys, 5 ~rls; ~ gestafioan age 32-+2.8 weeks, mera~ birth weight 1846 _+ 544 g, ~ 7.2 i" 17 at 5 minutes) with rds, requiring mv, received bov~e-suff~amt (survanta, ros~/aboti, laboratotie~ columbus, ohio) eadotracheally, as was recomm~aded by maaufacturer. as the c,~:ttrol group 19 o~bom premature infants (ot~ of 49; 39%, admitted with rds from euiy 1991 to eune 1991) were saelected ~d who did not receive surfaaam, compared with ~hctant ~'oup they were admitted for treatmeat e~'li~" aft~" daliv~:y (at the age 6.4::2.2 hours vs. 11.7+-13 hours), but they did not diff~ in othe~ baseline dam'a~eri~cs at ~ti~ion. entry crkeda for ~¢fa~aut ~hcadou were fractional i~firat o~ oxtgem r~emeats -fio2 > 0.50 -0.60, ratio au-lerlal to alveolar oxygea pre~are~ao2~ao2 < 0,20 ~ad oxyge~at,~ i~.dex -ol > 10. primary o~comes were deter~caned by ~hanges m exs'ge~ab, c~ ~r~d vmtilatic~ ~ the following variable~; (1) fi'aaic~ of i~spired oxtge~ (fio2); (2) mesa nnvay presmzre (map) (3) pag2 ~ao2 ratio, (4) oxyge~ion index (oi). commo~ comphcadces of prem,musty ~d con~ol mechamcal v~ati]al~on (pater dumas merios.s, intracr~nlal haemcrn:hage, air leak, br onchop ulmrmm'y dy~pl~a ~d death) were reg~ded as sec~d,~y outcomes. r~suas: in warfactaat group we observed slg~5.c~t improve~aeat (p<0.05) in oxygea~thia md veaatilation at 24 hours all~ e~try k~to the m~dy in compari~ion to nons~fa~m" group. compa~on of secondao' outcomes in ~ts with p,.ds showes table l we did not observe ~y major acute hfe fl:u-eattming complicatlola,s m sxlrlhct~mt grou~ tr/lmediately after stu'~actsmt rcplacemev_t therapy. the duramm of mechmucal ven~ation ~ad oxygen lreau~ent m survivals of both groups did not dafter 51gmficautl y a-ore ead~ other. condusion: l!a premature mthats with rds treated with surfaaaat replacemeaat therapy we observed decrease m mc~de~ce of tme'~m~o~oraces add de~th (p<0.01 and p<0.05), whe~e~s m othe~ observed variables thee was uo ,igmfi~t d~=ecce infectious complications during the therapy of respiratory insufficiency in neonates with birth weight less than 1500 g in the course of 3 yearsretrospective study. zitek infants on cmv, cppv, and imv were administered exosurf in dose of 50-60 mg/kg twice endotracheally (see table) . in 32 newborns (86.4%) 2 hours after surfactant admin fi02 value decreased by 20.8%, and after 6 hours -by 28.1% compared with initial value; pip and peep values decreased by 3-5 cm h20 and 1-2 cm h20 after 6 hours, and by 4-7 cm h20 and 2-3 cm h20 after 1 day, respectively accompanied by mean decrease of aado2 from 486,2 to 240.2 mmhg, qs/qt decrease from 24.9 to 13.2% (see table) . mean time of cmv, cppv was 7.8 days, imv-14-36 hours, cpap -10-24 hours. respiratory therapy in 5 newborns (13.5%) was complicated by pneumothorax (bilateral -in 2 infants chorioangioma is a rela~ively rare placentai malformation associated with considerable mortality and morbidity. a chorioangioma can be regarded as an arterio-venous shunt in the circulatory system of the fetus. this causes volume loading eventually resulting in cardiomegaly and high output cardiac failure. a female neonate (gest age 40 wk, birth weight 2290 g, -2.6 sd) was born with an apgar score of 4 and 7 after 1 and 5 rain respectively. the placenta showed multiple chorioangioma. ultrasound of the heart showed a hypertrophic cardiomyopathy. she developed severe hypertension (100/70 mm hg), treated with nitroglycerine and nitropruside. finally blood pressure decreased when enalaprillic acid was given (0.15 mg.kg4). we measuered the activity of the renin-angiotensinsystem. an elevation in renin-angiotensin system is shown probably to compensate for the low resistance circulation before birth, hypothesis: the instantaneous cut off of a large arteriovenous shunt did not result in a fast downregulation of the renin-angiotensin system resulting in hypertension. hypertension should be added to the list of complications of chorioangioma of the placenta. the authors studied 75 cases of children's septicemia with blood culture yielding staphylocucetts aurens. the age of patients varied from 2 months to 15 years (51,3% from 3 years downward), 74% of the children caught their disease in the hot season (may to october). the deaths also occured in this season: 87,5% (21/24). following were the anatomo-dinical lesions. -skin 42%, muscle 60,0%, bone 21,3%, joint 9.3%. -viscera : lung 50%, heart 33.3%, cerebrum 22.6%, kidney 60.6%, fiver 17,3%. -simple lesion skin-muscle-bone joint: 12%, no death in this group. the concomitant lesions of the soft tissue,bone-joint and viscera : 34% with one viscera, 26% with two viscera, 18% with three viscera and 9% with four viscera. -bone lesion : mainly on the long bones (50% on the tibia, 25% on the femur, the remainder being the mandible (3) and the humerus), inflammation of' the hip joint was the main one. -i,ung lesion had forms pneumatocele (4 cases), bronchopneumonia (6 cases), pleural effusion (7 cases), multimicroabcess bursting into the pleura (8 cases), most multimicroabcesses were lethal : 20/22 (90,9%), -heart: all thethreelay~rs got le@~r~, 20% had 2 or 3 layers alrected and death ensued. -cerebrum : the meninges had three forms of lesions purulent meningitis (13 cases), obturafing embolns of brain vessels (2 cases) and cerebral abcess (one case). the characteristic clinical sign was paralysis and meningismus, phlebothrombosis of eavcrnous finus (13 cases)was mually ther~sultofalxil vdfi:h burst there were 6 cases of death with lesion of the meninges and 2 cases of obturating embolns of brain vessels. -the main sign of lesion of the kidney was a change in the components of urine: 60% got proteinuria, 75% had leucocytes in their urine, 42% had erythrocytes in their urine, the urea in their blood increased (over 60rag%) in 21.4% of cases.the lesion of the kidney seemingly had little relation to death. seven cases of ictertts due to an increase of direct bilirubinemia and a decrease of blood-albumin. -the biological characteristics of the pathogen staphylococci showed that all the 75 isolated specimens had positive coagulaza ; the specimens from the dead patients were less semiti~e to, mad ~t to mali~ overag death rate was 34.7 % (24/75). the fungal infection to fusariun species in immunocompromissed child have been reported in the literature with a rare, severe and high, mortality rate in spite.of the use of antifungal drugs. we report a case of successful treatment of a severe disseminated fusariun infection in a ll-year-old boy with acute lymphocytic leukemia (lla-l3), after use a chemotherapy followed by absolute granuloeytopenia. the patient developed fever, skin lesions, pneumonia and fungaemia. fusariun species was cultured from the blood, necrotic skin lesions and lung secretion. the child developed multiple organ system disfunctiou in spite of use broad spectrum antibiotcs and antimycotic therapy needing. uci during 18 days. the patient receive suport treatment (mechanical ventilation, inotropie d~.ugs, diuretics, imunestimulants, blood components, a broad spectrum antibiotes and antifungal agents). we absorved a gradual recovery in the white blood cell count and regression on the sites of infection. the association of preeoce diagnostic and the terapentic with increase in the white blood cell count was the most important in a successful treatment. a 5 year old african-american child suffered a severe pulmonary injury in a house fire. initial survey revealed 1% total body surface burns, soot on the face, and bloody endotracheal secretions. initial chest radiograph revealed diffuse, bilateral infiltrates. severe respiratory failure with an oxygenation ratio of 73 rapidly developed. he developed a pneumomediastinum and subcutaneous emphysema. although transient improvement occurred with inverse i:e ventilation and surfactant, he became more hypoxic (sac2 as low as 47%) and acidotic. on day 2 post injury, he was placed on venc~venous extracorporeal life support (ecls). on ecls day 30 he was decannulated. chest radiograph on ecls day 15 showed an opacity in the left chest. ultrasound of the left chest was consistent with atelectasis rather than pleural fluid. flexible bronchoseopy failed to reveal any obstruction in the left lung. a computed tomography (ct) seen of the chest, which was performed after decannulation, revealed a large loculated collection of fluid in the left, anterior chest. under ct guidance, a 14 f cope loop catheter was inserted and 40 cc of thick blood was removed, follow-up ct performed immediately after this procedure revealed minimal change in the size of the fluid cavity. over the next 48 hr, we instilled urokinase 20,000 units over 20 minutes every two hours. a 30 minute dwell time was allowed before draining the fluid. repeat ct scan done at the end of the urokinase infusion showed a marked decrease in the size of the fluid cavity. act scan was not performed prior to decarmulation because the ecls circuit tubing was too short to allow appropriate positioning of the child in the ct scanner. after a ct scan revealed loculated pleural fluid, a simple drainage procedure was diagnostic but inadequate treatment. we were able to successfully dissolve the thrombus after 48 hr of urokinase therapy even though the thrombus was > 14 days old. we suggest that large loculated plenral thrombi which develop as a complication of ecls therapy may be successfully managed with urokinase infusion. introduction: haemorrhages, particularly intracranial, are major complications experienced in 10-35% of neonates treated with extracorporeal circulation. an induced thrombocytopenia and impaired platelet function play a key role in the increased bleeding tendency observed in these patients. the aim of the present study was to establish a dose-respons curve for the effect of a synthetic protease inhibiting agent, nafamostat mesilate (fut-175), on platelet membrane glycoprotein density and platelet activation during experimental perfusion. methods: two identical extracorporeal life support (ecls) circuits were primed with fresh, heparinized human blood and circulated for 24 h. four different concentrations of fut-175 (7.12 mg/l blood/h; 14.25 mg/l/h; 14.25 mg/l/h+25% bolus at the start of the perfusion and 2&5mg/l/h+25% bolus) were used in different perfusion experiments. a total of eight paired experiments were performed. platelet count, plasma betathromboglobulin levels and platelet membrane density of glycoprotein ib and lib/ilia were followed as well as plasma concentration of haemoglobin. results: a protective effect of the agent on platelet count, plasma concentration of btg and platelet membrane gpib could be observed during the first 3 hours of the perfusion when a bolus dose was added. no positive effect could be recorded with the two lower doses used. plasma concentration of haemoglobin was higher in all the fut-circuits compared to the control circuits. conclusion: the addition of a bolus dose of fut-175 at the start of the perfusion seem to induce a protective effect on platelets during the first hours of perfusion. extracorporeal membrane oxygenation (emco) is a form of invasive cardiopulmonary support that can provide imporary physiologic stabilisation in reversible circulatory failure and or respiratory failure. we reviewed our expierence with extra corporeal membrane oxygenetion in 4 children aged 1 day to 4 year between 1991 and 1995. two neonates was succesfully decanulated, but died 1-2 well after decanulation due to septic complictions. one child 4 years old, one neonates died on day 5 and day" 7 respectively while still on emco. complication which were and encountered were heavy bleeding in case 1 (child), 4 (neonate) and raceway rupture in case 2 (neonate). problems which are specific developing countries like indonisia are: high cost (20.000 us for 7 days) difficulty in transportation (transporting intubated baby) from the orgin hospital, lack of knowledge and understanding of the primary physician and nm-ses and difficulty organizing in 24 hours emco team. resnratory mon1tor/ng in picu z,zjvkovic, s. mihailovic, o, tosev respiratory monitoring in pediatric intensive care unit 0picu) provide the importartt informations for understanding of the pathophysiology of the clinical signs, aid with the diagnosis, and assist in therapeutic management and predicting prognosis. pien in children's hospital for ~flmonary diseases and tuberentosis remained for the t~s't two end a half years relatively limited for diagnomic tools and therapeutic regimens, mostly because of the poor fmnaeial suptx~rt. the number of children admitted for aurae asthmatic at.lzek~ severe pneumonias, bronehiolitis, complicated pulmonary tuberculosis, foreign bodies and exacerbations of ehronit'. pulatonary diseases was t362. for all patients the respirator' monitoring system means: physie~d examination, ehe~ x rays, capillary bltxxl gas mmlyses (vevv few ehiktren experienced itwasive arterial blt~.~'i gases), noninvasive oxyntctry, measuring of the vital capacity in coopo-able patients, as~d capnography. later on, after the imtial critical illness, a complete hmg fimction tests was performed, as well ,~s bronehoscopy in selected eases, (~lr experience revealed that abotrt 60% of ehil&en heos suecessthl outcome, without s~lllens , instead they had been tremted in limited conditions. ']'he rest of our patients were previously diagnosed ~s ettronie pulmonary patients, with high risk score system ibr having seqnells 'llae mortality rate were 0,5%. the continuous blood gas monitor, pasatrend 7 (biomedical sensors, ltd., high wycombe, bucks, england) has the capability of measuring ph, pco2, and po2 via an indwelling optical absorption optodelclark electrode sensor that is placed through an intra-arterial catheter. we evaluated the accuracy of the sensor in radial and femoral locations in critically ill pediatric patients. methods: the simultaneous values of ph, pcoz, and po2 recorded from the paratrend 7 monitor were compared to values measured by standard arterial blood gas analyzer (coming 278, ciba-corning diagnostics, medfield, ma). criteria for the elimination of data points included a core vs. sensor temp. gradient, and sensor pulled back beyond accepted insertion distance. mean time of monitoring per sensor was 108 hours (range 0.75-403.7 hrs). mean time of radial monitoring was 35 hrs (range 0.75-160.5hrs) and of femoral monitoring was 137.2 hrs (range 12.8-403.7 hrs.). linear regression and bland-altman analysis for bias and precision for each parameter were calculated. results: a total of 49 patients (age range 2 weeks to 18 years) had paired samples of ph, pens, and poz made by the sensor and blood g&s analyzer. the range of measurements were ph 6.99-7.66, pco, 16.0-i14.2 t(n r, and po2 34-480 torr. the paratrend 7 monitor demonstrated accuracy that is comparable to the accepted standard of blood gas analysis in a group of critically ill pediatric patients manifesting wide variation in ph, pen2, and poz..this technique appears m be very useful especially in the extreme values of the parameters measured. funding provided by biomedical sensors. understanding of pulse oximetry d.semple, l.e.wilson. royal hospital for sick children, edinburgh, eh9 1lf, scotland, uk. pulse oximetry is a useful, non-invasive monitor, routinely used on the itu and increasingly often on the general wards. we used a questionnaire incorporating questions on the theory and clinical uses of the pulse oximeter to assess understanding of pulse oximetry in medical and paramedical staff doctors indicated grade, speciality, pulse oximetry tuition and neonatology experience. 45 doctors, 15 itu nurses, t9 medical students and 4 physiotherapists completed the questionnaire. some confusion existed between the principles of pulse oximetry and transcutaneous oxygen measurement. wide variations in the lowest acceptable saturation in fit children were seen (80-95%), with around 20% of respondents in all groups accepting values of 90% or less. some potentially serious mistakes were made in the evaluation of oxygen saturations in the clinical scenarios. there were widespread variations in correct responses at all grades of medical staffing. nurses scored well on more clinically-orientated questions but relatively poorly on theory. only 15% of doctors (mostly senior grades) had received tuition in putse oximetry. neonatology rotations appeared to confer little additional knowledge on pulse oximetry. few doctors and nurses receive tuition in the use of pulse oximetry a significant proportion of nurses and doctors, of all grades, exhibited a lack o{" understanding of the principles of pulse oximetry. this may result in unsafe use of the equipment and put patients at risk. one can see from the table that blood composition in uv and ua differens in some characteristics, and similar in sgp magnitude. venous-asterlal gradients "gas functiomals" between uv and ua represent the measure of difference in this characteristics. the gradient cari be positive, zero -order or negative and change both in value and in sign but not reach apo2 (positive) and apco2 (negative) in absolute significance.minimization of "gas functionals" deviations atom the zero is achieved due to"mutual replacement acts" between po2 and pco2 in uv and ua blood. we suggest that presented tests can be useful in full evaluation of gas exchange in newborns. (pap) in the context of pulmonary hypertension is oft desired but rarely achieved. inhaled nitric oxide (no) has been shown to produce this desirable effect, but is relatively difficult to administer or monitor. we wondered whether np, chemicaily related to no but more stable in solution, would produce similar physiologic effects when administered in the convenient modality of nebulization. methods: 9 piglets were anesthetized, mechanically ventilated, and surgically instrumented. systemic blood pressure (bp), pap, and cardiac output (co) were monitored continuously. after postoperative stabilization, 0.9% nac} nebulization was begun, and pulmonary hypertensiorr was induced by reducing fio2 from 0.30 to 0.07. the piglets were monitored for 15 minutes during this hypoxic phase, next, without altering fio2 or ventilator settings, np (10 mg/ml, dissolved in 0.9% nacl, flow 4 ipm) was substitued for 0.9% nacl in the nebulizer circuit. np was nebulized for 15 mins. results: during hypoxia, pao2 fell from 159 to 29 mm hg. pap rose during hypoxia from 14 to 31 torr (p< 0.01). ,^fhile bp and co did not change significantly. pap fell during nebulized np in each piglet, (mean apap = 31 to 21 torr; p< 0.01; mean reduction of hypoxia-induced rise in pap = 61%; range: 36 to 78%; p < 0.01). pvr/svr fell by 28% during np nebulization (p< 0.01), while bp and co did not fall significantly (90 to 86 tort; 653 to 636 mllkg-min), the reduction in pap began within 2 minutes of the onset of nebulized np, and appeared to reach a plateau by 15 minutes. no tachyphylaxis to nebulized np was noted. nebulized np did not significantly affect pap, bp, or co under normoxic conditions. conclusions: 1) like no, np selectively reduced hypoxia-induced pulmonary hypertension without altering systemic bp, 2 ) unlike no, np can be administered by nebulizer, a technique familiar to virtually all health-care providers, and potentially adaptable to both intubated and non-intubated patients. 3 } nebulized np may be beneficial in clinical contexts where inhaled no is impractical. dang phuong kiet and nguyen xuan thu examining 6 cases of purulent pericarditis with various clinical forms treated by surgery, the authors drew the following experiences for their diagnosis. t. clinical factors. purulent pericarditis appeared like a cardiac tamponade in a septicemia due to staphylococci with dassieal symptoms: severe dyspnea, tachycardia, faint heartsound, big liver, prominent cervical vein ; rentgenography of the chest showing enlargement of the cardiac silhouette, a diminution of ventricular pulsations, ~i clear lung field. by an emergency operation, 500ml of diluted blood were drained. purulent pericarditis and pleural effusion appeared at the same time but at first tile symptoms of purulent pericarditis were masked by the predominant symptoms of plearal efihsion. after the pleura was drained, its pus was no more, the general state was relatively stabilized but there still were big liver, dyspnea, enlargement of the cardiac silhouette while central venous pressure increased. purulent pericarditis appeared late. in the first stage (about 2 weeks) there was no suspected sign. later on gradually appeared such symptoms as dyspnea (during serum transfusion for instance). central veinous pressure also raised. the heart chest diametre increased at first (up to 60-65%) then decreased (down to below 50% ) but the liver kept on swelling together with the particular changes of electroeaediegramme. now the pericardium had no more pus but get fibrous (up to 3ram) thus constricting the heart and its main arteries 0ike pick syndrome). 2. diagnostic values of electrocardiograms : common signs of ecg related of these purulent pericarditis were: a diminution of voltage, a widespread elevation of the st segment, the tf wave flattened and inverted. however, what should be stressed was : the diagnostic values of an electrocardiogram for purulent pericarditis was mainly in the dynamics of their signs: in the first week, the voltage diminished corresponding to a pericardium containing pus, while the st segment went up then seemed parallel to the fibrosis of the epicardium, the liver swelled, the central velnous pressure increased, the heart/chest dimension ratio decreased, the st segment went down, the t wave became more flat and inverted. between 1986 and 1995 23 neonates, aged 2 -23 days (median 5), weight 2,38 -4kg (median 3,28) with critical valvar pulmonary stenosis were scheduled for balloon dilation (psvp), 19 children (83%) were on pge1 and 13 (57%) needed mechanical ventilation. after stepwise dilation a final balloon : pulmonary valve (pav) ratio of 114% (25-150) was achieved, there was a significant correlation (p<0,01) between an adequately sized balloon and freedom of reintervention. two valves could not be passed, four neonates underwent surgical procedures (brock n = 3, commissurotomy n = 1), two children (10%) died of sepsis. 17/23 patients (73%) were successfully palliated by psvp in the first month of life. the rv : systemic pressure value fell from 132% (75-231) to 58% (40-87), complications included 2 transient dysrhythmias, 1 transient hypoxia, 3 vessel occlusions;-1 right ventricular outflow tract perforation. in 16/17 patients follow up data is available. the residual systolic peak doppler gradient over the pav on the last out patient visit (5-103 months after psvp) was 10-41 mmhg (median 20). four children needed repea.ted psvp 26 to 72 months after the initial intervention. conclusion: psvp of critically ill newborns is possible. the risk of mortality is relatively low. psvp in neonates with an adequately sized balloon is a challenging alternative to surgical treatment. post hypoxic-ischemic (hi) reperfusion induces the formation of non protein bound iron (npbi), leading to production of the reactive hydroxyl radical. it was investigated if the ironchelator deferoxamine (dfo) could reduce free radical production and improve neonatal myocardial performance after hi. severe hi was produced in 13 newborn lambs and changes from pre-hl values were measured at 15, 60 and 120 min post-hi for (mean) aortic pressure (mean pao), cardiac output (co) and stroke work (sw). left ventricular (lv) contractility and co were assessed by measuring lv pressure (tip-manometer) and volume (conductance catheter), using inferior caval vein occlusion to obtain slope (ees) and intercept of the end systolic pv relationship (v10). npbi, reduced and oxidized vitamine c ratio (vcred/ox) and lipid peroxidation (mda) were measured from sinus coronarius blood. 7 lambs received dfo (10 mg/kg i.v.) immediately post-hi, control lambs (cont) received a placebo. results: mean pao was stable, co and sw decreased up to 60 and 40% respectively in cont as compared to pre-hi. in both dfo-groups co and sw remained within the normal range. ees and v10 decreased in all groups post hi, but did not differ between groups. npbi and mda were higher at 15 min post hi (p<o.05), vcred/ox was lower at 15 min post-hi (p<o.o5) in cont as compared to dfo-group, indicating more oxidative stress in cont. conclusions: dfo reduced the oxidative stress of the heart and prevented co and sw to drop, suggesting a positive effect of iron chelation on the myocardium after h{. from 1987 we published reports with analysis of performance of the heart as a whole organ.while analysing the heart performance as a whole, we recognize three block in it located intrapericardially: l."atrial"block -left (la) and rigth (ra) atriums; 2."aorta-pulmonary" block-aorta 'bulb (a) and pulmonary artery(pa); 3.ventricular "three-chambered" block (vb) consisting of: -left (lv) and right (rv) myocardial chambers, both frith blood outflow into "aorta-pulmonary" block vessels, and -spongy (venous) myocardial chamber with the blood outflow through coronary sinus (cs) and thebesiau vein (tv) into "atrial" block. the following conceps are introduced for vb functions assessment "common" systole and "common" diastole of "three-chambered" vb. the process of normal "common" vb systole: -begins with blood ejection from vb spongy chamber into the "atrial" block; -continues with blood outflow from rv and lv into "aorta-pulmonary" block wi~ venous minimums -x-coiiapses -~btmation iu "a~riai" block; -completes with "three-chambered" vb general emptying. at this period the following blood volumes are transferring: -two-from rv and lv(their stroke volumes)into"aor~o-pulmonary"block; -two-from "spongy" chamber .into "atrial" block; -two-from systemic and pulmonary veins into "atrial" block during the process of so called "systolic" membrane suction (at pulling atrio-ventricular valves into rv and lv chambers as blood ejects out of them). it appears to be the regulation of blood inflow to "atrial" block by blond outflow from "three-chambered" vb into "aorto-pulmonary" block. objective: to evaluate the efficacy and complications of transpyloric enteral nutrition(ten) in critically ill children patients and methods~ from june 1994 to january 1996 23 children (i0 boys and 13 girls), aged from 5 days to 11 years (mean l.8 ± 3.4years) were treated with ten. 18 patients were included after cardiac surgery, 3 with acute respiratory failure, and 2 after cerebral surgery. the indication of ten was mechanical ventilation in 22 patients and failure of nasogastric nutrition in 1 patient.19 children (82 %) had previously received parenteral nutrition. 8 and i0 fg enteral tubes was inserted into duodenus through nasogastric intubation en 21 patients and by endoscopy in 2 patients. rx and ph determination were used as methods to control tube situation. 18 children received adapted formula, 7 caseine hidrolisate, and 3 enteral formulas. (in 5 patients the nutrition formula was changed during ten). 22 patients were supported with mechanical ventilation during ten, 17 received midazolamperfussion(range 2 to 15 mcg/kg/min), 15 fentanyl (i -12.5 mcg/kg/h), and 6 vecuronium (0.1 -0.3 mg/kg/h). resulte: ten was used during 3 to 73 days (mean 18.3 ± 19.3 days). mean maximmnvolume ad~iniateredwas 132 ± 47 ml/kg/day (range 34 208) .patients with midazolam, fentanyl and vecuronitml tolerated ten similar than children without sedatives. complications were diarrhoea 1 patient, abdominal distension and/or important gastric fed residuals 2 patients. pulmonary aspiration or respiratory complications were no registered. ten was ended in one patient due to diarrhoea, in 18 due to change to oral or nasogastric nutrition, 3 children continue yet with ten and 1 was discharged of picuwith ten. conolusion: ten is an useful method of nutritional support in critically ill children with mechanical ventilation and/or nasogastric intolerance. introduction: the enteral nutritional support of the critically ill child may reduce the morbidity and mortality by attenuating, the hypermetabolic and catabolic response, decreasing the gut translocation and the septic complications, improving the bowel mucosa integrity and the wound healing. the aim of this study was to show the safety and tolerance of en. methods: we enrolled 36 consecutive patients admitted to our unit from may to december 1995, mean age 3.4 years (range imonth to i7 years). the prism, mechanical ventilation, inotropic use, opiates and other drugs were recorded. the en was delivered by continuous infusion across a polyuretane tube.we kept the patients head elevated 30 °, and we used cisapride routinely. the gap betweeen admission and the en beginning, average time to meet the nutritional objective, en duration, selected formula, patients outcome and complications were recorded. results: prism groups were 0-1=0 patients; 1-5=1; 5-10=13; 10-15=7; 15-30=12. two patients with mof (multiorgan failure) died. 72% needed mechanical ventilation.the average time elapsed from picu admission to initiation of en was 2.1 days (range 1 to 7 ) and the average duration of the en was 7.3 days (range 3 to t4). the average time to meet the nutritional objective was 2 days (range 1 to 3). the selected formula were: lactose free infant formula in 10 patients, elemental diet in 8, pediasure in 16 and jevity in 2. we recorded vomiting in 4 patients, diarrhea in 5 and constipation in 4. none of them forced the en suspension. we didn't recorded any case of ~spiration or pneumonia. we found no relation between drugs, selected formula and complications. we think that en may be a safe and well tolerated procedure at the picu setting. generation of free radicals in iipid emulsions used in parenterai nutrition (pn) has been described recently. this seems to be due to high polyunsaturated fatty acid content. we studied lipoperoxidation status and antioxidant parameters presurgically and through the postoperative period (5 days) in a group of twelve children (ranging from 4 months to 12 years of age), who underwent heart surgery and received nutritional support with pn. malondiaidehyde (mda), an end product of lipid peroxidation was used as a marker of oxidative stress. it was determined by the yagi spectrofluorometric method. antioxidant activity was measured with an assay based on the inhibition of spontaneous autooxidation of a brain homogenate, and vitamin e in serum by the technique of hansen and warick. erythrocyte mda release fonowing incubation in hzo 2 in vitro was used as a functional measure of tissue vitamin e levels. data were compared using one-way variance analysis. there was not significant differences in the plasma mda production among the values obtained before surgery (3 :t: 0.5 nmol/ml), postoperative pre-pn (3.5 +_ 0.6 nmol/ml) and through the course of pn (3.5 5:0.6 nmol/ml). levels of vitamin e previous to surgery were 5,1 5:1.1 #g/ml and decreased to 3.2 _+ 1.4 #g/ml at 24 hours post-surgery before starting pn. while receiving pn, concentrations of vitamin e increased progressively up to 7 + 4. l #g/ml (p = 0.08). pre-surgicai plasma antioxidaut activity values (72 :i: 11%) dimiuished in the first postoperative day (56 _+ 9.5%) and then showed a clear increasing tendency, directly correlated with that observed in vitamin e levels. the measurements of the maximal percentage of mda release of erythrocytes in vitro, showed an inverse relationship with plasma vitamin e levels and with the plasma antioxidant capacity: preoperative 5.8 + 2.4 % and postoperative descending from 20.5 + 14% to 12 _+ 9.5%. our results indicate that during surgery and within 24 hours postsurgery, a decline in antioxidant defenses occurs, in the postoperative period, while patients are on pn and taldng into account our obtained data: the unaltered mda values, the rise of plasma vitamin e levels, the recovery of both functional erythrocyte membrane antioxidant protection and the plasma antioxidant activity, they do reflect that during the intravenous administration of lipid infusions, oxidative damage does not occur, probably due to the addition of vitamin e to pn solutions in order to avoid autooxidation of lipid emulsions. garnitine is one of the essential nutrient in the diet of newborn infants. mother's milk represents the natural eamitine source for the newborn infants. we studied enteral earnitine intake in 71 premature infants 35 eutrophic (eu) and 36 hypotrophic (hy), fed with mothers milk trough 5 consecutive days-from fifth to tenth day of life. mean gestational age was 33 (range 32-36) weeks for eu and 34 (range 32-36) weeks for hy. birth weight was ranged 1430-2450 g for eu and 1250-2300 g for hy (p < 0,05). breast milk carnitine concentration was 78,88 i.tmol/i in mothers milk delivered eutrophic babies, and singnificant higher mean coenetration 113,88 l.tmol/i, of hypotrophic premature infants (p<0,05). breast milk earnitine concentration was ranged between 39,15-163,71 ~moi,'l, and daily carnitine excretion was between 9,7-165,48 p.mol/i, mean 60,6 +-38,7i p.molll. the daily mean carnitine intake was from 1-1,93 mgkg/d, in eutrophic and 1,23-1,93 mg/kg/d, in hypotrophic premature infants, in milk volumene intake from 150-200 mllkgld., the results of this study suggest that premature infants should be fed with own mothers mitk in eady neonatal pedod in attempt to prevent camitine deficiency. keto-acids and parenteral lipid admlnistration. castorina m., antuzzi d., ricci r., rendeli c., polidori g. pediatric intensive care unit -catholic university -roma (italy). some metabolic studies have suggested that the administration of mediumchain tltgticerides (mct) might induce a marked increase in ketone body concentrations; the aim of this study is to evaluate the effective ketogenetic risk of infusing mct emulsions in total parenteral nutrition (tpn). two groups of seriously infected children were examined: a-12 septic infants were given to tpn with a 100 % of long-chain tryglicerides (100 lct). b-10 septic infants, in wich the lipid source in tpn consisted in a 50-50 mixture of lct and mct. the children in the different groups showed similar severity and therapeutic scores (prism, tiss, ~, f~2, f~3); the lipid intake was the same, corresponding to 1-1.5 g, ckg bodyweight/day. in all patients a sample of urine was taken six times a day for the whole time of tpn the urine samples were screened by the diphenylhydrazone test, and the ketoacids excretion was confirmed by a chromatography. the urinary excretion of 13-keto-acids equivalent to p-ohphenyl-pymvate (mcg/ml urine) is summarized in the no significant increases in frequence and/or in severity of acid-base disturbances were found in all studied patients in consequence of the lipid infusion. the patients of b-group showed a lower excretion of 13-ketoacids. the c~-keto-acids excretion was ever unsignificant and seemed to be correlated with the illness severity more than with the lenght of the tryglicerides chain. this observations let us suppose that the mct, at employed doses, can be safely administred also in childen with metabolic acidosis and/or serious illness. reye syndrome cayetano heredia hospital, lima, peru five patients with reye syndrome were studied; included were those diagnosed from january 1991 to march 1994 and treated at cayetano heredia hospital. the age of presentation varied from z.5 to 7 months. the syndrome occurred more frequently in males (4/5); the time of illness at the presentation varied from 2 to lo days, and the following features were found: fever (3/5), akeration in mental status (5/5), seizures (4/5), gastrointestinal illnesses (diarrhea) (4/5), and respiratory insufficiency (1/5) --in this case ventilator)-support was needed, in all cases hepatomegaly, intracranial hypertension, hypokalemia hyponatremia, metabolic acidosis, hyperammonemia and elevation of hepatic enzymes were found. coagulation blood tests were abnormal in three patients. cerebrospinal fluid (csf) showed hypocellularity in all cases (less than 8 cells/ram3). the cultives were negative, and the final diagnosis was confirmed by hepatic biopsy. no deaths were due to reye syndrome. a contribuhon to the study of reye's syndrome the aulhors analysed 38 records of patients meeting all the clinical, biochemical and anatomic criteria put forward by hutrealoch~ and samaha (1975) who were ireated at the emergency and resusdlation depammlt tithe ipch in 10 years. the average age of the children was 3 years (the 3~umgest 11 monks and the driest 7 year~ most of lhon (34/38) came from lhe countryside to lhe lmfia~ willfin 24 hours of file oulbreak. a few days previously the patients w¢~ld have fever (some of ahem wilh diarrhea or cough) ,,rod vomit then rapidly fidl into coma and c~nvuksien. they ~me to the imtia~ with the signs of typical increased inlracranial pressure ( stages 2-4 according to lovejiy for most cases) but the liver was unllkely big. the main biocheafical dmnge of the blood was acute hepatic thilure -the bllod glucose decreased : (ha the aver~ 23,46n~% (26) ~ not measured in five cases (glucose a"aces). -the blood ammonia ino~ased riven 158 to 275 microg/dl fin lhe average 220, n = 3) -the percentage of p~in was low, in the average 44,5% (n--9), 11% at the lowest (a case ofmekaa lasling five days was ctwed with vitamin k). -got and gpt enzymes increased 3 -4 times (n=14) besides, tht~re was decompcmated addt~is with lhe average valnes ph = 7.19; pcoz = 2~6mmhg and eli = -1~6 ~, (n~). the charadaislic dumge in the ¢~'ebrospinal fluid was a low glucose tx~acenwalien h the average of 1k65mg/dl (3 cases of glucose it'aces) ; meanwhile protein decreased in the average of 14mg/dl (4 cases below 10mg/dl). besides ~here was no inflammatory lesion ~tion) : very serious brain oedona and fatty degeneration of live~ scatteredly or parllally.microso~ically (biopsy and necropsy) in file brain appeared bright space around blood arteries and glioma extended v'lrdmw -robin space, with no inflammatory reaction. liver.-the sa~dure was intact there was no " "mtlammal~s3' cell, the kupffer cells did not show hyperplasia but show a heterogenous deg~nerafion~eart: also showed a fatty degencralion in some areas of the myocard eellkidney: the ihtty degenerati~l scattetedly in the tubular cell. pano~as:fatty degeneration scattetedly in file pancreatic cell. two cases of liver biopsy for lhe 2rid lime (check before leaving file inslitute) found that fatty degeneration was r~tta~ nearty ff~pletely. the exact muse remained unknown. objectives: to analyse whether there exists serum and urine electrolyte disorder in children with respiratory failure, methods: we have made prospective study of 48 children in our icu during a 12 month period, electrolyte concentrations were measured in serum and urine collected during 24 hours (sodium-na, potassium k, chloride-ci, calcium-ca, magnesium-mg and phosphorus-p). results: average values in serum were: na 139.28 +/-3,20 (rv:133-146) mmol/i, k 4.22 +/-0.55 (3.3-5.2)mmol/i), cl 95.20 +/-3.98 (93-106) mmol/l, ca 2.t2 +/-0.12 (2.20 2.65)mm01/i), mg 0.82 +/-0.22 (0.77-1.12)mmol/i. average electrolyte levels in 24 hours urine were: na 64.23 ~/~ 44.11 (42-202)mmol/day. k 22.14 +/-10.33 (22-112)mmol/day, ci 56.14 +/-27.22 (100-244)mmol/day, ca 1.17 +/-0.13 (1.68-6.8)mmol/day and p 11.89 +/-7.12 (11.7-32)mmol/day. conclusions: average serum ci and ca levels were decreased in children with respiratory failure. in urine, average k, ci, ca and mg levels were decreased and urine p concentration was increased. we concluded that serum and urine electrolite disbalance may be expected in children with respiratory failure. the nurse is going to kill me! (icu syndrome) anita duvndam ~, sonia v.d,sluis 2 ~dpt. of pediatrics, div. pediatric intensive care, sophia children's hospital, rotterdam 2dpt. of pediatrics, div. pediatric tntensive care, juliana children's hospital, the hague. content description: this presentation will provide the critical nurse with background information concerning the identification, the prevention and management of the critical ill child who has developed the icu syndrome. the results of a questionnaire concerning the occurrence of icu syndrome on a picu will be presented. behavioral objectives: at the end of this session the participant will be able to: 1. describe four clinical symptoms of the tcu syndrome, 2. identify major sources of picu environmental stress, 3. discuss nursing's unique role in helping the child to cope with the icu syndrome, 4. discuss nursing's unique role in preventing icu syndrome. content outline: the icu syndrome is a situation in which the individual is not able to deal with changes in observation and interpretation of both quantity and of patterns of sensory perceptions. in other words the borders between the inner and outer world becomes unclear for the person. the exact incidence of the icu syndrome in the critical care setting is unknown, we have limited knowledge of the occurrence of the icu syndrome in critical ill children. yet critical ill children in our hospitals sometimes show the symptoms of icu syndromes. to be able to identify the occurrence of icu syndrome we developed a questionnaire. the thesis of the questionnaire was: does the icu syndrome occur in critical ill children in the age between three and ten, who have been intubated and/or ventilated in a picu? eighteen questionnaires have been send out to parents of critical ill children in two hospitals. the response was 90 percent. conclusions: 1. most of the children were anxious (n=11), showed regression (n=8) or had sleeping problems (n-lo) during the stay on the picu. 2. children showed the same problems after discharge. 3. there is no relationship between environmental factors and symptoms of the tcu syndrome during the stay. 4. there is no relationship between preexistential behavior and symptoms of the icu syndrome during the stay. because the lack of a scientific definition for the icu syndrome in children and good criteria for diagnosis, we were not able to get an answer to our thesis. more research is needed. continuous veno venous haemofiltration (cvvh) was adopted as a first line renal replacement therapy in our paediatric intensive care unit (picu) some three years ago. ~/he process of introduction and development of cvvh proved to be an exciting challenge for nurses as indeed it was for the whole multidisciplinary team involved. we have successfully used cvvh in the treatment of over 20 infants and children, weighing between 3-85 kg. the range of conditions treated is ever increasing. to date we have not only used cvvh for patients in renal failure and fluid overload, but also to gain biochemical control in tumour lysis syndrome-and metabolic disorders. other distinct patient benefits in comparison with more conventional means of renal replacement therapies are that, it is a continuous controlled treatment being extremely effective in creating 'space' for nutrition, which is especially important in the fluid restricted, catabolic patient. of paramount importance in providing this level of service is the education and training necessary to impart the skills and knowledge for nurses to become expert practitioners in this field. we now have a teem of highly advanced practitioners who have developed their nursing skills to provide even more holistic care for their patients. this group are embracing new challenges and responding positively to the developing service whilst expanding their knowledge base. this paper will discuss the advantages and disadvantages of cvvh as we have experienced, relating i t to the wide variety of patients we have treated. we wish to share how cvvh has become an accepted role of the picu nurse and how the service has been successfully implemented. integrated care pathways (icp's) define the optimum course of events in the care of a patient with a specific condition, within a set timescale. aims and methods: icp's and case management (cm) were introduced to evaluate and improve clinical practice. icp's were developed for patients undergoing surgery for atrial septal defect (asd) ventricular septal defect (vsd) and fallots tetralogy. they were based on the median experiences of the last 31 patients. 108 patients have been managed using icp's. results: analysis of variation from the icp's shows a reduction in the following potentially avoidable causes of variation: delay in extubation has been reduced from 29% to 10%, and 31 patients (29%) were extubated earlier than the median. prolonged stay on the intensive care unit (icu) has decreased from 13% to 0%, and 24 patients (22%) were discharged to the ward a day earlier than the median. the number of patients receiving inadequate post operative analgesia has decreased from 35% to 15%. delayed feeding after operation has been reduced from 52% to 28%. unavoidable delays in extubation and discharge from icu occurred in 22 patients (20%) because of haemodynamic instability" or lung problems. cm utilising individualised pathways for these patients has reduced variation in care which can improve patient outcomes. pathways have been developed for other conditions and it is anticipated that approximately, 80% of patients will be treated using an icp revision of icp's results in continuous evaluation and improvement of the care provided, conclusions: the use of icp's and cm has improved patient care and decreased avoidable delays and variations. the future development of other icp's, combined with a case managed model for selected patients, is seen as a viable method of continuously improving patient care. this paper is a retrospective audit of 24 children who received continuous veno venous haemofiltration (cwh) whilst on the paediatrie intensive care unit (picu), data was collected over a 21 month period from may 1994 to january 1996. the 9 girls and 15 boys were aged from 1 day to 15 ½ years (median 4½ years) and weighed between 2,5 and 85 kg (median 17.75 kg). length of admission to picu was 2 to 47 days (median 9 days). sepsis syndrome was diagnosed in 11 cases: 4 of these had bowel necrosis and 3 meningoceccal disease. eight patients had an underlying haematological, oncolngical or immune disorder. the remaining children had inborn errors of metabolism (2), nephrotic syndrome (2), or cardiac failure (1). cvvh was instituted for a variety of reasons. muttiorgan dysfunction was present in 46% of patients, 29% had acute renal failure, 12.5% tumour lysis syndrome, 8.5% uncontrollable metabolic acidosis with hyperammoneemia and 4% required fluid management. treatment was continued for between 4 hours and 26 days (median 4½ days). haemofiltration was successful in achieving its desired effect in all except one patient. 87.5% of children with haematological, ontological or immune disorders died despite successful haemofiltration. survival was higher in cases of septicaemia (45.5%), nephrotic syndrome (50%) and errors of metabolism (50%). overall mortality was 62.5%. this was attributed to the severity of the underlying disease process rather than the effectiveness of cvvh as a renal replacement therapy. asynchronous defibrillation and synchronized cardioversion deliver direct currents of high amplitude through the chest, to stop ventricular fibrillation or to convert life-threatening arrhythmias. since the human body is partially conductant, it should be possible that after a brief delay of time, a derivation of this monophasic defibrillation pulse is measurable in regions of the defibrillated body, other than the traject between anodal ("sternum") and cathodai ("ape~;") paddles. one could also estimate that health care personnel in the immediate environment of defibrillatoty shocks, are always at risk to possible electric injury. accidents might occur when health care personnel do not stand clear from the patient during firing and create an additional electric path from this patient through their own body. most likely, the nonisolated parts creating a transversal eiectric path, will be the hands of the caretaker. since defibrillation generates touch voltages up to 5000 v and 60-70 a in ved, short delay's (5 to 10 millisec), the total body resistance during skin to skin or skin to paddle contact, is calculated to be as [ow as 750 ohm (percentile tank 50 for the entire population). an experimental protocol was developed to evaluate the conducted currents during defibrillation. mature pigs with a weight of 100 up to 150 kg. were used as animal models. the:,, had barbiturate anaesthesia, inotropic support with dobutamine at l 0 micmgrams/kg bodyweight and all had a sinasat rhythm at the begining of the test-rounds. synchronized defibrillation at 100 j (approx. 0.75 j/kg) and asynchronized -at 360 j (whenever ventricular fibrillation or ventricular tachycardia occurred), were attempted, through latero-lateral placed and firmly pressed defibrillation paddles and/or adhesive multi-function electrodes. gel pads were choosen as contactmedium to cross the skinpaddle barrier. subdermal electro-myography needle electrodes were put at different parts and regions of the pig's body, but not between the paddles. these electrodes were coupled to a resistance device of 800 ohm and a measuring computer. we measured monophasic and low current pulses up to 0,10 a during the defibrillation burst. thus conducted currents are high enough to produce accidental electric shocks when additional electric paths are created by the caretaker. "llaese are usually harmless, but in some "worst case" (wet or transpiring hands, rings,...) or in association with defectuous equipment, peak currents might be harmfull, and can produce pain, bums, apnae or cardiac (transient or persistant) arrhythmias. maria skogbv. karin mellgren, lars-g6ran friberg, g6sta mellgren, hans wadenvik. g6teborg, sweden. bleeding complications in extracorporeal circulation is partly due to perfusion-induced platelet dysfunction. the aim of this study was to evaluate an in vitro model for investigation of platelet function parameters in an extracorporeat system. study: two complete extracorporeal life support systems were perfused with fresh heparinized human blood for 24 hours. piatetet membrane glycoprotein changes, platelet granule release and platelet count were followed. eight paired experiments were performed. one of the circuits was perfused by a roller pump (polystan) and the other by a centrifugal pump (biomecicus), other components were identical. results: a continuous increase in glycoprotein (gp) ib-negative platelets was seen in both circuits. a marked increase of plasma beta-thromboglobulin concentration and a decrease of the intracellular pool of serotonin was observed, indicating a marked release of alpha as well as of dense granules. the plasma concentration of glycocalioin increased in parallel with a reduced platelet surface expression of gpib, suggesting that the loss of gpib is caused by proteolysis rather than by a downregulation of this receptor protein. conclusion: 24 hours of ecls perfusion induces pronounced platelet degranutation and causes changes of important membrane receptors. this is in accordance with clinical studies, suggesting that our in vitro model does mirror the platelet exhaustion observed in a clinical reality. no significant difference was observed between the two pumps. the purpose of this study was to examine signs of distress exhibited by several patients following the discontinuation of opioids and benzodiazepines (o & b) in a 7-bed pediatric intensive care unit. the authors compiled a list of all cases of possible withdrawal reactions reported by nurses in the study setting over a one-year period. five cases were selected for study in terms of their wide diversity of relevant circumstances. the 5 cases were examined through a retrospective chart review. data pertaining to analgesic and sedative administration, along with nurses' reports of behavioral distress, were transcribed and coded. a remarkable pattern of behavioral distress was clearly associated with discontinuation of o & b. cessation of benzodiazepines was associated with severe distress. these reactions were characterized by various combinations of crying, irritability, tremors, grimacing, gagging, vomiting, or feeding problems. some of these persisted in spite of large amounts of bolus drug administration. these signs were manifested for 2 to 9 days following cessation of o & b. these findings demonstrate that the rapid weaning of o & b infusions, sometimes following short-term therapy, can cause severe withdrawal reactions. the particular course that a specific patient will follow will likely be modulated by underlying manifestations of "icu psychosis" and unresolved pain and emotional distress. hermana tezano8 mt, pilaf orive j, latorre garcia j, lizarraga azparren ma, lopez bayon j ucip hospital de cruces. bilbao. spain a female child, one year old, was referred to our unit from another hospital because she had ischemic syntomsjn her right forearm wich started three days before. she had ~i downs syndrome and fallot's tetralogy with a systemic-pulmonary by-pass. nine days before the admission in our hospital she was undergone to a cardiac catheterization by arterial and venous femoral punctures with no incidences. on the admission to the picu her forearm was cold and pale with absence of cubital and radial pulses and slow capilary filling. treatment with heparine was started unsucessfully in the firsts 24 hours, so uroquinase was added that later was changed to rtpa plus brachial plexus blockade. by the time me arterial tlux became more and more evident (doppi~) il bccuute aiat) evident a regional aedema witch made necessary a fasciectomy &the wrist. she also developped necrotic delimitted areas that included her first and fifth fingers. she was discharged from the picu after 13 days and a week later she went to the operating room where an amputation of the distal phalange of the fifth finger was made. the etiolgy of this ischemic picture can't be attributed to the catheterization itself, but probably it should be due to some attempts in arterial puncture in its course, attempts that could be guess by the little marks noticed at the elbow flexure when she was admitted. long maintenance of cardiovascular funtion by assisted ventricular device with membrane oxigenator hermana tezanos mt, pilar orive j. latorre garcia j, lizarraga azparren ma lopez bay6n j. ucip. hospital de cruces. bilbao. spain a little girl of three and a half years came to the intensive care unit from the operating room. affected of great vessels transposition with a pulmona~' banding corrected vith rastelli technique, it became impossible to discharge her from the by-pass circulation. it was assumed that this factt was due to a transient myocardial disfunction dfter the surgeon x~ent through the technique and found no wrong step in it. at the picu admission she had an ejection fraction belox~ 20% (echncardiografy), and was manteined on veutricular assistance with a circuit consisting ofa plate's membrane oxigenator, a bio-medicus pump and pvc rubber tubes, with monitoring of pulmonary', right and left atrial pressures. initially she needed a ventncular support of 1.2 l/min with dopamine. dobutamine and adrenaline; gradually the ejection fraction rose to 60% aliowing a decrease in the mechanical and inotropic support. the organic function ,.'.-as preserved but x-ray of the thorax showed images of pulmonary aedema with an increase in the 02 needs until a fi02 of 0.4. even though she was placed in a transplant program, she was remouved when the ejection fraction rose to 50% on the 1 lth da 3 of the evolution. there were no signs of infection (profilactic coverage). on the fifhteen postsurgery day. with a good cardiac sound and an ejection fraction of at least 60%. it was considered the withdrawal of the ventficular support, but unsucessfully despite the increase ofinotropic drugs, so she died. we think that our patient is a good example of the posibiti~ to mantain x~ith ecmo during a prolonged period of time a potential transplant receiver without major complicatios. joan wierema; ma0)an mourik. sophia children's hospital, department of pediatric surger,j, rotterdam, the netherlands. the success of an ecmo program is heavily determined by the organizational structure and the daily availability of both well trained nurses and physicians. until now there is no general guideline to determine the optimal training schedule to set up an ecmo program. objective: to evaluate the set-up of an ecmo program in an area without direct availability of perfusionists of cardiothoracic surgeons. description of the progress: during the preparation of the ecmo program which started november 1991, different phases are identified. first phase, acquisition of experience by training abroad of one physician, staff member of the icu, physician acting as a fellow and one icu nurse with theprimary task to serve as an ecmo coordinator and train the nursing start. in the second phase 12 animal experiments were performed by 2 paediatric surgeons, 2 staff physicians, 2 fellows and 12 nurses. third phase, start of the actual ecmo program, priming by one trained nurse and ecmo fellow, daily care of the patient by 2 nurses, 1 icu nurse taking care of the patient related activities, 1 ecmo trained nurse taking care of the circuit. fourth phase, transition of the tasks for priming the system from the ecmo fellow to the ecmo nurse and changing daily care for the ecmo patient now including the circuit by 1 trained icu nurse with special legislation for ecmo. ongoing training of 6 to 8 nurses working for at least half a year in the icu. during an ecmo run direct contact between the ecmo nurse and one of the staff members; patient data: in a 4 year period 52 neonatal ecmo cases were performed with an overall mortality of 74%, ranging from over 95% in meconium aspiration to 45% in congenital diaphragmatic hernia. in 7 other patients pediatric ecmo (age range 1 month -14 months) mortality was 50%. conclusions: ecmo can be established without perfusionists or cardio surgical back up once a predetermined training schedule is available, resulting in comparable results with the international ecmo registry both for neonatal as well as for pediatric cases. mechanical ventilation at home makes normal development of the child with chronic respiratory failure possible. the parents must be encouraged to accept the treatment at home; therefore, they should be enabled to take care of their children all by themselves and the continuous professional support has to be offered to them. it must be stressed that the nurse is the link between the diseased child and his family introducing them into the process of the medical care at home. all the equipment needed for the mechanical ventilation of the child at home (ventilator, suction device, pulse oxymeter, oxygen tanks) has to be provided before the child is discharged. it is paid by the national insurance company. the maintainance service workers will make the necessary installations available and they will renew oxygen and compressed air weekly. the ventilation assembly must not interfere with the childs activities. the related dispensary and nursing service should be notified of the child's condition and the list of the required material (suction tubes, cannulas, desinfectants etc.) has to be made. our experience is so far -from the nursing and medical point of view -satisfactory, however the social status of the parents needs further regulations, omphalocele. mourik m, sophia children's hospital, department of pediatric surgery, rotterdam, the netherlands. in patients with (giant) omphalocde operative closure is impossible due to the lack of intra-abdominal space and a variable degree of pulmonary hypoplasia. consequently conservative treatment allowing epithelialization is the treatment of choice with a high risk of infection and/or sepsis due to a sometimes very long stay in the icu. this conservative treatment consists of daily application of an antibiotic containing powder on the cele which is covered by sterile gauzes. furthermore the enteral intake is started as soon possible. objective: to evaluate whether this treatment renders a high risk for serious infections. patient characteristics: in a 10 year period 23 patients with a giant omphalocele were admitted; mean birth weight 2800g (1500-3990g) mean gestational age 38.5 (33-42 weeks). overall mortality 6 (19%). 11 patients had to be ventilated for a median duration of t6 days (2,5-164). results: patients were discharged at the mean of 68 days following birth (17-260) following complete epithelialization of the omphalocele. following the initial stabilisation period parents were involved in daily care of all patients even during artificial ventialation. enteral intake was started at a median of 2 days (1-18). colonization of the omphalocele was observed within 2 -4 days in all patients. although infectious periods were observed at regular intervals, sepsis was the primarily cause of death in only 3 of the 6 patients who died, in conclusion: nursing care to prevent sepsis in patients with (giant) omphalocele is feasible for a long period of time and can be performed with simple measures, since june 1994, we have been using nasal cpap in the treatment of respiratory disorders in newborn infants (severe apnea syndrom, tachypnea, hyaline membrane disease). in newborns presenting hyaline membrane disease, we have used cpap and administered surfactant therapy to 25 premature babies out of 70. clinical data were : mean ga : 30.8 + 23 weeks. mean bw : 1686 + 551 g. mean administration time was 11 hours. before administration, mean fio2 was 0.4 + 0.07, mean pao2 6.2 + 1.8 kpa and mean a/ao2 ratio 0.21. all babies were intubated for administration of surfactant (curosurf) and were extubated after half an hour to 3 hours after administration. this treatment failed for seven babies and they were ventilated by oscillation ventilation ; all babies survived. complications : we have observed pneumothorax in 2 cases and 1 cerebral hemorrage. mean duration time of nasal cpap was 71 hours for the 18 babies without assisted ventilation. nurses in charge of babies with nasal cpap should be aware of neonatal care, of the possibility of surfactant administration and of complications during this type of treatment. therefore nurses should know very well the use of nasal cpap, the monitoring during this treatment, the fixation of the system on the baby, nursing of the babies during this treatment and finally should take care of the baby physical and psychological well being. as a conclusion, nurses in intensive care neonatal units should know the possibility of treatment of rds by nasal cpap and should be aware of baby-nursing. emco in the postcardiotomy infan_t : a simplified circuit and reduced management grillet lsabelle, bosson christa, goelz andrea, helmer pascale, icu nurses, tschaut rudy perfusianist aldo castaneda institute, clinique de genolier, suisse to improve postoperative survival of infants with repaired cardiac tessions but severe residual cardiopnlmonary dysfunction, emco was used in 4 infants, ranging in age from l0 days to 9 months and in weight from 2,8 kg to 7,8 kg. diagnosis included tga / intact ventricular septum (2) (both subiected to rapid arterial switch operation because of unsuitable lv function) and tga +vsd (2). "['he emco circuit included a sarns roller 15ump, a medtronic minimax plus hollow fiber oxygenator and tubing (1/4 -1/16 inch) with bioactive surfaces, an arterial canula 10 fr., 2 venous canulas : right atrial 20 fr, left atrial 12 fr., an air oxygen mixer and a sarns heater. the act was kept between 180-200 sec. after the infant was connected to emco by the surgical team and the perfusion technologist, the patients were being cared for and controlled exclusively by the 1cu nurse assigned to the patient and the physician on call for the icu, pump flows of 100-150ml / kg / mln were targeted to insure an adequate urine output, a brisk capillary fill and physiologic left and right atrial pressures. sodium nitroprusside was used to control systemic hypertension while all other inotropic drugs were discontinued. inspired oxygen fraction on the ventilator was reduced to 0,25 at a respiratory rate of 10-15 breaths per minute. body temperature was maintened close to normal. when the heart started to eject again, inspired oxygen was increased to about 0,40. the preparation for weaning from emco was done by the icu nurse: including oxygen fraction and ventilator rate. weaning from emcowas coordinated by the medical / surgical team and the perfusion technologist. the duration of emco in the 4 patients ranged from 2 to 9 days. three patients (75%) were saccessflly weaned from emco. one of the three died within 24 hours from neurologic complications, unrelated to the emco. the other 2 patients ( both with tga + vsd ) are long term survivors and are doing well. neither the survivors nor the patients who died bad hemorrhagic complications, despite the fact that 3 of the 4 were placed on emco because of failure to wean from cardiopulmonary bypass. the simplified emco circuit worked reliably throughout this experience and no complications occurred nor could any shortcoming be ascribed to the use of this reduced, cost effective management team. 10 years of praciical experience wiih exiracorporeal membrane oxygenation (echo) -the viewpoint of the nursing siaef monika schindler in 1985 a training program was started for the introduction of echo in the kinderklinik of mannheim/germsny. 2 years later (1987) the first european echo patient, a 3 days old newborn baby, ~as treated successfully in our institution. with echo, more treatment modalities ~ere available after failure of conventional therapy in eases of severe lung diseases like meeonium aspiration syndrome, congenital diaphragmatic hernia, sepsis/pneumonia, primary persistent pulmonary hypertension of the newborn and -in infants and children -ards (acquired respiratory distress syndrome) due to multiple underlying illnesses. in 1989 the first ards patient, a 5 years old girl, was treated successfully ~ith ecmo in mannheim. until now 137 patients (age: i day -10 years) were treated with echo in our institution; 111 additional patients were transferred to us from other institutions for therapy with echo, but could be treated ~ieh alternative moda]ities of therapy like improved conventional ventilatory support, high frequency oscillatory ventilation (hfov) and inhaled nieric oxide (no), unexpectedly the more ~ide-spread use of hfov and no -also in other ~ntensive care units -did not lead to a reduction of the echo frequency in our hospital. due to this long ecmo practice, which is helpful in many critical situations, a certain routine came up in the nursing staff of our intensive care units; but ecmo continues to be a maximum strain for all co-workers and implies an high personnel and material/financial-expense. but in our opinion the improved survival rates in severe respiratory failure-(70 % in neonates and 50 % in infants and children, estimated survival rates under continued conventional management: < 20 %) by the use of echo justifies this expense, even in times of less financial support for public health. • analyse the differences between each urdt. method : ni criteria were defined by the rt~a ped group according to cdc criteria. all data were collected by a nursing and medical team. data 3vere dealed with epi info software. all u-dections data were validated by an external mvestigatol : 4 525 patients who stay more than 48 hours in an icu were included over a 14 months period. 68 % were newborns, 19 % infants and ,13% children. 371 ni were idenlified among sll patients, the overall ni incidence rates was 8,2 % and 5,9 person day, septicemia was the first cause of ni (50 %). staphylococcus epidermidis were isolated in 60 % of septicemia cases. pneumonia was the other main ni (41%) with gram negatwe bacille isolated m 53 % (40 % of them beei,ng fseudomonas) accordmg to age, the septicemia mcidence rate varied from 6,8 ~., to 10.9 %,, catheter/day and pneumonia incidence rate from 3,9 %, to 7,3 % ventilation/daywith the lowest rate for newborns. : this survey was possible thanks to the collaboration between laboratories, bacteriologist, physicians and nurses, and allowed each concerned unit to work together in~tead of every one in his own field. 'lhe results of this survey bring changes in attlhades and empower the different team work the next step is to analyse the ditferent nurses procedures of indwelling central venous catheter in each unit ar)d implement a ni quatity care. program in all nicu anti picu. v~,~jl~j~'v~y : new born in~ra-v~6nous drugs ~md lheir pr~taration mcounter a lot of risk ~)f utters, due to inadapted conditionn~ng to podia)eric and minimal amount injec~.~d, th~ study t~ed to evaluate degrees of do.~age er~o~ dye tt) dru~ preparaek, n i~ nc,:,~atol~gy and to come out w~th ~ method of dihttion able to reduce risk to minima (errors _< 5 %) at the ~eme t~me, ¢o~,~x-tueaces on cost were studioj methnd~ : amikacine was choosen because this antibiotic is regularly ~,~l in neonatology unit and its dosage is easily used by standardiaud method, 30 amikacine doses (15, 23 mg doses and 15, 12 mg doses) were prepared" by 15 nurse~ form 50 mg for 1 m) vial. each dose w'a~ diluted to obtain 2 mi volume, tallowing usual ward method, for each dilution, amikacine con<entrati('m and it,s volume was measured and used equipment for preparation wa~ regi~tred. 10 "first s01utj(m" were prepared diluting ~t'~ mg amikacine pouder inn 51) ml glucnse solution bag (viaflex baxter bag with transfer cap), from tho~e "first solution" 15 doses at 23 mg and 15 doses of ~2 rng were prepared, amik~cine coc, cenir,)tmn for each preparabon and their volume were measured, ai,~n ntttl~,ber of seringe were tounted. first soiuhon staeiiity was conrroieo aher one week conservation at + 4'>c. amjkacine concentration were measured by fluorescence process (tdx abbott) after sample dilurion. on a 10 mg/l sample, tovhnical reliahility show~ > 9~ % of result mpmductlon and < 5 % of variation due to dilutions. results : when amikacine injection werv pro.pared from araikacme 5/) mg for 1 mt vial > 10 % do~ge, ermr~ were found in 19/40 cases ; ~ 30 % in ,t1,to cases. if preparation is done from amikacine "~it'st soltltion", les.--concenvr~tcd, it i~ more preci,,,e and only one dosage error ~ 5 % (6,3 %} is found in eli 30 studied doses. in add)inn to )hal if 10 doses were wep,m-'d from one "first soiatiol~' bag, the cost economy sl~ouid b~" of 32 fr~, and ii 20 dos~$ were prepared tram the same bag the saving mtmey should be o{ i72 its .cencluslon : .ur survey shows th~t h' ntu)nato|ogy the u~ of a "first sohation which can be kept fi~r one week is enable to reduce dosage erroes and i~ co,~tsavmg, regarding [,v. admimst'rahon method the survey is still on, introduction: so-called vein of galen m~iformations ale rare in~racranial embryologycal anomalies, repl~senti~g tess than 1 of symptomatic intracranied artefiovenoas l~alform~tions. the spontlneous prognosis is ~s~u~lly fatal, because of cardiac frilure due to left-to-right shunt thrq~ugh the fistula. recent developments of new techniques of treatment of the malformation and its cardiac consequence have led to a revolution in the practical approach of children w~th galen malformation. our fukfose is to contribute, with our persoaal series of 7s newborns and infal~ts admitted in our unit after endov~,scular embolization, to a better management of these children. such a management requ!res a rnultidisciplinary approach. intensive care are required prior to embollzation for patients with cardiac failure or cardiogenic shock and after cmbolization in order to insure cardiac and cerebral hemodyna.mic stabilities. this overlooking suppose for the nursing team to understand: prior to embolization : heart failure and cardiogenic shock. after cmbolization : evaluation of neurological and hemodynamic consequences of this proccdure, without forgetting the nursing and psychologic aspects, in concl'iision, this last ten yerrs, these new approaches give to the patients and their famitiy a good reason to hope a total recovew, in our exl)erience, the global mortality is 9 % aad 66 % of children #j-e neurologically normal after embolizafion, ii ii~ i ~ii i ii i i l i iiii~ i ~i iii i background: venous oxygen saturation (svo z) reflects the residuai oxygen after tissue oxygen extraction and represents the relation between tissue oxygen supply and demand. we studied svo 2 and arterial lactate during progressive isovolemic anemia to assess the relation between svo2 and tissue hypoxia. subjects: ten 8-10 day old anesthetized ventilated piglets sao 2 and svq were measured continuously by a fiberoptic catheter (oximetrix, abbott lab.) in the carotid and pulmonary a~epy tissue hypoxia was confirmed by a reduced vo, and an increase in lactate. conclusion: svo 2 reflects better a reduced dp obtained by progressive anemia surfactant replacement improves gas exchange in early-stage adult respiratory syndrome (ards) [1,2], but not in late-stage ards [3] . we report the first case of successfull treatment of ards after repeated instillation of surfactant.a ten year old boy, weighing 32 kg, presented with hemorragic shock. biphasic-positive-airways-pressure ventilation was performed (evita ii, dr~ger, germany). he had recieved nine units of packed red blood cells and underwent surgical exeresis of two bleeding gastric ulcus. post-operatively, a cardiac arrest required cardiopulmonary resuscitation for three minutes. hemodynamic status was subsequently stabilised. the chest-radiograph showed infiltrates of both lungs without signs of cardiac failure. on the third day, the patient became severely hypoxic with a pao2/fio 2 ratio of 30. gas exchange was not improved by high ventilator settings. peak inspiratory pressure (pip) and ventilatory rates were 40 cmh~o and 18 breaths/min respectively. inspiratory:expiratory time was 1:1 and the positive end expiratory pressure (peep) 8 cmh20. after increasing the peep level to 11 cmh20, we instilled over 2 minutes, 80 mg/kg of porcine surfactant (curosurf, serene france), in two equal volumes in both main bronchus,the spo~ rose to 97% within 15 rain, the fie 2 could be reduced to 0.6. twenty four hours later, gas exchange worsened again (pao2/fio2 ratio 90). we increased the peep from 8 to 11 cmh20, and instilled a second dose of surfactant (60 mg/kg). again, fie 2 could be reduced within 15 minutes (spo 2 95; fie 2 0.6.). the patient was weaned from the ventilator and extubated on the tenth day. follow-up at four month showed normal lung function.we demonstrate improvement in oxygenation after repeated exogenous surfactant administrations. we assume that in early-stage ards, surfactant may potentiate shunt-reducing effect of peep as it has been demonstrated in experimental model of ards [4] , and allow decrease in fie2. in case of secondary deterioration, we think that a second dose of surfactant should be administered. 1. weg jg, balk ra, tharratt rs, et al. ,lama 1994 : 272: 1433 -8. 2. spragg rg, gillard n, pdchman p, et al. chest t994: 105: 195-202. 3. haslam pl, hughes da, mcnaughton pd. et al. lancet 1994 343:1009 -11. 4. huang yc, caimulti sp, fawcett ta, et al. jappl physiol 1994 76:991-1001 43% (ref) . the aim of this study was to verify these data: patients/~lethods: all pts admitted to our multidisciplinary nicu/picu in 1995 were included if they were in respiratory failure recruiting conventional mechanical ventilation (cmv) with peep >_ 6 and 'fig2 -:2 50% or high-frequency oscillation ventilation (hfo) with mean airway pressure _> t8cm h20 for 12 or more houm. diagnosis, maximal ventilatory parameters, barotrauma, organ/ system failures, mechanism of death and glasgow oulcome scale (gos) 1 and 6 months after study entry were prospectively collected. results: 685 patients were admitted to the unit, o1 whom 337 required mechanical ventilation for a mean duration of 4.0 days. overall mortality was 5%, 22 patients fulfilled study criteria. 17 survivors had gos 5, 2 pts with preexisting neurological impairment survived with gos 3. neonatal diseases included hyaline membrane disease (7), meconium aspiration syndrome (4) and cardiovascular surgery (1), pediatric diseases included bacterial (1) and viral (5) pneumonia, aspiration (1) and cardiovascular surgery beyond the neonatal period (3). 1990 -1994) . patients and methods: cefotaxim was used as a prophylactic agent in 43 patients in life threatening situations (e.g. multitrauma, neurosurgery atc.). more than 85 % children required cefotaxim for the treatment of severe infections (epiglotitis, meningitis, sepsis, pneumonia mainly in immunodeficient and neutropenic patients) in monotherapy or in the combination with the other antimicrobial agents. results: cefotaxim as a prophylactic drug was successful in all 43 cases (100 %). the effectivity of treatment of infections was 82.8 % (313 patients). the change of antibiotic therapy required 9 patients (2.4 %). 40 patients (10.6 %) died, but only in 12 of them (3.2 %) the obduction confirmed infection. conclusion: we conclude that cefotaxim is very effective and safe antibiotic and represents "golden standard" in the treatment of severe infections in childhood. in order to improve nursing quality, we recently adapted nursing care to the "five nursing functions" (activities of daily living, accompagnment in crisis, treatment, prevention and research) as described by the swiss red cross in accordance to the new educational guidelines of the european community, the aim of this study was to document complications of "treatment nursing function".methods: all treatment complications were prospectively collected by the nursing and medical staff. the nursing staff included patient (pt) name, time of occurence and exact description of complication, proposal for prevention and information of parents. the medical staff reported type of complication together with pt information, diagnosis, medication, treatment and interventions, outcome and referral, all complications were discussed in monthly meetings including nursing and medical staff.results: from january until december 1995, 685 pts were admitted to the picu/nicu for 3233 nursing days (81% of total bed occupancy). 337 pts needed endotracheal intubation for an average of 4.0 days and 47 pts required nasal cpap. 26 complications in 21 pts were noted (1 per 26 pi): inadequate check-up of equipment 11; accidental extubation 4 (1 in 85 intubated pts); bedsores 3; false drug dosing 2; wrong drug 2; umbilical bleeding 2; wrong transfusion setup 1; nasal septal necrosis 1). there was no mortality due to these complications. exact documention of treatment complications and their meticulous discussion within the medical and nursing staff may improve "treatment nursing function". however, documentation and evaluation of nursing within all "five nursing functions" will be nessecary in order to achieve optimal nursing care. cardiac output determination by thermodilution, using iced injectate has been shown to be valid and reliable in pediatric patients. it has been demonstrated in adult patients that there is no difference in cardiac output values when using room temperature injectate as compared to iced temperature injectate. the purpose of this study is to examine the effect of injectate temperature on cardiac output values in pediatric patients. our study consisted of sixteen pediatric patients who had oximetric thermodilution catheters in place after cardiac surgery and who had cardiac output determined using both iced and room temperature injectate. with each patient, cardiac output was measured once on the day of surgery and again the following day. in each case cardiac output was measured using both iced and room temperature injectate. statistical analysis included a two-way, repeated measures analysis of variance for each individual injectate administered and no significant differences were found in cardiac output. no statistically significant differences were found between groups with regard to the order of injectate administration or volume of injectate used (i,e., 3 or 5 cc's). the correlation coefficients between groups for cardiac output measurements at each injectate administration time, and for the average measurements across times, ranged between 0.81 to 0.94 (p < .0005). preliminary data analysis suggests that cardiac output measurements for children are not effected by the temperature of injectate. a lenghty stay at a paediatric intensive care unit will always have sideeffects on a child's well-being and will put a high strain on the parents. in order to minimize the side-effects longterm intensive care unit opened in 1990 at the childrens' hospital. admitted children are all ~ongterm-ill and technically-dependent and the ventilatory support can alter from a tracheostoma to cpap or portable volume ventilator. nutritional support is applied by gastrostomies. a homelike atmosphere surrounds the children, they share a dormitory, a living-room and a dining-room the main purpose is to send the child home with or without technical equipment. this can only be implemented by giving structured education (theory and practice) to all categories involved. the multi-disciplinary team consists of one anaesthesiologist, head nurse, clinical specialist, rn nurses, nurses, one habilitation doctor, one social worker and therapists. twenty-four patients have been admitted to licu during these six years. length of stay was from one day to four years. four are presently staying at the trait. the assessment of pain in children (0-3 yrs) is still difficult, because children of this age have limited language and cognitive skills. to standardize the assessment of postoperative pain and distress in the intensive care unit an observational mstrument was needed that met several criteria. it should be easy to use in daily routine care. be suitable for the i.c. situation, and in children of 0-3 hrs of age. the comfort scale, an observational instrument designed to assess distress in infants in i.c. units, met these criteria. to accommodate the use of the comfort scale in the i.c. units and in research, nurses should be trained to use the scale. an additional requirement was that the inter-rater reliability should be sufficiently high, (cohen's kappa > .60). objectives: 1) to introduce the comfort scale in the i.c.u.; 2) to examine whether this instrument can easily, be incorporated into routine care; 3) to investigate the inter-rater retiabtlity. methods: the comfort scale is an 8-item instrument specifically designed for use in pediatric i,c, units and contains both physiological items (heart rate, blood pressure) and behavioral items (e.g., alertness behavior, calmness/agitation, body movement, facial expression respiratory response, muscle tension). the observation period is 2 minutes. the scale is supplemented with an item on crying tbr children who are not mechanically ventilated. groups of 8 t.c. nurses were trained by means of video's and observations at the wards. after the training, each nurse completed 10 scores with other nurses, after which the cohen's kappa was computed. when the kappa's for the items met or exceeded our .60 criterium, a new group of nurses was trained. results: to date, 30 nurses have been trained. nurses find the comfort scale easy" to administer and a valuable addition to routine care in the i.c. unit. the cohen's kappa's were higher than .60 for all items that the inter-rater reliability was high. the comfort scale is feasible in postoperative care in the i.v. and is considered a valuable instrument to improve and maintain high postoperative quality of care in the i.c. unit. introduction:children with neuro-muscular disease are believed to have a higher resting energy expenditure (ree), because of their increasedwork of breathing.the influence of nocturnal nasal mask ventilation on energy metabolism and nutritional state of these children has not been studied so far.objective:l,ls the ree inereased?2.1s there an influence of nasal mask ventilation on the ree?3.what is the nutritional state?4.what is the estimated total energy expenditure(ete) in relation to the caloric intake? methods:a pilot study of 4 patients(12-16 years) .the following measurements were performed:l.anthropometry.2.bioelectric impedance-3.ree was measured by indirect calorimetry during the day (in bed) with and without nasal mask ventuation,ree was compared with predicted ree according to schofield(pee),4.caloric-intake and activities were recorded during 48 hour before measurement.5.total energy expenditure was calculated as follows:measured ree x estimated activity factor. results:tin all children weight for height was too low, <p3.2.aii children had a low % of fat.3.according to the healthy group, ree is not increased.4,nasal mask ventilation lowered ree in 2 patients, in 1 patient we measured a clear decrease.conclueion:l.anthropometry and indirect calorimetry were helpful in evaluating the nutritional state.2.there is no increased ree,in t patient there is clearly effect of nasal mask ventilation.3.when caloric intake was related to ree and level of activity 2 patients had too low caloric intake.4.the influence of nocturnal nasal mask ventilation and nutritional assessment should be evaluated longitudinally. increasing awareness that the neonate can percept pain and suffer following surgical procedures, has major impact on pain management. in our unit continuous morphine infusion (t0/~g/kg/hr) was introduced from january 1990 as a standard medicatton following surgical procedures. before 1990 morphine was given as a bolus (i.m., i.v., rectal) of 100/~g/kg up till four times a day. repeated gifts of morphine were only gwen following observation by the nurse that the child experienced pain. aim of the study: to evaluate pain management before and tbllowing introduction of a continuous morphine infusion for postoperative pain in our icu. patients and methods: term born babies with isolated oesophageal atresia and tracheo-oesophageal fistula were included in the study. the total amount of morphine (/~£/kg/24 hours); number of days morphine was given; duration of artificial ventilation was evaluated in a case control study. res ults: 1985 -1990 1991 -1994 central venous catheters (cvc) have been increasingly used also at our picu, university medical centre ljubljana. besides advantages, the cvc has brought numerous risks of complications; the catheter sepsis occurs most often and can be fatal for the patient. for example, in 1995 we dealt with 400 children aged 0 -12 years or in other words 1.14 cvp per a child treated at our picu. a specialized unit for preparing parenteral natrition started to function in the university medical centre pharmacy in 1977. after that we equipped a special room for preparing other necessary infusion mixtures and organized a team of nurses -"catheter nurses".their tasks comprise: managing cvcs, replacing and installing infusion systems and mixtures made at pharmacy, preparing drugs and some necessary infusion mixtures in special room, permanent training and co-operation with related disciplines, pharmacy, epidemiology, microbiology, etc. all registered nurses at picu have been trained for this work. the routine is as follows: in the morningafter the doctor's visit, we first check the cvc and change the dressings; according to the nurse's observations, the doctor decides whether there is a need to remove or replace the cvc. if the catheter site is inflamed, swollen, or purulent, a swab is taken for microbiological culture and the area cleaned. in case of systemic infection signs, cvc has to be replaced, blood culture taken and the infection treated with antibiotics. the dressings are always changed by two tmcatheter nurses together. afterwards the infusion mixtures for the next 24 hours are prepared for each child. the infusion mixtures which will not be used immediately are kept on +4°c; before application they are, of course, warmed to at least room temperature. finally the "catheter nurse" supplies the cvc with new infusion mixtures made at our pharmacy. our observations show that such work requires responsibility and is demanding; but our pemanent care lessens the number of complications. thus the described scheme fulfilled our expectations. perl-0perative management of neonates with hypoplastic left heart syndrome muir warren, blondel maryse, foltz rhonda, farine martine, icu nurses, the aldo castaneda institute, clinique de genolier, switzerland both the pre-operative and post-operative physiology of patient with hypoplastic left heart syndrome is critically determined by the puhnonary-tosystemic resistance ratio. a high ratio results in minimum volume work for the right ventricle but is at the expense of important hypoxemia. a low ratio is reflected in minimum hypoxemia but an unfavorable increase in excess ventricular volume work. a resistance ratio of approximately 1 results in an arterial oxygen saturation of 75% -80%, a qp/qs of 1, and ventricular volume work only twice normal. this latter physiology appears best for oxygen delivery and systemic perfusion with tolerable volume work. carbon dioxide (co2) in the inspired gas is known to increase pulmonary vascular resistance without significantly influencing systemic resistance in the range of 1 to 20 tort partial pressure. hyperventilation with no co2 in inspired gas decreases pulmonary vascular resistance. thus, managing co2 may be useful to modulate the pulmonary-to-systemic resistance ratio, in the tast 16 months, 20 patients with hypoplastic left heart syndrome were managed by a norwood procedur e. 10 patients with endotracheal entubation pre-operatively had co2 added to inspired gas to modulate the pulmonary-to-systemic flow ratio. 17 patients had co2 added to inspired gas post-operatively. all but 1 patient had characteristically large pulmonaary-to-systemic flow ratio with oxygen saturation mean 90 % preoperatively, even in those patients receiving co2, co2 was accompanied by elimination of metabolic acidosis pre-operatively. the most comlnon partial pressure of co2 added post-operatively was 14 torr. po2 ranged from 25 mmhg to 40 mmhg. all surviving patients developed a metabolic alkalosis (10 mean) when treated with co2. there were 4 deaths, one from drug toxicity, one from excessive post-operative hemorrhage, two from sepsis or mocardial insufficiency. only one patient benefited from catecholamine support which can increase systemic resistance unfaborably. in this experience co2 was felt to be an important adjunct in some patients pre-operatively and most patients postoperatively to faborably modulate the critically important systemic-topulmonary flow ratio, to maximize oxygen delivery and systemic perfusion, while minimizing excessive right vantricular volume work. key: cord-005814-ak5pq312 authors: nan title: 8th european congress of intensive care medicine athens greece, october 18–22, 1995 abstracts date: 1995 journal: intensive care med doi: 10.1007/bf02426401 sha: doc_id: 5814 cord_uid: ak5pq312 nan objectives: evaluate the levels of tnf, il-6 and pai-i in different moments of the ards and the possible relationships among them. methods: 23 septic patients with ards were studied. also significant differences for: tnf, pai-i and il-6 in septic patients and both evaluations of ards with control gropup; pai-1 between septics and 2nd evaluation in ards, and between the ist and 2nd evaluation in ards; il-6 between septics and both evaluations in ards; and il-~ in both evaluations in ards patients in relation to mortality. conclusions: i) elevations of tnf, pai-i and il-6, with clinical signs, are suggestive of infection; 2) the persistent and progressive elevation of pai-i with any clinical criteria may suggest evolution to ards; 3) due to its own kynetics, il-6 takes part later in the acute phase, its levels being related to the magnitude of the injury in the tissues. objectives: the influence of long-term volume therapy with different solutions on plasma levels of circulating adhesion molecules was studied. methods: according to a randomized sequence, 30 patients with sepsis secondary to major surgery exclusively received either hydroxyethylstarch solution (10% hes, mean molecular weight (mw) 200,000 daltons, degree of substitution (ds) 0.5) or human albumin 20% (ha) for volume therapy for 5 days. plasma levels of circulating (soluble) adhesion molecules (endothelial leukocyte adhesion melecule-1 [selam -i] , intercellular adhesion molecule-1 [sicam -i] , vascular cell adhesion molecule-1 [svcam -i] , and p-selectin ) were serially measured on the day of admission to the intensive care unit (='baseline ' value) and during the next 5 days. results: selam-i, sicam-i, and svcam-i plasma levels were markedly higher than normal at baseline in both groups. in the hes-patients, selam-j decreased to normal range, whereas it further increased in the ha-group (from 89• to 106• during the study period, sicam-i and svcam-i plasma levels remained unchanged in the hes-patients, but further increased in the ha-group (from 626• to 1,329• sgmp-140 increased significatly only in the ha-group (483• to 683• only pao2/fio 2 was significantly correlated to plasma levels of adhesion molecules. conclusions: sepsis is associated with markedly elevated plasma levels of adhesion molecules indicating endothelial activation or damage. by long-term volume therapy with hes, these levels remained unchanged or even decreased, whereas volume therapy with human albumin did not have any beneficial effects on soluble adhesion. central venous catheters are frequently used in the care of the critically ill patient. the incidence of catheter related sepsis varies in the literature. we investigated the occurrence of contamination and sepsis compared to results of the epic study as part of quality assesment in our intensive care unit. from january until august 1994 all removed central venous catheters were examined for microbiological culture. the patients who showed signs of sepsis were also registered. the results of the contaminated catheters and septic patients were compared with results from the epic study. during the 8 month period ,2059 patients were hospitalized on our intensive care unit. 230 central venous catheters were examined for microbiological culture. 118 specimens appeared to be possitive (51%). 13 patients showed clinical signs of sepsis. the incidence of sepsis due to contaminated central venous catheters was 13/118 (11%). the incidence of sepsis due to the presence of all central venous lines was 13/230 (6%). the microorganisms responsible for the sepsis syndrom were : stapylococcus aureus (n=5), escherichia colt (n=7), others (n=6). in the epic study the percentage for sepsis on the icu was 17.6% for the netherlands and 17.8% for europe. despite a high number of positive culture from removed intravascular lines, a low percentage of sepsis was seen compared to results of the epic study. we recommend routine bacteriological culture of all removed central venous lines and recommend to look at colonization and sepsis due to intravascular lines as a measure of quality control in the intensive care unit. objectives: prognostic assessment of simplified acute physiology score (saps) in granulocytopenie patients with septic shock (ss). methods: the medical records of 59 admissions to an intensive care unit (icu) of granuloeytopenic patients with ss are reviewed. fiftytwo patients had haematological malignancies. seven patients had aplastie anaemia. patients were categorised as survivors (discharged from icl 0 and non-survivors (died in the icu). saps index was calculated for patients daily during their stay in icu. all patients were severe granulocytopenic (total white cell count less than 0,5 ]09]1). results: five patients (8,5%) were discharged from icu. fifty-four patients died in icu. non-survivors had saps on admission higher than survivors ( 20.9+4.6 and 16.5+3.0, respectively, p<0,01, mann-whitney u test). no patient with a saps greater than 20 survived. mortality among the 27 patients with saps from 9 to 20 was 81,5%o. the evolution of ss was rapid. the mean stay in icu among non-survivors was only 56 hours. an analysis of the saps index on admission of non-survivors showed an inverse correlation with the duration of their stay in icu (r=-0,52, p=0.001). all survivors recovered from granulocytopenia. they had normal white cell counts at the time of discharge from icu. there was inverse correlation in survivors between saps and white cell counts, when these parameters were evaluated daily. however, the saps index alone cannot be considered to be on individual predictor factor of mortality. patients who had failure of the malignancy to respond to chemotherapy and who had persistent granuloeytopenia died in icu despite saps index on admission and recovery from ss. conclusion: saps index greater than 20, failure of the malignancy to respond to chemotherapy and persistent leueopenia all point to a poor outcome of granulocytopenie patients with ss. introduction: antipyretics sometimes are used for fever control in febrile neutropenic patients with hematological malignancies(hm). we observed a dramatic fall of blood pressure(bp) and development of septic shock(ss) in some of the patients who received antipyretics. aim: to clarify can antipyretics provoke ss in neutropenic patients with infection. methods: retrospective review of medicat records of 52 neutropenic(wbc <0,5 109/1)patients with hm, admitted to the intensive care unit for ss, was performed. there was selected group of 8 patients receiving antipyretics shortly before a fall of bp. results: there was a definite causal relationship between receiving antipyretics and fall of bp in 4 from 8 patients. all patients had fever due to infection and had normal level of bp before receiving antipyretics. hypotension developed within 40 minutes up to 1,5 hours after administration of antipyretics. three patients received 0,5 g of metamisol and one 0,5 g ofparacetamol per os. in all cases we observed dramatic diaphoresis and the temperature fall to subnormal level (35.4+0.4~ accompanied'by hypotension. but in 8-12 hours the fever was coming back without blood pressure elevation. the fluid replacement was controlled by central venous or wedge pressures. there were required 1200+350 ml colloid and cristalloid solutions for volume loading. in spite of fluid administration the hypotension persisted and all patients required inotropic therapy. only one patient survived and is alive now. conclusion: it seems to us that our data offer to state that antipyretics administration can initiate ss in febrile neutropeuic patients with infection. objectives: to assess the agreement between cardiac output (co) measured by odm t and by 3 other methods used in icu patients. methods: we prospectively studied 12 adu t patients requiring hemodynamic monitoring with a pulmonary artery catheter. an esophageal doppler monitor provided measurements of co (odm), stroke volume and flow time (ft) used as an indirect evaluation of patient's volume status. patient hemodynamic status was evaluated by a modified fast response pulmonary artery catheter (baxter health care corporation, santa ana, ca), allowing co measurements by thermodilution 0"d) and an evaluation of right ventricular ejection fraction and end diastolic volume (rvef and rv-edv). in the last six patients co was measured by transthoracic echocardiography (echo) and oxygen consumption was measured by a deltatrack ii metabolic monitor (datex) allowing co calculation according to the fick formula (fick). the agreement between methods measuring co and their reproducibility, were evaluated by bland and altman analysis. results: agreement between co measurements is expressed as bias (d) and 95% limits of agreement (l of a = d_+2sd .15 td-fick -2.36 -8.06 to 3.34 fick-echo 0.60 -5.92 to 7.12 there was no correlation between ft and rv-edv. conclusions: although co measurements by odmil had the best reproducibility, the limits of agreement between the four methods tested were unacceptable for clinical purposes. further investigation is required in order to improve the accuracy of co measurement in the icu. phd, a. paltzev, v.bajbikov, b.dobryakov d.sc., a.ostanin phd, o.leplifia phd, h.chernykh phd munieip. hosp. n l, n 12; inst. of clin. immunol., novosibirsk, russia objectivies: efficiency of native cytokines used in the treatment of patients with severe surgical infections has been studied. methods: for two years 120 patients were treated with cytokine mixture (ssp) obtained by arterio-venous perfusion of swine spleen and contained the following cytokines: il-1, il-2, il-3, tnfa, ifny, gm-csf. results: ssp intravenous infusions were shown to accompany with mortality decrease from 23.4% to 12.5% in patients with abscessed pneumonia and lung abscesses and from 50% to 13% if disease course was complicated with sepsis. in patients with purulent peritonitis and sepsis efficiency of ssp was decreased due to endotoxieosis. thus, we used adoptive immunotherapy with mnc activated in vitro with ssp or recombinant il-2. intravenous infusions of such cells resulted in transformation of a pathologic process from destructive into productive one. moreover, clinical manifestations of sepsis were controlled in 81% and mortality was decreased from 46% to 19%. conclusions: the use of eytokines themselves as well as cytokine-treated lymphoeytes permits to control the disease and leads to the mortnlity decrease owing to stimulation of host defence mechanisms. background: although red blood cell transfusions (rbct) are used to increase oxygen availability in septic patients, several lines of evidence suggest that rbct may actually worsen tissue hypoxia. thus, rbct may negatively influence outcome of septic patients. objectives: to determine the association of 1) rbct ; 2) number of units transfused; and 3) mean age of the units transfused on the first day of transfusion with mortality of critically ill septic patients. methods: we prospectively identified patients who met strict criteria for sepsis syndrome (ss) seen in the icu of st. paul's hospital from 1992 to 1994 and excluded patients who died in the first 5 days after the onset of sepsis. we recorded clinical characteristics, multiple system organ failure score, and apache ii at onset of sepsis. then, we retrospectively recorded the total number and age of rbc units transfused during the first 5 days after onset of sepsis. overall 30-day mortality was 22%. results: the main results are shown in the table. the mortality of patients who received rbct was nearly double the mortality of those who did not receive rbct even after adjusting for severity of illness using apache ii. objectives: gastric mucosal acidosis is frequently observed in patients with sepsis. the aim of this study was to determine whether volume infusion using pentaspan| decreases abnormal gastric mucosal pco2 (pico2) in patients who have sepsis syndrome (ss) who have already been resuscitated using clinical endpoints. methods: we prospectively identified 5 patients who met strict criteria for ss, had a pulmonary artery catheter and a gastric tonometer in place, and pico2 > 50 mmhg. pentaspan| (500 ml) was infused in 30 rain. measurements of hemodynamics, hemoglobin, arterial lactate, blood gas analysis, and pico2 were performed before and repeated 30 miff and 2 hr after pentaspun| infusion. we calculated the pico2 -arterial pco'2 difference (pico2-paco2) and phi (using henderson-hasselbach equation). anova was used to assess statistical significance. results: all patients werereceiving adrenergie drugs. map was 73 :1:13 mmhg and lactate 1.2:1:0.6 mmol/l. pentaspan| increased ci by 22% (p<0.05) but did not change pico2 ( and increase m oxygen o* wery were simimny achieved in both groups. nevertheless, epinephrine was associated with a lactic acidosis and increased laetate/pyruvatemia ratio (l/p) that evoke a dysoxia rather than a metabolic effect. an higher gastric mucosal pco2 in the ep group compared to nor-rob suggests the hypothesis of an anaerobic production of co2 in favor of a splanchnic hypoxia. in both group, arterial ketone body ratio that reflects hepatic mitochondrial redox state, compared to a control group without shock was decreased but increased between 12 and 24 hours after restoration of arterial pressure. the association norepinephrine-dobutamine seems to be better for splanehnic circulation than epinephrine and should be used for dopamine resistant septic shock. moreover, the increase in arterial pressure with nor-dob improved gastric mueosal ph and hepatic mitochondrial redox state and argue to reconsider arterial pressure as a significant goal for resuscitation in septic shock. conclusion: significantly higher malondialdehyde and ghitathione levels and glutathione-peroxidase activity in group ns at the end of icu stay were related to mortality these findings indicate an increased generation of free oxygen radicals together with increased anfioxidant activity in this group and sapport the employment of antioxidant interventions in critically ill patients. oblecfives: to determine the role of nitric oxide (no) in the mechanism of septic shock induced by isolated limb perfuslen with recombinant tnfcr methods: we have measured tnfr~ and metebo~ites of no in 5 patients with signs ot septic shock following treatment with isolated limb perfusion for nonresectable soft tissue tumors and melanomas of a limb. perfuslen was carried out with melphalan (burroughs wellcome) and recombinant tnfcr (boehringer). tnfc~ was determined by specific radiometric assay (medgenix diagnostics), nitrate and nitrite were measured with a modification of the guess reaction ~. results: results are shown in the table. conclusions: during isolated limb pedusion with recombinant tnf~ very high levels of tnfcr were measured in arterial blood in 5 patients. they all showed signs of severe sepsis syndrome with shock from vasodilafion, probably due to leak of recombinant tnft~ from the peduslen circuit to the systemic circulation. tnfc~-induced vasodilation was not accompanied by a rise in serum no-metsbolites. our findings do not confirm the widely accepted theory, mainly based on animal experiments, that genera• of no is the key pathogenefic mechanism in septic vasodilafion 2, nor that tnfrt invariably induces forreafion of no. the precise mechanism of shock in these patients remains to be elucidated. references: 1. moshage h, kok b, huizenga jr, jansen plm nitrite and nitrate determinaiions in plasma: a critical evaluation. clin chem 1995: 41/6. 2. moncada s, higgs a. the l-argioine-nitrio oxide pathway. n engl j med 1993; 329: 2002 -2012 ec is a commonly used for prolonged, stable animal anesthesia. noting that the hypotension after iv lps was attenuated by ec, we hypothesized ec also protects against lps toxicity. sprague-dawley rats received ip saline (s), thiobutabarbita180 mg/kg (tb), or varied doses of ec, followed 2 hours later by bolus 30 mg/kg iv lps. 7-day survival is shown below: group: s tb ec(0.1gmikgi ec(0.sgm/kg) ec(i.2gm/kg) alive (n) t 0 0 3 9 ~ total (n) 10 s s 7 10 "signiflcant;y different from all other groups, p<0.0s 5/5 rats given lps followed 2 hours later by ec (1.2 gm/kg) also died. additional rats were treated with s (n=10) or 1 2 gm/kg ec (n=10) followed by 30 mg/kg lps, then sacrificed at 4 hours. blood glucose (bg, mg/dl),.hematocrit (hct), leukocyte count (wsc/mm~ platelet count (pltxl0~/mm3), bicarbonate (hco, mg/dl), gross bowel hemorrhage (bh, 0-4 scale) and lung myeioperoxidase activity (mpo, ~vmirvgm wet lung) are shown below ( we conclude that ec reduces the lethality and multiple organ toxit;~ty of lps. its diverse effects suggest asite of activity upstream from the cytokine cascade. these results are important for studies of lps which may use ec anesthesia and may have potential in the therapy of septic shock. [zo = 0 hz impedance (z; {dyn.sec.cm "5 }); zl = first harmonic z; zc = characteristic z; z1 ph. = t'trst harmonic phase angle {radians}; f, #, * at least p < 0.05 between fio2 0.4 and 0.12, fio2 0.4 and fio2 0. 4&no -0.46_+0.1 -0.26_+0.1 # -0.24+0.1 m -0.85+0.1 * -0.85+0.1 * -0.74+0.1 * -0.88_+0.1 * in hyperoxia, compared to dogs at the same q, minipigs had a higher ppa (26+1 rnmhg versus 16+1 mmhg; p < 0.01). hypoxia increased (ppa-ppao) at all levels of q by an average of 13 mmi-ig in minipigs and 2 mmhg in dogs. inhaled no inhibited hypoxia-induced (ppao-ppa)/q changes in both species. conclusions: we conclude 1 ~ that the minipig is an animal model of elevated pulmonary vascular resistance and impedance, and 2 ~ that hypoxia-induced alterations in pvz spectrum are due to changes of resistance in small arteries. objectives: 1) to determine the toxicity of ng-monomethyi-larginine (nma) administered by intravenous bolus to patients with refractory septic shock. 2) to investigate the biologic activity of nitric oxide synthase inhibitors in septic shock. methods: from august 1993 to january 1995, thirteen patients with vasopressor refractory septic shock received nma intravenously in escalating doses from 1 to 40 mg/kg. results: no hepatic, renal, gastrointestinal, or hematologic toxicity was observed at doses of nma as high as 40 mg/kg. significant biological activity was observed at all dose levels consisting of increased blood pressure (systolic blood pressure from 70.9 mm hg + 3.4 to 109.0 _+ 4.3 s.e.m., p=0.0004, systemic vascular resistance (430 + 57 to766 + 93 dyne.sec/ cm s, p=.002), and a decrease in vasopressor requirements. the magnitude and duration of these effect were dose dependent. decreased cardiac output (8.1 _+ 0.8 to 7.0 _+ 0.9 i/min p=.003) and increased pulmonary artery pressure (35.6 _+ 2.1 to 43.5 _+ 2.0 mm hg; p=.004) were also observed. no significant effects on heart rate, pulmonary capillary wedge pressure, or central venous pressure were observed. four of 13 patients survived for more than 28 days, 4 patients died of cancer complications (all 5 patients had maintained blood pressure for 24 h on nma) and 4 patients died of complication attributable to septic shock (mods, ards, dic, refractory hypotension), and 1 patient was unevaluable. conclusions: no adverse clinical effects have been observed in patients receiving bolus doses of nma as high as 40 mg/kg. the increased pulmonary artery pressures observed in septic shock patients is further augmented by nma and may limit the dose which can be administered by intravenous bolus. other schedules of drug dosing may attenuate this effect. glucose-insulin-potassium (gik) solutions have been shown to improve cardiac contractility and increase oxygen availability in experimental and clinical settings of septic shock. several mechanisms have been proposed to explain these effects including a direct improvemeut of the energy balance by glucose, a direct influence of insulin on cardiac performance or an increase in intravascular volume due to the hyperosmolarity of the solution. to explore the role of hyperosmolapity, we compared the effects of gik to those of a isoosmolar hypertonic saliue solutiou in endotoxin shock in dogs. methods : the study included 18 mongrel dogs (25• pentobarbitalanesthetized aud mechanically ventilated with air. thirty minutes after the intravenotls administration of 3 mg/kg of e. coli endotoxin, the dogs were randomized to receive a 2ml/kg infusion in 30 rain of a hypertonic (2895 mosm]l) solution iucludiug either a mixture of glucose 50 % with 750 u insulin and 2000 meq kcl/l (glk-group 1) or hydroxyethyl starch 7.5 % in naci 8.4 % (hes-group 2). in each dog, a 0.9 % saline infi~sion was continued to maintain the puhnonary arlery occluded pressure at baseline level. hemodynamic, blood gas aualysis and laboratory data were collecled at baseline and 30 miu, 60 rain, 120 rain, and 240 nunutes later.. results : eudotoxin administration was followed by a fall in mean arterial pressure (map) aud cardiac index (ci) and a rise in blood lactate levels. resuscitation with either gik or hes hypertoaic solutions resulted in similm increases in map, ci, oxygen delivery and left ventricular stroke index (table 1) . we conclude that during resuscitation from endotoxic shock the use of gik solutions is not superior to hypertouic hes solutions. the higher blood lactate levels observed in the dogs receiving gik can be attributed to the glucose metabolism. 1, 75 for group 2, 105 for group 3) were drawn and immediately analysed at 37~ using the abl500 radiometer for po2, pco2 and ph, and the osm3 radiometer for hbo2%, hbco% and methb%. psost (i.e. the ps0 at ph=7.40, pco2=40 mmhg and temperature at 37 ~ c) was calculated automatically by the instruments on mixed venous blood, as was the ps0"in vivo" (i.e. the ps0 at the patient's value of ph, pcoz and temperature), using siggaard-andersen's algorithm. the data were compared by the one-way anova test and by the t-test for paired and unpaired samples. results: the mean resulting values (in mmhg) with the statistical differences are shown in table i. in addition, the time series analysis shows the mean ps~st values as statistically below the psin vivo" in the septic patients while the opposite is shown for the cardiac patients. no differences in the time analysis are demonstrated for the second group. a possible clinical significance may be drawn from these different behaviours. objectives:toxemia degree and humoral immunity condition have been studied in 36 patients aged from 19 to 72 with progressive course of sepsis and polyorganic insufficience. methods: such toxemia and humoral immunity findings as lencositlcindex of toxication (lii), level of oligopeptides of the middle molecular mass registered at the wave length of 25nm(mmi) & 280nm (mm2), distribution index (id), immunoglobulins a,m,g, concentration of circulating immunocomplexes (cici & cic2) and also some clinical and biochemical findings on the 1,3,5 day after the operation serve as criteria for treatment effect. results: it was founded that in intensive therapy and detoxication, level of lii is successively decreased from 12.6~1.4 to 2.6+.5 on the 5-th day after the operation. true decrease of the level mm2 from .704~.09 to .402+.08 un & optimal density and increase of distribution index from .96 to 1.09 are argued. conclusions: in studlng the dynamics of the immunoglobulin's spectrum and the true increase of immunoglobulin g level from 8.4+.6g/i to i0.8+.7g/i on the 5-th day after the operation simultaneously with the decrease of cic2 from 806.9~60 to 624.7~54.8(p .05) were founded. some stages of the investigation true increase of lymphocytes from 9.0+2.6% to 15.0+1.8% was noted and it appeared to be a favourable prognosis finding for disease outcome. high correlation dependence between bacillus-and segmentonuclear neutrophils and immunoglobullns g & m (r=.5-.7 in p<.05) was discovered and it also showed positive dynamics of the course of the disease. a 34 year old male patient was admitted to the icu with severe paraquat poisoning. treatment consisted of gastic lavage and oral administration of fullers earth. because of very high plasma levels hemodialysis together with charcoal hemoperfusion was started within one hour after admission. this treatment was further continued by continuous veno-venous hemofiltration in order to remove the circulating paraquat and also circulating cytokines. nevertheless patient s condition worsened necessitating artificial. ventilation and hemodynamic support. patient died 24 hours after admission of acute multiple organ failure due to paraquat poisoning. serum levels of paraquat were determined by colorimetric method (table) . levels of interleukin 6 (il 6) and 8 (il 8), tumor necrosis factor (tnf-alpha), interleukin i receptor antagonist (il 1 ra) were determined both in plasma and ultrafiltrate ( q~!ectives : evaluate in critically ill patients the effects of tow-dose dopamine on gastric mucosal blood flow (gmbf) using laser-doppler flowmetry, a continuous non invasive method of assessing microcirculation. methods : 6 patients requiring both mechanical ventilation and pulmonary artery catheterization for multiple trauma (n=3), ards (n=2) and pancreatitis (n=l) were included. in each patient, the laser-doppler (ld) probe was inserted through a naso-gastric tube. the ld signal is proportional to the number of red blood cells moving in the measuring volume and the mean velocity of these cells. when the ld signal was satisfactory, an aspiration was created into a catheter which was fixed in parallel to the ld probe, to maintain the tip of the probe against the gastric wall at the site of measurement. data (systemic hemodynamic parameters and gmbf) were obtained at the end of a 30 rain resting period (baseline), then 30 min after dopamine (2 mcg/kg/min) infusion, and finally 30 rain after the end of dopamine infusion (recovery gmbf 185 _+ 23 (perfusion units) gmbf 0 ~a% vs baseline) * p < 0.05 vs "baseline" and "recovery". conclusions : 1) despite a slight increase in co (+13%), the dramatical increase in gmbf (+87%) with dopamine, strongly suggests a selective vasodilator effect of low-dose dopamine on gasaic mucosal perfusion. 2) laser-doppler flowmetry appears a promising method to assess gastric microcircalation in critically ill patients. increasing evidence suggests that the activation of inos is the final common pathway for vasodilation in human sepsis associated with endotoxic shock. activation of the cellular immune system induces the excessive release of the pteridines neopterin (n) and 7,8-dihydroneopterin (nh2) by human macrophages/monocytes. besides the well established diagnostic value of pteridines in several inflammatory diseases, it is speculated that these substances per se exhibit biochemical functions. thus we hypothesize that pteridines can modulate inos gene expression in vascular smooth muscle cells (vsmc) in vilro. cdtured rat aortic vsmc from female wistar kyoto rats were incubated with n (20 pm), nh2 (20 ilm), lipopolysaccharide (lps, 5 ~g/ml), and interferone-~/(ifn-~/, 100 u/ml) for 9 h, respectively, inos gene expression was measured by competitive reverse transcription polymerase chain reaction. the results are summarized in the table. the present study demonstxates a neopterin induced increase in inos mrna expression at the transcriptional level in vsmc. while coincuhation of cells with n + lps resulted in an additive effect on inos gene expression, n + ifn-7 seem to have a more than additive effect nh2 did not alter inos mrna synthesis, but it suppresses the lps as well as the ifn-yinduced augmentation of inos gene expression. we speculate that this pteridine-mediated modulation of inos gene expression is involved in the regulation of the vascular tone in endotoxic septic shock. the relationship of sepsis and coagulation abnormalities is well known, mainly in severe sepsis and septic shock. still farther, the extreme expression of hemostasis abnormalities (disseminated intravascular coagulation) in sepsis, has been extensively described. we studied the changes in several coagulation and fibrinolysis markers in septic patients, trying to correlate them with the evolution of the sepsis phenomenon, with an emphasis in its early stages, where therapeutic intervention might be more drastic. in 64 patients, 30 with sepsis, 22 with severe sepsis and 12 with septic shock, as well as in 14 healthy volunteers (control group) we measured : platelet (ptl), coagulation markers [fxii, fvii, fviii, fvw, fibrinogen (fibr) we conclude that all parts of the coagulation system are gradually changed during the evolution of sepsis phenomenon , even in the earliest stage of sepsis. the expression of an inducible nitric oxide (no) synthase (inos) plays a major role in the pathophysiology of septic shock (ss). inhibition of inos could therefore be of therapeutic value. however, such an inhibition has been shown to be detrimental, increasing tissue anoxia (and end-organ damage), possibly through the simultaneous blockade of constitutive nos (cnos). thus, selective inhibition of inos might be more suitable. we evaluated the effects of l-canavanine (can), a more potent inhibitor of inos than cnos, in an animal model of ss. method: in 30 anesthetized rats, catheters were placed in the femoral vein and artery. 23 rats were given an iv bolus of lipopolysaccharide (lps, 5 mg/kg), at baseline (to). after 1 h (t1), rats received at random an infusion of either can (20 mg/kg/h; can group, n=l 1) or an equivalent volume of 0.9% naci (2cc/kg/h; nac1 group, n=12), giyen over 4 h (t1-t5). a third group (sham group, n=7) received 0.9% nac1 in place of lps, and then was treated like the nac1 group. mean blood pressure (mbp), blood lactate and nitrates (no3) were measured each h. glucose, creatinine and asat were also measured in 18 rats (n=6 in each group). the can 98_+9* 304+52"t 3.2+1.1"~ 3.6+_1.4"t 138+46" 48+10" *p<0.05 can vs naci ?p<0.05 vs sham can suppressed the hypotension, reduced the hypoglycemia and hyperlactatemia, and attenuated the biological signs of renal and hepatic dysfunction induced by endotoxemia. these effects were associated with a lesser elevation of blood no3, confirming a partial inhibition of inos. conclusion: l-canavanine attenuates the hemodynamic and metabolic consequences of endotoxemia in the rat. these effects may be related to a partial inhibition of inos. they contrast with the deleterious effects described with non selective inhibitors of nos. l-canavanine could become a new tool for the treatment of septic shock. rocalc1tonin :marker of sepsis, ii~flammaiiur% t~ boifi .cheval*~ jf.timsit*, m.assicot**, b.misset*,/.carlet*, c.bohuon** saint joseph heap, paris**biochemistry institut g roussy, villejuif, ce bi~)l~i~ttectives_: high serum levels of procalcitoaln (proct) have been shown to be ~ss-ocinted with bacterial infection. however, few data exist about the ability of proct to differenciate septic shock and shock from other origin in which an activation of intlmmamtory mediators has been also demonstrated. methods: thirteen patients with bacterial septic shock (ss), 11 patients with non septic shock (nss), 14 patients with bacterial infection without shock (1nf) and 8 icu patients without shock and without infection (control) were compared for proct levels at dayl, 2, 3, 7, 14 . patients were classified blindly and independently fi'om proct results. twelve patients were excluded because any classification was impossible due to mixed pathology. proct was measured with ebemoluminescenee (brahms diagnostica-berlin). results: dayl, proct levels are significantly different between the four groups. dayl proct levels are correlated with saps (p=0.0002), infection (3.7+_3 vs 61_+25,p=0.0007), shock (13_+10 vs 60+.-27,p=0 .002), death at day28 (12_+7 vs 96_+44,p=0.003). when shock and infection are introduced in multifactor &nov& only infection remains correlated with day 1 proct levels (19=0.003) in patients with shock, dayl proct levels are correlated with saps, infection and death at day28, but not with arterial lactate levels (p=0.37), white blood calls (p=0.2) or fever (p=0.1). proct levels remain higher i~i septic shock patients at day 1, 2 and 3( figure) . i c0edpsion: procalcitonin levels in the first three days of shock are differen[" between septic and non septic shock patients. in patients with diseases known to induce acute an inflammatory process, procaldtonin seems to be a marker o~ infection. obiectives-to evaluate the effect of endotoxic shock on the distribution of blood flow between the mucosal and the muscular layer of the intestinal wall. methods: in 10 fasted pigs, mean aortic pressure (map, mm hg), cardiac output (co, ml/min-kg),superior mesenteric artery flow (q sma, ml/min.kg), and phi, where measured before (control) and after i.v. endotoxin (10 gg/kg). the blood flow to the mucosal and the muscular layer was measured in 3 regions (proximal jejunum (pj), mid-small intestine (mi) and terminal ileum (ti)) by colored microspheres, using 8 adjacent samples in each region. the muscular layer was separated from the mucosa by blunt dissection, and the flow determined independently in each layer. results: endotoxin with fluid resuscitation induced the expected decrease in map (95.8_+3.0 vs 53.9-+2.4, p<0.01), and phi (7.29!-_0.02 vs 7.13_+0.01, p<0.01), with a constant co (133_+4 vs 144_+8, p=0.28) and qst, aa (21.5_+0.8 vs 22.8_+0.9, p=0.09). the results of regional pertusion are presented in the table. (flow in ml/rain 9 100g of tissue; mean _+ sem ; * p<0.001 vs control by two-way anova) conclusions-these data indicate that the mucosal flow increased during septic shock. they suggest that a decrease in phi may be due to hypoper~usion of the muscular layer or to metabolic alterations within the mucosa, despite a 50% increase in flow. acute increase in wbc count (from a mean of lo.oo01mm a to 42 o00/mm~), between the 3rd and the 7th day of therapy. there was a decline of the wbc count to an average of about 25.0001mm a after decreasing the daily dose of the medication to 200 mcg there was no increase in tile absolute number of the eosinophils during the whole course of the medication. there was a slight decrease in the c3 complement between 0.26 to 0.29 g/i. normal values 0.5 to 0.9 g/i there was no change in c4 values. conclusions : an early increase in wbc count was observed (3rd day) without subsequent increase in the number of immature types from bone marrow, probably due to the mobilization of wbc from the periphery and this increase was dose dependent. there was a slight decrease in c3 fraction of complement, probably due to the consumption of this fraction in the process of opsonization. no adverse effects of the medication were observed, during the treatment with the above dose. these data sugest that cm csf may be a useful complement to tile main antimlcrobial treat,nent ~ of septic [cu patients. objectives: as part of a large multicentric, placebo-controlled, randomized clinical trial investigating the effects of interleukin-1 receptor antagonist (ii-lra) in the treatment of severe sepsis and septic shock, this substudy evaluated in dem.il the acute hemodynamic effects of ii-lra in patients who were invasively monitored. methods: in a total of 71 evaluable patients in whom vasoactive support was little altered, hemodynamic measurements were performed at baseline (twice), and i hour, 2 h, 3 h, 4 h, 8 h, and 12 h after the administration of 1 mg/kg (n=20) or 2 mg/kg (n=22) of i1-1 ra or the corresponding placebo (n =29). 58/71 patients (82 %) were treated with adrenergie agents and 66/71 (93 %) with mechanical ventilation. data were analyzed by a kruskal-wallis test. results: during the study, there was no significant difference with time or between groups in arterial pressure, cardiac filling pressures, cardiac index or left ventricular stroke work (figure). burmester, "~ man8 and h. djonlagic medical university (internal medicine, "cardiology, *'microbiology) and "**southern city hospital, lfibeck, germany obiectives: evaluation of the incidence of bacteremia and sepsis in patients with nontyphoidal salmonella (s.) infections, specification of risk factors, need of icu treatment, clinical course, and mortality in the group of the patients who developed septic complications. methods: data of all patients with microbiologically proven s. infections hospitalized in the medical university of lobeck and in the southern city hospital of l0beck from 1992 to 1994. results: within the observation period s. was isolated from the stool cultures of 748 patients. in 13 patients (g m, 4 f, median age 52 yrs) s. could be detected in blood cultures (9 s. enteritidis, 4 s. typhimurium). in addition, in 10 of these patients s. was also isolated from other specimens (urine, liquor, and tissue fluids derived from abscess punctures). in all 13 patients with positive blood cultures the clinical course of s, infection was complicated: ? patients developed mof (acute renal failure, ards, hemodynamic instability, dic) and required icu treatment for at least 4 up to 62 days, 4 of the 13 patients died. the predisposing disorders in the patients with s. bacteremia were (n=): aids (3), immunosuppressive drugs (2), chronic alcoholism (2), malignancies (2), none (4). septic complications in patients with nontyphoidal s, infections are relatively rare (in this study < 2 % of all hospitalized patients with microbiologically proven salmonellosis) but severe (mortality of approx. 30 %). patients at risk for a complicated clinical course are predominantly those with predisposing disorders but occasionally also patients without evidence for an underlying disease. age (yr) 65 + 9 58 + 14 death (n) 22 18 duration of shock (h) 27 + 37 54 + 44 noradrenaline (rag/h) 5,9 _+ 6 2 + 5 temperature (~ 38,2 + 2 38,1 + 1 pvr (dynxsecxcm -5) 1195 + 408 572 + 418 co (ljmin) 4,2 _+ 1,6 9,9 + 3,6 lactate (mmol/l) 7 + 5,9 9,2 + 9 interleukin-6 (pg/ml) 829 _+ 798 1331 + 888 interleukin-1 (pg/ml) 9,3 _+ 9,4 9,8 + 7,3 tnf-alpha (pg/ml) 23,5 + 31,7 166 + 209 neopterin (nmol/l) 43,2 + 43,7 218 + 193 crp (rag/l) 131 _+ 97 233 +_ 138 pro-ct (ng/ml) 22,6 + 50,5 77,9 + 160 there was no positive correlation between serum lactate levels, degree of shock, hypoxemia and pro-ct positivity. pts with septic shock of bacterial origin entirely developed hyperprocalcitoninemia, whereas pts with cardiogenic shock, who expired within 24 h did not. however, in late cardiogenic shock (>24h) all pts developed fever of unknown origin and consecutive hyperprocalcitoninemia. these data suggest bacterial inflammation and/or mucosal translocation of bacterial products in pts with prolonged cardiogenic shock. the use of a loading dose of quinine (16.7 mg/kg base in 4 h) is recommended in previously untreated patients (pts) with sfm, particularly in multi-drug resistance areas. this protocol is difficult to validate, since the viability of microorganisms is not assessed routinely in parasitology laboratories. objectives: to examine the evolution of parasite viability during the early phase of therapy of sfm. methods: from 02/1993 to 12/94, pts with sfm (who 1990) treated with iv quinine for less than 6 h were included prospectively. blood samples were collected at o, 6, 12, 18, 24, 36 and 48 h viability was assessed by culturing parasitized red blood cells in the presence of 3h-hypoxanthine, and radioactivity was determined at 42 h by scintillation counting. viability was expressed as the percentage of radioactivity compared to the initial sample. plasma quinine was determined by liquid chromatography. tile ratio plasma quinine (pmol/1)xlo00/icso for quinine (nmo]/]) was called the parasiticida/ index. results:5 pts were included, 42• saps1 18.6-+4.9. the initial parasitemia was 2t.4+7.2%. complications of malaria were coma (4 pts), shock (3 pts), renal failure (2 pts) and acute lung injury (2 pts). all strains were sensitive to quinine (icso 174--67 nmol/1). in 2 pts who were not given a loading dose, parasite viability increased by 63 and 157%, with concomitantly low quinine levels (22 and 19 #mow] at 12 h); 1 pt died. in 3 pts that received a loading dose (serum quinine at 12 h = 33.1--2.0 ~mol/]) a marked decrease of parasite viability (by 73+_10% at 12 h) was shown. viability was inversely correlated with plasma quinine (r=.677, p-.o11) and parasiticidal index (r=.678, p-.o1). conclusions: even with fully sensitive strains, the use of a loading dose of quinine seems warranted in severe falciparum malaria in order to reach rapidly adequate plasma quinine ]evels, necessary to inhibit significantly parasite viability. l nkka, e ruokonell j takala. critical care research program, department of intensive care, kuopio univ hospital, finland objective: to determine the incidence of positive blood cultures, their microbial subgroups and to evaluate the outcome of icu patients with different bacleremias. material and methods: we analysed all positive blood cultures in 3077 consecutive admission to a university hospital icu in 1992 -93 and the icu and hospital survival of the bacteremia patients. during these years 73 patients had 176 positive blood cultures that were considered as clinically relevant, excluding colonizations or contanfinations. results: patients with positive blood cultures had an icu survival of 65.8 % (vs. 92,7 % in all icu patients) and six month survival of 50.7 % (vs. 85.8 % in all icu patients). the most common bacteria were enterobacteriaceae (27,3 %), staphylococcus aureus ( 18,8 %) , coagulase negative staphylococci ( 14.2 %), pseudomonas ( 14.8 %) and slieptococci (9.1%). obiectives: to evaluate prognostic factors and mortality in consecutive patients (pts) with hiv infection and septic shock. methods: from 03-1991 to 12-1993 , records of consecutivepts with septic shock (crit care med 1992, 20: 864-74) admitted to the icu were reviewed retrospectively. results: among 76 pts with septic shock admitted during the study period, 28 had hiv infection-26 of whom had aids-(gr. i) and 46 were hiv-negative (gr. ill. ten gr. ii pts (21%) were irnmunosuppressed because of neoplastic or immune dlsease. mechanica] ventilation was required in 89% gr. i and 83% gr. ii pts in 8 gr . i pts (29%) a multivariate analysis demonstrated that hiv infection and sap5 i were independently predictive of death in pts with septic shock. ~onclusions: evidence of increased mortality, number of organ failures and higher severity scores (saps i does not take into account immunosuppression) is demonstrated in hi v-positive pts, infection with hiv appears to be an independent prognostic factor in pts with septic shock. the frequency of opportunistic infections (often responsible for delayed diagnosis and treatment) may contribute to the poor prognosis in this population. obiectives: to determine interleukin (il)-i 0 levels in plasma of patients with sepsis and septic shock. to analyze the relationship between plasma il-10 and the proinflammatory mediators, tumor necrosis factor-aifa (tnf) and il-6, the underlying severity of the disease and the evolution of patients with sepsis. methods: we studied 94 critically ill patients (63 men, 31 women; 18-86 years old) in three diferents groups. group i: 23 patients without evidence of infection, group i1:34 patients with sepsis and 37 with septic shock (group iii). we measured plasma il-lo, tnf and il-6 levels in the first 12 hours of diagnosis. severity of illness was estimated with the acute physiology and chronic health evaluation (apache ii) scoring sytem. results: plasma levels of il-10 were higher in group iii (median, 51 pg/ml; range, 5-6000 pg/ml) than in group ii (median, 10 pg/ml; range, 2-970 pg/ml; p <.001) and group i (median, 5 pg/ml; range, 2-133 pg/ml; p <.001). median il-10 concentrations did not differ among patients who survived (median 7 pg/ml; range, 2-6000 pg/ml) and those who died during the overall follow-up period (28 days) (median, 15; range, 5-5400 pg/ml); but patients who died in short-term (< 24 hours) with catecholamine-refractory hypotension showed the highest concentrations of il-io (median, 1200 pg/ml; range, 51-5400 pg/ml). in patients with bacteriemia (34%), levels of il-10 were higher (median, 51 pg/ml; range, 2-6000 pg/ml) than in those with negative blood culture (median, 8,5 pg/ml; range 2-5.400 pg/ml; p< .001). there was a good correlation between plasma il-io concentration and levels of tnf (r= .59; p < .001) and il-6 (r= .60; p < .001). the correlation between levels of il-10 and the apache ii score was significant only in the septic shock group (r= 0.48; p <.005). conclusions: in septic shock, il-io and proinflammatory citokines are released in high concentrations. the significant correlation observed in patients with septic shock between il-10 levels and apache ii, short-term death and bacteriemia can possibly be explained by the massive inflammatory response in septic shock with fulminant course. intensive care department -calmette hospital -59037 lille -france. in septic shock, inadequate splanchnic blood flow may play a prominent role in the pathogenesis of multiple organ failure. measurement of gastric phi has been propose to evaluate tissue oxygenation in splanchnic organs. objectives: to compare gastric phi values with hepatic icg clearance, an index of liver blood flow and function ; to determine if one of these two methods could be proposed to assess the entire splanctmic peffusion in septic shock. methods : 6 patients (age : 65• years ; saps ii : 46• were prospectively investigated (septic shock : bone criteria). following parameters were collected during 12 hours : systemic hemodynamic parameters (swan ganz catheter 93a434h -ref1 computer -baxter lab.), calculated systemic oxygen transport (do2), oxygen consumption (vo2) by indirect calorimetry (deltatrac datex lab.), gastric intramucosal pco 2 (pco2ss) and phi (trip -ngs catheter -tonometrics lab.) and plasma disappearance rate of icg (pdr dye) (femoral artery fiberoptic/thermistor catheter 2024, cold z021 computer -pulsian medizintechnik, germany). correlations were performed using a linear regression. elevated in all days with the highest value in second and third days of treatment. nonsurvivors had higher values of these parameters than survivors but differences did not reach statistical significance. another trend of changes were observed in selectin p (gmp-140) concentration. in all patients concentrations measured were elevated but in survivors after not significant decrease this parameter in second day another one had simmilar values. in patients who died we noted significant decrease in third day (p < 0.05) whereafter prominent increase, significant after seventh day, in comparison to third day value and value in survivors group. icam-1 concentrations in all patients reached high levels and in nonsurvivors after four day of treatment significant increase in comparison to survivors we found. conclusions: multiple trauma complicated with sepsis induce rapid elevation of concentrations of il-6, il-8 and increased expressior of adhession molecules (selectin e, p, icam-1) measure of icam-1 and selectin p concentration determine lung injury severity and prognosis as to health and life. (clp) .pathophysiology of cip is unclear, but changes in regional bloodflow may be a ~ignificant factor. nerve blood flow (nbf)is reduced in rat models of hemorrhagic shock (g),but no information is available in sepsis. we studied the comparative effect of acute endotoxemic shock {etx)& h on perfusion of rat sciatic nerve. methods: 20 male sprague-dawley rats were anesthetized with pentobarbital (ip), instrumented with a tracheostomy, carotid arterial & venous catheters and mechanically ventilated (fi02=0.5). the left sciatic nerve was surgically exposed. monitored variables included: a) mean arterial pressure (map,mmhg) ,b) nbf (ml/1o0 g/min) by laser doppler flow meter,c) nerve internal arterial diameter (id ~ m) by video image shearing and splitting method. after stable baseline measurements were obtained, acute hypotension was induced by randomly assigning the rats to etx (0.25 b6, difco) in saline at 1 mg/kg or h. both interventions produced 50% reduction in map within 3 min., which recovered to baseline values spontaneously in etx group, & by reinfusion of heparinized withdrawn blood in m. data were analyzed by linear regression, two-way repeated measures analysis of variance followed by bonferroni-t method. experimental stages were:(1)baseline, (2) mid-point of map reduction; (3) nadir of hypotension, (4)midpoint of map recovery, & (5) after stable recovery of map. both etx & h induced shock result in similar reduction in nbf consistent with lack of autoregulation in peripheral nerve vessels independent of etiology. since cip is primarily associated with sepsis, it is not likely that acute reduction in nbf alone causes cip. direct & indirect neurotoxic effects of mediators of sepsis need to be evaluated. .':_.~::::o4o:oc4., objectives : evaluate the relationship between il-10, a cytokine which inhibits tnf, production and protects mice from endotoxin toxicity, and the other proinflammatory cylokines, tnf~, il 6 and ils in severe sepsis and septic shock. methods : twenty-eight icu patients (19 m, 9 f, mean age 54 + 17 y) were studied as soon as they developped a severe sepsis (n = 16) or a septic shock episode (n= 12) as defined by a conference consensus in 1992 (1). tnf~, il 6, il s and il-10 plasma levels were measured by immuno-radiometrie assays from medgenix (fleurus, belgium). lc mean and range. results : the comparisons between cytokine levels in severe sepsis versus septic shock were made using the logarithm of the value in order to normalize the distribution of data, and student test. il-10 plasma levels were higher in patients with septic shock than in patients in severe sepsis. there was a significant correlation (p < 0.05) between il-10 and tnf a (r= 0.6), il-10 and il~ (r = 0.73) and il-10 and il s (r = 0.65) as well as between il-10 and apache n score (r= 0.52). patients who died (n = 13) had il-10 levels higher than patients who survived but this difference was not statistically significant (114 pg/ml vs 34.5 pg/ml; p>0.05). conclusions : during severe sepsis and sepsis shock, il-10 seems at least to follow the same evolution (increase in plasmatic level) with the severity of sepsis as the other cytokines. reference : (1) crit care med 1992;20:864-74. objectives: to evaluate the effects of steroids on hemodynamics and mortality in septic patients with konwn levels of cortisol concentration. methods: retrospectively we analyzed data ofpatients with documented septic shock who received steroids after assessment of adrenal function. in all patients hemodynamic parameters as well as the necessary vasoactive medication were assessed, before and 24 hours after corticosteroid medication. immediately before administration of corticosteroids adrenal function was evaluated with cortisol levels before and after synthetic corticotropin (0.250 mg). finally we studied mortality. we defined a positive respons on corticosteroids as an elevation of map of at least 30 mmhg and/or a decrease in the necessary vasoactive medication of at least 50% within 24 hours. adrenal insufficiency was defined as a cortisol level after stimulation of less than 500 nmol/l. results: 15 of 23 patients were found to respond to steroid medication, 8 did not. mean cortisol levels before and after corticotropin were 534 • 366 and 737 • 396 nmol/l in the responder group (rg) and 583 • and 907 • 301 nmol/l in the non responder group (nrg). in the rg 9 out of 15 (60%) were found to have an adrenal insufficiency, in the nrg 3 out of 8 (37%). in the rg 2-weeks mortality was 6.7% (l out of 15), the overall mortality 33% (5 out of 15). mortality in the nrg was 62% (5 out of 8) (p <0.01) and 75% (6 out of 8) (p <0.005) respectively. conclusions: in patients in septic shock there is a beneficial effect of steroids in case of adrenal insufficiency, but also in a subgroup with normal adrenal f{unction. obiectives: intercellular adhesion is a critical step in the accumulation of leukocytes. postischemic cardiac lymph has the capacity to stimulate icam-i. in the coronary microcirculation neutrophils can be trapped and in many cases obstruct capillaries, previously we found that troponin t (s-tnt) a marker for myocardial iechemia, was increased in septic patients. the aim of the study was to follow slcam-1 and s-tnt levels continuously starting at the beginning of sepsis. methods: 19 patients were ingluded in this institutionally approved study after relatives had given their informed consent. all patients were included within 24 hrs following the beginning of sepsis. blood was drawn every 4 hrs in the first ;~4 hrs, after 48 hrs, followed once per day for 7 days. s-tnt, icam-1, elam (elisa's, boehringer mannheim inc, r&d systems ltd.) arterial and venous blood gases were determined, an ecg and a complete hemedynamir measurement including cardiac output were obtained. all patients received adequate volume and catecholamine therapy (norepinephrine, dopamine, dobutamine; median (range) 0.6 (0.0-1.66), 3.12 (2.4-12), 6.29 (0.0-15.3) pg/kg/min, respectively). statistical analysis: wileoxon signed rank-sum test. .45 (0.06-7.6) 0.0003 13 patients had s-tnt levels >0.2pg/l. 11 of these died, whereas only 2 of 6 patients died with s-tnt values <0.2 pg/l (p=0.0296). all patients that died had elevated sjcam-1 levels (232 ilg/l:cut-off ) whereas in the survivor group only 50% had elevated icam-1 levels (p=0,043). conclusions: increased slcam-1 and s-tnt levels were found during early sepsis in the majority of patients, a high sicam-1 and s-tnt value was associated with a higher mortality. the research of the noninvasive haemodynamic monitoring accelerated recently all over the world. the aim of our study was to test whether the changes of the haemodynamk parameters measured by impedance cardiography (icg) were corresponded to clinical changes in septic patients. investigations were performed on 20 critically ill postoperative septic patients (their multiple organ failure score was 8-9/with icg monitor. in 9 cases the investigation~ were performed in septic shock. the measured parameters were: heart rate (hr), mean arterial pressure (map), cardiac output (co), peripherial resistance (svr),preejection period (pep), and ventricular ejection time (vet). these parameters were measured during 3-72 hours in every 30 minutes, depending on the patients cl~tnical condition. results: at the septic patients the hr and the co ]~reased. in septic shock the co was significantly higher the svr lower than in the septic group. in the hr there was no difference between the two groups. in septic shock noradrenalin influenced more effectively the measured parameters than dobutamin. conclusion: the trend of the measured icg parameters correlated with the clinical changes of septic patient's state. the noninvasive haemodynamic monitoring by impedance cardiography helps the planning and leading the adequate intensive therapy of these critically ill septic patients. to evaluate the development of sirs, sepsis and septic shock in hospitalized patients with fever, a prospective study was performed on 300 patients using previously defined criteria. methods: 300 normotensive patients with fever (temperature >38.0 ~ axillary), admitted to the department of internal medicine were evaluated for the existence of sirs during the first three days of the study and sepsis at inclusion. during a follow-up period of 7 days the patients were daily evaluated for the development of sepsis or septic shock. results: most patients (69%) had or developed sirs within the first three days, 16 patients (5%) did not. sepsis was present in 25% at inclusion. in patients with sirs, 76% did not progress to sepsis or septic shock, 24% progressed to sepsis (mean interval 2.55 • 1.97 days), and 1 patient (<1%) directly progressed from sirs to septic shock. in patients with sepsis, 17% progressed to septic shock (mean interval 2.08 • 1.56 days). sepsis was preceded by sirs in 40%. septic shock was preceded by sepsis in 92% and by sirs in 8%. conclusions: 94% of patients with fever in an internal medicine department develop sirs, or sepsis. furthermore, progression from sirs to sepsis or septic shock is poorly predicted by fever or sirs. nevertheless, all patients with septic shock were preceded bysirs or sepsis. taken together, this may indicate a severity hierarchy of the syndromes. however, fever, sirs and sepsis are relatively poor indicators of development of septic shock. this supports further research on additional predictors of septic shock. b. m.manuylov, v.b.skobelsky (moscow) in recent years sodium hypochlorite (sh) has been successfully used to eliminate pyo-septic complications. moreover, the mechanism of the sh effect on the immune system has not been sufficiently studied. the aim of the present investigation was to study the mechanism of sh effect in inflammatory pulmonary diseases. 20 patients with double pneumonia were subjected to the evaluation. sh in the concentration of 600 mg/l in the volume of 400-800 m1/24 hours was administered by drop infusion into the central vein. to evaluate one of the defence systems the leukocytes activity by the chemoluminescence technique was studied. in all the patients baseline secondary immunodeficiency which was indicated by the decrease in the luminescence level was established. even 1 hour after the sh administration the leukocytes activation exp-ressed by the enhancement of their chemoluminescence 0.5-5 times was observed. this supports the available findings that accumulation and liberation of the oxygen active forms (ol'oh, '02, h202) are accompanied by the increased phagocytosis, i,e. the signs of "the oxydation explosion" testify to the favourable sh effect on the course of inflammation processes. the use of sh permitted to decrease the percentage of lethality in double pneumonia by 15% in the intensive care unit over the year. at the same time, excessive activation of free radical oxygen may be a damaging factor. therefore, precise individual control over the choice of concentration, dosage and the preparation administration rate is required. prospective, double-blind, placebo-controlled, trial of atiii substitution in sepsis r. a. balk objective: pilot study to evaluate the efficacy and safety of atiii substimtion therapy in patients with sepsis. efficacy assessed using change in mortality or organ failure/dysfunction. adult patients meeting a definition of sepsis and cared for in a tertiary care academic medical center in chicago were identified and prospectively randomied to receive either atiii (kybernin p) or placebo in a double-blind treatment protocol. all other therapy and patient management were under the direction of the patient's attending physician. all patient's were followed for 28 days and the organ dysfunction/failure were scored using published scoring systems (jordan et al crit. care med. 1987 , goris et al arch. surg. 1985 , kuaus et al ann. surg. 1985 colldusions:wha~ we met the shomaeker objectiv% the mortality and the pro~os[s were i~ttc*. those criteria were obtained with file tradititmal t~ctor likr doht~mme, hut c.~vh ~,as ca1 in~aertam measure. they ac~s smxergically in the optimizatic~l of the fell vmtrictdar work index, tad fimdameatally cavh seox~s to have an impo.aat role in the better respiratory ev-altmtioa, leaving yet the possibility to coltrol the flui& r althou~l eomproved it's not aec~pt~xl file importmlce h* the diminution, of the sepsis modiat~lrs llke fnt and il-6 with h~wmotiltrafi(al, stopphlg the evolution to nmltiorganic failure mid de~easethe mortality. with ours clhlicals results, we could saythat cavii in multiol~atlie disfut~oa septic patieats, se~r~ to be an c4xilna] supoa or troatmeat maesure. of anaesthesia and intensive therapy, medical university of prcs, p~csf hungary. objectives: since some biological effects of bacterial endotoxin require an interaction between the lps molecule and a serum factor(s), we hypothesized that lps-induced no production and cgmp accumulation in vascular smooth muscle cells (vsmc), a mechanism ~thought to underlie cardiovascular collapse associated with septic shock, is modulated by serum factor(s). methods: cultured vsmc from rat aorta were challenged with e. coli lps for 4-6 hours either in the presence or absence of fetal calf serum (fbs), and no production was monitored by radioimmunoassay determination of cgmp content of hci extracts. results: in the absence of serum, 1o00 ng/ml lps was required to increase cgmp levels, whereas the presence of 10 % fbs shifted the lps concentration curve i00 times to the left. similarly to fbs, human serum also potentiated lps-induced cgmp accumulation. in contrast to lps, serum had no effect on cgmp accumulation elicited by sodium nitroprusside, a no releasing agent, suggesting that the sensitivity of vsmc to generate cgmp in response to exogenous no is not modulated by serum. heat inactivation (>80 ~ 30 min) but not removal of small molecules (<10,000 d) from the serum by dialysis, reduced the potentiation of cgmp accumulation by serum. time course studied indicated that serum is required within the first 120 min of lps exposure to increase cgmp levels. to investigate whether the effect of serum is specific for lps, we treated the cells with increasing concentration of interleukin 1-~ (il-i). 10% fbs shifted the il-iinduced cgmp responses five times to the left. conclusions: our study suggests that lower concentrations of e. cell lps and il-i require a heat labile macromolecule in the serum in order to elicit no production. this factor is present in the human serum and it may play a potentially important role during no synthesis induction in vsmc. objective: to evaluate the factors of acquisition and the outcome of methicillin resistant staphylococcus aureus (mrsa) bacteremia in an intensive care unit (icu). methods: all patients in which bacterermia due to staphylococcus aureus developed >72 hours following admission to our icu, during a 10 year period ( january 83 through january 94) were reviewed. 30 patients (pts) were included, mean age 68,1y (sd 13,1), saps 2 35,9 (sd 11,1), mac cabe (1 and 2) 53%, mortality directly due to sepsis 30%. 16 pts had mrsa bacteremia and 14 methicillin susceptible staph. aureus (mssa) . both groups were compared using the chi square (with correction of yates), fisher's exact, student's t or wilcoxon test. results: there was no statistically significant difference between mrssa and mssa regarding at age (71,8+ 4,8 vs 63,9+ 1,8) , saps2 (33,6+6,6 vs 38,2+14,1), use of vancomycin (94% vs 71%), mechanical ventilation (94% vs 100%), number of days (d) before the drawing of the first positive blood culture (median 20 d, range 7-150 d vs median 30 d, range 7-120 d). more mrsa than mssa pts had previous use of nonsteroidal anti-inflammatory drugs (nsaid) (25 % vs 0% p<0,001), central venous catheter infection due to staph.aureus (62,5% vs 14% p<0,01), but previous use of antibiotics was not significantly different (37,5% vs 21%). the outcome of the bacteremic pts was not statistically different: saps 2 at the first day of bacteremia (33,6+_.7,2 vs 40,7+14,5), severe sepsis and septic shock (31% vs 28%), persistence of the bacteremia (43% vs 78%), mortality directly due to bacteremia (25% vs 45%). conclusion: previous use of nsaid, infection of venous central catheter are more frequently associated with mrsa bacteremia. thus, similar to others studies (hershow infect control hosp epidemio11992; 13:587-593) , these results do not indicate that mrsa is associated with increased virulence. objectives: to closer definition of mosf formation mechanismes in nosocomial sepsis (ns) the complex clinicobiochemical, microbiological, immunological, functional exaroination of 62 cases with ns had been done. methods: examination of cellular and humoral immunity, nonspecific immunologic reactivity, systemic and hepatic circulation, microbiological examination of blood,electro-and echocardiography, sonography and computer tomography of chest and abdomen organs were obligatory. autopsy findings of 5 dead cases had been analized. results: in 45 cases (72,6%) opportunistic pathogen microscopic flora ( staphylococcus anreus,staphylococcus epidermidis, staphylococcus saprophyticus) had been found out in blood inoculations. in 36 cases (58%) side by side with destructive process in lungs the bacterial endo-and myocarditis with blood circulation failure had been determined.in 21 cases (34%) simultanious lesion of three organs (heart,lungs,liver) had been found. morphologic examinations of 5 dead cases (8%) internal revealed involvement of them in mosf-syndrome.hyperplasia of adenohypophysis;sclerosis of adrenal glands cortical layer;perivascular brain oedema,paralysis of brain capillaries and plasmorrhagia, cerebral thrombosis and cerebral abscess,necrobiosis of epithelium tubules of the kidney,pletora of hepar, fatty and granular degeneration of hepatocytes had been found.atrophy of white pulp and hyperplasia of red pulp, supress of lymphoid tissue, plethora and formation of infarctious had been found in spleen. mentioned changes in spleen were indispensable in ns. conclusion: in ns spleen can not secure it functions to support and appropriate detoxication potencial of organism,elimination of microbes,toxines,antoallergenes. insolvency of immunological link of antimicrobic defence is the starting mechanism of mosf developmentin ns. %neviere, jl. chagnon, b. vallet, d. mathieu, n lebleu, f. wattel ] ept of intensive care, hop calmette, lille, france ~everal studies have described tiypoperfusion of intestine during sepsis. 4owever, it is unknow whether the mesenteric blood flow is associated with nucosal hypoperfusion. additionally, the effects of resuscitation on the ntestinal microcirculation remain controversial. 3bjectives : to describe the effects of endotoxin in a porcine model during ~hock and resuscitation. ~ethods : ten pigs (30 kg) were anesthetized and instrumented for "neasurement of cardiovascular variables. gastric and gut oxygenation vere assessed by intra-mucosal ph and microvascular laser doppler lowmetry. after baseline data collection, a 30 minute intravenous infusion )f escherichia colt (serotype 34h4113, sigma, st. louis, mo) was begun ~t a rate of 150 pg/kg. an infusion of either saline at 1.7 ml/kg/min (group ; n=5) or saline and dobutamine at a rate of 5 pg/kg/min (group ii; n=5) vas begun 30 mn after the end of the endotoxin infusion. tesults : to td0 1120 t180 ~ fl0w fluid ioadin,q alone sfyras d, k perreas, e douzinas, k spanou, m pitaridis and c roussos critical care dpt, evangelismos hosp., athens univ, school of medicine. obiectives: much controversy exists concerning the beneficial effects of cvvh on sepsis. we studied the effects of cvvh application on septic patients with reference to the following parameters: i) survival rate ii) cytokines' removal and iii) timing of cwh onset. methods: 20 patients with sepsis (criteria according to accp/sccm, 1992) underwent cvvh as soon as they developed renal failure or dysfunction (urinary output<250 ml/8h, cr>2.5 mg/dl and bun>60 mgd'dl ). specimens were collected: blood samples before cvvh and therafter both blood and ultrafiltrate (uf) samples on 24, 48 and 72 hours. cytokines tnfa, i1-1 and ii-6 were measured by the immunoassay method in all specimens (uf and plasma -p) and sieving coefficient ([uf]/[p]) and 24 h solute mass transfer of tnf and i1-6 were calculated (v24 h x [uf] ). the apache ii score before cvvh onset, the duration of icu stay and the timing of cwh application related to the sepsis onset in days (ta) were recorded.with respect the mortality two groups were formed, i.e. group a (survivors) and group b (non-survivors) . the morbidity period in days of those septic patients who died in the past year and were not subjected to cwh (group c) was compared to that of group b. results: group a included 8 pts and group b 12 pts with mean+sd age (65 _+19 vs 64_+9, ns) and apache scores(24_+2 vs 24-+2.2, ns). the mean ta-+ sd was 3.6+2 vs 10-+6, p<0.05. the mean_+se morbidity period of group b vs group c was 20_+4 vs 8_+0.8 p<0.05 . the mean values of cytokines are presented in the following figures. the sieving coefficient for tnf was 0.2 and for i1-6 was 0.25. the solute mass tranfer was 6-fold the actual plasma content at a given time. . o conclusions: i) early application of cvvh seems to favourably affect the outcome of septic patients, ii) cytokine plasma levels do not decrease although cytokine removal is substantial, iii) it seems that cwh application in sepsis of any stage helps to buy time for further treatment. the most commonly monitored variables in shock stages idclude : arterial pressure, heart rate, central venous pressure, pulmonary artery wedge pressure and cardiac index. with vigorous therapy it is possible to bring these values back into the normal range in both survivors and nonsurvivors. therapeutic goal in septic shock stages is to maximize the values of cardiac index, 02 delivery (do2) and 02 consumption (c02). objectives: the main purpose of this article is to determine the relationship betwee~ 02 delivery an 02 consumption as a sign of hypoxia. fifteen patitents with septic shock were treated with intention to maximize the value of ci,d02 and v02. we compared the levels of these parameters between the survivors and nonsurvivors and found no significant differences after 24 hours. high levels of do2 and v02 may not guarantee against tissue hypoxia in early stage of septic shock. zjar~iic, dj janjic, lj. gvozdenovic, a.komareevic. t.petrovic, &marjanovic, institute of surgery, novi sad, yugoslavia objectives: evaluation and mutual comparison of clinical signs, laboratory data and microbiological monitoring in the patients with burn sepsis. method: retrospective analysis of the recorded data of all burn patients treated in our department between january 1989 and december 1994. specially attentions were given to data considering wound infection, positive haemocultures, positive urinocultures and characteristics of septic state. results: out of 372 patient there were 324 (87,09~) adults and 48 (12,9(3~) children. almost two thirds of the patients (238 -63,97~) were males. the predominantly cause (68,759~) of children's burns was scalding b~y hot liquids and flame burns ~63,97~) in adult patients. the most frequdntly species isolated from surface swat~ were pseudomonas aeruginosa (64"1796 in adult patients) and staphyloccocus epidermidis (78,57% in children). in only five patients (1,349~ 1 the haenmcultures were positive -pseudomonas aeruginosa was isolated in three and staphyloccocus aureus in two patients. urine infection was diagnosed in 6,72% of all patients. the treatment protocol included use of imipenem and polyvalent pseudomonas vaccine again~ pseudomonas aeruginosa and vancomycin and aminoglycosides against staphylococcus aureus. total mortality rate in this group of burned patients was 6,98~, but the mortality rate caused of sepsis was low (i 34%) . conclusions: early detection of any signs of wound infection and symptoms of septic state is a foundation for prevention and treatment of burn sepsis. the burn sepsis could be reliable detected by continuously monitoring the patient's status and by systematic microbacteriological monitoring of the burned patients. hyperdynamic vasoplegic septic shock p.f. laterre, p. goffette, j. roeseler, j.p, fauville, a. poncelet, p. lonneux, m.s. l~eynaert. dept. of intensive care, st. luc univ. hospital, brussels, belgium. splanchnic ischemia is described as a common feature of septic shock and could determine the development of msof. therapy such as noradrenaline (na) aiming at improving blood pressure is expected to worsen splanchnic ischemia by its vasoconstrictive effect and subsequent reduction in intestinal blood flow. ob[ective: evaluate the effect of na on splanchnic blood flow. material and method : in a patient admitted for variceal bleeding, ards and sepsis with positive blood culture, a fiberoptie catheter was positionned in the portal vein after recanalisation of its portosystemic stent shunt. blood pressure (bp-mmhg) , ci, svr, do 2 (vigilance ~ baxter), v02 (indirect colorimetry), arterial, mixed venous and portal vein blood gases, phi were determined before (to) and during (t1) na infusion (0, 1 to 0, 19 hcg/kg/min.) . changes in splanchnic flow were assessed by changes in portal oxygen saturation (sp02) and arterio-portal oxygen saturation gradient (sao, -spoe laterre, ,lp. pedgrim, th. dugernier, v. delrue, ph. hantson, p. mahieu, m.s. reynaert. dept. of intensive care, st. luc univ. hospital, brussels, belgium. aim of the study : prospective determination of plasma levels of in patients with ss and their correlation with the type of microorganism and outcome. material and methods : in 19 patients (pts) with ss and severe sepsis, plasma levels of tnfti, ill-b, il6 and il8 were determined every 8 hours for 3 days and on day 7 after fulfilling the criteria of ss and severe sepsis. results : in 9 pts, sepsis was caused by a gram (-) microorganism, in 6 pts by a gram (+) and in 4 pts no microorganism was identified. there were 12 survivors (63%) (s) and 7 non-survivors (37%) (ns) . cytokines profiles and levels were not different between gram (+) and gram (-) sepsis. ill-b levels were seldom elevated whatever the group studied. tnfot and il-6 were significantly higher in ns than in s ( objective: to evaluate the effects on the nitric oxide synthase inhibitor l-n~ hcl (546c88) on myocardial performance in human septic shock. method: septic shock was defined as severe sepsis with either persistent hypotension (mean arterial pressure; map<70mmhg) or the requirement for a noradrenaline (na) infusion >_ 0.i ]tg/kg/min with a map _< 90mmhg. cardiovascular support was limited to na _+ dobutamine (db), 546c88 was administered for up to 8 h at a fixed dose-rate of either 1, 2.5, 5, 10 or 20 mg/kg/h iv. during 546c88 infusion, na was to be reduced and if possible withdrawn, whilst maintaining map above 70 mmhg and the cardiac index (ci) as clinically appropriate. assessments were made at baseline (t = 0); at i h from the start of treatment (t = 1); and at the end of treatment (t = 8) with 546c88. conclusions: 546c88 can restore systemic vascular tone in patients with septic shock enabling na therapy to be reduced and/or removed. the ci tends to fall whilst lv performance is sustained over time. 546c88 is a novel vasoacfive agent for the treatment of septic shock, which is undergoing further clinical evaluation. laterre, f. thys, e. danse, j.p. pelgrim, e. florence, z roeseler, m.s. r eynaert. dept, of intensive care, st. luc univ, hospital, brussels, belgium. therapy aiming at improving blood pressure and cardiac index in septic shock (ss) might have deleterious effects on regional blood flow. objectives : compare the influence of volume loading (vl), dobutamine (dobu) and noradrenaline (na) on sushepatic oxygen saturation (shoe) and svoe-sho, gradient in treated ss. material and methods : in patients with ss, ci (thermodilution) , doe, svo,. sho,, svoe-sho e gradient and lactate (l) were determined before (to) and after (t1); vl, dobu and na. results: in 5 patients with treated ss, 16 tests were performed (vl n=8; dobu n=4; na n=4 method: septic shock was defined as severe sepsis with either persistent hypotension (mean arterial pressure; map<70 mmhg) or the requirement for a noradrenaline (na) infusion ~> 0.1 ~g/kg/min with a map _< 90mmhg. cardiovascular support was limited to na + dobutamine (db), 546c88 was administered for up to 8 h at a fixed dose-rate of either i, 2.5, 5, 10 or 20 mg/kg/h iv. during 546c88 infusion, na was to be reduced and if possible withdrawn, whilst maintaining map above 70 mmhg and the cardiac index (ci) as clinically appropriate. assessments were made at baseline (t = 0); at 1 h from the start of treatment (t = 1); and at the end of treatment (t -8) with 546c88. conclusions: 546c88 is a novel vasoactive agent that can sustain map in patients with septic shock, enabling na support to he reduced and/or removed. there is a tendency for the ci to fall during treatment, which may be reflex in response to the increase in systemic vascular tone. 546c88 is a promising new therapy for septic shock, which will now be evaluated in a randomised, placebo-controlled safety and efficacy study. k. guntupalli objective: to evaluate the acute effects of the nitric oxide synthase inhibitor l-n~ hc1 (546c88) on selected indices of organ function in patients with septic shock. method: septic shock was defined as severe sepsis with either persistent hypotension (mean arterial pressure; map < 70 mmhg) or the requirement for a noradrenaline (na) infusion --> 0.1 [xg/kg/ min with a map _< 90 mmirlg. cardiovascular support was limited to na + dobutamine. 546c88 was given for up to 8 h at a fixed dose-rate of either 1, 2.5, 5,10 or 20 mg/kg/h iv. during 546c88 infusion, na was to be reduced and if possible withdrawn, whilst maintaining map above 70 mmhg and the cardiac index (ci) as clinically appropriate. indices of organ function were assessed at baseline (t = 0); at the end of treatment (t = 8); and 12 h after treatment (t = 20) with 546c88. results. -median values (* assessment made at 8 h or when 546c88 discontinued). conclusions: there was no appareut dose-dependent adverse effect on these indices of organ function either during or after exposure to 546c88. the plmelet count tended to fall whilst creadnine appeared to increase over time in all dose cohorts. this novel and promising therapy for septic shock will now be evaluated in a randomised, placebo-controlled safety and efficacy sludy. pharmacokinetics of 546c88 in patients with septic shock preliminary results z. hussein, b. jordan, c. fook-sheung, k. guntupalli objective: to evaluate the pharmacokinetics of the nitric oxide synthase inhibitor l-n~ hc1 (546cg8) given by continuous infusion for 8 h in patients with septic shock. method: septic shock was defined as severe sepsis with either persistent hypotension (mean arterial pressure; map < 70 mmhg) or the requirement for a noradrenaline (na) infusion --> 0.1 ~tg/kg/min with a map _< 90 mmhg. cardiovascular support was limited to na • dobutamine. 546c88 was administered for up to 8 h at a fixed dose-rate of either 1, 2.5, 5,10 or 20 mg/kg/h iv. plasma was collected from each patient over a 24 h period and analysed for 546c88. pharmacokinetic parameters were derived from plasma concentration-time profiles using non-compartmental pharmacokinetic analysis. results: the (cm~ -maximum plasma concentration; auc -area under curve; cl -plasma clearance; v,, s -steady state volume of distribution; t'/2 -plasma elimination halflife). conclusion: the pharmacokinetics of 546c88 in patients with septic shock are dose-independent at infusion rates up to 2.5 mg/kg/h. at higher rates, clearance of 546c88 decreases without any marked change in volume of distribution. 546c88 metabolism may be partially saturable at dose-rates above 2.5 mg/kg/h. obiectives: investigate the effect of the no synthase inhibitor, l-nt-methylarginine hc1 (546c88) on the haemodynamics and survival rate in a conscious mouse model of endotoxin shock. methods: female cd-1 mice (25-35 g) were instrumented under gaseous anaesthesia (isofluorane, 2%) and connected to a swivel tether system for continuous monitoring of blood pressure and drug administration. results: after 24 h recovery, endotoxin administration (e. col• 026:b6, 6-12.5 mgkg -1 i.v.) elevated the plasma concentration of nitrite/nitrate (nox) and caused a progressive fall in mean arterial pressure (map) from 101 + 5 to 59 + 4 mmhg (n=5, p<0.05) at 12 h, with a survival rate at 24 h, 48 h and 72 h of 80%, 40% and 20% respectively. 546c88 administered as a 24 h continuous infusion (3 mgkg-th -t i.v., n=5), 4 h after endotoxin, inhibited the elevation of plasma nox and attenuated the fall in map from 105 + 2 to 70 + 3 mmhg (n=5) at 12 h, with an improved survival rate at 24 h, 48 h and 72 h of 100%, 100% and 60% respectively. conclusions: this study suggests that overproduction of no is involved in the hypotension and mortality characteristic of septic shock. inhibition of no synthase using 546c88 represents a novel and promising treatment for septic shock. cultures of e.coli (19,5%) and candida(8, 3%) were olso received from autopsy material of children;p.aeruginosa,unspored anaerobes,proteus sp.,s.aureus,b.pneumonia were found in the few cases. in adults the spectrum of bacterioflora was mo~ re limited speaking about the number of species and cultures. in generalized forms of bacterial pyo-septic pathology a wider specific spectrum of causative agents was revealed usua fly with associations. e.coli and k.pneumonia played the leading role in children as well as in adults. in general,k.pneumonia (23,7%cultures) and common e.coli(18,6%)prevailed according to the date of microbiological investigations of authopsy material in pyo-septfc pathology in 1994. objectives: .in spite of all clinical exertion sepsis is still the reason for high clinica! lethality. this study is characterizing the group of patients which survived a septi~ shock. methods: during a period of 12 months all surgical patients on icu were registrated prospectively, more than 270 parameters for each of them were documented'daily in a paradox file. results (see table 1): 20 of 286 patients fulfilled the criterion of a septic shock (r. bone, 1991 ) , 11 of them died at the 2 lth day, while the surviving group of patients stayed almost 51 days at icu. obiectives: to compare the effects of 6 and 10% pentastarch solutions to a human albumin solution on oxygen delivery (do2) in septic patients. methods: this stud}, included 41 septic patients with fever (t > 38~ tachycardia flqr > 90/rain), tachypnea (rr > 20/min) or mechanical ventilation, leukocytosis (wbc>12000/mm 3) or leukopcnla (wbc<400()/mm 3) and a clinical source of infection, who required a fluid challenge. in each patient the pulmonary arterial occlusion pressure (paop) was < 12 mmhg. patients were randomized to receive 400 ml of 4% albunun (n:i4), hydroxyethyl starch (hes -mw200/d.s. 0.5) 6% (n:14) or t0% (n=i3); 33 patients were also treated with adrenergic agents. results cardiac index (c1) increased significantly only in 10% lies (table) hemoglobin (hb) decreased significantly at 40 min in the same group. there was not significant change in oxygen delivery ( do2 ). baseline ci alb 3.7::1.3 (l'min/m 2) hes 6% 3.9=0.9 hes 10% 3. polyneuropathy of the critically ill (pci ) is a well recognized complication, acquired in the course of severe illness. we undertook a prospective study, to estimate the severity, extension and time of onset of pci in a selected group of 25 patient with established septic shock ( bone's criteria ). all patients received inotropic circulatory support and were mechanically ventilated. none received relaxants or aminoglycosides. pci was diagnose<by clinical investigation and by emg, and repeated weekly ( axonal degeneration ). after 3 days ( day 1 = onset of septic shock ) in 68% of the patients pci was demonstrable. after 14 days 80% of the patients had pci. there was a strong correlation between pci and other mof and between pci and encephalopathy ( eeg ). we conclude that i. pci is a common complication after septic shock. 2. pci develops early in the course of illness ( 85% after 5 days ). 3. pci can be considered as a part of mof, suggesting a common pathogenesis. objectives: we addressed two questions: 1) can serial measurements of vapco2 and avph also reflect the onset of tissue hypoxia during endotoxemia? 2) are whole body changes in vapco2 and avph associated with parallel changes in regional circulation? methods: in 12 anesthetized, mechanically ventilated dogs exhaled gases were sampled for determination of oxygen consumption (vo2). a catheter was positioned into the pericardial cavity to induce cardiac tamponade. ultrasonic flow probes were placed around superior mesenteric, left renal and left femoral arteries, and the corresponding veins were cannulated. group l served as control (n=6), group 2 (n--6) received 2 mg/kg of endotoxin. thirty min later, tamponade was induced to gradually reduce do2. results: systemic do2crit was higher (12.1_+2.2 vs 7.9+2.6 ml/kg.min, p <0.05) and critical oxygen extraction ratio (o2ererit) was lower (45.1+_9.7 vs 74.1+_9.1%, p<0.05) in the endotoxie than in the control group. meslenteric and femoral do2crit were higher in the endotoxic than in the control group (8.2_+2.5 vs 4.1_+0.5 mlll00 g tissue.min and 8.3_+2.3 vs 4.6_+0.9 ml/min, respectively, both p<0.05). regional o2ercrit were lower in we endotoxic than in the control group (mesenteric: 37.1_+15.4 vs 71.i_+7.4 %, renal: 30.7+ 24.6 vs 53.9-+28.7 % and femorah 48.1-+9.2 vs 75.3_+6.9 %, all p<0.05). vapco2 and avph followed a similar pattern in both groups, increasing slightly before do2crit was reached, but dramatically wh,en do2 fell below do2crit. in the presence like in the absence of endotoxin, systemic and regional do2crit calculated from vo2, from vapco2, or from avph were similar; do2crit was also significantly higher in the mesenteric and the femoral beds of the endotoxic than the control dogs. systemic and regional do2crit could be calculated from the blood lactate levels only in the control group. conclusions: during an acute reduction in blood flow i) vapco2 and avph can reflect hypoxic threshold in the presence like in the absence of endotoxemia. 2) alterations in organ oxygen extraction capabilities induced by endotoxin can be reflected by regional changes in vapco2 and avph. objectives: enhanced nitric oxide (no) release has been incriminated in the vasodilation and myocardial depression characterizing septic shock, but the administration of no blockers has yielded equivocal results. the present study explored the systemic and regional effects of a no donor linsidomine (sin-i) during endotoxic shock. methods: 14 anesthetized dogs received endotoxin (2 mg/kg iv), followed 30 min later by a saline infusion to keep cardiac filling pressures constant. oxygen uptake (vo2) was derived from the expired gas analysis. regional blood flow was measured by an ultrasonic flowmeter (transonic). results: the control endotoxie group (n=7) maintained low arterial pressure and systemic vascular resistance. the 7 dogs receiving a continuous infusion of 1,2,4 ~ug/kg.min of sin-1 starting 30 min following initial fluid resuscitation (each dose for 1 h), had a higher cardiac index (0.23-+0.03 vs 0.16_+0.08 l/min.kg, p<0.05) and lower systemic and pulmonary vascular resistance than the control group (1006-+170 vs 1460-+445 and 114-+38 vs 244-+84 dyne.sec/cm5, respectively, both p<0.05). arterial pressure and pulmonary artery pressure were not influenced. sin-1 significantly increased the mesenteric blood flow from 63-+17 to 148+49 % of baseline levels and renal blood flow from 67-+25 to 96-+24 % (both p<0.05) but did not influence femoral blood flow. the relative blood flow was increased in the mesenteric bed (at all doses), and reduced in the femoral bed (at highest dose). systemic oxygen delivery, vo2, oxygen extraction and blood lactate levels had similar course in the two groups. conclusions: in fluid-resuscitated endotoxic shock dogs, the no donor sin-1 increased cardiac index without deleterious effects on arterial pressure, and increased splanchnic blood fl0w. objectives: sepsis syndrome is associated with the release of a variety of inflammatory mediators, such as interleukins, tumor necrosis factor and platelet-activating factor (paf). administration of paf, a naturally occuring phospolipid, to laboratory animals has been demonstrated to resullt in pathological changes that closely resemble those found in sepsis. in addition, paf antagonists of various origins have been demonstrated to attenuate cardiovascular collaps and to protect against lethality in endotoxemia. in the present study the efficacy and safety of the paf-antagonist tcv-30g was investigated in sepsis syndrome patients. in addition, the putative inflammatory parameters tumor necrosis factor (tnf), interleukin il) -6 il-8, and soluble (s) e-selectin were measured in both tcv-309 and placebo treated patients. metho.~a randomized, double-biind, multi-center study was undertaken to compare the safety and efficacy of intravenously administered tcv 309 versus placebo (takeda chemical industries ltd., osaka, japan). tcv-309 was used as add-on to conventional therapy in the treatment of patients with severe sepsis and septic shock (1.0 mg/kg body weight intravenously over 2 hrs, twice a day for one week). day 28 mortality was registered, as well as vital signs, laboratory parameters, hemodynamic parameters, apache ii score and mof score. plasma samples for the determination of inflammatory parameters were collected twice a day for one week. the study was approved by each institutional review board and written informed consent was obtained for each patient. results: a total of 29 patients were included in the study. one patient had to be excluded as a result of protocol violation of the remaining 28 patients 12 patients were randomised to receive tcv-309 and 16 to be treated with placebo. there were no significant differences between the treatment group with respect to age. gender, and apache i~ score on admission. however, admission levels of il-6 and il-8, considered to reflect severity of disease, tended to be higher in tcv treated patients: 24.59. vs 2.37 ng/ml for il-6 (p.06) and 0.34 vs 0.04 ng/ml for il-8 (p.07) in tcv-309 and placebo treated patients, respectively. a remarkable difference in survival, however not significant, was observed at day 7 i.e. the end of tcv treatment (mortality: 2 out of 12 in the tcv group and 7 out of 16 patients in the placebo group). the inc;dence ui ~-,~verse events, 'n both treatment groups were comparable. plasma il-6 and plasma il-8 levels tended to decrease more rapidly in tcv treated patients compared to controls (p=.17 and p=.09, for il-6 and /l-8 respectively) conclusions: tcv-309 is safe as add-on treatment in sepsis syndrome patients. the data presented indicate that tcv-309 may enhance survival of sepsis syndrome which will be assessed in a phase ill study. objective: oxidation of lipids by free oxygen radicals playes an important role in sepsis. an important source of oxygen radicals is the respiratory burst of phagocytic leukocytes, especially~ polymorphonuclear leukocytes. clinically the hallmark of sepsis is the capillary leak, which is mediated in part by oxyradicals. the adult respiratory distress syndrom (ands} represents the most studied capillary leak phenomenon in sepsis. methods: in ii patients (df,~m, aged 20-81y.) with verified septicemia had been included in this study. beginning from day 1 oxidisable lipidautoantibodies (club), ~opterin and crp were determined in the patients serum and compared against the apache ii score during the days 1-7. 4 patients, 2f and 2m died @u= ring their stay at the icu, 3 of them in less than 48h~urs: after admission. at the begin surviving patients showed olab values between 75-119%, non-survivors between 68-104%, com= pared to neopterin values of 38-75 m~ol/l in survivors and 20-250 ~mol/l in non-survivors. apache ii score didn t show any difference. during the following days olab sbowed an in= crease in survivors, while in non-survivors olab stayed equal or showed a decrease. no difference between both groups was found in neopterin, crp and apache ii score. as result we conclude, tbat lipidperoxidation playes an important role during septicemia. olab as a marker of this process are predictive for the outcome of patients. obiectives : to evaluate the influence of cardiopulmonary bypass ongastrointestinal transit. meth~xls; gastrocoecal transit time (gtt) in 10 consecutive children on day 1 after uncomplicated open heart surgery was compared to gtt in 10 children after closed heart thoracic surgery and to healthy controls. gtt was measured by hydrogen breath testing, using lactulose (o,4g/kg in a 10% aequous solution) as nonabsorbable marker. exspiratory hydrogen content was measured using a electrochemical system (stimotron). patients in both groups had continuous infusion of morphine and midazolam and were comparable in respect to catecholamine dose. children with a history of toddler's diarrhoea and children with elevated central venous pressure were excluded. results: gastrocoecal transit was delayed in all patients after cpb, no transit was achieved within 480 minutes in 7/10. after closed heart surgery gtt was delayed 138(+-80) min vs. 56(+-20) compared to controls, but transit was achieved in all aptients within 285 rain. mean dopamine dose was 2,7mcg/kgmin after cpb and 4,8meg after closed heart surgery.. conclusions: after uncomplicated cpb gastmcoecal transit is extremely delayed. the degree of delay is not explained neither by the use of analgetic and sedative drugs neither by the use of catecholamines. factors directly related to cpb (e.g. low flow perfusion, hypothe,mia) may be responsible. these findings are of limited clinical relevance in ancomplicated cases. in critically ill patients delayed enteral feeding may contribute to infectious complications. studies of therapentic measures to accelerate gastrointestinal transit therefore are warranted. neonatal ecmo -czech experience. nekvasil r., penkova z, vasek l., plier p., bobula a novotna e., pavlicek v., hnilicka m. nicu and ecmo center brno and dept. pediatric surge13', children's hospital brno, czech republic. introduction. in spite of the fact that neonatal ecmo has been gradually considered a standard method of solving uncontrollable respiratol t failure in newborns in most countries of the western europe, the introduction of this sophisticated method in countries of the former communist block meets a lot of problem. material methods. in the course of two years, only 16 newborns, b.w. 2000 -4300 g, were reffered to neonatal ecmo because of uncontrollable respiratow failure. at the admission the average values of oxygenation index were 51 +12, and aado2 79,6 -+7,6 kpa. results. 4 newborns (25%) died shortly alter" admission or during transport without possibity to further is 5 newborns (31,3%) were treated using high-frequency ventilation (hfo or hfjv), one for conspicuous airway resistance paradoxically with low-frequency ippb. all ventilated newborns survived. ecmo was applied in 6 newborns (37,9%) and 3 of them survived the mortality rate of group mentioned was 44%. conclusion. the fact that in the czech republic (10 mil. population with natality rate of about 100.000 newborns/year) only small part of patients was reffered to neonatal ecmo has a number of causes. the most important are: unacquaintance of prognostic and indication criteria at forwarding workplaces and the aversion of neonatologist to all new.therapeutic interventions. last but not least, a negative role is also played by current change of health service systems when, under the influence of health-service insurance companies a newborn in serious condition given only a standard intensive care in primary and secondary nicu is literally "the golden calf", and therefore these workplaces either do not send him or send him too iate ~o an ecmo center. stndy aim: 8urfactant, which is being applied into the lungs for treatment of respiratory distress syndrome, can reach the circulatory blood system by absorption. it refinances local fimctiun of macrophages and lymphocytes and increases the accumulation of nentrophils into the hmgs this eunld influence the local acute phase reactiml in the hmgs after surfactant application. "ilia aim of the stud), was to investigate the influence of oatural and artificial surfactaat onto the acute phase reaction and the blood elottiog system. material :and methods: a) measurement of tnf-a, 11-6, ltb 4 and thromboxane b 2release in whole blood: each of 1.5 ml hepariaised blood of healthy adult volunteers was iucubated lbr 2 hears with eillier ill ug/ml lps, 1 mg/ml b&,ine smfactant (alveulhct/themae), 1 rag/nil artificial surfactant (exosurf/wellcome), 10 og lps + i mg/ml bovine surfactant or i0 ug lps + i mghal artificial sorfactant, tleparinised bhx-,d without additions was used for controls. '111e concentration of e)lokines and eicosanoids ".van measured semiquantilatively by eijsa or eia (dimmva/hamburg) after ceatrifagalion, b) measurement of platelet aggregation: each of 1.5 ml haparinised hltxxl of healthy adult vohmteers was inculmled lbr 2 hours with eilher 10 aghnl i,ps, i mg/ml bovine surfactant (alveafact/thomae), i mg/ml artificial snrlhctant (exosurf/wellcotne), it} ug lps + i mghnl bovine snrfactant or 10 ug lps + i mg/ml artificial surfaetant, tlepminised blood withont additions was nsed for controls. platelet aggregation in whole blood was measured by impedance in the aggregometer (g~nstaedt). c) measurement of the dotting activity: citrated bleed of healthy adult w~lunlccrs was mixed with difl'ereul couccalrations of imvine or artificial snrfactant fad then prolhrombiu tiaras and imrlial thrmnlx~plaslia filues were ineasnrcd. for measurement of the prokoagulatioo acfivily the reagent in the qnick test was replaced by surfaclant. results: dependmlt on the dose both surfactants stimulated the release of 11-6 but not or tnf-a io wlmle blood. "illere was no suppression of the lps-indaeed eytokine-release. both surfaclants did not directly influence the extrinsic blood coagulation system, while both activated the inniasie activity dependant on the dose. platelat aggregatiun was slightly stimulated by bovine snrfactunt and strongly inhibited by by artificial surthctant. in comhinafion with 1.1~ both factors showed no difft.'renca to lps ahme.'fhe reltu.tse of eiconasnid~." was stimolated more by artificial surfactant then by bovine surfilctant with regard to the cyehmxygelmse (thromboxsne b2) and the lipooxygenase (lencotrieae b4) pathways. conclusion: we cooclude: 1) i:lovine as well as artificial surfaelant stimulate the cellular inmnn]e response. 2) bovine as well as artificial surfnctant activate the intrinsic ctmgnlation cascade. 3) bovine surlilctant stimulates platelet aggregation, artificial sar-fijctant inhibits platelet aggregation.. introduction: although the use of modem noninvasive methods like measurement of tope2, tcpco 2 or san 2 leeds to an carly infonnation about pathologic alterations of the gas exdmage of ventilated patients, these methods cannot replace invasive bloodgas analysis. a new noninvasive method to monitor the capillary gas exchange is the reflection spectrophotometry. to record reflection spectra with a ldgh sensitivity on the surface of tissue we eonstnmted a special mierolightgnide photospectronmter (empito). this spectrometer enables the investigation of local helerogenities of llbo 2 and 1 lb content, capillary flow rate and oxygen uptake rate. aim of the study: 1 ) to compare the sousitivity and specifity ofthe empilo with other non-invasive method~ of monitoring the gas exchange in neonatal intensive care patients. 2) to investigate the possibility of obtainiug infonnations about the local 02nptake and the venous as well as arterial 02 satnration. patients and methods:we investigaled 16 non-selected patients of oar pediatric intensive care unit. for coulinnons monitoring we conslracted a special sappert to fix file end nf the microlightguide+ during the investigation tope2, tcpco 2 and san 2 were measured by standard methods. "fo investigate the local tisslm oxygen uptake we recorded 1000 spectres within 2 minutes by moving the lightgnide over an described area of the skin of the patient. 'lhe reselting i(gx) l ibo 2 vahms were sorted by a software package. on the assumption that the low values correspond to the tibo 2 value of the veaons ends of the capillaries and the high values to those of the arterial ends we 2) it is possible to gel infonnations about the local o2-uptake and arterial 02 saturation ia the peripheral tissue by photoslg'ctroscopy. 3) in combination with noninvasive pl i-measorement it seems possible to reduce withdrawal of blood for invasivn bhmd gas analysis. objectives:improve ventilation,tissue oxygenation and acid base balance among extremly low birth weightand premature infants. methods: fourteen cases were s~udied,their gestational age ranged from(27-32)ws.continnous positive air way pressure was applied to six cases at peep level from (3-6)cm h2o through nasal pronge,(group i),the other 8 cases were managed as routine,(group ii).blood gases, tcpo2,tcco2,resp.rate,depth and pattern were monitored for assessment of tissue oxygenation and ventilation, results: our rasults showed that early application of cpap improve ventilation among (83.3%)of cases,while (16.7%)of cases need imv.the cases of group ii need imv among (75%)of the studied cases during the second or the third day of life. conclusions:-this preliminary study showed the import-............ ance of early application of cpap before the onset of respiratory distress syndrom to improve spontaneous ve ventilation,tissue oxygenation and to gecrease the risk of intubation. comparative assessment of pediatric intensive care; a national multicenfre siudy. reinoud j.b.j. gemkc, gouke j. bonsel 2, the dutch picasso (pediatric intensive care assessment of outcome) study group. twilbelmina children's hospital and utrecht university, utrecht 2department of clinical epidemiology, academic medical center, : amsterdam, the netherlands the performance of all (10) pediatric intensive care units (icus) in the netherlands (6 tertairy and 4 non-tertiary) was analyzed in an open prospective muiticenter study. effectiveness of care was defined as the ratio of observed to prism-score derived expected mortality 0aenee, standardized mortality rate). efficiency was determined by 2 objective criteria (mortality risk > 1% or administration of at least 1 icu-dependent therapy)'. 1063 consecutive admissions aged < 18 years old were included. overall, observed and expected mortality were in good agreement (p > 0.5). between hospitals, crude mortality showed wide variations (mean 7.1%, range 1-10%). however, in each center, observed and expected mortality were similar (mean ratio 0.99, range 0.8-1.5). in tertiary care centres, severity of illness corrected mortality in high-risk patients was less than in non-tertiary care centres; paradoxically, in low-risk patients the opposite was found. probably the large proportion of low-risk tertiary care patients suffering from severe, incurable chronic disease, explains the higher mortality in this group. this indicates that simultaneous assessment of circumstances of dying and of long term morbidity in similar future studies is imperative. the average proportion of efficient icu days was 72%, however large variations between units were found (range: 22-95 %). in conclusion differences in mortality rates among pediatric icus were explained by differences in severity of illness. high efficiency rates in combination with adequate effectiveness, found in several centres suggest that admission and discharge decisions might be improved by a better selection of high risk patients requiring icu-dependent therapies, especially in less efficient centres. objectives: previously published studies showed that serum lactate levels correlated with outcome of severe ill adult, 'we hypothesized that critically ill newborns are often incurred hypopeffusion manifested by elevated lactate levels. these initial blood lactate levels should be related to nicu outcome. design: prospective study with ethical comfnittee approval. setting: the 14-bed neonatal intensive care unit of a university hospital material and method: a total of 209 consecutive outbem newborns admitted to nlod from 01,10.1991 to 31.,19.1992 were enrolled to the study. babies who died or were discharged from the unit within 48 hours of treatment were excluded from the study, mean birth weight was 2040g (+/-820r), mean gestatational age was 35 weeks (+/-3.5 wks), mean age at the admission was 50 h (+/-110hi. multiple (~_2j organ system failure occurred jn 38.3% of babies at the admission./~tertal lactates were measure/at the admission, among 22 -26 hour and 46 -50 hour of n[c'lj therapy. outcome was defined as a mortality and length of nicu stay. results" survival rate was 68.4%, mean length of nicu stay for survivors was 17.8 days (+/-15.1 day). we found high lactate levels at the admission in 82.8% babies (~9.2% with levels above 5.0 retool/i). the mean arterial lactate concentrations for nonsurvivors were signiftcahtly higher than for survivors durin~ consecutive da~ as follows: objectives: the purpose of our research was to analyze the frequency of bronchial asthma (b.a.) exacerbations in pregnant women and health status of infants. methods: the research was based on the epidemiological investigation and prolonged observation of 23 pregnant women with b.a. during the gestation period. remission of b.a. before the pregnancy in excess of 5 years was recorded in 9 patients (39.1%), 6 patients (26.1%) reported a 2-3 year remission and 8 patients (34.8%) had a remission lasting less than 12 months before they became pregnant. results: seven patients (30.4%) developed medium attacks in the second half of pregnancy, four patients (17.4%) experienced light attacks of b.a. asthma attacks were most frequently caused by acute respiratory diseases and stress factors. in two cases with grave manifestation of b.a., the pregnancy ended in abortion within the first 16-18 weeks due to the frequent and heavy choking attacks. to fight b.a. attacks, five patients used 132adrenomimetics (salbutamol, becotid) in sprays, six women were administered theophyllinum and salbutamol in the form of tablets during 1-2 weeks. a significant portion of pregnant women with b.a. (78%) exhibited frequent complications during pregnancy (toxemia, late gestosis, threat of miscarriage). our findings prove that babies born from women with b.a. of domestic and pollen origin had a low body weight (2800-2500 gr), functional immaturity and chronic antenatal and intranatal hypoxia twice as often as the infants born from healthy women without allergic background. conclusions: preventive treatment of women with b.a. prior to pregnancy is required to maintain a stable remission of the disease, which is a key to having healthy children delivered by mothers suffering from b.a. introduction. intracerebral hemorrhage (ich) is a common event in human prematudty, affecting about 25% of newborns weighing below 1500 g who are born before 32 weeks of gestation, however, little is known about the pathogenesis of ich with exception of the prematurity of the brain itself, (birth) trauma, and asphyxia. the postischemic production of oxygen free radicals (ofr) dudng reoxygenation as a cause of brain damage has been demonstrated in 9 animal research. since almost all preventive antioxidant activity of plasma is associated with ceruloplasmin and transferdn we investigated the association of such iron-oxidizing resp. iron-binding proteins and ich. we could demonstrate significantly reduced levels of both, iron-oxidizing and iron-binding proteins, in premature asphyxiated newboms pdor to development of ich. an increase of suparoxide after hypoxia in the presence of iron ions facilitates the formation ofthe highly reactive hydroxyl radicals. our data support the theory that ich may be caused by ofr, which can damage any sensitive tissue including growing endothelial cells. the estimation of transferrin-saturation and measurement of ceruleplesmin levels might help to identify an infant at dsk before the onset of ich. with the new medos | hia-vad | cardiac assist system the missing tool in the armamentarium of cardiac surgeons is available in two pediatric sizes: i0-ml and 25-ml pump volume. the right sided pumps are 10 % smaller for biventricular use. between february 1994 and may 1995 we implanted this assist system in 6 children. the indications and demographics are indicated in the following table (left ventricular assist device-lvad, right vad-rvad univentricular vad-uvad, post cardiotomy cardiac failure-pcf, dilated cardiomyopathy-cmr bland white garland syndrome-bwg, tetralogy of fallot-tof, hypoplastic left heart syndrome-hlhs). objectives: evaluate tile effeci'of inhaled nitric oxide (no) as puhnona] t vasodilating agent ill tile posloperalivc period after correclion of congenital heart defects in 3 infant. patient n.l: 4 kg, 7 lnonlhs, down syndrome undenvcnl rep~fir of atrioventricular septal defect (avsd). after surgery the puhnonary arlcry pressure (pap) slowly rose to tile syslemic dcspilc tnaximal eonvcnlional fllerapy (fentanyl 4 mcg/kg/h, hypocapnia of 27 mmhg and metabolic alcalinization). no was delivered into tile inspiratory branch of!be breathing circuit at 10 ppm, and the gas aoalyser for no and no2 (polylron dmger) were situated at the espiratory branch, a rapid dccrcasc of pap io i/3 of systemic was obtained with a dramalic improvement. no was continued at 5 ppm for six days and the baby was exlnbated if! days after surgery and discharged from the icu 5 days after. patient n.2 : 4.5 kg, 6 monlhs, onderwen! repair of avsd. the day after surgery the systemic oxygen salnralion was 76% wilh a pap at 75% of systemic. two hours of c01wenlional therapy failed 1o improve ihc patient and no administration was slarled at 10 ppm. so2 dramatically incrcased to 95%, but the pap dropped only to 50% of syslemic. nevertheless ihe clinical conditions improved and the no administration could be reduced at 5 ppm in the following 6 days. she was extubaled 8 days after surgery and discharged from the icu 20 days after. patient n.3:12 kg, 3 3'ears. underwen| hearl tral~splantalion for congenital heart disease with moderate hypoplasia of pulmonary arlcrics. at the end of cardiopulmonary bypass the transpnlnlonary al~erio-venoas gradient yeas higher than 7 mnfflg and we speculaled !hat w'ls due to a degree of puhnonary vasocostrictiont. the nsnal dose of no was otilised, however no significant modilicalion of pulmonary pressure or systemic oxygen saluralion was noled, and after 1 h no was discontinned. tile palienl was carried io the icu with maximal inotropic support, extubated after 4 d;b's and disclmrged from the icu after 15 days. in all patient no major adverse effect relaled to no admilfistration ",','as holed. conclusion: in our experience no ms a pulmonary vasodilaling agent is effective and easily adjustable to tile palienls requiemenls, however its use remains limited ill those palienl ill whoin tile alnonll! of fixed inlllllojliify vascular resistance is predominanl. we report the use of ecmo support in two unusual cases of severe tracheal disruption in which it had become impossible to achieve adequate ventilation. case 1: severe tracheal laceration due to aspiration of a share forelan bodv: a previously healthy 13 month old toddler was referred for ecmo following aspiration of a porcelain foreign body (with razor sharp edges) which had become embedded in the right mainstem bronchus with massive extrusion of air. this was removed on veno-arteda[ ecmo support, as the patient was unventilatable prior to bronchoscopy due to ongoing airieak. ecmg was continued after bronchoscopy to permit airway healing without the presence of an endotracheal tube. unfortunately, an extensive pulmonary haemorrhage on day 4 of ecmo necessited re-exploration of the airway. this revealed a posterior tracheal tear from the cricoid to the middle of the right lower lobe. following repair the patient was left on ecmo support together with high frequency oscillation ventilation (hfov), the latter being used to minimise potential aideak and maximise alveoli recruitment. ecmo was weaned after 17 days (420 hours) -the patient was extubated 7 weeks later. case 2: tracheal wound dehiscence due to seosls -tracheal transelant on ecmo: a 4 month old infant with a c[inically significant congenital long segment tracheal stenosis and left pulmonary artery sling underwent resection of the stenosis, followed by primary reanastomosis. this was complicated, 5 days later, by severe mediastinitis and complete dehiscence of the anastomosis. an autologous pericardial patch was used to repair this, however, the tracheal wound again dehisced 4 days later making mechanical ventilation impossible. in view of ongoing sepsis and a severely disrupted trachea ecmo was the only possible form of support. following resolution of the local sepsis (4 days) a definitive procedure in the form of a tracheal homograft (transplant) was undertaken on ecmo. the patient was managed on ecmo and hfov for a further 3 days, the hfov being used to optimize rapid lung inflation. unfortunately this patient died 9 months after weaning from ecmo due to complete disintegration of the homograft, which was not deemed reparable. conclusions: 1) ecmo can be used in the acute management of oxygenation when there is major airway disruption making mechanical ventilation impossible. 2) hfov was a useful adjunct in aiding recruitment of lung volume on ecmo in these two patients. backoreund: persistent pulmonary hypertension of the newborn (pphn) consists of a heterogenous group of diseases ranging from transient reversibte pulmonary hypertension to fixed primary malformations of the lung (primary pulmonary dyspfasia-ppd). inhaled nitric oxide (ino), a selective pulmonary vasodilator, has been proposed as a treatment for severe pphn. obiective and methods: ino was administered to 23 near term neonates with severe persistent pphn, oxygenation index > 25 and echocardiogrephic evidence of pulmonary hypertension, in order to further determine the clinical role of ino in the treatment of pphn. the response to ino was also analysed retrospectively to examine whether this could be of diagnostic value in differentiating at an early stage patients with reversible from fixed causes of pphn results: twenty one of the 23 patients studied responded to the initial trial of 1no (20ppm x 20 minutes), as defined by a greater than 20 percent improvement in pad2 as well as a fall in the el to < 40. these 21 patients were continued on ino therapy, with 3 patterns of response emerging: pattern 1 babies (n=8) continued to show a sustained response to ino and were successfully weaned from it within 5 days -all survived. pattern 2 babies (n=9) failed to sustain their response to ino over 24 hours, as definded by a rise in the el > 40. six survived, five with ecmo. pattern 3 babies (n=3) had a sustained dependence on ino for 3 -6 weeks. all three died and lung histology revealed severe primary pulmonary dysplasia (ppd). patients with ppd (pattern 3) not only required ino for longer periods of time than did the sustained responders (pattern 1), but also required significantly higher doses of ino we report on the air transport of 32 paediatric intensive care patients. these transports fall into three categories: 1) retrieval of critically ill neonates and paediatdc patients referred for either ecmo or inhaled nitric oxide (ino) (n = 12). one patient was transferred on ind. mean transfer time 2.2 hours (se +0.6hrs). 2) long distance international transport using chartered aircraft (n = 11). the indications for these transfers included both urgent retrievals for cardiac surgery and semi-elective transfer of stable patients back to their referring unit following treatment in tertiary centres. mean transfer time 4.4 hours (se +0.4hrs) 3) long distance international transport using commercial aircraft (n = 9). indications for transfer were either semi-elective retrieval for tertiary treatment or the return of stable chronically ventilated patients to their referring hospitals. mean transfer time 14 hours (se _+1 .fhrs, longest 24 hrs). the transport team consisted of a paediatric intensive care doctor of at least registrar grade and a registered sick chidrens nurse with intensive care experience. the administrative components of the transfer (ambulances, airlines, customs) were managed in collaboration with companies specializing in air ambulance transfers. outcome: all the patients were safely transported to their destination without mortality or morbidity. complications durino transfer ir~lv~; 1) patient complications -semielective endotracheal tube change and central access needed in the only patient brought to the commercial aircraft by the referring hospital (all others retrieved directly from referral hospital), seizure in patient with known encephalopathy, severe cyanotic spells in patient with fallots tetralogy who was retrieved for urgent surgery for this indication 2) mechanical compfications -ventilator failure, incubator battery failure, oxygen regulator failure -all occurred with equipment sent from referral hospital, this was unfamiliar and unchecked by our transport team -it was not the decision of the transfer team to use this equipment on this single occassion. 3) administrative complications -confiscation of incubator battery by airport security police, excessive delay by custom officials (2 hours) in the airport. the incidence of such problems were felt to be low and unpredictable. in conclusion: mechanically ventilated paediatric patients can be safely transported on both chartered and commercial airlines. these transports are best accomplished by trained intensive care medical and nursing staff with the backing of an air ambulance organization competent in arranging the necessary administrative details. it is essential to use your own equipment and to retrieve the patient _directly from the referrin(] hospital to minimise ootential complications. our experience with anaesthesia for paediatric electromyography _w_._pla_ti_k_a_n_o_v, r.eousseff, k.pavlova, d.marinova dpts. of anaesthesiology and int. care and clinika] neurophysiology, med. university, pleven, bulgaria ~)_b_j#~ti_v~. to t~st a " heavv sedation " regimen of anaest-es~a for the purpose of paediatric electromyography d#s~gil~ non-randomized,non-blinded human trial in the seting of an uriiversity hospetal. _m_a_t_eri_a_is_a_nd_ m_e_th_od_s_.110 children,asa i-if,median age 6 years,range 9-13 who undervent eleetrcmyography required anaesthesia. they recieved low-dose ketamine + i~iazepam or midazolam via musculary route( 25 children,age 0 -3 yrs,ketamine 2,5 mg/kg, diazepam 3-6 mg total dose ) or per os ( 85 children,ketamine 5-7 mg/kg,diazepam 0,3 mg/kg or midazclam 0,4 -0,5 mg/kg ) _resu_l_t_s. 20 -25 minutes after medication a state of heavy sedation with weak spontaneos and stimuli-provoked movements was achieved in all children, that lasted 30 -60 minutes and allowed adequate needle emg and nerve conduction investigation. 11 children recieved additional 0,6 -1,0 vol.% halothane during the placement of the needle. non -invasive blood pressure , breath and heart sounds and hb sad2 by pulse oxymetry were monitored.none of the older children disclosed memories of pain when asked after they regained adequate verbal contact.no complicationes were observed. antenatal maternal steroids reduce the risk of periventricular-intraventricular hemorrhage in very premature neonates treated with natural surfactants. i.apostolidou, c.papagaroufalis, g.touloumi, m.xanthou, n.kalpoyannis a' and b" neonatal icu "ag. sophia" children" s hosp. athens, greece. dept of hygiene and epidemiology, athens university, greece. obiectives: the aim of the study was to evaluate the association of periventricular-intraventricular hemorrhage (p-ivh) in surfactanl treated premature neonates with pre-and postnatal variables. methods: the population of the study was 88 neonates admitted during the years 1990 to 1992, with gestational age _< 32 weeks and severe respiratory distress syndrome (rds) (mechanical ventilation and arterialalveolar oxygen tension ratio (ajapo2) <0.22), who received rescue therapy of at least two doses of natural surfactants (alveofact or curosurf) and examined with ultrasound and/or autopsy for the presence of p-ivh (papile's classification). the examined factors in each neonate were the following: gestational age, birth weight, sex, multiple pregnancy, antenatal maternal steroids (complete and incomplete course of betamethasone), a/apo 2 before the administration of the 1st dose of surfeclant, delivery, apgar score at 5min, type of surfactant, pneumothorax and patent ductus arteriosus. the statistical methods used were x 2 and one-way analyses of variance followed by logistic regression medels, results: the incidence ot p-ivh was 31.8%. three factors were found to have an independent relation to p-ivh (final logistic regression model): gestalional age, a/apo 2 before surfactant administration, and antenatal administration of maternal steroids (complete and incomplete courses). for every 2 weeks of lower gestational age the neonates had an almost doubled associated risk of p-ivh (or: 1.92, 95% c1:1.14, 3.22). for every 0.02 on average decrease of a/apo 2 before surfactant administration the risk of p-ivh in the neonates was 1.27 times higher (95% ci: 1.02, 1.58). the neonates whose mothers received antenatally steroids had only one tenth of the risk of p-ivh of the neonates whose mothers had not (or: 0.10, 95% ci: 0.01, 0.82). conclusions: our results suggest that the antenatal administration of maternal steroids, even less than 24 hours before delivery, reduce the risk of pqvh in very premature neonates treated with natural surfactants, whereas the small gestational age and the lung immaturity still remain the main risk factors tor the development of p-ivh. we analysed retrospectively the management of 103 (51 boys, 52 girls) accidental ingestions of foreign bodies in children (mean age : 2.8 years, range : 7 months-10 years). no child had ingested more than 1 foreign object. the majority of the ingested foreign bodies were : coins (n : 44), toy parts (n : 11 ), jewellery (n : 3), batteries (n : 16), "sharp" materials such as needles and pins (n : 21), "large" amounts of food (n : 8). impaction of food occurs more frequently in children after oesophageal reconstruction in cases of oesophageal atresia. although according to literature "coca-cola" is reported to be effective, this was not seen in our experience. 28/103 patients had minor transient symptoms at the moment of ingestion, such as retrosternal pain. only 4 children experienced severe manifestations (cyanosis, dysphagia). in these children, endoscopy revealed oesophageal and gastric erosions. children were seen at the emergency ward within a few hours after the accident ( mean : 3 hours, range 20 min. -28 hours). chest and/or abdominal x-ray was performed as first-line investigation ( 93/103 objects were radio-opaque), and revealed an (unexpected) oeeophageal impaction in 6 children. in 87/103 the foreign body was in the stomach. batteries, sharp objects and objects trapped in the oesophagus were removed, either by endoscopy or by magnet-extraction whenever possible. the outcome of the patients was excellent. no complications were observed. extraction is recommended in symptomatic patients, and whenever the foreign body is trapped in the oesophagus, or if the foreign object is "sharp" or a battery. objectives: two strategies were used for management of malignant diphtheria in children aged from 0.5 to 13 years. methods: protocol n1 consisted of intravenous administration of diphtheria antitoxic serum, prednisolone (2 mg/kg bw/day), plasmapheresis and supportive care. protocol n2 included the use of antitoxic serum against the background of high-dose dexasone (2-3 mg/kg bw/day), hemocarioperfusion and a preventive use (before the clinical manifestation of myocardial damage) of inotropic medications, inhibitors of angiotensin-converting enzyme and pentoxyphylline. each of protocols included the monitoring of serum toxin (diphtherin) levels. results: the group of patients treated according to the protocol n1 consisted of 17 children with malignant diphtheria, 11 of them with severe malignant diphtheria (grade 2 and 3). all patients exhibited the circulation of toxin during at least three days after the start of treatment. all 11 patients with severe grade of disease demonstrated heavy cardiovascular disturbances associated with malignant diphtheria. of the 11 children in the group died seven. the children of the second group were treated according to the protocol n2. out of total of 22 patients of this group. 11 patients had severe malignant diphtheria. in all children a significant reduction in serum toxin level was revealed after hemocarboperfusion. in all but one case the satisfactory control of cardiovascular function on was achieved. of 22 children admitted to the trial 21 survived, one child with malignant diphtheria of grade 3 and congenital filbroelastosys of the left ventriculum died. the severity of neurological complications was similar in each of groups. conclusions: the use of hemocarboperfusion, high-dose dexasone and early prevention of heart failure as a adjunct to the standart treatment has been shown to be of benefit in the management of malignant diphtheria. t. schaible, i. reiss, j. m611er, l. gortner med. university of lqbeck, children's hospital, kahlhorststr. 31-35, 23538 l~beck, germany surfactant therapy seems a promising approach for the treatment of the biochemical and biophysical abnormalities of the pulmonary surfactant system in severe ards. patients and methods: over a 18 months period 10 non-neonatal pediatric ards patients (age 1-38 months) in a "pre-ecmo"-situation (oi 40 over 4 h) were treated with bovine surfactant (alveofact| the underlying conditions-of ards were pneumonia (5), sepsis (2), immunosuppression (1), near drowning (1), neurogenous ards (1). a total of 20-120 mg/kg b.w. was applied in several fractions. before surfactant therapy, we first tried different ventilation (best peep-finding, inversed i/e-ratio, hfo-ventilation) while monitoring the pulmonary mechanics. for hemodynamic stabilisation both norepinephrine and epoprostenol were used to optimize pulmonary perfusion for max. 4 hrs. if there was no improvement of the oi by at least 10, further treatment with surfactant was initiated. in addition to surfactant all patients received a treatment with dexamethasone of 1 mg/kg in 2 doses. patients with no benefit (oi remained unchanged or increased within the max. 2-4 hrs) were taken on ecmo. results: nine patients improved within 4 hours after surfactant therapy: the oi decreased from a level of 41 (mean, range 22-100) before our treatment to a level of 16 (mean, range 6-60) thereafter. in 6 patients we were able to continue the positive effects of our treatment and they could be weaned of the respirator within 3-10 days. the other 3 patients got worse despite respiratory improvement, they suffered of multiorgan failure of more than 3 organ systems. the last patient did not benefit from surfactant, he had to be put on ecmo, but died because of a complication (hemopericard)after 10 days. the autopsy of the ecmo-patient showed a pulmonary fibrosis, but the other 3 death were not due to pulmonary failure. conclusion: a different sequential ards treatment integrating surfactant therapy can reduce the number of patients requiring ecmo. but ecmo as a therapeutic tool should be available in centers involved in ards treatment. l.blindl, t.p.le, h.weinzheimer, centre for paediatrics, university of bonn, germany selective reduction of elevated pulmonary vascular resistance by inhaled prostacycliu (pgi) has been reported in adults with acute lung injury, neonates with persistent pulmonary hypertension and in one infant with idiopathic pulmonary hypertension. we report on the effect of aerosolized prostacyclin in two children with secondary pulmonary hypertension. patient 1: in a boy with down's syndrome an avsd had been surgically corrected at 11 month of age. at 5,6 yr of age a catheter examination revealed a pulmonary vascular resistance of 70 % of systemic vascular resistance in room air and at an fin2 of 1.0. prostacyclin (0.5 mcg/ml) was administered with a jet nebulizer at an fin2 of 0.21. pvr declined to 0.4 systemic vascular resistance and returned to baseline after stopping pgi-inhalation. subsequent intravenous infusion (5 ng/kg rain) had to be stopped after 5 minutes because of systemic arterial hypotension. patient 2: a 8 month old male infant with bronchopulmonary dysplasia developed suprasystemic right ventricular pressure inspire of therapy with oxygen and nifedipin. while he was spontaneously breathing 60 % oxygen via face mask pao2 was 37 mmhg, arterial ph was 7.35. systolic arterial pressure was 85 mmhg, a rv-ra gradient of 100 mmhg was measured by cw-doppler. while fio2 was maintained aerosolized prostacyclin was administered over 30 minutes. rv-ra gradient was 70 mmhg, systemic blood pressure 75 mmhg, pao2 58 mmhg. two hours later nitric oxide (20 ppm) was inhaled at an fio2 of (3,6. rv-ra gradient declined from 100 to 65 mmhg, systemic systolic blood pressure remained stable at 105 mlnhg. discussion: sporadic experience shows that aerosolized prostacyclin selectively reduces elevated pulmonary vascular resistance in some patients. in patient 2 the poor response to inhaled pgi1 compared to inhaled nitric oxide may be explained by the fact that the action of pgi is not independent from endothelial function, limiting it's effect in severe vascular disease. during the last two years (1993-94), 51 infants weighing less than 1000gr. admitted to our referral unit. thirty four of them (67%) survived, (28% of infants weighing 500-700g and 78% of infants weighing 701-1000gr survived) for the years 1986-87-88 the survival of these infants was 53% and for the years 1976-77-78, 14% (p<0.01). we analyzed the perinatal and neonatal factors influencing the outcome of these infants. the comparison among neonatal survivors (1) to neonatal deaths (2) shows: gestational age: 27.6 w (1) to 26.4 w (2) (s). birth weight: 923.5g (1) to 724.7 (2) (s). apgar score: 7,90 (1) to 7.56 (2) (ns). presentation and mode of delivery: breech presentation is associated with higher incidence of neonatal deaths. i.v.h. (at the age of 8 weeks): no one of the survival infants had evidence of i.v.h. respiratory problems: intubation, at the admittance of the infants 32.3",,(1) to 95% (2) (s) use of surfactant: 70% (1) to 95% (2). bpd observed in 62% of the babies and only one was dependent on oxygen at home. antenatal betamethasone was given in 20% of the mothers. in conclusion: 1) a great improvement in the survival rate observed in these infants the last 20 years in our unit. 2) factors with positive effect are increasing gestational age and birth weight, the absence of i.v.h. and the use of surfactant. the breech presentation and the severe respiratory problems increase the incidence of death. animal experiments demonstrated, that brain temperature determines the amount of neuronal damage caused by hypoxia and that mild hypothermia may have a protective effect. until now there is no method described and evaluated to measure brain temperature in neonatal intensive care units. we non-invasively measured brain temperature analogues, nasopharyngeal (tnasoph) and zero-heat-flux temperature (zht) at the temple whereby under zero heat flux surface temperature represents deep head and thus brain temperature. the aim of our study was to investigate the practicability of the method, the relationship of the two brain temperature analogues to rectal temperature (trect) and their dependence on insulation, thermal environment, body activity and time course. we investigated 19 healthy preterms less then 2 weeks postnatal age (gestational age 315 +_ 2.1 wks; x + sd, weight 1653 +_ 370 g) in an incubator. tnasoph was measured by a thermistor within a feeding tube, advanced to the nasopharynx, zht temple by a thermistor and a heat flux transducers both covered by an insulating pad, and trect thermal environment was characterised by operant temperature (tair .0.4 + twall 0.6). body activity was video taped. measurements were performed during the following interventions: i/ insulation increased by turning the temple with sensors onto the mattress (15rain). ii) insulation increased by a cap (30 min), iii) 30 min after its removal, iiii) increased operant temperature by 1.6 + 0.5~ (60min). results: seven children with ea had a gasless abdomen, the endoscopic procedure excluded (6) or diagnosticated an upper pouch fistula (1). in patients who suspected "h" fistula (2) broncoscopy has strong advocated method to make diagnosis and established cervical approach. from july 1992 14 newborns with ea and lower pouch tef received a selective transtracheal incannulation. we were not able to proceed just in 1 case with congenital subglottie stenosis. in these patients we provided gastric drainage by radiopaque and flexible 3-4 french catheter. the knowledge of the precise anatomic position of tef consent to adjust the tip of the endotracheal tube in order to achieve best ventilation. the presence of the catheter through the fistula helps the surgeon to identify, it quickly. no complications were correlated to the procedure and no babies had early pneumonia. alimentary continuity was achieved in all patients (30 primary anastomosis, 2 resections of tef, 6 oesophagocoloplasty and 1 died with gastrooesofagostomy). the late mortality 7.7% (3) was only directly related to the severity of associated malformations. conclusion: the advantages of this technical approach are unquestionable for the anaesthesiologist and the surgeon. in our experienc e the procedure improves perioperative management of babies and appears to be safe. relation between cytokines, prethrombotic markers and endotelial injury markers in children with septic shock objectives: to establish the relationship between cytokines (tnf, il-1, il-6) prethrombotic markers (d.d., pcam) and endothelial injury markers (tm, uwf) in pediatric patients with sepsis and bacteriemia without shock, and patients with septic shock. design and methods: prospective study, 18 children (9 months-16 years) were admitted in our picu in 1994 with the following diagnosis: bacteriemia (4) sepsis (4) and septic shock (10) according to jacob's r f criteria. measurements: il-1, il-6, tnf, tm, vnf, d.d. pcam and routine laboratory data on admision, 12, 24, 48 hours and on discharge. the prism (pediatric risk of mortality score) was also recorded. results and conclusions: two patients in the septic shock group died. significant differences were found between non-shock and septic shock patients in relation to tm, dd, pcam, il-2, il-6 and tne high levels of tnf and il-6 are closely associated with the severity of septic shock with purpura in children. low levels of pcam on admission were associated with severe shock. who underwent open hea~nt surgery, hypervotaemia with or without oliguria was the most frequent reason to start pd (77%). in 10 patients pd lasted less then one week and there were no complications; in 3 patients it lasted 13 -29 days (one child had a peritonitis). instillation of dialysis fluid into the peritoneal cavity was associated with a significant increase in central venous pressure. there were no significant changes in cardiac output or arterial oxygeu saturation. in all patients pd dhnjnished fluid overload or improved the metabolic status. 5 patients (38%) survived the postoperative course and all had complete reintegration of renal function. conclusion: pd is a useful method to treat the fluid overload and acute renal failure in paediatric patients following open heart surgery with file effects of little importance on the cardiovascular fimction. obieetives: with the marketing of computerised systems for lung function testing in newborns, there has been an increasing interest in clinical approaches. percentile curves of pulmonary parameters permit an appropriate and clinically useful interpretation. however, the manual evaluation of the results using different curves is an impractical technique. therefoi'e a computer programme was developed. methods: the percentiles (5%, 10%, 50~ 90%, 95%) of the most important pulmonary parameters were determined non-parametrically in 6 weight-classes. for the calculation we have taken results of our own as well as other laboratories using a meta-analysis of reference studies. in all, individual data of 300-600 healthy newborns ageing between 1-28 days were collated. using these percentiles, for every parameter in relation to the body-weight the cumulative distribution was calculated approximately using piecewise linear and exponential functions. as shown in the figure the results of computing are represented numerically as well as graphically and can be included in the patient report. conelusions: clinic~d experiences with the programme have shown that representation of all measured parameters on standardised 100% scales allows an easy interpretation at first sight and improves the detection of pathologic patterns in the parameters. ")supported by bmft, fp "risikoneugeborene" prism (pediatric risk of mortality) score is a well known, already validated scoring system that quantifies severity of illness based on 14 routinely clinical and laboratory variables measuring physiological instability. once computed the score by summing up the weights corresponding to the most abnormal value recorded during the first 24 hours, the overall risk of mortality can be predicted by using the coefficients estimated by a logistic regression where prism score is the main independent variable. (pollack mm et al, -pediatric risk of mortality (prism) score. crit. care med. 1988; 16:1110 -1116 . to assess the applicability and validity of prism in the italian setting we launched out a prospective data collection in a sample of 33 pediatric icus. measures of calibration (goodness of fit statistics) and discrimination (receiver operating characteristics and area under the roc curve) are planned to be adopted in the cohort of patients recruited during 1 year period. as the validation study started on july 94, data collection is still on going and validation analyses will be carried out on july 95. up to now 23 centers recruited 1116 cases. at present, characteristics of the sample recruited are the following: most of the patients were male (62%); the mean age is 3 years with 30% of patiens having less than 30 days; more than half were medical cases (59%) admitted from emergency room or from hospital floor (51%); 52% cases were admitted with an organ failure while 48% to be intensively monitored. icu-mortality was l 1%. the paper will present final results of calibration and discrimination analyses that will be carried out in the whole sample and across subgroups known to differ in terms of clinical relevance and prognosis. if calibration and discrimination assessment will produce not satisfactoty findings, a customization of the current coefficients will be made allowing a formal comparision of previous and new parameters. jf riera-faneao, m wells, j lipman. baragwanath intensive care unit, university of the witwatarsrand, south africa. [background the prism score is designed to assess the likelihood of death in ipaediatdc icu patients, using only acute physiological disturbances, age and [operative status to predict mortality. there is no evaluation of chronic health status, [including malnutrition. this may significantly affect its ability to accurately predict outcome in a population where malnutdtion is common. aim to determine the influence of nutritional insufficiency, as indicated by a low weight-for-age on outcome prediction by prism. patients & methods we analysed prism, weight and demographic data co ected prospectively from 1528 consecutive paediatdc icu admissions over a 6 year pedod. a proportional weight (pwt) was calculated as a percentage from the 50th centile of the who weight-for-age growth charts. the pwt was compared for survivors and nonsurvivors, and mortality compared for pwt categodes 0nho wellcome classification). multivariate statistical techniques were used to identity associations with non-survival and to develop a modified logistic regression equation including a measure of i nutdtional status. receiver operating characteristic (roc) analysis was performed including and excluding patients with low pwt for the odginal and modified equations. results non-survivors had a lower weight than survivors (6.8kg and 8.3kg medians p = 0 63) a lower pwt (85% and 90% medians p = 0.6"0. the incidence of malnutdtion , in our icu population was 33%. the mortality of manoudshed patients was' significantly increased (p = 0.001), with a good correlation with the degree of malnutrition. the accuracy of prism was significantly improved when malnourished patients were excluded from the analysis (roc value increased from 0.72 to 0.79). ! logistic regression and discriminant analysis identified a significant association between prism, pwt and outcome; age and operative status were not significantly related to mortality. the use of a modified equation including the raw prism score, pwt category and age can significantly improve the discriminatory power (az dm/elopmental sample 0.82, az validation sample 0.81). the modified formula is: legit = -2.864 +0.134*prism score -0.006*age + 0.463*weight category, where the probability of mortality is exp(iog/t)/1 + exp(iogio. discussion although we can improve the prediction of mortality by a modified or recelibrated formula, this still does not compare with the reference prism population. the need for validation of the score itself, in the association with outcome of the acute physiological variables themselves, is thus apparent. we conclude that while the odginal prism formula can be improved significantly, a modification of the basic variables in this and other third wodd populations may be essential. a high incidence of malnutrition is an independent risk factor of mortality, and an important cause of the poor discriminatory performance of prism. in order to improve the accuracy of prism, nutritional status should be taken into account. objectives: to assess the value of inhaled no to differentiate between pulmonary vascular constriction or fixed anatomical obstruction. methods: we assessed the response to 40 ppm inhaled no in 12 patients(9 m, 3 f, median age 4.5 months, range 1day to 17years) with signs of increased pulmonary vascular resistance, there were 5 pre and 7 postoperative patients. patients were divided into responders(+) or non-responders(-). a positive response was defined as a 20% reduction in pulmonary arterial pressure and pulmonary vascular resistance(pvr) or in the presence of a left to right shunt, a fall in pvr accompanied by increasing pulmonary blood flow. left atrioventricular valve atresia + 12 mustard pat: pulmonary atresia vsd: ventricular septal defect asd: atrial septal defect pda: patent ductus arteriosus tapvc: total anomalous pulmonary venous connection the responders(7/12) were characterised by left to right shunts or pulmonary venous hypertension(4/7). patient#11 was weaned from ecmo with inhaled no. patient#2, without congenital heart disease, underwent a lung biopsy which confirmed reversible pulmonary vascular changes. patient#1 had a pulmonary hypertensive crisis which responded to no. all non-responders(5/12) had evidence of anatomic obstruction to pulmonary blood flow (#7,10,12)or a low pvr(#8) on subsequent cardiac catheterisation. in patient #3, lung biopsy confirmed severe obliterative vascular disease. conclusions: inhaled no appears to be an effective pulmonary vasodilator. a failed response may be evidence of either irreversible pulmonary vascular disease or a residual anatomical obstruction which may be surgically remediable in the postoperative cardiac patient. therefore, inhalation of no may be a useful diagnostic test to differentiate between fixed anatomical obstruction and reversible vasoconstriction. results: during these 18 years, the incidence of sdra was 1.3% of the total of admissions. the most common etiology was meningococcic septic shock. since 1989, there is a decrease of its incidence. (from 24% to 11%) and an increase of pneumonia and immtmodeficiencies. mean age of our patients was 2,7 years (61% males, 39% females), total mortality by sdra was 59% and there is an increase up to 72% since 1989 mean time of stay of the dead was 4,3 days and 12,4 days those who survived. although during the late years we offer in the picu a better attendance quality to the patients with sdra and the mean stay is longer, both for those who die and for those who survive, mortality of patients with sdra have increased. the incidence of sdra secondary to the septic shock of a meningococcic etiology have decreased. on the contrary, the sdra secondary to infections by opportunistic germs in patients with congenital inmmunodeficiencies or acquired immuodeficiencies have a tendency to increase. in our series, this change of aetiology is the responsible for the increase in mortality. hospital infantil unlversitario "virgen de1 roclo". sevilla. espalqa aims:to assess the incidence, etiology, clinical course, sequelae and mortality of the patients admitted to a paedfiatic intensive care unit with the diagnosis of severe traumatism. material and method: 60 cases of severe traumatism in children admitted to our icu in the period from january 1990 to june 1990 were reviewed. age of patient ranged from 4 months to 9 years, 65% were males. in our series, 53% of cases suffered traumatism due to a traffic collision and 38% had a fall from a considerable height. only in one case was traumatism due to violence to the child. we assessed the first assistance received in 76% of cases: where was it performed, interval of time since the accident, and steps taken. these data were also studied in relation to the latter evolution. results: 75% of our patients suffered cranioencephalic traumadsm (ct); in 53 % it was an isolated picture and in 22 % of cases was associated to other lesions. there was participation of thoracic and/or abdominal organs in 16 % of cases. 10% of cases presented important maxillofacial involvement. only one case presented serious cervical medullar lesion. mortality in our series was 3.3%. in 8.3% important sequelae remained. all of these patients presented tepas on admission equal or lower than 4. 16% of those with traumatises had slight sequelae. 71.6% of the total evolve towards healing. a polytraumatized child is a patient that benefits considerably of it admission in a paedriatic !cu. the rapidity in receiving first aid and its quality are essential to avoid sequelae and to make mortality decrease. after unilateral lungtransplantation 20% of the patients develop a lung failure with decrease of perfusion and increase of pulmonary blood pressure in the transplantated lung. the improvement of perfusion is an importent task in the postoperative period. case report: a 14 year old girl with idiopathic pulmonary fibrosis received a left sided single lung transplantation. during the early postoperative period occured a higtter demand of oxygen and an increasment of the pulmonary vascular resistence in the left lung. the pulmonary ventilation and perfusion scintigraphy indicated in comparison with the right lung a reduced perfusion of only 30% in spite of a ventilation of 70% of the transplanted lung. to improve the perfusion of the transplant we administrated per inhalation prostacyclin in a maximal dose of 20 ng/kg/min. the arterial blood pressure decreased but the perfusion continued nearly at the same level. during the following administration of 10 ppm no in the respiratory air we achieved a significant reduction of the respiration pressure f~m 40 to 32 nun h20 and of the pulmonary arterial pressure. the perfusion in the transplanted lung increased to 70ca/of the total pulmonary perfusion. after 3 days of administration with no we were able to withdraw the axtifical respiration without any following complications. conclusions: the perfusion of transplanted lungs is a major proble_r~ in the postoperative period. this case demonstrated the advantage of no towards the inhalativ application of prostacyclin. no showed a significant improvement of perfusion in the transplanted lung of a 14 year old girl. results: a total of 11 children with ards were treated with bovine surfactant (alveofact| 9 cases were evalable. the median age was 1.3 years (range 2 weeks to 4,9 years). in six cases ards was associated with pneumonia, in two cases with lung hemorrhage; in one case isolated ards followed hemihepatectomy. the first surfactant application was performed with a median latency of 22 clays (range 3-68 days) after first symptoms of ards witha median doseof 84 mg/ kg (range 42-133mg/kg). in 9 patients 28 doses of surfactant were applied. during the hour before therapy, the median pao2 / fio2 -ratio was 70-65. within 30 min. after application of exogenous surfactant the pao2 / fio2 -ratio increased to 90 with successive decrease over a period of 8 hours to 75. accordingly, an increase in pao2 and oxygen saturation and (less significant) a decrease in ventilation parameters could be observed. analysis of broncho-alveolar lavage before surfactant application in children receiving repeated doses revealed in most examined cases either clear surfactant deficiency or pathological function. 2 of 9 treated patients survived (3 of the 11, respectively). 13 of the 28 surfactant doses were applied in the 2 surviving patients.conclusions: the application of exogenous surfactant in children with ards caused a significant increase in oxygenation, which declined over a period of 8-12 hours. the effect often could repeatedly reproduced, in one case after 11 applications. the increase in oxygenation often allowed the reduction of fio2 and/or the inspiratory pressure. no side effects were observed after exogenous surfactant application.in many cases the application of surfactant wag too late after first symptoms of disease (median latency 22 days). ards mostly due to pneumonia seemed to respond to surfactant therapy less well or not at all. permanent junctional reciprocating tachycardia (pjrt) is the most common incesant supraventricular tachycardia (svt) in children. it is usually drug resistant and its onset in early life has been associated with dilated eardiomyopathy. we report our clinical experience with 3 patients detected antenatally and another diagnosed at 2 months of age. method.diagnosis: negative p waves were detected in leads ii,iii and f, p'r > rp" and there was not warm-up at tachycardia onset.clinical records, ekg,x-rays, echo and holter were reviewed. ep studies were undertaken only with therapeutic purposes. results. in a 10 year period 13 patients under 14 y of age fullfilled diagnostic criteria; 3 were detected prenatally (25-34 weeks) and one was diagnosed at age 2 mo. the 3 fetuses had intermitent svt during gestation. all 3 of them had pjrt in the first month of life at rates between 180 and 240 bpm. they were admitted to the icu but did not develop signs of heart failure. they were controlled with digoxine (d); d and quinidine; d and propafenone in 2 to 20 days. one was in sinus rhytm until age 4y; he then showed persistent pjrt over 70% of the day on repeated holters and underwent successful radiofrecuency catheter ablation (rfca).the other two patients showed initially a lowering of tachycardia rate followed by sinus rhytm for over 90% of the day (follow-up 2ran and 4 y). the 2 mo. old infant was admitted to the icu in severe cardiac failure. echocardiogram showed marked systolic dysfunction (shortening fraction 15%) treatment with digoxine, amiodarone and propafenone were unsuccessful despite lowering heart rate to 185; rfca was performed at 3 m. of age with restoration of sinus rhytm and rapid recovery of contractility. all patients were given atp at admission with transient (15 to 35 see) recovery of sinus rhytm. ff,s162 clinical course of pjrt is variable. atp is useful only as a diagnostic tool. initial treatment with digoxine + amiodarone or propafenone is adviced. rfca is a very useful therapeutic modality and can also be performed in young infants twelve patients (5%) died. these were 4 meningitis, 2 head injury, 2 sub-arachnoid bleeds, 1 status epileptieus, 1 leukaemie, 1 drowning, and 1 multiple trauma. calculated from the a 2 admission day p edialric risk of mortality score (prism), the probability of death (p) ranged from 0-100%. of the 12 deaths, i 1 were predicted by prism analysis except for the leukaemie patient (p i%) who died from haematological complications following chemotherapy. two children predicted to die (p 43% & 73%) survived. the median length of stay was 2 days (range 1-34 days). 98 patlents(50%) received ventilatn~ support and 10 patienta(4%) were transferred to specialist units (5 neurosciences, 3 liver, 1 cardiac, 1 bums). this data supports the view that many paediatric patients are being adequately treated in a dgh icu. meningitis and other neurological illness caused the majority of deaths and respiratory problems caused most admissions. most deaths (9 of 12) occurred within a few hours of admission. ectopic junctional tachycardia (ejt) is one of the most dangerous arrhythmias in the postoperative setting of congenital heart defects since it does not respond to antiarrhythmics or defibrilation. the object of this presentation is to report on two patients who presented f_jt in the early postoperative period and developed intense congestive heart failure which could be controlled after treatment with moderate topical hypothermia. two patients, 8m and 2y, diagnosed of atdoventficular septal defect and tetralogy of fallot developed intense heart failure in the early postoperative period. taehyeardia rate was 215 and 225 bpm. medical drug therapy included weaning from vasoactive drugs, iv digitalization and iv amiodarone treatment. there was not response. they were both surfaced cooled by placing plastic bags filled with cold water over the patient's chest and abdomen. temperature was monitored to obtain a central temperature of 34~ there was a gradual decrease in heart rate in the following hours (130-150bpm) paralel to the degree of surface cooling and clinical course estabilized.both recovered normal sinus rhytm in 48 to 72 hours. there were not significant arrhytmias after the procedure and postop, was uneventful. conclusions. moderate hypothermia is a very useful manuever for the treatment of drug resistant ejt. since it lacks side effects of other antiarrthymics we beleave it should be the treatment of choice for the treatment of ejt in the postoperative patient. present understanding of the pathogenesis of sepsis, based on the theory of systemic inflammatory reaction, has risen new interest in the more invasive methods of treatment, like plasmapheresis, leucapheresis and exchange transfusion (et). obiectives: evaluate the effect of et in the treatment of neonatal sepsis. material and methods: from september 1 to december 31, 1994 a prospective study was carried out, where the severest cases of bacteriologically proven neonatal sepsis (n=9) were treated with et. in total 15 newborns were treated for culture positive sepsis in the intensive care unit during this study period. diagnosis of sepsis was based on the clinical criteria of suspected neonatal sepsis, used by mc harris et al., laboratory data and positive blood culture. newborns with severe congenital malformations were excluded. et was carried out with fresh (less than 24 hours old) adsol-conserved erythrocytes, from which buffy coat had been removed, and same donors plasma, using a slow continuous two-site technique. the mean volume of et was 164.3 ml/kg. the effect of et was assessed as a change in the score for acute neonatal physiology (snap), general treatment results were compared with a historical control group of 26 newborns, treated for culture-positive sepsis in the same icu during the first eight months in 1994. students ttest and chi-square test were used in statistical analysis of the data. results: with the use of el a significant decrease in mortality was achieved: 1 death of 15 cases during the study period, compared to 9 deaths among the 26 controls (p<0.05). no baby, receiving et, died. the incidence of severe complications did not differ in the two groups. the snap-score showed quick improvement by the first post-transfusion day (p<o.05) and the effect persisted during the five following days. conclusions: we conclude, that the use of et in the treatment of critical cases may help to lower the mortality of neonatal sepsis. it provides a quick improvement in the clinical condition of septic neonates. the granulocyte-colony stimulating factor (g-csf) is largely employed in granulocytopenic patients. experimental studies have' demonstrated the effectiveness and safety of the therapeutic use, nevertheless the human employ is reported almost exclusively in neoplastic patients submitted to chemotherapy and in patients with fungal or pseudomona~ spp infections. the authors report the data concerning three non granulocytopenic children: in the first, o girl 7 years old, affected by pseudomomm aerug/n~a pyeionephritis, the antibiotic therapy was not tolerated in consequence of renal and hepatic failure. the second patient, an infant 6 months old, has been treated with salbactam-ampiciilin, ceftriaxone and chioramphenicol because of haemepldlw iafluem~ meningitis; a late neurological aggravation required the antibiotic therapy recommencement the last, a 17 years old insulin dependent diabetic boy, was affected by cared/do spp systemic infection; the antifungnl therapy w~ not practicable in consequence of renal and hepatic involvement in all the g-csf was administred subcutaneously at the'dose of $ u for 7 days. a significant increase of granulocytes was ever noted already after the second administration until 24 hours after the last dose. clinical signs, fever and biochemical values are promptly influenced positively and all patients are restored to health. no side affects were noted. the granulocyte colony stimulating factor, at indicated doses, can prove of great help in critical patients, in who the antibiotic or antifangal therapies can result toxye or cannot be tolerated. the ~reptococcas p~, among the gram pmltlve~ represents the main causative organism of bacterial meningitis in children, with the majority of deaths occurring within hours of admission, the third generation cepohlosporim were indicated as antibiotic of first choice, and some literature reports have demonstrated no significant dlffereuces in dinical course of patients treated with ce~rlaxoae and of those treated with ampieibin and chlornmphenicol. the authors report the ease of on infant, male, 18 months old, submitted to a neurosurgical interventiol after 2 days the infant became febrile and lethargic and a bacterial meningitis by ~trepmcecctz ~ was diagnosed, in spite of he was given the eoftriaxone therapy.. an amelioration was found only when a systemic treatment with vaucomyein was added. after 2 days an intrathecal administration of vancomyein 40 m~day was started: the norbmlization of the spinal fluid profile was uoted two days later and the infant receve~d progressively. the follow-up after eight months doem't evidentiate motosensorini sequelae-the individuation' of ~cscc~ pmmmsm/ae strains heta-loctam antibiotics resistant requires alternative therapeutic regimens: the k~terity quead vitam and quoad valetudinem of these infections justifies an intrathecal therapy, especially failing o prompt answer to the systemic treatment objectives: to evaluate the incidence of the burnout (bo) of the staff in a picu in a university hospital. the bo is a syndrome charactedzed by many factors like emotional stress, lack of personality and reduced work ability. this syndrome represents a kind of abnormal response in the working habitat, above all, where psychological and personal characters are mostly involved. the symptoms of bo are represented by demoralisation, demotivation, incapability, boredom, isolation up to organic disturbances, like migraine, insomnia, dizziness and gastrointestinal disturbances. the major incidence of this syndrome was found in the hospital staff, especially among the nurses, working in an atmospher of high emotional dsk, just like in an intensive care unit. methods: from january '95 till april '95, all the staff of the picu of the a. gemelli hospital of rome, underwent a test to evaluate the bo. the test used was taken from the book "bumout: from tedium to personal growth", by pines and aronson. twenty seven people were studied (19 females, 8 males)i out of which 7 doctors, 7 residents and 13 nurses. it was considered sign!ficant as mild bo, a score between 3-4 and as severe, the score of bo >. 4. results: 10 subjects (37%) resulted positive for bo, out of which 8 were females (80%) and 2 were males (20%). the subjects with mild bo were 7/10:1 was a doctor, 3 residents and 3 nurses. the subjects with severe bo were 3/10, out of which 1 resident and 2 nurses. conclusion: the results obtained show that bo is a condition well represented in the staff of our picu. the category most at dsk seem to be the nurses (5 subjects), as well as residents (4 subjects), as in literature, which shows a major incidence of the syndrome in younger subjects and having a limited partecipation of functional decision. the results obtained obliged us to start a programme of serial controls so that the subjects most exposed can have a necessary psychological support to react adequately to this condition. the term systemic inflammatory response syndrome (sirs) was adopted by the consensus conference to denote a type of systemic response to severe infection or otherinsults in critically ill patients. when sirs occurs from infection it is called sepsis. sepsis occurs more frequently in persons with perexisting illness or severe trauma. there has been tremendous advances in prophylaxis, diagnosis, and treatment of sepsis. a comprehensive model of the disease progression from sirs to mods should be developed giving priority to severity of illness scoring system and other predictive methods. some recommendations for future clinical trials include: trials should not start with humans. before proceeding to human trials, animal studies should indicate an acceptable risk/benefit ratio. appropriate patient populations must be defined and treatment protocols should be standardized. full and rapid reporting of all results should be mandatory and a central repository of published and unpublished study results could be helpful. accrual at each center should be of sufficient size, and should include the number of patients accrued, mortality rates, and patient characteristics. pivotal trial should be preceded by sufficient pilot or phase ii studies. correct drug dosage and usage should be delineated in pilot studies. large, multicenter, trials should be used to enhance the unversality of trial results. analyses should be planned a priori. definitions for the target population should be explicit, reproducible, and include illness severity scores. outcomes should be relevant reproducible and include both measures of benefit and harm. mods and its reversal should be considered as an endpoint. quality of life should also be considered as an endpoint. the estimators of overall treatment effects should be controlled for base-line prognostic factors and subgroup anaiysis should only be used for hypothesis generation and not to modify the conclusoin of the trial. economic analysis should be included as part of clinical design. evaluatin of source control should be a critical component of any study. standardized clinical mediator assays should be pursued. placebo patients in clinical trials should be studied for a better understanding of the pathogenesis and epidemiology of sirs, evidence based medicine should be used to evaluate the validity of clinical. introduction: use of inhaled nitric oxide (no) as a modulator for optimizing ventilation-perfusion or lowering pulmonary artery pressure is becoming increasingly common. no is a free radical but little toxicological research has been published. clearance of nebulized 99mtc-dtpa is known to be, a sensitive indicator for early function impaimaent of the alveolocapillary barrier. we investigated whether exposure to no increased clearance of 9~tc-dtpa from the lung. methods: three groups of 5 white sealand rabbits (bw 3.5 kg) were anesthetized, tracheotomized and paralyzed. 2 groups were ventilated for six hours at pressure regulated volume control, set to deliver 10 ml/kg with a frequency of 30/rain, i/e ratio = 1:2 and peep = 3 cm hzo using a modified servo 300 ventilator (siemens, solna, sweden) with computerized no delivery system. gas mixture per group was either 0/25 or 20/25 [no (ppm) / fioz]. after six hours of ventilation in these 2 groups and immediately after anesthesia in group 3 (control), 99~tc-dtpa was nebulized into the inspiratory line of the breathing circuit and administered as a fine aerosol. gamma counting was measured for 20 minutes, monoexponential curves were fitted to the data and the clearance half-time (t 89 was calculated. the t~/2 mean • sd of the different groups were: t~a (mean 4-sd) h"e,i witl~ arf 1:14 di.ff:erent kinds, aged .q-ore 2 mon't.hes to [4 gears o11:1 (bodi weight .~rom 4.,5 to 45 kg), is presen .... "ed ( i,,~u::trl:e i:ibstraclive d:lse~se...14~ 2.ards'-8; :~,;,,arf o~ ::entral genes:i s .-3,0,~ :inc lud ing men ingeenceph 1 it :is-3~ reye ' s ~yrtdro~e-..#~,bri~:ln pes~.re~nimatior~ disease.."5). int:lrl~]. pa-. "iiulle'i,~s ariel regymes o+ l;mv,l;i"t"v were cle'l'.ermllled by ba'i~ier was. about 4. tuber,, dopamin tiara-:. t.io; was ~.".,,'.r:~r~led. cmv,cppv d~.!"~tion raniled -~rom f to 25 dayns.,~ <6.-:in 351, 6"t2-irl lo;and>12 davs'-in 6'l~atierr~{s i'i"ai3s:ltiol~ o4; patterers to imv, simv modee was per.r:)rmed, ~herl pif:' decrease.d to 16-17 ml~ar, fi02 ~ecreased to 0,4. lind less with 5a02=901/,,. i:lesq.lts:{ in pat:i.ents e{ group :l, who were tre,~d.ed w&th 2f'f'v, teoph :i. :1. l:i.r~ (is-24.mg/kg/day), g lucecdr t icostei~oids (2 .... :~;mg/kg/day), when r exceeded in 2,2-.];,4 times normal va i tea the e aqes/,'!:l"oln 2~j,, ite :i.~;::.!;, 8 ~ml"lrj), it was possible 't'(' ce 'e~ e aad]t:..~rom !1.20 2'.' 26i', 8 to !..';51,0-106, 1 mml-lg in ~}.. :~.[~ houi,!; ~d'l(:i to ru:}l",g'd!~l:i.2e i::h,:~e,'~c['el';i.stil obieetives : this chapter will describe what is knovca of the psychlogical responses of infant and children to hospiuiisation and attendant procedures. the factors which may modify these responses will he discussed and important considemtiorts will be outlined for optimal anaesthetic management and postoperative period of infants and children which will minimised the rise of emotional upset. methods : in this paper the autors will discttssed the probl 9 of: 1. health children (asa i, ii) facing single uncomplicated surgical elective procedures 2. various abnormal situations including neurotic children, children facing repeted operations, chronically ill, buaaes and tsaumatically impired ones 3. unfortunate young patient facing and often expoclting fatal outcome from le "ul'ukaemia, tumors, cystic fibroses or otheq" disease. : management of each child must vary greatly, ifi general the phases of emotional conditioning include home and preadmissiun preparation, admitiun preoperated and operative care and postoperative period. the authors would be happy if the child passes all stages without any trauma which could be prolonged in the future life. introduction ino is used to selectively reduce pulmonary vascular resistan(~e. we applied ino in the postoperative intensive care of patients with pulmonary hypertension and the risk of right ventricular failure after surgical correction of a congenital cardiac defect. methods 2-50 ppm no were added to the ventilatory gas mixture using a specially designed equipment (messer-griesheim, germany/austria). indications for application included pulmonary artery pressure >50% systemic pressure, critically depressed right, ventricular function or an oxygenation index >10. assessment of n oefficiacy consisted of on-off-on measurements according to the clinical stability of the patient including hemodynamic parameters, pulmonary gas exchange, continuous monitoring of ventitatory function and transesophageal echocardiography of the right heart. results in 29 situations (19 patients, age 3 days-71,1 years), ino was applied 0-628 h postoperatively. oxygenation was improved in 13 situations from 114_+51 to 171+53 mmhg pc2; pulmonary pressure was reduced in 17 situations from 70-*22% to 34_+17% of systemic pressure. in 7 situations, no reduction of pulmonary pressure was present, but measurement of cardiac output or echocardiographic analysis indicated an improvement of right ventricular function (right ventricular stroke volume +39-*12%, cardiac output +20-*11%). in 8 situations (immediately postoperativ with suprasystemic pulmonary artery pressures [n=4], multi-organ-failure [n=4]), no response to ino could be determined. conclusions for a special group of patients, the selective reduction of pulmonary vascular resistance by ino has become an important part of postoperative therapy. using this selective afterload reduction, postoperatively depressed right ventricular function can be improved. this effect of ino seems to be the most important one in the postoperative period. thus, ino appears justified to be appfleo when impaired right ventdcular function could be improved even when pulmonary artery pressure is not raised or remains unchanged. obiectives : premature infant are exposed to danger of apaea due to anaesthesia during their tirst months of life. it is yet unknown whether prematurity is corelated to any other kind of reslgratory disorder due to anaesthesia within the tirst year of life. methods : we theretbre researched retrospectively for respiratory disorders in all infants under 12 months of life belonging to asa group 1. they all had been anaesthetised in 1985.95 in our clinic for the following surgical reasons: ingvinal haemia, umbilical haemia, hydrocelae testis and phymosis. results : in 2350 cases we tbund: lafingospasm during induction in anaesthesia (0,5%), bronchospasm during induction in anaesthesia (0,22%), impaired intubation (0,1~ postanaesthetic laringospasm (0,1%), supposed aspiration (0,04%),postanaesthetic inspiratory stridor (0,05%), postinductional inngoedema (0,03%), death after 4 months in consequative of infection pneumonie (0,12%), none of these disorders was correlated the prematurity, 3 infants suffered of post anaesthetic apnea, 2 of them had premature medical history. concludions : prematurity does not enhance the risk of respiratory disorders due to anaesthesia within the first year of life, except the danger of postanaesthetic almea needs spetial cosideration. it could be demonstrated that aepgi 2 lowers pulmonary vascular resistance and indirectly improves cardiac function. this effect seemed to be selective, and was comparable to ino in the doses we have examined. therefore, aepgi 2 could represent a clinically useful alternate to inc. however, further research is necessary to work up the benefits of either therapeutic strategy. objectives: heat and moisture exchange filtem (hme) are used as artificial noses for intubated patients to prevent tracheo-bronchial or pulmonary damage resulting from dry and cold inspired gases. furthermore they are used for the prevention of bacterial contamination of the anesthetic apparatus by the patient's exspired air. so they are considered as a time-and money-saving device in anesthesia. filters are mounted directly on the tracheal tube, where they collect a large fraction of the heat and moisture of the exspired air, adding this to the subsequent inspired breath. the effective performance depends on the water-and bacteria-retention capacity of the filter. this study evaluates the efficiency of four different filters under clinical conditions. methods: four different types of filters ( dar hygrobac, gibeck humidvent, medisize hygrevent and pall bb 100 ) were investigated dudng mechanical ventilation over a pedod of 24 hours. 20 minipigs with hemorrhagic shock were intubated and ventilated for 5 days in an animal intensive care unit (icu). after 24 hours of mechanical ventilation the filter was randomly replaced maintaining the individual ventilatory conditions. the weight of the filter was determined before use and after removal after 24 hours. the airway pressure was monitored online to record changes during use. tracheal secretions and both sides of the filter were microbiolologically tested to see whether bacteria of the animal's respiratory system could be found on the patient's side of the filter or if they even would have penetrated the barrier. results and discussion: over a pedod of 24 hours 3 of 4 types of filters showed an increase in weight of 10 + 6% and airway pressure. bactedal celonisation 0ccured in nearly all fillers (93 of 100) on the patient's side, whereas only three of four types of filters showed identical bacterial colonisation on both sides. the only filter that did not show bacterial penetration, increase in weight or airway pressure was the pall-hme, a condensation humidifier without hygroscopic salts for moisture retention. with respect to our data one should use a condensation humidifier if airway conditions should remain stable dudng mechanical ventilation and desinfection of the anesthetic apparatus should be avoided after each patient. aim: to assess the clinical uses of, and experiences with, the hayek oscillator. this is a non-invasive device capable ef delivering not only continuous negative pressure (cnp) but also external oscillatory ventilation around a negative baseline (eov-nb) using an external cuirass. this type of ventilation avoids the need for intubation and intermittent positive pressure ventilation (ippv) and facilitates weaning in ventilator dependent patients. patients and methods: 21 patients in respiratory failure, age range 3 weeks to 15 years in a total of 29 patient episodes were treated using either cnp or eov-nb mode. duration of treatment varied from 4 hours to 8 days. indications for use ef the device were: 1) to facilitate weaning from ippv 2) prevent reintubation of patients following unsuccessful extubation, and 3) avoid intubation and ippv altogether using the hayek oscillator as the on[y means of respiratory support. results: there was an increase in pao2:fio2 ratio after cnp and eov-nb (p <0.0001, and p=0.01 respectively, wilcoxon signed rank test). patients who were in respiratory failure with hypercapnia showed a statistically significant reduction in paco2 both with eov-nb and cnp (p=0.02 and p=0.01 respectively) but the magnitude of change was individually greater in the patients who were treated with eov-nb. all patients, however, showed a fall in respiratory rate (p<0.0001) after the application of the cuirass in cnp mode. there was no physiological deterioration related to the application of external extrathoracic negative pressure in either cnp or eov-nb modes. conclusion: the improvement in pao2:fio2, the fall in paco2 and respiratory rate were indicators of an improvement in ventilation. the proposed mechanisms include improvement in frc, recruitment of additional alveolar units, and improvement in secretion clearance resulting in reduction in the work of breathing. meek to ~ month of the lifo,the bemodyuanicfacls were defined uitb the help of tetropolar reography method!. the excretion of !he catbocholauines fcfi] mith the urine gas detertend by taylor ll,laoorsy ~ iacg/dayl. hsaltl in the hypercuagulation stage of bic we deflorteeed the acliuutiun of the tbrubio and plasiin syaet~ mitb the increase of the inhihitnrs, in this case we registered in full uahe dot this process coabined uitb the dayl~ excreliou with lho urine epinopbr ne e], nor~pinopbr no tel and dophanine io], lbat shod the inlensificatiou of the s~nthosis prnoe-s~es and the release of ea in blood fron hissue deport the actffat on of the svnpathadrenui systen ]sfisl assisted to furl the b?perd~nanical rosins of the eircuidion and increase the ,icrocirculatinn, the klinicai sings of the insufissieutly of the circulalion have not defined,that has been associated the conpensatury character uf the ehan~es of ~ and heludy~enic status, t~e uun~u|p-lion ceugulupatby bus been donoustraled in the hypocougulatien stage ~bat man xauifosted b 7 the exhaust of lhe confulalion nod oessel-platel heuostasis, the consuxptton of cnnpononts tbronbln ,plnstin, kallek~eiu-kinln s~slots and the forniration eat in fell canoe clot uas accoqaued bs docrea,e of fl,nfl,o, the products of the xotabolisx of c~ and the activation of xonoaninoxydasu. the decrease of the extoll'on g and the exhaust deport co indicahd about t!e ]ou fund/anal reserve of ~fl~. it was one of the lain reason of ~bo heiod~uanic disbroed iheat insnfissient]~] and the uicrncireulaflion lintestinal codeme with the low effectife periferal flow] and nul[iplay organ failure,the distrued deport of sos mitb throubocytupenin no; be one of the nechanisn the dislrood of uessej-plalol heioshasis, the correlation bolueeo changes of boiostosis c~ and circulation ore reguired aduinistration nedidns, thai reslore the love s of c~ in the blood, prevent uulliplay organ failure and hetorrnge in children with sepsis, ~b~ectives: multi-measured correlative analysis of the most number of non-invasive indices of the cardiorespiratory system function was made to determine the structure of their interrelation and the ways of their adequate and effective correction. hethods: spiremetry, capno~raphy, oxygenography, indirect fick method at recurrent respiration, plethysmography, integral rheography -in all 52 indices were used. the received data were processed on a computer by a standard package of statistical bmdp programs. results: 70 women with ~h-gestosis (i group) and 48 somatically healthy pregnant women (ii group) were studied. cluster analysis has shown that the rate of the mean correlation connection between ventilation indices was 94% in the ist group and 90% in the iind group; gaseous metabolism -91% and 86%, respectively; central hemodynamics was 87~ in both groups. conclusion: cluster interpretation allowed to suggest that an increase of the rate of the mean correlation connection between the indices was characteristic of effective adaptation as the system was multi-component and well-regulated. on the contrary, the increase of the rate of strong correlation connection between the indices reveals the rigidity of the system and the tensity of adaptation mschaniams, i.e. the proximity to decompensation. it follows from this that in cases of eph-gestgsis, the reliability of regulating ventilation and gaseous metabolism decreases. seve/e hypoxemia in non intubated patients represents a major contraindicafion to fiberoptic bronehoscopy (fob) and bronehoalveolar levage (bal), but these procedures are often required for a correct diagnosis of the causative agent of pneumonia. aim of this investigation was to veaify the safety and efficacy of bronehoseopic procedures during pressure support ventilation administered through facial mask (fm-psv). five intensive care patients, all immunoeompromised, (3 males and 2 females; mean age 41.6• were enrolled in the study. all patients presented criteria for pneumonia with pao2/fio2 ratio ~ 100 and were responders to fm-psv. fob and bal were performed afte~ topical anesthesia with fm-psv ( ps = 16 em h20; peep = 5 emh20; trigger = -lemh20) continuously admires" tered ( 10' before fob fio2 = .7; during fob, fio2 =1 and for 90' alter fob, fio2 = 0.7). pao2/fio2 ratio as well as 02 saturation (sat) did not show signifteative changes during the procodure (fig.l) . no complication was observed and hemodynamic conditions were stable for all patients. cmv, pnenmoeystiis (2), legionella and mycobaetermm tuberculosis were identified from bal allowmg a prompt and targeted therapy. we concluded that mask psv can represent an excellea~ technique to pexform fob and bal in severely hypoxemic patients without deterioration of gas exchanges and avoiding endotraoheal intubation. intensive care unit, hospital general of albacete, albacet~ spain. objective: to analyze the current incidence and epidemiology of total parenteral nutrition (tpn) among critically ill patients placed on mechanical ventilation. design: prospective observational study. setting: medical intensive care unit in a tertiary hospital. patients: a total of 113 consecutive l'ritically ill patients with non-coronary related disease needing mechanical ventilation admitted in our icu during a 12 months period. measurements: data of sex, age, diagnosis, and outcome were recorded. severity of illness and therapeutic effort in the first 24 hours were measured using acute physiology score and chronic health evaluation (apache ii) and therapeutic intervention scoring system (ties). r~ults: 113 mechanically ventilated patients, 76 male and 37 female, were studied. only ten patients needed tpn and their main diagnoses were: five cases of multiple organ failure secondary to pneumonia (2), ards (2) and septic shock (1); two eases of acute panereatitis; and one mesenteric throngmsis, one status epilepticas, and one ,prolonged cholinergic crisis b~ suicidal organophnsphate insecticide subcutaneous injection. no statistically significant differences between both tpn and non-tpn groups were found: objectives: evaluate the efficacy of prone position in ards and determine its importance in the therapeutic algorithm. methods: 43 consecutive patients with severe ards (murray-score > 2,5; pao2/ fit 2 < 160 mmhg; 29 male, 14 female, mean age 62 years) were conventionally ventilated (pcv, peep 6-16 mbar, i:e=i:i, ppeak < 30 mbar). if after 24 hours pulmonary function did not improve patients were placed in prone position. change from prone to supine position was done every 12 hours. beside ultimate survival, parameters investigated were aado2, pao2/fio2, and venous admixture (qs/qt). results: during the first 12 hours in prone position 39 of 43 patients showed a significant decrease in qs/qt (25.3% vs. 17.8%) and aado 2 (235 vs. 187 mmhg), and an increase in pao2/fio2 (151 vs. 201 mmttg). changes were most pronounced in patients with high qs/qt, and in patients with an onset of ards less than 48 hours before first application of prone position. after an average of 6 position changes (2 to 16) 28 of 43 patients could be weaned from the ventilator. 22 patient could leave tile hospital. i11 the later course letality was primarily determined by additional organ failures and by the severity of the underlying disease. negative side effects were minor, including slight cardio-vascular depression and increase in p~co2, and never posed a limitation to continuation of prone position. especially in patients with septic shock skin lesions in exposed areas could not always be prevented, prone position could easily be combined with all ventilation modes and with all intensive care interventions. also immediately after major surgery and in patients with open packing prone position was possible. conclusions: in this investigation prone position proved to be an efficient and safe method in the treatment of severe ards. patients with a pronounced ventilation/ perfusion mismatch and patients in the early stages of ards appear to profit most from prone position. though the immediate effect on oxygenation is striking, still more the 40% of all patients die from multi organ failure and underlying diseases. a proposed therapeutic algorithm for ards is as follows: if under conservative ventilation (pcv, peep < 20 mbar, ppeak < 30 mbar) pulmonary function does not improve within 12 -24 hours prone position should be applied. when after 2 -3 position changes no lasting effect can be achieved further ventilation modes (e.g. pc-irv, aprv, no, etc.) should be used in addition to prone position. standard intensive care principles, such as fluid restriction and optimization of circulation, apply also to patients in prone position. objectives: nitric oxide reacts with superoxide to form peroxynitrite, an extremely reactive and toxic species. we quantified the presence nitrotyrosine, the stable product of the interaction ' of peroxynitrite with tyrosine residues in the lungs of pediatric patients that died with respiratory distress syndrome (rds). methods: paraffin embedded lung sections, obtained at autopsy, were incubated with a polyclonal antibody raised against nitretyrosine, followed by a secondary fluorescent antibody. alveolar structure-associated fluorescence was quantified using existing methods. results: tissue sections from patients who died with rds exhibited significant specific immunostaining which was uniformly distributed across the blood-gas barrier. in contrast only background levels of fluorescence were seen in the lungs of patients who died from non-pulmonary causes. intense staining was also seen in the lungs of rats that breathed 100% 02 for 60 h, a condition known to result in rds-type illness; no immunostaining was observed in air-breathing rats. conclusions: significant levels of peroxynitrite may be formed in the lungs of patients with acute lung injury. peroxynitrite may be contributing to the pathology of rds by damaging key components of the alveolar epithelium including the pulmonary surfactant system. mechanical ventilation time was prolonged 16,g • 10 days in patients with ardsvs 1,7 _+ l,4 days in control . mean staylcuwas lg _+ 10,g days in the ards group vs 4,9 • 2,7 days in control group postoperative mortality rate was 53% in ards patients vs 5,7% in those without respiratory failure. 1-ards incidence in liver transplantation is low (11,2% in our sene) but it causes high mortality (53%) page, gas ventilation of the perfluorocarbon-f'dled lung, supports gas exchange and circulation in small animals (<15kg) with lung disease. we hypothesized that large animals could be supported by page without adverse effects on bemodynamics. we first elucidated the determinants of gas exchange in normal sheep, and applied them to a model of adult respkatory distress syndrome (ards). methods: using the ventilator settings determined to be optimal in our pilot study (fio2 of 0.6, peep of 5 cm h20, imv of 6 bpm, it of 50%, and tv of 16 ml/kg), sheep weighing 58.9 ~ 8.3) kg had lung injury induced by instilling 2 ml/kg of 0.05n hc1 into the trachea. ten minutes after injury, sheep with pao2<100 ton" were randomized to continue gas ventilation (control, n=9) or to institute page (n=9). page was instituted by instilling 1.6 l of unoxygenated pefflubron into the trachea and resuming gas ventilation at the previous settings. abg's were drawn at baseline, 10 minutes after injury, 30 minutes after injury, and then every 30 minutes for 4 hours. objectives: inhaled nitric oxide (no) can improve oxygenation and decrease mean pulmonary artery pressure (papm) in hypoxemic patients with ards. in severe hypoxemic copd patients, it is not known whether inhaled no can exert a similar effect on hemodynamics and gas exchange. therefore, we investigated die response of inhaled no in hypoxemic copd patients and the results compared with those obtained in a group of ards patients. methods: ten copd patients (age 71_+2y;fev~ 0.98_+0.12l) and 11 ards patients (age 57_+5; lis 2.8_+0.1) mechanically ventilated were studied. hemodynamic parameters were measured using a swan ganz catheter. arterial and mixed venous blood gas determinations, sao2, svo2, hb and methb were measured (abl 500,osm3). mean intratracheal concentrations of no and no2 were continuously monitored using a chemiluminescence analyzer (nox 2000) . during the study the ventilatory pattern and fioz were kept constant. the protocol was for ards group: basalt, no loppm, basal~; copd group: basalz, no lo ppm, no 20 ppm, no 30 ppm and basal2 . after a steady state of 20 rain hemodynamic and gas exchange measurements were performed. a positive noresponse was defined as a 20% increment in pao 2. results: papm was similar in both groups and decreased significantly after no (ards, basal 33.6_+9.7 mmhg, no 29.7 +6.7 mmhg, p <0.01) (copd, basal 27.8_+6.3 mmhg, no-10 24.4_+5.3 nrmhg, p<0.01). all other hemodynamic variables remained unchanged after no. basal oxygenation was higher in copd group (paojfio 2 189_+53 mmhg) vs ards group (paojfio 2 100_+40 mmhg)(p<0.01). after no-10, pao2 increased (69_+20 mmhg to 97_+40 mmhg, p<0.01) and qs/qt decreased (37+11% to 31_+10%, p<0.01) only in ards group. in both groups, significant correlations between basal papm and inhaled no-induced decrease in papm were found. inhaled no-induced increase in pao2/fio2 was not correlated with basal paoflfio2. no responders were 8/11 (73 %) in ards group and 2/10 (20%) in copd group (p<0.05). conclusions. in hypoxemic ards and copd patients, inhaled no decreased mean pulmonary artery pressure. however, oxygenation only ameliorated in ards group because die number of responders to inhaled no were higher in ards group and this effect seems not to be related to the basal hypoxemia. these results might be explained by the v/q abnormalities present in copd patients. grant fis 95/1390. objectives: it has been recently reported that expired con slope as a function of time is modulated by total respiratory system resistance (rrs) in critically ill patients (chest 1994; 105:219-223) . in this study, we analyze the relative contribution of disease (dis), endotracheal tube resistance (rtube), airway resistance (rmin), additional resistance (~rrs), autopeep (peepi) and dylmmic/static elastance (ed/es) to the co2 elimination in different clinical conditions. methods: we have studied 37 adult patients (8 controls, 11 acute respiratory failure, 9 severe ards and 9 copd) mechalfically ventilated (servo 300 and 900c, siemens) without peep. we recorded tracheal pressure, airflow and capnograms. signals were analogic to digital converted for posterior data analysis. objectives: alveolar ejection volume (van) can be defined as the fraction of tidal volume (vt) with minimal dead space (vd) contamination. according to the classical paradigm: limvd_~ [vco2/vt] =facoz, vco2 vs vt relationship tends asyntotically to a constant slope when approaches end-tidal volume. we have defined van as the volume that defines this relationship until a limit of 5% variation. methods: six subjects with normal respiratory mechanics were studied during anesthesia for minor surgery. two subjects, otherwise normals but having high values of total resistance and dynamic compliance, were also studied. capnograms were recorded in steady-state at 3 levels of vt (0.3, 0.5 and 0.8 l) and four levels of peep (0, 5, 10 and 15 cmh20 objectives: patients with ards presented lung abnormalities which originate an increase in airway resistance (rmin), in additional resistance (~rrs) and in static elastance (ers). application of peep further increases ~rrs. capnographic indexes reflect lung ventilation]per fusion inhomogeneities. in these conditions, the effects of peep on lung mechanics could be better understood by simultaneous measurement of capnographic indexes. methods: we studied 3 groups of subjects. n: 8 normal subjects scheduled for minor surgery; arf: 9 critically ill patients with mild acute respiratory failure; ards: 8 patients with early ards (< 72 h). we recorded tracheal pressure, airflow and capnograms. signals were analogic to digital converted for posterior data analysis. respiratory system mechanics was assessed by constant end-inspiratory and end-expiratory occlusions technique. at equal tidal volmne (0.5l) a peep level of 0,5,10 and 15 cmh20 was applied in all patients. we calculated ers (cmh20/l), rmin, c~rrs (cmh20/l/s) and autopeep. capnographic indexes were alveolar ejection volume (vae)/vt ratio and expired co2 slope beyond vae (sipco2 in contrast to synthetic surfactant natural suffactants (alveofact|174 are able to inhibit pmn-activation. after incubation of activated neutrophils with surfactant, l-selectin expression is decreased. these effects depends on which preparation is used. we conclude, that natural surfactant (aveofact| can perhaps influence early recruitment (,,rolling") of pmn in patients with respiratory failure like ards. with ards hormann cb, baum m, putensen c, knapp r, lingnau w, putz g . clinic for anesthesia and general lntensiv care medicine, university of lnnsbruck, anichstrabe 35, 6020 innsbruck objectives: in thoracic ct scans of patients with severe ards atelectasis and pleural effusion can be found in the dependent lung regions. by rotating these patients from left lateral position to right lateral position a redistribution of the ct densities, a recruitment of atelectasis and therefore an improvement of gasexchange is possible within a few days (1, 2). the objective of this study was to find out the mechanism of alveolar recruitment during lateral positioning by ct scanning in left and right lateral position. methodes: after approvel by the local institutional reviewboard we investigated 7 ventilated patients with severe ards (entry criterias: murray score > 2,5) in the ct scann of the university hospital. after a stabilisation period of 30 minutes in supine position a thoracic ct scan slice 1 cm above diaphragm was taken. then two different positions of the patients were studied in a randomized order: a) 60 degree of left lateral position, b) 60 degree of right lateral position. each lateral position was held for 20 minutes. at the end of each of these periods a thoracic ct scan slice 1 cm above diaphragm was taken. quantitative analysis of ct scan data was based on the frequency distribution of the ct numbers. to quantify the alveolar recruitment during lateral positioning by means of ct scan we defined 3 compartments within the lungs: a) normaly inflated lung, b) poorly inflated lung, c) noninflated lung ( = atelectases) (3). results: independant of the side of lateral positioning (l) in the non-dependent upper lung a significant increase of the normaly inflated compartment (s: 45%; l: 65%) as well as a significant decrease of the noninflated compartment (s: 34%, l: 12%) was observed in comparison to supine position (s). in the dependant lower lung the normaly inflated compartment decreased significantly (s: 45%, l: 26%) whereas the noninflated compartment increased significantly (s: 34%, l: 51%). throughout the whole studyperiode we did not observe any significant change regarding gasexchange and hemodynamic parameters. conclusions: in lateral position the non-dependent upper lung is decompressed. therefore a significant recruitment of atelectases is observed in the upper lung within 20 minutes. on the other hand the dependent lung is compressed by the weight of the upper lung and the mediastinum. a great amount of the alveoli of the dependant lung collapse in this short time intervall. therefore the net effect of recruitment of one positioning maneuver is very small. when positioning patients one should be aware, that the patient is kept in each lateral position long enough to clean up the atelectases in the non-dependant lung and short enough to compress less lung tissue in the dependant lung. objective: to analyze effects of low-dose no inhalation ia patients with severe aeut~ respiratory distress syndrome (ards) over five days. methods: we prospectively studied 10 patients (9 men, 1 woman) with severe ards admitted to our icu between may 1994 and may 1995 who required no inhalation with a dose of 5 ppm for at least 5 days. entry criteria for no injaalafioa were murray score >i 2.5 aud pat/fie 2 < 125 nun hg with peep >~ 8 em i~o for at least 24 hours. all patients were sedated, intubated and mechanicauy vantil~ed with volume assist-control ventilation, and had indwelling arterial catheters (pulmonary artery, and radial or femoral artery) to measure cardiac output (by thermodilufion) and relevant intravaseular pressures, and to calculate derived parameters. no was administered between y piece of the ventilator and endotraeheal tube and flow was adjusted to obtain 5 ppm no in the inhaled gas. the no, no 2 and no x concentrations were continuously measured at the distal end of the endouacheal tube by the chemiluminiscence method (nox 4000, see-seres, france). metahemoglobinemia levels were mesured daily. no inhalation was manteined if paojfio ~ improved at least 20 % and was stopped when the change in pao2/fio ~ was below 20% or when the patient presented a paojf02 > 150 mm hg a~er 30 minutes without no inhalation. every day we made an on-off test to determine if no inhalation improved pao2/fio ~. statistics: analysis of vmiance. data: mean + standard deviation. results: the mean age was 60.1 +_ 10.2 years and mean lung injury score was 3.3 • 0.2. mortality was 60 % (6/10), metahemoglobinemia 1.1 • 0.2 %, and no2 concentrations zero. paojf~o 2 always improved significantly al~er 5 ppm no inhalation (see :~ conclusions: reintubation in salf-extubated patients strongly depends on the type of meehamcal venfilatory support: the probability of needing a reintabation ff ese occurs during fult vontilatory support is higher than ff ese occurs during weaning. these data suggest that some patients may remain under weaning from mechanical ventilation for unnecessarily prolonged periods of time. objective: the aim of this study was to evaluate the acute effects on gas exehonge and hemodynamics due to positional changes from supine (sp) to prone (pp) in patients with severe acute respiratory distress syndrome (ards). methods: nine intubated, sedated, paralyzed and mechanically ventilated patients with severe ards were prospectively studied. all had a murray score > 2.5, and a pao2/f~o 2 < 100 with peep ~8 cm h20 for at least 24 h. all patients had indwelling arterial catheters in the pulmonary artery as well as in the radial or femoral artery in order to measure cardiac output (by thermodilution) mad relevont pressures, and to withdraw blood samples. arterial blood gases and hemodynamie parameters were measured first in sp, and then in pp after 60 minutes of stabilization. vontilatoly parameters remaing unchanged during all the study. statistical analysis was done by the non parametric wdeoxon test. data are expressed as mean ~= sd. results: there were 6 men and 3 women with a mean age of 54.2 years (21-71) and mortality was 55 % (5/9). main results are shown below: objective: to describe and compare a new method for obtaining p-v loops (p-vcv) by using a two-way collins valve (twv) with thosu obtained by the supersyringe method (p-vss). methodology: we prospectively studied 14 patients who had an aeute lung injury and were intubated, sedated and paralyzed, and mechanieany ventilated. we performed the p-vev loops and p-vss loops in random order, and the static inflation pressure was limited to 35 emh20 with both methods. pressure (p) was measured at the airway opening by means of a differential p transducer, and volume was obtained from flow (measured with a pneumotacograph) integration. the p-vse method has already been described (h~trf a,et al.bepr 1975; 11:709-28) . the p-vev method consists in the following: the inlet of a twv is connected to the ventilator's y-piece, and both outlets are couneeted to the endotraeheal tube by means of an additional y-piece; one of this outlets has a one-way rudolph valve in order to allow inspiration but not expiration during the inflation maneuver. changing the twv tap position allows basal ventilation or progressiveinflation of the respiratory system. this maneuver is as follows: during an end-expiratory occlusion, the ventilatory settings are adjusted to deliver a 100 ml v r with a respiratory rate of 20/min and i/e ratio 1:4; at the same time the twv tap is ehonged in order to divert flow through the one-way valve. inflation then begins alter releasing the expiratory oonlusion. pressure and flow signals were digitized and acquired by a computer for subsequent data analysis. we analyzed the following parameters: inflation compllonee ( objective: to analyze the variables which eventually may differentiate ards patients who do and do not respond to low doses of inhaled no. we prospectively studied 10 patients (9 men, 1 woman) with severe ards admitted to our icu between may 1994 and may 1995 who were treated with no (5 ppm). the onta'y criteria for no inhalation were murray score >/2.5 and paojfo z < 125 mm fig and peep >/8 cm i~o for at least 24 hours. all patients were sedated, intubated and mechanically ventilated with volume assist-control ventilation. tidal volume was between 6 and 10 ml&g, with constant inspiratory flow, respiratory rate was 15-25/rain, and i/e ratio between 1:2 to 1:3. all patients had indwelling arterial catheters (pulmonary artery, and radial or femoral artery) in order to measure cardiac output (by thermodiintion) and relevant intravascular pressures, and to calculate derived parameters. no was administered between y piece of the ventilator and ondotracheal tube, and flow was adjusted to obi~a 5 ppm no in the inhaled gas. the no, no 2 and no x concentrations were continuously measured at the distal end of the endotracheal tube by the chemilumiinscenee method (nox 4000, see-seres, france). metahemogtobinemia levels were measured daily. we considered a response to no inhalation when an improvement in paoz/fo 2 above 20 % was observed after the inhalation of 5 ppm no (group r) . when the cha~age in paojfi0 z was below 20 % it was considered a lack of response (group non-r small airways functional abnormalities have been recognized as a common feature of lung pathology. however peripheral airways contribute relatively little (~ 10%) resistance to flow and there disturbances can not be adequately estimated by conventional measurements of respiratory mechanics. the purpose of the study was to evaluate the relationship between raw and small airways conductance following weaning from ventilator methods. 37 patients (age:24-62 years; 22 males) with no serious complications al~er mitral or multiple valves replacements and with more than 15 hrs on mechanical ventilation have been enrolled in this study. the modified flow interrupter technique (ptg "gould" with fleish head #2; differential pressure transducer pm-131-tc "statham" w amplifier "kistler 7251") and flow-volume recording of forced expiration (fleish head #4) have been applied before surgery and following operation on mechanical ventilation (my), after extubation (t:xtijb), on 2 (2 nay) and 3 (3 day) days. airways specific conductance (sg aw) has been calculated as a mean of 7-10 consequent measurements in each patient at each stage. the sac was estimated by max expiratory flow at 50 and 25% of vc on 3-4 f-v curves (mef .~0, mef 25) all the data were statistically analyzed with t-test introduction : noninvasive ventilation (niv) reduces the need for endotracheal intubation, the length of stay in icu and the mortality rate in acute exacerbation of copd. however, some patients failed to be ventilated with niv. .objectives...; to further delineate patients who failed to be ventilated with niv and to obtain predicted factors of failure. patients : a cohort of 51 patients (72 • 10 years) presenting with acute exacerbation of copd (fevi: 610 • 396 ml, paco2:62 • 17, ph: 7.33 • 0.08) and nonmvasively ventilated (pressure support through a full-face mask) between april 1990 and may 1994 twenty-seven (53%) were successfully ventilated with niv (discharged alive without the need for endotracheal intubation) while 24 (47%) failed, requiring endotracheal intubation. .methods : patients successfully ventilated and those who failed were compared according to 35 respiratory and nonrespiratory variables univariate analysis (wilcoxon rank-sum test and fisher-exact test) was performed to select variables included in a multivariate analysis by stepwise logistic regression. results : underlying disease assessed by the simplified acute physiologic score (15 • 3 vs 11 • 3, p = 0.0003), creatinine serum concentration (122 • 45 vs 86 • 25 gm/l, p = 0.005), blood urea nitrogen (bun : 12 • 6 vs 8 9 3 mm/l, p = 0.009), age (75 • 9 vs 69 • 10, p = 0.01) were higher and encephalopathy (71 vs 30%, p = 0.005) more frequent in patients who failed. multivariate analysis showed that encephalopathic patients (or (odd ratio) = 4, p = 0.001) older than 65 years (or = 4, p = 0.04) and presenting with bun >_ 10 mmyl (or = 3, p = 0.01) failed to be ventilated with niv. variables related to the respiratory" status (i.e. paco2, pao2, fev1) were unable to predict tile failure of niv. conclusion : copd patients older than 65 years, presenting with acute exacerbation, encephalopathy and bun > 10 ram/l, should be carefully monitored because of high probability of failure with niv. methods:from february to december 1994 we studied 30 pa_ timnts,25 males and 5 females(mean age 68+/-5);18 of the se had emphysema,lo chronic bronchitis,2 dilatative car diomyopatia,with tracheostomy and emphysema.mean pac02 at admission in icu was 95+/-8mmhg,while when weaningbegan, 60+/-5.mean autopeep was 8 cmh20(4-12).all patients were ventilated in crpv as long as four hours to calculate st8 tic and dynamic cmpliance and autopeep.then the ventila tion was continued with psv+cpap(peep 7cmh20 objectives: analysis of the incidence of neurogenic pulmonary edema (npe) in a population of headtrauma patients with acute respiratory failure (arf). npe can occur after a central nervous system insult. differential diagnosis: cardiogenic pulmonary edema and other forms of non eardiogenic pulmonary edema. true incidence and pathophysiohigy remain poorly defined, however the role of catecholamines seems undeniable. early onset npe (within 12 h after trauma) is characterised by hypoxemia, transient pulmonary hypertension and bilateral central fluffy infiltrates on chestx-ray. characteristics of cardiogenic edema or pneumonia are absent. late onset npe, (beyond 12 hours after trauma), is more insidious. the clinical and radiographic picture has to clear within 24 to 48 hours. (1) methods: all headtrauma patients admitted from january 1 to december 31, 1993 in a nearotrauma icu setting were retrospectively analyzed for arf with as sole criterinm a pao2-fio2 ratio < 250. results: 151 neurotrauma patients were admitted during 1993.94 patients (63%) presented with severe head injury (gcs<8), 42 patients (27.8%) with moderate (gcs 8-12) and 15 patients (9.9%) with minor head injury (gcs 12-15). overall mortulity was 19.2% early (within 12 h. after trauma) and delayed onset respiratory incidents were distinguished, counting for 29 (19.2%), respectively 27 patients (17.8%), 7 patients (4.6%) had early and late respiratory complications. early respiratory insufficiency was caused in 9 patients (25.0%) by aspiration, in 11 patients (30.1%) by lung contusion, in 1 patient (2.7%) by fat embolism and in 15 patients (41%) by npe. in the late onset group 31 patients (91.2%) presented with pneumonia, 1 (3.0%) with fat embolism and 2 (5.8%) with npe. the npe group, 17 patients, presented as follows: 15 patients (88.2%) developed early npe, and 2 (11.8%) delayed onset npe. 9 patients (53%) died within the first days after admission, showing high mortality. gcs was less than 8 in 16 patients (94.1%), indicating severity of head injuries. conclusions: high incidence of arf with various etiology (41,7~ was found in this population. in about 10% of all admitted hcadtrauma patients (26,9% of arf) npe was causing attetial hypoxemia. occurrence of npe seems to be related to the severity of the brain injury and thus to outcome. these data call for extreme vigilance in respect of the insidious occurrence of npe. were included if recovering from respiratory failure and if in the opinion of the primary physician were ready for extubation. patients were excluded if undergoing compassionate withdrawal of support or had tracheostomies. the attending physicians were blinded to the measurements. included patients were placed on pressure support (ps) of 0 em h20 with demand-flow continuous positive airway pressure (cpap) 5 cm h20. after a minimum of 30 minutes on the above sehiogs: gastric intramucosai pc'o2, abg, and a p0.1 were measured. the padents were then disconnected from the ventilator for a period of one minute and the patients" respiratory rate and minute ventilation were measured using a wrights respirometer to calculate the frequency to tidal volume ratio (f/vt). patients were then extubated. extubafion failure was defined as the inability to maintain spontaneous ventilation for 24 hours for any reason. results: twenty patients met criteria and were studied over one month period in october 1994. six of the twenty patients (30%) failed weaning. the mean and standard deviation is outlined in failure 7.01+/-0.10 6.8+/-3.9 74.3+/-43.3 87.5+/-43.6 comparison between roc areas shows phi and p0.1 to each show a statistically significant difference from an area of 0.5 (p<o.05). the area at which the test has no discriminative value. this is not the case for either the integrative index or the f/vt ratio. the differences between areas for each curve is not statistically significant. the area's for each roc curve is shown in table #'2. results. 9 of the 21 newborns referred to us for ecmo-therapy developed barotrauma, 6 of the 9 required ecmo and one of the three other patients who did not recieve ecmo-therapy died. to determine the correlation between the risk factor barotrauma and the severity of the pulmonary situation, we determined the oxygenation index of each patient referred to us for ecmo-therapy . the table demonstrates 36, d-35392 giessen, frg. inhalation of nitric oxide (no) and aerosulizatiun of prostaglandin (pg) i: have been suggested to achieve selective pulmonary vasodilation and improvement of arterial oxygenation in patients with acute respiratory distress syndrome (ards). we directly compared these two modes of transbronchial vasodilator therapy in 16 ards patients mechanically ventilated for 24 -96 h (mean murray lung injury score [1] 2.75 + 0.05). patients were randomized to receive either first no and then pgi2 (n=8), or vice versa (n=8), with an intermediate baseline period. each drug was titrated individually to find the lowest effective dose for maximum improvement of arterial oxygenation. gas exchange variables, including data from the multiple inert gas elimination technique (miget), and hemodymmaics under application of no/pgi2 were compared to pro-and past-challenge values. no (mean dose 17.8 + 2.7 ppm) increased the mean oxygenation index (pao2/fio2) from 115 + 12 to 144 + 15 mmi-ig (p < 0.01) and reduced the shunt-flow from 33.1 + 3.6 to 26.6 5:4.5 % (p < 0.05). aernselized pgi2 (mean dose 7.5 + 2.5" ng/kg rain) augmented pao2/fio2 from 114 + 12 to 135 + 12 mmhg (p < 0.01), and decreased shunt from 33.5 + 3.8 to 26.0 + 3.9 % (p < 0.05). the miget analysis showed predominant redistribution of blood-flow from shunt areas to regions with normal ventilation-perfusion ratios in response to both agents. in 10 patients, both no and pgi2 caused an increase in pao2/fio2 by at least 10 rnmhg. two further patients displayed a corresponding improvement of arterial oxygenation in response to either no or pgiz. the mean pulmonary artery pressure decreased from 34.8 5:2.2 to 33.0 5:1.8 mmhg in response to no, and from 35.0 5:2.2 to 31.9 5:1.7 mmhg (p < 0.05) in response to pgi2. cardiac output, systemic arterial pressure, and right and left heart filling pressures were not affected by either agent. we r that individually titrated doses of inhaled no and aerosolized pgi~ effect selective pulmonary vasodilation and redistribute blood-flow from shunt-areas to weu-ventilated regions with nearly identical efficacy profiles. obieetives: the use of extraeorporeal bypass systems for oxygenation and co2-removal is performed in patients with acute respiratory distress syndrome (ards) to reduce mortality and to improve restitution of pulmonary functions. high-dose heparin -commonly used in such patients to prevent thrombotic complications -might cause incurable bleadings as a major risk. this lead to the development of heparinized bypass systems; results of animal and first clinical studies proposed that such systems might run with low-dose heparin or even without any systemic anticoagulation. methods: since 1989, 47 patients with severe ards were treated with extracorporeal lung assist (ela) using heparin-coated systems (type maxima, medtronic, carmeda coating) in our icu. they received a moderate systemic heparinization. standard clotting assays (act, aptt, tt, ptt, at-ill, fbg, fsp) as well as special tests (tat, r~-tg, pf4, pal-l, d-dimers, protein c, cl-inhibitor) were performed. results: covalent heparin-eoating of the ela-systems combined with lowdose heparinization could not prevent major changes in coagulation: 1) in mean, platelets dropped from 305/nl to 76/nl within 60 h after onset of ela. 2) tat levels were already elevated before ela start (55 ug/]) and demonstrated an additional peak 2 h after onset of ela (up to 240 ug/[). 3) in parallel, there was a transient peak of pai-i levels (from 5 to 75 au/ml} 2 h after onset of ela. 4) in mean, fibrinogen levels dropped from 470 to 310 mg/100ml within 24 h after onset of ela. 5) in mean, cl-inhibitor levels dropped significantly from 132% to 115% after onset of ela and reached the initial levels within 48 h. 6) after membrane change, there were significant changes for c1-inhibitor, fibrin-d-dimers and tt. 7) global clotting tests such as aptt, ptt and tt were within normal range. conclusions: patients with severe ards develop a prethrombotic state already before they get connected to the ela bypass system. after onset of ela, they experience additional involvement of procoagulant, anticoagulant, fibrinolytic, and complement systems. in parallel to laboratory findings, clinical data suggest that the use of heparin-coated systems with accompanying systemic low-dose heparinization does not avoid thrombembolic and hemorrhagic complications in tong-term ela patients. thus, at the present, higher dosed individually adjusted heparin treatment has to be recommended. thereby, standard clotting monitoring is not sufficient. objective: poor muscle performance contributes to prolonged dependence on mechanical ventilation. longitudinal data on muscle function in icu patients is scarce. we evaluated changes in inspiratory and peripheral muscle performance during prolonged intensive care in patients with acute respiratory failure. methods: 22 patients requiring intensive care for more than one week were studied. maximum inspiratory pressure (pimax) was measured twice a week for a maximum of three weeks. handgrip power (dynamometer) and subjective fatigue (keldet score) were obtained in survivors only. one measurement was done on the date of extubation. pimax pressure was obtained by occluding the airway for 20 seconds or at least five inspiratory breath attempts. obiectives -measurement of peep-induced changes in lung volume helps to assess respiratory mechanics in acute lung injury (ali). we evaluated the accuracy and stability of a new respiratory inductive plethysmograph (rip), in the measurement of peep induced changes in end-expiratory lung volume (eelv) and tidal volume (vt) against volume reference, pneumotachograph (pnt), methods -two hours periods of basal ventilation and stepwise increases and reductions of peep (5-10-15-10-5 cm h20) were recorded in patients with all. in addition, the new rip device (respitrace plus tm) was compared to an older rip (respigraph tm, nims, fl, usa) to determine its thermal stability using cold and warm devices and a ventilated lung model. results -the difference between the new rip and pnt in the measurement of vt was -30 _+ 3 mi (x _+ se) or a -5.2 _+ 0.6 % error. increasing peep from 5 to 15 cm h20 induced a 511 + 93 ml change in eelv. a difference of 18 + 12 ml (3.5 % of max. eelv change) (ns) was found between rip and pnt eelv readings. during controlled ventilation with constant settings the mean change in eelv was 187 _+ 69 ml/120 min (fig.i) . validation of calibration showed a mean error of 1 -+ 1.78 % after 120 rain. the new rip had a higher drift when cold (cold 28 + 10 ml/30 min vs warm 3 _+ 1 ml/30 min), whereas the old rip was thermally stable (cold -3 + 3 ml/30 rain vs warm i + 1 ml/30 min). trauma icu, hospital traumatologia y rehabilitacidn. vall d'rebron. barcelona. spain. recent data suggest that the proportion of patients with relevant discrepancies between two jugular bulb (jb) oxygen saturation (sjo 2) values is higher than suspected. objectives: determine the incidence, the significance and the pro9nosis value of those differences,if they exist,in severe head injured-patients. material and methods: 35 severe head-injured patients, coma glasgow scale (gcs) 8, with diffuse brain injury according to marshall criteria in the first ct scan, icp recorded, with an interval from accident-double jb monitoring < than 12 hours, were studied. data from bilateral sjo 2 were obtained simultaneously, every 6 hours. discrepancies were considered significant, when differences in sjo 2 were > 10%. no chan9es in treatment protocol (hyperventilation, man• etc) were carried out due to this study. results: 30 men and 5 women were studied, aged 32• yrs. at arrival at hospital, gcs were < 5 in 20 and ) 5 in to. the incidence of high icp() 20 mmhg) were 7sz at the entry. the mean therapy index level required to control lop was 4~l all patients required vasopressor therapy to maintain upp over ds mmhg. in 20 patients a s.s f swan-ganz fiberoptic catheter was used to obtain a continuous recording of sjo 2. in the others 15, sj02 were intermittently controhed.the mean time of monitoring were d.8• days. ten patients died within this period. a total of 1.240 blood samples were analized. at arrival, sjo 2 discrepancies were found in 22 patients, b2%. at 48 hours, the incidence were lower, 18/35, 51.4%. at 4th day, were h/29, 38z and at day 7, when the catheters were retired, ii[25, 44z showed discrepancies. the ct showed new injuries in g4z of patients with differences > 10~ in sd02 values throughout treatment period. none of those were considered for neurosurgical treatment. no correlation was found between iop and sjo 2 values and sjo 2 differences. conclusions: the incidence of discrepancies between sjo 2 was higher than expected in severe head-injured patients. these situation could reflect disturbances between 02 demands. when differences are known, and those lend to change, the ct scan, nearly always, will show new injuries. platelet-activating factor (paf) is an inflamatory mediator implicated in the pathogenesis of bronchial asthma and acute respiratory distress syndrome (ards). its inhalation in healthy subjects produces transient bronchoconstriction and mild ventilation-perfusion mismatch, together with peripheral leukopenia as a result of intrapulmonary neutrophil (pmn) sequestration. likewise our group has shown in healthy subjects and asthmatic patients that aaibutamol (s) inhibits both pulmonary and systemic effects of paf, suggesting that s may inhibit paf-induced venoconstriction in pulmonary microoirculation. the aim of the present study was to investigate if s inhalation decreases pmn by lung sequestration induced by paf. we studied 8 healthy, non-atop• nonsmoking subjects (6m/2f, 24+4 yr), which were pre-treated with s (300,ug) or placebo, with a randomized, double-blind, crossover, design, before paf (24,ug) inhalation. we measured the respiratory system resistance (rrs) by forced oscillation, arterial btood gases and both total white cell and pmn count every 4 min over a 30 min. period. simultaneously, we recorded continuously the lung dynamics of inm-neutrophil and tc99m-erythrocytes activity, with a gammacamara. after placebo, paf inhalation decreased white cells (from 5410 2 1125 to 33022934x109/l), and pmn(from 29752693to 1222 _+ 767 x109/l), and increased aapo 2(from 2.1 _+9.5 to 14.7+ 12.2 mmhg, p<o.05) and rrs(from 3.0 2 0.6 to 3.7 2 0.7 cmh20.s.i q ) (p < 0.05 each). the fall in total white cell count correlated with the intrapulmonary pmn retention (r =0.83, p <0.05). by contrast, after s, paf did not induce any change in white ceil count (from 596321766 to 6147+1721 x109/i), pmn count (from 354321628 to 2953 2 1793 x109/i), aapo 2 (from 7.3 2 10 to 5.6 + 8.6 mmhg), and rrs (from 2.9 2 0.5 to 2.9 2 0.7 cmh20.s.i 1). compared with placebo, after s there was a lower ~lln-pmn lung activity (2.020.4 vs 1.320.7 cts/mci/pixel, p<o.01 ), lower intrapulmonary pmn retention (15.627.7 vs 11.1 24.8 %, p=o,09), and a faster washout (0.1420.08 vs 0.1720.007 s ~, p=0,4). our results indicate that s inhibits paf-induced intrapulmonary pmn sequestration, being consistent with the hypothesis that s may offset the venoconstriction of pulmonary microcirculadon caused by this mediator. supported inpiratory muscle fatigue with failure of force generation as def'med by a tensiontime index (tti)>0.15-0.20 has been shown to occur in normal volunteers and in stable copd patients with a specific imposed breathing pattern. its role, however, in hypercapnic respiratory failure is less certain. we studied 10 failed weaning trials in 5 copd patients in which breathing pattern, tension-time index (tti) of inspimtory muscles, dynamic peepi, dynamic lung elastance, lung resistance, and arterial paco2 and ph were measured at the beginning and end of a t-piece weaning trial. in addition, the change in esophageal pressure during a mueller maneuver (apes max) was measured. a weaning trail has been prospectively defined to have failed if one of the following criteria was met: a rise in pco2 >20mmhg from baseline accompanied by a fall in ph<7.35; a respiratory frequency (f) >30/min; excessive accessory inspiratory muscle recruitment; and a marked increase in dyspnea. values are expressed as mean • se. weaning failure was characterized by a more rapid, shallow breathing pattern, worsened mechanics, hypercapnia and respiratory acidemia despite an unchanged tri and pes max. we conclude that in this setting hypercapnic respiratory failure is not a consequence of inspiratory muscle fatigue. rather the adopted breathing strategy and resultant hypercapnia may represent an adaptation to forestall the onset of muscle fatigue. concerning the investigated elf-par~eters, no stadstically signhqcant differences were detected between the pgi2 and the control group. histopathologlcal changes occured in both groups and consisted in rare focal flaaaning 0f tracheal epithelium with loss of cilia and slight inflammatory cell infiltration, as well as slight swelling of alveolar typo4 pneumoeytes. sections of generation 5, 10 and 15 from bronchial tree were free of pathological changes. conclusion: alter 8h inhalation of p~ji2 no signs of respiratory-lract tissue damage caused by the aerosol could be detected. the minor pathological findings in the trachea are most likely due to mechanical irritation by bronchoscopy, changes of the alveolar epithelium are known for long-term mechanical ventilation 3. objectives: the aim of this study was to evaluate of efficiacy of ganglion stetlate blockade in patients with respiratory failure. methods: two groups of patients were investigated: group i (n = 15) trauma patients with acute lung injury (ali), group if (n = 15) patients with asthmatic status. in all cases continuous mandatory ventilation (cmv) was used with bennett 7200 ae. in both groups bilateral ganglion stellate blockade with antero-lateral approach was performed, using 0.375 % marcain. the following parameters were analysed: pao2, sao2, paco~, pip and c~t~t. results: in trauma patients with aij after bilateral ganglion stellate blockade short -lived and slight improvement of pao 2 and sao2, decrease of pacoz and pir and increase of static compliance of respiratory system were found. in second group bilateral ganglion stellate blockade interrupted the asthmatic status and significant statistical improvement of parameters of oxygenation, ventilation and respiratory system mechanics were observed. conclusions: we suggest that the bilateral ganglion stellate blockade is a very useful method in treatment of patients with obstructive respiratory insufficiency. the aim of the study was to analyse whether there exists serum and urine electrolyte disorder in patients(pts.) with acute respiratory insufficiency(ari). the study included t8 pts. with ari (pao2:8,24@1,49 kpa. paco2: 5,01i-0,77kpa, ph:7 42~:0,59, hco3: 26,3:~8,10 mmol/1, sao2 : 90,4~-7,42%) who were hospitally treated due to pneumonia(9 pts.),emboly of the pulmonary artery(3 pts.) and severe attack of bronchial asthma (6 pts). among tham there were 12(66,7%) males and 6(33,3%) females, average age 51,5~:16,1 years, otherwise previously healthy. electrolyte concentracions were measured at the onset of the disease in serum and urine collected during 24 hours (sodium-na,potassium-k, chlorine-c1, calcium-ca,magnesium-mgand phosphorus-p). the measured serum and urine electrolyte concentrations were compared with respective referent values (rv). by serum electrolyte analysis, the following average velues were obtained: na:l 4o,94 the object of our investigation was a group of 21 pts with massive pneumonias, 14 males (66.6%), 7 females (33.3%),mean age 55 yrs.thirteen (62%) of them were smokers,8(38%) nonsmokers. only 1 pt (4.7%) had pre-existing chronic respiratory disease, and 20 (95.2%) were admitted for the first lime,with no previous respiratory anamnesis. diagnose was based on anamnestic data of productive cough in 15 pts(71.4%),physicaly ~onchial breathing in 19 i~s (90.4%),white cell count onder 10 x 109 /l in 18 pts(85.7%). radiographicly, bilateral massive homogeneous shadows were found in 7 pts (33.3%), onilateral in 12 pts(57.1%),pleural effusion in 2 pts (9.52%). abnormal renal function was found in 14 pts (66.6%). sputum culture was positive in 8 pts (38%): slr.pneumoniae, str.pyogenes, pse'udomonas aerug, in 4, 2, 2 cases respectively. all patients had remarcable hypoxernia (pao2 range from 4,75 to 8,1 kpa) without hypercalmea. all patients needed oxygenotherapy together with antibiotics and other .symptomatic therapy. nineteen pts had anaelioration of general condition and normalization of blood gas analyses, while 2 pts with the lowest hypoxcmia died.in conclusion, massive pneumonias are frequently followed by respiratory insufficiency which is one of the markers of pneumonia severity. as existing hypoxemia complicates the course of the disease,prolonges the recovery, makes therapy more complexe and may be cause of death , frequent blood gas measurement is recomanded. we studied the effects of bosentan (bos), an eta and etb receptor antagonist, to examine if endogenous et mediates pulmonary hypertension in anesthetized and ventilated dogs with acute lung injury due to oleic acid (oa). the gradient between pulmonary artery pressure (ppa) and occluded ppa (ppao), and gas exchange (evaluated by arterial blood gases and sf6 intrapulmonary shunt) were measured at controlled flow. in 8 dogs (treatment), data were collected at baseline, during long injury (obtained 90 rain after intravenous administration of oa 0.06 ml/kg), and again after bos (10 mg/kg intravenously). in 5 dogs (pretreatment), data were obtained at baseline, after bos and then after oa. in treated dogs, oa increased (ppa-ppao, mmhg, table, means + sem, * p < 0.05 vs base) and deteriorated gas exchange. after oa, bos did not affect pulmonary vascular tone nor gas exchange. in pretreated dogs, bos had no effect on baseline pulmonary vascular tone but prevented the increase in (ppa-ppao) after oa. the deterioration in gas exchange after oa was not influenced by bos pretreatment. objectives: the alveolar 02 tension is measured by the application of the alveolar air equation in which the arterial pco 2 is used or by the simplified form of this equation in which the respiratory exchange ratio is taken at the value of 0.8. the purpose of this study was to estimate the effective alveolar 02 tension (pao2eff) during spontaneous breathing with a new bedside technique which is simple non-invasive in 14 normal subjects and 27 patients with chronic bronchitis-emphysema. we also compared these values with the ideal alveolar po 2 (pao2(i)), measured from the alveolar air equation in which paco 2 was substituted by the effective alveolar pco 2 (paco2eff) and with the alveolar po 2 measured from the simplified alveolar air equation (pa02). this study is complemantary to previous work for the estimation of paco2eff. methods: the subjects breathed quietly through the equipment assembly (mouthpiece monitoring ring, fleisch transducer head) connected to a pneumotachograph and a fast response 02 and co 2 analyzer. the method is a computerised calculation of the effective alveolar po2quite similar to that of paco2eff, obtained from the simultaneously recorded at the mouth expiratory flow, 02 and co 2 concentration versus time curves. results: the results showed a mean difference (pao2eff-pa02(i)) of -0.061 kpa in normal subjects and -0,711 in patients. the mean of the difference (pao2eff-paq 2) and (pad2(i]-pao z) was much greater than 0.281 in all subjects. the limits of agreement for the difference (paozeff-pa02(i))were -0.691 to 0.568 kpa in normal subjects and -2.040 to 0.596 in patients, while those for the differences (pao2eff-pad 2) and (pao2(i)-pad 2) were very large ( > -1.5 to > 1.7) in all subjects. conclusions: the effective alveolar po 2 is very close to the ideal one in normal subjects, tn patients pao2eff may excessively deviate from pa02(i) due to the observed significant difference between the alveolar/tidal volume ratio for o 2 and that for co 2. the alveolar po 2 measured from the simplified alveolar air equation (pao 2) differed substantially from pao2eff and pad2(i) in all subjects. the essential role of glucoprotein hormone erythropoietin is to control red cell production. hypoxemia, reduced blood 02-carrying capacity and increased affinity of hemoglobin for 02 are the primary stimuli for erythropoietin production. both anemia and hypoxemia induce rapidly erythropoietin secretion. kidney erythropoietin rna levels correlate inversely with hematocrit and directly with plasma erythropoietin level. similarly, hypoxemia increases kidney erythropoietin rna and plasma erythropoietin. the effect of hyperoxemia (pa02>lo0 mmhg) on erythropoietin secretion isn't very well understood. the purpose of this study was first to evaluate the erythropoietin secretion in patients with acute respiratory failure and second to determine the effect of hyperoxemia on erythropoietin secretion in patients with and without anemia. sixteen patients with acute or acute on chronic respiratory failure needed mechanical ventilation were included in this study. these patient were divided in two groups. the patient who developed anemia were included in group i and the patients without anemia in group i1. erythropoietin was estimated in venous blood in three stages. the first sample was taken during hypoxemia, the second during hyperoxemia and third during normoxemia. all the patients had high erythropoietin level during the hypoxemia period (mean value 98• mu/ml). during hyperoxemia etythropoietin levels were reduced in both groups ( mean value 21.6+15.2 mu/ml in group i, 36.8• mu/ml in group ii). in normoxemia stage, erythropoietin increased again in anemic patients, and decreased more in the patients of group i1. we conclude that hyperroxemia inhibit erythropoietin secretion in spite of anemia and tow arterial oxygen content. hyperoxemia may be a factor of the insisted anemia in with oxygen treated icu patients. the purpose of this study was to determine the relationship between clinical features of acute lung injury (all) and parameters like total proteins, total and individual phospholipids, the presence of paf, and acetylhydrolase activity in bal of mechanically ventillated patients. acetylhydrolase catalyses the cleavage of acetyl-group from the second position of the glycerylether backbone of paf, leading to its inactivation. mechanically ventillated patients were divided to three groups. group i includes patients without all; group ii, comprisespatients with moderate degree all, (1.0<g-i1<2.5); and group iii comprises 5 patients with severe ards (all score > 2.5). broncoalveolar lavage (bal) was obtained after infusion of normal saline at 37~ to intubated patients and cooled immediately. cells were removed after mild centrifugation (350 x g, 30 min, 4oc). aliquots from the supernatant were used for total protein, phospholipid and paf analysis and determination. acetylhydrolase activity was assessed after incubation of bal with 3h-paf labelled on the acetyl group. released label was measured by liquid scintillation counter in the supernatant after trichloroacetic acid precipitation of the non-reacted substrate. kinetic characteristics of the enzymes were also studied. total phospholipids appear reduced in bal of patients with all, while total proteins increase. these factors appear to correlate with the severity of all. paf was not present in bal samples pretreatad with equal volume of 20% acetic acid to denaturate acetylhydrolase. detection limit for paf under our experimental conditions: 60 pg paf/ml bal. instead, acetylhydrolase activity was detected in amounts increasing with the total protein content. background: intubated patients without lung injury or impaired breathing control normally display an inspiratory peak flow of below 1l/s. the aim of our study was to investigate the inspiratory peak flow generated by patients with acute respiratory insufficiency (ari). we had to take into account that both an inspiratory pressure support (ips) and the resistance of the endotracheal tube considerably influence the flow pattern generated by the patient. patients and methods: to investigate the non-influenced flow pattern we developed a new ventilatory mode which automatically compensates for the flow-dependent resistance of the endotracheal tube (automatic tube compensation, atc). furthermore, the mode maintains a constant tracheal pressure in inspiration and expiratio n . consequently, the measured flow pattern exactly corresponds to the flow pattern generated by the patient except that the ventilator modified for this mode (evita, driiger liibeck, germany) was not able to deliver a gas flow of more than 2l]s. we have investigated 10 patients with ari arising from different reasons. results: the inspiratory peak flow measured in the atc-mode was 1.7l/s _+0.3l/s. the maximal deliverable flow of 2l/s was obtained in 3 of 10 patients. the figure shows the flow pattern under atc and ips in [~s] oi:) one of these patients. conclusions: patients with ari display a highly increased inspiratory peak flow. ventilators used for spontaneous breathing should therefore be able to deliver a gas flow of more than 2l/s. an overproduction of no and reactive oxygen species (ros) has been demonstratred in septic shock. ros and nitric oxide (.no) are free radicals which are known to react together leading to peroxynitrite anions that can decompose to form nitrogen dioxide (no2) and hydroxyl radical (oh~ thus, no has been reported to have a dual effect on lipid peroxidation (prooxydant via the peroxinitrite or antioxidant via the chelation of ros). in the present study we have investigated in different models the in vitro and in vivo action of no on lipid peroxidation. copper-induced ldl oxidation was used as an in vitro model of lipid peroxidation. ldl (100 ~g apob/ml) was incubated with cu 2+ (2,5 ~tm) in presence or absence of no donor (sodium nitroprussiate or glutathione-no) from 10 to 500 ~m. oxidation of ldl was monitored continuously with conjugated diene formation (234 nm) and 4 hydroxy nonenal accumulation (hne). exogenous no prevents in a dose dependent maner the progress of copperinduced oxidation. ischaemia-reperfusion injury (i/r), characterized by an overproduction of ros, is used as an in vivo model. anaesthetized rats were submitted to 1 hour renal isehaemia following by 2 hours of reperfusion. sham operated rats (sop) were used as control. lipid peroxidation was evaluated by measuring the hne accumulated in rat kidneys in presence or absence of l-arginine or d-arginine infusion. l-arginine, but not darginine, enhances hne accumulation in i/r but not in sop (<0.05 nmol/g tissue in sop versus 0.6 nmol/g tissue in i/r), showing that in this experimental conditions, no produced from l-arginine, enhances the toxicity of ros. this study shows that the pro-or antioxydant effects of no are different in vivo and in vitro and could be driven by environemental conditions such as ph, relative concentration of no and ros, ferryl species...these conditions are impaired in circulatory shock. methods:" the diagnostic and therapeutic approach was standardized so that data collected over a 10-year period were comparable. a progressive deterioration of clinical conditions and/or pulmonary gas exchanges was considered as indication for my. variables potentially predicting the need for hv were derived from clinical and arterial gas data, extrapulmonary diseases, use of drugs, chest x-ray and ecg abnormalities. results: rv, performed with external and/or internal ventilators, was necessary in 130 patients (22%). at the hospital admission, pac02 was higher and ph was lower in patients requiring rv ( pneumomediastinum, pneumothorax, ateleetasis and myocardial infarction are rarely seen in bronchial asthma. these complications occur as a result of the severe asthma.the aim of our retrospective study was to analyse the complications seen in acute asthma attacks. during the years 1990 through 1994, 244 patients were admitted to hospital in acute asthma episode. there were 11 (4,5%) pts with complications; mean age of 27 yrs; 6 females (54%). clinical history, ecg and chest radiogr~hs were analysed. the mean duration of bronchial asthma was 14 yrs (range from 2 months to 17 yrs), all patients were atopics. there were four ex-smokem and one smoker. the worsening of asthma symptoms begun two days before the admission (range from 1 to 7 days). on ecg all patients had tschycardia. rightward shift of the qrs axis and st-t changes indicative of right ventrieutur strain were found in three pts. these were the transient fmdings that improved after curing the acute asthma attack. non-q myocardial infarction oeeured in one patlent and resulted from the hypoxaemia of asthma. hyperinfl~ion was the usual finding on the chest radiograpk pneumomediastinum and subcutaneous emphysema were apparent in five pts and required no additional treatment unilateral pneumothoraccs were present in two pts and needed eontimous intrapleural drainage; one of these patienst died in eardiorespiratory insufficiency. ateleetasis of right upper lobe was present in one patient. it oceured due to inspissated secretions and needed no additional treatment all these patients, except one who died, improved on lreaanent with oxygcr~ steroids, beta-two agonists, theophylline and antibiotics. in conclusion, complications occur in acute asthma episodes as a result of the severe asthma mediastir,*l emphysema and atelectasis are not serious complications. pneumothorax and myocardial infarction are very serious life-treatening complications and always have to i:m considered in taati~ts with sev~ asthma. acute bronchial asthmatic episodes represent one of the most common respiratory mnergendes, its maximmum expression "status asthmatiens" is one entity of low incidence, still it is a risk to the physical integrity of the patient. during 1993 a total of 52 patients with diagnosis of status asthmabcas were hospitalized. out of these palients six had a near-fatsl asthma and they were subjected to a complex examination. near-fatal asthma was defined as either respiratory arrest or acute asttuua with paco2 greater than 6,7 kpa and/or an altered state of consciousness. mean age was 56,2-d:16,2 yrs, four male and two female sex. at presentation two patients suffered from coma, others were confused. they exh'bited severe dystmoes, diffieul~ speaking, used accessory muscles of respiration, increased whee~tg while two cases had silent chest on auscultation. cyanosis indicated a very severe asthma attack in all six patients. mean respiratory rate was 28~4/min and puts rate 118.d: 12 bts/imn. arterial blood gases revealed a pao2 of 6,95~1,33 kpa, paco2 of 7,87• kpa and ph of 7,274-+-0,132. area-careful evaluation they received conventional therapy (immediately continuous oxygen, impelled nebulization with high doses of betatwo agonists and ipmtropium bromide, intmvanous st~oids and theophylline). in two eases signs and symptoms of deteriorating airflow and respiratory muscle fatigue determined the need for mechanical ventilation. out of six near-fatal attacks aggressive lrealanent was suscessfull in four patients and fatal in two eases. one patient admittcxl in coma died in severe hypoxae~a upon one hour and one mechanicaly ventilated died from cardiac arrhythmia. life-threatening attacks in asthmatics in our group developed gradual worsening despite neatment which r symptoms in most other patients. one patient had "brittle asthma", other long-standing acute episodes ireated with systemic steroids. conclusions: idantitiechon of fatality prone subjects may lead to fttrther muetion of seveze episodes. respiratory affest and coma upon admission, severe dyspnoca with silent chest on ausouhation, oyanusis and use of accessory muscles of respiration constitute the basic cfinieal picture. hypoxasmia must be immediately eon'ected.the patients and physicians should be able to assess the severity of asthma, a major factor in near-fatal and fatal asthma attacks. objectives :our purpose was to asses if the evolution of patients with a adult respiratory distress syndrome (ards) ,shows any relation to the pulmonary or systemic origin of the disease and whether or not there were differences in the frequency of the syndrome in both groups. methods : randomized prospective study in multidisciplinary icu. one hundred and sixteen patients with a high risk developing ards were distributed into two groups. one was named systemic origin group(so) and the other pulmonary origth group (po).ai1 patients only showed one cause (pulmonary or systemic) with potential risk of ards.the patient's hemodynamic and respiratory status was evaluated every 6 hours the first day and every 12 hours the second and third day. at the end of 72 hours the patients were diagnosed as ards or non-ards. measurements and main results : of the total 116 patients, 57 were finally included in the so group and 59 in the po group.patients in so group and po group had comparable ages (p<.01).peep in both groups was comparable (=.06) at the mmnent of admission to the study. there were no statistically significant differences for cardiac index and systemic vascular resistances. the pulmonary vascular resistances (pvr) showed significant differences at 48 h.(p<.05) and 72 h. (p<.03).the oxygen comsumption (vo) in patients of the so group showed statistically significant differences at 48 h. (p<.05) with respect to initial values.fifteen cases of ards (26.3%) in the so group and twenty five cases (42.3%) in the po group were identified. the time of onset of ards was 35_+ 14 hours in the so group and 11 + 4 b hours in the po group.the final outcome was very similar th both groups : mortality of 36% in the so group versus 37% in the pc group. conclusions : the pathogenesis of ards depends on whether the lesion is originated at or outside the lung. the po group showed a sborter thne of onset of ards, a faster and more severe increase of pulmonary shunt and a higher percentage of patients developing ards compared with patients of the so group.the so group showed a higher and faster increase in puhnonary resitances tbat po group and a decrease th oxygen comsumption earlier and more severe than in the po group. these data thus seem to show that there could be two mechanisms involved in the genesis of ards depending on the cause. the fact that the ards genesis is shorter in the cases of pulmonary etiology with faster impairment of pulmonary shunt, and a slower increase in pulmonary resistances in this pulmonary group, would indicate that the underlying mechanisms responsible for the hypoxemia are different to those which thitiate the increase in pulmonary resistances. finally, the exclusive inapairinent of oxygen consumption, which appears earlier than the onset of ards in the systemic origth group, could show the generalized character of the process in this group. perfusion of prostacyclin (pgi2) to treat pulmonary hypertension in adult respiratory distress syndrome (ards) worse pulmonary gas exchange due to a marked impairement of ventilation/perfusion mismatch. recently has been shown that if prostacyclin is given by aerosol instead of intravenous the net effect is an improvement of arterial oxigenation due to a redistribution of blood flow to well ventilated areas. objectives: to asses the effects of inhaled proatacyclin on pulmonary haemodynamics and gas exchange in patients with severe ards. methods : two patients with severe ards (murray score >3) recived inhaled pgi 2 at 15-20 ng.kg.min "1 using an ultrasonic nebulizer. haemodynamic measurements, arterial and mixed venous blood gas analysis were performed before and after 30 rain of pgi inhalation. results: short-terro p~i 2 inhalation improved pulmonary g-~ e-'~hange in both patients. arterial oxygen partial pressure (pao2) increased from 101 to 166 mmhg in patient 1 and from 87 to 108 in patient 2, the ratio pao to the fraction of inspired oxygen increased from 1262 to 207 (patient 1) and from 124 to 154 (patient 2). venous admixture decreased from 36% to 29% and from 34% to 27% in patient 1 and 2 respectively. mean pulmonary artery pressure decreased slightly from 25 to 23 mmhg in patient 1 and from 41 to 37 mmhg in patient 2. no effects on systemic haemodynamics were observed in any patient. conclusions: pgi 2 inhalation improves gas exchange and produces selective pulmonary vaaodilation, thus can be an alternative therapy for the treatment of pulmonary hypertension and hypexemia in patients with severe respiratory falllure. methods: we treated 67 ards-patients (age 41 yr (16-75) mean, range) during 1991-94. the lowest pao2/fio2-ratio was 74 (29-140), the worst murray score 3.0 (2.3-4.0), icu-stay 41 (1-121) days and hospital mortality 40%. the costs of intensive care were calculated according to intensivity of patient care as assessed by tiss-scoring (therapeutic intervention scoring system). the more intensive the care, the higher are the costs. costs per year of life saved (=life-year" in us $) were compaired by other medical treatments (1-4). it is assumed that the mean expected length of remaining life in ards-survivors after intensive care is 25 years. treatment life-year ($) ' bone marrow transplantation (acute leukemia) 65 000 lowering cholesterol using iovastatin 51 000 treating hypertension using nifedipine 32 900 heart transplantation 28 000 intensive care of ards-patients 3300 conclusions: intensive care of patients with severe ards is highly more cost-effective as compared with many other routinely used medical treatment strategies, the usually good recovery and the reasonable quality of life in survivors justifies investments to care of these patients (5). there is a close correlation between these two methods of measuring evlw. however there is an underestimation of 38.5 % in this kind of pulmonary edema ( oleie acid induced ) with the double dilution method. although the size of the sample is small, in normal lungs there appear not to be this underestimation. the effect of peep on evlw has been studied with contradictory results, probably as a consequence oft differences in methods of measuring evlw, variations in the type and severity of lung injury, and different timings of peep application. objective= 1) to analyse the effect of different levels of peep (0, 10 and 20omh20) on evlw during hpe; 2) to establish whether increases in intrathoracic pressure due to high peep levels can obstruct lymphatic drainage. material and methodet hpe was provoked in 3 groups of dogs by inflating a foley catheter in left auricular to a pressure of 24-26 r~uhg. peep levels of 0, i0 or 20 m~hg were applied. resultst objective: to assess the effect on extravascular lung water (evlw) of the application of peep and the reduction of vt in an oleic acid pulmonary edema model in pigs, using three ventila~ary strategies. material and methods: twelve adolescent pigs (weighing over 30 kg) were randomly divided in three gmups immediately alter infusing via a central vein 0.1 ml/kg of oleic acid to produce a permeability pulmonary edema. the ventilatory parameters for each group were as follows: group i (n=4) : vt: 10-15 ml/kg; zeep. group 2:(n=4) : vt: 10-15 ml/kg; peep: 10 cm h20. group 3:(n=4) : vt: 5-10 ml/kg; peep: 10 emil20. (resulting in permissive hypereapnla) after a four-hour period of ventilation the animals were killed and the lungs excised to calculate gravimetrically the extravascular lung water using a standardized procedure ( hemoglobin content method ). ill evlw (ml/kg) group obiective: in the postoperative period, maintenance of adeguate arterial oxygen tension is a major problem in morbidly obese patients probably because of a large reduction in functional residual capacity (frc). the aim of this study was to evaluate the effects of peep on respiratory mechamcs and gas exchange in this kind of patients. methods: in nine postoperative mechanically ventilated morbidly obese patients (bmi>40 kg/m 2) we partitioned the total respiratory system mechanics into its lung (1) and chest wall (w) components using the airway occlusion technique associated with the esophageal balloon, during constant flow inflation (jap 1989; 67: 2556) . at three different levels of peep (0, 5, 10 cmh20 ) we measured: compliance (cst), airway (rim) and "additional" (dr) resistance, frc and gas exchange. obiectives. to describe the use of prone position in our icu we analyzed the clinical records of all patients admitted in 1993-94, selecting adult patients with arf defined as: intubation and pao2/fio2<250 mmhg plus an fio2>0.5 or peep>5 cm i120. results. 146 patients met the arf criteria: 40 of them (27.4%) underwent prone positioning (p+). prone position use began in the early phase of arf (3.5• days from the beginning, range 1-32, median 2).25 out of 40 p+ pts were treated with controlled ventilation (cppv or pcv), while 14 were on assisted ventilation (simv+ps) and 1 on spontaneous breathing (cpap). only 2 pts were awake when turned prone, while 11 pts required adjuncts of sedation to tolerate the change of position. the duration of prone positioning was variable (average lenght 4.7• h, range 0.5-12 h). only minor side effects were observed (eyelids and facial edema, chest and facial pressure bruises). we consider responders (r+) those patients presenting at least 12.5 mmhg increase in pao2/fio2:35/40 patients (87.5 %.) were responders when first pruned. the pao2/fio 2 changes induced by prone position are reported in the figure. pao2/fio 2 increased when patients were pruned (*p<0.001) and remained higher than baseline values when returning supine(*p<0.001). paco 2 remained unchanged. prone positioning was used at least twice in 21/40 ( conclusions. this retrospective analysis confirms that prone positioning improves oxtgenation in the majorib' of arf patients. altough we have no available criteria to discriminate in advance r+ from r-pts, we now routinely consider the use of prone position in the treatment of severe arf. palo a, otivei m*, galbusera c, veronesi r, sala gallini g, zanierato m, iotti g, braschi a.servizio anest. e rianim. i, *laboratorio biotecnologie e tecnologie biomediche irccs s. matteo, pavia, italy inhaled no can improve arterial oxygenation and reduce pulmonary hypertension in ards patients; little information is, however, available about the dose-response curves. methods seven ards patients (lis 2.7+.5) submitted to mechanical ventilation randomly received 8 inhaled no doses in increasing or decreasing sequence: 0.5, 1, 5, 10, 20, 50 and 100 ppm. reference measurements were obtained before and after the entire period of no inhalation. hemodynamic parameters and blood gases were measured after 25 min in each condition. cmv was administered under sedation and paralysis, with constant ventilation, peep (lol-_2 cmh20) and fit2 (.56+.14). the changes in vt and fit2 due to the no (1000 ppm in n2) injection in the ventilator external circuit were compensated for. results .34 the dose of 0.5 ppm, ineffective on papm, significantly improved oxygenation. the increase of pat2 and the decrease of q'va/q' and papm were nearly maximal at 5-10 ppm. no deterioration of arterial oxygenation was observed at no doses as high as 100 ppm. co2 exchange was not influenced by no inhalation. systemic hemodynamic variables did not change throughout the study. these results suggest that a concentration around 10 ppm is adequate for obtaining maximum effects on hypoxemia and pulmonary hypertension in patients with ards. low-dose inhaled nitric oxide (no) induces redistribution of pulmonary perfusion in patients with severe ards and causes improvement of oxygenation [1] . however, addition of exogenous lowdose no in the inspiratory gas mixture might be only a replacement of missing atmospheric no (2-130 ppb) in hospital central-supplied medical air. [2] we have realised nitric oxide measurements in ten healthy volunteers, (4 smokers and 6 non-smokers) breathing with a mouthpiece and occluded nostrils through a ventilator circuit, with separation of inhaled and exhaled gases by a valve. no concentration was measured with a double-chamber chemiluminometer (environnement sa, france) and with charcoal/silicate purified compressed air. there was no nitric oxide detectable in the inspirat0ry limb of the ventilator. unfiltered central supply medical air contained :20 -50 ppb of no and 10 -30 ppb of no2, whereas central supplied oxygen was no/no 2 free. samples were taken after equilibration periods of 5 minutes, with increasing fit2 levels of 0.21, 0.50 and 1.0 for subsequent 5 minutes periods; paired values were recorded every 30 s. the mean no value was 4.57 ppb (sd 2.51) and n o significant differences were found for different fit2 levels both in smokers and non-smokers. these data suggest that the no concentration of pulmonary origin in the exhaled air of' healthy volunteers is probably lower than that reported by other authors [2] and that, previously reported, differences between smokers and non-smokers are not always striking [3] . we suggest the use of activated charcoal/silicate filters for clinical trials in order to achieve standard conditions. [ objective: to compare efficacy and safety of two doses of salbutamol. methods: sixteen adults who had severe acute a~hma were randomly assigned to receive either 10rag (n=9) or 5rag (n=7) of nebulized sulbutamol. both groups were similar with respect to age, duration of a~hma, duration of attack before arrival at the hospital and severity of a~hma according to baseline measurements (table) . evaluation was performed 30, 60, and 120 rain after the start of nebulization. results: compared with 10mg regimen, 5mg regimen resulted in the same improvement in peak-flow and fischl index (figure). the changes in heart rate, respiratory rate and pace2 did not differ significantly between both groups. the incidence of side effects, which included tremor, palpitations, cardiac arrythmlas and other symptoms, was not sj~ificanfly different in the two populations. conclusion:the results of this study suggest that nebulization of 10ng of salbutamol is not more effective than 5rag in the initial treatment of acute severe asthma in adult patients. the prognostic factors of neutropenic patients admitted to the icu remain poorly known. the aim of this study was to determine the respective weight of underlying malignancy and organ system failures on the outcome of these patients. patients and methods: the charts of 107 neutropenic patients (wbc < 1000/mm 3 and/or pmn < 500/ram3), admitted to the icu between 1986 and 1990, were retrospectively reviewed. the characteristics of the neoplastic disease (h~emopathy or solid tumor, tumoral evolution, duration of cancer disease and of neutropenia), the mac cabe's score, the organ system (respiratory, hemodynamic, renal, neurologic, hepatic) failures and the severity scores (saps, saps ii ,osf) were registred within the 1 st day in the icu. when discharged from the icu, the patients were classified as alive or dead. results: fifty-seven patients (53.3%) had a h~ematologic malignancy, and 50 (46.7%) a solid tumor. fifty-nine of the 107 patients died (55.1%); the mortality rate did not differ between both groups (61.4 and 48% respectively, p = 0.16). with univariate analysis, none of the tumoral features is linked to the prognosis; only the respiratory (p < 10 -4) and cardiovascular (p < 10 -3) failures, and the number of organ system failures (p < 10 -4) are associated to the risk of death. the saps (p < 10 -3) and saps ii scores (p < 10 -4) were higher in patients who died. with multivariate analysis (logistic regression), only the respiratory failure is correlated to the risk of death (p = 10-4); neither the features of the underlying malignancy (p > 0.8), nor the duration of neutropenia before admission in icu (p = 0.83), nor the severity scores figs ii: p = 0.068) are linked to the outcome. conclusions: the tumoral characteristics do not modify the prognosis after admission to the icu. they should not influence the decision to admit or refuse a cancer patient in the icu. respiratory failure at icu admission has the predominent weight on the risk of death in the icu. patients with respiratory acidosis due to asthma occasionally require levels of mechanical ventilation that place them at risk for barotrauma. a few case reports have described the use of an extra-corporeal membrane oxygenator(ecmo) circuit as an alternative means of co 9 removal. generally, this has been used for short periods of time (<24h) without serious complications and with low blood flows through the extra-corporeal circuit. we report a case of refractory asthma who could not tolerate even small-volume breaths from a mechanical ventilator due to severe bilateral airleak. ecmo therapy was initiated at the referring hospital prior to helicoptor transport. high blood flows were used (70% of the patient's cardiac output), sufficient to achieve both co 2 removal and oxygenation. satisfactory gas-exchanged was accomplished (pco2=50-60 mmhg) with nearly total lung rest for a prolonged period (60h). however, the long ecmo duration was associated with two severe complica-ti0ns:1) bilateral hemothoraces due to anticoagu!ation in the extra-corporeal circuit, and 2) prolonged weakness as a result of neuromuscular blockade for six days. the patient was discharged from the hospital in good condition. we present the respiratory and hemodynamic features of this case aw well as the potential complications of ecmo therapy in asthma. objectives: parameters derived from tidal expiratory flow ~e) and volume (vt) can be used to detect airflow obstruction in copd patients who might be unable to perform forced spirometry (e.g., icu). however, indices such as ave/v t and at/re are highly variable (thorax, 1981: 36; 135) . methods: we investigated whether the standardized for v m effective time (teff~) of a tidal breath, which is derived by asimple mathematical procedure (teff,= j'vdt/vt2), is a more reproducible and sensitive detector of airways obstruction, we studied nine normal subjects (5 male, 31 -+5yr) and 13 copd patients (4 male, 61 -+10yr) in the seated position, with a noseclip on. they breathed quietly, through a pneumotashograph to measure flow (v). volume was obtained by numerical integration of thellow signal. each subject had an initial 10-15 min trial run, in order to become accustomed to the apparatus and procedure. when regular breathing had been achieved, all breaths over a5 min time interval were recorded. the mean value of six consecutive breaths (ers criteria) for each subject was used for analysis under the condition that within session variation of tidal volume (vt) was <10%. lung function tests were: in normals (mean-sd), fevl%pred = 100• fevl/fvc%=81-+3% , and in copd patients, fev~%pred=53__.20 and fevi/fvc%=51 --.12%. results: values are shown as mean-..+-sd in the following a su~ve~ os literature sources p~oves that t~aditlona], i.e. medicinal medication and physiothe~apeutic methods os t~eatment often p~ove to be insufficientl~ effective both currently and in the ~emote future. the goal of this study was to investigate the efficacy os t~eatment of b~onchial asti~ma patients by means os speleo-and artificial sp~ay therapy. speleotherapy t~eatment was conducted in the conditions os mic~oclimate os salt mine in solotvino hospital. a~tis sp~ay the-~apy was conducted by means os a self-made device. ou~ method is based on the p~inci-~ le os using the majo~ facto~ of speleo-he~apy -highly dispe~sed sp~ay 0s sodium chloride. the obtained ~esults ~e~e analyzed in five g~adations. at the end os the speleothe~apy improvement and considerable improvement was observed in 75,0~ os patients; inconsiderable improvement -in 15,9~ os patients. having evaluated the e~s os t~eatment using a~tis sp~ay therapy the indices a~e 68,7h and 20,5~ ~espectively. remote ~esults of t~eatment a~e an important index os t~eatment, the ~esult os ~hich ~e~e studied by means 0s a ~uestionnaive-method. patients ~ho had been t~eated by speleothe~apy mo~e f~eguently ~e-po~ted a ~elapse in disease 3ust afte~ the course o~ t~eatment (29,3h). ho~eve~, in a ]ate~ phase the ~emission ~ould last ]on-~e~ (s 6 months in 84,5~ os patients, till one yea~ in 69~9~). in 12,5~ os patients who passed the co~se os a~tificial sp~ay therapy a ~elapse was ~egiste~ed immediately as the co~se os t~eatment. then thei~ condition stabilized ~hile in 73,5~ os patients a period os ~emission lasted s ha]s a yea~. 42,9~ of patients dida't ~epo~t a ~elapse of the disease du~in~ one yea~. evangelismos hospital, critical care department, athens, greece method#: 19 mechanically ventilated patients (5 copd, 6 ards, 8 other pulmonary diseases) were studied in two phases: 1) during the acute phase of respiratory failure; 2) during recovery 5-73 days later. we measured mip and monitored the pattern of breathing while the patients were breathing spontaneously through the respirator (pressure support mode with 3-8 cmh20) until either the point they were unable to sustain spontaneous breathing (sb) any longer (phase 1) or for two hours when they could sustain sb indefinitely (phase 2). subsequently the patients were sedated, paralyzed and mechanically ventilated. then we simulated the pattern of sb at the end of the sb trial by manipulating the variables of the ventilator and assessed respiratory mechanics b y the end-inspiratory and end-expiratory occlusion technique. 1. during recovery, a combination of reduced inspiratory load and increased venfilatory capability makes a patient previously unable to sustain sb to breathe spontaneously. 2. inspiratory load is reduced during recovery, mainly because both intrinsic peep and breathing frequency are diminished. obiectives: although elevated concentrations of a few cytokines have been shown to be present in the bronchoalveolar lavage (bal) fluid (balf) of patients with the adult (acute) respiratory distress syndrome (ards), the pethogenesis of ards is largely unknown. leukemia inhibitory factor (lif), a growth factor recently recognised as a polyfunctional cytokine integrated in cytokine networks was measured in unconcentrated balf of patients from different patient groups. methods: lif was measured in balf by means of a specific and sensitive elisa (detection limit 10 pg/ml)in balf (lavage of 3 x 50 ml in the right middle lobe). results: lif was not detected in the balf of 13 healthy control patients and in only one (34 pg/ml) out of 26 patients at risk for ards (after cadiopulmonary bypass surgery) who underwent bal 4 h after the end of the extracorporeal circulation. high and detectable levels were found in the unconcentrated balf of 10 out of 12 patients with full-blown ards (196 + 80, mean + sem, range 10-985 pg/ml). there was a good correlation between the level of lif in the balf and a number of markers of inflammation: neutrophils/ml (r:0.70, p= 0.01), albumin ( r:0.75, p=0.008) and protein level (r:0.74, p=0.006). conclusions:the biological role of lif in these balfs is not readily explained by its currently known actions and it is unkwon whether lif contributes to or is a response to local tissue damage. our results indicate that this cytokine with lots of interesting _functions is a pert of the inflammatory cytokine cascade in ards. background and obiective : we recently demonstrated that cisapride -a new prokinetic drug -enhanced enteral feeding in a heter0genoas group of ventilated icu patients by significantly accelerating their gastric clearance (crit care meal, 1995 ; 23 : 481-485) . it remains unknown, however, whether certain subgroups of patients might benefit more from adding cisapfide to their enteral nutrition regimen than others. patients with chronic obstructive pulmonary disease (copd) might represent such a subgroup since their illness and its specific treatment put them at risk for gastric emptying disorders. design and setting : prospective, consecutive sample study in an adult medical intensive care unit in a university hospital. patients : 10 mechanically ventilated and hemodynamically stable copd patients. interventions : gastric emptying was evaluated by bedside scintigraphy and expressed as the time at which 50% of a tcg~-labelled test meal was eliminated from the stomach (t 1/2). baseline data (do) were recorded after enteral nutrition reached 1500 to 2000 ml daily. scintigraphic measurements were repeated 4 days after cisapride (10 ml orally, q.i.d) had been added to this regimen (d4). patients were considered cisapride responders when gastric clearance improved by more than 50% from baseline. results : normal values for the test meal and for scintigraphic acquisitions obtained in the supine position were found to be 31 + 15 min. in healthy volunteers (crit care med, 1995 ; 23 : 481-485) . five patients responded to cisapride (t 1/2 : 81 + 31 rain vs. 26 + 10 min at do and d4, respectively) and five did not (t 1/2 : 36 + 18 min vs. 33 _+ 11 rain at do and d4, respectively). in contrast with non-responders, all five responders had clinically significant maldigestion at baseline (excessive (> 150 ml) gastric residues, vomiting (> 3 times/day and abdominal distension) which disappeared in 4 of them after the administration of cisapride. conclusion : copd patients who tolerate enteral nutrition well have basal gastric emptying times which are comparable with those of healthy volunteers and are not influenced by cisapride. however, cisapride treatment provides both scintigraphic and clinical improvement in those copd patients who exhibit clinically obvious gastric emptying disorders. cernv v., dostal p., zivny p., zabka l. dept. of anesth. and critical care, charles university, faculty hospital, i-irade~ kralove 500 36, czech republic objective: the aim of the study was to evaluate the effect of early entera nutrition started within 24 hours of injury on the incidence of multiple orgar failure (mof) in trauma patients requiring vantilatory support. methods: after institutional approval 25 patients were enrolled in the study enteral feeding was begun within 24 hours of injury in 14 trauma patients (en group) admitted to icu. nasuenteric tube was placed as soon as possible after admission into the distal duodenum under endoscopy. additional parenteral nutrition was used to meet patients energy and protein requirements. the control group (pn) consisted of 11 patients fed during this period paretuerally. severity score apache ii, trauma score, cumulative balance of nitrogen (g), incidence of mof (three and more organs) and length of ventilatury support (days) were calculated. values are expressed as mean + sd. results: tab introduction : parenteral nutrition (pn) is an important aspect in the optimal treatment of patients on gastroenterology or intensive care. the aim of this bi-center study in 38 patients has been to assess tolerence and efficacy of a new protein-lipid mixture for pn from a simple preparation. patients and m~hods : patients were selected in two hospitals (tenon and saint-lazare, paris) and were divided into two groups : group a (gastroenterology~ 1 l short bowel syndrome) and group b (intensive care, 27 surgical patients). all patients likely to require pig for a period of 10 days (group a) or 7 days (group b) were studied. the pn regimens administered were the following : combination with 50 g of mct/lct fat emulsion end 9,6 g of nitrogen, in 1 liter end glucose requirements were met by imfizsion of l liter of glucose 20-30 % via a "y " connection. lipid thus provided 3040 % of the non introgen calories. total daily calorie intake was 1540 to ] 940 kced. this study monitored, before and at the end of infusions, the sennn albumin (alb), preaiburtun (prealb), triglycendes (tg), cholesterol (cs), and the serum ammotransferases (sgot and sgpt) end alkaline phosphatase (alp) activities. statistical significances were calculated using the wilcoxon-tost. introduction: many 1cu patients present a catabolic illness in response to inflammation and infection, characterized by a rapid loss in skeletal-muscle mass despite optimal nutritional support. growth hormone (gh) is responsible for a rise of lipolysis, enhancing the energetic balance, and of protein synthesis. recombinant human gh (rhgh) is nowaday available for clinical use, but its cost is very high. therefore, rhgh should only be prescribed to icu patients when its efficacy can reasonably be anticipated (ie. when the patients are catabolic or stressed, but in order to avoid overprescription for unstressed patients and for those who are overly catabolic). hence, we, as others, recently demonstrated that rhgh had no favorable effect in highly stressed icu patients. objective: to detect on a clinical basis, low (ls), mild (ms) and severe stress (ss) states in icu patients and validate this clinical judgement by objective metabolic mesurements, in order to select early those icu patients potentially able to benefit from rhgh therapy. methods: 36 consecutive icu patients were prospectively stratified as ls, ms and ss by two experienced icu senior consultants (temperature; agitation; heart rate; arterial blood pressure; presence of an infection; respiratory rate; exogenous catecholamines). anabolic (insulin, igf-1, gh) and catabolic (cortisol, ghicagon) hormones, and nitrogen balance were determined for each patient within 8 hours after admission in the icu. metabolic and clinical data were then compared. the clinical stress states determined by icu physicians correlate with an objective metabolic assessment. therefore, the patients who will more likely benefit from adjuvant rhgh therapy can be detected simply and early. a prospective study on rhgh therapy in ms icu patients is in progress. berger mm md 1, chiolero r md 1, pannatier a phd 2, berger l 2, cayeux c 1, voirol p 2, hurni m md 3. 1 surgical icu, 2 pharmacy, and 3 cardiac surgery, chu vaudois, ch-iotl lausanne, switzerland objective. nutrition of the compromised cardiac surgical patient is challenging. numerous factors influence the gastrointestinal (gi) absorption function, among which gut perfusion, which depends largely on the systemic hemodynamic status. patients in hemodynamic failure are prone to organ failure, and may benefit from an early jejunal feeding. the study was designed to assess the absorption function after cardiac surgery in patients with adequate and altered hemodynamic status, using paracetamol as tracer of gi absorption. methods. after cardiac surgery, 24 patients, aged 63_+8 years (mean_+sd) were assigned to 2 groups (anaesthesia: fentanyl 20 gg/kg + midazolam): group 1 (n=10): reference group, with normal hemodynamic status, easy recovery. group 2 ('n=14): patients in low output syndrome, cardiac index < 2.5 i/m2 on day 1 (d1) after surgery, requiring prolonged intensive care, mechanical ventilation + nutritional support. paracetamol 1 g, was given intragastrically on d1 + d3: plasma levels measured (h.p.l.c), at administration (to), t30-60-90-120-180-240 and 480 rain. hemodynamic status assessed with pulmonary artery catheter. 5 healthy subjects served as controls. results. compared to healthy controls, absorption was strongly reduced on d1 in all patients (no difference between groups). on d3, peak paracetamol level was significantly lower in group 2 (low cardiac output): in group 2 the area under the curve on d1 and d3 were similar. there was a large inter-patient variability, reflecting the hemodynamic status. conclusion. gi absorption was decreased on d1 in all patients, and reverted to normal between d2 and d3 in case of normal cardiac function, but not in case of low output syndrome. the decrease on d1 can be attributed to fentanyl, known to slow down the gi transit. in patients with cardiac failure, correction of altered absorption was correlated with the hemodynamic status, suggesting that gi absorption is dependent on adequate splanchnic perfusion. the aim of the work was to define specific significance and evaluate efficiency of enteral component of infusion therapy in the intensive care of gastroenterotogic patients of surgical profile with pyo-septic complecations. there were used the methods of radial diagnostics and polyelectrography; the laboratory control on oxygen-transporting function, volumetric and hemodynamic state, changes in metabolic, hormonal and immunologic status was conducted. from january, [992 till november, 1994 there was carried out the randomized study of 155 patients with general purulent peritonitis; among them 70 persons constituted the control group and 85 -the main one. in the main g~oup the intestinal lavage, enterosorption, enteral introduction of nutrient solutions with gradual turn to enteral nutrition by equalized mixture "ovolaet" were started from the first hours after operation. the data obtained allowed to define the specifity of the program of artificial medical nutrition in the group of examined patients, based on necessity of individual selection of media for enteral introduction depending on the stages of intestinal insufficiency syndrome. it was shown that inclusion of enteral component into the program of infusion therapy during early periods stabilized circulation in the regime of moderate hyperdynamia, considerably decreases the deficiency of circulating blood volume, normalizes the values of oxygen transport, consumption an}d extraction, provides the optimal level of mycardial adaptive possibilities without tension of its compensatory functions and pulmonary circulation overload. due to combined application of parenteral and enteral nutrition the metabolic processes are shifted towards anabolism. this is supported by decrease to normal values in the contents of blood aggresive hormones (acth,hydrocortisone) and increase in somatotrophic hormone. the complete parenteral-andenteral nutrition influences positively on restoration of cellular and tumoral immunity, activates the factors of organism nonspecific protection and recovery from immunodepression, prevents the development of immunodeficiency. impact tm vs control. s atkinson, n maynard, r grover, e sieffert, r mason, m smithies, d bihari departments of surgery and intensive care, guy's hospital, london, u.k objectives: comparison of the effect of an immunonutrient enteral feed versus a control on the outcome of a mixed intensive care unit (icu) population. methods: admissions to this multidisciplinary adu)t icu thought likely to stay more than three days and with tube access to the gi tract ~r randomised to receive either impact tm, a feed with supplemental arginine, dietary nucleotides and omega-3 fatty acids, or an isocaloric and isonitrogenous control feed. study end points included mortality and icu stay. approval was obtained from the hospital ethics committee. rosults: 390 patients were entered into the trial. the two groups were well matched for age, sex, and admission apache ii with an overall mean admission risk of death of 30.8 (std. dev. -+ 22.9). on an intention to treat basis, there was a no significant difference in icu mortality, icu stay or standardised mortality ratio (s.m.r.) between the two groups (see table) . similarly, there were no differences after stratification for patients receiving 5 or more litres of feed. conclusion: there is no evidence of an effect of impact@, an enteral immunonutrient feed, on pre-determined end-points (icu mortality, icu stay or standardised mortality ratio) in a mixed intensive care unit population over that of an isocaloric, isonitrogenous control feed. objeeflves: evaluate changes of blood laatate levels according to patient medical status after cvvhd initj,~ion using dialysate solution containing lactate. method: review of medioal records of 20 consecutive patients ~eated by cvvhd (dialysate solution hmnosol lg2, hospal,uk, lactate concentration 40 retool/l). date obtained 1 hr before and 4 -6 hrs at~er cvvhd initiation were analysed. results: all data are presented as mean + sem. in one patient, pre end post filter lactate levds were measured during standard cvvhd setting (blood flow 100ml/mlu, dialysate solution flow i 1/hr), and approximate daily lactate flux into the patient was calculated to be as high as 920 mmol/d. lactate leveh measured after cvvhd initiation increased significenfly compared to baseline levels (3.80+0.67 axtd 2.88+0.68,respectively; p<0.01,paired t-test). when patiente with increased basal lactete (~-7) were compared to paliente with normal basal values (n=13), no difference in laotete increase was fmmd (p=0.22, manova). patiente with severe liver dysfunction (2 points in mop scomlg, n=9) had higher basal laotate levels than patiente with normal or slightly abnormal liver teste (0 or 1 point in mof scoring, n=ll), rite values being 4.04 + 1.17 and 1.84 + 0.23, respectively (p<0.05, student t-test). increase in blood lactate did not differ between these two groups after cvvhd was stetted (p=0.86, manova). in 11 pafiente with invasive hemedynamio mo~, no oorrelation batween changes in lactate levels and eitlm" changes in oxygen ddivery (t2=o.ol; p--o.81) or oxygen consumption (reversed fie, k) (r2-q).o7;p--0.66) were found after cvvhd initiation. conclusion: blood lactate increases on cvvhd with dialysate soh~on rich in lactate. this increase is predominantly caused by influx of lactate into the blood via the filter end does not seem to depend on the liver fimotion and/or oxygen metabolism changes. objectives: the study was designed in order to determine the effect on plasmatic proteins, of two types of aminoacids solutions of parenteral nutrition (pn) adapted to stress, having different concentration of branched chain aminoacids (bcaa), when applying to politraumatized critical patients. methods: a prospective study was performed using a randomized double blind design of 20 polytraumafized patients, split in two groups of ten patients each, with mean ages of 35 _+ 17 an 45 -+ 20 years. due to their condition, all patients required p.n. for at least 9 days. both groups were subjected to isocalorie and isonitrogenous solutions (45 ci/kg/ day and 0.24 g of nitrogen/ks/day), varying only in the concentration of bcaa; solution a having a 10 % concentration and solution b 23 %. blood samples determinations during days 0, 3, 6, 9 after the beginning of treatment with p.n. were total proteins., albumin, trandferrine, protein binding retinol; prealbumine and fibronectine. the anova test (one and two way) was used to compare the values between the two groups. results: the administration of solution a, showed statistically significant increases in the determinations of the values of protein binding retino] (p < 0.05) and prealbumin (p < 0.05). no significant increases were observed in the values of total protein, albumin, transferrine and fibronectin. solution b produced statistically significant increases only in the values of total proteins (p < 0.05). the remaining proteins did not changed from their control values during the whole period of pn administration. comparing both groups, no statistically significant differences were observed related to the type of diet. nevertheless, differences were found in total proteins, albumin, protein binding retinoi, fibronectin (p<0.05) and prealbumin (p < 0.005) in relation to the time course of pn therapy. only the albumin values showed significant differences (p < 0.01) when considering the interaction of both the type of diet and the time course of pn. conclusions: 1. solutions of pn adapted to stress, can maintain the control values of slow turnover proteins and improve the values of rapid turnover proteins. 2. no significant differences on plasma proteins were found between the two solutions having 10 % or 23 % concentration of branched chain aminoaeids. &determination of rapid turnover proteins does not seems useful for discriminating different solutions of bcaa during pn. obiectives; the hormonal changes in the post-traumatic situation often leads to an elevated blood glucose and a negative nitrogen balance. to reduce the elevated glucose production by aminoacids the apprication of xylitol may be an alternative energy source. in a double-blind randomized study we investigated the effects of a xylitol/glucose solution (group a: aminoacids 50 g/i; glucose/xylito180 g/40 g/l) on metabolism and particularly on pancreatic and liver enzymes compared to a glucose based nutrition solution regimen (group b: aminoacids 50 g/i; glucose 120 g/i). methods: the clinical trial was carried out after the approval by the local ethical committee on 31 patients with severe brain injury. there was no difference in body mass index bmi (group a: 25.9 +/-2.7 kg/m 2 and group b: 25.1 +/-2.4 kg/m=), age, and sex. daily individual energy expenditure was measured by indirect calorimetry (deltetrac "~). nutrition was started 24 -48 hours after trauma or surgery with carbohydrates and aminoacids. fat was added 24 h after nutrition had started. to analyze the effects on pancreatic and liver enzymes we investigated the following parameters for 4 days: blood gtucose, serum lipase, serum amylase, asat, alat, ~gt, ap, and serum cholinesterase (che). results: due to the daily indirect calorimetric measurements energy requirements were satisfied. there was no difference in blood glucose concentration and cumulative nitrogen balance between the two groups. neither were there any significant changes in asat, alat, ap, and che for 4 days in both groups. serum tipase steadily rose to 202 lull in group a and 320.2 lull in group b, respectively. conclusions: there was no measurable influence of either nutrition solution on liver enzymes. the xylitol/glucose nutrition regimen does not have any advantage over the glucose based nutrition solution concerning blood glucose level or nitrogen balance. the elevation of serum lipase to a 2-fold level in either group needs further investigation on trauma patients. the effects of fat emulsions in lung function, particularly in lungdamaged patients, have been attributed to alterations in pulmonary vascular tone caused by eicosanoid production modificatione. as the eicosanoid production may depend on the fatty acid profiles of the intravenous fat emulsion, haemodynamic, pulmonary gas exchange and plasma levels of prostanoids were investigated in acute respiratory distress syndrome (ards) patients, during different intravenous lipid emulsions (providing different prostanoid precursors). we studied in a randomized double-blind design 3 groups (n=7 each) with ards. group i (lct) received a fat emulsion with long chain triglycerids (lct-20%), group ii (mct) an emulsion containing a mixture of medium and long chain triglycerids (mct/lct 50/50-20%) and group iii placebo (control), during 12 h (2 mg/kg/min each). we measured before, at the end of 12 h infusion, and 12 h after the end of the infusion: lipaemia, arterial and venous blood gases, pulmonary and systemic haemodynamics, and plasmatic levels (arterial and in mixed venous sample) of eicosanoids (txb=, 6-keto pgf~,, and ltb4). at the end of the fat emulsion, groups (i and il) to 1,10• to 0,51 • mmol/i), the paoz/fio z remained unchanged in the three groups; no changes in intrapulmonary shunt (qs/qt) were shown; neither in the mean pulmonary artery pressure. in contrast, only in the lct group: cardiac output and oxygen consumption increased significantly (12.5% and 19%) (p<0.05). eicosanoids were increased at baseline compared to reference values (p<0,05). a decrease (p<o,05l in the arterio-venous difference of the vasodilatador prostanoid (6-keto pgf~=) was shown in lct group. our results indicate that fat emulsions in ards patient do not influence pulmonary gas exchange, provided that the perfusion rate were keept at 2 mg/kg/min. the present study was designed to evaluate the metabolic changes of a carbohydrate-based diet versus a fat-based diet on oxidation rate of substrates and energy muscle metabolism in patients with an acute exacerbation of chronic obstructive lung disease. patients were mechanically ventilated with a volume-cycled respirator. after an overnight fast, patients were randomnly treated with a continuous enteral feeding over a nasogastrie tube with a carbohydrate/fat ratio of 2/1 in group i (n=6) , and a carbohydrate/fat ratio of 1/2 in group i] (n = 6) during 8 consecutive days. indirect calorimetry was set at baseline and 24 hours later the enteral feeding was started. muscular quadriceps biopsy was performed on baseline and on day 8 of enteral feeding. urine was collected during 24 hours and was used to measure 24h urea nitrogen production. the caloric intake was set according to the measured resting energy expenditure. respiratory gas exchange rate were measured using a computerized open-circuit indirect calorimeter. quadficeps femoral muscle samples were obtained by muscular biopsy after local anesthesia. analyses of glycogen, glucose 6-phosphate, lactate, phosphocreatine and adenosine triphosphate (atp) and enzymes (glycogen synthase and glycogen phosphorylase) were determined. after 24 h of nutrition patients in group i showed an increase in carbohydrate oxidation (from 17.8% to 55.4%, p=0.06), and in group ii a decrease in protein oxidation (from 27% to 21%, p=0.03). after 8 days of enteral feeding a tendency to increase in glycogen concentration (group i, p=0 .06; and group ii,p=0.1) was observed. while patients in group i showed a tendency (p=0.06) to decrease glycogen phosphorylase, patients in group ii maintained these enzymatic levels. the increased in glycogen was correlated in group li with degradation enzyme (p =0.01 ). in group i, the pao2/fio 2 ratio increased after 8 days of treatment (from 235.9 to 284.5; p=0.03), but no changes in days of mechanically ventilation, length of stay in icu, or mortality rate was evidenced between groups. it is concluded that the oxidation rate of substrates used for energy production varies according to the caloric sources administered. with nutritional therapy muscle glycogen concentration increases in both diets, with different enzymatic influence. more studies are warranted to further assess the clinical implications of the different pathophysiologic behavior of the two diets. under certain experimental and human conditions (radiation and/or thermal injury) the gi mucosa is unable to perform this protective function and could play an important role in the pathophysiology of the syndrome of multiple organ failure (smof). the objective of the present multicenter study was to know the gi mucosal permeability in critically ill patients and the relationship with their outcome. the method used to assess the integrity of the gi mucosal barrier measured mucosal permeability to various hydrophilic compounds. specifically, we measured the urinary excretion ratio (uer) of two sugars (lactulose and mannitol) that had been previously administered through a nasogastric tube. urinary excretion rate was measured in 10 healthy humans (control) and in 30 patients admitted to the intensive care unit (icu) at days 1, 3 and 5. the apache ii of the patients studied was 15_+4. there was an increase of uer in the critically ill patients compared with controls (o.26_+0.1 vs 0.036_+0.01) (p<0.001). in contrast, no changes in uer between days 1, 3 and 5 were observed (0.26 -+ 0. . significant changes were also established in the different groups: major and minor surgery, general and epidural anaesthesia , transfused and non transfused patients except for iron and ferritin plasma levels which didn't change significantly in patients who had received blood. a positive significant correlation was established between crp and ferritin (r=0.89) for patients under general anaesthesia, but not on epidural, regardless of the type of surgery. conclusions: iron and transferrin plasma levels fell acutely in surgical patients while ferritin and crp rose. transfusion implied an acute iron load.the established correlation between cpr and ferritin in patients on general anaesthesia suggests that ferritin behave as an acute phase reactant in that setting, while epidural anaesthesia may ameliorate the inflammatory response. objectives: to analize the effect of gastrointestinal complicacions (gic) related to the enteral nutrition (en) in the real volume of diet administered to icu adult patients. a prospective multicenter study was designed (comgine study) in wich en application, gic definition and his management were defined by collaborative consensus.patients treated with en in one month pedod were included. in each case, daily volume of diet prescribed (pv) and administered (av) was recorded and the volume ratio calculated (vr=pvxl00/av). patients were divided for days of en according to the presence (group 1) or absence (group 2) of gic (abdominal distension, increase of gastdc residuals,vomits or regurgitation, diarrhea,constipation and bronchoaspiration). differences between groups were analized by anova and mann-whitney u test with p<0.05 for statistical significance. (vr%) 53 61 58 89 67 63 65 72 62 60 64 59 67 7461 group2 (vr%) 85 90 90 94 94 93 92 90 92 91 92 94 90 92 tolerance to the enteral nutrition (en) has been related to the severity of illness in icu patients. the aim of this study was to evaluate this relationship. methods. a prospective, multicenter, study was performed (comgine study) in wich en application and related gastrointestinal complications (gic) were defined by collaborative consensus. adult icu patients treated with en in one month pedod were included. gic were classified as: 1) abdominal distension (abd), 2) increase of gastdc residuals (igr), 3) vomits (vom), 4) diet regurgitation (reg), 5) diarrhea (dia) and 6) constipation (con). patients were divided in two groups: group a: patients with gic dudng their evolution. group b: patients without gic. differences between groups were analized for apache ii admission score or evolutive variables ( carbon dioxide production is a major determent of work of breathing. increase in co2 production during nutritional support may precipitate ventilatory failure and difficulty in weaning from mechanical ventilation. in the present study, co2 output was calculated while passive mechanically ventilated patient were fed with different amount of calories and different proportion of protein, carbohydrate and fat. four clinical stable and mechanically ventilated patients were studied. carbon dioxide was calculated while patients were paralyzed, sedated and mechanically ventilated. six nutritional regimens were used: a) no calories, b) t00gr glucose/24h, c) calories equal to rest energy expenditure in special formula with low carbohydrates and high fat (pulmocare) d) calories equal to rest energy expenditure in standard formula (nutdson) e) calories double to rest energy expenditure in standard formula (nutrison) and f) calories equal to rest energy expenditure parenterel (tpn). carbon dioxide output was low while no calories and/or only 100 gr/24h glucose was provided, the mean values of vco2 output were 134_+_25 and 140!-_26 ml/min respectively. there was no difference in vco2 output between with low carbohydrates and high fat regimen and standard regimen, mean values were 16t .5-+27 and 163_+26 respectively.the high calories regimen and tpn resulted in higher vco2 output, mean values 184_+25 and 211• respectively. we conclude that the special with low carbohydrates and high fat regimen did not reduce vco2 output, in the contrary, high calories intake as well as tpn increased vco2 output under tested conditions. klouche k., cristol j.p., vela c., canaud b., b6raud j.j. m6tabolic intensive care unit and nephrology department. lapeyronie hospital. 34059 montpeuier france. predictive factors for rhabdomyolysis (rh) -induced acute renal failure (arf) were studied in a retrospective study from january 92 to january 95. 33 patients (pts) (male:23, female:lo; mean age: 57+4 y.o.) were admitted with rh defined as cpk>1000 iu/1. etiologies were: traumatic and ischaemic 24, infectious 4, toxic 4, excess activity 1. factors studied were: simplified acute physiologic score (saps: 10.3+1.1), organ systemic failure (osf: 1.02_-!-0.2), diagnosis delay (d: 33+_5h), clinical parameters (sepsis, dehydration), blood chemistry data (cpk, bun, creatinine, potassium, phosphorus, calcium, proteins, hematocrit) and urinary ph. severity of rh was estimated by ward score determined according to phosphorus, albumin, potassium, cpk, dehydration and sepsis. urea appearance rate (uar) and creatinine index (ci*) were determined over a 24 hours period. arf was observed in 25 pts. in non-arf and arf groups respectively, saps (5.5_+0.5 vs 11.8+1.3), deshydratation (0 vs 11), sepsis (0 vs 12), phosphorus (1.03+0.16 vs 2.21-+0.21), calcium (2.1+0.07 vs 1.8_+0.07), ward score (4_+0.65 vs 11.8+0.8) were significantly different. however, no significance was observed in uar (310-+89 vs 210-+35) and ci (26_+7 vs 34_+3). 16 patients required hemodialysis (hd) (85:2 sessions) and 9 remained dialysis free. only osf (1.1_+0.1 vs 1.9-+0.23), ward score (9.2_-/-0.95 vs 13.25_+0.92) and ci (29+_3 vs 41-+3) appeared significantly higher in pts requiring hd. 5 pts died from associated disease. all patients suffering from arf recovered a normal renal function. we confwmed that an elevated ward score (over 7) is a good predictive index of arf. in addition we found that ci is a severity factor for arf requiring hd. thus, patients suffering for rh with elevated ward score and ci, have a fair chance of dialysis and should be treated more intensively. * ci (expressed in mg/kg) = (car + feces creatinine) / weight. where car: creatinine appearance rate; feces cr~t..= mean plasmatic creatinine x 0.043. tr~er k., cetin t.e., tugtekin i., georgieff m., ensinger h. universit~tsklinik flir an~sthesiologie, 89070 uim, germany introduction: endogenous as well as exogenous adrenergic agonists have a profound effect on carbohydrate metabolism in human critical illness. in this study the effects of noradrenaline (nor) and dobutamine (dob) on carbohydrate metabolism during a 4 hr infusion were investigated. methods: after approval by the local ethic committee 14 healthy volunteers were studied. hepatic glucose production (hgp [mg/kg/min]), using 6,6-d2glucose as stable isotope tracer, as well as plasma concentrations of glucose (glc [mmol/i]) and lactate (lac [mmol/i]) were measured prior and during infusion of nor (0.14 pg/kg/min) and dob (6 pg/kg/min). blood samples were drawn before and during the agonist infusion. results: no major changes in insulin and gtucagon plasma concentrations could be found during the study period. ::i:::: :iiiii~ 8~ i ::i: ~:: : :: i:ii. mean-+sd are shown. # p<0.01, anova for repeated measurments. conclusions: the effect of nor on hgp and glc were smaller as compared to adrenaline (i) with a similar time course. in contrast to the effects of adrenaline and nor, dob had a different effect on carbohydrate metabolism: a decrease in hcp and glc, which is uncommon for a /3-adrenoceptor agonist. since hgp is an energy consuming process that might deteriorate hepatic oxygen balance in critical illness, the differential effects of adrenergic agonists may be of importance and need further clarification. the nutritional insufficiency often accompanies post-operative hypercaloric states, inanition, serious infections and weakening chronic illnesses. that is why the early nutritional support, sufficient and appropriate for each individual base, is a fundamental component of intensive care unit as an indispensable factor for recovery. per this reason, our unit, developed a software for the implementation and nutritional control of t~e assisted patients. this software is incorporated is an expert system called ~i~su, designed and developed by the computational division of our unit. this system arrives to inferred diagnoses such as : respiratory, hepatic, renal(with and without dialysis) dysfunctions, pancreatitis, ards, decrease of consciousness, diabetes. according to these data objectives: to compare the effect of short term enteral feeding versus parenteral nutrition, when a isonitrogenous and isocaloric feeding solution is administered by either mute. methods: in a prospective controlled clinical trial 30 patients were studied; all exhibited moderate degree of malnutrition, normal liver and kidneys, and a functi6ning gastrointestinal tract. the patients were randomized to receive a free amino acid and small peptide diet (15 patients) or an isonitrogenous isocaloric parenteral support (tpn) (15 patients) (total energy: 2880 kcal, nitrogen: 14.5 g, carbohydrates: 380 g, fat: 112 g, n/non protein calories: 1/175) at least for 10 days. results: there were no significant changes in anthropometric parameters within either group. nitrogen equilibrium was aqhieved by day 3 in the tpn group and by day 5 in the enteral group (66.6% of the enterally fed patients and 80% of the tpn patients maintained in positive balance the day 10 of the study). there were no significant changes in serum albumin within either group. serum level of transferrin reached a significant increase in both groups (p=0.003). thyroxine-binding prealbnmin rose significantly in both groups as well (p=0.019 and 0.004 respectively). statistically significant rises in lymphocyte counts (p=0.003 and 0.001 respectively), in levels of c 3 (p=0.009 and 0.01)1 respectively), iga (p=0.002), igg (p=0.004 and 0.003 respectively) and igm (p=0.004) occurred in either treatment group. there was a high incidence of negative skin tests at the start of the study in the enteral group (73.3%) and the tpn group (60%). by the end of the study the incidence of negative responsiveness was 40.0% and 26.6% respectively. despite maintenance of similar glucose levels in both groups, tpn led to significantly higher serum insulin levels. the serum insulin increased almost linearly over the study period and eventually prevented fat mobilization and lipolysis, so that free fatty acid levels had fallen significantly. a significant elevation of the liver enzymes over the study period occurred in 73.3% of the tpn group, but not in the enterany fed patients. conclusions: the present findings provide no evidence that enteral diets containing free amino acids and small peptides, as their nitrogen sources, are in any way inferior to isonitrogenous isoealoric regimes parenterally given. aim: the aim of this study is to describe and explore the expectations of the functions of the critical care nurse to enable the formulation of guidelines for the scope of practice for the critical care nurse with a south african context, methods: phase i was to determine the expectations of the critical care nurse, the nursing service managers and the doctors with regard to the functions of the critical care nurse. a focus group interview was held with a group of experts in the field of critical care. the results were used to compile a questionnaire. this questionnaire was sent to the critical care nurses, the nursing service managers and the doctors in south africa for completion. from these results the functions of the critical care nurse were determined. phase ii was to formulate guidelines for the scope of practice for the critical care nurse within a south african context. through usage of the date (phase i) the scope of practice was formulated. guidelines were formulated for the practise, education and research regarding the limitations of the professional-ethical authoration and the implementation of the scope of practice for the critical care nurse. objectives : high output gastric aspirates arc occasionally observed during fasting in critically ill paticnts, preventing any attempt of feeding via the enteral route. although these patients are often said to suffer from "gastroparesia", the motor correlates of this condition arc lurgcly unknown. in this stud?', wc recorded the gastrointestinal motility of critically ill patients with abundant (>250 ml/24 hours) fasting gastric aspirates. methods : antral (4 sites separated each other from 1.5 cm), duodenal (1 site) and jejunal (1 site) contractions were recorded simultaneously by ~eans of a multihimen tube assembly positioned trader fluoroscopic control (perfused catheter technique). tracings from prolonged recordings were obtained on a multichannel recorder (7758a recorder, hewlett-packard) then anal)7,ed visually, with a special attention for the following abnormalities which are characteristic of intcstinal pseudoobstmctiou: l) absence or aberrant propagation of the migrating motor complex (mmc), 2) presence of bursts (> 2min) of nonpropagated phasic pressure and 3) presence of sustained (>30 min) uncnardinate pressure activity. 11 patients with a volume of gastric aspirates of 731 • 506 (sd) [median 5001 ml/24 hrs were investigated for 538 4-271 [median 4551 minutes. results : only one patient had no detectable motor abnormality. mmcs were either absent (n=4) or migrated abnormally (retrograde propagation : n=4; retrograde and stationnary : n=2) in 10 pts. bursts of nonpropagated phasic pressure activity were present in the duodenum in 9 pts and sustained uncoordinate pressure activity was found in 2 pts. additional abnormalities included episodes of prominent pyloric activity. (n=l) and sustained antral pressure activity (n=2}. conclusion : critically ill patients with large volume of gastric aspirates have manometric evidence of intestinal pseudoobstruction. prokinetic therapy in these patients should thus focus not only on enhancing gastric motility, but also on restoring a normal propagative contractile activity in the intestine. this prospective, open-label, randomized placebo-controlled study included 20 patients with hypokalemia in whom rapid potassium replacement (20 meq kci in 1 h) was performed: 14 patients received mg sulfate (6 g in 3 hours) and 6 patients received a corresponding saline infusion. measurements were made at time 0, +1, +3 and +6 hours results: k levels increased more in mg treated patients than in the patients who received saline infusion at time 1 and 3 h (p < 0.05 -students-newman-keuls). (table 1 ). introduction. dual lumen uaso-gastrojcjunal tubes are a major ads'ance in nutritional therapy of mechanically ventilated critically ill patients since the3" authorizc jejunal feeding with concurrent gastric decompression, there,, reducing the risk for aspiration. unfortunately, placcmem of these tubes in the jejunum regularly dictates to resort to endoscopy in order to facilitate pyloric intubation. recently, the remarkable gastrokinetic properties of the well known macrolide antibiotic er}lhromycin have been demonstrated in gastroparetic critically ill patients 1. aim. in the presem stu~,, we evaluated the feasibility of placing dual lumen naso-gastrojcjunal feeding tubes at the bedside without endoscopy, using edthromycin to help iranspy'loric migration of the tube under fluoroscopic control. methnd each patient admitted in our icu during a 2 months period and requiring artificial ventilation and enteral nutrition for a period of at least 3 days was included in the study.. after inserting the tube (stayput| sandoz, usa) in the gastric anmnn, e.rythromycin (200 rag) was aduunistored intravenously, to help fluoroscopic positioning of the tube into the jejunum. the total duration of the procedure (from nasal intabatiun to jejunal placement), as well as the duration of ftuoroscopy were recorded in each patient. results. 15 patients (male/female : 13/2: mean age : 56.9 + 22.2 years; mean apacbell score : 23.t • 7.0) wore enrolled into the study.the procedure was performed within the 2 dab,s following institution of mechanical ventilation. jejunal access was obtained in all 15 patients without resort to enduscopy in 10,81 • 7.31 min.(total duration of the procedure). mean duration of fluoroscopy was 3.54 + 2.97 rain. conclusion. we conclude that placement of dual lmnen naso-gastrojejunal tubes can be obtained in mechanically ventilated critically ill patients without resort to endoscopy., provided that e rythromycin is used as gastrokinetic agent to help pyloric intubation. the following ad and dis parameters were considered in all patients: -mid arm circumference, triceps skinfold thickness, serum transferrin, albumine and lymphoeites and urinary creatinine/height index. patients whose results were bellow 80% of normal values in 3 or more of the 6 above criteria were considered undernourished (und).statistical analysis was performed using %2 analysis.statistical significance was established at p<o.o5. results: a total of 129 patients(47 female, 82 male) -with a mortality rate (~) of 31,8% -were studied, aged 56-+8 years and a median ]enght of stay of 22 days. 1 -nourished (nou) at ad and at dis -22; ~ 0%; 2 -nou at ad and und at dis -17; ) 41,2% ; 3 -und at ad and und at dis -73; ~ 45.2%; 4 -und at ad and nou at dis -17; ~ 5,9%. we observed a p< 0.05 when we compared groups 1 and 2 (p=0.0012), and groups 1 and 3 (p=0.00026). variation in nutritional status and longht of stay: 1-nou at ad and nou at dis = > median lenght of stay 18 days; 2 und at ad and und at dis = > median lengbt of stay 22days; nutritional status and age at admission: 1-age > = 60 years : nou (13) , und (53) 2-age < 60 years: nou (26), und (37) nutritional status and age at discharge: 3-age > = 60 years : nou (12) , und (54) 4-age < 60 years: nou (36), und (27) we observed a p<o.05 when we compared groups 1 and 2 (p=o.01) and groups 3 and 4 (p =0.004) conclusion: such study permits to conclude that most of our patients (69.8%), staying in icu for more than 6 days, were und, namely the elder. we observed that this kind ef patient had a longer median length of stay in icu, associated with higher mortality rate. in conclusion, the nutritional management is an essential element in suportive care of critical)y ill patients. ohiectives: sirs is associated with hypercataholisnl and resistance to nutritional support, despite raised circu&ting levels of growth hormone and insulin. serum igf-1 levels however are low; thercti~re rhigf-i administration might produce nsefnl anabolic effects. the pharmacokinetics of exogenously administration rhlge-i in such patients are likely to differ from that seen in the less seriously ill due to changes in circulating blood volume, increased capillary permeability, poor nutritional stares, and alterations in binding proteins. the ol2jective of this study was to determine the clearance, apparent vohnne (if distribution (vd), time to peak concentration (tmax) and elimination half-like (tia) of a single subctkaneous il~ieetion of rhlgf-i in patients with sirs on the intensive care unit (icu). methods: 9 icu patients with sirs were entered into the study, which had been approved by the ethics comjnittee. in each patient 6 baseline hlood samples for circulating 1gf-i were obtained over a 24 hour period, prior to il!iection of 40 p.g/kg of rhlgf-1 subcutaneously. a further l4 samples were taken fi'om each subject over the next 48 hours. circulating ige-i was measured in serum at each time point by radioimmtme assay, and the area under the curve tbllowing administration of rhlgf-i was derived tbr individual patients fixm~ baseline a~!justed igf-i levels. clearance values were caietdated by dividing the administered dose of rh[ge-[ by the total area under the etnve. vd and tv2 were calculated by regression analysis of the terminal part nf the log sertnn concentration time profiles. results: results are expressed as median (range). age 35(29-73) years, apache ii score 18(12-34), and length of icu stay 26(10-92) days. baseiine circulating igf-1 levels were low 28( < 20-166) ng/ml reaching a peak of only 84( < 20-246) ng/ml. 2 of the 9 patients had basal levels predmnhaantly below the inwel-limit of detection of the assay, and showed no appreciable rise in serum igf-[ levels tbllowing rhigf-i injection. parmacokinetic variables could not therefi)re be determined in these two patients. following rhlgf-i injection in the remaining 7 patients tmax was at 3(2-6) hours, clearance was 89(31-452)0 ml/min, vd was 0.29(0.12-0.73) l/kg, and tia 9.2(4.2-30.1) honrs. conchlsions: there was marked patient wu-iability in response to rhlgf-i administration. vd was similar to that seen in poshq~erative maior surgery patients (0.29 vs 0.33 l/kg), but clearance was greater (89 vs 25 ml/min), tmax earlier (3 vs 5 hours) and t~a shorter (9.2 vs i0.8 homts). if riaigp-i is to be administered to dtically ill patients the dose should he nlodified m the light of these findings. baekgronnd: acute bacterial endoearditis continues to be a condition with high morbidity and mortality. the majority of patients are treated with high dose antibiotics, but a patient group requires surgical treatment. methods and results: from january 1991 to march 1994, 65 patients underwent surgery for acute bacterial endocarditis: 36 had native valve endocarditis and 29 had prosthetic valve endocarditis. there were 48 men and 17 women. streptococcus viridans was the predominant infecting agent in native ~alve endocarditis and staphylococcus epidermidis in prosthetic valve endocarditis. progressive congestive failure, uncontrolled sepsis and peripheral emboli were the most common indication for surgery. although the mitral valve is more commonly involved in acute bacterial endocarditis, the aortic valve most often requires surgical inlervetion. the postoperative bleeding in the no-aprotinin group was 1902+1450cc and in the aprotinin was 850:t:80 (p < 0,0001). the mortality in icu was 10,7%, the staphyloccocical endoearditis mortality was 18,2% and the no-staphyloccocical endocarditis mortality was 2,6% (p<0,05). conclusions: the early surgery is indicated if endocarditis has no response with medical treatment. the mortality of staphyloceocical endoearditis involving left valves was higher than caused by other infecting agents. aprotinin treatment improved prosta#andin metabolism and preserved pletelet function during open heart surgery and could be useful in this type of interventions. urgent surgery had a high mortality (24%). selective digestive decontamination (sdd) has increasingly become the subject of critical discussion sine~ the development of multiresistant organisms has become an issue. moreover, a selection of gram-positive pathogens must also be taken into account when using the sdd regimen, which is primarily directed at the gramnegative bacterial specumn, with the methicillin-resistant staphylococcus aureus strains (mrsa) being of particular importance. the objective of our study was to determine the extent to which colonization with mrsa strains is promoted by sdd. method: 65 patients (ventilation period > 5 days) were randomized and allocated to the sdd group (n=32) or the control group (n=33). in their general intensive care theraw, there were no differences between the groups. the sdd regimen consisted of the four times daily administration of 50 rag polymi~ 80 mg tobramycin and 500 mg amphotericin b in the nesc, mnoth and stomach. systemic prophylactic ~dmini~/rution of antibiotics was not part of the sdd regimen. smears were taken from the nose and the rectum twice wceldy and from the pharynx and trachea once wceldy, and tested for mrsa. further samples were taken as clinically reqnircr results: 625 smears were examined in the sdd group. mrsa strains were detected in 66 samples (10.5%) from 7 patients, and in 5 patients they were detected for a period of up to 4 weeks. the positive smears were districted as follows: tracheal 11/117 (9.4%), nasal 28/199 (14.0%), pharyngeal 15/111 (13.5%) and rectal 121198 (6.1%). severe mrsa-induced infections were observed in 2 patients (infection rate 28.6% of the colonized sdd patients). 560 smears were examined in the control group. ivlrsa swains were r in 15 samples (2.6%) from 6 patients, but only repeatedly over a period of up to 10 days in 3 patients. the po~tive snmars were distributed as follows: traclmal 1/114 (0.8%), nasal 8/174 (4.6%), pharyngeal 4/98 (4.0%) and rectal 2/174 (1.1%). there were no mrsa infections in the control group. conclusion: the data collected support the view that the use of sdd promotes a selection and persistence of mrsa strains. longer-term colonization with mrsa and sovere systemic inf~ons were only found in the sdd group. although the clinical and epidemiological impact of resistance develol~ng when sdd is applied ~maine unclear, this question should be given close scrutiny. tazobactam/piperacillin (taz/p1p) is a new broad spectrum antibiotic, in which the acylaminopenicillin piperaeillin is protected by the betatactamase inhibitor tazobactam from hydrolization by bacterial enzymes. taz/pip has shown to possess a high antibacterial activity against almost all clinically relevant bacteria and is a registered drug in germany. obiectives: purpose of this investigation was to evaluate, whether faz/pip 4.5g is suited for efficient antibacterial monotherapy of severe infections and what influence dosage frequency reveals on clinical efficacy. methods: 2151 hospitalized patients have been documented in this multicenter trial during a 2 year period. as this investigation should reflect the usual clinical treatment, the only criteria for enrolment were the typical signs of infection as e.g. temperature > 38~ leucocytosis or an isolated pathogen. exclusion criteria did not exist and the patients were treated in accordance to the severeness of infection, underlying diseases, risk factors etc. with taz/pip 4.5g t.i.d, or b.i.d. results: patients suffered in most cases from infections of the lower respiratory tract (n=926), followed by intraabdominal (n=765) and skin and soft tissue infections (n=460). 61% of the 926 lrtis wvre nosocomial acquired and in 75% the treatment was conducted as monotherapy. in 53% the lrti was treated with taz/pip b.i.d, and in 45% t.i.d. pseudomonas spp. (n=138) and staph..aureus (n=134) were the most isolated pathogens pretrcatment. the clinical response rates (cured/improved) after treatment with taz/pip 4.5g b.i.d, and t.i.d, were 89% and 81% respectively. results for intraabdominal-and skin and soft tissue infections will be presented. conclusions: in hospitalized patients with severe infections successful treatment with taz/pip in monotherapy is possible. in this population a reduction of the dosage frequency to 4.5g b.i.d, revealed equivalent clinical response rates. objectives. retrospective evaluation of 9 cases of severe generalized tetanus (sgt), treated in our icu the last 7 years. we review 9 cases of sgt (6m, 3f), mean age 66.7 years. in 5 eases the entry site of c.tetanus was a skin laceration, in 1 case it proved to be the external genitalia, while in the rest no portal of entry could be determined. in the first 6 cases incubation period was short (3-11days) and so was the period of onset (1-6 days). all patients needed mechanical ventilation (range 15-58 days), initally through an orotracheal tube,and later through a tracheostomy, performed 6• days after admission. clinical manifestations of sgt included muscle rigidity and i generalized spasms, persisting for up to 6 weeks in the most severe cases. significant autonomic nervous system dysfunction was present in 3 cases occurring 5-12 days after the admission and following the time course of generalized spasm. besides general supportive measures, specific treatment included passive +active immunization, penicillin g, magnesium sulphate and sedation in a variety of regimens. neuromuscular blockade was required in 5 cases. nosocomial infections occurred in 7 eases, with sepsis and mof in one. average stay in the icu was 18-62 days. one patient died with severe septic complications and one was discharged with severe 9 disability due to anoxaemie ancephalopathy, after a cardiac arrest on admission. ~ disinfectant in suspension test, without presence of organic load, 12 disinfectants showed efficacy on lm. in the carrier test, in the presence of organic load, 6 out of 14 examined disinfectants did not exposed efficacy on lm. the results of examinations clearly showed that evaluation of disinfectant's efficacy partly depend on the used test method. antun basi6, intensive care unit, kb firule split spin~ideva 1! jugoslavia bacteremia and sepsis are frequent complications encouuntered in severe icu patients.microorganism identification with hemoculture presents the basis for adequate and successful antibiotic treatment.in many patients damage and vulnerability of the peripheral veins presents an obstacle for obtaining the blood culture from the central venous (cv) catheter sample could be also used. material and methods blood cultures were perfomed in lo4 patients on blood samples simultaneously obtained from the peripheral vein and cv catheter three times in a 24-hour period.criteria for the suspected bacteremia were body temperature above 38 c and leucocytosis above ioooo leucocytes/dl. the site for venipuncture and the cv catheter stopcock port were cleansed with povidon iodine.after the initial 5 ml of blood were discarded,lo ml were used for the blood culture.standard laboratory technique for blood cultures was used. results and discussion in 76 (73%) patients hemocultures was negative at both sites,whereas in the remaining 28 (27%) they were positive.for twentyone (26~176 of the positive patients the same results were obtained at both sites (peripheral vein and cv catheter),whereas in 7 (6.7%) patients the blood culture were positive only for the cv catheter samples.the cv catheters were in place for less than 4 days in 81 patients and for more than 7 days in 23 patients.from 7 patients with positive blood culture from the cv catheter,one patient had the catheter for three days,whereas the other 6 had the catheter from 6-1o days. we neither found significant differences in hemodynamic dates : objectives: 1, to count and evaluate bacteria isolated from endotracheal (et) suctiori samples (with and without saline). 2. to establish the exogenous source(s) of pathogens isolated from carer's hands and the equipment involved in sampling in order to reduce the incidence of contamination and infection. method~: this prospective study included 20 consecutive ventilated patients (15 male and 5 female, 56_ + 16 yr; apache ii score 19-+7) over a period of 3 months. et aspirated samples with and without saline were taken daily from day 0 of intubation until pathogen~ were presented in counts of _> 105 per ml. at the same time, samples from both carer's hands were taken before and after et suction and a swab from the ventilator tube. results: the overall length of intubation varied between 3 to 65 days. bacterial transfer between staff and patients was noted in 80% of patients until day 5 of intubation. there was no significant correlation between severity score and appearance of colonization. the incidence of pneumonia in studied patients was 45% with an overall mortality rate of 30%. acinetobacter anitratas (no 15), staphylococcus aureus (no.15), klebsiella pna~moniae (no.9) and pscudomonas aeruginosa (no.4) isolates predominated in all our specimens. we noticed increased resistance to most antibiotics with the exception of imipenem for gram (-) bacteria and vancornycin for gram (+) bacteria. conclusions: i. tracheobronchial colonization appears directly in the maiority of intubated patients. 2. there is a close relationship between the microflora of personnel, patients and equipment. 3. bacteria transfer was noted both to and from patients. 4. strict hand disinfection policy remains an important measure for the proper care of mechanically ventilated patients to reduce respiratory infections. nnseeomial pneumonia is the most common nnsocomiai infection in the icu-settiag, reported in up to 20% of patients admitted to the icu following surgery. it is associated with significant mortality that ranges from 30~ to 70%. enteric gram-negative bacilli have been implicated in 75% to 85% of ventilntor-associated pneumonias and pseudomonas aeruginosa accounts for 27% to 40% of these pneumonias. importantly, epidemics of/3-1actamnse-pruducing enterobacter spp or klebsiella spp that are resistant to extended spectrum cephalosporins or penicillins, pose serious obstacles to effective antibiotic choices. carbapenems provide in ~tro activity against a wide range of enterobacteriaceaeand other gramnegative aerobic bacteria, except steaotrophomonns maltophilia. in vitro meropcnem is more active against pseudomonas spp than imipanem (especially p. aeruginosa and p. cepacia), imipenem and meropenem are effective against more than 95% of strains responsible for nnsocomial infections. all major pathogens associated with lrti are usually covered by the carbapenems, exceptions are pathogens involved in so-called atypical pneuomouia like mycoplasma, chlamydia and legionella. carbapenems are highly stable in the presence of most chromsomal and plasmid-mediated blactumases and usually offer a postantibiotie effect lasting for three hours against most of the enterubacteriaceae. reeent studies comparing imipenem/cilastatin with other ~-lactams and fluoroquinolones in severe lrti in icu patients resulted in favourable clinical cure rates and good tolerance, but development of resistance in p. aeruginosa and 5;. aureus during treatment were of some concern. meropenem offers the advantage of greater stability against enzymatic degradation, so no concomitant administration of an enzyme inhibitor is necessary, and meropenem appears to be associated with a lower risk of seizures, particularly when used at high doses. results from studies with meropenem in lrti, especially in critically ill patients with acute exacerbations of chronic bronchitis, demonstrated excellent cure rates and better gastrointestinal tolerance of this new carbapenem. both earbapenems are effective candidates for use as empiric monotherapy in nosucominl infections of critically ill patients. qbl~ctives a favourable effect of iv immunoglobulins in septic surgical patients has been reported, but not sufficiently validated. we conducted this study on trauma patients to: i) investigate the effect of ivig on septic complications and il) quantify this effect by means of serum bactericidai activity (sba) assessment and iii) to explore the effect of temperature increase (from 37 to 40 ~ c) on the sba methods: twenty trauma patierits matched on admission for age, sex, inju~ severity score and glasgow coma scale, were allocated to receive either wig (ivig group; i0 patients) or equal volumes of human albumin 20% (control group; 10 patients). wig (sandoglobulin) was administered in a total dose of 1 g/kg divided in a four time regimen on days 1, 2, 3 and 6 post-admission. three blood collections were performe& before the first dose (day 0) and 24 hours after the third and the fourth dose (days 4 and 7 respectively). complement, lgg fractions, the sba at 37 ~ and at 40 o c and clinical parameters were recorded. results-similar lgg and igg] serum levels were found in groups ivig and control on day 0 (743+_130 vs 898• ns and 394+103 vs 472+101, ns), whereas they were significantly higher (p<0 05) in the 1v1g group on days 4 (1700_+_274 vs 799+197, p<0 05) and 7 (1740_+227 vs 864+i64, p<0.05). the various complement-fractions increased in both groups without inter-group differences the mean (• sbas (37 ~ c) at 30 rain in ivig group vs control group were: -53_+32 vs -56• ns for day 0, 9_+46 vs -54_+46 p<005 for day 4 and 7_+34 vs -54+47 p<005 for day 7. the mean (+sd) sbas (40 ~ c) at 30 rain presented a significant improvement over those of 37 ~ c but for the control group remained negative a~d were respectively as following: -~8• vs -26+33, ns for day 0, 22+_39 vs -29_+35, p<0.05 for day 4 and 24_+31 vs -27_+36, p<0.05 for day 7. the increase of temperature induced a 3-fold improvement of sba in iv1g group and 2-fold ofcontrol-~oup positive blood cultures, and the product of the infectious episodes number multiplied by days of occurence, were significantly lower (p<0 05) in the ivig group than in the control (2 vs 7, and 440 vs 1900, respectively). conclusions: our study shows a significantly favourable effect of ivig administration on septic complications and on sba of trauma patients. the increase of temperature results in a significant improvement of sba of patients that received ivig, which theoretically means a farther prevention of infection in the febrile state. pharmaceutical microbiology, university of bonn, meckanheimer aune 168, d-53115 bonn, germany infectious diseases in intensive care patients are common in comparison to patients on other wards and out-patients. the main difference is that intensive care patients are much more sensitive even to less virulent bacteria. thus, the spectrum of infecting organisms is different. strains often regarded as pathogens with low virulence cause serious infections in these patients. strains such as serratia, however, have intrinsic resistance to most commonly used agents such as 3rd generation eephalosporins. furthermore, the common pathogens like staphylococci, psoudomonas aeruginosu, enterocneei and gram-negative bacteria, enterobacteriaeceae as well as the non-fermenters are less sensitive if isolated from intensive care patients. it is difficult to generalize on intensive care units as different patient groups are in different icus aud there are great changes from one hospital to another and from one country to another. if we take s. aurens strains from one study from 1990 the'overall resistance in intensive care units towards oftoxacin was 22 %, whereas in other hospital wards the percentage of resistance was 5.3 %, in out-patients, however, only 2.$ %. the same trend was true for entercnecus faecnlis, coagulase-negntive staphylococci, and other bacteria as well as other drugs. one most striking difference was found with klebsialla pneumoniae and gantamycin resistance, which was $ times higher in intensive care units as compared with outpatients, whereas in the same species no difference was to be seen with the resistance towards carbapenems. however, differences between countries seem to be even more striking, as example gantamycin resistance and staph. anrens is given. the extreme difference is more than 60 fold. thus, it is evident that there is a general trend towards higher resistance in intensive care units, but no generalizatiouis possible. therefore, surveillance studies in intensive care units are needed and the antibiotic policy has to be adapted to the specific needs of the unit. in the icu setting the most potent antimicrobial agents are required to address problem organisms including those resistant to penicillins, cephalosporins and aminoglycosides. carbapanems would appear to present a useful option in this setting. objectives of this study was the evaluation of systemic candid•177 in postoperative cardiac surgery patients (pts) with prolonged icu stay. methods: out of 2617 postoperative adults pts of mean age 61.1+8.9 years old, with a mean icu stay of 1.6_+0.6 days, following an open heart surgery from july 1993 to april 1995, 54 pts (2%) remained in icu for more than 10 days because of severe perioperative complications. patients were included in the protocol if they had clinical signs of infection or sepsis, and fungi isolated in blood culture or in culture from at least three different sites. the patients who developed systemic candidiasis received iv fluconazole (800 mg/day) (10 patients) or amphotericin-b for at least four weeks, and then they were closely monitored. results: out of 54 postoperative pts with prolonged jcu stay, 11 pts (20.3%) developed systemic candid•177 usually after the 20th postoperative day. they were 8 males and 3 females of mean age 64+_7.4 years old. this group of pts had prolonged bypass and aortic cross-clamp time compared to control group (119 min vs 84, and 64 vs 49 min). all these pts received inotropes per• (mean value=2.3). during their icu stay, 9 pts developed sepsis of bacterial origin, while the other two severe infection, and received antibiotic regimens for prolonged period. the patients were submitted to mechanical ventilation for a median period of 50 days. the median icu and hospital stay was 58 and 60 days respectively. all pts have been improved and finally negative cultures were obtained. conclusions: 1. a significant percentage of patients who remained in the postoperative icu for more than 10 days developed systemic candidiasis. 2. all patients who developed systemic candidiasis had received antibiotics because of sepsis or severe infection, for prolonged period. 3. fluconazole seems to be a very good alternative to amphotericin-b. 4. fluconazole is a safe antifungal agent with few side effects. botulism is the most severe and an odd food poisoning. although it is more commonly related to preserved meat derivatives, preserved fish and vegetables are also responsible for a number of cases. obiectives: to evaluate four familiar outbreaks of botulism . methods: we study the patients that were admitted in our hospital because of botulism from may 1982 to february 1995. results: the thirteen pacients involved had a previous history of home preserved beans ingestion. after a 24-hours incubation period, gastrointestinal symptoms (abdominal pain, vomits, constipation) appeared and lead them to hospital consultation in the 4th to 7th day after ingestion. two patients died (acute respiratory failure before admission), seven were admitted in icu, two in ward and two of them were discharged from emergency room. clinical symptoms and the previous history of the ingestion established the diagnosis, that was emg confirmed. in all cases, symptoms were consistent with b-toxin botulism. b-toxin was isolated in serum and food proceeding from the third outbreak, and the serum was negative in the other ones. neurological symptoms were predominant: midriasis (100%), dry mouth (100 %), dysfagia (100 %), asthenia (55 %), palpebral ptosis (55 %), accomodation paralisis (66%) and urinary retention (55%). muscle weakness lead to acute respiratory failure in three patients (one of them required mechanical ventilation). four patiens developed infections (respiratory, urinary and phlebitis). both died patients and one another presented severe hypertension. all admitted patients were treated with polivalent anti-toxin. the two patients who underwent a more severe muscle weakness received also guanidine hydrochloride, with no answer in one case and provoquing a cholinergic crisis in the other one. icu length of stay was 10 days. at hospital discharge, patients continued symptomatic, mainly with dry mouth, disfagia and impaired vision. conclusions: although botulism is a serious illness, the pronostic seems favorable if treatment and support measures are avaible. usually neurological symptoms we predominant and at discharge some of them could still persist. the arrow "hands-off" (aho) thermodilution catheter (tc) is completely shielded during balloon testing, preparation, and the insertion procedure. in order to assess the value of the aho thermodilution catheter in the prevention of systemic infections associated with pulmonary artery catheterization (siapa), we conducted a randomized prospective study over an 18-month period. methods : the patients (pts) were randomly assigned to two groups : group i for a standard tc customarily used in the department, versus group 2 for the aho thermodilution catheter. the diagnosis of siapa was determined on the basis of a positive culture of tc and bacteremia with the same organism, with out any other nearby focus, in association with regression or disappearance of the clinical signs of infection after removal of the thermodilution catheter. results ( objectives: the mortality rate (mr) of tb requiring mechanical ventilation (mv) is high (70-100%). the aim of the study was to evaluate mr, associated factors, and prognostic significance of mv and hemodynamic disorders from tb in icu in 35 patients with tb. methods: clinical parameters on admission, and complications in icu were related by univariate analysis to icu, hospital, and 6 month outcome. 18 patients required mv; 10 were immunocompromised (ic) including 8 hiv. tb was pleuropulmonary in 24, disseminated in 9 and meningeal in 2. results: mr was 31% in icu, 34% in hospital and 47% at 6 month. 12/16 (75%) <0.001 mortality was associated with a high saps score, initial shock, mv and nosocomial septicemia. the mr dramatically increased when ards occurred during illness, despite the lack of correlation between mr and initial po2/fio2 ratio or initial murray score. the site of infection did not influence the mr. surprisingly, the mean therapy delay was shorter for non survivors. mr was not related to ic status, nor hivstatus, but was only related to previous steroid therapy. conclusion: mr of tb requiring icu is high (47% at 6 month). need for mv increased mortality (72% vs 18%). general severity and respiratory dysfunction seem to be major prognostic factors in icu rather than tb per se or than therapy delay. in spite of the improvement in the prognosis of pneumococcal meningitis (pm) with third generation cephalosporins (tgc), this infection still presents a great mortality which could be increased with the appearance of antibiotic resistant streptococcus pneumoniae. objectives: to asses intensive care mortality and morbidity of pm and to define patients (pts) at risk of complicated evolution. patients and methods: a retrospective evaluation of pm cases (all diagnosed by csf culture) admitted in our icu from january 1985 tit march 1995. in all pts we analized: demographic data, underlying disease, apache ii score, clinical symtomps, treatment, complications and outcome. statistical analysis was done using bmdp sofware package. results:a total 0f42 pts were studied, 26 males; mean age 55,8 _+ 16 (16-81); apache ii score 16,6 + 7,9; glasgow coma scale (gcs) at admission 12,5 _+ 1,7; 17 (40%) pts suffer from cronic pathology; 5 (12%) pts diabetes mellitus (dm), 4 (9,5 %) pts had had a previous cranial traumatism. in 22 cases the source of infection was otic and also in 22 (52 %) episodes of pm there were bacteriemia. in 21 out of 26 (80%) pts that ct was performed no radiologic abnormalities were shown, 3 of them presented cerebral oedema and 1 pts a cerebral abscess. twenty-eight percent presented seixures, 14% hemiparesia, 46,3% respiratory failure, 17,5% shock, i5% renal failure, 5,1% multiple organ failure (mof). as for treatment refers 5,5% pts recieved only penicillin, 69,4% pts only tcg, 11,1% pts tcg followed by penicillin and 8,3% pts tcg+vancomycin. seventy-five percelat of pts recieved corticosteroids and 25,6% vasoaetive drugs. the mean icu stay was 7,5 5:6 days (1-28). twelve (28,5 %) pts died, two of them presented pm relapse (resistant streptococcus pneumoniae) and another two pts developed neurological sequelae. factors associated statistically with bad prognosis were dm, the use of vasoactive drugs, shock, mof, the apache ii score at admission, the gcs at the 48 and 72 hours from admission in the icu but not the gcs at admission. didn't resulted statistiealy signifcative age, previous eronie pathology, seizures, baeteriemia, renal failure and coagulation disorders. conclusions: mortality was high and associated to apache ii score at admission, to gcs at 48 and 72 hours after admission, shock, vasoaetive drugs and mof. objectives:the aim of the study was to analyse some of significant immunologycai changes in surgical patients,requiring intensive health care,and to determinate the possibility for evaluation,dynamical examination and importance of immunologycal problems for treatment. methodes:the study concerns a number of 30 patients with expanded surgical intervention or serious postoperative complications.the results has been carried out with fiowcytometryc analyses of lymphocytic suhpopulations and routins methods for investigation of humeral immunity.the"panel" for evaluation of 2(} immunologycal parameters has been offered:t-calls total/cd3+/;t-helper/cd4+/;t-supressor/cd8+/ th/ts ratio;b-cells/cd19+/;naturai kilier/nk/cells;skin test for cellular immune function;phagocytic and oxidative activity;serum levels of immunogiobulins-g ,a,m;protease inhibitors;c-reactive protein.all patients have been studied during suffering and after surgical procedures dynamicaly. results:there have been estimated significant changes in immunologycal parameters especially:decrease of t-cells: cd3+mean=37.62%/14.3%-47.9%/and cd4+mean=22.11%/9% -28.8%/;inverted th/ts ratio ,mean=o.72/0.37-0,90/;reduced or negative skin teste;reduced phagocytic and oxidative activity before septic complications. conclusions:dynamical examination of immunologycal parameters shows,that the prolonged t-total,t-helper lymphocytopenia with functional deficience of ceils-mediated immunity correlates with the stage of clinical condition of the patients and has prognostic importance.it's clear,that immunologycal monitoring gives a possibility for immunecorrection. patients (pts) with the human tmunodeficiency virus (hiv) infection have a decreased immune response and are particularly susceptible to infectious endocarditis (ie). the aim of our study was to analyze the prevalence of ie, its clinical and therapeutic implications in a hiv population 9 we prospectively studied 245 pts, 9.4% (23/245-group ie+) with ie during the clinical course of this disease. we analyzed the following parameters: age, gender, race, type of hiv, cdc classification, number of t4 and t8 type cell population and its ratio, therapeutic with azt, type and number of opportunist infections (inf, mycobacteriosis (mb), neoplasm's (nee) 9 the echocardiographic parameters were lv internal diastolic and systolic diameters, lv percentage of fractional shortening, interventricular and posterior wall thickness, the degree of valvular regurgitations and the presence of pericardial effusion. el was located at the mv in 2.7%, tv in 6.0%, av in 2% and pv in 0.9~ and was multiple in 2.0%. hiv el+ pts had larger lv diameters and more frequent significant valvular regurgitations (39% tr, pe 33%, mortality 32%). these two groups differed significantly in the following clinical parameters: the typical symptoms were watery diarrhea, high fever, tachycardia,luekocytopenia and oligouria within 7th postoperative days. the patients with mrsa enterocolitis had positive mrsa culture from the many materials except feces.mesa strains frequently had coagulase type 2,enterotoxin a and toxic shock syndrome toxin-1 .eight of 1 6 patients had postoperative organ failure.most of the mrsa strains in japan were similar in coagulase type to our hospital and our department.all of mesa strains were susceptible to vancomycin and arbekacin,tbough most of them showed resistant to many other antibiotics.we have employed guidelines for therapies such as oral or enteral administration of vancomycin and correction of the hemodynamics for dehydration and circulatory failure due to diarrhea from 1992.futhermore we have placed colonized or infected patients in private room,worn gown and mask,and carefully washed our hands from 1992. these countermeasures for prevention of nosocomial infections after 1992 significantly reduced the incidence of mrsa enterocolitis. conclusions:earlier diagnosis and treatment, and distric prophylactic measureres against mrsa infections are very important. -cdo ivda leptespiresls affects all the organs with widespread hemorrhage that is more prominent in skin, mucosa, skeletat muscles, liver and kidneys. lung involvement is usually mild and less common. suli, it is very uncommon acute respiratory failure to be the pr6sontirlg symptom. a case with leptosplrosl..,s which was presenting with acute respiratory failure is described. a 36 year-old man admitted to icu becauso of fever, myaigla, aevere c~, hemopty~s. his blood gases showed: pao2:46mmhg with fio2:1.0, pco2:27 mmhg, ph:7.4, hco3:20mecl chest x-ray film demonstrated diffuse bilateral alveolar pattern occupying beth lung4/4). trarmamlnase, bllllrubln, ~ and esr were elevated, wbc was 8.7001mm8, platelet: 40.0001ram3, hematesrlt:30%, hemoglobin: .sgrldl=. there was no clinical or ecttlographlc evidence of left heart failure.patient fulfilled the criteria for diagnosis ards he was found to have an ~lutinatlon tlter for leptoq~lral antigens(indirect he~lutlnatlon atomy, ilia} very high (1/800, negative<ill00) and iglm antibodies also very high (elisa, a=1.492, negative<0.150) strongly suggesting leptospirosls. treatment with tetracycline and non-tnvaslve mechanical ventilation by nasal mask were started. pre~jre support mode and cpap was usod. the following days the clinical picture,the oxygenation and the chest x-ray of the patients were improved, and patient dl~herged from the icu. the control of leptosplremlc phase as well as his good cooperation during nipsv contributed to rapid recovery and excellent outcome in hiv infections, pneumonias (pnm), mainly due to pne~ mocystis carinii (pc) have an outstanding place in pu ! monary complications. the aim of this work was to compare two group> of patients admitted with pnm in our icu during the same period (1990-94): group a, 19 patients hiv+, and group b, 152 patients hiv-. apache ii was identical in the 2 groups (p=ns). group a required more often mechanical ventilation (p=0,o07), had a higher p(a-a)o2 (p=0,004) and metabolic acidosis was more frequent (p=0,001). regarding laboratorial parameters group a had a lower no. of linfocytes (p=0,02), a higher ldh (p=0,04) and a more marked hypoalbuminemia (p=o,03). mortality was higer in group a (52,6%) than in group b (29,6%), (p=0,04). analysing the a group patients, we found no significant differences between alive and deceased patients, with exception for albuminemia, which was lower in the deceased patients (p=0,02). in conclusion, the hiv+ patient's pnm have a more agres sive behavior when compared with community acquired hiv-patient's pnm. the prognosis was not influenced by the apache ii. perhaps other parameters such as p(a-a)o2, metabolic acidosis, linfocytes, ldh and albumin shoud be more evaluated as possible predictive indices. some prognostic factors, usually accepted as predictive in the analysis of hiv+ patients do not seem to be worth in the late stages of aids, mainly when they reqquire intensive care. intensive care unit, onassis cardiac surgery center, athens, greece. objectives of this study was the comparison of two different antibiotic regimens as prophylaxis in cardiac surgery patients. methods: in a prospective randomised comparative study, two different forms of antibiotic regimens were investigated : a single dose of cefuroxime (zinacef, 3 gr) (group a) given during the induction of anaesthesia, versus a four days combination of amoxiculine (amoxil, 2 gr tid) plus netilmicin (netromycin, 150 mg bid) (group b). a total of 926 patients (pts) (767 males and 159 females, of mean age 60.6+8.7 years old) were included in the study over a period of one year; 424 in group a and 502 in the group b. patients were checked for the occurrence of infection during the first postoperative month. results: the total rate of infection in cardiac surgery pts was 5.8%; 5.4% in group a and 6.1% in group b (p=ns). 34 pts (4.7%) developed infection following cabg, 17 pts (7.9%) following valve replacement and 6 pts (17.6%) after other cardiac surgery. they were 43 males (5.6%) and 11 females (6.9%). endocarditis has occurred 0.4 % in group a and 0.2 % in group b. severe wound infection was recorded in 0.4% in group a and in 0.8% in group b. one case of sepsis (0.2%) in group a and in group b (0.2%). respiratory infection occurred in 11 pts of group a (2.6%) and in 11 pts of group b (2.2%). two cases of urinary tract infection was in group a and one in group b. catheterrelated infection was occurred in 5 (1.1%) in group a and 6 (1.1%) pts in group b. 3 pts (0.6%) had fever of unclear aetiology in group b. conclusions: there was no statistically significant difference regarding the rate of infection in both groups. a single dose administration of cefuroxime is accordingly just as effective as a four days regimen of amoxicilline plus netiimicin. legionella pneumophila is a common bacteria of the environment, and it is an agent responsible for severe community acquired pneumonia (cap). we analyzed the 8 patients with lpp admitted in our icu during the last 8 years (1986) (1987) (1988) (1989) (1990) (1991) (1992) (1993) (1994) . they represented 4.6% of cap. seven patients were males and 1 female, with mean age 46.7+12.1 years. tiss was 24.1+10.9 and apache ii 21.0+5.2. all, but 1 patient, were under mechanical yen tilation (mv) during a mean period of 11.7• (min-l, max-44) days. two pneumonias occurred beyond the season, while 4 patients had an epidemiological history. only 1 patient had no risk factor. in all the others tobacco smoking and alcohol abuse was quite frequent. diagnosis was based on serologic test and culture or direct fluorescent antibody staining of bronchial secretions. seven patients had a multisystemic disease with hepatic dysfunction in 5, renal failure in 4 (due to rhabdomy~ lysis in 3). one patient had a prosthetic valve endocarditis and another developped ards. nosocomial septicaemie occurred in 3 patients. mortality rate was 50%. deceased patients had initially higher apache ii, (a-a) 02, and lower natriemia. comparing lpp with the other cap (n=84), both submitted to mv, mortality rate was similar (57,1% versus 54.7%). in conclusion lpp can occur all over the year. there was a high incidence of severe complications and outcome was similar to the other cap when requiring mv. prospective specimen brash (psb) with culture > 10 cfu 10 3 cfu/ml. broncho-alv~lat lavage (bal) ~= 104 c'fu/rnl or positive blood culture. 10 were excluded for rapture of treatment ; 63 were analysed (shift with oral antibiotic8 : 3 ; prohibited antibiotics associations : 5 ; resistant germ : 2). clinical data : age 60,6 • 18,7 ; saps 12 • 2,86 ; mac cabe i : 76,2% -ii : 22,2 % -iii : 1,6. 63,5% of the patients were intubated and under mechanical ventilation. the pneumoaiae were : primitive in 35 (55,6%), copd 9 (14,3%), aspiration pneumonia 19 (30,2%). 75 germs were isolated (psb 67, bal 1, blood culture 7) : s. pneumoniac 28 (37,3%), h. influeazae 14 (18,7%), sttep~:occns 10 (13,3%), saar6ns 10 (13,3%), enterobaetdrindr 5 (6,7%), mosexella catarrhalis 2 (2,7%), othem 6. 71/75 (94,7%) were sensitive to freatment. the ltentment was 100 mg/kg/d of ampiclllin and 50 mg/kg/d of sulbactam in continuous iv adminisu'ation during at least 10 days. clinical eff~ienev : success 46 (73%), failures 17 (27%) with superinfeetion 7, worsening or relapse 3, dead 5, side effects 2. there was no difference between etiologies : primiti~;e~ 74,3%, copd 77,8%, aspiration pneamoniae 68,4%. the bacteriological effieieacy was evaluated only for 41 patients with eradication 30 (73,2%), eradication but super~ection 6 (14,6%) : with pseadomoaas a&ogiuosa 2, eater~ac~ 3 ; beeteriological failure 5 (12,2%). in conclusion, the aasor ampicillin -sulbactam is effective for the i~eatment of severe acquired community pneumonise. objectives : to assess the efficacy of chlorhexidine (cl) gel or suspension applied in the nose and in the op for the prevention of the tmcheobronchial colonization. methods : thirty-seven patients expected to be intubated for > 48h were randomized to received topical application oga cl suspension (2%) qshrs, a cl gel (1%) q6hrs or a placebo. in addition all vpts received a nasal and a op spray (2%) of either cl or placebo administrated according to the same schedule. semi-quantitative cultures of the anterior nares, the oropharynx (op) and the trachea were obtained on admission and once a day until extubation (just before the next application). the results were assessed according to the following criteria: success = no acquisition of gnb in the trachea ; failure = acquisition of gnb in the trachea. acquisition was defined by a follow-up culture positive for a gnb not present in the trachea on admission. results : success failure nosocomialpneumonia overall morality clsusp. placebo clgel placebo n=8 n=10 n=9 n=10 5/8 6/10 7/9* 3/10 3/8 4/10 2/9* 7/10 1/8 2/10 3/9 4/10 0/8 1/10 2/9 2/10 i *p = 0,03byfisher'sexacttest conclusions : these results suggest that topical cl gel administered q6hrs may prevent tracheal colonization by gnb. f. daumal*, m. daumal**, c. plot**, v. vurmmen ~ e.colpurt**, b. manonry** * hygiene hospitali&e, ** service de r6enmmtion, * service des admissiens-urgeuces centre hospitalier g-6ndral -02 321 saint-quentin -france obiectives: evaluate the nosocemial risk due to peripheral venous inserted short catheters, and the quality of care. patients-methods: the intensive tare unit (i.c.u.) is a 9 beds unit. the prospective study includes all the patients comn~ in from 01/01/1993 to 30/03/1995. the recruitemont uses an evaluation schedule of local clinical signs. the nurses aimed to create this evaluation data which includes the place of entry site, the duration of catheterization and the cause ot withdrawal. only patients staying longer than 2 days in the i.c.u. are accounted for. the diagnosis of uosoenmial infection is assured by the physician taking care of the patient and by the hospital epidemiologist on the next signs: evident pus at the catheter entry site, positive culture of the strain, with or without the same pathogen in the blood sla'uam,the patient having no other distant source of infection. analyses were performed on epi/nfo. results: the occurrence of 3 nosoeomjal inthrtions: i abcess and 2 bacteremia during the first part of the study lent the medical staff to modify the protocol of insertion end survey of the device. so we analysed 2 different periods: period 1( 1/01/93 to 31/10/93 ) and period 2 (01/11/93 to 30/03/95 ) for all 1 .e peripheral catheters inserted in the i.c.u. period 1 44,9 % 46,2 % en infection due to peripheral venous device is a daily threat. the severity of some clinical situations requiring admission in icu proves it. the motivation of nurses for rigid adherence to established protocol, the daily survey of the entry site, the withdrawal of the peripheral catheter every 72 hours aimed to reduce significantly the local signs of inflammation end infection of peripheral catheters inserted inside the i.c.u. objectives: to investigate the use of a new metabolic monitoring device for different ips levels by comparing oxygen consumption (vo2) to measurements of the mechanical work of breathing (web) and p0.1. methods: the study was approved by the institutiotml ethics committee. eight patients were investigated during weaning after prolonged mechanical ventilation (6-75 days) for various diagnoses when the clinical physician judged the patient to be ready fur weainag. ips was setto 15, 10, 5, 0 mbar far 20 rain periods each. all patients had a peep between 5-8 mbar.. respiratory frequency (f), tidal volume (tv), minute ventilation (ve) were read from the ventilator display (7200ae, puritan bennett, carlsbad, usa). flow and airway pressure were measured at the endotracheal tube site. esophageal pressure was measured using an esophageal balloon catheter (fa. ruesch, frg). web was determined as the area subtended by the pleural-pressure-vohime curve. p0.1 was determined by using standard occlusion technique and graphical analysis of the airway pressure tracing. vo2 and vco2 were measured using the pb 7250 metabolic monitor (puritan bennett, carlsbad, usa) connected to the pb 7200ae ventilator. all data are given as mean• deviation for each ips level. comparison between the different ips levels was performed using anova for repeated measurements. significance was considered at p<0.05, compared to ips 0 mbar. results: the values for breathing pattern, web, p0.1, vo2 and vco2 are given in the table for the different ips levels; significance is indicated by ~. objectives: fluidized beds are often used in the management of critically ill mechanically ventilated patients. critically ill patients are increasingly colonized with resistent pathogens [ie: p. aeruginosa, methicillinresistent s. aureus (mrsa), extended spectrum i~-iactamase producing enterobacteriaceae ] that can ultimately cause nosocomial infection. methods: we prospectively monitored bacterial colonization of mechanically ventilated patients and of the fluidized bed (clinitron) inwhich they were treated. multiple samples for quantitative bacterial cultures were taken from oropharynx, trachea, feces and bedsores. samples of ceramic beads from the bed were also taken both during and after patient stay (after bed operation in the absence of patient). re,~ults: 13 episodes in 12 consecutive patients (mean age: 57.6 years) were analyzed. all had bedsores and/or urinary catheters and fecal incontinence, 7 patients had nosocomial pneumonia, 6 had urinary tract infection [2 with extended spectrum imactamase producing k/ebsie//a pneumoniae (ki~lse)], one had positive blood cultures with mrsa, and one patient had a ki~lse found in high concentrations (103 -10 s cfu/ml) in 2 occasions in feces. patients were heavily colonized: the , samples from ceramic beads showed no growth or became sterile without any sterilisation procedure (even in one case of presence of kf~lse) during the patient stay. conclusions: fluidized beds do not put patients at high risk of acquiring nosocomin pathogens, and cross-contamination between patients seems unlikely, even when multiple resistent organisms were initially present. the recommandation from some manufacturers to undergo extensive sterilization of fluidized beds after use does not seem warranted, at least with the bed used in this study. ant. koutsoukou, a, tahmitzi, p. kithreotis, m. koutonlidou, k. stavrakaki, kainis e, g. vlahogiorgos and e. eliopoulos icu-centre for respiratory failure -chest diseases hospital of athens. the cost-effectiveness issue is becoming vital in modern medicine and may lead to moral dilemmas since sometimes certain groups of patients may not have access to highly specialised modalifies. objective: our study compared the mean daily cost for antimicrobial medication in copd patients treated in icu versus all other patients in the context of relevant epidemiological, prognostic and outcome data. methods: age, sex apache ii score, length of icu stay (los) and in -icu fatality were retrieved from the files of all icu admissions over 1994. mean daily cost for antimicrobial therapy per patient (dcat) was estimated. these variables were statistically compared between copd and non-copd patients. significance was assumed at p<0.05 results: 140 of the total 178 admissions were fully evaluable. 38 of them (27%) were copd patients. data (m---sd) results for statistical test are given in table i . copd patients were significantly older spent more time in the icu and presented with significantly higher apache ii scores. outcome and dcat were comparable in the two groups. objectives: the use of heat and moisture exchangers (hmes) during long term mechanical ventilation (mv) is increasing. in icu patients, they are routinely changed every day, according to the recommendations of the manufacturers, but the clinical basis for such a daily practice is lacking. we therefore prospectively assessed whether changing hmes (dar hygrobac, spa, mirandola, italy) every 48h only would affect their clinical and bacteriological efficiency. methods: two consecutive groups of patients requiring mv for >48h were compared: group1= hme replaced every day, n= 61 episodes of mv in 61 patients; group 2 = hme changed every 48h, n=68 episodes in 64 patients. tubings were not changed in the same patient during the whole length of ventilatory support. diagnosis of nosocomial pneumonia (np) was based on a positive quantitative culture (~103 cfu/ml) of a protected specimen brush in patients with clinical signs of pneumonia. quantitative cultures of pharynx, trachea and y-cannector were performed every 48h. results: the groups were similar in terms of age, indication for and overall duration of mv (10+_8.6 vs 10+_9days, p=0.9), and severity of illness (saps: 16---4.9 vs 16.4+_5.5, p=0.6). the maximal values for peak airway pressure were identical in both groups (33.4-+7.8 vs 33.7• cmh20, p=0.9). obstruction of the tracheal tube was observed in only one instance in a group 1 patient who had tracheal bleeding. circuit colonization was very rare, and of low grade in both groups. the level of patient colonization and the type of organisms were identical in both groups. more importantly, the incidence of np was the same (6/61 vs 8/68, p=0.7), as was duration of mv before the occurence of pneumonia (9• vs 10.5+_4.7, p=0.6) and overall mortality rate (17161 vs 17168, p=0.7). conclusions: the clinical efficiency of this hme does not seem altered after 2 days of use. indeed, replacing this hme every 48h only neither affect circuit and patient bacterial colonization nor the incidence of np. therefore, substantial savings could be obtained changing hmes every other day only. obiectives: to evaluate the usefulness of different paraclinical investigations for the diagnosis and prognosis of acute viral encephalitis in icu patients. methods: we reviewed 13 patients (pts) admitted to our icu from july 1989 to december 1993 with the diagnosis of acute viral encephalitis. all were in coma and were initially treated as presumed herpes simplex virus (hsv) encephalitis. the causative agents were: hsv (2 cases), herpes zoster varicellae (1), measle (1), rabies (1), unidentified (8). eleven pts survived and three presented neurologic sequelae. twelve pts were investigated by mri, and eleven also by spect and multi-modality eps. including brainstem auditory eps (baeps). these investigations were obtained as soon as possible following admission and were repeated during icu stay when possible. the clinical outcome was noted. results: six pts (6/12) had an abnormal mri. among them, 2 pts made a complete recovery, in comparison with 5/6 pts with a normal mri. in one hsv infected patient, mri remained normal despite clinical deterioration and bad outcome. when repeated, mri became abnormal in 3 cases (with poor outcome in one) and was improved in one. spect was found abnormal in 10/11 pts (among them, 4 pts had thus a normal mr/). the correlation regarding the topography of brain lesions was poor between mri and spect. the findings of spect could not be correlated with a poor outcome. the baeps confmned in 56% of the pts the clinical diagnosis of brainstem involvement. changes in visual and somatosensory eps were mild in all the pts and were not helpful for the prognosis. eps were otherwise interesting for the follow-up of the coma in these sedated and ventilated pts. conclusions: the value of mri and eps for the diagnosis of acute viral encephalitis is of limited interest. spect seems to show early modifications, even in pts with a normal mri, but this test is poorly specific and does not correlate with mri changes when present. concerning the prognosis, larger studies should probably confmn that a normal mri could usually result in a good outcome. this serie illustrates also that hsv encephalitis could be demonstrated only in a small number of cases and that the prognosis of non hsv encephalitis is not easily assessed. objectives: to study the influence of gram (-) bacterial lung infections on liver function i~ mv icu pts. pts and methods: we studied 102 pts, 68 # (66,7%), 34 (33,3%). hean age:48,3• years (16-82). mean stay in icu:13,3• days (8-75). they were divided in 2 groups: a(44 pts) who did not suffer from pneumonia and b (58 pts) who developed a gram(-) bacterial pneumonia. both groups were consisted of pts with same age, sex and disease distribution and same systemic failures. we measured sgot, sgpt, total bilirubin(tb), direct bilirubin (db), alk.phosphatase (al.ph.), v-gt and albumin (alb.) 3 times: on days o, 4 and 7 of the pneumonia for group b and respectively for g~oup a. conclusions: 1) in elderly intubated pts of an icu, kp is isolated more frequently than in icu pts<65 years (p<o,01). 2) the frequency of isolation of kp in icu pts during 1985-92 was 25-30%, but now (1992-95) kp becomes more and more rare (12-15%) and is isolated especially in elderly pts. 3) its sensibility to antibiotics remains always the same. 4) the prognosis of np in our study was independent from age (p<o,1), sex, presence of bacteremia (p<0,1) and type of invading microorganism. gram neg bacteria and renal failure (rf) from aminoglycoside toxicity are common and serious complications in critically ill patients. the aim of this prospective, pilot study was to compare the safety and adequancy of the endotrecheal (et) vs the intravenous (iv) administration of aminoglycosides. were measured in the plasma and the bronchial secretions at 1 and 4 hours after the iv (bolus) or the et (nebulized) administration of 80 mg of gm in seven criticcally ill patients (3 with established renal failure). safety was defined as peak and trough plasma gm levels _< than 8 and 2 ijg/ml respectively and adequancy as gm levels in bronchial secretions _> 0,5 ijg/ml. results: gentamicin was administered by the et and iv routes in 18 and 7 separate sessions respectively. a total of 107 samples were assayed, 69 in bronchial secretions (bs) and 38 in serum. the et route resulted in higher gm levels in the bronchial secretions compared to the iv route (3,26 + 2,86 vs 2,1 _+ 2,1 pg/ml respectively, p = ns ). adequate bronchial gm levels were achieved in 100% of patients after et administration, compared to 66% after iv aaministretion. the blood levels of gm were significahtly lower after the et vs the iv route (1,56 + 1,95 vs 5,56 • 1,96 pg/ml respectively, p _< 0.01). the et administration resulted in toxic bronchia~ gm levels in 47% of the specimens. 66% of these samples were from patients with renal failure, however toxic blood levels were reached in only 12% of these. gentamicin seems to be a safe and adequate alternative route of treatment for the lrti. however, in patients with renal failure the et administration of the aminoglycosides should also be modified and continuously monitored. in order to evaluate the pathogenic role of anaerobes in nosocomial pneumonia (np), we investigated the systemic humoral response in patients who developed a np with anaerobic bacteria, especially prevotella species. methods: blood samples from 4 groups of patients were tested. group i: 13 patients with a np in which prevotella spp. was isolated from protected specimen brush (psb), group ih a control group of 30 patients with a np without anaerobic bacteria, group ill: a control group of 27 patients with dental stumps but without pulmonary infection, group iv: a control group of 30 healthy voluntary people with prevotella spp. isolated from the dental plaque. an elisa was used to evaluate the total antibodies level against a mixture of four prevotella strains and a western-blot method was done to identify the antigenic proteins. results: data are expressed as means .+ sd. the antibody levels in patients of group i (63• was statistically higher (p=o.o05) than in the 3 control groups (respectively: 29+25, 32_+25, 31_+23). using western-blot method, the intensity of the response was roughly superposable to levels obtained by elisa and the profiles were different according to the prevotella species. the occurence of a np with anaerobic bacteria (prevotella species) isolated from psb leads to an antibody response which seems specific of the prevotella species isolated. fever is common in the intensive care unit, but is not always related to an infection. we sought to define the epidemiology of febrile patients in a general medical/surgical icu. methods: we prospectively analysed the source of fever (t >38.2 ~ c) in all adult patients admitted for >-48 hours in the icu during a two month period. these patients were studied for 14 consecutive days. and werc classified in 3 groups according to the evidence of infection (center for disease control criteria) after complete evaluation: documented infection: cdc criteria + isolation of pathogen (d); possible infectron: cdc criteria without isolation of pathogen (p); unlikely infection: patients who did nol meet the cdc criteria (u). results: of a total of 208 patients studied, 74 dec'eloped fever (35 6%). including (after complete evaluation) 39 d, 15 p and 20 u palients. both the highest temperature in tile first day of fever and the maximal temperature were higher in d than in u (38.7•176 versus 38.5•176 and 39.2-~0.9~ versus 38.64-0.4, respectively p= 0.05 and p= 0.003). most common sources of infection in d were the lungs in 25 patients (64%) and urina .ry tract in 4 (10%). 14 of these patients had positive blood cultures (36%). the overall mortality was 27% (23% in d, 40% in p and 25% in u. differences ns). antibiotics were given in 100% of d, 73% of p and 15% of u (3 patients). in p there was a non significant lower mortality." in patients who received antibiotics (3/11 (27%) versus 3/4 (75%) patients, respectively). conclusions: in febrile icu patients both the highest first day" temperaturc and maximal temperature are significantly higher in infected than in non infected patients, but the differences are too small to be useful clinicall). mortality rate is not significantly influenced either by the presence of an infection or by the administration of antibiotics, obiective: retrospective study to determine the influence of candida infection on icu outcome. methods: 126 patieet with a stay of more than 7 days in inteaasive care were screened for candida infection. 70 patients were treated with antifungal therapy due to either an increased antigen titre of -> 1:8 or clinical evidence of candida colonization. serological candida-antigens (ramco, pastorex) and antibody titres (hemagglutination, lgg-, igm-elisa) were examined routinely. seroconversion was defined as a threefold increase of antibody titre or a titre of 1:640 or higher. results: the median length of stay was 37 (ranging from 8 to 132) days, the mean apache ii score on admission was 18 (+_ 5.8 sd) points. of 126 patients 31 patients died (24.6%). in the group treated with antifungnls (71 patients) 19 patients died (26.7 %). although of the 126 patients only 51 (40.4 %) developed a candida infection as defined above the mortality in the group that showed signs of infection was significantly higher (37.2% vs. 14.6%, p < 0.05 [chi-square-test]). in 34 patients an antigen concentration-> 1:16 was measured. seroconversion was found in 41 patients. the most common fungus was candida albicans (66.4 %). furtberm0re, candida glabrata was found in 21.1%. most of the patients were treated with 2 x 200 mg fluconazole (66 patients). in 38 patients therapy was changed to amphotericin b/flucytosine. in 5 patients therapy was started with amphotericine b and flucytosine. in 40 patients a threefold decrease of candida antigen titre was found. 27 patients showed a decrease of candida antibody titre. conclusions: meticulous screening for eandida infection seems to be necessary since the number of patients with fatal outcome is significantly higher in the group with signs of fungal infections and thus requires immediate antifungal treatment. objective: early diagnosis of patients with ventilator-associated pneumonia (vap), and subsequent identification of causative microorganism, and selection of the appropriate therapy are critical important points that affect morbidity and mortality. the results of the quantitative bacterial cultures are not available for at least 24 hours, while a two hours period, since the specimen are obtained is enough to know the gram stain results. the aim of this study is to determine the usefulness of gram stain in specimens obtained by bronchoaiveelar lavage (bal), through the bronchoscope. material and methods: we studied 47 patients (36 males and 11 females, age 49 + 23) with suspected ventilator-associated pneumonia. the bal gram stain was considered positive when the specimen after a centrifugation at 1500 rpm for 15 min revealed: i) more than 20 leukocytes per optic field, ii) squamous epithelial cell less than 1 percent and iii) one or more microorganisms per optic field on 1000 magnification. all patients had been receiving antibiotics, with no change during the last 3 days, prior to bronchoscopy. results: 8 patients had vap and 39 patients did not. in 5 cases the bal specimens (quantitative bacterial cultures) established the diagnosis of vap in the remaining three patients the vap diagnosis was established by other procedures (blood or pleural fluid culture, clinical outcome, autopsy). apache fl score in patients with vap was 15,7 -+ 5,5, while in patients without vap was 17,9 + 6,4. there was a significantly higher incidence of vap in patients who had i) coma (gcs <8) and ii) been receiving neuromuscular blockade (p<0.05) . the sensitivity of the gram stain for vap diagnosis was 75%, the specificity 89,5%, the positive predictive value 60%, and the negative predictive value 94,6%. conclusion: our data indicate that the gram stain of bal specimens is useful for the early diagnosis of vap and the subsequent administration of the appropriate treatment. the role of anaerobes in mechanically ventilated patients with pneumonia (mvp) have been poorly investigated aim of the study : analyse the prevalence of anaerobic isolation in mvp. methods : between october 1992 and february 1995 all suspected mvp were investigated using protected specimen brush (psb) technique. brushes were rapidly transported in shaedler broth to laboratory. a special care was tooken for anaerobic isolation. results : among the 153 psb performed for suspected mvp (132 nosocomial and 21 community-acquired pneumonia), 81 yielded at least one micro-organism (positive psb : 53%). 63 of positive psb demonstrated only aerobic bacteria and 18 (23%) yielded with anaerobes. in 14 out 18 patients, anaerobes were associated with aerobic bacteria. anaerobes were mostly isolated in nosocomial pneumonia (17/76 positive psb). 27 strains of anaerobes were isolated. prevotella species represent 19 out these 27 strains (70%) the most frequent anaerobic species were prevotella oralis (6) p. intermedia (5) and p. buccae (4). comments:using adequate methods, anaerobic bacteria are frequently isolated in mvp. it could be off importance to take in account anaerobes in the choice of empirical antibiotic therapy in mvp. objectives: the majority of patients with multiple trauma are considered immunocompromised. the aim of this study was to identify risk factors of pneumonia in mechanically ventilated patients with multiple trauma or after surgery. methods: in this prospective study we studied 64 multi-trauma patients (mean age 58 + 19 years, apache ii 16.5 + 6), admitted to a general intensive care unit (icu). all patients were intubated and mechanically ventilated. we were considered that a patient had ventilator associated pneumonia (vap) when the specimens of bronchoalveolar lavage (bal) or protected specimen brush (psi?,), ebb'ned through the bronchoscope, had one or more microorganisms in concentrations greater than 105 and 103 cfu/ml respectively. all patients had been receiving antibiotics, with no change during the last 3 days, prior to bronchoscopy. results: 14 patients had vap, and 50 patients didn't. in the bivariate analysis, the glasgow coma scale (gcs)<8 (x2=4.15, p<0.05), the administration of neuromuscular blockade (x2=7.9, p<0.05), the duration of mechanical ventilation to be greater than 5 days (x2=5.5, p<0.05), the flail chest (x2=4.1, p<0.05), the parenteral nutrition (x2=5.6, p<0.05), the ards (x2=3.9, p<0.05), the abbreviated injury scale (ais) of more than 4 for thorax (:,:2=5.9, p<0.05), the pneumothorax (x2=5.1, p<0.05) were statistically significant related to development of vap. in multivariate regression analysis, using the stepwise technique, three of the seventeen studied factors showed to have an indepantent association with the development of vap:the administration of neuromuscular blockade (f: 4.8, p<0.001), flail chest (f:3.0, p=0.003), and gcs (< 8) (f:2.1, p= 0.039). conclusions: in patients admitted to icu for multiple trauma or major surgery, the administration of neuromuscular blockade, the flail chest, and the gcs (<8), in the population under study, were the indepedent risk factors for vap. mof is a sereous complication of differem states: infection, sterile inflamation, extensive fissure injure, intoxication, ets. there is close correlation between extension of mof and death, developement of nasocomial infection. immunologic disfunction. in order to prgnose probability of risk of mof development among the patients with sepsis and septic shock, we achived an eqation, allowing to recive a coeficient, closely connected with this probabiliti. we have used retrospective analisis of 160 cases of sepsis. diagnosis of sepsis was based according to bone's criterions of sepsis. mof was assessed as disfunction of 2 or more systems according to bone's classification of mof. having used correlation analisis we have estimated factors which have had high correlation coeficient with the probability of development of mof. there were: apache-ii score points, evidenceof septic shock, endocrinopathy. with the help of multyple regression analisis we acheved next equation: y= 0,2042 + 0,0243x~ +0,2931x 2 +0,1504x3 , were x i-apache-ii score points, x2-evidence of septic shock, x3-endocrinopathy. the explanatory power of this quation was evidenced by roc of 0.88, se (v 0 -0.033 introduction: the presence of liver dysfunction in the process of multiple organ failure is associated with an adverse outcome, particularly when it becomes progressive to liver failure. disturbances of liver function may occur early and their detection may be of significant importance for the further development of organ failure. routinely used liver function tests appear to be inconsistent indicators of hepatic damage. in this study, we used p_lasma disappearance rate (pdr) of indocyanin-green dye (icg) as an early estimate of liver function. methods: we serially evaluated pdr and routine liver function tests (serum bilirubin, sgot, sgpt), as well as acute phase and non-acute phase proteins (crp, transferrin) in 26 patients during the first week after trauma or the onset of sepsis. patients: group 1: (n = 11) multiple trauma iss > 30, group 2: (n = 15): abdominal sepsis, acute necrotizing pancreatitis (anp) grade iii. patients were selected on the basis of clin4cal estimates that these patients would require continued icu observation. pdr was determined by means of a fiberoptic catheter and a computerized system (cold z-021, pulsion), which permits repeated bedside measurements. the initial values of pdr, serum bilirubin and transaminases were not significantly different in trauma, sepsis and anp. in trauma patients pdr improved during the first week. in patients with sepsis and anp pdr remained low and worsened with time. the decrease in pdr preceeded an increase in biochemical liver function tests in these patients. 1+4.4 110&-_11 7(4-9) discussion: routinely available blood tests of liver function are usually altered several days after injury. however, they are generally non-specific indicators and they are influenced by extrahepatic factors. pdr seems to be useful to evaluate impaired liver function early after the onset of sepsis and trauma. objectives: to study frequency of organ system failure (osf) and it's influence on outcome in granulocytopenic patients with hematological malignancies and septic shock(ss). materials and method: retrospective review of medical records of 52 granulocytopenie(wbc<0,5xl09) patients with hematological malignancies and ss, who were admitted to the intensive care unit (icu). frequency of osf before and after ss was analysed. the patisnts were categorised on survival and non-survival. results: signs of osf were observed in 28.8% of patients before ss and in all patients after ss. only 3 patients presented with hypotension refractory to inotropic therapy. nevertheless there was a significant increase of frequency of acute respiratory failure (arf), acute renal failure (arenf) and liver injury (li) after ss occurred(showed on the figure). only 5 frequency of organ failure before and after objectives: statusmetria allows to define the effective level of oxygen status and accordance to it means of carbon dioxide and elec-trolyte8 in critical care. the conception of syndrome int~ive care (sic) is exhausted itself and invariable outcomes of sic of multiergan system failure (mosf) confirms that. therefore, an alternative to sic should be advanced. methods: efficlenoy of treatment has been asscsaed in 257 patients with mosf using value of metabolic rate and ability of an organism to cover it by oxygen and substrate supply. oxygen pulse (op) and index of efficacy of oxygen transport (ieto2) was monitored. ~lt~.lntenaive care is considered to be homeostasis-securing therapy (hst) if energostructure deficit is eliminated and necessary for recovery regeneration rate is .restored. op in patients with mosf was 0.8 mt-m "2, and le,~ and ie'i~ w~ 2.4 units in sic. we managed to maintain op of 1.0-1.7 ml.m "2 and ieto2 of 1.9-2.6 units in hst. 41 patients from 135 with mosf survived in sic and 96 patients from 122 survived in hst. efficiency of hst appeared to be two times as much as efficiency of sic. cr of homeostasia-se-'uring therapy is advancing. the conception provides restoration of regeneration rate due to effective then in sic elimination of en=gostructure deficit. the conception may be a basis of new technology for treatment of mosf. helen f goode phd, nigel r webster phd. anaesthesia & intensive care, university of aberdeen, ab9 2zd, uk. objectives: xanthine dehydmgenase is converted under conditions of ischemia, reperfusion and endothelial damage to xanthine oxidase, with superoxide anion as a co-product of its catalytic activity. multiorgan dysfunction syndrome is associated with splanchnic vasoconstriction resulting in significant and prolonged gut ischaemia. aggressive volume resuscitation with prompt restoration of blood flow results in reperfusion of the tissue and is likely to cause xanthine oxidase-mediated release of oxygen-derived radicals. this study investigates xanthine oxidase activation and oxygen-derived free radical-mediated damage in such patients. methods: fourteen consecutive patients on itu who met established criteria for septic shock and secondary organ dysfunction were studied. serum xanthine oxidase activity was measured using oxidation of a chromagen in a dual enzyme system and plasma malondialdehyde was measured using a specific spectrephctometdc assay. apache ii scores, blood pressure, svr, cardiac output and 28 day survival were also recorded. biochemical data were compared with results from 20 healthy subjects. results: xanthine oxidase activity was 6.30 + 1.59 units/i in patients (mean :t: sem) and 0.74 + 0.12 units/i in controls (p<o.01, mann whitney u test), and was highest in the 6 patients who survived (p<0.05). malondialdehyde concentrations were elevated (0.81 • 0.11 prnol/i compared with 0.29 • 0.05 pmol/i in controls, p<0.001). xanthine oxidase activity did not relate to apache score or any of the cardiovascular parameters recorded, and was not influenced by the degree of organ dysfunction. conclusions: patients with sepsis and secondary organ dysfunction have xanthine oxidase activation and evidence of free radical damage. the finding that activity was highest in those patients who survived suggests that reperfusion was incomplete in patients who died, with decreased tissue xanthine oxidase ~tash out' into the circulation. influence objective : evaluation of the feasibility of measuring the intragastrie partial pr~sure of carbon dioxide (pco2) using a chemilumin~nt optode and fibre~ptics originally developed for intrav~cular blood gas monitorlng(p7) methods the pco 2 electrode w~ calibrated i~-vitro according to the m~ufaeturers instructions the sensor w~ then stripped of its outer c~ing and threaded down the pile tube of a g~ c enema e (gt) so ha he ens ng portion sat well within the balloon. the modified gt was inserted n~og~trically after induction of anesthesia in ten patients undergoing c~diopuimonary byp~s throughout results: according to the datas of integral rheography 70% of patients were revealed to have hypodynamic type of blood circulation (cardiac output was decreased on 35-40% and general peripheral resistance increased on 80-100%). it was effective to use complamin (xantinoli nicotinas) in the complex therapy of su~h patients. the dosage was 10-15 mg/kg during 4-6 days. as a result of such therapy was also the oliguric stage shortening (ac n 1464083).in 9,6% of the cases unsatisfactory datas of central hemodynamic,combined with high (more than 15 cm h20) venous pressure,lung oedema.then nanipruss (0,5-1,5mkg/kg/min) was succesfutly used in complex with routine therapy.in the patients with low cardiac output and low general peripheral resistance mesaton (0,25-0,5 mg/kg) and dopamine (5-10 mkg/kg/min) were effective.change for the worse in the hemodynamics datas, inspire of therapy during 5-7 days,was prognostically unfavourable. conclusions:we consider early diagnostic of hemodynamic disorders and adequate correction of them is one of the most important factors, determined the outcome and prognosis in arf patients. obiective: to evaluate the results of renal replacement therapy (rrt) in surgical icu patients with acute renal failure (arf). arf in icu patients is associated with high mortality rates. we investigated the relation between mortality and organ failure in 4 different categories of surgical icu patients receiving renal replacement therapy (rrt) for arf. methods: the records of 76 surgical icu patients with arf requiring rri-(haemodialysis or cavhd) were examined. five different patient categories were defined; 1 .ruptured aneurysm of the abdominal aorta (raaa) n =20 2.elective vascular surgery (evs) n =20 3.abdominal sepsis n -17 4.trauma n = 8 5.others ( e.g. malignancy, obstetric emergencies) n-11 seven organ systems (cns,circulatory,respiratory,hepatic,renal,digestive,haematologic) were scored for possible failure using the mof score. results: mortality was as follows: all patients 72% , group 1 75% , group 2 70% , group 3 88% , group 4 38 % , group 5 75 % . mortality increased with the number of failing organ systems; mortality for all patients with > 4 failing organsysterns was 80% the only exception being the subgroup of trauma patients where mortality under these circumstances was 5o% conclusions: mortality in surgical icu patients receiving rrt for arf is high. no significant difference in mortality is found between raaa and evs. mortality increases with the number of failing organ systems. the subgroup trauma patients shows a lower mortality compared to the group as a whole, even with > 4 failing organ systems. to look for the most accurate scoring system to measure the severity of the complications occuring in the early phase ( first 30 day) of kidney transplantation and to asses their prognostic value. methods: in our retrospective study we applied the apache li and the goris scoring system for the kidney recipients who developed multiple organ failure (mof) as a consequence of their pulmonary and. cardiovascular complications following kidney transplantation. we evaluated the recipients the distribution of the women and men ( 60% ~ 40 % ) was the same as in the kidney recipients. applying the apache ii system most of the patients had their score between 10 and 19, and the function of 2,3 or 4 organs were affected at the time of the onset of mof. the apache ii system gave adequeate information about the disturbance of the function of other organs beside the kidney failure even at the time of the transplantation. the scores and the number of the affected organs correlated with the condition of the patients in the goris scoring system but not as sensitively as in the apache ii scoring system. conclusions: both the goris and the apache ii scoring system can be applied to measure the severity of the multiple organ failure occuring during the early phase of kidney transplantation. however the apache ii system is more suitable to follow not only the stateof the patients at the time of the admission but also the changes occuring in their condition during the complication. v.v.erofeev, v.v.ivleva scientific research institute for general reanimatulogy russian amsci, moscow, russia objectives: the analysis of ssc and results of their treatment in patients following critical states showed the necessity of developing a combined antibacterial therapy. methods: according to the protocol 97 patients (18-60 years old) with combined trauma and massive hemorrhagy following vast aml traumatic operations were examined. microflora's composition and resistence to up-to-date antibiotics was studied using the anaiyser iems reader by "labsisteme"(finland). general clinical, bacteriological, immunological indices, as weil as the duration of the treatment and recovering rate served as criteria of the combined antibacterial therapy effectiveness. results: it was proved expedient to administer antibiotics in staphylococcus infection in the following combinations: riphampizin with fluoroquinolones; i-ii degeneration, cephalosporins with aminoglycosides; cephalosporins with fluoroquinolones. in case of singling out the exciters of the euterobacteriaceae family, including the pseudomonas aereginosa, -fluoroquinolones combined with modern amynoglycosides; fluuroquinolones with ureidopenicillines; ureidopenicillines with amynoglycosides; amynoglycosides with the ii-iii generation cephalosporins; cephalosporins with fluoroquinolones. in severe ssc caused by combined infection (including anaerobes) clindamicin with modern amynoglycosides was prescribed. conclusion: the combined antibacterial therapy allows: 1) to increase the effect on microbic agents and the efficacy of treatment in combined infections; 2) to lessen the possibility of the exciters'resistence to antibiotics; 3) to prevent the development of superinfection: 4) to decrease the doses of medicine and its toxic effect. objectives: two methods of blood volume measurement in a group of critically ill patients were compared to investigate the practical possibilities of a new easy to use method based on carbon monoxide (co) uptake. methods: all patients had multi-organ failure and haemodynamic monitoring with a swan-ganz catheter. mean apache ii score was 19 (10-25). when indicated, 9 patients had blood volume measurements simultaneously based on the techniques of, i) dilution of 5~cr labelled red cells, and ii) inhalation of carbon monoxide gas with measurement of the rise of carboxyhaemoglobin produced. the co was administered via a newly designed, ventilator driven, fully closed circle system ensuring co retention and co2 removal with automatic addition of oxygen to m}ttch patient uptake. a portable computer performed all necessary calculations. results: volumes obtained by co uptake were compared with the "gold standard" radiolabelling method. mean blood volume determined by the co method was 6310ml (4710-7959ml) compared with 4690ml(3755-5778ml) with slcr labelled red cells (r=0.9). regression analysis produced an intercept at 769ml. the slope of the regression line was 0.62 (0.33-0.9, 95 % confidence limits). discussion: the co method produces volumes in excess of the radiolabelling method. there appears to be a systematic error, and one possible explanation is co binding to substances other than haemoglobin. conclusion: the co method is easier to use than radiolabelling and of the lower cost, since cohb measurement only is required. aceuraey is sufficient for clinical use and our preliminary findings suggest this system will meet the requirements. objectives: this study was conducted to determine the role of nitric oxide (no) in the pathophysiologic alterations and multiple organ damage, and the possible effects of " " " (l-n -monomethyl-l-arglnlne nmma) on hemodynamics and mortality in rats caused by a prolonged hypovolemic insult. methods: a prolonged hemorrhagic shock (30-35 mmhg for 180 rain) was induced in anesthetized rats followed by adequate resuscitation. l-nmma was administered intravenously at doses of 2.0 mg/kg or 20.0 mg/kg at the end of resuscitation. results: infusion of 2.0 mg/kg l-nmma diminished the fall in mean arterial pressure, significantly increased the cardiac index (ci) and stroke volume (sv), together with remarkable protection from multiple organ damage compared to the controls. the 48 h survival rate was significantly improved from 26.7% in the control group to 68.8% in the treatment group (p<0.05). in contrast, the high dose of 20.0 mg/kg l-nmma resulted in a strong blood pressure response but a marked reduction in ci and sv concomitant with an increased total peripheral resistance index within the observation period, and caused severe damage to various organs at 2 h after treatment. in addition, marked elevation in both endotoxin and tnf levels were observed in animals subjected to shock insult. conclusions: these results suggest that no induced by hemorrhagic shock in rats is an important mediator for pathophysiologic alterations associating with cardiovascular abnormalities, multiple organ dysfunction, and even lethality. thus, regulation of no generation and use of no inhibitors might provide new aspects in the treatment of hemorrhage related disorders, and the use of l-nmma would be either deleterious or salutary in a dose dependent manner. (hebert, chest-1993) . the purpose of this study was to assess the risk factors for hepatic dysfunction in mosf. methods: 733 patients have been hospitalized in our icu from january 1992 to may 1. 994, 198 (27%) with mosf. among mosf pati~ts, 57 (29%) have had hepatic dysfunction defined according to hebert (bilirubin ~ 60 ttmop1, chest 1993). thirty six of these 57 patients acquired hepatic dysfunction after admission in the icu. these 36 patients were compared with 36 mosf patients without hepatic dysfunction selected blindly. chrorfic diseases, severity scores, eanse of admission, clinico-biologieal and hemodyunrrfic parameters, use of vesopressors, use of hepaiotoxic drugs, use of nutritional support and mortality were compared for hepatic failare and non hepatic failure groups.twenty nine patients had postmortem hepatic histologic examination, results: univaciate analysis: only parameters with p _< 0.05 are pre~nted. including these paramet~'rs in a multivariate analysis, anly c~hosis and vascular surgery remain independent risk factors for hepatic dysfunction. in particular, pao2/fio2, arterial lactate, do2 were not different between the two groups, some de~'ee of histological abnormalities was found in all liver samples, despite a normal bilirubin level in 15 % of the cases conclusions: in our patients, conu'ary to previous studies, hypoxic and hemody~anfic parameters were not independent risk factors for hepatic dysfantion. this might be due to the inadequacy of the usual biologic definition of hepatic dysfunction as well as to the poor sensitivity of general hamodynamic parameters. critical states of various origin are complicated with the mldtiorgan farm (moi~ oceuzr~ce. due to their and functional features the lungs become the primmy damage target in various critical.states. ard8 that occurs in such states is associated with pulmonary edema development because of capillary permeability increase mediated by humeral and cenular responses to 0amag/~5 factors exposure. r nmst be emphasized that mediators and effecto~rs of this respo~e affect not only puknonary capillaries, but other organs capiu~es as wellenhancing their permeability. orsans edema is a conmm~ finding at the autopsy of patients died from mof.clinical and radiolosial findings allow to have a diagnosis of pulmonmy edema before ~mi!ar lesions in other organs occm. additionally, there are some techniques that permit quantitative assessment of pulmonary edema flv.id (evlw) volume. in conclusion, we suggest that evlw changes in .dyn~rmcs in patients with mof are considered as a critical state severity measure which reflects indirectly the edema in other organs. objectives: we compared three different dialysis membranes to find out whether or not there were differences between their clearance characteristics on substances such as inuline, creatinine, urea, and phosphate to be eliminated in acute renal failure (arf). moreover, if a loss of clearance did occur we were interested in whether this was due to heparinization and a high production of the thrombine-anti-thrombine-complex (tat). methods: we carried out a randomized controlled study on 13 consecutive critically ill patients presenting with arf, most of them in association with multi-organ failure, to be treated by continuous pump-driven arterio-venous renal replacement therapy on continuous low-dose heparinization. three different types of high-flux filter membranes (f 60 tm [fresenius] , ct 190 tm [baxter] , and filtra116 tm [hospal]) were assessed. each filter was changed intentionally after a 24 hours" use. together the data of 54 filters were evaluated, each at three different times (immediately after its onset [0 hi, after 8 h, and after 24 h). the clearances of creatinine, urea, phosphate, and inuline were measured. results: there were some significant differences in clearance characteristics of inuline, creatinine, urea and phosphate between the filters (p<0,05) showing the f 60 tm membrane excelling filtra116mand ct 190 tm the more. the loss of inuline clearance (3 mi/min/m 2) after 24 h, however, was insignificant for all 3 filter types. a continuous low-dose heparinization scheme was applied without any relevant prolongation of the aptt. even lower losses were noted for the clearances of creatinine, urea, and phosphate. we found the tat-producfion increased after 8 h (p<0,05), but it did not rise any further. conclusions: as we could demonstrate in our study the clearance data of different types of filter membranes applied during continuous renal replacement therapy do show significant differences. on the other side, no relevant loss of clearance occurs during a 24 hours" period indicating a high efficiency over time. to consider commercial aspects as well it shows that inexpensive conventional filter membranes can successfully be applied even for a longer renal replacement period, if needed. a retrospective study was performed on 100 patients with acute renal failure (arf). we analysed survival in continuous (cd) and intermittent dialysis (hi)). mean age of the patients was 60 years (y), 57 patients (57 %0) were <65 y, 43 patients (43%) were >= 65 y. the incidence of dialysed arf in our mixed intensive care departement is 3%/admission/y. statistics: fischer's exact test, mann-whitney-u test. efioloev: the contribution sepsis, cardiac failure and aminnglycosidcs was respectively 70%, 44 % and 35 %. treatment: cavh (cd) or cvvh (cd) was used in 40 patients (40%), hemedialysis (hd) was used in 60 patients (60 %). data: mean apache 2 scores were the same for cd and hd (27 for both groups), patients treated with continuous dialysis techniques had significantly (p<o,05) more need for ventilation (100 % vs 70 %), elevated bilirubin (82 % vs 57 %) and a higher vasopresser need (89 % vs 46 %@ than patients treated with hemedialysis. there was a trend towards more sepsis (83 % vs 62 %; p=0.06) mad coaguiation disorders (50 % vs 27 %; p=0.07) in cd. taere were sign/ficantly more younger patients (<65 y) treated with cd (70 % vs 30 %, p<0.os). mean apache 2 score was significantly lower in patiants<65 y than as patienta>=65 y (26 vs 30; p<0.05). patients<65 y had significantly (i}<0.05) more coagulation disorders (53 % vs 17 %) and elevated bilirabin (81% vs 52 %). there was no significant difference in vasopressur need and ventihatio~ between age groups. outcome:. hi) had a better sr compared to cd (43 % vs 15~ p<0.05). patiants>=65 y had a comparable sr vs patients<65 y (3") */e vs 28 %; p----a.s.). tha global survival rate (sr) was 32 % (32 patients). conclusions : diaiysed arf has a well known lowsurvival rate (32 %): hc~raedialysed patients had a better survival rate than patients treated with continuous dialysis. this can be explained by the fact that the latter were in a worse condition considering organ failure (more vantilatian, elevated bflirubin and need for vasepressurs), apache 2 score couldn't illustrate that. patient~65 y with arf have the same survival rate as patients<65 y: although patients >=-65 y have a higher apache 2 score they have less organ faille. the avacbe 2 score is not a good oredictor of survival in p with organ failure. departments of surgery and intensive care, guy's hospital, london, u.g-obiectives: a randomised controlled trial of a management protocol utilising the regular measurement of gastric intramucosal ph (phim) to control the administration of dopexamine. methods: patients admitted to a multidisciplinary teaching hospital intensive care unit (icu) undergoing insertion of a pulmonary artery catheter were managed according to a resuscitation protocol. randomisation was to either the protocol alone or to insertion of a nasogastric tonometer and subsequent management guided by phim. phim < 7.32 initiated volume and inotrope resuscitation and, if unsuccessful in elevating phim, dopexamine was commenced. approval was obtained from the hospital ethics committee. results: 94 patients were considered for analysis and the two groups were well matched for age and sex. overall, there was a high hospital mortality of 64.9%. there was no difference in icu or hospital mortality between the two groups (see table) . objectives: to compare cardiac output (co) measurements between continuous termodilution (cco) by thermal wire on pulmonary artery catheter (cco/svo2 vigilance. baxter critical care), and co measurement using a trans-esophageal doppler (dco) ultrasound system (odm ii, abbott laboratories), in the immediate postoperative period of cardiac surgery. methods: 15 patients undergoing myocardial revascularization were monitored with cco by a swan-ganz catheter and an intra-esophageal dco probe, after induction of anesthesia. exclusion criteria were: aortic valve disfunction, previous valvular surgery esophageal disease, absense of sinus cardiac rhythm, and need of ventricular or intraaortic assistance. hemodynamic parameters, co by both cco and dco, svo2. sao2, diuresis, pha, and hemoglobin were repeatedly registered during the first 6 hours after surgery, as the patients were kept under sedation and mechanical ventilation. results were compared using the method described by bland and altman. results: 176 measurements of co were obtained, ranging 2. objectives: a decreased tissue oxygen delivery is responsible for a higher morbi-mortality rate among surgical patients; this diminished oxygen delivery/consumption rate (dojvo2) may origin the lactic acidosis observed in the gastrointestinal tract, reported in patients undergoing hypothermic cardiopulmonary extra corporeal surgery, and can be registered by tonometry as result of the gastric mucose ph. the purpose of this study is to evaluate the reliability of the intramucosal ph (phi) measurement by a nasogastric catheter as indicator of the do2/vo > its co> relation to other parameters of do2/vo 2 disturbance, and with postoperative complications and clinical course. methods: 20 patients (16 male, 4 female) undergoing cardiac surgical procedures were included (16 myocardiai revascularizations, 3 valvular substitutions, 1 constrictive pericarditis). mean age was 63 + 12 years, mean weight 70 _+ 10 kg. a nasogastric probe (trie tonometrics) was placed after anesthesia induction; phi values were registered in the postoperative period (90', 120', 240", 360' and 18 h after surgery end). the corresponding hemodynamic parameters, venous oxygen saturation (svo2), diuresis and arterial ph (pha) were also recorded. results: phi values ranged 7.20 to 7.65 (mean 7.40 (0.8); the mean values of clinical evolution were: extubation time, 20 _+ 12 hr.; discharge from postoperative care unit, 88 4-50 hr.; and hospital total postoperative time, 10 _+2.2 days. complications registered were: 2 perioperative acute myocardial infarctions, 2 cases of respiratory insufficiency, 1 occlusion of coronary bypass, an 1 ease of hyperamilasemia. all patients with severe complications needing specific treatment showed either a low phi value, or a considerable descent in comparison with the initial register. statistic correlation between low phi and presence of complications was found; the low significance (p > 0.05) degree may be due to the low population size. conclusions: phi measurement in cardiac surgery patients is a non invasive, uncomplicated method for prediction of doz/vo 2 disturbances, thus reflecting risk of increased major complications, and may precede changes in other usual indicators (svo2, pha, cardiac output, ...). work-in-progress with a greater population size may offer more significant results. references: (1) gutidrrez g: lancet 1992; 339:195-202. (2) landow i: acta anaesthesiol scand 1994; 38: 629-639. the haemoglobin-level (hb) is besides the arterial oxygen saturation and the cardiac index one of the relevant parameters of oxygen supply to the tissue. in contrast to otherwise healthy patients, there is no agreement on tile so-called transfusion-trigger in critically ill patients. in i?ont of this background the question arises, whether and to what extent blood transfusion in critically ill patients improves oxygen supply io tile tissue. this study was performed in 34 critically ill/septic patients in the postoperative period alier an inlcclive/scptie revision operation of the hip or knee joint. on cardiac/seplic reasons monitoring consisted beside other measures of a pulmonary arlery catheter and of an indwelling arterial line li~r measurering/calculating standard haem~dynamic as well as systentic oxygen parameters. the indication for blood transfusion was given by hb together with the cliuical slatus of thc patienl (asa-scorc and multiple organ dysfunction (moi))). statistical analysis w~ks performed by mann-whitney-u-test. by fisher's exact-test and by wii.coxon-test: statistical significance was set with p<0.05. according tu the pretransfusion value of hb and of lactate (lac) palicnts ;,,'ere divided into groups as follows: a: hb<8 and b: >sg/dl: i: 1ac<2.8 and ii: >2.smm. in either group blood transfusion results in zt significant increase in hb (a: 7.5_+0.4 to 9.4+0.8 g/dl; b: 9.(~0.8 tt, 10.5+0.09 g/dl; i: 8.0-+1.0 to 10.2-+0.6 jdl; i1:8.4-+0.9 to 10.1+0.7 g/dl). wlailc, however, haemodynamic parameters do not difl)r significantly from each other before and alter blood transfusion, oxygen delivery (do, -ml/min x m-') increases significantly hi either group studied (a: 467-+86 to 581-+158; b: 521+125 to 577+137; 1:512 -+1 13 to 599-+141; i1:516-+123 to 632-+214), in contrast oxygen consumption (vo~ -ml/min x m e) does not change significantly in either group (a: i68-+148 to 158-+38; b: 180-+162 to 175-+52; i: 171-+38 tu 179-+35; 11: 199-+60 to 219+_71); oxygen exlraction ratio decreases. this study in critically ill/septic patients demonstrates, that in this group of patients studied blood transfusion at a base-line-value of >7.5-+0.4 g/dl expectedly rises do~, however, it does not improve vo=; even not in septic patients with elevated lac-values. paclitaxel in a new anticancer agent, extract from the bark of the yew tree (taxus brevifolia), employed against breast and ovarian cancers resistant to chemotherapy. it promotes the polymerization of tubuline, and disrupts the normal microtubule dynamics. hematologic toxicity, hypersensitivity reactions (bronchospasm, urticaria and hypotension), and peripheral neuropathy are the main reported toxic effects. cardiac side effects are rare: atrioventricular blocks of higher degree are reported in 0.1% of patients; congestive cardiotoxicity was discussed only in one trial in patients treated with paclitaxel and doxorubicin. we describe the history of a 48-years-old worn an with a breast cancer, diagnosed in 1989, initial staging t3nim0, treated with mastectomy, axillary lymphadenectomy, andchemotherapy with a cumulative dose of anthracyclines of 678 mg/m2 until august 1994. the patient complained of dyspnea and severe hypotension immediately after an intravenous infusion of 100 mg paclitaxel, given over 1 hour for the treatment of bilateral, malignant pleural effusion. at echocardiography die left ventricular ejection fraction was reduced to 20%. she died 20 days later because of a severe cardiac low output with hepatic and renal failure; an impressive hepatic cytolysis was observed. the post mortem examination confirmed the dilatation of the cardiac cavities, especially of the right ventricle, bilateral pleural fluid, and ascites. the histology was suggestive for a cardiomyopathy secondary to anthracyclines. the electron microscopy revealed a deposition of an unusual pathological pigment in the myocytes; subsarcolemmal deposition or membranous were absent. we hypothesize that paclitaxel was the cause of a major hypersensitivity reaction with shock and severe hepatic cytolysis, worsening the myocardial damage induced by anthracyclines. the possibility that a low doge of paclitaxel could directly increase anthracyclines cardiotoxicity -as decribed in the medical literature -will be discussed. objectives: activated endothelial cells release soluble intercellular adhesion molecule-1 (sicam-1), vascular cell adhesion molecule-1 (svcam-1), and e-selectin (selam-1). sicam-1, svcam-1, selam-1, and inflammatory cytokines were determined. methods: sicam-1, svcam-1, and selam-1 were determined by elisa. tnf-a, il-6, and il-8 were also measured by elisa. endotoxin was measured by an endotoxin-specific endospecy test after pretreatment of new pea method. results: the sicam-1 and s vcam-i levels were significantly higher in the septic multiple organ failure (mof) and sepsis groups than in the non-septic mof group. the selam-1 level was slightly higher in the septic mof group than in the sepsis withut mof group and non-septic mof group. the increases of soluble adhesion molecules were not in agreement with changes of plasma endotoxin level. levels of soluble adhesion molecules were correlated with the levels of plasma tnf-a and il-8, but the level of il-6. discussion and conclusion: the slcam-1 and svcam-1 levels in septic patients closely reflected the severity of the pathophysiological conditon. it was possible that the release of sluble adhesion molecules were not stimulated by plasma endotoxin, but endotoxin in the local infectious region. tnf-c~ and il-8 also were suggested to be involved in the release of these soluble adhesion molecules. obiectives: cardiopulmonary bypass (cpb) surgery is associated with a systemic inflammatory response attributable to the release of various inflammatory mediators and the activation of complement or coagulofibrinolytic system. in addition, adhesion molecules, such as icam-1, elam-1, and vcam-1, appear to be of central importance in the inflammatory process following cpb surgery. we previously reported the effects of a synthetic protease inhibitor, fut-175, reduced release of inflammatory cytokines (tnf, il-lg, il-6), activation of complement (c3a, c4a) or coagulofibrinolytic system (tat, pic, fpa) and protected platelet function (gpib, gpiib/llla) following cpb surgery. methods: in this study, we analyzed fut-175 on soluble adhesion molecules following cpb surgery. 20 patients undergoing cpb surgery were divided into two groups, group a consisted of 10 patients who received 1omg of fut-175 in priming solution, followed by a continuous infusion at 2mg/kg/hr during cpb in addition to initial heparin dose of 3mg/kg. group b, a control group, included 10 patients who were injected with heparin only. the plasma slcam-1, selam-1, and svcam-1 concentration was measured by elisa. results: every soluble adhesion molecules decreased during cpb in both groups, and rose after cpb. selam-1 and slcam-1 reached their peaks on 3 hours after cpb and on pod 1 respectively in both groups, but they remained lower in group a (selam-i: 32.1+11.5 vs. 38.6• ng/ml, p<0.05, slcam-i: 327• vs. 483• ng/ml, p<0.05), svcam-1, in both groups, remained lower than preoperative levels, but did much lower in group a. conclusions: fut-175 reduced adhesion molecules and suggested to be the effect on postoperative organ dysfunction. in the last few :,'ears the conditions of treatment in continuous hemofiltration/hemodiafiltration were discussed controversially. a significant removal of tnf-alpha and il-i could be demonstrated in cvvhd. the aim of our study was to investigate the elimination of tnf-alpha, 1l-2, il-6, il-8, s-cd-14 and ifn-gamma in cvvh by measurement in plasma and hemofiltrate of 10 critically ill patients with an acute renal failure. the patients of our study were treated with a continuous veno-venous-hemofiltration (polysulfone-filter, blood flow: 100-130 ml/h, filtration rate 500 ml/h). the samples, hemofiltrate and plasma, were taken one hour after the start of treatment. the patients suffered from septic shock (4), the so called hepatorenal s~aldrome (3) and a severe pancreatitis (3). the cytokine concentrations were measured with elisa-method. in contrast to elevated concentrations in plasma for tnf-alpha (5 cases), scd 14 (9 cases), il-2 (l case) and il-6 (4 cases), hemofiltrates contained no activities. only il-8 was removed in significant amounts with even higher levels in hemofiltrate than in plasma. this phenomenon was described so far for tnf-alpha and il-1 and may be due to the absence of metabolic properties (possibily enz~natic) in hemofiltrate. it can be shown, that tnfalpha, il-2, il-6 could not be eliminated in cvvh with a filtration rate to 500 ml/h. in contrast to findings of other investigators with a higher filtration rate (> 1000 ml/h), we found no significant concentrations of tnf-alpha and il 6 in hemofiltrate. we conclude, that for a significant removal of important cytokines higher filtration rates (>1000 ml/h) are necessary. objectives: multiple organ dysfunction syndrome including liver and renal impairment is a fatal complication in patients with the diagnosis of sever sepsis. this study focused to the effects of removing toxic substances from inflamnatory tissue by hemodiafiltration. ~4ethods: eleven patients were admitted to the icu in emergency center and met the criteria of systemic inflammatory response syndrome in association with infection. all patients developed liver and renal dysfunction and were treated by hemodiafiltration with high flux membranes (fb-u:nipro). the hemodiafiltration were performed 19 times using nafamostat mesilate as an anticoagulant in 5 hours with 12 l of substitution fluid (hf-b:fuso). the serdm levels of endotoxin, cytokines, endothelin-i (et-]), human neutrophil elastase ~1-proteinase inhibitor complex (hne-pi), fibronectin (fn), lactate, and amino acids were measured before and after the hemodiafiltration. the hemodiafiltration would be effective to renal dysfunction by reducing endothelin and beneficial to tissue metabolism represented in fisher's ratio, but might be harmful to respiratory function by activating neutropila in patients of severe sepsss. background : intermittent hd may be poorly tolerated in the early phase of arf in hemodynamically unstable patients (pts). this technic may fail to achieve steady state urea low levels in hypercatabolic pts. method : nt = 25 consecutive pts treated with hd; n 2 = 25 consecutive pts treated with cvvhf. hemodynamic unstability is defined by arterial hypotension and requirement of inotropie support despite adequate filling. rate of change in urea (u), ereatinin (cr), k + , ph were computed from a linear regression .analysis of data vs time in each treatment group during the 5 first days of application of the two technics (anova). dally worst values were recorded. results : hd-group : apach% score = 22 _+ 7; mean number of organ system failure (osf) = 1.5 -+ 1; mean blood pressure (mbp) = 75 • 22 mmhg (first day of application of hd). cvvhf-group : apachen score : 25 + 6; osf = 3 -+ 1; mbp = 57 + 20 mmhg (first day of application of cwhf discussion : during the 5 first days of application of hd/cvvhf, u and cr decreased much more rapidly in the cwhf-group. k* and ph were maintained within normal range in the two groups. initial mbp which was much lower in the cwhf-group significantly improved during the application of cvvhf while mbp remained unchanged in the hd-group. conclusion : despite higher severity of disease in cvvhf group (apachen score, osf, lower initial mbp), we obtained a better performanco with cvvhf regarding the decrease of u and cr and the improvement of mbp. in relation to the different and continuous renal replacement techniques, the continuous venovenous one is the alternative method to continuous arteriovenous for critical patients with acute renal failure (arf). we present you our experience with cvvh in patients with mof. in our intensive care unit (icu) 20 patients with mof were treated with cvvh in the period between january in 1992 to march in 1995. the mean (• age of our patient population was 52,1• years, being 65% male and 35% female the whole patient population was with mof iust at the moment the technique was accomplished; 75% was in mechanical ventilation, 90% needed vasopressor support and 65% required both of them (mechanical ventilation and vasopressor support) apache ii score mean of the patient population was 17,84~:6,97 (range 5-28) and ati of them were with arf oligoanudc. technique: cvvh was accomplished using a single-d~al iumen catheter, ptaced in either a temoral or subclavian vein by the stand ard seld{nger technique. pol{sultone hemofitiers were also used, and the extracerporeal circuit used standard arterial-venous blcod tubing. blood flow and hence oltrafiltration pressure, within the circuit was generated by a roller blood pump. the modulus has a roller pump, a pressure transducer connected in an arterious and venous line, such as an air-transducer which is adapted to a drip-chamber in the return way. the replacement used was a peritoneal dialysis solution. medicine 1, st. george's hospital medical school, london. england. hepatic sinusoidal endothelium shows a major inflammatory response in porcine sepsis that can be attenuated by the administration of dopexamine hydrochloride. dopexamine is a beta 2 and dopaminergic receptor agonist. the specific beta 2 adrenoceptor antagonist ici 118551 has been shown to reduce the protective effects of dopexamine. we investigated the effect of this antagonist on hepatic ultrastructure in porcine sepsis. six pigs (25-30kg) divided into 2 groups were anaesthetised and intubated. cardiac output and portal blood flow were measured using standard techniques. the 2 groups were; placebo, (peritonitis induced); blocker, (peritonitis induced and 200 pg/kg ici 118551 bolus infused then given hourly). caecal content was aspirated and peritonitis induced. colloid was infused to maintain pawp at 10-12mm hg for eight hours the animals culled, hepatic tissue removed and prepared for electron microscopy. in the placebo group hepatic endothelium was swollen and the sinusoids occluded by wbc. but in the ici 118551 blocker group, much of the sinusoidal endothelium was absent and there where large extra sinusoidal spaces among the hepatocytes. an assessment of the two groups showed worse hepatic architecture in the blocker group. the b2 antagonist blocked any protective effect of endogenous beta 2 adrenoceptor agonist (adrenaline) on hepatic endothelium in porcine sepsis. george's hospital medical school, london. england. dopexamine hydr0chloride, a beta 2 and dopaminergic receptor agonist reduces hepatic damage in porcine sepsis. we tested dopexamine's effect on cerebral oedema. the beta 2 adrenoceptor antagonist ici 118551 was infused to block any protective effect of dopexamine. nine anaesthetised pigs (25-30kg) were randomised into 3 groups; placebo, (peritonitis induced); dopexamine, (peritonitis induced and 5 ~tg/kgdar of dopexamine infused); blocker, (as in dopexamine group but in addition 200 pg/kg ici 118527 bolus given then infused at that rate hourly). caecal peritoneum was induced and colloid infused to maintain pawp at 10-12mmhg for eight hours when the animals were culled, cerebral tissue removed, prepared for electron microscopy and digitisation. digitisation of the area of oedema surrounding the blood vessel and expressed as a percentage of the micrograph. 30.5_+4.1, dopexamine 13.5+2.9", blocker 31.5+3.7. data expressed as mean + sd. significance p<0.05. * dopexamine compared to placebo and blocker. in the dopexamine group the area of tissue oedema was significantly lower than either the placebo or blocker groups. there were no significant differences between the placebo or blocker groups. the 62 antagonist completely blocked the protective effect of the drug on cerebral oedema in porcine sepsis. beta 2 adrenoceptor stimulation is protective of cerebral oedema in porcine sepsis. objectives: the hemodynamie~ of hepatic circulation during multiple organ failure (mof) have not been suffleienly studied. we investigated liver hemodynamics in two subgroups of patients with mof, those with either liver or lungs as the main organ of involvement. methods: three groups of patients were created: i) mof-hepatic involvement (mof-hi) (7 patients) with bilirubin >3.5 mg/dl and lung injury score <1.8, it) mof-ards (9 patients) with respective values <2.0 and >2, iii) 5 patients with head injury with respective values <2 and <1, served as group control. all patients were in haemodynamieally stable state with an oxygen delivery index >300 ml/min/m2 prior to measurements. two swan-ganz catheters 'were inserted, one in the hepatic veins and one in pulmonary artery and the following measurements were determined: the hepatic vein free pressure (hvfp), the hepatic vein wedge pressure (hvwp), cvp, paop and co. the gradient of hvwp-hvfp represents liver perfusion pressures. by injecting contrast media at dose of iml/lokg with the balloon inflated to achieve sinusoidai image, the hepatic blood flow (hbf) was concluded by the time in seconds of media removal after balloon deflation. results: the co, cwp and cvp were comparable to all three groups. namely, for mof-hi, mof-ards and control groups the mean (+sd) value of co was 7.2_+0.8 vs 6.9_+0.3 (ns) and 6.3_+0.6 respectively, of the paop was 8.7+_1.6 vs 10+:3 (ns) and 8.2+3.1 respectively and of the cvp was 12.+.2.3 vs 11.6+2.3 (ns) and 5.8 respectively. in contrast the two mof groups were different after the cut-offinclusion criteria ie the mean (+sd) value for bilirubin was 6.8+9.5 vs 1.20+0.7 (13<0.05) and 0.8_+0.2 respectively and lung injury score was 1. objectives: oxygen delivery (do2) and oxygen consumption (vo2) are increasingly monitored parameters in the icu. there still remain controversies about an oxygen supply dependency in critical illness particularly with respect to vo 2 determination by either indirect calorimetry (vo2m) or tick calculation (vo2c). the purpose of this study was to investigate the changes in vo2m and vo2c following do 2 increase. methods: the relatives of 24 critically ill patients (mean age 63 years, mean apache ii 24, mean mof-score 9) gave their written informed consent to participate in this institutionally approved, prospective study. do 2 was increased by fluid loading (hydroxyethylstarch 10%: mean volmne 750 ml, mean duration of infusion 80 min) and catecholamine support (dobutamine: mean dose 14,3 ~g/kg/min). changes in vo2m and v02c were recorded sinmltaneously before, during and following interventions. calorimetry was obtained with the metabolic monitor 7250 integrated in the ventilator 7200 (puritan bennett, carlsbad, ca adaptive endocrine response of organism to septic shock consisting in activation of the production of adrenal hormons, renin -angiotensin -aldosterone system (raas) and other hormonal systems has an influence over microvascular changes in these states and for development of multiple organ failure (mof). in 25 patients with peritonitis of different origins (18 nonsurvivors and 7 survivors) were followed the changes in cortisol level and raas by radioimmunological methods and many variables for evaluation of respiratory, renal, hepatic function, coagulation etc. as a signs of mof. it was observed significant increase of the level of cortisol (1099 +_ 4,83 nmol/ i), aldosterone (0,895 • 0,687 nmol/i). by factorial statistical analysis we found significantly high correlations between hormonal changes and respiratory function (for example r=-0,539, p < 0,02 between cortisol and pao2; r = 0,817, p < 0,001 between cortisol and d (a-v) 02; olso renin -cao 2 r=-0,824, p < 0,001, renin d ~,vl o2 r = 0,626, p < 0,001). such significant correlations was found and for raas with respiratory, renal function, byproducts of arachidonic acid thromboxan b 2 and p6fla, soluble fibrine degradation products etc. these correlations between the degree of endocrine changes and multiple organ failure in patients with septic shock produced by peritonitis suggest that their effects upon peripheral vascular resistance and constriction of the splanchnic, splenic, renal and other organ vasculatures are not always with physiologic expediency and there are perhaps the possibilities of therapeutic influence. intredu~on : dopexamlne has previously been shown to control hyperkalaemia ia patients with acdto renal failure (arf), however effects on the subsequent course of art are undomunente~ ob_iectlv~ : to evaluate clinical progress in patients with acute renal failure (arf) in an intensive care unit (icu) with regard to biochemical control, need for -and time to -dialysis, and outcome in patients receiving dopexamine. m~ods : 14 consecutive patients meeting standard criteria for diagnosis of arf were included in the study. full cardiovas~dar, biechemical and intervention/outcome details were recorded. dopex.~min~ was infilsed at a dose of 2 pg/kg/min in conjunction with a regimen of inotropir support and blood volume optimization. resn]~ : following the intzoduetion of dopc',~mine ilrinr vohlmes increased slightly over the next 24 hrs fzom 516 + 140 ml/24 hrs to 817 + 229 ml/24 hrs (ns). data expres,uxl as mean + sem. three patients (21%) became polyuric with urine output >100 ml/hr within 3 days and did not need dialysis. in the remaining patients the time to dialysis (to correct acid-base deficits or volume overload) was 5.09 + 0.84 days. serum potassium levels were well controlled. day 5 or immediate pre-dialysis levels were 4.48 + 0.19 mmol/l compared with pre-lreatment 4.67 + 0.2 mmol/l overall mortality in this series was 4/14 (28%). duration of acute dialysis in survivors with renal recovery was 16.8 +_ 1.82 days. 2 patients (14%) progressed into chronic renal failure and needed continuing renal replacement therapy. no adverse cardiovascular altects were seen at this low dopoxami~ dose although its competitive inhibition to adrenergic reuptake mechanisms meant that doses of pressor agents could often be reduced. : dopcx:~minr nsed in conjunction with inotropic support and blood volume oplimitntion, can safely postpone, or even avoid, the necessity for acute haemodialysis in icu patients. no evidence of tachyphylaxis to the effect on serum potassium levels was seen over the duration of the study. hen'era m., suarez g., dagn d., varela a., ramos j., garoia jm, aragdm c, jurado l, medina a. icu. hospital regional. malaga. spain. objective: to evaluate the haemodinamic tolerance to the veno-venous continuous hemefiltration (vvchf) system in patients with systemic inflammatory response sindrome (sirs), and the possible beneficial effect of this technique on the haemodinamics in these patients. material: 13 patient admitted to the icu, with diagnosis of sirs and monitored with a pulmonary artery catheter at the beginning of wchf. we performed a complete haemodinamic study to all these patients (cardiac output, vascular resistanoss, ph and co2 in arterial and mixed venous blood samples, saturation of pulmonary mixed venous blood, do2 and vo2 calculations and temperature) and determined the respiratory mechanics (compliance and pao2 /fie2 relatinship) before starting the procedure, after 10 minutes operating with the ultraflltrate branch closed (without filtered fluid production), afler 30 and 60 minutes of zero fluid balance bemofiltration and after 120 minutes of filtration with negative balanos adjusted to the patients conditions. for the statistical analisis we have performed the anova test over the mentioned variables. results: we have not detected statisticaly significant differences of the analyzed variables before the beginning after operating the pun'@ for 10 minutes without filtered fluid production and after 60 minutes of zero fluid balance hf. only temperature shows a meaningful decrease in time. objectives: among many organs, playing the important role in pathogenesis of multiple organ failure, the particular place is taken by the intestine. ~ethods: the study was carried out in 5 dogs !~n"~h pi was modelled by severe operative trauma (ot). the dcm was estimated by the indices values of work time (wt), contraction frequency (cf), mean amplitude of contractions (~ac) and motility index (mi) measured by method of tensography. "sl", created on the basis of sorbit and sodium lactate (1800 mosm/l), was injected in the dose of 10.o ml/ kg into v. cephalica antebrachii after 24 hrs of ot. the results of the present study are the evidence of "sl" stimulative action on dcm and are experimental ground for "sl" using in complex therapy of pi in clinic. with splanchnic venous blood pc02 p.f. laterre p. goffette, j.p. fauville, a. poncelet, p. loneux, m.s. reynaert. intensive care unit, st. luc univ. hospital, brussels, belgium. determination of gastric intramucosal ph (phi) by gastric tonometry using the henderson-hasselback equation is expected to allow the detection of splanchnic ischemia in critically ill patients. because of bicarbonate concentration and acidbase balance influences on the calculation of phi, it has been proposed to use arterio-gastric pco,_ gradient [p(gast-a)co,] to assess splanchnic perfusion. htpothesis : pcoz in the gastric mucosa is in equilibrium with intraluminal co z and with co, in the blood leaving the stomach (mesenteric and portal blood). objective: mesure pco; and ph in portal vein blood and compare its value with pco 2 and phi obtained simultaneously by gastric tonometry. material and method : in a patient (55 y.), a fiberoptic catheter (baxter r) was positionned in the portal vein after transhepatic stent shunt repermeabilisation. hemodynamic parameters, do, (vigilance n baxter), gastric co 2 and phi (tonometrics baxter) and portal blood gas were determined at regular intervals. results : 19 sets of data were obtained and are expressed in mean + sd. gastric pco z was 46,5+18 compared to 40,4+3.5 mmhg for portal pco 2. phi was 7.32+._0,17 vs 7.34+._o,04 for portal ph. no correlation was found for these 2 parameters. p (gast-a) c02 was 6.4+15 mm hg vs 2+1.6 mm hg for p (portal-a) coz (no correlation). there was a good correlation between do e and p (portal-a) co z (r = 0,61) [figure] but no correlation with p (gast-a) c02. obiectives: desaturation is a common finding during haemodialysis (hd). pulmonary oedema might be one cause for impaired gas exchange (1). the aim of this study was to quantitate the amount of extravascular lung water (evlw) and gasexchange in chronic renal failure patients during and after a regular hemodialysis session. methods: 10 chronic renal failure patients without symptoms or diagnosis of cardiac or respiratory disease were studied at the start (i), at the end (ii) and two hours after (iii) a regular bicarbonate hemodialysis session. the double-indicator 2 dilution method, with indocyanine green and the stable isotope h20 as tracers, was used to measure evlw (2). arterial bloodgases and endtidal co2 were registered. evlw data was compared to a group of 18 renal healthy patients (0). dcp n evlw, ml -pao2, mmhg h~o +, nmol/l control group 0 18 243 -2--61 l 10 332 _+ 91"* 13 -+ 3 38 _+4 crfgroup ii 10 269 -+ 84~ 11 +-2ns 34-+2"(" iii 10 283 +-78t 11 _+3ns 34-+2t ** p < 0.01 dcp i from dcp 0, t p < 0.01 dcp li or i1 from dcp i, :~ p < 0.001 dcp ii from dcp i the evlw at the start of dialysis was larger in the crf group than in the control group. the evlw decreased significantly to a level not different from the control group in response to the reduction in weight after hd. pao~ was normal at the start of hd and showed a nun-signficant reduction after hd. paco2 (5.3+0.6 kpa) and etco2 (5.2+0.8 kpa) were unchanged while h3 o+ decreased and bicarbonate increased significantly. conclusions: the elevated level of evlw at the start of hd did not impair gasexchange. the decrease in evlw did not inhibit the decrease in pao2. the reduction in h30 + followed by a fall in alveolar vantilation is the most plausible cause for the decrease in pao2 in bicarbonate dialysis. 1. prezant lung 1990; 168: 1-14.2. wallin j appl physio11994; 76: 1868-75. a. dona~ d. battis& l col~ r danieli, d. achill~ l viglienz;~ c. giov-anaini, p. piaropao~ oblectives: to verify if intraoperative modifications of mtramucosal gastric ph (phi) below the normal lowest value 7.32, can be predictive for important complications, as perforation, sepsis, mof or death. methocls: we have considered 20 patients who andenvent major abdominal surgery. all patients received the same drugs in pre-anaesthasia, the same type of anaesthesia (balanced anaesthesia) and the same treatment with h2-bloekers. after the induction of anaesthesia a gastric tonometer was positioned and a catheter was positioned in the radial artery. during the operation, every 30 minutes, the following parameters were measured at the same time: phi, arterial ph (pha), blood lactate, mean arterial pressure. in follow up we considered death and complications happened during the hospital stay, in relation to intraoperative phi falls below 7.32. results: among the 20 patients, 9 had a drop of phi below 7.32 during surgery. in three of them this fall was a single episode and happened within the first hour after the begiluting of the operation. after that phi rose to nomml values until the end of the operation these patients had a normal post-operative period, without complications, the other 6 patients had a fall of phi during the demolitive manoeuvres. two paticots of them died. the first had a lowest phi=7.25 and the second 6.97. the first one ~zs operated on for hepatic istiecitoma, suffered a complete del'dseenco of the surgical wound on the 20th day after operation and died on the 25th day, the second one was operated on for a hepatic carcinoma had an intraoperative haemorrhage and died ~vo hours after the end of the operation. the other 4 patients with a fall of phi had a lowest phi=7.24. 7.18. 7.26. 7.28 respectively.the first patient,operated onfor sigmoid carcinoma, underwent on a second operation for a transmural necrosis of the colic segment on the 25th day; the second one, operated for carcinoma of the right colon, had a cardiac ischelnia on the 5th pest-operative day and a dehiscence of the surgical wound on the 8th day: the third one, operated on for a sigmoid carcinoma, had melena in 41h post~ operative da b, and finally the fonrth patient, operated on for carcinoma of the tight colon, suffered a fistula of the surgical enteral anastomosis.all these 4 patients were discharged alive from the hospital. the other 11 patients, who had not reductions of phi ditring the operation, had a normal pest-operative period, without complications. conclusion: phi was able to predict the arising of some complications, probably due to intraoperative ischemic events. we can say that gastric tenometry, for its low invasivi.ty, can be included among the intraoperative monitoring in patients that tmdenvent on major abdominal surgery. (ttd),t"ea~rrerj.~ of 3 hours duraticn. all l:atients nm.'-~ms_(~lly va~2ated in eantrol wcde ard_ la':'ad a a,~m--ganz catheter, with optic fibers for contirums mmsuremmt of svo mic studies were performed, c~e before the hegir~ of hd, c~e 15 rain after the ~, ~ne at the middle, ~ne 15 rain before lhe erd ard one 30 rain after the erd of hd. paired t test ~as used far slatistical eval~ti~n. results: daring i~d there was a significant'reductton (p<o.~) of: c~tr~ venc~s eres~.re (0ve)emm 9 to 7.5 ~ ~, 2) ~arn'~w capil~ ~f~e pressure (i<~p) fr~n 13 to 11 ~mg hg, 3) i~09 from 33 to 30.5 mmg hg. ~here was also a less pmnameed bat also-slatistics/ly significant r~ucticn (p<o.05) of: i) s~o~ from 67 to patients undergoing surgical reconstruction of the aorta due to anenrysms of its abdominal part may develop transient and sometimes sustained episodes of dysoxia despite the conventional appeoreneas of being adequately resuscitated. indirect masurement of gastric mussel ph (phi) is being widely evaluated as a minimally invasive and sensitive means of assessing the adequaey of tissue oxygenation in these circumstances. the duration end degree of gastric intramucosal acidosis are related to the risk developing injured gastrointestinal tract end its putative consequences, i.e. translceation, cytokine release, organ dysfunction end failure, sepsis end death. measurement of phi is obtained indirectly by measuring pc02 in the lumen of the stomach with a silicone balloon tonometer (tonometrics, inc., worcester, ma, usa) and the bicarbonate concentration in arlz,,rial blood, end substituting these two values in the henderson-hasselbalch equation. objectives: to evaluate whether perioperative phi changes are predictive of complications end outcome end how they compare to standard haemodyuamic and oxygen trensport parameters. methods: 16 patients (15 males and 1 female) urtderwent surgical replacement of abdominal aortic anonrysm with the aortic simple or bifurcated grail. 14 patients were operated on eleetively, 1 had an emergency surgery for raptured enentysm. haemodynamic, arterial ph and bicarbonate concentration measurements were taken before and after induction of anaesthesia, after cross-clamping and declamping of the aorta end at admission to itu. phi calculations ware done at the sime time except before induction, as tonometers were inserted after patient had been asleep. we used pulmonary utilizing a computer billing survey as well as hospital service transfer data from a 67 month period (1/89 -7/94), all obstetric patients that were transferred to the med/surg icu were identified, as well as their diagnoses and procedures. twenty-eight obstetric patients were transferred to the med/surg icu, compared to 12,489 deliveries occurring during that time period for a .22% rate. the following procedural indications for icu transfer occured: 4 insertions of endotracheal tubes, 2 arterial catheterizations, 1 temporary tracheostumy, and 1 venous catheter. the following medical diagnoses were found: 5 hypertensive/eclamptic episodes, 2 hypotensive episodes, 1 pulmonary embolism, 1 mitral stenosis, and 1 coagulation deficiency. there were four episodes of respiratory failure (14%). the historical rates of respiratory failure from the medical literature seen at a university hospital ore higher than those at our community hospital despite comparable icu transfer rates for critically ill ob patients. objectives: to evaluate the usefulness of several isss used in order to predict risk of death (rd). design: prospective data collection for 7 months in a multidisciplinary 18-bed icu. methods: data from 270 consecutive patients admitted to the icu from 1/10/1994 to 1/5/1995 were studied. all the necessary informations were stored in a computer using special software for every day computation of three isss (saps, saps ii, apache ii) and of the rd predicted by saps ii, mpm0, mpm24 and odin. data from 2453 days of hospitalisation were used for comparative evaluation of isss and rd, while sensitivity and specificity in death prediction (cut off point of rd = 90%) were evaluated from data collected during the day of admission (saps ii, rpm0, odin) or after 24 hrs (mpm24). agreement between rd predictions was evaluated by bland and altman analysis. results:our results were the following: objectives: a) to create and validate software specially designed for the collection of demographic data and estimation of illness severity and predicted rd and b) to provide data necessary for the evaluation of the effectiveness and the efficiency of our icu. design: prospective data collection in a multidisciplinary 18-bed icu. methods: prospective data on specific physiological variables, selected demographic characteristics, comorbidity and the reason for icu admission were recorded every day for 270 consecutive patients admitted to the icu from 1/10/1994 to 1/5/1995. data were entered into a portable computer using specially designed software allowing the calculation of usual illness severity scoring systems (isss) and the corresponding predicted rd. results: we studied 176 men and 94 women, with a mean age of 55 years. we conclude that, although observed mortality was higher than predicted rd, this difference was not statistically significant. the fact that the sd of predicted rd was too large, limits the usefulness of isss predictions for a comparative evaluation of different icus. objectives :the importance of objective evaluation of patients prognosis at their admission to an emergency department is clear. the aim of this study was to appreciate the prognosis of patients with the simplified acute physiology score ii (saps ii), and correlate this score to the prognosis and orientations of patients. methods : the score which is a reduced form of saps ii was calculated from 6 clinical variables and 6 laboratory items for 200 patients. sensibili}y (se), specificity (sp) and accuracy (ac) were calculated to evaluate the performances of saps i1. thresholds were calculated with the youden's test (se+sp-1). results : 23 patients were admitted in the intensive care unit (icu)and 177 patients in medical units; 23 patients died. the saps ii was higher in patients which died than the survivors (31,0+11,4 vs. 1,9+9,3, p<0,0001 ; respectively), the best threshold was 24, se was de 86%, sp was 92%, the ac was 920/0. thus for patients admitted in icu than in medical unit (27,0:1:13,5 vs. 12,5+9,9 , p<0,0001; respectively); the best threshold was 21, se was 69%, the sp was 78%, the ac was 77%. conclusions : these preliminary results suggest that saps ii can be used to determine the short term prognosis and the destination of patients seen in an emergency department. design: data was collected on all admissions over a six month period. all patients had a screening hiv elisa test. confirmatory ifa tests, western blot and flow cytometry were performed on hiv positive patients. the institutional ethics committee considered the major clinical impact of the study sufficient cause to waive the patient's right to informed consent, icu staff and patients were blinded to hiv results. following discharge patients were given the option of being advised of their hiv status. setting: the study was conducted in a 16 bedded surgical icu at a tertiary care teaching hospital. patients, participants: all patients admitted during the period in question were included. interventions: "all patients were treated according to standard icu protocols. there were no interventions. results: most patients were admitted following trauma. there were no patients with aids, 52 hiv positive and 350 hiv negative patients. with respect to the latter two groups, the mean age and sex distribution was similar, there was a significant difference in organ failure (71% versus 49%, p < 0.05) and septic shock (39% versus 15%, p , 0:05) but no difference in icu/hosp[tal mortality or duration of icu/hospital stay. flow cytometry changes in hiv positive patients were consistent with the quiescent phase of hiv infection. conclusion: while morbidity is higher in hiv positive patients, there is no difference in mortality. hiv status should, therefore, not be an exclusion criterion for icu admission in this patient population. 1, n= 112) and january-march 1994 (period 2, n= 127) were studied. interventions: a resident micu team led by a trained intensivist took over the medical care from the primary physicians when the patients were admitted to the micu. the intensivist also vetted micu admissions and decided on micu discharge. in addition, there was a resident respiratory therapist to attend to vendlatory care during office hours. after office hours, the care of the micu was delegated to the on-call team on a rotational basis among the medical departments. results: the patients in the two groups were similar with respect to age, sex, race, source of admission and apache 11 scores. the icu and hospital mortalities decreased from 33.8% to 27.6% (p=ns) and 38.2% and 34.2% (p=ns) respectively in period 2. the mean icu and hospital stay was also decreased from 4.6 __+ 7.1 to 3.6 + 4.0 days (p=ns) and 19.3 __+ 357 to 17.2 • 22.2 days (p=ns) respectively. the improved micu length of stay for survivors in period 2 was statistically significant (4.2 • 4.2 days vs 2.8 • 2.8 days = 0.015). there was no significant differences in the vse of peritoneal dialysis (5.4% vs 6.3%) and mechanical ventilation (55.4% vs 49.6%). however, utilisation of intra-arterial lines and pulmonary artery catheters increased from 0% in both to 23.6% and 5.5% respectively in period 2. conclusion: the duration of critical illness was reduced in period 2. hence, we believe that the closed |cu which was staffed by an intensivist and had the services of a respiratory therapist was beneficial for the care of the critically ill. the intensivist, was also more likely to utilise invasive monitoring. in the absence of liver transplantation, intensive care for patients with acute hepatic decompensation arising from alcoholic liver disease (ald) is indicated only for those who are likely to benefit from conventional means of organ support. we have attempted to elucidate potential risk factors and the efficacy of organ support in relation both to hospital outcome and icu costs so as to allow the earlier identification of relatively futile intensive care in this group of patients. methods: over a period of one year, 59 ald patients (37 males; 22 females; median age 46 years, median apache ii score 19; hospital mortality 68%)with severe complications (30 [50%] with bleeding oesophageal varices, 16 [27%] with hepatorenel failure, 5 [9%] with respiratory failure, 3 [5%] with septic shock, and 5 [9%] others) were studied in our liver icu. organ system failure, daily therapeutic interventions and icu costs were assessed using a patient management system (riyadh intensive care program). results: whilst survivors had a significantly lower admission apache ii score compared with non survivors (medians 12 and 20, p<0.0003), 2 or more organ systems in failure in the first 24 hours of icu admission was associated with a 91% (21123) mortality. mortality was also related to child's grading and the numbers of organs supported. of the 29 patients who received more than one form of organ support only one survived. organs supported mortality a (n=2) 0% none 3/13; 23% b (n=6) 33.3% one 8/17; 47% c (n=51) 74.5% two or more 28/29; 97% the icu costs for the 59 patients were only s 440 but s 687 (75%, 216 patient days) was utilised by the 40 patients who died, giving an effective cost per survivor (ecps) of s 496. although the cost of treating the 23 patients with 2 or more organ systems in failure on the day of admission was s 510 (36% of the budget) the ecps was s 755. conclusion patients with ald who have 2 or more organ systems in failure early in their icu course have a poor prognosis and in the absence of liver transplantation, traditional strategies of organ support are ineffective. this begs the question whether such costly care is appropriate as although the ecps in this sub group is comparable with other patient groups with multiple organ failure, their outcome is considerably worse. objectives: to evaluate the economic impact to the healthcare sector of using dopexamine hydrochloride infusions to deliberately increase perioperative oxygen delivery in high-risk patients. methods: effectiveness data were obtained from a controlled clinical trial in which 107 surgical patients were randomly assigned to either a control group (n = 549 to receive best standard optimal fluid resuscitation perioperatively, or a protocol group (n = 53) to receive, in addition, an infusion of dopexamine hydrochloride to increase the perioperative oxygen delivery index to greater than 600 ml/min per square metre. resources consumed by patients during treatment were recorded prospectively and costs of resources at 1993/94 prices were obtained retrospectively. cost-effectiveness ratios were estimated by dividing the total cost for each treatment group by the total number of patients in each group and by the clinical outcome of mortality 28 days postoperatively. results: in the protocol group, there was a 75 % reduction in mortality 28 days postoperatively (p = 0.015), a significant reduction in the number of complications (p = 0.008) and a reduction in length of stay in the icu and surgical ward. the average cost per protocol patient to achieve a 94.3 % survival rate 28 days postoperatively was s 9,894, resulting in an average cost of s 10,488 per surviving protocol patient. the average cost of a control patient was s 11,331 to achieve a 77.8 % survival rate 28 days postoperatively, resulting in an average cost of s 14,568 per surviving control patient. the use of dopexamine to deliberately increase oxygen delivery perioperatively generated a cost saving to the nhs of s 1,436 per patient together with a 75 % reduction in mortality at 28 days postoperatively. hence, the cost per surviving protocol patient was s 4079.8 less than the cost of a surviving control patient. conclusion: the additional cost of dopexamine in protocol patients was offset by a lower incidence of complications and a shorter stay in the icu and on the surgical ward. hence, increasing oxygen delivery perioperatively in high-risk surgical patients saves both money and lives. objective: although king's college criteria are widely used, indication and timing for liver transplantation in acute liver failure is still far from certain. long-latency sensory evoked potentials -a proven measure of metabolic brain dysfunction (grimm et al. lancet 1988; 2: 1392-94; madl et al. lancet 1993; 341: 855-58 ) -are markedly affected in patients with acute liver failure. serial sensory evoked potential recording should provide very useful information for the management of patients with acute liver failure. methods: 90 recordings of standard bilateral sensory evoked potentials were performed in 25 patients with acute liver failure. findings were focused on cortical n70 peak -a stable negative deflection occuring 70 • 9 ms after median nerve stimulation in healthy subjects, which can be recorded by skull electrodes over the sensory cortex. results: nine patients recovered spontaneously, 8 patients were referred to emergency liver transplantation, and 8 patients died. in all 9 patients who recovered spontaneously, n70 peak was detectable between 74 and 162 ms. all 8 patients who were referred to liver transplantation and 7 of 8 patients who died, developed a loss of a prior detectable n70 peak during the course of acute liver failure. objective: to determine high risk arid low risk patient groups in a medical intensive care unit regarding short term and long term outcome. methods: data on all admissions of the year 1993 were collected prospectively. according to their mode of admission patients were classified as admissions by the physician staffed ambulance service (as), admissions through the emergency department (ed)i referrals from non-intensive care wards (ni), and secondary referrals from other hospitals (oth). outcome was assessed in terms of in-unit mortality, in-hospital mortality, and long time mortality. patient groups were compared using the \2-test. life table analysis were per{ormed by kaplan-meier method comparing patient groups by log-rank test. the software spss for windows 6.0.1 was used for statistical calculations. results: in-unit mortality: 184/1853 patients (9.9%) --oth 20.0% > as 10.6%> ni 9.1% > ed 6.8%; p = 0.01. in-hospital mortality: 365/1853 patients (19.7%) --oth 30.9% > ni 20.4% > as 19.6% > ed 16.2 %; p = 0,05. mean survival time in days after discharge: as 620 < ni 682 < oth 708 < ed 777; p = 0.66. conclusions: despite an excess in-unit mortality of secondary referrals from other hospitals the iongtime course of this special patient group is not different to others. solsuam, j, marrugat*, g, mirs, j, nolla, a, vazqu~z-sanchez, l alvamz, ~ioio s xndioina i~siw. ir~itate l(~icipal da l~sti~isn l~di~*, ~ospits dal objective: to study the influence of modifiable variables (complications derived from therapeutic activities) on the prognosis of ~atients admitted to the icu indapemently on thn severity of illnsss. patients am methods: between january 1990 asd ]lay 1993 data from 1,425 patients over 14 years of aqe who retained in the icu for mare than 24 hours ~ere pr~pectively regiatered. a cohort st~ly with follo~-~ nf patients durin~ ~eir stey in the hospital was deni~.el in all patients, reasons for a~issien, principal diagnosis sad severity of illn~s moasared by the saps scare vare recorded. fastens affecting patients' outcome that my be proventsd or modified included technical :omplisafioss, heapital-acqnired infections and in~pro~riate therapeutic decisions. a logistic regression model was used to assess the relative risk (l~} for in-heapital mortality adjusted for each variable. results: ic~ mortality ~s 17.2% and in-hospitul mortality 22.7%. patients who died showed a higher spas score then survivors (15,3 ~ i0,i). after adjusting hy severity of illness, co~;licetices that statistically increased the risk of in-hospital death were septic shock secomery to hoapitul-acqdired infection (1~ 7.18; 95% el, 1.9 to 27.1), pmo~othor~x related to mocasnical ventilation (@ 6.28; 95% cl, 1.7 to 22.3) and delay in the insertion of a fln~-quidod catheter (ii~ 5.49; 95% ic, i.i to 26.9). col~lusien: registration of complicaticas derived from therapeutic activities is a valuable tool far quality central in the icu. g, ~i~5, j.l mle~ma, j, ~amqat*, j..~lla, a, vazquez-saltemz, f, alvamz , servioia de nndicina l~siu. i~stitutu ~icipal de ln~sti~acidn ~4i:a*, hospital dsl objective: to dstsr~ine the incidence of self-extebatien and its effect on ~ortality. patients and ]~etheds: betveen january 1990 and april 1994, all i~tiente in whom selfextubatien w~s registered were inclnded in a prospective study. patients were divided into @nee who needed r~intabatinn within 24 hoers and those who did not. in all patients, dsmoqraphie and ciinical data were recorded as well as icii mortality, in-hoapital mrtality and severity of illness according to saps score. 0eta were analyzed usi~ the cbj-square test for cathgorical verinbls, the analysis of varianc~ (anva) for aontinuc~ ~ria~les and a leqi3tic regression anal~is to estimate the relative risk (iiii) for mortality as result of celt-nxtt~ation after adjusting for severity of illness. results: a total of 815 intnmtsd patients amre stndied. self-extu~atien occurred in 54 (6.6%) patients and 25.6% required reintuhot~pn. when a co,arise was made between patients who did not required reint@atinn and patien~.s who did, statistically significant differences in eqe (52.1 v_s 60.4 years, p = 0.~02), ~verity of illness (11.4 ~ 13.1 spas score, p = 0.02), dia~isstia category (4s.6% v_s 66.7% of patients with res~iratury conditiono, p =0,002} and mean length of stay (10,9 ~ 20,7 days~ p = 0.05) were fo~m, a~ter ad~sti~ for severity, patients with self-ext@atinn who did not reqnired reintalatien showed a 0.4 iir for mortality (95% ci, 0.i to 0.9) as co~arod with patients in when self-ext@ation did mot occur. conclnsien: self-~extamtice that does not require reint@ation is associated with a isamr in-hospital natality probably dt~ to a prolonged period of weaming. patients' admissions to ices am often delayed doe to the shortage of beds available. @ile amaltieq icu admission, these patients are treated in observation nits of @e emergency services which bare ,either tile structure nor the trained ~reomenl that are available in leb~. objective: to daterdno the effect on the patient's proqusis of a delay in tile admission to the icu when criteria for icij admission are fulfilled. ~terials and methods: between jme am l?ece~ber 1993 all patients who fulfilled criteria to be almittod to the ic0 who for waste~r reason retained in tile observation unit for more than 24 hours were included in a prospective stedy. in all patients, des~raphic end clinical dabs amre recorded as well as severity of illness aencrdi~j to saps score. a cesucontrol dasi~ was eend with a total ss~ln of 1,425 patients who suffered no delay is admission to icii over a period of 3 years. data wen analyzed using the chl.-squ~re test (to aeons the association hetwenn in-patienty mortality end categorical vari~lns) and a maltipln logistic reqression model to sstimta odds ratio for) for in-hospital mortality as result of delay in icy admission as compared with early ad~issi| after adjusting for severity of illness end use of assisted mchenical ventilation. ~9&ults: a total of 50 patients remained in the observation nit for more than 24 hours with a del w in igd admission of 55.8 _+ 25.1 hoers. assisted mechanical ventilation was requited in 22% of patients and only monitericatien in 46%. itsse patients were cspared with 112 ntients from the tet~l sample ratchod by age, sp~ score and rennoss of admission. in-hospital mortality for cases warn 16% as compared with 17.5% for controls (p = s). after adjamtilg fen spas, age and mobamioal ventihtien, no statistically significant differences between both ~renpa were foam, altho~b there was a tendency towards a higher mortality amen@ patients with delay in icu admission (or = 0.779; 95% ci, 0,2 to 2,5). conclnnien: ~se findings suggest that prognosis of critically-ill patients is no worse as a result of admission to the loll being deln~d for 24 borers. all data appropriate for the calculation of the apache ii score (aps) together wi'th other specific cardiac details relevant to these .patients were collected daily, verified and enter~ into a computer database. results: 150 patients were studied. six patients died and five of thee underwent cardiac surgery. the mean aps was 9 for survivors and t6 for non-survivors (p < 0.001). the mortality ratio was 1.1 and the major markers of mortality were apache ![ score, presence of chronic ill health, mean duration of ventiiation, mean length of icu stay and need for emergen~ surgery. sixteen percent (233) of icu bed days were occupied by 4% of patients (non-sarvivors) which resulted in cancellation of 60 cardiac sot#cat sessions in 6 momhs. conclusions: this study concludes that apache 1t could be used as an audit tool in a cardiac surgical icu and demonstrates the severe compromis~don of cardiac surgical throughput by a few non-survivors, organ to determine the number of organ failure free days (offd) in a cohort of survivors and non-survivors with sepsis syndrome followed over a 30 day period. 2) to determine sample size requirements for clinical trials utilizing a increase in the number of organ failure free days as the primary outcome as opposed to mortality. methods: beginning december 1990 through to april 1992, patients who met inclusion criteria of the "cardiopulmonary effects of ibuprofen in sepsis syndrome" and who did not have hiv/aids. brain death or moribund state were prospectively identified. presence or absence of failure of 7 organ systems (pulmonary, cvs, renal, hepatic, gi, hematologic, & cns) was recorded daily until death or until 30 days. a score of one was assigned to each organ system free of organ failure in patients still alive, ie, maximum daily off score=7, maximum 30 day off scorn=210, sample size estimations were performed for variable detectable differences in off scores (delta). alpha was set at 0.05 (two-sided), with n/group = 2[(z a +z b ) o conclusions: a clinically relevant increase in off days may be detected with as small a sample size as 30 to 50 patients per group. this represents a significantly smaller sample size than needed to detect a change in mortality from 40% to 30% (25% relative risk reduction) where the n/group=356. scoring patients in this manner prevents a lethal inte~entien from providing an improved organ failure score. in addition, an intervention that prolongs survival must also provide greater organ failure free days in order to be counted by this scoring method. survival as an outcome provides no information about the quality of that survival. off days provides a measurement of burden of illness. interventions which lessens this burden may be just as valuable as those that decrease mortality by providing a measure of the quality of survival and by decreasing costs of care. they may also prove to be an accurate surrogate marker of mortality. the advantage of this approach is that the event rote is much higher and sample size requirements are subsequently smaller. this would mean that clinical trials can be completed faster and at lower cost. outcomes such as mortality could then be assessed at a later date utilizing recta-analysis. we suggest that the use of off days is a valid outcome measure that may be utilized in clihieal trials of sepsis syndrome. the icu is perceived by many as being a stressful environment for both patients and staff. stress has been defined in three ways: a stimulus producing a particular response; the physiological and psychological response to a stimulus; an interaction butwom an individual and their environment. stress is currently thought to be a dynamic system of stimulus and. response which takes into account the individual's perception of the stimulus and their ability to respond effectively. stress may, therefore, be positive and allow personal development but an individual unable to respond effectively to a stimulus will experience negative effects or strain. critical illness is an intense stimulus to which the body needs to respond effectively. physiological responses are vital and most of intensive care involves supporting these. alternatively, blocking them, for instance with atom(date, increases mortality. psyehological responses are also vital but often poorly appreciated because of communication problems. many of the problems patients experience in an icu are evidence of psychological strain. this can be exhibited in various ways, for instance, anxiety, depression, passivity and confusion. dealing with critically ill patients is perceived as stressful. we recently studied occupational stress in our icu. most aspects of intensive care were not generally perceived as stressful indicating a self-selectien of icu staff. the most stressful aspects of icu work for nursing staff were the structure of the organization and career opportunities. medical and nursing staff had different stressors and different coping strategies. support for occupational stress, therefore, should focus on the individual and concentrate on information and communication. atmosphere, and especially at intensive care units, we face up to daily decision making. in most cases these are taken on the basis of personal opinion and the processing of a very limited amount of information. rising need to optimize the results of medical attendance becomes necessary to set structured system of d@cision making in which ethical basis have a sp@dial significance in view of next considerations: -we live into a pluralist society in which the importance of values is different. -most persons consider health as the first value only in the event of illness. -medical resources available are limited, whereas medical, attendance demand from population increases in a way many people consider it unlimited. in consequence, it becomes necessary to set up priorities in patients treatment. ehtical basis that rule decision making are essentially these ones: i. beneficence: to provide the patient that is being treated the highest profit. 2. non maleficence: it is our first duty to avoid hurting or damaging the patient."primum non nocere" 3. autonomy: in every particular medical attendance, the patient has ability to decide by himself. 4. justice: as equity: to provide the same treatment for those who have the same pathology, ignoring another factors such as age, sex or race. severe application of these principles can cause difficulty, which resolution requires a systematization of decision making. (1-84) . the lenght of stay between survivors and non survivors didn "t show statistical significance (p =0.51 ). the mean aiii score when considering all admissions was 59,9 (8-153) . the initial score between survivors and non survivors showed ststistical difference (48.6 vs 92.5) respectively (p < 0.0001). univariate logistic regresion analysis demostrated a 90% increment in death probability for every 10 points augmentation in the aiii score with a sensitlbity of 94.9% and specificity of 62.7%, the roc curve showed that the best cut off point for death prediction was 75 points with a sensitivity of 75.6% and specificity of 79.9%. if a patient is classified as high risk (> 75) the bayesian analysis showed a 52.8 probability of death and for one class(fed as low risk (<75) a death probability < 10%. conclusions: the first day aiii score in this population showed to be a good discriminator between survivors and non survivors, and the risk of death augments as the aiii does. in this population an aiii score > 75 points is asociated with a greater risk of death. using the aiii score in conjuntion with the clinical judgement will help clinicians reducing uncertainty in the every day decision making and better predict outcome, the results from this study should been taken with caution because the data were obtained from a small sample. objective: the quality of life has been considered a "uniquely personal perception" resulting from a mixture of health related factors and social circumstances [t. m. gill, jama 1994, 272: 619] . the aim of this study was to evaluate two measures of pqol in intensive care unit (icu) admitted patients. patients and methods: during icu stay and six-months after hospital discharge, 160 co-operative icu admitted patients were directly interviewed about their pqol. we administered ftrstly the uniscale (pqolu) [sage et al crit. care med. 1986, 14: 777-782] and then a 5 step verbal scale (pqolv): best, good, fair, poor, worst. of the 160 studied patients, at the first interview, 116 were able to use both scales, but 44 (27.5%) understood only the verbal one. at the second interview, 8 patients were not able to answer, 113 used both scales and 39 only pqolv. statistical analysis was performed using wilcoxon signed ranks, spearman rank correlation, student's t and chi square tests. results: of all cardiac surgery pts, 42 pts (1.6%) died in icu. they were 33 males (1.5%) and 9 females (1.6%). their mean age was 66 (+7) years and mean ef was 0.38 (+0.1). nineteen pts (45%) had low (<0.35) preoperative ef. mortality was 0.9% in the coronary artery bypass grafting (cabg) group (n=2014) and 2.8% in the valve replacement (vr) group (n=359). in the cabg +vr group, mortality was 8.4% (n=95), and 3.3% in the remaining pts (n=149). cardiogenic shock was the sole cause of death in 24 pts (57%), septic shock in 6 pts, whereas sepsis in combination with ards in 4 pts, sepsis and stroke in two pts. in addition, 6 pts died from cerebrovascular accidents, one from ards and one from pulmonary embolism. the pts who died in the icu had a significantly longer bypass and aortic cross clamp time and received more blood transfusions (p<0.001) than a matched control group that survived to icu discharge. the duration of mechanical ventilation and length of icu stay were greater in the pts who died in the icu than in the control group. conclusions: 1. although cardiogenic shock is the main cause of death (57%)in cardiac surgery pts, sepsis and cerebrovascular accident are relatively frequent causes. 2. patients who died in the icu had longer bypass and aortic cross clamp time and received more transfusions, compared with the control group. 3. although renal or hepatic failure contributed to death in some pts, they were not the primary cause of death in any patient. objectives: evaluate the acute and follow-up outcome of 27 patients (pts) treated with primary ptca (without prior thrombolysis) in acute myocardial infarction (ami) after 12 and up to 24 hours after onset of typical thoracic pain ("late" primary-ptca). methods and patients characteristics: from 12/89 to 4/95 364 consecutive pts with ami were treated by primary ptca in the wuppertal heart center 9 27 pts (7,4%) were admitted to our hospital > 12 hours and < 24 hours after symptom onset with ongoing chest pain and typical ecg-changes.mean age was 62 years (49-78). 23 pts were male, four female. 37% had an anterior wall myocardial infarction, 63% suffered an inferior/postero-lateral wall myocardial infarction.two pts were in cardiogenic shock at admission. singlevessel-disease was documented in 70.4%, multi-vessel-disease in 29.6%. average time of onset of pain to recanalisation was 929 min (720-1440). angiography revealed timi-flow 0 in 85.2% of the pts, timi-flow i in 11.1%, timi-flow ii in 3.7%. average follow-up (fu) period was 12 months (4-28 months). timi iii lv-ef ~ 30-day major late re-late flow p.i.* aeute/fu mortality bleeds infarction mortality 92.6% 58%/63% 7.4% 7.4% 3.7% 0% early mortality occured in the two pts, who were in cardiogenic shock at admission 9 no pt required emergency coronary artery bypass grafting.restenosis >50% was seen in 37% of the pts. conclusions: "late" primary ptca achieves a favourable high recanalisation rate of about 90% (timi ill-flow) in our study group. additionally, there seems to be a trend for lv-ef improvement in follow-up. early high mortality is influenced by the patients admitted in cardiogenic shock. there might be a trend for increased major bleeding complications. objective: to assess the validity of saps ii (new simplified acute physiology score), comparing it with the previous version, (saps), in a sample of patients recruited by giviti, a network of 128 icu's representative of the italian icu system 9 methods: measures of calibration (goodness-of-fit statistics) and discrimination (receiver operating characteristics curve and area under the curve) were adopted in the whole sample and across subgroups differing in relevant prognostic characteristics. of the 3004 patients recruited during one month period, a total of 1813 patients were included in this study. for the purpose of the comparison of the two scores, patients with less than 18 years, or having cardiac surgery or staying in the icu less than 24 hours were excluded. vital status at icu discharge in the whole sample and at hospital discharge in half cases wher adopted as outcome measure. re$01~: saps ii fits the data equally well compared to the older version (goodness-of-fit p=0.29 and 0 9 in the new and old versions, respectively) but its performance is somewhat better in terms of capability to distinguish patients who live from patients who die (areas under the curve 0.81 and 0.73, respectively). furthermore, saps ii is better in terms of uniformity of fit across relevant subgroups, although substantial over prediction of mortality was observed in trauma patients and in patients admitted without organ failure to be intensively monitored. saps ii performed very wet] also in the subsample where hospital mortality was the dependent variable.satisfactory measures of calibration (goodness-of-fit p--0.47) and discrimination (receiver operating characteristics area=0.80) were observed. c0nr saps ii, a multipurpose scoring system developed in an international study, retains its validity in this independent sample of patients recruited in a large network of italian icus. although it has shown a good performance when adopted to predict icu and hospital mortality in the entire sample, further investigations are warranted. the observed over prediction of mortality in a few subgroups indeed call for a through assessment of the impact of confounders and biases on model performance when saps ii is adopted in samples that do not reflect the "average" icu patient. objectives: 1) assess the effectiveness in a group of intensive care units by means of a quality performance index (qpi); 2) assess the efficiency by means of a resource use index (rui); 3) evaluate the performance of individual icus with respect to both indices (clinical and economical) while controlling for severity of illness. 1270 critical from 17 ucis in catalonia patients alearic islands have been included in the study. inhospital mortality and weighted hospital lenght-of-stay (los) have been considered the outcome variables. severity of illness has been measured with the mpm ii at admission. in each icu, expected mortality has been obtained adding the probabilities of dying for its patients. expected los has been estimated adjusting a second order polynomial to the severity of illness. performance indices have been obtained by dividing the observed by the expected outcomes. re~ult~: the overall qpi was 1.15 and it ranged from 0.58 to 2.05 in the 17 icus. the overall rui was 1 and it ranged l~ont 0.61 to 1.51. there was not a trade-offpattern between clinical performance and resource use. objectives: teaching hospitals often provide [cu care across a variety of specialized services. overall, this approach appears to result in the best risk adjusted survival rates, but at the highest cost (critical care medicine 1993;21:1432-42): recently, there has been increasing focus on markers of overall hospital performance. however, in large teaching institutions, such markers may fail to detect intra-institntional variation at a large tertiary care medical center. methods: first intensive care unit (icu) day, acute physiology and chronic health evaluation iii (apache iii) and active therapeutic intervention scoring system (tiss) data were collected on 1621 random admissions to 8 specialty icus with 90 beds (range 8-14) between february i and december 3 l, 1994. post-operative solid organ transplant recipients were excluded. units included 2 general medical, 2 general surgical, and trauma, neurosurgery, cardio-thoracic surgery, and coronary care units. data were analyzed for risk adjusted outcomes: icu and hospital mortality and length ef stay (los); risk of requiring active 1cu treatment; and icu readmissinn using apache iii risk prediction models. results: the study icus cared for a diverse group of patients. mean apache iii scores ranged from 36.9-55.5; predicted risk of hospital death ranged from 8.5-21.1%. standardized mortality ratios ranged from 0.40 to 1.24 with 4 icus performing significantly better and 1 performing worse than predicted (p<0,05). los ratios and icu readmission rates ranged from 0.95 to 1.09 (ns) and 2.1 to 13.2% respectively. patients predicted at low risk of requiring active icu treatment ranged from 6,6 to 45.8% conclusions: there was wide variation in the mean level of patient severity between icus. after controlling for this severity, outcomes also varied widely. no clear pattern of overall institutional performance was evident. these data suggest that efforts to assess performance, improve quality, and maximize efficiency must be focused within individual units. programmatic evaluation of outcome allows for focused review of the processes of care contributing to good outcome (best practices) and where to focus ongoing quality improvement and cost reduction activities. background and method : we compared icu mortality in different age groups presenting with the same severity of disease. we assessed severity of illness by the physiological day 1 -apache~ (physio-aa) score (thus excluding the age related points). for each of the following physio-a n score intervals (0-5; 6-10; 11-15; 16-20; > 20) , we compared tcu mortality within 6 age intervals (< 40; 41-50; 51-60; 61-70; 71-80; > 80 years -10, 11-15, 16-20) . in these groups mortality may be twice higher in the > 60 years patients than in the _< 60 years. mortality does not vary with age in low (physio a n = 0-5) and high (physio a n = > 20) risk groups. in the low risk group, mortality is low in all the age intervals because of the begninity of illness. in the high risk group, extreme severity of disease probably blunts the impact of age and leads to high mortality rates in all age intervals. introduction: to access the actual social/clinical outcome of the patients who undenvent intensive care therapy oct) is rather difficult, quality of lilr is not easih.' defined and ohserver subjectivity is a prime factor in the evaluation. mortality ratio after discharge must be established and its causes understood. obieetives: the propose of this stud)-is to look into the mortality ratio that occurred on a series of patients that undorwent ict at our unit from of the ~iew point of severity of the original illness and the diagnostic groups. material and methods: during the period of one )-ear (1994), 216 patients were treated at the unit, 45 of them died, and 16 ~ere not matched in our series because os incumpletc records. thirteen patients died in hospital after their reference to other departments, twelve patients were lost after discharge. thus. at the end. only 142 patients were evaluated on the fu. the, were classified into the follov4ng three groups: acute medical, elective surge d 9 and acute and emergency postoperative. the patients were seen at 3, 6 and 12 months after discharge. the, were evaluated in accordance to their abili~, to being self supported in their daily life and capecity to fully return and hold to their pre~4ous jobs. apache 11 scores were evaluated for each of the three groups and correlated to the icu dead, hospital dead, and mortality after hospital discharge, spss package was used for statistical analysis. remlts/conclasions: data shows that 19/142 patients died after discharge from the hospital, of ~itch nine died in the first three months. seventy-eight per cent of the patients were fully self supported in their daily life and 20% showed some kind of handicap. fosty-nine per cent of the patients wore on retirement either due to age or some form of chronic disease, when admilled to our unit. thirty-two peg cent had not been able to return to work, because the" were incapacitated on discharge. only 7% had return to their fully jobs but the period of the stu~, is not enough for all of them to be fully physically recovered. preliminmy statistical analysis shows us significant differences among groups. the aim of the present study is to compare the prognostic performance of five general severity indices ou coronary patienta and to find out if a proper ntatistical hundling of these indices could provide better results in these patients. methods: saps ii, mpm ii (mpm ii0 i mpmp ii24), apach ii end gaprik were evaluated o~ 456 patients with acute myocardial infurction admitted to 17 intensive care units from catulunye. calibration and discrimination were calculated for each index. calibration was calculated by th bosmer-lemeshow test. discrimination was evaluated by the area under the relative operating characteristic (roc)curve. if a model did not show a good performance it was customized using multiple logistic regression. finally, tworeduced models were developed, one fro~ the mpm series (mpm ii24cor) and one from the group apache-saps (sapsiicor).their performances were again evaluated. results: discrimination was high enough for all models. neverthelees, oelibration of apache ii, saps ii and mpm was not satisfactory. thus,mpm ii24, saps ii and gaprik were customized for coronary patients using the logits of both models, and obtaining good calibrations. mpm ii24, and apache-saps were adapted and reduced to 5 (mpm ii24cor) end to 4 variables (sapsiicor), respectively . both models showed better oalibrutions end discriminations than the original models. conolusion| models developed for multidisciplinary patients show a good discrimination when applied on aoronar i patients, but some needed customization in order to improve calibration. the number of variables of the principal model can be reduced (even to 5 or 4 variables) without loosing prognostic accuracy. objective: to compare the ability of two methods to predict outcome for intensive care patients. methods: we included 343 consecutive intensive therapy unit (itu) admissions with an itu stay>24 hrs in a 18 month prospective study (exclusion criteria: burn injury and age <16 yrs). data were couectsd applying the criteria described by the developers [1, 2] . the definition of coma (mpm24ii) was modified and the best assessment within 24 in's, rather than the admission score, was used. statistical analysis included classification tables and receiver operaung characteristics (roc) curves to assess discriminative power, and lemeshaw-hosmer statistics and calibration curves to test accuracy of prediction. results~ average abe was 58 yrs (ranse:16-92) with a male:female ratio of 1.6:1. the actual hospital mortality was 26.8%, mean predicted death rates were 22.8% (mpmz4ii) and 15.2% (ap hi). non-survivors had siguitlcanfly higher predicted risks than survivors applying both methods (p<0.000l, t-test). the total correct classification rates (tccr) for apache iii were bett~r for all decision criteria applied (tccr, decision criterion 50%: apache ]/i 77.1%, mpm24ii 71.4%). the area under the roc curve was 0.75 (ap iii) and 0.66 (mpm24ii) confirming the better discrimination of apache ill. accuracy of risk prediction was similar for both models (ap nl ~2-59, mpm24b ;(2-52, lemeslmw-hosmer). showing some fluctuation, calibration curves lay close to the ideal line for predicted risks -<50% with increasing deviation for higher risk groups (s. figure) . apache iii underestimated the risks of hospital death for almost all risk groups (curve above diagonal), whereas considerable overestimation for predicted risks >40%0 ceenred with mpm~ii. objective: to assess the goodness-of-fit of the apache iii model for british itu patients. methods: we prospectively studied a cohort of 715 adult patients consecutively admitted to a medical-surgical itu over a period of 18 months. patients with burn injury, age < 16 yrs and itu stay < 4 hrs were excluded. using a eomputerlsed database, we routinely recorded 24 hrs apache ill scores. predicted risks of hospital death were computed by critical audit ltd, london. accuracy of risk prediefion was assessed by hosmer-lemeshaw chi square (;(2) statistics and calibration curves [1 ]. discrimination was tested employing classification tables and receiver operating characteristics curves (roc). restths: the mean age of the 453 male and 262 female patients was 59 yrs (range: 16-92 yrs). of these patients, 64% were medical admissions, 17% were admired after emergency and 21% after elective surgery. the observed hospital mortality was 25.4%, the overall mean predicted death rate was 16.8%. mean predicted risks were siguifieanfiy greater for nonsurvivors (38.0 %o) than for survivors (9.6%, p<0.00l, t-test). apache iii showed good calibration (z2-~59, lemeshaw-hosmer). however, the calibration curve lay above the diagonal for almost all risk groups reflecting the tendency to underestimate actual mortality (s. figure) . the best total correct classification rate (tccr) was 89.3% (decision criterion: 50%). the area under the roc curve was 87.6% confirming the good discriminative ability of the model. objectives: the aim of this study is to point out the discrepancies between needs and actual treatment of less severely ili patients admitted in italian intensive cam units (icus) requiring only intensive monitoring, and verify the substantial likelihood of data comparing those collected from a national short term study with a regional long ternl use. ~: less severely ill patients ("observed patients") were only monitored; they did not require intubation, even if for a short period (less than 24 houm) or major cardioeiranlatory supports, and were neurologically normal. epidemiologieal national data were obtained from giviti group (gruppo italiano valutazione interventi in terapia intensiva); this cohort study, collected 5092 patients, in two months in summer in 1992 all over italy. regional data were echieved in a three years entlection (1990-i992) in lombardia' icus from archidia group (arehivio diagnostieo), including 10065 patients. mortality, severity score, diagnostic category and some typical intensive procedures were analysed and compared in both studies. patients' disgunstie categories were defined as surgical, medical and trauma, according to the main diagnosis and the presence/absence of surgical procedures. rr162 observed patients account for 23.2% and 22% of all icu's patients respectively in national and regional data. very tow mortality rate was found in national data (2.3%) and extremely low mortality in regional data (0.6%). in both studies mortality, s.a.p.s. and length of stay were much lowor in "observed patients" than in general icu's population (mortality: 25.7% and 22.3%; 8.a.p.s. score: 10.6 and 13; iength of stay: %7 and 9). homologous distribution of patients in the two studies was noted for what concern their diagnostic category, aside from a slight prevalence of tranmatised patients in the giviti study. in the two groups the surgical patients were respectively 47% vs. 57%, medical patients were 34% vs. 31% and traumatised were 19% vs. 13%. 92% of "observed patients" in national study and 93% in the regional did not received any intensive procedure. only a minority of these patients availed haemodynamie eonu'ol with swan-ganz or renal haemofiltration. conclusions: these results underline that about one fourth patients admitted in italian icus benefit an oversized slructure i, relation to the real needs of their pathology. in hot more than 90% did non received any advanced treatment and mortality and s.a.p.s. score were substantially lower respect to general population. the results obtained from these two studies are similar, suggesting an uniform distribution of the case mix in italy, even if a different recruitment period and a different gengraphieal distribution were used. some discrepancies in the two studies were found in the diagnostic categories moreover regarding the tranmatised patients (19% vs. 13%); this can be explained from the seasonal (summer) characteristic of the national study. mutuality, yet very low, is different in the two groups, but these data do not allow any definite explanation. finally these epidemiologieal survey suggest need of further studies settling more strict criteria of admission in icu. this study aims to evaluate patients outcome, quality of care and effectivity of therapy in our intensive care unit. the main goal was to indentify factors that the most influence that outcome. during 1994. the authors collected data of patients outcome and predictor variables. overall mortality rate was 39,3%. the most common causes of death were infection. the diagnosis of sistemic inflammatory response syndrome (sirs) and multiple organ dysfunction syndrome (muds) significantly correlate with death (90%). average length of stay was 6.6 days ~. 55% patients died in the first ten hosiptal days and only 18% after 30 days. age was directly correlated with death 50% of dead were older then sixty years. an analysis of physiological variables showed that serum levels of gl~cose (55%) and natrium (71%) were in optimal physiological values. serum proteins (72%) and haemoglobin (50%) levels were inversely related to death. multivariate showed that alveolo-arterio difference in 02 content was the most informative of all mortality predictors (mean value 22,4 mmhg in 90% patients io>mrnhg). factor that most influence the patients outcome was infection (sepsis) and muds. use of predictive indicators of outcome in critically ill patients may help to assess treatment regimens and to compare patient groups. acute physiology and chronic health evaluation (apache if) score (crit. care had. 1985; 13: 818-29) and the sepsis score of elebute and stoner (br. h surg. 1983; 70: 29-31) have been used, objectives: to compare sepsis score and apache ii score in predicting outcome of critically ill patients. methods: overall survival during the past 8 years for patients in our icu was calculated = 62% (prior probability). the outcome of 230 patients who were admitted to our icu for > 72 hours was observed. apache ii score on admission, patient predicted risk of death (apache ii risk) and the sepsis score on the first day of antibiotic course were prospectively recorded. discriminant function analysis of the scores in relation to outcome was performed. results: apache ii and sepsis scores in the survivors were significantly lower than in those who died (21.6 i 7.2 v~s 25.6 • 6.5 and 10.9 • 5 v's 15.2 • 5.9 respectively p < 0.001). correct prediction of outcome by each score is shown in discussion and conclusions: although both scores have been previously evaluated in predicting outcome of icu patients, studies of the sepsis score were conducted in small numbers of patients or involved additional measurements not routinely available. this study demonstrates that the sepsis score alone or in combination with apache ii score is more effective than apache ii score in predicting outcome. objective to test the hypothesis that resuscitation titrated against gastric intramucosal ph (phi) improves survival in critically ill patients as suggested by gutierrez et al~. method emergency admissions to the intensive care unit were randomized into control and intervention groups. in the control group phi was measured at 0, 12 and 24 h while in the intervention group phi measurements were made 4 hourly for 24 h. both groups were managed according to the same guidelines to achieve the following targets: mean arterial pressure >70 mmhg, systolic arterial pressure >90 mmhg, urine output >0.5/ml/kg, haemoglobin >8 g/dl, blood glucose < 12 mmol/1, arterial oxygen saturation >94% and correction of uncompensated respiratory acidosis. if the phi was < 7.35 after achieving these targets, or after maximal therapy to achieve the targets, patients in the intervention group were given fluid to ensure an adequate cardiac preload and then dobutamine at 5 then 10 mcg/kg/h, titrated against phi. this additional therapy was continued until 24 h after entry into the study. in each year patients were subdivided in two series with random selection, so that the 1st series contained abeat 2/3 and the 2nd 1/3 of the patients. the 1st series of all the years constituted the devdoping data set and the 2nd series the validation data set. with data of the 1st series (642 patients), we created the predictive model, using stepwise logistic regression (bmdp, usa). each patient has been evaluated in die 1st, 5th, 10th and 15th day, calculating for each lime the apache ii score (for a total of 1444 records), independent variables were, besides time and apache ii of the time ( michaloudia g,, melissaki a., alexias g., gogafi c., kolotoura a., krimpeni g., pamouktaoglou f, filias n. objectives: to determine the medical staff's attitude towards various ethical issues methods : between january 1994 and february 1995,185 anonymous questionnaires were sent to 20 intensive care units, all over greece. results : 107 questionnaires (57,7%) were replied and returned back. of them 58,9% were answered by male and 41,1% by female. the doctors replied in the following rate :81,2% aged up to 34,80% aged between 3544 and 94,4% aged over 45. 36 questions were answered and were divided into 3 main topics, as following: 1. admission criteria: limited bed availability was the main cause for refusing admission in 54,5% of icu's. 54,5% evaluated each case's viability and only 10,3% used some prognostic score system. 21,5% of icu's accepted all cases and a significant percentage (64%) gave in to pressure coming from their colleagues (72,7% female and 58,7% male). 2. informing the patient/relatives: only 6,5% was willing to tell the whole truth, while 39,2% had given selective information.. in the case of iatrogenic incident, 58,9% withheld it, because either they feared legal implications (34,6%), or lost of trust (46,7%). doctors are asking consent from the patient and/or his family, in order to include him/her in research protocols, in a rate of 82,3%, while only 55,1% found informed consent necessary for the proposed treatment procedure. 3. withdrawal of therapy/dnr orders/organ donation: 80,4% were willing to withdraw complex treatment in patients with short life expectancy, except of administi'ating intravenous fluids, feeding and analgesics. in 34,6% such a decis~n was unanimous, while the percentage of those carrying it out was 69,1% (72,2% female, 63,9% male). in case of brain stem death 87,8% (67,3% female, 85,7% male) withdrew any life support. 67,3% would like therapy withdrawal to be legally established, while only 12,1% would perform euthanasia, if there was substantial legal cover. for these cases, relatives' consent was considered to be necessary from a percentage of only 11,2%. 83,2% considered organ donation to be a necessary proposal, while 10,3% refused to ask the patients' relatives for an organ donation, either because they didn't have the psychological strength for it (3,7%), or because they doubted the procedures' objectivity (4,7%). note: in greece, icu beds are less than 1% from the total number of hospital beds available. only a percentage of 35-50% of these admissions comes from the same hospital, with a potentially direct evaluation. usually an icu doctor has to be informed through the telephone. finally, employment conditions in greece are such that any changes of the medical and nursing staffare limited. conclusions: the mathematical model we found has been validated also in the second series and the discrimination capability increases with time. using this model we can evaluate the probability of survive at every, time. its application at different times permits a better evaluation of haemodinamically instable patient trend. introduction: the feasibility to assess pulmonary capillary pressure (pcap) offers the opportunity to determine the longitudinal distribution of pulmonary vascular resistance (pvr). the purpose of this study was to measure pcap and to calculate pvr to determine whether relevant shifts in the distribution of pvr could be expected after routine cardiac surgery. methods: the study population consisted of 25 consecutively admitted patients after cardiac surgery. surgical procedures included coronary artery bypass graft (cabg) (n=14) and mitral valve replacement (mvr) (n=t 1). pcap was estimated by analysis of the pressure decay tracing after pulmonary artery occlusion. after estimation of pcap precapillary (ra) and postcapillary resistance (rv) was calculated. a complete set of hemodynamic variables was obtained at 1 hour and at 6 hours after operation. results: there were no significant hemodynamic changes during the first 6 hours after surgery. the mvr group maintained pulmonary hypertension and higher levels of pcap. ra/rv, reflecting the longitudinal distribution of resistances, remained unchanged. however, rv predominated ra during the postoperative period in both groups. objectives: evaluation of the influence of long-term continuous i.v. administration of the ace-inhibitor enalaprilat on regulators of circulatory homeostasis. methods: t9 trauma and 26 sepsis patients randomly received either 0.25 mg/h (group i, n=15) or 0.5 mg/h (group 2, n=15) of enalaprilat i.v. or saline solution (control, n=15) as placebo for 5 days. plasma levels of endothelin-1 (et), atrial natriuretic peptide (anp), renin, vasopressin, angiotensin-ii, and catecholamines were measured before injection of enalaprilat (='baseline' values) and during the next 5 days. results: except for et, plasma levels of all vasoactive substances exceeded normal range at baseline. angiotensin-ii significantly decreased during enalaprilat infusion (0.25mg/h: from 53.1• to 22.1• pg/ml; 0.50mg/h: 62.1• to 17.9• whereas it remained significantly elevated in the untreated control patients. vasopressin increased only in the control group (p<0.01) and decreased after 0.50mg/h of enalaprilat. et remained almostunchanged in group 2, whereas et increased significantly in the control patients (from 4.9• to 10.t• on the 5th day). catecholamine plasma levels (epinephrine, norepinephrine) markedly increased in the control group (p<0.01), but they did not change significantly throughout the study period in both enalaprilat groups. conclusions: continuous i.v. administration of the angiotensin-converting enzyme inhibitor enalaprilat beneficially influenced systemic and local vasoactive regulators of the circulation, which are normally increased in the critically ill. thus patients at risk of (micro-) circulatory abnormalities may profit from enalaprilat infusion. objectives: to determine the time taken for hemodynamic and gas exchange variables to a reach stady-state after a change from supine to trendelenburg position (trp). methods: we prospectively studied 8 adult patients with severe sepsis or septic shock requiring hemodynamic monitoring. usual cardiorespiratory parameters were measured at baseline, 15 min after the patient was placed in a trp and again 15 min after the return to a supine position. a fiberoptic pulmonary artery catheter (svo~ oximetrix, abbott) allowing continuous svo 2 monitoring wa~used. during the protocol we also continuously measured sao~ by pulse oximetry and vco~ and vo 2 by monitoring partial concentration of o2and co 2 ir~ inspiratory and expiratory gases (deltatrac metabolic monitor, datex). therefore, we were able to monitor cardiac output variations by dividing vo~ with arteriovenous difference according to the fick equation (co-fick). results: no significant difference in hemodynamic status was observed 15 min after the patients were placed in trp. despite the fact that no significant change was observed in co and vo~ estimated by thermodilution, co-fick had a tendency to dedrease continuously in trp and then to return to its initial value when patients regained supine position. respiratory gas analysis showed a small but persistent continuous increase in vco 2 without a similar trend in vo 2 values. conclusions: we conclude that no significant hemodynamic effect was detected in our patients after 15 min in trp. evaluation of vo 2 from respiratory gases analysis after a change in body's position should be interpreted with caution, since the patient may not yet have reached a stady-state after 15 rain. since vo 2 did not change, vco~ increase was probably due to position related changes in-pulmonary gas exchange and not to a change in patient's metabolic status. objectives: to determine whether changes in svo 2 and/or other hemodynamic parameters during weaning trials could be used to predict successful weaning. methods: we prospectively studied 10 adult patients with a history or clinical evidence of cardiovascular dysfunction, who were unable to tolerate spontaneous breathing (sb) for 3 hours. for all these patients right heart catheterisation was considered necessary in order to detect hemodynamic alterations during weaning. a fiberoptic pulmonary artery catheter (svo 20ximetrix, abbott) allowing continuous svo 2 monitoring was 5sod. hemodynamic status was evaluated ~t baseline and after one hour of spontaneous breathing through a t-piece. patients were assigned to one of two groups depending on whether they tolerated sb for 3 hours. data were analysed by analysis of variance and unpaired student's t-test we also used multiple linear regression analysis to determine which hemodynamic variables were correlated with the magnitude of svo~ change and multiple discriminant analysis to determine if asy of the above variables were associated with toleration of sb for 3 hours and/or successful weaning (s-w). (j physiol 1995; 78." 696-701) . we tested the hypothesis that the ventilatory stimulation by dead space (vd) loading and 3% co2 inhalation is accompanied by a proportionate cardiovascular change. methods: six healthy subjects, mean age, 25 year, performed three incremental exercise tests in a randomized order: 1) inspiring air without vd (air control, ac); 2) inspiring air with vd of 920 ml (avd); 3) inspiring 3% co2; 21% oxygen, balance nitrogen. the ventilatory responses were examined at matched heart rate (hr) equivalent to 90% peak hr. results: ventilation (vi) was significantly greater (p<0.0001) during the avd and co2 tests than during the ac test at the same work rates. end-tidal co2 (petco2) and estimated arterial co2 (paco2) were significantly greater (p<0.01) at 150 w and 200 w. oxygen saturation was significantly lower (p<0.05) during the avd test than during the ac and 3% co2 exerdse. at matched hrequivalent to 90% peak hr, vi was significantly greater (p<0.01) during the avd and 3% co2 tests than during the ac exerdse (123 l, 121 l, and 91/). conclusion: we conclude that the increase in xri and petco2 due to vd loading and 3% co2 inhalation is not associated with an acceleration in hr. sup.ported by mrc (canada). objeetlve: the production of large amounts of oxygen radicals from the onset of ~en may be responsible, st least in part, for peroxidative damage to myocardial tissue. the aim of this study was to evaluate the time dependence of plasma tbars in patients with am] receiving thrombolytie therapy (tt). patients and m~hods: filiy eight patients admitted in icu (46 men and 12 women; mean age 50.6 4-16.02 years) rec~ving systemic tt for possible am] were ~died. all patients received recorabinant haman tissue-type plasminogen activator (r-tpa). the mean time fi'om the onset of symptoms and the be~nning of tt was 3.01 4-2.13 hours. peripheral veao~s blood samples were obtained fi'om each patient before and serially after tt (0, 3, 6 and 9 hours). tbars levels woe determined by using a spectrophotometrie technique. rq~r fusion was identified by the timing of ereatine phosphate kkmse (cpk) peak (<15 hours). table i list the variation of plasma eoneenlrations of tbars (mean 4-sd) in groups (a,b, and c) as a function of time from the beginning of tr. co,arisen oftbe 0 time cuncentzatiens reveal a difference p<o.01 between group ami (a and b) versus group c. the between-groups (a-c) comparison showed significant differences (p<~).05) at 3,6 and 9 hours fi-om de begin~g of'it. comparison ofthe 3,6 and 9 hours eonocnlratiens did not reveafl any difference becween n~ (group b) and angor (group c). condmien: we have observed a maintenance of high level of tbars in reperfused patients enly. these results suggest that the increase of tbars levels may be due to phospholipid derangament of reperfused myocytes. objective: to detemline if tbars levels may be an early index reflecting peroxidative damage in ami patients. patients and methods: by using a spectrophotometric teclmiqtm, based on a modificatien oftbe buege and aust method, tbars levels ware determined in 51 healthy subjects (group i) , 43 patients with several risk factors of ischaemic cardiopathy (group if) and 99 icu patients with possible aim (87 were confirmed ami (group ma) and 12 were angor (group iiib)). peripheral blood was obtained at the time of the icu admission in the group i11. electrecardiographic data and eehocardiographic wall motion were used to identify ami location (anterior, inferoposterinr and indeterminate). statistical analysis was made with a standard statistical package (rsigma, horus hardware). , h 4, s, , h statistically significative increase of ' ix~e/ea.ee x~* tbars levels (p< 0.001) only in pstients with con/tuned ami (group ilia) . no differences were found between the rest of the groups. the figure shows the relationship between the tbars values and the time from the onset of the aim's symptoms. conclusion: we have found that in am/patients the tbars levels can be used as an early index reflecting peroxidative damage occurring to ischemie tissues. perioperative risk in patients with ischemic heart disease. the protective role of diltiazem. lg. hatziantoniou, p. tassiopoulos, c. fakiolas, ! g. foussas~ m. lycourinas perioperatlve morbidity and mortality are mainly of cardiac origin, particularly among elderly patients (pts) ~ith ischemic heart disease. we performed a randomized trial, in order to investigate the possible protective activity of diltiazem (d). methods: 58 pts (aged zo• with a history of coronary insufficiency were subjected to major urological operations. they were randomized to two groups: group i (29 pts) -p:ithout d and group ii (29 pts) -d ~as administered 0.15 mg/kg iv bolus 12 hours prior to anesthesia and 0.15 mg/kg/h upon the end of operation and for at least 12 hours post-operatively. iv was substituted by oval administration of 2x120 mg starting the next day and for the rest of hospital stay. all pts underwent daily clinical, electrocardiographic and laboratory (ck-mb) control. results: are shown in the following interactions between the heart-lung unit and a cardiac assist device are the crucial issues for successful outcome of patients under mechanical circulatory support. several factors have been indicated and we can diffrentiate between anatomical and hemodynamic interactions. especially right heart failure and pulmonary hypertension have been proven to limit left ventricutar assist device (lvad) performance. in order to study the effects of right heart failure (rhf) and pulmonary hypertension (pht) on mechanical circulatory support, we designed an experimental study in dogs and combined in six groups left heart failure (lhf) and rhf and pht, respectively. in the control group we induced lhf with subsequent occlusions of lad and cx followed by reperfusion: in the next group we supported with an lvad during 90 minutes starting 5 minutes after cx occlusion (group if). in the next group we added right ventricular ischemia by occluding the rca simultaneously to the cx (group iii). these both groups we repeated only with pre-existing acute pht induced by the injection of 1o0 fm glass beads (groups vi and v). a last group was similar to group v (lhf and rhf and pht), but instead of a lvad, we used bvad support. in i and v no animal survived the second perfusion period (4 hours). in ii and iii (no pht), all dogs survived in stable conditions. in iv (lvf, pht), 60 % survived, the remainder died of incomplete recovery. in vi with bvad, only 40 % survived. in iii and v, during occlusion of the rca, the right ventricle stopped contracting and right atrial pressure and mean pulmonary pressure equalized. this lead to a significant drop in inflow on the left heart side, which still provided sufficient lvad performance in case of absent pht (iii) and lead to death due to "low lvad output" syndrome in v. this "low lvad output" snydrome could be partly overcome by bvad support. we conclude that heart-lung interactions play a major role in lvad performance. by optimizing the perioperative management, they all can be overcome, except the occurrence of alveolar leakage syndrome. obiectives : we propose a new approach of transmural pulmtmary artery occlusion pressure (paoptm), in presence ef peep, from the folluwing hypothesis : if respiratory swings of paop are compared to those of alveolar pressure (apalv = plateau pressure -total peep), an index of transmission of airway pressures to pulmonary vessels is obtained (i t = apaop/apalv). the value of the product of it by peep can be substracted from end-expiratery paop (paopee) to obtain a calculated paoptm (paoptrnc). thus paoftmc = paopee -(apaop/apalv x peep). methods : we tested this hypothesis in 67 patients by comparing paopee, paoptmc, and paopnadir obtained within the first 3 seconds after disconnection from the ventilator, value of reference of paoptm in absence of intrinsic peep (peepi). at zeep, peepi was absent in 38 patients (group a) but present in 29 others (group b). patients were studied under the peep level chosen by the attending physician-(gr a : 11 +_ 2 ; gr b : 11 _+ 3 cmh20 ). results : l. gr a : paopee (12 _+ 6 mmhg) differed (p < 10 -3) from paopnadir (8 • 6 mmhg) and paoptmc (8 _+ 6 mrahg). gr b : paopee, paopnadir, paoptmc differed between themselves (14 "2-6, 12 + 6, 10 + 6 mmhg respectively). 2. good correlatkm (r (i.99 ) and agreement between paopnadir and paoptm were found in gr a, while there was no agreement in gr b (bland and altman analysis). 3. lung compliance (v t/bpah,) correlated well with i t in both groups (r -0.92), suggesting that our hypt)thesis was strtmg even in patients with peepi. conclusions : paoptm under peep ventilation can be obtained, even in patients with peepi, from parameters easily measured at the bedside. introduction: cardiopulmonary bypass (cpb) may cause ischemia in several organ systems like the heart, brain and kidneys. reactive oxygen species (ros) are formed by subsequent reperfusion and stimulation of neutrophils by cpb. ros are harmful to biological systems e.g. cellular membranes, enzymes~ dna. many natural antioxidant systems protect against the effect of ros. in patients undergoing cabg we evaluated the production of ros by total plasma antioxidant status (tas) and lipid peroxidation measured by lipofuscin flip). methods: tas (randox, uk) and concentrations ofllp (tsuchida et al.) were measured in 16 consecutive patients undergoing cabg. all the patients had a normal serum creatinine clearance (>50 ml/min). serum samples were obtained a) before operation, b) after removal of the aortic crossclamp, c) at admission to the icu, d) 4 hours after operation, e) 22 hours after operation. results: tas was significantly decreased after removal of the aortic crosselamp ( b, c and d lower than a), followed by a subsequent significant increase of lip ( c and d higher than b). the levels of tas and lip returned to baseline 22 hours after operation. methods: 10 patients with preoperative lvef<40% undergoing coronary artery bypass grafting were studied. after surgery, a 3f femoral artery catheter was inserted and connoted to a fiberoptic monitoring system (cold z-02t; pulsion medizintechnik, germany); this allows, with a double-indicator dilution technique, the calculation of cardiac index (ci,l/min/m2), intrathoracic bood volume (itbv,ml/m2), pulmonary blood volume (pbv,ml/m2) and extravascular lung water (evlw,ml/kg). with a 7f pulmonary artery catheter, wedge (w,nunhg) and central venous pressure (cvp,mmhg) were measured, while 02 extraction ratio (o2exr,%) and oxygen delivery (do2,ml/min/m2) was calculed. peak inspiratory pressure (pawp,cmh20) and mean airway pressure (mawp,cmh20) were measured with a varflex flow transducer (bicore,sensormedics,us). the patients were studied after 60 minutes (to) of volume controlled standard ratio ventilation (vc), and after 60 minutes (ti) of stabilisation period of pcirv (67% inspiratory time, 0 % pause). vt,ve and total peep were held constant in every mode of ventilation. 1+_0.4" *'p < 0,05 versus to conclusions: these data show that pcirv 2:1 is a safe ventilatory support also in cardiac patients with impaired ventricular function, and monitoring of itbv is more reliable to measure and optimise circulatory volume status, than w and cvp. c.ledeki-,g.rldisis,s.karotzai,c.micheilidis,m.agioutantb, g.beltapaulos. objeolivee:to evaluate the influence of lvswl on the well known correlation of sr and svo2. paw eight patients (12 melee end 16 females) were included in this study regerdlen of the icu ~h"niseion couse. all paints were ,'~theta~ with e fiboroptir pulmonary artery catheter connected with an oxymetfir (r)~ so2/co abbot computer.for any pulmonary artery catheter insertion, two pain= of sr and svo2 were obtained, one dudng inserlion and one during taking the catheter out. for any pair obtained, we eleo collected the deta concemig with the pedient's hemodynamir and oxygenation end we calculated the lvswi. were significantly (p<o.05) higher in group i. conclusion: breathing room air, the less hypoxemic patients had a rather satidactory do2 and tissue oxygenation (pro2) because they had properly adapted their ci. the absence of this mechanism (right heart impairement ?) in the hypoxemic patients, in spite of higher blood ne and e, was responsible for the poor do2 and pro2. objectives: acute massive pulmonary embolism (pe) increases pulmonary artery pressure (pap) and dght ventdcular aftedoad, which may lead to dght ventdcular failure. inhaled nitdc oxide (no) selectively dilates pulmonary vessels. thus, inhaled no may be of potential therapeutic value in the management of the acute phase of pe. methods: following institutional approval, ten piglets (body weight 17+2 kg) were anaesthetised (midazolam/piritramide) and ventilated using a modified servo ventilator (siemens elema, sweden; fio 2 0.3). the following variables were obtained: systolic pap (spap), mean pap (mpap), diastolic pap (dpap), mean artedal pressure (map), central venous pressure (cvp), pulmonary capillary wedge pressure (pcwp), cardiac output (co), and artedal (pao2) and mixed venous (pvo2) oxygen saturation. pulmonary vascular resistance (pvr) was calculated. to induce pe, 300 pm microspheres (sephadex g50 coarse, pharmacia biotech, freiburg, germany) were injected in an amount sufficient to increase mpap to 45 mmhg initially. 45 rain after embolisation when haemodynamics were in a steady state (control 1), inhaled no was administered. further measurements were taken 5 min after 40 ppm no (no 40), 5 min after 80 ppm no (no 80), and 10 min after discontinuation of no administration (control 2). the administration of inhaled no resulted in a significant decrease in spap, mpap, and pvr. the cessation of no inhalation led to a complete return to pretreatment control values (table) . co, map, cvp, pcwp, pao?, and pv~ remained unchan no administration. objectivss:evsluate the yield of recombinant tissue plasminogen activater(rt-pa, actiiyse ~ 9oehringer ingelheim) as a thrombolytic agent ie pulmonary embolism ie .kr~auma patients. methods: in 1994 five patients with diagnosed pulmonary embolism(blood gases, chest x-ray, echocardiography, perlesion lung scans) were given 100rag of actij.yse as ~ 2hrs infusion, followed by heparin~single 5000j. ihjaction snd iefusion ie doses 300-400j./kg/day).the effectiveness of rt-pg thrembo].ytic sctier~ was svaluated im 3.24. hrs(blood gases) amd on the third dayafter t~eat-,ment(~chocardiography and lung scan). results: three hrs after actilyse was given we observed significant clinical imprevemeet,confirmed by elevation of pao 2 and sao 2 in dlood.echocardiegr~m on the third day showed regression of pulmonary hypertension and lung scans showed 45% improvement in total lung perfusien. except slight bleeding from the site of injection nc~ more thromboiytic complicatioos were observed. conclusiens: actilyse used in five hrauma patients with pulmonary embol.ism was a very effsctive and safe thrombolytic agerlt, inducing rapid and evident clinical improvement. intermittent positive pressure ventilation (1ppv) by increasing pleural pressure, decreases the pressure gradients for systemic venous return and left ventricular (lv) ejection. we have previously shown that when inspiration is synchronized with systole using synchfjv in patients with severe cardiomyopathy, that cardiac output (co) was increased relative to both ippv and non-synchronized hfjv (1). however, it is not known if similar effects could be realized in ventilatordependent patients with lesser degrees of lv dysfunction and fluid overload. we thus compared synci-ifjv to ippv in stable patients following coronary artery bypas grafting. methods: 13 normothermic who were stable (< 20 % variance/h.) were studied following admission to the sicu. heart rate (hr), mean arterial (pa), pulmonary artery occlusion pressure(ppao), co by continuous thermodilutiun (vigilance, baxter, usa), mixed venous saturation (svoz) and arterial blood gases (abg) were measured after 30 rain. of each step. ippv was simv adjusted for a rate, tidal volume and fit2 to insure both normal abg and a peak airway pressure < 35 cm h,o. syncfifjv was delivered by a hfjv ventilator (acutronic) during systole with ventilator parameters adjusted to optimize abg. protocol: patients received three ippv runs (ippvi.3,5) with synchfjv runs interposed (hfjvz4). statistics: anova for repeat measures and paired t-test were used to compareruns and demonstrate variability. results:no significant difference in any of the measured hemodynamic variables were seen among the ippv and synchfjv runs (table) . post hoc separation of patients by preoperative ejection fraction (ef; ef > 40% ; n=10 and < 40%; n=4) did not alter these results. back~ound: in man, vascular endothelium-bound ace is expressed in concentrations greater than 50x that in serum and is believed to be the site of synthesis of circulating angioteusin il it is unclear whether ace inlubitors interact similarly with ace in different vascular beds. coronary vessels possess all the components of the renin-angiotensin system, including ace which may be involved in normalcardiac homeostasis, as well as in the pathogenesis of various cardiomyopathies. obiecfive: to develop a method for assaying the interaction of ace inkibitors with coronary endothelium-bunnd ace in man, methods: ace a~aty was meas~ed in five patients undergoing cabg surgery, from the transeuronary hydrolysis of the synthetic ace substrate 3h-bpap. trace mnou~ of ~fi-bpap (4gci) were injec~d as a bolus in the root of the aorta and simultaneously blood was withdrawn from a coronary sinus catheter into a syringe containing protease inhibitors which prevented the convession of umeaet~ ai-i-bpap by blood ace. the sample was later centrifuged to separate cells from plasma and the radioactivities due to formed product (~rl-bphe) and total sh were astimated in a [b-counter. two additional such determinations of ace activity were perform~ the second in the presence of 1.5pg/kg e (coinjected with ~-i-bpap) and the third ten minutes after e. results: all subjects were hemodynamically stable throughout the course of the there were no noticeable hemodynamic effects of e. control transcorunary metabolism of~-bpap averaged g0-a:3%, in agreement with previously reported data. in the presence of e, % metabolism of ~-bpap was reduced to 21• reflecting a 85• inhibition of normal ace activity. ten minutes after e, ~ri-bfap metabolism had partially recovered to 52:l:10%, representing a 50-a:15% inhibition of control ace activity. from this data, the dissociation constant of e for coronary ace in vivo was estimated as 6.8x10 "4 sec "l. conclusions: we have demonstrated the feasibility of repeated, reproducible measures of coronary endothelium-bound ace activity and of its inhibition by e. this procedure is safe and can be used to study the role of ace in normal cardiac function and in card pathologies. objectives. primary pulmonary hypertension (pph) is a progressive fatal disease of unlmown origin, with median life expectancy of less than three years after diagnosis. the responsiveness of pulmonary hypertension to a variety of vasodilator agents led to the speculation that, concomitant with vascular renmdelling processes, persistent vasoconstriction is an important feature of the disease. long term use of ca-channel blockers and intravenous pgiz may improve mortality in certain populations of pph patients, but both of these treatments lack selectivity for tire lung vasculature. the aim of this study was to test the efficacy of aerosolised prostacyclin and its stable analogue, [loprost for selective pulmonary vasodilatation in pph. methods: in three patients with pph, we compared aerosolisation of prostaglandin iz (pgi2) and iloprost to a battery of vasodilatory agents (diltiazem, nifedipin, inhaled nitric oxide, intravenous pgiz). results: nebulisation of pgi2 and iloprost tumed out to be most favourable for achieving effective and selective pulmonary vasodilatation. pulmonary vascular resistance decreased from 1664 + 75 to 1054 -+ 93dyn*s*cm 4 (p<0.001) and pulmonary artery pressure from 63.3 + 3.1 to 528 + 3.4 mmhg (p < 0.05), cardiac output increased from 2.66 + 0.11 to 3.57 _+ 0.16 i/rain (p < 0.001), mixed venous oxygen saturation from 49.6 _+ 2.2 to 63.3 + 2.8 % (p < 0.001) and arterial oxygen saturation from 87.9 + 2.6 to 93.6 _+ 2.2 % (mean _+ sem of 7 trials in 3 patients). 5-month iloprost nebulisation in one patient (100 gg/day in six aerosol doses) demonstrated sustained efficacy of the vasodilator r~men. conclusion: aerosolation of pgi2 or its stable analogue may offer as new strategy for selective pulmonary vasodilatation in pph. endothelial adhesion molecules may play an important role in the pathogenesis of myocardial cell damage, and may contribute to the progression of heart failure. we measured the plasma soluble intercellular adhesion molecule-1 (sicam-1), vascular cell adhesion molecule-1 (svcam-1), and e-selecfin (selam-1) levels in 27 patients with acute myocardial infarction admitted within 6 hours after onset. peripheral venous plasma-samples were collected at the time of admission, 12, 24, 36, 48, and 72 hours after onset. plasma soluble adhesion molecule concentrations were determined by elisa. patients were divided into 3 groups as follows: group 1; killip's class (k) 1 and 2 without thrombolytie therapy, group 2; k 1 and 2 with thrombolytic therapy and group 3; k 3 and 4. both plasma sicam-1 and svcam-1 concentrations in group 2 and 3 were elevated rapidly and significantly and maintained at a high level during the first 3 days. plasma selam-1 level did not change in any of the groups. these results suggest that the adhesion molecules icam-1 and vcam-1 may play a role in the pathogenesis of myocardial reperfusion injury and may indicate its severity in myocardial infarction. objectives: nitric oxide (no) is known to exert cytotoxic and negative inotropic effects on cardiomyocytes. no synthase activity has been reported to be increased in infarcted area in animal model of myocardial infarction. these findings suggest that no may be an important regulator for myocardial damage and cardiac function after myocardial infarction. we measured plasma no27no3-(nox) levels and estimated serial changes in acute phase of myocardial infarction. methods: subjects were 15 patients admitted within 3 hours after onset. venous blood samples were collected at 3-hour intervals on the first day, 6-bour intervals on the 2nd day and 12-hour intervals on the 3rd day and 4 th days after onset. plasma nox concentrations were determined by griess method. results: the time course of the plasma nox levels (mea~+sem) displayed a tendency to gradually increase and to make a biphasic pattern with two peaks about 24 hours and 2-3 days after onset (basal level; 32.8_+4.9, first peak; 42.0!-_7.0, second peak;47.0+7.6 ram/l). plasma nox concentration was not influenced by the thrombolytic therapy, and nox values at the time of 60 hours after onset were significantly correlated with maximal plasma creatine kinase level (r=0.83, p<0.01). the levels of plasma nox in the early stage of myocardial infarction (from admission to the 4th day after onset) did not correlate significantly with the hemodynamic parameters (left ventricular ejection fraction, pulmonary capillary wedge pressure). conclusion: the early and late increase in no production after myocardial infarction may be implicated in the deterioration of myocardial contractility and induction of myocardial damage in the early phase of myocardial infarction. range 36-85) fullfilling the high risk criteria of shoemaker (colectomy 13, gastrectomy 10, pancreaticoduodenectomy 4, others 6). patients were admitted to the icu preoperatively. arterial and pulmonary artery catheters were inserted and hemodynamics and oxygen transport were measured at admission and after stabilization to predetermined physiological end points. patients were considered stable when ci >2.5 l/min/m 2, pcwp >10 mmhg, hb >100 g/l, sat2 >.94. objectives: evaluate the acute effects of 0,08 mg ipratropium bromide and 0,2 mg fenoterol (ibf) inhaled dose on pulmonary function in 17 nonsmocers (nb:m) and 14 smocers (s) with sever (new york heart association class ii-iii), stabile congestive heart failure(chf) and 12 healthy subjects. methods: pulmonary function tests were performed < 3h postprandial. the tests consisted el arterial blood gas aspiration followed by routine spirometry and pletismography, and single-breath gas analysis. after performance of these maneuvers, the patients was administred 4 puffs-ipratropium bromide (0,08rag) and fenoterol (0,2 rag). for 0,5 h, spirometry was repeated. results: in resting, pulmonary abnormalities observer in the s group were more severe then abnormalities observere in the nsm group. after treatment with ibf the improvement in pulmonary function was even more marked in patients who had smoked. the mean changes by forced expiratory volume in 1 second(eevt) was 8,1% (p<0,00t) improvement and 3,9% (p<0,ob), forced expiratory flow betwen 25% and 75% of the forced vital capacity (fef25.75) was 30,8% (p<0,001) and 23,6% (p<0,001) and maxamal voluntary ventilation (mw) was 21,7% (p<0,05) and 16,2% (p<o,05) improvement. the mean changes in normal subjects were mild (fev 1 increased by 3,4%,fef 25-75 by 14,5% and mvv by 6,3%). we observed no adverse heamodinamie conseevence and no arrthytmogenicity. conclusion." in patients with chf the airway response to ibf is highly significant. further study is needed to determine whether therapy with ibf will load to improvement quality of life in patients with chf with dearly documented venti/atory defects. compared with unilateral ima grafts, usage of bilateral ima grafts in cabg patients causes greater thoracic trauma and further reduces blood supply to the sternum and intercostal muscles. these effects may be associated with increased postoperative respiratory complications obiectives: to study the effects of bilateral ima grafts on postoperative respiratory morbidity in cabg patients. methods: two groups of patients undergoing cabg were prospectively studied: group 1 (n=13) received both imas as grafts and group 2 (n=12) received only the left ima. the two groups were matched for age, smoking habits, total number of grafts and preoperative ejection fraction. pao2/fio2 ratio was measured in all patients in the icu, at 30,120,240 minutes on mechanical ventilation (mv), at 30, 120, 240 minutes after extubation (ex) and on postoperative day (pod) 5. in addition, radiografic scores of atelectasis and pleural effusion were assessed postoperatively. results: group 1 had significantly longer bypass (101_+23 vs 70+15. p<0,001) and aortic cross clamp time (45+-19 vs 28+7, p=0,009). pao2/fi02 ratio was significantly higher in group 1 only at 30 minutes after extubation (table) . there was no difference in the incidence or severity of atelectasis or pleural effusion between the two groups. the duration of mv was longer in group 2 (13+_3 vs 9_+2, p=0,004), but the length of icu stay and hospitalization were similar. there was one case of pneumothorax in group 2 and one case of pulmonary infection in each group. after extubation 30 m[n 120 min 240min 30min* 120rain 240min pod 5 1' 342_+87 302_+92 369+87 278-+67 2664-79 294-+102 376_+76 2 319_+105 324_+89 320_+76 223-+52 257_+94 249_+82 400-+43 mean_+sd, *p=o, o03 conclusions: compared with unilateral ima grafts, the usage of bilateral ima grafts in cabg patients is not associated with increased postoperative respiratory morbidity. the diagnosis of rv ischaemia is difficult in the critical care setting. the purpose of this study was to determine the ability of rv endomyocardial and interstitial ph electrodes to detect rv ischaemia in an animal model of rv infarction produced by fight coronary artery ligation. we hypothesized that changes in transmural and interstitial ph would accompany the induced tissue hypoperfusion. rv interstitial (phint) and transmural endomyocardial ph (phendo) were measured in 10 adult anaesthetized pigs before and serially after coronary ligation. phendo and phint fell progressively and significantly following coronary occlusion. this was accompanied by ischaemic ekg changes. the absolute and relative rates of change were greater for phendo compared to phint, particularly after 4 minutes of ischaemia, ph was unchanged in control experiments when the electrode was placed within the fight atrial or ventricular chambers, as well as when coronary ligation was not performed. these data suggest that rv myocardial ph measurements reflect coronary ligation induced rv ischaemia, ph recorded from an electrode placed against the rv endomyocardial wall via a central vein was as useful as an interstitial measurement that required a thoracotomy. this newly described technique may be helpful in clinical settings where differentiation between ischaemic and nonisehaemic causes of rv dysfunction is important. centre. depts of cardiac surgery and anesthesiology, university of bari, italy. lung injury secondary to cardiopulmonary bypass (cpb) is well recognized. nevertheless, mechanical respiratory changes after cpb have been poorly investigated. aim of the study was to evaluate the effects of cpb on respiratory mechanics. elastance of the lung (est l), chest wall (est cw), and total respiratory system (est rs) were measured in 8 patients without previous pulmonary disease undergoing elective cardiac surgery. there were 6 males and 2 females; no pleurotomy was done. cpb was carried out with membrane oxygenator. mean cpb and cross clamping times were 69_.27 and 39• min. flow (pneumothacograph), airway opening and esophageal pressures (pressure transducers and esophageal balloon) were measured on patients sedated and paralysed. measurements were obtained before sternotomy, at the end of cpb after chest closure, four and seven hours later. est rs, est l and est cw, were measured by occluding the airways and the end of a tidal breath. before end cpb-4 h after 7 h after stemotomy closed chest cpb cpb estrs (cmh20/l) 17.7+4.9 19.94-5.6" 22.5_+5.8* 22.64-4.3* est cw " 6.7_+4.1 6.3• 6.61+4.09 6.5-+4.1 est l " 10.9--4.3 13.54-5.4" 15.9-_4.9" 16.1_+5.1" x + sd. * p < 0.05: anova vs "before sternotomy". our data show that cpb induces increase in est l, whereas est cw remains unchanged despite sternotomy. impairment in est l after cpb evolves within the first 4 hours and stabilizes 7 hours later. during the clinical course of human imunodeficiency virus infection (hiv), patients (lots) can develop congestive and dilated cardiomyopathy syndrome (dcm). the aim of our study was to analyze the prevalence of dcm among an hiv population, its clinical and echocardiographic changes under a long term follow-up and therapy with an ace-inhibitor agent (captopril). we prospectively studied 220 pts, 23% (50/220) developed dcm during the follow-up study. among this group 26% (13/50 pts) were in class i and ii and 64% (37/50 pts) in class iii and iv of the nyha classification. we divided this population in a acei+ (22 pts) and acei-(28 pts), according to the administration of captopril. we analyzed the following parameters: age, gender, race, ivda, type of hiv, cdc classification, number of t4 and t8 type cell population and its t4/t8 ratio, therapeutic with acei, type and number of opportunist infections, mycobacteriosis and neoplasm's. we analyzed several echocardiographic parameters, which included the initial (i), final (f) and variation (a) of the lv internal diastolic (lvdd/mm) and systolic (lvsdimm) diameters, lv % of fractional shortening (lv%fs/%), ivs and posterior wall thickness and lv mass (lvm/g). the two groups differed significantly in the following parameters: left ventricular functional indices of patients (pts) with iscjhemic dilated cardiomyopathy (idcm) have a critical value in terms of clinical prognosis of this disease. three-dimensional echocardiography (3-de) is a recently developed method that gives a better evaluation of lv morphology and function. the purpose of our prospective study was to assess the clinical applicability of this new 3-de method in a population with ischemic dcm diagnosis and calculate its lv indices of global and regional function. we studied a group of 16 idcm pts, mean age 58_+11 yrs, 70% male gender, using a combined 3-de tee approach. all pts were in class ill/iv of the nyha classification. first, ekg gated images were acquired through a mechanical pullback of the tee probe, and each set of data was transferred to a conventional ibm-pc system using a dedicated 3-de software. computer processing and analysis of the tee images led to a 3-de reconstruction matrix and off-line calculation of several lv global and regional indices. in all idcm lots the lv cavity was clearly reconstructed and end-systole and diastole in several orthogonat projections, out in multiple planes and its function quantitated using 3-de derived indices. mean values of 3-de lvesv=149-+20 ml, lvedv=197_+32 ml, lvstv=48_+25 ml and lvef=24-+5% were obtained. for all these 3-de parameters, good correlations were observed with 2-de lv measurements obtained through biplane tee analysis of the lv cavity (r>.70; p<.01) as well as regional analysis of sequential 3-de cut planes. conclusion: in our group of patients with the diagnosis of ischemic dilated cardiomyopathy, this new 3-de method could be applied. our results show that this method allows a better assessment of the lv morphology and spatial geometry, with the calculation of global and regional indices with critical clinical and prognostic value in this particular cardiovascular pathology. simultaneous left atrial (la) and left ventricle (lv) inflow analysis assessed by pulsed doppler tee illustrate the loading conditions and reflect the hemodynamics of the left heart. we performed a prospective tee pulsed doppler study with recordings of the transmitral lv filling and pulmonary venous (pv) flow drainage in a group of patients with dilated cardiomyopathy (dcm). a group of 23 dcm patients, mean age 57_+11 yrs, 74% male were studied. this population was divided according to tee severe lv dysfunction (group slvd+ 62% pts; group slvd-38% pts) in each pt we measured the peak velocities (vel/m/sec) and time velocity integrals (vti/m) of the transmitral early (e) and late (a) filing waves, the vel and vti of the pv systolic (s), diastolic (d) and atrial contraction (c) reversal flows. 3-de tee evaluation of the lved, lves, lvst volumes and lvef were obtained. we calculated other parameters, such as e/a, s/d and a/c ratios and the sum of c+a vel, that refelect la systolic function and lv compliance. 19+5 305-_8 0.02 simultaneous and quantitative analytical approach of the pulmonary venous and transmitral flows and ventricular volumes improve the non invasive assessment and understanding of left ventricular diastolic function and cardiac performance in dilated cardiomyopathy patients. objectives : to assess the hemodynamic effects of fluid loading (fl) in acute circulatory failure (acf) due to acute massive pulmonary embolism. methods : hemodynamic measurements (fast-response thermistor pulmonary artery catheter) were performed at baseline (baseline) and after a rapid fluid loading with 250 (fl250) and 500 (fl 500) ml of dextl'an 40 (rhemacrodex| in 12 patients free of previous cardiopulmonary disease (66 • 3 yrs) with acf (ci < 2.5 l/rain/m2) due to angiographicalty proven mpe (miller score > 21) . results : are expressed as mean _+ sem and compared by anova. a significant negative correlation (r = 0.83) was observed between baseline rvedv[ and the effects of fl on ci. such correlation was not observed between baseline rap and the fl induced increased in ci. conclusion : fusibmificantly increases ci in acf due to mpe. however, the simultaneous decrease of arterial 02 content due to hemodilution, limits the benefits expected from improved ci on peripheral oxygenation. obiective: to examine the hemodynamic effects of external positive endexpiratory pressure (peep) on right ventricular (rv) function in acute respiratory failure (arf) patients. methods: incremental levels of peep (0-5-10-15 cmh20) were applied and rv hemodynamics were studied by a swan-ganz catheter with a fast response thermistor for right ventrieular ejection fraction (rvef) measurement in 20 mechanically ventilated arf patients (lis = 2.6 ~-0.45 sd). according to the response to peep 15, two groups of patients were defined: group a (9 pts.) with unchanged or increased rv end diastolic volume index (rvedvi) and group b (h pts) with decreased rvedvi. results: in the whole sample cardiac index (ci) and stroke index (sj) decreased at all levels of peep, while rvedvi , rv end systolic volume index (rvesvi) and rvef remained anchange d. at zeep the hemodynamic parameters of the two groups did not differ. in group a, ci decreased at peep5, rvef decreased at peep10 (~0.8%)~ rvesvi increased only at peep15 (+21.5%) and rvedv[ reded unchanged. in group b, ci and rvedvi started to decrease at peep5, 'rvesvi decreased only at peep15 (-21.4%), anf rvef was unchanged. individual behaviors of the hemodynamic parameters at the 4 levels& peep were studied. rvedvi and ci were significantly correlated in 10 out of:l 1 patients in group b, and in no patient of group a. on the contrary, mpap and rvesvi were significantly correlated in 5 out of 9 patients in group a, and in no patient of group b. the slope of the relationship between rvedvi and rv stroke work index (rvswi) expresses rv myocardial performance. this relationship was significant (no change in rv contractitity)in 8 patients of group b and in 2 patients of group a. in some patients of group a, increments of peep shifted the rvswi/rvedvi ratio rightward inthe plot (rv function decrease). conclusions: in arf patients peep causes more often a preload decrease with unclmnged rv conctraetility. on the contrary, the finding of increased rv volumes during the application of peep is related to a decrease in rv myocardial performance. thus, these data suggest that application of peep might be considered as a stress test to assess rv function. right introduction: after heart transplant (ht), the right ventricle can be subject to an acute pressure overload, especially in cases where there is a preexisting severe pulmonary hypertension. this provokes right ventricular failure and, occasionally, circulatory collapse in intensive care unit. desire the advances that have been made in systems for preserving the donor heart and in post-surgical management, we have failed in our attempts to totally avoid this problem. the right ventricular function, although it usually remains within tolerable limits in these patients during the post surgery period, represents a factor which limits the results achievable in clinical transplant programmes. objectives: to determine the maximum tolerance of the right ventricle (mxtrv) when faced with acute pressure overload. to study the function of both ventricles of the healthy heart (donor) when faced with different degrees of pulmonary hypertension. to detect possible interactions between the ventricles in the absence of the pericardium to approximate the experimental model to the clinical model of ht. materials and methods: the pulmonary artery is progressively constrained in an experimental model until biventricniar failure is detected. this experiment is performed in two diffferent situations: with and without pericardial integrity. results: when pericardial integrity is maintained the mxtrv faced with a pressure overload is 73.2 + 8.56 nun hg. when this pressure is exceeded there is a circulatory collapse with a sharp fall in the cardiac output and in the aortic pressure. however, when pericardectomy is performed (model similar to ht), only 52 • 6.71 nun hg is tolerated (p < 0.01). conclusions: with the pericardium open, as in heart transplant, the maximum pressure that the right ventricle can support is significantly less than with the pericardium closed. the pericardium has a positive effect in protecting the systolic ventricular interaction. it is, therefore, advisable to close the pericardium after heart transplant. jb prrez-bernal, a ordrfiez, a. heroandez, jm borrego, map camacho, c cruz, mac s~nchez, j monterrubio, c garcia, e. gonz~lez. hospital uulversitario " virgen del rocio ". sevilla. espaiqa. introduction: nowadays cardiomyoplasty isused incases of cardiac insufficiency as an alternative to cardiac transplant. after surgery the patients show a noteable improvement with the aid of this "biological circulatory assistance". some researchers suspect that the improvement could also be due to the formation of new blood vessels from the muscle that wraps the heart, nourishing the ischemic myocardium. objectives: our cardiovascular research group has proposed as an objective, the detection of any possible myocardial neovascularization through the muscle used for cardiomyoplasty. in the case that there are new blood vessels to the diseased myocardium through the wide dorsal muscle in which it is wrapped and which aids it mechanically, it would be possible to confirm the worldng hypothesis that cardiomyoplasty not only improves the cardiocirculatory funcfinn mechanically but also by facilitating a better blood flow to the ischemic myocardium. materials and methods: the cardiomyoplasty technique is described using an experimental model of myocardial ischemia. the vascular cast is achieved by injecting methacrylate simulataneously into both the coronary tree and the wide dorsal muscle, in five experiments the connections between the coronary vascular system and the vascular structure of the wide dorsal muscle are demonstrated, conclusions: we have demonstrated that cardiomyoplasty, as well as improving ventricular function, favours the revascularization of the myocardium. cardiomyoplasty could be indicated for cases of ischemic cardiopathy in patients in whom it is not possible to perform direct revacularization using conventional methods. a the therapeutic cardiological manouevres necessary in cases of ischeima reperfusion have increased considerably: fibrinolysis, transluminal angioplasty, coronary revascnlarization surgery and cardiac transplant. the appearance of a specific pathology ht acute reperfusion has been related to free oxygen radicals (for) generated by oxidative damage. objectives: to evaluate the appearance of for during a conti-olled process of ischemia-reperfusion in an experimental biological model and compare it with that in clinical cases. materials and methods: transitory cardiac ischemia was performed in five rabbits by reversible surgical ligation of the descending anterior coronary artery. after 15 minutes coronary reperfusion was performed. blood samples were taken in the basal situation, at the end of ischemia and at 5, 10 and 15 minutes after the start of reperfusion. malondialdehyde (mda) was measured to evaluate the degree of lipid peroxidation (oxidative damage to the membrane). in ten patients undergoing conventional cardiac surgery the production of for was measured after aortic clamping. results: we observed that after 5 minutes of reperfusion there was a highly significant increase (p < 0.001) in the mda values (mean =2.00 /zmols/l). these returned to basal levels after 10 and 15 minutes of reperfusion. conclusions: an "explosion" of oxygen free radicals was detected very quicldy, just a few minutes after post-ischemia reperfusion. thus, if antioxidant agents are to be used to reduce the toxic effects of the for, these will ordy have a therapeutic effect if they are administered in the early phases of reperfusion. introduction: aortic connterpulsation is a ventricular assistance widely used in intensive care units in patients with cardiogenic shock as a provisional ventricular assistance. paraaortic or external aortic counterpnlsation is been investigated as a definitive veutricular assistance in those cases of terminal congestive heart failure and when heart transplantation is counterindicated. aims: to assess the haemodynamic effects of an aortomyoplasty in a biological model of congestive heart failure. material and method: as specimens, we used 10 "large white" pigs. mean weight was 22 kg. after the administration of conventional anaesthesia, dissection of the ladssimns dorsi muscle was performed on the samples at the laboratory of experimental surgery of our hospital. then we performed a thoracotomy at the level of the fourth intercostal space to reach the thoracic aorta. the aorta is dissecated 7 centimetres from the exit of the subclavia and it is wrapped by the dissecated muscle. a cardiomyostimulator is provided in order to allow the synchronization between the diastole and the muscle contraction. the model of heart failure was provoked using verapamil plus propanolol i.v.. results: a significant increase of the aortic diastolic pressures and a significant decrease of the left ventricle telediastolic pressures were observed. this improvement in the parameters (dpti/tti) implies an increase of the coronary perfusion in a model of heart failure. conclusions: using the external aortic counterpulsation, the aortomyoplasty improves the coronary perfnsion and the heart efficiency in patients with heart failure in whom no conventional therapeutic action is possible. the permanent character of the paraaortic counterpulsation is it main advantage. the appearance of specific pathologies as a resuk of myocardial reperfasion has been related to the oxidative damage secondary to the release of oxygen derived free radicals (ofr). during the myocardial ischemia induced during heart surgery with extraeorporeal circulation, severalsubproducts of the oxygen are produced that shall cause toxic effects after the reperfusion which could be counteracted by the physiological antioxidant systems and/or provided by the medication. aims: to asses the ofr during heart surgery. to check whether an antioxidant treatment administered in the preoperative period make decrease the levels of ofr before and after the myocardial reperfusion and to verify whether its administration have any beneficial effect on the intra and extraoperative management. material and method: the study comprehends 20 patients studied as two groups of 10 individuals each (a and b). all patients underwent conventional heart surgery of valvniar substitmion or myocardial revaseularization. group a patients were administered 400 rag/8 hours of vitamin e (tocopherol acetate) 72 hours prior to the intervention as antioxidant treatment. group b patient were not administered vitamin e. we assessed the quantity of malondialdehido (mda) to assess the degree of lipidic peroxidation or oxidative damage of the membrane during the myocardial ischemia and 15 nm after the reperfusion. conclusion: patients who underwent heart surgery and were treated with tecopherol acetate in the preoperative period presented levels of rlo significantly lower than those who were not administered the drug, both during the intraoperative period and after myocardial reperfusion. we detected in these patients a need for antiarrhythmicals and pharmacoiogical support with catecholaminas, although not significant, both in the introaperative period and the immediate postoperative period. recommendations for the treatment of pulmonary embolism (pe) in the presence of right atrial thrombus (at) are conflicting. because of a significantly higher mortality rate due to fulminam or recurrent pe, there is a necessity to treat patients (pts) with mobile type a thrombi compared to pts with adherent type b thrombi. therapeutic strategies include anticoagulation, thrombolysis (t) or surgical thrombembolectomy. combination thrombolysis (cot), predominantly used for the treatment of acute myocardial infarction proved to prevent reocclusion of the infarct related artery at a comparable rate of hemorrhagia. benefit has been related to the alteration of hemostatic proteins by non-fibrinspecific thrombolytic s. administration of cot in pe has been performed sporadically. in the present case, a 55-year old male with no history of prior cardiovascular disease developed acute dyspnea which was related to pe in the presence of deep vein thrombosis of the left femoral vein. therapeutic anticoagulation was installed for a couple of days until there were several bouts of deterioration. biplane transesophageal echocardiography (tee) was performed and revealed a large, wormlike, hypermobile thrombus within the right atrium. computer tomography (ct) of the chest detected a saddle embolus in the bifurcation of the pulmonary tmnk almost occluding the entire left pulmonary artery (pa) and parts of the right pat consisted of 100 mg frontloaded rt-pa and the subsequent continuous administration of urokinase in a dosis of 200.000 u/hr for 24 hrs followed by therapeutic anticoagulation. symptoms, blood gases and ecg improved steadily during infusion, no adverse effects, i.e. minor or major hemorragia were registered. follow-up ct promptly after termination of t showed almost complete resolution of the saddle embelus, whereas tee showed complete dissolution of the at. ' finally, the patient was switched to oral anticoagulants and had an uneventful clinical course until he was discharged. conclusion: in the present case, cot was effective for the treatment of a complicated pe without any adverse effect. introduction: nowadays we can assist hearts with problems of insufficiency by techniques other than transplant. many researchers believe that the best way of assisting insufficient heart muscle is with another muscle from the patient. this technique of ventficular assistance is known as cardiomyoplasty. we describe the surgical technique of cardiomyoplasty using a biological model. the transformed skeletal muscle is transferred to the thoracic cavity where it wraps the heart and assists it. the choice and preparation of this muscle is currently under investigation. our group has focussed on the development of protocols for electrical stimulation to transform a skeletal muscle into a muscle which resists fatigue and which is functionally similar to the myocardium. we detect the optimum time at which this muscle has been transformed, by studying the transmembrane action potentials using intracellular electrodes. when the action potential of the trained muscle behaves like cardiac muscle we consider it ready for cardiomyoplasty. conclusions: cardiomyoplasty is an alternative surgical technique to cardiac transplant, which has a great future in the treatment of patients with advanced cardiac insufficiency. we describe methodology which, by intracellular techniques, allows selection of the optimum moment of transformation of a skeletal muscle trained to perform,like cardiac muscle, without suffering fatigue. purulent pericarditis is a rare disease. its treatment associate systemic antibiotics and drainage of the pericardium. we report a ease of purulent constrictive pericarditis in which intraperieardial fibrinolysis was use. a 38 years old patient admitted in our icu for a constrictive pericarditis as a complication of a purulent pericarditis diagnosed seventeen days before. he had also an aehalasia and the o'esogastric endoscopy had found an oesophageal neoplasm. a fistula was not seen, indeed pericardial of flora was the same that oropharyngeal. hemodynamie and echographic study had confirmed a constrictive pericarditis. because of the poor state of the patient an intraperieardial fibrinolysis was prescribed (250.000 ui of streptokinase on days 23, 25, 27, 29). fluid drainage was improved and cardiac output was also improved (day 23 : 2.53 1.min "i, day 27 : 3.58 l.min'l). no change ofhemostasis was noted. a pericardeetomy and an oesophagectomy were performed after 37 days of evolution. eighteen months latter the patient was still alive. intraperieardial fibrinolysis seems an interesting therapeutic way if rapidly prescribed in the purulent pericarditis course. the decrease in the systolic pressure following a mechanical breath, termed ddown (delta down), has been shown to be a sensitive indicator of preload (1,2) . however, the clinical use of this method necessitates the introduction of a short apnea. we have therefore developed a respiratory systolic variation test (rsvt) which obviates the need for apnea. the test is based on the delivery of 4 successive breaths of increasing magnitude (5, 10, 15, and 20 ml/kg). a line of best fit is drawn between the 4 minimal systolic values (one after each breath) and the downslope calculated as the decrease in blond pressure for each increase in airway pressure ( mmhg / cmh20). in 14 mechanically ventilated patients the rsvt was performed during controlled mechanical ventilation under sedation. the test was repeated after the administration of 7 ml/kg of plasma expander. the initial mean downslope of the rsvt was -.40 + .40 mmhg/cmh20. following volume loading the downslope decreased to -.23 + .44 (ns). at the same time, cardiac output (co) increased by .96 + 1.2 l/min (p<.02), end-diastolic area (determined by tee) increased from 18.5 + 6.9 to 20.3 + 7.1 cm2 (ns), and paop increased from 12 + 8 to 17 + 9 mmhg ( p < .001). the preinfusion downslope value of the rsvt correlated significantly with the increase in the co (r = .81) and the eda (r = .70). methods: an expert system has been constructed running on a multimedia computer with the two objectives in mind, viz training of inexperienced staff, and protocol guidance with treatment regimes for all staff. the system is based on experience gained from two previous systems, the one for dealing with acid-base and electrolyte problems in icu patients; the second for stabilisation of patients with heart rate and blood pressure abnormalities. the training section takes the form of a stage-by-stage account of the insertion of the pac and displays of correct waveforms, coupled with indications of possible incorrect placements, and guidance when failing to achieve the perfect positioning. the treatment protocol section extends an existing protocol for correcting abnormalities in heart-rate and blood-pressure, and now takes account of all the indices as measured by the pac. the system will suggest treatment to correct such things as abnormal wedge pressures concomitant with parameter values throughout the rest of the cardiovascular system. the type of patient eg post-operative cardiothoracic or i. c. u. trauma, will be taken into account when recognising abnormal parameter values and when prescribing treatment. results: a working system which will be improved by the finetuning being carried out. the results and lessons learnt will be presented at the conference. method: septic shock was defined as severe sepsis with either persistent hypotension (mean arterial pressure; map < 70 mmhg) or the requirement for a noradrenaline (na) infusion ~ 0.1 9g/kg/ rain with a map --< 90 mmhg. cardiovascular support was limited to na + dobutamine (db). 546c88 was given for up to 8 h at a fixed dose-rate of either 1, 2.5, 5, 10 or 20 mg/kg/h iv. during 546c88 infusion, na was to be reduced and if possible withdrawn, whilst maintaining map above 70 mmhg and the cardiac index (ci) as clinically appropriate. assessments were made at baseline (t = 0); at i h from the start of treatment (t -1); and at the end of treatment (t -8) with 546c88. conclusions: 546c88 does not appear to increase mpap or worsen pulmonary gas exchange in patients with septic shock, when given by infusion for up to 8 h. 546c88 is a novel vasoactive agent for the treatment of septic shock which will now he evaluated in a randomised, placebo-controlled safety and efficacy study. objectives : to compare cardiac output (q) data obtained for thermal indicators in pulmonary artery (qtpa) and aorta (qtao) and for the stable isotope 2hzo in aorta (q2v~2o) with indocyanine green (icg) in aorta (qicg) as reference. methods : an indicator solution of ice cold h20 (9.4 ml), 2h20 (0.6 ml) and icg (10 mg) was injected as bolus via the injection port of a swan-ganz catheter. qlco and qzmo was measured using a dual optical system (penn lab instruments, philadephia, pa, usa). qtpa and qtao was measured using a in contrast to the recoveries of thermal indicator in pa and 2h20 in aorta the :~covery of thermal indicator in aorta was significantly increased in group ii (n= 18 boluses) over group i (n=18 boluses) (1.3<-0.3 vs. 1.0+-0.1, p=0.04). conclusions: the "overrecovery" of thermal indicator in aorta is in agreement with " biscks deconvolution study (i) and results in erroneous values for q. the most pausible explanation is the distortion of the thermal curve caused by the slow response time of the thermal detection instrument as shown by ganz (2) objectives: to compare data obtained with the double indicator dilution method using indocyanine green (icg) and the stable isotope 2h20 for the estimation of extravascular lung water (evlw2hzo) to gravimetriu lungwater data (evlwg~). methods: an indicator solution oflcg (10 rag) and 2h20 (0.6 ml) was injected as bolus via the injection port of a swan-ganz catheter. dilution curves for icg and zh20 was registered in aorta with a dual optical system (penn lab instruments, philadephia, pa, usa). cardiac output and mean tranist time was measured for both tracers (qico, tlco, q2n2o, t202o) (1). data analysis: evlwg~av was reference for evlwzhzo calculated as q2hzo times the difference in mean transit time between t2nzo and rico (atm2n). as reference for atzn2o evlwg~,v was divided by q~cg to obtain atg~,. a reference distribution volume for 2h20 was calculated as the sum of central blood volume and evlwg=v. 54 boluses were administrated in a group (i) of 6 anaesthetized pulmonary healthy sheep while q was altered. another 18 boluses were administrated in a group (ii) of 6 anaesthetized sheep with stable oleic acid induced pulmonary oedema. evlwg~v measurement was performed postmortem. 2 results: for 72 boluses h20 parameters were not significantly different from their respective reference parameter: at2vao 5.3+_3.5 s vs. atg~, 5.4+3.2 s, evlwzh2o 332-+195 ml vs. evlwg~,~ 338+169 ml. in group i the ratio between 2hzo parameters and respective reference parameters (n=54) were independent of qlco from 1.4 to 7.1 l/min. obiectives: to assess the thermo dye method using indocyanine green (icg) and thermal indicator for the estimation of lung water (evlwt). methods: ice cold indicator solution of icg (10 mg) in water (10 ml the aim of the study was to assess left and right ventricular function in the early postoperative period after orthotopic heart transplantation to elaborate therapeutic approaches of heart function abnormalities correction. mathefial and methods. haemodynamic monitoring data of twenty one patients ( 19 men, 2 women ) age from 18 to 56 were studied. cardiac output, pulmonary artery, right atrium and pulmonary wedged pressure were measured with swan-gans catheter. central haemodynamic indices were calculated with the help of computer-based monitoring system. relations of ventricular stroke work index to it's end-diastolic pressure were used for ventficular function assessment. results. in most cases right ventricular disfunction was the main problem. isolated fight ventficular failure with high pulmonary vascular resistance (pvr) was observed in 24% (5pts), without high pvr-in 43% opts) and with left ventricular failure-in 33% (7pts). one of the most important reasons for fight ventricular failure was the time of heart ischemia more than 90min, which is of great importance in the ease of distance harvesting. the most effective treatment for cardiac failure was combination of dobutamine with i8oprotherenol, atrial pacing and vasodilatators in case of right ventfieular disfunction. all cases with isolated right ventricular failure were treated sucsessfully. biventricular heart failure was a sighn of bad prognosis and the reason of death in 2 cases. conclusion. right ventfieular disfunetion is the main problem during transplanted heart adaptation in the early postoperative period. optimal therapeutic management of cardiac disfunction includes infusion of dobutamine in combination with isoprotherenol, atrial pacing and vasodilatators. cardiology-department of clinical centre-kragujevac institution for occupational health "zastava"-kragujevac, sr yugoslavia the aim of the investigate is analisis five years survives patients with a.i.m.in dependence of locality and risk-factors. we ana~sed-39~-pat~e~ts (273 males and 118 woman), average 59,8 years. for statistic evaluation we used life-table slstem in oder to estimate prognostic determinants. patients with respkatory muscle paralysis may benefit from respiratory assistance by abdomino-diaphragmatie pneumatic belt. we used a non invasive technique, m-mode sonography, to assess the effect of this device on diaphragmatic excursion. we measured the amplitude of right diaphragm motion in seven patients with duehenne muscular dysl~ophy in supine position with various thoracic posture (0 ~ 45 ~ 75~ without and during pneumatic belt respiratory assistance. without respiratory assistance, the thoracic posture had no significant consequence on the amplitude of diapttragm motion, either in quiet or deep breathing. the pneumatic belt increased the diaphragm motion amplitude from 7.1 +__ 3.6 mm to 17.71 +_ 5.5 ram (p = 0.009) at 45 ~ tilt angle, and from 8.4 + 3.8 mm to 19.3 + 5.8 mm (p = 0.009) at 75" tilt angle. the tidal volume increased from 211 + 78 to 373 + 99 rut a145* tilt angle, and from 229 + 78 to 447 + 143 ml at 75* tilt angle (p = 0.009). two patients could not bear the horizontal position (0' tilt). in the five other patients, the pneumatic belt increased but not significantly the amplitude of diaphragm motion (9.2 + 4.9 mm to 15.5 + 7.3 ram). after an overnight respiratory assistance, pao2 increased from 66.4 +_. 8.7 to 73 + 10.1 mmhg (17 = 0.015), sao2 increased from 91.1 + 2.5 % to 91.9 +_. 3 % (p = 0.015), and paco2 decreased from 52 + 6.4 to 46.4 +_. 4 mmhg (p = 0.015) according to the ventilatory pattern result, m-mode sonography allows to measure non invasively the improvement of diaphragm kinetics obtained by pneumatic belt respiratory assistance, and may be helpful for its adjustment. objective: to study the effect of flow triggering (flow sensitivity 1 and 5 l/min) vs pressure triggering (-lcmh20) on inspiratory effort during pressure support ventilation (psv) and assited/controlled mode (a/c) in 8 stable copd patients non-invasively ventilated with a full face mask. methods: the patients were studied during randomized 15 min. runs using a bird 8400 st ventilator at zero peep (zeep). trigger values for pressure (-lcmh20) and flow (1 l/rain) were the lowest allowed by this ventilator. the transdiaphragmatic pressure time product per breath (ptpdi), dynamic intrinsic peep (peepi,dyn), maximal airway pressure drop during inspiration (apaw) andl ventilatory variables (ti,te,ttot,rr,vt and minute ventilation) were measured. results: no major problems due to airleaks or to auto-triggeriffg phenomena were observed in the patients, so that all of them were able to perform all the protocol runs. minute ventilation and respiratory pattern were not different using the two triggering systems. the ptpdi was significantly higher during both psv (10.6+6.8 cmh:0 x sec) and a/c (10.1+2.5) with pressure triggering, as respect to psv (8.9+7.5, p<0.02) and a/c (5.8+4.4, p<0.001) with flow triggering (1 l!m). no differences were observed between 1 and 5 l/min flow triggers. apaw was also significantly larger during pressure triggering; peepi,dyn was reduced during flow triggering being 5.5+1.5 cmh20 (psv flow trigger) vs 8.1+1.5 (psv pressure trigger) and 4.4+_1.3 (a/c flow trigger) vs'f~5+l (atc pressure trigger). conclusions: in stable copd patients non-invasively ventilated, flow triggering reduces the respiratory effort during both psv and aic mode as compared to pressure triggering. this may be partly due to a decrease in peepi,dyn using a flow-by system. objective. cardiac output is higher during alternating ventilation (av) (i.e. differential ventilation of the lungs with a phase shift of half a ventilatory cycle) than during synchronous ventilation (sv) of both lungs 1 . we verified the hypothesis that the higher cardiac output depended on a lower central venous pressure and intrathoracic pressure, due to a lower mean lung volume, which we attributed to part of the expansion of the inflated lung at the expense of the expiring, opposite lung 2. we studied this interaction between the lungs during one-sided inflation, which we called cross-talk. method. in 6 anaesthetized and paralyzed piglets we applied short periods (30 s) of one-sided ventilation (10 breaths per rain, bpm), while the other lung was open to the ambient air. the air flow into the non-ventilated lung during expiration of the ventilated lung was integrated to volume. we studied 1-to-r and r-to-i cross-talk at ventilatory rates of 10, 15 and 20 bpm. the amount of cross-talk was the volume displacement in the non-ventilated lung. results. during 10 bpm the r-to-i crosstalk was 23 _+ 4.7 % (mean +__ sd) of the tidal volume to the right lung and the 1-to-r crosstalk 31 _ 6.3 % of the left tidal volume. both values increased at 20 bpm to 30 _ 4.1% (p < 0.05) and 39 _ 7.7 % (p < 0.01) respectively. the values at 15 bpm were in between., conclusion. we concluded that the lower mean lung volume and lower thoracic expansion during av compared to sv depends on partial expansion of the inflated lung into the non-inflated lung, resulting in a lower mean intrathoracic pressure as the main reason for the higher cardiac output during av. obiective: natural surfactant given for rds in premature infants leads to a rapid improvement in oxygenation, but lung compliance did not improve in most studies. however, acute effects on lung mechanics during and immediately after surfactant administration have not been studied before. methods: a total of 13 administrations of bovine surfactant in recommended doses was given via a small catheter into the distal endotracheal tube either as a bolus (n = 8) or as a slow infusion (n = 5) in 10 infants with established rds. static compliance (c), resistance (r) and time constant (tc = cxr) of the lung were measured every 3 minutes with a lung function cart (sensormedics 2600) without interrupting ventilation. 3 infants receiving synthetic surfactant were studied as controls. results: after surfactant as a bolus or during infusion c first decreased but then increased, whereas r increased immediately with great fluctuations but did not return to baseline. this pattern was more pronounced in infusion than in bolus administration. change of c and r varied greatly in the individual case, maximum c was > 400 %, maximum r > 700 % of baseline value. retreatment was followed by an increase in r in all 3 patients, but c increased only in the one who was responder. patients receiving synthetic surfactant had no change of c or r and were non-responders. ob~i31ctives= acute lung injury (ali} sometimes induces severe hypoxernla which may be refractory to conventional modes of mechanical ventilation (mv). the elm of this study was to observe some cardio-pulmonary effects of an alternative method of ventilatory management of severe ali. five patients with severe ali (murray scores >3) requiring mv were studied. protocol inclusion was considered when a control-mode of mv (with a pzo~=l.0 and a peep level <15 cme=o} was not able to get either a p.ojf=o= ratio >55 or a s.o= >85%. patients were sedated, paralyzed, and a ventilator (serve 900c) was used for pressuz'e-control ventilation (pcv). fio= was maintained at 1.0 and peep removed. continuous gas flow (250• ml/kg] was humidified and jet delivered through a tube (7 ram id, 18 ml capacity, 0.09 ml/cm h=o compllancel ended in a nozzle (0.8 mm is) attached to the endotracheal tube connector. a thermodilution flcw-dlrected catheter was inserted in pulmonary artery. following variables were recorded 15 minutes before and after protocol started: tidal volume (vt), minute ventilation (vz), intratracheal pressures (p~w), wedge pulmonary artery pressure (wp), central venous pressure (cvp), mean arterial pressure (map), cardiac index (ci), arterial and mixed venous oxyhemoglobin saturation (sao=, svoa) , oxygen delivery (do~) , oxygen consumption (vo2) , intrapulmonary shunting (q./qt) , and oxygen extraction ratio (ero). this observation suggests that hfpv could allow to ventilate at lower fin2 and improve blood oxygenation during the acute phase after inhalation injury reducing toxicity risk related to high fin2. further studies are necessary to confima these results and evaluate the possible implications on mortality alter smoke inhalation and for other icu pts. objectives: to design a system for volume controlled high frequency ventilation (hfv) and to estimate the dependence of the tidal volume (vt) on frequency (f) in normocapnic ventilation in rats at frequencies 2 -25 hz. methods: a new system for volume controlled hfv was devised consisting of the generator of the constant flow during inspirium and the constant pressure during expirium. the ventilator allows ventilation at frequencies 2 -25 hz with the relative inspiratory time (ti) 0.2 -0.8. the airway pressure was measured at the proximal port of tracheostomic cannula , at the same site inspiratory and expiratory flow was measured using modified lilly-type of pressure-differential flow sensor. non-linearity of flow sensor was compensated on line by derived equation based on calibration at static and dynamic conditions. flow and pressure data were evaluated on line using original software. value of the positive end expiratory pressure (peep) was serve-regulated by analogous feed-back. in animal experiments white wistar rats (400-430 g) narcotized with ketamine/xylazine with cannulated carotid and femoral arteries were kept at the rectal temperature 37~ the arterial pressure was monitored. after traeheotomy the metal cannula (2mm [.d.) was inserted, animals were curarized and ventilated at the following condition: peep = 0.1 kpa, ti = 0.5. the dead space of ventilator including canula was 0.45 ml. the initial frequency was 2hz and 10 rain after each change of the ventitatory regimen the blood gases analysis was performed. the frequency was changed according to the following schedule : 2 hz-->4 hz-->8 hz-->4 hz-->16 hz-->4 hz--~25 hz-->4 hz. vt for each frequency was regulated to maintain normocapnie ventilation with arterial pco2 = 40 + 2 mm hg. the arterial po2 was always above 70 mm hg. results: for normocapnie ventilation in rats the following tidal volumes vt [ ml/kg] were found : vt1 = 5.91 --+ 0.30 ml/kg for ft = 2 hz, vt 2 = 4.31 +0.12 mukg for fz =4 hz, vt3 =3.30 +_0.27 ml/kg forf3 = 8 hz, vm4=2.61+0.08ml/kg forf4=16hz andvmt= 2.18 + 0.13 mukg for fs = 25 hz (presented as mean values _+ s.d., n = 6 ). the regression analysis using the mean values resulted in the equation for normocapnic vt in rats in our experiments : vtn = 37. 5 * f-e.30 . conclusions: the described system allowing ventilation in a wide frequency range 2 -25 hz with accurate measurements of airway pressures and vt might be useful for optimisation of artificial ventilation in new-barns with different lung pathologies. supported by grants iga mz cr nr 1448-3 and gacr nr 305. s122 intensive care unit. university. hospital of south manchester, uk. methods: measurements were conducted on 6 ventilated patients (puritan bennett 7200ac with metabolic monitor pb 7250 set to measure end tidal co2). all measurements were repeated with the patient stabilised at 5cm. 10cm and 15cm peep. inclusion criteria were: 1) haemedynamic stab(l( .ty for 1 hr; 2) pulmonad" anon" flotation catheter in situ: 3) volume control ventilation with plateau of 0.5s: 4) fio2~ > 11.6 to maintain pao~. > 10 kpa with 5em peep: 5) qs/ot > 20%; 6) pao2/fio2 ratio <150. measured variab!es included: r162 minute volume: plateau ainvay pressure: applied and intrinsic peep: fractional end tidal co2; arterial and mixed venous blood gases and hacmod).ttamic variables. results: statistical analysis was performed using repeated measures anova. significant decreases in cardiac index (ch p<0.01), compliance (p<t).05) and o,'gcn deliver, index (do2, p<0.02) occurred with increasing peep (table 1) . no other variable showed any significant change. methods : all consecutive patients orally intubated in the micu between january to april 1995 were studied. etts were positioned according to the reference marks mentioned above (measurements from upper incisors). the position of the ett tip in relation to the carina was measured on the chest radiograph done with the patient's head in neutral position. results : a total of 59 men and 46 women were studied (n= 105). the mean distance of ett tip to carina was 4.1 cm. using the reference markings, 29 cases (27.6%) had the ett tip < 3 cm from the carina and 5 cases (4.8%) > 7 cm. one case resulted in an endobronchial intubation. the mean height of all patients were 167 cm (153-187) for males and 155 cm (140-170) for females. of the patients with ett tip < 3 cm from carina, the mean height was 163 cm and 151 cm respectively. ~2onclusion : adopting the above quoted reference marks did not result in ideal positioning of the ett in a significant proportion of cases (32.4%). we postulate that [s because our asian population is generally shorter than those in previous studies. objectives: to measure the changes of pulmonary mechanics before and after tracheostomy in patients with prolonged mechanical ventilation and to determine factors that predict the outcome of liberation from mechanical ventilation. design: prospective. setting: respiratory intensive care unit (ricu) in a tertiary hospital. patients: twenty patients with chronic lung disease requiring long-term mechanical ventilation. tracheostomy is indicated for further care. intervention: tracheostomy. measurements and results: pulmonary mechanics including respiratory rate (rr), tidal volume (vt), peak inspiratory pressure (pip), intrinsic positive end ex~ piratory pressure (peepi), lung compliance (cld), mean airway resistance (rawm), work of breathing (wob), pressure time product (ptp) by bicore cp-100 pulmonary monitor were recorded 24 hours before and after tracheotomy. ventilator setting parameters remained the same during surgical intervention and were also recorded for comparison. generally, the mechanics including pir wob, raw~x and ptp showed improvment after tracheostomy. but only pip was significantly reduced (pre 33.4 _+ 11.8 to post 28.6 _+ 9.2, p < 0.05). changes of wobp showed significant correlation with pre-operation rr, minute volume (mv), wobp, and peep(. changes of raw m were also significantly correlated with pre-operation peep, vt, and raw m. the patients were divided into two groups according to their outcome after two week follow-up. group 1 included eight patients who were completely weaned from ventilator; group 2 included twelve patients who still remained ventilator-dependent or were mortality. there was no difference in age, duration of mechanical ventilation, pro, post or changes of several lung mechanics between the groups of patients. pre-tracheostomy peep i and cld showed significant difference between these two groups (1.1 _+ 1.6 vs 2.7 + 1.4 in peepi; 47.3 _+ 36.9 vs 28.8 _+ 16.5 in cld, p < 0.05). pre-tracheostomy ventilator setting in mode of assist/control also showed significant higher percentage in group 2 (3%5 % in group 1 vs 66.6 % in group 2). conclusion: in prolonged mechanical ventilation patients with chronic lung disease, tracheostomy will significantly improve pip and slightly reduce wobp, raw m and ptr patients who used pressure support mode before tracheostomy had better underlying lung conditions (lower lung compliance and auto-peep) will have better chance to wean from mechanical ventilation. forty-eight infants with congenital diaphragmatic hernia presenting within the first 6 hours of life, who underwent surgical rapair,were analysed prospectively in order to produce a reliable inde x of severity of disease that would reliably predict eventual outcome. there were 25 survivors and 23 deaths in this series (mortality 48%).using arterialpco 2 values measured 2 hours after surgical repairand correlating them with an index of mechanical ventilation,we have been able to clearly define two groups of diaphragmatic hernia based on their response to hyperventilation. the first group, with co 2 retention and severe preductal shunting,was unresponsive to hyperventilation with high rates and pressures the mortality was 90%. the second group responded well to hyperventilation and demonstrated reversable ductal shunting only. survival in this group was 97%. arterial co 2 accurately reflects the degree of lung development in this disease and separates those patients with severe pulmonary hypoplasia where the outcome is invariably fatal, from those with a well developed contralateral lung where there is excellent potential for survival. respiratory failure unit, dpt medicine, univ. thessaloniki, thessaloniki, greece the variability of arterial blood gases (po2, pc02) and the ph (abg) was examined in 20 stable icu patients, few hours before a successful weaning from the ventilator. all patients were lightly sedated and the ventgatory conti~ons were pressure support (ps) for 9 and ps plus intermitted mantatory ventilation in ii. [n each patient, 6 speciments of abg were measured at 10 min intervals during a 1-11 study period. at the same time with abg the arterial blood pressure (bp), the heart rate (cf), the tidal volume (tv) and the respiratory rate (n r were measured. for all the patients, the mean coefficient of variation (c) was 3.45 percent for po2, 3.53 percent for pco2 and 2.27 percent for hco3. the average sd for ph was 0.008, the corresponding c for systolic bp, diastolic bp, cf, tv, rf were 4.35, 5.21, 3.68, 2.51, 4.18 percent. we conclude that the spontaneous variability of arterial blood gases in icu patients is not substantial ~hen they have stable the heamodynamic and the ventilatory parameters. deptx?fa'aaesthesioiogy and reanimation, rhe sechenov medical academy, moscow, russia objective: ~he prevention and treatment of hypoxia in the critical patiems. methods: i~fusions of perphtoran -a blood substitute with gas-transporting fimclion based on perphtorhydrocarbon -in 496 patients with acute hypovolemia, microcirculatory distnrbance~ tissue gas exchange and metabolism; pulmonary iavage in 104; iongterm extrapulmonary oxigenation with tleoroearboa oxygenator in combination whb ~trafiltra!ion, hemosorption and hemodialysis -in 73 patients. results: pe~htoran increases blood volume, co,sv, decreases svr, improves capillary blood flow, increases the blood oxygen capacity, tissue oxygen tension, 102 del, vo2 by improving the rheologic properties of blood and plasma, normalizes 02 ext., prevents and eliminates fat embolisation and ards. decreases the need for blood transfusions and infusions of plasma expanders by 1.3-1.4 limes. alveolar venti!ation-perfusion ratio remains unchanged with its increased effective utilization. there was no surfactant destruction during lavage. extrapulmonary oxygenation of small volumes of venous blood eliminates venous destruction and then arterial hypoxia and increases pulmonary oxygenation. the use of lluorocarbon cxygenators during hemosorption and hcmodialysis provides the atraumatic and iongterm oxygenation of arterial blood and increases elimination of co2 which prevents the development of hypoxic complications. conclusions: perphtoran and fluorocarb~n oxygenators are effective in the correction of hypoxia in the criticat patients. objeqtives: to determine if there are differences in oxygen consumption (vo2) during weaning from mechanical ventilation (during total ventilatory support and spontaneous ventilation with cpap), and to compare different predictive parameters of weaning in predicting success of weaning. methods; prospective study in 20 critically ill patients treated with mechanical ventilation for at least 48h, who fulfilled at least 3 of 4 standard weaning criteria (vt>5ml/kg; respiratory frecuency (f) <35; pimax > 20 cm h20; pao2/fio2 > 150). baseline measurements: t, vt, p0.1, pimax, f/vt, p0.1*(f/vt), p0.1/pimax. study protocol: measurement of vo2, vco 2 (medgraphics), vt, f, ve, and arterial blood gases during total ventilatory support (cmv), and after 30 and 120 minutes of spontaneous ventilation with cpap 5 cm h20. the weaning trial was stopped, failure to wean diagnosed, and mv resumed it a patient presented significant tachypnea, tachycardia, bradycardia, cardiac rythm disturbances, hypertension, hypotension, hypoxemia or hypercapnia. results: four patients did not complete the weaning trial, 16 were extubatad, and 2 of them had to be reintubated before 48h, being considered also weaning failures. during cmv, vo2/kg was 4.07 + 0.2 ml/kg/min, and 5.09 _+ 0.4 mlo-2/kg/min after 30' on cpap 5 cm h20 (p < 0,01 ). 14 of 15 patients (93%) with 4 standard criteria were extubated, while only 2 of 5 (40%) with 3 criteria (p<0,01). next objectives: compare the extent and distribution of lung injury in dogs preinjured with oleic acid (oa) and ventilated with high tpp and adequate peep in the prone and supine position. methods: lung injury was induced with oa (0.06-0.09 ml/kg) in anesthetized, paralyzed, and intubated dogs (n=10) during volume controlled ventilation: rate=12/min, peep=5 cmh20, ti/ttot=0.3, fio2=0.6, vt=15 ml/kg. animals were rotated during the oa infusion and the following 90 minute stabilization period to assure uniform injury. in the supine position, peep was set 1-2 cmh20 above the lower inflection point (as determined by the pressure-volume curve), and vt was set to obtain a tpp of 35 cmh20: animals were ventilated in either the prone (n=5) or supine (n=5) position for four hours. pulmonary artery occlusion pressure was maintained constant (4-6 mmhg) with saline infusion. at the end of the protocol the lungs were removed and divided by template into dependent (d) and nondependent (nd) sections for wet weight/dry weight (v~n/dw) and grading of nstologic lung injury (hli; scale 0-3). oseillatron | is a pneumatic device that generates high frequency, oscillation by means of a reciprocating system in the form of a membrane. it generates sinusoidai wave form at 2(10 to 10(10 cycles/rain. the system does not deliver gas but must be adapted to the proximal respiratory, circuit of a conventional ventilator, resulting in ci-ifo. it was developed to enhance intrapnlmona~ diffusion during mechanical ventilation and to mobilise endebronchial secretions. methods. we measured arterial blood gases and haemedynamics during a first period of conventional ventilation (cppv) followed by. two 30 rain periods of chfo (sequences : 6(10 and 901) c/rain : group l, n = 1 l: 900 and 600 c/rain : group 2, n = 8). measurements were made at the end of each period. cardiac output was measured using thermedilution method: flu2 and peep were kept unchanged throughout the study. intrinsic peep was also evaluated by, means of an occlusive valve. results. pa02 is not significantly modified during chfo at 600 or 900 c/rain. paco2 is slightly decreased at 600 c/rain (p = 0.(16). however, intrinsic peep remains unchanged. there is no sequential effect (gr. l vs gr. 2). there is no more effect of chfo for patieets who are at a flu2 higher than 0.50 (n = 9). no changes in haemodynurmcs are observed except a slight increase in central venous pressure (cvp) during ci-ifo (p < 0.ol). obiectives: to examine the effects of inspiratory muscles unloading on neuromuscular output at controlled levels of chemical stimuli. methods: the ventilatory response to co 2 was examined in ten normal subjects using rebreathing method. ventilation ~) and respiratory muscle pressure output (pmus) at the same end-tidal partial pressure of co 2 (petco~) were compared with and without combined flow and volumeproportional pressure assist in two protocols (a and b). protocol a (n = 10): two levels of assist were studied; flow assist (fa) of 2 cmh20/i/sec and volume assist (va) of 2 cmh20/i (assist 1), and fa of 2 cmh20/i/sec and va of 4 cmh20/i (assist 2). all conditions were applied randomly. v~, tidal volume (vt) and breathing frequency (f) were measured breath by breath and plotted as a function of petco~. protocol b: in 5 subjects, in addition to above measurements, esophageal (pes) and gastric (pg) pressures were measured and the time courses of transdiaphragmatic pressure (pdi) and pmus were calculated. one level of assist (assist 2) was studied in this protocol. results: in both protocols inspiratory muscle unloading did not change the f response to c%. compared to control, with assist v t response was displaced upwards; at petco2 of 55 mmhg v t was increased significantly by 0.4+0.1 i and 0.7+0.2 i in protocol a with assist 1 end 2, respectively, and by 0.5_+0.1 i in protocol b with assist 2 (p<0.05). ~/~ responses showed similar changes as vtresponses. in both protocols the slope of v~ response (s did not change significantly with unloading. at low petco~ (50 mmhg), pdi and pmus waveforms did not differ with and without assist. with unloading, at high petco2 (59 mmhg), pdi and pmus at the end of neural inspiration decreased by 18.8-+8.3% and 11.7+15.7%, respectively, from control values. neither change was significant (p>0.05). by theoretical analysis we estimated the expected changes in vt and ~/~ when the levels of assist used in both protocols were applied in the absence of : any change in neural output response to co z. the predicted response was similar to that observed, indicating that the small difference in pdi and pmus between control and unloading runs was due to intrinsic properties of respiratory muscles end respiratory system. conclusions: these results suggest that when chemical stimulus is controlled, respiratory motor output is not downregulated with unloading. the determinants of the response of the respiratory output to inspiratory flow rates (v~) were examined in awake normal subjects. subjects were connected to a volume-cycle ventilator in the assist/control mode and v~ was increased in steps from 30 to 90 i/min and then back to 30 i/min. v~ pattern was square, and all breaths were subject-triggered. in six subjects the effects of breathing route (nasal or mouth) and temperature and volume of inspired gas (protocol a) and in 8 subjects the effects of airway anesthesia (upper and lower airways, protocol b) on the response of respiratory output to varying v~ were studied. in protocol b, in order to calculate muscle pressure during inspiration (pmus), respiratory system mechanics were measured using the interrupter method at end-inspiration. independent of conditions studied breathing frequency increased . significantly and end-tidal concentration of c% decreased as v~ increased. the response was graded and reversible and not affected by breathing route, temperature and volume of inspired gas and airway anesthesia. with and without airway anesthesia (protocol 8) neural inspiratory and expiratory time and neural duty cycle, estimated from pmus waveform, decreased significantly as v~ increased. at all conditions studied the rate of change in airway pressure prior to triggering the ventilator tended to increase as v~ increased. the changes in timing and drive were nearly complete within the first two breaths after transition with no evidence of adaptation during a given ~/~ period. we conclude that v~ exerts an excitatory effect on respiratory output which is independent of breathing route, temperature and volume of inspirate and airway anesthesia. the response most likely is neu~'al in origin, mediated through receptors not accessible to anesthesia such as those located in chest wall or below the airway mucosa. it has been shown, in mechanically ventilated awake normal humans, that increasing inspiratory flow rate (~/~) exerts an excitatory effect on respiratory output. it is not known if this effect persists during sleep. to test this seven normal adults were studied during wakefulness and nrem sleep. subjects were connected through a nose-mask to a volume-cycled ventilator in the assist/control mode and ~/t was increased in steps (3-4 breaths each) from 30 to 70 i/min and then back to 30 i/min. v~ pattern was square, and all breaths were subject-triggered. forty-one trials during nrem sleep and 10 during wakefulness were analyzed. both during sleep and wakefulness minute ventilation increased and total breath duration (ttot) decreased significantly in a graded and reversible manner as ~' increased. these changes were complete in the first breath after v{ transition. the response was significantly less during sleep than during wakefulness (p<0.05); at 30 i/min ttot, expressed as % of that at 70 i/rain, was 110.2+_1.3% during sleep and 127.8+_3.9% during wakefulness. during wakefulness, at 30 i/min, the rate of change in airway pressure prior to triggering the ventilator, an index of respiratory drive, was 60% of that at 70 i/min (p<0.05). the corresponding value during sleep, was 86% (p>0.05). in four sleeping subjects the increase in v~ was sustained for 1.5-2 min. there was no evidence for adaptation of the response; tro t, averaged over the last three breaths, did not differ from that obtained when vj was sustained for only 3-4 breaths. we conclude that 1) vt exerts an excitatory effect on respiratory output, mediated by a reflex neural mechanism and 2) the gain of this reflex is attenuated by sleep. chest radiographs is a common complementary technique for patients in critical care units, with a low cost and easily available. however, it has certain well-known limits in diagnosis, the most important derived from the low quality of some pictures. in this paper we make a general review of some new technical approaches developed for improving the quality of the images, and so incrensing the diagnostic value of conventional radiology. we begin deaeng with the correct positioning of the patient, trough the filtering techniques, the synchronization of radiology and ventilation, and we make reference to the new computerized systems for digital image processing. conclusions: the portable radiographic system is a device that probably with maintain for many years in critical care units as a basic non-invasive diagnostic tool. but we need an increase in the efficiency of it, applying means as simple as a correct positioning of the patient, or the use of fitlers or synchronizers. thus we should improve the general standards of portable radiography. "are circular circuits safe? quantifying undelivered tidal volume in pediatrics patients". objectives: to evaluate the overall influence of internal compliance of circular circuits on delivered tidad volume (vt). methods: we studied prospectively 14 asa i pediatrics patients (2 to 10 yr. old) scheduled for elective general surgery. mechanical ventilation was supplied by an ohmeda excel 210 (circular circuit). the internal compliance of the circuit (cc)-anesthesia machine plus external circuit-was determined by the supersyringe method: corrugated dar tubes of 10 mm. id and 1.5 m. long (children < 30 kg), and a corrugated dar set of 15 mm. id and 1.5 m. long (children > 30 kg) were respectively used for ccl an cc2 values of 9.3 and 9.5 ml/cm h20. a vtof 10 mlg/kg and respiratory frequency was adjusted for an end-tidal co2 (etpco2) between 30 35 mmhg. tidal volumes (measured by spirometry) and airway pressure (paw) data were recorded every ten minutes. volumes and thorax-lung compliances were calculated as follows: (vt delivered = vtadjusted-vol compressible, being vol. compressible = co x ppeak (aw). apparent compliance (ca) = vt adjusted/pplateau(aw), and true compliance (ct) = =vt delivered/pplatean(aw)). comparative statistics were separately designed between calculated compliance data and tidal volumes on a paired sample ~test basis. results: calculated values for volumes and thorax-lung compliances were: conclusions: due to the elevated internal compliance of the circular circuit there is a remarkable dilference between adjusted and delivered vt: mean undelivered vt was 38.8 % and reached as high as 64.1%. teere is also a significative error in calculating true thorax-lung compliance: its overestimation can be as high as 69.6 %. circular circuits are considered safe and cost-saving for anesthetical practice. nevertheless we conclude that anesthetists should bearin mind vt losses when using circular circuits, due to compressible volume. tracheal stenosis is one of the most serious complications of patients submitted to prolonged endotracheal intubation, in which the decrease in inner diameter of upper airway makes it very difficult to achieve a correct ventilation. objectives: compare the results of applying high frequency jet ventilation (hfjv) to some of these patients with conventional controlled ventilation (cmv). methods: we used a prototype of high frequency jet ventilator (santiago-2) developed in our university, and we developed a tracheal tube in wich we modified the distal tip (conic tip). we applied this system to two patients which were initially ventilated in the operating room with usuai controlled mecanical ventilation (cmv) following the standards of our department, and then intubated with the special endotracheal tube and ventilated with hfjv. results: we could verify a proper ventilation of both patients with cmv and hfjv. during hfjv, the airway pressures were lower than those recorded during cmv. a lower airway pressure prevents lesions due to high pressures. conclusions: hfjv is a good method of ventilation for patients with significative stenosis of the trachea, not only during surgical procedures, but also during ventilation for long periods in critically 111 patients. the ventilatory setting is pressure support mode. the pressure level and fit2 were kept constant during h/d. arterial blood gas, wbc count, and mean bp was checked according to the schedule: 0'(immediately before h/d), 15', 30', 60', 120', 180', 240'. respiratory drive (represented by poa), tidal volume(ti) and minute ventilation(ve) were continuously recorded by pulmonary mechanics monitor (bicore cp-100). the mean value of the breaths 5 minutes before blood sampling were used to represent the ventilatory status of that period. anova test is used for comparison between groups. for poa, hierarchical cluster method is applied to divide the cases into two groups of similar change. conclusions: our data suggest that pl is very useful, non invasive and low-expensive emergenc e support for arf, expecially in the elderly with severe chronic pulmonary disease and relative controindications to eti. pl seems to be an effective alternative when it is not immediatly possible to perform etl. the multiple inert gas elimination technique (miget) can be used to assess the effects of any given mode of mechanical ventilation on the pulmonary and systemic factors determining arterial po2 and pco> however, a potential problem in mechanically ventilated patients is that the 10 l mixing box (mb-10l) placed in series in the expiratory side of the circuit of the ventilator to sample mixed expired gas may provoke substantial discrepancies between the tidal votume set in the ventilator and the effective tidal volume delivered to the patient, due to the increase in the compression volume (vc) of the circuit. the effects of the mb-10l on the v c were compared with those produced by a new 1 l mixing box (mb-1 l) specifically designed to produce adequate gas mixing and to prevent loss of the two most soluble gases (ether and acetone) used in the miget. at any given peak cycling pressure (p~ak, cm h~o), the v c (ml) provoked by the mb-10l was substantially higher (vc= 7.4*ppeak) than that provoked by the new mb-1l (vc= 1.4*ppeak). at a ppeak = 50 cm h20 ~ the v c were 377 ml (mb-10l) and 67 m{ (mb-1l), respectively (p< 0.001). in a group of 6 subjects (4m/2f, 57_+6 years), for each of six the gases used in the miget, the regression line between the mixed expired partial pressures simultaneously obtained from mb-1 l and mb-10l fell on the identity line. it is concluded that the new mb-1l allows adequate assessment of the effect of different modalities of mechanical ventilatory support on pulmonary gas exchange, with less potential for gas compression and thus hypoventilation. objectives evaluate the influence of different pressure support ventilation (psv) levels on cardiovascular and respiratory funcion in icu polytrauma patients. metbed&we studied 15 polytrauma icu patients , who were in weaning process , after long term mechanical ventilation for acute respiratory failure . mean age 52 (37-71) yrs . they all were connected to servo ventilators siemens 900c , and all were in stable condition , without sedation , inotropes or diuretics. the hemodynamic studies were done with continuous svo2, swan ganz catheter (oximetrix, abbott). they all were in spontanuous mode (spent) with 5 cm h20 cpap for at least one hour. we turned them to psv with 0 inspiratory assistance (psv 0 cm h20) and after 60 rain we applied psv 10 cm h20, and after 60 min psv 20 cm h20 . hemodynamlo and respiratory measurements were done before and after the application of insiratory assistance. the results were statistically analyzed with anova. resets . respiratory variables . no significant changes in minute volume (ve). tidal volume (vt) and mean airway pressure (mpaw) increased statistically significant (p< 0.001 ) . respiratory rate (rr) decreased significantly (p<0.01) . blood gase showed no difference . cardiovascular variables. cardiac output (co) decreased ns , heart rate (hr) had no change , central venous pressure (cvp) , mean pulmonary artery pressure (mpap) , pulmonary capillary wedge pressure (pcwp) , increased ns , oxygen delivery (do2) decreased ns, oxygen consumption (vo2) decreased ns. conclusions. psv is a very useful respiratory mode helping patients to be weaned from long term mechanical ventilation . it has beneficial effects on respiratory function and oxygen consumption without affecting seriously the hemodynamic parameters, possibly due to a decrease of the work of breathing. a. michalopoulos, a. anthi, k. rellos, j. kriaras, s. geroulanos intensive care unit, onassis cardiac center, athens. objectives of this study was to examine the effect of different levels of peep on postoperative svo2 and pvo2 values in a group of patients, following open heart surgery. methods: upon transfer to icu, 67 patients (54 males and 13 females) of mean age 63_-+6 years, were randomly assigned to receive 0 (n=22), 5 (n=24), or 10 cm of peep (n=21). there were no statistically significant differences in demographic data or preoperative respiratory status among the three groups. all patients were ventilated on the assist control mode with a tidal volume of 10 ml/kg. the fraction of inspired oxygen (fio2) was adjusted to keep a pao2 around 100 mmhg. mixed venous po2 and svo2 were measured at 30 min, 4 and 8 hours after application of mechanical ventilation in the icu, just before extubation (be), half hour after extubation (ae), and at 4 hours post-extubation. differences at each study time were analysed by anova. results: mean svo2 and pvo2 values among the three groups, for all study intervals, are presented in the table. conclusion: we found no differences (p=ns) in tissue oxygenation (expressed by svo2 and pvo2) among the three groups, at any study interval, in the early postoperative course of patients following open heart surgery. intrinsic peep (peepi), and high elastance and resistance increase inspiratory work load in copd. cpap reduces work of breathing by counterbalancing peepi. pav provides flow (fa) and volume (va) assistance proportionally to patient resistance and elastance and inspiratory effort. we studied the effects of partitioned support (cpap-fa-va) on breathing pattern and inspiratory effort in five copd patients on pav compared to spontaneous ventilation (sv) and full support (fs: cpap+fa+va). flow, volume, minute ventilation (ve) respiratory rate (rr), inspiratory swing in esophageal pressure (apes), and its integral per breath (pti/b) and per minute (pti/m) were measured. objectives: to evaluate airway pressure fluctuation (apf) during spontaneous breathing in a high compliance cpap system. methods: the cpap system consisted of two 7l weighted balloons in a wedge shaped holder. ventilating gas flowed from one balloon through a low resistance one way valve into a tracheal tube (ett) provided with a pycor co 2 sensor to monitor rebreathing. the ett was connected to a piston drive mechanical lung. expired gas flowed through a low resistance valve into a second weighted balloon, from where it was exhausted through a peep valve connected in parallel with the second weighted balloon. we evaluated system performance at v r from 70 to 500ml, at rr from 10 to 120 bpm, while closely monitoring cpap airway pressure swings. at v v of 400 and 500ml the rr was limited to 60 bpm. for comparison we explored aps of a one 16l balloon cpap system, the cpap mode of the puritan bennett 7200, and siemens 300 ventilators, when connected to a healthy adult volunteer breathing through an ett. results: the compliance (cpl.) of one 7l balloon system was linear over a range from 1.0 to 3.3l, with a cpl. of 4.0 l/em h20.the cpl. of the 16 l balloon (0.5 l/em h20) was linear between a volume of 13 and 14.5 l. apf of the weighted balloon system was under 1 em h20 at all v r (except at a v r of 500ml aps was 1.5em h20), while the apf in the 16l balloon was up to 3 em h20. apf witli human volunteers with the two commercially available ventilators in the cpap mode was about 7 cm h20; while under identical conditions apf in the 16l balloon system was 1.5 emhzo; and in the two 7l balloon system was below lcm h20. conelusions: cpap using the two balloon system exhibits lower airway pressure fluctuations than a single balloon system; and is substantially lower than found in the two commercially available ventilators when used in the cpap mode. objective: to perform independent lung ventilation (ilv) with individual tidal volume (vt) set at a value generating a plateau airway pressure (pplat) < 25 crnh~o and to evaluate the usefulness of the continuous monitoring of endtidal co2 (etco2) as a guide to titrate individual lung vt during ilv and for the weaning from ilv. methods: in seven patients, ilv was performed with ttvo ventilators set with the same fio: and respiratory rate. each lung was ventilated with a vt that developed a pplat < 25 cmh~o. this setting led to a lower vt on pathological lung (pl). vt was increased in pl following etco~ and paco2-etco2 variations. ilv was discontinuated when etco~., vt and statical compliance (cst) were similar in both lungs. results: one hour after starting ilv (ti), pl mean vt was significantly lower than in normal lungs (nl) (224 + 46 ml vs 377 + 766 ml, p<0 001) two individual behaviours were observed on tl in pl: four patients presented low etco: (range 18 -31 mmhg)and normal pacoz (range 38 -42 mmhg), while three patients had normal etco2 (range 35 -45 mmhg) with high pac02 (range 44 -61 mmhg). one hour before stopping ilv (t2), vt, etc02 and paco2 were the same in each lung. the pao2/fio: ratio improved in all patients from the beginning ofllv cst of pl was 526 + 30 % of the normal lungs' cst on ti and improved to 97.6 + 27 % ofnl's cst on t2 (p<0.005 vs conclusions: setting vt of pl to a value not overcoming a pplat threshold does not impair oxygenation and is helpful in avoiding barotraumatism. measurements of differential etco2 and of the differential paco2-etco2 gradient can be used to titrate vt allocation during ilv and as a guide for the weaning from ilv. total respiratory resistance in mechanically ventilated patients exceeds values obtained in normal subjects, due to the added and highly flow dependent resistance of the endotracheal tube (rett). this can adversely effect the efficacy of pressure regulated modes of assisted ventilation, such as pressure support (psv) and proportional assist ventilation (pav). recent work demonstrates that the influence of rett during psv can be overcome by using tracheal (ptr) rather than airway opening (pao) pressure to regulate the pressure applied (intensive care med 20:$41, 1994) . the purpose of this study was to see if this approach would also be effective during pav. flow, volume, pao, ptr, and transdiaphragmatic pressure (pdi) were measured in 5 intubated patients in which either pao or ptt were used to regulate the pressure applied during pav where volume assistance was varied from 20 to 80% of respiratory elastance. representative results (mean + se) are shown below. compared to spontaneous breathing (pav 0%), pav increased tidal volume (vt) while reducing respiratory rate (rr) so that minute ventilation ('~e) also rose. this was associated with a reduction in inspiratory effort, as reflected by a decrease in the pressure-time integral ( [ p) of pes and pdi both per minute and per liter ~re. the effects on breathing pattern were similar for pao and ptr regulated pav. in contrast, the reduction in inspiratory effort was always greater for ptr regulated pav. in conclusion, the volume assistance provided by pav is more effective when ptr rather than pao is used to regulate the pressure applied. pav methods: retrospective data analysis of 596 adult patients with normal pulmonary function before operation and uneventful course following coronary artery bypass graft surgery over an 18 month period. we compared assist/controlled mandatory ventilation (s-cmv, 123 patients), synchronized intermittent mandatory ventilation with inspiratory pressure support (s-imv/psv, 431 patients) and biphasic positive airway pressure ventilation (bipap, 42 patients). results: patients ventilated with bipap had a significantly shorter mean duration of intubation (10.1 h, p< 0.05) than patients treated with s-imv/-psv (14.7 h) and s-cmv (13.2 hi. with s-cmv 39.9% of the patients required single or multiple doses of midazolam but only 13.5% in the s-imv-/psv group and 9.5% in the btpap group. the mean total amount of midazolam of these patients was significantly higher in the s-cmv group (8.8 mg) than in the s-imv/psv group (6.6 mg, p<0.05) and in the bipap group (4.3 mg, p<0.05). the consumption of pethidine and piritramide did not differ between s-cmv and s-imv/psv but was significantly lower during bipap (p<0.05). after extubation the paco2 patients was highest in the s-cmv group. conclusion: ventilatory support with bipap reduces the consumption of analgesics and sedatives and duration of intubation. unrestricted spontaneous breathing as well as fully ventilatory support allow adequate adaptation to the patients requirements. bipap seems to be an alternative to s-cmv and sqmv/psv ventilation not only in patients with severe ards but also in short term ventilated patients. _objectitives: after end-inspiratory airway occlusion we examined the ensuing gradual decrease in tracheal pressure (ptr) with the following equations proposed by bates et al. and hildebrandt: pv = p'v e'~cccl~2 +pst, rs (bates) [1] where p'tr is tracheal pressure immediately after occlusion, to= is occlusion time, "r 2 is viscoelastic time constant of respiratory system, and p t is static elastic recoil pressure of respiratory system. p~(t) = h 1 -h 2 log t (hildebrandt) [2] where h~ and h 2 are parameters depending on lung volume, and initial time is 1 s for analytical reasons. materials & methods: we studied 8 healthy patients intubated, anestethized with propofol, paralyzed with vecuronium, and mechanically ventilated with constant flow (0.5 i/s) at zeep for minor surgery. pressure was measured in the trachea. flow was measured with a pneumotachograph and volume was obtained by numerical integration. the rapid occlusions were produced by an external valve. the signals were sampled at a frequency of 200 hz and processed on a pc. the influence of the cardiac artifacts during the occlusion time (4 s) was reduced by a software low-pass filter kaiser finite duration impulse response of elevated order. results: the mean (+ sd) coefficient of correlation using eq. 1 was 0,912 -+ 0.168, and using eq. 2 was 0.884 + 0.045. the values ofz~ (eq. 1), however, decreased with increasing the tidal volume (vt) according to the following equation: "~2 = 1.52 -0.65 v t, similary, the values of h~ and h 2 increased with increasing v t according to the following functions: h~ = 4.4 + 13 v i and h 2 = 1.15 + 1.88 v t. conclusions: the behaviour of "% of eq. 1 suggests that the linear viscoelastic model is not sufficient to further describe the mechanical properties of the respiratory system over the vt range (6-14 ml/kg) in ventilated patients. infect this model predicts that "c 2 is constant and independent of tidal volume. on the other hand the plastoelastic model is not sufficient to further describe the mechanical properties of the respiratory system. in fact "r 2 obtained by fitting an exponential for data of eq. 2, is determined by the time of endinspiratory airway occlusion. obiectives: according to the viscoelastic model, the viscoelastic pressure of the respiratory system pv=rs during lung inflation with constant flow e~ is t/ r 1 2 1 wh t lsms ira tlmeand r given by:pv~c.~ = 2d~( -'e-~ )[ ] ere " ' p" tory " 2 and "r 2 are resistance and time constant of viscoelastic unit. in the past, the viscoaletic constants were determinated by performing a series of occlusions at different lung volumes, or a sedes of occlusions at a fixed lung volume achieved with various inflation flows. in the present study we have developed a new method for determining "c 2 and r 2 which requires a single constant flow inflation. our method is based on determination of pv~r, during a single breath constant flow inflation, and of z 2 during the ensuing end-inspiratory airway occiusion. dudng the occlusion the tracheal pressure p~, declines according the following function: ptr = p'lr e " too= " z2 + e~t.r= [2] where p'~r is tracheal pressure immediately after occlusion, toc c is occlusion time, p,i.rs is static elastic recoil pressure of respiratory system, and ~ is viscoelastic time constant. we first determinated "~2 by analyzing the time-course of ptr according to eq 2 and next determining r 2 according to eq. 1, using the expedmental values of p,i=~, ~ and ti, as well as "~2 obtained with eq. 2. materials & methods: we studied 8 healthy patients intubated, anestethized with propofol, paralyzed with vecurenium, and mechanically ventilated with constant flow (0.5 i/s) at zeep for minor surgery. pres-sure was measured in the trachea. flow was measured with a pneumniachograph and volume was obtained by numerical integration. the rapid occlusions were produced by an external valve. the signals were sampled at a fi'equency of 200 hz and processed on a pc. the influence of the cardiac artifacts dudng the occlusion time (4 s) was reduced by a software low-pass filter kaiser finite duration impulse response of elevated order. results: the mean coefficient of correlation with eq. 2 was 0.912. with v t of 7 ml/kg, the mean values (+ sd) of ':2 and r 2 of the 8 subjects amounted to 1.128 • 0.100 s and 3.990 • 0.890 cmh20 i "~ s. with the traditional multi breath method the corresponding values were 0.711 + 0.257 s and 4.445 _+ 1.474 cmh20 i "1 s, respectively. with the t-test the difference between new and traditional "~2 was statistically significant, between new and traditional r2 was not significant. conclusions: with the single breath method it is possible to compute ':2 and r 2. the mean values of r 2 with v t of 7 nd/kg, however, was slighuy different than those obtained with the traditional multi breath method. the application of modem principles of respiratory care and mechanical ventilation in icus has resulted in increased survival of critically ill individuals with neuromuscular, skeletal and irrevers~le pulmonary diseases. in these chronically ill individunts mechanical ventilation, long term 02 therapy (ltot) and continuous home care is considered a chronic life supporltng technique that can not be withdrawn after their discharge from an icu. the aim of this study was to present the results of a rehabilitation programme and home care that runs in our ward. twenw three patients were referred to our clinic f~om icus during 1993-94. a specific rehabilitation programme designed according to individual's needs was performed. patients that benefitted from this programme were grouped into the following disorders. 1) post tb respiratow failure 6(26%) 2) neuromuscular diseases, 3(21%) 3} undiagnosed sas 3{13%) 4) cope) 9(39%) (3 patients had a overlap syndrom). the programme consists of : 1) assessment and mechanical support ff needed of the respiratonj system with non invasive methods (nasal or via tracheostomy). 2) group and individual respiratory therapy 3) mobilization 4) nutritional support 5) educational classes for the members of the family. three from the patients passed away (during the year), 11 are under nippv during night with or without 02 supply, 13 pts recieve ltot. conclusion: the development of a programme for chronically ill individuals in especially designed wards in hospitals and the overall care at home is considered necessary at least in hospitals with icus. a rehabilitation programme and home care permits the fast but safe discharge of these patients from units of acute medicine that the cost of treatment is high and besides permits beds that are invaluable. we considered that the rehabilitation prod'amine and home care in our ward is the first performed in greek chronically ill pts and even though there is no special administxative support we think that the results are quite saltsfactory. objective: we postulated that the product of the respiratory frequency (f) and the ratio of inspiratory pressure (ip) to maximal inspiratory pressure (mip) would predict the weaning outcome in deeompensated copd patients better than either variable alone or other indices previously proposed. methods: in 28 decompensated copd patients with difficult weaning, we measured, daily, respiratory mechanics data both during mechanical ventilation and after ten minutes of spontaneous breathing. then we calculated weaning indices reported in literature and some new integrated indices. according to the results of the discriminant analysis, we considered the integrative index crop (acronym of compliance, rate, oxygenation and pressure), the rapid shallow breathing index f/vt, the load/capacity ratio ip/mip, and the following new index: f x ip/mip. we used receiver-operatingcharacteristic (roc) analysis by calculating the area under the curve considered as the overall probability of correct classification. results: main results are reported in the following objective: to evaluate the reliability of some indices of endurance in predicting the weaning outcome of decompensated copd patients. methods: in 28 decompensated copd patients with difficult weaning from mechanical ventilation (mv) we measured, daily, blood gas analysis, ventilatory and airway pressure pattern during mv, breathing pattern (frequency (f) and tidal, volume (v~)), inspiratory pressure (ip), and maximal ip (mip) during spontaneous breathing (sb). thereafter we calculated the following weaning indices: crop (compliance * mip * (pao2/pao2) / f), flvt, ip/mip. data obtained the day at which the patient was considered ready for a trial of sb on clinical grounds but weaning failed (wf) and those obtained the day of the successful weaning (ws) were compared statistically through the wilcoxon rank-sum pair analysis. in order to quantify the predictive accuracy for each index with respect to successful weaning we calculated sensitivity, specificity, and diagnostic accuracy according with the standard formulas. methods : five patients (64 + 6 yrs) suffering from ards (lung injury score > 2.5) for 48 hours or less entered into the study. irv (volume controlled, decelerating flow, 20 % inspiratory pause, lie = 2/1) was compared to conventional ventilation (cv) (volume controlled, constant flow, no inspiratory pause, iie= 1/2). these two modes were applied for 6 hours in a randomized order, with the same levels of total peep (peept = peep + peepi), tidal volume (8.0 • 0.7 ml/kg), respiratory rate (20 • 0"bpm) mad fit2 (63 • 2 %). measurements (respiratory mechanics, hemodynamics, arterial and mixed venous blood gases) were performed after 1, 2, 4 and 6 hours of application of each mode. rvsuils : are expressed as mean + sem and compared by anova. backeround and methods: periodic breathing (pb) is characterized by repetitive cyclic variation in minute ventilation. pb is considewxl to be provoked by an instability in the respiratory control. inintubated, spontaneously breathing patients conventional modes of pressure support ventilation, i.e., triggered inspiratory pressure support 0ps), do not allow patients to breathe with theirinherent breathing pattern. therefore, pb, if existing, will appear mainiy after extubation. since our new mode of pressure support ventilation" automatic tube compensation" (atc) continuonsly corrects for the flow-dependent tube resistance during insnmdon and expiration ("electronic" extubatim), it pemaits patients to maintain their own inherent breathing pattern. then, ff necessary, tracheal pressure can be additionally supported by volume-proportioead and/or by flow-proportional pressure support (proportional assist ventilation, pav). (~as~: we report the case of a 70-year-old male patient who was intubated due to acute respiratory insufficiency after acute myocardial infarction with left ventricular dysfunction. during ips of 10 mbar the patient showed a regular breathing pattem which became periodic during atc. in addition, proportional assist ventilation of 10 mbar/l increased periodic breathing in such a way that the typical cheyne-stokes breathing pattem occurred (see figure) . baqkground: the hering-breuer reflex (hbr) is characterized by an inhibition of inspiration during lung inflation. this response has been recognized as an important vagally mediated mechanism for regulating the rate and depth of respiration in newborn mammals. in adult man the hbr is considered to be active only at lung volumes well above functional residual capacity, i.e., at tidal volumes above 1000 ml. assessment of the hbr requires specialized methods such as single breath or multiple occlusion technique. methods; in the presence of desynchronization between ventilator and patient, which frequently occurs during triggered inspiratory pressure support ventilation (ips)(see figure) , prolongation of the interval between inspiratory efforts (indicated by negative deflection of the esophageal pressure) due to lung inflation exposes an active hbr. we examined the occurrence of hbr in intubated critically ill patients. strength of hbr was assessed by the formula: prolongation [%] = ((inspiratory interval of interest -preceding inspiratory interval)/preceding inspiratory interval) * 1(30. rr162 18 of 50 patients examined showed moderate to severe desynchronization. in 17 of these 18 patients a (re)activation of the hbr was found. the strength of hbr amounted to 134 + 51%. there was a significant correlation between tidal volume and strength of hbr. in contrast to previous reports, an active hbr was shown during lung inflation well below 1000 ml. b pck~round: triggered inspiratory pressure support ventilation (ips) is commonly used to support inspiration in intubated spontaneously breathing patients. despite its usefulness ips shows some disadvantages which can be deleterious in crificauy ill patients: -additional work of breathing to be performed by the patient due to the flow-dependent tube resistance -desynchronization between patient and ventilator due to inherent triggering failures of the ips mode suppression of the patient's inherent breathing pattern -inability to predict successful extubation in difficult-to-wean patients methods: based on the known flow-dependent tube resistance our new mode "automatic tube compensation" (atc) compensates for the pressure drop across the endotracheal tube ("electronic" extubation). then, if necessary, tracheal pressure can be supported by volume-proportional pressure support (vpps) and/or by flow-proportional pressure support (fpps). results: hitherto, we have examined 20 patients after open-heart surgery and 50 patients with acute respiratory insufficiency (ari) or ards using atc with/without vpps/fpps. preliminary results suggest that the new mode avoids additional work of breathing due to accurate compensation of the pressure drop across the endotracheal tube during in-/expiration prevents desynchronization between patient and ventilator allows patients to breathe with their inherent breathing pattern accurately predicts the outcome of extubation even in difficult-to-wean patients due to "electronic" extubation conclusions: the new mode atc with/without vpps/fpps allows to support ventilation in a more physiologic manner and overcomes the disadvantages of conventional modes of pressure support in intubated patients. backgound: cheyne-stokes respiration (cs) is characterized by regula]; recurring periods of hyperpnea and apnea. in normal subjects, cs may occur after hyperventilation, after arrival in high altitude, or during sleep. it has also been observed in patients with prolonged circulation time due to congestive heart failure, as well as in some neurological patients. there is no report about the influence of sedative drugs on periodic breathing (pb) and cs. methods: in intubated patients conventional modes of pressure support do not allow patients to breathe with their inherent breathing pattem. therefore, periodic breathing and cs are rarely seen. since our new mode of pressure support ventilation "automatic tube compensation" (atc) continuously corrects for the flow-dependent tube resistance during inspiration and expiration ("electronic" extubation) it permits patients to maintain their own inherent breathing pattem even if pathological, e.g., periodic. results: using this new mode of pressure support ventilation, periodic breathing was unmasked in 13 of 37 intubated patients, 6 of which showed cs. in 4 of these 6 patients the occurrence of cs was linked to impaired left ventricular function with increased circulation time. normal left ventricular and neurologic function was found in the remaining 2 patients. in 1 of these 2 patients cs disappeared after intravenous administration of the benzo-diazepine antagonist flumazenil (figure). consequently, in this patient cs was induced by benzodiazepine sedation. objecti',~s: in contrast to conventional rhodes for pressure supported spontaneous breathing, our newly developed ventilatow mode ,,automatic tube compensation" (atc) completely compensates for the flow-depandant pressure drop tlpm-r across endotracheal ttlbe (ett). in the atc mode, the ventilator supplies a flow v' in order to maintain a constant tracheal pressure p~,,~. to this end, pk,,= has to be oontinuousiy determined. since continued measurement of p,,~ by introducing a catheter via the ett is not reliable, we opted for its continuous calculation socordng to the following equation: p~ = p,,, -aperr, pw being the continuously measured airway pressure. this also requires the continual measurement .of flow v' to calculata apm-r using the non-fineer approximation: aport = kvv' + k2.w. the constant tube coefficients k~ and k2 are mathematically determined by mesns of a least-squares-fit procadum based on laboratory investigations. tracheal secretions, however, reduca the omss-saction of the ett. consequently, ~ values of ki end k2 are changed rendering the p~,ch calculations inaccurate. therefore, k1 and ~ have to be pedodcally updated to ensure an a~urete monitoring of pn,~ and a complete tube compensation under atc at any time. background: one of the first steps in weaning patients from controlled mechanical ventilation is to stop muscle relaxation and to reduce sedation. it can take several hours, however, until the patient is able to trigger the ventilator and to breathe spontaneously. during this period, many patients display a sudden increase in peak airway pressure of up to 30%. patients and methods: to investigate the reason for this potentially dangerous effect, we continuously measured lung and chest wall mechanics in post-operatively ventilated patients. lung mechanics (airway resistance and lung compliance) was measured using the esophageal balloon technique as described in [1] . chest wall mechanics (tissue resistance and chest wall compliance) was calculated from lung mechanics and total respiratory system mechanics as described in [2] . results: we found a decrease of chest wall compliance (cw) to be the main reason for episodes of sudden airway pressure increase while lung compliance (cl) remained unchanged. the decrease of c w can be intergil cano a, san pedro jm ~, sandar d, herntndez .1, carrizosa f, , herrero a. emergency and intensive care department, hospital of jerez, spain objective: 1) to determine the incidence of hypoteasion (h) associated with emergency intabatian of mechanical ventilation, and 2) to establish its relauonship with respiratory mechanics (rm) and arterial blood gases. mechanical ventilation performed in the emergency room, in a prospective eans~eative manner, were evaluated. data collected included patient demographics, diagnoses, blood pressure and arterial blood gas levels before and at~er intabatian, and p_m, including calculated pulmonary end-inspiratory volume above functional residual capacity (veic) and calculated dynamic hypetinflatien (dhc). all patients received midazolen and awaanrinm to facilitate tracheal intubatien and rm measurement. hypotension was defined as a decrease in systolic pressure higher than 40 mmhg or an absolute decrease in systolic blood pressure below to 90 mhg within 1 hour of intabatian. 14 patients were excluded because met at least one of the following exclusion criteria: preexisting shock or h (8), cardiac arrest (5) .1 there weren't any association between peepi or other airway pressures (paw) and h, but calculated pulmonary volitmes had tendency to be larger in patients with h (p < 0.1). high paco 2 before lrasheal intubatian (87.4 4-9 mmhg) with a quickly decrease alter starting mechanical ventilation was a usual finding (p < 0.01) in patients who developed h. paw. 3) thexe was a good relatienship between h and high arterial paco 2 before traqueal intahatian and its fast "washing" with mechanical ventilation. 4) because cao patients had the highest incidence of h, controned mechanicel hypoventilatien driven by paco 2 changes and pulmonary volumes monitoring instead paw, should be attempted in these patients to avoid this cemplication after tracheal intubatiert. introduction: the endotracheal tube (ett) and demand valve devices cause an added work of breathing (wobadd), which is the work necessary to overcome the resistive load of the ett and the breathing circuit (1). application of ips has been shown to partly compensate this added work (1). since tbe amount of wobadd is flow dependent, a fixed ips is not adequate to completly compensate the wobadd (2). therefore, atc has been developed as a new form of assisted spontaneous breathing (3), which provides a flow-dependent pressure support. thereby, it theoretically should compensate all the wobadd due to the tube. the purpose of this study was to evaluate the reduction of wobadd with ips and atc for different ett. methods: a mechanical lung model (ls 4000, dr*alger, liibeck, frg) was used to generate a constant spontaneous breathing pattern. the ls 4000 was connected to an artificial trachea (at, 10 cm long, 22 mm id). the at was intubated with three different tubes of 7.0, 8.0, 9.0 mm id and connected to an evita 2 ventilator modified to provide atc as an option (dfager, liibeck, frg). flow and airway pressure were measured between the y-piece and the ett for four different modes of ventilation: cpap, ips of 5 and 10 cm i420 and atc all with a peep of 10 cm h20. the tracheal pressure (ptrach) was measured in the at. total wobadd was calculated as the area subtended by the ptrach-volume curve below peep. results: the results for total wobadd in nd/1 are shown in the figure for the three different ett: breath/mln, s=success, f=failur% *~p<.05, **-p<01, ns = non significant, f versus s neveltheless, in 5/26 patients, invasive ventilation was necessary in mean 12.6_+12 hours after beginning of fmpsv. there was no significant difference between the two groups (success, failure) in following parameters : sex, age, previous histoly, medical treatment, saps1 & 2, clinical signs (rr, spo2, heart rate, blood pressure, glasgow score...), radiological and echocardiographic findings and standard biological parameters. only two parameters were related with failure : 1.a low value of pac02 on admission until the patients were intubated. 2. an increased level of cpk in relation with an acute myocardial infarction (4/5 cases in the failure group, vs 3/21 cases in the success group, x~(with continuity correction) : p<.05). conclusion : fmpsv is a noninvasive, safe, rapidly effective method of treatment in acpe, which may avoid tracheal intubation. further studies are necessary to precise if association of arf and low paco2 (<35mmhg) and/er acute myocardial infarction represents an indication of immediate invasive ventilation. introduction: since the added work of breathing (wobadd) imposed by the endotracheal tube (ets and the breathing circuit is regarded as an important contribution to the total work of breathing, considerable effort has been tmdettaken to compensate for this added work. ips has been fotmd to decrease the wobadd imposed by different ventilators (1, 2). because of the flow dependent pressure drop across the etf the tracheal pressure (ptr) should be measured to estimate the total imposed wobadd (wobtut) (3, 4). the aim of this study was to assess the circuit imposed work (wobcirc) and wobtot (including ett) for different demand valve ventilators during cpap and/ps. methods: a mechanical lung model (ls 4000, driiger, lfibeck, frg) generated a constant spontaneuus breathing pattern. the ls 4000 was connected to an artificial trachea (at), intubated with an 8.0 nun et]', end connected to one of four ventilators (servo 900c and servo 300, siemens,-elema, sweden; evita2, driiges, liibeck, frg; pb 7200ae, puritan bennett, carlsbad, usa). three different modes of ventilator settings were tested (cpap, ips 5 and 10 mbar; trigger set at maximal sensitivity, peep always 10 mbar). flow and airway pressure (paw) were measured between the y-piece and the etr; tracheal pressure (ptr) was measured in the at. wobtot was calculated as the area under the ptr-volume curve below peep, wobcirc was calculated as the area under the paw-volume curve below peep. results: in the foti g., patroniti n., cereda m., sparacino me., giacemini m., pesenti a. inst.of anesth.and intensive care-univ.of milan -sgh monza i aim of the study was to assess cpl,rs measurement obtained by the airway occlusion method during psv. we therefore studied 31 paralyzed cppv ventilated ali patients (lung injury score =2.25• that were weaned to psv. we performed end inspiratory and end expiratory airway occlusions using the hold function of the ventilator (siemens serve 900c), first during cppv and then within the 24th psv hour. airway pressure and flow signals were recorded (cpi00 bicore) for subsequent analysis. an airway pressure plateau was defined as a 0 flow tracing in which airway pressure was stable for at least 0.25 sec. end inspiratory (pel,rsi) and end expiratory (pel,rse) recoil pressures were then measured as the mean airway pressure during plateaus. cpl,rs was computed as tv/ (pel,rsi-pel,rse i) cpl,rs can be adequately estimated during psv using the airway occlusion method; 2) during psv inspiratory plateaus are longer than the expiratory ones; 3) the length of plateaus is negatively affected by the respiratory drive. foti g., de marchi l., *tagliabue m., gilardi p., giacomini m., sparacino me., pesenti a. inst.of anesth.and intensive care,-univ.of milan *dept.of radiology-sgh monza i we retrospectively compared ct scan and gas exchange findings between a group of patients successfully weaned from vcv to psv (group s = ii patients) and a group who failed the weaning (group f = 6 patients). we selected 17 ali patients (lis=2.5• in vcv mode who had available a chest ct scan performed within 4 days from the weaning trial. a psv trial was began as soon as the patient reached hemodynamic stability and a pao2 >80 mmhg, irrespective of fie2 (peep <15 cmh20). maximum psv level was < (pel,rs-peep) measured during vcv, where pel,rs was the respiratory system elastic recoil pressure at end inspiration. psv ventilation was considered successful if a respiratory rate <40 bpm, an increase in fie2 lower than 0.2 compared to vcv, a pace2 increase <20% of vcv value and hemodynamic stability were maintained during the next 48 hours of psv. if any of these conditions was not met the trial was declared a failure. interdisciplinary critical care unit, regional hospital lugano-ch *surgical critical care unit, university hospital, geneva-ch objective: to assess the degree of correlation of cardiac output measured by thoracic electrical bioimpedance and thermodilution in mechanically ventilated patients with different levels of positive end-expiratory pressure (peep). methods: prospective study with 10 ventilated patients, 7 after head injury and 3 with postoperative sepsis, with normal cardiac output: simultaneous determination of cardiac output by thermodilution and thoracic electrical bioimpedance performed with different levels of peep (0-5-15 cm h20). results: cardiac output measured by thermodilution during sequential increment of peep did not vary: 7.3 + 2.5 for peep 0, 7.4 + 2.7 for peep 5 and 6.9 + 1.7 l/rain for peep 15. simultaneously the bioimpedance device recorded a significant increase in cardiac output from 4.4 + 1.3 for peep 0 to 6.0 + 1.9 l/mi for peep 15. (p < 0,05). conclusion: cardiac output measured by bioimpedance cannot replace the invasive thermodilution methods of cardiac measurement output during mechanical ventilation with peep. we also isolated a subset (h) of 12 patients who had been hypercapnic (paco2>50mmhg) for at least 3 days (range 3 to 60 days) before the end of cv. the psv trial was started as soon as pao2 was > 80 mmhg, irrespective of fie2 and with peep < 15 cmh20 and the psv level had to be < (pplateau-peep) as measured during cv. pace2, pha, base excess (be) were collected before discontinuation of cv and on the ist day of psv: 05) . 2) weaning is more difficult in pts with head injury(p<o,05) and iss>30 (p<o,ol), in elderly(p<o,05) and in pts who need fi02>0,6 (p<o,05) and peep>io cm h20 (p<o,05). 3) pts with iss>30 need longer duration of mv (p<o,ol). 4) the mortality rate is higher in pts with iss>30 (p<o,o01), with head injury (p<o,ol) and in elderly (p<o,05). 5) paradoxally, compliance was found to be higher in pts>60 years than in pts<60 years (p<o,05). s. crotti, p.pelosi, d.mascheroni, m.cerisara, a.lissoni, l.gattinoni. istituto di anestesia e rianimazione -irccs, milano, italia. obiectives. we investigated by ct scan the effects of extrinsic peep (peepe) on lung inflation, tidal volume distribution and regional compliance in sedated, paralyzed chronic obstructive pulmonary disease (copd) patients with dynamic hyperinflation (peep• methods. two ct section (end expiration, end inspiration) were obtained at lung base level in 4 patients (individual analysis for each of the eight lungs) at 4 peepe levels: peepe = 0 cmh20 (zeep) and peepe amounting to 50%, 100% and 150% of the peep• at zeep (9.5 • 4 cmh20). tidal volume was kept constant (8 • 1.4 ml/kg). each lung section was divided into 3 zones equally spaced along the vertical axis: upper (the most ventral), middle and lower (the most dorsal) zone. for each zone we computed: gas volume at end expiration (gase) and at end inspiration (gas• tidal volume (dgas=gasi-gase) and compliance (cpl=dgas/dp;.dp=plateau pressureactual peep• we also computed the lung inflation as the entire ct section gas volume at end expiration (total gase). results. at each peep level both gase and gas• were higher in the upper than in the lower zone ( we cone!ude that in copd patients with dynamic hyperinflation only the application of peepe higher than peepi produces a further increase of lung inflation, decreasing cpl of the upper zone (the more expanded one) probably by stretching phenomena, and causing dgas redistribution from the non dependent to the dependent lung zones. i. ravagnan, p. vicardi, f. valenza, d. tubiolo. p. pelosi l. gattinoni. 1st . anestesia e rianimazione, universita' di milano, ospedale maggiore -irccs, italy objectives: to investigate the effects of peep and ps on respiratory. pattern and inspiratory effort during ps ventilation in patients with acute lung injury or acute exacerbation of chronic lung disease. methods: 4 pts with acute (a) (paco2= 39• mmhg, pao2/pao2= 0.4• and 4 pts with chronic (c)(paco2= 51• mmhg, pao2/pao2= 0.3:50.1) lung disease were studied in ps ventilation during the weaning phase. measuring airway pressure, esophageal pressure and gas flow we computed respiratory rate (ril bpm), tidal volume (tv, ml) and pressure time product (ptp, cmh20*s/min), which is an index of oxygen muscle consumption. measurements were collected, after 15 rain of stabilization, at 5 and 15 cmh20 ps and at two different peep levels (0 and 8 cndd20 conclusions: during psv: 1) breathing pattern and ptp are different between a and c; 2) in both groups peep does not modify breathing pattern; 3) peep reduces ptp in c but not in a; 4) ps modifies breathing pattern and reduces ptp in c but not in a. to test the performance of a new heat and moisture exchanger (hme): twistair, baxter. this is a hydrophobic-hydrophilic hme without hygroscopic salts. method: we evaluated the hme performance with a previously described method (i); it consists of a "lung simulator" connected to a mechanical ventilator and a flow divider, with a dry and a wet thermal probe inserted into inspiratory and expiratory limbs. on average ps level changes were associate( with opposed changes in both p0.1 and rr. changes in p0.1 were remarkable, homogeneous and highly significant (p<.002), while changes in rr were less pronounced, inhomogeneous and less significant (p<.02). it seems therefore that p0.1 is a better index than rr for estimating a change in load for the respiratory muscles. recently, the lsf method has been described as an interesitng alternative to conventional tecniques used to measure total respiratory mechanics, providing, over these latter, advantages such as no need for hold maneuvers and no need for particular flow patterns. data on the reliability of the lsf method, however, are still few. methods. 9 sets of measurements were obtained in each of 7 ards patients, paralyzed and ventilated in cmv with constant inspiratory flow and an end-inspiratory pause equal to 15% of ttot. the lsf method provided data for total compliance (ctotlsf) and total resistance (rtotlsf) by multiple linear regression analysis of airway pressure, flow and volume change, applied over the entire breath. the lsf measurements were automatic and continuous, and each value used for analysis was the average of 160 consecutive cycles. conventional measurements of dynamic.compliance (cdyn), quasistatic compliance (cqs), minimum inspiratory resistance (rmin) and maximum inspiratory resistance (rmax) were obtained by the constant flow, end-inspiratory occlusion method, each value being the average of 3 measuremts. ctotlsf was compared with cdyn and cqs, while rtotlsf was compared with rmin and rmax. results. ctotlsf showed a high correlation both with cdyn (cdyn=.87.ctotlsf+l.14, r2=.982), and with cqs (cqs =.94.ctotlsf +1.19, r2=.984). the average difference between ctotlsf and cqs, yet, was much lower (+.36+1.79 ml/cmh20) than the one between ctotlsf and cdyn (+2.43+2.40 ml/cmh20). rtotlsf correlated much better with rmax (rmax=.88*rtotlso+.98, r2=.914) than with rmin (rmin =.58.rtotlsf +. 1.71, r2=.804). the average difference between rtotlsf and rmin was +4.48+3.48 cmh20/1/s, while the one between rtotlsf and rmax was +.81+2.03, confirming the better agreement between rtotlsf and rrnax. conclusion.the agreement between rtotlsf and rmax suggests that rtotlsf takes into account both the airway and the tissue components of resistance, unlike rmin which only expresses airway resistance. the agreement between ctotlsf and cqs indicates that ctotlsf expresses the pure elastic components of the respiratory system. in the paralyzed patient, the expiratory time constant (rce) of the unit represented by total respiratory system and ventilator can be calculated from the slope of the expiratory flow-volume curve. recently it has been suggested that an estimate of this slope is provided by the ve/v'epeak ratio, ve being the exaled tidal volume and v'epeak the peak expiratory flow.the reliability of this method is still little known. methods. 9 sets of measurements were obtained in 7 mechanically ventilated ards patients during paralysis and cmv. measurements of ventilator expiratory resistance (rext) and of total respiratory mechanics were performed in order to calculate these 3 reference measurements for ve/v'epeak: rcdyn=(rmin+rext)ocdyn, rcstat=(rmax+rext)ocqs, rclsf=(rlsf+rext)~ cdyn (dynamic compliance), cqs (quasistatic compliance), rmin (minimum inspiratory resistance), rmax (maximum inspiratory resistance) were obtained by the constant flow, end-inspiratory occlusion method. clsf and rlsf respectively corresponded to measurements of total respiratory system compliance and resistance by the least squares fitting method. ve/v'epeak showed a high correlation with all 3 reference measurements, rcdyn (ve/v'epeak= 1.07orcdyn-.18, r2=.939), rcstat (ve/v'epeak = .97 9 rcstat -. 12, r2= .929), and especially rclsf (see fig the static pressure volume curve of the respiratory system (p/vst) provides valuable information, but the extensive data analysis prevents its use in clinical routine. dynamic p/vcurves are simpler to record. the objective of this study was to develop an automated method to obtain quasi-static p/v-curves (p/vqs) during low flow inflation as well as to measure resistance. preliminary tests were done in normal subjects and subjects with varying degree of lung disease. methods: a computer/ventilator interface (cvl) was constructed for control of a servo ventilator 900c during an automatic sequence: a) a normal breath, b) a prolonged expiration at zero peep, c) a low flow inflation of a predetermined volume, d) analysis of ventilator pressure and flow. pressure was corrected for tube resistance. airway resistance was calculated from a) and used to derive p/vqs from c). as reference, p/vst was obtained by the occlusion method. results: in non-obstructive diseases the method performed well at volume and pressure controlled ventilation. static and quasistatic compliance were virtually identical. p/vqs clearly reflected the lower inflection point and, when present, the upper inflection point (uip). in some patients uip was more evident in p/vqs, especially at an increased inflation flow rate. in obstructive diseases, the method for subtraction of resistive pressure was inadequate. conclusions: the system for automated computer controlled studies of lung mechanics based on a standard ventilator provides comprehensive information. differences between p/vst and p/vqs imply that the latter reflects also other factors than p/vst that contribute to impedance at hyperdistension. the clinical significance of the two curves remains to be shown. obiective: to evaluate the effect of combined high frequency jet ventilation (chfjv) in patients with severe respiratory insufficiency (sri). methods: in a retrospective study of the period 1985-1995 we analyzed patients suffering from sri who were refractory to conventional mechanical ventilation and were treated with combined high-frequency jet ventilation (chfjv). refractory to conventional mechanical ventilation was defined as a pao2/fio 2 < 100, despite maximal ventilator settings: peep > 8 cm hz0 , fit 2 > 0.6. a total of 43 patients matched these criteria, 27 males and 16 females with a median age of 44 (17-72) years. seventeen suffered from severe trauma. chfjv was started following a median period of 3 (1-22) days of conventional mechanical ventilation. prior to chfjv ventilation parameters expressed as median were the following: fit 2 0.8, pao2/fio 2 78, peep 12 cm h20 peak airway pressure (pap) 48 cm h20. chfjv consisted of high frequency jet ventilation with a frequency of 100 to 300 breaths/minute, driving pressure of 1.8 to 3.5 arm, and inspiration time of 20 to 30 percent, superimposed on the whole cycle of conventional mechanical ventilation with a frequency of l0 to 20 breaths/minute and tidal volumes of 100 to 400 ml. results: following two days of chfjv 31 of 43 patients showed an improvement of ventilatory parameters; peep could be reduced to < 8 cm h20 in 14 patients, the pap was decreased with > 5 cm h:o in 20 patients, fio 2 could be reduced to < 0.6 in 27 patients and finally the median pao2/fio 2 ratio changed from 78 to 133. during chfjv 23 patients died, 4 of respiratory failure and 19 due to multiple organ failure, 6 died within two days of chfjv. the median duration of chfjv in survivors and nonsurvivors was 6 days in both groups. conclusions: our data show that with chfjv in the majority of patients with sri who are refractory to conventional mechanical ventilatior" the ventilatory parameters can be improved. backeround and obiectives: although ventilation with peep above the inflection point (pinf) has been shown to reduce lung injury by recruiting previously closed alveolar regions, it carries the risk of hyperinflating the lungs. in the present study we set out to develop a new strategy to recruit the lung during ventilation with small vt, while maintaining peep levels as low as possible. we hypothesized that if the lung was recruited with a sustained inflation (si) to total lung capacity, recruitment would be maintained as long as the peep level was higher than the critical closing pressure of the lung, as observed on the deflation limb of the pv curve (ajrccm 1995; 151(4) :a432). the purpose of this study was to examine the hypothesis that a strategy using si and a peep<pinf would induce less lung injury during small tidal ventilation than ventilation at the same low peep level without using a recmitment strategy in a model of respiratory distress syndrome. methods: we used a randomized protocol with 4 groups to ventilate 40 nonperfused, lavaged rat lungs with small vt (5 to 6 ml/kg) for 2 hours and studied the effect on compliance and lung injury. group 1: peep>ping group 2: peep<pin f with an si, inflation to 30 on h20 over 30 sec, to recruit volume; group 3: peep<pinf without si; group 4: control group, lungs were inflated at peep<pin f, but not ventilated. results: in group 2 and group 4 static compliance did not change after ventilation (table) . in group 3 static compliance fell significantly. group 1 showed significant changes in the pv curve, which were less severe than in group 3. results from morphological examination are pending but preliminary results showed less lung injury in group 2, compared to group 3. conclusions: small tidal ventilation following a recruitment manoeuvre in a model of respiratory distress syndrome allows ventilation on the deflation limb of the pv curve at a peep below pinf. this strategy (1) minimizes lung injury as well as, or better than using peep above pint" and (2) ensures a lower peep to minimize the detrimental consequences of high lung volume ventilation. i~peep>pin~ 2 _objectives-this report is presenting the results of the clinical study for using eeg examination as a method of the evaluation of patients ability for weaning. methods: the study inclljqles 42 eeg examinations with fourier spectral analysis' of 37 patients ~vith respiratory insufficiency and prolonged control mechanical ventilation (cmv). all patients have had a-rhythm of eeg before weaning. we have followed respiratory rate, tidal volume, respiratory pa{tern, end-tidal co2 and blood gases during weaning. results: 13 patients had invariable eeg activity or short 13-waves period (till one hour). the weaning of this patients was fast arid sucsessful. other 24 patients have had a decreasing of a-activity, an appearence of 13-waves for an hour and more, a short episodes of a-and e-activity. after that this patients had gas exchange and respiratory disorders with regression of the weaning right up to cmv. conclusion: eeg could be used as a method of the evaluation of patients ability for weaning from cmv. some eeg signs shows the overstrain of compensatory systems before the change to the worse of gas exchange and respiratory pattern. s. elatrous, p. aslanian, d. touchard, d. corsi, h. lorino, l. brochard. medical intensive care unit, inserm u 296, hopital henri mender, cr~teil, france. in vitro comparison of flow triggering (ft) systems demonstrated advantages compared to pressure triggering (pt) systems for some ventilators (puritan bennett 7 200) but not others (siemens serve 300). we studied the two types of systems in two groups of 8 patients mechanically assisted with pressure support ventilation (15 + 6 cmh20). in the first group (pb 7 200) the effort of breathing, assessed by the esophageal pressure time index, was significantly lower with the ft than with the pt (139 + 40 cmh20.s/min -1 vs 158 + 32, p< 0.05). by contrast no significant difference appeared in the second group (serve 300), as predicted by the bench study despite marked interindividual differences (134 + 55 cmh20.s/min -1 vs 160 + 61, p = 0.1). we conclude that 1) rigorously performed bench studies can predict in vivo effects, 2) mild advantages can be found for the new triggering systems on some ventilators. objectives: pressore-volume curves (pv) of the respiratory system is of interest for the determination static compliance (cs0, lower (lip) and upper (uip) inflection points which indicate zones of airway recruitment and overdistension. this study aimed to compare an "automated low flow inflation" method (alfi) to the reference occlusion (oc) method. the ability of the former method to identify cst, lip and uip was tested in icu patients. me,otis: 16 (8 arf and 8 ards) sedated paralysed patients were studied using a serve 900c ventilator linked to a computer which automatically forced the ventilator to insufflate at a low constant flow a velum up to 1500-2000 ml or a maximum paw of 50 cm h20 (alfi). the quasistatic elastic pressure (pel,qs0 was obtained by subtraction of the resistive pressure of tubing and patient and related to volume for calculation of compliance cqst. for oc tidal volumes (v0 from 50 up to 1500-2000 ml were followed by a 3 s post-inspiratury pause for determination of static pal (pel,st) in relation to volume. compliance was defined from the linear part of the p/v curves. lip and uip were defined from the consistent deviation of p/v data from extrapolated the linear part. ~,~111i~: in ards, mean cst was 27.9 + 3.5 and cqst 29.7 + 3.9 ml/cm h20 (us), lipst 5.2 + 5.0 and lipqst 7.0 + 4.6 cm h20 (us), uipst 23.1 + 10.8 and uipqst 26.0 + 5~4 cm h20 (us). nosocomial pneumonias (np) are frequent and often unsuspected during ards (bell, !983). in the present study, we evaluated prospectively the onset of np during severe ards (group b of the european study). patients and methods: the charts of 15 patients with severe ards have been prospectively recorded. a plugged telescopic catheter (ptc) specimen has been systematically performed every 48 hours, for quantitative bacteriological analysis. the diagnosis of np was defined by a number > 103 colony forming units / ml. results: for the 15 patients studied, the mean saps score (+ sd) was 16+_2, the initial pao2/fio2 ratio was 100-&-_35, the duration of mechanical ventilation (mv) was 19+9 days. the mean delay before the onset of the first np was 8.6+5.6 days (5-12), and the mean pao2/fio2 ratio was 110+-28. respiratory symptoms (purulent aspirates, new pulmonary infiltrates, or gazometric changes) were present in 80% of the patients studied. alteration of gas exchange was present in 8 of the 15 patients (7 np) . a new pulmonary infiltrate was present in only 1 np (10%). an increase of fever was noted in 6 patients, an increase of leukocytosis > 20% in 8 patients, an increase of volume and purulence of sputum in 3 of the 10 patients with np. the degree ofgazometric worsening (pao2/fio2 before np minus pao2/fio2 during np) during the first episode of np was 44+17 mmhg. excluding the bacteriological criteria of np, the number of criterias of np present was 1 in 1/10 patients, 2 (5/10), 3 (2/10) or 4 (2/10). two patients only had a pulmonary colonization (ptc: < 1102 cfu / ml) before the first episode of np. the incidence of np is high (53%) during severe ards. the first episode occurs in average:at the 9 th day, and is the cause of a severe hypoxemia (pao2/fio2 110) . the onset of a np may contribute to the high mortality rate observed in our patients (93%). each worsening of hypoxemia during severe ards should induce to suspect a np. respiratory system during mechanical ventilation. the me~hod quantifies the dissipative energy consumption of the respiratory system in terms of energy loss aek, inefficiency ~k~ and respiratory dissipative resistance rk~ over a given partition of the tidal volume. the method can be applied in intensive care units with no interference to ventilatory support. it allows for monitoring the combined effects of inhomogeneities, non-linearities and visco-elastic effects, that are subject to change in the respiratory system. the method is studied on pigs~ in the presence of a log-dose response curve of methacholine (mch) induced disease. in healthy pigs~ we find a mean value of energy loss, ae, of 0.27 • j/l, a mean value of inefflency, ~ of 0.25 ~=0.05 and a mean value of resistance, 7~, of 4.40 • cm h20 s/1. the respiratory resistance, rk, shows a variation over the partition of tidal volume with armax ----3.90 • 0.66 cm h20 s/l. during methacholine provocation~ ae rises more than five-fold up to 1.48 • j/l~ doubles to 0.54 • and t~ increases to a maximum of 22 • cm h20 s/l, with armax : 15.1 • 7.0 cm h20 s/1. the variation in rk becomes more pronounced with higher doses of methacholine. methods: 10 ards patients were prospectively studied. initially they were ventilated in the amv (assist mechanical ventilation) mode with the settings prescribed by their primary physician. after stabilization, ventilatory gas exchange and hemodynamic variables were determined. patients were then ventilated in the mrv (mandatory rate ventilation) mode with 20 breaths as the target rate. in mrv the target rate is set and the ventilator autoregulates the pressure support level delivered ~o achieve this rate. after stabilization, the measurements done on amv were repeated. finally, patients were sedated and paralyzed and ventilated in cmv (control mechanical ventilation) with the ventilatory variables they had during mrv. measurements done in amv and mrv were repeated and respiratory mechanics were assessed with the constant flow end inspiratory occlusion method. results: two groups were recognized based on their response to mrv. tn group 1 patients responded to mrv by decreasing their v and increasing the t/t t ratio. ve, vo 2, and aado 2 decreased while paco 2 increased and tda vo ume and co remained unchanged. on the contrary, in group 2 v, vr and ve increased; ppeak and trr t remained unchanged, paco~ decreased while vo 2 and aado 2 increased with constant co, the pressure support level needed to achieve the target rate was much lower in group 1 than in group 2 (19,8-+1.3 vs 29.4_+2.0). obiectives : in the newly developed mode of ventilatory support ,,automatic tube compensation" (atc) the ventilator compensates for the flow-dependent pressure drop across the endetracheat tube (ett) thus allowing ,,e]ectronic extubation". the aim of the study is to investigate whether healthy subjects perceive atc in inspiration (atc-in) and in expiration (atc-in-ex) and whether atc provides an increase in subjective comfort compared with the conventional assisted spontaneous breathing mode (asb). methods : healthy volunteers (no preceding lung disease, non-smokers, male, 20-40 years)breathed spontaneously through an uncut ett of 7.5 mm id via a mouthpiece. the ett was connected with a prototype ventilator evita 2 modified by the manufacturer (drfiger, lebeck) for atc. flow and airway pressure were measured at the outer end of the ett. three ventilatory modes, (1) asb (10 mbarover 5 mbar peep), (2) atcin, (3) atc-in-ex were selected in random order. immediately following the transition from one mode to another the volunteers answered by hand sign how they perceived the new mode compared with the preceding mode: ,,better" (+1), ,,equal" (0) or ,,worse" (-1). inspiration and expiration were investigated separately by presenting 120 mode transitions (in total; including ,,placebo" transitions). results : the difference between atc and conventional asb is perceived in inspiration and in expiration. atc is positively judged; asb is nega ively judged. the diagrams show mean values _+ sd of five volunteers investigated up to now. the new mode atc is perceived as an increase in subjective comfort. our explanation is that atc preserves the natural breathing pattern better than conventional asb. objectives: to determine the role of cerebral vasoconstriction in the delayed hypoperfusion phase in comatose patients after cardiac arrest. to correlate the results with indices of cerebral oxygenation and the levels of several vasoactive hormones in the jugular bulb. methods: in comatose patients after cardiac arrest we measured the pulsatility index (pi) of the medial cerebral artery by transcranial doppler sonography. the pi is a reliable indicator of cerebral vascular resistance. we also sampled blood from the jugular bulb and measured cerebral oxygen extraction ratio and jugular bulb levels of endothelin, nitrate and cgmp. the first measurement was done within 4 hours after cardiac arrest and repeated 3, 6, 9, 12, 18 and 24 hours later. results: we studied 10 patients, 6 females, mean age 64,1+13,7 years. the pi decreased s!gnificantly between th~ first and the last measurement from 1.86 _+ 1.02 to 1.05 + 0.22 (p = 0.03). cerebral oxygen extraction ratio decreased also from 0.39+ 0.13 to 0.24 + 0.11 (.p = 0.015). endothelin levels were high, but didn't change during the studied period. nitrate levels varied in a wide range, but didn't change significantly. however, cgmp levels increased significantly from very low levels in the first measurement to very high levels 24 hours later, rasp. 2.95 pmol/ml (median; 25th 2.48-75th 5.43) and 7.5 pmol/ml (median; 25th 6.2-75th 14.00) (p = 0.02). eighteen and 24 hours after the first measurement we found a strong correlation between pi and cerebral oxygen extraction ratio ( r = 0.64, p = 0.05 and r = 0.76, p = 0.01). we.also found 12 hours after the first measurement a significant correlation between pi and cgmp levels ( r = 0.69, p = 0.03). we found no correlation between pi and endothelin or nitrate levels. conclusion.~; our results show a high cerebral vascular resistance in the first few hours after cardiac arrest, gradually decreasing during the next 24 hours. this is accompanied by an initially high cerebral oxygen extraction ratio and low cgmp levels, suggesting that the cerebral vascular resistance is induced by active vasoconstriction because of insufficient cgmp levels, leading to a decrease in cerebral blood flow and a compensatory ~ncrease in cerebral oxygen extraction. objectives: sudden cardiac arrest is a major cause of mortality in western countries accounting for over half of all cardiovascular deaths. in most cases the mechanism of death is prolonged cardio-circulatory arrest due to ver:tricular fibrillation (vf) preceding final asystole. recurrent syncopes due to idiopathic vf with good neurological prognosis have been reported in patients with and without cardiac etiology (1,2). in the past measurements of cerebral hemodynamics have been repeatedly done in humans during cpr, but until today no studies of cerebral blood flow velocity (cbfv) have been reported during controlled cardiac arrest in humans not under-going cpr. it was the purpose of our study to evaluate the acute hemodynamic effects of untreated vf on cbfv. methods: after approval by the local university ethics comittee, five male patients aged 34-48 years without evidence of cerebral disease were investigated during vf while undergoing implantation of a pacer cardioverter defibrillator system (model 7219d; medtronic| a standard anaesthetic regimen was used (propofol, fentanyl). after implantation of the automated cardiac defibrillator vf was induced by electrical countershock to test effective sensing, pacing, and defibrillation. to measure cerebral blood flow velocities (cbfvmca) the doppler probe was placed above the zygomatic arch between the lateral margin of the orbit and the ear and directed towards the m1 segment of the middle cerebral artery (mca). results: a total of 12 phases of vf were investigated. duration of vf ranged from 6 to 26 seconds, with cbfvmc a (mean_+sd, cm sec -1) flow pattern changing from pulsatile to laminar flow immediately after onset of vf. conclusions: the underlying mechanism of the laminar cerebral blood flow observed during vf in our patients is uncertain, but it may provide insight into the prognosis of patients with idiopathic vf. theoretically, the laminar cerebral blood flow observed in our pulseless patients may provide a substantial amount of cerebral perfusion even during clinical cardiocirculatory arrest objective: to investigate whether the intensive care nursing staff can inflate more accurately a specific air volume with the laerdal resuscitation bag when they receive feedback after each inflation about the delivered volume compared to no feedback. method: 42 icu nurses were asked to inflate a testlung model 10 times with a specific air volume (600 ml, 800,ml or 1000 ml) under three different conditions (normal, decreased compliance and increased resistance) without and with feedback. we measured the mean absolute difference from the specific airvolume after each ten inflations. results: the largest absolute difference was found when icu nurses inflated 600 ml (250 ml). the mean inflated volume for this group was 843 ml. when the icu nurses had to inflate 800 ml the mean absolute volume difference was 181 ml with a mean inflated volume of 913 ml. inflating 1000 ml produced an absolute volume difference of 131 ml with an mean inflated volume of 1042 ml. the absolute volume difference decreased when the compliance of the testlung was decreased and even more when the resistance of the used endotracheal tube was increased. when the icu nursing staff received volume feedback after each inflation the mean absolute volume difference was reduced between the 42 ml and 66 ml for all specific air volumes. 42% of the last 5 inflations with feedback were significantly smaller than 50 ml from the specific air volume (p < 0.05). conclusion: the majority of nurses overinflated the specific air volumes. the largest over inflation occurred when 600 ml and the smallest when inflating 1000 ml. when nurses were provided with volume feedback the performed significantly better. we concluded that icu nurses are not able to inflate a specific air volume with the laerdal resuscitation bag without receiving volume feedback. feedback is desirable in order to reduce the volume trauma. objectives: a pro_found impairment in systolic and diastolic myocardial function following successful cardiopulmonary resuscitation (cpr) has been demonstrated by using langerdorff method in rats. in the present study we have investigated post resuscitation myocardial dysfunction in a porcine model of cpr. methods: ventricular fibrillation (vf) was electrically induced by alternating current applied to the ep{cardium of the right ventricle in 11 domestic pigs. following 4 rain of untreated vf, precordial compression and mechanical ventilation was initiated and maintained for 8 min. electrical defibrillation was then attempted and 6 of 11 animals were successfully resuscitated. results: following successful cardiac resuscitation, stroke volume index (svi) decreased from prearrest value of 1.13 ml/kg to 0.74 ml/kg (p<0.05), and left ventricular stroke work index (lvswi) from 1.57 to 0.77 mmhg,ml/kg (p<0.05). both svi and lvswi remained depressed for another 3 hours. these decreases were associated with increases in heart rate from 145 bpm to 185 bpm (p<0.05). no significant changes from baseline in mean arterial pressure, mean pulmonary pressure, right atrial pressure and pulmonary artery wedge pressure were observed. prehospital resuscitation efforts c. k6ppel. g. fahron, h. lufft, a. kruger, c. th(jrk, f. bertschat, f. martens dept, of nephrology add medical intensive care, virchow-klinikum, humboldt-universit~t, d-13353 bedin, germany obiective: the success rate of prehospital resuscitation in patients with cardiocirculatory arrest in an emergency medical system (ems) may reach 30 -40% depending on the time of calling the ems, the distance to cover by the emergency ambulance and the training of the emergency physician and his staff. in the berlin ems, which is associated with the berlin fire brigade, the time between alarm and arrival at the scene ranges from 2 -31 min, mean 8 min. resuscftation is based on the advanced cardiac life support (acls) according to the guidelines of the american heart association. if resuscitation efforts fail to restore circulation, they are terminated after 30 -60 min, depending on duration of cardiocirculatory arrest, pre-existing disease, age, absence of an even transient response to cpr. however, there is a lack of practical criteria for termination of cpr in individual decision making. patients: we report 5 cases of prehospital cpr with primary asystolia terminated after 45 -60 rain of frustraneous cpr efforts including highdose epinephrine and dopamine. results: after termination of cpr, the ecg monitor remained connected and showed permanent asystolia in all patients while the emergency physician completed his records. spontaneous resumption of respiration and circulation was observed in these patients after 2 -5 min and cpr efforts were immediately resumed, nevertheless, 3 of the patients died at the scene, while 2 could be hospitalized with stable circulation. one of them died 3 hours after admission to the icu, the other survived for 3 weeks in a vegetative state. spontaneous resumption of circulation and respiration is most likely due to the development of extreme hypercapnia and acidosis, which -at least in some patients -seems to be a stronger stimulant of the circulatory and respiratory brainstem centers than cpr with high-dose catecholamines, conclusion: because of the legal and ethical implications of this rare phenomenon, emergency physicians should continue ecg monitoring for at least 5 rain. after termination of cpr efforts. pulmonary artery catheterezation is used for patient's monitoring [1]. we reported our results on such monitoring in 1969 [f.coaobbeb,r.fe6enb~-kap~monorm~,1969,n7,p.28-39] .however not all of the received criteria assessments meet demands that are necessary for early diagnosis of critical states. here we report the data on po2,pco2 (mm rg),so2,ph levels in femoral [af) and pulmonary (ap) arteries blood, as well as on summary gas pressure (sgp) calculated from pe=(po2+pco2) in mm hg in ap blood. these data were derived from:i)86 subjects free of cardiovascular pathology according to catheterization data during their spontaneous air breathing (n group in ap blood appears to be a measure of adequacy ratio between pc2 and sgp in ap blood during air breathing; partly its characteristics and variations ranges are presented earlier [2j. in control group it is equal to 1,91• mm hg. tests on sgp neither exclude nor substitute conventional (pc2 and pco2) tests, but rather include them as a part choosing only additive characteristic -pressure. they appear to be a part of general system of human metabolism regulation by pressure (arterial,venous,intracardiac, tissue,liquor,onco-osmotic,etc ietraabdeminal pressure produces perturbations of cardiac, pulmonary, and renal physiology. this most often occurs fonowing eeliotomy for peritonitis or intestinal obstruction; bowel edema and distention prevent wound closure without unacceptable compromise of blood pressure or pulmonary compliance. a variety of temporizing measures have been reported for managing wounds that cannot be closed: 1) using towel clips to reapproximate skin only, 2)i sewing silastic, marlex or other prosthetic grafts to the fascia to "enlarge" the peritoneal cavity, 3) using loosely tied retention sutures for partial closure, 4) simply packing the wound without attempts at c~osure. these techniques either traumatize the abdominal wall (complicating definitive closure), expose the bowel to damage, or allow excessive loss of fluid and heat. since 1989 we have evolved a suturelees technique which permits the abdomen to be partially closed in a quick, safe, sterile, sealed, atraumatic fashion -while providin! decompression of unphysiologic intraabdominal pressure. methods: whenever possible omentum is interposed between bowel and the open incision. viscera are covered by a layer of sterile, non-reactive plastic, placed deep to the fascia and extending we~t beneath the edges. sump tubes are placed above the plastic and covered in turn by two layers of an adhesive plastic drape which sticks to the skin and seals the wound in all directions, the patients remain intubated and paralyzed. results: we have used this technique in a total of 27 patients, four of whom suffered from compartment syndrome. all of the latter were males and ranged in age from 19 to 51. all four showed immediate physiologic improvement. all four incisions were eventually closed without complication. one compartment syndrome patient died 4t days later of multiple organ failure. there were no complications related to the closure technique in any of the 27 patients. conclusions; 1. selected patients with abdominal compartment syndrome will benefit from decompression using this temporary sutureless technique. the technique a) is quick, safe, sterile, sealed, and atraumatic, b) minimizes loss of fluid and heat, c) facilitates eventual definitive abdomina| closure. although m. brunner m. mitllncr objectives: to determine incidence and predisposing factors for cardiac arrest occurring during the first 24 hours after open heart surgery. methods: the study included patients who, following open heart surgery, had adequate cardiac function and in whom cardiac arrest was not anticipated. all data were prospectively recorded and analyzed. results: from 12/1993 through 3/1995, 2140 pts underwent open heart surgery at our hospital. of th~se, 23 pts (1%) (age 65_+9 yrs) had a cardiac arrest during the first 24 hours after transfer to icu. they were operated on for coronary artery bypass grafting (cabg) (17 pts), valve replacement (vr) (3 pts), cabg and vr (2 pts) and aortic aneurysm (1 pt). the preoperative ejection fraction was 44_+12% whereas bypass and aortic cross-clamp time were 127+70 and 72+42 rain, respectively. prior to arrest, they had a cardiac index of 2.23_+ 0.5 l/min/m 2 and were receiving 1.3+1 inotropes. arrythmias leading to cardiac arrest were ventricular tachycardia/fibrilation (10pts) and bradyarrythmia (9 pts). closed-chest cpr was initially performed on all pts and was followed by open-chest cpr in 12 pts. eighteen pts (78%) survived to icu discharge. causes of arrest included perioperative myocardial infarct (t2 pts, 52%), tamponade (3 pts, 13%), rupture of the proximal vein gra& anastomosis (1 pt, 4%), graft occlusion (2 pts, 9%); no cause was found in 5 pts (21%). conclusions: postoperative cardiac arrest in stable cardiac surgery pts is relatively infrequent (-1% incidence) and is associated with a high survival rate following successful cpr. perioperative myocardial infarct is the most common predisposing factor. group ~deptof anaesthesia and intensive care, semmelweis univ. medical school, 2 buda military hospital intensive care unit, budapest background: when a cardiac arrest occurs in-hospital, the outcome can be improved by a higher quality of basic life support provided by the witnessing health care workers until the code team arrives. this basic life ~pport (bls) should include the best available method for airway management as well. since not all medical staff are ready for carrying out endatracheal intnbation, we investigated the effieacy of the use of different airway management methods during bls. methods: we have investigated the efficacy of airway management of 25 doctors and 25 nurses from different hospital wards: internal medicine, department of surgery, trauma, urology and gynaecolagy. comparing the bag-valve-mask, laryngeal mask and the endotracheal intubafion, we have measured the following parameters: time needs for correct application (sec.), number of incorrect applications (out of ten trial), efficacy of artificial ventilation provided by the device. we used a computerised als trainer manikin for the evaluation of the performance. total performance score was created after the measurement between 0-10. after the first screening we held a 2 x 2 hours training. 8 doctors and 8 nurses were trained for the endotracheal intubation (group it1, 1t2) , 8 doctors and 9 nurses were trained to use the laryngeal mask (group lm1, lm2) . all respondent were trained to use the bag-valve-mask device. 1 day, 1 month and 3 month after the training we have carried out retention study using the same method. results: we have found that the efficacy of the artificial ventilation using the above mentioned devices were poor before the training. the average after-training performance scores of the groups are presented in the table below. (bls) should be initiated by the witnessing health care professional. the cpr study introduced a multi level code system, which means bls included sophisticated airway management, early defibrillation and early epinephrine administration provided before the code team arrives. our previous studies confirmed a poor level of cpr performance and a high demand for cpr training among health care professionals. method: we established a cpr training course centre, where doctors and nurses are being trained for in-huspital basic and advanced life support. 3 x 6 hours of training were held. after the theoretical introduction a step-by-step training method ws used for trainees to be familiar with all sequences of basic and advanced life support. then we synthetised all separated sequences. afterwards, a r01e play of rescue groups was taken in simulated situations. we also trained the multi level alarm system fur the in-hospital resuscitations. after the training all respondents had to sit for examination. the quality of performance was scored and compared to our previous results. semi-structured interviews were carried out before and aider the training among all respondents to collect information about the course. results: we have found a remarkably high interest among doctors and nurses in our cpr training courses. it was very important to use proper equipment for the training: audio-visual training facilities, computerised als trainer manikin, manual and automatic defibrillator units. the evaluation of the examination held immediately a~er the training course showed a significant higher quality of performance than before the training. the self.-eonfidence of the trainees for initiating and carrying out resuscitation had increased. their overall feeling about the course was positive and 100% responded the course "very useful". 73.6% of doctors and 79.4% of nurses claimed fur regular training facilities with als trainers, conclusion: the cpr training for health care werkers is mandatory including the training of sophisticated airway management and use of elad~l~ills~tt~r wlaa ~en ~r a~ti~atir ~nel r rm~a'*h*nr m~thnd for training will improve the efficacy, the satisfaction of trainees, therefore their compliance for further co-operation will also increase. s 144 objectives: the effect of reinfusion in emergency surgery and gynecology. methods: we had an experience of autologous blood transfusion in 22 patients whom was produce t an emergency surgical or gynecological interventions in occasion with break tubal pregnancies (45.5 %), penetrating abdominal wounds with injuries of mesenterial vessels (22.8 %), injuries of the liver (9.1%), blunt abdominal trauma with lien ruption (22.8 %). in 27.3 % patients had the previous somatic pathology. blood loss volume was 1500-4500 ml, & the reihfuside blood volume was 500-2000 ml, consisting 30-70 % of blood loss. it was needn't to fransuse donor blood in 18.2 % in further but 300-2500 ml of contanined erythrocytes were frasfused for supporting of hb concentration on the 80 g/l (8 g/dl) rate at the other patients with isovolemie hemodiluttion. results: the arterial blood pressure fast stabilisation on the perfusion level had noted after reinfusion, excluding the case, when the volume of reinfused blood had conisted just 40 % of blood loss at the patient with massive blood loss. complications have noted in two cases. one patient with slash wound, injury of arteria gastrica dextra and total blood loss of 4500 ml, has an episode of asystoly, dic (disseminated intravascular coagulation) syndrome, acute renal failure, and acute pancreatitis that we haven't connected to reinfusion. all the complications were successfully corrected and at thirty first day patient with subcapsular wound of the lien that has happened 14 days before complicated with external rupture of the capsull & massive intraabdominal bleeding, has the hemolytical shock, dic syndrome, acute renal failure developed after reinfusion. he was died. all another have no complications. posthemorrhagic anemia had corrected rapidly than in case when hemorrange corrected exclusively by donor blood. conclusions: we consider that simplicity, accessibility, high effectiveness, quite well further results of blood reinfusion, except the case of blood reinfusing that was for time-expired out of blood vessels (more than 10 days in our case) will promote to the wide spreading of this method, especially in emergency surgery, in massive injuries, & in disarters, all the cases of insufficiently of time for selection of lot of donor blood. objectives: study of a reaction of the oardioreepiratory system of pregnant women to i/v microperfusion of clophelinum which is known to eliminate hemodynsmic and endocrine nociceptive reactions and can be used for treating hypertensive syndrome in pregnancy and labor. methods: the following non-invasive methods were used: capnography, spirometry, oxygenography, indirect fick principle based on the circle breathing, plethysmography and integral rheography~ 52 functional indices of cardiorespiratory function were evaluated. results: 74 pregnant women with ~h-gestosis were examined before and after i/v infusion of i00 ml of 0.0001% clophelin solution, 0.005 mg/kg/hour. before the treatment intensification of carbohydrate metabolism, hyperventilation with moderate hypooapnia and complete respiratory compensation of metabolic acidosis~ increased alveolar ventilation, decreased alveolar volume, predomination of perfusion over ventilation, hypokinetio type of circulation with dominated load by peripheral vascular resistance to the blood flow was observed in this group of patients. microperfusion of clophelin imp~-oved the ventilation/perfusion ratio, ventilatory and gaseous exchange efficiency, resulted in a decrease of congestion in the pulmonary circulation, possibly owing to a decrease of peripheral vascular resistance by 17%, of the heart rate by io.5%, of the oardial output index by 9.5%. conclusionm: the resulted type of circulation with a decreased load on the heart both by resistance and volume allowed to improve the cardioreepiratory system function in pregnant patients. objectives: the injury severity score is a measure of severity of anatomic injuries. iss is a sum of squares of the highest degrees of the abbreviated injury scale (ais) for each of three most severity injured regions. the purpose of the study is to establish correlation between the iss values and mortality rate in older, polytraumatized patients. methods and results: iss was determined for 214 patients. the mean iss value was 27.65 + 17.36 while the median value was 21. minor injuries were present in 90 (42 %) patients with iss less than 21, while 124 (58 %) patients with iss more than 22 had severe injuries. increased mortality of the older patients was noted in the range 21-30. all patients older than 50 died while 20 % of patients below 50 yrs of age survived, indicationg correlation between iss and mortality rate in polytraumatized patients above 50 yrs of age. conclusions: this mode of evaluating severity of injuries may help in triage, determining appropriate level of care and as an indicator of future outcome of polytraumatized patients. objectives : tissue hypoxia is a non exclusive cause of hyperlactatemia. other serious medical situations induce hyperlactatemia. therefore, lactatemia could be a non specific indicator of severity in patients admitted in emergency unit. the aims of this study were to examine the correlations between lactatemia with the short term survival course prognosis and the unit of hospitalisation; intensive care unit (icu) or medicine unit, in patients admitted in our emergency department. methods -lactatemia was measured as soon as the admittance, in arterial blood sample of patients which needed arterial blond gas. sixty-one patients were included during 4 months. to assess the statistical performances of lactatemia, sensitivity (se), specificity (sp) and accuracy (ac) were calculated for the threshold determined by the youden's test (se+sp-1). results : fifteen patients were admitted in icu and 46 in a medical unit. fifteen patients died. a group of 35 patients had a lactatemia up to 2 mmol.l" 1. in this group of patients, 3 had acidocetosis, 3 had asthma, 3 had cerebral vascular ischemia, 3 had neoplasia, 2 had cardiogenic shock, 1 was epileptic, 8 had congestive heart failure, 6 had acute respiratory failure, 2 had septicaemia, 2 had hyperosmolar status finally 3 had medicinal intoxication. lactatemia was significantly higher in non survivor than survivor ( 5.5• vs. 2.3+1.0, p<o.oool, respectively), the cut point of lactatemia was 3.7 mmoll" 1 se was 60%, sp was 91% and the accuracy was 83%. on the other hand lactatemia was higher in patients admitted in icu than in medical unit (4.5+3.6 vs. 2.0• p<o.01, respectively). conclusions : this preliminary results suggest that lactatemia is a good indicator of the short term survival course in patients admitted in an emergency unit. furthermore, hyperlaotemia is present in several pathology seen in emergency. objective: to determine the pattern of injury, total peripheral white cell, neutrophil, and lymphocyte responses, and the outcome of trauma patients who are leucopaenic on admission to the icu. design: prospective, descriptive study of consecutive admissions over4 months. setting: a 16-bed surgical intensive care in a teaching hospital. patients: thirty consecutive adults admitted to the icu following trauma. leucopaenia was defined as a total peripheral white cell count of < 4 x 109, neulropaenia < 2 x 109, and lymphopaenia < 1.5 x 10 ~ cells per litre. measurement and results: the total peripheral white cell count was documented daily and the neurtophil and lymphocyte counts and differential percentages on days 0, 5, and 10. the incidence of leucopaenia was significanfiy higher in patients with gunshot wounds (p < 0.05) and hollow visceral intra-abdominal injury (p < 0.001). eight (27%) patients died. no significant differences were found in the initial mean total white cell, neutrophil, or lymphocyte counts nor in the differential percentages between survivors and non-survivors. the total peripheral white cell count increased significantly in survivors compared to those who died (p < 0.001) and significant differences were found in absolute neutrophil counts (p < 0.02) and differential percentages (p < 0.01) on days 5 and 10. no significant differences were found in lymphocyte counts or differential percentages. conclusions: there is a signficant association between leucopaenia, neutrepaenia, and intra-abdominal hollow visceral injury and peritoneal contamination. survival is related significantly to resolution of these white cell deficiencies. these findings suggest a possible role of granulocyte colony stimulating factor in the trauma patient with intra-abdominal infection and persistent neutropaenia. amelioration of organization emergency care team in order to improve caring for urgent cases. objectives: emergency care team (ect), as integral part of health, respectively as activity of primary health protection care all acute difficult conditions which vitaly danger patients and distressed. the aim of our investigations was to indicate on the possibilities of amelioration of organization ect in order to improve caring for urgent cases. methods: in our investigations we used epidemiologicai and statistical metods all informations and matherials from terrain ects. results: the number of traumatized in traffic accidents etc. have a epidemic character. it is well known that 58.1% traumatized died in first two hours. ect isn't enough qualified to care a traumatized on the place of accident and during transport. because that medical personnel must have an education with solid qualities. conclusions: 1. besides a physician specialized (vii --+ vii2 -~ viii gradus) in urgent medicine, it's necessary to introduce two (2) medical technicians instead of one as is existing now (1 ~ 2). 2.the medical technicians can drive cars and one of them always, drives the ambulance car ("b" category). 3.the technicians have been specially educated for urgent cases (iv ~ v ~ vi). 4. such a new team structure should be more capable in professional sense. 5.we can see an importance of team-work in ects. 6. such teams should also be more economic in comparison with existing one. 7. we think, that on the general medical studies need to include a new discipline-urgent medicine! objectives: to detoxicate an organism since 1983 we have used biological sorbents: isolated from liver and spleen of human or animal cells or tissue fragments. methods: cellular dialysis (164 dialyses) in patients with an acute hepatic insufficiency poisoning by hepatotropic poisons: cc14, dichloethane, mushrooms -were conducted using "artificial kidney" apparatus, by means of arteriovenous shunt, formed at forearm with disposable dialyzers. hepatocytes suspension was poured into dializing circuit of dialyzer at 5-10 mg of cells per i kg of patients weight. dialysis was performed during 1-2 hrs. 168 patients with purulent-septic states were treated as for hemoperfusion through prepared sorptional column: 40.0 ml of hemosorbent and 30.0 ml of fragmented spleen or hepatocytes suspension with the help of roller pump with the rate of 80-120 ml/min, during 90-120 min. average volume of hemoperfusion made 3.51. conclusions: in all cases we used both native and cryopreserved biomaterial. analysis of the dynamics of clinical, biochemical, scintigraphic and us-investigastions allows to conclude that application of cellular-sorptional method of detoxication enable to prolong life and reduce lethal outcome in some severe category of patients with endotoxicosis. after infusion of diazepam (0.2mg/kg),thiopentone (4mg/ kg) and vecuronium (lmg/kg),patient was intubated and mechanical ventilation was adjusted to mantain normoca= pnia. ct scan showed left parieto-occipital hypodensity with brain swelling. after admission in neurological icu,dexamethasone (24mg/ kg/die),mannitol (0.smg/kg) and diphenylydantoin (20mg/ kg/die) were administred. two hours later neurological status of the patien~ was normal and she was extubated. the risks of stereotactie radiosnkgety are related to: hemorrhgge during the latency interval;transient radia= tion effects;permanent radiatio~einduced complications; radiation induced neoplasia. no epilepsy is reported in pts with avm and no seizure before radiosurgery. in c0nclusion, epilepsy can be a posmib~e delayed eompli c~t~n, ~fe~r rgdi0surgery for cerebral avm. serum potassium deficiency is a frequently reported finding in the time course of acute myocardial infarction (ami). if this is also correct for patients with ami undergoing primary percutaneous transluminal coronary angioplasty (ptca) is unknown.for that reason we analysed in 20 patients (14 m.,6 f.,61 + 10 y.) serum potassium concentrations on ad ,mission and directly after ptca.the reference intervall for potassium in our clinical laboratory is 3,5 -5,0 mmol/l. although more than 90 % of the patients with ami undergoing ptca showed normal potassium serum concentrations on admission a measurable decrease of potassium could be found in 65 % of patients (13120) after ptca.therefore for the majority of patients with ami potassium supplementation prior to ptca can be justified to avoid electrolyte disturbances as a possible trigger factor for cardiac arrhythmias in the setting of ami and acute reperfusion due to primary ptca. objectives: diagnostic procedures are very important in the management of abdominal injures in trauma. the aim of this paper is to present our experience with peritoneal lavage as diagnostic procedure in abdominal injury. methods: we analyzed patients to whom peritoneal lavage was applied in the surgical emergency from 1st of january 1991 till 31st of december 1994. results; in this four years period there were 250 abdominal injures. there were 110 blunt injures. among them in 43 patients peritoneal lavage was applied. results were positive in 28 patients and negative in 11 patients. false positive finding were in 2 patients and false negative in two patients. in patients with positive lavage there were more than 90,000/mm3 red blood cells, more than 1,000/mm3 white blood cells and the level of amylase was up to 8 u/i. the gall, urine and stool were positive as well. in patients with negative lavage there were less than 40,000/mm3 red blood cells, less than 500/mm3 white blood ceils and amylase were negative. conclusion: peritoneal lavage is useful and important diagnostic procedure in the management of abdominal injures in trauma. our experience with urgent surgical management of the acute gastric and duodenal bleeding s.se6en md, z.milo~evi6 md, *s.srdi6 md, k.sar6ev md. clinic for abdominal and endocrine surgery, institute for surgery; *clinic for cardiology, institute for cardiovascular diseases; school for medicine novi sad, university of novi sad, yugoslavia objectives: acute gastric and duodenal bleeding are very common complication of gastric and duodenal ulcer. the aim of this paper is to present efficiency of our surgical management of these acute conditions. methods: in this paper results of surgical management of patients with acute gastric and duodenal bleeding, treated in our clinic from the 1st of january 1991 till 31st of december 1994, are presented. results: among 92 operated patients there were 31 females (33.70%) and 61 males (66.30%). there were 52 patients (56.53%) with gastric bleeding and 40 patients (43.47%) with duodenal bleeding. bleeding from gastric ulcer were treated with the following methods: excision of ulcer with suture in 23 patients (44.24%), ulcer suture in 12 patients (23.07%), billroth i in 9 patients (17.31%) and billroth ii in 8 patients (15.38%). heinecke-mikulicz-weinberger pyloroplastica was the most frequently used in the treatment of the duodenal ulcer bleeding. bilateral truncal vagotomy was done in 34 patients (85%), duodenotomy with ulcer suture was done in three patients (7.5%) and three patients underwent billroth ii operation. we had three complicationsduodenal dehiscence in pyloroplasty. there were no lethal complications. conclusions: based on our own experience we conclude that acute bleeding from gastric and duodenal ulcer can be safely managed with urgent and modern operative techniques. dept. of anaesthesiology. emergency center of serbia, belgrade, srj 0b0ectives and methods: cardiac contusion was evaluated by serial determination of cpk-mb fraction and co using non-invasive doppler technique in 60 patients with blunt trauma of the chest. results: miooardial contusion was diagnosed in 34(57%) patients, while in 26 (43%) it appeared unaffected. cpk-mb of 3% and more in comparison to total cpkwas 6.4+4,2% in 34 patients, and 1,9+0,6% in 26 pts (p<0.01).c0 of 5.8+1.17 l/min in patients with cardiac contusion, measured 16 hrs after admission at the latest, was significantly lower than in group of 26 patients with co of 4.8+0.86 l/min (p<0.01). conclusions: co values were significantly lower in patients with cardiac contusion and correlated with pathological values of cpk-mb fraction. objectives: extracorporal plasmophoresis (pph) was implemented by non-stop method in 28 patients with obstructive jaundice. methods: changes of serotonin (s) and histamine (n) in arterial (ab) and venous (vb) blood were investigated before and after one-time session of pph. s and h in blood serum were determined with fluoremetric method. results: before pph the average s contents in ab and vb did not differ. after one-time pph session s in the blood outflowing lungs was 23 % lower than in the blood inflowing. 24 hours after extracorporal detoxication implemented the indices kept the same level. similar dynamics after pph was observed in h. before the session h contens was equal both in the blood inflowing and in the blood outflowing lunge. after pph h contents in ab was 18 % lower compared to the vb and did not change for 24 hours. conclusions: dynamics of changes of biogene amines shows that under the influence of pph there takes place not only mechanical removal of their surpluses together with plasm, but there sharply grows inactivating role of lungs towards ba substances, which is confirmed by reduction of their concentration in the blood outflowing lungs. this effect is probably connected with the "deplasmation" of the lung cellular elements. simultaneously lungs' cells are freed not only from plasm but from toxic adsorbents on their surface. as a result the lung cells activate absorption of the surplus number of amines from the vessel channel. it is not excluded that s inactivation is due to growing activity of monoaminooxidase resulting in deminishing of intoxication. acute pulmonary embolism: thrombolytic therapy or pulmonary embolectomy? i.lodiena md, phd, s.shevchenko md, pphd., medical university, kharkov, ukraine objectives: pulmonary embolism (pe) remains a challenging problem for diagnosis and management for the emergency physician. the aim of this work is to identify the best treatment for massive pulmonary thromboembolism. methods: during recent 5 years 150 patients with symptomatic acute serious pe were reffered to our observations. selective pulmonary arteriography confirmed this diagnosis in all cases. 4 (2.7 %) underwent urgent surgery, of them 2 (1.33 %) with cardiogenic shock and 2 (1.33 %) with massive pulmonary embolism. 146 patients (97.3 %) underwent thrombolytic therapy. results: in the patients who received thrombolytic therapy successful results were achieved in 133 cases (91.1%), in 13 (8.9 %) patients subsequent pulmonary embolectomy was performed. mortality rate was 2 (1.36 %) in the patients who underwent pulmonary embolectomy after unsuccessful thrombolytic therapy and 4 (100 %) in the patients with cardiogenic shock and massive pe. lower extremity deep venous thrombosis caused pe in 89 % patients. pe mortality rate is repoted to be 4 %, but all patients with cardiogenic shock and massive pe died. this very high mortality rate related to the clinical status of the patients whose condition became worse despite intensive treatment. conclusions: the results of this study show that in most cases thrombolytic therapy is an effective rapid method of treating pe. however, surgical treatment is preferable when the patients reveal adverse effects to drug therapy, when medical therapy is unsuccessful or when the patients are seriously ill with reccurent cardiac arrest. high-quality pulmonary angiography remains the most accurate and reliable means of diagnosing pe. this prospective study was conducted to investigate the serum thyroid hormone levels in traumatized critical ill patients. serum thyroid stimulating hormene(tsh),total thyroxine(tr4),free thyroxine(ft4),total tri-iodothyronine(tr3),free tri-iodothyronine (ff3) levels were measured within 24 hr.of intensive care unit admission(first day) in 17 multiple tratmmtized male patients(injury severity score higher than 17). in fifth day the same hormone levels were repeated. tsh,et4,ft4,tr3 and ft3 levels were determined by radioimmueoassay(ria). the levels of 'i~sh,xt4,fr4,et3 and ft3 in survivors and nonsurvivors were compared with first and fifth days (table) . in conclusion, we are convinced that there was a high correlation between low ser~n thyroid hormone levels and mortality, serum thyroid hormone levels are prognostic value in traumatized critical ill patients. objectives: glucagon improves myokardium contractility, intensifies kidney blood flow, stops bronchospasm (1), these were the reasons of investigating its effect on hemodynamics and metabolism in hypovolemic shock expermentally and in clinic. methods : in experiments on white rats with traumatic shock glucagon (novo nordisk) was injected intraperitoneally in dose 20 rocg/kg. patients with hypovolemic shock (trauma, hemorrage) were injected glucagon intravenously in dose 1 mg on the background of infusion therapy. results : glucagon hightened recovery indexes of glucose in rats and patients with shock, whereas in groups without glucagon hyperglycemia developed. rats with glucagon injection had double times urea content than the norm ( 10,5+1,5 mmol& norm -4,9+0,3 mmol/i ), whereas rats without glucagon had urea content 30,3 +1,8 mmol/1 ( p < 0,001 ). osmolarity of blood plasma in rats having glucagon injections was 301,5 + 2,9 mosm/kg h20, without the drug -317,0 + 3,5 mosm/kg h20 ( p < 0,01), ldh activity -27,7 + 2.9 and 51,0 + 9,6 ( p < 0,01) mcmol cec-1 accordingly. lethality in the group of rats without glucagon was 70% /n=50/, with glucagon -0 (n = 70 ). in patients with hypovolemic shock in 15 minutes after glucagon administration stroke volume increased to 71% (sv), miocardium contractility -to 70% (mc), peripheral resistance of vessels ( vr ) decreased to 10%. in 1-1,5 hrs sv increased to 100%, mc-to 126%, pvr -decreased to 116%. conclusion : the study provides the evidence that glucagon has an anti-shock effect, improves hemodynamics and metabolism indexes in cases of hypovolemic shock. reference : picazo j. glucagon in acute medicine : pharmacological, clinical and therapeutic implications (norwell, mass:kluwer publishing,1993 ), 172 p. n, vasina (t) ,n.sebbota hph (2). dept; of acute endotoxicosis, n.v. s~lifosovslay scientific research institute of ermergency ltealth care, boscow, lk'ussia (i}. institute for problems of cryobiology and cryouledicine of the national academy of sciences of the f/maine, kl'k3r-key, [7maine (2), objectives: the investigation of isolated hepatocytes using's efficacy in patients with acute hepatic fai lln-e. methods: native and crioconsrved allo-and xeno-!mmatocztes fixed on semi;ermeable ~mbr~s were used. results: the artificial supporting system with isolated hepatocttes as metabolic ~its was. ap-plied in 85 patients. during tl~ treatmen~ with this cytotherapz an improvement of clinic state and biochemical rates were observed even inthe first 8 we~s. for instance, the kncreasing of glutamin acid's level in blood up to normal value and decreasing of ammontes -concentration from i9~26 mmol/l down to 89#15 mol/l were discovered. ~ae diminution of ence,%halo;athy was noted too. general lethality was' equal 37 z, there of 2i z concerned acute hepatic failure (/k~). conclusions: the using of sum~rting hepatic system is able to cope the ~nenomenon of ~i~. objectives: fat embolism (ee) is an important and insufficiently studied problem of the intensive care medicine. fe is clinically diagnosed in 25% and morphologically in 80-90% of the victims with severe multiple skeletal injury that is accompanied by the shock. methods: during the last 5 years 27 patients with the cerebropulmonary form of fe were treated in the icu. the prognosis of the probability of fe development was done using the computer system "cite-prognosis" in which the triss-method, dynamic and statistical prognosis of the injury outcome~ and the values of the most severe injury in points were realized. the treatment consisted of the surgical stabilization of the fractures with the authors' design of the rod apparatus in the acute period, long-term mechanical ventilation with peep via tracheostomy, complex fluid therapy using perfluorochemical emulsions (pe), and electrochemically active solution of na hypochlorite (snh) via the central vein. results: the treatment of 27 patients with fe gave a good result that was manifested by the complete regression of psychoneurologic and respiratory disorders. conclusions~taking into account the prognostic data~ it is necessary to provide early complex of intensive care to the polytraumatized patients. it includes the surgical stabilization of fractures, fluid therapy using pe and snh~ early long-term mechanical ventilation. dr. alexandr e. poladko, station of medical aid. kiev ukraine. objectives: the reason of investigation is to prove the necessity of beginning of the intravenous infusion of nitroglycerin before the hospitalisation. methods: from january i to june 30 1994 protocols of treatment myocardial infarction patients in kiev medical aid station. results: the results of treatment are better if infusion began early or prolonged during hospitalisation. conclusions: there were 30 patients with acute myocardial infarction treated before hospital by the cardiological groups. 100 patients were treated with the early infusion of nitroglycerin and 200 without. the results of treatment were: in the group with infusion: mortality in the first hours 5 % the full disappear of pain 90 % in the group without infusions mortality in the first hours 8 % the full disappear of pain 82 % that's why our opinion is that infusion of n. is necessary in the treatment of the patients with myocardial infarction from the first minutes of illness. dr. gennady d. kyrzhner, station of medical aid, kiev, ukraine. object: looking for safe medication which is good for the treatment of arterial hypertension and is simple for use. we inspected the effect of prazosin in the conditions of the cail during the year. method: we used 1 mg prazosin tablets sub lingua and measured blood pressure 4 ones during 2 hours. the parameters were registrated in protocol. result." one hundred patients took part in investigation. the middle age of patients was 69. the reason of hypertension was not taken into consideration. in two hours after beginning of the treatment we caught the reliable lowering of blood pressure in 90 % of patients. the general condition of patients became better in 10-15 min. it was only one accident of complication during the treatment -the orthostatic hypoter~sion, conclusion: prazosin hydrochloride in the dose 1 mg sub lingua is safe and simple for use in the urgent situations. objectives: hypomagnesemia is frequently observed in severely burned patients during the early period after injury. increased urinary excretion is insufficient to explain the magnitude of the magnesium (mg) depletion. the possibility of exudative cutaneous mg losses has been proposed, but not yet demonstrated. the study aimed at measuring the mg content of the cutaneous exudates and urine during the first week after major burns. methods: 16 patients, aged 34_+9 years (mean _+ sd), and burnt 37_+11% of body surface, were studied from day 1 (d1) to d20. serum samples were collected at do, serum and urine from d1 to d7, and on dio, d15, d20. cutaneous exudates were extracted from the textiles surrounding the patients, collected over 24h periods from d1 to d7, using bidistilled acidified water. mg supplements (8.2 to 123 mmol/24h) were prescribed from d1 to dio according to the severity of hypomg (serum mg < 0.7 mmol/i). results: mg serum levels decreased below reference ranges in 12 patients between d1 and d4, and normalised thereafter during supplementation. urinary mg excretion was increased in 11 patients, mean excretion being 4.8_+1.3 mmol/24h until d20. mean daily mg cutaneous losses, were 16 mmol/24h, i.e. 112 mmo; in 7 days. large inter-& intra-patient variability: the daily exudative losses ranged from 0 to 83 mmol/24h. conclusions: the mean daily mg cutaneous losses after burns were larger than the urinary losses, and equivalent to the recommended intakes for mg; the addition of the exudative and urinary losses largely explained the increased mg requirements after burns. complex immunologic alterations occur following thermal injury. the activation and consumption of the complement and dotting systems are suggested to play an important role in the development of the capillary leak syndrome, tn burned patients, a generalized inflammatory reaction as well as the impairment of the host defense are considered to be the major reasons for complications, such as sepsis and multiple organ failure. in a pig model we investigated a possible protective effect of cl-inhibitor (berinert, behring). before conducting the animal experiments the human cl-inhibitor concentrate was analyzed for its inhibitory capacity on purified pig cls and its pharmacoldnetic behaviour in pigs (t1/2,id50). pigs received anesthesia and analgesia and arterial, venous and pulmonary (swan ganz) katheters were placed into the carotis and jugular veins. the pigs were scalded for 25 seconds with 75~ hot water to achieve a 30% tbs partial-thickness burn. blood samples were taken prior and after surgery as well as 15, 45 minutes, 4, 8, 12 and every further 12 hours after thermal trauma. two control groups (each n=6) included animals which were either scalded or not scalded and treated only by resuscitation of ringers lactat solution. besides various parameters blood samples were analyzed for complement. the pigs (n=4) received c 1-inhibitor concentrate at an initial dose of 100 u/kg body weight either immediately or 12 hours after thermal trauma (n-2), followed by three further applications (of reduced amounts) every 12 hours. the clinical outcome as indicated by vital parameters and as well as demonstrated by reduced edema and inflammatory tissue destruction suggested a protective effect of cl-inhibitor. complement analysis revealed a faster recovery of the alternative and classical pathway activities in cl-inhibitor treated pigs as compared to the control group but only if the regulator was given immediately after thermal trauma. from these results a protective function of c 1 -inhibitor on thermal trauma can be assumed. objectives: the aim of study was to characterise the pattern of secretion of interleukin-6 (il-6) and tumor necrosis factor alpha (tnf alpha) in multiply injured or not injured critically ill patients and to relate these results to their outcome. methods: blood samples of 12 multiply injured ( 238 (73) 148 (46) 154 (57) il-6 (nis) 156 (81) 88 (54) 65 (39) il-6 (nin) 117 (68) 150 (76) 157 (04) conclusions: this data suggest tnf alpha is a better prognostic marker in patients with multiple injury, than in critically ill patients without injury. il-6 is not significantly higher in nonsurvivors of both groups. sepsis is associated with an increased production of nitric oxide (no), as reflected by a rise in plasma levels of nitrites (no2) and nitrates (no3). severe bum injury is associated with similar symptoms of inflammation like hypotension, high cardiac output low vascular resistance and increased vascular permeability so that an increased no production may be involved. the present study included 16 patients admitted to the intensive care unit of the burn center of brussels (age 35:1-18 years; burned surfaee area 374-19 %), and 6 control (non-septic) patients hospitalized in the neurological rehabilitation unit of the erasme hospital (age 64 4-18 years). the patients in both groups were fed enterally with 30 to 40 kcal/kg/day of a standard solution. in the burn group, daring the study period, 6 patients were mechanically ventilated, 4 required vasopressor (dopamine) therapy, and 5 received antibiotics; 13 patients were discharged alive from the intensive care unit, and no patient died during the study period. plasma was sampled for no2/no3 determinations (griess reaction) daily from day 1 to day 5 (n=ll) or to discharge (day 3, n=2; day 4, n=3) in the burn group, and once the control group. in the burn group, no2/no3 levels were elevated at day 1 (70_+ 12 #moles/i), reached a maximal value on day 2 (874-26 p.moles/l) and progressively decreased to day 5 (fig). these values were higher than in the control group (5.6:t:7.6 #moles/l, all differences p<0.01). however, no correlation was found between the plasma no2/no3 levels and the age of the patient or the total burned surface area; burned patients who became infected tended to have higher plasma no2/no3 levels than those who did not (177 + 131 vs 83 4-48 #moles/i, ns). the present study indicates that burn 120 noa/noa (llmol**/l) injury is associated with increasedloo] [__ ] ~ no2/no3 plasma levels, which are ao so consistent with an enhanced no pro-4o duction, ao obiectives:urapidil has been recommanded for therapy of hypotension in neurosurgical patients, because it was found not to increase intracranial pressure in patients with compromised intracranial dynamics (1). in contrast several authors reported an increase of icp after urapidil administration in patients with head injury (2). our study was designed to determin the influence of urapidil on mean lumbar cerebrospinal fluid pressure (csfp) in awake humans with uncompromised intracranial compliance. methods: after approval by the local university ethics comittee, six volunteers (asa 1, 4 male, 2 female) were studied in the lateral decubitusposition with the vertebral column positioned horizontally. a 25 gauge needle was inserted into the lumbar subarachnoid space at level l3/l4 and connected to a trancducer (mx860| medex inc.) for csfp measurements. the volunteers were advised to keep respiratory rate and etco2 constant. after a resting period of 10 minutes basline measurements were performed including csfp, cvp, map, hr, etco2 (cardiocap 2| datex). then the volunteers were given urapidil 0.2mg/kg over a period of 1 minute. 5 minutes later measurements were repeated. results: statistics: wilcoxon signed rank test, p<0.05 significant; values: mean_+sd, si = mmhg. 10-2_1" 74_+5* 64_+5* -3_+1" 35_+2 csfp: cerebrospinal fluid pressure, cpp: mean cerebral perfusion pressure. conclusions: during normoventilation urapidil leads to a decrease in map parallel by an increase in csfp and therefore in icp, most likley mediated by an autoregulatory dilatation of cerebral vessels. if this cerebrovascular response is suppressed by hyperventilation, no increase in icp is found despite a uredipil induced drop in map (2). after longterm hyperventilation cerebralbloodflow is normalized. this might be the reason why the drop in bloodpressure following urapidil administration again led to an increase in icp ( background and objectives: the intestine is one of the most sensitive tissues to ischemia-reperfusion injury. reperfusion of the intestine is often associated with increase in mucosal permeability and ulceration. d(-)-iactate is produced by indigenous bacteria found in the gastrointestinal tract and mammals do not possess the enzyme systems to rapidly metabolize it. the present study was designed to determine the kinetics of plasma d(-)-iactate alteration in rats paused by acute intestinal ischemia-reperfusion injury. methods: following the anesthesia, the superior mesentcric artery (sma) was exposed and it was occluded by a micro-bulldog clamp applied at its origin from the aorta. after 75 min of occlusion, the arterial clamp was removed and the supply area of the sma was allowed to be reperfused ( 6 h). plasma d(-)lactate levels were measured by an enzymatic spectrophotometric assay. the endotoxin content of plasma sample was assayed by the limulus amebocyte lysate test with a kinetic modification of the test kit procedure. results: intestinal ischemia for 75 min resulted in a significant increase in d(-)-lactate levels within the portal vein as compared to sham-operated animals. plasma d(-)-lactate levels had a tendency to further increase after reperfusion up to 6 h. it was showed that significant elevation of endotoxin concentrations in portal vein was alread~r detected at the end of 75-min ischemia, reaching peak at 0.5 h post-reperfusion and decreasing gradually throughout the study period. at the end of ischemia, marked mncosai damaged such as edema, hemorrhage and necrosis was observed. there was evidence of injury to ti, e muscuiaris propria together with diffuse mucosal damage and destruction following reperfusion, particular at 6 h postreperfusion. in addition, penetration of bacteria was commonly found in the intestinal wall at various time points following ischemia/reperfusion injury. conclusions: these data suggest that acute intestinal ischemia is associated with permeability change of mucosal barrier resulting in increased plasma d(-)qactate, which is also remarkably influenced by subsequent reperfusion. plasma d(-)-lactate may be a useful marker of intestinal injury following both ischemia and reperfusion insult. objective: to assess the clinical usefulness of two methods of intracranial pressatre (icp) monitoring: intraparonquimatous recording (icpc) and epidural recording (icpe), versus intraventricular icp (icpv) as a gold standard. we prospectively studied 13 patients with severe head injury (glasgow coma score < 8) admitted to our icu between fobmaly 1990 and december 1993. icpv was monitored in all pativrats by means of a multipzffolated catheter connected to a water coinnm. six of 13 patients were additionally icpc monitored by a fiber optic transducer (comma 420r~), and seven of 13 patients were icpe monitored by a couplvd fluid system (plastimedr). icpc was placed homolatetal to the focal, lesion m two cases and contralateral in three; icpe was placed homolateral to the focal lesiou in two cases and contralateral in three. all three systems were calibrated at the ear level. stali~cs: correlation coefficient and lineal regression were done between icpe and icpv, and between icpe and icpv with the hourly p~rs of values obtained dttting assistontial period. results: of the six icpc -icpv studied pa~onts, we observed a correlation coef~cieut r = 0.84 (p < 0.001) with a regres~on line y = 0.75 + 0.93x. four of ~hx lcpc -icpv patients had r > 0.85 when correlaliou eoet~dent was obtained indixddually. of the seven icpe -]cpv studied patients, we observed a cortelafiau ooeffioiont r = 0.47 (p < 0.001) with a regression line y = 7.2 + 0.43x. corralalmu eoetfieiont was inwer than 0.5 in all seven patients. corrdation eoelfieients for levals of icpv > 20 man hg, > 25 mm hg and > 30 tuna hg with icpe showed r = 0.89, r = 0.91 and r = 0.97 respectively; and with icpe r = 0.25, r = 0.13 and r = 0.09. the obtained values did not change during the study. conclusdns: in our study icpe was considered a good type of icp monitoring. /cpe signiticantly infravalorates icp values. we observed a good correlatinn between icpc and icpv values in patients with high inttacramal presanre. objective: midazolam is a benzodiazepine agonist widely used for sedation in emergency medicine. few studies in animals and humans point to a direct analgesic effect of midazolam probably mediated by spinal antinociceptive receptors and/or peripheral benzodiazepine receptors (1,2). in our experience in the berlin emergency medical system (unpublished results) with anecdotal cases of extreme chest pain due to binge drinking but no evidence of acute myocardial infarction or extreme abdominal pain due to peritonitis, acute intermittent porphyria, peutz-jeghers syndrome or testicular torsion, we found that small doses of midazolam (2 -5 mg i.v.) were much more effective in relieving pain than repeated administration of high doses of buprenorphine or morphine, which may be associated with a considerable respiratory depressant effect. the dose of midazolam required for pain relief in these patients is non-narcotic and allowed further communication on the character and localization of' the residual pain, which might be very important for the further diagnostic procedure. patients: ten patients with abdominal pain due to acute gastrointestinal bleeding, suspected pancreatitis, suspected acute porphyria, and chest pain with no evidence of acute myocardial infarction received first-line midazolam i.v. at an initial dose of 1 mg and were asked how it affected the intensity and character of pain. results: at the chosen dose of midazolam (2-8 mg), all patients were responsive to detailed questioning on basic orientation, the character, intensity and localization of the pain, and medical history. none of the patients required an additional opiate. all patients stated that the pain was tolerable after midazolam alone. conclusion: our preliminary clinical observations suggest that low-dose midazolam might be an alternative to opiates in extreme pain of presumably visceral odgin. objectives: it is known that severe head injury in elderly patients is associated with higher mortality than in younger patients. it remains however to be clarified whether the preinjury pathology which is frequent among these patients, affects the outcome. methods: in an attempt to investigate this hypothesis, 79 patients aged over 60 years suffering from head injury, with glasgow coma scale (gcs) of 8 or less, were studied retrospectively. twenty-six patients (32.9%) had preinjury pathology i.e. diabetes mellitus, arterial hypertension, heart failure, alcoholism, parkinson's disease etc. (group a) and fifty-three (67.1%) did not (group b). the following data were recorded: mortality in the i.c.u., duration of hospitalisation, incidence of infective complications and neurologic status at discharge. results: groups were comparable in terms of mean gcs (6.57 vs. 6.56) and median age (67.5 vs. 67). the incidence of brain pathology in the two groups was the following: epidural haematoma 7.69% vs. 11.32%, acute subdural! haematoma 30.7% vs. 30.19%, intracerebral haematoma 19.23% vs. 5.66%, subarachnoid haemorrhage 38.46% vs. 39.62%, diffuse haemorrhage 11.54% vs. 13.21%, contusion 26.92% vs. 49.06% and non-visible pathology (normal ct) 3.85% vs. 1.89%. unilateral pupilary dilatation was found to be 15.38% in group a and 18,87% in group b. the mortality during hospitalisation in the i.c.u. was almost the same: 50% iu group a and 47.2% in group b patients. however, group a patients had significantly more infective complications, required longer hospitalisation and had lower gcs at discharge. conclusions: the results show that the existence of preinjury pathology does not seem to affect the short-term outcome of elderly patients with severe head injury. it has however an impact on morbidity and perhaps long-term survival of these patients. the assessment of clinical development in intensive care patients with severe head injury still remains a problem. to optimize the monitoring of intracraniel prassure (icp) we rautlr~dly implant an eplduml measuring device in our hospital. the aim of this study was to prove the correlation of the icp-values with ct findings and clinical development. during a 12 month period (1993 -9r the icp was monitored in 23 p~,tients (14 male, 9 female) with severe head injury by an eplclural measuring device (epldyn~/$plegelberg| the mean age was 36.9 years (4 -83). the glasgow coma scale at admission was 6.9 (3 -15). in all cases the device was placed wfihln the first 10 hours after admission. the tcp was compared with physical examination, radioidglcal or intraoperatlve findings and cunlca! outcome. the average time of measuring was 7. 2 days (1 -19) . the traatment depended on the !cp values recorded. rising icp-valuea ~ed to radlologlcal c0ntra!s by ct-scan. in 1 case an intracranlai hemorrhage was detected and drained. the overall survival rate was 78.3 %. 113 showed a complete resolutl0n, in other 33.3 % psychological residuals like decreased mentatlon, in 17.4 % sensomotorlc residuals like cerebral nerve dysfunction and aphasia, and 11.1% of the injured remained in a comatous status. in 87 % of our cases the measured values correlated with clinical course and management. in 2 cases (8.6 %) we observed a displacement of the icp-pevice. there was no icp induced infecllon. istituto di anestesiologia e rianimazione, universit& ,,la sapienza", rome, italy * istituto superiore di sanit& -servizio di epidemiologia e biostatistica, rome, italy objectives: acute renal failure (arf) can be a severe complication of trauma. the current incidence of post-traumatic arf is associated with high mortality 1. identification of risk factors and prevention of this complication could improve the outcome of trauma patients. methods: one hundred fifty three consecutive trauma patients (age 37.6 _+ 19.6, injury severity score 28.3 +10.9) admitted to icu were studied. incidence of arf was 31.4 % (48/153). arf was defined as persisteat plasma creatinine >2mg/dl with or without oligoanuria 2. arf was defined as early when occurring within the first 96 hours (earf) and late when the onset was after the first four days (larf). results: earf occurred in 31 patients while larf developed in 17 patients. age, iss, and incidence of rhabdomyolysis and acute respiratory failure were not different in the two groups. an higher incidence of multiple organ failure (mof) and sepsis (76.6% for both) were observed in larf group, when compared to earf (25% and 23% respectively). abdominal trauma was more frequent in earf group (32% vs 18%). the gs for earf and larf were respectively 8_+4.4 and 9_+4.15 while in the group who not developed arf (narf) the gs was 10.5• conclusions: gs score difference seems suggestive and can be that an abnormal cerebral activity (hipofisary hormones?) may play a crucial role on onset of arf in these patients. moreover the frequency of acute respiratory failure in the group of arf was higher (91.7 versus 64.5) than narf group. the early ipoxia in the early phase of trauma, then, may be another crucial point for development organ failure. these are preliminary data. a more exact statistical analysis must be perform to have definitive conclusions. to compare the active compression-decompression cardiopulmonary resuscitation (acd-cpr) with the standard cardiopulmonary resuscitation (s-cpr) in out of hospital cardiac arrest patients. is a controlled, randomized study. two groups of patients with cardiac arrest out of the hospitalwere formed. group i, (acd-cpr) and group ii (s-cpr). for the acd-cpr groupweusedthecardiopumpdeviceofambulnternational. asfortherest, the erc (1992) algorithms for acls were followed. the utstein style (for out of hospitat cardiac errest) was used for listing and evaluating all cases of the study. the cpr was contucted by the crew and the doctors of our mobile intensive care units (micu). we studied 146 consequitive patients (75 in group i) and (71 in .group ii). demographics pre-cpr characteristics (e.g. ecg form of cardiac arrest) and procedures (eg bystanders or second tiers crew cpr, defibrillation, drugs) were quite similar for both groups. the mean arrival time of micu was 9min. in group i we recorded r.o.s.c. (return of spontaneous circulation) 17,5%, death 73%, continuation of cpr efforts 9,5%. while in group ii, 21%, 69%, and 9,9% respectively (recorded percentage until the admission to the hospital). no significant difference was found in anyofthe short term outcome parameters. no complications related to the acd-cpr technique, were noted. not any significant difference between the two methods was proven (from this small evaluated sample). the results of previous clinical studies are controversial (i) . more sophisticated studies proved the superiority, in a certain number of parameters (e.g pressures, flow, etc) of the new technique although there are many difficulties for establishing clinical results. in the pre-hospital setting that is related to many parameters (speed of the intervention, effectiveness of bystanders cpr, education ofparamedics, etc.)the evaluation is even harder. the superiority ofthe acd-cpr can be proven when it is performed in almost 0 times increased number of studied patients as w~ll as improvement of the technique could lead us to more established results. objectives; infectious morbidity is the major cause of mortality after burn injury, and is due to multiple factors. trace elements (te), which are involved in both humeral and cellular immunity, exhibit severely altered status after burns. te supplementation has been shown to be associated with increased leukocyte counts and shortened hospital stay. the trial aimed at studying the immune responses in severely burnt patients receiving normal te supplies or early large supplements. methods: 12 patients, aged 40_+16 yrs (mean_+sd), with burns covering 49+18 % of body surface were studied from day 1 (d1) to d30 post-injury, were randomised in 2 groups (g): g1-control receiving recommended te supplies + placebo; g2 -receiving in addition large supplements of cu, se and zn from d1 to d8. enteral nutrition was started within 12 hours of injury in all patients. immunological parameters: peripheral leukocyte counts, proliferation of mononuclear cells to mitogens, cell surface molecule expression, and neutrophil chemotaxis at d10 and d20. infectious episodes and micro-organisms were monitored until d30. results: the patients' characteristics were similar g1 & g2. the total leukocyte counts were higher in g2 between d10 and d20, due to increased neutrophils (significant from d13 to d15). total cd3+ and cdlg+ cells did not differ, whereas cd14+ (monocytes) were significantly increased at d20. proliferation to mitogens was significantly depressed in all patients. chimiotactism was not altered. the number of infectious episodes was significantly decreased in g2 with a mean of 2.0_+ 0.9 infections during the first 30 days versus 3.3_+ 0.8 in the control group (p < 0.03). conclusions: the large te supplements for 8 days was associated with a significant decrease of the number of infectious episodes. supplementation was associated with increases in total leukocyte, monoeyte and neutrophit numbers. further studies are required to determine the precise mechanism underlying the improved immune defences. objectives: evaluate the efficiency of local adsorption (la) with the use of carbon adsorbents in case of severe burns in expertment and clinic. methods: experimental studies on la were performed on a model of 20% body surface area iiib-iv burn in 335 rats. a burn eschar was excised on the 3rd day after burn, the wounds were dressed with the gauze bandages (control) or with adsorptive dressings (la), dressings were regularly changed. clinical investigations were carried out in the course treatment of 78 patients with severe thermal and radiation ilia-iv burn. in the dynamics of bum disease some indices of proteometabolism and intoyacation criteria were evaluated. results: the experiments have demonstrated that the application of la after early excision of a burn eschar exerts a pronounced normalizing effect on a protein electrophoregram and the activity of proteases and their inhibitors in burned tissues preserving vitality. thus, by the 14th day after burn infliction the activity of cathepsin d in injm'ed muscles is 6 times lower under an adsorptive dressing than under a gauze bandage (control) (p<0,05), the activity of trypsin-like proteases is 1.5 -3.4 times lower and the antitryptie activity does not differ significantly from the normal level. the cytotoxicity of extracts of burned tissues after the adsorptive dressing application fn vivo and adsorption in vitro is 25-35% and 7-20%, respectively, of the toxicity of control extracts. a similar normalizing effect of la is ok~rved for an intact muscular tissue and blood serum. the dectron-spin-resonance studies have demonstrated that la allows to normalize antitoxic activity of liver and functional activity of kidneys. the application of la in the treatment of patients with severe burns have been shown to localize a region of irreversible tissue changes, accelerate rejection of a burn eschar, attenuate an endogenous intoxication level and, as a result, shorten the time for grafting of a burn wound and accelerate wound heating. conclusions: proceeding from the obtained results, we can consider la as an effective method of localization of a region of irreversible tissue changes as well as of correction of local and general metabolism failures and overcoming burn autointoxication during burn disease. c de deyne, t vandekerckhove*, j. decruyenaere, b. vaganee, v vandewalle*, f colardyn depts of intensive care and neurosurgery*-university hospital gent-belgium. jugular bulb oximetry is the first bedside available cerebral monitoring technique providing an estimation of the adequacy of cerebral perfusion. its routine use in all patients suffering from severe head injury admitted to our ic unit enabled an extensive analysis of all very early cerebral perfusion data in order to evaluate the incidence of abnormal sjo~ data (and their possible causes) in this very eady period after traumatic insult and to search for possible implications as to the emergency management. these very early data were defined as the first 6 hours icu data and icu admission had to occur within 12h of traumatic insult. over the last 2 years, 150 pts with severe head injury (gcs<8) were monitored by jugular bulb oximetry, starting immediately after their arrival at the icu (mean of 4.8h after trauma, range between 2-9h). in a total of 85 pts (=56.6%), jugular bulb desaturatiens (<55%) were noticed during this early 6h period. in 24 pts (=16%), jugular bulb saturations higher than 75% were observed, whereas 41 pts (=27.4%) revealed no abnormal sjo 2 data (55-75%) during these first 6h. concerning the periods with too low jugular bulb saturations (n:85), we found the following correlation ; in 49 pts (=57.6%) cerebral perfusion pressure (cpp) was below 70 mmng, in 36 pts (=42.3%) paco~ was below 30 mmhg and finally in 6 pts (=7%) we found primary intracranial hypertension. for the high jugular saturations (n:24) we found a primary intracraniaf hypertension in f0 pts (=41%), and a pace 2 level above 40 mmhg in 6 pts (=25%). in all patients we could restore jugular bulb saturation within normal range (55-75%) with the correct!on of the presumed causative factor. we can conclude that ultra early jugular bulb saturation data revealed a high incidence of abnormal values, with a predominance of jugular bulb desaturations, confirming once again the high incidence of disturbed and too low cerebral perfusion within the first hours after severe head injury. these jugular bulb desaturations were especially correlated to systemic causes, as a too low cpp (caused in the vast majority by primary map insufficiency, and not by intracranial hypertension) and hyperventilation were the 2 major causes of the desaturation periods. as jugular bulb desaturatione are known to be significantly correlated to a worse neurological outcome after severe head injury, one might improve outcome by an emergency management avoiding these possible causes of jugular desaturation. therefore, extreme attention should be paid to the maintenance of an adequate mean arterial blood pressure (above 90 mmhg?) even duhng the few time spent at the emergency department. one should be as attentive to the maintenance of normoventilation during this very early period of admission and hyperventilation without any knowledge of icp or sjo2 should be abandonned. recently, indomethacine has been proposed for the treatment of therapy refractory intracranial hypertension in pts suffedng from severe head injury (1). indomethacine, a cyclo-oxygenase inhibitor, gives rise to a significant fall in cerebral blood flow by inducing cerebral vasoconstriction. therefore, its use could result in a drastic lowering of the intraeranial pressure (;cp) in pts suffering from intracranial hypertension secondary to cerebral hyperaemia and in whom the use of other cerebral vasoconstrictive drugs (barbiturates or hyperventilation) appears insufficient to control icp. for the last 18 months, we included the use of indomethacine in our therapeutic flow chart for severe head injury management. pts revealing intracranial hypertension (icp>20 mmhg) and cerebral hyperaemia (sjo~>75%) and in whom icp was not efficiently controlled by the combined use of hyperventilation and barbiturates were given indomethacine in a trial to control icp. a total of 98 head injured pts received treatment for intracranial hypertension over the last 18 months. six of them met the criteria set for the administration of indomethacine. in 2 pts, no decrease in icp or in sjo 2 was observed and both pts died due to therapy refractory intracranial hypertension. in the other 4 pts, a significant fall in icp and in sjo 2 was observed shortly after indomethacine administration. in 2 pts we observed a catastrophic fall of sjo= even below 55%, indicating an extreme cerebral vasoconstriction with the possible risk of inducing cerebral ischaemia. in one of the 4 pts, icp remained under control without further administration of indomethadne, but he died 3 days later in multiple organ failure. the other 3 pts, needed multiple indomethacine administrations (for 1 pt even during 4 consecutive days) to finally control icp. in all 3 pts, icp was finally controlled, but only 1 pt survived. both other pts died from systemic causes (multiple organ failure in 1 pt, massive gut infarction in the other tat, possibly due to the systemic vasoconsttictive effects of the indomethacine administration). in conclusion, indornethacine might have a role in the treatment of intraoranial hypertension, especially when caused by cerebral hyperaemia. we observed however a poor final outcome and a threatening high incidence of systemic events (multiple organ failure, gut infarction) in those pts receiving indomethacine for icp control. therefore, indomethacine in the treatment of intracranial hypertension should be reevaluated in controlled study settings, before its routine use can be considered. untill recently, intracranial hypertension (ich) in pts suffering from severe head injury was managed in a staircase approach, with csf drainage as first therapeutic step, mannitol as second step, hyperventilation as third step, and finally, barbiturates as the last rescue step for therapy refractory ich. this staircase approach for the treatment of tch was only guided by the intracraniat pressure, and not by other parameters such as e.g. the actual state of cerebral perfusion of the concerned pt. jugular bulb oximetry provides us with the first, bedside and continuous available, estimation of cerebral perfueion. its implementation in a rigourous flow chart, based on as well icp-as jugular bulb oximetry-data might result in an altered strategy for ich management. we adopted a '~ugular bulb saturation (sjo~)-guided approach" for ich management in 86 consecutive pts, suffering from severe head injury (gcs<8). we maintained csf drainage as first therapeutic step, but the decision for the second step was guided by sjo2 information. pts revealing ich and sjo=values above 75%, were treated with hyperventilation, and did not receive mannitol. if ich persisted, barbiturates were added as a third step. on the other hand, pts with ich and sjo= vales less than 75%, received mannitol administration as second step. hyperventilation and/or barbiturates were only added if ich persisted and if no cerebral hypoperfusion was discerned (sjo=>55%). our objectives were to prospectively analyze this new therapeuticstrategy, as compared to the formerly used staircase approach of ich. we managed 86 pts with ich, with an overall mortality of 13.7% due to therapy refractory ich. all pts received standard primary care with head elevation, full sedation and normovenfilation. fer 16 pts, csf drainage alone was sufficient to control ice of the remaining 70 pts, 38 pts received mannitol and 32 pts were hyperventilated as second approach. in the third line, 14 pts were managed with barbiturates, 12 with mannitol and 10 pts with hyperventilation. finally, barbiturates were used as the final rescue in 14 pts. these results reveal a less frequent use of mannitol as only 50 pts received mannitol, compared to the 70 pts that would have received mannitol using the former staircase approach. hyperventilalien was used much earlier in the treatment course, as 32 lots were already hyperventilated in the second line approach, were this was formerly exclusively reserved for the third line approach. finally, also barbiturates were used much eadier (14 pts received barbiturates as third approach). we may therefore conclude to a important change in the management of ich, induced by a sjo2-guided flowchart. however, future studies will have to elucidate if this new strategy for the intensive care management of severe head injury will also result in an improved outcome. obsectives: in a first series of experimental brain injury we investigated the course of brain po2, icp and cerebral blood flow after traumatic brain injury (tbi), whilst accordingly there are very few data available and the mechanisms leading to secondary brain damage are poorly understood. methods: in 6 piglets (14 days old, 3,3-5 kg) of either sex we produced a moderate brain injury (1,5 arm., 20 msec.) using a lateral fluid percussion {fp) device. complete measurements were made before and 5 min. after brain trauma and after 3, 5 and 24 hours including blood gases, cardiac output (htermodilution), heart rate, eeg, laser doppler flow probe (ldf} and icp values (camino), brain temp., po 2 by a clake type oxygen electrode (licox) and coloured microspheres for regional blood flow. results: immediately after the trauma a typical "cushing"response to the icp peak up to 130 mm hg being highly significant (before mean i0 mm hg, range 4-12 mm hg) could be observed: mean arterial blood pressure rose from appr. 85 mm hg to ii0 mm hg for 3-5 min. in two animals this was followed by an ischemic period lasting 15 min. accordingly icp values gradually returned to starting measures within 3 hours; in the ischemic animals they remained at a level of about 30 mm hg.-no secondary increase of icp could be observed, once icp dropped to starting values within 24 hours. cerebral blood flow (ldf) fell from mean values being i00 before trauma to appr. zero and recovered to around 50. brain po 2 started at mean values of 20 mm hg (range 15-30 mm hg) and fell to around zero depending upon the severity of the ischemic reaction. on average values of 15 mm hg were reached over the time course. conclusions: with our fp trauma model we can reproduce the well known "cushing"-response after brain injury; secondary icp elevations cannot be achieved, although local edema is observed. direct brain po 2 measurement seems to be a very sensitive variable for detection of cerebral ischemia and anticipates eventually following icp elevations by far. pulmonary aspiration s,traoaras. v. sgountzos, p. agouridakis, m eforakopoulou, e. ioannidou. intensive care unit (tcu) of "kat" hospital, athens, greece ob!e=ives: the reported mortality rate after pulmonary aspiration is variable in several series. the purpose of this study was to find out the influence of preexisting disease or situation on morbidity and mortality of intensive care unit (icu) patients with pulmonary aspiration. methods: patients who were treated in icu and had pulmonary aspiration, were studied, entrance's criteria in the study, all of them obliged, were: 1) suction of gastric contents from trachea during intubation, 2) presense of a predisposing factor, e.g. coma. 8) recent hypoxaemia or new infiltrates in xray. preexisting disease was recorded and correlated with complications and outcome. patients with glasgow coma scale 3, because of cerebral injury, and patients who died within 3 days from cause other than aspiration, were excluded from the study. method of statistical analysis: chi-square test, results: one hundred forty five patients were studied. the trauma patients were 96 and the non trauma patients 49. from the trauma patients, 77 had cerebral injury and 19 were polytreumatized without cerebral damage. from the non trauma patients, 13 had malignant neoplasms, 14 neurological diseases in terminal stage, 7 old age, 10 drug overdose, and 5 several diseases. eighty seven from 96 trauma patients (91%) and 45 from 49 non trauma patients (92%) manifested several complications (pneumonia, ards, etc), so there was no statistical difference in complications' frequency between the 2 groups (p>0,1). the severity of complications was also proportional in the 2 groups. eighteen deaths were recorded in the trauma patients (mortality 19%). only 7 deaths correlated directly or indirectly with the aspiration (7%). in non trauma patients, 32 deaths were recorded (71%). twelve deaths were recorded in 13 patients with neoplasms, 12 deaths in 14 patients with neurological diseases, 6 deaths in 7 aged patients, 1 death in 10 drug overdose patients, and 1 death in 5 patients with several diseases, the mortality difference in trauma and non trauma patients was statistically significant (p< 0,001). in patients with drug overdose the mortality was significantly lower from the other non trauma patients and the difference was statistically significant (p<0,001). conclusion: the preexisting disease or situation plays a major role in the outcome of the patients with pulmonary aspiration. the mortality of patients with aspiration seems to be caused by severe preexisting situations rather, that lead to death, than from the pulmonary aspiration per se, which may be a final happening in a predetermined course. obiectives; the purpose of this study was to compare fluconazole and amfotericin-b in the treatment of fungal infections in severe trauma patients. methods: thirty five severe trauma patients who were treated in intensive care unit (icu), were studied prospectively. they all developed fungal infections, prooved with blood positive cultures and at least one of the following: fever, positive urine or bronchial secretions cultures, infiltrates in xrays. the patients were separated randomly in 2 groups. the patients of group a (15 patients) received fluconazole 200 rag/day for 15 days. and the patients of group 8 (20 patients) amfotericin-b 50 rag/day for also 15 days. compaiison's criteria were the clinical responce to treatment (fever etc), the fungal elimination (blood and other cultures), the relapses of the disease, the side effects of drug, and the outcome of the patients. as method of statistical analysis was used the chi-square test. results: nine patients from 15 of the group a (60%), and 18 from 20 of the group b (90%), presented remission of fever (patients of group b had better clinical responce than patients of group a, and the difference was statistically significant, p<0,05). all the patients before treatment had positive for fungi blood cultures. after 10 days of treatment, 3 patients of group a and none of group b had positive cultures. eight patients (from 13 who had positive cultures of bronchial secretions before treatment) of group a. and 5 (from 17) of group 8. had positive cuttures of bronchial secretions after 10 days of treatment, so positive bronchial secretions were fewer in group b than in group a, but this difference wasn't statistically significant, (p<0,1 and p>0,05): ten patients (from 12) of group a and 7 patients (from 16) of group b had positive urine cultures, after 10 days of treatment (positive urine cultures were fewer in group b than in group a and this difference was statistically significant. (p<0,05). two patients of group a and none of group b had a relapse of fungal disease. in group a, no side effects were obsepced, while in group b were observed only minor side effects (small increase of serum creatinine in 2 patients, chills and fever during infusion in 3 patients, and hypokalemia in 12 patients). three patients of group a and 1 patient of group b died, because of sepsis. conclusion: amfotericin-b (even i~ short regimen of 15 days), is superior to fluconazole in the clinical and laboratory responce and also in the relapse of fungal disease, fluconazole is superior to amfotericin-b as it has no side effects. ob!ectives: flail chest after thoracic trauma is a serious injury. it is controversial if flail chest by itself orthe concomitant intrathoracic injuries e.g. pulmonary contusion, is the cause of the reported significant morbidity and mortality. in this study we searched the influence of concomitant thoracic injuries in the course and outcome of patients with flail chest. methods: eighty five patients with flail chest after isolated chest injuries were studied, for the purpose of analysis, we separated the patients into 4 groups, patients with isolated flail chest were included in group a, patients with flail chest and hemo-pneumothorax in group b, patients with flail chest and pulmonary contusion in group c, and patients with flail chest and hemo-pneumothorax and pulmonary contusion in group d. complications from the chest, duration of mechanical ventilation and mortality were compared in the 4 groups. statistical comparison of results belween groups was made using chi-square and t-studend tests. results: the patients were 85. all patients received mechanical ventilation, twenty eight patients were ihcluded in group a, 19 in group b, 20 in group c. and 18 in group d. seventy three patients manifested complications from the chest, especially pulmonary infections. there was no statistical difference among the 4 groups as to number of complications ( twenty four patients had chest complications in group a, 16in group b, 17 in group c, and 16 in group d. p>0,1}. the duration of mechanical ventilation was not statistically different among the 4 groups (the mean duration was 15,9 days in group a, 16,8 in group b, 16,5 in group c, and 17,5 in group d, p>0,1). there was also no statistical difference in mortality among the groups (six patients died in group a. 4 in group b, 4 in group c, and 5 in group d, p>0,1 ). conclusion: flail chest by itself is a serious thoracic damage with many complications, regardless of the presense of other thoracic injuries, which don't contribute to greater morbidity and mortality. the present study investigated the correlation between blood lactate mortality and organ failure in 129 trauma patients admitting between december 1, 1992 and july 31,1993 in the icu. road traffic accidents were the most common cause of trauma in this studded population. brain damage was the main cause of mortality .nevertheless, 29 of patients died from sepsis and multiple organ failure without significant brain damage and these deaths were potentially preventable. respiratory failure was the most common complication and was developed in 44 (44%) of survivors and in 26 (86%) of non survivors .we noted low fncidence 5 of renal failure may be do to the early and aggressive ittv'asive hemodynamic monitoring and cardiopulmonary support. as part of our routine case protocol serial blood lactate levels were measured in each patient at least 3 times a day until the valses returned within the normal range or until death. we analysed the blood lactate levels on admission, the highest value and the number of days until the first normal value (<l.smeq/i). initial lactate and highest lactate levels were significantly higher in patients with than without organ failure.(3.4 vs 2.4, 4.1 vs 2.8meq/l, p<0.01)the duration of hyperlactemia also was higher in the patients with organ complications. (2.2 vs 1.0 days, p<0.01). both initial lactate and highest lactate levels were higher in the non survivors than in the survivors.(4.0 vs 2.8,4.6 vs 3.4meq/l, p<0.01) the present study demonstrated that not only the degree but also the duration of hyperlactemia can be related to the development of posttraumatic complications. serial blood lactate measurements are reliable indicators of morbidity and mortality following trauma. s155 current evidence suggest that peep only affects intracrenisl pressure (zcp)when it interferes with systemic arterial pressure, although the effects of peep over cerebral oxygenation remains unknown. objective: determine the effects of peep over icp and cerebral metabolism measured by sj02, kjdo 2 and ceo 2 in severe head injured patients. meterial ~ methgds: patients with glasgow coma scale l 8 at arrival, with difusse brain injury in the first gt, according to marshall criteria,lop and 8jo 2 monitored, under analgesia and sedation, normoventilated with zeep, without mannitol treatment in the previous 4 hours, normal x ray chest and within the first 48 hours from injury, were considered candidates. peep value where increased fro~ zero to 15 cmh20, in three different steps, each one with 20 min of duration. all date were analized on line, pmax p'p ..... p.._, from the ventilator, systemlc haemodynem]c data, cerebra perfuslon pressure (cpp), icp and sjo 2, in the case of hemodynamic deterioration, during the study, ppc 6ommhg, extra-volume were administered. if persistence, dopamine were begun or increased dosage. if no good response were obtained, patients were retired from the study. results. 40 patients were candidates, 15 of them were exc;uded due to haemodynamic instability, k total of 25 completed all steps and were e~amined, 22 men and 3 women, age 32!7.9 yrs. at hospite; arrival, glasgow were < 5 in 18 patients, and >5 in the rest 7. 15 patients 20 mmhg at the beginning. zeep ob/ectives. critically ill patients are transpoded to an intensive care unit(icu), under conditions, which have not been systematically evaluated. therefore, we set suite investigate transportation and admission condition of these patients to our department. methods. we studied 36 patients(16 females), aged (mean-..+-sd) 56.2_ 17.3yrs, which were consecutively (from august 1994 to march 1995) admitted to the icu, through the greek national emergency transporta~on service. apache ii severity score upon admission was 17.4-+6.8 (range 4-31). the following data were evaluated: 1) number of medical departments, where health care was provided until final admission to the icu, 2) ambulance transportation conditions, 3) catheters and tubes inserted before admission, 4) vital signs upon admission 5) information provided by referring physician (scored on a 1 to 5 scale: history, electrocardiogram, chest x-ray, laboratory data, drug therapy already administered), 6) comparison of the state of the patient described by referring physicians, to the actual state u pen admission. resu/ts. one to four medical departments had provided health care before the palient was admitted the icu (1:22.2%, 2:47.2%, 3:27.7%, 4:4%). thirty/36 (83.4%) patients were escorted by a physician. twenty-six/36 (72.2%) were transported on oxyge n, fio2(mean__.sd): 46-+3%, pao2: 78.6-+35.2mmhg. five of the remaining 10, for whom no oxygen was provided, had pao2: 46.2-+7mmhg. twelve/36 (33.3%) were intubated and ventilated during transportation. thirtyfour/36 had a peripheral venous line, 5/36 had an arterial line, 13/36 had a nasogastdc tube, 20/36 had a urinary catheter. eleven/36 were sedated and 2/36 were paralysed. three/36 were on inotropes. vital signs upon admission were: arterial blood pressure, systolic 100.6-+44mmhg, diastolic 57-+23mmhg, heart rate 104-+22 bpm, temperature 36.3 -+2cc. patient information score was 217--. 1.7. the actual state upon admission was found substantially different, as compared to the description of the referring physician, in 28/36(77.7%) patients. conclusions. we conclude that several aspects of the greek national emergency transportation service to an icu should be reevaluated and further improved, i. e. ventilatory support, adequacy of information provided and accuracy of prior description of the patient's state. a new perspective must be applied for critically ill patients transportation since 78.8% of the patients were evaluated and treated in more than one, medical departments, mostly primary care, before they were finally admitted to our icu. dclhb is a human derived hemoglobin molecule that has been cross-linked to stabilize and permit heat pasteurization to remove residual proteins and inactivate viruses. dclhb is mixed with a lactated electrolyte solution to yield a total hemoglobin concentration of log/dl objective: to present an overview of four recently completed clinical safety studies of dclhb in the u.s. and europe, and to discuss the properties, actions and potential indications for dclhb. method: patient populations in the four studies included males and females ranging in age from 18 to 84 years. dosing ranged from 25mglkg to 300mg/kg. the controlled randomized safety studies were conducted in chronic renal failure patients, surgical patients undergoing total hip replacement or abdominal aorta repair and in hemorrhagic hypovolemic shock patients. these very diverse patient populations allowed safety evaluation of the product in patients who were generally elderly, often hypertensive with some degree of cardiovascular disease, and receiving medications for treatment of other conditions. results: over 150 patients received dclhb in the four:studies. no product related sarious adverse events occurred during the clinical trials. conclusion: results from phase itll safety studies of dclhb in patients undergoing chronic renal dialysis, abdominal aorta repair, or total hip replacement and in patients in hemorrhagic hypovolemic shock, indicate that the product was well tolerated in these distinct populations. although these studies were designed to evaluate safety, the data suggest clinical benefit. follow-up efficacy trials are indicated. prehospital emergency services represent the extension of emergency care into the community and constitutes the manpower, communications, transportations and facilities used to provide care for patients outside hospital. one of the main points of the system is how to decide the hospitalization of patients and what kind of facilities to provide : emergency medical service, fire brigade, locat general praclitionner or ambulance officers. objectives : to realize guidelines for using the prehospital emergency medical service in case of patient'calls outside hospital. methods : from 1st june 1994 to 14 july 1994, all the calls for emergency care were analysed using a questionnaire of 114 items (origin of the call, responses to the questions of an emergency practitionner, kind of emergency service provided and the issue of the patient). after taking account of the appropriatness of the decision, statistical method used was a logistic regression. results : 996 calls were analysed. the criteria, for prehospital emergency medical service using, given by the logistic regression were as following : existence of a call for emergency, thoracic pain, dyspnea, seizures, cyanosis, drug intoxication, fall of the patient, fracture, age, the state of consciousness and the neurologic reactivity. the minimal and maximal predictive values of the model given by the logistic regression are respectively 2% and 100%. the performance of the model is 88 %. conclusion : it seems possible to help medical decision of emergency medicine by using only some easy criteria and a predictive model. (italy) objective: to evaluate the incidence of blunt carotideal injury (bci) in patients admitted to our icu after head injury. methods: we reviewed the medical records of all patients diagnosed to have a bci. at admission, the severity of trauma was assessed either with glasgow coma scale (gcs) and with ct scan. bci was demostrated by doppler ultrasography (us) and by angiography (ang). results:since may 1991 to april 1995, 4 patients were admitted to our icu with bci (2m,2f, age 29+ 1 3). a history of direct trauma was present in 2 patients. admission gcs was 15 in all patients, and was associated with hemiparesis in 3 of them; the last became paretic 48 hours thereafter. two patients had concomitant injuries (a homoiateral clavicular and a controlateral zygomatic fracture, respectively). the initial ct scan was negative in every patient, and showed signs of ischemia after a variable timespan (2-4 days) after the onset of the symptoms. the bci was diagnosed with us and ang, which demonstrated a thrombosis of the internal carotid artery (ic). in two patients, an intimai dissection was also present. three patients were treated with heparin associated with antiaggregating agents and were discharged alive. the last patient was referred to our icu after the development of a massive hemispheric infarction, and died three days after the admission. at necropsy, the ic thrombosis was associated to an extensive homolateral extra and intracranial venous thrombosis. conclusions:the presence of focal neurological signs despite a negative ct scan should address the diagnosis toward a bci, thus implementing the diagnostic workup with us and/or ang. tab i: distribution of l~tients (%) in the 3 groups the outcome were monitorett results were sabmitted to statistical analysis using a continence table 3x2 in z2 test. res.cl~s: of 43 patients 34 were submitted to thrombolysts and 3 died. the higher incidence of bracb, ar~lhmias (ii degree gg p t39e 1 and 2 av block. 11i degree av block. avsb 6.141 <ii.05 sinus arrest) that requited the insertion of avsc 11.121 <cl005 temporar~ pace-maker was recorded in group a b vs c 0.004 ns in 64% of the cas6~. the rapid recognition of life tab li:;~ test. threatening conditions suggests the monitoring of v4r-lead r b' in the course of inferior ami. the authors report their own expirience in application of ringer lact.(rl)and 7,5%nacl/6%dextran 70. methods:in 40 polytraumatized patients with developed hae morrhagic shock,injures of the chest(qg)and abdomen(q2) prevalid.replacement of the volume loss 5egan in institutions of general medicine,frequently by rl and 69dextran 70 or haemaccel.after receiving necessary medical aid,polytraumatized patients wer transported to mma by helicopter,continuing replacing of circulation volume by the same solution. results:average quantities of the solution 8dministered ~o the injured wer~2375-2550ml.a group of the inoured which,after the admission to mma was resuscitated by adml nistration of the 7,5%nacl/6%dextran 7@(groupii)(4-sml/kg observed to the final surgical management,had statistically higher map(increase of 58% in relation to admission), cvp(average increase of 8,25cmh~o),diuresis(averagely 35omi after 30min.),better gas ~nalyses and acid-base status in relation to the group which was administered only rl(groupi)(increase of the map of q8%,average increase c~ of cvp 2,5cmh20 and average diuresis 20oml after 3omin). total quantity of the administered solutions was significantly smaller in the groupii(2800ml:4925ml).the only ob served complication was hyperhatr~f~a::and~moderat~hyper osmolerity of plasma which disappeared within 32hours. the quantity of replaced blodd was considerably smaller in the groupii(qooofnl:20ooml).in the group i interstitial pulmonary oedema was observed in two patients(qo%),while: in the group ii no complication was observed.coagulation i disorder and hypotension were not recorded because of the fast infusion of hypertonic/hyperoncotic solution in the groupil as the infusion of 7,5%nacl/6%dextran 70 lasted 5 minutes. w.scherzer(l~,k.lechner(1),r.simon(2). ll)dept.anaesthesiology,trauma hospital,vienna-meidlin 9 !(2)neurotraum.rehabilitation center,vienna-meidling both austrian workers'compensation board,vienna,austria what we usually call"unconcious" means only that the patient is not able to interact in any kind of way.experiences in intensive care medicine (robinson,1975) ,in general anaesthesia (bennet,1985) and with evoked potentials show,that unconcious patients are not automatically unhble to process and recall information.this implicates a challenge to start the efforts to restore the traumatica-!ly desintegrated brain function as early as possible in the acute phase ia (zieger,1992) .we present a method to ~repare the patient for the rehabilitation goals of the postacute phase ib,the phase of stabilisation ii and of rehabilitation iii within one concept. to achieve sensory stimulation in phase la we use: attempt at some kind of dialogue by speaking to and tou-ching the patient,to make him experience his body limits, ~acio-oral therapy by ice application,tast and smell pro4 vocations and trials of feeding,early adaption to circadian rhythm and guiding the patient in every-day-life-ac-tivities e.g.:in the brushing of teeth,washing etc. to achieve motor stimulation and orientation in terms of ~-ace,time and gravity in phase la we use: avoidance of perceptual impairment as produced by air-:sack-beds or habituation to supine position,using method~ according to affolter(1980) ,basal stimulation (bienstein, 1991) , bobath-therapy(1966) and facilitation of a physiological moving pattern(pnf)according to knott(1968) . conclusions: by integrating all these measures in the ~herapy and nursing even in the acute phase la one prei ares the comatose patient for multisensory stimulation nd motor training and all other rehabilitation activities in the following phases of therapy. s157 objectives." measurement of cardiac output (co) and stroke volume (sv) requires insertion of a central venous catheter and is associated with a considerable risk for complications. non-invasive methods for determination of hemodynamic parameters have been introduced recently. one of these methods is the aortic modelflow program, which provides co and sv continously using a three-element windkessel model. the aim of the study was to evaluate the accuracy of the aortic modelflow program in critically ill patients. methods: 21 patients (mean age: 69) admitted to the emergency department after cardiopuimonary resuscitation (n=ll) and for myocardial infaction (n=5), acute congestive heart failure (n=4) and anaphylactic shock (n=l) were included to the study protocol. after stabilization all patients received a swan-ganz catheter (baxter~; edwards swan-g-anz) via a central vein. co and sv were measured by the use of a cardiac output catheter and injection of 10rrd ice-cold glucose 5% non-invasive cardiac output measurement was done by using the continuous cardiac output software package modelflow (tno; portapres| results: overall, 159 paired measurements were used for the evaluation of the accuracy of non-invasive obstained sv and co. mean values, standard deviation and range of co and sv evaluated by invasive and non-invasive methods are summarized in the aortic modelflow provides reliable hemodynamic data in critically ill patients. it may be a useful alternative due to its non-invasive approach and continous measurement. objectives: to determine the feasability and accuracy of a rapid urine screening test (triage tm, merck-diagnostika) in an emergency department. methods: prospective analysis of the triage tm testkit (tt) in patients admitted because of suspected drug abuse during an observational period of 6 months. the tt is a eompetitve immunoassay which gives qualitative results within i0 minutes for methadone, benzodiazepines, cocaine, amphetamine, opiates, barbiturates and cannabis. urine samples were analysed with the tt and compared to the results of a enzyme-lihked tmmuno-assay (el.a) in our labratory (gold standard). results: during the study 25 patients were enrolled, in 19 of these patients substance abuse was proven by tt and verified by eia. we recorded no false positive or false negative results for benzodiazepines, barbiturates, opiates, cocaine and cannabis. two results were false positive and one false negative for methadone; one result was false negative for amphetamines. the triage-testldt had an overall sensitivity of 0.94 and specificity of 0.94. conclusions: the triage tm testkit seems to be a a useful rapid test device in addition to quantitative tests for drugs of abuse in an emergency department. objectives: the purpose of this study was to evaluate the influence of several factors of the renin-angiotensin-aldosterone system on blood pressure response in patients with hypertensive crises. methods: patients with systolic blood pressure >210 rorohg and diastolic blood pressure > 110 nunllg were included into the study. prior to treatment blood samples for determination of plasma renin activity (pra), angiotensin converting enzyme (ace), angiotensin ii (ang ii) and aldosterone (aldo) were collected. all patients received 5 rog enalaprilat intravenously. success of treatroent was defined as a reduction of systolic blood pressure below 180 mmi-ig and diastolic blood pressure below 95 mmi-ig within 75 minutes after start of treatment. results: 35 patients were included in our study, 20 (57%) patients responded successfully to treatment. mean arterial pressure decreased in responders by 36.5 mmhg and in non-respenders by 12.7 mmhg (p<0.001). responders and non-respenders differed signii'icantly concerning pra (p=0.001), ace (p=0.003) and ang ii (p=0.04). 0.003 0.04 the extent of blood pressure reduction correlated positively with the pretreatment pra and ang ii concentrations (correlation coefficient for pra: r=0.43; ang ii: r=0.66). conclusion: our data confirm that in patients with hypertensive crises blood pressure response to ace inhibition is mainly determined by circulatory pra, ace and ang ii. as the extent of blood pressure reduction correlates with pra, ace-inhibitors in patients with suspected high renin status cannot be recommended, as excessive blood pressure reduction, which carries a considerable risk for further organ damage, may occur. f. staikowsky, n. grillon, f.pevirieri, c.jedrecy, c. zanker, f. michard, a. haft medical emergency department. hospital bichat, paris epidemiology of acute intentional self medications-poisoning (smp) in france is especially known by data of poison control centei,s and intensive care units (icu). the purpose of this study is pro~,ided characteristics of this problem in a med for adults. method: july 1992 to june 1993, files of patients consulting to the ed for smp have been retrospectively analyzed. results: 727 patients, 482 women and 245 men, 33.3 + 12 years old (range 15-92) have been admitted for 804 episodes of smp (4% of all consultations) whose 77 relapses during the period of study. psychiatric disorders, drug addiction or hiv patients was found for respectively 42.6%, 9.1% and 2,9% of patients. the interval of time between the ingestion and emergency consultation was noted for 43% of smp (332 + 532 min, ranges 15-4320). the involved products name was known in totality in 89% of cases with an average number by episode of 1.7 + 1 drugs (ranges 1-8). the most often, 1 (52%) or 2 (21%) different products were interfered. the nonbarbiturate psychotropic drugs accounted for 76.7% of the products (benzodiazepines 67%, antidepressants 9.5%, neuroleptics 8%, carbamates 5.8%, imidazopyridines 5.1%, cyclqpyrrol0nes 2.7%). analgesics and nonsteroidal antiinflammatories represented 6.8% of all drugs, anticonvulsants 3.4%, cardiovascular drugs 2%, antiinfective agents 1.9%, drugs against cough 0.86%, muscle relaxants 0.86% and antihistamines h1 0.5%. the benzodiaz6pines were present in 531 episodes, alone in 316 episodes. in 36.5% of cases, there was a simultaneous intoxication with alcohol. the processing consisted of gastric lavage in 32.5% of cases, activated charcoal in 16.7% of cases, flumazenil in 16.9% of cases, naloxone and acetylcysteine in 3.4% of cases; orotracheal intubation was performed in 12 patients. admission in hospital was effective for 280 patients, in medical ward (n = 156), psychiatry (n = 63) or icu (n = 62); no fatal case was recorded. conelusion: smp to ed are often benign. the benzodiaz6pines are the most often incriminated but the new anxiolytics and hypnotics (imidazopyridines and cyclopyrrolones) take a growing place. the latsion burn center of athens. its planning constructive and functional refinements j. ioannovich, a. petalas-vourekus, d~ serbetis, h. carsin a 18 bed burns unit is under construction following a donation to the general hospital of athens. the plan of the unit, covering a surface of approximately 3.500 m2 is based on the principle of three identical 6 bed satelites which may function totally independent from each other. in the center of the unit the common facilities are installed, like operation theatres, storage rooms etc. this new modification in the plan of a burn unit is presented in this paper. the advantages from the fucntional, administrative and medical point of view are discussed. tiffs anisotropic conduodon could favour the ocenrence of a circular movement of the impulse that leads to tachyeardias by reentry. purposes of this work were to study, with the help of epicardial mapping, the influence of a trieyclie antidepressant, clomipramine (c), on the conduction velocity longitudinal (vl) and transverse (vt) to myocardial fiber orientation and on anisotropy (a = ratio vl/vt), and their modificutions by the sodium bicarbonate (13). method: a plaque of 64 electrodes, positioned on the left anterior ventricular wall of 9 anesthetized dogs, allowed to deliver, thanks to central electrodes, programmed electrical stimulations inducing vcuttienlar complexes, and to collect them. each entailed unipolar dectrogram was processed by a computer system that drew the isochrones and a map of activation allowing the calculation of v. the c was infused (0.5 mg/kg/min iv) during 75 rain; at t60, dogs received the b until the retuni of qrs to its initial value fro). a lengthening of qrs of at least 30% of its value at to was demanded before the administration of b. results: 1 dog was excluded because of an.~nsufficient prolongation of qrs before the administration of b. all values (map : mean arterial pressure, i-ir : heart rate, qrs andqt intervals, v) differed significatively (13<0.05) compared to values control fro)except qrs at t65. the b (7 + 6 ml/kg; ranges 2.8 and 20.5 ml/kg) modified no studied dements outside of the ( }rs. to ti5 t30 t45 t60 t65 t75 a 2,1 + 0,6 2,1 + 0,5 2,1 + 0,4 2,1 + 0,4 2,1 + 0,7 2,1 + 0,7 2,1 +-0,~ conclusion : the c slowed v l and v t without modify the anisotropy. the b did not modify the v of~conduction while the qrs prolongation was corrected. the c acts as a class i antiarrythmie drug on the inward sodium current during the phase 0 of action potential; the gap junctions have shown to be important in the conduction and an action on the gap junctions such as a modulation of the junctional resistivity, can not be rule out. is the doctor a heroe ? p. t.schies~.he, t. bauer, m. seyr dept. of anaesthesiology and intensive care, aokh krems, austria objectives: helicopter emergency services (hes) are getting popular more and more. the results concerning outcome are encouraging. however, some recent accidents with dead or badly wounded hescrew-members have shown the relatively high risk for the crews. therefore we were interested to eval0ate the motivation of physicians to participate in a hes. this survey was designed to investigate current concerns about safety and motivation of doctors on emergency call. methods: a questionnaire was sent to 205 doctors of the austrian emergency system. the survey consisted of multiple choice questions and subjective scoring tables from 1 (--full agreement) to 5 (=disagreement). overall, 64"/. of the active emergency physicians participated in the survey. results: 61.1% of the doctors assume the system is basically safe, experienced doctors tended to have less trust in safety. only 13% would not hesitate to go into action by dark. 14.8 % stdctly refuse night flights to accidents outdoors. although defibrillations are assumed to be safe dudng flight, only 29% would do it. 52.8% of the doctors would rather stop flying. the most common reasons for 9,uitting were wish of family and fear of an accident. 47.2% conclusioq: short transportation times help to avoid trauma related stress, pain and shock-induced organ complications. therefore the physiologic and economic advantages of hes are undebatable. however, the survey data indicate a considerable concern about safety of the medical personal in a hes. 14 crash landings within less than 10 years with 15 deadcases and 17 badly wounded crew members in a small country like austda make desire for safe flying conditions understandable. obiectives: to evaluate the clinical usefulness of trachlight. methods: trachlight is a new device facilitating endotracheal intubation. a stylet with a lightprobe is inserted into the endotracheal tube. intubation is guided by the light glowing through the neck tissues, thus rendering direct laryngoscopy unnecessary. intubation using trachlight was studied in 37 patients (age 21-68 years). the indication for intubation was elective surgery in 21 patients (asa i-ii) and emergency intubation in 16 patients. in the elective patients, anaesthesia was induced with thiopentone supplemented with fentanyl, and intubation was facilitated with vecuronium. the cause for intubation in the 16 emergency patients was dyspnea in 8, cardiac arrest in 2, trauma in,2 and unconsciousness due to drug overdose or seizures in 4 patients. intubation was facilitated with medication in 12 patients. results: of the elective 21 patients, 19 (91%) were successfully intubated. six patients (29 %) needed two attempts before successful intubation. the duration of intubation exceeded 30 seconds in 8 patients (38 %). of the emergency patients, 14 (88%) were successfully intubated. six patients (38%) needed two attempts, and the duration of intubation was more than 30 seconds in 9 patients (56 %). in 54 % of all 37 patients, intubation was assessed as easy. no or insufficient glow, prolonging intubation or necessitating two attempts, was noted in 11 patients (30 %). oesophageal intubation occurred in 2 patients. conclusions: trachlight may be a valuable adjunct for intubation in varoius settings provided that adequate training is provided. a learning curve was found to exist. objectives: to compare enoxaparin and standard heparin in cavhd and calculate the value of laboratory controls in the treaanent. patients and methods: twenty patients needing dialysis for acute renal failure participated in the study. the main exclusion criteria were massive bleeding or a thrombocyte level < 50 x e9/i. in each treatment the same type (av-400, fresenius ag, germany) of a polysulfone capillary haemofilter was used. the study scheme consisted of two consecutive four-day cavhd treatments, one course for each type of heparin. the order of heparin administration was counterbalanced between patients. the standard heparin was given as a continuous infusion aiming at an activated coagulation time between 200 and 250 s. the initial enoxaparin dose was 80 rag every 8:th hour intravenously, but was modified by any signs of coagulation in the dialysis blood lines or bleeding complications. results: the dialysis treatment was adequate in both treatment modes, with mean blood urea levels 24.3 and 25.2 mmol/l respectively (ns). the bleeding complications were moderate and similar in both treatment modes. the mean life-span of haemofilter using enoxaparin as an anticoagulant was some longer than using heparin (35.7 +31.6 h versus 22.3+26 h, ns). the mean aptt-levcl during heparin treatment was 79s and during enoxaparin treatment 54s (ref. 24 -34s). the mean daily dose of heparin was 422 nag, that of enoxaparin lg7mg. the mean anti-xa activities were 0.40 u/mi and 0.47 u/mi, respectively, reflecting a better bioavallability of enoxaparin. conclusions: both anticoagniation modes were equally effective and well tolerated. the amount of enoxaparin needed for a proper anticoagulation was, however, less than half of that of standard heparin. the changes in aptt level were too slight to make its use possible in controliing the dose of enoxaparin. the use of enoxaparin seems to be rather safe in cavhd even without laboratory controls. the adv~ucea in the management of computerized data of an intensive care unit have been petalled to the clinical advauces and the increasing sophistication of methods of diagnosis fop the clinical application an therapy. this has led our unit to design and develop a computational system called timbu which is used to help physicians assist patients. among its various uses, this system has a software for the hemodynsmic control of a critic patient. this program was carried out to get as fast as possible the hemodynamic data of the patients in an intensive care unit. as an example, we can mention that when we load 17 data obtained through direct measurement from the monitors and the lab, the program calculates 18 parameters that guide, intelligently, to the diagnosis and therapeutic behaviour of the hemodynamic problem through screen messages. the validation of this program in the unit of intensive care has demonstrated that its use allows a more efficient handling of the patient with serious hemodynamics and respiratory disorders. ohieetlve: traema is a heterogeneotm 'disease' that ecatr~ a~"o~s all age ~oupe with v~ying degrees of severity. this imerogeneity has made the di~e, trmma, diflkaflt to r the ehn of this stady wa~ to assr the fitaen of saps in ibis popeleties. methode: in order to compute the ~ probability, a model derived from logistic regression w~ developed. meam'e8 of calibration (goodaess-of-fit stetislj.r and di~'riminafion (roc ou~e) were adopted in developmm~ and validetlon set randomly taken from a database of 10065 pts eeeseemivety admitted in icu (arohidia). ~ witho= salm, p~ yom~ am is yam, with los ~horter thma 24 hotam wore exr fa'om thi~ mmly~ir thi~ model v~s then evahmed on the ~per ~mbgro~ (i.e., trmma pts). if'it did t~t fit the data well ~, new model wm developed rer the logit only on trm=~apm. reims: data were availabte for 8059 pts during aperiod of three .y~m , treama pts were 1156 04.3%), teats of calibration iadioaled probability model did mot provide m adequate refle~on of the mortality ezperieace in pm with ireutae, being the observed mortality lower flma the expected (figm'o). a aew model was then variable. this oastomized model fit~ the de~t of trmara pts very well (g 2=-8a7 p>0.25; roc = 0,94 ). the di:lferencea between the two modele were evident. conclusion: this ltudy shows that mortality in iramna pts is over wcfe~d when ~se~ed by menm of saps. however the r mode! meets high standmcd in terms of calibration mid dil~'iminat'~o~ ']"he advaatage of ~imd models meaas the colleotion of the ~ set of variables for all pm admitted in icu e~einat the ase of diasma specific ~oring syatex~. ("sl"): effects on cardiovascular and hemostasis systems (cvs, hss) a.oborin~ph, ~.~yndiuk~ph, b.kondratsky ~pt. of'""su~gery and transfusiology, research institute of hematology, lvov, ukraine objectives: great interest has been shown recently in the use of hoss for the initial resuscitation of hypovolemic shock. methods: the study was carried out in 6 dogs 1-~h hs was induced by jet momentary hemorrhage (h) from a. femoralls (the bloodloss volume made 29.8+1.9 ml/kg). the treatment was begun after 7.u+o.2 hrs of h. "sl", created on the basis of-sorblt and natrium lactate (1800 mosm/l) was injected into v. femofalls at the dose of io.0 ml/kg. results: it is established that before treatmen-~rterial blood and central venous pressures (abp, cvp) diminished to 30.0 mm hg and -0.6+0.2 cm h20 (p4.o01), while heart rate (hr)-increased to 190.0+9.6 per min (p<.o01). by this the indices of ~latelet counts (pic) and plasma fibrinogen (pf) lowered by 42.2% (p<.i) and 6.4% (p~.05), while fibrin degradation products (fdp) enlarged by 215.6% (p~ .001). after 30-40 min of treatment termination abp and cvp increased to 98.3+6.1 mmhg and 4.1+o.2 cm h20 (p<.o01), and ~[r diminished to t80.0+6.3 per min (p>.5). at the same time the indtces of pic and pf enlarged by 36.4% and 2.6% (p>.i), while fdp diminished by 8.2% (p>.i). one of 6 dogs survived. life duration of the other 5 dogs was 3.9+1.2 hrs. conclusions: the obtained data are ~he evidence of normalizing influence of "sl" on cvs and hss, and allow to recommend it as a mean of initial resuscitation of hs in clinic. oblectives: we prospectively studied 64 icu patients with severe head injury (hi), which cerebral lesions monitorized with sjo2 through opljcal fiber and the cerebral flux with tcd. methods: since january 1990 until june 1994, we collected 152 ht admitted to the icu, and 64 of them monitorized with optical fiber in the right jugular bulb and tcd. all patients needed mechanical ventilation related to gcs <__ 8, with ct in admission (classifing lesions according to marshall and al.) . we related the final results to the evolution of sjo 2 and tcd, with other monitorizing methods like gcs, ct and icp. ~sults: conclusions: in patients with gcs _< 8, sjo2 is useful to evaluate the evolution towards vegetative state, still more in cases with ct type ii in admission and higher apache ill. elevation of icp implies an evolutive nsk to brain death and data of tcd is a good indicator of brain death, the complete monitorization of these patients can improve the therapeutic control of this neurologic problem, , (16m,6f) , (m. age: 39+4 years), divided in two groups (a and b) under specific criteria(tremor and/or fever during admission in i.c.u., or not). the injury severity score was >25 in all studied patients. tbe group a (9 m, 41") had no tremor and/or fever on admisskm, while em group b (tin, 211 the above criteria were ix)sitive. bhx~d samplings were taken 2-9 hours after accident and 10-25 rain. after admisskm in i.c.u. micro-eli~ method was used for measuring cytokinc-levcls. statistic analysis was performed by studcnt-t test. as control group, 25 healthy people were examined. _resu!_ts-il-lct, il-ii~, il-2 and tnf-tt levels were similar to control group levels in both groups a and b. i!,-6 and g-csf levels were found increased in both groups (p<0jxjl), while il-6 levels were statistically significant comparing to group a. in con_tin_skin, during immediate post4raumatic period,proinflamatory cylokines il-i~, il-i~ and tnf.-ct, produced in an earlier stage than 11,.6, cannot be detected,whereas 11.-6 was increased significantly, especially in group b. g-csf was fimnd in increawal levels in both gr(mps, without statistically significant difference between gnmps a and i|. objectives-l~valantc proteolitic activity, disorders in" eariy, period after combined trauma and p(~.ssibilit, i' of their correction by injection of proteo[ysis inhibitors contrycal and s-fto~:nracil in combination with driving an isotonic snlu~ion of sodlum chloride and polig[ucine. methods: biochemicai studies of proteolitic activity in dogs with limited deep burn and acute bloodloss, . result:s: in case of deep 5% burn, cornplicated by bloodshed the of blood grows at 6-7 times. it; is the restdt of the pancreas glandischemi demage, caused by the centralised circulation of blood and intensifies the deviations of haemodiaamics and albumin exchange. the degree of endogene intoxication by mean mofecular peptides which are the products of albumin decay reses to 30%, and 77% in 3 hours. in 3 hours after the trauma the-process is accompanied b3! 59,6% lower inhibitory activity of blood, where as at the peak of the trauma it was 14,5~ higher. that proves the nnfavuurahle process of the shock in case a combined trauma. conclusion: the vein injection of 'proteolysis inhihitotz cnntrycal and 5-fforuraei[ in cumbination with driving an isotonic solution of sodium chloride and p.dligh]cine to refill lhe loss of blood helps to lower at 2 times the profeolitic activity of blood. but it still remains above the initial level. the degree of endogene intoxication lowers at 2 times; [15emodinamics aml albumin exchange stahilised. objectives: nimodipine, a known calcium antagonist, has been shown to dispose a beneficial effect on patients with subarachnoid hemorrhage, but its efficacy on traumatic or spontaneous intracerebral hematoma has not been justified. therefore, we studied the effect of nimodipine on the histopathological changes following an experimental intracerebral haematoma in rabbits. methods: twenty-three new zealand albin rabbits of both sexes, weighing 2-3,5 kgr and at age of 4-6 months were anesthetized and a small burr hold in the left parietal aerea was carried out under aseptic conditions. the dura was opened and 0.1 ml (this volume assuring a normal incranial pressure after kaufman 1985) of autologous blood was injected into a depth of 3 mm via a needle of 0.36 mm bore. the wound was closed and the animals were left to recover. nimodipine, of 2,1 mg/kgr of by weight per day was given via a nasogastric tube to fifteen animals for a period of time of fifteen days (group b). six rabbits were given water and served as control (group a). both groups of animals weie sacrified on the fifteenth day, their brains were removed and immersed into 10% formalin solution. tissue sections of 5 ~ were embedded into paraphin and stained with haematoxyline and eosin, mason and gfap stain for gliac cells. results: two animals died after the surgical procedure, because they developed large intracerebral bematoma. no animal developed neurological deficit except one of group a which manifested a right side hemiparesis. the results of the bistopathological changes are the following: i) the mean -+ sd diameter of the lesions in the group a was 260 --. 26 ~t while that of group b was 76 + 12 ~t (p<0,01) ii) secondary ischaemic neural tissue changes, characterized by the extravasatlon of red cells, the presence of haemosiderin-containing macrophages and signs of low grade inflammation zpredominated in the specimens of group a and were totaly absent from those of group b. iii) a ring of gliac hyperplasia and a low grade local fibrosis was found, encircling the lesions in the specimens of group a in contrast to those of group b. conclusions: nimodipine when administered in rabbits following the development of a non increasing the icp experimental intracerebral haematoma, prevents the extention and the severity of the lesion. objectives: to study the efficacy and side effects of adding intramuscular clonidine (clophelinum) to analgesic regimen in early management of patients with serious burn injury. methods: 20 pts with 20-40% bsa second to third degree flame burns (respiratory tact injury excluded) 19 to 61 yrs of age were randomised to study (n=10) and control (n=10) groups. burn shock was treated with hypertonic saline -bicarbonate solutions (250 mmol/l na +) 2ml/kg/%bsa for the first 24 hours and 1 ml/kg/%bsa for second day. analgesia in control group for the first 48 hours was provided by regular 6 hourly intramuscular administration of 20 mg of morphine sulphate and 500 mg of analgesic -antipyretic analgin with 10 mg of diphenhydramine (dimedrol). from the 3rd day regular administration of morphine was finished. in the study group 100 ixg of clonidine was added 8-hourly for 72 hours and dose of morphine halved. vas, verbal rating scale for sedation (vrs, 1 -5), sleeping time, spo2, hr, bp, diuresis, vomiting and other complications were comparatively evaluated during patients' stay in icu. results: addition of 300 ~g of intramuscular clonidine daily allowed to achieve better analgesia and sedation with halved consumption of morphine. mean vrs in study group for the first 3 days was 3.1 -3.3 vs 1.3 -1.7 in control group with twice longer sleeping time. there was significantly less tachycardia in study group; dynamics of bp for the first 24 hours did not differ considerably; later, there, was tendency for hypotension in study group without adverse effects on diuresis or other indices of tissue perfusion. because of high incidence of chronic ethanol abuse among study population 7 pts of control group suffered from psychomotor agitation or delirium, probably as a sign of alcohol withdrawal syndrome (aws). this made regular evaluation of vas impossible. in the study group only 1 pt showed sign of aws. mean vas score was in 2.4 -2.9 range for first 3 postburn days. 7 pts appeared excessively drowsy due to clonidine, but it had no adverse effect on their overall clinical course. mean spo 2 values in study group were in 95 -98 % range, among controls 90 -95 %; vomiting was absent in. cionidine group vs 4 cases among controls conclusions: clonidine could be a valuable addition to analgesic -sedative regimen in burns, especially for prevention of aws and deserves further study in this regard. hemodialysis -hemoflltration modifications and/or intratracheal gas insuflation have been recently used for blood gas exchange in several models of respiratory failure. objectives: evaluate the combination of cavh-m and igi for respiratory support in experimental acute lung injury. methods: five mongrel dogs (22-+4kgr) were mechanically ventilated inroom air, paralysed, heparinized, connected with a cavh-m system (diafilter-30 polysulphone membrane) and remained stable for one hour (pao~= 98.8• peco2=34-+8mmhg, ph=7.37-+0.07, bp=137-+12mmhg and pap=15-+2mmhg). all was induced two hours after oleic acid infusion (0.07ml/kgr) into the pulmonary artery (poo~=46.6_+6 -p<0.001, paco~-50.2_+10 -p<0.05, ph=7.10-+0.10 -p<0.01, bp=158-+25 -p=ns, and pap=29_+5 -p<0.01). fio 2 70% for the next 30 minutes did not significantly altered the b3ood gas abnormalities. afterwards, pure oxygen applied simultaneously a) through the inlet of the filtrate's compartment of the hemofilter (2l/min) while filtrate and gas were removed from the outlet port (bypass flow 220 ml/min) b) through a thin intratracheal catheter positioned 2cm above the carina (4l/min). the fio 2 given through the ventilator readjusted to 21%. results replacement fluids/filtrate during the next four hours were not exceed 0.7l/hour, whilst the blood gases and pressures were improved as follow: cavh-inlet:pao.=88. objective. to compare the changes in humoral immunity in trauma patients following massive transfusion of autologous and homologous blood. methods. we studied 3 randomised clinical groups of patients each containing 24 patients with trauma and operation of large arterial vessels. the amount of autologous or homologous blood transfused to the patients was exceeding 1 500 ml, while the patients in the control group did not recieve blood or blood products. results. we recorded most pronounced and characteristic changes on the 1-st and on the 7-th day in the group of patients recieving homologous blood transfusion, i.e. decreased amount of igg,iga,igm,c5 and c4 fractions of the complement system, haptoglobin and significant and sustained rise of circulating immune complexes up to the end of the study period. in the control group of patients the decrease was weaker and lasted only during the 1-st post-operative day; the dynamics of the circulating immune complexes level were almost the same as in the first group of patients. in the group of patients recieving autologous blood transfusion, the parameter values did not change significantly from preexisting levels after the 1-st day, while on the 7-th and on the 30-th day showed a tendency towards aslight rise. conclusions. autologous blood has a favourable effect upon humoral immunity and should be the transfusion medium of choice in cases where autologous blood reinfusion is technically possible. ivan petkov, m.d., rumen farashev, m.d. and dimitar terziiski, m. d. medicine, military medical academy, 3 g. sofiiski str.,1606 sofia, bulgaria objective. the amount of blood lost during trauma and operation could hardly be forseen and donor blood supplies are not always available in sufficient amounts. rare blood group types and/or unexpected haemorrhage pose a great challenge to the transfusion therapy and the methods of intraoperative autologous blood transfusion. methods. we report a case of a 18-year old male patient with extremely massive intraabdominal haemorrhage ( 7 300 m( blood loss ) during an abdominal aorta reconstruction following a traumatic injury of the abdominal aorta. we achieved a successful reinfusion of 6 600 ml of autologous blood using an original autotransfusion system developed by us ( pat. no 95311/ 11.10.1991) . results and conclusions. the autotogous blood in the case reported here was the only and the most suitable transfusion medium for the rapid intraoperative compensation of the acute haemorrhage and the favourable outcome of the patient. the post-operative period was smooth and no significant disorders in the clinical course as well as in the laboratory tests ( morphological,biochemical,coagulation and immunological) were recorded. there were no complications during the postoperative period despite the fact that the amount of blood reinfused to the patient was slightly exceeding his own volume of circulating blood. objective. the haemoglobin concentration and the perfusion pressure value could not be the only criteria for the early signs of tissue and organ dysfunction. because of this, we employed the extensive monitoring of oxygen transport during severe trauma in order to. achieve dynamic evaluation of physiologic compensatory mechanisms and to assess the efficacy of intensive care management. methods. we conducted a prospective controlled trial on the blood oxygenation, oxygen transport and tissue perfusion during the first 3 days after the trauma in 20 patients with polytrauma. we used a swan -ganz pulmonary artery catheter (beckton -dickinson, u.s.a.), deseret 1000 cardiac output computer (medical inc., u.s.a.) and hewlett -packard monitor (hewlett -packard, germany) to measure and calculate all the parameter values. the severity of the injury was assessed using the apache ii score system. all the patients had scores over 18. results. the results show a significant decrease in the arterial blood oxygen content and in the arterio-venous difference, as well as an increase in alveolo-arterial oxygen difference and in the transpulmonary right-to-left shunt. the tissue oxygen supply and the tissue oxygen consumption reveal a tendency towards a decrease below the physiologic minimum of adeqate values. the erythrocyte current velocity and the ratio between oxygen transport and erythrocyte current velocity also decrease inspite of the optimal blood rheology. conclusions. the dynamics in the parameters values are most pronounced between the 2-nd and the 18-th hr after trauma, which predisposes patients to the risk of developing stable hypoxemia and characterizes this period as the most critical for tissue metabolism and organ dysfunction. posttraumatic changes in immune mechanisms in lung compartment in trauma were analyzed in ao and da inbred strains of rats which differ in their immunological reactivity: the former being low responder and lat-~er hiperresponsive. methods: the levels of tnf-alpha activity in the 24 supernatants of cultured lung lobes and dynamics of cells migration from tissue explants in 6h lung cultures were assessed in ao and da rats subject ted to severe burn trauma. results: increased levels of tnf activity (160+3 pg/ml compared to 50+4.9 pg/ml in control) were found od day 3 following trauma in lung sups of ao rats while no changes in the levels of activity of this cytokine were found in lung-sups od da rats more pronounced extent and dynamics of cell emigration were noted in da rats, while almost unchanged in ao rats sharp rise in pmn percentages 3h following trauma (60-70% compared to rare pmns in control), followed by increase in lymphocyte numbers at later time points among lung cell emigrants was detected in ao rats. slower but persistent increase (25%, 3h following trauma and 60% and 50% on days 1 and 3 after trauma infliction, respectively) in pmn numbers among da lung cell emigrants was detected, which appeared to be activated, as judged by their nbt reduction capacity. increased percentages of peripheral blood pmns and increased state of leukocyte aggregation/adhesion were detected in both strains, but different levels of plasma tnf: increased levels in ao rats on days 1 and 3 following trauma, and initially but persistently high levels of plasma tnf alpha in da rats (4-5 fold higher compared to initial levels in ao rats). conclusions:different patterns of local (lung) and systemic changes in cell numbers and cytokine levels implicate differential posttraumatic migratory capacity of pmns vs. lymphocytes in lungs in ao and da rats. early diagnosis of acute intestinal ischemia by color doppler sonography e. danse, b.van beers, p.goffette, f.hammer,aav.dardenne, f.thys, p-f.laterre, m,s. reynaert, .lpringot dept of radiology (profb.maldague) and dept of intensive care ( prof m,s.reynaert), st.luc univ.hospital, brussels, belgium ob emergeny medical squad service is the most important segment in the process of saving the people, in the cases of mass accidents, like industrial accidents caused by the: explosion, fire, chemical poisoning, traffic accident, elemental catastrophes and the war. because of that, each emergency medical squad service needs to have in its motor-pool vehicle for the mass accidents/ for provoding at least 100 people, wounded as well as the people became ill/. objectives: presentation of such special vehicle, produced by "zastava-kamioni" and it's medical-technical equipment. methods: descriptive and comparative analysis of the medical and technical characteristics, based on the actual norms/din, 75080, iso 9001, yus.../ results: on the base of doctrinaired requirements of the emergency medical squad in the case of mass accidents, our researches resulted in the following medical and technical characteristics -the vehicles for mass accidents are gvw/with a payload off cca 5-8t, with the fixed, closed body, type: universal van, -technical equipment aggregates, stretches, anti-fire device, equipment for pitching the tent and for maintaing technical conditions of the work -medical equipment: linen bags with complete sets of bandage material, means for the reanimation and immobilization, for the infusion, medical instruments and remedies as well as the tent for lodging at least 50 wounded and sik people. in federal republic yugoslavia, it was proposed 24 such vehicles for the emergency medical squad needs. conclusion: we suggest to introduce this vehicle in the production range of the ambulance vehicles for saving, especially in the circles where can occur serious accidents. introduction : carbon monoxide (co) poisoning commonly generates central nervous system abnormalities though an important cardiac morbidity and mortality must be considered. long-term exposure to co with cohb levels < 30% may be more dangerous than short-term levels of 45-50%. we report a case of an adolescent who after prolonged exposure to co developed a severe reversible cardiac dysfunction with low levels of bloed cohe c a.ase history : a 15 year old boy was found comatose at home. his mother in the neighbouring bathroom died severn hours earlier of what was later proven to be a co intoxication. on arrival the gcs was 8/15 and the patient was breathing spontaneously. a postictal status with eventual postanoxic encephalopathy was suspected. a coh'b level of 10% was objectivated. the cardiorespiratory situation quickly deteriorated requiring mechanical ventilation. chest x-ray showed diffuse bilateral patchy infiltrates. ecg revealed signs of ischemia. severe left ventricular dysfunction was evidenced by pulmonary artery catheterisation and echecardiography and later by isotopic angiography (lvef 20%). treatment was intensified with inotropic support, intta-aortic balloon counterpulsation and oxygen therapy. the clinical course was further complicated by a crush syndrome and renal failure. the patient's condition gradually improved and he fully recovered without any residual lesions (lwf 80 %) conclusion : even after prolonged exposure cohb levels can be misleadingly low. high tissue levels of accumulated co can be associated with coma and fulminant cardiorespiratory failure requiring advanced life support facilities. introduction : both neuroleptics (nlp) and tricyclic antidepressive agents (tca) can induce arrhythmias, prolongation of the qt segment and the pr interval and hypotension. we report a case illustrating that combined overdose of these agents increases the toxicity of each compound and the risk for adverse cardiac events. .c, gse history : a 44 year old male ingested 2500 mg doxepin (sinequanr), a tca and 3500 mg prothipendyl (dominalr), a potent nlp in an attempted suicide. upon arrival in the emergency department the patient was unconscious (gcs 6/15), breathing superficially, and presenting signs of recent vomiting. physical examination revealed a taehycardia of 140 b.p.m., an arterial blood pressure of 90/70 mmh4g. ecg showed a brood qrs complex tachycardia. a chest x-ray revealed the presence of an aspiration pneumonia. laboratory investigation demonstrated increased levels of crcatine phosphokinase, lactate dehydrogenase and aspartate transaminase ; hyperglycemia and leucocytosis were present. the plasma concentrations of doxepin and prothipendyl were respectively 410 gg/l (toxic level 5130 #g/l) and 3900 i.tg/l (no reference). treatment consisted of mechanical ventilation, gaslric lavage and administration of activated charcoal and iv fluids and antibiotics. a hemodynamically well tolerated veatricular tachycardia developed 1 1/2 h later. nahco3 (250 meq/24 h) was administrated inducing an ectopic atrial tachycardia with a normal qrs complex and prolonged qt. 8 h after admission a normal sinus rhythm was present; the prolongation of the qt segment persisted for 2 days. the patient fully recovered. conclusion : the treatment with nahco~, alkalizing the blood and thus increasing the protein binding of the tricyclic antidepressant molecule, can readily correct the potentially life-threatening cardiac arrhythmias and therefore should be part of the routine treatment of combined tca-nlp overdose. ob/ectives: the development of diabetes insipidus (di) in patients with brain injury is a known negative prognostic sign. the aim of this study was to investigate whether this is also a reliable early prognostic sign of brain death. methods: this is a retrospective study of 85 patients treated" during a two year period (1-3-1992 to 1-3-1995) in our i.c.u who meeted the following criteria: (1) coma score _< 8 gcs within the first 24 hours, (2) positive brain ct scan on admission classified according to marshall's diagnostic classification (classes 1-6), (3) normal renal function during the entire icu stay. for the definition of di were used the usual di criteria plus hypematriaemia (serum na" >_ 155 meq/l). survival was defined up to the 30th postadmission day. conclusions: according to the findings of this study, the development of diabetes insipidus in brain injured patients seems to be a highly specific index for brain death (positive predictive value = 0.95). however, further prospective studies are needed for the definitive evaluation of these findings in such patients. emergency care in italy, despite all efforts, is still lacking a nationwide organized prehospital care system and, until today, there are only different regional solutions. the majority of these realities imply rather simple ambulance first-aid services without attending emergency physicians and without resuscitation equipment. the emergency medical service (ems) system in falconara m., italy, was implemented in august 1994 by a collaboration between the school of anesthesiology and intensive care of the university of ancona and the, already existing, volunteer rescuer organisation "yellow cross". according to the guidelines pubblished in 1992 [1] the pre-existing equipment of the volunteers was completed with type a ambulances and 1 special equiped motorcar (patient monitor, defibrillator) for ambulance indipendent physician transpur[. a special data collecting schedule was created to memorise every emergency intervention in a computerised data-base. the intraining members of the school of anesthesiology and intensive care provide 24 hour ready intervention. in this report the authors describe their experience concerning primary firstaid medical interventions. for a preliminary evaluation we considered, retrospectively, 300 consecutive emergency interventions in the time period from novembre 1, 1994 to april 30, 1995. the emergency physicians treated 131 male (44 %) and 169 female (56%) patients, 15 patients died before hospital admission and 75 patients (25%) were treated at home by the ambulance indipendent physician and did not need any further medical treatment. in the same time period 1 year earlier (november 1993 to april 1994) without attending physician the volunteer rescuers transferred all 257 first-aid interventions to near-by hospitals. we conclude that the presence of an attending, iudipendently motorised physician in emergency interventions is essential for the establishment of precise priorities and may be helpful to reduce hospital admissions by ambulance intervention, though reducing primary" health care costs. we have developed the method of liquor filtration which allows to purify the cerebrospinal liquor from blood and its decay products in the subarachnoid bloodstroke. the hemipermeable dialysis membrane was used as a filter, which lets only in water, electrolytes and substances with small molecular weight. the liquor filtration was used for the treatment of 19 patients with the subarachnoid bloodstrokes of different etiology. the perfusion of liquor was performed at the rate 3 ml/min in the recirculatory mode. its duration was 180 -240 min depending on the bloodstroke intensity. the filtration makes possible the most completely purifying of the hemorragic liquor, the reducing of the content of blood ceils and its decay products 80 -300 times as less. the monitoring of the patient's state during the perfusion didn't revealed the departure from the norm of the main vital part. the liquor filtration technique compares favo-~ rsbly with the routine method of cleaning by the absence of toxical effect of heterogenous solutions on the central nervous system. the filtrstion of the cerebrospinal liquor in the subarachnoid bloodstroke sllows to provide the the early cleaning of liqour, the regression of meningeal syndrome and to improve the patient's state of health. e3tabli~mczr 3bd ~ of rei~idnal medical first-aid zhoulittoing, ed., tan zi, m.d. dept. of sargery, the first teaching t[ospitat, 29 yejin-l)a-l)ao, wuhan 430080 fltlna objectives: the medical first-aid is the most important task of the public hc atth department. in general, single hospital model couldn't fatty, effective ly rescue mony severe patients who need mergant treatment in the scene. bub establishing the medical first-aid network, the severe patients can be given the most timely und the most scientific emergent treatment. so that, the suc cessfut rate of the saving wilt be greatly increased. methods..; our hospital is a general big hospital. through developing and cons tructlng for more than ten years, the medical first-aid network distributed art over the area under our jurisdiction has been set up. it consists of thr ee units: the medical first-aid unib center comartd and mnagment unit, co m~nlcation and tiaison unit. the principle of the network operation is with oat having to 90 far to mergoncy, specialized emergency and the best merge acy. results: the results of the network operation were notable. cmpari~ the to tat successful rate of the saving (91.6~), the successful rate of saving tra ma (93.~), the suscessfut rate of saving shock (98.~) and the successful rate of cardioputmonary resuscitation (52.4~) daring the three years after t he network operated with these before (86.6~), (91]. 4~), (92. ~) and (4ft. 5~), the successful rates after operating were remrk~iy higher ( p<i).o5 ). conctusions~ the above results showed estabiishmnt and mena0emont of region at medical first-aid network had very si~nificont action during mergentty s avin9 the severe patients. moreover, the regi~at medical first-aid network atso take an important part in prevention and health protection of the mass. objectives: se related mortality is due to the occurrence of both rv and lv dysfunction evoking cardiogenic shock and pulmonary edema (f. abroug, intensive care med 1995; s. nouira. chest 1995) . prospective evaluation of scorpion antivenon (sav), the only available specific treatment, is lacking. the aim of this study is to assess the effects of sav on the basis of a case-control study. methods:among 600 consecutive victims of se presenting to the emergency department, 135 were managed using sav (group sav+). these patients were matched to 135 envenomated patients who did not receive sav (group savchest injuries are the cause of death in 25% of trauma fatalities and a major contributing factor in an additional 50%. pneumothorax is a major complication of chest injuries and can be initially overlooked bringing to additional morbidity and mortality especially in patients who require mechanical ventilation. the real incidence of pneumothorax in severe blunt chest trauma hasn't yet been established, as many pneumothorax can be missed on the initial chest x-ray (cxr) and computed tomography (ct) has not been routinely used. to evaluate the incidence of pneumothorax in patients with severe blunt chest trauma. methods: a prospective study. from january 1 1993 to december 31 1994, all trauma patients with one of the followings conditions: fractures of four or more ribs, signs of lung contusion on the initial cxr or presenting a severe hypoxemia on admission (pao2/fio2 < 200) in the absence of pre-existing pathologies, were evaluated by initial cxr and subsequently submitted routinely to a ct. results: 52 severe trauma patients were considered. 8 of them, presenting a severe hypoxemia on admission, had no evidence of major chest trauma (ais<3). hypoxemia was the consequence of severe trauma in other districts; these patients were therefore excluded. 44 patients with severe thoracic trauma (ais>=3) were admitted into the study. the mean iss was 36.2 (16-75). thirty-six patients required artificial ventilation for at least 24 hours during the icu slay. three of them, who had a tension pneumothorax, were submitted to an emergency thoracic decompression on the field by the emergency helicopter team. in 7 cases pneumothorax was diagnosed an the initial cxr 14 more patients had a pnx which was identified only on the ct. in 4 cases a large pnx with lung collapse was missed on the cxr. in our group of severe blunt trauma patients, 54% (24/44) presented a pnx that required the insertion of a thoracic drainage. only one third (7/21) of the pneumothorax could be recognised on the initial cxr, while other 3 were decompressed before performing the cxr. as many as 58% of the cases of clinically significant pnx were missed on the cxr, and a ct performed soon after admission allowed an early diagnosis bringing to changes in the treatment. (as the patients were mechanically ventilated a chest tube was inserted in all these cases). in 4 cases, the initial cxr overlooked a huge tended pnx which was the cause of hemodynamie instability. conclusion: in patients with severe blunt chest trauma even large pnx can be missed on the initial cxr. moreover due to the non compliant compressible lung, a 20% pneumothorax which can be recegnised only on a ct, can bring to high intrapleural pressure altering eardiopulmonary function. n. andoeli6, 0.~osid, m.zesevid, m.risovid, d.stepi6, d.djokid b~rga~yc~qterclinicalcaqterafserbia, belgrade cb~ctives:~lis study ~ the use of ~rq]ofol earbired with k~t~ine (aq a~sjgh~ic s@~qt widn inirjrsic armlgesic pro~mities) or with fsqtmtyl,with psrtial azgmsis an hgenxlyn-a~ic ~ durirg ~ ~ re:~ver~ f~m ~ in hxh ~ of ~ti~. ~: yali~mial and ~bod:30 a~it p~tie~ts a~ i-ii were included in ibis shxly. patients were rsrd]nly dieided in two ~ns. all d~tie~ts ~me given 5-5 prcpofol bolus doses (o,5 ~gkg) for ird~iqn of ~. ~ia ~s m~sjn~ with an infusion 6 ~ ~ropafol. as sdflitianal were given fan-i~l (o,2 n]g) ~tely before ~ anj trad~e~ irfojoation followad by feasted bolus of o,i mg in ~ro4o l.patients in gr~4o 2 received i~ (an initial bolus dose of 35 rg slowly intcavax~ 8rd 25 mg as infusion over ~0 rain) .infusions of pro~fol or imcpofol with kg~mine ~ stopfsj 10-15 rain ]:~o~ extuhation.arterial blood ~ (sistolic arterial blood preassu-re~zap,mean ~rterial blood pr~,d~lic arterial preassure-[zp a~ h~art rate-~) ~ m~ before induction of a~ io, 30 snd 60 rain aftem ~ intutation. results: arterial blood preasstre ~s decreases duri~ irn~ction of sn~wd~sia in hy~ ~n~s,tnt mare in th~ ~ who r~eived fsqtanyl.~ere w~s statisticslly sifnific~ntly difemerme dmir~ m~ of an~ia. arterial blood r~easatre and heart rate were stable in the t-..e~min 9-~a4~. all th~,fl-e keta'nire grcqo hsd e~rly :~e~y time. ctrmlusi~s: ~e ombiretion of protxfol wilh keta/ne for irduorion a~d ~ of sn~sd~esis w~s yell accept~ by p~tierfcs anj coald he ~ as an alterrstive ~o ccnva~icrsl a~es -d~sia. objectives : assess the relation between cytokine or endotoxin release and indices of splanchnic malperfasion after hemorragic shock in multiple trauma patients. ]~r study was approved by the local ethical committee. trauma patients admitted to the emergency room who met the entrance criteria of more than 1 hour map < 60 mmhg or use of vasoactive agents or blood lactates > 5 mmol/1 were selected for study. a nasogastric tonometer (tonometrics, inc, plastimed, france) and a swan ganz catheter were placed on admission. phi, lactates, hemodynamics, plasma cytokine and endotoxin concentrations were measured on admission and at 3.6, 12, 24, 48 hrs. an immunoradiometric assay was used to determine plasma concentrations of il6 (n<0.03ng/ml) and tnfc~ (n<5pg/ml). plasma endotoxin concentrations were measured using a chromogenic limulus assay (n<0.1eu/ml)(1 endotoxine unit= 100pg). results : 9 severe multiple trauma patients (age = 42_+18 yrs, iss = 40-!-_15, saps = 19+'~, mean-+sd) were studied. they received 15+6 packed red cells during the first 24h. mean duration of collapsus before inclusion was 5.1_+2.9 hrs. death occm'red in ~tients. ~ pglml, *: ng/ml, etox : endotoxin(eu/ml), lact: lactate (retool/l) a significant correlation between initial il6 level and saps was observed. in the early post-injury period phi, sao2, svo2, vo2 were significantly associated with ;il6 release (p<0.05 at ho, h3, h6). later a significant correlation existed between lactates and ii6 (h6, h24). a peak of tnf was detected at 24 and 48 hrs. it was associated with low phi and low arterial ph of the early post-injury period (p<0.05 iat ho, h3, h6 ,h12, h24) and with high lactate levels of later period (_>h12). only the late release of endotoxins (i{48) was correlated significantly with initial !oxygea-delivered parameters. iconclusion : there was a marked increase in il6 in the early phase of trauma . i16 and tnf release after major trauma iwith hemorragic shock is associated with splanchnic malperfusion, as assess by the ivery low values of phi. lactates seem to be a later indice. toxic effects are a well-known complication of an overdosage of prescription theophylline. what is less known is that over-the-counter (otc) asthma medications contain theophylline, and that in some cases this might cause toxic effects. a case seen by us involved toxic effects from theophylline in an otc medication and to date is the only published case in the english literaturet the rationale for this study was to delineate the otc products containing theophylline from whatever data sources available. hyperthermia frequently occurs in intensive care treated patients and intentional application of whole body hyperthermia together with chemotherapy is a therapeutical access to treatment of malignant disorders. anaesthetic support is required in either condition. due to the marked decrease in systemic vascular resistance seen in hyperthermia an additional vasodilatory effect of the anaesthetic is unwanted. the vascular effects of anaesthetics in hypertherm organisms is not known in detail. therefore, we performed an experimental study to detect the effects of inhalational anaesthetics in whole body hyperthermia. in 30 sprague-dawley-rats katheters were inserted into trachea, jugular vein, and carotid artery. for continuous monitoring of cardiac output a flow probe was placed around the aortic arch. the rats were mechanically ventilated with different concentrations of inhalational agents in oxygen. we compared the effects of enflurane, isoflurane, and halothane in stepwise increased body temperature by submerging in a temperature controlled water bath. results: isoflurane lowers arterial pressure more than halothane or enflurane. the inhalational anaesthetics lower the cardiac output similarily and independently of temperature. isoflurane decreases systemic vascular resistance independently of core temperature and the decreasing effect of halothane on the resistance is completely abolished in hyperthermia. conclusions: the influence of hyperthermia on the systemic vascular resistance is dangerous. this allows no additional effect of the anaesthetic management. in spite of the vasodilating effect of inhalational agents in normotherm subjects, this effect is abolished in hypertherms using halothane. the condition of management of analgosedation in hyperthermia is different from normothermia. objectives: to evaluate a bedside computer processed cerebral function monitor for assessment of brain wave activity when clinical/visual clues are not present. methods: ten icu patients undergoing neuromuscular blockade monitored with the aspect 1000 brain wave monitor from january 1 to june 1, 1995. results: time to onset and depth of sedation were readily apparent to icu physicians not specifically trained in eeg reading. objectives: to determine whether non-depolarising neuromuscular blockade reduces oxygen consumption (vo2) in sedated, apnoeic patients. methods: haemedynamic. metabolic and oxygen transport variables were determined in 5 sedated, apnoeic patients with severe acute lung injury. all patients were ventilated using a puritan-bennett 7200ae ventilator with integrated 7250 metabolic monitor. inclusion criteria were; 1) stable cardiorespirator s" status; 2) systemic and pulmonary artery catheters already in situ; 3) inspired oxygen < 80%. patients were sedated with midazolam or propofol to abolish response to verbal stimuli, and sufficient morphine or alfentanil to abolish all spontaneous respiratory efforts. following baseline measurements, neuromuscular blockade was induced with intravenous vecuronium, 150 ug/kg, followed by an infusion of 80 ug/kg/h to maintain the train-of-four ratio at 0. a further four sets of measured and calculated variables were obtained at 20 min intervals. results: statistical analysis was by repeated measures anova. there were no significant changes in any variable over time. the changes in calculated oxygen consumption (vo2fick) , and measured oxygen consumption (vo2gas), and in energy expenditure (ee), are shown in the table. objetive: to study the effects on coronary hemodyrtamics and myocardiai metabolism of administering propofol during postoperation sedation of patients with normal coronary circulation and good ventricular function undergoing cardiac surgery. patients and methods: 18 patients (12 women and 6 men) undergoing aortic and/or mi~-a/ valvular cardiac surgery were selected, with an ejection fraction greater than 0.5 and normal coronary circulation. for postoperation sedation propofol was administered in 0.5 mg/kg i.v. bolus, followed by a 2.2 mg/kgth perfusion. all data were registered before administering propofol and after 20 minutes, the patients being hemodynamically stable and a rectal temperature of 34 _+ 0.5 -~ systemic and pulmonary hemodynamics, and global, as well as regional myocardial blood flow, and metabofic variables were measured. results: the patients studied were about 56 years old, and the average period of aortic cross-clamp was 77.50 min. the adminstering of propofol caused a decrease in the coronary blood flow (-9%), great curonary vein flow (-23%), myocardial oxygen consumption (-14%), regional myocardial oxygen constanption (-11%), myocardial oxygen extraction (-6%), regional myocardial ooxygen extraction (-10%), while coronary vascular resistances and global coronary vascular resistances did not change. oxygen saturation increased in the coronary sinus (+16%) as well as in the great cardiac vein (+32%). in no patient were significant changes suggestive of myocardial ischemia objectified. there was also found a decrease in systolic (-23%), diastolic (-20%) and mean (-25%) arterial pressure, systemic vascular resistance (-20%), and cardiac output (-8%). conclusions: in accordance with the clinical conditions of this study, the administering of propofol is not likely to cause changes in coronary autoregulation, oxygenation and myocardial metabolism. obietive: analyse the effects of 0.4% "end tidal" isoflurane (sedative dosage) on the metabolism and coronary hemodynamics during the postoperation period of patients undergoing cardiac surgery. patients and methods: 16 patients (12 women and 4 men) undergoing aortic and/or mitral valvular cardiac surgery, with an ejection fraction greater than 0.5 and normal coronary anatomy, were selected. after the surgical operation, 0.4 "end tidal" isoflurane was administered for postoperadon sedation. the determination of variables to be studied was carried out before and 20 minutes after administering isoflurane, die patients being hemodynamically stable and a rectal temperature of 34 _+ 0.5 -+c. systemic and pulmonary hemodynamics, and global, as well as regional myocardial blood flow, and metabolic variables were measured. results: the average age of the patients studied was 57 83 -+ 8.87 years. during surgical operation the period of aortic cross-clamp was 78.56 _+ 32.09 rain. the administering of isoflurane was followed by a statistically significant drop in coronary perfusion pressure (-26%), coronary vascular resistance (-29%), regional coronary vascular resistance (-29 %), regional myocardial oxygen consumption (-7%), regional myocardial oxygen extraction (-6%) and accompanied by a significant rise in oxygen saturation in the coronary sinus (+16%) and in the great cardiac vein (+32%). myocardial oxygen consumption, myocardial exu'action of lactate and regional myocardial lactate extraction did not change. in no patient were enzyme or electrocardiograph changes objectified. systolic (-23%), diastolic (-25 %), mean (-25 % ) arterial pressure, and systemic vascular resistances (-28%) decreased, while cardiac output did not. discussion: the administering of 0.4% "end ddal" isoflurane, in the clinical conditions of this study, produced a decrease in systemic arterial pressure due to a reduction of systemic vascular resistance without deteriorate cardiac output. at coronary circulation level, has and effect on coronary autoregulation but had no effect on oxygenation and myocardial metabolism. the idea of tiva implies the realisation of major anesthesia components (los of consciousness, neurovegetative inhibition, analgesia, myorelaxatiou, providing the adequate gas-exchange) through i.v. introduction of drugs exclasively. aim: providing for the main tiva components with minimal side effects of the drugs used, taking into consideration the patients characteristics and the surgery specific character. methods: 78 anaesthesias have been conducted in patients aged 15 75 years (28 females, 50 males), undergoing planned and urgent operations with the pathology of lower, extremities, perinaeum, small pelvis, hypogastrium and with reserved spontaneus respiration against a background of 100 % 02 insnffladon through mask. operations lasted from 0.5-1.5 h. anaesthesia adequacy was assested by constant monitoring: "cardiocap" (nibr hr, rr, sao2, t), through glykhaemia level and mimicry reactions. standart premedicatioo of m-cholinolytics (0.01 mg/kg) and h2-blockers (0.3 mg/kg) on the operational table was sumplemented by administration of 0.5-1.0 mg/kg of lidocaine, 1.50.0 mkg/kg of clonidine, 0.5-1.0 mg/kg of pentamidine by the tachifilaxia method. the premedication adequacy was assessed through haemodynamics characteristics. sedation: 0.05-0.1 mg/kg of droperidoi, 0.l-0.15 mglkg of diazepam and analgesia: 2-3 mkg/kg of phentanyl, 1.0--1.5 mg/kg of ketamine were introduced fractionally according to indications. infusion rate of ringer-lactat solution was 5-15 ml/kg/h and depended on the intraoperational blood loss volume and on the patients preoperational condition. the duration of postoperative analgesia was registered. results: clinical assessment of analgesia according to this techniques allowed to decrease the anaigetics dosage to the subauaesthetic levels. smooth stabilisation of haemodynamics (bp) at proper age norms in patients with the initial hypertension by the 30-th min. of anaesthesia as well as the absence of its increase in response to the additional introduction of anaesthetic have been achieved. (hr) had no abrupt changes and remained in the range of 70-80 per rain. adequate external breathing: decrease (rr) by 2-3 per rain., with sao2 increase from 9446 % to 98-100 %. hypoventilation was avoided by respirate ventilator. according to unauthentic data the glykhaemia level had been lowered by 10-t5 % to the end of the operation with the initial moderate hyperglykhaemia of up to 10 mmol/l the cutaneous covering grew warm and got pink colouring. no mimicry reactions. in the postoperative period patients were in the superficial sleep state (3-48) and analgesia lasted 6-8 b. there were no complications due to anaesthesia. conclusion: combined using of bz, opiates, neuroleptics potentiate the i.v. anaesthetics effects allowing lowering of each tiva component dosage and, as a consequence avoiding their negative influence on respiratory and heart vascular systems. complex application of adrenergetics (therapeutic doses of cionidine and pentamini with using of taehfilaxy effects) permitted to provide for analgetic and neurovegetative components of general anaesthesia under subanacsthetic doses of tiva main components, and manifestation of hyperdynamic reactions of haemodynamics decreased while using of lidocaine -the economicai activity of heart-vascular system. good level of muscle relaxation was achieved allowing for widening of surgical intervention extent without respirator ventilators and inhalation anaesthetics application. anaesthesia is easily controlled due to fractional introduction of drugs with quick recovery of cns functions after anaesthesia. postanaesthetic analgesia is increased while concurrent opiates doses are decreased. absence of marced haemodynamic, endocrine and metabolic reactions during the operation and after it resulted in shortening the period of patients staying in hospital. a 64 yo white man was admitted to hospital for dyspnea and a productive cough. he had cabg in past, but no recent cardiac ischemia. physical exam: decreased breath sounds over right lung. chest xray: consolidation of right lung. admission medications included diltiazem, furosemide (both were continued) and trazodone (which was discontinued). admission ecg: sinus rhythm, qt 0.44/qtc 0.49 sec, with st and t wave abnormalities similar to prior tracings. he required intubation and mechanical ventilation for progressive hypoventilation and hypoxemia. between icu days 8 and 16 he received haloperidol, 10-44 mg/d (cumulative dose 209 rag) for agitation and delirium. icu day 11: qt 0.46/qtc 0.57 sec. icu day 12: for better control of delirium, trazodone " 50 mg q hs was added. icu day 15: he developed frequent nonsustained ventdcular ectopy. icu day 16: qt 0.70/qtc 0.74 sec, pha 7.48, paco2 50 mm hg, pao2 72 mm hg, k 4.9 meq/l, mg 2.0 meq/l. later in icu day 16 the patient had 3 brief episodes of torsades de pointes, each responding to precordial thump, and finally rhythm stabilized with i.v. lidocaine and magnesium. haloperidor and trazodone were discontinued. ecg was unchanged and myocardial infarction was ruled out. next day, icu day 17: qt 0.42/qtc 0.53 sec. torsades de pointes, a form of ventricular tachycardia characterized by a twisting qrs axis, is commonly associated with qt prolongation. haloperidol is used frequently in icu for control of agitation and delirium, with reported doses up to 1000 mg/day. over past decade, 9 cases of torsades de pointes with prolonged qt related to haloperidol have been reported. trazodone may also prolong qt and cause ventricular arrhythmias, especially in patients with pre-existing cardiac disease. in this patient, trazodone likely exacerbated qt prolongation from halopeddol leading to torsades de pointes. critical care physicians must be aware of this interaction. it is imperative to follow the qt interval for patients receiving halopeddol, especially when another drug also known to prolong qt is added. one must consider discontinuing the drug when qt/qtc becomes prolonged. objectives: analgesics and intravenous anesthetic drugs are routinely used in critically fll patients, who often suffer from a secondary impairment of the immune system. previous in vitro studies have demonstrated inhibitory effects of these drugs on polymorpho nuclear cells (pmn). the potentially important role of endothelial cells (ec), however, was not investigated, since suitable test systems were not available until recently. therefore a physiologically more relevant in vitro migration assay through cultured human endothelial cell monolayers (ecm) we established. using this assay system, the comparative effects of fenlanyl, sufentanil, propofol and the known pmn inhibitor thiopontal were tested. methods: human umbilical vein endothelial cells (huvec) were isolated and cultured on microporous membranes (cyclopererm) until an ecm was grown. pmn from male and female volunteers were separated by standard procedures. ecm and pmn were preincubated with clinically relevant concentratious of thiopental (104 m), propofol (4p_g/ml), the solvent of propoful (intralipid), fentanyl (30ng/ml) and sufentanil (sng/ml). after preincubatiun (ecm 30 minutes, pmn 15 minutes) with the reslx~tive drug, leukocyte migration towards the chemoatfractant fmlp (1o 7 m) was measured in a two chamber 24 well system for 3 hours. the migration rate of untreated (untr.) and treated (treat.) pmn through untreated and treated ecm were determined. as a control untreated pmn and untreated ecm were used. results are given as means from 5 independent duplicate determinations and expressed as a percentage of control (table) . statistical analysis was done with student's t-test. results: clinical concentrations of fentanyl, sufentanil and prupofol showed similar inhibitor~ effects as the known pivin inhibitor thit e 1 ). 77% conclusions: for the first time we could show that analgesics and anesthetics exert their inhibitory effects not only on pmn, but mainly on the interaction of pmn with endothelial cells. moreover, we could shmv a significant suppressive effect of the opinids fentanyl and sufentanil on both ec and pmn. the known inhibitory effect of thiopental obtained in ec-free test systems were also confirmed in our physiologically more relevant assay system. objectives: to investigate when and how sedation is used in a consecutive cohort of patients admitted in a large sample of italian intensive care units (icus), gathered in a network named giviti, representative of the italian icus system. methods; the study called for a recruitment period of one month, from january 10 to february 8, 1994, data collection included age and other demographic variables, acute diagnostic broad profiles, severity of illness scores, treatments, lenght of stay and vital status at icu discharge. as concerned sedation, each patient was observed until discharge or for a maximum period of seven days. information on all the drugs used for analgesia/sedation, the route and modalities of administration, the timing, dosages and purpose of the administration have been recorded. results: the study involved the cooperation of 138 icus, 128 of which enrolled at least one case. the total sample included 2932 patients. overall, 60.7% of patients analyzed (t780/2932) received at least one prescription of sedative during their stay. globally, at least one sedative drug was prescribed to these 1780 patients in 5014 days in icu. although over 38 drugs were reported to be used, 10 pharmacological principles accounted alone for 89% of all prescriptions. opioids were actually used in 33% of prescriptions; propofol in 24% and benzodiazepine in 18.3%. as regards the way of administration, intravenous administration was applied in 74% of cases and, followed by intramuscular in 17.3%. moreover, non-steroidal anti-inflammatory drugs (nsald) were used in 19% of patients and neuromuscular blockade agents (nmba) in 23%. detailed analysis on certain subgroups (surgical, trauma, ventilated patients etc.) have been also carried out in order to describe the practice of sedation in these peculiar subgroups. findings will be widely discussed during the presentation. conclusions: these results should be interpreted keeping in mind how peculiar is the intensive care setting compared to many other less complex settings of hospital care. in conclusion we thought it was important to present the data currently available in the most neutral form, to start moving in a direction which will enable us -by means of more specific and detailed studies, and with the cooperation and involvement of all those participating in the project -to shed light on one of the many aspects of medical practice in the field of intensive care which deserve closer attention. introduction: the aged run perilously high risks in cardiac surgery: among others, of haemodynamic fluctuations, respiratory depresskm and organ failure. response to anaesthetics is a crucial determinant for post<)perative complications, none the less being reintubation due to mechanical ventilation difficulties which increase morbidity, mortality and intensive cdre unit (icu) stay. objective: we wanted to assess our a,aesthesia window (selection, and a view of the induction -extubation period) for predicting safe and swift awaking, thus: icu dismissal for the aged. methods: in 1994, 162 selected patients (pts) (>70y, 62f) followed a regular elective cardiac surgery protocol (propofol given at precisely designated time intervals). upon 1cu arrival, they were subjected to an admission protocol. our predictive criteria for early extubation at 8h included: a) alertness and ready response to commands; b) adequate gag reflex and sufficient protection for respirak)ry tract; c) pao2 >75 mmhg with flu 2 <0.4; d) stable ph>7.35 with spontaneous respiration; d) stable haemodynamics without dysrhythmias; e) adequate perfusion and diuresis (>1.(i ml/kg/h); f) mediastinal bfeeding<100ml/h for at least 2h; g) normothermia (core temp>36~ and no shivering). subsequent reintubation was for: 1) rr>35/min; 2) spontancx)us ventilation for 30 rain with paco2>50 mmhg; 3) pao2<50 mmhg with fio2>0.4; 4) ph>7.45; 5) heart rate>]20 bm; and/or 6) non mental alertness; and 7) other medical disorders, after which adequate weaning therapy was necessary. then, successful weaning after 24h was considered: 1) spontaneous breathing without any forrn of mechanical assistance; 2) stability in haemodynamics; and 3) elimination of fever threat. results: 122 pts (75%) were extubated at 8h without complication; 29 other pts (18%) at 8h but had to be reintubated because they were hypoxic and began weaning therapy; finally, they were all re-extubated by 48h. only 11 pts (7%) proved problematic. conclusion: a,aesthesia wimhlw options (selectkm, extubation, reintubation and weaning) predicted quick (times propofol administration) and safe (rigid criteria) extubation (75%=8h and 18%=24h), exempting pts with developed post-operative complications (7%=extubation<72h) unrelated to al~aesthesia window or icu protocol. dismissal and recovery then became an abbreviated question of time. fifisetll p, domeneg~i ~, sforzini i., veronesi i~, maconi a.g. *, breg~ massone p.p 30h [] ic+pca request conclusions:using e~aprenorphine, a synthetic,long-acting, ago-antagemist opinid drug as analgesic, in the major surgery we obtained the best clinic results with association of conttheus infusion of haft dose drug with bohts of pca in the first 15-20 hours and just pca in the secmad day after surgery when the patient is less sleepy. in this way we dent have a great sav~g of suppled drug but the major well-belng of patient without ~erious side-effects and quick mobilization; the dosage used don't compromise a good awake of patient: all patients are sleepy but ready for answer, no allueinatian, bradipnea but not less than 10 b/m without ipoxia. also the patient proffered this kind of truit meut than the traditional at demand. the ward staff feel it useful] and rehabl~ the negative feed-back technology of the electronic infuser system makes possible to use it safe in the ward with high drug's concentration too. the infusion rate of low dose of drug assure a continuative analgesic covering ~n the first postoperative periad; the pca mode involves the patient him-self in the managemenl of therapy and enables him to choose the best way to confront the dll~icuity of postoperative period without call medical stall using pca-device we have had no probicm~ no accident. analgesia during extracorporeal shook wave lithot ripsy a .levit, b.grinbezg regional hospital, ekaterinbu~g, russia 0b~ectives: our task was to compare ~he analgetic effect of norphin and tramel. methods: study was made of two groups of uro-li~patients aged 25-61. group a (23 patients) received baprenorphine hydrochloride (norphin) at dosages of #.52• mg/kg. group b (30 patients) received tramadel hydrochloride (t~aasl) st dosages of 1.50z0.38 mg/kg. before the procedure diazepam was administrated i.v. (0.24!0.03 mg/kg). blood saturation (spoz), hemodynamics incides (bp, hr,sv,co,sap,svr) were examined and the patients' subjective assessments of snsesthesis quality were analyzed. the hospital ethics committee approved the investigation. results: when using norphin hr increased by 17.7% on the onset of the procedure while sap and sv decreased by 8.%% and 9.6%, respectively (p<0.05). however, there were no reliable co chsnges. spoz ~educed by @.2% (p<0.05) and remained lower than the initial one after the procedure was oyez. when administrating tramsl 50 min. after ste~ting the procedure sap and svr increased by ~1.2% and 7.3% respectively. sv and co decreased insignificantly. nine patients in group b saffeting some dlscomfo~t needed additional tm~msl in~ection. in the course of the whole p~oced~e spo, was constant and was highez than that in ~he case of nozphin (p<o.05). conclusion: respiratory suppression by no~phin can limit its use in case of lithotzip@y while the advantages of tremsl a~e: lack of dyspnoe and good contact with the patti-b_ the role of uncommon agents of antinociception, placed epidurally, in the control of pain transmission in the spinal cord is well documented. somatostatin as an inhibitory agent is found in the dorsal horn of the gray matter of spinal cord. the effects of somatostatin is similar to the opiates if injected into the epidural space (ref. i, 2, 3, 4) . the aim of the present study is to present the clinical effect of above mentioned palliative therapy in the case of cancer and non cancer pain. informed consent had been obtained from every patient before treatment was initiated under the legalisation of the local ethics committee. using the permanent epidural catheter and continuous infusion pump, we administer somatostatin in the dose of 5 up to 20 microgram per hour for one up to three days. the patients paln feeling was evaluated using a vas (visual analogue scale). we found that the opiate resistant pain level decreased by 50%. the rest pain could be controlled by pereral opiate analgetics or by combination of somatostatin and an opiate plus a local anaesthetic agent epidurally. theoretically, it seems that epidural administration of somatostatin and local anaesthetic agent is useful in the control of acute pancreatic pain and systemic effect of somatostatin is beneficial in the treatment of acute pancreatitis. few studies have been performed in the critically ill (ci) and because of the complex pharmacodynamic and pharmacokinetic changes that occur, it is difficult to predict accurately drug responses and interactions in individual patients. midazolam (m) is a watersoluble benzodiazepine with a rapid onset and short duration of action in normal individuals; however its metabolism is decreased in the ci. critical care physicians every day experience suggest that among the ci there is a different pattern of response to m; here it is hypothized that such a response could be due to different metabolic behaviours of ci. m (i.v.infusion rate 1-7 rag/h) and antipyrine (1000rag p.o.) were infused to ci with (10 patients -group 1) and without (10 patientsgroup 2) endotracheal intubation. blood samples were collected at fixed times during and after m infusion. total urine nitrogen and antipyrine elimination half-life demonstrated different metabolic characteristics in group 1 chypermetabolizer") as compared to group 2 chypometabolizer"). results follow. the results of our study are preliminary and more kinetic data in critically ill patients are needed to statistically confirm our approach of "hypometabolizer" and "hypermetabolizer". objectives: some clinieai and experimental studies suggest that propofol decreases myocardial contractility in coronary artery bypass grafting (cabg) patients. the intrinsic negative inotropic action of the drug may be independent from changes of preload and afterload, whereas the myocardial depression induced by propofol may depend on changes in ca ++ ious availability. aim of this study was to verify the hemodynamic effects of propofoi in a group of cabg patients with uneompromised contractile function and to minimize myocardial depression, during induction of anesthesia, choosing to associate calcium chloride (cac12) in an other group and to define its role in producing this effect. methods: fourty patients, randomly divided in two groups (20 pts a and 20 pts b), undergone elective cabg, were enrolled in this study. anesthesia was induced by senior anesthetist in both groups by means of fentanyl 7 mg• kg < in 60", pancuronium 0.1 mg x kg -1 and propofol 2 mg • kg -1 in 60". a blind investigator administered via another venous way at same speed (60") saline in patients of group a and 10 mg x kg -1 cac12 in patients of group b. hemodynamic data (heart rate hr, mean arterial pressure map, mean pulmonary artery pressure pap, pulmonary capillary wedge pressure pcwp, central venous pressure cvp, cardiac index ci, stroke volume index svi, systemic and pulmonary vascular resistances indexed svri and pvri) were obtained at baseline (to), 2' after anesthesia induction (t1) and 2" after tracheal intubation (t2). results: hr decreased moderately in both groups (p = ns). map decreased as well, more markeatly in group a at ti and t2. this trend was statistical significant (p < 0.05) in both groups. pap, pcwp and cvp unchanged following induction in any of two groups. ci decreased in group a (p < 0.05) dramatically dropping at t2, while in group b it showed a negligible decrease at t1 (p = ns) and value similar to baseline at t2. svri and pvri did not exhibit statistically significant changes. conclusions: the use of propofol as a starter of anesthesia in cabg patients allows to reduce total dose of fentanyi, dramatically reducing post-operative intubation time. propofol-fentanyl association prevented the hyperdynamic response to stress (i. e. laryngoscopy, intubation) but severely depressed ci and svi. cac12 simoultaneous administration effectively minimized the negative inotropic effect of propofol. moreover no unwanted side-effects due to cac12 were observed. diseoordinated labor activity (dla) is one of most serious in obstetric pathology.medication sleep effect to a woman in birth is of limitation of pathologic impulsation from the central nervous system, but it does not influence processes resulting in and supporting dla on the organ level. constant discoordinated uterine contractions during medication sleep (ms) imerease disturbances of uterine blood flow, organ hepoxemia connected with hyperergia of vascular wall and myometrium to catecholamines, that reduces efficiency of anesthesiologic protection. we applied hexoprenalin in complex with ms to 20 primaparas. efficiency control was being accomlished through hysterograms, grade scale of analgetic effect where hemodynamics reaction to pain, significance of emotional reactions and their vegetative manifestations before and during ms were registered, and also through hydrocortisone level in plasma before and during ms. the examined women's average duration of ms was 2 hours more than in control and made 4 hours, though their average labor duration and in control was the same and made 12 hours. 98 % of the examined women experienced independant labor, as for the control group -only 68 %. supplementary methods of anesthesia were required in 73 % in control,that increased medicamentous depression of the fetus, but for the examined group -only in 16 %. the examinees's newborns experienced favourable terminations, in control 3 newborns were in state of light rate asphixia. analgetic effect was complete in control just in i5 %, and of examinees -in 82 %. on the women's hysterogrnms before ms discoordinated contractions were registered on the background of increased tonus. during ms in control similar uterine contractions were constantly registered, but for the examinees they were not marked at all or had the proper character. the examinees' hydrocortisone level was reduced for 42 % and in control only for 24 %. the study has allowed to make a conclusion that hexoprenalin applications in complex with ms in case of dla make anesthesiologic care safer and more effective, promote simultaneous removal of patholgic processes in the central nervous system and uterus, which lead to dla development. objectives: the aim of report is to present a clinical study results for the evaluation of anesthesia for a patients in unconscious states by eeg examination: methods: the study was conducted on 17 patients after a severe abdominal operations on stomach and intestine and on one patient after traumaticat exarticulation of upper extremity. all patients have different unconscious state levels from somnolence to coma ]. also thay hav~ had respiratory insufficiency and control mandatory ventilation have been used. we were used intrave,~ous injections of morphine hydrochloride from initial dose of 20 mg. than we were increased the dose to 80 mg i.v. by drops with an accompanying eeg monitoring and fourier spectral analysis. initially all patients have had 13-rhythm with slow 0and a-activity. an c~-activity index have been lower than 5%. results: after anesthesia the o-and a-activity have disappeared. the or-activity index have increased to 35%. also we have noted decreasing of the unconscious state levels to a possibility of a verbal contacts (13-14 points gcs). conclusion: the quality of anesthesia could be evaluated by eeg monitoring. a regaining consciousness afte~ the injections of high doses of morphine, probably, may be a result of an opiate deficiency (full or partial). anesthesia with the following drugs: clonidine, l-arginine, l-n-arginine-methyl-ester (l-name), and their combinations. tail-flick latency (tfl) was determined at time zero and 15 min after each treatment. clonidine (i-4 ~g/mouse) prolonged tfl in a dose-dependent manner. at a clonidine concentration of 4 gg/mouse, tfl increased 2.37-fold compared to time zero. l-arginine increased tfl at a high concentration (i00 gg/mouse). l-name suppressed tfl 1.2-fold compared to time zero at a concentration of 50 gg/mouse. conclusion: we have found that no is involved in clonidine spinal analgesia. studies are currently being undertaken to assess the effect of combination treatment of the above drugs on clonidine supraspinal analgesia. objectives: hemodynamic changes ussualy accompany laryngoscopy and tracheal intubation.the aim of this prospective study vas tu present the effectivness sufentanil in preventing reflex circulatory respone of laryngoscopy and tracheal intubation and provides stable hemodynamio condition for induction of balanced anaesthesia. methods: 30 adult asa i-ii patients sheduled for elective surgery.they were allocated randomly and divided into two groups:fifteen patients received single bolus of placebo prior to laryngoscopy.and tracheal intubation. anaesthesia induction was then performed with thiopental (smg/kg) and atracurium (0,5 mg/kg), followed by neurolept anaesthesia. mean arterial preassure (map) and heart rate (hr) were measured before, during and 5 min after intubation. althought ecg was recorded at the same time intervals. results: the groups were compared with regard to the changes in arterial blood pressure,heart rate and electrocardiogram. mean arterial preassure and heart rate were incresed significantly during and after laryngoscopy and tracheal intubation in control group but remained unohange the baselin values in the group who received sufentanil. control group ecg suggested more changes than sufentanil group. conclusions: the results indicated that 1,2 mg/kg sufentanil for anaesthesia induction reducing the pressor response to laryngoscopy and tracheal intubation. obiective: the aim of this experimental study was to evaluate morphologically the influence of anesthesia in liver as well as in isolated hepatocytes (hc). methods: a total of 16 female swine (20-22 kg) were used as liver donors and were randomly assigned to one of two groups regarding anesthesia protocol: group a (n=8): induction with intravenous sodium thiopental in a dose of 5 mg/kg, group b (n=8): propophol in a dose of 2 mg/kg for induction'and 10 mg/kg/h for maintainance of general anesthesia. both groups were under the same preanesthetic regimen (diazepam, ketalar and atropin) and received standarised doses of fentanyl and pancuronium during anesthesia. in beth groups total hepatectomy was performed and the liver was perfused with 4-5 it of cold (4oc) university of wisconsin solution for a period of 5 hours before hepatocyte isolation. liver tissue samples were fixed in formalin for the morphological study and stained with hematoxylin-eosin and pas. for hc isolation collagenase solution (type v, sigma, c-9263,1.3mg/ml) was infused via portal vein and the liver was incubated at 37oc for 45 rain. cells were filtered and then washed in cold hanks' solution. isolated hc were fixed and prepared for staining or simply cryopreserved (-20oc). results: histology was completed in 8/16 experiments (4 in group a and 4 in group b). mononuclear infiltration (halothane type injury) was found in all specimens (8/8). focal zonal necrosis and acidophilic bodies were found in specimens from group a (2/4 and 1/4 respectively) while portal inflammation with piece meal necrosis was found in one specimen from group b (1/4). smears of hepatocytes -beth formalin fixed and cryopreserved at -20oc -were stained with hematoxylin-eosin and showed morphologically intact hepatocytes. conclusion: pre[imineq~' study of our material reveals mote frequent livet injury in the thiopental group, but this finding has yet to be established by increasing the number of observations. objectives: in this paper we present our experience and point out the importance of sedation of those patients who suffered severe head injuries/contusic cerebri with signs of decelebration/and who were put on ventilatory machine to obtain better mechanical ventilation in neurosurgical intensive care unit of emergency center in belgrade. methods: 256 patients were treated from jan. 1994 till dec. 1994 results: in 157 patients we successed to reduce agitation and decelebration. conclusions: the major demand of sedation in neurosurgical patients are that the patient should be calm, comfortable and painless. references we compared three analogous groups of 20 patients in each, with orthopedic operations on lower extremities. in each group we applied different kind of analgesia: first group rece-ned local anesthetic (4 ml 0.5~ bupivacaine) when analgesia was first demanded; second group received 2 ml fentanyl via peridurai catheter when analgesia was first demanded, and third group received 2 m] fentanyi intravenously on demand for analgesia. we used visual analog scale (vas) for estimation of pain intensity and success of applied therapy. statistical data analysis was performed using student's t-test. tha best vas had group in which local anesthetic was applied periduraly. second was the group with periduraly applied fentanyl, and third was the group with intravenously applied fentanyl the longest duration of the effect of analgesia ~/as in the second group. there were no adverse effects and complications, apart from mild sedation in the third group. for orthopedic operations on lower extremities, application of peridural catheter for anesthesia and successful postoperative analgesia with local anesthetic, or opioid analgesics is acceptable. background and obiective : tympanic temperature measurement (ttm) is a relatively new procedure which provides accurate estimates of core temperature in stable postoperative patients and in patients admitted to the emergency ward. however, experience with this technique in hemodynamically unstable adult icu patients is limited. design : prospective study with patients serving as their own control. se:ttijlg : adult medico-surgical icu in a tertiary care hospital. patients : 51 consecutively enrolled patients with thermodilution catheters in place and without contraindieation for rectal temperature measurement and without hypothermia (core temperature <35~ interventions : tympanic temperature, measured by a commercialized tympanic thermometer (genius 3000 a first temp) was compared with pulmonary artery (pat) and rectal temperature (rt). within 10 minutes, core temperature was assessed in each patient by the same trained individual using the three techniques in random order. measurements and main results : mean bias (ix~ and its variability (sdr of method comparison pairs were calculated using the methods comparison analysis as described by bland and alunan (lancet, 1986 ; i : 307-310 objective: to measure the prevalence of pressure sores on the intensive care unit and the effect of risk calculation on pressure score prevalence and pressure score severity. method:in 1993 during a period of 5 months a series of prevalence studies was carried out on the sicu to investigate how many patient days with pressure sores were taken care for by the icu nursing staff, what kind of preventive interventions were done and what the patient risk was on developing a pressure score. results: on average there was a prevalence of pressure sores on the buttocks of 18.5% on the short stay unit and 42.8% on the long stay unit; preventive interventions increased when the pressure score was visible for the nurse and the majority of patients had a high risk or extra high risk for developing pressure sores according the pressure score risk calculator. as a result of these findings the nursing management decided in 1994 that nurses had to complete daily on every patient the pressure score risk assessment in order to be able to take adequate preventive interventions. when the results of the two studies were compared, the most important results were that: 1) there is no indication of a reduction in patient days with pressure sores (short stay unit: 14%, long stay unit: 55%); 2) there is a reduction in severity of pressure sores (grade iv decreased from 2.4 % to 0.7 %) because adequate preventive measurements are initiated by icu nurses in an earlier stage. possible explanations for the increase of patient days with pressure sores on the long stay unit are that the average pressure score risk score was increased from 17.8 to 19.8 and the mean frequency of nursing patients on alternate sides decreased from 1.4 to 0.6 times each day. conclusion: daily assessment of the individual patient risk on developing pressure sores does not decrease development but reduces the severity of the developed pressure sores. method: the conventional woundcare method of patients with an open abdomen is to change frequently the saline soaked ribbon gauze pads (4-6 times each day). this method is labour intensive for the itu nursing staff and rather uncomfortable for the patient. problems that often occur are: insufficient hygiene, frequent nursing interventions, a progressive risk for deterioration of the skin and underlying tissues, difficult to measure abdominal output and in case of bowel fistulas enteral feeding is hardly possible. in using the new method, stomahesive is applied on the skin surrounding the wound. a large size of surgical film dressing, opsite | is applied over the abdomen. two or more foley ~ catheters ch 22 are inserted through an opening in the surgical filmdressing. the application of several lateral openings in the catheters is advised. the drains are held firmly in place between two layers of sterile opsite | "flexigrid ~'' 6x7 with the so called bookend method and are connected to a low vacuum suction system (1-2 kph) results: we studied the frequency of dressings changes on 12 patients in the itu. in total they had 363 days with an open abdomen. during this period there were 63 dressing changes. this means one dressing change every 6 days. further advantages of the new method are; more hygiene in patient care, no maceration and irritation of the skin, nursing on alternate sides is possible, output can be measured, in case of bowl fistula, enteral feeding is possible and an abdominal lavage is possible on the sicu by opening the opsite | conclusion: this new method of woundcare management is more patient friendly, prevents several nursing complications and decreases the work load for the nursing staff. objective: to investigate the interaction between the ventilator and the closed suction system related to possible induced negative pressure by this sytem. method: we studied in a testlung model what the effect was of the different ventilators (evita: dr~iger; servo 900 c: siemens), ventilation modes 0ppv, simv) and ventilator settings (tv, trigger level, frequency, peep level) on the induced negative pressure by the closed suction system during suctioning. results: when using the evita the magnitude of the negative pressure increased when the ventilation frequency and tv decreased.. the largest negative pressure (-76 cm h20) was produced with a tv of 600 ml and a frequency of 6 /min in the ippv mode with trigger level on -3 cm h20 and a peep level of + 10 cm h20. the servo 900 c showed a complete different pattern. the largest negative pressures were measured when no trigger level was set (27 cm h20), the induced negative pressure was not depended on the tv or ventilation frequency but more depending on the ventilator mode. the simv mode produced the smallest negative pressures ( 1-4 cm h20 ) and the ippv mode the largest (16-27 cm h20 ). conclusion: the effectiveness of the closed suction system is very much depending on the kind of ventilator, ventilator mode and ventilator setting that is used. if the interaction with the used ventilator is not known by the icu nurse this piece of equipment can cause damage to the patient by inducing large negative pressure in the comprimised lung and there fore creating the possibility of alveolar collaps and atelectasis or oedema. introduction : ethical problems are daily and disturbing preoccupations for the icu staff. objectives : improving ethical management in icu by creation of an intelprofessional group of ethical reflexion ( iger ). methods : existing was evaluated by a preliminary formulated series of questions sended to the whole staff (q1, n = 43, 13 items) after two training sessions (laws, deontology, professional values, culture, norms of quality) followed by 38 persons (88 %) and directed by an external chairman, satisfaction and needs of the staff were evaluated by a second series of questions (q 2, n = 38, 12 items). at the issue, iger was created. results : q1 : 30 responders (70%). law's misinformation : 33 to 70 %, disagreement for limitation of intensive cares : 40 %, hope to improve collective decisions and creation of iger : 88% q 2 : 29 responders (76%) : satisfied by the training sessions : 65%, non satisfied : 31%, want to continue the project : 68 %, refuse : 10 %. iger was constitute by physicians, nurses, secretary, dietetic (n = 15). the first working theme was {< therapeutic's withholding and withdrawing decisions in icu ~>. four subgroups of iger's members (having access to an ethical library) worked independautly and submitted their reflexions in a tdmestrial plenary session of iger in the presence of an external chairman, allowing a synthesis. at the issue a report was writted to be used as a reference for bedside and individual decisions. conclusions : constitution of iger seems to improve ethical management in icu. the first result of iger is that it is now possible to began collectively a reflexion concerning therapeutic's withholding and withdrawing in icu. the work is going on and further subjects will be studied. objectives: 1) to compare the value of heat-moisture exchangers with bacterial filters (hmef) and without bacterial filters (hme) in the prevention of colonization of ventilator tubing and ventilator-associated respiratory infections. 2) to asses the temperature and relative humidity of inspired all using both types of heat-moisture exchangers. methods: 48 mechanically ventilated patients were randomized, to either hmef or hme. endotraeheal aspirates, pharyngeal swabs and samples from tubing were collected for bacterial cultures on the 1st, 2nd day mechanically ventilation and weekly thereafter. temperature and relative humidity were measured in 23 patients (13 hmef and 10 hme) 3 h and 24 h after placing the hme or the hmef. results: both groups were comparable as regards age, mechanical ventilation period, severity score (saps ii), leukocyte count, and number of patients with prior antibiotic treatment. from the hmef group, 10 (42%) ventilator tubing yielded microorganisms in, at least, one sample as compared to 7 (29%) of the hme group; p=ns. the incidence of respiratory infection was similar in both groups (25% vs 17%, p:ns, for hmef and hme respectively). among the 16 bacterial species isolated from ventilator tubing in the hmef group, 7 (44%) were not isolated from pharyngeal swabs. a similar ratio was shown in the hme group (6/15, 40%). both heat-moisture exchangers were efficacious in keeping a good relative humidity of inspired air (97% • vs 96% • 3.%; p=ns, for hmef and hme respectively). relative humidity was significantly higher after 3h of mechanical ventilation in the hme group as compared to hme group (28.5% • vs 26.5% • 2%; p=0.03). conclusions: both types of heat-moisture exchangers have the same effect on the prevention of colonization of ventilator tubing. similar relative humidities are achieved when using either type of heat-moisture exchanger. results: tumor and nontumer enhrgements of the thyroidea were present in 85~ of the operated, surgicel adrenal disease in io!, hyperplssle or persthyroid gland tumor in 2~ end endocrine pancreatic tumors in 3%. in the intensive oere unit, these patients wore screened by noninwsive monitoring in 85~ of cases: and invasive monitoring was applied in 15% of ceses.the basic noninvesive methods included: electrocardiogram with standard end precerdial leeds, percutaneous eutomotlc measurement of systolic, diastolic and mean arterial pressure, measurement of hourly diuresis and body temperature, frequency, hearing capacity and rhythm of one s own breathbng bs well as pulse oxymetry. a special plece in monitoring and control of vital parameters in postoperative period belonged to the nurse, thoroughly trained for enelysis end interpretation of the observed parameters which would be discussed in the paper. it has been believed that the leader sits at the pinnacle of power. over the years, this has proven to produce frustruation and anguish instead of the expected results. leaders have not been able to produce the changes they know are essential to their organization's survival with this command-and-control paradigm. through literature reviews and evaluating leadership styles, one can clearly see the most effective form is that of empowering people to a new level of performance -not ordering it. changing the leadership paradigm to a manner/style that has been shown to be effective and one of people empowerment shifts the focus to personal responsibility for performance. removing obstae}es~ stimulating self-directed actions, and determining focus and direction are just a few elements used to create the successful environment of empowerment. with increasing pressure in the health care arena, it becomes critical that a leader's job is to get the people to be responsible for their own performance. developing ownership, creating an environment where people want to be responsible, being a mentor or coach, and learning faster while encouraging others to do so demonstrates the commitment to effective leadership. this presentation will illustrate the critical components that are achieved when every person in the institution is empowered to perform at a level that is directed toward positive, effective results. herrera m. (md) . icu. hospital regional. malaga. spain. the systems of veno-vanous continuous haemofiltration (wchf) have a high cost and a limited life span. in an attempt of lengthening their mean life it has been proposed to accomplish programmed washes of the ~-stems. this practice supposes an increase in nursing workload. in order to evaluate the real efficiency of this practice we have accomplished this study. material: prospective randomized study of all the filters of vvchf used during the last year in our icu. we have determined two groups of filters, in the first (group a) we accomplished washed in a programmed way, and in the other (group b) only when the alarms of the system suggested a clotting of the filter. for the statistical analysis we used the kaplan-meier test for survival analysis. results: we have studied a total of 24 patient submitted to wchf during the last year. we used a total of 32 filters with this results. objectives. sounding out the nurses about the need to inform patients" relatives and the rigth kind of such information, like a preliminary approach to an information cuality assessment, methods: we inquired all the nurses of the intensive care unit of an regional hospital by an semiestructurated questionary which included personal data: age, sex, contractual relation, professional experience.., and opinion data: do you think to inform relatives is a nurse task?. which of the next informafions do you think is more important?, please, write others topics about information you think are relevant. we process the data on epi-info estatistical program and use x 2 test to compare the results. results" from 80 nurses of staff 5 refused to flu the quetionary, and 8 were not available. of the 67 remaining, 71%were v~men and 29% men. the mean age were 31.51% had an svable contract and 499( eventual, the mean professional experience were of 10 years and 44% worked in the unit since more than 6 years. the 88% answered that offer information to relatives is part of the nurse activities. we did not find differences with nurses who answered negatively comparing by sex, age, contractual relation or proffesional experience. the three information topics found out like more important were: 1) to inform about patient mood. 2) to inform about happenings from the last visit. 3) to inform about dressing instrument required by the patient, nurses who answered negatively think that to inform is a doctors task or that nurses are not competent. conclusion~ intensive care unit teams (nurses, doctors and auxiliar personnel) should get accord on who and how to inform relatives, we consider the nurses' role on information as unquestionable. objective: investigate the respiratory and cardiovascular response after discontinuing oxygen therapy durir~ intr~/]o~pital transport. desiqn: fifty-one patients (29 male and 22 female, aged 69+2,5 and 73,912,4 years respectively, ~+sym) being on 02 therapy were studied prospectively in two consecutive intrahospital transports. oxygen therapy was continued in the first transport while the second one was performed as usually, i,e, without 02. during transport each patient was monitored by pulse oxymeter and holter whereas arterlal blood gases were tested just before a~xl aft~-trar~portation. results: compared to daseline, pa02 and sa02 were signif~canthy decreased in the case of oxygen discontinuation (p<0,00i). paco2 was significantly inur~ds~i only in the subgroup of patients with obstructive lun[ disease (p<0,01) . heart rate increased in all phases of the transport when 02 administratlon was discontinued. blood pressure remained stable in either case. the percentage of supraventricu!ar extrasysto!es, ectopic v~r[hicui~r contractions and st-s6~ment depression was progressively increasing and became very high at the end of transport in the case of 02 therapy discontinuation. other arrhythmias did not change significantly. conclusion: discontinuation of oxygen therapy during intrahospital transport causes severe drop of pao2 and sa02, increases the heart rate and contributes to the appearance of arrhythmias which were not present before. methods:for evaluation of the functional state of brain the complex of methods was used,whieh included electro encephalngraphy ( brain mapping ), rheoencephalography, tetrapolar transtorax rheography. for the estimation of humoral status the level of histamine and serotonine, products of free-radical oxidation,enzimatic markers of ishemic damage of brain and of endogenous intoxication was investigated. results:92 patients with encephalopathies after resuscitation were observed.asystolia was as a result of:shock, trauma, asphyxia,poisonings,appiication of drugs, eclamp sia,injury of the heart,diseases of fhe cardiac vessels. all patients with postasystolic syndrome entranced in comafose condition.in the 1 group (reconvalescents) the depth of coma by glasgo~ pittsburg"s scale was 23,3+-1,78. the duration of coma was from 30 rain. to 48 hour,average 11,9+-4,sh.ln the 2 group (the deads) the depth of come was 13,8+-0,86.the artificial lung ventilation was used in all patients:in the 1 group 2,64+-0,92 days,in the 2~ 6,1 +-1,1 days.apallish syndrome developed in 5 cases,in 5 patients diagnozed <<brain death~. the 4th degree encephalopathy (coma) was found to be associated with disorganized eeg-pattern with predomi nant alpha-( 1 group) and slow (2 group) activity. the relative power of delta-and theta-ranges was about 77-87 per cent.the patients with apallich syndrome demonstrated greatly elevated theta-range power, that of beta -range was essentially decreased. the degree of disturbances of circulation in the both groups was different.cardiac index (ci) and stroke index (si) in the 1 group decreased on 19,5% and 44,3z, in the 2 -on 45,9% and (;3,3%.general peripheral resistance (gpr) increased in the 1 group on 40,5%,in the 2 -on (;9,4%.in the 1 group brain blood flow was increased, in the 2 -decreased on 37,6n.in the 1 group there was the normotonic type of reg-curve,in the 2 -atonic or hypotonic type (without reaction on pharmacologic influ ence).disturbances of permeability of cellular membranes (enhanced free-radical oxidation with an increase in di enic conjugates on 107 and 121n) and vascular ones (an increase of serotonin concentration on 90 and 110%, hi stamine content on 116 and 180%) resulted in hyperfermentia (an increase of concentration of creaune phosphoki nase in the 1 group on 59%,in the 2 -69%.the level of middle molecules (mm) increased in the 1 group on 79,2%, in the 2 -on 120,8%,of polyamines (p) (spermidin,spermin,putrescin).coefficient of correlation between mm and p was 0,891. immediate correction of neurocyte metabolism is impossible due to marked brain oedemaswelling,severe dis ordes of cerebral circulation, disturbances of membrane permeability.thus,the following treatment algorythm might be advisable: 1. protective inhibition of brain and reduction of its energy requirements. 2. recovery of cell and vascular membrane fuoctions.3.recovery of blood circulatiom4.oxygenation of nervous tissue and drainage of brain disintegration products.in 22 patientshbo carried out.5.active methods of detoxication (homo-and enterosorption).6.correction of cerebral metabolism.7. dehydrative therapy must be very cautious. conclusion: the activation of energetic metabolism in neurocytes must be carred out only under neurophysiolngical control and after definite preparation. (67). the clinical estimation of acute cerebral failure carried out by olasgo-pittsburg"s classification of coma. we studied such groups of patients: poisoning co (36), asystolia (8), eclampsia (5), acute renal failure (5), control group (13). methods: electroeneephalngraphy (brain mapping) was fulfilled by means of encephalograph <,medicor ~ in 8 monopolar channels. the estimation of eeg patterns by zhirmunskaya method (1984) was carried out. there were analyzed and changes of eegb by results of rapid transformation furie after rhythmical photostimulation(phts) 2,6,10,20 hz. we studied also the figures of absolute and relative power in such diapason : delta (1-4 hz), theta (5-7 hz), alpha (8-12 hz), betal(13-25hz), beta2(25 hz and more). results: all eeo changes were divided into following types: 1 -organized (3,4 groups); iii -asynehronic (8 group); iv -disorganized with prevalence of alpha-waves (12,13,14 groups); v -disorganized with prevalence of delta-and theta activity (16,17,18;19,20 groups) akkording to our model of hormonal-metabolic disbalance in pregnancy , one of the main reason of destosis is hypoergos , reesults in endotoxemia and neuro-endokrine disregulation. so, stady of central nervous system function give us the information about adaptation processes. the aim of our work is to study the degree of neurologikal deficit in destosis when olifen and lipin were used. there were studied 20 pregnants with different severe forms of gestosis. the degree of neurologikal deficit was valued by datas of neurologikal status . eeg with ,~brain mappinng~, , electrikal resistance of the skin. the komplex inteensive therapy with applikation of olifen and lipin allowed to improve the patient,s condition , decrease the degree of neurolodikal deficit and mortality . kostenko v.phd,kabanko t.,phd,galalu s.md,beliavtseva n. ,shramenko k. phd intensive care department,donetsk medical university, donetsk, ukraine plasmapheresis (pph),as other extracorporal methods of detoxicatin,has a great importance in contemporary medicine. the role of pph is special,because of its simplicity, effectiveness, atraumatic.we incalcated pph in obstetrical clinic. there are rough changes in agregate condition of blood in more of pregnants.these changes lead to disturbances of central,organ and peripheral hemodynamic.the therapeutic effect of pph is due to influence on agregate condition of blood. we considered that application of pph is very effective in pregnants with:bronchial asthma, severe forms of diabet, psorias, gestosis, allergic diseases. we used the membrane pph in pregnants: apparatus-,,hemos-pf>,, plasmofllter pmf-800,with effective area-800 cm,the volume of extracorporal contour-60 ml.such pph has no the ~ agressive effect,,, as in cases of application another extracorporal methods. this method was incalcated in our practice recently, so results will be reported in further publications. (2). post-operative cerebral neoplasm (1), post-operative subdural hematoma (1). icp was monitored via a catheter inserted in the lateral ventricle and values were continuously digitally recorded by means of a bedside computer data acquisition system (maclab). the fiberoptic tracheobroucosenpe, which guided the procedure, was passed between the nasotracheal tube and the trachea in order to avoid hypoventilalion. the patients had stable baseline hemodynaimcs. propofol infusion and fentanyl boli were administered to mantain stable mean arterial pressure values. peak (mean(sd)) icp duping the 30 minutes pre-ciaglia procedure (baseline values) were compared with values during ciaglia procedure, and the 30 minutes p0st-ciaglia procedure. data were compared with repeated measures anova. results: ciaglia procedure duration was (mean(sd)) 30 (14) objectives: transient global amnesia (tga) is a syndrome caracterized by impairment of short-term memory, inability to form new memories, retrograde amnesia and repetitive queries, without other neurological signs and symptoms. the pathophysiology of tga is unknown; thromboembolic, epileptic, migrainous and metabolic mechanisms have been suggested. to address some of these issues, we undertook a study of 25 cases of tga in whom we examined clinical, laboratory data, electroencephalogram, ct of the head, ultrasonography ecodoppler. methods: 25 patients were included in this study: 9 men and 16 women. the mean age was 64 years. all cases underwent a standard clinical examination, electrocardiogram, routinary humoral tests and x-ray, electroencephalogram (eeg), ct scan of the head, ultrasonography ecodoppler. results': the mean duration of amnesia was 5 h. 32 m. +/-7 h. 10 m. hypertension was found in 19 patients (76 %), ischemic heart disease in 4 patients (16 %), hypercholesterolemia in 10 patients (40 %), hypertrigliceridemia in 3 patients (12 %), smoking in 2 patients (8 %), atrial fibrillation in 1 patient (4 %), history of epilepsy in 1 patient (4 %), migraine history was not recorded. ct scans of the head showed multiple small deep infarcts in 4 patients (16 %), a single hypodense lesion in 4 patients (16 %). in 11 patients electroencephalogram was normal (44 %), in 8 patients there were widespread nonspecific electrical changes (32 %), in 6 patients there were focal nonspecific eeg abnormalities (24 %). conclusion: in our study tga was more common in women (64 %). we showed a prevalence of hypertension, hypercholesterolemia and cerebral infarcts compared to normal controls. we have demonstrated a higher incidence of nonspecific electrical changes in tga of lower length, while ischemic lesions in ct of the head were more frequent in tga of greater length. these data seem to be in agreement with the hypothesis that tga is a heterogeneous clinical syndrome, consisting of pure, epileptic, and ischemic types. however we did not find any correlation useful in discriminating pure from associated tga forms. from our study it is tempting to speculate that pure tga is a rare event, underlying still unknown mechanisms wich differ from ischemic, epileptic, migraineous causes. objectives: aneurysmal subarachnoid haemorrhage (sah) is special condition increasing intracranial pressure (icp) in various ways. at the other hand cerebral vasospasm and related delayed ischaemic deficit (did) could answer for the poor outcome. triple h therapy seems today a basic option to prevent did, but it may increase the icp worsening the altered intracranial pressure condition and thereby the cerebral perfusion pressure (cpp). is there any way to individualise the triple h therapy when it is necessary? methods: between sept. 94 march 95 thirty-seven patients with intracranial aneurysms were operated on within 48 hours following sah. five patients were in hunt-hess iv at admission. all patients received triple h therapy in a preventive fashion following surgery and were monitored by daily transcranial doppler ultrasonography (tcd). icp and cpp was measured in twenty-four cases. twenty-two of them received lumbar liquor drainage (lld) and nineteen were administered induced hypertension. the other group was treated by basic triple h therapy. results: in group with monitored icp the outcome was twenty-one excellent, one poor, two died (one of them died from extracranial decease). in the other group four had excellent, six moderate, two poor outcome, and one died. conclusion: according to our recent observation the patients can be divided into two groups of therapy. in group i, the patients with elevated tcd values and either low or high icp reacted to lld. we are concerned that haemodilution and slight hypervolaemia should dominate in the triple h therapy. in group ii patients having high icp with tcd and/or symptomatic vasospasm should be managed by the induced hypertensionhypervolaemia dominated therapy focusing on cpp (icp) and focal neurological signs. air emboli were detected in lo% (n=12) of natients undergoing coronary srtery bypass craftin~ (cabg). central nervous system ~ysfunction occured in 23~$ of the nstients with air embnli and in none of those ~ithhout air embo!i. hvtothermia is the classic form of oro-tect~on used dur~nc ~"~" " ~ ~ ca~.,~modu] :r, on~_,_.7 bj/oass. the surf~eon sho,;,ed thorough!~: evecnnte air from the heart, but the onesthesio!o[[ist can signifieamt!y influence the outcome by emt!oyin2~ methods to detect and treat air emboli. the changes in head rate are primarily due to alterations of autonomic tone. the heart rate variability (hrv), that express the degree of heart rate fluctuation around the mean heart rate, reflects somehow the condition of central nervous system. hrv may be measured by a number of techniques. short-term time-domain variables of hrv are reflect generally the vegal activity. in this study the changes in hrv variables of patients with brain damage, and in addition the changes in hrv measurements in comparison with the clinical evolution were evaluated. eight patient with brain damage and six normal individuals as control group were studied. a elecrocardiographer with availability of computation the sequence of beat-to-beat intervals for one minute was used. the following variables of hrv were measured: 1) standard deviation (sd) of beat to beat r-r interval differences that reflects the respiratory control, 2)the maximum/minimum (max/rain) interval that reflect variability related to baroreflex and thermoregulation and 3) the coel~cient of variation (cv), the results are shown in the in the patients with brain death and in vegetate state there were virtually no hrv. increased hrv pattern was found with clinical improvement, the changes of hrv precede of the changes of gcs, we conclude that time-domain hrv could reflects the degree of brain damage, it is good prognostic index of the brain damage and may change earlier than the gcs. objectives: cerebral co 2 vasoreactivity is an important determinant of cerebral blood flow (cbf) and has been shown to be of prognostic value in head trauma (acta anaesthesiol. scand. 1991; 35:113-122) . we wondered whether co 2 vasoreactivity could be selectively altered in one hemisphere in comatose patients. methods: 6 patients (5m/1f, age 32-65yrs, glasgow 4-8) in coma due an acute brain lesion (trauma, hemorrhage, or infection) were studied. cbf was measured bilaterally using jugular thermodilution at paco 2 25, 30, 35, and 40 mmhg by increasing pico 2 with mechanical ventilation kept constant. normal co 2 vasoreactivity was defined as an increase in cbf of at least i ml/min.100 g per mmhg paco 2. results: 2 patients had normal co 2 vasoreactivity bilaterally, 2 patients had altered co 2 vasoreactivity at both sides, and 2 patients had a normal response at one side (left or right) with an altered response on the other side (dght or left). for the 6 patients left cbf was in mean !7 ml/min.100g lower than right cbf (figure methods: following institutional approval 4 piglets (body weight 25:tl .5) were anaesthetized by 2% fluothane. a catheter was placed in the right femoral artery for blood pressure monitoring and a fiberoptic catheter (oxymetncs-3 abbott) was advanced via the right internal jugular vein to the jugular bulb for sjo 2 determinations. another catheter with a balloon on the tip was advanced in the right atrium via the right femoral vein. a mean arterial pressure (bp) at 25 mmhg was achieved by appropriate balloon inflation for 10 rain and two groups were cleated: i) the hypoxemic group by respirator disconnection (*) and it) the hyperoxemic group by fio2=l on respirator (o). samples were obtained at 0 time (1), 10' min at hypoperfusion (2) arid at reperfijsion at 1' (3), 3' (4) and 10' (5). pao2, pjo 2 and oxidative brain stress evaluation was performed from jugular bulb blood. the latter included: i) no synthase (nos) and xanthine oxidase (xo) activities by a method based on the oxidation of scopoletin detected fluorometrically, it) no levels estimated as onoo-by luminol enhanced chemiluminescence in the presence of 500~tm hydrogen peroxide (h202). resul'~s: the mean pao 2 was 34 mmt-ig for group i and methods: we retrospectively reviewed all 411 upper gi-endoscopies, performed in the period january 1992-july 1994 in 301 patients (199 men and 102 women) admitted at the 4 icu's of our hospital. results: it concerned 129 surgical, 103 medical, 50 eardiological and 19 neurological patients with a mean age of 57.9 yrs (range: 14-91). in 86%, the endoscopy was performed at the icu and in 14% at the endoscopy department. in 56% of the cases, the endoscopy was primarily diagnostic, of which 70% was performed for localization of upper gi blood loss. in 44 % the endoscopy was primarily thempentic, of which 89 % was performed for placement of a duodenal feeding canula. location of the upper gi bleeding was: variees (31%), duodenal ulcer (20%), oesophagitis (13%), gastric ulcer (11%), others (13%) and none (10%). as coincidental findings were noted: cesophagitis (37%), gastritis (16%), gastric deer (14%), duodenal ulcer (9%), duodenitis (8%), oesophageal ulcer (7%) and others (8%). conclusions: there were marked differences in indications and findings of endoscopy at the different icu's. these differences reflect an admission bias and differences in populations and treatment preferences. compared with cardiological and neurological icu's, substantially more endoscopies were performed at surgical and medical icu's. in a considerable number of cases, no source of upper gi blood loss could be found endoscopicaiiy. when upper gi blood loss was the icu admission diagnosis, the main cause was needing varices, which could be controlled endoscopically in the vast majority of cases. when upper gi blood loss was ndt the icu admission diagnosis, peigie ulcer and oesophagifis were the main causes of bleeding. because of the considerable number of coincidental almom~adities found at endoscopy, there is still room for debate whether antacid medication and/or motility stimulating agents should be given prophylactically at icu's. many studies have shown that blood lactate levels in survivors and nonsmvivors of traumatic and septic shock are significantly different. the degree of multiple organ failure is related to the duration of lactic acidosis (1). the aim of this study was to evaluate blood lactate level as a prognostic marker of high risk postoperative patients who may benefit from invasive hemodynamic monitoring and aggressive fluids administration and early inotropic support based on oxygen transport parameters. methods: 32 patients undergoing elective long term vascular and abdominal surgery (asa i-bi) were studied. blood lactate levels were measured after icu admission. in the case of blood lactate level above 2 mmoltl, measurement was repeated every 4 hours for 12 hours or until normaiisation (blood lactate level less than 2 mmol/1). type of surgery, length of surgery, amount of fluids delivered intraoperatively and postoperatively, hemoglobin levels, hemodynamic variables, diuresis, postoperative complications, length of icu stay and clinical outcome were recorded. because no attempts were made to randomisr therapy or change our standard therapy protocol institutional approval was not required. rebuts: the frequency of postoperative complications was 12,5 % and mortafity was 5,5 % in a group of patients with blood lactate level less than 2,5 mmol/l (n = 18). frequency of complications (62,5 %) was significantly increased in a group of patients with blood lactate levels 2,5-4 mmol/l (n = 8), mortality was 12,5 %. mortality (60 %) and frequency of complications (80 %) were significantly increased in a group of patients with blood lactate levels above 4 mmol/l (n = 5). conclusion: blood lactate levels can serve as early marker of high risk postoperalivr patients and may predict increased risk of postoperative complications mad ~e death. objective.~: investigated practicability and clinical value of the routine measurement of hepatic venous oxygen saturation (shvo2) after major liver surgery, as shvo 2 is considered an indirect parameter for splanchthc and hepatic blood flow. methods: 30 consecutive patients were included in this study after liver resections for primary or secondary liver tumors. 5 patients suffered from liver cirrhosis (childs a). immediately after post-operative admission on the icu a pa-catheter ,was inserted under fluoroscopy via the right jugular internal vein into the hepatic vein contralateral to the resection area. hepatic venous and arterial blood samples were drawn every two hours. shvo 2 was correlated to the clinical course, macro hemedynamics, abgs aug other established lab parameters. results: in 26 out of 30 attempts the catheter could be placed correctly. in four cases after right hemihepatectomy the left hepatic vein could not be intubated due to a dorso-lateral tilting of the left liver. this is also reflected in a significantly longer time of fluoroscopy for catheterization of the left hepatic vein (12.9 _+ %5 rain vs. 3.7 + 2.5 rain; p < 0.001). the procedure requires a total of between 45 and 75 minutes. relevant clinical complications were not observed except for short term supraventricular arrhythmias during passage of the catheter through the right atrium. hemodynamics and pulmonary function could be considered normal in all individuals at time of measurement. shvo 2 showed a span from 27.4% to 90.0% with a mean of 67.0% -+ 10.8%. the following statistically significant findings could be obtained: (a) patients with liver cirrhosis showed a significantly lower shvq than patients without (53.4% • 5.3% vs. 68.7% • 10.1%; p < 0.001). (b) a negative correlation between shvo 2 immediately after operation and the duration of intraoperative hepatic vascular occlusion could be observed (r = -0.58; p < 0.05). this correlation could also be seen for the first 12 post-operative hours (r = -0.42; p < 0.01). (c) a negative correlation between shvo2 and the difference between arterial and hepatic venous lactate levels was found (r = -0.39; p < 0.02). conclusions: the routine measurement of shvo 2 appears to be a promising extension of post-operative monitoring after major liver surgery. it is a safe method easily feasible on any major surgical icu though relatively time consuming. a further validation of this method is necessary in larger studies. therapeutic recommendations on the basis of shvo 2 findings cannot be given yet. methods: in 5 cases after major liver resection, in which abnormally low readings of shvo 2 suggested an impaired hepatic blood flow, pgi 2 was applied at a dose rate of 5 ng/kg/min. as shvo 2 can be considered an indirect parameter for hepatic blood flow, the effect of pgi 2 infusion on shvo 2 was measured. moreover, the changes of macro hemodynamics and pulmonary function were monitored. results: before the application of pgi z mean shvo 2 for all 5 patients .was 54.1% (47-9 -47-3). in three cases without major structural alteration of the remaining liver tissue the continuous intravenous administration of pgi 2 lead to a sustained increase of shvo z to an average of 67.1% (65.6 -69,1 ). the postoperative course in these three cases was uneventful. in two cases with compensated liver cirrhosis after hepatitis c no change in shvoz under pgi 2 infusion could be observed. both patients died 32 and 45 days respectively after operation in protracted liver failure. side effects of pgi 2 included a slight decrease of systemic and pulmonary vascular resistances. consequently map decreased by up to 10% as did intrapuimonary right-left shunt increase. in none of the observed patients did these side effects posed a limitation of continuous application of pgi z. conclusions: in patients without structural alteration of the liver the systemic application of prostacyclin at a dose rate of 5 ng/kg/min could significantly increase an abnormally low hepatic venous oxygen saturation after major liver resections, tn two cases of severe liver cirrhosis a similar increase could not be observed. after first clinical investigations and with the results of recent studies in animal further controlled clinical studies of prostacyclin in the postoperative management after liver surgery appear justified. any delay in gastric emptying can promote micro-aspiration and give rise to ventilator associated nosoarnnial pneumonia. h2-receptor antagonists have been suspected of promoting pneumonia by changing the gastric ph. in a few tri',ds on humans ranitidine was noted to delay gastric emptying. the aim of this prospective, randomised, blinded study was to evaluate in a ventilated icu population if there was a difference between cimetidine (c) and ranitidine (r) on the gastric filling index (gfi conclusion: in this population there was no difference in gfi between c and r; however the age and creatinine were significantly different and could have favoured the c group. also the very long t/2 could have hidden smaller differences between c and r as has been described in volunteers. between april 22, 1990 and april 19, 1993 , 102 patients with severe acute pancreatitis were admitted to 16 participating hospitals. patients were entered into the study if severe acute pancreatitis was indicated, on admission, by multiple laboratory criteria (imrie score >_ 3) and/or computed tomography criteria (balthazar grade d or e). patients were randomly assigned to receive standard treatment (control group) or standard treatment plus selective decontamination (norfloxacin, colistin, amphotericin; selective decontamination group). all patients received furl supportive treatment, and surveillance cultures were taken in both groups. results: fifty patients were assigned to the selective decontamination group and 52 were assigned to the control group. there were 18 deaths in the control group (35%), compared with 11 deaths (22%) in the selective decontamination group. (adjusted for imrie score and balthazar grade: p = 0.048). this difference was mainly caused by a reduction of late mortality (> 2 weeks) due to significant reduction of gram-negative panreatic infection (p = 0.003). the average number of laparotomies per patient was reduced in patients treated with selective decontamination (p < 0.05). failure of selective decontamination to prevent secondary gram-negative pancreatic infection with subsequent death was seen in only three patients (6%) and transient gramnegative pancreatic infection was seen in one (2%). in both groups of patients, all gram-negative aerobic pancreatic infection was preceded by colonization of the digestive tract by the same bacteria. reduction of gram-negative colonization of the digestive tract, preventing subsequent pancreatic infection by means of selective decontamination, significantly reduces morbidity and mortality in patients with severe acute necrotizing pancreatitis. ieco by sodium hypochlorite (nacio) infusion is considered to be a model of microsomal oxidation in liver on cytochrome p-450. active c10 provides oxidation of toxic metabolic products in the blood and exfused during plasmapheresis plasma, and also hydrophobic to hydrofilic transformation of substanses. sterile nacio in necessery concentrations was obtained by electrolysis of saline (0,85-0,9% naci solution) in electrochemical set e~io-4 (russin,moscow). methods: 1. the nacio in concentration 600 ragfl (400-800 ml/24h ) was administred into central veins in patients with extensive peritonitis and endotoxicosis 2-3/t. erytrocytes resistance to nacio, circulating blood volume glycemia and hemostasis were initially estimated. 2. after plasmapheresis exfused toxic plasma was mixed with nacio conccantration of i000 mg/t in 10:1 ratio in sterile "hemacons".the effectiveness of plasma detoxication and possibility of its reinfusion were evaluated by determination of albumin effective concentration (eca 35 g/l), the concanlration of medium molecular oligopeptides (mm 0,2) and other biochemical tests (bilimbin, creatinine, carbomide and so on). results: 1. the intravenous administration of nac10 excels detoxicative effect of hemosortion by 17-20% provides effictive presentation of protein components and blood cells and improves the transport function of albumin by 37%. 2. the return of exfused plasma after its purification ieco was 70-80%. only the remaning 20-30% of deficient plasma were compensated by fresh cryoplasma and albumin solutions. ischemic hepatitis (ih) is a severe complication in critically ill patients. acute circulatory failure of multiple etiology can lead to splachnic hypoperfusion and cause acute and reversible anoxic damage. over a period of 26 mos 12 pts, 8 m and 4 f, mean age 64+6.6 yrs developed liver disease compatible with ih. eight pts had a documented hypotensive episode (six pts with septic shock and two hypovolemic shock), while cardiogenic pulmonary edema in the absence of hypotension was responsible for ih in the remaining four pts. all the pts had a rapid striking elevation of ast, &lt and ldh with equally rapid resolution of these parameters to near normal wimin 9 days (mean 6.25). the mean peak level of ast, alt and ldh was 4340 iu/l (range 2105 to 7500), 3453 iu/l (range 1685 to 5150) and 2868 iu/l (range 1440 to 6960) respectively. serum total bilirubin levels rose transiently with a moan t:eak level of 1.95 mg/dl (range 1.1 to 2.7), while altered coagulation paran-,ete's (pt> 1.5 times normal) was observed in four pts and clinically significant coagulopathy with fibrin degradation products occurred in one pt (8.3%). renal impairment (cr>2.0 mg/dl) was manifest in all pts; six pts developed non-oliguric renal failure (50%) while two pts required hemodialysis. ten lots required vasoconstrictor inotropes [dobutamine (range 3-10pg/kg/min) and dopamine (range 7-25 pg/kg/min), while replacement of circulatory blood volume was performed in two pts with hypovolemic shock. eight lots expired (66.6%), but none died as a direct result of hepatic damage. the mortality rate was higher among pts with concurrent renal failure (75%). it is concluded that: 1) ih is not uncommon complication in the icu with the prognosis depending on the underlying disease. 2) clinically significant coagulopathy is uncommon complication of ih. 3) titration of inotropes is required to obtain optimal cardiac output support and subsequently liver blood flow. it is difficult to ascertain the perfusion of free flaps such as jejunal loops after surgery. objectives: to assess ischaemia as evidenced by intramural ph of jejunal free flaps used for reconstructive surgery following total pharyngolaryngectomy. methods: the sigmoid ph tonometer ( tonometrics inc.,usa ) was used to monitor intramural ph of the jejunal free microvascular flaps ( phig ) in 15 patients who underwent total pharyngolaryngectomy. a standard general anaesthetic was given and all patients were admitted to the icu for controlled ventilation and monitoring. all had similar postoperative care. phig was measured pre, post-revascularization of the flap and on icu admission, 4, 12 and 24 hours postrevascularization. objectives: to classificate the wide spectrum of itc of anp into distinct pathophysiological patterns according to presentation and course. patients (pts) and methods: 52 pts, 34 ~(65,4%), 18 (34,6%) were admitted in the icu because of anp and acute respiratory failure(arf), ilean age:54,3• years. hean stay in icu:29,2• days. 38 pts were operated, 15 of them twice. hean value of ranson's scale:4,4• (2-7). we analyzed hemodynamic measurements,arterial blood gases(abg), x-ray findings(xrf), ct-scans and operative records. results: 5 patterns of pleuropulmonary complications were identified: a)early hypoxia without xrf -33 pts. b)early ards with typical xrf -5 pts(1 died), c)early arf with xrf(atelectasis,infiltrates)-15 pts(9 died). d)late ards with typical xrf-32 pts(31 died), e)pleural effusions in various combinations with the above patterns -38 pts. overall mortality rate: 41/52 = 78,8%. conclusions: l)frequent x-rays and abg are important for the classification of itc of anp. 2)even though patterns of classification in anp are not clearly distinguishable,they facilitate an anticipatory management. 3)deterioration of abg and xrf indicates that preventive measures for arf must be intensified and agressive surgical therapy is required. 4)delay of surgical therapy is related to worse prognosis(p<o,05) and prolonged hospitalisation time(p<o,ol). in the contrary, the early timing of the operation before infection and extrapancreatic necrosis is essential for a better prognosis (p<o,05). objectives: the development of cholestasis during intensive care treatment is allied with an inferior prognosis. this study investigates the sensivity, specification and also the positive and negative forecast of patients survival -exemplary for bilirubine. methods: during a period of 12 months all surgical patients on icu were registrated prospectively (n = 1572). for documentation of disease development a standard foldcrform based on a computer-data-system was used. the results of this kind of documentation showed aapprax. 5,2 m!ll. of single patient informatioas. results: 1300 of 1572 patients didn't suffer from liver disease or have any operation on liver or bile tract. 1201 of them have had a normal scale of bilimbine in the postoperative period, a higher scale of bilirabine was regisu'atcd by 99 patients. at a ,,cut-off" of 4rag% (total-bilirubine) the sensitivity was 0,7, specivity 0,6, positive predicive level 0,8, negative predictive level 0,5 for survival criteria of a patient. the max. reached level of bilirubine, also the daily increase turned out almost the same results as they were found for other parameters nf cholcstasis like ap or gamma-gt. objective: the aim of this experimental protocol was to evaluate the morphological and functional parameters of isolated pig liver grafts, perfused in an extracorporeal liver support system. methods: the system consists of the graft liver, a membrane oxygenator (oxygenation surface 1.2 cm z, 02 flow=3 itlmin), a heater, a centrifuge pump and a fluid reservoir. twenty pig livers, mean weight 790 gr (min 610, max 870 gr) were obtained and after a mean cold ischemia time of 35 min were perfused fo} a mean of 7.5 h (min 4.5, max 9 h). perfusion solution consisted of r/l and hemacell. inflow to the graft liver was performed through the portal vein at a mean pressure of 16 (12-25) mmhg at 38~ while outflow was secured through the suprahepatic inferior vena cava. during perfusion the following parameters were evaluated through pre-and posthepatic hourly (to-t8) sampling: po2, 4" + +4" pco2, ph, hco3-, na , k , ca , osmolality, glucose, lactate, ast, alt, alp, u bilirubin and coagulation factors i, v and viii. biopsies were obtained periodicallty. b_p_~: results were as follows: mean ph fell from 7.45 at t o to 7.168 at t 8 while mean output po 2 fell from 527 at t o to 10 at t 8. mean output ast values increased from 361 at t o to >2500 at t 8 while mean output alp values increased from 3.66 at t o to 197 at t 8. mean output k + values increased from 3.93 at t o to >8 at t 8. histology revealed lesions of ischemic necrosis, more prominent after t 6. conclusion: results show that the isolated liver graft presents satisfactory function and morphology at least for a five hour perfusion period in the described extracorporeal circuit. correction of ph contributed to an increase in bile flow. between 1982 and 1993 the practice of transplantation has changed drasticaily in switzerland -besides kidneys also hearts, heart and lung, lung, iiver and pancreas transplantation has started in several centers. major information efforts have been made, organ exchange rules were set up and a national coordination center was initiated. the aim of this retrospective single center study was to assess the influence of transplantation on organ donation. in the past eleven years 205 organs were donated from 458 potential donors i139 single, 66 multi organ donations) analysis of refusal was evaluated categorized into medical and/or familiar reasons. the number of potential donors increased from 28 (1982) ,to 61 (1992) with a concomitant drastic reduction of donations from 64% in 1982 to 26% in 1992; amounting to a net unchanged number of donations over the last 10 years (1982 = 18; 1992 = 17) . the import and export of donor organs was balanced since the introduction of the national coordination center. in contrast multi organ donation increased from 0% in 1986 to 90% in 1993 despite of the more stringeant selection criteria, in conc]usion the introduction of a full range of transplantation procedures at several new university programs and the increase of multi organ donation has not had the forecasted impact on organ donation despite a sustained informative and promotional campaign, objective: monitoring hepatic venous oxygen saturation (svho2) provides online information about hepatic-splanchnic oxygen supply-demand ratio [1]. previously, x~ reported hepatic venous catheterization in patients undergoing orthotopic liver traru~lantation (olt) [2] . in the present study, we assessed the effects of nitroglycerin (ng), a vasudilator that affects the venous capacitance vessels more than arterial vessels and prostaeyclin (pgi2, flolan r~, wellcome, uk), an arterial and splanchnic vasodilator on hemodynamies and hepatic venous oxygen saturation (svho2) in human liver transplantation. methods: with institutional approval and informed consent, 14 consecutive patients, mean age 50-2-_10 years, were studied following olt. postoperatively, fiberoptic pulmonary artery catheter was inserted into the right hepatic vein. timed infusions of ng at a rate of 0.1 gg/kg/min and pgi2 at 5 ng/kg/min were initiated for a 45 rain period. each sequence was followed by baseline therapy for 45 rain. results are expressed as mean=tsd. statistical analysis was performed using friedman's-two-way-anova-test, significance was accepted at p<0,05. results: ng at 0.1 gg/kg/min induced a decrease of mean arterial pressure (map) (84_49 [baseline] vs. 75+9 mmhg) and pulmonary artery wedge pressure (pcwp) (8j:2 [baseline] vs. 65:1 mmhg). cardiac index (ci) (5-41 vs. 4+1 l/rain/m2), oxygen delivery index (do2i) (655-+108 vs. 618+123 mgnfin) and svho2 (74_~12 vs. 69-l-_19%) were decreased (p<0.05). pgi2 at 5 ng/kg/min induced a reduction in map (73• nm~. _g) and pcwp (6+1 mmhg). ci (6_+1 l/rain/m2), do2i (7555:135 ml/min) and svhoz (81+6%) were increased (!o<0.05). 9 vasedilatation induced by ng decreased systemic oxygen supply and impaired splanclmie oxygenation. 9 pgi2 increased systemic oxygen delivery in parallel with svho2, suggesting a corresponding improvement of hepatic-splanchnic okygenation. 9 thus, if vasedilator therapy is indicated in th6 15orient receiving liver grafting, pgi2 appears to be advantageous. however, due to its platelct aggregation inhibiting properties, the usefulness and safety of pgi2 in olt patients has still to be determined. objectives: to analyze the effect of steroid treatment given to donor on the early function of transplanted kidney. methods: from january, 1994 until now 56 donors were involved into this prospective study. every other donor was treated with 30 mg/kg solu-medrol one hour before organ retrieval. according to the steroid treatment of the donor the recipients were divided into two groups: group 1 -steroid pretreatment goup (y~=35), and group 2 -control group (n=37). the donors and the recipients were treated using the same kidney transplantation protocol onl~r the adults, and the first cadaver kidney transplanted patients were involved into the study. the daily routine parameters were analyzed pre-and intraoperafive, and on the 0-5th, 14th and 30th postoperative days. results: we could not show any clinically important differences between the two groups in respect of donor parameters. preoperative, the patients in group 2 had slightly lower ereatinin level (819 -+ 244 g.,non vs.923 -+ 254gmol/1) which persisted into the early postoperative phase. the values of the other examined pre-and intmoperativc parameters were almost the same. during the first 5 postoperative days the patients in group i needed less diuretics (furosemide and renal dose of dopamine) and their sodium excretion was closer to the physiological range than in group 2. the other parameters did not differ significantly. the less furosemide need in group ! pe~isted to the end of the first month. conclusions: according to our data the steroid treatment of the donors improves the early function of the transplanted kidney in some respects. to prove the real benefit of the donor steroid treatment needs more data and further analysis. objectives: severe infections may compromize the outcome of liver transplantation..determination of new parameters may increase the knowledge of pathophysiologic mechanisms and may lead to changes in postoperative therapeutic management of patients at risk. methods: between august 1993 and september 1994, 81 patients with 85 transplants were monitored for cytokines and extracellular matrix pammeters on a daily basis. serious infections (n=10) included microbiologic evidence and more than 2 secondary organ failures. patients with cholangitis (n=ll) or uneventful postoperative course (n=37) referred as control groups. results: 1-year patient survival was 88.9% (72/81): 5 patients died due to serious infections, while 4 died for other reasons. mean bilimbin, stnf-rii-, ifn-7-, il-4-, il-8-, il-10-, laminin-and neopterin levels were significantly elevated in patients with serious infections compared with patients experiencing mild cholangitis or with an uneventful postoperative course. a further increase of all parameters was observed in patients who subsequently died; tnf-ri/: 28310_+788 pg/ml vs 20452• pg/ml; ifn-7:466_+57 pg/ml vs 4.4-+1.8 pg/ml; il-4:214-+35 pg/ml vs 148-+29 pg/ml; il-8:667-+48 pg/ml vs 251_+26 pg/ml; il-10:149_+52 pg/ml vs 52• pg/ml; laminin: 3010-+312 ng/ml vs 1263-+ 117 ng/ml; neopterin: 247_+37 nmol/1 vs 96_+19 nmolb for non surviving vs-surviving patients. a significant decrease of sialic acid yeas observed in patients with serious infections; and a further decrease occurred in patients who subsequently died: 455-+31 mg/l vs 685• mg/1. conclusions: the increase or decrease of various cytokines and extracellular matrix parameters may be indicative for severity of infectiolx routine monitoring of these parameters may improve current diagnostic tools and poss~ly lead to changes in therapeutic management of patients at ~k. objectives: evaluation of the cytokine network after liver transplantation may give some insight in pathophysiologic mechanisms of rejection and may lead to detection of patients at high risk. methods: 81 patients with 85 transplants were monitored for various cytokines on a daily basis between august 1993 and september 1994. rejection was assessed by histology in combination with clinical signs of rejection and laboratory investigations. results: during the first postoperative month, 28 patients (34.6%) developed rejection; 14 patients were successfully treated with methylprednisolone (steroid-sensible rejection), while further 14 patients required additional treatment with fk506 or okt3 (steroid-resistant rejection). 4 patients subsequently developed chronic rejection. mean levels of various cytokines and extracellular matrix parameters including tnf-rii, ifn-7, il-ib, il-2r, il-4, il-6, il-8, hyaluronic acid and neopterin were significantly higher in patients with steroid-resistant than in patients with steroid-sensible rejection. a further increase of some parameters was observed in patients who subsequently developed chronic rejection; bilirubin: 18.2-+4.1 mg/dl vs 11.2-+1.7 rag/all; tnf-rii: 23374-+798 pg/ml vs 18246_+679 pg/ml; il-8:1024+-192 pg/ml vs 275-+67 pg/ml; neopterin 148_+37 nmol/1 vs 49-+21 nmol/1; hyaluronic acid: 290_+63 ~tg/l vs 223_+28 ~tg/l for patients with chronic versus patients with acute steroid-resistant ~ejection. sialic acid levels decreased in patients with acute steroidresistant rejection; and a further decrease was observed in patients who tieveloped chronic rejection: 437_+34 mg/l vs 671_+55 mg/1. ~onclusions: various cytokines and extraeeuular matrix parameters were indicative of severity of rejction. the extensive increase of bilirubin, tnf-ii, il-8, hyaluronic acid and neopterin may indicate subsequent chronic ection. monitoring of these parameters may, therefore, lead to changes in immunologic management after liver transplantation. background : combined kidney and pancreatic transplantation is being performed with increasing frequency in patients with diabetes mellitus and renal failure, as it offers more chances of success and better results than kidney transplantation alone. mycotic arterial aneurysm constitutes a devastating complication following pancreatic transplantation. all cases of mycotic arterial aneurysms have been however reported with exocrine pancreatic drainage into the gastrointestinal tract. intervention : we describe a series of 8 consecutive whole kidney-pancreas transplantation performed at the university of geneva hospitals (1500 beds) between december 1992 and may 1994. exocrine pancreatic drainage into the bladder (epdb) was performed to improve early detection of rejection episodes. epdb was hypothesized to reduce the risk of contamination from the gastrointestinal tract and the subsequent possible occurrence of potentially fatal infectious complication. in all patients the dual transplantation was performed through a median incision according to the procedure described by nghiem. results : two out of the 8 patients who received kidney-pancreatic transplant developed arterial mycotic aneurysms 15 and 35 days following surgery. aneurysms developed at the site of the arterial anastomosis used to rearterialize the homograft. both patients had peritonitis caused by candida albicans requiring surgical drainage and intravenous antifungal therapy. rupture with hemorragic shock occured in both patients leading to graft removal in one patient, and three episodes of lffetreateniug hemorragic shock followed by graft failure and removal 32 days after transplantation in the other. conclusion : arterial mycotic aneurysm constitutes an early, lifetreatening complication of kidney-pancreatic transplantation; it mandates graft removal. although exocrine pancreatic drainage into the bladder consitutes a definitive advantage for caller diagnosis of graft rejection, it does not eliminate the risk for retrograde colonization and subsequent severe infection in our experience. s. bocharov, i. teterina, regional clinical hospital, irkutsk, russia acute profound loss of blood can result from the very different injuries and hepato-pancreato-duodenai operations enter such a rank. ill-timed and inadeguate correction of operation hemorrage is one of the reasons for postoperation complications, including polyorganic insufficiency. the pathogenesis seems to be very complex. in early stages of bleeding the liquid enters the vessel bed, followed by hypoproteinosis and hematocrit fall. however, as decompensation develops, the fluid leaves the vessel system in the result of increasing postcapillary resistance and lowering col-ioidnooncotic blood pressure (cop). the resulting hypovolemia causes primarily acute disturbance of central hemodynamics and then of microcirculations and transcapillary exchange. central hemodynamic failure after acute loss of blood manifests itself through cardiac output lowering and capillary blood flow deceleration. taking into consideration, that 35 % is critical value for cpv loss and for cev it is 65 %, we consider arising the level of cop to the immediate task. cop raising allows to normalize transcapillary exchange, which we assess through cop and mcp (mean capilary pressure) gradient. the next task is to make up for globular volume till homeostasis providing level. considerable attention is given to catabolism inhibition and maximum possible enegry provision. control over high proteolitic activity of blood and callicreinkinin system activity implies direct proteases inhibitors. reologic, membrane stabilizing, antihypoxanthine and anticoagulant therapies are obligatory. virehow clinic, dept. of surgery, humboldt university berlin, germany regarding a high mortality up to 85 % of fulminant hepatic failure orthotopic liver transplantation seems to be the only promising therapeutic approach in many cases. this study shows experiences from a transplantation center. between june 1991 and april 1995 39 patients suffering fulminant hepatic failure were admitted to our surgical intensive care unit all patients showed severe liver dysfunction with grade ii to iv encephalopathy. after a period of diagnostics and conservative treatment ranging from few hours to 10 days (mean 2.4 days) we reported 22 of these patients as possible organ recipients to eurotransplant. all of these 22 patients were transplanted within 48 hours, 16 (73 %) of them even within 24 hours. the principal aetiologies were hepatitis b (7), hepatitis c (1), nanb hepatitis (5), mushroom poisoning (amanita phalloides 1). after transplantation 2 patients suffered from initial-non-function and underwent re-transplantation. the one-year-survival rate was 82 %, 5 patients died within 3 months after transplantation due to various reasons. 17 patients were not referred for liver transplantation. 10 of them never met transplantation criteria, improved by conventional therapy and could finally be discharged from hospital. the known reasons for liver failure in this group were mushroom poisoning (4), paracetamol intoxication (4) and fulminant hepatitis a (1). 7 patients suffering from fulminant hepatitis (6) or intoxication (1) were excluded from emergency liver transplantation for various contraindications. 6 of these 7 patients (86 %) died despite conventional intensive care. we don't know if some of the patients in the transplantation group would have survived without transplantation, because whenever we decided on transplantation we could perform the operation within 48 hours. but 9 the good survival rate in the transplantation group (82 %) 9 the 100 % recovery rate in the group, where there was no transplant-indication in our opinion 9 and the fatal outcome (86 % mortality) in patients with contraindications are an encouraging proof of a successful therapeutic strategy in acute liver failure. these results are based on a close cooperation between experienced transplant surgeons, hepatologists and intensive care doctors, using sophisticated laboratory and imaging techniques in a specialized center. introduction: during brain death patients suffer from multiple endocrinologic disturbances. one of the most important are those related with thyroidal axis. it is well described the euthyroid sick syndrome whose more frequent pattern consist of decreased triiodothyronine (t3), increased reverse t3 (rt3) with normal levels of tetraiodothyronine (2"4) and tsh, this lacking in "1"3 levels lead to a change from aerobic to anaerobic metabolism which results in tissular damage. objective: 1.to study thyroidal pattern in brain death patients potential organ donors. 2.to avoid organ impairment by administration of t3. 3.to study the hemodynamic and hormonal changes after the administration of t3 in these patients. material and methods:population: 22 brain death patients of any etiology potential organ donors admitted to the intensive care unit. patients were classified in hemodynamically stable (group 1) and unstable (group 2). group 2 received a bolus of 0.25p.gr/kg. and a perfusion at a dose of 2-3.5 p.gr]h of t3. hormonal assays: total t3 (tt3), total 2"4 (tt4), tsh. fxee t3 (ft3), free 1"4 (ft4) and rt3 were determine at the moment of clinical brain death (0 hrs) and in group two these assays were repeted at hours 4, 8 and 12. results: 22 patients (17 male) with a mean age of 33 years (range 17 to 71yrs.) were studied. the clinical brain death was confirm later with other explorations (eeg, doppler). there were 15 patients in group 1 (68,1%) and 7 patients in group 2 (31,8%). hormonal pattern: at the moment of brain death tt3 was normal in 21 cases (95,4%) and decreased in i (4,6%); tt4 was normal in 9 patients (40,9%) and decreased in 13 (59,1%); ft3 was normal in 3 cases (i3,6%), decreased in 19 (86,4%); fl'4 was normal in 19 patients (86,4%) , decreased in 3 (13,6%) .rt3 was normal in 17 cases (77,2%) and increased in 5 cases (22,8%). there were no statistically significant differences in hormonal pattern between the two groups. only t3 levels at hours 0, 4 and 8 were significant in group 2. in the 19 cases with ft3 decreased, the tt3 was normal in 18 (84%) and decreased in 1 (16%), tt4 was decreased in 11 (57,8%) and normal in 8 (42,1%), tsh was decreased in 1i (57,8%), normal in 7 (36,8%) and increased in i(5,2%) and ft4 decreased in 3 (15,7%) and normal in 16 (84,2%) and rt3 was normal in 14 (73,68%) and increased in 5 (26,3%). there were no statistically significant differences in cardiac index, vascular resistances and pulmonary shunt before and after the administration ef t3. conclusions: 1. the hormonal pattern most often find in brain death patients was: normal tt3, decreased tt4, normal tsh, decreased ft3, normal fr4 and normal rt3. 2 . there were discrepancies in the values of ft3 and tt3 3. there were no statistically significant differences in hemodynamic and pulmonary parameters. objectives: magnetic resonance angiographie (mra), a non-invasive procedure, provides flow-related information additionly to the anatomy of the vascular system. measurement of signal intensity and edge detection of vessel structures permits to calculate blood flow velocity and vascular diameters. we examined whether cerebral hemodynamic changes by altering the arterial pressure of carbon dioxid (pace2) could be detected by mra. methods: following institutional approval and informed consent, 10 mechanically ventilated patients without elevated intracraltial pressure underwent mra with defined periods of hyper-, hypo-and normoventilation (pace2: 30, 50, 40 mmhg; arterial blood gas probes; avl). mra was performed with a 1.5 tesla magnetom (vision, siemens). two different mra techniques were used: a conventional time-of-flight-3d-angiography (tr: 39 ms; te: 7 ms; fl: 20 deg; slab: 56 mm) for vessel diameter detection and a flash-2d-gradient-echo-sequence (tr: 28 ms; te: 5 ms; fl: 30 dog) for measurements of blood flow velocity. an axial view parallel to the ac-pc-iine (anteriorposterior-commissur-line) was used for repeated imaging of identical regions of interest 0toi) of the proximal part of the internal carotid (ica) and middle cerebral artery (mca) as well as of peripheral branches of the mca and the posterior cerebral artery (pca). results: changes of pace2 correlated with changing signal intensities, whereby under hyperventilation a decrease of 23,7% (p 0.01) and under hypoventilation an increase of 28.4% (p 0.01) was observed compared with normoventilation. blood pressures were stable throughout the whole study period, pace2 dependent changes in vessel diameters were more pronounced in peripheral branches of mca and pca. a change from normo-to hyperventilation produced a decrease in proximal vessel diameter of -3.5% (p _< 0.01) and in peripheral diameter of -22.2% (p _< 0,001). a change from normo-to hypoventilation produced an increase in proximal diameter of +6.1% (p < 0.05) and of +21.3% (p -< 0.001) in peripheral diameter. conclusions: pace2 related changes of cerebral vessel diameter can be easily detected by mra without injecting a contrast agent. the results confirm that co2-reactivity is more pronounced in peripheral cerebral vessels, which are subjected to greater changes in diameter than major basal arteries. hyperventilation leads to a decrease and hypoventilation to an increase in signal intensity thus reflecting the corresponding changes in blood flow velocity, intensive care unit (icu) of "kat" hospital, athens, greece, ob!ective$; the value of bronchoscopy in pulmonary atelectasis of icu patients is under question the presence of an air bronchogram sign in xrays, which is considered as evidence of central bronchus patency, is referred in several studies as a negative criterion for bronchoscopy, whereas its absence as a positive one. it is also referred that air bronchogram sign correlates with delayed resolution of atelectasis, probably because of obstruction of many periferal airways (not central). the purpose of this prospective study was the evaluation of the air bronchogram sign on frontal chest film as a negative criterion for bronchoscopy and as criterion of delayed resolution of atetectasis, methods: icu patients with atelectasis were studied prospectively. they underwent bronchoscopy, bronchoscopic findings, presense of air bronchogram sign, and outcome of atelectasis were recorded, correlations were made, between: 1) bronchoscopic potency of airways and air bronchogram sign 2} resolution time of atelectasis and broncoscopic potency of airways. 3) resolution time'of atelectasis and air bronchogram sign, methods of statistical analysis were the t-student test and the chi square test, results:the patients were 23, men 19 women 4, seventeen patients had atelectasis of whole lung, 3 of upper lobe, and 3 of lower lobe. ten patients had atelectasis in right and 13 in left lung. eight from 28 patients had air bronchogram sign in x-ray, there was no statistical correlation between air bronchogram sign and bronchoscopic potency of airways [6 from 8 patients with air bronchogram sign (75%) and 11 from 15 without air bronchogram sign (73%), had bronchoscopic potency of airways, p>0.1], resolution time of atelectasis didn't correlate statistically with bronchoscopic potency of airways (mean resolution time in patients with bronchoscopic potency 2,29 days and in bronchoscopically closed bronchi 2,33 days, p>0,1). there was also not a statistical correlation between resolution time of atelectasis and air bronchogram sign (mean resolution time in patients with air bronchogram sign 2,25 days, and without air bronchogram sign 2,33 days. p>0). conclusion~i; the presense of an air bronchogram sign in x-ray of icu patients with atelectasis, does not coexist obligatorily with bronchoscopic patency of airways and cannot be used as a negative criterion for bronchoscopy, neither as a criterion of delayed resolution of atelectasis. th. wertgen chest sonography (cs) is routinely used in our department to examine icu patients with clinical symptoms of pulmonary embolism, pneumonia, pleural effusion or unclear chest pain. we perform cs with a sector transducer (4.0 mhz) and a linear transducer (7.0 mhz) using acuson 128xp/10 c. the sonographic signs of pulmonary embolism and infarction are most well demarcated, mainly wedge shaped and triangular pleural based lesions, more roughly structured, observed with a hyperechoic reflex in the center corresponding to the bronchitic (fig. 1) . pneumonia is characterized by homogenously hypoechoic, wedge shaped parenchymal lesions, containing air or fluid bronchograms; they move with respiration (fig. 2) . pleural effusions are spaces of various echogenicities, from anechoic to homogeneously echogenic, which may contain floating strands or complex septa, located between visceral and parietal pleuras (fig. 3) . from march 1994 to april 1995 we did 55 examinations by cs in 34 icu patients (20 male, 14 female; age from 29-87). patients examinations pulmonary embolism 10 16 pneumonia 7 16 pleural effusion 13 19 us-guided thoracic punctions were performed in 7 patients. in two patients we found pneumonia or pleural effusion caused by a lung carcinoma. another two patients showed a normal cs (diagnosis: inflammation of the gall bladder, inflammation of the myocardium). conclusion: cs is a very useful method for icu patients with chest diseases. it takes less time and is less expensive than ctand sometimes of a higher diagnostic value than x-ray. last but not least cs is invaluable for the icu patient, because the examination is done save and quickly at bed side and the results of cs are very helpful in diagnoses and treatment. results : inter-observer reliability was evaluated as an 83 % concordance. results of the tee classification were : class 0 : n = 21 (34 %) ; class 00 : n = 13 (21%) ; class 1: n = 7 (12 %) ; class 2 : n = 8 (13 %) class 3 : n = 12 (20 %). therapeutic implications of tee in class 3 patients were : cardiac surgery in 5 patients (two cases of acute mitral regurgitation, two valvular abscesses and one hematoma compressing the left atrium), discontinuation of peep in one ventilated patient with an atrial septal defect, weaning of mechanical ventilation in one patient with an atrial septal defect, prescription of antimicrobial therapy in 8 patients with endocarditis and prescription of anticoagulant therapy in 2 patients with left atrial thrombus. the only noteworthy complication was a case of spontaneously resolving supraventrieular tachycardia. conclusion : tee is safe and well tolerated, and is useful in the management of icu patients with shock, unexplained and severe hypoxemia or suspected endecarditis. the aim of this study was to determine whether ultrasound guidance can help interns to improve the results of jugular vein access in icu. methods : in a prospective and randomized study, we compared, in 79 patients admitted to the icu, an ultrasound-guided method (ultrasound group : 37 patients) with an external landmark guided technique (control group : 42 patients). all jugular vein accesses were performed by young interns with an experience of < 5 procedures. results : internal jugular cannulatian vein was aci~ieved in all patients in the ultrasound group and in 10 patients (24 p.cent) in the control group (p < 0.01). average access time was longer in the control group (235 • 408 sec. vs 95 • 174 see. ; p = 0.06) and puncture of the carotid artery occurred in 5 patients in each group (p = 0.83). 32 patients (86 p.cent) in the ultrasound group and 23 patients (55 p.cent) ia the control group (p < 0.05) were cannulated in 3 rain. or less. the cannula was therefore unabie to be inserted within 3 minutes in 19 patients in the control group, with failure of eannulation in 10 of these patients (53 p.cent). failure was due to thrombosis (n = 1), small calibre of the internal jugular vein (< 4 ram) (n = 5), abnormal vascular relations (n = 3) or cervical irridation (n = 1). among the 10 primary failures of cannulation, an internal jugular vein catheter was able to be inserted in 4 cases by an experienced physician on the side initially selected and with ultrasound guidance in 2 cases. the catheter was inserted into the contralateral internal jugular vein under ultrasound guidance in the remaining 4 cases. jugular cannulation was obtained at the first attempt in 26 p.cent in the control group and 43 p.cent in the ultrasound group. conclusion : ultrasound guidance improved the success rate of jugular vein cannulation by inexperienced operators in icu patients. when the internal jugular vein has not been successfully eannulated within 3 minutes by the external landmark guided technique, the authors recommend the use of the ultrasound guidance. in the majority of cases right atrial or ventricular thrombi represent pulmonary emboli in transit. these may be fatal in patients (pts) treated conservatively with anticoagulation only. in literature the incidence of right heart thrombi in pts with proven pulmonary embolism (pe) is said to be in the range of 3-4%. extremely mobile, long, worm-shaped masses in the right heart cavities carry an especially high early thrombus-related mortality rate which ranges from 40-50%. current therapeutic strategies favour fibrinolytic therapy with consecutive anticoagulation. we report five cases (4 male, i female, 55-74 years) of right heart and pulmonary thromboembolism. in these pts diagnosis and regression of thromboemboli following systemic intravenous lysis therapy with recombinant tissue-type plasminogen activator (rt-pa) was documented by transesophageal echocardiography (tee). a submassive pe occured in 3 pts, a massive pe in 2 pts. one patient (pt) had a cardiac arrest. in all 5 cases tee clearly identified the extensive thrombns formation in the right-sided cavities of the heart and in the central pulmonary artery in 2 cases. all pts were treated with 100 mg rt-pa, 3 pts in a front-loaded regimen over 90 minutes, 1 pt over 120 minutes, and, due to the life threatening situation, in one case a bolus injection as ultima ratio was performed with no intracerebral bleeding complication. regression of thromboembolic masses after fibrinolytic therapy was demonstrated by transthoracic and transesophageal echocardingraphy after 1 to 15 hours. all pts survived and were put on coumadine, 1 pt developed an intracerebral bleeding with persistent hemiplegia. conclusions: the use of thrombolytic therapy is highly efficacious for the therapy of pts with pe and concomitant right or ventricular thrombus formation. transthoracic and especially transesophageal echocardiography are powerful bed-side diagnostic tools for the immediate diagnosis and follow-up of successful treatment in this life-threatening condition. although widely used, catheterisation of the femoral vein in the groin using "landmark" technique is frequently complicated by accidental arterial puncture. suboptimal hygiene and patient discomfort are also associated with this technique. with regard to these last two factors cannulation of the femoral vein 10-20 cm below the inguinal ligament would seem an attractive alternative. as "landmark" technique is not possible for the cannulation of the femoral vein in this part of the thigh, ultrasound was used to locate the vessel and the results of this technique were evaluated. methods: a portable compact ultrasound device (site rite,dymax corp.) featuring a 7.5 mhz transducer (ultrasound depth 4-5 cm) fitted with a needle guide and a 6 cm screen was used by residents with no previous experience in ultrasound guided cannulation. patients consisted of a surgical icu population. results: in 46 patients 55 catheters were introduced.in 6 cases more than one (2-4) attempt was made and in 3 patients the procedure was unsuccesfull due to the fact that the vessel was situated out of reach of the ultrasound (vessel depth > 4-5 cm), during the 55 procedures one accidental arterial punction was registered. the catheters remained in situ for a mean of 9 days (range 1-22) and were used for volume suppletion, medication, parenteral nutrition and haemodialysis.co-ionisation rates compared to those of subclavian catheters in our icu. in the first 20 patients 3 cases of asymptomatic thrombosis of the femoral vein were seer on ct-scans performed for other indications, in the following 26 patients duplex scanning performed after removal of the catheter yielded another 3 cases of asymptomatic femoral vein thrombosis. conclusions: ultrasound guided femoral vein catheterisation 10-20 cm below the inguinal ligament is a safe and simple technique that can easily be performed by residents without prior experience. the incidence and impact of thrombo-embolic complications associated with this technique are still subject to further investigation. objectives: to estimate the cost of antibiotherapy (ab-cost) in a multidisciplinary 8-bed greek icu and to correlate ab-cost with total cost of drugs and consumables and with patient's outcome, severity of illness and type of admission. methods: prospective data from 137 consecutive patients admitted to the icu from 1/10/1994 to 30/3/1995 were studied. a tick chart was designed to record all drugs, materials and consumables regularly used for icu patients, but did not include low price drugs and consumables, which are provided from hospital's pharmacy as stock and were included in a fixed icu cost calculated for a 12 month period. the chart also contained demographic details and data necessary for the calculation of several illness severity scoring systems. obiectives: over 3 years evaluate the necessary efforts and expenses to implement a cis in the routine of a 16-bed stcu. methods: in june 1992 a commercially available, unix-based cis was installed on a 16-bed surgical icu. the goal was a paperless documentation at the bedside. after more than 2 years clinical experience two aspects were investigated: what effort is necessary to install and support a cis, and what is the benefit for patients and personnel on the icu? results: the installation and support of a full-fledged cis requires a considerable effort: (a) the conceptual framework for the cis has to be defined. this includes the definition of documentation standards, as well as nursing and therapeutic standards, which is the essential basis for the configuration of any cis. (b) configuring a cis, i.e. "fine-tuning" it to the user's specific needs, is always a laborious task. moreover, constant maintenance is necessary. these tasks require the following personnel: experienced health care professionals for defining the conceptual framework, 1-3 trained health care professionals for configuration, 1 system administrator. on a single icu (12-20 beds) these are not considered full-time jobs. (c) training is best done employing the "train-the-trainers" approach. (d) beside the necessary amount of man power and money to install and purchase a cis, administrative and mis support is needed, especially when interfaces to the hospital and laboratory information systems have to be set up. in general, a cis needs the commitment of all people involved. without a really professional approach with a longterm goal any major cis can turn into an unnecessary but inevitable night mare. after 3 years clinical use and a thorough implementation of a cis on a major sicu it can be said that full-fledged cis offers an opportunity to dramatically improve the working environment on an icu. moreover, it adds to patient safety, quality of care and cost efficiency in one of the most advanced and expensive areas of medicine. conclusion: a major investment in man power and money is necessary to install and maintain a full-fledged cis. a sincere professional commitment to the goals of a cis is necessary. in exchange, a well configured and well maintained cis dramatically improves the quality of therapy and care on the icu. even return of investment and financial profitability of a cis seem feasible todayl from the clinical perspective it appears that the users themselves are the central determinant whether a cis makes a dream come tree or turns into a night mare. objectives: to establish a relationship between the activities of the staff and the occurrence of auditory alarms on the i. c.u. ard to evaluate confusion between auditory alarms. methods: laboratory based studies which investigated aspects of confusion between alarms in current use on the i. c. u. the observational studies were conducted over an 18 month period and examined the frequency and duration of alarms together with the concurrent activites being undertaken by staff on the unit. the laboratory based studies showed that there were enduring confusions between the alarms on various items of medical equipment, for example a ventilator alarm and an e. c. g. monitor alarm. the results of the observation studies demonstrated that alarms are activated when specific activities are being undertaken by staff. sounds could be used in future recommendations for alarms on medical equipment. suggestions are also discussed for improving and rationalising auditory warnings in the i. c. u. obiectives: we investigated inferior petrosal sinus (ips), the lowest affluent to jugular bulb (jb), as a possible source of contamination of samples in jb for monitoring oxyhemogiobin saturation (sjbo2). pulling back the catheter the oxyhemoglobin saturation usually rises indicating extracerebral contamination (jakobs en met al: j cereb blood flow metab 1989;9:717). methods: the study was carried out on patients undergoing ips sampling to differentiate cushing disease from ectopic acth syndrome and to lateralize any resulting pituitary lesion. we studied the value of oxyhemogiobkn saturation high in jb (sjbo2), at ips (sipso2) and at mid jugular vein (5th cervical vertebra) (smj 02) bilaterally. results: we found significant differences between right sjbo 2 and both right sipso 2 (p= 0.007) and right smjo 2 ( p= 0,017) and between left sjbo 2 and both left sipso 2 (p= 0.01) and left smjo 2 (p= 0.017) we did not fred any difference bilaterally. objectives: we studied various methods of receiving and editing of clinical datas in critically ill patients (different ethiology). 163 patients were investigated in regional intensive care center. methods : the following datas were studied : anamnesis, status praesens objectivus ( organs and systems ) ,. clinical and biochemical markers of critical condition , datas of eeg ,rheography . the medical information complex contained : 8channel electroencephalograph, 4-channel roencephalograph, ad-converter (16 analog inputs, 12 bit resolution, 60 k hz), ibm 486 dx2, software includes set of routines for spectral eeg analysis, eeg-mapping, correlative analysis, and brain bloodstream reg-monitoring (written in turbo pascal 4.0), expert programs for estimation objective and humoral patient status (written in clipper 5.0) and statistics. there were used following programme-language instruments : borland c++ 3.0, nantucket clipper 5.01, ca-clipper tools ii. as the methods of statistical processing of dates were used: t-students criterion , fisher criterion, methods of correlation analisis, calculation of the regression levels, dispersion analysis, results : there was created the optimal structure of hard and sofware complex of search steady objective regularity in dynamic of critically ill patients condition. conclusion : the created system allowed to value effectiveness of intensive care and give us new opportunities in study pathogenesis of systems disorders in critical condition . over a five year period a patient data management system has been installed which allows individualised patient data to be accurately collected. using this data a costing system has been developed which ascribes costs thus: 1. direct costs -drugs, fluids, consumables, interventions. these are ascribed to individual patients, according to data collected from the pdms. 2. indirect costs -energy, depreciation, admm costs, maintenance etc. these are summed for the year and ascribed as an overhead per patient day. n.b staffcusts contain art element of both cost types the aim is to make as many costs as possibie 'direct', hence 'activity costs' have been calculated winch comprise staff time, drugs and consumables -these are direct costs. these costs of patient care are then searnlessly integrated into the financial and budget management of the icu environment. it was found that by calculating costs in this manner 50% of the total cost of icu are captured within the 'direct' element, and so are able to be ascribed to individual patients. this is much more accurate than simply dividing the total costs of ~cu by the number of patient days. temporal costs (variations during patient stay) and cross sectional costs (cost differences between admitting specialities) were also noted with interest. results of the initial analysis of data captured by the system will be presented. little is known about the resource costs (not simply cash costs) of icu. even less is known about individual patient costs, with previous estimates of these costs varying widely. however, if cost effectiveness studies are to be undertaken accurate calculation of individual, group and total icu cost is an essential, prerequisite, which, via this system of costing, is now achievable. information about intensive care of cancer patients is limited in the literature, despite the increasing use of such facilities in oncology over the two last decades. in order to determine if and how critical care facilities can be used specifically for these patients, we performed a world-wide inquiry in anticancer centers selecting the hospitals by using the international directory of cancer institutes and organizations. we mailed a questionnaire to 146 centers and we received 84 responses (57.5 %). there was at least one uncological (i.e. with > 50 % of cancer patients) icu in 59 (% % an 18-year old woman with graves disease presents with sore throat, vomiting, diarrhea, sinus tachycardia at 155/minute and a temperature of 40~ several weeks before, treatment with propylthiouraeil had been stopped (rash and fever) and replaced by methimazole and ledide prior to a minor surgery. however, both drugs were discontinued by the patient two weeks before admission. shortly after arrival in hospital, patient's condition progressed to respiratory failure (upper airway edema), delirium and shock requiring icu admission, intubation and resuscitation with fluids and vasopressors. white blood count was 1300/mm ~ with 0 neutrophils. patient's hemodynamic data showed initial hyperdynamic profile followed by low output state with decreased sv02 (59%) (n 70-80%) and cardiac index (2,37) (n 2,5-3). echocardiogram confirmed cardiac chambers dilation as previously described in thyroid storm. lithium carbonate, corticosteroids, antibiotics and beta-blocker perfusion were given. plasmapheresis was started. free t& (n=9,2-21pmo/l) went from 143,6 to 16,6 after the first two pheresis. after a remarkable clinical recovery, sub-total thyroideetomy was done i0 days after admission. in life-threatening thyroid storm, plasmapheresis is a very effective therapy when anti-thyroid drugs are counterindicated. purpose: to compare the reliability of prognostic indexes in crhically iu patients admitted in an intesive care unit (icu) who had acute renal failure (arfi and were treated with different dialytic techniques. material and methods: 1087 patients were included in a prospective study from june 92 to november 94. 220 patients presented arf defined by creatinin serum leve(s greater than 150 pmol/l and previous normal levels. patients were divided in three groups. group i (control) : 156 patients with arf who did not receive substitutive techniques. group ih 21 patients under intermittent hemodialysis (hd) or peritoneal dialysis (pd). group ii1:43 patients under continuous hemodiafiltrstion (hf). the statistical analysis was chi-square test and analysis of variance. results: the table shows the results we obtained, we did not find any significant difference betwen the two groups of patients undergoing dialysis. d(fferences were observed only between group i and the other groups as shown below. we did not find any significant association between the theoretical mortality predicted and the observed mortality according to saps in the three groups. due to exposure to a wide variety of unpleasant stimuli, for example, tracheal suctioning, venipuneture and physiotherapy, most pataents admitted to the icu will require some form of sedation. this review will describe the suggested properties of an ideal sedative agent for use in the icu and review the current limitations of some of the available agents from this perspactive. methods used to quantify the level of sedation, such as the ramsay score, glasgow coma score, newcastle sedation score and visual analogue scores, and their deficiencies will be examined. consideration will be given to defining the optimal level of sedation and the circumstances under which sedation might be varied over the icu course will be discussed. preliminary results from an ongoing study examining the role of light versus heavy sedation and ischaemia in a cardiac surgical icu population will be presented. the pharmacceconomics of icu sedation will be briefly addressed. finally, the role that sedation may play in increasing morbidity, pastieuiarly nosocomial pneumonia, in the icu will be discussed. objectives : therapy cost(tc) in icu patients is a substantial component of total hospital care cost. estimation of tc during this year, partitioning to various groups of drugs used and attempt to minimise it, were considered practically useful. methods : in collaboration with the hospital pharmacy we were able to have a complete report of au drugs used for icu patients (including enteral and parenteral nutrition). mean apache ii severity score upon admission was 19.7 and mean length of tcu stay was 6.7 days. price per drug unit and cost per group of drugs were also available drugs were divided into two groups: antibiotics (1) cardiovascular drugs (2), gastrointestinal system drugs (3), enteral and parenteral nutrition (4), respiratory system drugs (5), sedative, analgesics and paralysing agents (6), parenteral solutions with electrolytes, vitamins and trace elements (7), anti-inflammatory agents (8), protein substitutes and immunomodulation agents (9), anticoagulative agents (10). antibiotics were further subdivided into those "freely" prescribed (a) and those whose prescription and administration requires filling of a relevant form (b). results : !) tc for icu patients/day was 30.530 drs ($122). total tc/patient was 295.195 drs ($1.108.7). ii) partitioning total tc per group of drugs reveals : (1) 43%, (2) 2.7%, (3) 2.7%, (4) 9.2%, (5) 0.3%, (6) 8.6%, (7) 9.3%, (8) 1.3%, (9) 15.8%, (10) 2.5%. t11) concerning antibiotics which consist the major cost component, group a and group b contributed by 29.1% and 13.9% to the total icu tc respectively. group b were administered to 13.9% of all icu patients. conclusions : i) for the above studied patient population antibiotics consist almost half of total tc followed by protein substitutes and immunomodulation agents. ii) if tc control could be attempted in the icu, prescription of beth groups must be reviewed. appropriate treatment should be prescribed and readily provided to any patient. clinical significance of routine protein substitution, currently controversial, should be re-evaluated. new antibiotics (third & fourth generation cephalosporins, quinolones, carbaponems) should be prescribed on the basis of strict diagnostic procedures using modern technology available. rationalisetion of antibiotic therapy will lead to cost control, redistribution of icu expenses and substantial contribution to infection policy in our country. objectives: i -to investigate the clinic efficiency of the monitoring of the rso2 cerebral, in relationship to the stroke prevention, in patient undergoing carotid surgery. 2-to determinate the variations of the rso2 during the different surgical and anesthetic procedures in these patients methods: ten patients undergoing carotid endarterectomy. precise neurological exploration previously to the surgery and in the immediate postoperative period. angiography evaluation to the extend of carotid artery disease. invasive blood pressure, ecg, pulse-oximetry ( pso2 ) and rso2 were collected previousty to the induction of anesthesia. the premedication was administered intravenously -midazolam (50 mcgr/kg) and fentanyl (i rncgr/kg) -. thiopental (4 mg/kg),fentanyl (3 mcgr/kg) and atracnrium (0,5 mg/kg) have been used for induction of anesthesia. co2te is monitoring al~er the orotraqueal intubation ! the anesthetic maintenance is accomplished with lsofluorane (0,5 -1,5%) and bolus of atracurium and fentanyh the surgical procedure is standard (without arterial shunt during the carotid cross-clamping). we register each 5 minutes: blood pressure, cardiac frequency, pso2, co2te and rso2. the rso2 cerebral variate in relation with: the anesthetic induction, blood ~ressure, co2te, cross-ulampping carotid and with the modifications of the head position. the maximum decrease of rso2 cerebral was in relation with the :ross-clampping carotid ( minimal value: 52 ). no patient had neurologic complications and postoperative stroke after carotid endarterectomy were not observed. objectives: there are more than 8000 anesthesia in chelyabinsk emergency hospital every year. to 80% patients of it emergency anesthesia is applied. more than 900 patients have ishemie heart disease (ihd), hypertansion (hp) and previos miocardial infarction (pmi). more than 5% of all patients are old patients (op). the resalts deep noninvasive bioimpedance monitoring (nbm) in surgical patients have been studied by us. methods: our nbm system "kentavr" includes 21 parameters of cardiac and vessels function. it is realised by monitors in operation theatres and computer network. moreover we are able to examine surgery patients before anesthesia and perioperatively by using special computers system for cardiovascular reflex control by fast fourie transform (fft) of 12 parameters simultaneously. results: 187 pathients extremly needed peryoperative monitoring of hemodinamics. from these 187 patients more 40% had stroke volume (sv) less than 30 ml, 18n -co less than 2.1/mim/m2, 25% -ejection fraction (ef) less than 65n and 32% -puls bioimpedans microvessels (pbm) less than 10 morn. 100 patient had intensive care in special department. 42 out of 187 died. comparing with survived with these patients before operation hr was larger, sv, co,ef, pbm and puls bioimpedance aortha was smaller. much more of these patients were with ihd, pmi, hd, op. even with survived patients these parameters decreased the towards the end of operation. surgery patients had different variability of 12 basic hemodinamical parameters with common tendency to increase power amplitude in low frequency by fft. conclusions: using of bioimpedanee noninvasive parameters allows to have criteria for corrections (infusies, vasodilatators, inotrops and others) and then us the final goal, to have more sucssesful surgery. with survived patients was perioperatively and postoperatively care more intensive. obiectives: the aim of the study was to compare the phi with the hemodynamically derived tissue oxygenation indexes as: oxygen delivery (do2), oxygen consumption (vo2), cardiac index (el), and arteriovenous difference in oxygen [(a-v)do2]. methods: 18 patients (15 males and 3 females) with major trauma or major abdominal surgery were studied. on admission, a nasogastric tube allowing phi measurement was introduced and a pulmonary artery catheter was inserted for optimal hemodynamic management. each phi measurement was accompanied with a complete hemodynamic study comprising systemic and pulmonary artery pressures, blood gases, and cardiac output measurements with the thermodilution method. derived parameters vo2, do2, ci, (a-v)do2 were measured according to the standard formula. hemodynamic parameters were opt• as soon as possible with fluids, inotrepes, and vasopressors according to repetitive hemodynamic measurements. all patients were under mechanical ventilation. after hemodynamic stabilisation phi and hemodynamic measurements were repeated every eight hours, during a 24-hour study period. a total number of 52 measurements were obtained and compared. statistics: results are presented as means + sd, correlations were performed between phi and the hemodynamically derived oxygenation parameters. a p<0.05 value was considered as significant. results: mean values were phi=7.19+0.1, do2=984+313, vo2=181+71, c.1= 3.7+ 1.2, (a-v)do2 = 4.47+1.2. no correlation was found between phi and do2, phi and vo2, phi and c.i, phi and (a-v)do2. on the contrary in 14 patients phi remained below 7.30 for more than 24 hours despite adequate hemodynamically derived tissue oxygenation parameters. mortality in this group of patients was very high (85%). conclusion: no correlation was found between phi and the hemodynamically derived tissue oxygenation parameters our data suggest that phi is a better oxygenation indicator than the hemodynamically derived tissue oxygenation parameters, because it is closely related to the patient's outcome. objectives: the pathogenesis of septic shock and multiorgan failure is believed to be related to tissue hypoxia of the gastrointestinal tract. therefore new monitoring techniques, preferably organ specific, are required to establish the adequacy of tissue oxygenation. peep is used to reduce pulmonary shunt volume and improve blood oxygenation, but is accused to impair splanchnic perfusion. we studied mucosal oxygenation and perfusion on the capillary level in the stomach and the duodenum. methods: we used the erlangen microlightguide spectrophotometer (empho ll) together with a specifically designed fibre probe (bodenseewerk ger~tetechnik, 0berlingen) in combination with a standard gastroscope. measurements were performed on 9 ventilated, traumatized patients (ages 17 -75 years), with no evidence of shock or severe infection, after informed consent was obtained from the relatives. all patients were hemodynamically stable without inotropic support. an area of 9 cm 2 was analysed in the gastric corpus, the antrum and in the duodenum. in three patients we simultaneously measured the muc0sal blood flow using a laser doppler flowmeter ( objectives: to investigate the influence of hb-o 2 affinity in the monitoring of svo~ during improvement of cardiac index (ci) in cardiogenic shock. design: to state whether changes in svo: were associated in changes in actual pso (p~0) and standard p~0 (ps0st) 22 consecutive measurements of artero-venous bga, before an.d after therapy-induced changes in ci, were evaluated in 11 patients (mean age 73-*7 y) suffering from cardiogenie shock, all under mechanical ventilation in psv modality. methods: together the hemodynamic measures, m~xed venous samples were analysed at 37 ~ c using the abl500 radiometer for po2, pco: and ph, and the osm3 radiometer for hbo2%, hbco% and methb%. psost (i.e. the p~0 at ph=7.40, pco:=40mmhg and temperature at 37 ~ c) was calculated automatically by the instruments on mixed venous blood as was the ps0"in vivo" (i.e. the pso at the patient's value of ph, pco2 and temperature), using siggaard-andersen's computerizated algorithm. mean time between paired measurements was 6.1 -* 1.2 houm. the data were compared by anova test for linear regression and t-test for paired samples. results: a dose linear relationship was found between svo2 and oxygen extraction ratio (oer), r=0.94,p=0.00000. the improvement of ci (1.41 -* 0.47 to 2.55 + 0.5 l/min/m 2, p<0.0000001) induced a significant increase in svo~ (0.495 -* 0.131 to 0.636 • 0.060 %, p<.0001). a significant decrease in p50 (32.5 • 6.7 to 27.9 • 2.5 mmhg, p<0.05) without any significant change in p~0st (29.1 • 2.2 to 28.7 • 2.3 mmhg, p=ns) was also found. these data show that either oer or the shift to the left of the oxygen dissociation curve account for increase in svo2 occurring with restoration of systemic blood flow. the program is intended to help the intensive care unit interne providing him with a practical tool when making decisions concerning patients in a critical condition. in his daily practice in intensive care unit, in this case the interne of the unit, uses this program for each patient as follows: on the first stage of data collection he should complete the following modules: (1)personal data (2)patient's pathology (3) laboratory and~ monitor lug data (4)drugs prescribed or toxic elements ingested. in this way, the system allows optionally the consult with a computerized data base about the drugs prescribed, standardized parameters and techinques performed by the central laboratory. (5)reference to an antibiotics guide regarding becterian sensitivety in our unit, whitch ee checked every six month (6) access to de questionnaired apache ii to load up new data. (7) statistcs about patient's admission and discharge. results: once all data collection is finished the system performs the followin duties: (1)detailed drugs interactions, including toxic elements (2)diagnosis starting from the clinical, laboratory and monitoring data. in some cases, it also establishes therapeutic strategies, e.g. a coagulopathy (3) give the l~narmacological incompatibilities between the drugs p~escribed and %he diagnosis established, and (4)perform dosage adjustments based upon the personal and pathological data. objeatve: to assess the power of diseri~,~ion ofa multiperpose severity score (sai~) when applied to subgroups ofpatieals (pta) according to their lemg~ of ~ay (los) in icu. design: in order to compute the saps probability, a model derived fi~m legible regression was developed. meaumree of calibration (goodmem..of.fit statistics) end discrimination (roc cm've and relative area under the cm've) were adopted in develotammtul asd validation set. the whole databue was ~ati~ed in five gronps reeked on los as follows: los = 2 days, los = 3-4 days, los = 5-7 da~, los = 8-14 days, los >15 day~. area under the carve (auc) was ud~ninted for each 8ro~. s~ing: 25 imlimlcus. patents: of 10~65 pts comec~ively admired ~ a period of three yeet~ (1990) (1991) (1992) , a total of 8059 was i~leded in this study. pts without saps, p~ yolmger them 18 yearn, p~ with los shorter ~ 24 hom'~ were excluded from this maly~is. iaterventinns: nose mema'onm~ end result: the logistic model developed gave good remits in terns of calibration md discrimin~on, both in developmental set (do.s g2: 9.24, p > 0.25; auc = 0.79i-0.01) and in validation ~t (g.o.g g2: 8.95, p > 0.50 ; auc = 0.78..+0.01). auc of each grottp showed a loss in di~zimination (i.e., prediaton) closely related with los, being 0.90i-0.01 in pts with los = 2 days el 0.59~.02 ia tm with los > 15 da~ (figure). following the present guidelines of integral management, in order to achieve optimization of sanitary resources and better use of facilities, we feel that the setting up of objetives is a key factor in the continuous process of improvement of quality care. postsurgical intensive care services maintain an interdepent relationship with other hospital services. within the general plan of the hospital it's of the utmost importance to delegate autonomy to the various depertments and service units in determining and achieving objetives. it's also necessary to establish mechanism for coordination of the activities in order to assure the succes of the program. the objetives cannot be improvised, they must be carried out in a specific manner in the following stages: 1.-analysis of the present situation (starting point). where are we?. defining objetives and making explicit the activities and methods to achieve them is to anticipate the future; it is of the utmost importance to comunicate said plans to all whom affect by encouraging them to attain the desired results. in the present paper we intend to show the guidelines to follow in carrying out a course of objetives. introduction:we presents results related to the quality of life (qol)of critical patients, from paeec project data. material and methods: the paeec project is a multicentre study define the type of patients cared for in spanish icus, and the therapeutic activity provided. ninety-five icus from spain are taking part. this study analyzes the qol of critical patients prior to their icu admission.for the evaluation of qol a questionnaire designed by our team for critical patients was used, with 15 items grouped in 3 sub-scales: physiological functions (4 items); functional capacity (8 items) and subjective aspects (3 items). qol is classified in 4 levels: normality (0 points); slight deterioration (1-4 points);moderate deterioration (5-9 points); significant deterioration (>i0 points). the we present results related to therapeutic activity in critical patients and their age, from the paeec project. material and methods: the paeec project is a multicentre study to define the type of patients in spanish icus, and the therapeutic activity provided. ninetyfive icus from spain are participating. this study analyzes therapeutic activity in the first 24 hours as evaluated by tiss, and related factors. results: the sample was 9,291 patients, sge 57.91~17.46 years. severity by apache ii system was 15.59• points. the tiss score was 19.87• points, distributed as follows: i (<i0 points): 14%; ii (i0 -19 points): 44%; iii (20 -39 points): 36%; iv (>39 points):5%.there is a positive correlation between the level of therapeutic activity and severity by apache ii (r = 0.46, p < 0.001), and a very weak but negative correlation between tiss and age (r = -0.048, p < 0.001), so that an increase in age corresponds to a lower level of therapeutic activity.patients the multivariate analysis of the relationship between tiss and age took into account: severity, existence of previous history, need for mechanical ventilation, size of hospital, diagnosis and mortality. it indicated that there continued to be a relationship between therapeutic activity and age, so that as age increased, therapeutic activity diminished. conclusions: therapeutic activity performed on critical patients is less in the oldest patients, in whom excessively aggressive procedures are limited. a relational data base management system in the icu. c. kotsavassiloglou*, d.matamis, g. dadoudis, j. kioumis, d. riggos. icu dep., g. papanicolaou gen. hosp., exohl, thessaloniki, and * a' neurological clinic of aristotelian university, thessaloniki, greece. objectives: the introduction of the information technology in the i. c. u seems to be unavoidable because of the large amount of produced data and the need for their systematic analysis. such an information system should be a) easy to use, b) friendly to the user, c) powerful and d) modular. on that basis, we created a patient data management system (pdms) according to the expectations of the medical staff of an eighteen bed multidisciplinary icu. methods: we selected paradox for windows v4.5 for the implementation of a relational data base because this program meets the above mentioned criteria. informations regarding the patients include a) demographic data, b) previous medical history, c)diseases upon admission, d)complications during hospitalization and e) outcome data. the diseases' registration consists of 421 items classified in 15 categories upon the principal system affected. specific informations about the need and duration of mechanical ventilation, nutrition, renal replacement, right heart catheterization and icp monitoring are also available. an extension was added concerning icu infections and related informations about antibiotic-resistant pathogens. all icu pathogens can be matched to their resistance or sensitivity and cost of antibiotics. the program can perform queries and various statistical analyses based on complex criteria. new modules can be added later according to the future needs and remarks of the users. results: the program was well accepted by the medical staff and 300 patients were registered as a test. the first analysis of the data related a) observed mortality versus the apache ii predicted mortality, b) mortality according to the age, gender, pathology aud duration of icu stay and c) pathology upon admission and icu related complications. conclusions: the long term use of this pdms can be an efficacious research tool. it can be used in retrospective or prospective studies by addition of necessary modules. the first data analysis revealed the iack of an international diseases' classification system. the development of a worldwide common classification system is essential for the compatibility of the data analysis among various icus. this will allow the realization of multicenter trials on a large scale. s. nanas= n. sphiris, a. precates, a. lymberis, m. pirounaki, and ch. roussos dept. of critical care, university of athens, athens, greece the complexity of the cases submitted to an icu, the variety of underline disease, tbe severity, as well as the large number of substances administered to each patient constitute obvious the need of support with an easy available dss. this system will assure the safety of the administered treatment will help to adjust the dose according to the situation of each patient and it will screen for possible interaction and incompatibilities between the administered drugs. the goal of the present effort is the design and development of a software system acting as a decision support tool to physicians of icu. the application is organised around a relation database management system (rdbms) that consist of: a) all available substances (1.850), b) all generic names of medications available in our country for each substance, c) incompatibilities (2.300 cases) and d) interactions with other substances (50.000 cases). the following figure shows the structure of the rdbms. y ta~ortato~ [ c~rs using the stored parameters for each patient the dose and the rate of administration of selected substances will be possible to calculate. the continuous monitoring of the treatment for each patient supports the medical staff to make the necessary changes of the prescriptions. the application is currently developing in wireless pen based computer systems which place patients at the centre of "islands of information" located throughout icu. in conclusion this dss is a powerful and useful tool for icu staff because it provides without additionai work to the routine of daily practice, the currently available information for each order concerning drug interaction and incompatibilities as well as treatment monitoring is to obsea~ among 113 critically ill pfdieats, stdjdivided following the diagn~s at the adn~ssio~ the diffmeax:es in the ~ and oxyplx~efic l~mmems bawe~ strvwors [s] and non sumvors ins] and to test the pc~'bih'ty to have soar survival criteria, as earliest as tx~able. method~ :we made a ~ study on 113 consexa~e ~ilically ill paliffas, subdivided in 3 series following the diastases at the admission: medical pafiea~ (29 s and 23 ns), surgical patients (19 s and 22 ns), a~d poliwauntas ( 14 s and 6 ns). follow up was done at 20 d,.ays from the admission in ice. all the patienls were ramitored with a ~ c~eter and 18 laeno:lymmi. "c and o .x.xyphorefic txuamaers va:~e couected at 7 fin~es (t): at fiae ~draission (t0), at 121x~ars from t0 (t1), at 24 (f2), 48 (y3), 72 (t4), % (t5) and 120 horus from t0 cf6). in~,h ~ies, for ~y ~ a all the lin'~ n~an and sandaid d~viation was ~ tx~h for s and for ns. th~ betw~ s and ns tl~ roeaas of ~h porarneter ~e ccmpared tt~ng t-lest and p < 0.05 w~ considered ska~ significant in each series in the t wheae the mast significative diffemx:as ~goeamd bet~en s and ns, we made a txedictive criterion, asamting as predictive indices for stnvival the i:r values, higher or lower than flae treans of the ~rar~ers of au flae patients, axx)rdhlg to those ones t~iatistically diff~'e~ betw~m s and ns. fhmlly xse co:weatxt onaong the 3 series the nrametees of the st~rs with the analysis of variance, to daserve the lxjsable differealt irea~ of sty1 hflices, following the diagn~s of admission: :nedkal, angical patient or poll~tam results: we c~ld not find ~ predictive criterion for politraonaas, perhaps ixx:ause of the few ntanber of l~fients. for high ri~ saw~cal patieras the following criterion at t2 has a sensitivi .ly of 100~ ,and a ~ecificity of 27.8%: sv1>32.89 nffmin/n~, map>92 mmhg, pmap<27 nmalqg cvp<i0 nunhg will4 nmah& lvswi>34 g m/m 2, sxo2>67~ do21>515 mlhnin/m2, o2er<31%. for lx~dical l~tienls at t5 the following criteric~a has a ser~tivi.ty of 100% and a ~zificity of 36.8~ cvp<7.5 mn~g, sao2>97%, s,g)2>74~ vo2i<133 ml/nfin/m 2, o2er<25%, shunt<19% survlvops' data of the 3 series ~ signitic~atly differenl~ both for the t~mody~nic a~ for fl~e ox3rphomfic lxlmn~s; moreover we ~ that the vatt~ of hemodynamic mad ox.~ho~tic indices were higher in politrautms. conclus'ions: acx~ording to the fftffe~mt patho!o~es, the ~ rnelabo~c needs are diffeten~ so that it is juslified to mash ~ the~alceutic goals, following the type oflmthology. 2hen~ we foru~d for high ~k mrgical pmka~ and for medical patier~s assme, ff mllslied, a good prognosis while, if n0[ ntljsfled~ the plinsliclioil ofdl~tth is no[ g(ioct finally, ab~ high iis~ supgical palieaats, according to what other atmhors say, txatws sh0~'n~ers ' therapeutic goalsvvould seem inadeqt~te, bec~jse they need a gear physiologic and themtx~ic elth~ in rdation to the rretabolic needs. figure 1) . thus, the smaller european nations had a greater participation than ~e larger ones, with the exception of norway. a similar result was evidenced for contributions to intensive care medicine (figure 2 ). these findings can be explained by different submission policies and language banners. however, there was no significant correlation with the gross national product of each country. conclusion: we conclude that the smaller european countries generally contribute more to international intensive care journals than the larger ones. objectives: to evaluate the agreement between a new and three old methods measuring ctp and to assess their reproducibility. methods: we studied 20 patients ventilated with a siemens 900c respirator. we measured ctp by dividing the tidal volume with the increase in airway pressure (paw), either with the respirator setting used (ca) or with a fixed setting (cf). by modifing the inspiratory time (ti) without changing inspiratory flow, we were able to deliver two series of 10 inflations (100, 200,... 1000 ml) before and after curarisation of the patient. the same volumes were also inflated in paralysed patients with a super syringe. at the end of each inflation a plateau of 3 sec was performed and paw was recorded. the above three sets of pressure-volume (pv) points were used to reconstruct the corresponding pv-curves ((31, c2, c3 the new method for ctp measurement without a super-syringe had the best reproducibility in paralysed patients and gave similar results without curarisation in the majority of them. however, agreement between the methods tested was unacceptable for clinical purposes. further investigation is required in order to improve the accuracy of ctp measurement in icu patients. m kunert, r.sorgenicht, l.scheuble, k.emmerich, h.g01ker med.clinic b (dept.of cardiology) i heart center of wuppertal/university witten-herdecke,germany objective to determine the accuracy of activated partial thromboplastin time (apl-l) and activated clotting time (act) studies when samples are drawn through heparinized central venous catheters (cvc). methods a total sample of 80 paired act/p't-/" values was analysed in 40 patients (28 m.,12 f.,66 + 12 y.) for monitoring heparin therapy.all patients had a cvc (certofix trio,braun,frg) in the internal jugular vein receiving a continous infusion of 20.000 u heparin via the central catheter.act (hr-act, hemotec,usa) and ap'i-f (neothromtin, behring,frg) samples were drawn from the cvc using the double syringe technique (removing and discarding 5 ml blood before drawing the sample). these blood samples were compared to act/ap'cf blood samples obtained by venipuncture (v.fem.) at the same time, act values were analysed directly in the intensive care unit (icu),api-i samples were measured in the hospital laboratory within 30 minutes. results ac-i -~ pi-f~ cact/~pi7 r = 0,62) cvc samples 132 88 +34 +22 . v.femoralis samples 1"28 84 +29 +24 p-value n.s. n.s. conclusion there is no difference in heparin anticoagulation studies drawn from heparinized central venous catheters compared to those obtained by femoral venipuncture,withdrawing 5 ml blood prior to obtaining the blood specimen is a safe way for eliminating heparin contamination.not only the aptt test but also the act test is a useful method for heparin anticoagulation assessment in the icu. objectives: evaluation of the delicate balance between filter-coagulation and patient-hemorrhage using heparin as anticoagulant in continuous renal replacement procedures. methods: from january 1991 through august 1994, we studied filter surviva[ and hemorrhagic complications during 240 filter periods in 78 critically d[ patients, treated with continuous arterio-venous hemo(dia)filtration, with special emphasis on the heparin dose, concurrent use of coumarins, systemic activated partial thromboplastin tirne(aptr), platelet count, mean arterial bloodpressure and the type of filter used. results: 141 filters (59%) were disconnected because of coagulation. mean survival of multiflow an69 filters was twofold shorter compared to survival of fh66 gambm filters. a total of 48 hemorrhagic complications occurred of which three patients died at aptt values of respectively 39, 48 and 56 seconds. after adjustment for mean arterial bloodpressure, platelet count and the type of the filter, the risk for filter-coagulation decreased 25% (relative risk 0.76, 95%c1 0.68-0.85) for each ten seconds increase in aptt. the risk for patient-hemorrhage increased 50% (relative risk 1.50, 95%ci 1.38-1.64) at an aptt-increase of ten seconds. the occurrence of filter-coagulation and patienthemorrhage was not correlated with the administered dose of heparin. concurrent use of cournarines had a positive effect on filter-survival, without increasing the overall incidence rate of patient-hemorrhage. conclusions: the systemic apt]" is a good predictor of the risk for filtercoagulation and patient-hemorrhage. heparine therapy seems optimal at an aptt between 35 and 45 seconds, although one should realize that fatal hemorrhagic complications still can occur. objectives: the alterations in vascular tone which are primarily regulated by adreno-sympathetic tone(ast) are compensatory responses in hemorrhagic patients. this study was designed to evaluate the correlation between vascular tone and ast in patients with hemorrhage, methods: the vascular tone was expressed by volume elastic modulus (ev) that is defined as; ev = ap/(av/v) (ap; the arterial pulse pressure, av/v; the volume change ratio). ev was measured using a non-invasive transmittance infrared photoelectric plethysmography (tipp) and a volume oscillometric sphygmomanometer . we prospectively studied 6 patients with hemorrhage. the initial ev measurement was performed on arrival and repeated for a 48hours duration. as a parameters of ast, serum concentrations of adrenalin (ad), noradrenalin (nor), plasma renin activity(pra) were measured simultaneously. we analyzed the correlation of ev and conventional parameters to ast by multivariate statistical analysis. results: ev values at transmural pressure 40mmhg on admission and 48hours later were respectively 864.2 + 249.5mmhg, 270.0 +_ 92.0 mmhg (mean + sd). systolic pressure(pas) and serum hormones on arrival and 48hours later were respectively, pas; 96.5_+20.4, 152+18.7mmhg, ad; 1.21_+1.02, 0.07_+0.04 ng/ml, nor; 1.60_+1.48, 0.65+0.39 ng/ml, pra; 26.6_+37.8, 2.5_+2.9ng/ml/hr. the ev values correlated significantly with ad (r=0.47, p=0.006, n=33), nor (r=0.47, p=0.005, n=33), pra (r=0.38, p=0.032, n=33). by multivariate statistical analysis, ev correlated more significantly with ad and nor and pra (p=0.00079) than the conventional parameters such as pas, heart rate and pulse pressure. conclusions: the alterations of ev correlates closely with ast. the compensatory mechanism in hemorrhagic patients can be detected noninvasively by ev monitoring. obiectives and method: autologous oxygenator blood was processed at the end of cardiopulmonary bypass (cpb) by either hemofiltration (hf 60, 1,2 m 2, fresenius) or by cell washing with a onntinous autologous transfusion system (cats, fresenius). prospectively the blood of 10 patients for each group was processed and then retransfused intravenously to the patient. besides, volume and time requirements, standard hematologic chemistry, coagulation and complement activation were measured. results (mean values for oxygenator blood at the end of cpb, and results of concentrate after processing by filtration or washing): both processing techniques show excellent hemoconcentration of the diluted cpb blood with a good transfusion effect for the patient. filtration retains all plasma proteins and large molecular weight plasma bound waste products. in contrast, cell washing with cats significantly depletes plasma proteins and waste products. the newely developped cats machine gives eonsisinnt laboratory result in a fully automatic continuous processing mode. in conclusion, both filtration and washing are effective for processing cpb blood. filtra tion yields a highly concentrated whole blood, whereas cats washing produces a high quality autologous erythrocyte concentrate. soluble fibrin has during the last years gained interest as a marker for the activation of the coagulation in connection with various clinical conditions, e.g. disseminated intravascular coagulation, deep venous thrombosis and myocardial infarction. elevated levels of soluble fibrin in plasma can be detected by the chromogenic assay coaset fibrin monomer, relying on the ability of fibrin to enhance the tpa-catalyzed conversion of plasminogen to ,plasmin. using this test, it has been shown that the level of soluble fibrin can be correlated to severeness of illness in critically ill intensive care unit patients. a revision of the coaset fibrin monomer kit has now been made and the new product, coatest soluble fibrin, is considerably more convenient to handle and gives higher resolution at low fibrin levels. the test is performed by the addition of a buffer dilution of the plasma sample to a microstrip well containing the colyophilized mixture of tpa, plasminogen and the plasmin specific cbromogenic substrate s-2403. the reaction is allowed to proceed at,. room temperature for 15 minutes before discontinuation. the absorbance at 405 nm, measured in a microplate reader, is proportional to the content of soluble fibrin in the sample. the assay is carefully standardized and calibration curves are provided in the kit. the convenient and rapid assay procedure makes the coatest soluble fibrin test well suited for single test analysis in acute situations. objectives : blood coagulation abnormalities have been reported in the systemic blood of patients with cerebral lesions. the physiopathology of such events is not yet completely understood. we compare the coagulation profile of blood from the right jugular bulb with systemic blood of patients with head injury. methods: we studied 4 patients, who were admitted to our neurosurgical intensive care unit between january and march 1995 with head injury and no other associated pathology (age 20-60 yrs), a glasgow coma score <= g, no abnormality in baseline coagulation profile and no history of coagulopaties. the patients did not undergo angiography. a one-way 16 gauge certofix catheter was inserted through the right internal jugular vein up to the jugular bulb. an identical catheter was inserted through a subclavian vein. blood was sampled from either catheter (a=atrial; j=jugular) 6-10 hours after trauma (t 1) and t 2 hours later (t2 the inddence dpontolx'rative thmmhi~e and haumord~gic complieatiom were assessed in padents treated with indobefen, heparin calcine caeca), low mollecolar weight heparin (lmwh) (f.nosheparin) and undergoing hemodiludun, blood predeposhing, intra mad postoperative blood saving. ]'he indolmfon tempota~.norks platelet aggregation through ,,elective inhibition of the cyclatygenasis and thus atacbldonicadd(1).tbe n'mimum effect occurs after 3 hours from the fast administration and is still present after 24 hours. ~-979 patients, mean age 62---11 yrs., weight 68---10 kg were studied. 321 (32.8%) were male and 658 (67.2%) female. 858 onderwent 713 hip prosthesis (7 previously plate and screw removal) 145 hip revim'un (10 stem, 33 cop and 102 stem + cop), 121 tutal knee prosthesis, in the 1st anaesthesidogy depl from 14-1992 to 30-6-1994. as for antithromboembolic ptephylam, apart from hemodihitiun 668 pts were with treated indobufen 0ndo), 199 with heparin ealdum caeca) and 112 with low mo!lecular weight hepam (lwr,91). as the slightest clinical and/or imtmmental suspidon of deep vein thrombosis (dv'i') or polmonary umbolism(pe), a phlebogram or sdndgram were respectively carried out. -the inddence of homologom transhisiom was significandy lower (p=0.000l) in the padeats treated with indobufen (4.256) compared 7.'ith heca (14.5%). the con~gency table shows statistical signifleance for the use of heca in patients with vein deficiency in the lower limbs, past dvr and/or pe, coronary heart disease (cdh'), while there is no correlation for renal, cardiac or liver defidency, obesity, systemic hypertemion, atrhythmy, diabetes, chronic bronchitis and rheumatoid arthritis. by comparing the postoperative cumplications with the risk factors, there ks a highly significant correlation (p=0.000l) between cdh and thrombotic and humord~agic complieatiom (pe, death, he~atoma, die use of hum_ologous blood). thee data show that hep~in, preferred in patients with c'dh, roost likely for leagal-tuedical reasons, did not have the de~'ed effect. conclusions -the stastisfical aar~ais shows ~nifieanfly different efflea~ (pro0.0001) between the therapies (see table) : it can be seen that in patients undergoing autotramfusiun and hemedihidon, indobufen produo~ a lower incidence of haemotrhagic complieatiens compared to heca and lmwh and is more effective in the prevention d ~c complications at clinical e~idence. the duration of i~toperadve hospital stay is signi~cantlylonger for patients transfused with homologous red ceils and treated with hec,7.a (13.7-+1.4 days) and lmwh (13.5+-1a days) compared with indo(ll.8_+1a days). one of the main causes for postoperative complications in major orthopaedic surgery is postopemtive bleeding with local effects in the operation site (hematomata, pain and delayed mobilization) and/or systemic and subsequent cardiodrculamry repercussions that are sometimes severe. the aim of this study is to assess the possibility to apply a new system of monitoring, control and saving postopemtive blood loss from the drainage. the bt797 recovery dideco (marandola, modena-italy) ~ used since it is the only apparatus capable of doing this. the apparatus consists of a pressure transducer, adjustable from -100 a +50 mmhg, which activates a peristaltic pump connected m drainage robes. the bt797 recovery display shows hourly bleeding in the first 8 hours, total bleeding, time passed since the start of monito~g and subsequent salvage and the aspimtioo pressure on the drainage robes; the latter is inserted at -10 mmhg and then modified according to bleeding/minute, g bt797 recovery also has an alarm that sounds automatically if.' blood loss is more than 300 ml/hour; air is in the circuit; the batteries are running low. materials and methods: 191 pts were studied (53 m and 138 ~), aged 63.5-+11.lyears, basal hemoglobin 13.1-+ 13(range 7.8-16.6)g/all, treated from 1st january, 1992 to mst december, 1994 in the 1st service of anesthesia and intensive care unit of our hospital. the patients underwent the following surgical treatment: total hip revision (132pts), cup revision (~ipts), stem revision (13 pts), total knee revision (2 pts). the average dumtion of the operations was 173-+58 min. intranpemtive monitoring and blood salvage was applied to all patients. genera! anesthesia was used on 57 pts. and integrated (epidural analgesia + light general) on the remaining t34. anttthromboembolic prophylaxis consisted of external pressure bandage, isovolemic hemodilution with iodobufen in 131(68.6%)pts., calalc heparin in 35(18.3%)pts., low molecular weight heparin in 25(13.1%)pts.; 1 pt did not give a predepoalt of blood, 4 gave 1 unit, 45 pts 2 units, 110 pts 2 units, 31 pts 4 units. the data obtained was statistically analysed using contingency tables and anova. results: average intmop salvage was 420-+345 ml, average postop salvage was 420-+265 mi the average intra+postop 909+-460 ml. average postop loss was 677-+359 ml. the global incidence of postop complications was: h~natomata 5.2%, dvt 1.1%, pulmonary thromboembolism 1,196, myocardiac ischemia 0.5%, acute myocardic infarction 0.5%, respiratory deflciecy 2.6%, arrhythmia 2%, cystitis 0.5% there were nn complications in 86.4% of pts. postop bleeding over 300 ml in under 60 minutes (with bleeding alarm activation) occurred in 30 pts (15.7%). this sta~tically correlates only with the type of operation performed (more frequently in total hip revision p=0.034) and with a significant decrease (p~0.003) in the pruthrombic activity detected about 8 hours after the operation. this bleeding, also made the alarm sound, calling the attention of staff who could act accordingly, by making the drainage pressure positive and incre~sthg the tension of the external pressure bandage. conclusions postop monitoring, control and blood loss salvage combined with predepoalting and intmop salvage has enabled allogenic transfusions in 16% of cases to be avoided in operations with high postop blood loss like hip or knee revision. the usefulness of the system can be seen by the fact that in the 30 patients with so much bleeding to set off the alarm, there was no significant difference in the incidence of allotransfusions and complications. references 1)borghi b., bassi a., de simone n., laguardia am., fonnaro g. an injury of the brain may result in various disorders of hemostasis caused by the release of • into the circulation through a damaged blood-brain bar tier. disseminated intravascular coagulation(dic) is one of these disorders. it is a freguent but relatively rare ly diagnosed complication of subaraohnoidal haemorrhage. the aim of this study was to evaluate some parameters of both blood coagulation and fibrynolisis in patients with sah.in addition one wanted to find out wh~ther potential changes correlated with the pa• condition in the acute phase of sah and whether they influenced the course of this disease. 60 patients with sah were studied. in 17 of them sah was due to closed eraniocerebral injury and in the rema ining 43 resulted from vascular malformation. the following parameters were evaluated:the prothrombine time,the activated partial thromboplastin time, the thrombine time,level of factor v,fibrinogen degrada tion products and fibrin monomers. the results let us show the presence of oic in 3 patients with closed craniocerebral injury and in 14 with vas. cular malformation despite the lack of clinical symptoms the tests in 5 posttraumatic patients and in 6 patients from second group showed incomplete dic.on admission patients with such changes in measured parameters were in poor condition.the course of the disease and the effe cts of treatment were also worse in these patients. the results showed ihal in patients with sah complex disorders of both coagulation and fibrynolisis occur, and they depend on clinical condition of the patient. they also influence the course of the disease. methods : charts of all patients admitted with d.i.c. over a ten year period (85-94) were reviewed. diagnosis of dic was based on the association of fibrinogen < 2 g/1 -platelets <150 109/1 -fpd > 40 ~tg/ml in the 24 hours of the admission. results : 40 patients -mean age 29+6 y -saps 8+_5 -gestanional age 35_+5weeks -the two first conditions associated with d.i.c. were placental abruption (35 %) and preeclampsia or eclampsia (22,5 %). bleeding episode was present in 22 pts (55 %) and surgical treatment has always been necessary. 26 pts (65 %) were given packed red ceils (12+10 u) and fresh frozen plasma (9+8 u). 6 patients were given platelets packs. heparin was never administered. 6 pts required mechanical ventilation and two patients hemodialysis. all the 40 patients survived. correction of prothrombin time (p.t.) and fibrinogen (f) was quick (p.t. at t24 h 69~2 % -f at t36 h 2,60+1,5 g/i). but platelets count remained low (plat. at t48 h 80 +42109/1) -no difference was observed in patients who received platelets. conclusion : prognosis of critically ill o.p. is good. blood loss is the main complication. correction of hypovolemia and anemia with concomitant surgical treatment are essential. the administration of coagulation factors or platelets is still under discussion. objectives: to evaluate the effects of antithrombin iii i at-iii) and a protease inhibitor, gabexate mesilate foy), on the coagulation and fibrinolysis in disseminated intravascular coagulation (dic). methods: after the approval of our institution and consent from patient's family, 40 patients with a dic score (1988, japan) more than 5 points (dic or having a risk for dic) entered this study. they were randomly divided into two groups, foy (i-2 mg/kg/h for 7 days or more) treated group and no foy group, each of 20 patients. platelet count (plt), fibrinogen (fen), at-iii fibrin degradation product (fdp), d-dimer (do), fibrin monomer (fm), thrombin-antithrombin complex (tat), plasmin-plasmin inhibitor complex (pic), and prothrombin time ratio (ptr) were measured before the start of treatment (at admission) and i, 3, 5 and 7 days after the admission. at-iii at 1500 units for 3 days was administered if the at-iii at admission was less than 70%. finally the patients were divided into four groups: group a, foy (+) and the at-iii ~ 70%; group b, foy (+) and the at-iii < 70%" group c, foy (-) and the at-iii 70%; group d, foy (~) anffthe at-iii <70%, each of 7 patients, to match the patients for backsrounds. all parameters, dic score and survival rate in a month following treatment were compared among the four groups. results: the at-iii and plt from day 3 to 7 were significantly higher in groups a and c than in groups b and d. the fdp, dd, tat, and pic after treatment decreased significantly from the baselines in groups a and c but not in groups b and d. the fgn and fm were not significantly different among the four groups. the ptr decreased in groups c and d but increased in group b. the dic score decreased significantly in groups a and c than in groups b and d. survival rates were 57%, 43%, 71% and 57% in groups a, b, c and d, respectively, although not significantly different. conclusions: in patients with dic or a risk for dic, foy had no expected effects but at-iii had suppressive effects on the coagulation and fibrinolysis mechanisms. a prognostic factor ? carbon monoxyde intoxication is a classical complication of inhalation injury. carbon monoxyda is also physiologically produced during the heme metabolism: heme is conversed to bi]irubin by the hemeoxygenase which is an intracellular stress protein. 20 icu patients (pts) were studied prospectively for apache ii score and carboxyhemnglobin (hbco) arterial level to assess if hbco level could be correlated with the severity of the pts. objective: to evaluate a new technique of non-surgical tracheotomy. patients: 55 adults, mean age 54 years and 11 children, mean age 18 months (2 me.-5 yrs). method: through a needle inserted in the trachea, a guide wire is retmgradely pushed out of the mouth and attached to a special device formed by a flexible plastic cone with pointed metal tip joined to an armoured tracheal cannula. this device is then pulled back through the oral cavity, larynx and trachea, and outwards across the neck wall by applying traction on the wire with one hand and counterpressure on the neck wall with the fingers of the operator's other hand. when the cone and 2/3 of the eannula have emerged, the cannula is cut off from the cone, straightened perpendicular to the skin, rotated and advanced caudally to its final position. results: endoscopic control facilitates and improves the safety of all manoeuvres. the pointed cone easily pierces the tissues, and the cannula is extracted without difficulty since it has the same outer diameter as the cone. tissue adherence around the cannula is absolute thus preventing local inflammation. the time in apnea required for dilation and cannula placement does not exceed 60 see., and it is well tolerated because within safety limits in patients hyperventilated with oxygen. only one case of bleeding occured in a patient on dialysis with severe coagulopathy. autoptic findings in subjects who died due to progression of primary disease showed a very regular stoma with an almost complete lack of hematic and flogistie infiltration in recent tracheotomies. .conclusions: translaryngeal tracheotomy (tlt), by virtue of its greater inherent safety and lower tissue trauma than percutaneous techniques, can also be carded out in infants and children, a severe test bench for any tracbeotomy technique. further specific indications are recently stemotomized patients, since tlt is associated with a low rate of infection, and short term tracheotomies after laryngeal surgery, to prevent obstructive complications. references: fantoni a., translaryngeal tracheotomy, apice, ed. gullo, trieste, 1993, 460 . background: inhalation of no has been shown to reverse hypoxic pulmonary vasoconstriction 1, to reduce pulmonary pressure in pulmonary hypertension of different origin and to improve gas exchange. in putmoflary embolism, pulmonary hypertension is caused by mechanical vascutar obstruction and by reactive vasoconstriction. the effects of inhaled no in putmonary embofism has been partiatly studied' the purpose of this study was to investigate and determine the effects of no inhalation on pulmonary hemodinamica and gas exchange in a hypoxic canine model of pulmonary embolism. methods: two groups of adult mongrel dogs were studied: group 1 (control} 5 dogs and group 2 (no inhaled) 6 dogs. both groups were anestesized with tiopental, mechanically normoventilated with an hypoxjc mixture of 02 and n~ (f[q2 0,16) and instrumented (swang-ganz catheter, femoral artery catheter) pulmonary embolism (pe) was induced by fisher's method s. no inhalation (80 ppm) in group 2 was started 15 rain. pdor to pe and kept constant throughout the experiment. no inhaled concentration was analyzecf by chemiluminiscence technique. pulmonary artery pressure (pap), central venous pressure and sistemic arterial pressure were continuosly recorded. cardiac output, artedat po~ (pan2) and mixed venous po~ were measured in both groups under hypo)dr conditions, before pe and 5, 15, 30 and 45 rain. after pe. pulmonary vascular resistance (pvr) and gas exchange (pao21fio:~ ratio), were calculate using standard formulas. data were process and analyzed with non pararnetdc test, and reported as mean -so and statistical significance was considered if p < 0,05. : no produced an increase in arterial oxigenation (pao2/fio~ ratio) and reduced pap before pe induction in group 2. after pe we found no significant difference with .respect to the time eour.se of pap, pvr and gas exchange between beth groups throughout the experiment. probably, the severe mechanical obstruction produced in pulmonary embolism masked the small effects of no inhaled. obiectives: blood volume measurement would be useful in critically ill patient management if it were easy to perform. this is not the ease and current methods are based on radiolabelled red cell dilution. inhalation and uptake of a known mass of carbon monoxide (co) gas and measurement of earboxyhaemoglobin increase can give results accurate enough for clinical use. this requires a rebreathing system providing oxygenation and carbon dioxide removal, yet complete retention of all carbon monoxide administer&l, and so most authors hand ventilate with a bag and waters soda-lime canister, adding oxygen as necessary. we aim to popularise this method by; i)design of an automatic co administration system driven by the itu ventilator and ii)writing of software for a portable computer to perform all necessary calculations 9 method: we show the computer is use estimating the co dose required and later estimating the blood volume. we also show the new gas administration system. this is a fully closed circle attached to a "bag in bottle", driven by the ventilator. the novel feature is the mechanism by winch driving gas (set to 100% 02) spills automatically into the circle, balancing o5 uptake by the patient, yet allowing no co loss. conclusions: this equipment is easy to use, reduces human error and allows optimum ventilator settings to remain. the operator merely administers the volume of co determined by the computer and takes blood on two occasions. carboxyhaemoglobin measurement is easy to perform, thus there is a cost saving also. with our modifications use of this technique may potentially become more widespread, the video demonstrates the method in use in our itu. -10 (25%) underwent conventional surgical therapeutics. 9" (90%) with resection of tracheal stenosis with end-to-end anastomosis(rts). i (10%) with broncoscopic dilatation. one patient died and the others still have stable patency(sp) without continued treatment. -29 (72,5%) have received endoscopic laser ablation with or without calibration tubes. 17 of them (58,6%) are receiving continued endotracheal treatment until now. 12 (41,4%) have sp wihout continued treatment. -i (2,5%) endoscopic laser therapeutic case turned to rts and is having sp. conclusion: conventional surgical aproach has been progressively replaced in our hospital by endoscopic laser ablation and silicone calibration tubes. this study suggests that these technics are effective and could be the elective treatment for iatrogenic stenosis. obiectives: hemorrhagic disorders due to thrombocytopenia and thrombocyiopathia remain one of the most serious complications during long-term extracorporeal membrane oxygenation (ecmo) in patients with severe acute respiratory distress ~drome (ards). in the presented study, nitric oxide (no), kwown as a potent endogenous platelet antiadhesive, disaggregating and antiaggregating compound, was evaluated for its possible antagonistic effect on platelet trapping when added to the gas compartment of membrane oxygenators (mo). meti~ods: two parallel separated extracorporeal circuits, consisting of heparin bonded hollow fiber oxygenators (minimax, medtronic, carmeda eioactive surface), tubing systems, low pressure reservoirs, and roller pumps were prepared. for each measurement, a pair of circuits was simultaneously filled blood from the same volunteer. low-heparinized fresh warm blood was obtained from four healthy volunteers, who had no drugs for at least two weeks. the gas inlets of both oxygenators received dry gas (21% oxxygen, 5 % carbon dioxide, 84 % nitrogen); gaseous no (20ppm) was added to the gas of one of the oxygenators (no-mo), whereas the other one (mo) was used as control. after 270 minutes no gas was switched off, so that the no-mo received no more no, and no was added to the gas inlet of the membrane, which had no no before_ to assure iutracircnit volume stability, drawn blood for measurements was replaced with saline, and platelet counts were corrected for dilution by hemoglobin values. the mean of four platelet counts (coulter counter) of each timepoint (start, 30, 90, 150, 210, 270, 330, 390 , and 450 minutes) was used for statistical analysis (paired sample t-test). results: in the no-mo platelets remained at 96 + 3,2 % (percentage of baseline value, mean -+ sd) until 270 min. in contrast, platelets of the mo continuously decreased after start and were significantly lower after 150 minutes ( 96,4 9 +3,5% vs90_+3,1%(p<0.05);210min. 95,9-+4,5%vs84,5_+2,2%(p< 0.05); 270 min. 82,7 _+ 2,5 % ( p < 0.05). after switching of no gas to the mo, further decrease of plateleta was stopped and platelets remained at 81,4 +_ 4,5 % until termination of circulation. platelets of the former no-mo decreased slightly after cessation of no gas to 92,6 _+ 5,4 %. conclusions: these data indicate that gaseous no significantly attenuates platelet trapping in hollow fiber oxygenators, when added to the gas compartment. this might be a new therapeutical approach for membrane oxygenator induced thrombocytopenia during long-term ecmd. objectives: nitric oxide (no) plays a pivotal role in regulation of vascular hemostasis. several studies elucidated the antiadhesive, antiaggregating, and disaggregating properties of endothelially synthesized no to platelets. additionally, agonist-induced no production in platelets by the l-arginine-no pathway was found as a negative feedback mechanism after platelet activation. although noplatelet interactions were intensively studied by several investigators, no data exist, about changes in platelet surface molecule expression in no-modulated platelets measured by flow cytometry using monoclonal antibodies (moabs). methods: p-selectin (alpha-granule-membrane protein, gmp-140, cd62p) and glycoproteiu 53 (gp53, lysosomal protein, cd63) are expressed only after platelet activation and degranulation. activation was quantified in thrombin (0.4 u/ml) and adp (0.1 ram) stimulated platelet rich plasma samples (prp). blood was obtained from healthy volunteers (n=7), who had no drugs for at least 14 days. for evahiation of no-modulated activation, the spontaneously noreleasing compound sin-i (0.1 mm) (3-morpholino-syndonimin-hydrochlorid) was added in parallel prepared samples prior to the addition of agonist. platelet surface molecule expression was evaluated with moabs directed against cd41a (gpilbliia, fibrinogen-receptor, phycoerythrin(pe)-conjugated), cd62p (fitcconjugated), and cd63 (fitc). only cd41a-positive signals were gated in sideangled light scatter, and assayed for activation marker expression (defined as percent of gated population). results: basal p-selectin expression was 1.6 + 0.7 %, and increased to 75.2 _+ 12.2 % after thrembin-activation, and to 26.7 + 5.3 % in adp-stimulated samples. addition of sin-1 attenuated p-selectin expression to 34.0 -4-193 % in thrombin (p<.001, two-tailed paired t-test), and 5.2 + 2.2 % (p<.001) in adpactivated platelets. basal gp53 expression was 1.7 _+ 0.5 % and increased to 63 .0 + 6.4 % in thrombin, and to 7.7 _+ 3.4 % in adp-stimulated samples. with sin-l, gp53 expression decreased to 346 _+ 10.4 % (p<.001) in thrombin, and 3.0 5:1.4 (p .001) in adp-stimulated samples. conclusions: these data implicate, that no leads to a significantly reduced activation of surface molecule expression in thrombin and adp-stimulated platelets. in addition, flow cytometry might be a useful tool for studying modulation of platelet activation by no or no-releasing compounds. introduction: acute cadmium poisoning is very rare. on initial presentation may mimic metal-fume fever, but acute inhalation cadmium toxicity may produce fatal chemical pneumonitis. case report: we present a case of acute fatal respiratory failure secondary to cadmium-fume irthalation. a 53 year old patient was trasferred from another hospital with acute respiratory failure presumably due to pneumonia. the last days before he had had commom cold symptoms. he had been cutting with a welder during one hour without any respiratory protective measure. three hours after exposure he developed progressive dispnea and was admitted to hospital. with presumtive diagnosis of respiratory infection, antibiotics were begun, however be failed to improve. all microbiological studies were negative. chest x-ray showed bilateral diffuse infiltrates. on seventh day he needed intubation and mechanical ventilation and on 10th he was admitted to our icu. antibiotics were stopped and new microbiological studies were performed including brochoalveolar lavage and virologic studies. all results were negative. he developed progressive hipoxemia and hipercapmia and finally, multiorganic disfunction syndrome. he died 19 days after exposure. the metal he had been working with was a 10% cadmium alleation. blood cadmilam concentration 15 days after exposure was 0.34 mcg cd/g cr, and urine cadmium concentration was 16.9 mcg/l. on postmortem examination, tissue cadmium concentrations were: blood 175 ng/ml, liver 823 ng/g, kidney 3571 ng/g and lung 1143 ng/g. these values confirm that cadmium was the cause of the fatal respiratory illness in this patient. conclusion: this case evidences the considerable hazard of acute poisoning after inhalation of eadmium-fume and stresses the need of appropiated safety measures against metal-fume poisoning. aim : lactic acidosis is considered the hallmark of cyanide poisonirig. however, the relationship between plasma lactate and blood cyanide levels has not been determined. the aim of this study was to determine the significance of plasma lactate concentration (plc) during the course of cyanide poisonings. methods : the patients were included according to the clinical suspicion of pure cyanide poisoning at the time of presentation. fire victims were excluded. serial blood samples were collected before and after intravenous hydroxocobalamin (hoco). blood cyanide concentration (bcc) was measured colorimetrically. plc was measured enzymatically. results : 8 patients were studied. on admission, plc ranged from 4.8 to 53 mmol/l, and bcc from 12.7 to 256 gmol/l. mean systolic blood pressure was 80 • 56 mm hg, mean arterial ph 7.34 • 0.14, mean anion gap was 29.7 + 7.7 mmol/l and mean pao 2 32.2 • 27.0 kpa. three patients died. before antidotal treatment, there was a significant correlation between plc and arterial ph (p = 0.008), anion gap (p = 0.008) and bcc (p = 0.016) but not with heart rate, pao2, paco 2 and blood glucose, or blood pressure. during the whole course of the poisoning, a plc _> 7 retool/1 was a sensitive and specific indicator of a blood cyanide concentration > 40 ~tmol/1. sustained catecholamine administration reduces the correlation coefficient. conclusion : baseline measurement of plc allows assessment of severity of acute cyanide poisoning. thereafter, plc may be used to assess the adequacy of antidotal treatment, more especially in patients not requiring sustained infusion of catecholamines. aim: the aim of this case report was [o study the correlation between the plasma lactate levels and several clinical, biological, and toxicological parameters serially measured during the course of a cyanide poisoning treated with a high dose of hydroxocobalamin. a 63-year-old male ingested potassium cyanide leading to cardiac arrest. cpr was performed prior to hospital arrival where the patient received 10 g hydroxocobalamin. sbp rapidly returned to normal allowing withdrawal of epinephrine. the patient remained comatose and died from brain injury 12 days after the ingestion. methods plasma lactate and blood cyanide levels were measured serially. blood cyanide levels were measured using a colorimetric method.~ plasma lactate levels were measured using an enzymatic method. for correlation spearman rank correlation test was used. results. initial plasma lactate and blood cyanide levels were 53 mmol/l and 256 gmol/l, respectively. there was no overall correlation between sbp and either blood cyanide or plasma lactate levels. similarly, there was no overall correlation between arterialvenous oxygen saturation difference with either blood cyanide or plasma lactate levels. in contrast there was a strong correlation between blood cyanide and plasma lactate levels (r=0.976, p<0.0001). the time-course of the blood cyanide concentrations was described by a mono-exponentiai decay (r2=0.968) with a blood half-life of 1.14 h. similarly, the time-course of plasma lactate levels was described by a mono-exponential decay (r2=0.986) with a blood half-life of 3.94 h. discussion. in this case of acute human poisoning, sbp was a much poorer indicator of continuing cyanide effect both before and after antidotal treatment, than was lactate production. this suggests a potential clinical role for following serial plasma lactate levels as a marker of the evolution of cyanide toxicity. aim : cyanide (cn) poisoning in fire victims is frequent and rapidly fatal. in a prospective study we tried to assess the clinical tolerance of a high dose of hydroxocobalamin (hoco) administered at the scene of the fire in fire victims suspected of cn poisoning. methods : inclusion criteria : soot in mouth or sputum ~ any degree of neurological impairment. exclusion criteria : children, pregnant women, burns of total surface body area > 20 %, multiple trauma. protocol desigrl following examination and the collection of a blood sample in dry heparin, a 5 g dose of hoco (10 g in case of cardiovascular collapse) was administered intravenously over 15 min. the systolic blood pressure was monitored before and after the administration of hoco, and one hour later. results : there were 28 females and 22 males. the mean blood cn concentration was 83 • 73 pmol/1. the mean blood carbon monoxide was 3.2• 2.1 mmol/1. nineteen fire victims eventually died. among the non-cn-intoxicated patients (blood cn < 40 ~mol/1), there was no significant change in arterial blood pressure. in the 33 cn-intoxicated patients (blood cn > 40 gmol/1) a significant increase in blood pressure was observed both immediately (p < 0.001) and 1 hour later (p < 0.001) after the admistration of hoco. no allergic reactions were observed. conclusions : in fire victims with cyanide poisoning, the administration of a high dose of hydroxocobalamin was associated with an improvement in systolic blood pressure. hydroxocobalamin is well tolerated in fire victims without cn poisoning. objectives: tricyclic antidepressant (tca) overdose can lead to serious complications including cardiac arrhythmias [ 1] . because of the known risk of early deterioration and the implication for management, emergent evaluation is essential. we determined the diagnostic usefulness of the electrocardiogram (ecg) in tca poisoning. methods: retrospective study of all patients with tca intoxication (pos. ,toxicology screening in urine and/or pos. history) in a 800-beduniversity hospital from 1989 through 1994. the severity was graded with mild= no symptoms or agitation; medium= disorientation, somnolence, tachycardia, or convulsions; and sever~ coma, significant arrhythmias or death. we analysed the first ecg after admission with a special emphasis on qrs-and qtc-intervals and the terminal 40ms frontal plane qrs-vector (tqrs), which, was reported to lie typically between +130 and 270* 798+310 915+453 5609• the best correlation with severity grade was found with qrs-and qtc-duration (p=0.0001), the tca-dose (p=0.0003) and hf (p= 0.027); tqrs did not correlate. 2 patients died (5.7%). conclusion: qrs-and qtc-prolongation in the admission ecg, and the reported dose of ingested drugs are useful predictors for severity of poisoning due to tricyclic antidepressants. we did not find additional benefit in determining the terminal 40ms frontal plane qrs-vector. objectives: since treatment of amphetamine poisoning is usually symptomatic and often associated with a fatal outcome, a search for specific drugs to help the amphetamine-intoxicated victim is sorely needed. methods: we report a case of a suicidal ingestion of large amounts of the amphetamine-derivative 3,4-methylenedioxy-ethamphetamine (mdea) and heroin (diacetylmorphine) and present the hypothesis that the two drugs produce opposing clinical effects. results: a 25 year old caucasian male was admitted to the emergency ward because of acute-onset confusion. at presentation, he was agitated and showed increased muscular rigidity. he had taken 40 tablets of "eve" (mdea, approx. 4 g) and 12 g of "smack" (heroin) by oral route approximately 2 h before admission. because of rapidly progressive tachypnea and exhaustion, the patient was intubated and ventilated. the serum concentration of "eve" on admission was 1400 ng/ml (lethal range 950-2000 ng/ml). trace amounts of cocaine and substantial amounts of heroin (115 ngtml; mean value in heroin-related deaths: 190 ng/ml) were also found in the serum. the patient was successfully weaned from the ventilator by day 4 and recovered without persistent neurobehavioral disturbance. despite high serum levels of both drugs, the patient did not present with the classic signs and symptoms normally seen during intoxication with these drugs. amphetamines in general, and mdea in particular, have opposite clinical effects to heroin or diacetylmorphine. none of these were however present in the case presented despite the high ingested doses and the serum levels in the lethal range. conclusions: the fascinating fact that, apart from the respiratory depression, none of the clinical signs reported after massive overdose with these two drugs were present, might be attributed to the opposite pharmacological effects of mdea and heroin. we believe that the patient unwittingly saved his own life by the oral coingestion of both mdea and heroin. our clinical data raise an interesting point about the pharmacological treatment of acute poisoning with amphetaminederivatives. introduction: the acute attack of aip still carries a significant risk of mortality of around 10 %. a succesful outcome depends on early diagnosis, removal of pricipitating factors and provision of intensive supportive therapy. objectives: twenty one patients (20 females, 1 male) with documented aip were seen over a 10-year period in the university hospital. 1 patient was in clinical remission and 20 were with the acute attack of aip, among them 4 with respiratory paralysis were required artificial lung ventilation and 4 -assistant ventilation with peee pathologic treatment during the attack was normosany, adenil, androgenes, glueosa, riboxin parenteral and enteral nutrition via nasogastric tube. symtomatic treatment -pethidine, propranoton, antibiotics, bronchoscopia. methods: intermittent phasmapheresis was performed on 15 patients. the following measurements were peformed: level of porphobilinogen (pbg) in the wire and delta-aminolevulinic acid in the blood. hematological and routine chemical evaluations, hepatic, hemodynamic and respiratory function. results: after plasmapheresis the median pbg excretion (normal range 1-2 mkg per/1 kgr creatinine) fill from 188 mkg on admission 140.8 mkg, then on 3-5 day raise to 193 mkg and then during treatment with normosong and prasmapheresis lowest level was 32.9 mgk. fatalities occured in two females during attacks with proforma cerebral involvement and 13 patients attained clinical remission. conclusion: after therapy with plasmapheresis normosong we found that there was consistently reduce the urinary excretion of pbg and shortening the duration of the acute attack. objectives: pigs has been reported to present with a higher pulmonary arterial pressure (ppa) and stronger pulmonary vascular reactivity than many other species, including man. aim of the present study was to compare pulmonary vascular impedance (pvz) before and after embolisation in weight-matched adult dogs and minipigs. methods: we investigated pvz spectra in 6 anaesthetized and ventilated (fio2 0.4) minipigs and 6 dogs. after baseline measurements the animals were embolised with autologous blood clots to reach a ppa above 35 mmhg. results: flow (03 and ppa matched pvz data (mean-+sem) are shown in the table. [zo = 0 hz impedance (z; {dyn.sec_em-5}); zl = first harmonic z; zc = characteristic z; z1 phase = first harmonic phase a@e {radians}; fmin = frequency of pvz the first m{n~mam; *, f p at least < 0.05 between dog and minipig, and before v~. after embolisation respectively]. before case report: a 53-yr-o]d woman affected by legs recurrent thmmbophlebitis, was admired in medmine department for tach.~pnea, chest pain, tachycardia and cyanosis. before starting two-dimensional transesophageal echocardiography (tee) to confirm the suspicion of pulmonary embolism, she suddenly had ventricular fibrillation. resuscitation and defibrillation were readily performed. when sinus rhythm was reinstituted she was in superficial coma with preserved corneal and light reflexes: right hemiplegia, poor perfusion and h~posphygrma of the left arm. tee showed dilation of rigth ventricle (rv), incomplete occlusion of pulmonary arter~ (pal at it~ hifurcation, severe tigth-to-left shunt through a patent foramen ovate, paradoxical embolism with incomplete occlusion of left subclavian artery mechanically ventilated with vt=800 ml, rr=12/mm, fio2=l, the patient had ph=7.28, pao2=57 mmhg and paco2=45.1 systemic bp was 130/80 mmhg and hr=80 b/min with low dose epinephrine (0.12 g/kg/min) a thrombolytic infusion (rtpa: 100mg/2h) through a peripheral vein was started tee imaging and clinical status 3 hours later were unmodified. a new rtpa infusion was performed through the pulmonary hole of a swan-ganz catheter with the tip close to the embolus. one hour later pa pressure decreased from 46/30 mmhg to 36/25 mmhg, etco2 increased from 26 to 30 mmhg and sao2 improved from 89% to 96% three days later the parietal, spontaneously breathing and with normalized tee scans of rv and pa, was transferred to rehabilitation service to perform physical therapy. conclusions: massive pulmonary embolism in a patient with patent foremen ovale, paradoxical embolism and refractory hypoxaemia was unaffected by systemic rtpa infusion, while intrapulmonary rtpa administration dramatically improved gas-exchange, hemodinamics and the general conditions of the patient. the presence of a large rigth-to-left _atrial shunt and the rapid rtpa metabolism could likely explain the effectiveness of its intrapulmonary administration in front of failure of systemic thrombolysis. introduction. cardiogenic shock during massive pulmonary embolism (blpe) is due to an acute increase of right ventricle (rv) afterload and possibly rv ischemia causing a failure of rv pump function. the rec~;mmended therapeutic strategies are: xoiume augmentation ~n ~rder m }ncrease rv pre-h~ad, adrenergic drugs to increase t'ontractillly and maybe coronary perfusion, fibrinolytic drugs to delermine clot lysis. there have been several reports of noradrenaline (na) as a useful drug in this setting for its sluing ~z, but also 1~, properties. case report.an obese 75 },ears old woman was transferred to our icu for tetanus. she was given the usual antibiotic and immunoglobuline therapy. l'wo thoracic epidural catheters were put in place at different levels and replenished with marcaine qid. a continous infusion of sedation (diazepam § was started together with mechanical ventilation. curarization ~,as given occasionally. fraxiparine 0.3/die was used for prophylaxis of thrombotic disease, on day 8th at 11.00 a.m. she started to be hypoxic (sa02 90%), tach3,tardic l1 l(i b/rain.), her blood pressure(rp) dropped frum norma~ values to 6r mm/hg, the central venous pressure (cvp) raised [rom lb to 27 mm/hg and the end tidal co2 was 7mm/hg lower than one hour before. the physical examination of the chest revealed a clear bilateral ventilation and the chest x-ray was normal apart from an elevation of the :tiaphragm as compared to the previous. an e.c.g. showed sinus tachycardia, right bundle branch block and a possible inferior necrosis (which was already present on admission). a trans-thoracic echozardiography was performed which showed "an acute overload of the right centricle wilh remarkable dilatation. tricuspidal regurgitation ++. paradoxical movement of septum. small left ventricle with normal wall kinetics". the cardiac enzymes were later shown to be normal. an acute massive pulmonary embolization was assumed m be present.. a bolus of streptokinase 750 x i(i 3 u. was given fonowed by a continous infusion . two liters of colloids were also given in a sh~rt time, two hours later the patient was still deeply hypotensive, hypoxemic and anurir(bp 54/32 mm/hg, cvs 23 mm/hg, spo2 90%) despite a cominnus infusion of dobutamine 20 fag/kg/min and adrenaline 0.5 ~tg/kg/min. at this stage a bolus of aoradrenaline 20 ,g was given followed by a cnntinous infusion of 0.05 !*g/kg/min. an immediate improvement of the hemodynamics was noticed and one hour later the bp was 149/77 mmhg, the cvp 24 mm/hg, the sao2 100% and a brisk diuresis started. the hemodynamics kept stable and weaning from vasoactive drugs was achieved within two days. one month iater the patient was discharged home in good conditions.. con c i u sio n.ne administration may help to restore rv coronary flow and ;~ump function during mpe. aeute putmonary t~omboembo~sm [ffe) cou16 be mamfeslated with either respiratory or cardiovascular syndromes or both. the arm of the study was to establish leading respn'atory symptoms, frequency and form of the roendganographic (rig) changes as well as blood gas disturbance degree in acute pte with dommam respiratory disease appearance. the study includes retrospeotive analysis of i 14 pte patients (pts), 63 males (average age 47,7 yrs) and .q females (average age 53,2 yrs). they were admitted at university, olinie" with suspection ofpleuropnlmonary disease, including pte. final diagnosis of pte was based o~ evident risk factors in 94,7% of the eases (deep venous thrombosis, surgery, trauma, imobilisation, malignancy ere), acceptable clinical, rtg, sdntigraphic and laboratory findings, as well as deep veins examination by dopple~-sonographie and radioisotopic -~enogmphy. respiratory symptoms appeared in all cases: sudden pleural pain (79%), dyspnea (64%), hemoptysis (49%), cough (39%) with association of two or more symptoms in 93%. chest xrays findings were abnormal in 92% with diaphragmal elevation (74,2~ lung opaeilies (69,5%), atelectasis (48,5%), plemal effusion (35,2%), main pulmonary brancah asimetry (22,8~ oligemia (19%), heart shadow changes (10,4%) and pulmonary arteries "cut off' (6,6%). the association of two or more abnormalities was found in 92,1% while normal chest x-rot was found in 8~ of the cases. hypoxemia with pao2<10,4 kpa was found in 64,4% followed with hypocapnia and respiratory alealosis in 34,6% in 27,8% of the gas exchage analysis were within normal limits. among cardiovascular symptoms short syn~cpa appeared in i0,5%, ecg changes-st q3t3 type in "~1,6%. results show high frequency of positive ~g findings in pte pts that is opposite to oppinion that chest x-ray in acute fie is the most ofran normal. leading symptoms are pleural pain and dyspnea, while hemoptysis were found in a half of the study group. blood gas changes were present in two thirds of the cases. kakkar, in his classic work ,clearly demonstrated the efficiency of low doses of heparin in prevention of deep vein thrombosis (lancet 2:669,1971) .after this first study the application of heparin prophylaxis became more and more diffused until to be considered a routine in many surgical departement.actually application of blood saving technique induces postoperative hemodilution effect. in that condition prophylaxis routinely applied seems a nonsense and can be at risk for postoperative hemorrhage. methods: to analize this problem we compared 100 patients arrived in our intensive care unit (i.c.u.) in.1980: (group a) with 100 arrived in 1994: (group b) .every patient was operated for major abdominal surgery.in each one we considered the hemoglobin (hb) value,hematocrit(hct), and coagulation pattern (c.p.) at the arrive in i.c.u. and 24 hours later. the patients was also divided in those receiving heparin prophylaxis (i) from not treated patients (ii) results:the application of blood saving technique clearly appears from the hb and hct level wich have a mean value of 11,4 +/-1,8 (hb) and 34 +/-2 (hct) in group a while in group b mean value are 9,7 4-/-1,2 (hb) and 29 +/-2(hct).patients of group a (ii) are the only one where a pathologycal c.p. with statistical significance has been demonstrated.in this goup we got four cases of evidence of venous thrombosis and one of pulmonary embolism.in patients of group b(i) we encontered the incidence of two cases of severe hemorrhage despite the absence of statistical significance in c.p.modifications. oxygen desaturation during broncho-alveolar lavage: role of oxygen saturation monitoring in prevention of acute respiratory insufficiency g. galluccio, b. valeri, s.batzella, m. di lazzaro*, servizio di endoscopia toracica, ospedale forlanini, rome, italy * servizio die anestesia a rianimazione, osp. forlanini the broncho-alveolar iavage is a diagnostic procedure employed in interstitial diseases of the lung. it requests the introduction through the working channel of a fiberoptic bronchoscope, after occlusion of a segmentary bronchus, of aliquots of saline solution at 37 c, subsequently gently reaspired, in order to remove cells and proteins from elf (endoalveolar lining fluid), which is related to interstitial medium. bronchoalveolar lavage induces deep effects on pulmonary function: -lowering of the alveolar surface of exchange; -shunt effect, depending on the perfusion of non-ventilated districts; -increased pulmonary arterial pressure, due to hypoxic vasoconstriction; -decrease of lung compliance. in this report the authors present the result of oxygen saturation monitoring in a group of patients with interstitial lung disease, who underwent diagnostic broncho-alveolar lavage. in most patients with severe interstitial involvement, the lavage performed without supplement of oxygen induced a severe fall in the oxygen saturation during the late phase of the procedure. if supplementary oxygen was delivered during bronchoscopy, since its beginning, only slight modifications of the curve were detected. in patients without thickening of interstitium, in whom the lavage was performed in order to obtain material for bacterial or cytologic examination, no modification of oxygen saturation was observed in standard procedure. as conclusion the authors strongly reccomend monitoring oxygen saturation in patients with radiologic evidence of interstitial involvement also in patients with no evidence of dyspnoea. g. galluccio, b.valeri, s.batzella, m. di lazzaro*, servizio di endoscopia toracica, ospedale forlanini, rome, italy * servizio die anestesia a rianimazione, osp. forlanini the treatment of choice in patients with alveolar proteinosis consists of pulmonary lavage. this procedure requests the introduction, through the working channel of a fiberoptic bronchoscope, segment by segment, of aliquots of saline solution at 37 c, subsequently gently reaspired, in order to remove the proteins deposited in the alveolar spaces. the method is very similar to that used in bronchoalveolar iavage, a diagnostic procedure used to obtain cells and substances from elf (endoalveolar lining fluid), which is related to interstitial medium. as known, bronchoalveolar lavage induces oxygen desaturation, because of shunt effect. understandably, one lung lavage has remarkably more deep effects on pulmonary function than bronchoalveolar lavage, for the amount of fluid introduced, the length of the procedure and the conditions of controlaterai lung. in this report the authors present the result of oxygen saturation monitoring in a patient who underwent pulmonary lavage for alveolar proteinosis. in the lavage performed without supplement of oxygen a severe fall in the oxygen saturation was observed during the late phase of the procedure. if supplementary oxygen was delivered during bronchoscopy, since its beginning, only slight modifications of the curve were detected. as conclusion the authors strongly reccomend the subministration of supplementary oxygen in pulmonary lavages, also in patients with excellent respiratory conditions. a. b. dublisky prof., m. r. isaakjan ass., v. a. zasukha, s. m. vinichuk prof., v. p. tserty ass. prof., chair of anaesthesiology, resuccitation and medicine of catastrophes, neurology of ukrainian state medical university, kiev, ukraine. objectives: detection of plasmophoresis's influence of results in treatment of ishemic insult. methods: we ve investigate 25 patients with ishemic insult, treated with reverse plasmopheresis in complex treatment. after primary infusive therapy we took 400 ml of patients' blood and separated it within 15 min with rotation frequensy of 2000/rain. after separation of erythrocytes from plasma, the latter has been returned to patients. we made 3-4 procedures during 3-4 days. hemoglobin, hematokrit, time of blood coagulation were determinated. the brain blood flow in internal carotid arteries, regional volum brain blood flow and total brain biood flow were evaluated with tetrapotar chest rheography and tetrapolar rheoencephalography. obtained date were comparised with control group after traditional treatment. results: it was found that after reverse plasmopheresis the hemoglobin and hematokrit levels decreased significantly in studied patients' plasma (from 140 + 3.2 g/l to 120 _+ 2.3 g/1 and from 44 + 2.1% to 35 _+ 1.8 % respectively). the time of blood coagulation by lee-white has increased by 2-2.5 times (up to 10-12 rain). the level of brain blood flow has been increased significantly after reverse plasmopheresis in comparison with control group. the following tests of brain blood flow have been increased: a) the total volume brain blood flow from 480.7 + 34.6 ml/min to 625.4 _+ 35.4 ml/min (p < 0.05); b) the regional brain blood flow from 52.2 _+ 2.8 ml/min to 87.1 + 6.2 ml/min (p < 0.01); c) the brain blood flow in internal carotid arteries from 166.1 _+ 12.2 ml/min to 206.3 + 14.6 ml/min (p < 0.05). conclusions: the use of reverse plasmopheresis in complex treatment of patients with ishemic insult aiiows to improve rheological blood patterns, helps to increase volume brain blood flow. it results in quicer reparation of neurological functions. objectives: a prospective evaluation of the efficacy of continuous infusion of verapamil in reducing the incidence of postoperative atrial fibrillation after pulmonary surgery. methods: a total of 199 consecutive patients, 100 on verapamil, 99 on placebo was included after lobectomy or pneumouectomy. a loading bolus of verapamil (10 mg over 2 minutes) was followed by a rapid loading infusion (0.375 mg/min) for 30 minutes and finally a maintenance infusion (0.125 rag/rain) for 72 hours. results: a mean plasma level of verapamil of 150 ng/ml was obtained only after more than 24 hours. atrial fibrillation occurred in five out of 78 patients who tolerated the verapamil infusion, and in 15 out of 99 patients on placebo (p = 0.08). verapamil infusion was not tolerated in 22 patients because of hypotension or a heart rate of less than 50/min, within 6 hours of the start of the therapy. when atrial fibrillation occurred, the ventricular response, mean _+ sd, was not significantly slower during verapamil infusion (132 + 22) compared to placebo (147 + 20). conclusions: because of its frequent side effects and the only modest efficacy verapamil should not be considered for prophylactic therapy of atrial fibrillation after pulmonary surgery, and is probably not a good first choice for slowing the heart rate in case of rapid ventricular response once atrial fibrillation has occurred in these patients. results: study of haemostasis in these patients has showed deep disturbances of blood coagulation. fibrogen level has reduced to 0.62 + 0.03 g/l, fibrinogen and/or fibrine degradation products concentration have enhanced to 0.40 _+ 0.04 g/l, monofibrin soluble complex concentration to 0.08-+ 0.04 g/l, blood plasmin level was enhanced to 34.0 + 0.2 mmol/1, plasminogen proactivator level was also enhanced to 153.0 + 0.60 ram, plateletes aggregation has decreased to 52 %. after plasmopheresis aggregation was decreased in 1.6 times. it has been connected with decrease of fibrin and/or fibrinogen degradation products level and level plasmin in 1.7 times, and plasminogtnt activator level in 4.6 times. at the same time we have observed increase in total antifibrinalitic activity of blood in 1.3 times. activity of activators plasmine and plasminogene proactivators has decreased in 1.2 times and in the same time activity of activation inhibitors and antiplasmines has increased in 7 times. conclusions: plasmapheresis leads to considerable improvement of a general condition and reduction of the haemorrhagic syndrom's sings (controlling of gastrointestinal haemorrage, reduction of intensity of subcutaneons haematoma). evaluation of continuous cardiac output (cc0) monitoring based on thermodilution technique in 35 critically ill patients. methods: cardiac output (co) was monitored continuously using a modified pulmonary artery (pa) catheter, on which a heating filament is located and by which energy is transmitted to the circulating blood. a microprocessor calculated co by a new algorithm. standard bolus thermodilution technique (10ml of ice-cold saline solution) was used to compare cc0 with intermittent bolus cardiac output (ic0) measurements. the following subgroups were prospectively studied: i. heart rate (hr) >120 beats/min, 2. cardiac output >10 i/min 3. cardiac output <4.5 i/min, 4. rectal temperature >39.0~ and 5. pa catheter was inserted for more than 4 days. results: a total of 404 pairs of ic0 and cc0 measurements were obtained from the 35 patients. bias (ico measurement minus cc0 measurement) of all measurements were 0.03• i/min and the 95% confidence limits (mean difference• were -1.01/1.06 i/min. also in the subgroups, cc0 measurement agreed closely with ico measurement (c0 >10 i/min: bias=0.16• i/min; co <4.5 i/min: bias=-0.17• i/mln). elevated temperature and prolonged lay-days of the pa catheter did influence agreement of cc0 measurement with ic0 measurement neither (>39~ bias=0.09• i/min). conclusions: monitoring of cc0 using a modified pulmonary artery catheter with a heated filament has proven to be accurate and precise also in the critically ill when compared with "standard" intermittent bolus thermodilution technique. this method enhances our armamentarium for more intensive monitoring of these patients under various circumstances. background: the number of patients who need coronary artery surgery was) grows every year. most of these surgical operations are with extrar eircuiation (ecc). since january 1994, this surgery is made without ecc in selected patients in our hospital. this technique is exceptional in spain. this type of surgery has proved useful in patients requiring revascularization of the left anterior descending, eireunflex or right coronary artery (not for grafting the pos~tefio~r descending branch}. blethods and results: since 1994, 30 patients aged 54 to 77 years (mean 66 years) underwent cas without ecc. the mortality in programmed surgery was 0%. no patient was reexplored for hemorrhage. the mean values of some clinics parameters v~ere: a) blood requeriments: 2 units per patient, b) need of mechanical ~entilation: i3,6 hours, c) postoperative bleeding: 900 cc, d) days at icui 2,5. we used the student % t test or fisber~s exact test to compare these results with the mean values of surgery with ecc: a) blood requeriments 4 per patient (p<0,0001), b) need of mechanical ventilation: 29 hours (p<0,0001), c) postoperative bleeding: 1300 cc (p<0,004), d) days at icu: 4 (p<0,001), e) programmed surgery mortality: 7% (p<0,05). conclusion: our limited experience shows that this surgery is an alternative in the treatment of coronary disease, especially for aged patients with associated pathology and in jehova's witness. the need of mechanical ventilation, days at icu, blood requeriments and morbi-mortality were fewer than surgery with ecc. to study the hemodynamic and antiarrhythmic influence of ace-inhibitor enalapril in acute myocardial infarction (mi). methods: holter ecg monitoring, heart rate variability analysis, echocardiography (3 and l0 days after beginning of the treatment), stress-echocardiography and stress ecg (8-10-th day after the onset of mi). enalapril was included into the treatment of 42 pts with mi (study group), with normal or increased blood pressure, from the 1-st day of the disease. the data were compared with 30 pts treated without enalapril (control group). results: silent ischemia during stress-test was registered in 6 pts of the study group and 8 of control group, the arrhythmia episodes during stress test -in 5 and 8 pts and episodes of silent nocturnal isehemia -in 7 and 12 pts correspondingly. enalapril importantly attenuated the hypertensi~re re~aetioh %0 stress test. in 10 pts of the study group the number of perifocal hypokinesis zones decreased; in the control group it didn't change. the quantity of ventricular extrasystoles in the patients of the study group decreased by 25%; the heart rate variability indices improved as well; in the control group the character of ventrieulir arrhythmias, heart rate and its va]~i~bili%y didn't change significantly. conclusions: the inclusion of enalapril into the treatment of mi is a useful t0ol to improve hemodynamie parameters and decrease the incidence of ventricular arrhythmias. objectives: to study left ventricular (lv) systolic function in the patients with acute myocardial infarction (ami) before and after peroral captopril test. methods: the original echocardiographic parameter of lv contractility, "coefficient of effective systolic function" (cesf), was proposed in the study. cesf is calculated from lv stroke volume (sv), obtained from doppler aortic flow in lv outflow tract and lv end-diastolic diameter (edd): cesf =sv/edd. the study included 60 patients with ami, who had local lv dyskinesia and global lv systolic dysfunction (ef<45%). besides cesf, the ejection fraction was calculated before and after administration of 25 mg eaptopril (on the fifth day of ami) by methods of bullet and simpson. results: the dynamics of these parameters, as well as heart rate (hr) and mean blood pressure (bp), is shown in the tabte. before cal~topril ef (bullet) 32.12 • 2.51 ef (simpson) 35 . introduction: the cold system is a monitoring system for measurement of right (copa) and left (coart) ventricular cardiac output, cardiac function index (cfi), fight ventricular ejection fraction crvef), fight ventricular cnddiastolic volume (rvedv), intrathoracic blood volume (!tbv), global enddiastolic volume (gedv), lung water (etv) and excretory liver function (pdr). patients and methods: 41 pts have been monitored by the cold system. above mentioned parameters are measured by thermal dye dilution and a fiheroptic femoral artery catheter. copa, rvef and rvedv measurements additionally were compared to measurements by the baxter explorer. :::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::: ;;;k;;;;i cov (%) explorer ! 6 13 ! 1 [ gedv, itbv and pdr showed a significant decrease dufing the first 12-24h after the operation, cfi and rvef si~canfly improved after 48k wheras etv showed a i~ in the early postoperative phase and fell to normal ranges at 48h. comparison of cold/explorer m~ements sb0wed good correlations. discussion: concerning m0~toring of ri,ght ventric~ar function cold and explorer can he seen as equal. rvef gives an ar report about the performance of the right ventricle without use o f echocardiography. measuring itbv and gedv ~ improve ~gement and con~ol of th.e volume status, monitoring etv helps preventing lung edema. pdr shows good corre|ati0n to liver blood chemistry and is bedside avai|ab|e. thus the cold system offers additional parameters for comprehensive m~nitofing of pts. ~e~ ~c surgery. obiectives: to evaluate the influence of an a!'~ered cardiac function on the cardiovascular response to the increase in oxygen demand induced by an increase in core temperature. methods: this preliminary study included 12 adult critica!ly ill patients monitored by arterial and pulmonary artery catheters in whom thermodilution cardiac index {ci) and arteria! and mixed-vef)ous blood gases measurements could be obtained before and after an acute change in core temperature of at least 0.5~ (max 60 rain apartl the patients were separated in two groups according to their cardiac function: 4 patients had an impaired cardiac function as defined by a history of cardiac disease and an ejection fraction below 45% and 8 patients had normal cardiac function. results: individual data are shown in the figure. in contrast to the control group (continuous line) in which c! increased without changes in oxygen extraction (02er), the q2er in patients with impaired cardiac function (dottled line) increased without changes in ci. conclusions: the increase in oxygen demand associated with changes in temperature is met by an increase in c! in patients with unaltered cardiac function and in an increase in o2er in patients with altered cardiac function. temperature should be taken into account in the assessment of the adequacy of cardiac output in patients with impaired cardiac function. objectives: to define the hemedynamic and metabolic response to physical therapy(pt) in relation to the type/level of sedation and the cardiac status in icu patients. methods: we studied 34 mechanically ventilated icu patients (64• years) in stable hemodynamic status (no change in vasoactive treatment for at least 2 hours), separated in 4 groups: group 1 = deep sedation, cardiac dysfunction required dobutamine (n=5)r group 2= deep sedation (barbiturates), unaltered cardiac function (h=lo), group 3= moderate sedation, altered cardiac function (h=7) and group 4= moderate sedation, unaltered cardiac function (n=12). complete hemodynamic data, arterial and mixed venous blood gases, respiratory gas analysis (metabolic cart ccm, medgraphics) were obtained at baseline (2x) and twice (q.10 min) during leg mobilization. data were analyzed by anova. calcium channel blockers were used in complex preoperative preparation of 59 hypertensive surgical patients. patients were allotted to 3 groups based on their hemodynamic profile: hypokinetic: ejection fraction (ef)<0.6, 29 patients; eukinetic (ef>0.5),i6 patients and hyperkinetic (ef>0.6),i4 patients. the most noticable change in hemodynamics was in the hypokinetic group: ef and cardiac output (co) were significantly decreased (p<0.001) while systolic arterial pressure (sap) (p<0.05) and peripheral resistance (pr) (p<0.01) were elevated. the results showed that in hypokinetic patients on nifedipine ef (p<0.00t) stroke volume (sv) (p<0.0l) and co (p<0.001) were increased while pr(p<0.0t), sap(p<0.001) and diastolic arterial pressure(p<0.05) were decreased. eukinetic type patients also showed an increase in ef,albiet to a lesser extent,than in the hypokinetic group. increased sv and co(p<0.01) were observed in eukinetic patients though this was to a lesser extent than in the hyperkinetic group. in the hyperkinetic group of patients nifedipine had no effect on the aforementioned parameters except for a decrease in sap(p<0.0 i). nifedipine increased ef in all hypokinetic patients. comparative results show that isoptin was less effective than nifedipine in decreasing peripl~eral vascular resistance and had a depressive effect on the myocardium. it can be concluded that the action of calcium channel blockers normalizing the circulation in the hypertensive surgical patient depends on: the condition of myocardium, the patients hemodynamic profile and their pharmacological properties. they were most effective in the hypokinetic group. zalo/nthinos e., daniil z. zakynthinos s., armaganidis a., kotanidou a., nikolaou ch..,roussos ch. critical care department, university of.athens, evangelismos hospital, athens, greece. introduction : surgical is the optimal treatrnent for ioculated effusions and the preferable procedure when multiple bands are seen in the pericardial sac by echo. patients : 6 palients, 4 post cardiac surgery, 2 uremic (3men, 3 women) with large pericardial effusion and clinical or echocardiographic findings of tamponade or both. these particular patients displayed numerous linear echo-dense bands and s~'ands crossing the pericardial space (in one of them a ioculated effusion compressed the left ventricule). one had aptt increased, four were mechanically ventilated. technklue : a 8fr polyurethane catheter with end and multiple side holes over 18 ga needle was echo-guided to the ideal site (fluid abundant and closest to the transducer). the catheter was attached to a close system with a heimlich valve for continuous drainage (pneumothorax kit). subcostal entry was selected in one patient and chest wall in five. the patient's position was changed every hour at least. (we believe that the small changes in the position of the catheter and the mechanical breaking of the bands in relation with the movement of the heart assist the pericardial fluid to remove). results : in all cases only a small quantity of fluid was withdrawn in the first minutes(30-70ml) with some clinical and echo-findings improvement. the fluid was bloody or serosanuginous with high protein content (ht=15% ,protein 5,1gr/dl) in all cases. in first 24 hours the mean volume of fluid removed was 550ml (350to 720ml). in that period echo showed no residual fluid. the catheter remained within the pericardium 1 to 3 days .. no complications are mentioned. conclusion : cardiac tamponade due to hemorrhagic high protein pericardial effusion in uremic and postcardiac surgery patients,, as it is revealed by echo dense bands, can be faced by 2-d echo guided perieardiocentesis. a 8-fr polyurethane catheter with multiple side holes, attached to a heimlich valve was effective to evacuate the pericardial fluid. no catheter was occluded though heparin infusions were not used. multiple changes of the patient's position may be fundamental. this 2-d echo guided pericardiocentesis performed in in~nsive care unit seems to be useful , safe and quick technique. determining the best inotropic drug represents a very serious problems. the use of more selective and potential inotropic and vasodilatative drugs does not always lead to improvement of hemodynamic parameters in patients with low cardiac output syndrome. this paper presents patients with acbp who need an inotropie support after extracorporeal circulation in first 24 hours. the patients were divided into dobutamin et dopamine groups. the heart rate (hr). mean sistemic arterial pressure [map), central venous pressure (cvp). and termodilution cardiac index (ci) were measured. the measurements were without using inotropic drugs, and then using them after 5 rain, 30 min, and finally with one hour rate, within first 24 hours. the statistical analysis shows that both drugs lead to an increase in hr in the first hour of the application. the final effect of dobutamine is no change in hr, whereas the effect of dopanime is very significant increase in hr. thus. an absence of taehyeardie response selects the dobutamine as a better choice. backeround: pulmonary vascular eadothelium possesses major metabolic functions, which when altered contribute to the development of serious pathologies such as ards. one such function is the conversion of angiotensin i to angiotensin ii, catalyzed by angiotensin converting enzyme (ace), located on the luminal surface of the endothelial cells. ace activity has been extensively studied in animals in vivo, by means of indicator-dilution techniques, providing: i) under toxic conditions, an early index of lung injury, and it) under normal conditions, estimations of dynamically perfused capillary surface area (pcsa). objectives: to validate the use of these techniques in matt: i) for pulmonary endothelial function assessment, and it) for pcsa estimation. methods: ace activity was estimated in ten adult haman volunteers, with no pulmonary medical history and normal pulmonary artery pressures, undergoing cardiac catheterization for coronary artery disease assessment. single-pass traspulmonary hydrolysis of the specific ace substrate 3hbenzoyl-phe-ala-pro (bpap; 30p.ci) was measured by means of indicatordilution techniques, and expressed as %metabolism (%m) and v=-hi(1-m). bpap was injected as a bolus i) into a main pulmonary artery, and it) inside the right atrium, to assess ace activity in one and both lungs. we also calculated a,~,/i~, an index of pcsa. pulmonary plasma flow (fv) was determined by thermodilution. fp in one lung was estimated as 0.5xf v. results: similar values of %m (69.6+3.8 vs 68.9• and v (1.29• vs 1.25• were observed in both and one lung respectively. a~k~ decreased from 4185• ml/min (both ltmgs) to 2122:~175 (one lung). conclusions: i) pulmonary endothelial ace activity and thus pulmonary endothelial function may be assessed in humans by means of indicator-dilution techniques, it) our data denote homogeneous pulmonary capillary ace coneentratious and capillary transit times in both haman lungs, iii) the 50% reduction of a=~/k~ in one lung suggests that this procedure can be used to quantify pcsa in man. (supported by the fonds de la recherche en saute du quebec and the national health system of greece). objective: verify whether antioxidant activity is higher in reperfused than in no-reflow myocardium after i.v. thrombolysis for acute myocardial infarction (ami). methods: 37 patients with ami were included. blood for estimation of catalase (cat), glutathione peroxidase (gpx) and mn-superoxide dismutase (sod) was drawn before initiation of i. the mechanism of myocardial cell defence against free radicals is probably identical in both reperfusion and no-reflow phenomena. therefore, antioxidants cannot be used as reperfusion markers. objectives_ to evaluate the precipitating factors of hypothermic phrenic nerve injury following cabg with lima. methods: fifty two consecutive patients (8 females), with a mean age of 59+8 (mean +sd) years were studied. during the ischemic arrest time topical hypothermia was obtained in al~ patients wffh ice slush and no cardiac insulation pad was used. all patients received a lima graft, with or whithout additional vein grafts. supramaximai, bilateral phrenic nerve stimulation was performed percutaneously preoperatively and whithin 24 hours postoperatively. square wave stimuli of 0.1 msec duration were applied at the posterior border of the sternomastoid muscle. the compound muscle action potential of the diaphragm was recorded, using surface electrodes on the anterior chest wall. the time interval from the application of stimulus to the onset of diaphragmatic activity, phrenic nerve conduction time (pnct), was measured. values exceeding 9.75 msec were considered as abnormal. besults: preoperatively, all patients had normal (mean+sd) pnct, 7.69• msec for the left nerve and 7.98• mseo for the right nerve. on the first postoperative day, right pnct was normal in atl patients (7.93• msec) , whereas left pnct was normal in 45 patients (7.86• msec) and abnormal in 7 patients (incidence 13.5%). in 6 patients the left phrenic nerve was inexcitable and in 1 patient left pnct was prolonged (10.50 msec). comparing patients with normal and abnormal pnct there was no difference in age, gender, number of grafts used, aortic cross-clamp and bypass time. however, patients with abnormal pnct had a lower preoperative ejection fraction (45• vs 53• p=0.03). moreover, in all of them lima was dissected from its origin ligating all upper arterial branches, which provide the blood supply to the left phrenic nerve, whereas in those with normal pnct the small vessels originating from the upper 2 to 3 cm of lima were preserved (p=0.001). conclusiojel~ a hypoperfused left phrenic nerve seems to be more susceptible to hypothermic injury during cabg with a lima conduit. objectives: to test if necessary interventions on systemic vascular resistance (svr) along with preset pump flew (q) during cpb could adversely affect autoregulatory response and cause vo 2 shifts. methods: we studied 26 males (554-7 yrs) who underwent cpb for cardiac surgery. at o oesophageal temperature 27-28 c we set pump flow at 2.1 i.m~2.min -1. when map was higher than 85 mmhg we calculated vo 2 by using fick equation. then we infused sodium nitropruaside (sn) to control map at 55-65 mmhg for 10 min and we calculated vq 2. without changing the sn infusion rate we set q at 2.5 i.m'2.min "1. ten min later we measured vo 2. we took vo 2 changes into consideration if greater than 15%. statistical analysis using students-t-test for paired data and analysis of variance was used as appropriate. results: depending on the biphasic vo 2 response to sn infusion during low and high q we classified pts in four groups (table). i. vo 2 increases with sn and increases further during high q unmasking hypoperfusion and supply dependency. ii. vo 2 increases with sn but the addition of high q results in systemic shunt. iii. vo 2 increase during high q proves that vasodilatation can turn flow insufficient. iv. vo 2 does not change with any intervention. the small number of pts and the wide standard deviation did not allow any statistical significance. conclusions: cpb is an interesting model for the behavior of microcirculation. intervention on svr and q can improve or impair effective regional oxygen delivery, resulting in either better perfusion or systemic shunt. vo 2 monitoring seems necessary during cpb. preoperative cardiovascular optimization (opt) to ci > 2.8 l/min/m 2, 8 _< paop < 18 mm hg,and svri __< 30 mmhg/ll/min/m 2 decreases cardiac events (events) and mortality (mort) in peripheral vascular surgery patients (pvs). objectives: to determine if opt to the same endpeints decreases events in patients undergoing abdominal aortic aneurysm repair (aaar) and to study the r predictive value in pvs patients. methods: 44 aaar patients and 41 pvs patients were admitted to the s cu monitored with e pa and arterial catheters and treated to achieve opt. patients underwent surgery independent of success of opt data included demograph cs, incremental risk factors, laboratory and hemodynamic data pre, intra, a~nd postoperatively events, and mort. events included arrhythmias requiring treatment or prolonging the sicu stay > 24 hours, a st depression > !mm or t wave inversion, an acute mr defined by a new q wave > 0.03 sec or cpk-mb > 5%. results are presented as means _4-. sd. opt was achieved in 32 of 41 (78%) and in 36 of 44 (82%) in the pvs and aaar group, respectively. events did nat differ between groups 9 of 41 (21,9%) and 12 of 44 (27,2%) in the pvs and aaar group, respectively (p>o.05). mort was 0 of 41 (0%) and 1 of 44 (2.2%) in the pvs and aaar group, respectively (p > 0.05), while there was no difference in endpoints of opt between patients with and with.out events in the aaar group, there was a significant difference in ci between patients with and without events in the pvs group. of note, 8 of 9 (89%) patients who developed events in the pvs group had a ci < 2.8 in contrast to 4 of 12 (33%)in the aaar group. the positive and negative predictive value were 89% and 97% in the pvs and 50% and 75% in the aaar group. conciusione: f. the endpoints of opt used for pvs patients cannot be ~sed to reduce events in aaar patients; 2. pvs patients who have net achieved opt are at extraordinary risk of perioperative events; 3. preoperative card ovascu ar opt in aaar patients makes no difference in cardiac related events, background : comparison of the right and left filling pressures (cvp/pcwp ratio) is considered as a useful diagnostic clue : the normal ratio is _< 0.6; ratio >_ 0.8 may suggest right ventricul~ infarction while equalization of the cvp and pewp is a classic sign of tamponade (1). however after cardiac surgery, many conditions (diastolic dysfunction, pulmonary hypertension, positive pressure ventilation) are susceptible to modify the '*normal" cvp/pcwp ratio. material and method : we determined cvp/pewp ratio in 100 consecutive patients (pts) after uncomplicated cardiac surgery (78 coronary artery bypass grafts; 22 valvular replacements) measurements were made before and after tracheal axtubation. results :cardiac index : 3.2 _+ 0.7 1/minlm~; laotate: 144 + 68 rag/i; cvp range : 4-17 rnmhg; pewp range : 2-16 mmhg. mean cvp/pcwp ratio before extubation is 0.94 (95 % confidence imerval : 0.86-1.02) and after extubation, 0.93 (95 % confidence interval : 0.83 -.1.03), (ns, paired t-test). in 25 % of the pts, cvp was higher than pewp. there are no correlation between the cvp/pcwp ratio and c! before (r = -0.04) and after extubation (r = -0.09) nor between the cvp/pcwp ratio and mean pulmonary arterial pressure (mpap), before (r = 0.08) and after extubation (r = -0.13), discussion : cardiac performance is adequate according to ci and lactate. however the cvp/pcwp ratio is markedly higher than the "normal" (_< 0.6) ratio. this difference is not related to mechanical ventilation because the ratio is similar before and after extubation, nor to pulmonary hypetaension because of absence of any correlation with mpap, post-cpb diastolic dysfunction of the right ventricle could be an alternative explanation. in this group of pts, increased cvp/pewp is not associated with any impairment of cardiac performance (absence of correlation with ci), conclusions : cvp/pcwp ratio as high as 1 within a large range of cvp (4-17 mmhg) and pcwp (2-16 mmhg) may still be considered as normal after cardiac surgery. this emphasizes the limitations of the hemodynamic monitoring after cardiac surgery (in comparison with echographic technics). careful analysis of the morphology of the cvp and right ventricular pressure curves (x descent, y descent, dip-plateau) is mandatory rather than relying on the quantitative assessment alone. reference : (1) ntensive care.-university hospital -m~laga (spaink introduction. fibrinolitic treatment (ft) permits the treatment of acute myocardial infarction (ami) addressing the etiology, thereby eading to mproved ventncular function and a marked reduction m mortality. the main clinical oroblem is the reduced time of application. delay in hospitalization, which can be from 50 to 120 minutes, is potentially the most avoidable delay. method. to reduce delays in hospitalization, the following was carried out in two chases. 1 audit: analysis of the time lapse from onset of symptoms to start of ft. showed that during "(he period 1 june to 31 december 1994, 79 patients with chest paros were treated within a 0eriod varying from 30 minutes to 6 hours from onset of symtoms. ages ranged from 33 to 86 (average 64,4), oelng 49 males and 30 females. they were glved initial ecgs to determine st mcreases suggesting ami. median t~me for this orocedure was l0 m.. 204 potentia ami patients were then admitted to the coronary unit, 103 [)atients, under age 75 with no contraindications received ft the median time apse from admission to corona-y care and administration of ft was 15 minutes (12. 17), -he total median delay was 58 minutes ~40-i h.45min,~ delays n start of this procedure are grouped as follows: extra-hosdita delays (from onset of symtoms to arrival at hospital) diagnostic delays (from hospital arrival to ecg). treatment delays (from diagnosis to ft). 2 objectives: protocol of procedure to implement a fast-track method. a protoco was drawn up with the object of reducing diagnostic delays to 8-i 0 minutes and treatment delays to less than i 5 minutes results. following rmplementatlon of this protocol in january 1995, 30 fts were glven, with an over all average delay of 24 minutes. this fast-track method did not reveal any inappropnate ft or any increase m complications, conclusions: detailed study of the various times taken for diagnosis ane treatment of ami patients, showed up weaknesses in the system and improvements througn the protocol based on performence orocedures which led to a 59% reduction in the start of ft background: the importance of the early use of thrombo!ytic agents in acute myocardial infarction (ami) is based in the better remaining ventrictjlar function and smaller mortality rate because of the greater reperfusion and sma!ler infarction size, therefore, it is very impodant to apply this treatment to the maximum number of patients without thrombolytic contraindicati0n, and within the minimun period of time. the "thrombolytic fast track" implementation allows to optimize the time to administrate thrombelytic agents avoiding multiple delays~ methodology: we anal!ze the application of thromboly0c agents to 73 patients with suspect of ami from the begin!ng of september 1994 until the end of february 1995. in this time there are two different periods, during the first 4 months thrombolytic agent were admin!strated at intensive care unit (icu), and during the second period we carried out a protocol of quick detection and thrombolysis therapy in susceptible patients at the emergency room in order to reduce the time to treatment. ma!n results are shown in the faffewins de ay h=hours m=minutes the implementation of the fast track does not need supplementary personal or equipment but a protocelized approach and training of the personal involved the main problem detected was the usual attendance overload of the emergency department that makes difficult to follow many structurated actions. conclusions: pratocqlized changes in the management of ami can significantly reduce the detay in the administration ef thrombolytic agents. it is not necessary to eomplet the procedure iq the emergency department, as the use of bolus schedules allows to begin the treatment in this area and to transfer the patient to icu afterwards. elective cardiac surgery. b calvet, f ryckwaert, p trinh duc, p colson. anesthesia -reanimation, hopital arnaud de villeneuve, montpellier, france. obhectives: the study was aimed at analysing the incidence of renal dysfunction following cardiac surgery and its prognosis (acute renal failure, post-operative morbidity and mortality). methods: two hundred and thirty seven patients (aged from 28 to 90) were consecutively operated on for elective cardiac surgery and retrospectively included in the study. patients with preoperative infections and operated on in emergency were excluded. each patient had preoperative invasive cardiac investigation with angiography and calculated ejection fraction (ef). anaesthesia, cardiopulmonary bypass (cpb) and cardiac arrest management were similar in all patients. general body temperature was reduced to 28 -30 ~ c. renal dysfunction was defined as a 20 % increase from baseline of serum creatinine. demographic data, asa, treatments, pre-operative creaunine level, cpb and clamping (axc) times, intra and postoperative use of inotrope, serum lactate level before surgery, at the end of cpb, at the time of admission in intensive care unit (icu) and on post operative day one and apache score were compared in patients with or without renal dysfunction using anova test for repeated mesures and x2 when appropriate. data are expressed as mean +__sd. p value less than 0.5 was considered statistically significant. results: thirtytwo patients (13,5 %) suffered from renal dysfunction. age, serum lactate level at the end of cpb, at admission in icu, at pod1 and apache level at admission in icu, intra-operative use of inotropes were statistically different in patients with or without renal dysfunction (p<0,05). mortality rate was statistically different in patients with or without renal dysfunction(~,2,5 % and 0 %, respectively, p=0,001). incidence of acute renal failure following renal dysfunction was 6,2 % (2 patients required hemodialysis). conclusions: although our cdteria for defining renal dysfunction were very sensitive, the incidence of renal dysfunction following elective cardiac surgery was lower than communly accepted in the litterature (1). however renal dysfunction appeared significantly associated with a poor prognosis. reference: 1-settergren g, ohqvist g current opinion in anaesthesiology 1994, 7:59-64 r 66; 159, 200 tzelepis, g. 112, 127, 142 late complications were observed in 14% of cannulations: local infection in 2 (i,7%), catheter displacement by the patient in 4 cases (3,5%), catheter displacement during nursing care in 5 (4,4%) and malfunction in 5 cases (4,4%). conclusions: central venous catheterizations are followed by immediate and late complications in almost the same percentage acute poisoning with amphetamines (mdea) and heroin: antagonistic effects between the two drugs methods: after institutional approval and informed consent, 33 selected patients (65_+9 years) undergoing peripheral vascular surgery (n=17) or carotid endarterectomy (n=16) were investigated. patients included had either documented cad (n=18) or two or more (n=15) dsk factors (age >65 years, smoking, diabetes meltitus, hypertension, hypercholesterolaemia >240 mg/dl). 12-lead ecg recordings were carded out preoperatively, on ardval in the postanaesthetic care unit, and 20 h, 48 h, 72 h, and 84 h postoperatively. ecg recordings were analysed by an independent blinded cardiologist for signs of pmi (new st segment depression >0.2 mv and/or new t inversion). in addition results: 22 of the patients investigated developed ecg-documented pmi, 86% occurdng in the immediate postoperative phase. troponin i levels >1.6 ng/ml were found in 19 of these 22 patients thus, comparing a cardiac troponin i cut-off level of 1 ng/ml with intermittent 12-lead ecg recordings, we found a sensitivity of 86% and a specificity of 91% methods: demographic, clinical and ecg data were analyzed. 53.8% of patients were male; 46.2% female. cad was the most common underlying cardiac disease (85.7%) and 58.4% underwent open heart surgery. 69% received proeainamide for supraventricular and 31% for ven~cular arrhythmias. 27% received a loading dose. maintenance was provided by iv route in 36.8% and by po in 63.2% (40.8%sr end 22.4% ir). 40.4% of patients were obese right ventricular function following cardiopulmonary bypass: is important the mode of myocardial protection we underwent this study in order to examine its safety and usefulness in pts with trustable coronary conditions (unstable angina ua the mean age for group a was 54 • 16 years, for group b 64 • 11 years, and for group c 59 • 13 years. a history of previous myocardial infarction was present in 6 pts of group a, in 3 of group b and in 54 of group c. three pts in group a, 5 in group b and 15 in group c had previous coronary artery bypass grafting. the median time between the onset of symptoms and a was 5 days (2 -19) for group a we used a continuous fixed intravenous a infusion at a dose of 140 the sn was 100% in groups a and b, 98% in c, and sp 100% for group a, (fixed defects included) and 50% for groups b and c. there was no difference of side effects among groups: chest pain (i pt -group a, 2 pts -group b, and 8 pts -group c), transient hypotension (1 pt -group c), headache (5 pts, group c), dyspnea (1 pt -group a), while st depression was seen in 2 pts of group b and in 2 pts in group c. the rate of a infusion was decreased to 70 7/kgr/min in one group b pt due to development of chest pain s228 five year follow up of humoral immunity in paced patients athens polyclinic hospital, department of cardiology athens, greece author index a abiad ch 133 bertschat, e 141 betbes6 75 blanch, l1 177 del nogal saez 93 e1-meneza 220 nolla, j. 98 nolla-salas 24 pilz~ u puig de la bellacasa e 38 scarpa, n. 77 217 van de wetering objectives: only 50% of patients suffering from acute guillain-barr@ syndrome (gbs) respond promptly to established therapies like plasma exchange or intravenous immunoglobulines. in contrast to serum, cerebrospinal fluid (csf) of gbs and ctdp patients contains enriched portions of antiexcitatory factors(i) and cytokines (2) able to induce pronounced conduction block (3). to reduce or remove such pathologic factors we introduced a technique with direct access to the subarachnoid space. methods: with informed consent we lumbally inserted 18 g catheters in 24 gbs-and 22 cidp -patients under sterile conditions. some of them had not responded very well to established therapies. 30-40 ml of csf were withdrawn and retransfused by a bidirectional pump (flofors) after passing newly developed filters (pall). daily filtrations with several cycles were performed (200 -300 ml) over one week. results: the 24 gbs patients improved after 19 days (median) for one grade (according to the gbs-scale from the gbs study group) . the ventilator dependent patients were weaned after 16 days (median). patients not at all treated before (16/24) responded better than patients that had been pretreated (8/24) with plasmaexchange or intravenous immunoglobulines. 18/22 cidp patients drew benefit from treatment, 10 stabilized iongterm. conclusions: csf-filtration is a relatively save and well tolerated additional procedure. the costs are considerably lower (1/3) than those for plasmaexchange or intravenous immunoglobulines. references:(1)wsrz aet al: csf and serum from patients with inflammatory polyradiculopathy have opposite effects on sodium channels. muscle nerve (1995) . (2) clinical observations were made in 24 patients admitted to the clinic. they were in coma associated with acute alcohol intoxication.standard evaluations (ecg-monitoring, electrocardiography, neuromonitoring, studies of acid-alkali condition, biochemical and toxicologic investigation of blood and urine) prior to and following the treatment conducted were undertaken in all the patients.to correct irreversible impairement of functions twofold laser blood irradiation by means of alok-01 apparatus, the exposure within 20 minutes, was carried out.the data obtained confirm more rapid coma withdrawal of the patients, reconstruction of the heart and central nervous system electrophysiologic indeces, reliable reduction in complications compared with the control group. objective: to know the actual incidence of the critical illness polyneuropathy(cip). setting: fourteen intensive/critical care unit beds, in 550 bed university hospital, covering 345.000 inhabitants (majority rural area). the icu patients are medical, surgical and coronary, excluded the neurotrauma and neurosurgical. design: a conseculive and prospective study. all the patients admitted during three months, from january lth to march 31th 1993, were eligible (patients with admittance diagnosis of polyneuropathy were excluded ). methods: patients with apache ii score >10, at the admission and six days after admissions were included into the study protocol. diagnosis of sepsis, mof, and all the drugs administered days before were recorded. a complete neurological exam, by a neurologist, in absence of ssdatives and muscles reliant (7th, 25~ and 60th days after icu admittance) was made. we evaluated the nerve and muscles function with and electromyography study in all patients, at same days. in some paeents with cip we performed a nerve biopsy. results: from 285 patients ( apache ii score: 12.82) admitted in the icu, 16 (5.6%) enter the study protocol. seven (2,45%) had an axonal polyneuropathy(cip), three very severe. only four of the patients with cip had pathologic clinical exam. apache ii score: cip vs non-cip was 22.6 vs 16.6. the incidence of cip by diagnosis (cip/diagnosis) was: sepsis, 5/9 and mof, 6/11. conclusions: 1. -we think that it is necessary to define the "critically ill" for some score, before designing a study to know the incidence of this syndrome. 2. -we think that the incidence of the cip is lower that the latest papers say. objectives:acute pancreatitis(ap)is becoming a more important problem among the elderly as the population ages. the increasing presence of gallstone disease,as well as the use of certain drugs,may also contribute to the occurrence of pancreatitis. methods:all patients(>60 years)admitted to our medical department over an eight year period were included.pancreatitis was confirmed by biochemical tests and imaging techniques.scores were developed using ranson's criteria and a multiple organ system failure(mosf)index . overall, 103 patients were evaluated; 21(23%)had pancreatitis of unknown etiology . results:(1)patients with pancreatitis of ~nlqnown etiology were sicker and had greater morbidity(48% vs 22%),mortality(24% vs 8%),and longer hospital stays than p~tierf~ with pancreatitis of known cause.(2)the best predicto~of severity and outcome was the mosf index and not ranson's criteria;the higher the score,the greater the associated disease,the worse the outcome.(3)curlously,no difference existed in associated medical conditions between patierts withknown and ur6~own causes of pancreatitis. conclusions:greater organ dysfunction exists in patients with pancreatitis of unknown etiology, even though age and associated medical conditions do not differ . the application of the total enteral nutrition in the burns disease has minimized the complication rate and consequently increased the survival rate of children and adults. time of initiation, composition, duration and way of administration are very important in obtaining the optimum beneficial effect from the treatment and diminishing the complication rate and side effects. the above features will be discussed in view of our experience in 240 cases. ta buckle?,, ra freebalm, c gomersall g joynt, r young. tg short. department of anaesthesia and intensive cm+e, prince of wales hospital. the chinese university of hong kong, shatin, hong kong introduction: gastric mucosal ph (phi) monitoring has been proposed as a relatively noninvasive index of the adequacy of aerobic metabolism in the gut. to examine the accuracy of gastric intramucosal pit measurements as a function of time and as a function of the catheter itself to determine whether the measurement error between catheters is clinically acceptable. patients with a gastric tonometer (trip tm, tonometrics, worcester. ma) insitu for >3 days were studied. following informed consent two new tonometers were inserted equidistantly & correct position was confirmed radiographically. measurements of intramucosal gastric ph were then performed over a 36 hr period. eight -ten measurements were made in each of ten critically ill patients.percent differences between the two new catheters were 1.6% ie at ph 7.3 _+0.12 (95% limits) and between old & new catheters were 2.2%, ie ph 7j3 _+0.16 (95% limits). conclusions: the results suggest that the function of the tonometer deteriorates over time and that the absolute values of phi m~ not ~ufficiently accurate. however as a trend monitor phi may be useful in the clinical setting. despite a continuous decline both in li'equency and severity of gastro-intestinal stress-lesion/-bleeding (gisb) due to both improvement in preclinical support and in intensive care medicine, patients with cerebral lesion are still considered at high risk for developing gis8. therefore the question arises, whether m> specific (}lsb-prophylaxis besides general and neurological intensive care, specific pharlnaeothcrapy or even the combination of two specific drugs reveals any protective efli~ct on frequency and severity of gisb.this pntspcclive randomized study has been perfornted in 173 patients snfrering t'rttna head-injury/cerebral lesion and with a glasgow-coma-scale on admission (gcs:,)of <9. according to randomization the patients have been grouped as tbllows: h analgesia/sedation (n=37); ih analgesiajsedation plus pirenzepine 60 mg/day (n=54); .[ih anatgcsia/sedalkm plus sncraltate 6 x [ g/day (n=47); iv: analgesidsedatkm plus pirenzcpine 60 mghlay plus sucralfate 6 x 1 e/day (n=35). slalislical analysis has been performed by chl:*tt~sl. rank correlatinn and unpaired t-test; statistical significance has been set with p <0.05. 28/173 patients (16.2 %) developed gisb. although the mean gcs~-value (x -+ sd) did not reach significance between patients with and without gisb (5.61 + 1.65 vs 6.12 -+ 1.65). a significant inverse correlation between gcs:, and the incidence of gtsb (rs~ = 0.89) has been shown. the frequency of gisb among the groups is as follows: h 18.9 %; lh 18.5 %; llh 17.0 %; iv: 8.6 % (ch1 -~ =1.94; not signilicant). no gisb-induced blood translusion or mortality, respectively, could be demonstrated. survival rate between the groups did not differ significantly (chi-" =5.86; p=0.1186) and reached an overall-value of 75.1%.drug-specific glsb-prophylaxis -administered either as monotherapy (pirenzepine, sueralfate) or in combination of these two specific-drugs -reveals no additional significant influence on the incidence of gisb in patients with cerebral lesion compared to no specific prophylaxis besides the general trauma-/disease-specific intensive care measures. critical care dpt, evangelismos hospital, athens university scho~" of medicine objectives: the correlation of longterm presence of nasogastric tube (ngt) to gastroesophageal reflux (ger) is still in question. in case of positive correlation, peg should represent an alternative to tube feeding in patients unable to be fed orally. therefore, we investigated: i) the correlation between ng and ger and ii) the effect of peg on ger. methods: a 24-h esophageal ph-metry was performed in 40 patients in recumbent position at 30 ~ who had a ngt for more than 10 days and were on sucralfate for gastric mucosal protection. the tip of the ph-probe was lied 5 cm over the esophagogasttie junction, confirmed by x-rays. patients who presented a percentage of ger-total (i.e. with a ph less or more than 4) (ger-t) more than 3%, underwent ~t peg. the presence of a creseent-notch on the esophagogastric junction persisting on inspiration and the grade os endoseopic and histologic esophagitis (scale=0-3) was noted. two ph-metrles repeated on 48 h and on 7 days post-peg were compared to the pre-peg one, with the followin~ parameters taken in consideration: i) % ger-t, ii) number of ger-total per hour (no/h ger-t) and iii) the duration that ph was less than 4 (tph<4). in case ot ger persistence at the ph-metry on ?th day post-peg (group ii) another endoscopy was performed, while patients with reduced ger (group i) were considered as esophagifis-free.results: 23 out of 40 patients presented a ger-t>3%. eleven out of 23 group i group (n=6) 1i ( objectives: the aim of the present study was to compare the performance of a specially modified version of a photo-and magnetoacoustic (pa/ma) gas analyzer (br~)el & kjaer, denmark) with a conventional quadrupole mass spectrometer (ms) (innovision, denmark) in inert gas rebreathing (rb) tests such as determination of functional residual capacity (frc), pulmonary capillary blood flow (pcbf) and lung tissue volume (vtc). methods : from simultaneous readings of inert gas concentrations with the ms and the pa/ma analyzer during rb experiments a comparison was made of the pcbf, vtc and frc values. the rb tests were performed during rest and exercise (0,50 and 100 w) in ten healthy subjects. results: the differences (mean +/-sd) between simultaneous estimates of rebreathing parameters were the following (pa/ma -ms) for pooled data, pcbf: 0.18 +/-0.38 i/min, vtc: -33 +/-108 ml and frc: 0.028 +/-0.048 liters. conclusions: smell but significant differences were found between the estimates of pcbf, vtc and frc using the ms and pa/ma, respectively. reference: p. clemensen, p. christensen, p. norsk, and j. gr~nlund. a modified photo-and magnetoacoustic multigas analyzer aplied in gas exchange measurements. j appl physiol 1994; 76: 2832-2839. objectives: because transcranial doppler (tcd) has been proposed to explore cerebral co 2 vasoreactivity in brain injury (stroke 1992;23:962-6), we compared this technique with the kety-schmidt reference method to assess cerebral vasoreactivity in comatose patients. methods: 17 mechanically ventilated patients (age 30-81 yrs, glasgow 3-10) in coma due to acute brain injury were investigated during stepwise changes in paco 2 (25, 30, 35, and 40 mmhg) by increasing inspired pco 2. middle cerebral artery velocity (vm) was measured by tcd. after insertion of a catheter in the ipsilateral jugular bulb, cerebral blood flow (cbf) was determined by the kety-schmidt method, using the inhalation of 10% n20 through the inspiratory line of the ventilator. for each patient a cerebral co~ vasoreactivity index was calculated as the slope of linear relationship between vm or cbf and paco2. objectives: after cardiac surgery the fluid shill, between interstitial and intravasal space may be marked. this is due either to the intraoperative volume loading by the extracorporeal circulation or the increased postoperative diuresis. therefore, infusion of a large amount &fluids is necessary during the first postoperative hours. it still remains unclear which of the substances at disposal is the best for this purpose. aim of the present study was to compare the different fluids with special regard to postoperative bleeding and rheological behaviour. methods: 93 patients undergoing cabg-surgery were investigated and randomizedly distributed to three different groups of postoperative volume replacement to stabilize the mean arterial pressure at 80 mm hg. 1. ringer's solution, 2. 3.5% gelatine solution, 3. 6% hydroxyaethylstarch (mean m.w. 70.000). we evaluated the following parameters within intervals of 30 min: arterial and central venous pressure, heart rate, postoperative bleeding, urinary output, volume replacement. results: there was no statistically significant difference between the groups with regard to urinary output and bleeding. in spite of larger amounts of fluids necessary in the ringer treated group patients of this group showed symptoms of hypovolemia. hematocrit was increased in the ringer patients. this was statistically significant. introduction: pulmonary wedge pressure (pcwp) and central venous pressure (cvp) are frequently used as parameters for cardiac preload, although it is known that both are poorly correlated to the cardiac index (ci). it has been claimed that intrathoracic blood volume (itbv) measured with the thermal dye dilution method reflects cardiac preload better than pcwp and cvp. we studied the correlation between itbv and ci in a mixed population of critically ill patients. methods: in 17 consecutive patients (6 sepsis/sirs, 2 acute heart failure, 3 ards, 6 transjugular intrahepatic portosystemic shunt) monitored with a pulmonary artery catheter, itbv was measured on regular intervals using the pulsion cold z-021 system (pulsion, munich, germany). ci, pcwp, and cvp were recorded simultaneously. results: a total of 1ol measurements was made. pcwp and cvp did not correlate to ci, nor did apcwp or acvp correlate to aci. itbv was correlated to ci in a non-linear fashion (f -139, df = 99, p < 0.001, (figure) ). aitbv was correlated to ac1 in a linear fashion (r = 0.76, f = 134, df = 99, p < 0.o01). a rapid and efficient circulatory support system may save a patient in cardiogenic shock. left heart bypass with percutaneous and transseptal placement of the aspiration canuia simplifies the circuit and avoids the need for an oxygenator. we assessed this preclinical set-up in 5 anaesthetized pigs using a centrifugal pump with a 14 f arterial catheter and a 16 f left atrial aspiration line. animals were supported for two hours at a mean flow of 3.1 liter (3'680 rpm), a mean hematocrit of 29% and low heparinisetion (act double baseline). hemodynamic and laboratory samples were taken at baseline (a), 10 minutes (b), one hour ( pulmonary hypertension (ph) usually involves obliteration and loss of functional pulmonary microvasculature. the microvaseular endothelium normally acts as a major metabolic organ, converting angiotensin i to angiotensin ii via the angiotensin-converting ectoenzyme (ace). it is unknown whether the loss of functional vasculature and altered pulmonary blood flow seen in ph will affect lung ace metabolic activity. we therefore estimated pulmonary vascular ace activity in 9 patients with ph of various causes: 2 primary; 1 post atrial septal defect closure (asd); 2 chronic thromboembolic (te); 1 anorexigen; 1 iv drugs; 2 collagen disease. single-pass transpulmonary hydrolysis of the specific ace substrate 3h-benzoyl-pbe-ala-pro (bpap) was measured and expressed as % metabolism (%me0. we also calculated an index of peffused functional capillary surface area (amax/km). all patients with ph had an abnormality of %met or amax/km, or both. as compared to 9 control humans (mean %met = 71.4% _+ 11.3% s.d.), the mean %met in ph patients was 54.3% _+ 14%. the %met in ph patients correlated inversely with cardiac output (r=0.74), possibly reflecting more complete bpap hydrolysis with longer pulmonary transit times. amax/km was markedly decreased in ph (1663 + 536 ml/min) as compared to controls (4225 _+ 1018 ml]min), consistent with a significant loss of functional capillary surface area. patients with collagen disease, asd and anorexigen-induced ph had the most marked abnormalities. in conclusion, patients with pulmonary hypertension have decreased pulmonary endothelial angiotensin converting enzyme activity, likely due to a loss of functional or perfused pulmonary microvaseulature. supported by the funds de la recherche en same du quebec and the national health system of greece. objective: to investigate adrenocortical function in patients with ruptured aneurysm of the abdominal aorta (raaa). studies investigating adrenocortical insufficiency in critically ill patients report an incidence ranging from 20% to less than 1%. this may in part be explained by difference in methods used (single cortisol measurement vs short acth stimulation test) and populations studied (heterogenous groups of patients with great individual variation in underlying disease as well as duration and severity of illness). methods: we investigated the adrenocortical function in 32 patients with (raaa).a short acth stimulation test (synacthen test; 250 ug 1-24 acth iv) was performed at 0800 hrs within 24 hrs of admission. plasma cortisol was measured before (cort basal) and after stimulation (cort stim). a plasma cortisol level > 0.55 umol\l before or after stimulation was considered normal, severity of illness was assessed using apache ii. results: of the 32 patients investigated 6 died and 26 survived. mean cort basal in nonsurvivors was significantly (p<0.o04) higher than in survivors; 1.03 (range 0.72-1.29) vs 0.69 (range 0.24-1,14). this difference between nonsurvivors and survivors was also present for cort stim but lacked significance; 1.30 (range 0.96-2.25) vs 1.00 (range 0.57-1.53). while 8 patients showed a cort basal < 0.55, no cort stim <0.55 was found. there was no significant difference in mean age or apache ii score between survivors and nonsurvivors; 70 vs75 and 19 vs 21. conclusions: single plasma cortisol levels were inadequate to assess the adrenocortical function in the patients studied, judged by a short acth stimulation test, our investigation in patients with raaa showed no adrenocortical insufficiency. mortality in raaa is associated with elevated plasma cortisol levels. obiectives: mortality in acute myocardial infarction (ami) prinicipally depends on hemedynamic impairment. thus, patients (pts) with elevated pulmonary wedge pressure (pwp) present high in-hospital mortality. however, the complete right heart catheterization is laborious, so the central venous pressure (cvp) alone is frequently used to assess the severity of ami. the accuracy of cvp in estimating pts with ami was tested in this retrospective study. methods: 131 pts. aged 68+14 years, admitted to our ccu from 1992 to 1994 with their first ami, were inctuded in this study. all had undergone right heart catheterization because of overt or suspected heart failure. swan-ganz catheters (7f, 85cm, abbott, il, usa) had been used, every treatment had been temporarily interrupted l h before the calheferization. based on ecg findings the pts were retrospectively divided into 3 groups. in group a we included 54 pts with anterior ami, in group b, 30 pts with inferior ami, and in group c, 47 pts with inferior and right ventricular ami. the initial values of cvp and pwp were considered for the linear regression of the pwp variable on cvp and p<0.05 was accepted as statistically significant.results: in g~oup a, the cvp and pwp vaiues were 8+3 mmhg and 14_+7 mmhg respectively. despite the signifanf correlation (p<0.01) between the two variables, it was not possible fo predict the exact value of pwp based on cvp value, 19 pts (35%) presented cvp>8 mrnhg and 16 of these (84%) had pwp_>15mmhg. in group 3, the cvp was 11_+8 mmhg and the pwp, 13_+6 mmhg. significant correlation (p<0.05) between the two variables also existed, however it was impossible to predict the pwp value. 6 pts (20%) had cvp>8 mmhg but only 2 of these (33%) had pwp>15 mmhg, similar was the relation between cvp and pwp in group c (p<0.01). cvp averaged 12+6 mmhg, and pwp, 17_+8 mmhg. 33 pts (70%) had cvp>8 mmhg and 25 from these (76%) presented pwp>15 mmhg,conclusions: a single measurement of cvp in ami does not ensure an accurate assessment of pwp. because every pt with ami needs optimal values of pwp in order to prevent pulmonary congestion or manifestations of low preload, the significance of complete right heart catheterization becomes apparent. in patients (pts) with advanced hf the need and the prognosis for heart transplantation (ht) can be predicted from vo= max. indirect measure of functional capacity with the six-minute walk test can also predict smvival in moderate hf. to predict vos max from indirect astinmtions of functional capadty such as 6-1~q~/, pulmonary and heart function tests, and to assess the prediddve value of the above parameters in hf pts survival. we evaluated 35 pts (age 48+12 yeats nyha class: 12 ii, 15 hi, 8 iv) with hf for pit. they underwent a pmgmmive exercise test on cycle ergometer for vo2 max determination, a 6-mw, a right heart catheterization and a spirometry and dlco estimation. introduction: brain death causes myocardial impairment by mechanisms that are not well understood yet. the aim of this work was to assess the echocardiographic features found in these patients from the clinical onset of brain death to somatic death, methods: seven brain dead patients were studied (patients" relatives refused to allow them to be used as donors). mean age was 23.5 (18-32) years old. four of the patients were female, none of the patients had any history of cardiac disease. transthoracic echocardiogram (echo) and electrocardiogram (ecg) were obtained at the onset of clinical brain death and were repeated every 24 hours until somatic death. we we detected severe diffuse hypokinesia (ef<50%) in 2 patients and mild hypokinesia in 3 others (ef 50-60%). systolic function was strictly normal in only 2 patients. corrected qt interval (qtc) in ecg was 54.6_+5.5 msec (normal range 38-43 msec) just before somatic death (b). conclusion: in patients with brain death we observed a significant increase of left ventricular mass due mainly to ivs "hypertrophy" without any important change in the dimensions of the left ventricle. to our knowledge, this finding has never been reported before and its importantance in heart transplantations may be of particular interest. predict right ventricular outcome. l. jacquet, r. dion, p. noirhomme. m. van dijck. m. goenen cardiothoracic intensive care unit, st-luc univ. hospital(ucl) we have registred: heart rate (hr), blood pressure (bp), pulmonary artery pressures (pap), central venous pressure (cvp), pulmonary capillary wedge pressure (pcwp), pulmonary and systemic vascular resistances (pvr, svr), right ventricle end-diastolic end end-systolic volume (redv, resv), right ejection fraction (ref), right sistolyc ventricular work (rsvw) and cardiac output (co) using a thermodilution thechnique and a microprocessor (model ref-1; baxter-edwards laboratory); duration of cpb and aortic clamping, and the requirements of haemodynamic support after cpb.results: in the c group an increase post-cpb of the fc (62 + 13 95.8 + 18.4, p < 0.01) was produced without significantly changes in the redv, resv, ref, rsvw neither co. in the w group, hr increased from 58.5 + 8.8 to 79.9 + 8.6 (p < 0.01); redv was reduced from 227.7 -+ 76 to 139.14 _+ 36.4 (p < 0.05); resv was reduced from 142 • 68.7 to 82 + 35.3 (p < 0.05). there were not changes in the other haemodynamyc parameters. there was a trend (no significantly) to an increase of ref in the w group (40.07 + 9.7|• 4.7) compared with the c"group (39 • 10.9($ 38.3 • 8.5) post-cpb. the need for haemodynamic support was similar in both groups.conclusions: the warm, continuous, anterograde-retrogade myocardial protection has obtained a decrease of preload, hr, and a trend to an increase in the ref, making an improvement in the right ventricular global performance when is compared with the classic form of cold myocardial protection. objective: to evaluate the effect of dobutamine on gastric mucosal ph (phi) after coronaly artery bypass surgery. design: prospective study in a university hospital intensive care unit (icu). subjects: 20 elective cardiac surgery patients. interventions: dobutamine was infused at 4 ug/kg/min for 3 hours immediately after admission to the icu. hemodynamics were measured every 30 minute periods until 3 hours and again 2 hours after stopping dobutamine. results: there were no significant differences in mean gastric phi between the groups but mean phi decreased in both groups during the study period. oxygen delivery and consumption both increased during dobutamine infusion but decreased to the control group level after stopping the dobutamine infusion. lactate levels did not change. baseline 90 objectives: the aim of the study was to evaluate the usefulness of a low dobutamine dose in conjunction with intraaortic balloon pumping and mechanical ventilation in cardiogenic shock. we studied 21 patients 58.9-+ t4.4 years of age suffered of post infarction cardiogenic shock characterized by a systolic arterial pressure< 80mmhg, urine output< 20 ml/h and mental confusion or purpueral signs of low output, non responded to dobutamine infusion up to 9 pg/kg/min. all patients underwent mechanical assistance by the intra-aortic balloon pump (iabp). five patients were additionally placed on mechanical ventilation due to blood gases disturbances. the end points in our study were: reversion of cardiogenic shock, improvement of patients survival or both on the 15th post infarction day and 6 months later. results: three patients refused iabp treatment and 0/3 survived on the 15th day. on the 15th day 3/13 supported by the iabp and 5/5 that underwent mechanical ventilation plus iabp were alive (p <0.01). on the 6th month 2/13 supported by the iabp and 5/5 that underwent mechanical ventilation plus iabp were alive (p<0.01). conclusions: in conclusion, the combined use of mechanical ventilation and iabp assistance in severe cardiogenic shock might improve survival. obiectives: the study was aimed at analysing predictive factors of swan ganz pulmonary catheter (pc) requiremen t during elective cardiac surgery according to the need of sustained inotropic support after surgery. methods: three hundred patients (aged from 27 to 85; 89 females and 211 males)were consecutively operated on for elective coronary artery bypass surgery (cabg, n=179), valvular replacement (vr, n=98), combination of both (vr-cabg, n=15), or others (n=6) and retrospectively included in the study. each patient had preoperative invasive cardiac investigation with calculated ejection fraction (ee). anaesthesia, cardiopulmonary bypass (cpb) and cardiac arrest managements were similar in all patients. pc requirement was estimated from the need of either dobutamine, adrenaline, dopamine or enoximone use during the first 48 hours after cardiac surgery. demographic data, asa and nyha classifications, preoperative ef and treatments, type of surgery, cpb and aortic cross clamping (axc) times, and postoperative incidence of complications were compared in patients with or without inotropic support using either student's t test or x2 with continuity correction when appropriate. results: seventy4hree patients (24.5%) required inotropic support after surgery. axc .and cpb times, mean stay in icu were significantly longer in patients with inotropie support (p<0.001). type of surgery, preoperative ef, and nyha classification are the first 3 significant factors related to inotropic support (p<0.005). most patients operated on for double-vr or vr=cabg required inotropic support (57 and 60%, respectively). postoperative mortality was higher in patients receiving inotropic support (8,2% vs 0,9% 'overall mortality, p=0.003). conclusions: since pc insertion is most.often justified because inotropes are required, these results suggest that elective rather than routine systemic pc insertion could be helped by considering several but selected preoperative factors. background: cardiovascular depression due to anaesthesia, old age and major gastrointestinal surgery is becoming an increasingly frequent challenge .to the anaesthesia-surgory team. deliberate preoperative manipulation of haemodynamics and oxygen transport parametres towards prede~t~mined optimal values may prove to be effective "in reducing 12 morbidity ~nd mortality in high risk surgical patients,. a new concept of using conlimaous perioperative measurement of cardiac'output to obtain and maintain supranormal oxygen delivery (do2i) is presented. methods: continuous measurement of cardiac output is a relatively new form of on-line monitoring, in which trains of impulses are emitted from a thermal filament mounted on a pulmonary artery catheter. computer software recognizes patterns generated by minute changes in blood temperature and ealoalates cardiac output every 30-60 seconds. cardiac 3 output and mixed venous blood oxygen saturation are displayed graphically on line. in tins tm study cardiac output was measured continuously by vigilance cardiac outpu t compl/ter (baxter). preoperative haemodynamic optimization was performed with the goal of increa-2 1 sing do2i to at least 600 ml/min/m accordfing to shoemaker's algorithm . this was.done by infusing colloids (albumin or hydroxy ethyl starch (haes-steril| until the desired do21 was reached. infusion was stopped if cardiac output ceased to increase with infusion, if there were signs of pulmonary oedema or if wedge pressure reached 18 mmhg. vasoactive or inotropic drugs were infused if the desired do21 was not reached by infusion alone. anaesthetic technique included continuous thoracic epidural and isoflourane anaesthesia. expected mol:bidity and mortality rates were calculated by the "possum" score aasing preoperative clinical and paradinical estimates of organ function as well as surgery characteristics 4. materials: 15 asa group ill-iv patients with a mean age of 75 years (range 60-92) and a mean weight of 67 kg (range 36-93)) scheduled for major abdominal surgery were included. results: 2 patients were excluded because do2i could not be raised at all. mean do2i was increased from 488 ml/min/m 2 (range 384-610) to 688 ml/min/m 2 (range 490-967). mean volume of preoperativdy infused colloid was 954 ml (range 0-2000). during surgery 1230 ml (range 5002300) of colloid was infused. mean length of surgery was 150 minutes (range 60-300). mean blood loss was 920 ml (range 04800). expected mortality and morbidity rates ("possum") were 56% and 92%, respectively, whereas patient follow up upon discharge or at death revealed mortality and morbidity rates of 8 % and 54%, respectively. conclusion: based on experience from the present study, continuous measurement of cardiac output has proved to be a valuable tool for perioperative optimization of do21 in asa group ili and iv patients during major surgery. however further studies including a greater number of patients are necessary to confirm the promising preliminary findings. we studied the hemodyn~c effects of three different combinations of positiv inotropic .agents, vasodilators, diuretics and av-filtration (av) in 16 patients (pts) with severe left heart faille (left veutrieul0x filling pressure (lvfp) >25 mmhg) due to acute myocardial infarction. hemodynamic measurements (intravascular pressures (lvfp), thermodilution (cardiac index (ci)) were made before (control) and after each therapy. in 11 furosemide (f) + d0butamin (d) + nitroglycerin (ni) reduced lvfp and a small increase of ci occurred. in 6 of these pts :(group a) nitroprusside (hip) instead of ni increased ci significantly, in the other 5 pts adding of amrinone (a) resulted in a pronounced increase of ci. group c (n=5): the combination of ni and av reduced lvfp but did not increase ci which was achieved by av+d+ni. in order to optimize the treatment of acute heart failure a combination of inotropic agents, vasodilators, diuretics and av-filtration should he used guided by hemodynamic monitoring. arias jr, miragaya d, sandard, san pedro dm ~, herndndez d, valenzuela 12 . objectives: to evaluate the variation in nomdrenaline (na) plasma concentrations in patients with acute myocardial infarction (am1) after thrombolytic therapy with noniltvasive reperfusion criteria (clinical, electrocardiographic and enzymatic), in relation to infarct size and location.methods: 42 consecutive patiens with ami, from october 1, 1994 to february 28, 1995, admitted within 6 hours alter onset of symptoms, undergone successfull systemic thrombolysis. 21 of them were anterior (group a) and 21 inferior (group b) . noradrenaline plasma levels at 0 (na1), 60 (na2) and 240 (na3) minutes after admission were compared with ck-peak plasma levels by linear regression. differences were tested for significance by student-t-test for paired and unpaired values. na plasma concentration was measured by high-presssure liquid chromatography. p< ns 0.01 ns means 4-sem (normal limit for our laboratory: na < 37/0 pg/ml; ck < 170 u/i ) conclusions: 1. the na plasma levels at admission (nai) are more increased in anterior than inferior amis, probably in relation to infarct size. 2. the decrease in na is more evidence in amis with anterior location. 3. this decrease is probably due to the major efficacy of thrombolytic therapy in amis with anterior location. arias jd, miragaya (group b) , probably due to certain degree of t~cg'rfueion. 3. there is not significant variation in na in conventional treated ami (group c). v.suchanov, a.levit, p.trofimov, icu, regional hospital, ekaterinburg, russiaobjectives: our task was to improve the technique of preservation of platelet rich plasma. methods: 38 patients scheduled for multiple cardiac valve replacement in 1994 were divided into two groups: group i (10 patients) -without pp; group ii (28 patients) -pp was performed preoperatively. the first pp was made ten days and the second -3 days before the operation. prp was preserved by cryoconservation. our technique of cryoconservation is distinguished by the speed of freezing (17-18~ and absence of dmso. this made it possible to preserve 90 % functionally active platelets during 20 days. the prp was transfused back after heparin neutralization. the hospital ethics committee approved the investigation.results: the blood loss through the 1st p. o. d. was significantly greatest in the group i (725 _+ 97 ml) and all the patients required transfusion of the donor blood (480 + 112 ml) whereas the blood loss in group ii was 489 +_ 75 ml and olny 12 patients required the donor blood. the number of platelets on the 1st p.o.d, was 107 _+ 12. 109/l (group i) and 153 + 19. 109/l (group ii), p < 0.05.conclusions: our technique of prp cryoconservation makes it possible to avoid the crystallization phase during freezing of prr thus the infusion of prp may improve hemostasis after open heart surgery and limit the use of the donor blood. in-hospital outcome of women suffering an ami is generally considered worse than that of men, but it is still debated whether female sex is per sea negative prognostic factor or is merely associated with other negative determinants of prognosis. the purpose of the present study is to evaluate the independence of the association between female sex and mortality (in the 567 patients of the swiss centers) and in the 36381 patients randomized in the isis-3 trail mortality rate in women was 14.8% (1421/9600) compared to 9.1% (2417/26480) in men; in switzerland: in-hospital mortality for women was 15.5% (20/129), for men 7.1% (31/438).the table shows the results of isis-3 in terms of odds ratios and their 95% confidence intervals either after unadjusted analysis or after adjustment for age, known to be the major confounding variable when prognosis of women after myocardial infarction is considered, and for all the available clinical and epidemiological characteristics collected at trial entry: these observations suggest that there is a small but independent effect of female sex on short-term mortality after acute myocardial infarction. (27) and bubble (13) oxygenators a, ere used. anaesthesia was balanced and pts were extubated 12 to 18 hrs after cpb. pts were monitored with swan-ganz catheters (sgc) for 24 hrs after cpb. at that time qs/qt was calculate( according to )be standard shunt equation. after the sgc had been removed, an estimated shunt was calculated. measurements of qs/qt were performed: before induction of anaesthesia (1), after induction of anaesthesia (i[), 5 mins after cpb (iii) 2 (iv) and 6 (v) hrs afiter cpb, 30 rains after extubation (vi), 24 hrs after cpb (v[1) and on the 2nd, 3rd, 5th, 8th and 13tb postoperative day (pd) (viii, 1 x, x, xi, xi1, respectively). analysis of data was performed by two-way analysis of variance, p < 0.05 being regard as significant.results: the figure shows the values for qs/qt expressed as means + sd. there was a significant increase in qs/qt above b~setine throughoul the whole investigated period except on the 13th pd. qs/qt reached maximum at 30 rains after extubation (vi). objectives: many stndies have shown advantages of membrane oxygenalors over 9ubbie type oxygenators. the aim of this study was to evaluate the influence of 3x3'genator type on pulmonary shunt (as/at) after coronary surgery. methods: 40 patients (pts) gave their informed consent to the study which was approved by the university ttuman research committee. pts were divided into two groups: a1 (n = 27) with a membrane o~genator and a2 (n = 13) with a bubble oxygenalor used during cardiopulmonary bypass (cpb). ths were monitored with swan-ganz catheters (sgc) for 24 hrs after cpb. at that tfme os/ot was calculated according to the standard shunt equation. alter the sgc had been removed, an estimated shunt was calculated..measurements of os/qt were performed: betore induction of anaesthesia (i), 30 mins after extubation (11), 24 hrs alter cpb (111) and on the 2nd, 3rd, 5th, 8th and 13th postoperative day (iv, v, vi, vii> viii, respectively). analysis of data was performed by one-way analysis of variance, p < 0.05 being regarded as significant.results: the figure shows the values for qs/qt expressed as means _+ sd. os/qt was significantly greater at 30 rains after extubation (ii) in a2 group. the difl'ereuce between the two groups was no more significant from 24 hrs after cpb (iii) to the end of the investigated period. ! i * p < a.0s betw~n ~o~ conclusions: membrane ox3'genation during cpb is accomplished by reduction in blood cellular destruction and less alteration in blood. the results of our study show the influence of oxygenator type on value of qs/ot only after extubation (12 to 18 hrs after cpb). the difference in qs/qt disappeared 24 his after cpb and since that time the oxygenator type had no influence on qs/qt. it may be of particular importance in patients with severe forms of cardiopulmonary disease who are at risk of higher postoperative morbidity and mortality. objectives: hypomagnesemia has been reported with a variable prevalence (20 to 61% ) in icu patients. magnesium deficiency can induce a number of climcal symptoms (primarily cardiovascular and neuropsychiatric) but can also be clinically silent (10-65% are asymptomadc), methods: we measured whole blood ionized magnesium (lmg++) in 74 patients on admission to the icu, using a nova 8 electrolyte analyzer (nova biomedical), containing an img++ electrode. blood was collected in syringes with dry heparin (radiometer qs 50 ). normal range of img++ was found between 0.45-0.55 mmot/l (healthy volunteers). results: for the entire population, we found a 61% prevalence (45/74) of hypomagnesemia (figure 1) . among the surgical patients, the prevalence was highest after cardiac surgery (85%) and after thoracic surgery (80%) and was lowest after neurosurgery (8%). hypomagnesemia was also common in patients after liver transplantation (lvtx) or with hepatic failure (100% for both groups). conclusion: our findings confirm that hypomagnesemia is common in acutely ill patients, especially in those after cardiothoracic surgery or those with liver disease. nevertheless. it is difficult to define the associated factors with sufficient specificity, so that measurements of img++ are warranted to diagnose hypomagnesemia. hepariu influences platelet function and may lead to thrombocytopenia called heparin-associated thrombocytopenia (hat) regardless of the dose and route of administration. additinnal venous and/or arterial thrombosis may lead to life-threatening complications. the incidence of so-calied heparin-associated thrombocytopenia and thrombosis (hatt) ranges between i-5%. hatt is confirmed by a heparin induced platelet activation assay (hipa). results: from 11/93 to 11/94 1146 consecutive patients of our icu were reviewed retrospectively. all patients were treated with heparim the incidence of hatt was 1% (12). in all cases diagnosis was proven by a positive hipa. 2/12 patients died. in 3/12 hatt could be confirmed before severe thromboembolic complications occured. 4/12 patients developed a deep vein thrombosis (dvt), 2/12 dvt and pulmonary embolism (pe), 2/12 dvt, pe and arterial thrombosis (at) and 1/12 a dvt, pe~ at and a sinus thrombosis. conclusion: the incidence of hatt in a r series of 1146 pts. is 1%. presence of thrombocytopenia and thrombosis of the great 'vessels is associated with a significant mortality (2/12). computed tom0graphy (ct) and transthoracic/transesophageal echocardiography (tte/tee) are important tools in diagnosing and monitoring the extent of cenlrai venous and arterial thrombosis. a. cabral md, m. shahla md c. meneses-oliveira md and jl vincenl md.phd. department of intensive care. erasme university hospital, brussels, belgium objective: to determine extreme hemodynanuc patterns in cardiogenic shock. although ~.~xdiogenic shock is characterized by a low cardiac index (ci), high systemic w~,scular resistance index (svri), and high cardiac filling pressures, some patients may develop art atypical pattern. we reviewed the hemodyuamic pattern of 73 patients with cardiogenic shock, as defined by an initial ct below 2.5 l/rain/m: in the presence of myocardial dysfimction attributed to ischemic heart disease (n=26), heart failure (n=7), valvulopathy (n=2) or recent cardiac surgery (n=38). after exclusion of 10 patients with concurrently suspected/documented infection, this study included 63 patients, of whom 23 (36.5%) survived. treatment of shock included dopamine (n=43), dobutamine (n=56), norepinephrine (n=18) and epinephrine (n=23). 39 patients with arterial hypertension (ah) and initially law plasnla renin activity (pra) had been studied. in all patient changes of arterial pressure (ap) after single administration of enap was studied. nypotensive reaction wiht deereasin e of average ap about 20-25 mm hg ayter single drug administration observed only in 4 patients. ezap monotherapy accomplished during one week with 20 mg daily dose. hypotensive effect observed in 5 patients including ones which were susceptible to single enap administration. after that first stage of therapy all patints began to combinate enap with hypothyazid in dose of 25 mg per day~ after week of treatment such drugs combination lead to veritable ap lowering in 3 addition patients. in the remaining resistant to such drug combination patients was add corinfar in daily dose of 40 mg. this new drug combination permits to lower ap in 23 patients. subsequent discontinuation of enap administration to such patients aid not connected with increasing of again.therefore the most of the patients with ah and law pra(78,7%)did not susceptible to enap therapy and enap and hypothyazid combination. on the contrary-combination of corinfar with hipothyazid was effective in 59% patients with ah and low pra. methods: in 35 patients with cardiogenic shock due to ischemic heart disease (n=26), heart failure (n=7) and valvulopathy (n=2), hemod31aamic data including measures of intravascular pressures, cardiac output and mixed venous gases were collected at regular times intervals, at least 3 times a da?. all measurements were obtamed in a relative steady state and in the absence of severe anemia or hypoxemia. treatment of shock included dobutamine (n=30), dopamine (n=24), norepinephrine (n=i2) and epinephrine (n=7 objective: based on our previous studies of the function of isolated liver grafts, this experimental protocol aims at developing a novel extracorporeal liver support circuit, with an incorporated pig liver. methods:the graft liver was obtained from pigs weighing 15-20 kg. under general anesthesia the aqimals underwent total hepatectomy,following cannulation of the portal vein, the infrarenal aorta and the infrahapatic vena cava and peffusion wit h 4 it of heparinised r/l solution at 4~ the circuit consisted of the graft liver connected to a fluid reservoir and a centrifuge pump. ten healthy pigs weighing 30-35 kgr were connected to the circuit as follows: the rt carotid artery was connected to the portal vein of the graft and the rt jugular vein was connected to the fluid reservoir, through the centrifuge pump. the fluid reservoir collected the outflow from the graft's suprahepatic inferior vena cava. the cystic duct of the graft was ligated and the bile.duct cannulated for bile collection and measurement. bridges were adapted to the circuit to bypass the graft liver when necessary, in cases of by pass blood perfusing the graft was oxygenated through a bubble oxygenator. mean total priming volume of the circuit was 600 ml. temperature was maintained at 38~ and portal vein pressure at 16 (12-20) mmhg. the flow was 0.5-0.7 ml/gr of graft liver mass per minute. observation period was 8 hours (t8). results: results of the hemadynamic and metabolic monitoring of the recipients [map (t0=124mmhg , t8=118mmhg), hr (t0=177, t8=201), rap (t0=11mmhg , t8=19mmhg), pap (t0=26mmhg, t8=31mmhg), pcwp (t0=14mmhg, t8=15~mhg), svr (t0=1940dyn'sec/cm '5, t8=2190dyn'seclcm~ pvr (t0=206dyn.sec/cm o, t8= 354 dyn.sec/cm ,'~), co (t0=4.631t/min, t8=3.61t/min), do 2 (t0=662ml/min, t8=261.6 ml/min), vo 2 (t0=118ml/min, t8=111ml/min), o2er (t0=17.8%, t8=42.5% ), ph (to= 7.48, t8=7.39 ), po 2 (t0=292mmhg, t8=371mmhg), pco 2 (t0=28mmhg, t8=30 mmhg), pvo 2 (t0=47mmhg, t8=36mmhg), svo 2 (t0=84%, t8=66%), be, na, k, ca ++, lactate, osmolality, ast, alt, pt, aptt, revealed hemodynamic and metabolic stability of the animal. 02 consumption, co 2 production and tissue oxygenation of the graft were also studied. conclusion; the described circuit proved to be safe and well tolerated by healthy animals but its value for temporary liver support is currently being estimated, in a surgically induced experimental fulminant hepatic failure modal. introduction: prosthetic materials like silikone, dacron, teflon e.tc. produce auto immune responses and may even trigger clinical syndromes like scleroderma, sjogren, sle el.c. in our study we followed the evolution of humorial immunity parametrs for up to five years in a cohort of paced pts with implanted metallic and silicone materials. method: 24 paced pts (mean age 55+-13 yrs) without clinical or laboratory findings of malignancy or immune disorders were included. we measured the immunoglobulins, the complement, the auto antibodies and the proteins involved in inflammatory reactions every 6 months. the initial and final mean values are shown in the obiectives: hsp, a systemic leucocytoclastic vasculitis and anaphylactoid purpura can be accompanied by abdominal pain and life-threatening intestinal bleeding. recently we could disclose, that these patients develop severe fxiii-deficiency and immense haemorrhagic oedema of the intestinal wall. by the following case report we will demonstrate and discuss the importance of fxiiideficiency for pathogenesis, therapy and outcome in hsp. case report: a 41 year old man developed typical skin manifestations of hsp following an episode of severe (biliary ?) pancreatitis and percutaneous draining of a pancreatic pseudocyst. two days later he had a paralytic "ileus with immense hemorrhagic wall-oedema and massive dilatation of the small bowel. he got fever up to 39.5 ~ and developed severe gastrointestinal haemorrhage (blood transfusions necessary). the coagulation data disclosed a severe fxhi-deficiency (activity 34%), whereas quickvalues, platelet count and atiii-level were found to be within the normal range. elastase was markedly elevated. substitution of fxiii to normal levels leeds to the cessation of bleeding symptoms and abdominal pain, later resulting in a restitutio ad integrum. conclusions: hsp with intestinal involvement is a life-threatening vasculitis, in which careful and frequent examinations of the coagulation system, especially of fxiii are necessary. detailed analysis of the coagulation data suggest, that the severe fxiiideficiency is due to a specific degradation by proteolytic enzymes (like elastase) as well as consumption within the immense haemorrhagic oedema of the intestinal wall. knowing these facts, even most severe cases of hsp with intestinal involvement can be successfully treated by substitution of fxih. a 49-year-old woman presented a 3 year history of occasional self-limited episodes of weakness, generalized edema and o!!~aria. the immunologic testing showed no~nnai levels of complements, clq inhibitor, and serum chemistry values, between or during a attack, she was not treated. she was a~mitted to the hospital with symptoms including nausea, vomiting, weakness and ol!guria. on examination, the patient presented facial and g~neralized edema. the systolic blood pressure was 60 mm hg, pulse 140 beats/mir~ute, hematocrit 0.59, seln~n protein 46 9/i, and se~um albumin 23 q/l. an leg-kappa pa[apfotein was demostrated (7.82 g/l) and urine was neaative for puotein. c~'stalloid and colloid don't increased the blaod pressure but resulted in anasarca, with a total of ii lit[as of in~ravenous fluids. therapy wink flozen plasma, 1.000 units of clq inhibitor, cortlcosteroids, annihistwnines and antifibrinolytic agents was uns~iccessfull. the a~minist~ation of dopamine, norepineph~ne and epinephrine was inefective. the patient died at the 48 bores, only a few cases have been reported, all had igg paraprotein, the pathophysio!o~] is urd~no~n% but is possible that the paraprotein may be zesponsib!e for the increased capillary pe~leabilityo despite efforts to res~scinate the patients during an acute attack, the syndrome is often fatal. the variable course of systemic uapiliary leak syndrome and the unpredictability and self-limited nature of attacks cloud assessment of therapeutic inte~-vention. the purpose of the present work is to provide some information about the nursing care and results from our experience in continous arteriovenus hemofiltration (cavh).cavh is an extracorporeal technique, especially applicable in the critically ill patients, for disturbances, and for the control of azotemia.we used this method in 30 critically ill patients 16 men and 14 women ages from 32-74 who had sepsis -arf 10 congestive heart failure 8 postoperative multiple organ failure 8 and polytrauma 4.this method was applied to these patients from 24 to 168 hours. 20 % of the patients recovered completely their kidney function, 50 % improved their kidney function and 30 % died.we concluded therefore that this method was very effective for the critically ill patients to whom it was applied, but it requires excellent and continuous nursing care; under the above mentioned circumstances the method works effectivelly. an animal model with rats undergoing a dialysis procedure was designed to test the hypothesis that recovery from ischemic acute renal failure (airf) may be affected by the type of membrane used in hemodialysis. male sprague dawley rats were allocated to 2 groups: in group i, (n=48) airf was inducted by bilateral renal artery clamping for 60 rain. group h (n=48) rats underwent a sham procedure. in each group, rats were dialyzed twice (4th and 8th day) with either a cuprophan (cupro), a hemophan (hemo) or a pan (an69) minidialyscr or stayed nondialyzed (no hi)). renal function was monitored daily by measuring urea and creatinine values and by two single shot inulin clearances on the days following dialysis. additionally hemolytical activity of complement was determined. inulin clearance on day 5 was reduced significantly but there was no difference in the degree of decrement in glomular filtration rate (gfr) between dialyzed and undialyzed rats, nor between the dialyzed animals with different membranes (gfr: no hi): 0.78_+0.54; cupro: 0.84_+0.9; hemo: 0.82_+0.28; an69: 0.77_+0.31). the evaluation of renal function by day nine revealed significant recovery for all airf-groups compared to day 5 (p<0.001), irrespective of wether they underwent dialysis or not, or the type of dialysis membrane. complement activation could be detected in all dialyzed groups but no statistical differences between the animal groups dialyzed with different membranes were noticed. our findings refute the hypothesis that in airf exposure to complement-activating cellulosic membranes impairs the recovery of renal function in rats. changes patients: 150 patients who underwent first cadaver kidney transplantation in our unit between january and december in 1994 were involved. the recipients were divided into 3 groups: group i." non functioning graft (n=27); group ii: delayed graft function (n=59), group ili: good graft function (n=64). the grouping criteria were: a/haemodialysis in the fii~t 5 postoperative days, b/diuresis in the i st postoperative day, c,' scram crcatininc difference between the 1st postoperative day and the preoperative level. all of the parameters were involved into the exarainatio, which we measllre in our every, day practice. results: the preoperative haematocrit level differed significantly between group i. (0.36) and croup ii. and iii. (0.31 and 0.30, p< 0.05). intmo!0emtive significant differences were found between the different groups in systolic blood pressure (group i. 110 hgrmn, group ii. 140 hgnnn, group iii. 165 hgmm, p<0.05), mean arterial pressure (group i. 66 hgmm, vs. group ii. 83 hgnun p<0.05, vs. group iii. 116 hgmm p<0.001), and pulse-amplitude and rate-pressure product too. the second warm ishaemic time in group iii. was significantly shorter than in the other two groups (group iii. 39 inin. vs. group ii. 43 rain. p< 0.05, vs. group i. 49 rain. p< 0.00!). the rejection rate was higher in the first 5 days in the patients with non-functioning grafts (group i. 53% and group ii. 24% vs. group iii. 12 %) . the other examined parameters have not differed significantly. conclusion: according to our results the success of the kidney transplantation is mnitifactorial. the most important factors of this relationship are: the perioperative fluid-balance, the maintenance of adequate perfusion blood pressure during the operation, good surgical technique and immunological problems. key: cord-299379-ch7a39d6 authors: de conto, flora; conversano, francesca; medici, maria cristina; ferraglia, francesca; pinardi, federica; arcangeletti, maria cristina; chezzi, carlo; calderaro, adriana title: epidemiology of human respiratory viruses in children with acute respiratory tract infection in a 3-year hospital-based survey in northern italy() date: 2019-01-17 journal: diagn microbiol infect dis doi: 10.1016/j.diagmicrobio.2019.01.008 sha: doc_id: 299379 cord_uid: ch7a39d6 acute respiratory tract infections (artis) are among the leading causes of morbidity and mortality in children. the viral etiology of artis was investigated over 3 years (october 2012–september 2015) in 2575 children in parma, italy, using indirect immunofluorescent staining of respiratory samples for viral antigens, cell culture, and molecular assays. respiratory viruses were detected in 1299 cases (50.44%); 1037 (79.83%) were single infections and 262 (20.17%) mixed infections. the highest infection incidence was in children aged >6 months to ≤3 years (57.36%). human respiratory syncytial virus (27.12%) and human adenovirus (23.58%) were the most common viruses identified. the virus detection rate decreased significantly between the first and third epidemic season (53.9% vs. 43.05%, p < 0.0001). the simultaneous use of different diagnostic tools allowed us to identify a putative viral etiology in half the children examined and to provide an estimate of the epidemiology and seasonality of respiratory viruses associated with artis. acute respiratory tract infections (artis) are a persistent public health problem (lu et al., 2013) . artis affect the upper respiratory tract, leading to colds, rhinosinusitis, pharyngitis, laryngitis, tracheitis, and otitis media, as well as the lower respiratory tract, causing tracheitis, bronchitis, bronchiolitis, and pneumonia (bicer et al., 2013) . although the majority of artis remain confined to the upper tract (tregoning and schwarze, 2010) , they can cause severe manifestations when affecting the lower tract (zappa et al., 2008) . viruses are frequently the cause of artis characterized by high risks of morbidity and mortality (taylor et al., 2017) . the viruses most frequently detected are: influenza viruses a and b (iav, ibv), human parainfluenza virus (hpiv), human adenovirus (hadv), human metapneumovirus (hmpv), and human respiratory syncytial virus (hrsv). this list is constantly evolving along with improvement in the performance of diagnostic tests (ljubin-sternak et al., 2016; tregoning and schwarze, 2010) and the discovery of more recent viruses, for example, human bocavirus (hbov) (allander et al., 2005) , human coronaviruses (hcovs) nl63 and hku1, and new human enterovirus (hev), parechovirus, and rhinovirus strains (berry et al., 2015) . the main diagnostic methods for respiratory virus infections are: virus isolation in cell culture, viral antigen/nucleic acid detection, and virus-specific serology. virus isolation is labor-intensive (landry, 1997) , making it impractical to determine a diagnosis during the acute infection phase. shell vial cultures and viral antigen detection reduce the time for diagnosis (engler and preuss, 1997; gardner and mcquillin, 1974) . nearly all these methods are being replaced by highly sensitive multiplex pcr (kim et al., 2009; tregoning and schwarze, 2010) . arti prevalence in children varies by geographic area, season, and year (goktas and sirin, 2016; moura et al., 2009; panda et al., 2017) . recent italian data are limited, apart from those relating to influenza (pariani et al., 2015; trucchi et al., 2017) . this three-year (october 2012-september 2015) hospital-based survey in parma (northern italy) aimed to determine the prevalence of respiratory virus infections, their seasonality, and any patterns of mixed infections in children with artis by using indirect immunofluorescent staining of respiratory samples for viral antigens, cell culture, and molecular assays. the study was conducted at the virology unit of the university hospital of parma (northern italy), a 1300-bed tertiary care center with more than 50,000 admissions per year from the city and surroundings with approximately 450,000 inhabitants. laboratory diagnosis was performed upon medical request. patients' identities and medical information were protected. the inclusion criteria were: patients ≤14 years old, acute fever, respiratory symptoms, and illness onset within 2 days. from october 1st, 2012 to september 30th, 2015, 2892 samples from 2575 children (1148 females, 44.58%, and 1427 males, 55.42%; 2311 inpatients, 89.75%, and 264 outpatients, 10.25%) were collected by bronchoalveolar lavages (bals), bronchial aspirates (bass), nasopharyngeal aspirates (npas), nasal swabs (nss), sputum (sp), and throat swabs (tss). npas, nss and tss were stored in viral transport medium (de conto et al., 2018) and all samples were kept at 4°c until submitted to laboratory (within 2 h of sampling). age was available for 2570 children (99.8%) and ranged from ≥2 days to 14 years (mean age: 3 years 6 months; median age: 2 years 1 month). children were divided into four age groups: 0 to ≤6 months (21.98%), n6 months to ≤3 years (37.78%), n3 to ≤6 years (18.95%), and n 6 to 14 years (21.28%). of note, 236 children (9.16%) were examined at least twice during the survey, with a time interval between care provider visits of 3 days to 8 months, presumably for either worsening symptoms of a current infection or infection recurrence. npas, nss, and tss were centrifuged (1000 rpm, 10 min, 4°c) and the pellets resuspended in phosphate-buffered saline (pbs, ph 7.4; 7 mm na 2 hpo 4 , 1.5 mm kh 2 po 4 , 137 mm nacl, 2.7 mm kcl), before cytocentrifugation (1000 rpm, 5 min). cell sediments on slides were dried in a laminar-flow hood and fixed in acetone (10 min, −20°c). these were then hydrated with pbs, blocked with 1% bovine serum albumin (bsa) in pbs, and the slides were incubated (1 h, 37°c) with anti-hrsv monoclonal antibodies (mabs) (1:40; argene/biomérieux, france) in 0.2% bsa in pbs. after washes with pbs, they were incubated with fluorescein isothiocyanate-conjugated anti-mabs (1:200; argene/ biomérieux) in 0.0001% evans blue in pbs (45 min, 37°c). after further washes with pbs, the sediments were mounted in glycerol and analyzed by epifluorescence microscopy (dmlb leica, germany). madin-darby canine kidney cells with 6-linked sialic acids enhanced expression (mdck-siat1) came from sigma-aldrich (italy), with human lung fibroblasts (mrc-5) from lgc standards (italy). human larynx epidermoid carcinoma (hep-2), embryonic human intestine (intestine 407), rhesus monkey kidney (llc-mk2), and african green monkey kidney (vero) cells came from the "istituto zooprofilattico sperimentale della lombardia e dell'emilia-romagna" (italy). cells were grown in earle's modified minimum essential medium (e-mem) supplemented with 2 mm l-glutamine, 10% fetal bovine serum, and antibiotics (100 u/ml penicillin, 100 μg/ml streptomycin), and maintained at 37°c in a 5% co 2 atmosphere. reagents were from life technologies (italy). after aspiration of the growth medium, mdck-siat1, hep-2, and intestine 407 cells grown in 8-well chamber slides (fisher scientific, italy) were incubated with samples (1 h, 37°c). after restoring the e-mem, incubation was carried out in e-mem for 24 h before iif with mabs against hadv, hpiv, hrsv, and influenza virus (iv) (argene/ biomérieux), as described above. hadv, hev, hpiv, hrsv, and iv isolation was performed in llc-mk2, mdck-siat1, hep-2, intestine 407, mrc-5, and vero cells grown in 24-well plates (sigma-aldrich). after aspirating the e-mem, cells were incubated with samples (1 h, 37°c). after restoring the e-mem, incubation in e-mem was continued until the appearance of cytopathic effect (cpe). if the cells showed no cpe after 7 days, they were scraped, and cell suspension was collected for a second inoculation. when cpe was observed, cells were scraped in pbs and cytocentrifuged, before iif. when cpe suggested hev infection, neutralization assays were performed. hevs serotype identification was performed with type-specific antisera (statens serum institut, denmark), according to lim and benyesh-melnick (1960) . samples were screened for hadv, hbov, hcov, hmpv, hpiv, hrsv, and iv nucleic acids with real-time (rt)-pcr or reverse transcription (rt)-rt-pcr assays. nucleic acid extraction was performed with the nuclisens® easymag® kit on the easymag extractor (biomérieux, italy) and rt with the rt-kit plus (elitechgroup molecular diagnostics, france) on the geneamp pcr system 9700 thermocycler (applied biosystems, thermo fischer scientific-waltham, usa). extracted nucleic acids were tested with influenza a/b q-pcr alert amplimix kit (elitechgroup) on the 7300 rt-pcr system (applied biosystems). the respiratory multi well system (mws) r-gene™ assay (argene/ biomérieux) was used to detect adv/hbov, hcov/hpiv, and rsv/ hmpv on the rotor-gene q system (qiagen, germany). assays were performed according to the manufacturer's instructions. a chi-square test was performed using graphpad prism software. p b 0.05 was considered statistically significant. a total of 2892 respiratory secretions from 2575 children were analyzed as described in the methods section; 1408 samples (48.69%) from 1299 (50.44%) children (714 males, 54.97%, and 585 females, 45.03%) were virus-positive (table 1 and supplementary table 1) . single infections were detected in 1128 (80.11%) samples from 1037 (79.83%) children. mixed infections were detected in 280 (19.89%) samples from 262 (20.17%) children. there was a significant difference in the frequency of single vs. mixed infections among positive samples (80.11% vs. 19.89%; p b 0.0001) as well as among total examined samples (39% vs. 9.68%; p b 0.0001). among mixed infections, two viruses were observed in 252 (90.00%) samples, three viruses in 26 (9.29%) samples, and four viruses in 2 (0.71%) samples. the percentage difference in the number of mixed infections with two viruses (90.00%) and four viruses (0.71%) was significant (p b 0.0001). overall, 1718 viruses were detected. hrsv (466/1718: 27.12%) and hadv (405/1718: 23.58%) were the most common viruses identified, followed by hcov (262/1718: 15.25%), iv (198/1718: 11.53%), hbov (161/1718: 9.37%), hpiv (114/1718: 6.64%), hmpv (76/1718: 4.42%), and hev (36/1718: 2.09%). hrsv and hadv were found in single (30.14% and 23.23%, respectively) and mixed infections (126/280: 45% and 143/280: 51.07%, respectively) ( table 1) the virus detection rate decreased from 53.9% (463/859) 34 (12.14) 2 (0.71) 0 (0) 0 (0) 0 (0) 0 (0) 36 (12.85) hrsv + hcov 13 (4.64) 22 (7.85) 0 (0) 0 (0) 0 (0) 0 (0) 35 (12.5) hadv + hbov 20 (7.14) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 20 (7.14) hrsv + hbov 11 (3.93) 5 (1.78) 0 (0) 0 (0) 0 (0) 0 (0) 16 (5.71) hrsv + iav 6 (2.14) 3 (1.07) 0 (0) 0 (0) 1 (0.36) 0 (0) 10 (3.57) hcov + iav 7 (2.5) 3 (1.07) 0 (0) 0 (0) 0 (0) 0 (0) 10 (3.57) hadv + hpiv 8 (2.85) 1 (0.36) 0 (0) 0 (0) 0 (0) 0 (0) 9 (3.21) hadv + hmpv 6 (2.14) 2 (0.71) 0 (0) 0 (0) 0 (0) 0 (0) 8 (2.85) hcov + hmpv 5 (1.78) 3 (1.07) 0 (0) 0 (0) 0 (0) 0 (0) 8 (2.85) hbov + hcov 6 (2.14) 1 (0.36) 0 (0) 0 (0) 0 (0) 0 (0) 7 (2.5) hbov + iav 6 (2.14) 1 (0.36) 0 (0) 0 (0) 0 (0) 0 (0) 7 (2.5) hrsv + ibv 7 (2.5) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 7 (2. for the 1295 children with viral artis and age available, the mean and median age were 2 years 9 months and 1 year 7 months, respectively. the infection rate was highest in children between n6 months to ≤3 years (57.36%; 43.01% among positive cases), followed by the 0 to ≤6 months (58.05%; 25.33% among positive cases) and n 3 to ≤6 years (49.9%; 18.76% among positive cases) groups (fig. 3a ). significant differences in the infection rate were evidenced among children aged 0 to ≤3 years and n 3 to ≤6 years (p b 0.05), among children aged 0 to ≤3 years and n 6 to 14 years (p b 0.0001), and among children aged n3 to ≤6 years and n 6 to 14 years (p b 0.0001), but not among children 0 to ≤6 months and n 6 months to ≤3 years (p n 0.05). hrsv had the highest prevalence (223/328: 67.98%) in children aged 0 to ≤6 months, followed by hcov (66/328: 20.12%) and hadv (42/328: 12.8%), while hadv prevailed in children n6 months to ≤3 years (218/557: 39.13%), followed by hrsv (167/557: 29.98%) and hcov (103/557: 18.49%) (fig. 3b) . in children n3 to ≤6 and n 6 to 14 years, hadv prevailed (94/243: 38.68% and 48/167: 28.74%, respectively), followed by iv (55/ of the 1408 virus-positive samples, 1373 were detected by molecular assays and 426 by culture (97.51% vs. 30.25%, p b 0.0001) ( table 2) . most of the culture-positive samples (421, 29.9%) were also positive by molecular methods, although different virus combinations were detected in 9 mixed infections. in discordant culture-negative samples molecular assays detected 262 hcov (18.6%), 161 hbov (11.43%), and 76 hmpv (5.4%). finally, of the 466 samples positive for hrsv by pcr, 233 (50%) were also detected by iif (100% vs. 50%, p b 0.0001). this survey carried out in parma (northern italy) from october 2012 to september 2015 describes the detection of viruses associated with respiratory samples from 2575 patients presenting with symptoms consistent with acute respiratory tract infections. overall, 50.44% of cases were positive by the simultaneous employment of different diagnostic assays. other studies have reported similar rates, although they refer to a single epidemic season and fewer number of cases examined exclusively by pcr (annan et al., 2016; pratheepamornkull et al., 2015; zuccotti et al., 2011) . however, different results have also been described (do et al., 2011; venter et al., 2011) . it must be considered that many factors could lead to prevalence variations, such as the virus panel examined, diagnostic methods, study setting, and geographic area. hrsv (27.12%) and hadv (23.58%) prevailed; hcov (15.25%), iv (11.53%), and hbov (9.37%) showed a higher frequency than hpiv (6.64%), hmpv (4.42%), and hev (2.09%). single infections predominated over mixed infections (80.11% vs. 19.89%, p b 0.0001), which was consistent with previous observations (martin et al., 2012; paranhos-baccalà et al., 2008) . of multiple infections, dual infection (90%) was predominant, in accordance with other authors (martin et al., 2012; peng et al., 2009) . hrsv, hadv, and hcov were mainly involved in mixed infections, as would be expected from their overlapping seasonal distribution. although hrsv and hadv are among the viruses most commonly involved in mixed infections (cantais et al., 2014; harada et al., 2013) , the relevance of rhinoviruses, hpiv, and iv has also been assessed (lu et al., 2013) . very complex combinations of mixed viral infections have been observed (lu et al., 2013; martin et al., 2012; paranhos-baccalà et al., 2008) and certain pathogens appear to have higher multiple infection frequency (brunstein et al., 2008) . in this scenario, superinfection exclusion mechanisms preventing a secondary infection with the same or a closely related virus should be envisaged (folimonova, 2012) . although mixed infections have been widely assessed in artis (moesker et al., 2016; seo et al., 2014; turner et al., 2013) , there is still no clear relationship with prolonged virus shedding, asymptomatic virus persistence, and disease severity. however, there is evidence that genome quantification could clarify the role of each virus in mixed infections (franz et al., 2010; martin et al., 2012) . accordingly, high viral loads have been related to severe diseases (franz et al., 2010; hasegawa et al., 2014; jansen et al., 2011) , but conflicting results have been reported (adams et al., 2015; wu et al., 2012) . in addition, some viruses have been detected in healthy children (advani et al., 2012; moe et al., 2016) . in this study, molecular assays showed greater sensitivity when compared to culture assays (97.51% vs. 30.25%, p b 0.0001) and hrsv antigen detection (100% vs. 50%, p b 0.0001). conversely, culture assays allowed the detection of infectious viruses in 426 (30.25%) of the 1408 positive samples, eventually providing a clue to discriminate the main causative agent in co-infections. nevertheless, cautious conclusions should be drawn, since the growth difficulties of some viruses may have decreased the number of those found by culture assays, while the long-term viral excretion after clinical recovery in children with frequent artis may have increased the mixed infection rate. the epidemic season of respiratory viruses ranged from 5 to 6 months, with peaks in february (2013 and 2014) or january (2015). the virus detection rate decreased significantly between the first and third seasons (53.9% vs. 43.05%, p b 0.0001), but environmental factors may have influenced the frequency and severity of artis (nenna et al., 2017) . hrsv showed a sharp winter/early-spring seasonality. when compared to previous reports, in the parma area the hrsv epidemic season was shorter (medici et al., 2006) . hadv circulated persistently throughout the study with higher rates in fall and spring of the first two years of surveillance, eventually indicating a reduction of circulation due to the accumulation of immunity in the population. the iv infection seasonality was consistent with previous reports (del manso et al., 2015a manso et al., , 2015b . no definite seasonality was shown by hbov, hcov, hmpv, hpiv, and hev. a correct viral diagnosis allows highlighting virus seasonality, which could favor both preventive measures and reduction of overuse of antibiotics. accordingly, in children with hrsv or iv, bacterial infections are less prevalent than in those without a viral infection (purcell and fergie, 2004; smitherman et al., 2005) . the virus detection rate was significantly higher in children aged 0 to ≤3 years (68.34%), when compared to the n3 to ≤6 years (18.76%, p b 0.05) and n 6 to 14 years (12.9%, p b 0.0001) groups, suggesting that immaturity of the immune system and absence of previous immunity could influence the severity of artis (hammond et al., 2007; monto, 2004) . hrsv prevailed (67.98%) in younger infants (0 to ≤6 months), while hadv was prevalent in all other age groups studied. accordingly, hrsv causes severe diseases in young children (moe et al., 2017; nguyen et al., 2017; shi et al., 2017) . for all the remaining respiratory viruses the highest detection rate was among children aged n6 months to ≤3 years. the attribution of an etiological role in artis to hbov has been extensively debated (debiaggi et al., 2012; schildgen et al., 2008) . in this study, hbov was most frequently found in single infections (55.9%) than in coinfections, strengthening the idea that it can be considered a pathogen. in conclusion, although this study has certain limitations, such as the lack of rhinovirus detection, it does provide relevant information on respiratory virus circulation in children with artis in an area of northern italy with a temperate climate. the simultaneous use of different diagnostic approaches made it possible to carry out in-depth analysis of a large number of cases spanning over 3 years. these findings contribute to estimate the disease burden associated with respiratory viruses, reinforcing the need for timely virologic diagnosis and continuous surveillance to optimize the prediction and control of artis in children. supplementary data to this article can be found online at https://doi. org/10.1016/j.diagmicrobio.2019.01.008. funding this study was funded by grants from the university of parma (fondi di ateneo fil 2014 -fil2014_deconto_mcs). this article does not describe any studies with human participants or animals performed by any of the authors. respiratory virus detection was performed according to the medical order; hence there was no need to obtain informed consent for the epidemiological analysis of the related data. comparison of human metapneumovirus, respiratory syncytial virus and rhinovirus respiratory tract infections in young children admitted to hospital detecting respiratory viruses in asymptomatic children cloning of a human parvovirus by molecular screening of respiratory tract samples similar virus spectra and seasonality in paediatric patients with acute respiratory disease, ghana and germany identification of new respiratory viruses in the new millennium virological and clinical characterizations of respiratory infections in hospitalized children evidence from multiplex molecular assays for complex multipathogen interactions in acute respiratory infections epidemiology and microbiological investigations of community-acquired pneumonia in children admitted at the emergency department of a university hospital mammalian diaphanous-related formin-1 restricts early phases of influenza a/nws/33 virus (h1n1) infection in llc-mk2 cells by affecting cytoskeleton dynamics the role of infections and coinfections with newly identified and emerging respiratory viruses in children influnet: sistema di sorveglianza sentinella delle sindromi influenzali in italia. rapporto sulla stagione influenzale 2013-2014 influnet: sistema di sorveglianza sentinella delle sindromi influenzali in italia. rapporto sulla stagione influenzale 2012-2013 viral etiologies of acute respiratory infections among hospitalized vietnamese children in laboratory diagnosis of respiratory virus infections in 24 hours by utilizing shell vial cultures superinfection exclusion is an active virus-controlled function that requires a specific viral protein correlation of viral load of respiratory pathogens and co-infections with disease severity in children hospitalized for lower respiratory tract infection rapid virus diagnosis: application of immunofluorescence prevalence and seasonal distribution of respiratory viruses during the 2014-2015 season in istanbul respiratory virus infection in infants and children does respiratory virus coinfection increases the clinical severity of acute respiratory infection among children infected with respiratory syncytial virus? respiratory syncytial virus genomic load and disease severity among children hospitalized with bronchiolitis: multicenter cohort studies in the united states and finland frequent detection of respiratory viruses without symptoms: toward defining clinically relevant cutoff values rapid detection and identification of 12 respiratory viruses using a dual priming oligonucleotide system-based multiplex pcr assay rapid viral diagnosis typing of viruses by combinations of antiserum pools. application to typing of enteroviruses (coxsackie and echo) etiology and clinical characteristics of single and multiple respiratory virus infections diagnosed in croatian children in two respiratory seasons epidemiology of human respiratory viruses in children with acute respiratory tract infections in jinan, china multiple versus single virus respiratory infections: viral load and clinical disease severity in hospitalized children four year incidence of respiratory syncytial virus infection in infants and young children referred to emergency departments for lower respiratory tract diseases in italy: the "osservatorio vrs" study respiratory virus detection and clinical diagnosis in children attending day care the burden of human metapneumovirus and respiratory syncytial virus infections in hospitalized norwegian children viruses as sole causative agents of severe acute respiratory tract infections in children occurrence of respiratory virus: time, place and person seasonality of influenza in the tropics: a distinct pattern in northeastern brazil respiratory syncytial virus bronchiolitis, weather conditions and air pollution in an italian urban area: an observational study acute respiratory infections in hospitalized children in vientiane, lao pdr -the importance of respiratory syncytial virus etiology, seasonality, and clinical characteristics of respiratory viruses in children with respiratory tract infections in eastern india (bhubaneswar, odisha) mixed respiratory virus infections ten years (2004-2014) of influenza surveillance in northern italy multipathogen infections in hospitalized children with acute respiratory infections causative agents of severe community acquired viral pneumonia among children in eastern thailand concurrent serious bacterial infections in 912 infants and children hospitalized for treatment of respiratory syncytial virus lower respiratory tract infection human bocavirus: passenger or pathogen in acute respiratory tract infections? etiology and clinical outcomes of acute respiratory virus infection in hospitalized adults global, regional, and national disease burden estimates of acute lower respiratory infections due to respiratory syncytial virus in young children in 2015: a systematic review and modelling study retrospective review of serious bacterial infections in infants who are 0 to 36 months of age and have influenza a infection respiratory viruses and influenza-like illness: epidemiology and outcomes in children aged 6 months to 10 years in a multi-country population sample respiratory viral infections in infants: causes, clinical symptoms, virology, and immunology fifteen years of epidemiologic, virologic and syndromic influenza surveillance: a focus on type b virus and the effects of vaccine mismatch in liguria region respiratory virus surveillance in hospitalised pneumonia patients on the thailand-myanmar border contribution of common and recently described respiratory viruses to annual hospitalizations in children in south africa severity of pandemic h1n1 2009 influenza virus infection may not be directly correlated with initial viral load in upper respiratory tract epidemiological and molecular surveillance of influenza and respiratory syncytial viruses in children with acute respiratory infections epidemiological and clinical features of respiratory viral infections in hospitalized children during the circulation of influenza virus a (h1n1)